ARTICLE INFO

Article Type

Original Research

Authors

Nobahari   M. (1)
Shahanipour   K. (*)






(*) Biochemistry Department, Falavarjan Branch, Islamic Azad University, Isfahan, Iran
(1) Biochemistry Department, Falavarjan Branch, Islamic Azad University, Isfahan, Iran

Correspondence

Address: Department of Biochemistry, Islamic Azad University, Kamarbandi Street, Falavarjan, Isfahan, Iran
Phone: +98 (31) 37420140
Fax: +98 (31) 37420136
shahanipour_k@yahoo.com

Article History

Received:  January  23, 2016
Accepted:  July 19, 2016
ePublished:  January 19, 2017

BRIEF TEXT


Chronic hyperglycemia in diabetes is associated with damage, disorder and disability of various organs, especially the eyes, kidney, nerves and cardiovascular system [1].

… [2-11]. Silymarin is the most important active ingredient in the Melanocytic herb called the Silybum marianum that is composed of flavonoligan [12]. The results of studies have shown that silymarin has a significant antioxidant effect which reduces free radicals and inhibits the peroxidation of lipids [13]. … [14-16].

The aim of this study was to determine the effects of methanolic extract of silybum marianum seeds on glucose level, oxidative indices, and biochemical factors in diabetic rats.

This is an experimental study.

White male Wistar rats weighing 250-300 gr were used.

24 male rats that under normal and fasting conditions had serum glucose level less than 130 mg/dl were used. Rats were randomly divided into four groups of six: healthy control (negative control), diabetic control without treatment with extract (positive control) and two diabetic groups treated with two doses of 100 and 150 mg/kg body weight of methanolic extract.

The seeds of silybum marianum were powdered and dried with dechlorethylene chloride at a temperature below 60 ° C in order to remove the oil from the powder. Then, not-fatty powder was extracted with methanol 70%. For induction of diabetes in rats, streptozotocin (Sigma, USA) was used as single-dose and intraperitoneal injection of 60 mg/kg as cold saline solution. After observing the symptoms of diabetes, the extracts were inseminated daily with insulin syringe for 4 weeks and intraperitoneal. Weight was determined at the beginning and end of the study. Serum glucose levels were also measured at the end of each week. 48 hours after the last infusion, rats were anesthetized by ketamine and blood samples were taken from their heart; after removal of serum, biochemical factors were evaluated. Measurement of cholesterol and HDL serum was done using enzymatic method and kit (Parsazmoon; Iran). In order to evaluate lipid peroxidation, MDA molecule was analyzed as an indicator of lipids. The barbituric acid method was used to measure MDA. Under acetic conditions and temperature of 95 °C, a monomeric aldehyde molecule reacted with two molecules of thiobarbituric acid and formed a pink complexion. At first, serum protein was precipitated using Trichloroacetic acid (TCA) and was isolated by centrifugation (Hermlez 320; Gersilybum marianumy) and the reclaimed solution was used to measure MDA. By spectrophotometric method (Behdad, Iran), optical absorption of a colored complex was read at 535 nm [17]. In order to measure the protein carbonyl content, after dilution of the serum, in two separate blank and sample micro tubes, DNPH (2 and 4-dinitrophenylhydrazine) (Sigma, USA) solubilized in acid chloride two normal was added to the sample Eppendorf. Only the two-normal chloride acid was added to the blank Eppendorf. The Eppendorfs were placed in the dark for an hour. In the next step, 20% trichloroacetic acid (Merck; Gersilybum marianumy) was added to both Eppendorfs which causes deposition of proteins. Then, the formed plate in each of the Eppendorf was centrifuged in three stages and was washed with ethanol/ethyl acetate. Finally, with the addition of 6 molars of guanidine hydrochloride (Sigma; Gersilybum marianumy) and incubation (Finec, South Korea) at 37 ° C, optical absorption of samples was read at 370 nm [18]. The activity of hydrophobic organophosphate (paraoxonase) of the PON-1 enzyme is determined by calculating the primary hydrolysis rate of Paraoxon substrate (Sigma, USA) to paranitrophenol. To this end, first based on the Beltowski protocol, a solution of 100 mM Tris-HCI (chloride acid) and 2 mM CaCl2 (Calcium chloride) were prepared at a final concentration of 2 mM paraoxan based on the Beltowski protocol, and its PH was adjusted to 8 and finally, by adding 20 μl of serum (containing the enzyme) to the mixture in a cuvette (under stable conditions), the level of paranitrophenol at 412 nm wavelength was monitored by using a spectrophotometer. In the end, using the following formula, the activity of the paroxysmal serum of PON-1 was calculated in μmoles per centimeter [19]: Paroxysmal enzyme activity=A/T.F F= (VT/VS) /ε412 VT=Total volume (μl), VS=sample size (μl), A=Change in absorption, T=Time (minute), ε412= molar extinction coefficient of paraoxonase per µmol/cm (0.01829). Data were analyzed by one-way ANOVA and repeated measure ANOVA.

The use of streptozotocin for induction of diabetes increased the level of malondialdehyde, carbonyl protein, cholesterol and blood glucose, and decreased paraoxonase activity and level of HDL in blood serum of diabetic rats compared with healthy control group (p<0.05). Mean glucose level was significantly different in the groups receiving methanolic extract at concentrations of 100 and 150 mg/kg compared to the negative control group (p<0.001; Table 1). Mean glucose level showed significant difference in the groups receiving methanolic extract at concentrations of 100 and 150 mg/kg compared to the negative control group (p<0.001; Figure 1) In all groups, except for the non-diabetic negative control group, the weight of the rats decreased at the end of the period (p<0.001). Negative no diabetic control group showed significant difference with all groups (p<0.05; Figure 2). Cholesterol serum level, carbonyl protein and paraoxonase enzyme showed significant difference in both diabetic groups treated with methanol extract of silybum marianum with the concentration of 150 mg/kg (high concentration) and concentration of 100 mg/kg (low concentration) compared to the diabetic positive control group. Regarding HDL, treatment with high concentration of methanol extract in high concentration showed significant difference with non-diabetic negative group and the treatment group with low concentration showed significant difference with two positive control and negative control groups. In the case of malondialdehyde, the diabetic group treated with high concentration of methanol extract was significantly different with the group treated with low concentration of methanol extract. Diabetic group treated with methanol extract with low concentration, also, showed significant difference with all other groups (p<0.05; Table 1).

… [20-26]. According to the results of this study, the level of malondialdehyde was significantly higher in the positive conFtrol group compared to negative (non-diabetic) control group. The association of increased blood glucose with lipid peroxidation has been studied in several studies [27]. … [28].

It is suggested that the duration of the treatment be increased and the oil of this plant is used for treatment. It is possible to use these extracts as a substitute for chemical drugs by conducting further researches into the clinical effect of these extracts.

Of the limitations of this study, a small number of rats as well as infections caused by them can be mentioned.

Methanolic extracts of silybum marianum seeds reduce the blood glucose level, cholesterol, malondialdehyde and carbonyl protein and increase the level of HDL and paraoxonase enzyme activity in diabetic rats.

Thanks to the staff of the Research Laboratory of Falavarjan Azad University who have been extremely involved in this research.

Non-declared

Observing all the rights of laboratory animals in research for husilybum marianum use was based on the international guidelines for the care and use of laboratory animals. Also, in all phases, the ethical rules and regulations of working with laboratory animals were also observed.

This article is based on a dissertation and has been conducted with personal finances.

TABLES and CHARTS

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