DESC:Priority Claim

[0001] This application claims priority from the provisional application numbered 202041001891 filed with Indian Patent Office, Chennai on 16th June 2020 entitled “A process for extraction and standardization of the active phytochemicals and nutrients of Moringa oleifera”, the entirety of which is expressly incorporated herein by reference.
Preamble to the description
[0002] The following specification particularly describes the invention and the manner in which it is to be performed:
Description of the invention
Technical field of the invention
[0003] The present invention generally relates to a process for extraction and standardization of the active phytochemicals of Moringa oleifera. More particularly, the invention relates to an extraction of nutraceutical and medicinal compounds from Moringa oleifera, which comprises standardization and isolation of the pharmacologically active phytochemicals and nutrients. In addition, the present invention also discloses a formulation of Moringa oleifera.
Background of the invention
[0004] Moringa oleifera also known as drumstick tree or horseradish tree, is a fast-growing deciduous, drought-resistant tree of the family Moringaceae that is native to tropical and subtropical regions of South Asia.
[0005] Moringa oleifera contains wide variety of proteins, vitamins, and minerals. The plant is known for centuries for its nutritive and medicinal properties. Moringa oleifera is a rich source of healthy antioxidants and bioactive plant compounds.
[0006] Many pharmacological studies have established the ability of the plant extract exhibiting properties such as anti-inflammatory, analgesic, anti-asthmatic, anti-diabetic, anti-urolithiatic, diuretic, local anesthetic, anti-allergic, anthelmintic, wound healing, antidiarrheal, immunomodulatory, antimicrobial, and anti-fungal etc.
[0007] Generally, a single extraction is sufficient to effectively extract the plant compounds based on the partition coefficient of the compound to be extracted from the solvent. However, a multi-step solvent extraction is often necessary to isolate the desired plant compound by extracting multiple times. The process is repeated two to three times or more in case the compound has low partition coefficient in the solvent.
[0008] Several extraction methods are employed to isolate the plant compounds, but methods for standardization of the extracts are very few. Some of the prior arts relating to extraction and isolation of the plant compounds from M. oleifera are discussed below.
[0009] The Patent Application “WO2014053944A1” titled “Extract from Morigaceae and a method to prepare the extract” discloses a pressurized hot water extraction method for preparing a plant extract from the family Moringaceae suitable for use as an ingredient in a food product, drink, dietary supplement, or as cosmetics. The extract comprises about 1000 mg/kg to 2000 mg/kg Total Phenolic Content (TPC), as measured according to the method described with an incubation period of about 2 hours. The invention further provides a food product, drink, dietary supplement, or a cosmetic product comprising the extract.
[0010] The Patent Application “WO2013183065A2” titled “Extraction of Phytochemicals from Plant Matter” discloses a method or a process for the extraction which enables wider range of plant constituents. The extraction of a first plant matters is carried out with an alcoholic solvent, the former is treated with a second plant matters containing acidic components that converts one or more of the insoluble constituents thereof into soluble forms. The plant species having the desired nutrients and the appropriate acidic components are reacted and extracted by a single extraction process with a single solvent to give a composition containing the desired vitamins, minerals, and other nutrients, in substantially desired proportions. The process eliminates the need for multi-step extractions unlike the conventional processes. The suitable acidic plant matters comprise amla fruits, lemon peels etc. The preferred solvent used for extraction is aqueous ethanol. The acidic treatment is carried out before or during the extraction process. The process further results in conversion of extracts into feasible forms exhibiting enhanced solubility and increased biological and metabolical activity or efficacy.
[0011] The US Patent Application “US20150209395A1” titled “Extracts from Plants of the Moringaceae Family and Methods of Making” discloses the materials and methods for producing extracts from a plant of the Moringaceae family having high concentrations of moringa isothiocyanates. The invention essentially relies on the fact that the enzyme myrosinase necessary to convert moringa glucosinolates (MGLs) into moringa isothiocyanates (MICs) is activated by simply injuring plant material (e.g., seeds, or fresh leaves or sprouts) of a plant of the Moringaceae family. The process does not require the plant material exposed to harsh conditions, such as harsh temperatures and drying conditions. The plant material is dried and stored, once the seeds, fresh leaves or sprouts are injured and subject to extraction at a later time to retrieve the isothiocyanates.
[0012] The US Patent Application “US20090098230A1” titled “Nutraceutical Moringa Composition” discloses a nutraceutical beverage comprising a portion from M. oleifera plant to provide a therapeutic effect. The leaves, seeds, and fruit of the M. oleifera plant are used in the invention to provide a biologically metabolized nutritional composition for health, wellbeing, and for the treatment of ailments.
[0013] Hence, in order to overcome the disadvantages that exist in the state of the art, the invention provides an extraction process and standarization of the extracts to isolate the active phytochemicals and nutrient from the M. oleifera. Thus, providing a customized extraction process with maximum intended medicinal efficacy of the obtained phytochemicals.
Summary of the Invention
[0014] The present invention overcomes the drawbacks of the prior arts by disclosing a process for extraction and standardization of the active phytochemicals of Moringa oleifera. The invention relates to multiple parts of extraction of nutraceutical and medicinal compounds from Moringa oleifera. The extracts are subjected to standardization to enrich the phytochemicals. The present invention also discloses a formulation of Moringa oleifera for various therapeutic activities.
[0015] The process of extraction according to the present invention comprises of three stages for extracting the phytochemicals based on their polarity and solubility in the solvents by employing a multi-step solvent partition separation, which results in isolating pure forms and eliminating impurities from the final extract. The extract thus obtained is subjected to standardization to enrich the phytochemicals and the standardized extract is subjected to quantitative and qualitative analysis.
[0016] The phytochemical screening through qualitative analysis using various tests and reagents identified various phytochemicals present in the extract of Moringa oleifera. The extract is analyzed for alkaloids, flavonoids, steroids, carbohydrates, glycosides including anthraquinone glycosides, cardiac glycosides, coumarin glycosides, cyanogenetic glycosides, saponin glycosides, tannins, proteins, phenolic compounds and amino acids.
[0017] The phytochemicals identified are quantified using various reagents and the amount of phytochemicals are identified in the standardized extract are higher in Part 1, Part 2, Part 3 extracts of Moringa oleifera.
[0018] The present invention also discloses a method of isolation of phytochemicals from standardized extract of Moringa oliefera. The method of isolation of phytochemicals from Moringa oliefera involves dissolving standardized Moringa oliefera extract in water and subjecting to liquid partitioning to obtain an ethyl acetate soluble fraction and an aqueous extract. The ethyl acetate layer and aqueous layer is separated in a separating funnel and the ethyl acetate layer is evaporated to obtain a dried and powdered form of ethyl acetate layer. The powdered form obtained in the previous step is sub-fractionated using silica gel column chromatography of particle size of 60-200 microns and the sample is eluted to separate the phytochemicals by using gradient elution with chloroform and methanol in a ratio of 100:1 to 80:20, ethyl acetate and methanol in the ratio of 80:20 to 40:60. As a result, totally eight fractions of phytochemicals are obtained and the ethyl acetate layer is subjected to silica gel column chromatography using gradient elution to separate the eight fractions.
[0019] The chemical characterization of the phytochemicals resulted in the presence of isothiocyanates such as moringin and moringinine, antioxidant such as isotrifolin, quercetin and kaempferol and beta-sitosterols.
[0020] The present invention also discloses a formulation with Moringa oleifera standardized extract as active ingredient. The formulation is useful as immune booster, immune booster protein powder and a formulation for weight management.
[0021] The formulation of Moringa oleifera standardized extract as immune booster comprises Moringa oleifera standardized extract, Spirulina extract, astaxanthin, ashwagandha extract and black pepper extract and the formulation is effective in enhancing the immune response. The same formulation also is useful as immune booster protein powder with addition of protein powder in the same formulation.
[0022] The formulation of Moringa oleifera standardized extract for weight management comprises Moringa oleifera standardized extract, Garcinia cambogia, green coffee bean extract and green tea extract.
[0023] Moringa oliefera extract of the present invention is useful in major industries of food and beverage sector in different forms such as capsules, aqua based supplement drinks, energy bars, nutrient supplements etc. The formulations are safe and does not induce any adverse effects.
Brief description of the drawings

[0024] The foregoing and other features of embodiments will become more apparent from the following detailed description of embodiments when read in conjunction with the accompanying drawings. In the drawings, like reference numerals refer to like elements.
[0025] Figure 1 illustrates the stage 1 of the extraction process to obtain active phytochemicals.
[0026] Figure 2 illustrates the stage 2 of the extraction process to obtain the active phytochemicals.
[0027] Figure 3 illustrates the stage 3 of the extraction process to obtain the active phytochemicals.
[0028] Figure 4 illustrates the process of standardization of the parts 1-3 from the extraction process.
[0029] Figure 5 tabulates the phytochemical screening of the standardized extract.
[0030] Figure 6 tabulates the results of the analysis of Part 1, Part 2, Part 3 extracts and the standardized extracts for phytochemicals.
[0031] Figure 7 illustrates a method of isolation of phytochemicals from Moringa oliefera.
[0032] Figure 8 illustrates the Thin-Layer Chromatography results of the separated fractions.
[0033] Figure 9 shows the structural formula of moringin.
[0034] Figure 10a shows the structural formula of isotrifolin.
[0035] Figure 10b shows the structural formula of quercetin.
[0036] Figure 10c shows the structural formula of kaempferol.
[0037] Figure 11 shows the structural formula of beta-sitosterols.
[0038] Figure 12 illustrates the formulation of standardized extracts of Moringa oliefera as immune booster.
[0039] Figure 13 illustrates the formulation of standardized extracts of Moringa oliefera as immune booster protein powder.
[0040] Figure 14 illustrates the formulation of standardized extracts of Moringa oliefera for weight management.
Detailed description of the invention

[0041] Reference will now be made in detail to the description of the present subject matter, one or more examples of which are shown in figures. Each example is provided to explain the subject matter and not a limitation. Various changes and modifications obvious to one skilled in the art to which the invention pertains are deemed to be within the spirit, scope, and contemplation of the invention.

[0042] In order to more clearly and concisely describe and point out the subject matter of the claimed invention, the following definitions are provided for specific terms, which are used in the following written description.
[0043] The term "Phytochemicals" refers to those biologically active compounds found in plants produced by primary or secondary metabolism exhibiting therapeutic properties.
[0044] The term "Micella or spent", also called as micelle or micelles or micellae is an aggregate or supramolecular assembly of surfactant molecules dispersed in a liquid colloid.
[0045] The term "HPLC" refers to High Performance Liquid Chromatography a separation technique in analytical chemistry to separate, identify, and quantify each component in a mixture, by pumping the sample mixture through a pressurized liquid solvent through a column filled with a solid adsorbent material.
[0046] The term "Centrifugation" is a separation technique to separate particles from a solution according to their size, shape, density, viscosity of the medium and rotor speed.

[0047] The term "Thin layer Chromatography (TLC)" is a chromatography technique used to separate non-volatile mixtures using a thin uniform layer of silica gel or alumina coated on to a piece of glass, metal, or rigid plastic.

[0048] The term "Solvent partitioning" refers to an extraction process requiring two solvents that are not miscible in each other, thus separating compounds based on their polarity.

[0049] The invention discloses a process for extraction and standardization of the active phytochemicals of Moringa oleifera. More particularly, the invention relates to an extraction of nutraceutical and medicinal compounds from M. oleifera which comprises standardization and isolation of the pharmacologically active phytochemicals and nutrients. The present invention also discloses a formulation of Moringa oleifera for various therapeutic activities.
[0050] Moringa oleifera plant used in the invention has a geographical origin from India. The invention requires selection of extremely good quality raw material comprising young and matured leaves, stem part and pods from the Moringa oleifera plant. Different parts of Moringa oleifera are used in unique ratios to obtain the predictable amount of pharmacological active phytochemicals.
[0051] The process of extraction comprises of three stages for extracting the phytochemicals based on their polarity and solubility in the solvents by employing a multi-step solvent partition separation, which results in isolating pure forms and eliminating impurities from the final extract.
[0052] The extracts obtained from three different stages are used to standardize the extract to enrich with bioconstituents. According to an embodiment, the standardized extract is subjected to qualitative and quantitative analysis.
[0053] Figure 1 illustrates the stage 1 of the extraction process to obtain active phytochemicals. The stage 1 of the extraction process (100) to obtain active phytochemicals comprises a step (101) of cleaning and drying the Moringa oleifera raw material comprising leaf, pods, and stem. At step (102), the raw material is coarsely powdered. At step (103), 300 g of the powdered raw material of Moringa oleifera is loaded into a stainless-steel extractor. At step (104), the powdered raw material is extracted with methanol of >98% purity at a ratio of 1:5 % w/v by circulating at a temperature of about 700C for a duration of 3 hours and the step is repeated thrice. At step (105), the liquid extracts obtained from repeating the step (104) are combined and filtered. The filtration is achieved using fine filter cloth of pore size 200. At step (106), the obtained micella or spent is kept aside for further extraction. At step (107), the filtered liquid extract is concentrated under vacuum at a temperature 700C to obtain a thick or dried semi solid extract as a thick paste. The extract obtain is in the form of dark green paste and is termed as Part 1 (P1) with a quantity of 65gms and % LOD is 52 with a yield on dry basis is 10.4%.
[0054] Figure 2 illustrates the stage 2 of the extraction process to obtain the active phytochemicals. The stage 2 of the extraction process (200) to obtain active phytochemicals comprises a step (201) of cleaning and drying the Moringa oleifera raw material comprising leaf, pods, and stem. At step (202), the raw material is coarsely powdered. At step (203), 300 g of the powdered raw material of Moringa oleifera is loaded into a stainless-steel extractor. At step (204), the powdered raw material is subjected to hydraulic extraction with 70% methanol i.e., at a ratio of 1:5 % w/v by circulating at a temperature of about 700C for a duration of 3 hours and the step is repeated thrice. At step (205), the liquid extracts obtained from repeating the step (204) are combined and filtered. The filtration is achieved using fine filter cloth of pore size 200. At step (206), the obtained micella or spent is kept aside for further extraction. At step (207), the filtered liquid extract is concentrated under vacuum at a temperature 700C to obtain a thick or dried semi solid extract as a thick paste. The extract obtain is in the form of green paste and is termed as Part 2 (P2) with a quantity of 155 gms and % LOD is 48 with a yield on dry basis is 26.8%.
[0055] Figure 3 illustrates the stage 3 of the extraction process to obtain the active phytochemicals. The stage 3 of the extraction process (300) to obtain active phytochemicals comprises a step (301) of cleaning and drying the Moringa oleifera raw material comprising leaf, pods, and stem. At step (302), the raw material is coarsely powdered. At step (303), 300 g of the powdered raw material of Moringa oleifera is loaded into a stainless-steel extractor. At step (304), the powdered raw material is subjected to aqueous extraction at a ratio of 1:5 % w/v by circulating at a temperature of about 700C for a duration of 3 hours and the step is repeated thrice. At step (305), the liquid extracts obtained from repeating the step (304) are combined and filtered. The filtration is achieved using fine filter cloth of pore size 200. At step (306), the obtained micella or spent is kept aside for further extraction. At step (307), the filtered liquid extract is concentrated under vacuum at a temperature 700C to obtain a thick or dried semi solid extract as a thick paste. The extract obtain is in the form of greenish brown colored paste and is termed as Part 3 (P3) with a quantity of 180 gms and % LOD is 55 with a yield on dry basis is 27.0%.
[0056] The parts P1, P2 and P3 obtained from the three stages 1, 2 and 3 of the extraction respectively subjected to standardization of bioconstitutents.
[0057] Figure 4 illustrates the process of standardization of the parts 1-3 from the extraction process. The process (400) of standardization starts with step (401) of adding the Part 1, Part 2 and Part 3 of the extracts into a clean reactor. The step includes addition of Part 1 extract at a weight percentage of 65% w/w, Part 2 extract at a weight percentage of 15% w/w and Part 3 extract at a weight percentage of 20% w/w. At step (402), the mixture of Part 1, Part 2 and the Part 3 is dissolved in 70% methanol at 1:1 %v/v ratio and mixed thoroughly for uniform distribution. At step (403), the mixture is subjected to cold precipitation at the temperature of 4°C for a duration of 10 to 12 hours. At step (404), the precipitate obtained from the cold precipitation is separated by using Hy-flow supercell bed filtration of the top supernated liquid. At step (405), the sediment is separated and the clear supernated liquid obtained by the filtration is collected in a separate vessel. At step (406), 10% to 12% w/w of bioavailability enhancers are added to the clear supernated liquid and is mixed thoroughly to form a uniform solution. At step (407), the solution is subjected to solvent recovery at 70°C under vacuum to obtain the extract in the form of green colored thick paste, which is further subjected to spray drying to obtain green to greenish brown colored fine powder. At step (408), spray dried powder is subjected to milling and sieving to obtain the powder in the form of uniform particle size. Finally, at step (409), the powder is considered for analysis of physicochemical, phytochemical and microbiological parameters.
[0058] The standardized extract is subjected to phytochemical screening for qualitative and quantitative analysis. The phytochemical screening is achieved through qualitative analysis using various tests and reagents to identify various phytochemicals present in the extract of Moringa oleifera. The extract is analyzed for alkaloids, flavonoids, steroids, carbohydrates, glycosides including anthraquinone glycosides, cardiac glycosides, coumarin glycosides, cyanogenetic glycosides, saponin glycosides, tannins, proteins, phenolic compounds and amino acids.
[0059] Figure 5 tabulates the phytochemical screening of the standardized extract. The specific parts of the extracts i.e., Part 1, Part 2, Part 3 and the standardized extract is subjected to phytochemical screening using various tests and reagents for the analysis of phytochemicals. Alkaloids are analyzed using Dragendroff’s test, Mayor’s test and Wagner’s Test. Flavonoids are analyzed using Alkaline reagent test and Shinoda Test. Steroids are analyzed using Salkowski test and Libermann-burchard test. Carbohydrates by Molisch’s test, anthraquinone glycosides are analyzed by Borntrager’s test, modified Borntrager’s test and by using hydroxyanthraquinones. Cardiac glycosides are analyzed by Kedde’s test, Raymond’s test, Legal’s test and Baljet test. Coumarin glycosides and cyanogenetic glycosides are analyzed by Guirnard reaction and saponin glycosides by foam test. Tannins are analyzed by Ferric chloride test, Lead acetate test whereas protein by Millon’s test and Biuret test. Folic acids are analyzed by Folin- Ciocaltaeu test and similarly amino acids by Ninhydrin test. The presence of phytochemicals in Part 1, Part 2, Part 3 extracts and the standardized extract are tabulated in Figure 5. The results interpret that the phytochemicals were higher in the standardized extract in comparison with the Part 1, Part 2, Part 3.
[0060] According to the embodiment of the invention, the Moringa oleifera extract is subjected to quantitative analysis for the identified phytochemicals. The quantitative analysis is achieved using various reagents based on the type of phytochemicals.
[0061] The following examples are offered to illustrate various aspects of the invention. However, the examples are not intended to limit or define the scope of the invention in any manner.
Example 1: Quantitative analysis of the extract for alkaloids
[0062] The extract is analyzed for the presence of alkaloids. 20 g of the extract is accurately weighed and transferred to 1-liter conical flask. To this, 400 ml of solvent ether and alcohol mixture (4: 1) and 20 ml of 5 % v/v ammonia solution is added. The solution is continuously stirred for one hour. The solution is filtered through cotton and the filtrate is transferred into a separator. The residue is washed with 2 x 50 ml of ether alcohol mixture (4: 1) and add 100 ml of 1N sulphuric acid to the combined ether alcohol solution. The solution is shaken well and allowed to separate, and the lower layer is collected into another separator. To the ether alcohol layer, add 100 ml of 0.5 N sulphuric acid and alcohol mixture (3: 1) and extract. Continue the extraction with 3 x 80 ml of the 0.5 N sulphuric acid and alcohol mixture (3: 1) until aqueous layer is colorless.
[0063] The acid solution is combined and washed with 40 ml of chloroform followed by 2 x 20 ml of chloroform. The combined chloroform layer is washed with acid alcohol mixture and the chloroform layer is discarded. The acid alcohol solutions are combined and made to alkaline with 5 % v/v ammonia solution and add 10 ml in excess.
[0064] The combined acid alcohol solution with 5% v/v ammonia treated part is extracted with chloroform of 3 x 100 ml in separating funnel. Further extraction is continued in 100 ml of chloroform, 2 more times if the extraction is incomplete.
[0065] The chloroform layers are combined and washed with 20 ml of distilled water. The chloroform layer is filtered through anhydrous sodium sulphate, previously moistened with chloroform, into a tared beaker. The residue in washed with little amount of chloroform and the chloroform layer is transferred to the same tared beaker and evaporated on a water bath. Finally, 5 ml of alcohol is added to the residue and dried to constant weight at 1050c.
Example 2: Quantitative analysis of the extract for flavonoids
[0066] Sample (1.0 ml) is mixed with 4ml of distilled water and subsequently with 0.30 ml of a sodium nitrate (NaNO2) solution (10%). After 5 min, 0.30 ml aluminum chloride (AlCl3) solution (10%) is added fallowed by 2.0 ml of sodium hydroxide (NaOH) solution (1%) to the mixture. Immediately, the mixture is thoroughly mixed and absorbance is then determined at 510 nm versus the blank. Standard quercetin is prepared (0-12mg/ml) and the results are expressed as quercetin equivalents (mg quercetin/gm dried extract).
Example 3: Quantitative analysis of the extract for saponins
[0067] The extract is analyzed for the presence of saponins. 5 gm of extract is accurately weighed and transferred into dry and clean 250 ml round bottom flask. The content is dissolved in 25 ml of 90% methanol and reflux for 30 mins in a water bath. The reflux is called and filtered, and the steps are repeated until the extraction is colorless. The methanol washes are combined and subjected to evaporation under vacuum. After evaporation, the residue is cooled, and 25 ml of petroleum ether is added at 40-600C and reflux for 30 mins after which the solvent is cooled and removed by decantation. The residue is treated with same process by 25 ml of chloroform and 25 ml of ethyl acetate. The solvents are discarded, and the residue is cooled, which is then dissolved in 5 ml of methanol and pour drop by drop with constant stirring in to 25 ml of acetone to precipitate saponins. Acetone is decanted and the precipitate is dried to a constant weight at 105°C.
Example 3: Quantitative analysis of the extract for sterols
[0068] The extract is analyzed for the presence of sterols. 5 gm of the extract is accurately weighed and transferred into a dry and clean 250 ml round bottom flask. The content is dissolved in 30 ml of chloroform and reflux for 30 minutes in water bath. The extract is cooled to room temperature of 25°C, filtered on cotton, and the residue re-extracted twice, using 30 ml of chloroform for 15 min. The two filtrates are collected and subjected to drying under reduced pressure at 40°C, rotary evaporator. The residue is then placed to an oven at 80°C until reached constant weigh.
Example 4: Quantitative analysis of the extract for tannins
[0069] The extract is analyzed for the presence of tannins. 2 g of the extract is added into a 250 ml conical flask. 200 ml water is added and subjected to sonication for 10 minutes. Allow the solution to settle and decant through a piece of cotton wool to a 500ml volumetric flask. 100 ml of water is added and subjected to sonication for 10 minutes and decanted. Further, 100 ml water is added and subjected to sonication for 10 minutes. In order to confirm the complete extraction of tannins, the procedure is followed: Add 3-4 drops of 8 % w/v in water ferric ammonium sulphate solution to 5ml of the extract and observed for a color change. If the tannins are completely extracted, blue color is not produced. If blue color is developed, the extract is again treated with 100ml water and check with ferric ammonium sulphate. Make up to the mark with water. Pipette out 25 ml from the above solution in to 250 ml conical flask and add 25 ml of indigo sulphonic acid solution. The solution is titrated with constant stirring against 0.1N potassium permanganate solution and observed for a golden yellow color as end point. Each ml of 0.1 N Potassium permanganate solution is equivalent to 0.004157g of tannin compound calculated as tannins. The blank test is achieved by titrating 25 ml of indigo sulphonic acid.
[0070] The results of the quantitative analysis from Part 1, Part 2, Part 3 extracts and the standardized extracts for alkaloids, flavonoids, sterols and tannins are tabulated.
[0071] Figure 6 tabulates the results of the analysis of Part 1, Part 2, Part 3 extracts and the standardized extracts for phytochemicals. The results indicated the higher percentage of alkaloids, flavonoids, sterols and tannins in standardized extract than Part 1, Part 2, Part 3 extracts of Moringa oliefera.
[0072] According to other embodiment of the claimed invention, the phytochemicals are isolated from Moringa oliefera using a specific method of isolation.
[0073] Figure 7 illustrates a method of isolation of phytochemicals from Moringa oliefera. The standardized extract of Moringa oliefera is used to isolate the phytochemicals. The method (700) of isolation of phytochemicals from Moringa oliefera starts with a step (701) by dissolving standardized Moringa oliefera extract in water and subjecting to liquid partitioning to obtain an ethyl acetate soluble fraction and an aqueous extract. At step (702), the ethyl acetate layer and aqueous layer is separated in a separating funnel and the ethyl acetate layer is evaporated to obtain a dried and powdered form of ethyl acetate layer. The evaporation of ethyl acetate layer is achieved using in a rotoevoporator. At step (703), the powdered form obtained in the previous step is sub-fractionated using silica gel column chromatography of particle size of 60-200 microns. At step (704), the sample is eluted to separate the phytochemicals by using gradient elution with chloroform and methanol in a ratio of 100:1 to 80:20, ethyl acetate and methanol in the ratio of 80:20 to 40:60. Finally, at step (705), totally eight fractions of phytochemicals are obtained.
[0074] The ethyl acetate layer is subjected to silica gel column chromatography using gradient elution with different ratio of chloroform and methanol ranging from 100:1 to 80:20. The results interpreted that silica gel column chromatography results in separation of eight fractions namely MOF-1 (Moringa oliefera Fraction-1) separated using 98% chloroform and 2% methanol, MOF-2 separated using 90% chloroform and 10% methanol, MOF-3 separated using 80% chloroform and 20% methanol, MOF-4 separated using 78% ethyl acetate and 22% methanol, MOF-5 separated using 65% ethyl acetate and 35% methanol, MOF -6 is separated using 65% ethyl acetate and 35% methanol, MOF -7 is separated using 45% ethyl acetate and 55% methanol and MOF-8 is the pure methanol eluted fraction layer. The separated fractions are subjected to qualitative analysis using Thin-Layer Chromatography (TLC) and High-Performance Liquid Chromatography (HPLC).
[0075] Figure 8 illustrates the Thin-Layer Chromatography results of the separated fractions. The thin-layer chromatography is performed using pre-coated with silica gel as stationary phase, methanol and ethyl acetate with trichloroacetic acid as developing solvent in a ratio of 2:8:1 v/v respectively. The visualization is achieved under various wavelengths namely UV 254 nm, UV 366 and normal light. The figure 7 interprets the results of thin-layer chromatography with separation of phytochemicals with Part 1 extract is lane 1, Part 2 extract in Lane 2, Part 3 extract in 3 and the standardized extract of Moringa oleifera in lane 4. The chemical characterization of the phytochemicals resulted in the presence of isothiocyanates such as moringin and moringinine, antioxidant such as isotrifolin, quercetin and kaempferol and beta-sitosterols.
[0076] Natural isothiocyanates are produced by enzymatic conversion of glycosylates in plants. The isothiocyanates extracted from Moringa oleifera include moringin and moringinine, which exert neuroprotective effects against neurodegenerative disease.
[0077] Figure 9 shows the structural formula of moringin. Moringin [4-(a-L-rhamnosyloxy)-benzyl isothiocyanate] with a chemical formula of C14H17NO5S exhibits chemopreventive properties. The moringin and moringinine extracted from Moringa oleifera exhibits anti-inflammatory and antioxidant effects thus protecting against neurodegenerative disorders.
[0078] Antioxidant prevents the damages caused by free radicals that may leads to oxidative stress, thus exhibiting free radical scavenging property. The antioxidants extracted from Moringa oleifera comprises isotrifolin, quercetin and kaempferol.
[0079] Figure 10a shows the structural formula of isotrifolin. Isotrifolin or isoquercitrin is a flavanoid with a chemical formula of C21H20O12 exhibiting anti-neoplastic properties.
[0080] Figure 10b shows the structural formula of quercetin. Quercetin is polyphenolic flavonoid with a chemical formula of C15H10O7 exhibiting chemopreventive activity.
[0081] Figure 10c shows the structural formula of kaempferol. Kaempferol is flavonoid with a chemical formula of C15H10O4 exhibiting antioxidant property and anticancer effect.
[0082] Beta-sitosterols are substances similar to cholesterol and aids in lowering the cholesterol levels and reduces the risk of some cancers.
[0083] Figure 11 shows the structural formula of beta-sitosterols. Beta-sitosterols or ß-sitosterol having a chemical formula of C29H50O is known to reduce blood cholesterol level in humans.
[0084] The selection of good quality raw material comprising young and matured leaves, pods and stem and their proper mixing is extremely essential. The process of extraction comprises selection of suitable solvents with water or a mixture of to extract specific phytochemicals followed by purification. Further, the highly purified, potent, and effective phytochemicals are targeted by customized extraction and partial purification technology. The amount of minerals, vitamins, amino acids, and essential elements are retained in the extract which is exponential to the amount present in the raw leaves. The invention is developed with the concept of food for medicine and is suitable for both nutraceutical and medicinal usage purpose.
[0085] The invention results in a unique product with different ratios of raw material from the Moringa oleifera to obtain a predictable amount of pharmacologically active phytocomponents. The multistage partition extraction process results in reducing the impurities thus enriching the phytochemical content.
[0086] The present invention also discloses the formulations comprising standardized extracts of Moringa oliefera for various therapeutic activities.
[0087] Figure 12 illustrates the formulation of standardized extracts of Moringa oliefera as immune booster. The formulation of the invention comprises standardized extracts of Moringa oliefera at a concentration in the range between 400-500 mg, Spirulina extract at a concentration in the range between 400-500 mg, astaxanthin at a concentration in the range between 2-3 mg, ashwagandha extract at a concentration in the range between 80-120 mg and black pepper extract at a concentration in the range between 2-4 mg. The formulation is useful and effective as immune booster.
[0088] Figure 13 illustrates the formulation of standardized extracts of Moringa oliefera as immune booster protein powder. The formulation of the invention comprises standardized extracts of Moringa oliefera at a concentration of 25 g, plant protein powder at a concentration of 40 g, spirulina extract at a concentration of 20 g, ashwagandha extract at a concentration of 10 g and black pepper extract at a concentration at 0.2 g. The formulation is useful and effective as immune booster protein powder.
[0089] According to an embodiment of the invention, the formulation is also considered for weight management.
[0090] Figure 14 illustrates the formulation of standardized extracts of Moringa oliefera for weight management. The formulation comprises standardized extracts of Moringa oliefera at a concentration in the range between 500-600 mg, Garcinia cambogia at a concentration in the range between 400-500 mg, green coffee bean extract at a concentration in the range between 50-100 mg, and green tea extract at a concentration in the range between 50-100 mg. The formulation is useful and effective for weight management.
[0091] Moringa oliefera extract of the present invention is useful in major industries of food and beverage sector in different forms such as capsules, aqua based supplement drinks, energy bars, nutrient supplements etc. Moringa oliefera extract is also useful in the pharmaceuticals, cosmetics, nutraceuticals due to its high minerals content such as calcium, potassium, magnesium, iron, sodium etc., and vitamins, carbohydrates, proteins and rich of fibres. Moringa oliefera extract is also used for making immune booster with other extracts of Spirulina extract, Astaxanthin and Ashwagandha in the form of capsules. The capsule exhibits very good immune boosting effect. The extract also exhibits hepatoprotective activity, anti-diabetic activity and effective against cough.
[0092] Moringa oliefera extract along with other herbal extracts formulation such as Garcinia cambogia extract, green coffee bean extract, green tea extract is used for weight management.

,CLAIMS:Claims:

We Claim,
1. A process for extraction of one or more phytochemicals from Moringa oleifera, the process comprises the steps of:
a. cleaning and drying Moringa oleifera raw material comprising
leaf, pods, and stem;
b. making the dried raw material of Moringa oleifera into coarse powder;
c. loading 300 g of the powdered raw material of Moringa oleifera into a stainless-steel extractor;
d. extracting the powdered raw material with a solvent at a ratio of 1:5 % w/v by circulating at a temperature of about 700C for a duration of 3 hours and repeating the step for at least three times;
e. combining the obtained liquid extracts and subjecting to filtering using a fine filter cloth;
f. setting aside the obtained micella or spent for further extraction; and
g. concentrating the filtered liquid extract under vacuum at a temperature 700C to obtain a thick or dried semi solid extract as a thick paste.
wherein the extraction process is achieved in three parts based on the use of the solvent.
2. The process as claimed in claim 1, wherein the solvent used is selected from methanol of 98 % purity, 70 % methanol and hot water such that the Part 1, Part 2 and Part 3 of Moringa oleifera are extracted respectively.

3. The process as claimed in claim 1, wherein the Part 1, Part 2 and Part 3 extracts of Moringa oleifera are standardized to enrich the phytochemical content.

4. The process as claimed in claim 1, wherein the process (400) of standardization of Part 1, Part 2 and Part 3 extracts of Moringa oleifera further comprises the steps of:

a. adding the Part 1 at a weight percentage of 65%, Part 2 at a weight percentage of 15% w/w and Part 3 at a weight percentage of 20% w/w of Moringa oleifera extracts into a clean reactor (401);
b. dissolving the mixture of Part 1, Part 2 and the Part 3 in 70% methanol at 1:1 % v/v ratio and mixing thoroughly to form a uniform solution (402);
c. subjecting the mixture to a cold precipitation at a temperature of 4°C for a duration of 10 to 12 hours (403);
d. separating the precipitate obtained from the cold precipitation by using Hy-flow supercell bed filtration of the top supernated liquid (404);
e. separating the sediment and collecting the clear supernated liquid obtained by the filtration into a separate vessel (405);
f. adding 10% to 12% w/w of one or more bioavailability enhancers to the clear supernated liquid and mixing to form a uniform solution (406);
g. subjecting the solution to solvent recovery at 70°C under vacuum to obtain an extract in the form of green colored thick paste and is subjected to spray drying to obtain green to greenish brown colored fine powder (407);
h. subjecting the spray dried powder to milling and sieving to obtain the powder with uniform particle size (408); and
i. considering the powder for analysis of physicochemical, phytochemical and microbiological parameters (409).

5. The process as claimed in claim 1, wherein the standardization process results in enrichment of one or phytochemicals the phytochemical screening showed the higher presence of alkaloids, flavonoids, saponins, sterols and tannins.

6. The process as claimed in claim 1, wherein the chemical characterization of isolated phytochemicals from standardized extract showed the presence of isothiocyanates such as moringin and moringinine, antioxidant such as isotrifolin, quercetin and kaempferol and beta-sitosterols.

7. A formulation of standardized extracts of Moringa oleifera extracted from said process, as immune booster, the formulation comprises:

a. standardized extract of Moringa oliefera at a concentration in the range between 400-500 mg;
b. Spirulina extract at a concentration in the range between 400-500 mg;
c. astaxanthin at a concentration in the range between 2-3 mg;
d. ashwagandha extract at a concentration in the range between 80-120 mg; and
e. black pepper extract at a concentration in the range between 2-4 mg.

8. A formulation of standardized extracts of Moringa oleifera extracted from said process, as immune booster protein powder, the formulation comprises:
a. standardized extract of Moringa oliefera at a concentration of 25 g;
b. plant protein powder at a concentration of 40 g;
c. spirulina extract at a concentration of 20 g;
d. ashwagandha extract at a concentration of 10 g; and
e. black pepper extract at a concentration at 0.2 g

9. A formulation of standardized extracts of Moringa oleifera extracted from said process for weight management, the formulation comprises:
a. standardized extract of Moringa oliefera at a concentration in the range between 500-600 mg;
b. Garcinia cambogia at a concentration in the range between 400-500 mg;
c. green coffee bean extract at a concentration in the range between 50-100 mg; and
d. green tea extract at a concentration in the range between 50-100 mg.