Studies On The Serial Chemical Composition And Bioactivities Of Mangrove Plant Rhizophora Stylosa

Mangrove plants are higher woody plants especially growing on the tidal flat of tropical and sub-tropical mangrove.They are one of rare or endangered species in the world.Mangrove plants not only have great ornamental value and scientific research value, but also play an important role in defending coastline against the strong wind and big waves,degradating pollution,regulating climate,keeping marine biodiversity,and so on. Many studies on mangroves have been mainly focused on ecology,ecological physiology, plant taxonomy and ecological community.However,there are only a few scattered studies on chemical composition and biological activities of a certain mangrove plant.Because there are some internal connections between what comprises the mangrove plant and the ecosystem environment it grows in.In order to reveal internal laws of chemical composition of mangrove plants to provide a scientific basis for further research on relationship between metabolites and the environment,preserving good ecosystem environment and protection and rational use of mangrove plant resources,the representative mangrove plant of Rhizophora stylosa,collected from Zhanjiang, Guangdong,China,was elected as subject.The contents of moisture,ash,acid infusible ash, sulphide,crude proteins and crude fats,trace elements,protein amino acids,the Maillard reaction products from the hydrolyzed amino acids,volatile oils,fatty acids, ploysaccharides and flavones were more systemically determined and analyzed. Meanwhile,inhibitory effect and anti-tumor activity of flavonoids extract in Rhizophora stylosa leaves and anti-tumor activity of the complex of Cu²⁺ and fusarium wilt toxins from blasted leaves of Rhizophora stylosa were studied.Main studies and conclusions summarized as follows:1.The basic chemical composition of leaves from Rhizophora stylosa were studied. The percentage contents of moisture,ash,acid infusible ash,sulphide,crude proteins and crude fats in leaves of Rhizophora stylosa were 69.18%,3.04%,0.094%,0.052%,7.91% and 7.41%respectively.Each of them is different from some terrestrial plants in varying degrees.2.Thirteen elements of A1,As,Ba,Cd,Cr,Cu,Fe,Mn,Ni,Pb,Sr,Ti and Zn of the leaves and fruits of Rhizophora stylosa were analyzed by ICP-AES.And the contents of the elements of them were determined.Zn,Fe,Cu and Zn,Fe,Cr are the comparatively rich elements in the leaves and fruits of Rhizophora stylosa respectively.The content of Zn, amounting to 183.0 mg/kg in the leaves and 218.4 mg/kg in the fruits,is the highest among the trace elements determined.The content of zinc in both of them are far more than common oil-bearing crops.3.Seventeen protein amino acids in leaves of Rhizophora stylosa with total content of 10.7g/kg were determined by HPLC,seven among which are essential amino acids, amounting to 25.75%of the total amino acids.They are Thr,Val,Met,Ile,Leu,Phe and Lys.Asp,amounting to 2.69 g/kg,was determined as the optimum amino acid.Forteen protein amino acids in fruits of Rhizophora stylosa with total content of 149g/kg were determined by HPIC-IPAD,six among which are essential amino acids,amounting to 46.15%of the total amino acids.And eight among which are unessential amino acids.The content of Glu,Phe,Arg and Val,amounting to 36.56g/kg,34.42g/kg,22.33g/kg and 15.47g/kg respectively were determined as the optimum amino acids.4.The Maillard reaction products were obtained from the reaction of complex amino acids from leaves of Rhizophora stylosa hydrolysised by concentrated hydrochloric acid and glucose and sucrose for six hours under the temperature between degree centigrade 105 and 108 respectively.The ether-soluble materials from the reaction product were extracted by ethyl ether and determined by GC-MS.There are some different components in both of the nine components.Both products have aromatic substances.The results presented that Maillard reactions can take place by sucrose without reductive character.5.The volatile oils from the leaves and fruits of mangrove plant Rhizophora stylosa were extracted by steam distillation and analyzed by GC-MS.Thirty-one volatile compounds in forty one separated from leaves of Rhizophora stylosa were identified, which accounted for 83.62%of the total volatile oil.BHT was determined as the optimum composition.2-Hydroxy-4-methylbenzoate,hentetracontyl alcohol and 2,6,10,15,19,23-hexamethyl tetracosane were discovered for the first time in the volatile oils from the leaves of Rhizophora stylosa.And all the volatile compositions separated from fruits of Rhizophora stylosa were identified,among which,3-Methoxy-1,2-propanediol,taking the proportion of 26.39%,was determined as the optimum composition.The six compounds of N,N'-di-benzoyloxy heptanediamide,2,3-Dihydro-benzofuran,1H-Imidazole-2-carboxaldehyde, Elemicin,3,7,11-Trimethyl-1,6,10-Dodecatrien-3-ol and 2,8-Dimethyl-4,6-nonanedione were discovered for the first time in the volatile oils from the fruits of mangrove plant Rhizophora stylosa.6.The fatty acids,with the yield of 3.272%and 2.530%respectively,were extracted by Soxhlet extraction from the leaves and the fruits of mangrove plant Rhizophora stylosa. The extracted fatty acids were esterified and analyzed by gas chromatography-mass spectrometry.Twelve fatty acids were detected and identified,amounting to 84.95%of the total fatty acids from the leaves of Rhizophora stylosa.The compounds mainly includ palmitic acid(40.49%),oleic acid(17.24%) and linoleic acid(11.14%).Eight fatty acids, mainly including palmitic acid,8,11,14-eicosatrienoic acid and linoleic acid,respectively amounting to 7.59%,4.25%and 3.22%were identified from the fruits of Rhizophora stylosa.And 8,11,14-eicosatrienoic acid was discovered for the first time.7.The polysaccharide,with the yield of 2%,was extracted from Rhizophpra stylosa leaves.And the polysaccharide extracted was tested and analyzed by IR.The IR spectrum indicates that there are characteristic absorption peaks of polysaccharides.Analyses by complete acid hydrolysis and aldoononitrile acetate derivatives and GC-MS indicated that it is composed of erythrose,D-arabinose and glucose.The results presented that the polysaccharide from Rhizophpra stylosa belongs to heteropolysaccharide.8.The extract of crud flavonoids in Rhizophora stylosa leaves were extracted by ethanol and then separated and purified by extraction,polyamide packed chromatography and crystallization and needle crystal compound separated out.The needle crystal compound was identified structurally by UV,IR,MS and element analyzing combined with physical and chemical property as rutin.The molecular formula of Rhizophora stylosa rutin is C₂₇H₃₀O₁₆ and its molecular weight is 610.5.The total flavonoids content of the crude flavonoids extract was determined as 58.29%by UV-spectrophotometry.9.The in vitro antimicrobial activity experiments of total flavonoids extract from the mangrove plant Rhizophora stylosa were studied by the plate diluting method.The results presented that the total flavonoids extract had varied inhibitory effects on the bacteria and plant pathogenic fungi.The extract was more distinctly sensitive to Escherichia coli, Colletotrichum musae and Fusarium oxysporum f.sp.cubense,and less to Colletotrichum sp.,Rhizotonia solani and Staphylococcus aureus and no to Aspergillus niger.The minimal inhibitory concentrations(MIC) were determined by plate diluting method.10.To evaluate anti-tumor activity of flavonoid extraction from Rhizophora stylosa, the anti-tumor activity of flavonoid extraction from Rhizophora stylosa against human lung cancer cell NCI-H460 were determined by MTT method.The result of the anti-tumor experiment presented that the flavonoid extraction from Rhizophora stylosa showed significant anti-tumor activity on human lung cancer cell NCI-H460 in vitro.11.A single crystal was obtained from the wilt leaves of Rhizophora stylosa by crystal induction method with Cu²⁺.The single crystal's structure information and the crystal data were present by X-Ray difference reflection technology.The single crystal belongs to the triclinic system with the molecular formula of Cu(C₁₀H₁₂NO₂)₂.It's space group is PI and the crystal data presented as follows:a=5.50520(10),(?),b=8.0683(3)(?), c=11.3008(5)(?),α=70.338(3)°,β=89.399(2)°,γ=78.7060(10)°,V=462.70(3)(?)³,Z=1, M_r=419,95,D_x=1.507mg·m^-3,μ=1.020mm^-1,F(000)=219,R=0.040,wR=0.107.It is proved that a ligand of the obtained complex is fusaric acid(C₁₀H₁₃NO₂).Then it is concluded the one of the reasons of the phenomenon is fusaric acid.The result of the anti-tumor experiment presented that the complex of fusaric acid and Cu²⁺ showed significant anti-tumor activity on human hepatoma cells BEL-7402 in vitro.