Chemicals and reagents
Zingiberis Rhizoma, Angelicae Sinensis Radix, Coptidis Rhizoma, Phellodendri Chinensis Cortex, Sanguisorbae Radix, and Granati Pericarpium were identified by Dr. Peng Guangtian of Guangzhou University of Chinese Medicine (Fig. 1). Zingiberis Rhizoma, Angelicae Sinensis Radix, and Granati Pericarpium were purchased from Anhui Jishun traditional Chinese Medicine Co., Ltd. (Bozhou, China); Coptidis Rhizoma and Sanguisorbae Radix were purchased from Anhui Jucao Traditional Chinese Medicine Co., Ltd. (Bozhou, China); Phellodendri Chinensis Cortex was purchased from Shanxi HongSen Traditional Chinese Medicine Co., Ltd. (Xian, China); Asini Corii Colla was purchased from Shandong Jishui Ejiao Co., Ltd. (Heze, China). Zingiberone, ferulic acid, palmatine, jatrorrhizine, berberine hydrochloride, gallic acid, ellagic acid, zingerone and ligustilide had a purity of 98%, as determined by HPLC or GC, and were provided by Chengdu Weikeqi Biotechnology Co. Ltd. (Chengdu, China). Methanol and acetonitrile were purchased from Merck & Co. Inc. (Darmstadt, Germany). Phosphoric acid was purchased from Aladdin Reagent Co., Ltd. (Shanghai, China). Dextran sulphate sodium salt (DSS) was provided by Yisheng Biotechnology Co., Ltd. (Shanghai, China). Salicylazosulfapyridine (SASP) enteric-coated tablets were provided by Fuda Pharmaceutical Co., Ltd. (Shanghai, China). All other chemicals were of reagent grade.
Five preparation methods of ganjiang decoction
GD can be divided into four combinations according to the property, flavor, and pharmacologic action of traditional Chinese medicine. Zingiberis Rhizoma and Angelicae Sinensis Radix are the combination 1, for the warm medicine group. Coptidis Rhizoma and Phellodendri Chinensis Cortex are combination 2, which is the cold medicine group. Combination 3 consist of Sanguisorbae Radix and Granati Pericarpium, which is the astringent group. Combination 4, there is Asini Corii Colla, which is the hemostatic group.
According to the property and flavor of these traditional Chinese medicine and the character of the ingredients contained, GD was prepared by five methods. Method 1 was to extract the whole compound prescription with distilled water. Coptidis Rhizoma and Phellodendri Chinensis Cortex, Sanguisorbae Radix and Granati Pericarpium were extracted respectively in methods 2-5 to prevent the reaction between components. Methods 2-5 were also investigated the effects of different solvents on the extraction efficiency of alkaloids. Some fine powder of Zingiberis Rhizoma and Angelicae Sinensis Radix was added in method 4 and method 5 to increase the volatile oil. The specific preparation method of GD was shown in Table 1.
Table 1 Different preparation method of ganjiang decoction.
Method
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Extraction
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Reflux extracted with distilled water for 30 min twice, decompressed subsequently, concentrated to a thickening paste, dried at 60℃.
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Reflux extracted with 50% alcohol for 60 min, decompressed subsequently, concentrated to a thickening paste, dried at 60℃.
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Fine powder
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1
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Extract Combination ①②③ together
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Combination ④
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2
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Extract Combination ①③ together,
extract Combination ② separately
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Combination ④
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3
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Extract Combination ①③ together
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Combination ②
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Combination ④
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4
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Extract Combination ①③ together,
extract Combination ② separately
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a little Combination ①, Combination ④
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5
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Extract Combination ①③ together
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Combination ②
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a little Combination ①, Combination ④
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Notes: Combination ①: Zingiberis Rhizoma and Angelicae Sinensis Radix; Combination ②: Coptidis Rhizoma and Phellodendri Chinensis Cortex; Combination ③: Sanguisorbae Radix and Granati Pericarpium. Combination ④: Asini Colla Corii. The total quantity of Combination ① in method 4 and method 5 was the same as that in other methods. The amount of fine powder added was one sixth of the amount extracted by solvent.
Fingerprint analysis of ganjiang decoction prepared by the five methods
The final sample obtained by the five extraction methods was the mixture of dried extract and dried medicinal powder. Methanol was added to the samples prepared by the five methods and ultrasonic for 30min. The supernatant solution was obtained by a 0.22 μm microporous filtration membrane.
The fingerprint and quantitative analyses of GD by HPLC were performed by an LC-20AT HPLC System and LabSolutions chromatographic work station (SHIMADZU Cooperation, Japan); Waters-C18 column (250 mm × 4.6 mm × 5 μm) (Waters Cooperation, US) was used. The mobile phases of the fingerprint analysis were composed of methanol (A)–0.1% phosphoric acid water (B), with gradient elution (0–5 min, A 10-15%; 5–25 min, A 15–39%; 25–38 min, A 39–45%; 38–40 min, A 45–48%; 40–50 min, A 48–48%; 50–55 min, A 48–60%) at a flow rate of 0.8 mL/min. The column temperature was 30°C. The detection UV wavelength was 254 nm, and the injection volume was 10 μL.
The data of 10 batches of samples for five extraction method were imported into the similarity evaluation system software of traditional Chinese medicine chromatographic fingerprint (Version 2012.130723). S1 was set as the reference map, the whole spectrum peak was automatically matched, the control map was generated, and the similarity was calculated.
The peak area of each component was standardized, and hierarchical clustering analysis was performed on the standardized data. In the hierarchical cluster analysis, Euclidean method was adopted. Distance was measured for similarity, and the clustering method was complete linkage. The clustering results were shown in heat map and dendrogram; the heat map represented the standardization coefficient of each component in each sample and the tree graph represented the clustering results.
Quantitative analysis of ganjiang decoction prepared by the five methods
For the quantitative analysis of ferulic acid, gallic acid, and ellagic acid, the chromatographic conditions were the same as those of the HPLC fingerprint analysis.
For the quantitative analysis of palmatine and berberine hydrochloride, the mobile phases were composed of acetonitrile (A)–0.1% phosphoric acid–0.2% triethylamine–water (B), with gradient elution (0–15 min, A 20–30%; 15–35 min, A 30–40%; 35–50 min, A 40–50%) at a flow rate of 0.5 mL/min. The column temperature was 25°C. The detection UV wavelength was 254 nm, and the injection volume was 10 μL. Sample preparation was the same as that of fingerprint analysis.
Quantitative analysis of zingerone and ligustilide was performed by GC-2010 plus GC System (SHIMADZU Cooperation, Japan) and a Hp-5 column (30 m × 0.25 mm × 0.25 μm). Gas column flow rate was 1.0 mL/min. For the column temperature, the initial temperature was 120°C, which was maintained for 2 min; subsequently, the temperature was increased to 220 °C at a rate of 12°C/min, which was maintained for 3 min. Injection port temperature was 250°C. Detector temperature was 260°C. Split-flow injection was employed, with a ratio of 20:1 and the injection volume of 1 μL.
Sample preparation of GC: 1.1g of five samples were taken in 11 mL petroleum ether. Ultrasonic extracted for 30min. After filtration, petroleum ether was added for extraction again. Two filtrates were combined, concentrated and fixed to 10ml with petroleum ether. Take the supernatant and pass the 0.22 μm microporous membrane to obtain the sample solution.
Animals
Male C57BL/6 mice (18–22 g) were provided by the Experimental Animals Center of Guangzhou University of Chinese Medicine. The animals were in specific pathogen-free laboratory (temperature 25°C, relative humidity 60%) with alternating light and dark for 12 h and had free access to tap water and food. This study was approved by the Animal Ethics Committee of Guangzhou University of Chinese Medicine (Approved No. S2018021).
Effect of the different preparation methods of ganjiang decoction on DSS-induced UC in mice
The mice entered the acute induction period after 7 days of adaptive feeding. Before the experiment, the mice fasted, but could not control the water for 12 h. The mice were provided 3% DSS solution to drink freely for 7 days to establish the UC model, except those in the control group. After 7 days, the UC mice were divided into the model group (i.g distilled water), SASP group (i.g SASP 0.0038 g/10 g), and another five groups for five extracts of the different methods of GD (i.g 0.1001 g/10 g) (group 1 (G1), group 2 (G2), group 3 (G3), group 4 (G4), and group 5 (G5)). Control group was given the same amount of distilled water. Each group was given gastric gavage once a day for 7 consecutive days.
The gavage drug was prepared according to the " Five preparation methods of ganjiang decoction". Extracts of methods 1-5 was concentrated, the corresponding fine powder was added, mixed into suspension and then gavage. During the modeling and administration period, daily dietary water intake, body weight, and disease active index (DAI) scores (fecal: 0 marks for normal, 1 marks for wet sticky, 2 marks for visible perianal fecal, 3 marks for runny bowel) were recorded. Animals were anesthetized with pentobarbital sodium 24 h after the last intragastric administration of GD. Blood samples were obtained from the aorta abdominalis and centrifuged at 3000 rpm for 10 min. Subsequently, the serum was aspirated and stored at −80°C. The spleen was quickly removed and weighed, and the entire colon was dissociated. The length of the colon was measured, 1 cm of the diseased colon tissue was cut, and the feces were rinsed; thereafter, the cut tissue was fixed in 4% paraformaldehyde and stored at 4°C. The rest of the colon tissue was frozen in liquid nitrogen and stored at −80°C for biochemical analysis.
Histopathological examination and evaluation of colons
The tissues were fixed in 4% paraformaldehyde and dehydrated successively by a series of ethanol solutions. The specimens were permeated and paraffin-embedded; thereafter, the specimens were cut (4 μm) and stained with hematoxylin–eosin. The stained slices were examined by a pathologist using an Olympus CX31 optical microscope (Olympus Corporation, Tokyo, Japan).
Stained tissues were scored according to the histopathological criteria referenced from Binabaj’s study [17]. Histopathological scores were obtained from the sum of the four scores of inflammatory infiltration, crypt loss, mucosal damage and the range of pathological change. The inflammation score was as follows: 0, no inflammatory infiltration; 1, mild inflammatory infiltration; 2, moderate inflammatory infiltration; 3, severe inflammatory infiltration. The crypt loss score was as follows: 0, normal crypt; 1, one-third loss; 2, two-thirds loss; 3, total crypt loss and intact endothelium; 4, total crypt loss and epithelial loss. Mucosal damage score was as follows: 0, intact mucosal; 1, only mucous layer damage; 2, submucosal injury; 3, muscle and serous membrane damage. The range score of pathological changes was as follows: 0, no pathological changed; 1, 25-50% pathological changed; 3, 51-75% pathological changed; 4, 76-100% pathological changed.
Biochemical analysis
Levels of TNF-a, IL-1β, IL-6, IL-10, and IL-1α in the colon tissue were determined by enzyme-linked immunosorbent assay (ELISA). The Detecting steps assessed using commercially available kits and quantified according to the manufacturers’ guidelines (R&D Systems, LXSAMSM-06).
Statistical analysis
Statistical analyses were conducted using SPSS 22.0 Statistical Software, and results were treated with GraphPad Prism 5.0. All data were presented as mean ± standard deviation. Comparison between two groups was examined using the least significant difference (LSD) t-test. One-way analysis of variance (ANOVA) followed by the LSD test was used to compare multiple groups. P < 0.05 was considered statistically significant.