Anti-hyperglycemic and Anti-hyperlipidemic Effects of Extract from Houttuynia cordata Thumb . in Streptozotocin-Induced Diabetic Rats

Aim: Various properties of Houttuynia cordata Thumb. has been reported. However, few studies on its pharmacological effects have been documented. To elucidate whether there are more pharmacological effects of this plant, this study was therefore, carried out to determine the anti-hyperglycemic and anti-hyperlipidemic effects of 80% ethanol extract of H. cordata (HCE). Their antioxidant activity and acute toxicity were also conducted. Methods: HCE at a dose of 250 mg/kg was oral given to Streptozotocin-induced diabetic rats daily for 8 weeks. DPPH assay and HCE at the doses of 1,000, 2,000 and 3,000 mg/kg were employed in antioxidant and acute toxicity studies. Results: HCE lowered FBG in the diabetic, but not in the normal treated rats. HCE did not affect the body weight of all rats, but recovered TP, Alb, Glob, BUN, CREA, UA, TB, AST, ALT, ALP, and reduced the elevated CHO, TG and LDL in the diabetic rats. HCE possessed relatively low antioxidant activity with IC50 of 115.98± 0.82 μg/mL compared to Vitamin C (42.54+1.37 μg/ml), but did not produce any symptoms of acute toxicity. Conclusions: The extract of H. cordata may have beneficial properties and is a new agent for diabetic treatment and improve renal and hepatic functions.

showed a potential antioxidant and anticancer properties. 17arious plants have been used for the treatment of diabetes.However, there are few scientific reports supporting the antidiabetic properties of H. cordata.The purpose of this study was therefore carried out to investigate the anti-hyperglycemic and antihyperlipidemic effects of the ethanolic extract from H. cordata in streptozotocin-induced diabetic rats.An antioxidant activity of the extract was also evaluated.To see whether the extract is safe for administration, acute toxicity of the extract was examined as well.

Plant Materials
The plant used in this study was H. cordata Thumb., purchasing from the local market in Chiang-mai Province, Thailand.The plant was identified by a Botanist in the department of Biology, Faculty of Science, Mahasarakham University, Thailand and the voucher specimens were deposited in the same University.

Plant Extract
The whole plants were washed thoroughly with tap water and dried at 50°C in an hot air oven.The dried plants (100 g) were powdered and macerated with 80% ethanol (1,000 mL) for 7 days.The mixture was The IC 50 value, the concentration of HCE required for 50% scavenging of the DPPH free radical, was determined from the curve of percentage scavenging plotted against the concentration, was also calculated.Each determination was done in triplicate.

Acute Toxicity Study
The rats were weighed and divided randomly into 4 groups with 6 rats in each; group 1; rats received 0.5% tween 80 (control group), group 2, 3 and 4; rats received 1,000, 2,000 and 3,000 mg/kg HCE respectively.HCE was once administered to the rats orally.Symptoms of toxicity (seizures, vomiting, diarrhea, and nausea) and rat mortality were observed within 24 h and over a further period for 14 days.On day 14, the rats were fasted overnight, weighed and sacrificed by using a cervical dislocation technique.The blood sample was then collected from the rat hearts for a determination of blood biochemistry and hematological values.

Statistical Analysis
All data 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 < 0.05.

Anti-hyperglycemic and anti-hyperlipidemic effects Fasting Blood Glucose Levels
At an initial stage, FBG of the diabetic controls was significantly (p<0.05)higher than that of the normal controls.At the end of the experiments, FBG in normal rats and normal HCE treated rats were not different.However, FBG was significantly (p<0.05)reduced in the diabetic HCE treated rats comparing to that in the diabetic controls (Table 1).

Body Weight
Figure 1, the initial body weight of normal controls, normal HCE treated rats, diabetic controls, and diabetic HCE treated rats were not different.However, at the end of experiments, the body weight of normal controls and normal HCE treated rats were significantly (p<0.05)higher than those of the diabetic controls and diabetic HCE treated rats which was not different.

Blood Biochemistry
As well recognized, blood chemistry; TP, Alb, Glob, BUN, CREA and UA involve renal function, while TB, AST, ALT and ALP involve in hepatic function. 20Table 1, TP, Alb, Glob and UA in normal controls, diabetic controls, normal HCE treated rats, and diabetic HCE treated rats were not different.BUN in the diabetic controls significantly (p<0.05)increased compared to that in normal controls.HCE significantly (p<0.05)reduced BUN in the diabetic treated rats compared to that in the diabetic controls.However, BUN in normal controls and normal rats treated with HCE was not different.CREA significantly (p<0.05)decreased in the diabetic controls compared to that in normal controls.Interestingly, HCE significantly (p<0.05)increased CREA in diabetic treated rats and closed to that in normal controls.TB, AST, ALT and ALP in diabetic controls significantly (p<0.05)increased compared to those in normal controls.HCE significantly (p<0.05)decreased TB, AST, ALT and ALP in the diabetic HCE treated rats.Fortunately, TB, AST, ALT and ALP in normal HCE treated rats did not differ from those in normal controls.

Lipid Profiles
Table 1, CHO and TG in diabetic controls significantly (p<0.05)increased compared to those in normal controls.Interestingly, HCE significantly (p<0.05)decreased CHO and TG in diabetic HCE treated rats.Fortunately, CHO and TG in normal HCE treated rats were not filtered using filter paper.The filtrate was evaporated by using a rotary evaporator (Heidolph Laborota 4000, Germany) and dried by using a freeze dryer (Christ Alpha 1-4, Germany).The obtained 80% ethanolic extract of H. cordata (HCE) was kept at 4°C until be used.

Animals
Animals used in this study were male albino Wistar rats weighing 250-300 g purchased from the National Laboratory Animal Centre, Mahidol University, Thailand.The rats were housed under the conditions of 25 + 2°C, 50 + 5 % RH with a 12 h D/L cycle and maintained with free access to water and instant food for rodent.The experimental protocol and performance of the rats were approved by the Institutional Ethical Committee for the Purpose of Use and Control, and a Supervision on Experiment in Animals, Mahasarakham University, Thailand (License No.0012/2558)

Induction of Diabetes
The rats were injected intra peritoneally with a single dose of 65 mg/kg b.w.streptozotocin (STZ; Sigma Chemicals, St. Louis, MO) dissolved in 20 mM citrate buffer pH 4.5.After the injection, they were provided with 2% sucrose solution for 48 h to alleviate the discomfort after initiating the hypoglycemic phase.The rats with FBG at or above 126 mg/dL were used as the diabetic rats. 18

Study on anti-hyperglycemic and anti-hyperlipidemic effects
The rats were randomly divided into 4 groups with 6 rats in each: group I: normal rats treated with 5% tween 80 (normal control group), groups II: diabetic rats treated with 5% tween 80 (diabetic control group), group III: diabetic rats treated with HCE (250 mg/kg b.w.), and groups IV: normal rats treated with HCE (250 mg/kg b.w.).Prior to the commencement of the experiment, HCE was suspended in 5% tween 80. HCE and 5% tween 80 were oral given to the rats once a day for 8 weeks.The volume of administration was 10 mL/kg b.w.

Determination of Fasting Blood Glucose
The normal and STZ-induced diabetic rats were fasted overnight and sacrificed before the collection of the blood samples from the tail vein of the rats.FBG was measured using Glucometer (Accuchek Adventage II, Roche, Germany).

Antioxidant activity Study
The antioxidant activity of HCE and of a standard solution (vitamin C) were determined using 1,1-diphenyl-2-picrylhydrazyl (DPPH) radical scavenging method. 19A total of 750 µL of HCE and vitamin C was added to 750 µL of DPPH in methanol solution.After incubation at 37°C for 20 min, the absorbance of each solution was determined at 517 nm using UV-VIS spectrophotometer.Corresponding blank readings were also taken and percent inhibition was then calculated as follows: different from normal controls.HDL in diabetic control rats significantly (p<0.05)decreased compared to that in normal controls.HDL in normal control, diabetic controls, normal HCE treated rats and diabetic HCE treated rats were not different.In addition, the difference of LDL in normal controls, diabetic controls, normal HCE treated rats, and diabetic HCE treated rats was not found.

Antioxidant activity
The study on antioxidant activity using DPPH scavenging activity revealed that HCE possessed relatively low potent antioxidant activity with IC 50 of 115.98± 10.82 µg/ml comparing to Vitamin C (42.54 + 1.37 µg/ml).

Acute toxicity study Symtoms of acute toxicity and body weight
All the doses of HCE did not produce any symptoms of acute toxicity or mortality of the rats during 24 h and over a further period lasting 14 days.The initial body weight of all rats was not different.And also at the end of the experiment the final body weight of HCE treated rats did not differ from those in control rats.(Figure 2)

Blood biochemistry
The toxicity study revealed that all the doses of HCE did not produce the alteration of TB, TP, Alb, Glob, BS; BUN, CREA, UA, AST, ALT, and ALP enzymes in the HCE treated rats when compared to those in controls (Table 2).showed that the major chemical components found in H. cordata are essential oil, flavonoid and alkaloids component . 6][13] The obtained results of the anti-hyperglycemic and anti-hyperlipedemic effects of the extract from H.cordata in the present study are in accordance with the effects of various plant extracts and/or plant products such as the 80% ethanolic extract of Pseuderanthemum palatiferum at a dose of 250 mg/kg b.w administered daily and orally to the diabetic rats for 14 days showed hypoglycemic activity by significantly (p<0.05)decreasing levels of fasting plasma glucose (FPG) compared to that in normal controls, 25 the leaf extract from Mimosa pudica which showed high hypo-lipidemic activity by significant (p<0.05)decreasing serum cholesterol, triglyceride and LDL levels, but increasing HDL level when compared to those positive controls (standard drug), 26 the ethanol extract from the whole plant of Mimosa pudica which showed a significant hypo-lipidemic effect by decreasing the lipid profiles such as cholesterol, triglyceride and LDL levels but increasing in HDL level in the serum which was similar to the standard drug. 27These results were also similar to the ethanolic extract of the aerial part of Salvadora oleoides Decne which produced a significant reduction (p<0.001) in blood glucose and also had beneficial effects ( P<0.001) on the lipid profile in euglycemic as well as alloxan-induced diabetic rats, although the reduction in the blood glucose and improvement in lipid profile was less than that achieved with the standard drug tolbutamide. 28The fresh leaf aqueous extract of Clerodendrum capitatum (CC) at the doses of 100, 400 and 800 mg/kg/day administering for 14 days significant (p<0.05,p<0.001) showed the dose dependent hypoglycemic and hypolipidemic effects in the treated rats. 29nd flower extract from Sphagneticola trilobata (L.) Pruski at the dose 250 mg/kg administering for 4 weeks significant (p<0.05)reduced the blood glucose levels, total cholesterol and low density lipoprotein in the diabetics but this is not in the normal rats.However, the extract had no effect on high density lipoprotein and triglyceride. 30HCE showed any

Hematological values
Hematological values including Hct, Hb, RBC, and WBC in HCE treated rats were not different from those in control rats.(Table 2).

DISCUSSION
The present study was designed to investigate the anti-hyperglycemic and anti-hyperlipidemic effects of Houttuynia cordata Thumb, and to support its traditional use for the treatment of diabetes.In the present study, STZ (65 mg/kg bw.) was selected in order to destroy the pancreatic β-cells, and consequently, the STZ-injected rats become the diabetic rats.HCE 250 mg/kg bw possessed a significant anti-hyperglycemic effect shown in the present study as it significantly reduced FBG in the diabetic treated rats.Unfortunately, HCE had no effect in normal rats, since FBG in the normal rats treated with HCE did not differ from that in normal controls.Previous study on H. cordata revealed the presence of essential oil in H. cordata and it has been shown to have an effect on insulin resistance of diabetes mellitus. 9,10Flavonoids, alkaloids, saponin, tannin, and triterpinoids have been reported to possess hypoglycemic activity. . 21herefore, the mechanism underlying the anti-hyperglycemic effect of this plant is partly due to the chemical compositions such as essential oil, alkaloids or flavonoids presence in H. cordata.
In the present study, the extract from H. cordata also showed the antihyperlipidemic effect as HCE significantly reduced CHO and TG which were increased in the diabetic control rats.In contrast, HCE slightly increased HDL which was reduced in diabetic control rats.β-myrcene, was frequently found in H. cordata 22 Lemongrass (Cymbopogon flexuosus) whose essential oil consists of equal amounts of myrcene and citral. 23emongrass intake provides a beneficial effect of reducing the blood cholesterol level. 24The underlying mechanism (s) on hypolipidemic effect of the extract from H. cordata may due to chemical constituent such as β-myrcene presence in H. cordata.HCE possesses an antioxidant activity but was less potent than Vitamin C. The antioxidant activity of HCE found in the present study is in line with the previous study which According to Duncan's multiple range test, values within the same column TP = total serum protein; Alb = albumin; Glob = globulin; BUN = blood urea nitrogen; CREA = creatinine; UA= uric acid; TB= total bilirubin; AST = serum aspartate aminotransferase; ALT = serum alanine aminotransferase; ALP = alkaline phosphatas, Hct = hematocrit, Hb = hemoglobin, RBC = red blood cells, WBC = white blood cell symptoms of acute toxicity in the present study suggesting that it is safe for administration.

CONCLUSION
In conclusion, H. cordata can be used as traditional medicine for diabetic treatment.HCE at a dose of 250 mg/kg shows a beneficial anti-hyperglycemic and anti-hyper-lipidemic effects in diabetic rats.HCE also improves the renal and hepatic functions resulting from diabetic state.HCE at the dose up to 3,000 mg/kg, did not produce any signs or symptoms of an acute toxicity and mortality of the rats throughout the period of observation for 24 h and a further period lasting 14 days, indicating HCE has no acute toxicity.Consummation of H. cordata may be a good resource for reducing a risk of diabetes, coronary heart diseases (CHD) and/or atherosclerosis.For further studies, isolation and investigation of the chemical constituents of H. cordata responsible for the anti-hyperglycemic and anti-hyperlipidemic effects should be undertaken in order to elucidate the underlying mechanism(s) of these activities.
%inhibition = [(Absorbance control-Absorbance of test sample)/ Absorbance of control)] × 100 where A blank is the absorbance of the control reaction and A sample is the absorbance of the test compound Pharmacognosy Journal, Vol 9, Issue 3, May-Jun, 2017

Figure 1 :
Figure 1 : Body weight in normal and STZ induced diabetic rats.Figure 2 : Body weight of the rats treated with HCE 1,000, 2,000 and 3,000 mg/kg) and controls from acute toxicity study

Figure 2 :
Figure 1 : Body weight in normal and STZ induced diabetic rats.Figure 2 : Body weight of the rats treated with HCE 1,000, 2,000 and 3,000 mg/kg) and controls from acute toxicity study