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 bacopa extract to be used in management of neurodegenerative disorders. The invention also discloses a process of preparation of bacopa extract composition, which yields higher concentrations of bioavailable bioactive components. The bacopa extract composition is safe and non-toxic.
Background of the invention
[0002] Bacopa monnieri commonly known as bramhi, is recognized for wide array of therapeutic properties. Bacopa is considered a potent tonic for the human brain. The herb bacopa has been utilized especially for the treatment of neurological disorders around the globe. Bacopa acts as an alternative and complementary medicine for multiple ailments.
[0003] Several phytochemical bioactive compounds have been extracted from bacopa. The extracts of bacopa are used as cognitive, anti-inflammatory, analgesic, spasmolytic, antiepileptic, anticancer, antischizophrenic, antidiarrheal, antiulcer, anticonvulsant, antipyretic, and antirheumatic agent.
[0004] The alcoholic extracts of bacopa comprises a number of bioactive compounds including bacosides A and B, saponins A, B and C, triterpenoid saponins, stigmastanol, β-sitosterol, betulinic acid, D-mannitol, stigmasterol, α-alanine, aspartic acid, glutamic acid, and serine and pseudojujubogenin glycoside.
[0005] Additionally, bioactive compounds of bacopa aid in the treatment for cardiovascular, hepatic, gastrointestinal, myocardial ischemia, neurological and respiratory ailments. The bioactive compounds of bacopa alleviate opioid-related nephrotoxicity and hepatotoxicity.
[0006] The diverse mechanisms of action for cognitive effects include that of acetylcholinesterase (AChE) inhibition, antioxidant neuroprotection, β-amyloid reduction, neurotransmitter modulation (acetylcholine [ACh], 5-hydroxytryptamine [5-HT], dopamine [DA]), choline acetyltransferase activation, and increased cerebral blood flow. The proposed activity is due to the individual and synergistic action of the bioactive compounds of bacopa.
[0007] The bioactive compounds extracted from the bacopa plant including parental bacosides are of high molecular weight. The high molecular weight of the parental bacosides restricts the intestinal absorption and penetration of blood brain barrier. Additionally, the hydrogen bonding capacity and molecular flexibility is also limited.
[0008] The Patent Application IN-DEL-2009-00705A entitled “An Improved Method for Manufacturing of Bacopa Monnieri Extract of Not Less Than 20% Bacosides” discloses a method for standardized extract of bacopa rich in bacosides. The extract of bacopa comprises 40% bacosides in the form of free flowing powder. The method for the extraction of bacosides from bacopa comprises steps of extraction of dried aerial parts of bacopa with 60% primary alcohol as solvent, subsequent solvent extraction for three times, mixing and filtering of extracts, distillation of alcohol, extraction of residual aqueous syrup with n-butanol followed by vacuum distillation. The method of extraction further involves refluxing of residual solids with n-hexane followed by vacuum drying. The obtained dried mass of the enriched bacopa extract is ground and sieved. The bacopa extract enriched with bacosides exhibits synergistic therapeutic activities. The bacopa extract enriched with bacosides aids in management of nervous disorders and for enhancing nerve impulse transmission, repair of damaged neurons by enhancing kinase activity, neuronal synthesis, restoration of synaptic activity and nerve impulse transmission.
[0009] The Patent Application AU2007257480B2 entitled “A Synergistic Herbal Composition from Bacopa Species for Management of Neurodegenerative Disorders and a Process of Preparation Thereof” discloses a synergistic composition comprising Bacoside A3 in the range of 0.1 to 25%, Bacopaside II in the range of 0.1 to 25%, Jujubogenin isomer of bacopasaponin C in the range of 0.1 to 25%, Bacopasaponin C in the range of 0.1 to 25%, Bacopaside I in the range of 0.1 to 25%, Bacosine in the range of 0.1 to 25%, Apigenin in the range of 0.05 to 5%, Luteolin in the range of 0.05 to 5% and Sitosterol-D-glucoside in the range of 0.05 to 5%. The process of preparation of the synergistic composition comprises steps of powdering plant parts, solvent extraction of the powdered plant parts, refluxing and concentrating the extract, centrifuging the residue followed by drying. The synergistic composition aids in management of neurodegenerative diseases including dementia, amnesia, alzhemier's disease, cognition, slow learning, attention deficit disorders and hyperactivity disorders. The synergistic composition further comprises granulating agents, binding agents, lubricating agents, disintegrating agents, sweetening agents, coloring agents, flavoring agents, coating agents, plasticizers, preservatives, suspending agents, emulsifying agents and spherization agents. The synergistic herbal composition is formulated into tablet, troches, lozenges, aqueous or oily suspensions, dispersible powders or granules, 35 emulsion in hard or soft gel capsules, syrups, elixirs, phyotceuticals, neutraceuticals and food stuffs.
[0010] The Patent Application WO2006097043A1 entitled “Bacopa Monnieri Extract and Process for Preparation and Use Thereof” discloses a composition of bacopa extract comprising total saponin of B. monnieri comprising 10% to 80%. The method of extraction comprises steps of drying B. monnieri, 40% - 80% ethanol solvent extraction by weight of the extract, in terms of bacoside A, concentration and drying of the extract, elution with ethanol followed by drying. The composition is in the form of oral preparation or injection.
[0011] The Patent Application IN-CHENP-2005-01477A entitled “A Process for Producing Enriched Fractions Containing Up to 100% Of Bacoside A and Bacoside B” discloses a process for extraction, concentration and separation of bacosides A and B from bacopa plant material. The process for preparation of enriched fractions comprises steps of subjecting bacopa plant material to solvent extraction using methyl alcohol or ethyl alcohol or alcohol or water, drying of the extract by non-polar solvent including petroleum ether or hexane, extraction of the residue by an organic solvent including ethyl acetate, n-butyl acetate, acetone, ethyl propionate, n-pentanol, or n-butanol, chromatographic separation and purification by silica gel columns using chloroform, acetone, methanol, ethyl acetate, water of the extract to elute bacosides A and B. The concentration of bacosides A and B is obtained in the concentration range of 80%-100%. The bacosides A and B are separated by chromatography to obtain fractions containing pure Bacoside A and Bacoside B.
[0012] Although the extracts of Bacopa monnieri exhibit notable neuropharmacological activities, the intestinal absorption of the bioactive compounds and hence permeability through the blood brain barrier is often limited. The factors such as high molecular weight, limited hydrogen bonding capacity and molecular flexibility are some of the challenges. Hence, there is a necessity for the process of preparation of bioavailable bacopa extracts.
Summary of the invention
[0013] The present invention overcomes the drawbacks of the existing compositions from bacopa extract. The process for preparation yields bacopa extract composition enriched with bacosides, jujubogenin and ebelin lactone. The bacopa extract composition exhibits enhanced absorption and permeability thus enhancing the bioavailability.
[0014] The present invention discloses a composition of bacopa extract and bitterless ebelin lactone and a process of preparation thereof. The process of preparation of baccopa extract composition comprises the steps of drying the bacopa plant material, subjecting the dried bacopa plant material to solvent extraction, filtering the first bacopa extract, subjecting the retentate of the bacopa extract to subsequent alcohol extraction, concentrating the bacopa extract to a total solid content of 30%, subjecting the concentrated bacopa extract to drying.
[0015] The process of preparation of bacopa extract composition further comprises steps of subjecting bacopa extract to solvent extraction with alcohol and water, filtering the precipitate in filter press and tray drying, washing the bacopa extract with an organic solvent, subjecting the bacopa extract to acid hydrolysis, subjecting the bacopa extract to cooling and adding water, subjecting the precipitate to filtration and washing with demineralized water and crystallizing the precipitate with alcohol and drying.
[0016] The process of preparation of bacopa extract disclosed in the present invention yields enriched bacopa extract composition comprising bacosides and aglycone derivative ebelin lactone. The bacopa extract composition comprises bacosides at a concentration in the range of 20% to 50% and ebelin lactone at a concentration of 5%.
[0017] The bacopa extract composition exhibits enhanced intestinal absorption in Cancer coli-2 (Caco-2) cell line. The enhanced absorption constitutes for the increased bioavailability. The bacopa extract composition exhibits 85% percentage inhibition of acetylcholine esterase (AChE) enzyme. The inhibition of AChE enzyme aids in management of neurodegenerative disorders.
[0018] The bacopa extract composition further exhibits 90% free radical scavenging activity at a lower concentration. Additionally, the synergistic effect of the bioactive compounds of the bacopa extract composition results in the inhibition of the cyclooxygenase-2 (COX-2) enzyme indicating the anti-inflammatory activity of the bacopa extract composition.
[0019] The bacopa extract composition inhibits the in vitro proliferation of cancer cell lines including MDA-MB-468 and HCT-116 at a lower concentration. The bacopa extract composition is safe and non-toxic against Kidney Epithelial cells (Hek293T) and Skin Fibroblast cells (HFF-1) at a concentration of 0.9 mg/ml.
[0020] The bacopa extract composition comprises bioactive components such as bacosides, jujubogenin, aglycone derivative ebelin lactone with therapeutic properties. The molecular weight, hydrogen binding capacity and molecular flexibility of the bioactive components makes the composition lipophilic enabling enhanced intestinal absorption and permeability through the blood brain barrier (BBB). The enhanced intestinal absorption and permeability of the bacopa extract composition accounts for the enhanced bioavailability. The bacopa extract composition is safe and non-toxic.
Brief description of the drawings
[0021] 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.
[0022] FIG 1 illustrates the flowchart for the process of preparation of bacopa extract according to an embodiment of the invention.
[0023] FIG 2 tabulates the bacopa extract composition.
[0024] FIG 3 illustrates the absorption of bacopa extract enriched with bacosides by Caco-2 cell line.
[0025] FIG 4 illustrates the absorption of bacopa extract enriched with bacoside A3 and jujubogenin by Caco-2 cell line.
[0026] FIG 5 illustrates the absorption of bacopa extract enriched with ebelin lactone by Caco-2 cell line.
[0027] FIG 6 illustrates the absorption of bacopa extract composition by Caco-2 cell line.
[0028] FIG 7 illustrates the chromatogram of bacopa extract composition enriched with bacosides absorbed by the Caco-2 cell line.
[0029] FIG 8 illustrates the chromatogram of bacopa extract composition enriched with ebelin lactone absorbed by the Caco-2 cell line.
[0030] FIG 9 illustrates the percentage acetylcholine esterase (AChE) inhibition by bacopa extract composition.
[0031] FIG 10 illustrates the percentage of radical scavenging activity of the bacopa extract composition.
[0032] FIG 11 illustrates the percentage glutathione reduction by bacopa extract composition.
[0033] FIG 12 illustrates the percentage cyclooxygenase-2 (COX-2) inhibition by the bacopa extract composition.
[0034] FIG 13 illustrates the percentage inhibition of cell proliferation of MDA-MB-468 cell line by bacopa extract composition enriched with bacosides.
[0035] FIG 14 illustrates the percentage inhibition of cell proliferation of MDA-MB-468 cell line by bacopa extract composition enriched with bacoside A3 and jujubogenin.
[0036] FIG 15 illustrates the percentage inhibition of cell proliferation of MDA-MB-468 cell line by bacopa extract composition enriched with ebelin lactone.
[0037] FIG 16 illustrates the percentage inhibition of cell proliferation of MDA-MB-468 cell line by bacopa extract composition.
[0038] FIG 17 illustrates the percentage inhibition of cell proliferation of HCT-116 cell line by bacopa extract composition enriched with bacosides.
[0039] FIG 18 illustrates the percentage inhibition of cell proliferation of HCT-116 cell line by bacopa extract composition enriched with bacoside A3 and jujubogenin.
[0040] FIG 19 illustrates the percentage inhibition of cell proliferation of HCT-116 cell line by bacopa extract composition enriched with ebelin lactone.
[0041] FIG 20 illustrates the percentage inhibition of cell proliferation of HCT-116 cell line by bacopa extract composition.
[0042] FIG 21 illustrates the percentage inhibition of cell proliferation of Hek293T cell line by bacopa extract composition enriched with bacosides.
[0043] FIG 22 illustrates the percentage inhibition of cell proliferation of Hek293T cell line by bacopa extract composition enriched with bacoside A3 and jujubogenin.
[0044] FIG 23 illustrates the percentage inhibition of cell proliferation of Hek293T cell line by bacopa extract composition enriched with ebelin lactone.
[0045] FIG 24 illustrates the percentage inhibition of cell proliferation of Hek293T cell line by bacopa extract composition.
[0046] FIG 25 illustrates the percentage inhibition of cell proliferation of HFF-1 cell line by bacopa extract composition enriched with bacosides.
[0047] FIG 26 illustrates the percentage inhibition of cell proliferation of HFF-1 cell line by bacopa extract composition enriched with bacoside A3 and jujubogenin.
[0048] FIG 27 illustrates the percentage inhibition of cell proliferation of HFF-1 cell line by bacopa extract composition enriched with ebelin lactone.
[0049] FIG 28 illustrates the percentage inhibition of cell proliferation of HFF-1 cell line by bacopa extract composition.
Detailed description of the invention
[0050] 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.
[0051] The term “Anti-inflammatory agent” refers to a substance which reduces inflammation in the body.
[0052] The term “Bacosides” refers to a group of triterpenoid saponins found in Bacopa monnieri.
[0053] The term “Bioavailability” refers to the ability of the drug to be absorbed by the body.
[0054] The term “Blood Brain Barrier” refers to a network of blood vessels shielding the brain from toxic substances.
[0055] The present invention discloses a composition of bacopa extract and bitterless ebelin lactone and a process of preparation thereof. The process of preparation of bacopa extract composition yields enriched bacosides and aglycone derivative ebelin lactone. The composition of bacopa extract and ebelin lactone exhibits enhanced absorption and membrane permeability.
[0056] FIG 1 illustrates the flowchart for the process of preparation of bacopa extract. The process (100) of preparing bacopa extract composition begins with step (101) of drying the bacopa plant material. At step (102), subjecting the dried plant material to solvent extraction with alcohol at a temperature of 70o C. The solvent is selected from a group of methanol and ethanol. At step (103), filtering the obtained first bacopa extract using a muslin cloth. At step (104), subjecting the retentate of the bacopa extract to subsequent steps of solvent extraction. The solvent extraction of the retentate is carried out at least three times. At step (105), subjecting the bacopa extract to concentration. The alcohol is removed from the obtained bacopa extract by the addition of water and is concentrated to a total solid content of 30%. At step (106), subjecting the concentrated bacopa extract to spray drying. The spray drying is achieved at a temperature range of 90o C to 110o C and at a flow rate of 100 L/hour.
[0057] At step (107), subjecting the spray dried bacopa extract to solvent extraction. The solvent extraction is achieved by mixing alcohol and water at a ratio of 1:3. The solvent for the extraction is selected from a group of methanol and ethanol. The obtained bacopa extract is allowed to precipitate for a duration of 8 hours to 10 hours. At step (108), subjecting the obtained precipitate to filtration by a filter press to obtain enriched bacopa extract. The filtered bacopa extract is tray dried at a temperature of 90o C. The obtained precipitate comprises bacopa extract enriched with bacosides. At step (109), washing the obtained enriched bacopa extract by an organic solvent. The organic solvent is selected from a group of chloroform, ethyl acetate, and acetone. The obtained bacopa extract is dissolved in acetone at a concentration of 50%-75% at a ratio of 1:4.
[0058] At step (110), subjecting the bacopa extract to acid hydrolysis with mineral acid. The mineral acid for acid hydrolysis is selected from a group of hydrochloric acid, sulphuric acid, and orthophosphoric acid at a concentration of 2N sulphuric acid. The bacopa extract is refluxed for a duration of 4 hours and at a temperature of 90o C by continuous stirring. At step (111), subjecting the bacopa extract to cooling at room temperature. The cooled bacopa extract is added with water at a ratio of 1:1 with constant stirring. The aglycone derivatives are precipitated. At step (112), subjecting the precipitate to filtration. The filtered precipitate is washed with demineralized water at a pH value of 7. At step (113), subjecting the precipitate to enrichment by crystallization. The precipitate is dissolved in alcohol at a ratio of 1:1 to 1:3 and at a temperature range of 50o C to 65o C. The enriched precipitate is dried at a temperature of 70o C to obtain bacopa extract enriched with ebelin lactone.
[0059] The present invention discloses a process of preparation of bacopa extract which yields in high concentration of bacosides, jujubogenin and bitter less aglycone derivative ebelin lactone.
[0060] FIG 2 tabulates the bacopa extract composition. The bacopa extract composition comprises bacosides at a concentration range of 20% to 50% and ebelin lactone at a concentration of 5%. The bacopa extract composition comprises bacosides and ebelin lactone at a ratio of 3:1 to 99:1. The enrichment of the bacopa extract composition with aglycones including ebelin lactone and jujubogenin exhibit desirable central nervous system (CNS) drug-like properties. The aglycones comprise removed sugar moieties from the parental bacosides which aid in decreased molecular weight and hydrogen bonding capacity and increased molecular flexibility and lipophilicity. The bacopa extract composition comprising enriched aglycones enable significant passive lipid-mediated transport through the blood-brain barrier (BBB) hence increasing the brain permeation. The aglycone derivate ebelin lactone aids in increased membrane permeability and absorption.
[0061] 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 bacopa extract enriched with bacosides and ebelin lactone
[0062] The bacopa extract composition is granulated into different forms but not limited to tablets, granules and capsules. The tablet comprises a mixture of bacosides and aglycone derivative at a ratio of 1:1 at a concentration of 500mg. The process of preparation of the tablet comprises steps of preparation bacopa extract composition enriched with bacosides and ebelin lactone, sieving and blending of the enriched bacopa extract, granulating the enriched bacopa extract using by granulation fluid, drying the granules in a tray dryer at a temperature range of 60oC to 70oC for 4 hours, milling and blending of the granules followed by compressing to obtain the tablet comprising enriched bacopa extract composition.
[0063] The tablet comprising bacopa extract composition exhibits improved compressibility and hence improved stability. The tablet comprising bacopa extract composition aids in management of neurological disorders such as Alzheimer's and various human cancers such as colon cancer, breast cancer.
Example 2: Analysis of intestinal absorption of the bacopa extract composition in vitro
[0064] The evaluation of pharmacokinetic analysis including absorption, distribution, metabolism and excretion is essential to regulate the accurate dosage of a drug molecule. The bacopa extract composition comprises diverse bioactive components with therapeutic properties. The absorption of bioactive components of bacopa extract composition including bacosides, jujubogenin, ebelin lactone were analyzed. The efficacy of the extract is dependent on the absorption and transportation through the intestinal epithelium. The analysis of intestinal absorption aids in determining the highest non-toxic concentration for orally administered drug molecules.
[0065] The intestinal absorption was evaluated by the analysis of bacosides and ebelin lactone absorption in Cancer coli-2 (Caco-2) cell line. The analysis of absorption correlates to the bioavailability of the bacopa extract composition.
[0066] 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 bacopa extract composition was analyzed by dissolving the bacopa extract composition in assay buffer and observing the direction of transport. The transport of the bacopa extract composition was analyzed by reverse phase chromatography and ultraviolet-visible detector for quantification.
[0067] 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 24 hours. The bacopa 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.
[0068] 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).
[0069] 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.
[0070] 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).
[0071] FIG 3 illustrates the absorption of bacopa extract enriched with bacosides by Caco-2 cell line. FIG 4 illustrates the absorption of bacopa extract enriched with bacoside A3 and jujubogenin by Caco-2 cell line. FIG 5 illustrates the absorption of bacopa extract enriched with ebelin lactone by Caco-2 cell line. The bacopa extract enriched with ebelin lactone exhibits an apparent permeability coefficient of 121 ± 11.3cm/s with an efflux ratio of 0.647. FIG 6 illustrates the absorption of bacopa extract composition by Caco-2 cell line. The bacopa extract composition exhibits the highest apparent permeability coefficient of 141 ± 18.7cm/s with an efflux ratio of 0.54
FIG 7 illustrates the chromatogram of bacopa extract composition enriched with bacosides absorbed by the Caco-2 cell line. FIG 8 illustrates the chromatogram of bacopa extract composition enriched with ebelin lactone absorbed by the Caco-2 cell line. The chromatogram indicates complete absorption of bacopa extract composition by the Caco-2 cell line at a time interval of 15 minutes.
Example 3: Analysis of anti-acetylcholinesterase (AChE) activity of the bacopa extract composition
[0072] The activity of the enzyme AChE was evaluated to determine the hydrolysis of acetylcholine. The inhibition of the activity of AChE enhances cholinergic neurotransmission. The inhibition of the activity of AChE is essential for treating neurological disorders. The bacopa extract composition was analyzed for the inhibition of the AChE enzyme.
[0073] The activity of the AChE enzyme was evaluated by AChE assay kit. The assay determines the hydrolysis of AChE to thiocholine using donepizil as standard. FIG 9 illustrates the percentage AChE inhibition by bacopa extract composition. The bacopa extract composition exhibits 85% AChE inhibition at a concentration of 1 mg/ml. The bacopa extract composition facilitates effective inhibition of the enzyme AChE. The inhibition of AChE by the bacopa extract composition is essential for treating neurodegenerative diseases.
Example 4: Analysis of antioxidant activity of the bacopa extract composition
[0074] The antioxidant activity is performed to analyze the free radical scavenging activity of the bacopa extract composition. The bacopa extract composition aids in minimizing the effect of reactive oxygen species (ROS), free radicals, deoxyribose nucleic acid (DNA) damage and ageing. The ROS oxidizes the cellular components increasing the oxidative stress and cell damage. The accumulation of ROS damages cells and tissues. The increase in oxidative stress is capable of causing inflammatory diseases, autoimmune diseases and neurodegenerative diseases.
[0075] 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 bacopa extract composition at concentrations of 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 bacopa extract composition and ascorbic acid and was allowed to incubate for 15 minutes at room temperature. The absorbance was measured, and respective percentage of inhibition was calculated.
[0076] FIG 10 illustrates the percentage of radical scavenging activity of the bacopa extract composition. The bacopa extract composition exhibits 90% radical scavenging activity at a concentration of 58.30 µg/ml. The bacopa 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 bacopa extract composition
[0077] The glutathione reductase activity was performed to analyze the pro-oxidant activity of the bacopa 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.
[0078] The generation of free radicals was measured by the reaction of Elman’s reagent with oxidized glutathione at 412 nm. The concentrations of bacopa 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.
[0079] FIG 11 illustrates the percentage glutathione reduction by bacopa extract composition. The standard ascorbic acid indicates ~7% glutathione reduction at a concentration of 1 µg/µl. The bacopa extract composition indicates 22% glutathione reduction at a concentration of 1 µg/µl. The bacopa extract composition comprising bacoside A3, jujubogenin and ebelin lactone exhibits synergistic effect in minimal ROS generation.
Example 6: Analysis of anti-inflammatory activity of the bacopa extract composition
[0080] The anti-inflammatory activity of the bacopa extract composition was evaluated for the analysis of inflammatory mediators by the bacopa 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 bacopa extract composition. The analysis was performed by COX-2 inhibitor screening kit for bacopa extract composition. Celecoxib was used as the standard for the analysis.
[0081] FIG 12 illustrates the percentage COX-2 inhibition by the bacopa extract composition. The standard celecoxib exhibited 91.1% COX-2 inhibition at a concentration of 100 nM. The bacopa extract composition exhibited 87.2% COX-2 inhibition at a concentration of 1 mg/ml. The bacopa extract composition exhibited a percentage COX-2 inhibition higher than that of the standard celecoxib. The synergistic effect exhibited by the bacosides, jujubogenin and ebelin lactone of the bacopa extract composition results in the inhibition of the COX-2 enzyme. The presence of bioactive components including bacosides, jujubogenin and ebelin lactone of the bacopa extract composition exhibits anti-inflammatory activity by the inhibition of COX-2 enzyme.
Example 7: Analysis of in-vitro anticancer activity of bacopa extract composition
[0082] The in-vitro anti-cancer activity was evaluated to analyze the effect of bacopa extract composition on proliferation of cancerous cells. The bioactive components of bacopa extract composition including bacosides, ebelin lactone and jujubogenin exhibit antitumor and immunomodulating properties.
[0083] The in-vitro anti-cancer activity of the bacopa 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 line MDA-MB-468 and colorectal cancer cell-line HCT-116. The concentrations of bacopa 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 4 hours, and the absorbance was measured at 570 nm.
[0084] FIG 13 illustrates the percentage inhibition of cell proliferation of MDA-MB-468 cell line by bacopa extract composition enriched with bacosides. The bacopa extract composition enriched with bacosides inhibits the proliferation of MDA-MB-468 cell line at a concentration of 0.2 mg/ml. FIG 14 illustrates the percentage inhibition of cell proliferation of MDA-MB-468 cell line by bacopa extract composition enriched with bacoside A3 and jujubogenin. The bacopa extract composition enriched with bacosides inhibits the proliferation of MDA-MB-468 cell line at a concentration of 0.03 mg/ml. FIG 15 illustrates the percentage inhibition of cell proliferation of MDA-MB-468 cell line by bacopa extract composition enriched with ebelin lactone. The bacopa extract composition enriched with ebelin lactone inhibits the proliferation of MDA-MB-468 cell line at a at a concentration of 0.21 mg/ml. FIG 16 illustrates the percentage inhibition of cell proliferation of MDA-MB-468 cell line by bacopa extract composition. The bacopa extract composition enriched with bacosides inhibits the proliferation of MDA-MB-468 cell line at a concentration of 0.11 mg/ml.
[0085] FIG 17 illustrates the percentage inhibition of cell proliferation of HCT-116 cell line by bacopa extract composition enriched with bacosides. The bacopa extract composition enriched with bacosides inhibits the proliferation of HCT-116 cell line at a concentration of 0.06 mg/ml. FIG 18 illustrates the percentage inhibition of cell proliferation of HCT-116 cell line by bacopa extract composition enriched with bacoside A3 and jujubogenin. The bacopa extract composition enriched with bacosides inhibits the proliferation of HCT-116 cell line at a concentration of 0.09 mg/ml. FIG 19 illustrates the percentage inhibition of cell proliferation of HCT-116 cell line by bacopa extract composition enriched with ebelin lactone. The bacopa extract composition enriched with ebelin lactone inhibits the proliferation of HCT-116cell line at a at a concentration of 0.055 mg/ml. FIG 20 illustrates the percentage inhibition of cell proliferation of HCT-116 cell line by bacopa extract composition. The bacopa extract composition enriched with bacosides inhibits the proliferation of HCT-116 cell line at a concentration of 0.055 mg/ml. The bacopa 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 bacopa extract composition
[0086] The analysis of toxicity indicates the safety dosage of the bacopa extract composition. The concentration of safety dosage was evaluated to analyze the therapeutic usage of the bacopa extract composition. The abcopa 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.
[0087] The safety analysis was performed by MTT assay, which indicates the mitochondrial activity of living cells. The Hek293T and human foreskin fibroblast (HFF-1) cells were incubated overnight. The incubated cells were added with the bacopa 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 48 hours and 72 hours, and the absorbance was measured at 570 nm.
[0088] FIG 21 illustrates the percentage inhibition of cell proliferation of Hek293T cell line by bacopa extract composition enriched with bacosides. The bacopa extract composition enriched with bacosides was found to be safe and non-toxic against Hek293T cell line at a concentration of 0.6 mg/ml. FIG 22 illustrates the percentage inhibition of cell proliferation of Hek293T cell line by bacopa extract composition enriched with bacoside A3 and jujubogenin. The bacopa extract composition enriched with bacosides was found to be safe and non-toxix agaisnt Hek293T cell line at a concentration of 10 mg/ml. FIG 23 illustrates the percentage inhibition of cell proliferation of Hek293T cell line by bacopa extract composition enriched with ebelin lactone. The bacopa extract composition enriched with ebelin lactone is safe and non-toxic against Hek293T cell line at a at a concentration of 0.9 mg/ml. FIG 24 illustrates the percentage inhibition of cell proliferation of Hek293T cell line by bacopa extract composition. The bacopa extract composition is safe and non-toxic against Hek293T cell line at a concentration of 0.9 mg/ml.
[0089] FIG 25 illustrates the percentage inhibition of cell proliferation of HFF-1 cell line by bacopa extract composition enriched with bacosides. The bacopa extract composition enriched with bacosides was found to be safe and non-toxic against HFF-1 cell line at a concentration of 1.026 mg/ml. FIG 26 illustrates the percentage inhibition of cell proliferation of HFF-1 cell line by bacopa extract composition enriched with bacoside A3 and jujubogenin. The bacopa extract composition enriched with bacosides was found to be safe and non-toxix agaisnt HFF-1 cell line at a concentration of 2.151 mg/ml. FIG 27 illustrates the percentage inhibition of cell proliferation of HFF-1 cell line by bacopa extract composition enriched with ebelin lactone. The bacopa extract composition enriched with ebelin lactone is safe and non-toxic against HFF-1 cell line at a at a concentration of 0.862 mg/ml. FIG 28 illustrates the percentage inhibition of cell proliferation of HFF-1 cell line by bacopa extract composition. The bacopa extract composition is safe and non-toxic against HFF-1 cell line at a concentration of 1.583 mg/ml.
[0090] The present invention discloses bacopa extract and bitterless ebelin lactone composition and a process of preparation thereof. The bacopa extract comprises enriched bacosides and aglycone derivative ebelin lactone. The bacopa extract composition comprising bacosides and enriched aglycone derivative ebelin lactone exhibits improved pharmacokinetic and biological properties. The bacosdies and aglycone derivative ebelin lactone are lipophilic as they have desired molecular weight, hydrogen-bonding capacity and molecular flexibility hence exhibt enhanced intestinal absorption and membrane permeabilty. The bacopa extract composition aids in management of neurodegenrative disorders as the bacopa extract composition is permeable and actively transported across the blood-brain barrier (BBB). The bacopa extract composition exhibits essential therapeutic properties including antioxidant, anti-cancer, anti-aceylcholinesterase and anti-inflammatory properties. The bacopa extract composition is safe and non-toxic.
, Claims:We Claim:

1. A process for the preparation of bacopa extract, the process (100) comprising steps of:
a) drying the bacopa plant material (101);
b) subjecting the dried bacopa plant material to solvent extraction with alcohol at a temperature of 70o C (102);
c) filtering the first bacopa extract through a muslin cloth (103);
d) subjecting the retentate of the bacopa extract to subsequent alcohol extraction (104);
e) concentrating the bacopa extract to a total solid content of 30% and (105);
f) subjecting the concentrated bacopa extract to drying at a temperature of 70o C (106);
g) subjecting the bacopa extract to solvent extraction with alcohol and water and precipitating for 8 to 10 hours (107);
h) filtering the precipitate in filter press and tray drying at a temperature of 90o C (108);
i) washing the bacopa extract with an organic solvent (109);
j) subjecting the bacopa extract to acid hydrolysis with a mineral acid for 4 hours at a temperature of 90o C (110);
k) subjecting the bacopa extract to cooling and adding water at a ratio of 1:1 to precipitate (111);
l) subjecting the precipitate to filtration and washing with demineralized water (112); and
m) crystallizing the precipitate with alcohol at a temperature range of 50o C to 65o C and drying at a temperature of 70o C (113).

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

3. The process (100) as claimed in claim 1, wherein said drying of the bacopa extract is achieved by a spray dryer at a temperature range of 90o C-110o C and at a flow rate of 100 L/hour.

4. The process (100) as claimed in claim 1, wherein said ratio of alcohol to water is 1:3.

5. The process (100) as claimed in claim 1, wherein said organic solvent is selected from a group of chloroform, ethyl acetate, and acetone.

6. The process (100) as claimed in claim 1, wherein said mineral acid is selected from a group of hydrochloric acid, sulphuric acid, and orthophosphoric acid at a concentration of 2N sulphuric acid.

7. A composition of bacopa extract and bitterless ebelin lactone, the composition comprising:

a) a bacoside at a concentration range of 20% to 50%; and
b) an ebelin lactone at a concentration of 5%;
wherein said bacoside and ebelin lactone are in the ratio of 3:1 to 99:1.
8. The composition as claimed in claim 7, wherein said bacopa extract composition exhibits apparent permeability coefficient of 141 ± 18.7cm/s with an efflux ratio of 0.54 and complete absorption at a time interval of 15 minutes by the cancer coli-2 (Caco-2) cell line.

9. The composition as claimed in claim 7, wherein said bacopa extract composition inhibits 85% acetylcholine esterase (AChE) activity at a concentration of 1 mg/ml.

10. The composition as claimed in claim 7, wherein said bacopa extract composition inhibits in vitro cell cytotoxicity in kidney epithelial (Hek293T) cell line at a concentration of 0.9 mg/ml against human foreskin fibroblast (HFF-1) cell line at a concentration of 1.583 mg/ml.