The Effect of Hordeum Vulgare L. Extracts on Blood Cholesterol Level and Lipid Peroxidation Activity in a Hypercholesterolemia Rodent Model

Hordeum vulgare L. (Barley) is an ancient and essential cereal grain crop with the claim that it has the potential to reduce cholesterol level and to lower oxidation activity in the liver.However, it hasn’t been proven scienti ically. Hence,this study was conducted to investigate the total phenolic content (TPC), total antioxidant activity (TAC) and liver peroxidation activity of barley aqueous and ethanol extractas well as assess the effect of ethanol extract on cholesterol level of high-fat diet rats. TAC of barley extract was determined by using ABTS (2,2-azinobis-(3-ethylbenzothiazoline-6-sulfonic acid) diammonium salt) assay andDPPH (2,2-diphenyl-1-picrylhydrazyl radical) assay. Meanwhile, Total Phenolic Content TPCwas determined by FolinCiocalteu assay. Atotal of 15 Sprague Dawley rats were tested for the lowering cholesterol properties in barley and its associationwith lipid peroxidation product (Melondialdhyhe level) by adding the barley into their daily diet. The result indicated that TAC and TPC value of ethanol barley extract was high. Barley ethanol extracts effectively lowering cholesterol level in Sprague Dawley rats. Meanwhile, the malondialdehyde level in the liver tissue was a significant difference between the high-fat diet group of rats and the high-fat diet group of rats treated with ethanol barley extract. Conclusion, ethanol barley extract possess more phenolic content, antioxidant component and reducing cholesterol level of high-fat diet rats.


INTRODUCTION
Cholesterol is a crucial biological molecule that has roles in membrane structure as well as being the precursor for the synthesis of the steroid hormone and bile acids (Nayak, 2008). There is no recommended intake for cholesterol in daily diet as the body can produce enough cholesterol and people do not develop cholesterol de iciency disease if it is not consumed as diet (Isaacs et al., 2011). However, the blood level tends to increase somewhat if the consumption of cholesterol is increased. (Brown, 2013). An individual with a healthy lifestyle also has the possibilities in having high cholesterol level in blood.
According to health guidance that is released by the National Institute of Health, an individual needs to keep track of their cholesterol level in the blood to reduce the risk of developing coronary disease. Cholesterol-lowering agent or product can be found in the food containing the speci ic added ingredient (National Institute of Health , 2005). Lowering cholesterol by adopting healthy habits will help in weight loss and increase energy as well as helping in preventing the cholesterol levels from becoming high. Weight reduction and exercise enhances the decrease the level of LDL-cholesterol that can also be achieved by reducing intake of saturated fats and cholesterol as well as contribute to the enhancement of energy level (Laclaustra et al., 2018).
A study done by (Chen et al., 2010) concluded that both the phytosterols and phytosterols diets signi icantly decrease the ratios of cholesterol in rat's plasma, red blood cells, liver, aorta and kidney. However, there is evidence that plant sterol and stanol help in reducing the cholesterol blood level, it is essential to remember that they are not a substitute for a healthy diet or a replacement for cholesterol-lowering medicines (Benelam, 2009;Trautwein et al., 2018).
Phytosterols and phytostanols, also referred to as plant sterols and stanols, are common plant and vegetable constituent with structurally related to cholesterol, but differ in the structure of the side chain (Cantrill and Kawamura, 2008). Due to the similar structure to cholesterol, phytosterols and phytostanols can act in the intestine to lower cholesterol absorption by displacing cholesterol from intestinal micelles and due to their poorly absorbed characteristics, blood cholesterol levels will likely drop owing to increase excretion (Murray and Pizzorno, 2012).
Barley is an excellent source of dietary ibre, protein, and complex carbohydrates, and is a good source of speci ic vitamins and minerals (Balch, 2003). Barley is also a rich source of tocols, including to cophenols and to cotrienols, which are known to reduce serum low-density lipoprotein cholesterol through their antioxidant action (Madhusweta and Sumeet, 2016). According to (Newman and Newman, 2008), nutritional components of barley are generally reported as averages, when in reality, barley may differ signi icantly in chemical composition due to genotype, cultural practices, environmental growing conditions and might also be affected by the type of solvent to extract barley. Hence, the main objective for this research is to determine the phenolic content and antioxidant activity as well as the lowering cholesterol level effect of barley extract on rats fed with a high-fat diet.

Preparation of Extract
Barley was purchased from a local wet market. Approximately 100g of barley was grounded and soaked in 1 Liter of aqueous and ethanol separately for seven days. The extractions were then iltered, driedusing Rota evaporatorand subsequently stored in the refrigerator before use. The working solution dilution of Barley extract was, according to (Jaafar et al., 2014).

Experimental animal design
The animal ethic was obtained from an animal ethic committee of UniKL MESTECH. A total of ifteen male Sprague Dawley with body weighed between 300 -400 g were used in this study. The rats were kept acclimatized for ive days under room temperature with 12 hour light and dark cycle and free access to water and standard basal diet.Hereafter, the rats were randomly divided into ive groups with three rats in each groupand were treated for 20 days with a high-fat diet, except for the control negative group. The high-fat diet formulation was adopted from the American Institute of Nutrition (Reeves et al., 1993). The treatment group of ratswas given 500µg/µL barley extract daily. (Jaafar et al., 2014).

Determination antioxidant activity of aqueous and ethanol extract
Antioxidant activity of both extracts (aqueous and ethanol extract) was determined by using ABTS (2,2-azinobis-(3-ethylbenzothiazoline-6-sulfonic acid) diammonium salt) assay (Re et al., 1999) and DPPH (2,2-diphenyl-1-picrylhydrazyl radical) assay (Brand-Williams et al., 1995). The test was done in triplicate. The percentage inhibition for both ABTS and DPPH assay was calculated by using the following formula where A o is the absorbance value of radical cation solution and is absorbance value after radical cation solution treated with extract or antioxidant.

Total phenolic content
Total phenolic content of both extracts was determined by using Folin-Ciocalteu assay (Singleton et al., 1999). Gallic acid was used as a standard, and the total phenolics were expressed as mg/g gallic acid equivalents (GAE). The test was performed in triplicate.

Total cholesterol level
By applying diet planning; throughout 49 days experiment, blood was aspirated from the tail vein of each rat for the determination of cholesterol level. Total cholesterol level was determined by using EasyTouch ® Blood Cholesterol Test Strips with the EasyTouch ® Cholesterol Meter. The cholesterol level was performed in triplicate according to manufacturer protocol.

MDA assay
The lipid peroxidation activity of the treated group of rats was evaluated by using malondialdehyde (MDA) production, according to (Ledwozyw et al., 1986) method. Total MDA concentration (nmol/mg) that represents lipid peroxidation activity was calculated by using the following formula MDA = (MDA X dilution factor) / (Protein) where dilution factor is 20.

Data Analysis
All statistical analysis was carried out using Statistical Package for the Social Sciences (SPSS) statistical software version 17.0. Data were expressed in mean ± standard error of the mean (SEM) (Mean ± SD) and p-value less than 0.05 considered as signi icant. For statistical analysis, one-way analysis (ANOVA) followed by Tukey's test was applied to compare means among groups. Paired-T-test was used for the comparison between before and after treatment.

Total Phenolic Content and Antioxidant Activity
Total phenols content in two different Hordeum vulgare L. extract was showed in Table 1. It was found that the TPC of ethanol extract of Hordeum vulgare L.is higher than the aqueous extract of Hordeum vulgare L. (Figure 1). Meanwhile, the TAC of Hordeum vulgare L. is also higher in ethanol extract compared with aqueous extract for both ABTS and DPPH radical. In this study, a higher level of TPC was found in ethanol extract may be attributable the complex formation of some phenolic compounds in the extract that is soluble in ethanol whereby these phenolic compounds may possess more phenol groups. Aof Hordeum vulgare L.antioxidant studies has been demonstrated in many studies (Anwar et al., 2010;Zimmermann et al., 2013;Oh et al., 2014) where barley contains much more signi icant amounts of phenolic compound (0.2 -0.4%) than other cereal grains which composed of polyphenols, phenolic acids, proanthocyanidins (PAs) and catechins (Quinde et al., 2004).

Food Consume, Body Weight Gain and Feed Ef iciency Ratio
In this study, the diet change (basal diet to high-fat diet) was slightly different for each group except rats from control negative group (basal diet) consistently ahead of other groups (data not shown). Meanwhile, rats from control positive group that received a highfat diet consumed more than group 2 and group 3, which received a high-fat diet with barley and highfat diet with simvastatin drug.
Initial weight and inal weight of every rat (n=15) was taken for the calculation of body weight gain (BWG). There was no signi icant difference between the diet given and the rat's body weight gain in each group.
During the experimental period, the average food consumption (g) per week was measured. The food consumption before and after treatment were equally similar for each group. In (Mahmoud, 2013), a decrease of food consumed was observed when the diet changed, which is contradict with this study. This could be the addition of barley extract was well accepted, and it did not in luence the acceptability of the diet by the rats.

Cholesterol Level of the Sprague Dawley
Mean cholesterol level after treatment for rats from control positive group that received high-fat diet was higher than rats from group 1 which receive  high-fat diet with barley ethanol extract (p = 0.007) and group 3 which received a high-fat diet with simvastatin drug (Figure 2). When the mean cholesterol level after treatment was compared between group 1 and group 3, the cholesterol level in group 3 was slightly decreased than group 1 (p = 0.18).
Studies indicate that increase in high fat dietary intake in an animal could lead to hypercholesterolemia (Kishida et al., 2002;Czerwiński et al., 2004). Hypercholesterolemia is characterized by increased blood cholesterol levels above the normal range (Steinberg, 2011). In this study, before treatment started, all group of rats showed an average increase in cholesterol level after some time. Yet, after the treatment started, group 1, group 2, and group 3 exhibited a decrease in cholesterol levels. Group 1 and group 2 received an additional supplement of barley in their diet. By examine on a group that received barley extract in the daily diet, the similar results with (Mahmoud, 2013) study were obtained. Whereby a cholesterol level of rats was successfully lowered.
Barley was found contains β-glucan that lowering blood cholesterol level (Federal Register, 2008). The effectiveness of β-glucan in barley food products in lowering blood cholesterol has been reported in numerous publications (Pins, 2006). Besides that, barley is also rich of tocols, including tocopherols and tocotrienols, which are known for reducing serum LDL through their antioxidant action (Qureshi et al., 1991). Hence, barley ethanol extract might contain a lot of β-glucan, tocopherols and tocotrienols.

Lipid Peroxidation
MDA level in the group supplemented with barley ethanol extract, group 1 and group 2, are signi icantly lower when compared to control positive (p = 0.04, Figure 3). A study by (Yang et al., 2008) reported that hypercholesterolemia responsible for oxidative modi ication of LDL, protein glycation, glucose-autooxidation with excess production of free radicals and lipid peroxidation products which represent a signi icant risk factor for cellular damage where MDA level is increased. MDA is a protein that involves in hyperlipidemia which will provoke free radical attacks on membrane lipoproteins and polyunsaturated fatty acids. It is also the primary source of oxidative stress in rat liver (Ming et al., 2009;Marczuk-Krynicka et al., 2009).

CONCLUSIONS
When the barley ethanol extract was subjected to the rat's diet as a supplement, decreasing cholesterol level was observed. The presence of higher phenolic content in barley ethanol extract might be the cause of decreased MDA level in rat's liver. However, these need to be further study by using histology analysis and HPLC analysis.