standardization of some plant-based formulations by modern analytical techniques

Standardization of formulations containing Bacopa monnieri

8

Standardization of Some Plant-Based Formulations By Modern Analytical Techniques 177

8.1 INTRODUCTION

8.1.1 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


8


(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 USA246, 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

.


8


(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 intellect248

.

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 insanity249

.

Bacopa is also used in the treatment of respiratory ailments including

bronchitis and asthma250

and has been listed as a diuretic251

.

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 conditions252

. In the Indian Herbal Pharmacopoeia

(1998) bacopa is listed as a 'brain tonic253

.

Fig. 8.1: Whole plant of Bacopa monnieri


8


(f) Pharmacology and Clinical Studies:

i. Sedative and Tranquillizing Properties

Earlier studies reported a sedative effect of glycosides named hersaponins254

.

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 dogs255

. On the other hand, it has been found that the alcoholic

extract of the plant and chlorpromazine improved the performance of rats in

motor learning256

.

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

258, 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 effect260

.

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 effect261-264

.


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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 activity268-273

and antistress activity274, 275

. Based on animal study results, bacosides were

shown to have antioxidant activity in the hippocampus, frontal cortex and

striatum276

. 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 brain277

. 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 rats278-286

.

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)287-

294.


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(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 placebo297-301

.

Anxiety and Depression302-304

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


8


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) Toxicity306

: Bacopa monnieri has a record of several hundred years of

safe therapeutic use in Ayurvedic medicine. A double-blind, placebo-

controlled 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 (100-200

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) Phytochemistry307-314

: 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


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


8


A and B.

(j) Active Principles:

Fig. 8.2: Structures of some saponins from Bacopa monnieri

Standardization of Some Plant

8.1.2 Bacoside A as marker c

Bacoside A is

Fig. 8.3: S

The nootropic activity has been attributed to the presence of two saponins,

namely Bacoside A and Bacoside B. From this two markers

considered and proved to be more pharmacologically active for nootropic

activity315

. Several pharmacological, clinical, analytical and agronomical

studies concerning

content of bacoside A. Chemi

bacoside A3, bacopaside II,

Standardization of formulations containing Bacopa

Standardization of Some Plant-Based Formulations By Modern Analytical Techniques

8.1.2 Bacoside A as marker compound for Standardization

one of the major phytoconstituents of Bacopa monnieri

Fig. 8.3: Structure of major saponins of Bacoside A


namely Bacoside A and Bacoside B. From this two markers



studies concerning B.monnieri have recently published316

with reports on the

content of bacoside A. Chemically it is a mixture of four saponins namely,

, bacopaside II, jujubogenin isomer of bacopasaponin

Bacopa monnieri

8

Based Formulations By Modern Analytical Techniques 186

ompound for Standardization

monnieri

tructure of major saponins of Bacoside A


namely Bacoside A and Bacoside B. From this two markers bacoside A is



with reports on the

cally it is a mixture of four saponins namely,

bacopasaponin C and


8


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.


8


8.2 ISOLATION OF BACOSIDE A

8.2.1 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.

8.2.2 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.

8.2.3 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 60F254 (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


8


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 Rf 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. 8.4)


Fig. 8.4

Track 1: Chloroform fraction



Fig. 8.4: Videoimage of chloroform fraction of Brahmi

the Bacoside A band at Rf 0.44

Track 1: Chloroform fraction of Brahmi

1 2

Bacopa monnieri

8


Brahmi showing

Band of Bacoside

A after spraying

with 10 % acid

alcohol

Rf = 0.44


8


1 2

Fig. 8. 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.

8.2.4 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


8


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 250.6°C. The yield obtained was

0.78% from the commercial methanol extract. The summarized data is

mentioned in Table 8.1.

Table 8.1: 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 250.6°C

6. Yield (%w/w) 0.78 --

8.2.5 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.


8


• HPTLC Studies: Component III was dissolved in chloroform and the

HPTLC analysis was carried out using the following densitometric

conditions:

� Stationary phase : Precoated plates of Silica Gel 60 GF254 (Merck)

� Mobile phase : Toluene: ethyl acetate: methanol: Gla. acetic

(3:4:3:1)

� Saturation time : 20 min

� Development time : 20 min

� Wavelength : 540 nm

� Lamp : Tungsten

� Visualizing reagent : Spraying with 10% Acid alcohol

� Band width : 7 mm

� Length of

Chromatogram

: 8 cm


8


• HPLC Studies: The component III of were analyzed by HPLC technique

using the following conditions:

• Column: C18 Phenomenex (250 x 4.60mm) - 5µ

• Mobile phase : Gradient

Acetonitrile (A) 0.05% (v/v) Orthophosphoric acid in water (B)

Time (min) Function Value

0 B. Conc 70

25 B. Conc 60

35 B.Conc 40

40 STOP

• Flow rate: 1.5 ml/min

• Wavelength : 205nm

• Injection loop capacity: 20µl

• Concentration of Samples: 1mg/ml of standard and isolated compound.


Fig.8.6:

Bacoside A

Fig .8.7:



.8.6: HPTLC chromatogram of reference standard

Bacoside A ( Rf = 0.44)

.8.7: HPTLC chromatogram of component III (R

Bacopa monnieri

8


of reference standard

I (Rf = 0.44)




Fig. 8.8: HPLC profile of reference standard

Fig. 8.9: HPLC profile of Component III

Bacopa monnieri

8


profile of reference standard

. 8.9: HPLC profile of Component III


8


The isolated component III showed a single peak at Rf 0.44 (Fig 8.6), which

perfectly matched with the standard bacoside A Fig 8.7) as reported in Indian

herbal pharmacopeia250

.

The HPLC profile of component III (Fig 8.9), also matched with Standard

bacoslde A (Fig 8.8). Four peaks with Rt: 19.2 min, 19.9 min 21.7min and

22.8 min which matched with standard profile with Rt: 19.17 min, 19.92 min

21.68 min and 22.81 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 278 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 A3, bacopaside II, Jujubogenin

isomer of Bacopasaponin C and Bacopasaponin C316,317

. 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:


1. 3-O-[b

O-(b-D

Aglycon Jujubogenin

RT: 19.1

LCMS



Fig. 8.10.1: Mass Spectrum of Peak 1

[b-D-glucopyranosyl-(1 - 3)-O-{a-L-arabinofuranosyl

D-glucopyranosyl)]

Aglycon Jujubogenin

RT: 19.1-19.4

LCMS – m/z 967.46, base peak 455.38, 475.25, 325.13.

Bacopa monnieri

8


arabinofuranosyl-(1 - 2)}-

m/z 967.46, base peak 455.38, 475.25, 325.13. (Fig.8.10.1)


2. 3-O-[a

glucopyranosyl]

RT: 19.8

Aglycon

LCMS

257.07




[a-L-arabinofuranosyl-(1 - 2)-{b-D-glucopyranosyl

glucopyranosyl]

RT: 19.8-20.2

Aglycon- Pseudojujubogenin

LCMS- m/z 967.46, base peak 473.39, 455.38, 437.36, 337.20,

257.07 (Fig.8.10.2)

Bacopa monnieri

8


glucopyranosyl-(1 - 3)}-b-D-

m/z 967.46, base peak 473.39, 455.38, 437.36, 337.20,


3. 3-O-[a

glucopyranosyl]

RT: 21.6

Aglycon

LCMS





glucopyranosyl]

21.6-22.0

Aglycon- Pseudojujubogenin

LCMS- m/z 937.39, base peak 473.39, 455.38, 437.36.

Bacopa monnieri

8


glucopyranosyl-(1 - 3)}-b-D-

m/z 937.39, base peak 473.39, 455.38, 437.36. (Fig.8.10.3)


4. 3-O-[a

L-arabinopyranosyl]

Aglycon jujubogenin

RT: 22.6

m/z 937.45, base peak 455.38, 437.37, 265.11.





arabinopyranosyl]

Aglycon jujubogenin

22.6-22.9

m/z 937.45, base peak 455.38, 437.37, 265.11. (Fig.8.10

Bacopa monnieri

8


glucopyranosyl-(1 - 3)-}-a-

Fig.8.10.4)


8


The HPTLC, HPLC, UV and LCMS studies confirmed that component III is

bacoside A comprising of acoside A3, bacopaside II, Jujubogenin isomer of

Bacopasaponin C and Bacopasaponin C.


8


8.3 METHOD DEVELOPMENT

8.3.1 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, 0.8, 0.9, 1.0, 2.0, 3.0, 4.0, 5.0, 6.0,

7.0, 8.0, 9.0, 10 µL of this solution were applied using LINOMAT 5

applicator with the band width of 8 mm., which gave different concentration

ranging 150- 2500 ng / spot respectively.

8.3.2 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

GF254 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 Rf value by altering the composition,

however the resolution of the peak was better (Fig 8.9 and Fig 8.10).

Band width: Three different band widths 6 mm, 7 mm, 8mm were tried.

Band width of 7 mm gave good separation without the over saturation of the

spot with sample.


8


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 : : Precoated plates of Silica Gel 60 GF254 (Merck)

Mobile phase : Toluene: ethyl acetate: methanol: Gla. acetic acid

(3:4:2.5:1)

Saturation time : 20 min

Development time : 20 min

Wavelength : 540 nm

Lamp : Tungsten

Spraying reagent : 10 % sulphuric acid in methanol

Band width : 7 mm

Length of

chromatogram

: 8 cm

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. (Rf value = 0.44) as

indicated in (Fig 8.11 and Fig 8.12).


Fig. 8.12

Fig. 8.11



Fig. 8.12: HPTLC profile of methanol extract of Brahmi vati

using optimized parameters

8.11: HPTLC Chromatogram of bacoside A using optimized

parameters

Bacopa monnieri

8


methanol extract of Brahmi vati

HPTLC Chromatogram of bacoside A using optimized


8


8.4 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.

8.4.1 Linearity:

Different concentrations of bacoside A were spotted and analyzed. The

analysis was done in triplicate and the concentration range showing regression

coefficient (r2) near to one with precise value of r

2 in all triplicate analysis was

chosen.

Linearity was evaluated in the range of 500-10000 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 500-2500 ng / spot of bacoside A. The correlation

coefficient of bacoside A was found to be 0.9998. The peak area (y) is

proportional to the concentration of bacoside A (x) following the regression

equation y = 15.267x - 7074.3 (Fig. 8.13). The experimentally derived LOD

and LOQ for bacoside A were determined to be 150 and 450 ng /spot

respectively.


8


y = 15.267x - 7074.3

R² = 0.9998

0

5000

10000

15000

20000

25000

30000

35000

40000

45000

0 500 1000 1500 2000 2500 3000 3500

Fig. 8.13: Calibration curve of Bacoside A

8.4.2 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 0.05-0.5

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.

8.4.3 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.


8


Precision data on repeatability (intra-day) and instrumental variation for three

different concentration levels are summarized in Table 8.2. Precision studies

showed R.S.D. less than 1%, indicating a sufficient precision

Table 8.2: Results of Precision Studies Bacoside A

Type of

Precision

Intra-day Inter-day

AUC for concentration of

Bacoside A (ng/spot)

AUC for concentration of

Bacoside A (ng/spot)

Sr. No 1000 1500 2000 1000 1500 2000

1 8565.01 15542.35 23544.58 8541.28 15748.49 23453.26

2 8578.19 15640.02 23457.65 8598.46 15684.15 23326.56

3 8588.16 15630.97 23315.16 8578.69 15840.86 23609.29

Mean 8577.12 15604.4 23439.1 8572.81 15757.8 23463

% RSD 0.13538 0.34585 0.49416 0.33874 0.49989 0.60358

8.4.4 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 80, 100 and 120 % of median concentration (1500

ng) of bacoside A standard solution. For sample preparation of formulations

please refer section 8.5. 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.


8


Except for two brands of Aristha (50.10%, 49.33%), the recoveries from other

formulations were in good range of acceptability (97.69-107.97 %) of

bacoside A. The proposed HPTLC method is accurate for the quantification of

bacoside A in five formulations containing Bacopa monnieri (Table 8.3).

8.4.5 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

2.625 (+5% of 2.5) and 2.375 (-5% of 2.5) and of Glacial acetic acid 1.05 and

0.95 (+5% and -5% of 1) was tried (Table 8.4.1). The chamber saturation

time was altered from 15 min to 30 min (Table 8.4.2). The altered conditions

did not cause any change retention factor and peak shape, thus the method is

robust.

8.4.6 Stability studies

Stability of the sample solutions was tested after 24, 48 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, 48, 72 hours. All formulations showed less

than 3.11% degradation at both investigated temperature. The highest non


8


degradant percent was found in Brahmi churna (99.90%) at 4°C in 24 hrs,

whereas the least observed in Brahmi Ghutika (96.89 %). (Table 8.5).

Table 8.3: Recovery studies for Bacoside A in various herbal products

Extracts

&

Formula-

tions

Amount

added

in µg

AUC

Recovery ±

R.S.D Formula-

tions

Standard

Standard

spiked

formulations

Brahmi

Vati

1200 775.4 1463.9 2368.5 105.77±0.02

1500 775.4 1820.8 2726.26 105.01±0.54

1800 775.4 2157.8 3084.25 105.15±0.17

Brahmi

Churna

1200 396.9 1463.9 1857.45 99.82±0.56

1500 396.9 1820.8 2211.71 99.73±0.94

1800 396.9 2157.8 2530.43 99.05±0.05

Brahmi

Gutika

1200 406.2 1463.9 1820.16 97.33 ±019

1500 406.2 1820.8 2159.96 96.99 ±0.11

1800 406.2 2157.8 2488.87 97.07 ±0.23

Brahmi

Capsules

1200 865.7 1463.9 2541.12 109.08±0.09

1500 865.7 1820.8 2936.07 109.29±0.63

1800 865.7 2157.8 3299.84 109.14±0.58


8


Brahmi

Tablets

1200 807.8 1463.9 2435.71 107.22±0.22

1500 807.8 1820.8 2815.75 107.12±0.44

1800 807.8 2157.8 3187.72 107.49±0.56

Brahmiarishta

(Brand 1)

1200 626.1 1463.9 2112.57 101.08±0.98

1500 626.1 1820.8 2487.27 101.65±0.45

1800 626.1 2157.8 2827.88 101.58±0.66

Brahmiarishta

(Brand 2 )

1200 673.6 1463.9 2208.67 103.33±0.12

1500 673.6 1820.8 2576.46 103.29±0.86

1800 673.6 2157.8 2918.32 103.07±0.52

Table 8.4.1: Robustness (Mobile phase variation) studies of Bacoside A

Sr.No

Mobile phase composition

(v/v)

Rf AUC

Toluene

Ethyl

acetate

Methanol

Formic

acid

1 3 4 2.5 1 0.44 15550.1 ±0.54

2 3 4 2.625 1 0.44 15487.66 ±0.47

3 3 4 2.375 1 0.44 15589.48 ±0.21

4 3 4 2.5 1.05 0.44 15471.06 ±0.61

5 3 4 2.5 0.95 0.44 15508.99 ±0.59

R.S.D. - -

- 0 0.31


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Table 8.4.2: Robustness (Chamber saturation time variation) studies of

Bacoside A

Sr. No

Chamber

saturation time

(min)

Rf AUC

1 20 0.44 15550.1 ±0.54

2 15 0.44 15598.14 ±0.23

3 25 0.44 15560.11 ±0.74

4 30 0.44 15591.15 ±0.48

5 35 0.44 15487.55 ±0.68

R.S.D. - 0 0.28

Table 8.5: Stability Studies of Bacoside A in formulations in various

herbal products

Formulations Temperature

4°C 25°C

24 hrs 48 hrs 72 hrs 24 hrs 48 hrs 72 hrs

Vati 99.86 99.23 98.11 98.99 98.58 97.23

Churna 99.90 99.81 98.69 99.01 98.56 97.09

Ghutika 99.14 98.61 97.30 98.02 97.14 96.89

Capsules 99.60 98.54 98.83 99.43 98.85 97.91

Tablets 99.79 98.69 97.86 99.39 98.78 98.12


8


8.5 Quantitative analysis of formulations containing Bacopa Monnieri for

content Bacoside A by HPTLC

8.5.1 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.

8.5.2 Preparation of Sample solutions

8.5.2.1 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.

8.5.2.2 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.

8.5.2.3 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.


8


8.5.2.4 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.

8.5.2.5 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.

8.5.2.6 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


8


altered by other substances present in the matrix.

The percent content of bacoside A for all seven formulations are indicated in

Table 8.6.

Table 8.6: 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 0.022 ± 0.11 250 mg 0.55 mg

Brahmi Churna 0.017 ±0.34 3.0 g (tbsp) 0.51 mg/tbsp

Brahmi Gutika 0.010 ±0.21 350 mg 0.35 mg

Brahmi Capsules 0.03 ± 0.42 200 mg 0.06 mg

Brahmi Tablets 0.079 ± 0.63 250 mg 0.1975 mg

Brahmiarishta (Brand 1) ---- 2 g --

Brahmiarishta (Brand 2) --- 2g --

This method could detect bacoside A with a wide range of concentration from

0.105 – 0.791%. 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.


8


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

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