Alloxan Recovered Rabbits as Animal Model for
Screening for Hypoglycaemic Activity of Compounds
Author: B. Venkanna Babu, Radha Moorti, S. Pugazhenthi, K.M. Prabhu, P.
Suryanarayana Murthy
Type of Publication: Pre-Clinical
Date of Publication: December 1988
Publication: Indian Journal of Biochemistry & Biophysics Vol. 25, pp 714-718,
December 1988
Organization/s: University of College of Medical Sciences
Abstract: A rabbit model has been developed for testing the hypoglycaemic activity
of plant extracts and drugs. Some rabbits given intraveously alloxan (80 mg/kg
body wt) showed initial increase in values of fasting blood glucose (FBG) up to 15
days which reverted to normal or near normal levels. In such rabbits, called “alloxan
recovered” (AR) rabbits, hypoglycaemic effect, could be assessed by improvement in
glucose tolerance tested by the prevention of elevation of blood glucose following
oral glucose load. In AR rabbit the effect, impaired glucose tolerance, was stable up
to 4 months. Hypoglycaemic effect of compounds having pancreatic and
extrapancreatic mechanism of action could even be quantitated from the area under
the curve.
Hypoglycaemic activity of plant extracts and other synthetic compounds is
conventionally assessed by inducing stable diabetes mellitus in a suitable animal and
observing the changes in either fasting blood glucose (FBG) or by the glucose
tolerance test (GTT). Diabetes is produced experimentally by stress, partial or toal
pancreatectomy and destruction of islets of Langerhans by chemical agents like
alloxan and streptozotocin. Anti-islets antibodies could also induce diabetes
mellitus1. Rats or rabbits are usually employed for such studies. Many plants
reported to be useful for the treatment of diabetes mellitus in Ayurvedic system of
medicine2 have been tested for their hypoglycaemic activity in diabetic animals by
various investigators3.4. Active principles have been partially purified from some
plant; for example charantin from Momordica charantia Linn5, trigonelle from
Trigonella foenum graecum6, bengalinoside from Ficus bengalensis7 and allicin from
Allium sativum8. None of these is in actual clinical use. Three medicinal plants, M.
charantia, T. foenum graecum and F. bengalensis, have been taken up for detailed
studies starting with extracts obtained with different solvents. However, with solvent
extracts, the hypoglycaemic effect, as judged by decrease in FBG levels in severe
alloxan diabetic rabbits, has been observed to be inconsistent and small. It was felt
that the conventional severely diabetic animal modelin which pancreas is completely
destroyed has some limitations because drug acting through pancreas would be
ineffective. By following the course of alloxan diabetes, abetter model which could
detect hypoglycaemic activity of crude extracts has been developed and the results
are presented here.
Materials and Methods
Animals—Male albino rabbits weighing between 1.0 and 1.5 kg were
purchased from the local animal breeders M/s All India Chemical and Scientific
Industries and kept in individual cages for two weeks from acclimatization. Rabbit
food (Hindustan Lever Ltd., Bombay) and water were given ad libitum.
Chemicals—Alloxan monohydrate, glucose oxidase (EC 1.1.34) and odianisidine hydrochloride were purchased from Sigma Chemical Company, St. Louis,
USA. Peroxidase was purchased from CSIR Centre for Biochemicals, Delhi.
Tolbutamide was a gift from M/s Hoechest Pharmaceuticals, Bombay. All the other
chemicals were purchased locally.
Fasting blood glucose (FBG) and glucose tolerance test (GTT) – For FBG,
glucose in the blood of overnight fasted rabbits was estimated by glucose oxidase
method of Hugget and Nixon9. For GTT, glucose (3 g/kg body wt) in water was given
orally. Blood samples were drawn at intervals of 30 min up to 150 min abd glucose
was estimated.
Alloxan recovered (AR) rabbits – Overnight fasted rabbits were given alloxan
intraveously (80 mg/kg body wt). FBG levels were determined at 5 day intervals.
After one month, the rabbits showing stabilized diabetes (FBG values 250 mg% or
above) were treated as severely diabetic group, and those showing initially higher
FBG values but returning to near normal or slightly above normal FBG values after
some days were kept as a separate group and GTT performed on them. Invariably,
all of them showed impaired glucose tolerance. Such rabbits with normal, near
normal or slightly elevated fasting blood glucose pattern were designated as “alloxan
recovered” (AR) rabbits. Rabbits which did not show any increase in FBG levels even
initially after alloxan injection were considered as totally resistant and excluded from
the studies.
F. bengalensis
Water extract – Fresh bark (50 g) cut into small pieces was homogenized for
10 min in a Waring blendor in 250 ml distilled water, kept overnight at room
temperature, and filtered. Water extract (1 g/kg) was administered orally.
Ethanol extracts – Sun dried bark was exhaustively extracted with ethanol
and the extract evaporated to dryness n vacuo.
Trigonella foenum graecum
Water extract – Fenugreek seeds were soaked overnight in distilled water
(300 ml/100 g). The extract (450 mg/kg) was administered orally.
Momordica charantia Linn
Ethanol extracts – Frest bitter gourds were cut, sun-dried, finely powdered
and extracted with distilled ethanol. The ethanol extract was evaporated to dryness
in vacuo. The residue was suspended in water containing few drops of alcohol and
Tween 80 and administered orally. Water containing same proportion of Tween 80
and alcohol was given to control animals. A. benzene extract was also prepared in a
similar way.
Assessment of hypoglycaemic activity in alloxan recovered (AR) rabbits – In
this test, the same animals also served as controls as indicated below in all groups
including untreated controls. Rabbits with stabilized AR status were used. Five to
seven days before the commencement of the experiment GTT was performed. This
pattern not only ensured the AR status but served as a basal or the pretreatment
glucose tolerance pattern. On the day of the test, fasting blood sample was drawn
from overnight fasted AR rabbits. Test drug was given to experimental group of
animals and equal volume of solvent to control group of animals orally. After 90 min
the glucose tolerance test was repeated as before. The degree of prevention of
elevation of blood glucose levels (especially in the peak value), i.e. suppression of
the glucose tolerance test curve, was taken as the criterion for the quantitative
assessment of hypoglycaemic activity.
Results and Discussion
Studies in normal and diabetic rabbits – Water extract of fenugreek seeds,
water and ethanol extracts of banyan tree bark and ethanol and benzene extracts of
bitter gourds were tried for their hypoglycaemic in normal, moderately and severely
diabetic rabbits. Statistically significant fall in the FBG levels and in blood glucose
levels at 1, 2 and 3 hr after administration of plant extracts was used as the criterion
of activity. With all the three plant materials reported to be active by others10-12, the
fall in blood glucose value was slight or found in some but not all animals thereby
leading to inconclusive results. The results of the studies with the extracts of the
three plants in moderately diabetic rabbits are shown in Table 1. There was some
indication of some hypoglycaemic effect with water extract fo fenugreek seeds as 3 3
hr and of slight but insignificantly activity with benzene extract of bitter gourds. The
drawback in this type of animals was the wide variation (large SD in Table 1) in the
blood glucose values even in controls and therefore was not suitable as method of
assessment of hypoglycaemic effect. The possibility that the hyperglyceamc
princple(s) is also present in the same plant extract and thereby interferes cannot be
excluded.
Coarse of Alloxan diabetes and AR rabbits – In order to devise a more
suitable animal model, the course of events in alloxan-induced diabetes was carefully
followed. In almost all the rabbits starting from the second day of alloxan injection,
there was a progressive elevation in the mean FBG values (Fig. 1) up to about 273 
43 mg% by about 15th day. Those not showing this increase (about 10% of the
animals) were considered as resistant and excluded from the study. After 15 days in
about 45% of the rabbits FBG continued to rise and reached values in the range 280400 mg% and got stabilized. These are severely diabetic rabbits. However, in some
other rabbits the FBG values which increased initially started decreasing and by
about 30-35 days reached normal or near normal values. Spontaneous reversal of
the diabetic state to normal is a known phenomenon and was observed in some
alloxan injected rats by Lazarow13 and in mice by Rerup14. However, these animals
were shown to suffer from abnormalities of glucose metabolism 15 and altered lipid
metabolism16. Keeping these observations in mind GTT was carried out in the
second group of rabbits. Their glucose tolerance pattern indicated that, in contrast
to healthy controls, the peak value reached slightly later, by about 90 min to a much
higher value of 290  12 mg% (Fig. 2) and remained at a higher level (207 V 12
mg%) at 2.5 hr instead of returning to fasting levels. The area under the curve
(AUC) for normal and alloxan recovered rabbits was 18,960  872 and 33,439  114
mg/dL/min, respectively and the increase was significant (P<0.001).
Although both the normal and alloxan recovered rabbits had comparable
fasting blood glucose levels, the latter group showed abnormal glucose tolerance
pattern i.e. impaired glucose tolerance pattern i.e. impaired glucose metabolism (Fig.
2). Therefore the fall in FBG values need not always be a reliable parameter and in
fact could be misleading for assessing hypoglycaemic activity. Under such
circumstances GTT could be used as a useful parameter provided the abnormal GTT
pattern is stable for a reasonable time and the drugs possessing hypoglycaemic
effect could prevent the elevation of blood glucose following oral glucose load. In
such rabbits this abnormality in GTT pattern and the mean AUC’s at the end of 1, 2
and 4 months did not differ significantly (results not shown). In one rabbit it
remained abnormal even after 9 months indicating that these rabbits remain in a
stable sub-diabetic state of non-insulin dependent diabetes mellitus type for at least
4 months, if left untreated. In view of this, these rabbits were called alloxan
recovered rabbits with impaired glucose tolerance pattern or simply AR rabbits.
Although obtained from a heterogenous population, the abnormal GTT is highly
reproducible.
Effect of tolbutamide on GTT pattern in AR rabbits – Before testing the
activity of plant extracts, it is to be ensured that a known drug, such as tolbutamide
showed suppression of GTT curve. At a dose of 500 mg/kg, tolbutamide showed
significant (P<0.001) suppression of the GTT curve at all points (Fig. 2, curve C).
There was about 40% reduction in the blood glucose level at 90 min. Overall
reduction in the AUC was 30%.
Hypoglycaemic effect of plant extracts in AR rabbits – the hypoglycaemic
effect of the extracts of the three plants was assessed from the prevention of
elevation of blood glucose in GTT curve after drug treatment. The blood glucose
values at the peak (usually at 90 min) and at 150 min and the area under the curve
were taken into consideration. Usually, changes in all the three parameters agreed
as well. All the three plants showed consistent reduction in blood glucose values
during GTT in all the animals tested. The water extract of T. foenum graecum (450
mg/kg) produced a fall of 22% (Table 2). With water and alcohol extracts of F.
bengalensis (1 g/kg), the fall was 20% and 43%, respectively while with the benzene
extract of M. charantia the fall was 23%. The hypoglycaemic potency of all the three
plants is thus established beyond doubt.
In view of this, purification of their active principle was undertaken. There
was one hypoglycaemic principle in each fenugreek seeds and banyan tree bark.
Three hypoglycaemic principles could be isolated from bitter gourd. Another
interesting observation was that in each of the three plants, there was another
hypoglycaemic principle which was found and removed in the isolation procedure.
This could explain, at least partly, why the crude solvent extracts of the three plants
showed only slight or inconsistent activity.
Thus the above studies with AR rabbits using the hypoglycaemic effect of a
drug or plant extracthave demonstrated that the very same extracts which gave
inconclusive or negative results in normal, in mildly and severely diabetic rabbits
gave highly reproducible and consistent results. An advantage of this AR model s
that the same animal serves as its own control before and after treatment
minimizing variations. Therefore this would offer better experimental model for
screening plant extracts with slight activity, or plants having both hypoglycaemic as
well as hyperglyceamic principles, or with plants or drugs, such as tolbutamide,
which require functional pancreas. The activity can be quantified by determining the
area under the curve and also by comparing it with standard drug. In the absence of
such a model, these palnts could have been easily missed as having only slight
activity and highly potent principles (5 from 3 plants) could not have been isolated.
It should be emphasized that ultimately active principles are to be isolated and
tested for their efficacy in all types of animals, viz. normal or with AR, mild and
severe diabetic status. It is hoped that this improved method of screening in AR
rabbits would unravel hypoglycaemic potential of many more plants.
Acknowledgement
This work has been carried out in a Department of Science and Technology Research
project granted to PSM, for which the authors are grateful.
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