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Worldwide Research Trends on Medicinal Plants

Esther salmerón-manzano.

1 Faculty of Law, Universidad Internacional de La Rioja (UNIR), 26006 Logroño, Spain; [email protected]

Jose Antonio Garrido-Cardenas

2 Department of Biology and Geology, University of Almeria, ceiA3, 04120 Almeria, Spain; se.lau@anedracj

Francisco Manzano-Agugliaro

3 Department of Engineering, University of Almeria, ceiA3, 04120 Almeria, Spain

The use of medicinal plants has been done since ancient times and may even be considered the origin of modern medicine. Compounds of plant origin have been and still are an important source of compounds for drugs. In this study a bibliometric study of all the works indexed in the Scopus database until 2019 has been carried out, analyzing more than 100,000 publications. On the one hand, the main countries, institutions and authors researching this topic have been identified, as well as their evolution over time. On the other hand, the links between the authors, the countries and the topics under research have been analyzed through the detection of communities. The last two periods, from 2009 to 2014 and from 2015 to 2019, have been examined in terms of research topics. It has been observed that the areas of study or clusters have been reduced, those of the last period being those engaged in unclassified drug, traditional medicine, cancer, in vivo study—antidiabetic activity, and animals—anti-inflammatory activity. In summary, it has been observed that the trend in global research is focused more on the search for new medicines or active compounds rather than on the cultivation or domestication of plant species with this demonstrated potential.

1. Introduction

Ten percent of all vascular plants are used as medicinal plants [ 1 ], and there are estimated to be between 350,000 [ 2 ] and almost half a million [ 3 ] species of them. Since ancient times, plants have been used in medicine and are still used today [ 4 ]. In the beginning, the trial and error method was used to treat illnesses or even simply to feel better, and in this way, to distinguish useful plants with beneficial effects [ 5 ]. The use of these plants has been gradually refined over the generations, and this has become known in many contexts as traditional medicine. The official definition of traditional medicine can be considered as “the sum total of the knowledge, skills and practices based on the theories, beliefs and experiences indigenous to different cultures, whether explicable or not, used in the maintenance of health, as well as in the prevention, diagnosis, improvement or treatment of physical and mental illnesses” [ 6 ].

It is a fact that all civilizations have developed this form of medicine [ 7 ] based on the plants in their own habitat [ 8 ]. There are even authors who claim that this transmitted knowledge is the origin of medicine and pharmacy. Even today, hundreds of higher plants are cultivated worldwide to obtain useful substances in medicine and pharmacy [ 9 ]. The therapeutic properties of plants gave rise to medicinal drugs made from certain plants with these benefits [ 10 ].

Until the 18th century, the therapeutic properties of many plants, their effect on the human organism and their method of treatment were known, but the active compound was unknown [ 11 ]. As an example, the Canon of Medicine written by the Persian physician and scientist Avicenna (Ibn Sina) was used until the 18th century [ 12 ].

The origin of modern science, especially in the Renaissance, in particular chemical analysis, and the associated instrumentation such as the microscope, was what made it possible to isolate the active principles of medical plants [ 13 ]. Since then, these active principles have been obtained synthetically in the laboratory to produce the medicines later [ 14 ]. The use of medicines was gradually expanded. Until today, the direct use of medicinal plants is apparently displaced in modern medicine [ 15 ]. Today’s medicine needs the industry producing pharmaceutical medicines, which are largely based on the active principles of plants, and therefore, these are used as raw materials in many cases [ 16 ]. Yet, today, the underdeveloped world does not have access to this modern medicine of synthetic origin, and therefore, large areas of the world continue to use traditional medicine based on the direct use of medicinal plants due to their low cost [ 17 ].

However, it should be noted that the possible trend to return to this type of traditional medicine may have two major drawbacks. The first is the use of medicinal plants without sanitary control, without thinking about the possible harmful aspects for health [ 18 ]. Although many plants do not have side effects like the aromatic plants used in infusions: chamomile, rosemary, mint, or thyme; however, others may have dangerous active principles. To cite an example, Bitter melon ( Momordica charantia L. ) used to cure fever and in cases of malaria [ 19 ], its green seeds are very toxic as they can cause a sharp drop in blood sugar and induce a patient’s coma (hypoglycemic coma) [ 20 ]; this is due to the fact that the components of bitter melon extract appear to have structural similarities to animal insulin [ 21 ]. Secondly, there has been a proliferation of products giving rise to false perspectives, as they are not sufficiently researched [ 22 ].

Examining the specialized literature of reviews and bibliometric studies on medicinal plants, three types of studies are found: those focused on a geographical area, those focused on a specific plant or family, and those focused on some type of medical interest activity. Regarding the studies of geographical areas, for example, there are the studies of Africa. Specifically, in South Africa, the plants that are marketed [ 23 ], as these plants of medical interest have been promoted [ 24 ], or for the treatment of specific diseases such as Alzheimer’s [ 25 ]. In Central Africa, the studies of Cameroon are remarkable, where for general bibliometric studies of its scientific output, the topic of medicinal plants stands out as one of the most important in this country [ 26 ]. Or those of Ghana, regarding frequent diseases in this country such as malaria, HIV/AIDS, hypertension, tuberculosis, or bleeding disorders [ 27 ]. Other countries that have conducted a bibliometric study of their medicinal plants have been Cuba [ 28 ] and China [ 29 ].

The other direction of the bibliometric studies mentioned, those that focus on specific plants, are those of: Artemisia annua L. [ 30 ], Aloe vera [ 31 ], Panax ginseng [ 32 ], Punica grantum L. [ 33 ], Apocynum cannabinum [ 34 ], or Andrographis paniculata [ 35 ]. The third line of the bibliometric research on medicinal plants deals with some kind of specific activity; there are studies for example for the activities of: antibacterial or antifungal [ 36 ], antioxidant [ 37 ], and anticancer [ 38 , 39 , 40 ].

As a common feature of the bibliometric studies published so far, none of them has a worldwide perspective. Furthermore, they are generally based on Web of Science and some of them on other more specific databases such as CAB Abstracts or PlantMedCUBA, but no work based on Scopus has been observed. Therefore, this paper aims to study what types of scientific advances are being developed around medicinal plants, what research trends are being carried out, and by which countries and research institutions. To this purpose, it is proposed to carry out a bibliometric analysis of all the scientific publications on this topic.

2. Materials and Methods

The data analyzed in this work have been obtained through a query in the Scopus database, which has been successfully used in a large number of bibliometric studies [ 41 ]. Due to the large amount of results, it was necessary to use the Scopus API to download the data, whose methodology has been developed in previous works [ 42 , 43 ]. In this study, the query used was: (TITLE-ABS-KEY(“medic* plant*”)). An outline of the methodology used is shown in Figure 1 . The analysis of the scientific communities, both in terms of keywords and the relationship between authors or between countries was done with the SW VosViewer [ 44 ].

Methodology.

3.1. Global Evolution Trend

From 1960 to 2019, more than 110,000 studies related to medicinal plants have been published. Figure 2 shows the trend in research in this field. Overall, it can be said that there was a continuous increase from 1960 to 2001, with just over 1300 published studies. From here, the trend increases faster until 2011, when it reaches a maximum of just over 6200 publications. After this period, publications stabilize at just over 5000 per year. These three periods identified are highlighted in Figure 2 .

Worldwide temporal evolution of medical plants publications.

3.2. Global Subject Category

If the results are analyzed according to the categories in which they have been published (see Figure 3 ), according to the Scopus database, it can be seen that most of them have been carried out in the Pharmacology, Toxicology and Pharmaceutics category with 27.1 % of the total. Other categories with significant relative relevance have been: Medicine (23.8%), Biochemistry, Genetics and Molecular Biology (16.7%), Agricultural and Biological Sciences (11%), Chemistry (8.7%), Immunology and Microbiology (2.5%), Environmental Science (2.1%), and Chemical Engineering (1.5%). All other categories are below 1%, such as: Nursing, Multidisciplinary, or Engineering.

Medicinal plants publications by scientific categories indexed in Scopus.

3.3. Distribution of Publications by Countries

If the results obtained are analyzed by country, a total of 159 countries have published on this topic. Figure 4 shows the countries that have published on the subject and the intensity with which they published has been shown. It is observed that China and India stand out over the rest of the countries with more than 10,000 publications, perhaps influenced by traditional medicine, although their most cited works are related to antioxidant activity, both for China [ 45 ], and for India [ 46 , 47 ], and in this last country also antidiabetic potential [ 4 ]. The third place is the USA followed by Brazil, both with more than 5000 publications. The most frequently cited publications from these countries focus on antioxidant activity [ 48 ], and antimicrobial activity [ 49 ] for the USA and anti-inflammatory activity for Brazil [ 50 , 51 ].

Worldwide research on medical plants.

As mentioned, the list of countries is very long, but those with more than 2000 publications are included: Japan, South Korea, Germany, Iran, United Kingdom, Pakistan, Italy, and France. If the overall results obtained are analyzed in their evolution by years, for this list of countries with more than 2000 publications, Figure 5 is obtained. From this point onwards, three groups of countries can be identified.

Temporal evolution on medical plants publications for Top 12 countries.

The first group is the leaders of this research, China and India, with between 800 and 1100 publications per year. China led the research from 1996 to 2010, and from this year to 2016, the leader was India, after which it returned to China. The second group of five countries is formed in order in the last year of the study: Iran, Brazil, USA, South Korea and Pakistan. This group of countries has a sustained growth over time, with a rate of publications between 200 and 400 per year. It should be noted that Brazil led the third place for a decade, from 2007 to 2016, since then that position is for Iran. The third group of five countries is made up of: Japan, Germany, United Kingdom, Italy, and France. They are keeping the publications around 100 a year, with an upward trend, but at a very slight rate.

If the analysis of the publications by country is made according to the categories in which they publish, Figure 6 is obtained, which shows the relative effort between the different themes or categories is shown. At first look, it might seem that they have a similar distribution. However, in relative terms the category of Pharmacology, Toxicology and Pharmaceutics is led by Brazil with 35% of its own publications followed by India with 33%. For the Medicine category, in relative terms it is led by China with 29 %, followed by Germany with 27 %. The category of Biochemistry, Genetics and Molecular Biology always takes second or third place for this ranking of countries, standing out especially for Japan and South Korea with 23% and for France with 22%. The fourth category for many countries is Agricultural and Biological Sciences, with Pakistan standing out with 20%, followed by Italy with 16%. The category of Chemistry occupies the fourth category for countries such as Japan with 20% or Iran with 14%. The other categories: Chemical Engineering, Immunology and Microbiology, Environmental Science, Multidisciplinary, or Engineering, are below 5 % in all countries.

Distribution by scientific categories according to countries.

According to these results, it can be seen the relative lack of relevance of the category of Agricultural and Biological Sciences for medicinal plants, compared to the categories of Pharmacology, Toxicology and Pharmaceutics, Medicine, or Biochemistry, Genetics and Molecular Biology.

3.4. Institutions (Affiliations)

So far, the distribution by country has been seen, but the research is done in specific research centers (institution or affiliations as are indexed in Scopus) and therefore, it is important to study them. Table 1 shows the 25 institutions with more than 400 publications, of which 13 are from China (including the first 7), 3 from Brazil, 2 from South Korea, and now with 1: Saudi Arabia, Pakistan, Iran, Mexico, Cameroon, France, and Malaysia.

Top 25 affiliations and main keywords.

If the three main keywords of these affiliations are analyzed, it can be seen that there are no great differences, and in fact, they are often the same: Unclassified Drug, Drug Isolation, Drug Structure, Chemistry, Controlled Study, Isolation And Purification, Chemistry, and Plant Extract. They only call attention to “Drugs, Chinese Herbal” which appears in two affiliations: China Academy of Chinese Medical Sciences, and Beijing University of Chinese Medicine, which of course is a very specific issue in this country.

3.5. Authors

The main authors researching this topic are shown in Table 2 , which are those with more than 100 publications on this topic. It is observed that they are authors with a significantly high h-index. On the other hand, it is noteworthy that the first two are not from China or India, which as we have seen were the most productive countries, and also had the most relevant institutions in this area. The lead author is from South Africa, J. Van Staden, and the second from Bangladesh, M. Rahmatullah. The author with the highest h-index is from Germany, T. Efferth.

Main authors in medicinal plants.

If the network of collaboration between authors with more than 40 documents is established, Figure 7 is obtained. Here, there are 33 clusters, where the most important is the red one with 195 authors, where the central author is Huang, L.Q. The second more abundant cluster is the green one, composed of 69 authors. In this cluster, there is no central author, but instead, a collaboration between prominent authors such as Kim, J.S., Lee, K.R. or Park, J.S. The third cluster, in blue, is composed of 64 authors, led by the authors M.I. Choudhary and M. Ahmad.

A collaborative network of authors with more than 40 publications on medicinal plants.

The fourth cluster, of yellow color is composed of 63 authors, the central authors are Y. Li and H-D. Sun. The fifth cluster, in purple, is also composed of 51 authors, the central author is W. Villegas. It should be noted that this cluster is not linked to the whole network, so they must research very specific topics in their field. The sixth cluster is composed of 48 authors and is cyan colored, the central author is Rahmatullah, M. The cluster of the main author of Table 2 , Van Staden, J., is composed of 23 authors, and would be number 17 in order of importance by number of authors, is light brown, and is located next to that of W. Vilegas but without any apparent connection.

3.6. Keywords

3.6.1. global perspective.

The central aspect of bibliometric studies is to study the keywords in the publications and, through the relationships between them, to establish the clusters or scientific communities in which the different topics associated with a field of study can be grouped together. If keywords are extracted from the total number of publications, an overview can be made of the most used keywords in relation to the subject of medicinal plants (see Figure 8 ). As expected, the search terms are the main ones, but then, there are two indexing terms, Human and Nonhuman, and then Unclassified Drug and Plant Extract.

Cloudword of keywords in medical plants publications.

If the keywords are analyzed by country, and we do not take into account the search terms, the results are obtained in Table 3 , where the four main keywords of the main countries that research this topic are shown. It can be seen that the terms: Unclassified Drug, Plant Extract, and Controlled Study, are the ones that dominate without a doubt.

Main keywords by country.

3.6.2. Keywords Related to Plants

If this keyword analysis is done by parts of the plant (see Table 4 ), which shows which parts of the plant have been most investigated. It should be noted that the number of documents is less than the sum of the individual keywords, since a publication contains more than one keyword. It has been obtained that the parts of the plant most studied in order of importance have been the value expressed in relative terms: Leaf-Leaves (33%), Root-Roots (22%), Seed (12%), Stem (10%), Fruit (10%), Bark (7%), and Flower (6%). The table also shows which plant families have been most used for the study of that part of the plant.

Main keywords related to plant parts and plant families studied.

To give an idea of the most studied plant families, see Table 5 . Although the first two are the same family, it has been left separately to indicate the indexing preferences of the two main affiliations that study them. This is also the situation with Compositae that correspond to the family of Asteraceae. This table lists for each plant family the main institution working on its study. However, it is curious that even if a country is a leader in certain studies related to plant families, most often it is found that the institution leading the issue is not from the country leading the study on that plant family. This helps to establish a certain amount of global leadership on the side of the institutions.

Plant families and Institutions.

3.7. Clusters

The analysis of the clusters formed by the keywords allows the classification of the different groups into which the research trends are grouped. A first analysis has been made with the documents published between 2009 and 2019 and in two periods, from 2009 to 2014 and from 2015 to 2019. Figure 9 shows the clusters obtained for the period 2009 to 2014, showing seven clusters, which can be distinguished by color, and in Table 6 its main keywords have been collected.

Network of keywords in medical plants publications: Clusters between 2009–2014.

Main keywords used by the communities detected in the topic in the period 2009–2014.

The first of these clusters, in red (1-1), is linked to traditional medicine. This is reflected in the main keywords associated with this cluster: phytotherapy, herbaceous agent, traditional medicine, ethnobotany. Within this cluster, the most cited publications are related to the antioxidant function of plants. This includes the prevention of hyperglycemia hypertension [ 52 ], and the prevention of cancer. Of the latter, studies suggest that a reduced risk of cancer is associated with high consumption of vegetables and fruits [ 53 ]. Another topic frequently addressed is the antidiabetic properties, as some plants have hypoglycemic properties [ 34 ]. It should be remembered that diabetes mellitus is one of the common metabolic disorders, acquiring around 2.8% of the world’s population and is expected to double by 2025 [ 54 ].

The second cluster, in green (1-2), appears to be the central cluster, and is related to drugs—chemistry. The main keywords are: drug isolation, drug structure, chemistry, drug determination, and molecular structure. Here, the most cited publications are the search for new drugs [ 55 ] or in natural antimicrobials for food preservation [ 56 ].

The third cluster, in purple (1-3), is focused on in vivo study through studies with laboratory animals, as shown by keywords such as mouse and mice. As it is known that in vivo drug trials are initiated in laboratory animals such as mice, in general studies focused on anti-inflammatory effect [ 57 , 58 ].

The fourth cluster, in yellow (1-4), is engaged in the search for drugs. The main keywords in this regard are unclassified drug and drug screening. Within this cluster, the studies of flavonoids stand out [ 59 ]. Flavonoids have been shown to be antioxidant, free radical scavenger, coronary heart disease prevention, hepatoprotective, anti-inflammatory and anticancer, while some flavonoids show possible antiviral activities [ 60 ].

The fifth cluster, in blue (1-5), is focused on the effectiveness of some drugs, and their experimentation on animals. Some of the most cited publications of this cluster over this period are those focused on genus Scutellaria [ 61 ], Epimedium ( Berberidaceae ) [ 62 ] and Vernonia ( Asteraceae ) [ 63 ].

The sixth cluster, in cyan (1-6), is aimed at the effect of extraction solvent/technique on the antioxidant activity. One of the most cited publications in this regard studies the effects on barks of Azadirachta indica , Acacia nilotica , Eugenia jambolana , Terminalia arjuna , leaves and roots of Moringa oleifera , fruit of Ficus religiosa , and leaves of Aloe barbadensis [ 64 ]. Regarding neuroprotection, some publications are the related to genus Peucedanum [ 65 ] or Bacopa monnieri [ 66 ]. This cluster is among the clusters of traditional medicine (1-1) and drug efficacy (1-5).

Finally, the seventh orange cluster (1-7) is of small relative importance within this cluster analysis and is focused on malaria. As it is known, malaria is one of the most lethal diseases in the world every year [ 67 ]. Malaria causes nearly half a million deaths and was estimated at over 200 million cases, 90 per cent of which occurred in African countries [ 68 ]. Of the Plasmodium species affecting humans, Plasmodium falciparum causes the most deaths, although Plasmodium vivax is the most widely spread except in sub-Saharan Africa [ 69 ]. On the other hand, this cluster cites Plasmodium berghei , which mainly affects mice, and is often used as a model for testing medicines or vaccines [ 70 ].

The second period under study, from 2015 to 2019, is shown in Figure 10 , where five clusters have been identified, Table 7 , as opposed to the previous period which was seven. Now, there is no cluster focusing on malaria. In Figure 10 , the colors of the cluster have been unified with those of Figure 9 , when the clusters have the same topic as in the previous period.

Network of keywords in medical plants publications: Clusters between 2015–2019.

Main keywords used by the communities detected in the topic in the period 2015–2019.

The first cluster in order of importance (2-1), the red one in Figure 10 , can be seen to be that of unclassified drug, which has gone from fourth place (1-4) to first in this last period. In this period, research works include one on the therapeutic potential of spirooxindoles as antiviral agents [ 71 ], or the antimicrobial peptides from plants [ 72 ].

The second cluster of this last period (2-2), the one in green in Figure 10 , is the one assigned to traditional medicine, which has now moved up to second place (1-1) in decreasing order of significance. It seems that this cluster of traditional medicine is now the merging with the drug efficacy cluster of the previous period (1-4). This cluster includes research such as oxidative stress and Parkinson’s disease [ 73 ].

The cluster from the previous period that was devoted to animals-in vivo study (1-3), we assume is now divided into three new clusters. The first of these would be the third cluster (2-3), blue in Figure 10 , which can be considered to be dedicated to cancer. One of the works in this cluster is “Anticancer activity of silver nanoparticles from Panax ginseng fresh leaves in human cancer cells” [ 74 ]. Then, the other two are committed to in vivo studies or with animals. The first one seems to be more engaged in vivo study at antidiabetic activity [ 75 , 76 ], would be the cyan-colored cluster 4 (2-4). The other cluster (2-5) involved in testing anti-inflammatory activity, with plants such as Curcumin [ 77 ], Rosmarinus officinalis [ 78 ], would be the purple cluster in Figure 10 .

3.8. Collaboration Network of Countries

Figure 11 shows the collaborative network between countries doing research on medicinal plants. Table 8 lists the countries of each cluster identified and the main country of each cluster. The countries that are most central to this network of collaboration between countries are India, Iran, Indonesia, and the USA. The largest cluster is led by Brazil, which is also not restricted to its own geographical area as it has strong collaborative links with European countries as well as with neighboring countries such as Argentina. The second cluster led by South Africa also presents the same features as the previous one, some collaborations with nearby countries, Tanzania, Congo, or Sudan, but also with European countries such as France, Belgium, or the Netherlands.

Countries network collaboration.

Countries collaboration in the period 2009–2019.

The third cluster is led by India and has very strong collaboration with Iran, but it could also be considered as the central country in the whole international collaboration network. The cooperation with European countries comprises mainly Eastern countries like Poland, Serbia, or Croatia.

The fourth cluster, led by Germany and Pakistan, includes Middle Eastern countries such as Jordan, Saudi Arabia, and United Arab Emirates, which are quite related to the cluster led by China. The fifth cluster seems to have a geographical consideration within Asia by including countries such as Indonesia, Malaysia, Thailand, and Australia. The sixth cluster includes very technologically advanced countries such as USA, UK, Japan, Canada, or South Korea. The seventh cluster is very small in the number of countries. It is made up of very different countries like some in Africa: Cameroon and Kenya; some of Europe as Denmark, and some from Asia like Nepal. In this sense, most of the research linked to African countries in general and to Cameroon particularly is linked to the most frequent parasitic diseases [ 79 ], such as African trypanosomiasis [ 80 ], diarrhea [ 81 ] or tuberculosis [ 82 ]. Finally, the China cluster is made up of nearby areas of influence such as Taiwan, Singapore, Hong Kong, Macau, or Taiwan.

4. Conclusions

The use of plants as a source of research in the search for active compounds for medicine has been proven to have a significant scientific output. An analysis of the scientific literature indexed in the Scopus database concerning medicinal plants clearly shows that in the last 20 years, progress has been rapid, with a peak in 2010. From this year onwards, publications have stabilized at just over 5000 per year.

The research of products derived from the plants shows great collaboration between the countries of the first world and the countries with a traditional use of these plants from Asia, Africa or Latin America, all this to produce new medicines with scientific tests of safety and effectiveness. Within the analysis of the different clusters of collaboration between countries, there are four from Asia, led by China, India, Indonesia and Pakistan; two from Africa, led by South Africa and Cameroon, and then one from Latin America, led by Brazil and another from North America, led by the USA. It has been proven that there is no cluster of European countries, but that they generally collaborate with countries with which they have a commercial relationship. The research of medicinal plants in Africa is greatly underdeveloped, in contrast with China and India. In fact, there is no African country among the countries that published the most in this field. Among the first 25 institutions there is only one that belongs to the African continent. From this top 25, 13 are from China (including the first 7), 3 from Brazil, 2 from South Korea, and 1 of Saudi Arabia, Pakistan, Iran, Mexico, Cameroon, France, and Malaysia.

The most widely used search terms by the main institutions researching in this field are Unclassified Drug, Plant Extract, and Controlled Study. From the study of the keywords in the period from 2009 to 2014, seven clusters have been found, those dedicated to: Traditional medicine, Drug determination, Animals-in vivo study, Unclassified drug, Drug efficacy, Effect of extraction solvent, and Malaria. Subsequently, from the period 2015 to 2019, the clusters are reduced to five, and those focused on: Unclassified drug, Traditional medicine, Cancer, In vivo study—antidiabetic activity, and Animals—anti-inflammatory activity.

This is proven by the fact that of the total number of publications analyzed, more than 100,000, only 11% are in the Agricultural and Biological Sciences category, while more than 50% are grouped in the Pharmacology, Toxicology and Pharmaceutics category and Medicine. This study highlights the scarce research from the agronomic perspective regarding domestication, production or genetic or biotechnological research on breeding of medicinal plants.

Acknowledgments

The authors would like to thank to the CIAIMBITAL (University of Almeria, CeiA3) for its support.

Author Contributions

E.S.-M., J.A.G.-C. and F.M.-A. conceived the research, designed the search, and wrote the manuscript. All authors have read and agreed to the published version of the manuscript.

This research received no external funding.

Conflicts of Interest

The authors declare no conflict of interest.

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Revitalizing the science of traditional medicinal plants

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As early as the Qin and Han Dynasty (roughly 221 BCE to 220 CE), Sheng Nong’s Herbal Classic recorded 365 medicines. By the time of the Ming Dynasty (1368–1644), the number of Chinese herbal medicines had grown to close to 2,000. Credit: Marilyna/iStock/Getty Images Plus

Plants can be frustratingly inconsistent. With so much dependent on environmental factors, even clones can produce foliage, roots and fruits of varying quantity and quality. Issues with consistency in plant studies have thwarted attempts to characterize the many botanical extracts used in traditional medicines. But traditional knowledge could be a rich resource for drug discovery, says Timothy Mitchison at Harvard Medical School’s Department of Systems Biology.

For example, pharmaceutical chemist, Youyou Tu, discovered artemisinin, an antimalarial extract from the plant Artemisia after being inspired by an entry in the sixteenth century tome, Compendium of Materia Medica . Used as an ancient remedy for fever, artemisinin was isolated and refined by Tu in the 1970s, and according to the World Health Organization, antimalarials containing artemisinin have saved more than three million lives since 2000. Tu was awarded a Nobel Prize for her work in 2015.

“The long history of human data we have for traditional Chinese medicine could be most valuable thing you can get to help characterize any drug,” says Mitchison. He adds that while traditional Chinese medicine-derived molecules typically exhibit poor pharmacology by the standards expected of a synthetic oral drug, that has implications that are under-explored. He says that short plasma half-lives could suggest these molecules have higher action in the liver or kidney, while low oral bioavailability could be the result of action in the gut, which, he says, might be useful for targeting gut diseases.

1,892: The number of herbs mentioned Compendium of Materia Medica. Credit: Lou-Foto/Alamy Stock Photo

In the case of a plant molecule, colchicine, Mitchison’s long-time study subject, its short half-life corresponds to local action in the liver. “These special features of plant-derived molecules cannot be achieved using standard synthetic drugs, which are systemically adsorbed,” he says. “I would encourage medical researchers to have an open mind regarding different medical traditions.”

In Tu’s lecture after winning the Nobel Prize in Physiology or Medicine, she recalled the difficulties of plant research, ranging from managing extraction and purification technologies, to the variables involved in the study of the six Artemisia species, such as accounting for origin, harvest season, and the use of different plant parts.

7,000: Roughly the number of samples in the traditional Chinese medicine collection at the Royal Botanical Gardens, Kew. Credit: Ileana_bt/Shutterstock

The technical and taxonomic challenges of plant research are a source of fascination for Monique Simmonds, director of the Commercial Innovation Unit at the Royal Botanical Gardens, Kew, in London, one of the world’s largest botanical collections. But increased scrutiny of plant research aimed at pharmaceuticals is crucial, she says.

In 1998, Simmonds helped raise funds to create a 7,000-sample traditional Chinese medicine plant collection at Kew, and she currently leads a 300-strong research team focused on unlocking potential drugs derived from plants.

“Some fellow scientists are rightfully cynical about traditional Chinese medicine − some of the research, unfortunately, hasn’t been done with the level of accuracy that you would need for a medicinal drug,” she explains. “A common mistake would be to study different plant species in the same family, such as mistaking Korean and Chinese ginseng.”

17,810: The number of plant species that have a medicinal use, out of some 30,000 plants for which a use of any kind is documented. Credit: Marilyna/iStock/Getty Images Plus

Improving plant study replication through more controlled global standards is part of Simmonds’ mission as the president of the Good Practice in Traditional Chinese Medicine Research Association. Established in 2012, the association now involves 112 institutions and 24 countries, who work on creating better guidelines.

“For example, we would recommend consultation with taxonomists to help independently verify the plants or plant parts being used in research,” says Simmonds. “While taxonomy has been the backbone of Kew’s scientific research, in the next 10 years accelerating taxonomy with machine learning and trait research − from genomic and chemical to morphological and ecological − will also be vital.”

Speeding up drug discovery

At Kew, drug discovery is also being accelerated by machine learning and high-throughput mass spectrometry that reveals the chemical structures of plant compounds. Kew’s Small Molecule Analysis Laboratory, for example, profiles small molecules produced by plants and fungi to help identify chemical structures that might be useful for drug development.

Kaixian Chen, a professor at the Shanghai University of Traditional Chinese Medicine (SUTCM), points out that these types of resources have radically sped up the shortlisting process for drug candidate study.

Chen was an early user of computer-aided drug design in the 1990s. “One of the biggest technological leaps during my career has been in virtual screening: we pair our small molecule libraries of traditional Chinese medicine bioactive components with protein structures that are most likely to bind to specific drug targets in our database, saving us a lot of research time and money,” he explains.

In 2021, for example, using high-throughput screening of natural product libraries, Chen’s colleagues at SUTCM discovered an agonist to bile acid receptor TGR5 that is a potential target for drugs to treat obesity. The agonist, notoginsenoside Ft1, is derived from Panax notoginseng , a ginseng species used for 2,000 years in traditional Chinese medicine to enhance circulation.

A small, early mouse model study has validated notoginsenoside Ft1’s potential in treating obesity. “But if we are to continue to make the most of accelerating drug screening technologies, we must ensure scientific rigour in traditional Chinese medicine studies,” Chen says.

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Genomic, transcriptomic, and metabolomic analyses provide insights into the evolution and development of a medicinal plant saposhnikovia divaricata (apiaceae).

Zhen-Hui Wang, Xiao Liu and Yi Cui, These authors made equal contribution to this work.

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Zhen-Hui Wang, Xiao Liu, Yi Cui, Yun-He Wang, Ze-Liang Lv, Lin Cheng, Bao Liu, Hui Liu, Xin-Yang Liu, Michael K Deyholos, Zhong-Ming Han, Li-Min Yang, Ai-Sheng Xiong, Jian Zhang, Genomic, transcriptomic, and metabolomic analyses provide insights into the evolution and development of a medicinal plant Saposhnikovia divaricata (Apiaceae), Horticulture Research , 2024;, uhae105, https://doi.org/10.1093/hr/uhae105

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Saposhnikovia divaricata , 2n = 2x = 16, as a perennial species, is widely distributed in China, Mongolia, Russia, etc. It is a traditional Chinese herb used to treat tetanus, rubella pruritus, rheumatic arthralgia and other diseases. Here, we assembled a 2.07 Gb and N50 scaffold length of 227.67 Mb high-quality chromosome-level genome of S. divaricata based on the PacBio Sequel II sequencing platform. The total number of genes identified was 42948, and 42456 of them were functionally annotated. A total of 85.07% of the genome was composed of repeat sequences, comprised mainly of long terminal repeats (LTRs) which represented 73.7% of the genome sequence. The genome size may have been affected by a recent whole-genome duplication event. Transcriptional and metabolic analyses revealed bolting and non-bolting S. divaricata differed in flavonoids, plant hormones, and some pharmacologically active components. The analysis of its genome, transcriptome, and metabolome helped to provide insights into the evolution of bolting and non-bolting phenotypes in wild and cultivated S. divaricata and lays the basis for genetic improvement of the species.

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• Published: 25 April 2018

Quantitative study of medicinal plants used by the communities residing in Koh-e-Safaid Range, northern Pakistani-Afghan borders

• Wahid Hussain 1 ,
• Lal Badshah 1 ,
• Manzoor Ullah 2 ,
• Maroof Ali 3 ,
• Asghar Ali 4 &
• Farrukh Hussain 5  

Journal of Ethnobiology and Ethnomedicine volume  14 , Article number:  30 ( 2018 ) Cite this article

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The residents of remote areas mostly depend on folk knowledge of medicinal plants to cure different ailments. The present study was carried out to document and analyze traditional use regarding the medicinal plants among communities residing in Koh-e-Safaid Range northern Pakistani-Afghan border.

A purposive sampling method was used for the selection of informants, and information regarding the ethnomedicinal use of plants was collected through semi-structured interviews. The collected data was analyzed through quantitative indices viz. relative frequency citation, use value, and family use value. The conservation status of medicinal plants was enumerated with the help of International Union for Conservation of Nature Red List Categories and Criteria (2001). Plant samples were deposited at the Herbarium of Botany Department, University of Peshawar for future reference.

One hundred eight informants including 72 male and 36 female were interviewed. The informants provided information about 92 plants species used in the treatment of 53 ailments. The informant reported maximum number of species used for the treatment of diabetes (16 species), followed by carminatives (12 species), laxatives (11 species), antiseptics (11 species), for cough (10 species), to treat hepatitis (9 species), for curing diarrhea (7 species), and to cure ulcers (7 species), etc. Decoction (37 species, i.e., 40%) was the common method of recipe preparation. Most familiar medicinal plants were Withania coagulans , Caralluma tuberculata , and Artemisia absinthium with relative frequency (0.96), (0.90), and (0.86), respectively. The relative importance of Withania coagulans was highest (1.63) followed by Artemisia absinthium (1.34), Caralluma tuberculata (1.20), Cassia fistula (1.10), Thymus linearis (1.06), etc. This study allows identification of novel uses of plants. Abies pindrow , Artemisia scoparia , Nannorrhops ritchiana , Salvia reflexa , and Vincetoxicum cardiostephanum have not been reported previously for their medicinal importance. The study also highlights many medicinal plants used to treat chronic metabolic conditions in patients with diabetes.

Conclusions

The folk knowledge of medicinal plants species of Koh-e-Safaid Range was unexplored. We, for the first time, conducted this quantitative study in the area to document medicinal plants uses, to preserve traditional knowledge, and also to motivate the local residents against the vanishing wealth of traditional knowledge of medicinal flora. The vast use of medicinal plants reported shows the significance of traditional herbal preparations among tribal people of the area for their health care. Knowledge about the medicinal use of plants is rapidly disappearing in the area as a new generation is unwilling to take interest in medicinal plant use, and the knowledgeable persons keep their knowledge a secret. Thus, the indigenous use of plants needs conservational strategies and further investigation for better utilization of natural resources.

The residents of remote areas mostly depend on folk knowledge of medicinal plants to cure different ailments. Plants not only provide food, shelter, fodder, drugs, timber, and fuel wood, but also provide different other services such as regulating different air gases, water recycling, and control of different soil erosion. Hence, phytodiversity is required to fulfill several human daily livelihood needs. Millions of people in developing countries commonly derive their income from different wild plant products [ 1 ]. Ethnomedicinal plants have been extensively applied in traditional medicine systems to treat various ailments [ 2 ]. This relationship goes back to the Neanderthal man who used plants as a healing agent. In spite of their ancient nature, international community has recognized that many indigenous communities depend on biological resources including medicinal plants [ 3 ]. About 80% of the populations in developing countries rely on medicinal plants to treat diseases, maintaining and improving the lives of their generation [ 4 , 5 ]. The people, in most parts of the world particularly in rural areas, rely on traditional medicinal plants’ remedies due to easy availability, cultural acceptability, and poor economic conditions. Out of the total 422,000 known angiosperms, more than 50,000 are used for medicinal purposes [ 6 ]. Some 75% of the herbal drugs have been developed through research on traditional medicinal plants, and 25% of prescribed drugs belong to higher plants [ 7 ]. Traditional knowledge has a long historical cultural heritage and rich natural resources that have accumulated in the indigenous communities through oral and discipleship practices [ 8 ]. Traditional indigenous knowledge is important in the formulation of herbal remedies and isolates bioactive constituents which are a precursor for semisynthetic drugs. It is the most successful criterion for the development of novelties in drugs [ 9 , 10 , 11 ]. Traditional knowledge can also contribute to conserve and sustain the use of biological diversity. However, traditional knowledge, especially herbal health care system, has declined in remote communities and in younger generations as a result of a shift in attitude and ongoing socio-economic changes [ 12 ]. The human communities are facing health and socio-economic problems due to changing environmental conditions and socio-economic status [ 13 ]. The tribal people have rich unwritten traditional medicinal knowledge. It rests with elders and transfers to younger orally. With rapid economic development and oral transmitted nature of traditional knowledge, there is an urgent need to systematically document traditional medicinal knowledge from these communities confined in rural and tribal areas of the world including Pakistan. The Koh-e-Safaid Range is one of the remote tribal areas of Pakistan having unique and century-old ethnic characteristics. A single hospital with limited insufficient health facilities is out of reach for most inhabitants. Nature has gifted the area with rich diversity of medicinal plants. The current advancement in the use of synthetic medicines has severely affected the indigenous health care system through the use of medicinal traditional practices in the area. The young generation has lost interest in using medicinal plants, and they are reluctant to practice traditional health care system that is one of the causes of the decline in traditional knowledge system. Quantitative approaches can explain and analyze the variables quantitatively. In such approach, authentic information can be used for conservation and development of existing resources. Therefore, the present research was conducted in the area to document medicinal uses of local plants with their relative importance, to record information for future investigation and discovery of novelty in drug use, and to educate the locals about the declining wealth of traditional and medicinal flora from the area.

Ethnographic and socio-economic background of the study area

Koh-e-Safaid Range is a tribal territory banding Pakistan with Afghanistan in Kurram Agency. It lies between 33° 20′ to 34° 10′ N latitudes and 69° 50′ to 70° 50′ E longitudes (Fig.  1 ).This area is federally administered by the Government of Pakistan. The Agency is surrounded on the east by Orakzai and Khyber agencies, in the southeast by Hangu district, and in the south by North Waziristan Agency and Nangarhar and Pukthia of Afghanistan lies on its west. The highest range of Koh-e-Safaid is Sikaram peak with, 4728 m height. The Agency is well-populated with many small fortified villages receiving irrigation water from Kurram River that flows through it. The weather of the Agency is mostly pleasant in summer; however, in winters, freezing temperature is experienced, and sometimes falls to − 10 °C. The weather charts website “Climate-Charts” ranked it as the fourth coldest location in Pakistan. Autumn and winter are usually dry seasons while summer and spring receive much of the precipitation. The total population of the Agency according to the 2017 censuses report is 253,478. Turi, Bangash, Sayed, Maqbal, Mangel, Khushi, Hazara, Kharote, and Jaji are the major tribes in the research area. The joint family system is practiced in the area. Most of the marriages are held within the tribe; however, there is no ban on the marriages outside the tribe. Marriage functions are communal whereby all relatives, friends, and village people participate with songs, music, and dances male and female separately. The death and funeral ceremonies are jointly attended by the friends and relatives. The people of the area follow Jirga to resolve their social and administrative problems. This is one of the most active and strong social institutions in the area. Economically, most people in the area are poor and earning their livelihoods by menial jobs. The professional includes farmers, pastoralists, shopkeepers, horticulturists, local health healers, wood sellers, and government servants. In the adjoining areas of the city, pastorals keep domestic animals and are considered a better source of income.

Map of the study area and area location in Pakistan

Sampling method

The study was conducted through purposive sampling by informants’ selection method. The selection of informants was primary based on the ethnomedicinal plants and their willingness to share the information. The selection criteria include people who prescribe recipes for treatment; people involved in buying, collection, or cultivation of plants; elder members of above 60 years age; and young literate members. The participants were traditional healers, plant collectors, farmers, traders, and selected knowledgeable elders above 60 years age and young ones. The interviews were conducted in local Pashto language in the local dialect. The informants were involved in the gathering of data with a consent of village tribe chieftains called Maliks.

Data collection

Semi-structured open-ended interviews were conducted for the collection of ethnomedicinal information from April 2015 to August 2017. Informants from 19 localities were interviewed including Sultan, Malikhail, Daal, Mali kali, Alam Sher, Kirman, Zeran, Malana, Luqman Khail, Shalozan, Pewar, Teri Mangal, Bughdi, Burki, Kharlachi, Shingak, Nastikot, Karakhila, and Parachinar city (Fig.  1 ). The objectives of this study were thoroughly explained to all the informants before the interview [ 14 ]. Data about medicinal plants and informants including local names of plants, preparation of recipes, storage of plant parts, informant age, occupation, and education were collected during face-to-face interviews. A questionnaire was set with the following information: informant bio-data, medicinal plant use, plant parts used and modes of preparation, and administration of the remedies. Plants were confirmed through repeated group discussion with informants [ 15 , 16 ]. For the identification of plants, informants were requested for transect walks in the field to locate the cited plant for confirmation.

Collection and identification of medicinal plants

The medicinal plants used in traditional treatment of ailments in the study area were collected with the help local knowledgeable persons, traditional healers, and botanists. The plants were pressed, dried, and mounted on herbarium sheet. The field identification was confirmed by a taxonomist in the Herbarium Department of Botany, University of Peshawar. The voucher specimens of all species were numbered and deposited in the Herbarium of Peshawar University (Fig.  2 ).

Landscape of Kurram Valley ( a winter, b summer). c , d Traditional healers selling herbal drugs on footpath. e Trader crushing Artemisia absinthium for marketing. f Principal author in the field during data collection. g , h Plant collectors in subalpine zone. i Lilium polyphyllum rare species distributed in subalpine zone. j Ziziphora tenuior endangered species of subtropical zone

Data analysis

The information about ethnomedicinal uses of plants and informants included in questionnaires such as botanical name, local name, family name, parts used, mode of preparation, use reports, frequency of citation, relative importance, and voucher number were tabulated for all reported plant species. Informants’ use reports for various ailments and frequency of citation were calculated for each species. The relative importance of species was calculated according to use-value formula (UV = UVi/Ni) [ 17 ], where “UVi” is the number of citations for species across all informants and “Ni” the number of informants. The citation probability of each medicinal plant across all informants was equal to avoid researchers’ biasness. Family use value was calculated using the formula FUV = UVs/Ns, where “UVs” represent the sum of use values of species falling within family, and Ns represents the number of species reported for the family. The conservation status of wild medicinal plants species was enumerated by applying International Union for Conservation of Nature (IUCN) criterion (2001) [ 18 ].

Informants’ knowledge about medicinal plants and their demography

A total of 108 including 72 male and 36 female informants were interviewed from 19 locations. The three groups of male respondents were falling in the age groups of 21 to 40, 41 to 60, and 61 to 80 years having the numbers of 19, 19, and 34, respectively. Among the female respondents, 10 aged 21 to 40, 14 aged 41 to 60, and 12 aged 61 to 80 years. Among the informants, 15 males were illiterate, 34 were matriculate, 13 were intermediate, and 10 were graduates. Among the females, 19 were illiterate, 16 were matriculate, and only 1 was graduate (Table  1 ). Informants were shepherd, healers, plant collectors, gardeners, and farmers. Twenty-eight informants of above 60 years age, living a retired life, were also interviewed. It was found that males were more knowledgeable than females. Furthermore, health healers were more knowledgeable.

Diversity of medicinal plants

A total of 92 medicinal species including 91vascular plant species belonging to 50 families and 1 mushroom Morchella of Ascomycetes of family Morchellaceae were reported (Table  2 ) . Asteraceae had eight species followed by seven species of Lamiaceae and Rosaceae. Three species were contributed by each of Moraceae, Asclepiadaceae, Polygonaceae, Brassicaceae, Solanaceae, Cucurbitaceae, and Liliaceae. Of the remaining eight families, namely, Poaceae, Pinaceae, Zingiberaceae, Chenopodiaceae, Plantaginaceae, Apiaceae, Fabaceae, and Zygophyllaceae, each one contributed two species [ 19 , 20 ]. Asteraceae, Lamiaceae, and Rosaceae were also reported with a high number of plants used for medicinal purposes. The reported plants were collected both from the wild (86.9%) and cultivated (13.1%) sources. However, greater percentage of medicinal plants from wild sources indicated higher species’ diversity in the study area. The 62 herbs species, 16 tree, 12 shrubs, and 2 undershrubs species were used in medicinal preparation for remedies.

Plant parts used in preparation of remedies

The plant parts used in the preparation of remedies were root, rhizome, bulbils, stem, branches, leaves, flowers, fruits, seeds, bark, resin, and latex. The relative use of these plant parts is shown in (Fig.  3 ). Fruits were frequently used plant part (26 species), followed by leaves (23 species) and remaining parts (21 species).

Plant parts used in the formulation of remedies

Preparation and mode of administration of remedies

The collection of data for the preparation of remedies from medicinal plants is extremely important. Such information is essential for identification of active ingredients and intake of relevant amount of drug. The present research observed seven methods for preparing recipes. It included decoction, powder, juice, infusions, roast, and ash methods (Fig.  4 ). The 37 species (40%) were most frequently used for the preparation of remedies. A plant part is boiled while infusion is obtained by soaking plant material in cold or hot water overnight. Eleven species (14%) are in powdered form, 11species (14%) in vegetable form, 7 species (9%) in juice form, 7 species (9%) in infusions form, 3 species (4%) in roasted form, and 1 species (2%) in ash form were used. Twenty-seven plant parts were used directly. It included wild fruits that were consumed for their nutritional and medicinal purpose. The most frequently used mode of administration of remedies was oral intake practice of 74 species (79%) followed by both orally and topically practice of 11 species (12%) and topically of 8 species (9%) (Fig.  5 ).

Different modes of drug formulation

Route of administration of drugs

Medicinal plants use categories

The inhabitants used medicinal plants in the treatment of 53 health disorders. The important disorders were cancer, diabetic, diarrhea, dysentery, hepatitis, malaria, and ulcer (Table.  3 ). These disorders were classified into 17 categories. Among the ailments, most plants were used for the treatment of digestive problems mainly as carminative (12 species), diarrhea (11 species), laxative (11 species), ulcer (7 species), appetizer (5 species), colic pain (4 species), and anthelmintic (4 species). Such higher use of plants for the treatment of digestive problems had been reported in ethnobotanical studies conducted in another tribal area of Pakistan [ 21 ]. The other categories (18 species) were used to treat respiratory disorders, followed by endocrine disorders (16 species); antiseptic and anti-inflammatory (15 species); circulatory system disorders (15 species); integumentary problems (15 species); antipyretic, refrigerant, and analgesic (9 species); and hepatic disorders (9 species). However, among the ailments, the highest number of plants were used in the treatment of diabetes (16 species), followed by antiseptic (11 species), cough (10 species), hepatitis (9 species), and ulcer (7 species). Among the remaining species, the informants reported three and two species used against malaria and cancer, respectively (Table  3 ).

Quantitative appraisal of ethnomedicinal use

Based on the quantitative indices, the analyzed data showed that few plants were cited by the majority of the informants for their medicinal value. Seventeen plant species with the highest citation frequency are shown in (Fig.  6 ). The highest citation frequency was calculated for Withania coagulans (0.96), followed by Caralluma tuberculata (0.90), and Artemisia absanthium (0.86). The high values of these species indicated that most of the informants were familiar with their medicinal value. However, the familiarity of these three plants could be linked to their collection for economic purposes [ 22 ]. Withania coagulans (1.63), Artemisia absinthium (1.34), Caralluma tuberculata (1.20), Cassia fistula (1.10), and Thymus linearis (1.06) were reported having the highest used values for medicinal purposes (Fig.  7 ). All these species were used for the cure of three or more diseases. The powdered fruit of Withania coagulans is used for the cure of stomach pain, constipation, diabetes, and ulcer. The next highest use value was calculated for Artemisia absinthium with five medical indications as diabetes, malaria, fever, blood pressure, and urologic problems. Among the remaining three plants, Caralluma tuberculata is used for diabetes, cancer, and stomachic problems, and as blood purifier; Cassia fistula for colic pain and stomach pain and as a carminative agent; and Thymus linearis for cough and as carminative and appetizer. Lowest use value was calculated for Rununculus muricatus (0.04) with next three species having same lowest use value: Abies pindrow (0.05), Lepidium virginicum (0.05), and Oxalis corniculata (0.05). Highest family use value was calculated for Juglandaceae (0.86), followed by Cannabaceae (0.78), Apiaceae (0.75), Asclepiadaceae (0.71), Fumariaceae (0.71), Berberidaceae (0.70), Fabaceae (0.67), Punicaceae (0.65), Solanaceae (0.64), and Asteraceae (0.61). This is the first study that presents a quantitative value of medicinal plants used in the investigated area.

Medicinal plants with highest relative frequency citation

Medicinal plants with highest relative importance

Conservation status of the medicinal flora

Plant preservation means the study of plant declination, their causes, and techniques to protect rare and scarce plants. Plant conservation is a fairly new field that emphasizes the conservation of biodiversity and whole ecosystems as opposed to the conservation of individual species [ 23 ]. The ex situ conservation must be encouraged for the protection of medicinal plants [ 24 ]. In the present case, the area under study is under tremendous anthropogenic pressure as well. Therefore, ex situ conservation of endangered species is recommended. The woody plants, cut down for miscellaneous purposes, are facing conservational problems. Sayer et al. [ 25 ] reported that large investments are being made in the establishment of tree plantation on degraded area in Asia [ 25 ]. Alam and Ali stressed that proper conservation studies are almost negligible in Pakistan [ 26 ]. Same is the case with the study area as no project has been initiated for the conservation of forest or vegetation so far. Anthropogenic activities, small size population, distribution in limited area, and specificity of habitat were observed as the chief threats to endangered species.

According to IUCN Red List Criteria (2001) [ 18 ] conservation status of 80 wild medicinal species have been assessed based on availability, collection status, growth status, and their parts used. The remaining 12 medicinal plants were cultivated species. Of these, 7 (8.7%) species are endangered, 34 (42.5%) species are vulnerable, 29 (36.2%) species are rare, 9 (11.2%) species are infrequent, and only 1 (1.3%) species is dominant. The endangered species were Caralluma tuberculata , Morchella esculenta , Rheum speciforme , Tanacetum artemisioides , Vincetoxicum cardiostephanum , Withania coagulans , and Polygonatum verticillatum.

Traditional medicines are a vital and often underestimated part of health care. Nowadays, it is practiced in almost every country of the world. Its demand is currently increasing rapidly in the form of alternative medicine [ 20 ]. Ethnomedicinal plants have been widely applied in traditional medicine systems to treat various ailments. About 80% of the populations in developing countries rely on medicinal plants to treat diseases, maintaining and improving the lives of their generation [ 19 ]. Traditional knowledge has a long historical cultural heritage and rich natural resources that has accumulated in the indigenous communities through oral and discipleship practices [ 8 ]. Traditional indigenous knowledge is important in the formulation of herbal remedies and isolates bioactive constituents which are a precursor for semisynthetic drugs. It is the most successful criterion for the development of novelties in drugs [ 11 ]. A total of 92 medicinal species including 91 vascular plant species belonging to 50 families and 1 mushroom Morchella of Ascomycetes of family Morchellaceae were reported (Table  2 ) . The current study reveals that the family Asteraceae represents eight species followed by seven species of Lamiaceae and Rosaceae each which showed a higher number of medicinal plants. Three species were contributed by each of Moraceae, Asclepiadaceae, Polygonaceae, Brassicaceae, Solanaceae, Cucurbitaceae, and Amaryllidaceae. While the remaining eight families, namely, Poaceae, Pinaceae, Zingiberaceae, Chenopodiaceae, Plantaginaceae, Apiaceae, Fabaceae, and Zygophylaceae, contributed two species each. Asteraceae, Lamiaceae, and Rosaceae were also reported with a high number of plants used for medicinal purposes. Indigenous use of medicinal plants in the communities residing in Koh-e-Safid Range of Pakistan is evident. Traditional health healers are important to fulfill the basic health needs of the economically poor people of the area. The high dependency on traditional healers is due to limited and inaccessible health facilities. Most people either take recipes from local healers or select wild medicinal plants prescribed by them. Some elders also knew how to preserve medicinal plant parts for future use. Traditional knowledge of medicinal plants is declining in the area due to lack of interest in the young generation to acquire this traditional treasure. Furthermore, most traditional health healers and knowledgeable elders hesitate to disseminate their recipes. Therefore, traditional knowledge in the area is diminishing as aged persons are passing away. Vernacular names of plants are the roots of ethnomedicinal diversity knowledge [ 27 ]. They can clear the ambiguity in the identification of medicinal plants within an area. It also helps in the preservation of indigenous knowledge of medicinal plants. The medicinal plants were mostly reported with one specific vernacular name in the investigated area. While Rosa moschata and Rosa webbiana were known by same single vernacular name as Jangle Gulab. Few species were known by two vernacular names: Curcuma longa as Korkaman or Hildi, Ficus carica as Togh or Anzer, Fumaria indica as Chamtara or Chaptara, Marrubium vulgare as Dorshol or Butaka, Solanum nigrum as Bartang or Kharsobay, Teucrium stocksianum as Harboty or Gulbahar, and Thymus linearis as Paney or Mawory. The informants also mentioned different vernacular names for species even belonging to single genus; Plantago lanceolata as Chamchapan or Ghuyezaba and Plantago major as Ghazaki or Palisepary. Majority of the species commonly had a single name. However, local dialects varied in few species, i. e., Withania coagulans was known by three names: Hapyanaga, Hafyanga, and Shapynga, Caralluma tuberculata as Pamenny or Pawanky, Foeniculum vulgare as Koglany or Khoglany, and Viola canescens was called as Banafsha or Balamsha. The species with high use value need conservation for maintaining biodiversity in the study area. However, in the present case, no project or programs for the conservation of forest or vegetation are operating. Grazing and unsustainable medicinal uses were observed as the chief hazard to highly medicinal plant species. The higher use of herbs can be attributed to their abundance, diversity, and therapeutic potentials as antidiabetic, antimalarial, antipyretic, antiulcerogenic, antipyretic, blood purifier, and emollient and for blood pressure, hepatitis, stomach pain, and itching. Aloe vera , cultivated for ornamental purpose, is used as wound healing agent. Among the plant parts, the higher use of fruit may relate to its nutritional value. The aerial parts of the herbaceous plants were mostly collected in abundance and frequently used for medicinal purposes. In many recipes, more than one part was used. The utilization of roots, rhizomes, and the whole plant is the main threat in the regeneration of the medicinal plants [ 28 ]. In the current study, decoction was found to be the main method of remedy preparation as reported in the ethnopharmacological studies from other parts [ 29 , 30 , 31 ]. Fortunately, we collected important information like preparation of remedies and their mode of administration for all the reported plants. However, the therapeutic potential of few plants are connected to their utilization method. A roasted bulb of Allium cepa is wrapped on the spine-containing wound to release the spine. The leaf of Aloe vera containing viscous juice is scratched and wrapped on a wound. The latex of Calotropis procera is first mixed with flour and then topically applied on the skin for wound healing. Infusion of Cassia fistula fruit’s inner septa is prepared for stomach pain and carminative and colic pain in children. The fruit of Citrullus colocynthis boiled in water is orally taken for the treatment of diabetes. Grains of Hordeum vulgare are kept in water for a day, and its extraction is taken for the treatment of diabetes. The decoction of Seriphidium kurramensis shoots are used as anti-anthelmintic and antimalaria. The leaves of Juglans regia are locally used for cleaning the teeth and to prevent them from decaying. Furthermore, its fruit is used as brain tonic, and its roasted form is useful in the treatment of dysentery. The roots of Pinus wallichiana are cut into small pieces and put into the pot. The cut pieces are boiled, and the extracted liquid is poured into the container. One drop of the extracted liquid is mixed with one glass of milk and taken orally once a day as blood purifier. An infusion of Thymus linearis aerial parts is prepared like hot tea and is drunk for cough and as appetizer and carminative. A decoction of Zingiber officinale rhizome is drunk at night time for relief of cough. Medicinal plants are still practiced in tribal and rural areas as they are considered as main therapeutic agents in maintaining better health. Such practices have been described in the ethnobotanical studies conducted across Pakistan. The current study reveals several plant species with more than one medical use including Artemisia absanthium , Cichorium intybus , Fumaria indica , Punica granatum , Tanacetum artemisioides , Teucrium stocksianum , and Withania coagulans . Their medicinal importance can be validated from indigenous studies conducted in various parts of the country. Amaranthus viridis leaf extract is an emollient and is used for curing cough and asthma as well [ 32 ]. Artemisia absanthium is used for the treatment of malaria and diabetes [ 33 , 34 , 35 , 36 ]. Cichorium intybus is used against diabetes, malaria, and gastric ulcer, and it is also used as digestive and laxative agent [ 28 , 37 , 38 , 39 , 40 , 41 ]. Leaves of Cannabis sativa are used as bandage for wound healing; powdered leaves as anodyne, sedative, tonic, and narcotic; and juice added with milk and nuts as a cold drink [ 42 ]. Whole plant of Fumaria indica [ 36 ] and Tanacetum artemisioides [ 43 ] is used for treating constipation and diabetes, respectively. Dried rind powder and fruit extract of Punica granatum are taken orally for the treatment of anemia, diarrhea, dysentery, and diabetes [ 44 , 45 , 46 , 47 ]. A decoction of aerial parts of Teucrium stocksianum is used for curing diabetes [ 29 , 48 ]. Withania coagulans is known worldwide [ 38 , 49 ] as a medicinal plant, whose fruit decoction is best remedy for skin diseases and diabetes. Its seeds are used against digestive problems, gastritis, diabetes, and constipation [ 21 , 28 , 50 ]. Our results are in line with the traditional uses of plants in the neighboring counties [ 8 ]. For example, Fumaria indica is used as blood purifier, and Hordeum vulgare grains decoction for diabetes; Juglans regia bark for toothaches and scouring teeth; Mangifera indica seed decoction for diarrhea; Solanum nigrum extract for jaundice; and Solanum surattense fruit decoction for cough have been documented in the study (40) . Such agreements strengthen our results and provide good opportunity to evaluate therapeutic potential of the reported plants. Three plants species Adiantum capillus-veneris , Malva parviflora , and Peganum harmala have been documented for their medicinal use in the ethnobotanical study [ 51 ]. According to this, the decoction of the aerial parts of Adiantum capillus-veneris is used for the treatment of asthma and dyspnea. Malva parviflora root and flower are used for stomach ulcers. Peganum harmala fruit powder and decoction are used for toothache, gynecological infections, and menstruation. The dried leaves of Artemisia absanthium is used to cure stomach pain and intestinal worm while an inflorescence paste prepared from its fresh leaves is used as wound healing agent and antidiabetic [ 52 , 53 ]. The bulb of Allium sativum is used in rheumatism while its seed vessel mixed with hot milk is useful for the prevention of tuberculosis and high blood pressure. The fruit bark of Punica granatum is used in herbal mixture for intestinal problems [ 54 ]. Avena sativa decoction is used for skin diseases including eczema, wounds, irritation, inflammation, erythema, burns, itching, and sunburn [ 55 ]. Foeniculum vulgare and Lepidium sativum are used for the treatment of diabetes and renal diseases [ 53 ]. Verbascum thapsus leaves and flowers can be used to reduce mucous formation and stimulate the coughing up of phlegm. Externally, it is used as a good emollient and wound healer. Leaves of Thymus linearis are effective against whooping cough, asthma, and round worms and are an antiseptic agent [ 21 ]. Berberis lycium wood decoction with sugar is the best treatment for jaundice. Chenopodium album has anthelmintic, diuretic, and laxative properties, and its root decoction is effective against jaundice. The whole plant decoction of Fumaria indica is used for blood purification. Dried leaves and flowers of Mentha longifolia are used as a remedy for jaundice, fever, asthma, and high blood pressure [ 36 ]. Morus alba fruit is used to treat constipation and cough [ 42 ]. Oxalis corniculata roots are anthelmintic, and powder of Chenopodium album is used for headache and seminal weakness [ 47 ]. Boiled leaves of Cichorium intybus are used for stomachic pain and laxative while boiled leaves of Plantago major are used against gastralgia [ 56 ]. Viola canescens flower is used as a purgative [ 32 ]. The above ethnomedicinal information confirms the therapeutic importance of the reported plants. The reported plant species show biological activities which suggest their therapeutic uses. The aqueous extract of Allium sativum has been studied for its lipid lowering ability and was found to be effective at the amount of 200 mg/kg of body weight. It also has significant antioxidant effect and normalizes the activities of superoxide dismutase, catalase, glutathione peroxidase, and glutathione reductase in the liver [ 57 ]. An extract of Artemisia absanthium antinociception in mice has been found and was linked to cholinergic, serotonergic, dopaminergic, and opioidergic system [ 58 ]. The ethanolic extract of Artemisia absanthium at a dose of 500 and 1000 mg/kg body weight has reduced blood glucose to significant level [ 59 ]. The hepatoprotective activity of crude extract of aerial parts of Artemisia scoparia was investigated against experimentally produced hepatic damage through carbon tetrachloride. The experimental data showed that crude extract of Artemisia scoparia is hepatoprotective [ 60 ]. Ethanolic and aqueous extracts from Asparagus exhibited strong hypolipidemic and hepatoprotective action when administered at a daily dose of 200 mg/kg for 8 weeks in hyperlipidemic mice [ 61 , 62 ]. The extract of Calotropis procera was evaluated for the antiulcerogenic activity by using different in vivo ulcer in pyloric-ligated rats, and significant protection was observed in histamine-induced duodenal ulcers in guinea pigs [ 63 ]. Cannabidiol of Cannabis sativa was found as anxiolytic, antipsychotic, and schizophrenic agent [ 64 ]. Caralluma tuberculata methanolic extract of aerial parts (500 mg/kg) in fasting blood glucose level in hyperglycemic condition decreased up to 54% at fourth week with concomitant increase in plasma insulin by 206.8% [ 65 ]. The aqueous and methanol crude extract of Celtis australis , traditionally used in Indian system of medicine, was screened for its antibacterial activity [ 66 ]. Cichorium intybus L. whole plant 80% ethanolic extract a percent change in serum glucose has been observed after 30 min in rats administrated with vehicle, 125, 250, and 500 mg notified as 52.1, 25.2, 39, and 30.9%, respectively [ 67 ]. Citrullus colocynthis fruit, pulp, leaves, and root have significantly decreased blood glucose level and restored beta cells [ 30 , 68 , 69 , 70 ]. The two new aromatic esters horizontoates A and B and one new sphingolipid C were isolated from Cotoneaster horizontalis . The compounds A and B showed significant inhibitory effects on acetylcholinesterase and butylcholinesterase in a dose-dependent manner [ 71 ]. The alkaloids found in Datura stramonium are organic esters used clinically as anticholinergic agents [ 72 ]. The methanolic extract of Momordica charantia fruits on gastric and duodenal ulcers was evaluated in pylorus-ligated rats; the extract showed significant decrease in ulcer index [ 73 ]. Antifungal activity of Nannorrhops ritchiana was investigated against fungal strains Aspergillus flavus , Trichophyton longifusis , Trichophyton mentagrophytes , Aspergillus flavus , and Microsporum canis were found susceptible to the extracts with percentage inhibition of 70–80% [ 74 ]. The inhibitory effects of Olea ferruginea crude leaf extract on bacterial and fungal pathogens have been evaluated [ 75 ]. The aqueous extract of Plantago lanceolata showed that higher doses provide an overall better protection against gastro-duodenal ulcers [ 76 ]. The oral and intraperitoneal management of extracts reduced the gastric acidity in pylorus-ligated mice [ 77 ]. The antiulcer effect of Solanum nigrum fruit extract on cold restraint stress, indomethacin, pyloric ligation, and ethanol-induced gastric ulcer models and ulcer healing activity on acetic acid-induced ulcer model in rats [ 78 , 79 ]. The antifungal activity (17.62 mm) of Viola canescens acetone extract 1000 mg/ml against Fusarium oxysporum has been observed [ 80 ]. Leaf methanolic extract of Xanthium strumarium has inhibited eight pathogenic bacteria at a concentration of 50 and 100 mg/ml [ 81 ]. Aqueous extract of the fruits of Withania coagulans in streptozotocin-induced rats at dose of 1 g/kg for 7 days has shown significant decrease ( p  < 0.01) in the blood glucose level (52%), triglyceride, total cholesterol, and low density lipoprotein and very significant increase ( p  < 0.01) in high density lipoprotein level [ 31 ]. This shows that further investigation on the reported ethnomedicinal plants can lead to the discovery of novel agents with therapeutic properties.

In the current study, conservation status of 80 medicinal species was reported which was growing wild in the area. The information was collected and recorded for different conservation attributes by following International Union for Conservation and Nature (2001) [ 18 ]. It was reported that seven species (8.7%) were endangered due to the much collection, anthropogenic activities, adverse climatic conditions, small size population and distribution in limited area, specificity of habitat, and over grazing in the research area. However, the below-mentioned species were found to be endangered: Caralluma tuberculata , Morchella esculenta , Rheum speciforme , Tanacetum artemisioides , Vincetoxicum cardiostephanum , Withania coagulans , and Polygonatum verticillatum . Unsustainable use and lack of suitable habitat have affected their regeneration and pushed them to endangered category. Traditional knowledge can also contribute to conservation and sustainable use of biological diversity [ 19 , 20 ].

Novelty and future prospects

Ethnomedicinal literature research indicated that five plant species, Abies pindrow , Artemisia scoparia , Nannorrhops ritchiana , Salvia reflexa , and Vincetoxicum cardiostephanum , have not been reported previously for their medicinal importance from this area. The newly documented uses of these plants were Abies pindrow and Salvia reflexa (antidiabetic), Artemisia scoparia (anticancer), Nannorrhops ritchiana (laxative), and Vincetoxicum cardiostephanum (chest problems). Adiantum capillus-veneris is reported for the first time for its use in the treatment of skin problems. These plant species can be further screened for therapeutic agents and their pharmacological activities in search of novel drugs. The study also highlights 16 species of antidiabetic plants Caralluma tuberculata , Momordica charantia , Marrubium vulgare , Artemisia scoparia , Melia azedarach , Salvia reflexa , Citrullus colocynthis , Tanacetum artemisioides , Quercus baloot , Olea ferruginea , Cichorium intybus , Artemisia absinthium , Hordeum vulgare , Teucrium stocksianum , Withania coagulans , and Abies pindrow . Except sole paper from District Attack, Pakistan [ 28 ], such a high number of antidiabetic plants have not been reported previously from any part of Pakistan in the ethnobotanical studies.

Traditional knowledge about medicinal plants and preparation of plant-based remedies is still common in tribal area of Koh-e-Safaid Range. People due to closeness to medicinal plants and inaccessible health facilities still rely on indigenous traditional knowledge of plants. The role of traditional healers in the area is observable in primary health care. The locals used medicinal plants in treatment of important disorders such as cancer, diabetes, hepatitis, malaria, and ulcer. The analyzed data may provide opportunities for extraction of new bioactive constituents and to develop herbal remedies. The study also confirmed that the communities residing in the area have not struggled for conservation of this traditional treasure of indigenous knowledge and medicinal plants. Medicinal plant diversity in the remote and backward area of Koh-e-Safaid Range has great role in maintaining better health conditions of local communities. Therefore, conservation strategies should be adopted for the protection of medicinal plants and traditional knowledge in the study area to sustain them in the future.

Abbreviations

Family use value

International Union for Conservation of Nature

The number of informants

Represent the number of species reported for the family

The number of citations for a species across all informants

Represent sum of use values of species falling within family

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Acknowledgements

This work is part of the Doctoral research work of the principal (first) author. The authors also acknowledge the participants for sharing their valuable information.

Authors’ contribution

WH conducted the collection of field data and wrote the initial draft of the manuscript. LB supervised the project. MU and MA helped in the field survey, sampling, and identification of taxon. AA and FH helped in the data analysis and revision of the manuscript. All the authors approved the final manuscript after revision.

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Field data of the research project Quantitative study of medicinal plants used by the communities residing in Koh-e-Safaid Range northern Pakistani-Afghan border. (XLSX 167 kb)

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Hussain, W., Badshah, L., Ullah, M. et al. Quantitative study of medicinal plants used by the communities residing in Koh-e-Safaid Range, northern Pakistani-Afghan borders. J Ethnobiology Ethnomedicine 14 , 30 (2018). https://doi.org/10.1186/s13002-018-0229-4

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• Published: 09 April 2024

Chloroplast genomes of Caragana tibetica and Caragana turkestanica : structures and comparative analysis

• LiE Liu 1 ,
• HongYan Li 1 ,
• JiaXin Li 1 ,
• XinJuan Li 1 ,
• Jing Sun 2 &
• Wu Zhou 1  

BMC Plant Biology volume  24 , Article number:  254 ( 2024 ) Cite this article

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The genus Caragana encompasses multiple plant species that possess medicinal and ecological value. However, some species of Caragana are quite similar in morphology, so identifying species in this genus based on their morphological characteristics is considerably complex. In our research, illumina paired-end sequencing was employed to investigate the genetic organization and structure of Caragana tibetica and Caragana turkestanica , including the previously published chloroplast genome sequence of 7 Caragana plants.

The lengths of C. tibetica and C. turkestanica chloroplast genomes were 128,433 bp and 129,453 bp, respectively. The absence of inverted repeat sequences in these two species categorizes them under the inverted repeat loss clade (IRLC). They encode 110 and 111 genes (4 /4 rRNA genes, 30 /31tRNA genes, and 76 /76 protein-coding genes), respectively. Comparison of the chloroplast genomes of C. tibetica and C. turkestanica with 7 other Caragana species revealed a high overall sequence similarity. However, some divergence was observed between certain intergenic regions ( matK-rbcL , psbD-psbM , atpA-psbI , and etc.). Nucleotide diversity (π) analysis revealed the detection of five highly likely variable regions, namely rps2-atpI , accD-psaI-ycf4 , cemA-petA , psbN-psbH and rpoA-rps11 . Phylogenetic analysis revealed that C. tibetica ’s sister species is Caragana jubata , whereas C. turkestanica ’s closest relative is Caragana arborescens .

Conclusions

The present study provides worthwhile information about the chloroplast genomes of C. tibetica and C. turkestanica , which aids in the identification and classification of Caragana species.

Peer Review reports

Caragana Fabr. comprises over 100 species and belongs to the family of Fabaceae. These plants are mainly distributed in arid and semi-arid regions of Asia and Europe. Of these species, 66 were found in China, 32 of which are endemic [ 1 ]. Caragana plants are renowned for their drought, infertile conditions, cold and heat tolerance [ 2 ]. They are widely cultivated due to their ability to adapt to dry conditions [ 3 ]. Similar to other Fabaceae family plants, these plants can convert atmospheric nitrogen into usable nutrients via nodules on their roots, playing a role in rejuvenating infertile soils, combating dust storms, and hindering desertification [ 4 ]. The distribution of various Caragana plants in China has been extensively studied (Table  1 ).

Additionally, previous studies have shown that this genus includes more than 10 plants with excellent pharmacological properties. These plants have been utilized to treat various diseases such as fever, inflammation, wound infections, headaches, rheumatoid arthritis, and cancer [ 4 , 14 , 15 ]. The C. tibetica studied in this article is mentioned in both Mongolian medicine and Tibetan medicine as a potential treatment for rheumatoid arthritis, wounds, hypertension, and anemia [ 4 ].

The current research indicates a limited availability of data on plants in Caragana , with only 14 chloroplast (cp) genomes reported . However, the evolutionary analyses using nuclear ITS (Internal Transcribed Spacer) and plastid marker sequence data ( matK , trnL-F , and psbA-trnH ) for studying the phylogenetic relationships of Caragana plants lack resolution, leaving unanswered questions about the classification of certain medicinal plants like Caragana changduensis Liou f. , Caragana frutex (L.) C. Koch , and Caragana polourensis Franch. [ 1 , 16 , 17 , 18 , 19 ]. These plants, representing different species within Caragana , exhibit varying morphological features in terms of flowers, leaves, stems, and other aspects. Additionally, their habitat preferences contribute to morphological adaptations and variations, as they inhabit diverse ecological environments. Thus, relying solely on morphology for identifying different Caragana species may introduce errors and uncertainties. Consequently, finding an accurate and convenient method for plant identification in Caragana is crucial.

According to reports, researchers have gained a deeper understanding of chloroplasts, including their origins, structures, evolution, and genetic engineering [ 20 , 21 ]. Chloroplasts contain their genetic system [ 22 ], and most plants have chloroplasts existing in the form of covalently closed circular DNA [ 23 ]. The rapid development of sequencing technologies has led to the discovery of more efficient molecular markers within the chloroplast genome, which are advantageous for accurate species identification. The chloroplast genome is an ideal choice for molecular identification, phylogenetic analysis, and species conservation research according to a previous study [ 24 ]. Unlike the nuclear genome, the chloroplast genome is particularly valuable for plant phylogenetic studies due to its unique features: it is typically inherited from only one parent, had a simpler structure, and contains multiple copies of each gene [ 23 , 25 ]. The plastid chromosome, which is circular and has a length of 120 ~ 160kb [ 25 ], consists of four regions containing two inverted repeat regions (IRs). These regions separate the large single copy region (LSC) and the small single copy region (SSC) [ 26 ]. Due to their high level of conservation and relative small size, plastid structure and gene content is easy to obtain completely and worth studying in species identification, population genetics, and phylogenetics [ 27 , 28 ]. Currently, various plants, such as Desmodieae, Picea, and Epimedium [ 29 , 30 , 31 ], utilize the chloroplast genome to study their phylogenetic relationships. Reports have indicated the occurrence of inverse repeated loss of the evolutionary branch (IRLC) in Fabaceae [ 32 , 33 , 34 , 35 , 36 ], including the 8 species of Caragana with IRLC that have been reported [ 1 , 16 , 17 , 37 ]. Therefore, it will be possible to study Caragana as a lineage representing a broad spectrum of IRLC with the improvement in the chloroplast genome data of Caragana .

In this study, we compared the complete chloroplast genomes of C. tibetica and C. turkestanica to those of other species within the Caragana genus. Additionally, we analyzed the structural characteristics and phylogenetic relationships of these chloroplast genomes with other species within the Fabaceae family. The results of this study have advanced the knowledge of chloroplast genome data within the genus Caragana , providing valuable insights for species identification, systematic evolutionary studies, and germplasm conservation and utilization.

Characteristics of Caragana chloroplast genomes

Draw gene maps using OGDRAW for C. tibetica (Fig.  1 A) and C. turkestanica (Fig.  1 B) based on the annotation results of their chloroplast genomes. The chloroplast genomes sizes of C. tibetica and C. turkestanica were found to be 128,433 and 129,453bp, respectively. With the loss of the IR region in the two plants, they do not have the typical quadripartite structure found in most flowering plants’ chloroplast genomes, and their lengths were accordingly shorter. Nevertheless, the cp genome structures, gene contents and direction were strongly comparable (Fig.  1 A and B).

The diagram illustrates the chloroplast gene maps of C. tibetica ( A ) and C. turkestanica ( B ). The genes located on the outer circle are transcribed counterclockwise, whereas those on the inner circle are transcribed clockwise. Different functional gene groups are represented by different color codes. In addition, changes in GC content are represented by light gray in the inner circle, while changes in AT content are represented by dark gray

The annotation results of the chloroplast genomes revealed that C. tibetica had a total of 110 specific genes in chloroplast genome, while C. turkestanica shared totally 111 specific genes, comprising 76 protein coding genes, 31 (30) tRNA genes and 4 rRNA genes (Table  2 ). The GC contents of the two species were very similar, with values of 34.30% and 34.71%, respectively. Seven cp genomes in the Caragana species ( C. arborescens, C. opulens, C. jubata, C. rosea, C. microphylla, C. kozlowii, C. korshinskii ) with missing IR regions were compared with C. tibetica (128,433 bp) and C. turkestanica (129,453 bp). The results revealed that the total sequence lengths ranged from 128,132 to 133,122 base pairs. The deletion of the IR region resulted in the shortest chloroplast genome length of 128,132 bp in C. jubata and the longest in C. rosea (133,122 bp). In addition, the number of genes in C. turkestanica , C. arborescens , and C. opulens was one more than that in other species (tRNA encoded by trnN-GUU gene). At the same time, the number of protein-encoding genes and ribosomal RNA genes was consistent among the nine plants.

From the standpoint of gene contents, the nine plants had the highest number of protein-encoding genes, which accounted for approximately half of the entire genome length. Following the most abundant genes were tRNA genes, which were shorter in length than other genes. In general, the sequence length and gene content of chloroplast genomes in the nine Caragana species were roughly consistent. We also analyzed the difference in GC content among the three types of genes. The GC content of rRNA genes was highest, exceeding 50% and were consistently so. GC content of tRNA genes were next in amount. The lowest GC content was observed for protein-coding genes, which was approximately 37%. Moreover, the average GC content of the nine species were around 34%, which suggested that the sequence of Caragana species was relatively conserved during the process of evolution.

The genes encoded by the chloroplast genomes of C. tibetica and C. turkestanica can be divided into three categories, similar to other species. There were 57 genes related to self-replication, including ribosomal RNA, transfer RNA, and three subunits (large, small, and DNA-dependent RNA polymerase) that encode chloroplast RNA polymerase; 44 photosynthesis-related genes; the remaining genes were categorized as other genes and unknown genes. In the chloroplast genomes of C. tibetica and C. turkestanica , 17 genes with introns were detected. Thereinto, C. tibetica had two genes ( clpP and ycf3 ) with two introns, while C. turkestanica only had one gene ( ycf3 ) with two introns, and the remaining 15 genes ( rpl16 , rpl2 , rps12 , rpoC1 , trnA-UGC , trnG-UCC , trnI-GAU , trnK-UUU , trnL-CAA , trnV-UAC , ndhA , ndhB , petB , petD , atpF ) had only one intron (Fig. 2 , Table 3 ).

Gene contents of C. tibetica and C. turkestanica chloroplast genomes. The color of each gene is unique, and the horizontal axis indicates that each box is proportional to the size (bp) of the gene

Analyses of repeats and simple sequence repeat (SSR)

Repetitive units played a critical role in evolution of the genome by facilitating genetic variation and diversity. Through size evolution and structural rearrangements, they promoted genomic mutations and diversity, providing the opportunity for organisms to adapt to new environments and develop new functionalities [ 38 , 39 , 40 ]. In the present study, we identified the repetitive sequences present in the cp genomes of C. tibetica and C. turkestanica , and analyzed content of the two plants. In the genomes of C. tibetica and C. turkestanica , a total of 119 (length range: 30–337 bp) and 128 (length range: 30–249 bp) repetitive sequences were identified, respectively, consisting of forward (F), palindromic (P), reverse (R), and complementary (C) repeats (Additional file 1 : Table S1). In C. tibetica and C. turkestanica , repetitive analysis detected 88 and 84 forward repeats, 30 and 36 palindromic repeats, 1 and 7 reverse repeats, and 0 and 1 complementary repeats, respectively (Fig.  3 A). Among all types of repeats, the frequency of occurrence was highest for sequences with a length ranging from 30 to 49 base pairs (bp). In C. tibetica , there were 44 forward repeats, 27 palindromic, and 1 reverse repeats with lengths ranging from 30 to 49 base pairs (Fig.  3 B-D). Similarly, in C. turkestanica , 53 forward repeats, 27 palindromic, 7 reverse repeats and 1 complementary repeats were 30–49 bp in length (Fig.  3 B-D).

Repeat sequences analysis of 9 Caragana cp genomes. A The total number of four types of repeat sequences in 9 Caragana species; B The frequency of forward repeats by length; C The frequency of palindrome repeats by length; D The frequency of reverse repeats by length

In addition, 129, 229, 80, 259, 178, 380, and 127 repeats were found in the reported C. arborescens , C. opulens , C. jubata , C. rosea , C. microphylla , C. kozlowii , and C. korshinskii cp genomes, respectively (Additional file 1 : Table S1, Fig.  3 A). This finding suggested that C. tibetica and C. turkestanica have a higher degree of similarity in repeat frequency with C. arborescens and C. korshinskii .

Simple Sequence Repeat (SSR) loci exhibit extensive and highly variable polymorphism within the genome. As a result, SSRs are considered as effective molecular markers for investigating genetic variations and individual genetic relationships within the genome [ 41 , 42 , 43 ]. In this study, we identified intact SSRs in the chloroplast genomes of C. tibetica and C. turkestanica together with seven additional Caragana species (Fig. 4 A-C). Based on the propensity of SSRs with 10 bp or longer to undergo slippage and mismatch in the DNA chain, specific parameters have been set to address this phenomenon, which is considered the primary mechanism for SSR polymorphism [ 43 ]. We detected a total of 27 types in the two Caragana plants, using software MISA (Fig. 4 A). Among these, 239 and 277 SSRs loci were detected in C. tibetica and C. turkestanica . Similarly, we found 277 SSRs in C. arborescens , 265 SSRs in C. opulens , 281 SSRs in C. jubata , 261 SSRs in C. rosea , 275 SSRs in C. microphylla , 287 SSRs in C. kozlowii , and 279 SSRs in C. korshinskii (Additional file 1 : Table S2). The SSRs in these chloroplast genomes were mainly composed of mononucleotide and trinucleotide repeats motifs. The mononucleotide repeats (A/T and C/G) varied from 150 (62.76%) in C. tibetica to 168 (63.64%) in C. opulens , while varying from 68 (28.45%) in C. tibetica to 86 (31.16%) in C. microphylla for trinucleotide repeats (AT/AT and AG/CT) (Fig. 4 B, Additional file 1 : Table S2). Among them, A/T repeat sequences were the most abundant SSRs. There were 146 and 158 SSRs containing A or T, respectively, in the sequenced species, while only 1 contains C or G.

Statistics of SSRs detected in the plastome of nine Caragana species. A Number of SSRs determined in different repetition types; B The amount of different SSR types found in nine Caragana species genomes; and C The number of SSRs were found in coding (CDS), and intronic regions, intergenic (IGS), Respectively

In addition, there were two pentanucleotide repeats in C. jubata , with one present in C. opulens , C. microphylla , C. kozlowii and C. korshinskii , one hexanucleotide repeats was found in C. jubata , C. rosea and C. kozlowii using our identification criteria (Fig. 4 B, Additional file 1 : Table S2). Furthermore, we analyzed the distribution of SSRs in the coding and non-coding regions. The results showed that the number of SSRs in protein-coding regions was significantly lower compared to the non-coding regions (Fig. 4 C). We have discovered that the clpP gene in C. tibetica contains the longest simple repeat sequence, which was a mononucleotide repeat sequence with a length of 49 bp, whereas, the longest SSR was found on the ycf1 gene in C. turkestanica , and it was a single nucleotide repeat sequence with a length of 46 bp ( Additional file 1 : Table S3).

Codon usage bias analysis

During the process of biological evolution, there is a widespread codon usage bias observed in plastids. By analyzing codon usage bias, it is possible to uncover phylogenetic relationships between organisms and molecular evolution of genes, thereby providing potential insights into the origins, mutation patterns, and evolution of species [ 44 , 45 ]. We have compiled the codon usage information for the protein-coding sequences of nine species (Additional file 1 : Table S4). C. tibetica and C. turkestanica presented the 63 RSCU, and composed of 21,965 and 22,035 codons. There were 21,998 ( C. arborescens ), 22,035 ( C. opulens ), 21,998 ( C. jubata ), 41,710 ( C. rosea ), 40,657 ( C. microphylla ), 41,030 ( C. kozlowii ) and 40,552 ( C. korshinskii ) codons, respectively. Leucine was the amino acid with the highest quantity (2,333 codons in C. tibetica , 2,347 codons in C. turkestanica , 2,327 codons in C. arborescens , 2,347 codons in C. opulens , 2,331 codons in C. jubata , 4,584 codons in C. rosea , 4,195 codons in C. microphylla , 4,270 codons in C. kozlowii and 4,116 codons in C. korshinskii ), while the least prevalent were cysteine (259 codons in C. tibetica , 261 codons in C. turkestanica , 257 codons in C. arborescens , 261 codons in C. opulens , 258 codons in C. jubata ) and Tryptophan (656 codons in C. rosea , 670 codons in C. microphylla , 630 codons in C. kozlowii and 639 codons in C. korshinskii .

Meanwhile, we also calculated the Relative Synonymous Codon Usage (RSCU) values to assess the codon usage preference in nine Caragana species. Using a threshold of 1, codons with RSCU values over 1 were considered as optimal codons. 30 preferred and 32 non-preferred (RSCU < 1.00) codon usages were detected in five species, which were C. tibetica (Additional file 2 : Fig. S1), C. turkestanica (Additional file 2 : Fig. S2), C. arborescens (Additional file 2 : Fig. S3), C. opulens (Additional file 2 : Fig. S4), and C. jubata (Additional file 2 : Fig. S5), 28 preferred and 33 non-preferred in C. rosea (Additional file 2 : Fig. S6) and C. korshinskii (Additional file 2 : Fig. S7), 29 preferred and 32 non-preferred in C. microphylla (Additional file 2 : Fig. S8), 31 preferred and 30 non-preferred in C. kozlowii (Additional file 1 : Table S4, Additional file 2 : Fig. S9). Furthermore, the RSCU values for most A/U-ending codons were > 1, while C/G-ending codons were < 1 (Additional file 1 : Table S2).

Sequence divergence analysis

To reveal the conservative character and divergence in Caragana species, we used mVISTA to compare the plastid sequences of C. tibetica and C. turkestanica studied in this paper and other seven species of Caragana plants that have been reported. The annotated chloroplast genome sequence of C. jubata served as the reference sequence (Fig.  5 ). The results revealed a high degree of similarity in the nine plastid genome sequences. However, the sequences were found to exhibit differences in the intergenic spacer (IGS) regions of certain genes, such as matK-rbcL , psbD-psbM , atpA-psbI , and etc. In addition, most of the protein-coding gene sequences were highly conserved, except for a few genes ( rpoC2 , accD , ycf2 , and ycf1 ). Furthermore, compared to the non-coding regions, the coding regions were more conserved. This suggested that the rapidly evolving regions in Caragana genus were located in IGS.

The chloroplast genome of nine Caragana species were compared by mVISTA. The gray arrow in the figure indicates the direction of gene translation; The x-axis represents the coordinates in the chloroplast genome; The y-axis represents the percentage between 50 and 100%; Blue indicates protein coding (exon); Light green indicates untranslated region (UTR); Orange indicates conserved non-coding sequences (CNSs)

We utilized the Mauve software to analyze the chloroplast DNA rearrangements in nine species of the genus Caragana . The alignment results revealed a high degree of consistency in the types, numbers, and arrangements of coding genes, including CDS, tRNA, and rRNA, across these plants, with no structural inversions or gene rearrangement events observed (Fig.  6 ).

Comparative genomic analysis of chloroplast genome of nine Caragana species. Note: Color bands represent genes, and different colors represent different blocks. The squares with the same color between different genes represent homologous regions. In each block, the similarity map of the genome sequence was drawn by Mauve software, and the height of the similarity map corresponded to the average conservative level of the genome sequence region. The two rows below the color band represent the gene. The upper side is on the positive chain, and the lower side is on the complementary chain. The white squares represent CDS, the thin lines in the white squares represent introns, and the green and red squares represent tRNA and rRNA

Then, we utilized DNAsp6 to detect nucleotide diversity, and identified highly mutated regions in the chloroplast genomes of nine Caragana species. The pi values range from 0 to 0.11847, with an average of approximately 0.01257 (Fig.  7 ), indicating significant differences among the sequences. We have identified five regions that were most likely to be variable, including rps2-atpI (π = 0.11847), accD-psaI-ycf4 (π = 0.05819), cemA-petA (π = 0.04949), psbN-psbH (π = 0.04144) and rpoA-rps11 (π = 0.04065). Among these regions, the rps2-atpI region had the highest π value (0.11847).

Nucleotide variability (π) values of nine Caragana plants. The linear gene graph spectrum of Caragana species is given below

Phylogenetic analysis

To investigate the phylogenetic positions of 9 species of genus Caragana in the family Fabaceae, we conducted phylogenetic analysis using Maximum Likelihood (ML) and Bayesian Inference (BI) methods. Except for the genus Caragana , the remaining 8 genus included Wisteria (1), Glycyrrhiza (2), Astragalus (1), Calophaca (1), Cicer (1), Medicago (3), Trifolium (3), and Lathyrus (4). The number in parentheses represents the number of species in the corresponding taxa.

The phylogenetic trees obtained from two methods showed similar topology, and the different datasets generally yielded consistent phylogenetic trees with strong support values. The phylogenetic analysis revealed that all samples were divided into three major branches. Four pairs of species showed closer relationships: C. tibetica and C. jubata , C. rosea and C. opulens , C. microphylla and C. korshinskii , and C. turkestanica and C. arborescens (Fig.  8 ). Notably, the close relationship between the genus Astragalus and genus Caragana (bootstrap: 100%) belonging to the Subtribe Astragaliinae was worth mentioning.

A phylogenetic tree based on the chloroplast genomes of 24 Fabaceae family and one outgroup Arabidopsis thaliana was constructed using BI and ML methods. The numbers following the nodes represent bootstrap values. GenBank accession numbers were provided after each species. C. tibetica and C. turkestanica were highlighted in red

This study presented the assembly and annotation of two complete chloroplast genomes. By analyzing these genomes, we gained in-depth insights into the chloroplast genomes of the Caragana genus and conducted comparative studies on seven previously reported Caragana species.

Reports indicate that researcher, by analyzing characteristics such as morphological features, chromosomes, and pollen morphology of 72 species within the Caragana genus and employing cladistic methods, successfully classified these species into 12 series and 5 groups [ 46 ]. However, this classification method based on morphology could be influenced by environmental changes and convergent evolution, leading to potential discrepancies in the classification outcomes. With the advancement of molecular marker technology, researcher further utilized DNA sequences from the rbcL gene, trnS-trnG introns and spacer regions, and the ITS region to study the phylogenetic relationships between 12 Caragana species and 48 other legume plants [ 47 ]. Although molecular markers have improved the accuracy of classifications in certain aspects, their limitations still exist, preventing the resolution of some evolutionary disputes. Given the chloroplast genome’s low mutation rate, ease of sequencing, and high sequence conservation, it has become a crucial tool for analyzing genetic differences among closely related species, helping to overcome the limitations of traditional methods and enhance classification accuracy [ 48 ].

In contrast to the majority of angiosperms, both C. tibetica and C. turkestanica , along with previously reported Caragana species, showed a notable absence of the inverted repeat (IR) region. This absence leaded to an unclear demarcation between the large single-copy (LSC) and small single-copy (SSC) regions. For example, C. rosea , C. microphylla , C. intermedia , C. jubata , C. erinacea , C. opulens , and C. bicolor [ 1 , 16 , 17 , 19 ]. Comparative analysis of chloroplast genomes within the Caragana genus was conducted. The lengths of the chloroplast genomes varied to some degree among the nine Caragana plants, with the longest being 133,122 bp in C. rosea and the shortest being only 128,132 bp in C. jubata . C. tibetica and C. turkestanica encoded 110 and 111 genes respectively, including 76/76 protein-coding genes, 4/4 rRNA genes, and 30/31 tRNA genes. In this study, we identified a unique gene, trnN-GUU , in the genome of C. turkestanica , which was missing in the genome of C. tibetica . The trnN-GUU gene encoded a tRNA that transports valine, representing a unique sequence within C. turkestanica ’s genome. Through codon usage bias analysis, we discovered that the RSCU of valine-related codons encoded by trnN-GUU in C. turkestanica genome exceeds 1, indicating that the use of valine in relation to trnN-GUU may be elevated during protein synthesis. In contrast, no homologous sequence for the trnN-GUU gene was identified in C. tibetica genome, suggesting that the two subspecies may have different evolutionary histories and paths of adaptive evolution. The presence or absence of trnN-GUU in the genomes of C. turkestanica and C. tibetica , respectively, may reflect their divergent evolutionary branches. Future research should investigate the functional role of the trnN-GUU gene in the adaptive evolution of C. turkestanica , as well as its evolutionary importance in adaptation to various environments. Additionally, 239 and 277 SSRs were found to be randomly distributed in their genomes. Furthermore, approximately 88/84 forward repeats, 30/36 palindromic repeats, 1/7 reverse repeats, and 0/1 complementary repeats were identified in both cp genomes. We have identified 17 genes containing introns in both chloroplast genomes, with 15 genes containing 1 intron each in C. tibetica (16 in C. turkestanica ), while the genes clpP and ycf3 contained 2 introns in C. tibetica , C. turkestanica only had one gene ycf3 contain 2 introns. Introns play a significant role in gene expression regulation, and recent studies have indicated that different introns can enhance the expression of exogenous genes at specific locations [ 49 , 50 , 51 ]. In transgenic mice, the addition of an intron from the rabbit-globin gene was found to enhance the expression of the human growth hormone gene, leading to increased levels of the hormone [ 52 ]. Similarly, research indicates that deleting the introns of the Drosophila alcohol dehydrogenase ( Adh ) gene leads to reduced expression levels of the Adh gene, whether measured by enzymatic activity or RNA levels. This accentuates the important role of introns in the regulation of gene expression [ 53 ]. It was found that ycf2 [ 54 ], rpl23 , and accD were frequently missing in some plants, but they were detected in chloroplast genome of Caragana plants reported in this paper [ 55 , 56 , 57 ]. Previous research has indicated that specific Caragana species’ chloroplast genome, including C. kozlowii , C. korshinskii , C. microphylla , and C. rosea , exhibit a loss of genes such as rps16 , infA , rpl22 , and ycf15 [ 1 ]. Similarly, this study also did not find the rps16 , infA , rpl22 , and ycf15 loci in C. tibetica and C. turkestanica . Among these genes, infA is an unusually unstable flowering plant chloroplast gene, whereas rpl22 encodes the ribosomal protein CL22 and has been eliminated from the chloroplast genome, relocating to the nucleus [ 58 , 59 ]. Nonetheless, recent studies have discovered that the infA gene is present in C. jubata , C. erinacea , C. opulens , and C. bicolor [ 4 ]. These findings strongly indicated that the infA gene might not be lost in some Caragana species. Due to the scarcity of available chloroplast genomic data for Caragana , it is imperative to conduct extensive experimental research in order to validate these conclusions.

A total of 119 and 128 repeats were detected in the chloroplast genomes of C. tibetica and C. turkestanica , respectively. These repeats include forward, palindromic, reverse, and complementary sequences. These repeats serve as crucial genetic markers and are closely associated with species emergence and development [ 60 ]. Repeat sequences are highly valuable in phylogenetic investigations and also contribute to genome rearrangements [ 61 , 62 ]. Furthermore, the analysis of various cp genomes has established the vital role of repeat sequences in indel and substitution events [ 63 ]. Moreover, no rearrangements have been observed in the plastids of C. tibetica and C. turkestanica . Previous studies have reported the absence of the IR region in several Caragana species, such as C. microphylla , C. bicolor , and C. jubata [ 1 , 16 ]. Similarly, the cp genomes of C. tibetica and C. turkestanica were found to lack the IR region in this study. Additionally, the G/C content of the chloroplast DNA (cpDNA) is crucial for determining inter-specific affinity [ 16 ]. The DNA G/C content of the two Caragana species discussed in this paper is highly alike. Additionally, SSRs are regarded as vital molecular markers for analyzing genetic variation within populations, and they are widely employed in assessing phylogenetic relationships, evolution, and genetic diversity [ 64 ]. A total of 239 to 277 SSRs were found in the chloroplast genomes of two Caragana plants, exhibiting a significant bias towards A/T. The majority of SSR types consist of single nucleotide repeats, and the highest number of SSRs was observed in the non-coding regions (IGSs) of the cp genomes. These SSRs are valuable starting points for the development of genetic markers in Caragana species, and their utilization is applicable in phylogenetic and ecological research.

Codon usage preferences are known to reflect the species of origin and the mutational model. Analyzing patterns of codon usage bias in chloroplast genomes can provide insights into plant phylogenetic relationships, gene expression mechanisms, and molecular evolution [ 44 ]. Leucine (Leu) is the most abundant amino acid in C. tibetica , C. turkestanica , and other Caragana species. Furthermore, our research found that the majority of synonymous codons preferred for RSCU values end with A/U, thereby contributing to a higher AT content in genes. Based on our analysis, we hypothesize that natural selection and gene mutation may be responsible for this codon usage pattern. Nevertheless, it is important to note that codon preference and utilization patterns provide only a partial reflection of the evolutionary relationship between species, and further research is necessary.

The plastid genome contains numerous variable nucleotides, which can be utilized as valuable DNA barcodes for determining the relationships between species or genera [ 65 , 66 ]. We simultaneously identified five intergenic spacer regions (IGSs) with relatively high differentiation values: rps2-atpI , accD-psaI-ycf4 , cemA-petA , psbN-psbH , and rpoA-rps11 . These variable regions have the potential to function as DNA barcodes for studying phylogenetic relationships, species identification, and population genetics research. Next, we compared the sequence variations of the nine assembled Caragana plants. Comparative analysis of the chloroplast genomes confirmed that the coding regions were more conserved than the non-coding regions, which is consistent with findings from other Caragana species [ 16 ].

Recently, the chloroplast genome has become a preferred option for studying the phylogenetic relationships of diverse plant species. For instance, the utilization of the complete chloroplast genomes from three Lycoris plants in phylogenetic analysis revealed a strongly supported relationship between Lycoris plants and the Narcissus genus [ 67 ]. Phylogenetic analysis of 23 Swertia plant species revealed that Swertia is paraphyletic rather than monophyletic [ 68 ]. The phylogenetic position of C. tibetica and C. turkestanica within Fabaceae was determined by constructing a comprehensive genomic dataset composed of 66 genes shared among 24 representatives from nine genera. The phylogenetic analysis indicated that Caragana species constituted a distinct clade, with C. microphylla and C. korshinskii showing a closer genetic relationship, which aligns with earlier findings [ 1 , 16 ]. The phylogenetic relationships inferred from the chloroplast genome offer novel insights and perspectives to advance our understanding of plant evolution.

For the first time, we assembled and analyzed the complete chloroplast genomes of C. tibetica and C. turkestanica , and compared them to other members of the Caragana genus. Our findings revealed that the sizes of their genomes, gene compositions, gene arrangements, GC content, and codon usage were similar to previously documented chloroplast genomes within the Caragana genus. Additionally, this study identified the positions and distributions of repetitive sequences in both species, and determined the sequence variability and nucleotide variation sites. The results of this study provide guidance for future studies on the phylogenetic evolution and species identification of the Caragana genus, as well as the development of new molecular markers. Ultimately, this discovery contributes to the augmentation of the chloroplast genome database for the Caragana genus.

Our study presents the initial assembly and annotation of C. tibetica and C. turkestanica and compares them with seven other Caragana species. Due to the absence of a pair of IRs, the plastomes of C. tibetica and C. turkestanica were shorter, with sizes ranging from 128,433 bp to 129,453 bp. The long repeats, SSR loci, and certain genes found in the IGS region ( matK-rbcL , psbD-psbM , atpA-psbI ) as well as five regions exhibiting high variability ( rps2-atpI, accD-psaI-ycf4, cemA-petA, psbN-psbH , and rpoA-rps11 ) identified in our study will promote future research efforts. These efforts include the development of new molecular markers and investigations of population genetics and phylogenetic analysis. By analyzing the sequence and structural information of the chloroplast genomes of two Caragana plants comprehensively, we have determined their genetic evolutionary position and relationships with other Caragana species. This information establishes a foundation for extensive and detailed research on the identification, genetic diversity, and phylogenetics of Caragana species. Furthermore, our study has significantly contributed to the enrichment of the chloroplast genome database for Caragana plants.

Plant material, DNA extraction and sequencing

The fresh and young leaves of C. tibetica , and C. turkestanica were gathered from eastern part of Qinghai Province (N36°43′24.80′′, E101°44′54.11′′), China. During outdoor sampling, the leaf tissues were temporarily stored in a low-temperature insulated box. After returning to the laboratory, the samples were immediately placed in a -80℃ ultra-low temperature freezer for storage. We used a modified cetyltrimethylammonium bromide (CTAB) [ 69 ] method to extract DNA from fresh tissue samples of Caragana plants. Ultrasound was used to fragment the DNA fragments, and the fragment size was selected by agarose gel electrophoresis. The selected fragments were amplified by PCR to form a sequencing library, and the qualified library was sequenced using the Illumina NovaSeq platform, with 150 bp pair-end reads.

Gene annotation, genome assembly and sequence analyses

Before assembly, we conducted a rigorous preprocessing of the raw data. The raw data were filtered using the Trimmomatic v 0.39 [ 70 ] tool to remove low-quality data. After that, we used SPAdes v3.10.1 ( http://cab.spbu.ru/software/spades/ ) [ 71 ] to assemble chloroplast genome sequences to obtain their SEED sequences, K-mer analysis was conducted on the seed sequence to obtain Contigs. We employed SSPACE v2 [ 72 ] for the assembly of contig sequences into scaffolds. Subsequently, Gapfiller v2.1.1 [ 73 ] was utilized to resolve any gaps within these scaffolds, thereby enhancing the coherence and completeness of the pseudo-genome assembly. Then, based on the structure of the chloroplast genome, the corrected pseudo genome sequences were reordered and aligned, resulting in two complete circular chloroplast genome sequences. After assembly, quality control of the final sequence was performed using the reference sequence for C. kozlowii in this study. The annotation information for the CDS, rRNA, and tRNA sequences in the chloroplast genome were gained using Blast v2.2.25 ( https://blast.ncbi.nlm.nih.gov/Blast.cgi ), hmmer v3.1b2 [ 74 ] ( http://www.hmmer.org/ ), and ARAGORN v1.2.38 [ 75 ] ( http://130.235.244.92/ARAGORN/ ) software, respectively. The chloroplast genome maps for C. tibetica and C. turkestanica were plotted by the online tool Chloroplot in OGDRAW [ 76 ]. Finally, the annotated chloroplast genome sequences of the genus Caragana were submitted to GenBank using the online submission tool BankIt, and the accession numbers OQ942026 and OQ942027 were obtained, respectively.

Repeats, simple sequence repeats and codon usage bias analysis

Vmatch [ 77 ] was chosen to search for forward, reverse, palindromic, and complementary repeats in the chloroplast genome sequences of Caragana species. Additionally, simple sequence repeats (SSRs) in this study were identified by MISA [ 78 ] with the following parameter settings: mono-nucleotide set as 8, di-nucleotide set as 5, tri-nucleotide set as 3, tetra-nucleotide set as 3, penta-nucleotide set as 3, and hexa-nucleotide set as 3, respectively. The program CodonW1.4.2 [ 79 ] was applied to calculate relative synonymous codon usage (RSCU) values of protein-coding genes under default settings.

Sequence divergence and comparative genome analysis

The chloroplast genome sequences of Caragana species were aligned using MEGA7 [ 80 ], and DnaSP6 [ 81 ] was used to calculate nucleotide diversity (π) values with the following parameter settings: window length of 600 bp and step size of 200 bp, which are commonly used in the literature. Comparison and visualization of complete Caragana chloroplast genomes using mVISTA [ 82 ] program (Shuffle-LAGAN mode). C. jubata plastome was labeled as reference. Utilizing Mauve software [ 83 ], an analysis was conducted on the chloroplast DNA rearrangements of nine species within the genus Caragana , aiming to identify changes in gene order, potential large-scale sequence rearrangements, and local tandem duplication events. This step employed the software’s recommended default parameters.

To determine the phylogenetic positions of Caragana species in this study, we downloaded plastid genome sequences of 24 legume species which belong to the IRLC, and one outgroup ( Arabidopsis thaliana ) from NCBI. Phylogenetic trees were constructed using PhyloSuite v1.2.2 [ 84 ]. First, we extracted 66 common protein-coding gene sequences from each of 25 chloroplast genomes. These protein gene sequences were then aligned in batches using the MAFFT [ 85 ] with auto strategy and normal alignment mode. This study constructed phylogenetic trees based on the chloroplast genome using two methods: Bayesian inference (BI) and maximum likelihood (ML). In the Bayesian Inference (BI) analysis, we employed Modelfinder [ 86 ] to determine the most suitable model, opting for the GTR + F + I + G4 nucleotide substitution model. Subsequently, phylogenetic trees were inferred using Bayesian inference [ 87 ], leveraging a partition model to enhance the accuracy of our findings. It runs in parallel with one execution, performing a total of 1,000,000 generations. During the analysis, the initial 25% of sampled data is discarded as a burn-in period. To ensure the convergence of the Markov Chain Monte Carlo (MCMC) algorithm, these samples are excluded from the final analysis. Additionally, the average standard deviation of split frequencies is set to a threshold greater than 0.01 [ 88 ]. In an evolutionary tree constructed using BI, a value of 1 represents the highest probability or maximum posterior probability for support, indicating that a particular structure or branch is highly reliable given the data and model. For ML, the IQ-TREE [ 89 ] tool was used, with automatic partition selection and 1,000 ultrafast bootstrap [ 90 ]replicates performed to assess the confidence of each branch. And we set the threshold for Bootstrap values at 70% as the criterion for evaluating support. Therefore, when the Bootstrap value of a particular clade reaches or exceeds 70%, it is considered to have strong support.

Availability of data and materials

The original sequencing data have been submitted to the NCBI database and received GenBank accession numbers OQ942026 ( C. tibetica ), OQ942027 ( C. turkestanica ). The data used in this study are already entirely in the public domain ( https://www.ncbi.nlm.nih.gov ). Voucher specimens of C. tibetica and C. turkestanica are stored in the herbarium of the College of Eco-Environmental Engineering at Qinghai University. The voucher specimen number for C. tibetica is QhST20190080, and number QhST20190081 for C. turkestanica .

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Acknowledgements

We would like to thank Dr. NH and Dr. JS for their help in researching the materials.

Our experimental research and field studies on plants comply with relevant institutional, national, and international guidelines and legislation.

National Natural Science Foundation (No. 32160386) of China and Qinghai Innovation Platform Construction Project.

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LL designed and executed experiments, completed data analysis, and wrote the first draft of the paper. HL, XL, and XL contributed to the experimental design and analysis. NH and JS assisted in sample collection and species identification. WZ was the project developer and leader, guiding the experimental design, data analysis, and paper writing and revision. All authors reviewed the manuscript.

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C. tibetica and C. turkestanica were collected in September 2019 from non-private land, and anyone is permitted to collect these wild plants for research purposes without causing ecological harm. Voucher specimens of C. tibetica and C. turkestanica are stored in the herbarium of the College of Eco-Environmental Engineering at Qinghai University. The botanical identification was performed by the corresponding author, Dr. Zhou. The voucher specimen number for C. tibetica is QhST20190080 and for C. turkestanica is QhST20190081.

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Supplementary Information

Additional file 1: table s1..

Types and numbers of Repeats in chloroplast genome of 9 Caragana spices. Table S2. Types and numbers of SSR in chloroplast genome of 9 Caragana spices. Table S3. Distribution of SSRs in cp genome of C. tibetica and C.turkestanica . Table S4. Analysis of coding ability and codon preference of chloroplast genome of C. tibetica and C.turkestanica .

Additional file 2: Fig. S1.

Amino acid frequencies of the chloroplast genomes of C. tibetica. The squares below represent all the codons that encode each type of amino acid; the height of the column above represents the total sum of RSCU values for all codons; the height of each column represents the RSCU value for each codon. Fig. S2. Amino acid frequencies of the chloroplast genomes of C. turkestanica. The squares below represent all the codons that encode each type of amino acid; the height of the column above represents the total sum of RSCU values for all codons; the height of each column represents the RSCU value for each codon. Fig. S3. Amino acid frequencies of the chloroplast genomes of C. arborescens. The squares below represent all the codons that encode each type of amino acid; the height of the column above represents the total sum of RSCU values for all codons; the height of each column represents the RSCU value for each codon. Fig. S4. Amino acid frequencies of the chloroplast genomes of C. opulens. The squares below represent all the codons that encode each type of amino acid; the height of the column above represents the total sum of RSCU values for all codons; the height of each column represents the RSCU value for each codon. Fig. S5. Amino acid frequencies of the chloroplast genomes of C. jubata. The squares below represent all the codons that encode each type of amino acid; the height of the column above represents the total sum of RSCU values for all codons; the height of each column represents the RSCU value for each codon. Fig. S6. Amino acid frequencies of the chloroplast genomes of C. rosea. The squares below represent all the codons that encode each type of amino acid; the height of the column above represents the total sum of RSCU values for all codons; the height of each column represents the RSCU value for each codon. Fig. S7. Amino acid frequencies of the chloroplast genomes of C. microphylla. The squares below represent all the codons that encode each type of amino acid; the height of the column above represents the total sum of RSCU values for all codons; the height of each column represents the RSCU value for each codon. Fig. S8. Amino acid frequencies of the chloroplast genomes of C. kozlowii. The squares below represent all the codons that encode each type of amino acid; the height of the column above represents the total sum of RSCU values for all codons; the height of each column represents the RSCU value for each codon. Fig. S9. Amino acid frequencies of the chloroplast genomes of C. korshinskii. The squares below represent all the codons that encode each type of amino acid; the height of the column above represents the total sum of RSCU values for all codons; the height of each column represents the RSCU value for each codon.

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Liu, L., Li, H., Li, J. et al. Chloroplast genomes of Caragana tibetica and Caragana turkestanica : structures and comparative analysis. BMC Plant Biol 24 , 254 (2024). https://doi.org/10.1186/s12870-024-04979-9

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Research Review and Literature Perception Towards Medicinal Plants Classification Using Deep Learning Techniques

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• Himanshu Kumar Diwedi 12 ,
• Anuradha Misra 12 ,
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The pharmaceutical industry is paying more attention to medicinal plants since they are less likely to cause undesirable responses and are less expensive than current medicines. These features have attracted researchers interest in automatic medicinal plant recognition. There are several ways to improve the powerful medicinal plant classifier that can be useful in real-time classification. This work uses several reliable and effective deep-learning techniques for classifying plants based on leaf images. The recent research work is collected systematically and categorized based on their techniques. Further, the dataset details, performance measures used for evaluation, tools utilized for implementation, and chronological review of the collected works are discussed. Finally, this research is concluded with an analysis of eminent ongoing opportunities and research perspectives for improvement in this domain.

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Diwedi, H.K., Misra, A., Tiwari, A.K., Mahmood, A. (2023). Research Review and Literature Perception Towards Medicinal Plants Classification Using Deep Learning Techniques. In: Borah, S., Gandhi, T.K., Piuri, V. (eds) Advanced Computational and Communication Paradigms . ICACCP 2023. Lecture Notes in Networks and Systems, vol 535. Springer, Singapore. https://doi.org/10.1007/978-981-99-4284-8_21

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