Preventive effect of small‐leaved Kuding tea (Ligustrum robustum) on high‐diet‐induced obesity in C57BL/6J mice

Abstract Small‐leaved Kuding tea (SLKDT; Ligustrum robustum) is a traditional Chinese tea. We systematically investigated the effect of SLKDT extract on obesity. SLKDT‐controlled weight gain in mice fed a high‐fat diet. Tissue specimen results showed that the SLKDT extract alleviated liver damage and fat accumulation. Meanwhile, SLKDT extract improved dyslipidemia by decreasing total cholesterol, triglycerides, and low‐density lipoprotein cholesterol levels and increasing high‐density lipoprotein cholesterol levels. Furthermore, SLKDT extract reduced alanine aminotransferase, alkaline phosphatase, and aspartate transaminase levels in the serum and liver tissues; decreased inflammatory cytokines, including interleukin (IL)‐1β, tumor necrosis factor‐α, interferon‐γ, and IL‐6; and increased the anti‐inflammatory cytokines, IL‐4 and IL‐10. The quantitative PCR results showed that SLKDT extract upregulated the mRNA expressions of peroxisome proliferator‐activated receptor (PPAR)‐α, lipoprotein lipase, carnitine palmitoyltransferase 1, and cholesterol 7 alpha hydroxylase and downregulated PPAR‐γ and CCAAT/enhancer‐binding protein‐alpha mRNA expressions in the obese mouse livers to reduce adipocyte differentiation and fat accumulation, promote fat oxidation, and improve dyslipidemia, thereby inhibiting the immune response and alleviating liver injury. SLKDT shows a potential for preventing obesity and regulating obesity‐related syndrome, so it is possible to be further developed as a novel treatment for fighting obesity.

, and oxidative stress (Savini, Gasperi, & Catani, 2016) are all possible mechanisms by which obesity occurs and develops. Many studies have shown that obesity can cause lipid and glucose metabolism disorders, thus inducing metabolic diseases such as diabetes, hypertension, atherosclerosis, and non-alcoholic fatty liver (Boulange, Neves, Chilloux, Nicholson, & Dumas, 2016;Despres & Lemieux, 2006;Haslam & James, 2005;Mozaffarian, 2016). Obesity seriously threatens peoples' health and living quality and increases financial burdens on families. Therefore, it is extremely important to study how to effectively prevent and control the occurrence of obesity and reduce the risk of its associated metabolic diseases.
Kuding tea is a traditional drink with a history spanning more than 2,000 years in China. It is mainly distributed in southwestern China (Sichuan, Chongqing, Guizhou, Hunan, and Hubei) and southern China (Jiangxi, Yunnan, Guangdong, Fujian, and Hainan) (Zhu et al., 2009).
Kuding tea is rich in natural active components such as triterpenoids (Wang et al., 2012), polyphenols, and flavonoids ; thus, it has remarkable biological activity and can regulate lipid metabolism , protect the cardiovascular and cerebrovascular systems (Chen, Li, & Xie, 1995), and exert hypoglycemic (Song, Xie, Zhou, Yu, & Fang, 2012), antioxidant (Zhang, Xu, Sun, Ye, & Zeng, 2010), and antibacterial effects (Lu & Huang, 2009). SLKDT is a common drink in daily life and is easily accepted by consumers. Compared with most tea leaves, SLKDT has high selenium and low caffeine contents (Yang & Zhu, 1996) and possesses good medicinal activity and health value.
Studies have revealed the effects of SLKDT on hypolipidemia and weight loss (Xie et al., 2015;Yang et al., 2015), but systematic research on the anti-obesity effects and mechanisms of SLKDT remains limited. Therefore, it is extremely significant to deeply study the active components of SLKDT and its anti-obesity effects and mechanisms, to develop SLKDT as a functional drug or food for preventing and treating obesity.
In this study, obesity models were established by a high-fat diets, and SLKDT extract was used to intervene. We observed body weights and pathological sections and measured the relevant biochemical indicators in the serum and tissue to verify the effect of SLKDT. Lipid metabolism-related gene expressions were tested to clarify the SLKDT mechanism of action. This investigation provided a theoretical basis and experimental support for developing SLKDT as a natural and efficient product for preventing and treating obesity.

| Preparation of the SLKDT extract
Dried SLKDT (100 g, from Hainan Province) was weighed and pulverized, and then 70% ethanol was added at a liquid/material ratio of 20:1. The mixed liquid was heated for 3 hr at 60°C, then cooled and filtered to obtain the crude extract. The extraction solution was purified via FL-3 macroporous resin with 70% ethanol as the eluant (Mao, Liu, & Ran, 2011). When the eluant became colorless, the eluted solution was collected and evaporated. The residue was dried and smashed to obtain the SLKDT extract.

| Animal models and treatment
Fifty healthy C57BL/6J mice (weighing 20 ± 2 g, half male and female) were acclimated for 1 week, then randomly divided into the normal, model, L-carnitine, low-concentration SLKDT extract (SLKDT-L), and high-concentration SLKDT extract (SLKDT-H) groups (n = 10/group [5 males and 5 females] per group). The normal group was supplied with drinking water and normal maintenance food; the other groups were fed a D1249251 high-fat diet (Chongqing Medical University).
Mice in the L-carnitine group were intragastrically administered 200 mg/kg L-carnitine daily; mice in the SLKDT-L and SLKDT-H groups were administered, respectively, 100 mg/kg and 200 mg/kg SLKDT extract by gavage daily. After 4 weeks, except the normal group, other groups were switched from regular drinking water to 10% sugar water. Eight weeks later, all mice were fasted for 24 hr and then euthanized. Blood was obtained via the orbits, and the livers and epididymal fat (from the males) were collected and recorded for further experiments. The formula was used to calculate the organ index: Organ index = Organ weight (g)/Mouse body weight (g) × 100.

| Histological analysis of the liver and epididymal fat
Pieces of both the liver and epididymal fat (~0.5 cm 2 each) were fixed in 10% formalin solution for 48 hr, then dehydrated, routinely processed, embedded in paraffin, sectioned, and stained with hematoxylin and eosin. Tissue histology was examined via an optical microscope (BX43 microscope; Olympus).

| Measurements of TC, TG, HDL-C, and LDL-C levels in the serum and liver
Serum was obtained by centrifuging the plasma at 730 g for 10 min, and then the supernatant was collected. Serum total cholesterol (TC), triglycerides (TG), low-density lipoprotein cholesterol (LDL-C), and high-density lipoprotein cholesterol (HDL-C) levels were measured via kits following the manufacturer's instructions (Nanjing Jiancheng Bioengineering Institute).
Liver homogenate (10%) was centrifuged at 730 g for 10 min to obtain the supernatant. TC, TG, LDL-C, and HDL-C levels in liver were determined following each kit's instructions (Nanjing Jiancheng Bioengineering Institute).

| Measurements of AKP, ALT, and AST levels in the serum and liver
Serum was obtained by centrifuging the plasma at 730 g for 10 min, and then the upper light-yellow liquid was collected. Alanine aminotransferase (ALT), aspartate transaminase (AST), and alkaline phosphatase (AKP) levels in serum were analyzed using kits (Nanjing Jiancheng Bioengineering Institute).
Liver homogenate (10%) was centrifuged at 730 g for 10 min to afford the supernatant. The levels of ALT, AST, and AKP in the liver were analyzed according to each kit's instructions (Nanjing Jiancheng Bioengineering Institute).

| Quantitative PCR assay
Liver tissue was homogenized, and 1 ml of RNAzol reagent (Invitrogen) was used to extract the total RNA from the liver. And then the total RNA extracted was diluted to the concentration of 1 μg/μl. One microliter of diluted total RNA extract was utilized for reverse transcription to synthesize the cDNA template as per the kit instructions (Tiangen Biotech Co., Ltd.). Ten microliter of SYBR Green PCR Master Mix and 1 μl of the upstream and downstream primers were added to the cDNA template (Table 1). The quantitative PCR cycles were performed under the condition: 95°C for 30 s, then 40 cycles of 95°C for 30 s, 55°C for 30 s, 72°C for 30 s, and a final extension at 95°C for 30 s, followed by 55°C for 35 s. The 2 −ΔΔCt method was utilized to calculate the relative gene expressions, in which GADPH served as the internal reference.

| Statistical analysis
The SPSS 17.0 (SPSS) and GraphPad Prism 7 statistical software (GraphPad Software Inc.) were used to analyze the data.
Experimental results are expressed as the mean ± SD. One-way analysis of variance or a t test were used for between-group comparisons. p < .05 was considered statistically significant.

| Compositional analysis of the SLKDT extract
The HPLC results showed that the chlorogenic acid and its derivatives were the main polyphenols in SLKDT extract ( Figure 1). The standard spectral data showed that the polyphenol contents were neochlorogenic acid (11.199 mg/g), chlorogenic acid (10.837 mg/g),

| Effect of the SLKDT extract on mouse body weight
Weight gain increased dramatically in the model group in the early stages but increased gradually in the normal group throughout the test period ( Figure 2). After experiment, the body weight of model group was obviously higher than that of normal group (p < .05).
And compared with the model group, the body weight of mice in L-carnitine group and SLKD-H was decreased (p < .05). The effect of SLKDT-H was similar to that of L-carnitine.

| Liver and epididymal fat indexes
The liver and epididymal fat indexes are shown in

| PPAR-γ, C/EBP-α, PPAR-α, LPL, CPT1, and CYP7A1 mRNA expressions in the liver tissue
To explore the molecular mechanism of SLKDT in preventing obesity and regulating lipid metabolism in mice, we investigated the expressions of lipid metabolism-related genes. Figure 4 shows that the high-fat diet enhanced the mRNA expression levels of PPAR-γ and C/ EBP-α and suppressed the mRNA expression levels of PPAR-α, lipoprotein lipase (LPL), CPT1, and CYP7A1 in the model group (p < .05).
SLKDT-H and L-carnitine downregulated the PPAR-γ and C/EBPα mRNA expressions and upregulated the PPAR-α, LPL, CPT1, and CYP7A1 mRNA expressions (p < .05). L-carnitine had a significant effect, and SLKDT-H was more effective than was SLKDT-L.

| D ISCUSS I ON
The main blood lipid components are TG and cholesterol. Triglycerides are mainly involved in energy metabolism, and cholesterol is mainly used to synthesize cell plasma membranes, steroid hormones, and bile acids. Long-term high-fat diets cause obesity (Hedegaard, 2016), and obesity is accompanied by dyslipidemia, which involves hypercholesterolemia, hypertriglyceridemia, and reduced HDL (Kotsis, Antza, Doundoulakis, & Stabouli, 2017). Dyslipidemia is closely related to the occurrence of diseases such as atherosclerosis and hypertension (Tonstad & Després, 2011 Note: Values presented are the mean ± SD (n = 10/group). L-carnitine, mice treated with 200 mg/ kg L-carnitine; SLKDT-L, mice treated with 100 mg/kg SLKDT extract; SLKDT-H, mice treated with 200 mg/kg SLKDT extract.
SLKDT controlled body weight gain and fat accumulation, significantly reduced the TC, TG, and LDL-C levels, and increased the HDL-C level in the serum and liver tissues of mice with lipid metabolism disorders. The phenolic acids may have played important roles in preventing obesity and regulating lipid metabolic disorders.
High-fat diets cause excessive fat accumulation in hepatocytes, which causes liver steatosis that develops into non-alcoholic fatty liver (Sookoian & Pirola, 2008). Obesity is the main cause of non-alcoholic fatty liver. When hepatocytes are damaged, the ALT, AST, and AKP levels increase significantly, reflecting the degree of liver damage Wong, Bach, Sun, Hmama, & Av-Gay, 2011). SLKDT reduced the liver weight and inhibited liver lipid accumulation to the same extent. Liver function test results showed that SLKDT significantly reduced the serum ALT, AST, and AKP levels to protect against highfat-diet-induced liver damage.
Studies have shown that obesity induces chronic low-level inflammation in the body, which differs from traditional inflammation that is characterized by redness, swelling, heat, and pain (Hotamisligil, 2006;Medzhitov, 2008). The long-term low-level inflammatory response is closely related to various metabolic syndromes, which can induce a series of related chronic diseases such as cardiovascular disease, type 2 diabetes, and malignant tumors (Gregor & Hotamisligil, 2011;Kolb, Sutterwala, & Zhang, 2016).
Adipose tissue is the earliest proven connection inflammation with obesity. In addition to storing energy, adipose tissue is an endocrine F I G U R E 4 mRNA expression levels of LPL,CPT1,and CYP7A1 in liver tissues of the different groups were investigated by RT-qPCR. The data are shown as mean ± SD (n = 10). a-e Mean values with different letters are significant difference (p < .05) according to analysis of variance. C/EBP-α, CCAAT/ enhances binding protein alpha; CPT1, carnitine palmitoyltransferase 1; CYP7A1, cholesterol 7 alpha hydroxylase; LPL, lipoprotein lipase; PPAR-α, peroxisome proliferator-activated receptor alpha; PPAR-γ, peroxisome proliferator-activated receptor gamma; SLKDT, small-leaved Kuding tea organ that secretes many hormones and cytokines such as leptin, adiponectin, resistin, and inflammatory factors (Hotamisligil, 2017;Hummasti & Hotamisligil, 2010). Hypertrophic adipose tissue increases the release of free fatty acids and inflammatory mediators (e.g., TNF-α, IL-6, IL-1, IFN-γ, and monocyte chemotactic protein-1 [MCP-1]), causing the tissue to become inflamed (Hotamisligil, 2017;Hummasti & Hotamisligil, 2010;Oh & Olefsky, 2012). Secreted cytokines can also activate immune cells and adjacent cells via the c-Jun N-terminal kinase and IκB kinase β/nuclear factor-κB signaling pathways to increase synthesis and secretion of chemokines, such as MCP-1, leading to proinflammatory macrophage infiltration (Oh & Olefsky, 2012). Compared with the normal group, the serum levels of proinflammatory factors TNF-α, IFN-γ, IL-6, and IL-1β in the model group were significantly increased, while the levels of anti-inflammatory factors IL-4 and IL-10 were decreased, indicating that the model group mice were in an inflammatory state. SLKDT intervention significantly inhibited obesity-induced inflammation by decreasing the proinflammatory factors IL-6, TNF-α, IFN-γ, and IL-1β and increasing the anti-inflammatory cytokines IL-4 and IL-10.
Carnitine palmitoyltransferase I (CPT-1) transports long-chain acyl-CoA to the mitochondrial inner membrane and is a key rate-limiting enzyme in β-oxidation of fatty acids (Huang et al., 2010).
Upregulation of CPT-1 promotes β-oxidation of fatty acids to reduce fat accumulation. As a rate-limiting enzyme involved in hydrolyzing TG, LPL catalyzes TG decomposition from chylomicron and very LDL into fatty acids and promotes increased HDL levels to regulate lipid metabolism (Graham et al., 2017;Tian et al., 2014). The genes that encode these two enzymes are the target genes of the PPARs signaling pathway (Ashish, Rader, & Millar, 2010;Niu, Yuan, & Fu, 2010;Yang et al., 2018), which can affect lipid metabolism and deposition.
Cholesterol 7 alpha hydroxylase (CYP7A1) regulates the conversion of cholesterol into bile acid in the liver and is the key enzyme in maintaining cholesterol and bile acid homeostasis (Liu, Pathak, Boehmie, & Chiang, 2016). SLKDT downregulated PPAR-γ and C/ EBP-α mRNA expressions and upregulated PPAR-α, CPT-1, LPL, and CYP7A1 mRNA expressions to reduce adipocyte differentiation and fat accumulation, accelerate fat oxidation, and improve dyslipidemia, then inhibit the immune response and alleviate liver injury.
In conclusion, we systematically studied the effects of SLKDT on high-fat-diet-induced obese mice, including the ability of SLKDT to prevent obesity, modulate dyslipidemia and inflammation accompanied by obesity, and prevent liver damage due to lipid metabolic disorders. SLKDT extracts upregulated mRNA expressions of PPAR-α, LPL, CPT1, and CYP7A1 and downregulated mRNA expressions of PPAR-γ and C/EBP-α, which reduced adipocyte differentiation and fat accumulation, promoted fat oxidation, and improved dyslipidemia. SLKDT shows great potential in preventing obesity and regulating obesity-related syndrome, which is conceivable to be further developed into anti-obesity product.

This research was funded by High-level Talents Project of Chongqing
University of Education (2013BSRC001), 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.