Antidiabetic Effects of Otostegia Persica Root in Alloxan-induced Diabetic Rats

Otostegia persica has been used in the Iranian traditional medicine for the treatment of diabetes, because of it, the antidiabetic effect of aqueous extract of Otostegia persica root was investigated in alloxan-induced diabetic rats. In the present study oral administration of the aqueous extract of Otostegia persica root (200, 300 and 400 mg/kg body wt.) were investigated for 28 days. The level of serum glucose, total cholesterol, triglyceride (TG), high density ipoprotein cholesterol (HDL-c), low density lipoprotein cholesterol (LDL-c), aspartate amino transferase (AST) and alanine amino transferase (ALT) were evaluated in diabet rats. The aqueous extract of Otostegia persica roots in all doses significantly decreased serum glucose and HDL levels when compared with diabetic control rats. In the dose of 300 mg/kg body wt, ALT and TG levels were significantly decreased. A comparison was made between the action of Otostegia persica root extract and metformin (50 mg/kg body wt.), the known antidiabetic drug. The antidiabetic effect of the extract was the same as the metformin. It is concluded that this part of plant might be considered as excellent candidate for future study on diabetes mellitus. 2 Ghodsieh Bagherzade et al.


Introduction
The world prevalence of diabetes among adults (aged 20-79 years) was estimated 6.4 %.Affecting 285 million adults, in 2010, and will increase to 7.7, and 439 million adults by 2030 (Shaw et al., 2010).
Several medicinal plants have been investigated for their beneficial use in different types of diabetes.The effects of these plants may delay the development of diabetic complications and correct the metabolic abnormalities using variety of mechanisms.A considerable number of plants were subjected to clinical trials and were found effective.Moreover, during the past few years many phytoconstituents responsible for Antidiabetic effects have been isolated from hypoglycaemic plants (Edwin et al., 2008).
The genus Otostegia belongs to family Labiate, which is an almost cosmopolitan family of about 180 genera and over 3500 species (Nasir and Ali, 1990).
Otostegia persica or ''Goldar'' is an endemic plant that can be found in southern provinces of Iran (Ghahraman, 1996).In Iranian traditional medicine the aqueous extract of aerial part of Otostegia persica is used as an antihistamin, antispasmotic and antidiabetic (Asghari et al., 2006).Recent studies on aerial parts of this plants suggested Otostegia persica could be considered as medicinal herbal candidate for treatment of diabetes (Akbarzadeh et al., 2012).The present study was investigated to study the antidiabetic and antihyperlipidemic effect of aqueous extract of Otostegia persica root in diabetic rats.

Collection of plants
The roots of Otostegia persica were collected during December 2011 from south Fars province, Iran.The plant and roots were identified by Ferdowsi university herbarium A voucher number specimen (23332) has been kept in herbarium in the department of Pharmacology, Ferdowsi University of Mashhad, Iran.

Preparation of extracts
Otostegia persica roots were cleaned and cut into small pieces and were allowed to dry in shade.Dried roots powdered by grinder (Moulinex AR1043-UK).Extraction was carried out by maceration of 500 g of dried powdered plant in 2500 ml of distilled water (48h) in magnetic shaker at the room temperature.After maceration, the resulting extract was filtered and concentrated in rotavapour under reduced pressure.The concentrated extract was lyophilized to get a powder (yield 2%, w/w).

Phytochemical screening
In order to determine the presence of alkaloids, glycoside, flavones, saponins and tannins a preliminary phytochemical study (colour reactions) with various plant extracts were performed (Tiwari et al., 2011).Polyphenol content was also determined spectrophotometrically by using Folin-Ciocalteu's method as described by Zivkovic et al (Zivkovich et al., 2009).Gallic acid was used as standard to measure the total polyphenol content in 3 extracts (petroleum ether, aqueous, ethanolic).

Experimental Animals
Male albino wistar rats of body weight 180-220 g were obtained from Pastor Institute, Tehran, Iran.Animal were housed in poly ethylene cages at temperature 21-25 degrees Celsius, 12 h light /12h dark cycle and relative air humidity 40-45%.Rats had continuous access to Standard commercial food (Javaneh co, Mashhad, Iran) and tap water.Permission for the study was obtained from ethical committee for animal research works of Birjand University of Medical Sciences.

Induction of diabetes in rats
Diabetes was induced by a single intrapritoneal injection of freshly prepared.Alloxan monohydrate (Sigma-USA) by the dose of 150 mg/kg to overnight fasted rats.After 7 days of alloxan administration rats by FBS levels upon 300 mg/dl were allocated as severe diabetic.(Kumar Sing et al., 2007).

Drug administration
Otostegia persica extract was suspended in normal saline and administrated orally through orogasteric tube at doses of 200, 300 and 400 mg/kg body wt.Same volume (1 ml) administered in all groups.Metformin (glucophage -Merck, co, France) was suspended in normal saline and preperated freshly before administration by dose 50 mg/kg body wt.

Experimental design
In the present study, 48 rats (40 diabetic and 8 normal rats) were used.The rats divided randomly into 6 equal groups, 8 rats in each group.
Group 1: Normal control rats were administered 1 ml of normal saline.Group 2: Diabetic control rats were administered 1 ml of normal saline.Group 3: Diabetic rats were administered metformin (glucophage-Merck co, France) in aqueous solution in the dose of 50 mg/kg orally by orogasteric tube.

Biochemical assay
In the first day, before treatment, 14 th and 28 th days, fasted blood glucose were measured by glucometer.After 28 days of treatment blood samples were collected from heart of overnight fasted rats.Total cholesterol, TG, (HDL-c), (LDL-c), (AST) and (ALT) levels were determined.

Statistical Analysis
All the data were expressed as mean ± S.E.M. Data were analyzed by using ANOVA and tukey post hoc test.Values were considered significantly different at P<0.05.

Preliminary phytochemical screening and total polyphenol content
The results of preliminary phytochemical screening of Otostegia persica root extracs showed (table 1) that all the root extracts (expect petroleum ether) contain flavonoids, Tannins and saponins.However alkaloid was absent in all extracts.The results of total polyphenol content (table 2) were different between each extracts.total polyphenol contents were highest in aqueous extract of otostegia persica root.

Hypoglycemic Effect of Otostegia persica root extract
The results of this study clearly indicated that the aqueous extracts of Otostegia persica root in all doses exhibit significant hypoglycemic activity in alloxan-diabetic rats, whilst there was no significant effect observed in glucose level of group 4 in 14 th days, however at the end of 28 th days of treatment there was a significant decrease (P<0.001) of serum glucose levels in this group (table 3).The standard drug metformin also significantly decreased serum glucose levels (P<0.001) compared with diabetic control group.

Effect of Otostegia persica on plasma lipid profile
The concentration of plasma lipids are presented in table-4 the concentration of total cholesterol were decreased significantly in the group 6 compared to the diabetic group on the 28 th days of study.Plasma triglyceride concentration was decreased significantly in group 5 compared to diabetic control.Plasma HDL levels was also significantly decreased in all Otostegia persica root extract groups.

Discussion
Otostegia persica commonly known as "Goldar'' which is an endemic plant that grows in the southern parts of Iran, especially in Fars, Kerman, Sistan and Baluchestan and Hormozgan provinces.There is a strong belief in antidiabetic effects of this plant in some regions of Iran (Ghahraman. 1996).
No pervious investigation on the antidiabetic effect of plant's root has been reported, but other studies such as: antioxidant activity (Sharififar et al., 2003), antimicrobial (Asghari et al., 2006), antidiabetic activity (Ebrahimpoor 2011, Akbarzadeh 2012) were found.All of these studies related aerial parts of Otostegia persica and present study is the first investigation of Otostegia persica root extract in diabetic rats.
The present data indicated that the aqueous extract of Otostegia persica root significantly decreased serum glucose and HDL in all doses, Otostegia persica in doses of 300 mg/kg body wt., decreased ALT and TG significantly. in agreement, two other studies that investigated aerial parts of Otostegia persica in diabeticrats, have shown the hypoglycemic effect and decreasing total cholesterol without change in HDL (Akbarzade 2012, Ebrahimi 2011).Most of the drugs that decrease total cholesterol also decrease HDL.(Wilson, P.W.F., 1990).
Phytochemical investigation of Otostegia persica root revealed the presence Flavonoids that are known to be bioactive for management of diabetes (Shammas, 1987;Rhemann and Zaman, 1989) and it is well known that certain flavonoids exhibit hypoglycemic activity (Pathak et al., 1991;Ahmed et al., 2000).Also Flavonoids are able to regeneration beta cell of pancreas (Chakravarti et al., 1981).Thus, the significant antidiabetic effect of aqueous extract of Otostegia persica root may be due to the presence of more than one antihyperglycemic principle and their synergistic properties.

Fig. 1 .
Fig. 1.Effect of aqueous extract of Otostegia persica root on Plasma AST (A) and ALT (B).AST: aspartate amino transferase; ALT: alanine amino transferase.Values are given as mean ± S.E.M. for 8 rats in each group.† P<0.05 compared with diabetic control group.

Table 3 .
Effect of Otostegia persica root on serum glucose level