ACADEMIA Letters

Phytochemical variability among herbal formulations of

Bacopa monnieri (L.) Wettst. (Brahmi) – A memory
enhancing herb
Munish Sharma, Department of Plant Science, Central University of Himachal
Pradesh
Ashok Ahuja, College of Agriculture, Rajmata Vijyaraje Scindia Krishi
Vishwavidyalaya
Sharada Mallubhotla, School of Biotechnology, Shri Mata Vaishno Devi University

Abstract
Extracts of the memory enhancing herb Bacopa monnieri are used in several commercial
ayurvedic formulations for a variety of health promoting effects. Adulteration in market sam-
ples and availability of sub-standard products is one of the greatest drawbacks in promotion
of herbal products from India. The most important challenge faced by herbal formulations
emerges because of their lack of complete evaluation. Therefore, the present study was un-
dertaken to assess the phytochemical products commercially available in the Indian market,
containing B. monnieri as the main ingredient were qualitatively and quantitatively analysed
for bacoside A3 and A2 (pharmacologically active constituents of B. monnieri) using Thin
layer chromatography (TLC) and High performance liquid chromatography (HPLC) tech-
niques respectively. The bacoside content ( bacoside A3 and A2) was found to vary in the
products analyzed. The study emphasizes the need for strict phytochemical standardization
of herbal products for better global marketability.
Keywords: Brahmi; Bacosides; Market adulteration; TLC; HPLC; Quality control
1. Introduction
The usage of plant based herbal drugs for the prevention and treatment of various health
related problems has been implicated from time immemorial (Ekor, 2013). Several herbal for-

Academia Letters, July 2021 ©2021 by the authors — Open Access — Distributed under CC BY 4.0

Corresponding Author: Munish Sharma, munishptc@gmail.com

Citation: Sharma, M., Ahuja, A., Mallubhotla, S. (2021). Phytochemical variability among herbal formulations
of Bacopa monnieri (L.) Wettst. (Brahmi) – A memory enhancing herb. Academia Letters, Article 1822.
https://doi.org/10.20935/AL1822.
1
mulations available in the market have health promoting effects and the demand of new plant
derived drugs increased likewise. Since ancient times, medicinal plants have been considered
as potential candidates for the drug derivation as they contains drug like activities and prop-
erties (Bernhoft, 2010). But the most important challenges faced by commercial formulations
arise because of their lack of complete evaluation which is necessary to ensure quality, purity
and consistency of bioactive components in the herbal products.
Bacopa monnieri (L.) Wettst. (Scrophulariaceae) commonly known as Brahmi, is one
of the most valued medicinal plants in Ayurveda and other traditional systems of medicine.
B. monnieri is regarded as the main rejuvenating herb for nerve and brain cells and there-
fore, has played a very important role in ayurvedic therapies for the treatment of cognitive
disorders of aging (Ernst, 2006; Russo and Borrelli, 2005). The plant has been reported
to have anti-inflammatory, analgesic, antipyretic, epileptic, anticancer and antioxidant ac-
tivities (Sinha and Saxena, 2006; Tripathi et al., 1996). Extracts of B. monnieri contain
different types of saponins, amongst which the steroidal tetracyclic triterpenoid saponin ba-
coside A is found to be the major active principle having biological activity (Basu et al.,
1967; Chatterji et al., 1965). The chemical composition of bacosides, contained in the po-
lar fraction, has been established on the basis of chemical and physical degradation stud-
ies. On acid hydrolysis, bacosides yield a mixture of aglycones, bacogenin (A1, A2, A3)
(Chandel et al., 1977; Kulshreshtha and Rastogi, 1973, 1974) and two genuine sapogenins,
jujubogenin and pseudojujubogenin (Rastogi et al., 1994). The saponin, bacoside B gener-
ally co occurs with bacoside A and is considered as an artefact produced during the process
of isolating bacoside A (Rastogi, 1990). Owing to the memory enhancing property of ba-
cosides, the demand of B. monnieri led to its extensive use in several commercial prepara-
tions. The average market price of B.monnieri whole plant is 148.6 (Rs/Kg) (http://www.e-
charak.in/echarak/avgmarketprice.do).
A variety of mono and poly herbal preparations of Brahmi are commercially sold in In-
dia. To our knowledge, no study has been reported on the bacoside contents in the products
available in the market. The present study was, therefore, undertaken to assess bacosides
composition in commercial herbal formulations of Brahmi.
2. Materials and methods
2.1 Reagents and chemicals
Standard bacoside (mixture of bacoside A3 and A2) was obtained from Indian Institute of
Integrative Medicine (CSIR), Jammu. Acetonitrile and Methanol of HPLC grade from RFCL
Limited (Rankem); Butanol , Acetic acid and Potassium dichromate from SD Fine-chem Lim-
ited ( SDFCL) ; Sulphuric acid from Loba chemie were used for the chemical analysis.
2.2. Plant material, Sample preparation and bacosides extraction

Academia Letters, July 2021 ©2021 by the authors — Open Access — Distributed under CC BY 4.0

Corresponding Author: Munish Sharma, munishptc@gmail.com

Citation: Sharma, M., Ahuja, A., Mallubhotla, S. (2021). Phytochemical variability among herbal formulations
of Bacopa monnieri (L.) Wettst. (Brahmi) – A memory enhancing herb. Academia Letters, Article 1822.
https://doi.org/10.20935/AL1822.
2
 Fresh aerial shoots of B. monnieri were collected from uniformly grown plants under shade
house conditions at Herbal Garden, Shri Mata Vaishno Devi University, Kakryal, Katra (J&K).
Five formulations containing B. monnieri were procured from the local market as detailed in
Table 1 along with their therapeutic and health promoting effects. The air dried plant, tablets
and capsule formulations were taken in equigram, ground to a fine powder using pestle- mortar
and extracted in a Soxhlet apparatus with methanol for 5 h at 600 C. The methanolic extracts
were filtered and dried overnight to concentrate the extract. Air dried samples were further
dissolved in HPLC grade methanol and filtered through 0.45µm PVDF syringe filter (Axiva)
prior to HPLC analysis. Each sample was prepared in triplicate.
2.3. Standard preparation
The standard bacoside (mixture of A3 and A2) was prepared in methanol to make a stock
solution of 1 mg/mL. and 1.0 mL of this solution was diluted to 10 mL with methanol to
get a 100µg/mL (w/v) working solution The standard solutions were sonicated/vortexed after
preparation and before using (injection in the HPLC) in order to ensure maximal solubilization
of each compound in the mixture.
2.4. Analytical techniques
The extracts were analyzed qualitatively and quantitatively by Thin layer chromatography
(TLC) and High Performance Liquid Chromatography techniques respectively to detect the
presence of bacosides.
2.4.1. TLC analysis
The concentrated methanolic extracts were spotted on precoated Silica gel 60 Merck plates
using solvent system [n-Butanol: Acetic acid: distilled water (4:1:5 v/v)] and spots visualized
by spray reagent (1 % potassium dichromate in 40% sulphuric acid).
2.4.2. HPLC procedure and Calibration
HPLC analysis was performed using an Agilent Technologies Series 1100, Waldronn,
Germany) system by the procedure mentioned earlier (Sharma et al., 2013)
3. Results and discussion
Though the plant botanicals based herbal medicines are widely accepted throughout the
world, questions related with their efficiency, quality and safety still limited their usage (Ma-
hady, 2001). In our study, qualitative and quantitative assessment of marketed formulations
of B. monnieri has been attempted.
The results of present study reveal wide variations in the amount of bacosides content
(bacoside A3 and A2) in the commercial herbal formulations. TLC analysis of B. monnieri
methanolic extract showed the presence of distinct spot (Fig. 1a). The spots with reten-
tion factor (Rf) value 0.5 was observed in sample F, B and D which coincided with standard
bacoside spot (Rf=0.5). Quantitative analysis of different formulations showed complete ab-

Academia Letters, July 2021 ©2021 by the authors — Open Access — Distributed under CC BY 4.0

Corresponding Author: Munish Sharma, munishptc@gmail.com

Citation: Sharma, M., Ahuja, A., Mallubhotla, S. (2021). Phytochemical variability among herbal formulations
of Bacopa monnieri (L.) Wettst. (Brahmi) – A memory enhancing herb. Academia Letters, Article 1822.
https://doi.org/10.20935/AL1822.
3
sence of bacoside A3 and A2 in samples A, C and E whereas sample B contain only bacoside
A3 (0.31±0.15 ). Sample D has been observed to contain varying amounts of bacoside A3
(0.43±0.27) and A2 (0.02±0.01) (Fig. 1b). The HPLC chromatograms of standard bacoside
and field sample are projected (Fig. 1c, d). Our results are in agreement with the earlier report
(Saini et al., 2012) wherein various commercial formulations retailed by different manufac-
turers of B. monnieri were analysed for assessing any variation in their biological activity.
They observed significant variation in composition and quality of herbal drugs. Giancarlo
et al. (2004) reported variability in the chemical composition and biological activity of Gly-
cyrrhiza glabra samples.Reports are available for comparative analysis of Withaferin A and
six unidentified molecules in commercial products of Withania somnifera containing Ash-
wagandha using HPLC technique (Sangwan et al., 2004). More than 70-fold variation in
Withaferin A was found in the commercial products. The phytochemical variations found in
commercial formulations containing Neem as the main ingredient have also been reported
(Gunasekaran and Anita, 2010).
Results clearly suggest that the commercial products vary by several orders of magnitude
with respect to their bacoside content. Therefore, there is need to enforce strict quality con-
trol measures in the pharmaceutical sector for increasing the marketability of Indian Herbal
drug formulations. Since phytochemicals, particularly unique ones, like bacoside in Brahmi
is related to health benefits, inordinate variations in the formulation results in blotchy and
irreproducible health benefits. Also the content of phytochemicals used in the formulations
is not mentioned on the packaging leading to ambiguity. Manufacturers of herbal products
should include marker compound concentration on labels instead of giving only the name of
the herb.
4. Conclusion
The growing interest for the bioactive compounds present in botanicals, and their health
benefits, necessitate the development of simple, highly sensitive and reliable method for their
analysis. Thus, a pharmaceutical formulation that is not standardized does not consistently
provide the required amount of drug that is required for therapeutic benefit. In such a situation,
a known therapeutically useful drug may cause more harm than benefit to the patient as it
exposes him to sub-or-over therapeutic concentrations which may lead to either failure-of-
therapy or unwanted adverse effects, respectively. A multi-step approach need to be adopted
that includes greater awareness amongst consumers and manufacturers, tough guidelines and
their positive implementation, in order to get the benefits of herbal products.
Acknowledgements
The authors are thankful to Director, Council of Scientific and Industrial Research (CSIR)
Indian Institute of Integrative Medicine, Jammu; for providing necessary facilities during the

Academia Letters, July 2021 ©2021 by the authors — Open Access — Distributed under CC BY 4.0

Corresponding Author: Munish Sharma, munishptc@gmail.com

Citation: Sharma, M., Ahuja, A., Mallubhotla, S. (2021). Phytochemical variability among herbal formulations
of Bacopa monnieri (L.) Wettst. (Brahmi) – A memory enhancing herb. Academia Letters, Article 1822.
https://doi.org/10.20935/AL1822.
4
present work. MS would also like to acknowledge Shri Mata Vaishno Devi University, Katra
for providing the financial assistance under Research Assistantship Programme during PhD
tenure.

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Academia Letters, July 2021 ©2021 by the authors — Open Access — Distributed under CC BY 4.0

Corresponding Author: Munish Sharma, munishptc@gmail.com

Citation: Sharma, M., Ahuja, A., Mallubhotla, S. (2021). Phytochemical variability among herbal formulations
of Bacopa monnieri (L.) Wettst. (Brahmi) – A memory enhancing herb. Academia Letters, Article 1822.
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Academia Letters, July 2021 ©2021 by the authors — Open Access — Distributed under CC BY 4.0

Corresponding Author: Munish Sharma, munishptc@gmail.com

Citation: Sharma, M., Ahuja, A., Mallubhotla, S. (2021). Phytochemical variability among herbal formulations
of Bacopa monnieri (L.) Wettst. (Brahmi) – A memory enhancing herb. Academia Letters, Article 1822.
https://doi.org/10.20935/AL1822.
6
Academia Letters, July 2021 ©2021 by the authors — Open Access — Distributed under CC BY 4.0

Corresponding Author: Munish Sharma, munishptc@gmail.com

Citation: Sharma, M., Ahuja, A., Mallubhotla, S. (2021). Phytochemical variability among herbal formulations
of Bacopa monnieri (L.) Wettst. (Brahmi) – A memory enhancing herb. Academia Letters, Article 1822.
https://doi.org/10.20935/AL1822.
7
Academia Letters, July 2021 ©2021 by the authors — Open Access — Distributed under CC BY 4.0

Corresponding Author: Munish Sharma, munishptc@gmail.com

Citation: Sharma, M., Ahuja, A., Mallubhotla, S. (2021). Phytochemical variability among herbal formulations
of Bacopa monnieri (L.) Wettst. (Brahmi) – A memory enhancing herb. Academia Letters, Article 1822.
https://doi.org/10.20935/AL1822.
8