Journal of E~~no~~aF~acology, 11 (1984) 275-281 275

Elsevier Scientific Publishers Ireland Ltd.

ANTI-HYPERGLYCAEMIC EFFECT OF AN EXTRACT OF MYRTUS

COMMUNE IN STREPTOZOTOCIN-INDUCED DIABETES IN MICE

MS. ELFELLAH, M.H. AKHTER and M.T. KHAN

Department of Pharmacology, Faculty of Medicine, P. 0. Box 1451, Uniuersity of
Garyounis, Benghazi (Libya)
(Accepted April 26, 1984)

summary
Intraperitoneal injection of streptozotocin (150 mg/kg) in mice produced
an initial transient hyperglycaemia at 2 h followed by a second hypergly-
caemic phase after 48-72 h which persisted throughout the 7-day obser-
vation period. An ethanol-water extract of Myrtus communis (2 g/kg)
administered intr~astric~y 30 min before streptozotocin abolished the
initial hyperglycaemia without affecting the second phase. Myrtus extract
given prior to streptozotocin and repeated at 24 h and 30 h, did not allow
hyperglycaemia to develop until after 48 h. Administration of Myrtus
extract 48 h after streptozotocin significantly reduced the hyperglycaemia
and this effect was maintained by its repeated administration. Myrtus
extract had no effect on the blood glucose level of normal mice. These
studies confirm the “folk-medicine” indication of Myrtus extract as poten-
tially useful in the treatment of diabetes mellitus.

Introduction

The plant Myrtus communis, family Myrtaceae, has a wide “folk-

medicine” reputation in Libya as a hypoglycaemic agent. The water extract
of its leaves and branchlets has long been used here for treating diabetes
mellitus and it is generally believed that it produces beneficial effects.
There appears to be no documented report on the antidiabetic activity of
Myrtus communis although a number of reports (Brahmachari and Augusti,
1961; Ratsimamanga et al., 1973; Oliver Bever and Zahnd, 1979) have
appeared on Eugenic jambolana, a plant belonging to the same family.
Many other plants have also been reported to have hypoglycaemic activity
(Deshmukh et al., 1960; Brahmachari and Augusti, 1962, 1963; Shani et al.,
1974; Oliver Bever and Zahnd, 1979). This paper reports on the antidiabetic
effect of Myrtus communis using streptozotocin-induced hyperglycaemic
mice as diabetic model.

0378-8741/84/$02.40 o 1984 Elsevier Scientific Publishers Ireland Ltd.

Published and Printed in Ireland
276

Experimental
Extraction of the plant material
The dried leaves and branchlets of Myrtus communis were soaked in
50% ethanol in water (v/v) for 10 days at room temperature, then filtered
and the filterate evaporated on a water bath. The residue thus obtained,
constituting 20% of the original material (referred to hereafter as “Myrtus
extract”), was suspended in water by gum tragacanth.
Production of experimental diabetes
Female Swiss albino mice (23-34 g) were used. Throughout the experi-
metal period the animals had free access to a standard pellet diet and tap
water. Streptozotocin (100-200 mg/kg; Boehringer-Mannheim) freshly
dissolved in citrate buffer (pH 4-4.5) was injected intraperitoneally.
Repeated blood samples (10 ~1) were collected from the tail vein by pin-
prick into end-to-end heparinized capillette.
Determination of blood glucose concentration
Blood glucose concentration was estimated spectrophotometricaly by
the hexokinase method using Boehringer-Mannheim Gluco Quant Kit
(Bergmeyer et al., 1974).
Statistical analysis
Statistical analysis was carried out by Student’s t-test, P-values of 0.05
or less being taken to indicate significance.

Study of the effect of Myrtus extract on the streptozotocin-induced hyper-

glycaemia
Myrtus extract was administered to mice intragastrically with the help
of a cannula. One group fo mice received Myrtus extract (2 g/kg) 30 min
before streptozotocin once only, another group received the same dose
30 min before streptozotocin and repeated at 24 h and 30 h after strepto-
zotocin. In another experiment, groups of mice received different doses
(0.5-5 g/kg) of Myrtus extract twice daily for 2-3 days starting 48 h after
streptozotocin treatment. One group of normal mice was administered
Myrtus extract (2 g/kg) twice daily for 3 days. Control mice groups received
only the vehicle.
Results
Table 1 shows dose-related effect of streptozotocin on blood glucose
concentration in mice. At the lowest dose (100 mg/kg) studied, no hyper-
glycaemic response occurred. A dose of 150 mg/kg significantly increased
the blood glucose level to 278 f 31.8 mg% from the control value of 127 *
5.3 mg% (P < 0.001). Increasing the dose to 200 mg/kg, streptozotocin
 277

TABLE 1
EFFECT OF DIFFERENT DOSES OF INTR~ERITONEAL INJECTION OF
STREPTOZOTOCIN ON THE BLOOD GLUCOSE CONCENTRATION IN MICE

Results expressed as mean + S.E.M.

Dose of No. of Blood glucose (mg%)at time after streptozotocin injection (h)
streptozotocin mice
(mg/kg i.p.1 0 24 48 72

Vehicle only
(citrate buffer) 8 138 * 2.35 140 f 1.40 136 f 2.54 146 + 3.18
100 6 143 f 3.88 - 157 f 4.07 141 f 4.16
150 13 127 + 5.31 139 r 4.13& 256 c_30.56s 278 + 31.81*
200 8b 147 f 3.71 187 t. 9.51 374 f: 36.59” 408 t 56.61*

aFigure from 5 mice only.

bOne mouse died after 24 h and three died after 48 h.
* Significantly different from control values (P S: 0.001).

produced a greater rise in blood glucose concentration (408 + 56.6 me) as

compared to the control value (147 rt:3.7 me) but resulted in the mortality
of 50% mice within 72 h. A dose of 150 mg/kg was, therefore, used through-
out this study. In the control group receiving only the vehicle (citrate
buffer), the blood glucose concentration remained almost near the control
values throughout the 72-h observation period.
Study of the temporal course of streptozotocin-induced hyperglycaemia
showed that a dose of 150 mgfkg produced a sharp and transient hype&y
caemia after 2 h. At 4 h the blood glucose level returned to normal but
rose again after 8 h, reaching maximum within 48-72 h (Figs. 1 and Z),
which then persisted at almost the same level for the whole observation
period of 7 days (Fig. 3).
Myrtus extract (2 g/kg) treatment once only 30 min before streptozo-
tocin injection abolished the initial acute hyperglycaemic effect. However,
after 24 h, the blood glucose concentration started rising and became higher
than the initial acute hyperglycaemic phase within 48 h (Fig. 1). In mice in
which Myrtus extract (2 g/kg) was administered 30 min before streptozo-
tocin, and repeated at 24 h and 30 h after streptozotocin, the blood glucose
concentration remained relatively constant and close to pre-strep~zotocin
level for the whole observation period of 48 h (Fig. 2). The blood glucose
concentrations of treated group at 2,24 and 48 h after streptozotocin were
133.3 f 8.37, 124.2 + 5.54 and 138.1 + 6.1 mg% which were significantly
less than the corresponding values of the control group, i.e. 170.9 + 9.7,
147.1 it 6.3 and 182.4 + 7.3 mg% (P < 0.001, P < 0.02 and P < 0.001,
respectively).
When Myrtus extract administration was started 48 h after streptozotocin
injection, a significant (P < 0.05) reduction in hyperglycaemia was achieved
24 h after the initiation of Myrtus extract treatment (Fig. 3). This effect
278

260-

240 .

220 -
2
m 200 -
5
8 180.
%
d 160-
8
2 l&O .

120 - STREPTOZOTOCIN + VEHICLE

w--u STREPlOZOTOClN + MYRTUS EXTRACT
t
I 1
I
8 i- 48
-9 2 4 TIME AFTER STREPTOZOTOCIN INJECTION th)

Fig. 1. Effect of pretreatment with Myrtus extract on streptozotocin-induced hypergly-

caemia in mice. Streptozotocin (150 mg/kg Lp.) was given at time zero (Q). Myrtus
extract (2 g/kg) or vehicle was given at t in a single oral dose 30 min before streptozo-
tocin. Each point represents mean f S.E.M. from 10 mice.

180 -

160 -

,,. 1 , , , , -i’““““““:r’H”‘:, ~: ,
x-- -u STREPTOZOTOCIN MYRTUS EXTRACT

-1 0 2 4 8 L8
2Gr t
tf TIME AFTER STREPTOZOTOCIN INJECTION (h)

Fig. 2. Effect of pre- and post-treatment with Myrtus extract on streptozotocininduced

hyperglycaemia in mice. Streptozotocin (150 mg/kg i.p.) was given at time zero (f ). At
each time point indicated by t , Myrtus extract (2 g/kg) or vehicle was given orally. Each
point represents mean + S.E.M. from 9-10 mice.
 279

350 -

m
- STREPTOZOTOCIN + MYRTUS EXTRACT VEHICLE
So- * --* STREPTOZOTOCIN + MYRTUS EXTRACT
- STREPTOZOTOCIN VEHICLE ONLY (CONTROLS)

,
m 60 ’ ’ 60 ’ T80 ’ b0 120 140 160 180
t”
TIME AFTER STREPTOZOTOCIN INJECTION (h)

Fig. 3. Hypoglycaemic effect of Myrtus extract in streptozotocin-induced hyperglycaemia

in mice. Streptozotocin (150 mg/kg i.p.) was given at time zero (f ). At each time point
indicated by t , Myrtus extract (2 g/kg) or vehicle was given orally (except to controls).
During the period of Myrtus extract administration, the blood glucose concentration was
determined just before and 3 h after the first dose of the extract on the first and second
day, and once only 3 h after the first dose of the extract on the third day. Each point
represents mean r S.E.M. from 8-10 mice.

TABLE 2
EFFECT OF ORAL ADMINISTRATION OF DIFFERENT DOSES OF MYRTUS
EXTRACT ON THE HYPERGLYCABMIA INDUCED BY STREPTOZOTOCIN
(150 mg/kg i.p.) IN MICE

Myrtus extract given at 48, 54 and 69 h, mean f S.E.M. from 8 mice in each group.

Dose of Myrtus Blood glucose (mg%) at time after streptozotocin injection (h)
extract (g/kg)
0 48 72

Control
(vehicle only) 122.8 i 6.66 261.3 c 35.62 259.4 t 25.66
0.5 138.5 * 3.21 292.3 f 34.83 277.9 r 38.37
1 125.8 f 4.28 270.3 f 26.61 224.3 f 23.53
2 141.9 f 3.62 257.4 r 27.36 187.2 i 11.34*
5 141.3 ? 4.02 261.7 + 23.79 191.2 c 11.22*
-
*Significantly different from the value of control group (P < 0.05).
280

of Myrtus extract was maintained by its repeated administration till 96 h.

At this stage, when the Myrtus extract treatment was withdrawn, the blood
glucose concentration in the mice again increased.
Table 2 shows that 0.5 and 1 g/kg doses of Myrtus extract were ineffective
in reducing streptozotocin-induced hyperglycaemia. A dose of 2 g/kg signifi-
cantly reduced the hyperglycaemia but the effect was not increased at the
higher dose of 5 g/kg.
Myrtus extract (2 g/kg twice daily) had no significant effect on the blood
glucose concentration of normal mice (results not shown).

Discussion

In the present work, collection of blood samples (10 ~1) by pinprick for
glucose estimation was found convenient. This allowed repeated collection
of blood samples from the same mouse. For this purpose, earlier workers
(Rerup and Tarding, 1969) used orbital puncture in mice which is more
difficult.
Rerup and Tarding (1969) reported that streptozotocin in mice showed
triphasic response - first sharp and transient hyperglycaemia at 45 min then
hypoglycaemia at 8 h and finally followed by chronic hyperglycaemia at
48 h. In the present work, a biphasic hyperglycaemic effect was obtained
with no evidence of hypoglycaemic phase. Streptozotocin treatment pro-
duced an acute hyperglycaemia at 2 h with a return to normal at 8 h and
a second phase of hyperglycaemia at 48 h which persisted throughout the
7day observation period. The absence of hypoglycaemic phase in the
present work might be because of the intraperitoneal injection of lower
dose (150 mg/kg) of streptozotocin unlike Rerup and Tarding (1969) who
used a higher dose (200 mg/kg) by intravenous route.
The present findings showed that Myrtus extract significantly reduced the
hyperglycaemia induced by streptozotocin in mice. It was effective in
inhibiting the acute phase of hyperglycaemia when a single dose was given
30 min before streptozotocin. However, the blood glucose concentration
again started rising after 24 h resulting in chronic hyperglycaemia. This
indicates that hypoglycaemic effect of a single administration of Myrtus
extract does not last more than 24 h. Inhibition of the acute phase of
streptozotocin-induced hyperglycaemia by pretreatment with tolbutamide
and nicotinamide have been reported (Rerup and Tarding, 1967; William
et al., 1969). However, unlike Myrtus extract, the hypoglycaemic effect of
single dose treatment of nicotinamide lasted for more than 24 h (Masiello
and Bergamini, 1977).
Myrtus extract given 48 h after streptozotocin effectively reduced the
hyperglycaemia. This effect of Myrtus extract could be maintained by its
repeated administration but after stopping the treatment, the blood glucose
concentration again increased within 24 h which further indicates that the
effect of the Myrtus extract does not last for more than 24 h.
 281

These findings so far indicate the need for further work on the isolation
and purification and study of the mechanism of action on the hypogly-
caemic principle(s) of MYrtus communis.

Acknowledgements

We thank Prof. I.H. Stevenson and Prof. Aklaque N. Khan for critical
reading of the manuscript and Messrs. Abdus Salam and M. Sulaiman for
technical assistance. This work was supported by the Research Centre,
University of Garyounis.

References

Bergmeyer, H.U., Bernt, E., Schmidt, F. and Stork, H. (1974) Determination with hexo-
kinase and glucose-6-phosphate dehydrogenase. In: H.U. Bergmeyer (Ed.), Methods of
Enzymatic Analysis, 2nd English edn. (translated from 3rd German edn.), Academic
Press, New York, p. 1196.
Brahmachari, H.D. and Augusti, K.T. (1961) Hypoglycaemic agents from Indian plants.
Journal of Pharmacy and Pharmacology 13,381-382.
Brahmachari, H.D. and Augusti, K.T. (1962) Orally effective hypoglycaemic agents
from plants. Journal of Pharmacy and Pharmacology 14, 254-255.
Brahmachari, H.D. and Augusti, K.T. (1963) Orally effective hypoglycaemic principles
from Coccina indicn Wight and Am. Journal of Pharmacy and Pharmacology 15,
411-412.
Deshmukh, V.K., Shrotri, D.S. and Aiman, R. (1960) Isolation of a hypoglycaemic
principle from the bark of Ficus bengalensis Linn. Indian Journal of Physiology and
Pharmacology 4,182-185.
Dulin, W.E., Wyse, B.M. and>Kalamazoo, M.S. (1969) Studies on the ability of com-
pounds to block the diabetogenic activity of streptozotocin. Diabetes 18, 459-466.
Masiello, P. and Bergamini, E. (1977) Nicotinamide and streptozotocin diabetes in the
rat. Factors influencing the effectiveness of the protection. Experientia 33, 1246-
1247.
Oliver Bever, 0. and Zahnd, G.R. (1979) Plants with oral hypoglycaemic action. Quarterly
Journal of Crude Drug Research 17, 139-196.
Ratsimamanga, A.R., Loiseau, A., Ratsimamanga-Urverg, S. and Paulette Bibal-Prot
(1973) Nouvelle contribution a l’btude de l’action d’un principle hypoglycemiant
mis en evidence dans l’icorce jeune de Eugenia jumbolana (Myrtacbes) sur l’hyper-
glycemic provoqdee du lapin et poursuite de sa purification. Comptes Rendus de
1’Academie des Sciences 277, 2219-2222.
Rerup, C. and Tarding, F. (1969) Streptozotocin- and alloxan-diabetes in mice. European
Journal of Pharmacology 7, 89-96.
Shani (Mishkinsky), J., Goldschmied, A., Joseph, B., Ahronson, Z. and Sulman, F.G.
(1974) Hypoglycaemic effect of Trigonella foenum Graecum and Lupinus termis
(Leguminosae) seeds and their major alkaloids in alloxan diabetic and normal rats.
Archives Znternationales de Pharmacodynamie 210, 27-37.