Antidiabetic and Antihyperlipidemic Activities of Methanolic Leaf Extract of Stephania japonica in Alloxan Induced Diabetic Rats

This present study was aimed to investigate the antidiabetic and antihyperlipidemic activities of methanolic leaf extract (LE) of Stephania japonica alone and in combination with metformin in alloxan induced diabetic rats. Primarily acute toxicity study and oral glucose tolerance test were performed. Diabetes was confirmed after 12 days of single intraperitoneal injection of alloxan (120 mg/kg BW) in albino male rats. Rats were divided into six groups’; normal control (Group I) and diabetic induced groups as (Group II, III, IV, V and VI). Group III & IV were treated with leaf extract of S. japonica (200 mg/kg BW & 350 mg/kg BW). Group V (Met 850 mg/70 kg BW) and group VI: (combination of Met 425 mg/70 kg BW and LE 250 mg/kg BW) for four weeks. Body weight of each rat in the different groups was recorded at 0, 7, 14, 21 and 28 day of treatment. TC, TG, LDL-C and HDL-C were measured analytically after 28 days of treatment. Alloxan induction also caused left ventricular hypertrophy. LE of S. Japonica showed a good result in OGTT. Oral treatment of different doses of LE and combination therapy reduced elevated level of BG, TC, TG, LDL-C and increased HDL-C level significantly (p < 0.001) but extract (350 mg/kg BW) therapy was the most effective. Moreover, LE reduced LVH and improved BW of each rat. Extract from S. japonica (Thunb.) Miers showed antihyperlipidemic and antidiabetic effect and hence could be suggested as a potential therapeutic agent for diabetic treatment.


Introduction
Diabetes mellitus (DM) is a chronic metabolic disorder that occurs when the body cannot produce sufficient insulin or cannot utilize insulin effectively.Early symptoms concern hyperglycemia and encompass excessive thirst, excessive eating, production of excessive amount of dilute urine and blurred vision.Later complications encompass an abnormal condition of the blood vessels, peripheral neuropathy, nephropathy and tendency to suffer from infection.Almost every organ system in the body can be affected by diabetes mellitus [1].
According to World Health Organization there were 171 million people with diabetes throughout the world in the year 2000 and this is often predicted to extend to 366 million by 2030 [2].The American Diabetes Association (ADA) estimated the national expenses of diabetes in the USA for 2002 to be US132 billion, increasing to US192 billion in 2020 [3].Recently, biguanides, thiazolidinediones, sulfonylureas, D-phenylalanine and α-glucosidase inhibitors along with insulin are extensively used in the treatment of diabetes by a sustained reduction in hyperglycemia.However, due to enormous unwanted side effects and in addition to high cost of allopathic drugs the effectiveness of these compounds are not acceptable and there is an increasing demand for both effective as well as safer drug compounds for the treatment of diabetes Mellitus [4] [5].To avoid side effects of insulin and oral hypoglycemic agents, patient's interest are expanding to use traditional plants with antidiabetic activity [6] [7] [8].
It is generally found in native to eastern and southern Asia and Australia.In Bangladesh, it is grown in all over the country.The whole plant is bitter in test and the leaves and roots of these plants are used in fevers, diarrhoea, dyspepsia, rheumatism and urinary disease [16] [17].Leaves and roots are bitter and astringent; used in fever, diarrhoea, urinary diseases and dyspepsia.Different studies have shown that a crude methanolic extract exhibited good analgesic activity, vital cytotoxic activity and moderate antioxidant activity [18].Study of Stephania japonica roots showed diuretic activity [19].Multidrug-resistance-re- versing action was shown by alkaloidal extract of vines of Stephania japonica [20].On all phases of carrageenan-induced inflammation Stephania japonica showed vital anti-inflammatory effect [21].Previous study have shown that it has anti-diarrheal effect as in a castor oil-induced diarrheal model the extract reduced a total number of stool [21].Recent study of fresh fruits obtained a hasubanan ester-ketal alkaloid, stephabenine [22].
Methanolic extract of S. japonica leaves showed antinociceptive result in the treatment of assorted painful conditions [23].To find new hypoglycemic agents from various plants and to evaluate their activity and toxicity on experimental animals and human beings, several studies have been performed [24] [25].This research paper reports about the assessment of the hypoglycemic activity of methanolic leaf extracts of Stephania japonica (Thunb.),Miers, from the family of Menispermaceae to the standard reference metformin and with the combination with metformin.

Chemicals
Alloxan was purchased from Sigma chemicals, Germany.Glucose was purchased from Glaxo Smith Kline.Triglycerides, total cholesterol, HDL-cholesterol and LDL-cholesterol kits were purchased from LINEAR CHEMICALS S.L., Spain.Metformin was obtained from Chad well Heath Essex, England.All other chemicals used in the study were of analytical grade.All solutions were prepared on the same day of experiments.

Plant Materials
The leaves of S. japonica (Thunb.),Miers were collected from surrounding area of Hazrat Shahjalal International Airport, Kurmitola, Dhaka-1229, in March, 2015.The plants were identified by expert of Bangladesh National Herbarium, Mirpur, Dhaka, Bangladesh, where the voucher specimen has been deposited.

Preparation of Extracts
After collecting, the leaves were thoroughly washed with water and were well sun-dried for one week.The leaves were ground into coarse powder with the help of a suitable grinder.Dried leaf powder (500 g) was soaked in 2000 ml methanol in a separate clean, round bottomed flask for about 7 days at room temperature with occasional shaking.After 7 days the solution was filtered using cotton filter and Whatman's filter paper.After completion of filtration the filtrate was concentrated using a rotate evaporator at 40˚C until the extra solvent completely dried.A semi solid S. japonica (Thunb.)extract was obtained after complete elimination of alcohol under reduced pressure.

Selection of Animal
Six-weeks-old male and Long-Evans rats (115 -120 g) were purchased from animal's house of Jahangir Nagar University, Department of Pharmacy.Before initiation of the experiment, the rats were acclimatized for a period of 7 days under standard environmental conditions at 25˚C, humidity (50% ± 5%) and 12 hrs.light & 12 hrs.dark cycles).Before and during the experimental rats were fed with standard pellets supplied from ICDDR, B and fresh drinking water [26].
All the animal studies were carried out in accordance with the Animal Ethical Committee of Southeast University, Pharmacy Department.

Acute Oral Toxicity Studies
For the determination of acute toxicity studies the animals were divided into five groups (n = 5) and fasted overnight.All groups' animals were fed with different doses of methanolic extract of S. japonica in increasing dose level 100, 250, 300, 400 and 500 mg/kg body weight.The animals were periodically observed for 72 hrs for the mortality and general behavior.After one week it has been observed that 2 out of 3 rats taking 500 mg/kg methanolic extract of S. japonica died.The method of Litchfield and Wilcoxon were followed to determine acute toxicity [27].

Oral Glucose Tolerance Test (OGTT)
Oral glucose tolerance test was performed in overnight (18 h) starved normal Long-Evans rat.The rats were randomly divided into five groups, each consisting of five rats (n = 5).
Group I: rats were administered 0.9% (w/v) saline Group II: rats were administered methanolic leaf extract of S. japonica (200 mg/Kg BW) Group III: rats were administered methanolic leaf extract of S. japonica (350 mg/Kg BW) Group IV: rats were administered standard drug metformin (850 mg/70 Kg BW) Group V: rats were administered combination of leaf extract (250 mg/Kg BW) + Metformin (425 mg/70 Kg BW) Glucose 2 g/kg BW was fed 30 min after the administration of different doses of leaf extract and combination drugs.Blood was withdrawn from the tail vein at 0, 30, 60, 90 and 120 min of glucose administration, blood glucose level were estimated by using glucometer (Tyson Bioresearch, Based Industrial Park, Chu-Nan, Taiwan).

Grouping of Experimental Animals
Long-Evans rats were randomly assigned into group I, II, III, IV, V and VI.Five rats in each group for the respective four weeks treatment protocol for the determination of blood glucose level and lipid profile test studies.

Experimental Induction of Diabetes
Here in this study alloxan is used as a diabetogenic agent.Except normal control rats, Group-II to group-VI animals were allowed to fast for 12 hours followed by injecting a freshly prepared solution of alloxan (120 mg/kg BW) in saline water intraperitoneally after base line glucose estimation was done.The alloxan treated animals were not allowed to food overnight and drink 10% glucose solution to overcome initial drug induced hypoglycemia.Normal control grouped rats are allowed to take saline water only as vehicle.The fasting blood glucose level was estimated after 72 hrs from alloxan administration.The fasting blood glucose level was estimated by using glucometer (Tyson Bioresearch, Based Industrial Park, Chu-Nan, Taiwan) by the blood sample which was collected from the tail vein from the rats.Animals with blood glucose levels above 15.0 mmol/L were selected for the study.

Preparation of Leaf Extracts Solution
Leaf extract was semisolid and very slightly soluble in water.The dosage was prepared in suspension from using methanol in such a concentration that, each 0.1 ml of solution contains leaf extract according to the dose of 200 mg/kg body weight & 350 mg/kg body weight [28].

Preparation of Metformin Solution (850 mg/70 Kg BW)
Metformin was in white crystal form and freely soluble in water.The dosage was prepared in solution form, using water in such a concentration that, each 0.1 ml of solution contained Metformin according to the dose of 850 mg/70 kg BW, since Metformin is effective in such dose in humans.

Combination of Leaf Extract (250 mg/Kg BW) + Metformin (425 mg/70 Kg BW)
The dosage was prepared separately & such a way that, each 0.1 ml of solution contained plant extract and Metformin according to the dose of 250 mg/kg body weight and 425 mg/70 kg body weight respectively.

Glucose Level Determination
In all experimental rats fasting blood glucose levels were determined initially to determine the level of diabetes and thereafter every week during the four weeks study period.Blood samples were collected from end tail vein by pricking with a sharp needle and blood glucose level was determined by using glucose monitoring system (TysonBio Evolve, Based Industrial Park, Chu-Nan, Taiwan).

Blood Serum Collection
After completing four weeks treatment the rats were at first anesthetized with diethyl ether.Then after cutting the abdominal skin, thoracic artery was opened.3 -4 ml of blood was collected directly from thoracic artery by heparinized syringe.The blood samples were centrifuged at 4000 rpm for 20 minutes at 22˚C (Digisystem Laboratory Instruments Inc., Taiwan) and the plasma sample were frozen up at −4˚C until biochemical estimations.Rats were dissected; heart were removed and cleaned of the surrounding tissues (Right atrium).The organ weights were measured immediately and the ratio of organ weights to body weight ratio (g/kg) were calculated.

Lipid Profile Test
Leaf extract, Metformin and the combination of (leaf extract & Metformin) were administered daily for four weeks in the alloxan-induced diabetic rats.After completing drug treatment for 28 days serum was collected and the concentration of TC, TG, LDL and HDL Cholesterol were measured by taking absorbance by UV spectrophotometer using diagnostic kits (LINEAR CHEMICALS, Spain).The ratio of LDL to HDL cholesterol was calculated.

Measurement of Left Ventricular Hypertrophy
The beating heart was excised from the chest cavity and immersed briefly in phosphate buffer solution at room temperature in order to wash out blood from the chambers.Later on, the atria were separated completely from the ventricles.The right and left ventricles were then separated such that the left ventricle was composed of the left ventricular free wall plus the septum.The weight of the left ventricle was taken.Left ventricular hypertrophy was determined for each animal by calculating the ratio of left ventricular weight (including the septum) and body weight (LV/BW) (an index of cardiac hypertrophy).Then left ventricular hypertrophy is calculated by the following formula.

Statistical Analysis
Values were expressed as mean ± SEM for five rats in the each group.The above estimations were analyzed statistically by applying One-way analysis of variance (ANOVA) followed by Dunnet's test for multiple comparisons.The differences were considered significant when P < 0.05.

Effect on Oral Glucose Tolerance (OGTT) of Normal Rats
To evaluate the acute study of different doses of methanolic leaves extract of Stephania japonica (200 mg/kg BW & 350 mg/kg BW), Metformin (850 mg/70 kg BW), and combination of methanolic extract (250 mg/kg BW) & Metformin (425 mg/70 kg BW), when administered 30 min, before to glucose loading caused significant reduction (P < 0.05) in the rise in blood glucose levels, after glucose administration (Table 1).It was observed that, the different doses of methanolic extract (200 and 350 mg/kg BW) produced 8.82%, 22.06% reduction in blood glucose level at 120 min when compared to the normal control.In case of Metformin and the combination of metformin and extract blood glucose level decreased at 26.47% and 20.59% as compared to the normal control groups (Figure 1).

Effect of Methanolic Leaf Extract and Metformin on Blood Glucose Level
In administration of different doses of methanolic extract of Stephania japonica  mg/kg BW) & Metformin (425 mg/70 kg BW)] is 61.18% compared to DC group.But, the methanolic extract (200 mg/kg BW) did not significantly decrease blood glucose level, which is 42.32%.Here the maximum reduction of blood glucose level by 62.22% was observed for methanolic extract (350 mg/kg BW), which is indicated with the arrow sign (Figure 2).

Effect on Lipid Profile
It had been reported that alloxan treatment not only increased blood glucose levels but also increased the levels of TC, TG and LDL cholesterol and decreased the levels of HDL cholesterol in diabetic rats.In our study it was found that the TC level (39.13%),TG level (93.24%),LDL-Cholesterol level (50.88%) were increased and HDL-Cholesterol level (23.6%) was reduced in alloxan induced diabetic rats in comparison with their respective normal rats after four weeks (Table 3) and (Figures 3-6).It has been observed that a significant reduction in TC,     TG, LDL-Cholesterol and increase in HDL-C levels in alloxan induced diabetic rats treated with either methanolic extract of Stephania japonica or metformin after four weeks treatment.The evidence represented that both the methanolic extract and combination therapy produces beneficial effects on TC, TG, LDL and HDL-Cholesterol level in ADRs after four weeks treatment.As well as standard drug metformin showed significant result in correcting dyslipidemia and normalizing blood glucose level.

Effect on Left Ventricle
Left ventricular hypertrophy (LVH) is the thickening of the myocardium (muscle) of the left ventricle of the heart.In our study it was observed that all treatment    their combination is 32.56% and 34.88% respectively.Here all the treatment groups showed significant result.But the effect of combination therapy showed greater result than extract and metformin alone.

Effect on Body Weight
In the present study, in comparison with normal rats alloxan induced diabetic rats showed significant (P < 0.01) reduction in body weight.At the end of 28 days treatment, the body weight of normal rats, diabetic control, different doses of methanolic leaf extract and metformin treated rats were observed (Table 4 & Figure 8).After four weeks treatment with methanolic leaf extract of S. japonica    8), displays the effect of the different doses of S. japonica leaves extract and metformin on the body weight on the alloxan induced diabetic rats.Diabetic control group continued to decrease the weight till the end of the study.

Discussion
The pathogenesis and management of diabetes mellitus by existing antidiabetic agents without any side effects have raised great interest in recent years among the scientists [29].Alternative or complementary medicines have been used for a long time for the treatment of diabetes.Now-a-days, with an alarming rise in the prevalence of this disease and associated treatment costs, interest in plant medicine has grown [30].
Stephania japonica (Thunb.),Miers, lie geographically in southern China [31] are well known for the medicinal uses like the tuberous root is astringent [32] in nature used in the treatment of diarrhoea and dysentery; fevers; stomach ache and dyspepsia; hepatitis; and urinary diseases [32] [33].The root is said to be used in the cure of itches [33] [34].This plant is used medicinally for the treatment of fevers, diarrhoea, urinary diseases and stomach-ache [35].The crushed leaves in water form a slightly gelatinous mass which is applied to breast infections [33] [35].Extracts from the leaves have shown mild insecticidal properties against fruit flies in Thailand [33].Until now this plant is under extensive experiment to evaluate that either it has antidiabetic activity or not.Sultana et al.; demonstrated that, methanolic extract of Stephania japonica (Thunb.)Miers, tendril (MSJT) has potent antihyperglycemic and anti lipid peroxidative activity in alloxan induced diabetic rats [36].
Since the phytochemical constituents of leaves and tendrils are different, we have selected the leaves of Stephania japonica for evaluating antihyperglycemic and antihyperlipidemic activity in our present study.In this current study alloxan is used for the initiation of diabetes.Alloxan (2,4,5,6-pyrimidinetetrone) is an oxygenated pyrimidine by-product.Alloxan has a destructive effect on the beta cells of the pancreas.Alloxan induces diabetes by destroying the insulin-producing beta cells of the pancreas [37] [38].In vitro studies have shown that alloxan induces cell death due to its poisonous effect on pancreatic beta cells [39].The cytotoxic action of alloxan is mediated by reactive oxygen species; however, alloxan and the product of its reduction, dialuric acid, establish a redox cycle with the formation of superoxide radicals.These radicals endure dismutation to hydrogen peroxide.Thereafter, extremely reactive hydroxyl radicals are fashioned by the Fenton reaction.The action of reactive oxygen species with a coincidental large increase in cytosolic calcium concentration causes fast destruction of β cells [40].
It was observed that, alloxan caused a definite rise in blood insulin and glucose levels with a subsequent drop of free fatty acids.In the liver of alloxantreated rats the amount of sulfhydryl groups was diminished and glutathione peroxidase activity was considerably higher which may be indicative of β cell damage by alloxan as well as its direct effect on other tissues.It was also observed that, alloxan in vivo does not merely employ detrimental effect on pancreatic β cells [41].It has been reported earlier that, due to muscle destruction or degradation of structural proteins there was a loss in body weight in alloxan induced diabetic rats [42].Oral administration of different doses of methanolic leaf extract of Stephania japonica and metformin significantly improved the body weight compared to the diabetic control rats.On the other hand, untreated diabetic control rats were gradually losing their body weight.Hence all doses of methanolic leaf extract, metformin and combination therapy showed a protective response in regulating muscle exhausting.Before starting the treatment blood glucose level increased significantly in all groups when compared with normal control rats.In administration of different doses of methanolic extract of Stephania japonica (350 mg/kg BW) & amp; Metformin (850 mg/kg BW) and combination of them (Metformin 425 mg/70 kg BW + LE 250 mg/kg BW) for four weeks, BGL is decreased significantly in ADRs in comparison with diabetic control rats.But the methanolic extract (200 mg/kg BW) didn't significantly decrease blood glucose level.The significant antihyperglycemic activity of methanolic leaf extract of Stephania japonica may be due to the presence of hypoglycemic saponins, tannins, triterpenes, alkaloids and flavonoids etc. and it is reported that Stephania japonica leaves extract is the rich source of flavonoids and phenolic compounds [43].
Present study suggests that, methanolic extract of Stephania japonica (350 mg/kg BW) and combination therapy have the properties to stimulate or regenerate the β-cells for the secretion of insulin.Leaves of Stephania japonica lowered hyperglycemia and it may be useful for the treatment of diabetes and associated complications.On the other hand, the mechanism of action of reference drug metformin is well documented [44] [45].In addition, Diabetes affects both glucose and lipid metabolism [46].The most common lipid abnormalities in diabetes are hypercholesterolemia and hypertriglyceridemia.In this study, diabetic control rats showed significant changes in lipid abnormalities [47].Furthermore, high plasma cholesterol and triglyceride levels are major risk factors of cardiovascular disease [48].Alloxan induced diabetic rats showed elevated plasma cholesterol, triglyceride levels and LDL-Cholesterol level but decreased level of HDL-Cholesterol level due to hyperglycemia and insulin resistance [49].In this study, alloxan induced diabetic rats also showed significant changes in lipid abnormalities [47].After oral administration of particular doses of methanolic leaf extract of Stephania japonica caused a momentous reduction of serum lipid levels in diabetic rats, viz.total cholesterol, triglyceride, LDL-Cholesterol level and improved the level of HDL-Cholesterol level after four weeks treatment.Metformin and combination therapy as well as showed significant result in correcting abnormal lipid profiles.Methanolic leaf extract of Stephania japonica may lead to conversion of the pancreatic β-cells and the resultant loss in BGL may lead to blockage of lipid peroxidation.
Plant having flavonoids, terpenoids, alkaloids, and glycosides claimed to possess antioxidant and antidiabetic activity.Flavonoids present in the plant reproduce the damaged beta cells of pancreas, and the saponin and polyphenolic compounds being within the plants suppress glucose transport by suppressing sodium glucose co-transporter-1 (S-GLUT-1) in intestine [50] [51].Antihyperglycemic and antidyslipidemic effect of methanolic extract of the leaves of Stephania japonica might be due to the presence of flavonoids, phenolic com- pounds, tannins and saponin etc. Metformin corrected dyslipidemia mainly by correcting abnormal glucose metabolism [52].As a result of decreased hepatic synthesis of VLDL it also causes moderate reduction in the triglyceride levels [53].In our present study a similar observation has been reported.Earlier studies showed that long-term induction of diabetes without any taking of hypolipidemic agent produced cardio vascular disease [54].Treatment with different doses of extract and combination therapy (metformin and extract) noticeably (p & lt; 0.001) reduced LV hypertrophy in AIDRs after four weeks treatment.The LV/BW ratio was significantly lower in extract treated rats than in untreated diabetic control rats.

Conclusion
According to our data it can be terminated that Stephania japonica have significant glucose lowering activity as well as lipid lowering activity in alloxan induced diabetic rats when compared with metformin.In addition, this medicinal plant might be considered to be effective, complementary and alternative treatment for diabetes with cardiovascular disease due to improvement in left ventricular hypertrophy.Moreover, methanolic extract of S. japonica in combination with oral hypoglycemic agents may be a valuable novel therapy for the treatment of diabetes and could prevent the early onset of diabetic complications.Finally, further analysis is required to identify the exact compounds that are responsible for its antidiabetic activity and to determine their mechanism of action.rials.In addition, there is no possible conflict of interests about the content, submission and publication of the manuscript.
Group I: Normal Control rats administered saline water only Group II: Diabetic Control rats administered saline water only (Alloxan treated group) Group III: Diabetic tested rats administered methanolic leaf extract of S. japonica (200 mg/Kg BW) Group IV: Diabetic tested rats administered methanolic leaf extract of S. japo-nica (350 mg/Kg BW) Group V: Diabetic tested rats administered standard drug metformin (850 mg/70 Kg BW) Group VI: Diabetic tested rats administered combination of leaf extract (250 mg/Kg BW) + Metformin (425 mg/70 Kg BW).

Table 1 .
Effect of methanolic leaf extract and metformin on oral glucose tolerance (OGTT) after 28 days treatment on normal rats.

Table 2 .
Effect of different doses of methanolic leaf extract of Stephania japonica, metformin and their combination on blood glucose level in alloxan induced diabetic rats after 28 days of treatment.
All values are expressed as mean ± SEM (n = 5) in each group.*P < 0.05; **P < 0.01; ***P < 0.001; ANOVA, followed by Dunnett's multiple comparison test.a Compared to normal control and b Compared to diabetic control.

Table 3 .
Effects of different doses of methanolic leaf extract of Stephania japonica, metformin and their combination on lipid profile & LVH in alloxan induced diabetic rats after 28 days of treatment.

Table 4 .
Effect of different doses of methanolic leaf extract of Stephania japonica, metformin and their combination on body weight in normal and alloxan induced diabetic rats.