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ex vivo hepatoprotective activity of medicinal plants on rat hepatocytos
lyl)-~~yl~o~alpha-[2-(4-fluorophenoxy~~yl]-l-pipe~~~e~~ol) and R 59494 (N-methyl-N-[l+-phenoxy-butyl)-3-pyrrolidinylE2-~~o~~ol~e) veratridine was used to induce an intracellular Na overload and the a~m~~ng “Ca movements were detected. In the isolated rat aorta veratridine (10m6 - 10m4 M) dose-dependently increased the 4SCa uptake maximally by above unstimulated “‘Ca uptake. The veratridine-induced “‘Ca uptake was abolished by tetrodotoxin (10B6 M) but remaind unaffected by amiloride (10m5 - 10s3 M). Nifedipine (lo-’ - 10q6 M) and verapamil (lo-’ - 10s5 M) diminished the veratridine (10e4 M)-induced 45Ca uptake maximally by 60% whereas the depolarization-induced 45Ca uptake was completely blocked at lower concentration ranges of 10m9 - 10s6 M and lo-* - lo-’ M, respectively. R 56865 inhibited the veratridine-induced %a uptake in the concentration range of lo-* - lo-’ M maximally by 70%. The K-induced 45Ca uptake was attenuated at higher concentrations (lo-’ - lo-’ M). R 70608 (lo-* - lo-’ M) blocked the veratridine-induced 45Ca uptake completely, although the K-induced 45Ca uptake was diminished in the concentration range lo-’ to lo-’ M with a maximal effect of 70%. In electrically driven (1 I-Ix) isolated left atria of the rat veratridine (ItY4 M) induced an increase in 4sCa uptake which is accompanied by mechanical dysfunction. In the presence of veratridine the 45Ca uptake amounted to 250% of the unstimulated 45Ca uptake. Tetrodotoxin (10s6 M) totally abolished the veratridine-induced 45Ca uptake, whereas amiloride (6 x low3 M) was ineffective. Nifedipine (lo-’ M) exhibited no effect on veratridine-induced “Ca uptake. verapamil (lo-’ - 10B6 M) suppressed the veratridine-induced 45Ca uptake, however the unstimulated “Ca uptake was significantly diminished in the same concentration range. R 70608 (10T5 M), R 56865 (lo-* - 10m5 M) and R 59494 (lo-9 - lo-’ M) completely blocked the veratridine-induced 45Ca uptake without influencing the unstimulated 45Ca uptake. In conclusion, in the isolated aorta and the isolated left atrium of the rat veratridine induces a Na overload which is accompanied by a 45Ca uptake. The pathway by which Ca enters the cell is distinct from the L-Type Ca channel and a major contribution of the Ma/Ca exchanger can be excluded. R 56865 and R 59494 effectively suppressed the veratridine-induced“Ca uptake, but exhibited no influence on unstimulated 45Ca uptake. It is suggested, that R 70608, R 56865 and R 59494 exert their protective effects by inhibiting cellular Na and Ca overload.
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Dhawan, B.N., Visen, P.K.S., Kapoor, N.K. and Patnaik, G.K. ICMR Centre for Advanced Pharmacological Research on Traditiomd Remedies, Central Drug Research Institute, Chattar Manril, Lucknow 226 001, ?ndia
Hepatocytes offer a good model for in vitro evaluation of hepatoprotective agents. The medicinal plants, however, have to be initially tested as crude extracts which makes in vitro testmg difficult due to nonspecific effects and insoluble nature of some of the constituents. Hence an ex uiuo model has been standardised and successfully used to evaluate hepatoprotective activity of plants used in Ayurvedic system of medicine. Silymarin has been used as a standard for comparison. Rats were pretreated orally for 5-7 days with the test compound or extract before being sacrificed. Hepatotoxicity was induced by paracetamol(2 g/kg p.o.), or thioacetamide (200 mg/kg subcut.). The hepatocytes were prepared by standard procedures following collagenase perfusion of the liver. The effect of hepatotoxic or protective agents was assessed in term of changes in viability (evaluated by Trypan blue exclusion and oxygen uptake tests), levels of selected enzymes (the transaminases-GOT and GRT- and alkaline phosphatase) and specific binding of I’25-low density lipoproteins (LDL). Crude extracts of Picrorhiza kurrooa, (root and rhizome). Phyilanthus amarus (aerial parts) and Ricinus communis (leaf) have been screened. In addition Picroliv (a standardised active fraction from P.kurrooa), n-demethyl-ricinin (from Rcommunis), andrographolide (from Andrographis paniculata) and sdymarin (from S&bum marianum) were also tested. Four groups of 5 animals e;rch were en@ -,oyed in each experiment, one group served as the control, the second received the test agent, the third was treated with the hepatotoxin and the fourth was administered the test drug before the hepatotoxin. Wherever necessary, the serum levels of the enzymes were also monitored.
607
In normal animals > 95% hepatocytes could be isolated in a viable form. The oxygen uptake aas 4.3 u litre/hr, GOT 0.42 units, GPT 0.73 units and alkaline phosphate activity 0.17 units per mg protein respectively. Treatment with the hepatotoxic agents reduced the viable cell count by 4650% accompanied with 50-70% decrease in enzyme activities of hepatocytes and similar elevation in serum enzyme levels. Significant dose related protection %as observed with crude extracts of the 3 plants against the deleterious effects of both the hepatotoxic agents in the hepatocyte and serum parameters; Picrorhizu kurroou being the most potent and Ricinus communis having lowest activity. Among the purified preparations andrographolide and Picroliv were most effective (upto 0.75 mg/kg) followed by n-demethyl ricinin (3 mg/kg) and silymarin (6 mg/kg). Picroliv and silymarin were also found effective against similar damage with galactosamine (400 mg/kg) but a higher dose was required. Picroliv also antagonised paracetamol induced decrease in specific binding of Ii2’-LDL to isolated hepatocytes. Further, the biochemical effects of Picroliv collaborated well with protection observed histologically. The technique offers excellent possibility for assessment of hepatoprotective effect of natural products at various stages of chemical fractionation as well as of purified active constituents.
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Teng, C.-M., Ko, F.-N., Yu, S.-M., Chen *, C.-C., Wu * *, T.-S. and Lin * * *, C.-N. Pharmacologicalinstitute, College of Medicine, National Taiwan University, Taipei, * National institute of ChineseMedicine, Taipei, * * Department of Chemistry National Cheng-Kong University, Tainan and * * “‘Schoolof Pharmacy, KuohsiungMedical College, Kuohsiung, Taiwan, People’s Republic of China
A large scale screening test of the vasorelaxing actions of the extracts from Chinese herbs has been performed and the active principles were isolated: osthole from Angelica pubescens. protopine from Corydalis tubers, norathyriol from Gentianaceae, denudatin B and fargesone B from Magnolia fargesii, apigenin from Apium graveolens, and magnolol from Magnolia officinales. In this experiment, we tried to study the effects of these Chinese herbal principles on the vasoconstriction of rat thoracic aorta induced by high potassium solution, norepinephrine (NE) and caffeine, and to elucidate their modes of action. All the seven principles suppressed the contraction of rat thoracic aortic rings caused by cumulative concentrations of calcium (0.03-3 mM) in high potassium (60 mM) medium. They also relaxed the NE (3 PM)-induced contraction, and their pretreatment inhibited both the phasic and tonic contractions elicited by NE in the absence or presence of calcium (1.9 mN) in the medium. Indomethacin (20 FM) did not have any antagonizing effect on these relaxations. Osthole and magnolol also inhibited caffeine (10 mM)-induced phasic contraction, -while others did not have any effect. In qrv’n-Zloaded cultured rat vascular smooth muscle cells, NE-indt:. -od rises of intracellular calcium were suppressed by denudatin B and fargesone B. 45Ca++ uptakes induced by either NE or high potassium were inhibited by the seven vasorelaxants in a concentration-dependent manner. All these vasorelaxants did not change the levels of prostacyclin or CAMP of rat aorta. Both osthole and magnolol caused formation of cGMP in aorta, while other vasorelaxants did not have any effect or were effective only at higher concentrations than those relaxing the muscle. The cGMP increase and initial relaxation caused by magnolol were disappeared after the aorta was denuded. This endothelium-dependent relaxation by magnnolol was antagonized by methylene blue (50 PM) and hemoglobin (10 PM) which were known to inhibit activation of guanylate cyclase and inactivate endothelium-derived relaxing factor (EDBF), respectively. In conclusion, seven vasorelaxants isolated from Chinese herbs inhibited vascular muscle contraction of rat aorta by suppressing the calcium influx through both voltage-gated and receptor-operated calcium channels. EDRE release by magnolol and cGMP increase by osthole may enhance their relaxing effects in rat aorta.
t
rote&e
activity
0
Dhawan, B.N., Visen, P.K.S., Kapoor, N.K. and Patnaik, G.K. ICMR Centre for Advanced Pharmacological Research on Traditiomd Remedies, Central Drug Research Institute, Chattar Manril, Lucknow 226 001, ?ndia
Hepatocytes offer a good model for in vitro evaluation of hepatoprotective agents. The medicinal plants, however, have to be initially tested as crude extracts which makes in vitro testmg difficult due to nonspecific effects and insoluble nature of some of the constituents. Hence an ex uiuo model has been standardised and successfully used to evaluate hepatoprotective activity of plants used in Ayurvedic system of medicine. Silymarin has been used as a standard for comparison. Rats were pretreated orally for 5-7 days with the test compound or extract before being sacrificed. Hepatotoxicity was induced by paracetamol(2 g/kg p.o.), or thioacetamide (200 mg/kg subcut.). The hepatocytes were prepared by standard procedures following collagenase perfusion of the liver. The effect of hepatotoxic or protective agents was assessed in term of changes in viability (evaluated by Trypan blue exclusion and oxygen uptake tests), levels of selected enzymes (the transaminases-GOT and GRT- and alkaline phosphatase) and specific binding of I’25-low density lipoproteins (LDL). Crude extracts of Picrorhiza kurrooa, (root and rhizome). Phyilanthus amarus (aerial parts) and Ricinus communis (leaf) have been screened. In addition Picroliv (a standardised active fraction from P.kurrooa), n-demethyl-ricinin (from Rcommunis), andrographolide (from Andrographis paniculata) and sdymarin (from S&bum marianum) were also tested. Four groups of 5 animals e;rch were en@ -,oyed in each experiment, one group served as the control, the second received the test agent, the third was treated with the hepatotoxin and the fourth was administered the test drug before the hepatotoxin. Wherever necessary, the serum levels of the enzymes were also monitored.
607
In normal animals > 95% hepatocytes could be isolated in a viable form. The oxygen uptake aas 4.3 u litre/hr, GOT 0.42 units, GPT 0.73 units and alkaline phosphate activity 0.17 units per mg protein respectively. Treatment with the hepatotoxic agents reduced the viable cell count by 4650% accompanied with 50-70% decrease in enzyme activities of hepatocytes and similar elevation in serum enzyme levels. Significant dose related protection %as observed with crude extracts of the 3 plants against the deleterious effects of both the hepatotoxic agents in the hepatocyte and serum parameters; Picrorhizu kurroou being the most potent and Ricinus communis having lowest activity. Among the purified preparations andrographolide and Picroliv were most effective (upto 0.75 mg/kg) followed by n-demethyl ricinin (3 mg/kg) and silymarin (6 mg/kg). Picroliv and silymarin were also found effective against similar damage with galactosamine (400 mg/kg) but a higher dose was required. Picroliv also antagonised paracetamol induced decrease in specific binding of Ii2’-LDL to isolated hepatocytes. Further, the biochemical effects of Picroliv collaborated well with protection observed histologically. The technique offers excellent possibility for assessment of hepatoprotective effect of natural products at various stages of chemical fractionation as well as of purified active constituents.
1s.ln0.09.5 ( Action mechanisms of s
ts isola
fro
nese
s
Teng, C.-M., Ko, F.-N., Yu, S.-M., Chen *, C.-C., Wu * *, T.-S. and Lin * * *, C.-N. Pharmacologicalinstitute, College of Medicine, National Taiwan University, Taipei, * National institute of ChineseMedicine, Taipei, * * Department of Chemistry National Cheng-Kong University, Tainan and * * “‘Schoolof Pharmacy, KuohsiungMedical College, Kuohsiung, Taiwan, People’s Republic of China
A large scale screening test of the vasorelaxing actions of the extracts from Chinese herbs has been performed and the active principles were isolated: osthole from Angelica pubescens. protopine from Corydalis tubers, norathyriol from Gentianaceae, denudatin B and fargesone B from Magnolia fargesii, apigenin from Apium graveolens, and magnolol from Magnolia officinales. In this experiment, we tried to study the effects of these Chinese herbal principles on the vasoconstriction of rat thoracic aorta induced by high potassium solution, norepinephrine (NE) and caffeine, and to elucidate their modes of action. All the seven principles suppressed the contraction of rat thoracic aortic rings caused by cumulative concentrations of calcium (0.03-3 mM) in high potassium (60 mM) medium. They also relaxed the NE (3 PM)-induced contraction, and their pretreatment inhibited both the phasic and tonic contractions elicited by NE in the absence or presence of calcium (1.9 mN) in the medium. Indomethacin (20 FM) did not have any antagonizing effect on these relaxations. Osthole and magnolol also inhibited caffeine (10 mM)-induced phasic contraction, -while others did not have any effect. In qrv’n-Zloaded cultured rat vascular smooth muscle cells, NE-indt:. -od rises of intracellular calcium were suppressed by denudatin B and fargesone B. 45Ca++ uptakes induced by either NE or high potassium were inhibited by the seven vasorelaxants in a concentration-dependent manner. All these vasorelaxants did not change the levels of prostacyclin or CAMP of rat aorta. Both osthole and magnolol caused formation of cGMP in aorta, while other vasorelaxants did not have any effect or were effective only at higher concentrations than those relaxing the muscle. The cGMP increase and initial relaxation caused by magnolol were disappeared after the aorta was denuded. This endothelium-dependent relaxation by magnnolol was antagonized by methylene blue (50 PM) and hemoglobin (10 PM) which were known to inhibit activation of guanylate cyclase and inactivate endothelium-derived relaxing factor (EDBF), respectively. In conclusion, seven vasorelaxants isolated from Chinese herbs inhibited vascular muscle contraction of rat aorta by suppressing the calcium influx through both voltage-gated and receptor-operated calcium channels. EDRE release by magnolol and cGMP increase by osthole may enhance their relaxing effects in rat aorta.