Journal of Ethnopharmacology, Elsevier Scientific Publishers
29 (1990) 51Ireland Ltd.
51
5’7
PRELIMINARY EVALUATION OF EXTRACTS OF ALSTONIA SCHOLARIS BARK FOR IN VIVO ANTIMALARIAL ACTIVITY MICE
MANOJ
GANDHI
and VIRENDER
KUMAR
Department of Experimental Medicine, Research, Chandigarh 160-012 IIndia, (Accepted
November
IN
VINAYAK
Postgraduate
Institute
of Medicinal
Education
and
3, 1989)
Summary
The petroleum either extract and methanol extract of the bark of Alstoniu scholaris were found to be devoid of antiamalarial activity in mice infected with Plasmodium berghei However, a dose-dependent improvement of conditions and delayed mortality amongst animals receiving methanol extract of was noticed. Studies with A. constricta and alstonine shall help resolve the antimalarial status of the bark in question.
A. scholaris
Introduction
Although the developed nations have successfully eradicated or controlled malaria, it still remains a common tropical disease in Southern Asia, China and the African and the South American continents, with a population of 150 million around the world affected annually (Davidson, 19821. The situation is complicated further by the development of a resistance to chloroquine by Plasmodium falciparum, a species responsible for the occurrence of cerebral malaria. Claims regarding the use of indigenous plants and folk remedies for maleria have been made in China, the Southeast Asian countries (Burkhill, 19661, the Philippines (de Padua et al. 19811, Africa (Watt and Breyer-Brandwijik, 1962; Sofowora, 19801, South and Central Americans (Morton, 19811 and India (Manjunath, 1948; Chopra et al. 1956; Antarkar and Vaidya, 19831. In the Indian context, the Ayurvedic practitioners employ plants in addition to other treatment modalities such as fasting, specific dietary regimens and defined activity status. Among the plants used are: Alstonia scholaris R. Br. (Desai, 19271, Tinospora cardifolia_ Willd.,_ Andrographis paniculata Nees., Correspondence
to: Dr. V.K. Vinayak.
0 1990 Elsevier 0378-8741 $02.80 Published and Printed in Ireland
Scientific
Publishers
Ireland
Ltd.
52
Swertia chirata Buch-Ham., Picrorrhiza kurroa Linn. and Caesalpinia crista Linn. All have bitter constituents and are commonly used in the treatment of pyrexial paroxysms known in India as “jwara” (Antarkar and Vaidya, 19831. The recent discoveries of artemisinin from the Chinese herb Artemisia annua L (Jiang et al., 1982) and of active extracts from the fruits of Brucea javanica (L.1 Merr. (O’Neill et al., 19871 have rejuvenated interest in herbal remedies for malaria. AZstonia scholaris (L.1 R. Br., a tall evergreen tree of the family Apocynaceae is found in abundance in Bengal and South India and the bark (dita bark) is used to prepare a bitter tonic for the treatment of fevers and skin diseases (Hawkins and Elderfield, 1942; Chopra et al., 19581. Philippinos commonly employ crude extracts of the bark of Alstonia species as a “cure” for malaria. Two species, A. scholaris and A. constricta F. Muell., were also formerly recognised in the I.P. and the B.P. of 1914 (Sharp 1934; British Pharmaceutical Codex, 19491. A bitter principle, ditaine (now known as echitarnine), has been credited with the febrifuge properties of the bark. The chief constituents from A. scholaris are ditamine and echitamine (Bacon, 1906; Goodson and Henry, 19251. Another non-nitrogenous bitter, alstonine, has been reported from A. constricta (Sharp, 19341. These alkaloids are thought to be very useful in malaria and other fevers reported in the Philippines. Previous observations at Manilla Hospital, those from India in 1921 (Chopra et al., 19581 and the recent open uncontrolled clinical trial (Antarkar and Vaidya, 19831 of A. scholaris (also known as Saptaparna Ghanvati) reported satisfactory activity, but were rather inconclusive with reference to the drug’s intrinsic antimalarial capabilities. Because of reported problems in the isolation of pure crystalline bases (Hawkins and Elderfield, 19421, it was decided to test crude extracts of the bark of A. scholaris for antimalarial activity against Plasmodium berghei in albino mice. Materials
and methods
Plant material Bark pieces (1.5 kg1 were procured from the Regional Research Laboratories, Jammu, and another 2 kg from Eastman and Company, Calcutta, which were confirmed for identity by the pharmacognosist of Panjab University, Chandiagarh (Fig. 1). The materials from the two sources were pulverized separately and processed independently. Sequential organic extractions with petroleum ether and methanol were made using a Soxhlet apparatus and the solvents quickly removed by rotary vaccum evaporation (Ghosal, 19771. The petroleum ether extracts prepared from the Jammu and Calcutta samples gave yields of 3.5% and 3.7% (w/w), respectively, and the syrupy methanol extract was 8.3% and 8.0% (w/w) with respect to the dry weight of crude bark. The water-insoluble waxy petroleum ether extract was warmed, mixed in 30% gum acacia and suspended in water to get a 50 mglml suspension. The water-soluble methanol extract was suitably diluted in water before use.
53
Fig. 1. Bark specimens of Alstonia scholaris.
The test materials were administered polyethylene feeding cannula. Animal
to mice as a single oral dose, using a
studies
Inbred weighing
mice 3-4 weeks old (Naval Medical Research Institute, U.S.A.) 15-20 g each of both sexes were infected intraperitoneally with PZusmodium berghei parasites (National Institute of Communicable Diseases, New Delhi) contained in a O.l-ml aliquot. The animals were kept on a 24% protein diet commercially obtained from Himachal Agro Industries, Parwanoo (Himachal Pradesh) and after infection were randomly allotted to test or control treatments. Experiment 1. The animals of the test batch W = 81 received the petroleum ether extract of the Jammu sample. Another batch of 6 mice (controls) received only the dosing vehicle. The extract was administered at 1.25 g/kg (42 g/kg crude equivalent) at a dosage volume of 25 ml/kg as a maximum ingested single dose, daily for 3 days, starting on the 6th day after i.p. inoculation with 0.5 x lo6 P. berghei parasites/O.1 ml/mouse. Experiment 2. The methanol extracts at a dose of 3.4 g/kg (42 g/kg crude equivalent) from both sources were administered independently to two groups of 8 mice each. The extracts were given daily for 6 days, beginning one day before the i.p. inoculation with 1.8 x lo6 P. berghei parasites/O.1 ml/ mouse. An equal number of mice receiving water only served as vehicle controls for the two test batches.
54 Experiment 3. In order to study a three-fold higher dose level, the methanol extracts from both sources were administered at 10.2 g/kg (126 gl kg crude equivalent) using a 408 mglml solution to 8 mice per batch. The extracts were administered for 7 days, beginning one day before i.p. inoculation with 0.6 x lo6 P. berghei parasites/O.1 ml/mouse. An equal number of infected control mice were given water only.
General procedures After daily examination of Giemsa-stained blood films (Hudson and Hay, 19801 of all animals, the percent parasitized RBCs were expressed as mean + S.E.M. Averages and peak percent parasitemia of test and control batches were compared. The animals were observed closely after drug administration for adverse changes in spontaneous exploratory behaviour, appearance, gait, reflexes, reactivity to external stimuli and respiration and a qualitative assessment of their condition was made. Percent mortality at regular intervals after day 1 (day of inoculation) to the end of the experiment was also calculated. The end points for assessment of efficacy were: (i) prolongation of pre-patent period, (ii1 reduction in levels of parasitemia, (iii) improvement in the behavioural patterns and (iv) prolongation in survival time with reduction in percent mortality. The differences between test and control data were tested for statistical significance using Student’s t-test (Snedecor and Cochran, 19761. Results
and discussion
Experiment 1. The mice treated with an oral dose of the petroleum ether extract equivalent to 42 g/kg crude bark showed no difference from control in peak percent parasitemia (test = 65.7 2 5.2; control = 50.5 rt 4.91, nor any change in mortality patterns (Fig. 2). The mean survival time in the test batch (14.0 f 1.0 days) was comparable to that of controls (12.1 + 0.8 days). Treated animals exhibited a depressed normal exploratory behaviour and activity for 2-4 h after the administration of the extract. Experiment 2. Treatment of animals with methanol extracts from Jammu and Calcutta bark respectively at the same dose level (42 g/kg crude equivalent) did not alter the pre-patent period and also showed no change in peak percent parasitemia (Jammu 18.3 +- 1.3; Calcutta = 19.2 -C 3.5; control = 22.8 + 6.5). The acute depressant effects after daily oral administration of extract were over by 2-3 h. There was no significant difference in the mean survival time among the test (Jammu = 8.8 -+ 1.3 days; Calcutta = 12.2 +2.2 days) and controls (12.5 f 2.2 days). The mice administered with the methanol extract from Calcutta bark, however, exhibited better reactivity and appeared healthier than the controls, with 418 animals exhibiting a delayed onset of mortality (Fig. 2). This difference was evident only by day 15. Since the levels of parasites continued to increase, there were no survivors by the end of the 23-day observation period.
55
6 10
17
DAYS
15 DAYS
23
Ib
15
28
DAYS
Fig. 2. Effect of A. scholaris bark extracts on mortality patterns in P. berghei-infected mice. A: 0, 1.25 g/kg petroleum ether extract of Jammu bark (42 g/kg crude equivalent); 0, control. B: A, 3.4 g/kg of the methanol extract of Calcutta bark; Cl, equivalent treatment using Jammu bark extract (42 g/kg crude equivalent). C: A, 10.2 g/kg of the methanol extract of Calcutta hark; 0, equivalent treatment using Jammu bark (126 g/kg crude equivalent).
Experiment 3. A three-fold increase in dose of the methanol bark extracts from both sources respectively exhibited a greater reduction in mortalities for the first 15 days (Fig. 2). There was an increase in the mean survival time of the treated mice from 11.7 -t 2.3 days in controls to 16.0 + 2.6 days in Jammu batch (not significant1 and 20.0 + 2.7 days in the Calcutta batch P < 0.051. It was notable that the treated animals survived in spite of elevated peak parasitemia (Jammu = 16.8 f 4.4; Calcutta = 25.0 f 5.0) whereas untreated controls having peak parasitemia (17.4 + 5.51 died rapidly during this period. Treatment of mice with the extracts had no effect on the prepatent period. Therefore, it seems apparent that the methanol extracts were ineffective in reducing the parasitemia and only marginally improved the endurance of the infected mice. Higher equivalent dosages that the 3.3 g for 2 weeks total reported human dose were arbitrarily given to ensure the administration of the active ingredients. It was speculated that if significant schizontocidal activity appeared, then at least effective dose would be explored further. It became evident that even at an equivalent dose of 126 g/kg crude bark, the methanol extract was inactive against rodent malaria. However, a dose-dependent improvement in the condition of infected animals was observed. No higher
56
doses of methanol extract were tolerated, and mortality occurred within 1 h of such administration. This preliminary investigation may point to a previously reported use of AZ&on&z alkaloids in that the bark has been used as a source of bitters (Bacon, 1906; Goodson and Henry, 19251 and can act as a febrifuge. Clearly the methanol extracts have been shown here to have a toxic potential with no effect on the progression and multiplication of P. berghei in mice and that the petroleum ether extract of A. scholaris bark from Jammu is both toxic and inactive against P. berghei. Previous attempts to demonstrate the “antimalarial” activity of echitamine and other alkaloids have been frustrating (Goodson and Henry, 1925; Sharp, 1934; Goodson et al., 19301. Alstonine sulfate, A. constricta bark and several of its alkaloidal fractions have been found to be inactive in avian malaria (Sharp, 1934; Hawkins and Elderfield, 19421. According to Mukerji et al. (19421, the total alkaloids of A. scholaris bark showed little or no demonstrable action in malaria induced in monkeys or as naturally occurring in humans. The bark appeared to have value only as a febrifuge (Mukerji, 19461. It is possible that in the reported human trials (Chopra et al., 1958; Antarkar and Vaidya, 19831 the aqueous extracts improved the condition of the patients only by virtue of their temperature-lowering activity (mechanism unknown). It is difficult at present to comment on the claimed effective clearance of parasites in those patients reported in such studies. Situations of conflicting reports often stem from inadequate recording of the exact methods for extract preparation and inadequate details of the conditions under which the various remedies were used. A. scholaris and allied species have been stated to be useful in “jwara” (Antarkar and Vaidya, 19831 or as a “healing agent” (Chopra et al., 19581 useful in malaria and “other fevers”. Such information can lead to therapeutic misinterpretations. There seems to be no alternative to well designed and fully documented testing of ethanomedical remedies to distinguish between myths from facts. Alstonine has been stated to be the active antimalarial principle in A. scholaris and is being developed as a drug at the Walter Reed Institute (Antarkar and Vaidya, 1983). The present study would appear to disagree with its efficacy but, in actuality, it only rules out A. scholaris bark as a useful antimalarial substance. In order to substantiate this conclusion one must recapitulate that (i) both A. scholaris and A. constricta have been widely used in the far east as a “cure” for malaria, (ii) alstonine has been demonstrated only in A. constricta and botanically, A. constricta differs markedly from other members of the same genus, (iii) A. constricta also does not appear to grow in India (Chopra et al., 19581. Therefore, it becomes obvious that the results of the present study confirm the older negative reports on the efficacy of Alstonia scholaris extracts with reference to an intrinsic antimalarial activity. Studies on the efficacy of A. constricta and alstonine need to be carried out to substantiate the usefulness of this Alstonia species against malaria.
57
References Antarkar, D.S. and Vaidya, A.B. (1983) Therapeutic approach to malaria in ayurveda. In: D. Subrahmanyam and V. Radhakrishna (Eds.), Symposium on Recent Advances in Protozoan Diseases. Hindustan Ciba-Geigy Research Centre, Goregaon, Bombay, India, pp. 96 - 101. Bacon, R.F. (1906) The physiologically active constituents of certain Philippine medicinal plants. Philippine Journal of Science 1, 100’7-1036. As cited in Chemical Abstracts (190’7) 1, 1162. British Pharmaceutical Coder (1949) Council of the Pharmaceutical Society of Great Britian, The Pharmaceutical Press, London, p. 1441. Burkhill, I.H. (1966) Dictionary of the Economic Products of the Malay Peninsula Vols. 1 and 2. Ministry of Agriculture and Cooperatives, Kuala Lumpur, Malaysia. Chopra, R.N., Nayar, S.L. and Chopra, I.C. (1956) Glossary of Indian Medicinal Plants. Council of Scientific and Industrial Research, New Delhi. Chopra, R.N., Chopra, I.C., Handa, K.L. and Kapur, L.D. (1958) Alstonia scholaris R. Br. In: Chopra’s Indigenous Drugs of India. Academic Publishers, New Delhi, pp. 276-278. Davidson, G. (1982) Who does not want to eradicate malaria? New Scientist 96, 731-736. de Padua, L.S., Lugod, G.C. and Pancho, J.V. (1981) Handbook of Philippine Medicinal Plants, Technical Bulletin, Vol. V (2). Documentation and Information Section, University of the Philippines, Los Banus. Desai, V.G. (1927) Aushadhi-Sangraha Y.T. Acharya, Bombay, pp. 471-472. As cited by Antarkar and Vaidya (1983). Ghosal, S. (1977) Some aspects of phytopharmacological evaluation of medicinal plants. In: B. Mukkherjee (Ed.), The Indian Pharmacological Society Souvenir, Decennial Conference (29 December). Calcutta, pp. 33-41. Goodson, J.A. and Henry, T.A. (1925) Echitamine. Journal of the Chemical Society 127, 16401648. Goodson, J.A., Henry, T.A. and Macfie, J.W.S. (1930) The action of cinchona and certain alkaloids in bird malaria, Biochemical Journal 24, 874 - 890. Hawkins, W.L. and Elderfield, R.C. (1942) Alstonia alkaloids. II: A new alkaloid ‘alstonine’ A. constricta Journal of Organic Chemistry 7, 573-580. Hudson, L. and Hay, F.C. (1980) Practical Immunology, 2nd Edn. tions, London, p. 28. Jiang, J.-B., Guo, X.-B., Li, G.-Q. and Kong, Y.-C. (1982) Antimalarial quinghaosu. Lancet 2 (8293). 285-288.
Blackwell activity
Scientific
other from
Publica-
of mefloquine
and
Manjunath, B.L. (1948) Alstonia scholaris R. Br. In: The Wealth of India A Directory of Indian Raw Materials and Industrial Products, Raw Materials, Vol. 1. Publications and Information Directorate, Council of Scientific and Industrial Research, New Delhi, pp. 63-64. Morton, J.F. (1981) Atlas of Medicinal Plants of Middle America - Bahamas to Yucatan, Charles C. Thomas, Springfield, Illinois. Mukerji, B. (1946) Antimalarial drugs of the indigenous materia medica of China and India. Nature 158, 170. Mukerji, B., Ghosh, B.K. and Siddons, L.B. (1942) Search for an antimalarial drug in the indigenous materia medica. I: A. scholaris R. Br. IndianMedical Gazette 77,723-725. O’Neill. M.J., Bray, D.H., Boardman, P., Chan, K.L., Phillipson, J.D, Warhurst, D.C. and Peters, W. (1987) Plants as sources of antimalarial drugs. IV: Activity of BrzLcea javanica fruits against chloroquinine-resistant Plasmodium falciparum in vitro and against Plasmodium berghei in vivo. Journal of Natural Products 50, 41-48. Sharp, T.M. (1934) The alkaloids of Alstonia barks. Part I: A. constricta F. Muell. Journal of the Chemical Society 1, 287-291. Snedecor, G.W. and Cochran, W.G. (1976) Statistical Methods, 6th Edn., Oxford and Indian Book House Publishing Co., New Delhi, pp. 91- 104. Sofowora, A. (1980) The present status of knowledge of the plants used in traditional medicine in Western Africa: A medicinal approach and a chemical evaluation. Journal of Ethnophamnacology 2. 109- 118. Watt, J.M. and Breyer-Brandwijk, and Eastern Africa, 2nd Edn.,
M.G. (1962) The Medicinal E 8 S Livingstone, London,
and Poisonous p. 80.
Plants of Southern
29 (1990) 51Ireland Ltd.
51
5’7
PRELIMINARY EVALUATION OF EXTRACTS OF ALSTONIA SCHOLARIS BARK FOR IN VIVO ANTIMALARIAL ACTIVITY MICE
MANOJ
GANDHI
and VIRENDER
KUMAR
Department of Experimental Medicine, Research, Chandigarh 160-012 IIndia, (Accepted
November
IN
VINAYAK
Postgraduate
Institute
of Medicinal
Education
and
3, 1989)
Summary
The petroleum either extract and methanol extract of the bark of Alstoniu scholaris were found to be devoid of antiamalarial activity in mice infected with Plasmodium berghei However, a dose-dependent improvement of conditions and delayed mortality amongst animals receiving methanol extract of was noticed. Studies with A. constricta and alstonine shall help resolve the antimalarial status of the bark in question.
A. scholaris
Introduction
Although the developed nations have successfully eradicated or controlled malaria, it still remains a common tropical disease in Southern Asia, China and the African and the South American continents, with a population of 150 million around the world affected annually (Davidson, 19821. The situation is complicated further by the development of a resistance to chloroquine by Plasmodium falciparum, a species responsible for the occurrence of cerebral malaria. Claims regarding the use of indigenous plants and folk remedies for maleria have been made in China, the Southeast Asian countries (Burkhill, 19661, the Philippines (de Padua et al. 19811, Africa (Watt and Breyer-Brandwijik, 1962; Sofowora, 19801, South and Central Americans (Morton, 19811 and India (Manjunath, 1948; Chopra et al. 1956; Antarkar and Vaidya, 19831. In the Indian context, the Ayurvedic practitioners employ plants in addition to other treatment modalities such as fasting, specific dietary regimens and defined activity status. Among the plants used are: Alstonia scholaris R. Br. (Desai, 19271, Tinospora cardifolia_ Willd.,_ Andrographis paniculata Nees., Correspondence
to: Dr. V.K. Vinayak.
0 1990 Elsevier 0378-8741 $02.80 Published and Printed in Ireland
Scientific
Publishers
Ireland
Ltd.
52
Swertia chirata Buch-Ham., Picrorrhiza kurroa Linn. and Caesalpinia crista Linn. All have bitter constituents and are commonly used in the treatment of pyrexial paroxysms known in India as “jwara” (Antarkar and Vaidya, 19831. The recent discoveries of artemisinin from the Chinese herb Artemisia annua L (Jiang et al., 1982) and of active extracts from the fruits of Brucea javanica (L.1 Merr. (O’Neill et al., 19871 have rejuvenated interest in herbal remedies for malaria. AZstonia scholaris (L.1 R. Br., a tall evergreen tree of the family Apocynaceae is found in abundance in Bengal and South India and the bark (dita bark) is used to prepare a bitter tonic for the treatment of fevers and skin diseases (Hawkins and Elderfield, 1942; Chopra et al., 19581. Philippinos commonly employ crude extracts of the bark of Alstonia species as a “cure” for malaria. Two species, A. scholaris and A. constricta F. Muell., were also formerly recognised in the I.P. and the B.P. of 1914 (Sharp 1934; British Pharmaceutical Codex, 19491. A bitter principle, ditaine (now known as echitarnine), has been credited with the febrifuge properties of the bark. The chief constituents from A. scholaris are ditamine and echitamine (Bacon, 1906; Goodson and Henry, 19251. Another non-nitrogenous bitter, alstonine, has been reported from A. constricta (Sharp, 19341. These alkaloids are thought to be very useful in malaria and other fevers reported in the Philippines. Previous observations at Manilla Hospital, those from India in 1921 (Chopra et al., 19581 and the recent open uncontrolled clinical trial (Antarkar and Vaidya, 19831 of A. scholaris (also known as Saptaparna Ghanvati) reported satisfactory activity, but were rather inconclusive with reference to the drug’s intrinsic antimalarial capabilities. Because of reported problems in the isolation of pure crystalline bases (Hawkins and Elderfield, 19421, it was decided to test crude extracts of the bark of A. scholaris for antimalarial activity against Plasmodium berghei in albino mice. Materials
and methods
Plant material Bark pieces (1.5 kg1 were procured from the Regional Research Laboratories, Jammu, and another 2 kg from Eastman and Company, Calcutta, which were confirmed for identity by the pharmacognosist of Panjab University, Chandiagarh (Fig. 1). The materials from the two sources were pulverized separately and processed independently. Sequential organic extractions with petroleum ether and methanol were made using a Soxhlet apparatus and the solvents quickly removed by rotary vaccum evaporation (Ghosal, 19771. The petroleum ether extracts prepared from the Jammu and Calcutta samples gave yields of 3.5% and 3.7% (w/w), respectively, and the syrupy methanol extract was 8.3% and 8.0% (w/w) with respect to the dry weight of crude bark. The water-insoluble waxy petroleum ether extract was warmed, mixed in 30% gum acacia and suspended in water to get a 50 mglml suspension. The water-soluble methanol extract was suitably diluted in water before use.
53
Fig. 1. Bark specimens of Alstonia scholaris.
The test materials were administered polyethylene feeding cannula. Animal
to mice as a single oral dose, using a
studies
Inbred weighing
mice 3-4 weeks old (Naval Medical Research Institute, U.S.A.) 15-20 g each of both sexes were infected intraperitoneally with PZusmodium berghei parasites (National Institute of Communicable Diseases, New Delhi) contained in a O.l-ml aliquot. The animals were kept on a 24% protein diet commercially obtained from Himachal Agro Industries, Parwanoo (Himachal Pradesh) and after infection were randomly allotted to test or control treatments. Experiment 1. The animals of the test batch W = 81 received the petroleum ether extract of the Jammu sample. Another batch of 6 mice (controls) received only the dosing vehicle. The extract was administered at 1.25 g/kg (42 g/kg crude equivalent) at a dosage volume of 25 ml/kg as a maximum ingested single dose, daily for 3 days, starting on the 6th day after i.p. inoculation with 0.5 x lo6 P. berghei parasites/O.1 ml/mouse. Experiment 2. The methanol extracts at a dose of 3.4 g/kg (42 g/kg crude equivalent) from both sources were administered independently to two groups of 8 mice each. The extracts were given daily for 6 days, beginning one day before the i.p. inoculation with 1.8 x lo6 P. berghei parasites/O.1 ml/ mouse. An equal number of mice receiving water only served as vehicle controls for the two test batches.
54 Experiment 3. In order to study a three-fold higher dose level, the methanol extracts from both sources were administered at 10.2 g/kg (126 gl kg crude equivalent) using a 408 mglml solution to 8 mice per batch. The extracts were administered for 7 days, beginning one day before i.p. inoculation with 0.6 x lo6 P. berghei parasites/O.1 ml/mouse. An equal number of infected control mice were given water only.
General procedures After daily examination of Giemsa-stained blood films (Hudson and Hay, 19801 of all animals, the percent parasitized RBCs were expressed as mean + S.E.M. Averages and peak percent parasitemia of test and control batches were compared. The animals were observed closely after drug administration for adverse changes in spontaneous exploratory behaviour, appearance, gait, reflexes, reactivity to external stimuli and respiration and a qualitative assessment of their condition was made. Percent mortality at regular intervals after day 1 (day of inoculation) to the end of the experiment was also calculated. The end points for assessment of efficacy were: (i) prolongation of pre-patent period, (ii1 reduction in levels of parasitemia, (iii) improvement in the behavioural patterns and (iv) prolongation in survival time with reduction in percent mortality. The differences between test and control data were tested for statistical significance using Student’s t-test (Snedecor and Cochran, 19761. Results
and discussion
Experiment 1. The mice treated with an oral dose of the petroleum ether extract equivalent to 42 g/kg crude bark showed no difference from control in peak percent parasitemia (test = 65.7 2 5.2; control = 50.5 rt 4.91, nor any change in mortality patterns (Fig. 2). The mean survival time in the test batch (14.0 f 1.0 days) was comparable to that of controls (12.1 + 0.8 days). Treated animals exhibited a depressed normal exploratory behaviour and activity for 2-4 h after the administration of the extract. Experiment 2. Treatment of animals with methanol extracts from Jammu and Calcutta bark respectively at the same dose level (42 g/kg crude equivalent) did not alter the pre-patent period and also showed no change in peak percent parasitemia (Jammu 18.3 +- 1.3; Calcutta = 19.2 -C 3.5; control = 22.8 + 6.5). The acute depressant effects after daily oral administration of extract were over by 2-3 h. There was no significant difference in the mean survival time among the test (Jammu = 8.8 -+ 1.3 days; Calcutta = 12.2 +2.2 days) and controls (12.5 f 2.2 days). The mice administered with the methanol extract from Calcutta bark, however, exhibited better reactivity and appeared healthier than the controls, with 418 animals exhibiting a delayed onset of mortality (Fig. 2). This difference was evident only by day 15. Since the levels of parasites continued to increase, there were no survivors by the end of the 23-day observation period.
55
6 10
17
DAYS
15 DAYS
23
Ib
15
28
DAYS
Fig. 2. Effect of A. scholaris bark extracts on mortality patterns in P. berghei-infected mice. A: 0, 1.25 g/kg petroleum ether extract of Jammu bark (42 g/kg crude equivalent); 0, control. B: A, 3.4 g/kg of the methanol extract of Calcutta bark; Cl, equivalent treatment using Jammu bark extract (42 g/kg crude equivalent). C: A, 10.2 g/kg of the methanol extract of Calcutta hark; 0, equivalent treatment using Jammu bark (126 g/kg crude equivalent).
Experiment 3. A three-fold increase in dose of the methanol bark extracts from both sources respectively exhibited a greater reduction in mortalities for the first 15 days (Fig. 2). There was an increase in the mean survival time of the treated mice from 11.7 -t 2.3 days in controls to 16.0 + 2.6 days in Jammu batch (not significant1 and 20.0 + 2.7 days in the Calcutta batch P < 0.051. It was notable that the treated animals survived in spite of elevated peak parasitemia (Jammu = 16.8 f 4.4; Calcutta = 25.0 f 5.0) whereas untreated controls having peak parasitemia (17.4 + 5.51 died rapidly during this period. Treatment of mice with the extracts had no effect on the prepatent period. Therefore, it seems apparent that the methanol extracts were ineffective in reducing the parasitemia and only marginally improved the endurance of the infected mice. Higher equivalent dosages that the 3.3 g for 2 weeks total reported human dose were arbitrarily given to ensure the administration of the active ingredients. It was speculated that if significant schizontocidal activity appeared, then at least effective dose would be explored further. It became evident that even at an equivalent dose of 126 g/kg crude bark, the methanol extract was inactive against rodent malaria. However, a dose-dependent improvement in the condition of infected animals was observed. No higher
56
doses of methanol extract were tolerated, and mortality occurred within 1 h of such administration. This preliminary investigation may point to a previously reported use of AZ&on&z alkaloids in that the bark has been used as a source of bitters (Bacon, 1906; Goodson and Henry, 19251 and can act as a febrifuge. Clearly the methanol extracts have been shown here to have a toxic potential with no effect on the progression and multiplication of P. berghei in mice and that the petroleum ether extract of A. scholaris bark from Jammu is both toxic and inactive against P. berghei. Previous attempts to demonstrate the “antimalarial” activity of echitamine and other alkaloids have been frustrating (Goodson and Henry, 1925; Sharp, 1934; Goodson et al., 19301. Alstonine sulfate, A. constricta bark and several of its alkaloidal fractions have been found to be inactive in avian malaria (Sharp, 1934; Hawkins and Elderfield, 19421. According to Mukerji et al. (19421, the total alkaloids of A. scholaris bark showed little or no demonstrable action in malaria induced in monkeys or as naturally occurring in humans. The bark appeared to have value only as a febrifuge (Mukerji, 19461. It is possible that in the reported human trials (Chopra et al., 1958; Antarkar and Vaidya, 19831 the aqueous extracts improved the condition of the patients only by virtue of their temperature-lowering activity (mechanism unknown). It is difficult at present to comment on the claimed effective clearance of parasites in those patients reported in such studies. Situations of conflicting reports often stem from inadequate recording of the exact methods for extract preparation and inadequate details of the conditions under which the various remedies were used. A. scholaris and allied species have been stated to be useful in “jwara” (Antarkar and Vaidya, 19831 or as a “healing agent” (Chopra et al., 19581 useful in malaria and “other fevers”. Such information can lead to therapeutic misinterpretations. There seems to be no alternative to well designed and fully documented testing of ethanomedical remedies to distinguish between myths from facts. Alstonine has been stated to be the active antimalarial principle in A. scholaris and is being developed as a drug at the Walter Reed Institute (Antarkar and Vaidya, 1983). The present study would appear to disagree with its efficacy but, in actuality, it only rules out A. scholaris bark as a useful antimalarial substance. In order to substantiate this conclusion one must recapitulate that (i) both A. scholaris and A. constricta have been widely used in the far east as a “cure” for malaria, (ii) alstonine has been demonstrated only in A. constricta and botanically, A. constricta differs markedly from other members of the same genus, (iii) A. constricta also does not appear to grow in India (Chopra et al., 19581. Therefore, it becomes obvious that the results of the present study confirm the older negative reports on the efficacy of Alstonia scholaris extracts with reference to an intrinsic antimalarial activity. Studies on the efficacy of A. constricta and alstonine need to be carried out to substantiate the usefulness of this Alstonia species against malaria.
57
References Antarkar, D.S. and Vaidya, A.B. (1983) Therapeutic approach to malaria in ayurveda. In: D. Subrahmanyam and V. Radhakrishna (Eds.), Symposium on Recent Advances in Protozoan Diseases. Hindustan Ciba-Geigy Research Centre, Goregaon, Bombay, India, pp. 96 - 101. Bacon, R.F. (1906) The physiologically active constituents of certain Philippine medicinal plants. Philippine Journal of Science 1, 100’7-1036. As cited in Chemical Abstracts (190’7) 1, 1162. British Pharmaceutical Coder (1949) Council of the Pharmaceutical Society of Great Britian, The Pharmaceutical Press, London, p. 1441. Burkhill, I.H. (1966) Dictionary of the Economic Products of the Malay Peninsula Vols. 1 and 2. Ministry of Agriculture and Cooperatives, Kuala Lumpur, Malaysia. Chopra, R.N., Nayar, S.L. and Chopra, I.C. (1956) Glossary of Indian Medicinal Plants. Council of Scientific and Industrial Research, New Delhi. Chopra, R.N., Chopra, I.C., Handa, K.L. and Kapur, L.D. (1958) Alstonia scholaris R. Br. In: Chopra’s Indigenous Drugs of India. Academic Publishers, New Delhi, pp. 276-278. Davidson, G. (1982) Who does not want to eradicate malaria? New Scientist 96, 731-736. de Padua, L.S., Lugod, G.C. and Pancho, J.V. (1981) Handbook of Philippine Medicinal Plants, Technical Bulletin, Vol. V (2). Documentation and Information Section, University of the Philippines, Los Banus. Desai, V.G. (1927) Aushadhi-Sangraha Y.T. Acharya, Bombay, pp. 471-472. As cited by Antarkar and Vaidya (1983). Ghosal, S. (1977) Some aspects of phytopharmacological evaluation of medicinal plants. In: B. Mukkherjee (Ed.), The Indian Pharmacological Society Souvenir, Decennial Conference (29 December). Calcutta, pp. 33-41. Goodson, J.A. and Henry, T.A. (1925) Echitamine. Journal of the Chemical Society 127, 16401648. Goodson, J.A., Henry, T.A. and Macfie, J.W.S. (1930) The action of cinchona and certain alkaloids in bird malaria, Biochemical Journal 24, 874 - 890. Hawkins, W.L. and Elderfield, R.C. (1942) Alstonia alkaloids. II: A new alkaloid ‘alstonine’ A. constricta Journal of Organic Chemistry 7, 573-580. Hudson, L. and Hay, F.C. (1980) Practical Immunology, 2nd Edn. tions, London, p. 28. Jiang, J.-B., Guo, X.-B., Li, G.-Q. and Kong, Y.-C. (1982) Antimalarial quinghaosu. Lancet 2 (8293). 285-288.
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Manjunath, B.L. (1948) Alstonia scholaris R. Br. In: The Wealth of India A Directory of Indian Raw Materials and Industrial Products, Raw Materials, Vol. 1. Publications and Information Directorate, Council of Scientific and Industrial Research, New Delhi, pp. 63-64. Morton, J.F. (1981) Atlas of Medicinal Plants of Middle America - Bahamas to Yucatan, Charles C. Thomas, Springfield, Illinois. Mukerji, B. (1946) Antimalarial drugs of the indigenous materia medica of China and India. Nature 158, 170. Mukerji, B., Ghosh, B.K. and Siddons, L.B. (1942) Search for an antimalarial drug in the indigenous materia medica. I: A. scholaris R. Br. IndianMedical Gazette 77,723-725. O’Neill. M.J., Bray, D.H., Boardman, P., Chan, K.L., Phillipson, J.D, Warhurst, D.C. and Peters, W. (1987) Plants as sources of antimalarial drugs. IV: Activity of BrzLcea javanica fruits against chloroquinine-resistant Plasmodium falciparum in vitro and against Plasmodium berghei in vivo. Journal of Natural Products 50, 41-48. Sharp, T.M. (1934) The alkaloids of Alstonia barks. Part I: A. constricta F. Muell. Journal of the Chemical Society 1, 287-291. Snedecor, G.W. and Cochran, W.G. (1976) Statistical Methods, 6th Edn., Oxford and Indian Book House Publishing Co., New Delhi, pp. 91- 104. Sofowora, A. (1980) The present status of knowledge of the plants used in traditional medicine in Western Africa: A medicinal approach and a chemical evaluation. Journal of Ethnophamnacology 2. 109- 118. Watt, J.M. and Breyer-Brandwijk, and Eastern Africa, 2nd Edn.,
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