Yellow tea was purchased from Sichuan Mengding Huangcha Tea Industry Co., Ltd., China. The yellow tea was stored at −80°C and freeze-dried to produce a powder. A twenty-fold volume of boiling water was added to the powdered sample and extraction was conducted twice. The aqueous extract was evaporated using a rotary evaporator (Eyela N-1100, Tokyo, Japan), concentrated and then dissolved in dimethylsulfoxide (DMSO; Amresco, Solon, OH, USA) to adjust to the stock concentration (20%, w/v).

The DPPH free radical-scavenging activity was determined according to the method of Blois (10). Four milliliters of 100, 200 and 500 μg/ml concentrations of the sample solution were added to 1.0 ml DPPH methanol solution (1.5×10⁻⁴ M). After storing at room temperature for 30 mins, the absorbance of the solution was determined at 520 nm using a spectrophotometer and the remaining DPPH was quantified. The results expressed are the means of triplicate values (11).

The OH radical-scavenging activity was assessed as described by Banerijee et al (12). The reaction system (1.4 ml), contained yellow tea extract, deoxyribose (6 mM, 0.2 ml), 0.2 ml sodium phosphate buffer solution (20 mM, pH 7.4), 0.2 ml anhydrous iron chloride (FeCl₃; 400 μM), 0.2 ml FeSO₄-ethylenediaminetetraacetic acid (EDTA; 400 μM), 0.2 ml H₂O₂ (3 mM), 0.2 ml ascorbic acid (400 μM) and 0.2 ml yellow tea extract solution (50, 100 and 200 μg/ml). After incubation at 37°C in a water bath for 60 min, the reaction was stopped by adding 1 ml trichloroacetic acid and 1 ml 2-thiobarbituric acid in a 1.4-ml reaction system. The solution was boiled for 20–25 min at 90°C in a water bath. The absorbance was measured at 532 nm. All analyses were run in triplicate and averaged.

Male Sprague-Dawley rats (n=50, 7-weeks-old) were purchased from the Experimental Animal Center of Chongqing Medical University (Chongqing, China). The rats were maintained in a temperature-controlled facility (temperature, 25±2°C; relative humidity, 50±5%) with a 12-h light/dark cycle and free access to a standard rat chow diet and water.

The experimental design was as follows: the normal and control groups received 14-day repeated oral administration of distilled water and a single dose of the vehicle (2 ml/kg b.w. olive oil, p.o.); the sample groups received 14-day repeated oral administration of 250, 500 or 1,000 mg/kg yellow tea extract. Then, the control and sample group rats were administered 1 ml HCl/ethanol (60% in 150 mM HCl) p.o. through esophageal intubation and were then sacrificed 1 h later under deep ether anesthesia. The stomachs were removed, inflated by injecting 10 ml 1% formalin for 10 min to fix the tissue walls and opened along the greater curvature. The area (mm²) of hemorrhagic lesions developed in the stomach was measured using a digital camera (D550; Canon, Tokyo, Japan) with a square grid and the images were analyzed by ImageJ software (National Institutes of Health, Bethesda, MD, USA). These experiments followed a protocol approved by the Animal Ethics Committee of Chongqing Medical University (Chongqing, China).

For the serum cytokine assay, blood from the inferior vena cava was collected in a tube and centrifuged (1,370 × g for 10 min at 4°C). The serum was aspirated and assayed as follows: Concentrations of the inflammatory-related cytokines IL-6 and TNF-α in serum were measured by ELISA according to the kit manufacturer’s instructions (BioLegend, San Diego, CA, USA). Briefly, after the biotinylated antibody reagent was added to 96-well plates, supernatants of homogenized serum were incubated at 37°C in CO₂ for 2 h. After washing with phosphate-buffered saline (PBS), horseradish peroxidase (HRP)-conjugated streptavidin peroxidase solution was added and the plate was incubated for 30 min at room temperature. The absorbance was then measured at 450 nm using a microplate reader (13).

Data are presented as mean ± standard deviation (SD). Differences between the mean values for individual groups were assessed with one-way analysis of variance (ANOVA) with Duncan’s multiple range test. P<0.05 was considered to indicate a statistically significant difference. SAS version 9.1 (SAS Institute Inc., Cary, NC, USA) was used for statistical analyses.

The radical-scavenging activity of yellow tea was investigated using DPPH radicals (Fig. 1). Yellow tea showed scavenging activity on DPPH radicals at various concentrations. At 100, 200 and 500 μg/ml, the radical-scavenging activities were 10.8, 41.0 and 72.6%, respectively. This indicates that the radical-scavenging activity of yellow tea increased as the concentration of its extract increased.

The ability of yellow tea to scavange OH radicals was evaluated by measuring the deoxyribose damage induced by the Fe³⁺/ascorbate/EDTA/H₂O₂ system using the thiobarbituric acid (TBA) method (14). Deoxyribose degrades into fragments that react with TBA upon heating at a low pH to form a pink color. The inhibitory effects of yellow tea on deoxyribose damage are shown in Fig. 2. The inhibitory rate of a 200 μg/ml extract was 57.6%, which is higher than those of 100 μg/ml (32.2%) and 50 μg/ml (13.2%) extracts.

The IL-6 level of normal rats was 52.1±4.5 pg/ml; however, that of control rats was significantly increased to 271.2±10.2 pg/ml following the induction of gastric injury. The levels of IL-6 in rats fed with 250, 500 and 1,000 mg/kg yellow tea were 250.7±11.2, 206.3±9.8 and 144.3±12.2 pg/ml, respectively (Fig. 3). The TNF-α levels in normal, control and 250, 500 and 1,000 mg/kg yellow tea-treated rats were 127.5±14.6, 843.3±22.2, 715.3±18.5, 635.4±20.6 and 438.4±19.4 pg/ml, respectively (Fig. 4). The serum IL-6 and TNF-α levels in the rats in the yellow tea-treated groups were significantly lower than those in the control group.

The administration of yellow tea to rats prior to the induction of gastritis led to reduced gastric injury. The rats of the control group demonstrated a gastric injury area of 14.2±3.4 mm². Treatment with 250 and 500 mg/kg yellow tea resulted in gastric injury inhibition rates of 11.3% (gastric injury area, 12.6±2.2 mm²) and 56.3% (gastric injury area, 6.2±1.6 mm²), respectively, while 1,000 mg/kg yellow tea (inhibition rate, 74.6%; gastric injury area, 3.6±1.1 mm²) demonstrated the best gastritis preventive effect (Table I and Fig. 5). The results suggest that yellow tea has a strong preventive effect on gastric injury.