Preventive effect of flavonoids from Wushan Shencha (Malus doumeri leaves) on CCl4‐induced liver injury

Abstract Wushan Shencha (Malus doumeri leaf) is a unique tea‐like drink. Herein, the effect of flavonoids from Wushan Shencha (FWSSC) on carbon tetrachloride‐induced liver injury was studied. The serum and liver tissues of experimental mice were analyzed by kits, a slice technique, and qPCR assay. The liver index is a calculated liver‐to‐body weight ratio, and the experimental results showed that FWSSC reduced the liver index of the model group with liver injury, which was the highest. Sections stained with H&E showed that FWSSC reduced stem cell necrosis caused by liver injury. FWSSC reduced the serum levels of AST, ALT, TG, and TC, as well as the levels of IL‐6, TNF‐α, and IFN‐γ cytokines in the serum of mice with liver injury. Liver biochemical tests also showed that FWSSC increased the SOD activity and decreased TC, TG, and MPO levels in mice with liver injury. It was found that FWSSC upregulated the expression of Cu/Zn‐SOD, Mn‐SOD, CAT, and IκB‐α, and downregulated the expression of NF‐κB, COX‐2, TNF‐α, and IL‐1β in the liver tissue of mice with liver injury by detecting the expression of mRNA in liver tissue. It is concluded that FWSSC is an active substance with hepatoprotective effects. The activity of FWSSC increased with increasing concentration, and the hepatoprotective effect of FWSSC at 100 mg/kg concentration was stronger than that of silymarin.

abnormal, it will have a severe impact on the body. With increased economic growth, people's dietary habits have also changed, and there has been a steady increase in fatty liver, alcoholic liver, drug-damaged liver, and other liver diseases that is closely related to numerous factors such as a high-fat diet, excessive alcohol consumption, indiscriminate use of medication, drug abuse, smoking, and lack of sleep, which will cause liver damage (Lee et al., 2007).
The pathological basis of chemical liver injury is lipid peroxidation and free radical production. As an intermediate product of energy transfer, free radicals participate in various physiological and biochemical reactions in the human body. The dynamic balance of the amount of free radicals is of great significance for maintaining the stability and health of the human internal environment. When stimulated by external environmental factors or aging, the accumulation rate of free radicals in cells is faster than that of scavenging, resulting in oxidative stress, which is manifested by damage to cells, tissues, and organs as well as loss of function, chromosomal variation, and even death (Weber, Boll, & Stampfl, 2003).
Carbon tetrachloride (CCl 4 ) is often administered to laboratory rats and mice to create an animal model of liver injury. The main mechanism of injury is driven by the free radical metabolites of CCl 4 , whereby CCl 4 produces toxic metabolites through cytochrome P4502El metabolism in the liver (Manibusan, Odin, & Eastmond, 2007). The lysozyme effect of CCl 4 itself could lead to hepatocyte damage, although the process of hepatic damage caused by free radicals of CCl 4 is considered to be the main mechanism (Lettéron et al., 1990). Reactive oxygen species (ROS)-induced oxidative stress causes a certain degree of damage to hepatocytes, which may play a key role in the pathogenesis of various liver injuries. ROS mainly comes from the process of ATP production by electron transfer in the mitochondrial respiratory chain complex. The mitochondria are abundant in the liver, and thus, the liver becomes the main organ that is attacked by ROS. The resulting oxidative stress can cause acute liver injury and negatively affect the body (Pramyothin, Janthasoot, Pongnimitprasert, Phrukudom, & Ruangrungsi, 2004).
Flavonoids have a wide range of functions and strong antioxidant activity (van Acker et al., 1996). By eliminating a great number of oxygen free radicals from the body, they can slow down the aging and degeneration of cells to prevent the occurrence of cancer (Trueba, Sánchez, & Giuliani, 2004). Flavonoids promote blood circulation, reduce cholesterol, and ameliorate cardiovascular and cerebrovascular diseases or reduce their incidence (Knekt et al., 2000(Knekt et al., , 2002. Flavonoids also promote wound healing by inhibiting the exudation of inflammatory enzymes. Some studies have found that the flavonoids in different plants have preventive effects on carbon tetrachloride-induced liver injury (Xie et al., 2018;Zhang et al., 2012;Yuan et al., 2008). However, the preventive effect of flavonoids from Wushan Shencha on carbon tetrachloride-induced liver injury has not yet been reported.
In this study, the flavonoids from Wushan Shencha were extracted, and their preventive effect on acute liver injury in mice was tested by establishing a CCl 4 -induced liver injury model. The serum and liver tissues of mice were tested, and biochemical and molecular biology experiments showed that the flavonoids of Wushan Shencha had a good preventive effect on experimental acute liver injury.
These experimental results will provide the theoretical basis for further utilization of this resource.

| Extraction of flavonoids
A mixture was created of 1.3 g of Na 2 CO 3 , 2.6 g of Na 2 B 4 O 7 , and 60 g of crushed Wushan Shencha leaves, which was then extracted by adding 600 ml of 40% ethanol solution and incubating at 50°C for 30 min. The solution was centrifuged, the supernatant was removed, and 600 ml of 40% ethanol solution was added to the precipitate, which was incubated at 50°C for 30 min. The supernatant was separated by centrifugation. After two extractions, the supernatant was adjusted to pH 4.0 with 12% hydrochloric acid, and then, the flavonoid extract was obtained by rotary evaporation (R-1001-VN; Zhengzhou Greatwall Scientific Industrial and Trade Co., Ltd.) (Qian et al., 2018).

Experimental Animal Center of Chongqing Medical University)
were fed with standard laboratory diet, and pads to absorb waste in the cages were changed every 2 days. After adaptive feeding for a week, the mice were divided into five groups: normal group, model group, FWSSCL group, FWSSCH group, and silymarin group, with 10 mice for each group. Normal and model mice were fed with gastric perfusion. FWSSCL and FWSSCH mice were fed 50 and 100 mg/ kg of flavonoids, respectively, for 14 days. The silymarin mouse group was fed with silymarin at a concentration of 100 mg/kg for 14 days. On the 14th day, all mice except those in the normal group were injected with CCl 4 inducer (the volume ratio of CCl 4 to olive oil was 1:1, 0.1 ml/10 g) . After intraperitoneal injection of CCl 4 solution, all experimental mice underwent fasting for 24 hr and were euthanized, and the liver and blood were then taken for use. Additionally, the liver tissue index (%) = liver mass (g)/ body mass of mice (kg) × 100 was determined. Diet and drinking of mice water were recorded. This study was approved by the Animal Ethics Committee of Chongqing University of Education (201903001B).

| Liver tissue levels in mice
The livers from the mice were processed into 10% homogenates and centrifuged at 730 × g for 10 min. The supernatant was removed, and the levels of TG, TC, superoxide dismutase (SOD, A001-3-2), and myeloperoxidase (MPO, A044-1-1) in the liver tissue were determined according to the kit instructions (Nanjing Jiancheng Bioengineering Institute).

| Pathological observation
The liver tissue was removed from the fixative, and the target tissue was smoothed. The trimmed tissues and corresponding labels are dehydrated in the dehydration box, and the wax-soaked tissues were embedded in the embedding machine (Histocentre 3, Thermo Fisher Scientific). The embedded slices were cooled on −20°C freezing table, and the wax was solidified; then, the wax blocks were removed and repaired. Then, the wax blocks were placed on the paraffin slicer (Finesse E+, Thermo Fisher Scientific) and sliced into the 60°C oven to bake the slices. Sections were baked and dried, and then were stored at room temperature. The sections were put into xylene for 20 min, absolute ethanol for 10 min, then into ethanol with volume fractions of 95%, 90%, 80%, and 70%, respectively, for 5 min, and then washed with distilled water. The sections were dyed in hematoxylin dye solution (H&E, ab245880; Abcam) for 8 min, washed with distilled water, and then differentiated with 1% hydrochloric acid and alcohol for 10 s. The sections were washed with distilled water, then returned to blue with 0.6% ammonia water, and rinsed with running water. The sections were dyed in eosin dye for 3 min, then dehydrated, dried, and sealed with neutral gum. The morphological changes in the liver tissues were observed under an optical microscope (BX43; Olympus) and analyzed by drug-induced liver injury (DILI) method. Hepatocyte steatosis score was 3 (bullous 1, vesicular 2), hepatocellular cholestasis score was 1, apoptotic body score was 1, eosinophilic leukocyte infiltration score was 2, above total score was 7; epithelial granuloma score was 1, and iron deposition score was 1 in necrotic area.

| Quantitative PCR (qPCR) assay
The liver tissue of mice was crushed, and then, the general RNA in tongue tissue was extracted by RNAzol (Invitrogen). The RNA concentration was diluted to 1 μg/μl. Then, a diluted general RNA solution of 5 μL was extracted and retrieved by a reverse transcription kit to obtain the cDNA template. Next, 2 μL of cDNA template was mixed with 10 μL of SYBR Green PCR Master Mix and 1 μL upstream and downstream primers (

| Western blot
The 100 microgram liver tissues were homogenized (2,190 × g for 5 min at 4°C, Hangzhou Allsheng Instrument Co Ltd.) with 1 ml RIPA and 10 L PMSF (Thermo Fisher Scientific). After the intermediate protein layer solution was removed, the protein was quantified by BCA protein quantitative kit (Beijing Solarbio Technology Co. Ltd.). The protein (50 g/ml) was mixed with the sample buffer (Thermo Fisher Scientific) at 4:1, then heated for 5 min at 100°C, and ice bath for 5 min. Then, mixing acrylamide, resolving buffer, stacking buffer, distilled water, 10% APS, and TEMED (Thermo Fisher Scientific) were mixed to make SDS-PAGE separating and concentrating glue and (Thermo Fisher Science) for observation ).

| Statistical analysis
Three parallel experiments were carried out on the serum and tissue indexes of each mouse, and then, the average values were taken. The data were analyzed by SAS 9.1 statistical software. The one-way analysis of variance (ANOVA) method was used to determine whether there were significant differences among the groups at the level of p < .05.

| Purity of flavonoids
The experimental results showed that the regression equation of the standard curve of rutin standard solution is Y = 823.15X−0.2432 (R 2 = .9925, Figure 1). Y indicates the concentration of rutin, and

| Diet and drinking water of mice
As can be seen from Figure 2, the mice in each group had normal diet and drinking water in the first 14 days, and there was no significant difference. After carbon tetrachloride was induced on the 14th day, the diet and drinking water of mice in each group decreased significantly (p > .05) compared with the normal group, the model group decreased the most, and the FWSSCH group decreased the least.
It can be seen that FWSSC had no effect on the diet and drinking water of mice before inducing liver injury. After inducing liver injury, FWSSC could alleviate the abnormality of mice caused by carbon tetrachloride and alleviate the drastic decrease in mice's intake of diet and drinking water.

| Liver index
As shown in Table 2 Table 3 shows that the serum levels of AST, ALT, TG, and TC in normal mice were the lowest, but those in the model mice were the highest. The levels of AST, ALT, TG, and TC in mice with liver injury decreased after treatment with flavonoids from Wushan Shencha, and the effect was more pronounced with increasing concentration. There was no significant difference between the effect of flavonoids from Wushan Shencha and silymarin at 100 mg/kg concentration (p > .05).

| AST, ALT, TG, and TC levels in mice
The enzymes AST and ALT are the most sensitive indicators for the diagnosis of hepatocyte damage. Under normal conditions, the blood levels of ALT and AST are very low, and therefore, the activities of the two enzymes in normal serum are very low. When the liver tissue is damaged and the permeability of the cell membrane increases, these two enzymes infiltrate into the blood. The activity of these enzymes in serum then significantly increases, and thus, the increase in AST and ALT in serum reflects the degree of damage of hepatocytes and the extent of liver injury (Dong et al., 2013). The experimental data in this study also confirm that CCl 4 increases AST and ALT levels in mice. The flavonoids of Wushan Shencha reduce the levels of AST and ALT in the serum of mice, thus playing a role in preventing liver injury. Table 4 shows that the serum levels of IL-6, TNF-α, and IFN-γ cytokines in normal mice were the lowest, while the levels of IL-6, TNF-α, and IFN-γ cytokines in mice with CCl 4 -induced liver injury were significantly increased. Wushan Shencha flavonoids and silymarin significantly inhibited the increase in cytokine levels caused by liver injury (p > .05). The levels of IL-6, TNF-α, and IFN-γ cytokines in the silymarin group were close to those in the FWSSCH group, and the levels of cytokines in the FWSSCL group were only lower than those in the model group.

| Serum cytokine interleukin (IL)-6, tumor necrosis factor (TNF)-α, and interferon (IFN)-γ levels in mice
TNF-α is a polypeptide mediator with extensive biological activity, and it mediates liver injury caused by various reasons. The increase in TNF-α is directly related to liver injury (Zhao, Qian, Li, & Tan, 2015). IL-6 stimulates hepatocytes to synthesize acute phase proteins and participate in inflammation. IL-6 also effectively promotes cachexia induced by TNF and IL-1 and aggravates tissue damage (Luckey & Petersen, 2001). IFN-γ mediates the antiviral effect of nontarget cell injury, and TNF-α participates in the injury response, promoting liver injury and inflammation of liver tissue (Shim et al., 2009). This study also confirmed that the flavonoids from Wushan Shencha prevent and alleviate liver injury by reducing IL-6, TNF-α, and IFN-γ cytokines in mice.  in the liver tissue of mice with liver injury so that they were almost the same as that of the normal group, and the higher the concentration of the flavonoids, the more obvious the effect. The higher the concentration of Wushan Shencha flavonoids, the more similar the observed effect is to that of silymarin.

| Liver tissue TG, TC, SOD, and MPO levels in mice
Carbon tetrachloride poisoning can damage the endoplasmic reticulum, resulting in a significant reduction in protein synthesis, while the TG generated in liver tissue is excreted in the form of very low density lipoprotein (VLDL)-TG. When apolipoprotein synthesis is insufficient, VLDL is insufficient to transport TG. As a result, TC will also accumulate in the liver (Pan, Long, Yi, & Zhao, 2018). This study shows that the amount of TG and TC in the  However, the degree of edema was significantly less than that of the control group, and no balloon-like changes were observed. The degree of hepatocyte necrosis was also significantly reduced.  free radicals and prevent liver injury (Sipes, Sisi, Sim, Mobley, & Earnest, 1991;Bonthius, Winters, Karacay, Bousquet, & Bonthius, 2015). CAT is an antioxidant enzyme that inhibits oxidative stress by clearing the body of hydrogen peroxide, slowing the oxidation caused by carbon tetrachloride, and thus inhibiting liver injury (Lee, Hyun, Jenner, & Halliwell, 2001). After liver injury, reactive oxygen species (ROS) lead to lipid peroxidation of cell membrane, hepatocyte necrosis, and imbalance of antioxidant defense system, which directly affect the behavior of hepatic stellate cells and myofibroblasts, accompanied by the decrease in SOD level.

| Pathological observation of liver tissue
Studies have shown that flavonoids from some plants can effectively control the effects of oxidative stress on liver (Mi et al., 2019). In the current study, the flavonoids of Wushan Shencha enhanced the expression of Cu/Zn-SOD, Mn-SOD, and CAT in mice with liver injury, thus might playing a role in preventing liver injury. In this study, flavonoids of Wushan Shencha might also regulate these genes, thereby further controlling oxidative stress and inhibiting liver injury.

| Nuclear factor kappa-light-chainenhancer of activated B cells (NF-κB) and inhibitor (I)κB-α of mRNA and protein expression in the liver tissue of mice
3.10 | Cyclooxygenase (COX)-2, TNF-α, and IL-1β mRNA expression in the liver tissue of mice of inflammatory cytokines can be used as a marker of liver injury, and inhibition of these inflammatory cytokines can help restore liver function. Overexpression of COX-2 occurs when liver tissue is damaged. TNF-α and IL-1β are related to the pathogenesis of hepatotoxicity. Inflammatory cytokines TNF-α and IL-1β increased significantly within 24 hr of liver injury. Therefore, blocking the production of TNF-α or IL-1β may attenuate liver injury (Hu et al., 2019). In this study, the flavonoids of Wushan Shencha effectively enhanced the expression of IκB-α and reduced the expression of COX-2, TNF-α, and IL-1β in mice with liver injury. Activation of NF-κB will lead to multiple verification processes, including oxidative stress induced by TNF-α, including GSH-Px, T-SOD, ROS, XOD, and MDA abnormalities induced by TNF-α. The regulation of TNF-α by regulating the NF-κB pathway may also play a role in inhibiting inflammation by enhancing the activity of antioxidant enzymes in vivo and reducing the harmful products produced by oxidative stress .
Through the influence of these expressions, the effects of inflammation on the organism could be alleviated, which may be able to inhibit the abnormal expression of tissue caused by liver injury.

| CON CLUS IONS
This study shows that the flavonoids of Wushan Shencha inhibit inflammation and oxidative stress in mice with liver injury. The flavonoids of Wushan Shencha can regulate liver function indexes and levels of inflammatory cytokines, and influence oxidative stress in liver tissues as well as verify the expression of related genes, so as to prevent liver injury induced by carbon tetrachloride in an all-encompassing manner. It proves that the flavonoids of Wushan Shencha are beneficial for the liver. The bioactive components responsible for visceral protection have value and can be further developed and utilized. Based on basic animal experiments, this study has proved the role of the flavonoids in Wushan Shencha. In the future, in-depth human trials will be needed to comprehensively verify the biological activity of flavonoids in Wushan Shencha.

This research was funded by The Program for Innovation Team
Building at Institutions of Higher Education in Chongqing [CXTDX201601040], China.

CO N FLI C T O F I NTE R E S T
The authors of this manuscript state that they do not have conflict of interest to declare.

E TH I C A L R E V I E W
This study has not any potential sources of conflict of interest.
This study was conducted in accordance with the Declaration of Helsinki, and the protocol was approved by the Ethics Committee