Effects of several tea extracts on nonalcoholic fatty liver disease in mice fed with a high‐fat diet

Abstract Nonalcoholic fatty liver disease (NAFLD) is considered as a severe threat to human health. It has been reported that tea has abundant bioactive compounds and beneficial effects. In our study, the effects of 12 tea extracts on NAFLD were assessed and compared at the dose of 200 mg/kg body weight in mice fed with a high‐fat diet (HFD) for 15 weeks. Enshi Yulu Tea, Fenghuang Narcissus Tea, and Yihong Tea showed strong effects in suppressing the accumulation of epididymal and perirenal adipose tissue as well as the increases of body weight and liver weight. The histopathological analysis revealed that hepatic steatosis and adipocyte hypertrophy induced by a HFD could be ameliorated by tea supplementation. In addition, Enshi Yulu Tea and Qing Brick Tea exerted more remarkable functions on decreasing the level of serum triglyceride and preventing hepatic fat accumulation, respectively. Furthermore, Fenghuang Narcissus Tea, Enshi Yulu Tea, and Qing Brick Tea could reverse the abnormal change in the levels of glutathione and superoxide dismutase. Moreover, 13 phytoconstituents were detected and quantified in these teas with high‐performance liquid chromatography (HPLC) method. The correlation analysis demonstrated that gallic acid might decrease MDA level, and the reduction of liver weight might be attributed to ellagic acid. However, it should be paid attention to some teas that showed hepatotoxicity with elevated levels of aspartate transaminase and alanine aminotransferase. Several teas showed strong effects in the prevention of NAFLD, which could be developed into functional foods against NAFLD.

accumulation of excessive triglyceride (TG) in hepatocytes (hepatic TG > 5% of liver weight) (M. H. Pan et al., 2014). Thus, the prevention of NAFLD is important for the control and management of the liver disease. Additionally, overweight and obese people are more susceptible to NAFLD (Stefan et al., 2019). Obesity is closely associated with the development of NAFLD (Meng et al., 2018). Excessive intake of dietary fat and energy could increase the TG store in adipocytes, which results in obesity. The lipolysis of adipose tissue increases the level of free fatty acids (FFAs), which could promote the synthesis of TG in the liver (Pan et al., 2014). Meanwhile, overloaded FFAs are oxidized in the liver, which causes excessive production of reactive oxygen species (ROS), resulting in oxidative stress (Shin et al., 2018). Furthermore, oxidative stress can give rise to lipid peroxidation as well as hepatocyte injury, thus promoting the progress of NAFLD (Pan et al., 2014). Therefore, some natural products which possess strong anti-obesity and/or antioxidant activity could be a good alternative for the prevention of NAFLD because of the limited efficacy and potential side effects of chemical drugs.
Tea (Camellia sinensis) is famous all over the world and its unique taste makes it a popular drink. Tea has multiple biological activities, such as antioxidant, anti-cancer, anti-obesity, and antidiabetic properties, because it is abundant in bioactive compounds such as catechins Meng et al., 2019;Xu et al., 2020;Zhao et al., 2019). Catechins mainly includes epigallocatechin gallate (EGCG), gallocatechin gallate (GCG), gallocatechin (GC), epigallocatechin (EGC), epicatechin (EC), epicatechin gallate (ECG), catechin (C), and catechin gallate (CG). Due to the diverse fermentation degrees, tea can be categorized as white, green, yellow, oolong, black, and dark teas . A meta-analysis showed that green tea consumption could decrease the risk of NAFLD (Yin et al., 2015). Moreover, a clinical trial demonstrated that 700 ml/ day of green tea could decrease the body fat as well as improved liver function of patients with NAFLD (Sakata et al., 2013). In addition, the treatment of epigallocatechin gallate (EGCG) alleviated obesity as well as hepatic steatosis in a mouse model induced by high-fat diet (HFD) (Bose et al., 2008). However, previous studies generally focused on one category of tea. This research was designed to assess and compare the effects of 12 kinds of teas selected from six categories on NAFLD induced by HFD. Most of these teas were firstly evaluated for their effects against NAFLD.
The findings might provide useful information for the development of dietary supplements or functional foods against NAFLD, and guidance for the public to choose the most effective tea for the prevention and management of NAFLD.  Table 1 showed the details of 12 kinds of teas, including the category, fermentation degree, and production place. The extraction process of tea was based on the published literature . Firstly, 10 g of tea was placed in 100-mL boiling deionized water and extracted in a 98°C water bath for 10 min. The tea leave was extracted for three times and the tea extracts would be collected and filtered.

| Extraction of teas
Then, the tea extracts could be concentrated to about 15 ml by a vacuum rotary evaporator at 60°C. The concentrated infusions were frozen into powders by a lyophilizer in the end. The powder of tea extracts was kept at − 80°C, and it was dissolved in deionized water to obtain tea extracts with a concentration of 20 g/L (w/v) when needed.

| Animal study
C57BL/6J male mice (18-20 g) were obtained from the Experimental Animal Center of Guangdong province, Guangzhou, China. The experimental protocols of mice used within this study complied with the ethical guidelines suggested by the Animal Care Committee at the School of Public Health, Sun Yat-Sen University (No. 2019-002;28 February 2019). The mice were housed in a room of specific pathogen-free condition and the room kept at temperature of 22 ± 0.5°C and humidity of 40%-60% with a 12-hr light/dark cycle. After one-week acclimatization, 8-week-old mice were randomly divided into 14 groups (n = 10), which includes a normal diet (ND) control, a HFD model group, and 12 tea-treatment groups. The mice in the control group received a ND (3.6 kcal/g, 12% calories from fat), which was obtained from Jiangsu Xietong Pharmaceutical Bioengineering Co., Ltd. (Nanjing, China). The other groups were fed with HFD (5.0 kcal/g, 60% calories from fat, TP 23,400), which was obtained from TROPHIC Animal Feed Hightech Co., Ltd. (Nantong, China). High doses of EGCG might cause liver injury. According to some published literature, the intervention groups in our study were administrated with 200 mg/(kg•d) tea extracts for 15 weeks by intragastric gavage (Bae et al., 2018;Liu et al., 2019). Meanwhile, the control and model groups were given deionized water (10 ml/kg) intragastrically. All mice had free access to diet as well as water. The consumption of food as well as body weight was recorded daily and weekly, respectively. When the experiments come to end, all mice were weighed and anesthetized after fasting for 12 hr. Then removing the eyeballs of mice, the blood samples were obtained and stored at 25°C for 1 hr. Then, the blood samples were centrifuged at the condition of 3,000 × g as well as 4°C for 15 min to obtain the serum, which was kept at 4°C before further biochemical assays. Moreover, the liver, perirenal adipose tissue as well as epididymal adipose tissue were harvested.
A part of the liver as well as a piece of epididymal adipose tissue were cut for histopathological analysis, and the rest of liver was kept at − 80°C for further use.

| Biochemical analysis of serum
The contents of AST, ALT, TC, TG as well as LDL-C in serum were measured by kits obtained from Roche diagnostics (Shanghai, China).
Briefly, the contents of TG, TC as well as LDL-C were determined via enzymatic tests. Additionally, the contents of AST as well as ALT were detected through velocity tests.

| Determination of hepatic TG and total protein contents
The 25 mg of hepatic tissue was mixed with 500 μL of lysis buffer, and then the mixture was homogenized. The supernatant of liver homogenate was heated at 70°C for 10 min, and the product was centrifuged to obtain the supernatant (2,000 × g, 25°C, and 5 min). The detection kits of hepatic TG and total protein were obtained from Apply-gen Technologies Inc (Beijing, China).

| Determination of GSH, SOD as well as MDA levels in liver
The liver tissue was mixed and homogenized with 0.9% NaCl to prepare 10% (w/v) hepatic homogenate. The liver homogenate was centrifuged at the condition of 2,500 × gas wells at 4°C for 10 min, and the supernatant was taken for the detection of glutathione and superoxide dismutase. We purchased the kits of GSH and SOD from Nanjing Jiancheng Bioengineering Institute (Nanjing, China). The detection kits of MDA were obtained from Apply-gen Technologies Inc (Beijing, China).

| Histopathological analysis
A part of the liver as well as a piece of epididymal adipose tissue was fixed by 4% paraformaldehyde for a few days before embedding in paraffin. Subsequently, the embedded samples could be sliced to obtain 5µm-thick sections, which were deparaffinized, rehydrated, and finally stained with hematoxylin and eosin. At last, a light microscope was used for the image capturing of the liver and epididymal adipose tissue.

| Statistical analysis
The data and values were shown as mean ± standard deviation (SD). Comparisons of statistical significance between groups were conducted via one-way analysis of variance plus a posthoc least significant difference test with SPSS version 25.0 (IBM SPSS,

| Effects of tea on body weight
The effects of 12 teas on energy intake as well as body weight in mice receiving HFD are shown in Figure   which have been reported that the consumption of tea could reduce food and energy intake, like black tea and green tea (Du et al., 2005;Huang et al., 2014;Pan et al., 2018). Furthermore, previous studies pointed out that EGCG in teas contributed to the decrease in food consumption as well as body weight via the suppression of appetite (Kao et al., 2000).

| Effects of tea on fat mass
The

| Histopathological evaluation of liver
The preventive effect of several tea treatments against hepatic steatosis was further verified by histopathological assay (Figure 5).
No obvious damage was found in the liver of ND group (Figure 5a).
However, in HFD fed mice, the hepatocytes did not arrange orderly and there was extensive hepatocyte steatosis with many lipid droplets, indicating that simple liver steatosis occurred ( Figure 5b). As displayed in Figure 5c and d, tea supplementation significantly attenuated the accumulation of lipid droplets as well as alleviated hepatic steatosis led by the HFD. However, Figure 5e as well as Figure 5f revealed that inflammatory cell aggregation was seen in White Peony Tea and Pu-erh Tea groups.

| Effects of tea on serum lipid profiles
The results of serum TG, total cholesterol (TC), and low-density lipoprotein cholesterol (LDL-C) levels are displayed in Figure 6.

| Effects of tea on liver redox state
As displayed in Table 2 (Leung & Nieto, 2013). The activity of SOD could be elevated and the level of MDA could be reduced by 0.5%-1% green tea extract in a leptin-deficient (ob/ob) mouse model (Park et al., 2011).
However, our findings were not in accordance with some studies.
The content of MDA was not markedly elevated in the HFD group, we were unclear whether it resulted from the activation of SOD.
We speculated that two factors might account for the inconsistency. On the one hand, liver antioxidant enzymes were activated in the model group when the oxidative stress existed (Perlemuter et al., 2005). The low activity of SOD in the treatment groups meant that tea extracts might defend against ROS through the antioxidant property of the polyphenols like free radical-scavenging ability and ferric-reducing antioxidant power .
On the other hand, the TG content in the treatment group was low, indicating that the oxidation of FFA in tea-treatment group was less than that in the HFD group and less ROS was produced.
Thus, the activity of SOD was decreased in the groups treated with tea extracts in comparison with the HFD group. In conclusion, Fenghuang Narcissus Tea, Enshi Yulu Tea, and Qing Brick Tea might have in vivo antioxidant property, which were critical for the prevention of NAFLD.

| Bioactive components of teas
HPLC method was used for the measurement of main phytoconstituents in 12 kinds of tea extracts. Figure 7a and b displayed the chromatograms of standard components and Enshi Yulu Tea at 254 nm.
The results of main constituents in 12 teas are displayed in Table 3. with the values from to 1.11 ± 0.01 to 7.79 ± 0.03 mg/g DW.
Astragalin was found in six kinds of tea extracts, among which   were consistent with some studies. For example, a study found that the treatment of caffeine could increase the level of MDA in the zebrafish brain (de Carvalho et al., 2019). Several studies showed that ellagic acid and gallic acid could ameliorate liver steatosis in mice (Sousa et al., 2020;Zhang et al., 2019).

| CON CLUS IONS
In our research, we assessed and compared the effects of 12 teas on NAFLD in mice led by a HFD. It was found that several tea extracts could prevent NAFLD through significantly decreasing energy consumption, body weight gain, liver weight, epididymal and perirenal fat mass, lowering the content of TG in serum and liver, and improving hepatic steatosis as well as liver redox state. However, the effects varied in different teas. Enshi Yulu Tea and Fenghuang Narcissus Tea significantly reduced body weight, and Yihong Tea was the most effective tea in preventing the elevation of epididymal and perirenal fat mass. Moreover, tea treatments might exert hypolipidemic effects by significantly decreasing serum TG level, which was particularly obvious in Enshi Yulu Tea and Fried Green Tea. More importantly, Qing Brick Tea could protect against NAFLD by evidently alleviating the increase of liver TG content, which was confirmed by the results of the liver histopathological analysis. Furthermore, Fenghuang Narcissus Tea, Enshi Yulu Tea, and Qing Brick Tea might have potential in vivo antioxidant activity. However, the increased contents of AST as well as ALT in some tea-treatment groups indicated that some teas at a dosage of 200 mg/kg b.w. might cause liver injuries, such as Gongmei White Tea and Pu-erh Tea. The correlation analysis results indicated that ellagic acid and gallic acid contributed to the decrease of liver weight and MDA level, respectively, and thus prevented the development of NAFLD to some extent. To sum up, these findings indicated that some teas had the potential to be developed into functional food for preventing and managing NAFLD, and the results from this study could provide a reference for the public to select teas.

E TH I C A L A PPROVA L
The authors declare that they do not have any conflict of interest.
The study's protocols and procedures were ethically reviewed and approved by the Animal Care Committee at the School of Public Health, Sun Yat-Sen University.