Antidiabetic and Antioxidant Activities of 70% Ethanol-Diluted Extract of Piper Crocatum Leaves in Stretozotocin Induced Diabetic Rats

Red betel plant (Piper crocatum) has been empirically used to cure diabetes mellitus. However, data regarding this plant are still limited. The aim of this study was to find antidiabetic and antioxidant activities of 70% ethanol-diluted extract of Piper crocatum leaves. Thirty Wistar rats aged 4 months were divided into 6 groups (I-VI) randomly. The groups of I-IV were injected with 40 mg i.p streptozotosin to induce DM. All groups were treated for 21 days. Group I (normal control group) and group II (diabetic control group) were given normal saline, while group III, IV, and V were treated with various doses of 70% ethanol-diluted extract of Piper crocatum leaves (50, 100, 200 mg/kg.b.w/day). Group VI (positive control group) was given glibenclamide 0,9 mg/kg.b.w/day. On day 21, the body weight, fasting blood glucose, plasma MDA, and insulin levels of all rats were examined. The data were processed using statistics to determine the effectiveness of antidiabetic and antioxidant activities of the extract. A significant decrease in fasting blood glucose and plasma MDA levels were observed in group III, IV, and V (p=0,00), when compared to group II (diabetic control group). A significant increase in body weight (p=0,04) and plasma insulin levels (p=0,008) occurred in group V only. In conclusion, seventy percent ethanol-diluted extract of Piper crocatum leaves has potential antidiabetic and antioxidant activities.

DM (11)(12)(13).STZ was freshly dissolved in 0,1M citrate Management of DM without side effects is still a challenge buffer (pH 4,5).The STZ was purchased from Sigma in the medical community.DM therapy with insulin or oral chemical company, St Louis, Missouri, USA.STZ caused hypoglycemic agents have failed to prevent complications degeneration of pancreatic ß cells, the clinical symptoms in some patients (3,4).In addition, the drug ingredient has of DM will appear on two to four days after injection.The been being an imported commodity in Indonesia, so the animals with blood glucose levels exceeding 200 mg/dl drug prices are relatively expensive.Consequently, were considered as diabetic ( 14).ongoing research is needed to find an alternative herbal Animals studied were separated into six groups and given medicine for DM treatment.Traditional medicinal plants treatment for 21 days after the STZ injection, as below: contain mixtures of compounds which may possess -Group I : (Normal Control Group): The rats were given efficacious antidiabetic activity and work synergistically.
normal saline.Besides, the price will be less expensive and profitable, -Group II : (Diabetic Control Group): DM + normal especially for people living in developing countries (5,6).saline Although the traditional treatment of DM in -Group III : DM + 70% ethanol-diluted extract of Piper complementary medicine has been recommended, the crocatum leaves 50mg/kg.b.w/day.mechanism of some herbs has been unknown (7).
-Group IV : DM + 70% ethanol-diluted extract of Piper Traditional antidiabetic medicinal plants were to be crocatum leaves 100mg/kg.b.w/day studied to uncover their abilities to improve -Group V : DM + 70% ethanol-diluted extract of Piper hyperglycemia in DM (4).WHO has strongly emphasized crocatum leaves 200mg/kg.b.w/day.the rational use of traditional and indigenous natural -Group VI : (Positive Control Group): DM + medicines for treating DM (8).glibenclamide 0,9mg/kg.b.w/day Indonesia, as a tropical country, is rich in medicinal plants

Sample Collection potentially efficacious as antidiabetic and antioxidant
After 21 days of treatment, rats were fasted for 12 hours, agents.One of the traditional medicinal plants that has then blood long been believed to be efficacious against DM is leaves of Piper crocatum.Seventy percent ethanol-diluted extract of Piper crocatum leaves contains phytochemical compounds, including some classes of fatty acids, minutes.Plasma samples flavonoids, alkaloids, terpenoids, steroids, pyrimidine, were used for the measurement of glucose, insulin, and essential oils, polyphenols, and vitamin E. The compound TBARS in different studied thought to have antidiabetic and antioxidant activities are polyphenols, alkaloids, flavonoids, terpenoids, and vitamin E (9,10).
Fasting blood glucose levels were measured using the Ethiopathogenesis of DM closely associated with enzymatic colorimetric test "GOD PAP".Lipid peroxide oxidative stress, so the antioxidant agents is necessary in levels were examined by measuring the plasma MDA levels its treatment (7).This study is aimed to examine the using the spectrophotometric method of TBARS (units in antidiabetic and antioxidant activities of 70% ethanolmmol /l).While measurement of plasma insulin levels was diluted extract of Piper crocatum leaves.The antidiabetic performed with Rat/Mouse Insulin ELISA Kit.96-well Plate activity was observed by identifying its effects toward (Cat.# EZRMI-13K).body weight, antihyperglycemia, and stimulation of Statistical Analysis insulin secretion, while the antioxidant effect was examined by measuring the decrease in plasma MDA Data for various biochemical parameters were expressed (malondialdehyde) levels.
as mean ± SD and compared using one way analysis of variance (ANOVA) test.Values were considered statistically METHODS significant when p<0,05.Statistical analysis was conducted using SPSS for Windows version 17.

Extraction of Piper crocatum Leaves
Piper crocatum was extracted in 70% ethanol through a RESULTS percolation method performed at the Laboratory of A significant body weight loss and increase in fasting blood Pharmaceutical Mathematics and Science of Sebelas glucose and plasma MDA were observed in diabetic Maret University.control group when compared to normal control group Animals Tested (Table I).Changes in these parameters indicate the clinical and laboratory DM , so that the animal can be used as a rat Thirty Wistar male rats (body weight of 150-200 grams) model of DM.Administration of 70% ethanol-diluted were maintained at a temperature of 25-30°C and 45-55% samples were collected.Retro-orbital blood samples were collected from the inner canthus of the eye under ether anesthesia using capillary tubes containing sodium fluoride.Plasmas were separated by centrifugation at 3000rpm for 10 groups.

Biochemical Measurements
Jurnal Kedokteran Brawijaya, Vol. 29, No. 1, Februari 2016 extract of Piper crocatum leaves to diabetic rats endogenous antioxidant activity due to hyperglycemia (17).significantly increased the body weight of rats in group V Piper crocatum leaves contain a number of compounds (p=0,04).Significant decrease in fasting blood glucose and possessing antioxidant activities, such as polyphenols, plasma MDA were also observed in group III, IV, and V alkaloids, flavonoids, terpenoids, and vitamin E. This study (p=0,00).However, a significant increase in serum insulin estimated that the antioxidant activity is an important only occurred in group V (p=0,008) (dose 200mg/kg.b.w efficacious factor toward diabetes (3).Polyphenol is a /day).
group of chemical substances in plants.These substances have a lot of phenol groups in their molecules.Some DISCUSSION studies suggest that the group has a role as an antioxidant which is good for health.These polyphenols protect body Streptozotocin (STZ) causes degeneration of the ß cells of cells from free radical damage by binding to free radicals pancreatic Langerhans island, consequently inducing and preventing inflammation processes in the cell body impaired insulin secretion (15).Low dose STZ-induced (18).Provision of antioxidant-rich polyphenol affects to diabetic rat would represent a model for good non-insulinup-regulate antioxidant activity by increasing the dependent diabetes mellitus (NIDDM) experimental concentration of Total Glutathione (GSH), Antioxidant diabetic state and provide a relevant example of Capacity (AOC), and plasma HDL, decreasing plasma MDA, endogenous chronic oxidative stress due to hyperglycemia LDL and hydrogen peroxide (H2O2) levels (19).Some (4).Induction of diabetic animal models was confirmed by flavonoids have also been known to be able to cope with the appearance of DM signs, including body weight loss and some diseases caused by free radicals, as DM (4).increased fasting blood glucose level.Fasting blood glucose levels were examined on day four after injection of 40 Vitamin E also acts as an antioxidant that is quite effective mg/kg.b.w STZ.At this dose, STZ has caused hyperglycemia against hyperglycemia.Vitamin E has a role in (blood glucose >200mg/dl) at groups II to VI.
strengthening insulin activity and glucose metabolism (17,20).Vitamin E can increase the activity of glutathione Injection of STZ led to increased plasma MDA levels (7).
peroxidase (GPX) and Superoxide Dismutase (SOD), and There was an increase in plasma MDA levels in diabetic decrease plasma MDA level (21)(22)(23).Natural antioxidants control group (group II) when compared to the normal from plants have an important role in maintaining cell group (group I) (p=0,00), suggesting thatthat the rats membrane integrity by preventing peroxidation of experienced oxidative stress (Table I).Administration of polyunsaturated fatty acid (18).70% ethanol-diluted extract of Piper crocatum leaves in group III, IV, and V increased body weight (especially in This complete extract contains a mixture of active group V) and decreased blood glucose levels.The compounds.It is difficult to determine the best administration of the extract decreased the plasma MDA component of the extract, in which some of these levels significantly compared to group II (p=0,00).The components possibly affect the glucose metabolism greatest decrease occurs in group V (200mg/kg.b.w/day), synergistically through several mechanisms, such as which is even greater than group VI (positive control through inhibition of carbohydrate digestion and glucose group).These results indicate that 70% ethanol-diluted absorption in the intestine, stimulation of insulin secretion extract of Piper crocatum leaves is able to act as a potential from the pancreatic ß-cells, modulation of glucose release antioxidant material that can reverse oxidative stress from liver, activation of insulin receptors and glucose conditions.It increased insulin secretion significantly at uptake in the insulin-sensitive tissues, and modulation of dose 200mg/kg.b.w/day.It also poses the greatest hepatic glucose output (24,25).hyperglycemia reduction among the groups studied.
This study suggests that 70% ethanol-diluted extract of The generation of reactive oxygen species (ROS) has an Piper crocatum leaves has potential antidiabetic and important role in the development of atherosclerosis and antioxidant activities, especially at dose 200 complications of DM.The exposure of hyperglycemia mg/kg.b.w/day.The antidiabetic activity is able to improve condition to endothelial cells leads to an increase in weight loss, reduce hyperglycemia levels, and stimulate production of superoxide anion, which potentially reduces the secretion of insulin hormone.Besides, the antioxidant nitric oxide levels, a potent endothelium derived activity of this extract also plays an important role in the vasodilator (16).There are two mechanisms that explain mechanism of against DM.This study still requires some the rise of oxidative stress in DM: 1) the rise of free radicals advanced research stage to be applied as an anti-diabetes during auto-oxidation of glucose, 2) reduction of drug in humans .