Materials and chemicals
2,4,6-Tri(2-pyridyl)-1,3,5-triazine (TPTZ), 1,1-Diphenyl-2-picrylhydrazyl Free (DPPH), 2,2′-azino-bis (3-ethylbenzothiazoline-6-sulfonic acid) (ABTS), Nutrient Broth, Agar Powder, and Trypsin (1:250) are from Beijing Solarbio Science & Technology Co., Ltd. Pepsin (1:3000) from BioFroxx, Germany. Escherichia coli (E. coli) is used as standard quality control strain ATCC 25922 and Staphylococcus aureus (S. aureus) is used as standard quality control strain ATCC 25923. Penicillin sodium is from Shanghai yuanye Bio-Technology Co., Ltd, Lot: S15J7Y9073, mass fraction 97%. Gallic acid is from Chengdu Kelong Chemical Reagent Factory, mass fraction 99%. Gallic acid ethyl ester is from Chengdu Prefa Technology Development Co. Lot: PRF20090442, mass fraction 98%. Folin–Ciocalteu’s phenol reagent is a homemade laboratory product. Other chemical reagents are all of analytical purity grade. Methanol is chromatographically pure grade. Water is purified water.
Raw material
The Guyinye was prepared by extracting Turkish Gall with a certain concentration of ethanol. The Guyinye residues from Xinjiang Qimu Medical Research Institute were extracted with 60% ethanol to obtain secondary residues (Zeng et al. 2019). Secondary residues were dried, crushed, and sieved through 200 mesh to obtain the raw material (Fig. 1).
Preparation of working solutions
Dilute hydrochloric acid: Concentrated hydrochloric acid (234 mL) was diluted to 1000 mL with water.
Potassium dihydrogen phosphate buffer: Potassium dihydrogen phosphate (6.8 g) was added with water to obtain 500 mL buffer solution. The pH of the buffer solution was adjusted to 6.8 by using 0.1 mol/L sodium hydroxide.
Artificial gastric juice (pH = 2): The dilute hydrochloric acid (16.4 mL), 800 mL water, and 10 g pepsin were added to volumetric flask (1000 mL). The mixtures were shaken well and diluted to 1000 mL with water (Zhu et al. 2019).
Artificial intestinal solution (pH = 6.8): Potassium dihydrogen phosphate buffer (500 mL) and trypsin (10 g) were added to volumetric flasks (1000 mL). The solutions were mixed and diluted to 1000 mL with water (Chen et al. 2018).
Hydrochloric acid buffer (pH = 2) was prepared in the same way as artificial gastric juice without the addition of digestive enzymes.
Phosphate buffer (pH = 6.8) was prepared in the same way as artificial intestinal solution without the addition of digestive enzymes.
Treatment procedure
SRTG (1 g) was added with 50 mL of working solutions (Lin et al. 2021). The mixtures were kept in a constant-temperature and lightproof water bath at 37 °C for 30 min and filtered to obtain extracts. The extracts were diluted to determine the total polyphenols content (TPC). The extracts were volatilized, dissolved with sterile water, passed through 0.22 μm sterile filter membrane, and used for bacteriostatic experiments. The extracts were extracted with ethyl acetate three times (25 mL), and then upper clear layer obtained from the three extractions was combined, evaporated, dissolved in methanol, passed through 0.22 μm membrane, and used for high-performance liquid chromatography (HPLC) experiments.
Determination of the TPC
We referred to the Pharmacopoeia of the People’s Republic of China (The Commission of Pharmacopoeia 2020). Sample solutions (2 mL), Folin-Ciocalteu’s phenol reagent (1 mL), and 15% Na2CO3 (13 mL) were added to a 25 mL volumetric flask, and diluted to the mark with water, shaken well, and placed for 30 min. The absorbance was measured at 760 nm. Blank controls were prepared using the method above.
HPLC analysis
Standard solutions of gallic acid and gallic acid ethyl ester were prepared, passed through 0.22 μm filter membrane, and stored at 4 °C. Separation was performed using HPLC with a diode array detector (HPLC-DAD) apparatus equipped with the WondaSil C18 column (4.6 × 250 mm, 5 μm) and set column temperature as 30 °C. The mobile phase was modified referred previous method (Majumdar et al. 2021). The solvent was consisted of solutions A (methanol) and B (0.2% phosphoric acid water). The gradient program was as follows: 0–3 min, 80% B–57% B; 3–7 min, 57% B–55% B; 7–11 min, 55% B–54% B; 11–14 min, 54% B–51% B; 14–15 min, 51% B–45% B; and 15–20 min, 45% B–35% B. Analyses were performed at a flow rate of 0.8 mL/min, and the detected wavelength was 273 nm with an injection volume of 2 μL.
Inhibitory activity of SRTG against Escherichia coli and Staphylococcus aureus
E. coli and S. aureus are the main pathogenic bacteria that cause gastrointestinal diseases. The inhibition effects of the extracts were observed using the perforation method (Soliman and El-Sayed 2021). A single colony was collected from a blood plate culturing E. coli and S. aureus, placed in 2 mL nutrient broth culture, and incubated at 5% CO2 and 37 °C for 24 h. Suspensions of E. coli and S. aureus were diluted for counting. The bacterial solution was adjusted to 108 CFU/mL. Suspensions of E. coli and S. aureus (100 μL, 108 CFU/mL) were collected and spread onto agar plates with a glass triangular stick. 6 mm holes were punched on the petri dish by using a hole puncher (Javed et al. 2020). Sample solutions (40 µL) were injected into the well. Penicillin sodium solution was positive control solution and solvent was blank control solution. Petri dish was placed in incubator 24 h to measure the diameter of the inhibition zone.
Assessment of the minimum bactericidal concentration (MBC)
The double dilution method and streak plate method were used to determine the minimum bactericidal concentration (MBC) against E. coli and S. aureus (Wang et al. 2022). The suspensions of E. coli and S. aureus were diluted to obtain a concentration of 106 CFU/mL and used in test. The lowest drug concentration for sterile growth was called the MBC. Each experiment was performed parallel in three times.
Antioxidant capacity
The experiments were carried out with some modifications according to the previous methods (Wu et al. 2021). The antioxidant capacity of the samples was determined using the microplate reader. ABTS working solution preparation: 10 mL of 7 mmol/L ABTS solution and 5 mL of 2.45 mmol/L K2S2O8 were mixed to obtain ABTS stock solution. Then ABTS stock solution was kept out light for 14 h. The ABTS working solution was obtained by diluting the stock solution with ethanol to have an absorbance of 0.70 ± 0.02. The samples were diluted with water to 1250, 625, 312.5, 156.25, and 78.125 μg/mL, respectively. The above dilutions (100 μL) and ABTS working solution (100 μL) were pipetted into a 96-well plate, mixed well, and kept away from light for 10 min and the absorbance was measured at 734 nm. The scavenging rate was calculated in the following equation (Eq. (1)). A0 was the absorbance value of the control group (no samples), A1 was the test group (samples and reagent), and A2 was the absorbance value of the sample group (no reagent).
$$\mathrm{Scavenging rate }\left(\mathrm{\%}\right)=\frac{{A}_{0}-\left({A}_{1}-{A}_{2}\right)}{{A}_{0}}.$$
(1)
The DPPH scavenging assay followed the relevant literature with some modifications (Takatsuka et al. 2022; Chen et al. 2022). Samples of 5000, 2500, 1250, 625, and 312.5 μg/mL were prepared. The extracts solutions (100 μL) and 0.1 mmol/L DPPH-ethanol solution (100 μL) were taken into a 96-well plate, respectively, shaken well, and kept out light for 30 min. The absorbance of the sample solution was measured at 517 nm. The scavenging rate was calculated in the same way as Eq. (1).
Ferric ion reducing antioxidant power (FRAP) assay referenced to relevant literature with some modifications (Mocan et al. 2020). Preparation of FRAP working solution is as follows: FRAP1: Take 0.18 g of sodium acetate and 1.6 mL of acetic acid to 100 mL volumetric flask and fix to volume. FRAP2: Take 78 mg of TPTZ and fix it to 25 mL with 40 mmol/L hydrochloric acid. FRAP3: Fix 270.3 mg of 6H2O·FeCl3 in water to 50 mL volumetric flask. FRAP1: FRAP2: FRAP3 = 10:1:1, which were mixed to obtain the FRAP working solution. Pipette 10 μL of extracts solution and 200 μL of FRAP working solution into a 96-well plate, shake well, and keep away from light for 30 min. Absorbance values were measured at 593 nm. FeSO4 was used as a standard substance and its antioxidant capacity was expressed in terms of FeSO4 concentration (μmol/L).
OH radical scavenging experiment was based on the literature with some modifications (Li et al. 2022a). The extracts solutions (70 μL), 6 mmol/L FeSO4 solution (60 μL), and 6 mmol/L salicylic acid-ethanol solution (60 μL) were added in a 96-well plate sequentially, mixed, and placed for 10 min, and 60 μL of 6 mmol/L H2O2 solution was added, mixed, and kept out light for 30 min. Absorbance values were measured at 510 nm. The scavenging rate was calculated in the same way as Eq. (1).
Statistical Analysis
Each group of experiment was repeated three times, and the values are expressed as mean ± standard deviation. The OriginPro version 8.5.1 was used for image reporting, and the SPSS version 21 was used for statistical data analysis. The one-way analysis of variance was used to analyze differences between samples. For the multiple comparison analysis, the Tukey test for significant differences was used. Pearson correlation analysis was also used. p < 0.05 indicated a significant difference.
