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ObjectiveTo develop a quantitative analysis of multi-components by single marker （QAMS） for determination of bufalin， cinobufagin and resibufogenin in Shexiang Baoxin pills， and to provide a method for improving the national standard of the pills. MethodHigh performance liquid chromatography （HPLC） was developed for simultaneous determination of bufalin， cinobufagin and resibufogenin in Shexiang Baoxin pills and the methodology validation was carried out. The chromatographic separation was performed on a Nucleosil 100-5 C18 column （4.6 mm×250 mm， 5 μm） with the mobile phase of acetonitrile -0.1% potassium dihydrogen phosphate aqueous solution （pH adjusted to 3.2 with phosphoric acid） （48∶52）， and the flow rate was 0.6 mL·min-1， the detection wavelength was set at 296 nm and the column temperature was 35 ℃. Taking cinobufagin as the internal standard， the relative correction factors （RCFs） of bufalin and resibufogenin were calculated， and the key influencing factors of RCFs were investigated. Relative retention time was used for the chromatographic peak location of the analyte， combining with the on-line ultraviolet spectroscopy and accurate relative molecular weight obtained by ultra-performance liquid chromatography-quadrupole time-of-flight mass spectrometry （UPLC-QTOF/MS）. The external standard method was used to verify the contents of three components obtained by QAMS. ResultQAMS was established for the determination of bufalin， cinobufagin and resibufogenin in the samples， and RCFs of cinobufagin to bufalin and resibufogenin were 0.922 and 1.01， respectively. The total content of the three marker compounds in 11 batches of Shexiang Baoxin pills was 33.7-36.0 µg per pill. There was no significant difference between the quantitative results of QAMS and external standard method. ConclusionThe established method can be used for the quality control of bufalin， cinobufagin and resibufogenin in Shexiang Baoxin pills. It is suggested that bufalin should be considered as one of three marker compounds， and the sum of bufalin， cinobufagin and resibufogenin should be used for the content limit of this preparation.
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This manuscript aims to investigate the effects of resibufogenin on the proliferation, migration and invasion of human hepatocellular carcinoma cells and its related mechanisms. MTT assay was used to determine the inhibitory effect of resibufogenin on the growth of four hepatocellular carcinoma cells in vitro. Wound-healing assay and Transwell assay were used to evaluate the migration and invasion ability of resibufogenin on MHCC-97H cells. Western blot assay was used to detect the expression of migration and invasion related proteins in MHCC-97H cells treated with different concentrations of resibufogenin. The results showed that resibufogenin significantly inhibited the proliferation of hepatocellular carcinoma cells in vitro. The half maximal inhibitory concentration (IC50) values on MHCC-97H, HepG2, SK-Hep-1 and Huh-7 cells were 0.55 ± 0.06, 2.83 ± 0.24, 5.25 ± 0.49, 14.89 ± 2.28 μmol·L-1, respectively. Resibufogenin also suppressed the migration and invasion of MHCC-97H cells in a concentration-dependent manner. The protein expression of integrin α2, integrin α6, integrin β1, N-cadherin, matrix metalloproteinase 2 (MMP2) and transcription factor Twist in MHCC-97H cells were decreased significantly with the increase of the concentration of resibufogenin, while the protein expression of E-cadherin increased. In addition, we found that p-PI3K/PI3K and p-AKT/AKT ratios were significantly reduced after treatment with resibufogenin. In conclusion, resibufogenin can inhibit the proliferation, migration and invasion of hepatocellular carcinoma MHCC-97H cells in vitro, which is related to the regulation of intracellular migration and invasion protein expression and PI3K/AKT signaling pathway.
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As a new strategy capable of uncovering the characteristics of traditional Chinese medicines, the quantitative analysis of multi-components by single-marker(QAMS) has been widely employed for the quality evaluation of Chinese medicinal materials, slices, and extracts. However, its application in the assessment of Chinese patent medicines is yet to be explored. By referring to the determination of three bufogenins in Bufonis Venenum by QAMS described in Chinese Pharmacopoeia(2020 Edition), this paper selected seven representative preparations containing Bufonis Venenum and explored whether the relative correction factors(RCFs) of cinobufagin(CB) to bufalin(BF) and resibufogenin(RB) could be directly used for the quality control of Bufonis Venenum-contained preparations. Based on the qualitative analyses under the same chromatographic conditions as used for toad venom, combing specificity test, five preparations such as Yatong Yili Pills, Houzheng Pills, Xiongdan Jiuxin Pills, Liushen Pills and Niuhuang Xiaoyan Pills, were expected to use validated RCFs for the direct determination of three components. Taking Houzheng Pills as an example, the methodological validation of bufalin, cinobufagin and resibufogenin was carried out, and the recoveries of bufalin, cinobufagin and resibufogenin were 90.64%-106.1%. The obvious difference was not observed between the contents of bufalin and resibufogenin in 24 batches of preparation samples by QAMS and external reference method. In the tested samples, the content of bufalin, cinobufagin and resibufogenin were 1.27-2.61, 2.44-5.66 and 0.988-3.16 mg·g~(-1) in 10 batches of Liushen Pills samples. The contents of bufalin, cinobufagin and resibufogenin were 0.760-1.32, 1.35-2.39 and 0.600-1.55 mg·g~(-1) in 10 batches of Houzheng Pills samples from three manufacturers. The obtained data contribute to improving the quality standard of Bufonis Venenum-contained preparations, and they also provide some ideas for the application of QAMS in the quality evaluation and control of Chinese patent medicines.
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China , Chromatography, High Pressure Liquid , Medicine, Chinese Traditional , Nonprescription Drugs , Quality ControlABSTRACT
Objective: To optimize the extraction and purification technology of liposoluble constituents from Bufonis Venenum. Methods: The total content of cinobufagin and resibufogenin was taken as the index, and the ethanol potency, solvent multiplication and extraction time were taken as the investigation factors. The optimum extraction process parameters was determined by orthogonal test. Through single factor investigation experiment combined with Box-Behnken response surface method, taking the expansion agent ratio, ratio of diameter to height, adsorbent and sample quantity as the investigation factors, the optimum purification process was selected and determined. Results: It was determined that the optimum extraction process for the addition was 10 times 85% ethanol for 90 minutes’ extraction, the best purification process for the expansion agent cyclohexane-chloroform- acetone was 4:3:3, column height and diameter was 7:1, adsorbent and sample volume was 5.5:1. The content of two kinds of toad poison base purified was up to 66.51%. Through the verification of three batches of amplification process, it was shown that the model fit well, and the separation and purification of toad poison ligand by silicone column chromatography had the advantages of simple operation, fast separation speed, and good separation effect. Conclusion: The selected process is reasonable and feasible, which provides technical reference and basis for the industrialization application of the product in the future.
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Objective: To develop a rapid, simple, sensitive, and accurate high performance liquid chromatography-tandem mass spectrometry (HPLC-MS) method for determining the contents of cinobufagin and resibufogenin from Bufo Corium. Methods: The contetns were determined by Zorbax SB-C18 column and tested in positive electrospray ionization with multiple reaction monitoring of HPLC-MS. The mobile phase was conswasted with the mixture of acetonitril-0.1 mol/L formic acid water solution (41:59), flow rate 200 μL/min, column temperature 30 oC. Mass spectrometry conditions: nitrogen temperature 350 oC, nitrogen flow rate 12 L/min, nebulizer pressure 101.316 kPa (35 psi). The shredded voltage of cinobufagin was 160 V, the collwasion energy was 15, the parent ion and the daughter ion were 443.2 and 364.8 respectively; The fragmentation voltage of resibufogenin was 130 V, the collwasion energy was 15, and the parent ion and the daughter ion were 385.2 and 366.9 respectively. Results: There was good linearity in the range of 0.99-7.92 μg/mL and 1.04-8.32 μg/mL for the cinobufagin and resibufogenin. The detection limit (S/N ≥ 3) was 0.3 ng/g. The recoveries of cinobufagin and resibufogenin ranged from 97.96% to 103.7% and 96.86% to 102.4%, respectively. The intraday and daytime precwasion were both less than 3%. Conclusion: The results showed that the method was sensitive and reliable, which can meet the needs of analyse of toxic substances in B. Corium.
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Objective To study the changes of chemical constituents and pharmacodynamics with different drying methods (sun- drying, 50 ℃ vacuum-drying, 50 ℃ and 80 ℃ heat-drying, freeze drying method) in Bufonis Venenum. Methods HPLC method and TLC method was established for studying the changes of chemical constituents from B. Venenum before and after being dried, and determine the inhibitory effect of dried samples on five different tumor cell lines proliferation by MTT assay. Results The B. Venenum processed by five different methods were different in character, while no significant differences in the types and content of chemical constituents; The total content of resibufogenin and cinobufagin was more than 6%, which was consistent with the 2015 edition of Chinese Pharmacopeia; 50 ℃ and 80 ℃ heat-drying of B. Venenum showed more effective inhibitiory effect than other dry methods. Conclusion The appearance of B. Venenum met the criterion of pharmacoperia by sun-drying and 50 ℃ heat-drying method. Although the color of B. Venenum under the vacuum-drying and freeze drying method did not meet the requirements of Chinese Pharmacopeia, the main effective components have a few changes.
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OBJECTIVE:To study the in vitro uptake of Resibufogenin(RBG)lactic acid glycolic acid copolymer-water solu-ble vitamin E (PLGA-TPGS) in human liver cancer HepG2 cells,mouse ascites-type lymphatic metastasis of tumor HCa-F cells, and the toxicity on HepG2 cells. METHODS:RCPTN loading RBG and coumarin-6(C6)were prepared. Fluorescent inverted mi-croscope was used to observe the in vitro uptake by RCPTN HepG2,HCa-F cells. It was divided into negative control group,blank PLGA-TPGS nanoparticles(EPTN)group,5-fluorouracil solution(FS)group,RBG solution(RS)group,RBG/PLGA nanoparti-cles(RPN)group and RPTN group. WST-1 was conducted to investigate the optical density at 450 nm wavelength of HepG2 cells after 24,48,72 h incubated by FS,RS,RPN and RPTN with different final concentrations (1.25,2.5,5,10,20 μg/mL);the cell viability (CV) and half inhibitory concentration (IC50) were calculated. RESULTS:RCPTN distributed around the nucleus of HepG2,HCa-F cells. CV was decreased by RBG concentration increased in RPN group and RPTN group,and decreased by time prolonged;compared with FS group,CV in RPTN group was decreased(PFS>RPN>RPTN;IC50 incubated by RPN and RPTN for 48,72 h was obviously less than that of FS and RS(P<0.05 or P<0.01). CONCLUSIONS:RPTN can deliver RBG in-to HepG2,HCa-F cells,showing inhibition effect on HepG2 cells which is stronger than RPN,RS and FS.
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OBJECTIVE:To evaluate the quality of solid lipid nanoparticle of the skin extract of Bufobufo gargarzans. METH-ODS:The morphology of solid lipid nanoparticle of the skin extract of B. gargarzans was observed by TEM. The particle size was determined by laser scattering particle size analyzer. The contents of cinobufagin and resibufogenin, encapsulation efficiency, drug-loading amount and accumulative release rate of cinobufagin were determined by HPLC. The stability of nanoparticle was in-vestigated within 24 h at 60,25 and 4 ℃. RESULTS:The solid lipid nanoparticle of the skin extract of B. gargarzans were uni-form in particle size and showed round and spheroidicity shape;average particle size was(138.5±4.2)nm,The encapsulation effi-ciency of cinobufagin and resibufogenin were 90.60% and 91.51%,and drug-loading amount were 35.82% and 44.15%. The accu-mulative release rate of cinobufagin was 50%at 4 h and reached 88%at 48 h,which was in line with Weibull equation(r=0.9438). Under 3 kinds of temperature conditions,encapsulation efficiency decreased gradually as the holding time of nanoparticle pro-longed;the decrease degree was the smallest at 4 ℃.CONCLUSIONS:The quality evaluation results of solid lipid nanoparticle of the skin extract of B. gargarzans are in line with the standard,and prepared nanoparticles show sustained-release effects and should be kept under low temperature.
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OBJECTIVE:To establish an HPLC method for the determination of the contents of cinobufagin and resibufogenin in Yigankang capsule.METHODS:The samples were separated on Spherigel ODS C18(150 mm?4.6 mm,5 ?m)chromatographic column with 0.5% KH2PO4-acetonitrile(50∶50,pH was adjusted to 3.2 by phosphoric acid)used as a mobile phase at a flow rate of 1.0 mL?min-1.The detection wavelength was set at 296 nm and the column temperature was kept at 40 ℃.RESULTS:The linear ranges of cinobufagin and resibufogenin were 0.22～1.09 ?g(r=0.999 8)and 0.26～1.30 ?g(r=0.999 9),respectively,and the average recoveries of cinobufagin and resibufogenin were 99.4%(RSD=1.9%,n=9)and 98.7%(RSD=1.4%,n=9),respectively.CONCLUSION:This method is proved to be sensitive,simple,accurate and specific,and it is applicable for the quality control of Yigankang capsule.
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Objective To estalish an accurate method for the determination of cinobufagin and resibufogenin in Jiuxin Tablet. Method Waters Symmetry Shield RP18 (3.9 mm?250 mm, 5 ?m) column in an oven at 30 ℃ was need, with a mobile phase consisting of acetonitrile-0.5mol/L kalium dihydrogen phospate (phosphoric acid preparating pH=3.2) (42∶58) and a UV detector at 296 nm, the flow rate was 1.0 mL/min. Results The linear ranges of Cinobufagin was 0.0696～0.8352 ?g. The RSD of measurement precision test was 0.53%, the average recovery was 101.86% (RSD=1.81%, n=5), the linear ranges of Resibufogenin was 0.1576～0.9456 ?g, the RSD of measurement precision test was 0.15%, the average recovery was 102.07% (RSD=1.62%, n =5). Conclusion The method was simple and accurate, and can be used for the quality control of Jiuxin tablet.
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AIM: To estalish a HPLC method for the determination of cinobufagin and resibufogenin in Niuhuangxiaoyan Tablet(Calculus Bovls, Radix et Rhizoma Rhei, Concha Margaritifera Usta, Venenum Bufonis, etc.). METHODS: Waters Symmetry Shield RP18(3.9mm?250mm,5?m) column at 30℃ was used with a mobile phase consisted of acetonitrile -0.5mol?L -1 potassium dihydrogen phosphate(solution was adjusted by phosphoric acid to pH=3.2) (42∶58) and a UV detector at 296nm. The flow rate was 1.0mL?min -1 . RESULTS: The linear range of cinobufagin was 0.0696～0.8352?g. The RSD of measurement precision test was 0.53%; The average recovery was 100.06% ( RSD =1.25%, n =5). The linear range of resibufogenin was 0.1576～0.9456?g. The RSD of measurement precision test was 0.15%. The average recovery was 100.54%( RSD =1.49%, n =5). CONCLUSION: The method is simple and accurate, and can be used for the quality control of Niuhuangxiaoyan Tablet.
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Objective: To study the effect of Azone on the transdermal kinetics of Resibufogenin from toad venom. Methods: The transdermal constants of Resibufogenin var hairless mice skin are determined with various concentration of Azone as transdermal enhancers. Results: The transdermal constants are 81.25, 114.92, 169.63 , 196.31, 208.27, 261.35 (?g/cm 2?h) while the concentration of Azone are 0%, 2%, 4%, 6%, 8%, 10%, respectively. Conclusion: Azone can enhance Resibufogenin penetrate skin and the optimum concentration of Azone is 10%.
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Objective: To determinate the content of bufalin,resibufogenin and cinobufagin in Cinobufacini Injection. Methods: Liposoluble components in cinobufacini were extracted with ethyl acetate and determined by HPLC using a C 18 column, acetonitrile water(50∶50) as a mobile phase and UV detection wavelength at 299nm. Results: The method showed the good resolution, high sensitivity, satisfactory accuracy and specificity. Quantitatively analyze results showed that the concentration of bufalin, cinobufagin and resibufogenin in Cinobufacini Injection were 0.333 ?g?mL -1 , 0.159?g?mL -1 and 0.110?g?mL -1 , respectively. Conclusion: Bufalin in Cinobufacini Injection reached effective concentration, which was regarded as one of anti cancer components.
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Objective: To establish the determination of Shexianbaixin Pill. Methods: HPLC was applied to determine cinobufagin and resibufogenin. The determination was carried out using an ODS column with a solvent system: acetonitrile 0.5KH 2PO 4. The flow rate was 1.0mL?min -1 , detection wavelength was at 296nm and the column temperature was at 35℃. Results: The linear ranges of cinobufagin and resibufogenin were 0.148?g～0.746?g and 0.116?g～0.580?g, respectively. The average recoveries of cinobufagin and resibufogenin were 100.90%( RSD =1.004%, n =5), and 100.64%( RSD =1.006%, n =5), respectively. Conclusion: This method is simple, accurate with strong specificity and can be used to control the quality of Shexiangbaoxin Pill.
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Objective: To determine the contents of cinobufagin and resibufogenin in Qianglijiuxin Dripping Pills. Methods: The HPLC method was used at Kromasil C 18 column of 250mm?4.6mm, 5?m, 0.5% Dipotassium Hydrogen Phosphate Acetonitrile (50∶50) as the mobile phase, 1.0mL/min as the flow rate at detection wavelength of 296nm. Results: For cinobufagin, the linear range was 0.67?g～4.7?g and the average recovery rate was 97.78% with RSD=1.2%. For resibufogenin, the linear range was 0.33?g～2.3?g and the average recovery was rate 99.61% with RSD=2.0%.Conclusion: The method can be applied to content determination of cinobafagin and Resibufogenin in Qianglijiuxin Dripping pills.
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AIM: To isolate three kinds of bufadienolides composition from Venenum bufonis in order to investigate their antitumor activity in vitro.METHODS: Alcohol extraction and isolation by Silica gel colume were employed in this study.Their antitumor activities in vitro were evaluated by MTT colorimetric method.RESULTS: With such methods,three kinds of bufadienolides,including Bufalin,cinobufagin,and resibufogenin with purity above 90% were prepared.The in-vitro results showed that three kinds of bufadienolides had prominent inhibition effect on human lung cancer A549 cells,human breast cancer MDA-MB-435 cells in the range of 0.001 ?g/mL ～ 100 ?g/mL.CONCLUSION: Isolation by Silica gel colume can be used to separate three kinds of bufadienolides from Venenum bufonis and its constituents has prominent anti-cancer effects.
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AIM: To establish the method for quality control of Chansu Tongbi Ointment(Venenum Bufonis,Borneolum Syntheticum,Radix Stephanial Tetrandrae etc.) METHODS: Radix Stephaniae Tetrandrae in Chansu Tongbi Ointment was identified by TLC,and the content of cinobufagin and resibufogenin were determined by HPLC. RESULTS: Radix Stephaniae Tetrandrae could be identified by TLC.Cinobufagin showed a good linear relationship at the range of 1.15 ?g-5.75 ?g,r=0.999 7.The RSD of measurement precision test was 1.05%,The average recovery was 97.95%(RSD=1.63%,n=5).Resibufogenin showed a good linear relationship at the range of 1.05 ?g-5.25 ?g,r=0.999 6.The RSD of measurement precision test was 1.02%,The average recovery was 97.45%(RSD=0.61%,n=5. CONCLUSION: The method is accurate,reliable and available with a reproducibility and can be used to control the quality of this ointment effectively.
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AIM: To establish the method of determining cinobufagin and resibufogenin in Xinkening Capsules(Venenum Bufonis,etc.). METHODS: HPLC was applied to determining cinobufagin and resibufogenin.The determination was carried out using an ODS column with a solvent system: methanol-water(50∶40).The flow rate was 1.0 mL/min,detection wavelength was at 296 nm. RESULTS: The linear ranges of cinobufagin and resibufogenin were 3.5 ?g-0.28 ?g and 3.75 ?g-0.30 ?g,respectively.The average recoveries of cinobufagin and resibufogenin were 99.86%(RSD=1.8%,n=5),and 99.84%(RSD=1.2%,n=5),respectively. CONCLUSION: This method is simple,accurate with strong specificity and can be used to control the quality of Xinkening Capsules.
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OBJECTIVE:To establish a RP-HPLC method for determination of the contents of resibufogenin and cinobufagin in Meihuadianshe pills METHODS:The condition of HPLC was Kromasil C18 column,acetonitrile-0 5%KH2PO4(50∶50) as a mobile phase and detecting wavelength 296nm RESULTS:This method showed good linearity The average recoveries of resibufogenin and cinobufagin were 97 40% with a RSD of 2 2% and 97 78% with a RSD of 2 6% respectively CONCLUSION:The method is simple,reliable and satisfactory in resolution It can be used in quality control of Meihuadianshe pills