Evalution of anti-inflammatory and bronchodilator effects of Dinh Suyen P/H extract on experimental animals

JOURNAL OF MEDICAL RESEARCH<br /> <br /> EVALUTION OF ANTI-INFLAMMATORY AND<br /> BRONCHODILATOR EFFECTS OF DINH SUYEN P/H<br /> EXTRACT ON EXPERIMENTAL ANIMALS<br /> Nguyen Thi Chau Loan1, Pham Thi Van Anh2, Pham Vu Khanh3,<br /> Nguyen Thi Thanh Loan2<br /> 1<br /> Thai Binh University of Medicine and Pharmacy,<br /> 2<br /> Department of Pharmacoloy, Hanoi Medical University,<br /> 3<br /> Ministry of Health of Vietnam<br /> The purpose of this study was to evaluate the anti-inflammatory and the bronchodilator effects of<br /> Dinh Suyen P/H extract in experimental animals. The anti-inflammatory activity of Dinh Suyen P/H<br /> extract was evaluated in an induced peritoneal inflammation model in rats and amiant-induced<br /> granuloma model in mice. To evaluate the bronchodilator effect, the bronchospasmolytic activity<br /> in anesthetized guinea pig model was used. The present study demonstrated that Dinh Suyen<br /> P/H extract at dose 6.22 g/kg per day and 18.66 g/kg per day inhibited acute inflammatory activity<br /> in rats. The results obtained in the amiant-induced granuloma in mice did not show evidence of<br /> chronic anti-infammatory effect of Dinh Suyen P/H extract at dose 12.44 g/kg per day or 37.32 g/<br /> kg per day. In addition, the findings of this study indicated that Dinh Suyen P/H extract at dose 8.29<br /> g/kg per day inhibited the bronchocontriction in bronchospasmolytic activity in a guinea pig model.<br /> <br /> Keywords: Dinh Suyen P/H extract, anti-inflammatory, bronchodilator<br /> <br /> I. INTRODUCTION<br /> Asthma affects an estimated 300 million<br /> individuals worldwide. It is a serious global<br /> health problem affecting all age groups, with<br /> increasing prevalence in many developing<br /> countries, rising treatment costs and a rising<br /> burden for patients and the community<br /> [1; 2]. Inhaled corticosteroid is the most<br /> effective anti-inflammatory medications for<br /> persistent asthma. When a medium dose of<br /> inhaled corticosteroid alone fails to achieve<br /> good symptom control, the addition of a<br /> Corresponding author: Nguyen Thi Thanh Loan, Department of Pharmacoloy, Hanoi Medical University<br /> Email: nguyenthanhloan@hmu.edu.vn<br /> Received: 27 June 2017<br /> Accepted: 16 November 2017<br /> <br /> 44<br /> <br /> long-acting beta 2 agonist bronchodilator<br /> improves<br /> symptoms,<br /> lung<br /> function<br /> and reduces asthma exacerbations. In<br /> addition, inhaled short-acting beta 2<br /> agonist bronchodilators and short-acting<br /> anticholinergics are medications of choice<br /> for acute relief of asthma symptoms and<br /> bronchoconstriction [1; 3; 4].<br /> Currently, phytotherapy is gaining<br /> popularity. The use of complementary<br /> traditional herbal medicines in the treatment<br /> of various diseases has expanded rapidly in<br /> both developed and developing countries.<br /> This rapid expansions is attributable to<br /> affordability, accessibility and efficacy [5; 6].<br /> The traditional remedies, Hebra Ephedrae,<br /> JMR 111 E2 (2) - 2018<br /> <br /> JOURNAL OF MEDICAL RESEARCH<br /> Rhizoma Pinelliae, Fructus Schisandrae,<br /> Folium Eriobotryae, Radix Glycyrrhizae,<br /> and Rhizoma Zingiberis can help relieve<br /> asthma [7 - 9].<br /> Dinh Suyen P/H extract contains<br /> the abore cemedies, and is a traditional<br /> medicinal formula used in the treatment<br /> of asthma. The present study hoped<br /> to evaluate the anti-inflammatory and<br /> bronchodilator effects of Dinh Suyen P/H<br /> extract in experimental animals.<br /> <br /> II. MATERIALS AND METHODS<br /> 1. Materials<br /> Plant materials<br /> A bottle of 250mL Dinh Suyen<br /> P/H contains 20g Hebra Ephedrae,<br /> 30g Rhizoma Pinelliae, 20g Fructus<br /> Schisandrae, 20g Folium Eriobotryae,<br /> 20g Radix Glycyrrhizae, 6g Herba Asari<br /> sieboldi, 20g Rhizoma Zingiberis, 20g<br /> Semen Armeniacae amarum, 20g Bulbus<br /> Fritillariae, 20g Pericarpium Citri reticulatae<br /> perenne, and 20g Ziziphus mauritiana. All<br /> herbal materials were decocted in water to<br /> obtain 40g Dinh Suyen P/H extract.<br /> Dinh Suyen P/H was provided by<br /> Phuc Hung Company Limited, and meets<br /> the pharmaceutical quality standards of<br /> manufacturers.<br /> Experimental animals<br /> Wistar albino rats weighing between<br /> 180 gram – 220 gram and Guinea pig<br /> weighing between 350 gram – 400 gram<br /> were obtained from animal center of Đan<br /> Phuong, Ha Noi, Viet Nam. Swiss albino<br /> mice weighting between 18 gram – 22 gram<br /> were obtained from the National Institute<br /> <br /> JMR 111 E2 (2) - 2018<br /> <br /> of Hygiene and Epidemiology, Hanoi,<br /> Viet Nam. The animals were allowed an<br /> acclimatization period of 7 days to laboratory<br /> conditions prior to the initiation of the study.<br /> They were maintained for 12 hours in light<br /> and dark cycle in a well ventilated house,<br /> with free access to food and water ad<br /> libitum.<br /> 2. Methods<br /> Induction of Peritoneal Inflammation<br /> (Peritonitis) in Rats:<br /> A total of thirty Wistar albino rats were<br /> divided into four groups of ten animals each<br /> [10]. The animals were orally administered<br /> one of the following solutions:<br /> - Group 1 (control group) served as the<br /> vehicle control and received 1 ml/100g per<br /> day sterile distilled water;<br /> - Group 2 (positive control group) were<br /> orally administered methylprednisolone at<br /> dose 10 mg/kg;<br /> - Group 3 (treated group) were orally<br /> administered Dinh Suyen P/H extract at<br /> dose 6.22 g/kg per day;<br /> - Group 4 (treated group) were orally<br /> administered Dinh Suyen P/H extract at<br /> dose 18.66 g/kg per day.<br /> Carrier solution, methylprednisolone and<br /> Dinh Suyen P/H extract were administered<br /> daily for a period of 5 days to all animals.<br /> One hour after treatment on the 5th<br /> day, rats were injected intraperitoneally<br /> into peritoneum with 1ml/100g of 0.05%<br /> carrageenan and 1.4% formaldehyde<br /> suspension in 9‰ sodium chloride. After<br /> 24 hours, the animals were killed and<br /> the peritoneal cavity exposed. Peritoneal<br /> exudates were collected by aspiration<br /> 45<br /> <br /> JOURNAL OF MEDICAL RESEARCH<br /> and the volumes recorded; the amount of<br /> leukocytes per 1 ml exudation were counted<br /> and protein in the exudation quantified. The<br /> difference of the average values of these<br /> parameters was calculated and compared<br /> to the control group.<br /> Amount of leukocytes were quantified<br /> using automatic hematological analyzer<br /> Exigo - Boule Medical AB. Total protein<br /> content was quantified using biochemical<br /> analyzer Erba Chem.<br /> Amiant-induced granuloma in mice<br /> Mice were divided into four groups of<br /> ten animals [12]. The animals were orally<br /> administered one of the following solutions:<br /> - Group 1 (control group) served as the<br /> vehicle control and received 0.2 ml/10g per<br /> day sterile distilled water;<br /> - Group 2 (positive control group) were<br /> orally administered methylprednisolone at<br /> dose 20 mg/kg per day;<br /> - Group 3 (treated group) were orally<br /> administered Dinh Suyen P/H extract at<br /> dose 12.44 g/kg per day;<br /> - Group 4 (treated group) were orally<br /> administered Dinh Suyen P/H extract at<br /> dose 37.32 g/kg per day.<br /> A sterilized amiant fiber weight 6 mg<br /> (soaked in 1% carrageenan) was implanted<br /> in the back of the neck of mice in different<br /> groups. They were treated with Dinh suyen<br /> P/H extract daily for consecutive 10 days.<br /> One hour after treatment on the 10th day<br /> after implantation, all mice were killed.<br /> Then, the granulomas were removed.<br /> After removing extraneous tissues, they<br /> were dried overnight at 56°C and weighed.<br /> The mean net dry weight of the pellets of<br /> the groups, which was after subtracting<br /> 46<br /> <br /> the initial weight of the pellet (6mg) was<br /> calculated and compared to the control<br /> group.<br /> Bronchospasmolytic<br /> activity<br /> in<br /> anesthetized guinea pigs model<br /> Guinea-pigs were divided into three<br /> groups of six animals. They were orally<br /> administered one of the following solutions<br /> before receiving histamine for 30 minutes<br /> [12].<br /> - Group 1 (control group) served as the<br /> vehicle control and received 1ml/100 g per<br /> day sterile distilled water;<br /> - Group 2 (positive control group) were<br /> orally administered salbutamol at dose 6<br /> mg/kg per day;<br /> - Group 3 (treated group) were orally<br /> administered Dinh Suyen P/H extract at<br /> dose 8.29 g/kg per day;<br /> Guinea-pigs were anaesthetized with<br /> pentobarbital and xylazin. The trachea<br /> was cannulated by means of a two way<br /> cannula, one arm of which was connected<br /> to the respiratory pump and the other to<br /> the transducer. Excess air, not taken up<br /> by the lungs due to bronchospasm, was<br /> measured and recorded on a polygraph.<br /> The internal jugular vein was cannulated for<br /> the administration of spasmogens and test<br /> compounds.<br /> The tracheal cannula was clamped<br /> (equivalent to clamping the trachea) ̣to record<br /> the maximum bronchospasm in millmeters<br /> (Amax). Then after giving histamine 20µg/<br /> kg<br /> intravenously, the excess air was<br /> recorded immediately to determine the<br /> bronchial response to histamine (0’) and at<br /> 5’, 10’, ..., 60’ after histamine administration<br /> (At). The percentage of bronchospasm was<br /> JMR 111 E2 (2) - 2018<br /> <br /> JOURNAL OF MEDICAL RESEARCH<br /> calculated for each animal at each time<br /> point where: Pt = (At/Amax)*100<br /> Pt (%): the percentage of bronchospasm<br /> at minute t<br /> At (mm): the average respiratory<br /> amplitude at minute t.<br /> Amax(mm): the maximum respiratory<br /> amplitide<br /> Statistical analysis<br /> Data was analysed using Microsoft<br /> Excel software version 2007. The levels of<br /> significance between the experimental group<br /> and the control were made using student's<br /> <br /> t-test. Image J, a public domain Java<br /> image processing program, was analyzed<br /> to measure the width of the respiratory<br /> amplitude in bronchospasmolytic activity in<br /> the anesthetized guinea pigs model. Data<br /> are shown as mean ±  standard deviation.<br /> All data were considered significant at p <<br /> 0.05.<br /> * p < 0.05; ** p < 0.01; *** p < 0.001;<br /> compared with control group<br /> Δ p < 0.05; ΔΔ p < 0.01; ΔΔΔ p < 0.001;<br /> compared with positive control group<br /> <br /> III. RESULTS<br /> 3.1. Anti-inflammatory effect<br /> 3.1.1. Acute anti-inflammatory effect<br /> Table 1. The effect of Dinh Suyen P/H extract on volume, protein content,<br /> and amount of leucocytes in inflammatory exudation<br /> Group<br /> Group 1<br /> (Control group)<br /> <br /> Exudate volume<br /> (ml/100g)<br /> <br /> Total amount of<br /> leukocytes (G)<br /> <br /> Total protein<br /> content (mg/dl)<br /> <br /> ( X ± SD)<br /> <br /> ( X ± SD)<br /> <br /> ( X ± SD)<br /> <br /> 2.03 ± 0.50<br /> <br /> 35.52 ± 24.65<br /> <br /> 10.37 ± 3.82<br /> <br /> Group 2<br /> (Positive control group)<br /> <br /> 1.43 ± 0.71*<br /> <br /> 6.83 ± 5.28**<br /> <br /> 6.71 ± 3.75*<br /> <br /> Group 3<br /> (Dinh Suyen P/H extract<br /> at dose 6.22 g/kg/day)<br /> <br /> 1.26 ± 0.62**<br /> <br /> 16.79 ± 12.66*Δ<br /> <br /> 7.14 ± 3.71<br /> <br /> Group 4<br /> (Dinh Suyen P/H extract<br /> at dose 18.66 g/kg/day)<br /> <br /> 1.08 ± 0.94*<br /> <br /> 12.40 ± 12.97*<br /> <br /> 5.30 ± 4.79*<br /> <br /> As shown in tabe 1, methylprednisolone at dose 10 mg/kg decreased significantly the<br /> exudate volume, amount of leukocytes and protein level in inflammatory exudate when<br /> compared to the control. Dinh Suyen P/H extract at dose 6.22 g/kg per day decreased<br /> significantly the exudate volume and amount of leukocytes in inflammatory exudate compared<br /> to the control group; but had a non-significant veffect on protein levels in inflammatory<br /> exudation compared to the control group. Dinh Suyen P/H extract at dose 18.66 g/kg per<br /> JMR 111 E2 (2) - 2018<br /> <br /> 47<br /> <br /> JOURNAL OF MEDICAL RESEARCH<br /> day also decreased significantly the exudate volume, amount of leukocytes and protein level<br /> in inflammatory exudate compared to control group. No significant difference was observed<br /> between groups treated Dinh Suyen P/H extract at dose 18.66 g/kg per day and the positive<br /> control group treated with methyl prednisolone.<br /> 3.1.2. Chronic anti-inflammatory effect<br /> Table 2. The effects of Dinh Suyen extract on the weight of granuloma<br /> Group<br /> <br /> n<br /> <br /> Net dry weight of granuloma (mg)<br /> ( X ± SD)<br /> <br /> Group 1<br /> (Control group)<br /> <br /> 10<br /> <br /> 17.18 ± 9.93<br /> <br /> Group 2<br /> (Positive control group)<br /> <br /> 10<br /> <br /> 6.79 ± 3.74**<br /> <br /> Group 3<br /> (Dinh Suyen P/H extract at dose<br /> 12.44 g/kg/day)<br /> <br /> 10<br /> <br /> 14.49 ± 5.51 ΔΔΔ<br /> <br /> Group 4<br /> (Dinh Suyen P/H extract at dose<br /> 37.32 g/kg/day)<br /> <br /> 10<br /> <br /> 23.44 ± 8.51ΔΔΔ<br /> <br /> Observing in table 2, methylprednisolone at dose 20 mg/kg per day caused a significant<br /> reduction in the dry weight of granuloma when compared to the control group (p < 0.05). Dinh<br /> Suyen P/H extract at at both doses did not reduce the dry weight of granuloma compared to<br /> the control group (p > 0.05).<br /> 3.2. The bronchodilator effect<br /> Table 3. Effect of Dinh Suyen P/H extract on bronchospasmolity activity<br /> in a guinea pig model<br /> Group 1<br /> Control group (%)<br /> <br /> Group 2<br /> Positive control group (%)<br /> <br /> 98.15 ± 7.11<br /> 92.31 ± 6.39<br /> 80.9 ± 12.37<br /> 80.39 ± 11.68<br /> 77.26 ± 12.54<br /> <br /> 85.19 ± 12.08*<br /> 60.09 ± 12.5***<br /> 54.72 ± 14.85**<br /> 57.06 ± 12.24**<br /> 51.1 ± 14.03**<br /> <br /> Group 3<br /> Dinh Suyen P/H<br /> extract (%)<br /> 97.51 ± 3.31Δ<br /> 74.47 ± 11.09**<br /> 66.34 ± 9.55*<br /> 73.17 ± 8.68Δ<br /> 69.89 ± 10.97Δ<br /> <br /> 25<br /> 30<br /> 35<br /> 40<br /> <br /> 75.3 ± 13.73<br /> 73.32 ± 14.23<br /> 62.81 ± 15.73<br /> 64.44 ± 17.76<br /> <br /> 51.75 ± 15,16*<br /> 52.02 ± 14,96*<br /> 53.86 ± 14.79<br /> 53.58 ± 14.47<br /> <br /> 62.76 ± 14.15<br /> 62.47 ± 10.83<br /> 59.61 ± 9.74<br /> 57.4 ± 9.85<br /> <br /> 45<br /> <br /> 61.05 ± 15.46<br /> <br /> 50.85 ± 13.8<br /> <br /> 57.36 ± 8.82<br /> <br /> Group<br /> Minutes<br /> 0<br /> 5<br /> 10<br /> 15<br /> 20<br /> <br /> 48<br /> <br /> JMR 111 E2 (2) - 2018<br /> <br />