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Endothelium modulated vasorelaxant response of a polypharmaceutical herbal drug (lipotab) and its individual constituents
Journal of Ethnopharmacology 66 (1999) 97 – 102
Short communication
Endothelium modulated vasorelaxant response of a polypharmaceutical herbal drug (lipotab) and its individual constituents M. Zahid Ashraf a, M.S.Y. Khan b, Hakeem Abdul Hameed b, M. Ejaz Hussain c, M. Fahim a,* a
Department of Physiology, VP Chest Institute, Uni6ersity of Delhi, PO Box 2101, Delhi 110007, India b Jamia Hamdard, Hamdard Nagar, New Delhi 110067, India c Centre for Biosciences, Jamia Millia Islamia, New Delhi 110025, India Received 5 June 1998; received in revised form 5 October 1998; accepted 25 November 1998
Abstract The present study was undertaken to examine the endothelium modulated effects of polypharmaceutical drug lipotab and its individual ingredients in isolated aortic rings of rat. Endothelium intact and denuded aortic rings were precontracted with phenylephrine 10 − 6 M and drugs were added in cumulative manner in concentration ranging from 1 to 50 mg/ml. The results demonstrated an endothelium-dependent vasorelaxant effect of lipotab and its individual ingredients, with the exception of nicotinic acid. The dose dependent relaxant response of nicotinic acid was not altered significantly in the endothelium-denuded rings, suggesting a direct effect of the drug on the vascular smooth muscle. Vasorelaxant effect of lipotab and its individual constituents suggests the therapeutic potential of these compounds in certain cardiovascular diseases. © 1999 Elsevier Science Ireland Ltd. All rights reserved. Keywords: Endothelium; Vascular smooth muscle; Alium sati6um; Lipotab; Curcuma longa; Nepeta hindoostana
1. Introduction Vascular endothelium plays an important role in modulating cardiovascular functions by releasing vasoactive substances e.g. endothelium * Corresponding author. Fax: +91-11-7257420. E-mail address: [email protected] (M. Fahim)
derived relaxing factors (EDRFs): nitric oxide (NO), prostacyclins, hyperpolarizing substances and vasoconstrictor substances (Luster et al., 1990). The major role of EDRFs in cardiovascular system includes modulation of contraction of underlying vascular smooth muscle, interaction between blood components and blood vessel wall, vascular growth etc. (Badimon et al., 1992). Al-
0378-8741/99/$ - see front matter © 1999 Elsevier Science Ireland Ltd. All rights reserved. PII: S 0 3 7 8 - 8 7 4 1 ( 9 8 ) 0 0 2 2 6 - 8
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M.Z. Ashraf et al. / Journal of Ethnopharmacology 66 (1999) 97–102
tered endothelial functions and related processes are known to be involved in the pathogenesis of many cardiovascular diseases including hypertension and atherosclerosis (Rubnayi, 1993). Impaired endothelial regulation is an important factor in the development and progression of these diseases. Recent studies have indicated the possibility of some existing therapies (angiotensin converting enzyme inhibitors) to protect endothelium (Rubnayi 1993). Although the involvement of endothelium has been clearly demonstrated during vascular diseases, there is no convincing evidence that the drugs generally used, are targeted for the treatment of endothelial dysfunction. Most of the synthetic drugs in use are in their pure and active forms and many of these compounds are known to have some adverse effects on heart and other organs of the body. Those looking for an alternative therapy have tried natural herbs. Certain indigenous drug preparations with naturally occurring herbs, have been in use for many decades for the treatment of certain cardiovascular diseases. One such drug is lipotab, a polypharmaceutical herbal drug, which has Alium sati6um L. (Family: Liliaceae) (Bulbs), Curcuma longa L. (Family: Zingiberaceae) (Rhizomes), Nepeta hindoostana (Roth) Haines (Family: Labiatae) (aqueous extract of flower and oil) as main ingredients and nicotinic acid as a vitamin source (Rajkumar et al., 1998) is known to reduce cholesterol and triglycerides in hyperlipidemic patients (Arora et al., 1985). The individual constituents of lipotab are also effective in reducing serum cholesterol in experimental animals kept on high cholesterol diet (Arora et al., 1985). For standardisation of such herbal drugs, the knowledge of its therapeutic as well as protective mechanism of action is essential. Therefore, the present study was undertaken to investigate the effect of lipotab and its individual constituents on in vitro vascular smooth muscle preparations and to examine the endotheliummodulated response of this drug and its individual constituents.
2. Material and methods
2.1. Isolated 6ascular preparation White albino rats of either sex weighing between 200–300 gm were sacrificed with an overdose of pentobarbitone sodium (80 mg/kg). Abdominal aorta was carefully dissected, cleaned of fat and adhering connective tissue, then cut into rings of 3–4 mm length in Kreb’s–Henseliet solution. Aortic rings were mounted horizontally between metallic hooks in 10 ml organ bath filled with Krebs–Henseliet solution and bubbled with 95% O2 and 5% CO2 (pH 7.49 0.1, 379 1°C). The composition of Krebs–Henseliet solution was (mM): NaC1, 118; KCl, 4.8; MgSO4, 1.2; KH2PO4, 1.2; NaHCO3, 25; CaCl2, 2.5; and glucose, 11 (Sabouni et al., 1991). Changes in isometric tensions were measured with force transducers (Grass FT-03 c) connected to a polygraph through a strain gauge amplifier (Lectromed, UK). Aortic rings were equilibrated for 1 h under initial tension of 2 gm and buffer was changed after every 15 min. After equilibration, rings were challenged with 10 − 6 M phenylephrine until a constant and reproducible contraction was achieved. For the removal of endothelium the lumen of arterial segment was gently rubbed with a pair of forceps before setting up in the organ bath and removal of endothelium was confirmed by the loss of vasorelaxant response to acetylcholine. The dose-response relationship of lipotab and its individual constituents (A. sati6um, C. longa, N. hindoostana and nicotinic acid) was examined by adding these agents in cumulative manner in concentration ranging from 1 to 50 mg/ml. After adding the highest concentration of drugs the rings were washed two to three times. From the preliminary observations, the same concentrations of drug were selected. These compounds in concentrations lower than 1 mg/ml did not show any significant effect and concentrations higher than 50 mg/ml did not show any further increase in response. Viability of the intact endothelium aortic rings were tested with acetylcholine at the end of the experiment while the rings with denuded-endothelium were tested with sodium nitroprusside, in the end of the experi-
M.Z. Ashraf et al. / Journal of Ethnopharmacology 66 (1999) 97–102
ments. From each animal four aortic rings were obtained for observations, two were with intact endothelium and other two were endothelium denuded.
2.2. Preparation of the plant extracts The process yield of A. sati6um, C. longa and N. hindoostana was done according to the methods described earlier (Aqel et al., 1991). The filtrates were divided into 5 ml aliquots and stored at −20°C until use. Solutions were prepared with distilled water on the day of experiment.
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relaxation of aortic rings at 1, 5, 10 and 20 mg/ml concentration of the drug, respectively. A maximum of 45.739 5.72% relaxation was observed at a dose of 50 mg/ml, which is nearly 50% of response produced by same dose of drug in endothelium-intact rings.
3.2. Effect of A. sati6um
Phenylephrine hydrochloride, acetylcholine chloride and sodium nitroprusside were purchased from Sigma Chemicals, USA. A. sati6um, C. longa, N. hindoostana, nicotinic acid and lipotab were supplied by Hamdard (Wakf) Laboratory, India.
In aortic rings with intact-endothelium, 5 mg/ml of A. sati6um produced 24.489 4.42% relaxation, which was enhanced to 50.169 5.83% at 20 mg/ml concentration. Greater relaxation 66.319 2.56% was observed at 50 mg/ml dose of A. sati6am, which persisted for a long time. Endothelium-denuded rings showed a substantially attenuated response. 5 mg/ml of drug did not produce any appreciable change in tension of the aortic rings. At 10 mg/ml concentration, the relaxation was 19.279 2.59%. Larger relaxant response of 44.279 3.46% was observed at 50 mg/ml concentration of the drug.
2.4. Statistical analysis
3.3. Effect of C. longa
The results were expressed as mean9 SE. The statistical significance was tested with the paired or unpaired Student’s t-test. P value B 0.05 were considered to be significant. Increase/decrease in tension were expressed as percent of maximal response to phenyephrine (10 − 6 M).
3. Results
In the intact-endothelial tissues the response to lower doses of C. longa was relatively low. However, 18.439 5.45% relaxation was observed at 20 mg/ml which increased to 39.37 9 5.56% at 50 mg/ml concentration. In endothelium-denuded tissues there was no clear cut response up to 5 mg/ml. 20 mg/ml produced 14.4493.61% relaxation and 21.4894.53% relaxation of tissues was observed at 50 mg/ml.
3.1. Effect of lipotab
3.4. Effect of N. hindoostana
In tissues with intact endothelium, lipotab in concentration upto 5 mg/ml produced small relaxation (28.5393.33%), while higher dose of lipotab (20 mg/ml) produced significant (58.38 9 2.25%) relaxation of aortic smooth muscle. A total of 50 mg/ml concentration of the drug produced much stronger (84.495.45%) and long lasting relaxant response. In tissues with denudedendothelium, there was a significant reduction in the relaxant effect of lipotab, producing 2.779 1.28, 11.9192.53, 21.0694.39 and 30.229 3.91%
N. hindoostana showed a significant endothelium-mediated effect which was reduced to half after removal of endothelium. Intact-endothelial preparations showed a consistent relaxation of 21.369 3.97% at 10 mg/ml, which increased to 34.879 5.12% at 20 mg/ml. A larger, 47.37 9 3.39% relaxation was observed at 50 mg/ml concentration. Endothelium-denuded tissues did not show any clear cut response up to 5 mg/ml. Relaxation gradually increased to 6.269 3.03 and 11.869 5.64% at 10 and 20 mg/ml concentrations,
2.3. Drugs
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respectively. In comparison to endothelium-intact rings significantly lower, 21.1692.98% relaxation was observed in endothelium-denuded rings at 50 mg/ml.
3.5. Effect of nicotinic acid Endothelium-intact tissues showed consistent relaxant response of nearly 8% at 5 mg/ml concentration of the drug and 15.279 3.05% at 10 mg/ ml. This response significantly increased to 33.7792.83% at 50 mg/ml concentration. In the endothelium-denuded tissues, there was 7.259 2.01% relaxation at 5 mg/ml and 14.459 2.89% at 10 mg/ml. Relaxant effect increased to about 24.6494.68% at 20 mg/ml and 31.369 3.25% at 50 mg/ml concentration.
4. Discussion and conclusions For many years, lipotab has been successfully used for lowering cholesterol and triglycerides in hyperlipidemic patients (Arora et al., 1985). Its individual constituents have also been reported to have the same ability of lowering serum cholesterol and triglycerides in animals kept on high cholesterol diet (Rajkumar et al., 1998). The therapeutic dose of the drug recommended is 1 – 3 gm per day or more, depending on the severity of the disease. The toxicity of lipotab and its constituents is reported to be negligible (Arora et al., 1985). In the present study, lipotab has shown a prominent vasorelaxant effect, which appears to be a synergistic effect of all its constituents. One of the possible mechanism of protective action of lipotab has been suggested to be either by maintaining the balance of intracellular K + ions and acting as an exogenous source of potassium or by stabilizing cell membrane, thus preventing the loss of intracellular potassium (Siegal et al., 1992). The vasorelaxant response to lipotab was reduced in endothelium denuded vascular tissues, suggesting the involvement of endothelium mediated mechanisms e.g. EDRFs release. Further endotheliumderived hyperpolarizing factor (EDHF) which is linked to increase in K + efflux, via cyclic nucle-
otide pathways (Foushee et al., 1982) is also known to play a role in the vasorelaxation. A. sati6um was found to be the most potent vasorelaxant among all the constituents of lipotab which supports its antihypertensive (Lee et al., 1994) and antiatherosclerotic (Orekhov et al., 1995) actions. It is known that thioallyl compounds which are natural constituents of garlic, inhibit malignant cells. It can also reduce proliferation of normal cells (Aqel et al., 1991) indicating the possibility of garlic interfering with the concentration of cytoplasmic free Ca2 + (Aqel et al., 1991). Aqueous garlic extract, causes a concentration-dependent hyperpolarization and vasodilation by opening K + channels and closure of Ca 2+ channels, mediated via cA-PK or cG-PK pathway (Siegal et al., 1991). The membrane of endothelial cells possibly serves as a flow sensor in vasodilation, hence there was a significant inhibition of vasorelaxant response to garlic in endothelium-denuded tissues. C. longa, has an anti-oxidant activity (Sinha et al., 1972) as well as a sharp transient hypotensive effect (Azuine and Bhide, 1992). The anti-inflammatory action of curcumin in stomach and skin tumours has been reported (Srivastava et al., 1986). Turmeric inhibits platelets aggregation without inhibiting synthesis of prostacyclins (PG12) (Ahmad et al., 1985) and it also inhibits lipopolysaccharides and interferon-gamma induced nitrite production from NO in mouse peritoneal cells (Chan et al., 1995). Endothelium-dependent vasorelaxant effect of turmeric may involve either or both of the above mentioned mechanisms. N. hindoostana is an important drug in Unani system of medicine which contains a tripenoid compound nepeticine. It has beneficial effects in myocardial necrosis and has an antifungal activity (Hallahan et al., 1995). N. hindoostana showed a very significant endothelium-mediated relaxation, but there is a need for evaluation of its exact mechanism of action. An acyclic monoterpene primary alcohol: NADP-oxidoreductase is reported to be present in N. racemosa leaves, its effect on vascular smooth muscle is not known. Nicotinic acid used in the drug as a vitamin source is known to be hypolipidemic (Altschul et
M.Z. Ashraf et al. / Journal of Ethnopharmacology 66 (1999) 97–102
al., 1995).The vasorelaxant effect shown by niacin which seems to be independent of endothelium, possibly mediates through NAD/ NADP, physiologically active forms of nicotinic acid. NAD/NADP act as coenzymes in the oxidation–reduction reactions and transfer ADP-ribosyl moieties to protein via guanine nucleotide binding regulatory protein (G-protein) which stimulates adenylase cyclase (Gillman et al., 1990) and causes the relaxation of vascular smooth muscle. The present study suggests that intact endothelium is one of the greatest source of natural protection against vascular diseases, reduced release of EDRFs and enhanced liberation of EDCFs are common in the pathological conditions. The alteration in endothelial functions are likely to contribute to the pathogenesis as well as progression of cardiovascular disorders. Indigenous drugs are the natural source of protection against certain disorders which can be used more effectively by knowledge of its active ingredients as well as its mechanism of action. Lipotab, a potent vasorelaxant can be very helpful for the treatment of certain cardiovascular disorders related to the conditions of increased vascular resistance.
Acknowledgements Authors thank Dr Anita Pawar and Dr Prasanna K. Reddy for assistance in typing and proof reading the manuscript. This research work was supported by Hamdard National Foundation, India.
References Ahmad, J., Siddiqi, T.O., Arora, R.B., Pillay, K.K., 1985. Pharmacognostical, elementological and cardiovascular studies on the flowers of N. hindoostana (Roth) haines. Asian Congress of Pharmacology 1, 14–15. Altschul, R., Hoffer, A., Stephene, J.D., 1995. Influence of nicotinic acid on S. cholesterol in man. Archives of Biochemistry and Biophysics 54, 558–559.
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Aqel, M.B., Gharaibah, M.N., Salhab, A.S., 1991. Direct relaxant effects of garlic juice on smooth and cardiac muscles. Journal of Ethnopharmacology 33, 13 – 19. Arora, R.B., Roy, S., Qadri, I.Z., Kesar, D.K., 1985. Long term follow-up of patients of hypercholesteremia and hyperlipidemia with herbal polypharmaceutical-lipotab. Hamdard National Foundation Monograph 175 – 184. Azuine, M.A., Bhide, S.V., 1992. Chemopreventive effect of turmeric against stomach and skin tumours induced by chemical carcinogens in Swiss mice. Nutrition and Cancer 17 (1), 77 – 83. Badimon, L., Badimon, J.J., Penny, W., Webstar, M.W., Chesebro, J.H., Fuster, V., 1992. Endothelium and atherosclerosis. Journal of Hypertension 10 (2), 43 – 50. Chan, M.M., Ho, C.T., Huang, H.I., 1995. Turmeric on inflammation-induced nitrite production. Cancer Letters 96 (1), 23 – 29. Foushee, D., Ruffin, J., Banerjee, U., 1982. Garlic as a natural agent for the treatment of hypertension: a preliminary report. Cytobiosis 34, 145 – 152. Gillman, A.F., Rall, T.W., Nics, A.S., Taylor, P., 1990. In: Goodmann Gillman A (Ed.), The Pharmacological Basis of Therapeutics. Pergamon, Oxford, pp. 1539 – 1540. Hallahan, D.L., West, J.M., Wallsgrove, R.M., Smiley, D.W., Dawson, G.W., Pickett, J.A., Hamilton, J.G., 1995. Purification and characterization of an acyclic monoterpene primary alcohol: NADP + oxidoreductase from catmint (Nepeta racemosa). Archives of Biochemistry and Biophysics 318 (1), 105 – 112. Lee, E.S., Steiner, M., Lin, R., 1994. Thioallyl compounds: potent inhibitors of cell proliferation. Biochemica et Biophysica Acta 1221 (1), 73 – 77. Luster, T.F., Derich, D.D., Buhler, F.R., Vanhoutte, P.M., 1990. Interaction between platelets and the vessel wall: role of endothelium derived vasoactive substances. In: Hypertension: Pathophysiology. Raven Press, New York, pp. 637 – 648. Orekhov, A.N., Tertov, V.V., Sobenin, I.A., Pivovarova, E.M., 1995. Direct anti atherosclerosis related effect of garlic. Annals of Medicine 27 (1), 63 – 65. Rajkumar, S., Pillai, K.K., Balani, D.K., Hussain, S.Z., 1998. Antiatherosclerotic effect of lipotab forte in cholesterol-fed rabbits. Journal of Ethnopharmacology 59, 125 – 130. Rubnayi, G.M., 1993. The role of endothelium in cardiovascular homeostasis and diseases. Journal of Cardiovascular Research 22 (4), 1 – 14. Sabouni, M.H., Cushing, D.J., Makujina, S.R., Mustafa, S.J., 1991. Inhibition of adenylate cyclase attenuated adenosine receptor mediated relaxation in coronary artery. Journal of Pharmacology and Experimental Therapeutics 59 (2), 508 – 512. Siegal, G., Emden, J., Wenzel, K., Mirrneau, J., Stock, G., 1992. Potassium channel activation in vascular smooth muscle. Advances in Experimental Medicine and Biology 311, 53 – 72.
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Siegal, G., Walter, A., Schnalke, F., Schmidt, A., Buddecke, E., 1991. Potassium channel activation, hyperpolarization and vascular relaxation. Zeitschrift fu¨r Kardiologie 80 (7), 9–24. Sinha, M., Mukherjee, B.P., Sikdar, S., Mukherjee, B., Dasgupta, S.R., 1972. Further studies on the pharmacological
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properties of curcumin. Indian Journal of Pharmacology 4, 135 – 136. Srivastava, R., Puri, V., Srimal, R.C., Dhawan, B.N., 1986. Effect of curcumin on platelets aggregation and vascular prostacyclin synthesis. Arzneimittel-Forschung 36, 715.
Short communication
Endothelium modulated vasorelaxant response of a polypharmaceutical herbal drug (lipotab) and its individual constituents M. Zahid Ashraf a, M.S.Y. Khan b, Hakeem Abdul Hameed b, M. Ejaz Hussain c, M. Fahim a,* a
Department of Physiology, VP Chest Institute, Uni6ersity of Delhi, PO Box 2101, Delhi 110007, India b Jamia Hamdard, Hamdard Nagar, New Delhi 110067, India c Centre for Biosciences, Jamia Millia Islamia, New Delhi 110025, India Received 5 June 1998; received in revised form 5 October 1998; accepted 25 November 1998
Abstract The present study was undertaken to examine the endothelium modulated effects of polypharmaceutical drug lipotab and its individual ingredients in isolated aortic rings of rat. Endothelium intact and denuded aortic rings were precontracted with phenylephrine 10 − 6 M and drugs were added in cumulative manner in concentration ranging from 1 to 50 mg/ml. The results demonstrated an endothelium-dependent vasorelaxant effect of lipotab and its individual ingredients, with the exception of nicotinic acid. The dose dependent relaxant response of nicotinic acid was not altered significantly in the endothelium-denuded rings, suggesting a direct effect of the drug on the vascular smooth muscle. Vasorelaxant effect of lipotab and its individual constituents suggests the therapeutic potential of these compounds in certain cardiovascular diseases. © 1999 Elsevier Science Ireland Ltd. All rights reserved. Keywords: Endothelium; Vascular smooth muscle; Alium sati6um; Lipotab; Curcuma longa; Nepeta hindoostana
1. Introduction Vascular endothelium plays an important role in modulating cardiovascular functions by releasing vasoactive substances e.g. endothelium * Corresponding author. Fax: +91-11-7257420. E-mail address: [email protected] (M. Fahim)
derived relaxing factors (EDRFs): nitric oxide (NO), prostacyclins, hyperpolarizing substances and vasoconstrictor substances (Luster et al., 1990). The major role of EDRFs in cardiovascular system includes modulation of contraction of underlying vascular smooth muscle, interaction between blood components and blood vessel wall, vascular growth etc. (Badimon et al., 1992). Al-
0378-8741/99/$ - see front matter © 1999 Elsevier Science Ireland Ltd. All rights reserved. PII: S 0 3 7 8 - 8 7 4 1 ( 9 8 ) 0 0 2 2 6 - 8
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M.Z. Ashraf et al. / Journal of Ethnopharmacology 66 (1999) 97–102
tered endothelial functions and related processes are known to be involved in the pathogenesis of many cardiovascular diseases including hypertension and atherosclerosis (Rubnayi, 1993). Impaired endothelial regulation is an important factor in the development and progression of these diseases. Recent studies have indicated the possibility of some existing therapies (angiotensin converting enzyme inhibitors) to protect endothelium (Rubnayi 1993). Although the involvement of endothelium has been clearly demonstrated during vascular diseases, there is no convincing evidence that the drugs generally used, are targeted for the treatment of endothelial dysfunction. Most of the synthetic drugs in use are in their pure and active forms and many of these compounds are known to have some adverse effects on heart and other organs of the body. Those looking for an alternative therapy have tried natural herbs. Certain indigenous drug preparations with naturally occurring herbs, have been in use for many decades for the treatment of certain cardiovascular diseases. One such drug is lipotab, a polypharmaceutical herbal drug, which has Alium sati6um L. (Family: Liliaceae) (Bulbs), Curcuma longa L. (Family: Zingiberaceae) (Rhizomes), Nepeta hindoostana (Roth) Haines (Family: Labiatae) (aqueous extract of flower and oil) as main ingredients and nicotinic acid as a vitamin source (Rajkumar et al., 1998) is known to reduce cholesterol and triglycerides in hyperlipidemic patients (Arora et al., 1985). The individual constituents of lipotab are also effective in reducing serum cholesterol in experimental animals kept on high cholesterol diet (Arora et al., 1985). For standardisation of such herbal drugs, the knowledge of its therapeutic as well as protective mechanism of action is essential. Therefore, the present study was undertaken to investigate the effect of lipotab and its individual constituents on in vitro vascular smooth muscle preparations and to examine the endotheliummodulated response of this drug and its individual constituents.
2. Material and methods
2.1. Isolated 6ascular preparation White albino rats of either sex weighing between 200–300 gm were sacrificed with an overdose of pentobarbitone sodium (80 mg/kg). Abdominal aorta was carefully dissected, cleaned of fat and adhering connective tissue, then cut into rings of 3–4 mm length in Kreb’s–Henseliet solution. Aortic rings were mounted horizontally between metallic hooks in 10 ml organ bath filled with Krebs–Henseliet solution and bubbled with 95% O2 and 5% CO2 (pH 7.49 0.1, 379 1°C). The composition of Krebs–Henseliet solution was (mM): NaC1, 118; KCl, 4.8; MgSO4, 1.2; KH2PO4, 1.2; NaHCO3, 25; CaCl2, 2.5; and glucose, 11 (Sabouni et al., 1991). Changes in isometric tensions were measured with force transducers (Grass FT-03 c) connected to a polygraph through a strain gauge amplifier (Lectromed, UK). Aortic rings were equilibrated for 1 h under initial tension of 2 gm and buffer was changed after every 15 min. After equilibration, rings were challenged with 10 − 6 M phenylephrine until a constant and reproducible contraction was achieved. For the removal of endothelium the lumen of arterial segment was gently rubbed with a pair of forceps before setting up in the organ bath and removal of endothelium was confirmed by the loss of vasorelaxant response to acetylcholine. The dose-response relationship of lipotab and its individual constituents (A. sati6um, C. longa, N. hindoostana and nicotinic acid) was examined by adding these agents in cumulative manner in concentration ranging from 1 to 50 mg/ml. After adding the highest concentration of drugs the rings were washed two to three times. From the preliminary observations, the same concentrations of drug were selected. These compounds in concentrations lower than 1 mg/ml did not show any significant effect and concentrations higher than 50 mg/ml did not show any further increase in response. Viability of the intact endothelium aortic rings were tested with acetylcholine at the end of the experiment while the rings with denuded-endothelium were tested with sodium nitroprusside, in the end of the experi-
M.Z. Ashraf et al. / Journal of Ethnopharmacology 66 (1999) 97–102
ments. From each animal four aortic rings were obtained for observations, two were with intact endothelium and other two were endothelium denuded.
2.2. Preparation of the plant extracts The process yield of A. sati6um, C. longa and N. hindoostana was done according to the methods described earlier (Aqel et al., 1991). The filtrates were divided into 5 ml aliquots and stored at −20°C until use. Solutions were prepared with distilled water on the day of experiment.
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relaxation of aortic rings at 1, 5, 10 and 20 mg/ml concentration of the drug, respectively. A maximum of 45.739 5.72% relaxation was observed at a dose of 50 mg/ml, which is nearly 50% of response produced by same dose of drug in endothelium-intact rings.
3.2. Effect of A. sati6um
Phenylephrine hydrochloride, acetylcholine chloride and sodium nitroprusside were purchased from Sigma Chemicals, USA. A. sati6um, C. longa, N. hindoostana, nicotinic acid and lipotab were supplied by Hamdard (Wakf) Laboratory, India.
In aortic rings with intact-endothelium, 5 mg/ml of A. sati6um produced 24.489 4.42% relaxation, which was enhanced to 50.169 5.83% at 20 mg/ml concentration. Greater relaxation 66.319 2.56% was observed at 50 mg/ml dose of A. sati6am, which persisted for a long time. Endothelium-denuded rings showed a substantially attenuated response. 5 mg/ml of drug did not produce any appreciable change in tension of the aortic rings. At 10 mg/ml concentration, the relaxation was 19.279 2.59%. Larger relaxant response of 44.279 3.46% was observed at 50 mg/ml concentration of the drug.
2.4. Statistical analysis
3.3. Effect of C. longa
The results were expressed as mean9 SE. The statistical significance was tested with the paired or unpaired Student’s t-test. P value B 0.05 were considered to be significant. Increase/decrease in tension were expressed as percent of maximal response to phenyephrine (10 − 6 M).
3. Results
In the intact-endothelial tissues the response to lower doses of C. longa was relatively low. However, 18.439 5.45% relaxation was observed at 20 mg/ml which increased to 39.37 9 5.56% at 50 mg/ml concentration. In endothelium-denuded tissues there was no clear cut response up to 5 mg/ml. 20 mg/ml produced 14.4493.61% relaxation and 21.4894.53% relaxation of tissues was observed at 50 mg/ml.
3.1. Effect of lipotab
3.4. Effect of N. hindoostana
In tissues with intact endothelium, lipotab in concentration upto 5 mg/ml produced small relaxation (28.5393.33%), while higher dose of lipotab (20 mg/ml) produced significant (58.38 9 2.25%) relaxation of aortic smooth muscle. A total of 50 mg/ml concentration of the drug produced much stronger (84.495.45%) and long lasting relaxant response. In tissues with denudedendothelium, there was a significant reduction in the relaxant effect of lipotab, producing 2.779 1.28, 11.9192.53, 21.0694.39 and 30.229 3.91%
N. hindoostana showed a significant endothelium-mediated effect which was reduced to half after removal of endothelium. Intact-endothelial preparations showed a consistent relaxation of 21.369 3.97% at 10 mg/ml, which increased to 34.879 5.12% at 20 mg/ml. A larger, 47.37 9 3.39% relaxation was observed at 50 mg/ml concentration. Endothelium-denuded tissues did not show any clear cut response up to 5 mg/ml. Relaxation gradually increased to 6.269 3.03 and 11.869 5.64% at 10 and 20 mg/ml concentrations,
2.3. Drugs
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M.Z. Ashraf et al. / Journal of Ethnopharmacology 66 (1999) 97–102
respectively. In comparison to endothelium-intact rings significantly lower, 21.1692.98% relaxation was observed in endothelium-denuded rings at 50 mg/ml.
3.5. Effect of nicotinic acid Endothelium-intact tissues showed consistent relaxant response of nearly 8% at 5 mg/ml concentration of the drug and 15.279 3.05% at 10 mg/ ml. This response significantly increased to 33.7792.83% at 50 mg/ml concentration. In the endothelium-denuded tissues, there was 7.259 2.01% relaxation at 5 mg/ml and 14.459 2.89% at 10 mg/ml. Relaxant effect increased to about 24.6494.68% at 20 mg/ml and 31.369 3.25% at 50 mg/ml concentration.
4. Discussion and conclusions For many years, lipotab has been successfully used for lowering cholesterol and triglycerides in hyperlipidemic patients (Arora et al., 1985). Its individual constituents have also been reported to have the same ability of lowering serum cholesterol and triglycerides in animals kept on high cholesterol diet (Rajkumar et al., 1998). The therapeutic dose of the drug recommended is 1 – 3 gm per day or more, depending on the severity of the disease. The toxicity of lipotab and its constituents is reported to be negligible (Arora et al., 1985). In the present study, lipotab has shown a prominent vasorelaxant effect, which appears to be a synergistic effect of all its constituents. One of the possible mechanism of protective action of lipotab has been suggested to be either by maintaining the balance of intracellular K + ions and acting as an exogenous source of potassium or by stabilizing cell membrane, thus preventing the loss of intracellular potassium (Siegal et al., 1992). The vasorelaxant response to lipotab was reduced in endothelium denuded vascular tissues, suggesting the involvement of endothelium mediated mechanisms e.g. EDRFs release. Further endotheliumderived hyperpolarizing factor (EDHF) which is linked to increase in K + efflux, via cyclic nucle-
otide pathways (Foushee et al., 1982) is also known to play a role in the vasorelaxation. A. sati6um was found to be the most potent vasorelaxant among all the constituents of lipotab which supports its antihypertensive (Lee et al., 1994) and antiatherosclerotic (Orekhov et al., 1995) actions. It is known that thioallyl compounds which are natural constituents of garlic, inhibit malignant cells. It can also reduce proliferation of normal cells (Aqel et al., 1991) indicating the possibility of garlic interfering with the concentration of cytoplasmic free Ca2 + (Aqel et al., 1991). Aqueous garlic extract, causes a concentration-dependent hyperpolarization and vasodilation by opening K + channels and closure of Ca 2+ channels, mediated via cA-PK or cG-PK pathway (Siegal et al., 1991). The membrane of endothelial cells possibly serves as a flow sensor in vasodilation, hence there was a significant inhibition of vasorelaxant response to garlic in endothelium-denuded tissues. C. longa, has an anti-oxidant activity (Sinha et al., 1972) as well as a sharp transient hypotensive effect (Azuine and Bhide, 1992). The anti-inflammatory action of curcumin in stomach and skin tumours has been reported (Srivastava et al., 1986). Turmeric inhibits platelets aggregation without inhibiting synthesis of prostacyclins (PG12) (Ahmad et al., 1985) and it also inhibits lipopolysaccharides and interferon-gamma induced nitrite production from NO in mouse peritoneal cells (Chan et al., 1995). Endothelium-dependent vasorelaxant effect of turmeric may involve either or both of the above mentioned mechanisms. N. hindoostana is an important drug in Unani system of medicine which contains a tripenoid compound nepeticine. It has beneficial effects in myocardial necrosis and has an antifungal activity (Hallahan et al., 1995). N. hindoostana showed a very significant endothelium-mediated relaxation, but there is a need for evaluation of its exact mechanism of action. An acyclic monoterpene primary alcohol: NADP-oxidoreductase is reported to be present in N. racemosa leaves, its effect on vascular smooth muscle is not known. Nicotinic acid used in the drug as a vitamin source is known to be hypolipidemic (Altschul et
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al., 1995).The vasorelaxant effect shown by niacin which seems to be independent of endothelium, possibly mediates through NAD/ NADP, physiologically active forms of nicotinic acid. NAD/NADP act as coenzymes in the oxidation–reduction reactions and transfer ADP-ribosyl moieties to protein via guanine nucleotide binding regulatory protein (G-protein) which stimulates adenylase cyclase (Gillman et al., 1990) and causes the relaxation of vascular smooth muscle. The present study suggests that intact endothelium is one of the greatest source of natural protection against vascular diseases, reduced release of EDRFs and enhanced liberation of EDCFs are common in the pathological conditions. The alteration in endothelial functions are likely to contribute to the pathogenesis as well as progression of cardiovascular disorders. Indigenous drugs are the natural source of protection against certain disorders which can be used more effectively by knowledge of its active ingredients as well as its mechanism of action. Lipotab, a potent vasorelaxant can be very helpful for the treatment of certain cardiovascular disorders related to the conditions of increased vascular resistance.
Acknowledgements Authors thank Dr Anita Pawar and Dr Prasanna K. Reddy for assistance in typing and proof reading the manuscript. This research work was supported by Hamdard National Foundation, India.
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