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Hepatoprotective activity of Thunbergia laurifolia Linn extract in rats treated with ethanol: In vitro and in vivo studies
Journal of Ethnopharmacology 102 (2005) 408–411
Hepatoprotective activity of Thunbergia laurifolia Linn extract in rats treated with ethanol: In vitro and in vivo studies Pornpen Pramyothin a,∗ , Hemvala Chirdchupunsare a , Anudep Rungsipipat b , Chaiyo Chaichantipyuth c a
Pharmacological Action of Natural Products Research Unit, Department of Pharmacology, Faculty of Pharmaceutical Sciences, Chulalongkorn University, Bangkok, Thailand b Department of Pathology, Faculty of Veterinary Science, Chulalongkorn University, Bangkok, Thailand c Department of Pharmacognosy, Faculty of Pharmaceutical Sciences, Chulalongkorn University, Bangkok, Thailand Received 11 October 2004; received in revised form 14 June 2005; accepted 22 June 2005 Available online 8 August 2005
Abstract Primary cultures of rat hepatocyte and rats were used as the in vitro and in vivo models to evaluate the hepatoprotective activity of aqueous extract from Thunbergia laurifolia (TLE). Ethanol was selected as hepatotoxin. Silymarin (SL) was the reference hepatoprotective agent. In the in vitro study, MTT reduction assay and release of transaminases (ALT and AST) were the criteria for cell viability. Primary cultures of rat hepatocyte (24 h culturing) were treated with ethanol (96 l/ml) and various concentrations of TLE (2.5, 5.0, 7.5 and 10.0 mg/ml) or SL (1, 2 and 3 mg/ml) for 2 h. Ethanol decreased MTT (%) nearly by half. Both TLE and SL increased MTT reduction and brought MTT (%) back to normal. Ethanol induced release of ALT and AST was also reduced by TLE (2.5 and 5.0 mg/ml) and SL (1 mg/ml). In the in vivo study, serum transaminases, serum triglyceride (STg) together with hepatic triglyceride (HTg) and histopathological examination were the criteria for evidences of liver injury. Ethanol (4 g/(kg day), po for 14 days) caused the increase in ALT, AST, HTg and centrilobular hydropic degeneration of hepatocytes. TLE at 25 mg/(kg day), po, or SL at 5 mg/(kg day), po, for 7 days after ethanol enhanced liver cell recovery by bringing HTg, ALT and/or AST back to normal. These results suggest that TLE and SL possess the hepatoprotective activity against ethanol induced liver injury in both primary cultures of rat hepatocyte and rats. © 2005 Elsevier Ireland Ltd. All rights reserved. Keywords: Thunbergia laurifolia; Hepatoprotective study; Ethanol
1. Introduction Scientific research in herbal medicine with hepatoprotective activity may be a great benefit as an alternative therapy in alcohol induced liver diseases. Over consumption of alcohol is now a serious problem in Thai society and worldwide. Three pathologically life-threatening liver diseases induced by alcohol abuse are fatty liver (steatosis), hepatitis and cirrhosis. Thunbergia laurifolia Linn. (Acanthaceae) is known in Thai as “Rang Jued”. It has been commonly used
∗
Corresponding author. Tel.: +66 22188322; fax: +66 22188326. E-mail address: [email protected] (P. Pramyothin).
0378-8741/$ – see front matter © 2005 Elsevier Ireland Ltd. All rights reserved. doi:10.1016/j.jep.2005.06.036
as antipyretic and antidote in Thai traditional medicine for many years. Treatment of alcoholism is also claimed using its aqueous extract. Alcohol and hexane extracts from Thunbergia laurifolia also possess anti-inflammatory activity against carageenin-induced paw edema in mice (Charumanee et al., 1998). Its leaves extract protects mice from hepatic injury induced by ethanol (Chanawirat et al., 2000). This investigation is aimed to investigate the hepatoprotective effect of aqueous extract from Thunbergia laurifolia, firstly, to estimate its hepatoprotection against ethanol in primary cultures of rat hepatocyte and secondly, to examine its promotion of liver recovery in ethanol treated rats. These effects are compared to silymarin, the reference hepatoprotective agent.
P. Pramyothin et al. / Journal of Ethnopharmacology 102 (2005) 408–411
2. Materials and methods 2.1. Animal Male Wistar rats (180–200 g) were obtained from National Laboratory Animal Center, Mahidol University, Salaya, Nakornpathom. They were housed in animal care facility at the Faculty of Pharmaceutical Sciences, Chulalongkorn University, under controlled environmental conditions (room temperature 25 ± 1 ◦ C with 12-h light:12-h dark cycle, relative humidity of approximately 60%) with free standard rat pellets and tap water. Rats were acclimatized for 3 days before experimentation.
409
(MTT) was dissolved in phosphate-buffered saline (PBS) at a concentration of 5 mg/ml and added to the cell culture to the final concentration of 100 l/ml. After 1 h, medium was removed and the remaining MTT crystals were dissolved in 100 l DMSO. Optical density was assessed using a microplate reader at 570/620 nm. 2.5.2. Determination of ALT and AST release ALT and AST in medium were assayed by kits from CPT diagnostics. Transaminases activity were measured as kinetic reaction using IFCC method. The absorbance of reaction was determined at 340 nm by spectrophotometer. 2.6. In vivo study
2.2. Test compounds Aqeous extract from leaves of Thunbergia laurifolia (TLE) was prepared by Assoc. Prof. Chaiyo Chaichantipyuth, Faculty of Pharmaceutical Sciences, Chulalongkorn University. Silymarin (SL) was kindly provided by The British Dispensary (L.P.) Co. Ltd. 2.3. Primary cultures of rat hepatocyte Hepatocytes were isolated from male Wistar rat using methods of Berry and Friend (1969) as modified by Stacey and Priestly (1978) and Pramyothin (1986). Cell viability was estimated immediately after the isolation by Trypan blue exclusion test using the exclusion index of >90%. Hepatocytes were washed and suspended in Eagle’s MEM supplemented with 10% inactivated fetal bovine serum, 10−8 M insulin, 10−6 M dexamethasone, penicillin (100 IU/l) and streptomycin (100 g/ml). Cells were placed in 24-well culture plates (Greiner, Germany) with a cell density of 1 × 105 cells/0.8 ml at 37 ◦ C, under carbogen (95% air, 5% CO2 ) atmosphere. 2.4. Hepatoprotective evaluation of TLE and SL against ethanol in primary cultures of rat hepatocyte After 24 h of culturing, hepatocytes were treated with ethanol (96 l/ml) and various concentration of TLE (2.5, 5.0, 7.5 and 10.0 mg/ml) or SL (1, 2 and 3 mg/ml) for 2 h. After 2 h incubation, cell viability was tested using MTT assay, ALT and AST release. All experiments were performed at least three times using different cell preparations. 2.5. Determination of cell viability in primary cultures of rat hepatocyte 2.5.1. MTT assay MTT assay is based on the ability of viable cells to reduce MTT from a yellow water-soluble dye to a dark blue insoluble formazan product (Mossmann, 1983). In brief, 3(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide
Rats were divided into five groups with eight rats in each group. Group 1 was the control, groups 2–5 received ethanol at 4 g/(kg day), po for 14 days. Then, groups 1 and 2 were sacrificed, blood was collected for ALT, AST and serum triglyceride determination; livers for hepatic triglyceride and histopathological examination. Group 3 was left for 7 days while groups 4 and 5 received SL at 5 mg/(kg day), po and TLE at 25 mg/(kg day), po, for 7 days, respectively. On day 21, rats (groups 3–5) were sacrificed. Blood and livers were collected for the above mentioned assays. 2.6.1. Determination of serum ALT and AST Serum ALT and AST was measured as described in Section 2.5.2. 2.6.2. Determination of serum triglyceride (STg) Using TG Kit (Human Gesellschaft fur Biochemica und Diagnostica mbH, Germany), 10 l of serum was incubated with 1 ml TG reagent at 37 ◦ C for 5 min. The absorbance was determined at 500 nm by spectrophotometer. 2.6.3. Determination of hepatic triglyceride (HTg) Liver (4 g) was homogenized with 6 ml ice-cold 0.1 M phosphate buffer pH 7.4. Liver homogenate was used for the determination of hepatic triglyceride by the method of Mendez et al. (1975). 2.6.4. Histopathological examination Liver was cut approximately 1 cm × 1.5 cm × 0.5 cm and fixed in 10% buffered formalin then embedded in paraffin. Haematoxylin–Eosin (H&E) staining was performed according to standard procedures on 4 m sections. Necrotic cells were quantified by image analysis under light microscope. The pathological changes of rat liver were graded: 0, normal; +1, mild degree; +2 moderate degree and +3, severe degree. 2.7. Statistical analysis Data are expressed as mean ± S.E.M. Statistical significance was assessed by one-way ANOVA followed by Tukey’s test.
P. Pramyothin et al. / Journal of Ethnopharmacology 102 (2005) 408–411
410
Table 1 Hepatoprotective effect of TLE or SL given together with ethanol in primary cultures of rat hepatocyte Treatment
MTTa (%)
MTTa (%)
ALTa (U/L)
Control Ethanol (E) E + TLE 2.5 mg/ml E + TLE 5.0 mg/ml E + TLE 7.5 mg/ml E + TLE 10.0 mg/ml Control Ethanol (E) E + SL 1 mg/ml E + SL 2 mg/ml E + SL 3 mg/ml
100 59.4 ± 6.7* 113.9 ± 7.6** 120.4 ± 9.7** 115.8 ± 7.0** 107.9 ± 5.5** 100 43.7 ± 1.6* 98.5 ± 4.0** 138.6 ± 2.7*,** 138.1 ± 3.7*,**
– 100 199.7 ± 12.2** 209.0 ± 10.0** 204.4 ± 14.1** 191.7 ± 14.7** – 100 225.1 ± 3.5** 298.6 ± 3.7** 336.6 ± 4.6**
3.3 24.6 12.1 8.8 14.3 36.3 3.3 24.6 11.3 29.1 34.1
a * **
± ± ± ± ± ± ± ± ± ± ±
ASTa (U/L) 15.0 ± 2.3 51.1 ± 2.4* 30.3 ± 7.3** 24.5 ± 1.5** 20.5 ± 3.7** 32.9 ± 6.2 15.4 ± 1.2 51.1 ± 2.4* 36.0 ± 4.2*,** 48.4 ± 2.3* 59.5 ± 4.2*
0.5 0.8* 1.4*,** 1.2*,** 1.5*,** 6.2* 0.5 0.8* 0.5*,** 3.2* 0.9*,**
Values are mean ± S.E.M., n = 8. Significant difference from control (p < 0.05). Significant difference from ethanol (p < 0.05).
Table 2 Promotion of rat liver recovery by TLE (25 mg/(kg day)) and SL (5 mg/(kg day)) given daily for 7 days after 14 days of ethanol (4 g/(kg day)) treatment Treatment
HTga (mg/g liver)
STga (mg/dl)
ALTa (U/L)
ASTa (U/L)
Severity of liver injuryb
Control Ethanol 14 days (E) E + 7 days E + SL 7 days E + TLE 7 days
3.4 ± 0.4 6.8 ± 0.8* 3.8 ± 1.0** 2.8 ± 0.2** 2.4 ± 0.6**
169.0 ± 11.6 148.2 ± 40.4 158.4 ± 50.0 150.0 ± 33.3 162.6 ± 34.6
24.2 ± 2.8 33.2 ± 2.6* 31.4 ± 0.9 25.2 ± 1.5 24.8 ± 1.2**
54.4 ± 2.1 69.4 ± 5.0* 58.2 ± 3.9 53.8 ± 2.6** 52.6 ± 2.7**
0 +2.5 +2.5 +1.5 +1.5
a b * **
Values are mean ± S.E.M., n = 8. 0, Normal; +1, mild; +2, moderate; +3, severe. Significant difference from control (p < 0.05). Significant difference from ethanol (p < 0.05).
3. Results 3.1. Hepatoprotective evaluation of TLE and SL against ethanol in primary cultures of rat hepatocyte TLE at 2.5, 5.0 and 7.5 mg/ml increased MTT (%) about two-folds and about three-folds for SL at 1 mg/ml when compared to effect of ethanol alone. Both TLE and SL at the above mentioned doses decreased the release of ALT and AST caused by ethanol. TLE at 10 mg/ml and SL at 2 and 3 mg/ml increased MTT (%) but not the decrease in ALT and AST release (Table 1). 3.2. Promotion of rat liver recovery by TLE and SL after 14 days of ethanol induced liver injury After 14 days of ethanol, HTg, serum ALT and AST increased with focal necrosis or centrilobular hydropic degeneration with the severity of +2.5. TLE at 25 mg/(kg day) or SL at 5 mg/(kg day), for 7 days after ethanol brought back the HTg, ALT and AST to normal with the severity of liver injury of +1.5. Group of rats with self-recovery still showed liver injury of +2.5 and high levels of ALT and AST (Table 2). 4. Discussion and conclusions TLE and SL at appropriate doses increase viability of primary cultures of rat hepatocyte treated with ethanol up to
two- to three-folds as shown by the increase in MTT (%) and the decrease in ALT and AST release. TLE and SL also promote rat liver recovery after 14 days of ethanol treatment demonstrated by the decrease in severity of rat liver injury and the normalization in the levels of HTg, ALT and AST. These results suggest that TLE and SL possess hepatoprotective activity both in primary cultures of rat hepatocyte and rats. Acknowledgements This research work is partially supported by Research Donation from The British Dispensary (L.P.) Co. Ltd. and Grant to Support High Potential Research Unit, Ratchadaphiseksomphot Endowment Fund, Chulalongkorn University. References Berry, M.N., Friend, D.S., 1969. High yield preparation of isolated rat liver parenchymal cells; a biochemical and fine structure study. Journal of Cell Biology 43, 506–520. Chanawirat, A., Toskulkao, C., Temcharoen, P., Glinsukon, T., 2000. Protective effect of Thunbergia laurifolia extract on ethanol-induced hepatotoxicity in mice. Thesis, Faculty of Graduate Studies, Mahidol University, Bangkok, Thailand. Charumanee, S., Vejabhikul, S., Taesotikul, T., Netsingha, W., Sirisaard, P., Leelapornpisit, P., 1998. Development of topical anti-inflammatory
P. Pramyothin et al. / Journal of Ethnopharmacology 102 (2005) 408–411 preparations from Thunbergia laurifolia Linn. Phase 1. Research Report, Faculty of Pharmacy, Chiangmai University, Chiangmai, Thailand. IFCC methods for the measurement of catalytic concentration of enzymes, 1986. Journal of Clinical Chemistry and Clinical Biochemistry 24, 481–495. Mendez, J., Feanklin, B., Gahagan, H., 1975. Simple manual for determination of serum triglycerides. Clinical Chemistry 21, 768–770.
411
Mossmann, T., 1983. Rapid colorimetric assay for cellular growth and survival: application to proliferation and cytotoxic assays. Journal of Immunologic Method 65, 55–63. Pramyothin, P., 1986. Isolated liver cell preparations. Journal of the National Research Council 18, 1–9. Stacey, N., Priestly, B.G., 1978. Dose dependent toxicity of CCl4 in isolated rat hepatocytes and the effects of hepatoprotective treatments. Toxicology and Applied Pharmacology 45, 29–39.
Hepatoprotective activity of Thunbergia laurifolia Linn extract in rats treated with ethanol: In vitro and in vivo studies Pornpen Pramyothin a,∗ , Hemvala Chirdchupunsare a , Anudep Rungsipipat b , Chaiyo Chaichantipyuth c a
Pharmacological Action of Natural Products Research Unit, Department of Pharmacology, Faculty of Pharmaceutical Sciences, Chulalongkorn University, Bangkok, Thailand b Department of Pathology, Faculty of Veterinary Science, Chulalongkorn University, Bangkok, Thailand c Department of Pharmacognosy, Faculty of Pharmaceutical Sciences, Chulalongkorn University, Bangkok, Thailand Received 11 October 2004; received in revised form 14 June 2005; accepted 22 June 2005 Available online 8 August 2005
Abstract Primary cultures of rat hepatocyte and rats were used as the in vitro and in vivo models to evaluate the hepatoprotective activity of aqueous extract from Thunbergia laurifolia (TLE). Ethanol was selected as hepatotoxin. Silymarin (SL) was the reference hepatoprotective agent. In the in vitro study, MTT reduction assay and release of transaminases (ALT and AST) were the criteria for cell viability. Primary cultures of rat hepatocyte (24 h culturing) were treated with ethanol (96 l/ml) and various concentrations of TLE (2.5, 5.0, 7.5 and 10.0 mg/ml) or SL (1, 2 and 3 mg/ml) for 2 h. Ethanol decreased MTT (%) nearly by half. Both TLE and SL increased MTT reduction and brought MTT (%) back to normal. Ethanol induced release of ALT and AST was also reduced by TLE (2.5 and 5.0 mg/ml) and SL (1 mg/ml). In the in vivo study, serum transaminases, serum triglyceride (STg) together with hepatic triglyceride (HTg) and histopathological examination were the criteria for evidences of liver injury. Ethanol (4 g/(kg day), po for 14 days) caused the increase in ALT, AST, HTg and centrilobular hydropic degeneration of hepatocytes. TLE at 25 mg/(kg day), po, or SL at 5 mg/(kg day), po, for 7 days after ethanol enhanced liver cell recovery by bringing HTg, ALT and/or AST back to normal. These results suggest that TLE and SL possess the hepatoprotective activity against ethanol induced liver injury in both primary cultures of rat hepatocyte and rats. © 2005 Elsevier Ireland Ltd. All rights reserved. Keywords: Thunbergia laurifolia; Hepatoprotective study; Ethanol
1. Introduction Scientific research in herbal medicine with hepatoprotective activity may be a great benefit as an alternative therapy in alcohol induced liver diseases. Over consumption of alcohol is now a serious problem in Thai society and worldwide. Three pathologically life-threatening liver diseases induced by alcohol abuse are fatty liver (steatosis), hepatitis and cirrhosis. Thunbergia laurifolia Linn. (Acanthaceae) is known in Thai as “Rang Jued”. It has been commonly used
∗
Corresponding author. Tel.: +66 22188322; fax: +66 22188326. E-mail address: [email protected] (P. Pramyothin).
0378-8741/$ – see front matter © 2005 Elsevier Ireland Ltd. All rights reserved. doi:10.1016/j.jep.2005.06.036
as antipyretic and antidote in Thai traditional medicine for many years. Treatment of alcoholism is also claimed using its aqueous extract. Alcohol and hexane extracts from Thunbergia laurifolia also possess anti-inflammatory activity against carageenin-induced paw edema in mice (Charumanee et al., 1998). Its leaves extract protects mice from hepatic injury induced by ethanol (Chanawirat et al., 2000). This investigation is aimed to investigate the hepatoprotective effect of aqueous extract from Thunbergia laurifolia, firstly, to estimate its hepatoprotection against ethanol in primary cultures of rat hepatocyte and secondly, to examine its promotion of liver recovery in ethanol treated rats. These effects are compared to silymarin, the reference hepatoprotective agent.
P. Pramyothin et al. / Journal of Ethnopharmacology 102 (2005) 408–411
2. Materials and methods 2.1. Animal Male Wistar rats (180–200 g) were obtained from National Laboratory Animal Center, Mahidol University, Salaya, Nakornpathom. They were housed in animal care facility at the Faculty of Pharmaceutical Sciences, Chulalongkorn University, under controlled environmental conditions (room temperature 25 ± 1 ◦ C with 12-h light:12-h dark cycle, relative humidity of approximately 60%) with free standard rat pellets and tap water. Rats were acclimatized for 3 days before experimentation.
409
(MTT) was dissolved in phosphate-buffered saline (PBS) at a concentration of 5 mg/ml and added to the cell culture to the final concentration of 100 l/ml. After 1 h, medium was removed and the remaining MTT crystals were dissolved in 100 l DMSO. Optical density was assessed using a microplate reader at 570/620 nm. 2.5.2. Determination of ALT and AST release ALT and AST in medium were assayed by kits from CPT diagnostics. Transaminases activity were measured as kinetic reaction using IFCC method. The absorbance of reaction was determined at 340 nm by spectrophotometer. 2.6. In vivo study
2.2. Test compounds Aqeous extract from leaves of Thunbergia laurifolia (TLE) was prepared by Assoc. Prof. Chaiyo Chaichantipyuth, Faculty of Pharmaceutical Sciences, Chulalongkorn University. Silymarin (SL) was kindly provided by The British Dispensary (L.P.) Co. Ltd. 2.3. Primary cultures of rat hepatocyte Hepatocytes were isolated from male Wistar rat using methods of Berry and Friend (1969) as modified by Stacey and Priestly (1978) and Pramyothin (1986). Cell viability was estimated immediately after the isolation by Trypan blue exclusion test using the exclusion index of >90%. Hepatocytes were washed and suspended in Eagle’s MEM supplemented with 10% inactivated fetal bovine serum, 10−8 M insulin, 10−6 M dexamethasone, penicillin (100 IU/l) and streptomycin (100 g/ml). Cells were placed in 24-well culture plates (Greiner, Germany) with a cell density of 1 × 105 cells/0.8 ml at 37 ◦ C, under carbogen (95% air, 5% CO2 ) atmosphere. 2.4. Hepatoprotective evaluation of TLE and SL against ethanol in primary cultures of rat hepatocyte After 24 h of culturing, hepatocytes were treated with ethanol (96 l/ml) and various concentration of TLE (2.5, 5.0, 7.5 and 10.0 mg/ml) or SL (1, 2 and 3 mg/ml) for 2 h. After 2 h incubation, cell viability was tested using MTT assay, ALT and AST release. All experiments were performed at least three times using different cell preparations. 2.5. Determination of cell viability in primary cultures of rat hepatocyte 2.5.1. MTT assay MTT assay is based on the ability of viable cells to reduce MTT from a yellow water-soluble dye to a dark blue insoluble formazan product (Mossmann, 1983). In brief, 3(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide
Rats were divided into five groups with eight rats in each group. Group 1 was the control, groups 2–5 received ethanol at 4 g/(kg day), po for 14 days. Then, groups 1 and 2 were sacrificed, blood was collected for ALT, AST and serum triglyceride determination; livers for hepatic triglyceride and histopathological examination. Group 3 was left for 7 days while groups 4 and 5 received SL at 5 mg/(kg day), po and TLE at 25 mg/(kg day), po, for 7 days, respectively. On day 21, rats (groups 3–5) were sacrificed. Blood and livers were collected for the above mentioned assays. 2.6.1. Determination of serum ALT and AST Serum ALT and AST was measured as described in Section 2.5.2. 2.6.2. Determination of serum triglyceride (STg) Using TG Kit (Human Gesellschaft fur Biochemica und Diagnostica mbH, Germany), 10 l of serum was incubated with 1 ml TG reagent at 37 ◦ C for 5 min. The absorbance was determined at 500 nm by spectrophotometer. 2.6.3. Determination of hepatic triglyceride (HTg) Liver (4 g) was homogenized with 6 ml ice-cold 0.1 M phosphate buffer pH 7.4. Liver homogenate was used for the determination of hepatic triglyceride by the method of Mendez et al. (1975). 2.6.4. Histopathological examination Liver was cut approximately 1 cm × 1.5 cm × 0.5 cm and fixed in 10% buffered formalin then embedded in paraffin. Haematoxylin–Eosin (H&E) staining was performed according to standard procedures on 4 m sections. Necrotic cells were quantified by image analysis under light microscope. The pathological changes of rat liver were graded: 0, normal; +1, mild degree; +2 moderate degree and +3, severe degree. 2.7. Statistical analysis Data are expressed as mean ± S.E.M. Statistical significance was assessed by one-way ANOVA followed by Tukey’s test.
P. Pramyothin et al. / Journal of Ethnopharmacology 102 (2005) 408–411
410
Table 1 Hepatoprotective effect of TLE or SL given together with ethanol in primary cultures of rat hepatocyte Treatment
MTTa (%)
MTTa (%)
ALTa (U/L)
Control Ethanol (E) E + TLE 2.5 mg/ml E + TLE 5.0 mg/ml E + TLE 7.5 mg/ml E + TLE 10.0 mg/ml Control Ethanol (E) E + SL 1 mg/ml E + SL 2 mg/ml E + SL 3 mg/ml
100 59.4 ± 6.7* 113.9 ± 7.6** 120.4 ± 9.7** 115.8 ± 7.0** 107.9 ± 5.5** 100 43.7 ± 1.6* 98.5 ± 4.0** 138.6 ± 2.7*,** 138.1 ± 3.7*,**
– 100 199.7 ± 12.2** 209.0 ± 10.0** 204.4 ± 14.1** 191.7 ± 14.7** – 100 225.1 ± 3.5** 298.6 ± 3.7** 336.6 ± 4.6**
3.3 24.6 12.1 8.8 14.3 36.3 3.3 24.6 11.3 29.1 34.1
a * **
± ± ± ± ± ± ± ± ± ± ±
ASTa (U/L) 15.0 ± 2.3 51.1 ± 2.4* 30.3 ± 7.3** 24.5 ± 1.5** 20.5 ± 3.7** 32.9 ± 6.2 15.4 ± 1.2 51.1 ± 2.4* 36.0 ± 4.2*,** 48.4 ± 2.3* 59.5 ± 4.2*
0.5 0.8* 1.4*,** 1.2*,** 1.5*,** 6.2* 0.5 0.8* 0.5*,** 3.2* 0.9*,**
Values are mean ± S.E.M., n = 8. Significant difference from control (p < 0.05). Significant difference from ethanol (p < 0.05).
Table 2 Promotion of rat liver recovery by TLE (25 mg/(kg day)) and SL (5 mg/(kg day)) given daily for 7 days after 14 days of ethanol (4 g/(kg day)) treatment Treatment
HTga (mg/g liver)
STga (mg/dl)
ALTa (U/L)
ASTa (U/L)
Severity of liver injuryb
Control Ethanol 14 days (E) E + 7 days E + SL 7 days E + TLE 7 days
3.4 ± 0.4 6.8 ± 0.8* 3.8 ± 1.0** 2.8 ± 0.2** 2.4 ± 0.6**
169.0 ± 11.6 148.2 ± 40.4 158.4 ± 50.0 150.0 ± 33.3 162.6 ± 34.6
24.2 ± 2.8 33.2 ± 2.6* 31.4 ± 0.9 25.2 ± 1.5 24.8 ± 1.2**
54.4 ± 2.1 69.4 ± 5.0* 58.2 ± 3.9 53.8 ± 2.6** 52.6 ± 2.7**
0 +2.5 +2.5 +1.5 +1.5
a b * **
Values are mean ± S.E.M., n = 8. 0, Normal; +1, mild; +2, moderate; +3, severe. Significant difference from control (p < 0.05). Significant difference from ethanol (p < 0.05).
3. Results 3.1. Hepatoprotective evaluation of TLE and SL against ethanol in primary cultures of rat hepatocyte TLE at 2.5, 5.0 and 7.5 mg/ml increased MTT (%) about two-folds and about three-folds for SL at 1 mg/ml when compared to effect of ethanol alone. Both TLE and SL at the above mentioned doses decreased the release of ALT and AST caused by ethanol. TLE at 10 mg/ml and SL at 2 and 3 mg/ml increased MTT (%) but not the decrease in ALT and AST release (Table 1). 3.2. Promotion of rat liver recovery by TLE and SL after 14 days of ethanol induced liver injury After 14 days of ethanol, HTg, serum ALT and AST increased with focal necrosis or centrilobular hydropic degeneration with the severity of +2.5. TLE at 25 mg/(kg day) or SL at 5 mg/(kg day), for 7 days after ethanol brought back the HTg, ALT and AST to normal with the severity of liver injury of +1.5. Group of rats with self-recovery still showed liver injury of +2.5 and high levels of ALT and AST (Table 2). 4. Discussion and conclusions TLE and SL at appropriate doses increase viability of primary cultures of rat hepatocyte treated with ethanol up to
two- to three-folds as shown by the increase in MTT (%) and the decrease in ALT and AST release. TLE and SL also promote rat liver recovery after 14 days of ethanol treatment demonstrated by the decrease in severity of rat liver injury and the normalization in the levels of HTg, ALT and AST. These results suggest that TLE and SL possess hepatoprotective activity both in primary cultures of rat hepatocyte and rats. Acknowledgements This research work is partially supported by Research Donation from The British Dispensary (L.P.) Co. Ltd. and Grant to Support High Potential Research Unit, Ratchadaphiseksomphot Endowment Fund, Chulalongkorn University. References Berry, M.N., Friend, D.S., 1969. High yield preparation of isolated rat liver parenchymal cells; a biochemical and fine structure study. Journal of Cell Biology 43, 506–520. Chanawirat, A., Toskulkao, C., Temcharoen, P., Glinsukon, T., 2000. Protective effect of Thunbergia laurifolia extract on ethanol-induced hepatotoxicity in mice. Thesis, Faculty of Graduate Studies, Mahidol University, Bangkok, Thailand. Charumanee, S., Vejabhikul, S., Taesotikul, T., Netsingha, W., Sirisaard, P., Leelapornpisit, P., 1998. Development of topical anti-inflammatory
P. Pramyothin et al. / Journal of Ethnopharmacology 102 (2005) 408–411 preparations from Thunbergia laurifolia Linn. Phase 1. Research Report, Faculty of Pharmacy, Chiangmai University, Chiangmai, Thailand. IFCC methods for the measurement of catalytic concentration of enzymes, 1986. Journal of Clinical Chemistry and Clinical Biochemistry 24, 481–495. Mendez, J., Feanklin, B., Gahagan, H., 1975. Simple manual for determination of serum triglycerides. Clinical Chemistry 21, 768–770.
411
Mossmann, T., 1983. Rapid colorimetric assay for cellular growth and survival: application to proliferation and cytotoxic assays. Journal of Immunologic Method 65, 55–63. Pramyothin, P., 1986. Isolated liver cell preparations. Journal of the National Research Council 18, 1–9. Stacey, N., Priestly, B.G., 1978. Dose dependent toxicity of CCl4 in isolated rat hepatocytes and the effects of hepatoprotective treatments. Toxicology and Applied Pharmacology 45, 29–39.