Ja~~~~ of ~~~~o~ha~acolog~, 29 (1990) 207 - 211 Elsevier Scientific Publishers Ireland Ltd.
207
PROTECTIVE EFFECTS OF GA~C~~~A KOLA SEED EXTRACT AGAINST PARACETAMOL-INURED HEPATOTOXICITY IN RATS
ALADE AKINTONWA and AN1 R. ESSIEN Toxicology unit, Lkpartment Lagos unaged
of Pharmacology,
College
of Medicirae,
~~~veTs~ty of
Lagos,
(Accepted January 4, 19901
The hepatoproteetive effect of GarcinM kolu seed extract was investigated in rats treated with high doses of paracetamol. The extracts when administered at 100 mg/kg three times a day for five consecutive days reduced paracetamol- (800, 1000, 1200 mg,kg) induced lethality from 50, 90 and 100% to 0, 20 and 40%, respectively. There was a significant reduction in the liver enzymes SGOT and SGPT and histology scores. The hepatoprote~tive effect of the extract may be due to inhibition of cytochrome P-450 which normally converts paracetamol to the toxic intermediate metabolite N-aeetyl-p-benzoquinoneimine (NAP&I).
Introduction
Paracetamol (aeetaminophenl is widely used as a non-narcotic analgesic and antipyretic agent. Its antipyretie and analgesic properties have been widely abused. Indiscriminate ingestion can lead to accidental poisoning and potentially lethal hepatotoxicity (Prescott et al., 19711. Several compounds such as i%aeetyl-cysteine, methionine, dithioearb and, recently, propylthiouracil have been shown to decrease paraeetamoi-induced hepatotoxicity (Mitchell et al., 19731. Seeds of Gurc~~~ ~0~ Iieekel (Fam. Guttiferae; synonmy: Cola u~~~~u~u Schott Endl.1 are believed to have a general antidotal effect in folk medicine in Africa. Known as “bitter kola” or false kola, the seeds are believed to possess aphrodisiac properties and have been used for the treatment of catarrh and abdominal colicky pain. In addition, their use is believed to improve the singing voice and relieve cough (Irvine, 19611. Extracts of the plant have been shown to be effective as antihepatotoxi~ agents (fwu and Xgboboko, 19821.Kolaviron, a fraction of the defatted ethanol Published and Printed in Ireland
208
extract, and two biflavones of the kola seeds (GBl and GB2) significantly modify the hepatotoxicity actions of carbon tetrachloride, galactosamine, samanitin and phalloidin (Iwu, 19851. One of the problems of the management of paracetamol poisoning in this country is the non-availability of methionine and other recommended sulfhydryl-containing compounds. In an attempt to find a possible substitute for treatment of paracetamol-induced hepatotoxicity, the effect of the kolaviron extract of G. kola was investigated on paracetamol-induced hepatotoxicity in rats. Materials and methods Plant materials Coarsely powdered G. kola seeds (3.2 kg) were successively extracted with light petroleum spirits (b.p. 40-60°1, acetone and distilled water using a Soxhlet extractor (Iwu and Igboboko, 19821. The acetone extract was partitioned between chloroform and water. The aqueous fraction was further extracted with n-butanol until the aqueous layer gave a negative aluminum chloride test. The organic fractions were concentrated under reduced pressure in a rotary evaporator to a golden residue, here termed kolaviron (65% yield) (Iwu, 19851. Animal toxicity studies Male Wistar rats weighing 200-230 g obtained from the Animal House of the College of Medicine, University of Lagos, were used for all experiments. The animals were allowed animal chow (Pfizer Nigeria Ltd., Lagos) and water ad libitum. Paracetamol (Sigma Chemical Company, St. Louis MO) was prepared as a supersaturated solution (50 mglmll in distilled water at 40°C. The animals were divided into seven groups, each consisting of 10 rats. Groups I, II, III and IV were administered 0.9% saline (10 ml/kg) orally three times daily for 5 consecutive days prior to the oral administration of a single dose of paracetamol (600, 800, 1000 or 1200 mglkg, respectively). Kolaviron was suspended in normal saline with 4% Tween 20 and administered orally (100 mglkgl three times a day for 5 consecutive days to the animals in groups V, VI and VII. An hour after the last dose of kolaviron, animals in groups V, VI and VII were administered a single oral dose of paracetamol (800, 1000 or 1200 mg/kg, respectively). The animals were observed for lethality for a 24-h period. Blood samples were obtained 6 h post-treatment by cardiac puncture using sterile disposable syringes. Serum glutamic oxaloacetic transaminase @GOT) and serum glutamic pyruvic transaminases (SGPTl were determined by the methods of Tuchweber et al. (19791. Sections of the liver were obtained from six randomly selected animals in each group. The sections were fixed in 10% formalin and embedded in paraffin and stained with hematoxylin and eosin for histological studies. The histological sections were examined under a light microscope and the extent of
209
necrosis graded as follows: histologically normal sections (01, minimal centrilobular involvement ( + 11,extensive necrosis confined to centrilohular region (+ 21, necrosis extending from central zones to portal triad (+3), massive necrosis of most hepatocytes ( + 41. Results and discussion
Table 1 shows the effect of kolaviron on paracetamol-induced lethality. Kolaviron premeditation significantly reduced paracetamol-induced mortality. Table 2 shows the effect of kolaviron on SGOT and SGPT levels in rats similarly treated with toxic doses of paracetamol. The results show that animals pretreated with the extract for five consecutive days before administration of toxic doses of paracetamol had SGOT and SGPT blood levels reduced significantly relative to comparable animals not receiving the extract. The SGOT levels were reduced by an average of 30% while SGPT levels were down about 34%. The hepatic necrosis score was reduced from +3to +lor +2. The present study shows that an extract of Garciniu kolu seed can protect against paracetamol-induced lethality and hepatotoxicity in rats. Several workers have indicated that Garcinia koh extract may have antihepatotoxic properties (Decker and Kepler, 1972; Iwu and Igboboko, 1982; Iwu, 1984, 19851. The protective action of kolaviron against galactosamine-induced hepatotoxicity may be of clinical importance since galactosamine-induced
TABLE EFFECT TAMOL
I OF GARCZNZA KOLA
EXTRACT
(KOLAVIRON)
Treatment
I
0.9oh Saline (30 ml/kg/day for 5 days) + paracetamol (600 mg/kg) 0.9% Saline (30 ml/kg/day for 5 days) + paracetamol(800 mg/kg) 0.90/b Saline (30 ml/kg/day for 5 days) + paracetamol(1000 mg/kg) 0.9% Saline (30 ml/kg/day for 5 days) + paracetamol(1200 mg/kg) Extract (300 mglkglday for 5 days) + paracetamol(800 mg/kg) Extract (300 mglkglday for 5 days) + paracetam010000 mg/kg) Extract (300 mglkglday for 5 days) + paracetamol(1200 mg/kg)
III IV V VI VII
ON PARACE-
Lethality (%)
Group
II
PREMEDICATION
LETHALITY
x* analysis of data (groups II-
(total dosage)
IV vs. V-VII):
P < 0.01. N per
0 50 90 100 0 20 40
group
=
10.
210 TABLE
2
MEAN + S.E.M. EFFECT SGOT AND SGPT LEVELS
OF GARCINIA KOLA EXTRACT (300 mg/kg/day IN RATS PRETREATED WITH PARACETAMOL
Group
Treatment
SGOT (Units/l)
C I II III V VI VII
Saline control Saline + paracetamol (600 mg/kgl Saline + paracetamol (800 mglkgl Saline + paracetamol(1000 mglkgl Extract + paracetamol (800 mglkgl Extract + paracetamol (1000 mg/kgl Extract + paracetamol(1200 mglkgl
135 1266 1824 1990 921 1067 1295
f 2 + + 2 f +
for 5 days) ON
SGPT 0Jnitslll 9 42 28 48 24 20* 29*
Student’s t-test analysis of data: *P < 0.01. N per treatment which were based on 6 randomly selected rats per group.
=
4225 806 f 1056 f 1302 f 553 f 670 + 859 -c 10, except
Histology score 22 35 17 12 19* 22*
2.0 3.0 3.0 0.0 1.1 1.0
histology
f 0 f 0 f 0 -r- 0 r?r.0 f 0 scores
hepatotoxicity simulates the lesions of viral hepatitis in humans (Decker and Keppler, 19721. The protective effect of kolaviron was believed to be due to its membrane stabilization properties (Iwu, 19851. The cytotoxicity of paracetamol is believed to be mediated by N-acetyl-pbenzoquinoneimine (NAP&I), a reactive metabolite formed by cytochrome P450 mixed-function oxidase (Mitchell et al., 1973, 1974, 19811. NAP&I, a strong electrophile and oxidizing agent, is normally detoxified by reduced if intracellular glutathione becomes glutathione in the liver. However, depleted after paracetamol overdosage, covalent binding to nucleophilic cell macromolecules results, and leads to cell death. The protective effect of kolaviron against the lethality and hepatotoxicity induced by paracetamol may be due to inhibition or reduction of the production of the reactive hepatotoxic intermediate metabolite NAP&I. It is postulated that the effect of kolaviron on paracetamol toxicity may be due to the inhibition of the cytochrome P-450 mixed-function oxidase system. Management of paracetamol poisoning is still a major medical problem in developing countries. Exogeneous glutathione is expensive and permeates cell membrane poorly. Cysteamine (mercaptaminel, an effective protective agent, inhibits NAPQI formation but causes nausea, vomiting and drowsiness. Acetylcysteine, methionine and other agents have proved useful in paracetamol poisoning. Unfortunately, most of these therapeutic agents are not available in this region, thereby necessitating the need for an alternative treatment. It appears that Garcinia kolu extract may be an effective alternative. References Decker, K. and Keppler, D. (1972) Galactosamine-induced liver injury. In: H. Popper and F. Schaffner (Eds.1, Progress in Liver Disease, Vol. 4, Grune and Stratton, New York, pp. 183199.
211 Irvine, F.R. (1961) Woody Plants of Ghana, Oxford University Press, Oxford, pp. 146- 147. Iwu, M.M. and Igboboko, A.O. (1982) Constituents of Garcinia kola seeds. Journul of Natural Products 45,650-651. Iwu, M.M. (1984) Antihepatotoxicity of biflavonoids of Garcinia koiu 92nd Ann& Congress for Medicinal Plant Research, Antwerp, Belgium, Abstract 248, p. 61. Iwu, M.M. (1985) Antihepatotoxic constituents of Garcinia kolu seeds. Experientiu 41, 699-700. Mitchell, J.R., Jollow, D.J. and Potter, W.Z. (1973) Acetaminophen-induced hepatic necrosis. I: Role of drug metabolism. Journal of Pharmacology and Experimental Therapeutics 187, 185- 194. Mitchell, J.R., Thorgeirson, S.S. and Potter, W.Z. (1974) Acetaminophen-induced hepatic necrosis. II: Role of covalent binding in vivo. Journal of Pharmacology and Experimental Therapeutics 16,676-684. Mitchell, M.C., Schenker, S. and Avant, G.R. (1981) Cimetidine protects against acetaminophen hepatotoxicity in rats. Gastroenterology 81, 1051- 1060. Presscott, L.F., Wright, W. and Roswe, P. (1971) Plasma paracetamol half-life and hepatic necrosis in patients with paracetamol overdosage. Lancet 1, 519-522. Tuchweber, B., Sieck, R. and Trost, W. (1979) Prevention by sibybin of phalloidin-induced acute hepatotoxicity. Toxicology and Applied Pharmacology 51,265275.
207
PROTECTIVE EFFECTS OF GA~C~~~A KOLA SEED EXTRACT AGAINST PARACETAMOL-INURED HEPATOTOXICITY IN RATS
ALADE AKINTONWA and AN1 R. ESSIEN Toxicology unit, Lkpartment Lagos unaged
of Pharmacology,
College
of Medicirae,
~~~veTs~ty of
Lagos,
(Accepted January 4, 19901
The hepatoproteetive effect of GarcinM kolu seed extract was investigated in rats treated with high doses of paracetamol. The extracts when administered at 100 mg/kg three times a day for five consecutive days reduced paracetamol- (800, 1000, 1200 mg,kg) induced lethality from 50, 90 and 100% to 0, 20 and 40%, respectively. There was a significant reduction in the liver enzymes SGOT and SGPT and histology scores. The hepatoprote~tive effect of the extract may be due to inhibition of cytochrome P-450 which normally converts paracetamol to the toxic intermediate metabolite N-aeetyl-p-benzoquinoneimine (NAP&I).
Introduction
Paracetamol (aeetaminophenl is widely used as a non-narcotic analgesic and antipyretic agent. Its antipyretie and analgesic properties have been widely abused. Indiscriminate ingestion can lead to accidental poisoning and potentially lethal hepatotoxicity (Prescott et al., 19711. Several compounds such as i%aeetyl-cysteine, methionine, dithioearb and, recently, propylthiouracil have been shown to decrease paraeetamoi-induced hepatotoxicity (Mitchell et al., 19731. Seeds of Gurc~~~ ~0~ Iieekel (Fam. Guttiferae; synonmy: Cola u~~~~u~u Schott Endl.1 are believed to have a general antidotal effect in folk medicine in Africa. Known as “bitter kola” or false kola, the seeds are believed to possess aphrodisiac properties and have been used for the treatment of catarrh and abdominal colicky pain. In addition, their use is believed to improve the singing voice and relieve cough (Irvine, 19611. Extracts of the plant have been shown to be effective as antihepatotoxi~ agents (fwu and Xgboboko, 19821.Kolaviron, a fraction of the defatted ethanol Published and Printed in Ireland
208
extract, and two biflavones of the kola seeds (GBl and GB2) significantly modify the hepatotoxicity actions of carbon tetrachloride, galactosamine, samanitin and phalloidin (Iwu, 19851. One of the problems of the management of paracetamol poisoning in this country is the non-availability of methionine and other recommended sulfhydryl-containing compounds. In an attempt to find a possible substitute for treatment of paracetamol-induced hepatotoxicity, the effect of the kolaviron extract of G. kola was investigated on paracetamol-induced hepatotoxicity in rats. Materials and methods Plant materials Coarsely powdered G. kola seeds (3.2 kg) were successively extracted with light petroleum spirits (b.p. 40-60°1, acetone and distilled water using a Soxhlet extractor (Iwu and Igboboko, 19821. The acetone extract was partitioned between chloroform and water. The aqueous fraction was further extracted with n-butanol until the aqueous layer gave a negative aluminum chloride test. The organic fractions were concentrated under reduced pressure in a rotary evaporator to a golden residue, here termed kolaviron (65% yield) (Iwu, 19851. Animal toxicity studies Male Wistar rats weighing 200-230 g obtained from the Animal House of the College of Medicine, University of Lagos, were used for all experiments. The animals were allowed animal chow (Pfizer Nigeria Ltd., Lagos) and water ad libitum. Paracetamol (Sigma Chemical Company, St. Louis MO) was prepared as a supersaturated solution (50 mglmll in distilled water at 40°C. The animals were divided into seven groups, each consisting of 10 rats. Groups I, II, III and IV were administered 0.9% saline (10 ml/kg) orally three times daily for 5 consecutive days prior to the oral administration of a single dose of paracetamol (600, 800, 1000 or 1200 mglkg, respectively). Kolaviron was suspended in normal saline with 4% Tween 20 and administered orally (100 mglkgl three times a day for 5 consecutive days to the animals in groups V, VI and VII. An hour after the last dose of kolaviron, animals in groups V, VI and VII were administered a single oral dose of paracetamol (800, 1000 or 1200 mg/kg, respectively). The animals were observed for lethality for a 24-h period. Blood samples were obtained 6 h post-treatment by cardiac puncture using sterile disposable syringes. Serum glutamic oxaloacetic transaminase @GOT) and serum glutamic pyruvic transaminases (SGPTl were determined by the methods of Tuchweber et al. (19791. Sections of the liver were obtained from six randomly selected animals in each group. The sections were fixed in 10% formalin and embedded in paraffin and stained with hematoxylin and eosin for histological studies. The histological sections were examined under a light microscope and the extent of
209
necrosis graded as follows: histologically normal sections (01, minimal centrilobular involvement ( + 11,extensive necrosis confined to centrilohular region (+ 21, necrosis extending from central zones to portal triad (+3), massive necrosis of most hepatocytes ( + 41. Results and discussion
Table 1 shows the effect of kolaviron on paracetamol-induced lethality. Kolaviron premeditation significantly reduced paracetamol-induced mortality. Table 2 shows the effect of kolaviron on SGOT and SGPT levels in rats similarly treated with toxic doses of paracetamol. The results show that animals pretreated with the extract for five consecutive days before administration of toxic doses of paracetamol had SGOT and SGPT blood levels reduced significantly relative to comparable animals not receiving the extract. The SGOT levels were reduced by an average of 30% while SGPT levels were down about 34%. The hepatic necrosis score was reduced from +3to +lor +2. The present study shows that an extract of Garciniu kolu seed can protect against paracetamol-induced lethality and hepatotoxicity in rats. Several workers have indicated that Garcinia koh extract may have antihepatotoxic properties (Decker and Kepler, 1972; Iwu and Igboboko, 1982; Iwu, 1984, 19851. The protective action of kolaviron against galactosamine-induced hepatotoxicity may be of clinical importance since galactosamine-induced
TABLE EFFECT TAMOL
I OF GARCZNZA KOLA
EXTRACT
(KOLAVIRON)
Treatment
I
0.9oh Saline (30 ml/kg/day for 5 days) + paracetamol (600 mg/kg) 0.9% Saline (30 ml/kg/day for 5 days) + paracetamol(800 mg/kg) 0.90/b Saline (30 ml/kg/day for 5 days) + paracetamol(1000 mg/kg) 0.9% Saline (30 ml/kg/day for 5 days) + paracetamol(1200 mg/kg) Extract (300 mglkglday for 5 days) + paracetamol(800 mg/kg) Extract (300 mglkglday for 5 days) + paracetam010000 mg/kg) Extract (300 mglkglday for 5 days) + paracetamol(1200 mg/kg)
III IV V VI VII
ON PARACE-
Lethality (%)
Group
II
PREMEDICATION
LETHALITY
x* analysis of data (groups II-
(total dosage)
IV vs. V-VII):
P < 0.01. N per
0 50 90 100 0 20 40
group
=
10.
210 TABLE
2
MEAN + S.E.M. EFFECT SGOT AND SGPT LEVELS
OF GARCINIA KOLA EXTRACT (300 mg/kg/day IN RATS PRETREATED WITH PARACETAMOL
Group
Treatment
SGOT (Units/l)
C I II III V VI VII
Saline control Saline + paracetamol (600 mg/kgl Saline + paracetamol (800 mglkgl Saline + paracetamol(1000 mglkgl Extract + paracetamol (800 mglkgl Extract + paracetamol (1000 mg/kgl Extract + paracetamol(1200 mglkgl
135 1266 1824 1990 921 1067 1295
f 2 + + 2 f +
for 5 days) ON
SGPT 0Jnitslll 9 42 28 48 24 20* 29*
Student’s t-test analysis of data: *P < 0.01. N per treatment which were based on 6 randomly selected rats per group.
=
4225 806 f 1056 f 1302 f 553 f 670 + 859 -c 10, except
Histology score 22 35 17 12 19* 22*
2.0 3.0 3.0 0.0 1.1 1.0
histology
f 0 f 0 f 0 -r- 0 r?r.0 f 0 scores
hepatotoxicity simulates the lesions of viral hepatitis in humans (Decker and Keppler, 19721. The protective effect of kolaviron was believed to be due to its membrane stabilization properties (Iwu, 19851. The cytotoxicity of paracetamol is believed to be mediated by N-acetyl-pbenzoquinoneimine (NAP&I), a reactive metabolite formed by cytochrome P450 mixed-function oxidase (Mitchell et al., 1973, 1974, 19811. NAP&I, a strong electrophile and oxidizing agent, is normally detoxified by reduced if intracellular glutathione becomes glutathione in the liver. However, depleted after paracetamol overdosage, covalent binding to nucleophilic cell macromolecules results, and leads to cell death. The protective effect of kolaviron against the lethality and hepatotoxicity induced by paracetamol may be due to inhibition or reduction of the production of the reactive hepatotoxic intermediate metabolite NAP&I. It is postulated that the effect of kolaviron on paracetamol toxicity may be due to the inhibition of the cytochrome P-450 mixed-function oxidase system. Management of paracetamol poisoning is still a major medical problem in developing countries. Exogeneous glutathione is expensive and permeates cell membrane poorly. Cysteamine (mercaptaminel, an effective protective agent, inhibits NAPQI formation but causes nausea, vomiting and drowsiness. Acetylcysteine, methionine and other agents have proved useful in paracetamol poisoning. Unfortunately, most of these therapeutic agents are not available in this region, thereby necessitating the need for an alternative treatment. It appears that Garcinia kolu extract may be an effective alternative. References Decker, K. and Keppler, D. (1972) Galactosamine-induced liver injury. In: H. Popper and F. Schaffner (Eds.1, Progress in Liver Disease, Vol. 4, Grune and Stratton, New York, pp. 183199.
211 Irvine, F.R. (1961) Woody Plants of Ghana, Oxford University Press, Oxford, pp. 146- 147. Iwu, M.M. and Igboboko, A.O. (1982) Constituents of Garcinia kola seeds. Journul of Natural Products 45,650-651. Iwu, M.M. (1984) Antihepatotoxicity of biflavonoids of Garcinia koiu 92nd Ann& Congress for Medicinal Plant Research, Antwerp, Belgium, Abstract 248, p. 61. Iwu, M.M. (1985) Antihepatotoxic constituents of Garcinia kolu seeds. Experientiu 41, 699-700. Mitchell, J.R., Jollow, D.J. and Potter, W.Z. (1973) Acetaminophen-induced hepatic necrosis. I: Role of drug metabolism. Journal of Pharmacology and Experimental Therapeutics 187, 185- 194. Mitchell, J.R., Thorgeirson, S.S. and Potter, W.Z. (1974) Acetaminophen-induced hepatic necrosis. II: Role of covalent binding in vivo. Journal of Pharmacology and Experimental Therapeutics 16,676-684. Mitchell, M.C., Schenker, S. and Avant, G.R. (1981) Cimetidine protects against acetaminophen hepatotoxicity in rats. Gastroenterology 81, 1051- 1060. Presscott, L.F., Wright, W. and Roswe, P. (1971) Plasma paracetamol half-life and hepatic necrosis in patients with paracetamol overdosage. Lancet 1, 519-522. Tuchweber, B., Sieck, R. and Trost, W. (1979) Prevention by sibybin of phalloidin-induced acute hepatotoxicity. Toxicology and Applied Pharmacology 51,265275.
