Study On The Isolation And Mechanism Of The Components In Ligustri Lucidi Fructus With Immune-enhancing Activity

Ligustri Lucidi Fructus（LLF）is the dry and mature fruit of Ligustrum lucidum Ail.It is sweet and bitter in flavor,cool in nature and attributes to the liver and kidney meridians.Its main effects are to invigorate liver and kidney,strengthen muscles and bones,and improve the eyesight.It can be used to cure fever due to the deficiency of Yin,weakness of the waist and knees,the deficiency of kidney manifested as spermatorrhea,failure of eyesight,etc.The results showed that LLF mainly contained oleanolic acid,ursolic acid,specnuezhenide,nuzhenide and other chemical components,which had the effects of immune regulation,liver protection,anti-inflammatory,antibacterial and antiviral.However,most of the studies on immunoregulation of LLF were still on the preliminary exploration of its activity,and there is no systematic study on the main components of its immune-enhancing activity,and the research on its mechanism is even less.The purposes of this study are to detect the immune-active polar parts from LLF,isolate,purify and identify the immune-enhancing chemical constituents from LLF extracts and explore the immune-enhancing activity and mechanism of the active components of LLF on macrophages and lymphocytes.The following results have been achieved so far:（1）Detection of polar parts from LLF with immune-enhancing activity.The five polar fractions of LLF were prepared by ethanol extraction and liquid-liquid extraction.The immune activities of the five fractions were detected by the phagocytic activity of RAW264.7 macrophages and proliferation activity of spleen lymphocytes.The results showed that the yields of petroleum ether（LLF-Pe）,chloroform（LLF-CH）,ethyl acetate（LLF-Ea）,n-butanol（LLF-Bu）and aqueous（LLF-Aq）were 1.64%,1.28%,1.13%,11.15% and 26.96% respectively.Moreover,the immune activities of LLF-Bu and LLF-Ea were better than those of other parts.Therefore,LLF-Bu and LLF-Ea were proved as the major extracts with immune activity.（2）Isolation,purification and structure identification of compounds from LLF with immune-enhancing activity.The LLF-Bu was isolated and purified by macroporous resin,silica gel,Sephadex LH-20 column chromatography and preparative thin layer chromatography.The structures of the compounds were identified by ¹H-NMR,¹³C-NMR and HR-ESI-MS.The immunological activities of the alcohol fractions and the compounds were detected by the phagocytic activity of RAW264.7 macrophages and proliferation activity of spleen lymphocytes.The results showed that the yields of different ethanol extracts of LLF-Bu named LLF-Bu-Aq,LLF-Bu-30%,LLF-Bu-50%,LLF-Bu-70% and LLF-Bu-90% were 8.54%,25.62%,58.34%,1.42% and 1.07%.Immunoactivity experiments showed that LLF-Bu-30%had the strongest activity in promoting macrophage phagocytosis and lymphocyte proliferation.Ten compounds namely oleuropeinic acid（1）,nuezhenide（2）,isonuezhenide（3）,salidroside（4）,isoligustrosidic acid（5）,ligulucidumoside A（6）,8（E）-nuezhenide（7）,hydroxytyrosol（8）,oleuropein（9）,and p-hydroxyphenethyl 7-β-D-glucosideelenolic acid ester（10）were isolated and identified from LLF-Bu-30%.Immunoactivity experiment showed that hydroxytyrosol had the strong activity in promoting macrophage phagocytosis and lymphocyte proliferation.Other compounds oleuropeinic acid,salidroside,8（E）-nuezhenide,oleuropein,and p-hydroxyphenethyl 7-β-D-glucosideelenolic acid ester could also enhance the immune cell activity.（3）Effect and mechanism of hydroxytyrosol in LLF on enhancing immune activity of macrophages.The antigen uptake ability of RAW264.7 macrophages was observed by fluorescence microscope.The secretion of cytokines was detected by ELISA.The release of nitric oxide（NO）was detected by Griess.The production of reactive oxygen species（ROS）was detected by fluorescence microscope and flow cytometry.The expressions of cell surface molecules were detected by flow cytometry.The expression of NF-κB and TLR4 signaling pathway related target proteins was detected by Western blot.The results showed that hydroxytyrosol（HT）can promote macrophage activation by enhancing phagocytic activity,antigen uptake,cytokine secretion,NO release and ROS production of RAW264.7 macrophages.In addition,HT can promote the activation of RAW264.7 macrophages by up regulating the expression of MHC-Ⅱ,CD80 and CD86.The activation of RAW264.7 macrophages by HT could through up regulating the expression of TLR4/My D88（TRIF）/IKKβpathway,and then activating the NF-κB signaling pathway,resulting in the increase of NF-κB p65 expression in the nucleus and the decrease of IκB-αexpression in the cytoplasm,thus realizing the regulation of macrophage immune activity.（4）Effect and mechanism of hydroxytyrosol in LLF on enhancing immune activity of lymphocytes.The secretion of cytokines and immunoglobulin was detected by ELISA,the proportion of cell subsets was detected by flow cytometry,the concentration of intracellular Ca²⁺was detected by fluorescence microscope and flow cytometry,and the activity of calcineurin was detected by microplate method.The expression of NFAT and NF-κB signaling pathway related target proteins were detected by Western blot.The results showed that hydroxytyrosol（HT）could significantly increase the secretion of IL-4,IFN-γand TNF-α,and increase the ratio of CD3⁺and CD4⁺/CD8⁺in lymphocyte subsets.In addition,HT could significantly up regulate intracellular Ca²⁺concentration,increase the activity of calcineurin,significantly up regulate the expression of NFAT and NF-κB p65 in the nucleus and down regulate the expression of NFAT and IκB-αin the cytoplasm,which indicated that HT could activate splenic lymphocytes by promoting the cascade reaction of Ca²⁺/calcineurin/NFAT and NF-κB signaling pathway.In conclusion,this study showed that LLF-Bu and LLF-Ea were proved as the major extracts with immune-enhancing activity,and several compounds with immune-enhancing activity were isolated and screened,among which the activity of hydroxytyrosol was the best.Further studies revealed that hydroxytyrosol could enhance the immune activity of macrophages and lymphocytes by affecting the transmission of cell signal pathway and then inducing NF-κB p65 and NFAT to enter the nucleus.Our research provided a theoretical basis for the development of LLF as an immune enhancer,and provided ideas for the development of Traditional Chinese Veterinary Medicine as feed additives that can replace antibiotics.