BACOPA MONNIERA - STUDY The human brain remains a mystery today, even after years of research into the

biological processes underlying its specific actions. In addition to controlling and coordinating vital life processes, the brain regulates higher level operations such as memory and cognition, functions that provide us with the capacity to think, reason and act. These mental abilities, however, have been observed to diminish significantly with advancing age as early as in the fifth decade of life, and factors such as emotional stress could precipitate these effects even earlier in life. Gradual deterioration may progress to a point where the person, often in good physical health, is unable to perform even simple daily tasks. In recent years, researchers have identified a number of natural compounds that could potentially help to retard mental deterioration. Most of this research is based on plants used in the ancient systems of medicine to promote brain health. One plant that has been used in mental conditions and illnesses is Bacopa monniera Wettst. (syn Herpestis monniera). It is commonly known as Indian water hyssop or Brahmi and belongs to the family Scrophulariaceae. Brahmi has been used by Ayurvedic medical practitioners in India for almost 3000 years. The earliest chronicled mention is in the Ayurvedic treatise, the Charaka Samhita (100 A.D.), in which Brahmi is recommended in formulations for the management of a range of mental conditions including anxiety, poor cognition and lack of concentration. According to the Charaka, Brahmi acts as an effective brain tonic that boosts ones capabilities to 6 think and reason. The Sushruta Samhita (200 A.D.) attributes the plant with efficacy in maintaining acuity of intellect and memory. The Bhavprakasha Nighantu, commonly known as the Indian Materia Medica (1500 A.D.), 7 cites the plant as a brain tonic that is effective in maintaining vigor and intellect . In India, Brahmi is currently recognized as being effective in the treatment of mental illness and epilepsy. The name Brahmi is derived from the word "Brahma", the mythical "creator" in the Hindu pantheon. According to Hindu concepts, the brain is the center for creative activity. Thus, any compound that improves this faculty of the brain is called Brahmi. Other Sanskrit names for this plant are "Bahuphene", Atiphena" and "Phenavati". The word "Phena" means "foaming property". When mixed with water, Bacopa plant parts produce a stable froth that is attributed to the saponins present in the plant. Pharmacologically, it is understood that Brahmi has an unusual combination of constituents that are beneficial in mental inefficiency and illnesses and useful in the management of convulsive disorders like epilepsy. Bacosides, Brahmi's active principles responsible for improving memory related functions, are attributed with the capability to enhance the efficiency of transmission of nerve impulses, thereby strengthening memory and cognition.
1,2,3,4,5

The mechanism of memory The hippocampus, the brain's seat of memory, is located in the temporal (left and right) sides of the brain. It processes signals sent to the brain by the senses into the templates of memory, which are then stored in other parts of the brain, creating a long-term memory. Signals are converted into electrical impulses in the nerve cells due to a rapid change in protein composition. These impulses are then conducted across neurons (nerve cells) and through synapses, which connect nerve cells. This process continues until the bonds between the nerve cells strengthen, and memory is created. Normal synaptic activity is a process mediated by neurotransmitters. Each neuron is a single nerve cell. It has one or more arms called axons that send signals (impulses) and one or more arms called dendrites that receive signals. When a signal is transmitted through an axon terminal, spherical bodies called vesicles fuse with its membrane. Neurotransmitters are released when the vesicles burst open into the synaptic space, the minute space between the sending and receiving cells used to discharge neurotransmitters ("passengers"). To end the signal, the axons reabsorb some neurotransmitters; and the enzymes in the synapse neutralize the other neurotransmitters 9. (Fig. 1.2)

Figure 1.2 Schematic representation of the mechanism of synaptic activity It is evident that a disruption in any part of normal synaptic activity would affect memory. This normally occurs with advanced age and continuous electrical activity, which wears out the synapses. As a result, new memory creation is impaired and memory loss occurs.

Chemical substances and plant extracts that are known to restore the memory work in different ways. The bacosides are the memory chemicals in Brahmi. According to scientists at the Central Drug Research Institute located in Lucknow, India, the bacosides help to repair damaged neurons by adding muscle to kinase, the protein involved in the synthesis of new neurons to replace the old ones10, 11. Depleted synaptic activity is thus restored, leading to augmented memory functions. The possible mode of action of Brahmi is schematically represented in Fig. 1.3.

A pyramidal cell from the hippocampus : the sites of memory are located on the "branches" How Bacopa Boosts Memory

1.

On receiving the signals from the sensory organs, the receptors in each neuron of the hippocampus trigger an electric pulse, mediated through a change in protein composition. The pulse is transmitted to the next neuron through the synapse. The process continues till the bonds between the neurons become strong and the memory is created. However, continuous electrical activity wears out the synapses, impairing new memory creation and causing loss of Memory. The Bacosides help in restoring the synaptic activity of neuron. Bacopa monniera and Centella asiatica: two distinct species

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3.

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In India, Bacopa monniera, known as "Brahmi", is revered in the indigenous system of medicine as a nerve tonic. In early literature, the name Brahmi was also used to refer to another plant species, Centella asiatica Linn., known as Indian penniwort. However, these plants are distinctly different. The name Jala-brahmi or water-brahmi assigned to Bacopa monniera in ancient Sanskrit writings provides the differentiation8. The vernacular name "mandukaparni", often confused with Brahmi, in fact refers to Centella asiatica. A critical study of the comparative phytochemistry, pharmacology and therapeutic properties of these two botanicals also support the view that they are distinct:

The Charaka Samhita 3 considers them both to be promoters of cognitive functions, but it suggests that Brahmi is superior to mandukaparni. Brahmi is used to treat specific mental disorders such as insanity and epilepsy, while mandukaparni is a general rejuvenative tonic which improves mental health. Brahmi promotes fertility and sustains implantation of the embryo in the uterus, while mandukaparni tends to reject the embryo. This suggests that the plant materials have opposite effects on uterine functions. The Sushruta Samhita6 defines the properties of the herbs. Brahmi belongs to tikta rasa (bitter), while mandukaparni belongs to kasaya rasa (astringent). Comparative modes of action of the major cognition-facilitating nutraceuticals In this context, it is interesting to compare the mechanism of action of Bacopa monniera with that of other plant extracts and compounds that possess neuropharmacological activity such as Ginkgo biloba and the well-known, brain cell nutrient, phosphatidylserine. Ginkgo possesses efficacy in alleviating disturbances of the central nervous system of both the primary degenerative type (early stages of degenerative dementia) and of vascular origin. Several fractions of Ginkgo biloba extract have been proven to be pharmacologically active. They function through two major mechanisms12: Improvement of cerebral circulation, a function related to the anti-ischemic activity of the extract. Thus, patients suffering from CNS insufficiency states or peripheral vascular and neurosensory disorders benefit. The extract furnishes a protective effect on neural tissue via free radical scavenging, increased energy metabolism or through inhibition of neurotransmitter degrading enzymes. Both these mechanisms indirectly contribute to enhancing alertness, awareness and cognition. However, direct effects on retention and memory are not indicated. The well-known, brain cell nutrient, phosphatidylserine, is a phospholipid that forms one of the large "lipid" molecules that hold the other large molecules in the cells membrane systems together. The cell membrane plays a

vital role in the entry of nutrients into the cell, the exit of waste products, inter-cell communications, ion transport and cell movement. Phosphatidylserine is a vital part of the cell membrane that helps to attenuate these functions, thereby helping to maintain the cells internal environment, enhancing signal transduction mediated through protein kinase C and adenylate cyclase and promoting secretory vesicle release, a process essential for the release of neurotransmitters. All these effects contribute to phosphatidylserines protective effect on the hippocampus, the seat of memory. The loss of dendrite connections, a normal occurrence of advanced age, is thereby prevented13. Thus, phosphatidylserine indirectly affects memory.

Traditional uses of Bacopa: contemporary relevance Brahmi has been used in Ayurvedic formulations for conditions ranging from catarrhal complaints, gastrointestinal disturbances due to excessive tobacco use, habitual abortions and high blood sugar due to anxiety disorders and epilepsy1-8, 14. In certain parts of India, Brahmi is believed to be an aphrodisiac8 . In Sri Lanka, under the name of Loonooweela, Brahmi is prescribed for fevers8. In the Phillipines, it is used as a diuretic15. The traditional use of the plant, of particular relevance to contemporary medicine, is its validated efficacy in promoting memory functions and providing relief to patients with anxiety neurosis. In Ayurveda, Brahmi is described as "medhya rasayana" or brain tonic with the ability to promote mental functioning along with providing general rejuvenative effects as discussed below: Eight ml of plant juice or 1/2 gm of plant powder once a day has been traditionally used to increase the speed of learning and extent of memory power. It is also useful in increasing the sharpness of perception by the sense organs. Brahmi is believed to be particularly useful for the promotion of memory in children. In addition, Brahmi was found to be useful in the prevention and alleviation of convulsions. In adults, it helps to relieve insomnia. Brahmi has a bitter taste. Traditionally, the fleshy leafs and stems were made into a paste or pressed for juice extraction. Sugar, jaggery or honey was added to make it more palatable. Some of the known preparations with Brahmi are Brahmi Ghrita (in clarified butter), Sarasvatarishta (a decoction used as a brain tonic), Brahmi Rasayana (a rejuvenating formulation with other herbs), Brahmi taila (medicated oil), and Brahmi sarbat (a cooling drink). In recent years, "Memory Plus", a product that contains the standardized extract of bacosides from Brahmi, has been marketed in India16. Under the definition of herbal drugs in the Guidelines of Herbal Medicines issued by the World Health Organization (WHO) in 1991, an herbal product that has been used traditionally without demonstrable harm does not require specific regulatory action unless new evidence demands a revised risk/benefit analysis. Subsequently, several formulations containing Bacopa monniera extracts standardized for bacosides content, have appeared in the global marketplace. Other pharmacological effects of Bacopa monniera Besides mental functioning, Ayurvedic texts advocate the use of Brahmi in other physiological conditions. As a result, researchers have evaluated the antioxidant, anti-inflammatory, cardiotonic and other pharmacological effects of Bacopa monniera preparations/extracts. Antioxidant action Since free radical-induced damage is implicated in most of the disease conditions treated with Brahmi in Ayurveda, recent researchers attempted to evaluate the antioxidant action of the whole plant and the isolated bacosides17. Preclinical research supports the partial role of the bacosides antioxidant action in the frontal cortex, striatum and hippocampus on the cognition-facilitating action of Bacopa monniera18.

Anti-inflammatory and analgesic activities. The anti-inflammatory effect of Brahmi rasayan, a rejuvenating preparation containing Bacopa monniera was studied in rodents19. The effectiveness of Brahmi rasayan in inhibiting experimentally induced inflammation was compared with that of indomethacin, a known anti-inflammatory drug. The results of this study showed that Brahmi rasayan effectively suppressed experimentally induced inflammatory reactions and did not cause gastric irritation at anti-inflammatory doses. The mechanism of anti-inflammatory action, in part, was proven to be similar to that of aspirin and other nonsteroidal anti-inflammatory drugs (NSAIDs), which are mediated through inhibition of prostaglandin synthesis and partly through the stabilization of lysosomal membranes. Cardiotonic properties Preclinical studies on frogs demonstrated the cardiotonic and neuromuscular blocking actions of an extract of the whole plant 19. Anticancer effects An alcoholic extract of the entire plant was found to have anticancer activity against Walker carcino sarcoma 256 (intramuscular) in rats20. It is speculated that Bacopas anticarcinogenic acitivity is furnished by inhibition of DNA replication. However, further research needs to be performed to confirm the validity of these findings. Use of Bacopa monniera in the management of irritable bowel syndrome An indigenous preparation containing Bacopa monniera and Aegle marmelos correa (Bilva) was clinically evaluated for its efficacy in the management of Irritable Bowel Syndrome (IBS) in a 6-week, double blind randomized trial. 21 IBS is a painful condition accompanied by a variety of gastrointestinal symptoms, including gas, diarrhea and constipation. About 169 patients with irritable bowel syndrome (IBS) received either standard therapy with clidinium bromide, chlordiazepoxide and isaphaghulla or the Ayurvedic preparation of Aegle marmelos correa and Bacopa monniera Linn along with a matching placebo. The Ayurvedic preparation (57 patients) was found to be effective in 64.9% of the cases, while standard therapy (60 patients) was useful in 78.3% of the cases. Only 32.7% of the patients receiving the placebo(52 patients) showed improvement. In cases where diarrhea was predominant, the Ayurvedic preparation was found to be better than the conventional therapy. In contrast, the conventional therapy was found to better than the Ayurvedic one in cases of predominant constipation and painful IBS. Overall, the Ayurvedic preparation appeared to be effective and safe in the management of the diarrheal form of IBS for which long-term use of antidiarrheals with possible side effects is prescribed. However, neither the Ayurvedic nor conventional forms of therapy were found to be better than the placebo in preventing a relapse after six months.

Characteristic Features Of Brahmi The plant source for Brahmi is Bacopa monniera, a small herb with light purple flowers. It grows in wet and sandy areas and near streams in tropical regions. It is a creeping herb with numerous branches and small fleshy, oblong leaves. Flowers and fruits appear in summer. The whole plant is medicinally useful24. Synonyms for Bacopa monniera Wettst. 1. Bacopa monniera (L.) Pennell.yes 2. Lysimachia monnieri L. Cent. 3. Gratiola monnieri (L.) L. 4. Monniera cuneifolia Michaux 5. Herpestis monniera (L.) Kunth

Cultivation The plants grow on wet soil or in very shallow water. They are generally found in marshy places growing near reservoirs, canals and waterways. In India, the herb is found throughout the country from sea level to altitudes of 4400 ft. It can be easily cultivated in damp areas. The land should be ploughed and the field cleaned for weeds. Irrigation and drainage are provided by narrow canals dug across the fields. In traditional practice, natural fertilizer in the form of cow dung manure is added at levels of 2 to 3 tons per acre25. Planting Seeds or rooted plants planted in 24" x 24" patches are used for propagation. The planting is done in the period between mid June and early July, roughly at the onset of the monsoon season. About 440 lbs. of fresh plants are required for cultivation in an acre. The plants may be grown for three consecutive years at a single location. Vegetative propagation is predominant. Irrigation & Harvesting The plant requires adequate water. The irrigation requirement depends upon the soils moisture content. Weeding is done periodically. The whole plant can be removed after 120-150 days. The planting material for the next crop is ensured by retaining 24" x 24" patches of the cultivated crop. Good quality final produce is green in color. The method of drying affects the final quality. The plants should be washed properly before drying. For bulk drying, the harvested crop should be immediately spread in thin layers in well-ventilated shaded areas. In order to ensure quick and uniform drying, the produce should be stirred at least twice a day. The drying process takes about 8 to 10 days.

Description of the Plant The plant is a profusely branched herb, rooting at the nodes and forming dense mats. The leaves are fleshy. Flowering and fruiting occur throughout the year in brief, successive durations. The plant is diagrammatically represented in Fig. 2.1. The salient botanical features are described below: Stem: prostrate, (sub)succulent, herbaceous. Leaves: decussate, simple, oblong, 1 x 0.4 cm, (sub)succulent, punctate, penninerved, margin entire, apex obtuse, (sub)sessile. Flower(s): axillary, solitary, bracteate (2 bracteoles), linear, pedicel to 0.5 cm., purple in color. Calyx:5 lobes (unequal); outer 2 lobes larger, ovate, 7 x 3.5 mm; inner 2 lobes linear, 5.5 x 0.7 mm; median 1 lobe oblong, 5.5 x 2 mm, imbricate, (sub)succulent, punctate, obtuse, acute. Corolla: white with violet and green bands inside the throat, 0.8 cm across, 5 mm tube; 5 lobes, obscurely 2-lipped, 2+3, (sub)equal, obtuse or emarginate. Stamens: 4, didynamous; filament pairs I and 2.5 mm anthers oblong, contiguous, 1. 5 mm. Ovary: Oblong-globose, 2 mm; style slightly deflexed, 5.5 mm; stigma flat capsule, oblong-globose, 5 x 2.5 mm, septicidal or locilicidal, or 4-valved. Seeds: oblong; testa striate.

Chemical Constitutents The pharmacological effects of Bacopa monniera are attributed to the presence of a number of biologically active compounds, including alkaloids, saponins and sterols. The compounds responsible for the memory enhancing effects of Bacopa monniera are triterpenoid saponins called "Bacosides", Isolation of Alkaloids and other components In view of the importance of this plant in the indigenous system of medicine, systematic chemical examinations of the plant were carried out by several groups of researchers. Detailed investigations were first documented in 1931 when Bose and Bose26 reported the isolation of the alkaloid "Brahmine" from Bacopa monniera. This alkaloid was shown to be toxic at concentrated levels, but it had beneficial effects at dilutions of 1 in 200,000 to 1 in 500,0004. Later, in 1947, Basu and Pabrai27 isolated another alkaloid "herpestine" (C34H46O6N2) from Bacopa monniera. A mixture of three other alkaloids was isolated from the leaves of Bacopa monniera by Basu and Walia25. The isolation of D-mannitol, a saponin, hersaponin (m.p. 232-234C) and potassium salts by Sastry and coworkers in 1959 provided further details about the chemical components of Bacopa monniera29.

In 1960, Rastogi and Dhar30 isolated betulic acids and a saponin. Datta and Basu31 of the Bengal Immunity Research Institute in Calcutta, India isolated and identified acid A (m.p. 284-285C) and a triterpene saponin, monnierin (m.p. 263-265C), from Bacopa monniera grown under controlled conditions. Acid A was obtained in flakes from the petroleum ether extract. After crystallization from methanol, the flakes were converted to silky needles. Purification of the alcohol extract of the dried plant by conventional methods, yielded a crude saponin. On repeated fractional crystallization from methyl alcohol, fine needles of monnierin were obtained. The saponin could be hydrolyzed by refluxing for 12 hours with hydrochloric acid (5%) in aqueous methanol (50%). After neutralization, the hydrolysate indicated two sugars, glucose and arabinose, on paper chromatograms.

Isolation and identification of Bacosides A & B A milestone in the elucidation of the pharmacologically active principles in Bacopa monniera was achieved in 1965 by Chatterji and coworkers32, whose detailed investigations led to the isolation of the following constituents (Table 3.1): Table 3.1: Chemical Constituents of Bacopa monniera Compound Bacoside A Bacoside B Betulinic acid D-Mannitol Stigmastanol b-Sitosterol Stigmasterol Melting point C 250-251 (decomposes) 203 (decomposes) 315 166 170 137 141 Yield (%) on dry basis 1.54 0.65 0.11 0.02 0.0013 0.0014 0.005

Bacoside A and Bacoside B, the saponins now considered to be responsible for the characteristic neuropharmacological effects of Bacopa, were obtained in crystalline form from the whole plant. The plant material was extracted with ethyl alcohol, and a fraction soluble in acetone was used to obtain the Bacosides.

The chemical composition of Bacoside A has been established on the basis of chemical and physical degradation studies33. A double beam spectrophotometer (Shimadzu, U.V., visible 260 ) was used to perform the analysis. Bacoside A, on acid hydrolysis for 4 hours, gave an aglycone (non-sugar) fraction and a mixture of three sugars. The sugar components were separated by ion exchange chromatography over Dowex 50W. The sugars were identified as D-glucopyranose and L-arabinopyranose by optical rotation, osazone formation and paper chromatography. The third component was found to be a disaccharide, because it produced glucose and arabinose on hydrolysis .

Basu and others34 (1967) reported the results of investigations on bacoside B. This compound was obtained as colorless needles (m.p. 203C) from methanol extract. It was sparingly soluble in ethanol, methanol and water. It could not be hydrolyzed with emulsion or distilled ethanolic sodium hydroxide solution. However, under similar conditions, bacoside A could be hydrolyzed with distilled, methanolic sulfuric acid. The neutral, aqueous hydrolysate showed two spots, glucose and arabinose, by paper chromatography. The total sugars in the molecule of bacoside B were estimated iodometrically to be 35.1%. It is clear that bacosides A and B are identical with respect to their respective carbohydrate and aglycone moieties. Therefore, bacoside B has the same gross structure as that which has been proposed for bacoside A. However, bacoside B is dextrorotatory, and bacoside A is levorotatory. This may be explained on the assumption that the configuration of the carbohydrate chain is different in the two glycosides. The bacosides obtained in the ethanolic extract of Brahmi are essentially triterpenoid saponins. On acid hydrolysis, they yield aglycones called bacogenins A1 , A2, A3 and A4, whose structures have been elucidated by earlier researchers. Bacogenin A4 was shown to be ebelin lactone. Recently, minor saponins, bacoside A1 and bacoside A3, were isolated and characterized as 3-O-(a1-arabinofuranosyl(1,3)-a-1-arabinofuranosyljujubogenin and 3-b-(O-b)-D-glucopyranosyl-(1,3)-O-(a-Larabinofuran-osyl(1,2)-O-b-D-glucopyranosyloxyjujubogenin, respectively32. The chemical structures of some of these compounds are as follows35:

BACOSIDE A (Levorotatory)

BACOSIDE B (Dextrorotatory)

(Triterpenoid saponin)

(Bacogenin A4)

Proximate composition of the leaves of Bacopa monniera: Chemical analysis of the leaves by standard methods gave the values indicated in Table 3.220: Table 3.2: Proximate composition of the leaves of Bacopa monniera Component Moisture Protein Fat Carbohydrate Crude fiber Ash Analytical results 88.4 g/100g 2.1 g/100g 0.6 g/100g 5.0 g/100g 1.05 g/100g 1.9 g/100g

Calcium Phosphorus Iron Ascorbic acid Nicotinic acid Energy

202 mg/100g 16 mg/100g 7.8 mg/100g 63 mg/100g 0.3 mg/100g 38 cal/100g

Pharmacological Properties The biological effects of Bacopa monniera are well documented in traditional as well as scientific literature. The most important of these are the effects of the plant, plant extracts and isolated bacosides on cognition and memory functions, their anxiolytic effects and their role in the management of convulsive disorders. Preclinical studies 1. Antioxidant action : The results of a recent study suggest that the cognition promoting functions of Bacopa monniera may be partially attributed to the antioxidant effects of the bacosides. Currently, a number of deteriorative processes in the body are attributed to the action of damaging free radicals. During the physiological process of respiration, inhaled and tissue incorporated oxygen oxidizes cellular components and biomolecules. This process results in the generation of oxidative products, free radicals and reactive oxygen species, when titled towards excessive activity by external factors (e.g. improper nutrition) or internal factors (e.g. disease). These free radicals are highly reactive chemical molecules, which react with biological compounds causing tissue damage by a process called "free radical pathology". The aging process exemplifies the cumulative result of deterioration of individual cells, tissues and organs caused and promoted by free radicals. The human body has built-in mechanisms to counteract free radicals. These mechanisms are collectively known as the bodys antioxidant defense. Unfortunately, in most instances the antioxidant defense is gradually overwhelmed by the aging process, disease or a combination of both. The inflammatory processes associated with microbial or viral infections, the progression of cancer and neurological disorders are just a few disease conditions which contribute to depletion of the antioxidant defense system of the body. This defense system is constituted by the enzymes superoxide dismutase (SOD), catalase (CAT) and glutathione peroxidase (GPX). Lipid peroxidation is essentially a "free radical chain reaction" involving the following stages: 1. Initiation Hydrogen is abstracted from a polyunsaturated fatty acid side chain by a free radical species: CH+R*------- C*+RH C*+O2 ---------- -CO2*

2. Propagation The fatty acid side chain peroxyl radical -CO2* attacks adjacent fatty acid side chains. CO2*+-CH-------- -CO2H + -C* The propagation continues, leading to accumulation of lipid peroxides in the membranes, destabilization of the membranes and the entry of damaging ions. Peroxyl radicals attack ions as well as membrane proteins. An antioxidant terminates the propagation of free radicals either by accepting and quenching them or by retarding the "initiation" step by reducing the generation of free radicals. One study determined the effects of alcohol and hexane extracts of Brahmi on lipid peroxidation induced by ferrous sulfate and cumene hydroperoxide in rat liver homogenate. The alcohol fraction of Bacopa monniera furnished greater protection against both inducers, ferrous sulfate and cumene peroxide17. Table 4.1 indicates the effective inhibitory doses.

Extract Alcoholic extract Hexane extract

ED50 (mg) ferrous sulfate ED50 (mg) cumene hydroperoxide 100 290 177 400

Fig. 4.1 Comparative effects of Vitamin E and Brahmi on lipid peroxidation in rat liver homogenate

The results were also compared with known antioxidants, tris (2-amino-2-hydroxymethyl-1,3-propanediol) (a hydroxyl trapper), EDTA (a metal chelator) and the natural antioxidant, Vitamin E. (Note: 100 mg of the alcoholic extract of Brahmi was equivalent to 247 mg of EDTA (0.66mM) and 58 mg of Vitamin E.) Tris did not show any protective effect, while Brahmi (alcoholic extract), EDTA and Vitamin E, offered dose-dependent protection against ferrous sulfate-induced peroxidation (Fig. 4.1).

The authors also observed that Brahmi only slightly protected against the oxidation of reduced glutathione at doses lower than 100 mg/ml, while higher concentrations enhanced the rate of oxidation. The authors concluded that Brahmis mechanism of antioxidant action could be attributed to metal chelation at the initiation level of the free radical-induced, chain reaction or the quenching of free radicals at propagation level17. A more recent study explored the antioxidant activity of Bacopa monniera in the rats frontal cortex, striatum and hippocampus. The levels of the antioxidant enzymes, SOD, CAT and GPX, were measured following the administration of a standardized extract of Bacopa monniera (bacoside A content 82 5%, administered in doses of 5 mg/kg and 10 mg/kg orally) for 7, 14 and 21 days. These results were compared with the effects induced by ()deprenyl (selegiline hydrochloride, a well-known neurological antioxidant, 2 mg/kg orally) for the same time period. The enzyme activities were investigated in the frontal cortex, striatum and hippocampus after 14 and 21 days. After 14 and 21 days, Bacopa monniera extract induced a dose-related increase in SOD, CAT and GPX activities in all brain regions investigated, while (-)deprenyl induced an increase in the frontal cortex and striatum but not the hippocampus. The results suggest that Bacopa monniera extract exhibits significant antioxidant effects, and these effects extend to the hippocampus, the

seat of memory and cognition, unlike deprenyl. The authors concluded that increased free radical scavenging may explain, at least in part, the cognition-facilitating action of Bacopa monniera extract documented in the ancient Ayurvedic texts and in modern scientific literature. Bacopa monniera supports the functions of the central nervous system Bacopa monniera has been studied in animals for its purported effects on the functions of the central nervous system in its various forms, crude form, standardized extract, and compounded formulation known as Brahmi Rasayan (Table 1) 36,37. Table 1: Constituents of Brahmi Rasayan Herb Plant part used 10 2 1 1 40 Ratio (w/w)

Bacopa monniera Leaves Eugenia caryophyllus (cloves) Flower Piper longum (long pepper) Stalks Elettaria cardamomum (cardamom) Seed Sucrose

Beneficial effects on learning and memory: Preliminary studies established the role of Brahmi in improving learning and memory capabilities. Treatment with the plant produced improvement in maze learning in albino rats38. In a detailed study, the effects of an alcoholic extract of Bacopa plant fed to rats was evaluated on the learning ability of the animals39. The rats were faced with three different situations which required learning new skills and memorizing the newly learned skills. One groups of rats received Bacopa extract at a dose of 40 mg/kg/day, while the other group served as the control (untreated) group. After three consecutive days of feeding with Bacopa extract, it was observed that the treated rats significantly improved in the acquisition of new skills compared to the control group. They were also able to retain the skills acquired for the entire duration of the three-day treatment. However, the animals forgot these skills once treatment with Bacopa extract was discontinued. In order to sustain these beneficial effects, the animals had to be fed a maintenance dose of the extract for a number of days. The

authors of this report suggest that Bacopa extract might facilitate learning ability in rats due to qualitative improvements in the neurons and their message-carrying connections. Based on these experiments, Bacopa was proven to exert significant but short-lived effects on memory in rats (Fig. 4.2 (a), (b)). The ease of acquisition of new skills indicated by the decrease in time taken to learn was enhanced by about 50% on treatment with Bacopa. In addition, the Bacopa treatment enhanced the retention of learning indicated by ease of "relearning" after a specific time interval by about 200%.

(a) Learning skills: ease of acquisition

(b)Relearning Index

Figure 4.2 The effect of Bacopa administered orally to rats on acquisition, retention and extinction of the newly learned skills In a follow-up experiment, the same group of researchers evaluated the potential of the active saponin fractions of Bacopa monniera, Bacosides A and B, to enhance learning abilities in rats40 . The bacosides were fed to rats at a single dose of 10 mg/kg 90 minutes prior to teaching the animals a new skill. The treated animals were able to learn within a shorter time compared to the control group. In addition, their relearning ability was enhanced

significantly even after the treatment was withdrawn. This implies that administration of bacosides improved learning abilities in the long run.

In another test, rats were taught to avoid drinking water containing lithium chloride, a chemical that causes unpleasant gastrointestinal symptoms. Rats treated with bacoside A and bacosides A & B at doses of 2.5 - 7.5 mg/kg showed a significant decrease in lithium chloride intake compared to the control group. The results of the experiment revealed that the group of animals receiving bacoside A (I to V, fig 4.3) and bacosides A & B (VI to X, fig. 4.3) learned to avoid lithium chloride faster and in a dose dependent manner. The authors of this report commented that the positive effects of bacoside A and bacosides A & B is manifested in learning enhancement with negative reinforcement (punishment) of the newly learned skill as well as with positive reinforcement (reward) of the new skills. This is a significant finding suggesting that bacosides are effective in enhancing learning ability under two different imposed conditions, viz., punishment and reward. Summary of dosage regimen Group treatment Bacoside A I Saline II Bacoside III Bacoside IV Bacoside V Bacoside Saline 0.5 ml 2.5 5.0 7.5 5.0 (no LiCl incubation on day 1) Dose of bacosides (mg/kg oral)

Bacosides A and B VI Saline VII Bacoside VIII Bacoside IX Bacoside X Bacoside Saline 0.5 ml 2.5 5.0 7.5 5.0 (no LiCl incubation on day 1)

a : p <0.01; b : p < 0.05 Figure 4.3 The dose dependent effect of bacosides administered orally to rats on learning to choose sucrose drink (outer columns) rather than lithium chloride drink (inner columns)

Mode of action of the Bacosides Learning: An early study employed a sleep deprivation model to investigate the effect of Brahmi on the learning process in rats. When deprived of sleep, the levels of the stress hormone, serotonin, increased in rats. There was also an increase in glutamate levels, while GABA (gamma amino butyric acid), a chemical involved in the transmission of nerve impulses, showed marked reduction. Discrimination learning was significantly reduced following sleep deprivation stress. Brahmi significantly reduced the levels of stress hormones following sleep deprivation and improved discrimination learning in the animals. The authors concluded that Brahmi helps to regulate the altered levels of biogenic amines following stress, thereby improving learning. Memory: An important feature of memory formation in various animal species is its progression from a short-lived labile form to a long lasting stable form, probably by consolidation through a multiphasic pathway. During this period of consolidation, memory can be disrupted by administering agents that induce amnesia. Electroconvulsive shock, hypothermia and hypoxia are conditions that induce retrograde amnesia non-invasively. Other chemical agents (e.g. diethyldithiocarbamate) could induce temporary amnesia. Earlier studies on the processing of memory have revealed that memory consolidation involves both serial and parallel processing of information.

A recent study tested the ability of rats to remember skills learned before amnesia was induced in order to confirm the mode of action of the bacosides on the memory process11. Retrograde amnesia was induced in rats by immobilization stress administered for 18 hours or by administering electroconvulsive shock (0.5 mA, 50 Hz, 0.5 sec). Both treatments were administered immediately after completion of training. The training schedule involved a brightness discrimination reaction. The animals were divided into two groups; one group served as the untreated controls. Bacosides were administered to the second group at an oral dose level of 20 mg/kg for three days before

induction of amnesia. The amnesic treatments led to a significant disruption of consolidation in control groups compared to the groups pretreated with bacosides (Fig. 4.4). .

Fig. 4.4 Effect of bacosides (20 mg/kg oral dose for 3 days) on immobilization-induced amnesia

Anxiolytic effects of Bacopa monniera The current interest in the anxiolytic properties of Bacopa monniera extract assumes greater relevance in view of the fact that Bacopa monniera promotes cognition unlike the amnesic action of benzodiazepine anxiolytics. One study compared the anxiolytic effect of standardized Bacopa monniera extract (25.5% bacosides) with that of benzodiazepine. The effects of Bacopa monniera extract at levels of 5, 10 and 20 mg/kg administered orally to rats were compared to

those elicited by lorazepam (0.5 mg/kg administered intraperitoneally). The higher doses of Bacopa monniera extract produced significantly greater anxiolytic effects compared to lorazepam41. A recent conference presentation summarized the anxiolytic profile of standardized Brahmi extract. Two batches of the extract were subjected to a number of CNS tests in animal models. The authors of this presentation concluded that the mild anxiolytic effects exhibited by Brahmi extracts were partly responsible for the role of Brahmi in improving cognitive functions. Standardized extracts of Brahmi were found to exhibit nootropic effects. Besides enhancing cognitive functions, they neither showed classical stimulant nor depressant effects on the central nervous system. The extracts were found to increase barbiturate-induced sleeping time, increase survival time under low oxygen levels, reduce clonidine-induced fighting and biting behavior and reduce hyperactivity in amphetamine-treated animals. In addition, the extracts also blocked secondary conditioned response in a conditioned avoidance response,

suggesting a mild anxiolytic effect. (Conditioned avoidance response is a type of behavior in which the animal avoids stimuli that it associates with anxiety-provoking symptoms, thereby reducing anxiety. This becomes a positive reinforcer for continuing avoidance.) The lack of secondary conditioned avoidance shown by the animals suggests that the extract lowers anxiety levels.

Anti-convulsive action Another important property of Bacopa, dependent on its effects on the central nervous system, is its anticonvulsive action. One study evaluated the anti-convulsive effects of Bacopa monniera in the prevention of epileptic attacks in animals. Aqueous and alcoholic extracts of Bacopa administered to rats for 15 days significantly reduced mortality due to seizures as well as the intensity of the seizures (Fig. 4.6). (Seizure intensity was measured by increased time of seizure onset. The longer the time of onset, the greater the protective effect of the therapeutic compound.)22 However, the dose used in these experiments was up to 50% of the LD50 values. The dose was administered intraperitoneally at levels of 600mg/kg of the aqueous extract and 7000mg/kg of the alcoholic extract. At the doses used, Bacopa extracts decreased spontaneous activity of tested animals, but they did not produce a sedative effect or cause uncoordinated movement in the animals.

Figure 4.6 Effect of 15-day administration of Bacopa extracts on mortality in rats with chemically induced epileptic convulsions Another experiment studied the beneficial effects of Brahmi Rasayan on convulsions in mice. When the formula was fed to mice at doses of 1 to 30 gm/kg, it showed a significant protective effect against seizures in animals (Fig. 42 4.7) and significant pain relieving action (Fig. 4.8), preventing painful stimuli from reaching the brain . The protective effect of the formula was evaluated 30 minutes after its administration. At doses of 10 and 30 gm/kg, Brahmi Rasayan induced sedation in treated animals. Another interesting finding in the second part of this study (using rats) was that the compound formula at doses 10 and 30 gm/kg was effective in preventing drug (haloperidol)-induced catalepsy (Fig. 4.9). Catalepsy is a serious condition in animals and humans that is characterized by sudden loss of consciousness, muscle tone and coordination.

Figure 4.7 Effect of oral administration of Brahmi Rasayan on convulsive seizures in mice (Increase in response time to seizure stimulus indicates greater protective effect.)

Figure 4.8 Pain relieving effect of oral administration of Brahmi Rasayan in mice (The greater the reaction time, the better the pain relieving effect.)

Figure 4.9 Effect of oral administration of Brahmi Rasayan (30 g/kg) on haloperidol induced catalepsy in rats (The lower the cataleptic score, the greater the protective effect of the compound.) Protection from phenytoin-induced cognitive deficit The side effects of phenytoin, the commonly used anti-epileptic drug, include impairment of cognitive functions. 43 Bacopa monniera extract was evaluated for efficacy in reversing this side effect in animal models . Evaluations with regard to passive avoidance response task (PA), maximal electroshock seizures and locomotor activity were performed in mice. The mice received phenytoin (25 mg/kg orally for 14 days). Bacopa monniera (40 mg/kg for 7 days) given along with phenytoin in the second week of the two-week regimen significantly reversed PHT-induced impairment of cognitive function as determined from the PA results. Both acquisition and retention of memory were improved without affecting the anti-convulsant activity of PHT. These effects were independent of motor stimulation. The authors concluded that Bacopa monniera has potential use in correcting the cognitive deficit induced by PHT. Postulated mode of action: It is postulated that the anti-convulsive effects could be mediated through GABA (gamma amino butyric acid) which is involved in neural impulse transmission, because substances which stimulate GABA are known to possess 44 anticonvulsant, pain relieving and sedative activity . A more recent study demonstrated the calcium antagonistic activity of an alcoholic extract of Bacopa monniera on vascular and intestinal smooth muscles of rats and guinea pigs. The plant extract inhibited the spontaneous movements of both guinea pig ileum (IC50: 24 4 mcg/ml) and rabbit jejunum (IC50: 136 9 mcg/ml) Contractions in the guinea pig ileum induced by acetylcholine were inhibited by the extract at levels of 100-700 mcg/ml in a 46 concentration dependent manner . These results indicate the direct action of the extract on smooth muscles. The authors also observed that calcium chloride-induced responses in the rabbits blood vessels and jejunum were reduced in the presence of the plant extract (10-700 mcg/ml), suggesting direct interference with the influx of calcium ions. However, since the extract did not affect contractions induced by noradrenaline or caffeine, the authors concluded that the extract had no appreciable effect on the mobilization of intracellular calcium. Based on the results of the experiment, the authors postulated that the spasmolytic effect of Bacopa monniera extract in smooth muscles is predominantly due to the inhibition of calcium influx, applicable to both electrical impulse-mediated and receptor-mediated calcium channels in the cell membrane.

One research paper provided an exhaustive summary of the preclinical pharmacological studies on Bacopa monniera and the isolated bacosides. The cognition facilitating activity of the extract is attributed to the saponins, Bacoside A and Bacoside B, which are effective in much lower doses in various model studies, including tests for 45 conditioned taste aversion and conditioned shock avoidance response Beneficial effects in the management of animal models of Alzheimers disease The effect of subchronic (14 days) administration of a standardized extract of Bacopa monniera (bacoside A content 82%) was evaluated on two animal models of Alzheimers disease induced by intracerebroventricular administration of colchicines and by lesioning of the nucleus basalis magnocellularis (nbm) with ibotenic acid47.

Alzheimers disease is believed to be associated with impaired cholinergic functions. It is indicated by depleted concentrations of the neurotransmitter, acetylcholine, a reduction in choline esterase activity and decreased muscarinic cholinergic receptor binding in the frontal cortex and hippocampus in rats. Colchicine administration triggered these effects. Lesioning with colchicines or ibotenic acid induced marked deficits in the retention of active avoidance learning on day 7 and increased progressively by day 14. Subchronic administration of Bacopa monniera at a dose of 10mg/kg significantly reduced the magnitude of memory deficits induced by both compounds as observed on days 7 and 14, while the effects were evident with the lower dose only on day 14 . The dose of 10 mg/kg reversed the detrimental effects induced by the colchicines in the frontal cortex and the hippocampus. The effects of the lower dose of 5 mg/kg were evident only after 14 days of administration. The authors concluded that the memory-facilitating effects of Bacopa monniera and its active constituents, the bacosides, may be due to their effects on central cholinergic modulation of memory functions. Treatment of anxiety neurosis The beneficial effects of Brahmi on memory retention and its effectiveness in alleviating anxiety neurosis have been validated by clinical trials on normal subjects as well as those with specific mental disorders. Preliminary clinical trials performed over thirty years showed encouraging results. Only recently have studies on Brahmi been extended to patients suffering from mild to moderate mental deficiencies. Study design Singh and Singh48 performed clinical studies on the anti-anxiety effect of Bacopa monniera. Thirty-five patients suffering from anxiety neurosis were selected for this study. They were divided into two groups. One group was followed for two weeks from the commencement of the therapy, and the other group was followed for four weeks. After clinical diagnosis and baseline evaluation, the patients received a regimen of treatment with Brahmi syrup at a dose of 30 ml per day in two divided doses (the total daily dose provided 12 g of dry, crude Bacopa monniera) for a period of one month. The patients followed a uniform diet. Clinical assessment of the Brahmi treatment was based on the following performance parameters evaluated at weekly intervals: Anxiety level Adjustment level Mental fatigue rate Immediate memory span Disability level Physiological changes (viz., pulse rate, blood pressure, body weight, rate of respiration and breath holding time).

Biochemical changes (blood chemistry) Results: (1) Anxiety reduction The mean total anxiety level of each patient over the selected time period (two or four weeks) was assessed using the Sinha anxiety scale49. The mean, total, anxiety level in the group of patients evaluated during two weeks of therapy was 34.88 compared to the corresponding baseline value of 53.22 (p<0.01). Similarly, the mean total anxiety level in the patients followed after 4 weeks was 40.70 compared to the baseline pretreatment value of 49.05. (p < 0.01) (Fig. 4.10). These results suggest that treatment with the extract helped to lower the anxiety levels significantly.

Figure 4.10: Effect of Brahmi extract on anxiety levels

(2) Behavioral Improvements: Anxiety neurosis often manifests itself in behavioral problems, such as inability to adjust to situations encountered in daily life, including social interactions and work-related activities (maladjustment). The mean total maladjustment level in patients was assessed using the guidelines provided by Asthana50. The degree of maladjustment in patients after two weeks of treatment was 138.17, compared to its corresponding pretreatment value of 166.0 (p<0.01). The mean maladjustment level in patients after four weeks of therapy was 53.58, which was significantly (p<0.01) lower than the corresponding pretreatment value of 166.28. (Fig. 4.11)

Figure 4.11 Effect of treatment with Brahmi extract on levels of maladjustment (3) Improved disability level Anxiety neurosis is characterized by the inability to manage personal, social and occupational challenges effectively. It is classified as a disability in psychology. The disability level and also its personal, social and occupational components were found to decrease after treatment with Brahmi as assessed by the guidelines provided by Asthana50. The mean disability score in patients after two weeks of therapy was 40.17 compared to the corresponding pretreatment value of 61.58 (p<0.01). A similar trend of reduction in the level of disability was observed in patients after four weeks. (4) Effects on Mental fatigue rate. The mental fatigue rate was determined in terms of total work output for a given time and the number of mistakes that a person made during work. The assessment was based on the results of Joshis digit cancellation test51. The mean work output in patients followed up for two weeks under treatment was 801.65 compared to the corresponding pretreatment value of 688.15 (p<0.01). Similarly, the mean work output in patients followed for four weeks was 855.80 compared to the corresponding pretreatment value of 711.15 (p<0.01) (Fig. 4.12).

Figure 4.12 Effect of treatment with Brahmi extract on work output/mental fatigue

(5) Effects on Immediate memory span. Brahmi has been shown to significantly increase immediate memory span determined by the Joshis digit retention test51. The mean memory span in patients after two weeks was 5.89 compared to the corresponding pretreatment value of 5.43, (p<0.01). The mean immediate memory span in patients after 4 weeks was 6.37 compared to the initial value of 5.94 (p < 0.01) (Fig. 4.13).

Figure 4.13 Effect of treatment with Brahmi extract on immediate memory span 6. Other improvements: The patients reported relief from symptoms such as insomnia, headache, fatigue, irritability, lack of concentration, anorexia, dyspepsia, tremors, palpitation and nervousness. 7. Physiological changes There was a significant increase in body weight at the end of 4th week of treatment with Brahmi. The body weight changes at the end of the 2nd week of treatment were not statistically significant. Similarly, a significant lowering in the rate of respiration was noted, and the breath holding time significantly increased after both two and four weeks of the evaluation. Pulse rate and blood pressure lowering were not statistically significantly during the course of the treatment. 8. Biochemical changes The stress-related increased secretion of the adrenal hormones was moderated in the course of the treatment with Brahmi. Levels of excreted urinary metabolites of adrenal hormones were lowered as a result of the treatment. This finding suggests that treatment with Brahmi helped to reduce psychosomatic stress levels in the subjects, leading to lowered secretion of stress hormones. The authors of this study concluded that Brahmi produces significant anti-anxiety effects in humans without subjectively or objectively reported side effects.

Treatment of impaired mental functioning A clinical trial was performed to determine the efficacy of Brahmi in the management of impaired mental functioning52. One hundred and seventy two patients with mild, moderate or severely impaired mental abilities received Brahmi in syrup form (three doses per day, each containing 500 mg of crude Brahmi). The placebo was administered to 114 patients over the same time period. Electronic, memory span apparatus was used to measure the mental abilities of the subjects initially and after one year of the therapy. Brahmi significantly increased the concentration ability, memory span and overall mental performance of the subjects with mild to moderate degrees of mental deficiency. No adverse side effects were reported.

Efficacy of Bacopa monniera in revitalizing intellectual functions in children

A study was conducted to determine the effects of Bacopa monniera in revitalizing the mental capacities of 40 elementary school children (6-8 years old)53. Study design: Group I : Twenty children were given one teaspoonful of Bacopa monniera syrup three times daily for three months. (Each teaspoonful was equivalent to 350 mg of crude Brahmi.) Group II : Twenty children received the placebo, syrup with no Bacopa, three times daily for three months.

The psychological measures of efficacy of Bacopa monniera were based on the following tests: Test 1: WISC maze to measure discrimination learning by way of visual motor performance Test 2: WISC digit span test to approximate immediate memory skills Test 3: Ravens colored progressive matrices to measure cognitive ability Test 4: Bender Gestalt test which records activities related to brain functions as a measure of cognitive skills. Results: Test 1. A statistically significant increase in raw scores and their corresponding total quotient (p < 0.001) and a statistically significant decrease in reaction time, (p < 0.001), performance time (p < 0.01) and error incidence (p < 0.1) were observed in the Bacopa-treated group. These changes were insignificant in the placebo group. Test 2. Statistically significant (p < 0.01) improvement was observed in the Bacopa-treated group. Test 3. There was significant improvement in reaction time in the Bacopa-treated group (p < 0.001). Insignificant changes were observed in the placebo group. Test 4. There was a significant decrease in scores (positive response, p < 0.1) in the Bacopa-treated group. Insignificant changes were observed in the placebo group. No direct comparison between the herbal treatment group and the placebo group was calculated. However, significant improvements were observed in maze learning (visual motor function) and the digit span test (immediate memory) for the treated group. No improvement was observed in the placebo group.

With regard to perceptual organization and reasoning ability, the effects of both treatments were comparable. These results show that the Brahmi treatment significantly improved discrimination learning, memory and cognitive functions in the subjects. These findings validate the potential use of Bacopa monniera in formulations to improve visual motor functions and immediate memory in children

Improved cognitive functions in stroke victims Mentat, an indigenous, Ayurvedic preparation containing Bacopa monniera, Centella asiatica, Withania somnifera, Evolvulus alsinoides and Nardostachys jatamansi, was tested for its efficacy in patients suffering from functional impairment following stroke. Twenty-four patients participated in the study. Thirteen patients received Mentat , and eleven patients received placebo for a 12-week period. The Mentat patients had better EMG (electromyographic) responses compared to the placebo group after 12 weeks54.

Memory enhancing effects in children suffering from ADHD (attention deficit hyperactivity disorder)

Study 1 Design: The efficacy of Memory Plus, a formulation containing bacosides, was determined in a double-blind, placebocontrolled, randomized trial55. Thirty-six children (mean age: 8.3 years in the Bacopa-treated group, 9.3 years in the placebo group; male: female ratio 5:1 in the Bacopa group and 3:1 in the placebo group) participated in the trial. Nineteen subjects received the Bacopa preparation (50 mg twice a day for 12 weeks), and 17 subjects received the placebo. Active drug treatment was followed by 4 weeks placebo treatment, making the total time period for the study 16 weeks. The children were evaluated based on personal information, mental control, sentence repetition, logical memory, word recall (meaningful and non-meaningful), digit span test, delayed response learning, picture recall and paired associate learning before drug administration and after 4, 8, 12 and 16 weeks. Results: Data analysis revealed significant improvement on sentence repetition, logical memory and paired associate learning following 12 weeks of administration of the Bacopa preparation. This improvement was maintained in the 16th week evaluation, 4 weeks after the withdrawal of the drug. There were no reported adverse side effects and drug tolerability was excellent. The authors of this study concluded that treatment with the Bacopa monniera preparation is safe and beneficial for children with ADHD. Study 2 Design: An Ayurvedic formulation (containing Bacopa monniera, Centella asiatica, Asparagus racemosus, Acorus calamus, Celastrus paniculatus and Xanthium strumarium) was tested in a double-blind, placebo-controlled trial on 50 normal school children over a period of six weeks. The daily dose of the composition was 10 g and contained 1 g mixed herbs (0.2 g of Bacopa monniera powder). The effects on attention, concentration and memory were observed. Significant improvement in mean reaction time (measuring attention) was observed in the group treated

with the Ayurvedic formulation compared to the group receiving the placebo (p < 0.001). However, no significant improvement in terms of enhanced concentration and memory was observed in either group56.

The effect of Bacopa monniera on convulsive disorders Study 1 Design: An early trial57 tested the efficacy of an aqueous and an ethanolic extract (defatted) of Bacopa monniera on 24 subjects suffering from convulsive disorders (grand mal epilepsy and/or diseases other than epilepsy such as cerebral palsy, intellectual disability, behavioral disorders and schizophrenia). The subjects received either 64 ml of the aqueous extract containing the equivalent of 50 g of the fresh plant or 2-4 mg of the defatted, ethanolic extract (1 mg equivalent to 200 mg fresh plant) by oral administration daily for 5 months. Results: The aqueous extract was reported to be effective in 50% of the cases as manifested in lowered frequency or occurrence of seizures. The defatted, ethanolic extract was effective in 60% of the cases, and the seizures ceased in two patients over the 5-month, treatment period. Administration in combination with anticonvulsive medications was also beneficial. In some non-epileptic patients improvement was observed in behavior, insomnia, intellectual capabilities and mood swings. However, no beneficial effects were observed in schizophrenics. Hematological and urinary biochemical parameters were found to be normal in all treated subjects.

Study 2-summary A study on 13 epilepsy patients using a defatted, ethanolic extract of Bacopa monniera was performed over a fivemonth period. The patients showed improvement in the frequency of seizures over a period of two to five months. The seizures ceased in five cases. One patient reported weakness, loss of concentration and dizziness58.

Toxicology and Recommended Doses Toxicology The LD50 of aqueous and alcoholic extracts of Bacopa monniera in rats were 1000 mg and 15 g/kg by the intraperitoneal route, respectively22. The aqueous extract given orally at a dose of 5 g/kg did not show any toxicity. The LD50 of the alcoholic extract was 17 g/kg given orally22. Both extracts did not produce any gross behavioral changes at these levels. A phase I, double-blind, placebo-controlled trial investigated the safety of pharmacological doses of isolated bacosides in healthy, male, human volunteers. Single (20-30 mg) and multiple (100-200 mg) daily doses of bacosides for 4 weeks were well tolerated and devoid of adverse side effects.11 Except for an isolated case in one uncontrolled trial, no untoward side effects were reported in any of the clinical trials conducted.59 Bacopa monniera has a record of several hundred years of safe use in Ayurvedic medicine. The daily doses generally recommended in traditional practice are 8-16 ml of the infusion or 5-10g of the powder. The

recommended daily dose of Bacopa (standardized to contain a minimum of 50% Bacosides A and B) for adults and children are as follows: Adult: 200 mg - 3 times daily; Children: 100 mg - 3 times daily Bacopa monniera can be combined with Ginkgo biloba, Ashwagandha, antioxidants such as rosemary, Centella asiatica and other nutraceuticals to yield comprehensive formulations beneficial in the management of cognitive dysfunction. A recent publication lists Bacopa monniera as one of the five substances (the others being Ginkgo biloba, phosphatidylserine, acetyl-carnitine and vinpocetine) offering interesting contributions (specifically antianxiety, anti-fatigue and memory-strengthening effects) to a personalized approach in the integrative management of cognitive dysfunction60. The adaptogenic41,48 properties of the herb would be beneficial in the management of stress related conditions. Analysis of Bacopa Identification By Thin Layer Chromatography61: Mobile phase: n-Butanol : Ethyl acetate : Water 4:1:5 Stationary phase: Silica gel GF254 precoated TLC plates of 0.2 mm thickness Standard Preparation Weigh accurately about 10 mg of Bacoside A of 90% purity in a 10 ml volumetric flask. Dissolve and dilute to volume with methanol.

Sample preparation Weigh accurately about 500 mg of sample, previously powdered, and transfer it into a 250 ml round bottom flask fitted with a ground glass joint. Add 70 ml of methanol and reflux on a water bath for 1 hour. Cool, wash the condenser with 10 ml of methanol and filter. Adjust the volume to 100 ml with methanol. Procedure Apply 10 l of the sample preparation and 10 l of the standard preparation separately (side by side) on the chromatoplate. Allow the spots to dry. Develop the plate in the previously saturated chamber containing the mobile phase. After developing, remove the plate and dry in a current of air. Spray the plate with anisaldehyde sulfuric acid spray reagent. Heat the plate at 105C for 5 minutes. Compare the Rf value of the spot corresponding to Bacoside A in the sample preparation with Bacoside A in the standard spot. Fig. 6.1 shows a sample chromatogram. Mobile phase: n-butanol: Water:Ethyl acetate 5:4:1 Standard preparation: 10mg in 10 ml methanol Sample preparation: 200 mg in 5 ml of warm methanol, filter and spot Volume: 10 mL Detection: Spray with anisaldehyde sulfuric acid reagent and heat plate at 105 C. Rf: value: 0.42 *Note: Only major spots are shown.

Quantitative Estimation of Bacosides content61: By High Performance Liquid Chromatography: Mobile phase: Prepare a filtered and degassed solution containing methanol and water in the ratio of 9:1. Adjust the mobile phase composition, if required, for better resolution.

Standard preparation: Weigh accurately about 2g of Working standard (50% Bacoside A & B) into a 100ml flask. Add 10ml of methanol to dissolve the standard. Then add 50ml of 10% v/v sulfuric acid. Reflux at 110C for 4 hours. Cool and transfer to a separating funnel. Extract with chloroform three times using 30 ml of the solvent for each extraction. Collect all the chloroform layers. Add 0.5 ml of 0.01% v/v ammonia solution and wash with water until neutral. Collect the chloroform layer through sodium sulfate into a beaker. Evaporate the chloroform on a water bath. Dissolve the residue in methanol and make the volume up to 100 ml. Sample preparation: Carry out the procedure as per the standard preparation using 2 g of finely powdered sample. Chromatographic system: The liquid chromatograph is equipped with a 262 nm detector and 250 mm x 4.6 mm column that contains ODS(C18) packing [SIGMA- ALDRICH HYPERSIL ODS 5 mm will be suitable]. The flow rate is about 1.8 ml per minute. Chromatograph the standard preparation and record the peak responses for the bacosides. The relative standard deviation for replicate injections is not more than 2.0%. Procedure: Separately inject equal volumes (20 l) of the standard preparation and sample preparation into the chromatograph, record the chromatograms and measure the responses for the peaks corresponding to Bacosides A & B . Calculate the content of the total Bacosides in the sample as follows. Sum of areas of Bacosides A & B in sample x Std. concentration in mg /ml x Std. Purity in % x 100 ----------------------------------------------------------------------------------------------------------------------------------Sum of areas of Bacosides A & B in standard x sample concentration in mg / ml x (100 LOD)

= Percentage of Bacosides

References Chopra, R.N., Chopra, I.C. Handa K.K. and Kapur, L.D. (1994). Indigenous Drugs of India. Academic Publishers, Calcutta, India Kirtikar, K.R. and B.D. Basu, (1993). Indian Medicinal Plants. Periodical Experts Book Agency, New Delhi, India. Nadkarni, A. K., (1954). Indian Materia Medica, Vol. 1, Popular Book Depot, Bombay. Chopra, R.N., (1958). Indigenous drugs of India, 2nd Edition, U.N. Dhur and Sons, Calcutta. P.341. Chopra, I.C., Handa, K.L. and. Sobti, S.N (1956). Indian J. Pharm. 18: 369. Bhishagratna K.K., editor-translator. (1991) Sushruta Samhita., Chowkhamba Sanskrit Series Office: Varanasi, India. Bhavamishra - Bhavprakash Nighantu, Sastu Sahitya Vardhak Karyalaya, Ahmedabad, India. Dymock, W, Warden, C.J.H. and Hooper, D. Pharmacographia Indica : A history of the principal drugs met with in British India, Vol II, Reprinted by the Institute of Health and Tibbi Research, Pakistan., 332-333. Swerdlow, J.L., (1995) Quiet Miracles of the Brain, The National Geographic, June: 2.-21 Chhachhi, V., (1996) Business Today, March 22- April 6 issue, page 18. Singh, H.K. and Dhawan, B.N. (1997) Neuropsychopharmacological effects of the Ayurvedic nootropic Bacopa monniera Linn. (Brahmi). Indian Journal of Pharmacology. 29:S359-S365. DeFeudis, F.V. (1991) Ginkgo biloba Extract (EGb 761): Pharmacological activities and clinical applications. Elsevier. Kidd, P.M. (1995) Phosphatidylserine (PS) a remarkable brain cell nutrient. Lucas Meyer, Inc. Satyawati, G., Gupta, A.K. and Tandon, N. (1987) in Medicinal plants of India, Vol. 2, (ICMR, New Delhi), 50. Uphof, J.C. Th. (1968) Dictionary of economic plants, Verlag von J. Cramer, (N.Y.), 62. "Wonder memory drug will hit Indian market" : News report in The Times of India, March 3, 1996 Tripathi, Y.B. et al. (1996) Bacopa monniera Linn. as an antioxidant : mechanism of action. Indian Journal of Experimental Biology, Vol. 34, pp. 523-526. Bhattacharya SK, et al. (2000) Antioxidant activity of Bacopa monniera in rat frontal cortex, striatum and hippocampus. Phytother Res 14(3):174-9 Jain, P.,. Khanna, N.K, Trehan, T., Pendse,V.K. and Godhwani, J.L. (1994). Antiinflammatory effects of an Ayurvedic preparation, Brahmi Rasayan, in rodents. Indian J. Exp. Biol. 32: 633-636. Bhukani, D.S., Dhar, M.L., Dhar, M.N., Dhawan, B.N. and Mehrotra, B.N. (1969) Screening of Indian plants for biological activity II Indian J. Exp. Biol., 7, 250. Yadav, S.K., A.K. Jain, S.N. Tripathi and Gupta, J.P. (1989). Irritable bowel syndrome:therapeutic evaluation of indigenous drugs. Indian J. Med. Res.,90: 496-503. Martis, G., Rao, A. And Karanth, K.S. (1992) Neuropharmacological activity of Herpestis monniera. Fitoterapia, 63(5), 399-404. Barrett, S.C.H. and Strother, J.L. (1978) Taxonomy and Natural History of Bacopa in California. Systematic Botany, 5(4) : 408-419. Mathew, K. M., (1984). The Flora of Tamil Nadu and Carnatic, Rapinat Herbarium, St. Joseph's College, Tiruchirappalli, India. Report on Herbal Industry: Brahmi Bose, K.C. and N.K. Bose, (1931). Observations on the actions and uses of Herpestis monniera J. Indian Medical Association., 1: 60. Basu, N.K. and Pabrai, P.R. (1947). Quart. J. Pharm.,20: 137. Basu, N.K. and Walia, J.N.(1944). Indian J. Pharm., 4: 84. Sastry, M.S., Dhalla N.S. and Malhotra, C.L. (1959). Chemical investigation of Herpestis monniera Linn. (Brahmi). Indian J. Pharm., 21: 303-304. Rastogi, R.P. and M.L. Dhar,(1960). J. Sci. Industr. Res., 19B: 455. Datta, T., and U.P. Basu, (1963). Terpenoids: Part II- Isolation of a new triterpene saponin, Monnierin, from Bacopa monniera Wettst. Indian J. Chem., 1: 408-409. Chatterji, N., R.P. Rastogi and M.L. Dhar, (1965). Chemical examination of Bacopa monniera Wettst.: Part IIsolation of chemical constituents. Indian J. Chem., 1: 212- 215.

Chatterji, N., R.P. Rastogi and M.L. Dhar, (1965). Chemical examination of Bacopa monniera Wettst.: Part II- The constitution of bacoside A. Indian J. Chem., 3: 24-29. Basu, N., Rastogi, R.P. and Dhar, M.L.(1967). Chemical examination of Bacopa monniera Wett. Part 111. Bacoside B. Indian J. Chem., 1: 212. Rastogi, S., Pal, R. And Kulshreshta, D.K. (1994) Bacoside A3 - a triterpenoid saponin from Bacopa monniera. Phytochemistry 36(1) : 333-337. Aithal, H.N.and Sirsi, M. (1961). Pharmacological investigations on Herpestis monniera H.B. & K. Indian J. Pharm., 23: 2-5. Ganguly, D.K. and Malhotra, C.L.(1967). Some behavioral effects of an active fraction from Herpestis monniera Linn. (Brahmi). Indian J. Med. Res., 55: 473-482. Singh, H.K. and Dhawan, B.N. (1982) Effect of Bacopa monniera (Linn.) extract on avoidance responses in rat. J. Ethnopharmacology, 5(2) : 205-214. Malhotra, C.L. and Das, P.K. (1959). Pharmacological studies of Herpestis monniera Linn. (Brahmi). Indian J. Med. Res., 47: 294-305. Singh, H.K., Rastogi, R.P., Srimal, R.C. and Dhawan, B.N. (1988) Effect of Bacosides A and B on avoidance responses in rats. Phytotherapy Research, 2(2) : 70-75. Bhattacharya, S.K. and Ghosal, S. (1998) Anxiolytic activity of a standardized extract of Bacopa monniera: an experimental study. Phytomedicine, 5(2):77-82. Shukla, B.,. Khanna N.K and Godhwani, J.L. (1987). Effect of Brahmi Rasayan on the central nervous system. J. Pharmacol. 21: 65-74. Vohora, D. et al. (2000) Protection from phenytoin-induced cognitive deficit by Bacopa monniera, a reputed nootropic plant. Shanker, G. and Singh, H.K. (2000) Anxiolytic profile of standardized Brahmi extract. Abstract of paper presented at the conference on "Frontiers in Pharmacology and Therapeutics in the 21st century" Dar, A. and Channa, S. (1999) Calcium antagonistic activity of Bacopa monniera on vascular and intestinal smooth muscles of rabbit and guinea pig. J. Ethnopharmacol. 66(2):167-74. Agrawal, A., Gupta, U., Dixit, S.P., Dubey, G.P. (1993) Changes in brain biogenic amines under the influence of an Ayurvedic drug, Brahmi ,and its effect on discrimination learning. Pharmacopsychoecologia, Vol 6(1) 15-19. Bhattacharya, S. et al. (1999) Effect of Bacopa monniera on animal models of Alzheimers disease and perturbed central cholinergic markers of cognition in rats. Res. Commun. In Pharmacol and Toxicol. 4(3&4), 11/1-11/2. Singh, R.H. and Singh, L.(1980). Studies on the anti-anxiety effect of the Medhya Rasayana drug Brahmi (Bacopa monniera Wet.)- Part 1. J. Res. Ayurveda and Siddha 1:133-148 Sinha, D. (1978). Anxiety scale, Rupa Psychological Corporation, Varanasi, India. Asthana, H.S. (1968). Adjustment Iinventory, Rupa Psychological Corporation, Varanasi, India. Joshi, M.C., (1974). Cancellation sheet for testing mental fatigue. Rupa Psychological Corporation, Varanasi, India. Agrawal, A., Gupta, U., Dixit, S.P., Dubey, G.P. (1993) Management of mental deficiency by an indigenous drug Brahmi. Pharmacopsychoecologia, Vol 6(1) 1-5. Sharma, R. et al. (1987), Efficacy of Bacopa monniera in revitalizing intellectual functions in children J. Res. Ind. Med. Agarwal, U, Dixit, S.P and Dubey,G.P. (1994) The management of post-stroke functional disability by an indigenous formulation Mentat. The Indian Practitioner. XLVII(9):759-761. Negi, K.S. et al. (2000) Indian Journal of Psychiatry, Vol 42 April, Supplement http://www.ijponline.org/indIJP%20PsychoPharmacology.html Kaur, B.R. et al. (1998) Effect of an Ayurvedic formulation on attention, concentration and memory in normal school going children. Indian Drugs. 35(4):200-203. Mukherjee, GD and Dey, CD (1968) Comparative study on the antiepileptic action of some common phytoproducts. Journal of Experimental Medical Sciences.11(4):82-85 Mukherjee,GD and Dey, CD (1966) Clinical trial on Brahmi I Journal of Experimental Medical Sciences. 10(1):5-11. Morgan, M. and Bone, K. (1999) Bacopa. Modern Phytotherapist. Vol 5(2):21-27. Kidd, P.M. (1999) A review of nutrients and botanicals in the integrative management of cognitive dysfunction. Alternative Medicine Review. 4(3):144-161.