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compounds 3-6 were isolated for the first time from the ge- nus of Begonia. The potential of the compounds 1-6 to influence biologi- cal targets was assessed with several in vitro test systems. For the determination of cytotoxic activity cultured KB (hu- man nasopharyngeal carcinoma), 3T3 (murine embryonic fi- broblasts), PC3 (human prostrate carcinoma), and MethA (murine methylcholanthrene-induced fibrosarcoma) cells were tested with the isolated compounds and processed as described previously (3). In addition, potential immunomod- ulation was investigated by evaluating the influence on pro- liferation of mitogen ConA activated, interleukin-2 depend- ent murine lymphoblasts and mitogen ConA activated murine spleen cells (3). Compounds 1-3 showed cytotoxic activity against KB, 3T3, PC3, and MethA cell lines. The immuno- modulatory activity observed in vitro is not of interest due to the activity pattern and its combination with the results of the compounds‘influence on growth of permanent cell lines. Compounds 4-7 showed no activity (IC50 > 20µg/ml) in the test systems mentioned. Due to their cytotoxicity against various tumor cell lines (4) with unusual potency, cucurbitacins have been investigat- ed extensively. Recently, pharmacological studies on their possible mode of action (e.g., actin disruption, anti- proliferative, antimitotic) heve been reported (5-7) showing evidence of a strucure activity relationship. The structural sites specifically associated with cytotoxicity are: α,β- unsaturated ketone in position C-22, a 25-acetate group (5-7), free 16α-OH, free 20β-OH, ring A with either diosphenol or 3-ketol structure (5). 101

The biological results presented here strongly support previous assumptions based on pharmacological studies on cucurbitacins: The importance of the side chain at C-17, me- diating specific affinity interactions by its α, β-unsaturated ketone in position C-22 and/or the 25-acetate group (1, 2). Yet the here presented results additionally outline the strong influence. Yet the here presented results additionally outline the strong influence of the substitution pattern in positions C- 2 and C-3 of ring A (3-6). It seems that not only the presence of a 3-keyol alone but particularly its combination with a 2- OH is essential as the compound does not show any activity when hydroxylated in position C-3 (4) or glycosidated posi- tion C-2 (5,6). This is further supported by the fact that cu- curbitacins with a glycosidic bond in position C-16 still show strong activity (8). Polygonum sp. From Ladakh, Science citation: Surinder Kitchlu 102

Pharmacological and biological effects of cucurbitacins including cytotoxicity have been reviewed recently (9) and are numerous. The data presented in this study indicate a sig- nificant cytotoxic effect of three of the isolated cucurbitacins, but cannot yet conclusively demonstrate the usefulness of B.heracleifolia in the treatment as described by Zapotecs. Further isolation and structure elucidation yielded the compounds stigmasterol, the C-24 epimers spinasterol (24α) and chondrillasterol (24β), 3-O- β-D-glucopyranosyl- stigmasterol. The esters of six fatty acids (arachidic, behenic, linoleic, plasmatic, steraic acid) were elucidated by GC-MS analysis and a similarity search in a database. All isolated compounds showed no activity (> 20 µg) against several bacterial, fungal and yeast targets in an agar overlay method (10) [Bacillus subtilis (ATCC 6633)], Mi- crococcus luteus (ATCC 9341), Escherichia coli (ATCC 25922), Bacillus cereus (ATCC 10720), Mycobacterium for- tuitum (Inst. Of Microbiology, University of Zurich), Staphy- lococcus aureus (ATCC 25923), Staphylococcus epidermidis (ATCC 27853), Pseudomonas aeruginosa (ATCC 27853), Candida albicans (H29 ATCC 26790)]. The antibacterial ac- tivity of the crude extract (3) must therefore be due to other compounds. The dry root of Bupleurum falcatum (Radix Bupleuri) is listed officially in the Chinese and Japanese pharmacopoeias and is used in Asian traditional medicine to treat certain dis- orders that are accompanied by inflammation. The main anti- inflammatory constituents of this drug are the saikosaponins A and D (1). 103

Some species of Bupleurum growing in Europe have been studied for their chemical composition (2, 3) and /or their an- ti-inflammatory activity (4, 5, 6). From the aerial parts of B.fruticescens , we have recently reported the isolation and identification of three new lupane saponins, designated as fruticesaponin A,B and C (7). According to revised literature, the lupane saponins are found much less commonly than the oleanane derivatives, and are scarcely isolated from plants or other natural sources (8). Moreover, there have been no reports about the phar- macological activities of these saponins, although anti- inflammatory, antitumoral, antiviral and cytotoxic activities have been reported for the lupase aglycones (9). In the context, we previously established the anti- edematous effects of betulin and betulinic acid, both isolated from the leaves of Diospyros leucomelas (Ebenaceae), against different irritant inducers. The results obtained led us to conclude that betulin and betulinic acid could have mech- anism of action related to that of the glucocorticoids, for their anti-inflammatory activity is blocked in the presence of mRNA or protein synthesis inhibitors (10). In this chapter, we report on the anti-inflammatory activi- ty of fruticesaponin A, B and C against different experi- mental models of acute inflammation in mice. The results al- lowed us to suggest some relationship between chemical structure and anti-inflammatory activity. Cirsium subcoriaceum (Less.) Schultz-Bip. (Asteraceae), known as ―cardosanto‖ in Latin America and ―ktqeqnihuke‖ by the Totonaco people of Mexico, is a me- dium-sized perennial herbs that grows from central Mexico 104

to Panama(1,2). Practitioners of folk medicine have used the infusion of the whole plant and applied it tropically to treat breast inflammation, while the infusion of the flowers has been ingested to alleviate various ailments of the respiratory system (1,3), Buddleia cordata HBK. (Buddlieaceae), a small tree known as ―tepozan‖ is used for treatment of various ailments, including rheumatic diseases (4). In the present investigation we have evaluated the poten- tial existence of analgesic and anti -inflammatory activities in the aqueous extracts from the aerial parts of C.subcoriaceum and B.cordata and their principle glycosides pectolinarin and linarin, respectively. Analgesic effects were evaluated in mice using the writhing test described by Woolfe (6), while the anti-inflammatory activity was investigated using the car- rageenan-induced rat paw edema describe by Winter et al.(7). Considering these results and those previously reported for analgesic activities of linarin (4), we compare the profile of pectolinarin and linarin as analgesic and anti-inflammatory agents. Conclusion: The conventional radiotherapy and chemotherapy with synthetic drugs evoke severe side effects including severe immunosuppression and in majority of cases patient die due to organ failure. Edible phytochemicals are inexpensive, ef- fective and are readily applicable and accessible approach to cancer control and management. Little is known about the Phytochemical and their mode of chemopreventive action. Some of several distint mechanisms of these dietary phyto- chemicals yet to be understood. 105

Pharmaceuticals properties and bioavailability are key problems in investigating the dietary prevention of cancer. Potentialities of several phytochemicals are already proved in laboratory and in some cases by clinical trial. A better under- standing of their mode of action and specificity to particular cancer will help to develop a multitargeting anticancer drug with natural dietary phytochemical with minimal side effects. Aglaia (family Meliaceae) plants are used in traditional medicine (e.g., in Vietnam) for the treatment of inflammatory skin diseases and allergic inflammatory disorders such as asthma. Inflammatory diseases arise from inappropriate acti- vation of the immune system, leading to abnormal expression of genes encoding inflammatory cytokines and tissue- destructive enzymes. The active compounds isolated from these plants are derivatives of rocaglamide. In this study we show that rocaglamides are potent immunosuppressive phy- tochemicals that suppress IFN-γ, TNF-α, IL-2, and IL-4 pro- duction in peripheral blood T cells at nanomolar concentra- tions. Rocaglamides, inhibit cytokine gene expression at the transcriptional level. At the doses that inhibit cytokine pro- duction, they selectively block NF-AT activity without im- pairing NF-κB and AP-1. We also show that inhibition of NF-AT activation by rocaglamide is mediated by strong acti- vation of JNK and p38 kinases. The heartwood of Dalbergia odorifera T. Chen.(Leguminosae) is a traditional Chinese medicine, known as jiangxiang in China. It has been used to treat blood disorder, ischemia, swelling, necrosis and rheu- matic pain (1), indicating possible antiplatelet aggregation and anti-inflammatory activities. This plant had been report- ed to effect inhibition of leukotriene biosynthesis in mam- mals (2), inhibition of prostaglandin biosynthesis (3), and an- 106

ti-inflammatory activities (4). In our continuing search for antiallergic and anti-inflammatory agents from natural sources, we found that the methanolic extract of the heart- wood of D.odorifera inhibited the release of β-glucuronidase and lysozyme from rat neutrophils, and the release of β- glucuronidase from rat mast cells. We did not determine the effect of the methanolic extract on the release of histamine from rat mast cells because frac- tions A-D affected the histamine assay system. From the methanolic extract of D.odorifera, we isolated three new compounds, (3R)-4‘-methoxy-2‘,3,7- trihydroxyisoflavanone(11), 7-methoxy-3,3‘,4‘,6- tetrahydroxyflavone (18),and 2‘,7-dihydroxy-4‘,5‘- dimethoxyisoflavone (22),together with twenty-two known compounds. The genus Guettarda (Rubiaceae) comprises plants widely distributed throughout tropical areas. They are known in South America as folk medicines for the treatment of wound and inflammation (1). In previous studies we re- ported on the constituents and biological activity of Guettar- da playpoda plant used in Brazilian folk medicine as febri- fuge (2). Although Guettarda acreana is a popular anti- inflammatory and anti-pasmodic remedy in Bolivia, no pre- vious phytochemical study is reported on this plant in the lit- erature. In the course of a systematic screening of medicinal plants which produce alkaloids with a wide range of biologi- cal activities (3, 4, and 5), here we report the results of a phy- tochemical and pharmacological investigation on Guettarda acreana. Extract from Bupleurum fruticescens were examined for oral and topical anti-inflammatory activities. The BuOH ex- 107

tract proved to be active against carrageenan and tetradeca- noylphorbol acetate acute edemas and allowed the isolation of three saponins identified by spectroscopic techniques as 3β-O-(O-α-L-rhamnopyranosyl-(1→4)-O- [β-D- glucopyranosyl-(1→6)]-O-β-D-glucopyranosyl)lup-20(29)- ene-23,28-dioic acid (fruticesaponin A),3β-O-(O-α-L- rhamnopyranosyl-(1→4)-O-β-D-glucopyranosyl)lup-20(29)- ene-23,28-dioic acid 28-O-β-D-glucopyranosyl ester (fruticesaponin B), and 3β-O-(O-α-L-rhamnopyranosyl- (1→4)-O-[β-D-glucopyranosyl-(1→6)]-O-β-D- glucopyranosyl)-lup-20(29)-ene-23,28-dioic acid 28-O-β-D- glucopyranosyl ester(fruticesaponin C). These compounds were studied against carrageenan, tetradecanoylphorbol acetate, arachidonic acid and ethyl phenylpropiolate acute edemas. Fruticesaponin B, a bidesmosidic saponin with an unbranched saccharide moiety was the most active in all the tests applied. The dried aque- ous extract of aerial parts of Cirsium subcoriaceum (Aster- aceae) and its major flavonoid glycoside, or pectolinarin, have been evaluated for analgesic and anti-inflammatory ef- fects in mice and rats, respectively. Both the extract and pec- tolinarin exerted significant and dose-dependent analgesic and anti-inflammatory activities. Also, the anti-inflammatory activities of an aqueous ex- tract of Buddleia cordata and its principal glycoside linarin were evaluated. The results of pharmacological testing proved that linarin is a better anti-inflammatory agent that pectolinarin and indomethacin. On the other hand, pectolinar- in exerted a better analgesic effect than linarin. References: Appendix III Contact: Dr. Anpurna Kaul - [email protected] 108

Chapter 4 Bioactive Potential of Endophytic Microorganisms & Bio prospecting Microorganisms for Drug Discovery and Development Dr. Sarojini Johri, co-author Dr. Asha Chaubey & co- author Dr. Vidushi Abrol Introduction: The ‘German botanist’ named Anton de Bary (father of plant pathology), discovered the term endophytes that are present inside the plant tissues like stem, leaves and roots in 1886. Most of the researchers generally termed this as mi- crobes that are available in the intra or intercellular tissue re- gion of the host plant. Endophytes are also known biological source that have been found for pharmaceutical importance (Brader, Compant, Mitter, Trognitz, & Sessitsch, 2014). There are almost 297,326 species of Gymnosperms, Mono- cotyledons, Dicotyledons, Ferns, Mosses and allies are known from International Union for Conservation of Nature and Natural Resources but very are reported from endophyte microbiota (G. Strobel & Daisy, 2003). Researchers observed that plants act as reservoirs for a number of microbial species that are known as endophytes(Kaul, Gupta, Ahmed, & Dhar, 2012). Studies shown that plants act as repository for number of microbial species that are known for bioactive product formation, commonly referred as secondary metabolites (Makkar, Siddhuraju, & Becker, 2007). The unknown and important compounds that gives assis- tance and pharmacological importance in the present scenario of the pandemic is the need of the hour. The resistance of drugs in the microbes, the life-threatening viruses, the recur- rent various diseases issues in the mankind along with multi- ple organ transplants, and the massive hike in the occurrence 109

of fungal diseases in the globe causing stressful environment due to our repeated failures in treating these health issues. From onset of the COVID pandemic, it has become clear that the economic globalization has inadvertently aided, abetted and triggered biological pollution that has contributed to cat- astrophic economic and ecological ramifications. All microorganisms that inhabit, at least for one period of their life cycle, the interior of a vegetable, may be considered as an endophyte(Azevedo, Maccheroni Jr, Pereira, & De Araújo, 2000). Mycorrhyzae and nitrogen-fixing bacteria al- so live in an intimate relationship with their hosts and could be considered as endophytic microorganisms. However, my- corrhyzae are distinguished from other root endophytes by the fact that they possess external structures as hyphae. Likewise, nitrogen-fixing endophytic bacteria such as Rhizo- bium, which form external structures called nodules, are dis- tinguished from other root endophytic bacteria (Favre-Godal, Gourguillon, Lordel-Madeleine, Gindro, & Choisy, 2020). Microorganisms on roots and in the rhizosphere benefit from root exudates, but some bacteria and fungi are capable of en- tering the plant as endophytes that do not cause harm and could establish a mutualistic association (X.-F. Huang et al., 2014). Plants constitute vast and diverse niches for endophyt- ic microorganisms (Batra et al., 2018). Endophytic fungi and bacteria have been isolated from a large diversity of plants as reviewed by Sturz and associates (Sturz, Christie, & Nowak, 2000). It seems there is not a single plant species devoid of endophytes. Endophytic microorganisms only received considerable attention in the last 20 years, when their capacity to protect their hosts against insects-pests, pathogens and even domes- tic herbivores as sheep and cattle was recognized. As endo- 110

phytic fungi and bacteria could confer other important char- acteristics to plants, such as greater resistance to stress con- ditions (i.e. water), alteration in physiological properties, production of phyto-hormones and other compounds of bio- technological interest (i.e. enzymes and pharmaceutical drugs). It has also come to know that certain microbes, se- lected from their natural ecological settings, may provide new and useful leads for industrial, medicinal and agricultur- al product discovery(G. Strobel, Daisy, Castillo, & Harper, 2004). Endophytic symbionts can have profound effects on plant ecology, fitness and evolution (RJ Rodriguez, White Jr, Ar- nold, & Redman, 2009). The fossil record indicates that plant have been associated with endophytic fungi, for >400 million years and were likely associated when plants colonized land, thus having an important role in driving the evolution of life on land (Rusty Rodriguez & Redman, 2008). Endophytic fungi are major subunit that amplify in healthy tissues of plants and can be isolated. They behave as source of novel bioactive products like terpenoids, alkaloids, quinones, phenolic acids, saponins, steroids, and tannins that act as a potential applicant for antimicrobial, anticancer, anti- insect and many other pharmacological properties (Gouda, Das, Sen, Shin, & Patra, 2016). Their extensive diversity can provide a good compass for the search in the area of novel drug-like molecules (Figure 1). This study aims to grasp at- tention in the contribution and utilization of endophytes and their relationship with their host medicinal plants. 111

Alkaloids Perylene Terpenoids derivatives Quinones Shikimates Isocoumarins Secondary Furandiones Depsipeptides Metabolites Steroids Glycosides Phenols Plant microbe Isoprenoids Polyketides interaction Host Xanthones Plant Bio-prospection Antibiofilm Antiinflammatory Antioxidant Antimicrobial Anticancer Figure 1 Schematic diagram of Natural Products from Endophytes Endophyte-Host Interaction : Endophytes interact with host plants with the symbiotic, mutualistic or parasitic association inside the living part of the host plant. The number of difficulties arises during the interaction between host and microbe. Generally, microbes enter into the plant through soil into the seed and travels to the different parts of the host plant. In this association, mi- crobes developed into the plants and takes the source of nu- trition through the host plant and synthesize several com- pounds that gives support to their host from various stresses like biotic and abiotic. Bioactive Metabolites from Endophytes Natural therapy with the advent of flora and fauna availa- ble at our planet has been the main source of cure since cen- turies. Our Indian systems of medicine i.e. Ayurveda has been the source of inspiration towards use of herbals as med- icines. Eventually other source have been emerged time to time to combat with newer diseases and challenges. During the last century, majority of new drugs have been discovered from organic natural products (secondary metabolites) isolat- ed from plants and microbial species. Large numbers of US- FDA approved drugs available in the market are natural 112

products or their semi-synthetic derivatives/analogues of nat- ural products(Divyashree et al., 2020). Despite several success stories, the pharmaceutical re- search on natural products has declined during last few dec- ades. During this period research has been inclined towards combinatorial chemistry and high throughput screening. However, this shift in strategy led to dramatic decline in number of new drug approvals in the last decades. Hence, the focus is bound to shift back to natural product-based drug discovery. Natural products may be extracted from tissues of terrestrial plants, marine organisms or microorganism fer- mentation broths. A crude (untreated) extract from any one of these sources typically contains novel, structurally diverse chemical compounds, which the natural environment is a rich source (S. V. Kumar, Saravanan, Kumar, & Jayakumar, 2014). Chemical diversity in nature is based on biological and geographical diversity, so researchers travel around the world obtaining samples to analyse and evaluate in for medicinal aspects or bioassays. This effort to search for natural prod- ucts is known as bioprospecting. Plants have always been a rich source of lead compounds e.g alkaloids, morphine, nico- tine etc. Several lead compounds are useful drugs in them- selves (e.g. Alkaloids, morphine and quinine), and others have been the basis for synthetic drugs (Lahlou, 2013). Clinically useful drugs which have been isolated from plants include the anticancer drugs, such as paclitaxel from the yew tree, and an anti-malarial drug artemisinin from Ar- temisia annua. Generally, plants provide rich, complex and highly varied structures which are not easy to synthesize the laboratory. However, as a source of the number of plants that 113

have been extensively studied is relatively very few and the vast majority have not been studied at all. With the emerging sense of deforestation and its effects on environment, activists as well as various forest depart- ments have some strict regulations for plant material collec- tions for research purposes also (Bergman, Davis, & Phillips, 2019). Natural products remain a consistent source of drug leads with more than 40% of new chemical entities (NCEs) reported from 1981 to 2005 having been derived from micro- organisms (Newman & Cragg, 2012). Further, more than 60% of the anticancer and 70% of the antimicrobial drugs currently in clinical use are natural products or natural prod- uct derivatives (Kinghorn, Chin, & Swanson, 2009). This is not surprising in the light of their evolution over millions of years in diverse ecological niches and natural habitats. In comparison to other natural sources like plants, microorganisms are highly diverse but underexplored. Stud- ies based on estimation of microbial populations have re- vealed that only about 1% of bacteria and 5% of fungi have been characterized and the rest remain unexplored for their contribution to the human welfare (Qadri et al., 2013). (Table 1) The Microbial World: Alexander Fleming began the microbial drug era in 1928, when he discovered accidently discovered that a compound produced by a mold killed the bateria Staphylococcus aureus (Diggins, 1999). The mold was identified as Penicillium no- tatum, produced an active agent, penicillin (Gaynes, 2017). Using this method, other naturally occurring substances e.g. chloramphenicol and streptomycin, were also isolated (Yil- maz, Sova, & Ergün, 2018). Naturally occurring antibiotics 114

are produced by fermentation. Microorganisms such as bac- teria and fungi have been invaluable for discovering drugs and lead compounds. These microorganisms produce a large variety of antimicrobial agents which have evolved to give their hosts an advantage over their competitors in the micro- biological world (Mohr, 2016). The screening of microorgan- isms became highly popular after the discovery of penicillin. Soil and water samples were collected from all over the world in order to study new bacterial or fungal strains, lead- ing to an impressive arsenal of antibacterial agents such as the cephalosporins, tetracyclines, aminoglycosides, ri- famycins, and chloramphenicol (Wright, Seiple, & Myers, 2014). There are distinct groups of organisms, namely unicellular bacteria, eukaryotic fungi and Actinomycetes being the most frequent and versatile producer of novel antimicrobial agents. The filamentous Actinomycetales species produces over 10000 bioactive compounds, 7600 derived from streptomy- ces and 2500 from rare Actinimycetes species, represent the largest group 45% of bioactive microbial metabolites. Vast numbers of these antimicrobial agents are discovered from Actinomycetes by screening natural habitat such as soil and water bodies (Berdy, 2005). Although most of the drugs derived from microorganisms are used in antibacterial therapy, some microbial metabolites have provided lead compounds in other fields of medicine. For example, asperlicin - isolated from Aspergillus alliaceus - is a novel antagonist of a peptide hormone called cholecys- tokinin (CCK) which is involved in the control of appetite. CCK also acts as a neurotransmitter in the brain and is thought to be involved in panic attacks (Roques, 2006). Other examples include the fungal metabolite lovastatin, which was 115

the lead compound for a series of drugs that lower cholester- ol levels, and another fungal metabolite called ciclosporin which is used to suppress the immune response after trans- plantation operations (Amedei & M D'Elios, 2012). Impact of New chemical entities (NCEs): Natural products remain a consistent source of drug dis- coveries; this is not surprising in the light of their evolution over millions of years in diverse ecological niches and natu- ral habitats. The potential of microorganisms is further lim- ited by the presence of orphan biosynthetic pathways that remain unexpressed under general laboratory conditions (Omeje et al., 2017). However, the vast choice of techniques pertaining to the growth and manipulation of microorganisms like media engineering, co-culture(Arora et al., 2016), chem- ical induction (Abrol, Kushwaha, Arora, Mallubhotla, & Jaglan, 2021), epigenetic modulation and metabolite remod- elling (Bhat, Riyaz-Ul-Hassan, Ahmad, Srivastava, & Johri, 2013), coupled with the fermentation technology for scale up, make them suitable for production of useful natural prod- ucts, both known and novel (Qadri et al., 2013). Thus, it has become imperative to explore microorganisms for NCEs (New chemical entities) and lead-drug-molecules to run sustainable programs like drug discovery. Consequent- ly, bio-prospecting of microorganisms is carried out from every possible source, including extreme environments like ocean beds, geothermal vents, cold desserts etc., in search of novel strains with promising bioactivities (Aswani, Tijith, George, & Jisha, 2017; Qadri et al., 2013). Endophytic microorganisms are excellent sources of bio- active natural products that can be used to satisfy demand of pharmaceutical, medical agriculture and industries (Jalgaon- 116

wala, Mohite, & Mahajan, 2011). Interest in endophytes in- creased immensely with the discovery of an endophytic fun- gus Taxomyces andreanae, from taxol producing plant Taxus brevifolia, producing the billion dollar anti-cancer drug, tax- ol. Numerous bioactive molecules have been isolated from endophytic fungi since this ground breaking discovery (Ti- wari & Bae, 2022). Endophytes are metabolically more active than their free counterparts due to their specific functions in nature and ac- tivation of various metabolic pathways to survive in the host tissues (Hamilton, Gundel, Helander, & Saikkonen, 2012). As the previous research on endophytes mainly focused on search for the host-plant metabolites in the endophytic part- ner, the theory of horizontal transfer from the host plant to its microbial symbiont received much impetus (Skillings, 2016). However, the sequencing of the Taxadiene synthase gene from the taxol-producing endophyte revealed that endophytes possess biosynthetic pathways independent of the plant host (Yang et al., 2014). This indicates that microorganisms have much more bio- synthetic proficiency than previously thought. Thus, micro- organisms may be screened for a wide range of biological ac- tivities and explored for useful chemical entities consistently produced by them. Establishment of microbial repositories from various ecological niches is an important step towards tapping their potential for human welfare, including drug dis- covery and sustainable agriculture (Rana et al., 2019). Re- searchers assumed that endophytic fungi’s have the ability to synthesize compounds associated with those of their host plants for example, camptothecine, hypericin, vinblastine, paclitaxel, podophyllotoxin, and diosgenin (V. Kumar, Rai, Gaur, & Fatima, 2014). 117

The recent study has shown that volatile organic com- pounds (VOC’s) i.e. green chemicals such as hydrocarbon like substances isolated from endophytic fungi, have a poten- tial to be a bio-diesel, called myco-diesel. Furthermore, Strobel et al (2010) research group studied VOCs of G. roseum and found them to be constituting of lower mass ali- phatic alkane derivatives similar to the makeup of diesel fuel and therefore they suggested this VOC mixture of G. roseum fungus has been dubbed as mycodiesel (G. A. Strobel et al., 2010). VOCs produced by endophytic fungi are secondary me- tabolites such as mono-alkanes, cyclohexanes, cyclopen- tanes, terpenoids, alkyl alcohols/ketols, benzenes, and poly- aromatic hydrocarbons (Babu & Sarma, 2019). Most of these VOCs are closely related to the compounds that are present in petroleum diesel and interestingly VOCs are produced from celluloses and hemicelluloses found in all plant-based agricultural wastes (G. Strobel, 2014). A broad spectrum of VOCs is produced by endophytic fungi and most of the com- pounds are related volatiles that are present in petroleum dis- tillate fuels. The biological production VOCs by fungi is dynamic. Depending on the substrate, nutrients, incubation periods, and environmental factors, the profiles of VOCs vary (Mor- ath, Hung, & Bennett, 2012; Thorn & Greenman, 2012). Hy- drocarbons and their derivatives produced by endophytic fungi have fuel potential (Naik, 2018). Mixtures of straight- chain hydrocarbons and acetyl esters of straight and branched hydrocarbons, long-chain alcohol and acids, and various mono- and sesquiterpens have been dubbed as mycodiesel (Babu & Sarma, 2019). 118

Conclusion : Endophyte comprise of variety of species present in dif- ferent ecosystems and have capacity to synthesize bioactive products that will be referred as novel drug discovery. They can produce same or similar metabolites isolated from their host plants. It act as a reservoir of genetic diversity. Search for novel drugs is the need of the hour as many microorgan- isms have developed resistance against the various drugs and diseases. It is also important for the development of efficient and effective antibiotic against deadly pathogens like in the era of COVID pandemic. However, it is necessary to understand the host plant en- dophytes, physical and chemical characteristics, its pathways, defensive mechanism, and secondary metabolite production. Moreover, the modern biotechnological applications and its advantages uses in different fields such as the medical, pharmaceutical, food and cosmetics to obtain different natu- ral products that are required in the present scenario and en- courages the researcher to contribute. Future Prospective Despite of exhaustive reports has been done in the last few decades, the sustainable industrial processes for the produc- tion of pharmaceutical products is still pending. There are number of untapped resources for the discovery of NCEs like temperate rainforest having major microbial biodiversity. Moreover, conventional techniques like isolation and cultiva- tion are laborious and time-consuming. Also, these microbes are growth specific as it can only be grown if selective com- ponents similar to their environment is available. Hence, well established in-vitro conditions will give new way to natural products with pharmacological importance. The host- 119

microbe interaction generally affects their metabolic path- ways in natural ecology. Therefore, identification and selec- tion of capable endophyte and evolution of sustainable pro- cesses using the selected microbe have always been a chal- lenging task in the research. It has been evaluated that only 1% of endophytes are cur- rently discovered and identified, recommended that number of natural products and their natural sources are yet to be studied. Also, the presently known culture strategies are competent in discovering only a micro fraction of microbes from the different ecological niches. Henceforth, evolution of more effective isolation and numerate techniques is the need of the hour. The study of the endophytes, their interactions with the host plant and other microbes, and their biotechno- logical applications can be further studied using various bio- logical and bioinformatics. Table 1: List of bioactive metabolites from natural source S.No. Host Plant Endophytes Secondary Me- References tabolites 1. Taxus cus- Alternaria Paclitaxel (Swamy et al., 2022) pidata sp 2. Tripterygi- Rhinocladi- Cytochalasin (Hridoy et Cytochalasin H al., 2022) um wil- ella sp Cytochalasin J Epoxycyto- (Kusari, fordii chalasin Zühlke, & Podophyllotoxin Spiteller, 3. Juniperus Aspergillus 2011) communis fumigates Podophyl- Phila- lum pelta- locephala tum fortinii 120

Juniperus Fusarium Polyketide (S. Kumar, recurva oxysporum Aharwal, 4. Annona Fusarium Vincristine Shukla, squamosa oxysporum Topotecan Rajak, & Sandhu, 5. Catharant Mycelia 2014) hus roseus Sterilia (Kharwar, 6. Camp- Fusarium Verma, Strobel, & totheca solani Ezra, 2008) (Shweta et al., 2010) acuminata 7. Nothapo- Entro- Camptothecin (Pur et al., 2007) dytes foeti- phospora da infrequens 8. Torreya Pestalo- Torreyanic acid (Talukdar, Paul, & taxifolia tiopsis mi- Taxol Tayung, Pestacin 2021) crosporum Isopestacin (Swamy & Vasamsetti, 9. Taxus Taxomyces 2022) (Tiwari & brevifolia andreanae Bae, 2022) 10. Terminalia Pestalo- Isobenzo- (X.-Z. moroben- tiopsis mi- furanone Huang et al., sis crospora 2012) Graphislactone (Song, 11. Sinarundi- Cephalo- A Huang, Sun, naria ni- sporium sp. Wang, & tida Tan, 2005) 12. Trachelo- spermum jasmi- noides 121

13. Nerium Chaetomium Flavonoids (W.-Y. oleander sp. Phenolic Huang, Cai, acids Hyde, Corke, & Sun, 2007) 14. Ginkgo Xylaria sp. (Liu et al., biloba 2007) 15. Cajanus Fusarium sp. Cajaninstilbene (Zhao et al., cajan acid 2012) 16. Medicinal Cytonaema Cytonic acid A (Idris, Al- Plant sp. &B tahir, & Idris, 2013) 17. Juniperus Aspergillus Podophyllotoxin (Dar et al., communis fumigates 2013) Podophyl- Phila- lum pelta- locephala tum fortinii Juniperus Fusarium recurva oxysporum 18. Lannea Colleto- 9- octadecena- (Premjanu & co- trichum mide, hexade- Jaynthy, rammen- gloeospori- canamide, di- 2015) dalica oides ethyl pythalate, 2-methyl-3- methyl-3-hexene and 3-ethyl-2,4- dimethyi- pentane 19. Swietenia Aspergillus Di-n-octyl (Yin, macro- terreus phthalate Ibrahim, & phylla Lee, 2017) 20. Tec- Diaporthe Fatty acid phe- (Sharma, tonagran- phaseolorum nolic Sharma, dis L.f Abrol, 122

Panghal, & Jaglan, 2019) 21. Ipomoea Aspergillus Asperamide A (Frisvad, batatas niger and Cerebroside Møller, C Larsen, Kumar, & Arnau, 2018) 22. Rauwolfia Cladospori- Fusarubin- me- (Khan et al., serpentina um sp. thyl ether 2016) 23. Ocimum 2L-5 Ergosterol (Haque et basilicum Cerevisterol al., 2005) 24. Calyptous Stemphylium Tannins, phe- (Hateet, radicinum nols and amino 2016) acid 25. Buxus sini- Colleto- Colletotrichones (Wang et al., ca trichum sp. A, Colleto- 2016) BS4 trichones B and Colletotrichones C 26. Opuntia Fusarium sp. Equisetin (Zhang, dillenii Wang, Zhu, Yu, & Gong, 2018) 27. Laguncu- Paecilomy- Viriditoxin (Barakat & laria rac- ces variotii Saleh, 2016) emosa 28. Pteris pel- Emericella Toluene (Goutam, lucida qaudriline- Kharwar, ata Tiwari, Mishra, & 123

29. Grewia Alternaria Alternariol and Singh, 2016) asiatica alternata 3,7-dihydroxy- (Sriravali, 9-methoxy-2- Jindal, methyl- Singh, & 6Hbenzo[c]chro Paul) men-6-one 30. Entada ab- Epicoccum Beauvericin (Dzoyem et yssinica nigrum al., 2017) 31. Achy- Aspergillus Terrein (Goutam, ranthus terreus Kharwar, aspera Tiwari, Singh, & Sharma, 2020) 32. Carthamus (22E,24R)- (Elkhayat, lanatus stigmasta- Ibrahim, 5,7,22-trien-3- Mohamed, β-ol & Ross, 2016) 33. Siparuna Diaporthe Phomosine A, (Xu et al., gesneri- sp. Phomosine C 2021) oides 34. Salvia Alternaria Alternariol 9- (Tian et al., miltiorrhi- sp. methyl ether 2017) za 35. Garcinia Phomopsis 18- (Deshmukh kola sp. methoxycyto- et al., 2022) chalasin J, cyto- chalasins H and cytochalasins J 36. Rauvolfia Purpureocil- Purpureone (Kaaniche et macro- lium lilaci- al., 2019) 124

phylla num 37. Ipomoea Aspergillus Asperamide A, (Wu, batatas niger Cerebroside C Ouyang, Su, & GUO, 2008) 38. Rauwolfia Cladospori- Fusarubin- me- (Wulandari, serpentina um sp. thyl ether Examinati, Huspa, & Andayanings ih, 2018) 39. Emblica Nigrospora Griseofulvin (Rathod, officinalis oryzae Dar, Gade, Rai, & Baba, 2014) 40. Allium sa- Trichoderma Trichodermin (Shentu, tivum brevicom- Zhan, Ma, pactum Yu, & Zhang, 2014) 41. Senecio Penicillium 4- (Elkhayat & flavus sp. hydroxymellein, Goda, 2017) 6- hydroxymellein 42. Phyl- Trichotheci- Trichothecinol- (Taware et lanthus um sp. A al., 2014) amarus 43. Eugenia Myco- (2S,3R,4R)-(E)- (Carvalho et bimargina- sphaerella 2-amino-3,4- al., 2019) ta sp. dihydroxy-2- (hydroxyme- thyl)-14- oxoeicos-6,12- dienoic acid, myriocin 44. Rhododen- Penicillium Outovirin C (Kajula et 125

dron to- raciborskii . al., 2016) mentosum 45. Centaurea Trichoderma α-viridin, β- (Abdou, stoebe sp. Viridin, Shabana, & Adenosyl 9a-D- Rateb, 2020) arabino- furanoside 46. Seaweed Xylaria sp. Cytochalasin D (Singh et al., 2018) 47. Litsea hy- Phomopsis Phomocyto- (Hsiao et al., pophaea theicola chalasin, 2016) cytochalasin H, cytochalasin N, RKS-1778, dankasterone B, Cyclo(L-Ile-L- Leu) 48. Entada ab- Epicoccum Anthraquinone (Braga, yssinica nigrum quinizarin Padilla, & Araújo, 2018) 49. Elaeocar- Pestalo- Terreic acid, (Midhun & pus syl- tiopsis sp. 6- Jyothis, vestris methylsalicylic 2021) acid 50. Ficus re- Dendryphion Naphthoquinone (Murugesu, ligiosa nanum (Herbarin) Selamat, & Perumal, 2021) 51. Hintonia MEXU Thielavins A, J (Rivera- latiflora 27095 and K Chávez, González- Andrade, del Carmen González, 126

Glenn, & Mata, 2013) 52. Salvia Alternaria Alternariol 9- (Lou et al., miltiorrhi- sp. methyl ether 2016) za 53. Panax no- Penicillium Brefeldin (Xie et al., toginseng sp. 2017) 54. Quercus Alternaria Altertoxin (Bashyal et emoryi tenuissima I,II,III,V al., 2014) 55. Cephalo- Homoharring- (Hu et al., taxus hai- tonine 2016) nanensis Li Contact: Dr. Sarojini Johri - [email protected] Dr. Asha Chaubey - [email protected] Dr. Vidushi Abrol - [email protected] 127



Chapter 5 The Super food: Spices Dr. Rekha Sapru Dhar & Dr. Vidushi Mahajan Introduction: For the betterment of life, humans have a long history of exploring and utilizing several natural resources in their lives in the form of nutracueticals and pharmacueticals. These re- sources (Nature's cure) include plants, bacteria, herbs, spices and others. Plants are the prominent and efficient source of secondary metabolites/phytochemicals, which have been uti- lised / exploited to develop several drugs for various diseas- es. Such medicinal properties of plants led to the investiga- tion now from more than a decade in the scientific world due to their potential therapuetic activities and, less toxicity. How extraordinary it is to know that herbs and spices such as spirilluna, turmeric, cinnamon, ginger, ginseng dating mil- lions of years ago, afford us to have affordable life saving medicines that have helped us to maintain our ecosystem bal- ance as well as maintain our health. Nature has always had & will continue to have abundant of natural & organic antidotes to our disease-creating lifestyle. Every seed, every fruit, eve- ry leaf, every root, every flower, every herb, and every tree tells a story that is profound and reveals how each of them contribute in healing human suffering caused by diseases. Even algae’s found in the ocean’s, that are million years old - the oldest form of life-forms on the planet, is a super food that is studied to heal forty four diseases, that was ini- tially dismissed by the medical practioniers as “pond scum”, also known as cyano-bacteria. Algae’s such as spirulina or chorella are known to prevent or cure allergic rhinitis, arthri- tis, cataracts, corneal disease, inflammation, obesity, hyper- tension, HIV, & cholesterol. 129

The Indian traditional systems of medicines have devel- oped many formulations from the plant extracts. Plants have been used on the basis of a single molecule or a group of molecules which showed an overall activity. Similarly, spices are also one of the main resources and an integral part of diet especially in India, which has been used for food flavourings, food preservatives and for medicinal purposes. The name spice is derived from the word species, which was applied to groups of exotic foodstuffs in the middle Ages. Aromatically scented herbal products have been used since ancient times to flavor foods and for preparing incenses and perfumes. Exotic imports obtained from Asia were particularly ap- pealing to Greeks and Romans, who spent vast fortunes on trade with Arabia, which was the center of the spice trade. Rare spices were utilized in cooking as a sign of wealth in Rome, and later in Medieval and Renaissance times, and the privileged developed an exaggerated taste for spicy foods. Today, many of the valued old spices, such as nutmeg, have lost their fabulous attraction, while the more lowly garlic, peppers and other commonplace kitchen herbs have become, paradoxically, increasingly popular. It is now impossible to give a strict definition of a spice: the word suggests an im- ported tropical herbal plant or some part of it that is valued for providing color and aromatic flavoring along with stimu- lating odor for use in cooking and in condiments, as well as in candies, cosmetics, fragrances and medications. In the late 18th century, British ships began carrying citrus fruits on their ships in order to prevent deadly scurvy disease to their sailors. At that time no one knew that it was Vit. C that was responsible for preventing scurvy, only until 1928 Dr. Albert Szent-Györgyi and later 1933 Dr. Haworth dis- covered it and its structure respectively in their laboratories, that affirmed this intuition & observation from 18th century British mariners. It was in 1948 that scientist started to take 130

note & discover these “invisible” compounds within fruits, vegetables, herbs and marine plants, and called them phyto- nutrients or phytochemicals. It was only in 1980’s, National Cancer Institute chemoprevention program began studying the role of different phytochemicals in its medicinal proper- ties to either prevent or cure diseases. The consumption of herbs and spices has increased tre- mendously worldwide. From 1995 to 1999, annual world im- ports of spices averaged 500,000 tonnes, growing at an aver- age 8.5% a year. This strong growth rate is a good indicator of the increase in consumption of spices. The sale growth in UK and USA has increased by 20-25% for the last five years. In Australia the market for local fresh-cut culinary herbs was estimated to be worth over $62 million per year in 2004 and continues to grow at 20% per annum. Information from ma- jor supermarket sales in 2003 estimates suggest that total re- tail sales of fresh herbs and spices were valued at $54 mil- lion, and a further $107 million for dried products. The world market for imported spices and culinary herbs is large, val- ued at just over US$ 2.3 billion. But, India is the largest producer, consumer, and exporter of spices in the world. The demand scenario for major spices in India has been comprehensively examined. (Shinoj P. and V.C.Mathur, Agricultural Economics Research Review Vol. 19 July-December 2006 pp 367-376). The annual growth rates in area and production have been estimated to be 3.6 per cent and 5.6 per cent, respectively for the year 2003 (Survey of Indian Agriculture, 2004). In the year 2002, the production of spices in India had reached a level of 3.08 mil- lion tonnes on 2.60 million hectares of land (Economic Sur- vey, 2002-03). India has certain natural comparative advantages with re- spect to production and utilization of spices; these include 131

diverse agro-climatic production environments, availability of innumerable varieties and cultivars of each spice suitable for different climatic conditions, cheap labour, large domes- tic market and a strong tradition of using spices and their products in food, medicine and cosmetics. This is the reason that in almost all the states and union territories of India, at least one spice is grown in abundance. India is not only the largest producer but also the largest consumer of spices in the world. Historically, spices have enjoyed a rich tradition of use for their flavour enhancement characteristics and for their medic- inal properties. The rising prevalence of chronic diseases worldwide and the corresponding rise in health care costs is propelling interest among researchers and the public for mul- tiple health benefits related to these food items, including an- timicrobial, antioxidant, a reduction in cancer risk and modi- fication of tumor behaviour. Spices can improve the palata- bility and the appeal of dull diets or spoiled food. The Pi- quant flavours stimulate salivation and promote digestion. Pungent spices can cause sweating, which may even cause a cooling sensation in tropical climates; on the other hand they can add a sense of inner warmth when present in cooked foods used in cold climates. Spices also fitted into philosophic concepts of improving health, since it was understood that they could affect the four humors (blood, phlegm, yellow bile and black bile) and in- fluence the corresponding moods (sanguine, phlegmatic, choleric and melancholic). Thus, ginger would be used to heat the stomach and improve digestion; clove was believed to comfort the sinews; mace would prevent colic and bloody fluxes or diarrhea; nutmeg would benefit the spleen and re- lieve any bad cold. Cinnamon, one of the most popular fla- vours in cooking, was considered to be particularly good for digestion and for sore throats Even so, a little data does exist 132

with regards to their medicinal values. Here, we tried to dis- cuss some very important herbs and spices for their valuable medicinal attributes which can be taken by researchers to ex- plore them for their molecules to develop drugs. This chapter has featured only top five most important spices that are super foods - arsenals of natural protection & contribute to boosting our immunity. These herbs and spices are 1. curcumin, 2. garlic, 3. ginger, 4. cinnamon, and 5. pepper. Curcumin: Curcumin contributes to anti-aging, and reducing inflam- mation.”Ageing is manifested by the decreasing the health status and increasing probability to acquired age-related dis- ease such as cancer, Alzheimer, atherosclerosis, metabolic disorders and others. Studies suggest that turmeric can im- prove brain function, fight Alzheimer’s, reduce the risk of heart disease and cancer, and relieve arthritis. These diseases are on-set due to low grade inflammation driven by oxygen stress and manifested by the increased level of pro- inflammatory cytokines. It is believed that aging is plastic and can be slowed down by caloric restrictions as well as by some nutraceuticals. Ac- cordingly, slowing down ageing and postponing the onset of age-related diseases might be achieved by blocking the NF- kappa B-dependent inflammation. Researchers consider the possibility of the spice curcumin, a powerful anti-oxidant and anti-inflammatory agent contributing towards anti aging”. (Sikora, Scapagiini Ageing, 2010 January, 17:7). Researchers have observed that curcumin extends life span, improves immunity, and modulates the expression of genes associated aging in Drosophila melanogaster. The abil- ity of curcumin to mitigate the expression levels of gene is a cause, rather than an effect, of its life span-extending effects. 133

(Lee, KS, Lee BS, Semnani S, et al. Rejuvenation Res. 2010 Oct. 13(5), 561-70). It has come to understand that oxidative stress plays a pivotal role in triggering neuro degenerative disorders such as Alzheimer. Researchers have noticed that curcumin from turmeric has anti-oxidant property, a promising compound; for the development of Alzheimer disease therapies. (Hama- guchi T, Ono K, Yamada M, CNS Neurosci Ther. 2010 Oct; 16(5), 285-97.) This has been confirmed by researchers stud- ying curcuminoids in an amyloid infused rat model of alz- heimer disease. (Ahmed T, Enam SA, Gilani AH, Neurosci- ence 2010, Sept 1, 169(3): 1296-306.) This is further af- firmed by a study which demonstrates that curcumin im- proves the “memory ability of Alzheimer diseased mice and inhibits apoptosis in cultured PC12 cells induced by AICI, whose mechanism might involve enhancing the level of Bcl- 2”. (Pan R, Qui S, Lu DX, Dong J, Chin Med J (English), 2008 May 5, 121(9):832-9.) Researchers have also studied that turmeric has increased detoxification systems apart from being a strong anti-oxidant, which assists in mitigating the effects of several dietary car- cinogens. (Goud VK, Polasa K, Krishnaswamy K, Plant Foods for Human Nutrition, 1993, July ; Vol 44, number 1:87-92). Curcumin is known to be essential in both redox reactions and scavenging of oxygen radicals which is im- portant for maintaining good immunity. ((Hjorth TH, Green- hill JV, International Journal of Pharmaceutics, 1992, vol 87, number 1-3: 79-87). “The activation of nuclear transcription factor Kappa-B has now been linked with a variety of inflammatory diseases, including cancer, atherosclerosis, myocardial infarction, dia- betes, allergy, asthma, Crohn’s disease, multiple sclerosis, Alzheimer’s disease, osteoporosis, psoriasis, septic shock, and AIDS. Researchers have studied that the pathway that 134

activates this transcription factor can be interrupted by phy- tochemicals derived from spices such as turmeric (curcumin), red pepper (capsaicin), cloves (eugenol), ginger (gingerol), cumin, anise, and fennel (anethol), basil and rosemary (ur- solic acid), garlic (diallyl sulphide, S-ally-mercapto-cysteine, ajone), and pomegranate (ellagic acid). Researchers therefore believe there is every reason for seasoning our food, with these herbs and spices. (Aggrawal BB, Shishodia S, NY Academy of Science, 2004, December, 1030:434-41). Garlic: Garlic is known to have medicinal properties for disease prevention and healing, that puts it in the top ten super-foods category. Researchers have documented that garlic can pre- vent or treat thirty two health conditions. Researchers have further studied that garlic can significantly reduce risk of at- least fifteen different cancer, making it one of the Nature’s greatest cancer fighter. Compelling evidence has indicated that garlic’s health benefits include reducing the risk of car- diovascular disease, stroke, cancer and aging. (Borek C, Journal of Nutrition, 2001, March 131(3s):1010S-5S.) The anti-cancer properties of garlic have contributed to lowering of serum cholesterol and triglycerides levels through the inhibition of their bio-synthesis in liver, and to inhibit oxidation of low density lipoprotein, that are respon- sible for onset of cardiovascular diseases. Furthermore, in- vivo & in-vitro studies have revealed that aged garlic extract stimulated immune functions such as proliferation of lym- phocytes, cytokine release, NK activity, and phagocytosis. (Sumiyoshi H, Nippon Yakurigaku Zasshi (Japan) 1997, Oc- tober; 110 Suppl 1, 93P-97P.) The anti-bacterial – anti- staphylococcal properties of gar- lic are more potent than an equal amount of allicin. The oxy- 135

gen in allicin, functions to liberate the s-ally moiety which destroys the growth of bacteria. (Fujisawa H, Watanabe K, Suma K, Origuchi K, Matsu- fuji H, Seki T, Ariga R, T. Biosci Biotechnol Biochem 2009, Sept; 73(9): 1948-55.) Researchers have also confirmed that garlic used as a dietary supplement may be beneficial in in- creasing the antioxidant capacity of the body. (Koseoglu M, Isleten F, Atay A, Kaplan YC Phytother Res 2009, August 3). The thiacremonone, a novel isolated compound from gar- lic, exerts anti-inflammatory and anti-arthritic properties through the inhibition of NF KappaB activation via interac- tion with the sulfhydryl group of NF- kappaB molecules, could be a useful drug to treat inflammation and arthritis. (Ban JO, Oh JH, Kim TM, Kim DJ, Jeong HS, Han SB, Hong JT. Arthritis Rs Ther.2009, Sept 30, 11(5):R145). An important garlic derivative, Diallyl disulfide has demonstrated to exert a potential molecular target against dif- ferent human cancers. Researchers studied diallyl disulfide induced expressions in breast, prostate, and lung cancer cells, showing caspase-dependent apoptosis in human cancer cells through a Bax-triggered mitochondrial pathway. (Nagraj NS, Anilakumar KR, Singh OV, Journal of Nutr Biochem, 2010, May, 21(5): 405-12.) Researchers have also studied the important role of garlic oil to inhibit cancer cell proliferation, which occurs by inhib- iting cyclin E expression in cultured SGC7901 cells as well as TGF alpha treated cells, indicating that it inhibits TGF al- pha autocrine and paracrine loops that are responsible for cell decay. (Liang WJ, Yan X, Zhang WD, Luo RC, Nan Fang, Yi Ke, Da Xue, Xue Bao, (China) 2007 Aug, 27(8): 1241-3.) It has come know to the scientific community, that the an- ticancer property within garlic is brought about by number of mechanisms such as the scavenging of radicals, increasing 136

glutathione levels, increasing the activities of enzymes, inhi- bition of cytochrome p4502E1, DNA repair mechanisms, and prevention of chromosomal damage etc. (Khanum F, Anil- akumar KR, Viswanathan KR, Crit Rev Food Sci Nutr, 2004, 44(6): 479-88.) Researchers have also found that garlic compounds has been found to block covalent binding of carcinogens to DNA, enhances degradation of carcinogens, exhibits anti- oxidative and free radical scavenging properties, and regu- lates cell proliferation, apoptosis and immune responses. This has paved way to a new field i.e. cancer chemoprevention. (Das S, Asian Pacific Journal Cancer Prevention 2002, 3(4):305-311.) Significant research has been conducted on the common herbs & spices such as garlic, black cumin, cloves, cinna- mon, thyme, all spices, bay leaves, mustard, and rosemary that exhibit anti-microbial property. While, other spices such as saffron, turmeric, green or black tea, & flaxseeds do con- tain phytochemicals such as carotenoids, curcumin, cate- chins, and lignin which exhibit anti-cancer property. (Lai PK, Roy J. Curr Med Chem. 2004 June; 11(11): 1451-60.) Researchers have found that garlic boosts human body’s own product of hydrogen sulphide, which relaxes blood ves- sels, increases blood flow, and prevents blood clots and oxi- dative damage. This occurs when allicin and other com- pounds in garlic are metabolized, a chemical messenger hy- drogen sulphide is produced which is responsible for cellular signalling that are responsible for cardiovascular protection. (Benavides GA, Squadrito GI, Mills RW, Patel HD, Isbell TS, Patel RP, Darley-Usmar VM, Doeiler JE, Kraus DW. Proceedings of National Academies of Science 2007, No- vember 13, 104(46):17977-82. 137

Cinnamon: Cinnamon is a traders spice whose route is seen through old testament of the Holy Bible in Jerusalem, as both as a perfume and as spice, to Egypt used by pharaohs as a most demanding spice in daily cuisine, to Roman Empire as a medicine and spice. Its use has been recorded in the twentieth century for treating diabetes, immune booster, hypertension, and treating cardiovascular ailments. Researchers have studied the cinnamon oil isolated from its twigs and leaves, and have found it to be exhibiting excel- lent anti-inflammatory property. (Tung YT, Chua MT, Wang SY, Chang ST, Bioresour Tchnol 2008, June; 99(9): 3908- 13.) Researchers have also confirmed the hypothesis that if you intake cinnamon dissolved in water, it will reduce the risks related to diabetes and cardiovascular diseases in obese people. (Roussel AM, Ininger I, Benaraba R, Ziegenfus TN, Anderson RA, Journal of American Coll Nutrition, 2009 Feb;28(1):16-21.) Researchers have also confirmed cinnamon is effective, and induces active cell death against tumor cell proliferation in lymphoma, melanoma, cervix, colorectal cancers, through inhibition of NF Kappa B and AP1. (Kwon HK, Hwang JS, So JS, et al BMC Cancer, 2010 July 24; 10:392.) Cinnamon intake with food is associated with decreasing & preventing the metabolic syndrome, which is a cluster of conditions that occur together increasing the possibility of one getting cardiovascular related diseases, and diabetes type 2. This syndrome is associated with insulin resistance, ele- vated glucose & lipids, inflammation, decreased antioxidant activity, increased weight gain, and increased glycation of proteins. (Qin B, Panickar KS, Anderson RA, Journal of Di- abetes Sci Technol, 2010 May 1; 2(3):685-93.) 138

Ingestion of cinnamon with water has shown to improve glucose tolerance and insulin sensitivity. This indicates that the anti-oxidant property of cinnamon and its water soluble compounds have reduced the risk factors associated with di- abetes and cardiovascular disease. (SolomonTP, Blannin AK, European Journal of Applied Physiology, 2009, April; 105(6); 969-76.) Ginger: Ginger has been present in the late twienth century and early twenty first century, as the healing food, primarily be- cause it has shown to be an antidote to certain cancers, such as ovarian and skin. It has also shown effective against alz- heimer disease, atherosclerosis, cancer, cholesterol, ulcera- tive colitis, crohn’s disease, & obesity. The extensive research on ginger has shown that the pathway that activates nuclear transcription factor kappaB can be interrupted by phytochemicals derived from spices such as ginger (gingerol), cloves (eugenol), red pepper (cap- saicin) etc. It was observed by the researchers that it is this activation factor that is linked to variety of inflammatory dis- eases such as cancer, atherosclerosis, myocardial infarction, diabetes, allergy, asthma, arthritis, multiple sclerosis, alz- heimer, osteoporosis, psoriasis, septic shock & AIDS. (Ag- garawal BB, Shishodia S; Annual NY Academy of Science, 2004; Dec; 1030:434-41.) This research confirms the “rea- soning for seasoning”. Researchers have further confirmed through their study that ginger has exhibited the anti-alzheimer properties, as it has shown inhibition of acetyl cholinesterase activities and prevention of lipid peroxidation in the brain. (Oboh G, Ademilyui AO, Akinyemi AJ; Exp. Toxicol Pathol. 2010, Oct 15.) 139

Researchers have confirmed that consumption of ginger in daily diet, has reduced plasma cholesterol, inhibits LDL oxi- dation, and attenuates development of atherosclerosis lesions. This was concluded by the experiments performed on athero- sclerotic, apolipo-protein E-deficient mice, whose atheroscle- rotic lesions reduced significantly. (Fuhrman B, Rosenblat M, Hayek T, Coleman R, Aviram M; J Nutr 2000, May; 130(5):1124-31). 6-Shagoal, the active constituent isolated from ginger, is known to inhibit breast cancer MD-MB-231 cell invasion by reducing matrix metalloproteinase-9 expression via blockade of nuclear factor-kB activation. (Ling H, Yang H, Tan SH, et al Br J Pharmacol 2010 December; 161(8):1763-77.) Researchers have confirmed that 6-gingerol compound in ginger, possess apoptotic potential, associated with the modulation of p53 and involvement of mitochondrial signal- ling pathway in B(alpha) 9 P-induced mouse skin tumorigen- esis, as a mechanism of chemoprevention. (Nigam N, George J, Srivastava S, et al Cancer Chemother Parmacol, 2010, March 65(4):687-96.) Researchers have also concluded that dietary consumption of ginger, significantly reduces plasma & LDL cholesterol levels, reducing as well as preventing, the risk of cardiovas- cular diseases. (Fuhrman B, Rosenblat M, Hayek T, Coleman R, Aviram M; J Nutr 2000, May; 130(5):1124-31). Researchers have confirmed that curcumin-induced altera- tions reverse insulin resistance, hyperglycemia, hyper- lipidemia and other symptoms linked to obesity. Other struc- turally similar homologous nutraceuticals, derived from red chilli, cinnamon, cloves, black pepper, and ginger also exhib- it effects against obesity and insulin resistance. 140

Pepper: Capsicum is a genus of a flowering plant, known to be first cultivated and grown in the Americas. The fruits of this plant include chilli peppers & bell peppers. Cayenne peppers and paprika powder are made from grinding dried peppers. Capsaicin is the main flavanoid in pepper that imparts the an- ti-cancer, antioxidant, anti-proliferative, & anti-inflammatory properties to it. Researchers have confirmed capsaicin’s anti-cancer prop- erty on different cancer cell lines due to its ability to induce apoptosis. This was confirmed by treating capsaicin to mouse to neuro 2a cells results in the inhibition of proteasome activ- ity in a dose and time dependent manner, which seems to correlate with its effect on cell death. (Maity R, Sharma J, Jana NR; J Cell Biochem, 2010 April 1; 109(5): 933-42.) Researchers have also confirmed that black pepper exerts immunomodulatory role, and anti-tumor bioactivity through there –in-vivo experiments. (Majdalaweih AF, Carr RI; J Med Food 2010 April, 13(2):371-81.) Researchers have also confirmed that pepper also exhibits anti-cancer properties against tongue, colon, hematoma, pan- creas, breast, gall bladder, & prostate cancers. Capsaicin in- duces apoptosis and decreases the percentage of viable cells in a dose dependent manner and produced DNA fragmenta- tion and GO/GI phase arrest in tongue cancer SCC-4 cells. (Ip SW, Lan SH, Huang AC, et al, Environ Toxicol, 2010 October 5.) A researcher has studied that pepper – capsaicin in it, in- duces apoptosis in colon carcinoma cells by elevation of ni- tric oxide, which activates caspase leading to complete knockout of p53-WT cells and effectively stops the spread of two isogenic HCT-116 human colon carcinomas cells. These findings offer an exciting opportunity to treat colon cancer. 141

(Kim My, Trudel LJ, Wogan GN; Anticancer Res. 2009, Oct 29th; (10):3733-40. Researchers also confirmed that Lupeol, a dietary triter- pene microtubule targeting agent, found in pepper, has thera- peutic & chemo preventative properties for the treatment of inflammation and cancer. This was further confirmed that it overcomes resistance to TRAIL-mediated apoptosis in chemo resistant human pancreatic cancer cells. It also dis- rupts surviving / CFLIP activation in prostate cancer cells. (Saleem M, Murtaza I, Witkowsky O, Kohl AM, Maddodi N, Biochem Biophys Commun, 2009 Oct 23; 388(8); (Saleem M, Cancer Lett. 2008 Nov 28, 285(2):109-15.) Pepper’s anti-cancer property was further confirmed by researchers, by studying capsaicin induced apoptosis in hu- man breast cancer MCF-7 cells through caspase independent pathway. The researchers noted that reactive oxygen species and intracellur calcium ion fluctuation has a minimal role in this process. (Chou CC, Wu YC, Wang YF, Chou MJ, Kuo SJ, Chen DR, Oncol Rep. 2009 Mar, 21(3): 665-71.) Researchers have also confirmed that capsaicin effectively inhibited tumor growth and induced apoptosis in-vivo through oxidative stress, using NOD/SCID mice with no tox- ic side effects on leukaemia cells, making it a potential thera- peutic agent for the treatment of the disease. ( Ito K, Nakaza- to T, Yamato K, Miyakawa Y, Yamada T, Hozumi N, Sega- wa K, Ikeda Y, Kizaki M; Cancer Res 2004 Feb 1, 64(3):1071-8.) Pepper – capsaicin has also been found to be effective on lowering cholesterol levels due to its marked modulation on it as well as increasing resistance of serum lipoproteins / LDL cholesterol, to oxidation. (Ahuja KD, Ball MJ, Br J Nutr, 2006, August; 96(2): 239-42.) Pepper is also proven to be effective against obesity & in- sulin resistance. It is also known to have suppressive effect 142

on fat intake. (Yoshioka M, Imanaga M, Ueyama H, Yamane M, Kubo Y, Boivin A, St Amand J, Tanaka H, Kiyonaga A, Br J Nutr, 2004 Jun; 91(6):991-5.) Contact: Dr. Rekha Sapru - [email protected] Dr. Vidushi Mahajan - [email protected] 143

Peganun harmala, Ladakh, Science citation: Surinder Kitchlu 144

Chapter 6 Bioprospecting Marine Plant:Window to the Blue World Dr. Suphala Gupta & Dr. Niha Dhar Introduction: Oceans cover most of the earth's surface, inhabiting ap- proximately five million species, most being distinctive and unclassified. The diverse marine environment and its associ- ated chemical diversity amount to indefinite resource of nov- el compounds. Marine resources are classified as the princi- pal reservoir of natural molecules with unique chemical scaf- folds. The distinctiveness in the chemistry of metabolites produced is imparted due to adaptation in very difficult, competitive, dynamic and aggressive surroundings, which is diverse in several aspects from the terrestrial environment (Bowen et al. 2013). Oceanic plants range from single-celled organisms to large, complicated organization inhabiting near water surfaces, deep black Ocean to ice. They grow and evolve under high pressure, predominantly surrounded by sa- line water and aquatic animals adjusting to specific condi- tions, such as limited light making them phosphorescent and high flow remodelling body shape etc. Seagrasses, algae and seaweeds correspond to the majority of marine plants (Bian- chi and Morri 2000). Algae and seaweeds represent simpler forms and are often microscopic, while Seagrasses have complex organization. It is hard to imagine the evolution of marine animals or their survival if marine plants had not ex- isted. Through ages, these marine plants have provided ele- mentary nourishment to the food chain. Phytoplanktons, the smallest single-celled marine along with algae develop the base of the unique marine food chain which is vital for the balanced ecosystem. Marine plants constitute an imperative position in the formation and upholding of the coral reefs, providing nourishment and shelter for animals wherein algae 145

living inside marine animals consume nitrogen from coral waste and provide nutrients in a symbiotic relationship. De- spite having crucial functions in the ecological network, they are increasingly becoming vulnerable to pollution. Dredging and harvesting coral has drastically injured their wholesome assortment. The excess use of fertilizers and pesticides in the fields, oils exploration and spillage in the oceans, radioactive material, sewage and hazardous wastes drainage from cities into oceans have damaged Seagrass beds and reefs extensive- ly. Explosives used by tropical commercial fishers to stun fishes have destroyed marine plant habitats hampering a range of symbiotic associations as in Chesapeake Bay in Maryland where Seagrasses have witnessed enormous wreckage (Rick et al. 2017). Some scientists speculate grow- ing ozone hole, along with other alarming consequences, might cause irreparable damage to Antarctica’s marine plants. Further, marine plants transported by shipping vessels can occasionally dominate the native plant species growing in distant areas resulting in newer challenges. Several man- made changes in ocean composition also alter marine biodi- versity at large. The presence of high nitrogenous waste al- lows the algae population to develop fungi quite frequently depleting carbon supplies. This can lead to algal blooms or toxic red tide which in due course can smother coral reefs due to nitrogen imbalance and starve marine plants and or- ganisms of oxygen. As was seen in 1996 when a red algal tide killed many Florida manatees. Global warming has its share in contributing to this mess. Increased water tempera- ture in the oceans may result in corals expelling out algae thereby appearing bleached white because the algae is the reason behind coral's energy and colour. 146

Uses Marine plants are the largest oxygen resource on earth contributing to approximately 70% of oxygen generation and regulation in the atmosphere. The oceans encompass about three-fourth of the surface of our planet representing over 99% of the biosphere inhabiting in the extremes of salinity, temperature, light, and pressure conditions. Adaptation to harsh environments has led to a rich, diverse and unexplored marine bioresource with potential biotechnological applica- tions for developing new resources and industrial processes. The marine organisms are the biomarkers of the environment and the organisms including the residents of land and under water health indicates environmental problems that humans and land organisms might encounter in the longer run. Hu- mans have been using marine plants for medicinal uses since ages. The marine plant based biotoxins are a valuable source for developing pharmaceuticals. Oceanographers and ocean research scientists have joined hands in exploring seas using latest deep-diving techniques to gather samples in the quest to identify and understand the unexplored resource hidden in the blue seas. The promising research outcomes are advanta- geous for the pharmaceutical manufactures as well to seek putative new chemical compounds for drug development. The diversity, novelty and uniqueness of marine plants have raised hopes of proposing new treatments for diseases re- sistant to existing non marine-plant-derived drugs as in mi- crofibrous collagen sheets which are a promising drug carrier for cancer treatment (Sato, Kitazawa, Adachi, & Horikoshi, 1996) due to its two desirable qualities of maintenance of drug concentration for long-term and controlled release at target site, besides, playing a critical role in the formation of cells, tissues and organs. Clinical investigations have demon- strated collagen/gelatin hydrolysates intake as pain-reliever in osteoarthritis and cartilage matrix synthesis, and as a gene 147

delivery agent promoting bone and cartilage formation (Nak- agawa and Tagawa 2000). The pharmaceutical companies are marketing collagen/gelatin as a supplement for maintaining bone integrity and nourishing scalp hair. Various classes of compounds including polyphenols, polysaccharides, alka- loids, and peptides are the potent bio actives predominantly having anticancer properties. United States Food and Drug Administration (USFDA) has approved marine resourced an- ticancer drugs including cytarabine, trabectedin for mye- logenous leukemia and advanced soft tissue sarcoma, re- spectively. Several other drugs like Eribulin mesylate, bren- tuximab vedotin for breast cancer and liposarcoma, and an- aplastic large cell lymphoma respectively extracted from ma- rine source have been marketed. The examples of marine- derived drugs include an antibiotic from fungi, two closely related compounds from a sponge that treat cancer and the herpes virus, and a neurotoxin from a snail that has painkiller properties making it 10,000 times more potent than morphine without the side effects. There are several more marine- resourced compounds currently in clinical trials and it is like- ly that many more will advance to the clinic as more scien- tists look to the sea for these biotechnological uses. Chitosan Chitosan is an chemical substance present in the exoskele- ton of marine shell fishes like snails and crabs. Research has shown it to be an excellent nano-delivery system for anti- cancer targets due to its hydrophilic nature and biocompati- bile, and biodegradable properties making it a desirable anti- cancer drug delivery system. Stable, porous, and bioactive chitosan nanocarriers have been successfully designed which has enabled extensive use of chitosan in the distribution of anticancer medicines. In addition to new medicines, other uses for marine-derived compounds includes cosmetics (al- 148

gae, crustacean and sea fan compounds), nutritional supple- ments (algae and fish compounds), artificial bone (corals), and industrial applications (fluorescent compounds from jel- lyfish, novel glues from mussels, and heat resistant enzymes from deep-sea bacteria). Generation of the immense quantity of underutilized marine processing by-products has long been recognized as waste, and efforts are on to use these materials in various applications. Researches on marine based by- products have resulted in the identification of biologically ac- tive compounds with application for human utilization. Po- tential applications of proteins, lipids, chitin and minerals in marine bio-processing leftovers as bioactive materials have increased the value of processing by-products in recent years. Marine plants like algae, Seacucumber, Seagrasses and Red Alga have also been used as a source of nutrients, cosmaceu- tical and pharmaceutical properties. The two most important long-chain polu unsaturated fatty acids omega-3 (ω-3), namely Eicosapentaenoic acid (EPA) and docosahexaenoic acid (DHA) are the involved in human physiology, and vital to infants' brain development are almost exclusively based on microalgae. Approximately 40% of baby formula is made from these algae. The plant has an advantage of easily grown under controlled conditions with a constant biochemical composition and ensuring sustainable supply making it an obvious choice for the biotechnology industry. Dunaliella bardawil biosynthesizes the orange pigment beta-carotene, which the human body converts into vitamin A. Commercial production of this alga manufactures carotene. Similarly, the red algae are the used in seaweed drinks and cooking. Other commercialization of marine plants includes post harvest value addition of seaweed for various products, including foods and fertilizer as in Chlorella and Spirulina. Research- ers aspire to transfer gene responsible to tolerate extreme saltiness and sun exposure identified from D. bardawil on to 149

land plants cultivated in places with high salinity and sun- light. Hopes are high in achieving results from studies on the physiological relationship of algae and water for optimum cell growth and photosynthesis to reduce crop losses, and to understand how terrestrial plants as corn can tolerate droughts. Researchers are also investigating molecular exam- inations of marine and land plants to comprehend relation- ship between water supply, growth rate and metabolism in underwater plants. The cells of the alga Chara corallina are large enough to observe how dehydration affects them over a short period of time. Evolutionary adaptations of marine species resulting in unique modifications both physiologically and morphologi- cally to thrive in these environments have resulted in a living library of biological and genetic diversity with limitless bio- technological potential. Biodiversity is essentially adaptation to constant changes in the environment. Diverse forms have evolved in response to various environmental factors; distinct species arose and diversified to occupy and exploited new niches including the Oceans. The simple unicellular forms to the complex multi-cellular organization are more diverse and uniquely adapted than the terrestrial forms. Also, the limita- tions of the desired exploration vehicles (manned or un- manned) to the deepest parts of the ocean is hampering the exploration of the diversity. The oceans truly are among the last frontiers of exploration on the planet. Yeasts have received enormous consideration for over one hundred years as these can produce numerous kinds of bioac- tive substances. In this regard, terrestrial yeasts have been mined more than their marine counterparts. However, various studies have reported bioactive substances from wide marine yeast varieties, which escalate their remedial significance. Using marine yeast as a potential bioactive resource would help save freshwater resources as seawater, the most abun- 150