| Tea(Camellia sinensis L.),an important crop has been cultivated in Asian countries such as China,Japan,India,and Thailand for more than 5,000 years.For a long time,people have been using leaves of tea to make tea drink,but little attention has been paid to the flowers of tea plant,causing a waste of rich resources.In the past two decades,it was discovered that tea flowers have similar functional molecules to tea leaves,and thus showed various biological activities such as antioxidant,anti-inflammatory,anti-tumor,anti-cancer and immunostimulation.In 2013,tea flower was listed as a new resource food by the Ministry of Health of China,thereby the research value of tea flowers was established.Polysaccharide is one of the most representative active ingredients in tea flowers.Studies have found that tea flower polysaccharides(TFPS)have the ability to resist tumors and stimulate immunity,but the mechanism of their action is still unknown.Indeed,most polysaccharides with immunoregulatory functions are indigestible and are involved in shaping and modulating the organism's immune response through gut microbes.Additionally,short-chain fatty acids(SCFAs),common gut microbiota metabolites from polysaccharide,are one of the critical ways for polysaccharides to participate in immune regulation.Therefore,gut microbiota might be the main target for polysaccharides to exert immunomodulatory effects.In this thesis,the modulation effect of tea flower aqueous extract(TFE)on immunoreactions and gut microbiota was explored in cyclophosphamide(Cy)-induced immunosuppressed mice,and then TFPS was isolated and purified and the immunoregulatory activity of purified polysaccharide fractions were identified with cell model in vitro.Furthermore,the digestibility of the purified polysaccharide fraction with the relatively optimal immunomodulatory activity was probed in saliva,simulated gastrointestinal conditions in vitro and its effect on gut microbiota were also studied.Based on in vitro explorations,Cy-induced immunosuppressive mice were used as model to investigate the effects and mechanisms of TFPS fraction on immunoreactions.This work is of great significance in identification of active ingredients contributing to immunomodulatory property of TFE and elucidating of the mechanism of immnuno-regulation effect of TFPS fraction.The main findings are as follows:1.Effects of TFE on immune response and gut microbiota in miceTea flowers are extracted by hot water and lyophilized to obtain TFE.TFE was mainly composed of carbohydrates,phenolic compounds,proteins,amino acids,saponins and some mineral elements.The effects of TFE intervention on immune response and gut microbiota were studied with Cy-induced immunosuppressed mice.The results showed that TFE at a dose of 200 mg/kg·BW/d for 10 days could effectively alleviate the immunodeficiency,intestinal barrier damage,microecological imbalance and liver peroxidation caused by Cy in mice.Spearman correlation analysis revealed that the effects of TFE on the improvement of intestinal barrier damage,immunosuppression and oxidative stress were related to its role in regulating the gut microbiota.2.Isolation,purification,physicochemical properties and in vitro immunomodulatory activity of polysaccharides from tea flowerTFPS was obtained and firstly separated by a 0.1 ?m ultrafiltration membrane to obtain a retentate,namely TFPS-1,and a permeate,namely TFPS-2.These two components were then separated by a DEAE-Sepharose Fast Flow anion exchange chromatography column to obtain three components,namely TFPS-1-1,TFPS-1-2,TFPS-1-3,TFPS-2-1,TFPS-2-2 and TFPS-2-3,respectively.The main components of which are TFPS-1-2,TFPS-1-3,TFPS-2-2 and TFPS-2-3 were further purified by Sephadex G-100 gel chromatography column to obtain six purified components TFPS-1-2a,TFPS-1-2b,TFPS-1-3p,TFPS-2-2a,TFPS-2-2b and TFPS-2-3p.The protein and sulfate content of purified components decreased,and the monosaccharide composition changed from complex to relatively simple due to the purification,It was found that both TFPS-1-2a,TFPS-1-2b,TFPS-1-3p,TFPS-2-2a,TFPS-2-2b and TFPS-2-3p can effectively promote the proliferation of RAW264.7 cells,improve their phagocytosis and acid phosphatase activity,and promote their release of immune effector molecules nitric oxide(NO)and cytokines including tumor necrosis factor(TNF)-?,interleukin(IL)-1? and IL-6(indicating their immune-promoting function in vitro.In terms of the production amount of NO,TNF-? and IL-6 at 500 ?g/mL,the promoting effect of TFPS-1-3p was significantly higher than other components.3.Digestibility of TPFS-1-3p and its effect on gut microbiota in vitroThe digestive characteristics of TPFS-1-3p in the oral cavity,stomach,small intestine and large intestine were explored by simulating digestion and anaerobic fermentation system in vitro.Meanwhile,the effect of TPFS-1-3p on gut microbiota in vitro were studied by high-throughput sequencing technology.The results showed that TPFS-1-3p could not be degraded by saliva,simulated gastric juice and small intestinal fluid,however could be degraded by gut microbiota in the large intestine to promote the formation of SCFAs.TPFS-1-3p could modulate the composition of gut microbiota and significantly increase the relative abundance of Bacteroides thetaiotaomicron,Bacteroides plebeius and Collinsella aerofaciens at the species level,which may be related to its own degradation.4.Study on the immunomodulatory effect and mechanism of TPFS-1-3pThe effect and mechanism of TFPS-1-3p intervention on the immunoreactions were studied in Cy-induced immunosuppressed mice.The results show that TFPS-1-3p could effectively alleviate the decrease of Cy-induced body weight and immune organ index,and increase the levels of serum TNF-?,IFN-?,IL-1?,IL-2 and IL-6.Improving intestinal health is a key way for TFPS-1-3p to regulate the host's immune response.Firstly,TFPS-1-3p improved the intestinal barrier and activated TLR4/MyD88/NF-?B p65 and JAK2/STAT3 pathway in colonic tissues at the mRNA level.Secondly,TFPS-1-3p regulated gut microbiota and its metabolites,SCFAs,in response to the intestinal immune system.Especially,TFPS-1-3p intervention could reverse 62 of 68 disorder genera and in 17 of 20 altered bacterial function KEGG pathways in Cy-induced immunosuppressed mice.Spearman correlation analysis showed that the immunomodulatory effect of TFPS-1-3p was significantly or extremely significantly related to certain gut microorganisms.Taken together,both TFE and TFPS-1-3p have the effect of regulating immunoreactions and gut microbiota.Spearman correlation analysis revealed that gut microorganisms responding to immune enhancement after TFPS-1-3p intervention were consistent with changes in response to that of TFE,to some extent,reflecting that the regulation effect of TFE on gut microbiota may be due to the polysaccharide component. |
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