Chemicals and Drugs
Acetylcholine chloride was from Chengdu Huaxia Chemical Testing Co., Ltd. (Chengdu, China). Sodium bicarbonate, magnesium sulphate, potassium chloride, glucose, sodium dihydrogen phosphate, calcium chloride and sodium chloride were produced by Chengdu Chemicals Co., Ltd. (Chengdu, China). Distilled water was used for the preparation of reference solutions, sample solutions, diluents, and physiological salt solutions (Tyrode’s sulutions). Verapamil was from MedChemexpress Co., Ltd. (USA). The castor oil was from Hualong Pharmaceutical Co., Ltd. (Henan, China). The quercetin was from Chengdu Alpha Biotech Co., Ltd. (Chengdu, China). All research-grade chemicals were used in experimental work.
Plant material and preparation of EWOV
The whole plant of OV was collected from Nanchong (Sichuan, China) in the month of June 2017. In the process of collection, attention should be paid to the protection of ecological environment. On the premise of “keeping roots and preserving species”, appropriate amount of plants should be collected for this study. OV was identified by teacher Lan Yang, who was from School of Pharmacy, North Sichuan Medical College and the voucher specimen (CBY-2017-0003) was deposited in the herbarium of the same institution. The whole plant of OV was dried in an electric oven at constant temperature (50 °C) and pulverized into a coarse powder (shredding machine: FW177, Taisite, Tianjin, China). The preparation process of EWOV was as follows: OV powder(100 g) were immersed with 75% ethanol and performed ultrasonic-assist extraction for 3 times (1 h each). All the extracting solution was merged for rotatory evaporation till no ethanol under reduced pressure(50 °C) (Rotary evaporator: RE-52AA, Yarong, Shanghai, China).The extract was further dried thoroughly in a vacuum decompression drying oven (ZK 6050B, Opson, Wuhan, China) and preserved in a vacuum desiccator at 4 °C until further use. Obtained EWOV was dark greenish brown solid and weighed 21.06 g, the percentage yield of OV powder was 21%.
Adult male Kun Ming mice weighing 18–22 g and locally bred rabbits weighing 2.0-2.5 kg (License No. : SYXK (Chuan) -2018-076) were provided by the Animal Laboratory Center of North Sichuan Medical College (Sichuan, China). The 12 h light-dark cycle (temperature 23–26 °C, humidity 70 ± 5%) was maintained for the animals, and the white wood chips were used as bedding while animals were given free access to water, but fasted for 24 h before the experiment.
Ethics and Consent to Participate
The animal research conformed to the requirements of Ethical Review Committee of SLAS (Sichuan Association for Laboratory Animal Sciences) Assessment agencies and followed the requirements of animal welfare and experimental practices.
Preparation of the reference solution and the sample solution
Quercetin was accurately weighed and with 75% ethanol as solvent, to prepare a reference solution with 8.928 µg/mL quercetin. The solution was passed through a 0.22 µm nylon microporous membrane and kept at 4 °C before use.
Appropriate amount of EWOV (0.2127 g/g DW) was accurately weighed and with 75% ethanol as solvent, to prepare a sample solution with 40.05 mg/mL (Crude drug concentration) EWOV, The solution was filtered by a 0.22 µm nylon microporous membrane, and then used for HPLC analysis.
The reference and sample solutions were analyzed by an Agilent-1220 high performance liquid chromatograph system (Agilent, American). The column was Kromasil 100-5-C18 (250 mm × 4.6 mm, 5 µm). 0.2% acetic acid was used as mobile phase A, chromatographic acetonitrile was used as mobile phase B. Then the mobile phase was filtered by passing through a 0.45 µm filter membrane. The column loaded with the reference solution and the sample solution was run with a mobile phase consisting of acetonitrile and 0.2% acetic acid water (27 : 73, pH = 2–3) for the determination of quercetin from EWOV. The detection wavelength was 360 nm, and the flow rate was 1.0 mL/min. The column temperature was 30 °C, and a sample of 10 µL of the solutions was directly injected.
Total flavonoid content
The Total flavonoid content of EWOV was determined by a reported method (Shima et al. 2019). Appropriate amount of EWOV in 25 mL brown glass flask volumetric was mixed with 1.0 mL 5% NaNO2 (w/v), after 6 min interval, 1.0 mL 10% AlCl3 (w/v) was added, then 10 mL 4% NaOH (w/v) solution was added in order. The reaction mixture was allowed to incubate for 15 min at room temperature before the absorbance was detected at 510 nm. Water (2 mL) was used to substitute aluminum chloride for blank. Rutin was used as a standard for the calibration curve. The result was expressed as rutin mg/g rutin equivalents after triplicate analysis.
Total polyphenol content
The content of total polyphenols in EWOV was determined by Folin-Ciocalteu (F-C) colorimetry as Gallic acid was used as the reference material. Appropriate amount of EWOV in 25 mL brown glass flask volumetric was mixed with 1.0 mL F-C reagent, after 3 min interval, 1.0 mL 10% Na2CO3 (w/v) was added, ultrapure water for volume fixing and shake well, reaction for 2 h in 25 °C constant temperature water bath, then the absorbance was measured at the wavelength of 765 nm.
In vivo experiments
Acute oral toxicity test
Mice were kept on fasting for 24 h before the experiment, and were divided into 6 groups with 10 mice in each group. EWOV was diluted with normal saline to different concentrations. According to the dosage of 500, 1000, 2000, 4000, 8000, 16000 mg/kg body weight of EWOV, each mouse in the experimental group was given 0.2 mL EWOV normal saline diluent orally. Any signs of toxicity and death were strictly documented for 14 days after administration. During these days, the mice were free to access water and food. A dose-response curve for the determination LD50 was established. The safety of EWOV was assessed with the single maximum dose .
Castor oil-induced diarrhea
Reference to the method of Gong et al. (2017) and Guo et al. (2014), this study first carried out a preliminary experiment. The mice were screened by giving 0.4 mL castor oil and those presenting with diarrhea were randomly divided into 5 groups with 10 mice in each. Random numbers were generated using the standard = RAND() function in Microsoft Excel. The negative control group was given 0.2 mL normal saline (20 mg/kg). While the positive group was given 0.2 mL verapamil (50 mg/kg) and the test groups were administrated orally with EWOV (125, 250 and 500 mg/ kg) respectively. Each mouse was caged individually and blotting paper placed under it. Castor oil (20 mL/kg) was provided orally, the subsequent onset of castor oil-induced diarrhea was observed after half an hour of treatment. The amount of solid feces, semi-solid feces, liquid feces and the time of initial semi-solid appearance was recorded within 4 h after castor oil. The following formula was used to evaluate the severity of diarrhea. Evacuation Index (EI) = solid feces × 1 + semi-solid feces × 2 + liquid feces × 3 (The distribution was as follows: 1 referred to solid feces, 2 referred to semi-solid feces, and 3 referred to liquid feces)
In vitro experiments
Preparation and calibration of isolated tissues (Rabbit jejunum)
The local bred rabbits were killed after 24 h of fasting then the tissues (jejunum) were isolated. Isolated rabbit jejunum smooth muscle was used to study the spasmolytic action and likely mechanism of action of EWOV. Rabbits were sacrificed by skull impingement. The isolated jejunum was cut into 2–3 cm and was flushed in 4 °C Tyrode’s solution. Then it was mounted vertically in a tissue bath (20 mL) comprising of Tyrode’s solution, which was kept at normal body temperature of 37 ± 0.5 °C with a mixture of 95% O2 and 5% CO2 aerated. After preloading 1 g, the jejunum was balanced to reach a stable level before the active compounds were added. Jejunum activity was measured with a force sensor and recorded by BL-420F Physiological Signal Collection and Handling System.
Effect of EWOV on spontaneous contraction and ACh/KCl-induced contraction of rabbit jejunum
Rabbit isolated jejunum smooth muscle showed spontaneous contraction under the standard experimental settings. After an equilibrium period of jejunum in Tyrode’s solution, cumulative concentrations of EWOV (0.01, 0.03, 0.1, 0.3, 1, 3, 10 mg/mL), quercetin (0.3, 1, 3, 10, 30, 100 µM), verapamil (0.01, 0.03, 0.1, 0.3, 1, 3, 10 µM) were added to determine the effect on the spontaneous contraction.
To investigate the spasmolytic activity and mechanism of EWOV, the isolated jejunum smooth muscle was pretreated with ACh (10− 5 M) and KCl (60 mM). When the ACh/KCl-induced contraction reached a stable level, EWOV (0.01, 0.1, 1, 3, 10 mg/mL) was added in the test group. While the positive group was treated with verapamil (0.003, 0.03, 0.3, 1, 3 µM).
Effect of EWOV on CaCl2-induced cumulative contraction
To determine the effect of EWOV on Ca2+ influx, the jejunum smooth muscle was stabilized in Tyrode’s solution and was then incubated with Ca2+-free and high-K+ (60 mM) solution containing EDTA (0.1 mM) for 30 min in order to eliminate Ca2+ from tissue, followed by a Ca2+-free and high-K+ (60 mM) for 15 min. The samples were then treated in the absence and presence of EWOV (0.3, 1 mg/mL) and verapamil (0.03, 0.1 mM), Ca2+ was added in a cumulative manner (3 ⋅ 10− 5-3 ⋅ 10− 2 M) to obtain the concentration-response curves of CaCl2. The contraction induced by 3 ⋅ 10− 2 M CaCl2 was regarded as 100% in the absence of EWOV and verapamil.
Being expressed as mean ± standard error (SEM), all data were analyzed by single-line statistical significance variance analysis (ANOVA) followed by the Dunnett’s test. SPSS 19.0 system was used for testing. P ≤ 0.05 was considered statistically significant.