| Styela plicata (lesueur) is an ascidian which belongs to class Ascidiacea, subphylum urochordata .More than 1500 kinds of ascidians have been known in the nature and they mainly distribute over the tropical zone and subtropical zone. There are abundant ascidian resources in the South China Sea. Studying on bioactivity components from ascidian is a popular domain in marine drugs research during these years. Many chemical compositions such as Alkaloids, peptide benzopyrrole, heavy metal chelating reagent, polysulfide, macrolide, terpenoid, and so on, have been found in ascidian. And most of them have strong biological activity, such as anti-tumor, anti-virus, antibiosis, inducing sarcoplasmic reticulum to dispel calcium,etc.At present ascidian research is concentrated on anti-tumor and anti-virus compositions separation, synthesis and reconstruction. But there are few reports about anti-HBV compositions from ascidian. In this report, 2.2.15 cell transfected HBV is used as screening model in vitro to find anti-HBV compositions from Styela plicata (lesueur). And HPLC finger printing of anti-HBV effective fraction from Styela plicata has been established. This report has three parts.1 Separation and identification of anti-HBV compositions from Styela plicataFresh Styela plicata was soaked in 95% alcohol and extractum was obtained by evaporating the solvent. The extractum was resolved in water and the water solution was extracted by equal dichlormethane, n-butanol. The water extraction was separated by macroporous resin chromatography, gel silica chromatography, andSephadex LH20 chromatography. Two pure substances were obtained and identified as polypeptide by qualitation essay. Their molecular mass was identified by MALDI-TOF MS and their ammo acid sequences were identified by EDMAN degradation.Sub 1: Its molecular mass was 711.4 assayed by MALDI-TOF MS and the amino acid sequences were identified as Lys-Gly-Lys-Ile-Glu-His by EDMAN degradation. Its theory molecular mass was 710.83 and it was the same as the trial molecular mass.Sub 2: Its molecular mass was 663.3 assayed by MALDI-TOF MS and the amino acid sequences were identified as Ser-Ser-Leu-Ser-Ala-Ala by EDMAN degradation. Its theory molecular mass was 662.74 and it was the same as the trial molecular mass.2 Anti-HBV activity assay of different fractions of Styela plicata2.2.15 cell is the general model in anti-HBV drug screening in vitro. Different fractions of Styela plicata were merged in 2.2.15 cell. Pharmacodynamic effect was measured by assaying tite of HBsAg and HBeAg in cell culture and toxicity was determined by MTT. And these two indexes could be used to screening anti-HBV drugs.According to the pharmacodynamic results, the fractions of Styela plicata,such as A(crude alcohol extract), C(water fraction after extracted), F(10% alcohol part after macroporous resin) , G(30% alcohol part after macroporous resin), H(50% alcohol part after macroporous resin), 1(70% alcohol part after macroporous resin), J(95% alcohol part after macroporous resin),K(Sub l),L(Sub 2) and positive control drug Lamivudine were shown to have inhibit HBsAg and HBeAg secretion. And other fractions,such as B(dichlormethane fraction), D(n-butanol fraction), E(water part after macroporous resin) had no anti-HBV effect. By analyzing the experiment result, we know anti-HBV active constitutents of Styela plicata concentrated on water-solubility fractions. And part H(50% alcohol part after macroporous resin) was shown to have the strongest anti-HBV effect. By separating this part, we have obtained two pure substances which have strong anti-HBV effect.From the MTT data, we know all fractions of Styela plicata have no cytotoxicity. IC50(HBsAg) of Subl was 3.65mg/ml and Sub 2 was 3.28mg/ml andof Subl was 4.40mg/ml and Sub 2 was 4.48mg/rnl, respectively. They were shown some toxicity in high concentration and their TC50 were more than 64mg/ml. Their TI(HBsAg) were more than 17.53and 19.51,and TI(HBeAg) were more than 14.54 and 14.29,respectively. On the basis of their TI, these two substances can be presumed to have inhibitory effect on HBsAg and HBeAg expression in 2.2.15 cell and have little cytotoxicity.3 Study on finger printing of anti-HBV effective fraction of Styela plicata HPLC technology was used to establish finger printing of anti-HBV effective fraction of Styela plicata. This method was on the base of effective pure substance separated from Styela plicata. And anti-HBV effective fraction of Styela plicata was the quality control target. Linear gradient elution was used in this experiment and the chromatographic condition could be controlled easily. Every peak was separable and the result had good precision and repeatability.Ten chromatographic peaks of this anti-HBV effective fraction could be detected on this chromatographic condition. In the figure, chromatographic peak S which was regarded as reference peak was the peak of Sub 1 and peak 5 was the peak of Sub2. Contrasting to this reference peak, relative retention time and peak area ratio could be calculated. Relative retention time of ten peaks was rather stable in HPLC figures. The average relative retention time of seven samples was 0.359(l),0.680(2), 0.857(3),0.911(4),1.000(S),2.284(5),2.774(6),2.980(7),3.241(8),4.927(9),respectively. And relative deviation was less than 3%. The average relative retention time could be considered as a quality identification index. Main HPLC peak areas of seven samples were 29198.97, 29210.75, 29243.47, 29244.18, 29190.55, 29094.68, 29240.23 and average peak area was 29203.26. The relative deviation between main HPLC peak areas of different sample and average peak area could also be calculated. The absolute amount of effective fraction could be measured by main HPLC peak areas and the relative amount of effective fraction in different samples could be measured by the relative deviation. The sample which had small relative amount could be abandoned and ten general peaks of the effective fraction could be definite. As key feature peaks in finger printing, they could be used to identify the effective fraction of Styela plicata. |
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