[Research progress in quality control of Bufonis Venenum in preparations].

Bufonis Venenum, an animal medicinal material, is widely used for treating cardiovascular diseases and pain induced by rheumatics or malignant tumors. In view of the high activity and high toxicity, it is of great significance to pay attention to the quality control of Bufonis Venenum to ensure the safety and effectiveness of its preparations. China's drug standards involve 102 preparations(474 batch numbers) containing Bufonis Venenum approved for sale, including 14 preparations in the Chinese Pharmacopoeia(2020 edition) and 68 preparations in the standards issued by the Ministry of Health Drug Standard of the People's Republic of China. Bufonis Venenum is mostly used in pill and powder preparations in the form of raw powder, with the main functions of clearing heat, removing toxin, relieving swelling and pain, replenishing qi, activating blood, opening orifice, and awakening brain. Except the high level of quality control for Bufonis Venenum in the preparations in the Chinese Pharmacopoeia(2020 edition), the quality control standards of Bufonis Venenum in other preparations are low or even absent. Therefore, it is urgent to conduct research on the improvement of quality standards for the preparations containing Bufonis Venenum. This study retrieved the reports focusing on the quality evaluation and quality control of the preparations containing Bufonis Venenum from CNKI, PubMed, and Web of Science. Qualitative and quantitative analysis methods for 64 preparations containing Bufonis Venenum have been reported, mainly including thin-layer chromatography, HPLC fingerprint, and multi-component content determination. The index components mainly involved bufadienolides, such as gamabufalin, arenobufagin, bufotalin, bufalin, cinobufagin, and resibufogenin. According to the literature information, this paper suggests that attention should be paid to the correlations between the analysis methods and detection indexes of medicinal materials, decoction pieces and preparations, the monitoring of indole alkaloids, and the content uniformity inspection for further improving the quality standards for the preparations containing Bufonis Venenum.

Research progress in quality control of Bufonis Venenum in preparations / 中国中药杂志

Bufonis Venenum, an animal medicinal material, is widely used for treating cardiovascular diseases and pain induced by rheumatics or malignant tumors. In view of the high activity and high toxicity, it is of great significance to pay attention to the quality control of Bufonis Venenum to ensure the safety and effectiveness of its preparations. China's drug standards involve 102 preparations(474 batch numbers) containing Bufonis Venenum approved for sale, including 14 preparations in the Chinese Pharmacopoeia(2020 edition) and 68 preparations in the standards issued by the Ministry of Health Drug Standard of the People's Republic of China. Bufonis Venenum is mostly used in pill and powder preparations in the form of raw powder, with the main functions of clearing heat, removing toxin, relieving swelling and pain, replenishing qi, activating blood, opening orifice, and awakening brain. Except the high level of quality control for Bufonis Venenum in the preparations in the Chinese Pharmacopoeia(2020 edition), the quality control standards of Bufonis Venenum in other preparations are low or even absent. Therefore, it is urgent to conduct research on the improvement of quality standards for the preparations containing Bufonis Venenum. This study retrieved the reports focusing on the quality evaluation and quality control of the preparations containing Bufonis Venenum from CNKI, PubMed, and Web of Science. Qualitative and quantitative analysis methods for 64 preparations containing Bufonis Venenum have been reported, mainly including thin-layer chromatography, HPLC fingerprint, and multi-component content determination. The index components mainly involved bufadienolides, such as gamabufalin, arenobufagin, bufotalin, bufalin, cinobufagin, and resibufogenin. According to the literature information, this paper suggests that attention should be paid to the correlations between the analysis methods and detection indexes of medicinal materials, decoction pieces and preparations, the monitoring of indole alkaloids, and the content uniformity inspection for further improving the quality standards for the preparations containing Bufonis Venenum.

A novel cathelicidin from Bufo bufo gargarizans Cantor showed specific activity to its habitat bacteria.

Toad Bufo bufo gargarizans Cantor is still used in China as traditional Chinese medicine. However, present investigations on its skin secretions were mainly focused on the bufadienolides, the proteins/peptides contained in the secretions are largely unknown. A cDNA encoding a novel cathelicidin termed BG-CATH was identified by analysis of the toad skin transcriptome. The BG-CATH precursor was predicted to have 2 possible cleavage sites following dibasic cleavage signals at its C-terminal, which will generate two mature peptides, BG-CATH37 and BG-CATH(5-37). Phylogenetic analysis suggests that amphibian cathelicidins might evolve from common ancestors. The two predicted mature cathelicidins from B. bufo gargarizans were synthesized and both of them showed weak antimicrobial activities against human pathogenic bacteria Escherichia coli, Pseudomonas aeruginosa and Staphylococcus aureus (MIC ≥ 200 µg/mL). However, BG-CATH37 and BG-CATH(5-37) had strong antimicrobial activities against aquatic bacteria of Vibrio splendidus, Streptococcus iniae and Aeromorus hydrophila, which were common microorganisms in the habitat of B. bufo gargarizans (MIC 3.125-40 µg/mL). BG-CATH37 and BG-CATH(5-37) showed no hemolytic activity even at high concentrations (400 µg/mL). CD spectra analysis suggested that structure rigidity of BG-CATH37 and BG-CATH(5-37) might play an important role to regulate their biological activities. Selective antimicrobial activity against habitat microorganisms might reflect the adaptation of amphibians to their living environments.

Purification of bufadienolides from the skin of Bufo bufo gargarizans Cantor with positively charged C18 column.

As a kind of promising anticancer compounds, the preparation of bufadienolides is a hot study spot. However, due to the complexity of biological sample, the purification of bufadienolides from a crude sample (toad skin) is a tough work. In this paper, we reported a new way based on positively charged C18 material (XCharge C18) to quickly separate and purify bufadienolides from toad skin. By this method, the different ionic feature of the amino acid conjugated bufadienolides (AACBs) and the free form bufadienolides (AAUBs) was firstly utilized to obtain distinct separation selectivity on the XCharge C18 column. Additionally, the peak tailing problem of AACBs on conventional C18 was resolved and better resolutions were achieved on the XCharge C18, thus, two kinds of bufadienolides on one column were successfully purified respectively. Taking F13 as an example, the method was validated by liquid chromatography-mass spectrometry (LC-MS), and then 4 AACBs as well as 4 AAUBs were simultaneously purified by preparative XCharge C18. In addition, the application of this method in other fractions was also validated. The results suggested that the developed method is a practical and promising tool for efficient separation and purification of bufadienolides from toad skin.

Comparison of toad skins Bufo bufo gargarizans Cantor from different regions for their active constituents content and cytotoxic activity on lung carcinoma cell lines.

BACKGROUND: The skin of Bufo bufo gargarizans Cantor, rich in bufadienolides, peptides, and alkaloids, has approved pharmacological activity for preliminary anti-liver and lung tumor treatment. However, few studies have systematically focused on the influence of the producing regions on the content and antitumor activity of the active constituents in toad skins. OBJECTIVE: This study aims to compare toad skins obtained from six different regions in China (Jiangsu, Anhui, Henan, Hebei, Jiangxi, and Shandong province) for their bufadienolide and alkaloid content, and their cytotoxic activity on two lung carcinoma cell lines (SPC-A-1 cells and A549 cells). MATERIALS AND METHODS: High performance liquid chromatography (HPLC) was used to quantificationally determine four bufadienolides, which included bufotalin, bufalin, cinobufagin, and resibufogenin in toad skins, from six different regions, respectively. In addition, an ultraviolet (UV) spectrophotometer was also employed to identify the content of the total alkaloids using 5-hydroxytryptamine (5-HT) as the reference substance. An MTT assay was performed to compare the antiproliferative effects of the toad skins' ethanolic extracts from the different regions against SPC-A-1 and A549 cells. RESULTS: In this study, the toad skins from Jiangsu province had the highest amount of bufadienolides (472.6 µg/g crude drug) and alkaloids (1.51 mg/g crude drug). Meanwhile, according to the extract, it exhibited the strongest cytotoxic effect against the lung carcinoma cell line (SPC-A-1 cells and A549 cells) with IC50 values of 24.82 ± 0.76 and 23.77 ± 0.63 µg crude drug/mL, respectively. CONCLUSION: The toad skins that originated from the Jiangsu province, have comparatively greater advantages over samples from other regions as far as active constituent content and potential anti-lung cancer activity is concerned, suggesting that it can be a promising chemotherapeutic agent in lung cancer therapy, in further studies.

Cyto-/genotoxic effects of the ethanol extract of Chan Su, a traditional Chinese medicine, in human cancer cell lines.

ETHNOPHARMACOLOGICAL RELEVANCE: Chan Su, an ethanolic extract from skin and parotid venom glands of the Bufo bufo gargarizans Cantor, is widely used as a traditional Chinese medicine for cancer therapy. Although the anti-cancer properties of Chan Su have been investigated, no information exists regarding whether Chan Su has genotoxic effects in cancer cells. The aim of the present study was to examine the cyto-/genotoxic effect of Chan Su in human breast carcinoma (MCF-7 cells), human lung carcinoma (A-549 cells), human T cell leukemia (Jurkat T cells), and normal human lymphocytes. MATERIALS AND METHODS: Effects on the viability of MCF-7, A-549, Jurkat T cells, and normal lymphocytes were evaluated by Trypan blue exclusion assays. The DNA content in the sub-G1 region was detected by propidium iodide (PI) staining and flow cytometry. The genotoxicity of Chan Su was assessed by single-cell gel electrophoresis (comet assay) and the cytokinesis-block micronucleus assay (CBMN assay). RESULTS: Chan Su significantly inhibited the viability of MCF-7, A-549, and Jurkat T cells dose dependently, but had no effect on normal human lymphocytes. Apoptotic death of the cancer cells was evident after treatment. Chan Su also induced genotoxicity in a dose-dependent manner, as indicated by the comet and cytokinesis-block micronucleus assays. CONCLUSIONS: These findings suggest that Chan Su can induce apoptotic death of, and exert genotoxic effects on, MCF-7, A-549, and Jurkat T cells.

Rapid and quantitative determination of multiple quality indicators (indole alkaloids, etc.) in concentration process of Bufo bufo gargarizans Cantor extract using fourier transform near infrared spectroscopy and chemometrics / 中国药学杂志

OBJECTIVE: To investigate the application of near infrared spectroscopy in rapid and quantitative determination of multiple quality indicators in concentration process of Bufo bufo gargarizans Cantor extract. METHODS: NIR transmission spectra of concentrated solutions were collected in rectangular quartz cuvette with different optical pathlengths (2 and 5 mm). Calibration models of density, water content and indole alkaloids were developed by partial least squares regression (PLSR). The established models were applied to predict the unknown samples to test the performance of the models. RESULTS: The correlation coefficients of the density, water content and indole alka. The residual predictive deviations (RPD) were all above 3, and the relative standard errors of predictions(RSEP) were less than 10%, indicating the satisfactory model performance and predictive ability. Moreover, better calibration results were obtained with an optical path length of 2 mm for density and water content, and 5 mm for indole alkaloids. The results revealed that the optical path length has influence on the NIR analysis results, and the appropriate path length for the NIR analysis should be determined by comparison analysis. CONCLUSION: NIR spectroscopy technique provides a novel efficient and environmental friendly approach for the fast simultaneous determination of three key quality indicators (density, water content and indole alkaloids) in the concentration process of Bufo bufo gargarizans Cantor extract. Copyright 2012 by the Chinese Pharmaceutical Association.