HPLC Separation And Purification Of Peptide Toxin And It's Antitumor Effect Of Amanita Subjunquillea.S.lmai

The biology of Amanita subjunquillea.S.lmai from Chang bai mountains was studied by fieldobservation, fruitbody'shape observation and different from temperation PDA culture medium. The majorobjevtive, peptide toxins were separated and purificated by HPLC (high pressure liquid chromatography),furthermore it's antitutor effect of A. subjunquillea. S. lmai.The biology characters showed that it occurried in August and September, and the basidiocarp wascharacteried by its yellow color, a apicalobvious annulus and a firm volva. The detail description of A.subjunquillea. S. lmai. was the same Yang zhu liang's description. The mycelium grew extremely showly indifferent temperation PDA culture medium.Using HPLC, it was researched methyl alcohol crude extracts of A. subjunquillea.S.lmai from indifferent dry condtion, at the same time eparated and purificated of methyl alcohol crude extract. Twokinds of eluents A and B. The eluent A: 10% Acetonitrile+90% 0.02M Ammonium Acetate, AmmoniumAcetate adjusts the pH value to 5.0; The eluent B: 30% Acetonitrile+70%0.02M Ammonium Acetate,Ammonium Acetate adjusts the pH value to 5.0. The elution gradient : 0→15min,B 0→5%, A100%→95%; 15→35min,B 5→80%, A 95%→20%,; 35→40min,B 80%, A.20%; 40～45minB.80→100%, A 20→0%; 45～50minB.100%; 50→55min,B 100→0%, A 0→100% and maintains10min.The results indicated that (1) the toxins content were same in drying in the shade with thefreeze-drying fruitbodies, the dry method was not affect its toxins content. (2) it obtained to 9 kinds ofcompounds, they were determined by high pressure liquid chromatography(HPLC), mass spectra (MS) andnuclear magnetic resonance (NMR) identified 6 kinds of compounds. The compounds 1～6:β-Amanitin(β-AMA),α-Amanitin(α-AMA),phallisacin (PSC),phallisin(PHS),phallacidin(PCD) andphalloidin(PHD).As the content of compound7, compound 8 and compound 9 were all to little. It obtainedthe molecular weight both are only 131,The compound 9 obtained the molecular weight587.They are notthe 22 kind of peptide toxins.By SY Shun's method, two kinds for the medicine ways were fill stomach and inject abdominalcavity, methyl alcohol crude extract carry on the acute toxicity test. The result showed take fill the stomachto the medicine, the measurement as 600mg/kg, observed two weeks not to the death, their right eyeablepsia. Take injection the abdominal cavity to the medicine, obtained lethal does (LD₅₀) was 65.4mg/kg.Two kinds for the medicine way discovered that the thing enable raises the Kunming mouse's liver thicklyto have the fat stack. The effects of phallisacin (PSC) and phalloidin(PHD) on cellular proliferation was proved with theMTT assay..All data were use the SPSS14.0 statistics software to carry on processing. The result indicatedthat all they could not affect the growth of SGC cell and SMMC-7721 cell.Inhibitory effects of the methyl alcohol crude extract and the compound 6(phalloidin,PHD) on thegrowth of H₂₂ tumor cell lines in Kunming mice were detected. All data were use the SPSS14.0 statisticssoftware to carry on processing, take inject abdominal cavity to the medicine. The result indicated that themethyl alcohol crude extract 10mg/kg/d and 20mg/kg/d had the obvious inhibitory action on on H₂₂ mice.The inhibition rate of the methyl alcohol crude extract (10mg/kg/då’Œ20mg/kg/d) were 41.30% and 43.48%in a concentration-dependent manner, and was not remarkable compared with the control group. Itaffected the body weight obvious increase compared with 5-FU group. Through the thymus and the spleenindex were obvious increases heavily, it did affect the immune organ in H₂₂ mice markedly. The compound6 had the obvious inhibitory action on on H₂₂ mice, concentration-dependent manner, the inhibition rate ofthe different medicine dosage (0.15mg/kg/d and0.3 mg/kg/d ) were 53.03% and 65.15% in aconcentration-dependent manner. It affected the body weight obvious increase compared with 5-FU group.Through the thymus index was obvious increases heavily compared with control group and 5-FU, it didaffect the immune organ in H₂₂ mice markedly. But the dissection had not discovered the organ has changesthe black phenomenon obviously, explained it was small to the immune system negative influence.In all, A.subjunquillea.S.lmai' methyl alcohol crude extract were research by high pressure liquidchromatography (HPLC), .separation and purification obtained to 9 kind of compounds, identified 6 kind ofcompounds. The compounds 1～6:β-Amanitin(β-AMA),α-Amanitin(α-AMA),phallisacin (PSC),phallisin(PHS),phallacidin(PCD) and phalloidin(PHD). The methyl alcohol crude extract was takeinjection the abdominal cavity to the medicine, obtained lethal does (LD₅₀) between 65.4mg/kg by SYShun's method. In vitro in vivo anti-tumor experiment, all data were use the SPSS14.0 statistics software tocarry on processing. The result indicated phallisacin (PSC) and phalloidin(PHD) could not affect thegrowth of SGC cell and SMMC-7721 cell. The methyl alcohol crude extract and phalloidin(PHD) had theobvious inhibitory action on on H₂₂ mice, the inhibition rate of the methyl alcohol crude extract andphalloidin(PHD) were 43.48% and 65.15%.