Endophytic Fungi Residing in Medicinal Plants Have the Ability to Produce the Same Or Similar Pharmacologically Active Secondary Metabolites As Their Hosts

Hellenic Plant Protection Journal 10: 51-66, 2017 DOI 10.1515/hppj-2017-0006

REVIEW ARTICLE

Endophytic fungi residing in medicinal plants have the ability to produce the same or similar pharmacologically active secondary metabolites as their hosts

A. Venieraki, M. Dimou and P. Katinakis*

Summary Medicinal plants have been used for thousands of years in folk medicines and still are used for their health benefi ts. In our days medicinal plants are exploited for the isolation of plant-derived drugs as they are very eff ective and have relatively less or no side eff ects. However, the natural resources of medicinal plants are gradually exhausted and access to plant bioactive compounds is challenged by the low levels at which these products accumulate in native medicinal plants. For in- stance, to meet the market demands of 3 Kg per year of vinca alkaloids, powerful plant-derived anticancer drugs, 1.5x106 Kg dry leaves are required. In this regard, this review aims to highlight the fact that endophytic fungi residing in medicinal plants are capable to biosynthesize pharmacologically active secondary metabolites similar or identical to those produced by their host medicinal plant. Fur- thermore, the evolutionary origin of the genes involved in these metabolic pathways as well as the approaches designed to enhance the production of these metabolites by the isolated endophytic fungi are also discussed.

Additional key words: metabolites from endophytic bacteria and actinomycetes, chemical ecology

Introduction present in virtually all organs of a given plant host, and some are seed borne. Endophytes Plant endophytes consist of bacterial and often confer considerable benefi ts to the fungal communities that colonize and spend host plant they inhabit, since they can pro- the whole or part of their life cycle inside mote the growth of host plants, enhance re- the plant tissues, without instigating any sistance to biotic and abiotic stresses (Rodri- noticeable symptoms of infection or visible guez et al., 2009; Hardoim et al., 2015), and manifestation of disease to their hosts (Pe- accumulate bioactive secondary metabo- trini and Fisher, 1990). Evidence of plant-as- lites (Kusari et al., 2012). The ecological role sociated microorganisms found in the fossil- of secondary metabolites produced by en- ized tissues of land plants stems and leaves dophytes is not clear. However, recent stud- suggests that endophyte–plant associa- ies have shown that these metabolites are tions may have evolved along with the evo- involved in deterrence of herbivory (Pannac- lution of higher land plants (Krings et al., cione et al., 2014), protection against fungal 2007). Nearly all vascular plant species stud- (Soliman et al., 2015) or bacterial pathogens ied were found to harbor endophytic bac- (Mousa et al., 2017) and amelioration of plant teria and/or fungi (Rodriguez et al., 2009; abiotic stress (Hamayum et al., 2016). Hardoim et al., 2015). They are found to be Bioactive secondary metabolites derived from medicinal plants are Laboratory of General and Agricultural Microbiology gradually decreasing - Alternative Department of Crop Science, Agricultural University approaches for their production of Athens, Iera Odos 75, GR 118 55 Votanikos, Athens, Greece * Corresponding author: [email protected] Medicinal plants, as a rich source of nat-

© Benaki Phytopathological Institute 52 Venieraki et al. ural products, have been used to treat vari- this review, we aim to show that the large ous ailments and have been the foundation number of medicinal plants used for the for discovery and development of modern isolation of medically important bioactive therapeutics (Pan et al., 2013). Up to 80 % of compounds harbor endophytic fungi capa- people in developing countries are totally ble of host-independent biosynthesis of the dependent on herbal drugs for their prima- same or similar bioactive secondary metab- ry healthcare. More than 51% of small mole- olites as their hosts. This review will also dis- cule drugs approved between 1981 and 2014 cuss the evolution and origin of pathways were based on natural products, the rest be- involved in the biosynthesis of these bioac- ing synthetic (Chen et al., 2016). With the in- tive compounds and potential approaches creasing demand for herbal drugs, natural aiming to enhance their production. health products and secondary metabolites, the use of medicinal plants is growing rapid- ly throughout the world (Chen et al., 2016). Medicinal plants harbor endophytic However, we are facing the accelerated loss fungi capable of mimicking their host of wild medicinal plant species; one third plant secondary metabolite profi le- of the estimated 50.000-80.000 medicinal Case studies on medicinal plants plant species are threatened with extinction producing metabolites of known from overharvesting and natural anthropo- medical importance genic habitat destruction (Chen et al., 2016). Furthermore, the feasibility of access to Since the fi rst report of endophyte Tax- plant bioactive compounds is challenged by omyces andreanae that produces the same the low levels at which these products accu- bioactive secondary metabolite taxol (pacli- mulate in native medicinal plants, the long taxel) as its host Taxus brevifolia in 1993 (Sti- growth periods required for plant matura- erle et al., 1993), several studies have shown tion, and the diffi culty in their recovery from that plant-derived secondary metabolites other plant-derived metabolites (Staniek et are produced by endophytes (Zhao et al., al., 2014). For example, the taxol concentra- 2011). In this section, we will present a liter- tion is about 0.001–0.05% in Taxus brevifolia, ature survey aiming to show that medicinal which is the most productive species. Thus, plants used for isolation of medically impor- 15 kg of Taxus bark, three trees, are required tant secondary metabolites usually harbor for production of 1 g, while every cancer pa- endophytic fungi which are capable of host- tient requires about 2.5 g (Malik et al., 2011). independent biosynthesis of these metabo- Therefore, it is important to fi nd alter- lites. In each one of the presented case stud- native approaches to produce the medici- ies, emphasis will be placed on the plant nal plant-derived biologically active com- species, the organ where the bioactive compounds, in particularly those derived from pound is accumulated and the organ from endangered or diffi cult-to-cultivate plant which the active compound-producing fun- species, to meet the medical demand. This gi were isolated. can be achieved by the application of plant cell and tissue culture, heterologous pro- Salvia sp. (Lamiaceae) duction, total chemical synthesis, semi-syn- Salvia species have many important thesis, or by starting with a microbially - pro- medicinal properties with proven pharma- duced or plant-extracted natural product cological potential. Some of these proper- occurring more abundantly in nature (Ata- ties may be mediated by biologically active nasof et al., 2015; Rai et al., 2016; Ramirez-Es- polyphenols or terpenoids (Wu et al., 2012). trada et al., 2016) or by exploiting the abili- Two kinds of bioactive compounds, tanshi- ty of endophytic fungi residing in plants to nones (tanshinone I, tanshinone IIA, tanshi- produce the same or similar bioactive com- none IIB, isotanshinone I, and cryptotanshi- pounds as their hosts (Zhao et al., 2011). In none) and salvianolic acids (salvianolic acid

© Benaki Phytopathological Institute Endophytic fungi & secondary metabolites in medicinal plants 53 and rosmarinic acid) have been found in the the endophytes for the production of vinca roots and leaves of S. miltiorrhiza, respec- alkaloids revealed that only endophytic fun- tively. Tanshinones belong to diterpenoid gi residing in the leaves of C. roseus were ca- quinones, and are considered as potent an- pable of producing vinblastine and vincris- ti-carcinogenic, antiatherosclerosis, and an- tine. These endophytic fungi were identifi ed tihypertensive, whereas salvianolic acids are as Fusarium oxysporum, Talaromyces radicus phenolic acids, which are mainly respon- and Eutypella spp. The drugs were purifi ed sible for benefi cial eff ects on cardiovascu- by TLC and HPLC and authenticated using lar and cerebrovascular diseases (Chun-Yan UV-Vis spectroscopy, ESI-MS, MS/MS and et al., 2015). Several Salvia species produce 1H NMR. Culture fi ltrates of the fungi yield- the bioactive phenolic labdane-type diter- ed >55 μg/L of vinblastine or vincristine, re- penes rosemarinic acid, carnosic acid and spectively (Kumar et al., 2013; Palem et al., carnosol. These compounds show distinct 2015; Kuriakose et al., 2016). anti-oxidant activity with carnosic acid carnosol being approved food additives (Wu et Coleus forskohlii (Willd.) Briq. (Lamiace- al., 2012). Salvia divinorum produces a novel ae) diterpenoid, salvinorin A, which is a power- Coleus forskohlii or Indian Coleus is a trop- ful hallucinogen in humans and shows a se- ical perennial shrub of the Lamiaceae fam- lective, high effi cacy agonist activity (Butel- ily and grows in the subtropical temperate man and Kreek, 2015). climates of South-east Asia and India. The Eighteen endophytic fungal strains have plant is extensively cultivated in southern been isolated from the roots of Salvia milti- India and the roots are used in Indian folk orrhizae, the site of tanshinones accumula- medicine for treating a broad range of hu- tion, and 58 fungal strains from the leaves, man health disorders (Kavitha et al., 2010). the main site of salvianolic acid accumula- The roots of the herb contain a pharmaco- tion. Liquid culture extracts of all the fungi logically active compound called forskolin were screened for the presence of tanshino- that accumulates in the root cork (Patera- nes or salvianolic acid, respectively. One fun- ki et al., 2014). The approved and potential gus in each case was proven to produce tan- applications of forskolin range from allevia- shinones or salvianolic acid compared with tion of glaucoma, anti-HIV or antitumor ac- authentic standards. However, the yield was tivities, treatment of hypertension and heart quite low; about 4μg/L for tanshinones and failure to lipolysis and body weight control 47μg/L for salvianolic acid (Ming et al., 2013; (Pateraki et al., 2017). Li et al., 2016). Screening of endophytic fungi isolated from inner tissues of root and stems of Catharanthus roseus (L.) G.Don (Apocyn- C. forskohlii for the production of forskolin aceae) revealed that one of the endophytic fun- Catharanthus roseus is well known for the gi identifi ed as Rhizoctonia bataticola was production of several anticancer vinca alka- able to stably synthesize forskolin and inter- loids such as vincristine, vindesine, vinorel- estingly, release it into the broth (Mir et al., bine, vinblastin and the recently discovered 2015). vinfl unine (Kumar et al., 2014). The two major anticancer vinca alkaloids, vincristine Macleaya cordata (Willd.) R.Br. (Papaver- and vinblastine, used in chemotherapy reg- aceae) imens, have been isolated from leaves (Ku- Sanguinarine (SA) is a benzophenanthri- mar et al., 2014). dine alkaloid isolated from Macleaya cor- The diff erent C. roseus plant organs har- data leaves, and is known to have a wide bour a plethora of endophytic fungi (Khar- spectrum of biological activities, such as war et al., 2008; Kumar et al., 2013; Palem et antibacterial, antihelmintic, antitumor and al., 2015; Kuriakose et al., 2016). Screening all anti-infl ammatory (Wang et al., 2014). SA is

© Benaki Phytopathological Institute 54 Venieraki et al. used in feed additives for livestock produc- leaves of Cephalotaxus have been used in tion (Kantas et al., 2014). Most of the SA cur- Chinese folk medicine as anticancer agents, rently used in herbal supplements and med- and its biological active constituents were icines is extracted from M. cordata. Recently, proved to be alkaloids. Among these alka- SA has gained increasing attention as a po- loids, homoharringtonine (HHT) was shown tential agent in the treatment of cancer (Yu eff ective against acute myeloid leukemia et al., 2014). and has recently been approved by the Screening of endophytic fungi isolated Food and Drug Administration for the treat- from leaves of M. cordata revealed that one ment of chronic myeloid leukemia (Hu et al., of 55 isolates has the capacity to produce SA 2016). (Wang et al., 2014). A large number of endophytic fungi have been obtained from Cephalotaxus Cajanus cajan (L.) Millsp. (Fabaceae) phloem. The bioactive compounds isolated Cajanus cajan (pigeon pea) is a grain le- from their culture extracts were character- gume crop in semitropical and tropical ar- ized as sesquiterpenoids, anthraquinones eas of the world. The extract of pigeon pea and aromatic compounds, which exhibited leaves exhibit therapeutic eff ects on sickle cytotoxic and antibacterial activities (Lu et cell anemia, plasmodiosis, and hepatic dis- al., 2012; Xue et al. 2012; Zheng et al., 2011). orders. Moreover, pigeon pea roots are used The hunt for an HHT-producing endophytic as a sedative, a vulnerary preparation. The fungus was eventually successful following active constituents of pigeon pea are fl a- the screening of 213 strains isolated from vonoids and stilbenes. Cajaninstilbene acid the bark of Cephalotaxus trees grown in Chi- (CSA) is one of the major stilbenes found in na and Thailand. The fungus was identifi ed pigeon pea. Pharmacological studies have as Alternaria tenuissima and stably produced revealed that CSA exhibited anti-infl amma- 100 μg/L HHT (Hu et al., 2016). tory and analgesic eff ects. In addition, CSA has an antioxidant activity similar to that of Cinchona spp. (Rubiaceae) the natural antioxidant resveratrol (Liang The bark of the stem and roots of vari- et al., 2013). Cajanol is a isofl avone isolat- ous trees of the genus Cinchora produce ed from pigeon pea roots. Pharmacological quinine alkaloids (quinine, quinidine, cin- studies have shown that cajanol has anti- chonidine and cinchonine), which were the plasmodial, antifungal and antimicrobial only eff ective treatment of malaria for more activities. In addition, cajanol has been de- than four centuries. Cinchona bark and its scribed as a novel anticancer agent, which alkaloids remained the most effi cient treat- induced apoptosis in human breast cancer ment of malaria until the 1940s when chlo- cells (Luo et al., 2011). roquine and other synthetic antimalarial A total of 245 endophytic fungi isolated compounds were developed (Kaufman and from roots, stems and leaves of pigeon pea Ruveda, 2005). With the development of re- plants were screened for the production of sistant malaria strains, the quest for new an- cajaninstilbene acid or cajanol. Three fungal timalarial compounds was successful with strains isolated from leaves were capable of the discovery of artemisinin from a Chinese producing CSA and one strain isolated from herbal medicine based on Artemisia annua roots stably produced cajanol at a concen- L. (Tu, 2011). tration of 500μg/L (Zhao et al., 2012; Zhao et Twenty-one endophytic fungi have been al., 2013). isolated from Cinchona ledgeriana young plant stems and screened for the presence Cephalotaxus hainanensis H.L.Li (Cepha- of Cinchora alkaloids. These fungi com- lotaxaceae) prised of Phomopsis, Diaporthe, Schizophyl- Cephalotaxus hainanensis H. L. Li is an in- lum, Penicillium, Fomitopsis and Arthrinium digenous conifer tree of China. The bark and species while fermentation studies demon-

© Benaki Phytopathological Institute Endophytic fungi & secondary metabolites in medicinal plants 55 strated that all produce quinine and quini- 3β-D-glucoside whereas a second endo- dine, as well as cinchonidine and cinchonine phytic fungus was found to secrete pei- (Maehara et al., 2011; Maehara et al., 2013). misine and peiminine. Interestingly, a large number of the remaining endophytes were Passifl ora incarnata (Passifl oraceae) able to produce large amounts of antioxi- Passifl ora consists of 500 species that dants, such rosemarinic acid (Pan et al., 2014; are found mostly in warm and tropical re- Pan et al., 2015; Pan et al., 2017). gions. Passifl ora incarnata leaves were found to contain several active compounds, in- Huperzia serrata (Thunb. ex Murray) Tre- cluding alkaloids, phenols, glycosyl fl a- vis (Huperziaceae) vonoids, and cyanogenic compounds. The Huperzia serrata is a traditional Chinese major compounds present in P. incarnata herb medicine and has been extensively are C-glycosyl fl avonoids (vitexin, isovitex- used for the treatment of a number of ail- in, orientin and chrysin) and b-carbolinic al- ments, including contusions, strains, swell- kaloids (harman, harmin, harmalin, harmol, ings, schizophrenia, myasthenia gavis and and harmalol). Among these natural prod- organophosphate poisoning. These phar- ucts, chrysin has shown interesting biolog- maceutical applications of H. serrata are ical activities, including antibacterial, an- mainly due to its biologically active lycopo- ti-infl ammatory, anti-diabetic, anxiolytic, dium alkaloids. Among the lycopodium al- hepatoprotective, anti-aging, anticonvul- kaloids, huperzine A (HupA) was found to sant and anticancer eff ects (Seetharaman et possess potent acetylcholine esterase inhi- al., 2017). bition (AChEI) and is clinically used for the Three endophytic fungi identifi ed as Al- treatment of Alzheimer’s disease (Zhao et tenaria alternata, Colletotrichum capsici, and al., 2013). The content of HupA in the leaf is C. taiwanense were isolated from leaves of P. richer than that in the stem and root of H. incarnata and production of fungal chrysin serrata (Gu et al., 2005). was confi rmed through UV-vis spectrosco- Several groups have isolated endophyt- 1 py, FT-IR, LC-ESI-MS, and H1 NMR analysis of ic fungi from leaves, stems and roots of H. their extracts. The quantitative HPLC analy- serrata. Screening culture extracts of these sis revealed that the yield of chrysin from A. fungi for HupA production revealed that alternata was higher when compared with most of HupA-producing fungi were iso- previously reported bioresources (Seethara- lated from leaf tissues (Su et al., 2017). The man et al., 2017). HupA-producing endophytic fungi belong to Penicillium griseofulvum, Penicillium sp., Fritillaria cirrhosa D.Don (Liliaceae) Aspergillus fl avus, Mycoleptodiscus terrestris, Bulbus Fritillaria have been used in tra- Trichoderma sp., Colletotrichum gloeospori- ditional Chinese medicine for more than oides strain ES026 and Shiraia sp.. The pro- 2000 years, and at present, they are among ductivity of these strains is less than 60-90 the most widely used antitussive and ex- μg/L, with Shiraia sp. Slf14 being the best pectorant drugs. The major biological ac- producer (327.8 μg/L) (Su et al., 2017). Inter- tive ingredients of Bulbus Fritillaria cirrho- estingly, many H. serrata endophytic fungi sa are steroidal alkaloids, such as peimisine, with AChE inhibitory activity did not contain imperialine-3β-D-glucoside, and peimine HupA in their extracts (Su et al., 2017; Wang (Wang et al., 2011). et al., 2016) suggesting that some endophyt- Several dosens of endophytic fungi were ic fungi produce new compounds with ac- isolated from fresh bulbus of Fritillaria uni- tivity against AChE. bracteata var. wabensis. One of these fungal endophytes, Fusarium redolens 6WBY3 was Rhodiola spp. (Crassulaceae) capable of producing and secreting in the Rhodiola rosea is a perennial herbaceous culture medium peimisine and imperialine- plant that belongs to the family Crassu-

© Benaki Phytopathological Institute 56 Venieraki et al. laceae. This species is mainly distributed in is possible, however it does not appear to be high altitudes of >2,000 m in the Arctic and practical as the overall yield was only 10.5%, mountainous regions throughout Asia and requiring 13 steps (Wei et al., 2011). Europe. This typical alpine plant has been Three fungal endophytes have been iso- widely used as an important food crop and lated from S. nigrum stems, leaves and fruits. folk medicine since ancient times by many Their culture extracts were screened for the countries, such as Sweden, Russia, India, potential production of steroidal alkaloids. and China (Chiang et al., 2015). Rhodiola rhi- The stem derived endophytic fungal strain zome, as a traditional folk medicine, stimu- A. fl avus was able to steadily produce sola- lates mental and physical endurance, coun- margine with a titer of about 250–300 μg/ L teracts depression, improves sleep quality, which is higher than the plant callus culture and prevents high-altitude sickness. Mod- method (El-Hawary et al., 2016). ern pharmacology research suggests that Rhodiola rhizome has received consider- Piper longum L. and Piper nigrum L. (Pip- able attention because of its biological be- eraceae) havior, including antioxidant and anti-aging Piperine is a major alkaloid present in the properties, anti-microwave radiation, anti- fruit of Piper longum and Piper nigrum and it hypoxia and adaptogenic activities. Most of is known to have a wide range of pharma- these eff ects are ascribed to phenolics, such ceutical properties including antibacterial, as salidrosides and p-tyrosol, and glycosides antifungal, hepato-protective, antipyretic, like rosavins (Chiang et al., 2015). anti-infl ammatory, anti-convulsant, insec- Screening of 347 endophytic fungal ticidal and antioxidant. The amount of pip- strains isolated from rhizomes of R. crenula- erine varies in plants belonging to the Pip- ta, R. angusta and R. sachalinensis revealed eraceae family; it constitutes 2% to 7.4% of that four endophytic fungi were capable of both black pepper and white pepper (Cor- producing salidrosides and p-tyrosol (Cui gani et al., 2017). Screening of endophyt- et al., 2015). One of these endophytic fungi ic fungi isolated from both plant species re- identifi ed as Phialocephala fortinii was able vealed the presence of piperine in culture to stably produce large amounts of salidro- extracts of endophytic Periconia strains iso- sides and p-tyrosol, 2.3 and 2 mg/ml of cul- lated from leaves of P. longum (Verma et al., ture medium, respectively (Cui et al., 2016). 2011) and Colletotrichum gloeosporioides from the stems of P. nigrum (Chithra et al., Solanum nigrum L. (Solanaceae) 2014). Solanum nigrum L., family Solanaceae is a well-known medicinal plant which possess- Digitalis lanata Ehrh. (Plantaginaceae) es several biological activities such as anti- Glycosides from plants of the genus Dig- oxidant, hepato-protective, antiinfl amma- italis have been reported to be cardiotonic tory, antipyretic, diuretic, antimicrobial and and are widely used in the treatment of var- anticancer activities due to its fl avonoid and ious heart conditions namely atrial fi brilla- steroidal alkaloids content (Jain et al., 2011). tion, atrial fl utter and heart failure. The bio- Solamargine, one of the major steroidal al- active glycosides accumulate in the leaves kaloids in S. nigrum has been demonstrated and to a less extent in other organs of the to exhibit potent anticancer activity against plant (Alonso et al., 2009). colon, prostate, breast, hepatic and lung A total of 35 fungal endophytes were iso- cancer cell lines (Jain et al., 2011). Solamar- lated from stems and leaves, and screened gine is always found in a complex mixture for the production of secondary metabo- with other glycoalkaloids such as solasonine lites. Crude extracts of fungal cultures re- and solanine, which makes solamargine iso- vealed the production of glycoside digox- lation from the plant quite diffi cult (Milner et in from cultures of fi ve endophytic strains al., 2011). Chemical synthesis of solamargine (Kaul et al., 2013).

Capsicum annuum L. (Solanaceae) seven fl avolignans (silybin A, silybin B, isosi- Capsaicin, the pungent alkaloid of red lybin A, isosilybin B, silychristin, isosilychris- pepper (Capsicum annuum), is present in tin, and silydianin) with reported chemopre- large quantities in the placental tissue, the vention and hepatoprotective properties internal membranes and, to a lesser extent, (Feher and Lengyel, 2012). the other fl eshy parts of the fruits of Cap- Twenty one endophytic fungi were iso- sicum. The pharmacological properties of lated from stems, leaves, roots, and seeds of capsaicin include cardio protective infl u- S. marianum and were examined for produc- ence, anti-lithogenic eff ect, anti-infl ammation of fl avolignans (El-Elimat et al., 2014). tory and analgesia, thermogenic infl uence, Two of these compounds, silybin A and si- and benefi cial eff ects on gastrointestinal lybin B, have been extracted as fermenta- system (Srinivasan et al., 2016). tion products of two strains of Aspergillus ii- An endophytic fungal strain identifi ed as zukae isolated from the leaves and stems of Alternaria alternata has been isolated from S. marianum, respectively. Subcultur of one fruits of C. annuum and has been found to fl avonolignan-producing strain revealed an produce and secrete capsaicin up to three attenuation of the production of fl avono- generations (Devari et al., 2014). lignans. However, when autoclaved leaves of the host plant were added to the growth Ginkgo biloba L. (Ginkoaceae) medium, the production of fl avonolignans Ginkgo tree contains in bark and leaves could be resumed (El-Elimat et al., 2014). fl avones and terpenoide lactones, among which, bilobalide and ginkgolides (terpe- Vinca minor L. (Apocynaceae) and Neri- noide lactones) have been shown to be um indicum Mill. (Apocynaceae) benefi cial to human health (Usai et al., 2011). Vincamine indole alkaloids (vincamine, Ginkgolide B has revealed potent antagonis- tabersonine and catharanthine) are widely tic eff ects on platelet activating factors in- found in plants of the Apocynaceae family volved in the development of a number of and show benefi cial properties for human, renal cardiovascular, respiratory and central such as prevention of cerebrovascular, pre- nervous system disorders (Usai et al., 2011) caution of chronic ischemic stroke, and re- while bilobalide was found to exert neuro- duction of vascular dementia or memory protective eff ects (Kiewert et al., 2008). impairment (Saurabh and Kishor, 2013). Vin- Screening of 27 endophytic fungal camine is a precursor compound for other strains isolated from the bark of G. biloba medicinal alkaloids such as 11-bromovin- trees revealed that only one isolate F. ox- camine, ethyl-vincamine and vinpocetine, ysporum SY0056, was capable of produc- which have shown potential clinical thera- ing Ginkgolide B (Cui et al., 2012). The search peutic eff ect (Manda et al., 2015). Vincamine for bilobalide -producing endophytic fun- is accumulated in the leaves and stems of gi was far more copious; a total of 57 fungal Vinca minor and Nerium indicum. Though strains were isolated from stem, root, leaf, abundant chemical synthesis and semi-syn- and bark of the plant G. biloba and their ex- thesis research results have been reported, tracts were evaluated for the presence of bi- the main sources of vincamine indole alka- lobalide. Only the isolate Pestalotiopsis uvi- loids are stems and leaves of Vinca minor L. cola GZUYX13 residing in leaves was proven Eleven fungal strains have been isolated to be a bilobalide-producing fungus (Qian from the stems and roots of Nerium indicum et al., 2016). and fungal culture extracts were screened for the presence of indole alkaloids (Yin and Silybum marianum (L.) Gaertn. (Aster- Sun, 2011). One fungal strain, CH1, produces aceae) vincamine alkaloids as its host plant as de- Silymarin is a bioactive extract of the termined by TLC, HPLC and LC–MS analy- fruits of Silybum marianum and contains sis. The yield of vincamine, ethyl-vincamine,

© Benaki Phytopathological Institute 58 Venieraki et al. and tabersonine was 1.279, 1.279 mg/L, 0.102 strain Colletotrichum gloeosporioides isolat- mg/L, respectively (Na et al., 2016). In a simi- ed from the fruits was found to produce the lar study, 10 endophytic fungal strains were active constituent phillyrin as was judged by isolated from the roots, stems and leaves of TLC, HPLC and HPLC-MS analysis (Zhang et the plant V. minor. One fungal strain isolat- al., 2012). ed from the stems was found to produce vincamine although with a relatively lower Miquelia dentata Bedd. (Icacinaceae), yield as compared to that of another fungal Camptotheca acuminata Decne. (Nys- strain isolated from N. indicum (Yin and Sun, saceae) and Nothapodytes nimmoniana 2011). (Graham) Mabb. (Icacinaceae) Camptothecine (CPT), a quinoline indole Rheum palmatum L. (Polygonaceae) alkaloid and its analog, 10-hydroxy camp- Rheum palmatum is a medicinal plant tothecine (10-OH-CPT) are potent inhibitors and its air-dried roots have been used in of the eukaryotic topoisomerase I and are the traditional medicine. R. palmatum pres- currently used as effi cient anticancer drugs ents cathartic eff ect on the digestive move- against a broad band of tumor types such ment of the colon, protects the damaged as small lung and refractory ovarian can- liver, and has antibacterial, anti-infl amma- cers. (Kai et al., 2015). CPT and 10-OH-CPT tion, and anti-aging properties. The most ef- are naturally produced by several plant spe- fective biologically active compounds in the cies of the Asterid clade. Among them how- roots of the genus Rheum are anthraquino- ever, the major sources of commercial CPT nes including emodin, rhein, physcion, al- in the world market are Camptotheca acum- oe-emodin. Pharmacological tests revealed inata and Nothapodytes nimmoniana (Uma that rhein can alleviate pain and fever and Shaanker et al., 2008). Exceptional high lev- inhibits infl ammation (You et al., 2013). els of CPT and 10-OH-CPT are also found Fourteen endophytic fungal strains have in the fruits and seeds of Miquelia dentata been isolated from R. palmatum: 12 strains (Ramesha et al., 2013). were isolated from the root, 2 strains from Twenty-three fungal isolates were ob- the stem. The strain R13, isolated from the tained from diff erent fruit parts of M. denta- roots, was capable to produce the bioactive ta. All fungal isolates produced CPT though compounds rhein and emodin. The yield of in varying quantities (Shweta et al., 2013). rhein in R13 can reach 5.67 mg/L (You et al., Three fungal species, A. alternata, Phomop- 2013). sis sp. and Fomitopsis sp., were identifi ed as CPT-producers with the highest yield of CPT Forsythia suspensa (Thunb.) Vahl. being obtained from A. alternata (73.9 μg/g (Oleaceae) DW) (Shweta et al., 2013). CPT-producing en- The main chemical constituents of F. sus- dophytic fungi have also been isolated from pensa are composed of lignans including C. acuminata (Pu et al., 2013) and N. nimmo- phillyrin and forsythiaside, triterpenic ac- niana (Bhalkar et al., 2016). ids including oleanolic acid and ursolic acid. Phillyrin was reported to have various biological activities such as antioxidant, anti-in- Biochemical convergence or horizontal fl ammatory, anti-hyperlipidemia and anti- gene transfer confer the ability to the pyretic activities (Qu et al., 2008). Studies on endophytic fungi to produce the same phillyrin have shown its presence mainly in bioactive compounds as their host the leaves and fruits of the plant F. suspensa (Piao et al., 2008). The discovery of endophytic fungi pro- A total of 24 fungal strains were isolated ducing the same or similar bioactive com- from stems, leaves and fruits of F. suspensa pounds as their hosts raises the question as and screened for phylirin production. One to whether parallel pathways evolved sim-

© Benaki Phytopathological Institute Endophytic fungi & secondary metabolites in medicinal plants 59 ply because each lineage has benefi tted as PCR analysis did not reveal the presence from making a given compound completely of Taxus ts gene sequences in SSM001 (Soli- independently of the other or whether hori- man et al., 2013). Hence, the divergence of zontal gene transfer (HGT) events took place the two biosynthetic pathways is support- between the fungi and the plant. ed with conservation only in specifi c en- There is precedent for the indepen- zyme sites to be important for the activi- dent development of the same biosynthet- ty rather than the whole protein structure. ic pathway (biochemical convergence) in Similar fi ndings have been reported in the fungi or plants and other organisms. For in- case of huperzine A producing endophytes. stance, although higher plants and endo- Their fungal amine oxidase genes have phytic fungi produce structurally identical been found to present low similarities to the GAs, profound diff erences have been found corresponding plant genes, and only con- in the GA pathways and enzymes of plants served consensus sequences were present and fungi (Hamayum et al., 2016), e.g. 7-m- by the fungal and plant functional amine ethyl-cyercene-1 found in both the fungus oxidase proteins (Yang et al., 2014; Yang et Leptosphaeria maculans (anamorph Pho- al., 2016; Zhang et al., 2015), which supports ma lingam) and the marine mollusk Ercol- the co-evolution theory rather than the HGT ania funereal is produced by distinct en- theory. This has been well established in the zymes (Cutignano et al., 2012). Cyanogenic case of gibberellin biosynthetic pathways in glucosides linamarin and lotaustralin found fungi and higher plants where diff erences in both the moth Zygaena fi lipendulae and in genes and enzymes indicated converged their food plant Lotus japonicus are biosyn- evolution of GA metabolic pathways (Böm- thesized by distinct enzyme systems(Jensen ke and Tudzynski, 2009). et al., 2010). However, a horizontal gene The list of taxol producing endophyt- transfer event between plants and fungi, al- ic fungi is large and encompasses numer- though rare, should not be excluded (Rich- ous fungi belonging to diverse genera (Sti- ards et al., 2009). erle and Stierle, 2015). A similar situation Several studies have reported the pres- appears to hold for CPT-producing fungi (Pu ence of Taxus tree key genes (ts,dbat and et al., 2013) and HupA-producing endophytic bapt) which are involved in plant paclitaxel fungi (Su et al., 2017) suggesting a horizontal biosynthesis in taxol-producing endophytic transfer of large secondary metabolism gene fungi. These results stimulated the conjec- clusters between fungi. Several studies off er ture that the origin of this pathway in these support to this idea; the complete sterigma- two physically associated groups could have tocystin gene cluster in Podospora anserine been facilitated by horizontal gene transfer was horizontally transferred from Aspergil- (Kusari et al., 2014). Other studies, howev- lus (Slot and Rokas, 2012). Furthermore, it has er, provided evidence that microbial taxol been shown that CTP is also produced by a genes exist independent of the plant genes diverse group of endophytic bacteria (Shwe- (Xiong et al., 2013). Recent data support the ta et al., 2013; Pu et al., 2015) suggesting that latter proposal; genome sequencing and bacterial CPT biosynthesis may represent analysis of the taxol-producing endophyt- an independently assembled pathway from ic fungus Penicillium aurantiogriseum NRRL that in fungi or plants. This may be surpris- 62431 revealed that out of 13 known plant ing since converged evolution of the diter- Taxol biosynthetic genes, only 7 showed low pene GA metabolic pathway in plants, fungi homology(>30%) with genes identifi ed in P. and bacteria is well established (Tudzyns- aurantiogriseum (Yang et al., 2014). Further- ki et al., 2016). Therefore, extensive genome more, polyclonal antibodies against Yaxus sequencing of the various endophytic fun- TS strongly cross-reacted with a protein of gi will provide an opportunity for a compre- the taxol-producing fungus Paraconiothyri- hensive study on the phylogenetic origin of um SSM001 grown in liquid culture, where- fungal and bacterial metabolic pathways.

Exploring endophytes for sustainable tive in stimulating the transcription of atten- and enhanced production of secondary uated biosynthetic gene clusters of endo- metabolites phytic fungi (Vasanthakumari et al., 2015; Magotra et al., 2017), thereby resulting in The discovery of endophytic fungi ca- the enhancement of the production of de- pable of producing the same bioactive sired secondary metabolites. Bioprocess en- compounds as their host medicinal plant gineering strategies such as manipulation has raised the expectation that these com- of media and culture conditions, co-culture pounds could be produced in large scale condition, epigenetic modulation, elicitor through fermentation processes, thus and or chemical induction, mixed fermen- meeting the growing demand of the mar- tation, and fermentation technology, have ket, while relieving the dependence on been proven promising in alleviating to their respective endangered host plants some extent these obstacles (Venugopalan for the metabolites. However, this expecta- and Srivastava, 2015). tion remains hampered primarily by the low Upon availability of the endophytic fun- yields as well as the attenuation of metabo- gal genomes, the putative genes encoding lites production after sub-culturing of fungi the enzymes involved in the biosynthesis (Kusari et al., 2011; Kumara et al., 2014; El-Eli- of bioactive compounds could be identi- mat et al., 2014). The reasons for the attenua- fi ed and their function could be verifi ed tion could be attributed to factors that stem through transcriptomic, proteomic and me- from loss of presumed signals provided by tabolomic, RNA interference, gene knock- the host or co-existing endophytes, result- out, and gene over expression. Genome ing in the silencing of genes in axenic mon- editing technologies implemented for met- ocultures (Sachin et al., 2013). abolic engineering of fi lamentous fungi may Passage of attenuated CPT-producing be applied for triggering the biosynthesis endophytic fungi from the host plants re- of metabolites. Alternatively, the identifi ed stored CPT production in the re-isolated en- biosynthetic pathway of the correspond- dophytic fungi (Vasanthakumari et al., 2015) ing bioactive compounds can be assem- suggesting that a certain critical signaling bled, engineered and then introduced in may be necessary for the fungus to main- other genetically tractable microorganisms tain its endogenous production. Co-culti- to increase their yields (El-Sayed et al., 2017; vation studies of taxol producing fungus Wakai et al., 2017). Paraconiothyrium SSM001 with endophytic fungi isolated from Taxus tree revealed an eightfold increase in fungal Taxol pro- Medicinal plant endophytes in Greece duction from SSM001 (Soliman and Raizada, 2013). Co-cultivation of the endophytic fun- Greece is endowed with a rich biodiver- gus Fusarium tricinctum with the bacterium sity of medicinal plant species with a long Bacillus subtilis, led to an up to 78-fold en- tradition in herbal medicines, and their hancement in the accumulation of the con- complex endomicrobiome may be directly stitutively present fungal metabolites (Ola and indirectly responsible for the produc- et al., 2013). Co-cultivation (mixed fermentation of a wealth of explored and unexplored tion) under optimized conditions of the two bioactive compounds. Thus, it is expected CPT-producing fungal species Colletotri- that many new or known products for medi- chum fruticola and Corynespora cassiicola cine may emerge through the exploration of isolated from the same host tree N. nimmo- the endophytes of these medicinal plants. niana enhanced the yield of produced CPT We are currently isolating fungal and bac- (Bhalkar et al., 2016). terial endophytes from indigenous medici- Epigenetic modifi cations using chemical nal plant species in the genera such as Fritil- inhibitors have also been found to be eff ec- laria, Hypericum, Teucrium, Calendula, Salvia

© Benaki Phytopathological Institute Endophytic fungi & secondary metabolites in medicinal plants 61 as well as Olea europaea and the exotic Ni- Chiang, H.M., Chen, H.C., Wu, C.S., Wu, P.Y. and Wen, gella sativa aiming to identify such bioactive K.C. 2015. Rhodiola plants: chemistry and biological activity. Journal of Food and Drug Anal- compounds. ysis, 23: 359-369. Chithra, S., Jasim, B., Anisha, C., Mathew, J. and Rad- hakrishnan, E.K. 2014. LC-MS/MS based nidenti- Conclusions fi cation of piperine production by endophytic Mycosphaerella sp. PF13 from Piper nigrum. Ap- plied Biochemistry Biotechnology, 173: 30–35. Medicinal plants off er an extensive biore- Chun-Yan, S.U., Qian-Liang, M.I.N.G., Rahman, K., source of new bioactive compounds that Ting, H.A.N., and Lu-Ping, Q.I.N. 2015. Salvia mil- have signifi cant potential as antiparasit- tiorrhiza: Traditional medicinal uses, chemistry, ics, antibiotics, antioxidants, and anticancer and pharmacology. Chinese Journal of Natural agents. During the last 10 years it became Medicines, 13: 163-182. apparent that endophytes are capable to Cui, J., Guo, T., Chao, J., Wang, M. and Wang, J. 2016. Potential of the endophytic fungus Phialo- produce the same bioactive secondary me- cephala fortinii Rac56 found in Rhodiola plants tabolites as their hosts and therefore there to produce salidroside and p-tyrosol. Molecules, is a tremendous interest of the scientifi c 21: 502. community towards isolation, characteriza- Cui, J.L., Guo, T.T., Ren, Z.X., Zhang, N.S. and Wang, tion and exploitation of endophytic fungi M.L. 2015. Diversity and antioxidant activity of culturable endophytic fungi from alpine plants from medicinal plants as was judged by the of Rhodiola crenulata, R. angusta, and R. sacha- amount of publications and number of pat- linensis. PloS one, 10: e0118204. ents (Gokhale et al., 2017). Cui, Y., Yi, D., Bai, X., Sun, B., Zhao, Y. and Zhang, Y. 2012. Ginkgolide B produced endophytic fungus (Fusarium oxysporum) isolated from Ginkgo Literature cited biloba. Fitoterapia, 83: 913-920. Cutignano, A., Villani, G. and Fontana, A. 2012. One Atanasov, A.G., Waltenberger, B., Pferschy-Wenzig, metabolite, two pathways: convergence of E.M., Linder, T., Wawrosch, C., Uhrin, P., Temml, polypropionate biosynthesis in fungi and ma- V., Wang, L., Schwaiger, S, Heiss, E.H., Rollinger, rine molluscs. Organic letters, 14: 992-995. J.M., Schuster, D., Breuss, J.M., Bochkov, V., Mi- Devari, S., Jaglan, S., Kumar, M., Deshidi, R., Guru, S., hovilovic, M.D., Kopp, B., Bauer, R, Dirsch, V.M. Bhushan, S. and Taneja, S.C. 2014. Capsaicin pro- and Rollinger, J. M. 2015. Discovery and resup- duction by Alternaria alternata, an endophytic ply of pharmacologically active plant-derived fungus from Capsicum annum; LC–ESI–MS/MS natural products: A review. Biotechnology ad- analysis. Phytochemistry, 98: 183-189. vances, 33: 1582-1614. Dziggel, C., Schäfer, H., and Wink, M. 2017. Tools of Bhalkar, B.N., Patil, S. M. and Govindwar, S.P. 2016. pathway reconstruction and production of eco- Camptothecine production by mixed fermenta- nomically relevant plant secondary metabolites tion of two endophytic fungi from Nothapodytes in recombinant microorganisms. Biotechnology nimmoniana. Fungal Biology, 120: 873-883. Journal, 2:1666145. Bömke, C. and Tudzynski, B. 2009. Diversity, regu- El-Elimat, T., Raja, H.A., Graf, T.N. Faeth, S.H., Cech, lation, and evolution of the gibberellin biosyn- N.B. and Oberlies, N.H. 2014. Flavonolignans thetic pathway in fungi compared to plants and from Aspergillus iizukae, a fungal endophyte of bacteria. Phytochemistry, 70: 1876-1893. milk thistle (Silybum marianum). Journal of Nat- Butelman, E.R. and Kreek, M.J. 2015. Salvinorin A, a ural Products, 77: 193–199. kappa-opioid receptor agonist hallucinogen: El-Hawary, S.S., Mohammed, R., AbouZid, S.F., Ba- pharmacology and potential template for novel keer, W., Ebel, R., Sayed, A.M., and Rateb, M.E. pharmacotherapeutic agents in neuropsychiat- 2016. Solamargine production by a fungal en- ric disorders. Frontiers in Pharmacology, 6: 285. dophyte of Solanum nigrum. Journal of Applied Carrier, D.J., van Beek, T.A., van der Heijden, R., and Microbiology, 1201: 143–50. Veroorte, R. 1998. Distribution of ginkgolides El-Sayed, A.S., Abdel-Ghany, S.E. and Ali, G.S. 2017. and terpenoids biosynthetic activity in Ginkgo Genome editing approaches: manipulating of biloba. Phytochemistry, 48: 89–92. lovastatin and taxol synthesis of fi lamentous Chen, S.L., Yu, H., Luo, H.M., Wu, Q., Li, C.F. and Stein- fungi by CRISPR/Cas9 system. Applied Microbiol- metz, A. 2016. Conservation and sustainable ogy and Biotechnology, 101: 3953-3976. use of medicinal plants: problems, progress, Feher, J. and Lengyel, G. 2012. Silymarin in the pre- and prospects. Chinese Medicine, 11: 37. vention and treatment of liver diseases and pri-

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Ενδοφυτικοί μύκητες που διαβιούν εντός των φαρμακευτικών φυτών έχουν την ιδιότητα να παράγουν τους ίδιους ή παρόμοιους δευτερογενείς μεταβολίτες με τους ξενιστές τους

Α. Βενιεράκη, Μ. Δήμου και Π. Κατινάκης

Περίληψη Τα φαρμακευτικά φυτά χρησιμοποιούνται εδώ και χιλιάδες χρόνια στην παραδοσιακή φαρ- μακολογία και ιατρική. Στις μέρες μας, τα φυτά αυτά αξιοποιούνται για την απομόνωση ιδιαίτερα απο- τελεσματικών φυτικών φαρμακευτικών ουσιών, με καθόλου ή ελάχιστες παρενέργειες στο χρήστη. Οι φυσικές πηγές φαρμακευτικών φυτών εξαντλούνται σταδιακά με αποτέλεσμα πέραν της οικολογικής διατάραξης από την εξαφάνιση του φυτικού είδους, να κινδυνεύει δραματικά η απόκτηση του βιοδρα- στικού προϊόντος, το οποίο ούτως ή άλλως βρίσκεται σε χαμηλή συγκέντρωση στο φυτό. Επί παρα- δείγματι, η ποσότητα των αλκαλοειδών που προέρχονται από φυτά βίνκας και τα οποία χρησιμοποι- ούνται ως ισχυρά αντικαρκινικά φάρμακα, ανέρχεται στα 3 κιλά ανά έτος δηλαδή απαιτούνται 1.5x106 κιλά ξηρού βάρους φύλλων. Από αυτήν την άποψη, η παρούσα βιβλιογραφική ανασκόπηση αποσκο- πεί στο να τονίσει τη σημασία των ενδοφυτικών μυκήτων που διαβιούν εντός των φαρμακευτικών φυ- τών και οι οποίοι είναι ικανοί να βιοσυνθέτουν τους ίδιους ή παρόμοιους δευτερογενείς μεταβολίτες με τους ξενιστές τους. Επιπλέον, συζητείται η εξελικτική προέλευση των γονιδίων που εμπλέκονται σε αυ- τές τις μεταβολικές οδούς καθώς και οι προσεγγίσεις που αποσκοπούν στην ενίσχυση της παραγωγής αυτών των μεταβολιτών από ενδοφυτικούς μύκητες.

Hellenic Plant Protection Journal 10: 51-66, 2017