Functional food mixtures: Inhibition of lipid peroxidation, HMGCoA reductase, and ACAT2 in hypercholesterolemia‐induced rats

Abstract Mixtures of selected functional foods (MSFF) were composed of nattokinase (fermented soybean), red yeast rice extract, Ginkgo biloba, oat fiber, garlic, bee pollen, and propolis as anti‐hypercholesterolemic were studied. The goal of this study was to determine the bioactive compounds in these mixtures and their cholesterol‐lowering potential effects (biochemical profiles, lipid peroxidation, liver tissue histopathology, and enzymatic activity analysis; HMGCoA reductase and ACAT2. The LC‐MS/MS analysis showed that bioactive compounds such as Monacolin K, naringin, tocopherol, and glutamate, which have potential as anti‐hypercholesterolemic agents, were present in these functional food mixtures. MSFF supplementation at 50 mg/kg 100 mg/kg and 200 mg/kg showed substantial reductions in serum lipid profiles (TC and LDL) (p < .05). The serum liver profiles of AST (115.33 ± 8.69 U/L) and ALT (61.00 ± 1.00 U/L) were significantly reduced (p < .05) with MSFF supplementation at 200 mg/kg. MDA lipid peroxidation has also decreased significantly (p < .05) in serum (3.69 ± 0.42 μmol/L) and liver (15.04 ± 0.97 μmol/mg) tissues and has been shown to protect against hepatic steatosis. The significant (p < .05) inhibition activity of HMGCoA reductase (163.82 ± 3.50 pg/ml) and ACAT2 (348.35 ± 18.85 pg/ml) was also attributed by the supplementation of MSFF at 200 mg/kg.

Hypercholesterolemia is a condition that refers to a metabolic disorder that may result in elevated concentration of plasma low-density lipoprotein (LDL) cholesterol (Adekiya et al., 2018;Mu et al., 2017). Hypercholesterolemia has become a primary risk factor for the pathogenesis of cardiovascular diseases such as the development of atherosclerosis, hyperlipidemia, coronary heart diseases, ischemic heart diseases, and stroke due to the presence of high levels of cholesterol in the blood (Cheong et al., 2018).
Nowadays, conventional synthetic lipid-lowering drugs such as fibrates, statins, and bile acid sequestrants had been acknowledged for the treatment of hypercholesterolemia. However, these medications still have limited efficacy and severe side effects including myopathy, rhabdomyolysis, and polyneuropathy (Moosmann & Behl, 2004). It is important to find an alternative for the treatment of hypercholesterolemia from natural sources due to their potential as anti-hypercholesterolemic agents compared to synthetic drugs that have side effects for long-term consumption.
Natural ingredients such as nattokinase (fermented soybean product), red yeast rice extract, Ginkgo biloba, oat fiber, garlic, bee pollen, and propolis have potential effects as anti-hypercholesteraemic agents. The bioactive compounds such as Monacolin K, a product from fermented red yeast rice resembled similar properties as lovastatin potentially decrease the level of HMGCoA reductase activity (Ajdari et al., 2014). Besides, naringin from bee pollen and propolis as antioxidant ability to scavenge against free radicals and block the activity of HMGCoA reductase (Sobral et al., 2017).
Tocopherol derivatives, tocotrienols also provide hepatoprotective properties against fatty liver diseases and interact with plant sterols as soybeans and oat fiber to retard the synthesis of cholesterol (Guo et al., 2014;Hoene et al., 2018). Glutamate derivatives, glutamic acid that mainly found in soybeans and oat help to down-regulate the cholesterol biosynthesis by suppressing LDL level in rats (Wan Saidatul Syida et al., 2018).
Previous studies also reported the anti-hypercholesterolemic properties of the single ingredients as nattokinase (fermented soybean) is regarded as an anti-atherosclerotic agent to suppress intimal thickening in rats (Chen et al., 2018). Besides, red yeast rice extract can decrease blood cholesterol by reducing lipid peroxidation (Yeap et al., 2014). Ginkgo biloba may stimulate the production HDL level (Kang, 2017). Oat fiber aid in lowering total cholesterol levels due to its role in altering the metabolism of bile acids. Garlic has allicin compound that may reduce streaks formation (atherosclerosis) and bee pollen control the elevation of lipid and cholesterol at the normal level and prevent clumping of blood platelets (Komosinska-Vassev et al., 2015). Propolis can suppress the level of triglyceride in the rats (Albokhadaim, 2015).
Previous studies have shown promising effects of each constituents of these functional foods in alleviating hypercholesterolemia in vivo (Bharti et al., 2017;Desamero et al., 2017;Weng et al., 2017;Xu et al., 2014;Yang et al., 2018). Predicated upon that the present study aimed to determine synergistic effects of bioactive compounds in mixtures of these selected functional foods in hypercholesterolemia-induced rats.

| Composition of 1% HCD diet
The diet was based on a purified atherogenic diet that consisted of 1% (10 g/kg) of cholesterol, 0.5% (5 g/kg) of sodium cholate.

| Identification of bioactive compounds by LC-MS/MS
Briefly, 10 mg extract from mixtures of selected functional foods (MSFF) were macerated with 1 ml of aqueous solutions of methanol and filtered using a 0.2 mm chromatographic filter and LC-MS/ MS operating system that coupled to HPLC (Matei, Gatea, & Radu, 2015). The chromatographic separations were accomplished by C18 column with particle size at 1.9 µm, (length: 100 mm × diameter: 2.1 mm) and the column temperature was kept at 40°C. Solution A (Water + 0.1% Formic Acid) and Solution B (Acetonitrile) were served as mobile phases at flow rate of 0.4 ml/min. The injection volume was 2.0 µl and the column temperature was set at 40°C. The mass spectrometer was coupled with heated electrospray ionization (HESI) sources that run in negative and positive ion modes with high sensitivity. Data were analyzed and searched by comparing parent ion fragmentation pattern m/z with database (PubChem, Mass Bank, and Chem Spider).

| Measurement of body weight changes and absolute liver weight
The measurement of the body weight of all groups was taken regularly at week 0, week 4, and week 8. The absolute liver weight of all groups was measured at end of week 8 based on the formula according to Adeneye et al., (2010).

| Histopathological analysis of liver tissues
Liver tissues were fixed in 10% formalin phosphate buffer solution, dehydrated, paraffin-embedded, and archived. All samples had been undergone sectioning process and mounted on aminopropyltriethoxysilane-coated slides. Next, deparaffinization in xylene, sections rehydrated, stained with hematoxylin and eosin (H & E), and examined by light microscopy (Yeh et al., 2010).

| Enzymatic assays (HMGCoA reductase and ACAT2)
Enzymatic assays of HMGCoA reductase and ACAT2 in rat's liver were determined respectively using ELISA Kits according to the manufacturer's instruction and measured at 450 nm.

| Data analysis
All results in this experiment were expressed as mean ± standard error (SEM). One-way ANOVA (Tukey test) was used for all experiments. Two-way repeated-measures ANOVA by post hoc Bonferroni test was analyzed for body weight changes and all values with p < .05 were considered significant.  Table 1 listed the peak, retention time, compounds, integrated peak area, ion mode, and precursor ion for m/z value, molecular weight, and chemical formula respectively. About twenty-four (24)   revealed that 1% HCD group was showed a significant (p < .05) increased (433.00 ± 11.41 g) of body weight than normal group (340.00 ± 10.87 g). There were no significant different (p > .05) of body weight changes of all treated rats with MSFF at 50 mg/ kg (421.16 ± 16.51 g) 100 mg/kg (422.83 ± 16.26 g) , 200 mg/ kg (415.17 ± 20.63 g) and treatment with SVS at 10 mg/kg (414.83 ± 14.04 g) compared to 1% HCD group (433.00 ± 11.41 g).

| Biochemical analysis of serum lipid profiles (TC, TG, LDL, and HDL)
As shown in Figure 4 1% HCD group exhibited a significant (p < .05) increased of TC level (2.91 ± 0.19 mmol/L) and LDL (1.35 ± 0.33 mmol/L) than normal group. Treatment of MSFF at different doses (50 mg/kg, 100 mg/kg, and 200 mg/kg) was found a significant (p < .05) decreased of TC and LDL levels compared to 1% HCD group. Treated rats with SVS at 10 mg/kg showed no  HCD group (Figure 4).

| Serum and tissue malondialdehyde (MDA) levels
Serum MDA level was found a significant increased (p < .05) in 1% HCD group (25.06 ± 1.49 µmol/L) compared to normal group For tissue MDA level, 1% HCD group had a significant (p < .05) increased (74.42 ± 5.72 µmol/mg) compared to normal group (7.97 ± 0.30 µmol/mg) (Figure 7).Treated rats with all doses of MSFF and SVS showed significant (p < .05) decreased of MDA levels F I G U R E 2 Body weight changes during week 0 (initial weight), week 4 (after induction) and week 8 (after treatment) for all experimental rats. Values are expressed as mean ± standard error, SEM (n = 6). Significance was measured by performing one-way ANOVA followed by Bonferroni's post hoc test, p-value for ANOVA for repeated measures was given (p < .05)

| Histopathological analysis of liver tissues
The hepatic strands of hepatocytes indicated the normal alignment in absence of ballooned fat vacuoles surrounded central vein (CV) in the liver tissue of normal rats (Figure 8a) (Brunt et al., 1999;Takashi & Fukusato, 2014). Normal group had been scored as 0 due to absence of hepatic steatosis indicated as healthy (<5%) ( Table 2)

| Enzymatic activities of HMGCoA reductase and ACAT2
HMGCoA reductase activity was significant ( decreased compared to 1% HCD group (Figure 9). The activity of HMGCoA reductase was found a significant (p < .05) decreased in MSFF treated rats at 100 mg/kg and 200 mg/kg when compared to SVS groups but no significant different (p > .05) when compared to normal group.
As shown in Figure 9, ACAT2 activities showed a signifi-  Feeding with 1% of high cholesterol diet had induced hypercholesterolemia with contributed to body and liver weight gain, impaired serum lipid profiles, the elevation of liver enzymatic activities, and oxidative stress. Composition of this diet that rich in 62% saturated fatty acid may cause greater body fat deposition in rats (Kai et al., 2015). Besides, 1% cholesterol would disrupt the normal physiological function of the liver to metabolize lipid and triggered the accumulation of fatty acid on hepatocytes (Cheong et al., 2018).

| D ISCUSS I ON
Therefore, these conditions eventually elevated total cholesterol production and LDL in circulating blood.
Administration of MSFF at 50 mg/kg, 100 mg/kg, and 200 mg/ kg were found to maintain body weight gain due to the availability of tocopherol and lignoceric acid compound. Tocopherol would possess hepatoprotective effect against fatty liver disease based on previous studies (Górnicka et al., 2019). Besides, lignoceric acid was classified as very long saturated fatty acids (VLSFA) found to be beneficial in improving metabolic disorders and cardiovascular diseases through weight gain control (Lee et al., 2015). Simvastatin (SVS) treatment at 10 mg/kg also involved in weight control but the mechanism of weight reduction was till unclearly defined because it mainly involved in the structural damage in muscles (Suzuki et al., 2018). Means values with different letters were significantly different at level (p < .05) between all groups. 1% HCD, 1% high cholesterol diet, MSFF, mixtures from selected functional foods, SVS, simvastatin. Scoring of hepatic steatosis based on percent of hepatocytes in the biopsy involved (<5%: 0 (healthy), 5%-33%:1 (mild) , 34%-66%:2 (moderate) and >66%: 3 (severe) (Brunt et al., 1999).
High cholesterol feeding at 1% becomes as an extrinsic inducer to catalyst elevation of serum TC, LDL, and TG. The additional bile salts derived from (0.5%) of sodium cholates which act as primary bile acids and deoxycholic acid as secondary bile acid thus, increasing the capacity of the liver to down-regulate bile acid synthesis that facilitates the excretion of cholesterol (Hofmann & Hagey, 2008). Besides, the increment of cholesterol levels in the blood may trigger inflammation at kidneys and stimulate renal oxidative stress (Rajeswari et al., 2017). The deposition of fatty acid on the liver had triggered the elevation of serum AST and ALT in hypercholesterolemic conditions to indicate as liver injury (Adekiya et al., 2018).
Besides, the production of cholesterol biosynthesis could be suppressed by inhibiting the enzymatic activities of HMGCoA reductase activity. Inhibition of ACAT2 enzymatic activities also may prevent the esterification of free cholesterol before being transported in form of VLDL into LDL to be delivered through all parts of the body system (Nguyen et al., 2019). Oxidative stress had triggered the production of MDA levels in serum and tissue in hypercholesterolemic rats that might facilitate the development of hepatic steatosis and toxic to cells (Repetto et al., 2012).
Cholesterol-lowering effects of bioactive compounds produced by combination of MSFF may exert curative action on hypercholesterolemia at different doses (50 mg/kg, 100 mg/kg, and 200 mg/kg).
Monacolin K was known as a secondary metabolite from a fraction of red yeast rice extract had possessed lipid-lowering properties via inhibition of HMGCoA reductase activities to retard cholesterol production (Ajdari et al., 2014). Besides, naringin majorly found in propolis and bee pollen exhibited strong antioxidant properties that belong to the flavonoid compound would share a similar mechanism as Monacolin K with availability of tocopherol in reducing cholesterol level (Pengnet et al., 2019;Sobral et al., 2017). In our study,  (Liu et al., 2019).This could be revealed that previous study reported that up to 200 mg/ kg administration of single fermented soybean extract was found to inhibit HMGCoA reductase activity by 28.30% in treated rats which represented only at almost half percentage of reduction compared to our data (Pyo & Seong, 2009).
Naringin has its part of aglycone, naringenin might interact together with tocopherol, isoflavones and phytosterols content from soybean fraction in MSFF to exert greater inhibition of ACAT2 activities range from 49.7% until 59.0%. Previous study reported that ACAT2 activities were inhibited at smaller range for treatment using single naringenin (35.3%), and oat extract (17.0%) at 100 mg/kg dose (ElRabey, 2013;Wilcox et al., 1999). It revealed that combination of these compounds had powerful ability to suppress the expression of microsomal triglyceride transfer protein (MTP) that involved in packaging cholesterol ester (CE) into chylomicrons from ACAT2 activity (Borradaile et al., 2002;Zeka et al., 2017). Hence, this process directly lowers the total cholesterol level and cholesterol absorption in the regulation of hypercholesterolemia.
The administration of simvastatin (SVS) at 10 mg/kg, a cholesterol-lowering drug also had demonstrated no significant effect on serum lipid profiles (TC, LDL, and TG) that mainly attributed by origin source, initial weight, feeding sessions, duration of the study, stress level and environmental conditions of experimental rats. The primary site of simvastatin action was at the liver to block the activity of HMGCoA reductase. However, the long-term consumption of this drug may affect liver function, loss of appetite, myopathy with an elevation of creatinine kinase and rhabdomyolysis (Tiwari & Khokhar, 2014).

| CON CLUS ION
Effects of bioactive compounds such as monacolin K, naringin, tocopherol in this combination of selected functional food mixture (MSFF) may exert curative action on hypercholesterolemia via inhibition of lipid peroxidation, HMGCoA reductase and ACAT2 activities compared to the treatment using single ingredient. The beneficial effects of these functional ingredients should be advocated as an alternative treatment to combat hypercholesterolemia.

ACK N OWLED G EM ENTS
The authors would like to thank the sponsor for providing the investigational product (mixtures of functional foods).

CO N FLI C T O F I NTE R E S T
The authors declare no conflict of interest.

DATA AVA I L A B I L I T Y S TAT E M E N T
The data that support the findings of this study are available from the corresponding author upon reasonable request.