Antihyperlipidemic and Antioxidative Potentials of Onion (Allium cepa L.) Extract Fermented with a Novel Lactobacillus casei HD-010

The purpose of this study was to investigate antihyperlipidemic and antioxidative potentials of onion (Allium cepa L.) extract fermented with a novel Lactobacillus casei HD-010. In general, fermented onion extract is used for its antioxidative activity (ORAC), inhibitory effect on adipocytes differentiation, quercetin contents, and antihyperlipidemic activities. However, the effect of fermented onion extract on hyperlipidemia after oral administration using ApoE-deficient mice has not been reported yet. To understand the effect of fermented onion extract on hyperlipidemia, we used benzafibrate (10 mg/kg, bw/day) as a positive control in the present study. Serum was collected every week to analyze levels of low density lipoprotein (LDL), high density lipoprotein (HDL), triglyceride (TG), and cholesterol, 3-hydroxy-3-methylgutaryi-CoA (HMG-CoA) reductase activity, and cholesterol ester transport protein (CETP) activity. In the fermented onion-treated group, HDL level was significantly increased while levels of TG and LDL were significantly decreased compared to those in the control group. In addition, the inhibition activity of HMG-CoA reductase was increased 20% in the fermented onion-treated group at 100 mg/kg. CETP activity has been observed to be significantly inhibited in the fermented onion-treated groups compared to that in the control group. These results suggest that fermented onion has a preventive/therapeutic effect on hyperlipidemic disease. It might have potential to be developed as a functional food.


Introduction
Recently, food consumption pattern has considerably changed from traditional fermented food-based intake (Kimchi, fermented soy bean, etc.) to fat containing westernized diet (meat, fats, etc.) in Asia, including Korea [1][2][3]. Westernized food intake pattern is known to increase risks of obesity, high blood pressure, diabetes, and hyperlipidemia [4][5][6][7]. Hyperlipidemia is a risk factor of cardiovascular diseases. Controlling hypercholesterolemia is important to prevent hyperlipidemia. Reducing triglyceride level in blood stream is one of treatments for patients with cardiovascular related disease through inducing LDL receptors and limiting VLDL secretion with certain drugs [8].
There are several medicines for reducing hyperlipidemia symptoms, such as HMG-CoA reductase inhibitor (statins), PPAR-alpha activator (fibrate), CETP inhibitor, bile acid sequestrants, and ACAT inhibitor [9][10][11]. However, long-term treatment with these medications has side effects. Thus, many studies have tried to increase drug efficiency [12,13].  Reducing cholesterol concentration in blood is an important research issue for functional food and drug development to decrease the risk of cardiovascular related diseases. Natural components from plant or organisms are potential candidates to decrease the risk of disease outbreak. Onion (Allium cepa L.) has been used for decreasing blood cholesterol levels [14]. In Asia, it was traditionally used as a medicine due to its fever-reducing, antiparasitic, detoxification, and intestinal anti-inflammation effects [5,[15][16][17]. Major compounds in onion are flavonoids (quercetin, quercitrin, and rutin) and sulfuric compounds (allyl propyl disulfide, diallyl disulfide) with health improving effects [18]. Another method to reduce cholesterol is by using Lactobacillus for fermentation. Lactobacillus has been studied for its cholesterol-reducing effect. Klaver et al. [19] have reported that Lactobacillus can deconjugate bile acid and inhibit the function of cholesterol. However, the effect of fermented onion extract on hyperlipidemia after oral administration using ApoE-deficient mice has not been reported yet. Therefore, the purpose of this study was focused on antihyperlipidemic and antioxidative potentials of fermented onion (Allium cepa L.) with a novel Lactobacillus casei HD-010 in lipid metabolism.

Selection of Bacterial Strain and Culture
Condition. Ten strains were identified from fermented onion and the main strain was Lactobacillus casei HD-010 (Table 1). We used L. casei KCTC 2180 from Korean Collection for Type Cultures as a positive control. The identified strain L. casei HD-010 was cultured at 30 ∘ C for 10 days to ferment onion extract. Onion extract was prepared with minced clean onion after washing with double distilled water three times. Autoclaved onion extract at 121 ∘ C for 15 minutes was used for fermentation. Strain identification medium was prepared using 5.5% MRS broth (Difco, France) with 2.0% agar (Difco, France). Liquid culture media was prepared as the strain identification media without 2.0% agar.

Preparation of Fermented Onion Extract.
A 30-liter fermenter (Biostat C Plus, Sartorius, Sweden) [20,21] was used for onion extract fermentation with 100% onion extract under sterile condition. After cooling the onion extract, 1% HD-010 which was incubated at 37 ∘ C with shaking (200 rpm) for 24 hours inoculated into the fermenter and cultured at 37 ∘ C with shaking (25 rpm) for 10 days. After filtering the fermented onion extract with a filter (0.2 m pore size), the extract was lyophilized (PVTFD20RS, Ilshin Lab. Co. Ltd., Korea) and kept at -80 ∘ C until the experiment was performed. As a positive control, L. casei KCTC 2180 was used. It was prepared with the same method as L. casei HD-010.

Assay of Oxygen Radical Absorbance Capacity (ORAC).
Antioxidant capacities of fermented onions, layers of organic solvents, fractions, and subfractions were determined using ORAC assay as described by Gillespie et al. [22]. Briefly, samples or Trolox (0, 6.25, 12.5, 25, 50, and 100 g/ml) were mixed with phosphate buffered saline (75 mmol/L, pH 7.4, Thermofisher scientific, Waltham, MA, USA). After the addition of -phycoerythrin (0.2 mmol/L) and 2,2'-azobis(2amidinopropane) dihydrochloride (AAPH, 200 mmol/L, Wako Pure Chemical Industries, Ltd., Osaka, Japan) as radical generators were added to wells of a 96-well plate. Fluorescence was measured with a fluorescence ELISA reader (VICTOR5, PerkinElmer, USA) every two minutes for sixty minutes (excitation wavelength: 535 nm, emission wavelength: 590 nm). The equation used to obtain AUC (area under the curve) was as follows: where f0 was the initial fluorescence reading at 0 min and fi was the fluorescence reading at i (from 1 to 60) minutes. Next, 3T3-L1 preadipocytes cells were cultured in differentiation medium (MDI) containing 10% fetal bovine serum (FBS, Gibco), 10 g/ml insulin (Sigma-Aldrich), 0.5 mM 3isobutyl-1-methylxanthine (IBMX, Sigma-Aldrich), and 1 M dexamethasone (Sigma-Aldrich). Two days after stimulation with differentiation inducer (MDI, including 0.5 mM IBMX, 1 M dexamethasone, and 10 g/ml insulin), the medium was switched to DMEM containing 10% FBS and 10 g/ml insulin. Two days later, the medium was changed again to 10% FBS/DMEM. Cells were cultured in 10% FBS/DMEM every two days. Full differentiation was achieved by day 8. Onion extract samples were added to 3T3-L1 cells culture at various concentrations (6.25 ∼ 100 g/ml) on four days after differentiation induction. Intracellular lipid content was measured in 96-well plates using AdipoRed6 assay reagent (Cambrex, MA, USA). On day 8, the treatment medium was removed and the cells were fixed in a 4% formaldehyde solution at room temperature (25 ∘ C) for 5 hrs. After rinsing cells with PBS, each well was added with 200 l PBS and 5 l of AdipoRed reagent. Following incubation at room temperature for 10 min, plates were measured with a fluorescence ELISA reader (VICTOR5, PerkinElmer, USA) at an excitation wavelength of 485 nm and an emission wavelength of 535 nm. Values from each group were used to calculate the 50% effective inhibition concentration (EC 50 ) for reducing adipocyte differentiation. As positive controls, benzafibrate and simvastatin were used.

Separation and Fractionation of Onion Extract
Fermented with L. casei HD-010. Freeze-dried fermented onion extracts were resuspended in distilled water and partitioned with four different organic solvents (n-hexane, CH 2 Cl 2 , ethyl acetate, and n-butanol) and residual H 2 O. These fractions were subjected to decompression enriching and freeze-drying to remove residual solvent. The CH 2 Cl 2 layers were sequentially applied to HP-20, silica gel and RP-C 18 open column chromatography under the same column conditions (3.8 x 60 cm, 300 g) [23][24][25] to obtain the active compound from fermented onions with L. casei HD-010 (LFAc).

Animal Experiments.
Male ApoE-deficient mice (five weeks old) were supplied from Central Laboratory Animal Inc. in Korea and housed at 23 ± 0.5 ∘ C with 55 ± 7% humidity and light-dark cycle (12 hrs : 12 hrs). All animals were acclimated at least one week. They were caged and fed a low fat, low cholesterol control diet D12336 (Central Laboratory Animal Inc., Seoul, Korea). All animal studies were performed in a pathogen-free barrier zone at Kyungpook National University. All procedures used in this study were approved by the Animal Care and Use Committee of Kyungpook National University (IACUC approval number: KNU2012-136).
The control group was fed with a high fat diet. The positive control group was fed with benzafibrate (10 mg/kg). Fermented onion extract was fed into three groups with different amounts by oral administration in 0.5 ml of saline (low dose, 25 mg/kg; medium dose, 50 mg/kg; and high dose, 100 mg/kg). Saline alone group was used as a negative control (N=10/group) [24,27]. Animal experiment design of this study is shown in Figure 1.

Lipid Contents Measurement.
Blood was collected from mouse by retroorbital sinus bleeding method using intraorbital venous plexus every week for six weeks. Blood samples were incubated at room temperature for 30 minutes and centrifuged at 600 g for 10 minutes at 4 ∘ C. Sera samples were prepared and kept at -80 ∘ C until assay. HMG-CoA reductase and CETP inhibition activities were measured using sera samples collected at the last experimental point (6 th week samples). HMG-CoA reductase and CETP inhibition activities were measured using HMG-CoA reductase assay kit (Sigma, USA) and CETP assay kit (Biovision, USA), respectively. Serum was measured for contents of total cholesterol (TC), LDL-cholesterol (LDL-C), HDL-cholesterol (HDL-C), triglyceride (TG) using Asan kit (Asan medical company, Korea) and a Beckman Coulter biochemical analyzer.

Statistical Analysis.
The results are presented as mean ± standard deviation (mean ± SD). The statistical analyses of data were determined by using two-tailed Student's t-test.

Fermented Onion Exhibits Antioxidative Activity.
We have investigated the anti-oxidative activity of onion extract fermented with L. casei HD-010 (LFAc) by ORAC assay. LFAc had higher ORAC value than Trolox, a positive control (ORAC PE of LFAc extract = 1.02).
In order to determine which fractions of the onion extracts fermented with L. casei HD-010 contained antioxidative ingredients, we further separated the extract using four different organic solvents as described in the Materials and Methods section. The LFAc-EtOAc fractions had the highest ORAC value (ORAC PE of LFAc-EtOAc = 1.12) (Figure 2), suggesting that the EtOAc fractions of the onion extract

Adipocyte Differentiation Inhibition.
Fermented onion with L. casei HD-010 (LFAc) showed an inhibitory effect on adipocytes differentiation compared to fresh onion or autoclaved onion (> 20%). The inhibitory effect of LFAc was specifically observed in the CH 2 Cl 2 layer (> 45%) ( Figure 3). As a positive control, benzafibrate had no effect on differentiation. However, simvastatin treatment showed more than 90% of differentiation inhibition. Therefore, LFAc has an inhibitory function by blocking HMG-CoA reductase activity.

Dichloromethane Layers of Onion Extract Fermented with L. casei HD-010 (LFAc) Have Both Adipocytes Differentiation
Inhibiting and Antioxidative Effects. In order to purify and identify the active compound in LFAc for the induction of physiological activity, CH 2 Cl 2 layers were subjected to several isolation procedures (HP-20, Silica gel, and RP-C 18 open column). HLFAc-30 and SLFAc-4 fractions with strong adipocyte differentiation inhibiting activities were sequentially obtained (date not shown) after further isolation of SLFAc-4 fractions in an RP-C 18 open column.
In order to investigate hyperlipidemia inhibitory function after LFAc, MC fraction was subjected to HP-20, silica gel, and RP-18 open chromatography. Antioxidant and adipocytes differentiation inhibition effects were observed for LFAc-HP3 fraction from HP-20, LFAc-S4 fraction from silica gel, and LFAc-C3 from C1 (Table 2).

Quercetin Contents.
Thin layer chromatography (TLC) was used to separate components from raw onion extract * * (FO), sterilized onion extract (AO), and fermented onion extract (LFAc). The pattern was not different between samples and four major spots were found (data not shown). LFAc-C4 fraction showed the best adipocytes differentiation inhibitory effect and an effective single fraction was identified. Quercetin, one of major onion components, Evidence-Based Complementary and Alternative Medicine 5   was identified from LFAc-C4 fraction. FO, AO, LFAc, and LFAc CH 2 Cl 2 were examined with HPLC. Quercetin contents in these fractions were FO, 3.90 ± 0.041 mg/ml; AO, 7.13 ± 0.009 mg/ml; LFAc, 2.89 ± 0.064 mg/ml; and LFAc CH 2 Cl 2 , 20.53 ± 0.304 mg/ml. Quercetin content was not altered by fermentation procedure. However, following fermentation with probiotics, quercetin content was enhanced by almost 10-fold in LFAc-CH 2 Cl 2 (Figure 4).

Body Weight.
The effect of fermented onion extract on body weight was tested for six weeks using mice with a high fat diet. Any significant decrease in body weight has been observed in the fermented onion extract feeding group.
Dietary fiber, flavonoids, and sulfuric components in onion efficiently reduced their body weights compared to the high fat diet alone group (data not shown). This result suggests that oral administration of fermented onion extract has no direct effect on body weight, consistent with other studies [18].

Serum Lipid Contents Measurement.
Serum was collected every week for six weeks and assessed for changes in LDL-C, HDL-C, TG, and TC contents. At the end of the experiment, serum was tested for HMG-CoA reductase and CETP inhibition effect. Fermented onion extract feeding groups (low, medium, and high) showed significant decreases in LDL-C level from the fifth week. The medium and high fermented onion extract feeding groups showed continuous decrease in their body weights (Table 3). In addition, HDL-C level was increased from the first week until the sixth week after administration ( Table 4). The LSP-11 supernatant feeding group showed considerable changes in HDL-C and LDL-C levels at the third and fifth weeks. These data suggest that fermented onion extract might have synergic effects on functions of secondary metabolites from Lactobacillus casei HD-010.
Serum TG level was slightly decreased in all groups compared to that in the control. However, such decrease was not statistically significant. Specifically, high fermented onion extract fed group showed significant decrease in TG level at the first, second, third, and fifth week (Table 5). TC level was reduced in the fermented onion extract fed group from the fifth week (Table 6). However, the positive control group that was fed with benzafibrate and Lactobacillus supernatant did not show any significant difference in TC level, when compared to the control group. Evidence-Based Complementary and Alternative Medicine   Data are presented as mean ± SD (10 animals per group; three independent experiments were performed). Statistical significance between control and treated values was determined by two-tailed Student's t-testand is given as a p value; * p value < 0.05 and * * p value < 0.001. Data represent mean ± SD(10 animals per group; three independent experiments were performed). Statistical significance between control and treated values was determined by two-tailed Student's t-testand is given as a p value; * p value < 0.05 and * * p value  Figure 5: Effect of fermented onion with L. caseiHD-010 on serum CETP and HMG-CoA reductase activity in ApoE-deficient mice at six weeks. Data are presented as mean ± SD (10 animals per group; three independent experiments were performed). Statistical significance between untreated and treated values was determined by two-tailed Student's t-test and is given as a p value; * p value < 0.05 and * * p value < 0.001(versus control group); #p value < 0.05 and ##p value < 0.001 (versus L. casei KCTC 2180 and benzafibrate treated group).
We used ApoE-deficient mice model in order to assess the efficiency of fermented onion extract on reducing lipid accumulation, HMG-CoA reductase inhibition, and CETP inhibition. HMG-CoA reductase is involved in cholesterol synthesis [13,28]. It was reduced following fermented onion extract administration. However, such decrease was not statistically significant ( Figure 5). CETP protein works as a carrier for HDL and LDL into the body [3,29] and HMG-CoA reductase is involved in cholesterol synthesis [13,28]. As shown in Figure 5, CETP activity and HMG-CoA reductase were significantly following fermented onion extract administration ( Figure 5). These data suggest that fermented onion extract can efficiently block intestinal fat adsorption through inhibition of CETP activity.
Hyperlipidemia is an important issue in healthcare. It is involved in many serious cardiovascular diseases. Many Evidence-Based Complementary and Alternative Medicine 9 experimental and clinical studies have shown that hyperlipidemia may cause hypertension, diabetes, and obesity [4,6,7,19].
Many studies have reported that onion components or Lactobacillus can lower lipid contents in blood. Onion is a well-known traditional medicine. It has been investigated for many epidemiologic studies [19,30]. In Asian countries, onion and garlic plants that contain diallyl sulfide and quercetin are used to prevent cardiovascular diseases. Onion contains about 90% water, 7 ∼8% sugar (mainly fructose), and minor amounts of vitamins [29,31]. S-methyl-L-cysteine sulphoxide is one of the components in onion. It can reduce lipid contents in blood [14,16]. Quercetin has a similar effect in reducing lipid production and synthesis in animal experiment [32]. Lactobacillus can also reduce cholesterol level in blood. Many studies have shown that Lactobacillus can inhibit bile acid readsorption and attachment of cholesterol onto cell wall [33,34]. However, fermented onion extract has not yet been studied well. Few research groups have attempted to develop fermented onion extract drink product.
In this study, we attempted to identify a proper bacterium for onion fermentation and determine its effect on blood lipid level. Our data suggest that fermented onion extract has an effect on lipid metabolism by oral administration.

Conclusions
The main active material responsible for the antihyperlipidemia effect of fermented onion was quercetin. Our results suggest that fermented onion has preventive/therapeutic effect on hyperlipidemic disease. It might have potential to be developed as a functional food.

Data Availability
The data are linked to online repositories of http://www.nodagi .net.