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Antiamoebic activity of Piper longum fruits against Entamoeba histolytica in vitro and in vivo
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~ Journal of ~ ETHNO PHARMACOLOGY
E LSEVl ER
Journal of Ethnopharmacology50 (1996) 167-170
Antiamoebic activity of Piper longum fruits against Entamoeba histolytica in vitro and in vivo S h e e l a G h o s h a l *a, B . N . K r i s h n a P r a s a d a, V. L a k s h m i b aDivision of Microbiology, Central Drug Research Institute, Lucknow 226001, India bDivision of Medicinal Chemistry, Central Drug Research Institute, Lucknow 226001, India
Received 3 July 1995; revised 8 January 1996; accepted 8 January 1996
Abstract
The fruits of Piper longum used in traditional remedies against intestinal distress have been tested for their efficacy against experimental caecal amoebiasis of rats. The ethanolic extract, hexane fraction, n-butanol soluble fraction exerted in vitro amoebicidal action at 1000 gg/ml and the chloroform fraction at 500 #g/ml. The ethanolic extract and piperine, a pure compound, from this plant material cured 90% and 40% of rats with caecal amoebiasis, respectively. Keywords: Antiamoebic; Entamoeba histolytica; Piper longum; Piperine
1. Introduction
Amoebiasis caused by the protozoan parasite Entamoeba histolytica affects approximately 10% of the world's population, the prevalence being higher in tropical and subtropical countries (Walsh, 1986). A common feature of this disease is the occurrence of morbidity affecting the quality of life and the pace of developmental activities. A number of agents possessing potent amoebicidal action against trophozoites of E. histolytica are available for therapeutic use. These are known to provide prompt symptomatic relief in cases suffering from intestinal dysentery. This is evidenced by the return to normal condition of * Corresponding author.
form and frequency of the evacuated stools. However, the benefits occuring are short lived. Relapses, despite adequate treatment, often occur (Singh, 1975). Further, the toxicity of the antiamoebic agents (Martinez Palomo, 1982) along with the reported occurance of metronidazole resistance of the human pathogenic bacteria Helicobacter pylori (van Zwet et al., 1994) are yet reasons against repeated use of an anti-amoebic agent in cases of relapse and reinfections, thus reinforcing the need for developing safe and effective alternative anti-amoebic agents. This communication deals with the antiamoebic action of the plant Piper longum Linn, 1753, family Piperaceae, which is used in the traditional system of medicine against intestinal ailments (Satyavati, et al., 1987).
0378-8741/96/$15.00 © 1996 ElsevierScience Ireland Ltd. All rights reserved PII 0378-8741(96)01382-7
168
S. Ghoshal et al./Journal of Ethnopharrnacology 50 (1996) 167-170
2. Materials and methods
2.1. Test agents Piper longum is an indegenously growing plant in India (Chopra et al., 1956; Satyawati et al., 1987). Its voucher specimen is kept at the Division of Botany, CDRI, Lucknow, and its reference number is Botany Registration Serial No. 6250. Its dried fruits were purchased from the local market. The powdered fruit material (1.0 kg) was extracted with 95% ethanol (4 x 3.0 1) and the combined ethanolic extract after filtration was concentrated under reduced pressure below 50°C to a viscous oil (50 g), which was finally dried under high vacuum to remove any traces of the solvent. The ethanolic extract was fractionated into hexane soluble (19 g), chloroform soluble (5 g), nbutanol soluble (10 g) and n-butanol insoluble (11 g) fractions by extracting with different solvents successively. A pure compound, piperine, was isolated from the hexane and chloroform fractions. All the above fractions were screened for antiamoebic activity both in vitro and in the rat caecum. The stock solutions of the test materials were prepared by dissolving in distilled water using small quantities of dimethyl formamide and sterilized by autoclaving at 1.1 kg/cm 2 for 15 min. Serial dilutions were prepared aseptically for in vitro tests.
and Krishna Prasad, 1973). To 8.0 ml of the medium in 16 × 125 mm screw-capped test tubes, 1 ml of the test material was added and 1 ml of the amoebic inoculum containing about 10 x 105 trophozoites of E. histolytica counted on a haemocytometer was added. The tubes were incubated at 37°C and the walls of the tubes examined microscopically for amoebae at 24 h intervals up to 72 h. Also, the sediments from the tubes were examined for any viable amoebae at the end of the incubation period. Viable amoebae remained refractile and adhered to the sides of the test tube, while the dead ones rounded up, shrank in size and settled at the bottom of the test tube. In cases of doubt, the sedimented amoebae were inoculated into fresh medium and examined for growth. Cent percent death of the amoebae was used to establish the amoebicidal action of the test agents.
2.2. E. histolytica Axenic culture of E. histolytica, NIH 200, was originally obtained from Dr. L.S. Diamond and maintained in this Institute since 1972, and a polyxenic culture, VP, freshly isolated from the faecal sample of a patient suffering from acute amoebic dysentery, were used in the study. E. histolytica, NIH 200, was maintained axenically in modified TPS-1 medium containing 0.2% Lcysteine hydrochloride (Singh, Das and Dutta, 1973) and strain VP in modified Boeck and Drobohlav medium (Dutta and Rao, 1966).
2.4. Experimental production of caecal amoebiasis of rats The therapeutic efficacy of a test material was determined in the rat model. Druckray rats (21 days old) weighing 18-20 g, obtained from the animal house of the Institute, were fed autoclaved rice for seven days. They were then inoculated intracaecally during laparotomy under ether anaesthesia with 0.2 to 0.3 ml of amoebic inoculum pooled from 24 h-old cultures containing about 10 x 105 trophozoites of E. histolytica. It has been our experience that E. histolytica infection in rat caecum is determined by the occurance of mildly acidic pH of caecal contents, which ensures absence of free ammonia (Krishna Prasad and Indu Bansal, 1982, 1983; Krishna Prasad et al., 1984). These observations have been confirmed by Leitch (1988). Based on these observations, efforts were made to create conditions in the rat caceum suitable for the development of E. histolytica infections and this was achieved by feeding the rats with autoclaved rice diet (Krishna Prasad et al., 1986). The rats weighed about 30 g when drug administration was initiated.
2.3. In vitro action of test agents The in vitro action of the test materials against trophozoites of E. histolytica was determined in accordance with the method described earlier (Das
2.5. Therapeutic evaluation of test agents The test material was suspended in gum acacia solution in distilled water and administered orally at 900 mg/kg body weight with the help of a
S. Ghoshal et al. / Journal of Ethnopharmaeology 50 (1996) 167-170
blunted 18 gauge hypodermic needle attached to a syringe. Each rat received 0.5 ml containing 27 mg of the test agent once daily for 5 consecutive days, beginning 48 h after infection. The pure compound, piperine, was tested at a dose of 100 mg/kg body weight (3 mg/rat). This dose was selected to compare it with the ED 100 of metronidazole which was used as a drug standard (Krishna Prasad et al., 1986). The results were evaluated 24 h after administering the final dose of the test material by sacrificing the animals with excess of ether anaesthesia and examining the contents and scrapings of the caecal wall for trophozoites of E. histolytica. The caecal scoring was determined in accordance to the criteria of Neal (1951). In cases of doubt, the caecal contents and scrapings of the caecal wall were cultured in fresh media and examined for amoebae. 3. Results and discussion The effect of P. longum against trophozoites of E. h&tolytica in vitro and against experimental caecal amoebiasis of rats is described in Table 1. In vitro activity was recorded for all test samples except for the n-butanol insoluble fraction and the pure compound, piperine. The therapeutic efficacy of the test agents against caecal amoebiasis of rats presented interesting results. The crude ethanolic extract exhibited the highest efficacy with 90%
169
cures. The in vitro and in vivo results for the hexane fraction and piperine did not corelate with each other. The former showed only in vitro amoebicidal activity, while the pure compound, piperine, despite being non-amoebicidal in vitro, effected 40% cures. The caecum of rats receiving ethanol extract appeared normal. The walls were thin and comparable to those treated with metronidazole (dose 100 mg/kg body weight). The contents, however, were also normal but less formed as compared to those receiving metronidazole treatment. Chloroform fraction, n-butanol soluble fraction and piperine helped to restore near normality to the caecum of the infected rats and the results were comparable to those receiving metronidazole, 50 mg/kg body weight. These results are significant when compared with the untreated rat caecum, which was shapeless due to localised thickenings, ulcers on the walls and presence of mucus with very little faecal matter as contents. It is apparent from these results that P. longum possesses therapeutic efficacy against caecal amoebiasis of rats despite its low in vitro amoebicidal activity. Such results are not uncommon in tests with agents used in traditional remedies. The root powder and ethanolic extract of Coleus forskohlii did not possess in vitro amoebicidal action against tropohozoites of E. histolytica but were effective against caecal
Table I Effect of P. longum against trophozoites of E. h&tolytica in vitro and against experimental caceal amoebiasis of rats Test materials
In vitro amoebicidal activity (/zg/ml)
Dose mg/kg/b.w.
EtOH ext. Hexane fr. Chloroform fr. n-BuOH (soluble fr) n-BuOH (insoluble fr.) Piperine Metronidazole
1000 1000 500 1000 NA a NA 8
Untreated control
-
900 900 900 900 900 100 50 100 --
alnactive at 1000 t~g/ml; bFor rats showing E. histolytica infection.
Caecal amoebiasis of rats No. of rats cleared/treated (% cured)
Average caecal score b Contents
Wall
Total
18/20 (90) 0/10 (0) 14/20 (70) 12/20 (60) 0/20 (0) 8/20 (40) 12/20 (60) 20/20 (100) 0/20 (0)
0.5 2.0 1.5 1.25 2.00 1.33 1.00 0.0 3.4
0.0 1.0 1.25 0.75 2.00 1.00 1.00 0.0 3.4
0.5 3.0 2.75 2.0 4.0 2.33 2.00 0.0 6.8
170
S. Ghoshal et al./ Journal of Ethnopharmacology 50 (1996) 167-170
amoebiasis of rats (Varma et al., 1990). Similarly, the ethanolic extracts of seeds, leaves, roots, flowers and stem of Nyctanthes arbor-trist showed therapeutic efficacy against caecal amoebiasis of rats despite their lack of in vitro amoebicidal activity (Chitravanshi et al., 1992). It is interesting to add here that exposure of N. arbor-tritis materials to 1 N HCI makes them amoebicidal in vitro, indicating that these are predrugs and their intestinal transit modifies them to become amoebicidal (unpublished data). In the present study, Piperine appears to have acquired amoebicidal property and the mechanism responsible for this transformation needs to be worked out. Apart from this, the crude P. longum may possess certain compounds other than piperine which is/are responsible for their enhanced efficacy.
Acknowledgements The authors thank Dr. V.P. Kamboj, FNA, Director, CDRI, Lucknow, Dr. A.P. Bhaduri, Head, Division of Medicinal Chemistry, Dr. N.B. Singh, Head, Division of Microbiology for their support in carrying out this work. Our thanks are also due to Dr. B.N. Mehrotra for supplying the dry fruits of P. longum. Thanks are also due to Mr. A.K. Gupta, Mr. A.P. Singh and Mr. U.C. Pandey for providing technical assistance. Lastly, it is a pleasure to thank Prof. B.N. Dhawan, FNA, and former Director, CDRI, for his valuable comments and to Mrs. Padma. V. Nair for typing the manuscript.
References Chitravanshi, V.C., Singh, A.P., Ghoshal, S., Krishna Prasad, B.N., Srivastava, V. and Tandon, J.S. (1992) Therapeutic action of Nyctanthes arbor-tristis against caecal amoebiasis of rat. Int. J. Pharmacog. 30(1), 71-85. Chopra, R.N., Nayar, S.L. and Chopra, I.C. (1956) Glossary of Indian Medicinal Plants CSIR Publication, New Delhi, 194. Das, S.R. and Krishna Prasad, B.N. (1973) A reliable and reproducible method for testing direct acting amoebicides against axenic Entamoeba histolytica. Curt. Sci., 42, 796-797.
Dutta, G.P. and Rao, M.V.K. (1966) Use of bovine serum as a substitute for horse serum for growing Entamoeba histolytica in modified Boeck and Drbohlav medium. Indian J. of Microbiol. 6, 83-86. Krishna Prasad, B.N. and lndu Bansal (1982) Effect of caceal pH of rat on the development of experimental Entamoeba histolytica infection. Indian. J. Parasitol. 6, 97-98. Krishna Prasad, B.N. and Indu Bansal (1983) Interrelationship between faecal pH and susceptibility to Entamoeba histolytica infection of rats. Trans. Roy. Soc. Trap. Med. Hyg. 77, 271-274. Krishna Prasad, B.N., lndu Bansal, Nandi, R.C., Singh, S. and Sarin, J.P.S. (1984) Correlation of naturally occuring concentration of free ammonia in the caecum of rats with resistance to experimental Entamoeba histolytica infection. Curr. Sci. 53, 371-374. Krishna Prasad, B.N., Indu Bansal, Nandi, R.C., Singh, S. and Sarin, J.P.S. (1986) Effect of caecal pH on the efficacy of drugs against Entamoeba histolytica in caecal amoebiasis of rats. Indian J. Parasitol. 10(1), 33-38. Leitch, G.J. (1988) Intestinal luminal and mucosal microclimate H- and NH3 + concentrations as factors in the etiology of experimental amoebiasis. Am. J. Trop. Med. Hyg. 38, 480-496. Martinez Palomo, A. (1982) The Biology of Entamoeba histolytica. Published by Research Studies Press, A division of John Wiley and Sons Ltd., Chichester, New York, Brisbane, Toronto, Singapore. Neal, R.A. (195 I) Some observations on variation of virulence and response to chemotherapy of strains of Entamoeba histolytica. Trans. Roy. Soc. Trop. Med. Hyg, 44, 439-452. Satyavati, G.V., Gupta, A.K. and Tandon, N. (1987) Medicinal Plants of India, ICMR Publications, New Delhi, 2, 426-456. Singh, B.N., Das, S.R. and Dutta, G.P. (1973) Importance of O-R potential in initiating cultures of axenically grown Entamoeba histolytica from small inocula. Curr. Sci. 42, 227-230. Singh, B.N. (1975) Pathogenic and non-pathogenic amoebae. Published by the Macmillan Press Ltd., London and Bassingstoke. Van Zwet, A.A., Thijs, J.C., de Vries, W.S., Schiphuis, J. Snijder, J.A.M. (1994) In vitro studies on stability and development of metronidazole resistance in Helicobacter pylori. Antimicrob. Agents Chernother. 38 (2), 360-362. Varma, N., Srivastava, V., Tandon, J.S., Krishna Prasad, B.N. and Chitravanshi, V.C. (1990) Effect of Coleus forskohlii against caecal amoebiasis of rats. Int. J. Crude Drug Res. 28(1), 1-3. Walsh, A. (1986) Problems in recognition and diagnosis of Amoebiasis: Estimation of the global magnitude of morbidity and mortality. Rev. Inf. Dis. 8(2), 228-238.
•
~
~
+
:~
!
~ Journal of ~ ETHNO PHARMACOLOGY
E LSEVl ER
Journal of Ethnopharmacology50 (1996) 167-170
Antiamoebic activity of Piper longum fruits against Entamoeba histolytica in vitro and in vivo S h e e l a G h o s h a l *a, B . N . K r i s h n a P r a s a d a, V. L a k s h m i b aDivision of Microbiology, Central Drug Research Institute, Lucknow 226001, India bDivision of Medicinal Chemistry, Central Drug Research Institute, Lucknow 226001, India
Received 3 July 1995; revised 8 January 1996; accepted 8 January 1996
Abstract
The fruits of Piper longum used in traditional remedies against intestinal distress have been tested for their efficacy against experimental caecal amoebiasis of rats. The ethanolic extract, hexane fraction, n-butanol soluble fraction exerted in vitro amoebicidal action at 1000 gg/ml and the chloroform fraction at 500 #g/ml. The ethanolic extract and piperine, a pure compound, from this plant material cured 90% and 40% of rats with caecal amoebiasis, respectively. Keywords: Antiamoebic; Entamoeba histolytica; Piper longum; Piperine
1. Introduction
Amoebiasis caused by the protozoan parasite Entamoeba histolytica affects approximately 10% of the world's population, the prevalence being higher in tropical and subtropical countries (Walsh, 1986). A common feature of this disease is the occurrence of morbidity affecting the quality of life and the pace of developmental activities. A number of agents possessing potent amoebicidal action against trophozoites of E. histolytica are available for therapeutic use. These are known to provide prompt symptomatic relief in cases suffering from intestinal dysentery. This is evidenced by the return to normal condition of * Corresponding author.
form and frequency of the evacuated stools. However, the benefits occuring are short lived. Relapses, despite adequate treatment, often occur (Singh, 1975). Further, the toxicity of the antiamoebic agents (Martinez Palomo, 1982) along with the reported occurance of metronidazole resistance of the human pathogenic bacteria Helicobacter pylori (van Zwet et al., 1994) are yet reasons against repeated use of an anti-amoebic agent in cases of relapse and reinfections, thus reinforcing the need for developing safe and effective alternative anti-amoebic agents. This communication deals with the antiamoebic action of the plant Piper longum Linn, 1753, family Piperaceae, which is used in the traditional system of medicine against intestinal ailments (Satyavati, et al., 1987).
0378-8741/96/$15.00 © 1996 ElsevierScience Ireland Ltd. All rights reserved PII 0378-8741(96)01382-7
168
S. Ghoshal et al./Journal of Ethnopharrnacology 50 (1996) 167-170
2. Materials and methods
2.1. Test agents Piper longum is an indegenously growing plant in India (Chopra et al., 1956; Satyawati et al., 1987). Its voucher specimen is kept at the Division of Botany, CDRI, Lucknow, and its reference number is Botany Registration Serial No. 6250. Its dried fruits were purchased from the local market. The powdered fruit material (1.0 kg) was extracted with 95% ethanol (4 x 3.0 1) and the combined ethanolic extract after filtration was concentrated under reduced pressure below 50°C to a viscous oil (50 g), which was finally dried under high vacuum to remove any traces of the solvent. The ethanolic extract was fractionated into hexane soluble (19 g), chloroform soluble (5 g), nbutanol soluble (10 g) and n-butanol insoluble (11 g) fractions by extracting with different solvents successively. A pure compound, piperine, was isolated from the hexane and chloroform fractions. All the above fractions were screened for antiamoebic activity both in vitro and in the rat caecum. The stock solutions of the test materials were prepared by dissolving in distilled water using small quantities of dimethyl formamide and sterilized by autoclaving at 1.1 kg/cm 2 for 15 min. Serial dilutions were prepared aseptically for in vitro tests.
and Krishna Prasad, 1973). To 8.0 ml of the medium in 16 × 125 mm screw-capped test tubes, 1 ml of the test material was added and 1 ml of the amoebic inoculum containing about 10 x 105 trophozoites of E. histolytica counted on a haemocytometer was added. The tubes were incubated at 37°C and the walls of the tubes examined microscopically for amoebae at 24 h intervals up to 72 h. Also, the sediments from the tubes were examined for any viable amoebae at the end of the incubation period. Viable amoebae remained refractile and adhered to the sides of the test tube, while the dead ones rounded up, shrank in size and settled at the bottom of the test tube. In cases of doubt, the sedimented amoebae were inoculated into fresh medium and examined for growth. Cent percent death of the amoebae was used to establish the amoebicidal action of the test agents.
2.2. E. histolytica Axenic culture of E. histolytica, NIH 200, was originally obtained from Dr. L.S. Diamond and maintained in this Institute since 1972, and a polyxenic culture, VP, freshly isolated from the faecal sample of a patient suffering from acute amoebic dysentery, were used in the study. E. histolytica, NIH 200, was maintained axenically in modified TPS-1 medium containing 0.2% Lcysteine hydrochloride (Singh, Das and Dutta, 1973) and strain VP in modified Boeck and Drobohlav medium (Dutta and Rao, 1966).
2.4. Experimental production of caecal amoebiasis of rats The therapeutic efficacy of a test material was determined in the rat model. Druckray rats (21 days old) weighing 18-20 g, obtained from the animal house of the Institute, were fed autoclaved rice for seven days. They were then inoculated intracaecally during laparotomy under ether anaesthesia with 0.2 to 0.3 ml of amoebic inoculum pooled from 24 h-old cultures containing about 10 x 105 trophozoites of E. histolytica. It has been our experience that E. histolytica infection in rat caecum is determined by the occurance of mildly acidic pH of caecal contents, which ensures absence of free ammonia (Krishna Prasad and Indu Bansal, 1982, 1983; Krishna Prasad et al., 1984). These observations have been confirmed by Leitch (1988). Based on these observations, efforts were made to create conditions in the rat caceum suitable for the development of E. histolytica infections and this was achieved by feeding the rats with autoclaved rice diet (Krishna Prasad et al., 1986). The rats weighed about 30 g when drug administration was initiated.
2.3. In vitro action of test agents The in vitro action of the test materials against trophozoites of E. histolytica was determined in accordance with the method described earlier (Das
2.5. Therapeutic evaluation of test agents The test material was suspended in gum acacia solution in distilled water and administered orally at 900 mg/kg body weight with the help of a
S. Ghoshal et al. / Journal of Ethnopharmaeology 50 (1996) 167-170
blunted 18 gauge hypodermic needle attached to a syringe. Each rat received 0.5 ml containing 27 mg of the test agent once daily for 5 consecutive days, beginning 48 h after infection. The pure compound, piperine, was tested at a dose of 100 mg/kg body weight (3 mg/rat). This dose was selected to compare it with the ED 100 of metronidazole which was used as a drug standard (Krishna Prasad et al., 1986). The results were evaluated 24 h after administering the final dose of the test material by sacrificing the animals with excess of ether anaesthesia and examining the contents and scrapings of the caecal wall for trophozoites of E. histolytica. The caecal scoring was determined in accordance to the criteria of Neal (1951). In cases of doubt, the caecal contents and scrapings of the caecal wall were cultured in fresh media and examined for amoebae. 3. Results and discussion The effect of P. longum against trophozoites of E. h&tolytica in vitro and against experimental caecal amoebiasis of rats is described in Table 1. In vitro activity was recorded for all test samples except for the n-butanol insoluble fraction and the pure compound, piperine. The therapeutic efficacy of the test agents against caecal amoebiasis of rats presented interesting results. The crude ethanolic extract exhibited the highest efficacy with 90%
169
cures. The in vitro and in vivo results for the hexane fraction and piperine did not corelate with each other. The former showed only in vitro amoebicidal activity, while the pure compound, piperine, despite being non-amoebicidal in vitro, effected 40% cures. The caecum of rats receiving ethanol extract appeared normal. The walls were thin and comparable to those treated with metronidazole (dose 100 mg/kg body weight). The contents, however, were also normal but less formed as compared to those receiving metronidazole treatment. Chloroform fraction, n-butanol soluble fraction and piperine helped to restore near normality to the caecum of the infected rats and the results were comparable to those receiving metronidazole, 50 mg/kg body weight. These results are significant when compared with the untreated rat caecum, which was shapeless due to localised thickenings, ulcers on the walls and presence of mucus with very little faecal matter as contents. It is apparent from these results that P. longum possesses therapeutic efficacy against caecal amoebiasis of rats despite its low in vitro amoebicidal activity. Such results are not uncommon in tests with agents used in traditional remedies. The root powder and ethanolic extract of Coleus forskohlii did not possess in vitro amoebicidal action against tropohozoites of E. histolytica but were effective against caecal
Table I Effect of P. longum against trophozoites of E. h&tolytica in vitro and against experimental caceal amoebiasis of rats Test materials
In vitro amoebicidal activity (/zg/ml)
Dose mg/kg/b.w.
EtOH ext. Hexane fr. Chloroform fr. n-BuOH (soluble fr) n-BuOH (insoluble fr.) Piperine Metronidazole
1000 1000 500 1000 NA a NA 8
Untreated control
-
900 900 900 900 900 100 50 100 --
alnactive at 1000 t~g/ml; bFor rats showing E. histolytica infection.
Caecal amoebiasis of rats No. of rats cleared/treated (% cured)
Average caecal score b Contents
Wall
Total
18/20 (90) 0/10 (0) 14/20 (70) 12/20 (60) 0/20 (0) 8/20 (40) 12/20 (60) 20/20 (100) 0/20 (0)
0.5 2.0 1.5 1.25 2.00 1.33 1.00 0.0 3.4
0.0 1.0 1.25 0.75 2.00 1.00 1.00 0.0 3.4
0.5 3.0 2.75 2.0 4.0 2.33 2.00 0.0 6.8
170
S. Ghoshal et al./ Journal of Ethnopharmacology 50 (1996) 167-170
amoebiasis of rats (Varma et al., 1990). Similarly, the ethanolic extracts of seeds, leaves, roots, flowers and stem of Nyctanthes arbor-trist showed therapeutic efficacy against caecal amoebiasis of rats despite their lack of in vitro amoebicidal activity (Chitravanshi et al., 1992). It is interesting to add here that exposure of N. arbor-tritis materials to 1 N HCI makes them amoebicidal in vitro, indicating that these are predrugs and their intestinal transit modifies them to become amoebicidal (unpublished data). In the present study, Piperine appears to have acquired amoebicidal property and the mechanism responsible for this transformation needs to be worked out. Apart from this, the crude P. longum may possess certain compounds other than piperine which is/are responsible for their enhanced efficacy.
Acknowledgements The authors thank Dr. V.P. Kamboj, FNA, Director, CDRI, Lucknow, Dr. A.P. Bhaduri, Head, Division of Medicinal Chemistry, Dr. N.B. Singh, Head, Division of Microbiology for their support in carrying out this work. Our thanks are also due to Dr. B.N. Mehrotra for supplying the dry fruits of P. longum. Thanks are also due to Mr. A.K. Gupta, Mr. A.P. Singh and Mr. U.C. Pandey for providing technical assistance. Lastly, it is a pleasure to thank Prof. B.N. Dhawan, FNA, and former Director, CDRI, for his valuable comments and to Mrs. Padma. V. Nair for typing the manuscript.
References Chitravanshi, V.C., Singh, A.P., Ghoshal, S., Krishna Prasad, B.N., Srivastava, V. and Tandon, J.S. (1992) Therapeutic action of Nyctanthes arbor-tristis against caecal amoebiasis of rat. Int. J. Pharmacog. 30(1), 71-85. Chopra, R.N., Nayar, S.L. and Chopra, I.C. (1956) Glossary of Indian Medicinal Plants CSIR Publication, New Delhi, 194. Das, S.R. and Krishna Prasad, B.N. (1973) A reliable and reproducible method for testing direct acting amoebicides against axenic Entamoeba histolytica. Curt. Sci., 42, 796-797.
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