Effect of Thai Folklore Recipe from Abutilon indicum and Mimosa pudica in Streptozotocin-Induced Diabetic Rats

Cite this article: Konsue A, Talubmook C. Effect of Thai Folklore Recipe from Abutilon indicum and Mimosa pudica in Streptozotocin-Induced Diabetic Rats. Pharmacog J. 2018;10(3):480-85. ABSTRACT Context: Abutilon indicum and Mimosa pudica were a folklore recipe in Northeastern of Thailand. The recipe was reported that claim to diabetic treatment. Aims: The studies were evaluated to hypoglycemic effect, serum insulin secretion and blood biochemistry in streptozotocin (STZ)-induced diabetic rats. Materials and Methods: The recipe were composed of whole plants from A. indicum and M. Pudica (1:1 w/w) powder. The pound plants were macerated with aqueous (AMA), hydro-ethanol (AMHE) and 80% ethanol (AME) to crude extracts. The AMA, AMHE and AME at the doses of 125, 250 and 500 mg/kg body weight (b.w.) were administered orally daily in diabetic rats during eight weeks. Fasting blood glucose levels (FBG) were measured at weekly. The serum insulin levels and blood biochemical data including blood urea nitrogen (BUN), creatinine (CREA), total protein (TP), albumin (Alb), serum aspartate aminotransferase (AST), serum alanine aminotransferase (ALT), alkaline phosphatase (ALP), triglycerides (TG), total cholesterol (TC), high-density lipoprotein (HDL) and low-density lipoprotein (LDL) were estimated at the end of experiment. Results: All doses of the extracts were showed significantly (p<0.05) decreasing percent age of FBG in diabetic rats. Especially, AME 125 mg/ kg b.w. was showed more potent significantly (p<0.05) decreasing percentage of FBG at week of 2, 5, 7 and 8. The serum insulin levels of all doses administered with the extracts were significantly (p< 0.05) higher than diabetic control group. On the other hand, all doses of the extracts were significantly (p< 0.05) decreasing ALT and ALP lower than diabetic control group. While, AMA and AMHE at the doses of 250 and 500 mg/kg b.w. were increased HDL, but decreased TC, TG and LDL. Conclusion: The study was proved to diabetic treatment and improvement of diabetic stage and blood biochemical parameters. In addition, the experiment was confirmed to folklore traditional use.


INTRODUCTION
Diabetes mellitus (DM) is a group of chronic metabolic disease characterized by hyperglycemia resulting from defects in insulin secretion, often combined with insulin action, or both.The chronic hyperglycemia of diabetes is associated with long-term damage, dysfunction and failure of various organs and systems. 1 Diabetes requires continuous medical care and patient self-management education in order to prevent acute complications and to reduce the risk of long-term complications. 2 Oral anti-diabetic agents are not only expensive, but also have undesirable side effects or contraindications. 3Diabetic patients often seek complementary therapy such as herbal medicines.In Thailand, several herbal medicines have been documented support as diabetic treatment. 4any Thai medicinal plants have been used as the traditional medicines for management of diabetes.A Thai folklore recipe consist with M. pudica (MAI-YA-LAP) and A. indicum (KHOB-FUN-SRI) have also been reported as being effective in diabetic treatment.
M. pudica or the sensitive plant is a medicinal plant belonging to the family Fabaceae.The plant was found to grow in undisturbed shady areas, under trees or shrubs. 5The most important biologically active constituents of this plants are alkaloids, steroids, tannins, flavonoids, glycosides and saponins compounds. 6The wide range of pharmacological activities of this plant have been reported including wound healing, 7,8 analgesic, anti-inflammatory, 9 anticonvulsant, 10 anti-diarrhea, 6,11 anti-oxidant, 5,9,12,13 hepatoprotective, 13,14 hypolipidemic, 15,16 diuretic property, 17 hypoglycemic 5,18-20 and hyperglycemia activities. 21. indicum Sweet is belonging to the family Malvaceae.The plants is an erect, woody, shrubby plant, widely distributed in the tropical countries and has a long medical history of being used as an anti-diabetic remedy. 22The components contained the natural substances such as amino acids, aliphatic compounds, sugars, polyphenols, flavonoids, alkaloids and glycosides. 23The plant is also reported to possess analgesic, 24 hypoglycemic, [25][26][27][28][29] hepatoprotective 30 and antibacterial activity. 31With respect to its traditional use in Thai traditional recipe, A. indicum and M. pudica should contain an active ingredient against diabetes and was believed to reduce some symptoms of diabetic complications. 32owever, any signs or symptoms on diabetic treatment has not yet been demonstrated.Therefore, the purposes of this study were designed to determination of hypoglycemic effect and blood biochemical parameters using an different doses of a Thai folklore recipe that consist both whole plants of A. indicum and M. pudica in diabetic rats.

Collection of plant materials
The fresh of whole plants (roots, stems, leaves, flowers and fruits) were harvested from Kalasin Province, Northeastern of Thailand.It was deposited at the Faculty of Medicine, Mahasarakham University, Thailand (code :: M. Pudica as MSU.MED-MP0001/AK ; A. Indicum as MED-AI0001/AK).The fresh plants were dried at 50°C for 48 hr in a hot air oven then powdered.

Preparation of crude extracts
The aqueous extracts (AMA) were prepared by boiling dried plant powder from A. indicum and M. pudica (1:1 w/w) in distilled water (1:10 w/v) at 100 o C for 30 min for 3 times.The hydro-ethanolic (AMHE) and ethanolic (AME) extracts were prepared by macerating dried plant powder in 50% ethanol and 80% ethanol (1:5 w/v) for 7 days.The residue powder was excluded by using the filter papers.The extract was dried using a rotary evaporator (Heidolph Laborota 4000, Germany) followed by a freeze dryer (Christ Alpha 14, Germany) to get obtain dark brown extracts.The extracts were kept in the fridge at 4°C until be used.

Preparation of animals
Healthy adult male albino Wistar rats weighing between 150-200 g purchasing from the National Laboratory Animal Centre, Mahidol University, Thailand were used in this study.They were acclimatized in an air conditioned room at 23±2 °C, 12 h light/12 h dark cycle (350-400 Lux) and relative humidity of 50-55%, and given with a standard chow and watered ad libitum for 7 days prior to the commencing experiment.The rats were maintained in accordance with the guidelines of the Committee Care and Use of Laboratory Animal Resource, National Research Council Thailand and approved in accordance with the advice of the Institutional Animal Care and Use Committee, Mahasarakham University (IACUC-MSU), Thailand (License No. 0005/2017).

Induction of diabetic rats
The animals were injected intra-peritoneally with a single dose of 65 mg/ kg b.w.STZ (Sigma Chemicals, St. Louis, MO) dissolved freshly in cold 20 mM citrate buffer (pH 4.5). 33After injection, they were provided with 2% sucrose solution for 48 h to alleviate the discomfort after initiating the hypoglycemic phase.Three days after injection, the rats were examined for FBG levels to confirm their diabetic stage.The rats showing FBG levels greater than 126 mg/dL were used in the experiments. 34

Experimental designs
The animals were randomly divided into the following twelve experimental groups with eight animals in each.Group I was normal control rats treated orally with 2% Tween 80; group II was diabetic control rats treated orally with 2% Tween 80; group III was diabetic rats administrated orally with glibenclamide 0.5 mg/kg b.w.; group IV-VI were diabetic rats administrated orally with AMA at the doses of 125, 250 and 500 mg/kg b.w.; group VII-IX were diabetic rats administrated orally with AMHE at the doses of 125, 250 and 500 mg/kg b.w.; group X-XXII were diabetic rats administrated orally with AME at the doses of 125, 250 and 500 mg/kg b.w.The extracts and glibenclamide were suspended in 0.5% Tween 80 and administered orally once a day for 8 weeks using an orogastric tube.The volume of administration was 1 mL for each animal.

Determination of fasting blood glucose
The rats were fasted overnight and then the blood samples were collected from the tail vein of the rats.FBG was measured weekly for 8 weeks with Glucometer (Accu-chek Performa, Roche, Germany).

Determination of serum insulin and blood biochemical data
After 8 weeks of treatment, the rats were fasted overnight.They were sacrificed by cervical dislocation technique, and then blood samples were drawn from the rat's heart.The blood samples were centrifuged at 3500 rpm for 20 min to separate blood serum.The serum insulin levels was estimated using the radioimmunoassay kit (MP Biomedicals-Orangeburg, USA) and detected by an automatic gamma counter (Wallac 1470 Wizard, Perkin Elmer Instrument, Germany).The blood biochemical parameters include total cholesterol (TC), triglycerides (TG), high-density lipoprotein (HDL), low-density lipoprotein (LDL), blood urea nitrogen (BUN), creatinine, total protein, albumin, serum aspartate aminotransferase ( ALT), serum alanine aminotransferase (ALT) and alkaline phosphatase (ALP) which were measured by an automatic blood chemical analyzer (BT 2000 plus, Germany).

Statistical analysis
The results were expressed as mean±standard error of mean (SEM).Statistical analysis was carried out using One-Way ANOVA followed by Duncan's new multiple range test.The criterion for statistical significance was at a p-value less than 0.05.

Fasting Blood Glucose Levels
Table 1 shows the effect of various doses of AMA, AMHE and AME from Thai folklore recipe on percentage decreasing of FBG levels in rats.AME at the dose of 125 mg/kg b.w.showed significant highest percentage reduction of FBG levels in diabetic rats especially at the week 5, 7 and 8 which was greater than antidiabetic drug, glibenclamide and diabetic control groups.While, AMHE at the dose of 250 mg/kg b.w.produced a significant effect in lowering FPG which was showed at week 2 (12.22%) and 4 (9.31%)respectively.Pleasingly, at the week 4, AMA at the dose 500 mg/kg b.w. and AME at the dose 125 mg/kg b.w were significant decreasing percentage of FBG higher than normal control, diabetic control and glibenclamide.

Serum Insulin Levels
Figure 1 shows the effect of various doses of AMA, AMHE and AME from Thai folklore recipe on serum insulin levels in rats after 8 weeks of administration.All doses of AMA, AMHE and AME showed serum insulin levels significant similar anti-diabetic drug, glibenclamide and were more potent higher than diabetic control group.

Blood Biochemistry
Table 2 shows the effect of various doses of AMA, AMHE and AME from Thai folklore recipe on the blood biochemistry : renal functions (BUN and CREA) and liver functions (TP, Alb, AST, ALT and ALP).AMA at the dose of 500 mg/kg bw and AMHE at the dose of 250 mg/kg bw showed significantly (p<0.05)reducing BUN compared to the diabetic control group.All the doses of AMA and AMHE (125 and 250 mg/kg bw) were decreased CREA in diabetic rats, On the other hand, all the doses of AME and AMHE 500 mg/kg bw were increased when compare with diabetic control and glibenclamide groups.The liver function data showed that TP and Alb in diabetic rats administered at the all doses of extract were not different from diabetic control rats.The AST in rats at the doses of AMA and AMHE 500 mg/kg bw were potent significantly Pharmacognosy Journal, Vol 10, Issue 3, May-Jun, 2018 The results show the mean ± SEM. a represents statistical significance (P < .05)compared with normal control.b represents statistical significance (P < .05)compared with diabetic control.c represent statistical significance (P < .05)compared with DM GB 0.5 mg/kg.lowering than diabetic control and glibenclamide rats.Especially, ALT in rats at the doses of AMHE 500 mg/kg bw showed produced similar to normal control rats.Moreover, all the doses of extracts were showed significantly decreasing ALP different from compare group, diabetic control and glibenclamide rats.

Lipid Profiles
Table 3 shows the effect of various doses of AMA, AMHE and AME from Thai folklore recipe on lipid profiles (TC, TG, HDL and LDL).All doses of the extracts were more potent significantly increasing HDL, decreasing LDL different from diabetic rats.TG in rats administered with all of the extracts exclude AME 250 and 500 mg/kg bw showed significantly (p<0.05)lower than the diabetic group which was almost equal to glibenclamide.
Only dose of AME 250 mg/kg bw was produced significantly decreasing TC different from diabetic control and glibenclamide.

DISCUSSION
The present study was designed to investigate hypoglycemic effect of the Thai folklore recipe which contain of A. indicum and M. Pudica (1:1 w/w).The experiments were treated to comparing with current traditional medicine, anti-diabetic drug, glibenclamide to promote its traditional use for diabetic treatment.Furthermore, it was also tested for its any side effect or symptom on the renal functions, liver functions and lipid profiles in the rats.STZ (65 mg/kg bw) was selected in order to partially destroy the pancreatic β-cells.Under these conditions, insulin was secreted but not in an amount sufficient to regulate blood glucose levels and consequently, the rats became permanently hyperglycemic stage.STZ is a broad-spectrum antibiotic that separated from Streptomyces acromogenes.STZ has the cytotoxic effect on β-cells of pancreas. The result showed that AM had a significant FBG reduction observed in STZ-induced diabetic rats by decreasing blood glucose levels but not in a dose dependent manner.AME at the dose of 125 mg/kg bw showed higher effect than glibenclamide (0.5 mg/kg bw) especially at the 2, 5, 7 and 8 weeks.This result shows that AME at the dose of 125 mg/kg bw is approximately dosage which may be absorb into rat's digestive system.This assumption was proved in a further study by testing with various doses of AME below 125 mg/kg bw.8][39] The mechanism of this effect is probably to regeneration of pancreatic β-cells which are destroyed by streptotozin. 35The recipe, A. indicum and M. Pudica, the chemical components of the plants were revealed that any phytochemical of these composed with some antidiabetic substances including alkaloids, flavonoids, glycosides, saponins and tannins. 6,23,35Saponins and alkaloids could be inhibit glucose uptake, while flavonoids could be protect various cell types from oxidative stressmediated cell injury. 36The flavonoids such as kaempferol as well known chemical compound which improve insulin stimulating glucose uptake in mature 3T3L1 adipocytes and protect the body against free radicals, it is biologically plausible relation between flavonoids and low risk of diabetes. 37Glycosides could be stimulated insulin secretion as well. 25These chemical substances in plants may be responsible for the anti-diabetic effects were observed in the study.Moreover, the blood biochemical data were found that improve some renal and liver function such as BUN, AST, ALT and ALP. 385] Our data showed improvement of lipid profiles which increased HDL, decreased TC, TG and LDL in the diabetic rats. 15,16,41

CONCLUSION
The study confirms the traditional uses of Thai folklore recipe from M. pudica (MAI-YA-LAP) and A. indicum (KHOB-FUN-SRI) to improvement of diabetes.The recipe also prevents complications resulting from diabetes and improve the blood biochemistry.Further studies, isolations, purifications and investigation of the chemical constituent(s) of both plants responsible for the hypoglycemic effect should be undertaken in order to confirm and clarify the mechanism behind this activity.

Figure 1 :
Figure 1: Effect of Thai folklore recipe at 8 weeks serum insulin levels in animal.The results show the mean ± SEM. * Represents statistical significance (p < 0.05) compared with normal control.# Represent statistical significance (p < 0.05) compared with diabetic control.X Represent statistical significance (p < 0.05) compared with DM GB 0.5 mg/kg.

Table 3 : Effect of Thai folklore recipe on Lipid profiles in rats. Treatments and doses Lipid profiles (mg/dL)
b represent statistical significance (p<0.05)compared with diabetic control.c represent statistical significance (p< 0.05) compared with DM GB 0.5 mg/kg.