DIABETES AND ITS ASPECTS
1. Introduction Diabetes is a disorder of metabolism due to absolute deficiency or diminished effectiveness of insulin. Due to lack of insulin, hyperglycemia and glycosuria almost invariably occur (Hyder et al., 1998-99). It is a fatal health problem in the present world. Diabetes is the fourth- leading cause of death ( Saha et al., 2006). The diabetic population is rapidly increasing globally, particularly in the developing countries. South Asian region including, Bangladesh is the most vulnerable focus. The estimated diabetes prevalence for 2010 worldwide is 285 million people corresponding to 6.4% of the world’s adult population. By 2030, the number of people with diabetes is estimated to have risen to 438 million (IDF, 2010). The 10 countries estimated to have the highest numbers of people with diabetes in 2000 and 2030 are estimated. In 2000, Bangladesh is listed at 10 positions but for 2030, it will be raised at 7 positions reflecting anticipated changes in the population size and structure in these countries between the two time periods (Wild et al., 2004). 2. History of Diabetes mellitus History of Diabetes mellitus dates back to thousands of years. With discovery of insulin in 1921 and glucose reducing drugs in 1955, treatment of diabetes was certainly boosted up. But complications due to this disease still continue. More importantly, in the third world countries like Bangladesh the prevalence of diabetes is raising, partly because of the insulin treatment, lack of knowledge, inadequate medical facilities and insufficient health care. The treatment of diabetes is at present neither simple nor cheap. Insulin, the main antidiabetic agent is extracted commercially from animal (cattle or pig) pancreas. It is the principal agent to fight against the disease. Insulin is indicated for ketosis prone juvenile-type diabetics as well as for those adult-onset diabetics with insulinopenia who do not respond to diet therapy either alone or in combination with oral hypoglycemic drugs. Ideal replacement therapy would provide insulin in a manner comparable to the secretor pattern of normal individuals. It is not possible to completely reproduce the physiologic patterns of insulin secretion
with subcutaneous injections of soluble or longer-acting insulin suspensions or combinations of insulins. Even so, with the help of appropriate modifications of diet and exercise, it has been possible to achieve acceptable control of blood glucose by using variable mixtures of short- and longer-acting insulin (Hyder et al., 1998-99). Thus, the investigation of proper alternatives of insulin is very urgent and proper dietary adjunct is necessary for diabetics. A significant proportion of these patients obviously fail to get proper treatment and medication. Indigenous drugs, since long, have been used for the treatment of diabetes (Said, 1969). Hundreds of plants are known to be useful in treating diabetes in different corners of the world. Bangladesh is abundant in antihyperglycemic plants. These species may represent a source of new hypoglycemic compounds for developing better remedies to treat diabetic patients without serious side effects. 3. Diabetes: general consideration
Diabetic mellitus is a clinical syndrome characterized by persistent hypoglycemia with or without glycosuria due to deficiency or diminished effectiveness of insulin. The metabolic disarrangement is frequently associated with permanent and irreversible functional and structural change in the cells of the body and lead to many complications. The complications of diabetes most characteristically affect the eye, the kidney and the nervous system (Davidson, 1977).
4. Types of diabetes: On the basis of etiology two main categories of diabetes are recognized, namely primary (idiopathic) diabetes and secondary diabetes. 4.1. Primary Diabetes:
It consists of two main clinical typesi. Juvenile-onset or insulin dependent diabetes and ii. Maturity-onset or insulin independent diabetes. Although the precise etiology in still uncertain, several contributing factors are known to be involved which differ in younger and older-onset diabetes. Age: Diabetes is a disease of the middle aged and elderly because 80 percent of cases occur after the age of 50 years. Sex: Both sexes suffer equally but in lower age groups males and in middle age groups females are affected more. Heredity: In both types of diabetes a familial tendency exists and twins are more often both diabetic when they are identical than when they are non-identical. Auto-immunity: Diabetes is seen to be associated with other autoimmune diseases such as pernicious anemia, hyperthyroidism and Addition’s disease. Infection: There is some evidence that viral infection may be involved in the etiology of juvenile insulin-dependent diabetes. Obesity: The association of obesity and diabetes has long been recognized but it is still uncertain whether obesity is the result or the cause of diabetes. Diet: Overeating, especially when combined with under activity, is associated with a rise in the incidence of diabetes in the middle aged and elderly.
4.2. Secondary Diabetes:
a) Pancreatic diabetes is caused by the destruction of the pancreases due to pancreatitis, carcinoma and pancreatic calculi. b) Non-pancreatic diabetes occurs due to the abnormal concentrations of hormones in the circulation i.e., in acromegaly, Cushing’s syndrome, thyotoxicosis etc. c) Iatrogenic diabetes occurs after the prolonged use of thiazide, diuretics, steroids, contraceptive pills etc. Clinical features: Onset is usually gradual but rarely there may be acute onset, the probable clinical features of diabetes are as follows: i) polyurea ii) polydipsia iii) polyphagia iv) rapid emaciation v) dryness of mouth and threat vi) constipation. Diagnosis: Whenever diabetes is suspected, the diagnosis should be confirmed by glucose tolerance test (GTT), by random blood sugar sedimentation and by urine testing especially for glycosuria. 5. Method of treatment: i) Diet regime ii) Diet and oral hypoglycemic drugs and iii) Diet and insulin. 6. Drug management of diabetes These are best suited for middle-aged obese diabetics who would otherwise require insulin. There are several groups of oral hypoglycemic agents. These agents with their derivatives are given below: i. Insulin secretagogues (emission prompter of insulin): Sulphonylureas- 1. Aceloberamide, 2. chlorpropamide, 3. Glibenclamide, 4. Glibornuride, 5. Glimepiride, 6. Glimepiride amyral, 7 glipizide, 8. Glyburide, 9. tolazamide, 10. Tolbutamide, 11. Gliclazide, 12. Repaglinide, 13. nateglinide (last two are not chemically sulfonylureas). ii.
Insulin sensitizers
(activity prompter
of
insulin):
1.
Biguanides-
metformin,
Thiazolidinediones- pioglitazone, rosiglitazone, troglitazone. iii. Disaccharides Inhibitors (inhibitors intestinal glucose absorption): 1. Acarbose, 2. miglitol.
2.
iv. Drug modalities Incretins exendin-4: 1. Liraglutide (subcutaneous injection), 2. vildagliptin, 3. sitagliptin, 4. pramlintide (amylin analogues-subcutaneous injection), 5. saxagliptin, 6. alogliptin. Mechanism of action Although the mechanisms of action of these drugs are different, the action of first two groups depends upon a supply of endogenous insulin. Sulphonylureas: These drugs act either by supressing the alpha cells secretion, glucagons or by stimulating the beta cells to secret insulin. Biguanides: Mrtformin reduces hepatic glucose production and increases peripheral glucose utilization.The mechanism of action is still poorly understood (DeFronzo, 1991). Thiazolidinediones:.This class of agents works by increasing insulin sensitivity. Acarbose: It is effectively compensate for defective early-phase insulin release by inhibiting the breakdown of disaccharides to monosaccharides in the intestinal epithelium without any side effects. The cell surface serine amino peptidase enzyme, DPP-4, rapidly degrades and inactivates GLP-1, GIP, and other peptides in vivo via cleavage of the N-terminal two amino acids. Inhibition of this enzyme leads to an increase in circulating endogenous GLP-1 and GIP levels, making it a promising therapeutic strategy for type 2 diabetes. The orally administered DPP-4 inhibitors available and in development delay the breakdown of incretin hormones, prolonging and enhancing their activity and thereby increasing glucose-mediated insulin secretion and suppressing glucagon secretion (Drucker, 2003). Insulin (11) Insulin is needed to keep the blood glucose within reasonable level without undue risk of hyperglycemia. Two main therapeutic forms of insulin are available, namely 1. Rapid-onset, short-acting and 2. Delayed-onset, long-acting or depot preparation Briefly they are known as short-acting insulin and long-acting insulin. Mechanism of action The basic mechanism of insulin action is to increase the permeability of the cell membranes of glucose. It is suggested that this change in permeability is induced either by facilitating the conversions of glucose through phosphorylation or oxidation into more lipid soluble compounds or by altering the physical structure of the cell wall through the combination of the insulin protein with the membrane protein, so that glucose can pass more easily into the cell (Davidson, 1977). Diabetes and the oxidative damage The body maintains a balance between the amount of reactive oxygen species generated and its antioxidant defense. The balance may be tipped, however, by conditions that greatly increase the
generation of reactive oxygen species (such as cigarette smoke in the lungs) and/or lack of antioxidant defense due to malnutrition. There is substantial evidence that people with diabetes tend to have increased generation of reactive oxygen species, decreased antioxidant protection, and therefore increased oxidative damage. Hyperglycemia, or a high blood glucose level, has been shown to increase reactive oxygen species and end product of oxidative damage in isolated cell cultures, in animals with diabetes, and in humans with diabetes. Measurement of the end products of oxidative damage to body fat, proteins and deoxiribonucleic acid (DNA) are commonly used to assess the degree of oxidative damage to body cells and tissues. Most studies show that these measures are increased in people with diabetes. The activities of key antioxidant enzymes are also to be abnormal in people with diabetes. In many studies, these enzymes are also found to be lower than normal, suggesting a compromised antioxidant defense, while other studies show higher activity, suggesting an increased response to oxidative stress. Few studies demonstrate that oxidative damage is greater in people with Type 2 diabetes compared with Type 1, especially people with Type 2 diabetes and the metabolic syndrome, which involves central obesity, hypertension (high blood pressure) and high blood fat levels along with insulin resistance (decreased effectiveness of insulin metabolizing blood glucose) suppressor (Internet III-I). 7.0. Survey of literature Hypoglycemic agents are the materials which are generally have opposite test to sweet and biologically possess insulin like properties or act as a suppressor of glycemic level in blood. The agents may be obtained from synthetic or plants (natural) sources. 7.1. Hypoglycemic agents from synthetic origin The hypoglycemic agents are either synthetic compounds or of animal origin (Table 3.1). Table 3.1: Chronological list of synthetic hypoglycemic agents
1 2 3 4 5 6 7
Hypoglycemic agents In 1908 the structural modification of sulfanilamides discovered from the dye ‘prontosil’ lead to synthesis of drugs for the treatment of diabetes mellitus. Guanidine which is generally administered in the form of its hydrochloride is an effective hypoglycemic agent of the pre-insulin era. With discovery of insulin by Frederiech Grant Banting and Charles Herbert Best in 1921 treatment of diabetes was certainly boosted up. Jabon et al in 1941-42 first observed that isopropyl-thiazole, a sulfonamide derivative produced hypoglycemia in patients receiving the drug. Cyclopropyl derivative of sulfonamide has hypoglycemic effect in normal rabbit but due to its goiterogenic side effect the drug was not studied further. Sodium salicylate and acetyl-salicylic acid were among the earliest drugs which effectivelyreduce the total daily urinaryglucose output in diabetic patients. A group of German researchers in 1954 found a sulfonamide derivative called ‘Carbutamide’ as the first effective synthetic anti-diabetic agent. But
References/year Zuelzer, 1908 (Williams, 2002) Wantanbe, 1918 1921, Banting and Best Jabon et al. in 1941-42 Chen et al., 1946 Gross et al., 1948 German researchers, 1954
8 9 1 0 1 1 1 2 1 3 1 4 1 5 1 6 1 7 1 8 1 9 2 0 2 1 2 2 2 3
the agent had severe toxicity problems in a small percentage of the patients treated with this drug diminishing its application. With discovery of glucose reducing (hypoglycemic) drugs carbutamide in 1955, treatment of diabetes was certainly boosted up. Aspirin has improved in the state of some of the diabetic patients given fairly large doses. 2, 4 Dinitrophenol reduces the fasting blood sugar in some diabetic patients and increases glucose uptake by isolated rat diagragm. Discovery of other synthetic agent known as ‘Tolbutamide’ in the same year with little or no side effects became the first therapeutic agent in ‘adult-onset diabetic patients Isopropyl-thiazole opened up a new horizon in the field of treatment of diabetes mellitus with synthetic agents Thiocarbonate is as much effective hypoglycemic agent as tolbutamide when administered at a dose of 25 mg/kg body weight It was observed that 1-phenyl-3-methyl-4-aryl hydrazone-2-pyrazolin-5ones has effective antidiabetic activity in experimental animals ‘Benzimidazole’ derivatives of C 12H16N4O3S, having an aromatic nucleus with a side chain of ‘-SO2NHCONRR’ was capable to reduce blood glucose level singnificantly after six hours of administration Carburide, a synthetic hypoglycemic agent has reported that the drug had no serious side effect and enhanced the early release of insulin after oral glucose load in patients The hypoglycemic action of gliclazide, a sulfonylurea derivative was studied by Furman and coworker. They found that glibenclamide when administered in fasting rats decreases The plasma glucose and plasma FFA concentration were decreased by glibenclamide when administered in fasting rats (N-(1-methyl-2-pyralidinyl-1-pyrralidine carboximidamide) had hypoglycemic effect in non-diabetic rats, dogs, mice and monkeys, which was structurally unrelated to sulfonylureas or phenformin It was experimentally found that pyrogliride potentials glucose induced insulin secretion from isolated islets and facilitates glucoronic metabolism Saliman et al. studied the antidiabetic action of new 3-methyl-5phenylpyrozole sulfonylurea derivative in diabetic rats First biosynthetic human insulin is introduced Insulin pen delivery system is introduced Troglitazone [Rezulin, Resulin, Romozin] was introduced in the late 1990s but turned out to be associated with an idiosyncratic reaction leading to drug-induced hepatitis.
Franke 1955
et
al.,
Reid et al., 1957 Raudle, 1957 McMahan, 1963 McMahan, 1963 Doglas 1968
et
al.,
Garg et al., 1970 Deshpande et al., 1970 Joyce, 1971 Furman 1977
et
al.,
Thomas 1978
et
al.,
Zawalich et al., 1980 Saliman et al., 1981 1983 1986 Internet III-II (2010)
All these drugs control the pancreatic function. These drugs may causes hypoglycemia due to toxic and adverse effect. This limits their use and prompted many researchers to look into herbs and plants for less toxic hypoglycemic agents for adult-onset diabetes or insulin non-dependent diabetes in particular.
7.2 Hypoglycemic agents from plant origin The hypoglycemic agents discussed so far are either synthetic compounds or of animal origin. But a search for the discovery of hypoglycemic agents in plant kingdom has shown promising results. Ivorra et al studied the structure of 78 different compounds isolated from plants with attributed hypoglycemic activity. They classified these compounds according to the following chemical groups: I-
Polysaccharides and proteins (59 compounds).
II-
Steroids and terpenoids (7 compounds).
III-
Alkaloids (7 compounds).
IV-
Flavonoids and related compounds (5 compounds) (Rahman et al., 1989).
(List of some antidiabetic compounds is given in the last column of Appendix-5 against the respective plant/plant part(s) from which it was isolated). Some plant originated hypoglycemic agents are listed in Table 3.2. Table 3.2: Chronological list of hypoglycemic agents from plant origin. 1 2 3 4 5 6
Hypoglycemic agents Glucokenin, an extract isolated from Cephalandra indica has the property of reducing the amount of sugar in the blood. Galegine (8), a guanide isolated from the seeds of Galega officinalis, was found to have a significant blood sugar lowering effect when administered by mouth. Gymnemic acid (10), a glycosidic acid isolated from Gymnema sylvetre (Fam. Asclepiadaceae) was found to have a significant blood sugar lowering effect when administered by mouth. When gymnemic acid is administered with insulin a prompt response was observed. Antihyperglycemic effect of andrographolide in streptozotocin-induced diabetic rats. Andrographolide-lipoic acid conjugate (AL-1) had both hypoglycemic and beta cell protective effects which translated into antioxidant and NF-ÎşB inhibitory activity. AL-1 is a potential new anti-diabetic agent.
References Collip, 1923 Muller, 1925 Muller, 1925
H H
Morris et al., 1976 Yu et al., 2006 Zaijun, et al., 2009
Plants in treating diabetes Dolichos lablab commonly known as field bean reduced the blood glucose level of diabetic patient when administered for a long time (Nivasam, 1957). The tablets made from leaves of Coccinia indica demonstrated the hypoglycemic actions when administered at a dose of 3 tablets daily (Khan et al., 1980).The hypoglycemic effect of the seeds Cyamopses tetragonoloba Taub (Gower) on rabbits and found its action similar to tolbutamide at a dose of 20 mg/kg bw. The effect, which was found to be due to extra pancreatic action, was remarkable at 40 mg/kg.bw (Pilloi et al., 1980). Similar investigations proposed that hundreds of plants are known to be useful in the treatment of diabetes in different corners of the world. According to Tanira more than 400 plants incorporated in
approximately 700 recipes are used to treat diabetes mellitus in almost two thirds of the world population. In addition, clinical trials have shown some plants as useful antidiabetic agents. The pure chemical compounds isolated from the crude extracts of these plants do not bear structural resemblance to the antidiabetic drugs in current clinical use nor have they similar mechanisms of action. The search for a novel antidiabetic drug advocates the utilization of plants as a potential source. Some research approaches are suggested to increase the likelihood of isolating novel hypoglycemic agents from plant sources. Effectiveness of various herbs and their products in the treatment of diabetes mellitus in our country are well known. The scientific name of available indigenous plants seems to have hypoglycemic properties is listed in Appendix-4 and another detailed list of antidiabtic plants is enclosed in Appendix-5. 7.3. Hypoglycemic studies with selected plants 7.3.1 Materials Plant materials: For scientific investigation of hypoglycemic activity, the selected plant materials are listed in Table 1.1. These plants have been used traditionally as folk medicines in treating various diseases especially in diabetes by the common people but enough evidence was not available to confirm the hypoglycemic activity of the various extracts on different hyperglycemic conditions. Some evidences are listed in Table 3.3 with references. Table 3.3: List of hypoglycemic (antidiabetic) evidence of the studied plants. Plants/compound Evidence Screening for antihyperglycemic activity in several local herbs of Malaysia Antidiabetic property of methanolic extract of A. A. paniculata niculata in streptozotcin rats Hypoglycemic (diabetes), febrifuge, cholagogue, anthelmintic Antidiabetic Hypoglycemic Andrographolide potentially reduced serum glucose in Andrographolide streptozotocin-induced diabetic rats. (isolated from A. Andrographolide-lipoic acid conjugate (AL-1) had both paniculata) hypoglycemic and beta cell protective effects which translated into antioxidant and NF-ÎşB inhibitory activity. AL-1 is a potential new anti-diabetic agent Root bark shows hypoglycemic and anthelmintic properties Hypoglycemic, astringent, stomachic, antimalarial A. chinensis
S. sesban
Stem bark shows hypoglycemic and anthelmintic properties Diabetes, anthelmintic, catarrh, skin deases, stimulant, emmenagogue and galactogogue
References Husen et al., 2004 Zhang et al., 2000 Ahmed et al., 1977 Ghani, 2003 Ghani, 2003 Yu et al., 2006 Zaijun, 2009
et
al.,
Acharyya et al., 2010 Kitagawa et al., 1996 Ghani, 2003 Yadava et al., 1996
M. oleifera
M.oleifera Lamk. (Moringaceae) is commonly used as healing herb to treat diabetes
Dolly 2009
et
al.,
Others Materials Animal: Rats- Albino, Long Evans rats and Swiss mice Albino rats and Swiss albino mice were collect from Animal Breeding Center of BCSIR Laboratories, Chittagong. Long Evans rats were procured from Animal House, BCSIR Laboratories and from International Center for Diarrheal Disease Research, Bangladesh (ICDDR, B), and naturalized in the animal house of the Faculty of Pharmacy department and Institute of Nutrition and Food (INFS), University of Dhaka.. Each of the selected rats was housed in a screen-bottomed cage and maintained at room temperature. Generally male and female Long Evans rats weighing about 200 to 250 gm (of age 100 to 150 days) were selected from a colony of rats. The rats were fed on a good quality basal diet and water ad libitum. The diet supplied to each rat was about 20 g of diet per day that was approximately isocaloric. The detailed composition of the basal diet used for rats in the study has been given in appendix-B. Market drugs: Diactin (glipizide), daonil (glibenclamide), glimepiride (SK& F),. Diabetic inducers: Alloxan (Sigma Chemical Co., St. Lowis, MO), streptozotocin Glucose kit: Human (Germany and Accu-Check Active Meter, sanofi aventis) Glucometer: EPS (Easy Pain Supreme, Bio Technology Corp. Belgium] Spectrophotometer: SPEKOL 1300, analylik jena Basal diet: (Composition Appendix-6) Saline, syringe, feeding tube, chloroforn, diethyl ether, cotton, Al-foil, gloves and many more chemicals and laboratory apparatus. 7.3.2 Methods Induction of diabetes in rats: Alloxan momohydrate/ tetrahydrate was dissolved in normal saline and injected in the experimental rats to produce permanent diabetes (juvenile type). The doses of alloxan, the degree of occurrence of diabetes in rats and estimating blood/serum glucose level have been described thoroughly at the related plant section.
Albino rat
Long Evans rat
Figure. 3.1 Photographs of rats
Figure 3.2: Photos of Swiss mice
Figure 3.3: Administrationof test sample in animals
Figure 3.4: Measuring of serum/blood glucose by spectrophotometer and glucometer 7.3.3 Statistical analysis (Calculation): Students `t’ test was formulated for analysis of data from each experimental group. Percentage change in glucose level (increased or decreased) was determined by using formula: (detailed in Appendix 7). (Average from control groups -_average from treated groups) Average from control groups
Ă—100
7.4. Hypoglycemic study with Andrographis paniculata 7.4 .1 Materials and its processing Collection of plant material: The aerial part of A. paniculata was collected from the plantation area and neighboring hilly regions of BCSIR, Chittagong, in the month of July 2006. The plants were cut into small pieces, dried in the sun and then in an oven at 50 oC for several hours. It was then ground by means of an electric grinder to obtain a coarse powder.
Extraction of plant materials: Hot water extracts (WE): For preparing water extract the powder of A. paniculata was mixed with distilled water (1:12), boiled for 5-7 minutes, cooled at room temperature and filtered through a filter paper. The liquid (aqueous extract) was then administered to rats through feeding needle. Ethanol extracts (EE): The powder (900 g) of powder was soaked in ethanol (4.5 L) in a large glass bottle that bearing a tap at the lower part. It was left e for 7 days with occasional shaking. Then the liquid extractive was passed through the tap and was filtered by using a filter paper. The extract, so obtained was concentrated under reduced pressure at about 45-50 oC with a rotary vacuum evaporator.
Animal and diet: Adult male and female albino rats obtained from the Animal Breeding Center, BCSIR Laboratories, Chittagong Bangladesh weighing 200-230 g were used for the study. The rats were acclimatized to standard laboratory conditions (relative humidity 55 ±5%, temperature 24±1oC and a 12 h diurnal photoperiod) in galvanized cages (3-6 rats/cage) with replaceable wire-meshed net lid for 7 days before the commencement of the experiment. During the study, all animals were maintained on normal laboratory chow and water ad libitum.
Induction of diabetes in rats: In glucose-loaded study, rats were fasted overnight (18 h) before oral administration of glucose. Glucose at a concentration of 1.5 g/kg b.w. was dissolved in distilled water immediately administratered through feeding needle. In alloxan induced study, rats were injected with alloxan solution (40 mg/kg bw) intraperitonially and then fasted for 18 hours.
7.4 .2 Experimental design Glucose-loaded experiments: 28 rats were randomly divided into four groups (7 rats in each gr.) as followsGroup I: Vehicle control, received only distilled water Group II: Negative control, diabetic untreated (glucose, 1.5 g/kg bw) Group III: Positive control, diabetic treated with daonil (4 mg/kg bw) Group IV: Positive control, diabetic treated with water extract (1 g/kg bw)
All the animals were primarily fasted for 18 hours (given only distilled water) and then glucose solution (group II to group IV) were given through feeding tube. After 2 hours, distilled water, drug solution and sample (water extract for one day’s experiment and ethanol extract for another day’s experiment) were given orally according to rats of respective group. Two hours later, all the animals were anesthetized with diethyl ether and sacrificed and blood sample were collected from cardiac vessel by syringe for every observation in each study. Alloxan induced experiments: 28 rats were divided randomly and evenly as followsGroup I: Vehicle control, received only distilled water Group II: Negative control, diabetic untreated (alloxan, 40 mg/kg bw) Group III: Positive control, diabetic treated with diactin (4 mg/kg bw) Group IV: Positive control, diabetic treated with ethanol extract (1 g/kg bw).
The rats of gr-II to gr-IV were injected with alloxan and all groups were fasted for 18 hours. Then standard drug and sample (water extract for one day’s experiment and ethanol extract for another
day’s experiment), were given orally to the rats group wise in every experiment. Two hours after treatment, blood samples were collected as described earlier.
Estimation of blood/serum glucose level (BGL): The level of glucose in blood samples from each of the experimental and control rat was determined by using standard glucose kit essentially following the glucose oxidase-peroxidase (GOD-POD) method (Barham, et al., 1972; Trinder, 1969). The blood was taken by syringe from cardiac vessels of sacrificed rats. It was then centrifuged to get a clear supernatant (serum). 2µl of serum was taken in 2ml of standard glucose kit solution in a separate test tube. The intensity of the color of the solution was measured with a spectrophotometer at 546 nm for quantification of the glucose initially present in the blood specimen. 7.5. Results and discussion Effect of water extract on glucose-loaded hyperglycemic (GLH) rats: The effect of hot water extract on BGL of glucose-loaded rats is presented in Table-3.4. Administration of glucose increased the BGL of rats by 89.47% as compared to vehicle control rats while the hot water extract A. paniculata
significantly (p<0.001) decreased the elevated BGL by 41.51% as
compared to diabetic control (glucose-loaded) rats. In the case of standard drug, daonil treatment, the percent of BSL decrease was 44.70. Effect of EE on GLH rats: The effect of EE on BGL of glucose-loaded rats has been summarized in Table-3.5. Administration of glucose increased the rats BGL by 87.07% when compared to vehicle control rats. On the other hand, rats treated with ethanol extract of A. paniculata significantly (p<0.001) lowered (41.82%) the enhanced BGL as compared to diabetic control rats. In the case of drug (diactin) treatment group, the glucose level was lowered by 45.63%. Effect of hot WE on alloxan-induced diabetic (AID) rats: Table 3.6 depicts the effect of hot water extract on BGL of alloxan-induced diabetic rats. Administration of alloxan increased the rats BGL by 104.69% as compared to vehicle control-control group. On the other hand, rats treated with hot water extract of significantly (p<0.001) lowered the elevated BGL by 46.21% when compared to diabetic control group. In this situation, the standard drug reduced the BGL by 49.66%.
Effect of EE on AID rats: Table 3.7 shows the serum blood sugar level in vehicle-control, diabetic control (alloxan), standard drug and sample treated groups. Table 3.4: Effect of hot water extract of A. paniculata in GLH rats. Blood glucose levela Percent change Group Treatment Mean ± S.D (mg/dl) [Increased(↑)/decreased (↓)] I Vehicle control 60.77 ± 3.28 --II Diabetic control 115.14 ± 2.36 89.47(↑)
Drug treated 63.67 ± 3.05 44.70(↓) (Diactin) IV Sample treated 67.35 ± 2.17 41.51(↓) a Values are Mean ± S.D., (n=7) S.D.= Standard deviation, n = number of animal III
Table 3.5: Effect of ethanol extract of A. paniculata on GLH rats. Percent change Blood glucose levela Group Treatment Mean ± S.D (mg/dl) [Increased(↑)/decreased(↓)] I Vehicle control 61.40 ± 3.34 II Diabetic control 114.86 ± 1.62 87.07(↑) Drug treated III 62.45 ± 4.02 45.63(↓) (Diactin) IV Sample treated 66.83 ± 2.36 41.82(↓) a Values are Mean ± S.D. (n=7), S.D.= Standard deviation, n = number of animal Table 3.6: Effect of hot water extract of A. paniculata on AID rats. Percent change Blood glucose levela Group Treatment [Increased(↑ )/decreased(↓ ) Mean ± S.D (mg/dl) ] I Vehicle control 60.22 ± 3.19 II Diabetic control 123.27 ± 3.68 104.69(↑) Drug treated III 62.31 ± 5.18 49.66(↓) (Diactin) IV Sample treated 66.31 ± 4.93 46.21(↓) a Values are Mean ± S.D. (n=7), S.D.= Standard deviation, n = number of animal Table 3.7: Effect of ethanol extract of A. paniculata on AID rats. Percent change Blood glucose levela Group Treatment [Increased(↑ )/decreased(↓ ) Mean ± S.D (mg/dl) ] I Vehicle control 61.21 ±3 .25 II Diabetic control 125.24 ± 3.19 104.61(↑) III Drug treated (Diactin) 61.30 ± 3.06 51.05(↓) IV Sample treated 68.72 ± 5.02 45.13(↓) a Values are Mean ± S.D. (n=7), S.D.= Standard deviation, n = number of animal
Graphical representation of the results is shown in Figure 3.5 to 3.8.
Blood Glucose Level (mg/ dl)
Effect of hot WE of AP in GLH rats 140
140
120
120
100
60
115.14
40
63.67
60.77
67.35
20 0 e Co
l ntr o
tr ol Con et ic b a Di
l ntro g co u r D a A.p
( l at a ni cu
ted) trea
Figure 3.5
80 60 40
62.45
66.83
Drug control
A.paniculata (treated)
61.4 20
Vehicle Control Diabetic Control
Figure 3.6
100
100
80
80
123.27
62.31
60.22
66.31
20
Blood Glucose Level (mg/ dl)
120
40
Effect of EE of AP in AID rats
140
120
60
114.86
0
Effect of WE of AP in AID rats
140
Blood Glucose Level (mg/ dl)
Blood Glucose Level (mg/ dl)
100
80
icl Veh
Effect of EE of AP in GLH rats
125.24
60 40 61.21
61.3
68.72
20 0
0 Vehicle Control
Figure 3.7
Diabetic control
Drug control
A.paniculata (treated)
Vehicle Control
Diabetic control
Drug control
A.paniculata (treated)
Figure 3.8
Figure 3.5: Chart for effect of water exact in glucose-loaded hyperglycemic rats Figure 3.6: Chart for effect of ethanol exact in glucose-loaded hyperglycemic rats Figure 3.7: Chart for effect of water exact in alloxan-induced diabetic rats Figure 3.8: Chart for effect of ethanol exact in alloxan-induced diabetic rats
Table 3.8 Comparison of the effect of hot WE and EE of A. paniculata on GLH rats hot water extracta ethanol extract Group Treatment (Mean ± S.D), (mg/dl) (Mean ± S.D), (mg/dl) I Vehicle control 60.77 ± 3.28 61.40 ± 3.34
II Diabetic control 115.14 ± 2.36 114.86 ± 1.62 IV Sample treated 67.35 ± 2.17 66.83 ± 2.36 % decreased -----41.51 41.82 a Values are Mean ± S.D. (n=7), S.D.= Standard deviation, n = number of rat Table 3.9 Comparison of the effect of hot WE and EE of A. paniculata on AID rats hot water extracta ethanol extract Group Treatment (Mean ± S.D), mg/dl) (Mean ± S.D), mg/dl) I Vehicle control 60.22 ± 3.19 61.21 ± 3.25 II Diabetic control 123.27 ± 3.68 125.54 ± 3.19 IV Sample treated 66.31 ± 4.93 68.72 ± 2.02 % decreased -------46.21 45.13 a Values are Mean ± S.D. (n=7), S.D.= Standard deviation, n = number of rat (N.B: All the above results are average of 7 time experiments in each case) Alloxan enhanced the BGL by 104.61% when compared with vehicle-control rats. On the other hand, treatment of rats with ethanol extracts were significantly (p<0.001) decreased 45.13% the alloxan elevated BGL. Here, the blood glucose lowering effect of the standard drug, diactin was 51.05%. It is clearly evident from the study that the aqueous and ethanol extractives were capable to exhibit significant blood sugar lowering effects in both the glucose-loaded and alloxan-induced diabetic rat (Table 3.4-3.7 and Figure 3.5-3.8). The lowering of blood glucose levels by the aqueous extract was also comparable to methanol extract. Both the extractives were found to able to reduce the sugar level almost identically as evident from tables 3.8 and 3.9. 8.0. Hypoglycemic Study with Anthocephalus chinensis 8.1 Materials and its processing Collection of plant materials: The stem bark of A. chinensis was collected from Faridpur and was identified at Bangladesh National Herbarium where a voucher specimen (# DACB 31749) has been maintained. The barks were cut into small pieces, dried at room temperature and then ground to a coarse powder. Extraction of plant materials: The powdered bark was soaked in methanol in a closed container for 7 days with occasional shaking. Then the extractive was filtered by using a filter paper and concentrated under reduced pressure at about 45-50 oC with a vacuum rotary evaporator. The concentrated extract so obtained was suspended in distilled water at 125 and 250 mg/kg b.w. (according to need) with the help of Tween 80. The aqueous slurry was then administered to rats through feeding needle.
Animal and diet: Long Evans rats of either sex (100-200 g) were used for the investigation. The rats were housed in standard conditions (relative humidity 55±5%, temperature 21±2 oC) and a 12 h light-
dark cycles and were given standard pellet diet and water ad libitum. Animals were acclimatized to their environment for one week prior to experimentation. 8.2 Experimental Design The 30 rats were divided into five groups evenly as follows: Group 1: Normal control: given normal diet only Group 2: Negative control: diabetic untreated (alloxan, 150 mg/kg bw) Group 3: Diabetic treated with Glimepiride (200 Âľg/kg bw /body) Group 4: Diabetic treated with methanolic extract (125 mg/kg bw/day) Group 5: Diabetic treated with methanolic extract (250 mg/kg bw/day) All rats except normal-untreated group (gr-1) were injected with alloxan solution (150 mg/kg bw) intraperitonially. After 48 h of injection, hyperglysomia was confirmed by using electronic glucometer. The animals were treated with methanolic extract for consecutive 7 days only as dose variation test and blood glucose was measured every 24 hours after treatment. Estimation of blood glucose level (BGL): The level of blood glucose (sugar) of the experimental and control rats was rapidly determined by using an electrochemical detection technique (Cass et al., 1984).
8.3 Results and discussion In the present study, the hypoglycaemic potential of the methanolic extract of A. chinensis was determined in alloxan-induced rats for 7 consecutive days (Table 3.10). The study demonstrated that, the extracts were capable to reduce the elevated blood sugar and the hypoglycaemic activity of the extract increased with the increment of doses of the extractive. The glucose levels obtained in blood of normal and experimental rats are given in table 3.10 for plant extract. From table 3.11, it is evident that the methanolic extract of A. chinensis exhibited a significant (p<0.05) reduction of blood glucose by 26.24% and 30.36% on 1 st day (24 hr after 0 time) for the dosing of samples 125 and 250 mg/kg bw/day respectively, in diabetic rats as compared to the untreated diabetic rats (i.e. 1 st day BGL of respective group). The BGL reduction capacity was successively increased and at the end of 7 th day, the highest values obtained and it were 38.95% and 40.60% for the dosing of samples 125 and 250 mg/kg bw/day respectively, in diabetic rats as compared to the untreated diabetic rats. Moreover, the lowest value (%BGL reduction) was obtained only by 26.24% for 125 mg/kg bw/day at the end of 1 th day and the highest value was 40.60% for 250mg/kg b.w/day at the end of 7th day.
Table 3.10: Effect of methanol extract of A. chinensis on BGL in AID rats
Group
Gr-1 Normal (untreated) Gr-2: Diabetic control Gr-3: Glimepiride treated Gr-4: meth. ext. treated (125 mg/kg b.w.) Gr-5:meth. ext. treated (250 mg/kg b.w. )
mmol/L Initial sugar level 5.85±0 .45 19.8.± 1.2 20.47± 1.8
1st day
2nd day
3rd day
4th day
5th day
6th day
7th day
5.9±0. 6 19.32± 1.4 12.4±1 .09
5.72±0 .61 19.92± 0.9 10.43± 0.4
5.77±0 .52 20.68± 1.3 9.32±1 .03
5.82±0 .57 20.4±0 .78 8.82±1 .0
5.88±0 .64 20.12± 1.2 7.32±1 .35
5.84±0 .93 19.59± 1.3 7.18±2 .01
5.76±0 .74 20.04± 0.9 7.53±1 .7
19.82± 1.8
14.62± 1.6
14.09± 0.9
13.33± 1.3
13.14± 1.5
12.95± 1.6
12.46± 0.8
12.1±1 .9
20.32± 3.2
14.15± 1
13.83± 1.6
13.5±0 .74
13.1±1 .12
12.93± 1.2
12.33± 2.4
12.07± 1.8
Table 3.11: Percentage changes of BGL in AID rats by A. chinensis extractives. Treating Group and doses
% Reduction 1st 2nd day day 39.42 49.05
3rd day 54.47
4th day 56.91
5th day
6th day 64.92
7th day
Gr-3: Glimepiride 64.24 63.22 Gr-4: Methanolic extract (125 26.24 28.91 32.74 33.70 34.66 37.10 38.95 mg/kg) Gr-5: Methanolic extract (250 30.36 31.94 33.56 35.53 36.37 39.32 40.60 mg/kg) [N.B: % changes (reduction) of BGL of any days were calculated to compare with 1 st day BGL (mmol/L) In conclusion the experimental data suggests that the methanolic extract of A. chinensis has significant capability in reducing the elevated blood glucose level (Table 3.11). The results so obtained, justify the folk use of the plant for treatment of diabetes. Further comprehensive pharmacological investigations are needed to elucidate the extract mechanism of the hypoglycemic potential and long-term effects of the use of extractives in treating diabetes.
9. Hypoglycemic Study with Sesbania sesban 9.1 Materials and its processing Collection of plant material: The leaves of S. sesban were collected from plantation area of the BCSIR Laboratories, Chittagong and were identified at the Plant Taxonomy Division and Bangladesh national herbarium, where a voucher specimen has been deposited. The leaves were dried at room temperature for some days, followed by drying in an oven and were then ground to a coarse powder. Extraction of plant material: The powder leaves were soaked in methanol in a closed container for 7 days. Then the extractive was filtered using a filter paper. The extract obtained was concentrated under reduced pressure with a rotary vacuum evaporator.
Animal and diet: Adult male and female Long Evans rats obtain from Bangladesh Council for Scientific and Industrial Research, Dhaka weighing 120-210 g were used for the study. During the study, all animals were maintained on normal laboratory chow, water ad libitum. Induction of diabetes in rats: Diabetes was induced in rats by injecting intraperitonially a freshly prepared aqueous solution of alloxan (100 mg/kg b. w.), after a base line glucose estimation was done. After 48 hours of alloxan administration, rats with blood sugar levels above 14 mmol/L were selected for the study.
9.2 Experimental design A total of 42 rats were randomly divided in equal number into 7 groups. Group A: Normal untreated rats Group B: Alloxan induced diabetic (AID) rats (100 mg/kg bw) Group C: AID rats given glimepiride orally (200 Âľg/kg bw) Group D: AID rats given plant extract orally (50 mg/kg b w) Group E: AID rats given plant extract orally (100 mg/kg bw) Group F: AID rats given plant extract orally (200 mg/kg bw) Group G: AID rats given plant extract orally (300 mg/kg bw) In this study, at 0 time, all the rats of gr-B to gr-G were injected alloxan and 24 hours later, BGL of all group rats were measured. After every 24 hours, rats of gr-C (glimeperide) and rats of gr- D to gr-G were given drug and plant samples of respective amounts. The body weight of all rats was assessed weakly. The blood samples of rats were drawn after an over night fasting (12 hrs) from tail tip at different time intervals i.e., 1, 7, 14, 21 and 28th day for study with plant extract. Estimation of blood glucose level: The blood glucose level of all were rapidly determined by using a glucometer that act as an electrochemical detection technique (Cass et al., 1984) and described in the respective section of A. chinensis. 9.3 Results and discussion Diabetes induced rats produced a significant reduction of body weight in all hyperglycemic rats. The administration samples improved the body weight significantly (p<0.05) after 28 days as compared to diabetic control groups (Table 3.12). It was evident that the daily administration of different doses of methanolic extract for 28 days revealed a statistically significant (p<0.05) increase in body weight when compared with diabetic control rats. On the other hand, no significant body weight anomaly was observed between glimepiride and the samples treated groups. The glucose levels obtained in blood of normal and experimental rats are given in table 3.13 for plant extract. From table 3.14, it is evident that the methanolic extract of S. sesban showed a significant (p<0.05) reduction of blood glucose by 10.0%, 22.61%, 24.17% and 27.80% on 7 th for the dosing of samples 50, 100, 200 and 300 mg/kg bw/day respectively, in diabetic rats as compared to the
untreated diabetic rats (i.e. 1 st day BGL of respective group). We observed that the glucose lowering activity was higher in higher doses of samples. In the same way, reduction capacity was successively increased day by day and at the end of 28 th day, the highest values were obtained and it was 25.48%, 46.29%, 51.88% and 53.12% for the dosing of samples 50, 100, 200 and 300 mg/kg bw/day respectively, in diabetic rats as compared to the untreated diabetic rats. Table 3.12: The Changes in body weight of treated and untreated rats. Body weight in g Dose Groups mg/kg 1st day 7th day 14th day 21st day 175.0±2.9 179.6±3.6 183.7±3.6 186.85±3. A- Normal- untreated ---8 3 6 9 181.25±4. 172.32±4. 167.08±3. 159.65±5. B- Alloxan treated 100 3 7 9 1 CGlimepiride 175.32±5. 171.26±4. 166.65±5. 162.33±5. 0.2 treated 2 8 3 1 194.81±1. 188.22±2. 181.65±3. D- zMeth ext treated 50 200±3.65 9 4 7 172.87±1. 167.51±2. 162.14±2. 156.09±3. E- ,, ,, 100 8 2 3 3 183.22±3. 177.76±3. 172.25±2. 166.65±2. F- ,, ,, 200 5 2 8 3 166.78±2. 161.40±2. 155.97±3. 150.55±2. G- ,, ,, 300 7 9 0 5
28th day 190.86±3. 8 149.43±4. 2 161.16±5. 5 176.01±2. 8 149.88±2. 1 161.23±2. 2 145.12±3. 1
Values are given in average body weight g±SEM for groups of six animals each. *p<0.05 Table 3.13: The effect of 4 weeks treatment of methanol extract S. sesban on BGL in AID rats. Groups Auntreated
Normal-
B- Alloxan treated C-Glimepiride treated D- Met. ext. treated E-
,,
F- ,, G- ,,
Dose mg/kg
BGL in mmol/L 1st day 7th day
14th day
21st day
28th day
----
5.17±0.38
5.35±0.45
5.38±0.45
5.23±0.48
5.18±0.39
100
15.4±0.12
15.7±0.09
16.02±0.1 1
16.37±0.0 8
16.54±0.07
6.25±0.22
5.32±0.19
4.65±0.26
4.20±0.23
50
15.86±0.2 0 15.7±0.57
,,
100
15.08±1.77 11.67±1.96 10.13±1.43 9.27±1.27
8.1±1.39
,, ,,
200 300
14.4±1.73
6.93±4.32
0.2
14.13±0.24 13.28±0.46 12.25±1.15 11.7±0.51 10.92±0.7
9.38±0.52
7.86±0.69
14.1±0.56 10.18±2.0 8.32±0.61 7.15±1.03 6.1±0.95 Values are given as mean ±SEM for six animals in each group. Diabetic control (Group B) was compared with normal group (Group A) on corresponding day. Experimental groups (Groups D-G) were compared with diabetic control group (Group B) on corresponding day.*p<0.05; **p<0.001 Table 3.14: Effect of leaves methanolic extract of S. sesban on BGL of AID rats Group Dose % Reduction mg/kg 7th day 14th day 21st day 28th day C: Glimepiride treated 0.2 60.63 66.46 70.11 73.54 D –Met. ext. treated 50 10.0 15.41 21.97 25.48
E - ,, F - ,, G- ,,
,, ,, ,,
100 200 300
22.61 24.17 27.80
32.82 34.86 40.99
38.53 45.42 48.26
46.29 51.88 53.12
Experimental groups (Groups D-G) were compared with diabetic control group on corresponding day. The above experimental data suggests that the methanolic extract of S. sesban was highly capable in lowering the blood glucose level in alloxan-induced hyperglycemic rats. Thus the folk use of the plant for treatment of diabetes is justified. However extensive pharmacological investigations are needed to elucidate the exact mechanism of the hypoglycemic potential and long term effect of the use of samples in treating diabetes. 10. Hypoglycemic Study with Moringa oleifera 10.1 Materials and its processing Collection of plant materials: The stem bark of M. oleifera was collected from Rajbari and was identified at the Plant Taxonomy Division of Dept. of Botany, University of Dhaka. The chops of bark were dried at room temperature for some days, followed by drying in an oven and were then ground to a coarse powder. Extraction of plant materials: The powdered M. oleifera was soaked in methanol in a closed glass bottle for 7 days. The extractive was filtered byusing a filter paper. The extract so obtained was concentrated under reduced pressure with a rotary vacuum evaporator. Animal and diet: Adult male and female Long Evans rats purchased from Bangladesh Council of Scientific and Industrial Research, Dhaka weighing 120-180 g were used for the study. During the study, all animals were maintained on normal laboratory chow and water ad libitum. Induction of diabetes in rats: Diabetes was induced in rats by injecting intraperitonially a freshly prepared aqueous solution of alloxan monohydrate (150 mg/kg b. w.) after a base line glucose estimation was done. 10.2 Experimental design A total of 36 rats were randomly divided in equal number into 6 groups as following protocol. It is dose dependant study. Group I: Normal untreated rats Group II: Alloxan induced diabetic (AID) rats (150 mg/kg bw) Group III: AID rats given glimepiride orally (200 Âľg/kg bw/day) Group IV: AID rats given plant extract orally (100 mg/kg bw/day) Group V: AID rats given plant extract orally (200 mg/kg bw/day) Group VI: AID rats given plant extract orally (400 mg/kg bw/day)
The study was carried over for 3 consecutive weeks and the body weight was assessed weakly. The blood samples of rats were drawn after an over night fasting (12 hrs) from tail tip at different time intervals i.e., 1st (after 48 hrs of 0 time or base line measure), 7 th , 14th and 21st day for study with plant extract.
Estimation of blood glucose level: The level of blood glucose (sugar) of the experimental and control rats was determined by using standard glucose test kit based on the glucose oxidase method. The blood of every rat was taken on strip by puncturing the tail tip. 10.3 Results and Discussion Diabetic rats showed a significant reduction of body weight in all hyperglycemic rats. The administration of M. oleifera extract improved the body weight significantly (p<0.05) after 21 days as compared to diabetic control groups (Table 3.15). The glucose levels obtained in blood of normal and experimental rats are given in table 3.16 for plant extract. From tables 3.17, it was evident that the methanolic extract of M. oleifera showed a significant (p<0.05) reduction of blood glucose by 16.47%, 21.62% and 22.66% on 1 th for the dosing of samples 100, 200 and 400 mg/kg bw/day respectively, in diabetic rats as compared to the untreated diabetic rats (i.e. after 48 hrs BGL of diabetic induction of respective group). We observed that the glucose lowering activity was higher in higher doses of samples. In the same way, reduction capacity was successively increased day by day and at the end of 21 th day, the highest values were obtained and it was 24.47%, 27.10% and 28..02% for the dosing of samples 100, 200 and 400 mg/kg bw/day respectively, in diabetic rats as compared to the untreated diabetic rats. Moreover, the lowest value (%BGL reduction) was obtained only by 16.47% for 100 mg/kg bw/day at the end of 48 hrs base measuring (1st day) and the highest value was 28.02% for 400-mg/kg b.w/day at the end of 21 th day. Table 3.15: The changes in body weight of treated and untreated rats Body weight in g Dose mg/kg 1st day 7th day 14th day 21st day I- Normal-untreated ------178.1±1.98 182.6±3.60 186.1±2.56 189.86±4.9 II- Alloxan treated 150 184.26±4.32 174.32±3.44 168.09±3.35 158.61±4.1 III Glimepiride treated 0.2 173.34±5.21 171.22±4.8 164.75±5.35 157.38±5.2 IV-Met. ext. treated 100 203±4.45 196.83±1.75 189.26±2.37 180.65±3.8 V- ,, ,, 200 175.77±1.8 169.53±2.28 163.15±3.2 155.09±3.6 VI- ,, ,, 400 186.43±3.5 179.56±3.1 173.45±2.8 165.67±2.4 Values are given in average body weight g±SEM for groups of six animals each. *p<0.05 Groups
Table 3.16: The effect of 3 weeks treatment of ME of M. oleifera on BGL in AID rats. Groups Dose BGL in mmol/L
mg/kg INormal- as untreated reqd II- Alloxan treated 150 III Glimepiride treated IV-Met. ext. treated V- ,, ,, VI- ,, ,,
0.1 100 200 400
After 48 h st 1 day of dosing
7th day
14th day
21st day
4.88±0.49
5.60±0.60
5.32±0.61
4.91±0.52
5.4±0.57
18.35.±1.21
19.22±1.4 0
19.72±0.95 20.60±1.12
19.59±1.73
13.40±1.09 9.43±0.48
18.72±1.46
15.62±1.62 15.09±0.93 14.33±1.6
8.32±1.03
20.12±0.5 3 7.82±1.00 14.14±1.10
19.33±2.27 19.42±1.31
15.15±1.33 14.83±1.62 14.5±0.97 14.1±1.81 15.02±2.17 14.74±1.01 14.11±0.96 13.97±1.52 Values are given as mean ±SEM for six animals in each group. Diabetic control (Group II) was compared with normal group (Group I) on corresponding day.Experimental groups (Groups IV-VII) were compared with diabetic control group (Group II) on corresponding day. *p<0.05; **p<0.001 Table 3.17: Effect of stem bark extracts of M. oleifera on BGL in AID rats. Reduction, % Dose Group mg/kg 1st day 7th day 14th day 21th day III –Drug 0.1 31.60 51.86 57.53 60.08 IV-Sample 100 16.56 19.39 23.45 24.47 V - ,, 200 21.62 23.27 23.99 27.10 VI - ,, 400 22.66 24.10 27.34 28.02 Experimental groups (Groups IV-VII) were compared with diabetic control group on corresponding day. The experimental data has shown that the higher dose of methanolic extract of M. oleifera was moderately capable in reducing the blood glucose level in hyperglycemic rats. 11. Conclusion Water and ethanolic exrtact of A. paniculata, methanolic extracts of A. chinensis, S. sesban and M. oleifera were subjected to investigate the hypoglycemic activity. In the case of glucose-loaded rats; hot water extract of A. paniculata, exhibited the glucose lowering efficacy (41.51%) and ethanol extract by 41.82%. In AID rats blood glucose decreasing efficacy was 46.21% by hot water extract and 45.13% by ethanol extract. After consecutive five days treatment with the 125 mg/kg.bw/day and 250 mg/kg.bw/day of bark extract of A. chinensis, the BGL lowering capability in the elevated blood glucose level were found 38.95% and 40.60%, respectively. Leaves extract (300-mg/kg/day) of S. sesban possess excellent blood glucose reducing properties (53.12%). From table 3.9 & 3.10, it is evident that the methanolic extract showed a significant blood glucose reduction by 25.48%, 46.29%, 51.88%, and 53.12% on 28th day, respectively for 50, 100, 200 and 300 mg/kg bw/day in diabetic rats as compared to the untreated diabetic rats. Bark exact of M. oleifera exhibited moderate hypoglycemic activity in this experiment with maximum fall of blood glucose of 28.02% for 400 mg/kg bw/day at the end 21th day. Diactin (glipizide, marked
drug) and glimepiride (active) were used as positive control in this study. At a dose of 4 mg/kg bw, diactin showed a fall of glucose level by 51.05%, whereas glimepiride exhibit a decrease of glucose level by 60.08% to 73.54% in different experiment. In this study, some of the parameters and doses of extractives are different in some extent. Moreover the experiments with above extractives, two isolated compounds from S. sesban leaves exact, like oleanolic acid and 7-methoxy genistein (isoflavone) are known to possess potential antidiabeic and antioxidant properties (Matsuda et al., 1998, Mapanga et al., 2009). Long-term oral administration of genistein significantly inhibits retinal vascular leakage in experimentally induced diabetic rat (Masami et al., 2001). In in vivo studies also, pretreatment of rats with oleanolic acid displayed significant (p<0.05) antihyperglycemic activity in starch tolerance test however, administration of starch fortified with oleanolic acid to the rats could not exhibit antihyperglycemic activity (Tiwari et al. 2010). Oleanolic acid glycosides exhibit their hypoglycemic activity by suppressing the transfer of glucose from the stomach to the intestine and by inhibiting glucose transport at the brush border of the small intestine [Chem Pham Bull (Tokyo ), 1998].