A Study Effects of Licorice Plant and Prednisolone on Some Physiological and Immunological Parameters in Experimental Model

Zaid Al-Nuaimi,Nasser Almukhtar,Safaa Alturaihy
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Keywords : Licorice; Glycyrrhiza glabra; flavonoids; IL-5; asthmarabbit’s model
Medical Journal of Babylon  14:1 , 2017 doi:1812-156X-14-1
Published :17 July 2017

Abstract

The present study conducted thirty of adult male white rabbits to uncover the impact of Licorice in minimize the signs associate degreed symptoms after induction of allergic bronchial asthma in rabbits and elucidate what extent it will show an beneficial effects herbs therapy, in state use corticosteroids medical care. The rabbits haphazardly divided into 5 groups and albumen sensitizer was used to stimulate allergic reaction beside to use sensitization boost challenge dose that emphasize by the presence of the clinical symptoms of innate reflex, wheezing, chest tightness and shortness of breath. As well as, to changes within the level of total white blood corpuscle count, Eosinophils measurements, Interleukin-5 estimation and body temperature measurements. Meanwhile, eight days after induction allergic reaction. Extraction of Licorice was given in a variety dose of (160 and 80) mg/kg orally was administrated for eight days. Blood collected samples were done on the day sixteen and thirty, for investigate the above previous parameters through collected blood serum samples. statically analysis fore results were showed that the albumen action considerably (p? 0.05) induce allergic response in those of experimental rabbits associate degreed show an elevation with total white blood corpuscle, Eosinophils and Interleukin-5 levels. Whereas orally treating rabbits with root extract observe a big decrease (p? 0.05) within the total white blood corpuscle, Eosinophils and with the degree of Interleukin-5. As these effects of (the plant extract) wherever analyzed in comparison with recorded noticeable effects in those of glucocorticoid treated group and those healthy management teams severally.

Introduction

?sthma is one of the most current chronic diseases, with an estimated 300 million patients afflicted by this disease worldwide. The Global Initiative for Asthma [1] 2004 estimated that more than 10% of the population in Australia, Brazil, Canada, New Zealand, Peru, England, and United States had asthma. The prevalence of asthma is expected to increase, and there may be an additional 100 million peoplewith asthma by 2025 [1]. A 2012 survey revealed that 25.5 million people in the United States alone [2]. Moreover, asthma prevalence has increased from 3.1% in 1980 [3] to 8.3% in 2012 [4, 5]. Asthma is a disease involve a several distinct endotypes manifested by contribution of cellular and molecular biomarkers. The joining of endotypes character with clinical phenotypes character has extremely preceding our recognizing of asthma pathophysiology and the process of novel targeted therapeutics [6]. One of the best-characterized endotypes of asthma is the patient population with eosinophilic airway inflammation, which accounts for approximately 40–60% of patients with severe asthma [7,5]. Several important observations in human initially supported the hypothesis that eosinophils play a critical role in the pathogenesis and severity of asthma. An increased eosinophil count is associated with increased asthma severity, frequency of exacerbations and mortality in patients with asthma [8]. Several studies have demonstrated that sputum eosinophils increase during exacerbations and that increased numbers of eosinophils in the peripheral blood and airways of patients with asthma correlate with disease severity. Perhaps most importantly, persistent, eosinophilic airway inflammation increases the risk of subsequent exacerbations [9]. For example, markers of eosinophilic airway inflammation increase well before the onset of exacerbations induced by corticosteroid withdrawal, and sputum eosinophil number predicts loss of asthma control after corticosteroid reduction or discontinuation [12]. The root of licorice (Glycyrrhiza glabra) is one of the most frequently used natural medicines in the world, and has been described as ‘the grandfather of herbs [12]. It has been used medicinally in both Western and Eastern countries for more than 4000 years [13]. It has been traditionally used for respiratory, gastrointestinal, cardiovascular, and skin disorders. The biological and pharmacological activities of Glycyrrhiza glabra have been widely studied as its long history and biologically active constituents are still interesting to many research groups [15]. A large number of clinical and experimental studies reported its useful biological properties such as antioxidant, immuno-modulatory, cardioprotective, anti-inflammatory, antiviral and anticacer effects [18]. Recently, Glycyrrhiza glabra is reported to have neurological properties such as antidepressant, anxiolytic, and anticonvulsant effects [19]. Chemical analysis of Glycyrrhiza glabra root extract showed the existence of triterpenes (glycyrrhizin, glycyrrhetic acid and liquirtic acid), flavonoids (liquirtin and formononetin) and various other substances [20]. The pattern of inflammation in asthma is characteristic of allergic diseases, with similar inflammatory cells seen in the nasal mucosa in rhinitis (Figure 2). An indistinguishable pattern of inflammation is found in intrinsic asthma, perhaps reflecting local rather than systemic IgE production. Acute-on-chronic inflammatory episodes, corresponding to exacerbations of asthma, are usually triggered by upper respiratory tract virus infections or allergen exposure. Although the common pattern of inflammation is eosinophilic, some patients with severe asthma and asthmatics who smoke show a predominantly neutrophilic pattern that is less sensitive to corticosteroids. Several inflammatory cells are involved in asthma [21].

Materials and methods

Laboratory animals:
The experimental model used of this search were consisted of thirty male adult rabbits with ranging weighing: (2.100-2.600) kg and its age were ranging from(11-16) months. The rabbits were house within the animal house in school of medicine / University of Babylon. Animals were left one month for an adaptation prior the experiments. Animals were kept in optimized stainless steel standard cages as 6 rabbits in each cage. With freely feeding at fresh green vegetables, chaw pellets and free of water. The rabbits were kept under a same circumstances of the temperature range of (22-25°C), relative humidity of 75+5% and light regime of 12 hours light, and 12 hours darkness divided into five groups, each group was consists of six male rabbits used according to parts of experimental design.
Experimental Protocol:
The experimental protocols are adopted from (Kamaruzaman et.al.,2014). As the Induction of allergic airway inflammation is performed by intraperitoneal (i.p) injection of the Ovalbumin (OVA) 0.1 mg and 10 mg of aluminium hydroxide disolve in 2 ml of phosphate buffer saline (PBS) in day 1 and a booster challenge dose through second sensitization in day 14 of the immunization program shown in the following groups.

Control Group (Group1):
Control group comprised of six rabbits injected (i.p) twice / experiment with phosphate buffer saline (PBS) first injection had been done at the day 1 and the second injection had been done at the day 14 of the experiment and treatment starting from day 23 to 30 orally with 2 ml (PBS), and the blood sample were collected then all animals are sacrificed on day 31 of the experiment.
Asthma Stimulate Group (Group2):
OVA group comprised of six rabbits injected (i.p) twice / experiment with OVA 0.1mg and 10mg of aluminum hydroxide dissolve in 2ml of PBS by immunizationat day 1 of the experiment and booster challenge dose at day 14 of the experiment, and blood sample were collected then all animals are scaron day 15 of the experiment.
Prednisolone 1mg/kg B.W (Group 3):
Prednisolone group comprised of six rabbits injected (i.p) twice / experiment with OVA 0.1mg and 10mg of aluminum hydroxide disolve in 2ml of PBS by immunization at day 1 of the experiment and booster challenge dose at day 14, and  then treatment starting from day 23 to 30 orally with 1mg/kg prednisolone (Davis, 1971).  And blood sample were collected then all animals are sacrificed on day 31 of the experiment.

Licorice Root 160 m/kg B.W (Group 4):
Licorice  group comprised of six rabbits injected (i.p) twice / experiment with OVA 0.1mg and 10mg of aluminum hydroxide disolve in 2 ml of PBS by immunization at day 1 of the experiment and booster challenge dose at day 14, andtreatment starting from day 23 to 30 orally with 160 mg/kg of Licorice Root extract, and  blood sample were collected then all animals are sacrified on day 31 of the experiment.
Licorice root + prednisolone (80+0.5) mg/kg B.W (Group5):
A combination of Licorice root plus prednisolone group comprised of six rabbits injected (i.p) twice / experiment with OVA 0.1mg and 10mg of aluminum hydroxide disolve in 2ml of PBS by immunization at day 1 of the experiment and  booster challenge dose at day 14, and treatment starting from day 23 to 30 orally with Licorice Root 80 mg/kg plus prednisolone 0.5mg/kg together, and blood sample were collected then all animals are sacrified on day 31 of the experiment.
Statistical Analysis:
The data were analyzed by using computer Statistical Package for Social Science Software (SPSS, version 20) program. As well as, to one Way ANOVA test expressed data and mean± SD for a continuous variable. The p-value of less than 0.05 was statistically significant and highly significant for a p-value of less than 0.05 [22].




Results

Impact of Prednisolone on Total WBCs estimation in the Control, Asthma Motivate and Treated Rabbits. The results in table (1) show a significant increment of total WBCs estimation for both of motivate ovalbumin rabbits group in a dose of (ova 0.1ml and 10mg of aluminum hydroxide in 2 ml of PBS) and for asthma treated (administer) prednisolone groups (6.380±1.190; 11.880±1.044) in comparison with control group (4.340±0.808) respectively as were it daily treated with prednisolone dose of (1mg/kg of body weight) along time such period according to planning program which continue for 8 days, start from the days 23 until the days 30 of the experiment. Effect of bisector doses of Licorice, Prednisolone and medley doses on Total WBCs estimation in Asthma Motivate Rabbits. Presented data in Figure (3) observe, that asthma motivate rabbits when treated with 2 different doses of Licorice plant root extract as (160 mg/kg) as a first dose and as a combination of second dose of (80 mg/kg of Licorice plus 0.5 mg/kg of prednisolone to given be together ) of body weight are appear with a significant decrease (P ? 0.05) of total WBCs measurement of (4.840±1.221 and 4.960±1.289) respectively in comparison with the asthma motivate , prednisolone treated groups and in reflect with control group (4.340 ± 0.808) in consistence with the planning program which continue for 8 days from the days 23 until the days 30 prior the sacrificing day of the experiment.

Discussions

Effects of oral Licorice intake on the total WBC and Eosinophils estimation of Treated groups: Presented data in Figures (3) and (4) are pointing a significant increase in total WBC, neutrophils and eosinophils measurements and expand their role in exhibiting of asthma signs and symptoms through releasing of inflammatory mediators of cytokines, chemokines as reflect to use of OVA and going to play a big role in distinguishing allergy and asthma characterizations. Meanwhile, it shows a significant decrease (p ? 0.05) in neutrophils and eosinophils that have estimated contribute to the inhibitory effect of flavonoids of Licorice, with the present work. Asthma is a chronic respiratory disease, and such therapy as corticosteroid is inadequate and has many adverse effects. Therefore it needs better therapeutic agents that to be preferable of natural origin, manifested by modest or no adverse effects. As the Glycyrrhizin (GRZ), is the active constituents of the (Licorice root), which show efficacy on relief asthmatic features. Rabbits sensitized and challenged with (OVA) to induction of asthmatic features, such as allergic asthma and inflammatory response development. Treated asthmatic group after OVA-stimulation has been evaluate for reversal effect, presented on those of asthmatic rabbits. As well as, for other parameters associated with asthma like interleukin (IL-5) and immunoglobulin OVA-specific (IGE) were also detected by ELISA. Results record decrease in IL-5 and eosinophil. Addition to, it reduce level of OVA-specific IgE in serum. So results recorded that Licorice of the asthmatic features in rabbits and it could naturally have high value towards developing a better therapeutic plant agent after time [23]. Most of the activated cells in the pathogenesis of allergic asthma, is eosinophils that present as prominent cell type, and an increased number in the airways is correlated with the severity of asthma ; accordingly, eosinophils is often a target for the therapy of various inflammatory diseases including allergic asthma. A former study illustrated that recruitment of eosinophils into bronchus-associated lymphoid tissue (BALF) was clearly observed as expected in OVA-sensitization asthma. and from here study found elevate leukocyte infiltration, goblet-cell hyperplasia, and mucus hypersecretion, and these finding in a good agreement with previous study of [24]. Eosinophilic cells concentrate in high numbers through the lungs of asthmatic patients, and are consider of importance in the pathogenesis of asthma. A potent eosinophil chemoattractant is produced in the asthmatic lung. These are of a small protein, and called chemokine eotaxin, that produce in a number of different cell types, and is stimulated by interleukin-4 and interleukin-13, which are synthesized by T-helper (TH) 2 lymphocytes. Low molecular weight compounds have been developed that can block the eotaxin receptor C-C chemokine receptor (CCR) 3, and prevent stimulation by eotaxin [25]. However, all the mentioned improved data were coincided in good relation with our outcomes of the present findings this search Licorice effects on the hematological parameter and particularly with total WBC and eosinophils. In a prior study of Arjun et al. [26]pointed that flavonoids played the major role in asthma and in quite possible that GRZ may block eotaxin production in vivo as well to blocked infiltration of the eosinophils in the lungs. In conclusion, GRZ important soften the asthmatic features such as OVA induced immediate airway constriction, airway hyperreactivity, and eosinophilia and lung inflammation.

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