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Hepatoprotective properties of Crepis rueppellii and Anisotes trisulcus: Two traditional medicinal plants of Yemen
105
Journal ofEthnopharmacology, 16 (1986) 105-111 Elsevier Scientific Publishers Ireland Ltd.
HEPATOPROTECTIVE PROPERTIES OF CREPZS RUEPPELLZZ AND ANZSOTES TRZSULCUS: TWO TRADITIONAL MEDICINAL PLANTS OF YEMEN
J. FLEURENTINs,
C. HOEFLER*, A. LEXAs, F. MORTIERb
and J.M. PELTa
aLabomtoire de Pharmacognosie, Centre des Sciences de I’Enuironnement, Universitk de Metz, 1 rue des R kcollets 57000 Metz and bLaboratoire de Pharmacognosie, Faculte de Pharmacie, Universitd de Nancy, rue Albert Lebrun, 54000 Nancy (France) (Accepted
December 12,1985)
Summary Pharmacological investigations were carried out to evaluate the hepatoprotective effects of Crepis rueppellii and Anisotes trisulcus. Ethanolic extracts of these plants were investigated for their ability to reduce mortality of mice after ethanol intoxication and to lower the activities of plasma glutamic-pyruvic transaminase (GPT) after carbon tetrachloride-induced hepatitis in rats. Crepis and Anisotes extracts and a 50 : 50 mixture of both at 200 mg/kg presented significant hepatoprotective effects in both experimental situations. The traditional therapeutic indications of these plants have been largely confirmed.
Introduction From the traditional pharmacopoea of Yemen (Fleurentin and Pelt, 1982, 1983), two medicinal plants have been selected for pharmacological investigation baaed on botanical, chemical, pharmacological and cultural uniqueness (Fleurentin et al., 1983a,b). Crepis rueppellii Sch. Bip. (Compositae) and Anise tes trisulcus (Forssk.) Nees (Acanthaceae) are considered by traditional healers as treatments for all hepatic syndromes, including jaundice and hepatitis, and for gallstones and other hepatic disorders. Although there are no available data on their chemical composition, a previous pharmacological study has demonstrated that both extracts induce hypercholeresis with Anisotes stimulating the bile acid-dependent fraction and Crepis stimulating the bile acid-independent fraction (Lanhers et al., 1986). The purpose of the present work is to study the hepatoprotective action of these plants on two different experimental models of liver disease: an acute intoxication with ethanol in mice resulting in a well known major perturbation of a metabolic pathway leading to death (Lieber, 1977; Comporti, 1978) 0378-8741/86/$02,40 0 1986 Elsevier Scientific Publishers Ireland Ltd Published and Printed in Ireland
106
and an intoxication with carbon tetrachloride (Ccl,) in rats producing a toxic metabolite resulting in a reversible necrosis (Slater, 1978; Plaa and Hewitt, 1982). Materials and methods Plants The leaves of A. trisulcus (4 kg) and C. rueppellii (2 kg) were collected in four areas around Taez in Yemen in July and August of 1981. Initial extraction of dried plant material in a Soxhlet with chloroform allowed chlorophyll removal; this extract was eliminated. Further extraction with 95% ethanol produced an ethanol extract which was then concentrated in vacua at 40°C with a rotary evaporator. In both cases the extractions were stopped when the solvents in the Soxhlet apparatus became colourless. Doses in this study will be expressed in mg of concentrated ethanolic extract: 1 mg of Crepis extract corresponds to 9.6 mg of dried leaves and 1 mg of Anisotes extract to 7.9 mg of dried leaves. These two plants present a very low toxicity in Swiss mice: the LD5,, of Anisotes extract was 920 mg/kg i.p. while the Crepis extract had no lethal effect up to 1000 mg/kg i.p. Traditional chronic therapeutic doses in Yemen are equivalent to about 30 mg/kg daily for Crepis and 60 mg/kg daily for Anisotes for 2 weeks of therapy. Experimental doses used in the present study were determined after preliminary screening. Ethanol-induced
hepatotoxicity
Male Swiss mice (Laboratoire de Psychopharmacologic, Strasbourg, France) weighing 35-40 g were fed ad libitum (Croquettes Extralabo, Provins, France) and allowed free access to water. The animals received intraperitoneally a single injection of ethanol (6.4 g/kg in 0.9% NaCl; 10 ml/kg). The extracts of Crepis (100,200 and 400 mg/kg), Anisotes (100, 200 and 400 mg/kg) and a 50: 50 mixture of Crepis and Anisotes (100 + 100 and 200 + 200 mg/kg) were administered intraperitoneally 30 min before ethanol intoxication. The mortality was noted 48 h after ethanol administration. Carbon tetrachloride-induced
hepatotoxicity
Male SpragueDawley rats (Iffa Credo, L’arbresle, France) weighing 250300 g were allowed free access to water and food (Croquettes Extralabo, Provins, France) until CCll administration and then starved for 24 h. The animals received intraperitoneally a single injection of CC& (Rectapur, Prolabo, France) (0.3 ml/kg in olive oil; 5 ml/kg). The extracts of Crepis (100, 200 and 400 mg/kg), Anisotes (100, 200 and 400 mg/kg) and the 50 : 50 mixture of Crepis and Anisotes (100 + 100 and
107
200 + 200 mgfkg) (2 ml/kg in 0.9% NaCl) were injected ~~a~~tone~y 30 min before CCL intoxication, Silymarine (Bellon, Neuilly-so-Seine, France), a natural active constituent of Silybum marianum, administered under the same conditions at 100 mg/kg, was used as a reference product for its hepatoprotective effects. Twenty-four hours before CCL intoxication and 24 h and 48 h after intoxication, rats were slightly anesthetized intramuscularly with 100 mg/kg of ketamine (Imalgene 1000, Iffa Merieux, France) to collect 150 ~1 of blood from the orbital sinus in heparinized vials. Plasma was obtained by centrifugation (3000 X g, 5 min) and GPT activity was determined by enzymatic method of Wrobleswski and La Due (Kit Boehringer, Mannheim, F.R.G.). Plasma trilirubin was measured by the Jendrassik method (Kit Boehringer, Ma~heim, F.R.G.) and total plasma proteins by the classic Biuret method (Kit Boehringer, Mannheim, F.R.G.). Calculations Percentage mortality of treated mice was compared to percentage mortality of placebo animals using the x2 test with the Yates correction (Schwartz, 1963). Results of GPT and bilirubin are reported here as geometric meahs accompanied by a range equivalent to + 1 S.D. as calculated from the logarithms of the values (Heath, 1967). Plasma protein is expressed here as arithmetic mean values + 1 S.D. After testing for homogeneity of variance by Bartlett’s test, an analysis of variance using the Fmex test was used to indicate significant differences (P < 0.05). The means of each treated group were compared for significant differences relative to placebo using the Student’s t-test. The percentage of protection was calculated by dividing the means of placebo group minus means of treated group by the means of placebo group minus normal GPT values (20 units/l). ReSUltS
Ethanol-induced
lethality in mice
As shown in Table 1, a single dose of ethanol (6.4 g/kg) induced a mortality of 67%. A significant protective action of Crepis and Anisotes was noted when they ‘flvereadministered at 200 mg/kg; mortality was, respectively, 20% and 15% (P < 0.001). At lower or at higher doses, the hepatoprotective action of both extracts was absent. When both extracts were injected together, the protective effect was significant at 200 mg/kg (5% mortality, P < 0.001) and at 400 mg/kg (40% mortality, P < 0.05). CC&-induced hepatotoxicity
in rats
As shown in Table 2 and in Fig. 1, a single injection of CCL (0.3 ml/kg) induced 24 h later a significant increase of plasma GPT (P < 0.001) and of
108 TABLE
1
PROTECTIVE EFFECTS LETHALITY IN MICE Treatment ethanol
+
OF CREPZS AND ANZSOTES
Treatment dose (w/k)
ON ETHANOL-INDUCED
Quantal lethality
P
30145
-
-~~ Control
%repis
100 200
400 Anisotes
100
200 400 Crepis + Anisotes
100 + 100
200 + 200
9110
4120 lo/lo
8110 3120 5110 l/20 8120
NS < 0.001 NS NS < 0.001 NS
-co.05
plasma bilirubin (P < 0.05) and 48 h later a significant decrease of plasma proteins (P < 0.01). Under the same conditions, corn oil-treated rats (control group) presented normal physiological values. When Crepis (200 mg/kg), Anisotes (200 mg/kg) and a 50 : 50 mixture of Crepis and Anisotes (400 mg/kg) were administered 30 min before CCL !‘intoxication, GPT levels were significantly decreased. The hepatoprotective effect of the mixture of Anisotes and Crepis at 400 mg/kg was slightly more than the effect of silymarine, a known hepatoprotective medicine, at 100 mg/ kg. At 200 mg/kg, Crepis appeared more active than Anisotes. Plasma bilirubin was significantly decreased in rats only after treatment with the 50 : 50 mixture of Crepis and Anisotes at 400 mg/kg and after a treatment of Crepis and Anisotes at 100 mg/kg. Total plasma proteins were not modified by the administration of the extracts. Discussion Crepis and Anisotes extracts at 200 mg/kg show hepatoprotective properties against two known hepatotoxic agents, ethanol and CC14.They reduced lethality in mice after injection of 6.4 g/kg of ethanol and they prevented an increase of plasma GPT activity in rats given CCl,. At lower and at higher doses, these hepatoprotective effects were absent. Moreover, a mixture of these extracts at 200 mg/kg was more active than the plants administered separately; this indicates that both extracts may act in synergy. Previous experiments have shown that Crepis and Anisotes can induce hypercholeresis in the rat; however, the mechanisms of bile formation are different since Anisotes has been reported to stimulate the bile acid-dependent fraction and Crepiq the bile acid-independent fraction (Lanhers et al., 1986). These results
109 TABLE
2
INFLUENCE OF CREPIS AND ANISOTES EXTRACTS PROTEIN AFTER Ccl, INTOXICATION IN RATS Agents
Dose
N
(mg/kg)
Placebo
Control
-
5
Crepis
100
5
Ankotes
100
5
Placebo
-
10
Crepis
200
10
Anisotes
200
10
Placebo
Bilirubin
GPT (units/l) +24
h
ON GPT BILIRUBIN
Protein
(mg/l)
Control
+24
h
AND
(g/l)
Control
+ 48h
15 13-18 14 12-17 17 13-22
74 39-140 33 12-91 68 35-133
0.9 0.5-1.3 0.4 0.24.8 0.4 0.2-0.9
7.3 6.8-8.6 4* 2.5-5.7 5.1. 4.6-5.8
66.2
i 2.2
65.0
f 2.3
66.5
i 1.5
60.5
* 4.2
65.9
i 2.2
61.9
f 4.7
17 14-22 19 16-24 18 15-22
185 108-317 48* 29-80 87* 51--150
1.2 0.8-1.7 1.2 1.0-1.5 1.4 0.7-2.7
2.6 1.7-3.9 2.7 2.0-3.7 2.8 2.2-3.5
62.4
f 3.2
58.1
f. 2.3
64.4
f 3.8
58.3
+ 3.0
59.8
r 3.4
57.1
f 2.8
1.1 0.8-1.5
2.3 1.9-2.8
62.7
* 3.4
53.5
i 4.8
1.4 1.1-1.7 1.3 1.0-1.8 1.5 1.1-2.0
2.7 1.9-4.0 3.4 3.0-3.7 3.2 2.54.2
63.0
* 2.3
53.4
f 1.9
60.4
* 4.4
51.8
r 2.5
62.0
i 2.0
53.1
f 8.5
-
10
17 13-22
Crepis
400
5
Anisotes
400
5
Crepis + Anisotes Crepis + Anisotes
100 + 100
10
18 16-21 15 13-18 19 17-23
469 1811212 216 124-376 245 162-370 369 169-804
200 + 200
5
19 16-22
65* 40-106
1.3 0.9-1.8
1.4** l.l--1.7
60.2
zt 7.3
57.0
r 2.7
Mymarine Control
100
5
1.1 0.9-1.4 1.6 1.4-1.9
2.6 1.74.0 1.4 0.8--2.4
i 6.1
53.5
i 3.5
10
73* 11493 14 11-17
58.7
-
16 13-19 14 12-17
66.4
i 6.8
64.4
+ 4.4
Statistically
different
from
placebo:
lP < 0.01;
*P < 0.001;
**P < 0.05 N = no. of rats.
suggest that Crepis and Anisotes act on hepatocytes at different levels and through different mechanisms. Their antinecrotic action may involve differ& biochemical mechanisms which reinforce one another when they are administered simultaneously. This hypothesis cannot be correlated with specific active principles because their chemical compositions have not yet been investigated. A norsesquiterpene la&one has been identified in certain species
110
100
50
100
400
mg.
g
Fig. 1. Protective effects of Crepis, Anisotes and silymarine on plasma GFT increase after CC&-intoxication. m, silymarine;;m;?, Crepis;m, Anisotes;EX$Crepis + Anisotes. Significance relative to placebo : *P < 0.01; *P < 0 .OOl.
of Crepis (Casinovi et al., 1982) and alkaloids derived from quinazolone (Arndt et al., 1967; Gibbs, 1974) in other species. The performance of ethanolic extracts of Crepis and Anisotes at 200 mg/kg appeared to be comparable to the therapeutic activity of siiymarine at 100 mgfkg which is the active component of the seeds of Si~y~~~ ~ar~~~~~ (seeds contain about 10% of silymarine). The traditional therapeutic indications of Crepis rueppellii and Anisotes trisukus have been largely confirmed in this study. The active doses of Anisotes and Crepis are higher than the doses recognized by traditional healers, but this may represent species variation. Certainly, traditional therapeutics involves chronic treatment rather than acute treatment. Applied research confirming the effectiveness of traditional herbal treatments should be expanded since it introduces new remedies into modem therapy and represents the first step of an integration of traditional medicine into national health programs as recommended by the World Health Organization. Such research can also lay the cornerstone for a ph~ma~utic~ industry in Yemen and bring economic benefits to the country.
111
Acknowledgements The authors wish to thank “Fondation pour la Recherche MBdicale, Cornit Lorrain” for supporting the transport of plants from Yemen to France in 1981, and the Ministry of Health of Yemen Arab Republic. References Amdt, R.R., Eggers, S.H. and Jorden, A. (1967) The alkaloids of Anisotes sessiflorua C.B. Cl. (Acanthaceae). Five new 4-quinazolone alkaloids. Tetrahedron 23, 3521-3532. Casinovi, C.G., Fardella, G. and Rossi, C. (1982) 4,5-Dihydro-11-nor-11-hydroxy-A-7,11santonin, a new norsesquiterpene-lactone found in Crepis pygmaea. Planta Medica 44, 186-187. Comporti, M. (1978) Ethanol-induced liver injury. In: T.F. Slater (Ed.), Biochemical Mechanisms of Liver Injury, Academic Press, London, New York, San Francisco, pp. 469-516. Fleurentin, J. and Pelt, J.M. (1982) Repertory of drugs and medicinal plants of Yemen. Journal of Ethnopharmacology 6,86-108. Fleurentin, J. and Pelt, J.M. (1983) Additional information for a repertory of drugs and medicinal plants of Yemen. Journal of Ethnopharmacology 8, 237-243. Fleurentin, J., Mazars, G. and Pelt, J.M. (1983a) Cultural background of the medicinal plants of Yemen. Journal of Ethnopharmacology 7,183-203. Fleurentin, J., Mazars, G. and Pelt, J.M. (1983h) Additional information for the cultural background of drugs and medicinal plants of Yemen. Journal of Ethnopharmacology 8,335-344. Gibbs, R.D. (1974) Chemotaxonomy of Flowering Plants, McGill Queen’s University Press, Mont&al. Heath, D.F. (1967) Normal of log-normal: appropriate distributions. Nature 213,11591160. Lanhers, M.C., Bertrand, I., Fleurentin, J., Lehr, P.R. and Pelt, J.M. (1986) Influence of Anisostes trisulcus and Crepis rueppellii extracts on sites of bile formation in rat. Arzneimittel Forschung (Drug Research), in press. Lieher, C.S. (1977) Pathogenesis of alcoholic liver disease, an overview. In: M.M. Fischer and J.G. Rankin (Eds.), Alcohol and the Liver, Plenum Press, New York, London, pp. 197-256. Plaa, G.L. and Hewitt, W.R. (1982) Quantitative evaluation of indices of hepatotoxicity. In: G.L. Plaa and W.R. Hewitt (Eds.), Toxicology of the Liver, Raven Press, NewYork, pp. 103-120. Schwartz, D. (1963) Methodes statistiques d l’usage des mCdecins et des biologistes, Flammarion Medicine Sciences, Paris. Slater, T.F. (1978) Biochemical studies on liver injury. In: T.F. Slater (Ed.), Biochemical Mechanisms of Liver Injury, Academic Press, London, New York, San Francisco, pp. l-44.
Journal ofEthnopharmacology, 16 (1986) 105-111 Elsevier Scientific Publishers Ireland Ltd.
HEPATOPROTECTIVE PROPERTIES OF CREPZS RUEPPELLZZ AND ANZSOTES TRZSULCUS: TWO TRADITIONAL MEDICINAL PLANTS OF YEMEN
J. FLEURENTINs,
C. HOEFLER*, A. LEXAs, F. MORTIERb
and J.M. PELTa
aLabomtoire de Pharmacognosie, Centre des Sciences de I’Enuironnement, Universitk de Metz, 1 rue des R kcollets 57000 Metz and bLaboratoire de Pharmacognosie, Faculte de Pharmacie, Universitd de Nancy, rue Albert Lebrun, 54000 Nancy (France) (Accepted
December 12,1985)
Summary Pharmacological investigations were carried out to evaluate the hepatoprotective effects of Crepis rueppellii and Anisotes trisulcus. Ethanolic extracts of these plants were investigated for their ability to reduce mortality of mice after ethanol intoxication and to lower the activities of plasma glutamic-pyruvic transaminase (GPT) after carbon tetrachloride-induced hepatitis in rats. Crepis and Anisotes extracts and a 50 : 50 mixture of both at 200 mg/kg presented significant hepatoprotective effects in both experimental situations. The traditional therapeutic indications of these plants have been largely confirmed.
Introduction From the traditional pharmacopoea of Yemen (Fleurentin and Pelt, 1982, 1983), two medicinal plants have been selected for pharmacological investigation baaed on botanical, chemical, pharmacological and cultural uniqueness (Fleurentin et al., 1983a,b). Crepis rueppellii Sch. Bip. (Compositae) and Anise tes trisulcus (Forssk.) Nees (Acanthaceae) are considered by traditional healers as treatments for all hepatic syndromes, including jaundice and hepatitis, and for gallstones and other hepatic disorders. Although there are no available data on their chemical composition, a previous pharmacological study has demonstrated that both extracts induce hypercholeresis with Anisotes stimulating the bile acid-dependent fraction and Crepis stimulating the bile acid-independent fraction (Lanhers et al., 1986). The purpose of the present work is to study the hepatoprotective action of these plants on two different experimental models of liver disease: an acute intoxication with ethanol in mice resulting in a well known major perturbation of a metabolic pathway leading to death (Lieber, 1977; Comporti, 1978) 0378-8741/86/$02,40 0 1986 Elsevier Scientific Publishers Ireland Ltd Published and Printed in Ireland
106
and an intoxication with carbon tetrachloride (Ccl,) in rats producing a toxic metabolite resulting in a reversible necrosis (Slater, 1978; Plaa and Hewitt, 1982). Materials and methods Plants The leaves of A. trisulcus (4 kg) and C. rueppellii (2 kg) were collected in four areas around Taez in Yemen in July and August of 1981. Initial extraction of dried plant material in a Soxhlet with chloroform allowed chlorophyll removal; this extract was eliminated. Further extraction with 95% ethanol produced an ethanol extract which was then concentrated in vacua at 40°C with a rotary evaporator. In both cases the extractions were stopped when the solvents in the Soxhlet apparatus became colourless. Doses in this study will be expressed in mg of concentrated ethanolic extract: 1 mg of Crepis extract corresponds to 9.6 mg of dried leaves and 1 mg of Anisotes extract to 7.9 mg of dried leaves. These two plants present a very low toxicity in Swiss mice: the LD5,, of Anisotes extract was 920 mg/kg i.p. while the Crepis extract had no lethal effect up to 1000 mg/kg i.p. Traditional chronic therapeutic doses in Yemen are equivalent to about 30 mg/kg daily for Crepis and 60 mg/kg daily for Anisotes for 2 weeks of therapy. Experimental doses used in the present study were determined after preliminary screening. Ethanol-induced
hepatotoxicity
Male Swiss mice (Laboratoire de Psychopharmacologic, Strasbourg, France) weighing 35-40 g were fed ad libitum (Croquettes Extralabo, Provins, France) and allowed free access to water. The animals received intraperitoneally a single injection of ethanol (6.4 g/kg in 0.9% NaCl; 10 ml/kg). The extracts of Crepis (100,200 and 400 mg/kg), Anisotes (100, 200 and 400 mg/kg) and a 50: 50 mixture of Crepis and Anisotes (100 + 100 and 200 + 200 mg/kg) were administered intraperitoneally 30 min before ethanol intoxication. The mortality was noted 48 h after ethanol administration. Carbon tetrachloride-induced
hepatotoxicity
Male SpragueDawley rats (Iffa Credo, L’arbresle, France) weighing 250300 g were allowed free access to water and food (Croquettes Extralabo, Provins, France) until CCll administration and then starved for 24 h. The animals received intraperitoneally a single injection of CC& (Rectapur, Prolabo, France) (0.3 ml/kg in olive oil; 5 ml/kg). The extracts of Crepis (100, 200 and 400 mg/kg), Anisotes (100, 200 and 400 mg/kg) and the 50 : 50 mixture of Crepis and Anisotes (100 + 100 and
107
200 + 200 mgfkg) (2 ml/kg in 0.9% NaCl) were injected ~~a~~tone~y 30 min before CCL intoxication, Silymarine (Bellon, Neuilly-so-Seine, France), a natural active constituent of Silybum marianum, administered under the same conditions at 100 mg/kg, was used as a reference product for its hepatoprotective effects. Twenty-four hours before CCL intoxication and 24 h and 48 h after intoxication, rats were slightly anesthetized intramuscularly with 100 mg/kg of ketamine (Imalgene 1000, Iffa Merieux, France) to collect 150 ~1 of blood from the orbital sinus in heparinized vials. Plasma was obtained by centrifugation (3000 X g, 5 min) and GPT activity was determined by enzymatic method of Wrobleswski and La Due (Kit Boehringer, Mannheim, F.R.G.). Plasma trilirubin was measured by the Jendrassik method (Kit Boehringer, Ma~heim, F.R.G.) and total plasma proteins by the classic Biuret method (Kit Boehringer, Mannheim, F.R.G.). Calculations Percentage mortality of treated mice was compared to percentage mortality of placebo animals using the x2 test with the Yates correction (Schwartz, 1963). Results of GPT and bilirubin are reported here as geometric meahs accompanied by a range equivalent to + 1 S.D. as calculated from the logarithms of the values (Heath, 1967). Plasma protein is expressed here as arithmetic mean values + 1 S.D. After testing for homogeneity of variance by Bartlett’s test, an analysis of variance using the Fmex test was used to indicate significant differences (P < 0.05). The means of each treated group were compared for significant differences relative to placebo using the Student’s t-test. The percentage of protection was calculated by dividing the means of placebo group minus means of treated group by the means of placebo group minus normal GPT values (20 units/l). ReSUltS
Ethanol-induced
lethality in mice
As shown in Table 1, a single dose of ethanol (6.4 g/kg) induced a mortality of 67%. A significant protective action of Crepis and Anisotes was noted when they ‘flvereadministered at 200 mg/kg; mortality was, respectively, 20% and 15% (P < 0.001). At lower or at higher doses, the hepatoprotective action of both extracts was absent. When both extracts were injected together, the protective effect was significant at 200 mg/kg (5% mortality, P < 0.001) and at 400 mg/kg (40% mortality, P < 0.05). CC&-induced hepatotoxicity
in rats
As shown in Table 2 and in Fig. 1, a single injection of CCL (0.3 ml/kg) induced 24 h later a significant increase of plasma GPT (P < 0.001) and of
108 TABLE
1
PROTECTIVE EFFECTS LETHALITY IN MICE Treatment ethanol
+
OF CREPZS AND ANZSOTES
Treatment dose (w/k)
ON ETHANOL-INDUCED
Quantal lethality
P
30145
-
-~~ Control
%repis
100 200
400 Anisotes
100
200 400 Crepis + Anisotes
100 + 100
200 + 200
9110
4120 lo/lo
8110 3120 5110 l/20 8120
NS < 0.001 NS NS < 0.001 NS
-co.05
plasma bilirubin (P < 0.05) and 48 h later a significant decrease of plasma proteins (P < 0.01). Under the same conditions, corn oil-treated rats (control group) presented normal physiological values. When Crepis (200 mg/kg), Anisotes (200 mg/kg) and a 50 : 50 mixture of Crepis and Anisotes (400 mg/kg) were administered 30 min before CCL !‘intoxication, GPT levels were significantly decreased. The hepatoprotective effect of the mixture of Anisotes and Crepis at 400 mg/kg was slightly more than the effect of silymarine, a known hepatoprotective medicine, at 100 mg/ kg. At 200 mg/kg, Crepis appeared more active than Anisotes. Plasma bilirubin was significantly decreased in rats only after treatment with the 50 : 50 mixture of Crepis and Anisotes at 400 mg/kg and after a treatment of Crepis and Anisotes at 100 mg/kg. Total plasma proteins were not modified by the administration of the extracts. Discussion Crepis and Anisotes extracts at 200 mg/kg show hepatoprotective properties against two known hepatotoxic agents, ethanol and CC14.They reduced lethality in mice after injection of 6.4 g/kg of ethanol and they prevented an increase of plasma GPT activity in rats given CCl,. At lower and at higher doses, these hepatoprotective effects were absent. Moreover, a mixture of these extracts at 200 mg/kg was more active than the plants administered separately; this indicates that both extracts may act in synergy. Previous experiments have shown that Crepis and Anisotes can induce hypercholeresis in the rat; however, the mechanisms of bile formation are different since Anisotes has been reported to stimulate the bile acid-dependent fraction and Crepiq the bile acid-independent fraction (Lanhers et al., 1986). These results
109 TABLE
2
INFLUENCE OF CREPIS AND ANISOTES EXTRACTS PROTEIN AFTER Ccl, INTOXICATION IN RATS Agents
Dose
N
(mg/kg)
Placebo
Control
-
5
Crepis
100
5
Ankotes
100
5
Placebo
-
10
Crepis
200
10
Anisotes
200
10
Placebo
Bilirubin
GPT (units/l) +24
h
ON GPT BILIRUBIN
Protein
(mg/l)
Control
+24
h
AND
(g/l)
Control
+ 48h
15 13-18 14 12-17 17 13-22
74 39-140 33 12-91 68 35-133
0.9 0.5-1.3 0.4 0.24.8 0.4 0.2-0.9
7.3 6.8-8.6 4* 2.5-5.7 5.1. 4.6-5.8
66.2
i 2.2
65.0
f 2.3
66.5
i 1.5
60.5
* 4.2
65.9
i 2.2
61.9
f 4.7
17 14-22 19 16-24 18 15-22
185 108-317 48* 29-80 87* 51--150
1.2 0.8-1.7 1.2 1.0-1.5 1.4 0.7-2.7
2.6 1.7-3.9 2.7 2.0-3.7 2.8 2.2-3.5
62.4
f 3.2
58.1
f. 2.3
64.4
f 3.8
58.3
+ 3.0
59.8
r 3.4
57.1
f 2.8
1.1 0.8-1.5
2.3 1.9-2.8
62.7
* 3.4
53.5
i 4.8
1.4 1.1-1.7 1.3 1.0-1.8 1.5 1.1-2.0
2.7 1.9-4.0 3.4 3.0-3.7 3.2 2.54.2
63.0
* 2.3
53.4
f 1.9
60.4
* 4.4
51.8
r 2.5
62.0
i 2.0
53.1
f 8.5
-
10
17 13-22
Crepis
400
5
Anisotes
400
5
Crepis + Anisotes Crepis + Anisotes
100 + 100
10
18 16-21 15 13-18 19 17-23
469 1811212 216 124-376 245 162-370 369 169-804
200 + 200
5
19 16-22
65* 40-106
1.3 0.9-1.8
1.4** l.l--1.7
60.2
zt 7.3
57.0
r 2.7
Mymarine Control
100
5
1.1 0.9-1.4 1.6 1.4-1.9
2.6 1.74.0 1.4 0.8--2.4
i 6.1
53.5
i 3.5
10
73* 11493 14 11-17
58.7
-
16 13-19 14 12-17
66.4
i 6.8
64.4
+ 4.4
Statistically
different
from
placebo:
lP < 0.01;
*P < 0.001;
**P < 0.05 N = no. of rats.
suggest that Crepis and Anisotes act on hepatocytes at different levels and through different mechanisms. Their antinecrotic action may involve differ& biochemical mechanisms which reinforce one another when they are administered simultaneously. This hypothesis cannot be correlated with specific active principles because their chemical compositions have not yet been investigated. A norsesquiterpene la&one has been identified in certain species
110
100
50
100
400
mg.
g
Fig. 1. Protective effects of Crepis, Anisotes and silymarine on plasma GFT increase after CC&-intoxication. m, silymarine;;m;?, Crepis;m, Anisotes;EX$Crepis + Anisotes. Significance relative to placebo : *P < 0.01; *P < 0 .OOl.
of Crepis (Casinovi et al., 1982) and alkaloids derived from quinazolone (Arndt et al., 1967; Gibbs, 1974) in other species. The performance of ethanolic extracts of Crepis and Anisotes at 200 mg/kg appeared to be comparable to the therapeutic activity of siiymarine at 100 mgfkg which is the active component of the seeds of Si~y~~~ ~ar~~~~~ (seeds contain about 10% of silymarine). The traditional therapeutic indications of Crepis rueppellii and Anisotes trisukus have been largely confirmed in this study. The active doses of Anisotes and Crepis are higher than the doses recognized by traditional healers, but this may represent species variation. Certainly, traditional therapeutics involves chronic treatment rather than acute treatment. Applied research confirming the effectiveness of traditional herbal treatments should be expanded since it introduces new remedies into modem therapy and represents the first step of an integration of traditional medicine into national health programs as recommended by the World Health Organization. Such research can also lay the cornerstone for a ph~ma~utic~ industry in Yemen and bring economic benefits to the country.
111
Acknowledgements The authors wish to thank “Fondation pour la Recherche MBdicale, Cornit Lorrain” for supporting the transport of plants from Yemen to France in 1981, and the Ministry of Health of Yemen Arab Republic. References Amdt, R.R., Eggers, S.H. and Jorden, A. (1967) The alkaloids of Anisotes sessiflorua C.B. Cl. (Acanthaceae). Five new 4-quinazolone alkaloids. Tetrahedron 23, 3521-3532. Casinovi, C.G., Fardella, G. and Rossi, C. (1982) 4,5-Dihydro-11-nor-11-hydroxy-A-7,11santonin, a new norsesquiterpene-lactone found in Crepis pygmaea. Planta Medica 44, 186-187. Comporti, M. (1978) Ethanol-induced liver injury. In: T.F. Slater (Ed.), Biochemical Mechanisms of Liver Injury, Academic Press, London, New York, San Francisco, pp. 469-516. Fleurentin, J. and Pelt, J.M. (1982) Repertory of drugs and medicinal plants of Yemen. Journal of Ethnopharmacology 6,86-108. Fleurentin, J. and Pelt, J.M. (1983) Additional information for a repertory of drugs and medicinal plants of Yemen. Journal of Ethnopharmacology 8, 237-243. Fleurentin, J., Mazars, G. and Pelt, J.M. (1983a) Cultural background of the medicinal plants of Yemen. Journal of Ethnopharmacology 7,183-203. Fleurentin, J., Mazars, G. and Pelt, J.M. (1983h) Additional information for the cultural background of drugs and medicinal plants of Yemen. Journal of Ethnopharmacology 8,335-344. Gibbs, R.D. (1974) Chemotaxonomy of Flowering Plants, McGill Queen’s University Press, Mont&al. Heath, D.F. (1967) Normal of log-normal: appropriate distributions. Nature 213,11591160. Lanhers, M.C., Bertrand, I., Fleurentin, J., Lehr, P.R. and Pelt, J.M. (1986) Influence of Anisostes trisulcus and Crepis rueppellii extracts on sites of bile formation in rat. Arzneimittel Forschung (Drug Research), in press. Lieher, C.S. (1977) Pathogenesis of alcoholic liver disease, an overview. In: M.M. Fischer and J.G. Rankin (Eds.), Alcohol and the Liver, Plenum Press, New York, London, pp. 197-256. Plaa, G.L. and Hewitt, W.R. (1982) Quantitative evaluation of indices of hepatotoxicity. In: G.L. Plaa and W.R. Hewitt (Eds.), Toxicology of the Liver, Raven Press, NewYork, pp. 103-120. Schwartz, D. (1963) Methodes statistiques d l’usage des mCdecins et des biologistes, Flammarion Medicine Sciences, Paris. Slater, T.F. (1978) Biochemical studies on liver injury. In: T.F. Slater (Ed.), Biochemical Mechanisms of Liver Injury, Academic Press, London, New York, San Francisco, pp. l-44.