Identification of the Allergenic Ingredients in Reduning Injection by Ultrafiltration and High-Performance Liquid Chromatography.

Reduning injection is a traditional Chinese medicine injection which has multiple functions such as clearing heat, dispelling wind, and detoxification. Although Reduning injection was widely utilized, reports of its allergenicity emerged one after another. However, there is little research on its allergenic substances. The aim of this study is to evaluate the sensitization of Reduning injection and explore the underlying cause of the anaphylactic reaction. The main ingredients in Reduning injection were analyzed before and after ultrafiltration. Ultrafiltrate Reduning injection, unfiltered Reduning injection, egg albumin, Tween-80, and nine effective components in Reduning injection were utilized to sensitize guinea pigs. The serum 5-hydroxytryptamine level was used to assess the sensitization effect of Reduning injection. We found a significant decrease in Tween-80 content comparing to other components in the injection after ultrafiltration. Unfiltered Reduning injection, Tween-80, chlorogenic acid, and cryptochlorogenin acid caused remarkable anaphylactoid reaction on guinea pigs while ultrafiltration Reduning resulted in a significantly lower degree of sensitization. Our results suggest that ultrafiltration could significantly reduce the sensitization of Reduning injection, which is likely due to the decrease of Tween-80. We also conjectured that the form of chlorogenic acid and cryptochlorogenin acid within the complex solution mixture may also affect the sensitizing effect.

Study on the anaphylactoid of three phenolic acids in Honeysuckle.

ETHNOPHARMACOLOGICAL RELEVANCE: Chlorogenic acid (CA) is one of the major ingredients in Honeysuckle which exhibits anticancer, antibacterial, antiviral, hypoglycemic and anti-HIV activities. However, with the frequent emergence of anaphylactoid reactions of traditional Chinese medicine (TCM) injections which contains Honeysuckle in recent years, many researchers found that CA exhibited allergenicity. AIM OF THE STUDY: To explore the changes of content of CA, neochlorogenic acid (NCA) and cryptochlorogenic acid (CCA) during the preparation of Honeysuckle and evaluate the anaphylactoid of these three ingredients. MATERIALS AND METHODS: Two extracts of Honeysuckle were prepared by different methods and the content of CA, NCA and CCA were determined by high performance liquid chromatography (HPLC) analysis. Guinea pig and mast cells RBL-2H3 were utilized as the animal and cell model to investigate the anaphylactoid of these three ingredients. RESULTS: The content of CA decreased while CCA and NCA increased during the preparation of Honeysuckle. In vitro and in vivo studies showed that CA and CCA could significantly increase the plasma serotonin and ß-hexosaminidase levels in guinea pigs and induce the degranulation of RBL-2H3 cell, while NCA did not show such properties. In addition, CAA had a stronger effect than CA. CONCLUSION: We believed that both CA and CCA could cause anaphylactoid reaction while NCA could not. Moreover, the anaphylactoid of CCA is higher than CA. Our result demonstrated that CA is not the only cause of anaphylactoid reactions of TCM injections which contains Honeysuckle.

Contribution of serum IL-4 and IgE to the early prediction of allergic reactions induced by chlorogenic acid.

Chlorogenic acid (CA) is one of the active ingredients in some Chinese herbal injections, which may cause allergic reactions in clinic therapy. However, the criterion of test for allergen had not been employed in current Pharmacopeia of United States, European Pharmacopeia, Japanese Pharmacopeia and British Pharmacopeia. In order to find a new way to predict allergic reactions induced by CA earlier, the guinea pigs were sensitized successively by injecting CA intravenously once a day for three times, the results were compared that of Chinese Pharmacopeia by injecting CA intraperitoneally once every other day for three times, serum IL-4 and total IgE were detected by method of enzyme linked immunosorbent assay (ELISA) before guinea pigs were challenged once by injecting the same drug intravenously. The time-effectiveness and dose-effect of allergic reactions induced by CA were also studied. We found that contents of serum IL-4 and total IgE increased significantly before guinea pigs were challenged, either in D8 after intravenous sensitization (1.5 g/l CA, 0.5 ml) or in D14 and D21 after intraperitoneal sensitization (1.5 g/l CA, 0.5 ml), and allergic reactions occurred in all guinea pigs after challenged once by injecting CA (1.5 g/l, 1.0 ml) intravenously. It provides a new way to predict whether CA (or Chinese herbal injections contained CA) can provoke allergic reactions by detecting serum IL-4 and total IgE earlier; the examination period is reduced by 1-2 weeks. It has a good prospect of application in drug emergency test.

[Study on sensitization and mechanism of CGA-BSA].

OBJECTIVE: To investigate the sensitization and mechanism of artificial antigen of chlorogenic acid (CGA-BSA). METHOD: Using intensive immunization to establish allergy animal model on guinea pig and preparing antiserum and tissue for further test. Using HE staining to observe pathology change of lungs, trachea, liver. Using passive mast cell (PMC) degranulation test to observe the immunogenicity of CGA-BSA and using ELISA to detect IgE and histamine in plasma. RESULT: There established allergy animal model on guinea pig, which include a increase cell degranulation by a ratio (63.58 +/- 10.23)% in PMC test, increase of specific antibody IgE and increase of histamine in plasma after provocation by ELISA. CONCLUSION: Allergen CGA-BSA could provoke allergenic response in guinea pig, and the allergic response belongs to type I allergy.

Evaluation of the immunosensitizing potential of chlorogenic acid using a popliteal lymph node assay in BALB/c mice.

It has yet to be established whether chlorogenic acid (CGA), a common xenobiotic with potential exposure risk to humans, is associated with immune-mediated hypersensitivity reactions (HRs). The primary limitation in evaluating this potential relationship is the lack of an effective animal model for use in predicting the immunosensitizing potential of low molecular weight compounds (LMWCs). Currently, the popliteal lymph node assay (PLNA) is considered a very promising tool for assessing immunosensitizing potential of LMWCs. To determine whether CGA may possess an intrinsic capacity to stimulate or dysregulate immune responses, and if so, what mechanisms may be involved, we characterized the popliteal lymph node reaction induced by CGA in naive female BALB/c mice using both a direct PLNA (d-PLNA) and a reporter antigen PLNA (RA-PLNA) method. Our results show that CGA failed to induce immunoreactivity following a single subcutaneous injection either alone or when combined with TNP-OVA or TNP-Ficoll. These results indicated that CGA lacks the intrinsic capacity to sensitize or stimulate immune responses in BALB/c mice. Moreover, these results suggest that exposure to CGA may not represent a safety concern for humans and that removal of CGA from Traditional Chinese Medicine Injections may not significantly decrease the prevalence of HRs.

[Study of immunogenicity of chlorogenic acid-BSA with different number of CGA].

OBJECTIVE: To investigate the immunogenicity of chlorogenic acid-BSA(CGA-BSA) and the foundation for the allergization of CGA-BSA. METHOD: The CGA-BSA with different number of CGA was synthesized to allergize the BALB/c mice and SD rats to get antiserum. The level of IgE and histamine was analyzed by ELISA, and the passive cutaneous anaphylaxis (PCA) test was carried to analyze the antibody titer. RESULT: When the coupling rate of CGA-BSA is 20, the highest IgE and histamine level in BALB/c mice and the antibody titer in PCA are the highest. CONCLUSION: It has better immunogenicity when the coupling rate of CGA-BSA is between 10 to 20, while the CGA-BSA 20 with the best immunogenicity.