Description:Preamble to the Description
[0001] The following specification describes the invention and the manner in which it is to be performed:
DESCRIPTION OF THE INVENTION
Technical field of the invention
[0001] The present invention relates to a composition of ashwagandha root extract to be used in enhancing the brain function and protecting cognitive health. The invention also discloses a process of preparation of ashwagandha root extract composition, which yields higher concentrations of bioactive components. The ashwagandha root extract composition is safe and non-toxic.
Background of the invention
[0002] Withania somnifera commonly known as ashwagandha, is a natural herb recognized for several therapeutic properties. Ashwagandha is well known as Indian winter cherry or Indian cherry. Ashwagandha has diverse applications in Ayurveda. Ashwagandha has been used for various kinds of diseases specifically as a nerve tonic.
[0003] Ashwagandha comprises several bioactive components including alkaloids, withanolides, withaferins and saponins. The bioactive components constitute the properties including immunomodulatory actions, anti-stress activity, antioxidant activity, anti-arthritic activity, anti-cancer activity and so on.
[0004] The root and orange-red berry of ashwagandha are used as a tonic in treating medical conditions. Ashwagandha roots exhibit potent neurodegenerative and neuroregenerative potential. Additionally, leaves, flowers and seeds of ashwagandha are known for their astringent, anthelminthic, diuretic, aphrodisiac and anti-pyretic properties.
[0005] The neuropharmacological effects of ashwagandha root and leaf extracts include notable anti-stress and anti-anxiety activity. The extracts aid in alleviating symptoms of depression, insomnia by modulating the pituitary through various pathways. The extracts further exhibit neuropsychiatric advantages.
[0006] The withanolides extracted from the roots of ashwagandha exhibit several pharmacological properties. The efficacy of the withanolides is dependent on the absorption and transportation of the withanolides. The bioavailability of the withanolides is limited due to the restricted permeability of the withanolides through the intestinal epithelium. Withanolides have a narrow range of paracellular permeability with high molecular weight.
[0007] The Patent Application US201615326450A entitled “A Process to Enhance The Bioactivity of Ashwagandha Extracts” discloses a process for preparation of enriched ashwagandha extract comprising glycosides and saponins. The process comprises steps of cleaning roots of ashwagandha with ammonia, extraction of the root with methylene dichloride, washing the residue with water, drying and powdering to obtain powder of the extract. The process further comprises steps of preparing granules, enteric coating of the granules. The ashwagandha root extract comprises 3% withanolide glycosides and 1% saponins. The composition comprises excipients including disintegrant, diluents, binders, tillers, a carrier, adsorbents, emulsifiers, lubricants, stabilizing agents, antiadherents, galidants, and antioxidants. The enteric coating composition comprises ashwagandha extract and a second extract comprising amla extract, turmeric extract, grape seed extract, green tea extract, pomegranate extract, amaranth extract, costus extract, cocoa extract, coconut root extract, rosemary extract, mint leaf extract, star anise, sweet basil extract, cinnamon extract/clove extract, ginger extract, cumin seed extract, black pepper extract, fenugreek extract, and nitrate-rich vegetables.
[0008] The Patent Application CA2022050048W entitled “Withanolide-Enriched Compositions & Methods of Use Thereof” discloses a composition comprising ashwagandha extract and withanolides including Withanolide A, Withanolide B, Withanolide D, Withaferin A at a concentration of 1.5%, Withanoside IV, and Withanoside V. The composition comprises 10%-15% of withanolides. The composition comprises combination of extracts of ashwagandha whole plant, root and leaf. The composition comprises carriers including alcohols, propylene glycol, water, and surfactants. The composition is formulated as a capsule, tablet, pill, powder, granule, emulsion, microemulsion, solution, suspension, syrup, or elixir. The composition is used as topical scalp or skin application along with essential oils.
[0009] The Patent Application IN201911034479 entitled “A Method For The Extraction Of Consolidated Constituents of Withanolides from Ashwagandha (Withania somnifera) Roots” discloses a method for the extraction of withanolides from ashwagandha. The method of extraction comprises steps of sorting and sieving of roots, pulverizing the roots, extracting the pulverized roots with polar solvent, collection of residues, centrifugation, and crystallization of the extract in the presence of a non-polar solvent. The product comprises Withanolide A, Withanolide B, Withanone, Withaferine A, Withastramonolide, Withanoside IV, Physaqulin D, 27-Hydroxy withanone, Withanoside V, Withanoside VI. The polar solvents used for the extraction are methanol, ethanol, acetone, isopropyl alcohol and ethyl acetate. The non-polar solvents used are hexane, chloroform, dichloromethane and dichloroethane. The withanolides from ashwagandha are used for the treatment of anxiety, amnesia, cancer, stress and for immunomodulatory activity.
[0010] The Patent Application WO2019IN50774 entitled “Withania somnifera Composition, Method of Preparation and Use Thereof” discloses an ashwagandha extract enriched with withanolide glycosides and withanolide aglycones. The extract comprises lesser concentrations of withaferin-A. The composition comprises 3%-15% withanolide glycosides 2.5-8% of withanolide aglycones, and 0.01-0.7% of withaferin-A. The composition comprises additional biological agents comprising pharmaceutically acceptable active ingredients, vitamins, minerals, pharmaceutically acceptable, excipients, carriers or diluents. The composition is formulated into a tablet, a capsule, a soft chewable tablet or gummy bear. The composition comprises extracts of roots, stems, leaves, tender stems, tender twigs, aerial parts or whole plant or mixtures of Withania somnifera. The method of preparation comprises steps of extraction of powdered Withania somnifera root with an organic solvent, concentration under vacuum, adjusting pH with organic bases, re-extracting the extract followed by drying. The organic solvent used for extraction comprises ethanol, methanol, n-propanol, isopropyl alcohol or water. The inorganic bases used for adjusting pH are potassium carbonate, potassium hydroxide, sodium bicarbonate, sodium carbonate and sodium hydroxide. The composition aids in improving testosterone levels, energy levels, sustained energy, vigor, stamina, muscle mass and muscle strength.
[0011] Although ashwagandha extracts have been known for the therapeutic effect of the bioactive components, the bioavailability of those bioactive components is often limited. Hence, there is a necessity for a process for the preparation of ashwagandha root extract resulting in higher concentrations of bioactive components with enhanced absorption.
Summary of the invention
[0012] The present invention overcomes the drawbacks of the existing compositions from ashwagandha root extract. The process for preparation yields higher concentrations of bioactive compounds from ashwagandha root including withanolides with enhanced cell permeability and absorption.
[0013] The present invention discloses a composition of bioabsorbable ashwagandha root extract and a process of preparation thereof. The process of preparation of ashwagandha root extract composition comprises steps of drying and pulverizing ashwagandha root material, solvent extraction of the pulverized root material by hydroalcohol, filtering the extract, concentrating the extract to 30% total solid content, subjecting the concentrated extract to drying, adding 2.5 volumes of cow’s milk to the dried extract, subjecting the milk extract to drying, subjecting the spent residue from the solvent extraction to water extraction, filtering the water extract, concentrating the water extract, subjecting the concentrated water extract to spray drying, blending the milk extract and water extract in the ratio of 2:1 to 10:1 and drying the blended extract under heat and vacuum.
[0014] The present invention further discloses a composition of bioabsorbable ashwagandha root extract. The composition comprises ashwagandha milk extract at a concentration range of 67%-91% and ashwagandha water extract at a concentration range of 9%-33%. The ashwagandha root extract composition comprising ashwagandha milk extract and ashwagandha water extract is capable of being granulated to produce tablets, granules and capsules.
[0015] The process of preparation of ashwagandha root extract disclosed in the present invention yields higher concentrations of withanolides. The bioactive component withanolide exhibits several therapeutic properties. The ashwagandha root extract composition comprises withanolides at a concentration range of 3%-5%. The percentage ratio of aglycones and glycosides is 60:40 to 65:35.
[0016] The ashwagandha root extract composition exhibits enhanced cell transportation across intestinal epithelium in various directions in Cancer coli-2 Caco-2 cell line. The enhanced absorption constitutes for the increased bioavailability. The ashwagandha root extract composition exhibits 83% percentage inhibition of acetylcholine esterase (AChE) enzyme. The inhibition of AChE enzyme promotes cognitive and memory enhancing functions.
[0017] The ashwagandha root extract composition further exhibits 90% free radical scavenging activity at a lower concentration. Additionally, the synergistic effect of the ashwagandha milk extract and ashwagandha water extract results in the inhibition of the cyclooxygenase-2 (COX-2) enzyme indicating the anti-inflammatory activity of the ashwagandha root extract composition.
[0018] The ashwagandha root extract composition inhibits the in vitro proliferation of cancer cell lines including breast cancer cell lines MDA-MD-231 and MDA-MB-468, pancreatic cancer cell line MIA PaCa-2 and colorectal cancer cell-line HCT-116 at a lower concentration. The ashwagandha root extract composition is safe and non-toxic against Hek293T and HFF-1 cell lines at a concentration of 1 mg/ml.
[0019] The ashwagandha root extract composition comprises higher concentrations of essential bioactive molecule withanolides. The ashwagandha root extract composition exhibits enhanced cell permeability and absorption, hence increasing the bioavailability. The ashwagandha root extract composition is safe and non-toxic.
Brief description of the drawings
[0020] 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.
[0021] FIG 1 illustrates the flowchart for the process of preparation of ashwagandha root extract.
[0022] FIG 2 tabulates the composition of ashwagandha root extract.
[0023] FIG 3 illustrates the absorption of ashwagandha root extract composition by Caco-2 cell line.
[0024] FIG 4 illustrates the chromatogram of withanolides absorbed by the Caco-2 cell lysate.
[0025] FIG 5 illustrates the chromatogram of withanolides absorbed by the Caco-2 cell media.
[0026] FIG 6 illustrates the percentage of AChE inhibition by the ashwagandha root extract composition.
[0027] FIG 7 illustrates the percentage of radical scavenging activity of the ashwagandha root extract composition.
[0028] FIG 8 illustrates the percentage of glutathione reduction by the ashwagandha root extract composition.
[0029] FIG 9 illustrates the percentage of COX-2 inhibition by the ashwagandha root extract composition.
[0030] FIG 10 illustrates the percentage inhibition of cell proliferation of MIA PaCa-2 cell line by the ashwagandha root extract composition.
[0031] FIG 11 illustrates the percentage inhibition of cell proliferation of MDA-MB-231 cell line by ashwagandha root extract composition.
[0032] FIG 12 illustrates the percentage inhibition of cell proliferation of MDA-MB-468 cell line by ashwagandha root extract composition.
[0033] FIG 13 illustrates the percentage inhibition of cell proliferation of HCT-116 cell line by ashwagandha root extract composition.
[0034] FIG 14 illustrates the percentage inhibition of cell proliferation of Hek293T cell line by the ashwagandha root extract composition.
[0035] FIG 15 illustrates the percentage inhibition of cell proliferation of HFF-1 cell line by the ashwagandha root extract composition.

Detailed description of the invention
[0036] 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.
[0037] The term “Anti-inflammatory” refers to a substance which reduces inflammation in the body.
[0038] The term “Withanolides” refers to a group of naturally occurring steroidal lactones.
[0039] The term “Bioavailability” refers to the ability of the drug to be absorbed by the body.
[0040] The present invention discloses a composition of bioabsorbable ashwagandha root extract and a process of preparation thereof. The composition of ashwagandha root extract comprises a blend of ashwagandha milk extract and ashwagandha water extract. The process of preparation of ashwagandha root extract results in obtaining higher concentrations of withanolide glycosides and aglycones. The composition of ashwagandha root extract exhibits increased permeability and bio absorption.
[0041] FIG 1 illustrates the flowchart for the process of preparation of ashwagandha root extract. The process (100) of preparing ashwagandha root extract begins with step (101) of drying the ashwagandha root material and the dried ashwagandha root material is pulverized to a coarse powder. At step (102), subjecting the powdered root material to solvent extraction at a temperature of 70o C. The solvent is selected from a group of methanol, ethanol or other hydroalcohols. At step (103), filtering the obtained exact by muslin cloth. The retentate is collected after filtration of the extract. At step (104), subjecting the retentate to subsequent solvent extractions to obtain the complete extract. At step (105), concentrating the extract to obtain total solid content of 30% by removing alcohol from the extract. The concentrated extract is allowed to precipitate for 12 to 15 hours. At step (106), subjecting the concentrated extract to drying at a temperature not exceeding 70o C.
[0042] At step (107), 2.5 volumes of cow’s milk is added to the dried ashwagandha root extract. The mixture is subjected to constant stirring for a duration of 3 to 4 hours at a temperature of 60o C. At step (108), subjecting the milk extract by a tray dryer at a temperature of 70o C. At step (109), subjecting spent residue from the solvent extraction to water extraction by percolation for 12 to 24 hours to obtain water extract. At step (110), filtering the extract by muslin cloth and concentrating the water extract. The extract is concentrated to obtain desired total solid content. At step (111), spray drying the concentrated water extract to obtain free-flowing dry extract powder.
[0043] At step (112), the obtained milk extract and water extract are blended in the ratio of 2:1 to 10:1. At step (113), subjecting the blended milk extract and water extract to drying under heat and vacuum. The process of preparation results in an ashwagandha root extract with withanolides at a concentration range of 3%-5%. The percentage ratio of bioactive components aglycones and glycosides is in the range of 60:40 to 65:35.
[0044] The present invention discloses a process of preparation ashwagandha root extract which yields in high concentration of withanolides. Additionally, the composition of ashwagandha root extract comprises <0.5% of withaferin. The consumption of higher concentrations of withaferin has toxic effects.
[0045] FIG 2 tabulates the composition of ashwagandha root extract. The composition comprises ashwagandha milk extract in the concentration range of 67%-91% and ashwagandha water extract in the concentration range of 9%-33%.
[0046] The ashwagandha root extract composition comprising ashwagandha milk extract and ashwagandha water extract exhibit synergistic activity in enhancing the properties of the composition. The addition of milk to the composition enhances the lipophilicity hence increasing the membrane permeability and absorption of the ashwagandha root extract. The enhancement of membrane permeability and absorption thus increases the bioavailability of the ashwagandha root extract composition.
[0047] Having generally described this invention, a further understanding can be obtained by reference to certain specific examples, which are provided herein for purposes of illustration only and are not intended to be limiting unless otherwise specified.
Example 1: Tablet composition of ashwagandha milk extract and ashwagandha water extract
[0048] The ashwagandha root extract composition comprising ashwagandha milk extract and ashwagandha water extract is granulated into different forms but not limited to tablets, granules and capsules. The blend of ashwagandha milk extract and ashwagandha root extract was granulated by ethanol solvent system. The granules were autoclaved for microbial control. The autoclaved granules were compressed into tablets and capsules. The optimum granulation ratio of ashwagandha milk extract and ashwagandha water extract is 1:1 to 1:10. The granules were standardized by high performance liquid chromatography (HPLC). The hardness of the granules was found to be in the range of 3kg/cm to 4kg/cm. The friability of the granules was found to be <1%. The disintegration time of the granules was found to be <30 minutes.
Example 2: Analysis of bioabsorption of withanolides of ashwagandha root extract composition in vitro
[0049] The roots of ashwagandha comprise diverse bioactive components with therapeutic properties. The bioactive component withanolides accounts for the pharmacological applications of ashwagandha root extract. The efficacy of the extracts is dependent on the absorption and transportation of the withanolides through the intestinal epithelium. The intestinal absorption was evaluated by the analysis of withanolide absorption in Cancer coli-2 (Caco-2) cell line. The analysis of absorption correlates to the bioavailability of the withanolides from the ashwagandha root extract composition.
[0050] The analysis of absorption was evaluated in apical-basolateral (A-B) and basolateral-to-apical (B-A) directions at 37 °C for 2hours in a shaking incubator at 100rpm. The ashwagandha root extract composition was analyzed by dissolving the ashwagandha root extract composition in assay buffer and observing the direction of transport. The transport of the ashwagandha root extract composition was analyzed by reverse phase chromatography and ultraviolet-visible detector for quantification
[0051] The in vitro cell culture of Caco-2 cell line involves seeding of 80,000-1,00,000 cells in 35mm cell culture dishes to adhere for 12 hours. The ashwagandha root extract composition was administered at a concentration of 1mg/ml. The absorption was evaluated at time intervals of 0 minutes, 5 minutes, 10 minutes, 15 minutes, 30 minutes, 60 minutes, 90 minutes, 180 minutes, and 360 minutes. The cells were frozen at -80o C. The cells were washed with phosphate buffer saline (PBS) and trypsinized. The trypsinized cells were centrifuged and suspended in lysis buffer. The cells were further centrifuged, and acetone was added to the supernatant. The cells were centrifuged and vacuum dried to remove acetone.
[0052] The apparent permeability coefficient (Papp) was evaluated by using the formula:
Papp = (dQ / dt) / (A x C)
where dQ / dt (µg/s) is the rate of drug transportation; A is the surface area of the cell monolayer (cm2); and C is the initial concentration of the drug on the administered side (µg/ml).
[0053] The efflux ratio indicates the transport of the compound from the basolateral compartment to the apical compartment. The efflux ratio aids in assessing transport in both directions (apical to basolateral (AB) and basolateral to apical (BA)) across the cell monolayer which in turn indicates the rate of active efflux of the substance.
[0054] The efflux ratio was further evaluated by using the formula:
Efflux ratio = Papp (BA)/ Papp (AB)
where Papp (BA) is the apparent permeability coefficient substance's transport from the basal to the apical side and Papp (BA) is the apparent permeability coefficient for the substance's transport from the apical to the basal side is Papp (ab) (absorptive transport).
[0055] FIG 3 illustrates the absorption of ashwagandha root extract composition by Caco-2 cell line. The ashwagandha root extract composition comprising ashwagandha milk extract and ashwagandha water extract with high concentration of withanolides exhibit enhanced membrane permeability and absorption. Further, the ashwagandha root extract composition exhibits increased apical to basolateral transport and basolateral to apical transport. FIG 4 illustrates the chromatogram of withanolides absorbed by the Caco-2 cell lysate. FIG 5 illustrates the chromatogram of withanolides absorbed by the Caco-2 cell media. The chromatogram indicates complete absorption of withanolides from the ashwagandha root extract composition at a time interval of 5 minutes
Example 3: Analysis of anti-acetylcholinesterase (AChE) activity of the ashwagandha root extract composition
[0056] The anti-AChE activity was evaluated to analyze the effect of the ashwagandha root extract composition in improving memory and cognitive functions. The extracts of ashwagandha aid in promoting cognitive functions and enhancing memory. The ashwagandha root extract composition comprising ashwagandha milk extract and ashwagandha water extract with withanolides was analyzed for the inhibition of the AChE enzyme.
[0057] The activity of the AChE enzyme was evaluated by AChE assay kit. The assay determines the hydrolysis of AChE to thiocholine. Donepizil was used as standard for the assay. FIG 6 illustrates the percentage AChE inhibition by ashwagandha root extract composition. The ashwagandha root extract composition exhibits 83% AChE inhibition at a concentration of 3.57 mg/ml.
Example 4: Analysis of antioxidant activity of the ashwagandha root extract composition
[0058] The antioxidant activity is performed to analyze the free radical scavenging activity of the composition. The ashwagandha root extract composition aids in minimizing the effect of reactive oxygen species (ROS), free radicals, deoxyribose nucleic acid (DNA) damage and ageing.
[0059] The antioxidant activity was screened by the analysis of conversion of free radical 1,1-diphenyl-2-picryl hydroxyl (DPPH) to 1,1-diphenyl-2-picryl hydrazine. DPPH is a purple-colored substance and upon the action of antioxidants DPPH gets converted into colorless 1,1-diphenyl-2-picryl hydrazine. The change of color was measured by the rate of absorbance at 517 nm. The antioxidant activity was analyzed for ashwagandha root extract composition at concentrations of 1 µg/µl, 2.5 µg/µl, 5 µg/µl and 10 µg/µl. The control for the analysis was ascorbic acid at concentrations of 200 µg/µl, 400 µg/µl, 600 µg/µl, 800 µg/µl and 1000 µg/µl. DPPH was added to different concentrations of ashwagandha root extracts and ascorbic acid and was allowed to incubate for 30 minutes at room temperature. The absorbance was measured, and respective percentage of inhibition was calculated.
[0060] FIG 7 illustrates the percentage of radical scavenging activity of the ashwagandha root extract composition. The ashwagandha root extract composition exhibits 90% radical scavenging activity at a concentration of 32.16 µg/ml. The ashwagandha root extract composition exhibits a higher percentage of radical scavenging activity by the inhibition of production of ROS at a lower concentration.
Example 5: Analysis of glutathione reduction activity of the ashwagandha root extract composition
[0061] The glutathione reductase activity was performed to analyze the pro-oxidant activity of the ashwagandha root extract composition. The generation of free radicals enables ROS-induced cell damage in cancerous cells. The analysis of reduced glutathione (GSH) and oxidized glutathione (GSS) relates to the generation of free radicals.
[0062] The generation of free radicals was measured by the reaction of Elman’s reagent with oxidized glutathione at 412 nm. The concentrations of ashwagandha root extract composition used was 1 µg/µl and the concentration of ascorbic acid standard used was 1mg/ml. The contents for the analysis comprising sample, glutathione, Elman’s reagent and tris buffer were mixed and incubated for 30 minutes. The absorbance was measured at 412 nm.
[0063] FIG 8 illustrates the percentage glutathione reduction by ashwagandha root extract composition. The standard ascorbic acid indicates ~7% glutathione reduction at a concentration of 1 µg/µl. The ashwagandha root extract composition indicates ~15.6% glutathione reduction at a concentration of 1 µg/µl.
Example 6: Analysis of anti-inflammatory activity of the ashwagandha root extract composition
[0064] The anti-inflammatory activity of the ashwagandha root extract composition was evaluated for the analysis of inflammatory mediators by the withanolides of the ashwagandha root extract composition. The onset of inflammation results in activation of cyclooxygenase pathway where arachidonic acid gets converted to prostaglandins. The reaction is mediated by cyclooxygenase-2 (COX-2) enzyme. The screening of COX-2 enzyme correlates to the anti-inflammatory activity of the ashwagandha root extract composition. The analysis was performed by COX-2 inhibitor screening kit for ashwagandha root extract composition. Celecoxib was used as the standard for the analysis.
[0065] FIG 9 illustrates the percentage COX-2 inhibition by the ashwagandha root extract composition. The standard celecoxib exhibited 69% COX-2 inhibition at a concentration of 100 nM. The ashwagandha root extract composition exhibited 87.2% COX-2 inhibition at a concentration of 1 mg/ml. The ashwagandha root extract composition exhibited percentage COX-2 inhibition higher than that of the standard celecoxib. The synergistic effect of the ashwagandha milk extract and ashwagandha water extract results in the inhibition of the COX-2 enzyme. The presence of bioactive components including withanolides of the ashwagandha root extract composition exhibits anti-inflammatory activity by the inhibition of COX-2 enzyme.
Example 7: Analysis of in-vitro anticancer activity of ashwagandha root extract composition
[0066] The in-vitro anti-cancer activity was evaluated to analyze the effect of ashwagandha root extract composition on proliferation of cancerous cells. The evaluation also analyzes the effectiveness of the ashwagandha root extract composition on improving the immune response, eliminating free radicals, and inhibiting cell division and inflammation. The bioactive components of ashwagandha root including withanolides, withaferins exhibit antitumor and immunomodulating properties.
[0067] The in-vitro anti-cancer activity of the ashwagandha root extract composition was evaluated by 3-(4, 5-dimethylthiazolyl-2)-2, 5-diphenyltetrazolium bromide (MTT) assay. The MTT assay determines the mitochondrial activity of living cells. The MTT assay was performed on breast cancer cell lines MDA-MD-231 and MDA-MB-468, pancreatic cancer cell line MIA PaCa-2 and colorectal cancer cell-line HCT-116. The concentrations of ashwaganda root extract composition used was 0.1 mg/ml, 0.25 mg/ml, 0.5 mg/ml, and 1 mg/ml. Dimethyl sulfoxide (DMSO) was used as the negative control and doxorubicin was used as the positive control for the assay at a concentration of 0.5 mg/ml. The cells were cultured for 24 hours, and the absorbance was measured at 570 nm.
[0068] FIG 10 illustrates the percentage inhibition of cell proliferation of MIA PaCa-2 cell line by the ashwagandha root extract composition. The ashwagandha root extract composition inhibits the proliferation of MIA PaCa-2 cell line at a concentration of 8.52 µg/ml. The ashwagandha root extract composition exhibits higher percentage of inhibition of cell proliferation of MIA PaCa-2 cell line at a lower concentration.
[0069] FIG 11 illustrates the percentage inhibition of cell proliferation of MDA-MB-231 cell line by ashwagandha root extract composition. The ashwagandha root extract composition inhibits the proliferation of MDA-MB-231 cell line at a concentration of 1.72 µg/ml. The ashwagandha root extract composition exhibits higher percentage of inhibition of cell proliferation of MDA-MB-231 cell line at a lower concentration.
[0070] FIG 12 illustrates the percentage inhibition of cell proliferation of MDA-MB-468 cell line by ashwagandha root extract composition. The ashwagandha root extract composition inhibits the proliferation of MDA-MB-468 cell line at a concentration of 1.46 µg/ml. The ashwagandha root extract composition exhibits higher percentage of inhibition of cell proliferation of MDA-MB-468 cell line at a lower concentration.
[0071] FIG 13 illustrates the percentage inhibition of cell proliferation of HCT-116 cell line by ashwagandha root extract composition. The ashwagandha root extract composition inhibits the proliferation of HCT-116 cell line at a concentration of 1.68 µg/ml. The ashwagandha root extract composition exhibits higher percentage of inhibition of cell proliferation of HCT-116 cell line at a lower concentration.
Example 8: Analysis of toxicity of the ashwagandha root extract composition
[0072] The analysis of toxicity indicates the safety dosage of the ashwagandha root extract composition. The concentration of safety dosage was evaluated to analyze the therapeutic usage of the ashwagandha root extract composition. The ashwagandha root extract composition was evaluated for the safety dosage by in-vitro toxicity studies against Kidney Epithelial cells (Hek293T) and Skin Fibroblast cells (HFF-1). The cytotoxicity displayed determines the cell growth, function and survival.
[0073] The safety analysis was performed by MTT assay, which indicates the mitochondrial activity of living cells. The Hek293T and HFF-1 cells were incubated overnight. The incubated cells were added with the ashwagandha root extract composition at concentrations of 0.1 mg/ml, 0.25 mg/ml, 0.5 mg/ml, and 1 mg/ml. DMSO was used as the negative control and doxorubicin was used as the positive control for the assay. The cells were cultured for 24 hours, and the absorbance was measured at 570 nm.
[0074] FIG 14 illustrates the percentage inhibition of cell proliferation of Hek293T cell line by the ashwagandha root extract composition. The ashwagandha root extract composition was found to be safe and non-toxic against Hek293T cells at a concentration of 1 mg/ml.
[0075] FIG 15 illustrates the percentage inhibition of cell proliferation of HFF-1 cell line by the ashwagandha root extract composition. The ashwagandha root extract composition was found to be safe and non-toxic against HFF-1 cells at a concentration of 1 mg/ml.
[0076] The present invention discloses bioabsorbable ashwagandha root extract composition and a process of preparation thereof. The ashwagandha root extract composition comprises ashwagandha milk extract and ashwagandha water extract. The ashwagandha root extract comprises crucial bioactive component withanolides at a higher concentration. The ashwagandha root extract comprises withaferin at a minimum concentration <0.5% which when consumed at higher concentrations induce toxic effects. The addition of cow’s milk enhances the lipophilicity of the ashwagandha root extract composition hence enhancing the cell permeability, absorption and bioavailability. The ashwagandha root extract composition exhibits essential therapeutic properties including antioxidant, anti-acetylcholinesterase activity, anti-cancer, and anti-inflammatory properties. The ashwagandha root extract composition is safe and non-toxic.

, Claims:We Claim:

1. A process for the preparation of ashwagandha root extract, the process (100) comprising steps of:
a) drying and pulverizing ashwagandha root material to a coarse powder (101);
b) subjecting the powdered ashwagandha root material to solvent extraction using hydroalcohol at a temperature of 70o C (102);
c) filtering the extract through a muslin cloth (103);
d) subjecting the retentate of the extract to subsequent hydroalcohol extraction (104);
e) concentrating the extract to a total solid content of 30% and precipitating for a duration of 12 to 15 hours (105);
f) subjecting the concentrated extract to drying at a temperature of 70o C (106);
g) adding 2.5 volumes of cow’s milk to the dried extract with continuous stirring (107);
h) subjecting the obtained milk extract to drying (108);
i) subjecting spent residue from solvent extraction to water extraction by percolation to obtain water extract (109);
j) filtering the extract by muslin cloth and concentrating the water extract (110);
k) subjecting the concentrated water extract to a spray drying (111);
l) blending the milk extract and the water extract in the ratio of 2:1 to 10:1 (112); and
m) subjecting the blended extract to drying under heat and vacuum (113).

2. The process (100) as claimed in claim 1, wherein said hydroalcohol is selected from a group of methanol and ethanol.

3. The process (100) as claimed in claim 1, wherein said drying of the milk extract is achieved by a tray dryer at a temperature of 70°C.

4. The process (100) as claimed in claim 1, wherein said water extract is percolated for a duration of 12 hours to 15 hours.

5. A composition of ashwagandha root extract, the composition comprising:

a) an ashwagandha milk extract at a concentration range of 67% to 91%; and
b) an ashwagandha water extract at a concentration range of 9% to 33 %;

6. The composition as claimed in claim 5, wherein said ashwagandha root extract composition comprises withanolides at a concentration range of 3% to 5%.

7. The composition as claimed in claim 5, wherein said ashwagandha root extract composition exhibits absorption of withanolides by Cancer coli-2 (Caco-2) cell line at a time interval of 5 minutes.

8. The composition as claimed in claim 5, wherein said ashwagandha root extract composition inhibits 83% acetylcholine esterase (AChE) enzyme activity at a concentration of 3.57 mg/ml.

9. The composition as claimed in claim 5, wherein said ashwagandha milk extract and ashwagandha water extract exhibit synergistic activity in inhibiting cyclooxygenase-2 (COX-2) enzyme with a percentage inhibition of 87.2%.

Bindu Sharma
Agent for the Applicant
Regn no: IN/PA 1055
Dated: 12th January 2023