IOSR Journal of Pharmacy (IOSRPHR), www.iosrphr.org

IOSR Journal of Pharmacy

Vol. 2, Issue 1, Jan-Feb.2012, pp. 052-056

IOSR

SCREENING AND STANDARDISATION OF BACOPA MONNIERI
USED AS MEDICINE IN HOMOEOPATHY USING HPTLC METHOD

AMIT KHANDAGALE 1 and DAMODAR SHANBHAG 2

1 Department of Chemistry, D. G Ruparel College, Mahim, Mumbai - 400016, India,
2 Department of Chemistry, D. G. Ruparel College, Mahim, Mumbai - 400016, India.

ABSTRACT:

In this study, we have chosen HPTLC as a method of analysis to develop a standard procedure based on fingerprinting
characteristics for the evaluation of homoeopathic formulations. A simple and accurate HPTLC method has been developed
for the quantification of Bacoside-

A and fingerprinting of the in-house mother tincture considered here to be a standard with that of different marketed samples
available from manufacturers of homoeopathic medicines in India. This HPTLC method was quantitatively evaluated in
terms of stability, repeatability, accuracy and calibration providing the utility in the analysis of the mother tincture.

Keywords: HPTLC, Standardisation, Mother Tincture, Bacoside-A, Fingerprint, MWL, Spectra

I. INTRODUCTION:

Bacopa Monnieri is belongs to the family of "Scrophulariaceae". It is a medicinal herb found throughout the Indian
subcontinent in wet damp and marshy area [1]. It is used as traditional Indian medicine for the treatment of anxiety, and in
improving intellect and memory of the several country [2,3]. In addition to the memory boosting activity, it also claimed to
be useful in the treatment of cardiac, respiratory, neuropharmacological disorder like insomnia, insanity, depression,
psychosis, epilepsy and stress [4,5]. It was reported to posses anti -inflammatory, analgesic, antipyretic, sedative free radical
scavenging and anti-lipid peroxidative activities [6,7]. The pharmacological properties of Bacopa monnieri were studied
extensively and the activities were attributed mainly due to the presence of characteristics saponins called as "bacosides" [8].

II MATERIALS AND METHODS

1.1 Chemicals and Materials:

Authentic dried plant of Bacopa Monnieri was used to prepare the mother tincture. Bacoside-A (C 4 iH 68 0i 3 m.p. 251°C,
purity >99% w/w by TLC) was purchased from SPIC Pharma, Chennai. The solvents 99.9% absolute ethanol,
dichloromethane, methanol, HPLC water were of analytical grade purity (MERCK Ltd.).

1.2 Preparation of Standard Mother Tincture:

The dried plant was coarsely powdered, 10 g of this powder was used and the requisite amount of alcohol and water was
added as specified in HPI and the standard mother tincture was prepared by the percolation method. This tincture was
transferred to suitable glass container and stored for further study [9].

1.3 Preparation of Standard Bacoside-A:

Ten milligram of Bacoside-A was weighed in a 10 mg volumetric flask. To this 10 ml of ethanol was added.

1.4 Standardization of Standard Mother Tincture:

Camag HPTLC [10] system comprising of Linomat 5 as sample applicator and TLC Scanner3 controlled by winCATS
software version 1.3.4 was used for quantitative evaluation. Stationary phase used was MERCK precoated TLC Aluminium
foil silica gel 60 F254 and the mobile phase used was Dichloromethane : Methanol : Water (4.5 : 1.0 : 0.1) v/v [11-12].
Samples and standard were applied as 8 mm bands with 6 mm distance between the tracks. Tank saturation and plate
equilibrium was given with filter paper for 10 min. Ascending development for a distance of 80 mm in a twin trough
chamber was completed in approximately 15 min. Volume of standard MQ was first optimized at 6 ul for fingerprinting.
The A^ax of Bacoside-A was found to be 225 nm after taking the spectra of the standard of Bacoside-A [fig-1]. Quantitative
measurement in the absorbance mode was done at 225 nm using a slit dimension of 6.00 x 0.45 mm.

ISSN: 2250-3013 www.iosrphr.org 52 I P a g e

IOSR Journal of Pharmacy

Vol. 2, Issue 1, Jan-Feb.2012, pp. 052-056

IOSR

1.5 Linearity response:

The volume of the std. mother tincture was optimized to 6 ul for quantification. It was then simultaneously applied with
different concentration of standard Bacoside-A. The method was found to be linear with a regression of 0.99945 and a
standard deviation of 1.82% and the amount of Bacoside-A was calculated in the mother tincture [fig-2].

1.6 Standardisation of the standard mother tincture by fingerprint method:

Standardisation [13] of the mother tincture was done by evaluating its fingerprint characteristics, using HPTLC method.
Standard mother tincture was chromatographed simultaneously along with four other mother tinctures available in market at
6 ul on the same plate for comparison [TABLE- 1]. Multi wavelength (MWL) scan was done for finding the optimum
wavelength for scanning. The optimum wavelength was found to be 225 nm. The entire plate was further scanned at this
wavelength for quantification and spectral match. Many fractions of std. mother tincture were matched with the help of its
characteristic spectra with that of other marketed samples [fig-3]. Individual A, max of each fraction was also found with the
help of spectral scanning and then the plate was scanned with these selected wavelengths in MWL mode. The pattern of the
peaks was compared for the std. mother tincture and marketed samples. It was observed that the response for various
concentrations of standard Bacoside-A was linear in the range of 200 ng to 1000 ng with a coefficient of variation of 0.99958
and a standard deviation of 1.79% [fig-4]. Bacoside-A was quantified and the amount was calculated in individual mother
tinctures. With this method we compared all available mother tinctures and the active principle was also quantified. Thus
the method can be said to be standardised.

1.7 Quantification of Bacoside-A in market samples and standard mother tincture:

The amount of Bacoside-A was calculated in standard mother tincture (A) and market samples (Al to A4) and was found
asgivenin[TABLE-2].

Ill RESULTS AND DISCUSSIONS

The decomposition of the analyte during application or development was confirmed by two-dimensional
chromatography. The chromatogram did not show any extra fractions. Repeatability of the method was checked by scanning
15 tracks of 6 liI volume std. mother tincture. The co-efficient of variation (CV) was found to be 0.368. The percentage
recovery of Bacoside-A was calculated using the above method. The average recovery values obtained were 92.0% to
97.0%, which confirms that the method is validated.

The HPTLC Fingerprinting characteristics of "Bacopa MonnierF mother tinctures obtained from manufacturer (Al to A4)
and the in-house std. MQ (A) had been scanned at 225 nm wavelength. The scanning report as well as the fingerprint
characters obtained after integration has been shown in [TABLE- 1].

From the results obtained after densitometric scanning, it was observed that the Std. MQ (A) of Bacopa Monnieri shows 9
peaks. The marketed samples Al shows 9 peaks, A2 shows 8 peaks, A3 shows 9 peaks, A4 shows 8 peaks.

Value of the four marketed tinctures (Al to A4) was found to show minimum 8 different peaks with Rf values similar to std.
MQ (A) and they are similar within themselves. So from this study it was confirmed that Bacoside-A tincture contains
different components with Rf values (0.04-0.05-0.06, 0.18-0.19, 0.21-0.22, 0.27-0.28, 0.36-0.37, 0.42, 0.44-0.45, 0.48,0.49-
0.50-0.51-0.52-0.53, 0.76, 0.78-0.79). These components must be considered to determine quality of any further sample of
the same. Also spectral analysis indicates that spectra with particular Rf values of various components (0.06, 0.19, 0.27,
0.37, 0.48, 0.76) have similar pattern within themselves. It may be concluded that samples procured from the market that are
showing lesser peaks may not be up to the standard level.

Based on this approach our aim was to develop a standardised procedure to evaluate the mother tinctures for its accuracy,
sensitivity and reproducibility. This standardisation may lead to a solution to the factors which are responsible for variation
in the homoeopathic formulations. The above HPTLC method is powerful, rapid, reliable and cost effective with respect to
the accuracy of the result based on both qualitative and quantitative analysis.

ISSN: 2250-3013 www.iosrphr.org 53 I P a g e

IOSR Journal of Pharmacy

Vol. 2, Issue 1, Jan-Feb.2012, pp. 052-056

IOSR

IV REFERENCES

[I] Kapoor L. D., Handbook of Ayurvedic medicinal plants. Boca Raton: CRC Press; 1990.

[2] Singh H. K., Dhawan B.N., Neuropsychopharmacological effects of the Ayurvedic nootropic Bacopa

monneira Linn. (Brahmi), Indian J. Pharmacol 1997; 29: 359-65.
[3] Singh H. K., et.al, Effects of Bacoside A and B on avoidance response in rats. Phytother Res 1998;

2:70-4.
[4] Nadkarni K.M., The Indian material medica. Columbia MO: South Asia Books; 1988.

[5] Russo A, Borrelli R, Bacopa monniera, a reputed nootropic plant: An overview. Phytomedicine 2005;

12:305-17
[6] Kishore K., Singh M., Effects of bacosides, alcoholic extract of Bacopa monniera Linn, (brahmi) , on

experimental amnesia in mice. Indian J Exp Biol 2005; 43: 640-5
[7] Anbarasi K. et.al, Creatine kinase isoenzyme patterns upon chronic exposure to ciagarette smoke:

Protective effects of Bacoside A. Vascul Pharmacol 2005; 42: 57-61.
[8] Deepak M, Amit A, The need of establishing identities of 'bacoside A and B', the putative major

bioactive saponins of Indian Medicinal plant Bacopa Monnieri. Phytomedicine 2004; 11: 264-8.
[9] Verma P. N and Vaid I.: Encyclopaedia of Homoeopathic Pharmacopoeia, B. Jain Publishers Pvt. Ltd.,

New Delhi; 1995, p.770-771.
[10] Sethi P.D: High Performance Thin Layer Chromatography (HPTLC), CBS Publishers, New Delhi;

1996, p.3-62.

[II] Wagner H., Bladt S. and Zgainski E.M.: Plant Drug Analysis, Springer Verlag, Berlin; 1984, p.70.

[12] Shanbhag D. A., HPTLC standardization of homoeopathic mother tinctures and comparison with

products in market: Thesis, 2003, p.53 1-536.
[13] Raina M.K, Recent Trends in Standardisation of Herbal Materials, Indian J. of Natural Products, 1993,

9, 18-22.

FIG. 1 ABSORPTION SPECTRUM OF STANDARD BACOSIDE-A

ISSN: 2250-3013

www.iosrphr.org

54 I P a g e

IOSR Journal of Pharmacy

Vol. 2, Issue 1, Jan-Feb.2012, pp. 052-056

IOSR

Substance: Bacoside - A

Regression mode: Linear

Regression via area Y = 218.019 + 6.613*X c=0. 99945 sdv=1.82%

0.00 100.00 200.00 300.00

400.00 500.00

FIG. 2 CALIBRATION CURVE OF BOCOSIDE-A (AREA)

FIG. 3 OVERLAY OF ABSORPTION SPECTRA OF STANDARD, STANDARD MQ AND MARKETED SAMPLES

Substance: Bacoside-M 1 225nm Regression mode: linear

1 Y=-70.260 + 6.777*X

:=0. 99958 sdv=1.79%

FIG. 4 CALIBRATION CURVE OF BACOSIDE-A

ISSN: 2250-3013

www.iosrphr.org

55 I P a g e

IOSR Journal of Pharmacy

Vol. 2, Issue 1, Jan-Feb.2012, pp. 052-056

IOSR

Peak

A

Al

A2

A3

A4

Rf

Max.
Ht.

%
area

Rf

Max
Ht.

%
area

Rf

Max.
Ht.

%
area

Rf

Max.
Ht.

%
area

Rf

Max.
Ht.

%
area

1

0.06

23.5

2.70

0.05

83.8

6.12

0.04

17

1.51

0.05

29.8

2.08

0.05

27.6

1.61

2

0.18

10.2

1.76

0.18

26.3

1.42

0.18

14.1

1.21

0.19

70.1

10.29

0.18

26.1

2.43

3

0.22

14.7

2.63

0.21

52.1

4.95

0.22

33.2

6.34

0.27

41.3

7.06

0.28

37.7

4.95

4

0.28

12.7

2.34

0.36

365.1

39.59

0.27

24

2.92

0.36

150.3

24.98

0.37

428.
7

59.36

5

0.37

217.2

47.95

0.42

50.2

4.81

0.36

343.9

50.57

0.44

38.3

9.13

0.44

38.2

6.45

6

0.45

27.6

7.50

0.44

40.6

2.63

0.48

96.3

15.81

0.49

47.5

8.54

0.49

82.1

13.13

7

0.50

42.5

10.13

0.49

94.4

11.31

0.51

33

4.81

0.52

31.7

5.44

0.61

14.6

2.22

8

0.53

27.9

5.32

0.52

48.9

6.09

0.78

56.2

17.46

0.59

11.8

2.68

0.79

35.4

9.85

9

0.79

39.7

19.66

0.76

115.0

23.08

-

-

-

0.76

91.9

29.79

-

-

-

TABLE - 1 : ANALYSIS OF DIFFERENT BACOPA MONNIERI MOTHER TINCTURES AT SCANNING
WAVELENGTH 225 NM

Sr. No.

Name of sample

Wt. of Bacoside-A in 100 ml sample

1.

A

5.41 mg

2.

Al

8.43 mg

3.

A2

1.57 mg

4.

A3

4.82 mg

5.

A4

2.82 mg

TABLE - 2 : AMOUNT OF BACOSIDE-A IN BACOPA MONNIERI MOTHER TINCTURES

A- Standard mother tincture of Bacopa Monnieri prepared in our laboratory.
Al - A4 - Four samples of Bacopa Monnieri tincture from manufacturer.
Rf corresponds to maximum peak height.

ISSN: 2250-3013

www.iosrphr.org

56 I P a g e