Journal of Ethnopharmacology 80 (2002) 109– 113

www.elsevier.com/locate/jethpharm

Hypoglycaemic effect of the lyophilised aqueous extract of Ajuga

i6a in normal and streptozotocin diabetic rats
Jaouad El Hilaly, Badiâa Lyoussi *
Faculty of Sciences Dhar El Mehraz, UFR Physiology – Pharmacology, Laboratory of Animal Physiology, Department of Biology, USMBA,
BP 1976, Atlas, Fez, Morocco
Received 11 September 2001; received in revised form 28 September 2001; accepted 23 November 2001

Abstract

The purpose of this study was to examine the hypoglycaemic effect of the lyophilised aqueous extract of the whole plant of
Ajuga i6a (L.) Schreber (Labiatae) in normal and streptozotocin-induced diabetic rats. Single and repeated oral administration of
the extract of Ajuga i6a L (AI) at a dose of 10 mg/kg produced a slight and significant decrease in plasma glucose levels in normal
rats 6 h after administration and after 3 weeks of treatment. AI reduced plasma glucose levels of streptozotocin diabetic rats from
33799.3 to 102.2 917.7 mg/dl after 6 h of oral administration (PB 0.001). Repeated oral administration of AI to streptozotocin
diabetic rats significantly decreased the plasma glucose levels after 1 week of treatment (112 9 14.4 mg/dl at 1 week vs 337 99.3
mg/dl at the baseline values, (P B0.001). It continuously decreased thereafter and showed a rapid normalisation after 1 week of
AI treatment. It is concluded that these results demonstrated that the water extract of the whole plant of AI possess a strong
hypoglycaemic effect in diabetic rats, and support therefore, its traditional use in diabetes mellitus control. © 2002 Published by
Elsevier Science Ireland Ltd.

Keywords: Ajuga i6a; Lyophilised aqueous extract; Streptozotocin rats; Oral administration; Plasma glucose levels; Hypoglycaemic effect

1. Introduction Several scientific studies have been conducted on

Hypoglycaemic plants are still prevalent in develop-
ing countries, where they have been used to treat
diabetes for many centuries (Lewis and Elvinlewis,
1977; Akhtar, 1985; Bailey and Day, 1989; Lin, 1992;
Jahodar, 1993). More than 1200 species of plants have
been used empirically for their alleged hypoglycaemic
activity (Marles and Farnsworth, 1995). This fact is
attributed to the high cost and the lack of availability
of current therapies for the majority of patients in
developing countries. It is worth stating that phytother-
apy is widely adopted by the Moroccan population
(Bellakhdar, 1997; Ziyyat et al., 1997; Hassar, 1999;
Hmamouchi, 1999). Nevertheless, many medicinal
plants claimed effective by folk medicine require scien-
tific investigation to ascertain their effectiveness, toxic-
ity and then provide alternative drugs and therapeutic
strategies (Xavier, 1997).
* Corresponding author.
many species of the genus Ajuga, which are ecologically
related and some of their active compounds have been
identified (e.g. Cantrell et al., 1999; Chen et al., 1997;
Kuria and Muriuki, 1984; Takasaki et al., 1999; Breschi
et al., 1992). Nevertheless, to our knowledge, no phar-
macological investigation has been conducted in Ajuga
i6a (L.) Schreber (Labiatae) (AI), although numerous
beneficial effects are claimed by folk medicine. It is used
as an anthelmintic, against intestinal disorders (Bel-
lakhdar et al., 1991), and as a diuretic agent (Alliotta
and Pollio, 1994). According to ethnobotanical data
collected in oriental Morocco by Ziyyat et al. (1997),
AI, locally known as ‘Chendgora’, is also alleged to
possess hypoglycaemic activity and it is believed by
many Moroccan diabetics that the decoction of AI
consumed over a long time removes the cause of dia-
betes. The anti-diabetic effect of this plant has never
been experimentally demonstrated. The purpose of this
investigation was to determine the acute toxicity of this
plant and to ascertain evidence of a scientific basis for
this claim using normal and STZ-induced diabetic rats.

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110 J.E. Hilaly, B. Lyoussi / Journal of Ethnopharmacology 80 (2002) 109–113
2. Material and methods
2.1. Experimental animals
Healthy male and female adult Wistar rats, weighing
200–260 g were used in this study. Animals were
housed in an air-conditioned animal room at 239
2 °C, with 12 h/12 h light/dark photoperiod, and main-
tained with free access to water and ad libitum feeding.
2.2. Plant material
The whole plant of AI was harvested in the septentri-
onal Moroccan province named Taounate in April–
May (1998), and authenticated by Prof. M. Fennane
from the Scientific National Institute (Rabat), where a
voucher specimen was deposited (H 63).
2.3. Preparation of the extract
The whole plant was ground and 50 g of powder
mixed with 500 ml of distilled water (10%). The mixture
was heated and boiled under reflux for 30 min. The
decoction obtained was centrifuged, filtered, frozen at
− 20 °C, and then lyophilised (FreeZone®Dry 4.5,
USA).
2.4. Experimental design
2.4.1. Normal rats
The hypoglycaemic effect was evaluated orally by
force-feeding in 18 normal rats, previously fasted for 16
h and randomly divided into three groups: (n =6 per
group).
Group 1: received distilled water and served as a
control group.
Group 2: treated with extract of AI at a dose of 10
mg/kg.
Group 3: treated with Glibenclamide (GLB)
(BENCLAMID® PROPHARM) at a dose of 2.5
mg/kg, and served as a reference standard drug.
2.4.2. Induction of experimental diabetes
Adult rats were artificially made diabetic by intra-
venous injection of STZ (Sigma, St. Louis, MO)
through the tail vein (60 mg/kg dissolved in citrate
buffer, 0.1 M, pH 4.5).
After 3 days, hyperglycaemia was confirmed spec-
trophotometrically using the glucose oxidase method
and Glucometer (GlucoMenGlyco, A. Menari Diagnostic
MTB, Germany). Only rats with fasting blood glucose
levels greater than 250 mg/dl were selected and used in
this study. Then, animals were classified into three
groups treated as described for groups 1–3, normal
rats, (n=6 per group).
2.5. Blood samples
Blood was obtained from the retro-orbital puncture
of all Wistar rats using capillary tubes. Blood samples
were centrifuged at 4000 rev/min for 10 min at 4 °C,
then the plasma was conserved until analysis.
2.6. Parameters determination
Plasma glucose levels were measured by the glucose
oxidase method (Biosystem, Spain). Plasma insulin con-
centrations were determined by radioimmunoassay kits
using Beta Matic Comptor.
2.7. Acute toxicity
The lyophilised aqueous extracts lethality in mice was
tested orally using graded doses (0, 2, 4, 6, 10, 14 g/kg).
Furthermore, the general behaviour of mice was
recorded continuously for 12 h, and daily for a further
2 weeks for any eventual mortality.
2.8. Statistical analysis
All data are presented as mean9 SEM. Student’s
t-test was used to assess statistically significant differ-
ences. Comparisons between group means were carried
out using analysis of variance. Mean values were con-
sidered significantly different if P B0.05.
3. Results
3.1. Acute toxicity
The lyophilised aqueous extract of AI did not show
mortality, nor any remarkable symptoms of toxicity
and/or any significant changes in general behaviour in
mice, at the doses used. However, administration of
doses over 14 g/kg was not possible as the aqueous
suspensions of the lyophilised plant were too thick to
be administered orally by feeding needle.
3.2. Acute hypoglycaemic effect
Fig. 1 summarises the data of plasma glucose levels
in normal rats (Fig. 1a) and diabetic rats (Fig. 1b),
following single oral administration of test materials.
The lyophilised aqueous extract of AI (10 mg/kg)
significantly lowered the plasma glucose levels in nor-
mal rats as compared to the untreated groups and to
the pre-treatment levels (0 h) (799 3.96 mg/dl at 6 h vs
100.79 3.34 mg/dl at 0 h, PB0.01). While, oral admin-
istration of GLB did not cause any significant reduction
of plasma glucose levels within the 6 h of treatment.
 J.E. Hilaly, B. Lyoussi / Journal of Ethnopharmacology 80 (2002) 109–113 111

Fig. 2. Plasma glucose changes after sub-chronic oral administration

) control;
Fig. 1. Plasma glucose changes after single oral administration of
lyophilised aqueous extract of AI within 6 h in (a) normal rats and
(b) diabetic rats. Data are expressed as mean 9 SEM; n= 6 per
group; (*) P B 0.05; (**) P B0.01; (***) P B0.001; (
) control;
A. i6a; (a) GLB.
In STZ rats, oral administration of the lyophilised
aqueous extract reduced mostly the mean plasma glu-
cose levels as compared to the baseline value (1839
34.03 mg/dl at 2 h vs 337 99.34 mg/dl at 0 h,
PB 0.01). It was found to be more effective than
GLB (3039 15.23 mg/dl at 2 h vs 3789 20.16 mg/dl
at 0 h, PB 0.05), which also gradually and signifi-
cantly decreased hyperglycaemia without reaching the
same normal value obtained with AI at 6 h. Plasma
glucose level of animals treated with GLB was signifi-
of lyophilised aqueous extract of AI within 21 days in (a) normal rats
and (b) diabetic rats. Data are expressed as mean 9SEM; n= 6 per
group; (*) PB 0.05; (**) P B0.01; (***) P B0.001; (
A. I6a; (a) GLB.
cantly reduced compared to AI administration (PB
0.05) throughout the 6 h sampling period (Fig. 1b).
The aqueous extract of AI did not produce any
significant changes in plasma insulin concentrations
between the levels before and after 6 h of treatment
in normal rats (37.391.05 vs 39.190.67 mU/ml at
the baseline value) (Table 1).
Similarly, no remarkable changes were noticed be-
tween the untreated and treated diabetic rats, before
and after treatment, for the plasma insulin concentra-
tions (7.49 0.46 mU/ml at 6 h vs 7.49 0.27 mU/ml at
the baseline level (0 h) in treated diabetic rats, and
7.190.39 mU/ml at 6 h vs 7.0 90.41 mU/ml at the
pre-treatment level) (Table 1).

Table 1
Effect of single and repeated oral administration of A. i6a lyophilisate on plasma insulin concentrations (mU/ml) in normal and diabetic rats

Experimental groups Single oral administration (h) Repeated oral administration (days)

0 6 0 6

Normal rats
Control 34.59 0.77 34.9 9 1.02a 34.5 9 0.77 34.5 90.93a
A. i6a 39.19 0.67 37.3 9 1.05a 39.1 9 0.67 37 9 1.04a
Diabetic rats
Control 79 0.41 7.1 9 0.39a 7 90.41 7 90.30a
A. i6a 7.49 0.27 7.4 90.46a 7.4 90.27 7.6 9 0.40a

Tabular values represent the mean 9 SEM; n = 6 per group.

a
Not significant when compared to the baseline values for the single and repeated oral administration.
112 J.E. Hilaly, B. Lyoussi / Journal of Ethnopharmacology 80 (2002) 109–113
3.3. Sub-chronic treatment
As shown in Fig. 2a, normal rats treated by AI
exhibited a slight and significant reduction of plasma
glucose levels until the 21st day (849 2.5 mg/dl vs
96 9 2.9 mg/dl, P B0.05). GLB (2.5 mg/dl) also pro-
duced a significant decrease in the plasma glucose levels
at 15–21 days after the administration (889 2.32 mg/dl
at 15th day and 8392.95 mg/dl at 21st day vs 999
3.16 mg/dl at the baseline value, P B 0.05).
In diabetic rats, the repeated oral administration of
the lyophilised aqueous extract elicited a highly signifi-
cant decrease of plasma glucose levels which attained
normalisation at the 7th day (1129 14.4 mg/dl vs
3379 9.3 mg/dl, PB 0.001), and continued to fall until
the peak effect at the 21st day (779 5.9 mg/dl, PB
0.001).
The hypoglycaemic potency of GLB (at 2.5 mg/kg)
appeared, in this study, less effective than the
lyophilised aqueous extract of AI at the dose of 10
mg/kg (PB 0.01). GLB also reduced hyperglycaemia at
the 7th day (2259 13.5 vs 316918.76 mg/dl, P B 0.01),
and it reached its maximum reduction at the end of the
study (11899.97 mg/dl, P B0.001) (Fig. 2b).
The normal rats treated with AI at a dose of 10
mg/kg showed no significant variation in the plasma
insulin concentrations after 3 weeks of treatment (379
1.04 vs 39.190.67 mU/ml at the pre-treatment level
(Table 1). Furthermore, in STZ-induced diabetic rats,
there is no remarkable changes in plasma insulin con-
centrations after 3 weeks of treatment (7.69 0.4 vs
7.4 90.3 mU/ml before AI treatment) (Table 1).
4. Discussion
The results of the present study showed that single
oral administration of the lyophilised aqueous of AI
decreased significantly plasma glucose levels over 2–6 h
either in normoglycaemic rats (PB 0.01) or in STZ-hy-
perglycaemic rats (P B 0.001) (Fig. 1). However, daily
treatment with the same extract repeated for 21 days,
decreased plasma glucose levels after the first week only
in STZ-induced diabetic rats (P B0.01), while the nor-
moglycaemic rats showed a slight decrease only in the
third week (P B0.05) (Fig. 2).
Theoretically, hypoglycaemic plants act through a
variety of mechanisms. At the present juncture, it is not
possible either to pinpoint the exact mechanism of the
hypoglycaemic effect of AI or to identify the active
principle(s). Subsequently, some hypothetical sugges-
tions can be made. For these studies, any stimulation of
insulin secretion is likely to have taken place at less
than 6 h after administration of the extract, typically
within 30 min for the first phase and maybe up to 2 h
for the second phase. However, it does show no pancre-
atic recovery in STZ-diabetic rats. The plasma glucose
lowering effect in the absence of significant change in
plasma insulin concentration (Table 1), suggests that AI
treatment may involve an insulin independent mecha-
nism (Dabis et al., 1984), like some species either in the
same family (Labiatae) (Jimenz et al., 1986), or in
others (Jouad et al., 2000; Benwahhoud et al., 2001).
Also, it can act by enhancing glucose utilisation in the
peripheral tissues (Naik et al., 1991; Obatomi et al.,
1994; Peungvicha et al., 1998).
A preliminary phytochemical analysis of the aqueous
extract of AI was made according to the Paris and
Nothis method (1969). It revealed that flavonoids are
the major constituents of the aqueous extract. These
natural compounds could be responsible for the hypo-
glycaemic effect of AI (Meiselman et al., 1976; Choi et
al., 1991). It has been reported that AI contains
ecdysones and ecdysterones (Khafagy et al., 1979; Ikan
and Ravid, 1971; Fujimoto et al., 2000), which are
known for their anabolic action (Syrov, 1984). Further-
more, other active compounds have been identified in
the genus Ajuga, like triterpenes, diterpenes (Chen et
al., 1996; Ben Jannet et al., 2000; Cantrell et al., 1999),
flavones, anthocyanins (Terahara et al., 1996), gly-
cosides (Takasaki et al., 1999) and Withanolides.
Toxicological studies have shown that the aqueous
extract of AI could be considered as free of toxic effects
at hypoglycaemic doses since the extract did not pro-
duce any lethality or any changes in general behaviour
in mice at 2– 14 g/kg (Horn, 1956).
In conclusion, the present study demonstrates experi-
mentally the hypoglycaemic activity of AI and corrobo-
rate the empirical use of AI to treat diabetes mellitus in
Moroccan folk medicine (Ziyyat et al., 1997). Further-
more, comprehensive chemical and pharmacological re-
search is required to reveal the mechanism of the
hypoglycaemic effect and to identify the active con-
stituent(s) responsible for this effect.
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