Hypotensive effect of the methanolic extract of Mimusops elengi in normotensive rats

Hypotensive effect of the methanolic extract of Mimusops elengi in normotensive rats

Phytomedicine, Vol. 6(5), pp. 373-378 © Urban & Fischer Verlag 1999 http://www.urbanfischer.de/journals/phytomed Hypotensive effect of the methanoli...

1MB Sizes 2 Downloads 16 Views

Phytomedicine, Vol. 6(5), pp. 373-378

© Urban & Fischer Verlag 1999 http://www.urbanfischer.de/journals/phytomed

Hypotensive effect of the methanolic extract of Mimusops elengi in normotensive rats A. Dar, S. Behbahanian, A. Malik and N. jahan

Summary The methanolic extract of Mimusops elengi caused hypotensive activity in anaesthetized rats. On intravenous administration (i.v.) at a dose range of 2-16 mg/kg, it produced about a 7-38% fall in mean arterial blood pressure, in a dose-dependent manner. The effect was independent of adrenergic, muscarinic and histaminergic receptors. The hypotension was also unchanged after autonomic ganglion or angiotensin-converting-enzyme blockade. Administration of calcium channel blockers, however, including nifedipine (0.9 mg/kg) and verapamil (3.9 mg/kg), caused corresponding reductions of 81 and 64% in extract-induced hypotension. These data imply M. elengi might possess calcium-blocking activity which would explain its hypotensive effect. Key words: Mimusops elengi, hypotension, Ca 2+ channels.



Introduction

Mimusops elengi L. belongs to the Sapotaceae and is

Preparation of extracts

indigenous to Indo-Pakistan (Core, 1955; Hills, 1952). The local name is "Maulsari" and the plant is used in folk medicine for treatment of asthma, diarrhea, fever, headache, rheumatism, eye pain and the strengthening of gums (Perry, 1980). Phytochemical work on this plant has revealed the presence of various sugars, amino acids, free triterpenes, fatty acids, flavonoids and free sterols (Misra, Nigam and Mitra, 1974; ]ahan, Ahmad and Malik, 1996). Pharmacological studies showed that this plant possesses diuretic (Kanjanapothi et al., 1971), spermicidal (Banerji et al., 1979), spasmolytic (Banerji et al., 1982) and antimycotic (Deshmukh, 1986) activity. Preliminary studies indicated hypotensive effects for this plant (Dar et al., 1998). The present study was undertaken to determine the possible mode of hypotensive action of M. elengi.

The shade-dried plant material (10 kg) was extracted four times with methanol (14.3 liter, 96h each) at room temperature and the combined methanol extract was evaporated under reduced pressure to obtain a dark brown extract (245 g), a part of which was used for pharmacological studies.

Materials and Methods Plant materials

Acute toxicity test

Plant extract lethality was determined in mice of either sex weighing 25-30 g (n = 5, per dose) by i.p. administration of 25, 50, 100, 500 and 2000 mg/kg body weight. A group of five mice receiving the vehicle (DMSO 10%, 10 ml/kg body wt.) was used as control. Animals were observed for behavioral effects and mortality for 7 days after treatment. Lethal dose causing 50% mortality (LDso value) of mice was determined by plotting the percent of mortality versus the concentration of the plant extract (mg/kg body wt.) used. The plant extract was also tested orally at 2000 mg/kg body wt. (n = 5).

Mimusops elengi was collected from Karachi and identified by a taxonomist from the Department of Botany, University of Karachi. 0944-7113/99/06/05-373 $ 12.00/0

374

A. Dar et al.

Studieson arterial blood pressure and heart rate Albino rats of either sex (233.3 ± 3.5 g) provided by the animal house facility of H.E.]. Research Institute of Chemistry were used throughout this study. Rats were anaesthetized using sodium pentothal (60 mg/kg body wt.) by i.p. injection and trachea was intubated to facilitate spontaneous respiration. Animal body temperature was maintained at 37°C using an overhead lamp. The right carotid artery and left jugular vein were cannulated with polyethylene tubing PE-50 to monitor blood pressure changes via a pressure transducer (P23 ID) connected to a Grass model 79D polygraph, and for i.v. injection, respectively. After 20 min of stabilization and steady base line, noradrenaline and acetylcholine (1 ~M/kg) were administered and those animals showing normal responses were selected to receive different doses of M. elengi. Changes in the blood pressure and heart rate were registered after successive administration of increasing doses of plant extract (2-25 mg/kg body wt.) to rats. Control animals received the same volume of the saline. Systolic, diastolic and mean arterial blood pressures (MABP) were measured in mmHg and heart rate in beats/min; the results were expressed as percent change induced by the extract as compared with the control values. For each dose tested (2-25 mg/kg), decline in MABP (mmHg) was calculated with their respective control values. The ED 30 value indicates the dose required to achieve a 30 mmHg decline in the MABP. The ED 30 value was determined by plotting the fall in MABP against the concentration of the plant extract (mg/kg body wt.) for four individuals and the mean was obtained. To elucidate the mechanism of action of hypotensive response, a dose of 10 mg/kg body wt. of the methanolic extract (a dose shown previously to produce detectable effect) was added in the presence of the following various antagonists: atropine (2 mg/kg body wt.), cimetidine (15 mg/kg body wt.), hexamethonium (10 mg/kg body wt.), phentolamine (2 mg/kg body wt.), propranolol (2 mg/kg body wt.) and pyrilamine (15 pg/kg body wt.). These were added 10 min prior to the addition of the plant extract, however, captopril (40 Ilg/kg body wt.) was administered by slow infusion (10 ul/rnin/kg body wt.) within 30 min. Parallel experiments were also conducted using appropriate agonists, including acetylcholine (2 ug/kg body wt.), histamine (2 ~g/kg body wt.), methoxamine (50 ug/kg body wt.), isoproterenol (1.2 ug/kg body wt.) and angiotensin I (100 ng/kg body wt.). The changes in MABP induced by the agonists and plant extracts were analyzed in the presence or absence of the respective antagonists. The changes in MABP caused by the plant extract (10 mg/kg) was also evaluated in the presence or absence of Ca 2 + channel blockers, i.e. nifedipine (0.3 mg/kg body

wt.) and verapamil (1.3 mg/kg body wt.). The methanolic extract was first administered and changes in MABP served as control values. As soon as the blood pressure returned to its original level and became steady, verapamil or nifedipine was administered. Both Ca 2 + channel blockers caused about a 20% decrease in MABP. After a steady response (within 10 min), plant extract was added and changes in MABP were monitored. This procedure was repeated 3 times. Statistical analysis The significance of differences between the control and experimental groups were tested by Student z-test, A probability level of at least P < 0.05 was considered statistically significant. Confidence interval was also calculated for ED 30 values for MABP (Walpole, 1982).



Results

The toxicity trials for the methanolic extract in mice, via i.p. route, showed an LD so value of 100 mg/kg body wt. although oral administration of the plant extract (2000 mg/kg) showed no mortality during the course of experiment. The methanolic extract of M. elengi (2-16 mg/kg body wt.) elicited a dose-dependent decrease systolic, diastolic, and mean arterial blood pressures in rats (Table 1, Figure 1). The decrease systolic blood pressure at 2-4 mg/kg body wt. (10.8 and 14%) was more pronounced than that of diastolic blood pressure, which was significant at higher doses, reaching a maximum of 69.3 ± 5.2 mmHg at 16 mg/kg body wt. At 6 mg/kg body wt. and higher doses the plant extract elicited a significant decline in mean arterial blood pressure (18-38%). The duration of hypotensive action was lengthened progressively as the dosage was increased upto to 16 mg/kg. This effect of the plant extract reached its full extent within 10 sec, and was completely reversed in about 2 min. Dose of 25 mg/kg body wt. caused profound and sustained hypotension which was, in some cases, followed by death of the animals (Figure 1). Although there was a reduction in heart rate, it bore no relation to the dose of the extract used and did not reach statistical significance in any case. The mean ED 30 value was found to be 8.3 ± 1.1 mg/kg body wt. (1.6-14.9 mg/kg, 99% confidence limits). The hypotensive activity induced by the methanolic extract did not change significantly in the presence of various antagonists such as atropine, captopril, cimetidine and pyrilamine, hexamethonium, phentolamine and propranolol. However, the effects elicited by their respective agonists were reduced significantly (Table 2). Cumulative administration of nifedipine (0.3 mg/kg

Hypotensive effects of the methanolic extract of Mimusops elengi

375

Table 1. Effect of the methanolic extract of Mimusops elengi on systolic, diastolic, and mean arterial blood pressure, and heart rate in normotensive rats. Treatment

Control (n = 67)

M. elengi

Dose

Heart rate

(mg/kg)

(beats/min)

Systolic blood pressure (mmHg)

-

360

7.0

142.1

30.0"'" 20.0 11•5 34.611•5 • 30.0".5 . 26.011•5 •

126.7 122.0 116.0 113.2 94.7

2 4 6 10 16

330 340 300 300 280

± ± ± ±

± ±

Diastolic blood pressure (mmHg)

±

1.5

112.9

± ±

1.3"* 2.3 *,} 3.4"" 5.7"" 8.7*"

110.7 ± 4.0 106.7 ± 5.P·s. 94.7 ± 5.7" 78.8 ± 3.8* 69.3 ± 5.2'f*

± ± ±

±

1.6

124.4 11 5 • •

Values represent the mean ± SEM of 3-67 experiments. Asterisks indicate significant change at P < 0.05* and P < 0.01 ** from control values. 11.5. non-significant difference. III' 1911

Figure 1. Representative tracings showing dose-dependent (4-25 mglkg body wt.) hypotensive effect of the methanolic extract of M. elengi in anaesthetized

rats.

"nnll!:

Mean arterial Percent fall in blood pressure MABP (MABP) (mmHg) ±

116.0 ± 111.8 ± 101.7 ± 90.3 ± 77.8 ±

1.3

0

2.8 4.5 11•5• 4.9" 4.3" 5.9*'} 11 5 • •

6.8 10.2 18.2 30.9 37.5

±

2.211•5•

±

3.6 11•5• 3.9* 5.5'} 4.8**

± ± ±

376

A. Dar et al.

Table 2. Effect of Mimusops elengi in the absence or presence of various chemicals, on mean arterial blood pressure of anaesthetized rats. Treatment

Mean arterial blood pressure (MABP) (mmHg)

Changes in MABP (mmHg)

o

Control Methanolic extract (10 mg/kg)

124,4

±

1.3 (67)

87.8

±

2.5 (29) -36.6

Acetylcholine (1 ]lM) Atropine (2 mg/kg) Atropine + acetylcholine Atropine + extract

104,4 112.3 120.6 87.0

±

2.3 2.5 3.7 2.1

± ± ±

(3) -19.9 (3) -12.0 (3) -3.8 (3) -37,4

±

2.5

±

2.3 2.5 3.7* 2.1 n.s.

± ± ±

Angiotensin 1(100 ng/kg) 143.8 Captopril (40 ug/kg) 120,4 Captopril + angiotensin I 130.8 91.3 Captopril + extract

±

±

4.6 (5) 2.5 (5) 2.1 (5) 12.8 (5)

Histamine (2 ug/kg] Cimetidine + pyrilamine (15 mg/kg each) Cimetidine + pyrilamine + histamine Cimetidine + pyrilamine + extract

112.3

±

4.6 (5) -14.1

±

4.6

lOLl

±

7.9 (5) -23.3

±

7.9

122.1

±

2.9 (5) -3.3

2.9<-

84.7

±

2,4 (5) -39.7

±

2,4n.s.

Isoprotrenol (1.2 pg/kg] Propranolol (2 mg/kg) Propranolol + isoprotrenol Propranolol + extract

80.2 118.7

±

10.7 (3) -44.2 4.3 (3) -9.0

±

10.7 4.3

4.3 (3) -9.7 8.7 (3) -41.5

±

115.8 82.9

Methoxamine (50 ug/kg] 144.5 95.2 Phentolamine (2 mg/kg) Phentolamine + methox127.8 amme 90.5 Phentolamine + extract Hexamethonium (10 mg/kg) 106.8 Hexamethonium + extract 92.0

± ±

± ±

±

+19,4 ± 4.6 -5,4 ± 2.5 +6,4 ± 2.1 * -33.1 ± 12.8 n.s.

±

±

±

4.3" 8.7n. s.

±

5.3 (4) +20.1 ± 5.3 12.9 (4) -29.6 ± 12.9

±

2.1 (4)

±

±

± ±

+3.4 ± 2.1" 3.8 (4) -33.9 ± 3.8n .s.

3.3 (9) -17.6 4.6 (9) -29.2

± ±

3.3 4.6 n. s.

Values represent the mean ± SEM, and number of experiments are shown within parentheses; (-) and (+) indicate decrease and increase in MABP, respectively. Asterisks indicate significant difference at P < 0.05" as compared to respective controls. n.s. non significant difference as compared to response of methanolic extract. body wt. X 3) and verapamil (1.3 mg/kg body wt. X 3) reduced significantly the plant-extract-induced hypotension (Table 3, Figure 2). Nifedipine induced 32, 59 and 81 % reductions in hypotensive action of the plant extract after sequential administration. Similarly, verapamil elicited 31, 54 and 64% reductions in plant extract-induced responses.



Discussion

The methanol extract of M. elengi induced hypotensive effects when administered intravenously to anaesthetized rats. This may be due to its direct or indirect effect on cardiac pump or vascular tissues (Ebeigbe and Ezimokhai, 1988) responsible for reduction in vascular muscle tone, leading to vasodilation (Kimura et al., 1986; Ebeigbe and Ezimokhai, 1988). Keeping this in mind, plant-induced effect was analyzed in the presence of various antagonists. It appeared that prior treatment with agents which block ex or ~ adrenergic receptors, cholinergic and histaminergic receptors, angiotensin-converting enzyme and ganglionic-blocking drugs did not modify the effect of the plant extract, indicating that its action is due to relaxation of the peripheral vascular smooth muscles (Donald et al., 1973). The response of agonists were found to be suppressed in the presence of their respective antagonists, proving that no experimental error existed. It is well-established that contractions of vascular smooth muscle involve influx of extracellular Ca 2+ through calcium channels as well as its release from intracellular sources (Endo et al., 1990; Rasmussen et al., 1990; Greenberg et al., 1991). Consequently, calcium-channel blockers can relax smooth muscle, reduce peripheral vascular resistance and lower arterial blood pressure by interfering either with calcium entry or its release. Of the three calcium channel blockers, diltiazem, nifedipine and verapamil, nifedipine is the most potent in vascular tissue, both in vivo and in vitro (Kalsner, 1997). Similar results were obtained in the present investigation. It appears that the hypotensive effect of M. elengi may be due partly to interruption in calcium mobilization through Ca 2 + channels. This vascular effect could be attributed to compounds such as saponins and triterpenoids reported to be present in the members of Sapotaceae family, and has been suggested to be responsible for nonspecific spasmolytic activity (Banerji et al., 1982). The methanolic extract, which we used in this study, was a crude one and contains a variety of substances. Thus the possibility that the hypotensive property of M. elengi is due to the presence of saponins cannot be ignored and may be an important question for further study. In conclusion, the present data show that the methanolic extract of Mimusops elengi exhibits marked hypotensive activity in rats and that plant extract possibly possesses calcium blocking activity. Further invitro experiments and bioassay - directed fractionation will be required to elucidate the exact mode of action and to identify the principles responsible for this activity.

Hypotensive effects of the methanolic extract of Mimusops elengi

377

BP mmHg 190

A

140

1 min

90

b

a Figure 2. Effect of calcium channel blockers (nifedipine and verapamil) on hypotensive activity of M. elengi. The figure shows: A) Hypotensive activity of M. elengi (10 mg/kg) in anaesthetized rats; B) Inhibitory effect of nifedipine (a: 0.3, b: 0.6 and c: 0.9 mg/kg); or C) Verapamil (d: 1.3, e: 2.6 and [: 3.9 mg/kg body wt.) on M. elengi-induced hypotension.

c

B

• •

e

d

C

f

Table 3. Effect of cumulative administration of nifedipine (0.3-0.9 mg/kg body wt.) and verapamil (1.3-3.9 mg/kg body wt.) on systolic, diastolic and mean arterial blood pressures in the presence of the methanolic extract (10 mg/kg) from Mimusops elengi in normotensive rats. Treatment

Control (n =67) M. elengi M. elengi + nifedipine

M. elengi + verapamil

(mg/kg)

Systolic blood pressure (mmHg)

Diastolic blood pressure (mmHg)

Mean arterial Fall in blood pressure MABP (MABP) (mmHg) (mmHg)

10 0.3 0.6 0.9 1.3 2.6 3.9

142.1 ± 113.2 ± 128.8 ± 136.4 ± 136.0 ± 129.7 ± 137.9 ± 137.6 ±

112.9 ± 1.6 78.8 ± 3.8 87.2 ± 4.5 n .s. 98.4 ± 3.9 n .s . 113.0 ± 8.1" 85.2 ± 4.1 n .s . 95.2 ± 5.7 n .s. 99.6 ± 4.4"

124.4 ± 1.3 90.3 ± 4.3 101.0 ± 4.3 n.s 111.1 ± 4.2" 118.3 ± 5.3"" 100.0 ± 4.4 n .s. 109.4 ± 5.3*" 112.3 ± 4.2""

Dose

1.5 5.7 5.0 n .s. 6.0" ':. 12.5" 5.4" 5.2"" 4.4**"

0 33.4 23.4 13.8 8.2 24.4 15.0 12.1

± ± ± ± ± ± ±

5.1 4.3 3.8 3.5 4.4 5.3 4.2

Inhibition of fall in MABP (%)

o 32.3 ± 58.7 ± 80.6 ± 30.5 ± 54.3 ± 63.6 ±

11.0 n .s. 11.0" 11.3 ,." 10.7 n .s. 13.5" * 12.5''''

Values represent the mean ± SEM of 5-67 experiments. Asterisks indicate significant changes at P < 0.05", P < 0.01 *" and P < 0.005""" as compared to M. elengi responses. significant difference.



References

Banerji, R., Srivastava, A. K., Misra, G., Nigam, S. K., Singh, S., Nigam, S. c., Saxena, R. c.: Steroid and triterpenoid saponins as spermicidal agents. Indian Drugs 17: 6-8, 1979. Banerji, R., Prakash, D., Patnaik, G. K., Nigam, S. K.: Spasmolytic activity of saponins. Indian Drugs 20: 51-54,1982.

n.s.

non-

Core, E. L.: "Plant Taxonomy". Prentice Hall, Englewood Cliffs, N] pp. 390, 1955. Dar, A., Behbahanian, S., ]ahan, N., Malik, A.: Hypotensive effect of Mimusops elengi on anaesthetized normotensive rats. XIIIth International Congress of Pharmacology, P 38 (70),1998. Deshmukh, S. K.: A note on mycotoxicity of some essential oils. Fitoterapia 57: 295-297, 1986.

378

A. Dar et al.

Donald, W. D., Walter, A. E, Boyd, E. G., Robert, G. c. Pharmacological profiles of minoxidil, a new hypotension agent.]. Pharmacal. Exp. Ther. 184: 662-670, 1973. Ebeigbe, A. B., Ezimokhai, M.: Vascular smooth muscle responses in pregnancy-induced hypertension. Trends Pharmacal. Sci. 9: 455-457, 1988. Endo, M., lino, M., Kobayashi, T., Yamamoto, T.: "Control of calcium release in smooth muscle cells", in: Frontiers in Smooth Muscle Research (Ed. N. Sperelakis and J. D. Wood), Alan R. Liss, Inc., New York, pp. 193-204, 1990. Greenberg, S. S., Wang, Y, Xie, J., Diecke, E P. J., Curro, G. A., Smartz, L., Rammazzatto, L.: "Calcium-dependent mechanisms of contraction in canine lingual artery to U46619", in: Regulation of Smooth Muscle Contraction (Ed. R. S. Moreland), Plenum Press, New York, pp. 491-498, 1991. Hills, A. E: Economic Botany. McGraw-Hill, London, pp. 147-433,1952. Jahan, N., Ahmad, W., Malik, A.: Phytochemistry of the Genus Mimusops.]. Chem. Soc. Pak. 18: 51, 1996. Kalsner, S.: Vasodilator action of calcium antagonists in coronary arteries in vitro. ]. Pharmacal. Exp. Ther. 281: 634-642, 1997. Kanjanapothi, Duangta, Tjasen, Panee: Mechanism of diuretic action of Pikhum (Mimusops elengi) flower extract. Chiang Mai Med. Bull. 10: 89-97, 1971.

Kimura, Y, Fukui, H., Tanaka, M., Okamoto, M., Morino, A., Miuru, A., Kimura, A., Enomoto, H.: Antihypertensive effects of a new dihydropyridine calcium antagonist. Arzneimittelforsch. Drug Res. 36: 1329-1335, 1986. Misra, G., Nigam, S. K., Mitra, C. R.: Studies on Mimusops species. Planta Med. 26: 155, 1974. Perry, L. M.: Medicinal Plants of East and South East Asia. Cambridge University Press London, pp. 376-377, 1980. Rasmussen, H., Haller, H., Takuwa, Y, Kelley, G., Park, S.: "Messenger Ca2 +, protein kinase C, and smooth muscle contraction", in: Frontiers in Smooth Muscle Research, (Ed. Sperelakis and J. D. Wood), Alan R. Liss, Inc., New York,pp. 89-106, 1990. Walpole, R. E.: Introduction to Statistics. Macmillan Publishing Company, Inc., New York, Third Edition, pp. 247-304, 1982.



Address

A. Dar, H. E. J. Research Institute of Chemistry, International Center for Chemical Sciences, University of Karachi, Pakistan E-mail: [email protected]