Studies On The Separation And Pharmacological Activities Of Middle And High Polar Constituents From Camellia Oleifera Cake

Camellia oleifera Abel is a genus of Camellia Linn plant belonging to the family Theaceae,which widely cultivated in the southwest and southeast of China.The seed cake of Camellia oleifera is a by-product of camellia oil extracted from Camellia oleifera Abel.Despite its yield was huge,it was often discarded or used as fertilizer by people,and can not be used comprehensively effectively,resulting in a great waste of resources.In order to make better use of the abundant resources of Camellia oleifera cake adequately in our country and give full play to its valuable value,the Camellia oleifera cake was taken as the research object in this paper.The extraction,isolation,structure identification and pharmacological activity of small molecular compounds and polysaccharide components of Camellia oleifera cake were research.The NF-κB activities of each extracts fraction from Camellia oleifera cake were tested.The separation,purification and activity of n-butanol and water fraction were further studied by activity-guided method.Crude saponins were obtained and nine compounds were separated from the n-butyl layer respectively by means of open column chromatography and HPLC.Including 2 flavone:Tricin（1）and Kaemperol-3-O-α-L-rhamnoside（2）;2oxyneolignans:Nitidanin（3）and（7’E）,（7R,8S）-3’,5’-dimethoxy-3-7-epoxy-8-oxyneolignan-4’,9,9’-triol（4）,5 pentacylic triterpennoids:Camelliagenin B（5）,Camelliagenin E（6）,Oleanolic acid（7）,16β,21β,23α,28-tetrahydroxyolean-12-en-3-one（8）,4α-methyl-21β,22α,28-trihydroxyolean-12-en-3-one（9）.Compound1,3 and 4 were isolated from Camellia oleifera Abel for the first time.Compound 8and 9 are new compounds.The antitumor activity of saponins was preliminary studied.The experimental in vitro results show that the total saponin can inhibit cell proliferation by blocking them in S phase,induce cell apoptosis by improving the intracellular ROS,Ca²⁺concentration,lead mitochondrial dysfunction by reduce mitochondria membrane potential.Total saponin demonstrated potential antitumor activity.The in vitro anti-inflammatory effects of compounds 5-9 were investigated in LPS-stimulated murine macrophage RAW264.7 cells.The results indicated that compound 6 has the best anti-inflammatory activity.OTAPS-1 was isolated from the water extract of Camellia oleifera cake,its average Mw of OTAPS-1 was 4.08×10⁴Da,mainly make up of eight sugars.Its immunomodulative activity and molecular mechanism were studied by establishing a pharmacological experimental model using macrophage RAW264.7 cell.OTAPS-1exhibits the ability to enhance immunostimulatory activities obviously as it could activate macrophages and enhance phagocytosis of cells,inducing the release of toxic molecules（NO and ROS）,the secretion of cytokines（IL-6,IL-1βand TNF-α）in concentration-dependent manner.Meanwhile,the IL-6,NO and TNF-αm RNA expression can thus be upgraded by OTAPS-1 through the activation of signal protein in MAPKs,AKT and NF-κB via intracellular receptors TLR2,TLR4 and CR3 in RAW264.7 murine macrophage in vitro.In this paper,the separation,structure identification and pharmacological activity of n-butanol parts and chemical components of water extract of Camellia oleifera cake were studied in order to provide some scientific theoretical guidance and experimental basis for the in-depth development and rational utilization of Camellia oleifera Abel.