Aged green tea reduces high-fat diet-induced fat accumulation and inflammation via activating the AMP-activated protein kinase signaling pathway

Ruohong Chen, Xingfei Lai, Limin Xiang, Qiuhua Li, Lingli Sun, Zhaoxiang Lai, Zhigang Li, Wenji Zhang, Shuai Wen, Junxi Cao* and Shili Sun*

Tea Research Institute, Guangdong Academy of Agricultural Sciences/Guangdong Provincial Key Laboratory of Tea Plant Resources Innovation and Utilization, Guangzhou, China These authors contributed equally to this work.

Popular scientific summary


Background: Obesity is a global public health concern and increases the risk of metabolic syndrome and other diseases. The anti-obesity effects of various plant-derived bioactive compounds, such as tea extracts, are well-established. The mechanisms underlying the anti-obesity activity of Jinxuan green tea (JXGT) from different storage years are still unclear.

Objective: The aim of this study was to evaluate the effects of JXGTs from three different years on the high fat diet (HFD)-fed mouse model.

Design: The mice were divided into six groups, the control group received normal diet and the obese model group received HFD. We analyzed the effects of JXGTs from 2005, 2008, and 2016 on HFD-fed obese mice over a period of 7 weeks.

Results: The JXGTs reduced the body weight of the obese mice, and also alleviated fat accumulation and hepatic steatosis. Mechanistically, JXGTs increased the phosphorylation of AMP-activated protein kinase (p-AMPK)/AMP-activated protein kinase (AMPK) ratio, up-regulated carnitine acyl transferase 1A (CPT-1A), and down-regulated fatty acid synthase (FAS), Glycogen synthase kinase-3beta (GSK-3β), Peroxisome proliferator-activated receptor-gamma co-activator-1alpha (PGC-1α), Interleukin 6 (IL-6), and Tumour necrosis factor alpha (TNFα). Thus, JXGTs can alleviate HFD-induced obesity by inhibiting lipid biosynthesis and inflammation, thereby promoting fatty acid oxidation via the AMPK pathway.

Discussion: The anti-obesity effect of three aged JXGTs were similar. However, JXGT2016 exhibited a more potent activation of AMPK, and JXGT2005 and JXGT2008 exhibited a more potent inhibiting glycogen synthase and inflammation effect. Furthermore, the polyphenol (–)-epicatechin (EC) showed the strongest positive correlation with the anti-obesity effect of JXGT.

Conclusions: These findings demonstrate that JXGT treatment has a potential protection on HFD-induced obesity mice via activating the AMPK/CPT-1A and down-regulating FAS/GSK-3β/PGC-1α and IL-6/TNFα. Our study results also revealed that different storage time would not affect the anti-obesity and anti-inflammation effect of JXGT.

Graphical abstract


Keywords: anti-obesity; anti-inflammation; AMPK; aged green tea; metabolism


Citation: Food & Nutrition Research 2022, 66: 7923 - http://dx.doi.org/10.29219/fnr.v66.7923

Copyright: © 2022 Ruohong Chen et al. This is an Open Access article distributed under the terms of the Creative Commons Attribution 4.0 International License (http://creativecommons.org/licenses/by/4.0/), allowing third parties to copy and redistribute the material in any medium or format and to remix, transform, and build upon the material for any purpose, even commercially, provided the original work is properly cited and states its license.

Received: 19 May 2021; Revised: 30 November 2021; Accepted: 4 January 2022; Published: 10 March 2022

Competing interests and funding: The authors declare no conflict of interest. This study was funded by the “14th Five-Year Plan” team-building projects of Guangdong Academy of Agricultural Sciences [Grant Nos. 202126TD]; Guangdong Basic and Applied Basic Research Foundation [Grant Nos. 2020A1515011266, 2021A1515010958]; Guangzhou Science and Technology Plan Projects [Grant Nos. 202102020047, 202002030202]; Key-Area Research and Development Program of Guangdong Province [Grant Nos. 2020B0202080003]; Maoming Science and Technology Program (Grant Nos. mmkj2020045); Zhanjiang Science and Technology Program (Grant Nos. 2020A03014); Innovation Fund projects of Guangdong Academy of Agricultural Sciences (Grant Nos. 202115, 202035); Special fund for scientific innovation strategy-construction of high level Academy of Agriculture Science (Grant Nos. R2019PY-JX004); the Innovation Fund projects of Guangdong Key Laboratory of Tea Plant Resources Innovation and Utilization (Grant Nos. 2021CX02). Funders did not have any role in study design, data collection, and data analysis.

*Junxi Cao, Tea Research Institute, Guangdong Academy of Agricultural Sciences, No. 20, Jinying Rd., Guangzhou 510640, P.R. China. Email: junxic@126.com

*Shili Sun, Tea Research Institute, Guangdong Academy of Agricultural Sciences, No. 20, Jinying Rd., Guangzhou 510640, P.R. China. Email: sunshili@zju.edu.cn

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Obesity results from the imbalance between high-energy intake and low-energy expenditure, and is currently a global health concern (1). It is a major risk factor of hypertension, type 2 diabetes, cancer, rheumatoid arthritis and cardiovascular diseases (25). Studies show that several plant-derived bioactive compounds can alleviate obesity without the side effects of conventional weight loss drugs (69).

Tea brewed from the fresh leaves of Camellia sinensis contains a variety of bioactive compounds including polysaccharides, polyphenols, and so on (10, 11). Several studies have demonstrated the anti-obesity effects of green tea, Fubrick tea, and black tea (1214). The composition of the bioactive compounds in the different types of tea depends on the processing and fermentation. Green tea is a non-fermented tea, although its prolonged storage induces a slight natural fermentation that may alter its active components.

AMP-activated protein kinase (AMPK) controls lipid metabolism by modulating the CPT-1A and FAS pathways (15, 16). The AMPK activation also correlates with GSK-3β downregulation (17) and mitochondrial biogenesis via PGC-1α (18). In our previous studies, we found that different types of tea can alleviate obesity via AMPK activation. Furthermore, the weight-loss effect of green tea is associated with the AMPK/CPT-1A/FAS and GSK-3β/ PGC-1α pathways.

The aim of this study was to evaluate the effects of Jinxuan Green teas (JXGTs) from three different years on the high-fat diet (HFD)-fed mouse model for determining the effect of its prolonged storage on the anti-obesity components of green tea. We found that JXGTs alleviated HFD-induced weight gain by elevating the p-AMPK/AMPK ratio, and the activated AMPK mitigated lipid synthesis and balanced energy metabolism through the CPT-1A/FAS and GSK-3β/PGC-1α pathways, respectively. Furthermore, JXGTs inhibited obesity-induced inflammation by downregulating pro-inflammatory factors, such as IL-6 and TNFα. Taken together, JXGT mediates anti-obesity and anti-inflammatory effects that warrant further investigation.

Materials and methods

Preparation and characterization of lyophilized JXGT extract

Dried JXGT leaves from the years 2005, 2008, and 2016 were obtained from the Tea Research Institute, Guangdong Academy of Agricultural Sciences in China. As reported previously (19), the JXGT leaves were pulverized and extracted by boiling in water for 30 min (tea/water = 1:20 w/v). The tea extracts were concentrated by rotary evaporation to one-fifth of the original volume and dried by a vacuum freeze dryer. The content of free amino acids, total soluble sugar, polyphenols, caffeine, and catechin were measured by the ninhydrin method, anthrone-sulfuric acid colorimetric assay, Folin-phenol method, and high-performance liquid chromatography (HPLC), respectively, as previously reported (2022).

Establishment of obesity model in mice and treatment regimen

Male C57BL/6J mice (7 weeks old) were purchased from Beijing Huafukang Bioscience Co. Ltd. (Beijing, China) All experimental procedures were approved by the Ethics Committee of the institute, and performed according to the institutional guidelines for the care and use of laboratory animals. The protocols were approved by the Ethical Committee of Tea Research Institute. The mice were individually housed at 23 ± 2°C and 60 ± 15% humidity on a 12-h light/dark cycle, with free access to deionized water and basic feed. After a week of adaptation, the mice were randomly divided into the following six groups (n = 8 each): control (basic diet), model (HFD), positive control (HFD + 10 mg/kg/day atorvastatin), JXGT 2005 (HFD + 1000 mg/kg/day JXGT 2005), JXGT 2008 (HFD + 1000 mg/kg/day JXGT 2008), and JXGT 2016 (HFD + 1000 mg/kg/day JXGT 2016). The mice were given intragastric administration once a day for 7 weeks. The normal diet consisted of 18% proteins, 4% fats, 62% carbohydrates, 5% fiber, 8% minerals, and 3% vitamins for the control group. The calorific contribution of fats, proteins, and carbohydrates in the HFD were 45, 20, and 35% respectively for HFD-induced groups. Both feeds were prepared by the Guangdong Medical Laboratory Animal Center. Each group was provided with distilled water, and the body weight, food and water intake were recorded once a week.

Tissue processing

After 7 weeks of treatment, the mice were anesthetized with 40 mg/kg pentobarbital following overnight fasting and euthanized by cervical dislocation. The whole blood was collected into heparinized tubes, and the sera were separated by centrifuging at 3,000 rpm for 10 min. The adipose tissues (including abdominal fat, intestinal fat, and perirenal fat) and liver were removed, washed with PBS, weighed, and frozen at –80°C for further analysis.

Biochemical analysis

The serum levels of triglycerides (TGs), total cholesterol (TC), high-density lipoprotein cholesterol (HDL-C), and low-density lipoprotein cholesterol (LDL-C) were measured using commercially available kits (Nanjing Jiancheng Bioengineering Institute, China) according to the instructions.

Protein extraction and Western blotting

Total protein was extracted from the liver using a protein extraction kit (Jiancheng Bioengineering Institute, Nanjing, China). Equal amounts of protein per sample were resolved by 10% SDS-PAGE and transferred to Polyvinylidene fluoride (PVDF) membranes. After blocking with 5% skimmed milk in Tris Buffered Saline with Tween-20 (TBST) for 1 h at room temperature, the proteins were incubated overnight with primary antibodies against AMPK (#2532S, Cell Signaling Technology, Danvers, MA, USA), p-AMPK (#2535S, CST), CPT-1A (15184-1-AP, Proteintech Group, Rosemont, USA), FAS (Abp51334, Abbkine, CA, USA), GSK-3β (#9315, CST), PGC-1α (2178S, CST), IL-6 (bs-0379R, Bioss, Beijing, China), TNFα (ab6671, Abcam, Cambridge, UK), and β-actin (Sigma-Aldrich, St Louis, MO, USA) at 4°C. The membranes were then probed with anti-rabbit secondary antibody IgG (HRP) (ab6721, Abcam) or anti-mouse secondary antibody IgG (HRP) (ab197767, Abcam) for 1 h at room temperature. After washing thrice with TBST, the blots were developed using a chemiluminescence reagent (P0018A, Shanghai Beyotime Biotechnology Co., Ltd, China), and the positive bands were visualized with a Gel Imaging System (General Electric, Fairfield, CT, USA). The band intensities were measured using the ImageJ software.

Statistical analysis

All statistical analyses were performed using SPSS 16.0 (IBM, USA), and GraphPad Prism 7.0 (USA) was used to plot graphs. Multiple groups were compared by one-way analysis of variance (ANOVA) followed by Dunnett’s test. Independent Student’s t-test (two-tailed) was used for pairwise comparison. The correlation between factors was evaluated by Pearson correlation analysis. All data are presented as the means ± SD of at least three independent experiments, P < 0.05 was considered to be statistically significant.


Prolonged storage affects the composition of JXGTs

As shown in Table 1, JXGT2005 and JXGT2008 had a higher water content compared with JXGT2016. Due to time-dependent degradation and oxygenation during storage, the content of free amino acids, soluble sugars, and tea polyphenols was significantly lower in the aged JXGT, as reported in our previous studies (23, 24).

Table 1. The components of Jinxuan Green teas from three different storage years
Constituent JXGT2005 JXGT2008 JXGT2016
Free amino acid (%) 1.62 ± 0.03a 1.65 ± 0.06a 2.30 ± 0.04b
Soluble sugar (%) 6.61 ± 0.02a 6.25 ± 0.11a 7.80 ± 0.03b
Tea polyphenols (%) 30.77 ± 2.79a 30.54 ± 2.52a 32.65 ± 1.52a
GA 13.16 ± 1.23a 11.25 ± 1.88a 55.98 ± 3.04b
GC 5.87 ± 0.03a 5.86 ± 0.04a 9.04 ± 0.21b
EGC 1.11 ± 0.00a 1.11 ± 0.00a 1.06 ± 0.00b
C 1.54 ± 0.01a 1.64 ± 0.05a 2.31 ± 0.05b
CAFF 19.73 ± 0.29a 20.38 ± 0.14a 17.59 ± 0.32b
EC 3.53 ± 0.10a 4.15 ± 0.16a 4.72 ± 0.25b
EGCG 31.23 ± 0.41a 32.07 ± 0.13a 28.32 ± 0.26b
GCG 8.31 ± 0.14a 7.48 ± 0.20b 8.68 ± 0.30a
ECG 1.21 ± 0.02a 1.14 ± 0.04a 0.87 ± 0.04a
CG 5.89 ± 0.05a 5.87 ± 0.21a 5.88 ± 0.17a
Water (%) 9.50 ± 0.02a 9.20 ± 0.02b 4.64 ± 0.01c
The value is mean ± SD (n = 3). Values marked with different lower case letters in superscript format indicate significant difference, values marked with the same lower case letters in superscript format indicate no significant difference.
Note: (−)-epicatechin (EC), (−)-epigallocatechin (EGC), (−)-epicatechin gallate (ECG), (−)-epigallocatechin gallate (EGCG), (+)-catechin (C) and (+)-gallocatechin (GC), (−)-catechin gallate (CG) and (−)-gallocatechin gallate (GCG). a,b,c The value of ingredients contents is mean ± SD (n = 3). Means followed by the same letter are not significantly different at P < 0.05.

JXGTs reduced body weight in HFD-fed obese mice

As shown in Fig. 1A, HFD feeding for 7 weeks significantly increased the body weight of the mice compared with the normal diet-fed controls. In contrast, intragastric administration of JXGTs during the 7-week regimen significantly inhibited the HFD-induced weight gain (P < 0.01; Fig. 1B). The daily food and water intake did not show any marked differences among all groups (Fig. 1C and D). Taken together, JXGTs can prevent HFD-induced obesity without suppressing calorific intake.

Fig 1
Fig. 1. Effect of JXGTs on the body weight (A), weight gain (B), diet consumption (C), and water consumption (D) of HFD-fed obese mice. Data are presented as means ± SD (n = 8). **P < 0.01 and *P < 0.05.

JXGTs attenuated fatty liver and adiposity in the HFD-fed obese mice

The effects of JXGTs on fat accumulation were evaluated in terms of anatomical and biochemical indices. JXGTs markedly reduced the accumulation of white fat in the HFD-fed obese mice compared with the untreated mice (Fig. 2A). As shown in Fig. 2B, yellowish-brown fatty livers characterized by uneven surface were observed in mice fed with the HFD for 7 weeks compared with the healthy controls. JXGTs treatment protected the liver of the HFD-fed mice from steatosis. Furthermore, JXGTs also significantly decreased the size of the abdominal (Fig. 2C) and perirenal (Fig. 2D) fat tissue masses, especially in the JXGT2008 group. Consistent with this, JXGTs also reduced the total amount of white fat, and that of epididymal, intestinal and pararenal fat in the HFD-fed mice to near-baseline levels, and the effect was similar for the JXGTs from different storage years (P < 0.01; Fig. 2E–H). Thus, JXGT treatment can effectively attenuate HFD-induced fatty liver and adiposity. Furthermore, HFD markedly increased the serum levels of TGs, TC, high-density lipoprotein (HDL), and low-density lipoprotein (LDL). The supplementation of JXGT reversed the HFD-induced increment in TG (Supplementary Fig. 1A) but did not affect the other indices (Supplementary Fig. 1B–D).

Fig 2
Fig. 2. JXGTs attenuate fatty liver and adiposity in HFD-induced obese mice. Representative images of whole body (A), fatty liver (B), abdominal (C), and perirenal (D) white fat in all groups. The indices of total (E), abdominal (F), intestinal (G), and perirenal (H) white fat relative to body weight. Data are presented as the means ± SD (n = 8). **P < 0.01 and *P < 0.05.

JXGTs activate AMPK-driven metabolic pathways

AMPK plays an important role in energy metabolism by stimulating fatty acid oxidation. The HFD-fed obese mice had significantly a lower level of p-AMPK in the liver, which was reversed by JXGT treatment (Fig. 3A). Consistent with this, HFD decreased the p-AMPK/AMPK ratio by 44% compared with that in healthy controls, and was restored by JXGTs from the different storage years (Fig. 3B). CPT-1 is the rate-limiting enzyme of fatty acid oxidation, and FAS is a key enzyme involved in fatty acid synthesis. As shown in Fig. 4A, CPT-1 and FAS were, respectively, downregulated and upregulated in the liver of obese mice, and their expression levels were significantly reversed by JXGT treatment (Fig. 4B). GSK-3β and PGC1-α are the key protein kinases involved in energy metabolism. As shown in Fig. 5A, GSK-3β was up-regulated in the HFD-fed mice and decreased by JXGT treatment. In addition, PGC1-α was down-regulated in the JXGT-treated groups (Fig. 5B and C).

Fig 3
Fig. 3. JXGTs activate AMPK phosphorylation. (A) Immunoblot showing AMPK protein levels in the liver of differentially treated mice and (B) densitometric quantification. Data are presented as means ± SD (n = 3). **P < 0.01 and *P < 0.05.

Fig 4
Fig. 4. JXGTs upregulate CPT-1 and inhibit FAS expression. (A) Immunoblot showing expression levels of CPT-1 and FAS protein in mouse liver and densitometric quantification of (B) CPT-1 and (C) FAS. Data are presented as the means ± SD (n = 3). **P < 0.01, and *P < 0.05.

Fig 5
Fig. 5. JXGTs upregulate PGC-1α and inhibit GSK-3β expression. (A) Immunoblot showing expression levels of GSK-3β and PGC-1α protein in mouse liver and densitometric quantification of (B) GSK-3β and (C) PGC-1α. Data are presented as means ± SD (n = 3). **P < 0.01 and *P < 0.05.

JXGTs inhibit IL-6 and TNF-α expression

Obesity is usually associated with an increase in inflammation, and high in situ levels of IL-6 and TNFα, which can aggravate liver injury and weaken the hepatic glucolipid and lipid metabolism (25). As shown in Fig. 6, IL-6 and TNFα levels were significantly higher in the liver tissues of obese mice. Treatment with the different JXGTs significantly decreased the levels of both factors compared with that in the untreated obese mice (P < 0.01).

Fig 6
Fig. 6. JXGTs inhibit IL-6 and TNF-α expression. (A) Immunoblot showing IL-6 and TNF-α protein levels in mouse liver and densitometric quantification of (B) IL-6 and (C) TNF-α. Data are presented as means ± SD (n = 3). **P < 0.01 and *P < 0.05.

Correlation analysis

To further evaluate the role of tea-derived phytochemicals against obesity, we analyzed the Pearson correlation between the phytochemical composition of JXGTs and various parameters of obesity and inflammation (Fig. 7). The content of tea polyphenols, amino acids and soluble sugar was positively correlated with AMPK pathway activation, as well as most anti-inflammatory parameters. Furthermore, GCG (GCG) and catechin gallate (CG) were positively correlated with the loss of body weight.

Fig 7
Fig. 7. The correlation between the phytochemicals and obesity parameters and inflammation indices in the different experimental groups. (−)-epicatechin (EC), (−)-epigallocatechin (EGC), (−)-epicatechin gallate (ECG), (−)-epigallocatechin gallate (EGCG), (+)-catechin (C) and (+)-gallocatechin (GC), (−)-catechin gallate (CG) and (−)-gallocatechin gallate (GCG). Significant correlations are annotated by **P < 0.01 and *P < 0.05.


Obesity is primarily a result of increased consumption of sugars and fats, and lack of physical exercise, along with aberrant fatty acid biosynthesis and degradation (26). It is a major health problem worldwide (27), and is accompanied by several hormonal and inflammatory disturbances that increase the risk of diabetes (28), hypertension (3), dyslipidemia and metabolic syndrome (29). The commonly prescribed weight-loss drugs like orlistat, sibutramine, and rimonabant cause side effects, such as oily stools and flatulence (30). Several studies have identified plant-derived bioactive compounds with significant anti-obesity and weight-loss effects (3134). For instance, tea brewed from fresh leaves of C. sinensis has several beneficial effects. Depending on the extent and method of fermentation, tea is classified into the non-fermented green tea, lightly fermented yellow tea and white tea, partially fermented oolong tea, completely fermented black tea, and post-fermented dark tea (35). Green tea, in particular, has exhibited protective effects against skin photoaging, stress, neurodegeneration, hypertrophy, hypolipidemia, inflammation, and obesity (3639).

Atorvastatin is one of the most widely prescribed drugs and the most widely prescribed statin in the world (40), which is widely used as a positive control to lower elevated lipid levels and anti-obesity by difference dosages (1–80 mg/kg/day) treatment in the HFD induced model (19, 41, 42). Therefore, positive control group is treated with a relatively low-dose atorvastatin (10 mg/kg/day) in this study. In this study, we compared the potential anti-obesity effects of JXGTs from different storage years on HFD-fed mice. Although the JXGTs had little effect on the calorific intake of the mice, they significantly reduced body weight and fat accumulation at multiple anatomical sites, and the duration of storage had no significant effect on the anti-obesity effects of JXGT (23).

The liver is the central organ of lipid storage and metabolism. The consumption of high amount of dietary fat leads to liver steatosis (43). AMPK is the main sensor of energy status in eukaryotic cells, and thus, coordinates the growth and metabolism of specific tissues. The AMPK/p-AMPK is highly sensitive to energetic stress, and the liver-specific AMPK activation reprograms lipid metabolism and mitigates diet-induced obesity in mice (44). Studies show that the green tea extract and specific bioactive compounds like maslinic acid and EGCG can reduce obesity in mouse and zebrafish obesity models, respectively, through AMPK activation (4547). In our previous studies, we found that aged oolong tea and Hakka stir-fried tea protected mice against obesity by activating the AMPK signaling pathway (19, 23, 24). As the JXGTs also markedly induced AMPK phosphorylation and the p-AMPK/AMPK ratio in liver tissues, their anti-obesity effects are also likely mediated via the AMPK signaling pathway.

The limiting factor of lipogenesis is malonyl-CoA, which is also an important precursor of the lipid biosynthetic pathway. AMPK activation decreases cellular malonyl-CoA levels, which, in turn, upregulates CPT1 (48). And FAS is a major regulator of lipogenic protein, and its activity is also regulated by AMPK. Then we demonstrated that aged JXGT significantly increased the expression of CPT1A (Fig. 4A and B) and inhibited the protein level of FAS (Fig. 4A and C). Our data established that the activation of AMPK/CPT-1A pathway and the inhibition of FAS pathways might be potential targets for JXGT treatment to prevent hepatic lipid accumulation.

The activation of AMPK not only inhibits the lipid synthesis and increases lipid oxidation but also the glucose synthesis in liver (49). GSK3β is a key enzyme of glycogen synthesis, and is elevated in both human subjects and animal models with diabetes. AMPK activation is associated with inhibition of GSK3β (50), and JXGT-mediated activation of AMPK in our HFD-fed model also decreased the obesity-induced overexpression of GSK3β. Another downstream target of AMPK is the transcription factor PGC-1α, which increases the expression of genes involved in mitochondrial biogenesis (51). Studies show that the activation of AMPK can up-regulate PGC-1α and ultimately promote mitochondrial biogenesis (52, 53). In this study, the aged JXGTs decreased the levels of IL-6 and TNFα, which is indicative of their potent anti-inflammatory effect. The effects of these three JXGTs on body weight were similar (Figs. 1 and 2). However, the ratio of p-AMPK/AMPK in the JXGT2016 group was higher than that in the JXGT2005 and JXGT2008 groups (Fig. 3). And JXGT2005 and JXGT2008 groups decreased the levels of GSK-3β (Fig. 5B), IL-6, and TNFα (Fig. 6) much lower than the JXGT2016 group, which is indicative of their potent inhibited glycogen synthesis and anti-inflammatory effect. Our study results showed that different storage years of JXGT can significantly attenuate body weight gain by HFD through its increased lipid metabolism, inhibited glycogen synthesis, and anti-inflammatory functions related to p-AMPK activation.

The major bioactive component in green tea are the polyphenol compounds that constitute 24−36% of the dry weight, followed by protein (15%), lignin (7%), amino acids (3−4%), caffeine (2−4%), organic acids (2%) and chlorophyll (0.5%) (54). Most of the beneficial effects of green tea are attributed to the high polyphenol content (55). We found that the content of free amino acids, soluble sugar, and tea polyphenols was positively correlated with p-AMPK levels and negatively correlated with the serum levels of TG, HDL-C, and LDL-C, whereas no significant correlation was observed with fat accumulation (Fig. 7). The major polyphenols of green tea include EC, (−)-epigallocatechin (EGC), (−)-epicatechin 3-gallate (ECG), (−)-epigallocatechin 3-gallate (EGCG), (+)-catechin (C), and (+)-gallocatechin (GC), along with smaller amounts of (−)-catechin gallate (CG) and (−)-c (GCG) (12). Daily consumption of green tea extracts, especially EGCG, has been shown to increase fat oxidation and energy expenditure (55, 56). In addition, CG, EGC, ECG, and EGCG can suppress intracellular lipid accumulation in 3T3-L1 cells (57). Gallic acid (GA) inhibits lipid accumulation via the activation of AMPK in HepG2 cells (58). In this study, we analyzed the levels of specific polyphenols in the JXGTs by HPLC-MS (Table 1), and then revealed that GA, GC, C, EC and GCG were positively correlated with the AMPK pathway (Fig. 7). The correlation between the phytochemicals and obesity and inflammation indices in the different treatment groups was in agreement with the previous study. Whereas EGC, EGCG, ECG, and caffeine (CAFF) had a negative correlation, EC showed the strongest correlation with weight gain, and GCG and CG were positively correlated with fat accumulation (Fig. 7). Our data revealed that EGCG, as a portion of phytochemicals of JXGT, might have a opposite dose-dependent effect of AMPK activation in our used dose range. However, it does not mean that EGCG inhibits the activation of AMPK. However, the exact anti-obesity and anti-inflammatory effects of the different polyphenols need to be explored further. Furthermore, the possible synergistic effects of the different bioactive compounds of JXGTs need to be investigated. For instance, the consumption of caffeine and EGCG synergistically increased fat oxidation and energy expenditure (59).


JXGTs reduced white fat accumulation, increased lipid metabolism, and inhibited glycogen synthesis in the HFD-fed obese mice by targeting FAS, GSK-3β, and the AMPK/CPT1A pathway. In addition, JXGT reduced inflammation by downregulating IL-6 and TNFα. The storage duration had no significant effect on the activity of JXGT. Finally, the polyphenol EC showed a significant positive correlation with AMPK activation and weight gain Taken together, JXGT is a promising therapeutic agent against obesity and metabolic disorders, and different storage time would not affect the anti-obesity and anti-inflammation effects of JXGT.

Authorship contributions

All authors contributed to the design and conduct of the study, data collection and analysis, data interpretation, and manuscript writing.


  1. Lam DW, LeRoith D. The worldwide diabetes epidemic. Curr Opin Endocrinol Diabetes Obes 2012; 19: 93–96. doi: 10.1097/MED.0b013e328350583a
  2. Pichard C, Plu-Bureau G, Neves ECM, Gompel A. Insulin resistance, obesity and breast cancer risk. Maturitas 2008; 60: 19–30. doi: 10.1016/j.maturitas.2008.03.002
  3. Seravalle G, Grassi G. Obesity and hypertension. Pharmacol Res 2017; 122: 1–7. doi: 10.1016/j.phrs.2017.05.013
  4. Vecchie A, Dallegri F, Carbone F, Bonaventura A, Liberale L, Portincasa P, et al. Obesity phenotypes and their paradoxical association with cardiovascular diseases. Eur J Intern Med 2018; 48: 6–17. doi: 10.1016/j.ejim.2017.10.020
  5. Luo Y, Blackledge WC. Microbiome-based mechanisms hypothesized to initiate obesity-associated rheumatoid arthritis. Obes Rev 2018; 19: 786–97. doi: 10.1111/obr.12671
  6. Zhang WL, Zhu L, Jiang JG. Active ingredients from natural botanicals in the treatment of obesity. Obes Rev 2014; 15: 957–67. doi: 10.1111/obr.12228
  7. Hu S, Xu Y, Gao X, Li S, Jiang W, Liu Y, et al. Long-chain bases from sea cucumber alleviate obesity by modulating gut microbiota. Mar Drugs 2019; 17: 455. doi: 10.3390/md17080455
  8. Sheng Y, Liu J, Zheng S, Liang F, Luo Y, Huang K, et al. Mulberry leaves ameliorate obesity through enhancing brown adipose tissue activity and modulating gut microbiota. Food Funct 2019; 10: 4771–81. doi: 10.1039/c9fo00883g
  9. Chen G, Ni Y, Nagata N, Zhuge F, Xu L, Nagashimada M, et al. Lycopene alleviates obesity-induced inflammation and insulin resistance by regulating M1/M2 status of macrophages. Mol Nutr Food Res 2019; 63: e1900602. doi: 10.1002/mnfr.201900602
  10. Khan N, Mukhtar H. Tea polyphenols in promotion of human health. Nutrients 2018; 11: 39. doi: 10.3390/nu11010039
  11. Chen G, Chen R, Chen D, Ye H, Hu B, Zeng X, et al. Tea polysaccharides as potential therapeutic options for metabolic diseases. J Agricult Food Chem 2019; 67: 5350–60. doi: 10.1021/acs.jafc.8b05338
  12. Huang J, Wang Y, Xie Z, Zhou Y, Zhang Y, Wan X. The anti-obesity effects of green tea in human intervention and basic molecular studies. Eur J Clin Nnutr 2014; 68: 1075–87. doi: 10.1038/ejcn.2014.143
  13. Ling W, Li S, Zhang X, Xu Y, Gao Y, Du Q, et al. Evaluation of anti-obesity activity, acute toxicity, and subacute toxicity of probiotic dark tea. Biomolecules 2018; 8(4): 99. doi: 10.3390/biom8040099
  14. Jing N, Liu X, Jin M, Yang X, Hu X, Li C, et al. Fubrick tea attenuates high-fat diet induced fat deposition and metabolic disorder by regulating gut microbiota and caffeine metabolism. Food Funct 2020; 11: 6971–86. doi: 10.1039/d0fo01282c
  15. Ronnett GV, Kleman AM, Kim EK, Landree LE, Tu Y. Fatty acid metabolism, the central nervous system, and feeding. Obesity 2006; 14(Suppl 5): 201S–7S. doi: 10.1038/oby.2006.309
  16. Fang K, Wu F, Chen G, Dong H, Li J, Zhao Y, et al. Diosgenin ameliorates palmitic acid-induced lipid accumulation via AMPK/ACC/CPT-1A and SREBP-1c/FAS signaling pathways in LO2 cells. BMC Complement Alternat Med 2019; 19: 255. doi: 10.1186/s12906-019-2671-9
  17. Abbas NAT, Kabil SL. Liraglutide ameliorates cardiotoxicity induced by doxorubicin in rats through the Akt/GSK-3beta signaling pathway. Naunyn-Schmiedeberg’s Arch Pharmacol 2017; 390: 1145–53. doi: 10.1007/s00210-017-1414-z
  18. Timm KN, Tyler DJ. The role of AMPK activation for cardioprotection in doxorubicin-induced cardiotoxicity. Cardiovasc Drugs Ther 2020; 34: 255–69. doi: 10.1007/s10557-020-06941-x
  19. Liu C, Guo Y, Sun L, Lai X, Li Q, Zhang W, et al. Six types of tea reduce high-fat-diet-induced fat accumulation in mice by increasing lipid metabolism and suppressing inflammation. Food Funct 2019; 10: 2061–74. doi: 10.1039/c8fo02334d
  20. Zhang XB, Du XF. Effects of exogenous enzymatic treatment during processing on the sensory quality of summer tieguanyin oolong tea from the Chinese Anxi county. Food Technol Biotechnol 2015; 53: 180–9. doi: 10.17113/ftb.
  21. Du H, Wang Q, Yang X. Fu Brick tea alleviates chronic kidney disease of rats with high fat diet consumption through attenuating insulin resistance in skeletal muscle. J Agricult Food Chem 2019; 67: 2839–47. doi: 10.1021/acs.jafc.8b06927
  22. An R, Wen S, Li DL, Li QH, Lai XF, Zhang WJ, et al. Mixtures of tea and citrus maxima (pomelo) alleviate lipid deposition in HepG2 cells through the AMPK/ACC signaling pathway. J Med Food 2020; 23: 943–51. doi: 10.1089/jmf.2020.4706
  23. Li Q, Lai X, Sun L, Cao J, Ling C, Zhang W, et al. Antiobesity and anti-inflammation effects of Hakka stir-fried tea of different storage years on high-fat diet-induced obese mice model via activating the AMPK/ACC/CPT1 pathway. Food Nutr Res 2020; 64: 1681. doi: 10.29219/fnr.v64.1681
  24. Yuan E, Duan X, Xiang L, Ren J, Lai X, Li Q, et al. Aged oolong tea reduces high-fat diet-induced fat accumulation and dyslipidemia by regulating the AMPK/ACC signaling pathway. Nutrients 2018; 10: 187. doi: 10.3390/nu10020187
  25. Tiegs G. Cellular and cytokine-mediated mechanisms of inflammation and its modulation in immune-mediated liver injury. Zeitschrift fur Gastroenterologie 2007; 45: 63–70. doi: 10.1055/s-2006-927397
  26. Dorresteijn JA, Visseren FL, Spiering W. Mechanisms linking obesity to hypertension. Obes Rev 2012; 13: 17–26. doi: 10.1111/j.1467-789X.2011.00914.x
  27. Grundy SM. Metabolic syndrome update. Trends Cardiovasc Med 2016; 26: 364–73. doi: 10.1016/j.tcm.2015.10.004
  28. Diabetes Prevention Program Research G, Knowler WC, Fowler SE, Hamman RF, Christophi CA, Hoffman HJ, et al. 10-year follow-up of diabetes incidence and weight loss in the Diabetes Prevention Program Outcomes Study. Lancet 2009; 374: 1677–86. doi: 10.1016/S0140-6736(09)61457-4
  29. Jung UJ, Choi MS. Obesity and its metabolic complications: the role of adipokines and the relationship between obesity, inflammation, insulin resistance, dyslipidemia and nonalcoholic fatty liver disease. Int J Mol Sci 2014; 15: 6184–223. doi: 10.3390/ijms15046184
  30. Padwal RS, Majumdar SR. Drug treatments for obesity: orlistat, sibutramine, and rimonabant. Lancet 2007; 369: 71–77. doi: 10.1016/S0140-6736(07)60033-6
  31. Misawa K, Hashizume K, Yamamoto M, Minegishi Y, Hase T, Shimotoyodome A. Ginger extract prevents high-fat diet-induced obesity in mice via activation of the peroxisome proliferator-activated receptor delta pathway. J Nutr Biochem 2015; 26: 1058–67. doi: 10.1016/j.jnutbio.2015.04.014
  32. Hocayen Pde A, Grassiolli S, Leite NC, Pochapski MT, Pereira RA, da Silva LA, et al. Baccharis dracunculifolia methanol extract enhances glucose-stimulated insulin secretion in pancreatic islets of monosodium glutamate induced-obesity model rats. Pharm Biol 2016; 54: 1263–71. doi: 10.3109/13880209.2015.1067232
  33. Wang S, Wang Y, Pan MH, Ho CT. Anti-obesity molecular mechanism of soy isoflavones: weaving the way to new therapeutic routes. Food Funct 2017; 8: 3831–46. doi: 10.1039/c7fo01094j
  34. Choi EO, Park C, Shin SS, Cho EJ, Kim BW, Hwang JA, et al. Zanthoxylum schinifolium leaf ethanol extract inhibits adipocyte differentiation through inactivation of the extracellular signal regulated kinase and phosphoinositide 3-kinase/Akt signaling pathways in 3T3-L1 pre-adipocytes. Mol Med Rep 2015; 12: 1314–20. doi: 10.3892/mmr.2015.3463
  35. Lyu C, Chen, C, Ge F, Liu D, Zhao S, Chen D. A preliminary metagenomic study of puer tea during pile fermentation. J Sci Food Agricult 2013; 93: 3165–74. doi: 10.1002/jsfa.6149.
  36. Roh E, Kim JE, Kwon JY, Park JS, Bode AM, Dong Z, et al. Molecular mechanisms of green tea polyphenols with protective effects against skin photoaging. Crit Rev Food Sci Nutr 2017; 57: 1631–7. doi: 10.1080/10408398.2014.1003365
  37. Prasanth MI, Sivamaruthi BS, Chaiyasut C, Tencomnao T. A review of the role of green tea (Camellia sinensis) in antiphotoaging, stress resistance, neuroprotection, and autophagy. Nutrients 2019; 11: 747. doi: 10.3390/nu11020474
  38. Jochmann N, Baumann G, Stangl V. Green tea and cardiovascular disease: from molecular targets towards human health. Curr Opin Clin Nutr Metabol Care 2008; 11: 758–65. doi: 10.1097/MCO.0b013e328314b68b
  39. Okuda MH, Zemdegs JC, de Santana AA, Santamarina AB, Moreno MF, Hachul AC, et al. Green tea extract improves high fat diet-induced hypothalamic inflammation, without affecting the serotoninergic system. J Nutr Biochem 2014; 25: 1084–9. doi: 10.1016/j.jnutbio.2014.05.012
  40. Kogawa AC, Pires A, Salgado HRN. Atorvastatin: a review of analytical methods for pharmaceutical quality control and monitoring. J AOAC Int 2019; 102: 801–9. doi: 10.5740/jaoacint.18-0200
  41. Thatiparthi J, Dodoala S, Koganti B, Kvsrg P. Barley grass juice (Hordeum vulgare L.) inhibits obesity and improves lipid profile in high fat diet-induced rat model. J Ethnopharmacol 2019; 238: 111843. doi: 10.1016/j.jep.2019.111843
  42. Kuang W, Zhang X, Lan Z. Flavonoids extracted from Linaria vulgaris protect against hyperlipidemia and hepatic steatosis induced by western-type diet in mice. Arch Pharm Res 2018; 41: 1190–8. doi: 10.1007/s12272-017-0941-y
  43. Chung KW, Kim KM, Choi YJ, An HJ, Lee B, Kim DH, et al. The critical role played by endotoxin-induced liver autophagy in the maintenance of lipid metabolism during sepsis. Autophagy 2017; 13: 1113–29. doi: 10.1080/15548627.2017.1319040
  44. Garcia D, Hellberg K, Chaix A, Wallace M, Herzig S, Badur MG, et al. Genetic liver-specific AMPK activation protects against diet-induced obesity and NAFLD. Cell Rep 2019; 26: 192–208 e196. doi: 10.1016/j.celrep.2018.12.036
  45. Li F, Gao C, Yan P, Zhang M, Wang Y, Hu Y, et al. EGCG reduces obesity and white adipose tissue gain partly through AMPK activation in mice. Front Pharmacol 2018; 9: 1366. doi: 10.3389/fphar.2018.01366
  46. Lio, CJ, Dai YW, Wang CL, Fang LW, Huang WC. Maslinic acid protects against obesity-induced nonalcoholic fatty liver disease in mice through regulation of the Sirt1/AMPK signaling pathway. FASEB J 2019; 33: 11791–803. doi: 10.1096/fj.201900413RRR
  47. Zang L, Shimada Y, Nakayama H, Kim Y, Chu DC, Juneja LR, et al. RNA-seq based transcriptome analysis of the anti-obesity effect of green tea extract using Zebrafish Obesity Models. Molecules 2019; 24: 3256. doi: 10.3390/molecules24183256
  48. Lally JSV, Ghoshal S, DePeralta DK, Moaven O, Wei L, Masia R, et al. Inhibition of acetyl-CoA carboxylase by pPhosphorylation or the inhibitor ND-654 suppresses lipogenesis and hepatocellular carcinoma. Cell Metabol 2019; 29: 174–82 e175. doi: 10.1016/j.cmet.2018.08.020
  49. Long YC, Zierath JR. AMP-activated protein kinase signaling in metabolic regulation. J Clin Investig 2006; 116: 1776–83. doi: 10.1172/JCI29044
  50. Weikel KA, Cacicedo JM, Ruderman NB, Ido Y. Knockdown of GSK3beta increases basal autophagy and AMPK signalling in nutrient-laden human aortic endothelial cells. Biosci Rep 2016; 36: e00382. doi: 10.1042/BSR20160174
  51. Anderson R, Prolla T. PGC-1alpha in aging and anti-aging interventions. Biochim Biophys Acta 2009; 1790: 1059–66. doi: 10.1016/j.bbagen.2009.04.005
  52. Lan F, Cacicedo JM, Ruderman N, Ido Y. SIRT1 modulation of the acetylation status, cytosolic localization, and activity of LKB1. Possible role in AMP-activated protein kinase activation. J Biol Chem 2008; 283: 27628–35. doi: 10.1074/jbc.M805711200
  53. Beh BK, Mohamad NE, Yeap SK, Ky H, Boo SY, Chua JYH, et al. Anti-obesity and anti-inflammatory effects of synthetic acetic acid vinegar and Nipa vinegar on high-fat-diet-induced obese mice. Sci Rep 2017; 7: 6664. doi: 10.1038/s41598-017-06235-7
  54. Juneja LR, Kapoor MP, Okubo T, Rao T. Green tea polyphenols: nutraceuticals of modern life. Herbalgram, 2013. pp. xiii, 348 p.
  55. Xing L, Zhang H, Qi R, Tsao R, Mine Y. Recent advances in the understanding of the health benefits and molecular mechanisms associated with green tea polyphenols. J Agricult Food Chem 2019; 67: 1029–43. doi: 10.1021/acs.jafc.8b06146
  56. Suzuki T, Pervin M, Goto S, Isemura M, Nakamura Y. Beneficial effects of tea and the green tea catechin epigallocatechin-3-gallate on obesity. Molecules 2016; 21: 1305. doi: 10.3390/molecules21101305
  57. Furuyashiki T, Nagayasu H, Aoki Y, Bessho H, Hashimoto T, Kanazawa K, et al. Tea catechin suppresses adipocyte differentiation accompanied by down-regulation of PPARgamma2 and C/EBPalpha in 3T3-L1 cells. Biosci Biotechnol Biochem 2004; 68: 2353–9. doi: 10.1271/bbb.68.2353
  58. Tanaka M, Sato A, Kishimoto Y, Mabashi-Asazuma H, Kondo K, Iida K. Gallic acid inhibits lipid accumulation via AMPK pathway and suppresses apoptosis and macrophage-mediated inflammation in hepatocytes. Nutrients 2020; 12: 1479. doi: 10.3390/nu12051479.
  59. Berube-Parent S, Pelletier C, Dore J, Tremblay A. Effects of encapsulated green tea and Guarana extracts containing a mixture of epigallocatechin-3-gallate and caffeine on 24 h energy expenditure and fat oxidation in men. Br J Nutr 2005; 94: 432–6. doi: 10.1079/bjn20051502