[Systematic comparison of two kinds of Bufonis Venenum derived from different Bufo gargarizans subspecies based on metabolomics and antitumor activity].

This paper compared the differences between two kinds of Bufonis Venenum produced by Bufo gargarizans gargarizans and B. gararizans andrewsi, and verified the rationality of the market value orientation of Bufonis Venenum based on the zebrafish mo-del. Twenty batches of Bufonis Venenum from Jiangsu province, Hebei province, Liaoning province, Jilin province, and Liangshan, Sichuan province, including B. gargarizans gargarizans and B. gararizans andrewsi, were collected. The UHPLC-LTQ-Orbitrap-MS combined with principal component analysis was used to compare the differences between two kinds of Bufonis Venenum. According to the limiting conditions of VIP>1, FC<0.5 or FC>2.0, and peak total area ratio>1%, 9 differential markers were determined, which were cinobufagin, cinobufotalin, arenobufagin, resibufogenin, scillaredin A, resibufagin, 3-(N-suberoylargininyl)-arenobufagin, 3-(N-suberoylargininyl)-marinobufagin, and 3-(N-suberoylargininyl)-resibufogenin. The content of 20 batches of Bufonis Venenum was determined according to the Chinese Pharmacopoeia(2020 edition) by high-performance liquid chromatography, and the 2 batches of Bufonis Venenum, CS7(8.99% of total content) and CS9(5.03% of total content), with the largest difference in the total content of the three quality control indexes of the Chinese Pharmacopoeia(bufalin, cinobufagin, and resibufogenin) were selected to evaluate their anti-liver tumor activity based on the zebrafish model. The tumor inhibition rates of the 2 batches were 38.06% and 45.29%, respectively, proving that only using the quality control indexes of the Chinese Pharmacopoeia as the value orientation of Bufonis Venenum market circulation was unreasonable. This research provides data support for the effective utilization of Bufonis Venenum resources and the establishment of a rational quality evaluation system of Bufonis Venenum.

Cytotoxic compounds from the leaf of Bersama abyssinica subspecies abyssinica.

From the leaves of Kenyan medicinal plant Bersama abyssinica Subspecies abyssinica, four previously undescribed compounds namely, three bufadienolides, 10ß-formylpaulliniogenin B, 10ß-formylpaulliniogenin A and 1ß-acetoxy-3ß,5ß-dihydroxy-15-methoxy-16,19-dioxobufa-14(15),20,22-trienolide, and a phenolic compound 2,6,4'-trihydroxybenzophenone-4-O-(6‴-cinnamoyl)-ß-D-glucoside were isolated together with four known compounds. The structural elucidation of the compounds was based on 1D and 2D NMR spectroscopy and HRMS data analyses. The relative configurations were defined by NOESY correlations. Cytotoxic activities on L929 and KB3.1 cell lines of the isolated compounds were investigated using MTT assay. The 1ß-acetoxy-3ß,5ß-dihydroxy-15-methoxy-16,19-dioxobufa-14(15),20,22-trienolide showed significant cytotoxic activity against KB3.1 cell lines with IC50 of 3.9 ± 0.99 µM.

Quantitative analysis of fourteen bufadienolides in Venenum Bufonis crude drug and its Chinese patent medicines by ultra-high performance liquid chromatography coupled with tandem mass spectrometry.

ETHNOPHARMACOLOGICAL RELEVANCE: Venenum Bufonis, a product of the secretions of Bufo gargarizans Cantor or B. melanostictus Schneider, possessed an array of pharmacological activities, such as cardiotonic, anti-tumor, antinociceptive, anti-inflammatory, anesthetic and antimicrobial activities. However, there were few efficient methods for quality evaluation of Venenum Bufonis medicinal materials and its related Chinese patent medicines. AIM OF THE STUDY: To establish an effective method for quality assessment of crude drugs and Chinese proprietary medicines of Venenum Bufonis, and explore the relationship of primary compounds - target - pathway - disease through a series of network databases. MATERIALS AND METHODS: An ultra-high performance liquid chromatography coupled with tandem mass spectrometry (UHPLC-QqQ-MS/MS) method was developed and validated to simultaneously determine 14 bufadienolides for quantitative analysis of 71 batches of crude drugs and 20 kinds of Chinese patent medicines of Venenum Bufonis. Multiple reaction monitoring with good specificity and accuracy was applied to monitor the 14 bufadienolides in positive mode. RESULTS: The methodology was validated with good specificity, precision, stability, repeatability and recovery. The low limits of quantification were in the range of 0.1-2.7 ng/mL. The relative standard deviation values for intra- and inter-day precisions ranged from 0.98% to 6.3% and from 2.39% to 6.76%, respectively. The recovery was varied from 87.78% to 110.57% for crude drugs and 88.32%-100.96% for Chinese proprietary medicine (Shexiang Baoxin Pill). The contents of 14 analytes in 71 batches of crude drugs and 20 sorts of Chinese proprietary medicines were procured, the results showed that the contents of crude drugs collected from the market exhibited great variations. Furthermore, 13 batches of crude drugs were identified as counterfeit with no bufadienolides detected. In addition, the total contents of bufadienolides in the same drug showed great difference among products from various manufacturers or brands. Subsequently, 9 bufadienolides with the higher contents were applied to screen the anti-tumor effect by network pharmacology, and 8 pathways which had prior correlation with bufadienolides were disclosed. CONCLUSION: This method could be used for quality assessment of crude drugs and Chinese patent medicines of Venenum Bufonis, and the data could be served as the fundamental basis for drug research and development of Venenum Bufonis.

High Resolution Mass Profile of Bufadienolides and Peptides Combing with Anti-Tumor Cell Screening and Multivariate Analysis for the Quality Evaluation of Bufonis Venenum.

In order to evaluate the quality of Bufonis Venenum commercial herbs, a three-step qualitative and quantitative research study was performed. Firstly, we tried to identify small molecules and peptides in Bufonis Venenum using pre-fractionation chromatography and high-resolution mass spectrometry. The database search of the small molecules and peptides of Bufonis Venenum revealed that the dried venom consisted of free/conjugated-type bufadienolides and peptides with a mass range of 0.4-2 kDa. Secondly, we used partial least squares (PLS) multivariate statistical analysis to screen bufadienolides markers (VIP > 1.5) responsible for the anti-tumor cell activity of Bufonis Venenum, including 21 identified bufadienolides and 7 unknown compounds. It is noticeable that these bufadienolide markers could not be recognized by traditional HPLC-UV based spectrum-effect relationship analysis (correlation coefficient ranging from -0.24 to 0.40). Finally, we proposed a weight coefficient-based corrected total contents of 9 bufadienolides as a quality evaluation indicator, which had good correlation with inhibitory effects on tumor cells of commercial Bufonis Venenum. The correlation coefficient increased from 0.4 to 0.6. Thus, our pre-fractionation chromatography and mass spectrometry strategy had significant advancement over the traditional spectrum-effect relationship method for chemical marker identification. These results could be crucial and helpful in the development of a quality evaluation method that could reflect the pharmacological activity of Bufonis Venenum.

Quantitative analysis of multicomponents by a single marker and quality evaluation of Venenum Bufonis from different geographical origins.

Bufadienolides are the main bioactive components of Venenum Bufonis (VB) and have been widely used to treat different types of human cancers for decades. The bufadienolide content in VB varies significantly in materials from different geographical origins. In this work, a new strategy for the quality assessment of VB was developed through quantitative analysis of multi-components by single marker (QAMS). Cinobufagin was selected as the internal reference substance; seven bufadienolides were separated and simultaneously determined based on relative correction factors. The correlation coefficient value (r ≥ 0.9936) between QAMS and the normal external standard method proved the consistency of the two methods. According to the outcomes of 30 batches of VB samples, the contents of the seven bufadienolides were used for further chemometric analysis. All of the samples of VB from various geographical origins were divided into three categories based on hierarchical cluster analysis and radar plot, which indicated the crucial influence of geographical origins on VB. This study showed that QAMS combined with chemometristry could be used to comprehensively evaluate and effectively control the quality of VB from different geographical origins.

Implementation of a Single Quadrupole Mass Spectrometer for Fingerprint Analysis: Venenum bufonis as a Case Study.

The mass spectrometry (MS) has been widely used for profiling chemical components of traditional Chinese medicine (TCM). However, there are few studies reporting quality control of TCM based on mass spectrometry fingerprint (MSF) due to its complicated operation and high cost. The aim of this study was to extend the application of MSF for quality evaluation of TCM. In this study, an MSF based on single quadrupole mass spectrometry method was established, and was successfully used for the quality control of Venenum bufonis (VB), a famous TCM which was used clinically for cancer treatment in China. The results showed that the superiority of MSF for more chemical information exposure and the finding of more potential chemical markers (eight versus four) compared with the traditional photo-diode array (a kind of ultra violet detector, PDA). Besides, the performance of MSF was also validated by similarity and principle component analysis (PCA) of MS data acquired on two other mass spectrometry (low-resolution, triple quadrupole, QQQ, and high-resolution, quadruple time-of-flight, Q-TOF), showing high consistency with QQQ and Q-TOF, but robustness with few parameters' settings. Based on our study, MSF could be widely applied for the quality control of TCM.

Quantification of bufadienolides in the poisons of Rhinella marina and Rhaebo guttatus by HPLC-UV.

Bufadienolides are the main active compounds in the Bufonidae family of frogs. Recent studies have demonstrated cytotoxic and/or antitumor activity in these molecules. A HPLC-DAD method was developed and validated to quantify three bufadienolides (telocinobufagin, marinobufagin and bufalin) in ethyl acetate extracts of the cane toad poison frogs and smooth-sided toad. The chromatographic analysis was performed on Phenomenex Luna C18 (250.0 × 4.6 mm, 5 µm), using gradient elution with acetonitrile and water, at a flow rate of 1.0 mL min(-1) and detection at 296 nm. The method showed linearity (r > 0.999) and adequate recovery values (86%-111%). The limits of quantification of bufadienolides were 7.4 µg mL(-1) for telocinobufagin, 4.2 µg mL(-1) for marinobufagin and 4.0 µg mL(-1) for bufalin. Intraday and interday values of the method were evaluated and presented standard deviation values lower than 5%. The method was successfully applied to quantify the bufadienolides in the venom extract of the cane toad, which showed a content of 60% of marinobufagin. The same method was not selective for the venom extract of the Rhaebo guttatus, despite being linear, accurate and precise, requiring the development of a technique that presents a greater selectivity.

[Identification of bufadienolides profiling in cinobufacino by HPLC-DAD-FT-ICR-MS method].

Cinobufacino injection is a significant anti-tumor medicine for the treatment of various tumors in clinic, which was made from water extraction of the skin of Bufo bufo gargarizans. In present paper, HPLC-DAD-FT-ICR-MS method was used to identify the major bufadienolides in cinobufacino for the first time. Solid-phase extraction with dichloromethane and silica was used to enrich the total bufadienolides in cinobufacino. Based on the UV and high resolution MS/MS data, 33 bufadienolides were analyzed and characterized. Among them, eight compounds were identified by comparing with standard references unambiguously. This study elucidated the major bufadienolides in cinobufacino, which provided material foundation of cinobufacino and will be benefit for the further pharmacological research.

Both high and low maternal salt intake in pregnancy alter kidney development in the offspring.

In humans, low glomerular numbers are related to hypertension, cardiovascular, and renal disease in adult life. The present study was designed 1) to explore whether above- or below-normal dietary salt intake during pregnancy influences nephron number and blood pressure in the offspring and 2) to identify potential mechanisms in kidney development modified by maternal sodium intake. Sprague-Dawley rats were fed low (0.07%)-, intermediate (0.51%)-, or high (3.0%)-sodium diets during pregnancy and lactation. The offspring were weaned at 4 wk and subsequently kept on a 0.51% sodium diet. The kidney structure was assessed at postnatal weeks 1 and 12 and the expression of proteins of interest at term and at week 1. Blood pressure was measured in male offspring by telemetry from postnatal month 2 to postnatal month 9. The numbers of glomeruli at weeks 1 and 12 were significantly lower and, in males, telemetrically measured mean arterial blood pressure after month 5 was higher in offspring of dams on a high- or low- compared with intermediate-sodium diet. A high-salt diet was paralleled by higher concentrations of marinobufagenin in the amniotic fluid and an increase in the expression of both sprouty-1 and glial cell-derived neutrophic factor in the offspring's kidney. The expression of FGF-10 was lower in offspring of dams on a low-sodium diet, and the expression of Pax-2 and FGF-2 was lower in offspring of dams on a high-sodium diet. Both excessively high and excessively low sodium intakes during pregnancy modify protein expression in offspring kidneys and reduce the final number of glomeruli, predisposing the risk of hypertension later in life.

Comparison of toad venoms from different Bufo species by HPLC and LC-DAD-MS/MS.

ETHNOPHARMACOLOGICAL RELEVANCE: Toad venom, called Chansu in China, has been widely used for the treatment of heart failure, sores, pains, and various cancers for a long time in clinic. AIM OF THE STUDY: The aim of the study is to investigate the chemical differences among a variety of toad venoms from different geographic locations and related Bufo species. MATERIALS AND METHODS: Ten batches of commercial toad venom collected from different regions in China, one batch of fresh toad venom obtained from Bufo bufo gargarizans, and six batches of related Bufo species were analyzed by HPLC and LC-DAD-MS/MS. Individual components were identified by comparison of retention times, UV spectra, and mass spectra with authentic compounds, standard addition, as well as summarized MS fragmentation rules. Based on the profile of identified constituents and the content of cinobufagin and resibufogenin, the chemical differences observed among different samples are discussed. RESULTS: Overall, 43 compounds were identified in the methanolic extracts of the different samples of toad venom. Besides of suberoyl arginine, several free bufadienolides, bufadienolide sulfates, and suberoyl esters of bufadienolides were found. The total amounts of cinobufagin and resibufogenin, which are the only two control markers according to the current Chinese Pharmacopoeia, varied widely from 0.7% to 10.9% in the commercial Chansu samples collected in the different locations in China. Low levels of resibufogenin, but no cinobufagin was observed in the samples from Bufo melanosticus and Bufo marinus, and even neither of both compounds was found in the sample from Bufo viridis. CONCLUSIONS: The chemical profiles of the different commercial and collected toad venoms from related Bufo species differed significantly, not only in the absolute and relative contents, but also in the number and type of the constituents. The main reason for this variation are species-specific differences, but additional factors, such as the harvest and post-harvest processing, and adaption to environmental factors in different geographic locations, also seem to contribute.

Rapid structural identification of cytotoxic bufadienolide sulfates in toad venom from Bufo melanosticus by LC-DAD-MS(n) and LC-SPE-NMR.

Toad venom, namely, "Chansu" in China, has been widely used for the treatment of heart failure, sores, pains, and various cancers. Upon LC-MS analysis of the venom from Bufo melanosticus collected in Indonesia, new bufadienolide sulfates were identified. For a complete characterization, the MeOH extract of the toad venom from B. melanosticus was fractionated by preparative HPLC, and the structures of five new buadienolide sulfates (1-5) along with one new bufogenin (6) were rapidly elucidated on the basis of LC-DAD-MS(n) and LC-SPE-NMR data. The in vitro growth inhibitory activity of these six compounds along with hellebrin (positive control) has been assayed by means of the MTT colorimetric assay in four human and two mouse cancer cell lines. Compound 3 and hellebrin displayed similar and marked in vitro cytotoxicity.

Simultaneous determination of cytotoxic bufadienolides in the Chinese medicine ChanSu by high-performance liquid chromatography coupled with photodiode array and mass spectrometry detections.

ChanSu (toad venom) is a traditional Chinese medicine for the treatment of serious liver and gastric cancers. The major cytotoxic compounds in ChanSu are bufadienolides. In this paper, a strategy combining qualitative LC/MS analysis and quantitative HPLC determination of major bufadienolides was used for global quality control of ChanSu crude drug. Majority of the bufadienolides in methanol extract of ChanSu were unambiguously characterized by high-performance liquid chromatography coupled with atmospheric pressure chemical ionization tandem mass spectrometry (HPLC/APCI-MS/MS), and by comparing with pure compounds. In addition, eight major bufadienolides were simultaneously determined in one single HPLC run within 30 min with photodiode array detection (DAD). All compounds showed good linearity in a wide concentration range, and their limits of detection (LOD) were around 1 ng. Thus, > 95% of the bufadienolides in ChanSu could be characterized, and > 90% of them were quantitated. The established method is rapid, simple and sensitive, and could be used for the routine analysis of ChanSu crude drug and its preparations. This research sets a good example for the comprehensive quality control of traditional medicine.

Analysis of bufadienolides in the Chinese drug ChanSu by high-performance liquid chromatography with atmospheric pressure chemical ionization tandem mass spectrometry.

The qualitative analysis of bufadienolides in the Chinese drug ChanSu was performed using high-performance liquid chromatography with atmospheric pressure chemical ionization tandem mass spectrometry (APCI-MS/MS). Bufadienolides are the major bioactive constituents of ChanSu, which is used to treat heart failure and cancer in traditional Chinese medicine. The APCI-MS fragmentation behavior of bufadienolides was studied. For bufadienolides with only hydroxyl substituents, the fragmentation was characterized by successive eliminations of H(2)O and CO molecules, and the profile of MS/MS product ions was correlated with the number of hydroxyl groups. If a C-16 acetoxyl group was present, the fragmentation of [M+H](+) ions was triggered by initial loss of 60 Da (HOAc). The elimination of CO was significant for bufadienolides with a 19-formyl group, and the 19-hydroxyl group could be characterized by the loss of 30 Da (HCHO). These fragmentation rules were applied to the identification of bufadienolides in a methanolic extract of ChanSu, which was separated on a C(18) column with gradient elution. A total of 35 bufadienolides were identified, including four new constituents. The method established here facilitated the convenient and rapid quality control of ChanSu crude drug and its pharmaceutical preparations.

Study on mechanism of action of Chinese medicine Chan Su: dose-dependent biphasic production of nitric oxide in trophoblastic BeWo cells.

BACKGROUND: Chan Su, a traditional Chinese medicine, is used for treating the heart diseases and other systemic illnesses. Our studies with animal model have revealed its role in increasing intracellular calcium concentration in cardiomyocytes. Nitric oxide (NO), a second messenger molecule, and its metabolites have been demonstrated to maintain and modulate multiple physiologic functions including the cardiovascular and reproductive systems. In order to explore the mechanism of action of Chan Su, we studied the ability of Chan Su to stimulate NO production in cultured trophoblastic BeWo cells. MATERIALS AND METHODS: BeWo cell is a cloned established cell line purified from human choriocarcinoma. These cells have some similarities in biological behavior with endothelial cells. Therefore, BeWo cell line may act as a model system for production of nitric oxide by Chan Su both in placenta and in cardiovascular tissue, and the results can easily be extrapolated to cardiomyocytes. Very small amount of ethanol extract of Chan Su was added to the cultured cells in KBM buffer and a chemiluminescence system was used for the measurement of nitric oxide. The amounts of Chan Su extract added to cultured cells were comparable to expected level of Chan Su in human serum after ingestion. We also repeated these experiments with bufalin, the active component of Chan Su. RESULTS: The ethanol extract of Chan Su (5 and 10 microg/ml) significantly increased NO production up to 110% of basal control value, but higher concentrations (40 and 80 microg/ml) of Chan Su (as expected in an overdose) resulted in decreased NO production below the control level. This biphasic effect on nitric oxide production was also observed with bufalin, the active component of Chan Su responsible for its digoxin-like immunoreactivity. The presence of bufalin in Chan Su preparation was confirmed by thin layer chromatography (TLC) analysis. CONCLUSIONS: Chan Su as well as bufalin is able to modulate the production of NO in BeWo cell line.

Endogenous marinobufagenin-like immunoreactive substance. A possible endogenous Na, K-ATPase inhibitor with vasoconstrictor activity.

Vasoconstrictor and Na/K pump inhibitory properties of a bufodienolide Na/K-ATPase inhibitor, marinobufagenin, were studied in isolated rings of 2 to 3 order branches of human pulmonary arteries respectively. Marinobufagenin displayed concentration-dependent vasoconstrictor activity (0.01 to 10 mmol/L). In sarcolemma membranes prepared from pulmonary artery marinobufagenin inhibited Na/K-ATPase (IC50 = 50 nmol/L). In eight healthy male Caucasians, concentrations of marinobufagenin-like immunoreactive material in C-18 extracted plasma were 1.38 +/- 0.60 nmol/L. Twenty-four-hour urinary release of marinobufagenin-like immunoreactive material in eight healthy males was 1.20 +/- 0.95 nmol/day. Chloroform extract of human urine was fractionated using reverse-phase high-performance liquid chromatography (32% acetonitrile, Deltapak). The HPLC fraction coeluting with marinobufagenin in 7 min, cross reacted with antimarinobufagenin and antidigoxin, but not antiouabain antibody. These results demonstrate that human plasma and urine contains a bufodienolide vasoconstrictor EDLF, marinobufagenin-like immunoreactive Na,K pump inhibitor.

Structures of novel bufadienolides in the eggs of a toad, Bufo marinus.

In this paper, we report chemical structures of five compounds including four novel polyhydroxylated cardiac steroids in the eggs of a toad, Bufo marinus. These cardiac steroids were purified by high-performance liquid chromatography, and their structures were determined to be 11 alpha,19-dihydroxy-telocinobufagin (I), 11 alpha-hydroxytelocinobufagin (II), 11 alpha,19-dihydroxymarinobufagin (III), 11 alpha-hydroxymarinobufagin (IV) and 19-hydroxytelocinobufagin (V) on the basis of spectral data of nuclear magnetic resonance and mass spectroscopy. All the five compounds showed biological activity, as tested by inhibition of Na+,K(+)-ATPase activity and of [3H]ouabain binding to the receptor on Na+,K(+)-ATPase. This is the first finding of bufadienolides as cardiac steroids in animal eggs.

Identification of digitalis-like compounds in human cataractous lenses.

Human cataractous lens nuclei extract inhibited, in a dose-dependent fashion, [3H]ouabain binding to rat brain synaptosomes and microsomal Na(+)- and K(+)-dependent adenosine triphosphate (Na+, K(+)-ATPase) activity and interacted with anti-digoxin antibodies. The compounds responsible for these activities, termed digitalis-like compounds (DLC), were also detected in bovine, rat, cat and rabbit, normal, transparent lenses, but the levels were only 0.7-5.4% of the average levels in the cataractous human lenses. DLC from the human cataractous lenses were purified by a procedure consisting of organic extractions and batch chromatography followed by filtration through a 3000 Da cut-off filter and subsequent separations using reverse-phase high-performance liquid chromatography. The presence of DLC in the different fractions obtained in the chromatograms was monitored by their ability to inhibit [3H]ouabain binding and Na+, K(+)-ATPase activity. Based on chemical ionization mass spectrometry together with ultraviolet spectrometry and biological characterization, it is suggested that new bufodienolides, 19-norbufalin and 19-norbufalin peptide derivatives are responsible for the endogenous DLC activity. It is proposed that these compounds may regulate Na+, K(+)-ATPase activity in the lens under some physiological and pathological conditions.