A 500-g sample of Dragon-pearl tea dust was added to hot water (90°C, 10 litres) for 60 min. Sodium chloride (600 g) was subsequently added into the extract solution to achieve a mass fraction of 6%, dialyzing in distilled water for 1.5 h. Following filtration, 15 g NaHSO₃ and 100 ml aluminum sulfate saturated solution were added to the extract solution. The extract solution was heated to 80°C and 15% NaHCO₃ solution was added until the pH value of the solution reached a range of 5.0–6.0. A large quantity of precipitates was filtered and washed three times with 70°C water of the same volume (10 litres). The precipitates were dissolved in pH 4.0 hydrochloric acid solution (1.5 L) for 50 min, and a small quantity of gelatinous precipitate was removed by centrifugation at 25°C for 20 min, 4,000 × g. Next, 9 g NaHSO₃ was added to the acid solution, which was further extracted through administration of ethyl acetate (0.5 times of the solution's volume) three times, with each extraction occurring for 10 min and the three extractions were mixed. Vitamin C of 2% of the tea's weight was added to water of 0.4 times the extraction's volume, and the pH value was adjusted to 3.0 through the addition of citric acid. The solution was washed twice with the ethyl acetate extraction, and the DTCP extracts were finally obtained using rotary evaporation (RE-52A; Shanghai Yarong Biochemical Instrument Factory, Shanghai, China).

Samples of 4-ml DTCP solution (25, 50 or 100 µg/ml) were added to 1.0 ml DPPH methanol solution (1.5×10⁻⁴ M). Following storage at room temperature for 30 min, the absorbance of the solution was determined at 520 nm using a spectrophotometer (UV-5100; Shanghai Metash Instruments Co. Ltd., Shanghi, China), and the remaining DPPH was quantified [(OD_DPPH-OD_sample)/OD_DPPH]×100%, where OD is optical density. The results are expressed as the mean of triplicate experimental values (10).

A 1.4-ml reaction system was constructed, containing 0.2 ml deoxyribose (6 mM), 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 (400 µM), 0.2 ml H₂O₂ (3 mM), 0.2 ml ascorbic acid (400 µM) and 0.2 ml DTCP solution (25, 50 or 100 µg/ml). Following incubation in a water bath for 60 min at 37°C, the reaction was stopped by adding 1 ml trichloroacetic acid and 1 ml 2-thiobarbituric acid. The solution was subsequently boiled in a water bath for 20–25 min at 90°C. The absorbance was measured at 532 nm by ultraviolet spectrophotometer (UV-5100). All analyses were run in triplicate and average values were calculated (10).

Institute for Cancer Research mice (male, n=50, 7 weeks old), purchased from Chongqing Medical University (Chongqing, China), were allocated into five groups: Normal, control and 50, 100 and 200 mg/kg DTCP (n=10 per group). The mice in the normal and control groups received 0.2 ml distilled water per day by gavage for 4 weeks. However, the DTCP group mice were orally administered 50, 100 or 200 mg/kg DTCP solutions (0.2 ml) everyday for 4 weeks. Subsequently, the control, 50 100 and 200 mg/kg DTCP group mice were administered a single intraperitoneal injection of 10 mg/kg body weight/day reserpine (Sigma-Aldrich, St. Louis, MO, USA) for 3 days. Following the final injection, all the mice were fasted for 24 h. The mice were sacrificed using CO₂ and the stomach was removed and inflated by injecting 10 ml formalin (1%) for 10 min to fix the tissue walls, and the stomach was opened along the greater curvature (11). The gastric ulcer inhibitory index of the hemorrhagic lesions developed in the stomach was measured using a D550 digital camera (Canon, Tokyo, Japan) with a square grid, and the images were analyzed using ImageJ software (National Institutes of Health, Bethesda, MD, USA). The gastric ulcer inhibitory index was calculated as follows: Inhibitory index (%) = (gastric ulcer area of the control mice - gastric ulcer area of the treated mice)/gastric ulcer area of the control mice. In addition, the gastric secretion volume of each mouse was measured using a 10-ml measuring cylinder, and the pH of the gastric juice was measured using a SevenEasy pH meter (Mettler-Toledo Ltd., Schwerzenbach, Switzerland) following dilution 10 times with distilled water. Experiments followed a protocol approved by the Animal Ethics Committee of Chongqing Medical University (Chongqing, China).

Serum levels of SOD, GSH-Px, MDA, LPO, CAT, PGE2 and NO were determined using assay kits for SOD (#A001; WST-1 method), GSH-Px (#A005; colorimetric method), MDA (#A003; thiobarbituric acid method), LPO (#A106), CAT (#A007; visible light method), PGE2 (#H099) and NO (#A012; nitrate reductase method), respectively (Nanjing Jiancheng Bioengineering Institute, Jiangsu, China).

Total RNA from the gastric tissues was isolated using TRIzol® reagent (Invitrogen Life Technologies, Carlsbad, CA, USA), according to the manufacturer's instructions. The RNA was digested with RNase-free DNase (Roche Diagnostics, Basel, Switzerland) for 15 min at 37°C, and purified using an RNeasy kit (Qiagen GmbH, Hilden, Germany), according to the manufacturer's instructions. The total RNA (2 µg) was incubated for l h at 37°C with avian myeloblastosis virus reverse transcriptase (GE Healthcare Life Sciences, Little Chalfont, UK) and random hexanucleotides to form cDNA, in accordance with the manufacturer's instructions. Primers (Thermo Fisher Scientific, Waltham, MA, USA) for epidermal growth factor (EGF; forward: 5′-GCC AAG CTC AGA AGG CTA C-3′; reverse: 5′-CAG GCC AGC CTC GTC TCA T-3′), epidermal growth factor receptor (EGFR; forward: 5′-TCG GTG CTG TGC GAT TTA-3′; reverse: 5′-TTT CTG GCA GTT GCT CCT C-3′), gastrin (Gas; forward: 5′-CCT ACT GCC ACA ACA GTT AA-3′; reverse: 5′-CAT CCA TCC GTA TGC TTC-3′), vascular endothelial growth factor (VEGF; forward: 5′-CCT GGC TTT ACT GCT GTA CCT-3′; reverse: 5′-GTG CCA AAA GGG TCA TCA TCT C-3′) and vascular endothelial growth factor receptor 1 (Flt-1 or VEGFR-1; forward: 5′-CAA GTG GCC AGA GGC ATG GAG TT-3′; reverse: 5′-GAT GTA GTC TTT ACC ATC CTG TTG-3′) were used to specifically amplify the genes. GAPDH was used as a control (forward: 5′-CGG AGT CAA CGG ATT TGG TC-3′; reverse: 5′-AGC CTT CTC CAT GGT CGT GA-3′). Amplification was performed in a thermal cycler (denaturation at 94°C for 1 min, annealing at 56°C for 1 min and elongation at 72°C for 1 min for 30 cycles; Eppendorf AG, Hamburg, Germany). The PCR products were separated on a 1.0% agarose gel and visualized using ethidium bromide staining (12).

Analysis of variance (ANOVA) was performed and the results are presented as the mean ± standard deviation. Differences between the mean values of the individual groups were assessed using one-way ANOVA and Duncan's multiple range test. P<0.05 was considered to indicate a statistically significant difference. SAS software, version 9.1 (SAS Institute, Inc., Cary, NC, USA) was used to perform all the statistical analyses.

DTCP exhibited notable DPPH and OH radical-scavenging activity in vitro (Fig. 1). A DTCP concentration of 100 µg/ml exhibited the highest DPPH and OH radical scavenging activity, at 72.5 and 85.2%, respectively. Furthermore, at doses of 25 and 50 µg/ml, DTCP treatment exhibited 21.3 and 48.7% DPPH radical scavenging activity and 33.6 and 57.1% OH radical scavenging activity, respectively.

Gastric ulcer levels in the mice from the experimental groups were shown to decrease following DTCP treatment (Fig. 2). The gastric ulcer area was the largest in the untreated control mice at 11.47±3.13 mm². However, the 50, 100 and 200 mg/kg DTCP-treated mice presented with reduced gastric ulcer areas of 8.68±2.66, 6.71±1.89 and 3.14±1.53 mm², while the gastric ulcer levels were 24.32, 41.50 and 72.63%, respectively.

In the reserpine-induced gastric ulcer control mice, the highest gastric secretion volume (0.82 ml) and the lowest pH value (1.04) were observed (Fig. 3). Conversely, the mice in the normal group exhibited the lowest gastric secretion volume (0.15 ml) and the highest pH value (3.62). DTCP treatment was shown to improve these levels, with the 50, 100 and 200 mg/kg DTCP-treated mice exhibiting gastric secretion volumes of 0.63, 0.41 and 0.27 ml, respectively, and pH values of 2.03, 2.68 and 3.10, respectively.

Control group mice presented with the lowest levels of SOD, GSH-Px and CAT, and the highest levels of MDA and LPO (Table I). Mice in the three DTCP treatment groups exhibited increasing levels of SOD, GSH-Px and CAT, and decreasing levels of MDA and LPO levels. The highest concentration of DTCP (200 mg/kg) produced the maximal change in these levels in the reserpine-induced gastric ulcer mouse model, resulting in levels that were comparable with the normal group mice.

Control group mice exhibited the lowest levels of PGE2 and NO, at 57.80 ng/l and 48.29 µmol/l, respectively (Fig. 4). However, treatment with DTCP was demonstrated to increase these levels when compared with the control mice. The 50, 100 and 200 mg/kg DTCP-treated mice exhibited increasing PGE2 levels of 66.23, 69.38 and 72.65 ng/l, respectively, and increasing NO levels of 53.47, 55.71 and 57.08 µmol/l, respectively. Among the groups, the PGE2 and NO levels were highest in the normal group mice, at 77.86 ng/l and 60.26 µmol/l, respectively.

Expression levels of a number of genes in the gastric tissue of the mice were determined using RT-qPCR. DTCP was demonstrated to alter the mRNA expression levels of EGF, EGFR, Gas, VEGF and Flt-1. DTCP at concentrations of 50, 100 and 200 mg/kg was shown to increase the mRNA expression levels of the epidermal growth-related EGF and EGFR (Fig. 5) Furthermore, DTCP treatment increased the mRNA expression levels of vascular endothelial growth-related VEGF and Flt-1 (Fig. 6). However, the mRNA expression levels of Gas were reduced in the mice following DTCP treatment at 50, 100 and 200 mg/kg (Fig. 7). The highest concentration of DTCP (200 mg/kg) exerted the most marked effect, resulting in mRNA expression levels of the various genes that were comparable to those of the normal group mice.