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ejpmr, 2015,2(6), 141-146 Research Article
EUROPEAN JOURNAL OF PHARMACEUTICAL
Gopalakrishnan et al. European Journal of Pharmaceutical and Medical Resea
N 3 294-3211
AND MEDICAL RESEARCH
HEPATOPROTECTIVE ACTIVITY STUDIES OF CUCUMIS TRIGONUS ROXB.
AGAINST RIFAMPICIN-ISONIAZID-INDUCED TOXICITY IN RATS.
Subarayan Bothi Gopalakrishnana*, Thangaraj Kalaiarasib
a*Senior Professor of Chemistry and Controller of Examination, Noorul Islam University, Kumaracoil,
Kanyakumari-629 180, Tamil Nadu, India. bAssistant Professor of Chemistry, Kalasalingam University, Kalasalingam Academy of Research and Education,
Anand Nagar, Krishnankoil-626126, Tamil Nadu, India. *Correspondence for Author: Subarayan Bothi Gopalakrishnan
Senior Professor of Chemistry and Controller of Examination, Noorul Islam University, Kumaracoil, Kanyakumari-629 180, Tamil Nadu, India. Article Received on 22/08/2015 Article Revised on 15/09/2015 Article Accepted on 07/10/2015
The fruits of Cucumis trigonus Roxb. was analysed for the hepatoprotective activity against albino rats with liver damage induced by rifampicin-isoniazid. Rifampicin (RIF) plus isoniazid (INH) treated rats showed significant increase in the levels of serum enzyme activities, reflecting the liver injury. The ethanolic extract of the fruits of Cucumis trigonus showed normalization of body weight, biochemical parameters like serum glutamic oxaloacetic transaminase (SGOT), serum glutamic pyruvic transaminase (SGPT), serum alkaline phosphatase (SALP), γ- glutamyl transpeptidase (GGTP), total bilirubin (TB), total protein (TP) as well as the levels of liver homogenates, Lipid peroxidase (LPO), glutathione peroxidase (GPx), glutathione reductase (GRD), superoxide dismutase (SOD), catalase (CAT) and reduced glutathione (GSH). The effects of ethanolic extract of the fruits of Cucumis trigonus (100 mg/kgbw ip 250 mg/kgbw ip & 500 mg/kgbw ip) was compared with that of the standard drug, silymarin. The ethanolic extract showed significant hepatoprotective activity in 500 mg/kg ip dose. The hepatoprotective activity has also been supported by histopathological studies of liver tissue. KEYWORDS: Hepatoprotective, Cucumis trigonus, Histopathology.
Cucumis trigonus (Fam. Cucurbitaceae) is commonly Rifampicin (RIF) and isoniazid (INH), the two front-line known as "Thummittikai" in Tamil, "Bitter gourd" in drugs have been used in the treatment of tuberculosis, English, and "Vishala" in Sanskrit. The fruits of that is known to be potentially hepatotoxic. Rifampicin, Cucumis trigonus are reported to be useful in treating which is generally co-administered with isoniazid in the leprosy, jaundice, diabetes, and other abdominal treatment of tuberculosis, is toxic to hepatocytes. A meta disorders. Cucumis trigonus fruit is shown to possess analysis of studies involving the use of a multiplicity of various activities such as antidiabetic activity, anabolic antituberculosis drug regimens predominantly in adults activity, cardioprotective activity, analgesic and anti- have shown an incidence of liver injury of 1.1 % in inflammatory, and diuretic activity. patients with RIF alone, 1.6 % in patients with INH alone, and 2.55 % in patients with RIF plus INH. Hepatoprotective activity of the ethanolic extracts of the Reactive oxygen species play a key role in RIF-INH- fruits of Cucumis trigonus and Cucumis sativus against induced hepatotoxicity. Since oxidative stress has been paracetamol-induced toxicity in albino rats have been regarded as the major mechanism of antituberculosis already performed by our group.[12, 13] In the present drug-induced hepatotoxicity, antioxidants might be study hepatoprotective activity studies of the ethanolic used as potential antihepatotoxic drugs against RIF-INH extract of the fruits of Cucumis trigonus on RIF-INH- caused liver injury. induced liver toxicity in albino rats have been carried A major defense mechanism involves the antioxidant enzymes, including SOD, CAT and GSH-Px, which MATERIAL AND METHODS
convert active oxygen molecules into non-toxic Collection of plant materials
compounds. The pathogenesis of the hepatotoxicity is The fruits of Cucumis trigonus was collected in the involved in all the hepatic cell types via death and month of March from Alangulam, Tirunelveli District, regeneration processes, and liver diseases often progress Tamil Nadu and identified by Prof. P. Jayaraman, Plant from subclinical icteric hepatitis to hepatic fibrosis, Anatomy Research Centre, West Thambaram, Chennai- cirrhosis and hepatocellular carcinoma. 600 045, Tamil Nadu, India (Reg.No of the authentification certificate: PARC/2013/2048). Gopalakrishnan et al. European Journal of Pharmaceutical and Medical Research
Group VI: Received 2.5 mg/kgbw ip of silymarin
Male wistar albino rats weighing 150-200 g were used (Standard drug) and simultaneously received 50 for hepatoprotective studies. The animals were fed with mg/kgbw ip per day of RIF + INH each by ip route for standard pellet diet supplied by Hindustan Lever Ltd., Kolkata, India and fresh water ad libitum. They were housed in standard stainless-steel cages at a 12 h cycle of At the end of 21 days, all the animals were sacrificed by light and dark. Room temperature was kept at (25° ± cervical decapitation. Blood samples were collected, and 3°C), humidity maintained at 50 %. the serum was separated by centrifuging at 2500 rpm for 15 min and analyzed for the various biochemical Drugs and chemicals
parameters. Body weights of the rats were measured Rifampicin and isoniazid were purchased from Micro daily for 21 days. Daily changes in body weights were Labs, India. Silymarin was obtained as gift sample from Ranbaxy (Devas, India), Standard kits of SGPT, SGOT, SALP, bilirubin and total protein were obtained from Assessment of liver damage
Jain Scientific Industries, Moradabad, India. All other Liver damage was assessed by the estimation of serum reagents used for the study were of analytical grade. activities of serum glutamic oxaloacetic transaminase (SGOT), serum glutamic pyruvic transaminase (SGPT), Preparation of extract
serum alkaline phosphatase (SALP), γ- glutamyl The collected fruits were cut into pieces, shade-dried at transpeptidase (GGTP), total bilirubin (TB), conjugated room temperature and powdered. The dried fruit bilirubin, unconjugated bilirubin, total protein (TP), albumin, and globulin according to the method by using petroleum ether (40°- 60°C), benzene, chloroform, commercially available test kit.[16-22] Lipid peroxidase ethanol and water by using Soxhlet apparatus. The last (LPO) glutathione peroxidase (GPx) glutathione trace of solvent was removed under reduced pressure reductase (GRD) superoxide dismutase (SOD) distillation and then vacuum dried. The dried crude catalase (CAT) and reduced glutathione (GSH) ethanolic extract was used for the study. were estimated in liver homogenate. Acute toxicity
Acute toxicity study was performed for the ethanolic The livers were removed from the animals and the extract of the fruits of Cucumis trigonus as per OECD tissues were fixed in 10 % formalin for at least 24 h. guidelines. Albino rats received 2000 mg/kgbw ip of Then, the paraffin sections were prepared (Automatic the ethanol extract. The animals were observed for toxic tissue processor, Autotechnique) and cut into 5 µm thick symptoms continuously for the first 4 h after dosing. The sections using a rotary microtome. The sections were rats were continuously observed for their mortality and then stained with Haematoxylin-Eosin dye and studied behavioural response for 48 h and thereafter once in a for histopathological changes, such as fatty changes, day for 14 days. There was no mortality recorded. necrosis, vacuole, space formation, loss of cell Therefore the drug should be free from toxicity. boundaries for microscopic observations. Statistical analysis
Induction of experimental hepatotoxicity
The values were expressed as Mean ± SD. Statistical Each 50 mg/kgbw ip of RIF + INH solutions were analysis was performed by one way analysis of variance prepared separately in sterile distilled water. Rats were (ANOVA) followed by Tukey multiple comparison test divided into nine groups, each group consisting of six and data on liver weight variations were analyzed using Student‟s „t‟ test. The levels of significance were Group I: Control received the vehicle viz. normal saline
mentioned as * P ≤ 0.05, ** P ≤ 0.01. Group II: Received 50 mg/kgbw ip per day of RIF +
RESULTS AND DISCUSSION
INH each by ip route for 21 days. Rifampicin is a first line drug used in the treatment of Group III: Received 100 mg/kgbw ip of the ethanolic
tuberculosis and leprosy. It possesses the ability to extract of the fruits of Cucumis trigonus and eliminate semi dormant or persisting organism. Short simultaneously received 50 mg/kgbw ip per day of RIF + course chemotherapy containing rifampicin and isoniazid INH each by ip route for 21 days. (Low dose). in combination has proved to be highly effective in the Group IV: Received 250 mg/kgbw ip of the ethanolic
treatment of tuberculosis, but one of its adverse effects is extract of the fruits of Cucumis trigonus and hepatotoxicity. RIF-induces cytochrome P450 enzyme simultaneously received 50 mg/kgbw ip per day of RIF + causing an increased production of toxic metabolites INH each by ip route for 21 days. (Moderate dose). from acetyl hydrazine (AcHz). RIF can also increase the Group V: Received 500 mg/kgbw ip of the ethanolic
metabolism of INH to isonicotinic acid and hydrazine, extract of the fruits of Cucumis trigonus and both of which are hepatotoxic. The plasma half life of simultaneously received 50 mg/kg ip per day of RIF + AcHz (metabolite of INH) is shortened by RIF and AcHz INH each by ip route for 21 days. (High dose). is quickly converted to its active metabolites by increasing the oxidative elimination rate of AcHz, which Gopalakrishnan et al. European Journal of Pharmaceutical and Medical Research
is related to the higher incidence of liver necrosis caused Administration of RIF + INH combination only, showed by INH and RIF in combination. Damaged hepatocytes a significant derangement of liver function as assessed or biliary epithelium may release cell constituents (e.g. by change in serum enzymes SGOT, SGPT, SALP, enzymes) into blood resulting in increased levels of these GGTP, TB, TP, albumin, globulin as well as the levels of analytes. The more commonly measured „liver‟ enzymes liver homogenates, LPO, GPx, GRD, SOD, CAT, and are alanine aminotransferase (ALT, formerly SGPT), GSH and also liver histopathology. aspartate aminotransferase (AST, formerly known Table-1 shows the levels of SGOT, SGPT, SALP, GGTP SGOT), serum alkaline phosphatase (SALP), and γ- in the serum and bodyweight. There was a significant glutamyl transpeptidase (GGTP). increase in the levels of SGOT, SGPT, SALP, GGTP in Although there will be an increase of AST and ALT in serum of rats treated with RIF + INH when compared heart and liver diseases, total bilirubin (TB), a byproduct with that of the control rats. Whereas the levels of body of the breakdown of red blood cells in the liver is a good weight in RIF + INH treated rats were decreased. There indicator of liver function. High levels will cause icterus is a gain in body weight in all the drug treated groups. and are indicative of damage to the liver and bile duct. Pretreatment of rats with the ethanolic extract of the The estimation of GGTP level is a valuable screening fruits of Cucumis trigonus caused a significant reduction test with high negative predictive value for liver disease. in the levels of enzymes leading to a significant reversal of hepatotoxicity.
TABLE 1: Effect of the ethanolic extract of the fruits of Cucumis trigonus on the body weight and other
biochemical parameters on rifampicin-isoniazid-induced hepatotoxicity in rats.
Final weight (g)
/ loss () (g)
Values are Mean ± SD of 6 animals in each group. Statistical analysis ANOVA followed by Dunnett t-test. *P < 0.05; **P < 0.01 as compared with normal control to liver damaged control; #P<0.05; ## P<0.01 as compared with liver damaged control to drug treated animal. ns: not significant. Table-2 shows the changes in the levels of total bilirubin, protein, albumin, and globulin (p < 0.05). Interestingly, conjugated bilirubin, unconjugated bilirubin, total in the RIF + INH-induced rats, the levels of total protein, albumin, and globulin in the serum of different bilirubin, conjugated bilirubin, unconjugated bilirubin, experimental rats. In comparison with the control group, total protein, albumin, and globulin in the liver could be in the RIF + INH treated rats, significant increase in the normalized by the pretreatment with the ethanolic extract of the fruits of Cucumis trigonus. 500 mg/kgbw ip of unconjugated bilirubin (p < 0.01) were noticed. Cucumis trigonus showed better activity. There was a significant reduction in the levels of total
TABLE 2: Effect of the ethanolic extract of the fruits of Cucumis trigonus on the biochemical parameters on
rifampicin-isoniazid-induced hepatotoxicity in rats.
Values are Mean ± SD of 6 animals in each group. Statistical analysis ANOVA followed by Dunnett t-test. *P < 0.05; **P < 0.01 as compared with normal control to liver damaged control; #P<0.05; ## P<0.01 as compared with liver damaged control to drug treated animal. ns: not significant.
Gopalakrishnan et al. European Journal of Pharmaceutical and Medical Research
Table-3 shows the levels of LPO, GPx, GRD, SOD, dependent. The hepatoprotective role of the ethanolic CAT, and GSH in the liver homogenate. The level of extract of the fruits of Cucumis trigonus might be due to lipid peroxide sharply increased (6.93±0.013 nm the antioxidant potential of the drugs. MDA/mg protein (p < 0.01) after RIF + INH intoxication. However, the levels of GPx, GRD, SOD, The ethanolic extract of the fruits of Cucumis trigonus CAT, GSH decreased after RIF + INH intoxication. The improved liver function by decreasing the serum administration of all the three doses, viz, the low dose, enzymes SGOT, SGPT, SALP, GGTP, TB, conjugated moderate dose, and high dose of Cucumis trigonus bilirubin, unconjugated bilirubin, LPO. However, the decreased the level of LPO and increased the levels of levels of total protein, albumin, globulin, GPx, GRD, GPx, GRD, SOD, CAT, GSH (p < 0.01). Among the SOD, CAT, and GSH are increased. This indicates the three different doses, 500 mg/kgbw ip dose showed protective effect over liver and improvement in its better activity than the standard drug, silimarin, in the functional efficiency. case of LPO and GRD. The protective effect was dose-
TABLE 3: Effect of the ethanolic extract of the fruits of Cucumis trigonus on the liver homogenate biochemical
parameters on rifampicin-isoniazid-induced hepatotoxicity in rats.
Values are Mean ± SD of 6 animals in each group. Statistical analysis ANOVA followed by Dunnett t-test.
*P < 0.05; **P < 0.01 as compared with normal control to liver damaged control;
#P<0.05; ## P<0.01 as compared with liver damaged control to drug treated animal; ns: not significant.
In histopathological studies of liver [Figure 1], the
control showed normal gross appearance; dark maroon color of liver having smooth surfaces, microscopically normal lobular appearance having normal central vein, normal hepatic cells each with well-defined cytoplasm, prominent nucleus, well brought out central vein, normal architecture of liver, radiating cords of hepatocytes, and normal portal tract. RIF + INH treated rats showed moderate to severe liver damage characterized by disarrangement of normal hepatic cells, vacuolization, loss of cell boundaries, space formation, and crowding of central vein marked level of fatty changes or Control group
degeneration and centrilobular hepatic necrosis of the liver cells. RIF + INH and low dose (100 mg/kgbw ip) of the ethanolic extract of the fruits of Cucumis trigonus showed minimal necrosis, mild inflammation and less steatosis. RIF + INH and moderate dose (250 mg/kgbw ip) of the ethanolic extract of the fruits of Cucumis trigonus showed slight recovery and evidence of regeneration in some hepatocytes. RIF + INH and high dose (500 mg/kgbw ip) of the ethanolic extract of the fruits of Cucumis trigonus showed significant recovery showing absence of necrosis, space formation and vacuoles. RIF + INH and silymarin (2.5 mg/kg ip) showed normal liver architecture and occasional inflammatory cells with no traditis or necrosis. RIF + INH (100 mg/kg ip)
Gopalakrishnan et al. European Journal of Pharmaceutical and Medical Research
EE - Ethanolic extract, CT - Cucumis trigonus, RIF- Rifampicin, INH – Isonizid, CV - Central vein, H -
Hepatocyte, N - Nucleus, FC - Fatty changes, NC -
Necrosis, V - Vacuole, SF - Space formation, LCB -
Loss of cell boundaries.
Hepatotoxicity occurs significantly with anti-TB drugs.
The present study proves that the ethanolic extract of the
fruits of Cucumin trigonus shows significant protective
action against the hepatotoxicity induced by the drugs
used in the treatment of tuberculosis. However, treatment RIF + INH + EE of CT (100 mg/kg ip)
of these extract completely protected the liver cells. GC- MS analysis indicated the presence of bile acids, carotenoids, antibiotics, steroids and phorbol ester in Cucumis trigonus. Hence the hepatoprotective effect of the extract may be due to the presence of one or more phytochemical constituents present in the Cucumis trigonus which scavenged the free radical offering hepatoprotection. Thus the ethanolic extract of the fruits of Cucumis trigouns which are useful in controlling hepatic injury in drug induced hepatotoxicity. Isolation and characterization of the active principles may yield good hepatoprotective drugs. REFERENCES
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AP® STATISTICS 2011 SCORING GUIDELINES (Form B) Question 2 Intent of Question The primary goals of this question were to assess students' ability to (1) distinguish an experiment from an observational study; (2) critique statistical information, in particular whether or not researchers are justified in making a specific conclusion based on the given information; (3) recognize and describe a potential problem with a study that lacks random assignment or blinding. Solution Part (a):