FIELD OF THE INVENTION

[0001] The present invention relates to a nutraceutical or pharmaceutical composition obtained from 80 unmilled, colored rice varieties, a process for identification and characterisation of said composition, processes for preparing rice seed extract, extraction of protein from said rice varieties and uses of obtained composition or products in making formulations or preparations for nutraceuticals including foods, or pharmaceuticals including fiber, digestive, dietary supplements for particular nutritional and/or medical purposes, and the like.

BACKGROUND OF THE INVENTION

[0002] Consumption of food products containing high amount of polyphenols, flavonoids, lignans, and edible useful tannins have been reported to lower the risk of various disorders such as obesity/hypercholesterolemia, diabetes, cardiovascular disease, cancers and metabolic syndromes (Jones, 2006; Jones et al., 2002). These components are widely distributed in frequently used food grains like wheat, rice, cereals, fruits, vegetables, and beverages like wine, tea, and cocoa etc. (Jones, 2006; Jones et al., 2002).

[0003] Rice is a versatile grain that is a major part of several ethnic cuisines. Rice is one of the important food grains, which is having extraordinary benefits to act as an important diet and commercial food product because of a) it is consumed as a whole grain, making it a very good source of dietary fibers, vitamins, minerals and phytochemical (health-promoting components), b) it constitutes more than 80% of daily diet of approximately 2/3 of world's population.

[0003] Rice comes in different sizes, forms and colors. Rice can be categorized on the basis whether or not the grains have been subjected to milling. Milling is a process where the outer layers of the grains are removed, exposing the white kernel inside. Unmilled rice has only the husk removed. The husks are removed but bran layer and germs are still intact. The unmilled rice is more nutritious as it also contains lot of minerals, proteins, vitamins and fibers. Unmilled rice may also be called as colored rice. There are number of colored varieties of rice available throughout the world, which are rich in specific health-promoting components such as flavanoids, lignans, polyphenols, micronutrients, macronutrients, proteins, vitamins etc.

[0004] Colored rice is reported to have various, polyphenols, flavonoids and pigments, which are good natural antioxidants (Matilla et al., 2005; Hu et al., 2003; Roher and Siebenmorgen, 2004). Several medicinal properties of rice based on old ayurvedic literature have been nicely reviewed, recently (Ahuja et al., 2008). Rice bran, which is composed of pericarp, seedcoat, nucellus, aleurone layer and germ is considered as the main source of these metabolites (Roher and Siebenmorgen, 2004). The bran contains several fat-soluble (Khatoon and Gopalakrishna, 2004; Gopalakrishna, 2002; Gunstone et al., 1986) and water-soluble (Hu et al., 2003; Chiang et al., 2006; Zhang et al., 2005) pigments, vitamins, proteins, fiber, minerals, oils and other nutraceuticals (Orthoefer and Eastman, 2004; Yokoyama, 2004). Rice has been reported to inhibit chloride channels, thus very useful to decrease intestinal water loss during diarrhea (Goldberg and Sultzman, 1996). Rice-based oral rehydration solutions (ORS) have also been preferred
over glucose-based ORS by world health organization (WHO) (Gore et al., 1992).

[0005] Polyphenols such as phenolic acids, flavonoids serve as derivatives of conjugated ring structures and hydroxyl groups that have the potential to function as antioxidants by scavenging superoxide anion, singlet oxygen, lipid peroxy-radicals, and/or stabilizing free radicals involved in oxidative processes through hydrogenation or complexing with oxidizing species (Ren et al., 2003). In rice, bran color is reported to have highly statistical significance to the total contents of phenolics in it (Goffman and Bergman, 2004; Shen et al., 2009). Increased consumption of phenolic compounds has been associated with the reduced risk of chronic, degenerative diseases and certain types of cancers due to its potent antioxidant nature (Liu, 2004, 2007; Dykes and Rooney, 2007). The flavonoids are chemically one-electron donors, which are acting as reductants of these highly oxidizing/damaging radicals into harmless or less harmful products (Ren et al., 2003; Kandaswami et al., 1994

[0006] Rice protein obtained from unmilled, colored rice is a protein supplement which cannot be a cause of digestive problems unlike whey powder made from dairy and soy. Most people digest rice protein more easily and it is also a gluten-free diet for diet conscious people. Rice protein is less expensive than whey protein powder and the overall amount of calories present in rice-based powders is less than other types of protein powders, making it a better option for people trying to lose weight and being more desirable in daily diet and medicinal formulations.

[0007] More than 4000 flavonoids, other polyphenols, and other useful components are known to present in plants, the list is constantly expanding. These constitute an important part of the human diet and are considered as active principals of many medicines. However, some phytochemicals, including phenolics and flavonoids, have not received as much attention as other compositions in rice especially in colored rice grains.

[0008] Many open literature and patent documents in the prior art discuss about phytochemicals, including phenolics and flavonoids, vitamins, proteins and their anti-oxidative capacities in other plants and much attention has not received to rice especially in colored rice grains in case of said components present in plants.

[0009] Patent No. US7972633 discloses, compositions for healthy memory and optimizing mental energy and methods for improving, preventing, and treating mental disorders or deterioration and such compositions can be formulated as nutritional or dietary supplement.

[0010] Patent Application No. US2007104855 discloses milling products and whole grain wheat flour and products including the same. Patent Application No. US2011 0136245 discloses synergistic interactions of phenolic compounds found in food.

[0011] Given that a number of popular consumer products made with milled rice are low in polyphenols, protein, fiber, micronutrients, macronutrients, vitamins etc. and do not have the nutritional and pharmaceuticals benefits of their respective whole grain counterparts, it would be desirable to develop an unmilled, colored rice composition, and processes to produce high protein, high anti-oxidative capacities, high fiber, high in vitamin content, highly nutritious and low calorie products that can be used in nutraceutical as well as in pharmaceutical area.

[0012] Although few patent documents reveals nutritional and pharmaceutical properties of unmilled, colored rice compositions and related processes, today's consumer continue to demand products with additional or improved properties, making it highly desirable to develop such improved compositions or products.

OBJECT OF THE INVENTION

[0013] The principle object of the present invention is to generate of a comprehensive metabolic and nutritional profile of 80 unmilled, colored rice varieties, selection of the most useful varieties on the basis of their specific medicinally useful metabolites, their anti-oxidative capacities, polyphenol, protein and vitamin contents.

[0014] Another object of the present invention is to provide isolation and comprehensive listing of all physiologically important metabolites in said rice varieties by using mass spectrometry in combination to liquid chromatography (LC-MS) and the process for identification and characterization of said metabolites by using the tandem LC/MS/MS fragmentation approach using soft ionization method /. e. electrospray ionization.

[0015] Yet another object of the present invention is to provide an easy and efficient process for extraction of protein and an efficient process for preparing rice seed extract obtained from 80 unmilled, colored rice varieties.

SUMMARY OF THE INVENTION

[0016] According to one aspect of the invention, the present invention describes a nutraceutical or pharmaceutical composition, in combination of 80 unmilled, colored rice varieties. The composition comprises various metabolites, and such said metabolites comprise phenolic acids, vitamins, flavanols, flavonols, flavanones, flavanoids, and terpenes derivatives, complete metabolic profiling and study of anti-oxidative capacities of 80 unmilled, colored rice varieties by performing various biotechnology techniques that includes mass spectrometry coupled to liquid chromatography (LC-MS). The anti-oxidative properties are assigned to the presence of polyphenols such as phenolic acids, flavonoids, pigments and vitamins in the bran/grain of these colored rice varieties. The antioxidative capacities were measured performing standard free radical scavenging assays such as l,l-diphenyl-2-picrylhydrzyl (DPPH) and total oxygen radical absorbance capacity (ORAC) assays followed by the measurements of total polyphenols contents in all these rice varieties. The metabolites also have a high amount of micronutrients and macronutrients and a high amount of rice protein. The composition is having therapeutic properties for the mitigation of skin disorders, cancer and other related diseases including cosmetic properties.

[0017] According to another aspect of the invention, the present invention describes a process for identification and characterization of a nutraceutical or pharmaceutical composition, wherein the isolation, identification and characterization of metabolites using soft ionization method such as electrospray-ionization and fragmentation approach (LC/MS/MS) of all the parent ions revealed the presence of various important metabolites. On the basis of known specific anti-cancer, anti-inflammatory, anti-diabetic and lipogenic properties of observed compounds, seven best varieties are identified which contains high nutritional value and health-protective compounds.

[0018] According to yet another aspect of the invention, the present invention describes a process for preparing rice seed extract obtained from 80 unmilled, colored rice varieties. The rice seed extract possess medicinal properties for treatment of cardiovascular, diabetes diseases and other diseases.

[0019] According to one aspect of the invention, the present invention describes a process for extraction of protein from 80 unmilled, colored rice varieties.

[0020] The details of one or more embodiments of the present invention will emerge from the following description.

DETAILED DESCRIPTION OF THE INVENTION

[0021] An embodiment of the present invention will be explained with reference to given examples.

[0022] In one embodiment, the present invention describes a nutraceutical or pharmaceutical composition, in combination of 80 unmilled, colored rice varieties comprising of metabolites. The metabolites comprising of phenolic acids, vitamins, fiavanols, flavonols, fiavanones, flavanoids, and terpenes derivatives possessing high anti-oxidative capacities, a high amount of micronutrients, macronutrients and a high rice protein. The composition is a component of a food product, a nutritive supplement, a fiber supplement, a digestive supplement, dietary supplement, a functional food, and a medical supplement.

[0023] The composition is a component of rice based eatables individually and in combination with other food stuff and the fiavanols, flavonols, terpenoids, vitamins are identified in said 80 unmilled, colored rice varieties on the basis of anti-cancer, anti-inflammatory, anti-diabetic, and lipogenic properties of observed compounds. The composition is having therapeutic properties for the mitigation of skin disorders, cancer and other related diseases including cosmetic properties. The composition is formulated with specific combinations of compositions approximately 10 to 80% of 6 unmilled, colored rice varieties and approximaely 20% of other colored rice varieties. The nutraceutical or pharmaceutical composition can be formulated in dosage forms like tablet, troches, lozenges, aqueous or oily suspensions, dispersible powders or granules, emulsions in hard or soft gel capsules, syrups, elixirs, phytoceuticals, nutraceuicals and foodstuff.

[0024] In second embodiment of the present invention, 80 unmilled, colored rice varieties have been extensively studied and examined for their complete metabolic profiling and net antioxidant capacity. Here, a process for identification and characterization of a nutraceutical or pharmaceutical position comprising different metabolites. Isolation and listing of all the physiologically important metabolites in said rice varieties have been done by performing numerous biotechnology techniques which includes mass spectrometry in combination to liquid chromatography (LC-MS). Further, identification, characterization of various components has been done using tandem LC/MS/MS fragmentation approach using soft ionization method i.e. electrospray ionization. The anti-oxidative capacity of these rice samples has been measured using standard free radical scavenging assays such as l,l-diphenyl-2-picrylhydrzyl (DPPH) and total oxygen radical absorbance capacity (ORAC) assay. Total protein, carbohydrates, fats, moisture content, ash content, energy content has been studied and examined in the selected seven varieties. From the observations of antioxidant assays and metabolic profile six varieties were selected for detailed analysis, multiplication and cultivation.

[0025] Eighty unmilled, colored rice varieties have been procured from the rice germplasm bank of International Rice Research Institute, Manila (IRRI) (Table 1). These were stored at room temperature in dark. Example 1:

[0026] Extraction of metabolites from unmilled, colored rice varieties (procedure): The rice seeds were ground to a fine homogenous powder, using a pestle and mortar. The extraction of metabolites from rice seeds has been done using 100 mg of rice powder in 1 ml of 80% ethanol by 3 cycles of together, 5 min vortex followed by 15 min of sonication. The extract was then centrifuged at 13000 rpm for 15 minutes, using a free angle rotor centrifuge (5180 R, Eppendorf, Germany) and the clear supernatant was collected (Table 1).

Example 2:

[0027] DPPH assay: The free radical scavenging activity of rice seed extracts were determined by the DPPH assay, as described (Brand-Williams et al., 1995). Briefly 36 ^1 of DPPH (1 mM) (Sigma-Aldrich) prepared in 100% methanol and various concentrations of rice seed extracts (80% ethanol) were mixed and incubated for 30 min at 37° C. After the incubation, the absorbance of reduced DPPH was measured at 517 nm using microplate reader (Synergy HT, Biotek). The standard curve was obtained by using various concentrations of ascorbic acid (1 to 10 ug/ml). The activity has been expressed as the EC50, the amount of various rice varieties (mg) required for scavenging 50% of DPPH radical in the solution (Table 1)

Example 3:

[0028] Polyphenol assay. Total polyphenol contents were determined using Folin-Ciocalteu (FC) assay, following the method of (Singleton and Rossi 1965). Said rice seed extracts 200 jul) were mixed with FC reagent (1000 p.1) and incubated at room temperature for 5 min. Subsequently, 800 ^1 solution of sodium carbonate (7.5% w/v) was added to it and incubated further for 30 min at room temperature. After the reaction period, the absorbance of the reaction mixture was measured at 750 nm using UV-1700 UV-Visible spectrophotometer (Shimadzu). Various concentrations (1 to lODg/ml) of the gallic acid were also subjected to the same reaction and a standard curve was derived. The concentrations of total polyphenols in said rice varieties were expressed as % Gallic acid equivalent (%GAE) (Table 1). Example 4:

[0029] ORAC assay: The antioxidant capacity of said rice seed extracts has been measured using Oxygen Radical Absorbance Capacity hydrophilic test (ORAC) (Cao et al., 1993). The stock solutions of fluorescein (4.2 mM) (Sigma) and 2,2'-azobis (2-methylpropionamidine) dihydrochloride (AAPH) (240 mM) (Calbiochem) was freshly prepared in potassium phosphate buffer (75 mM, pH 7.0) and stored on ice bath in dark, until use. The reaction mixture containing 150 ul diluted fluorescein (10 nm) in potassium phosphate buffer (75 mM, pH 7.0) and 25 ul sample/standard/phosphate buffer was incubated at 37 °C for 30 min in micro-titer plate. The measurements conditions were Ex. 485 nm, Em. 520 nm. After incubation, 25 ul of AAPH stock solution was added in the reaction mixture. The fluorescence intensity was measured upto 90 min using a multi-mode microplate reader (Synergy HT, Biotek, US). Trolox (6-Hydroxy-2,5,7,8-tetra-methylchroman-2-carboxylic acid) (Cayman) has been used as a standard to prepare calibration curve (6.25 to 50 uM). Results are expressed as micromoles of trolox equivalents (TE) per gram or per milliliter of sample (umol of TE/g or umol of TE/ml) (Tablel).

Example 5:

[0030] Protein estimation: The protein estimation has been done as described (AOAC 2001.11 method) by using Kjeldahl apparatus, based upon the determination of the total nitrogen. (Tablel)

[0031] In same embodiment of the present invention, the comparative analysis of the antioxidant capacity of 80, unmilled, colored rice varieties has been studied. The bran of colored rice is rich in polyphenols such as phenolic acids, flavonoids, vitamins and pigments contents, which possesses high antioxidant properties. Thus, the ORAC values of all these 80 colored rice varieties together with RASI (uncolored, white rice variety) were determined using hydrophilic ORAC assay. The highest ORAC value observed was 99.7 in a red color rice variety (IRRI ACC.NO.64661-R07-MET) (Table 1). It was observed that all the dark colored extracts (red and black) were having good ORAC values. On contrary, ORAC value of seed extract of the colorless rice variety RASI was 11.2. However, one brown rice variety namely, (IRRI acc.No.56744-R07-mef) was observed with lesser ORAC value (4.49) than colorless variety. Thus, On the basis of ORAC values, 6 best varieties (IRRI acc.No.27665-R07-met), IRRI acc.No.27726-R07-met, (IRRI acc.No.50783-R07-met), (IRRI acc.No.51618-R07-met) together with one premium variety (IRRI acc.No.57861-R07-met) having ORAC values 88.07, 86.99, 80.66, 80.13 and 74.58, respectively, have been selected for detail investigations.

[0032] The estimation of the total polyphenol contents of all the 80 unmilled, colored rice seed extracts has been done using standard FC method (Table 1). It has been observed that the average polyphenol content of seed extract from colored rice varieties with the mean value of 0.15 % GAE (Table 1). The maximum value was 0.37 % GAE in the rice variety (IRRI acc.No.27665-R07-met) and 0.06 % GAE in three rice varieties. However, the total content of polyphenol in colorless variety RASI was 0.01 % GAE, which was the minimum among all the rice seed extracts. Six best varieties, selected by their ORAC values were also shown to have the polyphenol content in maximum range i.e. 0.37, 0.31, 0.31, 0.29, 0.23 and 0.18. IRRI acc.No.51618-R07-met, IRRI ACC.NO.64661-R07-MET, IRRI acc.No.57861- R07-met and IRRI acc.No.50783-R07-met, respectively. Although some other varieties were also observed to have higher polyphenol contents yet it was not considered among the best varieties because of their lower antioxidant capacities determined on the basis of respective ORAC values.

[0033] The DPPH radical scavenging activity of six selected rice varieties also shows high free radical scavenging functions. Among these rice varieties, red colored variety (IRRI acc.No.51618-R07-met) has shown the highest scavenging activity of DPPH radical with an EC50 value of 0.23 mg ml"1. However, the colorless variety, RASI has shown 15.8 EC50 value. The other selected black and red varieties like (IRRI acc.No.57861-R07-met), (IRRI acc.No.27665-R07-met), (IRRI acc.No.27726-R07-met),(IRRI ace No.64661-R07-MET), and (IRRI acc.No.50783-R07-met) has the EC50 values 5.93, 1.96, 1.96,2.23 and 3.34, respectively.

[0034] The total protein content of these rice varieties using AOAC method (2001.11) shows that the maximum protein content was 16.3 g per 100 g of rice sample of variety, 14090700016. All the six selected varieties have shown higher protein contents among all the tested varieties i.e. 14.7, 13.9, 13.6, 13.1, 12, 10.7 and 10.3 in IRRI ace. No.64661-R07-MET, IRRI acc.No.57861-R07-met, IRRI acc.No.27726-R07-met, IRRI acc.No.27665-R07-met, IRRI acc.No.50783-R07-met, IRRI acc.No.51618-R07-met and RASI (Tablel).

Table 1: The Name, IRRI accession numbers, their repective ORAC values, total polyphenol and protein content of the all the 80 varieties and 'Rasi'. The total polyphenol content, The ORAC values and the total protein were estimated as described in 'materials and methods' section.

Example 6:

[0035] Liquid chromatography and Mass spectrometry

Measurement parameters and analysis details (HPLC): All the samples were filtered through a 0.1 to 0.5-\i- 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 CShimadzu. Prominence.

The temperature of the autosampler was maintained at 6°C to 10°C throughout the experiment. The samples were eluted from UFLC by a binary gradient through a5 fi 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 0.2 to 0.5 ml /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).

[0036] Measurement parameters and analysis details (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, GS1 40, GS2 60, and declustering potential of 35 for acquisition of TIC of EMS in negative ionization whereas, 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, GS1 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.

[0037] The acquisition of Enhanced Product Ion (EPJ) by LC-MS/MS: The enhanced production 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 4000.00 GS1 40, GS2 60 with interface heater and the dynamic fill time on. For negative polarity mode the curtain gas was set to 20, Collision Energy -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.

Data processing

[0038] For the processing, the total ion chromatogram (TIC) of blank (solvent) and test sample were Gaussian smoothened, 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 were further extracted to obtain Extracted ion chromatogram (XIC) or amplified. The metabolites were primarily identified by comparing their molecular mass with HMDB, KEGG, NIST database and were confirmed finally based on their individual fragmentation pattern.

[0039] Analysis and identification of major masses using LC-MS: The diversity in the structures and physical/chemical properties of various metabolites requires several runs with numerous instrumental settings and approaches. However, in our experiments, these metabolites were resolved using a combination of HPLC reverse-phase RP-18 column with a large gradient of solvent and ESI-MS. These methods were optimized on LC-MS prior to the final run as these samples have an array of metabolites i.e. vitamins, flavons, flavonols, flavanols, phenolics etc. The LC-ESI-MS provided extremely sensitive identification and detection of structural analysis of various metabolites in different colored and colorless rice varieties. Approximately, 2000 m/z values were obtained together in negative and positive ionization modes of ESI-MS/MS. However, all the values above 1000 m/z have not been considered to avoid interference of larger polypeptides and minimize the data set. In addition to the excellent sensitivity and selectivity of HPLC-MS, these measurements provided structural information of compounds using fragmentation approach (EPI). Fortunately, many metabolites are fragmented by this method in ways that provided a great deal of information about their structures, hence assisted in confirming the compound identification. On the basis of significance of the metabolites for health, the identified metabolites have been classified into three categories i.e. vitamins, phenolic acids, and flavonoids.

Vitamins:

[0040] Rice bran is rich in bioactive compounds including vitamins like thiamine (Bl), riboflavin (B2), niacin (B5), pyridoxal/pyridoxine (B6), folic acid (B9), tocopherol (E) and retinal (A) etc. It has been found that tocopherol and its isomers (Vit. E) were invariably present in all the selected seven varieties namely (IRRI acc.No.27665-R07-met), (IRRI acc.No.27726-R07-met), (IRRI acc.No.51618-R07-met), (IRRI ace. No.64661-R07-MET), (IRRI acc.No.57861-R07-met), (IRRI acc.No.50783-R07-met) and RASI, respectively (table 2). However, vitamin B complexes are present in selective varieties. Thiamin (Vit. Bl) was observed in (IRRI acc.No.27726-R07-met)and RASI, however, presence of riboflavin was observed in all the rice varieties including RASI except one red variety (IRRI ace. No.64661-R07-MET). Vitamin B6 was present in the form of either pyridoxine or pyridoxal in all the red/brown varieties i.e. (IRRI acc.No.50783-R07-met), (IRRI acc.No.51618-R07-met) and (IRRI acc.No.64661-R07-MET), and also in one black variety (IRRI acc.No.27726-R07-met). Either of the forms of vitamin B6 was not found in other black varieties and RASI. Vitamin B5 was present in a single red variety (IRRI acc.No.51618-R07-met), as a high intensity peaks. The presence of folic acid (B9) was reported in all the red and black varieties, however, it was absent in colorless variety RASI. Presence of vitamin 'A' could not be observed in any of the rice varieties. It can not be detected in LCMS studies due to the unstable ionizing states of natural pro-vitamin 'A' i.e. carotenoids, xanthophylls etc. (Table 2)

Phenolic acids:

[0041] Presence of several phenolic compounds is reported in food grains and vegetables.
Phenolic compounds are known to have high antioxidant characteristic/properties. Phenolic compounds are broadly separated into two group flavonoids, which are polyhydroxyl polyphenolic compounds and non-flavonoids, which are carboxylic acid derivatives of phenols also called as Phenolic acids. In rice two major types of Phenolic acids are reported i.e. hydroxybenzoic acid and hydroxycinnamic acids. In rice bran, presence of gallic acid, vanillic acid, syringic acid and procatechuic acid types of hydroxybenzoic acids were already reported earlier. However, the hydroxycinnamic acids found in the rice includes ferulic acid, caffeic acid, /?-coumaric acid and sinapic acid etc. In the current study, procatechuic hydroxybenzoic acid is found in all the seven varieties including colorless variety RASI. Similarly, ferulic and caffeic hydroxycinnamic acids was also found in all the selected seven varieties (table 3) It has been observed that the relative peak intensities of the masses of Phenolic acids were higher in RASI than other colored rice varieties. (Table 2)

Flavonoids:

[0042] Flavonoids are ketone or non-ketone containing polyhydroxy polyphenolic pigmented compounds. These possess high antioxidant properties. Various classes of flavonoids have been reported in colored rice varieties i.e. flavanols, flavonols, flavanones, flavonoids and terpene derivatives. Flavanols namely catechins, epicatechins were present in the brown/red varieties IRRI acc.No.51618-R07-met and IRRI ace. No.64661-R07-MET but absent in all other black and red varieties including RASI. The flavonols quercetin and its derivative were found in all the black varieties i.e. IRRI acc.No.27665-R07-met), IRRI acc.No.27726-R07-met and IRRI acc.No.57861-R07-met; it was present in trace amounts in two red varieties i.e. IRRI acc.No.51618-R07-met and IRRI ace. No.64661-R07-MET but was absent in other varieties and RASI. The methylated derivatives of quercetin such as 7 methylquercetin (Rhamnetin), 3-methylquercetin (Isorhamnetin) and 3-methyl-3-glucopyranoside quercetin (Isorhamnetin glucosylpyranoside) were also reported in all the above mention three black varieties. The other derivatives of quercetin like dihydroquercetine, dihydroquercetine-glucopyranoside, quercetine methyl ether glucopyranoside have been found exclusively in the black rice varieties (IRRI acc.No.27665-R07-met), (IRRI acc.No.27726-R07-met) and (IRRI acc.No.57861-R07-met). These metabolites were absent in red varieties and RASI. Another important metabolite of these black rice varieties is a bioflavonel luteoline and its derivatives like 8-Hydroxy luteolin methyl ether, and 8-C glucoside of luteoline which is called as orientin. These were also absent in RASI and all other red varieties. (Table 2)

Table 2: List of all the important metabolites of selected 6 varieties of rice and RASI (control)

Example 7:

[0043] Elemental analysis: Iron, Copper, Zinc, Manganese, Cadmium, Lead, Arsenic, Mercury content were analyzed by using Atomic Absorption spectrometry, Selenium, Phosphorous by colorimetry, Sodium, Potassium by flame photometery Magnesium , calcium and aluminum by chemical methods and the content are as given in the (Table -3) below:

Table 3: Mineral and metal Amount in six rice varieties and control RASI

Example 8:

[0044] Total fat estimation: The total fat has been determined using AOAC 2003.05 method (J. AOAC Int., 2003), which is based upon the extraction of crude fat from the total grain by boiling with the solvent (Table 4).

Example: 9

Moisture determination:

[0045] The moisture content has been determined (AOAC: 925.10 method) by heating a pre weighed sample (100 g) in an oven at 105 °C for 5 hours till it attained constant weight. The total weight loss after five hours of heating represents the moisture content of the sample (Table 4).

Example 10:

The ash content:

[0046] The ash content is a measure of the total amount of minerals present within a food. The most widely used methods for the determination of ash content are based on the fact that minerals are not destroyed by heating, and that they have a low volatility compared to other food components. The ash content has been determined following the method (AOAC: 923.03) (Table 4).

Example 11:

Total carbohydrate:

[0047] The total carbohydrate content was determined using simple mathematical calculation based upon the fact that any biological material consists of carbohydrate, fat, protein, minerals and moisture. Thus, the remaining weight of each 100 g grain after subtracting the total fat, protein, ash and moisture content, represents total carbohydrate (Table 4).

Example 12:

[0048] The energy determination: The amount of calories obtained from each 100 g of various rice grains has been calculated using the formula (Table 4).

Energy (Kcal) = Carbohydrates (g/lOOg) * 4 + Protein (g/lOOg) * 4 + Fat (g/lOOg) * 9
Table 4: Total fat, carbohydrate, moisture, ash and the energy content of selected rice varieties.

[0049] In third embodiment of the present invention, the process for preparing rice seed extract obtained from 80 unmilled, colored rice varieties is described. For this process, seeds from 80 rice varieties which are unmilled, colored are obtained, and weighed. Seed from each said 80 unmilled, colored rice varieties is weighed as 100 gm in 1 litre (1:10 wt/vol). Seeds from each variety are ground forming a homogenous seed powder.Said rice seed powder is put for an extraction with a polar to non-polar solvents for distillation wherein the ratio of solvent is 100 gm in 1 litre (1:10 wt/vol).

[0050] The individual natural compounds from said rice seed extract are separated at each step of extraction based on the polarity of said polar or non-polar solvents by means of solvent partitioning, followed by column flash chromatography, preparatory HPLC, TLC , finally followed by HPLC NMR LC-MS for structural elucidation. The temperature required for the process is in between ambient to 100°C. This rice seed extract ccomprises natural compounds like a polyphenol, a polysaccharide, amino acids, terpenoids, alkaloids which have medicinal properties for treatment of cardiovascular, diabetes diseases and other like diseases.

[0051] In fourth embodiment of the present invention, the process for extraction of protein from 80 unmilled, colored rice varieties is described. Extraction of protein from 80 unmilled, colored rice varieties is done by alkaline extraction method.

Example 13: The % protein yield by alkaline extraction method.

[0052] All 80 unmilled, colored rice samples were de-husked and weighed, approx 500 mg of each rice variety. Added 25ml of 0.2% NaOH solution to the rice flour and mixed well. The mixture was stirred (Rocker) at room temperature for overnight. The mixture was then centrifuged at 3000xg for 10 minutes and the supernatant was collected. The precipitate was extracted again with 0.2% NaOH and the second supernatant were combined with the first one. The pH of combined extracts was adjusted to 6.0 with IN HC1 and the rice crude protein was precipitated. The precipitate was collected by centrifugation at 3000xg for 10 minutes and washed three times with distilled water. Finally the precipitate was completely dried.

[0053] This rice protein can be used in different food products and medicinal supplements. This rice protein can be a substitute for milk protein due to its easily digestible property and also in other medicinal formulations.

[0054] Accordingly, while only a few embodiments of the present invention have been shown and described, it is obvious that many modifications and variations may be made to the present invention without departing from the scope of the present invention. Thus, it is intended that the present invention covers the modifications and variations of this invention provided they come within the scope of the appended claims and their equivalents thereof.

REFERENCES

Ahuja, U., Ahuja, S. C, Thakrar, R., & Singh, R. K. (2008). Rice - A nutraceutical. Asian Agriculture History, 12, 93-108. Brand-Williams, W., Cuvelier, M. E., & Berset, C. (1995). Use of a free radical method to evaluate antioxidant activity. Lebensmittel-Wissenschaft und-Technologie/Food Science and Technology, 28,
25-30. Cao, G., Alessio, H. M., & Cutler, R. G. (1993). Oxygen-radical absorbance capacity assay for antioxidants. Free Radical Biology & Medicine, 14, 303-311. Chiang, A. N., Wu, H. L., Yeh H. I., Chu, C. S., Lin, H. C, & Lee, W. C. (2006). Antioxidant effects of black rice extract through the induction of superoxide dismutase and catalase activity. Lipids, 41, 797-803. Dykes, L., & Rooney, L. W. (2007). Phenolic compounds in cereal grain and their health benefits. Cereal Foods World, 52, 105-111. Goffman, F. D., & Bergman, C. J. (2004). Rice kernel phenolic content and its relationship with
antiradical efficiency. Journal of the Science of Food and Agriculture, 84,1235-1240. Goldberg, E. D., & Saltzman J. R. C. (1996). Rice inhibits intestinal secretions. Nutrition Reviews, 54, 36-37. Gopala Krishna, A. G. (2002). Nutritional components of rice bran oil in relation to processing. Lipid Technology, 14, 80-84. Gore, S. M., Fontaine, O., & Pierce, N. F. (1992). Impact of rice-based ORS on stool output and duration
of diarrhea: Meta-analysis of 13 clinical trials. British MedicalJournal, 304, 287-291. Gunstone, F.D., & Harwood, J.L. (2007). Occurrence and characterization of oils and fats. In F. D.

Gunstone, J. L. Harwood & A. J. Dijkstra, (Eds), The lipid handbook, (p. 37-141), Chapman and Hall, London. Hu, C, Zawistowski, J., Ling, W., & Kitts, D. D. (2003). Black rice (Oryza sativa L. indica) pigmented fraction suppresses both reactive oxygen species and nitric oxide in chemical and biological model systems. Journal of Agriculture and Food Chemistry, 51, 5271-5277. Jones, J. M. (2006). Grain-based foods and health. Cereals food world, 51,108-113.

Jones, J. M., Reicks, M., Fulcher, G., Marquart, L., Adams, J. F., Weaver, G., & Kanter, M. (2002).

Taking no action to move forward with the message about whole grain. In L. Marquart, J. Slavin & R. G., Fulcher (Eds.), Whole-grain foods in Health and Disease, (p. 359-369) AACC International, St. Paul, MN. Kandaswami C, & Middleton E., Jr. (1994). Free radical scavenging and antioxidant activity of plant flavonoids. Advances in Experimental Medicine and biology, 366, 351-376. Khatoon, S., & Gopala Krishna, A. G. (2004). Fat-soluble nutraceuticals and fatty acid composition of selected inian rice varieties. Journal of the Oil chemists' Society, 81, 939-943. Liu, R. H. (2004). Potential synergy of phytochemicals in cancer prevention: mechanism of action.

Journal of Nutrition, 134, 3479S-3485S. Liu, R.H. (2007). Whole grain phytochemicals and health. Journal of Cereal Science, 46,207-219. Mattila, P., Pihlava, J. M., & Hellstrom, J. (2005). Contents of phenolic acids, alkul- and alkenylresorcinols, and avenanthramides in commercial grain products. Journal of Agriculture and Food Chemistry, 53, 8290-8295. Orthoefer, F.T., & Eastman, J. (2004). Rice bran and oil. In E. T. Champagne, (Ed.), Rice Chemistry and Technology, (p. 569-593) AACC, St. Paul, Minnesota, U.S.A. Ren, W., Qiao, Z., Wang, H., Zhu, L., & Zhang, L. (2003). Flavonoids: promising anticancer agents.

Medicine Research Reviews, 23, 519-534. Roherer, C. A., & Siebenmorgen, T. J. (2004). Neutraceutical concentrations within the bran of various rice kernel thickness fractions. Biosystems Engineering, 88, 453-460. Shen, Y., Jin, L., Xiao, P., Yan L. Y., & Bao, J. (2009). Total phenolics, flavonoids, antioxidant capacity in rice grain and their relations to grain color, size and weight. Journal of Cereal Science, 49, 106— 111. Singleton, V. L., & Rossi, J. A. Jr. (1965). Colorimetry of total phenolics with phosphomolybdic-
phosphotungstic acid reagents. Americal Journal ofEnology and Viticulter, 16, 144-158. Yokoyama, W., (2004). Nutritional properties of rice and rice bran. In: Champagne, E.T. (Ed.), Rice

Chemistry and Technology (p. 595-609), AACC, St. Paul, Minnesota, U.S.A. Zhang, M. W., Guo, B. J., Chi, J. W., Xu, Z. H., Zhang, Y., & Zhang, R. F. (2005). Antioxidations and their correlation with total flavonoids and anthocyanin contents in different black rice varieties.

Scientia Agricultura Sinica, 38, 1324-1331.

What is claimed is:

1. A nutraceutical or pharmaceutical composition, in combination of 80 unmilled, colored rice varieties comprising of metabolites, wherein said metabolites comprising of at least one selected from the group consisting of phenolic acids, vitamins, flavanols, flavonols, flavanones, flavanoids, and terpenes derivatives.

2. A nutraceutical or pharmaceutical composition according to claim 1, wherein said metabolites have high anti-oxidative capacities.

3. The nutraceutical or pharmaceutical composition according to claim 1, wherein said composition further comprising of a high amount of micronutrients and macronutrients.

4. The nutraceutical or pharmaceutical composition according to claim 1, wherein said composition additionally comprising of a high rice protein.

5. The nutraceutical or pharmaceutical composition according to claim 1 to 4, wherein out of 80 said unmilled colored rice varieteies, 6 elite unmilled, colored rice varieties are selected for fixing percentage thereof in said composition based on said high amount of micronutrients and macronutrients, said high rice protein, and said high anti-oxidative capacities, wherein said 6 selected unmilled, colored rice varieties are selected from the group consisting of IRRI Acc.No.27665-R07-met, IRRI Acc.No.27726-R07-met, IRRI Acc.No.57861-R07-met, IRRI Acc.No.50783-R07-met, IRRI Acc.No.51618-R07-met, and IRRI Acc.No.64661-R07-met.

6. The nutraceutical or pharmaceutical composition according to claim 1, wherein said composition is a component of at least one selected from the group consisting of a food product, a nutritive supplement, a fiber supplement, a digestive supplement, dietary supplement, a functional food, and a medical supplement.

7. The nutraceutical or pharmaceutical composition according to claim 1 and 6, wherein said composition is a component of rice based eatables individually and in combination with other food stuff.

8. The nutraceutical or pharmaceutical composition according to claim 1, wherein flavanols, flavonols, terpenoids, vitamins are identified in said 80 unmilled, colored rice varieties on the basis of anti-cancer, anti-inflammatory, anti-diabetic, and Hpogenic properties of observed compounds.

9. The nutraceutical or pharmaceutical composition according to claim 1, wherein said composition is having therapeutic properties for the mitigation of skin disorders, cancer and other related diseases including cosmetic properties.

10. The nutraceutical or pharmaceutical composition according to claim 1 and 5, wherein said composition is formulated with specific combinations of compositions approximately 10 to 80% of 6 unmilled, colored rice varieties and approximaely 20% of other colored rice varieties.

11. The nutraceutical or pharmaceutical composition according to claim 1, wherein said composition is formulated in dosage forms wherein, said dosage form is selected from a group consisting of tablet, troches, lozenges, aqueous or oily suspensions, dispersible powders or granules, emulsions in hard or soft gel capsules, syrups, elixirs, phytoceuticals, nutraceuicals and foodstuff.

12. A process for identification and characterisation of a nutraceutical or pharmaceutical composition according to claim 1, comprising the steps of:

a. Obtaining 80 varieties of unmilled, colored rice seed;

b. Grinding said rice seed in order to obtain a homogenous rice powder;

c. Subjecting said rice seed powder to extraction of metabolites with an aqueous or organic solvent or a combination thereof, using 100 to 110 mg of said rice powder in 1 to 2 ml of 80% ethanol by 2-3 cycles of together, 3-5 minutes of vortex followed by 13 to 15 minutes of sonication, followed by centrifugation at 12,000 to 16,000 rpm for 13 to 18 minutes in order to obtain rice seed extract, wherein said rice seed extract is obtained optionally for a defined period of time;

d. Collecting rice seed extracts for using them as rice seed extract samples;

e. Optionally, lyophilizing said rice seed extracts;

f. Filtering said rice seed extract samples through 0.1 to 0.5-p.- syringe filter;

g. Subjecting said rice seed extract samples to an autosampler attached to HPLC at a temperature ranging from 6°C to 10°C;

h. Eluting said rice seed extract samples from UFLC by a binary gradient through a 3 to 7 H particle size RP 18 column (4 .6 mm D X 250 mm X L) held at 30-50°C in a temperature controlled column oven at a flow rate of 0.2 to 0.5 ml/min over 50 to 70 min;

i. Directing said UFLC eluent into mass spectrometer for identifying chromatography and mass spectrometry peaks from said rice seed extract sample run, wherein said mass spectrometry peak is one of an MS peak and MS/MS peak (50 amu to 1500 amu);

j. Using nominal or exact mass in order to generate a list of said rice seed extract sample data having said identified peaks;

k. Performing chemometric analysis on said rice seed extract sample data in order to identify major classes of metabolites;

1. Analysing said rice seed extract sample data using chemometry and said analysis is performed without loss of retention time data by same application performimg the programmatic identification of said Liquid chromatography and mass spectrometry peaks; and

m. Analysing and identifying major classes of metabolites using Liquid Chromatography and Mass Spectrometry (LC-MS).

13. The process for identification and characterisation of a nutraceutical or pharmaceutical composition according to claim 12, wherein isolation, identification and characterization of metabolites is carried out by using soft ionization method, wherein selected method is electrospray-ionization and fragmentation approach (LC/MS/MS) of all the parent ions revealing the presence of various important metabolites.

14. A process for preparing rice seed extract obtained from 80 unmilled, colored rice varieties comprising the steps of:

a. Obtaining 80 varieties of colored rice seed;

b. Weighing 1: 5 to 10 gm of seeds of each said 80 unmilled, colored rice varieties;

c. Grinding said rice seed of 80 unmilled, colored varieties in order to obtain a homogenous rice seed powder;

d. Subjecting said rice seed powder to an extraction with a polar to non-polar solvents for distillation wherein the ratio of solvent is 100 gm in 1 litre (1:10 wt/vol); and

e. Separating individual natural compounds from said rice seed extract at each step of extraction based on the polarity of said polar or non-polar solvents by means of solvent partitioning, followed by column flash chromatography, preparatory HPLC, TLC , finally followed by HPLC NMR LC-MS for structural elucidation.

15. The process for preparing rice seed extract obtained from 80 unmilled, colored rice varieties according to claim 14, wherein said polar solvent is selected from the group consisting of acetone, ethanol, methanol, and acetic acid.

16. The process for preparing rice seed extract obtained from 80 unmilled, colored rice varieties according to claim 14, wherein said non-polar solvent is selected from the group consisting of non-polar solvent comprises chloroform, and petroleum ether.

17. The process for preparing rice seed extract obtained from 80 unmilled, colored rice varieties according to claim 14, wherein said process is carried out at temperatures ranging from ambient to 100°C.

18. The process for preparing rice seed extract obtained from 80 unmilled, colored rice varieties according to claim 14, wherein said rice seed extract comprises at least one said natural compound selected from the group consisting of a polyphenol, a polysaccharide, amino acids, terpenoids, alkaloids and a combination thereof.

19. The process for preparing rice seed extract obtained from 80 unmilled, colored rice varieties according to claim 14, wherein said rice seed extract is having medicinal properties for treatment of cardiovascular, diabetes diseases and other diseases.

20. A process for extraction of protein from 80 unmilled, colored rice varieties comprising the steps of:

a) Dehusking of seed from 80 colored rice varieties;

b) Weighing of seeds approximately 500mg of each said 80 colored, rice varieties;

c) Grinding of seeds from said 80 colored rice varieties in order to obtain rice flour;

d) Adding 25 to 50 ml of 0.2-10% NaOH solution to said rice flour;

e) Mixing well in order to obtain a mixture;

f) Stirring said mixture at room temperature for over nigh;

g) Centrifuging said mixture at 3000xg for 10-20 minutes;

h) Collecting a first supernatant;

i) precipitating said first supernatant;

j) extracting said precipitate again with 0.2% NaOH in order to obtain second supernatant;

k) Combining said second supernatant with said first supernatant in order to get a combined extracts; 1) Adjusting said combined extracts to pH 7.0 with IN HC1; m) Precipitating a rice crude protein;

n) Collecting said precipitate by centrifugation at 3000xg for 10 minutes;

o) Washing said precipitate for three times with distilled water in order to get washed precipitate;
and

p) Drying completely said washed precipitate in order to obtain a rice protein.

21. The process for extraction of protein from 80 unmilled, colored rice varieties according to claim 20, wherein the % protein yield is 14.46 for IRRI- Ace. No. 27726-R07-met, 11.52 for IRRI- Ace. No. 27665-R07-met unmilled, colored rice variety.