There are a wide range of underutilized plants with well known nutritional and medicinal properties. They have the potential for further promotion as they are locally available or are byproducts or waste materials. They are culturally accepted and often well established component of life systems and diets. Some of the isolated phytochemicals from them have commercial value.
Seabuckthorn (SBT), (Hippophae L.) is a deciduous shrub of the genusHippophae, (family Elaeagnaceae). In addition to the nutritionally active constituents, many clinically important activities of seabuckthorn (SBT), such as beneficial effects on skin, cardiovascular diseases, diabetes, wound healing, cancer chemoprevention, antiviral activity, sun screen and radioprotection have been established. Unfortunately, most of the reported studies have been carried out with ill-defined crude extracts (e g “flavone”, “hydro alcoholic extracts”, “SBT oil”, “aqueous extracts”).
`Due to non-uniformity, the results are often not reproducible. Therefore there is urgent need for identification of active principles. Presently, work in the laboratory is directed towards the isolation and characterization of active principles/fractions from bioactive fractions of SBT. Isolated compounds are useful for the treatment of diseases such as diabetes, dermatitis, CVD, peptic ulcers, sun-screen and radio protective formulations, wound healing agents. Wherever isolation presents difficulty within a reasonable time frame, the extracts are standardized with respect to bio-markers and fingerprinting.
Many of the medicinal properties of SBT have been attributed to the polar constituents.Our earlier work had established that most of the activities are present in leaves of SBT. A detailed study on leaves was carried out. The polar constituents mainly consist of glycosides of quercetin, isorhamnetin, kaempferol, gallic acid, ellagic acid etc.
CNSL is a byproduct of cashew industry (Anacardiumoccidentale). It is a rich source of non- isoprenoid phenols. Traditionally CNSL has been used for healing cracked foot. Anacardic acids, cardol and cardanol have been isolated from CNSL.UV, NMR and MS. Individual components have been separated and characterized by UV, IR, NMR. LC-MS.In-house Studies shows that anacardic acids inhibit the catalytic activity of matrix metalloproteinase-2 and matrix metalloproteinase-9. This has relevance in the cancer therapy.Cardol and cardanol are important industrial raw materials which are useful for polymer synthesis.
Sesuviumportulacastrumcommonly known as sea purslane, is a sprawling perennial herb that grows as weed in coastal areas throughout the world. It has been found to be a good source for ecdysterone, which has many pharmaceutical and nutraceutical properties such as improves lipid metabolism, has immunomodulatory effects and effects on CNS. Cosciniumfenestrum leavesare by products from the ayurvedic industry. It has been found to be a good source of ecdysterone.Gomphrenacelosioides,Diploclisiaglaucescens and Cyathulacapitataare the other sources of ecdysterone and other related compounds. Many known pharmacologically important compounds were isolated and characterized from the underutilized plants. In-house investigationreveal that it has good wound healing activities.This source of ecdysterone are used in field experiments.
Compounds isolated or purified fractions obtained from different plant sources are subjected to a series of bioassays for potential compounds for target dependent bioactive studies. Purified compounds are subjected to antioxidant studies DNA protection and nitric oxide scavenging assays. In house bioassays such as cell proliferative studies, wound healing studies, enzyme inhibition assays are also performed.
In the Phytochemistry laboratory, development, analysis, and optimization of technical processes for target dependent bioactive compound isolations and assays are carried out. A brief account is given below.
An eco-friendly process for isolation of phytoecdysteroids and related compounds has been developed( patent pending). It utilizes very small quantity of solvents. Taking advantage of presence of both hydrophilic and hydrophobic functionalities within the molecule, a convenient method has been developed by use of judicious matrices.The processinvolves selective isolation for obtaining phytoecdysteroid depending upon the accompanying contaminants present in the plant extracts.
A simple and quick process for isolation of non-isoprenoidphenolics such as anacardic acids and their analogs from plant sources such as cashewnut shell liquid (Anacardiumoccidentale)has been developed. It involves selective isolation of components namely anacardic acids, cardol, cardanol and related compounds could be recovered by careful use of adsorbant and mixture of solvent. The processdoes not require elaborate infrastructure and ordinary glass wares and equipments are used. It is energy efficient, eco-friendly,simple and quick.
Quantative analysis of naringenin in the presence of its glycoside is a tedious process. A spectroscopic method in conjunction with shift reagent has been developed.It is a quick, simple method for the analysis and hasdirect application in food industry. Process for selective hydrolysis of disaccharides has been developed.
Sericulture is a labor intensive agro based cottage industry engaging marginal farmers; mainly women.Sericulture serves as an important tool for rural reconstruction, benefiting the weaker sections of the society. It does not require elaborate establishment. It provides assurred income to marginal farmerswithin short period.Using insect growth regulators (IGR’s) isolated from the local plants such as juvenile and moulting hormones or their analogs (juvenoids and ecdysoids), field trials have been carried out in the farms in Kuttanoor village. By judicious application in correct time and developmental stage, an increase in yieldupto(15-20% of silk fibre has been obtained.
Divergent oriented synthesis is a strategy which aims to the synthesis of compounds with diverse chemical structures. It is often an alternative to convergent synthesis or linear synthesis. With this intention, the diversity oriented synthesis was developed. In Phytochemistry laboratory our aims is to generate a library of bioactive oxygen heterocyclic compounds by first reacting with a easily available starting material to form set of intermediates, e.g. chalcones, 1,3-diketones. The next target compounds are generated by suitable transformations of intermediates, e.g. flavones, flavonols, flavanones, isoflavones and biflavones. This methodology quickly diverges to large numbers of different classes of compounds from simple starting materials. It is also efficient synthesis. The scheme methodology is given below.
Some examples of compounds synthesised are given below:
1) Chalcones, 2) Dihydroflavonols, 3) Flavones 4) Flavonol 5) Isoflavones
After broad separation of tannins as lead complex from the SBT extract , flavonoid glycosides (in the supernatant) were subjected to column chromatography using SiO2 and LH-20. Partially purified fractions were put for preparative HPLC and glycosides were isolated. The amounts of individual compounds were small. The UV spectra of the isolated fractions before and after the hydrolysis show a bathochromic shift of 16 nm (in Band II) suggesting that positions 3 of flavonoids were blocked. Therefore it was inferred that the 3-hydroxyls were blocked by glycosidation or conjugation. Use of UV shift reagents (AlCl3, NaOAc, H3BO3) gave information on the positions of other hydroxyls in the molecules. LC-MS/MS gave the molecular weights of the glycosides. Acid hydrolysis of the glycosides furnished information on the aglycones and the carbohydrates. The qualitative information on the carbohydrates could be obtained by TLC/paper chromatography. A careful analysis of MS/MS data gave quantitative data on the sequence of attachment of carbohydrates and structure of the aglycone. As an illustration of this strategy, analysis of peak at m/e 771 is 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 suggest the presence of two consecutiverhamnosyl residues. Ion 479 give 317 with the loss of 162 which represents a glucosyl entity. The remaining ion 317 was identified as isorhamnetin by MS/MS. Taking consideration of the fragmentations of the peak at 771 it is concluded that it represents isorhamnetin 3-glucodirhamnoside Similarly the structures of other glycosides were derived as given in the following table:1.
LC-MS FRAGMENTATION OF ION 771
Table : 1
|No.||COMPOUNDS||MOLECULAR WEIGHT/ FRAGMENTS|
|1||Kaempferol -3-diglucoside||MW=611, fragment 611-162-162=287.|
|2||Isorhamnetin -3-rhamnodiglucoside||MW=787, fragment 787-162-162-146=317|
|3||Kaempferol-3- rhamnoglucoside||MW=595, fragment 595-162-146=287.|
|4||Isorhamnetin-3- glucorhamnoside||MW=595, fragment 595-162-146=287.|
|5||Kaempferol-3- glucorhamnoside||MW=595, fragment 595-162-146=287.|
|6||Isorhamnetin-3- rhamnoglucoside||MW=625, fragment 625-146-162=317|
|7||Quercetin-3- diglucoside||MW= 627, fragment 627-162-162=303.|
|8||Isorhamnetin-3- rhamnoglucoside||MW= 641, fragment 641-162-146=317|
|9||Quercetin-3- diglucoside||MW= 627, fragment 627-162-162=303.|
|10||Quercetin -3-diglucorhamnoside||MW=773, fragment 773-162-146-162=303.|
Following similar strategy, tannins were also characterized. For example one of the fractions from Sephadex LH-20 colunm, on LCMS gave an ion M++1 at 751 which lost 152 to give 599 ion indicating loss of galloyl unit. Further loss of 162 from 599 suggests the loss of glucosyl moiety to give 303 which was identified as ellagic acid by the analysis of its MS/MS fragments. Thus 751 ion represent :Ellagi-gluco- gallate. About half a dozen tannins were thus identified.
LC-MS FRAGMENTATION OF M+ ION 751
Docking studies are computational techniques for the exploration of the possible binding modes of a substrate to a given receptor, enzyme or other binding site. The application of computational methods to study the formation of intermolecular complexes has been the subject of intensive research during the last decade. The department of phytochemistryhas a collection of compounds structures. Our aim is to find a target molecules for different Matrix metalloproteinase (MMPs) in cancer cells, diabetic wound healing and carbonic anhydrase inhibitors. We presently focusing on, the docking interactions on carbonic anhydrase with 40 different flavonoids (flavones, flavonols and biflavones). A few lead molecules have been identified. Some representative are given below.
The Phytochemistry lab at the Amrita school of Biotechnology maintains a chemical library which is a unique source of diverse compounds for hit/lead discovery. The library originates from the well regarded prolonged research expertise of Dr. AsokeBanerji. A wide collection of compounds from different class such as Terpenes, Steroids, Alkaloids,Coumarins, Flavonoids, Phenols, Tannins, Glycoside remains as an asset.Few of the compounds and their bioactive studies are:
|1||Ecdysterone||Cell proliferation , Wound healing, Sericulture.DNA protection, enzyme inhibition assays, Antioxidant,nitric oxide scavenging assay, Antidiabetic, Anticancer studies etc|
|16||Anacardic acid monoene|
|17||Anacardic acid diene|
|18||Anacardic acid triene|