Bufothionine

Abstract: Objective To kinds of establish a HPLC method for determining contents of indole alkaloids and bufadienolides contained in toad medicines, and analyze two kinds of components contained in toad venom, toad skin and toad periostracum. Method As for alkaloids, Nucleosil C18 column was adopted with acetonitrile and water containing 0.5% potassium dihydrogen phosphate (6: 94, adjust pH to 3.2 with phosphate acid) as the mobile phase. The flow rate was 0.8 mL x min(-1), the detection wavelength was 275 nm, and the column temperature was 30 degrees C. As for bufadienolides, Alltima C18 column was adopted with acetonitrile and water containing 0.3% acetic acid (B) as the mobile phase. The gradient process was as follows: a linear gradient from 28% to 54% acetonitrile in the first 15 min, then kept at 54% for additional 20 min. The flow rate was 0.6 mL x min(-1), the detection wavelength was 296 nm, and the column temperature was 30 degrees C. Result The linear ranges were 0.079 6-0.796 microg for serotonin, 0.097 2-1.945 microg for N-methylserotonin, 0.074 4-0.744 microg for N,N-dimethylserotonin, 0.103-2.05 microg for N,N,N-trimethylserotonin, and 0.067 2-0.672 microg for bufothionine, respectively. The average recoveries of serotonin and N-methylserotonin were 98.6% and 91.3%, respectively. The linear ranges of gamabufotalin, bufotalin, bufalin, cinobufagin and resibufogenin were 0.004 83-0.614, 0.007 9-1.006, 0.007 95-1.016, 0.009 7-1.24 and 0.009 6-1.22 microg, respectively, and their average recoveries were 101.6%, 102.5%, 101.0%, 99.1% and 98.9%, respectively. Conclusion Toad venom has the highest contents of indole alkaloids and bufadienolides, followed by toad skin, and toad periostracum showed the lowest contents and even no detection result.

Abstract: OBJECTIVE To establish a HPLC method for bufothionine in the skin of Bufo bufo gargarizans and Huachansu injection. METHOD The samples were separated using a Lichrosob C18 column with CH3CN-H2O (10:90) as mobile phase. Flow rate was at 1.0 mL x min (-1) and the detection wavelength was at 225 nm. RESULT The calibration curve of bufothionine was linear over the range of 0.0772-0.4632 microg and the average recovery was 99. 2%. The contents of bufothionine were fluctuated from 36.4-641.8 microg x g(-1) in the skin of Bufo bufo gargarizans and 22.47-33.16 microg x mL(-1) in Huachansu injection, respectively. CONCLUSION The contents of bufothionine were greatly different between cultured and wild species. The method was suitable for the quality control of the skin of Bufo bufo gargarizans and its preparation.

Abstract: Objective: To determine bufothionine in different parts of the dried toad skin from two species.Method: A quantification method was developed by HPLC with photodiode array(PDA) detection.Bufothionine was identified unambiguously by HPLC-DAD 3D and HPLC-MS-MS analysis in comparison with reference standard.The extraction method was optimized as 50 times 50% MeOH ultrasonic 60 min.The analysis was performed on WondsilTM C18 column(4.6 mm×250 mm,5 μm) with ACN-H2O(6∶94) as mobile phase and detection wavelength was at 226 nm.Result: The samples were analyzed with good linear regression relationship(r=0.999 7),precisions(RSD,0.59%),repeatability(RSD,1.56%),stability(RSD,0.73%) and recovery(98.57%;RSD,2.03%).Bufothionine was detected in different parts of toad skin from Bufobufo gargarizans cantor and Bufo melanostictus Schneider.The contents were from 0.07% to 0.17%.Conclusion: The content of bufothionine revealed no significant differences among the different parts in these two species of toad skin.

Abstract: Fifteen compounds were isolated from the toad skin by a combination of various chromatographic methods including macroporous resin, silica gel, ODS and semi-preparative HPLC. Their structures were identified as 4,5-dimethyl-1,3,4,5-tetrahydropyrrolo[4,3,2-de]quinolin-6-ol(1), serotonin(2), N-methyl serotonin(3), O-methyl bufotenine(4), 1,2,3,4-tetrahydro-6-hydroxy-β-carboline(5), O-methylserotonin(6), glycinebetaine(7), caffeine(8), bufotenine(9), shepherdine(10), tryptophan(11), (5-hydroxy-1H-indol-3-yl)acetic acid(12), 5-hydroxy tryptophol(13), 2-methyl-6-hydroxy-1,2,3,4-tetrahydro-β-carboline(14), bufothionine(15). Among them, compound 1 was a new compound,compound 5 was a new natural product. Compounds 4-8 and 10-14 were separated from toad skin for the first time.