a preliminary study on the effect of traditional ayurvedic purifying ...

Journal of Herbal Medicine and Toxicology 4 (2) 237-247 (2010)
ISSN : 0973-4643 Original Article
A PRELIMINARY STUDY ON THE EFFECT OF TRADITIONAL
AYURVEDIC PURIFYING METHODS OF SEMECARPUS
ANACARDIUM LINN. NUTS – A PHYSICO-CHEMICAL AND
POWDER MICROSCOPIC STUDY
Venkateshwarlu G, Saraswathi P, Shantha T R, Shiddamallayya,
Kishore K R, Sridhar B N
Drug Standardization research Unit, National Ayurveda Dietetics Research Institute, (Central Council for
Research in Ayurveda & Siddha, Dept. of AYUSH, Ministry of Health & F. W, Govt. of India, New Delhi),
Ashoka Pillar, Jayanagar Bangalore-11
email: nadribengaluru@gmail.com
Abstract : Bhallataka - Semicarpus anacardium Linn seeds form one of the important
ingredients used in many medicinal formulations in Ayurveda for therapeutic and
Rasayana (rejuvenative) effects. They are reported to be potent antioxidants
corroborating their usage as rasayana (rejuvenator) in Ayurveda. Nonetheless, if used
without processing known as shodhana as per Ayurveda they have toxic effects. The
procedure of shodhana has been mentioned in ancient classics as a procedure that
removes external and internal doshas (impurities) and makes the material more potent,
effective, safe, assailable and homogeneous with out any adverse effects. But there
have been no studies to look into the changes in physiochemical and phytochemical
properties of the drug before and after shodhana. Hence in order to observe and
interpret the changes that might occur during shodhana and present it in the
contemporary light this <strong>study</strong> was planned incorporating Physicochemical analysis,
TLC and powder microscopy studies.
Keywords: Bhallataka, Semicarpus anacardium, Marking nut, Shodhana, Physico
chemical and Phytochemical studies, Powder microscopy
INTRODUCTION
Ayurveda is an ancient medical science for the
restoration of health and prevention of diseases
(patwardhan et al., 2009; Sharma et al., 2007).
Semecarpus anacardium (SA) nut finds in use in
variety of Ayurvedic medicinal preparations and is
effective against many diseases especially those that
are not amenable to allopathic medicines. It is called
as bhallataka in Ayurveda and commonly known as
marking nut in India. Chemical and phytochemical
analyses reveal the presence of biflavonoids, phenolic
compounds, bhilawanols, minerals, vitamins and amino
acids. A variety of nut extract preparations from this
source are effective in various diseases, viz. arthritis,
infections, tumours etc., (Premalatha., 2000;
Ramprasath et al., 2006), SA has been shown to
possess property of inducing apoptosis (Thatte et a.l,
2000). It has potent antioxidant properties (Verma et
al, 2009). Clinically compound preparations with SA
have been found to be safe including in pregnant
women (Murty, 1974). However, studies also have
reported cytotoxic effect of SA (Smith et al., 1995)
and toxic at dose dependent levels in animal studies
(Kesava Rao et al., 1979). Spermicidal effect of SA
in rats also has been reported (Sharma et.,al
2003).Toxic Phenols in SA causes dermatitis and
rashes (Goldsmith and Norman, 1943). A comparative
<strong>study</strong> of various purifying methods including the
traditional one using brick powder has been reported
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Journal of Herbal Medicine & Toxicology
and it has been found that the oily compound of the
SA nut has pro oxidant properties (Tripathi et al.,
2008). But there has been no conclusive studies
looking into physicochemical and powder microscopy
analysis of SA nuts before and after traditional
Ayurvedic purification method. Processing of
medicinal ingredients used in Ayurveda to detoxify
them is known as shodana (Mohapatra et al., 2007).
shodhana not only reduces the toxic effects but also
enhances the therapeutic effect of the drugs at times
imparts additional qualities* (CS,su.1). Hence this
<strong>study</strong> was planned.
Brief description of the drug in Ayurveda
Pharmacodynamics and pharmacokinetics in
Ayurveda is explained in terms of attributes of an
ingredient used as medicine or food the description in
brief are as follows; Rasa [tastes viz., Madhura
(sweet), Amla (sour) Lavana (saline), Katu (pungent)
and Kashaya (astringent)]. Guna [properties (effect
it has on the body after ingestion and assimilation
including the nature of its interaction with digestive
juices), viz., laghu (light for digestion), guru (heavy
for digestion). ruksha (dryness) and so on]. Veerya
[potencies (release or conservation of energy during
digestion and metabolism) viz., ushna veerya (releases
energy during digestion and metabolism) and sheeta
veerya (conserves energy during digestion and
metabolism)]. Vipaka [post digestive effect on
metabolism viz., madhura (sweet), amla (sour), katu
(pungent)]. Based on these the probable action of a
drug or food can be predicted and understood in terms
of action on a dosha (vata pitta or kapha) known as
Doshaghnata. Karma is systemic action
vyadhiharatva or prabhava is specific action on a
particular disease. The details of bhallataka are given
in table1.
3. Known facts about bhallataka
The pericarp of the fruit contains a bitter and powerful
astringent principle. The black corrosive juice of the
pericarp has tarry oil consisting of 90% of an oxyacid
named Anacardiac acid and 10% of a higher
non-volatile alcohol called Cardol (Naidu et al., 1925).
The crude extracts were found to be very toxic and
after purification, the toxicity was found to increase
as evident from LD50 values (Patwardhan et al.,
1986).
238
Ayurvedic method of administration after purification
has shown reduction in toxicity with maintained
efficacy (Patwardhan B.K.,et al 1988).
In order to verify the claims about the beneficial effect
of Shodhana, a <strong>preliminary</strong> <strong>study</strong> on the effect of
shodhana on the Bhallataka seeds have been
undertaken and also studied its physicochemical &
phytochemical changes of the drug before and after
shodhana with its comparative studies and detoxified
findings
4. Aim of the <strong>study</strong>
To identify change in phytochemical (TLC) and
botanical parameters (microscopy) in bhallataka seeds
with traditional purificatory procedures called
shodhana
MATERIALS AND METHODS
Materials used
Ballataka seeds -250gm.
Ishtika Churna (Brick powder)- Q.S. for gharshana
Jala (Water)- Q.S. for kshalana
Gunny bag for pottali,
5.2. Purification method
The Bhallataka seeds (250gms) were purchased
from the local market and the thalamus part of the
Bhallataka seeds was removed and made in to four
pieces of each seed. The cut pieces of Bhallataka
seeds were mixed with sufficient quantity of ishtika
churna (coarse brick powder) and made in to a pottali
by using a gunny bag. The mixtures was kept for 7
days then after these seeds were rubbed thoroughly.
The purified seeds were then washed in hot water
and dried in sun light and stored in moisture free
container (Ref. Rasmritam-Parishishtam-8,prishta-
147). The final quantity of sodhita bhallataka seeds
was 200gms.
5.3. Physico-Chemical Analysis
Both samples of seeds i.e. 1). Unpurified. 2). Purified
in Ishtika curna (coarse brick powder) were powdered
and used for the analysis. Physico-chemical &
<strong>preliminary</strong> phytochemical analysis of two samples
were carried out employing standard procedures and

Table 1 : Ayurvedic description of Bhallataka
S. No. Ayurvedic characteristic Properties
1 Rasa (taste) Katu (pungent)
2 Guna (quality) Laghu (light)
3 Veerya (potency) Ushna (hot)
4 Vipaka (post digestive effect) Madhura (sweet)
5 Doshagnata (effect on doshas) Vata kapha hara (pacifying)
using GPR grade reagents [(WHO, 1996) British
Pharmacopoea, Indian Pharmacopoea ].
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6 Karma (action) Medhya (intellect promoting) agnivardhaka (digestive & metabolic
corrective)
7 Vyadhiharatva (indications)
8
Bhallataka Yoga (preparations
with SA)
Kushta (skin disorders), gulma (space occupying lesions in the
abdomen), krimi(parasitic infestations) arshas (hemorrhoids)
Bhallataka rasayana, Bhallataka modaka, Amritha Bhallataka leha,
Sanjeevani vati, Bhallataka ghritha and Bhallataka avaleha
Table 2 : Showing values of physicochemical parameters before and after shodhana
S. No Parameters (%) Fresh seed (Before shodhana) Seed (After shodhana)
1 Weight 250 gms 200 gms
2 Ash content 1.88 13.98
3 Water soluble ash 0.69 6.68
4 Acid insoluble ash 0.50 3.76
5 Solubility in ethanol 10.50 9.34
6 Solubility in water 4.35 7.59
7 pH 6.2 6.9
8 Loss on drying at 105ºC 1.98 2.15
Table 3 : Rf values of the SA seeds before and after shodhana (for polyphenols)
S.No. Sample Extractive
s
1 Semicarpus
anacardium Seeds
(Before purification)
2 Semicarpus
anacardium Seeds
(After purification)
Adsorben
t
Methanol Silica gel
60 F254
Methanol Silica gel
60 F254
Solvent
system
Toluene: Ethyl
acetate (93:7)
Toluene: Ethyl
acetate (93:7)
Viewing
reagent
Rf Values
Iodine vapour 0.10, 0.24,
0.47, 0.71,
0.76, 0.85
Iodine vapour 0.18, 0.27,
0.30, 0.36,
0.43, 0.57,
0.71, 0.91
5.4. Thin layer chromatographic studies (TLC)
Comparative TLC was done using two solvent
systems a) 93:7- Toluene: Ethyl acetate solvent

Journal of Herbal Medicine & Toxicology
Table 4 : Rf values of the SA seeds before and after shodhana (for oils and fats)
S.No. Sample Extractives Adsorbent Solvent
system
1 Semicarpus
anacardium Seeds
(Before
purification)
2 Semicarpus
anacardium Seeds
(After purification)
Methanol Silica gel
60 F254
Methanol Silica gel
60 F254
Table 5 : Organoleptic characteristics of bhallataka seeds
240
Benzene:
Ethyl acetate
(6:1)
Benzene:
Ethyl acetate
(6:1)
Viewing
reagent
Iodine
vapour
Iodine
vapour
Rf Values
0.10, 0.18,
0.23, 0.32,
0.38, 0.58,
0.82, 0.90
0.10, 0.18,
0.23, 0.32,
0.38, 0.58,
0.90
S. No. Parameter Characteristic (before shodhana) Characteristic (after shodhana)
1 color Brownish black dark brown
2 touch Greasy coarse Dry coarse
3 odour odourless odourless
4 consistency Very hard Moderately hard
Table 6 : Showing description of microscopy images
S. no Figure no. Description
1 1-4 Different fragments of tissues
2 5-6 Elongated small brown colored epicarp cells of epidermis.
3 7 Elongated stone cells with heavily lignified cell walls
4 8 Parenchyma cells and xylem vessels with helical thickenings
5 9 Parenchyma cells with reddish tannin contents
6 10 Thin walled rounded parenchyma cells.(Mesocarp cells)
7 11 Elongated ,thin walled parenchyma cells
8 12 Xylem vessels with helical thickenings
9 13 Xylem vessels with helical to reticulate thickenings
10 14 Rounded to elongated stone cells with heavily lignified cell walls.
11 15 Parenchyma cells with oilgloubles
12 16 Brown colored thick walled parenchyma cells
13 17 Parenchyma cells with reddish tannin content
14 18 Thin walled parenchyma cells
15 19 Fragments of endocarp cells, which are elongated, covered with thin cuticle and with
reddish content of tannin.
16 20 Reticulate to spiral xylem vessel.
17 21 Abundant small oilgloubles
18 22 Simple, oval to rounded starch grains
19 23 Compressed, elongated stone cells with lignified thickenings, lumen narrow

system for characterization of polyphenols and (b)
6:1- Benzene: Ethyl acetate solvent system for
characterization of oils and fats (Igon & Stahl, 1969).
The iodine vapour and long wave length (365 nm)
ultra violet images were evaluated.
5.4. Powder macro and microscopy
Sodhita bhallataka seeds were then powdered and
organoleptic characteristics were noted. Powder was
then treated with chloral hydrate solution and water,
observed under the microscope for diagnostic and other
features and photomicrographs were taken before and
after shodhana.
RESULTS
6.1. Physicochemical analysis of bhallataka seeds
before and after shodhana
The values of physico-chemical parameters before
and after shodhana are given in Table 2
6.2. Thin Layer Chromatography studies before and
after shodhana in 93:7 -Toluene: Ethyl acetate solvent
medium
The iodine vapour view and U-V view of T L
Chromatograms are shown in images 1, 1a, 2, 2a and
Rf values in Table 3.
6.3. Thin Layer Chromatography studies before and
after shodhana in 6:1 Benzene: Ethyl acetate solvent
medium
The iodine vapour view and U-V view of T L
Chromatograms are shown in images 3, 3a and Rf
values in Table 4.
6.4. Powder Macroscopy of Bhallataka seed powder
The macroscopy of bhallataka powder is shown in
image 4 and characteristics in table 5.
6.5. Powder microscopy
The diagnostic features of bhallataka seed powder
(Prasad et al, 1968) were identified in the sample and
are as follows.
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Presence of fragments of small, thin walled epidermal
cells.
Presence of fragments of elongated parenchyma cells
filled with reddish content of tannin of endocarp cells.
Presence of oilgloubles in parenchymatous cells.
Presence of compressed stone cells, lumen narrow,
with lignified walls .
Presence of fragments of xylem vessels with pitted ,
reticulate and helical thickenings. .
Presence of thinwalled parenchymatous cells with
reddish content of tannin.
The images and detailed descriptions of powder
microscopy of bhallataka seeds are shown in figures
1-23 and detailed descriptions are depicted in table 6
DISCUSSION
The objective of shodhana in Ayurveda is to make
the medicinal ingredient fit for a formulation and for
human consumption in anticipation of a desired disease
modifying effect. This concept hence is indicative of
quality control awareness of ancient scientists.
Nevertheless, the conventional interpretation of a
reductionist approach in detoxifying process, of late
can lead to misunderstanding of the intentions of
processes like shodhana with which they were
originally designed. This is because process like
shodhana might bring key changes that are beyond
just presence or absence of some chemical
constituent.
7.1. Physicochemical analyses
Physicochemical analyses showed the following; Loss
of 50 gms after shodhana might be attributed to the
washing and transfer of contents during physical
cleansing. Increase in water soluble ash and
corresponding increase in pH value turning it towards
neutral suggests that increase in alkaline salts
associated with shodhana making it less corrosive.
Increased water insoluble ash might be attributed to
increased amount of silica when the seeds were mixed
with brick powder. Insignificant change in ethanol
solubility might suggest that the process has not
brought in any change in the ethanol soluble fraction
of the polyphenols that have been attributed with
antioxidant activity. Whereas increased water
solubility was seen. This might be because of
increased fraction of water soluble tannins that most
of the times possess free radical scavenging activity.
Change in values of loss on drying was also
insignificant. The above results hence suggests that
shodhana procedure brings about increasing the
alkalinity of a more acidic form, apart from maintaining
the phenolic constituents of the seeds that may be
useful in bringing about free radical scavenging activity
usually noted in bhallataka seeds.
7.2. Thin Layer Chromatography
In the present <strong>study</strong> Methanol extracts were subjected
to TLC in Toluene Ethyl acetate (93:7) solvent system
which is specific among others, for poly-phenols. The
compounds corresponding to certain Rf values present
before shodhana (0.10, 0.24, 0.47, 0.76, 0.85) were
absent after shodhana. Similarly compounds
corresponding to certain Rf values (0.18, 0.27, 0.30,
0.36, 0.43, 0.57, 0.91) were present only after
shodhana. This suggests that shodhana must have
brought extensive rearrangement in the phenolic
constituents of bhallataka that might contribute to the
multidimensional attributed effects.
Further, TLC of Methanol extracts of the seeds before
and after shodhana in Benzene: Ethyl acetate (6:1)
solvent system specific to oils and fats revealed that
almost all the compounds corresponding to certain
Rf values (0.10, 0.18, 0.23, 0.32, 0.38, 0.58, 0.82, 0.90)
present before shodhana were present after shodhana
except that corresponding to 0.82. Besides, the
intensity of the chromatogram was reduced after
shodhana which means that shodhana has brought in
some change in the oily fraction of the constituents
of bhallataka by removing a certain compound that
makes it non toxic. Hence suggesting shodhana does
not necessarily mean complete removal of the tarry
oily portion, rather change in the chemical architecture
of this portion to make it non toxic and safe for human
consumption.
7.3. Powder Microscopy
Powder Microscopical studies after shodana revealed
that, all the microscopical characters are found to be
present in powder except calcium oxalate crystals
are not present in powder form. The powder <strong>study</strong> of
the fruit after purification has been described
highlighting its important characters, which will be
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Journal of Herbal Medicine & Toxicology
useful in identification of the fruit in powder form after
purification. The powder <strong>study</strong> revealed that, when it
is compared with that of the microscopical studies of
the fruit (S.Prasad etal 1968) it appears to be same in
having all the characters in powder form, even after
purification. It can be concluded that, all the characters
appears to be similar before and after purification of
the drug except in the absence of calcium oxalate
crystals in purified powder. Calcium oxalate crystals
are common biomineralization product in higher plants
(Jáuregui et al., 2003). They have been used as
microscopic markers in authentication of certain herbs
like ginseng (Yap et al., 2009). It also helps in
differentiating as well as characterization of herbal
products like in Fourier Transform Infra Red
Spectroscopy (FTIR) (Liang et al., 2009).
crystallochemical characterization of calcium oxalate
crystals can prove to be useful in identification of
different morphologies shown by these crystals grown
inside specialized cells in plants (Jáuregui et al., 2003).
Hence presence and absence of Calcium oxalate
crystals can be promising in its utility as a authentic
markers for bhallataka before and after shodhana.
Further, after absorption cannot be metabolized and
is excreted by the kidney into urine, where it binds to
calcium forming an insoluble salt that may precipitate
to form kidney stones (Massey et al., 2001). Hence
removal of calcium oxalate crystals after shodhana
is strong evidence of purification that makes bhallataka
safe for human consumption.
7.4. Summary and conclusion
Bhallataka seeds form one of the important ingredients
used in many medicinal formulations in Ayurveda.
They are reported to be potent antioxidants
corroborating their usage as rasayana (rejuvenator)
in Ayurveda. Nonetheless, if used without processing
known as shodhana as per Ayurveda they have toxic
effects. The procedure of shodhana has been
mentioned in ancient classics as a procedure that
removes external and internal doshas (impurities) and
makes the material more potent, effective, safe,
assailable and homogeneous with out any adverse
effects. But there have been no studies to look into
the changes in physicohemical and phytochemical
properties of the drug before and after shodhana.
Hence in order to observe and interpret the changes
that might occur during shodhana and present it in
the contemporary light this <strong>study</strong> was planned.
246
Physicochemical analysis, TLC and powder
microscopy studies on the drug before and after
shodhana revealed that the ancient detoxification
procedure brings about changes in pH, alters
phytochemical composition and removes calcium
oxalate crystals respectively making the drug fit for
human consumption.
ACKNOWLEDGEMENT
Authors are thankful to Director General, C.C.R.A.S,
New Delhi for providing facilities and encouragement.
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