DESC:Priority Claim:

[0001] This application claims priority from the provisional application numbered 202221069283 filed with Indian Patent Office, Chennai on 1st December 2022 and post-dated to 1st June 2023 entitled “A composition to enhance the bioavailability and efficacy of Bacopa”, the entirety of which is expressly incorporated herein by reference.
Preamble to the description
[0002] 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
[0003] The present invention relates to a composition to enhance the bioavailability and the efficacy of bacosides. More particularly, the invention relates to a composition of bacoside-calcium complex, which has ability to cross the Blood-Brain-Barrier (BBB) thus increasing the bioavailability and efficacy of bacosides. The invention also discloses a process for preparation of the composition by complexing bacosides with the mineral source.
Background of the invention
[0004] Bacosides are the putative bioactive components of the Indian medicinal plant Bacopa monnieri. Bacoside, being a folk-medicinal substance believed to exhibit therapeutic properties, particularly enhancing cognitive functions and improving memory.
[0005] Bacopa monnieri, popularly called Brahmi is a water hyssop, thyme-leaved gratiola, and herb of grace, is a staple plant in traditional Ayurvedic medicine. The beneficial and therapeutic effects of Bacopa monnieri are attributed due to the presence of the bacosides.
[0006] Bacosides are used as a medicine for mental disorders and loss of memory. They also exert its activity with other pharmacological properties such as antioxidant, antidepressant, antiulcer, hepatoprotective, anticancerous, vasodilator, smooth muscle relaxant, mast cell stabilizer, and various other functions. Increasing clinical trials indicate the potential role of bacosides even in Alzheimer’s disease and in epilepsy.
[0007] Bacosides attribute to the neuroprotective function mainly through modulating antioxidant enzymes, namely, Superoxide Dismutase (SOD), catalase, etc. Bacosides also regulate the levels of different neurotransmitters in the brain.
[0008] In spite of vast therapeutic activities, the use of bacosides is challenging because of poor absorption either due to their large molecular size or due to their low lipid solubility, which cannot be absorbed by passive diffusion. As a result, there is a limitation in their ability to transport across lipid-rich biological membranes, thus resulting in their poor bioavailability.
[0009] In order to overcome this, various technological strategies are reported in the literature, including micronization, solid dispersions and microencapsulation etc.
[0010] Bioavailability as well as binding of bacosides to the receptors is controlled by the Blood Brain Barrier (BBB). However, nano conversion of these drug candidates easily resolves the BBB restriction and carries a promising role in future therapies.
[0011] One of the available formulations Bacognize® is a Bacopa extract standardized to bacosides for adaptogenic support targeting cognitive wellness, memory, learning, focus, attention, mood, stress management, emotional balance, and circadian rhythm support for improved quality of life and quality of sleep. Bacosides are complexed with beta-cyclodextrin, and encapsulation technology is employed.
[0012] Similarly, the formulation Cognique® is prepared through the preservation of bacosides with complete multiple natural matrices including gingerols and rosemary extracts containing rosemary essential oil, rosmarinic acid and carnosic acid by using Polar-Nonpolar-Sandwich (PNS) technology. Cognique® is utilized as a natural nutritional supplement for supporting neuroprotective effect and cognition improvement through maintenance of gut-brain axis.
[0013] However, among the available technologies, the self-micro-/nano-emulsifying drug delivery systems have attracted more attention due to its improved oral bioavailability that allows for reduced dosing more consistent with temporal drug absorption profiles, selective drug targeting to a specific absorption window in the gastrointestinal tract (GIT), and protection of molecules from the hostile environment in the gut. However, compared to traditional metastable emulsions, self-nano emulsifying drug delivery system is a thermodynamically stable formulation with high solubilization capacity for lipophilic drugs and may also be filled directly into soft or hard gelatin capsules for easy oral administration.
[0014] The blood vessels that vascularize the central nervous system (CNS) possess unique properties, termed the blood–brain barrier, which allow these vessels to tightly regulate the movement of ions, molecules, and cells between the blood and the brain. The selective transport of molecules in BBB represents a major obstacle to the delivery of active compounds to the CNS. Thus, overcoming the struggle of transporting therapeutic agents to specific regions of the brain through the use of different technologies plays a major challenge for treatment of most brain disorders.
[0015] The Patent Application No. WO2017021974A2 entitled “Novel and synergistic composition of lecithin and lysolecithin for improving bioavailability and solubility of hydrophobic compounds and extracts” discloses composition of hydrophobic plant molecule and/or extract with enhanced bioavailability comprising hydrophobic active and synergetic mixture of lecithin and lysolecithin and to the process for preparation.
[0016] The Patent Application No. IN201721019368A entitled “Microemulsion compositions of herbal extracts using food grade oils for enhancement in oral bioavailability” discloses micro-emulsion or nano emulsion, self-emulsifying formulation including at one or more of an active ingredient. The active ingredient includes one or more of a food grade oil mixed with an aqueous phase along with one or more of a surfactant and one or more of a co-surfactant with by an extract of one or more of an active herbal constituent. The dissolution study in-vitro showed sufficient release of herbal actives from formulation could be dissolved in pH 1.2 or pH 6.8 buffer solutions. Validated RP-HPLC methods were used for quantifying in vitro and in vivo samples for estimation of the concentrations of the actives for oral bioavailability of the developed formulation versus the commercially available formulation and pure extracts was done by oral administration in Sprague Dawley rats in vivo.
[0017] The unique approach of mineral complexation is the technology behind complexing triterpenoid saponins especially bacosides using mineral source for making bacosides more bio-absorbable or “bioavailable” to the body, without changing their molecular form and structure.
[0018] Hence, there is a need for a composition of bacosides which overcomes the challenge of passage through blood brain barrier to enhance the bioavailability thus executing the enhanced therapeutic activity of bacosides.
Summary of the Invention
[0019] The present invention discloses a composition to enhance the bioavailability and the efficacy of bacosides. The invention relates to a composition of bacosides complexed with a mineral source to increase the permeability through the Blood-Brain-Barrier (BBB) thus increasing the bioavailability and efficacy of bacosides.
[0020] The composition of the present invention comprises a combination of bacosides at a concentration in a range between 15% to 60% w/w, calcium as mineral source at a concentration in a range between 0.5% to 5% w/w and the non-bacoside mixture including but not limited to flavonoids, sterols, terpenes, polyphenols, glycosides etc. at a concentration in a range between 40% to 80% w/w. The complex is prepared using methyl alcohol as a solvent at a ratio of 1:0.5 to 1:50 w/v and the mineral source between 1:0.1 to 1:10 moles with respect to bacoside respectively in an alkaline pH medium.
[0021] The invention further discloses a process for preparation of the composition by complexing bacosides with the mineral source.
[0022] The process of complexation according to embodiment of the invention is initiated by extracting bacosides from whole plant of Bacopa monnieri using methyl alcohol as solvent. The extraction is achieved at room temperature and for 3 cycles. The alcoholic extract thus formed is subjected to liquid extraction using combination of butanol and water resulting in the formation and separation of butanol fraction and aqueous fraction. The butanol fraction is concentrated to obtain butanol extract, which is subjected to purification using acetone at room temperature. This results in the formation of two fractions namely acetone soluble and acetone insoluble fractions. The acetone insoluble fraction thus obtained comprises more than 50% of bacosides comprising bacoside A3, bacopaside I, bacopaside II, bacopaside X and bacopasaponin C. Bacosides are subjected to complexation with calcium at a concentration in a range between 0.5% to 5% w/w using alcohol as solvent and in alkaline pH medium. Bacopa extract rich in bacosides is added to a 500 ml round-bottom flask containing 130 ml of ethanol and mixture is stirred for 15 minutes such that the solid bacosides are completely dissolved. pH of the mixture is adjusted to a range between 8.2 – 8.8 using sodium methoxide and the mineral source solution is added to the mixture. The reaction mixture is refluxed for a duration of 8 hours resulting in the formation of Bacopa-calcium complex, which is collected by filtration, washed using a mixture comprising ethanol and water at a ratio of 1:3 and finally the complex is dried using a vacuum tray dryer.
[0023] The uniqueness of the present invention is complexation of bacoside with mineral source in order to enhance the bioavailability and efficacy of bacosides. The bacoside-calcium complex is the mineral complex of bacoside, which is capable of crossing the blood-brain-barrier because of enhanced permeability and also exhibits high efficacy with small doses.
[0024] Bacopa composition of the present invention exhibited improved permeability using paracellular marker 100 µg/mL of lucifer yellow in colon carcinoma cell line. The composition also exhibited improved pharmacokinetic profile with respect to the pharmacokinetic parameters such as peak plasma concentration (C max), Time to peak Concentration (t max), Area under the plasma concentration-time curve (AUC 0-t and AUC 0-8), elimination rate constant (Keli), Elimination half-life (t ½) are calculated based on plasma concentration over time.
[0025] The bacoside-calcium complex of the present invention is also analyzed for therapeutic activity with respect to the antioxidant and anti-inflammatory activities. The bacoside-calcium complex of the present invention exhibited antioxidant activity against 2, 2-axino-bis (3-ethylbenzothiazoline-6-sulfonic acid) diammonium salt (ABTS) and 2, 2-Diphenly 1-picryl hydroxyl solution (DPPH) free radical’s method in rat glial cells.
[0026] Further, the bacoside-calcium complex of the present invention at a concentration of 500 µg/ml and 1000 µg/ml exhibited dose dependent inhibition of TNF-alpha in rat macrophage cell line thus proving the anti-inflammatory activity.
[0027] The mineral complexation of the present invention is effective in enhancing the bioavailability of bacosides and to improve the permeability of bacosides through blood brain barrier, which allows a selective transport of molecules and for selective delivery of drugs or molecules to the central nervous system.
Brief description of the drawings
[0028] 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.

[0029] FIG 1 tabulates the composition according to an embodiment of the invention.
[0030] FIG 2 illustrates a flowchart for a process of preparation of bacoside composition by complexation of bacosides.
[0031] FIG 3 illustrates the results of quantification of bacosides in Bacopa extract and bacoside-calcium complex of the present invention.
[0032] FIG 4 illustrates the results of evaluation of calcium in Bacopa extract and bacoside-calcium complex of the present invention.
[0033] FIG 5 interprets the results of FTIR spectrum of Bacopa extract and bacoside-calcium complex of the present invention.
[0034] FIG 6 interprets the results of DSC analysis of Bacopa extract and bacoside-calcium complex of the present invention.
[0035] FIG 7 interprets the results of TGA analysis of Bacopa extract and bacoside-calcium complex of the present invention.
[0036] FIG 8 indicates the permeability of Bacopa composition of the present invention in colon carcinoma Caco-2 cell line.
[0037] FIG 9 tabulates the pharmacokinetic parameters of Bacopa composition of the present invention in Wistar albino rats.
[0038] FIG 10 tabulates the Bacoside levels in brain homogenates of Wistar albino rats.
[0039] FIG 11A illustrates the effect of Bacopa composition of the present invention against 2, 2-axino-bis (3-ethylbenzothiazoline-6-sulfonic acid) diammonium salt.
[0040] FIG 11B illustrates the effect of Bacopa composition of the present invention against DPPH.
[0041] FIG 12 tabulates the anti-inflammatory activity of Bacopa composition of the present invention.
Detailed Description 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 “Bioavailability” refers to the extent a substance or drug becomes completely available to its intended biological destination.
[0044] The term “Bacoside” refers to class of chemical compounds as dammarane-type triterpenoid saponins isolated from Bacopa monnieri.
[0045] The present invention discloses a composition to enhance the bioavailability and the efficacy of bacosides. The invention relates to a composition of bacosides combined with a mineral source to increase the permeability through the Blood-Brain-Barrier (BBB) thus increasing the bioavailability and efficacy of bacosides. The invention also discloses a process for preparation of the composition by complexing bacosides with the mineral source.
[0046] The composition of the present invention comprises bacosides namely bacoside A3, bacopaside I, bacopaside II, bacopaside X, bacopasaponin C, a mineral and an alcohol as solvent.
[0047] FIG 1 tabulates the composition according to an embodiment of the invention. The composition comprises a combination of bacosides at a concentration in a range between 15% to 60% w/w, calcium as mineral source at a concentration in a range between 0.5% to 5% w/w and the non-bacoside mixture including but not limited to flavonoids, sterols, terpenes, polyphenols, glycosides etc. at a concentration in a range between 40% to 80% w/w. The complex is prepared using alcohol as a solvent at a ratio of 1:0.5 to 1:50 w/v and the mineral source between 1:0.1 to 1:10 moles with respect to bacoside respectively in an alkaline pH medium.
[0048] The composition of the present invention is a complex of bacosides with a mineral source using the mineral complexation process. The complexation of bacoside with mineral is unique to the present invention to enhance the bioavailability of bacoside.
[0049] The structure of bacoside is complex saponin derivative with active hydroxyl groups. This creates a barrier to permeability of bacoside due to high molecular weight, which is overcome by the complexation of bacoside with the mineral source. Due to positive charge of the mineral source and upon complexation, electrostatic interactions are formed and is associated with Vander wall force of attraction between the mineral source and the hydroxyl groups of the bacosides. Further, the presence of combination of bacosides in the matrix, weak forces of attraction is created that results a complexation between the bacosides and the mineral source.
[0050] The present invention also discloses a process of complexation of bacosides. The bacosides are complexed with a mineral source to enhance the bioavailability of bacosides.
[0051] FIG 2 illustrates a flowchart for the process of preparation of bacoside composition by complexation of bacosides. The process (200) starts with step (201) of extracting bacosides from Bacopa monnieri. The whole plant powder of Bacopa monnieri is extracted using methyl alcohol as solvent. The extraction is achieved at room temperature and for 3 cycles. At step (202), the alcoholic extract thus formed is subjected to liquid extraction using combination of butanol and water. This step results in the formation and separation of butanol fraction and aqueous fraction. At step (203), the butanol fraction is concentrated to obtain butanol extract. At step (204), the butanol extract is subjected to purification using acetone at room temperature, which results in two fractions namely acetone soluble and acetone insoluble fractions. At step (205), the acetone insoluble fraction thus obtained comprises more than 50% of bacosides. The bacosides comprises bacoside A3, bacopaside I, bacopaside II, bacopaside X, and bacopasaponin C.
[0052] The bacoside fraction thus obtained exhibits low bioavailability. In order to enhance the bioavailability, bacosides are subjected to complexation with the mineral source especially calcium at a concentration in a range between 0.5% to 5% w/w using alcohol as solvent and in alkaline pH medium. At step (206), Bacopa extract rich in bacosides is added to a 500 ml round-bottom flask containing 130 ml of ethanol. At step (207), the mixture is stirred for 15 minutes such that the solid bacosides are completely dissolved. At step (208), the pH of the mixture is adjusted to a range between 8.2-8.8 using sodium methoxide. At step (209), the mineral source solution is added to the mixture. The mineral source solution is prepared by dissolving 1 mmol of calcium in 20 ml of ethanol. At step (210), the reaction mixture is refluxed for a duration of 8 hours resulting in the formation of Bacopa-calcium complex. Finally, at step (211), the Bacopa-calcium complex is collected by filtration and subjected to washing using a mixture comprising ethanol and water at a ratio of 1:3 and finally the complex is dried using a vacuum tray dryer.
[0053] The bacoside-calcium complex prepared according to the process of the present invention exhibits enhanced bioavailability and the efficacy than the natural bacoside extract without affecting the stability. The bacoside-calcium complex is the mineral complex of bacoside, which is capable of crossing the blood-brain-barrier because of enhanced permeability and also exhibits high efficacy with small doses.
[0054] The bacoside-calcium complex of the present invention is analyzed for the quantification of bacosides. Bacosides namely bacopaside I, bacoside A3, bacopaside II, bacopaside X and bacopasaponin C are quantified in Bacopa extract and bacoside-calcium complex of the present invention. The quantification is performed using HPLC using column LC column USP method. The mobile phase of the analysis comprises Solution A comprising 0.14 g anhydrous potassium dihydrogen phosphate and 0.5 mL of phosphoric acid in 1 L of water and acetonitrile as Solution B. The detection is carried out in an isocratic flow at a wavelength of 205 nm using a photodiode array detector and the elution is carried out using isocratic method with 1.5 mL/min flow rate.
[0055] FIG 3 illustrates the results of quantification of bacosides in Bacopa extract and bacoside-calcium complex of the present invention. The HPLC analysis indicated the presence of bacosides such as bacopaside I, bacoside A3, bacopaside II, bacopaside X and bacopasaponin C in Bacopa extract and bacoside-calcium complex of the present invention.
[0056] The bacoside-calcium complex of the present invention is evaluated for the total calcium content using Inductively Coupled Plasma Optical Emission Spectrometry (ICP-OES). The ICP-OES instrument is used for elemental analysis after selecting a suitable wavelength and calibration using standard solutions from 0.05 to 10?mg L-1. The Bacopa extract and bacoside-calcium complex of the present invention with a blank are introduced into the plasma and analyzed for calcium content. Bacoside-calcium complex is prepared using different ratio of calcium with bacosides namely Bacoside-calcium complex I in a ration 1:0.5, Bacoside-calcium complex I in a ration 1:1 and Bacoside-calcium complex I in a ration 1:2.
[0057] FIG 4 illustrates the results of evaluation of calcium content in Bacopa extract and bacoside-calcium complex of the present invention. The results confirmed the presence of calcium and percentage of calcium is dependent on the ration of calcium used in the preparation of the complex.
[0058] The Bacopa extract and bacoside-calcium complex of the present invention is analyzed using different techniques such as Fourier-transform infrared spectroscopy (FTIR), Differential Scanning Calorimetry (DSC) analysis the Thermal Gravimetric Analysis (TGA).
[0059] 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: Analysis of Bacopa extract and bacoside-calcium complex of the present invention using FTIR.
[0060] The Bacopa extract and bacoside-calcium complex of the present invention are subjected to FTIR analysis.
[0061] FIG 5 interprets the results of FTIR spectrum of Bacopa extract and bacoside-calcium complex of the present invention. The FTIR analysis spectrum of the Bacopa extract interpreted a broad band at 3432 cm-1 (–OH stretching), bands at 2925 cm-1 and 2855 cm-1 (C–H stretching), and a band at 1730 cm-1 (carbonyl group stretching of an aliphatic ketone in the bacoside molecule). Also, bands at 1616 cm-1 and 1440 cm-1 correspond to C=C and C–C stretching, respectively. Further, the presence of additional bands at 1368 cm-1, 1162 cm-1, and 1264 cm-1 attributed to –OH bending, C–O ether stretching, and C–O aliphatic stretching. The bands at 1076 cm-1 and 1028 cm-1 indicated C–O stretching in the furanosyl and pyranosyl groups of the bacosides.
[0062] The FTIR analysis spectrum of the bacoside-calcium complex of the present invention Bacoside-calcium complex I in a ration 1:0.5, Bacoside-calcium complex I in a ration 1:1 and Bacoside-calcium complex I in a ration 1:2 displayed similar characteristic bands confirming the presence of bacoside. In addition, the reduction in band intensity indicated interaction of bacosides with calcium.
Example 2: Analysis of thermal stability of Bacopa extract and bacoside-calcium complex of the present invention using DSC.
[0063] The Bacopa extract and bacoside-calcium complex of the present invention are analyzed for thermal stability using DSC to analyze the interactions between bacosides and calcium.
[0064] FIG 6 interprets the results of DSC analysis of Bacopa extract and bacoside-calcium complex of the present invention. The Bacopa extract and bacoside-calcium complexes Bacoside-calcium complex I in a ration 1:0.5, Bacoside-calcium complex I in a ration 1:1 and Bacoside-calcium complex I in a ration 1:2 exhibited significant differences in melting endotherms. The results indicated that Bacopa extract exhibited decreased melting point from 284°C to 268°C in its calcium complex form. Sharp peaks at 284.51°C are noted in the Bacopa extract and the FIG indicates a noticeable reduction in the height of the endothermic peak and the heat of fusion, suggesting a transition in the physical state from crystalline to amorphous.
Example 3: Analysis of thermogravimetric behavior of Bacopa extract and bacoside-calcium complex of the present invention using TGA
[0065] Bacopa extract and bacoside-calcium complex of the present invention are analyzed for thermogravimetric behavior using TGA.
[0066] FIG 7 interprets the results of TGA analysis of Bacopa extract and bacoside-calcium complex of the present invention. The TGA spectra of Bacopa extract exhibited a weight loss of over 50% at approximately 330.41°C. In contrast, Bacoside-calcium complex showed a weight loss of 55.50% at around 313.66°C. These findings indicate that the thermal stability of bacoside-calcium complexes Bacoside-calcium complex I in a ration 1:0.5, Bacoside-calcium complex I in a ration 1:1 and Bacoside-calcium complex I in a ration 1:2 exhibited is assessed alongside the Bacopa extract.
[0067] The Bacopa extract and bacoside-calcium complex is subjected to accelerated stability. This is achieved by subjecting the both the samples to simulated conditions of long-term storage using elevated temperature and humidity. The results indicated that both Bacopa extract and bacoside-calcium complex are stable under accelerated conditions suggesting the robustness and suitability for use of bacoside-calcium complex over extended periods.
Example 4: Evaluation of permeability of Bacoside-calcium complex of the present invention in the Caco-2 cell line
[0068] Bacoside-calcium complex of the present invention is evaluated for the permeation in colon carcinoma Caco-2 cell line by analyzing the in vitro permeation co-efficient. Caco-2 cells are seeded onto inserts (0.4µ) and are cultured for 25 days and once the membrane integrity was established, the permeability coefficient of the Bacopa composition of the present invention is evaluated after 0 minutes and 120 minutes incubation with the test substance in a uni-directional manner using HPLC method.
[0069] 10 mg of the Bacoside-calcium complex of the present invention is dissolved in DMEM-HG supplemented with 2% inactivated Fetal Bovine Serum (FBS), and volume is made up with media to obtain a stock solution of 1 mg/ml concentration and sterilized by filtration.
[0070] The cytotoxicity of the Bacoside-calcium complex of the present invention is analyzed by assessing the cell viability. Bacopa composition is added to cells and incubated at 37°C in 5% carbon dioxide atmosphere.
[0071] The permeability of the Bacoside-calcium complex of the present invention is assessed using paracellular marker 100 µg/mL of lucifer yellow, which is an indicator for monolayer integrity. The permeability of the monolayers to lucifer yellow is assessed using a microplate reader. The permeability coefficient of all the samples were determined using the standard formula.
[0072] FIG 8 indicates the permeability of Bacoside-calcium complex of the present invention in colon carcinoma Caco-2 cell line. The results indicated that Bacopa-calcium complex of the present invention exhibited significantly higher permeability by 3, 2 and 1.5 folds over the Bacopa extract 1, Bacopa extract 2 and Bacopa extract 3 respectively.
Example 5: Evaluation of bioavailability of Bacoside-calcium complex of the present invention in Wistar albino rats
[0073] The Bacoside-calcium complex of the present invention is evaluated for the bioavailability by analyzing the pharmacokinetic profile in Wistar albino rats. The rats are divided in to two groups containing 6 animals in each group. Group–I received Bacopa extract at 1000 mg/kg and Group –II received Bacopa-calcium complex of the present invention at 1000 mg/kg body weight of the animals. After administration of test substances, 500µL of blood sample is collected at different time intervals of 0 min, 15 min, 1 hour, 2 hours, 4 hours, 6 hours, 8 hours and 12 hours. Bioanalysis is performed using HPLC method for the quantification of the reference standard in rat plasma.
[0074] The pharmacokinetic parameters such as peak plasma concentration (C max), Time to peak Concentration (t max), Area under the plasma concentration-time curve (AUC 0-t and AUC 0-8), elimination rate constant (Keli), Elimination half-life (t ½) are calculated based on plasma concentration over time and are tabulated as in FIG 9.
[0075] The results indicated that Bacopa-calcium complex of the present invention exhibited the highest peak concentration (Cmax) of bacosides in both the plasma (73.35 µg/ml) as well as in the brain homogenates (1.0112 µg/ml) when compared to Bacopa extract.
[0076] Further, Bacopa-calcium complex of the present invention exhibited higher bioavailability when compared to the Bacopa extract as measured in terms of AUC0-t and AUC0-inf both in terms of plasma concentration and the brain homogenate concentration of Bacosides. The relative oral bioavailability of Bacopa-calcium complex of the present invention when measured in terms of the mean Cmax achieved in the plasma is 26.65% higher at 73.35 µg/ml compared to the Bacopa extract measured at 41.52 µg/ml.
[0077] FIG 10 tabulates the Bacoside levels in brain homogenates of Wistar albino rats. The results indicated that maximum concentration of Bacopa-calcium complex of the present invention is 73.35 µg/ml at 4th hour and maximum concentration of Bacopa extract is 41.52 µg/ml at 4th hour. The mean concentration of Bacopa extract is 1.0007 µg/ml as detected in brain and mean concentration of Bacopa-calcium complex of the present invention is 1.0112 µg/ml as detected in brain interpreting that Bacopa-calcium complex of the present invention showed statistically significant higher bioavailability in plasma when compared to the Bacopa extract.
[0078] The Bacopa-calcium complex of the present invention exhibits increased permeability.
[0079] The Bacopa-calcium complex of the present invention is analyzed for efficacy with respect to antioxidant and anti-inflammatory properties.
Example 6: Evaluation of antioxidant activity of the Bacoside-calcium complex of the present invention in rat glial (C6) cell line
[0080] The Bacopa-calcium complex of the present invention is evaluated for the antioxidant activity against lipopolysaccharide induced cell damage in rat glial cell line.
[0081] The modulation of antioxidant properties is estimated by determining the levels of antioxidant enzymes reduced glutathione and superoxide dismutase against LPS induced cell damage. The sample is prepared by weighing 10 mg of Bacopa composition and dissolved in DMEM medium supplemented with 2% inactivated Fetal Bovine Serum (FBS) to obtain a stock solution of 10 mg/mL.
[0082] The rat glial cells are cultured in DMEM supplemented with 10% inactivated FBS, 100 IU/mL penicillin, 100 ?g/mL streptomycin and 5 ?g/mL amphotericin B in an humidified atmosphere of 5% CO2 at 37?C until confluent. The cells are dissociated with Trypsin-EDTA solution containing 0.2% trypsin, 0.02% EDTA and 0.05% glucose in PBS. The cell cultures are grown in 25 cm2 culture flasks and all experiments are carried out in 96 microtiter plates.
[0083] Glial cells are grown to confluency in 60mm petri dishes. The Bacopa-calcium complex at concentrations of 250 µg/ml and 500 µg/ml along with lipopolysaccharide as control to the culture except untreated cell control and incubated for 24 hours. The cells are washed after incubation and centrifuged at 1000g for 10 minutes. The supernatant is discarded and the sediment is retained. The cells are homogenized in Phosphate Buffered Saline (PBS) by sonication for 10 minutes. Cell homogenates are then subjected to freeze thaw cycle followed by centrifugation at 1500g for 10 minutes at 4 °C. The clear supernatant is collected, which is used for determination and the protein concentration of the supernatant is determined by Bradford Protein assay using bovine serum albumin as a protein standard.
[0084] The cells are analyzed for Glutathione peroxidase (GSH-Px), which promotes the reaction of hydrogen peroxide and reduced glutathione to produce hydrogen peroxide and oxidized glutathione. Glutathione peroxidase reacts with dinitro benzoic acid to produce 5-thio-dinitrobenzoic acid anion, which is analyzed by a stable yellow color measured at 412 nm.
[0085] The activity of superoxide dismutase is analyzed by catalyzing the xanthine oxidase reaction to generate water soluble formazan dye and the activity of superoxide dismutase is negatively correlated with the amount of formazan dye which is measured at 450 nm.
[0086] Lipopolysaccharide induced cell damage is associated with free radical injury and oxidative stress, which is characterized by increased lipid peroxidation and altered non-enzymatic and enzymatic antioxidant systems. Further, lipopolysaccharide also resulted in decrease in activities of glutathione peroxidase and superoxide dismutase indicating stress in glial cells.
[0087] The effect of Bacopa-calcium complex of the present invention against 2, 2-axino-bis (3-ethylbenzothiazoline-6-sulfonic acid) diammonium salt (ABTS) and 2, 2-Diphenly 1-picryl hydroxyl solution (DPPH) free radical’s method in rat glial cells.
[0088] Bacopa-calcium complex at a concentration of1000µg/ml is analyzed for radical scavenging activity. 0.2 ml of Bacopa-calcium complex and ascorbic acid as standard are taken separately in Eppendorf tube. To this 1.0 ml of PBS and 0.16 ml of ABTS solution is added to make a final volume of 1.36 ml. For control, 0.2ml of methanol is taken in place of test substance and 0.16ml of distilled water is taken for test blank and control blank. The tubes are incubated for 20 minutes. After incubation 0.1ml of reaction mixture is pipetted out to microtiter plate. The absorbance is measured in ELISA reader at 734nm and values are recorded. The procedure is repeated for standard by replacing test sample with standard. Test and control are performed in triplicate and test blank and control blank are conducted in singlet.
[0089] FIG 11A illustrates the effect of Bacopa-calcium complex of the present invention against 2, 2-axino-bis (3-ethylbenzothiazoline-6-sulfonic acid) diammonium salt. Bacopa-calcium complex exhibited higher antioxidant activity when compared to Bacopa extract with IC50 value 862.06±14.11 µg/ml against ascorbic acid exhibited activity with IC50 value 91.68±4.15 µg/ml.
[0090] Bacopa-calcium complex at a concentration of1000µg/ml is analyzed for radical scavenging activity. 0.01ml of Bacopa-calcium complex are loaded into 96 well micro titer plate and standard is added separately in the test and test blank wells. 0.01ml of DMSO is taken for control and control blank. 0.2 ml of DPPH is added to the test and control, whereas to the test blank and control blank 0.2ml of methanol is added in place of DPPH. The tubes are incubated for 20 minutes. After incubation 0.1ml of reaction mixture is pipetted out to microtiter plate. Test and control are performed in triplicate and test blank and control blank are conducted in singlet. The micro titer plate is incubated at 37°C for 30 minutes and absorbance is measured at 490 nm using microplate reader.
[0091] FIG 11B illustrates the effect of Bacopa-calcium complex of the present invention against DPPH. Bacopa-calcium complex and Bacopa extract exhibited antioxidant activity with IC50 values 114.23±7.25µg/ml and 341.60±9.90µg/ml, respectively against the standard ascorbic acid exhibited activity with IC50 value 78.85±1.06 µg/ml. The results indicated better antioxidant activity of Bacopa-calcium complex with low IC50 value.
[0092] The results interpreted that the Bacopa extract and Bacopa-calcium complex increased the activities of glutathione peroxidase and superoxide dismutase. Further, Bacopa-calcium complex exhibited better activity with higher levels of glutathione peroxidase and superoxide dismutase by neutralizing the lipopolysaccharide induced stress in glial cells.
Example 7: Evaluation of anti-inflammatory activity of the Bacoside-calcium complex of the present invention in rat macrophage cell line
[0093] The Bacopa composition of the present invention is evaluated for the anti-inflammatory activity against lipopolysaccharide induced Tumor Necrosis Factor- a (TNF-a) production and nitric oxide inhibition in rat macrophage cell line (RAW 264.7).
[0094] The study is performed by seeding macrophage cell line into 6 well culture dishes at a cell population 1.5x105 cells/ml in DMEM with 10% FBS. After 24 hours. The cells are treated with known non-toxic concentration of Bacopa-calcium complex of the present invention at a concentration of 500 µg/ml and 1000 µg/ml along with 1?g/mL of lipopolysaccharide and incubated at 37 °C with 5% CO2 for 24 hours. The culture media or supernatant and cells are collected separately after the treatment. The level of TNF-a in the supernatant is determined by ELISA using specific mouse antibody and ELISA Kit. The level of nitric oxide is determined by nitric oxide colorimetric assay kit.
[0095] FIG 12 tabulates the anti-inflammatory activity of Bacopa-calcium complex the present invention. The results indicated that Bacopa-calcium complex of the present invention at a concentration of 500 µg/ml and 1000 µg/ml exhibited dose dependent inhibition of TNF-alpha in rat macrophage cell line. The Bacopa-calcium complex of the present invention and Bacopa extract exhibited significant protection by 64.48% and 90% at dose 1000µg/ml over control in rat macrophage cell line.
[0096] Further, the Bacopa-calcium complex of the present invention and Bacopa extract exhibited reduction of nitric acid by 80.94% and 91.63% at dose 1000µg/ml over control in rat macrophage cell line.
[0097] The mineral complexation of the present invention is the unique approach to enhance the bioavailability of bacosides and to improve the permeability of bacosides through blood brain barrier, which allows a selective transport of molecules and for selective delivery of drugs or molecules to the central nervous system. The complex allows the co-delivery of bacosides, and the mineral source synergistically improve the passage across the blood brain barrier with enhanced bioavailability and targeted delivery of the actives to the intended site of action in the brain and maintain gut brain axis.
[0098] The Bacoside-calcium complex of the present invention enhances the stability of bacosides, increases the bioavailability, improving the permeability through blood brain barrier finally enhancing the efficacy of bacosides with low doses without inducing any side effects.
,CLAIMS:
We Claim,

1. A composition of Bacopa monnieri mineral complex, the composition comprises:
a. bacosides at a concentration in a range between 15% to 60% w/w;
b. calcium as mineral source at a concentration in a range between 0.5% to 5% w/w; and
c. non-bacoside mixture at a concentration in a range between 40% to 80% w/w.

2. The composition as claimed in Claim 1, wherein bacosides comprises bacoside A3, bacopaside I, bacopaside II, bacopaside X and bacopasaponin C.

3. The composition as claimed in Claim 1, wherein the non-bacoside mixture comprises flavonoids, sterols, terpenes, polyphenols and glycosides.

4. The composition as claimed in Claim 1, wherein methyl alcohol is used at a ratio of 1:0.5 to 1:50 w/v with a mineral source between 1:0.1 to 1:10 moles with respect to bacoside respectively and in an alkaline pH medium.

5. The composition as claimed in Claim 1, wherein bacoside-calcium complex is the mineral complex of bacoside and is capable of crossing the blood-brain-barrier because of enhanced permeability.
6. A process for preparation of the Bacopa monnieri composition, the method comprising the steps of:
a. extracting one or more bacosides from Bacopa monnieri;
b. subjecting the extracted bacosides to liquid extraction using combination of butanol and water to result in butanol fraction and aqueous fraction);
c. concentrating the butanol fraction to butanol extract; and
d. subjecting the butanol extract to purification using acetone at room temperature, resulting in two fractions acetone soluble and acetone insoluble fractions;
e. acetone insoluble fraction thus obtained comprises 50% of bacosides;
f. complexation of bacosides with a mineral source at a concentration in a range between 0.5% to 5% w/w by adding Bacopa extract rich in bacosides to a 500 ml round-bottom flask containing 130 ml of ethanol;
g. stirring the mixture is stirred for 15 minutes such that the solid bacosides are completely dissolved;
h. adjusting the pH of the mixture to a range between 8.2-8.8 using sodium methoxide;
i. adding the mineral source solution to the mixture wherein the solution is prepared by dissolving 1 mmol of mineral in 20 ml of ethanol;
j. refluxing the reaction mixture for a duration of 8 hours resulting in the formation of Bacopa-calcium complex;
k. collecting the Bacopa-calcium complex by filtration and subjecting to washing using a mixture comprising ethanol and water at a ratio of 1:3 and drying the complex using a vacuum tray dryer.

7. The process as claimed in Claim 6, wherein whole plant powder of Bacopa monnieri is extracted using methyl alcohol as solvent.

8. The process as claimed in Claim 6, wherein extraction is achieved at room temperature and for at least 3 cycles.

9. The process as claimed in Claim 6, wherein the mineral source is calcium.

10. The process as claimed in Claim 6, wherein the complexation of bacosides with calcium enhances the bioavailability by increasing the permeability.