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Antihepatotoxic activity of p-methoxy benzoic acid from Capparis spinosa
Journal of Ethnopharmacology 66 (1999) 187 – 192
Antihepatotoxic activity of p-methoxy benzoic acid from Capparis spinosa Chhaya Gadgoli, S.H. Mishra * Pharmacy Department, Faculty of Technology and Engineering, M.S. Uni6ersity of Baroda, Baroda 390001, Gujarat, India Received 5 August 1998; received in revised form 5 December 1998; accepted 6 December 1998
Abstract p-Methoxy benzoic acid isolated from the methanolic soluble fraction of the aqueous extract of Capparis spinosa L. (Capparidaceae) was found to possess significant antihepatotoxic activity against carbontetrachloride and paracetamol induced hepatotoxicity in vivo and thioacetamide and galactosamine induced hepatotoxicity in isolated rat hepatocytes, using in vitro technique. The compound was characterized through physicochemical and spectral studies. Isolation, identification and antihepatotoxic activity of the compound is reported for the first time in the plant. HPTLC analysis of methanol soluble fraction indicated that the compound constitutes 33% w/w of the active fraction. © 1999 Published by Elsevier Science Ireland Ltd. All rights reserved. Keywords: Capparis spinosa; Capparidaceae; p-Methoxy benzoic acid; Antihepatotoxic activity
1. Introduction The total aqueous extract of aerial parts of Capparis spinosa (Capparidaceae) forms one of the constituents of polyherbal formulations to treat liver ailments listed by Handa et al. (1986). The different successive and total aqueous extracts of the plant are reported to possess significant activity (Gadgoli and Mishra, 1995). Therefore the total aqueous extract of the plant
* Corresponding author.
was subjected to detailed investigations of the antihepatotoxic activity of its constituent(s) in order to justify its utility profile as antihepatotoxic agent. The present study deals with the isolation, identification of the active compound(s) and evaluation of these substances for antihepatotoxic activity on rats against carbontetrachloride (CCl4) and paracetamol (Pcl) induced toxicities in vivo and thioacetamide (Th) and galactosamine (Galn) induced toxicities in vitro. The activity of the compound against CCl4 and Pcl was compared with silymarin (50 mg/kg b.w. i.p.), a wellknown antihepatotoxic agent.
0378-8741/99/$ - see front matter © 1999 Published by Elsevier Science Ireland Ltd. All rights reserved. PII: S 0 3 7 8 - 8 7 4 1 ( 9 8 ) 0 0 2 2 9 - 3
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2. Materials and methods
2.1. Plant material The aerial parts of Capparis spinosa L. Capparidaceae were procured from the Botany Department, Faculty of Science, M.S. University of Baroda, and the plant was identified by Dr. Pradeep, Taxonomy Laboratory, Botany Department.
2.2. Extraction and isolation of acti6e compound The total aqueous extract (63.35 % w/w) was prepared by decoction. The extract was evaporated under vacuum and 100 gm was fractionated into 500 ml of methanol to obtain methanol soluble (FI, 42.28 g) and methanol insoluble (FII, 54.72 g) fractions. Fraction I, being found active, was subjected to TLC studies. A total of 10 g of FI was dissolved in 50 ml methanol and chromatographed on TLC plates with silica gel G as adsorbent and benzene:acetone (9.5:0.5) as mobile phase. The TLC studies revealed the presence of three spots when observed under UV 365 hm: Rf of 0.89, pale blue fluorescent spot; Rf of 0.73, blue fluorescent spot; and Rf of 0.23, dark absorbing spot. An attempt was made to isolate all three spots using preparative TLC technique but only one spot of Rf 0.89 could be isolated in a better yield. The compound was recrystallized in methanol and subjected to physiochemical and spectral studies (UV, IR, NMR, MS). The isolated compound was characterized as p-methoxy benzoic acid based on the following findings and by comparing them with the available data (Grasselli, 1974; Aldrich Library, 1975). Colourless to cream coloured crystals of needle shape, m.p. 182–183°C, l max (MeOH) 205 and 256 hm. Soluble in methanol, chloroform, insoluble in water. C, 56; H, 6.1; N, 0; O, 27.2%. Mol. wt from MS, M + = 152, C8H8O3. FT IR 3200, 1695, 1150–1085, 1170 – 1115, 1224, 1040, 1585, 1575 –1400, cm − 1. 1H NMR 3.89, OCH3 (S), (7.94, 7.97) (d) orthocoupled H (AB), 7.26 (S), – OH, 13C NMR 51.95 ( – OCH3), 131.908 (C2, C6), 115.22 (C2, C6), 60.117 (COOH), m/e – 152
M + , 121 (OCH3), 93 (C6H5O + ). The HPTLC analysis of FI, revealed that FI contains 33% w/w of p-methoxy benzoic acid.
2.3. Animals Either sex albino rats of Wistar strain (150–200 g) were used. The animals were maintained on normal diet and tap water ad libitum with temperature 24 9 2°C, RH 40–70% and light/dark cycle of 12 h (06:00–18:00 h).
2.4. In 6i6o antihepatotoxic acti6ity testing The isolated compound p-methoxy benzoic acid was suspended in 4% aqueous acacia solution and was administered in a dose of 30 mg/kg b.w. i.p. The protocols described by Handa and Sharma (Handa and Sharma, 1990a; Handa and Sharma, 1990b) were followed for evaluation of the activity against CCl4 and Pcl induced hepatotoxicities, respectively. Silymarin, a gift sample from M/s Ranbaxy Research Laboratory, New Delhi, was suspended in 4% w/v aqueous acacia solution and administered in a dose of 50 mg/kg b.w. i.p. for antihepatotoxic testing. The three groups of rats were treated in the following manner.
2.4.1. Control Vehicle, 1 ml/kg b.w. i.p. was administered on 1st, 2nd and 3rd day+ olive oil (Oilo Sasso, Italy), 1 ml/kg b.w. s.c. on the 2nd and 3rd day, 30 min later the administration of the vehicle, for CCl4 induced hepatotoxicity. In case of Pcl induced toxicity instead of olive oil, 60% w/v sucrose solution, 1 ml/kg b.w. p.o. was administered on the 3rd day, followed 2 h later by the administration of the vehicle. 2.4.2. Toxicant Vehicle 1 ml/kg b.w. i.p. was administered on 1st, 2nd and 3rd day+toxicant, 2 ml/kg b.w. s.c of CCl4 on the 2nd and 3rd day, 30 min later the administration of vehicle while Pcl suspension, 3 g/kg b.w. p.o. was administered on the 3rd day, 2 h later the administration of the vehicle.
C. Gadgoli, S.H. Mishra / Journal of Ethnopharmacology 66 (1999) 187–192
2.4.3. Test substance (T.S.) The test substance p-methoxy benzoic acid, 30 mg/kg b.w. i.p., silymarin, 50 mg/kg b.w. i.p. on the 1st, 2nd and 3rd day +toxicants (CCl4 on 2nd and 3rd day, 30 min later the administration of the T.S. and Pcl on the 3rd day, 2 h later the administration of T.S.). CCl4 solution: 50% v/v solution in olive oil Vehicle: 4% w/v aqueous acacia solution Pcl solution: Pcl was suspended in 60% w/v aqueous sucrose solution. Blood was withdrawn on the 4th day in case of CCl4 and on the 5th day in case of Pcl induced toxicities. 2.5. Assessment of antihepatotoxic acti6ity Blood was collected from retroorbital plexus and serum was separated by centrifuging at 2000 rpm for 10 min. The activity of serum glutamyl pyruvate transaminase (SGPT) and serum glutamyl oxalacetate transaminase (SGOT) was assayed using the method as described by Reitman and Frankel (1957). The levels of alkaline phosphatase (Alkp) and total bilirubin (T.Bil) were assayed using the methods described by Bessay et al. (1946) and Jendrassik and Groff (1938), respectively The antihepatotoxic activity was confirmed through histopathological studies on liver of rats treated with toxicants as well as the compound. The liver of one rat from each group was removed and processed suitably to stain with hematoxylin-eosin (Bhagwan and Kandswami, 1988) to assess its architecture before and after treatment with the compound.
2.6. In 6itro antihepatotoxic testing In vitro studies involved isolation of hepatocytes and examination of the effect of toxicants along with the compound under investigation. The rat hepatocytes were isolated according to Seglen (1975) with slight modifications (Visen et al., 1991) by recirculating enzymatic perfusion technique (in situ). The hepatocytes thus isolated were incubated for 24 h following the protocol given below:
189
Control: 0.1 ml of hepatocyte suspension+ 0.1 ml vehicle (distilled water)+ 0.1 ml PBS (phosphate buffer saline, pH=7.4.) Toxicant: 0.1 ml of hepatocyte suspension+ 0.1 ml of toxicant (Galn 40 mg/ml, Th 20 mg/ml in distilled water)+ 0.8 ml PBS Test substance: 0.1 ml of hepatocyte suspension+ 0.1 ml of toxicant (Galn 40 mg/ml, Th 20 mg/ml in distilled water)+ 0.1 ml test substance (1000, 100, 10 mg/ml, suspended in distilled water)+ 0.7 ml PBS. Each set contained three samples.
2.7. Assessment of antihepatotoxic acti6ity Assessment of antihepatotoxic activity was done by determining % viability of hepatocytes using trypan blue dye exclusion test and oxygen uptake of hepatocytes using Geilson’s Oxygraph, described by Estabrook (1967).
2.8. Statistical analysis Analysis of variance (ANOVA) was employed for estimation of variations in a set of data followed by individual comparison within the groups using least significant square method (Randolph and Ciminera, 1980).
3. Results
3.1. In 6i6o antihepatotoxic acti6ity Administration of CCl4, and Pcl led to increase in the serum enzyme levels by two- to threefold as compared to control group. Treatment of rats with p-methoxy benzoic acid (30 mg/kg b.w. i.p.) resulted in 89.68, 105.28, 78.91, 56.55 and 137.4, 86.30, 92.91, 62.55% reductions in SGPT, SGOT, Alkp and T.Bil levels as compared to CCl4 and Pcl groups, respectively (Table 1).
3.2. In 6itro antihepatotoxic acti6ity Incubation of hepatocytes with Th and Galn resulted in 74.77, 76.27, and 51.00, 47.95% decrease in the % viability and oxygen uptake of
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Table 1 Effect of p-methoxy benzoic acid on biochemical parameters in rats intoxicated with CCl4 (2 ml/kg b.w. s.c.) and Pcl (3 gm/kg b.w. p.o.)a Groups
Control (group I) CCl4 (group II)b CGCaI+CCl4 (group III) Sil+CCl4 (group IV) Pcl (group V) CGCaI+Pcl (group VI)c Sil+Pcl (group VII)
SGPT (U/l), mean 9S.D. (% reduction)
SGOT (U/l), mean 9S.D. Alkp (U/l), mean 9 S.D. T.Bil (mg/dl), mean 9 S.D. (% reduction) (% reduction) (% reduction)
65.06 95.42
110.679 28.70
43.73 93.55
0.208 9 0.034
302.47 9 17.54
464.85943.58
188.72 912.8
2.035 9 0.438
89.08 920.83 (89.88)**
91.94 9 40.65 (105.28)**
74.30 942.02 (78.91)**
1.000 90.059 (56.65)**
78.08 96.83 (94.40)**
169.479 17.81 (83.39)**
120.67 9 8.46 (46.96)**
0.791 9 0.038 (68.08)**
124.57 916.43 47.78 98.51 (137.45)**
243.799 7.19 128.099 28.15 (86.30)**
174.50 9 30.50 53.00 9 24.80 (92.91)**
1.682 9 0.138 0.760 9 0.098 (62.55)**
63.41 9 2.67 (102.85)**
143.219 12.81 (75.55)**
106.83 9 4.62 (51.74)**
0.582 9 0.023 (74.42)**
a Values (U/l and mg/dl) are mean 9S.D. of six animals. CGCaI refers to the isolated compound p-methoxy benzoic acid. Sil refers to silymarin. b Groups II and IV are compared with group I: PB0.001. c Groups VI and VII are compared with group V: PB0.001 for all values. ** PB0.001, groups III and IV are compared with group II .
hepatocytes, respectively (Table 2). The maximum increase in the % viability (95.44%) and oxygen uptake (79.41%) was obtained at concentration of 100 mg/ml against Th induced toxicity. In case of Galn induced toxicity maximum % increase in % viability (73.93%) and oxygen uptake (96.16%) was observed at concentration of 1 mg/ml of p-methoxy benzoic acid. 4. Discussion The isolated compound was characterized through physicochemical and spectral data as pmethoxy benzoic acid which is reported for the first time in the plant. Pretreatment of the rats with the compound prevented toxic effects of both CCl4 and Pcl, indicated by significant % reduction in the elevated levels of SGPT, SGOT, Alkp and T.Bil. In the present study the toxicity induced by these toxicants is indicated by significant elevation in the biochemical parameters, due to liver injury and disturbances in the transport functions of hepatocytes, thus resulting in leakage of enzymes in serum.
The compound significantly restored the changed enzyme levels at the dose of 30 mg/kg b.w. i.p. The results were found comparable to silymarin (50 mg/kg b.w. i.p.). The structure of the compound shows some resemblance to paracetamol, hence it can be presumed that the compound may be acting by the mechanism of competitive inhibition of metabolism of paracetamol. The compound is metabolised completely and the metabolites are non-toxic (Tecwin, 1959). In vitro studies on the other two models confirm activity of the compound on four different models. Thioacetamide leads to induction of toxicity by altering semipermeable character of cell membrane of hepatocytes resulting in increased influx of Ca2 + and finally death (Gallgher et al., 1956). Galactosamine induces toxicity in hepatocytes which simulates the condition of viral hepatitis in humans (Keppler et al., 1968). Induction of toxicities are indicated by a significant decrease in % viability and oxygen uptake of hepatocytes. The protective effect was indicated by restoration of marked changes. The maximum protective effect was observed in concentrations of 100 mg/ml and 1 mg/ml against Th and Galn
C. Gadgoli, S.H. Mishra / Journal of Ethnopharmacology 66 (1999) 187–192
191
Table 2 Effect of different concentrations of CGCaI on % viability and oxygen uptake of rat hepatocytes intoxicated with Th and Galna Groups
Concentration (mg/ml) mean 9 % Viability, S.D. mean 9S.D.
Oxygen uptake (ml/h per mg of protein), mean9 S.D.
Control (group I) CGCaI Th (group II)b CGCaII+Th (group III) CGCaI+Th (group IV)c CGCaI+Th (group V)c Galn (group VI)c CGCaI+Galn (group VII)b CGCaI+Galn (group VIII) CGCaI+Galn (group IX)
– 1000 20 1000
98.059 1.25 95.48 9 1.25 24.739 2.54 85.08 9 1.61**
4.13 9 0.108 3.92 9 0.520 0.98 9 0.032 2.94 9 0.073**
100
93.58 9 6.60**
3.28 90.096**
10
72.88 97.88**
2.54 9 0.150**
40 1000
50.019 5.01 72.49 93.89**
1.98 9 0.053 3.99 90.031**
100
60.34 9 2.58*
–
10
52.59 9 1.27
–
a Values (% viability and oxygen uptake) are mean 9 S.D. of three readings in each group. CGCaI and Sil refer to p-methoxy benzoic acid and silymarin, respectively. b Groups II and VII are compared with I: PB0.001 c Groups IV, V, VI compared with II: PB0.001. * PB0.01, groups VIII, IX compared with VI. ** PB0.001, groups VIII, X compared with VI.
induced toxicities, respectively. The compound itself was found non-toxic at 1 mg/ml as indicated by nearly normal levels of % viability and oxygen uptake values. The findings therefore substantiate the claims made in the traditional system of medicine and incorporation of plant extract in polyherbal formulations for liver ailments in this country.
References Aldrich Library of IR Spectra (1975). Pachaurt, C.J., 2nd Edition, Aldrich Chemical Company. Bessay, O.A., Lowry, O.H., Bross, M.J., 1946. A method for rapid determination alkaline phosphatase with five cubic millilitres of serum. Journal of Biological Chemistry 164, 321 – 329. Bhagwan, B., Kandswami, 1988. Laboratory Techniques in Histology, In: Mukharjee, K. (Ed.), Medical Laboratory Technology — A Procedure Manual for Routine Diagnostic Tests, vol. 3, Tata MacGraw-Hill, New Delhi, 1124–1177. Estabrook, J.E., 1967. Mitochondrial respiratory control polarographic measurement of ADP: O ratio. Methods in Enzymology 10, 41 – 47.
Gadgoli, C.H., Mishra, S.H., 1995. Preliminary screening of Achillea millefolium, Cichorium intybus and Capparis spinosa for antihepatotoxic activity. Fitoterapia 66 (4), 319 – 323. Gallgher, C.H., Judah, J.D., Rees, K.R., 1956. Mechanism of thioacetamide toxicity. Journal of Pathology of Bacteria 72, 193 – 201. Grasselli, J.G., 1974. Atlas of Spectral Data and Physical Constants of Organic Compounds. CRC Press, Boca Raton, FL, p. 1064. Handa, S.S, Sharma, A., 1990a. Hepatoprotective activity of andrographaloid from Andrograhis paniculata. Indian Journal of Medical Research 92, 276 – 283. Handa, S.S, Sharma, A., 1990b. Hepatoprotective activity of andrographaloid against galactosamine and paracetamol intoxication in rats. Indian Journal of Medical Research 92, 284 – 292. Handa, S.S, Sharma, A., Chakraborti, K.K., 1986. Natural products and plants as liver protecting drugs. Fitoterapia 57, 307 – 349. Jendrassik, L., Groff, P., 1938. Simplified photometric method for determination of blood bilirubin. Biochemical Journal 297, 18 – 19. Keppler, D., Lesch, R., Reutler, W., Decker, K., 1968. Experimental hepatitis induced by D-galactosamine. Experimental and Molecular Pathology 9, 279 – 290.
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Randolph, K.L., Ciminera, J.L., 1980. Statistics. In: Osol, A. (Ed.), Remington’s Pharmaceutical Sciences, 16th ed. Mac Publishing, New York, pp. 104–136. Reitman, S., Frankel, S., 1957. A colorimetric method for determination of serum oxalacetic acid and glutamyl pyruvate transaminase. American Journal of Clinical Pathology 28, 56 – 58. Seglen, P.O., 1975. Preparation of isolated rat liver cells. Cell
Biology 13, 29 – 83. Tecwin, W.R., 1959. The metabolism of aromatic acids. In: Detoxication Mechanisms, 2nd ed. Chapman and Hall, London, p. 357. Visen, P.K.S., Shukla, B., Patanaik, G.K., Kaul, S., Kapoor, N.K., Dhawan, B.N., 1991. Hepatoprotective activity of Picrorrhiza kurroa on rat hepatocytes against paracetamol toxicity. Drug Development Research 22, 209 – 219.
.
Antihepatotoxic activity of p-methoxy benzoic acid from Capparis spinosa Chhaya Gadgoli, S.H. Mishra * Pharmacy Department, Faculty of Technology and Engineering, M.S. Uni6ersity of Baroda, Baroda 390001, Gujarat, India Received 5 August 1998; received in revised form 5 December 1998; accepted 6 December 1998
Abstract p-Methoxy benzoic acid isolated from the methanolic soluble fraction of the aqueous extract of Capparis spinosa L. (Capparidaceae) was found to possess significant antihepatotoxic activity against carbontetrachloride and paracetamol induced hepatotoxicity in vivo and thioacetamide and galactosamine induced hepatotoxicity in isolated rat hepatocytes, using in vitro technique. The compound was characterized through physicochemical and spectral studies. Isolation, identification and antihepatotoxic activity of the compound is reported for the first time in the plant. HPTLC analysis of methanol soluble fraction indicated that the compound constitutes 33% w/w of the active fraction. © 1999 Published by Elsevier Science Ireland Ltd. All rights reserved. Keywords: Capparis spinosa; Capparidaceae; p-Methoxy benzoic acid; Antihepatotoxic activity
1. Introduction The total aqueous extract of aerial parts of Capparis spinosa (Capparidaceae) forms one of the constituents of polyherbal formulations to treat liver ailments listed by Handa et al. (1986). The different successive and total aqueous extracts of the plant are reported to possess significant activity (Gadgoli and Mishra, 1995). Therefore the total aqueous extract of the plant
* Corresponding author.
was subjected to detailed investigations of the antihepatotoxic activity of its constituent(s) in order to justify its utility profile as antihepatotoxic agent. The present study deals with the isolation, identification of the active compound(s) and evaluation of these substances for antihepatotoxic activity on rats against carbontetrachloride (CCl4) and paracetamol (Pcl) induced toxicities in vivo and thioacetamide (Th) and galactosamine (Galn) induced toxicities in vitro. The activity of the compound against CCl4 and Pcl was compared with silymarin (50 mg/kg b.w. i.p.), a wellknown antihepatotoxic agent.
0378-8741/99/$ - see front matter © 1999 Published by Elsevier Science Ireland Ltd. All rights reserved. PII: S 0 3 7 8 - 8 7 4 1 ( 9 8 ) 0 0 2 2 9 - 3
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C. Gadgoli, S.H. Mishra / Journal of Ethnopharmacology 66 (1999) 187–192
2. Materials and methods
2.1. Plant material The aerial parts of Capparis spinosa L. Capparidaceae were procured from the Botany Department, Faculty of Science, M.S. University of Baroda, and the plant was identified by Dr. Pradeep, Taxonomy Laboratory, Botany Department.
2.2. Extraction and isolation of acti6e compound The total aqueous extract (63.35 % w/w) was prepared by decoction. The extract was evaporated under vacuum and 100 gm was fractionated into 500 ml of methanol to obtain methanol soluble (FI, 42.28 g) and methanol insoluble (FII, 54.72 g) fractions. Fraction I, being found active, was subjected to TLC studies. A total of 10 g of FI was dissolved in 50 ml methanol and chromatographed on TLC plates with silica gel G as adsorbent and benzene:acetone (9.5:0.5) as mobile phase. The TLC studies revealed the presence of three spots when observed under UV 365 hm: Rf of 0.89, pale blue fluorescent spot; Rf of 0.73, blue fluorescent spot; and Rf of 0.23, dark absorbing spot. An attempt was made to isolate all three spots using preparative TLC technique but only one spot of Rf 0.89 could be isolated in a better yield. The compound was recrystallized in methanol and subjected to physiochemical and spectral studies (UV, IR, NMR, MS). The isolated compound was characterized as p-methoxy benzoic acid based on the following findings and by comparing them with the available data (Grasselli, 1974; Aldrich Library, 1975). Colourless to cream coloured crystals of needle shape, m.p. 182–183°C, l max (MeOH) 205 and 256 hm. Soluble in methanol, chloroform, insoluble in water. C, 56; H, 6.1; N, 0; O, 27.2%. Mol. wt from MS, M + = 152, C8H8O3. FT IR 3200, 1695, 1150–1085, 1170 – 1115, 1224, 1040, 1585, 1575 –1400, cm − 1. 1H NMR 3.89, OCH3 (S), (7.94, 7.97) (d) orthocoupled H (AB), 7.26 (S), – OH, 13C NMR 51.95 ( – OCH3), 131.908 (C2, C6), 115.22 (C2, C6), 60.117 (COOH), m/e – 152
M + , 121 (OCH3), 93 (C6H5O + ). The HPTLC analysis of FI, revealed that FI contains 33% w/w of p-methoxy benzoic acid.
2.3. Animals Either sex albino rats of Wistar strain (150–200 g) were used. The animals were maintained on normal diet and tap water ad libitum with temperature 24 9 2°C, RH 40–70% and light/dark cycle of 12 h (06:00–18:00 h).
2.4. In 6i6o antihepatotoxic acti6ity testing The isolated compound p-methoxy benzoic acid was suspended in 4% aqueous acacia solution and was administered in a dose of 30 mg/kg b.w. i.p. The protocols described by Handa and Sharma (Handa and Sharma, 1990a; Handa and Sharma, 1990b) were followed for evaluation of the activity against CCl4 and Pcl induced hepatotoxicities, respectively. Silymarin, a gift sample from M/s Ranbaxy Research Laboratory, New Delhi, was suspended in 4% w/v aqueous acacia solution and administered in a dose of 50 mg/kg b.w. i.p. for antihepatotoxic testing. The three groups of rats were treated in the following manner.
2.4.1. Control Vehicle, 1 ml/kg b.w. i.p. was administered on 1st, 2nd and 3rd day+ olive oil (Oilo Sasso, Italy), 1 ml/kg b.w. s.c. on the 2nd and 3rd day, 30 min later the administration of the vehicle, for CCl4 induced hepatotoxicity. In case of Pcl induced toxicity instead of olive oil, 60% w/v sucrose solution, 1 ml/kg b.w. p.o. was administered on the 3rd day, followed 2 h later by the administration of the vehicle. 2.4.2. Toxicant Vehicle 1 ml/kg b.w. i.p. was administered on 1st, 2nd and 3rd day+toxicant, 2 ml/kg b.w. s.c of CCl4 on the 2nd and 3rd day, 30 min later the administration of vehicle while Pcl suspension, 3 g/kg b.w. p.o. was administered on the 3rd day, 2 h later the administration of the vehicle.
C. Gadgoli, S.H. Mishra / Journal of Ethnopharmacology 66 (1999) 187–192
2.4.3. Test substance (T.S.) The test substance p-methoxy benzoic acid, 30 mg/kg b.w. i.p., silymarin, 50 mg/kg b.w. i.p. on the 1st, 2nd and 3rd day +toxicants (CCl4 on 2nd and 3rd day, 30 min later the administration of the T.S. and Pcl on the 3rd day, 2 h later the administration of T.S.). CCl4 solution: 50% v/v solution in olive oil Vehicle: 4% w/v aqueous acacia solution Pcl solution: Pcl was suspended in 60% w/v aqueous sucrose solution. Blood was withdrawn on the 4th day in case of CCl4 and on the 5th day in case of Pcl induced toxicities. 2.5. Assessment of antihepatotoxic acti6ity Blood was collected from retroorbital plexus and serum was separated by centrifuging at 2000 rpm for 10 min. The activity of serum glutamyl pyruvate transaminase (SGPT) and serum glutamyl oxalacetate transaminase (SGOT) was assayed using the method as described by Reitman and Frankel (1957). The levels of alkaline phosphatase (Alkp) and total bilirubin (T.Bil) were assayed using the methods described by Bessay et al. (1946) and Jendrassik and Groff (1938), respectively The antihepatotoxic activity was confirmed through histopathological studies on liver of rats treated with toxicants as well as the compound. The liver of one rat from each group was removed and processed suitably to stain with hematoxylin-eosin (Bhagwan and Kandswami, 1988) to assess its architecture before and after treatment with the compound.
2.6. In 6itro antihepatotoxic testing In vitro studies involved isolation of hepatocytes and examination of the effect of toxicants along with the compound under investigation. The rat hepatocytes were isolated according to Seglen (1975) with slight modifications (Visen et al., 1991) by recirculating enzymatic perfusion technique (in situ). The hepatocytes thus isolated were incubated for 24 h following the protocol given below:
189
Control: 0.1 ml of hepatocyte suspension+ 0.1 ml vehicle (distilled water)+ 0.1 ml PBS (phosphate buffer saline, pH=7.4.) Toxicant: 0.1 ml of hepatocyte suspension+ 0.1 ml of toxicant (Galn 40 mg/ml, Th 20 mg/ml in distilled water)+ 0.8 ml PBS Test substance: 0.1 ml of hepatocyte suspension+ 0.1 ml of toxicant (Galn 40 mg/ml, Th 20 mg/ml in distilled water)+ 0.1 ml test substance (1000, 100, 10 mg/ml, suspended in distilled water)+ 0.7 ml PBS. Each set contained three samples.
2.7. Assessment of antihepatotoxic acti6ity Assessment of antihepatotoxic activity was done by determining % viability of hepatocytes using trypan blue dye exclusion test and oxygen uptake of hepatocytes using Geilson’s Oxygraph, described by Estabrook (1967).
2.8. Statistical analysis Analysis of variance (ANOVA) was employed for estimation of variations in a set of data followed by individual comparison within the groups using least significant square method (Randolph and Ciminera, 1980).
3. Results
3.1. In 6i6o antihepatotoxic acti6ity Administration of CCl4, and Pcl led to increase in the serum enzyme levels by two- to threefold as compared to control group. Treatment of rats with p-methoxy benzoic acid (30 mg/kg b.w. i.p.) resulted in 89.68, 105.28, 78.91, 56.55 and 137.4, 86.30, 92.91, 62.55% reductions in SGPT, SGOT, Alkp and T.Bil levels as compared to CCl4 and Pcl groups, respectively (Table 1).
3.2. In 6itro antihepatotoxic acti6ity Incubation of hepatocytes with Th and Galn resulted in 74.77, 76.27, and 51.00, 47.95% decrease in the % viability and oxygen uptake of
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Table 1 Effect of p-methoxy benzoic acid on biochemical parameters in rats intoxicated with CCl4 (2 ml/kg b.w. s.c.) and Pcl (3 gm/kg b.w. p.o.)a Groups
Control (group I) CCl4 (group II)b CGCaI+CCl4 (group III) Sil+CCl4 (group IV) Pcl (group V) CGCaI+Pcl (group VI)c Sil+Pcl (group VII)
SGPT (U/l), mean 9S.D. (% reduction)
SGOT (U/l), mean 9S.D. Alkp (U/l), mean 9 S.D. T.Bil (mg/dl), mean 9 S.D. (% reduction) (% reduction) (% reduction)
65.06 95.42
110.679 28.70
43.73 93.55
0.208 9 0.034
302.47 9 17.54
464.85943.58
188.72 912.8
2.035 9 0.438
89.08 920.83 (89.88)**
91.94 9 40.65 (105.28)**
74.30 942.02 (78.91)**
1.000 90.059 (56.65)**
78.08 96.83 (94.40)**
169.479 17.81 (83.39)**
120.67 9 8.46 (46.96)**
0.791 9 0.038 (68.08)**
124.57 916.43 47.78 98.51 (137.45)**
243.799 7.19 128.099 28.15 (86.30)**
174.50 9 30.50 53.00 9 24.80 (92.91)**
1.682 9 0.138 0.760 9 0.098 (62.55)**
63.41 9 2.67 (102.85)**
143.219 12.81 (75.55)**
106.83 9 4.62 (51.74)**
0.582 9 0.023 (74.42)**
a Values (U/l and mg/dl) are mean 9S.D. of six animals. CGCaI refers to the isolated compound p-methoxy benzoic acid. Sil refers to silymarin. b Groups II and IV are compared with group I: PB0.001. c Groups VI and VII are compared with group V: PB0.001 for all values. ** PB0.001, groups III and IV are compared with group II .
hepatocytes, respectively (Table 2). The maximum increase in the % viability (95.44%) and oxygen uptake (79.41%) was obtained at concentration of 100 mg/ml against Th induced toxicity. In case of Galn induced toxicity maximum % increase in % viability (73.93%) and oxygen uptake (96.16%) was observed at concentration of 1 mg/ml of p-methoxy benzoic acid. 4. Discussion The isolated compound was characterized through physicochemical and spectral data as pmethoxy benzoic acid which is reported for the first time in the plant. Pretreatment of the rats with the compound prevented toxic effects of both CCl4 and Pcl, indicated by significant % reduction in the elevated levels of SGPT, SGOT, Alkp and T.Bil. In the present study the toxicity induced by these toxicants is indicated by significant elevation in the biochemical parameters, due to liver injury and disturbances in the transport functions of hepatocytes, thus resulting in leakage of enzymes in serum.
The compound significantly restored the changed enzyme levels at the dose of 30 mg/kg b.w. i.p. The results were found comparable to silymarin (50 mg/kg b.w. i.p.). The structure of the compound shows some resemblance to paracetamol, hence it can be presumed that the compound may be acting by the mechanism of competitive inhibition of metabolism of paracetamol. The compound is metabolised completely and the metabolites are non-toxic (Tecwin, 1959). In vitro studies on the other two models confirm activity of the compound on four different models. Thioacetamide leads to induction of toxicity by altering semipermeable character of cell membrane of hepatocytes resulting in increased influx of Ca2 + and finally death (Gallgher et al., 1956). Galactosamine induces toxicity in hepatocytes which simulates the condition of viral hepatitis in humans (Keppler et al., 1968). Induction of toxicities are indicated by a significant decrease in % viability and oxygen uptake of hepatocytes. The protective effect was indicated by restoration of marked changes. The maximum protective effect was observed in concentrations of 100 mg/ml and 1 mg/ml against Th and Galn
C. Gadgoli, S.H. Mishra / Journal of Ethnopharmacology 66 (1999) 187–192
191
Table 2 Effect of different concentrations of CGCaI on % viability and oxygen uptake of rat hepatocytes intoxicated with Th and Galna Groups
Concentration (mg/ml) mean 9 % Viability, S.D. mean 9S.D.
Oxygen uptake (ml/h per mg of protein), mean9 S.D.
Control (group I) CGCaI Th (group II)b CGCaII+Th (group III) CGCaI+Th (group IV)c CGCaI+Th (group V)c Galn (group VI)c CGCaI+Galn (group VII)b CGCaI+Galn (group VIII) CGCaI+Galn (group IX)
– 1000 20 1000
98.059 1.25 95.48 9 1.25 24.739 2.54 85.08 9 1.61**
4.13 9 0.108 3.92 9 0.520 0.98 9 0.032 2.94 9 0.073**
100
93.58 9 6.60**
3.28 90.096**
10
72.88 97.88**
2.54 9 0.150**
40 1000
50.019 5.01 72.49 93.89**
1.98 9 0.053 3.99 90.031**
100
60.34 9 2.58*
–
10
52.59 9 1.27
–
a Values (% viability and oxygen uptake) are mean 9 S.D. of three readings in each group. CGCaI and Sil refer to p-methoxy benzoic acid and silymarin, respectively. b Groups II and VII are compared with I: PB0.001 c Groups IV, V, VI compared with II: PB0.001. * PB0.01, groups VIII, IX compared with VI. ** PB0.001, groups VIII, X compared with VI.
induced toxicities, respectively. The compound itself was found non-toxic at 1 mg/ml as indicated by nearly normal levels of % viability and oxygen uptake values. The findings therefore substantiate the claims made in the traditional system of medicine and incorporation of plant extract in polyherbal formulations for liver ailments in this country.
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