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Stevia rebaudiana regulates blood sugar, prevents hypertension and tooth decay. Other studies have shown that it has antibacterial as well as antiviral property.
Preliminary phytochemical screening of aqueous, ether and methanolic extracts of S. rebaudiana was done. Acute and sub-acute toxicity were conducted on twenty four Albino rats, divided into one control (Group I) and three treatment groups viz. aqueous extract (Group II), ether extract (Group III) and methanolic extract (Group IV). For the study of antidiabetic effect of S. rebaudiana rats were divided into seven groups (n=6). Diabetes was induced by a single dose of 5% alloxan monohydrate (125 mg/kg, i.p.) after 24 hour fasting.Blood samples were analysed on day 0, 1, 5, 7, 14 and 28.
Phytochemical tests showed presence of different kinds of phyto-constituents in aqueous, ether and methanol extract of Stevia rebaudiana leaves. Daily single dose (2.0 g/kg) administration of aqueous extract (A.E.) , ether extract (E.E.) and methanol extract (M.E.) for 28 days of S. rebaudiana could not show any significant change in ALT and AST levels in rats. Blood sugar level was found to be decreased on day 28 in groups of rats treated with A.E., E.E. and M.E. of S. rebaudiana.
The extracts of Stevioside rebaudiana could decrease the blood glucose level in diabetic rats in time dependent manner.
Stevia rebaudiana Bertoni commonly known as sweet leaf is a perennial shrub and is a member of Asteraceae family. It is native to the valley of the Rio Monday in highlands of Paraguay, between 25 and 26 degrees south latitude where it grows in sandy soils near streams. Its medicinal use includes regulating blood sugar, preventing hypertension, treatment of skin disorder, and prevention of tooth decay. It also possesses antibacterial and antiviral properties. Standard extracts of S. rebaudiana are used as natural sweetener or dietary supplements in different countries for their content of stevioside or rebaudioside A. These compounds possess upto 250 times the sweetness intensity of sucrose and they are noncalorigenic.
The principles of S. rebaudiana are due to natural sweet active components present in the leaves that is stevioside and rebaudiosides A, B, C, D, and E; dulcoside A; and steviolbioside. Stevioside has a slight bitter aftertaste and provides 250–300 times the sweetness of sugar. The sweet diterpenoid glycoside, rebaudioside F has been isolated from leaves and its structure was established by chemical and spectral studies.[2,3]
In Japan, cultivation of stevia is done as an alternative to artificial sweeteners such as cyclamates saccharine, which are suspected carcinogens. The plants leaves, the aqueous extract of leaves, and purified steviosides are used as sweeteners. Japan currently consumes more stevia than any other country, with stevia accounting for 40% of the sweetener market. Today stevia is cultivated and used in food elsewhere in East Asia, including in China, Korea, Thailand, and Malaysia. China is the world's largest exporter of stevioside.
In US, stevia is mostly employed as sugar substitute. About one-fourth teaspoon of the natural ground leaves is equivalent to one teaspoon of sugar. In South America, a standard infusion is sometimes used as a natural aid for diabetes and hypertension. The difference between stevia and sugar is that stevia does not cause tooth decay. It has been reported that stevia kills the bacteria Streptococcus mutans, which is the prime factor in teeth plaque.
Active principles of many plant species are isolated for direct use as drugs, lead compounds, or pharmacological agents. Different species of medicinal plants are used in the treatment of diabetes mellitus. For diabetes treatment, before the discovery of insulin, the only options were those based on traditional practices. Till today, metformin is the only ethical drug approved for the treatment of noninsulin-dependent diabetes mellitus patients, which is derived from a medicinal plant Galega officinalis. Among those plants used traditionally for the treatment of diabetic complications is S. rebaudiana Bertoni. Hence, the present experiment was undertaken to study the antidiabetic effect of S. rebaudiana in rats.
Rats of Wister strain (180–200 g) of both sex and guinea pigs were used in this experiment after approval of the protocol by Institutional Animal Ethics Committee. Rats were kept in cages (2–3 rats per cage) under standard laboratory conditions (light period of 12 h per day and temperature 27 ± 2°C). They were fed standard pelleted feed and access to water ad lib. The rats were acclimatized to the animal house conditions. Prior to each study, the animals were made to fast for 12–14 h but had free access to water.
Fresh mature leaves of authenticated S. rebaudiana leaves were obtained from Directorate of Research, BAU, Kanke, Ranchi. It was air-dried under shed at room temperature and finely powdered with the help of grinder. Leaves powders were always prepared fresh for aqueous, ether, and methanolic extraction.
Standard screening tests of three extracts were carried out for various plant constituents. The crude extracts were screened for the presence or absence of secondary metabolites such as alkaloids, steroidal compounds, phenolic compounds, flavonoids, saponins, tannins, and anthraquinones using standard procedures.
Froth test – 0.5 g extracts were dissolved in 10 ml of distilled water in a test tube. The test tube was stoppered and shaken vigorously for about 30 sec. The test tube was allowed to stand in a vertical position and observed over a 30-min period of time. If a “honey comb” froth above the surface of liquid persists after 30 min. the sample is suspected to contain saponins.
The acute oral toxicity studies of all the three extracts were undertaken as per the Organization for Economic Co-operation and Development (OECD) guidelines for testing of chemicals by up-and-down procedure. The rats were fasted overnight and the weight of each rat used was recorded just before use. Animals were divided randomly into a control and three treatment groups for each extract, each group consisting of four mice (two males and two females). Control group received only the vehicle and each treatment group received orally the EE , ME, and AE of the studied plant in the limit test at a rate of 2000 mg/kg body weight was conducted and terminated after four survivals out of four animals.
Again a higher dose of 5000mg/kg of all extracts were given to three groups of rats. Animals were kept under close observation for 4 h after administering the extracts, and then they were observed daily for 3 days for any change in general behavior and other physical activities
Subacute toxicity of AE, EE, and ME of S. rebaudiana leaves was studied in albino rats of either sex (n = 24). Rats were divided randomly into four groups. Group I (n = 6) served as control and the other three groups were used as experimental groups. Group II, AE (n = 6) III, EE (n = 6), and IV, ME (n = 6) were given 2 g/kg, i.p. of S. rebaudiana leaves per day for 4 weeks. The blood samples were collected on day 0, 14th, and 28th by heart puncture after anesthetizing the rats by ethyl alcohol. The biochemical parameters (ALT and AST) were measured by kit supplied by ERBA chemicals on semiautoanalyzer.
Diabetes in rats was induced by a single dose of 5% alloxan monohydrate (125 mg/kg, i.p.) after 24 h fasting. Induction of diabetes was confirmed after a week of alloxan treatment by estimation of fasting blood glucose level. Only those rats with blood glucose level between 200–300 mg/dl were included in the study. These rats were further divided into seven groups (I-nondiabetic control; II-diabetic control; III-Aqueous extract, IV-Ether extract, V-Methanolic extract, VI-Glibenclamide, VII-Glibenclamide + Aqueous extract) of six rats each. Groups III–V were subgrouped (IIIA, IIIB, IVA, IVB, VA, VB). Groups I and II (control) received comparable volume of NSS. Groups III–V received lower and higher daily doses of AE, EE, and ME at a rate of 50 and 100 mg/kg p.o., respectively, once daily for 4 weeks. VIth group was administered hypoglycemic drug glibenclamide (5 mg/kg, p.o.) once daily for 4 weeks and group VII was administered daily dose of glibenclamide (50 mg/kg) and 100 mg/kg AE, p.o., respectively. The blood glucose levels were measured by glucometer on day 0, 1, 5, 7, 14, and 28. The blood samples were collected from tail vein puncture and blood glucose levels were analyzed.
In order to determine the presence of chemical constituents, phytochemical tests were performed, which revealed the presence of phytoconstituents in aqueous, ether, and methanol extracts [Table 1], which is in consonance with the report ofRef.
A preliminary toxicity study was designed to demonstrate the appropriate safe dose range that could be used for subsequent experiments rather than to provide complete toxicity data on the extract. Acute toxicity studies conducted revealed that the administration of graded doses of three crude aqueous, ether, and methanol extracts (up to a dose of 5000 mg/kg) of S. rebaudiana did not produce significant changes in behaviors such as alertness, motor activity, breathing, restlessness, diarrhea, convulsions, coma, and appearance of the animals. No death was observed up to the dose of 5 g/kg body weight. The mice were physically active. These effects were observed during the experimental period (72 h). The result showed that in single dose, the plant extracts had no adverse effect, indicating that the medium lethal dose (LD50) could be greater than 5 g/kg body weight in mice. Search for the available literature revealed the nontoxic effect of the leaves of S. rebaudiana in mice.
[Table 2] shows the mean concentration of ALT and AST. ALT was estimated to be 40.00 ± 1.40, 40.33 ± 1.39, 40.21 ±1.23 unit/ml in AE-, EE-, and ME-treated groups, respectively. On 28th day, it was found to be 40.75 ± 1.42, 39.75 ± 0.97, and 40.15 ± 1.00 unit/ml in all the three groups. No significant difference in the mean concentration was found.
The AST level was recorded as 20.15 ± 1.33, 20.26 ± 1.35, 20.75 ± 1.15 in AE, EE, and ME, respectively. On day 28, the level of AST was recorded as 20.12 ± 1.00, 20.22 ± 1.11, and 20.16 ± 1.10 units/ml in AE, EE, and ME, respectively. Mean values did not show significant difference on day 0 and 28.
The blood glucose levels were 220.16 ± 8.63, 220.00 ± 11.20 mg/dl in AE-, 209.66 ± 4.15, 220.83 ±09.24 mg/dl in EE-, 218.66 ±4.93, 232.00 ± 11.81 mg/dl in ME-treated rats on day 0 [Table 3]. In the glibenclamide-treated group, the blood glucose was 211.00 ± 5.10 mg/dl on day 0, whereas in glibenclamide + AE treated rats the blood glucose level on day 0 was found to be 208.16 ± 9.23 mg/dl. It may be noted in the above table that a significant decrease in the mean blood glucose levels was found on day 28 in AE-, EE-, and ME-treated rats, both after 50 and 100 mg/kg daily dose administration. The results obtained in this study for extracts of S. rebaudiana showed decrease in the mean blood glucose levels which were in agreement with the observations of Abdula et al.
The study also showed that the rats which had been given the extracts of AE and EE at higher dose (100 mg/kg) exhibited greater decrease in mean blood glucose level as compared to those given at a rate of 50 mg/kg b.w. on day 28. Therefore, it is obvious from the results obtained in this study that antihyperglycemic activity of AE and EE were dose-dependent. The findings obtained in this investigation are similar to that of Jeppesen et al., who pointed that stevioside and steviol dose dependently enhanced insulin secretion. The data showed that there was significant decrease in the mean blood glucose level (100.50 ± 0.22) in the group VII where AE was given with glibenclamide. However, it did not differ with the blood glucose level on day 28 (101.83±0.30) as compared to that group which received only glibenclamide. Therefore, it is obvious that glibenclamide and AE both are working differently in rats.
The stevia leaves powder has also been reported to reduce the blood glucose concentration of diabetic rats. The findings of this experiment are similar to the reports of Chang et al. However, it was observed that as hypoglycemic drug, glimepiride, was better, though powdered form of stevia (S. rebaudiana Bertoni) leaves at a rate of 250 mg/kg body weight showed very potent hypoglycemic efficacy, but comparatively less effective than glimepiride. It is known that sulfonylureas like glimepiride produce hypoglycemia by increasing the secretion of insulin from the pancreas and these compounds are active in mild streptozotocin-induced diabetes, whereas they are inactive in intense streptozotocin diabetes (nearly all β-cells have been destroyed).[14,15] Since our results showed that glimepiride reduces the blood glucose levels in hyperglycemic animals, so it can be postulated that the state of diabetes was not severe. It may be mentioned that stevioside regulates blood glucose level by enhancing insulin secretion and also enhances glucose utilization in peripheral tissues and muscles in diabetic rats.
It was concluded that the extracts of S. rebaudiana could decrease the blood glucose level in diabetic rats in time-dependent manner. The antidiabetic effect might be due to steviosides counteracting the glucotoxicity in β-cells or also by suppressing the glucagon secretion by α-cell of pancreas; both the mechanisms have been depicted by Shibata et al. and Chen et al.
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Conflict of Interest: None declared.