Materials and Methods
Plant extract
Terminalia glaucescens fresh leaves were harvested
from Mbalmayo in center province of Cameroon. Terminalia glaucescens
was identified by Dr Simeon Tchoulagueu of "Teacher Training High School (ENS)
of the University of Yaoundé I" who botanically studied the plant and
kept a voucher specimen in the laboratory. Two kilograms of the sun-dried
powdered leaves were macerated in a mixture of methanol/methylene chloride
(1:1) for 7 days (with occasional stirring) at room temperature. The mixture
was filtered with Whatman No. 1 filter paper. The filtrate was concentrated
under reduced pressure to obtain 125 g of a dark solid. This extract was
dissolved in 10 % dimethyl sulfoxide (DMSO) solution. The volume of
administration was 5 uL/g body weight for each experimental dose.
Animals of experiment
The study was carried on normal male albino wistar mice (23-27
g weight, 6-8 weeks old) raised in the animal house of the laboratory in
natural conditions were allowed free access to water and regular rodent chow.
For experiment, the mice were weighted and randomly divided into 6 groups of 7
animals each.
- 1 group of control (NC) receiving 10 % DMSO p.o.,
- 3 groups of mice receiving per os 100 mg/kg body weight
(NE100), 200 mg/kg bw (NE200) or 300 mg/kg bw (NE300) plant extract.
Animal housing and In vivo experiments were done
according to the guidelines of the European Union on Animal Care (CEE Council
86/609) that was adopted by the Institutional Committee of the Ministry of
Scientific Research and Innovation of Cameroon.
Body weight, food and water intakes
measurement.
Food and water intakes were monitored on day 0, 3, 6, 9, 12,
15, 18, 21, 24 and 27. Body weight was measured on day 0, 3, 6, 9, 12, 15, 18,
21, 24, 27 and 30. Food and water consumption were measured as the difference
between the amount given and that removed from the cage. Metabolic efficiency
was determined using the formula:
ME: metabolic efficiency; bwg: total body mass gain
within a period; FI: amount of food consumed during the period.
Naso-anal length was measured to calculate adiposity index (Lee
index) as follow:
bw
3
Li = Lna
Li: Lee index; bw: body weight; Lna: naso-anal length
Glucose tolerance test
After 28 days of treatment, a fasting blood sample was taken
from the tail tip for glucose determination by using a glucometer (Accu-Check,
Roche). Four more blood samples were collected at 30, 60, 90 and 120 minutes
intervals after oral administration of glucose (2 g/kg bw)6.
Blood parameters and tissue dissection.
At the end of treatment (Day30), blood samples for glucose
determination were obtained from the tail tip of 4 h fasted mice using a
glucometer (Accu-Check, Roche). Mice were weighed and anesthetized with sodium
pentobarbital (60 mg/kg i.p). All the sacrifices were performed between 9:00
and 12:00 h. Blood was rapidly collected by cardiac puncture in syringes
containing EDTA. Blood samples were centrifuged (1min, 8000 g),
plasmas were collected, aliquoted and snap frozen in liquid nitrogen.
4 Plasma parameters were assayed using commercially available
kits according to the manufacturer' s recommendations: triglycerides (TG: PAP
bioMérieux, Marcy l'Etoile, France), non esterified fatty acids (NEFA,
NEFA-C, Wako), cholesterol (Cholesterol RTU, bioMérieux), HDL
cholesterol (HDL-cholesterol direct, bioMérieux). LDL-Cholesterol
(LDL-C) level was determined using this formulae7:
LDL-C = TC - (HDL-C +
TG )
n
n = 2 when values are expressed in mmol/L and n = 5 when values
are expressed in g/L
LDL: LDL cholesterol; TC: total cholesterol; HDL: HDL
cholesterol; TG: triglycerides
Plasma leptin levels were measured by radioimmunoassay method
using kit of mouse leptin (mouse leptin RIA Kit, LINCO Research, Inc St
Charles, MO) with Guinea pig anti-mouse leptin serum; precipitation was
obtained with goat gamma immunoglobulin anti-guinea pig.
Parametrial (pWAT), retroperitoneal (rWAT), inguinal (ingWAT)
white adipose tissues, were removed and weighted. Heart, kidney, and liver were
quickly excised and weighed.
Openfield test
Twenty seven days after the beginning of the treatment (D27),
effect of T glaucescens administration on mouse motor activity was
evaluated using the open-field test. The animals were individually placed
inside a square Plexiglas area (125×125×50 cm) from which it cannot
escape, locomotors pattern and behaviours such as rearing, grooming and
defecation were estimated. Each animal was placed in the centre of the square
and allowed to explore for two minutes.
Statistical analysis
The results are expressed as mean #177; standard error of mean
(X #177; S.E.M). Mean values were obtained by one way analysis of variance
(ANOVA) using computer program StatView 4.5. The significance of difference
between and within various group was determined. Values of p< 0.05 were
taken to imply statistical significance.
| 
