Antidiabetic activity of the methanol and acetone extracts of twigs of Combretum molle in dexamathasone induced-insulin resistance in rats

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1 World Journal of Pharmaeutial Sienes ISSN (Print): ; ISSN (Online): Published by Atom and Cell Publishers All Rights Reserved Available online at: Original Artile Antidiabeti ativity of the methanol and aetone extrats of twigs of Combretum molle in dexamathasone indued-insulin resistane in rats David Miaffo,2, Sylviane Laure Kamani Poualeu 2, Albert Kamanyi 2 Department of Life and Earth Sienes, Higher Teahers Training College, University of Maroua, Far North Region-Cameroon 2 Laboratory of Animal Physiology and Phytopharmaology, Department of Animal Biology, Faulty of Sienes, University of Dshang, West Region-Cameroon ABSTRACT Reeived: / Revised: / Aepted: Combretum molle plant has appliations in Afrian traditional mediine against pain, diabetes mellitus and mirobial diseases. In present study, we investigated the antidiabeti potential of the methanol and aetone extrats of twigs of C. molle in dexamethasone indued-insulin resistane diabetes mellitus. Single of extrats (250 and 500 mg/kg, p.o.) was administered to normal and hyperglyemi animals. Insulin resistane was indued by daily sub-utaneous injetion of dexamethasone at dose of ml/kg. The anti-diabeti effets were also evaluated, by measuring glyeamia, body weight, serum levels of lipid parameters and atherogeni indies and oronary artery risk. These studies revealed that oral administration of both extrats (methanol and aetone) to normal and hyperglyemi rats signifiantly suppressed the rise in blood gluose level, as glibenlamide. Oral treatment with dexamethasone injetion for days was assoiated with signifiant weight loss, hyperglyemia, hyperlipidemia and atherogeni indies. However, pretreatment with extrats signifiantly prevented inrease in any of these measured parameters. Results of this study suggest that the hypoglyemi, anti-hyperlipidemi and anti-atherogeni effets of theses extrats are mediated through inreased peripheral gluose uptake and improvements in insulin resistane, thus, validating its ethnomedial use in the traditional management of diabetes mellitus. Keywords: Dexamethasone, insulin resistane, antidiabeti ativity, Combretum molle twigs, methanol and aetone extrats. INTRODUCTION Diabetes mellitus (DM) is a group of metaboli diseases haraterized by hroni hyperglyemia or inreased blood gluose levels with disturbanes in arbohydrates, protein and fat metabolism resulting from insulin defiieny, impaired effetiveness of insulin s ation, or to a ombination of both []. The disease has reahed epidemi proportions in the urrent entury. DM is a ommon health problem worldwide, and the prevalene and inidene of this disease are rapidly inreasing [2]. The world s prevalene of diabetes among adults (aged years) was 6.4%, affeting 285 million adults in 20 and will inrease to 7.7% ie.439 million adults by 2030 [3]. India leads the world today with the largest number of diabetis with a mean annual inrement of.8 million [3]. Of all these diabeti populations, about 90% aount of type 2 diabetes [4]. Clinial studies on different speies of animals have shown that onsuming less food redues the risk of diabetes and heart disease. Current treatment of type 2 diabetes remains inadequate; prevention and leaving are preferable weapon [4]. One therapeuti approah for treating DM is derease postprandial hyperglyemia. Several antihyperglyemi drugs suh as biguanides, sulfonylureas, thiazolidinediones and alphagluosidase inhibitors are the ornerstone of diabetes treatment; they have important adverse effets and annot always maintain euglyemia and prevent diabetes ompliations signifiantly [5]. If diabetes is not duly treated, it will lead to long term damage, dysfuntion and failure of various organs Corresponding Author Address: Miaffo David, Department of Life and Earth Sienes, Higher Training Teaher s College, University of Maroua, Far North Region-Cameroon; david_miaffo@yahoo.fr

2 and systems, espeially the eyes, kidney, nerves, limbs and ardiovasular system [6]. The ompliations of diabetes are the main auses of morbidities and mortalities [7,8]. However, all these treatments have limited effiay and have been reported to be assoiated with undesirable side effets [9]. In order to overome the side effets assoiated with diabetes, interest has been shifted to use of other alternative mediine. Traditional mediines and extrats from mediinal plants have been extensively used as alternative mediine for better ontrol and management of diabetes mellitus. Mediinal plants have ontinued to be a powerful soure for new drugs, now ontributing about 90% of the newly disovered pharmaeutials []. In fat, many developing ountries around the world use traditional mediine, beause it is sometimes the only affordable soure for healthare. As for the developed ountries, the use of herbal mediine for hroni diseases is enouraged beause there is onern about the adverse effets of hemial drugs and treatment using mediines of natural origin appears to offer more gentle means of managing suh diseases. Herbal drugs are presribed widely beause of their effetiveness, fewer side effets and are relatively low in ost. Plants have long been used for the treatment of diabetes, partiularly in developing ountries where most people have limited resoures and do not have aess to modern treatment. Ethnobotanial studies have reported more than 200 plant speies with potential antidiabeti effets []. Combretum molle (Combretaeae) is a shrub or small, graeful, deiduous tree 3-3 m high; trunk rooked or leaning, oasionally swollen at the base, up to 30 m in diameter. It is found espeially in savannah vegetation that uts aross from Senegal to West Cameroon, but generally exists in tropial Afria [2]. C. molle is widely used in Afrian traditional mediine for the treatment of various diseases. It is reported to possess antifungal, antimirobial, antiparasiti, antioxidant, and anti-inflammatory and antidiabeti pharmaologial properties [3]. Therefore there is a depth of information regarding the bioativity of the different parts of this plant whih merits further investigation. This study was therefore undertaken to find out whether in the ase of C. molle twigs aetone and ethanol extrats ould be effetive in the management of diabetes in dexamethasone-indued insulin resistane in rats. MATERIALS AND METHODS Chemials and drugs: Alloxan, aarbose, and kits for biohemial dosages were purhased from David et al., World J Pharm Si 204; 2(9): Sigma-Aldrih, St. Louis, USA. Starh, D-gluose and surose were purhased from Edu-Lab Biology Kit, Bexwell, Norfolk PE38 9GA, UK. All hemials and drugs were obtained ommerially and were of analytial grade. For dilutions, metformine, glibenlamide, starh, aarbose, gluose and surose were dissolved in normal saline. The extrats were dissolved in physiologial saline by DMSO (dimethyl sulfoxide). All drugs were prepared immediately before use. Experimental animals: Nulliparous and nonpregnant healthy albinos female rats aged 8-2 weeks, weighing between g were used for toxiologial test and albinos males rats aged 3-4 months, average weight 250 g were used for pharmaologial tests. These animals were raised in the animal house of the Department of Animal Biology, Faulty of Sienes at the University of Dshang, Cameroon, were used. The animals were housed in plasti ages and maintained in ambient temperature of 24 ± 0 C, relative humidity of 55-65% and normal light/dark yle. They were fed standard laboratory diet and water ad libitum. The rats were alimatized to laboratory ondition for one week before ommenement of experiments. The Ethial learane for the usage of rats was obtained from the Institutional Animal Ethial Committee (IAEC) prior to the beginning of the study [4]. Plant material: The fresh twigs of Combretum molle were olleted in the month of Deember from village Moutourwa, situated near Maroua ity (Far North Region, Cameroon). The plant material was authentiated in the National Herbarium, Yaoundé where vouher speimen was deposited. After authentiation, the olleted fresh twigs were leansed thoroughly under running tap water, dried under shade and ground into fine powder. Preparation of extrats: The dried powder of C. molle (200 g) was soaked in 00 ml of eah solvent (methanol and aetone) for 72 h in old ondition. The whole mixture was suessively filtered through a piee of lean, white otton material and No. Whatman filter paper. The filtrate obtained was evaporated at 80 C using rotary evaporator to obtain 9.35 g and 4.2 g representing a yield of 9.67 % and 7.48% for methanol and aetone extrats respetively. Qualitative phytohemial tests: The extrat obtained was subjeted to the preliminary phytohemial analysis, whih inluded tests for alkaloids (Dragendoff reagent), saponins (frothing test), tannins (FeCl 3 ), glyosides (Legal test), flavonoids (NaCl and HCl), reduing sugars (Fehling liquor), steroids (hloroforme and H 2 SO 4

3 onentrate), terpenoïdes (hloroforme and H 2 SO 4 onentrate) and phenols (FeCl 3 and K 3 Fe (CN) 6 ). These were identified by harateristi olor hanges using standard proedures [5]. Aute toxiity: Aute toxiity studies were performed aording to the Organization for Eonomi Co-operation and Development (OECD) guidelines 425 [6]. Ten animals were divided in two groups of 5 animals eah. The rats were fasted for 6 h with free aess to water only. The aetone extrat and DMSO were administered orally in doses of 2000 mg/kg and ml/kg to different groups of female rats. Observations were made and reorded systematially 30 min, 4 h, 24 h, 48 h, 7 days and 4 days after dose administration for skin and fur hanges, eyes, salivation, lethargy, sleep, oma, onvulsion, tremors, diarrhea, mortality, mobility, aggressivity, sensitivity of the sound, touh and pain as well as respiratory movement. Normoglyemi study: Overnight fasted rats were divided 6 groups onsisting of 6 animals of eah group. Group I rats reeived normal saline ( ml/kg, p.o) as vehile. Group II rats reeived glibenlamide (0.3 mg/kg, p.o) as standard drug. Groups III and IV rats reeived methanol extrat of C. molle (MECM) at the doses of 250 and 500 mg/kg body weight, p.o. in a single dose. Groups V and VI rats reeived aetone extrat of C. molle (AECM) at the doses of 250 and 500 mg/kg body weight, p.o. in a single dose. The tail was snipped and blood gluose level of eah rat was tested using the gluometer (One touh R ultra 2, Blood gluose meter, Life San Europe, 6300zug, Switzerland). Blood sample was tested just before oral administration of substanes (0 hour) and at 2 and 4 hours in eah ase. Oral arbohydrate hallenge tests in normal rat [7]: The oral arbohydrate tolerane tests were arried out using gluose, starh and surose separately in normal groups of rats and were equally divided into various treatment groups. In oral gluose tolerane test, rats were divided into 6 groups onsisting of 6 rats in eah group. The rats were fasted overnight for 6 hours but had free aess to water. Group I rats were treated orally with normal DMSO 5% ml/kg (normal ontrol). Group 2 rats were treated orally with glibenlamide mg/kg body weight (positive ontrol). Groups III and IV animals reeived 250 and 500 mg/kg MECM. Groups V and VI rats reeived 250 and 500 mg/kg AEMC. The rats in all the groups were administrated with D-gluose (3 mg/kg, p.o), 60 min after extrat administration. Blood samples were olleted from the tail at 30, 60, 90 and 20 min after gluose administration. Blood gluose levels were measured immediately David et al., World J Pharm Si 204; 2(9): with a gluometer one touh. The oral starh and surose tolerane tests were arried out with DMSO 5%, aarbose, MECM and AECM in the same way as above, but in these tests, starh and surose at a dose of 4 g/kg body weight eah was used. Dexamethasone indued insulin resistane: The study was arried out for days to aess the effet of various treatments on biohemial parameters of different tissues in dexamethasone indued insulin resistane in rats. The rats were divided into 7 groups, onsisting of 5 animals eah. Animals were kept for overnight fasting 6 hours prior to experiment. Rats in the first group reeived normal saline ( ml/kg, p.o. + ml/kg, s..) and served as normal ontrol. The seond group of rats reeived normal saline ( ml/kg, p.o.) and served as diabeti ontrol group (negative ontrol group). Rats in the third group were treated with metformin (40 mg/kg, p.o.) and served as positive ontrol group. Rats in experimental group 4 and 5 were treated with MECM (250 and 500 mg/kg p.o. respetively). Rats in the group 6 and 7 reeived AECM (250 and 500 mg/kg p.o. respetively). One hour after pretreatment, all rats in the treatment groups II-VII were subutaneously ingested with ml/kg of dexamethasone sodium phosphate. The animals were weighed every day during all the treatment. Glyeamia of eah overnight fasted animal was estimated as above but, this time at the beginning and at the end of the experimental period. Oral gluose tolerane in diabeti rat: After an overnight fast on the days of the experiment, the fasting blood gluose levels of rats in all the treated groups were determined using one touh ultragluometer. 90 min afterwards, oral tolerane test was onduted in all the drug treated rats with 3 g/kg of D-gluose. The blood gluose levels were monitored at 30, 60, 90 and 20 min after administration of gluose. Following the oral gluose tolerane test, the animals were deprived of food overnight (2 h), anesthetized with diazepam/ketamine, i.p and sarified. Whole blood samples were taken from the abdominal artery by atheterization method. The blood samples left for 5 minutes at 37 C for serum separation, then entrifugated at 3000 rpm for 20 minutes, then sera were separated and kept in eppendorf tubes at -20 C until analyses. Just after the blood olletion samples, organs suh as kidneys, heart, panreas, liver, spleen and lungs were removed and weighed. Biohemial Analysis: Total holesterol (TC) was determined in serum aording to the enzymati

4 olorimetri method desribed by [8] using DIALAB kit. Triglyerides (TG) were determined in serum aording to the enzymati olorimetri method desribed by [9] using INMESCO kit. Serum high density lipoprotein holesterol (HDL) onentration was determined in serum aording to the enzymati olorimetri method desribed by [20] using INMESCO kit. Serum low density lipoprotein holesterol (LDL-) onentration was alulated by redution of the sum HDL- and LDL- onentrations from that of TC, using equation: LDL- = TC (TG/5) HDL. Determination of atherogeni and oronary artery risk indies: Atherogeni index (AI) was alulated aording to the methods of [2] and expressed as AI = (TC-HDL-)/HDL-. Coronary artery risk index (CRI) was also alulated using the formula TC (mg/dl)/hdl- (mg/dl) [22]. Statistial analysis: Statistial analysis was performed using the statistial funtions of the Graph pad Prism version 4.. All the results were expressed as mean ± SEM. The signifiane of differene between mean values for the various treatments were tested using one-way analysis of variane (ANOVA) followed by Turkey test and two-way analysis of variane followed by Bonferroni test. Statistial signifiane was onsidered at p < 0.05; p < 0.0 and p < RESULTS Preliminary phytohemial sreening: The preliminary phytohemial sreening revealed the presene of glyosides, saponins, flavonoids, phenols, triterpenoids and tannins. Aute oral toxiity study: Aute oral administration of a dose 2000 mg/kg of aetone extrat of C. molle twigs produed no mortality after 24 hours and after 4 days of observation period. The extrat treated female rats did not produe any grossly negative physial and behavioral hanges. The LD50 value was found to be more than 2000 mg/kg body weight. Hene, about one tenth of this dose (250 mg/kg) was seleted as the level for examination of antidiabeti potential. Normoglyemi study: The normoglyemi study showed that all rats treated with glibenlamide (0.3 mg/kg) produed a signifiant (P < 0.0 and P < 0.05) redution in blood gluose level as ompared to ontrol group, 2 and 4 hours after administration. Similar results were obtained with AEMC at two doses 250 and 500 mg/kg. However, MECM at a dose of 250 mg/kg orally did not produed any David et al., World J Pharm Si 204; 2(9): blood sugar level while a dose of 500 mg/kg of the same extrat signifiantly (p < 0.05) dereased the blood gluose level after 4 hours, as ompared with ontrol group (Table ). Oral gluose tolerane test in normal rat: The administration of glibenlamide and graded doses of MECM and AECM orally showed improved gluose tolerane in normal rats (Figure ). In fat, in glibenlamide group rats, oral gluose administration was assoiated with a signifiant and time-dependant redution in blood gluose level at 60 (P < 0.05), 90 (P < 0.00) and 20 min (P < 0.00) when ompared to vehile ontrol animals. Similar pattern of postprandial gluose hanges was reorded for rats treated with AECM at dose 500 mg/kg while in the ase of AEMC a dose of 250 mg/kg, redution effet was observed min after gluose load (P < 0.05). However, MECM at a dose of 250 mg/kg indued a signifiant (P < 0.05) and transitory derease in blood gluose level at 90 min while the higher dose of the same extrat redued blood gluose level signifiantly at 90 (P < 0.0) and 20 min (P < 0.05), in omparison with ontrol group rats. Oral starh tolerane test in normal rat: In normal rats, MECM at two doses and AECM at dose 250 mg/kg did not show any signifiant derease in blood gluose level ompared to ontrol rats (Figure 2). However, AECM at dose 500 mg/kg, redued blood gluose level signifiantly (P < 0.05) at 90 min. Aarbose signifiantly redued the blood gluose level at 60 (P < 0.05), 90 (P < 0.0) and 20 min (P < 0.0). Oral surose tolerane test in normal rat: In normoglyemi rats, none of the MECM-treated groups showed any redution in blood gluose level as ompared to normal ontrol group (Figure 3). However, AECM at dose 250 mg/kg showed a signifiant (P < 0.05, P < 0.0 and P < 0.05) derease in blood gluose at 60 min, 90 min and 20 min respetively. At 60 min, 90 min and 20 min, AECM at dose 500 mg/kg redued blood gluose level signifiantly (P < 0.0). Both AECM at dose 500 mg/kg and aarbose failed to signifiantly (P < 0.05) suppress the lower blood gluose level at 60 min, 90 min and 20 min; but at 30 min after surose administration, aarbose produe signifiant derease (P < 0.05) in blood gluose level ompared to ontrol group rats. Effet of C. molle extrats on blood gluose level in dexamethasone-indued insulin resistane in rat: The effet of methanol and aetone extrats on blood gluose level of dexamethasone indued insulin resistane in rats is presented in Figure 4. Results showed that the administration of

5 dexamethasone ( ml/kg) resulted in insulin resistane as evidened by the signifiant (P < 0.00) inrease in mean gluose (45.60 ± 2.63 mg/dl) of the diabeti ontrol when ompared with the normal ontrol. Continuous treatment with metformine and extrats of rats of the respetive groups for days along with dexamethasone showed signifiant derease in blood gluose level when ompared to diabeti ontrol group. Metformine, MECM at dose of 500 mg/kg and AECM at doses of 250 and 500 mg/kg along with dexamethasone produed a signifiant (P < 0.00) derease in mean gluose (38.4 ±.9 mg/dl, ± 0.07 mg/dl, ±.63 mg/dl and ±.98 mg/dl) respetively, when ompared to diabeti ontrol group. However, all animals treated with MECM at dose of 250 mg/kg along with dexamethasone redued glyeamia by about 4.80 ±.8 mg/dl signifiantly (P < 0.0) as ompared to diabeti ontrol group. Oral gluose tolerane test in diabeti rat: As shown in Figure 5, the diabeti ontrol group rats showed a signifiant inrease in post-prandial gluose onentration at 60 (P < 0.0), 90 (P < 0.00) and 20 min (P < 0.00) in omparison to normal ontrol group, whereas dexamethasone + MECM 250 mg/kg treated group showed tendeny in postprandial blood level redution at 90 min (P < 0.05). On the other hand, metformine and AECM at dose of 500 mg/kg along with dexamethasone demonstrated a signifiant (P < 0.0, P < 0.0 and P < 0.0) drop in blood gluose level at 60, 90 and 20 min when ompared to diabeti ontrol, while the lower dose of AECM along with dexamethasone shifted and delayed the blood gluose onentration from 90 (P < 0.0) to 20 min (P < 0.0). Furthermore, diabeti rats treated with a ombination of dexamethasone and MECM at dose of 250 mg/kg, showed a signifiant (P < 0.0) derease in blood gluose level at the end of 20 th min as ompared to diabeti non-treated animals. However, at 60, 90 and 20 min, the higher dose of MECM dereased blood gluose level signifiantly (P < 0.05; p < 0.00; P < 0.00) in experimental rats respetively, when ompared to dexamethasone group. Effet of C. molle extrats on body and organs weights in dexamethasone-indued diabeti rats: Table 2 show the effet of the days of oral treatment with the methanol and aetone extrats on the body weight and organs weight of treated rats. The results showed that, metformine and AECM at dose of 500 mg/kg produed about 29% and 25% inrease in body weight (P < 0.0) at the end of th day of their treatment respetively in omparison to diabeti ontrol. However, the body weights of rats treated as above were reverted to David et al., World J Pharm Si 204; 2(9): near normal when ompared to normal ontrol. There was a 5.40 % redution in body weight (P < 0.00) in dexamethasone treated rats when ompared to normal ontrol. The same results were observed with 250 mg/kg AECM and both MECM at doses 250 and 500 mg/kg; this reduing and dose-dependent effet was about.09 % (p < 0.05),.80 % (p < 0.0) and 9.22 % (p < 0,05) respetively. Furthermore, administration of metformine, dexamethasone and different extrats of C. molle prior to dexamethasone injetion resulted in a nonsignifiant differene between the weight of kidneys, heart, lungs and panreas of this group and the normal ontrol group. However, negative ontrol rats produed a signifiant (p < 0.00 and p < 0.0) inrease in the weight of liver and spleen to about 37.0 % and 50 % respetively. In omparison to diabeti ontrol, all rats pretreated with metformine, MECM at doses of 250 and 500 mg/kg and with AECM at doses of 250 and 500 mg/kg demonstrated a signifiant and dosedependent drop in liver weight of about 6.22 % (p < 0.0), 8.92 % (p < 0.0), % (p < 0.00), % (p < 0.00) and % (p < 0.00) respetively. Spleen weight of animals pretreated with both AECM at doses of 250 and 500 mg/kg was also signifiantly (p < 0.0) lower (about % and 3.25 % respetively) than those of the diabeti group, whereas MECM at doses of 250 and 250 mg/kg did not produe any signifiant variation in spleen weight. Effet of C. molle on serum lipids and ardiovasular risk indies in dexamethasoneindued insulin resistane in rats: In Table 3, the lipid parameters, AI and CRI of extrat treated groups were ompared with the diabeti ontrol group. Results indiate that, in omparison to normal ontrol, dexamethasone group produed a signifiant (p < 0.00) mean differene in the TC (7.98 ±.34 mg/dl), TG (20.09 ± 0.47 mg/dl), HDL- (8.23 ± 0.84 mg/dl), LDL- (32.9 ± 0.43 mg/dl), AI (.70 ± 0.24 mg/dl) and CRI (.70 ± 0.24 mg/dl) values. Animals pretreated with metformine for days at a dose of 40 mg/kg showed a signifiant (P < 0.00) derease in serum TC (26.08 ± 0.53 mg/dl, TG (25.74 ± 3.20 mg/dl), LDL- (39.9 ± 0.56 mg/dl), AI (.90 ± 0.25 mg/dl) and CRI (.90 ± 0.25 mg/dl) levels, and a signifiant (p < 0.00) inrease in serum HDL- (8.97 ±. mg/dl), omparatively to diabeti ontrol. Similar effet was also observed on the serum TC, TG, HDL-, LDL-, AI and CRI of pretreated rats groups with both extrats.

6 DISCUSSION Insulin resistane and hyperinsulinemia are often assoiated with a group of risk fators as obesity, atheroslerosis, dyslipidemia, hypertension and impaired gluose tolerane. Any interventions therefore to derease insulin resistane may postpone the development of diabetes and its ompliations. Treatment with herbs has been a better hoie beause they are effetive with fewer side effets and are affordable as ompared to presently used syntheti oral antidiabeti drugs. The present study was undertaken to investigate the antidiabeti effets of Combretum molle twigs extrats in dexamethasone indued diabeti rats as an experimental model of type 2 diabetes mellitus. Aute toxiity test at 2000 mg/kg body weight of the AECM produed no mortality after 4 days of observation whih indiates that the mean lethal dose (LD50) of the extrat is greater than 2000 mg/kg body weight. Generally, aute toxiity did not produe any grossly negative behavioral hanges in the rats it instead redued reation to noise was observed suggesting that, the extrat may have depressant effet on the entral nervous system [23]. The ability of MECM and AECM to effetively ontrol inreased blood gluose level in normal and diabeti rats may be attributed to its hypoglyemi and antihyperglyemi effets. In fat, normoglyemi study revealed that the high dose (500 mg/kg) of MECM and both doses (250 and 500 mg/kg) of AECM redued blood gluose level signifiantly (p < 0.05) 2-4 hours post treatment. Oral administration of both extrats to rats fed with gluose signifiantly (p < 0.05; p < 0.0 and p < 0.0) suppressed the rise in blood gluose level in both normal and diabeti rats, as glibenlamide or metformine. These results suggest that animals treated with extrats have better gluose utilization apaity suggesting its mehanism being almost similar to referene produts (glibenlamide or metformine). They promote tissue gluose uptake and redue hepati gluose output, thereby produing hypoglyemi and antihyperglyemi effets. However, both doses of AECM only were administered to surose loaded normal fasted rats resulting in hypoglyemia (p < 0.05; p < 0.0 and p < 0.0), whereas MECM did not affet the rise in glyeamia. Moreover, onerning oral starh tolerane test, AECM at dose 500 mg/kg only redued blood gluose level signifiantly (P < 0.05) at 90 min. The above results show a striking similarity to the effets of aarbose. These findings suggested that AECM ould derease the postprandial gluose level by inhibiting the ativity of α-amylase (weakly) and α-gluosidase, whih David et al., World J Pharm Si 204; 2(9): are important enzymes in the digestion of the omplex arbohydrates into absorbable monosaharides in the food [24]. Reently, some reports showed that glyosides and triterpenoids ould effetively inhibit the ativity of α-amylase and/or α-gluosidase to derease the absorption of arbohydrates from food [25,26]. So the glyosides and triterpenoids in AECM might take the responsibility for the postprandial antihyperglyemi effet of extrat. It was very interesting to point out that AECM was more effiient for omplex arbohydrate surose than the starh. Aarbose like drug that inhibit α- gluosidase present in the epithelium of the small intestine, have been demonstrated to derease postprandial hyperglyemia and improve impaired gluose metabolism without promoting insulin seretion in type 2 DM patients [27]. These mediations are most useful for people who have just been diagnosed with type 2 diabetes and who have blood gluose levels only slightly above the level onsidered serious for diabetes. The present study indiated that daily administration of C. molle (250 and 500 mg/kg) up to days showed anti-diabeti and hypolipidemi effets in diabeti rats. In fat, high exposure to gluoortioids impairs insulin sensitivity, ontributing to the generation of metaboli syndrome inluding insulin resistane, hyperglyemia and dyslipidemia in human and experimental animals [28]. The mehanism by whih dexamethasone indues peripheral insulin resistane is by inhibiting GLUT-4 (gluose transporters) transloation from intraellular ompartments to the plasma membrane partiularly of skeletal musles. The extrats ould at by reversing the gluoortioid mediated transloation of the gluose transporters from the plasma membrane to the intraellular ompartment. The twigs extrats of C. molle, therefore, appeared to have improved insulin resistane through enhaned insulin sensitivity in peripheral tissues, as was evident from the dereased gluose levels. Moreover, dexamethasone ation is mediated by the gluoortiortioid reeptor, a nulear reeptor that regulates physiologial events through ativation or repression of target genes involved in inflammation, gluoneogenesis and adipoyte differentiation [29]. It is therefore, probable that the C. molle extrats may ontain substanes that ompete with dexamethasone for the gluoortioid reeptor (ompetitive antagonism) or they may ontain substanes that stimulate the prodution of repressor elements that inhibit the transription. That is, they may bind to negative gluoortioid response elements that mediate the repression of gene transription. However, phytohemial analysis of the twigs of the plant shows the presene of saponins, steroids, flavonoids, phenols,

7 terpenoids, and tannins. It is likely that the gluose lowering property ould be due to the ombined effet of these bioative onstituents. For instane, molli aid gluoside, a ompound isolated from C. molle leaf, possesses hypoglyaemi and antidiabeti properties in rodents [30]. Furthermore, phenoli onstituents of Pteroarpus marsupium signifiantly lowered the glyemia of diabeti rats in a manner omparable to that of metformine [3]. DM is assoiated with profound alteration in the serum lipid and lipoprotein profile with an inreased risk in oronary heart disease [32]. Hyperlipidemia is a reognized ompliation of DM haraterized by elevated levels of holesterol, triglyerides and phospholipids, and hange lipoprotein omposition [33]. Thus, redution in TC and TG through dietary or hypoholestrolemi agent has been found benefiial in preventing ardiovasular disease risk fators (atherogeni and oronary artery indies) as well as improving lipid metabolism in diabeti patients [34]. In the present study, the inreased TG, TC and LDL- fration and dereased HDL- fration were observed in diabeti rats. This abnormally variation of serum lipid levels is mainly due to uninhibited ations of lipolyti hormones on the fat depots, mainly due to impairment of insulin sensibility at diabeti state. After treated with C. molle extrats (250 and 500 mg/kg) or metformine (40 mg/kg) for days, serum TG, TC and LDL- levels were signifiantly dereased while serum HDL levels was inreased; however, the inreased HDL- (ardioprotetive lipid) level was omparable to the standard drug, metformine. Therefore, C. molle extrats have potential role to prevent formation of atheroslerosis and oronary heart disease. Several authors reported that seondary metabolites suh as saponins, flavonoids, phenoli ompounds, and triterpenoids have hypolipidemi ativity [35]. Hene, the hypolipidemi properties of this plant may be due to different types of ative seondary metabolites presents in the plant extrats. Moreover, in the liver, gluoortioids inrease the ativities of enzymes involved in fatty aid synthesis and promote the seretion of lipoproteins. The hepati lipogeni effet of gluoortioids results in aumulation of triglyerides in the liver; this redues insulin sensitivity in the liver [36]. The plant extrats ould also at by inhibiting the expression of phosphoenolpyruvate arboxykinase and gluose-6-phosphatase and by inreasing lipase ativity in adipose tissue their by ausing impairment of endothelium-dependent vasodilatation [37]. There exist a link between obesity, oronary heart disease and insulin resistane and weight loss has been reported to lower the risk of oronary heart disease and insulin resistane; in fat, weight loss of 5% or more has David et al., World J Pharm Si 204; 2(9): been linked to improvements in glyeami ontrol, lipid parameters and quality of life [38]. In the present study, a severe loss in body weight observed in diabeti ontrol might be the results of protein wasting to unavailability of arbohydrate for utilization as an energy soure [39]. Metformine and AECM at dose of 500 mg/kg prevented this augmentation while the others dose of C. molle treated rats did not normalized the body weight ompletely. An inrease in the body weight of diabeti treated rats might be due to an enhanement in glyeami ontrol and inreased synthesis of strutural proteins [40]. Moreover, a signifiant inrease in liver and spleen weights was observed in non-treated diabeti rats. This inrease of liver weight might be due to intensity metaboli ativities in this organ, whih is onsidered as detoxifiation organ. However, metformine and plant extrats prevented this inrease in weight of organs. CONCLUSION From the present study, it an be onluded that, oral administration of twigs of C. molle as aute dose even 2000 mg/kg in albinos rats is relatively safe. Moreover, the hypoglyemi, antihyperglyemi, anti-hyperlipidemi and antiatherogeni potentials of the extrats in type 2 DM model were onfirmed. From preliminary phytohemial analysis it was found that the major hemial onstituents of the C. molle extrats were glyosides, saponins, flavonoids, phenols, triterpenoids, and tannins so it is possible that ertain of these onstituents may be responsible for the observed antidiabeti ativity and redue lipid parameters and ardiovasular disease risk fators. If these results are extrapolated to humans, then C. molle might prove useful to be a soure of potent type 2 DM and/or therapeuti priniples ating also in preventing insulin resistane in non-diabeti states suh as obesity and impaired gluose tolerane. This assertion lends redene to its suggested folklori use in the ontrol and/or management of insulin resistane DM in ertain ommunities of Cameroun. Further pharmaologial and biohemial investigations are underway to find out the ative onstituents responsible for antidiabeti ativity and to eluidate its mehanism of ation. ACKNOWLEDGMENTS This work was arried at the Laboratory of Animal Physiology and phytopharmaology, University of Dshang and Laboratory of Life and Earth Sienes, Higher Teahers Training College of Maroua. Authors are highly thankful to Heads of Departments for providing the failities, and H. Foyet and D.E. Tsala for their valuable enouragements and suggestions.

8 David et al., World J Pharm Si 204; 2(9): Table : Effet of methanol and aetone extrats of C. molle on normoglyemi rats. Groups 0 h 2 h 4 h Control ( ml/kg) ± ± ±.0 Glibenlamide (0.3 mg/kg) ± ± ±.82 MECM (250 mg/kg) 8.05 ± ± ±.67 MECM (500 mg/kg) ± ± ±.93 AECM (250 mg/kg) ± ± ±.64 AECM (500 mg/kg) 82.7 ± ± ±.54 Values are expressed as mean ± SEM (n = 6). P < 0.05, P < 0.0 ompared to ontrol. Table 2: Effet of C. molle extrats on body weight and relative organs weight in dexamethasone indued diabeti rats. Body weight (g) Organs weight (%) Groups 0 th day th day Liver Kidneys Heart Lungs Spleen Panreas Normal ontrol ± ± ± ± 0.3 ± 0.6 ± 0.42 ± 0.32 ± Diabeti ontrol 22.8 ± ± ± ± 0.44 ± 0.80 ± ± ± Dexa + MET (40mg/kg) ± ± 7.73b 2.97 ± 0.09b 0.68 ± ± ± ± ± 0.05 Dexa + MECM (250 mg/kg) 28.4 ± ± ± 0.09b 0.64 ± ± 0.75 ± 0.68 ± ± Dexa + MECM (500 mg/kg) 27.4 ± ± ± ± 0.46 ± 0.68 ± 0.72 ± ± 0.00 Dexa + AECM (250 mg/kg) 29.0 ± ± ± ± 0.30 ± ± 0.4 ± b 0.28 ± Dexa + AECM (500 mg/kg) ± ± 5.b 2.84 ± 0.6 ± 0.34 ± ± ± 0.05b 0.35 ± Values are expressed as mean ± SD of 6 rats in eah group. P < 0.05; P < 0.0; P < 0.00 ompared to ontrol. a P < 0.05, b P < 0.0; P < 0.00 ompared to diabeti ontrol. Table 3: Effet of C. molle on serum lipid and ardiovasular risk indies in dexamethasone-indued insulin resistane in rats Groups TC (mg/dl) TG (mg/dl) HDL- LDL- AI CRI (mg/dl) (mg/dl) Normal ontrol 9.05 ± 8.70 ± ± ±.07 ± 2.08 ± Diabeti ontrol ± ± ± ± ± ± 0.3 Dexa + MET (40mg/kg) 0.95 ± ± ± ± ± ± 0.07 Dexa + MECM ( ± ± ± ±.34 ± 2.43 ± 0.3 mg/kg) 3.44a Dexa + MECM ( ± ± ± ± 0.98 ±.89 ± mg/kg) Dexa + AECM ( ± ± ± ±. ± 2. ± 0.5 mg/kg) b Dexa + AECM (500 mg/kg).27 ± ± ± ± ± ± 0.06 Values are expressed as mean ± SEM (n = 6). P <0.0 ompared to ontrol. a P < 0.0; b P < 0.0; P < 0.00 ompared to diabeti ontrol. 962

9 Blood gluose level (mg/dl) Blood gluose level (mg/dl) Blood gluose level (mg/dl) David et al., World J Pharm Si 204; 2(9): Control mg/kg Gliben 0,3 mg/kg MECM 250 mg/kg MECM 500 mg/kg AECM 250 mg/kg AECM 500 mg/kg Time ourse (min) Figure : Effet of the extrats of Combretum molle on gluose tolerane test in normal rat Control ml/kg Aarbose mg/kg MECM 250 mg/kg MECM 500 mg/kg AECM 250 mg/kg AECM 500 mg/kg Time ourse (minute) Figure 2: Effet of the extrats of Combretum molle on starh tolerane test in normal rat Control ml/kg Aarbose mg/kg MECM 250 mg/kg MECM 500 mg/kg AECM 250 mg/kg AECM 500 mg/kg Time ourse (min) Figure 3: Effet of the extrats of Combretum molle on surose tolerane test in normal rat. 963

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