Hypoglycemic effect of Sclerocarya birrea {(A. Rich.) Hochst.} [Anacardiaceae] stem-bark aqueous extract in rats.

Summary

This study was undertaken to evaluate the hypoglycemic effect of Sclerocarya birrea {(A. Rich.) Hochst.} subspecies caffra (Sond.) Kokwaro [family: Anacardiaceae] stem bark aqueous extract in normal (normoglycemic) and in streptozotocin (STZ)-treated, diabetic Wistar rats. In one set of experiments, graded doses of S. birrea stem-bark aqueous extract (SB, 100-800 mg/kg p.o.) were separately administered to groups of fasted normal and fasted diabetic rats. In another set of experiments, a single dose of the plant aqueous extract (SB, 800 mg/kg p.o.) was used. The hypoglycemic effect of this single dose (SB, 800 mg/kg p.o.) of S. birrea stem-bark aqueous extract was compared with that of chlorpropamide (250 mg/kg p.o.) in both fasted normal and fasted diabetic rats. Following acute treatment, relatively moderate to high doses of S. birrea stem-bark extract (SB, 100-800 mg/kg p.o.) produced dose-dependent, significant reductions (P < 0.05-0.001) in the blood glucose concentrations of both fasted normal and fasted diabetic rats. Chlorpropamide (250 mg/kg p.o.) also produced significant reductions (P < 0.05-0.001) in the blood glucose concentrations of the fasted normal and fasted diabetic rats. Administrations of the single dose of S. birrea stem-bark aqueous extract (SB, 800 mg/kg p.o.) significantly reduced (P 0.01 < 0.001) the blood glucose levels of both fasted normal (normoglycemic) and fasted STZ- treated, diabetic rats. The results of this experimental animal study indicate that aqueous extract of Sclerocarya birrea possesses hypoglycemic activity, and thus lend credence to the suggested folkloric use of the plant in the management and/or control of adult-onset, type-2 diabetes mellitus in some African communities.

Key words: Sclerocarya birrea stem-bark, aqueous extract, hypoglycemic effect

* Introduction

Sclerocarya birrea {(A. Rich.) Hochst.} subspecies caffra (Sond.) Kokwaro [family: Anacardiaceae], popularly known in South Africa as 'marula tree', is a medium-sized, single-stemmed tree of up to 15 metres in height. The rough stem-bark is flaky, with a mottled appearance due to contrasting grey and pale-brown patches. The leaves are divided into 10 or more pairs of leaflets, each about 60 mm long, dark-green above, much paler below, with the tip abruptly narrowing to a sharp point. The flowers are borne in small, oblong clusters. Male and female flowers occur separately, usually but not always on separate trees. The flowers are small, with red sepals and yellow petals (Van Wyk et al. 1997). The fruit, which is the size of a small plum, is pale yellow when ripe. The rounded, slightly flattened fruit is about 30 mm in diameter and borne in profusion in late South African summer to mid-winter. The fruits are aromatic and edible, and are much sought after by baboons, monkeys, elephants and human beings for their delicious pulp and edible nuts. The outer skin of the fruit has a rather pungent, apple-like odour, and its flavour has been described as resembling that of litchi, apple, guava or pineapple. It makes an excellent conserve. The whole fruit is used in many parts of Southern Africa for the brewing of beer, and spirit is often distilled from it. In Mozambique, the fruit is used for making a "national" fermented beverage. The nut has a very thick shell, containing a kernel. The kernel is edible and very tasty, especially when cooked. Its flavour resembles that of the groundnut. The fruit pulp contains citric and malic acids, vitamin C and sugar, while the nut is rich in a non-drying oil, protein and some iodine. The gum from the tree is rich in tannin, and is sometimes used in making an ink substitute. In Zimbabwe and South Africa, the wood of S. birrea is used for making dishes, mealie stamping mortars, drums, toys, curios, divining bowls and carvings (Watt and Breyer-Brandwijk, 1962). In South Africa, Sclerocarya birrea has become a commercial fruit crop in recent years, the fruit pulp being used to produce a jelly and to flavour liqueur (Van Wyk et al. 1997).

In Southern Africa sub-region, the stem-bark of S. birrea is used for an array of human ailments, including: malaria and fevers, diarrhoea and dysentery, stomach ailments, headaches, toothache, backache and body pains, infertility, schistosomiasis, hypertension, epilepsy, proctitis, gastric and duodenal ulcers, diabetes mellitus, asthma, urinary tract infections, arthritis and other inflammatory conditions, and so forth. The Zulus of South Africa use a decoction of Sclerocarya birrea bark as a prophylactic remedy against gangrenous rectitis, and the fruit for the destruction of ticks. The Zulus also regard the fruit as a potent insecticide. In South and East Africa, the stem-bark of S. birtea is used as a potent remedy for dysentery and proctitis. The Vendas of South Africa usually administer powdered bark of the plant to pregnant women to regulate the sex of babies. The bark from a male tree is administered for a boy, while the bark of a female tree is administered for a girl. The Zulus and Thongas of South Africa also use a decoction of S. birrea bark as a ritual cleansing emetic before marriage. Branches of the tree are also used in the funeral rites of the Thongas. The divining dice of the Shangana diviners include a Sclerocarya nut which represents the 'vegetable kingdom' or 'medicine' (Van Wyk et al. 1997; Hutchings et al. 1996; Pujol, 1993).

A number of African traditional healers have claimed that Sclerocarya birrea stem-bark extracts are effective in the management and/or control of adult-onset, type2 diabetes mellitus. However, there are no reports in the medical literature to support this claim. The present study was, therefore, undertaken to evaluate the hypo glycemic effect of S. birrea stem-bark aqueous extract in laboratory animals, with a view to providing a pharmacological justification (or otherwise) for the folkloric use of the plant in the management and/or control of adult-onset, type-2 diabetes mellitus in some African communities.

* Materials and Methods

The experimental protocol used in this study was approved by the Ethics Committee of the University of Durban-Westville, Durban 4000, South Africa; and conforms with the "Guide to the care and use of animals in research and teaching" [published by the Univerity of Durban-Westville, Durban 4000, South Africa].

Plant material

Pieces of fresh stem-barks of S. birrea were collected from the University of Durban-Westville's campus in Durban, South Africa, between January 2000, and June, 2001. The stem-barks were identified by Prof. H. Baijnath, the Chief Taxonomist/Curator of the University of Durban-Westville's Department of Botany, as those of Sclerocarya birrea {(A. Rich.) Hochst.} subspecies caffra (Sond.) Kokwaro [family: Anacardiaceae].

* Preparation of the Extract: One kg of the fresh stem-bark of S. birrea was air-dried at room temperature, cut into small pieces, pounded and then homogenized in a Waring blender. The powdered stem-bark was Soxhlet extracted twice, on each occasion with 2.5 1 of distilled water at room temperature for 24 hours with shaking. The combined aqueous extracts were filtered and concentrated to dryness in vacuo under reduced pressure at 30[+ or -]1 [degrees]C. The resulting aqueous extract was freeze-dried, finally yielding 48.5 g (4.85%) of dark-brown, powdery, S. birrea stem-bark crude aqueous extract. Aliquot portions of the plant extract residue were weighed and dissolved in distilled water for use on each day of our experiment.

* Animal Material: Balb C mice (Mus domesticus) of both sexes, and young adult, male Wistar rats (Rattus norvegicus) were used. The animals were kept and maintained under laboratory conditions of temperature, humidity, and light; and were allowed free access to food (standard pellet diet) and water ad libitum. The animals were divided into drug-treated 'test' and distilled water-treated 'control' groups of 8 animals per group. All the animals were fasted for 12 hours, but still allowed free access to water, before the commencement of our experiments. The mice were used for the 'acute toxicity testing' of the plant extract, while the rats were used for the hypoglycemic investigation of the stem-bark extract.

* Acute Toxicity Testing: Balb C albino mice (Mus domesticus) weighing 20-25 g were used for the acute toxicity testing experiments. The median lethal dose ([LD.sub.50]) of S. birrea stem-bark aqueous extract was determined in the mice according to the method of Lorke (1983). Mice fasted for 12 hours were randomly divided into groups of 8 mice per group. Aqueous extract of S. birrea (SB) 50, 125,250, 500, 1000 and 1500 mg/kg were separately administered orally to the mice in each of the 'test' groups. Each of the mice in the 'control' group was treated with distilled water (2 ml/kg p.o.) only. The mice in both the 'test' and 'control' groups were then allowed free access to food and water, and observed over a period of 24 hours for signs of acute toxicity. The number of deaths (caused by the extracts) within this period of time was recorded. Log-dose response plots were constructed for the plant extract, from which the median lethal dose ([LD.sub.50]) of the aqueous extract was determined.

* Determination of blood glucose levels: Young adult, male Wistar rats (Rattus norvegicus) weighing 250-300 g were used. The animals were randomly divided into two (A and B) groups of 'test' and 'control' rats. Diabetes mellitus was induced (in Group A diabetic 'test' rats) by intraperitoneal injections of streptozotocin (STZ, 90 mg/kg). Diabetes was allowed to develop and stabilize in these STZ-treated rats over a period 3 to 5 days. The 'control' (Group B) normal (normoglycemic) rats were treated with distilled water (2 ml/kg p.o.) only. All the animals were kept and maintained under laboratory conditions of temperature, humidity, 12-hour day:12-hour night cycle; and were allowed free access to food (standard pellet diet) and water ad libitum. Before the commencement of our experiments, both the 'control' normal (normoglycemic) and STZ-treated, diabetic (hyperglycemic) rats were fasted for 12 hours, but still allowed free access to water throughout. Fasted STZ-treated rats with blood glucose concentrations [greater than or equal to] 500 mg/dl were considered to be diabetic, and used in this study. At the end of the 12 hours fasting period--taken as zero time (i.e., 0 hour), blood glucose levels (initial glycaemia--Go) of the fasted normal (normoglycemic) and STZtreated, diabetic rats were determined and noted. For both the normoglycemic and hyperglycemic animals, chlorpropamide (250 mg/kg p.o.) was used as reference antidiabetic (hypoglycemic) agent for comparison. The test compounds [i.e., S. birrea stem-bark aqueous extract (100-800 mg/kg p.o.) and chlorpropamide (250 mg/kg p.o.)] were administered orally to the groups of fasted normal and fasted diabetic 'test' rats by means of oesophageal catheter. 1, 2, 4 and 8 hours following administration of the 'test' compounds to the animals, blood glucose concentrations (G[sub.t]) were determined. Blood samples were collected from the "tail vein" of each rat for blood glucose analysis. Blood glucose concentrations were determined by means of Bayer's Glucometer Elite and compatible blood glucose test strips. The percentage of glycemic variation was calculated as a function of time (t) by applying the formula:

% glycemic change = [G.sub.t]- [G.sub.0] x 100 / [G.sub.0]

where [G.sub.0] and [G.sub.t] represent initial (zero time--0 hour) glycemic values before, and glycemic values at 1, 2, 4 and 8 hours after, oral administrations of the 'test' compounds respectively. At the same time, rats treated with distilled water alone (2 ml/kg p.o.) were used as 'controls'.

Data Analysis

Blood glucose concentration data obtained from the blood samples of S. birrea stem-bark aqueous extractor chlorpropamide-treated fasted 'test' rats, as well as those obtained from distilled water-treated fasted 'control' rats were pooled, and expressed as means ([+ or -] SEM). The difference between the plant extract- or chlorpropamide-treated 'test', and distilled water-treated 'control' means was analysed statistically by using "Student's t-test" (Snedecor and Cochrane, 1967). Values of P [less than or equal to] 0.05 were taken to imply statistical significance.

* Results

In the mice, intraperitoneal injections of graded doses of S. birrea stem-bark aqueous extract in our acute toxicity tests produced a median [LD.sub.50] value of 1215 [+ or -] 38 mg/kg. This finding probably suggests that the plant extract is safe in mammals.

In a separate set of experiments involving normal 12-hour fasted and normal non-fasted rats, the mean blood glucose levels were found to be 65 [+ or -] 7.2 mg/dl and 108 [+ or -] 5.31 mg/dl respectively. In our 'control' set of experiments, pre-treatment of the animals with distilled water (2 ml/kg p.o.) did not significantly modify (P > 0.05) the blood glucose concentrations of either the fasted normal, or fasted diabetic rats. In these animals, pre-treatment with distilled water (2 ml/kg p.o.) for 1, 2, 4 and 8 hours either slightly but insignificantly (p > 0.05) decreased, increased, or did not affect at all, the blood glucose concentrations of the fasted control animals. The distilled water-induced changes in the blood glucose levels of the fasted rats varied by values ranging between 0.1% and 0.8% of the mean basal blood glucose concentrations (data not shown). However, compared with the distilled water-treated 'control' rats, pre-treatment of the fasted animals with relatively moderate to high doses of S. birrea stem-bark aqueous extract (SB, 100-800 mg/kg p.o.) produced dose-dependent, significant reductions (p < 0.05-0.001) in the blood glucose concentrations of both fasted normal and fasted diabetic 'test' rats. The maximal reductions in the blood glucose concentrations of the fasted 'test' rats occurred at the plant extract dose of 800 mg/kg p.o. (see Fig. la, b). Pre-treatment of fasted normal (normoglycemic) and STZ-treated diabetic rats with chlorpropamide (250 mg/kg p.o.) or with S. birrea stembark aqueous extract (SB, 800 mg/kg p.o) for 1, 2, 4 and 8 hours also produced significant reductions (P < 0.05-0.001) in the blood glucose concentrations of the animals, compared with distilled water-treated fasted 'control' rats (see Table la, b). The hypoglycemic effect of the plant extract became significant (P < 0.05) 1 hour following oral administration, reaching the peak of its hypoglycemic effect 4 hours after oral administration. However, the hypoglycemic effect of the plant extract was still significant 8 hours after oral administration of the plant extract (see Table 1 a, b). Thereafter, the blood glucose concentrations of the animals gradually returned to normal levels at the end of the 24th hour.

* Discussion and Conclusion

The major classes of synthetic oral hypoglycemic agents currently available for the management and/or control of adult-onset, NIDDM, type-2 diabetes mellitus include the sulphonylureas, biguanides, thiazolidinediones, alpha-glucosidase inhibitors, and so forth.

Chlorpropamide, used as the standard hypoglycemic agent in this study, is a member of the 'first-generation' sulphonylureas. As a class, sulphonylureas enhance and increase the release of endogenous insulin from pancreatic [beta]-cells. They also promote and facilitate peripheral tissue uptake and utilization of glucose. It has been proposed (Jackson and Bressler, 1981) that sulphonylureas produce their hypoglycemic effects via three main mechanisms, viz:

1. increased insulin release from pancreatic [beta]-cells: sulphonylureas bind to pancreatic receptors associated with potassium ([K.sup.+]) channels [[K.sup.+]-channels] on the surface of the pancreatic [beta]-cells. This binding inhibits [K.sup.+] efflux from the [beta]-cells through the [K.sup.+]-channels. Consequently, depolarization of pancreatic [beta]-cells ensues. This depolarization opens the 'voltage-gated' calcium ([Ca.sup.2+]) channels [[Ca.sup.2+]-channels]. This process allows and/or facilitates [Ca.sup.2+] influx into the pancreatic [beta]-cells, subsequently resulting in the release of preformed insulin from pancreatic [beya]-cells;

2. potentiation of insulin's action on target tissues and increased glucose removal from the blood: sulphonylureas increase the binding of insulin to peripheral tissue insulin receptors, thereby enhancing glucose uptake and utilization by peripheral tissues;

3. reduction of blood glucagon levels: administration of sulphonylureas may lead to reduced blood glucagon concentrations. This effect can be attributed, at least in part, to the hypoglycemic action of sulphonylureas, since the primary actions of glucagon are to enhance the metabolism of stored glycogen, and to facilitate and/or increase gluconeogenesis and ketogenesis. Enhanced release of insulin (and somatostatin) inhibits glucagon secretion via inhibition of pancreatic [alpha]-cell secretion. Thus, any plant chemical constituent which is capable of affecting the pancreatic [beta]- or [alpha]-cell secretion in any of the three ways illustrated above will be a good mimicker of sulphonylureas, and will produce hypoglycemic effects in mammals via mechanisms similar to those of sulphonylureas.

Although the use of herbal remedies for the treatment of diabetes mellitus has greatly declined in Europe and many other Western countries since the introduction of insulin and synthetic oral hypoglycemic agents in the 1920s and 1950s respectively (SwanstonFlatt et al. 1989, 1990), approximately 80% of the peoples in the rural African communities still rely on the use of plant remedies to control and/or manage diabetes mellitus. In Africa, hundreds of plants are used traditionally for the management and/or control of diabetes mellitus. To date, however, only a few of such African medicinal plants have received scientific scrutiny, despite the fact that the World Health Organization has recommended that medical and scientific examinations of such plants should be undertaken (WHO Expert Committee on Diabetes mellitus, 1980). Sclerocarya birrea is one of such neglected medicinal plants commonly used in African traditional medicine.

Since streptozotocin is known to destroy insulin-producing pancreatic [beta]-cells, the STZ-treated rat model, therefore, appears to represent a good laboratory NIDDM experimental diabetic state, with residual or remnant insulin production by the pancreatic [beta]-cells. The diabetic state of STZ-treated diabetic rats is, therefore, not the same as that obtained by total pancreatectomy, as daily administration of insulin is not required for survival in STZ-treated diabetic animals.

Acute treatment of the 'control' Wistar rats with distilled water alone did not produce any significant change in the blood glucose concentrations of either the fasted normal or the fasted STZ-treated, diabetic rats (data not shown). However, the aqueous plant extract, like chlorpropamide--a sulphonylurea antidiabetic agent, produced significant reductions in the blood glucose levels of fasted normal and fasted STZ-treated diabetic rats. The plant extract examined in this study appears to act promptly and markedly in both fasted normal and fasted STZ-treated diabetic rats. The hypoglycemic effect of S. birrea stem-bark aqueous extract thus appears to be most probably exerted via a mechanism that is similar to that of chlorpropamide, and/or probably related to insulin secretion from pancreatic [beta]-cells.

Sclerocarva birrea has been widely reported to contain many chemical compounds, including: tannins and flavonoids, alkaloids, steroids including [beta]-sitosterol, coumarins, triterpenoids, sesquiterpene hydrocarbons, ascorbic acid, oleic acid, myristic, stearic and amino acids with a predominance of glutamic acid and arginine (Van Wyk et al. 1997; Hutchings et al. 1996; Watt and Breyer-Brandwijk, 1962). A number of investigators have shown that coumarins, flavonoids, terpenoids, and a host of other secondary plant metabolites, including arginine and glutamic acid, possess hypoglycemic effects in various experimental animal models (Akah and Okafor, 1992; Marles and Farnsworth, 1995; Ross, 1999, 2001; Ojewole, 2002). Although the hypoglycemic effect of terpenoids appears to involve stimulation of pancreatic [beta]-cells and subsequent secretion of preformed insulin, the mechanism of the hypoglycemic action of coumarins probably involves hepatotoxicity (Marles and Farnsworth, 1995). Coumarins are hepatotoxic in rats and dogs, where they are metabolized through 3-hydroxycoumarin pathway to reactive quinone metabolites that bind covalently to microsomal proteins. In humans and other primates, however, coumarins are metabolized through 7-hydroxycoumarin route to glucuronide conjugates that are rapidly excreted from the body, and consequently, no hepatotoxicity occurs in human beings (Cohen, 1979; Marles and Farnsworth, 1995). Furthermore, since Sclerocarya birrea contains arginine and glutamic acid in addition to its several other chemical constituents, it is not unlikely that these chemical compounds have contributed, at least in part, to the hypoglycemic effect of S. birrea stem-bark aqueous extract observed in this study. For instance, possibilities exist that arginine could have increased the ability of the plant extract to stimulate and increase preformed insulin secretion from pancreatic [beta]-cells by depolarizing pancreatic [beta]-cells. Moreover, insulin secretion by the pancreatic [beta]-cells could also have been increased by glutamic acid metabolism.

The exact chemical constituent/s of the plant extract that is/are specifically responsible for the hypoglycemic effect of S. birrea stem-bark aqueous extract remains speculative at present. However, if the hypothesis of Marles and Farnsworth (1995) which stipulates that "plants which contain terpenoids and/or coumarins possess hypoglycemic activities in diabetic and normal mammals" is anything to go by, it would seem reasonable to assume that, in part at least, the hypoglycemic effect of S. birrea stem-bark aqueous extract may probably be due to the coumarins and/or terpenoids present in the plant. One or more of the other chemical constituents of the plant, especially flavonoids, arginine, glutamic acid, and so on, is/are also likely to have played a crucial role in the hypoglycemic action of the plant extract. Nevertheless, the exact mechanism of the hypoglycemic action of the plant extract remains largely speculative.

The next step in our series of experimental studies will be to probe the chemical constituents of S. birrea stem-bark aqueous extract for hypoglycemic activity, and thereafter establish the mechanism/s of their hypoglycemic action. In conclusion, the experimental evidence obtained in this laboratory animal study indicates that S. birrea stem-bark aqueous extract possesses hypoglycemic activity, and thus lends credence to the suggested folkloric use of the plant in the management and/or control of adult-onset, type-2 diabetes mellitus in some African communities.

Table 1a. Effects of Sclerocarya birrea stem-bark aqueous extract
(SB, 800 mg/kg p.o.) and chlorpropamide (250 mg/kg p.o.) on blood
glucose concentrations (mg/dl) of normal (normoglycemic) rats.
Values given represent the mean ([+ or -] SEM) of 8 observations.

Treatment          Before           After Treatment
                   Treatment
                   0 hr             l hr

Control (2 ml/kg   110.12           111.05
distilled water)   [+ or -] 3.56    [+ or -] 2.75

SB                 109.43           101.36
(800 mg/kg p.o.)   [+ or -] 4.21    [+ or -] 3.55 *

Chlorpropamide     110.25           96.50
(250 mg/kg p.o.)   [+ or -] 4.13    [+ or -] 3.37 **

Treatment          After Treatment

                   2 hr                4 hr

Control (2 ml/kg   109.35              110.55
distilled water)   [+ or -] 4.15       [+ or -] 3.50

SB                 92.70               78.81
(800 mg/kg p.o.)   [+ or -] 4.19 **    [+ or -] 3.76 ***

Chlorpropamide     81.41               69.15
(250 mg/kg p.o.)   [+ or -] 4.40 ***   [+ or -] 3.62 ***

Treatment          After Treatment     Maximal      %
                                       Reduction    Maximal
                   8 hr                             Reduction

Control (2 ml/kg   111.15              0.77         0.70
distilled water)   [+ or -] 4.68

SB                 94.53               30.62 ***    27.98 ***
(800 mg/kg p.o.)   [+ or -] 4.15 **

Chlorpropamide     83.35               41.10 ***    37.28 ***
(250 mg/kg p.o.)   [+ or -] 4.21 ***

* P < 0.05; ** P < 0.01; *** P < 0.001 [Student's t-test]

Table 1b. Effects of Sclerocarya birrea stem-bark aqueous extract
(SB, 800 mg/kg p.o.) and chlorpropamide (250 mg/kg p.o.) on blood
glucose concentrations (mg/dl) of STZ-treated, diabetic rats.
Values given represent the mean ([+ or -] SEM) of 8 observa-tions.

Treatment          Before           After Treatment
                   Treatment
                   0 hr             1 hr

Control (2 ml/kg   560.45           558.51
distilled water)   [+ or -] 20.48   [+ or -] 24.55

SB                 550.51           464.37
(800 mg/kg p.o.)   [+ or -] 25.41   [+ or -] 23.28 *

Chlorpropamide     558.55           455.42
(250 mg/kg p.o.)   [+ or -] 23.30   [+ or -] 25.40 *

Treatment          After Treatment

                   2 hr                4 hr

Control (2 ml/kg   556.35              558.50
distilled water)   [+ or -] 25.36      [+ or -] 25.41

SB                 376.22              281.56
(800 mg/kg p.o.)   [+ or -] 21.55 **   [+ or -] 20.45 ***

Chlorpropamide     340.63              232.45
(250 mg/kg p.o.)   [+ or -] 27.15 **   [+ or -] 24.50 ***

Treatment          After Treatment     Maximal      %
                                       Reduction    Maximal
                   8 hr                             Reduction

Control (2 ml/kg   558.21              4.10         0.73
distilled water)   [+ or -] 27.26

SB                 355.61              268.95 ***   48.85 ***
(800 mg/kg p.o.)   [+ or -] 21.38 **

Chlorpropamide     283.35              326.10 ***   58.38 ***
(250 mg/kg p.o.)   [+ or -] 26.44 **

* P < 0.05; ** P < 0.01; *** P < 0.001 [Student's t-test]

Acknowledgements

The author is grateful to Prof. H. Baijnath for the identification of Sclerocarya birrea stem-bark examined in this study. He also wishes to thank Prof. C. O. Adewunmi for his valuable and constructive comments; Dr. E. K. Mutenda for her assistance in the extraction processes; Mrs P. Koloko for her interest in this study; and the Council of the University of Durban-Westville for the provision of a Research Grant [R642] to carry out a part of this study.

* References

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Lorke D (1983) A new approach to practical acute toxicity testing. Archieves of Toxicology 54:275-287

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* Adress

John A. O. Ojewole

Department of Pharmacology, University of Durban-Westville

Private Bag X5400, Durban 4000, South Africa

Tel.: ++27-31-204 4356; Fax: ++27-31-204 4907;

e-mail: [e-mail]