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Cholinesterase inhibition activity of

Marsilea quadrifolia Linn. an edible
leafy vegetable from West Bengal, India
a a b
Santanu Bhadra , Pulok K. Mukherjee & A. Bandyopadhyay
a
School of Natural Product Studies, Department of
Pharmaceutical Technology, Jadavpur University, Kolkata 700 032,
India
b
Indian Institute of Chemical Biology, Kolkata 700 032, India

Available online: 06 Oct 2011

To cite this article: Santanu Bhadra, Pulok K. Mukherjee & A. Bandyopadhyay (2011):
Cholinesterase inhibition activity of Marsilea quadrifolia Linn. an edible leafy vegetable from West
Bengal, India, Natural Product Research, DOI:10.1080/14786419.2011.565006

To link to this article: http://dx.doi.org/10.1080/14786419.2011.565006

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 Natural Product Research
2011, 1–4, iFirst

SHORT COMMUNICATION
Cholinesterase inhibition activity of Marsilea quadrifolia Linn. an edible
leafy vegetable from West Bengal, India
Downloaded by [National Agency of Science and Technology Information] at 06:36 11 December 2011

Santanu Bhadraa, Pulok K. Mukherjeea* and A. Bandyopadhyayb

a
School of Natural Product Studies, Department of Pharmaceutical Technology, Jadavpur
University, Kolkata 700 032, India; bIndian Institute of Chemical Biology, Kolkata 700 032, India
(Received 13 September 2010; final version received 17 February 2011)

Maesilea quadrifolia Linn. (Marsileaceae) is a leafy vegetable well known in India.

The current study aims to explore the phytochemical profile of M. quadrifolia and
investigate its anti-cholinesterase potential. The methanol extract of the plant was
subjected to qualitative and quantitative phytochemical screening (total alkaloi-
dal content, saponin content and phenol content) and its anti-cholinesterase
potential was tested by TLC bioautography and other screening methods using
acytylcholinesterase (AChE) and butyrylcholinesterase (BChE). The study
revealed that the extract contains various classes of phytoconstituents including
steroids, saponins, alkaloids and other polyphenols. Total alkaloid, phenolic and
saponin contents were found to be 19.3 mg g 1 and 158.5  1.02 mg g 1 as gallic
acid equivalents and 2.63 mg g 1 of the extract, respectively. The TLC
bioautography method exhibited the inhibition of both enzymes. In a microtiter
plate assay, the IC50 values of the extract for AChE and BChE were found to be
51.89  0.24 mg mL 1 and 109.43  2.82 mg mL 1, respectively. These findings
suggest that M. quadrifolia is a potential lead as an AChE and BChE inhibitor,
which may be useful in the management of Alzheimer’s disease.
Keywords: Marsilea quadrifolia; acetylcholinesterase; butyrylcholinesterase;
Alzheimer’s disease; total phenolic content; total saponin content

1. Introduction
Alzheimer’s disease (AD) is one of the most common forms of age-related neurodegen-
erative disorder, manifests by progressive memory impairments and emotional stress
(Mukherjee & Houghton, 2009; Mukherjee & Saha, 2003). According to the cholinergic
hypothesis and evidences from the clinical trials it has been observed that the therapeutic
approach to enhance cholinergic neurotransmission by the use of cholinesterase (AChE)
inhibitors has been proven to be the most successful means of balancing the cholinergic
deficit and stabilising the symptoms in mild AD, thus many AChE inhibitors are marketed
today for the treatment of AD (Choudhary, Devkota, Nawaz, Ranjit, & Rahman, 2005;
Giacobini, 2004). Recent advancements in research revealed that both the enzymes,
Acetylcholinesterase (AChE) and butyrylcholinesterase (BChE), play a critical role in
cholinergic transmission by hydrolysing the excitatory transmitter acetylcholine (Ach;
Ucar, Gokhan, Yesilada, & Bilgin, 2005). Though, specificity of AChE towards ACh is

*Corresponding author. Email: naturalproductm@gmail.com

ISSN 1478–6419 print/ISSN 1478–6427 online

ß 2011 Taylor & Francis
http://dx.doi.org/10.1080/14786419.2011.565006
http://www.tandfonline.com
 2 S. Bhadra et al.

more than BChE, but at later stage with lower AChE concentration in brain, BChE
aggravates the disease condition (Houghton et al., 2004). Thus inhibition of both the
enzymes is important in the therapy of AD.
Many natural compounds such as galanthamine, rivastigmine etc. are well-established,
clinically approved AChE inhibitors found in market for the treatments of AD
(Mukherjee & Houghton, 2009). In our laboratory we have reported many plant derived
AChE inhibitors and the search is still on (Mukherjee, Ahamed, Kumar, Mukherjee, &
Downloaded by [National Agency of Science and Technology Information] at 06:36 11 December 2011

Houghton, 2007a; Mukherjee, Kumar, & Houghton, 2007b; Mukherjee, Kumar, Mal, &
Houghton, 2007c; Satheesh, Mukherjee, Bhadra, & Saha, 2009a). From this point of view
we have selected Maesilea quadrifolia (Marsileaceae), a common Indian hydrophytic
species growing abundantly in deep water or erect in low water or on land. In the Indian
traditional system of medicine it is used as a sedative and the local people in West Bengal,
India, use this plant along with their meal for the treatment of insomnia and other mental
disorders. Among other traditional uses, juice of this plant is used as anti-inflammatory,
diuretic, depurative, febrifuge, refrigerant, in the treatment of snakebite and abscesses
(Ripa, Nahar, Haque, & Islam, 2009). Presence of some chemical constituents were
reported previously in this plant, these include ployphenolics, terpinoids and steroids,
namely Quercetin-3-rutinoside and naringenin 7-rhamnoglucoside (Lal, Gupta, & Garg,
1983; Venkataramaiah, Venkataramaiah, Ramana, & Prasad, 1981). It also contains an
enzyme named Thiaminase (Ripa et al., 2009). Present work was undertaken to investigate
the cholinesterase inhibition activity of M. quadrifolia, based on its prominent effect on
central nervous system and its use in traditional Indian medicine.

2. Results and discussion

The present study was an effort to determine the phytochemical profile and anticholin-
esterase potential of M. quadrifolia. Currently, AChE inhibitors are clinically approved
first line of treatments for the management of mild to moderate AD. Natural products
contributed a lot in the development of potential inhibitors for AChE and BChE for the
management of AD. There are several reports on different classes of phytoconstituents
responsible for inhibition of brain cholinesterases. Alkaloids, terpinoids, flavonoids from
different plants showed potential Anticholinesterase (Mukherjee & Houghton, 2009).
Supplies of M. quadrifolia were purchased from local supplier in Kolkata, West Bengal,
India and authenticated (voucher specimen no. SNPS-1059). Plant materials were
extracted with 90% methanol (per cent yield 10.78% w/w). Qualitative phytochemical
screening (Wagner, Bladt, & Zgainski, 1984) of the extract revealed that the plant contains
various classes of phytoconstituents, steroids, flavonoids, alkaloids, terpinoids and
saponins. Total saponin content in the extract was determined by the method as described
by Mukherjee (2002) and found to be 2.63 mg g 1 of extract. Estimation of total phenolic
content was determined by Folin–Ciocalteu (FC) reagent (Spanos & Wrolstad, 1990). The
phenolic content was found to be 158.5  1.02 mg g 1 as gallic acid equivalent (GAE).
Similarly the total alkaloid content of the extract was found to be 19.3 mg g 1 of extract
(Mukherjee, 2002).
Extract of M. quadrifolia (10 mg mL 1) showed prominent inhibiting spots in TLC
bioautography by cholinesterase inhibition methods (Ellman, Lourtney, Andres, &
Gmelin, 1961; Satheesh, Mukherjee, Bhadra, Saha, & Pal, 2009b). The true inhibition was
confirmed by matching the false positive plates. It was observed that the spots developed
on extract plates for each enzyme was not corresponding to the false positive spots. This
experiment concluded the preliminary confirmation of cholinesterase inhibitory activity of
the extract. For further confirmation of the cholinesterase inhibition activity, microplate
 Natural Product Research 3

assay method was applied (Ellman et al., 1961; Satheesh et al., 2009b), where the extract
showed good inhibition against both AChE and BChE enzymes and the IC50 values of
were found to be 51.89  0.24 mg mL 1 and 109.43  2.82 mg mL 1, respectively. The
inhibition activity of the crude extract was lower compared to galanthamine. It was also
observed that the affinity of M. quadrifolia was more towards the AChE than the BChE.
Dose response curve of the methanol extract and galanthamine has been depicted in
Figures 1 and 2. This finding emphasises that extract of M. quadrifolia has potential
Downloaded by [National Agency of Science and Technology Information] at 06:36 11 December 2011

anticholinesterase activity.

3. Conclusion
M. quadrifolia showed potent AChE and BChE inhibition activity and it can be explored
further for the lead development from natural resources with reference to AD
management.

80
Methanol
70
Galantamine
60
% Inhibition

50
40
30
20
10
0
–5 –4 –3 –2 –1 0
Log concentration

Figure 1. Dose response curve M. quadrifolia extract and galanthamine against AChE, calculated
from the prism sigmoidal dose response curve (variable slope) obtained by plotting the percentage
of inhibition vs. the concentrations.

45
40 Methanol
35 Galantamine
30
% Inhibition

25
20
15
10
5
0
–5 –4 –3 –2 –1 0
Log concentration

Figure 2. Dose response curve M. quadrifolia extract and galanthamine against BChE, calculated
from the prism sigmoidal dose response curve (variable slope) obtained by plotting the percentage
of inhibition vs. the concentrations.
 4 S. Bhadra et al.

Supplementary material
Experimental details relating to this article are available online.

Acknowledgements
The authors wish to express their gratitude to the Department of Science and Technology,
Government of India for financial support through SERC project grant [F. No.-SR/SO/HS-11/
2008].
Downloaded by [National Agency of Science and Technology Information] at 06:36 11 December 2011

References

Choudhary, M.I., Devkota, K.P., Nawaz, S.A., Ranjit, R., & Rahman, A. (2005). Cholinesterase inhibitory
pregnane-type steroidal alkaloids from Sarcococca hookeriana. Steroids, 70, 295–303.
Ellman, G.L., Lourtney, D.K, Andres, V., & Gmelin, G. (1961). A new and rapid colorimetric determination of
acetylcholinesterase activity. Biochemistry and Pharmacology, 7, 88–95.
Giacobini, E. (2004). Cholinesterase inhibitors: new roles and therapeutic alternatives. Pharmacological Research,
50, 433–440.
Houghton, P.J., Agbedahunsi, J.M., & Adegbulugbe, A. (2004). Choline esterase inhibitory properties of
alkaloids from two Nigerian Crinum species, Phytochemistry, 65, 2893–2896.
Lal, S.D., Gupta, V.M., & Garg, R.K. (1983). Quercetin-3-rutinoside and naringenin 7-rhamnoglucoside in
Marsilea sporocarps. Current Science, 52, 263–264.
Mukherjee, P.K. (2002). Quality control on herbal drugs. New Delhi: Business Horizons.
Mukherjee, P.K., Ahamed, N., Kumar, V., Mukherjee, K., & Houghton, P.J. (2007a). Protective effect of
biflavones from Araucaria bidwillii Hook in rat cerebral ischemia/reperfusion induced oxidative stress.
Behaviour Brain Research, 178, 221–28.
Mukherjee, P.K., & Houghton, P.J. (2009). The worldwide phenomenon of increased use of herbal products:
Opportunities and threats. In P.K. Mukherjee & P.J. Houghton (Eds.), Evaluation of herbal medicinal
products - perspectives of quality, safety and efficacy (pp. 3–12). London: Pharmaceutical Press.
Mukherjee, P.K., Kumar, V., & Houghton, P.J. (2007b). Screening of Indian medicinal plants for
acetylcholinesterase inhibitory activity. Phytotherapy Research, 21, 1142–45.
Mukherjee, P.K., Kumar, V., Mal, M., & Houghton, P.J. (2007c). Acetyl cholinesterase inhibitors from plants.
Phytomedicine, 4, 289–300.
Mukherjee, P.K., & Saha, B.P. (2003). Quest for GMP in the production of quality botanicals.
In P.K. Mukherjee & P.J. Houghton (Eds.), GMP for botanicals (pp. 165–109). New Delhi: Business
Horizons.
Ripa, F.A., Nahar, L., Haque, M., & Islam, Md.M. (2009). Antibacterial, cytotoxic and antioxidant activity of
crude extract of Marsilea quadrifolia. European Journal of Scientific Research, 1, 123–129.
Satheesh, N.K., Mukherjee, P.K., Bhadra, S., & Saha, B.P. (2009a). Acetylcholinesterase enzyme inhibitory
potential of standardized extract of Trigonella foenum graecum L and its constituents. Phytomedicine, 17,
292–295.
Satheesh, N.K., Mukherjee, P.K., Bhadra, S., Saha, B.P., & Pal, B.C. (2009b). Acetylcholinesterase inhibitory
potential of a carbazole alkaloid, Mahanimbine, from Murraya koenigii. Phytotherapy Research, 24,
629–31.
Spanos, G.A., & Wrolstad, R.E. (1990). Influence of processing and storage on the phenolic composition of
Thompson seedless grape juice. Journal of Agricultural and Food Chemistry, 38, 1565–1571.
Ucar, G., Gokhan, N., Yesilada, A., & Bilgin, A.A. (2005). 1-N-Substituted thiocarbamoyl-3-phenyl-5-thienyl-2-
pyrazolines: a novel cholinesterase and selective monoamine oxidase B inhibitors for the treatment of
Parkinson’s and Alzheimer’s diseases. Neuroscience Letters, 382, 327–331.
Venkataramaiah, C., Venkataramaiah, V., Rao, K., Ramana, V., & Prasad, S.V. (1981). Studies on phenolic acid
pattern in Marselia quadrifolia, L. Comparative Physiology and Ecology, 6, 302–304.
Wagner, H., Bladt, S., & Zgainski, E.M. (1984). Plant drug analysis: A thin layer chromatography atlas. Springer-
Verlag: Berlin Heidelberg.