Bulletin of Faculty of Pharmacy, Cairo University (2015) 53, 23–28

HO ST E D BYCairo University

Bulletin of Faculty of Pharmacy, Cairo University

www.elsevier.com/locate/bfopcuwww.sciencedirect.com

ORIGINAL ARTICLE

Hepatoprotective potential of Saraca ashoka(Roxb.) De Wilde bark by carbon tetrachloride

induced liver damage in rats

* Corresponding author.

E-mail address: manjushachoudhary@gmail.com (M. Choudhary).

Peer review under responsibility of Faculty of Pharmacy, Cairo

University.

http://dx.doi.org/10.1016/j.bfopcu.2014.11.0021110-0931 ª 2014 Production and hosting by Elsevier B.V. on behalf of Faculty of Pharmacy, Cairo University.

Bharti Aroraa, Manjusha Choudhary

a,*, Preeti Aryaa, Sunil Kumar

a,

Nitesh Choudhary b, Surender Singh c

a Institute of Pharmaceutical Sciences, Kurukshetra University, Kurukshetra 136118, Haryana, Indiab RP Inderprastha Insttitue of Technlogy, Karnal 132001, Haryana, Indiac All India Institute of Pharmacology, New Delhi 110029, India

Received 27 August 2014; accepted 13 November 2014Available online 6 December 2014

KEYWORDS

Hepatotoxicity;

Saraca ashoka;

Carbon tetrachloride;

Hepatocytes

Abstract Objective: Saraca ashoka [SA] (Family: Caesalpiniaceae) is widely used in skin infection,

CNS function, uterus pain during painful periods, in bacterial infection and for hepatoprotective

activity. The present study was carried out to investigate possible hepatoprotective activity of meth-

anolic and hydroalcoholic extract of Saraca ashoka stem bark.

Methods: Hepatoprotective activity of hydroalcoholic and methanolic extract of stem bark of S.

ashoka (HAESA and MESA) was demonstrated by using CCl4 induced hepatotoxicity model. Nor-

mal group was given only vehicle and CCl4 was given at 0.5 mL/kg, s.c. as toxic dose to induce hep-

atotoxicity. Liv-52 was given as standard drug (1 mL/kg/day, p.o.). Two doses of MESA and

HAESA (200 and 400 mg/kg/day, p.o.) were tested for hepatoprotective activity for nine days.

Results: Administration of hepatoxin CCl4 showed significant biochemical and histological deteri-

oration in the liver of experimental animals. Pretreatment with methanolic extract more signifi-

cantly and to a lesser extent hydroalcoholic extract reduced the elevated levels of serum enzymes

like serum glutamic-oxaloacetic transaminase (SGOT), serum glutamic-pyruvic transaminase

(SGPT) and alkaline phosphatase (ALP) and reversed the hepatic damage which evidenced the

hepatoprotective activity of stem bark of S. ashoka.

Conclusion: The results suggest that MESA and HAESA extracts at doses of 200 and 400 mg/kg,

p.o. have a significant effect on the liver of CCl4 induced hepatotoxicity animal model.ª 2014 Production and hosting by Elsevier B.V. on behalf of Faculty of Pharmacy, Cairo University.

1. Introduction

The liver plays a pivotal role in the regulation of vital physio-logical functions such as metabolism, secretion of bile, storage

of vitamins and detoxification of drugs.1 Liver diseases are

24 B. Arora et al.

among more serious ailments affecting a large population ofworld and can be classified as acute or chronic hepatitis andcirrhosis. Hepatotoxicity implies chemical-driven liver damage

and can occur due to overdose and sometimes even in thera-peutic ranges, may injure the organ. Other chemical agents,such as those used in laboratories and industries can also

induce hepatotoxicity.2 Drug induced liver injury is responsiblefor 5% of all hospital admissions and 50% of all acute liverfailures.3

In spite of tremendous strides in modern medicine, thereare very less drugs that stimulate liver function, offer protec-tion to the liver from damage or help regeneration of hepa-tocytes.4 Herbal drugs have gained importance and

popularity in recent years because of their safety, efficacyand cost effectiveness. Traditional medical systems such asAyurveda, Siddha and Unani are predominantly based on

the use of plant materials. Many formulations containingherbal extracts are sold in the Indian market for liver disor-ders. One such formulation is Liv-52 which is very com-

monly used as hepatotonic.5

Saraca ashoka is an evergreen plant belonging to familycaesalpiniaceae6,7 and found throughout India, especially

in Himalayas, Kerala, Bengal and whole South region.The plant exhibit hepatoprotective, antibacterial and anti-cancer activities8,9 as well as is used to treat skin infection,CNS function, uterus pain during painful periods, clots and

ammenorhoea.10–12 Phytochemical study of the bark extractshowed the presence of phytosterols, carbohydrates, tanninsand glycosides.13 Glycosides (polyethanoid) are known to

possess anti-inflammatory, hepatoprotective and neuropro-tective activities.14 In some of the plants of family caesal-piniaceae biological activities such as anti-leukemic, anti-

inflammatory and hepatoprotective have been reported.Some of the important plants under this family are Cae-salpinia bonducella, Delonix regia, and Bauhinia racemosa.

Literature reviews indicated that the hepatoprotective activ-ity of the bark of S. ashoka has not been scientifically eval-uated so far. In view of this, the present study was aimedat evaluating the hepatoprotective activity of the bark of S.

ashoka plant against CCl4 induced hepatotoxicity in albinorats.

2. Materials and methods

2.1. Chemical reagent

Methanol and petroleum ether were purchased from SD FineChemical Ltd. Carbon tetra chloride was obtained from Hime-

dia Laboratories Pvt. Ltd. and all biochemical estimation kitswere purchased from Erba diagnostic kits, Transasia biomed-icals Ltd.

2.2. Plant material

The bark of S. ashoka was collected from Haryana AgricultureUniversity, Hisar district, Haryana and authenticated by Dr.

H.B Singh, Head, Raw Material Herbarium & Museum,(RHMD), National Institute of Science Communication andInformation Resources, New Delhi, (India). Voucher specimen

Ref. No. NISCAIR/RHMD/CONSULT/-2010-11/1639/237has been preserved there for future references.

2.3. Preparation of plant extracts

The bark was washed under running tap water so as to removeany type of contamination. Then washed plant material wasair dried under shade, powdered in grinder and passed through

the sieve of mesh size No. 40 The bark powder was first defat-ted by petroleum ether and then successive extraction wasdone with methanol and hydroalcohol by the hot Soxhletextraction method. The extracts were filtered and concentrated

by rotary evaporator under reduced pressure and preserved inairtight container at 4–8 �C for further investigation.

2.4. Preliminary phytochemical screening

Qualitative phytochemical investigation of MESA andHAESA was carried out using standard procedure given by

Kokate and Harbone.15,16

2.5. Experimental animals

Wister albino rats (100–150 g) were purchased from the animalhouse of Chaudhary Charan Singh Haryana Agriculture Uni-versity, Hisar, Haryana (India). Animals were housed in ani-mal house, Institute of Pharmaceutical Sciences, Kurukshetra

University, Kurukshetra, Haryana (India) and were fed withavailable rat feed. The standard condition of temperature(25 �C± 5 �C) humidity (55 ± 10%) and light/dark cycle

(12:12) was maintained properly. The study protocol wasapproved by Institutional Animal Ethics Committee (IAEC)(Register Number: 536/02/a/CPCSEA) and was conducted

according to the CPCSEA (Committee for the Purpose ofControl and Supervision of Experimental on Animals)guidelines.

2.6. Selection of doses and administration

Doses of 200 and 400 mg/kg were selected based on the litera-ture survey of previous research.17

2.7. Hepatoprotective activity

Hepatoprotective effect of MESA and HAESA extracts of

bark of S. ashoka was demonstrated by using the CCl4 inducedhepatotoxicity model and phenobarbitone induced sleepingtime. CCl4 activates by Cytochrome P-450 into a free radical

(CCl3) which causes peroxidative degradation in the adiposetissue resulting in fatty infilteration of the hepatocytes.18 Livertoxicity also affects the metabolism of barbiturates thus

enhancing the sleeping time due to delay in excretion of pheno-barbitone. So, the present study was aimed to investigate thehepatoprotective activity of the bark of S. ashoka againstCCl4 induced hepatotoxicity in rats.

2.7.1. Carbon tetrachloride induced hepatotoxicity model

The effects of MESA and HAESA extracts were studied inCCl4 induced hepatotoxicity. Wistar albino rats of either sex

weighing from 100 to 150 g were used in this method. Animalswere divided into seven groups containing six animals each. Allthe groups were treated orally for 9 days. Rats in group I (nor-

mal control) received only distilled water (1 mL/kg, p.o.);

Hepatoprotective potential of Saraca ashoka 25

group II (toxic group) received distilled water and CCl4(0.5 mL/kg, s.c.) for 9 days. Group III received standard drugLiv-52 (1 mL/kg/day, p.o.) with CCl4 (0.5 mL/kg, s.c.) for

9 days. Group IV and V received MESA extract (200 and400 mg/kg/day, p.o.) and CCl4 (0.5 mL/kg, s.c.) for 9 days;group VI and VII received HAESA extract (200 and 400 mg/

kg/day, p.o.) and CCl4 (0.5 mL/kg, s.c.) for 9 days.19 On the10th day, all the animals were sacrificed under anesthesiaand blood as well as liver samples were collected for biochem-

ical and histopathological investigation.20,21

2.7.2. Phenobarbitone induced sleeping time

Liver toxicity affects the metabolism of barbiturates thus

enhancing the sleeping time due to delay in excretion of pheno-barbitone. In this model, phenobarbitone sodium (40 mg/kg)was administered after 48 h of last treatment of both the

extracts. The time interval between onset of sleep and regainof righting reflex was measured.

2.7.3. Biochemical investigation

Blood samples were taken by retro orbital plexus and allowedto clot at room temperature for 45 min. Centrifugation wasdone at 1200–1500 rpm for 20 min for separation of serum.

The serum was used for the estimation of biochemical param-eters namely serum glutamic-oxaloacetic transaminase(SGOT), serum glutamic-pyruvic transaminase (SGPT) andalkaline phosphatase (ALP) by using autoanalyzer.

2.7.4. Histopathology

After draining the blood, liver samples were excised andwashed with normal saline and processed separately for histo-

logical observations. Initially the samples were fixed in 10%buffered neutral formalin for 48 h and then with bovine solu-tion for 6 h. Liver tissues were dehydrated with alcohol and

their paraffin section were cut in 5 mm thickness, and werestained with alum hematoxylin and eosin.22 The sections wereexamined microscopically for histopathological parameters at

Anupam Memorial Garg Diagnostic Center, Sirsa.

2.8. Statistical analysis

Statistical analysis was carried out by a one-way analysis ofvariance (ANOVA) followed by Dunnett’s test. Results were

Table 1 Chemical constituents present in MESA and HAESA extr

S. No. Phytoconstituents Method

1 Alkaloids Dragendorff’s test

Mayer’s test

Hager’s test

2 Carbohydrates Molish’s test

Fehling’s test

3 Steroids Liebermann–Burchard

4 Tannins Ferric chloride test

5 Saponins Foam test

6 Proteins and amino acids Millon’s test

Ninhydrin test

7 Flavonoids Shinoda test

Lead acetate test

+ indicates positive; � indicates negative.

expressed as mean ± S.E.M for six rats in each group. pvalue < 0.05 was considered significant.

3. Results

3.1. Preliminary phytochemical screening

Preliminary screening revealed the presence of carbohydrates,steriods, tannin, saponin, flavonoids, protein and amino acids

in methanolic and hydroalcoholic extracts (Table 1).

3.2. Pharmacological evaluation

3.2.1. CCl4 induced hepatotoxicity

In CCl4 treated rats, the levels of SGOT, SGPT and ALP

increased as compared to the vehicle control group. Pretreat-ment with Liv-52 reduced SGOT significantly (p< 0.01) ascompared with diseased control. Both MESA and HAESAextracts also decreased the SGOT significantly (p< 0.01) at

both the doses but effect of MESA at dose 400 mg/kg wassuperior to dose 200 mg/kg and comparable to the standardgroup. MESA and HAESA extracts at the dose of 400 mg/

kg showed significant changes (p< 0.01) and reduced the levelof SGPT and ALP (Table 2).

3.2.2. Phenobarbitone induced sleeping time

Due to hepatic injury, barbiturate metabolism is delayed andexcretion is slow. The CCl4 control group has prolonged sleep-ing time when compared to the vehicle control group. MESA

extracts reduced the sleeping time (p < 0.05) at the dose of400 mg/kg significantly as compared to CCl4 induced hepato-toxic group (Table 3).

3.3. Histopathological studies

Vehicle control group showed normal central vein and hepato-

cytes. CCl4 toxic group showed degeneration of hepatocyteswith fatty changes (Fig. 1). Liv-52 treated group showed nor-mal central vein and mild changes in hepatocytes. MESA andHAESA (200 mg/kg) treated animals showed normal hepato-

cytes and less or mild fatty changes. MESA and HAESA(400 mg/kg) showed dilated vein and mild degeneratehepatocytes.

acts of S. ashoka.

Methanolic extract Hydroalcoholic extract

� �� �� �+ +

+ +

reaction + �+ +

+ +

+ +

++ ��++ +

Table 3 Effect of pretreatment of Liv-52, MESA and HAESA extracts of S. ashoka on phenobarbotone induced sleeping time in CCl4induced liver injury.

Groups Treatment Dose (mg/kg, p.o.) Duration of phenobarbitone induce sleep in min (% reduction)

Group 1 Vehicle Control – 278.78 ± 8.0*

Group 2 CCl4 Control – 339.02 ± 7.0

Group 3 CCl4 + Liv52 – 277.95 ± 3.0*

Group 4 CCl4 +MESA 200 291.28 ± 15.4

Group 5 CCl4 +MESA 400 277.95 ± 14.8*

Group 6 CCl4 + HASEA 200 304.61 ± 18.6

Group 7 CCl4 + HASEA 400 289.61 ± 17.6

Values were expressed as mean ± SEM (n = 6 rats/group). Percentage reduction in parameters compared to Liv-52 and difference between

CCl4 & Liv-52 treatment group *(p< 0.05) (One-way ANOVA followed by Dunnett’s Test).

Table 2 Effect of MESA and HAESA extracts of S. ashoka on serum SGOT, SGPT and ALP in CCl4 induced liver toxicity.

Groups Treatment Dose (mg/kg, p.o.) SGOT(IU/L) SGPT(IU/L) ALP(IU/L) Liver weight (g)

Group 1 Vehicle control – 95.04 ± 3.3 37.41 ± 2.1 17.05 ± 0.8 2.98 ± 0.1

Group 2 CCl4 control – 189.43 ± 12.8 134.43 ± 8.2 100.53 ± 3.8 4.08 ± 0.1

Group 3 CCl4 + Liv 52 – 109.43 ± 5.8** 47.13 ± 2.8** 35.04 ± 1.3** 3.09 ± 0.2**

Group 4 CCl4 + MESA 200 129.43 ± 9.1** 75.03 ± 3.3** 62.76 ± 5.3** 3.02 ± 0.2**

Group 5 CCl4 + MESA 400 103.53 ± 7.8** 43.46 ± 2.3** 39.66 ± 2.9** 3.60 ± 0.0

Group 6 CCl4 + HAESA 200 149.83 ± 5.9** 73.63 ± 6.8** 70.03 ± 5.8** 3.54 ± 0.1

Group 7 CCl4 + HAESA 400 128.03 ± 8.3** 48.74 ± 4.8** 44.87 ± 3.4** 4.15 ± 0.0

Values was mean ± S.E.M, n= 6 in each group, all groups were compared with disease control, *(p< 0.05), **(p < 0.01) (One way ANOVA

followed by Dunnett’s Test).

26 B. Arora et al.

4. Discussion and conclusion

Liver diseases like hepatotoxicity remains as one of the serious

health problems and every year approximately 18,000 deathsoccur due to liver cirrhosis. It is considered to be one of themost prevalent causes of human sufferings and death.23,24

Unfortunately synthetic drugs used in the treatment of liverdiseases are inadequate and sometimes can have serious sideeffects.25

Herbs are one of the most promising sources of new drugs

as these are free of or having very less side effects and adversereactions. Various plant extracts and polyherbal formulationsplay a specific therapeutically active role in the management of

liver disorder. Liv-52 is a polyherbal used traditionally in thetreatment of various liver disorders. It is a polyherbal formu-lation consisting of powders of Capparis spinosa, Cichorium

intybus, Solanum nigrum, Cassia occidentalis, Terminaliaarjuna, Achillea millefolium, Tamarix gallica, and Mandurabhasma.26 The hepatoprotective activity of esculetin and

p-methoxybenzoic acid, the main constituents of Cichoriumintybus and Capparis spinosa, respectively, has been reportedby previous researchers, in chemically-induced hepatotoxicityin experimental animals.27–29 The hepatoprotective effects of

Mandura bhasma and Cassia occidentalis, and the other twocomponents of Liv-52 were also investigated against chemi-cally-induced liver damage in experimental animals.30,31 Keep-

ing in view the above facts, Liv-52 was selected as standard.The hepatoprotective effects of both MESA and HAESAextracts of S. ashoka were studied in rats by using the CCl4induced hepatotoxicity model at the doses of 200 and400 mg/kg.

CCl4 is a potent hepatotoxin32 activated by CytochromeP-450 into a free radical (CCl3) which causes peroxidative

degradation in the adipose tissue resulting in fatty infilterationof the hepatocytes. CCl3 radical further interacts with

molecular oxygen to form trichloromethyl peroxy radical.Both CCl3 and peroxy radical are capable of binding to proteinor lipid which elicit lipid peroxidation leading to functional

and structural disruption of hepatocytes.33,34 During hepaticdamage, cellular enzymes like SGOT, SGPT and ALP will leakinto the serum resulting in elevation of their serum concentra-

tions. Hepatology of the damaged liver showed histologicalchanges, such as fatty changes in hepatocytes and perivenularfibrosis. We observed that pretreatment with Liv-52 and barkextracts of S. ashoka showed a significant protective effect

against CCl4 induced liver damage and significantly reducedthe elevated liver enzymes indicating the hepatoprotectiveaction. Both the extracts of the bark of A. ashoka prevented

the histological changes caused by CCl4 which furtherconfirmed its hepatoprotective activity against CCl4-inducedhepatic damage. The possible mechanism of action may be

associated with scavenging of free radicals responsible forCCl4 toxicity.

In this investigation, we observed that there is marked liverdamage due to CCl4 intoxication with elevation in various

markers like SGOT, SGPT and ALP. Pretreatment with barkextracts for 9 days and Liv-52 has significantly reduced lipidperoxidation and elevated biochemical markers.

These results showed that the treatment with MESA andHAESA extracts of S. ashoka possessed significant protectionagainst liver damage caused by CCl4. Hepatoprotective activ-

ity was confirmed by the results of biochemical, and histolog-ical studies. The hepatoprotective activity of S. ashoka may bedue to flavonoids which were present in both the methanolic

and hydroalcoholic extracts. Flavonoids are known for theirpowerful free radical scavenging activity.35 Superoxide anion

Group 1 (Vehicle group) Group II (CCl4 intoxicated group)

Group III (Liv-52 +CCl4) Group IV (MESA 200 mg/kg+CCl4)

Group V (MESA 400 mg/kg+CCl4) Group VI (HAESA 200 mg/kg+CCl4)

Group VII (HAESA 200 mg/kg+CCl4)

Figure 1 Histopathology of MESA and HAESA extracts of S. ashoka leaves on CCl4-induced hepatotoxicity in rats.

Hepatoprotective potential of Saraca ashoka 27

and hydroxyl ion present in flavonoids show antioxidant prop-erty which effectively helps detoxify the toxic compounds.36

Thus these results showed that the extracts possess hepatopro-

tective activity.Thus the result showed that the MESA and HAESA

extracts of S. ashoka have hepatoprotective activity against

CCl4 induced hepatotoxicity at both the doses of 200 and400 mg/kg. The antioxidant property of phytoconstituents ofthe bark of S. ashoka may be the possible mechanism for itshepatoprotective activity.

5. Conflict of interest statement

We declare that we have no conflict of interest.

Acknowledgement

The authors are highly thankful to Institute of PharmaceuticalSciences, Kurukshetra University, Kurukshetra for providingnecessary research facilities.

28 B. Arora et al.

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