Evalution of anti - inflammatory and bronchodilator effects of dinh suyen p / h extract on experimental animals

Nội dung text: Evalution of anti - inflammatory and bronchodilator effects of dinh suyen p / h extract on experimental animals

1. 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 1Thai Binh University of Medicine and Pharmacy, 2Department of Pharmacoloy, Hanoi Medical University, 3Ministry 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 or37.32g/ 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 long-acting beta 2 agonist bronchodilator individuals worldwide. It is a serious global improves symptoms, lung function health problem affecting all age groups, with and reduces asthma exacerbations. In increasing prevalence in many developing addition, inhaled short-acting beta 2 countries, rising treatment costs and a rising agonist bronchodilators and short-acting burden for patients and the community anticholinergics are medications of choice [1; 2]. Inhaled corticosteroid is the most for acute relief of asthma symptoms and effective anti-inflammatory medications for bronchoconstriction [1; 3; 4]. persistent asthma. When a medium dose of Currently, phytotherapy is gaining inhaled corticosteroid alone fails to achieve popularity. The use of complementary good symptom control, the addition of a traditional herbal medicines in the treatment Corresponding author: Nguyen Thi Thanh Loan, De- of various diseases has expanded rapidly in partment of Pharmacoloy, Hanoi Medical University both developed and developing countries. Email: nguyenthanhloan@hmu.edu.vn This rapid expansions is attributable to Received: 27 June 2017 affordability, accessibility and efficacy [5; 6]. Accepted: 16 November 2017 The traditional remedies, Hebra Ephedrae, 44 JMR 111 E2 (2) - 2018
2. JOURNAL OF MEDICAL RESEARCH Rhizoma Pinelliae, Fructus Schisandrae, of Hygiene and Epidemiology, Hanoi, Folium Eriobotryae, Radix Glycyrrhizae, Viet Nam. The animals were allowed an and Rhizoma Zingiberis can help relieve acclimatization period of 7 days to laboratory asthma [7 - 9]. conditions prior to the initiation of the study. Dinh Suyen P/H extract contains They were maintained for 12 hours in light the abore cemedies, and is a traditional and dark cycle in a well ventilated house, medicinal formula used in the treatment with free access to food and water ad of asthma. The present study hoped libitum. to evaluate the anti-inflammatory and 2. Methods bronchodilator effects of Dinh Suyen P/H Induction of Peritoneal Inflammation extract in experimental animals. (Peritonitis) in Rats: II. MATERIALS AND METHODS A total of thirty Wistar albino rats were divided into four groups of ten animals each 1. Materials [10]. The animals were orally administered Plant materials one of the following solutions: A bottle of 250mL Dinh Suyen - Group 1 (control group) served as the P/H contains 20g Hebra Ephedrae, vehicle control and received 1 ml/100g per 30g Rhizoma Pinelliae, 20g Fructus day sterile distilled water; Schisandrae, 20g Folium Eriobotryae, - Group 2 (positive control group) were 20g Radix Glycyrrhizae, 6g Herba Asari orally administered methylprednisolone at sieboldi, 20g Rhizoma Zingiberis, 20g dose 10 mg/kg; Semen Armeniacae amarum, 20g Bulbus - Group 3 (treated group) were orally Fritillariae, 20g Pericarpium Citri reticulatae administered Dinh Suyen P/H extract at perenne, and 20g Ziziphus mauritiana. All dose 6.22 g/kg per day; herbal materials were decocted in water to - Group 4 (treated group) were orally obtain 40g Dinh Suyen P/H extract. administered Dinh Suyen P/H extract at Dinh Suyen P/H was provided by dose 18.66 g/kg per day. Phuc Hung Company Limited, and meets Carrier solution, methylprednisolone and the pharmaceutical quality standards of Dinh Suyen P/H extract were administered manufacturers. daily for a period of 5 days to all animals. Experimental animals One hour after treatment on the 5th Wistar albino rats weighing between day, rats were injected intraperitoneally 180 gram – 220 gram and Guinea pig into peritoneum with 1ml/100g of 0.05% weighing between 350 gram – 400 gram carrageenan and 1.4% formaldehyde were obtained from animal center of Đan suspension in 9‰ sodium chloride. After Phuong, Ha Noi, Viet Nam. Swiss albino 24 hours, the animals were killed and mice weighting between 18 gram – 22 gram the peritoneal cavity exposed. Peritoneal were obtained from the National Institute exudates were collected by aspiration JMR 111 E2 (2) - 2018 45
3. JOURNAL OF MEDICAL RESEARCH and the volumes recorded; the amount of the initial weight of the pellet (6mg) was leukocytes per 1 ml exudation were counted calculated and compared to the control and protein in the exudation quantified. The group. difference of the average values of these Bronchospasmolytic activity in parameters was calculated and compared anesthetized guinea pigs model to the control group. Guinea-pigs were divided into three Amount of leukocytes were quantified groups of six animals. They were orally using automatic hematological analyzer administered one of the following solutions Exigo - Boule Medical AB. Total protein before receiving histamine for 30 minutes content was quantified using biochemical [12]. analyzer Erba Chem. - Group 1 (control group) served as the Amiant-induced granuloma in mice vehicle control and received 1ml/100 g per Mice were divided into four groups of day sterile distilled water; ten animals [12]. The animals were orally - Group 2 (positive control group) were administered one of the following solutions: orally administered salbutamol at dose 6 - Group 1 (control group) served as the mg/kg per day; vehicle control and received 0.2 ml/10g per - Group 3 (treated group) were orally day sterile distilled water; administered Dinh Suyen P/H extract at - Group 2 (positive control group) were dose 8.29 g/kg per day; orally administered methylprednisolone at Guinea-pigs were anaesthetized with dose 20 mg/kg per day; pentobarbital and xylazin. The trachea - Group 3 (treated group) were orally was cannulated by means of a two way administered Dinh Suyen P/H extract at cannula, one arm of which was connected dose 12.44 g/kg per day; to the respiratory pump and the other to - Group 4 (treated group) were orally the transducer. Excess air, not taken up administered Dinh Suyen P/H extract at by the lungs due to bronchospasm, was dose 37.32 g/kg per day. measured and recorded on a polygraph. A sterilized amiant fiber weight 6 mg The internal jugular vein was cannulated for (soaked in 1% carrageenan) was implanted the administration of spasmogens and test in the back of the neck of mice in different compounds. groups. They were treated with Dinh suyen The tracheal cannula was clamped P/H extract daily for consecutive 10 days. (equivalent to clamping the trachea) ̣to record One hour after treatment on the 10th day the maximum bronchospasm in millmeters after implantation, all mice were killed. (Amax). Then after giving histamine 20µg/ Then, the granulomas were removed. kg intravenously, the excess air was After removing extraneous tissues, they recorded immediately to determine the were dried overnight at 56°C and weighed. bronchial response to histamine (0’) and at The mean net dry weight of the pellets of 5’, 10’, ..., 60’ after histamine administration the groups, which was after subtracting (At). The percentage of bronchospasm was 46 JMR 111 E2 (2) - 2018
4. JOURNAL OF MEDICAL RESEARCH calculated for each animal at each time t-test. Image J, a public domain Java point where: Pt = (At/Amax)*100 image processing program, was analyzed Pt (%): the percentage of bronchospasm to measure the width of the respiratory at minute t amplitude in bronchospasmolytic activity in At (mm): the average respiratory the anesthetized guinea pigs model. Data amplitude at minute t. are shown as mean ± standard deviation. Amax(mm): the maximum respiratory All data were considered significant at p < amplitide 0.05. Statistical analysis * p < 0.05; ** p < 0.01; *** p < 0.001; Data was analysed using Microsoft compared with control group Excel software version 2007. The levels of Δ p < 0.05; ΔΔ p < 0.01; ΔΔΔ p < 0.001; significance between the experimental group compared with positive control group and the control were made using student's III. RESULTS 3.1. Anti-inflammatory effect 3.1.1. Acute anti-inflammatory effect Table 1. The effect ofDinh Suyen P/H extract on volume, protein content, and amount of leucocytes in inflammatory exudation Exudate volume Total amount of Total protein Group (ml/100g) leukocytes (G) content (mg/dl) ( X ± SD) ( X ± SD) ( X ± SD) Group 1 (Control group) 2.03 ± 0.50 35.52 ± 24.65 10.37 ± 3.82 Group 2 (Positive control group) 1.43 ± 0.71* 6.83 ± 5.28** 6.71 ± 3.75* Group 3 (Dinh Suyen P/H extract 1.26 ± 0.62** 16.79 ± 12.66*Δ 7.14 ± 3.71 at dose 6.22 g/kg/day) Group 4 (Dinh Suyen P/H extract 1.08 ± 0.94* 12.40 ± 12.97* 5.30 ± 4.79* at dose 18.66 g/kg/day) 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
5. 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 2. The effects of Dinh Suyen extract on the weight of granuloma Net dry weight of granuloma (mg) Group n ( X ± SD) Group 1 (Control group) 10 17.18 ± 9.93 Group 2 (Positive control group) 10 6.79 ± 3.74** Group 3 (Dinh Suyen P/H extract at dose 10 14.49 ± 5.51 ΔΔΔ 12.44 g/kg/day) Group 4 (Dinh Suyen P/H extract at dose 10 23.44 ± 8.51ΔΔΔ 37.32 g/kg/day) 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 3. Effect of Dinh Suyen P/H extract on bronchospasmolity activity in a guinea pig model Group 3 Group Group 1 Group 2 Dinh Suyen P/H Control group (%) Positive control group (%) Minutes extract (%) 0 98.15 ± 7.11 85.19 ± 12.08* 97.51 ± 3.31Δ 5 92.31 ± 6.39 60.09 ± 12.5*** 74.47 ± 11.09** 10 80.9 ± 12.37 54.72 ± 14.85** 66.34 ± 9.55* 15 80.39 ± 11.68 57.06 ± 12.24** 73.17 ± 8.68Δ 20 77.26 ± 12.54 51.1 ± 14.03** 69.89 ± 10.97Δ 25 75.3 ± 13.73 51.75 ± 15,16* 62.76 ± 14.15 30 73.32 ± 14.23 52.02 ± 14,96* 62.47 ± 10.83 35 62.81 ± 15.73 53.86 ± 14.79 59.61 ± 9.74 40 64.44 ± 17.76 53.58 ± 14.47 57.4 ± 9.85 45 61.05 ± 15.46 50.85 ± 13.8 57.36 ± 8.82 48 JMR 111 E2 (2) - 2018
6. JOURNAL OF MEDICAL RESEARCH 50 62.53 ± 7.42 46.86 ± 13.15 58.43 ± 7.43 55 52.61 ± 11.11 48.19 ± 12.94 56.99 ± 3.65 60 52.07 ± 8.38 42.84 ± 15.36 55.63 ± 3.53 Percent of bronchospasm (%) 120 Δ 100 * ** Δ Δ 80 * *** Dinh Suyen P/H 60 ** ** ** * * Salbutamol Control 40 20 0 his 5 10 15 20 25 30 35 40 45 50 55 60 Minute Figure 1. Effect ofDinh Suyen P/H extract on bronchospasmolity activity in a guinea pig model The results in table 3 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 0 , 5, 10, 15, 20, 25 and 30 compared to the control group at same time points. - The treated group: At minute 5 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 induced granuloma model in mice. important mediating processes in asthma Acute inflammation is characterized [1]. To determine the acute and chronic by classical symptoms, including heat, anti-inflammatory efects of this extract, we redness, swelling and pain. Edema used a rat peritonitis model, induced by (swelling) is therefore a good measure of carrageenan/formaldehyde and an amiant- experimentally-induced acute inflammation JMR 111 E2 (2) - 2018 49
7. JOURNAL OF MEDICAL RESEARCH [12]. In experimental animals, peritonitis The present study demonstrated that can be induced by physiological mediators the Dinh Suyen P/H extract had an acute of inflammation (histamine, bradykinin) anti-inflammatory effect at dose 6.22 g/kg or nonspecific irritants (carrageenan, per day and 18.66 g/kg per day. The extract formaldehyde). The structure of inhibited inflammation. Shown by reducing carageenan is a polysaccharide like a the exudate volume, amount of leukocytes bacterial capsular polysacharide. It can and/or protein level in inflammatory exudate. induce acute inflammation by triggering The amiant-induced granuloma the release of inflammatory mediators model was used to evaluate chronic anti- (histamine, serotonin, bradykinin). These inflammatory effects of Dinh Suyen P/H mediators increase vascular permeability extract. Thymus dependent antigen elicits and prostaglandin production. This results antibody production by B lymphocytes in a gradual increase in fluid leakage and with involvement of Th cells. Additionally, more cells migrating into the animal’s these antigens can also induce the second peritoneal cavity, especially neutrophils immune response (cell mediated immunity) [13; 14]. Adding formaldehyde can increase that involves mostly T lymphocytes. the volume of exudate. Carrageenan/ The foreign body (cotton, amiant), once formaldehyde induced peritonitis is often implanted in the skin of animal produced used in the study of acute inflammatory undifferentiated connective tissue around it. process in Viet Nam [11]. The adhesion of macrophages, neutrophils, Methylprednisolone is a synthetic fibroblasts and collagen leads to granuloma glucocorticoid used to reduce inflammation formation. This amiant induced granuloma in asthma. Multiple mechanisms mediate model shows the proliferative phase of the in the suppression of inflammation by inflammation [13; 14].To test the chronic glucocorticoids. They inhibit leukocyte anti-inflammatory effect, amiant-induced infiltration at the site of inflammation, granuloma was used in this study. The interfere with mediators of inflammatory results of this study showed that the extract response, and suppress humoral immune at dose 12.44 g/kg per day and 37.32 g/ responses. The anti-inflammatory actions kg per day did not show anti-inflammatory of glucorticosteroids are thought to involve effect due to non-reduction in the dry weight phospholipase A2 inhibitory proteins, of granuloma after treatment. In this study, lipocortins, which control the biosynthesis the chronic anti-inflammatory effect has not of potent mediators of inflammation been clearly demonstrated yet. such as prostaglandins and leukotriene. In the other studies, some compounds Methylprednisolone is a powerful anti- in the extract were found to inhibit inflammatory agent [6]. Therefore, inflammation such as pseudoephedrine, methylprednisolone can be used as a ephedroxan (Herba Ephedrae) [8], positive control drug in acute or chronic schizandrin (Fructus Schisandrae) [15], inflammatory models. glycyrrhizin (Radix Glycyrrhizae) [17], 50 JMR 111 E2 (2) - 2018
8. JOURNAL OF MEDICAL RESEARCH gingerols, 6-shogaol (Rhizoma Zingiberis) V. CONCLUSION [18]. These compounds were known to The present study demonstrated that effect the activities of cyclooxygenase and/ Dinh Suyen P/H extract at dose 6.22 g/kg or lipoxygenase, and PGE2 in inflammation per day and 18.66 g/kg per day inhibited reactions, resulting in decreased production acute inflammatory activity in rats. The of inflammatory mediators and pro- results did not show evidence of any chronic inflammatory cytokines such as gingerols, anti-infammatory effect of Dinh suyen P/H glycyrrhidin, and schizandrin [15 - 17]. extract at dose 6.22 g and 18.66 g/kg per Histamine is an important mediator of day in mice. acute allergic and imflammatory reactions. The findings of this study indicate that It causes bronchoconstriction by activating Dinh Suyen P/H extract at dose 8.29 g/kg H1 receptors [4; 12]. Bronchospasmolytic per day inhibited the bronchocontriction in activity in anesthetized guinea pigs bronchospasmolytic activity in guinea pig. permits the evaluation of a drug's bronchospasmolytic effect by measuring Acknowledgments the volume of air, which is not taken by the The authors would like to thank Phuc lungs during bronchospasm. Salbutamol is Hung Company Limited for financial support. a highly selective β2-adrenergic receptor agonist that has a bronchodilator effect REFERENCES [4]. It is used to as the positive control in 1. Global Initiative for Asthma this study. The results of this study showed (2015). A Pocket Guide for Physicians and that Dinh Suyen P/H extract at dose 8.29 Nurses Updated 2015. g/kg per day had the bronchodilator effect 2. Global Asthma Network (2014). by inhibiting bronchospam at at minutes The Global Asthma Report. 5 and 10. Other studies have found that 3. Ministry of Health (2014). some herbs in the 11-herb formula can Pharmacology, Vietnam Education act as bronchodilators, such as Herba Publishing House Limited Company, Ha Ephedrae (ephedrin, pseudoephedrin), Noi. Rhizoma Pinelliae (ephedrin), Fructus 4. Dao Van Phan (2012). Clinical Schisandrae (schizandrin, schizandrol), pharmacology, 469 - 486. and Radix Glycyrrhizae (isoliquiritingenin) 5. World Health Organization (2000). [9; 14; 16; 18]. Ephedrine may be the Working group on the safety and efficacy of main bronchodilating agent because it is a herbal medicine. Report of regional office sympathomimetic agonist at both α and β for the western pacific of the World Health adrenergic receptors. It also displays indirect Organization. sympathetic activation, by enhancing the 6. World Health Organization release of norepinephrine from sympathetic (2007). Guidelines for Assessing Quality neurons [8]. of Herbal Medicines With Reference to JMR 111 E2 (2) - 2018 51
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