Indian Journal of Experimental Biology Vol. 43, May 2005, pp. 430-436

Effect of Emblica officinalis (Gaertn) on CC14 induced hepatic toxicity and DNA synthesis in Wi star rats

Sarwat Sultana, Salahuddin Ahmad, Naghma Khan & Tamanna Jahangir

Section of Chemoprevention and Nutrition Toxicology, Department of Medical Elementology and Toxicology, Jamia Hamdard (Hamdard University), Hamdard Nagar, New Delhi 110 062, India

Received 15 September 2004; revised 21 February 2005

A single dose of CCl4 (1 mllkg body weight, po in corn oil) increased the levels of SGOT (serum glutamate oxaloacetate transaminase), SGPT (serum glutamate pyruvate transaminase), LDH (lactate dehydrogenase), glutathione-S-transferase and depletion in reduced glutathione, glutathione peroxidase and glutathione reductase. It also caused enhancement in the levels of lipid peroxidation (LPO) and DNA synthesis. There was also pathological deterioration of hepatic tissue as evident from multi vacuolated hepatocytes containing fat globules around central vein. The pretreatment of E. officinalis for 7 consecutive days showed a profound pathological protection to liver cell as depicted by univacuolated hepatocytes. Pretreatment with E. officinalis at doses of 100 and 200 mg/kg body weight, prior to CCl4 intoxication showed significant reduction in the levels of SGOT, SGPT, LDH, glutathione-S-transferase , LPO and DNA synthesis. There was also increase in reduced glutathione, glutathione peroxidase and glutathione reductase. The results suggest that E. officinalis inhibits hepatic toxicity in Wi star rats.

Keywords: CCI4 ; Emblica officillalis; Hepatic toxicity.

Liver is a versatile organ of the body that regulates internal chemical environment. Liver injuries induced by various hepatotoxins have been recognized as a major toxicological problem for years 1 • Carbon tetrachloride (CCI4), an established hepatotoxicant for both animals and humans and has been used as solvent to coal tar, oil, resins and raw material of fluorocarbon for coolant and as fire extinguishing agent2

• CCl4 is metabolized in the liver by cytochrome P450 to trichloromethyl radical that reacts with GSH to form a GSH containing radical and causes various pathological and toxicological manifestations3

. Early events associated with the cytotoxicity of CCI4

following activation includes Iipidperoxidation and covalent binding to proteins and lipids followed immediately by diminished protein and lipid turnover and alterations in calcium homeostasis4

• CCl4 has been shown to acti vate Kupffer cells by increasing intracellular Ca2+concentration, causing release of harmful cytokines that contribute to the death of hepatocytes and oxidative stress5

. The cellular infiltration of activated neutrophillic leukocytes

*Correspondent author: Phone: 0091-11-26059688 Ext: 5565/5566 Fax: 0091-11-26059663 E-mail: sarwat786@rediffmail.com

amplifies inflammatory response and cellular injury or death due to release of superoxide anions and other toxic mediators6

. Free radicals bind covalently to membrane proteins and lipids causing enzyme inactivation and promotion of lipidperoxidation as a result of interaction with membrane unsaturated fatty acids?

Regenerating liver, a model of synchronized cellular proliferation, provides an opportunity to study the factors that can suppress or intensify the manifestations involved in the regenerative process8

.

Historically plants have been used as folk medicine against various types of diseases9

. Experimental work on several plants has been carried out to evaluate their efficacy against chemically induced toxicitylo.I' . In the present study the efficacy of extract of fruits of Emblica officinalis (Gaertn) has been elucidated against experimental tissue injury, oxidative stress and proliferative response induced by hepatotoxic agent, CCI4 •

Phytochemical evaluation of the plant E. officinalis showed the presence of quercetin, ascorbic acid and ellagic acid. The major principle is quercetin 12. The polyphenolic components of higher plants reportedly act as antioxidant or as agents of other mechanisms contributing to anti carcinogenic action I3. Flavonoid

SULTANA eta/.: EMBLICA OFFICINA LIS & CC14 INDUCED HEPATIC TOXICITY IN RATS 431

and other plant phenolics possess multiple biological activity including anticarcinogenic, anti-inflammatory and anti-allergic activity 14. Flavonoids are reported scavanger of superoxide radicals '5, peroxyl radicals and inhibitor of lipidperoxidation '6. Quercetin has been shown to mediate the down regulation of mutant PS3 in human breast cancer cell lines and modulation of TGF in ovarian cells 17. Ellagic acid has been shown to inhibit carcinogen-induced neoplasia in mouse skin, rat mammary gland and mouse forestomach l8

•

The most typical reaction leading to endogenous formation of mutagens is between nitrite and nitrosamines or amides, which can be inhibited by , vitamin C, vitamin E, gallic acid, ferrulic acid and caffeic acid l9

. The above information and present status of the dietary intervention prompted the present work on the evaluation of the efficacy of the fruit extract of Emblica offlcinalis against CCl4 induced hepatic toxicity and DNA synthesis.

Materials and Methods Chemicals-Oxidized and reduced glutathione

(GSSG and GSH), NADPH, H20 2, dithionitrobenzene (DTNB), l-chloro-2, 4-dinitrobenzene (CDNB), dinitrophenyl hydrazine (DNPH), glutathione reductase, reduced nicotinamide adenine dinucleotide (NADPH), ethylenediamine tetra acetic acid (EDTA), pyridoxal phosphate, phenyl methyl sulphonyl fluoride (PMSF), 2-mercaptoethanol, dithiothreitol, Tween 80, Brij 3S, ethanolamine, methoxyethanol, citric acid, y-glutamyl p-nitroanilide, diethyl nitrosamine, 2-acetylamino­fluorine and quercetin were purchased from Sigma Chemical Co. (St. Louis, Mo. USA). eH] thymidine (specific activity 73.0 Ci/m mol) was purchased from Amersham Corporation, UK. All other chemicals and reagents used were of highest purity commercially available.

Plant extractioll----Dried fruits of the plant E. offlcinalis were procured from Jamia Hamdard campus and authenticated at source by Prof. M Iqbal, Department of Environmental Botany, Jamia Hamdard, New Delhi, India. Freshly collected fruits were shade­dried and coarsely powdered in a grinder. The extraction procedure was followed as described by Didry et apo. Powdered dried fruits (SOO g) were extracted in round-bottomed flask 'with 2000 ml petroleum ether, benzene, ethyl acetate, acetone, methanol and double distilled water. The residues yielded from each solvent (14, 10, 16,30, 42 and 22 g respectively) were stored at 4°C. The extracts were evaporated to dryness under reduced pressure in a

rotatory evaporator (Buchi Rotavapor, Switzerland). The methanol fraction obtained (42 g) was suspended in water to prepare the required dilutions at the time of dosing.

Animals-Male albino Wistar rats (130-1S0 g) were obtained from Central Animal House of · Hamdard University, New Delhi, India. They were housed in polypropylene cages in groups of six rats per cage and were kept in a room maintained at 2So ± 2°C with a 12: 12 hr L:D cycle. They were given free access to standard laboratory feed. (Hindus tan Lever Ltd." Bombay, India) and water ad libitum. The animals were sacrificed according to the guidelines of the current laws of CPCSEA (Ethical Committee for the purpose of control and supervision of experiments on animals), India.

Experimental protocol To study the biological, serological and

pathological changes 24 rats were divided into 4 groups of (; each. Group I served as saline treated control. Rats in Group II were given CCl4 [1 ml/kg body weight, po, in corn oil (1: 1)]. Groups III and IV animals were given E.offlcillalis extract at doses of 100 and 200 mg/kg body weight, respectively for 7 consecutive days followed by CCl4 intoxication on the seventh day. Animals in all the groups were sacrificed 24 hr after CCl4 or saline administration. The doses of the plant extracts were selected after performing in vitro assays like microtonal lipid peroxidation and cytochrome P450. Serum was separated and stored at 4°C for the estimation of serum glutamate oxaloacetate transaminase (SGOT), serum glutamate pyruvate transaminase (SOPT), lactate dehydrogenase (LDH). Tissue was processed for the estimation of glutathione (GSH) content, microsomal lipid­peroxidation and actIVities of glutathione-S­transferase (GST), glutathione peroxidase (GPx) and glutathione reductase (GR). Sections of liver from each group were cut and preserved in buffered formalin for histopathological studies.

For eH] thymidine incorporation study, grouping of animals was same as described above. All the animals were given intraperitoneal eH] thymidine (25 Ilci/0.2 mlsaline/l00 g of animal) 2 hr before killing. All animals were sacrificed 48 hr of CCl4 intoxication and liver sections were quickly excised, rinsed with ice cold saline, freed of extraneous material and processed for th~ quantification of eH] thymidine incorporation into the hepatic DNA.

432 INDIAN J EXP BIOL, MAY 2005

Post-mitochondrial supernatant (PMS) and microsome preparation--The samples were homogenized in chilled phosphate buffer (0.1 M, pH 7.4) using a Potter Elvehjen homogenizer. The homogenate was centrifuged at 3000 rpm for 5 min at 4°C by Eltek Refrigerated Centrifuge. The aliquot obtained was centrifuged at 12000 rpm for 20 min at 4°C to obtain post-mitochondrial supernatant (PMS). PMS was centrifuged for 60 min by ultracentrifuge at 34,000 rpm at 4°C. The pellet was washed with phosphate buffer (0.1 M, pH 7.4).

Biochemical estimations-Tissue processing and preparation of post mitochondrial supernatant (PMS) were done immediately after animal sacrifice. For all biochemical estimations, serum samples were utilized. All the biochemical estimations were completed within 24 hr of animal sacrifice. SGOT21 , SGPT21 , LDH22, LP023

, GST24, GPX25, GR26, DNA27.28 were estimated as

per standard procedures. Protein estimation-Protein content in all samples

was estimated by the method of Lowry et al.29 using bovine serum albumin as standard.

Statistical analysis-The level of significance between different groups is based on Dunnett's t test, followed by the ANOV A test.

Results and Discussion The effect of prophylactic administration of the

extract on CCl4 mediated leakage of liver marker enzymes in serum is shown in Table 1. CC4 administration resulted in a significant rise in the levels of SGOT, SGPT and LDH in serum by 278%. 349% and 155% of saline treated control respectively. Pretreatment with E. officinalis extract at higher dose showed marked alleviation of SGOT, SGPT and LDH by 94%, 127% and 31% respectively as compared

with CCl4 treated group. Hepatoprotective effects of the extracts can be explained on the basis of decreased liver enzyme leakage in serum on pretreatment and significant recovery of the cell membrane towards stabilization as evident from pathological recovery30. CCl4 is metabolized in liver by cytochrome P450 dependent electron transport chain system yielding trichloromethyl radical that in aerobic condition rapidly gets converted to its peroxyl radical4. These radicals bind directly to lipids and proteins through covalent bonds and also interact with membrane phospholipids leading to promotion of lipid peroxidation7. Apart from the studies of mechanism of CCI4 induced injury, recent studies have shown the involvement of Kupffer c-ells, cytokines, and neutrophilic leukocytes31 . A recent study has also shown the protective effect of plant against CCl4 induced hepatic damage32.

The effect of pretreatment of extract on the GSH metabolizing enzymes GST, GPx and GR is shown in Table 2. The administration of CCI4 resulted in significant (P<O.ool) elevation of GST activity by 168% and a concomitant decrease in the activities of GPx and GR by 42% and 39% as compared with saline treated control group. The pretreatment of rats with E. officinalis resulted in marked down regulation of GST by 39% at higher dose of the plant. The concurrent alleviation in the GPx was observed on pretreatment of E. officinalis by 27% and of GR activity by 31 % respectively as compared with CCl4 treated group. CC4 administration resulted in increase in GST activity with concomitant decrease in GPx and GR activities that were significantly modulated on prophylactic pretreatment of the extract. Liver possesses growth ability after cellular loss in hepatotoxicant induced liver injury. Regenerating

Table 1-Effect of pretreatment of the extract of E. officinalis on CC14 induced release of liver damage markers in serum [Values are mean ± SE from 6 animals in each group]

Treatment groups

Saline CCI4alone

CCI4+EO (D1) CCI4+EO (02)

SGPT (IU/litre)

27.6 ±0.86 76.8 ± 1.97"

62.28 ± 2.51 b 50.79 ± 2.38b

P values: "<0.01 (compared to saline treated group) b<O.OOl (compared to CCI4 treated group) c<O.OOI (compared to CCl4 treated group)

SOOT (IU/litre)

20.72 ±0.62 72.28 ± 2.12a 59.18 ± 2.15b

45.98 ± 2.23b

LOH (nmol NAOH

oxidized/min/mg protein)

356.8 ± 31.2 553.8 ± 37.9" 496.4 ± 22.6b

443.6 ± 24.9b

EO=E. officinalis; 01= 100 mg/kg body weight of E. officillalis; D2= 200 mgikg body weight of E. officillalis.

SULTANA etal.: EMBLICA OFFICINALIS & CCI4 INDUCED HEPATIC TOXICITY IN RATS 433

liver provides a model to study the factors that modulates the process of regeneration. Acute hepatic necrosis on CCl4 administration is followed by proliferative response that reaches peak at 48 hr, with scarce mitotic cells.

The effect of pretreatment of animals with crude extract of E. officinalis plants on CCl4 induced GSH depletion, enhanced MDA formation and DNA synthesis is shown in Table 3. Pretreatment of rats with extract produced a marked inhibitory effect on GSH depletion with concomitant alleviation of MDA levels. E. officinalis showed a significant protection of GSH content by 30% and decrease in MDA formation by 111 % as compared with CCl4 treated group. Administration of CCl4 resulted in significant (P<O.OOI) 263% increase in the rate of eH] thymidine incorporation into hepatic DNA, which is a marker of DNA synthesis as compared with saline group. The pretreatment of rats with E. officinalis showed a marked (P<O.OI) suppressing effect on the rate eH]

thymidine incorporation into hepatic DNA by 102% at higher dose of the plant as compared with CCl4 treated group. Cellular thiol status is an important consideration for the study of hepatotoxicity, particularly in conditions of oxidative tissue injury33. GSH is a tripeptide (y-glutamyl cysteinyl glycine) non-protein thiol abundant in liver that plays pivotal role in the detoxification processes including conjugation of reactive intermediates and maintenance of GSH redox cycle as antioxidant armoury34. The enhancement of hepatotoxicity by GSH depletion has been noted during the metabolism of several compounds35. CCl4 administration resulted in marked depletion of tissue GSH content consequently increasing vulnerability of tissue to free radical attack causing lipidperoxidation. ProteCtive agents have shown to exert their action against CCl4 mediated lipidperoxidation either through decreased production of free radical derivatives or due to antioxidant activity of the agent itself36. The

Table 2-Effect of pretreatment of the extract of E.officillalis on glutathione metabolizing enzymes against CCI4 induced oxidative stress in rat liver

[Values are mean ± SE from 6 animals in each group]

Treatment Groups

Saline CCI4 alone

CCI4 +EO (01) CCl4 +EO (D2)

GST (nmol conjugate

formed/min/mg protein)

865.91± 34.63 1454.78±65.19" 1271.88 ± 34.94c

1096.22 ± 46.68b

P values: °<0.01 (compared to saline treated group) b<O.OOI (compared to CCl4 treated group) c<O.OOI (compared to CCl4 treated group)

GPx (nmol NADPH oxidized

min/mg protein)

210.56 ± 6.27 122.12 ± 6.42" 154.82 ± 7.93c

179.27±6.2 1b

EO=E. officinalis; Dl= 100 mg/kg body weight of E. officillalis; D2= 200 mg/kg body weight of E. officillalis

GR

189.54 ± 4.92 115.65 ± 3.47" 155.2 ± 6.97b

174.92 ± 7.43b

Table 3--Effect of pretreatment of the extract of E. officinalis on CCl4 induced depletion of GSH and in·crease in the levels of LPO and DNA synthesis

[Values are mean ± SE from 6 animals in each group]

Treatment GSH Groups (nmol GSHlg tissue)

Saline 0.62 ± 0.05 CCl4alone 0.32 ± 0.04" CCI4+EO (Dl) 0.45 ± 0.05b

CCI4+EO (D2) 0.52 ± 0.03b

P values: "<0.01 (compared to saline treated group) b<O.OOI (compared to CCl4 treated group) c<O.OOI (compared to CCl4 treated group)

LPO (nmol MDA formedlhr/g

tissue)

8.42 ± 0.8 24.31 ± 1.0" 18.1 ± 0.9b

15.4 ± 1.2b

EO=E. officillalis; Dl= 100 mg/kg body weight of E. officinalis; D2= 200 mg/kg body weight of E. officinalis

DNA synthesis (dprnlJ-lg DNA)

31.3 ± 5.8 82.4 ± 12.9" 65 .9 ± 8.7b

50.4 ± 6.5b

434 INDIAN J EXP BIOL, MAY 2005

pretreatment of animals with E. officinalis significantly ameliorated GSH content with concomitant decrease in lipid peroxidation. It could be suggested that antioxidant presence of several active constituents including quercetin, ascorbic acid and ellagic acid in E. officinalis may have counteracted the free radicals through effective scavenging and blocking the conjugation of reactive intermediates to GSH as evident from ameliorated GSH content and decreased MDA formation. Similarly, Vitamin E has been shown to suppress free radical mediated chain initiation and consequently protecting tissue from pathophysiological insult". Induction of GST activity has been correlated to increased GSH consumption and 4-hydroxynonenolm (4-HNE) levels in tissue37

• The sudden elevation of hepatocytes growth factor (HGF) in plasma provides mitogenic signals that trigger DNA synthesis during liver regeneration38

. In the present, study CCl4

administration resulted in enhancement in the rate of DNA synthesis when compared to normal group.

Pretreatment of rats with E. officinalis extracts significantly suppressed the rate of eH] thymidine incorporation into hepatic DNA in a dose dependent manner. The enhancement of DNA synthesis has been shown as a consequent step to the severe necrosis induced by hepatotoxicants39

.

The protective effect of plant E. officinalis on the basis of histopathological findings is shown in Fig. 1. Figure 1a shows the normal pathology of liver section. Administration of CCl4 resulted in pathological deterioration of hepatic tissue as evident multi vacuolated hepatocytes containing fat globules showing fatty changes around central vein and large vacuolated cells with central nuclei at the periphery of lobule showing necrosis. (Fig. 1b). The pretreatment of animals with E. officinalis fruit extract showed a profound pathological protection to liver cells as depicted by univacuolated hepatocytes surrounding multivacuolated hepatocytes, mild infiltration and fatty changes around vascular channels (Fig. 1 c, d). In summary, the present data suggest that pretreatment

Fig. I-Effect of pretreatment of rats with E. ojJicinalis on CCl4 induced pathological changes in rat liver. a-d: H & E: (a) saline only (x 50), (b) only CCI4• (c) E. ojJicinalis (01) + CCI4 and (d) E. ojJicinalis (D2) + CCI4• b-d x 250.

SULTANA et al.: EMBLICA OFFICINA LIS & CCI4 INDUCED HEPATIC TOXICITY IN RATS 435

of animals with E. officinalis markedly inhibited oxidative stress and lipidperoxidation induced by necrogenic dose of CCl4 and also suppressed tissue injury and hepatotoxicant post necrotic regeneration process.

Acknowledgement The author (S S) is thankful to CSIR, New Delhi,

India for financial assistance.

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