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Original Article

Satwa from three Tinospora species exhibits differentialhepatoprotective activity against repeated acetaminophendosing in rats

Tejaswi Chavan a, Suresh Khadke a, Shubhangi Harke a, Abhijit Ghadge a,Manjiri Karandikar b, Vijaya Pandit c, Prabhakar Ranjekar a, Omkar Kulkarni a,Aniket Kuvalekar a,d,*a Interactive Research School for Health Affairs (IRSHA), Bharati Vidyapeeth Deemed University, Pune-Satara Road, Pune, Maharashtra

411043, IndiabDepartment of Pathology, Bharati Vidyapeeth Medical College, Bharati Vidyapeeth Deemed University, Pune-Satara Road, Pune,

Maharashtra 411043, IndiacDepartment of Pharmacology, Bharati Vidyapeeth Medical College, Bharati Vidyapeeth Deemed University, Pune-Satara Road, Pune,

Maharashtra 411043, IndiadScientist C, Nutrigenomics and Functional Foods Laboratory, Interactive Research School for Health Affairs (IRSHA), Bharati Vidyapeeth

Deemed University, Pune-Satara Road, Pune, Maharashtra 411043, India

a r t i c l e i n f o

Article history:

Received 16 August 2012

Accepted 6 November 2012

Keywords:

Alkaline phosphatase

Bilirubin

Guduchi satwa

Heptotoxicity

Silymarin

* Corresponding author. Nutrigenomics and FVidyapeeth Deemed University, Pune-Satara

E-mail address: kuaniket@gmail.com (A.0974-6943/$ e see front matter Copyright ªhttp://dx.doi.org/10.1016/j.jopr.2012.11.026

a b s t r a c t

Tinospora cordifolia (Willd.) Miers ex Hook. F., and Tinospora sinensis (Lour.) Merrill, are

herbaceous vines found all over India and are well known herbal medicines. Since, ancient

time T. cordifolia, T. sinensis and Neem-giloe (T. cordifolia growing on Azadirachta indica A.

Juss.) individually have been used in the ayurvedic medicine system in the form of Guduchi

satwa to treat liver disorders. In the present studies, hepatoprotective potential of satwa

prepared from three Tinospora species against hepatotoxicity induced by repeated dosing of

acetaminophen was assessed. Repeated acetaminophen dosing produced elevations in the

level of liver marker enzymes and changes in the lipid profile status of the animals. T.

cordifolia had a specific action on maintaining lipid profile with improvements in the levels

of total cholesterol, HDL and LDL. T. sinensis exhibited a positive effect on SGPT and ALP

activities apart from improvements in VLDL and triglyceride levels. Neem guduchi had

specific beneficial effect on SGOT and bilirubin levels. The differential hepatoprotective

effect of these three different satwa was also evident from liver histology. T. cordifolia

exhibited normalization of periportal hepatocytes while T. sinensis showed prominent

hepato-regenerative activity with increase in the normal hepatocytes in the treatment

group. The Neem guduchi treated group showed strikingly normal liver histology without

any anatomically detectable anomalies. The results reveal that these satwa have their

actions at different physiological targets and hence exhibit differential hepatoprotective

activity. Due to this differential hepatoprotective activity these three satwa may be used in

unctional Foods Laboratory, Interactive Research School for Health Affairs (IRSHA), BharatiRoad, Pune, Maharashtra 411043, India. Tel.: þ91 20 24366929; fax: þ91 20 24366931.

Kuvalekar).2012, JPR Solutions; Published by Reed Elsevier India Pvt. Ltd. All rights reserved.

j o u rn a l o f p h a rma c y r e s e a r c h 6 ( 2 0 1 3 ) 1 2 3e1 2 8124

combination as a liver tonic. It is also required that the effect of these satwa on the acute

acetaminophen hepatotoxicity should be assessed.

Copyright ª 2012, JPR Solutions; Published by Reed Elsevier India Pvt. Ltd. All rights

reserved.

1. Introduction during month of FebruaryeApril 2012 from Pune and Dapoli,

Liver diseases are still a worldwide health problem. Use of

medicinal plants and their formulations are common for the

treatment of liver diseases.1 Lever is known to be a unique

organ with self-regenerative ability and serves a dual purpose

of secretory and excretory functions.2 The central role of liver

in detoxification of endogenous and exogenous compounds,

and consequently, its continuous exposure to various xeno-

biotics, therapeutic agents and pollution contributes toward

compromised health of this vital organ.3

Acetaminophen (Paracetamol) is one of the safe and reliable

antipyretic and analgesic drugs when used at recommended

therapeutic doses.4 Overdose of acetaminophen may lead to

hepatotoxicandnephrotoxiceffectswithseriousconsequences.5

Due to paucity of reliable hepatoprotective drugs in

modern medicine, herbal drugs are being recommended for

the treatment of liver diseases.6 However, no scientific

evidence is available to support these claims and for their

mechanism of action. Tinospora (Guduchi) is one of such herbs

which is most commonly practiced and is prescribed for

various disorders for its curative as well as preventive role. In

Indian sub-continent, Tinospora occurs in four different

species, viz. Tinospora cordifolia (Willd.) Miers ex Hook. F. &

Thoms, Tinospora sinensis (Lour.) Merr., Tinospora crispa (L.)

Miers ex Hook. f. & Thoms and Tinospora glabra (Burm f.)

Merrill. The plant is locally known as Amrita, Amritavalli,

Chinnobhava, Chakralakshanika, Guduchi, Gulvel, Gurch, Kaduvel,

Kundalini, Madhuparni, Sudarsana Tantrika, Vatsadani etc.7

The reports of hepatoprotective potential of T. cordifolia

include normalization of altered liver functions8; anti-

hepatotoxic activity in CCL4 induced liver damage9; significant

increment in the functional capacities of rat peritoneal

macrophages10; as preventive antitubercular drugs11 for

jaundice12 and activity against hepatitis B and E.13 Themature

stem of T. sinensis has been used to treat fever, jaundice and

burning sensation.14 In china, the fresh leaves and stems are

used in the treatment of chronic rheumatism15 and for

treatment in piles and ulcerated wounds.16 The scientific

validation studies on T. sinensis report anti-inflammatory16

and anti-diabetic17 activities.

The present study was undertaken to assess comparative

hepatoprotective activity of satwa of three most common

Tinospora species. This is the first report of comparative hep-

atoprotective activity of satwa of three Tinospora species.

2. Materials and methods

2.1. Collection of plant material

Stem of T. cordifolia, T. sinensis and Neem-guduchi [Guduchi

plant growing on treeAzadirachta indica (neem)] were collected

Maharashtra, India.

2.2. Preparation of Guduchi Satwa

Fresh stems of selected three variants of Tinospora species

were used for the preparation of Guduchi Satwa. The prepa-

ration as defined in Ayurvedic literature18 is a sediment extract

which is predominantly starchy in nature. In brief, freshly

collected stem parts were washed thoroughly with water and

outer brownish white colored peel was removed. It was then

cut into small pieces and pounded slightly in pounding

machine. The crushed stem pieces of three species were

separately suspended in a quantity of water 4 times of their

weight. This mixture was kept undisturbed for 24 h. Next day,

Guduchi was rubbed with hand till it became slimy and foam

appeared on water. This homogenized mixture was then

filtered through several layers of sterile muslin cloth and

filtrate was left undisturbed for 24 h. On the next day, the

water was decanted carefully without disturbing the sedi-

ment. The sediment was again suspended in half liter water

and kept undisturbed for 2 h. The water was then carefully

decanted, satwa was collected and sun dried for two days.

White colored satwa thus formed was stored in air-tight

containers till further use.

2.3. Experimental animals

Three months old Male albino Wistar rats weighing between

150 and 200 g were procured from the institutional animal

house. They were acclimatized to animal house facilities for

seven days and were maintained under standard condition

(Temperature 25� 2 �C, 12-h light: 12-h dark cycle) throughout

the experimentation. The animals were fed with standard

pellet diet (Nutrivet life science, Pune, M.S., India) and water

was supplied ad-libitum. The studies were carried out as per

the CPCSEA guidelines and after approval of the Institutional

Animal Ethical Committee (Ref.No.: BVDUMC/443/2012-2013).

2.4. Hepatoprotective activity

Rats were randomly selected and divided into six groups of six

animals each. The inter and intra groupweight difference was

below 20%. Hepatotoxicity was induced in rats by orally

feeding 1000 mg/kg b.w. acetaminophen suspended in water.

The dose of satwa was finalized on the basis of the earlier

studies carried out in the laboratory.

The treatment protocol, as mentioned below, was

followed:

Group I: Control (n ¼ 6); received feed and water normally for

15 days

Group II: Negative Control (n ¼ 6); administered acetamino-

phen (1000 mg/kg b.w./day, p.o.)

es,

bilirubin

andlipid

pro

file

ofc

ontrola

ndexperim

entala

nim

als.G

roupIishealthyco

ntrolw

hileGro

upIIandIIIs

erv

edasnegative

upIV

,V

andVIwere

treatedwith200m

g/kgb.w

.T.cord

ifolia,T.sinen

sisandNeem

guduch

isa

twa(p.o.)resp

ectively.

PT(U

/mL)

ALP(U

/mL)

BIL

(mg/dL)

Lipid

pro

file

Totalch

olestero

l(m

g/dL)

HDL(m

g/d

L)LDL(m

g/dL)

VLDL(m

g/dL)

TG

(mg/dL)

.64�

6.3

29.94c**

�5.7

0.84c*�

0.2

59.00c**

�4.6

22.75�

5.2

20.38a**

�7.7

15.87a*�

2.1

79.36a*�

10.5

.40b**

�3.9

53.96�

4.4

1.56�

0.1

87.45�

7.9

6.49b**

�0.7

53.33�

4.9

27.62�

3.6

138.12�

18.1

.12b**

�3.7

26.31c**

�4.1

1.12�

0.3

42.65c**

�5.4

9.16b**

�0.9

18.11�

3.2

15.38�

4.1

76.89�

20.6

.10b**

�4.6

36.77�

2.9

1.39�

0.1

46.57c**

�6.1

11.86b*�

2.4

16.07a**

�8.6

18.64�

3.3

93.22�

16.3

.60b**

�4.4

22.13c**

�6.5

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0.2

56.40c**

�2.6

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1.2

28.39a*�

4.8

16.43�

2.6

82.15�

13

.10b**

�1.1

33.56c*�

2.9

1.05�

0.1

54.37c**

�4.5

9.29b**

�0.5

25.39a*�

6.7

19.68�

2.5

98.43�

12.3

Ms

Ss

Ms

Ss

Ms

Ss

Ms

Ss

Ms

Ss

Ms

Ss

Ms

Ss

Ms

6.5

677.30

3112.1

622.41

2.163

0.4327

6249.7

1249.9

807.02

161.40

4702.5

940.50

522.27

104.45

13057

2611.4

7.8

91.575

2558.3

106.60

4.319

0.1799

3559.1

148.29

721.49

30.062

4704.2

196.01

1164.2

48.510

29106

1212.8

4.3

NA

5670.4

NA

6.482

NA

9808.8

NA

1528.5

NA

9406.7

NA

1686.5

NA

42163

NA

96

5.839

2.404

8.429

5.369

4.798

2.153

2.153

eatm

ents

¼5;Residuals

¼24;Total¼

29;SGOT:se

rum

glutamic

oxaloace

tictransa

minase

;SGPT:se

rum

glutamic

pyru

vic

transa

minase

;ALP:Alkaline

density

lipopro

tein;LDL:Low

density

lipopro

tein;VLDL:Very

low

density

lipopro

tein;TG:Triglyce

rides;

Ss:

Sum

ofsq

uares;

Ms:

Meansq

uare.

j o u r n a l o f p h a rm a c y r e s e a r c h 6 ( 2 0 1 3 ) 1 2 3e1 2 8 125

Group III: Positive Control (n ¼ 6); administered Silymarin

(100 mg/kg b.w./day, p.o.) and after 30 min acetaminophen

(1000 mg/kg b.w./day, p.o.)

Group IV: Treatment group 1 (n ¼ 6); administered T. cordifolia

satwa (200 mg/kg b.w./day, p.o.) and after 30 min acetamino-

phen (1000 mg/kg b.w./day, p.o.)

Group V: Treatment group 2 (n ¼ 6); administered T. sinensis

satwa (200 mg/kg b.w./day, p.o.) and after 30 min acetamino-

phen (1000 mg/kg b.w./day, p.o.)

Group VI: Treatment group 1 (n ¼ 6); administered Neem

guduchi satwa (200 mg/kg b.w./day, p.o.) and after 30 min

acetaminophen (1000 mg/kg b.w./day, p.o.)

The animals were observed daily for any signs of discom-

fort and/or infection. After 15 days of continuous treatment,

animals were fasted overnight, blood was collected by retro-

orbital puncture and animals were humanely sacrificed.

Liverwas excised immediately, washed in saline, weighed and

stored in 10% neutral buffered formalin for histological anal-

ysis. Blood was allowed to clot at R.T. for 30 min and serum

was collected after centrifugation at 2000 rpm for 15 min.

2.5. Blood biochemistry

Marker enzymes of liver damage (serum glutamic oxaloacetic

transaminase (SGOT), serum glutamic pyruvic transaminase

(SGPT) and alkaline phosphatase (ALP)), total bilirubin, total

Cholesterol, HDL Cholesterol, total Triglycerides were esti-

mated using commercial kits (Coral clinical system, Goa,

India). LDL Cholesterol (mg/dL) was estimated by using the

formula: (Total CholesteroleHDL Cholesterol)e triglycerides/

5 and VLDL Cholesterol was estimated by using the formula:

Triglycerides/5.

2.6. Histological analysis

Paraffin-embedded liver tissues were cut at 4 mm and stained

with hematoxylin-eosin. The slides were examined under

microscope and photographed.

2.7. Statistical analysis

Results are presented as Mean � Standard Error (SE). Dunnett

Multiple Comparison Test and one way Analysis of Variance

(ANOVA) was done to estimate the statistical significance

between groups.

Table

1eLevels

ofliverm

ark

erenzy

mandpositiveco

ntrolresp

ectively.Gro

Gro

up

SGOT(U

/mL)

SG

Gro

upI

129.38c*�

11.8

151

Gro

upII

186.10�

9.7

183

Gro

upIII

128.14c*�

20.4

175

Gro

upIV

148.98�

11.3

178

Gro

upV

150.71�

8.5

176

Gro

upVI

147.43�

18.9

182

ANOVA

table

Sourceofvariation

Ss

Ms

Ss

Treatm

ents

10076

2015.2

338

Residuals

22676

944.83

219

Total

32752

NA

558

FStatistic

2.133

7.3

*P�

0.05;**P�

0.01;Degreesoffreedom:Tr

phosp

hatase

;BIL:Totalbilirubin;HDL:High

aSignifica

ntagainst

PC.

bSignifica

ntagainst

HC.

cSignifica

ntagainst

NC.

3. Results

In the present study, comparative hepatoprotective potential

of T. cordifolia, T. sinensis and Neem-guduchi Satwa were evalu-

ated by assessing activities of serum enzymes SGOT, SGPT,

ALP and total bilirubin. The animals of paracetamol treated

group showed elevated levels of SGOT, SGPT, ALP and bili-

rubin, as compared with normal control group (Table 1). The

results of comparative hepatoprotective potential of T. cordi-

folia, T. sinensis and Neem-guduchi Satwa on paracetamol

treated rats indicate differential activity of three different

species in hepatoprotection.

j o u rn a l o f p h a rma c y r e s e a r c h 6 ( 2 0 1 3 ) 1 2 3e1 2 8126

T. cordifolia was found to have a specific action on main-

taining lipid profile. The experimental group treated with T.

cordifolia exhibited improvements in the contents of total

cholesterol (46.57 � 6.1 mg/dL) with approx. 47% decrease,

HDL (11.86 � 2.4) with 45% increase and LDL (16.07 � 8.6 mg/

dL) with approx. 70% decrease over acetaminophen treated

group and being comparable with the group treated with

Fig. 1 e Hematoxylin and eosin stained cross sections of paraffi

experimental groups (40X). Liver from healthy (a) group shows

treated group (b) shows mild conjunction of central vein, hepat

hyaline globules are seen around central vein, ballooning and d

mild nucleomegaly is seen. Liver from silymarin treated group

hepatocytes. The liver histology of animals treated with Tinosp

acetaminophen treated group (d) but with intermittently swolle

appear normal. The liver histology of the group treated with Ti

prominent hepato-regeneration as evident from distribution of

hepatocytes and normal periportal hepatocytes. The liver histo

normal without any histologically detectable anomalies.

silymarin. Tinospora sinensis had specific effect on improve-

ments in SGPT (176.60� 4.4 U/mL), ALP (22.13� 6.5 U/mL) with

58% decrease, VLDL (16.43� 2.6 mg/dL) and Triglyceride levels

(82.15 � 13 mg/dL) with 40% decrease when compared with

acetaminophen treated group. It may be noted that the levels

of VLDL and triglycerides in Tinospora sinensis treated group

are found to be statistically insignificant when compared to

n embedded liver tissues of rats from control and

normal architecture. Sections of liver from acetaminophen

ocytes show apoptotic death, few intra and extracellular

egeneration of hepatocytes around central vein along with

(c) shows near normal liver architecture with few swollen

ora cordifolia satwa exhibit improvements over

n centrilobular hepatocytes while periportal hepatocytes

nospora sinensis exhibits near normal histology (e) with

normal hepatocytes among degenerating swollen

logy of Neem guduchi satwa treated group (f) is strikingly

j o u r n a l o f p h a rm a c y r e s e a r c h 6 ( 2 0 1 3 ) 1 2 3e1 2 8 127

silymarin treated group, and hence are comparable to positive

control. Neem guduchi was found to have specific effect on

SGOT (147.43 � 18.9) and bilirubin (1.05 � 0.1) levels.

The differential hepatoprotective effects of guduchi satwa

prepared from these three Tinospora species are also evident

from liver histology (Fig. 1 aef). The liver histology of the

animals treated with T. cordifolia satwa exhibit improvements

over acetaminophen treated group (Fig. 1d) but with inter-

mittently swollen centrilobular hepatocytes which are more

prone to ischemic injury while periportal hepatocytes appear

normal. The liver histology of the group treatedwith T. sinensis

exhibits near normal histology (Fig. 1e) with prominent

hepato-regeneration as evident from distribution of normal

hepatocytes among degenerating swollen hepatocytes. This

group also shows normal periportal hepatocytes. The liver

histology of Neem guduchi satwa treated group (Fig. 1f) is strik-

ingly normal without any histologically detectable anomalies.

4. Discussion

The liver disorders are treated with an aim to prevent

degeneration of hepatocytes and consequent metabolic

derailments and to promote regeneration of hepatocytes.3

Overdose of acetaminophen is known to have hepatotoxic

effects which is reflected at the biochemical as well as histo-

logical level in the form of altered liver function tests andmild

to severe alterations in the histological architecture of

hepatocytes.

Tinospora is known to exhibit potent hepatoprotective and

immunomodulatory activities.19e22 Themajority of studies on

hepatic injury are found to be based on acute dosing of hep-

atotoxicant23e26 and indicating the effect of Tinospora or other

phytomedicines in alleviating hepatic injury. It is also known

that the repeated dosing of acetaminophen, even for four days

in male SpragueeDawley rats leads to development of phys-

iological adaptation to overdose of acetaminophen.27 Hence

care must be taken to design the animal experiments when

considering acetaminophen as heptotoxicant, in order to

avoid the dosage levels leading to development of physiolog-

ical adaptation which may be mistaken as a hepatoprotective

effect of the agent under investigation.

In the present study, the animals were dosed repeatedly

with hepatotoxicant (acetaminophen) and simultaneously

treated with different Tinospora satwa. This study is the first

report where three satwa prepared from three different Tino-

spora species was used to assess the hepatoprotective efficacy

in repeated acetaminophen dosing to animals. The dosage

level of hepatotoxicant was specifically selected to avoid

development of physiological adaptation. The study indicates

that the satwa prepared from three different Tinospora species

has varying modes of hepatoprotective action through recti-

fying the liver marker enzymes, bilirubin content and

controlling the lipid profile status of the animals.

5. Conclusion

This is a first report of its kind wherein the hepatoprotective

effect of guduchi satwa, prepared as per ayurvedic guidelines,

from three different Tinospora species was assessed. It is

evident from the present study that the satwa from these

Tinospora species have potent hepatoprotective activity. The

results reveal that these satwa have their actions at different

physiological targets and hence exhibit differential hep-

atoprotective activity. Such differential hepatoprotective

activity is also evident fromhistological improvements in liver

sections of the treated animals. Neem guduchi satwa treated

group exhibited strikingly normal liver histology. Hence it

may be concluded that these satwa have differential hep-

atoprotective activity and may be used in combination as

a liver tonic. It is also required that the effect of these satwa on

the acute acetaminophen hepatotoxicity should be assessed.

Conflicts of interest

All authors have none to declare.

Acknowledgments

The authors sincerely thank Prof. S. Mahadik, Medical College

of Georgia, USA for his kind support and suggestion.

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