Memoire Online - Effect of methanol/methylene chloride extract of Terminalia glaucescens

Effect of methanol/methylene chloride extract of Terminalia glaucescens
leaves on glucose level, weight gain and lipids parameters on normal mice.

GBS Nya Njomen¹, R Kamgang^1*, PR Nkomo Soua¹, JL Essame Oyono², Njifutie Njikam¹.

¹General Endocrinology and Metabolism Systems (GEMS), Laboratory of Animal Physiology, Faculty of Sciences, University of Yaounde 1, Cameroon

²Faculty of Medicine and Biomedical Sciences, University of Yaounde 1 and IMPM - Yaounde, Cameroon

*For correspondence: GEMS, Laboratory of Animal Physiology, University of Yaounde I, P. O. Box 8127 Yaounde - Cameroon.

Abstract

Terminalia glaucescens is traditionally used to improve obesity conditions. This study aimed to assess the effect of the leaves extract of that plant on obesity parameters on normal mice. During 30 days mice were treated with methanol/methylene chloride extract of T glaucescens leaves (100, 200 and 300 mg/kg body weight). We have investigated the effect on body weight gain, food and water intakes, fat pad weight, blood glucose and lipids parameters levels. he methanol/methylene chloride extract of T glaucescens leaves, in dose dependant manner, significantly (P< 0.0 1) reduced adipose tissue mass and decreased body weight gain. The extract has increased the glucose tolerance, lowered blood levels of leptin, triglycerides, NEFA and total cholesterol (P< 0.05); tended to enhance HDL cholesterol and to decrease LDL cholesterol levels. The metabolic efficiency was markedly reduced (P< 0.05). These results indicate that the methanol/methylene chloride extract of T glaucescens leaves could have anti-obesity and could prevent cardiovascular diseases.

Introduction

Because of the increasing of obesity and related diseases, obesity is recognized as a major public health problem in industrialized as well as in developing countries, and the factors that regulate white adipose tissue are of great interest. An excess of body fat predisposes to the development of insulin resistance and metabolic syndrome, especially when concentrated within the abdomen. Classified as a disease, obesity diminishes both quality of life and life expectancy. The metabolic deregulation ultimately may lead to complications such as diabetes mellitus, coronary heart disease, hypertension, and some types of cancer¹. The number of drugs approved for the treatment of obesity is very limited compared to other multifactorial diseases such as hypertension. This situation urged scientists to elucidate the mechanisms of adipose tissue development and to find natural substances able to antagonize excessive fat deposition. Medicinal products obtained from a variety of plants are

currently being used in the treatment of many diseases. Amongst the natural substances inhibiting fat deposition, recent studies gave pride of place to flavonoids, a group of polyphenolic products ubiquitously present in the plant kingdom². Flavonoids exert antioxidant, anti-inflammatory and lipid lowering effects3, ⁴. In Cameroon, Terminalia glaucescens (Combretaceae) is claimed to be useful in the treatment of obesity, diabetes mellitus and some bacteria diseases. The preliminary phytochemical screening of T glaucescens extract revealed the presence of tannins, alkaloids, flavonoids and saponosides⁵. Although many plants have certain medical benefits to humans, many of these claims are unproven scientifically.

The objective of the present study was to test in vivo the effect of Terminalia glaucescens extract on some obesity parameters such as white adipose tissue growth and lipids parameters in adult mice.

Materials and Methods

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.

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.

- 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.

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; bw_g: 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:

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.

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:

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.

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.

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.

Results.

Body weight of all groups was not significantly different from normal control group before the
extract administration. From the 12^th days of treatment, NE200 and NE300 body weight gain

decreased progressively (Fig. 1). At the end of the treatment (Day30), as refer to control, significant decrease was observed in weight gain: -11% and -19% (P< 0.01) respectively in NE200 and in NE300.

Food and water intakes did not exhibit significant variation, but metabolic efficiency (ME) was significantly altered: -43 % and -69 % (P< 0.05) respectively in NE200 and NE300 (Table 1). The animal length, the Lee index and the heart weight decreased in dose dependant profile but not significantly. No weight variation of the liver, the kidney and the carcass was observed with any extract dose. The fat pad weight weights were reduced in dose dependant manner. Compared with vehicle the parametrial and the total fat mass were significantly reduced by the extract at 200 mg/kg and 300 mg/kg: respectively -45% (p<0.05) and -64% (P< 0.01) for the parametrial fat mass, -34 % (P< 0.05) and -48% (P< 0.0 1) for the total fat mass. The inguinal (ingWAT) and retroperitoneal (rWAT) fat mass lowering was significant (P< 0.05) with 300 mg/kg extract dose

In the openfield test no significant difference in behaviour (rearing, grooming and defecation) was noticed between control and extract treated mice. Motor activity apparently increased dose dependently (Table 2).

No change was observed in blood glucose level. Compared to the control group, total cholesterol decreased significantly in all treated groups: -25%, -24%, -38% (P< 0.05) respectively for 100 mg/kg, 200 mg/kg and 300 mg/kg extract doses, while the extract apparently and dose dependently enhanced the HDL cholesterol and lowered the LDL cholesterol levels (Table 3). Triglyceridemia and NEFA decreased significantly (P< 0.05) at the doses 200 mg/kg and 300 mg/kg. Leptin level decreased in dose dependant manner, and the decrease was significant with 300 mg/kg extract dose (-28%, P< 0.05).

Increase in plasma glucose levels after glucose administration was lowered at 30, 60, 90 minutes in all mice treated with extract 200 mg/kg and 300 mg/kg bw (Fig 2). Compared with control group, the difference in plasma glucose level was significant (P< 0.05) with 300 mg/kg extract at 30 min (- 12%), 60 min (-15%) and 90 min (-11%).

Discussion

The study was carried out in the aim of assessing the effect of Terminalia glaucescens
(Combretaceae), plant used traditionally by corpulent persons against weight gain. A key

observation of the present work is that in adult male mice, methanol/methylene chloride leaves extract of T glaucescens in dose dependant manner decreased adipose tissue mass, body mass and all lipids parameters. The parametrial adipose tissue was significantly reduced, suggesting that the extract principally affects intra-abdominal adipose depots. The decrease in body mass may be correlated to the decrease of fat mass since organs weights (liver, heart and kidney) and carcass weight did not change markedly. Body mass and specially adipose tissue mass, result from the equilibrium between energy intake and energy expenditure⁸. The decrease in adipose tissue mass may results from either the decrease in food intake or increase in energy expenditure. Since the variation in food and water intakes in treated mice compared with control was not significant, the decrease in body mass could mainly result from the increase of energy expenditure. Locomotors activity which may be considered as a good index of energy expenditure was found notably changed during the open-field test in animals treated with extract, but the metabolic efficiency was affected by the 300 mg/kg extract, so the indirect calorimetry should be required to assess energy expenditure and to clarify this hypothesis. The preliminary phytochemical screening of T glaucescens extract revealed the presence of tannins, alkaloids, flavonoids and saponosides⁵. Flavonoids stimulate lipolysis in isolated rat adipocytes and decrease adipose deposition in mice9, 10. The presence of flavonoids and other compound in the extract might explain his effect on fat reduction.

Lipids parameters such as cholesterolemia, triglyceridemia and non esterified fatty acids (NEFA) were significantly reduced. These changes are coherent with the reduction in fat deposition. There is a risky relationship between serum lipids and cadiovascular disease11, ¹² . Thus, lowering the serum cholesterol, (LDL cholesterol) and triglycerides levels with the enhancing of HDL cholesterol level, is important for preventing high mortality life style-related cardiovascular diseases. T glaucescens can therefore be expected to help to prevent such disease and by this may explain the use of this plant in the treatment of diabetes and hypertension by tradipractitioner.

Elevated plasma free fatty acids (FFA) levels account for up to 50% of insulin resistance in obese patients with type 2 diabetes mellitus¹³. Hepatic lipid accumulation in diabetes has been linked to the development of hepatic insulin resistance¹⁴. T glaucescens extract by reducing plasma FFA will be benefit in obesity and type 2 diabetes cases, and may prevent cardiovascular diseases. It has been generally accepted that â-oxidation is increased in the liver of obese and diabetic patients¹⁵. The elevated FFA levels lead to excessive â-oxidation that eventually results in impaired glucose utilization in liver. Thus, a potential approach to decrease blood glucose levels in type 2 diabetic patients is to reduce excess â-oxidation. In this study the decrease of plasma FFA may result in the reduction of â-oxidation.

In genetic models of obesity in rodents leptin plays a major role as a controller of obesity,. a central role in the regulation of food intake, body weight and energy expenditure16, ¹⁷ . Plasma leptin level is positively correlated with fat mass, body weight and plasma insulin level¹⁸. The decrease in plasma leptin in this study is coherent with the reduction of body weight and fat mass.

The 300 mg/kg extract markedly lowered the level of plasma glucose in the glucose tolerant test and tended to decrease fasted plasma glucose, suggesting that the extract increases cellular sensitiveness to glucose. Flavonoids, triterpenoids, alkaloids and phenolics are know to be bioactive antidiabetic principles19, ²⁰. The effect of T glauscecens on glucose metabolism after glucose administration may be due to the presence of more than one antihyperglycaemic principles and their synergistic properties.

The present study has shown that T glaucescens extract improved blood lipids parameters, enhanced cellular glucose sensitiveness, and reduced the body fat mass and body weight gain. These properties might justify the usefulness of T glaucescens in obesity conditions.

References

Figure 1: Body weight mass (expressed as % of initial values iV = 100 %) over 30 days of treatment (once a day) with methanol/methylene chloride leaves extract of T glaucescens 100 mg/kg (NE100), 200 mg/kg (NE200), 300 mg/kg (NE300) bw. Data are mean #177; SEM, (n=7 per group). Significant difference: *P< 0.05, **P< 0.01 compared with control values.

Figure 2: Plasma glucose levels during oral glucose tolerance test after 28 days of treatment (once a day) with methanol/methylene chloride leaves extract of T glaucescens 100 mg/kg (NE100), 200 mg/kg (NE200), 300 mg/kg (NE300) bw. Data are mean #177; SEM, (n=7 per group). Significant difference: *P< 0.05, **P< 0.01 compared with control values.

Table 1: Anthropometric parameters of mice after 30 days of treatment (once a day) with 100 mg/kg (NE 100), 200 mg/kg (NE200), 300 mg/kg (NE300) bw methanol/methylene

chloride extract of	T glaucescens leaves.
chloride extract of	Control	NE 100	NE200	NE300
Inguinal WAT (mg)	16.14 #177; 1.35	13.64 #177; 1.49	12.43 #177; 0.86	8.23 #177; 0.60**
Retroperit WAT (mg)	16.57 #177; 1.51	14.71 #177; 0.65	13.29 #177; 0.60	11.14 #177; 0.56*
Parametrial WAT (mg)	38.00 #177; 2.79	36.71 #177; 1.68	21.00 #177; 0.28*	13.29 #177; 0.46**
Total fat (mg)	70.71 #177; 4.95	65.07 #177; 1.93	46.71 #177; 1.42*	32.66 #177; 0.75**
Liver (g)	2.20 #177; 0.05	2.17 #177; 0.03	2.18 #177; 0.03	2.19 #177; 0.03
Heart (mg)	124.71 #177; 2.58	121.57 #177; 2.52	120.33 #177; 2.18	118.67 #177; 3.35
Kidney (mg)	225.71 #177; 0.83	221.86 #177; 1.62	222.33 #177; 1.98	224.33 #177; 1.89
Carcass (g)	22.57 #177; 0.61	22.28 #177; 0.52	21.71 #177; 0.52	21.86 #177; 0.40
Length (cm)	9.64 #177; 0.10	9.57 #177; 0.09	9.56 #177; 0.09	9.54 #177; 0.08
Weight gain (g)	34.93 #177; 0.28	33.50 #177; 0.12	31.10 #177; 0.1 1**	28.55 #177; 0.16**
Lee index (x 1000)	340.00 #177; 8.13	338.00 #177; 8.67	332.29 #177; 8.01	322.43 #177; 5.65
Metabolic efficiency	0.074 #177; 0.007	0.061 #177; 0.010	0.042 #177; 0.01 5*	0.023 #177; 0.01 9*

Table 2: Mice Openfield test after treatment (once a day during 27 days) with 100 mg/kg (NE100), 200 mg/kg (NE200), 300 mg/kg (NE300) bw methanol/methylene chloride extract of T glaucescens leaves.


	Control		NE100		NE200		NE300
Locomotors activity (AU)	137.29 #177; 19.27		144.28 #177; 23.85		159.57 #177; 19.68		168.29 #177; 18.98
Rearing (Nb per 2 min)	0.43	#177; 0.20	0.43	#177;0 02	0.43	#177; 0.30	0.71	#177; 0.36
Grooming (Nb per 2 min)	32.86	#177; 6.31	30.86	#177; 8.53	22.14	#177; 4.09	31.29	#177; 7.52
Defecation (Nb per 2 min)	0.43	#177; 0.30	0.86	#177; 0.46	0.43	#177; 0.30	0.57	#177; 0.30

Table 3: Plasma parameters levels of mice after the treatment (once a day during 30 days) with methanol/methylene chloride leaves extract of T glaucescens 100 mg/kg (NE 100), 200

mg/kg (NE200), 300	mg/kg (NE300) bw.
mg/kg (NE200), 300	Control	NE100	NE200	NE300
Leptin (mg/L)	5.86 #177; 0.50	5.37 #177; 0.36	4.94 #177; 0.26	4.23 #177; 0.46*
Glycaemia (mmol/L)	6.64 #177; 0.35	6.42 #177; 0.42	6.36 #177; 0.35	6.03 #177; 0.26
Triglycerides (mmol/L)	0.91 #177; 0.03	0.86 #177; 0.03	0.78 #177; 0.03*	0.79 #177; 0.02*
Cholesterol (mmol/L)	1.33 #177; 0.08	1.00 #177; 0.04*	1.00 #177; 0.10*	0.83 #177; 0.11*
NEFA (mmol/L)	0.38 #177; 0.03	0.34 #177; 0.02	0.28 #177; 0.01*	0.23 #177; 0.01*
HDL-c (mmol/L)	0.065 #177; 0.004	0.068 #177; 0.002	-	0.077 #177; 0.007
LDL-c (mmol/L)	0.860 #177; 0.109	0.714 #177; 0.197	-	0.682 #177; 0.092

NEFA: non esterified fatty acids; HDL-c: HDL-cholesterol; LDL-c: LDL-cholesterol.

Data are mean #177; SEM (n=7 per group). Significant difference: *P< 0.05 compared with control values.