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Preliminary study of an extract from Diodia scandens on some toxic effects of Echis carinatus venom
Journal of Ethnopharmacology, 26 (1989) 189- 196 Elsevier Scientific Publishers Ireland Ltd.
189
Short Communication
PRELIMINARY STUDY OF AN EXTRACT FROM DIODIA DENS ON SOME TOXIC EFFECTS OF ECHIS CARINATUS VENOM
SCAN-
G. ONUAGULUCHI Department of Pha~~c~logy Enugu (Nigeria
and Th~~apeut~c~, College of Medicine, University of Nigeria,
(Accepted November 10,1988)
Introduction
Snake bite is a major medical problem in Nigeria and in many tropical countries. It is estimated that between 30,000 and 40,000 persons die from snake bite world wide (Dreisbach, 19801. In the grassland areas of Nigeria, the most common snake is the saw-scaled viper @&his c~~~~~~~~). The clinical data on viper bites in Nigeria are reported by Onuaguluchi (19601and Warrell et al. (19’771show that a large proportion of the victims die in spite of energetic treatment with polyvalent antiserum and other modern supportative treatment. A considerable proportion of victims die within a few hours, probably from cardiovascular shock, while a number die some days later after envenomation from the combined effects of prolonged internal bleeding and cardiovascular shock. In Nigeria, many people live far away from hospitals and many hospitals do not always have ample supply of the antiserum; consequently, many victims of snake bite have to be treated by traditional medicine practitioners versed in the use of herbs. My father, an educated Warrant Chief was well known in Achi, Anambra State, and the neighbouring towns for his herbal remedy for snake bites. Although Achi is situated in the Southern part of Nigeria, it has grassland vegetation, and is notorious for its large population of saw-scaled vipers, locally called NVUTUAchi I accompanied my father several times into the bush to collect the herb and on those occasions helped to prepare the herb for use by snake-bite victims. In the treatment of the snake-bite victim, the area of the bite is scarified with a clean razor blade and a thick paste of the ground-up herb (leaves) is applied over the wound. A segment of the stem is fastened a little below the 0378-87~1/89/$03.15 0 1989 Elsevier Scientific Pubfishers Ireland Ltd. Published and Printed in Ireland
190
wound in the belief that it will cause the venom to gravitate down and remain below the wound! The rest of the herb is then chopped up and boiled for approximately 1 h in a pot of water and the patient is given a cup (approx. 200 ml) of the infusion to drink. He continues to take this quantity of the infusion three or four times daily for another 3 days or more. The herb used for this study was collected from the bush in Achi. It was collected in September 1985 and was identified botanically by Dr. J.C. Okafor of the Enugu Forest Herbarium as Diodia scundens (Gronov ex L.1. A specimen of the herb was planted in my garden at Enugu where it grows luxuriantly and thus affords one a ready access to the herb for future studies. Enugu is approximately 50 km east of Achi. Diodia scandens is a member of the Rubiaceae and is a creeping or climbing herbacious plant. Figure 1 shows the stem, leaves and flowers of the
Fig. 1. A branch of Diodia scandens showing placement of the leaves aggregated in large numbers at the node and at the apex of the branch.
and the white flowers
191
plant. The flowers are white and are aggregated in large numbers nodes and at the end of the branches. The plant grows luxuriantly the rainy season but withers somewhat in the dry season. Materials Extraction
at the during
and methods methods
After collection from the bush, the aerial parts were stored overnight in a refrigerator wrapped in paper and sealed in a polythene bag. The next day, the herb was chopped and weighed. The material was then extracted in a soxhlet apparatus with boiling 95% ethanol for 12 h. Upon removal of the solvent, a dark green residue was obtained. Clarification of this residue was achieved by dissolving it in 50% aqueous ethanol and passing the solution through a column of activated charcoal. The filtrate obtained was colourless and deposited colourless crystals on standing. Further crystallisation occurred when the filtrate was kept overnight in a refrigerator in a covered container. The crystals (fraction A) were removed by filtration using Whatman filter paper, dried in the air and weighed. The yield was 0.40% (w/w) in terms of fresh starting material. The mother liquour was evaporated under reduced pressure to dryness in a water bath maintained at 40°C and yielded a dark brown residue (fraction B). This residue was kept in a desiccator in a glass container of known weight. The container with the residue was weighed every 2 days until the weight remained constant. The weight of the residue was then determined (yield = 3.4%). Finally, the charcoal column was eluted with 50% aqueous ethanol containing 0.05% glacial acetic acid. Evaporation of the eluate yielded a brown residue (fraction CL The weight of fraction C was determined (yield = 0.35%) by using the procedure described in respect to fraction B. Fraction A was insoluble in water while fraction B and C were water soluble. Because fraction B was obtained in the largest amounts and because of its solubility in water, it was the fraction used for this study. Protection
of mice from
venom
Preliminary studies were performed to determine the toxic potency of the specimen of the Echis carinatus venom obtained from Sigma Chemicals, USA. Albino mice of either sex weighing between 20 and 25 g were divided into four groups of ten. Each mouse in each group received the same dose of the venom intraperitoneally (i.p.1: 2, 4, 8 or 12 mglkg. The animals were observed closely during the first 4 h after venom administration. Thereafter, each group was kept in a separate cage and left overnight in the laboratory with the usual supply of water and pellets of food. The number of deaths 24 h after administration of the venom was noted and the percent mortality calculated for each dose of venom. From this preliminary study it was clear that the surely lethal dose of
192
venom should not be more than 4 mg/kg as there was 100% mortality at this dose within 1 h. Consequently, for the protection study, the doses of venom used were 0.5, 1, 2, 3 and 4 mglkg. Mice weighing between 20 and 25 g were divided into ten groups of ten animals each. Five groups received only venom while the remaining five groups were pretreated i.p. with fraction B (1.5 mg/ kg), 30 min before venom administration. The mice were observed very closely for 4 h after administering the venom and were left in cages as already described. The number of mice dead in each group was noted 24 h after venom injection and the percent mortality due to each dose of venom in the presence and absence of the herbal extract was calculated. Log doseresponse curves showing the percent mortality due to venom in the presence and absence of the herbal extract were constructed and the LD,, of the venom in the presence and absence of the herbal extract were calculated. Statistical analyses were performed using the $test with a P value of < 0.05 taken as indicating significance. Acute toxicity of fraction B in mice Because of the protective effect obtained with the fraction B at 1.5 mglkg, it was necessary that its level of toxicity should be determined. Preliminary studies showed that no mouse died even after receiving 400 mg/kg i.p. Therefore, the following doses of fraction B were used for the primary study: 0.4, 1, 1.5, 2, 3 and 4 kg/kg i.p. Mice of either sex weighing between 20 and 25 g were divided into groups of ten animals each. The animals were closely observed as already described. The number of mice dead 24 h after administering the herbal extract was noted and the percent mortality due to each dose was calculated. Effect of fraction B on venom-induced effects in the anaesthetised cat Three cats weighing between 1 and 2 kg were anaesthetised with thiopentone sodium (50 mg/kg i.p.1 and tied in a supine position on a BrownSchuster myographic table. The trachea was cannulated with a metallic tracheal tube and connected to a tambour writing on smoked paper on a kymograph drum. One common carotid artery was cannulated and connected to a mercury manometer in the conventional manner. A femoral vein was exposed and cannulated as an injection site. The cannula was filled with heparinized normal saline and connected to a syringe using a three-way stop cock which allowed for the injection of saline, venom or drugs (fraction B or adrenaline). The intravenous effects of adrenaline 50 pg/kg (Gedeon Richter Ltd., Budapest, Hungary) and venom 0.5 mgkg were studied. When the effect of the venom on the blood pressure (BP) had disappeared and the BP had returned to the pre-injection level, the animal was given the extract 10 mgkg dissolved in 1 ml of saline. Ten minutes later, the dose of the venom was reported. The effects of the venom on the BP and respiration before and after giving the herbal extract were then compared.
193
Results Toxicity studies Table 1 shows the mortality seen after various doses of the venom alone and after pretreatment with fraction B (1.5 mg/kg i.p.) When the dose of the venom was 2 mg/kg i.p., the extract reduced the mortality due to the venom from 50% to 10%. The reduction narrowly missed the criterion for statistical significance. However, when the dose of the venom was 3 mg/kg, the extract reduced the mortality from 80% to 30%. This reduction in mortality was statistically significant P < 0.05). Although fraction B appeared not to have ultimately offered protection when the dose of venom was 4 mg/kg, it was observed that deaths occurred within 10 min in all mice not pretreated with the extract, but no mouse pretreated with the extract died in less than 4 h. Moreover the extract prevented the venom-induced clonic convulsions which normally preceded death. The LD, due to the venom was 1.09 mg/kg + 0.61 i.p. (95% confidence limits) but this was increased to 3.0 mg/kg f 0.77 i.p. (95% confidence limits) following pretreatment with fraction B. Fraction B had no deleterious effect on the mice up to doses of 2 g/kg i.p. However, it caused 30% mortality at 3 g/kg i.p. Effect of the fraction B on venom-induced effects in the anaesthetised cat Adrenaline (50 pg/kg i.v.1 produced a 20 -30 mmHg rise in blood pressure which returned to the pre-injection level in approximately 5 min. The venom at 0.5 mg/kg i.v., produced a lo-20 mmHg pressor effect with the blood pressure returning to pre-injection levels in approximately 2 min. Fraction B (10 mg/kg i.v.1 given 10 min after administering the venom had no effect on
TABLE
1
EFFECT OF FRACTION B (1.5 mg/kg i.p.) ON THE PERCENT RECEIVING VARIOUS DOSES OF ECHIS CARINATUS VENOM Venom dose (mg/kg)
Mortality
0.5 1.0 2.0 3.0 4.0 *Statistically
significantly:
P < 0.05.
MORTALITY
IN MICE
(%I)
Venom alone
Venom + extract
10 40 50 80 100
10 10 10 30* 100
194
Fig. 2. Effect of the fraction induced in the anaesthetised
B 10 mg/kg i.v. (B) on changes in blood pressure cat by 0.5 mg/kg i.v. of Echis carinatus venom (VI.
and
respiration
the blood pressure in the cats. However, it prevented the rise in blood pressure when the venom (0.5 mg/kg i.v.1 was repeated 10 min later. On respiration, the venom at 0.5 mg/kg i.v. caused a marked reduction in the respiratory rate and there were long periods of apnoea. Fraction B reduced the duration of the apnoeic periods and the respiration became regular approximately 4 min after extract injection. When the dose of the venom was repeated 10 min after injecting fraction B, the apnoeic periods induced by the venom were less pronounced than when the cat had not been treated with the extract. Figure 2 shows the effect of the venom (0.5 mglkg i.v.1 on blood pressure and respiration in the absence and presence of fraction B. It also shows the salutary effects of fraction B on the Cheyne-Stokes type of respiration induced by the venom. Discussion
The results
of the toxicity
studies
in mice showed that the extract
from
Diodia scandens tended to protect mice from the lethal effects of the venom of Echis carinatus. Although no protection from death was observed when
the dose of venom was 4 mg/kg i.p., the extract delayed the clonic convulsions produced by the venom. At this dose level, all untreated mice died within 10 min but no animal pretreated with the extract died before 4 h had elapsed. Venom-induced convulsions were also observed by Warrell et al. (1977) but have not been observed clinically in man. This may be due to the fact that for a 60-70 kg human victim, the maximum dose of the venom which the snake could possibly introduce would be approximately 0.5 mg/kg and indeed could be appreciably less since the approximate yield of dry venom from Echis carinatus is 20-35 mg (Dreisbach, 1980). The effects of the venom on blood pressure and respiration are of considerable interest. The venom at 0.5 mg/kg caused a small rise in blood
195
pressure (lo-20 mmHg1 but induced a marked Cheyne-Stokes disturbance of the rhythm of respiration. The rise in blood pressure was surprising, because clinically, when the snake bite victim presents, the blood pressure is often low from shock. However, it is possible that even in human subjects, a rise in blood pressure could occur first to be followed some minutes later by a state of shock with peripheral vascular collapse due to the liberation of large amounts of kinins by the venom (Cohen et al., 19691.The rise in blood pressure in the cat experiments may be due to the effect of increased PCO, from the Cheyne-Stokes type of respiration induced by the venom or to a direct vasoconstrictive effect on the vascular smooth muscles or to some combination of these factors. Further work on the effects of the venom and the extract of Diodia scandens on the mammalian cardiovascular system is clearly indicated. It is of great interest that fraction B of Biodia scandens at 1.5 mg/kg partially protected mice from the lethal effects of the venom but had no deleterious effect itself on the mice up to doses of 2 g/kg i.p. This indicates that its safety margin should be very high. The dose of the extract used in this study to protect mice would appear to be small but it was chosen because in a separate study on the in vitro effects of fraction B on the blood coagulation defects induced by the venom, it was found that a l:lO,OOO solution of the extract statistically significantly inhibited some of the defects induced by a l:lO,OOOsolution of the venom (unpublished data). Since the minimum lethal dose of the venom was approximately 0.5 mg/kg and its LD, was 1.09 mglkg, the dose of 1.5 mglkg which was chosen for the extract appeared appropriate for this preliminary study. Another plant reputed to be of some value in the treatment of snake bite in Nigeria is Schumanniophyton magnificum Harms (family Rubiaceael. However, this is a shrub and requires many years to grow. In contrast, Diodia scandens is a quick growing herb and could be exploited for commercial production of a safe cheap drug for treating snake bites. This plant could, therefore, be of much interest to the many Third World countries in the tropics who cannot afford, because of scarce financial resources, to produce or purchase antisera to treat snake bites. It is noteworthy that heat destroys the antivenom effect of the active principle from the stem bark of Schumanniophyton magnificum (Akunyili and Akubue, 1986; 19871 so that drying and storage may adversely affect its antivenom activities. On the other hand, the antivenom effect of Diodia scandens is apparently not affected by boiling the plant. Acknowledgements
I wish to thank Mr. Paul Okeke for technical assistance, Mrs. B. Ukonu for typing the manuscript, the Medical Illustration Unit for the photographs and Dr. J.C. Okafor for identifying the plant botanically.
196
References Akunyili, D.N. and Akubue, P.I. (1986) Schumanniofoside, the antisnake venom principle from the stem bark of Schummanniophyton magnificum Harms. Journal of Ethnopharmacology 18, 167- 172. Akunyili, D.N. and Akubue, P.I. (1987) Antisnake venom properties of the stem bark juice of Schumanniophyton magnificum. Fitoterapia 58, 47-49. Cohen, M., Zur, E.M. and De Vries, A. (1969) Isolation and characterization of kinin-releasing enzyme of Echis coloratus venom. Biochemical Pharmacology 19, 785-793. Dreisbach, R.H. (1980) Handbook of Poisoning, Lange Medical Publications, Los Altos, California, p. 457. Onuaguluchi, G.O. (1960) Clinical Observations on snake bite in Wukari, Nigeria. Transactions of the Royal Society of Tropical Medicine and Hygiene 54, 265-269. Warrell, D.A., Davidson, N.M., Greenwood, B.M., Ormerod, L.D., Pope, H.M. Watkins, B.J. and Pretence, C.R.M. (1977) Poisoning by bites of the saw-scaled or carpet viper Wchis carinatus) in Nigeria. Quarterly Journal of Medicine 46, 33-62.
189
Short Communication
PRELIMINARY STUDY OF AN EXTRACT FROM DIODIA DENS ON SOME TOXIC EFFECTS OF ECHIS CARINATUS VENOM
SCAN-
G. ONUAGULUCHI Department of Pha~~c~logy Enugu (Nigeria
and Th~~apeut~c~, College of Medicine, University of Nigeria,
(Accepted November 10,1988)
Introduction
Snake bite is a major medical problem in Nigeria and in many tropical countries. It is estimated that between 30,000 and 40,000 persons die from snake bite world wide (Dreisbach, 19801. In the grassland areas of Nigeria, the most common snake is the saw-scaled viper @&his c~~~~~~~~). The clinical data on viper bites in Nigeria are reported by Onuaguluchi (19601and Warrell et al. (19’771show that a large proportion of the victims die in spite of energetic treatment with polyvalent antiserum and other modern supportative treatment. A considerable proportion of victims die within a few hours, probably from cardiovascular shock, while a number die some days later after envenomation from the combined effects of prolonged internal bleeding and cardiovascular shock. In Nigeria, many people live far away from hospitals and many hospitals do not always have ample supply of the antiserum; consequently, many victims of snake bite have to be treated by traditional medicine practitioners versed in the use of herbs. My father, an educated Warrant Chief was well known in Achi, Anambra State, and the neighbouring towns for his herbal remedy for snake bites. Although Achi is situated in the Southern part of Nigeria, it has grassland vegetation, and is notorious for its large population of saw-scaled vipers, locally called NVUTUAchi I accompanied my father several times into the bush to collect the herb and on those occasions helped to prepare the herb for use by snake-bite victims. In the treatment of the snake-bite victim, the area of the bite is scarified with a clean razor blade and a thick paste of the ground-up herb (leaves) is applied over the wound. A segment of the stem is fastened a little below the 0378-87~1/89/$03.15 0 1989 Elsevier Scientific Pubfishers Ireland Ltd. Published and Printed in Ireland
190
wound in the belief that it will cause the venom to gravitate down and remain below the wound! The rest of the herb is then chopped up and boiled for approximately 1 h in a pot of water and the patient is given a cup (approx. 200 ml) of the infusion to drink. He continues to take this quantity of the infusion three or four times daily for another 3 days or more. The herb used for this study was collected from the bush in Achi. It was collected in September 1985 and was identified botanically by Dr. J.C. Okafor of the Enugu Forest Herbarium as Diodia scundens (Gronov ex L.1. A specimen of the herb was planted in my garden at Enugu where it grows luxuriantly and thus affords one a ready access to the herb for future studies. Enugu is approximately 50 km east of Achi. Diodia scandens is a member of the Rubiaceae and is a creeping or climbing herbacious plant. Figure 1 shows the stem, leaves and flowers of the
Fig. 1. A branch of Diodia scandens showing placement of the leaves aggregated in large numbers at the node and at the apex of the branch.
and the white flowers
191
plant. The flowers are white and are aggregated in large numbers nodes and at the end of the branches. The plant grows luxuriantly the rainy season but withers somewhat in the dry season. Materials Extraction
at the during
and methods methods
After collection from the bush, the aerial parts were stored overnight in a refrigerator wrapped in paper and sealed in a polythene bag. The next day, the herb was chopped and weighed. The material was then extracted in a soxhlet apparatus with boiling 95% ethanol for 12 h. Upon removal of the solvent, a dark green residue was obtained. Clarification of this residue was achieved by dissolving it in 50% aqueous ethanol and passing the solution through a column of activated charcoal. The filtrate obtained was colourless and deposited colourless crystals on standing. Further crystallisation occurred when the filtrate was kept overnight in a refrigerator in a covered container. The crystals (fraction A) were removed by filtration using Whatman filter paper, dried in the air and weighed. The yield was 0.40% (w/w) in terms of fresh starting material. The mother liquour was evaporated under reduced pressure to dryness in a water bath maintained at 40°C and yielded a dark brown residue (fraction B). This residue was kept in a desiccator in a glass container of known weight. The container with the residue was weighed every 2 days until the weight remained constant. The weight of the residue was then determined (yield = 3.4%). Finally, the charcoal column was eluted with 50% aqueous ethanol containing 0.05% glacial acetic acid. Evaporation of the eluate yielded a brown residue (fraction CL The weight of fraction C was determined (yield = 0.35%) by using the procedure described in respect to fraction B. Fraction A was insoluble in water while fraction B and C were water soluble. Because fraction B was obtained in the largest amounts and because of its solubility in water, it was the fraction used for this study. Protection
of mice from
venom
Preliminary studies were performed to determine the toxic potency of the specimen of the Echis carinatus venom obtained from Sigma Chemicals, USA. Albino mice of either sex weighing between 20 and 25 g were divided into four groups of ten. Each mouse in each group received the same dose of the venom intraperitoneally (i.p.1: 2, 4, 8 or 12 mglkg. The animals were observed closely during the first 4 h after venom administration. Thereafter, each group was kept in a separate cage and left overnight in the laboratory with the usual supply of water and pellets of food. The number of deaths 24 h after administration of the venom was noted and the percent mortality calculated for each dose of venom. From this preliminary study it was clear that the surely lethal dose of
192
venom should not be more than 4 mg/kg as there was 100% mortality at this dose within 1 h. Consequently, for the protection study, the doses of venom used were 0.5, 1, 2, 3 and 4 mglkg. Mice weighing between 20 and 25 g were divided into ten groups of ten animals each. Five groups received only venom while the remaining five groups were pretreated i.p. with fraction B (1.5 mg/ kg), 30 min before venom administration. The mice were observed very closely for 4 h after administering the venom and were left in cages as already described. The number of mice dead in each group was noted 24 h after venom injection and the percent mortality due to each dose of venom in the presence and absence of the herbal extract was calculated. Log doseresponse curves showing the percent mortality due to venom in the presence and absence of the herbal extract were constructed and the LD,, of the venom in the presence and absence of the herbal extract were calculated. Statistical analyses were performed using the $test with a P value of < 0.05 taken as indicating significance. Acute toxicity of fraction B in mice Because of the protective effect obtained with the fraction B at 1.5 mglkg, it was necessary that its level of toxicity should be determined. Preliminary studies showed that no mouse died even after receiving 400 mg/kg i.p. Therefore, the following doses of fraction B were used for the primary study: 0.4, 1, 1.5, 2, 3 and 4 kg/kg i.p. Mice of either sex weighing between 20 and 25 g were divided into groups of ten animals each. The animals were closely observed as already described. The number of mice dead 24 h after administering the herbal extract was noted and the percent mortality due to each dose was calculated. Effect of fraction B on venom-induced effects in the anaesthetised cat Three cats weighing between 1 and 2 kg were anaesthetised with thiopentone sodium (50 mg/kg i.p.1 and tied in a supine position on a BrownSchuster myographic table. The trachea was cannulated with a metallic tracheal tube and connected to a tambour writing on smoked paper on a kymograph drum. One common carotid artery was cannulated and connected to a mercury manometer in the conventional manner. A femoral vein was exposed and cannulated as an injection site. The cannula was filled with heparinized normal saline and connected to a syringe using a three-way stop cock which allowed for the injection of saline, venom or drugs (fraction B or adrenaline). The intravenous effects of adrenaline 50 pg/kg (Gedeon Richter Ltd., Budapest, Hungary) and venom 0.5 mgkg were studied. When the effect of the venom on the blood pressure (BP) had disappeared and the BP had returned to the pre-injection level, the animal was given the extract 10 mgkg dissolved in 1 ml of saline. Ten minutes later, the dose of the venom was reported. The effects of the venom on the BP and respiration before and after giving the herbal extract were then compared.
193
Results Toxicity studies Table 1 shows the mortality seen after various doses of the venom alone and after pretreatment with fraction B (1.5 mg/kg i.p.) When the dose of the venom was 2 mg/kg i.p., the extract reduced the mortality due to the venom from 50% to 10%. The reduction narrowly missed the criterion for statistical significance. However, when the dose of the venom was 3 mg/kg, the extract reduced the mortality from 80% to 30%. This reduction in mortality was statistically significant P < 0.05). Although fraction B appeared not to have ultimately offered protection when the dose of venom was 4 mg/kg, it was observed that deaths occurred within 10 min in all mice not pretreated with the extract, but no mouse pretreated with the extract died in less than 4 h. Moreover the extract prevented the venom-induced clonic convulsions which normally preceded death. The LD, due to the venom was 1.09 mg/kg + 0.61 i.p. (95% confidence limits) but this was increased to 3.0 mg/kg f 0.77 i.p. (95% confidence limits) following pretreatment with fraction B. Fraction B had no deleterious effect on the mice up to doses of 2 g/kg i.p. However, it caused 30% mortality at 3 g/kg i.p. Effect of the fraction B on venom-induced effects in the anaesthetised cat Adrenaline (50 pg/kg i.v.1 produced a 20 -30 mmHg rise in blood pressure which returned to the pre-injection level in approximately 5 min. The venom at 0.5 mg/kg i.v., produced a lo-20 mmHg pressor effect with the blood pressure returning to pre-injection levels in approximately 2 min. Fraction B (10 mg/kg i.v.1 given 10 min after administering the venom had no effect on
TABLE
1
EFFECT OF FRACTION B (1.5 mg/kg i.p.) ON THE PERCENT RECEIVING VARIOUS DOSES OF ECHIS CARINATUS VENOM Venom dose (mg/kg)
Mortality
0.5 1.0 2.0 3.0 4.0 *Statistically
significantly:
P < 0.05.
MORTALITY
IN MICE
(%I)
Venom alone
Venom + extract
10 40 50 80 100
10 10 10 30* 100
194
Fig. 2. Effect of the fraction induced in the anaesthetised
B 10 mg/kg i.v. (B) on changes in blood pressure cat by 0.5 mg/kg i.v. of Echis carinatus venom (VI.
and
respiration
the blood pressure in the cats. However, it prevented the rise in blood pressure when the venom (0.5 mg/kg i.v.1 was repeated 10 min later. On respiration, the venom at 0.5 mg/kg i.v. caused a marked reduction in the respiratory rate and there were long periods of apnoea. Fraction B reduced the duration of the apnoeic periods and the respiration became regular approximately 4 min after extract injection. When the dose of the venom was repeated 10 min after injecting fraction B, the apnoeic periods induced by the venom were less pronounced than when the cat had not been treated with the extract. Figure 2 shows the effect of the venom (0.5 mglkg i.v.1 on blood pressure and respiration in the absence and presence of fraction B. It also shows the salutary effects of fraction B on the Cheyne-Stokes type of respiration induced by the venom. Discussion
The results
of the toxicity
studies
in mice showed that the extract
from
Diodia scandens tended to protect mice from the lethal effects of the venom of Echis carinatus. Although no protection from death was observed when
the dose of venom was 4 mg/kg i.p., the extract delayed the clonic convulsions produced by the venom. At this dose level, all untreated mice died within 10 min but no animal pretreated with the extract died before 4 h had elapsed. Venom-induced convulsions were also observed by Warrell et al. (1977) but have not been observed clinically in man. This may be due to the fact that for a 60-70 kg human victim, the maximum dose of the venom which the snake could possibly introduce would be approximately 0.5 mg/kg and indeed could be appreciably less since the approximate yield of dry venom from Echis carinatus is 20-35 mg (Dreisbach, 1980). The effects of the venom on blood pressure and respiration are of considerable interest. The venom at 0.5 mg/kg caused a small rise in blood
195
pressure (lo-20 mmHg1 but induced a marked Cheyne-Stokes disturbance of the rhythm of respiration. The rise in blood pressure was surprising, because clinically, when the snake bite victim presents, the blood pressure is often low from shock. However, it is possible that even in human subjects, a rise in blood pressure could occur first to be followed some minutes later by a state of shock with peripheral vascular collapse due to the liberation of large amounts of kinins by the venom (Cohen et al., 19691.The rise in blood pressure in the cat experiments may be due to the effect of increased PCO, from the Cheyne-Stokes type of respiration induced by the venom or to a direct vasoconstrictive effect on the vascular smooth muscles or to some combination of these factors. Further work on the effects of the venom and the extract of Diodia scandens on the mammalian cardiovascular system is clearly indicated. It is of great interest that fraction B of Biodia scandens at 1.5 mg/kg partially protected mice from the lethal effects of the venom but had no deleterious effect itself on the mice up to doses of 2 g/kg i.p. This indicates that its safety margin should be very high. The dose of the extract used in this study to protect mice would appear to be small but it was chosen because in a separate study on the in vitro effects of fraction B on the blood coagulation defects induced by the venom, it was found that a l:lO,OOO solution of the extract statistically significantly inhibited some of the defects induced by a l:lO,OOOsolution of the venom (unpublished data). Since the minimum lethal dose of the venom was approximately 0.5 mg/kg and its LD, was 1.09 mglkg, the dose of 1.5 mglkg which was chosen for the extract appeared appropriate for this preliminary study. Another plant reputed to be of some value in the treatment of snake bite in Nigeria is Schumanniophyton magnificum Harms (family Rubiaceael. However, this is a shrub and requires many years to grow. In contrast, Diodia scandens is a quick growing herb and could be exploited for commercial production of a safe cheap drug for treating snake bites. This plant could, therefore, be of much interest to the many Third World countries in the tropics who cannot afford, because of scarce financial resources, to produce or purchase antisera to treat snake bites. It is noteworthy that heat destroys the antivenom effect of the active principle from the stem bark of Schumanniophyton magnificum (Akunyili and Akubue, 1986; 19871 so that drying and storage may adversely affect its antivenom activities. On the other hand, the antivenom effect of Diodia scandens is apparently not affected by boiling the plant. Acknowledgements
I wish to thank Mr. Paul Okeke for technical assistance, Mrs. B. Ukonu for typing the manuscript, the Medical Illustration Unit for the photographs and Dr. J.C. Okafor for identifying the plant botanically.
196
References Akunyili, D.N. and Akubue, P.I. (1986) Schumanniofoside, the antisnake venom principle from the stem bark of Schummanniophyton magnificum Harms. Journal of Ethnopharmacology 18, 167- 172. Akunyili, D.N. and Akubue, P.I. (1987) Antisnake venom properties of the stem bark juice of Schumanniophyton magnificum. Fitoterapia 58, 47-49. Cohen, M., Zur, E.M. and De Vries, A. (1969) Isolation and characterization of kinin-releasing enzyme of Echis coloratus venom. Biochemical Pharmacology 19, 785-793. Dreisbach, R.H. (1980) Handbook of Poisoning, Lange Medical Publications, Los Altos, California, p. 457. Onuaguluchi, G.O. (1960) Clinical Observations on snake bite in Wukari, Nigeria. Transactions of the Royal Society of Tropical Medicine and Hygiene 54, 265-269. Warrell, D.A., Davidson, N.M., Greenwood, B.M., Ormerod, L.D., Pope, H.M. Watkins, B.J. and Pretence, C.R.M. (1977) Poisoning by bites of the saw-scaled or carpet viper Wchis carinatus) in Nigeria. Quarterly Journal of Medicine 46, 33-62.