Quercetin 3-Glucoside-7-Rhamnoside from Hippophae rhamnosides L. Fruits Attenuate Allergy in Ovalbumin-Induced Allergic Mouse

Allergy are a serious health topic today in many countries [1].
Allergy are very patient peculiarity and include various allergies and
skin symptoms.


Introduction
Allergy are a serious health topic today in many countries [1].
Allergy are very patient peculiarity and include various allergies and skin symptoms [2]. Allergy have come to epidemic proportions in international world and incidence of allergy is continuing to growth in relation with the present lifestyles [3]. Therefore, it is urgent to understand the pathophysiology and care strategies of the various allergic diseases influencing the population of the world.
Pharmacotherapy of allergy includes anti-allergic drugs, bronchodilators, and corticosteroids [4,5]. These products are clinically limited because of their side effects such as hyperlipidemia, susceptibility to pathogens and hyperglycemia [6]. Ayurvedic medicines include several plants possessing medicinal efficacy in its formulations for the care of allergic diseases [7]. Thus, it can be undoubted that the therapy using herbal medicines could be a potential alternative in the pharmacotherapy of allergic diseases.
Hippophae rhamnoides L. (HR) is a hardy, deciduous shrub belonging to family Elaeagnaceae [8]. It grows in Central Asia and Europe [9]. This plant has lately gained world's attention because of its various potential. A HR fruits, seed and other parts is primarily valued for its very rich physiological compounds [10]. Fruit of HR is one of the few exclusions among carotenoid-rich plants that contain abundant lipids [11].
For centuries, the people of Asia have used HR as a candidate of alternative medicine to prevent various diseases. The HR fruits were used as a fountainhead of herbal medicines and skin care in the world including Europe and Asia [9]. A wide spectrum of pharmacological effects of HR have been recently published, including antioxidant efficacy, immunomodulatory effect, and hepatoprotective function Various studies have shown that flavonoids have several pharmacological activities.
In the present study, the anti-allergic activities of 3-O-glucoside-7-O-rhamnoside (Q3G7R) from Hippophae rhamnoides L. fruits was investigated in ovalbumin (OVA)induced mouse allergic model. Allergy was induced by intraperitoneal injection of ovalbumin into balb/c mice. After 4 weeks, IgE level by mouse IgE ELISA Kit, histamine level by fluorometric method and tryptase level by Immuno CAP Tryptase reagents in serum, and then the levels of T h 2 cells-derived cytokines interleukin (IL)-4 and IL-13 by ELISA kit in spleencyte were observed. As the results, Q3G7R showed significant a reduction of OVA-specific IgE levels in serum, decrease of histamine and tryptase level in serum, and then the decrease of the levels of T h 2 cells-derived cytokines interleukin IL-4 and IL-13 in spleencyte. In histological analysis for lung, Q3G7R excellently reduced eosinophil infiltration with inhibition of characteristic lesions and inflammation including ovalbumin (OVA)-induced necrosis, numbers of inflammatory cells and pulmonary edema. Therfoure, Q3G7R showed anti-allergy effect in mouse allergic model and may be a potential agent for the treatment of allergic disease.
anti-tumor activity and antioxidant activity and have been widely used in the study of natural medicine as well as in practical clinic [24][25][26].

Extraction and Isolation
The parched fruits of Hippophae rhamnoides L. were

Animal and Application
Five-week-old male Balb/c mice were purchased from Charles River Japan (Kanagawa, Japan). Mice were housed at 21 ± 2 with 55 ± 15% humidity of a 12 hrs light-dark cycle (light on 7 AM, 7 PM). All experimental protocols were approved by the Institutional Animal Care and Use Committee (IACUC) of Daejeon University, South Korea (DJUARB2013-028). All procedures were executed according to the Guide for the Care and Use of Laboratory Animals.
Balb/c male (6-week-old) was used and ovalbumin was injected in intraperitoneally of them for 0, 7, 14, 21 and 28 days to Balb/c. The Q3G7R containing 100 mg of Q3G7R/kg body weight per day, dissolved in 0.4% polyethylene glycol in 0.5% EtOH, were applied topically, on a daily basis, for 4 weeks.

Measurement of IgE Level
To measure IgE level in serum, animals were anesthetized for euthanasia with the following reagents: isoflurane and sodium pentobarbital. Isoflurane was administered by inhalation at an overdose of 4% with oxygen as a carrier gas for 5 min in a sealed chamber at a pressure of 6 psi by using inhalation anesthesia

Histamine and Tryptase Assay in Serum
The heparinized blood was processed immediately and/ or kept at 4°C (a maximum of 24-48 hrs) after its extraction and subsequently centrifuged to separate the plasma that was processed afterwards to quantify the amount of histamine. Plasma histamine was determined using the fluorometric method following the technique modified by Siraganian [28] with an AutoAanalyzer 3 (Bran Luebbe, SEAL Analytical Inc, Mequon, WI, USA). Serum tryptase levels were determined with the B12 assay [29], using Immuno CAP Tryptase reagents and the Phadia 250 analysis device (Phadia AB, Uppsala, Sweden).

Measurement of Cytokines Level
Animals were killed by decapitation one day after measurement of IgE level, histamine and tryptase levels. To measurement of cytokines level, OVA/alum was conducted by intraperitoneal injection. And then, spleen cells were seperated from each mouse after 4 weeks. Cytokine measurement in the spleen spleen cells (2 × 10 6 /mL) were cultured for 48 hrs in the culture dish (Corning Inc, USA.), which had been coated with CD3 mAb of 0.5 μg/mL in 12 hrs before. Then, IL-4 and IL-13 were determined by using ELISA kit. Then the plate was washed twice, and 100μL of Avidin-HRP conjugated antibody was treated, washed, and TMB solution was treated for 30 min in the dark. After stopping the reaction with 50μL of stop solution, the absorbance at 450 nm was measured by using ELISA reader.

Histological Analysis
Histological evaluation was performed on lungs. Lungs were inflated with PBS containing heparin, tied off, and removed from the mice and placed in 10% formalin. After 10 days in formalin, the lungs were initially assessed macroscopically for affected percentage, and then embedded into paraffin, sectioned at 5μm thickness, and stained with hematoxylin and eosin as described before [17]. Histological analysis was carried out under a light microscope (Leica DM 1000, Germany) at high magnification (250×). All macroscopic and microscopic lesions were analyzed independently by a resperatory experter blinded from the study.

Statistical Analysis
Results are expressed as means ± standard deviation. The data were analyzed using ANOVA and Duncan's multiple range tests.

Q3G7R Decrease Histamine and Tryptase Level in Serum
Serum histamine levels were significantly higher in the OVA-CT group compared with the Nr group (p<0.05) and were significantly smaller in the Dexa group (p<0.01; 2mg/kg), and Q3G7R (200mg/ kg) group compared with the OVA-CT group (p<0.05; (Figure 3A)).
This finding indicated that Q3G7R treatment significantly improved the serum histamine contents in mice with significant difference from the control groups. Serum tryptase levels were significantly

Q3G7R Reduce T h 2 Cells-Derived Cytokines IL-4 and IL-13
Ovalbumin was to be intraperitoneal injection.

Inhibitory Effect of Characteristic Lesions and Inflammation by Q3G7R
Histologic analyses were performed of lung samples from mice in 4 weeks.

Discussion
Allergic diseases are a significant health problem today in both developed and developing countries [30]. They have reached epidemic proportions worldwide and their incidence is continuing to increase in association with modern lifestyles [31].
The mechanism underlying the recurrence of allergic diseases is far from resolved [32][33][34], making it urgent to explore the mechanisms of allergic diseases and to develop new drugs that effectively reduce the rate of recurrence.
To determine whether the Q3G7R can be used to alleviate the allergy-caused inflammatory reactions as an alternative, Q3G7R were used to treat the OVA-induced allergy in a mouse model.
Generally, animals demonstrated similar hallmarks to acute human allergy characterized by pathophysiological alterations in airways, mucus secretion, production of allergen specific IgE and the increase of T h 2 cytokines level [35]. Both clinical and animal experiments have confirmed the production of T h 2 cytokines in allergy, such as IL-4, IL-5 and IL-13, which can induce the activation of initial effector cells such as eosinophils and mast cells, and subsequently activate many inflammatory mediators, resulting in chronic airway inflammation [36][37][38]. The results demonstrated that mice treated with Q3G7R significantly reduced OVA-specific IgE, histamine and tryptase levels in the serum of mice compared with OVA-CT group without Q3G7R treatment. Treatment of Q3G7R significantly reduced IL-4 level and increased IL-13 in serum of OVA-challenged mice. In these results, Q3G7R significantly improved the serum histamine contents in mice with significant difference from the OVA-CT group and significantly decreased the tryptase level of mice serum.

The infiltration of lung inflammatory cells (mast cells,
lymphocytes and eosinophils), especially those of eosinophils, is representative of inflammatory events of asthma, the severity of which therefore is directly related to the degree of eosinophilia [39]. It has been shown that patients with allergic rhinitis induced by allergen showed an obvious increase in the number of eosinophil peroxidase-stained nasal cavity mucosal eosinophils [40]. Our data showed that treatment with Q3G7R was able to suppress OVAinduced allergic lesions, inflammation and eosinophils in lung.
There alleviating effects will be correlated with the regulation of inflammatory cytokines via reduction of OVA-specific IgE, histamine and tryptase levels.

Conclusion
In conclusion, results in the present study confirmed that Q3G7R possess anti-allergy activities. Further investigation on the mode of action of this Q3G7R will be continued in our future projects.

Conflicts of Interest
No potential conflicts of interest relevant to this article were reported.