The purpose of this study was to evaluate the anti-inflammatory and the bronchodilator effects of Dinh Suyen P/H extract in experimental animals. The anti-inflammatory activity of Dinh Suyen P/H extract was evaluated in an induced peritoneal inflammation model in rats and amiant-induced granuloma model in mice.
JOURNAL OF MEDICAL RESEARCH EVALUTION OF ANTI-INFLAMMATORY AND BRONCHODILATOR EFFECTS OF DINH SUYEN P/H EXTRACT ON EXPERIMENTAL ANIMALS Nguyen Thi Chau Loan1, Pham Thi Van Anh2, Pham Vu Khanh3, Nguyen Thi Thanh Loan2 Thai Binh University of Medicine and Pharmacy, Department of Pharmacoloy, Hanoi Medical University, Ministry of Health of Vietnam The purpose of this study was to evaluate the anti-inflammatory and the bronchodilator effects of Dinh Suyen P/H extract in experimental animals The anti-inflammatory activity of Dinh Suyen P/H extract was evaluated in an induced peritoneal inflammation model in rats and amiant-induced granuloma model in mice To evaluate the bronchodilator effect, the bronchospasmolytic activity in anesthetized guinea pig model was used The present study demonstrated that Dinh Suyen P/H extract at dose 6.22 g/kg per day and 18.66 g/kg per day inhibited acute inflammatory activity in rats The results obtained in the amiant-induced granuloma in mice did not show evidence of chronic anti-infammatory effect of Dinh Suyen P/H extract at dose 12.44 g/kg per day or 37.32 g/ kg per day In addition, the findings of this study indicated that Dinh Suyen P/H extract at dose 8.29 g/kg per day inhibited the bronchocontriction in bronchospasmolytic activity in a guinea pig model Keywords: Dinh Suyen P/H extract, anti-inflammatory, bronchodilator I INTRODUCTION Asthma affects an estimated 300 million individuals worldwide It is a serious global health problem affecting all age groups, with increasing prevalence in many developing countries, rising treatment costs and a rising burden for patients and the community [1; 2] Inhaled corticosteroid is the most effective anti-inflammatory medications for persistent asthma When a medium dose of inhaled corticosteroid alone fails to achieve good symptom control, the addition of a Corresponding author: Nguyen Thi Thanh Loan, Department of Pharmacoloy, Hanoi Medical University Email: nguyenthanhloan@hmu.edu.vn Received: 27 June 2017 Accepted: 16 November 2017 44 long-acting beta agonist bronchodilator improves symptoms, lung function and reduces asthma exacerbations In addition, inhaled short-acting beta agonist bronchodilators and short-acting anticholinergics are medications of choice for acute relief of asthma symptoms and bronchoconstriction [1; 3; 4] Currently, phytotherapy is gaining popularity The use of complementary traditional herbal medicines in the treatment of various diseases has expanded rapidly in both developed and developing countries This rapid expansions is attributable to affordability, accessibility and efficacy [5; 6] The traditional remedies, Hebra Ephedrae, JMR 111 E2 (2) - 2018 JOURNAL OF MEDICAL RESEARCH Rhizoma Pinelliae, Fructus Schisandrae, Folium Eriobotryae, Radix Glycyrrhizae, and Rhizoma Zingiberis can help relieve asthma [7 - 9] Dinh Suyen P/H extract contains the abore cemedies, and is a traditional medicinal formula used in the treatment of asthma The present study hoped to evaluate the anti-inflammatory and bronchodilator effects of Dinh Suyen P/H extract in experimental animals II MATERIALS AND METHODS Materials Plant materials A bottle of 250mL Dinh Suyen P/H contains 20g Hebra Ephedrae, 30g Rhizoma Pinelliae, 20g Fructus Schisandrae, 20g Folium Eriobotryae, 20g Radix Glycyrrhizae, 6g Herba Asari sieboldi, 20g Rhizoma Zingiberis, 20g Semen Armeniacae amarum, 20g Bulbus Fritillariae, 20g Pericarpium Citri reticulatae perenne, and 20g Ziziphus mauritiana All herbal materials were decocted in water to obtain 40g Dinh Suyen P/H extract Dinh Suyen P/H was provided by Phuc Hung Company Limited, and meets the pharmaceutical quality standards of manufacturers Experimental animals Wistar albino rats weighing between 180 gram – 220 gram and Guinea pig weighing between 350 gram – 400 gram were obtained from animal center of Đan Phuong, Ha Noi, Viet Nam Swiss albino mice weighting between 18 gram – 22 gram were obtained from the National Institute JMR 111 E2 (2) - 2018 of Hygiene and Epidemiology, Hanoi, Viet Nam The animals were allowed an acclimatization period of days to laboratory conditions prior to the initiation of the study They were maintained for 12 hours in light and dark cycle in a well ventilated house, with free access to food and water ad libitum Methods Induction of Peritoneal Inflammation (Peritonitis) in Rats: A total of thirty Wistar albino rats were divided into four groups of ten animals each [10] The animals were orally administered one of the following solutions: - Group (control group) served as the vehicle control and received ml/100g per day sterile distilled water; - Group (positive control group) were orally administered methylprednisolone at dose 10 mg/kg; - Group (treated group) were orally administered Dinh Suyen P/H extract at dose 6.22 g/kg per day; - Group (treated group) were orally administered Dinh Suyen P/H extract at dose 18.66 g/kg per day Carrier solution, methylprednisolone and Dinh Suyen P/H extract were administered daily for a period of days to all animals One hour after treatment on the 5th day, rats were injected intraperitoneally into peritoneum with 1ml/100g of 0.05% carrageenan and 1.4% formaldehyde suspension in 9‰ sodium chloride After 24 hours, the animals were killed and the peritoneal cavity exposed Peritoneal exudates were collected by aspiration 45 JOURNAL OF MEDICAL RESEARCH and the volumes recorded; the amount of leukocytes per ml exudation were counted and protein in the exudation quantified The difference of the average values of these parameters was calculated and compared to the control group Amount of leukocytes were quantified using automatic hematological analyzer Exigo - Boule Medical AB Total protein content was quantified using biochemical analyzer Erba Chem Amiant-induced granuloma in mice Mice were divided into four groups of ten animals [12] The animals were orally administered one of the following solutions: - Group (control group) served as the vehicle control and received 0.2 ml/10g per day sterile distilled water; - Group (positive control group) were orally administered methylprednisolone at dose 20 mg/kg per day; - Group (treated group) were orally administered Dinh Suyen P/H extract at dose 12.44 g/kg per day; - Group (treated group) were orally administered Dinh Suyen P/H extract at dose 37.32 g/kg per day A sterilized amiant fiber weight mg (soaked in 1% carrageenan) was implanted in the back of the neck of mice in different groups They were treated with Dinh suyen P/H extract daily for consecutive 10 days One hour after treatment on the 10th day after implantation, all mice were killed Then, the granulomas were removed After removing extraneous tissues, they were dried overnight at 56°C and weighed The mean net dry weight of the pellets of the groups, which was after subtracting 46 the initial weight of the pellet (6mg) was calculated and compared to the control group Bronchospasmolytic activity in anesthetized guinea pigs model Guinea-pigs were divided into three groups of six animals They were orally administered one of the following solutions before receiving histamine for 30 minutes [12] - Group (control group) served as the vehicle control and received 1ml/100 g per day sterile distilled water; - Group (positive control group) were orally administered salbutamol at dose mg/kg per day; - Group (treated group) were orally administered Dinh Suyen P/H extract at dose 8.29 g/kg per day; Guinea-pigs were anaesthetized with pentobarbital and xylazin The trachea was cannulated by means of a two way cannula, one arm of which was connected to the respiratory pump and the other to the transducer Excess air, not taken up by the lungs due to bronchospasm, was measured and recorded on a polygraph The internal jugular vein was cannulated for the administration of spasmogens and test compounds The tracheal cannula was clamped (equivalent to clamping the trachea) ̣to record the maximum bronchospasm in millmeters (Amax) Then after giving histamine 20µg/ kg intravenously, the excess air was recorded immediately to determine the bronchial response to histamine (0’) and at 5’, 10’, , 60’ after histamine administration (At) The percentage of bronchospasm was JMR 111 E2 (2) - 2018 JOURNAL OF MEDICAL RESEARCH calculated for each animal at each time point where: Pt = (At/Amax)*100 Pt (%): the percentage of bronchospasm at minute t At (mm): the average respiratory amplitude at minute t Amax(mm): the maximum respiratory amplitide Statistical analysis Data was analysed using Microsoft Excel software version 2007 The levels of significance between the experimental group and the control were made using student's t-test Image J, a public domain Java image processing program, was analyzed to measure the width of the respiratory amplitude in bronchospasmolytic activity in the anesthetized guinea pigs model Data are shown as mean ± standard deviation All data were considered significant at p < 0.05 * p < 0.05; ** p < 0.01; *** p < 0.001; compared with control group Δ p < 0.05; ΔΔ p < 0.01; ΔΔΔ p < 0.001; compared with positive control group III RESULTS 3.1 Anti-inflammatory effect 3.1.1 Acute anti-inflammatory effect Table The effect of Dinh Suyen P/H extract on volume, protein content, and amount of leucocytes in inflammatory exudation Group Group (Control group) Exudate volume (ml/100g) Total amount of leukocytes (G) Total protein content (mg/dl) ( X ± SD) ( X ± SD) ( X ± SD) 2.03 ± 0.50 35.52 ± 24.65 10.37 ± 3.82 Group (Positive control group) 1.43 ± 0.71* 6.83 ± 5.28** 6.71 ± 3.75* Group (Dinh Suyen P/H extract at dose 6.22 g/kg/day) 1.26 ± 0.62** 16.79 ± 12.66*Δ 7.14 ± 3.71 Group (Dinh Suyen P/H extract at dose 18.66 g/kg/day) 1.08 ± 0.94* 12.40 ± 12.97* 5.30 ± 4.79* As shown in tabe 1, methylprednisolone at dose 10 mg/kg decreased significantly the exudate volume, amount of leukocytes and protein level in inflammatory exudate when compared to the control Dinh Suyen P/H extract at dose 6.22 g/kg per day decreased significantly the exudate volume and amount of leukocytes in inflammatory exudate compared to the control group; but had a non-significant veffect on protein levels in inflammatory exudation compared to the control group Dinh Suyen P/H extract at dose 18.66 g/kg per JMR 111 E2 (2) - 2018 47 JOURNAL OF MEDICAL RESEARCH day also decreased significantly the exudate volume, amount of leukocytes and protein level in inflammatory exudate compared to control group No significant difference was observed between groups treated Dinh Suyen P/H extract at dose 18.66 g/kg per day and the positive control group treated with methyl prednisolone 3.1.2 Chronic anti-inflammatory effect Table The effects of Dinh Suyen extract on the weight of granuloma Group n Net dry weight of granuloma (mg) ( X ± SD) Group (Control group) 10 17.18 ± 9.93 Group (Positive control group) 10 6.79 ± 3.74** Group (Dinh Suyen P/H extract at dose 12.44 g/kg/day) 10 14.49 ± 5.51 ΔΔΔ Group (Dinh Suyen P/H extract at dose 37.32 g/kg/day) 10 23.44 ± 8.51ΔΔΔ Observing in table 2, methylprednisolone at dose 20 mg/kg per day caused a significant reduction in the dry weight of granuloma when compared to the control group (p < 0.05) Dinh Suyen P/H extract at at both doses did not reduce the dry weight of granuloma compared to the control group (p > 0.05) 3.2 The bronchodilator effect Table Effect of Dinh Suyen P/H extract on bronchospasmolity activity in a guinea pig model Group Control group (%) Group Positive control group (%) 98.15 ± 7.11 92.31 ± 6.39 80.9 ± 12.37 80.39 ± 11.68 77.26 ± 12.54 85.19 ± 12.08* 60.09 ± 12.5*** 54.72 ± 14.85** 57.06 ± 12.24** 51.1 ± 14.03** Group Dinh Suyen P/H extract (%) 97.51 ± 3.31Δ 74.47 ± 11.09** 66.34 ± 9.55* 73.17 ± 8.68Δ 69.89 ± 10.97Δ 25 30 35 40 75.3 ± 13.73 73.32 ± 14.23 62.81 ± 15.73 64.44 ± 17.76 51.75 ± 15,16* 52.02 ± 14,96* 53.86 ± 14.79 53.58 ± 14.47 62.76 ± 14.15 62.47 ± 10.83 59.61 ± 9.74 57.4 ± 9.85 45 61.05 ± 15.46 50.85 ± 13.8 57.36 ± 8.82 Group Minutes 10 15 20 48 JMR 111 E2 (2) - 2018 JOURNAL OF MEDICAL RESEARCH 50 55 60 62.53 ± 7.42 52.61 ± 11.11 52.07 ± 8.38 46.86 ± 13.15 48.19 ± 12.94 42.84 ± 15.36 58.43 ± 7.43 56.99 ± 3.65 55.63 ± 3.53 Percent of bronchospasm (%) 120 Δ 100 * ** * 80 Δ Δ *** ** 60 ** Dinh Suyen P/H ** * Salbutamol * Control 40 20 his 10 15 20 25 30 35 40 45 50 55 60 Minute Figure Effect of Dinh Suyen P/H extract on bronchospasmolity activity in a guinea pig model The results in table show that: - The control group: Giving histamine at dose 20 µg/kg intravenously induced potent bronchospasm immediately and the maximum bronchospasm of giving histamine was over 98 percent in comparison to clamping the trachea cannula Then the bronchospasm showed a decreasing tendency during 60 minutes of monitoring - The positive control group: The bronchodilator effect of salbutamol at dose 6mg/kg was observed at all time points The bronchospasm significantly decreased at minute , 5, 10, 15, 20, 25 and 30 compared to the control group at same time points - The treated group: At minute and minute 10, Dinh Suyen P/H extract at dose 8.29 g/ kg per day showed a bronchodilator effect by significantly decreasing the bronchospasm compared to the control group The extract preformed similarly to the salbutamol at these times At later time points, the extract did not show a brochodilator effect compared to the control group IV DISCUSSION Inflammation is one of the most important mediating processes in asthma [1] To determine the acute and chronic anti-inflammatory efects of this extract, we used a rat peritonitis model, induced by carrageenan/formaldehyde and an amiantJMR 111 E2 (2) - 2018 induced granuloma model in mice Acute inflammation is characterized by classical symptoms, including heat, redness, swelling and pain Edema (swelling) is therefore a good measure of experimentally-induced acute inflammation 49 JOURNAL OF MEDICAL RESEARCH [12] In experimental animals, peritonitis can be induced by physiological mediators of inflammation (histamine, bradykinin) or nonspecific irritants (carrageenan, formaldehyde) The structure of carageenan is a polysaccharide like a bacterial capsular polysacharide It can induce acute inflammation by triggering the release of inflammatory mediators (histamine, serotonin, bradykinin) These mediators increase vascular permeability and prostaglandin production This results in a gradual increase in fluid leakage and more cells migrating into the animal’s peritoneal cavity, especially neutrophils [13; 14] Adding formaldehyde can increase the volume of exudate Carrageenan/ formaldehyde induced peritonitis is often used in the study of acute inflammatory process in Viet Nam [11] Methylprednisolone is a synthetic glucocorticoid used to reduce inflammation in asthma Multiple mechanisms mediate in the suppression of inflammation by glucocorticoids They inhibit leukocyte infiltration at the site of inflammation, interfere with mediators of inflammatory response, and suppress humoral immune responses The anti-inflammatory actions of glucorticosteroids are thought to involve phospholipase A2 inhibitory proteins, lipocortins, which control the biosynthesis of potent mediators of inflammation such as prostaglandins and leukotriene Methylprednisolone is a powerful antiinflammatory agent [6] Therefore, methylprednisolone can be used as a positive control drug in acute or chronic inflammatory models 50 The present study demonstrated that the Dinh Suyen P/H extract had an acute anti-inflammatory effect at dose 6.22 g/kg per day and 18.66 g/kg per day The extract inhibited inflammation Shown by reducing the exudate volume, amount of leukocytes and/or protein level in inflammatory exudate The amiant-induced granuloma model was used to evaluate chronic antiinflammatory effects of Dinh Suyen P/H extract Thymus dependent antigen elicits antibody production by B lymphocytes with involvement of Th cells Additionally, these antigens can also induce the second immune response (cell mediated immunity) that involves mostly T lymphocytes The foreign body (cotton, amiant), once implanted in the skin of animal produced undifferentiated connective tissue around it The adhesion of macrophages, neutrophils, fibroblasts and collagen leads to granuloma formation This amiant induced granuloma model shows the proliferative phase of the inflammation [13; 14].To test the chronic anti-inflammatory effect, amiant-induced granuloma was used in this study The results of this study showed that the extract at dose 12.44 g/kg per day and 37.32 g/ kg per day did not show anti-inflammatory effect due to non-reduction in the dry weight of granuloma after treatment In this study, the chronic anti-inflammatory effect has not been clearly demonstrated yet In the other studies, some compounds in the extract were found to inhibit inflammation such as pseudoephedrine, ephedroxan (Herba Ephedrae) [8], schizandrin (Fructus Schisandrae) [15], glycyrrhizin (Radix Glycyrrhizae) [17], JMR 111 E2 (2) - 2018 JOURNAL OF MEDICAL RESEARCH gingerols, 6-shogaol (Rhizoma Zingiberis) [18] These compounds were known to effect the activities of cyclooxygenase and/ or lipoxygenase, and PGE2 in inflammation reactions, resulting in decreased production of inflammatory mediators and proinflammatory cytokines such as gingerols, glycyrrhidin, and schizandrin [15 - 17] Histamine is an important mediator of acute allergic and imflammatory reactions It causes bronchoconstriction by activating H1 receptors [4; 12] Bronchospasmolytic activity in anesthetized guinea pigs permits the evaluation of a drug's bronchospasmolytic effect by measuring the volume of air, which is not taken by the lungs during bronchospasm Salbutamol is a highly selective β2-adrenergic receptor agonist that has a bronchodilator effect [4] It is used to as the positive control in this study The results of this study showed that Dinh Suyen P/H extract at dose 8.29 g/kg per day had the bronchodilator effect by inhibiting bronchospam at at minutes and 10 Other studies have found that some herbs in the 11-herb formula can act as bronchodilators, such as Herba Ephedrae (ephedrin, pseudoephedrin), Rhizoma Pinelliae (ephedrin), Fructus Schisandrae (schizandrin, schizandrol), and Radix Glycyrrhizae (isoliquiritingenin) [9; 14; 16; 18] Ephedrine may be the main bronchodilating agent because it is a sympathomimetic agonist at both α and β adrenergic receptors It also displays indirect sympathetic activation, by enhancing the release of norepinephrine from sympathetic neurons [8] JMR 111 E2 (2) - 2018 V CONCLUSION The present study demonstrated that Dinh Suyen P/H extract at dose 6.22 g/kg per day and 18.66 g/kg per day inhibited acute inflammatory activity in rats The results did not show evidence of any chronic anti-infammatory effect of Dinh suyen P/H extract at dose 6.22 g and 18.66 g/kg per day in mice The findings of this study indicate that Dinh Suyen P/H extract at dose 8.29 g/kg per day inhibited the bronchocontriction in bronchospasmolytic activity in guinea pig Acknowledgments The authors would like to thank Phuc Hung Company Limited for financial support REFERENCES Global Initiative for Asthma (2015) A Pocket Guide for Physicians and Nurses Updated 2015 Global Asthma Network (2014) The Global Asthma Report Ministry of Health (2014) Pharmacology, Vietnam Education Publishing House Limited Company, Ha Noi Dao Van Phan (2012) Clinical pharmacology, 469 - 486 World Health Organization (2000) Working group on the safety and efficacy of herbal medicine Report of regional office for the western pacific of the World Health Organization World Health Organization (2007) Guidelines for Assessing Quality of Herbal Medicines With Reference to 51 JOURNAL OF MEDICAL RESEARCH Contaminants and Residues World Health Organization, Geneva Do Tat Loi (2015) Vietnamese medicinal plants and substances Medical Publshing House, Ha Noi Abourashed E.A., El-Alfy A.T., Khan I.A et al (2003) Ephedrae in perspective- a current views Phytotherapy research, 17, 703 - 712 Kim H., Ahn Y.T., Kim Y.S et al (2014) Antiasthmatic effects of schizandrae fructus extract in mice with asthma Pharmacognosy magazine, 10, 80 - 85 10 Bekemeier H., Hirschelmann R (1982) Pharmacology, biochemistry and immunology of the inflammatory reaction, Halle : Martin-Luther-Universität HalleWittenberg 11 Dang Diem Hong, Hoang Minh Hien, Hoang Thi Lan An (2011) Studies on the analgesic and anti-inflammatory activities of Sargassum swartzii (Turner) C Agardh (Phaeophyta) and Ulva reticulata Forsskal (Chlorophyta) in experiment animal models African Journal of Biotechnology, 10(12), 2308 - 2314 12 Gerhard Vogel H (2008) Drug discovery and development: evalution pharmacological assays, Springer, Berlin 13 Ha Noi Medical University (2007) 52 Pathophysiology and immunology Medical Publishing House, Ha Noi 14 Dhalendra G., Satapathy T., Amit Roy A (2013) Aniamal model for inflammation: a review Asian Journal of Pharmaceutical Sciences, (4), 207 - 212 15 Kang Y.S., M.H.H., S.H.H et al (2014) Anti-inflammatory effects of Schisandra chinensis (Turcz.) Baill Fruit Through the Inactivation of Nuclear FactorκB and Mitogen-activated Protein Kinases Signaling Pathways in Lipopolysaccharidestimulated Murine Macrophages Journal of Cancer Prevention, 19 (4), 279 - 287 16 Yang N., Patil S., Zhuge J et al (2013) Glycyrrhiza uralensis flavonoids present in anti-asthma formula, ASHMI™, inhibit memory Th2 responses in vitro and in vivo Phytotherapy Research, 27 (9), 1381 - 1391 17 Dugarani S., Pichika M.R., NadarajahV.D (2010) Comparative antioxidant and anti inflammatory Journal of Ethnopharmacol., 127 (2), 515 - 520 18 Wang H.L., Li Y.X., Niu Y.T (2015) Observing Anti-inflammatory and Anti-nociceptive Activities of Glycyrrhizin Through Regulating COX2 and Proinflammatory Cytokines Expressions in Mice Inflammation, 38 (6), 2269 - 2278 JMR 111 E2 (2) - 2018 ... cemedies, and is a traditional medicinal formula used in the treatment of asthma The present study hoped to evaluate the anti-inflammatory and bronchodilator effects of Dinh Suyen P/H extract in experimental. .. treated Dinh Suyen P/H extract at dose 18.66 g/kg per day and the positive control group treated with methyl prednisolone 3.1.2 Chronic anti-inflammatory effect Table The effects of Dinh Suyen extract. .. effect of Dinh suyen P/H extract at dose 6.22 g and 18.66 g/kg per day in mice The findings of this study indicate that Dinh Suyen P/H extract at dose 8.29 g/kg per day inhibited the bronchocontriction