Protective Effect of Hypericum Triquetrifolium Aqueous Extract on Biochemical and Histopathological Parameters in Hyperlipidemic Male Rats

Hypericum triquetrifolium is an herbaceous perennial plant, numerous of the bioactive compound are present in it that is responsible for several biological functions. Aim of this research the protective effect of aqueous extract of Hypericum triquetrifolium on some biochemical parameters and histopathological study in hyperlipidemic male rats. Thirty-six male albino rats were divided into six groups, control group (fed with standard pellet), hyperlipidemia group , three ,four and five groups hyperlipidemic rats treated with aqueous extract of Hypericum triquetrifolium in different dosage (1000,2000,3000) mg/kg body weight, respectively, and last group hyperlipidemic rats treated with rosuvastatin drug 10 mg/kg for 60 days . The results showed that the body weight, serum cholesterol, triglyceride, low-density lipoprotein, very low-density lipoprotein, cholesterol/high-density lipoprotein ratio, and hepatic enzymes increased significantly (p˂0.05) in hyperlipidemia group when compared with control group, but Hypericum triquetrifolium have ability to reduction the level of body weight, serum cholesterol, triglyceride, low-density lipoprotein, very lowdensity lipoprotein, cholesterol/high-density lipoprotein ratio, as well as the low dose of H. triquetrifolium nonsignificantly (p>0.05), prevent to increase liver enzymes, conversely the level of liver enzymes in rosuvastatin group treatment increased when compared with other treated and untreated groups. Noticeable damage of histology in testis showed degeneration in the germinal layer and fat change in liver of hyperlipidemia and rosuvastatin group, the low dose of H. triquetrifolium has beneficial effect on sperm parameters and histology of testis and liver. The quantity of aqueous extract of Hypericum triquetrifolium and antioxidant power of this plant have an essential role in their activity. Kirkuk University Journal /Scientific Studies (KUJSS) Volume 14, Issue 4, December 2019, pp. (63-88) ISSN: 1992-0849 (Print), 2616-6801 (Online) Web Site: www.uokirkuk.edu.iq/kujss E-mail: kujss@uokirkuk.edu.iq, kujss.journal@gmail.com 64


Introduction:
Hyperlipidemia is a metabolic disorder considered that the proportion of total cholesterol, triglyceride, and low-density lipoprotein in bloodstream are increased. Hyperlipidemia has been one causes of obesity, diabetes, and eventually renal failure and other cardiac diseases, for instance, coronary heart disease, arteriosclerosis or myocardial infarction [1]. Generally, the classification of Hyperlipidemia could be included either familial Hyperlipidemia or acquired Hyperlipidemia. The features of familial hyperlipidemia are triggered by particular genetic abnormalities such as familial hypercholesteremic by mutating genes that encoding LDL receptor in both hepatic and extra hepatic tissues which is due to an increase in the level of plasma LDL-C and elevated risk to heart disease [2]. However, the acquired hyperlipidemia is not inherited, mainly defect in lipoprotein lipase (LPL) activity via many diseases such as diabetes, hyperthyroidism and some kinds of liver disease that cause reduction excretion cholesterol to bile [3]. Herbal natural medicines are becoming progressively common in the population and have been much used because mostly demonstrated it is natural, as well as assumed harmless than allopathic medicine, limited side effects, and easier availability. However, pharmaceutical research frequently focused at discovery drug therapies to prevent or treat many heart diseases that caused by hyperlipidemia [4]. The genus Hypericum has belonged to the Hypericaceae family, which comprise further than 500 species, spread in various location in the world such as Europe, Asia, and Africa [5]. Many species of the Hypericum genus in traditional used as medicinal plants. Hypericum triquetrifolium Turra is one of these species, Hypericum triquetrifolium is an herbaceous perennial plant, grow in many places of Iraq, contain numerous of the bioactive compound responsible to several biological functions comprise anti-inflammatory, antibacterial, antifungal, anti-tumor, antinociceptive, anti-depression, and Antisedative effect [6]. The common component of this plant is Hyperforin, hypericin, and pseudohypericin and flavonoids. The flavonoids compound such as kaempferol, rutin, hyperoside, quercitrin, quercetin [7]. The process against oxidation was represented antioxidant; usually, organisms developed a series of defense mechanisms after exposure to free radical, numerous constituents of the plant act as an antioxidant, especially phenol class. One of the crucial antioxidants is flavonoids, which represent a range of polyphenolic compounds naturally occurring in plants [8]. The study of Kladar,Srđenović [9] believed that the process of lipid peroxidation inhibited by the antioxidant potential of Hypericum sp. The ability of Hypericum sp to reduce Fe3+ to Fe2+ is estimated based on a method by Lesjak et al. [10]. Problems with sperm production and maturation are the most prevalent factor of male infertility leading to in a low sperm count, impaired sperm morphology or decline sperm motility [11]. Many factors are affecting sperm production and maturation may be the environment, genetic or combine both, drugs can influence negatively on infertility, these effects might include incorporating spermatogenesis impairment and change of epididymal development or other impact induce sexual dysfunction through different mechanisms, maybe by variation hormones or no hormonal mechanism [12]. Statins also called HMG-CoA reductase inhibitors represented the group of medical therapy to decline cholesterol concentration by supperssing the enzyme HMG-CoA reductase, the synthesis of cholesterol take place in the liver by activity of HMG-CoA reductase enzyme [13]. The purpose of this research to evaluate the effect of Hypericum triquetrifolium on the hyperlipidemic male albino rat to treat hyperlipidemia and compared with lipid-lowering drug rosuvastatin and influence on the male reproductive system.

Plant Material and Extraction
Hypericum triquetrifolium Turra freshly collected from Jadida Zab (a town of Erbil city

Experimental Animals:
Ethical guidelines were followed for handling and performing experiments on animals, adult male Wistar rats, Rattus norvegicus weighing (210 ± 30) grams procured from the animal house center (College of Education, University of Salahaddin-Erbil). They kept in spacious polypropylene cages (50 cm x 30 cm x 10 cm) bedded with wood sawdust in the animal house center, standard conditions to the animal room were applied such as temperature (24 ±3 °C) light control (12 h light/12 h dark cycle). The rats feed on standard rat chow and water ad libitum.

Induction of Hyperlipidemia:
Induced hyperlipidemia did via feeding rat cholesterol-rich high-fat diet prepared according to the method of [15] by add 5gm of cholesterol, 5 gm deoxycholic acid and 150 butter to 700 gm of powdered rat chow diet.

Drug study:
Rosuvastatin drug 10 mg was used which manufactured by Turkey (ROSUFIX 10 mg) purchased from the clinical pharmacy in Erbil province.

Experimental design:
All rats randomly allocated to six groups of six rats of each group for 60 days fed high-fat diet except the control group.
doses for all groups were orally administrated by gavage once daily.

Body Weight and Blood Collection:
The body weights of rats were recorded for the calculation the body weight gain. Rats fasted overnight before the day blood sample was obtained. The procedure in progress by anesthetizing the rats by an injected mixed of ketamine and xylazine, Then, The blood samples drawn by puncture of heart from each rat in each group and gathering in nonheparinized tubes, the blood centrifuged at 3000 rpm for 15 minutes to prepared serum and determination of biochemical parameters like lipid panel involving (cholesterol, triglyceride, high -density lipoprotein, Low-density lipoprotein, very low-density lipoprotein levels) and liver function assay involving (Alanine aminotransferase (ALT), Aspartate aminotransferase (AST), Alkaline phosphatase (ALP), Gama Glutamyl Transferase (GGT))were determined by colorimetric method using Roche diagnostic kits (Germany) using the Auto analyzer biochemistry Cobas Integra 400 plus. However, Serum testosterone concentration was determined by the colorimetric method using Roche diagnostic kit (Germany) via Auto analyzer biochemistry Cobas E411 [16].

Epididymal Sperm Analysis:
The suspension of epididymal sperm was prepared in 3 ml of phosphate-buffered saline (PBS) at the temperature 37 °C and pH 7.2. The caudal epididymis was cut and collected in a PBS tube, and the tube contains sperm enriched epididymal fluid. The spermatozoa were analyzed for their motility, concentration, and abnormalities [17].

Sperm Concentration:
The concentration of spermatozoa was prepared via diluting the sperm suspension with PBS (1:20), then mixed, after that a drop (10 µl) of them transfer into the Neubauer hemocytometer in each side of the counting chamber. [19].

Sperm Abnormality:
For the analysis of morphological abnormalities, Thin smears prepared and stained by sperm suspension in one volume was mixed with two-volume of 1% eosin&nigrosen stain.

Ferric Reducing Antioxidant Power (FRAP) Assay:
This test was performed based on the technique described by [21]. The FRAP solution was freshly prepared, and analysis was performed by placing 100 μL of the plant extract and 2 mL of the FRAP reagent, the samples were continuously shaken and leave in the dark location for thirty minutes. Then the absorbance at 593 nm was recorded. The standard curve of ascorbic acid was prepared for comparison using various concentrations, as shown in Fig. 1.   Animals in each group dissected, and the liver and testis were removed and fixed the tissue in 10% neutral buffered formalin solution, then the sections were prepared and stained with hematoxylin and eosin, then examined by using a light microscope [22].

Statistical Analysis:
Results are presented as mean ± SE. All analyses were carried out using Statistical Package for Social Science (SPSS/Version 23.0) software. Data were analyzed using one-way analysis of variance (ANOVA) followed by Duncan Alpha test for comparison between groups. Probability values less than 0.05 were considered statistically significant.

Results:
Rats with high-fat diet group significantly (p<0.05) enhanced the rate of serum The altered letters mean statistical differences and same letters mean no statistical differences.
In the hyperlipidemia group, the activates of aspartate aminotransferase (AST) and alkaline phosphatase (  The altered letters mean statistical differences, and the same letters mean no statistical differences. On the other hand, the Table 3 indicated that the sperm count, motility in hyperlipidemia group declined significantly (p< 0.05) but abnormal sperm increased when compared with the control group. However, in treated groups with H.triquetrifolim, the sperm count increased significantly, motility increased nonsignificantly, abnormal sperm increased significantly in high dose when compared with hyperlipidemia group. Rosuvastatin drugs had a significant special effect on sperm count and abnormal sperm, as well as a nonsignificant effect on motility. Bodyweight of rats changed along treated, and non-treated groups, the weight gain of hyperlipidemia group of rats elevated significantly (p<0.05) compared with the control group.
However, the treated groups had an adverse effect, especially in group 3 treatment, which reduced significantly, group 4 and group 5 decline nonsignificantly. However, Rosuvastatin group increased non-significantly when compared with hyperlipidemia groups Fig. 2. The antioxidant activity of the Hypericum triquetrifolium extract and ascorbic acid were measured by FRAP assay and has been presented in Table 4. It showed that electron-donating groups increased reducing power. The Hypericum triquetrifolium indicates that they are the most operative electron donor, and the FRAP value 6787.7 µM/g slightly decreased when compared with ascorbic acid.

Histopathological Study :
Normal structure of seminiferous tubules in the testis of rats with a huge number of sperms in the lumen of the tubules with normal germinal layers in control group Fig. 4

Discussion:
High-fat diet normally encourages an elevated calorie intake, high energy density, since associated with increasing body weight, circulating serum lipid profile as well as elevated adipose tissue, hepatic steatosis and glucose intolerance [23]. In our study the results illustrated that the group of rats receiving three various quantity of Hypericum triquetrifolium significantly affected to reduce the level of lipid profile comparison with induction hyperlipidemia rats, the LDL cholesterol extremely impressed through the low quantity of H.
triquetrifolium. This finding is in agreement with the researcher, which elucidated the In our study the TCH, TG and LDL, VLDL Ch/LDL ratio significantly reduced in group 6 compared with hyperlipidemia group, this finding agreement with many studies that illustrated the mechanism action of statin by blocking the active site of hydroxyl methyl glutaryl CoA reductase enzyme in the mevalonate pathway. This action due to blocked the conversion HMG-CoA to mevalonic acid, also may by increased cell surface LDL receptor illustrated that the orally administrated 6 mg\kg rosuvastatin for 6 week lead to liver steatosis by more than 66 % of hepatocyte affected, as well as showed this fat accumulation by induced