Pu-erh tea has become a focus of research due to its reported biological activities, including anti-oxidation, anti-inflammation and anti-immunosenescence. The present study was performed to evaluate the potential gastroprotective function of Pu-erh tea extracts against ethanol-induced gastric mucosal damage in rats. Sprague Dawley rats were randomly divided into seven groups: A normal control, a model control, a cimetidine (0.08 g/kg) group, three Pu-erh tea extracts groups (low, moderate and high-dose; 0.50, 1.00 and 1.50 g/kg, respectively, and a green tea powder (1.00 g/kg) group. The normal and model groups were pre-treated with distilled water while the other groups were respectively administered cimetidine, Pu-erh tea extracts and green tea powder for 14 days. Then, absolute ethanol was orally administered to the rats of all groups excluding the normal controls. The effects of the pretreatments on gastric mucosal injury were evaluated by gross assessment of gastric lesions, examination of histopathology and determination of myeloperoxidase (MPO) activity and asymmetric arginine (ADMA) concentration in gastric mucosal homogenate. Pre-treatment with cimetidine or Pu-erh tea extracts markedly suppressed the formation of ethanol-induced gastric lesions. Furthermore, clear decreases in MPO activity and ADMA concentration in the gastric mucosal homogenate were observed following pretreatment with cimetidine or Pu-erh tea extracts. The anti-gastric ulcer activity of green tea was less than that of Pu-erh tea. Overall, these effects of Pu-erh tea extracts may be due to potential functions in protecting the gastric mucus layer and suppressing inflammation.
Pu-erh tea is a type of fermented tea that incorporates microorganism metabolites during the fermentation process (
It is established that gastric mucosal injury occurs due to an imbalance between mucosal defensive and aggressive factors (
The biological functions of Pu-erh tea in the stomach are broad according to previous literature, and include detoxification, promotion of food digestion, regulation of gastrectasia and removal of fats (
Sprague-Dawley male rats (n=126) were obtained from the Vital River Laboratories Co., Ltd. (Beijing, China), the weight of which ranged from 180 to 220 g. They were group-housed (5 rats per cage) in standard and pathogen-free environmental conditions (22±1°C, 60±5% humidity, 12-h light/dark cycle) with free access to a standard commercial diet and water ad libitum. Animals were acclimatized to the environment for at least one week. The study protocols were approved by the Tasly Laboratory Animal Welfare and Ethics Committee of Tasly Pharmaceuticals, Inc. (Tianjin, China), and conducted according to the rules of animal experimentation and the guide for the Care and Use of Laboratory Animals of Tasly Pharmaceuticals, Inc. The rats were randomly divided into seven groups (n=18 per group).
An aqueous extract of Pu-erh tea was provided by Tasly Pharmaceuticals, Inc., which was dissolved in distilled water and prepared as described previously (
The ingredients in fermented Pu-erh tea include caffeine, polyphenols, γ-aminobutyric acid, theanine, statin, polysaccharides (
Cimetidine, used as a reference drug in this study, was obtained from GlaxoSmithKline (Shanghai, China) and dissolved in distilled water. A standardized powder of green tea was purchased from Damin Foodstuff (Zhangzhou) Co., Ltd., (Zhangzhou, China). Hematoxylin and eosin were provided by Muto Pure Chemicals Co., Ltd. (Tokyo, Japan). Absolute ethanol, formalin, paraffin and dimethylbenzene were supplied by Rionlon (Tianjin) Industry Co., Ltd. (Tianjin, China). ELISA kits for rat MPO (catalogue no. CK-E30635) and rat ADMA (catalogue no. CK-E30769) were provided by Shanghai Bogoo Biotechnology Co., Ltd. (Shanghai, China). Other reagents used in the study were of analytical grade or higher without further purification.
The rats in normal and model groups were intragastrically (i.g.) administered 10 ml/kg distilled water, while those in other groups were administered 0.08 g/kg cimetidine (i.g.), Pu-erh tea extracts at 0.50, 1.00 and 1.50 g/kg, and 1.00 g/kg green tea powder, respectively. The animals were administered with the test drugs or distilled water between 8:00-9:00 am once a day for 14 consecutive days. Acute gastric lesions were created by intragastric application of absolute ethanol according to a common method (
After 15 min, the stomachs in 10% buffered formalin were removed. Photographs of the gastric mucosa were taken, and the mucosal lesions were scored by a laboratory animal technician blinded to the experimental protocol. The length and width of each injured area of the gastric mucosa were measured with a vernier caliper. Gastric mucosal ulcer index (UI) was determined according to
The formalin-fixed stomach tissues were embedded in paraffin wax and gradient dehydrated in increasing concentrations of ethanol (70–100% v/v). The specimens were sectioned (4-µm thick) and stained with hematoxylin (~1.5 min) and eosin (30 sec) at room temperature for histopathological examination. An epithelial damage scoring system (
The segments of stomach tissue with ulcers were processed into 20% tissue homogenate in cold saline with a UP400S ultrasonic processor (Ningbo Xinzhi Bio-tech Co., Ltd., Ningbo, China). The rat MPO and ADMA ELISA kits were then used to determine MPO activity and ADMA concentration in the homogenate, according to the manufacturer's protocols. Optical absorbance (O.D.) at 450 nm was recorded with a Tecan Infinite 200 Microplate Reader (Tecan Group, Ltd., Mannedorf, Switzerland). MPO activity or ADMA concentration in the samples was then determined by comparing the O.D. value of the samples to the standard curve.
The results from each group were expressed as the mean ± standard error of mean. The data were analyzed by one-way analysis of variance with Fisher's least significant difference post hoc analysis. Statistical analysis was performed with SPSS 16.0 (SPSS, Inc., Chicago, IL, USA), and P<0.05 was considered to indicate statistical significance.
Cimetidine and moderate-to-high-dose Pu-erh tea extracts (1.00 and 1.50 g/kg) administered prior to alcohol-induced gastric injury significantly decreased the ulcer index in rat gastric mucosa, compared with the model control (P<0.01;
Histopathological assessment of the gastric tissues was subsequently performed (
MPO activity in gastric tissue homogenate of the model control group was significantly increased compared with that of the normal control group (2,032.59±69.63 vs. 1,778.13±20.58 U/l, P<0.05;
Gastric mucosal damage is caused by an imbalance between the protective and aggressive mechanisms in the mucosa, and is considered the net result of the actions of several endogenous factors and aggressive exogenous factors (
Ethanol is commonly used to induce ulcers in experimental animals, and causes acute gastric mucosal damage (
Gastric mucus when secreted in sufficient quantity is an important factor for the functioning of the gastric mucosa. It consists of a viscous, elastic, adherent and transparent gel, formed by water and glycoproteins, that covers the surface of the gastrointestinal mucosa (
MPO, a biomarker for neutrophil-dependent inflammation, is mainly released from neutrophils, and therefore is also an essential marker for normal neutrophil function. MPO and other tissue-damaging substances including reactive oxygen metabolites and cytotoxic proteins are released into the extracellular space when neutrophils are stimulated (
ADMA is the endogenous inhibitor of nitric oxide synthase (NOS), and has been implicated in pathophysiologies of the upper gastrointestinal tract (
In conclusion, the study present focused on the protective effect of Pu-erh tea extracts in the gastric mucosa. Absolute ethanol was used to induce gastric mucosal injury and the action of Pu-erh tea extracts was investigated. The extracts exerted significant protective effects against the gastric mucosal damage. The human equivalent of the rat dose 1.00 g/kg is 0.16 g/kg, according to the dose conversion relationship of the Food and Drug Administration (
Not applicable.
The present study was supported by the China National Science and Technology Support Program (grant no. 2013Bad26q01).
The analyzed data sets generated during the study are available from the corresponding author on reasonable request.
JY and WZ were the principal investigators responsible for the study and contributed to the design of the experiments, and JY wrote the original manuscript. YG and JD were responsible for analyzing the experimental data. XL, PT and YL were responsible for collecting experimental data. XM and YZ was the leader of the research group, and was responsible for the study design and guidance, and for checking the accuracy and authenticity of the manuscript.
The study protocols were approved by the Tasly Laboratory Animal Welfare and Ethics Committee of Tasly Pharmaceuticals, Inc. (Tianjin, China), and conducted according to the rules of animal experimentation and the guide for the Care and Use of Laboratory Animals of Tasly Pharmaceuticals, Inc.
Not applicable.
WZ, XL, PT and YL are research fellows and XM is vice director of Tasly Pharmaceuticals, Inc., Tianjin, China. All other authors declare no competing interests.
Gross appearance of the gastric mucosa of rats. Representative images are shown (n=9). (A) Treatment with distilled water (normal control). No disturbance in the gastric mucosa was observed. (B) Alcohol-induced gastric lesion pretreated with distilled water (model control). Severe injuries were observed in the gastric mucosa. Absolute ethanol induced extensive visible hemorrhagic necrosis in the gastric mucosa of the rats. (C) Alcohol-induced gastric lesion pretreated with 0.08 g/kg cimetidine. Injury in the gastric mucosa was reduced compared with that in the model control. (D) Alcohol-induced gastric lesion pretreated with 1.00 g/kg green tea powder. (E-G) Alcohol-induced gastric lesions pre-treated with Pu-erh tea extracts at doses of 0.50, 1.00 and 1.50 g/kg, respectively. Gastric mucosal injury was reduced in a dose-dependent manner. Blue and red arrows indicate areas of spot erosion and area erosion, respectively; yellow arrows indicate mucosal folds.
Test drugs reduce ulcer index dose-dependently in rats with alcohol-induced gastric ulcer. Data were expressed as means ± standard deviation (n=9). *P<0.05 and **P<0.01 as indicated. The inhibitory rate of each test drug is presented above the bars.
Histopathological evaluation of the gastric mucosa in rats. Representative images are shown (n=9). (A) Treatment with distilled water (normal control). No disturbance in the gastric mucosa or damage in the mucous superficial layer was observed. (B) Alcohol-induced gastric lesion pretreated with distilled water (model control). Severe pathological changes including hyperemia, bleeding and epithelial cell degeneration and necrosis were observed in the gastric mucosal layer. (C) Alcohol-induced gastric lesion pretreated with 0.08 g/kg cimetidine. The pathological changes in the gastric mucosa layer were reduced compared with those in the model control. (D) Alcohol-induced gastric lesion pretreated with 1.00 g/kg green tea powder. (E-G) Alcohol-induced gastric lesions pretreated with Pu-erh tea extracts at doses of 0.50, 1.00 and 1.50 g/kg, respectively. The pathological changes in the gastric mucosa layer were inhibited in a dose-dependent manner. The rat stomach sections were stained with hematoxylin and eosin. Images are shown at magnification, ×400. Scale bars, 60 µm. Red arrows indicate areas of mucosal damage.
Effect of the tested drugs on MPO activity in the stomach tissues of rats with alcohol-induced gastric ulcer. Data were expressed as the mean ± standard deviation (n=9). *P<0.05 and **P<0.01 as indicated. MPO, myeloperoxidase.
Effect of the tested drugs on ADMA concentration in the stomach tissues of rats with alcohol-induced gastric ulcer. Data were expressed as the mean ± standard deviation (n=9). *P<0.05 and **P<0.01 as indicated. ADMA, asymmetric arginine.
Gross scoring system for gastric mucosal lesions.
Points | ||||
---|---|---|---|---|
Gastric mucosal lesion | 1 | 2 | 3 | 4 |
Spot erosion (no.) | 1 | – | – | – |
Erosion length (mm) | 1–5 | 6–10 | 10–15 | >15 |
Erosion width (mm) | 1–2 | >2 | – | – |
Scoring system for histopathological changes in gastric mucosal epithelia.
Points | |||||
---|---|---|---|---|---|
Pathology, area affected | 1 | 2 | 3 | 4 | 5 |
Hyperemia | <1/5 | 1/5-2/5 | 2/5-3/5 | 3/5-4/5 | All over the epithelia |
Bleeding | <1/5 | 1/5-2/5 | 2/5-3/5 | 3/5-4/5 | All over the epithelia |
Degeneration and necrosis | <1/5 | 1/5-2/5 | 2/5-3/5 | 3/5-4/5 | All over the epithelia |
Histopathological scores of the gastric mucosa of rats with alcohol-induced gastric lesions pre-treated with the tested drugs.
Group | Hyperemia | Bleeding | Epithelial cell degeneration/necrosis | Pathological changes, total score |
---|---|---|---|---|
Normal | 0.38±0.20 | 0.00±0.00 |
0.00±0.00 |
0.38±0.20 |
Model | 1.22±0.16 |
2.11±0.37 |
3.33±0.25 |
15.44±0.56 |
Cimetidine (g/kg) | 1.11±0.12 | 2.00±0.31 | 1.56±0.31 |
9.78±0.79 |
Green tea powder (g/kg) | 1.11±0.12 | 1.67±0.35 | 3.11±0.41 | 13.78±1.50 |
Pu-erh tea extracts(g/kg) | ||||
0.5 | 1.11±0.12 | 1.78±0.42 | 3.44±0.26 | 13.89±1.26 |
1.0 | 1.25±0.25 | 1.25±0.37 | 2.00±0.76 |
9.75±1.16 |
1.5 | 1.11±0.12 | 0.56±0.19 |
1.33±0.18 |
6.22±0.77 |
Data were expressed as the mean ± standard deviation. (n=9).
P<0.05
P<0.01 vs. normal control group
P<0.05
P<0.01 vs. model control group.