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 JErom 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; Comer, 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 etal. 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 in 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 flexibiUty. 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-alcohoUc 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 UP1.C, 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 metabohte 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 Uquid; (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 specfra in negative ionization mode of Deschampsia antarctica sample 1,
sample 2 (Ca35), sample 3 (Ca36), from m/z 900-1005
Figure 16: Overlap mass specfra 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 product ion mass spectrum of 5 - hydroxyferulic acid from Deschampsia antarctica
Figure 19: Enhanced product ion mass spectrum of 7, 5- dimethoxyflavone from Deschampsia antarctica
Figure 20: Enhanced product ion mass spectrum of 2 -o- arabinopyranosylorientin from Deschampsia
antarctica
Figure 21: Enhanced product ion mass spectrum of Caffeic acid from Deschampsia antarctica
Figure 22: Enhanced product ion mass spectrum of Cinnamaldehyde from Deschampsia antarctica
Figure 23: Enhanced product ion mass spectrum of Citrate from Deschampsia antarctica
Figure 24: Enhanced product ion mass spectrum of Coumaric acid from Deschampsia antarctica
Figure 25: Enhanced product ion mass spectrum of Cyanidin from Deschampsia antarctica
Figure 26: Enhanced product ion mass spectrum of Cyanidin -3- rutinoside from Deschampsia Antarctica
Figure 27: Enhanced product ion mass spectrum of Cyanidin 3, 6 dimalonylglucoside from Deschampsia
antarctica
Figure 28: Enhanced product ion mass spectrum of Diacetylgalactopyranosylorientin from Deschampsia
antarctica
Figure 29: Enhanced product ion mass spectrum of Dihydroxykaemferol from Deschampsia antarctica
Figure 30: Enhanced product ion mass spectrum of Dihydroxyacetonephosphate from Deschampsia
antarctica
Figure 31: Enhanced product ion mass spectrum of Dihyrdoxiindole -2- carboxylic acid from
Deschampsia antarctica

Figure 32: Enhanced product ion mass spectrum of Dihydroxyphenylarabinosyl- dihydroxybenzopyran
from Deschampsia antarctica
Figure 33: Enhanced product ion mass spectrum of L-DOPA from Deschampsia antarctica
Figure 34: Enhanced product ion mass spectrum of Ferulic acid from Deschampsia antarctica
Figure 35: Enhanced product ion mass spectrum of Fructose from Deschampsia antarctica
Figure 36: Enhanced product ion mass spectrum of Galacturonic acid from Deschampsia antarctica
Figure 37: Enhanced product ion mass spectrum of Gluconic acid from Deschampsia antarctica
Figure 38: Enhanced product ion mass spectrum of Glucopyranosyltrihydroxymethylflavone from
Deschampsia antarctica
Figure 39: Enhanced product ion mass spectrum of Glucose from Deschampsia antarctica
Figure 40: Enhanced product ion mass spectrum of Glyceraldehyde 3-phosphate from Deschampsia
antarctica
Figure 41: Enhanced product ion mass spectrum of Hespertin from Deschampsia antarctica
Figure 42: Enhanced product ion mass spectrum of Homovaratic acid from Deschampsia antarctica
Figure 43: Enhanced product ion mass spectrum of 4 (l-hydrxy-2-methylaminoethyl 2-methoxyphenol
from Deschampsia antarctica
Figure 44: Enhanced product ion mass spectrum of Indole acetic acid from Deschampsia antarctica
Figure 45: Enhanced product ion mass spectrum of Immidazolone -5- propionic acid from Deschampsia
antarctica
Figure 46: Enhanced product ion mass spectrum of Isowerticin 2 -arabinose from Deschampsia antarctica
Figure 47: Enhanced product ion mass spectrum of Isowertiajaponica from Deschampsia antarctica
Figure 48: Enhanced product ion mass spectrum of Isowertisin from Deschampsia antarctica
Figure 49: Enhanced product ion mass spectrum of Kaempferol 3- o - rhamnosyl glucoside from
Deschampsia antarctica
Figure 50: Enhanced product ion mass spectrum of Kaempferol from Deschampsia antarctica
Figure 51: Enhanced product ion mass spectrum of Luteolin from Deschampsia antarctica
Figure 52: Enhanced product ion mass spectrum of malonylgalactosylpyranosylorientin from
Deschampsia antarctica
Figure 53: Enhanced product ion mass spectrum of o/m/p hydroxybenzoic acid from Deschampsia
antarctica
Figure 54: Enhanced product ion mass spectrum of Octadecanoic acid from Deschampsia antarctica
Figure 55: Enhanced product ion mass spectrum of Orientin from Deschampsia antarctica
Figure 56: Enhanced product ion mass spectrum of Orotic acid from Deschampsia antarctica

Figure 57: Enhanced product ion mass spectrum of dihydroxybenzoic acid (protocatechuic acid) from
Deschampsia antarctica
Figure 58: Enhanced product ion mass spectrum of 3,7,3,4,5pentahydroxy 5- methoxyflavihum from
Deschampsia antarctica
Figure 59: Enhanced product ion mass spectrum of pentahydroxy flavilium malonylglucose from
Deschampsia antarctica
Figure 60: Enhanced product ion mass spectrum of pyranxylvitexin from Deschampsia antarctica
Figure 61: Enhanced product ion mass spectrum of Quercetin from Deschampsia antarctica
Figure 62: Enhanced product ion mass spectrum of Quercetin 2,3,4 triacetylglucoside from Deschampsia
antarctica
Figure 63: Enhanced product ion mass spectrum of Rhamnose from Deschampsia antarctica
Figure 64: Enhanced product ion mass spectrum of Sinapinic acid from Deschampsia antarctica
Figure 65; Enhanced product ion mass spectrum of Uracil -5-carboxylate from Deschampsia antarctica
Figure 66: Enhanced product ion mass spectrum of Vitexin from Deschampsia antarctica
Figure 67: Enhanced product ion mass spectrum of Proline from Deschampsia antarctica
Figure 68: Enhanced product ion mass spectrum of Serine from Deschampsia antarctica
Figure 69: Enhanced product ion mass spectrum of Threonine from Deschampsia antarctica
Figure 70 Enhanced product ion mass spectrum of Leucine from Deschampsia antarctica
Figure 71: Enhanced product ion mass spectrum of Glycine from Deschampsia antarctica
Figure 72: Enhanced product ion mass spectrum of Alanine from Deschampsia antarctica
Figure 73: Enhanced product ion mass spectrum of Cysteine from Deschampsia antarctica
Figure 74: Effect of Deschampsia antarctica extract Ca 35 (sample2) on cell viability on WS-1 cells at
24hrs of treatment Figure 75: Effect of Deschampsia antarctica extract (sample 3) Ca 36 on cell viability on WS-1 cells at
24hrs of treatment
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 cosmeceutical, 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

(foodstufO 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 fiirther 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 fruits 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 diseases, 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.
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 metaboUtes 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, phenoUcs, tannins, proteins and amino acids) and water (alkaloids, carbohydrates, glycosides, saponins, phenoUcs, tannins, proteins, amino acids, gums and mucilage) at 65°C. These fractions will be lyophilized and stored in amber colored bottles at 4°C.
Metabolic fingerprinting of all the direct and successive extracts from Deschampsia antarctica plant parts is done by 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 ^ 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 progranuned 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 (Shimadzu) for spectral analysis using both LC-Solution and Analyst software (Applied Biosystems) for mass analysis to determines the molecular weight of chemical compounds by ionizing, separating and measuring molecular ions according to their mass-to-charge ratio (m/z). The Full survey scan of TIC of +EMS and -EMS of the Deschampsia antarctica (Da) yielded respective EMS spectrum from m/z 50 through m/z 1500. The +EMS and the -EMS spectrum of Deschamapsia antarctica showed a total of 1999 and 2012 spectral features respectively including isotopes. The similar +EMS and -EMS spectra of the extract of Ca35 showed 1126 (+EMS) and 1203 (-EMS) spectral features respectively including isotope patterns whereas, the extract of Ca36 showed 1209 and 1358 spectral features. The above features, after removal of noise and the isotopes in the extract of Da, revealed the presence of 1019 and 1382 molecules in the +EMS and -EMS spectrum respectively where as, a total of 608 and 642 molecules; and 442 and 518 molecules have been found to be present in the negative ion spectra and positive ion spectra of the extracts of Ca35 and Ca36 respectively.
10

The invention further describes the biotherapeutic potential of various extracts of Deschampsia antarctica3& 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.
Procedure 1: Extraction o{ 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 -80oC was grinded into a fine powder using
Uquid 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 65oC.
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
11

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 lyophilized extract (after drying) * 100
Wt. of Plant material (initial)
II. 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
III Fractionation of extract of Deschampsia antarctica: Deschampsia antarctica extract fractionation SiUca gel Column:
Deschampsia antarctica extract Ca35 was loaded on to silica gel column previously equilibrated with petroleum ether. 20 ml of the pet ether eluents /fractions were collected until the fractions were colourless. Then the column was sequentially eluted with Chloroform, Acetone, Ethanol, and Water and around 10ml of the elution is collected per fraction. Finally the siUca colunm was taken in a conical flask with 80% ethanol to remove all uneluted compounds.
TLC profile - fractions of Ca35
2-4 ml from the above fractions were spotted on a TLC plate and allowed to air dry. They were eluted
with different eluent solvent combination for different fractions. After complete drying the plates were
exposed to Iodine vapours to better visuaUze spots since it has high affinity for both unsaturated and
organic compounds. In most cases there were clear spot/band along with trail. Few plates were developed
with p-anisaldehyde or vanillin reagent. Compilations of all visible bands/spots are given in the table
below.
12

Ca 35 Fractions from silica gel column on TLC
Fraction number Rf Elution Solvent
F2 0.81 Acetic Acid: H20 : EA: Hexane
F3 0.83

F4 0.00

F5 0.00 Acetic Acid: H20 ; EA : Hexane
F6 0.81

F7 0.86

F8 0.79

F9 0.61 Acetic Acid: H20 : EA: Hexane

0.76

FIO 0.61

0.76

Fll 0.63

0.78

F12 0.61

0.83

F13 0.59 Acetic Acid : H20 : EA : Hexane

0.77

F14 0.59

0.77

F15 0.59

0.77

F16 0.59

0.77

F17 0.57 Acetic Acid: H20 : EA: Hexane

0.75

F18 0.57

0.77

F19 0.59

0.77

F20 0.59

0.75

F21 0.57 Acetic Acid: H20 : EA : Hexane

0.75

F22 0.61.

0.80

F23 0.45

0.59

0.82

F24 0.50

0.64

0.80

F25 0.45 Acetic Acid: H20 : EA : Hexane

0.61

13

0.80
F26 0.45

0.61

0.77

F27 0.50

0.61

0.82

F28 0.48

0.61

0.82

F29 0.70 Acetic Acid : H20 : EA : Hexane
F30 0.73

F31 0.73

F32 0.70

F50 0.00 Acetic Acid : H20 : EA : Hexane
F53 0.11

0.18

0.30

0.39

0.50

F57 0.00

F62 0.00

F68 0.00

F72 0.00

F73 0.00

F76 0.00

F80 0.00

F73 0.00 MeOH : EA : H20 : Phosphoric Acid
F76 0.62

F80 0.62

F104 0.00

F87 0.56 MeOH : EA : H20 : Phosphoric Acid
F87 0.85

F89 0.62

F89 0.85

F93 0.92

FlOO 0.87

F73 0.00 MeOH: EA : H20: Phosphoric Acid
F76 0.77

1.00

F80 0.79

1.00

F104 0.00

14

Example 3:
A qualitative TLC-DPPH test - Fractions of Ca35
TLC plates were spotted with various fractions (representative from different solvents fractions) and air-dried. After complete drying the plates were dipped in 0.035% DPPH solution for about 5 seconds and quickly removed. The plate images were captured by scanning the plates. The fi-actions with antiradical activity appear as yellow/pale or bleached spots against the purple-violet background. This a qualitative test serves as a screening for fractions (using very minute amount of sample) with anti-oxidant potency. The TLC -DPPH images for the representative fractions are given below
Example 4:
Liquid chromatography and Mass spectrometry oi Deschampsia antarctica Ca35, Ca36
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 oi Deschampsia antarctica leaves were ground in Uquid Nitrogen and extracted with I ml
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 centrifiiged for 15 min at 14000 rpm and 4°C to remove any
suspended particles.
Sample 2: Extract of Deschampsia antarctica Ca35 was extracted with 1ml of methanol: chloroform:
water (6:2:2). The extracts were vortexed for 5 min and was kept at A°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 Deschampsia antarctica Ca36 was 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
All the 3 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 (SIL20AC) attached to HPLC (Shimadzu, Prominence). The temperature of the autosampler was maintained at 8°C throughout the experiment. The samples were eluted from UFLC
15

by a binary gradient through a 5 ^ 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 0.4mymin 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 (AppUed 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"' Torr, curtain gas 20, Collision Energy (CE) 5, GSl 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"' Torr, curtain gas 20, Collision Energy (CE) 5, GSl 40, GS2 60, and declustering potential of 35. The scan rate was set at ICXX) 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 0.4 mL min'' 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 4000.00 GSl 40, GS2 60 with interface heater and the dynamic fill time on. For negative polarity mode the curtain gas was set to 20, CoUision Energy -30, -40, CES 10, ion spray voltage was set at -2750.00, temp 275.00, GSl 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.
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Example 5:
Estimation of PolyphenoUcs 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 FoUn 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 P2Wi8062'' to H4P2Wi8062'* and the 2-, 4-, and 6-electron reduction
products in the molybdate series H2P2M018O62"* to H6P2M018O62'*.
Procedure:
Polyphenol assay is carried out by Using singleton, V., Rossi, J.A. Jr, method (1965). To a 200|jl of 50% Methanol / Standard / test sample with various concentrations, add 1000|al of FC reagent, mixed and incubate at Room Temperature for 5min. Add 800|xl 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 PhenoUcs of Deschampsia antarctica

Polyphenols as % Gallic acid equivalent GAE (g/lOOg)
Deschampsia antarctica extracts Total Phenolic Simple Phenolic Tannin 1.07
Ca35 5.57 4.50

Ca36 4.56 4.03 0.53
Example 6: 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.
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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 centrifiiging at 5000 rpm for 10 - ISminutes. The clear supernatant is taken for the assay.
Reaction
Various concentration of fresh Deschampsia antarctica extract or standard ascorbic acid taken in 215 ^,1
of methanol were mixed with 36 ^1 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

DPPH assay
Samples Ascorbic acid standard EC50 ng/ml

5.6
Deschampsia antarctica wild type (fresh wt) 1700
D. antarctica extract Ca35 259
D. antarctica extract Ca36 220
Example 7:
ORAC assay: The antioxidant capabilities of Deschampsia antarctica are evaluated using ORAC is
exhibiting a value of 274.05 Tp n mol/g Sample. (Table 3)
Table 3: ORAC assay of Deschampsia antaratica

ORAC assay
Samples TE |imol/g
D. antarctica extract Ca35 274.05
Example 8:
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 ng/ml. (Figure 74 and75)

We claim:
1. 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 and mass spectrometry peaks.
2. A method of preparing a deschampsia 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 ethanohc 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.
3. The deschampsia species extract of claim 2, wherein the fraction comprises a compound selected from the group consisting of a polyphenol, a polysaccharide, amino acid, terpenoids, alkaloids and combinations thereof
4. A deschampsia species extract comprising a fraction having a mass spectrometry chromatogram of any of Figures 1 to 73.
5. The deschampsia species extract of claim 3, wherein the fraction comprises of 5 - hydroxyferulic acid from Deschampsia antarctica.
6. The Deschampsia species extract of claim 3, wherein the fraction comprises of 7, 5-dimethoxyflavone.
7. The Deschampsia species extract of claim 3, wherein the fraction comprises of 2 -o-arabinopyranosylorientin from Deschampsia antarctica.
8. The Deschampsia species extract of claim 3, wherein the fraction comprises of Caffeic acid.
9. The Deschampsia species extract of claim 3, wherein the fraction comprises of Cinnamaldehyde.
10. The Deschampsia species extract of claim 3, wherein the fraction comprises of Cyanidin.
11. The Deschampsia species extract of claim 3, wherein the fraction comprises of Cyanidin -3-rutinoside.
12. The Deschampsia species extract of claim 3, wherein the fraction comprises of Cyanidin 3, 6 dimalonylglucoside.
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13. The Deschampsia species extract of claim 3, wherein the fraction comprises of Diacetylgalactopyranosylorientin.
14. The Deschampsia species extract of claim 3, wherein the fraction comprises of Dihydroxykaemferol.
15. The Deschampsia species extract of claim 3, wherein the fraction comprises of Dihydroxyacetonephosphate.
16. The Deschampsia species extract of claim 3, wherein the fraction comprises of Dihyrdoxiindole -2-carboxyUc acid.
17. The Deschampsia species extract of claim 3, wherein the fraction comprises of Dihydroxyphenylarabinosyl-dihydroxybenzopyran.
18. The Deschampsia species extract of claim 3, wherein the fraction comprises of L-DOPA.
19. The Deschampsia species extract of claim 3, wherein the fraction comprises of Ferulic acid.
20. The Deschampsia species extract of claim 3, wherein the fraction comprises of Glucopyranosyltrihydroxymethylflavone.
21. The Deschampsia species extract of claim 3, wherein the fraction comprises of Hespertin.
22. The Deschampsia species extract of claim 3, wherein the fraction comprises of Homovaratic acid.
23. The Deschampsia species extract of claim 3, wherein the fraction comprises of 4 (l-hydrxy-2-methylaminoethyl 2-methoxyphenol.
24. The Deschampsia species extract of claim 3, wherein the fraction comprises of Immidazolone -5-propionic acid.
25. The Deschampsia species extract of claim 3, wherein the fraction comprises of Isowerticin 2 -arabinose.
26. The Deschampsia species extract of claim 3, wherein the fraction comprises of Isowertiajaponica.
27. The Deschampsia species extract of claim 3, wherein the fraction comprises of Kaempferol 3- o -rhamnosyl glucoside.
28. The Deschampsia species extract of claim 3, wherein the fraction comprises of Kaempferol.
29. The Deschampsia species exfract of claim 3, wherein the fraction comprises of malonylgalactosylpyranosylorientin.
30. The Deschampsia species extract of claim 3, wherein the fraction comprises of o/m/p hydroxybenzoic acid.
31. The Deschampsia species extract of claim 3, wherein the fraction comprises of Octadecanoic acid.
32. The Deschampsia species extract of claim 3, wherein the fraction comprises of Orientin.
33. The Deschampsia species extract of claim 3, wherein the fraction comprises of Orotic acid.
34. The Deschampsia species extract of claim 3, wherein the fraction comprises of 3,7,3,4,5pentahydroxy 5- methoxyflavihum.
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35. The Deschampsia species extract of claim 3, wherein the fraction comprises of pentahydroxy flavilium malonylglucose.
36. The Deschampsia species extract of claim 3, wherein the fraction comprises of pyranxylvitexin.
37. The Deschampsia species extract of claim 3, wherein the fraction comprises of Sinapinic acid.
38. Food or medicament comprising the Deschampsia species extract of claim 2 for the therapeutic use
for the mitigation of skin disorder, cancer and other related disorder.