8.1 INTRODUCTION Plant profile of Bacopa monnieri

Transcription

1 .1 INTRODUCTION.1.1 Plant profile of Bacopa monnieri Bacopa monnieri, has been used for centuries in the Ayurveda, a holistic system of medicine originating from India. The name Brahmi has been derived from the word 'Brahma', the mythical 'creator' in the Hindu pantheon. Because the brain is the centre for creative activity, any compound that improves the brain health is called Brahmi, which also means 'bringing knowledge of the supreme reality' In India; Bacopa monnieri is largely treasured as a revitalizing herb used by Ayurvedic medical practitioners for almost 3000 years. It is classified as a medhyarasayana, a drug used to improve memory and intellect (medhya). The herb has been mentioned in several ancient Ayurvedic treatises including the 'Charaka Samhita' since sixth century AD, in which it is recommended in formulations for the management of a range of mental conditions including anxiety, poor cognition and lack of concentration, as a diuretic and as an energizer for the nervous system and the heart 245. Bacopa monnieri Linn. (a) Classification: Kingdom: Plantae Division: Magnoliophyta Class: Magnoliopsida Order: Lamiales Family: Scrophulariaceae Genus: Bacopa Species: B. monnieri Standardization of Some Plant-Based Formulations By Modern Analytical Techniques 177

2 (b) Vernacular names: Sanskrit : Brahmi, Nira-brahmi Hindi : Brahmi Marathi : Neer brahmi Kannada : Nirubrahmi Tamil : Neer brahmi Telugu : Neer brahmi English : Water hyssop, Pennell, Herb-of-Grace (c) Part used : whole plant (d) Origin and distribution B. monnieri is a herb found in wetlands throughout the Indian subcontinent in damp and marshy or sandy areas near streams in tropical regions. The genus Bacopa includes over 100 species of aquatic herbs distributed throughout the warmer regions of the world. Apart from India, Nepal, Sri Lanka, China, Taiwan and Vietnam and is also found in Florida state and other southern states of USA 246, 247. (e) Botanical description: Bacopa monnieri is a small herb with 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. Stem is prostrate, (sub) succulent and herbaceous. Leaves are decussate, simple, oblong, 0.4 cm, succulent, punctuate, penninerved, margin entire, apex obtuse, sessile. Flowers are axillar, solitary, bracteate, linear, pedicel to 0.5 cm, purple in colour 246. Standardization of Some Plant-Based Formulations By Modern Analytical Techniques 17

3 (f) Traditional uses: Brahmi belongs to a group of medicinal plants classified as Medhyarasayana in Ayurveda; these are nervine tonics used to promote mental health and improve memory and intellect 24. Brahmi is also considered to promote youthful vitality and longevity. In Ayurvedic medicine it is described as being cold, sweet, astringent, diuretic, laxative and as a tonic for the heart and nerves. Brahmi is said to clear the voice, improve digestion and dispel poisonous affections, splenic disorders and blood impurity. In Ayurveda, these are indicated in dermatosis, dyspepsia, emaciation and insanity 249. Bacopa is also used in the treatment of respiratory ailments including bronchitis and asthma 250 and has been listed as a diuretic 251. In India and Pakistan, Bacopa is used internally as a febrifuge, nervine and cardiac tonic, and a hot poultice of the plant is applied in acute bronchitis, cough and children's chest conditions 252. In the Indian Herbal Pharmacopoeia (199) bacopa is listed as a 'brain tonic 253. Fig..1: Whole plant of Bacopa monnieri Standardization of Some Plant-Based Formulations By Modern Analytical Techniques 179

4 (f) Pharmacology and Clinical Studies: i. Sedative and Tranquillizing Properties Earlier studies reported a sedative effect of glycosides named hersaponins 254. A subsequent study has found that the alcoholic extract, and to a lesser extent the aqueous extract of the whole plant exhibited tranquilizing effects on albino rats and dogs 255. On the other hand, it has been found that the alcoholic extract of the plant and chlorpromazine improved the performance of rats in motor learning 256. ii. Cognitive effects It has been reported that a standardized bacosides-rich extract of Bacopa monnieri, reversed the cognitive deficits induced by intracerebroventricularly administered colchicines and injection of ibotenic acid into the nucleus Basalis magnocellularis 257. The cognition facilitating activity of the Bacopa monnieri 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 conditioned taste aversion and conditioned shock avoidance response 25, 259. Antioxidant properties of Bacopa monnieri s and its ability to balance super oxide dismutase (SOD) and catalase levels were postulated to account for this effect 260. iii. Antidepressant and Antianxiety Effects Research using a rat model of clinical anxiety demonstrated that a Bacopa monnieri extract containing 25% bacoside A content exerted anxiolytic activity comparable to lorazepam, a common benzodiazepine anxiolytic drug. It was noted that the Brahmi extract did not induce amnesia, side effects associated with lorazepam, but instead had a memory-enhancing effect Standardization of Some Plant-Based Formulations By Modern Analytical Techniques 10

5 iv Anti-Epileptic Effects Although Bacopa monnieri has been indicated as a remedy for epilepsy in Ayurvedic medicine, 265 research in animals showed anticonvulsant activity only at high doses over extended periods of time. Early research in India demonstrated that hersaponin (an active constituent) exhibited protection against seizures in mice and mentioned the possibility of its use as an adjuvant in treatment of epilepsy 266,267. v Antioxidant and Adaptogenic Properties Bacopa monnieri extract or bacosides have shown antioxidant activity and antistress activity 274, 275. Based on animal study results, bacosides were shown to have antioxidant activity in the hippocampus, frontal cortex and striatum 276. Animal research has shown that the Bacopa monnieri extracts modulate the expression of certain enzymes involved in generation and scavenging of reactive oxygen species in the brain 277. It was suggested that the adaptogenic properties of the herb would be beneficial in the management of stress related conditions as Bacopa monnieri showed the potential to be effective in stress in a study on rats vi Gastrointestinal Effects Some in vitro, animal and human studies have investigated the effects of Bacopa monnieri extract on the gastrointestinal tract. In vitro studies have demonstrated direct spasmolytic activity on intestinal smooth muscle, via inhibition of calcium influx across cell membrane channels. This property suggests that Bacopa monnieri extract may be of benefit in conditions characterized by intestinal spasm such as irritable bowel syndrome (IBS) Standardization of Some Plant-Based Formulations By Modern Analytical Techniques 11

6 (g)clinical Studies: Cognition Numerous clinical studies have been carried out to date to establish the efficacy of Bacopa monnieri in memory and attention disorders and to study its acute and chronic effects clinically on cognitive function. A study was conducted to measure the effect of Bacopa monnieri extract on human memory. Seventy-six adults aged between 40 and 65 years volunteered for the double-blind randomized, placebo control study. The results showed a significant effect of Bacopa monnieri extract on the test for the retention of new information. In the follow-up tests, it was found that the rate of learning was unaffected, suggesting that Bacopa monnieri decreases the rate of forgetting of newly acquired information. In adults, only chronic administration was shown to enhance cognitive effects. On the other hand, significant cognitive-enhancing benefits have been demonstrated with more chronic administration of Bacopa monnieri extract, as demonstrated in a double-blind, placebo-controlled, 12-week trial utilizing the same patient selection criteria and same dose of Bacopa monnieri extract (300 mg daily) containing 55% combined bacosides 295, 296. At the end of the 12-week study, results indicated a significant improvement in verbal learning, memory consolidation and speed of early information processing in the treatment group compared to placebo Anxiety and Depression In one latest study, effects of a standardized Bacopa monnieri extract (300 mg/day) on cognitive performance, anxiety and depression in the elderly were evaluated in a randomized, double-blind, placebo-controlled clinical trial with a placebo run-in of 6 weeks and a treatment period of 12 weeks. Bacopa Standardization of Some Plant-Based Formulations By Modern Analytical Techniques 12

7 monnieri participants had enhanced Auditory Verbal Learning Test (AVLT), delayed word recall memory scores relative to placebo, decreased Center for Epidemiologic Studies Depression scale (CESD10) depression scores, combined state plus trait anxiety scores and heart rate over time compared to that of the placebo group. This study provided further evidence that Bacopa monnieri has a good potential for safely enhancing cognitive performance in the ageing. Gastrointestinal Disorders A double-blind, randomized, placebo-controlled trial of 169 patients with irritable bowel syndrome (IBS) compared the effects of an Ayurvedic preparation containing Bacopa monnieri to standard therapy (clidinium bromide, chlordiazepoxide and psyllium) 305. (h) Toxicity 306 : Bacopa monnieri has a record of several hundred years of safe therapeutic use in Ayurvedic medicine. A double-blind, placebocontrolled clinical trial of healthy male volunteers investigated the safety of pharmacological doses of isolated bacosides over a 4-week period. Concentrated bacosides given in single (20-30 mg) and multiple ( mg) daily doses were well tolerated and without adverse effects. The LD 50 of aqueous and alcoholic extracts of Bacopa monnieri in rats was 1000 mg and 15 g/kg by the intraperitoneal route, respectively. The aqueous extract given orally at a dose of 5 g/kg did not show any toxicity. (i) Phytochemistry : Compounds responsible for the pharmacological effects of Bacopa monnieri include alkaloids, saponins and sterols. Detailed investigations first reported the isolation of the alkaloid 'brahmine' from Bacopa monnieri. Later, other alkaloids like nicotine and herpestine have also Standardization of Some Plant-Based Formulations By Modern Analytical Techniques 13

8 been reported. Subsequently, the isolation of D-mannitol and a saponin, hersaponin and potassium salts was reported. The major chemical entity shown to be responsible for neuropharmacological effects and the nootropic action or antiamnestic effect of Bacopa monnieri is bacoside A, assigned as 3-(a-L-arabinopyranosyl)-O-b-D-glucopyranoside- 10,20-dihydroxy-16-keto-dammar-24-ene. Bacoside A usually co-occurs with bacoside B; the latter differing only in optical rotation and probably an artefact produced during the process of isolating bacoside A. On acid hydrolysis, bacosides yield a mixture of aglycones, bacogenin A1, A2, A3, which are artefacts, and two genuine sapogenins, jujubogenin and pseudojujubogenin and bacogenin, A4, identified as ebelin lactone pseudojujubogenin, were isolated. Successively, a minor saponin bacoside A1 and a new triperpenoid saponin, bacoside A3, were isolated. Later, three new dammarane-type triterpenoid saponins of biological interest, bacopasaponins A, B and C, pseudojujubogenin were isolated and a new dammarane-type pseudojujubogenin glycoside, bacopasaponin D, were identified by spectroscopic and chemical transformation methods. Five new pseudojujubogenin glycosides designated as bacopaside I from II were isolated from glycosidic fraction of the methanol. In addition, the isolation of three new phenylethnoid glycosides, viz. monnierasides I-III along with the known analogue plantainoside B was reported from the glycosidic fraction of Bacopa monnieri. Moreover, an isolation of a new saponin, a jujubogenin, named bacopasaponin G, and a new glycoside, phenylethyl alcohol was also reported Standardization of Some Plant-Based Formulations By Modern Analytical Techniques 14

9 A and B. (j) Active Principles: Fig..2: Structures of some saponins from Bacopa monnieri Standardization of Some Plant-Based Formulations By Modern Analytical Techniques 15

10 .1.2 Bacoside A as marker compound for Standardization Bacoside A is one of the major phytoconstituents of Bacopa monnieri Fig..3: Structure of major saponins of Bacoside A The nootropic activity has been attributed to the presence of two saponins, namely Bacoside A and Bacoside B. From this two markers bacoside A is considered and proved to be more pharmacologically active for nootropic activity 315. Several pharmacological, clinical, analytical and agronomical studies concerning B.monnieri have recently published 316 with reports on the content of bacoside A. Chemically it is a mixture of four saponins namely, bacoside A 3, bacopaside II, jujubogenin isomer of bacopasaponin C and Standardization of Some Plant-Based Formulations By Modern Analytical Techniques 16

11 bacopasaponin C. As bacoside A is the major constituent from Bacopa monnieri responsible for the therapeutic activity, it is used as a Marker compound for Marker Based Standardisation of formulations containing Brahmi. Standardization of Some Plant-Based Formulations By Modern Analytical Techniques 17

12 .2 ISOLATION OF BACOSIDE A.2.1 Procurement of commercial extract of Bacopa monnieri (Linn.) Pennel Commercial methanol extract of Bacopa monnieri (Linn.) Pennel was used to isolate bacoside A. Bacopa monnieri extract was provided as gift sample by Amsar Pvt. Ltd..2.2 Fractionation of methanol extract of Brahmi The methanol extract was dissolved in methanol solvent and was refluxed for 4 hrs with 100 ml of water. After refluxing 400 ml of water was added and allowed to cool. The solution was filtered and was evaporated to its half the volume. The concentrated solution was then extracted with 4 x 300ml of chloroform. All portions of chloroform fractions were pooled together. The final fraction was washed with water to remove any water soluble matter. The washed fraction was dried completely and weighed. The yield of chloroform fraction obtained was of 9.07% w/w..2.3 Procedure of isolation of bacoside A by Preparative Thin Layer Chromatography Stationary phase: Preparative thin layer chromatography was carried out using precoated silica gel aluminium plate 60F 254 (20 cm 10 cm with 250 µm thickness; E. Merck, Darmstadt, Germany, supplied by Anchrom Technologists, Mumbai). Development of plates: The plates were pre-washed by methanol and activated at 60 C for 5 min prior to chromatography. The mobile phase consisted of toluene: ethyl acetate: methanol: formic acid (3:4:3:1) (v/v) and 15 ml of mobile phase was Standardization of Some Plant-Based Formulations By Modern Analytical Techniques 1

13 used for each analysis. Linear ascending development was carried out in 20 cm x 10 cm twin trough glass chamber (Camag, Muttenz, Switzerland) saturated with the mobile phase. The optimized chamber saturation time for mobile phase was 20 min at room temperature (25 C ± 2) at relative humidity of 60% ± 5. Sample preparation: The chloroform fraction was used for isolation of bacoside A. The chloroform fraction of Brahmi was dissolved in chloroform to make a stock solution of 50mg/ml. Location of spots Chloroform fraction was spotted manual by means of a glass capillary in the form of a band. The chromatographic conditions reported in Indian herbal pharmacopeia were used and detection was carried under 540 nm 251. The band with R f 0.44 matching with standard bacoside A, was scrapped and the silica was dispersed in chloroform. The chloroform solution was then sonicated for 30 min and then filtered. The filtrate was dried and TLC studies were carried out. The TLC profile showed a single band indiacting a fairly pure compound (COMPONENT III, Fig..4) Standardization of Some Plant-Based Formulations By Modern Analytical Techniques 19

14 Band of Bacoside A after spraying with 10 % acid alcohol R f = Fig..4: Videoimage of chloroform fraction of Brahmi showing the Bacoside A band at R f 0.44 Track 1: Chloroform fraction of Brahmi Standardization of Some Plant-Based Formulations By Modern Analytical Techniques 190

15 1 2 Fig.. 5 : Videoimage of TLC plate showing Component III and chloroform fraction of Brahmi Track 1: Component III Track 2: Chloroform fraction of Brahmi Component III showed a single pink spot on TLC plate after spraying with 10 % methanolic shlphuric acid indicating presence of bacoside A..2.4 Physicochemical analysis of component III The component III was evaluated for different physicochemical parameters such as melting point, solubility and elemental analysis. The parameters were compared with that of the reported data of bacoside A. Solubility was tested in different solvents such as chloroform, methanol and water. It was observed that component III was readily soluble in methanol. The Lassaigne sodium Standardization of Some Plant-Based Formulations By Modern Analytical Techniques 191

16 fusion test was carried out for checking the presence/ absence of elements. The elements present were Carbon, hydrogen. Nitrogen, halides and sulphur were found to be absent. The melting point was found to be 249 C which was very close to melting point of the standard C. The yield obtained was 0.7% from the commercial methanol extract. The summarized data is mentioned in Table.1. Table.1: Physicochemical analysis of component III Sr. Parameters Component III Standard (Reported in literature ) 1. Color White White 2. Solubility Methanol Methanol 3. Elements present C, H, (O) C,H, (O) 4. Melting point 249 C C 6. Yield (%w/w) Confirmation of identity of isolated compound as Bacoside A The isolated component II was confirmed to be bacoside A by comparing with reference standard by HPTLC, HPLC, UV and LC-MS studies. Standardization of Some Plant-Based Formulations By Modern Analytical Techniques 192

17 HPTLC Studies: Component III was dissolved in chloroform and the HPTLC analysis was carried out using the following densitometric conditions: Stationary phase Mobile phase : Precoated plates of Silica Gel 60 GF 254 (Merck) : Toluene: ethyl acetate: methanol: Gla. acetic (3:4:3:1) Saturation time Development time Wavelength Lamp Visualizing reagent Band width Length of Chromatogram : 20 min : 20 min : 540 nm : Tungsten : Spraying with 10% Acid alcohol : 7 mm : cm Standardization of Some Plant-Based Formulations By Modern Analytical Techniques 193

18 HPLC Studies: The component III of were analyzed by HPLC technique using the following conditions: Column: C 1 Phenomenex (250 x 4.60mm) - 5µ Mobile phase : Gradient Acetonitrile (A) 0.05% (v/v) Orthophosphoric acid in water (B) Flow rate: 1.5 ml/min Time (min) Function Value 0 B. Conc B. Conc B.Conc STOP Wavelength : 205nm Injection loop capacity: 20µl Concentration of Samples: 1mg/ml of standard and isolated compound. Standardization of Some Plant-Based Formulations By Modern Analytical Techniques 194

19 Fig..6: HPTLC chromatogram of reference standard Bacoside A ( R f = 0.44) Fig..7: HPTLC chromatogram of component III (R f = 0.44) Standardization of Some Plant-Based Formulations By Modern Analytical Techniques 195

20 Fig..: HPLC profile of reference standardd Fig..9: HPLC profile of Component IIII Standardization of Some Plant-Based Formulations By Modern Analytical Techniques 196

21 The isolated component III showed a single peak at R f 0.44 (Fig.6), which perfectly matched with the standard bacoside A Fig.7) as reported in Indian herbal pharmacopeia 250. The HPLC profile of component III (Fig.9), also matched with Standard bacoslde A (Fig.). Four peaks with Rt: 19.2 min, 19.9 min 21.7min and 22. min which matched with standard profile with Rt: min, min 21.6 min and 22.1 min. UV Spectroscopy: The UV spectrum of the standard bacoside A and isolated component IIII was recorded in methanol. The UV λ max of standard bacoside A and also of component III was obtained at 27 nm. LC-MS Analysis: The four compounds detected in HPLC studies were separated and mass to charge ratio was determined by LCMS. M. Deepak et.al and Cillara S et.al., have reported that bacoside A is a mixture of four isomers namely bacoside A 3, bacopaside II, Jujubogenin isomer of Bacopasaponin C and Bacopasaponin C 316,317. HPLC profile of component III matched with the reported four isomer s profile, Thus it was confirmed with the fragmentation pattern of all four peaks. The values are as follows: Standardization of Some Plant-Based Formulations By Modern Analytical Techniques 197

22 Standardization of formulations containing Bacopa monnieri Fig..10.1: Mass Spectrum of Peak O-[b [b-d-glucopyranosyl-(1-3)-o-{a-l-arabinofuranosyl arabinofuranosyl-(1-2)}o-(b-d D-glucopyranosyl)] Aglycon Jujubogenin RT: LCMS m/z , base peak 455.3, , (Fig..10.1) Standardization of Some Plant Plant-Based Based Formulations By Modern Analytical Techniques 19

23 Standardization of formulations containing Bacopa monnieri Fig..10.2: Mass Spectrum of Peak O-[a [a-l-arabinofuranosyl-(1-2)-{b-d-glucopyranosyl glucopyranosyl-(1-3)}-b-dglucopyranosyl] RT: Aglycon Pseudojujubogenin AglyconLCMS m/z , base peak , 455.3, , , LCMS (Fig..10.2) Standardization of Some Plant Plant-Based Based Formulations By Modern Analytical Techniques 199

24 Standardization of formulations containing Bacopa monnieri Fig..10.3: Mass Spectrum of Peak O-[a [a-l-arabinofuranosyl-(1-2)-{b-d-glucopyranosyl glucopyranosyl-(1-3)}-b-dglucopyranosyl] RT: Aglycon Pseudojujubogenin AglyconLCMS m/z , base peak , 455.3, (Fig..10.3) LCMS- Standardization of Some Plant Plant-Based Based Formulations By Modern Analytical Techniques 200

25 Standardization of formulations containing Bacopa monnieri Fig..10.4: Mass Spectrum of Peak O-[a [a-l-arabinofuranosyl-(1-2)-{b-d-glucopyranosyl glucopyranosyl-(1-3)-}-al-arabinopyranosyl] arabinopyranosyl] Aglycon jujubogenin RT: m/z , base peak 455.3, , (Fig..10 Fig..10.4) Standardization of Some Plant Plant-Based Based Formulations By Modern Analytical Techniques 201

26 The HPTLC, HPLC, UV and LCMS studies confirmed that component III is bacoside A comprising of acoside A 3, bacopaside II, Jujubogenin isomer of Bacopasaponin C and Bacopasaponin C. Standardization of Some Plant-Based Formulations By Modern Analytical Techniques 202

27 .3 METHOD DEVELOPMENT.3.1 Preparation of standard Bacoside A solutions Reference standard solutions were prepared by dissolving 5.0 mg of bacoside A in 10 ml methanol, yielding stock solution of concentration = 0.5 mg ml 1. From this 0.1, 0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0., 0.9, 1.0, 2.0, 3.0, 4.0, 5.0, 6.0, 7.0,.0, 9.0, 10 µl of this solution were applied using LINOMAT 5 applicator with the band width of mm., which gave different concentration ranging ng / spot respectively..3.2 Chromatographic conditions The different parameters of HPTLC such as mobile phase, band width, and detection wavelength were optimized. Stationary phase: The commonly used stationary phase i.e, Silica Gel 60 GF 254 was used as stationary phase. The plates were pre-washed by methanol and activated at 60 C for 5 min prior to chromatography. The compound was well separated and resolved in the extracts of Brahmi. Mobile phase: Mobile phase reported in Indian herbal pharmacopeia is toluene: ethyl acetate: methanol: gla. acetic acid, 3:4:3:1. This mobile phase composition was slightly modified to toluene: ethyl acetate: methanol: gla. acetic acid, 3:4:2.5:1. The change was done as the peak of bacoside A was not very clear and not well resolved when it was observed in matrix like extract. There was no significant change in the R f value by altering the composition, however the resolution of the peak was better (Fig.9 and Fig.10). Band width: Three different band widths 6 mm, 7 mm, mm were tried. Band width of 7 mm gave good separation without the over saturation of the spot with sample. Standardization of Some Plant-Based Formulations By Modern Analytical Techniques 203

28 Detection: The TLC plate was scanned under 540 nm (visible range) after spraying with 10 % acid alcohol using tungsten lamp. The following densitometric conditions were used for HPTLC studies: Stationary phase : Mobile phase : Precoated plates of Silica Gel 60 GF 254 (Merck) : Toluene: ethyl acetate: methanol: Gla. acetic acid (3:4:2.5:1) Saturation time Development time Wavelength Lamp Spraying reagent Band width Length of : 20 min : 20 min : 540 nm : Tungsten : 10 % sulphuric acid in methanol : 7 mm : cm chromatogram Linear ascending development technique was carried out in 20 cm x 10 cm twin trough glass chamber (Camag, Muttenz, Switzerland) saturated with the mobile phase. Densitometric scanning was performed with Camag TLC scanner III in the reflectance-absorbance mode and operated by Win CATS software (1.3.0 Camag). Concentrations of the compound chromatographed were determined from the intensity of diffusely reflected light. Evaluation was carried out by comparing peak areas with linear regression. The proposed method gave very good separation and resolution of the bacoside A in the methanol extract of Brahmi vati. (R f value = 0.44) as indicated in (Fig.11 and Fig.12). Standardization of Some Plant-Based Formulations By Modern Analytical Techniques 204

29 Fig..11: HPTLC Chromatogram of bacoside A using optimized parameters Fig..12: HPTLC profile of methanol extract of Brahmi vati using optimized parameters Standardization of Some Plant-Based Formulations By Modern Analytical Techniques 205

30 .4 METHOD VALIDATION Validation of HPTLC method as per ICH guidelines Analytical method validation is a process of performing several tests designed to verify that an analytical test system is suitable for its intended purpose and is capable of providing useful and valid analytical data. The developed method was validated for various parameters like linearity, limit of detection (LOD), limit of quantitation (LOQ), accuracy, precision, robustness and system suitability as per ICH guidelines..4.1 Linearity: Different concentrations of bacoside A were spotted and analyzed. The analysis was done in triplicate and the concentration range showing regression coefficient (r 2 ) near to one with precise value of r 2 in all triplicate analysis was chosen. Linearity was evaluated in the range of ng / spot of bacoside A. Peak area versus concentration was subjected to least square linear regression analysis and the slope, intercept and correlation coefficient for the calibration were determined. Under the above described experimental conditions, linear correlation between the peak area and applied concentration was obtained in the concentration range of ng / spot of bacoside A. The correlation coefficient of bacoside A was found to be The peak area (y) is proportional to the concentration of bacoside A (x) following the regression equation y = x (Fig..13). The experimentally derived LOD and LOQ for bacoside A were determined to be 150 and 450 ng /spot respectively. Standardization of Some Plant-Based Formulations By Modern Analytical Techniques 206

31 y = x R² = Fig..13: Calibration curve of Bacoside A.4.2 Limit of detection (LOD) and limit of quantitation (LOQ): LOD and LOQ was determined by using standard deviation method. A calibration curve was prepared using concentrations in the range of µg/spot which is below linearity range. Standard deviation of residuals was measured and kept in the following equation for determination of detection limit and quantitation limit. Detection limit =3.3σ /S and quantitation limit=10 σ /S where σ is the residual standard deviation of a regression line and S is the slope of the calibration curve. The experimentally derived LOD and LOQ for bacoside A were determined to be 150 and 450 ng /spot respectively..4.3 Precision studies Precision of the method was evaluated by repeatability (intra-day) and instrumental precision. Each level of precision was investigated by three sequential replicates of spotting of bacoside A at concentrations of 1000, 1500 and 2000 ng / spot. Standardization of Some Plant-Based Formulations By Modern Analytical Techniques 207

32 Precision data on repeatability (intra-day) and instrumental variation for three different concentration levels are summarized in Table.2. Precision studies showed R.S.D. less than 1%, indicating a sufficient precision Table.2: Results of Precision Studies Bacoside A Type of Precision Intra-day AUC for concentration of Bacoside A (ng/spot) Inter-day AUC for concentration of Bacoside A (ng/spot) Sr. No Mean % RSD Accuracy studies In order to evaluate the validity of the proposed method, accuracy was evaluated through the percentage recoveries of known amounts of bacoside A added to solutions of extracts and all commercial products. The analyzed samples were spiked with 0, 100 and 120 % of median concentration (1500 ng) of bacoside A standard solution. For sample preparation of formulations please refer section.5. A constant application volume of 10.0 µl/spot was employed for all the sample solutions. Accuracy was calculated from the following equation: [(spiked concentration mean concentration)/spiked concentration] 100. Standardization of Some Plant-Based Formulations By Modern Analytical Techniques 20

33 Except for two brands of Aristha (50.10%, 49.33%), the recoveries from other formulations were in good range of acceptability ( %) of bacoside A. The proposed HPTLC method is accurate for the quantification of bacoside A in five formulations containing Bacopa monnieri (Table.3)..4.5 Robustness For the determination of the robustness of method, chromatographic parameters, such as mobile phase composition and chamber saturation time, were intentionally varied to determine their influence on the retention time and quantitative analysis. The mobile phase composition was altered by ± 5 % changes in the ratio of methanol and glacial acetic acid. The two composition of methanol were tried (+5% of 2.5) and (-5% of 2.5) and of Glacial acetic acid 1.05 and 0.95 (+5% and -5% of 1) was tried (Table.4.1). The chamber saturation time was altered from 15 min to 30 min (Table.4.2). The altered conditions did not cause any change retention factor and peak shape, thus the method is robust..4.6 Stability studies Stability of the sample solutions was tested after 24, 4 and 72 hours after preparation and storage at 4.0 C and 25.0 C separately. Stability was assessed by comparing the chromatographic parameters of the solutions after storage with the same characteristics of freshly prepared solutions. There should not be more than 5% of degradable content. The results were calculated as the percentage of non-degraded content of bacoside A standard at the 21, 4, 72 hours. All formulations showed less than 3.11% degradation at both investigated temperature. The highest non Standardization of Some Plant-Based Formulations By Modern Analytical Techniques 209

34 degradant percent was found in Brahmi churna (99.90%) at 4 C in 24 hrs, whereas the least observed in Brahmi Ghutika (96.9 %). (Table.5). Table.3: Recovery studies for Bacoside A in various herbal products Amount added in µg Extracts & Formulations Formulations AUC Standard Standard spiked formulations Recovery ± R.S.D ±0.02 Brahmi Vati ± ± ±0.56 Brahmi Churna ± ± ±019 Brahmi Gutika ± ± ±0.09 Brahmi Capsules ± ±0.5 Standardization of Some Plant-Based Formulations By Modern Analytical Techniques 210

35 ±0.22 Brahmi Tablets Brahmiarishta (Brand 1) Brahmiarishta (Brand 2 ) ± ± ± ± ± ± ± ±0.52 Table.4.1: Robustness (Mobile phase variation) studies of Bacoside A Mobile phase composition Sr.No Toluene (v/v) Ethyl acetate Methanol Formic acid R f AUC ± ± ± ± ±0.59 R.S.D Standardization of Some Plant-Based Formulations By Modern Analytical Techniques 211

36 Table.4.2: Robustness (Chamber saturation time variation) studies of Bacoside A Chamber Sr. No saturation time R f AUC (min) ± ± ± ± ±0.6 R.S.D Table.5: Stability Studies of Bacoside A in formulations in various herbal products Formulations Temperature 4 C 25 C 24 hrs 4 hrs 72 hrs 24 hrs 4 hrs 72 hrs Vati Churna Ghutika Capsules Tablets Standardization of Some Plant-Based Formulations By Modern Analytical Techniques 212

37 .5 Quantitative analysis of formulations containing Bacopa Monnieri for content Bacoside A by HPTLC.5.1 Procurement of Brahmi formulations Four formulations of Brahmi namely Gutika, Vati, Churna, Arishta (two different brands), Tablets and Capsules were procured from the local market..5.2 Preparation of Sample solutions For Brahmi Vati Vati is the traditional solid dosage form similar to tablet which is modern dosage form. The extract or the powdered drug is compressed into vati. 2 g of powdered vati was transferred to 100 ml volumetric flask containing 50 ml of methanol and the mixture was macerated on a shaker for 24 hrs at room temperature and then the volume was made up to 100 ml with methanol. Then 1.0 ml of this extract was diluted to a 10 ml with methanol For Brahmi Chruna Churna is a traditional form of Ayurvedic medicine in powder dosage form. 2 g of chruna was transferred to 100 ml volumetric flask containing 50 ml of methanol and the mixture was macerated on a shaker for 24 hrs at room temperature and then the volume was made up to 100 ml with methanol. Then 1.0 ml of this extract was diluted to a 10 ml with methanol For Brahmi Gutika Gutika is the traditional solid dosage form similar to vati but bigger in size. The extract or the powdered drug is compressed into gutika. 2 g of powdered Gutika was transferred to 100 ml volumetric flask containing 50 ml of methanol and the mixture was macerated on a shaker for 24 hrs at room temperature. Then 1.0 ml of this extract was diluted to a 10 ml with methanol. Standardization of Some Plant-Based Formulations By Modern Analytical Techniques 213

38 For Brahmi Capsule Capsule is a modern solid dosage form in which the powdered crude drug or extract of the herb is filled into the shells of the capsules. Sample solutions of capsule formulation were prepared same as that of vati by transferring 2 g of capsule contents For Brahmi Tablet Tablet is a modern solid dosage form where the powdered crude drug or extracts are compressed in form of tablet. Sample solutions of tablet formulation were prepared by transferring 2 g of powdered tablet to 100 ml volumetric flask containing 50 ml of methanol and the following the procedure was same as that of vati For Brahmiarishta Arishta is a galenical form of medicine. It is also a traditional form which is prepared by natural fermentation process. Two brands of Brahmiarishta namely Brand 1 and Brand 2 were analyzed and the sample preparation was as follows. 10 ml of Brahmiarishta (each brand) was evaporated to dryness. 2 g of residue was dissolved in 50 ml methanol in a volumetric flask and then the volume was made up to 100 ml with methanol. 2.5 ml of this extract was diluted to 10 ml with methanol. A constant application volume of 10.0 µl/s was employed for all the sample solutions. Validity of the proposed method was applied to standardization for both traditional and modern dosage forms viz. Brahmi Vati, Churna, Gutika, Capsule, Tablet, Arishta Brand 1 and Brand 2. The shape of the peaks was not Standardization of Some Plant-Based Formulations By Modern Analytical Techniques 214

39 altered by other substances present in the matrix. The percent content of bacoside A for all seven formulations are indicated in Table.6. Table.6: Percent Content of Bacoside A in various formulations containing Brahmi Extracts & Formulations Percent Content ± S.D. (%) Weight of per unit Content per unit dosage form (mg) Brahmi Vati ± mg 0.55 mg Brahmi Churna ± g (tbsp) 0.51 mg/tbsp Brahmi Gutika ± mg 0.35 mg Brahmi Capsules 0.03 ± mg 0.06 mg Brahmi Tablets ± mg mg Brahmiarishta (Brand 1) g -- Brahmiarishta (Brand 2) --- 2g -- This method could detect bacoside A with a wide range of concentration from %. The bacoside A content was highest in Brahmi capsules (modern dosage form) and least was in Brahmi vati. Both the brands of Brahmiarishta did not show any peak corresponding to bacoside A. It was observed that both the brands of Arishta labelled as Brahmi did not contain Bacopa monnieri but the substituent. This may be due to the addition of substitute ie; Centella asiatica instead of Bacopa monnieri. Standardization of Some Plant-Based Formulations By Modern Analytical Techniques 215

40 A simple, convienent, accurate method was developed and validated for bacoside A using HPTLC. The method was used to standardise formulations containing Brahmi. The analysis enabled to quantify the marker compound and even to detect the adulterant. The method can very well empolyed for the analysis of Brahmi products for routine quality control analysis to detect the percent content of the bioactive compound. Standardization of Some Plant-Based Formulations By Modern Analytical Techniques 216