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AbstractFungal endophytes are the microorganisms that live

asymptomatically within the plant tissue without caus-ing any apparent damage to the plant body. These endo-phytes are the epitomes of numerous natural products and secondary metabolites, which have an application in the medicinal, agricultural, or industrial arena. Since the advent of taxol production from endophytic fungi Taxus brevifolia, fungal endophytes have generated significant interest in the healthcare industry. From antibiotics to an-tiviral compounds; antioxidants to anticancer agents, the secreted products of fungal endophytes have emerged as a potential substitute for the conventional medicine. It is noteworthy that, the changing lifestyles have shown an up-surge in ailments like cancer, asthma, chronic obstructive pulmonary disease, and cardiovascular diseases (CVD). Although these diseases have been curbed to a large ex-tent, but CVD is still a global burden which alone claims 17.5 million lives every year. The conventional synthetic drug therapy has proven vague, hence, there is a general call to identify new cardioprotective agents from micro-organisms that are highly effective with minimal side ef-fects. It is estimated that there are nearly 20,00,000 natural products of microbial origin, of which nearly 23% have cardio-protective properties. Hence, the intent of this re-view is to provide an insight on novel aspects of some nat-ural products produced by fungal endophytes in nature that may have significant therapeutic potential in CVD management and hence, facilitate the product discovery processes.

Chapter 2

Natural Products of Fungal Endophytes and their Therapeutic Potential: A Fo-cus on Cardiovascular Disease

Riyaz Ahmad Rather and Veena Dhawan*

Department of Experimental Medicine and Biotechnology, Postgraduate Institute of Medical Education and Research, India

*Corresponding Author: Veena Dhawan, Room No 2014, Department of Experimental Medicine and Biotechnol-ogy, 2nd Floor, Research Block-B, PGIMER, Chandigarh, 160012, India, Tel: +91 172 2755235; Fax: +91 172 2744401; Email: [email protected]

First Published October 20, 2016

Copyright: © 2016 Riyaz Ahmad Rather and Veena Dhawan.

This article is distributed under the terms of the Creative Commons Attribution 4.0 International License (http://creativecommons.org/licenses/by/4.0/), which permits unrestricted use, distribution, and reproduction in any medium, provided you give appropriate credit to the original author(s) and the source.



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IntroductionThe burden of cardiovascular disease (CVD) is ever

growing and it is estimated that 17.5 million people die every year due to this disease. Due to the diverse risk fac-tors (high LDL-cholesterol, diabetes, hypertension, physi-cal inactivity, smoking, obesity or use of alcohol), the CVD is the leading cause of mortality in the United States for both men and women [1]. Though with the use of con-ventional medicines the death rate has declined to a sig-nificant level, but the burden of this disease still remains high. As conventional therapy evolved a placid response, a systematic search for an alternative form of medicine or new drug entity with an aim to manage the CVD in a bet-ter way, is a need of an hour. In a plethora of alternative medicines, secondary metabolites or natural products are tried as anticancer drugs, antiviral agents and few have emerged as a worthy immunomodulators [2,3]. However, the use of fungal natural products as therapeutic agents in CVD management is still a matter of discussion.

Fungal endophytes are the major producers of natu-ral products besides plants and marine macro-organisms. The term “fungal endophytes” includes a group of fungi that grow and colonize the internal plant tissues beneath the epidermal cell without causing any apparent symp-toms or damage to the host plant and are recognized as the rich sources of bioactive natural products [4]. The mu-tual interaction between endophytes and the host plant results in fitness benefits for both partners and may pro-

vide a defense and survival conditions to the host plant by generating a plethora of metabolites which, when isolated and characterized, may serve as a potential substance for use in medicine, agriculture, and industry. The making of bioactive compounds by endophytes is directly associated with the independent evolution of these microorganisms within the host plant, which may have assimilated the ge-netic information from the host tissues through the hori-zontal gene transfer, permitting them to better acclimatize within the host plant. Fungal endophytes produce varied bioactive natural products including steroids, terpenoids, alkaloids, tetralones, benzopyranones, phenolic acids, be-sides xanthones, chinones, quinones, flavonoids, and oth-ers, which are promising in human health concerns [3]. Such bioactive metabolites find a wide-ranging applica-tion like antibiotics, immunosuppressants, antiparasit-ics, antioxidants, and anticancer agents, though a few of them like statins, have been used as golden standards in cardiovascular research and have evolved as an alternative therapeutic agent for CVD management. Statins are won-der drugs that have redesigned the treatment of hyper-cholesterolemia and allied cardiovascular diseases. These wonder drugs are produced as natural products by certain endophytes like simvastatin by Aspergillus terreus; mevas-tatin by Penicillium citrinum; and lovastatin by Pleurotus ostreatus. Although statin production from endophytes is in dearth, they have been successfully used as derivatives for the synthesis of other statins. The intent of this chapter



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is to deliver insights into the presence of various natural products produced by the endophytes in nature and how some of these natural products could be used for targeted therapeutics in CVD management.

Fungal Endophytes as Producers of Natural Products

The advent of fungal endophytes came into attention only when Freeman in 1904 presented pieces of evidence about the presence of fungus in an annual grass. Later scientists around the world started to explore the folklore of this new entity from almost all plant populaces in na-ture from different plant parts, geographical niches and diverse environmental conditions and sites [6–8]. Subse-quent studies have shown that the endophytic association with the host plant influences the ecology and evolution of both fungal endophytes and their host plant which em-powered them to create the wide host range. Fungal endo-phytes are not host specific rather a single endophyte may colonize different host plants or even can be isolated from different plant tissues of the same plant [9] [Figure 1].

Reported estimates say that there are more than 20,000 bioactive metabolites which are of microbial origin [10]. Among all producers, fungal endophytes are the most im-portant recognized eukaryotes that are well-known pro-ducers of numerous putative and novel metabolites which are straightway used as drugs or function as lead struc-tures for synthetic modifications [11–13]. According to

the estimates of Hawksworth and Rossman (1987), there may be as many as 1 million different fungal species, yet only about 100,000 have been defined. Though evidence of endophytes being recognized as major producers of metabolites accumulates day by day, yet only 8.9% of de-fined endophytes are explored in the natural product in-dustry [14]. Thus, it seems that the discovered percentage of economically viable fungal metabolites is scanty and further needs a good technological approach.

Figure 1: An assortment of endophytic fungi recovered from the foli-age of Asparagus racemosus.

The successful discovery of numerous drugs from mi-crobial origin like antibiotic penicillin from Penicillium sp., the immunosuppressant cyclosporine from Cylindro-



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carpon lucidum, the antifungal agent griseofulvin from Penicillium griseofulvum, micafungin, an anti-fungal me-tabolite from Coleophoma empetri, mycophenolate used as immunosuppressant in renal transplant produced by Penicillium brevicompactum, illudin-S, an anti-cancer ses-quiterpenoid from Omphalotus illudens, β-lactam antibi-otics from various fungal taxa, and/-or cefditoren pivoxil, a broad-spectrum antibiotic derived from Cephalospori-um sp., has shifted the focus of drug discovery from plants to microorganisms [15–18]. Most of the microbial origin drugs established are either anticancer, immunosuppres-sant, antifungal, antiviral, or antioxidants but very few have been established in CVD treatment. For example, the cholesterol biosynthesis inhibitor lovastatin from As-pergillus terreus, rosuvastatin from Penicillium brevicom-pactum and Penicillium citrinum, used for treating dyslipi-demias [19] and paracin from Cephalosporium sp., used as a cyclooxygenase-2 inhibitor in atherosclerosis-mediated inflammation are just a token of endophytic origin drug leads which are used to treat CVD. The described popula-tions of endophytic strains with respect to CVD treatment are few, however, the opportunity to find new strains and targeting their natural products of cardioprotective inter-est, will pave new avenues and milestones in treating vari-ous ailments of CVD.

Natural Products as Alternative Med-icines

Ancient people, in past times, realized that different plant parts like root, leaf, or stem concoctions had an im-pending effect in curing some diseases. These plant con-coctions improved the quality of life, acted like an anes-thetic and reduced the pain, and provided long-term relief in various ailments. Although these plant mixtures had a propounding effect on the human health, the chemical na-ture of bioactive compounds in these complex mixtures and how they functioned, was elucidated much later. It was not until Pasteur postulated that fermentation pro-cess is caused by some living entities that people seriously initiated to investigate these living entities as a source for bioactive natural products. Then, observational serendip-ity and the command of opinion provided the impetus to Fleming to usher in the antibiotic era via the discovery of penicillin from Penicillium notatum. Since then, people have been engaged in the discovery and application of mi-crobial natural products with activity against both plant and human pathogens.

Natural products are chemical compounds or by-products derived from microorganisms, plants, or ani-mals [5]. Endophytes provide a broad variety of bioactive secondary metabolites with a unique structure, such bio-active metabolites find wide-ranging application such as antibiotics, immunosuppressants, antioxidants, antipara-



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sitics, and anticancer agents besides acting as cardiopro-tective agents [11]. An exemplary progress has been made in product chemistry for the last few decades, which lead to an impression on contemporary medicine since about 40% of prescribed drugs are based on natural products. Additionally, 49% of the new chemical products registered by the Food and Drug Administration (FDA) are natu-ral products or derivatives thereof [20]. As per the recent reports, between 1989 and 1995, 60% of approved drugs from FDA and pre-new drug candidates were natural de-rivatives [21], and from 1983 to 1994, over 60% of drugs approved as cancer drugs or cancer drugs candidates were of natural origin [22]. In fact, the world’s first billion-dol-lar drug, paclitaxel (Taxol) an anticancer drug, is a natural product derived from the yew tree and few fungal endo-phytes from the group Taxomyces [23]. Literature regard-ing the use of natural products from fungal endophytes as cardioprotective agents is scanty and only a few have been explored in cardiovascular research till date. Products like lovastatin and rosuvastatin are lipid-lowering drugs isolated from fungal endophytes [19]. Likewise, pheno-lics and flavonoids of Xylaria sp., Colletotrichum sp., and Pestalotiopsis microspora exhibit a strong cholesterol low-ering effect. Besides the hypolipidemic effect, these prod-ucts have strong antioxidant effects and prevent further reactive oxygen species (ROS) generated cellular damage within the intima of the vascular wall [24]. Most of the cardioprotective natural products are bestowed with anti-oxidative properties. In one of our study, we have shown

that the aqueous extract of Terminalia arjuna displays anti-atherogenic effects in an atherogenic rabbit model system [25]. Whether, metabolites produced by the endophytes within the tissues of Terminalia arjuna, do exert a role in anti-atherogenesis, needs to be evaluated. In the next sec-tion, are highlighted and illustrated few endophytic origin natural products which are being used as cardioprotective agents in the modern civilization.

LovastatinLovastatin (C24H36O5), [Figure 2] is a polyketide-

derived natural product isolated from Aspergillus terreus and Pleurotus ostreatus with a commanding inhibitory ef-fect on 3-hydroxy-3-methyl glutaryl coenzyme A (HMG-CoA) reductase [26]. Lovastatin production is also report-ed in Aspergillus niger, Aspergillus flavus, Penicillium sp., Trichoderma viride, Monascus ruber, and Monascus sp., endophytes [27]. Immediately after its discovery in 1970, this metabolite was taken into clinical development as an impending drug for lowering LDL cholesterol. After its successful clinical trial, lovastatin received the approval in the USA in 1987 and is believed to be the first statin approved by the FDA, which comes under various brand names like Mevacor, Altocor, Altoprev, and Advicor [28–29]. Experimental evidence from literature reveals that at least 18 proteins are involved in the lovastatin biosynthe-sis process and acetate is the major initiator molecule in the production process [30].



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Figure 2: The structures of some compounds isolated from endo-phytic fungi which are used in CVD treatment and management. A) Lovastatin; B) Pestacin; C) Simvastatin; D) Ternatin; E) Nicotinamide

riboside; F) Mevastatin; G) Isopestacin and H) Graphislactone A.

Lovastatin is a pro-hypolipidemic metabolite that inhibits HMG-CoA reductase, a key enzyme that cata-lyzes the conversion of HMG-CoA to mevalonate in the cholesterol biosynthesis pathway. Mevalonate is an obli-gate precursor required for cholesterol biosynthesis and lovastatin impedes with its production by binding to the HMG-CoA reductase as a reversible competitive inhibitor of HMG-CoA. Lovastatin in its native state is an inactive lactone-form pro-drug, once hydrolyzed, forms an active form. The in vivo hydrolysis of the gamma-lactone closed

ring-form releases the β-hydroxy acid open ring form, which is the active form of this drug [31]. Though lovas-tatin is a well-tolerated drug in the body from the physi-ological point of view, it still incurs few side effects and contraindications. For example, its continued use elevates creatine phosphokinase levels, causes flatulence, indiges-tion, constipation and muscle pain. Unlike other statins it rarely causes dermatomyositis, hepatotoxicity or severe myopathy. Lovastatin is contraindicated during pregnan-cy as it may cause birth defects such as bone abnormalities or learning disabilities due its potential to disrupt the lipid metabolism. Lovastatin has been studied for their chemo-therapeutic effects in certain types of cancers. The clinical data from these studies could not demonstrate any en-couraging results, however, these studies have shown that lovastatin reduces the proteasome activity within the ac-tive dividing breast cancer cell lines, independent of their HMG-CoA reductase inhibition [32].

CompactinMevastatin (ML-236B) or Compactin (C23H34O5)

[Figure 2] is a cholesterol-lowering agent isolated from Penicillium citinium, Pythium ultimum, Penicillium cyclo-pium, and a few strains of Colletotrichum sp., [30]. Apart from its hypolipidemic action, mevastatin reduces stroke damage and augments endothelial nitric oxide synthase, while few studies found it a vital antioxidant in their stud-ies [33]. Akira Endo and his associates carried an in vitro studies on more than thousand different strains of endo-



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phytes for their ability to produce an inhibitor of sterol synthesis. The investigating team discovered three active compounds, which were later designated as ML-236C, ML-236A, and ML-236B in the culture broth of the fun-gus Penicillium citrinum. The main compound ML-236B, also called as mevstatin, is identical to another compound which was isolated later from Penicillium breuicompactum by an individual British group [34]. ML-236B is a specific inhibitor of HMG-CoA reductase and highly effective in lowering plasma cholesterol levels.

Mevastatin competitively inhibits HMG-CoA reduc-tase with a binding affinity, counted 10,000 times great-er than the HMG-CoA substrate. Mevastatin, like other naturally occurring statins, is an inactive prodrug that is activated by in vivo hydrolysis of the lactone ring [35]. The hydrolyzed lactone ring imitates the tetrahedral interme-diate produced by the reductase allowing the agent to bind with greater affinity than its natural substrate. The bicyclic portion of the mevastatin binds to the coenzyme A por-tion of the active site and hence imparts its function. Me-vastatin has served as one of the lead metabolites for the development of the synthetic compounds like pravastatin, and even few fungal strains like Penicillium citinium and Penicillium cyclopium have been genetically mutated to enhance the mevastatin production in fermentation batch cultures [36].

It is presumed that mevastatin is considerably safe when compared to other types of statins because of its

simple structure and hence causes least contraindications and adverse events. Accept myopathy, no other worrying side effects were reported in study subjects with the con-tinuous use of this drug.

SimvastatinSimvastatin (C25H38O5) [Figure 2] , sold under the

trade name Zocor, is a lipid-lowering cardiovascular drug produced by an endophytic fungus Aspergillus terreus, as a fermentation product [37]. Alike other statins of endo-phytic origin, simvastatin inhibit hepatic HMG-CoA re-ductase in a competitive fashion and reduces the serum lipids to a larger extent. Evidence from the literature sug-gests that its intake along with exercise and low lipid diet, display propounding results in lowering the elevated cho-lesterol levels. Apart from acting as hypolipidemic agent, this product also acts as an immunomodulatory agent and modulates immune response via suppression of major histocompatibility complex II on antigen-presenting cells like human vascular endothelial cells [38].

The main use of simvastatin is to treat dyslipidemia and to prevent atherosclerosis-related complications like stroke, myocardial infarction or heart attacks in individu-als who are at high risk. However, few studies suggest that it is also used as a trial candidate in the management of cancer [39]. In the 4S (Scandinavian Simvastatin Surviv-al Study) study, a randomized clinical trial carried for 5 years, simvastatin reduced overall mortality in individuals



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with existing CVD and high LDL-cholesterol by 30% and cardiovascular mortality by 42%. The study also shows that the risks of heart attack, stroke, or requiring a coro-nary revascularization procedure were reduced by 37%, 28%, and 37% respectively [40]. In the Heart Protection Study carried for 4.5 years on a diverse set of subjects in-cluding the women, the elderly, and patients with diabe-tes, evaluated the effects of simvastatin in people with risk factors including existing CVD, diabetes, or stroke but having relatively low LDL cholesterol. This trial showed that simvastatin reduced the overall mortality by 13% and cardiovascular mortality by 18% [41]. The same study also demonstrated that people receiving simvastatin experi-enced 38% fewer non-fatal heart attack.

Simvastatin is contraindicated with pregnancy caus-ing severe birth effects like lung immaturity and mental retardation, whereas it is highly recommended that simv-astatin uptake should be avoided during breastfeeding, as it severely disrupts the lipid metabolism of the baby [42]. Other side effects may include joint pain, muscle cramps, indigestion, and eczema.

TernatinNatural products are known to influence several cel-

lular processes in the body by modulating protein targets. Besides Clitoria ternatea plant, various fungal endophytes like Aspergillus sp., Penicillium sp., Chaetomium sp., ac-cumulate a group of polyacylated anthocyanins, named

ternatins [Figure 2]. These compounds suppress hypergly-cemia, hence exhibit its antidiabetic activity via increased insulin release and enhanced antioxidant defense. This way ternatins reduce the influence of the one of the risk factor of CVD [43].

Apart from hypoglycemic properties, ternatin poly-phenols have exhibited an anti-inflammatory property in lipopolysaccharide (LPS)-stimulated RAW 264.7 mac-rophage cells, which was demonstrated by Nair et al (2015) [44]. A strong inhibition of COX-2 activity and partial inhibition of reactive oxygen species (ROS) was demon-strated by a flavonol fraction of ternatin. The anthocyanin fraction was shown to inhibit iNOS protein expression, and nitric oxide production independent of ROS inhibi-tion and also suppressed nuclear NF-κB translocation. The authors suggested their use as drugs or nutraceuticals for protection against chronic inflammatory diseases by suppressing the excessive production of pro-inflammato-ry mediators from macrophage cells [44]. This study pro-vides a possible hint that ternatins could act as a potent mediator in the macrophage-laden inflammasome milieu.

Ternatin is shown to possess cytotoxic and anti-adi-pogenic properties. Shimokawa et al (2007) demonstrated that a highly methylated cyclic heptapeptide (-)-Ternatin (1), significantly inhibited fat accumulation in 3T3-L1 cells (EC50=0.14 microg/ml). In high-fat-fed mice, treat-ment with 1 at 5 mg/kg/day significantly suppressed an increase in body weight and fat accumulation [45]. Ito et



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al examined the mechanism used by (-)-ternatin and [l-Ala(4)]ternatin, an inactive analog of (-)-ternatin to in-hibit adipocyte differentiation. The authors determined the expression of adipocyte markers and lipogenic en-zymes and found that (-)-ternatin significantly attenuated the mRNA expression of several adipocyte markers in a dose-dependent manner, while [l-Ala(4)]ternatin showed no effects. The authors suggested that (-)-ternatin showed its effects on mid-to-late differentiation stages of adipo-cytes. Also, (-)-ternatin potently inhibited triglyceride synthesis in adipocytes and primary hepatocytes [46]. The role of ternatin has also been studied in ischemia/reper-fusion injury. Guimaraes, et al in their study induced is-chemia/reperfusion injury in rat testis and demonstrated that bioflavonoid ternatin (TTN) pretreatment before I/R injury showed antiperoxidative and antioxidative proper-ties [47].

From the above-mentioned evidence available from in vitro and in vivo studies in literature, it is clear that ternatin possesses potent anti-inflammatory, antioxidative and an-tiadipogenic properties besides inhibiting migration and proliferation of cells. Based on these properties, research-ers have advocated its use in stress-related and metabolic disorders. So far its role in cardiovascular diseases has not been explored. Since inflammation and oxidative stress, as well as obesity, are the key factors implicated in the pathophysiology of CVD, it is natural that studies should be conducted with this natural cyclic peptide to exploit its

virtues in experimental and clinical studies and find its therapeutic targets.

Nicotinamide RibosideNicotinamide, nicotinic acid, nicotinamide riboside

(NR) [Figure 2] and nicotinic acid riboside (NAR) are the major precursors for nicotinamide adenine dinucleotide (NAD) biosynthesis in humans. NAD biosynthesis is a highly regulated process, responsive to many biological cues and NAD is essential for cellular homeostasis and serves as a sensor of the metabolic state of the cell. This way NR impacts several biological pathways eg., cell sur-vival, energy metabolism, aging and cell signaling [48]. NR is isolated from few endophytes of Bacopa monnieri and Azadirachta indica plants. Besides Saccharomyces cer-evisiae, the most prominent among NR producers are Pri-formospora sp., Colletotrichum sp., and Epichole sp., [49].

Although, NR does not play a solo role in cell regula-tion or metabolism, it exerts its beneficial effects via NAD. The latter is mediated by the enzymes like poly(ADP-ri-bose) polymerases, mono-ADP-ribosyltransferases, and the sirtuins. All these enzymes are involved in protein modifications, stress resistance, metabolism, and endo-crine signaling, suggesting that these enzymes and/or NAD(+) metabolism could be targeted for the therapeutic benefit of CVD. However, till date there is no practical ad-vance on this hypothesis, whether the enzymatic pathway, mediated by NR or its enzymes involved in the patho-genesis of CVD, could be utilized as a therapeutic ap-



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proach towards CVD management. Despite scarce leads, few studies do suggest that NR is involved in promoting insulin sensitivity, mitochondrial biogenesis, and sirtuin functions. It is shown to negate the effects of high–fat diet and is also shown to be neuroprotective [50]. Low-grade chronic inflammation (metaflammation) is a character-istic feature of metabolic diseases, such as type 2 diabe-tes, obesity, cancer and cardiovascular disease. Lee et al. (2015) demonstrated that NR in a dose of 100 mg/kg/day given for 7 days to a rat model of type 2 diabetes attenu-ated hepatic metaflammation. Further, NR treatment was shown to significantly improve hepatic proinflammatory markers, including tumor necrosis factor-alpha, inter-leukin (IL)-6, and IL-1 [51]. In a mice model of cardio-myopathy lacking transferrin receptor Tfr1 in the heart, administration of nicotinamide riboside was shown to prevent against cardiomegaly, poor cardiac function, mi-tochondrial respiration, and impaired mitophagy.

As outlined above, the evidence in the literature clear-ly suggests that NR being a natural precursor of NAD+ has the powers of being a very effective nutrient and can provide protection against several metabolic and age-re-lated disorders eg. cardiovascular disease, obesity, diabe-tes, cancer and neurodegenerative that are influenced by high-fat diet, inflammation, mitochondrial dysfunction, etc. and thus NR shows the promise to be used as a treat-ment strategy towards the protection of CVD.

Antioxidant CompoundsFungal endophytes are considered as the finest anti-

oxidant producers after herbs. This potential is attributed to their ability to produce phenolics, terpenoids, and fla-vonoid compounds, which have the scavenging effects on free radicals and reactive oxygen species. The significance of natural products possessing antioxidant activity basi-cally lays in the fact that they are exceptionally effective against the damage caused by ROS and oxygen-derived free radicals. The generation of the free radicals and ROS within the different cells of the heart contributes to vari-ous pathophysiologies of CVD, for instance, atheroscle-rosis, ischemia/reperfusion injury, hypertension, or dia-betes mellitus. The antioxidant compounds sequester the generated reactive ROS and free radicals, which otherwise lead to the oxidative modification of lipoproteins that may lead to damage the cellular structure. These antioxidant compounds besides acting as cardioprotective agents, also possess anti-inflammatory, antitumor, antiviral, an anti-bacterial activities [52].

In a study, Liu et al (2007), isolated Xylaria sp., from the medicinal plant Ginkgo biloba, that exhibited a strong antioxidant activity. The methanolic extract of this endo-phyte exhibited a strong antioxidant potential because of the presence of phenolics and flavonoids compounds [53]. Pestacin and isopestacin are the two important an-tioxidants which are produced by the endophytic fungus Pestalotiopsis microspore, isolated from the plant, Termi-



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nalia morobensis [Figure 2]. Due to their structural simi-larity with the flavonoids, both the compounds scavenge superoxide and hydroxyl free radicals in the presence of water molecules. Besides their antioxidant activity, these two compounds also also shown to exhibit antimycotic and antifungal properties [54]. In a similar fashion Graphis-lactone A, a phenolic metabolite [Figure 2] isolated from the endophytic fungus Cephalosporium sp., was shown to exhibit a strong antioxidant and free radical-scavenging activities in vitro more efficiently than the celebrated ef-fects of ascorbic acid and butylated hydroxytoluene [55].

Natural Product Collection and Isola-tion Techniques

Plants bearing ethnomedicinal properties, possess higher endophytic load than those plants which own less or no medicinal properties, the rationale of which is still a matter of deliberation. Endophytes can be isolated from any plant part, like leaf, stem or root, though the former is the most preferred plant part, due to the rich endophytic diversity within its tissues.

Once the plant sample (part of a root, leaf or stem) is selected, it is cut into small pieces and placed in a steri-lized sealed plastic bag, excess moisture is removed and stored at 4°C, if not proceeded on the same day. The plant material is thoroughly surface sterilized with 70% etha-nol, to remove the epiphytes and finally they are air dried

under a laminar-flow hood. Then, with a sterile blade, tis-sues are cut into small pieces with 1–3 mm3 dimensions and placed onto a potato dextrose agar (PDA) medium amended with chloramphenicol to inhibit the bacterial growth. After several days of incubation, hyphal tips of the fungi are removed and transferred to the PDA, identified via morphological and molecular biology techniques and finally grown in pure cultures. Eventually, the identified endophyte is tested for its ability to produce secondary metabolites or natural product, by growing in a shake or still culture with varying media and growth conditions. Ultimately, once appropriate growth conditions are found, the endophyte is fermented, extracted and the bioactive compound(s) is isolated by polar/non-polar or neutral solvents in different propositions and finally characterized by various spectroscopic techniques. It is important to mention that all sorts of common or advanced procedures are explored for the product isolation, identification, and characterization, in order to obtain the product(s) of im-portance. Needless to say, at this point, one cannot put an emphasis on the isolated compound unless and until it is not tested for any bioassay method. Because, central to the isolation process, bioassays guide the compound purifica-tion processes and further help in establishing a transla-tional approach. Further, bioassays are important in de-coding and depicting the natural- product activity within the target system. The target system can involve organ-isms, animal models, tissues, enzymes or their analogs, or model chemical systems that relate to the purpose for



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which the new compound is needed [56]. The workflow depicting the discovery of a natural product from endo-phytic fungi is depicted in Figure 3.

Figure 3: The practical workflow depicting the framework for the dis-covery and identification of a natural product/secondary metabolite

from endophytic fungi.

ConclusionFungal endophytes are the least studied and inves-

tigated group than the other microorganisms of same ecological niche. They act as a rich and reliable source of novel bioactive compounds with a prospective for exploi-tation in medical, agricultural, or industrial arenas. It is imperative to understand the ways through which endo-phytes exist and affiliate to their surroundings, in order to be more accurate and extrapolative about which host plants to search for the study and spend time isolating en-dophytic microflora components. This may expedite the natural product discovery processes.

Although, work on the utilization of natural products in the medicinal arena has begun, but their exploration towards the CVD management is still lukewarm. Natural products of fungal endophytes are widely used as antibi-otics, antiviral, anticancer, antimicrobial, and antifungal agents, but barring few statins like lovastatin, mevastatin, and simvastatin, fungal metabolites are the least studied compounds in cardiovascular research. However, the nat-urally occurring statins have proved as a reliable standard for the synthesis of other synthetic statins like atorvasta-tin, cerivastatin, or fluvastatin. In this context, the use of natural products of endophytes as mediators in the pro-cess of CVD treatment and management assumes greater importance. Nevertheless, much of the natural products exhibit an antioxidant activity, but only a few have yet been tested for their possible role in CVD prevention and



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treatment. As the studies on this huge poorly exploited re-source of microorganisms have just begun, it has already become evident that a huge potential for the organism, its products, and an impending functional discovery towards CVD management holds a great promise.

AcknowledgementWe acknowledge the help of our colleagues in prepar-

ing this chapter.

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