Study On The Preparation Process Of ILPTM And Its Immunomodulatory And Safety

ILPTM contains two components: longan polysaccharide (LYP) andGui-Yuan-Yi-Qi-Bu-Xue-Tang (QXXP). Gui-Yuan-Yi-Qi-Bu-Xue-Tang, alsocalled Guiyuanyiqibuxue decoction (composed of five herbs, i.e., ArillusLongan, Radix Astragali, Radix Angelica sinensis, Rhizoma Dioscoreae andRadix Glycyrrhizae) is a traditional Chinese medicine, which has been used topromote blood metabolism, increase the immunomodulatory, etc. Longanpolysaccharide is a class of macromolecular substance that recognizes a broadspectrum of biological response modifiers existing widely in organism.Therefore, it is significant to research the synergistic effect of combinedtreatment with QXXP and LYP.Objective: To study on preparation technology and quality control of QXXP,and to evaluate the immunomodulatory and toxicological safety of ILPTM,which will provide scientific evidence for health food registration.Methods:1. Orthogonal design was used to optimize the decoction conditions ofQXXP，with the content of multi-target ingredients such as astragaloside，ferulicacid，the yield of dry extractum and the yield of polysacchride. Three factorswere investigated including the amount of the water，decoction time and decoction times.2. Radix Astragali, Radix Angelica sinensis and Radix Glycyrrhizae wereidentified by TLC. The content of astragaloside IV and ferulic acid weredetermined by HPLC. Adopting HPLC-DAD to establish the fingerprint ofQXXP.3. Crude longan polysaccharides were obtained with the water extraction andalcohol precipitation method. The proteins in the extract were removed by themethod of papain hydrolytic. Crude polysaccharides were purified byDEAE-cellulose, and LYP was obtained. The polysaccharides content wasdetermined by phenol-sulfuric acid method.4. We investigated the synergistic immunostimulating activity of combinedtreatment with QXXP and LYP in cyclophosphamide-treated mice.5. We investigated the synergistic immunostimulating activity of combinedtreatment with QXXP and LYP on the function of immunocyte in mice.6. We evaluated the immunomodulatory and toxicological safety of ILPTMaccording to health food registration regulation of Ministry of Public Health.Results:1. The optimum decoction conditions of QXXP were as follow：decocted2times with12fold water，36minutes each time.2. The identification of TLC was exclusive, and spots of Radix Astragali,Radix Angelica sinensis and Radix Glycyrrhizae were clear and well separatedwithout interference in the negative reference. Ferulic acid and astragaloside IVshowed a good linearity (r>0.999) in a determination range．The HPLCfingerprints of QXXP was established in the research, and the similarity of10batches of QXXP was larger than0.9. There were23common peaks in the fingerprint of10samples, and peak8was ferulic acid.3. The yield of longan polysaccharides was7.62%which obtained by waterextraction and alcohol precipitation method.4. In the study of enhancement of immunity in the immunosuppressive miceby different dosage of QXXP, the effect of immunomodulatory was: QXXP(36g/kg)> QXXP(18g/kg)> QXXP(9g/kg)> QXXP(72g/kg).5. The present study had demonstrated that the combination of QXXP andLYP acts synergistically in enhancing the immunostimulating effect andregulating INF-γ/IL-4ratio in cyclophosphamide-induced immunosuppressedmice.6. QXXP and LYP significantly increased Con A-induced spleen cellproliferation, increased the phagocytosis of mouse peritoneal macrophages andenhance the production of cytokines (TNF-α, IL-6, IFN-β, IL-12p40and IL-1β)and NO release compared with the normal control (p <0.05).7. The results of humoral immunity test, natural-killer(NK) cell test, cellularimmunity test and monocyte-macrophage cell function test are positive.8. The oral MTD in both male and female mice were more than20g/kg BW,which showed that ILPTM was nonvenomous. The result of Ames test,micronucleus test of born marrow in mice, sperm shape abnormality in micewere negative, which indicated ILPTM had no genetic toxicity. The results ofthe30days feeding test were negative. ILPTM showed no significantly toxiceffects on body weight, food consumption, food availability, organ/body weightratio, haematology parameters, blood biochemistries and histological pathologyin rats compared with the normal control.Conclusion: 1. The optimum decoction conditions of QXXP is stable and reproducible.2. TLC and HPLC are simple, feasible and reproducible. They can be usedfor the quality control of QXXP．3. The methods of QXXP’fingerprint meet the requirement of the quality,which include stability, precision and repeatability.4. The most effective dosage of QXXP was36g/kg.5. Combination of QXXP and LYP acts synergistically in enhancing theimmunostimulating effect, balance the ratio of Th1/Th2cytokines, and actedmore effectively than QXXP in some aspects.6. Our present researches indicate that QXXP and LYP can improve theimmune function of immunocyte in mice and their immunomodulatorymechanism maybe via active NF-κB signaling.7. The results indicate that ILPTM has immunomodulatory and no toxicity,and it might be useful for developing safe health food.