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Ancient Science of Life Vol: XXIV (3) January, February, March - 2005 Pages 143 - 151
Evaluation of Oxidative Stress Status Following Polyherbal Formulation
Therapy In Patients of Cholelithiasis with Choledocholithiasis
PANKAJ SRIVASTAVA, M.SAHU,SHRUTI KHANNA AND HARI D. KHANNA
Department of Shalya Shalakya and Biophysics
Institute of Medical Sciences, Banaras Hindu University, Varanasi - 221005
Received: 16-7-2004 Accepted: 12-12-2004
ABSTRACT:
Free radicals produce persistent oxidative stress in biological system and are highly reactive
molecules produced as a byproduct of metabolism. A reactive free redical generated in the body
reacts with non-radical molecules and results in free radical chain reaction leading to formation
of new free radicals. If the defense mechanism of body fails to combat them or they are not
properly utilized in the body -these silent killers pose a threat by injuring tissues, their proteins
and fat contents. Lipids in the cell membrane undergo degradation to form hydroperoxides(1, 2, 3).
Polyunsaturated fatty acids, PUFA, are especially liable to lipid peroxidation. Lipid
hydroperoxids decompose to form a variety of products including malondialdehyde (MDA)
which is used as an indicator of oxidative damage of cells and tissues(4).
The present investigations involve the study of oxidative stress in the bile juice from the patients
of cholecystitis/cholelithiasis with choledocholithiasis treated by cholecystectomy with
choledochotomy (CBD exploration) with T-tube drainage. Results of malondialdehyde status in
the bile juice of these patients pre-operatively and following polyherbal formulation therapy
from 3rd
to 10th
post operative day are discussed.
Key Words: Oxidative stress, MDA status, Bile, Polyherbal drug therapy.
INTRODUCTION
Phaltrikadi Kwath is a well known and
commonly used drug in the management of
(Jaundice) kamala roga. This decoction
comprises of eight different plants viz.
Haritaki, Vibhitak, Amalaki, Guduchi,
Kutki, Chirayita, Vasa and Neem. The
chemical constituents and pharmacological
actions of these drugs are well established.
These are potent cholagogue,
immunomodulator, antiallergic, antioxidant,
bitter, anti-inflammatory, antiallergic,
antioxidant, antipyretic and hepatoprotective
drugs. Therefore the present study was
carried out to evaluate the efficacy of "
Phaltrikadi Kwath" in the patients before an
after administration of the drug following
the cholecystectomy.
MATERIAL AND METHODS
Selection of Cases
Thirty cases of cholelithiasis with
choledocholithiasis were selected from the
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patients attending to shalya OPD/IPD of
University Hospital, BHU. Each patient was
selected on the basis of the diagnostic
criteria which included a detailed history in
the form of chief complaint-occurrence of
abdominal pain, it site, character, duration,
associated features Viz. nausea,vomiting,
fever, chills, jaundice and flatulence. Apart
from this, history of systemic disorders like
tuberculosis, hypertension and diabetes
mellitus was taken. History of previous
treatment particularly previous surgery (type
of operation), family history, occupation and
dietary habits was recorded. Blood, urine,
stool and radiological investigations was
done for each patient.
Selection Clinical Model
The selection of clinical model was done in
such a way so that it should be easy, non,
invasive, less expensive and patient friendly
to meet our the needs of the research work.
Therefore, we selected the patients (age
group 30-60 years) of cholecystitis/
cholelithiasis with choledocholithiasis
treated by cholecystectomy with
choledochotomy (CBD exploration) with T-
tube drainage.
In these patients T-tube drainage of bile was
the usual procedure and the bile was
collected either directly from the T-tube or
collection bag without doing anything
specific to the patients for at least 10 days.
In routine, postoperative bile samples were
taken by T-tube whereas intra-operative
samples were taken by direct aspiration of
common bile duct.
Thirty cases of cholelithiasis with
choledocholithiasis were selected for the
present study and they were divided into two
groups off fifteen each. Group B was the
drug treated where as group A was without
any drug and served as control.
Drug and Dosage
Table 1 illustrates the composition of drug
material of polyherbal formulation known as
"Phaltrikadi K wath" depicting the name of
ingredients, part taken alongwith its
properties for preparation of decoction. All
the ingredients were dried in shade, taken in
equal proportion and chopped into small
pieces. All materials were thoroughly
mixed and soaked into water for 3-4 hours
and then boiled in the stainless steel
container at 70o
-80o
c till only once fourth
(1/4th
) of the initial volume remained.
During this period intermittent stirring of the
contents was done. The decoction was
finally filtered through muslin cloth and than
allowed to cool at room temperature. For
preparation of 100ml of drug (decoction)
approximately 22 gms of crude drug mixture
was taken.
Freshly prepared decoction was given orally
to each patient of group (B)at the dosage of
30ml twice daily and the duration was 3rd
to
10th
post operation day whereas no drug (A)
cases during this period.
Physico-chemical studies of the drug
(decoction) were made to record its
absorption spectrum by using a recording
Beckman spectrophotometer, pH with pH
meter, viscosity in relation to water with a
Ostwald's type of viscometer, relative
density with a specific gravity bottle,
refractive index with a refractometer,
conductivity with a conductometer,
Osmolarity with a Osmometer and polarity
with a polarimeter.
Assay of Lipid peroxidation (LPO):
The assay of LPO in the serum was
performed by the technique of Philpot (5)
with the suggested modifications (6).
A fresh
stock regent of TBA-TCA-HCI was
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prepared at the time of assay. 0.01%
butylated hydoxy toluene was added to the
stock regent to abolish the metal catalysed
autoxidation of lipids (7).
Standard
malondialdehyde bis (dimethyl) acetal
solution obtained form Aldrich chemical
Co., USA. 2ml of stock regent was added
separately to 1ml test sample, standard and
black respectively. All the samples of test,
standard and blank were heated for 20
minutes at 80oc and then allowed to cool at
room temperature. Thereafter, the pink
pigment from all the samples was extracted
with 4ml of n-butanol and their optical
absorbance was recoded at 530 nM in a
SICO spectrophotometer.
RESULTS
All the patients in both the groups were age
and sex matched. The patients were in the
age group of 30-60 years. The patients were
distributed equally in both the groups
according to their habits viz., vegetarian or
non vegetarian, occupation, socio-economic
and bowel habits. Maximum number of the
patients were tobacco chewers followed by
smoking and alcohol whereas 27% of the
patients had no addiction. The presenting
features of the patients varied from upper
abdominal pain with nausea and fever.
Jaundice was observed in more than 90% of
cases in both the groups.
Assessment of preoperative hematological
parameters of patients showed routine TLC,
DLC, hemogram, fasting blood sugar level,
blood urea and serum creatinine were within
normal range with lower levels of
hemoglobin values. Liver function tests
were also preformed in all patients. Serum
bilirubin levels were higher in group in
group B cases.
Physico-chemical characteristics of drug
"Phaltrikadi K wath" shown in the table 2
points that it contains approximately 5%
total solid content, reducing sugar and
tannins were present in all samples where as
polysaccharides were absent; with mean
values of pH 3.52 0.14, viscosity 1.465 
0.024 centipoise, specific gravity 1.008
0.0013, refractive index 1.333  0.0007,
conductivity 2.406  0.234 m Mhos/cm and
Osmolarity 139.65  4.0688 mOsm/kg. The
decoction (Kwath) is optically inactive.
The effect of Phaltrikadi Kwath (decoction)
was assessed in the randomly selected 30
patients. The drug was given orally in the
form of freshly prepared decoction to all the
patients of treated cases (group B from 3rd
post-operative day to 10th
post operative day
whereas no drug was given to the control
cases (group A) during this period.
Lipid peroxidation levels in the bile juice of
patients of cholecystitis/cholelithiasis with
choledocholithiasis treated by
cholecystectomy with choledochotomy
(CBD exploration) and Kehr's T-tube
drainage were assessed in all the treated
patients immediately on day 0(sample I), on
3rd
day (sampleII)and on 10th
day (sample
III). Bile samples were taken from all the
patients irrespective of the size, number and
type of stones. Bile samples were analysed
for malondialdehyde levels immediately
after operation or were stored at 20oc until
the time of analysis. The results were
compared with their control cases (table3).
The observed lipid peroxidation levels show.
Difference between the levels of sample I
(day0) and sample II (day 3) in both the
groups was significant (p<0.001)
Difference between the levels of sampleII
(day3) and sample III (day 10) in both the
groups was significant (p<0.0001)
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Difference between the levels of sample I
(day 0) and sample III (day 10) in control
and treated groups was also significant at
P<0.01 and p<0.001 respectively.
Observed increase in lipid peroxidation
levels in sample I and II followed by a
decrease in MDA levels in sample III on day
10 of drug therapy with respect to control
cases points that the drug "Phaltrikadi
Kwath" has antioxidant potential.
DISCUSSION
This study was designed to evaluate the
oxidative stress status following the
polyherbal drug formulation therapy in
patients of cholelithiasis with
choledocholithiasis. In Ayurveda
"Phaltrikadi Kwath" is described for
management of obstructive jaundice
shakhashrita kamala roga8. Phaltrikadi
Kwath contains eight herbal drugs which are
having predominantily kamalahara
properties like pittkapha shamaka,
yakriduttejaka, shothahara, pandurogahar,
rechan, and Deepan etc.
The objective of the present study therefore
was to evaluate antioxidant potential of
polyherbal formulation "Phaltrikadi Kwath"
in biliary stone diseases.
To assess the effect of phaltrikadi Kwath,
bile samples were analyzed at different
intervals and the study points that the drug
significantly lowers the oxidative stress in
bile. As free radical injury is proved to be
implicated in gall stone formation, reduction
of free radical formation (oxidative stress)
improves the biochemistry of bile and thus
prevents the stone formation.
CONCLUSION
 Surgical trauma exerts significant
ocidative stress in the body as it is seen
in bile of both the groups.
 Free radical injury in gall bladder alone
is not responsible for gallstone
formation; this is the liver bile which
may contribute to the gallstone
formation.
 The evaluated polyhedral formulation
(PHALTRIKADI KWATH) has got
antioxidant potential as it decreases
oxidative stress more as compared to
control group.
 The drug can be used prophylactically to
prevent the recurrence of stones and
oxidative stress in bile.
 Drug is cheap, freely available, easily
prepared and cost -effective.
Table 1: Composition of DRUG MATERIAL
In Ayurvedic classics (Sarangdhar Samhita - Madhyama Khan; 257) the polyherbal
Formulation is known as "PHALTRIKADIKWATH (=KASHAYA)", decoction (water extract )
of eight different plants taken in equal quantity.
S.
No
Name of Ingredients Part Taken Properties
1. Haritaki [Terminalia
chebula]
Fruit without
seed
Antiseptic Antispasmodic, Anti-
inflammatory
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2 Vibhitaki [ Terminalia
belerica]
Fruit without
seed
Choleretic Antipyretic, Antiemetic,
Anti-inflammatory
3 Amalaki [Emblica
officinalis]
Fruit without
seed
Antipyretic,
Antispasmodic,Antioxidant
4 Guduchi [Tinospora
cordifolia]
Stem Anticomplementary,
Antispasmodic,Antioxidant
Immunomodulator
5 Kutki [picrorrhiza kurroa] Rhizomes Choleretic, Anticholestatic,
Antiallergic, Antianaphylactic,
Anti -inflammatory,Immunostimulant
Potent Antioxidant
6 Chirayita [Swertia chirayita] Whole plant Tonic, Antispasmodic, Antipyretic,
anti-inflammatory
7 Vasa [Adhatoda vasica] Whole plant Astringent, Antispasmodic,Tonic,
Styptic
8 Neem[ azdirechta indica] Stem bark Atipyretic, Anti-inflammatory, Liver
tonic
Table 2: Physico - chemical parameter of Phaltrikadi Kwath:
S.No pH Viscosity
Centipoise
at 20oc
Specific
gravity
Refractive
Index
Conductivity
mMhos/cm
Osmolarity
mOsm/kg
Polarity
1 3.4 1.458 1.008 1.334 2.38 135 NP
2. 3.4 1.446 1.008 1.333 2.38 135 NP
3. 3.8 1.459 1.006 1.334 2.40 138 NP
4. 3.6 1.462 1.008 1.334 2.42 142 NP
5. 3.4 1.462 1.006 1.334 2.44 144 NP
6. 3.5 1.458 1.008 1.332 2.42 145 NP
7. 3.4 1.458 1.010 1.334 2.42 135 NP
8. 3.4 1.466 1.006 1.334 2.44 145 NP
9. 3.5 1.462 1.008 1.332 2.38 138 NP
10. 3.4 1.462 1.010 1.334 2.40 144 NP
11. 3.6 1.466 1.010 1.334 2.38 138 NP
12 3.4 1.458 1.008 1.333 2.38 142 NP
13 3.4 1.459 1.006 1.333 2.38 145 NP
14 3.5 1.460 1.010 1.334 2.40 142 NP
15 3.6 1.462 1.008 1.334 2.42 136 NP
16 3.8 1.460 1.008 1.334 2.38 135 NP
17 3.6 1.467 1.010 1.334 2.44 138 NP
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Table 3: Lipid peroxidation levels, MDA in mmol/L,in bile juice at different
intervals:
Group Sample I
(Day 0) Mean
SD
Sample II
(Day 3) Mean
SD
Sample III
(Day 10) Mean
SD
I vs II I vs III II vs III
P-value
Group A
Control
(n=15)
3.02401.008 3.4941101109 2.666010.9016 <0.001 <0.01 <0.001
Group B
Treated
(n=15)
3.52521.5677 4.47021.5059 2.48550.9020 <0.001 <0.001 <0.001
18 3.4 1.462 1.008 1.334 2.44 136 NP
19 3.6 1.458 1.008 1.332 2.42 135 NP
20 3.8 1.467 1.008 1.334 2.40 145 NP
Mean
SD
3.525
0.1446
1.465
0.024
1.008
0.0013
1.333
0.0007
2.406
0.234
139.65
4.0688
-
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ACKNOWLEDGEMENT:
The Preparation of this article was made possible by the financial support of the project
45/3/2000 BMS/TRM, provided by Indian Council of Medical research, New Delhi.
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