research study about herbal plants

REVIEW article

Medicinal plant analysis: a historical and regional discussion of emergent complex techniques.

• 1 Herbal and East Asian Medicine, School of Life Sciences, College of Liberal Arts and Sciences, University of Westminster, London, United Kingdom
• 2 Pharmacognosy and Phytotherapy, UCL School of Pharmacy, London, United Kingdom

The analysis of medicinal plants has had a long history, and especially with regard to assessing a plant’s quality. The first techniques were organoleptic using the physical senses of taste, smell, and appearance. Then gradually these led on to more advanced instrumental techniques. Though different countries have their own traditional medicines China currently leads the way in terms of the number of publications focused on medicinal plant analysis and number of inclusions in their Pharmacopoeia. The monographs contained within these publications give directions on the type of analysis that should be performed, and for manufacturers, this typically means that they need access to more and more advanced instrumentation. We have seen developments in many areas of analytical analysis and particularly the development of chromatographic and spectroscopic methods and the hyphenation of these techniques. The ability to process data using multivariate analysis software has opened the door to metabolomics giving us greater capacity to understand the many variations of chemical compounds occurring within medicinal plants, allowing us to have greater certainty of not only the quality of the plants and medicines but also of their suitability for clinical research. Refinements in technology have resulted in the ability to analyze and categorize plants effectively and be able to detect contaminants and adulterants occurring at very low levels. However, advances in technology cannot provide us with all the answers we need in order to deliver high-quality herbal medicines and the more traditional techniques of assessing quality remain as important today.

Introduction

Medicinal plants have been a resource for healing in local communities around the world for thousands of years. Still it remains of contemporary importance as a primary healthcare mode for approximately 85% of the world’s population ( Pešić, 2015 ), and as a resource for drug discovery, with 80% of all synthetic drugs deriving from them ( Bauer and Brönstrup, 2014 ). Concurrently, the last few hundred years has seen a prolific rise in the introduction, development, and advancement of herbal substances analysis. Humans have been identifying and selecting medicinal plants and foods based on organoleptic assessment of suitability and quality for thousands of years, but it is only in the span of the last seven decades since the invention of basic analytical techniques, e.g., paper chromatography, that has seen rapid development from sight, touch, and smell to using sophisticated instrumentation. Though this mechanization of the senses has appeared relatively recently, historically conceptual expansion has been building throughout the scientific revolution, outwards toward the universe and inwards to a scale below recognition capable with a human eye, leading to development of some of the earliest analytical tools assisting the senses, the telescope and microscope. From the initial discovery of new microscopic worlds, through structural, chemical, and atomic levels, the sensitivity and range of human perception has been extended and enhanced.

Rapid progress is especially evident considering that the concept of a laboratory was only formally formed in Europe during the early 1600s. First as an extension of philosophers’, doctors’, and scientists’ workrooms, it becomes a space to study nature and gather empirical evidence ( Wilson, 1997 ), where studies could be conducted at the analyst’s convenience rather than at specific times when daylight or weather permitted. This was a small but important step towards more formalized analytical investigations.

In modern analysis, single techniques such as paper chromatography and much earlier colorimetry appeared. It was followed by a greater range and wider application of these techniques until early hyphenations such as LC-UV emerged, culminating more recently in multiple combinations of multi-hyphenated instrumentation, availing of the analytical advantages inherent in each individual technique. The emergence of hyphenated analytical techniques in many aspects is analogous to the organoleptic synthesis that occurs when selecting a medicinal plant; viewing, smelling and tasting it to use combinations of different senses, increasing the points of reference/statistical degrees of freedom to improve the probability of correctly identifying and assessing its quality. The emergence and application of these hyphenated techniques only became possible and useful as computer systems and data management tools developed, enabling rapid and selective synthesis of information from the large amount of instrumental and analytical data signals generated.

Probably the single greatest influence in recent times in the advancement of the analysis of herbal materials (and arguably analysis generally) is, though, how large amounts of data can be collected, assimilated, and used more meaningfully in human readable forms. Similar to the historical advancements in combinatorial hyphenated instrumentation, now combinatorial data processing techniques like fingerprinting, metabolomic profiling, and pattern recognition algorithms have emerged, further increasing analytical capabilities, while reducing operator time and expertise required. This trend has further accelerated the pace and rate of advancement of analytical techniques and has led to an increase in the pace and capability of the associated research. In this paper, we analyze publication trends and pharmacopoeial developments in order to better understand the role and progression of analytical techniques. Since their initial discovery and development, with a particular focus on China, an Asian country with both deep cultural and long-term historical roots in plant medicine, to more modern day developments and applications.

Publication Trends

Increasing interest in medicinal plant research and analysis is reflected in the number of recent publications, with more than a three-fold increase from 4,686 publications during the year 2008 to 14,884 in 2018. Output published during the 8 years of the present decade alone outnumbered all those combined before 2000, since the included database records began in 1800 ( Figure 1 ).

Figure 1 The herbal substance analysis publications trend since search records began in 1800. A keyword search was conducted using the combination “medicinal plant” OR “herbal medicine” AND “analysis” chosen for the maximum retuned records after exploring a list of similar topic and combination of keywords such as “photochemical analysis,” “traditional medicine,” and “herbal.” The Web of Science or collection, KCI- Korean Journal database, MEDLINE ® , Russian Science Citation index, and SciELO Citation index databases were included in the search.

The largest proportion of publications cited in current databases over the last 10 years for medicinal plant analysis reports are in the disciplines of pharmacology and pharmacy ( Figure 2 ). With plant sciences, biochemical molecular biology and agriculture research following closely behind, together comprising almost 70% of the total publications.

Figure 2 Herbal substance analysis publications by discipline, 2008–2018 (169,917 records).

Regional Trends—Last 10 Years

The majority (about 58%) of medicinal plant analysis publications in the last 10 years have collectively emerged from mainland China, India, USA, and South Korea ( Figure 3 ). This may be an expression of the strong medicinal plant traditions in Asia in addition to the USA’s dominant presence as an international user of herbal products ( Hu et al., 2013 ). The major East Asian regions, in particular, China, Japan, South Korea, together with Taiwan, contribute more than half of the total citations (55%). This may be indicative of the rapid economic progress and technological capability of these countries. China is the major contributor, with a 15% increase in its dominance of research outputs in the last 10 years. This influence has also been seen in the effect of China’s growing involvement in aiding the development of pharmacopoeias around the world and as a leader in the analysis of Chinese medicinal plants ( Figure 3 ).

Figure 3 Herbal analysis publications by region, 2008–2018.

Regulation and a Changing Analytical Landscape

From a regulatory perspective, the pharmacopoeial requirements are the central reference point for the analysis of medicinal plants. Though internationally many pharmacopoeias exist, the most comprehensive of these relating to herbal medicinal materials is the Chinese Pharmacopoeia (ChP). The current ChP introduced in 2015 is the 10th iteration presented in three volumes and includes 5,608 drugs, a 10-fold increase from its first edition in 1953. More than half of the current monographs ( Hamid-Reza et al., 2013 , 598) relate to CHM specifically including raw plants, slices, herbal mixtures, and oils. A noticeable inclusion in the current version compared with the previous version is the addition of 400 herbal mixtures ( Qian et al., 2010 ).

Pharmacopoeia Monographs—Their Influences and Challenges

Though more recently the ChP is playing an increasing role in influencing medicinal plant analysis, the development of the ChP has been heavily influenced by Western pharmacopoeias. Historically the identification, preparation, and analysis of medicinal plants were based on classic texts such as the Shengnong Bencao Jing (Shengnong Materia Medica, 25–220 CE), where the category and quality of 365 plants and 113 prescriptions were assessed by taste. Organoleptic sensing of bitterness, sweetness, saltiness, and even neutral tastes were thought to indicate the function and application of the medicine. Arguably, the most influential Chinese pharmacy monograph is the Bencao Gangmu (Compendium of Materia Medica, 1368–1644 CE) containing 1,892 plant descriptions and 11,096 prescriptions sorted in 16 divisions and 60 orders, emphasizing appearance, taste, and odor as a key to authentication and quality.

However, the main precursor to the modern format of the current Chinese Pharmacopoeia was printed in the 1930s with 670 drugs. Even at this early stage, the then dominant Western powers such as Britain, Germany, America, and Japan found challenges in understanding and forming consensus for recognizing, categorizing, and assuring the quality of Chinese medical materials. At this time a difficulty emerged in securing materials for the more Western styled “scientifically run” hospitals. Initially it was though that as Japan had adopted a translation of the German pharmacopoeia in 1886, the Chinese could follow suit using the British Pharmacopoeia, which in 1927 had been translated into Chinese as a joint effort by the London and British Chambers of Commerce. However, some differences in opinion between the four occupiers had to be first resolved.

Many of the technological demands necessary to produce and maintain the pharmacopoeial standards required by the Americans was beyond the ability and technological capability of the Chinese at that time. America had recently just printed a Chinese translation of its United States Pharmacopeia (10 th edition) published in 1926. The strict American standards for aconite, digitalis, adrenalin, and insulin were purported to be managed by new or foreign trained pharmacists ( Read, 1930 ). Preparations such as liniments found in the British and U.S. Pharmacopoeias were included in the Chinese version. Syrups such as those of codeine and glucose and tinctures of cannabis were from the British influence. Foreign residents in China found it difficult to ingest local food and stated an “extensive need for bowel remedies.” Therefore, drugs of the time, albuminis, aspidium, and emetin, were included. Vaccines for diphtheria, tetanus, and smallpox were maintained through the instruction of the USP.

German chemists had already gained a reputation for the isolation of chemical compounds, many of which were used medicinally and were already included in the Japanese Pharmacopoeia such as oxalic acid, pyrogallic acid, and bromine. Therefore, the existing German-Japanese analytical methods were generally utilized for these areas, which comprised about 25% of the new Chinese Pharmacopoeia. Whereas more British and American derived analytical methods and preparations were included for vegetable- and animal-based materials.

Agreement over the correct translation and naming of chemical compounds also proved problematic, e.g. when attempting to resolve disagreement between German-Latin and Anglo-American descriptions such as “natrium chloratum” and “sodii chloridum.” The shared Latin common language elements aided European and American common understanding; however, translation into Chinese was troublesome. A potentially easier route would have been to adopt the Japanese Pharmacopoeia names and descriptions, often possessing the same Asian (Hanzi) character as that in China, however, this was resisted due to the strong nationalistic sentiment at the time in mainland China ( Read, 1930 ).

Though the Japanese favored direct foreign phonetic transliterated terms for drugs, about 60 original Chinese materia medica entries had persisted in the Japanese Pharmacopoeia including entries for camphor, ginger, aloes, cardamom, and star anise.

Difficulty in plant identification and common naming was not confined to Asia. During the early 1900s period of European and American political expansion, attempts were being made in Europe to catalogue multilingual terms for similar plants such as the publication of “the illustrated polyglot dictionary of plants names” in Latin, Arabic, Armenian, English, French, German, Italian, and Turkish languages ( Bedevian, 1936 ), cataloguing 3,657 plants in eight languages.

Chronology of Pharmacopoeial Developments in China

1900–1949.

Medicinal plant publications during the early 1900s, before the formation of the People’s Republic of China in 1949, were greatly influenced by the previous “age of exploration.” Many scientific societies were set up by explorers, their peers, and investors as forums to communicate knowledge and acknowledge ownership of findings and discoveries ( Fyfe and Moxham, 2016 ). The rise in fashion of the “gentleman scholar” engaging in academic pursuits supported the occupation of writing. During this time, many publications focused on the identification and classification of ethnic/indigenous medical plants, such as Aztec medicinal plants still in use in modern Mexico ( Braubach, 1925 ; Heinrich et al., 2014 ), Algonquians from nowadays, Canada, ( Speck, 1917 ), Micronesians ( St John, 1948 ), Babylonians and Assyrians, ( Jastrow, 1914 ), Native American Indian tribes ( Castetter et al., 1935 ), Persia, ( Garrison, 1933 ) and India, ( Chopra, 1933 ). Publications in English describing the history and use of Chinese medicine in the context of Western orthodox also appeared ( Chan, 1939 ).

Periods of advancements in TCM research after 1949 to the present day have been described as occurring in three defined phases lasting about 20 years each. The first was 1950–1970, springing from the rapid development of TCM in universities, research, and hospitals in China during this time. The second phase took place during 1980–2000s, where we see the construction of legal, economic, and scientific networks. The third phase, from 2000 to date, is defined by a focus on elucidating the scientific basis and scientific clinical practice of TCM using cross-disciplinary and global collaborations ( Xu et al., 2013 ).

1950–1969

Political context.

This period immediately followed the formation of the People’s Republic of China and saw a rise in nationalism and political introspection. International relationships cooled and a closer connection with the Soviet Union was officially forged with the Sino-Soviet Treaty of Friendship, Alliance, and Mutual Assistance in 1950.

Regulatory and Pharmacopoeial Developments

This period saw the launch of the first edition of the People’s Republic of China Pharmacopoeia (ChP) in Chinese launched in 1953. It contains 531 monographs and mainly retains the information of the previous precursor published in the 1930s, compiled from foreign influences. It guided both identification and quantification of synthetic drugs and medicines together in one issue. Some crude herbal materials were listed, but not in analytical detail. Internationally post-World War II, good-will fostered a sense of cooperation and collaboration. This was also reflected by the World Health Organization’s release of the international pharmacopoeia (Ph. Int) issued by the World Health Organization in 1951, produced in two volumes. It contained 344 monographs and 84 tests, with an aim to provide a harmonized international reference for pharmacopoeial methods. The first European Pharmacopoeia Ph. Eur. was produced in 1967, with a more European focus, but combining many common elements of the long-existing British Pharmacopoeia and the United States Pharmacopeia.

Medicinal Plant Research and Analytical Development

Research publication output during the 1950s was varied but the most cited publication trends concerned identification of plant species using electron microscopy ( Watson, 1958 ), the use of plant tissue staining methods ( Bergeron and Singer, 1958 ; Fernstrom, 1958 ), and use of plant extracts for colorimetric analysis ( Holt and Withers, 1958 ; Lillie, 1958 ). Though originating in the 19 th century, the analytical tradition of extraction, purification, and separation of chemical plant components, e.g., the alkaloids, became increasingly sophisticated during this period ( Svoboda et al., 1959 ). Toxicity studies during this time were still basic, exposing mainly mice to plant extracts and using mortality rate counting and organ biopsy and cell station techniques, e.g., quercetin, podophyllotoxin, and podophyllin extract toxicity studies ( Leiter et al., 1950 ) and induced liver lesions with Pyrrolizidine alkaloid extracts ( Schoental, 1959 ).

Chemical screening of plants for their medicinal effects in various chemical and clinical trials is featured ( Farnsworth, 1966 ) as did their use in derivatized forms for the treatment of nerve inflammation ( Jancso et al., 1967 ) and in human metabolism studies ( Pletscher, 1968 ). Studies into the use of medicinal plants for their potential use in cancer treatments were encouraged by the first isolation of paclitaxel from the pacific yew, Taxus brevifolia Nutt.

Older basic chromatographic techniques that had been already in use remained commonly used analytical techniques, e.g., paper chromatography applied to the analysis of common broom [ Cytisus scoparius (L.) Link.] ( Jaminet, 1959 ) and in medicinal plant quality control ( Paris and Viejo, 1955 ). Separation of alkaloids e.g. in Duboisia myoporoides R. Br. ( Hills and Rodwell, 1951 ) remained a common interest and the analysis of other important metabolites including scilliroside in red squill, Drimia maritima. (L.) Stearn ( Dybing et al., 1954 ). An investigation of Cannabis sativa L. for its antibacterial activity was also conducted during this timeframe ( Krejci, 1958 ).

Much of the medicinal plant research of this period concerned the extraction and isolation of single compounds from plants. Basic colorimetric tests, UV-visible and infrared spectroscopy, and paper chromatography had previously supported this type of analysis. Spectroscopic techniques such as UV-Vis spectrometry with chart recorders had been in use since the 1920s ( Hardy, 1938 ). These were being increasingly used for quantitative applications, such as in the analysis of glucoside in walnuts and monitoring the chemical composition of plants in relation to seasonal variations ( Daglish, 1950 ).

However, the 1950–1970s was a golden period for the development of analytical technology. A time when the techniques of mass spectrometry (MS), nuclear magnetic resonance (NMR) spectroscopy, and gas chromatography (GC) techniques had come of age. Mass spectrometry, which had been invented in the late 1800s and used in a more analytical form during the 1910s, had now come into a relatively more advanced era. It was during the period 1950–1970 that the ion trap technique was developed, for which Dehmelt and Paul later received a Noble prize. The Purcell and Bloch groups at Harvard and Stanford University, respectively, developed NMR techniques and in 1952 also received a Nobel Prize (in Physics). In 1952, Archer John Porter Martin and Richard Synge also shared a Nobel Prize (in chemistry) for inventing partition chromatography, the basis of modern GC. Gas–liquid separations solved the problem of separating sugar-based molecules, which tended to bond with traditional stationery phases such as silica and volatile compounds, such as volatile oils, which are lost through evaporation during collection, preparation, and analysis. GC was applied for the first time to resolve 17 difficult to separate plant glycosides from a broad range of chemical classes, including phenolic, coumarin, isocoumarin, isoflavone, anthraquinone, cyanogenic, isothiocyanate, and monoterpene ( Furuya, 1965 ), 15 kinds of valerian sesquiterpenoids in valerianaceous plant oils ( Furuya and Kojima, 1967 ), and the extraction and analysis of rose oil ( Minkov and Trandafilov, 1969 ).

Publications included well-applied examples where visible, ultra-violet (UV), and infrared (IR) spectral data were combined to elucidate structural characteristics of plants while undergoing chemical degradation, e.g., the stereochemical discrimination of lignin components paulownin and isopaulownin from Paulownia tomentosa Steud. ( Takahashi and Nakagawa, 1966 ), the alkaloids of the Orchidaceae ( Lüning et al., 1967 ), and terpenoids of Zanthoxylum rhetsa DC ( Mathur et al., 1967 ).

MS was also used side-by-side with NMR, resulting in the structural elucidation of key metabolites, e.g., the characterization of the opium papaverrubine alkaloids and their N ‐methyl derivatives in the genus Papaver ( Brochmann-Hanssen et al., 1968 ), the analysis of three new coumestan derivatives from the root of licorice, Glycyrrhiza spp., ( Shibata and Saitoh, 1968 ), and the isolation and purification of polyprenols from the leaves of Aesculus hippocastanum L. (horse chestnut) ( Wellburn et al., 1967 ).

Up to this time, China had played a very marginal role in international research and development activities, a situation that was to change significantly in the following period.

1970–1989

1971 saw China’s introspection from the Mao era revert to more external international engagement with the “People’s Republic of China” (PRC) elected as a permanent member of the United Nations’ General Assembly. This followed the American government’s extension of political relations with PRC after the Richard Nixon presidential visit that catalyzed an “Opening up to the West” phase in Chinese history. This opening began in 1978, orchestrated by the interim leader Deng Xiaoping, who initiated support for wide sweeping economic reforms. On a local level this manifested as individuals within China being allowed to make personal economic decisions, with the tightly governed communes being dissolved. Rural markets were replaced by open markets, resulting in a dramatic increase in international trade, supporting Xiaoping’s wish to fund economic growth from foreign investment. In the context of medicine, China’s ambition to look outward was highlighted over a decade earlier by a University College London anatomy Professor, Derrick James, when a British delegation visited China in 1954 and in his subsequent Lancet article outlined China’s intention to introduce a more scientific, modernized TCM ( James, 1955 ).

As international trade from China expanded, so did the trade in medicinal plants from Asia and with it, increased access for Chinese scientists to modern analytical instrumentation. Internally by the mid-1980s, 25 Chinese medicine colleges were formed in a reportedly scientific and modern style with an almost 30-fold increase of TCM hospital beds to 2.5 million since the formation of the state in 1949 ( Cai, 1988 ).

The establishment in 1985 of the China State Administration of Traditional Chinese Medicine began the formal organization of TCM research and development nationally and internationally, sowing the seeds for the formal cooperative global links that would provide the backbone for the future of international Chinese medicinal plant research. China’s motivation to secure international links was also manifest in the publication of the PRC’s first dual Chinese and English language Pharmacopoeia, ChP, 4 th edition in 1997, which began its new 5-year publication cycle trend.

Medicinal Plant Research and Analytical Developments

The newly fostered R&D investment and cooperation during this period globally is represented by the leap in sophistication and complexity of the research published, with a shift from basic to more advanced biochemical investigations and more emphasis focused on disease and diagnosis strategies such as in cancer and infectious disease. The most widely cited articles of this time include advanced biomedical research on Forskolin, from the roots of Plectranthus barbatus Andrews as a diterpene activator in nucleotide metabolism. Even though basic biochemical equipment and colorimetric methods and spectrometric enzymatic assays were used, a more complex understanding of plant metabolites is apparent ( Seamon et al., 1981 ).

This is also evident in the investigation of lectins as cell recognition molecules and their involvement in a wide range of molecular processes and potential pathologies, e.g., in metabolic regulation, viral, and bacterial infection processes ( Sharon and Lis, 1989 ). In addition to plants playing a role as phytochelants in complexing heavy metals ( Grill et al., 1985 and Grill et al., 1987 ), licorice was studied in greater depth using a conceptually new approach of assessing the mineral-corticoid activity of licorice and its role in sodium retention ( Stewart et al., 1987 ) and the radical scavenging properties of its flavonoids ( Hatano et al., 1988 ).

Awareness of plants having a role in cancer with both causative and curative effects emerged, with a highly cited review of potential causes of esophageal cancer in China. Particular concerns were linked to effects of fungal growth and associated nitrosamines due to poor storage conditions ( Mingxin et al., 1980 ). This was a precursor to later studies on aflatoxins, which are now acknowledged as causing serious health problem linked to poor storage and processing. From a therapeutic perspective, the interest in antileukemia and anti-tumor agents, e.g., in Taxus brevifolia Nutt. stem bark, first investigated some decades before, continued and ultimately resulted in the introduction of a completely new therapeutic approach ( Wani et al., 1971 ).

One of the landmark discoveries in medicinal plant history was reported to the west during this period. The antimalaria effect of artemisinin, derived from Artemisia annua L., for which the Chinese scientist Youyou Tu later received a Nobel Prize in Medicine ( Klayman, 1985 ), described a conceptual shift in the approach to treating malaria, illustrating both a change in approach from using quinoline-based drugs, which parasites were showing increasing resistance to, and paving the way for the development of new classes of drugs e.g. with potential in antiviral and anticancer treatment ( Su and Miller, 2015 ).

1990–2008

This period in China was characterized largely by economic, political, and academic success delivering on the earlier aspirations of Deng Xiaoping through focused planning and the tight administrative grip of three successive presidents (Chairpersons) and state administration. An unusually high-performing economy producing more than a 10% sustained gross domestic profit (GDP) created a stable base for China to successfully join the world trade organization in 2001, marking its arrival on the world stage as a competent economic power and its transition to a market economy ( Morrison, 2013 ). This, however, came with challenges to families and the environment.

On a local level as communes of the last decades had dissolved, a system of “household responsibility” was adapted as a kind of contract that guaranteed agricultural family holdings to provide a certain level of food (and herb) output ( Ash, 1988 ). This ensured that levels of agricultural production were optimized for the land available. Because families were now allowed to sell grown products in an open market that mirrored the economic national trend, food and medicinal herbs began to take on more distinct financial attributes. This combined with mass migration of rural workers to rapidly developing industrialized cities away from countryside homes without sufficient locally produced food in urban surrounds created a situation of widespread supply and demand, leading to new value chains for food and medicinal plant products, along with potential motivation for the substitution or adulteration of these products.

As industrialization occurred so too did environmental pollution, with increased volume and concentration of raw materials and waste presenting greater potential for pollution of medicinal plant material. The PRC at this stage had gone through a period of prolonged political stability. Economic policy became more flexible and governance developed an increasingly regulatory role compared with that of previous, more rigid enforcement. Regulation and safety testing of medical products saw further guidance through the production of four further volumes of the ChP in both Chinese and English culminating in the 8 th edition in 2005, listing 3,217 monographs, almost double that of the 1990 edition. This period saw China’s confidence increase and extend to regulatory and guidance aspects, with the ChP undergoing the greatest leap in analytical sophistication and rate of change to date. The 1990 edition was a significant step in the acceptance and introduction of modern instrumental analytical techniques for standard herbal substance testing. Since the 1985 edition, specific identification tests were introduced using mainly thin layer chromatography (TLC). Now chromatogram images of the crude and test samples were included and required for testing. Basic identification was expanded to require quantitation where high-performance liquid chromatography (HPLC) and GC were now included for the first time and TLC extended for content analysis. More instrumental techniques replaced older ones such as the introduction of spectrophotometric determination of the alkaloid content of berberine, which had been gravimetrically analyzed in previous editions. Quantification moved from measuring simpler marker components to more specific active compounds like anthroquinone from He Shou Wu, Polygonum multiflorum Thunb [now Reynoutria multiflora (Thunb.) Moldenke]. The 2000 edition introduced assays for residues of organic chlorine pesticides for Gan Cao, Glycyrrhiza uralensis Fisch. ex DC. and Huang Qi, Astragalus membranaceus Fisch. ex Bunge ( Kwee, 2002 ). Another leap occurred in the 2005 edition with an expansion of the acceptance of HPLC-MS, LC-MS-MS, and DNA molecular markers and chemical fingerprinting, setting the stage for 21 st century pharmacopoeial trends and the ChP as a central global influence for the analysis of medicinal plants.

The fruition of investment in external academic relations from the “opening up” phase and internal support for the now formed TCM structures of the previous decades state initiatives were borne out by the publication output in this period, with a six-fold increase in output compared with that of the previous equivalent 20-year period. Much of the output from this time demonstrated a refinement of thought around the effect of plant compounds on humans as a holistic system rather than the more singular metabolic pathway thinking of previous years. It also shows a tremendous emphasis on obtaining large datasets especially of the known metabolites and a wide exploration of acclaimed effects. Whole plant extracts and combinations of metabolites rather than single ones became a core theme, as became a medicinal plant’s effect on longer term health and preventative medicine. This ignited a resurgence of interest in the analysis of medicinal plants as a source of lead compounds for drug discovery.

The role of medical plants in coronary disease analysis becomes topical during this phase, e.g., long-term studies on elderly demonstrating the reduced risk of death from sustained flavonoids intake via inhibition of the oxidation of low-density lipoprotein ( Hertog et al., 1993 ). More sophisticated quantitative analysis and differentiation appeared during this time such as HPLC of mulberry leaves containing four varieties of flavonoids (including rutin and quercetin), and their antioxidant properties ( Zhishen et al., 1999 ). Flavonoid coronary disease risk prevention and cancer roles were advanced by the characterization and analysis studied in a wide range of fruits, seeds, oils, wines, and tea ( Middleton et al., 2000 ). A greater awareness of the potency and efficacy of drugs and medicinal plants became evident as in the studies and analysis of the effect of fluorine on drug binding and potency ( Purser et al., 2008 ). Cancer research also demonstrated further advances through combining previous findings on receptor binding with advancements in DNA extraction, amplification techniques, and cloning techniques. Resveratrol became a key area of interest for its chemoprotective effects ( Jang et al., 1997 ).

Many of the most cited publications of these two decades were detailed reviews, which brought together the findings of previous research on individual plant research.

21 st Century

China’s growing influence was marked in 2011 with the Chinese State Administration of TCM (SATCM) forming an official relationship with the European Directive on the Quality of Medicines (EDQM) to share expertise and knowledge in addition to raising the standards of testing in China and Europe through cooperation. These include translation of historical TCM documents, information relating to preparation of products, process, and sourcing. Europe, seen as an aggregate, has an approximately 16% representation in the last decades’ research output, higher than the USA. The European Pharmacopoeia (Ph Eur) manages CHM’s by allowing importation of CHM’s to countries who have signed up to the European Pharmacopoeia convention. Currently there are 43 CHMs included in the Ph Eur, 8th edition, 34 from the Ph Eur TCM Working Party, 21 of which have been included as full monographs ( Wang and Franz, 2015 ). New Ph Eur CHM monographs are being developed based, in part, on the ChP. This was facilitated by a working party on TCM (Ph Eur WP) and was officially introduced in 2005. It included 38 member states with a delegation from the EU (a representative from DG Health & Food Safety and the European Medicines Agency). Additional observers are composed of 27 countries/regions/organizations [which include 7 European countries, the Taiwan Food and Drug Administration (TFDA), and World Health Organization (WHO)] ( EDQM, 2017 ). The WHO, through participation in the PhEur, additionally has led efforts to develop a harmonized international pharmacopoeia ( WHO, 2018 ).

The monographs for medicinal plants in Ph Eur have developed from standard western drug monographs with an emphasis on chemical and physical testing, while those in the ChP have formed from revisions of older traditional texts.

As pharmacopoeial monographs expand and develop, so too does the range and complexity of analytical methods and analytical hardware needed to meet the regulatory demands and expectations of quality.

These emerging research trends and pharmacopoeial directives have paved the way for the development of a broad range of analytical techniques, mainly centering around the use of liquid chromatography (LC), GC, MS, and established UV/visible spectrophotometric techniques.

We present a selection of these analytical techniques and give examples of their applications in the analysis of medicinal plants and medicinal plant products.

Analytical Hardware, Attested and Emerging Methods

High-performance liquid chromatography.

HPLC is one of the most developed and widely used analytical techniques. It is built on a historical knowledge base amassed from TLC and optical chemistry experience. HPLC chromatography elements rely on similar principles of TLC/HPTLC, where separation of components is dependent on selective affinities to stationary supports and liquid phases.

Detection employs a photomultiplier system able to detect individual wavelengths of light, a range (spectrum) and/or multiple simultaneous wavelengths in its different iterations, combined in an enclosed automated instrument system with sample injectors; this has significantly increased the precision and reproducibility of the chromatography when compared with older chromatographic methods. The widespread use of HPLC has made it more affordable for laboratories. High operator skill level is not required; it is robust and sensitive to low level detection and is particularly used for the quantification of components (active substances and adulterants).

HPLC applied to herbal products is well developed, and it has been successfully applied to the analysis of complex mixtures of similar compounds, both for the separation of individual compounds and for the differentiation of medicinal plant species. The high resolution of the technique has supported the development of the concept of a characteristic “fingerprint” developed for medicinal plants and herbal products to aid identification and authentication, e.g., Li et al. (2010) demonstrated differentiation of the same type of medicinal plant product from 40 different manufacturers, while simultaneously separating nine marker chemical compounds (berberine, aloe-emodin, rhein, emodin, chryso- phanol, baicalin, baicalein, wogonoside, and wogonin).

High-Performance Thin Layer Chromatography

HPTLC has become a common addition to the method section of new monographs, replacing the widely used TLC tests; it has shown to be a reliable and reproducible method of analysis that provides essential information regarding the compositional quality of an herbal substance.

Some advantages of this technique include low cost and a relatively simple test method. It does not require advanced sample preparation methods or high levels of expertise. Sample amounts are relatively small, and it is a more sensitive technique compared with HPLC, well suited to detecting contaminants. However, some disadvantages are that the reproducibility is dependent on a variety of external factors, and although more sensitive than HPLC, it is not able to sufficiently detect compounds at very low concentrations (PPB) where LC-MS (or HPTLC-MS) may be more suitable. HPTLC relies on the same principle as TLC and uses similar TLC plates and mobile phases, although relatively small amounts of solvents are required compared with standard TLC. The process of adding the sample to plates (spotting) has been made more reproducible and precise by spraying the sample onto the plate to form a band of compound rather than a spot. Retention factors for individual compounds are more reproducible due to controlled humidity during development. Derivatizing the analysis plates is completed mainly by machine and the visualization is captured by modern camera systems connected to powerful software. The software allows further manipulation of images to optimize visualization in a way that would be very difficult chemically. Another advantage is that the HPTLC system can be easily linked to a scanning densitometer; this not only allows for more precise quantitative work to be carried out but also the data can be exported for multivariate analysis. It is likely that more of the monographs with TLC requirements will be upgraded to HPTLC in the future.

Gas Chromatography

GC in respect to medicinal plant analysis is mainly used for the analysis of compounds with higher volatility, e.g., compounds found within essential oils, and more volatile adulterants, e.g., pesticides. While single GC column chromatography and its hyphenated derivatives have been use for many years, 1991 saw the introduction of 2D-GC or GC x GC, where the eluents of a standard separation are trapped and recirculated for another round of separation. This allows not only greater resolution and better separation but also the ability to purge undesired or interfering compounds so that more specific areas of the separation can be targeted ( Liu and Philips, 1991 ). This led the way for multidimensional gas chromatography (MDGC) and the advances of the modules and valve systems that trap, control, and divert sample streams. These improvements extend to the thermal control and valve systems allowing greater thermal flow and split streaming ( Bahaghighat et al., 2019 ). One key problem with GC is the introduction of sample into a gas stream. Historically squeezing, boiling, and later distillation of herbal materials were used for the collection and production of volatile compounds such as oils. However, the inherent instability of volatile components and losses as well as the poor recovery of these substances presented difficulties. This situation has somewhat been overcome by advances in extraction techniques such a solvent-free microwave extraction, e.g., for citrus peel oils [Citrus sinensis (L.) Osbeck]. No solvents or water are necessary for high recoveries with this method, and it allows for highly efficient, compatible sample introduction without the need for interfering solvents ( Aboudaou et al., 2018 ). This sample extraction method commonly known as headspace analysis for GC has undergone many iterations ( Gerhardt et al., 2018 ). It has now developed to the stage where it is increasingly used for bacterial and microorganism detection such as in Commiphora species ( Rubegeta et al., 2018 ).

Microextraction techniques are essential for the introduction of small sample volumes into the GC gas stream. Needle-based extraction techniques have the advantage of automation, ease of interface to other instruments, and compatibility with miniaturization. Advances in solid phase dynamic extraction (SPDE), In-tube extraction (ITEX), and needle trap extraction (NTE) have refined the use of these techniques for natural and herbal compounds ( Kędziora-Koch and Wasiak, 2018 ), e.g., SPDE and ITEX for pesticide residues in dried herbs ( Rutkowska et al., 2018 ), herbal mint aromas compounds in commercial wine ( Picard et al., 2018 ), and volatiles in Chinese herbal formula Baizhu Shaoyao San ( Xu et al., 2018 ).

Supercritical Fluid Chromatography

Another liquid-based chromatographic technique based on pressurized low viscosity (supercritical) fluids, often carbon dioxide, is supercritical fluid chromatography (SFC). Since its introduction by Klesper in 1962, it has made large advances mainly due to improvements in its initially troublesome instrumentation ( Desfontaine et al., 2015 ). Its main advantage over other techniques is in its usefulness for separating complex components characteristic of natural compounds. Selection of the correct conditions of SFC mobiles phases and modifiers can be finely tuned across a wide range of polarities from non-polar to polar allowing a broad selection of separations ( Gao et al., 2010 ). Early analysis of natural products with SFC was when it was first hyphenated with gas chromatography ( King, 1990 ). Recently, it has been more fully developed to analyze a range of natural compounds in herbal substances, notably, focusing on terpenes, phenolics, flavonoids, alkaloids, and saponins. This has been achieved with hyphenation to MS, diode array detectors, SFC-ELSD, in addition to the development of novel stationary phases such as cyanopropyl, pentaflouro phenyl (PFP), and imidazolyl. An example of this is with the separation of coumarins in Angelica dahurica (Hoffm.) Benth. & Hook.f. ex Franch. & Sav. roots and anthraquinones in rhubarb root ( Pfeifer et al., 2016 ).

Near-Infrared Spectroscopy

Although commonly used within industry since the 1990’s, near-infrared (NIR) spectroscopy was not the method of choice for medicinal plant analysis mainly due to overlapping peaks making interpretation of data problematic, and consequently, it never became the instrumentation of choice within the quality control laboratory in the same way that HPLC and TLC developed. However, with the addition of new computational software, NIR is re-emerging as an affordable and useful analytical technique used in the analysis of medicinal plants and has been particularly favored by Chinese companies in routine quality control analysis due to its ability to both rapidly differentiate between species and provide quantitative information on metabolite content ( Li et al., 2013 ; Zhang and Su, 2014 ).

As with HPTLC and NMR data, NIR also provides an opportunity for multivariate analysis and it appears capable of resolving very small variations in metabolite content. It is argued that more traditional TLC or HPLC techniques can be more subjective in the data interpretation stage and require a high degree of operator skill and that NIR is more suitable for high volume analysis in the routine quality control laboratory ( Wang and Yu, 2015 ). However, this has partly been addressed by the introduction of the fully automated systems available for HPTLC analysis and the inclusion of scanning densitometry equipment that reduce the need for operator interpretation. The main advantages of NIR appear to be the preservation of sample integrity, little sample preparation needed, and no need for solvents, and it has shown to perform well comparable to HPLC for species differentiation and quantification of metabolites ( Chan et al., 2007 ). Probably the main drawback in NIR compared with other methods, and especially, TLC, HPTC, LC-MS is in its sensitivity and some reports suggest that this technique may only be suitable for detecting compounds that exist at a concentration above 0.1% ( Lau et al., 2009 ). Another consideration is that variation in NIR data is dependent both on the chemical and physical properties of the sample, with the physical properties, e.g., particle size, having greater effects on the variation than the chemical. Therefore, before multivariate analysis can take place some pre-treatment of the spectral data is necessary, e.g., to reduce baseline noise, light scattering, and consequently enhance any chemical variation in the sample set ( Chen et al., 2008 ). Some advantages of NIR certainly are apparent, although it may not be appropriate for all situations and all types of samples. The technology has made a huge leap forward since its first introduction and now it needs to establish itself more widely as a useful tool in the quality analysis of medicinal plants.

Hyphenated Techniques

Combinations of techniques with modern developments in metabolomic analysis and computational pattern recognition programs open up a wider scope of applications to medicinal plant analysis. Tandem combinations of analytical instrumentation such as MS with HPLC has proved a productive route to expanding analytical medicinal plant applications. Not only in identification and fingerprinting but further chemical characterization of individual compounds e.g., Liu et al. (2011) , characterized a spectrum of alkaloid components in the Chinese herb Ku Shen ( Sophora flavescens Aiton). Further combinations and permutations of MS and NMR in combination with HPTLC have been demonstrated, such as the detection of acetylcholinesterase inhibitors in galbanum in a search for natural product drug candidates ( Hamid-Reza et al., 2013 ), and mass spectroscopy (MS) HPTLC-MS shown for Ilex vomitoria Aiton with the use of a sampling probe following HPTLC combined with MS with Electrospray Ion Trap ( Ford and Van Berkel., 2004 ) and Hydrastis canadensis L., with HPLTLC-MS atmospheric pressure chemical ionization ( Van Berkel et al., 2007 ).

Analytical combinations including ESI-IT-TOF/MS-HPLC-DAD-ESI-MS have been demonstrated for the analysis of coumarin patterns in Angelica polymorpha Maxim. roots ( Liu et al, 2011 ) and multihyphenated techniques such as SPE-LC-MS/MS-ABI quadrupole trap have been used for the analysis of six major flavones in Scutellaria baicalensis Georgi ( Fong et al., 2014 ) and 38 saponins in the roots of Helleborus niger L. by LC-ESI-IT-MS ( Duckstein et al., 2014 ).

Merging the separation ability of HPTLC or HPLC with the analysis power of NMR and MS has significant benefits for analyzing complex samples in complex matrices such a blood, soil, and plants. However, each technique also possesses its inherent disadvantages. MS being complex, expensive, and time-consuming, requiring high analytical skill levels, it may not be suitable for a general quality assurance laboratory. Though powerful, extensive method development and post analysis data processing is required when applied to natural compounds with broad complex compositions in contrast to simpler synthesized pharmaceutical ingredients. Similarly, NMR is also expensive and sensitive to variations in sample preparation and composition. It is not fully applicable to all natural compound samples and signals generated from NMR analysis often overlap making data analysis for individual compounds problematic. However, the relative speed, rich information output, and insight into the overall composition of medicinal plants from both MS and NMR far outweigh the disadvantages. These techniques allow the detection of compounds into the parts per billion analytical range (MS) and allow a detailed fingerprint of metabolites across differing polarities (NMR) and so for research and for larger companies they are highly applicable analytical hardware.

Metabolomics

Pharmacopoeial methods focus on authentication and quality of herbal materials; however, metabolomics allow us to go a step beyond authentication and look in more detail at a broad range of secondary metabolites. By coupling analytical data to multivariate software, this allows us to develop statistical models to firstly differentiate between species but also to get a better idea of a typical metabolite composition for a particular species. The advantage of this is that it can help to inform any laboratory test or clinical intervention. There has been great emphasis on making sure that any experiment or intervention uses plant material that is authenticated, with a herbarium specimen deposited. However, the requirements do not stipulate that a good representative of the species should be used. This is where metabolomics can provide essential information—by collecting a wide range of samples from different geographical locations, altitudes, growing conditions, it allows us to map their metabolite differences and highlight how diverse or how similar metabolite composition is. When an experiment is performed, we have the choice to use a specimen that may be typical, i.e., contains an average composition or we can look at compositions that are atypical, containing greater amounts of specific metabolites or even different metabolites. Moreover, if a particular experiment produces positive results and we want to reproduce the data, a metabolomic model allows us to choose species that have a similar composition.

This approach has important economic implications as a detailed understanding of metabolomic analysis allows us to inform industry as to how to grow plants that will be of the best composition and so help to support local livelihoods of farmers and primary processors in developing economies, e.g., Chachacoma ( Senecio nutans Sch. Bip. ) cultivation in the high altitude regions of Chile where metabolomics has helped to establish the best altitude for growing plants with the highest content of the anti-inflammatory acetophenone ( Lopez et al., 2015 ).

This strategy also has applications in product development, where metabolomics can help to determine the quality of products based on their metabolite content, e.g., Curcuma longa L. (Turmeric products) ( Booker et al., 2014 ), and also help to provide evidence that can lead to value addition of a product and greater confidence in its quality and safety.

Nanoparticles

Nanoparticles 1–100 nm sized ions or organic/inorganic molecules have proven to be important in the development of new analytical testing ( Tao et al., 2018 ), occupying the analytical regions of space between the ionic dimensions and small molecules.

Recent developments in nanoparticle research has led to an increased focus on chemo-bio sensing, as DNA has become the most used biological molecule to functionalize nanoparticles. Nanoparticles have provided many advantages to more consistent and specific testing including providing a more reproducible stable matrix for research and development, more controllable and reliable basis for designing and conjugating to functional molecules, and a wide rebate of flexibility for purification, selection, and modification of analytes. Nanoparticles have been used in creating a biological bar code for trace analysis of mycotoxins in Chinese herbs e.g. conjugated nanoparticles with DNA fragments to bind and target Chinese medicinal plants, e.g., Jue Ming Zi [Cassia seeds— Senna obtusifolia (L.) H.S.Irwin & Barneby], Yuan Zhi ( Polygala tenuifolia Willd.), and Bai Zi Ren [ Platycladus orientalis (L.) Franco] ( Yu et al., 2018 ).

The next steps in analytical advancement in combination with technological improvements will most likely occur in the realm of artificial intelligence. Neural networks have already shown promise in consumer electronics and online search engine optimization. Self-learning algorithms have been in development for decades, with great potential for the application of self-synthesizing, auto-creating, and auto-adapting algorithms, which can optimally recognize and synthesize analytical data into meaningful and useful patterns. This goes beyond what a single human mind could hope to achieve in lifetimes, now possible in seconds with current and more so with future technology. This extends not only the human potential of thinking and observation but also prediction and design. This could potentially play a role in self-design of analytical instrumentation and its modules, self-optimizing of methods in real-time, saving time that would perhaps take an analyst weeks or months of human work-hours to complete.

The greatest challenge with AI is its opacity and computational complexity. With self-learning systems already self-generating codes and pathways that would take decades for a single human to decode and understand, if ever possible. This presents a great challenge for use in reproducible, validated quality-driven, audit-trailed regulated orientated environments. This is where natural compounds such as herbal substances can play a significant role i.e. data from the same plants species with variable composition can help verify the input and outputs of complex analysis and recognition software. In AI-driven systems, natural substances are ideal candidates for testing the analytical attributes such as accuracy, precision, and robustness of whole AI-instrumentation systems.

Conclusions

As pharmacopoeial requirements continue to develop and instrumental technology advances, it is clear that we will be able to delve further and further into the chemical composition of medicinal plants and develop more advanced techniques for the detection and quantification of adulterants and contaminants. However, it should be considered that although these technological advances give us this opportunity, more traditional organoleptic analysis also provides us with essential sensory information regarding medicinal plant quality.

We have shown the emergence and historical importance of complex analytical techniques used in medicinal plant analysis. However, any analytical approach, can only provide a partial perspective on complex multicomponent preparations. So future improvements in this area may not entirely rely on developing ever more complex analytical techniques, but in implementing best practice throughout all stages of the production and supply of herbal medicines.

Author Contributions

AB wrote the sections on applications of metabolomics, NIR, parts of the introduction, and conclusions. MF wrote most of the instrumentation, trends in publications and history, part of the introduction and conclusions. MH contributed towards the methodological design of the study and assisted with the data analysis.

MF scholarship is funded by Brion Research Group (Sun Ten Pharmaceutical Co) and Herbprime, UK.

Conflict of Interest

The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.

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Keywords: herbal medicine, medicinal plant, analysis, quality, pharmacopoeia, complexity, advances

Citation: Fitzgerald M, Heinrich M and Booker A (2020) Medicinal Plant Analysis: A Historical and Regional Discussion of Emergent Complex Techniques. Front. Pharmacol. 10:1480. doi: 10.3389/fphar.2019.01480

Received: 04 September 2018; Accepted: 14 November 2019; Published: 09 January 2020.

Reviewed by:

Copyright © 2020 Fitzgerald, Heinrich and Booker. This is an open-access article distributed under the terms of the Creative Commons Attribution License (CC BY) . The use, distribution or reproduction in other forums is permitted, provided the original author(s) and the copyright owner(s) are credited and that the original publication in this journal is cited, in accordance with accepted academic practice. No use, distribution or reproduction is permitted which does not comply with these terms.

*Correspondence: Anthony Booker, [email protected]

Disclaimer: All claims expressed in this article are solely those of the authors and do not necessarily represent those of their affiliated organizations, or those of the publisher, the editors and the reviewers. Any product that may be evaluated in this article or claim that may be made by its manufacturer is not guaranteed or endorsed by the publisher.

Herbal Teas and their Health Benefits: A Scoping Review

Affiliations.

• 1 Department of Applied Sciences, University of the West of England, Frenchay Campus, Bristol, BS16 1QY, UK.
• 2 Pukka Herbs, The Chocolate Factory, Bristol, BS31 2GN, UK.
• 3 Centre for Research in Biosciences, University of the West of England, Frenchay Campus, Bristol, BS16 1QY, UK.
• 4 Pukka Herbs, The Chocolate Factory, Bristol, BS31 2GN, UK. [email protected].
• PMID: 31243622
• DOI: 10.1007/s11130-019-00750-w

Herbal teas are used as therapeutic vehicles in many forms of traditional medicine and are a popular global beverage. The purpose of this scoping review was to examine the evidence relating to the clinical efficacy and safety of herbal teas, and to identify the main research themes and gaps in knowledge to inform further work. A scoping review methodology was followed that set out the research question and described the sourcing, selection and analysis of studies. Overall, a total of 145 research publications were retrieved from global bibliographic databases, and after applying exclusion criteria, 21 remained. These studies looked at herbal tea use in female health, diabetes, heart disease and weight loss, with plant species including lavender, chamomile, fenugreek, stinging nettle, spearmint, hibiscus, yerba maté, echinacea and combinations of herbs. Observational studies explored associations between herbal tea consumption and cancer risk, liver health, and the risks linked to the consumption of environmental contaminants in the plant material. Despite plant materials being the basis for drug discovery, and the popularity of herbal teas, the number of articles exploring clinical efficacy and safety is small. In this review we discuss how herbal teas may be beneficial in some areas of clinical and preventative health, and what further research is required to understand whether regular consumption can contribute to healthy living more generally.

Keywords: Herbal tea; Infusions; Phytochemicals; Plant biodiversity; Plant medicine.

Publication types

• Biodiversity
• Medicine, Traditional*
• Phytochemicals / analysis*
• Plants, Medicinal*
• Preventive Medicine
• Teas, Herbal / analysis*
• Phytochemicals
• Teas, Herbal

• Open access
• Published: 24 March 2022

Ethnomedicinal study of medicinal plants used by Mizo tribes in Champhai district of Mizoram, India

• T. B. C. Laldingliani 1 ,
• Nurpen Meitei Thangjam 1 ,
• R. Zomuanawma 2 ,
• Laldingngheti Bawitlung 1 ,
• Anirban Pal 3 &
• Awadhesh Kumar   ORCID: orcid.org/0000-0003-4751-1142 1  

Journal of Ethnobiology and Ethnomedicine volume  18 , Article number:  22 ( 2022 ) Cite this article

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Medicinal plants have been used countless times for curing diseases mainly in developing countries. They are easily available with little to no side effects when compared to modern medicine. This manuscript encompasses information on ethnomedicinal plants in Champhai district, located in the North East Region (NER) of India. The region lies within Indo-Burma biodiversity hotspot. This study will be the first quantitative report on the ethnomedicinal plants used by the local tribes of this region. Knowledge of medicinal plants is mostly acquired by word of mouth, and the knowledge is dying among the local youths with the prevalence of modern medicine. Hence, there is urgency in deciphering and recording such information.

Information was gathered through interviews with 200 informants across 15 villages of the Champhai district. From the data obtained, we evaluate indices such as used report (UR), frequency of citation (FC), informant consensus factor ( F ic ), cultural values (CVs) and relative importance (RI) for all the plant species. Secondary data were obtained from scientific databases such as Pubmed, Sci Finder and Science Direct. The scientific name of the plants was matched and arranged in consultation with the working list of all plant species ( http://www.theplantlist.org ).

Totally, 93 plant species from 53 families and 85 genera were recorded. The most common families are Euphorbiaceae and Asteraceae with six and five species representatives, respectively. Leaves were the most frequently used part of a plant and were usually used in the form of decoction. Curcuma longa has the most cultural value (27.28 CVs) with the highest used report (136 FC), and the highest RI value was Phyllanthus emblica . The main illness categories as per Frequency of citation were muscle/bone problem (0.962 F ic ), gastro-intestinal disease (0.956 F ic ) and skin care (0.953 F ic ).

The people of Mizoram living in the Champhai district have an immense knowledge of ethnomedicinal plants. There were no side effects recorded for consuming ethnomedicinal plants. We observed that there is a scope of scientific validation of 10 plant species for their pharmacological activity and 13 species for the phytochemical characterisation or isolation of the phytochemicals. This might pave the path for developing a scientifically validated botanical or lead to semisyntheic derivatives intended for modern medicine.

Plants have been known to be a major source of diverse chemical compounds possessing both medicinal properties and commercial value. There have been several reports on medicinal plants as a source for drug discovery. However, new diseases will likely continue to emerge along with drug-resistant pathogens. This dynamic nature of pathogens has constantly challenged researchers to look for alternatives. The past few decades have witnessed the surge in ethnomedicinal plant research [ 1 ], one of the reasons being that the natural products have played an important role in the development of drugs, contributing more than 50% of clinical drugs in the pharmaceutical industry [ 2 ]. Further, the rapid growth in human population has raised the demand which in turn has increased the quest for novel plant resources, triggering a threat to natural resources [ 3 ].

Traditional knowledge and practices of herbal remedies have been passed on to new generations over the centuries and will continue to do so, with some variations taking place every generation. Plants have been the essential source for therapeutic regimes since ages, and traditional practices are proved to have little known side effects besides their low cost and easy availability. India has been well known worldwide for its indigenous traditional pieces of knowledge and practices from ancient times, through different systems of medicine such as Ayurveda, Siddha and Unani [ 4 ]. Although more than 427 tribal communities are having vast diversity of ancient traditions, still there has been criticism of ethnomedicines due to regional variation, political and socio-economic challenges [ 5 ]. Reports are stating that several plants have been increasingly utilised by the indigenous people of India [ 1 ]. Generally, in India, it was estimated that 6,000 species are used in traditional and herbal medicine which represent about 75% of the needs of the third world, and meanwhile, 3,000 plants were officially acknowledged due to their medicinal values [ 6 ].

The healthcare system of India witnesses a wide variation encompassing urban and rural populations which rely on both modern and traditional systems of medicine. The recently implemented Ayushman Bharat Pradhan Mantri Jan Arogya Yojana from the Commonwealth Fund enables cashless secondary and tertiary care at private facilities [ 7 ]. Besides, health insurance schemes also exist for institutions and factories. Catering to the huge population has its limitations, and thus, many of the ailments are treated either by traditional healers or through traditional knowledge and practices, especially in remote areas. One such state in the North-Eastern part of India is Mizoram.

Although some researchers [ 8 , 9 , 10 , 11 , 12 , 13 ] have documented and identified several ethnomedicinal plants of Mizoram mentioning their mode of preparation, usage, distribution and habitat, they mostly reported from the core areas of the cities. Their studies highlighted the qualitative data. However, there are no in-depth ethnobotanical studies recorded in Champhai district. Therefore, the present study aims to carry out a quantitative study using different cultural importance indices to assess the most valued plants and document the ethnomedicinal practices involving medicinal plants of the Champhai district of Mizoram, India. Their practical knowledge has been established based on more than a century of credence and observation.

Description of the study area

Mizoram lies within the Indo-Burma biodiversity hotspot region and shares two international borders with Bangladesh in the west and Myanmar in the east. According to Champion and Seth (1968), Mizoram forests are classified into Tropical semi-evergreen forests, tropical wet evergreen forests and mountain sub-tropical pine forests [ 14 ]. The study area, i.e. the Champhai district, is classified as a rural area where healthcare facilities are relatively poor which drives the people to rely on traditional medicines. The traditional healers using medicinal plant-based formulations for various ailments indicate that traditional medicines are still one of the mainstays in their contemporary health care. It is felt that prospection and research on the medicinal plants that play such an important role in the health care of Mizo tribes need a more intensified effort.

Champhai is one of the 8 districts in Mizoram, amidst the North-East Region of India. It is located in the eastern part of Mizoram, internationally bordered by Myanmar and therefore becoming the main gate of trading for India and Myanmar. It lies between 23.456° N latitude and 93.328°E longitude. The average annual rainfall is approximately 1814 mm, and the temperature remains around 18.6 °C which is slightly colder than the rest of the state during winter. The total land area is 3185.83 sq kilometres at an elevation around 1678 m above sea level, population density is 10 per sq kilometres (32,734). According to an official Census (2011), Champhai reported a population of 1,26,000, of which male and female were 62,357 and 63,388, respectively [ 15 ]. The study area was divided into 15 village council areas (Vengthlang, Vengthlang North, Venglai, Vengsang, Electric veng, Kanan, Kahrawt, Bethel, New Champhai, Zotlang, Hmunhmeltha, Tualcheng, Ngopa, Khawzawl and East Lungdar) for extensive data collection (Fig.  1 ). The majority of people living in this area are Mizo tribe and use the Mizo dialect in common.

Location of the present study area: Champhai district, Mizoram, India

Investigative method

In the field study, formal questionnaires were distributed to each participant while having face to face interviews at their residence. At least 16 people were interviewed in each village council area. Only those people who have knowledge in the art of preparing medicines either for their families or their neighbourhood were considered for the interaction. The interactions primarily focussed on their experience, type of dosage form, duration of usage, any adverse effects observed and the source of their knowledge about the plant and their parts used. This information was then correlated with the scientific data curated from related databases (Pubmed, SciFinder and Science Direct). In most of the cases, the voucher specimens were deposited (Herbarium, Mizoram University, Aizawl, Mizoram, India) for their authentication and archiving.

Characteristics of demographic data

This demonstrated the socio-economic information of the informant including qualities like age, sex, education level and occupation. Using random sampling method, 200 people (12–14 individuals from each village) in the ages group of 18–71 years were interviewed, of which 112 and 82 were males and females, respectively. Respondents belonged to various professions while some were students. Most of the informants do not engage in full-time ethnomedicinal practice or as a profession. The feature of demographical characteristics obtained in the study is tabulated below (Table 1 ).

Quantitative analysis

Frequency of citation

Frequency of citation was used to further examine the primary data by finding the sum of total citations/usage reports for a particular species. The usage report is the quotation of one plant by an informant [ 16 ].

Use value or UV is used to express the correlative importance of each particular plant species locally known and was calculated by the following equation [ 17 ].

where ‘ U i ’ represents the number of citations of each species by the informants and ‘ n ’ represents the total number of informants in the study area. The larger the number of citations, the greater is the use-value.

Informant consensus factor

F ic or ICF is used to represent the consistency of the information among the informants, indicating whether there were shared knowledge and concurrence in the use of plants for treating the ailment category among the plant’s users in the study area. It was calculated by the following equation [ 18 ].

where ‘ N ur ’ refers to the number of users reports in each illness category and ‘ N t ’ refers to the number of plant species used for a particular illness category by all the informants.

Further, F ic value with 1 or either close to 1 indicates that a large number of informants had agreed on using few plants for curing an illness category while low F ic value signified that there was an argument on using medicinal plants to treat illness amidst the category.

Relative importance

When calculating RI, both the informants who mentioned the useful plant species and their various kinds of uses are considered. So, it was calculated by the following equation [ 19 ].

where ‘NUC’ refers to the number of illnesses use category of each species divided by the total number of most use categories among the species and ‘NT’ refers to the number of illness types of uses of each species divided by the total number of most types of uses among the species.

Cultural values

In this index, the use category is taken into account and it was calculated by using the following equation [ 20 ].

where ‘UCs’ is the number of the used reports for each species divided by the total number of use categories of that species. ‘ICs’ is the number of informants who mention each plant as effective divided by the total number of informants, and ∑IUCs is the number of informants who report the use of each species divided by the total number of informants.

Demographic characteristics

All the 200 respondents were randomly selected from 15 village council areas interviewing at least 16 persons in each area with no equal separation of male–female ratio. Amongst them, the elderly in their seventies and above occupied 6.5% only, while people between 31 and 50 years old occupied 34.5%. The average age among the informants was 54 years. Mizoram is the second most literate state in the country (2011 census), and all the informants were literate having at least primary school level education. Out of the total informants, 32.5% were engaged in government jobs like teachers, officers, while 35% were self-employed like farmers, carpenters, skilled workers, small businesses and the rest 32.5% of the informants were unemployed including students and housewives (Table 1 ).

Taxonomy identification

In the present study, 93 medicinal plant species belonging to 53 families and 85 genera have been reported for treating various kinds of ailments. The most prominent families were Euphorbiaceae with 6 plant species followed by Asteraceae with 5 plant species and 4 species each among Cucurbitaceae and Zingiberaceae. Liliaceae, Fabaceae, Verbenaceae, Solanaceae, Rutaceae, Anacardiaceae are with 3 species each while Orchidaceae, Combrethaceae Theaceae, Arecaceae, Apocynaceae, Musaceae, Rubiaceae, Scrophulariaceae, Lamiaceace, Mimosaceae, Smilacaceae are with 2 species each and other 34 families with one species each as shown in Table 2 . The high usage report of this large family like Euphorbiaceae (6 species), Asteraceae (5 species) and Zingiberaceae (4 species) occupied 10.8%, 9.2% and 8.35% of the total used report, respectively, indicating that most people in the study area are inclined to use plants that are easily available and abundant around them (Table 2 ).

Frequency of usage of parts of plants

The most commonly used medicinal plants fell under herbs (35.5%) followed by trees (33.3%), shurbs (18.3%) and creepers (12.9%) as shown in (Fig.  2 ). Among the parts, leaves, fruits and barks were mainly utilised by the informants (Fig.  3 ). A detailed analysis concluded that leaves (47%) followed by fruits (14%), barks (11%), seeds (10%), rhizomes (6%), stems (4%), young shoot (2%), oil (1%) and in some cases the whole plant (3%) were used for ethnomedicinal purposes.

Percentage of plants habit

Percentage of parts used

Mode of preparation and administration

The mode of formulation preparation or administration was observed to be in the form of decoction (44.2%) followed by paste (23%), raw (19.5%), juice (9.73%), powder (1.77%) and others like maceration and oil (1.77%) (Fig.  4 .).

Distribution of formulation usage

Usage analysis based on the treatment of ailments

The total number of user reports documented in this study was 2717, in which all different illnesses were categorised into 16 groups using International Classification of Primary Care (ICPC) with a slight modification. Among the illness category, the gastro-intestinal disease has the highest usage report (940) followed by skincare (259) cardiovascular (222), kidney disease (196), hyperglycaemia (175), ENT (159), genito-urinary disease (139) and so on as shown in Table 3 .

Data analysis

Among the total number of user reports (UR) cited, Curcuma longa L. (136 FC), Flueggea virosa (Roxb. ex Willd.) Royle (126 FC), Psidium guajava L. (98 FC), Chromolaena odorata (L.) R.M. King & H. Rob. (87 FC), Mikania micrantha Kunth. (82 FC), Citrus limon (L.) Osbeck (68 FC), Carica papaya L. (53 FC), Ananas comosus (L.) Merr. (49 FC), Sarcococca pruniformis Lindl. (49 FC), Phyllanthus emblica L. (48 FC), Rhus chinensis Mill. (45 FC), Clerodendrum glandulosum Lindl. (44 FC), Senecio scandens Buch- Ham. ex D. Don (43 FC) were those species having the highest FC (Table 4 ).

Plant use value

From the UV value evaluation, Curcuma longa L. (0.68), Flueggea virosa (Roxb. ex Willd.) Royle (0.63), Psidium guajava L. (0.49), Chromolaena odorata (L.) R.M. King & H. Rob. (0.43), Mikania micrantha Kunth. (0.41), Citrus limon (L.) Osbeck (0.34), Carica papaya L. (0.26), Ananas comosus (L.) Merr. (0.24), Sarcococca pruniformis Lindl. (0.24), Phyllanthus emblica L. (0.24), Clerodendrum glandulosum Lindl. (0.22), Rhus chinensis Mill. (0.22), Senecio scandens Buch- Ham. ex D. Don (0.21) were reported to have the highest use value (UV).

We calculated the informant consensus factor by categorising the reported illness into 16 ailment groups along with the number of users report and taxa (Table 5 ). In our study, F ic values ranged from 0.866 to 0.962 which were all close to 1.

Relative importance and cultural value

Results of top-ranking species in terms of both indices of relative importance and cultural value are given in Table 6 . This study elucidates the highest cultural valued species and relative importance species utilised by the inhabitants of the study area. In general, the evaluated values were quite high in case of CVs and an average value of RI (0.607 ± 0.38) clarified that the versatile species, i.e. Phyllanthus emblica (RI = 2) was 3.3 times more relevant than the rest of the listed species.

Correlation and validation studies

An attempt was made to compare the use of all the medicinal plants reported by the informants with the previous papers published for their biological activity or ethnomedicines (Table 4 ). According to the studies conducted by Cakilcioglu et al., 2011, it was stated that if a medicinal plant has been reported for similar use in other parts of the world, its pharmacological effect could be more easily known [ 209 ].

The use of crude juice of Allium cepa L. showed a significantly higher hair growth rate than tap water when applied twice a day for two months which corroborated the present report of hair regrowth [ 22 ]. Metallothionein, an antioxidant protein present in Aloe vera (L.) gel, has been reported to have a protective effect against UV and gamma radiation damage to the skin. It scavenges free radicals by preventing the suppression of glutathione peroxidase and superoxide dismutase in the skin [ 30 ]. So, this validated the used of A. vera for skin care and burning by the Mizo tribes. In the present study, Betula alnoides Buch- Ham. ex D. Don has been used as toothpaste for whitening teeth while it was proved that 80% methanolic bark extract had the potential α-glucosidase inhibitory effect that prevent the (98.4%) at 40 µg/mL concentration [ 2 ]. Cajanus cajan (L.) Millsp is used effectively in Champhai district to treat jaundice and intestinal worms. To certify this, the methanolic extracts showed hepatoprotective activity in Swiss albino mice by inducing carbon tetrachloride (CCl 4 ) that cause liver damage. It lowers the serum levels of glutamate pyruvate transaminase (SGPT), or alanine aminotransferase (ALT) aspartate aminotransferase (AST) or serum glutamate oxaloacetate transaminase (SGOT) significantly [ 58 ].

When the aqueous extracts of Carica papaya L. and Ananas comosus L. were given to Spraque Dawley rats orally at doses of 5 and 10 mg/kg, both possessed mild to strong diuretic activity. Careful measure should be taken when using these plants as increase in the level of urinary K + , serum BUN and creatinine were mentioned [ 71 ]. This validated the used of C. papaya and A. comosus in kidney disease and urinary infection. The contemporary reports showed that Drymaria cordata (L.) was used as an instant pain killer for rheumatism; meanwhile, the scientific study also demonstrated that the aqueous whole plant extract exhibited analgesic and antipyretic properties at doses of 100, 200, and 400 mg/kg p.o mediated through peripheral and central mechanisms [ 210 ]. The latex water-soluble fraction of Euphorbia royleana Boiss. showed dose-dependent anti-arthritic and anti-inflammatory effects in rats and mice administered through gavage at doses of 50–200 mg/kg having more than 1500 mg/kg oral LD 50 in both [ 135 ]. Dose-dependent and significant decline in the number of abdominal constrictions induced by intraperitoneal administration of acetic acid was observed in methanol extract of Lablab purpureus (L.) Sweet. at a dose of 200 mg and 400 mg exhibited far better analgesic activity than 200 mg aspirin per kg of body weight [ 211 ].

Colocasia esculenta (L.) Schott and Elaeagnus caudata Schltdl. ex Momiy. were declared to use to discharge placenta after birth and to treat vaginal discharge (Lochia) for women in present study. Besides this record, in Cachar hills district of Assam, India, 5 ml of Elaeagnus caudata fresh root extract diluted in 10 ml of fresh water was also administered orally once a week to prevent miscarriage during pregnancy although there is no scientific study to backup this claim [ 118 ]. Apart from present report in Jamaica, Mikania micrantha Kunth. was most popularly used too for wound healing and its extract showed anti-inflammatory and antimicrobial activity against common pathogens, namely Escherichia coli , Staphylococcus aureus and Streptococcus sp. [ 212 ]. The decoction of Psidium guajava leaf was effectively used for diarrhoea which already proved to have antidiarrhoeal and protein conservative effects in diarrhoeal rats at a dose of 50 and 100 mg/kg of body weight. It increased the kidney weight and concentration of sodium, potassium and chloride significantly [ 213 ]. In the animal study of anti-urolithialic activity of Solanum nigrum , the fruit hydroalcoholic extract elicited potent activity against calcium oxalate urolithiasis effected by ethylene glycol through tumour necrosis factor adiponectin stimulation and alpha inhibition, also maintained the balance between stone promoter and inhibitor such as calcium and magnesium, respectively [ 214 ]. Thus, this authenticated the used of S. nigrum for removing kidney stone by the Mizo tribes in India.

Anoectochilus brevilabris Lindl. , Begonia inflata C.B. Clark , Dysoxylum excelsum Blume , Embelia vestita Roxb , Ensete glaucum (Roxb.) Cheesman , Gomphogyne cissiformis Griff. , Helicia robusta (Roxb.) R. Br. ex Blume , Laurocerasus undulata (Buch- Ham. ex D. Don) M. Roem. and Lobelia angulata G. Forst., Sarcococca pruniformis Lindl. were the plants that did not have biological activity reported previously which means that there is no scientific validation to support their application. Therefore, these plants were especially recommended in carrying out further investigation.

In addition, we compiled the secondary metabolite isolated chemical constituents done by several researchers for all the documented plants in the present study. Further investigation revealed that secondary metabolites from 13 plant species that have neither less nor none chemical compound isolated or identified— Anoectochilus brevilabris Lindl., Begonia inflata C. B. Clark., Castanopsis tribuloides (Sm.) A. DC., Combretum wallichii DC, Elaeagnus caudata Schltdl. ex Momiy., Embelia vestita Roxb., Ensete glaucum (Roxb.) Cheesman, Gomphogyne cissiformis Griff., Helicia robusta (Roxb.) R. Br. ex Blume, Laurocerasus undulata (Buch- Ham. ex D. Don) M. Roem., Lobelia angulata G. Forst., Pandanus odorifer (Forssk.) Kuntze, Sarcococca pruniformis Lindl. (Table 4 ) which will surely have great potent on ethnopharmacological study.

According to our findings, women practitioners (44%) were less than men (56%) which may be explained partly by the low sex ratio of the district; however, it can be assumed that women play lesser role in ethnomedicinal practices [ 215 , 216 ]. Among self-employed, farmers account for 58.5%, business persons 34.2% and carpenters were 21.4%. Farmers represented the highest percentage as they often lack access to modern healthcare facilities due to various issues ranging from financial, transportation and higher education. These issues forced them to rely on traditional medicines, cultivating and utilising them more regularly than others and somehow playing a big role in conservation too. Through this study, we observed that young informants like students around 18 to 25 years old have little expertise in practicing ethnomedicine and utilised them rarely as compared to elder informants. This may be due to change in mentality brought by education to rely only on prescribed medicines. Further, the results of the usage of plants dominated by the families were followed and confirmed the work done by some researchers stating that greater the plants grew in the study area the more it will be favourably and commonly used [ 217 ]. This supports the non-random plant selection hypothesis by Moerman 1979 [ 218 ]. Large families such as Asteraceae and Euphorbiaceae were most utilised while Orchidaceae and Poaceae were underutilised (low used report). However, due to non-random selection, small families like Cucurbitaceae and Zingiberaceae became over-represented (high used report). Thus, this implies that medicinal plants are not selected randomly by the inhabitants of Champhai district but are utilised based on their cultural and traditional knowledge [ 219 ]. In the present study, we laid out the only accepted botanical names by ‘The Plant List’ and their family, local name, habit, mode of preparation and ailments as illustrated in Table 4 .

Out of 93 species, 40 were cultivated species, whereas 53 were found in the wild. There were also 6 invasive alien species most notably Chromolaena odorata and Mikania micrantha which were commonly used to treat wounds topically. This is because wounds are the most common form of injury and these two species can be found almost everywhere [ 219 ]. The frequent use of herbaceous plants as medicines among the informants was due to their richness, abundance as well as their ability to grow easily in nature. Meanwhile, many parts of the world have been commonly using herbs as their medicinal ingredients due to their wide range of medicinal properties [ 220 ]. Leaves are the most utilised part of the plants due to their ease off collection as compared to their underground part. It is also the active site of photosynthesis accompanied by the production of metabolites [ 1 , 221 ]. In addition, leaves can be easily prepared and stored. It can be dried quickly under the sun in lesser amount of time than other parts like stem, bark and rhizome.

Similarly, it is also reported that decoction was the most common preparation method for herbal medicine while in some other tribal community [ 3 ], preparation of paste was the most common method applied [ 1 , 216 , 222 ]. For decoction the plant part was washed thoroughly and boiled with water administering the juice orally, whereas for paste the materials were crushed or rubbed within palms and applied topically. To make fine powder plant parts were shade dried and ground. Intake of oral administration and external topical formulation were the main mode of administration used in traditional herbal medicines which has also been previously reported [ 215 , 223 ]. Regarding the duration of consumption of herbal medicine, it depends on the illness whether it was short term or long term. For instance, short-term illness like cold, flu, stomach upset and skin problem, the consumption period did not last more than 1 week. On the contrary, the long-term illness like diabetes, kidney failure and heart diseases, the consumption period of plants (e.g., Flueggea virosa ) was much longer and last more than a month and so on.

The inhabitants of the study area extensively exploited medicinal plants to treat various illnesses and other needs which have not been previously reported. For instance, Anoetochilus brevilabris was used for pile treatments, Betula alnoides as toothpaste, Capsicum annuum to soothe and prevent scars from skin burns. Colocasia esculenta to expel lochia, Euphorbia milii as antidiarrhoea, Lablab purpureus as a pain reliever, Mussaenda macrophylla to stop internal bleeding and Parkia timoriana for treating baby umbilical cord. From this study it was clear that among the informants, stomach problems like ulcer, indigestion, diabetes, hypertension and kidney problems were common illness resulting in high user rate of consuming herbal medicines and similar record was reported by Mahwasane et al., [ 224 ]. Further, skin problem like dermatitis which was the second highest usage report was the highest ailment in most other tribal communities like Malda district in West Bengal reported by Saha et al. [ 225 ].

Generally, majority of the informants did not consume the medicines prescribed by the Doctor’s prescribed medicines along with their herbal medicine and claimed that many plants like Sarcococca pruniformis (tonsil), Psidium guajava (diarrhoea), Mikania micrantha (cut/wound), Flueggea virosa (chicken pox), Elaeagnus caudata (veginal discharge) were really effective and most importantly, none of them reported any adverse effect such as vomiting, headache, nausea, allergic reactions and/or skin rashes. Moreover, regarding the expenditure on buying medicines, 38% of the informants usually purchased their herbal medicines either in raw form ( Allium sativum, Allium cepa, Beta vulgaris ) or in processed form like juice ( Citrus limon, Phyllanthus emblica, Citrus aurantiifolia ), fruits ( Punica granatum, Phyllanthus emblica, Cucumis sativus ), and powder ( Curcuma longa ). Concerned about the source of their knowledge, all the informants reported that they have heard and learned some of their information from their elders, family and/or acquaintances. Besides these, 30% of the informants have also gathered additional information through social media and 10% through books, magazines and newspapers. This documentation clearly showed that knowledge and cultural practices of herbal medicines had been shared through the indigenous community through word of mouth.

Frequency of citation showed the sociocultural importance of medicinal plants to identify their therapeutic value [ 16 ]. The FC value is directly proportional to the use value (UV), the more FC value will increase the used value significantly.

Curcuma longa L. is one of the main commercially grown as seasoning plants in India. In Southeast Asia including India and China, turmeric powder has been used extensively for spice and colouring food material. It had a wide range of medicinal value that curcumin was the main bioactive chemical constituents [ 226 ]. C. longa was a mandatory spice that each and every household kept it that’s why the reason used report (UR) for medicinal value and cultural value (CVs) were high among the informants. In case of CVs, the high value was due to a greater number of the used report with lesser-used categories. The informants in present study reported that Flueggea virosa have a prominent effectiveness against diabetes (59 UR) and chickenpox (50 UR). The Mizo tribes extensively used F. virosa and Embelia vestita Roxb. plant to treat chickenpox and measles by bathing once a day with the decoction of leaf mixed with water. Apart from the degree of the used report, this index also attributed to the effectiveness of their use and importance.

Higher in the UV value indicates the more rate of agreeing and sharing their knowledges and practices of the medicinal plants among the informants [ 216 ]. Among the Terai forest of western Nepal Curcuma longa L. was also reported as the highest used value [ 227 ] similar to this result.

The plants with low UV value were Colocasia esculenta (L.) Schott, Eulophia nuda Lindl. and Ocimum americanum L., Maesa indica (Roxb.) A. DC, Morus macroura Miq, Tectona grandis L.f., Hibiscus sinensis Mill, Elaeis guineensis Jacq, Smilax perfoliata Lour with less than 0.05 UV as shown in Table 4 . Tectona grandis L.f. was also described with very low UV value by Ayyanar and Ignacimuthu as relevant to this result [ 1 ]. According to Chaudhary et al. 2006, the plants with low used value were in at risk of misrecollecting and passing on to the young generation which might be gradually disappearing [ 228 ]. On the other hand, the relevance of knowing the plant used value was for the convenience of pharmacological study and their used reliability [ 229 ].

However, Rajakumar and Shivanna had mentioned that the value of F ic depends on the accessibility of the taxa for the treatment of various diseases in the study area. Muscle/Bone problem with 81 UR have the highest F ic value of 0.962 followed by gastro-intestinal disease (GID) with 940 UR and skin care (SC) with 259 UR (Table 5 ). The lowest F ic value in the present study was the General Health (GH) category (Cold, fever, immunity boost) with 0.887 which was still more than the previous maximum F ic value report in Shimoga district, Karnataka, India i.e. 0.77 in Liver complaints [ 230 ]. Most RI value ( Phyllanthus emblica ) is considered to be versatile on its uses which would also increase the importance of the plant when it is used to treat more illnesses. The high RI values of some species may be attributed to their abundance and availability in the study area [ 19 ].

Overall, the quantitative analysis revealed that Curcuma longa was the most relevant species with the highest used value, frequency of citation and cultural value except in relative importance. This is due to the fact that the RI value is independent of the number of informants used report. On the conflict of these report, our study indicated that there was high consistency of the indigenous informant knowledge in the practices of ethnomedicines and utilised the same plants to treat it.

Conclusions

The present study concluded that the native people in the study area have their unique way of utilising medicinal plants to treat different kinds of ailments. We documented 93 valuable medicinal plants belonging to 55 families and 85 genera in which Euphorbiaceae and Asteraceae family were the most widely used in the area. This study supported the non-random selection of medicinal plants hypothesis. Among the plants part, leaves were the most commonly used. No new medicinal taxa were reported, but this study is a first quantitative report of ethnomedicine in this region and no informant had reported an adverse effect of herbal medicines. Their traditional pieces of knowledge had been passed on from their elders mostly through word of mouth. This study also revealed that younger generations between the ages of 18 and 30 have little to no knowledge of preparation of herbal medicines and their use as compared to the older age groups. This is mostly due to the availability of modern clinical drugs in the villages. Therefore, the traditional knowledge and practices of medicinal plants in the study area are somehow at risk of dying. This is why it is important to document the valuable knowledge as well as for conservation of the taxa.

The use of quantitative indices was essential in the field of ethnobotany to determine the most valuable plants along with their role played in a particular culture and to develop conservation initiatives. The plants which have high usage report and frequency of citation were known to possess numerous phytochemical compounds. The calculated informant consensus factor was extremely high, which means that the acquired data can be used as reference and reliable for ethnopharmacological study in the future. Even though the remedial value of many high cited plants has already been verified, there are still some plants that need to be validated. Hence, they are strongly recommended for further studies to develop alternative drugs.

Availability of data and materials

All data generated or analysed during this study are included in this published article.

Abbreviations

Used reports

Whole plants

Young shoot

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Acknowledgements

The authors were thankful to the department of Horticulture, Aromatic and Medicinal Plant, Mizoram University, for providing the necessary facilities to complete our work. We also offer our heartfelt gratitude to all the local people of Champhai district, Mizoram, who shared their valuable knowledge and precious time for this research.

This research did not receive any specific grant from funding agencies in the public, commercial or not-for-profit sectors.

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T. B. C. Laldingliani, Nurpen Meitei Thangjam, Laldingngheti Bawitlung & Awadhesh Kumar

Department of Botany, School of Life Science, Mizoram University, Aizawl, 796004, India

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Bioprospection and Product Development, CSIR-Central Institute of Medicinal and Aromatic Plants, CIMAP, Lucknow, 226015, India

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TBCL and AK carry out ethnobotanical survey, write the manuscript and analyse the data; TBCL, NMT, RZ, LB and AK were study proposer, design the questionnaire and revise the manuscript; NMT and AP design the graphical abstract; RZ and AP were proof reader. All authors read and approved the final manuscript.

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Laldingliani, T.B.C., Thangjam, N.M., Zomuanawma, R. et al. Ethnomedicinal study of medicinal plants used by Mizo tribes in Champhai district of Mizoram, India. J Ethnobiology Ethnomedicine 18 , 22 (2022). https://doi.org/10.1186/s13002-022-00520-0

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Medicinal plants and natural products for treating overactive bladder

• Huanxian Chen 1 ,
• Maggie Pui Man Hoi 1 , 2 &
• Simon Ming Yuen Lee 1 , 2 , 3  

Chinese Medicine volume  19 , Article number:  56 ( 2024 ) Cite this article

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Overactive bladder (OAB) presents a high prevalence of 16–18% worldwide. The pathophysiology of OAB is still poorly understood while effective therapy or countermeasure are very limited. On the other hand, medicinal plants and herbal remedies have been utilized for treating lower urinary tract symptoms (LUTS) in both Eastern and Western cultures since ancient times. In recent years, accumulating progress has also been made in OAB treatment research by using medicinal plants.

Relevant literature on the studies of medicinal plants and herbs used to treat OAB was reviewed. The medicinal plants were summarized and categorized into two groups, single-herb medications and herbal formulations.

The present review has summarized current understanding of OAB’s pathophysiology, its available treatments and new drug targets. Medicinal plants and natural products which have been used or have shown potential for OAB treatment were updated and comprehensively categorized. Studies on a wide variety of medicinal plants showed promising results, although only a few phytochemicals have been isolated and identified. Until now, none of these herbal compounds have been further developed into clinical therapeutics for OAB.

Conclusions

This review provides the basis for discovering and designing new phytopharmaceutical candidates with effective and well-tolerated properties to treat OAB. Increasing evidences indicate new strategies with alternative herbal treatment for OAB have high efficacy and safety, showing great promise for their clinical use. Future studies in a rigorously designed controlled manner will be beneficial to further support the eligibility of herbal treatment as OAB therapeutics.

Introduction

Lower urinary tract symptoms (LUTS) impact over 50% of the global adult population [ 82 ]. These symptoms emcompass storage, voiding, and post-micturition symptoms. Notably, overactive bladder (OAB) syndrome is classified as a specific subset within the domain of storage symptoms [ 2 ]. According to the International Continence Society (ICS), OAB describes the symptom complex of urinary urgency, often accompanied by increased frequency and nocturia, with or without urgency urinary incontinence, occurring in the absence of urinary tract infection or other obvious pathological coditions [ 54 ]. OAB is reconginized as a highly prevalent, troublesome and distressing condition. Its incidence tends to rise with age and it exerts a remarkable impact on quality of life (QOL). OAB affects both male and female equally, resulting in a large economic burden on individuals and society, in terms of the direct health care costs and lost productivity.

Presently, clinical strategies for pharmacotherapy of OAB are still limited to antimuscarinics and β3 agonists. Due to associated risks and adverse effects of conventional medicines, the use of alternative therapies to treat diseases nowadays has witnessed a rapid increase. Compared to synthetic chemicals, phytochemical compounds from medicinal plants are usually less expensive, less toxic and present less side effects. Ethnopharmacology has been applying traditional medicine and natural products for disease management. For instance, some currently used drugs, like aspirin, artemisinin, and digoxin, are deprived from plant extracts. Some regimens and single-herb medications of Traditional Chinese Medicine (TCM) have exhibited efficacy in managing symptoms associated with OAB. Natural phytochemicals enable the maintenance of a biological balance and avoid drug accumulation in the body [ 12 ]. Additionally, a remarkable increase in the use of medicinal plants and natural products to treat OAB has been observed. [ 12 , 34 , 96 ]. Herein, we review and summarize the body of evidence, obtained through traditional application and modern scientific methodology, which supports the use of medicinal plants, natural products and herbal formulations for the treatment of OAB. The summary and consolidation of the existing scientific data would greatly facilitate improvement of future research and offer OAB patients a wider range of potentially improved alternative therapies.

Pathophysiology of OAB

At present, the etiology of OAB is largely considered idiopathic, and its underlying pathophysiology remains poorly comprehended, necessitating ongoing research endeavors. Two possible origins of OAB symptoms were proposed by the ICS: (1) reduced ability to process the afferent signals in the brain (the neurogenic hypothesis); and (2) abnormally enhanced afferent signals from the bladder and/or urethra: elevated afferent activity is considered to be associated either with aberration in the urothelium receptor function and neurotransmitter release (the urethrogenic hypothesis) or with aberration in myocyte excitability (the myogenic hypothesis) [ 123 ] (Fig. 1 ).

Pathophysiology and underlying mechanisms of OAB

The urotheliogenic hypothesis

The urotheliogenic hypothesis refers to dysfunctions in signal molecules and ion channels within the urothelium. The urothelium not only acts as a protective barrier but also functions as a sensor to thermal, mechanical, and chemical stimuli. In the absence of a healthy urothelium, there may occur an elevation in spontaneous detrusor activity [ 107 ]. Through different receptors and transient receptor potential (TRP) channels, the urothelial cells respond to mechanical and chemical stimuli such as bradykinin, purines, norepinephrine, and acetylcholine (Ach), stimulating nearby afferent nerves. Abnormalities in the function of urothelium receptor and the release of neurotransmitter as well as in the sensitivity and coupling of the suburothelial interstitial result in involuntary contractions and manifestation of OAB symptoms [ 123 , 166 ].

The myogenic hypothesis

The myogenic hypothesis posits that impaired function of the myocytes in the detrusor muscle may cause elevated excitability, hence leading to the occurrence of uncontrolled contractions [ 123 ]. Studies have demonstrated that myocytes obtained from the bladders of patients with detrusor overactivity (DO) display enhanced excitability and an amplified response to stimuli [ 23 ]. According to Drake et al. [ 37 ], it is hypothesized that DO could arise from histological alterations in the detrusor, resulting in abnormal electrical coupling of smooth muscle cells. Consequently, physiological micromotions get synchronised and transform into active involuntary contraction in the detrusor [ 59 ]. Bladder smooth muscle cells are interconnected through gap junction channels, predominantly composed of connexins, enabling their coupling. Although the involvement of connexins (Cxs) in the pathophysiology of OAB requires further study, Cx45 and Cx43 appear to be the most prominent Cxs expressed in human detrusor smooth muscle (DSM) cells [ 37 , 111 ], and an increased Cx43 expression has been detected in individuals with neurogenic DO and urinary symptoms [ 8 , 119 ].

The neurogenic hypothesis

The neurogenic hypothesis considers the abnormal management of afferent signals. In this case, urgency originates from the brain and spinal cord or peripheral afferent terminals in the bladder. Suprapontine lesions including cerebrovascular and neurodegenerative diseases like stroke and Parkinson’s disease, could cause aberrant central nervous system (CNS) activation and then lead to inappropriate excitation of the detrusor. Urinary retention occurs as a result of a spinal cord lesion, which eliminates voluntary and supraspinal control over the process of micturition [ 117 , 123 ]. On the other hand, several specific neurotransmitters and related receptors participate in afferent signal transduction, such as muscarinic receptors and β3 adrenergic receptors. Among muscarinic receptors, M 2 and M 3 are the major subtypes expressed in the bladder [ 20 ]. Patients with idiopathic DO and painful bladder syndromes exhibit an elevated expression of M 2 and M 3 receptors in the bladder, and there is a notable correlation between the density of suburothelial M2 and M3 receptors and clinical urgency scores [ 109 ]. β3 adrenergic receptors is the dominant β-receptor subtype in the bladder [ 163 ], and expression of β3 adrenergic receptors in the bladder was strongly correlated with OAB-related symptoms [ 164 ].

The integrative hypothesis

The integrative hypothesis includes each of the above hypotheses since each of the components is likely to contribute to the pathophysiology of OAB considering the sophistication of mechanisms involved in micturition. Various potential triggers could induce local contractions (micromotions) in detrusor which are then transmitted to the bladder wall [ 35 ], resulting in occurrence of the sense of urgency. On the other hand, different other pathological and physiological conditions such as bladder outlet obstruction (BOO), inflammatory reactions, metabolic syndrome (such as diabetes), depression and anxiety also take part in the pathophysiology of OAB [ 106 , 137 ]. Therefore, when patients present OAB symptoms, it is of utmost importance to conduct a comprehensive evaluation aimed at identifying and ruling out potential underlying causes.

Current treatments for OAB

Various treatment options exist for OAB considering it involves multiple mechanisms. It is governed mainly by the severity of symptoms and the the extent to which it negatively affects patient’s quality of life (Table  1 ). General lifestyle changes have been suggested as a first-line therapy in all OAB patients; while pharmacotherapy or medications, including anticholinergics/antimuscarinics and β 3 adrenergic agonists, are the mainstream treatment for OAB, based on their pharmacological efficacy both in theory and clinical application. They are regarded as second-line therapy for OAB. Third-line OAB therapy refers to neuromodulation of the nerves that control bladder function.

Lifestyle intervention, behavioral treatments and bladder training

The lifestyle intervention includes smoking termination, body weight reduction, timed voiding, modifying fluid intake, eliminating bladder irritants such as carbonated drinks and caffeine, adjusting bowel movements to avoid constipation and sprains during bowel movements and sleep [ 61 , 101 ]. As for behavioral treatment, patients are trained to improve their ability to control and thereby disrupt or inhibit detrusor contraction. Pelvic floor muscle training (PFMT) could help inhibit pelvic floor contraction on the detrusor, therefore, ameliorating urgency and urge-related incontinence [ 99 ]. Bladder training is appled for cognitively and physically capable adults to further improve detrusor overactivity and regain continence by training them to gradually increase time interval between urinations [ 105 , 121 ]. These practices mentioned must be accomplished daily with motivation and patience. If they fail to improve or control the symptoms, then pharmacotherapy or medications are added.

Anticholinergic or antimuscarinics drug

Drugs that inhibit acetylcholine-induced involuntary detrusor contractions are currently the mainstay treatment options for OAB. Anticholinergics were the first OAB pharmacotherapy on the market and present the largest available dataset [ 142 ]. It is widely recognised that normal bladder contraction in human is modulated primarily by stimulation or activation of muscarinic receptors within the detrusor muscle [ 17 , 165 ]. The predominant cholinoceptors present in urinary bladder are M 2 receptors, while its contraction is mainly mediated by the minor population of M 3 receptors [ 165 ]. Therefore, M 3 -selective antimuscarinic agents (such as darifenacin and solifenacin) offer the first-line treatment for OAB, acting through competitively antagonizing acetylcholine at the M 3 receptors in the DSM to inhibit DO [ 10 ]. However, regrettably, these M 3 -selective anti-muscarinic agents clinically result in various significant adverse reactiosns in many patients, including paralysis of accommodation, tachycardia, constipation, and dry mouth [ 11 ].

β3-adrenergic agonistic drug

During the urine storage stage in the bladder, norepinephrine (noradrenaline) is released by the sympathetic nerves and binds to the β3-adrenergic receptors on the DSM, exhibiting inhibitory action, which results in the bladder relaxation [ 43 , 57 ]. Mirabegron is the only approved drug that acts as a effective and selective β3-adrenoceptor agonist. It is thought to relax the DSM by directly activating the β3 adrenergic receptors, which subsequently results in elevated cAMP and adenylyl cyclase levels in the tissue. In OAB rat models, mirabegron increases the average voiding volume in each urination, decreases the frequency of non-voiding contraction and enhances the bladder capacity without deranging discharge. Mirabegron could contribute to reduced annulled incidence; therefore, it can be applied in patients who discontinue previous anticholinergic/antimuscarinic therapy [ 14 , 39 , 124 , 125 , 141 ]. Common adverse effects of mirabegron include urinary tract infections, tachycardia, headache, and diarrhoea [ 150 ].

Neuromodulation and onabotulinum toxin A (Botox ® ) therapy

When patients fail first- and second-line OAB therapy, their conditions are regarded as refractory. Particularly, these patients show inadequate response to behavioral therapy. They also demonstrate either lack of response to medications (at least two types) or intolerance of medications (due to contraindications or adverse effects). Neuromodulation or onabotulinum toxin A (Botox ® ) therapy may be used for the carefully selected refractory patients. There are three different types of neuromodulation therapy, namely: peripheral tibial nerve stimulation (PTNS), sacral neuromodulation (SNS), and temporary chemodenervation of the bladder detrusor muscle. However, the treatment benefits are often counteracted by frequent and moderately severe adverse events such as lead migration, pain at the stimulator and lead sites, infection/irritation, electric shock, the requirement for additional surgeries, urinary tract infections (UTIs), dysuria, and hematuria etc. Onabotulinum toxin A (Botox ® ) therapy refers to injecting a chemodenervation agent, Botox ® (Allergan, Inc., Irvine, CA, USA), into the bladder, which was approved by FDA for idiopathic OAB in 2013. Onabotulinum toxin A is originated from a bacterium Clostridium botulinum and it binds to peripheral cholinergic terminals at the presynaptic membrane of the neuromuscular junction to inhibit acetylcholine release. This action causes muscle fibers paralysis until new fibers grow, thus temporarily affecting the myocytes in the bladder wall [ 94 , 130 , 153 ].

The current treatments for OAB present various limitations or side effects in clinical use. Hence, there is a strong rationale for the exploration and development of novel treatment strategies that aim to optimize therapeutic efficacy against OAB while simultaneously minimizing adverse reactions and side effects associated with effective dosages. Medicinal plants play an important role in this aspect since they have a long tradition in LUTS treatment. The screening and mechanistic elucidation of herbal drugs will greatly value OAB therapy.

New drug targets for OAB treatment

Increasing research suggests that the etiology of OAB is multifactorial, necessitating the continued exploration of new drug candidates. Several new drug targets and cellular pathways in the urinary bladder to treat OAB have been proposed recently, as follow: (1) Modulators of cyclic nucleotide (cyclic adenosine monophosphate and cyclic guanosine monophosphate) that mediate adenosine triphosphate (ATP) release from bladder wall tissues, such as P2X3 receptor antagonists and nitric oxide (NO)-sensitive soluble guanylyl cyclase (sGC) activator; (2) new targets for β3 agonists, including the bladder muscularis mucosa and bladder wall blood vessels; (3) Various TRP channels (TRPV 1 , TRPV 4 , TRPM 8 , TRPA 1 , and TRPM 4 ) and the effects of their mediators (antagonists) on detrusor overactivity; (4) Large and small conductance Ca 2+ ‐activated K + channels (BK and SK channels, respectively) and their impacts on spontaneous contractions; (5) Antioxidants that function to inhibit oxidative stress pathways; (6) Antifibrosis agents that directly or indirectly modulate the TGF‐β pathway, namely the canonical fibrosis pathway [ 9 , 47 , 158 ].

Recent research shows that medicinal plants and herbs could provide more efficacious treatment options with less side-effects. And they have been found to act on different targets or pathways compared to synthetic drugs. Current scientific evidence has demonstrated that a fair number of medicinal plants and natural products with efficacy of treating OAB act via one or a few of the targets mentioned above. Effects and mechanisms of various medicinal plants on OAB treatment from existing studies are summarized in Tables  2 and 3 .

Medicinal plants and natural products for OAB treatment

Literature reviews show a significant amount of research addressing the potential application of medicinal plants and natural products in OAB treatment [ 12 , 34 , 96 ]. A wide variety of medicinal plants have been employed to treat OAB in various regions and countries throughout different historical periods. Clinical evidence as well as preclinical in vitro and in vivo investigation have demonstrated that medicinal plants and/or their active ingreidents are efficacious for alleviating OAB. Additionally, herbs or medicinal plants are usually combined into therapeutic formulas in accordance with the theories of traditional medicine. TCM practitioners believe in “synergistic” or “emergence” effect when using herbal formulations, which are considered more effective and comprehensive in the treatment of diseases than single herbs alone. In this section, single-herb medications and herbal formulations for OAB treatments are reviewed and summarized in Tables  2 and 3 respectively. And the illustration of their possible interverntion mechanisms in the unrinary bladder is shown in Fig.  2 .

Illustration of possible intervention mechanisms of medicinal plants and natural products on OAB in the urinary bladder. a Promotion of the NO synthesis and release. b Reduction of the ATP synthesis and release. c Inhibition of the expression of the M receptors. d Up-regulation of the expression of the adrenergic receptors. e Down-regulation of the expression of P2X receptors. f Inhibition of transmembrane Ca 2+ influx and stimulation of Ca 2+ release from intracellular stores. g Activation of K + channels. h Inhibition of TRP channels. i Improvement in the barrier function of urothelium. j Regulation of excess blood flow. k Increase in intracellular cAMP in the bladder smooth muscles. l Inhibition of the release of inflammatory mediators, such as Substance P, CGRP etc.

Single-herb medications for OAB treatment

Alpinia oxyphylla.

Alpinia oxyphylla is known as “yì zhì (益智)” in Chinese. For centuries, the capsular fruit of this plant has been widely utilized in TCM to address specific symptoms of urinary incontinence, including frequency, urgency, and nocturia. Izalpinin, a flavonoid isolated from the fruit of Alpinia oxyphylla , was found to antagonized Carbachol-induced contractions concentration-dependently on rat bladder detrusor strips, which is due to its muscarinic receptor antagonistic action [ 168 ].

Artemisia monosperma (Wormwood)

This plant thrives extensively in the Arabian desert and has been deemed as an antispasmodic and anthelmintic in traditional medicine. It is also applied to treat hypertension. An isolated flavanone from Artemisia monosperma , 7- O -Methyleriodictyol, inhibited the amplitude of the phasic contractions in a dose-dependent fashion, and lowered the tone of ileum, uterus, and urinary bladder in rats. It also relaxed the phenylephrine-precontracted pulmonary artery and the acetylcholine-precontracted trachea [ 3 ].

Artemisia vulgaris

Artemisia vulgaris is widely distributed across natural habitats gloablly, spanning Asia, Europe, North and South America, as well as Africa [ 38 ]. It has been employed as a culinary spice in the food industry in different regions worldwide. For many centuries, A. Vulgaris has been utilized in traditional Chinese, Hindu, and European medicine to regulate the gastrointestinal system function and address a range of gynecological diseases [ 38 ]. In traditional system of medicine, this herb is considered a beneficial therapeutic agent in alleviating smooth muscle spasms [ 68 ]. In a recent research, it was found that A. Vulgaris extract inhibited carbachol-induced urinary bladder contractions via dual, anticholinergic and Ca 2+ antagonist mechanisms, by blocking muscarinic receptors and influx of extracellular calcium [ 78 ]. This indicates its potential for treatment of bladder overactivity.

Aspalathus linaeris

Aspalathus linearis , commonly know as Rooibos (meaning “red bush”) is a leguminous shrub indigenous to the Cape Floristic Region of South Africa [ 7 ]. The species complex composes of several different growth forms, and among them the Red type has been cultivated for producing rooibos herbal tea. In the last decades, A. linaeris has become very popular for its antioxidant and medicinal attributes [ 122 ]. In traditional medicines, A. linaeris is generally accepted as a helpful therapeutic in relieving smooth muscle spasms [ 68 ]. In a study with rabbit urinary bladder strips, A. linaeris extract relaxed the bladder and inhibited carbachol-induced urinary bladder contractions via a dominant opening of ATP-sensitive potassium-channels and a weak blockade of calcium channels [ 78 ]. This reveals its medicinal usefulness in hyperactive bladder disorders.

Astragalus membranaceus (Huangqi)

Astragalus membranaceus , also called huáng qí (黄芪), it is an herb widely used in TCM and diet, and it has been widely studied in western medicine for various disease treatment. Astragalus polysaccharide (APS) is a type of water-soluble heteropolysaccharide deprived from the stems or dried roots of Huangqi [ 174 ]. Astragaloside IV (AS-IV), a small molecular saponin, is another major component from the aqueous extract of  Astragalus membranaceus [ 170 ]. An in vivo study reported that both APS and AS-IV ameliorated urinary frequency on a CYP-induced OAB female mice model via modulation of urothelial wound healing, possibly via the increased expression of tight junction protein ZO-2 [ 32 ].

Bletilla striata

Bletilla striata is distributed widely in eastern Asian countries, including China, Japan, North Korea, and Myanmar. As widely used in TCM for thousands of years, its functions of hemostasis, detumescence, and improving one’s health have been recorded in Chinese Pharmacopeia (2015) [ 69 ]. Besides having been employed to treat hemoptysis, traumatic bleeding, chapped skin, swelling, and ulcer bleeding [ 157 ], it has also been used in TCM empirically to treat interstitial cystitis (IC). Study on zymosan‐induced cystitis in female rats [ 97 ] showed that treatment of B. striata extract solution decreased abdominal withdrawal reflex (AWR) scores and amplitude of bladder detrusor electromyogram (EMG). Furthermore, it demonstrated notable improvements in OAB by effectively prolonging the micturition interval and enhancing urine storage capacity. These results implied the possible efficacy of B. striata on treating OAB. However, more research on OAB models and patients is required for further validation.

Bridelia ferruginea

This tropical plant is native to Africa and has been utilized in African traditional medicine to treat intestinal and bladder ailments. Ethanolic extract of B. ferruginea leaves inhibited KCl-induced contractile response in rat urinary bladder smooth muscle. This effect might be ascribed to the blockade of purinergic neurotransmission [ 116 ]. It can be speculated that B. ferruginea leaf exact could be useful in bladder overactivity although further research is required to better evaluate its potential therapeutic application.

Bryophyllum pinnatum

Bryophyllum pinnatum is a succulent perennial plant originating in Madagascar, whose leaf press juice was shown to reduce the electrical- or carbachol-induced contractile response in porcine DSM [ 129 ]. The flavonoid fraction of this plant lowered the porcine detrusor contractility in dose- and time-dependent fashion [ 48 ]. In addition, results from a clinical trial with twenty postmenopausal women demonstrated that the leaf extract improved OAB symptoms and reduced micturition frequency after 8-week treatment [ 18 ].

Camellia sinensis (Green tea)

Green tea is a widely consumed healthy beverage around the world, known for its potent anti-inflammatory/antioxidant properties. Previous studies have identified these beneficial properties and attributed them to the presence of polyphenols in green tea. As the predominant catechin in green tea, epigallocatechin gallate (EGCG), is a well-established polyphenol flavonoid with renowned antioxidant activities. It was reported that EGCG restored ovariectomy (OVX)-induced bladder dysfunction dose-dependently through antioxidant, anti-fibrosis and anti-apoptosis effects on a surgical menopause-induced OAB rat model [ 72 ]. In addition, studies have revealed that EGCG can enhance bladder storage function and provide protection against interstitial fibrosis induced by Metabolic syndrome (MetS) and OVX in rats, potentially through the mitochondria and endoplasmic reticulum (ER) apoptosis pathways [ 89 ].

Cananga odorata

Ylang ylang essential oil is extracted from Cananga odorata , a medicinal plant grows in the north-east of Brazil. It possesses myorelaxant and antispasmodic properties, and it is used to relieve intestinal spasms as a smooth muscle relaxant [ 100 ]. In vivo and in vitro studies showed that ylang ylang essential oil inhibited urinary bladder contractile response induced by EFS and various agonists on rat and rabbit urinary bladders. The relaxing effect on bladders is thought to be mediated by c-AMP pathway [ 79 ].

Cannabis sativa

The plant Cannabis sativa , originated from Central Asia, has been used medicinally and recreationally, also as a source of textile fiber for thousands of years. It has been evaluated for its protective effect against LUTS and OAB caused by multiple sclerosis (MS). Clinical studies consistently revealed that standardized extracts derived from Cannabis sativa , such as Sativex, containing delta-9 tetrahydrocannabinol (THC) and cannabidiol (CBD), have been effective in reducing incontinence episodes in individuals with MS [ 24 , 46 , 149 ]. Later, a multicenter double-blind randomized clinical trial reported that Sativex reduced the number of nocturia episodes, voids per day and daytime voids, and improved Patient’s Global Impression of Change (PGIC) in patients with OAB due to MS [ 76 ]. It was found that extract enriched with cannabidiol inhibited cholinergic-mediated urinary bladder smooth muscle contraction in rats and human bladders. This effect is modulated by TRPV1 in rats but not in human. The inhibition was not observed in bladder contractions induced by potassium chloride (KCl), electrical field stimulation (EFS), or α,β-methylene adenosine triphosphate (α,β-MeATP) [ 26 ].

Capsicum annuum (Chili pepper)

Capsicum annuum fruit, usually termed as chili pepper, has been broadly used as food vegetables, natural colorants and flavoring ingredients, as well as a crude drug in many traditional medicine systems since ancient times [ 56 ]. Capsaicin is an active ingredient originated from chili peppers, found in the fleshy part of the pepper, rather than the seeds. Intravesical instillation of capsaicin can induce desensitization of TRPV1 receptors on afferent nerves, leading to a reduction in neural firing, and inhibition of the micturition reflex. As a TRPV1 channel agonist, capsaicin attaches to and activate the vanilloid receptors, leading to calcium entry through the neuronal membrane, and then resulting in desensitization of the nerve, hence, analgesia. Also, binding of the receptor causes a decrease in substance P, a major pain neurotransmitter [ 55 ]. But capsaicin often causes local irritation and edema. Attempts to translate these desensitization activity have been used to the bladder control. In a meta-analysis comprising eight open-label and two placebo-controlled clinical trials involving 200 patients with lower urinary tract disorders, intravesical capsaicin treatment for neurogenic hyperreflexic bladder demonstrated clinical or urodynamic symptom improvement in 84.3% of the patients. However, side effects appear during or immediately after instillation [ 36 ]. Application of capsaicin in overactive bladder is very much limited.

Citrus depressa

Citrus depressa (shekwasha), is a kind of citrus fruit produced in southern part of Japan. A study by Ito et al. discovered that nobiletin, a polymethoxy flavonoid present in shekwasha abundantly, significantly improved hyperactive urodynamic symptoms in CYP-induced cystitis rats by decreasing micturition frequency. The beneficial effect may be partly attributed to the potency of raising intracellular cAMP level in the bladder smooth muscles [ 67 ].

Cucurbita pepo (Pumpkin)

Pumpkin seed oil obtained from Cucurbita pepo presents a strong antioxidant activity and valuable nutritional benefits. It is useful for treating a range of diseases, such as benign prostatic hyperplasia (BPH), urinary disorders, hypertension, hyperlipidemia, diabetes, and cancer [ 115 ]. In an in vivo urodynamics study on rabbits, it was reported that a preparation of pumpkin seed oil reduced bladder pressure, improved bladder compliance, and reduced urethral pressure in rabbits [ 173 ]. In a study involving 45 patients with symptoms of OAB, pumpkin seed oil was investigated and found to significantly decrease the Overactive Bladder Symptom Score (OABSS) without any observed adverse reactions over a 12-week treatment period [ 113 ]. In a recent single-blind randomized clinical trial involving 73 subjects with BPH aged ≥ 50 years, pumpkin seed oil decreased International Prostate Symptom Scores (IPSS) and improved QOL in patients, with BPH symptoms alleviated and no side effects [ 169 ].

Cyclotrichium niveum

It is a Turkish flora widely used as tea. The essential oil of C. niveum relaxed of carbachol precontracted rabbit bladder strips, which showed the antispasmodic activity of C. niveum . The essential oil also exhibited radical scavenging effects [ 29 ]. Results showed its possible effects on reducing OAB symptoms, however, further studies must be undertaken to verify this speculation.

Euphorbia resinifera (Resin spurge)

A cactus-like plant commonly found in Morocco, Euphorbia resinifera contains the naturally occurring constitutent resiniferatoxin (RTX), which is a potent functional analog of capsaicin and is a potent TRPV1 agonist. By selectively binding to the TRPV1 receptor, intravesical RTX effectively obstructs the afferent nerves responsible for transmitting pain sensations to the brain. A meta-analysis of seven trials involving 355 patients diagnosed with either interstitial cystitis (IC) or DO reported that RTX resulted in an increase in maximum cystometric capacity (MCC) in patients with DO and provided relief from bladder pain in patients with either IC or DO. Nevertheless, no significant improvement in frequency, nocturia, incontinence or first detrusor contraction (FDC) was observed [ 52 ]. On the other hand, other study has presented an opposite result of higer pain sensation, lower bladder capacity, increased urinary frequency and nociceptive behaviors (such as licking and freezing) after instillation of RTX into rat bladders [ 126 ]. RTX is rarely used clinically due to its inconsistency in efficacy, difficulties in delivery and acute pain.

Galium aparine

It is found in North America, and it’s an annual herbaceous plant that grows on roadsides, pastures and uncultivated places. Due to presence of hooks, this plant can’t be consumed raw and is usually used in teas. It has been employed in cystitis traditionally although it hasn’t been demonstrated pharmacologically [ 25 ]. Its possible efficacy of treatment OAB might be worth exploring with scientific studies.

Ganoderma lucidum

Medicinal benefits of the fungi Ganoderma lucidum has been widely recognized for thousands of years in East Asia (mainly China, Japan and Korea). Its fruiting body is called “Reishi” in Japan and “líng zhī (灵芝)” in China. Traditionally, it is known as “longevity-promoting-tonic” and has been used in China in the Qi replenishment, mind relaxation, as well as easing the cough and asthma [ 5 ]. And in the modern medicine systems, it has been applied to cure different ailments such as hepatitis, hypertension, hypercholesterolemia, diabetes and various cancers [ 127 ]. In a randomized, double-blind, placebo-controlled and dose-ranging clinical study on 50 male volunteers (≥ 50 years old), the extract of Ganoderma lucidum demonstrated an improvement in IPSS among men with LUTS with no major adverse effects reported [ 114 ]. Through an in vivo rabbit model of ischemia/reperfusion (I/R), it was observed that Ganoderma lucidum exhibited antioxidant effects, effectively mitigating the detrimental impact of I/R-induced oxidative stress on bladder compliance and contractile responses [ 90 ].

Glycine max (Soybean)

Glycine max is a species of legume native to East Asia, also termed as the soybean or soya bean [ 152 ]. On isolated strips of rabbit detrusor, genistein, a major dietary phytoestrogen from soybean exhibiting tyrosine kinase inhibitory activity, were shown to induce relaxation in detrusor muscle contracted by the muscarinic receptor agonist bethanechol (BE) and the purinergic P2X receptor agonist α, β-methylene ATP (α, β-MeATP). Genistein exhibited a selective reduction in peak contractions induced by α, β-MeATP and steady-state contractions induced by BE, possibly through inhibition of voltage operated Ca 2+ channels (VOCCs) [ 120 ]. A cross-sectional study involving a substantial sample of 2000 elderly Chinese men has shown that dietary intake of soy isoflavones was linked to a lower risk of LUTS [ 155 ]. Later in another study, two soy isoflavones genistein and daidzein were reported to dose-dependently decrease detrusor contractions induced by EFS probably via activation of large and small conductance K + (Ca) channels [ 147 ].

Hippophae rhamnoides (Searberry)

Seaberry or sea buckthorn obtained from Hippophae rhamnoides , which grows in Northern Europe, Western Asia, China and Canada, has shown inhibitory effect on carbachol induced contractions in rat bladder strips and on TGF-β-induced constrictions in human bladder smooth muscle cells. Triterpenoids and flavonoid glycosides in seaberry which include 3-O-coumaroyl 2,23-dihydroxy oleanolic acid, ursolic acid, uvaol, pomolic acid, oleanolic aldehyde and isorhamnetin 7-O-rhamnoside, might contribute to this protective activity. Among them, ursolic acid (1–100 μM) and isorhamnetin 7-O-rhamunoside (10 μM) remarkably inhibited carbacol-induced bladder constraction [ 132 ]. In a clinical trial of seaberry extract supplementation in Japanese men and women with mild urinary dysfunction, several emotional parameters associated with urinary dysfunction were improved, suggesting that it may be beneficial for relieving moderate urinary symptoms [ 140 ].

Hypericum perforatum

Hypericum perforatum is a herbaceous perennial plant originating in Asia and Europe, and it has been introduced into the United States [ 16 ]. St John’s wort (SJW), obtained from the leaves and flowering tops of H. perforatum , is a well-known successful herbal antidepressant. The investigation on its effect on bladder contractions showed that it inhibited EFS-contractile response in isolated rat bladder smooth muscle strips. Opioid receptors might be involved in inhibitory activity on excitatory transmission of SJW [ 27 ].

Perilla frutescens

Perilla frutescens is an aromatic plant exhibits a wide distribution in East Asian countries, such as China, Japan, Korea, and Vietnam. It has been cultivated as an edible cropand used in TCM since ancient times [ 58 ]. Perilla leaves have served in preparation of vegetable curries, chutneys and pickles. Perilla has also been applied in medical and pharmacological terms, for its anti-oxidant, anti-inflammatory and anti-allergy activities. An in vivo study [ 84 ] showed that 2-week perilla extract treatment notably enhanced the micturition interval in female spontaneously hypertensive rats (SHRs) without significantly affecting maximal pressure, suggesting that perilla improves frequent urination, without suppressing contraction of the detrusor muscle. In comparison to the control group, the perilla group displayed a reduction in expression of nerve growth factor (NGF), tumor necrosis factor- α (TNF-α), interleukin-1β (IL-1β) and TRPV1, as well as an increased level of uroplakin 3A (UPK3A). Thin or defective urothelium was detected in the control group, while the perilla group displayed nearly complete preservation of the urothelial integrity. Additionally, being the main components of perilla extract, perillaldehyde and perillic acid, inhibited the induction of NGF and TNF-α by IL-1β in vitro [ 84 ]. These results imply effects of perilla on OAB is likely be modulated, at least partly, by improvement of the urothelial presence and by the anti-inflammatory activities of perilla.

Peucedanum japonicum

The root of Peucedanum japonicum is traditionally used to treat inflammatory diseases in southern parts of Japan. Preclinical studies showed that extract from Peucedanum japonicum inhibited agonist-induced rabbit bladder contractile response and improved urodynamic symptoms in hyperactive rat bladders with a reduction of micturition frequency, while its active ingredient, isosamidin, demonstrated the ability to reduce or mitigate phenylephrine‐activated contractions in isolated human prostate tissue strips [ 65 , 139 ]. On the other hand, clinical effects of the extract were investigated in male patients with LUTS. It improved urodynamic parameters and subjective symptom scores without reported adverse drug reactions [ 74 ]. These investigations suggest its possible therapeutic application in OAB treatment.

Potentilla chinensis

Potentilla chinensis is a perennial herb can be found extensively across Korea, Japan and China. In Korea, it has been traditionally utilized as a medicinal remedy to treat inflammation, myalgia, scabies, and dysentery [ 53 , 143 ]. Wróbel et al. found that the aqueous extract of P. chinensis alleviated retinyl acetate (RA)-induced DO in rats, and the mechanisms may involve the extract’s ability to inhibit the release of transmitters from both afferent and efferent fibers that innervate the urinary bladder, and its influence on the exocytotic process depending on SNARE (soluble N-ethylmaleimide-sensitive-factor attachment protein receptor) protein activity [ 156 ]. More recently, aqueous extract of P. chinensis (PCE) was found to attenuate DO in rats suffering from cyclophosphamide (CYP)-induced hemorrhagic cystitis. Considering PCE contains an abundance of antioxidants, the protective activities of PCE appear to be related to preventing oxidative stress-dependent dysfunction of the urinary bladder [ 73 ].

Puerariae lobatae (Gegen)

The Chinese herb Gegen (gé gēn 葛根) is the dried root of Puerariae lobatae (wild), and has been traditionally employed to treat a wide range of symptoms including diarrhea, acute dysentery, deafness and cardiovascular diseases. In an ex vivo experiment on isolated rat bladder strips, Gegen water extract caused relaxation of detrusor muscle in urothelium-independent fashion, and it acted synergistically with the water extract of Salviae Miltiorrhizae Radix (Danshen) [ 92 ]. Later, an in vivo study [ 175 ] showed that water extract of Gegen reduced carbachol-induced tonic contractions in male spontaneously hypertensive rats (SHR) but did not alter the amplitude of phasic contractions. Combination of Gegen and darifenacin exhibited synergetic effect on inhibition of electric field stimulation (EFS)-induced contractions. Gegen improved DO through neurogenic and anti-muscarinic action, more specifically, on M 3 receptor.

Rhois aromatica

Rhois aromatica is originated from Northern America. Its extract was shown to inhibit carbachol and KCl-induced contractile response in rat and human bladders. This reduction of contractile response involved direct antagonistic effect on muscarinic receptors and receptor-independent mechanisms [ 21 ].

Salvia cinnabarina

It is an American species of the genus of Salvia , which is used in folk medicine for its various bioactivities. It has been shown that a secoisopimarane diterpenoid from Salvia cinnabarina , 3, 4-secoisopimar-4(18), 7, 15-triene-3-oic acid significantly inhibited EFS-contratile response in isolated rat urinary bladder in a dose-dependent manner, with a mechanism related to, at least partly, NO production [ 28 ].

Serenoa repens (Saw palmetto)

Serenoa repens , commonly accepted as saw palmetto, is a small, low-growing, dwarf-palm tree endemic to the south eastern America and West Indies [ 4 ]. Traditionally, the berries were used as a staple food and medicine. It has been used to treat diarrhoea and stomach ache, and served as a diuretic and sexual tonic [ 154 ]. Several clinical studies showed that administration of saw palmetto ( Serenoa repens ) extract improved the IPSS in patients with LUTS and alleviated urodynamic symptoms including urination frequency and nocturia [ 41 , 51 , 74 ]. In a clinical research encompassed 591 patients with inflammation assosciated chronic benign prostate conditions, saw palmetto extract (SPE) mitigated bladder voiding, LUTS as well as erectile function, and improved IPSS and National Institute of Health—Chronic Prostatitis Symptom Index (NIH-CPSI) [ 51 ]. Another study on 20 male patients (≥ 50 years old) with untreated LUTS and no serious complications showed that 4-week SPE administration improved IPSS-QOL score, nocturia, and OABSS-2 [ 74 ]. The efficacy and safety of SPE (12-week treatment) were studied in 76 adult women with urinary symptoms and the results showed SPE significantly alleviated daytime frequency and nocturia with high safety [ 162 ]. In line with this, preclinical study on obese male Wistar rats showed SPE improved smooth muscle fiber structure and reduced cell proliferation in the bladder, indicating its potential beneficial effects on LUTS [ 41 ]. Several mechanisms of action have been proposed for SPE, which include anti-inflammatory/anti-oedematous effect, anti-androgenic effect, prolactin signal mediation, and anti-proliferative action via inhibiting growth factors [ 50 ].

Silybum marianum

Silybum marianum, known as milk thistle, is indigenous to Northern Africa, Southern Europe, Southern Russia and Anatolia and also distributed in South Australia, North and South America [ 102 ]. For thousands of years, it has been used as a remedy for a range of liver dysfunctions and gallbladder disorders [ 19 ]. Silymarin, a mixture of flavonolignans obtained from S. marianum , is the active component of this herb. It includes mainly silybin A, silybin B, isosilybin A, isosilybin B and other flavonolignants such as silychristin, neosilyhermin, silyhermin and silydianin, which are predominantly found in the fruits and seeds of the plant compared to other parts [ 75 ]. It was shown to reduce cyclophosphamide-induced enhanced contractile response in CYP-induced cystitis rat model, suggesting its possible application in treating bladder overactivity in cystitis. This action may be associated with its antioxidant and anti-inflammatory activities [ 40 ].

Solanum lycopersicum (Saladette tomato)

Saladette tomato, scientifically referred to as Solanum lycopersicum , holds significant importance as one of the key vegetable plants globally. It orginated in western South America, with its domestication considered to have taken place in Central America [ 83 ]. In an in vivo study on high-carbohydrate diet induced obese Wistar rats (male), lipidic extract of saladette tomato reduced hyperplasia and contractility, improved fiber structure of smooth muscle and decreased cell proliferation in the bladder, revealing its potential protective effects on LUTS and OAB [ 41 ].

Solidaginis virgaurea

It is native to middle Europe, and it is the most frequently used plant for extraction to produce preparations in phytotherapy to treat bladder dysfunction including the OAB syndrome. Extract from the Solidaginis virgaurea showed inhibitory action on carbachol and KCl-induced contractile response in rat and human bladders, suggesting its possible efficacy on treating OAB. Additionally, direct antagonistic effect on muscarinic receptors and receptor-independent mechanisms of Solidaginis virgaurea are related to the reduced contractility [ 21 ].

Uncariae Ramulus Cum Uncis

Uncariae Ramulus Cum Uncis (Gambir Plant or gōu ténɡ 钩藤 in Chinese) is a herbal medicine that has enjoyed broad usage in China and Japan for thousands of years. It mainly grows in tropical regions, such as Southeast Asia, Southeast America and Africa [ 171 ]. Rhynchophylline, as the main active component of Uncariae Ramulus Cum Uncis , has been discovered to inhibit the constriction of isolated rat urinary bladder strips ex vivo, and improved urodynamic parameters in rats in vivo, through activating calcium-activated potassium channels and blocking L-type calcium channels [ 70 , 71 ]. Rhynchophylline inhibited the intracellular-calcium-induced contractions of rat bladder strips at a low concentration (10 μmol/L), whereas it inhibited both intracellular- and extracellular-calcium-induced contractions at a high concentration (20 μmol/L) [ 70 ]. Based on these findings, it can be inferred that Rhynchophyllineplays has the potential to serve as an alternative therapeutic agent for OAB treatment.

Vaccinium corymbosum (Blueberry)

Blueberries ( Vaccinium corymbosum ) have a rich historical background of being utilized both as a food source and for medicinal purposes in Europe and North America. Nowadays they are widely consumed as a health food worldwide. They are commonly considered as one of the most abundant sources of antioxidant phytonutrients among the fresh fruits and vegetables that have been researched [ 134 ]. A study by Miyazaki et al. reported that blueberries successfully averted the onset of bladder dysfunction resulting from BOO in rats via antioxidative effects and inhibiting bladder remodeling, and that these two effects might act synergistically to exert a preventive activity [ 108 ].

Vanilla planifolia

It is a precious orchid originating in Mexico and Central America, and has been cultivated in various tropical regions worldwide for production of natural vanilla flavor [ 22 ]. The scent of vanilla has been applied to treat sleep disorders thanks to its relaxing effect. It has been reported that vanilla oil decreased serum catecholamine (adrenaline, noradrenaline and dopamine) levels, increased intervals between bladder contractions, decreased urination frequency in rats in a sleep-like state induced by light urethane anesthesia, indicating that it may reduce nocturia by reducing sympathetic activity [ 138 ].

Vitis vinifera (Grape)

Vitis vinifera is a climbing vine cultivated worldwide, and the largest fruit crop in the world [ 131 ]. It has served as a nutritional supplement or food colouring additive in the food industry [ 42 ]. Thanks to its antibacterial activity, V. vinifera has been proposed as an alternative to chemical preservatives [ 118 ]. The fruits of V. vinifera , namely grapes, have extensive application in the production of juices, wines, and raisins [ 133 ]. In the pharmaceutical industry, V. vinifera serves as a valuable source of raw materials renowned for their antioxidative, hepatoprotective, cardioprotective, anticancer, antiviral and antibacterial effects. The potential utilization of V. vinifera or the derived active compounds as environmentally friendly agents with antibacterial or anticancer properties have been also reported [ 13 , 87 ]. In addition, the production of cosmetics with grape extract is specifically popular worldwide nowadays. Raw materials deprived from V. vinifera have gained significant recognition and widespread use in cosmetics, especailly for their antioxidant, anti-ageing, UV-protection and skin-whitening activities. Furthermore, the safety of V. vinifera has been well-established, further enhancing its appeal for cosmetic applications. [ 131 ].

The protective effects of grape suspension and resveratrol against increased contractions and voiding frequency have been confirmed in multiple animal studies [ 6 , 44 , 167 ]. Resveratrol is considered to be the basic active ingredient responsible for the antioxidant properties of grapes [ 60 ]. In studies carried out by Francis et al. on H 2 O 2 -induced oxidative stress in rabbit urinary model, it was observed that whole grape suspension produced a higher reduction of citrate synthase activity in muscle and mucosa compared to resveratrol. Whole grape suspension also showed higher protective effect than resveratrol against choline acetyltransferase activity, which is responsible for the synthesis of Acethylcholine and finally causes bladder smooth muscle contractile response. It was concluded that whole grape suspension were more effective in ameliorating oxidative stress than resveratrol, suggesting combinational benefits of the active components [ 44 , 45 ]. In addition, resveratrol was reported to improve overactive bladder via downregulation of the protein expression level of SCF, c-Kit and p-AKT in the bladder of rats with chronic prostatitis (CP), and the combination of resveratrol and solifenacin strengthened the improvement in overactive bladder, indicating potential pharmacological synergy as a theraputic strategy for CP patients [ 167 ].

Zea mays (Cornsilk)

Cornsilk is obtained from the female flower of corn ( Zea mays ). Its application in cystitis that involves OAB symptoms has been reported [ 30 , 34 ]. However, there are no pharmacological or clinical studies that have been carried out regarding this effect. It might be worth exploring.

Natural products are often classified into four groups according to their biosynthetic origins and major structural features: alkaloids, phenylpropanoids, polyketides, and terpenoids[ 136 ]. From the herbs mentioned, a number of phytochemicals have been isolated and identified for their pharmacological activity against OAB. Summary and classification of these compounds are shown in Fig.  3 .

Structures of phytochemicals used for treating OAB

Herbal formulations for OAB treatment

Tcm formulations, bu-zhong-yi-qi-tang.

Bu-Zhong-Yi-Qi-Tang (BZYQT) is an herbal formula described in the Pi Wei Lun (脾胃论, Treatise on Spleen and Stomach), a medical text dating back to 1249 AD. This formulat primarily targets spleen and stomach deficiencies, which are are considered as the origin of various health disorders. Translated typically as “the decoction for strengthening the center and enhancing qi,” the name Bu-Zhong-Yi-Qi-Tang encapsulates the essence of this herbal formula (Kim et al., 2017b). This formula consists of ten herbs, including Radix Astragali (huáng qí 黄芪), Rhizoma Atractylodis (bái zhú 白术), Radix Panacis Ginseng (rén shēn 人参), Radix Angelicae (dāng guī 当归), Radix Bupleuri (chái hú 柴胡), Fructus Zizyphi (dà zǎo 大枣), Aurantii Nobilis Pericarpium (chén pí 陈皮), Radix Glycyrrhizae (gān cǎo 甘草), Rhizoma Cimicifugae (shēng má 升麻), Rhizoma Zingiberis (shēng jiāng 生姜). In a few clinical studies carried out in China, BZYQT showed significant efficacy in treating OAB, both alone [ 91 ] and in combination of Sang-Piao-Xiao-San [ 95 ] or propiverine [ 33 ]. BZYQT is believed to stimulate the flow of Qi, which serves as essential energy for the body to nourish the internal organs and maintain physical activities.

Ba-Wei-Di-Huang-Wan (Hachi-mi-jio-gan)

Ba-Wei-Di-Huang-Wan (BWDHW), also known as Hachi-mi-jio-gan in Japanese, is traditionally applied to warm the kidney yang and alleviating frequent urination. It is one of the most frequently used TCM prescription for treating abnormal thirst, polyuria, polydipsia, and urinary frequency with diabetes-like symptoms. The BWDHW regimen was created by the renowned Dr. Zhongjing Zhang (张仲景) over 1800 years ago. This herbal mixture contains eight ingredients, including Rehmanniae radix (dì huáng 地黄), Cornus officinalis (shān zhū yú 山茱萸), Dioscoreae rhizoma (shān yào 山药), Alismatis rhizoma (zé xiè 泽泻), Porica cocos (fú líng 茯苓), Moutan radicis cortex (mǔ dān pí 牡丹皮), Cinnamomi cortex (guì pí 桂皮), and heat-processed Aconiti radix (fù zǐ 附子). Nowadays, the granules of BWDWH manufactured by modern pharmaceutical companies are widely used by practitioners in Asia.

BWDHW exhibits potential in treatment for bladder overactivity associated with diabetes and metabolic syndrome in rats. In streptozotocin-induced diabetic rats, Tong et al. investigated the effects of BWDHW on the cholinergic function of the bladder and reported that BWDHW lowered the plasma glucose and reversed the hyper-contractility in the bladder. Results from this study indicated that BWDHW inhibits M 2 receptors overexpression and attenuats bladder overactivity [ 144 ]. Hachi-mi-jio-gan extract (HARNCARE ® , HE), a galenical made from BWDHW, was reported to inhibit acetylcholine-induced contraction of rat bladder strips and exert significant binding activity to muscarinic receptors, 1,4-dihydropyridine (DHP) receptors and purinergic receptors in the bladder [ 66 ]. Moreover, the effects of THC-002, an ethanol extraction of BWDHW (HARNCARE®), were tested on SHRs and it was reported that THC-002 effectively down-regulates the expression of tachykinins and P2X 3 and TRPV1 receptors in the bladder, thus inhibiting adenosine triphosphate (ATP)-induced DO [ 63 ]. Later on, Lee et al. demonstrated that BWDHW treatment ameliorated CYP-induced ongoing bladder hyperactivity by suppressing protein overexpression of mucosal P2X 2 , P2X 3 , M 2 , and M 3 receptor, as well as detrusor M 2 and M 3 receptor in rat bladders exposed to CYP treatment. In addition, BWDHW pretreatment reduced acidic ATP solution-induced bladder hyperactivity in rats by preventing hypersensitization of TRPV1 receptor in bladder mucosa activated by acidic stimulation and supressing overexpression of P2X 3 receptor in naïve bladder mucosa [ 88 ]. Furthermore, Tsai et al. demonstrated that BWDHW, through its active ingredient loganin, alleviates bladder overactivity as well as modulates substance P (SP)-induced oxidative injury and inflammation by inhibiting SP/neurokinin-1 receptor and nuclear factor kappa B/intercellular adhesion molecule 1 (NF-kB/ICAM-1) signaling pathways [ 145 ].

Fangjihuangqi Tang

Fangjihuangqi Tang (FHT) is composed of four herbs: Radix Stephaniae Tetrandrae (fáng jǐ 防己) 12 g, Radix Astragali Mongolici (huáng qí 黄芪) 15 g, Rhizoma Atractylodis Macrocephalae (bái zhú 白术) 9 g, and Radix Glycyrrhizae (gān cǎo 甘草) 6 g. It was first described in the ancient Chinese medical classic Jin Gui Yao Lue (金贵要略, Synopsis of Golden Chamber), and has been one of the most common TCM prescription to treat dysuria and edema for more than 1800 years. A study on benign prostatic hyperplasia (BPH) rats showed that 4-week FHT treatment could alleviate symptoms of lower urinary tract dysfunction by modulating smooth muscles in the bladder and urethra instead of lowering the volume of the enlarged prostate itself. It was speculated that one of the mechanisms of FHT might be related to active ingredients with calcium channel blocking effects and thus the constraint of the internal flow of Ca 2+ [ 31 ].

Ji‐Sheng‐Shen‐Qi‐Wan (Gosha-jinki-gan)

Ji-Sheng-Shen-Qi-Wan (JSSQW), also called Gosha-jinki-gan, has gained wide application as a treatment for patients suffering from LUTS, diabetic neuropathy, and nocturia in China and Japan. Traditionally, it is used to treat water accumulation caused by kidney yang deficiency. It was first recorded in the Southern Song Dynasty (AD 1127–1279). Containing 10 ingredients, JSSQW is a modification of BWDHW with the addition of Plantaginis semen (chē qián zǐ 车前子) and Achyranthis radix (niú xī 牛膝).

In basic research, JSSQW was found to improve bladder hyperactivity induced by intravesical installation of acetic acid (AA) through inhibition of resiniferatoxin (RTX)-sensitive bladder afferent neurons in rats [ 172 ]. In a rat model of AA induced OAB, Gosha-jinki-gan-fed rats exhibited longer intercontractile intervals and smaller contraction amplitudes in cystometry, and also lower dopamine and serotonin levels in plasma, compared with untreated controls. These results imply that JSSQW may have an impact on both the afferent and efferent micturition reflex and may exert central effects on micturition mechanisms. By regulating the balance of sympathetic and parasympathetic nervous systems to maintain a lower level of activity, it effectively restrains bladder overactivity [ 112 ]. Moreover, a later study demonstrated that pretreatment with JSSQW may prevent the bladder urothelium from overexpressing tachykinins (including neurokinin A, neurokinin B and substance P) and TRPV1 and P2X 3 receptors in rats subjected to intravesical AA stimulation. Besides, the authors further indicated that JSSQW may reduced expression of transmitter proteins and sensory receptors, without damaging nerve fibers [ 62 ]. A clinical trial reported that patients with nocturia exhibited decrease in both nocturia episodes and the IPSS after 12-week JSSQW treatment [ 161 ]. Thus, as addressed in literature, JSSQW mixtures show potential in offering alternative therapeutic option for OAB.

Modified Ojayeonjonghwan (Wuzi Yanzong Wan)

Ojayeonjonghwan (Wuzi Yanzong Wan) consists of five different fruits and seeds obtained from five types of berries: Psyllium, Cuscuta chinensis Lam , Lycium chinense Miller , Rubus coreanus Miquel , and Schisandra chinensis Baillon . A new berry mixture formula was developed in South Korea, and it is a modification of Ojayeonjonghwan with Psyllium replaced by Cornus officinalis Sieb . Bae, Lee et al. used a partial BOO animal model to demonstrate that the modified Ojayeonjonghwan (MO) exhibited similar pharmacologic activities to solifenacin in controlling DO induced by BOO via anti-inflammatory effect and anti-oxidant effects, as well as the increase of the NO pathway [ 15 ].

Sang-Piao-Xiao-San (Mantis Formula)

Sang-Piao-Xiao-San (Mantis Formula, 桑螵蛸散, SPXS) was first described in Ben Cao Yan Yi (本草衍义, Augmented Materia Medica) in the Northern Song Dynasty (AD 960–1127) for its effectiveness in addressing conditions characterized by heart and kidney deficiency, such as urinary incontinence accompanied by spermatorrhea. It is composed of Mantidis ootheca (sānɡ piāo xiāo 桑螵蛸), Radix Polygalae (yuǎn zhì 远志), Rhizoma Acori Tatarinowii (shí chāng pú 石菖蒲), Fossilia Ossis Mastodi (Dragon’s Bone, lóng gǔ 龙骨), Radix Panacis Ginseng (rén shēn 人参), Radix Angelicae (dāng guī 当归), and Carapax Testudinis et Plastrum. Sānɡ piāo xiāo means “mantis eggs in a foamy pouch”. Mantidis ootheca is the main herb in this formula. It has been discovered to induce relaxation of vascular smooth muscle via endothelium-dependent activation of PI3K/AKT-mediated NO-cyclic guanosine 3′,5′-monophosphate (cGMP)-protein kinase G (PKG) signaling pathway, upregulating NO production, and via possible involvement of K + channel [ 80 ]. A clinical study in patients with OAB showed co-treatment of SPXS and BZYQT decreases urinary frequency, urgency and urge incontinence, and enhances the voided volume and patient QOL [ 95 ]. Lu et al., studied the effects of SPXS and solifenacin in patients with OAB after menopause. They reported that SPXS combined with solifenacin improved TCM symptom scores, as well as the scores of urination, nocturnal urination, urgency of urination and urgency incontinence compared to treatment with solifenacin alone. It improved bladder compliance, maximum urinary flow rate and maximum bladder capacity, the initial urine volume (VFD), and also lower NGF and NGF/Cr level to a greater extent than using solifenacin alone.

Suo-Quan-Wan

Suo-Quan-Wan (SQW) is a classical phytotherapeutic formula in TCM consisting of three herbs: Alpinia oxyphylla Miq (yì zhì rén 益智仁), Lindera radix (wū yào 乌药), and Dioscorea rhizoma (shān yào 山药). SQW was first recorded in the Southern Song Dynasty (AD 1127–1279) and has been frequently applied in relieving nocturnal enuresis and frequent urination. Lai et al . reported that SQW reduced TRPV1 receptors expression in rat bladders and slowed down the advancement of bladder overactivity in a rat model of partial BOO [ 85 ]. In a later study with TRPV1 knockout mice, they provided additional evidence that SQW enhances bladder function by regulating TRPV1 receptors [ 86 ]. In another study by Xu et al., SQW was found to enhance bladder control, storage capacity and contraction ability in aging mice by increasing the sensitivity and expression of β 3 -adrenoceptor (β 3 -AR) [ 160 ]. Recent research discovered SQW’s protective effects against OAB and bladder dysfunction in diabetic mice. SQW treatment remarkably improved urodynamic urination with reduced non-voiding contraction (NVC) frequency, maximum bladder capacity (MBC), residual volume (RV), and bladder compliance (BC), and enhanced voided efficiency (VE). It also attenuated thickened bladder wall in diabetic mice, decreased DSM strips contraction response for stimuli including α, β-methylene ATP and carbachol. The mechanism involves upregulating motor protein myosin Va and transporter protein SLC17A9 in the bladder [ 151 ].

Wenglitong capsule

Wenglitong capsule (WLT) is a medicinal preparation that contains 11 herbal ingredients: Semen Coicis (Ma‐yuen jobstears seed, yì yǐ rén 薏苡仁), Bulbus Fritillariae Thunbergii (Chekiang fritillary bulb, zhè bèi mǔ 浙贝母), Caulis Clematidis Armandii (Armand clematis stem, chuān mù tōng 川木通), Fructus Gardeniae (Cape jasmine, zhī zǐ 栀子), Flos Lonicerae Japonicae (Japanese honeysuckle flower bud, jīn yín huā 金银花), Flos Inulae Japonicae (Japanese inula flower, xuán fù huā 旋覆花), Lycopi Herba (Hirsute bugleweed herb, zé lán 泽兰), Verdigris (tóng lǜ 铜绿), Radix Glycyrrhizae (Liquorice root, gān cǎo 甘草), Radix Astragali Mongolici (huáng qí 黄芪) and Rheum officinale (dà huáng 大黄). In a clinical trial on 182 female OAB patients, treatment with WLT reduced urgency incontinence, urinary frequency and significantly improved OAB symptoms, while exhibiting a slower onset and lower effectiveness compared to tolterodine, the treatment presented a favorable profile with fewer adverse effects. In addition, The combined administration of WLT and tolterodine demonstrated greater effectiveness in alleviating symptoms of OAB compared to using tolterodine alone [ 159 ].

Non-TCM formulations

Choreito (crt).

As a traditional Japanese (Kampo) medicine, Choreito (CRT) finds extensive application in Japan to treat OAB and other lower urinary tract symptoms. CRT consists of five ingredients including aluminum silicate hydrate with silicon dioxide, Alisma rhizome, Polyporus sclerotium, Poria sclerotium, and donkey glue. In rats, CRT demonstrated the ability to reduce DO induced by intravesical AA instillation, which appears to be mediated via alleviating urothelial damage and regulating excess blood flow [ 146 ].

The phytotherapeutic agent Eviprostat is a herbal remedy, consists of a mixture of whole plant extract deprived from several plants including Chimaphila umbellate (umbellate wintergreen), Populus tremula (aspen), Pulsatilla pratensis (small pasque flower) and Equisetum arvense (horsetail). This combination is blended with wheat-germ oil. With a history of over 50 years of prescription in Germany and Japan, it is one of the most extensively utilized phytotherapeutics to treat LUTS in BPH. And it is well known to exhibit antioxidant and anti-inflammatory activities, which have been investigated and validated in multiple studies. It was reported that Eviprostat was found to reduce the levels of 8-hydroxy-2’ –deoxyguanosine (8-OHDG), a urinary marker of oxidative stress, in both a rabbit model of surgically induced partial BOO and patients with LUTS related to BPH [ 104 ]. In another animal study on a rat model of bladder overdistension and emptying (OE), Eviprostat reduced inflammation and oxidative stress in OE rat bladders. It effectively prevented the reduction bladder blood flow (BBF) and the elevation in bladder weight, malondialdehyde levels (a marker of oxidative stress), proinflammatory cytokines and myeloperoxidase activity [ 77 ]. It has also been found that Eviprostat improved overactive bladder contractile response and down-regulated the increased levels of cytokines associated with cyclophosphamide-induced cystitis [ 110 ]. Clinical studies on Eviprostat demonstrated clea improvements in various parameters including IPSS, QoL score, and maximum and average urinary flow rates, increased prostatic volume, and reduced prostatic inflammation, without occurrence of any severe adverse effects [ 64 , 104 , 135 ].

Granu Fink femina

Granu Fink femina is obtained from seed oil from the combination of Uromedic pumpkin (cultivar of Cucurbita pepo ), Rhus aromatica (fragrant sumach) bark extract, and Humulus lupulus (hop) cone extract. A clinical study [ 49 ] on 117 women (age: 21–78 year) with OAB showed that Granu Fink femina exhibited the ability to decrease urination frequency and reduce the average frequency of leakages and pad usage among patients. Notably, it significantly improved all aspects of quality of life related to OAB after just 1 week of use, with further enhancements observed at 6 and 12 weeks. In addition to these positive effects, the observed excellent tolerability profile makes it a very promising therapeutic option for OAB.

Kubiker (Naturmed, Montegranaro, FM, Italy) is a new complementary and alternative medicine (CAM), which has been proposed to treat OAB, containing vitamins (C and D), herbas (cucurbita maxima, capsicum annum, polygonum capsicatum) and amino acid L-Glutammina. In a randomized, controlled clinical study on 90 consecutive women (mean age 65 year; range 40–75) with symptoms of OAB, Kubiker was found to reduce daily micturitions, nocturia and episodes of urge incontinence, improve Patient Perception of Intensity of Urgency Scale (PPIUS), Overactive Bladder questionnaire Short Form (OAB-q SF) and Patient Global Impression of Improvement questionnaire (PGI-I), in a more effective level compared to Solifenacin Succinate [ 148 ].

Urox is a herbal mixure of 3 phytomedicines that have well established traditional uses, including Crataeva nurvala (used in Ayurvedic Medicine ) , Equisetum arvense (an herbal remedy traced back to ancient Roman and Greek eras) and Lindera aggregata (wū yào 乌药, used in TCM). Efficacy of Urox on a range of bladder symptoms were studied clinically in a randomized, double-blind, placebo controlled trial with 150 participants. The treatment group exhibited a decrease in episodes of nocturia, symptoms of urgency, and total incontinence. After the treatment, notable enhancements in quality of life were reported, accompanied by minimal adverse effects [ 128 ].

Discussion and conclusion

In recent years, there have been notable accomplishments in utilizing medicinal plants to treat OAB. This article reviews the current application as well as pharmological and clinical studies of medicinal plants or hebal formulations in OAB treatment, hoping to bring novel perspectives for the management and investigation of OAB.

Effective OAB therapy options are still very limited while patient compliance of therapeutic regimens is quite low. Alternative therapies are pursued by patients to mitigate symptoms of OAB. Accumulating evidence indicates the promising potential of medicinal plants and natural products in prevention and treatment of OAB, showing greater effectiveness and tolerability. Due to the differences in context and focus of natural product research and the complexity of ancient medicinal theories, the acceptability and clinical application of medicinal plants and natural products in Western medicine are greatly hindered. It is thus essential to conduct pharmacological evaluation using modern scientific strategies. Isolation and identification of the active ingredient(s) present in plants responsible for their biological or therapeutic activities are foundamentally important for understanding the underlying mechanisms. The present review shows that most of the plant extracts used to treat OAB are aqueous extracts, while the remaining are lipidic extracts or essential oils. Until now, only a few phytochemicals have been isolated and characterized for their pharmacological activities against OAB, and it is necessary to put more efforts into making progress in this aspect.

Limitation in study design is another obstacle for OAB herbal treatment. Firstly, the plant species utilized in the same treatment regimen may differ across publications, and it is important to note that a TCM herb may not consistently originate from a single plant species.Thus, the botanical Latin names of the plant species should be carefully confirmed. In addition, the quality of herbs/plants could directly influence their pharmacological activities and clinical effectiveness. Hence, it is crucial to accurately identify authentic herbs/plants. Geo-authentic plants are cultivated in their native regions, where the climate and ecological conditions are optimal for producing high-potency herbal products. The desired parts of these plants are harvested at the peak of their season and in a specific manner to ensure the preservation of their medicinal properties without compromise. To ensure the quality and safety of herbs, it is important to choose GAP suppliers with legitimate sources of geo-authentic herbs and GMP pharmaceutical manufacturers with established standardization procedures.

What’s more, various types of animal models for OAB are utilized, each with distinct applications and scopes [ 93 ]. Hence, how to choose an appropriate and suitable model for medicinal plants targeting different mechanisms needs further exploration.

Based on the review of existing evidence of medicinal plants, natural products and herbal formulations used in treating OAB, we summarized their possible interverntion mechanisms in the unrinary bladder (Fig.  2 ). Considering the complicated and elusive pathophysiology of OAB, the mechanisms of treatment of OAB with medicinal plants or natural products are complex and the exact mechanism of different agents remains to be fully elucidated and verified.

Previous studies indicated phytochemicals from natural sources has a higher pharmacokinetic stability than synthetic drugs in the body. Due to their nature as small molecular compounds, natural products exhibit rapid absorption and high permeability. It is suggested different plant extracts components have synergistic effects beneficial for their bioavailability. The precise mechanism of action and pharmacokinetic properties including absorption, distribution, metabolism and excretion (ADME) of biologically active ingredients should be thoroughly investigated. Moreover, the combination of traditional medicine or natural products with conventional chemical drugs has emerged as a novel avenue of research and their synergistic actions have been increasingly studied in recent years. Herbal formulations have a long-standing history of utilization in the management of OAB, both in traditional practices and contemporary times. In recent years, a plethora of scientific studies have been conducted to enhance our comprehension of the efficacy and underlying mechanisms of these formulations. The efficacy-enhancing and toxicity-reducing effects of different kinds of herbal combinations are an attractive strategy, considering the multifactorial pathogenesis of OAB and the limitations of current available therapeutic options.

In conclusion, this review yielded an evidence basis of various medicinal plants and natural products used for OAB treatment. However, none of these herbal drugs have advanced to become novel clinical therapy options for OAB. Consequently, there is still a significant demand for effective and well-tolerated phytopharmaceuticals in this regard. Promising new alternative herbal treatment strategies are emerging with high safety level and efficacy worthy of clinical application. There is a growing aspiration to foster the development of OAB management. Nevertheless, the existing cases of medicinal plants or natural products treating OAB are still relatively limited, and some of them have been only used in traditional ways without modern scientific research evidence. This review would serve as a reference basis for researchers investigating OAB and encourage more relevant pharmacological and clinical research on those understudied natural medicines. Clinical trials using phytochemicals or hebal formulations against OAB are still in infancy. Future rigorously designed controlled studies will allow the validation of their place in the therapeutic arsenal for OAB.

Availability of data and materials

All data are fully available without restriction.

Abbreviations

Acetic acid

Alpha, beta-methylene ATP

Abdominal withdrawal reflex

Bladder blood flow

Bethanechol

Cyclophosphamide

Dihydropyridine

Detrusor overactivity

Detrusor smooth muscle

Electrical field stimulation

Electromyogram

Endoplasmic reticulum

First detrusor contraction

Intercellular adhesion molecule 1

Interleukin-1β

International Prostate Symptom Scores

Lower urinary tract symptoms

Maximal cytometric capacity

Multiple sclerosis

Nuclear factor kappa B

National Institute of Health—Chronic Prostatitis Symptom Index

Nerve growth factor

Neurogenic detrusor overactivity

• Overactive bladder

Overactive Bladder questionnaire Short Form

Overactive bladder symptom score

Patient’s Global Impression of Change

Patient Global Impression of Improvement questionnaire

Patient Perception of Intensity of Urgency Scale

Quality of life

Retinyl acetate

Resiniferatoxin

Stem cell factor

Solute Carrier Family 17 Member 9

Soluble N-ethylmaleimide-sensitive-factor attachment protein receptor

Traditional Chinese Medicine

Tumor necrosis factor- α

Transient receptor potential

Transient receptor potential V1

Uroplakin 3A

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Chen, H., Hoi, M.P.M. & Lee, S.M.Y. Medicinal plants and natural products for treating overactive bladder. Chin Med 19 , 56 (2024). https://doi.org/10.1186/s13020-024-00884-3

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The tug-of-war on iron between plant and pathogen

• Jiaying Sun 1 ,
• Shuqin Xiao 1 &
• Chunsheng Xue   ORCID: orcid.org/0000-0003-4271-0599 1  

Phytopathology Research volume  5 , Article number:  61 ( 2023 ) Cite this article

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Iron participates in various crucial metabolic processes as an essential cofactor of many enzymes, which are vital to the survival of plants and their pathogens. However, excessive iron is toxic to the cells of plants and pathogens. Iron plays a complex role in the interactions between plants and pathogens. Plants and pathogens have evolved sophisticated mechanisms to modulate iron status at a moderate level for maintaining fitness. Iron competition extensively exists on both sides of plants and pathogens during infection. Plants employ iron withholding, local iron accumulation, or iron deficiency to trigger resistance against pathogens. Pathogens counteract host-derived iron stress or interfere with plant iron homeostasis to ensure virulence during infection. This review focuses on the recent progress in understanding the roles of iron in plant-pathogen interactions and proposes prospects for future studies.

Iron (Fe) is essential for most living organisms, including plants and pathogens (Philpott 2006 ). Iron exists as reduced, ferrous Fe 2+ and oxidized, ferric Fe 3+ , making them essential cofactors of enzymes that mediate redox reactions in a variety of key cellular metabolic processes such as respiration, tricarboxylic acid cycle, DNA and lipid synthesis, electron transfer, and cell proliferation (Aznar et al. 2015 ; Camprubi et al. 2017 ; Verbon et al. 2017 ).

Iron is mainly present in the Earth’s crust as ferric hydroxides, which has extremely limited bioavailability due to its poor solubility under neutral aerobic conditions (Mori 1999 ). The growth and virulence of pathogens are defective under iron-deficiency conditions (Johnson 2008 ; Braun and Hantke 2011 ), as are chlorophyll synthesis and photosynthesis of plants, resulting in chlorosis and severe growth defects (Hänsch and Mendel 2009 ; Ravet et al. 2009 ). However, excess ferrous Fe 2+ inside cells easily combines with oxides or peroxides to form toxic hydroxyl radicals via the Fenton reaction, causing damage to proteins, DNA, and lipids (Pierre and Fontecave 1999 ; Papanikolaou and Pantopoulos 2005 ; Dixon and Stockwell 2014 ). As a result, plants and pathogens have evolved various mechanisms for tightly regulating iron uptake, transport, and storage.

Competition for iron is a pivotal issue of plant-pathogen interactions (Verbon et al. 2017 ; Liu et al. 2021 ; Herlihy et al. 2020 ). For one thing, host plants redistribute iron at the cellular level during pathogen infection to initiate iron immunity, modulate reactive oxygen species (ROS) bursts, or directly activate the immune system (Weinberg et al. 2008 ; Kehl-Fie and Skaar 2010 ; Ganz and Nemeth 2015 ; Soares and Weiss 2015 ; Xing et al. 2021 ). For another, some pathogens counteract plant immunity by extracting iron from host iron storage proteins, switching iron uptake strategies to overcome host-derived iron stress, or secreting effectors to interfere with plant iron homeostasis during infection (Singh et al. 2016 ; Xing et al. 2021 ; Wang et al. 2022 ). These manipulations are primary participants in the iron tug-of-war between plants and pathogens. In this review, we focus on recent progress in the regulatory mechanism of iron homeostasis in plants and pathogens, as well as the fundamental role of iron on plant immunity and pathogen virulence, emphasizing the unique role of iron in plant-pathogen interactions.

Iron homeostasis in plants

Plants have developed sophisticated mechanisms to ensure an adequate supply of iron in a fluctuating environment. Plants sense iron status and modulate the transcription of iron uptake-associated genes to regulate iron uptake from soil to root.

Iron acquisition strategies in plants

To adapt to Fe-deficient environments, plants evolved two different iron uptake mechanisms, known as reducing (Strategy I) and chelating strategies (Strategy II) (Römheld and Marschner 1986 ). Non-grass plants employ Strategy I to mobilize and acquire iron, which includes acidification of the rhizosphere by root-released H + involving H + -ATPase, such as AHA2 (Santi and Schmidt 2009 ). Fe 3+ is reduced to Fe 2+ by plasma membrane protein Ferric Reduction Oxidase 2 (FRO2) before being transported to the root epidermis by high-affinity iron transporter Iron-Regulated Transporter 1 (IRT1) (Eide et al. 1996 ; Robinson et al. 1999 ; Brumbarova et al. 2015 ). The grass family represents Strategy II plants, which release phytosiderophores (PS) from roots to solubilize and chelate Fe 3+ in soil by Transporter of Mgineic Aid1 (TOM1) (Nozoye et al. 2011 ). Fe 3+ -phytosiderophores chelates are then taken up by specific transporters, such as Yellow Sytripe1 transporter (YS1) or YS1-like (YSL) in plants (Curie et al. 2001 ; Kobayashi and Nishizawa 2012 ).

Regulation of iron homeostasis in plants

Iron uptake from the soil is essential for maintaining plant iron homeostasis, but it is not the sole mechanism involved in the above process. Plants have developed mechanisms for regulating gene expression in response to iron availability to maintain iron homeostasis. Multiple basic helix-loop-helix (bHLH) transcription factors are involved in regulating plant iron homeostasis. The FER-like Iron deficiency-induced Transcription factor (FIT) and POPEYE (PYE) modules are the two critical regulatory networks of iron homeostasis in the Strategy I plant (Long et al. 2010 ; Ivanov et al. 2012 ). Upon iron deprivation, the FIT (bHLH29) is activated at the transcriptional and post-translational levels after it interacts with the Ib subgroup of bHLH transcription factors (bHLH38/39/100/101) (Yuan et al. 2008 ; Sivitz et al. 2012 ; Wang et al. 2013 ) to activate downstream iron-uptake genes, such as AHA2, FRO2, and IRT1 (Colangelo and Guerinot 2004 ; Santi and Schmidt 2009 ). Independent of FIT, the expression of the IVb subgroup of bHLH transcription factor PYE/bHLH47 is also upregulated upon iron deficiency. Like FIT, PYE interacts with IVc bHLH transcription factor ILR3/bHLH115, activating FRO2/IRT1 or ferritins FER/nicotianamine synthase 4 (NAS4) to facilitate iron transportation (Long et al. 2010 ). E3 ubiquitin-protein ligase BRUTUS (BTS) interacts with ILR3/bHLH105 and bHLH115 to facilitate their degradation via the 26S proteasome pathway, negatively regulating the expression of FIT and PYE to prevent iron overload in Fe-sufficient environments (Long et al. 2010 ; Selote et al. 2015 ; Rodrı´guez-Celma et al. 2019 ). In addition, UPSTREAM REGULATOR OF IRT1 (URI/bHLH121) has been recently identified and characterized as a positive regulator of plant iron homeostasis that directly or indirectly regulates the expression of most of the known genes participating in FIT and PYE regulatory networks (Kim et al. 2019 ; Gao et al. 2020 ; Lei et al. 2020 ).

Iron homeostasis in rice, a model plant for studying the strategy II mechanism, is also regulated by the bHLH transcription factors. The rice OsbHLH156/ Oryza sativa FER-Like Fe Deficiency Induced Transcription Factor (OsFIT), OsbHLH56/Iron Related Transcription Factor 2 (OsIRO2), and OsbHLH63/OsIRO3 directly regulate genes involved in the iron uptake of Strategy II plant (Ogo et al. 2007 ; Liang et al. 2020 ; Wang et al. 2020 ). OsbHLH56/OsIRO2 positively regulates phytosiderophore biosynthesis and the expression of iron (III)-Deoxymugineic acid transporter YSL15 (Ogo et al. 2006 ); OsFIT/OsbHLH156 interacts with OsIRO2/OsbHLH56 and promotes its accumulation in the nuclear (Liang et al. 2020 ; Wang et al. 2020 ). In contrast, OsIRO3 negatively regulates iron deficiency responses (Zheng et al. 2010 ). Iron deficiency induces an increase in transcript abundance of OsIRO2 and OsIRO3, which is mediated by Positive Regulator of Iron Homeostasis OsPRI1/OsbHLH060, OsPRI2/OsbHLH058, and OsPRI3/OsbHLH059 (Zhang et al. 2017 , 2020 ; Kobayashi et al. 2019 ). Two more transcription factors, the Iron Deficiency-Responsive Element-binding Factor 1 and 2 (IDEF1 and IDEF2) from the ABI3/VP1 and NAC families, are also vital in regulating iron homeostasis (Kobayashi et al. 2007 , 2009 ). OsIDEF1 functions upstream of OsIRO2, forming a transcriptional cascade that enhances the expression of genes involved in Fe(III)-DMA uptake and translocation, whereas OsIDEF2 regulates iron transport by binding to the promoters of several genes involved in iron homeostasis (Kobayashi et al. 2007 , 2009 ). Haemerythrin Motif-containing Really Interesting New Gene (Ring) and Zinc-Finger Protein 1 and 2 (OsHRZ1 and OsHRZ2), two rice ubiquitin E3 ligases displaying high homology with BTS, have been reported as potential iron sensors that play a negative role in iron acquisition under iron-sufficient conditions (Kobayashi et al. 2013 ). The in-depth study of the transcriptional regulation of iron homeostasis in strategy II plants is mainly focused on rice, which remains to be further explored.

Plant signals and hormones are also involved in the regulation of iron homeostasis. Salicylic acid (SA), gibberellin (GA), nitric oxide (NO), and ethylene (ET) play key roles in the Fe-response signaling pathway (Graziano et al. 2002 ; Lingam et al. 2011 ; Meiser et al. 2011 ; Wild et al. 2016 ). SA has been found to upregulate the expression of Fe-responsive transcription factor genes bHLH38 , bHLH39 , and the Fe transport gene YSL3 (Kang et al. 2003 ). The SA levels increase, and the expression of SA-responsive genes is upregulated in Arabidopsis under iron deficiency conditions (Chen et al. 2014 ; Shen et al. 2016 ). DELLAs, the repressors of the GA signal pathway, directly bind to FIT, thereby inhibiting the expression of the downstream IRT1 gene under Fe-abundant conditions (Wild et al. 2016 ). Iron deficiency stimulates NO accumulation in plant roots, which upregulates iron uptake genes (Graziano et al. 2007 ). In tomato plants, treatment with an NO donor to Fe-deficient roots induces the upregulation of FRO1, IRT1, and FER (Graziano et al. 2007 ). Applying NO to maize mutants defective in Fe uptake can revert the chlorosis phenotype (Graziano et al. 2005 ). Furthermore, NO has also been identified as a stabilizing stimulus of FIT protein abundance implicated in post-translational regulation of FIT (Meiser et al. 2011 ). In Arabidopsis , tomato, and cucumber, supplementary ethylene induces the Fe deficiency response (Romera and Alcantara 1994 ; Lucena et al. 2006 , 2015 ; Waters et al. 2007 ; Garcia et al. 2010 ). Ethylene induces physiological and morphological responses in plant roots under Fe-deficient conditions (Lucena et al. 2015 ). The addition of ethylene, ACC, or ethephon, plants show physiological changes, such as enhanced ferric reductase activity, Fe 2+ uptake capacity, rhizosphere acidification, and flavin excretion (Romera and Alcántara 2004 ; Lucena et al. 2006 ; Waters et al. 2007 ; García et al. 2010 ). In addition to physiological responses, ethylene also regulates morphological responses to Fe deficiency, such as enhanced root hairs, surface area of root epidermal transfer cells, and cluster roots (Schmidt and Schikora 2001 ; Schikora and Schmidt 2002 ; Zaid et al. 2003 ; Romera and Alcántara 2004 ). Reciprocally, Fe deficiency influences ethylene biosynthesis and signaling pathways (Wu et al. 2011 ; Lucena et al. 2015 ; Ye et al. 2015 ). FIT promotes stability and assists iron acquisition by interacting with transcription factors EIN3 and EIL1 in the ET signaling pathway (Lingam et al. 2011 ). Thus, there is positive feedback between Fe deficiency responses and ethylene biosynthesis. Recently, it has been reported that Fe deficiency induces the high expression of SAM1 and SAM2 in a FIT-bHLH Ib module-dependent manner in plant roots (Lu and Liang 2023 ). These findings reveal that SA, NO, and ET are positive regulators of the Fe uptake, whereas GA is a negative regulator. The fact that hormones and signals contribute to iron homeostasis indicates that fine-tuning Fe transport, storage, and uptake is crucial for immunity. ROS and Ca 2+ signals also play a vital role in the regulation of plant development and stress response (Castro et al. 2021 ; Dong et al. 2021 ; Luan and Wang 2021 ). ROS-inducible transcriptional regulator ZAT12 interacts with FIT to prevent FIT degradation (Brumbarova et al. 2015 ). Calcium-dependent protein kinase CIPK11 interacts with FIT and activates FIT via phosphorylation at Ser272, allowing for FIT-dependent Fe deficiency responses (Gratz et al. 2019 ). Under iron-deficient conditions, calcium-dependent protein kinases CPK21 and CPK23 interact with and phosphorylate IRT1, promoting the transport of Fe from the extracellular space to the intracellular space (Wang et al. 2023 ). These findings indicate that plants fine-tune iron homeostasis at transcriptional and post-transcriptional levels.

Iron homeostasis in plant pathogens

During pathogen infections, iron is closely combined with plant ferritin, which makes it extremely low and unable to be absorbed and utilized by pathogens. Pathogens have developed various iron uptake strategies to successfully uptake iron from host plants for infection. In addition, pathogens have evolved precise iron-responsive regulatory systems to maintain iron homeostasis to adapt to iron-scarce or abundant host environments.

Iron acquisition of plant pathogens

The iron uptake strategies of plant pathogens have classically been divided into low-affinity and high-affinity uptake pathways (Haas 2014 ). Low-affinity uptake pathways encompass iron-containing protein uptake pathways and ferrous iron absorption pathways, mainly employed when iron is sufficient. The high-affinity uptake pathways play essential roles in acquiring iron under iron deficiency conditions, among which the siderophore-mediated iron uptake pathway is the most well-studied (Haas et al. 2008 ).

The high-affinity uptake pathways play substantial roles in the iron acquisition of phytopathogenic fungi during infection, including the reductive iron assimilation (RIA) pathway and the siderophore-mediated iron assimilation (SIA) pathway (Haas et al. 2008 ; Albarouki and Deising 2013 ). The RIA pathway is characterized by two redox steps at the plasma membrane. Iron reductases reduce the extracellular insoluble or chelator-complexed ferric Fe 3+ to soluble ferrous Fe 2+ (Dancis et al. 1992 ). Subsequently, Fe 2+ is oxidized to Fe 3+ and translocated into the cytoplasm by the synergistic complex multicopper ferroxidase (Fet3) and iron permease (Ftr1) (Marvin 2004 ; Albarouki and Deising 2013 ). Extracellular siderophores are a group of low molecular weight (ranging from 500 to 1500 Da) ferric-iron-specific chelators that positively influence the iron uptake of pathogens (Chu et al. 2010 ). Ornithine is catalyzed by the L-ornithine-N 5 -monooxygenase SidA and the non-ribosomal peptide synthase NPS to synthesize siderophores (Philpott 2006 ; Haas et al. 2008 ; Johnson 2008 ). The Fe 3+ -siderophore complexes are transported into the cell by the siderophore iron transporter (ARN/SIT) subfamily (Haas 2014 ).

Various extracellular siderophores are widespread in most phytopathogenic bacteria (Chu et al. 2010 ; Hider and Kong 2010 ). For Gram-negative bacteria, siderophores are secreted into the extracellular space and specifically bind to Fe 3+ to form Fe 3+ -siderophore complexes, delivered into the periplasm by TonB-dependent transporters (TBDTs) located in the outer membrane. Then, the Fe 3+ -siderophore complex is transported into the cytoplasm via an ABC transporter in the inner membrane. For Gram-positive bacteria, due to the absence of the outer membrane system of the bacteria, the Fe 3+ -siderophore complex uptake is implemented in one step, which is performed through an ABC-like transport system (Andrews et al. 2003 ; Pandey et al. 2023 ). For some Gram-negative bacteria, the ferrous iron uptake system is vital for iron acquisition in some anaerobic-microaerophilic environments. The ferrous iron is transported to the periplasm by Fe 2+ -specific porins. Then, the FeoB complex (FeoABC) transporter transports the ferrous iron to the cytoplasm (Janakiraman and Slauch 2000 ; Marlovits et al. 2002 ; Hantke 2003 ).

Transcriptional regulation of iron homeostasis in plant pathogens

Regulation of iron homeostasis is indispensable to ensure optimal cellular metabolism and avoid iron toxicity in phytopathogenic fungi. A negative feedback loop consisting of transcription factors Sre and HapX tightly regulates iron homeostasis in plant fungal pathogens (Canessa and Larrondo 2013 ; John et al. 2021 ). GATA-type transcription factor Sre (Siderophore biosynthesis repressor) is a core transcriptional regulator of iron homeostasis in phytopathogenic fungi (Voisard et al. 1993 ; Chao et al. 2008 ). Under iron sufficiency conditions, Sre binds to the consensus sequence ATCWGATAA and represses RIA and SIA pathways to avoid iron toxicity (An et al. 1997a , b ; Chung et al. 2020 ). Under iron starvation conditions, the transcriptional repression by Sre is disinhibited in the pathogen, thereby initiating the iron uptake pathway to rapidly acquire iron from the host plant (Schrettl et al. 2010 ). The bZIP-type transcription factor HapX is highly conserved among phytopathogenic fungi and has a basic leucine zipper domain that specifically binds to the 5′-CCAAT-3′ motif (Schrettl et al. 2010 ; Wang et al. 2019 ). Under iron starvation conditions, HapX spares iron by repressing iron-consuming pathways involved in processes of respiration, amino acid metabolism, citric acid cycle, DNA replication, and DNA repair (Jung et al. 2010 ; Schrettl et al. 2010 ; Chen et al. 2011 ; Hsu et al. 2011 ; López-Berges et al. 2012 ). Sre represses the expression of hapX under iron sufficiency conditions, while HapX represses Sre under iron starvation conditions (Mercier et al. 2006 , 2008 ; Jbel et al. 2009 ; Jung et al. 2010 ). Additionally, both Sre and HapX are regulated post-translationally with iron to inhibit HapX and activate Sre (Haas 2012 ). Recently, histone H2B deubiquitination (H2B deub1) and the deposition of histone variant H2A.Z and histone 3 lysine 27 trimethylation (H3K27 me3) have been found to be involved in the networks of HapX- and Sre-mediated iron homeostasis regulation. Under iron excess conditions, HapX activates iron storage by promoting H2B deub1 at the promoter of the responsible genes. Meanwhile, Sre inhibits iron acquisition by facilitating the deposition of H2A.Z and H3K27 me3 at the first nucleosome after the transcription start site (Sun et al. 2023 ).

Although Sre and HapX have been identified to be involved mainly in iron homeostasis regulation, they are also members of a larger transcriptional network in which other transcription factors modulate their expressions, and several of their targets are also subject to additional transcriptional regulation. Recent findings show that pH-responsive transcription factor PacC and nitrogen metabolism regulator AreA are involved in the regulation of iron homeostasis in phytopathogenic fungi (Gu et al. 2022 ; Wang et al. 2019 ). This finding indicates the potential existence of a regulatory network tandem between iron homeostasis, nitrogen metabolism, and pH response pathways in phytopathogenic fungi.

Fur (Ferric uptake regulator) is a global regulatory transcription factor that plays a core role in maintaining bacterial iron homeostasis, and its function depends on the availability of Fe 2+ (Baichoo et al. 2002 ; Fuangthong and Helmann 2003 ). Under iron excess conditions, Fur dimer and its corepressor Fe 2+ form a complex that binds to the conserved fur-box located in the promoter of many iron-uptake-related genes, including the biosynthetic genes of siderophores, to suppress their expression (Jittawuttipoka et al. 2010 ; Troxell and Hassan 2013 ). In contrast, under iron deficiency conditions, Fur dissociates with Fe 2+ , disengages from the promoter regions of target genes, and initiates iron uptake (Pandey 2023 ). XibR ( Xanthomonas iron-binding regulator) is another novel iron-binding transcriptional repressor of siderophore-biosynthetic genes (Pandey and Chatterjee 2022 ). Under iron-replete conditions, the Fe 3+ -XibR complex directly binds to the promoter region of genes involved in siderophore synthesis, thereby repressing gene expression. Meanwhile, under iron-deplete conditions, XibR transcriptionally activates the expression of genes related to iron storage and outer membrane receptors for enhancing iron uptake (Pandey et al. 2016 ). These findings indicate that the regulation of iron homeostasis in phytopathogenic bacteria is a complex system.

The role of iron in the interactions between plants and pathogens

Iron is a key microelement with multiple roles throughout plant-pathogen interactions. Both sides of the interaction have evolved a variety of strategies to limit the rival’s iron availability or disrupt iron homeostasis.

Iron and plant immunity

A defense strategy in vertebrates is withholding Fe by ferritin to limit iron availability for pathogen proliferation (Soares and Weiss 2015 ). The growth and developmental restriction of pathogens conferred by plant iron availability is called “iron immunity” (Xing et al. 2021 ). Host plants hide iron with iron storage proteins and defensin to create a locally extremely deficient iron host environment, inhibiting pathogen growth, development, and virulence (Kieu et al. 2012 ; Hsiao et al. 2017 ). The iron-binding ferritin gene ( FER ) expression is upregulated in plants after pathogen infection (Dellagi et al. 2005 ). During the interaction between Arabidopsis thaliana and Dickeya dadantii , the iron content of infected sites is significantly lower than that of non-infected sites. The expression of the iron storage gene FERRITIN1  ( AtFER1 ) is upregulated to initiate iron immunity that sequesters free Fe 3+ in the cytoplasm and prevents Fe 3+ entering apoplasts against pathogen invasion (Kieu et al. 2012 ; Aznar et al. 2015 ). Arabidopsis plants deficient in FER expression are more susceptible to D. dadantii (Dellagi et al. 2005 ). The role of plant defensins (PDF1.1, 1.2, and 1.3) is consistent with FER in iron immunity (Thomma et al. 2002 ; Sels et al. 2007 ). The AtPDF1.1 is secreted into the apoplast to chelate Fe 3+ , limiting the iron supply and reducing pathogen virulence (Hsiao et al. 2017 ). Moreover, multiple plant immune-signaling pathways commonly suppress the pathogenic bacterial iron acquisition pathway (Nobori et al. 2018 ). The above examples suggest that iron immunity is one of the essential defense responses in plant-pathogen interactions.

Local iron accumulation is also an iron-immune strategy in plants. Recruitment of iron in infection sites induces ROS bursts through the Fenton reaction, which is a critical immune response for plants, particularly poaceae. In wheat- Blumeria graminis pathosystems, Fe 3+ accumulates and ROS bursts at cell wall appositions (CWAs) (Greenshields et al. 2007 ; Liu et al. 2007 ). In rice- Magnaporthe oryzae pathosystems, Fe 3+ is overaccumulated at infectious sites, dramatically suppressing the pathogen’s growth by host ROS bursts. Fe 3+ and ROS bursts at infected sites and phytochemical accumulation significantly enhance host resistance with high iron treatment. In agreement with these findings, in the pathosystems of maize and Colletotrichum graminicola , sufficient iron induces ROS accumulation and enhances host resistance (Ye et al. 2014 ). In addition, in the incompatible rice- M. oryzae interaction, iron redistribution and ROS accumulation lead to iron- and ROS-dependent ferroptotic cell death, which is an important plant immune response (Dangol et al. 2019 ; Liang et al. 2021 ). These data indicate that the mechanistic connection between the recruitment of iron and a successful immune response is explained, at least in part, by ROS bursts dependent on iron.

Iron deficiency confers plant resistance as an important immune response. Under iron deficiency conditions, A. thaliana confers resistance to the bacterial pathogen D. dadantii and the necrotrophic fungus Botrytis cinerea (Kieu et al. 2012 ). The inoculation of B. cinerea activates the Fe deficiency response of plants, which further induces ethylene synthesis and then resistance to B. cinerea (Lu and Liang 2023 ). In addition, iron deficiency induces the production of secondary metabolites involved in plant immunity. Under iron-deficient conditions, Arabidopsis roots secrete coumarin with antimicrobial ability and function as defense compounds in host plants against pathogen infection (Verbon et al. 2017 ; Beyer et al. 2019 ). In rice, iron deficiency triggers the secretion of protocatechuic acid, which has antimicrobial activity and confers resistance to the fungal pathogen C. circinans in onions (Tzin and Galili 2010 ; Ishimaru et al. 2011 ). These data show that iron deficiency can trigger the plant immune system (Fig.  1 ).

Iron-mediated plant immunity. A strategy is to withhold Fe by chelating to limit iron availability necessary for pathogen proliferation (upper left). The second strategy is to suppress pathogens at infection sites by local iron accumulation, which induces ROS bursts (lower left). The third strategy is to induce the production of defense compounds or ethylene synthesis via iron deficiency to activate the expression of defense genes (right)

In addition, plant iron homeostasis is inextricably linked to plant immunity. Pathogen infections significantly modulate the high expression of iron homeostasis genes in host plants. Meanwhile, the expression of IRT1 , FRO2 , and AtNRAMP3 (an iron transporter located in vacuole membrane) is upregulated in roots by D. dadantii inoculation in Arabidopsis leaves (Dellagi et al. 2005 ; Segond et al. 2009 ; Aznar et al. 2014 ). In rice, miR7695 expression is regulated by M. oryzae infection, with subsequent downregulation of an alternatively spliced transcript of iron transporter OsNramp6 (natural resistance-associated macrophage protein 6) (Li et al. 2019 ; Sánchez-Sanuy et al. 2019 ). These results suggest that IRT1, FRO2, and NRAMP are involved in plant immunity as positive regulators. PYE/ILR3 plays a key role in ROS accumulation by facilitating cluster shuttling between proteins in intracellular organelles and the cytosol via a conserved 2Fe-2S protein NEET (Nechushtai et al. 2012 ; Zandalinas et al. 2020 ). In addition, ILR3 also interacts with the Alfalfa mosaic virus coat protein and positively regulates accumulations of ROS, pathogenesis-related protein 1 (PR1), SA, and JA (Aparicio and Pallás 2017 ). BTS, an iron-binding E3 ligase, interacts with AtVOZ1 and AtVOZ2, which are NAC transcriptional regulators that activate the defense responses against fungal and bacterial infection in A. thaliana (Nakai et al. 2013 ; Selote et al. 2018 ). Thus, iron homeostasis has an intrinsic role in coordinating plant growth, development, and disease resistance.

Iron and virulence of plant pathogens

Siderophore-mediated iron uptake pathway is essential for the full virulence of many phytopathogenic bacteria, such as D. dadantii , Erwinia amylovora , E. carotovora , Pseudomonas syringae pv. tabaci , and Xanthomonas oryzae pv. oryzicola (Barnes and Ishimaru 1999 ; Franza et al. 2005 ; Taguchi et al. 2010 ; Rai et al. 2015 ; Müller et al. 2022 ). However, siderophores exhibit no obvious impact on virulence in some phytopathogenic bacteria, such as P. syringae pv. tomato DC3000, and Ralstonia solanacearum AW1 (Bhatt and Denny 2004 ; Jones and Wildermuth 2011 ). The ferrous iron uptake pathway also plays an important role in the virulence of some phytopathogenic bacteria. For example, the full virulence of X. oryzae pv. oryzae is dependent on the ferrous iron uptake pathway rather than on the siderophore-mediated iron uptake pathway (Pandey and Sonti 2010 ).

The ability to uptake iron from the host is indispensable to the virulence of phytopathogenic fungi. High-affinity iron assimilation pathways RIA and SIA are essential for full virulence. The RIA pathway is necessary for U. maydis and Microbotryum violaceum to ensure their full virulence (Birch and Ruddat 2005 ; Eichhorn et al. 2006 ). SIA pathway is essential for the full virulence of C. miyabeanus , A. brasicicola , A. alternata , F. graminearum , M. grisea , C. graminearum , and Aspergillus fumigatus (Oide et al. 2006 ; Greenshields et al. 2007 ; Hof et al. 2007 , 2009 ; Chen et al. 2013 ; Haas 2014 ; Lu et al. 2021 ). In addition, the low-affinity iron assimilation pathway also plays a role in virulence (Grinter et al. 2018 ; Zheng et al. 2017 ; Yu et al. 2023 ).

A unique correspondence between the high-affinity uptake pathways and lifestyles exists in maize pathogenic fungi, and biotrophic and necrotrophic fungi have been found to rely on RIA and SIA pathways, respectively, for iron acquisition during pathogenesis (Mei et al. 1993 ; Eichhorn et al. 2006 ; Oide et al. 2006 ; Condon et al. 2014 ). Interestingly, the maize hemibiotrophic fungi utilize RIA and SIA pathways at biotrophic and necrotrophic stages, respectively (Albarouki et al. 2014 ). In typical maize biotrophic fungus U. maydis , the deletion of SIA pathway gene Sid1 has no impact on virulence, while inactivation of the multicopper oxidase gene Fer 1 and the iron permease gene Fer2 in the RIA pathway results in virulence reduction (Mei et al. 1993 ; Eichhorn et al. 2006 ). In maize necrotrophic fungus C. heterostrophus , the siderophore biosynthesis gene ChNPS6 is required for full virulence, while the iron permease gene FTR1 is dispensable for virulence (Oide et al. 2006 ; Condon et al. 2014 ). Meanwhile, the two life stages of maize hemibiotrophic fungus C. graminicola coincide with the two distinct iron uptake pathways. Inactivation of the RIA pathway gene fet1-3 in C. graminicola impairs the infection structure differentiation and the appressorial penetration in the biotrophic phase, while the deletion of SIA pathway genes sid1 and nps6 , results in necrotrophic hyphae expansion and virulence reduction (Albarouki and Deising 2013 ; Albarouki et al. 2014 ). These findings suggest a correlation between the iron assimilation pathways switch and the trophic style transition in hemibiotrophic fungi in maize.

Extracellular siderophores trigger plant immunity

In addition to contributing to pathogen virulence, extracellular siderophores are able to trigger plant immunity. Fungal extracellular siderophores initiate plant immunity in two different ways. Extracellular siderophores activate the salicylic acid pathway in A. thaliana by scavenging Fe, whereas siderophore-Fe complexes are ineffective (Dellagi et al. 2009 ). Similarly, treatments of barley leaves with deferrioxamine (DFO) upregulate the expression of PR and Fe homeostasis genes (Liu et al. 2007 ). These results indicate that iron scavenging is precisely a mechanism of immunity. Fungal extracellular siderophores also directly activate plant immune responses. Pretreatment with the coprogen primes maize defenses against the hemibiotroph pathogenic fungus C. graminicola (Albarouki et al. 2014 ). Pre-treated maize leaves enhance PR gene induction and ROS accumulation upon subsequent attack by pathogens. Similar results are obtained upon treatments with apo-coprogen or Fe-coprogen, indicating that priming, in this case, is independent of Fe scavenging. As the priming effect of coprogen on immune responses in maize does not rely on Fe scavenging, coprogen may be recognized by a receptor to activate downstream responses, as in the case of MAMPs that are recognized by pattern recognition receptors. In addition, extracellular siderophores modify Fe distribution at the cellular level in plants. Siderophores induce ROS production and activate immunity by directly transferring iron from apoplast to cell wall accumulation (Aznar et al. 2015 ).

Plant pathogens overcome “iron-related immunity” using diverse strategies

Well-adapted pathogens have evolved mechanisms for extracting iron from host iron storage proteins (Fig.  2 a).  Verticillium dahliae chelates iron from host plants through Asp-type CFEM family members in iron-deficiency xylem and Asn-type CFEM members to suppress immunity, for successful colonization and propagation in host plants (Wang et al. 2022 ). The xfeA (iron receptor gene) in X. oryzae pv. oryzicola senses extra-cytoplasmic iron by adenosine-to-inosine (A-to-I) RNA editing, suggesting that bacteria may use A-to-I editing as an alternative strategy to promote the uptake of metabolic iron and improve their competitiveness (Nie et al. 2021 ). This observation has revealed a new mechanism by which bacteria use A-to-I RNA editing to adjust iron concentrations. The hemibiotrophic pathogens shift lifestyle from biotrophs during the early stage of infection to the necrotrophic phase at the late stage. The lifestyle transition in their infectious cycle suggests that the hemibiotrophic fungi switch their iron uptake strategies to counteract host iron immunity during infection. C. graminicola utilizes the RIA pathway to efficiently acquire iron in the biotrophic stage and overcome host low iron stress for pathogens invasion and development (Albarouki and Deising 2013 ). Subsequently, pathogens secrete extracellular siderophores to chelate Fe 3+ . Furthermore, extracellular siderophores plunder iron from host ferritin to limit oxidative stress caused by iron accumulation in plants (Albarouki et al. 2014 ). Biosynthetic genes of extracellular siderophores are specifically downregulated during the biotrophic phase, possibly regulating the production of siderophores at the early stages of infection to circumvent the elicitation of host immune responses.

Pathogens employ multiple mechanisms to overcome plant iron-regulated immunity. a One strategy is to extract iron from host iron storage proteins. For example, under plant iron-depleted conditions, V. dahliae employs the Asp-type CFEM-containing VdSCPs (VdSCP33, VdSCP41, VdSCP43, VdSCP72, VdSCP99, VdSCP116, and VdSCP120) to sequester iron to counteract host resistance; under iron-depleted conditions or in planta , xfeA in Xanthomonas oryzae pv. oryzicola senses extra-cytoplasmic iron and changes the hydrogen bonding network of ligand channel domains by adenosine-to-inosine (A-to-I) RNA editing; C. graminicola utilizes the RIA pathway to efficiently acquire iron in the biotrophic stage, overcomes host low iron stress. b Pathogens secrete extracellular siderophores to chelate Fe 3+ to limit host oxidative stress. Another strategy is to interfere with plant iron homeostasis. For example, effectors delivered by pathogens interact with and target the plant iron homeostasis protein to facilitate iron uptake or inhibit ROS production

Interfering with plant iron homeostasis is another mechanism by which pathogens counteract iron-regulated immunity (Fig.  2 b). AvrRps4, an effector protein delivered by P. syringae , interacts with and targets the plant iron sensor protein BTS to facilitate iron uptake and pathogen proliferation in A. thaliana . AvrRps4 resulted in iron accumulation, especially in the plant apoplast (Xing et al. 2021 ). M. oryzae suppresses ROS accumulation by secreting effector AVR-Pii and interferes with ferroptosis to overcome host immunity (Singh et al. 2016 ). These findings suggest that secreting effector proteins is an important strategy by which pathogens interfere with plant iron homeostasis.

Conclusion and Perspectives

Iron should be considered as a crucial microelement with complex roles in plant-pathogen interactions. Many topics remain to be addressed about the role of iron in the interaction between plants and pathogens, which will influence future research directions.

Iron is a conserved factor as a microelement and signal that potentially modulates defense response against invaders in animal and plant kingdoms during evolution. Plant iron status is a key indicator of plant-pathogen interactions and particular defense responses. Fe supply modulation leads to different outcomes depending on the strategy of pathogen infection. In some cases, Fe directly contributes to the amplification of plant ROS production. Moreover, Fe supply indirectly affects plant metabolic activity, thereby allowing the production of antimicrobial compounds or other defenses that require Fe-dependent enzymes. However, Fe deficiency also causes the accumulation of antimicrobial compounds. These lines of evidence suggest that iron status has multiple effects on plant immunity depending on specific plant-pathogen interactions. However, studying how the host plants determine to defend the pathogens by withholding iron or over-accumulating iron is still needed.

Microbial extracellular siderophores initially secreted by pathogens to acquire Fe from their environment have been shown to trigger defense responses. Depending on the host, defense responses by extracellular siderophores are involved in either their Fe scavenging property or as MAMPs (microbe-associated molecular patterns). In the soil environment, plants are exposed to a variety of beneficial or pathogenic microorganisms, all of which are likely to produce extracellular siderophores. A better understanding of how extracellular siderophores affect plant immunity holds promise for designing new crop protection strategies.

Hemibiotrophic pathogens switch their iron uptake strategies to adapt to host iron status. For example, maize plants recruit free iron to the infection site, which induces ROS accumulation during C. graminicola infection (Ye et al. 2014 ). The iron assimilation pathway of C. graminicola switches from RIA to SIA in response to host iron status changes (Albarouki and Deising 2013 ; Albarouki et al. 2014 ). This evidence shows that the iron status of host plants substantially determines iron assimilation pathways utilized by fungal pathogens. A comprehensive study of biotrophic, hemibiotrophic, and necrotrophic pathogens would allow a better understanding of the link between the pathogen lifestyle and plant iron status. In addition, it would be interesting to know whether the plant Fe status affects pathogen lifestyle, which could also affect pathogenesis. In addition, it is meaningful to study how the pathogens sense the intracellular and extracellular iron status. Iron homeostasis genes are considered crucial elements with complex roles in plant immunity, whereby iron homeostasis genes could be underlying resistance-related genes in crops. Crops could be engineered to overexpress Fe homeostasis genes that positively affect plant disease resistance without yield loss.

Iron-limiting soils are widespread, causing significant losses in plant growth and productivity. Rhizosphere microbes have great potential for improving plant iron nutrition under iron-limited conditions. Under iron-limited conditions, plant-secreted coumarin compounds are beneficial mediators of plant-microorganism interactions. These specialized metabolites alter the composition of root microbiota and are necessary for microbiota-mediated plant iron uptake and immune regulation (Verbon et al. 2017 ; Schmidt et al. 2020 ). In Arabidopsis , variation in coumarin production has been shown to correlate with performance under iron limitation (Siwinska et al. 2014 ; Tsai et al. 2018 ). Rhizosphere microbes improve the performance of iron-limited plants dependent on plant iron import and secretion of the coumarin (Harbort et al. 2020 ). These findings show that the root microbiota is an integral component of plant edaphic adaptation to growth in iron-limiting soil. Root-secreted coumarins are inducible under iron starvation and mediate an interaction between the host and commensals that improves host iron nutrition. A better knowledge of these complex interactions and their monitoring will aid the improvement of crop production in iron-limiting soil.

Availability of data and materials

The datasets used and/or analyzed during the current study are available from the corresponding authors on request.

Abbreviations

Cell wall appositions

Deferrioxamine

Gibberellin

Phytosiderophores

Reductive iron assimilation pathway

• Reactive oxygen species

Salicylic acid

Siderophore-mediated iron assimilation pathway

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Preparation of corrosion inhibitor from natural plant for mild stil immersed in an acidic environmental: experimental and theoretical study

• Maryam Pourmohseni 1 ,
• Alimorad Rashidi 2 &
• Mehrnoosh Karimkhani 1  

Scientific Reports volume  14 , Article number:  7937 ( 2024 ) Cite this article

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• Mathematics and computing
• Plant sciences

In the present study, the inhibition performance of some medicinal plants (i.e. Yarrow, Wormwood, Maurorum, Marjoram, and Ribes rubrum ) was theoretically and experimentally investigated for mild steel immersed in 1M HCl. In this way, the obtained extracts characterized by Fourier transform infrared spectroscopy (FT-IR) and the electrochemical and theoretical techniques were used to study the inhibition mechanisms of the extracts for the immersed electrode in the acidic solution. In addition, the microstructure of the electrode surface immersed in the blank and inhibitor-containing solutions characterized by field emission scanning electron microscopy (FE-SEM), and Violet-visible (UV–Vis) spectroscopy was used to confirm the adsorption of the compounds on the electrode surface. The obtained electrochemical results revealed that the inhibition performance of the green inhibitors increased by increasing their dosage in the electrolyte. In addition, it was proved that Marjoram plant extract possessed the most inhibition efficiency (up to 92%) among the under-studied herbal extracts. Marjoram extract behaved as a mixed-type inhibitor in the hydrochloric acid solution, and the adsorption process of the extract on the steel surface followed the Langmuir adsorption model. Adsorption of the compounds on the steel surface was also studied using density functional theory (DFT), and it was found that the protonated organic compounds in the extract have a high affinity for adsorption on the electrode surface in the acidic solution.

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Introduction

It is asserted that natural disasters, including tornadoes, lightning, floods, fires, and earthquakes, have lower yearly costs than corrosion 1 . Therefore, safeguarding alloys from the phenomena is an alluring topic in both the industrial and academic spheres 2 , 3 , 4 . Acid solutions are frequently used in industrial cleaning, acid pickling, and oil-well acidizing 5 , 6 . Corrosion inhibitor compounds are one of the most important ways to avoid alloys submerged in corrosive solutions from degrading 7 , 8 .

It is well known that inhibitors’ electronic structure and spatial orientation have been linked to their efficacy 9 . Generally, inhibitors containing π-bonds and heteroatoms (P, S, N, and O) with lone pair electrons are the most potent and efficient 10 . Because both inorganic and organic inhibitors are usually hazardous, researchers are looking for more environmentally friendly substances, often known as green or natural inhibitors (NIs) 11 , 12 . NIs, which comprise medication and plant extracts, are regarded as safe substances devoid of heavy metals 13 . The use of medicinal plants as NIs for submerged alloys in aggressive media has been experimentally described previously, in addition to their conventional use as medicines 14 , 15 . Antimalarial, analgesic, antibacterial, antiviral, antifungal, and antineoplastic medications have all been reported to be employed as NIs for submerged alloys under different conditions 16 , 17 .

For hundreds of years, people have used Urtica dioica L. extract as an effective ancient medicine to treat anemia, eczema, arthritis, gout, and painful muscles and joints. Today, it is utilized to address urinary issues when prostate enlargement is still in its early stages 18 . Nasibi et al. 19 investigated the corrosion inhibition performance of the extract for mild steel (MS) immersed in 1M HCl. The obtained electrochemical analysis results revealed that the maximum inhibition efficiency of the extract was 92.24% in the presence of 300 ppm of the NI at 40 °C. Malva sylvestris L. is another ancient medicine with antimicrobial, hepatoprotective, anti-inflammatory, and antioxidant properties 20 . Naghi Tehrani et al. 15 used the herbal medicine as NI for MS and found that the inhibition efficiency of the extract was about 91% at the concentration of 2000 ppm in a saline media. Mentha suaveolens L. extract has many health advantages, including lowering fevers and easing asthma and depression. Mint is frequently used as a tea as a home remedy to help relieve stomach discomfort. In addition, the extract was originally used as a medicinal herb to treat stomach and chest aches 21 . Salhi et al. 14 used the plant’s essential oil and aqueous extract as a potent NI for MS in 0.5M H 2 SO 4 . The obtained EIS results showed that 2000 ppm of the essential oil and the aqueous extract provided inhibition efficiencies of 81.9% and 91.3% in the corrosive media, respectively. Foeniculum vulgare M., which is usually known as fennel, has been used for respiratory, reproductive, endocrine, and digestive systems. It is additionally utilized by breastfeeding women as a galactagogue agent 22 . Lahhit et al. 23 and Bouoidina et al. 24 used essential oil of the herbal medicine as an NI for carbon steel in an HCl electrolyte. Both studies proved that the essential oil's inhibition performance increased in the presence of more inhibitor dosages but decreased with the increase in temperature.

Yarrow is a helpful plant to keep on hand for various illnesses since it possesses antiseptic, astringent, antibacterial, and anti-inflammatory qualities. To aid in stopping bleeding, Yarrow leaves can be applied topically or powdered into a styptic powder 25 . Wormwood has historically been considered a helpful treatment for issues with the liver and gallbladder. Absinthin and Anabsinthin, two potent bitter substances in Wormwood, stimulate the gallbladder and digestive systems. According to popular belief, Wormwood helps to ease intestinal spasms and stimulate digestion 26 . In folk medicine, Maurorum treats piles, migraine, warts, and rheumatism as purgative, diaphoretic, expectorant, and diuretic 27 . Marjoram has a long history of usage in both traditional medicine and cookery. It may reduce inflammation, ease gastric discomfort, and regulate the menstrual cycle, among other potential advantages 28 . Lycopene, an anti-oxidant carotenoid, is found in Ribes Rubrum. Heart disease and cancer risk are both decreased by lycopene, particularly prostate cancer. Furthermore, it shields the body from the effects of free radical stress, which can harm DNA and other cellular structures 29 .

In the present study, extracts of five different herbal medicines (i.e., Yarrow, Wormwood, Maurorum, Marjoram, and Ribes rubrum ) were used as NIs for MS exposed to 1M HCl solution, and their inhibition performance was investigated using electrochemical techniques. The results of this research can open a window to using of traditional herbal medicines as new and powerful NIs to prevent corrosion damage.

Materials and methods

HCl solution (1M) was prepared using hydrochloric acid (37% Merck) and distilled water. Steel panels (St-12) in the dimension of 0.5 × 5 × 15 cm were obtained from Foolad Mobarakeh Company (Iran) with the following chemical composition: C (0.190 wt%), Si (0.288 wt%), Mn (1.388 wt%), Cr (0.03 wt%), Mo (0.016 wt%), Co (0.386 wt%), Cu (0.299 wt%), Nb (0.354%), and Fe (balance). Ribes rubrum (collected from Arsbaran forests, Iran), Wormwood (collected from Mazandaran, Iran), Marjoram (collected from Astara, Iran), Yarrow (collected from Esfahan, Iran), Maurorum (collected from Tabriz, Iran). Picture of the plants are shown in Fig.  1 .

Picture of the plants ( a ) Marjoram, ( b ) Yarrow, ( c ) Ribes rubrum , ( d ) Maurorum, ( e ) Wormwood.

Preparation of the extracts

At first, the medicinal plants, including Yarrow, Wormwood, Maurorum, Marjoram, and Ribes rubrum were powdered, and then 50 g of powders was mixed with 500 ml of distilled water, followed by heating and stirring on a magnetic stirrer at 70 °C for 5 h. afterward, the mixtures were centrifuged at 5000 rpm for 5 min to remove undissolved particles. Then, the solvent (water) was evaporated by incubating the filtered solution in an oven at 500 °C for 48 h and the obtained dry powders were used as the NIs for the electrochemical analyses.

Steel surface preparation

To prepare the surface of mild steel (MS) panels for the electrochemical tests, the sheets were degreased with acetone to remove surface contamination and then polished with 400, 600, 800, and 1200 sandpapers to remove the oxidized layers. Then, they were rewashed with acetone and dried at 50 °C.

Characterization methods

To investigate the chemical compounds in the used extracts, FT-IR analysis was performed in the range of 400–4000 cm −1 using a Perkin-Elmer spectrometer. UV–VIS spectra were carried out using a UV–Vis spectrophotometer (Hitachi U-3010) in the solution medium. Using an emission scanning electron microscope (FE-SEM) model Mitra3, the surface of the immersed electrodes was studied in the presence and absence of the NIs.

Electrochemical tests

The corrosion inhibition of the extracts for MS immersed in the 1M HCl was evaluated in the presence of different dosages of the NIs (0, 200, 400, 600, and 800 ppm) at different immersion times (0.25, 2, 4 6, and 24 h).

Electrochemical analyses (EIS and PP) were done using a 3-electrode system including a saturated calomel reference electrode, platinum as an auxiliary electrode, and the prepared steel panels with a diameter of 1 cm 2 as the working electrodes by an Ivium Compactstat instrument. Before performing the electrochemical analyses, the steel electrodes were submerged in the corrosive solutions for 15 min to reach steady state conditions. EIS measurements were performed in the frequency range of 10 kHz to 10 mHz with a sinusoidal voltage of ± 10 mV. PP tests were also carried out in the potential range of + 250 mV to – 250 mV with a 0.001 V s −1 scan rate.

Theoretical studies

The ORCA program package, module version 4.0, was used to carry out the quantum-based calculations. Prior to completing geometry optimization, the calculations began with no geometry constraints. The global minimum for geometry optimizations has been found after careful consideration. The hybrid B3LYP functional level with a higher basis set, indicated as 6-311G(d,p), was used for all calculations with complete geometry optimization 30 . Self-Consistent Reaction Field (SCRF) theory and the Polarized Continuum Model (PCM) have been used to analyze the effect of solvent 31 . Koopman’s theory states that the energies of the HOMO and LUMO orbitals were used to calculate the ionization energy and the electronic affinity 32 , 33 .

Ethical statement

The plant collection and use was in accordance with all the relevant guidelines.

Results and discussion

Ft-ir investigations.

FT-IR spectroscopy was used to investigate the chemical compounds in the under-studied extracts. Other plant extracts are presented in the supplementary information except for the FT-IR result of the Marjoram extract. The FT-IR spectrum of herbal extract from Marjoram and the present chemical compounds in the extract (described by Lopez et al. 34 ) are shown in Figs.  2 and 3 , respectively.

FT-IR spectrum of the Marjoram extract used in this research.

The chemical compounds found in the extract of Marjoram medicinal plant described by Lopez et al. 34 .

As demonstrated in Fig.  2 , the marjoram extract contains chemical compounds with C–H, C–OH, O–H, and aromatic rings. The broad band located at 3428 cm −1 and the sharp peak centered at 1623 cm −1 belong to the stretching and bending vibrations of O–H bonds in the hydroxyl groups and surface adsorbed water, respectively 35 . The stretching vibrations peak of C–H bonds in aliphatic groups is located at 2830 cm −1 36 . Absorption peaks at 1521 cm −1 and 1401 cm −1 are related to the stretching vibration of C=C bonds in aromatic rings and the bending vibration of C–H bonds, respectively 37 . The peak of stretching vibration of C–O–C and C–OH bonds appeared at 1064 cm −1 38 . The peak centered at 539 cm −1 is related to the bending vibration of the C–H bonds connected to the aromatic rings and the bending vibration of the rings 37 .

EIS measurements

Nyquist and Bode curves of MS electrodes immersed in the 1M HCl electrolyte with different dosages of the NIs for various exposing times are presented in Figs. S6 and S7 , respectively, in supplementary information . The charge transfer resistance of the samples can be directly related to the diameter of the Nyquist semicircles. The Nyquist curves clearly show the increased diameter of the semicircles in the presence of the herbal extracts compared to the inhibitor-free samples, indicating the effect of the medicinal plant extracts on the anodic and cathodic reactions of MS corrosion and showing their inhibition effect. The increased diameter of the semicircles is also observed with increasing time, suggesting that the adsorption of the NI’s compounds on the immersed MS and the formation of the protective layer on the electrode surface is a time consuming process. In addition, it can be seen that the largest diameter of the semicircles in all concentrations and all immersion times belongs to the sample containing Marjoram extract, indicating that the herbal extract has a more significant effect on the corrosion reactions of the immersed MS in the acidic environment than other extracts.

The impedance at the lowest frequency (|Z 10mHz |) can be considered an index for a system's total corrosion resistance 39 , 40 . A comparison of the |Z 10mHz | values observable from the Bode curves (Fig. S7 ) revealed that the index values for the NI-containing samples were more than those obtained for the blank samples. Furthermore, the total corrosion resistance values increased with the immersion times and the concentration of the NIs. It is also observed that the immersed MS in the solution containing the Marjoram extract possessed more |Z 10mHz |values than the other samples.

Due to the fact that only one peak can be seen in the phase angle curves, it can be concluded that the under-studied samples can be electrochemically modeled with a simple Randels equivalent electrical circuit. In this circuit, Rs is solution resistance, R ct is charge transfer resistance, and Q dl is a constant phase element (CPE) of the double layer, which is used instead of an ideal capacitor to better modeling of the EIS data. According to Figs. S6 and S7 , the modeled data fitted well with the experimental results, suggesting the accuracy of the obtained electrochemical data reported in Table S1 (supplementary information) . In this table, Y 0 and n are the admittance and power values of Q dl, respectively. The “n” value can represent the degree of heterogeneity of the metal surface (0 ≤ n ≤ 1). According to literature 41 , 42 , the higher n values in the inhibitor - containing solutions proves the creation of a protecting film on the steel surface.

In Table S1 , the C dl value, which corresponds to the capacity of the double-layer capacitor, is estimated with Eq. ( 1 ) 43 .

As the concentration of the extract increases, the C dl value decreases. The decline can be attributed to a decrease in the dielectric constant due to substituting the extract’s chemical compounds with water molecules on the MS surface. Indeed, since the value of the water dielectric coefficient is high than organic molecules, the adsorption of the organic molecules on the MS surface and desorption of water molecules results in the increment of the capacitance values. In addition, the capacitance of a capacitor has an inverse relationship with the distance between the plates 44 . Thus, replacing of the large inhibitor molecules instead of water molecules on the metal surface increases the thickness of the electric double layer, causing a decrease in the C dl value.

According to the results reported in Table 1 , the C dl values were significantly reduced by increasing the dosage of the NIs and exposing time. In other words, due to the adsorption of the natural compounds on the immersed surface, the formed layer leads to a decrease in the C dl values. Among the herbal extracts, it can be again seen that the lowest values of the C dl belong to the Marjoram sample, which also confirms the formation of a more protective layer on the steel surface in the presence of this inhibitor.

Since the obtained R ct values in Table S1 can be considered as the electrochemical resistance against corrosion reactions 45 , the column diagrams of the parameter are shown in Fig.  4 for a better comparison of the results.

Column diagrams of the reported R ct values in Table S1 for the under-studied samples in various dosages of the NIs and different immersion times.

From the graphs shown in Fig.  4 , it can be seen that the charge transfer values increased by increasing the dosage of the NIs, indicating that the adsorption of the chemical compounds on the MS surface is the main mechanism for the inhibition behavior of the used NIs. In addition, unlike the blank samples, the R ct values for the NI-containing samples increased by increasing the immersion time, as seen in Nyquist and Bode diagrams. Furthermore, the higher charge transfer resistance of the sample containing Marjoram extract can be clearly observed, followed by the sample containing Yarrow extract, which confirms the results of the previous results. Moreover, the lowest corrosion resistance values among the NI-containing samples belonged to those containing the extracts of R. Rubrum and Maurorum.

The value of inhibition efficiency (%IE) in Table S1 is obtained from Eq. ( 2 ).

where, R ct in and R ct 0 are the charge transfer resistance in the presence and absence of the NIs, respectively. The obtained results confirm the higher inhibition performance of the Marjoram-containing sample, followed by the samples containing Yarrow, Wormwood, Ribes rubrum , and Maurorum herbal extracts.

PP measurements

PP curves of the samples containing different amounts of the herbal extracts are represented in Fig. S11 . Electrochemical parameters obtained from Tafel extrapolation of the PP curves are reported in Table S2 (in supplementary information) . The PP curves show that by increasing the concentration of the NIs the corrosion current density (corrosion rate) decreases, confirming the obtained data from EIS analysis. In fact, in the presence of more dosages of the NIs, and the formation of a thicker adsorbed layer on the immersed electrodes, the electron transfer process from the anode regions to the cathode regions disrupts, which can inhibit the corrosion reactions 46 .

In Table S2 , the slopes of the anodic and cathodic branches are represented by βa and βc. E corr is the corrosion potential and i corr is the corrosion current density of the electrodes. R p is the polarization resistance and %IE is the inhibition efficiency, which are respectively calculated using Eqs. ( 3 ) and ( 4 ) 47 .

In Eq. ( 4 ), θ is the coverage degree of the NIs obtained from Eq. ( 5 ).

where i 0 corr and i corr are the corrosion current densities of the samples without and with the Nis, respectively. Column graphs of the obtained polarization resistance and inhibition efficiency values from the Tafel extrapolation of the PP curves are shown in Fig.  5 .

Column graphs of ( a ) R p and ( b ) %IE values for the under-studied samples at different dosages obtained from the PP curves.

It can be seen from Fig.  5 that the samples with the extracts of Marjoram and Yarrow have the highest polarization resistance and inhibition efficiency values. Accordingly, the highest inhibition efficiency (about 90%) and the lowest corrosion rate among the studied samples were obtained for the sample with 800 ppm of Marjoram. These results confirm those obtained in the EIS test and indicate that the inhibition performance of Marjoram extract is optimal compared to the other samples. Indeed, the formation of a more protective layer in the presence of the NI on the surface of the MS electrode led to providing a higher polarization resistance value than other herbal extracts.

At low concentrations, the surface coverage of the NIs is inadequate. So, with the increase of the NI dosages and increase of the surface coverage, the inhibition efficiency of the NIs arises (Fig.  5 b). Despite the higher i corr value in the blank solution than the NI-containing samples, the Tafel slopes remained almost unchanged, indicating that the extracts had no significant effect on the reaction kinetics of the hydrogen gas evolution and the mechanism of iron dissolution. Therefore, the main mechanism of corrosion inhibition is simple adsorption of the compounds in the extracts on the electrode surface.

Due to the negligible alterations in the values of β a and β c in the presence of different dosages of the NIs, as well as the slight shift of the corrosion potential in these samples (less than ± 85 mV compared to the blank sample), it can be concluded that the NIs acted as mixed-type inhibitors 16 , 48 .

By adding the compounds shown in Fig.  5 to the corrosive electrolyte, the protonation of these compounds in the acidic electrolyte causes them to be absorbed on the steel surface because the steel surface has gained a negative charge due to the absorption of chloride anion on it. On the other hand, the formation of a complex between the iron cation produced by the anodic reaction with the organic compounds in the extract causes the formation of insoluble compounds, which create a protective layer by depositing on the surface of the immersed MS. In addition, creating a covalent bond between lone pair electrons of heteroatoms and multiple bonds in the compounds of the extract with the empty orbital of iron atoms on the surface of the electrode leads to the formation of a chemical bond between the electrode and the compounds. So, the inhibition mechanism of the extracts can be considered physical, chemical, or physical–chemical. In this regard, investigating adsorption isotherm models can help identify the inhibition mechanism.

Adsorption isotherms

The mechanism of surface adsorption and the interaction between molecules of a NI and the metal surface can be characterized using models called adsorption isotherms 49 , 50 . Langmuir (Eq.  6 ) and Freundlich (Eq.  7 ) adsorption isotherms are the main models widely employed to investigate the adsorption of an inhibitor on an electrode surface 51 .

where C is the concentration of the NI, and θ is the coverage degree. Langmuir and Freundlich absorption constants are respectively represented by K ads ads and K F . The affinity of a NI adsorption can be achieved by the coefficient 1/n in the Freundlich isotherm equation 51 . According to Eq. ( 6 ), if C/θ is plotted versus C, and the linear regression (R 2 ) is close to one, it can be concluded that the NI followed the Langmuir isotherm model. Likewise, a linear regression near to one for plotting ln (θ) versus ln C shows that the inhibitor followed the Freundlich model.

According to Fig.  6 , the immersed MS in the electrolyte solution with 800 ppm Marjoram extract (the optimum sample based on the electrochemical analyses) follows the Langmuir adsorption model with R 2  > 0.99, indicating that the adsorption is in a monolayer form and all active adsorption sites have the same tendency to adsorb the inhibitor compounds 49 , 50 .

( a ) Langmuir and ( b ) Freundlich adsorption isotherm plots for the MS immersed in the electrolyte with 800 ppm Marjoram extract.

The values of K ads and ΔG ads (obtained from Eq.  8 ) are equal to 5786.03 L mol −1 and − 31.309 kJ mol −1 , respectively.

In Eq. ( 8 ), R is the universal gas constant (3.314 kJ mol −1 ) and T is the absolute temperature (K).

The negative value of ΔG ads indicates the spontaneity of the inhibitor adsorption on the MS surface in the corrosive media. Additionally, the high value of K ads suggests that the NI has a high thermodynamical affinity to adsorb on the surface of the immersed metal 52 , 53 . Generally, the adsorption mechanism can be determined by the absolute value of ΔG ads. When ΔG ads  < 20 kJ mol −1 , the adsorption mechanism is physisorption, and when ΔG ads  > 40 kJ mol −1 , chemisorption can be considered the main adsorption mechanism. 20 kJ mol −1  < ΔG ads  < 40 kJ mol −1 indicates that the physicochemical adsorption is the main interaction between the NI and the immersed metal 54 , 55 . So, for the under-studied sample, it can be concluded that the adsorption of the NI on the MS surface is in the form of the physico-chemical model.

Microscopic studies

Figure  7 displays the morphology and microstructure of the MS surface exposed to the acid solution with and without the optimum NI (800 ppm Marjoram extract). The highly rough surface of the soaked MS in the blank solution indicates the surface degradation of the electrode due to the dissolution of iron atoms as Fe 2+ and Fe 3+ cations into the electrolyte. From the high magnification micrograph (Fig.  7 b), it can be clearly seen that the surface was strongly damaged by the direct attack of the acid solution.

FE-SEM micrographs of MS surface immersed for 24 h in the corrosive electrolyte ( a , b ) without and ( c , d ) with 800 ppm Marjoram extract.

In the presence of the NI, a compact adsorbed layer can be seen on the surface of the exposed MS. The protection layer led to the reduction of Fe dissolution in the acidic environment and created a smoother surface. In fact, the formation of covalent bonds between the iron atoms and the extract’s compounds reduced the iron dissolution in the HCl electrolyte. Therefore, it can be concluded that the presence of the NI affects the corrosion reactions of the MS and reduces the rate of anodic and cathodic reactions, confirming the electrochemical analyses. SEM image of yarrow plant extract as an organic inhibitor in the sample containing 800 ppm inhibitor is provided for a comparison in Fig.  8 .

FE-SEM micrographs of MS surface immersed for 24 h in the corrosive electrolyte with 800 ppm Yarrow extract.

UV–VIS spectroscopy

The interaction between the immersed MS surface in the NI-containing medium and the inhibitor molecules in an acidic medium can be studied using UV–Vis spectroscopy. To confirm the formation of an adsorbed inhibitor-based protect layer on the immersed metal surface, UV–Vis analysis was performed before and after exposing the MS electrodes to the 1M hydrochloric acid solution with 800 ppm Marjoram extract (Fig.  9 ). Generally, the appeared peaks at 250 and 325 nm in the spectrum of the MS-free electrolyte can be related to π–π* and n–π* electron transition of C=C and C=O bonds, respectively 56 . It is clear from Fig.  9 that the intensity of the peaks decreased significantly after 24 h immersion of the MS electrode in the solution, indicating the adsorption of the extract’s compounds on the surface of the electrode, which decreased the concentration of organic compounds in the solution. These observations indicate the formation of a bond between inhibitor molecules and the surface of the steel sample, which reduces the opening of organic compounds in the electrolyte to absorb on the surface of the electrode. The lone pair electrons of heteroatoms, as well as ring π electrons, have been successfully shared with d orbitals of Fe atoms located on the steel surface. These reactions create a single nanometer layer on the surface, which can absorb and reduce the speed reactions of the steel in the active sites of the metal.

UV–Vis spectra of the HCl solution with 800 ppm Marjoram extract before and after immersion of the MS electrode.

A schematic of absorption mechanisms of the Marjoram extract compounds on the surface of an MS electrode immersed in an HCl environment.

Schematic of the adsorption process

A schematic of the adsorption mechanisms of the Marjoram extract compounds on the MS surface immersed in an acidic environment is shown in Fig. 10 .

In the first stage, the anodic reaction of the corrosion produces a positively charged region near the immersed electrode surface due to the presence of Fe 2+ and Fe 3+ in this region. The positively charged region causes the attraction of Cl − from the electrolyte toward the metal surface and form a negatively charged layer on the surface of the immersed electrode 41 . At the next stage, the protonated organic compounds in the solution adsorb on the MS surface through an electrostatic attraction and forms a compact layer on the electrode/electrolyte interface 57 , 58 . Finally, unsaturated pair electrons on oxygen atoms and π-bonds in the chemical structure of the Marjoram extract compounds can be shared with vacant iron orbitals on the electrode surface and lead to chemical adsorption. Therefore, the physicochemical adsorbed layer on the surface of the MS prevents direct contact of the electrode surface with the corrosive environment and reduces the corrosion rate of the metal 47 , 59 .

Theoretical investigations

DFT is one of the most powerful tools in quantum chemistry. It is the shooting star in theoretical modeling. The optimized chemical structures of the present compounds in the optimum extract (Marjoram) are presented in Table S3 (in supplementary information) . In the literature 60 , 61 , quantum calculations for the main substances present in the extract are performed to predict the mechanism of the compounds to protect the working electrode surface from corrosion reactions. In this study, DFT calculations were performed for 8 active substances in the Marjoram called Thymol, Carvacrol, β_Myrcene, Linalool, Caryophyllene, Cymene, Terpinene, and Sabinene as described by Lopez et al. 34 . Calculated parameters such as the energy of the highest orbital of the occupied E HOMO molecule, the lowest of the non-occupied molecular orbital of E LUMO , the energy distance between HOMO and LUMO (ΔE), the ionization potential (IP), the electron affinity (E a ), the electronegativity (χ), hardness (σ), and ΔN (the fraction of electrons that move from the molecule to the metal surface) for the molecules are given in Table 1 . Parameters IP, E A , χ, σ, and ΔN are obtained using the following equations 62 , 63 .

In the last equation, χ Fe is 7 eV, and σ Fe is equal to zero.

Large amounts of E HOMO indicate that the molecule tends to give electrons to the low-energy empty orbitals of the using electrode 64 . The increase in values of E HOMO facilitates the adsorption process by affecting the transfer process between the adsorption layers. Therefore, the effectiveness of an inhibitor can be improved by enhancing the transferring process. From Table 1 , it is clear that the E HOMO values for the under-studied inhibitors decrease in the order; Sabinene > Thymol > Carvacrol > Terpinene > β_Myrcene > Linalool > Caryophyllene > Cymene. However, inhibiting molecules does not only give electrons to the empty orbital of the Fe atom, but electrons are also accepted from the occupied orbitals of the electrode. This process leads to the creation of the feedback bond. Thus, E LUMO indicates the ability of an inhibitor to accept electrons from the electrode surface, which would definitely improve the adsorption and inhibition efficiency of the anti-corrosion agent on the steel surface 65 . The E LUMO for the under-studied molecules followed the order: Sabinene > Linalool > Caryophyllene > Carvacrol > Thymol > Cymene > β_Myrcene > Terpinene, indicating that the Terpinene has a higher affinity to electron accept from the immersed electrode. ΔE = E HOMO −E LUMO is another parameter whose low value indicates higher inhibition efficiency of the corrosion inhibitor 66 . The obtained results (Table 1 ) revealed that the Cymene molecule has a fewer ΔE than the other molecules. Therefore, the Cymene molecule has a greater effect on protecting the immersed electrode from corrosion in the acidic environment. ΔN is another important quantum parameter in the study of anti-corrosion agents. Generally, positive and negative ΔN values indicate electron transfer from the anti-corrosion agent to the electrode surface and from the electrode surface to the anti-corrosion molecule, respectively 65 , 67 . It has also been reported that values of less than 3.6 indicate a higher electron-donating power of the molecule resulting in better molecular inhibition performance. Based on the results in Table 1 , the values of ΔN are positive for all molecules (except for β_Myrcene), and their value is less than 3.6 (except for Sabinene). Thus, all molecules (except for β_Myrcene and Sabinene) are capable of forming covalent bonds to the immersed electrode surface and act as an effective protection agent for the used mild steel against corrosion reactions.

In an attempt to further evaluate the molecular reactivity and stability of the inhibitors, the absolute hardness (η) and softness (σ) were determined. The absolute softness and hardness are related to soft and hard solutions through the theory of HSAB 68 , 69 . Chemical hardness indicates the polarization resistance of the electron cloud of molecules, atoms, or ions with minor perturbations of the chemical reaction. The absolute hardness for the under-studied molecules was reduced in the following order: Sabinene > Caryophyllene > Linalool > Cymene > Carvacrol > Thymol > β_Myrcene > Terpinene. Terpinene, with the lowest hardness value (1.805 eV) compared with other compounds, had the lowest ∆E, while Sabinene with the highest hardness value (3.165 eV), had the highest ∆E. The softness followed a reverse trend of hardness values. This result was consistent with the general belief that hard compounds should have a large ∆E and soft molecules should have small ∆E. Therefore, Terpinene is expected to have higher inhibition performance than other compounds since the lowest global hardness value (i.e., the highest global softness) was likely the highest inhibition performance.

The dipole moment (µ) of compounds gives information about the polarity in the bond of a molecule and the distribution of electrons in the molecules. It is well known that a higher dipole moment value of an inhibitor tends to more adsorption tendency of the compound on the immersed electrode surface 70 , 71 , 72 . The µ value for the under-studied compounds was reduced in the following order: Carvacrol > Linalool > Thymol > Terpinene > Caryophyllene > β_Myrcene > Sabinene > Cymene. Although there is inconsistency with the use of the parameter to predict the direction of a corrosion inhibition reaction in literature; however, it is well known that the adsorption of polar molecules with more values of µ on the surface of the electrode should enhance inhibition performance.

The distribution of HOMO and LUMO orbitals can also be used to investigate how the chemical structure is adsorbed on the immersed electrode surface. The distribution of the HOMO and LUMO orbitals for the under-studied molecules is shown in Table S3 . From the distribution of the HOMO and LUMO orbitals of all molecules, it can be concluded that the HOMO orbitals are more focused on double-bonded carbon atoms (C=C) and oxygen atoms. As a result, this portion of the molecule is adsorbed to the immersed electrode surface.

Electrochemical analyses revealed that aqueous extracts of Wormwood, Maurorum, Yarrow, R. rubrum, and Marjoram herbal plants are suitable NIs for MS in 1M HCl electrolyte and their efficiencies increase with dosage in the order of Marjoram > Yarrow > Wormwood > Maurorum > R. rubrum.

The highest inhibition performance belonged to the Marjoram extract with an inhibition efficiency of about 92%.

The obtained results from the adsorption isotherm investigations proved that the adsorption of the organic compounds in the Marjoram extract acted as a mixed-type NI and obeyed the Langmuir model.

It was demonstrated that the organic compounds formed a protective layer on the electrode surface via physicochemical interactions.

UV–Vis and FE-SEM results confirmed the adsorption of the NI from the corrosive electrolyte on the MS surface.

DFT computation showed that the oxygen atoms and π-bonds are suitable sites for sharing their lone pair electrons with empty iron orbitals on the soaked MS surface.

Data availability

All data generated or analysed during this study are included in this published article [and its supplementary information files ].

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Maryam Pourmohseni & Mehrnoosh Karimkhani

Nanotechnology Research Center, Research Institute of Petroleum Industry (RIPI), Tehran, Iran

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Maryam Pourmohseni: collected the data, Doing the lab work and collecting data, Contributed data or analysis tools, Performed the analysis, Wrote the paper. Alimorad Rashidi: Conceived and designed the analysis, Supervision, Contributed data or analysis tools, Lab work and materials, Supervision, Validation, Visualization, Performed the analysis, Wrote the paper, Revised and editing the manuscript with help the other authors, Other contribution, Corresponding Author. Mehrnoosh Karimkhani: Conceived and designed the analysis, Supervision, Contributed data or analysis tools, Lab work and materials, Validation, Visualization, Performed the analysis.

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Pourmohseni, M., Rashidi, A. & Karimkhani, M. Preparation of corrosion inhibitor from natural plant for mild stil immersed in an acidic environmental: experimental and theoretical study. Sci Rep 14 , 7937 (2024). https://doi.org/10.1038/s41598-024-58637-z

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Plants 'scream' while they're being harvested, new study finds

Vegetarians, we're afraid we've got some news that you're probably not going to like…

Plants emit sounds, akin to ‘screams’, when they’re distressed, according to a new study.

They allegedly produce clicking noises that humans can’t hear without the use of scientific equipment, research has found.

The research, which was published in Cell back in 2023, showed that plants produce these noises in times of acute distress.

Lilach Hadany is an evolutionary biologist at Tel Aviv University. Hadany said : "Even in a quiet field, there are actually sounds that we don't hear, and those sounds carry information. There are animals that can hear these sounds, so there is the possibility that a lot of acoustic interaction is occurring.”

"Plants interact with insects and other animals all the time, and many of these organisms use sound for communication, so it would be very suboptimal for plants to not use sound at all."

The findings show that plants which are distressed have incredibly high-pitched popping noises, while unstressed plants do not emit noises. The study’s definition of distressed included plants that were having their stems cut or were dehydrated.

However, it’s not yet clear how the plants produce the noises.

"Now that we know that plants do emit sounds, the next question is – 'who might be listening?'" Hadany said.

"We are currently investigating the responses of other organisms, both animals and plants, to these sounds, and we're also exploring our ability to identify and interpret the sounds in completely natural environments."

It comes after a new study suggested that western, industrial diets may be changing the way humans digest plants .

As modern diets lack fibre the way our bodies break down cellulose, found in fruits and vegetables , is changing.

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Plant-based plastic releases nine times less microplastics than conventional plastic

A newly developed plant-based plastic material releases nine times less microplastics than conventional plastic when exposed to sunlight and seawater, a new study has found.

The research, led by experts from the University of Portsmouth and the Flanders Marine Institute (VLIZ), in Belgium, looked at how two different types of plastic break down when tested in extreme conditions.

A bio-based plastic material made from natural feedstocks held up better when exposed to intense UV light and seawater for 76 days -- the equivalent of 24 months of sun exposure in central Europe -- than a conventional plastic made from petroleum derivatives.

Professor of Mechanical Engineering, Hom Dhakal, from the University's School of Mechanical and Design Engineering, and member of Revolution Plastics said: "Bio-based plastics are gaining interest as alternatives to conventional plastics, but little is known about their potential source of microplastics pollution in the marine environment.

"It's important to understand how these materials behave when they're exposed to extreme environments, so we can predict how they'll work when they're used in marine applications, like building a boat hull, and what impact they might have on ocean life.

"By knowing the effect of different types of plastics on the environment, we can make better choices to protect our oceans."

According to the Plastic Oceans International Organization, the equivalent of a truckload of plastic is poured into the oceans every minute of the day. When this plastic waste is exposed to the environment, it breaks down into smaller particles which are less than 5mm in size.

These particles are known as 'microplastics' and have been observed in most marine ecosystems, posing a serious threat to aquatic life.

"We wanted to look at a conventional industrial polymer, polypropylene, which is non-biodegradable and difficult to recycle, against polylactic acid (PLA), a biodegradable polymer," Professor Dhakal explained.

"Although our findings show that the PLA released less microplastics, which means using plant-based plastics instead of oil-based ones might seem like a good idea to reduce plastic pollution in the ocean, we need to be careful as microplastics are still clearly being released and that remains a concern."

The research also found that the size and shape of the tiny plastic pieces released depended on the type of plastic. The conventional plastic released smaller pieces and had fewer fibre-like shapes compared to the plant-based plastic.

Professor Dhakal added: "Overall our research provides valuable insights into the behaviour of different plastic types under environmental stressors, which is important for our future work to tackle plastic pollution.

"There is a clear need for continued research and proactive measures to mitigate the impact of microplastics on marine ecosystems."

Professor Dhakal is a member of the Revolution Plastics initiative that has been instrumental in informing national and global policies on plastics, pioneering advanced enzyme recycling techniques and contributing to critical discussions on the UN treaty to end plastic pollution.

Revolution Plastics operates as a network of interconnected researchers and innovators across the University, consolidating and expanding a world-leading plastic-focused research, innovation and teaching community.

The study was led by experts from the Flanders Marine Institute (VLIZ), in Belgium, under collaborative international work within the SeaBioComp project, which received funding from the Interreg 2 Seas Programme co-funded by the European Regional Development Fund.

The paper is published in Ecotoxicology and Environmental Safety.

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Materials provided by University of Portsmouth . Note: Content may be edited for style and length.

Journal Reference :

• Zhiyue Niu, Marco Curto, Maelenn Le Gall, Elke Demeyer, Jana Asselman, Colin R Janssen, Hom Nath Dhakal, Peter Davies, Ana Isabel Catarino, Gert Everaert. Accelerated fragmentation of two thermoplastics (polylactic acid and polypropylene) into microplastics after UV radiation and seawater immersion . Ecotoxicology and Environmental Safety , 2024; 271: 115981 DOI: 10.1016/j.ecoenv.2024.115981

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L.A. County faces $12.5 billion in climate costs through 2040, study says

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A first-of-its-kind report has estimated that Los Angeles County must invest billions of dollars through 2040 to protect residents from worsening climate hazards, including extreme heat, increasing precipitation, worsening wildfires, rising sea levels and climate-induced public health threats.

The report, published this week by the nonprofit Center for Climate Integrity, identified 14 different climate adaptation measures that authors calculated would cost L.A. taxpayers at least $12.5 billion over the next 15 years, or approximately $780 million per year. The vast majority of those costs — more than $9 billion — will be incurred by local municipal governments, including the cities of Los Angeles, Long Beach and Santa Clarita, the report said.

“These numbers don’t include the costs of recovering from disasters — from extreme weather events that knock out power or damage infrastructure or do all the kinds of things they do,” said Richard Wiles, president of the Center for Climate Integrity. “So it’s a very conservative estimate, and yet it’s a really big number.”

Aggressive and impactful reporting on climate change, the environment, health and science.

Wiles said the costs for L.A. County are nearly as high as for the entire state of Pennsylvania, which faces about $15 billion in climate adaptation costs over roughly the same period.

“This is a big number, but this is going to happen,” he said. “These costs will be incurred at some point, and it’s just much better to pay now than it is to pay later. I can’t emphasize that enough.”

The most expensive adaptation categories are related to precipitation and heat, including an estimated $4.3 billion for improved stormwater management, $2.5 billion for cool pavement investments and $1.4 billion for tree canopies to combat urban heat islands, the report found. Other costs include wildfire mitigation; coastal defense and infrastructure protection; building upgrades for cooling and air conditioning; and responses to vector-borne diseases such as West Nile virus.

County officials said the findings weren’t surprising and agreed that they may even be conservative given the scale of the threats.

“The impacts of climate have become more and more visible over the past few years in particular,” said Rita Kampalath, L.A. County’s chief sustainability officer. “We know that we’re facing really, really huge needs in terms of how we prepare our communities to face those, and to be resilient in the face of increased climate impacts. It’s only going to increase from here.”

Stormwater capture in particular has been on the minds of many Angelenos this winter as record-breaking rainfall pounded the region . A monster storm in February saw the Los Angeles River roar to life and funnel millions of gallons into the Pacific Ocean .

But the river — which was encased in concrete nearly a hundred years ago — and other local flood channels will be no match for climate change-enhanced storms of the future. Though the long-term trend in the West is toward hotter and drier conditions, Los Angeles will still see bouts of severe storms and extreme wet years that will increase flood risk significantly, according to the state’s fourth climate change assessment .

To mitigate these impacts, the county must expand its stormwater drainage infrastructure by installing bioswales, porous pavement and other opportunities for stormwater to seep into the ground, the report found. It noted that these “green infrastructure” upgrades are the least expensive option to cope with extreme rainfall events, as opposed to increasing the size and scale of hard infrastructure such as drain pipes.

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The county is making progress on this work through its Safe Clean Water Program, passed by voters as Measure W in 2018 , Kampalath said. The program allocates about $280 million annually to stormwater capture projects, although recent reports have found that progress to date has been slow .

“While it is a big need, I do actually feel like the county has been investing, and our residents and voters in particular have shown that this is a high priority,” she said. “We’re not as far as we would like to be — it’s hard to say that about much of anything when it comes to climate — but I do think that we have resources available to try to address some of these needs.”

Meanwhile, extreme heat continues to pose a significant threat to L.A. County residents, and it is predicted to only get worse in the years and decades ahead. The region is expected to experience an average of 48.5 days above 90 degrees per year between 2024 and 2040, the report says. That’s about 12.5 more hot days per year than communities experienced between 1994 and 2013.

Some of the best methods to combat the dangers of rising heat include installing cool pavements , expanding urban green space, painting railway tracks with reflective paint to keep them at operable temperatures, and upgrading cooling systems for public buildings such as schools, the report says. Converting public parking lots to cool pavements that reflect instead of absorb sunlight can also help lower ambient temperatures.

Heat is “the impact that affects communities of color the most, and people less able to adapt themselves and their personal lives,” Wiles said. He noted that some urban areas can simmer up to 20 degrees hotter than surrounding neighborhoods with heavy tree canopies.

“From a public health perspective, these types of adaptations are increasingly critical just to make neighborhoods livable,” he said.

The report comes at a moment when the state is facing a significant $37.9-billion budget deficit , which has prompted Gov. Gavin Newsom to slash $2.9 billion from California climate programs , delay an additional $1.9 billion and shift $1.8 billion to other funds.

Kampalath said it’s too soon to say whether those cuts will trickle down to L.A. County’s climate efforts, but that they could potentially affect funds officials were hoping to take advantage of through grants and other programs.

However, she noted that many of the county’s climate adaptation strategies can have multiple benefits, such as tree canopy programs that help combat heat and improve stormwater management simultaneously.

“As we’re looking at how to address these impacts, we do need to think about a multi-benefit approach, and what kind of strategies we can put in place that are really going to address a wide range of things — not only climate, but biodiversity and health impacts and the well-being of our communities as well,” she said.

As climate hazards converge, more Californians are living in harm’s way

When wildfire smoke and extreme heat combine, they create a “synergistic effect” or an additional burden on people’s health, researchers say.

Feb. 3, 2024

Ultimately, funding for the projects outlined in the report will come from taxpayers, whether at the municipal, state or federal level, Wiles said. But he also hopes that oil and gas companies will be held accountable for their role in the worsening climate crisis, as fossil fuel emissions are by far the largest driver of global warming.

Last year, California filed a bombshell lawsuit against five of the largest oil and gas companies for their alleged “decades-long campaign of deception” about the risks posed by fossil fuels, which have forced the state to spend billions of dollars to address environmental-related damages. State Atty. Gen. Rob Bonta is seeking to create a nuisance abatement fund to finance climate mitigation and adaptation efforts, among other outcomes.

“Each and every community in Los Angeles County should consider bringing similar legal actions to hold climate polluters accountable and ensure that taxpayers aren’t left to pay the bill alone,” the report says.

Indeed, there are other climate hazards that will cost Angelenos billions in adaptation expenses over the next decade and a half, the report found.

They include an increase in vector-borne diseases such as West Nile virus as more mosquitoes are drawn to the area’s changing temperatures and precipitation patterns. About 500,000 new cases of the virus are expected in the county through 2040, which will cost an estimated $993 million to treat. Climate change will also lead to more pediatric asthma cases due to an increase in pollen, with about 160,000 new cases expected through 2040.

The county also needs about $680 million in road improvements as heat and rain contribute to more cracks, erosion and soft surfaces. A foot of sea level rise along the coast of L.A. County will require at least $576 million for berms, flood walls, bank stabilization and other infrastructure measures to prevent flooding and to avoid infrastructure damage by 2040.

Wildfires, already getting larger, faster and more frequent across California , will necessitate nearly $1 billion just to clear vegetation and other fuels from land around the county’s infrastructure, the report found. It noted that L.A. County will face an average of 36 more high-fire days through 2040 when compared to the 1994-to-2013 baseline.

The estimated $919-million wildfire cost does not account for fighting fires or repairing damage from blazes. The 2018 Woolsey fire racked up an estimated $3 billion to $5 billion in insured losses alone.

Wiles said the expenses outlined in the report won’t solve climate change but will help “hold things where they are today,” or least prevent the hazards from getting worse.

He said he hoped the report would help guide county officials as they face difficult choices about where, how and to what limited funds should be allocated. Investing in climate adaptations now can save money — and lives — later, he said.

“These costs are still coming,” Wiles said. “The next disaster will happen. This is just what it’s going to cost to prepare.”

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