Antidiarrheal Activity of Ethanol Extract of Ophioglossum Vulgatum In Mice And Spasmolytic Effect on Smooth Muscle Contraction of Isolated Jejunum In Rabbits

Background Methods The chemical constituents of EWOV were analyzed by high performance liquid chromatography (HPLC). In vivo, the antidiarrheal activity of (125, 250 and 500 mg/kg; orally) in castor oil-induced Kun Ming mice was evaluated. In vitro, the effect of EWOV (0.01-10 mg/mL) on the spontaneous contraction of isolated rabbit jejunum smooth muscle was studied. Verapamil positive and possible spasmolytic effect and containing EDTA.

detoxifying toxins, activating blood circulation and dispersing blood stasis, treating hepatitis and pneumonia, etc. [6]. Studies show that the characteristic chemical constituents of OV are avonoids, glycerides and amino acids [5]. Quercetin is a avonoid compound, which widely exists in vegetables, fruits and Chinese herbal medicines. It has been shown that quercetin has many biological activities such as antioxidation, anti-in ammation, antibacterial, and antimutation [7,8,9,10]. In addition, quercetin can inhibit the metastasis of gastric, colon and lung cancer [11,12,13]. It is extremely important that quercetin also has spasmolytic effect on intestinal smooth muscle, which can delay intestinal transport and inhibit intestinal peristalsis [14], it provides clues for us to assess the antidiarrheal and spasmolytic effect of EWOV.

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 identi ed 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%.

Animals
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.

Phytochemical study
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 ltered by a 0.22 µm nylon microporous membrane, and then used for HPLC analysis.

Chromatographic conditions
The reference and sample solutions were analyzed by an Agilent-1220 high performance liquid chromatograph system (Agilent, American). The column was Kromasil 100-5-C 18 (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 ltered by passing through a 0.45 µm lter 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 ow 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 avonoid content
The Total avonoid content of EWOV was determined by a reported method (Shima et al. 2019). Appropriate amount of EWOV in 25 mL brown glass ask volumetric was mixed with 1.0 mL 5% NaNO 2 (w/v), after 6 min interval, 1.0 mL 10% AlCl 3 (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 ask volumetric was mixed with 1.0 mL F-C reagent, after 3 min interval, 1.0 mL 10% Na 2 CO 3 (w/v) was added, ultrapure water for volume xing 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 [15].

Castor oil-induced diarrhea
Reference to the method of Gong et al. (2017) and Guo et al. (2014), this study rst 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 sacri ced by skull impingement. The isolated jejunum was cut into 2-3 cm and was ushed 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% O 2 and 5% CO 2 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-

Effect of EWOV on CaCl 2 -induced cumulative contraction
To determine the effect of EWOV on Ca 2+ in ux, the jejunum smooth muscle was stabilized in Tyrode's solution and was then incubated with Ca 2+ -free and high-K + (60 mM) solution containing EDTA (0.1 mM) for 30 min in order to eliminate Ca 2+ from tissue, followed by a Ca 2+ -free and high-K + (60 mM) for 15 min.

Statistical analysis
Being expressed as mean ± standard error (SEM), all data were analyzed by single-line statistical signi cance variance analysis (ANOVA) followed by the Dunnett's test. SPSS 19.0 system was used for testing. P ≤ 0.05 was considered statistically signi cant.

Chemical content of EWOV
Under the optimal liquid chromatography conditions, the separation between quercetin and other chemicals peaks were excellent. The reference solution and the sample solution chromatography pro les were shown in (Figure. 1). The reference solution ( Figure. 1A) and the sample solution ( Figure. 1B) had corresponding chromatographic peaks at the same retention time. Figure. 1B illustrates the chromatograms obtained from EWOV. The content of quercetin in EWOV was 0.1924%. Meanwhile, the total avonoids content of EWOV was 2.74% (27.4 mg/g), and the total polyphenols content of EWOV was 1.47% (14.70 mg/g).

Acute oral toxicity test
In the LD50 test, after EWOV was given in the increasing does (500, 1000, 2000, 4000, 8000, 16000 mg/kg) of intragastric administration, there were no death or changes in physical behavior during the observation period in the single maximum dose trial. Based on these results, the LD50 value was estimated to be > 16000 mg/kg. Castor oil-induced diarrhea EWOV (125, 250 and 500 mg/kg; orally) exhibited dose-dependent antidiarrheal activity. The negative control group (saline) did not show protection against diarrhea. The onset time of semi-solid feces in negative control group and the positive group were 57.9 ± 3.70 and 80.80 ± 3.77 min, while the EI scores were 13.00 ± 1.05 and 6.5 ± 1.08. The EI scores in test group were 12.79 ± 1.70, 10.10 ± 1.37 and 8.70 ± 1.16, respectively (p > 0.05, p < 0.05, p < 0.01), while the onset time of semi-solid feces were 73.00 ± 3.88, 77.00 ± 3.43, 85.20 ± 3.99 min (p < 0.01, p < 0.01, p < 0.01). The results showed that the EI scores decreased and the onset time of semi-solid feces prolonged in the test group, same to the positive group

Effect of EWOV on CaCl 2 -induced cumulative contractions
Our results indicated that EWOV antagonized the contraction of isolated tissue induced by cumulative concentration of CaCl 2 . EWOV (0.3, 1 mg/mL) could move the cumulative CaCl 2 curve to the right with a decrease in the maximum. Verapamil, the calcium antagonist, also moved the CaCl 2 curve to the right with a decrease in the maximum. Compared with the negative group, EWOV (0.3, 1 mg/mL) and verapamil (0.03, 0.1 µM) reduced the maximum contraction induced by 0.3 mM CaCl 2 to 48.26 ± 2.27%, 69.53 ± 1.93%, 59.06 ± 1.11% and 73.14 ± 1.63% (p < 0.001) respectively ( Figure. 6).

Discussions
As mentioned in the introduction, diarrhea has a high incidence rate and is a worldwide public health problem, we will elaborate further on its classi cation and pathogenesis. The classi cation of diarrhea can be roughly divided into osmotic diarrhea, secretory diarrhea, exhaling diarrhea, absorptive disturbance diarrhea and gastrointestinal motility disorder diarrhea [16]. Diarrhea is an intestinal disease caused by multiple pathogens and factors, and its pathological basis is in ammation and edema of mucosa, hyperactivity of intestinal secretion and motor function, the occurrence of loose stools, frequent stools and other symptoms under the stimulation of infection or non-infection factors of the intestinal tract [17]. In recent years, Chinese herbal medicines have been widely used in the medical system [18]. Compared with chemical substances, Chinese herbal medicines are safer [19]. Chinese herbal medicine also has the advantages of rapid curative effect, less toxic and side effects and low cost in the treatment of diarrhea [20]. Therefore, it is necessary to develop natural Chinese herbal medicines for the treatment of diarrhea. OV is one of the commonly used Chinese medicinal materials in clinics, mainly distributed in the provinces of Yunnan, Sichuan, Hubei, Guizhou and Tibet [21], which has anti-in ammatory, antibacterial, antitumor, and other pharmacological effects [22]. It is widely used in clinical practice and has high medicinal value.
In the acute toxicity experiment, the single dose of EWOV was 16000 mg/kg, and the mice did not cause any death or toxic effects during the observation period. According to Lorke [23], any 5 g/kg non-toxic substance can be considered relatively safe. The above conclusions demonstrate the safety of EWOV and provide necessary theoretical basis for subsequent experiments. Castor oil is from castor seed and is a kind of effective laxative, which can reduce the absorption of small intestine and increase the secretion of it [24]. The active ingredient of castor oil inducing diarrhea is ricinoleic acid, which produces irritating and in ammatory actions on the intestinal mucosa and leads to the release of prostaglandins [25]. In addition, it also can promote the release of nitric oxide (NO) and the activation of adenylate cyclase, reduce the activity of ATPase pump, so as to reduce the absorption of potassium sodium ions, which leading to the accumulation of electrolytes and water, then diarrhea phenomenon appears [3]. Therefore, the castor oil-induced diarrheal model including secretory and motility diarrhea [25]. In the castor oilinduced diarrhea model in mice, EWOV can delay the onset time of semi-solid and reduce the diarrhea index, showing signi cant antidiarrheal effects. Chemical composition studies show that the characteristic chemical composition of EWOV are avonoids and glycoside compounds, particularly, avonoids can prevent diarrhea by reducing intestinal peristalsis and hydro-secretion [26] and inhibiting the release of autacoids and prostaglandins [27]. Based on the experimental results, we can infer that EWOV may inhibit the castor oil-induced diarrhea in mice by inhibiting the release of prostaglandin and reducing gastrointestinal smooth muscle propulsion.
In order to explore the mechanism of EWOV, we extended it to rabbit isolated jejunum smooth muscle experiment. In the experiment of spontaneous contraction of isolated jejunum smooth muscle, both EWOV and quercetin showed concentration-dependent inhibition. Intestinal smooth muscle has low excitability and self-discipline, and its contraction is mainly regulated by intracellular Ca 2+ concentration [28]. Intracellular Ca 2+ is increased mainly through voltage-dependent Ca 2+ channel in ow or release from intracellular storage in the sarcoplasmic reticulum [25]. Therefore, the blocking of Ca 2+ channels or the reduction of Ca 2+ release from intracellular storage will weaken the contraction of intestinal smooth muscle. ACh is one of the most important neurotransmitters in gastrointestinal tract, which can bind to muscarinic M receptor and involve the activation of PLC, leading to the generation of inositol-1, 4, 5trisphosphate (IP3), which induces Ca 2+ release and leads to small intestine contraction [29,30]. The experimental results showed that EWOV could signi cantly relax the contraction of ACh (10 − 5 M) induced jejunum smooth muscle in vitro. Therefore, we believed that EWOV's antispasmodic effect on the jejunum tract might be related to blocking M receptor and reducing Ca 2+ internal owing. High concentrations of K + can horizontally open voltage-dependent L-type calcium ion channels and allow extracellular Ca 2+ to enter the cell, thereby promoting contraction of intestinal smooth muscle [31,32]. Therefore, the existence of calcium antagonists is suggested as the substance that relaxing the contraction induced by high-K + . To verify this hypothesis, as verapamil is an L-type calcium channel blocker [33], we used KCl (60 mM) solution to induce jejunum contraction and used verapamil as the control group. The results showed that EWOV could relax the contraction of jejunum smooth muscle induced by KCl (60 mM) solution, and with the increase of EWOV dose, the concentration-response curves of CaCl 2 moved to the right and down, which was similar to verapamil, suggesting that EWOV inhibited the contraction of jejunum smooth muscle may be related to blocking calcium channel.

Conclusion
The results showed that EWOV had signi cant antidiarrheal effect in castor oil-induced diarrheal mice. In addition, EWOV could relax the spontaneous contraction of jejunum and the contraction induced by ACh