| Objects:With the development of extraction, separation and purification of traditional Chinese medicine by new technology, preferable Chinese materia medica preparation which has good therapeutic action now can be required. Kinds of pharmaceutical dosage form of traditional Chinese medicine have been out-of-date, and is not meet medical needs. In order to change the present situation, many conventional dosage forms are improved into injectable dosage forms, such as Qingkailing injection, Shuanghuanglian injection, Yuxingcao injection and so on. These new injections which were base on ancient prescription are not only cheap, but also can be used for mergencies and acute disease. But the incidence of side effects happened during injection process is high, which include allergy, cardinal symptoms, even drug-induced allergic shock and death. Since the prevalence of adverse event and intercurrent illnesses continue to rise,FDA had decided to stop using some traditional Chinese medicine, such as Qingkailing injection and Yuxingcao injection. In view of the above-mentioned facts, we have to find a way to solve the problems existing in traditional Chinese medicine injection to improve the security and extend clinical application of Chinese medical injection. Multiple data-sources, methods, analyses or theories should be combined to overcome the bias that comes from single informants, single-methods or single theory studies. The global property of Chinese medicine should be also paid more attention. In this study we designed to investigate the antigen in the murine allergic with Qingkailing injection.Methods:1 Big weight molecule is the most common antigen in the medication, for instance:protein. First of all, we inspect the probability of sensitization of big weight molecule. SDS-PAGE gel electrophoresis detects the proteins which precipitate by trichloroacetic acid (TCA) and ethanol. Meanwhile, nucleic acid was be detected by agarose gel electrophoresis. Apply the HPLC/MS to estimate the molecular weight range of Qingkailing injection.2 6-week-old female BALB/c mice were randomly divided into 4 groups: Qingkailing injection(QKL), Qingkailing injection with Freund's adjuvant (QKL+FCA), Ovalbumin(OVA)group and physiological saline group,6 mice in each group. Qingkailing injection were dissolved in sterilized saline and emulsified with an equal volume of Freund's adjuvant, The emulsion solutions were injected 0.3mL of subcutaneously into BALB/C mice in QKL+FCA group; BALB/c mice of QKL and OVA group received subcutaneous injection of 0.3mL Qingkailing injection and OVA (0.1mg) respectively. Booster injections were carried out on the 14th day after the primary doses. QKL+FCA group followed subcutaneous injection with the same quantity of immunogen emulsified with incomplete Freund's adjuvant at 14 days intervals. QKL and OVA group were received the some doses by subcutaneous injection with Qingkailing injection and OVA. Saline group were administered the same volume of physiological saline. Mice were bled from the retro-orbital plexus just at 7,14,21days after Booster injections. After collecting, sera were allowed to repose for 30 min at 37℃and thus slightly centrifuged in a bench centrifuge during 10 min at 4℃and the clean material stored at-20℃until use.The samples were taken to obtain plasma for analyzing the IgE by enzyme-linked immuno sorbent assay(ELISA) and Passive Cutaneous Anaphylaxis Reaction (PCA).3 Hapten is a class of small molecules and is immunogenicity obtained only by coupling with carrier protein. The Mannich reaction is a familiar an amino-alkylated reaction in which compounds containing active hydrogen, such as esters, phenols, ketones etc., can be condensed with formaldehyde and an amido in weak acidity. The carrier protein is bovine serum albumin that has been modified by substituting anionic carboxyl groups with cationic aminoethyl-amide groups. When used as an immunogen, this cationized BSA (cBSA) stimulates a much higher antibody response than native BSAThe preparation of QKL-cBSA base on the Mannich reaction. Briefly, a quantity of QKL (2,4,6,10 mg) and 2mg of cBSA were dissolved in physiological saline and conjugation buffer (0.1 mol/1 MES pH 4.8) respectively. After dropwise addition of QKL solution into the protein solution, the mixture was stirred gently, then 50μl of formaldehyde were added into the mixture and immediately stirred gently for 24 h at 37℃. Apply the entire hapten-carrier reaction mixture directly onto the top disc of the desalting column while collecting the flow-through in a test tube. When the sample has completely entered the column and the flow has stopped, transfer the column to a new tube and add 0.5 ml of the purification buffer.Finally the sample was detected by ultraviolet spectrophotometer.4 BALB/c mice were randomly divided into 3 groups:QKL-cBSA group, OVA group and physiological saline group,6 mice in each group. Groups of QKL-cBSA obtained QKL-cBSA conjugate was emulsified with an equal volume of Alum adjuvant. OVA group received subcutaneous injection of 0.3mL OVA (contain O.lmg OVA).Saline group were administered the same volume of physiological saline. Booster injections were carried out according to the primary doses on the 14th day after the first injection.Mice were bled from the retro-orbital plexus just at 30min and 7,14,21,days after Booster injections. After collecting, sera were allowed to repose for 30 min at 37℃and thus slightly centrifuged in a bench centrifuge during 10 min at 4℃and the clean material stored at-20℃until use. The samples were taken to obtain plasma for analyzing the IgE and histamine by ELISA:In consideration of the cBSA as carrier protein which molecular weight is 68KD, if cBSA mix with carrier-hapten, it also can induce hypersusceptibility in mice. So we use cBSA as antigen to stimulate mice in dose of experiment by two ways: six-week-old female BALB/c mice intraperitoneal injection and boosted by vein of trail with the 0.05mg cBSA on the 14th day after the first injection. Aother group was administrated by subcutaneous injection with 0.05mg cBSA was emulsified with an equal volume of Alum adjuvant. Booster injections were carried out according to the primary doses on the 14th day after the first injection,too. Mice were bled from the retro-orbital plexus just at 7,14,21, days after Booster injections.The samples were taken to obtain plasma for analyzing the IgE by ELISA and PCA.Results:1 Protein content in medicine used to induce antibodies synthesis was estimated according the method of Greg. Protein profile of QKL was evaluated by polyacrylamide gel electrophoresis. According to the results, The poorest protein fraction extracted by TCA and ethanol has been not exhibits in previous works. nucleic acid was analyzed by gelose gel eelctrophoresis. As observed under the UV, There is lack of nucleic acid in QKL.The range of molecular weight is 1-1.5KD. 2 None of the fractions induced antibodies level increases when mice received QKL and QKL+FCA by subcutaneous route and thus, did not develop allergy. Nonetheless, anti-sera of mice sensitized with OVA by subcutaneous route displayed considerable immunological response while QKL and QKL+FCA did not. IgE level augmented consistently against OVA tested Possible allergenic effects induced by QKL,QKL+FCA and OVA were investigated by PCA in rats and mice to estimate IgE synthesis, respectively. The QKL,QKL+FCA were not able to induce synthesis of IgE by subcutaneous route.However, synthesis of IgE was estimated to occur blue points in animals that reCeived OVA by subcutaneous stimulation. As a rule, the OVA induce immunological response and animals receiving OVA via subcutaneous were sensitive to produce IgE.3 Two important parameters in preparing conjugates are the hapten utilized ratio and the molar ratio between hapten and carrier protein. The former is known to estimate the efficiency of hapten utilization, which assists in the choice of a suitable initial molar ratio; the latter play an important role, with regard to the degree of immune reactivity, solubility of the final conjugate, and the sensitivity of the immunoassay based on such antibodies elicited by the corresponding conjugate. In the current study,2,4,6,10 mg of Qingkailing were mixed with 2mg of cBSA in conjugation buffer to prepare the conjugates. In summary, QKL coupled with cationized protein cBSA has been demonstrated by UV-Vis. The maximum absorbing wavelength in cBSA UV-Vis spectra, which are characteristic of protein, were slightly blue-shifted from 280 to 277 nm compared with BSA. This might be explained in terms of increased cBSA polarity due to induction of excessive cationic groups, amino-ethylaminegroups, into BSA. The spectra of QKL-cBSA conjugates showed a broad characteristic absorption band around 313 nm, attributed to QKL, and another band around 277 nm, indicating a combination band of 277 nm and 313 nm due to QKL and cBSA, respectively, but it was interesting that the absorbency of the ratio of 1:2 is higher than others When the initial molar ratio of QKL to cBSA was increased, there was a marked precipitate for the conjugate molar ratio to increase, This implies that the best molar ratio is 1:24 Both the total IgE and specific IgE levels of mice which stimulated with QKL, QKL+FCA, and cBSA shown that sensitizer do not make the mice allergy. But the total IgE and histamine of cBSA-QKL conjugate group which had statistically significant effect vs physiological saline group (P<0.05). According to the results, the conjugates of cBSA-QKL can provoke allergy by subcutaneous route.Conclusion:QKL is composed of salts and many other low molecular substances some of such already described. The very low concentration of such molecules in QKL could certainly explain absence of detectable immunological response. Using a method based on Mannich-type principles. The coupling effects were investigated with different initial molar ratios of QKL to cBSA. The conjugate molar ratio was 1:2 The cationized proteins and their conjugates were identified by UV-Vis spectra, which showed the characteristic bands of cBSA and QKL, respectively. After BALB/c mice were immunized with QKL-cBSA, a quicker immunological response and a similar sensitivity of antisera against QKL were observed, compared with immunization by QKL and QKL+_adjuvant. This suggests that the Mannich-type reaction might be an alternative method of preparation for QKL-cBSA. As a rule, the conjugate induce immunological response and animals receiving conjugate samples via subcutaneous were more sensitive to produce IgE and histamine... |
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