By V. J. DHAR,V. S. MATHUR and S. K. GARG
Abstract
Daucus carota Linn. (seeds) has been reported to possess antifertility activity 1966). GARGand GARG(1971) have reported (CHOPRAet al., 1958; CHAUDHURY, antifertility activity in the alcoholic and aqueous extracts of the seeds of this plant in female albino rats. These two extracts and the perroleurn ether extract were subjected to chromatographic fractionation as GARG and MATHUR(1972) reported encouraging antifertility activity in five chromatographic fractions. The present paper deals with the general pharmacology of the chloroform fraction of the petroleum ether extract of this plant possessing significant antifertility activity in rats.
Experimental The seeds of Dauctrs carota were air dried, powdered and extracted with petroleum ether (b. p. 60-80' C) in a soxhlet apparatus. The extract obtained was resolved chromatographically by eluting the column from non-polar solvents. The solvents used for elution were petroleum ether, benzene, chloroform, methanol and their mixtures. The chloroform fraction (CF) was dried on a water bath and was used for the present study. The CF was dissolved in 10% alcohol. For each parameter, control experiments were done with 10% alcohol. The effect of this fraction was studied on the following preparations.
Isolated rat rrterus: Isolated rat uterus preparation was put up as described by HOLTON(1948). It was suspended in a bath containing Dejalon's solution at 30' C. The records were obtained with a frontal writing lever on a smoked drum. The magnification being 5-7 times. The changes produced by CF on (a) spontaneous activity (bath temperature 37O C), (b) on the responses p r ~ d ~ by ~ elow a doses of oxytodn (final concentration 0.02 mylrnl) and (c) on the responses produced by higher doses of oxytocin (final concentration 0.2 mplml) were studied, for these observations oxytocin was allowed to remain in the bath for ten minutes. One minute after the addition of oxytocin, the CF was added, each observation was made for ten minutes.
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The chloroform fraction (CF) of the petroleum ether extract of the seeds o f Daucus carota showed inhibitory effect on spontaneous uterine motility and oxytocin induced contractions. Histamine induced contraction of guinea pig ileum was inhibited. No significant effect was observed on skeletal muscle, isolated guinea pig heart, ECG of guinea pigs and rabbit blood pressure.
Pharmacological Studies o n Daucus carota
13
Isolated guinea pig ileum: Isolated guinea pig ileum preparation was set up as per the technique described by PERRY(1970). T h e magnification was 7 times. T h e effect of CF was studied o n (a) responses produced by histamine (final concentration 0.02 pg/ml) and (b) on the tetanic contractions produced by higher doses of histamine (final concentration 0.2 pg/ml). lsolated frog rectus abdominis muscle: This preparation was set up as described by PERRY(1970). T h e effect of CF on the responses of acetylcholine was studied. Isolated guinea pig heart: T h e isolated guinea pig heart was set up (PERRY,1970). The solution used was Ringer Locke. T h e solution was oxygenated and temperature kept a t 37' C. T h e effect of CF was studied a t different dose levels.
Rabbit blood pressure: Healthy rabbits weighing between 1.5 to 2.0 kg of either sex were anaesthetized with 10-15 ml of 25% urethane. T h e jugular vein was cannulated for the administration of saline and CF. T h e carotid artery was cannulated and connected to the manometer.
Results T h e effect of CF on isolated rat uterus is presented in Table I. Both spontaneous activity and oxytocin evoked responses were inhibited by the CF. T h e inhibition was dose dependent, 0.5 mglml of the CF produced a marked inhibition on oxytocin induced response of the rat uterus (Fig. 1).
'OCIN
6 2 mu OXY
A L C O +4 0 L
10
'/
w
0 2 m u OXY
W
I
O2mu
o x YL
.5mg
c F-
..
1
WO2rnu OXY I .I m~ A
W
i
Figure 1. Action of chloroform fraction (CF) of the petroleum ether extract of Dauccis cnrotn (seeds) on the isolated rat uterus (spasmodic contractions)
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ECG of guinea pigs: Guinea pigs: Guinea pigs weighing behveen 750 to 1000 grams of either sex were anaestetized with urethane intra-peritoneally (25% urethane, 5-6 mllkg). T h e jugular vein was cannulated for administration of saline and CF. ECG records from lead I and lead 11 were obtained.
14
Dhar, Mathur and Garg
Planta medica Vol. 28 1975 Table I
Effect of chloroform fraction (.CF) on spontaneous and oxytocin induced responses of isolated rat uterus
Spontaneous contractions
Alcohol - 10% Responses produced by lower doses of oxytocin Alcohol - 10% Responses produced by higher doses of oxytocin Alcohol - 10%
0.1 0.2 0.4 0.1 rnl 0.1 0.2 0.4 0.1 ml 0.4 0.5 0.1 ml
Average height of contraction in mm*" Before After
Oxytocin" mufrnl
-
64.66 63.33 56171 27.34 36.00 35.70 50.00 51.00 113.10 106.00 106.00
-
0.02 0.02 0.02 0.02 0.10 0.20 0.20
w
63.33 57.40 Nil 20.29 34.00 13.50 Nil 47.00 Nil 29.50 98.50
Percentage inhibition 2.05 9.36 100.00 25.78 5.55 62.10 100.00 7.84 100.00 72.17 7.75
" Final concentrattion in 10 ml bath "" T h e values are the means obtained from three observations in each case T a b l e I1 Effect of chloroform fraction (CF) on the responses produced by histamine pig ileum
Responses produced by histamine
Alcohol - 10% Tetanic contractions Alcohol - 10% . -
Dose of CF" mglml
Histamine" ~ d m l
0.1 0.2 0.4 0.5 0.1 ml 0.5 0.1 ml
0.02 0.02 0.02 0.02 0.02 0.20 0.20
-
-
-
-
011 isolated
Average height of contraction in rnrnSg Before After 51 51 51 60 63 104.66 87.33 -
37.0 26.5 8.5 6.0 59.0 17.17 59.66
guinea
Percentage inhibition 27.45 48.03 83.33 90.00 6.34 93.14 32.83
-
" Final concentration in 10 ml bath '* T h e values are the means obtained from three observations in each cases
Table I1 shows the effect of graded doses of CF on histamine evoked responses on isolated guinea pig ileum. Here again a dose dependent reduction was observed. When tested on the acetylcholine evoked responses on isolated frog rectus abdominis muscle, 0.5 mg/ml of CF did not have any effect.
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Dose of CF" mg/ml
Pharmacological Studies on Daucus carota
15
CF a t a dose of 0.2 mg/ml did not produce any effect o n isolated guinea pig heart. However, when the dose of CF was increased, there was a progressive reduction in the amplitude of the heart. T h e reduction with CF and the solvent (10% alcohol) was of the same magnitude. No change was observed in the ECG of guinea pigs with 2.5-25 m g m l of the CF. At a dose of 0.4 and 0.8 mg/ml, CF did not produce any change in the rabbit blood pressure.
T h e chloroform fraction (CF) of the petroleum ether extract of Daucus carota (seeds) was found t o possess marked inhibitory effect on oxytocin induced contractions o n isolated rat uterus and histamine induced contractions on isolated guinea pig ileum. T h e CF also inhibited the spontaneous activity of the isolated rat uterus. No significant effect was seen on the skeletal muscle, isolated guinea pig heart, ECG of guinea pigs and rabbit blood pressure. GARGand MATHUR (1972) have reported that the CF possess antifertility activity in female albino rats. T h e inhibitory effect seen o n the rat uterus with the CF may contribute towards this activity. This could be brought about by delayed transport of the fertilized egg in the uterine lumen so that it does not reach the site o f nidation a t the precise time o r may be due to inhibition of sperm transport. W e are now in process of studying acute and sub-acute toxicity and possible teratogenic effects. If all these are favourable, then the drug (CF) would be put for.clinica1 trial. Thanks are due to Indian Council of Medical Research, New Delhi, for the financial support.
References CHAUDBURY, R. R.: Plants with Possible Antifertility Activity (p. 5), Special Report Series No. 55, Indian Council of Medical Research, New Delhi (1966) CHOPRA,R. N., CHOPRA,I. C., HANDA,K. L., and KAPUR,L. D.: Indigenous Drugs of India, (2nd ed., p. 568), U. N. Dhur & Sons Private Ltd., Calcutta, India (1958) GARG,S. K. and GARC,G. P.: Ind. Jour. Med. Res. 59,302-306 (1971) GARG,S. K. and MATHUR,V. S.: Jour. Reprod. Fertil. 31,143-145 (1972) HOLTON, P.: Brit. J. Pharmacol. 3,328 (1948) PERRY,W. L. M.: Pharmacological Experiments on Isolated Preparations (2nd ed., p. 38, 62 & 116), E & S Livingstone, Edinburgh and London (1970) Address: Dr. S. K. Garg, Department of Pharmacology, Postgraduate Institute of Medical Education and Research, Chnndigarh, lndin
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Discussion
Abstract
Daucus carota Linn. (seeds) has been reported to possess antifertility activity 1966). GARGand GARG(1971) have reported (CHOPRAet al., 1958; CHAUDHURY, antifertility activity in the alcoholic and aqueous extracts of the seeds of this plant in female albino rats. These two extracts and the perroleurn ether extract were subjected to chromatographic fractionation as GARG and MATHUR(1972) reported encouraging antifertility activity in five chromatographic fractions. The present paper deals with the general pharmacology of the chloroform fraction of the petroleum ether extract of this plant possessing significant antifertility activity in rats.
Experimental The seeds of Dauctrs carota were air dried, powdered and extracted with petroleum ether (b. p. 60-80' C) in a soxhlet apparatus. The extract obtained was resolved chromatographically by eluting the column from non-polar solvents. The solvents used for elution were petroleum ether, benzene, chloroform, methanol and their mixtures. The chloroform fraction (CF) was dried on a water bath and was used for the present study. The CF was dissolved in 10% alcohol. For each parameter, control experiments were done with 10% alcohol. The effect of this fraction was studied on the following preparations.
Isolated rat rrterus: Isolated rat uterus preparation was put up as described by HOLTON(1948). It was suspended in a bath containing Dejalon's solution at 30' C. The records were obtained with a frontal writing lever on a smoked drum. The magnification being 5-7 times. The changes produced by CF on (a) spontaneous activity (bath temperature 37O C), (b) on the responses p r ~ d ~ by ~ elow a doses of oxytodn (final concentration 0.02 mylrnl) and (c) on the responses produced by higher doses of oxytocin (final concentration 0.2 mplml) were studied, for these observations oxytocin was allowed to remain in the bath for ten minutes. One minute after the addition of oxytocin, the CF was added, each observation was made for ten minutes.
Downloaded by: Universite Laval. Copyrighted material.
The chloroform fraction (CF) of the petroleum ether extract of the seeds o f Daucus carota showed inhibitory effect on spontaneous uterine motility and oxytocin induced contractions. Histamine induced contraction of guinea pig ileum was inhibited. No significant effect was observed on skeletal muscle, isolated guinea pig heart, ECG of guinea pigs and rabbit blood pressure.
Pharmacological Studies o n Daucus carota
13
Isolated guinea pig ileum: Isolated guinea pig ileum preparation was set up as per the technique described by PERRY(1970). T h e magnification was 7 times. T h e effect of CF was studied o n (a) responses produced by histamine (final concentration 0.02 pg/ml) and (b) on the tetanic contractions produced by higher doses of histamine (final concentration 0.2 pg/ml). lsolated frog rectus abdominis muscle: This preparation was set up as described by PERRY(1970). T h e effect of CF on the responses of acetylcholine was studied. Isolated guinea pig heart: T h e isolated guinea pig heart was set up (PERRY,1970). The solution used was Ringer Locke. T h e solution was oxygenated and temperature kept a t 37' C. T h e effect of CF was studied a t different dose levels.
Rabbit blood pressure: Healthy rabbits weighing between 1.5 to 2.0 kg of either sex were anaesthetized with 10-15 ml of 25% urethane. T h e jugular vein was cannulated for the administration of saline and CF. T h e carotid artery was cannulated and connected to the manometer.
Results T h e effect of CF on isolated rat uterus is presented in Table I. Both spontaneous activity and oxytocin evoked responses were inhibited by the CF. T h e inhibition was dose dependent, 0.5 mglml of the CF produced a marked inhibition on oxytocin induced response of the rat uterus (Fig. 1).
'OCIN
6 2 mu OXY
A L C O +4 0 L
10
'/
w
0 2 m u OXY
W
I
O2mu
o x YL
.5mg
c F-
..
1
WO2rnu OXY I .I m~ A
W
i
Figure 1. Action of chloroform fraction (CF) of the petroleum ether extract of Dauccis cnrotn (seeds) on the isolated rat uterus (spasmodic contractions)
Downloaded by: Universite Laval. Copyrighted material.
ECG of guinea pigs: Guinea pigs: Guinea pigs weighing behveen 750 to 1000 grams of either sex were anaestetized with urethane intra-peritoneally (25% urethane, 5-6 mllkg). T h e jugular vein was cannulated for administration of saline and CF. ECG records from lead I and lead 11 were obtained.
14
Dhar, Mathur and Garg
Planta medica Vol. 28 1975 Table I
Effect of chloroform fraction (.CF) on spontaneous and oxytocin induced responses of isolated rat uterus
Spontaneous contractions
Alcohol - 10% Responses produced by lower doses of oxytocin Alcohol - 10% Responses produced by higher doses of oxytocin Alcohol - 10%
0.1 0.2 0.4 0.1 rnl 0.1 0.2 0.4 0.1 ml 0.4 0.5 0.1 ml
Average height of contraction in mm*" Before After
Oxytocin" mufrnl
-
64.66 63.33 56171 27.34 36.00 35.70 50.00 51.00 113.10 106.00 106.00
-
0.02 0.02 0.02 0.02 0.10 0.20 0.20
w
63.33 57.40 Nil 20.29 34.00 13.50 Nil 47.00 Nil 29.50 98.50
Percentage inhibition 2.05 9.36 100.00 25.78 5.55 62.10 100.00 7.84 100.00 72.17 7.75
" Final concentrattion in 10 ml bath "" T h e values are the means obtained from three observations in each case T a b l e I1 Effect of chloroform fraction (CF) on the responses produced by histamine pig ileum
Responses produced by histamine
Alcohol - 10% Tetanic contractions Alcohol - 10% . -
Dose of CF" mglml
Histamine" ~ d m l
0.1 0.2 0.4 0.5 0.1 ml 0.5 0.1 ml
0.02 0.02 0.02 0.02 0.02 0.20 0.20
-
-
-
-
011 isolated
Average height of contraction in rnrnSg Before After 51 51 51 60 63 104.66 87.33 -
37.0 26.5 8.5 6.0 59.0 17.17 59.66
guinea
Percentage inhibition 27.45 48.03 83.33 90.00 6.34 93.14 32.83
-
" Final concentration in 10 ml bath '* T h e values are the means obtained from three observations in each cases
Table I1 shows the effect of graded doses of CF on histamine evoked responses on isolated guinea pig ileum. Here again a dose dependent reduction was observed. When tested on the acetylcholine evoked responses on isolated frog rectus abdominis muscle, 0.5 mg/ml of CF did not have any effect.
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Dose of CF" mg/ml
Pharmacological Studies on Daucus carota
15
CF a t a dose of 0.2 mg/ml did not produce any effect o n isolated guinea pig heart. However, when the dose of CF was increased, there was a progressive reduction in the amplitude of the heart. T h e reduction with CF and the solvent (10% alcohol) was of the same magnitude. No change was observed in the ECG of guinea pigs with 2.5-25 m g m l of the CF. At a dose of 0.4 and 0.8 mg/ml, CF did not produce any change in the rabbit blood pressure.
T h e chloroform fraction (CF) of the petroleum ether extract of Daucus carota (seeds) was found t o possess marked inhibitory effect on oxytocin induced contractions o n isolated rat uterus and histamine induced contractions on isolated guinea pig ileum. T h e CF also inhibited the spontaneous activity of the isolated rat uterus. No significant effect was seen on the skeletal muscle, isolated guinea pig heart, ECG of guinea pigs and rabbit blood pressure. GARGand MATHUR (1972) have reported that the CF possess antifertility activity in female albino rats. T h e inhibitory effect seen o n the rat uterus with the CF may contribute towards this activity. This could be brought about by delayed transport of the fertilized egg in the uterine lumen so that it does not reach the site o f nidation a t the precise time o r may be due to inhibition of sperm transport. W e are now in process of studying acute and sub-acute toxicity and possible teratogenic effects. If all these are favourable, then the drug (CF) would be put for.clinica1 trial. Thanks are due to Indian Council of Medical Research, New Delhi, for the financial support.
References CHAUDBURY, R. R.: Plants with Possible Antifertility Activity (p. 5), Special Report Series No. 55, Indian Council of Medical Research, New Delhi (1966) CHOPRA,R. N., CHOPRA,I. C., HANDA,K. L., and KAPUR,L. D.: Indigenous Drugs of India, (2nd ed., p. 568), U. N. Dhur & Sons Private Ltd., Calcutta, India (1958) GARG,S. K. and GARC,G. P.: Ind. Jour. Med. Res. 59,302-306 (1971) GARG,S. K. and MATHUR,V. S.: Jour. Reprod. Fertil. 31,143-145 (1972) HOLTON, P.: Brit. J. Pharmacol. 3,328 (1948) PERRY,W. L. M.: Pharmacological Experiments on Isolated Preparations (2nd ed., p. 38, 62 & 116), E & S Livingstone, Edinburgh and London (1970) Address: Dr. S. K. Garg, Department of Pharmacology, Postgraduate Institute of Medical Education and Research, Chnndigarh, lndin
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Discussion
