FIELD OF INVENTION;
The present invention relates to the field of Bacopa mormieri extract comprising of a defined percentage of bacoside. More specifically the present invention describes a method for obtaining an extract of Bacopa monneiri rich in bacoside, with therapeutic properties.
BACKGROUND AND PRIOR ART;
Bacopa monnierii plant contains crystalline compound and its one of the components that crosses the blood brain barrier. The occurrence of two saponins designated as Bacoside A & B has bee reported which are present in a concentration of over 2% in the dry plant (Chatterji N., Rastogi R.P. and Dhra, M.L. (1963). Chemical Examination of Bacopa monniera wettst: Part I-Isolation of Chemical Constituents, Indian J. Chetn., 1, 212-215). Bacopa monniera has been used in India since ancient times in ayurvedic preparations as a brain and a nerve tonic and also for the treatment for epilepsy and asthma. Laboratory studies on rats indicate that extracts of the plant improve memory capacity and motor learning ability. Studies in humans show that an extract of the plant has antianxiety effects. It has antioxidant properties, reducing oxidation of fats in the bloodstream.
Bacoside rich leaf extract of Bacopa monniera shows a therapeutic potential of in improving the memory functions in hypobaric conditions. Bacoside administration was seen to enhance learning ability in rats along with augmentation in memory retrieval and prevention of dendritic atrophy following hypoxic exposure. In addition, it decreased oxidative stress, plasma corticosterone levels and neuronal degeneration. Bacoside administration also increased cytochrome c oxidase activity along with a concomitant increase in ATP levels. Hence, administration of bacosides could be a useful therapeutic strategy in ameliorating hypobaric hypoxia induced cognitive dysfunctions and other related neurological disorders (Sunil Kumar Hota, Kalpana Barhwal, Iswar Baitharu, Dipti Prasad, Shashi Bala Singh and Govindasamy Ilavazhagan. 2009. Bacopa monniera leaf extract ameliorates hypobaric hypoxia induced spatial memory impairment,, Neurobiology of Disease, Volume 34, Issue 1, Pages 23-39) The neuroprotective role of B. monnieri extract in alteration of glutamate receptor

binding and gene expression of NMD A Rl in hippocampus of temporal lobe epileptic rats has been demonstrated. In association with pilocarpine-induced epilepsy, there was significant down regulation of NMDA Rl gene expression and glutamate receptor binding without any change in its affinity. B. monnieri treatment of epileptic rats significantly reversed the expression of NMDA Rl and glutamate receptor binding alterations to near-control levels. Also, in the epileptic rats, we measured a significant increase in the activity of glutamate dehydrogenase, which neared the control level after B. monnieri treatment.
Bacopa monniera dried herb has been moistened with water and then extracted with alcohol. The extract is concentrated under reduced pressure and repeatedly macerated with benzene for defatting [Chatterji R. Rastogi R.P. and Dhra. M.I.. (1963). Chemical Examination of Bacopa monniera wettst: Part I-Isolation of Chemical Constituents, Indian J. Chem., 1, 212-215; Chatterji N., Rastogi R.P. and Dhar, M.L. (1965). Chemical Examination of Bacopa monniera wettst: Part II. The constitution of Bacoside A.. Indian./. Chem.. 3. 24]. The filtrate is diluted to 60% concentration of alcohol and is treated with an excess of lead acetate. The lead salts are filtered and the residual lead was removed from the filtrate with hydrogen sulphide. The pH of the filtrate is adjusted to 6.4 with sodium carbonate and concentrated at 50.degree. C. under vacuum to one third of its volume and partitioned repeatedly with butanol & water. The butanol fraction on concentration under vacuum deposits a powder containing bacosides A&B. The powder is then crystallized from alcohol as colourless needles. An additional amount of bacosides is obtained from the filtrate by freeing it from solvent and macerating the residue with acetone. In another method by Singh et al. (H K Singh and B N Dhawan Journal of Bhanopharmacology. Vol 5: 205-214. 1982 1982) the air dried plant material is extracted with 90% ethanol by soxhlet extraction apparatus and the extract obtained is mixed with 10% gum acacia. Singh et al. (H K Singh, R P Rastogi, R C Srimal and B N Dhawan. Phytotherapy Research , Vol 2 (2): 70-75, 1988) reported the alcoholic extraction of the plant after defatting with benzene and lead acetate treatment and is extracted with n-butanol. The n-butanol soluble fraction after processing yielded a powder. In another method by Elangovan et al. (V. Elangovan, S Govindasamy, N Ramamoorthy and K. Balasubramanian, "In vitro studies on the anticancer activity of Bacopa monniera. Fitoterapia . Vol I.XVI (3): 211-215. 1995). the

extraction process comprises of soaking the powdered plant material in 95% ethanol for 48 hr, concentrating the extract under vacuum and drying it by lyophilisation. In majority of the extraction process the main problem is the difficulty of obtaining the stable final product in the form of a dry free flowing powder as the active constituents (bacosides) are highly hygroscopic.
Nine different methods of extraction of Brahmi was compared for obtaining a commercially viable method. Among nine methods, the highest yield (27.89+0.48 %) was found in the extract obtained from maceration of the plant material in methanol at room temperature for 3 days. However, the extract containing the highest amount of total saponins (19.28+ 0.12 %) was obtained from percolation with ethanol after the plant material was soaked in water (Watoo Phrompittayarat , Waraporn Putalun, Hiroyuki Tanaka,Kanchalee Jetiyanon,Sakchai Wittaya-areekul and Kornkanok Ingkaninan. 2007. Naresuan University Journal 2007: 15,1,29-34).
Publication No. WO 2007141807 relates to a potent synergistic herbal composition [BacoMind ™ ] from the plant species Bacopa monnieri and its beneficial effects in learning, memory, cognition and Attention Deficit Hyperactivity Disorder [ADHD] or Attention Deficit Disorder [ADD].
US Patent No. 6,833,143 provides a process for the preparation of Bacopa monniera extract with bacosides enriched fraction in a non-hygroscopic form. The freshly harvested herbs are dried in a hot air oven at 37-42.degree. C, powdering and sieving the dried herb to obtain powder of 30-40 mesh size, defatting the powdered herb with hexane, extracting the defatted powdered herb with acetone, again extracting the same herb with methanol to obtain an extract containing bacosides, concentrating the extract to one twentieth of its original volume under reduced pressure, gradually adding the concentrated extract to acetone for precipitating the bacosides, filtering the bacosides in a Nutsche type vacuum filter, dissolving the crude bacoside mass into 2-10 parts water, extracting the bacoside solution with n-butanol to selectively transfer the bacosides to the solvent phase, separating and concentrating the solvent phase under vacuum to obtain semi-dry mass, dissolving the semi dried mass into 2-10 parts water, adding and stirring 1-5% of .beta.-cyclodextrin to stabilize the bacosides, spray drying the stabilized bacoside solution by maintaining hot air temperature at 90-

110.degree. C, to obtain a stable free flowing fraction of Bacopa monniera rieh in bacosides.
US Publication No. 20050209445 describes a process of extraction concentration and separation of fractions containing Bacosides A and B from plant materials of Bacopa species. The dried plant materials are subjected to alcoholic or hydroalcoholic or water extraction and concentration of the extract. This solid mass is washed with a nonpolar organic solvent to remove fatty materials. The solid obtained thereafter is extracted with an organic polar solvent; the solvent extract is washed with water to remove water soluble contaminants there from and then dried in vacuum. This factor which is enriched with bacosides A and B. It is then further subjected to chromatographic separation and purification to obtain Bacoside A and Bacoside B.
US Publication No. 20080132455 relates to a process of producing a fraction from Bacopa species wherein the total Bacopa saponin concentration is up to 100%.
Thus in view of the above prior art, this invention disclosure provides a improved method with reduced method steps for obtaining a extract of Bacopa monnieri comprising of the active constituents, bacoside in a defined percentage useful for pharmaceutical formulations.
OBJECTS OF THE INVENTION:
The principal object of the present invention is to provide an improved method with reduced process steps for obtaining Bacopa monnieri extract.
Another object of the present invention is to obtain a standardized extract of Bacopa monnieri enriched with a defined percentage of bacoside.
Yet another object of the present invention is to obtain a standardized extract of Bacopa monnieri comprising of synergistic ingredients useful for therapeutical applications.
Still another object of the present invention is to obtain a standardized extract of Bacopa monnieri in an easy to use free flowing powder form.

Another object of the present invention is to eliminate the use of hazardous solvents in the extraction process.
Yet another object of the present invention is to provide a method of extraction of bacoside from Bacopa monnieri which is economical and is with reduced process time.
Still another object of the present invention is the use of the standardized extract of Bacopa monnieri, comprising of defined percentage of bacoside in different pharmaceutical formulations related to nervous disorders.
SUMMARY:
• Accordingly the present invention provides a novel improved method with reduction in method steps for the extraction of bacoside from Bacopa monnieri in a defined quantitative value. The aerial parts of the herb Bacopa monnieri is extracted with alcohol. The solvent is filtered and distilled out under vacuum to a syrupy solution. The syrupy solution is extracted with n-butanol. The n-butanol fraction, after separation is distilled under vacuum till complete solvent removal. The dried mass is refluxed with n-hexane. The n-hexane phase is separated-out. The precipitate is dried, grinded, sieved and packed in nitrogen atmosphere. The precipitate comprises of a percentage of bacoside not less than 40%. In a preferred embodiment of the present invention, a method is provided for the extraction of Bacopa monnieri wherein the extract comprises of bacoside not less than 40% and the said method comprises
a. extraction of dried aerial part of Bacopa monnieri with 4 times
solvent (w/v) in absence of light, wherein the said solvent is a 60%
primary alcohol selected from the group comprising ethanol,
methanol, propanol and the process of extraction is carried out at a
temperature in the range from 45.degree.C to 65.degree.C the
extraction process is repeated thrice;
b. extracts from step a. is mixed and filtered:
c. the alcohol phase from the filtrate of step b. is distilled at a
temperature in the range from 40.degree.C to 60.degree.C under
vacuum;
d. the residual aqueous syrup from step c. is further extracted with n-
butanol;

e. the n-butanol phase from the extraction of step d. is vacuum
distilled at a temperature in the range from 55.degree.C to
75.degree.C;
f. the residual solid from step e. is refluxed with n-hexane for a
duration of one hour;
g. the solid mass from step f. is vacuum dried at a temperature of
60.degree.C to 75.degrec:C for a duration ranging from 6 hours to
9 hours;
h. the dried mass from step g. is grounded and sieved with a mesh size of 40.
In another embodiment of the present invention, the method of extraction of Bacopa monnieri comprises
a. extraction of dried aerial part of Bacopa monnieri in absence of
light with solvent wherein the quantity of the said herb is ranging
from 1.0 kg to 2.0 kg and the said solvent comprises 60% primary
alcohol selected from the group comprising ethanol, methanol,
propanol further the said solvent volume ranges from 4.0litres to
S.OIitres at a temperature in the range of 45.degree.C to
65.degree.C for duration ranging from 3 hours to 4 hours with
stirring;
b. the solvent from the extraction of step a. is filtered and the process
is repeated twice under similar conditions;
c. the alcohol phase of filtered extracts of step b. is distilled under
vacuum at a temperature in the range of 40.degree.C to
60.degree.C till the volume is reduced to 2.0litres;
d. the resjdual aqueous phase of step c. is further extracted with n-
butanol of volume ranging from 1 .Olitres to 2.0litres;
e. the n- butanol phase of step d. is distilled completely under
vacuum at temperature in the range from 65.degrec.C to
75.degree.C;
f. the residual thick aqueos syrup of step e. is refluxed with n-hexane
of volume ranging from 100 ml to 300 ml for time duration
ranging from 1 hour to 2 hour;

g. the solvent phase of step f. is separarted and the residual solid is dried under vacuum at temperature in the range of 60.degre.C to 75.degree.C for time duration ranging from 6 hours to 8 hours; h. the dried mass from step g. is grounded and sieved with a mesh size of 40. In yet another embodiment of the present invention, the method of extraction of Bacopa monnieri comprises
a. extraction of dried aerial part of Bacopa monnieri in an amount of
about 1.0 kg with 60% primar\ alcohol selected from the group
comprising ethanol, methanol, propanol and the said solvent
volume is about 4.0L at a temperature of 60.degree.C for a
duration of 3 hours with stirring;
b. the solvent from the extraction of step a. is filtered and the process
is repeated twice under similar conditions;
c. the alcohol phase of the extracts of step b. is distilled under
vacuum at a temperature of 55.degree.C till the volume is reduced
to 2.01itres.
d. the residual aqueous phase of step c. is further extracted with n-
butanol of volume of about 1.5litres;
e. The n- butanol phase of step d. is distilled completely under
vacuum at temperature of 70.degree.C;
f. the thick residual aqueous phase from step e. is refluxed with
200ml n-hexane for one hour and solvent phase is separated:
g. the residual solid from step f. is dried under vacuum at temperature
of 60.degree.C for 9hours;
h. the dried mass from step g. is grinded and sieved through 40 mesh. In still another embodiment of the present invention, the method of extraction of Bacopa monnieri wherein free flowing powder of bacoside is obtained with bacoside content ranging from NLT 40% to NLT 43%.
In another embodiment of the invention, the said method of extraction eliminates the use of hazardous solvents
In yet another embodiment of the invention, the said method of extraction is with less processing time
In still another embodiment of the invention, the Bacopa monnieri extract comprises of synergistic ingredients having therapeutic activities.

In another embodiment of the present invention, the extract of Bacopa monnieri based on the said method of extraction has applications as nerve tonic and in pharmaceutical formulations effective for selected from the group comprising 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.
BRIEF DESCRIPTION OF TABLES:
It is to be noted, however, that the appended drawings and tables illustrate only
typical embodiments of this invention and are therefore not to be considered for
limiting of its scope, for the invention may admit to other equally effective
embodiments.
Table 1 shows the comparative summary of the examples.
DETAILED DESCRIPTION OF THE INVENTION:
The present invention discloses a method for the production of Bacopa monnieri extract having not less than 40% Bacosides. The purity of the extract is assessed by HPLC as well as UV- Vis. Spectrophotometer method.
The extract is obtained from the aerial parts of the plant. The Bacopa monnieri extract comprises of tetracyclic tri- terpenoids glycosides (bacoside-A and B). which are reported to be memory enhancer. The extraction is by primary alcohol with water. The solvent mixture is filtered and alcohol is distilled-out. The aqueous phase is exchanged with n- butanol. The n-butanol fraction after separation is distilled under vacuum till complete solvent removal. The dry softmass is refluxed with n-he.\ane. The n-hcxane phase is then scparatcd-out. The precipitate is dried at 60.degree.C for 8 hours, grinded, sieved and packed in nitrogen atmosphere. The standardized extract is used in the treatment of mental retardation, epilepsy and insanity as well as nerve tonic.
The plant for the present invention is obtained from the Amity herbal garden. Manesar, Haryana, India.
The plant Bacopa monnieri commonly known as Brahmi, has been used in herbal formulations of the Ayurvedic or Indian system of medicine for memory enhancing. Bacopa monnieri (Coastal Waterhyssop, Brahmi, Thyme-leafed gratiola. Water hyssop) is a perennial, creeping herb whose habitat includes wetlands and muddy shores. It commonly grows in marshy areas throughout

India, Nepal, Sri Lanka, China, Taiwan, and Vietnam, and is also found in Florida and other southern states of the USA.
In the present invention method dry aerial part of Bacopa monnieri at room temperature to the exclusion of light is extracted with the a combination of water and low molecular weight primary alcohol at a temperature in the range of 45.degree.C to 65.degree.C with 4 times solvent (w/v) each time, the method being repeated thrice.' It is established that under the following aforesaid operating parameters after third extraction, the herb is completely exhausted of the desired ingredients. The extraction method is monitored b> TLC. using pre-coated TLC Plate (E-Merck), Silica gel 60 F254 -The mobile phase comprises of ethyl acetate: methanol: water:: 60:14:10, v/v/v. visualizing is by using 7.5% H2SO4, and heating at 1 lO.degree.C for 10 minutes.
Filter the extract, mix and distill the filtrate at a temperature in the range of 40.degree.C to 60.degree.C under vacuum. The aqueous syrup is extracted with n-butanol and n-butanol phase is vacuum distilled at temperature ranging from 55.degree.C to 75.degree.C. The resulting solid is refluxed with n-hexane for one hour. The solid mass is vacuum dried at temperature ranging from 60.degree.C to 75.degree.C, for 6hours to 9 hours, grounded and sieve with 40 mesh, packed in nitrogen atmosphere.
The extract has not less than 40% bacosides, and is analysed by HPLC (Pal, R. et. al.1998) as well as spectrometric (Pal, R and Sarin J.P.S., 1992) method. Estimation of Bacoside by HPLC comprises the following steps
Step I
1 (a) Preparation of the sample is by weighing about 250 mg of finally powdered sample in 200ml r b flask and dissolving it in 2 ml of methanol and 10ml of 10% sulphuric acid.
1 (b) The solution of the extract prepared in Step I (a) is refluxed at 1 lO.degree.C for 4 hours, cooled and transferred completely to a separating funnel.
1 (c) The solution after Step 1 (b) is extracted with chloroform for three times with 10ml volume each time.
1 (d) The chloroform layer of Step 1 (c) is collected and washed with 0.5 ml of 0.01% (v/v) ammonia solution initially and then with demineralised water till neutral.
1 (e) The chloroform layer after Step I (d) is passed through sodium sulphate into

beaker. 1 (f) The chloroform after Step 1 (e) is evaporated into water bath.
1 (g) The residue after Step 1 (f) is dissolved in methanol and volume is made up
to 100ml. Step 2
2 (a) Preparation of the standard is by weighing about 50 mg of standard Bacoside
A in 200ml r b flask and dissolving it in 2 ml of methanol and 10ml of 10% sulphuric acid.
2 (b) The solution of the extract prepared in Step 2 (a) is refluxed at I lO.degree.C for 4 hours, cooled and transferred completely to a separating funnel.
2 (c) The solution after Step 2 (b) is extracted with chloroform for three times with 10ml volume each time.
2 (d) The chloroform layer of Step 2 (c) is collected and washed with 0.5 ml of 0.01% (v/v) ammonia solution initially and then with demineralised water till neutral.
2 (e) The chloroform layer after Step 2 (d) is passed through sodium sulphate into
beaker.
2 (f) The chloroform after Step 2 (e) is evaporated into water bath.
2 (g) The residue after Step 2 (f) is dissolved in methanol and volume is made up
to 100ml.
Step 3
3 (a) The sample prepared in Step 1 is analyzed by HPLC against standard
prepared in Step 2 using C 18 Column Thermohypersil. (250 x 4.6 mm. 5µ) by injecting equal volumes.of both standard and sample and the peak area of corresponding bacosides is recorded and the percentage content in the sample is calculated.

3 (b) The mobile phase of the HPLC analysis of Step 3 (a) is the mixture of methanol and water 8.3:1.2, well degassed, flow rate is 1.3 ml/ min and is detected at wave length of 278nm.
The invention is described in detail with reference to the example given below. The example is provided just to illustrate the invention and therefore, should not be construed to limit the scope of the invention.
EXAMPLE 1:
1.0kg dried aerial portion of Bacopa monnieri was extracted with 4.0litres solvent having 1.60litres water with primary alcohol such as ethanol, at 45.degree.C for three hours, with stirring. Filter the solvent and repeat the cycle at twice under similar conditions. Collect all the extract together and distill-out the solvent under vacuum at 50.degree.C till the total volume is about 2.01itres. The aqueous phase was exchanged with n-butanol, 1.51itres and separated. Distill-out the n- butanol completely at 65.degree.C temperature, under vacuum. The thick syrup was refluxed with 200ml n-hexane for one hour and solvent phase was separated. The solid was dried under vacuum at 70.degree.C for 7 hours, grinded, sieved through 40 mesh and pack under nitrogen atmosphere. Free flowing powder, 22.35 gm, having 40.98% total bacosides, by HPLC. Chromatographic conditions: mobile phase; The mixture of methanol and water 8.3:1.2, well degassed. Column: C-18. Thermohypersil. (250 \ 4.6 mm. 5u). wave length: 278nm, Flow rate 1.3 ml/ min.
Sample preparation; Accurately weigh about 250 mg of finally powdered sample in 200ml r b Flask. Add 2ml of methanol to dissolve and 10ml of 10% sulphuric acid, reflux at 110.degree.C for 4 hours, cool and transfer completely to a separating funnel. Extract it with chloroform, three times with 10ml volume, at each. Collect the chloroform layers. Wash the chloroform layer with 0.5 ml of 0.01% (v/v) ammonia solution, initially and then with DM water till neutral. Collect the chloroform layer through sodium sulphate into beaker. Evaporate the chloroform into water bath. Dissolve the residue in methanol and make-up volume 100ml.
Standard preparation: weigh about 50 mg of standard Bacoside A in 200ml r b Flask. Add 2ml of methanol to dissolve and 10ml of 10% sulphuric acid, reflux at 110.degree.C for 4 hours, cool and transfer completely to a separating funnel.

Extract it with chloroform, three times with 10ml volume, at each. Collect the chloroform layers. Wash the chloroform layer with 0.5 ml of 0.01% (v/v) ammonia solution, initially and then with DM water till neutral. Collect the chloroform layer through sodium sulphate into beaker. Evaporate the chloroform into water bath. Dissolve the residue in methanol and make-up volume 100ml.
Analysis: Separately inject equal volumes of standard preparation and sample preparation, record the peak area of corresponding bacosides and calculate the percentage content in the sample, (these results were further authenticated by following the method of UV Spectroscopy as per Pal R. and Sarin .IPS. 1992. lnd. J. pharma. Sci. 54, 17-18.)
EXAMPLE 2;
1.0kg dried aerial portion of Bacopa monnieri was extracted with 4.01itres solvent having 1.60litres water with primary alcohol such as ethanol, at 50.degree.C for three hours, with stirring. Filter the solvent and repeat the cycle at twice under similar conditions. Collect all the extract together and distill-out the solvent under vacuum at 40.degrre.C till the total volume is about 2.0litres. The aqueous phase was exchanged with n-butanol, 1.5litres and separated. Distill-out the n-butanol completely at 70.degree.C temperature, under vacuum. The thick syrup was refluxed with 200ml n-hexane for one hour and solvent phase was separated. The solid was dried under vacuum at 65.degree.C for 8 hours, grinded, sieved through 40 mesh and pack under nitrogen atmosphere. Free flowing powder. 22.61 gm. having 41.67% total bacosides. Chromatographic conditions: mobile phase; The mixture of methanol and water 8.3:1.2, well degassed: Column: C-18, Thermohypersil. (250 x 4.6 mm, 5µ), wave length: 278nm, Flow rate 1.3 ml/min.
Sample preparation; Accurately weigh about 250 mg of finally powdered sample in 200ml r b Flask. Add 2ml of methanol to dissolve and 10m! of 10% sulphuric acid, reflux at 110.degree.C for 4 hours, cool and transfer completely to a separating funnel. Extract it with chloroform, three times with 10ml volume, at each. Collect the chloroform layers. Wash the chloroform layer with 0.5 ml of 0.01% (v/v) ammonia solution, initially and then with DM water till neutral. Collect the chloroform layer through sodium sulphate into beaker.

Evaporate the chloroform into water bath. Dissolve the residue in methanol and make-up volume 100ml.
Standard preparation: weigh about 50 mg of standard Bacosidc A in 200ml r b Flask. Add 2ml of methanol to dissolve and 10ml of 10% sulphuric acid, reflux at 110.degree.C for 4 hours, cool and transfer completely to a separating funnel. Extract it with chloroform, three times with 10ml volume, at each. Collect the chloroform layers. Wash the chloroform layer with 0.5 ml of 0.01% (v/v) ammonia solution, initially and then with DM water till neutral. Collect the chloroform layer through sodium sulphate into beaker. Evaporate the chloroform into water bath. Dissolve the residue in methanol and make-up volume 100ml.
Analysis: Separately inject equal volumes of standard preparation and sample preparation, record the peak area of corresponding bacosides and calculate the percentage content in the sample, (these results were further authenticated by following the methodof UV Spectroscopy as per Pal R. and Sarin JPS, 1992. Ind. J. pharma. Sci. 54, 17-18.)
EXAMPLE 3:
1.0kg dried aerial portion of Bacopa monnieri was extracted with 4.0litres solvent having 1.601itres water with primary alcohol such as ethanol, at 65.degree.Cfor three hours, with stirring. Filter the solvent and repeat the cycle at twice under similar conditions. Collect all the extract together and distill-out the solvent under vacuum at 60.degree.C till the total volume is about 2.0litres. The aqueous phase was exchanged with n-butanol, 1.51itres and separated. Distill-out the n-butanol completely at 75.degree.C temperature, under vacuum. The thick syrup was refluxed with 200ml n-hexane for one hour and solvent phase was separated. The solid was dried under vacuum at 60.dcgrcc.C for 9 hours, grinded, sieved through 40 mesh and pack under nitrogen atmosphere. Free flowing powder, 22.45 gm. having 42.15% total bacosides. Chromatographic conditions: mobile phase; The mixture of methanol and water 8.3:1.2, well degassed. Column: C-18. Thermohypersil. (250 x 4.6 mm. 5µ). wave length: 278nm, Flow rate 1.3 ml/ min.
Sample preparation; Accurately weigh about 250 mg of finally powdered sample in 200ml r b Flask. Add 2ml of methanol to dissolve and 10ml of 10% sulphuric acid, reflux at 1 lO.degree.C for 4 hours, cool and transfer completely

to a Separating funnel. Extract it with chloroform, three times with 10ml volume, at each. Collect the chloroform layers. Wash the chloroform layer with 0.5 ml of 0.01% (v/v) ammonia solution, initially and then with DM water till neutral. Collect the chloroform layer through sodium sulphate into beaker. Evaporate the chloroform into water bath. Dissolve the residue in methanol and make-up volume 100ml.
Standard preparation: weigh about 50 mg of standard Bacoside A in 200ml r b Flask. Add 2ml of methanol to dissolve and 10ml of 10% sulphuric acid, reflux at 110.degree.C for 4 hours, cool and transfer completely to a separating funnel. Extract it with chloroform, three times with 10ml volume, at each. Collect the chloroform layers. Wash the chloroform layer with 0.5 ml of 0.01% (v/v) ammonia solution, initially and then with DM water till neutral. Collect the chloroform layer through sodium sulphate into beaker. Evaporate the chloroform into water bath. Dissolve the residue in methanol and make-up volume 100ml.
Analysis: Separately inject equal volumes of standard preparation and sample preparation, record the peak area of corresponding bacosides and calculate the percentage content in the sample, (these results were further authenticated by following the methodof UV Spectroscopy as per Pal R. and Sarin J PS. 1992. Ind. J.pharma. Sci. 54, 17-18.)
EXAMPLE 4:
1.0kg dried aerial portion of Bacopa monnieri was extracted with 4.01itres solvent having 1.60litres water with primary alcohol such as ethanol. at 60.degree.C for three hours, with stirring. Filter the solvent and repeat the cycle at twice under similar conditions. Collect all the extract together and distill-out the solvent under vacuum at 55.degree.C till the total volume is about 2.0litres. The aqueous phase was exchanged with n-butanol, 1.51itres and separated. Distill-out the n-butanol completely at 70.degree.C temperature, under vacuum. The thick syrup was refluxed with 200ml n-hexane for one hour and solvent phase was separated. The solid was dried under vacuum at 60.degree.C for 9 hours, grinded, sieved through 40 mesh and pack under nitrogen atmosphere. Free flowing powder, 23.12 gm, having 42.60% total bacosides. Chromatographic conditions: mobile phase: The mixture of methanol and water 8.3:1.2, well degassed. Column: C-18, Thermohypersil, (250 x 4.6 mm, 5µ), wave length: 278nm, Flow rate 1.3 ml/ min.

Sample preparation; Accurately weigh about 250 mg of finally powdered sample in 200ml r b Flask. Add 2ml of methanol to dissolve and 10ml of 10% sulphuric acid, reflux at 1 lO.degree.C for 4 hours, cool and transfer completely to a separating funnel. Extract it with chloroform, three times with 10ml volume, at each. Collect the chloroform layers. Wash the chloroform layer with 0.5 ml of 0.01% (v/v) ammonia solution, initially and then with DM water till neutral. Collect the chloroform layer through sodium sulphate into beaker. Evaporate the chloroform into water bath. Dissolve the residue in methanol and make-up volume 100ml.
Standard preparation: weigh about 50 mg of standard Bacoside A in 200ml r b Flask. Add 2ml of methanol to dissolve and 10ml of 10% sulphuric acid, reflux at 110.degree.C for 4 hours, cool and transfer completely to a separating funnel. Extract it with chloroform, three times with 10ml volume, at each. Collect the chloroform layers. Wash the chloroform layer with 0.5 ml of 0.01% (v/v) ammonia solution, initially and then with DM water till neutral. Collect the chloroform layer through sodium sulphate into beaker. Evaporate the chloroform into water bath. Dissolve the residue in methanol and make-up volume 100ml.
Analysis: Separately inject equal volumes of standard preparation and sample preparation, record the peak area of corresponding bacosides and calculate the percentage content in the sample, (these results were further authenticated by following the methodof UV Spectroscopy as per Pal R. and Sarin .IPS. 1992 Ind. J. pharma. Sci. 54, 17-18.)

Table-1: Comparative summary of the aforesaid examples
Dried aerial portion of Bacopa monnieri, as above examples, is processed as;
(Table Removed)
Numerous modifications and adaptations of the system of the present invention will be apparent to those skilled in the art, and thus it is intended by the appended claims to cover all such modifications and adaptations which fall within the true spirit and scope of this invention.

WE CLAIM:
1. A method is provided for the extraction of Bacopu monnieri wherein the extract comprises of bacoside not less than 40% and the said method comprises
a. extraction of dried aerial part of Bacopa monnieri with 4 times
solvent (w/v) in absence of light, wherein the said solvent is a 60%
primary alcohol selected from the group comprising ethanol,
methanol, propanol and the process of extraction is carried out at a
temperature in the range from 45.degree.C to 65.degree.C, the
extraction process is repeated thrice;
b. extracts from step a. is mixed and filtered;
c. the alcohol phase from the filtrate of step b. is distilled at a
temperature in the range from 40.degree.C to 60.degree.C under
vacuum;
d. the residual aqueous syrup from step c. is further extracted with n-
butanol;
e. the n-butanol phase from the extraction of step d. is vacuum
distilled at a temperature in the range from 55.degree.C to
75.degree.C;
f. the residual solid from step e. is refluxed with n-hexane for a
duration of one hour;
g. the solid mass from step f. is vacuum dried at a temperature ol
60.degree.C to 75.degree.C for a duration ranging from 6 hours to
9 hours;
h. the dried mass from step g. is grounded and sieved with a mesh size of 40.
2. The method of extraction of Bacopa monnieri as claimed in claim 1 comprises
a. extraction of dried aerial part of Bacopa monnieri in absence of light with solvent wherein the quantity of the said herb is ranging from 1.0 kg to 2.0 kg and the said solvent comprises 60% primary alcohol selected from the group comprising ethanol, methanol, propanol further the said solvent volume ranges from 4.0litres to 5.0litres at a temperature in the range of 45.degree.C to
65.degree.C for duration ranging from 3 hours to 4 hours with stirring;
b. the solvent from the extraction of step a. is filtered and the process
is repeated twice under similar conditions;
c. the alcohol phase of filtered extracts of step b. is distilled under
vacuum at a temperature in the range of 40.degree.C to
60.degree.C till the volume is reduced to 2.0litres;
d. the residual aqueous phase of step c. is further extracted with n-
butanol of volume ranging from 1 .Olitres to 2.01itres;
e. the n- butanol phase of step d. is distilled complete!) under
vacuum at temperature in the range from 65.degree.C to
75.degree.C;
f. the residual thick aqueos syrup of step e. is refluxed with n-hexane
of volume ranging from 100 ml to 300 ml for time duration
ranging from I hour to 2 hour;
g. the solvent phase of step f. is separarted and the residual solid is
dried under vacuum at temperature in the range of 60.degre.C to
75.degree.C for time duration ranging from 6 hours to 8 hours;
h. the dried mass is grounded and sieved with a mesh size of 40. 3. The method of extraction of Bacopa monnieri as claimed in claim I Comprises
a. extraction of dried aerial part of Bacopa monnieri in an amount of
about 1.0 kg with 60% primary alcohol selected from the group
comprising ethanol. methanol, propanol and the said solvent
volume is about 4.0L at a temperature of 60.degree.C for a
duration of 3 hours with stirring;
b. the solvent from the extraction of step a. is filtered and the process
is repeated twice under similar conditions:
c. the alcohol phase of the extracts of step b. is distilled under
vacuum at a temperature of 55.degree.C till the volume is reduced
to 2.01itres.
d. the residual aqueous phase of step c. is further extracted with n-
butanol of volume of about 1.5 litres:
e. The n- butanol phase of step d. is distilled completely under
vacuum at temperature of 70.degree.C;
f. the thick residual aqueous phase from step e. is refluxed with
200ml n-hexane for one hour and solvent phase is separated:
g. the residual solid from step f. is dried under vacuum at temperature
of 60.degree.C for 9hours:
h. the dried mass from step g is grinded and sieved through 40 mesh.
4. The method of extraction of Bacopa monnieri as clamed in claim 1 wherein
free flowing powder of bacoside is obtained with bacoside content ranging from
NLT40%toNLT43%.
5. The Bacopa monnieri extract based on the method of extraction as claimed in claim 1 comprises synergistic ingredients having therapeutic activities.
6. The extract of Bacopa monnieri based on the said method of extraction as claimed in claim 1 has applications as nerve tonic and in pharmaceutical formulations effective for selected from the group comprising 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.
7. A method of extraction of bacoside from Bacopa monnieri substantially as
herein described with reference to the tables and examples accompanying this
specification.