A herb mixture to ameliorate non-alcoholic fatty liver in rats fed a high-fat diet

This study was performed to investigate the effects of an herb extract mixture (HM) in ameliorating non-alcoholic fatty liver disease (NAFLD). The HM contained equal amounts of 70% ethanol extracts from Zingiber officinale, Centella asiatica, and Boehmeria nivea. In vitro, the HM significantly inhibited lipid accumulation in oleic acid-stimulated HepG2 cells. We further evaluated the anti-NAFLD activities of the HM in vivo in an animal model. Rats were fed two different amounts of the HM (50 and 200 mg/kg body weight) along with a high-fat diet for 6 weeks. HM supplementation reduced liver weight; epididymal, peri-renal, and intra-abdominal fat content; and serum triglyceride, total cholesterol, and low-density lipoprotein cholesterol levels as well as increased high-density lipoprotein cholesterol levels in a dose-dependent manner. Histological evaluation of liver specimens further demonstrated that administration of HM significantly prevented hepatic lipid accumulation and subsequent development of hepatic steatosis. These findings suggest that HM can be used as an alternative nutraceutical for ameliorating NAFLD.


Introduction
Non-alcoholic fatty liver disease (NAFLD) is a condition associated with the accumulation of lipids in the liver and can progress into simple steatosis, steatohepatitis, fibrosis, cirrhosis, and hepatocellular carcinoma [1,2]. Global NAFLD prevalence and incidence are higher than previous estimated [3]. Especially, NAFLD prevalence is rapidly increasing in certain subpopulations such as obese and metabolic syndrome [4]. Several molecular and metabolic changes with the 'multiple hit' such as insulin resistance, hormones, nutritional factors and gut microbiota take place in NAFLD [5]. Current study showed that anti-diabetic agents, glucagon-like peptide-1 modulators, sodium-glucose transport protein inhibitors, and thiazolidinedione insulin sensitizers can be potentially used to NAFLD medicine [6]. Modulation of glucose and lipid metabolism by natural products alleviated NAFLD [7,8]. However, there is no FDA approved the medicine for NAFLD treatment. Development of medicine for NAFLD is urgently needed.
Development of a single herb extract for treating certain diseases has been the main focus of research and development to date [9][10][11][12]. Zingiber officinale (the rhizomes of ginger) is a herbal medicine used to relieve inflammation, high cholesterol Abbreviations: eWAT, Epididymal white adipose tissue; HDL, High density lipoprotein; HFD, High-fat diet; HM, Herb extract mixture; LDL, Low density lipoprotein; NAFLD, Non-alcoholic fatty liver disease; OA, Oleic acid; ORO, Oil Red O; TC, Total cholesterol; TG, Triglyceride.
(hypercholesterolemia), obesity, and diabetes [13,14]. Ginger treatment was shown to significantly decrease the levels of both serum cholesterol and triglycerides in mice [15]. Centella asiatica is Asian traditional herb that has protection role from hepatotoxicity through inhibition of oxidative stress, inflammation and apoptosis [16]. Centella asiatica was also reported to ameliorate early stage of hyperlipidemia in rats [17]. Boehmeria nivea has been used for its anti-inflammatory and anti-oxidative properties [18,19].
Herb mixtures (HM) is useful approach to development therapeutic agents [20,21]. We previously explored the functionality of herbs by evaluating changes in gene expression profiles before and after herb extract addition to various cell lines. This system is unique in terms of inferring functionalities of herb mixtures compared to typical research which focuses on the genetic effects of individual herb extracts. A prerequisite for selecting an appropriate herb mixture is that it is expected to have better functionality than the individual herb components based on the functionality inferring system. In this study, we investigated the effect of Zingiber officinale, Centella asiatica, and Boehmeria nivea mixture on hepatic lipid accumulation using HepG2 cells and in NAFLD rat models.

Preparation of herbal extracts
The three herbs, Z. officinale, C. asiatica, and B. nivea, were purchased from DAMAONYAKCHO (Yeongcheon-si, Gyeongsangbukdo, Republic of Korea). To prepare the extracts from 100 g of pulverized powder, each powder was suspended in 2 L of 70% aqueous ethanol and ultrasonicated for 1 h in a 750-W ultrasonic processor (VCX 750, Sonics and Materials, Inc., Newtown, CT, USA). Extraction in the ultrasonic processor was repeated three times. The undissolved residue was filtered out using quantitative Whatman No. 1 filter paper (Whatman, Maidstone, UK) and centrifugation. The extracts were evaporated on a rotary vacuum evaporator (R205, Buchi, Fostfach Switzerland) and lyophilized to yield a dry powder.

Cell viability assay
For cell viability tests following exposure to oleic acid (OA) and herbal extracts, HepG2 cells were seeded at a density of 5 × 10 4 cells/well in 96-well plates and incubated with various concentrations of OA (Sigma-Aldrich, St. Louis, MO, USA) or herbal extracts in 1% bovine serum albumin (BSA, Sigma, USA)-supplemented DMEM for 24 h. The medium containing only 1% BSA was used as the control. After incubation, the medium was discarded, and 0.1 mg/ml MTT reagent was added and incubated for 4 h. The solution was removed, and dimethyl sulfoxide was added to dissolve the formazan crystals. The absorbance of the solution at 570 nm was measured with a spectrophotometer (Molecular Devices, Sunnyvale, CA, USA).

ORO staining
HepG2 cells were seeded at a density of 2 × 10 5 cells/well in 24-well plates and incubated with OA (0.6 mM) and/or individual herbs (Z. officinale, C. asiatica, B. nivea; 100 μg/ml) or the combinational herb mixture (HM; 50 or 100 μg/ml) in 1% BSA-supplemented DMEM for 24 h. An equal proportion of extracts obtained from Z. officinale, C. asiatica, and B. nivea was mixed and used as the HM. After washing the cells twice with Dulbecco's phosphate-buffered saline, they were fixed with 10% formalin for 30 min. The cells were washed again with Dulbecco's phosphate-buffered saline and stained with a freshly prepared solution of ORO (Sigma-Aldrich) for 15 min at room temperature in the dark. After washing several times, the cells were observed under a microscope (Nikon, Tokyo, Japan). To quantify the ORO content, isopropanol was added to each sample followed by shaking at room temperature for 10 min. The optical density of the isopropanol-extracted sample was measured with a spectrophotometer at 510 and 630 nm.

Animal model of NAFLD
Seven-week-old male Sprague-Dawley rats (n = 32, 280 ± 10 g) were purchased from Orient (Seungnam, Korea). The animals were kept under controlled environmental conditions (22 ± 3 • C with a 12/12-h light/dark cycle) for one week prior to the experiment. The rats were divided into the following four groups (n = 8 per group): ND, HFD, HFD + 50 mg/kg HM (HM50), and HFD + 200 mg/kg HM (HM200). An equal proportion of extracts obtained from Z. officinale, C. asiatica, and B. nivea was mixed and used as the HM. All rats were provided food and water ad libitum. All groups except the ND (Teklad rodent diet 2918C; ENVIGO, Barcelona, Spain; 5.77% fat, 44.2% carbohydrate, 17.7% protein) were provided a 60% fat calorie diet (Teklad Custom diet TD.06414; ENVIGO, Barcelona, Spain; 60.3% fat, 21.4% carbohydrate, 18.3% protein). The HM was orally administered daily for 6 weeks to the two HM groups along with the HFD. Feed intake and body weight were measured weekly for 6 weeks. At the end of the experimental period, the animals were fasted for 14 h prior to anesthetization with intraperitoneal injection of ketamine (80 mg/kg) and xylazine (8 mg/kg) and sacrifice. The blood from abdominal aorta (800 μl), liver, and parametrial adipose tissues (epididymal fat, peri-renal fat, and intra-abdominal fat) were quickly removed and measured weight. All animal studies were performed in accordance with international regulations of the S.K. Ha et al. usage and welfare of laboratory animals and protocols approved by the Institutional Animal Care and Use Committee in Chonbuk National University Hospital in terms of ethic procedures and scientific care (IACUC, cuh-IACUC-2017-18).

Biochemical analysis
The collected blood was left at room temperature for 30 min and then centrifuged at 3000 rpm for 10 min to obtain the serum. The serum TG and TC levels were measured with commercial enzymatic assay kits from Asan Pharmaceutical Co. (#AM202, Seoul, Korea), and HDL and LDL levels were measured using commercial enzymatic assay kits from BioVision (#K613-100, Milpitas, CA, USA).

Histopathological examination
Harvested livers and eWAT were fixed in 10% formalin. After fixation, the tissues were washed in tap water and then paraffinized.
Paraffinized tissues were embedded in a paraffin block then cut into 5 μm thick sections with a rotary microtome. The sections were deparaffinized in xylene, rehydrated through a descending alcohol series (100%, 95%, 90%, 80%, and 70%), and hydrated sections were washed in PBS. After removing PBS by wiping, the sections were stained with Harris' hematoxylin for 5 min and eosin for 3 min. The sections were mounted using a cover glass and DPX Mountant for histological analysis. The mounted sections were imaged using an upright microscope (Olympus, Tokyo, Japan). The diameter of cells (n = 100 cells) was measured from four sections for each group and analyzed using ImageJ software (NIH, Bethesda, MD, USA).

Analytical method condition
Analysis was performed on the High-performance liquid chromatography (HPLC) system (Waters Corporation, Milford, MA, USA). The HPLC system was equipped with a Waters Alliance e2695 Separations Module and UV/VIS detector (Waters, 2489). The output signal of the detector was recorded using a Empower® 3 software. The separation was executed on a YMC Hydrosphere C 18 column

Statistical analysis
Data are expressed as the means ± standard error of the mean for the number of replicates indicated. The significance of differences between groups was assessed by one-way ANOVA with Tukey correction using Prism 7.0 software (GraphPad Software, La Jolla, CA, USA).

Effect of HM on lipid accumulation in HepG2 cells
We used OA to prepare an in vitro model of NAFLD in HepG2 cells to explore the potential lipid-lowering effects of the HM. The cytotoxicity of OA and HM was first assessed. OA and HM did not affect the viability of HepG2 cells at up to 0.6 mM and 200 μg/ml, respectively, based on the results of MTT assays (data not shown). To verify the inhibition of OA-induced lipid accumulation by HM or the individual herb extracts, HepG2 cells were treated with 100 μg/ml concentrations of individual herb extracts or the HM in the presence of OA (0.6 mM) for 24 h. The cells were then stained with ORO, and lipid accumulation was quantified by measuring the absorbance at 510 nm. HM reduced the OA-induced ORO signal in a dose-dependent manner (Fig. 1A). Specifically, lipid accumulation was reduced by 30% and 55% at HM concentrations of 50 and 100 μg/ml, respectively (Fig. 1B). Whereas the individual herbs Z. officinale, C. asiatica, and B. nivea did not show any inhibitory activity on OA-induced lipid accumulation in HepG2 cells (Fig. 1B).

Effects of HM on the body fat index of rats
To further investigate the ameliorating activity of HM against NAFLD, HM was supplemented to rats fed HFD. After 6 weeks of HM supplementation, the liver, epididymal fat, peri-renal fat, and intra-abdominal fat tissues were harvested from the rats, and their weights were measured. There were no significant differences in feed intake and body weights between the HFD and HM groups after 6 weeks ( Fig. 2A and B). However, the weights of the epididymal fat, peri-renal fat, and intra-abdominal fat tissues were significantly decreased in the HM group compared to those in the HFD group (Fig. 2C-F).

Effects of HM on serum lipid profiles of rats
Treatment with HM caused a significant decrease in serum TG, TC, and LDL levels of rats fed the HFD (Fig. 3A-D). The levels of serum TG in both HM groups (50 and 200 mg/kg) were significantly lower than those in the HFD group, whereas only 200 mg/kg HM resulted in a significant decrease in the serum TC level ( Fig. 3A and B). The HFD caused a significant decrease in HDL levels after 6 weeks, whereas daily co-supplementation with 200 mg/kg of HM alleviated HFD-induced HDL reduction. Both HM groups (50 and 200 mg/kg) showed a significant reduction in serum LDL compared to that in the HFD group after 6 weeks of feeding ( Fig. 3C and D). These results indicate that supplementation of HM helps to maintain a normal serum lipid profile under HFD feeding.

Effects of HM on histology of rat tissues
To examine whether the reductions in liver weight was attributed to decreased hepatic lipid accumulation, the liver samples were fixed, sectioned, and stained with hematoxylin and eosin. The staining results revealed that lipid droplet in the HFD group was observed, whereas HFD-induced lipid droplet was hindered by HM supplementation (Fig. 4A). The HFD group also displayed a significantly increased eWAT mass and adipocyte area than the ND-fed group, which was also significantly suppressed by  supplementation with both 50 and 200 mg/kg body weight/day of HM (Fig. 4B). These data suggest that supplementation of HM prevents HFD-induced lipid accumulation in liver and eWAT.

Analytical method development and validation
A simple and reliable analytical method has been developed and validated for simultaneous determination of the three major components (kaempferol-3-O-rutinoside, asiaticoside, 6-gingerol) in herb mixture ( Fig. 5A and B). Method validation was executed by linearity, precision and accuracy test on the basis of ICH guidelines [22]. The calibration curve of three components showed good linearity (R 2 > 0.9962) ( Table 1). The limit of detection (LOD) and limit of quantification (LOQ) were observed within the ranges 0.013 to 0.07 and 0.04-0.21 μg/μL, respectively. The relative standard deviation (RSD) values of intra-and inter-day testing were indicated that less than 3%. The results of recovery test were 96.28-99.99% and RSD range was measured from 0.03 to 1.74% (Table 2). In conclusion, this analytical method has been successfully applied to the simultaneous determination of three components in herb mixture.

Discussion
There is many studies to develop the NAFLD drugs using single herb extract [23,24]. Z. officinale, C. asiatica, and B. nivea have anti-inflammatory effects in various inflammatory diseases respectively [18,25,26]. However, single dose treatment of each of these are not significantly suppressed lipid accumulation in HFD-induced NAFLD rat model in our previous study (data not shown). In this case, herb mixtures may be more useful as therapeutic approach. In the present study, we evaluated effect of herbal mixture from Z. officinale, C. asiatica, and B. nivea on NAFLD rat model. Treatment of HM suppressed OA-induced lipid accumulation in HepG2 cells and HFD-induced lipid droplet in NAFLD rat model. Especially, HM treatment improved HFD-induced body fat index in NAFLD model. NAFLD is the most common form of chronic liver disease in parallel with the obesity characteristic, with the fastest emerging manifestations of the metabolic syndrome worldwide [27]. Multiple hits by insulin resistance, hormones and epigenetic factors in fatty liver accelerate NAFLD progression [5]. Current study showed that gut microbiota alternation causes NAFLD [28]. Progressive form of NAFLD, nonalcoholic steatohepatitis, leads to cirrhosis and liver cancer [29]. Accumulating evidence indicates that NAFLD patients are at potential risk for the development of hypertension, coronary heart disease, and cardiomyopathy that clinically result in increased cardiovascular mortality [30]. Many studies showed that herbal extracts have suppression role for NAFLD [28]. However, knowledge on the pathogenesis and therapeutic strategy of NAFLD is still unclear. OA is not an essential fatty acid that is endogenously synthesized by Stearoyl-CoA desaturase 1 in humans [31]. Excessive accumulation of OA through de novo fatty acid synthesis leads to lipogenesis and NAFLD in HepG2 cells [32,33]. Treatment of HepG2 cells with OA induced morphological changes similar to steatotic characteristics [34]. Single treatment of herbal extract from Z. officinale, C. asiatica, or B. nivea not suppressed OA-induced lipid accumulation in HepG2 cells. However, HM treatment suppressed OA-induced lipid accumulation in HepG2 dose dependently. Z. officinale and C. asiatica have anti-hyperlipidemic activity [35,36]. Especially Z. officinale suppressed 3% cholesterol and 15% butter containing diet induced SCD1 expression in rodent liver [35]. These results indicate that HM may suppress OA-induced lipid accumulation through SCD1 suppression and is a potent nutraceutical which may useful for treating NAFLD.
The HFD-induced NAFLD rat model has been widely used for evaluating the pharmacological effects of drugs on NAFLD [37,38]. HFD increased TC, LDL, and TAG levels and suppressed HDL levels in rodent model [39][40][41]. HFD-induced NAFLD model is more accurately reflects the human clinical NAFLD pathologies, including obesity, insulin resistance, and high serum triglycerides as well as the typical hepatic steatosis, and inflammation [42]. Thus, this model was used for further study to validate the effects of HM on NAFLD. Liver histology analysis showed that HFD induced typical steatosis based on ballooning degeneration and numerous lipid droplets. Adipose tissue mass and dyslipidemia were increased by HFD. This is mainly attributed to adipocyte hypertrophy caused by increased levels of serum TG, TC, and free fatty acids. Supplementation of the HM effectively suppressed HFD-induced body fat indices such as liver weight, peri-renal fat weight, intra-abdominal fat weight and epididymal fat weight induction. HFD-induced serum TG, TC, and LDL induction ameliorated by HM supplementation. HFD-induced lower HDL levels were recovered by HM supplementation. Administration of bioactive compound from natural product reduced HFD-induced TG, TC, and LDL levels and ameliorated HDL levels [43,44]. Z. officinale, C. asiatica, and B. nivea suppress lipid accumulation, blood glucose levels, and fat accumulation in liver respectively [45][46][47]. Therefore, HM supplementation may improve lipid profiles against NAFLD. Histological observation of liver specimens also demonstrated that HFD-induced lipid accumulation and development of hepatic steatosis were suppressed by HM supplementation. These results indicate that HM have anti-hypolipidemic activity which suppressed HFD-induced NAFLD model. HPLC analysis revealed that keampferol-3-O-rutinoside, asiaticoside and 6-gingerol were identified as major compound in HM. Keampferol-3-O-rutinoside has anti-adipogenesis activity [48]. Asiaticoside have protective effects on acute liver injury [49]. 6-Gingerol suppressed HFD-induced NAFLD through activating LKB1/AMPK pathways in mice [50]. These results indicate that HM containing each compounds may suppress HFD-induced NAFLD in rat.
In conclusion, present study has limitation about action mechanism of HM. However, HM treatment markedly suppressed HFDinduced pathological changes in both HepG2 cells and the HFD-induced NAFLD rat model. These data demonstrate that the HM from Z. officinale, C. asiatica, and B. nivea may a potent nutraceutical for alleviating NAFLD.

Ethics statement
All animal studies were performed in accordance with international regulations of the usage and welfare of laboratory animals and protocols approved by the Institutional Animal Care and Use Committee in Chonbuk National University Hospital in terms of ethic procedures and scientific care (IACUC, cuh-IACUC-2017-18).

Author contribution statement
Sang Keun Ha, Guijae Yoo: Performed the experiments; Analyzed and interpreted the data. Jin Ah Lee: Performed the experiments; Analyzed and interpreted the data; Wrote the paper. Donghwan Kim: Analyzed and interpreted the data; Wrote the paper. Inwook Choi: Conceived and designed the experiments; Analyzed and interpreted the data.

Data availability statement
Data included in article/supplementary material/referenced in article.

Declaration of competing interest
The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper.