Haemagglutination as a rapid tool to differentiate Saraca asoca bark from the adulterant
Polyalthia longifolia
C BEENA* AND V V RADHAKRISHNAN
All India Coordinated Project on Medicinal , Aromatic Plants and Betalvine, College of Horticulture, Kerala Agricultural
University, P.O. Vellanikkara, Thrissur -680656, Kerala, India.
E mail: beenac2@gmail.com
ABSTRACT
Saraca asoca(Roxb.) Wilde, the asoka tree is one of the red listed plants of the Western Ghats. . The bark of asoka tree is
the source of the ayurvedic medicine “asokarishtam” used in the treatment of gynecological disorders. The rising
demand has led to its widespread adulteration. It is widely adulterated with the bark of Polyalthia longifolia an
ornamental tree. This paper presents a quick and easy method to determine the adulteration in asoka bark.
Haemagglutination method using the phosphate buffered saline ( PBS) extract of the stem barks and o positive
human erythrocytes was proved to serve as an effective ,quick, easy and cheap tool in differentiating the raw bark of
asoka from its major adulterant Polyalthia longifolia .This can be recommended as a tool for the floor level checking of
the market samples for ensuring the quality .
Key words:
Adulteration, haemagglutination, Saraca asoca, Polyalthia longifolia, PBS ( phosphate buffered saline) .
Saraca asoca (Roxb.) de Wilde commonly known
as Asoka (Figure 1) is a sacred tree of India, famous for its
use in the treatment of gynaecological disorders. Asoka
belongs to the family Caesalpiniaceae. It is one of the red
listed plants of the Western Ghats. Asoka is especially relied
upon as an astringent to treat excessive uterine bleeding from
various causes including hormone disorders, fibroids and for
regulating the menstrual cycle. It was estimated that the
domestic demand of the bark of Saraca asoca was more than
15,000 tonnes for the year 2007-08. This high annual demand
of the bark needs to be obtained from this medicinal tree
which is now in an endangered stage. As there is a wide gap
between demand and availability, it is clear that some other
plant material is collected and utilized instead of Saraca
asoca. There are reports that the bark of asoka is widely
adulterated with the bark of Polyalthia longifolia (Sonn.)
(Figure2) which is known as Bangali ashok. belonging to the
family Annonaceae. Polyalthia is having different medicinal
properties and uses and it cannot be used as a substitute to
asoka. Active ingredients that contribute to the medicinal
property of asoka are phenols and tannins where as that of
polyalthia are alkaloids. Substituting asoka with polyalthia
may not be effective in treating gynaecological disorders or it
may lead to some serious health hazards whose symptoms
will develop only later.
of common major adulterant of the important ayurvedic
herbal drug asoka bark and the results of the study are
presented here.
MATERIALS AND METHODS
Stem barks of Saraca asoca and Polyalthia
longifolia were collected from College of Horticulture,
Kerala Agricultural University, Thrissur, Kerala, India.and
authenticated by the botanists. The samples were shade dried.
1 g sample of each was put in 10 ml Phospahte buffered
saline ( PBS, pH 7.4) overnight ( 10%). This extract was used
for the HA ( haemagglutination) assay using standard
methodology10. Double fold serial dilutions of 50 ul extract
in 50 ul Phosphate buffered saline( PBS) was prepared in
micro titer plates (ELISA plates)and mixed with 50ul of 2%
PBS washed human erythrocytes of O positive blood group
taken from human volunteer. Microtiter(ELISA) plates were
incubated at room temperature for about 2 hours and the HA
titer for each sample was recorded. Haemagglutination titer
(HA titer) is the maximum dilution of the sample giving a
visible agglutination. Agglutination is the clumping together
of blood cells due to the network like linkage between the
Red Blood Cells (RBCs) and the specifically reacting
molecules present in the samples. As the RBCs are coloured
there is no need of any other colouring agents. It was noted
that all the S.asoca samples (4 different tree samples taken)
gave positive haemagglutination with an HA titer ranging
from 8 to 36 where as no agglutination was given by any of
the four different Polyalthia longifolia bark samples ( HA
=0) tried .(Figure-3). This revealed that the genuine S.asoca
bark can be easily differentiated from the adulterant
Polyalthia. using this technique.
Adulteration of herbal products has clinically
relevant effects. Health problems related to herbal drugs are
observed too often due to the contaminants rather than the
declared ingredients. As these adulteration cause serious
health hazards later ,it is important to have a floor level
checking for the market samples for avoiding the adulterants.
Under this circumstances we have taken up this study to find
out an easy ,quick and reliable method for the identification
Figure.1. Saraca asoca
Figure.2.Polyalthia longifolia
1
Haemagglutination assay (Figure 3)
2
4
8
16
32
6
6
A1 to A 4
P1 to P4
- asoka samples with 8, 8, 32, 8 as HA titer respectively.
- polyalthia samples showing no Haemagglutination.
reliable and effective tool for the authentication and quality
assessment of S. asoca and this method can be recommended
for the floor level checking of market adulterant of the
important herbal raw drug Saraca asoca. The work was
carried out during 2009- 2010.
RESULTS AND DISCUSSION
In most of the cases of drug adulteration, the
adulterant will have similar morphology as that of the
genuine samples. It is very difficult to distinguish them
physically. If the drug in question is spurious or adulterated,
or is from an entirely different biological source it may still
contain similar confusing compounds. Hence chemical
fingerprints also will be confusing. Fingerprinting
experiments by TLC conducted showed that there were a lot
of similarities between asoka and polyalthia rather than
differences. Remashree et al has reported that the
comparative anatomical study can be taken up for the
differentiation between the original and spurious bark
samples of asoka. Very recently S.Khatoon et al has reported
that HPTLC profile studies using the methanol extract of
bark samples can be depended. All these techniques require
costly equipments , chemicals and cumbersome procedures.
But the present study revealed that HA assay using O +
human RBCs is a good technique,practically very simple,
cheap and less cumbersome. It can be used as a quick
Haemagglutination technique has never before tried
adulterant identification in herbal drugs. Usually
chromatographic techniques are reported for standardization
and to control the quality of both the raw material and the
finished products. We tried a different biological technique
that can be used for differentiating asoka from polyalthia.
The presence of an entity- a haemagglutinin- was found in
the stem barks of saraca asoca which causes agglutination
of RBCs whereas it was found to be absent in polyalthia .
Detailed studies are required to find out the specific
molecule causing haemagglutination in asoka samples.
Acknowledgement
Authors thank the financial support from ICAR.
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