Salvigenin has Potential to Ameliorate Streptozotocin-induced Diabetes Mellitus and Heart Complications in Rats

Objectives: Flavonoids are the active ingredients, found in herbal remedies for amelioration the variety of disorders. Salvigenin is a plant flavenoid, which is found in Salvia officinalis . Salvigenin has an antioxidant, anti-inflamatory, anti-tumor and vascular relaxant activities. This study was conducted to evaluate the possible antidiabetic and cardioprotective effects of salvigenin. diabetic streptozotocin Rats were divided into four groups that treated with salvigenin at doses of 5, 10 or 25 mg/kg. All the treatments were administered orally for 4 weeks. At the end of the experiment, the blood samples were collected for determining the FBS, HbA1C, insulin, triglyceride, cholesterol and HDL. After 72 hrs, animals were anesthetized; hearts were removed quickly and mounted on Langendorff apparatus. The max pressure, heart rate, max dP/dt, contractility index and coronary flow were measured. Results: Administration of salvigenin exhibited a significant reduction in fasting serum glucose, triglycerides, total cholesterol, HbA1c and increased level of plasma insulin and HDL in diabetic rats (P<0/05). Salvigenin could significantly increased hemodynamic indices such as max pressure, max dP/dt, contractility and coronary flow (P<0/05). Conclusion: Salvigenin improved diabetes through decreasing blood glucose, lipid profile, HbA1c. Increased insulin secretion can be a mechanism for antidiabetic effect of salvigenin. Regarding the antidiabetic and cardioprotective effects of salvigenin, it can be concluded that this flavonoid compound can be potentially used to reduce diabetes and it’s cardiovascular complications.


INTRODUCTION
Diabetes is an increasing metabolic disorder resulting from a defect in increased insulin secretion, insulin action or both. Defects in insulin secretion or signaling leads to chronic hyperglycemia associated with disturbances in the metabolism of carbohydrates, fats and proteins [1]. Hyperglycemia and glycation and the subsequent neuronal dysfunction, dysfunction of kidney and other organs are the long-term consequences of blood-sugar during diabetes [2,3]. Diabetes causes secondary patho-physiologic changes in multiple organs of the body, and cardiovascular diseases are one of the consequences of the disease. Increased incidence of congestive heart failure, myocardial infarction, reduced muscle strength and arrhythmias have been observed in people with diabetes [4]. This disorder is due to coronary artery disease and cardiomyocyte dysfunction [5]. Now, the main treatment for diabetes is the effective use of drugs such as insulin and amino analogues, sulfonylureas and Biguanides. In recent years, in order to control, prevent and delay the development of diabetes outcomes, the use of herbs and drugs derived from them is increasing [6]. Manyactive compounds are found in plants, such as guanidine in Galga and myricetin in okra, that a synthetic form of them has been identified and even used to treat diabetes [5]. One of the plants, that has antidiabetic effects and there is considerable evidence for proving that in traditional medicine [6] and also in scientific papers [7][8][9][10][11], is Salvia officinalis, which naturally grows in Asia and the Mediterranean [12,13]. Salvia, the largest genus of the Lamiaceae family, includes about 900 species, spread throughout the world. Many species of Salvia, including Salvia officinalis L. (sage), have been used as traditional herbal medicine against a variety of diseases [7]. In its leaves and root, there are compounds such as caffeic acid, gallic acid and flavonoids such as Salvigenin, terpenes and polyphenol such as tannins [14,15]. Salvigenin (5-hydroxy 6, 7, 4 tri methoxy flavones) is an active flavonoid in the leaves and aerial part of this plant and other plants that has anti-diabetic effect [11,15]. So far, direct evidence for anti-diabetic effects of this flavonoid hasn't been reported yet. Eidi et al. [16] have studied the effect of oral administration of wild garlic, which contains flavonoid, on bloodglucose level and blood lipids of rats with type 2 diabetes. This study showed that oral administration of wild garlic in the diet of diabetic rats significantly reduced the blood glucose, cholesterol and triglycerides. In another study, Raafatian et al. in [17], have studied on salvigenin protective effect on SH-SY5Y cells against hydrogen peroxide treatment and found out that Salvigenin protects cells against the oxidative stress induced by hydrogen peroxide by increasing Otto phage and by weakening apoptosis. Anti-tumor effects [18], neuron-protection ] 17 [ and compounding with DNA [18] of this flavonoid have been reported in previous studies. On the other hand, in many studies, the effect of flavonoids in food, on increased insulin sensitivity and on increased insulin secretion have been expressed. In this study, the effect of salvigenin extracted from Salvia officinalis, on type 1 diabetes and cardiovascular dysfunction have been evaluated.

Extraction of Methanolic Plant Material
Sage leaves were collected from Mashhad city, IRAN, in summer and identified in ferdowsi Department of mashhad university (Voucher number: 043722, deposited in: Farabi Herbarium, Director: Dr. B. Hamidi). The plant was cleaned, shed dried at 25°C, and the dried leaves of the plant were ground with a blender, and the powder was kept in nylon bags in a deep freezer until the time of experiments. About 60 g of powder were submitted to extraction with 300 ml methanol (80%) in a Soxhlet apparatus for 72 h. After extraction, the solvent was filtered and then evaporated under reduced pressure by Rotavapor. The H-NMR spectrum of the extract showed that the extract contains flavonoids.

Extract Purification
For isolation and purification of compounds in the plant extracts, column chromatography (CC) was used. For this purpose, a column of silica gel (230-400 mesh ASTM) (100 cm × 2 cm with gel 60 andparticle size from 0.040 to 0.063 mm, Merck product), mixed with hexane were used.

Animals
Thirty two Wistar rats weighing 300±25 that bred in the animal house of Shahid Sadoughi Medical School, Yazd, Iran were selected. Animals were housed at controlled temperature (22± experiments reported 2°C) with a 12 h-light/dark cycle and with standard lab chow and tap water ad libitum were kept in special cages. This study was carried out in accordance with current ethical guidelines for the investigation of experimental pain in conscious animals [19].

Induction of Diabetes
The overnight fasted adult wistar ratswere induced diabetic with streptozotocin (STZ, ENZO, Argentina) 65 mg/ kg, i.p (The STZ was dissolved in the citrate buffer 0.01 M, PH:4.5).Diabetes was proved by measuring the fasting blood-glucose concentration 72 h after injecting STZ. The wistar rats with the bloodglucose level above 300 mg/dl were considered to be diabetic.

Treatment Groups
Diabetic rats were randomly divided into four groups of eight rats: control group administrated normal salin daily and treatment group receivedorally 5, 10 and 25 Salvigenin mg/kg daily for four weeks. four weeks after gavage, cardiovascular parameters were measured in all groups.

Blood Sampling
After 4 weeks of treatment, blood samples were drawn from the retro-orbital sinus. Fasting blood sugar (FBS) were determined by enzymatic method of glucose oxidase. The serum insulin levels were estimated by using the radioimmunoassay kit (Diasorin, Italy). hemoglobin A1C, lipid profile and body weight were measured [20,21]. Triglycerides [22], serum cholesterol [23], high-density lipoprotein [24] and glycosylated hemoglobin A1C were measured too.

Langendorff Apparatus
The Langendorff apparatus was used for perfusion of isolated rat heart [25,26]. Briefly, treated rats were anesthetized with sodium thiopental (75 mg/kg, i.p.) and heparinized with heparin (1000 IU, i.p). Then, The hearts were removed Immediately and aortas were cannulated and perfused retrograde with Krebs-Ringer solution containing (mM): NaCl 118.0, KCl 4.7, CaCl2 1.25, KH2PO4 1.2, MgSO4 1.2, NaHCO3 25.0, and glucose 11.0 that equilibrated with 95% O2 and 5% CO2 at about 37°C. The PH of perfusing solution was controlled 7.35-7.45. The constant pressure of perfusion was 100 cm H2O. Ventricular pressure was recorded continuously by an adjustable water filled probe that inserted into the left ventricle via left atria and linked to pressuring transducer (AD instruments, Australia). Electrocardiogram (EKG) was recorded permanently through two electrodes placing on apex and one placing on the base of ventricles (AD instruments, Australia). The temperature of cardiac jacket was maintained about 37°C using warm water recycle. Coronary flow was collected manually at various times (at baseline and at 5, 15, 30, 60, 90 minutes of reperfusion).

Statistical Analysis
Data were expressed as mean ± SEM and were analyzed by statistical software (version 5.0, Statview, St. Louis, MO) using one-way analysis of variance-(ANOVA) followed by Tukey's test and cardiac index by two-way ANOVA (Two-Way ANOVA).p <0.05 were considered significant.

RESULTS
As shown in Fig. 1, Salvigenin administered at doses of 5, 10 and 25 milligrams per kilogram,as a significant and dose-dependent, decreased fasting glucose in diabetic animals (p <0.05). Also Salvigenin at all doses have no significant dose-dependent effect on glycated hemoglobin (Hb A1c) (p <0.05). Insulin in the group receiving 5 mg (p <0.05) and 10 mg Salvigenin significantly increased (p <0.01) (Fig. 2). Fig. 3 shows that long-term administration of Salvigenin at doses of 10 and 25 mg per kg were significantly reduced body weight of diabetic animals (p <0.05).
Furthermore, Salvigenin effect on blood lipid profile is shown in Fig. 4. The levels of triglycerides, total cholesterol and LDL cholesterol were significantly reduced at all doses (p <0.01). On the other hand, only at a dose of 25 mg per kg could significantly raise HDL cholesterol levels in the blood (p <0.01).

Effect of Salvigenin on Cardio Metabolic Risk Factors in Diabetic Rats
Below the maximum pressure, at times before ischemia, immediately after ischemia, 15 and 30 minutes after ischemia, Different doses of Salvigenin has a maximum pressure in the left ventricleafter ischemia times, but this increase was not significant compared with the control group (p<0.001) ( Table 1).
The heart beat Ratebefore ischemia, 15 and 30 minutes after ischemia weremeasured. heart rate increased after administer of doses 10 and 25 mg Salvigenin after ischemia but this was nosignificant compared to control group (p <0.05) ( Table 2).
The maximum rate of pressure change in different periods, before ischemia, immediately after ischemia, 15 and 30 minutes after ischemia is shown in Table 3. at doses of 10 and 25 mg, Salvigenin has a maximum rate of pressure change at different times. This increases of maximum rate of pressure change at doses of 25 mg before ischemia ,and 10 mg at 30 minutes after ischemia was significant compared to controls (p <0.01) ( Table 3).
Ventricular contraction time before ischemia, immediately after ischemia, 15 and 30 minutes after ischemia is shown in Table 4. Salvigenin at doses of 10 and 25 mg can increase contraction of the left ventricle at different times significantly compare to the control group (p <0.001) ( Table  4).
The results show that the discounted rate on coronary flow in all groups after ischemia and ischemia in all groups, the more time passes the coronary flow rate decreases. Salvigenin coronary flow rate for the group that received a dose of 10 mg kg at all times to find a significantly increased compared to controls (p <0.001) (Fig. 5).

DISCUSSION
The results show that Salvigenin, flavonoids extracted from Salvia officinalis, reduced blood sugar and glycosylated hemoglobin in rats with type 1 diabetes. This flavonoid increases plasma insulin level in diabetic rats. Consumption of salvigenin can reduce the level of triglyceride, cholesterol and level of LDL in plasma. Consumptionof salvigenin can increase level of HDL in plasma in animals with type 1 diabetes. In addition, salvigenin protects rats with type 1 diabetes from severe weight loss.
However, there have been studies in scientific resources on anti-diabetic and metabolic effects of Salvia officinalis, from which salvigenin has been extracted, but no studies have been reported on salvigenin metabolic effects on diabetic and non-diabetic animals. Medium-and long-term administration of hydro-alcoholic extract of leaves of Salvia officinalis, reduced fasting glucose in diabetic animals. Another study showed that oral administration of infused Salvia officinalis will increase liver cells respond to insulin and can inhibit new glucose production of these cells and will reduce blood glucose of health animals [11]. Eidi  The results of this study suggest that weight loss in type 1 diabetes is controlled by Salvigenin.
Other studies have shown that weight lose in type 1 diabetes are due to protein and fat catalysis and dehydration caused by poly uriea [28]. Also, salvigenin has important role in preventing proteins and fat degradation and dehydration by decreasing fasting blood sugar and glycosylated hemoglobin [29,30].
Effects of flavonoids on reducing glycated hemoglobin levels have been reported in many studies [31,32]. Glycated hemoglobin level shows the average amount of glucose in the past 2 or 3 months [33,34] and its great importance in the prevention of diabetes complications and early glycation products [34]. Increased level of glycated products stimulates specific receptors of these products on the surface of macrophages and increases the production of inflammatory mediators such as IL-6 and TNFα [35]. In this study, salvigenin could control the progress of this process by reducing glycosylated hemoglobin level and, therefore, control diabetes. Studies have shown that most of the flavonoids control inflammation by controlling the activity and production of enzymes that produce eicosanoids [36]. As salvigenin is not a flavonoid, its anti-diabetic effect can be due to induced inflammation caused by streptozotocin and highfat food. evidence indicates that oxidative stress plays an important role in the pathogenesis of chronic diseases such as diabetes. Free radicals that are the base of oxidative stress, are produced in the case of diabetes, because of the oxidation of glucose, non-enzymatic glycosylation of proteins and oxidative decomposition of this glycosylated products [37]. Flavonoids, due to the phenolic groups, have a higher anti-oxidant effect than other compounds present in herbs [38]. Since it is widely accepted that the antioxidant compounds have beneficial effects on chronic diseases such as diabetes [31,39] and the antioxidant effect of salvigenin has been proved [40], it is possible that part of its anti-diabetic effects are caused by its anti-oxidant effect.
Furthermore, in previous studies and the present study show that flavonoids increase insulin secretion, insulin response and the number of beta cells in diabetic animals [41]. It is likely that increases of blood insulin level, maybe related to flavonoid effects on increases of insulin secretion, cell response to insulin and increased number of beta cells. The numbers of beta cells and cell sensitivity to flavonoids have not been studied in this study but insulin level have been evaluated.

Studies
have shown that polyphenolic compounds reduce cardiovascular complications of diabetes by eliminating free radicals and controlling diabetes parameters [42][43][44][45][46][47]. Free radicals are produced by different mechanisms. Increased activity of xanthine oxidase in reperfusion Injury phase produces superoxide ions that are damaging free radicals. Flavonoids, such as salvigenin, with their antioxidant effect [48], can reduce oxidative damage of ischemiareperfusion injury by inhibiting the activity of xanthine oxidase [49,50].
Polyphenolic compounds increase insulin resistance by increasing bioavailability of nitric oxide in the endothelium and by reducing the formation of reactive oxygen and nitrogen [51]. Several studies suggest a relationship between insulin resistance and endothelial dysfunction [52]. A number of flavonoids improve endothelial function [53]. Antioxidants improve the function of endothelial cells in patients with diabetes, expressing that oxidative stress plays an important role in the dysfunction of endothelial cells [54,55].
Flavonoid intake may reduce the risk of death from coronary disease [56][57][58]42] . Daily consumption of flavonoid-rich plants such as tea may reduce the risk of atherosclerosis and coronary disease, as well as a preventive effect against stroke [59][60][61].
The protective effects of polyphenols against cardiovascular disease in general, is attributed to the ability of these compounds to modulate endothelial function, antioxidant and antiinflammatory properties, induced nitric oxide production and expansion of blood vessels, inhibiting the hyperactivity of platelets, inhibiting proliferation and angiogenesis [62][63][64]. The epidemiological evidence and clinical studies on this subject in recent years has emphasized that Polyphenols have a significant impact on prevention of cardiovascular disease, especially coronary heart disease and myocardial infarction [65][66][67].
Aides Dugan et al. have studied on the relaxant effect of salvigenin flavonoid on rat aorta. Their results indicated that salvigenin is a flavonoid affecting vessel relaxant and this action is mediated by nitric oxide and endothelium-derived prostaclyclin. Vessel relaxant effect of this flavonoid is like apigenin and both of them do it by NO. Flavones effectively overcome the contraction of the entry of calcium into cells or released intracellular calcium. In addition, other mechanisms, such as inhibition of intracellular protein kinase C, or nucleotide phosphodiesterase or activated potassium channels may attend by vascular relaxant effect of these flavonoids. Recent studies show a relationship between the protective effects due to their antioxidant activity that causes the production of endothelial NO. As a consequence of this vessel relaxant effect, salvigenin that is mediated by endothelium, can prevent Oxidative damage and cardiovascular consequences of diabetes [68].

CONCLUSION
In general it can be concluded that salvigenin as a flavonoid can do its anti-diabetic effects through reduction of blood glucose and by decreasing glycosylated hemoglobin, increasing insulin secretion and improving lipid profiles. To know more about its anti-diabetic mechanisms and its use in treatment, further studies are necessary. According to previous studies and positive effects of salvigenin flavonoid on hemodynamic parameters in diabetic hearts, it can be concluded that this flavonoid compound can be used to control oxidative stress and cardiovascular complications of diabetes.

CONSENT
It is not applicable.

ETHICAL APPROVAL
As this study performed on animals, human ethical approval is not applicable.