The multidisciplinary researches in the Phytochemistry laboratory at Amrita School of Biotechnology are directed towardthe exploration of natural wealth for the development of novel bioactivities. The clues from natural products lead to syntheses of bio-rationally selected analogues. “Fragment approach” has been used for bioprospection, structural analysis, as well as developing bio-inspired syntheses. A brief account some of the activities is discussed below.

  1. Wealth from waste
    A review of literature indicates that after theutilization of the valuable parts of plants, remaining partsare often discarded and lead to waste generation and create environmental pollution.For example, in the preparation of Ayurvedic drugs, often a part of the plant is used while the rest of the portions are discarded.Daru haridra (Cosciniumfenestratum)is extensively used in Ayurveda, but only the stem is used for the formulations. Leaves are not used and discarded. A phytochemical investigation on the leaves revealed thatthese are rich source of a high value chemical, ecdysterone, which has a prominent use in sericulture (see below) and also as a nutraceutical.Similarly, the waste products are generated from many other sources after using the valued portion.Our studiesare directed towards the exploration of unutilized parts of these materials as possible source of phytochemicals. In an ongoing project (supported by Amrita Vishwa Vidyapeetham)value addition to some of the waste or underutilised plant products is carried out. Studies on cashew nut shell liquid (CNSL), a by-product(20,000 ton/year)of cashew industry, led to isolation and characterizationof industrially important products such as alkenyl phenols (cardol, cardanol, anacardic acid). A convenient process for their separation and isolation of CNSLhas been developed (described below).It is estimated that huge amount of wasteis generated from usage of onion globally. Three important compounds viz.quercetin, spiraeoside and anthocyanin are obtained from onion peels.Phytochemical investigationof other wastes such as flowers from temples and or from social functions, like Tagetes,a halophytic weed from coastal India,viz.Sesuvium portulacastrum,coconut shell, Seabuckthorn (a Himalayan plant)etc., are some of the other examples of value addition.Spiraeoside(quercetin-4-glucoside) isolated from onion peel wastewas utilized as a starting material to synthesise a rare flavone,pachypodol. Pachypodolshows anti-viral, anti-tumour and MMP-2 inhibitory activities. Quercetin, another compound isolated from onion peels, was used for the synthesis of azaletin, a natural product from Rhodedendron. Quercetagetin(3,5,6,7,3’,4,’-hexahydroxyflavone) was isolated from the marigold flowers.It is usefulin the metabolic disorders such as cardiovascular disease, diabetics,obesity etc. It is a good antioxidant and a natural dye. Ecdysterone, isolated from weeds, Sesuvium portulacastrum and Diploclisia glaucescens, is an insect growth regulator and has anabolic activities. Oxyresveratrol,an excellent NF-kB inhibitor was isolated from coconut shell.
  2. Bioinspired, diversity oriented syntheses of oxygen heterocycles
    Discovery of new chemical entities with potential bio-applications could be achieved either by developing new synthetic routes or by exploring natural sources. There is a continued demand for new drug molecules due to the development of resistance by many pathogens to the increasing number ofexisting drugs.Also there have been emergence of new diseases which require new drugs to treat. The drug development process is very expensive and time consuming, Ttherefore, attempts have been made all over the world to develop more efficient and rapid strategies to accelerate the drug-discovery process. 'Fragment based' approach could be an effective and novel paradigm for the drug discovery. This is based on choosing 'biologically relevant templates' and rationally modify them to improve their bioactivities and pharmacokinetic properties. Thisis in contrast to the combinatorial syntheses which are based on structural modification around the pharmacophores.Unfortunately,results obtained using combinatorial synthesisare not very encouraging in the past few years, possibly due to the limited chemo-diversity offered by combinatorial approach. Our studies suggest that fragment approach offers better route for drug discovery with possibly higherrateof hits. It has better coverage of the 'chemical space' and involve substantially smaller libraries as compared to high throughput screenings.A perusal of the literature indicates that though very large number of molecules are available in nature, most of the biological studies have been restricted onlyto very few candidate molecules; may be due totheir vast and easy availability. For example, amongst flavonoids, the studies are directed ona very few compounds such as quercetin, luteolin and genistein etc. Of late it has been realized that many of these molecules are not the actual active principles but are prodrugs and express their activities after metabolic transformations or activations. The actual active principles may be the metabolites rather than the original compounds. Therefore, an easy access to the metabolites is very desirable activity for drug discovery programs.Though partially alkylated flavonoids and related compounds occupy substantial 'chemicalspace', but there is scant information on their biological activities. Since they are comparatively less accessible from the natural sources, the development of general synthesis protocols is desirable. Taking clues from nature, a bio-inspired, eco-friendly syntheses of polyhydroxy and partially alkylated flavonoids have been developed in the phyto medicine group (Fig 1).

    Fig1: Synthesis of bioactive flavones from onion.
    Conventional methods use selective methylation involving extensive protection/deprotection steps using benzoyl or benzyl or methyl derivatives, thereby adding extra steps to syntheses. We have chosen methoxy methylation(MOM)for the protection of hydroxyl groups. Preparation and deprotection of MOM derivatives are achieved under very mild conditions. Using selective protection/deprotection method alibrary of flavones,flavonols,isoflavones and biflavones was synthesised. The synthesized compounds were subjected to bioassays. This led us to discover several hitherto un-reported inhibitors of biomarkers for MMP’s, NFkB, carbonicanhydrase, anti-diabetic and anti-microbial compounds. In Nature, chalcones play pivotal role in the biosynthesis of an array of phytochemicals. Taking advantage of their multiple functionalities, many transformations could be carried out. MOM derivatives were compatible with reaction conditions used. Using chalcones as starting point, following innovative syntheses were developed:
    1. Synthesis of Flavones.
    2. Synthesis of Flavonols
    3. Synthesis ofBiflavones
    4. Synthesis of Isoflavones.
    About 80 different molecules were prepared. Some of the compounds were bio-assayed using in-house facility of Cell Biology Section. Screening results showed that some the simple phytochemicals have remarkable bioactivities (Fig2).

    Fig 2: Examples of synthetic bioactive compounds
    A simple flavone namely,chrysoriol was found to be a better inhibitor of NFkB than quercetinwhich is used generally as a standard.Plumbagin(Plumbago rosea)and oxyresveratrol (from coconut shell) alsoexhibited NFkBand COX inhibitory activities and are prospective candidates for trials for anti-tumour activity. Examples of bioactive compounds isolated or synthesized are: anacardic acid (from coconut shell liquid CNSL), biacacetin(synthesis by biorational approach),pachypodiol(precursor obtained from onionpeel),plumbagin from the medicinal plant, Plumbago rosea, diadzein(biofilm inhibitor, anti- atherosclerosis, synthetic), acacetin (synthetic), arjunolic acid (from Arjunaterminalia) as inhibitors for carbonic anhydrase (Table 1).

    Table 1: Examples of bioactive compounds isolated or synthesized in our lab.
  3. Development of Eco-friendly methods for isolation of bioactive compounds
    For natural product research, it is desirable to develop eco-friendly, economic methods for the purification of compounds in significant quantities from the crude extracts. The isolation and purification of active principles depends on the structure, stability, and quantity of the compounds in the mixture. Chromatographic methods have been used extensively for their separation. However, most of these methods are expensive, not environmentally friendly, requires long time and invlove multiple stages of purification steps. A simple method for isolation and purification of phytochemicals from plant extracts or crude mixtures has been developed in our group (patent pending). Separation of components depends on the physical and chemical properties of the individual component. The separation is based on the differences between the adsorption affinities of the individual component of the mixture towards the surface of the adsorbents and are crucial to achieve significant rate of separation. In contrast to column chromatography, in the present method, the whole matrix is utilized for fixation of the mixture. In conventional column chromatography, the fixing of the mixture is carried out only on the top portion of the column: elution is done by using appropriate solvents or solvent mixtures sequentially. In contrast, in the present method the individual component is desorbed by using appropriate solvent or solvent mixtures. Conventional chromatography consumes large volume of solvents and involves collection of numerous fractions; each fraction is individually worked up and analyzed making the process very labor intensive.
    As mentioned above, a large quantity of cashew nut shell liquid (20,000 tons/ton) is generated during the processing of cashew nut. Using the present protocol, all the three important phytochemicals namely, anacardic acid, cardol, cardanol were isolated from CNSL within 3-4 hours, thus adding value to CNSL. Easy method of extraction led to the isolation of significant quantities of anacardic acid and many of its activities such as anti-fungal, anti-bacterial, anti- biofilm,anti-fungal activities could be evaluated. Similarly, plumbagin isolated from the medicinial plant, Chitrak (Plambago rosea) could be obtained,in highly crystalline form, using SiO2 as adsorbent and a solvent mixture of1:1 petroleum ether: chloroform. Huge quantities of flower waste is generated in temples and social functions in India. Marigold flower, Tagetes erecta is one most commonly used flower used in religious and social functions. Two important phytochemicals namely lycopene and quercetagetin are isolated from the flowers by the procedure developed in our lab. A large quantity of onion is used in the households, restaurants and food processing industries. Outer peels of onion are the waste products and its disposal pose environmental problems. The peels are rich source of flavonoids. Depending upon the variety, they are excellent source of quercetin, quercetin-4’-glucoside (spiraeoside) and anthocyanin. The peels provide natural antioxidants and natural food colorant. It also provides advanced starting compound for synthesis of other bioactive compounds. Azaleatin, which occurs in Rhododendron, has been synthesised using quercetin obtained from onion peels. A synthesis of pachypodol was carried out for the first time starting from quercetin-4’-glucosideas starting material (Fig. 1). Oxyresveratrol isolated from coconut shell, is a good source of inhibitor of cell signal compound, NFkB. Two important flavonoids, gossypetin (antioxidant, radioprotector) and hibifolin (neuroprotective) are known to occur in the common weed, Hibiscus vitifolius. Usual method using multiple chromatography for separationof individual components is very tedious and time consuming. Using the method developed in the Phytomedicine group, a short eco-friendly procedure has been developed which allows easy access to these phytochemicals. Seabuckthorn (Hippophae spp) is known for source of high amounts of vitamins ( A, E, B, C), antioxidants (flavonoids and tannins), phytosteroids, rare fatty acids. It is an underutilized plant occurring in Himalayan regions. Processes for isolation of these phytochemicals have been developed. The eco-friendly and short method for isolation of natural products was successfully utilized for the isolation of different high value phytochemical from diverse complex mixtures of plant extracts (Fig 4).

    Fig 4: Compounds isolated using the simple isolation method from different plant sources.
  4. A new approach for molecular characterization: Molecular characterization of phytochemicals form an integral part of natural product research (NPR).
    Identification of known compounds can be carried out by dereplication. Analyses of crude extracts require separation of individual compounds before the analysis can be undertaken. This can be long drawn procedure. This could be avoided by using conventional methods in conjunction with modern techniques.It may start with the age old colour reactions, followed by IR, UV-VIS spectroscopy and LC-MS/MSn.
    Our approach is illustrated in the analysis of polar fractions of seabuckthorn leaves which contain intractable mixture of tannins and flavonoid polyglycosides. A broad separation of tannins and the polyglycosides was carried out by preferential precipitation of tannins with lead acetate. The information on the major fractions of the extract could be obtained by acid hydrolysis of the extracts which furnished information on the total aglycones and carbohydrates. These components (fragments) were characterized as quercetin, isorhamnetin, kaempferol, ellagic acid, gallic acid, glucose and rhamnose by standard analysis. The UV-VIS spectra of the intact fractions before and after acid hydrolysis show a bathochromic shift of 16 nm (in Band II). From this, it was inferred that positions 3 of flavonoids present in the mixture were blocked. Therefore, it was inferred that the hydroxyls at position 3 of the flavonoids were blocked by glycosylation or conjugation. Using LC-MS/MS, the molecular weights of the individual glycosides could be obtained. A careful analysis of MS/MS data gave information on progressive loss of sugar moieties. From which, the quantitative data on the number and sequence of attachment of carbohydrates and structures of the aglycone could be obtained. The position of the attachments of sugars to the aglycone could be derived using shift reagent in UV-VIS spectroscopy. The use of this strategy is illustrated by the analysis of HPLC peak at m/e 771 as given below: The peak at 771 (M+ +1) on MS loses a mass of 146 to give an ion at 625 . This ion on further MS/MS show loss of another fragment of 146 to give an ion at 479. Successive loss of two 146 moieties suggests the presence of two consecutive rhamnosyl residues in the molecule. Ion 479 on further fragmentation give ion at 317 with the loss of 162 which represents a loss of glucosyl entity. The remaining ion 317 was identified as isorhamnetin by MS/MS data. Taking consideration of the fragmentations of the peak at 771 it is concluded that it represents isorhamnetin 3-gluco-dirhamnosideFig 5. Following this fragment approach analysis of 20 other glycosides was carried out without recourse to tedious isolation procedures. The tannin glycosides were also analysed following the same logic.

    Fig 5: isorhamnetin 3-glucodirhamnoside identified using molecular fragmentation analysis.
  5. Indigenous formulations
    Sericulture can contribute substantially to augment the income of marginal farmers. Since women are employed in the sericulture, this can be good option for Kerala farmers. Insect growth regulators (IGR) form important component in the sericulture management. Bio prospection of the regional flora resulted in the recognition of many bio sources of IGR’s. Indigenous formulations of IGR from the local weed viz.Diploclisia glaucescens, were developed in our lab. Successful trials were carried out at the farmers’ fields with very encouraging results. This product can replace the imported IGR presently used by the Indian sericulture farmers Fig.6.
    In parellel studies, we also discovered that, anacardic acid preparations can providegood protection activity to agricultural crop against rice blast disease.
    Lack of access to safe drinking water is still a major problem in India. To address this, a cellulose (cotton) based cost effective method for domestic water purification by ultra-filtration has been developed by our group. This is ideally suited for people living in remote villages.
    Formulations prepared from cashew nut shell liquid and Hygrophila auriculata extracts in our lab are undergoing clinical trials.

    Fig. 6: Field studies using Indigenous IGR formulations on sericulture.
    Apart from theses, we were also able to isolate and identify the active principle of the ayurvedic medicine, phaltaka (Semecarpus anacardium) as urshiol.

Conclusions

In search of bioactive compounds, several phytochemicals (50) and natural-like compounds were assayed for bioactivities using in-house facilities. About 100 compounds were synthesized by bio-rational approach. Fragment approach was adopted for bio screening which resulted in the discovery of new bioactive compounds (e.g. inhibitors of MMP’s, NFkB, carbonic anhydrases, COX-2, anti-bioflms, anti- radiation, antioxidants). Studies on unutilized or underutilized plants led to isolation of high value phytochemicals. An innovative method for isolation and purification, with possible application in industry, was developed. For plant based drug discovery approach bio prospection of flora led to identification of about a dozen of promising medicinal plants. A combination of classical and modern techniques was very useful for the analysis of extracts containing difficult labile phytochemicals, without recourse to tedious separation techniques. This approach is likely to be useful for metabolomics studies of secondary metabolites (natural products).

ACCORDING TO AMMA “NATURE IS A BOOK TO STUDY, EACH OBJECT IN NATURE IS A PAGE IN THAT BOOK”

WE AT PHYTOCHEMISTRY GROUP HAVE TURNED JUST FEW OF THE PAGES OF NATURE’S BOOK. WHILE A VAST MAJORITY REMAINS UNTAPPED.

Acknowledgements

Thework of the “phytochemistry Group “is multidisciplinary and involves participation of several biological Laboratories. Work was carried out in close collaboration with the Cell Biology Laboratory. Authors are grateful to the members of the Cell Biology Laboratory. Mass spectral determinations were carried out at the Amrita Agilent Analytical Centre (AAAC). Thanks are due to Prof. Walter Schrenk and Prof. Sudarslal of AAAC for extending all the necessary help for mass spectral work. We are also grateful to Dr. Ram Manohar, Director, Amrita Centre for Advanced Research in Ayurveda and Prof. Sanjit Dey, (Radioprotection), Head of Physiology Dept. Calcutta University for their contributions. Finely, we express our gratitude to Prof. Bipin Nair for all the support during the course of this work. Financial support from Amrita Vishwa Vidyapeetham is gratefully acknowledged.

References

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  2. Athira Omanakuttan, Jyotsna Nambiar, Rodney M. Harris, Chinchu Bose, Nanjan Pandurangan, Rebu K. Varghese, Geetha B. Kumar, John A. Tainer, Asoke Banerji, J, Jefferson P. Perry and Bipin G. Nair. Anacardic acid inhibits the catalytic activity of matrix metallo proteinase-2 and matrix metallo proteinase-9. Molecular Pharmacology, 2012, 82, 614-622.
  3. Amitava Khan, Krishnendu Manna, Chinchu Bose. Mahuya Sinha, Dipesh Kr. Das, Swaraj Bandhukesh, Anindita Chakrabarty. Gossypetin, a naturally occurring hexahydroxy flavone, ameliorates gamma radiation- mediated DNA damage. International Journal of Radiation Biology, 2013, 11, 965–9.
  4. Khan Amitava, Manna Krishnendu, Chinchu Bose, Das Dipesh Kr, Sinha Mahuya, Kesh Swaraj Bandhu, Das Ujjal, Dey Rakhi Sharma,Banerji Asoke, Dey Sanjit Seabuckthorn (Hippophae rhamnoides L.) leaf extract ameliorates the gamma radiation mediated DNA damage and hepatic alterations. Indian Journal of Experimental Biology, 2014, 52, 952-964.
  5. Sreejit Chittiyath Madhavan, Chinchu Bose, Thomas Mathew Perakathusseril, Asoke Banerji. Indian medicinal plant, Coscinium fenestratum- A new bio source for the multifunctional bio-active molecule–ecdysterone. International Journal of Herbal Medicine, 2014, 2, 9-12.
  6. Suhail Muzaffar, Chinchu Bose, Ashok Banerji, Bipin G. Nair, Bharat B. Chattoo. Anacardic acid induces apoptosis-like cell death in the rice blast fungus Magnaporthe oryzae. Applied Microbiology and Biotechnology. 2015, 9, 1-13.
  7. Amrutha, K.; Pandurangan Nanjan; Shaji, S.K.; Sunilkumar, D.; Subhalakshmi, K.; Rajakrishna, L.; Asoke Banerji. Discovery of lesser known flavones as inhibitors of NF-κB signaling in MDA-MB-231 breast cancer cells-A SAR study. Bioorganic and Medicinal Chemistry Letters, Pergamon, Volume 24, Issue 19, p.4735-4742 (2014)
  8. Athira Omanakuttan, Jyotsna Nambiar, Rodney M. Harris, Chinchu Bose, Nanjan Pandurangan, Rebu K. Varghese, Geetha B. Kumar, John A. Tainer, Asoke Banerji, J, Jefferson P. Perry and Bipin G. Nair. Anacardic acid inhibits the catalytic activity of matrix metallo proteinase-2 and matrix metallo proteinase-9. Molecular Pharmacology, 2012, 82, 614-622.
  9. Pandurangan Nanjan; Jyotsna Nambiar; Bipin G. Nair; Asoke Banerji. Synthesis and Discovery of (I-3,II-3)-Biacacetin as a Novel Non-zinc Binding Inhibitor of MMP-2 and MMP-9. Bioorganic & Medicinal Chemistry, Volume 23, Number 13, p.3781 - 3787 (2015)
  10. Amitava Khan, Krishnendu Manna, Chinchu Bose. Mahuya Sinha, Dipesh Kr. Das, Swaraj Bandhukesh, Anindita Chakrabarty. Gossypetin, a naturally occurring hexahydroxy flavone, ameliorates gamma radiation- mediated DNA damage. International Journal of Radiation Biology, 2013, 11, 965–9.
  11. Subhalakshmi Kalyanavenkataraman’ PanduranganNanjan, AsokeBanerji, Bipin G.Nair, Geetha B.Kumar. Discovery of arjunolic acid as a novel non-zinc binding carbonic anhydrase II inhibitor. Bioorganic Chemistry Volume 66, June 2016, Pages 72-79
  12. Khan Amitava, Manna Krishnendu, Chinchu BosE, Das Dipesh Kr, Sinha Mahuya, Kesh Swaraj Bandhu, Das Ujjal, Dey Rakhi Sharma,Banerji Asoke, Dey Sanjit Seabuckthorn (Hippophae rhamnoides L.) leaf extract ameliorates the gamma radiation mediated DNA damage and hepatic alterations. Indian Journal of Experimental Biology, 2014, 52, 952-964.
  13. Sreejit Chittiyath Madhavan, Chinchu Bose, Thomas Mathew Perakathusseril, Asoke Banerji. Indian medicinal plant, Coscinium fenestratum- A new bio source for the multifunctional bio-active molecule–ecdysterone. International Journal of Herbal Medicine, 2014, 2, 9-12.
  14. Suhail Muzaffar, Chinchu Bose, Ashok Banerji, Bipin G. Nair, Bharat B. Chattoo. Anacardic acid induces apoptosis-like cell death in the rice blast fungus Magnaporthe oryzae. Applied Microbiology and Biotechnology. 2015, 9, 1-13.

"This article is authored by Chinchu Bose, N. Pandu Rangan and Dr. Asoke Banerji , (School of Biotechnology, Amrita Vishwa Vidyapeetham, Amritapuri)

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