WO2011058423A2

WO2011058423A2 - Identification and characterization of natural chemical entities by liquid chromatography and mass spectrometry lc-ms/ms and uses thereof

Info

Publication number: WO2011058423A2
Application number: PCT/IB2010/002886
Authority: WO
Inventors: Villoo Morawala Patell; Renuka Jain; Manohar Shinde; Laksmi Settu
Original assignee: Avesthagen Limited
Priority date: 2009-11-13
Filing date: 2010-11-12
Publication date: 2011-05-19
Also published as: WO2011058423A3

Abstract

The present invention further relates to extracts which are isolated from Deschampsia antarctica plant, the preparation of such extracts, the medicaments containing said extracts, and the use of these extracts and constituents for the preparation of a medicament.

Description

Identification and characterization of Natural Chemical Entities by Liquid chromatography and Mass spectrometry LC-MS MS and uses thereof

FIELD OF INVENTION

The present invention pertains to cosmeceutical, nutritional or pharmaceutical compositions comprising extracts or concentrates of plants and the mixtures thereof belonging to Deschampsia sp. with specific reference to Deschampsia antarctica. The invention further relates to screening and characterization of extracts for their activity in preventing, mitigating, or treating various disorders pertaining to skin. The disclosure relates to extractions derived from Deschampsia species, having an elevated essential oil amount, an elevated phenolic acid amount, methods of preparing such extractions, and methods for use of such extractions.

BACKGROUND OF THE INVENTION

Deschampsia antarctica Desv. (Poaceae) is the only native Gramineae found in the Antarctic. It is mostly found in Antarctic Peninsula and its offshore islands. This plant survives in a very harsh climate, therefore it has attracted scientists to search for the freeze-tolerance genes from this plant. (Alberdi et al. 2002). Few research papers have reported the micro-propagation of this plant by tissue culture. Gidekil et

al., 2007 reported the antineoplastic activity from the extracts of Deschampsia antarctica.

Various studies in the fields of biology: ecology, taxonomy, morphology, anatomy, reproduction, physiology, biochemistry and molecular biology, tissue culture, about D. antarctica are reported (Greene, 1970; Moore, 1970; Corner, 1971; Greene and Holtom, 1971; Edwards, 1972, 1974,1975; Jellings et al., 1983; Edwards and Lewis Smith, 1988; Zu'n~iga et al., 1994, 1996; Convey, 1996; Barcikowskiet al., 1999; Day et al., 1999; Romero et al., 1999; Bravo et al., 2001; Bystrzejewska, 2001; Nkongolo et al, 2001;Alberdi et al., 2002; Zwolska and Rakusa-Suszczewski, 2002; Lewis Smith, 2003; Chwedorzewska et al., 2004; Gielwanowska and Szczuka, 2005 Marley Cuba etal., 2005, Gidekil et.al. 2007).

D. antarctica is physiologically and biochemically well adapted to various, rapidly changing growth conditions and the effects of various abiotic factors such as high and low radiation, deficient precipitation and drought, flooding, salinity, variable sometimes extremely low) temperatures accompanied by frost, frozen ground, snow- and ice-cover (Zu'n~iga et al., 1996; Barcikowski et al., 1999; Day et al, 1999; Bravo et al., 2001; Bystrzejewska, 2001; Nkongolo et al., 2001; Alberdi et al., 2002; Zwolska and Rakusa-Suszczewski, 2002; Lewis Smith, 2003; Chwedorzewska et al., 2004). The unusually high accumulation of sucrose and fructans mainly at the end of the Antarctic summer is considered one of the protective mechanisms against low temperature in D.antarctica. In the present investigation metabolomics approach has been used to identify and characterize the metabolites present in this plant Metabolomics, a new "omics," joining genomics, transcriptomics, and proteomics as a tool employed toward the understanding of global systems biology, has become widespread since 2002. Metabolomics focuses on the comprehensive and quantitative study of metabolites in a biological system. In contrast to genomics, transcriptomics and proteomics which, address macromolecules with similar chemical properties, such as DNA, RNA and proteins, metabolomics analysis deals with diverse properties of low molecular weight bio-compounds. Metabolomics offers a means of deciphering cellular metabolism and metabolic regulation. As metabolomics is the downstream product of genomics and proteomics, metabolomics is also complement of other "omics" for interpretation of gene function (functional genomics). Due to a wide range of metabolites in the metabolic network, e.g., approximately 600 metabolites m Saccharomyces cerevisiae, 1692 metabolites in Bacillus subtilis and up to 200000 metabolites in plant kingdom, it is a very challenging task to establish analytical tools for identifying and quantifying all of them.

A typical metabolomics study includes the collection of samples of interest, which follows the extraction of small molecules (low molecular weight metabolites) from the sample and is analyzed using techniques that separate and quantitate the molecules of interest. The analysis of the spectrum of metabolites are carried out by sophisticated separation and analytical techniques however, more precisely the hypenation techniques such as HPLC-MS/MS (high resolution mass spectrometry), GC- MS/MS, HPLC-NMR, are frequently being used by numerous investigators. The greatest advantage of LC-MS for application to metabolomic studies in pharmacology and toxicology is its flexibility. Different combinations of mobile phase and columns make it possible to tailor separations to the compounds of interest, including chiral compounds when appropriate conditions are used. As a result, most compounds can be analyzed by LC-MS. Instruments exist that enable low, medium, or high mass accuracy, and linear ion traps can enable MS", providing fragmentation profiles specific for given molecules.

This background information is provided for the purpose of making known information believed by the applicant to be of possible relevance to the present invention. No admission is necessarily intended, nor should be construed, that any of the preceding information constitutes prior art against the present invention. OBJECT OF THE INVENTION

The principle object of the present invention is to provide an active extract and bioactive fraction obtained from different parts of Deschampsia antarctica plant.

Another object of the invention is to provide a process for isolating bioactive fraction from Deschampsia antarctica using aqueous, alcoholic and/or hydro-alcoholic solvent, the preparation of such extracts, evaluating bioenhancing bioavailability of Deschampsia antarctica extract or bioactive fraction in combination with nutraceuticals or herbal drugs/products.

Still another object of this invention is to develop a comprehensive method using Liquid chromatography - mass spectrometry (MDS SCIEX 4000 Q-Trap MS/MS, Applied Biosystems, synchronized with Shimadzu UFLC, Prominence), to obtain a wide spectrum of low molecular weight chemicals (LMC) from Deschampsia antarctica.

Further, object of this invention is to develop the method for the sample preparation, separation and the detection of the entire spectrum of LMC including construction of specific mass spectrum metabolite library and to understand the role of bioactivity of this extract.

This invention contributes significantly to identify metabolites present in this frost resistant species - Identification and characterization of various Low Molecular Mass chemicals

Acquisition of + EMS in full scan mode from m/z 50 amu to 1500 amu

Acquisition of - EMS in full scan mode from m/z 50 amu to 1500 amu

Acquisition MS/ MS/ MS or EPI of selected ions

The instrument used for the identification and characterization of LMCs is LC-MS/MS (MDX SCIEX, 4000QTrap LC/MS/MS, Applied Biosystem coupled to UFLC, Shimadzu Prominence) Yet, another object of the invention is to provide composition comprising active principles of Deschampsia antarctica, and the use of these extracts and constituents for the preparation of nutritional and nutraceutical application.

Still another object of the present invention is to provide Deschampsia plant extract capable of treating various disorders in more than one mode of action.

Still another object of the present invention is to provide Deschampsia plant extract, which is easily and safely administrable to children and adults. SUMMARY OF THE INVENTION

Accordingly, the present invention deals with Deschampsia antarctica extracts, the process of isolation, extraction, separation and identification of low molecular mass chemicals (LMC) by Liquid chromatography - mass spectrometry (LC-MS/MS) and polyphenols content, antioxidant activity and cell toxicity, the said process comprising steps of (a) size-reducing plant parts to obtain powder; (b) extracting the bioactives with a solvent and/or combination of solvents by heating at temperature ranging from 21° to 105° C to obtain a mixture; (c) clarifying the mixture to arrive at clear liquid; (d) concentrating the clear liquid to achieve a concentrated extract; (e) solubilizing the concentrated extract in a solvent and re-concentrating it to obtain further concentrated extract, followed by drying the treated extract to obtain the plant bioactive

BRIEF DESCRIPTION OF THE ACCOMPANYING

Figure 1: TIC and enhanced mass spectrum of Deschampsia antarctica sample 1 in positive ionization mode

Figure 2: TIC and enhanced mass spectrum of Deschampsia antarctica sample 1 in negative ionization mode

Figure 3: TIC and enhanced mass spectrum of Deschampsia antarctica sample 2 (Ca35) in negative ionization mode

Figure 4: TIC and enhanced mass spectrum of Deschampsia antarctica sample 3 (Ca36) in negative ionization mode

Figure 5: Overlap mass spectra in negative of Deschampsia antarctica sample 1, sample 2 (Ca35), sample 3 (Ca36), from m/z 50 -105

Figure 6: Overlap mass spectra in negative of Deschampsia antarctica sample 1, sample 2 (Ca35), sample 3 (Ca36), from m/z 100 -185

Figure 7: Overlap mass spectra in negative of Deschampsia antarctica sample 1, sample 2 (Ca35), sample 3 (Ca36), from m/z 185 -240

Figure 8: Overlap mass spectra in negative of Deschampsia antarctica sample 1, sample 2 (Ca35), sample 3 (Ca36), from m/z 240 -295

Figure 9: Overlap mass spectra in negative of Deschampsia antarctica sample 1, sample 2 (Ca35), sample 3 (Ca36), from m/z 258 -310

Figure 10: Overlap mass spectra in negative ionization mode of Deschampsia antarctica sample 1, sample 2 (Ca35), sample 3 (Ca36), from m z 310-346 Figure 11: Overlap mass spectra in negative ionization mode of Deschampsia antarctica sample 1, sample 2 (Ca35), sample 3 (Ca36), from m/z 340 - 485

Figure 12: Overlap mass spectra in negative ionization mode of Deschampsia antarctica sample 1, sample 2 (Ca35), sample 3 (Ca36), from m/z 480-555

Figure 13: Overlap mass spectra in negative ionization mode of Deschampsia antarctica sample 1, sample 2 (Ca35), sample 3 (Ca36), from m/z 550-820

Figure 14: Overlap mass spectra in negative ionization mode of Deschampsia antarctica sample 1, sample 2 (Ca35), sample 3 (Ca36), from m/z 820-950

Figure 15: Overlap mass spectra in negative ionization mode of Deschampsia antarctica sample 1, sample 2 (Ca35), sample 3 (Ca36), from m/z 900-1005

Figure 16: Overlap mass spectra in negative ionization mode of Deschampsia antarctica sample 1, sample 2 (Ca35), sample 3 (Ca36), from m/z 1000-1200

Figure 17: Overlap mass spectra in negative ionization mode of Deschampsia antarctica sample 1, sample 2 (Ca35), sample 3 (Ca36), from m/z 350-1500

Figure 18: Enhanced mass spectrum of 5-hydroxyferulic acid from Deschampsia antarctica

Figure 19a: Enhanced mass spectrum of 5, 7-dimethoxyflavone from Deschampsia antarctica

Figure 19bUV- visible spectrum of 5, 7-dimethoxyflavone from Deschampsia antarctica

Figure 20a: Enhanced mass spectrum of 2-o-arabinopyranosylorientin from Deschampsia antarctica

Figure 20b:UV- visible spectrum of 2-o-arabinopyranosylorientin from Deschampsia antarctica

Figure 21a: Enhanced mass spectrum of Caffeic acid from Deschampsia antarctica

Figure 21b:UV- visible spectrum of Caffeic acid from Deschampsia antarctica

Figure 22: Enhanced mass spectrum of Cinnamylaldehyde from Deschampsia antarctica

Figure 23a: Enhanced mass spectrum of Apigenin from Deschampsia antarctica

Figure 23b: UV- visible spectrum of Apigenin from Deschampsia antarctica

Figure 24: Enhanced mass spectrum of Coumaric acid from Deschampsia antarctica

Figure 25: Enhanced mass spectrum of Cyanidin from Deschampsia antarctica

Figure 26: Enhanced mass spectrum of Cyanidin -3- rutinoside from Deschampsia antarctica

Figure 27: Enhanced mass spectrum of Cyanidin 3, 6 dimalonylglucoside from Deschampsia antarctica

Figure 28: Enhanced mass spectrum of Diacetylgalactopyranosylorientin from Deschampsia antarctica

Figure 29: Enhanced mass spectrum of Dihydroxykaempferol from Deschampsia antarctica

Figure 30: Enhanced mass spectrum of Dihydroxyacetonephosphate from Deschampsia antarctica Figure31: Enhanced mass spectrum of Dihyrdoxyindole -2- carboxylic acid from Deschampsia antarctica

Figure 32: Enhanced mass spectrum of Dihydroxyphenylarabinosyl- dihydroxybenzopyran from Deschampsia antarctica

Figure 33: Enhanced mass spectrum of L-DOPA from Deschampsia antarctica

Figure 34: Enhanced mass spectrum of Ferulic acid from Deschampsia antarctica

Figure 35: Enhanced mass spectrum of Galacturonic acid from Deschampsia antarctica

Figure 36: Enhanced mass spectrum of Gluconic acid from Deschampsia antarctica

Figure 37: Enhanced mass spectrum of Glucopyranosyltrihydroxymethylflavone from Deschampsia antarctica

Figure 38: Enhanced mass spectrum of Hespertin from Deschampsia antarctica

Figure 39: Enhanced mass spectrum of Homovaratic acid from Deschampsia antarctica

Figure 40: Enhanced mass spectrum of 4 (l-hydroxy-2-methylaminoethyl 2-methoxyphenol from Deschampsia antarctica

Figure 41: Enhanced mass spectrum of Immidazolone -5- propionic acid from Deschampsia antarctica

Figure 42a: Enhanced mass spectrum of Isowerticin 2 -arabinose from Deschampsia antarctica

Figure 42b:UV- visible spectrum of Isowerticin 2 -arabinose from Deschampsia antarctica

Figure 43a: Enhanced mass spectrum of Isowertiajaponin from Deschampsia antarctica

Figure 43b: UV- visible spectrum of Isowertiajaponin from Deschampsia antarctica

Figure 44: Enhanced mass spectrum of Isowertisin from Deschampsia antarctica

Figure 45a: Enhanced mass spectrum of Kaempferol from Deschampsia antarctica

Figure 45b: UV- visible spectrum of Kaempferol from Deschampsia antarctica

Figure 46a: Enhanced mass spectrum of Kaempferol 3-o-Rhamnosyl glucoside from Deschampsiaa antarctica >

Figure 46b: UV- visible spectrum of Kaempferol 3-o-Rhamnosyl glucoside from Deschampsia antarctica

Figure 47a: Enhanced mass spectrum of Luteolin from Deschampsia antarctica

Figure 47b: UV- visible spectrum of Luteolin from Deschampsia antarctica

Figure 48a: Enhanced mass spectrum of malonylgalactosylpyranosylorientin from Deschampsia antarctica

Figure 48b: UV- visible spectrum of malonylgalactosylpyranosylorientin from Deschampsia antarctica

Figure 49a: Enhanced mass spectrum of o/m/p hydroxybenzoic acid from Deschampsia antarctica Figure 49b: UV- visible spectrum of o/m/p hydroxybenzoic acid from Deschampsia antarctica Figure 50: Enhanced mass spectrum of dihydroxybenzoic acid (protocatechuic acid) from

Deschampsia antarctica

Figure 51 : Enhanced mass spectrum of Octadecanoic acid from Deschampsia antarctica

Figure 52a: Enhanced mass spectrum of Orientin from Deschampsia antarctica

Figure 52b: UV- visible spectrum of Orientin from Deschampsia antarctica

Figure 53: Enhanced mass spectrum of Orotic acid from Deschampsia antarctica

Figure 54: Enhanced mass spectrum of 3,7,3,4,5pentahydroxy 5- methoxyflavilium from

Deschampsia antarctica

Figure 55: Enhanced mass spectrum of pentahydroxy flavilium malonylglucose from Deschampsia antarctica

Figure 56a: Enhanced mass spectrum of pyranxylvitexin from Deschampsia antarctica

Figure 56b: UV- visible spectrum of pyranxylvitexin from Deschampsia antarctica

Figure 57: Enhanced mass spectrum of Quercetin from Deschampsia antarctica

Figure 58: Enhanced mass spectrum of Quercetin 2,3,4 triacetylglucoside from Deschampsia antarctica

Figure 59: Enhanced mass spectrum of Rhamnose from Deschampsia antarctica

Figure 60: Enhanced mass spectrum of Sinapinic acid from Deschampsia antarctica

Figure 61: Enhanced mass spectrum of Uracil -5-carboxylate from Deschampsia antarctica

Figure 62a: Enhanced mass spectrum of Vitexin from Deschampsia antarctica

Figure 62b: UV- visible spectrum of Vitexin from Deschampsia antarctica

Figure 63a: Enhanced mass spectrum of Rhamnosyl - 4-methylorientin from Deschampsia antarctica Figure 63b: UV- visible spectrum of Rhamnosyl-4-methylorientin from Deschampsia antarctica Figure 64a: Enhanced mass spectrum of 2-o-L Rhamnosyl orientin from Deschampsia antarctica Figure 64b : UV- visible spectrum of 2-o-L Rhamnosyl orientin from Deschampsia antarctica Figure 65a: Enhanced mass spectrum of 2-o-L Rhamnosyl vitexin from Deschampsia antarctica Figure 65b :UV- visible spectrum of 2-o-L Rhamnosyl vitexin from Deschampsia antarctica Figure 66a Enhanced mass spectrum of 2-o- Rhamnosyl 4- methylvitexin from Deschampsia antarctica

Figure 66b: UV- visible spectrum of 2-o- Rhamnosyl 4- methylvitexin from Deschampsia antarctica Figure 67a: Enhanced mass spectrum of Dimethylpyranxyl vitexin from Deschampsia antarctica Figure 67b: UV- visible spectrum of Dimethylpyranxyl vitexin from Deschampsia antarctica Figure 68a: Enhanced mass spectrum of Isoorientin from Deschampsia antarctica

Figure 68b: UV- visible spectrum of Isoorientin from Deschampsia antarctica Figure 69a: Enhanced mass spectrum of pyranxyl orientin from Deschampsia antarctica

Figure 69b UV- visible spectrum of pyranxyl orientin from Deschampsia antarctica

Figure 70a: Enhanced mass spectrum of Taxifolin 3-Rhamnoside from Deschampsia antarctica Figure 70b: UV- visible spectrum of Taxifolin 3-Rhamnoside from Deschampsia antarctica Figure 71: Effect of Deschampsia antarctica extract Ca 35 (sample2) on cell viability on WS-1 cells at 24hrs of treatment

Figure 72: Effect of Deschampsia antarctica extract (sample 3) Ca 36 on cell viability on WS-1 cells at 24hrs of treatment

Figure 73 Effect of. Ca35 extract on cell viability and UV-C irradiated 3T3L1 cells

Figure 74 Effect of Dal fraction of the extract Ca35 on cell viability and UV-C irradiated 3T3L1 cells

Figure 75 Effect of Da2 fraction of the extract Ca35 on cell viability and UV-C irradiated 3T3L1 cells

Figure 76 Effect of Da3 fraction of the extract Ca35 on cell viability and UV-C irradiated 3T3L1 cells

Figure 77 Effect of Da5 fraction of the extract Ca35 on cell viability and UV-C irradiated 3T3L1 cells

Figure 78 Effect of Da7 fraction of the extract Ca35 on cell viability and UV-C irradiated 3T3L1 cells

Figure 79 Effect of Da8 fraction of the extract Ca35 on cell viability and UV-C irradiated 3T3L1 cells

Figure 80 Effect of Da9 fraction of the extract Ca35 on cell viability and UV-C irradiated 3T3L1 cells

Figure 81 Effect of DalO fraction of the extract Ca35 on cell viability and UV-C irradiated 3T3L1 cells

Figure 82 Effect of Dal 1 fraction of the extract Ca35 on cell viability and UV-C irradiated 3T3L1 cells

Figure 83 Effect of Da 12 fraction of the extract Ca35 on cell viabiUty and UV-C irradiated 3T3L1 cells

/ DETAILED DESCRIPTION OF THE INVENTION

The present invention is in relation to efficiency of the bioactive component of the plant extract for therapeutic use, wherein said extract from Deschampsia antarctica is for comoceutical, pharmaceutical, nutritional and nutraceutical applications.

In one aspect of the invention, there is a provided a prophylactic method for preventing the occurrence of a disease state in a mammal which comprises administering to the said mammal an effective non-toxic amount of an extract from Deschampsia antarctica as defined herein in the preparation of a comestible (foodstuff) for prophylaxis against the occurrence of Skin disorder. Preferably the mammal is human and the said extract comprises a single extract from a plant part of Deschampsia antarctica or a combination of extracts there from as detailed herein. Thus the present invention further relates to extracts, which may be isolated from fruits of the Deschampsia antarctica plant, the preparation of such extracts, medicaments comprising such extracts, and the use of these extracts and constituents for the preparation of a medicament.

One of the embodiments of the present invention provides, extracts that are isolated from plant part of Deschampsia antarctica, using conventional organic solvent extraction and supercritical fluid extraction technology. Generally, extracts of the invention capable of functioning in a prophylactic or therapeutic manner as outlined herein can be extracted from any Deschampsia plant, depending on the end purpose that is required of the extract.

In some of the embodiments of the present invention there is provided a process for preparing extracts of the invention from plant parts of Deschampsia antarctica that comprises:

• Pulverizing selected plant material to a powder;

• Subjecting the powdered plant material to solvent extraction;

• Lyophilizing the obtained extracts.

The choice of selected plant material may be of any type but is preferably the fruits of the Deschampsia antarctica plant.

The solvent extraction process may be selected from direct types such as extraction from plant parts in reflux extractor apparatus or in flasks at room temperature or at higher temperature with polar and/or non-polar solvent(s)._. Typically, the extraction process is as outlined herein. In another embodiment of the invention, the compositions for preventing, treating, or managing cardiovascular diseases and related disorders, comprises of direct composite extract of plant species with alcohol, water and hydroalcohol solvent and successive extract of plants with alcohol, water and hydroalcohol solvent, alone or in combination thereof. The compositions/medicaments may contain a pharmaceutically acceptable carrier, excipient, or diluent.

It will be apparent to the skilled addressee that the selection of solvent, or mixtures of solvents for each step in the isolation of extracts of the invention showing activity can be guided by results of bioassay analysis of separate fractions, for example as indicated herein and/or as shown in the examples.

Some of the embodiments of the invention will describe the HPLC profiles and Mass spectrums of direct and successive solvent extracts of Deschampsia antarctica plant parts thereby giving each extract an identity of itself.

Extracts will be analyzed using HPLC and GC/LC ESI- MS.

The successive extraction from plant extract will be carried out using soxhlet extractor. The solvents used, will be based on their sequential polarity starting from non-polar to polar, wherein, various classes of metabolites will be extracted viz. petroleum ether (phytosterols, fixed oils and fats), benzene (fixed oils and fats), chloroform (alkaloids), acetone (phytosterols, phenolics and tannins) ethanol (alkaloids, carbohydrates, glycosides, phytosterols, saponins, phenolics, tannins, proteins and amino acids) and water (alkaloids, carbohydrates, glycosides, saponins, phenolics, tannins, proteins, amino acids, gums and mucilage) at 65°C. These fractions will be lyophilized and stored in amber colored bottles at 4°C.

Phytochemical investigations will be also carried out on these extracts using various tests like Mayer's and Dagendorf s tests for alkaloids; Molisch, Fehling and Benedict tests for carbohydrates; Lieberman Buchard's test for phytosterols and triterpenes; spot test for fixed oils and fats; Ferric chloride and Lead acetate test for phenolic compounds and tannins; Ninhydrin and Biuret tests for protein and aminoacids; alcoholic precipitation followed by Molisch test for gum and mucilages.

Metabolic fingerprinting of all the direct and successive extracts from Deschampsia antarctica plant parts is done by HPLC and LC/MS technique. The extracted fractions will be subjected to HPLC using μ bondapak C i8 column (Waters Alliance 2695 Separation Module) to separate the constituent metabolites. The fractions will be eluted using a combination (80:20, 60:40, 50:50, 40:60, 20:80) of methanol: water / acetonitrile: water. The gradient run was also be carried out wherever required. 5- lOul of sample was injected with flow rate of 1 ml/min and HPLC run was performed for 30 minutes. The detection will be done on photodiode array and the analysis of the results will be done with the help of Millennium™ software.In some embodiments of the present invention, the analysis of out put. of LC-PDA and LC/MS MS will be done using both LC-Solution and Analyst software of Applied Biosystems along with the script that is developed by Avesthagen to determines the molecular weight of chemical compounds by ionizing, separating and measuring molecular ions according to their mass-to-charge ratio (m z).In some of the embodiments of the present invention, mammalian cell based efficacy tests are conducted by growing Human hepatoblastoma cell line (Hep G2), in a flask with EMEM containing 10% FBS, 1% glutarnine-penicillin-streptomycin and 1% fungizone in a humidified incubator at 37°C in an atmosphere of 5% CO₂ and 95% air. It is further subcultured when cell become 80% confluent they are subjected to treatment with bioactive under investigation. The incubation is followed by estimating levels of bio-molecules like Apo AI, total cholesterol, HDL cholesterol, triglyceride levels between the bioactive treated and untreated sets.

The invention further describes the biotherapeutic potential of various extracts of Deschampsia antarctica&s described above, by studying their performance in cell based assay models.

In a third aspect of the invention there is provided a method for treating a disease in a mammal, which comprises administering to the said mammal an effective non-toxic amount of at least an extract from Deschampsia antarctica as defined herein. Preferably the mammal is a human being. The skilled addressee will appreciate that "treating a disease" in a mammal means treating, that is to say, alleviating symptoms of the disease and may also mean managing a disease in the sense of preventing such a disease state either advancing i.e. getting worse or becoming more invasive, or slowing down the rate of advance of a disease.

The compositions/medicaments may contain a pharmaceutically acceptable carrier, excipient, or diluent. The compositions can be included as unit dosage suitable for parenteral, oral, or intravenous administration to a human. Alternatively, the compositions are dietary supplements, food compositions or beverage compositions suitable for human or animal consumption.

The frozen plant material was procured from Coppermine Peninsula on Robert Island, South Shetland Island, Antarctica and was exported to us by Instituto Antarctico Chileno. Procedure 1: Extraction of Deschampsia antarctica:

Extraction of Deschampsia antarctica plants was carried out by Ethanolic extraction method, 15 mins at 4° C in reflux extractor apparatus followed by lyophilization under vacuum. The solvents used for extraction is 80% Ethanol.

Example 1: Extraction of Deschampsia antarctica grass - Wild type

I. Extraction procedure for Ca35

Reflux extraction:

Deschampsia antarctica wild type grass (fresh) stored at -80°C was grinded into a fine powder using liquid nitrogen in a mortar and pestle. This was transferred to a round bottomed flask and two - three volumes of 80% ethanol was added, few ceramic chips or glass beads were taken in the flask and kept on a heating mantle. A condenser with cold water circulating through it was placed on the flask before setting the temperature to 65°C.

The solvent vapor from the flask passes through the inlet of the extractor and condenses. The condensed solvent extracts the polar compound from the plant material. This process is continuous as long as there is stable heat and water circulation to condense the vapors. The extraction was continued for 2 hours and extracted at least for two times. After 2 hours the mantle was switched off and the water flow was stopped. After cooling, the extract was collected separately and centrifuged. The extract was concentrated by drying under vacuum.

The percentage yield of the extract is calculated with respect to the initial weight of the plant material taken before extraction.

Percent Yield = wt. of lyophihzed extract (after drying) * 100

Wt. of Plant material (initial)

Π. Extraction procedure for Ca36

This extraction was carried out at Room temperature

Deschampsia antarctica wild type grass (fresh) stored at -80 °C was grinded into a fine powder using liquid nitrogen in a mortar and pestle. This was transferred to a glass conical flask with 3 volumes of 80% ethanol. A magnetic bead was added and kept on a magnetic stirrer and the conical flask mouth was covered to avoid vaporization if any. Extraction was carried out with constant stirring at RT for 2 hours and repeated for 3 times for maximum yield. After the plant material settles down, the liquid extract is filtered through filter paper to obtain clear extract solution, which is dried under vacuum. The percent yield is calculated as given above

Example2: Fractionation of 80% Ethanolic Extract of Deschampsia antartica (Ca35) in Silica gel column using VersaFlash :

10 grams of Ca35 extract was weighed in a beaker and 100ml of petroleum ether (petether) was added directly and sonicated for 30 minutes. The clear pet ether fraction was collected by filtering it through filter paper. This procedure was repeated for another 3 times and totally 400ml of pet ether was used for de-fatting before subjecting Ca35 for silica gel fractionation.

Ca35 after pet ether treatment was dissolved in 80% ethanol and adsorbd on Silica -gel. (60 - 120, Merck) in vacuo and loaded on the Silica Cartridge (40 X 75mm, SUPELCO, Sigma-Aldrich Co) packed with Silica gel (60 - 120, Merck) and was run usinf VersaFlash, with a flow rate of lOml/min The fractions were collected using non-polar and polar solvents sequentially Chloroform, acetone, ethanol, 80% ethanol 50% ethanol water, water with 0.1% acetic acid, water with 0.5% acetic acid and finally to watenacetonitrile (45:55). All the fractions of Ca35 were subjected to TLC profiling with different elution solvent system All the collected fractions were pooled into 13 fractions based on TLC pattern

Example 3:

Liquid chromatography and Mass spectrometry of Deschampsia antarctica Ca35, Ca36 and fractions Dal - Dal3 from Ca35)

Identification and characterization of various phytochemicals present in Deschampsia antarctica by LC-MS/MS

i) Acquisition of + EMS in full scan mode from m/z 50 amu to 1000 amu

ii) Acquisition of - EMS in full scan mode from m/z 50 amu to 1000 amu

iii) Acquisition MS/ MS of selected ions

iv) Analysis of the mass peaks and characterization of the metabolites Sample Preparation:

Sample 1 : 20mg of Deschmapsia antarctica leaves were ground in liquid Nitrogen and extracted with lml of methanol: chloroform: water (6:2:2). The extracts were vortexed for 5 min and was kept on 4°C for 1 hour. The vials containing extract were then centrifuged for 15 min at 14000 rpm and 4°C to remove any suspended particles. Sample 2: Extract ot Ueschmapsia antarctica Ca 3 was extracted with lml ot methanol: cniorororm: water (6:2:2). The extracts were vortexed for 5 min and was kept on 4°C for 1 hour. The vials containing extract were then centrifuged for 15 min at 14000 rpm and 4°C to remove any suspended particles

Sample 3: Extract of Deschmapsia antarctica Ca36 was extracted with lml of methanol: chloroform: water (6:2:2). The extracts were vortexed for 5 min and was kept on 4°C for 1 hour. The vials containing extract were then centrifuged for 15 min at 14000 rpm and 4°C to remove any suspended particles

Sample 4: The fractions collected from Ca35 extract were dissolved methanol. Except Da9-10 in methanol :water (50:50)).

All the 4 samples were. filtered through a 0.2-^-syringe filter, the clarified extracts were carefully transferred into respective autosampler vials (1.5 mL capacity, Shimadzu, Prominence). The extracts were subjected to an autosampler (SEL20AC) attached to HPLC (Shimadzu, Prominence). The temperature of the autosampler was maintained at 8°C throughout the experiment. The samples were eluted from UFLC by a binary gradient through a 5 μ particle size RP- 18 column, (4.6 mm D x 250 mm x L) held at 40°C in a temperature controlled column oven (CTO 20AC) at a flow rate of 0.4ml/min over 60.01 min. The gradient system consisted of 0.1% aqueous formic acid (A) and 0.1% formic acid in acetonitrile (B). The gradient was programmed to attain 95% (B) over 52 min, remains same till 56 min and decreases instantly to 5% at the end of 57 min. The 5% (B) remains till 60 min and the UFLC stops at 60.01 min. The UFLC eluent was further directed into mass spectrometer (Applied Biosystems MDS SCIEX 4000 Q Trap MS/MS).

The Mass spectrometer was operated in an EMS positive and negative polarity mode. The ion spray voltage was set to 2750, source temperature 275°C, vacuum 4.6^"5 Torr, curtain gas 20, Collision Energy (CE) 5, GS 1 40, GS2 60, and declustering potential of 35 for acquisition of TIC of EMS in negative ionization where as, for the acquisition of TIC of positive EMS the ion spray voltage was set to 4000, source temperature 400°C, vacuum 4.6^"5 Torr, curtain gas 20, Collision Energy (CE) 5, GS 1 40, GS2 60, and declustering potential of 35. The scan rate was set at 1000 amu/ s with the interface heater 'on', 967 scans in a period and a dynamic LIT fill time (20 m sec) was on.

The acquisition of Enhanced Product Ion (EPI) by LC-MS/MS. The enhanced product ion and MS/MS was performed at LC flow rate of 0.5 mL min^"1 over a period of 60.01 min,. The fragmentation (EPI) was done for the selected ions both in positive and_. negative polarity mode. For positive polarity mode the curtain gas was set to 20, Collision Energy 30, 40, CES 10, ion spray voltage was set at 4UUU.00 iS 1 4U, (iS2 60 with interlace heater and the dynamic till time on. tor negative polarity mode the curtain gas was set to 20, Collision Eriergy -30, -40, CES 10, ion spray voltage was set at -2750.00, temp 275.00, GS1 40, GS2 60 with interface heater and the dynamic fill time on.

For the processing, the total ion chromatogram (TIC) of blank (solvent) and test sample were Gaussian smooth, base line subtracted and noise was set to 1%. The TIC of blank was subtracted from that of the TIC of test and the spectrum was generated using Analyst Software 1.4.2. The noise level of spectrum was set to 1%. The processed spectrum is also manually verified. The data list is then generated to check the number of ions present with their m/z, centroid m z, peak intensities, resolution, peak area and their charge specification. Next level of processing involves the elimination of the multiple charge ions by checking their singly charged ions. The low intense ions are further extracted to obtain Extracted ion chromatogram (XIC) or amplified. (Figl- 18,19a- 21a,23a, 42a,43a,45a-49a,52a,56a,62a-70a).

Apart from the molecules given figure other primary metabolites viz., glucose fructose, citrate, serine threonine, proline, leucine.glycine alanine and cysteine.

Example 4:

Estimation of Polyphenolics in tissue cultured Deschampsia antarctica

Phenolic compounds in alkaline condition (sodium carbonate) dissociate to yield a proton and phenolate anion, which is capable of reducing Folin ciocalteu reagent. FC reagent is an oxidizing agent comprised of heteropolyphosphotungstate-molybdate. Sequences of one or two electron reduction reaction lead to blue color species. The blue colored product is a mixture of the 1-, 2-, 4-, and 6-electron reduction products in the tungstate series P₂W_lg0₆₂ ^"7 to H₄P₂W₁₈0₆₂ ^"8 and the 2-, 4-, and 6-electron reduction products in the molybdate series H₂P₂Mo₁₈0₆₂ ^"6 to H₆P₂Mo₁₈0₆₂ ^"6.

Procedure:

Polyphenol assay is carried out by Using singleton, V., Rossi, J.A. Jr, method (1965), In brief to a 200μ1 of 50% Methanol / Standard / test sample with various concentrations, add lOOOul of FC reagent, mixed and incubate at Room Temperature for 5min. Add 800μ1 7.5% sodium carbonate, mix and incubate at Room Temperature for 30min. Read the absorbance at 750nm against blank by spectrophotometer, Colour Correction: Contains the same concentration of the test sample in 50% Methanol without FC reagent. Observation and results are given in the Table 1. Table 1: Total Phenolics of Deschampsia antarctica

Example 5: DPPH assay:

DPPH is a free radical, has got free electron, very unstable generally. When this free radical reacts with antioxidant, antioxidant donates electron to free radical and makes it stabilize. This reduced DPPH gives change in colour from black to yellow and the change in absorbance at 517nm is followed which can be measured spectrophotometrically.

Procedure

Samples: Deschampsia antarctica grass wild type and in-house Deschampsia antarctica leaves stored at -80°C were used.

Sample preparation: One gram of fresh tissue was grinded into a fine powder using liquid nitrogen in a mortar and pestle and extracted in 5 ml of 80% ethanol. Sonicated for 2 minute before centrifuging at 5000 rpm for 10 - 15minutes. The clear supernatant is taken for the assay. Various concentration of fresh Deschampsia antarctica extract or standard ascorbic acid taken in 215 μΐ of methanol were mixed with 36 μΐ of ImM DPPH solution in a 96- well plate. Incubated at 37 °C for 30 minutes. Appropriate colour corrections were run simultaneously. Reduction of DPPH was followed at 517nm in a biotech reader.

Observation and results are given in the DPPH assay of Deschampsia antarctica Table 2

Table 2: DPPH assay of Deschampsia antarctica

Example6:

ORAC assay: The antioxidant capabilities of Deschampsia antarctica are evaluated using ORAC is exhibiting a value of 274.05 TEmmoVg Sample. (Table 3)

Table 3: ORAC assay of Deschampsia antaratica

Example 7:

Viability assay: Viability assays were carried out to check cytotoxicity of Ca35 and Ca36 extracts on human fibroblast cell culture WS-1.

Ca35 and Ca36 extracts did not show any cytotoxicity at all the studied doses of 0.1-500 μg/ml. (Figure 7 land72)

Example 8:

HPLC and UV- visible Spectrum of metabolites from Deschmapsia antarctica :

HPLC analysis of Deschampsia antarctica extract was done using UFLC (Shimadzu, Prominence). Deschampsia antarctica leaves (20 mg) were ground in liquid Nitrogen and extracted with 1ml of methanol: chloroform: water (6:2:2). The extracts were vortexed for 5 min and was kept at 4°C for 1 hour. The vials containing extract were then centrifuged for 15 min at 14000 rpm and 4°C to remove any suspended particles. The extract was subjected to autosampler that was maintained at 8°C throughout the experiment. The samples were eluted from UFLC by a binary gradient using RP- C18 column (Phenomenex, Gemini 4.6 mm D x 250 mm x L, 5,μ particle size) held at 40°C in a temperature controlled column oven (CTO 2UA J) at a flow rate or U.4ml/min over 60.01 mm. Ihe gradient system consisted of 0.1% aqueous formic acid (A) and 0.1% formic acid in acetonitrile (B). The gradient was programmed to attain 95% (B) over 52 min, remains same till 56 min and decreases instantly to 5% at the end of 57 min. The 5% (B) remains till 60 min and the UFLC stops at 60.01 min. The UFLC eluent was further directed into LC-PDA detector (SPD-M20A, Shimadzu) and fluorescence detector (RF 10 AXL, Shimadzu) for spectral analysis using LC-Solutions The UV visible spectrum of identified molecule are given in (Fig 19b- 21b,23b, 42b,43b,45b-49b ,52b,56b,62b-70b). The metabolites mentioned in the figure are chromophores and these metabolites fall under the flavonoid group.

Example 9 :

Photoprotection activity screening of Deschampsia antarctica

The in vitro 3T3L1 photoprotective test is used to identify the photoprotective potential of test samples on exposure to UV light. 3T3-L1 cells are exposed to UV radiation in the presence and absence of test material and the photoprotective potential of the test material is determined by comparing the cell viability of cells. Substances identified by this test are likely to be photoprotective in vivo, following systemic application to the skin.

Protocol for phototoxicity assay:

Cells were seeded in a 96 well tissue culture plate at 1 X 10⁵ cells per well. The plate were incubated at 37°C for 24 hrs (5 % C0₂). Once the cells were confluent, media was discarded and cells were washed with HBSS. The cells were brought to quiescence by placing them in DMEM. The control plates were placed in the dark while the experimental plates were exposed to UV-C radiation for 2 hrs. After treatment, HBSS was removed and cells were washed with DMEM and re-fed with complete, media and incubated for a further 24 hrs. Ca35 extract and all the 13 fractions (DA 1-13 ) extracts were analysed for phototoxicity..100 μΐ 'οί DMEM is added to the cell pellet, ΙΟμΙ of WST-8/ MTS is added to each well The colour developed is measured at 450/490 nm

The results from phototoxicity indicates that Deschampsia extact Ca35 is a good cell prohferator as seen from the increase in cell number at 100 μg/ml but there is no UV protective activity as it is not showing. any significant difference in cell number compared to the cell viability graph at 100 μg/ml (Fig 73). The fraction DAI from Ca35 extract is a good cell proliferator as seen from the increase in cell number at 100 μg/ml and also exhibited UV protective activity by showing a significant difference in cell number compared to the cell control (DMSO0.1 ) at 100 μg/ml. (Fig 74).

The fraction DA2 from Ca35 extract is non cytotoxic at all the studied doses of 0.1-100 μ^πιΐ, however, exhibited UV protective activity by showing a significant difference in cell number compared to the cell control (DMSO0.1%) at 100 μg/ml. (Fig 75) -

The fraction DA3 from Ca35 extract is not cytotoxic at the studied doses of 0.1-100 μ^ητΐ, however, exhibited UV protective activity by showing a significant difference in cell number compared to the cell control (DMSO0.1 ) at 1 μ^πιΐ. (Fig 76)

The fraction DA5 from Ca35 extract is non cytotoxic at all the studied doses of 0.1-100 μg/ml, however, exhibited UV protective activity by showing a significant difference in cell number compared to the cell control (DMSO 0.1%) at 100 μ^ητΐ. (Fig 77)

The fraction DA7 from Ca35 extract is not cytotoxic at the studied doses of 0.1-100 μg/ml, however it exhibited UV protective activity by showing a significant difference in cell number compared to the cell control (DMSO0.1%) at 100 μg/ml (Fig 78).

The fraction DA8 from Ca35 extract is not cytotoxic at all the studied doses of 0.1-100 μg/ml, however it exhibited UV protective activity by showing a significant difference in cell number compared to the cell control (DMSO0.1%) at 1 μg/ml (Fig 79).

The fraction DA9 is non cytotoxic at all the studied doses of 0.1-100 μg/ml, however it exhibited UV protective activity by showing a significant difference in cell number compared to the cell control (DMSO0.1%) at l(X^g/ml (Fig 80).

The fraction DA10 from Ca35 extract is non cytotoxic at all the studied doses of 0.1-100 μ^ητΐ, however, exhibited UV protective . activity by showing a significant difference in cell number compared to the cell control (DMSO 0.1%) at 100 μg/ml (Fig 81 ).

The fraction DA11 from Ca35 extract is non cytotoxic at all the studied doses of 0.1-100 μ^πιΐ, however, exhibited UV protective activity by showing a significant difference in cell number compared to the cell control (DMSO0.1 %) at 100 μg/ml (Fig 82)..

The fraction DA12 from Ca35 extract is non cytotoxic at all the studied doses of 0.1-100 μg/ml, however it exhibited UV protective activity by showing a significant difference in cell number compared to the cell control (DMSO0.1%) at 100 μ^ητΐ (Fig 83). References:

1. Alberdi M, Bravo LA, Gutie 'rrez A, Gidekel M, Corcuera LJ (2002) Ecophysiology of Antarctic vascular plants. Physiol Plant 115:479-486

2. Manuel Gidekel, Luis Destefano-Beltra'n, Patricia Garci'a, Lorena Mujica, Pamela Leal,Marely Cuba, Lida Fuentes, Leo' n A. Bravo.Luis J. Corcuera , Miren Alberdi, Eoria Concha, Ana Gutie 'rrez, Identification and characterization of three novel cold acchmati on- responsive genes from the extremophile hair grass Deschampsia antarctica Desv. Extremophiles (2003) 7:459^69

3. Greene SW. 1970. Studies in Colobanthus quitensis ( unth) Bartl. and Deschampsia antarctica Desv. I. Introduction. British Antarctic Survey Bulletin 23: 19-24

4. Moore DM. 1970. Studies in Colobanthus quitensis (Kunth) Bartl. and Deschampsia antarcica Desv. Π. Taxonomy, distribution and relationships. British Antarctic Survey Bulletin 23: 633-680.

5. Corner RWM. 1971. Studies in Colobanthus quitensis (Kunth.) Bartl. and Deschampsia antarctica Desv. IV. Distribution and reproductive performance in the Argentine Island. British Antarctic Survey Bulletin 26: 41-50.

6. Greene DM, Holtom A. 1971. Studies in Colobanthus quitensis (Kunth)Bartl. and Deschampsia antarctica Desv. ΙΠ. Distribution habitats and performance in Antarctic Botanical Zone. British Antarctic SurveyBulletin 26: 1-29.

7. Edwards JA. 1972. Studies in Colobanthus quitensis (Kunth) Bartl. And Deschampsia antarctica Desv. VI. Reproductive performance on Signy Island. British Antarctic Survey Bulletin 39: 67-86.

8. Edwards JA. 1974. Studies in Colobanthus quitensis (Kunth) Bartl. and Deschampsia antarctica Desv. Ph.D. Thesis, University of Birmingham.

9. Edwards JA. 1975. Studies in Colobanthus quitensis (Kunth) Bartl. And Deschampsia antarctica Desv. VTL Cycling changes related to age in Colobanthus quitensis. British Antarctic Survey Bulletin 40: 1-6.

10. Jellings AJ, Usher MB, Leech RM. 1983. Variations in the chloroplasts to cell index in Deschampsia antarcica along a 16_ latitudinal gradient.British Antarctic Survey Bulletin 61:

. 13-23.

11. Edwards JA, Levis Smith RI. 1988. Photosynthesis and respiration of Colobanthus quitensis and Deschampsia antarctica from the maritimeAntarctic. British Antarctic Survey Bulletin 81: 43-63. Zu'rfiga GE, Alberdi M, Fernandez J, Mo^'ntiel P, Corcuera LJ. 1994. Lipid content in leaves of Deschampsia antarctica from the maritimeAntarctic. Phytochemistry 37: 669-672.

Zu'nlga GE, Alberdi M, Corcuera LJ. 1996. Non-structural carbohydrates in Deschampsia antarctica Desv. from South Shetland Islands, Maritime Antarctic. Environmental and Experimental Botany 36: 393-398.

Convey P. 1996. Reproduction of Antarctic flowering plants. Antarctic Sciences 8: 127-134. BarcikowskiA, Ly_zwin'ska R, Zarzycki K. 1999. Growth rate and biomass production of Deschampsia antarctica Desv. in the Admirality Bay region. South Shetland Islands

Antarctica. Polish Polar Research 20: 301-31 1.

Barcikowski A, Czaplewska J, Gielwanowska I, Loro P, Smyka J. 2001. Deschampsia antarctica (Poaceae) - the only native grass from Antarctica. In: Frey L, ed. Studies on grasses in Poland. Krako'w: W. Szafer Institute of Botany, Polish Academy of Sciences, 367-377.

Zwolska I, Rakusa-Suszczewski SJ. 2002. Temperature as an environmental factor in the Arctowski Station Area (King George Is., South Shetlands Iss.). Geographia Polonica Special Issue, Global Change 9:51-65.

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Nkongolo KK, Deck A, Michael P. 2001. Molecular and cytological analyses of Deschampsia caespitosa populations from Northern Ontario (Canada). Genome 44: 818-825. Chwedorzewska K, Bednarek PT, Puchalski J. 2004. Molecular variation of Antarctic grass Deschampsia antarctica Desv. from King George Island (Antarctica). Acta Societatis Botanicorum Poloniae 73: 23-29.

New extracts of Deschampsia antarctica desv. With antineoplastic activity (WO/2009/064480), PCT/US2008/012819

Claims

We claim:

1. A method of preparing a Plant extract comprising the steps of : a) Obtaining plant material from one or more parts of the plants, b) Obtaining an extract from the plant material by contacting the plant material with an aqueous, an ethanolic or an organic solvent, or a combination thereof, , optionally for a defined period of time thereby providing one or more plant extracts, c) Removing the plant material from the supernatant obtained in step b, d) Optionally, lyophilizing said supernatant.

2. A method of preparing fractions from Deschampsia antarctica plant extract as in claim 1 using non polar and polar solvents petroleum ether.chioroform, acetone, methanol, ethanol , water and acetic acid

3. In a metaboUte analysis system, a method, comprising the steps of: Solvent composition used in extraction of the metabolites from Deschampsia antarctica for identifying chromatography and mass spectrometry peaks from the sample run; mass spectrometry peak being one of an MS peak and MS/MS peak (50 amu to 1500 amu) and using nominal or exact mass; generating a list of sample data having said identified peaks; performing chemometric analysis on said sample data to identify metabolites said chemometric analysis performed without loss of retention time data by the same application performing the programmatic identification of said chromatography arid mass spectrometry peaks.

4. The Deschampsia species extract of claim 1, 2 and 3, wherein the fraction comprises a natural chemical entity selected from the group consisting of a polyphenol, a polysaccharide, amino acid, terpenoids, alkaloids and combinations thereof.

5. The plant extract of claiml, 2 and 3„ wherein the fraction comprises of 5 - hydroxyferulic acid

6. The plant extract extract of claim 1, 2 and 3„ wherein the fraction comprises of 5, 7- dimethoxyflavone

7. The plant extract of claim 1, 2 and 3„ wherein the fraction comprises of 2 -o- arabinopyranosylorientin

8. The plant extract of claim 1, 2 and 3, wherein the fraction comprises of Caffeic acid

9. The plant extract of claim 1, 2 and 3, wherein the fraction comprises of Cinnamaldehyde

10. The plant extract of claim 1, 2 and 3, wherein the fraction comprises of Apigenin

11. The plant extract of claim 1, 2 and 3, wherein the fraction comprises of Coumaric acid

12. The plant extract of claim 1, 2 and 3, wherein the fraction comprises of Cyanidin

13. The plant extract of claim 1,2 and 3, wherein the fraction comprises of Cyanidin -3- rutinoside

14. The plant extract of claim 1,2 and 3, wherein the fraction compnses of Cyanidin 3, 6 dimalonylglucoside

15. The plant extracts of claim 1, 2 and 3, wherein the fraction comprises of Diacetylgalactopyranosylorientin.

16. The plant extract of claim 1, 2 and 3, wherein the fraction comprises of Dihydroxykaempferol.

17. The plants extract of claim 1, 2 and 3, wherein the fraction comprises of

Dihydroxyacetonephosphate.

18. The plant extract of claim 1, 2 and 3, wherein the fraction comprises of Dihyrdoxy indole -2- carboxylic acid.

19. The plant extract of claim 1, 2 and 3, wherein the fraction comprises of Dihydroxyphenylarabinosyl- dihydroxybenzopyran.

20. The plant extract of claim 1, 2 and 3, wherein the fraction comprises of L-DOPA.

21. The plant extract of claim 1, 2 and 3, wherein the fraction comprises of Ferulic acid.

22. The plant extract of claim 1, 2 and 3, wherein the fraction comprises of Galacturonic acid

23. The plant extract extract of claim 1,2 and 3, wherein the fraction comprises of Gluconic acid

24. The plant extract of claim 1, 2 and 3, wherein the fraction comprises of Glucopyranosyltrihydroxymethylflavone.

25. The plant extract of claim 1, 2 and 3, wherein the fraction comprises of Hespertin.

¹26. The plant extract of claim 1, 2 and 3, wherein the fraction comprises of Homovaratic acid.

27. The plant extract of claim 1, 2 and 3, wherein the fraction comprises of 4 (l-hydroxy-2- methylaminoethyl 2-methoxyphenol.

28. The plant extract extract of claim 1, 2 and 3, wherein the fraction comprises of Immidazolone -5- propionic acid.

29. The plant extract extract of claim 1, 2 and 3, wherein the fraction comprises of Isowerticin 2 - arabinose.

30. The plant extract of claim 1, 2 and 3, wherein the fraction comprises of Isowertiajaponin

31. The plant extract of claim 1, 2 and 3, wherein the fraction comprises of Isowertisin.

32. The plant extract of claim 1, 2 and 3, wherein the fraction comprises of Kaempferol.

33. The plant extract of claim 1, 2 and 3, wherein the fraction comprises of Kaempferol 3- o - rhamnosyl glucoside.

34. The plants extract of claim 1, 2 and 3, wherein the fraction comprises of Luteolin.

35. The plants extract of claim 1, 2 and 3, wherein the fraction comprises of malonylgalactosylpyranosylorientin.

36. The plant extracts of claim 1, 2 and 3, wherein the fraction comprises of o/m/p hydroxybenzoic acid.

37. The plant extract extract of claim 1, 2 and 3, wherein the fraction comprises of dihydroxybenzoic acid.

38. The plant extract extract of claim 1, 2 and 3, wherein the fraction comprises of Octadecanoic acid.

39. The plant extract extract of claim 1, 2 and 3, wherein the fraction comprises of Orientin.

40. The plant extract of claim 1, 2 and 3, wherein the fraction comprises of Orotic acid.

41. The plant extract of claim 1,2 and 3, wherein the fraction comprises of 3,7,3,4,5pentahydroxy 5- methoxyflavilium.

42. The plant extract of claim 1, 2 and 3, wherein the fraction comprises of pentahydroxy flavilium malonylglucose.

36. The plant extract of claim 1, 2 and 3, wherein the fraction comprises of pyranxylvitexin.

37. The plant extract of claim 1, 2 and 3, wherein the fraction comprises of Quercetin

38. The plant extract of claim 1, 2 and 3, wherein the fraction comprises of Quercetin 2,3,4 triacetylglucoside

39. The plant extract of claim 1, 2 and 3, wherein the fraction comprises of Rhamnose

40. The plant extract of claim 1, 2 and 3, wherein the fraction comprises of Sinapinic acid.

41. The plant extract of claim 1, 2 and 3, wherein the fraction comprises of Uracil-5-carboxylate

42. The plant extract of claim 1, 2 and 3, wherein the fraction comprises of Vitexin

43. The plant extract of claim 1, 2 and 3, wherein the fraction comprises of Rhamnosyl 4- methylorientin

44. The plant extract of claim 1, 2 and 3, wherein the fraction comprises of. 2-o- L Rhamnosyl orientin

45. The plant extract of claim 1, 2 and 3, wherein the fraction comprises of. 2-o- L Rhamnosyl Vitexin.

46. The plant extract of claim 1, 2 and 3, wherein the fraction comprises of. 2-o- L Rhamnosyl 4 methyl vitexin.

47. The plant extract of claim 1, 2 and 3, wherein the fraction comprises of. Iso orientin

48. The plant extract of claim 1, 2 and 3, wherein the fraction comprises of. Pyranxyl orientin

49. The plant extract of claim 1, 2 and 3, wherein the fraction comprises of. Taxifolin 3- Rhamnoside

50. The method and the composition where in claims 1-49 comprising photo protection activity (Fig 73-83) . ,

51. The method and the composition where in claims 1-49 comprises of chromophores.

52. The method and the composition where in claims 1-49 comprising cell proUferator Activity(Fig 73-83)

53 : Food or medicament comprising the plant species extract of claim 1 - 49 for the therapeutic use for the mitigation of skin disorder, cancer and other related disorder.