Allergen-Denaturing Activities of Constituents Identified in Eriobotrya japonica leaves against Egg Allergen Ovalbumin

Abstract

Egg allergy is the second most common cause of food allergies in children. It causes an overreaction of the immune system which may lead to severe physical symptoms for millions of infant around the world. Most cases are derived from egg white which induces allergic conjunctivitis, itchiness, and a runny nose. Early studies reported that one of the major allergen ovalbumin (OVA, Gal d 2, 45 kDa) constitutes about 54 % of egg white proteins. It is very difficult to control of ovalbumin physically or chemically because their stability to heat and digestive enzymes, reflecting their capacity to stimulate a specific immune response. Medication such as anti-histamines, oral corticosteroids and epinephrine has been used to reduce symptoms of allergic disorders. They have not only low efficacy but also side effects. As an allergen denaturant, promethazine is affordable but their residual property is criticized. There is therefore a critical need for the development of new improved allergen denaturants with novel target sites. Eriobotrya japonica has been used in Korean and Chinese traditional medicine for inflammatory disease and chronic bronchitis. A traditional therapy using it in an ancient book has shown that it is effective to treat asthma, coughs, and high fever as to drink its tea. This research is focused on isolating allergen denaturing activity compound from the leaves of E. japonica and an assessment was made of allergen-denaturing activities of constituents identified in E. japonica leaves against egg allergen ovalbumin. The allergen denaturing activities of active principle from E. japonica are compared with those of the commercial allergen denaturant, promethazine. The activities were analyzed by measuring of intensity and IC50 about Immunoblot and Competitive Indirect enzyme-linked immunosorbent assay (ciELISA), respectively. Moreover, fragmentation of protein induced by treatment of natural products was examined, allergenicity and antigenicity of residual materials was compared between human sera from allergic patients and commercial rabbit antibodies. The active principles of E. japonica leaves were separated using column chromatography, glass thin layer chromatography and preparative HPLC. In immunoblot using rabbit anti-OVA antibodies, active principle exhibited antigenic inhibition. The results from the immunoblot bioassay demonstrated that the allergen denaturing activity of compound 1 (relative antigenicity, 99.18 % at 100 μg/mL) did not differ significantly from that of promethazine (99.34 % at 100 μg/mL), whereas compound 2 (95.83 % at 10 μg/mL) and compound 3(83.33 % at 25 μg/mL) treatments were much higher than that of promethazine. In E. japonica derived compounds, ellagic acid suppressed the binding of OVA and specific IgG effectively followed by lactic acid, hydrobenzoic acid, rutin trihydrate, (D)-(-)-tartaric acid, riboflavin and palmitic acid (85.2–96.7 %). The activities of these compounds were comparable to that of promethazine. In the ciELISA inhibition, the concentration of treated compounds, which were reduced to 10 μg/mL. Tartaric acid was the most active compound (IC50, 114.18 μg/mL) as compared with that of native OVA (11.49 μg/mL) and ellagic acid treatments (13.11 μg/mL) did not inhibit the antigenicity of the OVA effectively as that of promethazine (12.81 μg/mL). However, in isolated compounds, compound 3 has been shown to be most effective, which was more active than compound 1 and compound 2. The antigenicity was reduced to 1/10 along with oleanolic acid, palmitic acid, lactic acid and p-coumaric acid (106.63-100.52 μg/mL). Other compounds failed to show good effects on the reduction of the antigenicity of OVA. In the SDS-PAGE bioassay, treatment with urea clearly indicates that E. japonica leaf extract and promethazine caused appearance of ovalbumin protein and residual material bands in pellet solutions. In addition, high concentration of each sample extracts strongly increased the content of protein by exposing particular four amino acids. In the immunoblot assay, allergenicity of residual materials treated with E. japonica leaf extract remained, whereas antigenicity of residual materials treated with E. japonica leaf extract was removed. However, the three isolated compounds either physically or chemically modified egg white whole protein and ovalbumin, their binding activities to allergic patients specific IgE and rabbit-specific IgG were removed. Moreover, the denaturing activity of E. japonica leaf extract was higher than that of promethazine. E. japonica leaves principle as well as its constituents possess allergen denaturing activity against OVA protein, respectively. They merit further study as potential allergen denaturants or lead molecules which control OVA populations through suppression of IgG-mediated allergic response for the prevention or eradication of egg allergic disorders. In addition, in the light of global efforts to reduce the level of toxicity and side effects of drugs, it is suggested that the use of plant materials could considerably modify symptoms of allergic disorders as allergen denaturants are warranted.