standardization of some plant-based formulations by modern analytical techniques
TRANSCRIPT
Standardization of formulations containing Bacopa monnieri
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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
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(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
.
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(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
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(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
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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
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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
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A and B.
(j) Active Principles:
Fig. 8.2: Structures of some saponins from Bacopa monnieri
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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,
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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
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
. Several pharmacological, clinical, analytical and agronomical
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
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ompound for Standardization
monnieri
tructure 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
. Several pharmacological, clinical, analytical and agronomical
with reports on the
cally it is a mixture of four saponins namely,
bacopasaponin C and
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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.
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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
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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)
Standardization of Some Plant
Fig. 8.4
Track 1: Chloroform fraction
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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
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Brahmi showing
Band of Bacoside
A after spraying
with 10 % acid
alcohol
Rf = 0.44
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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
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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.
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• 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
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• 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.
Standardization of Some Plant
Fig.8.6:
Bacoside A
Fig .8.7:
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Standardization of Some Plant-Based Formulations By Modern Analytical Techniques
.8.6: HPTLC chromatogram of reference standard
Bacoside A ( Rf = 0.44)
.8.7: HPTLC chromatogram of component III (R
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of reference standard
I (Rf = 0.44)
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Fig. 8.8: HPLC profile of reference standard
Fig. 8.9: HPLC profile of Component III
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profile of reference standard
. 8.9: HPLC profile of Component III
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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:
Standardization of Some Plant
1. 3-O-[b
O-(b-D
Aglycon Jujubogenin
RT: 19.1
LCMS
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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.
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arabinofuranosyl-(1 - 2)}-
m/z 967.46, base peak 455.38, 475.25, 325.13. (Fig.8.10.1)
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2. 3-O-[a
glucopyranosyl]
RT: 19.8
Aglycon
LCMS
257.07
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Fig. 8.10.2: Mass Spectrum of Peak 2
[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)
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glucopyranosyl-(1 - 3)}-b-D-
m/z 967.46, base peak 473.39, 455.38, 437.36, 337.20,
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3. 3-O-[a
glucopyranosyl]
RT: 21.6
Aglycon
LCMS
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Fig. 8.10.3: Mass Spectrum of Peak 3
[a-L-arabinofuranosyl-(1 - 2)-{b-D-glucopyranosyl
glucopyranosyl]
21.6-22.0
Aglycon- Pseudojujubogenin
LCMS- m/z 937.39, base peak 473.39, 455.38, 437.36.
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glucopyranosyl-(1 - 3)}-b-D-
m/z 937.39, base peak 473.39, 455.38, 437.36. (Fig.8.10.3)
Standardization of Some Plant
4. 3-O-[a
L-arabinopyranosyl]
Aglycon jujubogenin
RT: 22.6
m/z 937.45, base peak 455.38, 437.37, 265.11.
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Fig. 8.10.4: Mass Spectrum of Peak 4
[a-L-arabinofuranosyl-(1 - 2)-{b-D-glucopyranosyl
arabinopyranosyl]
Aglycon jujubogenin
22.6-22.9
m/z 937.45, base peak 455.38, 437.37, 265.11. (Fig.8.10
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glucopyranosyl-(1 - 3)-}-a-
Fig.8.10.4)
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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.
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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.
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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).
Standardization of Some Plant
Fig. 8.12
Fig. 8.11
Standardization of formulations containing Bacopa
Standardization of Some Plant-Based Formulations By Modern Analytical Techniques
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
Based Formulations By Modern Analytical Techniques 205
methanol extract of Brahmi vati
HPTLC Chromatogram of bacoside A using optimized
Standardization of formulations containing Bacopa monnieri
8
Standardization of Some Plant-Based Formulations By Modern Analytical Techniques 206
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.
Standardization of formulations containing Bacopa monnieri
8
Standardization of Some Plant-Based Formulations By Modern Analytical Techniques 207
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.
Standardization of formulations containing Bacopa monnieri
8
Standardization of Some Plant-Based Formulations By Modern Analytical Techniques 208
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.
Standardization of formulations containing Bacopa monnieri
8
Standardization of Some Plant-Based Formulations By Modern Analytical Techniques 209
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
Standardization of formulations containing Bacopa monnieri
8
Standardization of Some Plant-Based Formulations By Modern Analytical Techniques 210
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
Standardization of formulations containing Bacopa monnieri
8
Standardization of Some Plant-Based Formulations By Modern Analytical Techniques 211
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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Standardization of Some Plant-Based Formulations By Modern Analytical Techniques 212
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
Standardization of formulations containing Bacopa monnieri
8
Standardization of Some Plant-Based Formulations By Modern Analytical Techniques 213
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.
Standardization of formulations containing Bacopa monnieri
8
Standardization of Some Plant-Based Formulations By Modern Analytical Techniques 214
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
Standardization of formulations containing Bacopa monnieri
8
Standardization of Some Plant-Based Formulations By Modern Analytical Techniques 215
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.
Standardization of formulations containing Bacopa monnieri
8
Standardization of Some Plant-Based Formulations By Modern Analytical Techniques 216
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.