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ObjectiveTo investigate the efficacy and safety of cinobufagin tablets combined with thalidomide/dexamethasone （TD） regimen in the treatment of newly diagnosed multiple myeloma （NDMM） with phlegm and stasis obstruction. MethodsThe clinical data of 50 patients with NDMM of phlegm and stasis obstruction who were hospitalized at the Jiangsu Province Hospital of Chinese Medicine from June 1st， 2015 to July 31th， 2019 were retrospectively analyzed， and they were divided into a control group （bortezomib/dexamethasone-containing regimen， 27 cases） and an observation group （cinobufagin tablets combined with TD regimen， 23 cases）. The clinical efficacy and safety were compared between the two groups after two or three courses of treatment. The primary outcomes were clinical remission rate including overall response rate and deep remission rate， one-year and two-year overall survival rate， and adverse effects. The secondary outcomes were the proportion of plasma cells in bone marrow， hemoglobin， β2-microglobulin， lactate dehydrogenase， serum creatinine， blood urea nitrogen， bone pain score， and KPS functional status score （KPS score） before and after treatment. ResultsIn terms of clinical efficacy， there was no statistically significant difference （P＞0.05） in the overall response rate ［the observation group 69.57%（16/23） vs the control group 70.37% （19/27）］ and deep remission rate ［the observation group 56.52% （13/23） vs the control group 55.56% （15/27）］ between groups after the treatment. The one-year overall survival rates of the observation group and the control group were 90.9% and 92.4%， and the two-year overall survival rates were 81.8% and 80.9% respectively， with no statistically significant differences between groups （P＞0.05）. During the treatment， no renal function injury occurred in both groups. The incidence of peripheral nerve injury in the observation group was 8.70%， which was lower than 48.15% in the control group （P＜0.01）. After the treatment， the proportion of myeloma plasma cells， β2-microglobulin， serum creatinine level， and bone pain score decreased， while the hemoglobin level and KPS score increased in both groups （P＜0.05 or P＜0.01）. Compared between groups after treatment， the bone pain score of the observation group was lower than that of the control group， while the KPS score was higher than that of the control group （P＜0.05）. ConclusionThe clinical efficacy of cinobufagin tablets combined with TD in the treatment of NDMM is equivalent to bortezomib/dexamethasone-containing regimen， but the former is more helpful in relieving the pain and improving the quality of life， and has better safety.
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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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Huachansu is a traditional Chinese medicine widely used in the clinic for cancer therapy, while the underlying mechanism is not fully clarified. This study was to investigate the targets and mechanisms of cinobufagin (CBG), an active component of Huachansu, in terms of blocking mitosis of cancer cells. Propidium iodide (PI) DNA staining was used to analyze the effect of CBG on cell cycle. The effect of CBG on mitosis of cancer cells was examined by α-tubulin and pericentrin staining after synchronization by a double thymidine block. Tubulin turbidity, tubulin polymerization and α-tubulin immunofluorescence assays were used to evaluate the effect of CBG on microtubule polymerization. CRISPR/Cas9 gene-editing technology was used to knockout microtubule-severing protein Katanin regulatory subunit B1 (KATNB1) in HCT116 cells, and the inhibitory effect of CBG on wild-type cells and knockout cells was measured by CCK-8. The engagement of CBG with KATNB1 was measured by CETSA and DARTS assays. The effect of CBG on KATNB1 protein and mRNA level was examined by Western blot and real-time PCR, respectively. Our data showed that CBG arrested HCT116 cell cycle at the G2/M phase, disrupted mitosis and induced centriole overduplication. CBG significantly inhibited tubulin polymerization in vitro and in vivo. The cytotoxicity of CBG inhibition on HCT116 was significantly attenuated upon KATNB1 depletion. Moreover, CBG bound to KATNB1 and decreased its protein level, while mutated KATNB1 weakened this effect. In conclusion, CBG inhibited microtubule polymerization via targeting KATNB1, thereby disrupting mitosis in cancer cells.
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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 establish the HPLC fingerprint of Bufonis Venenum pulp and the determination methods of eight components in Bufonis Venenum pulp, and compare the differences of components of Bufonis Venenum pulp in different origins for quality evaluation. Methods: The mobile phase was acetonitrile and water with a gradient elution program. The detection wavelength was set at 296 nm. The flow rate was 1.2 mL/min. The column temperature was set at 30 ℃, and 10 μL of the test solution was injected. HPLC fingerprint of Bufonis Venenum pulp was established and eight components were determined. The results were analyzed by cluster analysis and principal component analysis. Results: There were 12 common peaks in the HPLC fingerprints of 18 batches of samples, and the similarity of each sample was different. The linear relationship of eight components was good (r2 > 0.999 5), RSD of precision and repeatability was less than 0.5%, and the stability was also good within 30 h (RSD < 0.7%). The average recoveries of gamabufotalin, arenobufagin, telocinobufagin, bufotaline, cinobufotalin, bufalin, cinobufagin and resibufogenin were 103.7%, 103.0%, 102.9%, 103.0%, 103.9%, 100.3%, 103.4%, and 103.2% respectively, and RSD was all less than 1.2%. The results of the content determination, cluster analysis and principal component analysis of eight components showed that Bufonis Venenum pulp in different habitats were different from each other. Conclusion: The method is simple and reliable, which can provide some basis and reference for quality control of Bufonis Venenum pulp.
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Objective: To investigate the analgesic mechanism of cinobufagin in rats with bone cancer pain. Methods: Female SD rats meeting the conditions of pain threshold were selected to construct cancer-induced bone pain (CIBP) model. On the 7th day after modeling, the sham group and the model group were administrated by saline, while the treatment groups were administrated with the low, medium and high concentrations of cinobufagin for consecutive 7 d. The pain behavior (mechanical withdrawal threshold and thermal pain threshold) was tested before modeling and after modeling, and single injection of cinobufagin after 0.5, 1, 2, 4, 6, 8 and 24 h at the first day. The expression of MAPKs protein was detected by Western Blotting, and the content of spinal cytokines (IL-1β, TNF-α, MCP-1) was detected by ELISA. Results: The mechanical pain threshold and thermal pain threshold were significantly decreased in the model group, compared with the sham group (P 0.05). Protein levels of MAPKs were increased in the model group, while the levels of JNK and p38 were decreased in the cinobufagin group (P 0.05). ELISA results showed that cinobufagin significantly decreased the content of cytokines in the spinal cord, when compared with the model group (P < 0.05). Conclusion: Cinobufagin can inhibit the expression of MAPKs proteins in the spinal cord of the rat model with bone cancer pain, ultimately decrease the content of IL-1β, TNF-α, and MCP-1 to alleviate the pain during the process of cancer pain.
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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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As known,simultaneous determination of various chemical indicators is one of the future trends in quality control of traditional Chinese medicines because of the extremely complex chemical compositions. This project is to screen the quality markers that can accurately control the quality of the Bufonis Venenum by exploring the intrinsic correlation of components. In this study,venom of Bufo bufo gargarizans from 17 different sources were used as research samples,and the contents of 7 bufogenin were determined by HPLC-DAD. Then,the data obtained were analyzed by Spearman correlation analysis and principal component analysis( PCA). In addition,a stepwise regression analysis was used to establish a predictive model for the contents of the seven bufogenin components( independent variable) and the total contents of the bufogenin( dependent variable). The results indicated that there is a significant positive correlation between the contents of telocinobufagin and cinobufotalin,and there is a significant positive correlation between the contents of bufalin,cinobufagin and resibufogenin. In contrast,the contents of telocinobufagin and cinobufotalin are negatively correlated with the contents of bufalin,cinobufagin and resibufogenin. However,the correlation between gamabufotalin and bufotalin and other components are not obvious. Furthermore,further study found that there is a correlation between the sum of the contents of bufalin,cinobufagin and telocinobufagin and the total contents of the bufogenin. In fact,the application of bufalin,cinobufagin and telocinobufagin as the quality control indicators of the Bufonis Venenum can better reflect the quality characteristics of the Bufonis Venenum compared with the previous quality control indicators. The conclusions will provide a reference for the revision of the quality standards of the Bufonis Venenum.
Subject(s)
Animals , Amphibian Venoms , Chemistry , Bufanolides , Bufo bufo , Chromatography, High Pressure Liquid , Medicine, Chinese Traditional , Quality ControlABSTRACT
OBJECTIVE@#To observe the effect of cinobufagin on transient outward potassium current () in rat dorsal root ganglion cells of cancer-induced bone pain (CIBP) and explore the possible analgesic mechanism of cinobufagin.@*METHODS@#Whole cell patch clamp technique was used to examine the effect of cionbufagin on in acutely isolated dorsal root ganglion (DRG) cells from normal SD rats and rats with bone cancer pain.@*RESULTS@#The DRG cells from rats with CIBP showed obviously decreased current density, an activation curve shift to the right, and an inactivation curve shift to the left. Cinobufagin treatment significantly increased the current density and reversed the changes in the activation and inactivation curves in the DRG cells.@*CONCLUSIONS@# current is decreased in DRG neurons from rats with CIBP. Cinobufagin can regulate the activation and inactivation of current in the DRG cells, which may be related to its analgesic mechanism.
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Animals , Rats , Analgesics , Pharmacology , Bufanolides , Pharmacology , Cancer Pain , Drug Therapy , Cells, Cultured , Ganglia, Spinal , Patch-Clamp Techniques , Potassium Channels , Metabolism , Rats, Sprague-DawleyABSTRACT
Aim To investigate the effect of cinobufagin on the migration and invasion of esophageal cancer Kyse-520 cells, and to explore the underlying molecular mechanism. Methods The rates of inhibition after treated with different concentrations of cinobufagin 12, 24, 48 h were detected by CCK-8 method, and the changes of cell migration and invasion were observed with wound healing and transwell assay. The mRNA expressions of FAK, Akt, PTEN, VEGF-A, MMP-2 and MMP-9 were detected by quantitative real-time polymerase chain reaction ( RT-qPCR ). The protein expressions of FAK, p-FAK ( Tyr397 ), Akt, p-Akt (Sei473 ), VEGF-A, PTEN, MMP-2 and MMP-9 were measured by Western blot. Results The results of CCK-8 showed that cinobufagin could inhibit the proliferation of Kyse-520 cells in a time- and concentration-dependent manner, and cinobufagin significantly inhibited the cell invasion and migration. Meanwhile, data from RT-qPCR and Western blot suggested that cinobufagin had no significant effect on mRNA and total protein of FAK and Akt, but it reduced the expression of p-FAK(Tyr397), p-Akt(Ser473), VEGF- A, MMP-2 and MMP-9, and increased the PTEN expression. Conclusions Cinobufagin significantly inhibits the invasion and migration of esophageal cancer Kyse-520 cells through inducing PTEN expression and down-regulating FAK/PI3K/AKT pathway.
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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 investigate the effect and mechanism of cinobufagin combined with Sorafenib on the proliferation and apoptosis of hepatocellular carcinoma Huh7 cells. Methods The proliferation of Huh7 cells was measured using MTT assay; The apoptosis morphological changes of Huh7 cells were detected using Hoechst33342/PI fluorescence staining; The cells cycle was detected by flow cytometry; The expression of Ki67 protein was detected by immunocytochemistry; The expressions of Bax, Bcl-2, Caspase-8, AURKA, Ras, Raf, ERK, and p-ERK proteins were measured using Western blotting. Results Cinobufagin, Sorafenib, and combination therapy inhibited the proliferation of Huh7 cells, and the inhibitory effect of the combination group was more obvious with synergistic effect. Fluorescence staining showed morphological changes of apoptosis. Sorafenib induced the cell cycle S phase arrest, cinobufagin and combination therapy induced the cell cycle G2/M phase arrest, combination group had more obvious cell cycle arrest in G2/M phase than single drug groups. Both cinobufagin and Sorafenib attenuated the expression of Ki67, and the effect of combination group was more significant. Cinobufagin, Sorafenib, and combination therapy up-regulated the expression of Bax and Caspase-8 proteins; down-regulated the expression of Bcl-2 protein; up-regulated the ratio of Bax/Bcl-2; had no obvious effect on the expression of ERK protein; significantly down-regulated the expression of AURKA, Ras, Raf, and p-ERK proteins; And the effect of combination group was more significant (P < 0.05). Conclusion Cinobufagin combined with Sorafenib could inhibit the proliferation and induce the apoptosis of hepatocellular carcinoma Huh7 cells through AURKA/Ras/Raf/ERK signaling pathway.
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Aim To investigate the effect of the active ingredients of toad venom (bufalin and cinobufagin) combined with sorafenib on the growth of hepatocellular carcinoma HepG2 cells,and to explore the possible mechanism.Methods The rates of inhibition after treated with drugs 12,24,48 h were detected by MTT assay.The changes of cell morphology were detected by Hoechst 33342 fluorescent staining.The changes of cell cycle were detected by flow cytometry.The expressions of proteins such as Akt,p-Akt (Ser473),IκB,NF-κB,p-NF-κB p65,Bcl-2,Bax,cyclin A,PCNA were detected by Western blot.Results Bufalin,cinobufagin and sorafenib could inhibit the proliferation of HepG2 cells,presenting a dose-and time-dependent manner.Meanwhile,it could significantly increase the inhibitory rate of cells compared with those of single treatment,and they performed a synergistic activity in sorafenib combined with cinobufagin or bufalin by Jin Formula after 24 h treatment (P ＜ 0.01).The results of fluorescence staining showed the observation of the morphological features of nuclear condensation.Sorafenib induced the cell cycle G0/G1 phase arrest (P ＜0.01),and bufalin,cinobufagin and the combination treatment generated the cell cycle S phase arrest (P ＜0.01).The results of Western blot showed that the expressions of Akt,NF-κB were not obviously changed between control and all other treatment.The expression levels of p-Akt (Ser473),p-NF-κB p65,Bcl-2,PC-NA and cyclin A in combination treatment significantly decreased,and the expression levels of IκB and Bax significantly increased compared to those in single treatment (P ＜ 0.01).Conclusion The active ingredients of toad venom (bufalin and cinobufagin) combined with sorafenib performs a synergetic effect on the anti-cancer of HepG2 cells by down-regulating Akt/ NF-κB signaling pathway.
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Objective To investigate the function of aurora kinase (AURK) in liver cancer and the mechanism of cinobufagin and bufalin-induced liver cancer HepG2 cells growth inhibition by down-regulating AURK family. Methods Kaplan-Meier survival method analyzed the relationship between mRNA expression levels of AURKA and AURKB and survival periods. The viability and cell cycle of HepG2 cells were detected by MTT method and flow cytometry. Western blotting analyzed the expression levels of spindle-associated protein AURKA, AURKB, TPX2, SMC2, TOP2A, and cyclin-dependent kinase CDK1. Results Kaplan-Meier survival analysis presented a significantly negative correlation between mRNA expression levels of AURKA and AURKB and survival periods. Cinobufagin and bufalin inhibited the growth of HepG2 cells in a time- and dose-dependent manner, and induced the cell cycle G2/M phase arrest. They all down-regulated the expression of AURKA, AURKB, TPX2, SMC2, TOP2A, and CDK1 (P < 0.05). Conclusion There is a significantly negative correlation between mRNA expression levels of AURKA and AURKB and survival periods. Cinobufagin and bufalin could induce HepG2 cells growth inhibition and cell cycle arrest by down-regulating the expression of AURKA and AURKB and other mitosis-regulating proteins.
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Objective To assess the consistency of 30 batches of Xueshuan Xinmaining Tablets (XXT), we establish the UPLC-PDA fingerprint of XXT and provide reliable scientific basis for the Chinese medicine quality consistency. Methods Methanol was used to extract the XXT samples, and the extracts were measured by UPLC with the absorption wavelength at 280 nm. Waters Acquity UPLC HSS T3 reversed-phase column (50 mm × 2.1 mm, 1.8 μm) was used for determining the extracts at a flow rate of 0.5 mL/min and column temperature of 40 ℃. The mobile phase condition was acetonitrile-0.1% formic acid aqueous solution with gradient mode. The methodology, similarity calculation and cluster analysis of 30 batches of XXT samples were investigated. The common peak of XXT was belonged to single medicinal materials and common peaks were identified by comparing with the reference. Results The UPLC fingerprint of XXT was established. Twenty-seven common fingerprint peaks were indicated with wavelength 280 nm, Among them, peaks 1, 10, 12-16, 18, and 21-28 belonged to Salviae Miltiorrhizae Radix et Rhizoma (SMRR), peaks 2-4, 6, 8, 9, and 17 belonged to Chuanxiong Rhizoma (CR), peaks 3, 7, 9, 11, and 16 belonged to Ilicis Pubescentis Radix (IPR), peak 5 belonged to Sophora Flos (SF), peaks 19 and 20 belonged to Bufonis Venenum (BV). Peaks 4, 5, 13, 19, 20, and 28 were ferulic acid, rutin, salvianolic acid B,resibufogenin, cinobufagin, tanshinone IIA, respectively, compared with reference. The analyzed samples were classified into two classes and the similarity was greater than 0.960. Conclusion The method of XXT UPLC-PDA fingerprint was established with good precision, stability and repeatability, supplying helpful information for the comprehensive evaluation of XXT.
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Aim To explore the relationship between Mre11/Rad50/Nbs1 ( MRN ) complex focus formation and DNA double-strand breaks( DSBs) caused by cinob-ufagin in human hepatocellular carcinoma HepG2 cells. Methods The Na+,K+-ATPaseα1 subunit expression level in liver cancer tissues was detected by immunohis-tochemistry. After HepG2 cells were treated with 5μmol·L-1 cinobufagin for 6, 12 and 24 h, the drug-in-duced DSBs were assessed by single cell gel electro-phroesis ( SCGE ) , the gene transcription and protein levels of Mrel1, Nbs1, Rad50 and p53 were evaluated by Real time-PCR and Western blot. The cell cycle in parallel was analyzed by flow cytometry. Results The Na+, K+-ATPase α1 subunit expression level in liver cancer tissues was significantly increased compared with the tissue adjacent to carcinoma ( P <0. 05 ) . The 5μmol · L-1 cinobufagin could induce the DSBs in a time-dependent manner (P <0. 05), and it could up-regulate the gene expression levels of Mre11, Nbs1, Rad50 and p53 in HepG2 cells ( P<0. 05 ) . The pro-portions of HepG2 cells in S phase were ( 21. 32 ± 4. 21) % in the control group, and (33. 25 ± 5. 72) %, (56. 72 ± 6. 29) % and (67. 32 ± 9. 42) % in HepG2 cells treated with 5 μmol · L-1 cinobufagin for 6, 12 and 24 h, respectively. The proportions of cells in S phase in cinobufagin groups were significantly increased compared with the control group ( P<0. 05 ) . Conclu-sion Cinobufagin could induce the cell cycle arrest in liver cancer HepG2 cells by activation of Mre11/Rad50/Nbs1 Complex.
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Objective: To establish an optimized method to determine the entrapment efficiencies (EE) of cinobufagin(CBG) and resibufogenin(RBG) in the toad skin extract loaded with solid lipid nanoparticles. Methods: Dialysis, high-speed centrifugation and low-speed centrifugation were selected to determine the entrapment efficiencies of toad skin extract loaded solid lipid nanoparticles. The effects of different dialytic media on the entrapment efficiencies were investigated, then the EE of every method were compared. Results: Different methods had different results of EE, the EE of low-speed centrifugation, high-speed centrifugation and dialysis method in CBG and RBG were (74.00±1.69)% and (75.01±2.05)%,(83.60±0.99)% and (82.51±1.56)%,(91.01±0.75)% and (89.22±0.88)%, respectively. The EE determined by different dialytic media of dialysis were different, but the results did not have the significant differences. Conclusion: Different determination methods of EE have some significant influences on the results of EE, dialysis method is more suitable for the determination of EE for CBG and RBG in the toad skin extract loaded solid lipid nanoparticles.
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To investigate the effect of ultrasound-guided percutaneous injection of cinobufotalin or anhydrous ethanol in the treatment of portal vein tumor thrombus (PVTT) through a comparative analysis. MethodsA total of 56 patients with PVTT after liver cancer surgery, who were admitted to the 94th Hospital of PLA from October 2009 to December 2011, were enrolled. Among these patients, 27 underwent ultrasound-guided percutaneous injection of cinobufotalin (cinobufotalin group) and 29 underwent ultrasound-guided percutaneous injection of anhydrous ethanol (anhydrous ethanol group). The clinical outcome and survival time were compared between the two groups after treatment, and the levels of total bilirubin (TBil), alanine aminotransferase (ALT), and aspartate aminotransferase (AST) before and after treatment were compared. The t-test was used for comparison of continuous data between groups, the chi-square test was used for comparison of categorical data between groups, the rank sum test was used for comparison of ranked data, and the Kaplan-Meier method was used for survival analysis. ResultsThe response rate showed no significant difference between the cinobufotalin group and the anhydrous ethanol group (63.0% vs 58.6%, χ2=0.111, P＞0.05). After treatment, the cinobufotalin group had significantly lower levels of TBil, ALT, and AST than the anhydrous ethanol group (t=2.24, 2.40, and 2.39, all P＜0.05). The 6-month, 1-year, and 2-year survival rates showed no significant differences between the cinobufotalin group and the anhydrous ethanol group (81.5%/63.0%/29.6% vs 82.8%/586%/31.0%, χ2=0.016, 0.111, and 0.013, all P＞0.05). ConclusionUltrasound-guided percutaneous injection of cinobufagin or anhydrous ethanol has similar short- and long-term effects in the treatment of PVTT and can inhibit the growth of tumor thrombus and prolong survival time, but cinobufagin is superior to anhydrous ethanol in the aspect of protecting liver function.
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Objective To establish an optimized method to determine the entrapment efficiencies(EE)of cinobufagin(CBG) and resibufogenin(RBG)in the toad skin extract loaded with solid lipid nanoparticles. Methods Dialysis,high-speed centrifugation and low-speed centrifugation were selected to determine the entrapment efficiencies of toad skin extract loaded solid lipid nanoparti?cles. The effects of different dialytic media on the entrapment efficiencies were investigated ,then the EE of every method were com?pared. Results Different methods had different results of EE,the EE of low-speed centrifugation,high-speed centrifugation and dialysis method in CBG and RBG were(74.00±1.69)%and(75.01±2.05)%,(83.60±0.99)%and(82.51±1.56)%,(91.01±0.75)%and(89.22± 0.88)%,respectively. The EE determined by different dialytic media of dialysis were different,but the results did not have the signifi?cant differences. Conclusion Different determination methods of EE have some significant influences on the results of EE,dialysis method is more suitable for the determination of EE for CBG and RBG in the toad skin extract loaded solid lipid nanoparticles.