Multi-component identification and target cell-based screening of potential bioactive compounds in toad venom by UPLC coupled with high-resolution LTQ-Orbitrap MS and high-sensitivity Qtrap MS

Traditional Chinese medicines (TCMs) are undoubtedly treasured natural resources for discovering effective medicines in treating and preventing various diseases. However, it is still extremely difficult for screening the bioactive compounds due to the tremendous constituents in TCMs. In this work, the chemical composition of toad venom was comprehensively analyzed using ultra-high performance liquid chromatography (UPLC) coupled with high-resolution LTQ-Orbitrap mass spectrometry and 93 compounds were detected. Among them, 17 constituents were confirmed by standard substances and 8 constituents were detected in toad venom for the first time. Further, a compound database of toad venom containing the fullest compounds was further constructed using UPLC coupled with high-sensitivity Qtrap MS. Then a target cell-based approach for screening potential bioactive compounds from toad venom was developed by analyzing the target cell extracts. The reliability of this method was validated by negative controls and positive controls. In total, 17 components in toad venom were discovered to interact with the target cancer cells. Further, in vitro pharmacological trials were performed to confirm the anti-cancer activity of four of them. The results showed that the six bufogenins and seven bufotoxins detected in our research represented a promising resource to explore bufogenins/bufotoxins-based anticancer agents with low cardiotoxic effect. The target cell-based screening method coupled with the compound database of toad venom constructed by UPLC-Qtrap-MS with high sensitivity provide us a new strategy to rapidly screen and identify the potential bioactive constituents with low content in natural products, which was beneficial for drug discovery from other TCMs.ᅟGraphical abstract

Toad venom; Multi-component analysis; Target cell-based screening; UPLC-Orbitrap-MS; UPLC-Qtrap-MS

For thousands of years, traditional Chinese medicines (TCMs) have been applied for the treatment of many diseases including various kinds of cancers in China and beyond [[1] ]. TCMs are undoubtedly treasured natural resources for discovering effective medicines in treating and preventing various diseases [[2] , [3] ]. However, the bioactive compounds in TCMs are usually ambiguous and trace, which lead to a blurry pharmacodynamic mechanism, therefore, hindering the developments and applications of TCMs. So far, it is still extremely difficult for screening the bioactive compounds in TCMs because there are plenty of constituents making it full of challenges to single out “the good one or ones” in TCMs.

The conventional approach for screening bioactive constituents includes extraction, isolation, and then pharmacological evaluation in vivo and in vitro, which is time and sample consuming, labor intensive, expensive, and inefficient. Recently, based on the hypothesis that drugs should bind with some receptors or enzymes on cell membranes or enter the target cells to elicit activity, the target cell-based screening method has been used for screening of potential bioactive components in the extract of plant herbs or TCMs [[4] -[6] ], exhibiting potential to screen for bioactive constituents in TCMs. However, for traces of chemicals in plant herbs or TCMs, they may be ignored if the analytical tools do not have enough detection sensitivity. Meanwhile, the analytical tools used in the screening method have undergone evolution from common liquid chromatography (LC) [[7] ], liquid chromatography mass spectrometry (LC-MS) [[8] -[10] ] to liquid chromatography high-resolution mass spectrometry (LC-HR-MS) [[6] , [11] ], and liquid chromatography tandem mass spectrometry (LC-MS/MS) [[12] , [13] ]. LC-HR-MS has been used extensively in compound identification owing to its high mass accuracy and reliable constituent identification [[14] , [15] ]. In addition, coupling of LC with targeted multiple reactions monitoring (MRM) mode from triple quadrupole or Qtrap MS shows high sensitivity, specificity, and selectivity in the simultaneous identification and quantitation of compounds in complex matrices [[16] ].

Among the TCMs, poisonous Chinese herbal medicine toad venom gained increasing attention for cancer therapy [[17] -[19] ]. Toad venom can induce apoptotic death of various kinds of cancer cell lines in vitro and in vivo [[20] -[22] ]. In clinic, the toad venom preparation cinobufacini was effective for the treatment of hepatocellular carcinoma, nonsmall-cell lung cancer, and pancreatic cancer [[23] -[26] ]. Toad venom contains several classes of constituents including indole alkaloids, amino acids, cardioactive steroids (bufogenins), and amino acid-conjugated bufogenins (bufotoxins) [[27] -[29] ]. The compounds in toad venom were reported to possess a great potential to search for new compounds with a wide range of biomedical applications [[30] ], which could constitute a promising bio-resource for the development of potential drugs and value-added products [[31] ]. However, due to the complicated components, it is difficult to rapidly identify the active ingredients, and the number of constituents identified in toad venom is limited [[32] -[34] ]. Thus, comprehensive characterization and identification of chemical constituents and screening of the potential active compounds in toad venom are essential for scientifically robust investigation of its pharmacological activity and targeted development of potential therapies.

This work aims to screen for the potential bioactive components in toad venom that act against cancer with the target cell-based screening method. HepG2 and MCF-7 cells were selected as the target cells. Seventeen compounds were detected to bind with the cancer cells by the constructed compound database of toad venom that contains 93 constituents. Further, an MTT assay was performed to verify the anti-cancer activity. Among the 17 detected compounds, 6 bufogenins and 7 bufotoxins in toad venom represented a promising resource to search for anticancer agents, which should be further investigated.

The dried toad secretion derived from Bufo melanostictus Schneider was collected from Beijing newborn toad breeding center (Beijing, China) and was authenticated as toad venom (voucher specimen number: TV-0811) by professor Shilin Hu from Institute of Chinese Materia Medica, China Academy of Chinese Medical Sciences (Beijing, China). Captopril (CA), nifedipine (NI), paclitaxel (PA), and sorafini (SO) were purchased from Sigma Aldrich (St. Louis, MO, USA). Nominin (NO), serotonin, arenobufagin, bufalin, cinobufagin, and resibufogenin were purchased from Baoji Herbest Bio-Tech Co., Ltd. (Baoji, China). Hellebrigenol, 16-desacetyl-19-oxo-cinobufotalin, Ψ-bufarenogin, gamabufotalin, hellebrigenin, desacetylcinobufotalin, bufotalinin, argentinogenin, telocinobufagin, bufotalin, desacetylcinobufagin, and cinobufotalin were isolated from the toad skin in our laboratory and identified by HR-ESI-MS and NMR techniques. Purities of all compounds were above 96% by HPLC analysis.

HepG2 cells and MCF-7 cells were obtained from the national experimental cell resource sharing platform (Beijing, China). Dulbecco’s modified eagle medium (DMEM, LOT: 8116489), fetal bovine serum (FBS, LOT: 1527494), and trypsin (LOT: FGK0652) were purchased from Invitrogen Gibco (Carlsbad, CA, USA). HPLC grade methanol, acetonitrile, and MS grade formic acid were purchased from Thermo Fisher Scientific (Pittsburgh, PA, USA). Ultrapure water was obtained from a Milli-Q water purification system (Millipore Corporation, Billerica, MA, USA). Other chemicals and solvents were of analytical reagent grade.

A mixed standard solution containing 2 mg/mL of CA, 10 mg/mL of NI, 20 mg/mL of PA, and 1 mg/mL of SO was prepared in dimethyl sulfoxide (DMSO), and then the mixed standard was diluted with DMEM to the required concentrations prior to the target cell-based bioactivity screening. NO (internal standard, IS) was dissolved in methanol at a concentration of 5 μg/mL. Serotonin was dissolved in pure water at a concentration of 10 μg/mL. Arenobufagin, bufalin, cinobufagin, resibufogenin, hellebrigenol, 16-desacetyl-19-oxo-cinobufotalin, Ψ-bufarenogin, gamabufotalin, hellebrigenin, desacetylcinobufotalin, bufotalinin, argentinogenin, telocinobufagin, bufotalin, desacetylcinobufagin, and cinobufotalin were dissolved in DMSO respectively at a concentration of 1 mg/mL and diluted with methanol to a concentration of 10 μg/mL.

Toad venom was pulverized into powder. Then, 40 g of the powder was extracted twice by heat-reflux with ethanol (400 mL, 1 h; 320 mL, 1 h) and once with pure water (400 mL, 1 h). The combined extract was evaporated under vacuum and lyophilized (26.8 g, extract rate: 67%). The working solution of toad venom (200 mg/mL) was prepared by dissolving the freeze-dried powder of toad venom extract in DMSO. Two parallel samples of toad venom extract were prepared at a final concentration of 1 mg/mL. One was diluted in methanol, and then passed through a 0.22 μm filter prior to analysis by UPLC-HR-MS. The other was diluted in DMEM under a sterile environment, which was used for the target cell-based screening.

The ultimate 3000 hyperbaric LC system coupled with high-resolution LTQ-Orbitrap XL MS via an electrospray ionization (ESI) interface from Thermo Fisher Scientific (Bremen, Germany) was used for a comprehensive analysis of the constituents in toad venom extract. The chromatography system was equipped with an auto-sampler, a diode-array detector, a column compartment, and two pumps. The chromatographic conditions were optimized and a CSH C18 column (1.7 μm, 2.1 mm ID × 100 mm, Waters) maintained at 35 °C was finally chosen for separation of toad venom extract. The mobile phase was composed of water (0.1% formic acid, A) mixed in gradient mode with acetonitrile (B), at a flow rate of 150 μL/min. The elution gradient was optimized as follows: 0-3 min, 3% B; 3-5 min, 3% to 28% B; 5-12 min, 28% B; 12-22 min, 28 to 35% B; 22-30 min, 35 to 100% B; 30-32 min, 100% B. The injection volume was 3.0 μL and the sampler was set at 4 °C.

For identification of the components in toad venom, full scans mode within the range of m/z (mass/charge ratio) 100-1500 at a resolution of 30,000 was used for acquisition of accurate molecular ion (mass error < 5 ppm). The fragment ions in MS/MS data obtained by collision-induced dissociation (CID, collision energy: 35 eV) were further utilized for confirmation of the structures of the components. In addition, standards were also used for assistance of identification of the components, especially the isomers.

To achieve a comprehensive screening with high sensitivity, a compound database of toad venom was developed using MRM mode from an AB Sciex QTrap® 4500 tandem MS, (Foster, CA, USA) equipped with an ESI source connected to the UPLC system (I-class Acquity ultra performance liquid chromatography, Waters). Firstly, an instrument method in MRM (Q1 = Q3) information-dependent acquisition (IDA)-enhanced product ion (EPI) mode of toad venom as reported in our previous research [[16] ] was established on the basis of the identification of compounds in toad venom by UPLC-HR-MS. Ultra-pure nitrogen (N2) was used as the nebulizing and sheath gas. Product ion scanning experiments were conducted using ultra-high-purity N2 as collision gas and the collision energy (CE) was optimized for each analyte to generate the most abundant product ions. The product ion spectra were further used to select the precursor-product ion pairs for the development of MRM assays. Further, a compound database of toad venom was established based on the quasimolecular ion in Q1 and its characteristic fragment ions in Q3 as ion pair in MRM mode to screen for the active compounds in toad venom. In addition, quantification of CA, NI, PA, and SO using UPLC-Qtrap-MS was also established to validate the reliability of the cell-based bioactivity screening method. The declustering potential (DP) was optimized based on the standards. The liquid chromatographic conditions were the same as those of UPLC-HR-MS analysis. Typical operating Qtrap MS parameters were set as follows: collision gas (CAD) medium, curtain gas (CUR) 25, ion source gas1 (GS1) 45, ion source gas2 (GS2) 50, temperature 550 °C, and electrospray voltage 5500 V. All of the experiments were operated in the positive ionization mode.

After overall identification of constituents and successful compound database construction of toad venom, the target cell-based screening assay was performed. The screening scheme (shown in Fig. 1) includes incubation (drug-cell interaction), washing (unbounded compounds on the cell surface were washed away), extraction (the potential active components were extracted with organic solvent), and LC-MS analysis [[6] , [35] -[37] ].Scheme of the analytical procedure for the identification of the target cell-based screening of bioactive constituents from toad venom extract

HepG2 cells and MCF-7 cells in the logarithmic growth phase were seeded into cell culture flasks at a density of 1.0 × 106 cells/mL, and were cultured in DMEM medium in a humidified 5% CO2 incubator at 37 °C for 24 h. The culture medium was replaced by 3 mL of toad venom extract diluted in DMEM (free of serum) at a final concentration of 1 mg/mL, and incubated at 37 °C for 4 h. The incubation solution was discarded and then the cells were washed five times with PBS to remove unbounded components. The eluates were discarded except for the last one, which was collected as a control for LC-MS analysis. Finally, the cells were collected and extracted with 200 μL of methanol containing 5 μg/mL of NO by ultrasonic extraction for 5 min. After centrifugation at 12,000 rpm for 10 min, the obtained supernatant was used for UPLC-Qtrap-MS analysis. The control samples free of drugs were prepared using the same procedures as above. The mixed standard solutions containing CA, NI, PA, and SO at various concentrations were prepared using the same procedures as above.

For cytotoxicity assay, MCF-7 cells and HepG2 cells in logarithmic growth were plated in 96-well plates at a density of 6.0 × 103 cells per well in 100 μL of culture medium and were allowed to adhere for 24 h before treatment. Serial concentrations of each sample (serotonin, arenobufagin, cinobufagin, and resibufogenin) were then added (100 μL per well). After treated for 48 h, MTT solution (10 μL per well, 5 mg/mL) was added to each well and incubated for 4 h at 37 °C. The supernatant was then carefully removed, and 150 μL of dimethyl sulfoxide was added to each well. After the formazan crystals had dissolved completely, optical density at 570 nm was determined with a SpectraMax 190 microplate reader (Molecular Devices, Sunnyvale, CA, USA). Dose-response curves were obtained, and the IC50 values were calculated by OriginPro 8.0.

In the UPLC-HR-MS analysis, the accurate mass and composition for the precursor ions and fragment product ions were analyzed using Xcalibur 3.0 software package. Internal calibration by infusion of a calibrant achieved typical mass accuracy within 5 ppm before the experiment. The identification of the compounds in toad venom extract was performed based on the retention time, high-resolution Orbitrap MSn data, isotope abundance, fragment product ions, literature data, and standard substances. In our present work, constituents in toad venom extract in the negative and positive ion modes were investigated, and the positive ion mode was more suitable for the component analysis in toad venom (see Electronic Supplementary Material (ESM) Fig. S1). In total, we characterized 93 compounds in toad venom in the positive ion mode by UPLC-HR-MS, which were much more than those reported in the previous reports [[29] , [32] , [38] -[40] ]. Among the 93 compounds detected, 17 compounds were confirmed definitely by standard substances (Fig. 2) and 8 compounds including 4, 12, 14, 18, 19, 82, 89, and 90 identified as minor constituents in toad venom were reported for the first time. The identification results and their UPLC-HR-MS characteristics are listed in Table S1 in the ESM.Total ion chromatograms (TICs) of toad venom extract and extracted ion chromatograms (XICs) of the 17 standard substances in the positive ion mode using UPLC-HR-MS

In total, nine amino acids were detected and/or tentatively identified based on their HR-MSn data. Compounds 3, 10, 11, 13, 16, and 17 showed the diacid loss leading to the arginine moiety (1) and were identified as succinyl (C4 diacid), adipoyl (C6 diacid), pimeloyl (C7 diacid), suberoyl (C8 diacid), sebacyl (C10 diacid), and azelayl (C9 diacid) arginine, respectively. To be specific, compound 11 (tR = 5.7 min) was detected with [M + H]+ ion at m/z 317.1819 (C13H25O5N4, Cal. 317.1820, error − 0.3 ppm). Its product ions at m/z 282.1453, 264.1348, 236.1405, and 175.1193 were detected (Fig. 3a). By comparing its HR-MSn data with the literatures [[39] -[41] ], compound 11 was characterized as pitneloyl arginine. Accurate mass measurement showed that the molecular formulas of compounds 4 and 12 were C10H16O4N4 and C20H29O5N6, respectively, based on their [M + H]+ and [M + Na]+ ions in the full mass spectra (ESM Table S1). The MS/MS fragment of compound 12 at m/z 175.1195 (ESM Fig. S2A) indicated that 12 was a arginine derivative. The structures of the seven amino acids were shown in Fig. 4a.The representative HR-Orbitrap MS/MS spectra of a 11 (Pitneloyl arginine), b 5 (Serotonin), c 85 (Cinobufagin), and d 80 (Cinobufotoxin)Chemical structures of representative amino acids a, alkaloids b, bufogenins (c: 14-hydroxy bufogenins; d 14,15-epoxy bufogenins; e other bufogenins) and bufotoxins (f 14-hydroxy bufotoxins; g 14,15-epoxy bufotoxins) identified in toad venom

Among the 93 compounds detected, 7 indole alkaloids (5-9, 15, and 18) were observed and 6 out of them were identified as indolealkylamines based on their HR-MS/MS spectra. Compounds 5-8 showed the loss of alkylamine side chain leaving a propene group to give the ion at m/z 160.0757 and were identified as serotonin, N′-methyl serotonin, bufotenidine, and bufotenine, respectively. Among them, compound 5 was confirmed as serotonin by standard. It was detected at retention time (tR) = 2.6 min with [M + H]+ ion at m/z 177.1022 (C10H13ON2, Cal. 177.1021, error − 0.05 ppm). In the MS/MS analysis, the product ion of compound 5 at m/z 160.0757 by the loss of NH3 was detected (Fig. 3b). The alkaloids 9 and 15 were detected in agreement with dehydrobufotenin previously reported from the Amazonian toad Rhinella marina [[39] ] and bufothionine from species of the family Bufonidae [[30] ]. Compound 18 was diluted at tR = 8.6 min with [M + H]+ ion at m/z 277.1182 (C14H17O4N2, Cal. 277.1183, error − 0.45 ppm) and [M + Na]+ ion at m/z 299.1001 (C14H16O4N2Na, Cal. 299.1002, error − 0.43 ppm) in the full mass spectrum. In its MS/MS analysis, the product ion at m/z 160.0760, which was the characteristic fragment of indole alkaloid in toad venom, was detected (ESM Fig. S2B), indicating that compound 18 was an indole alkaloid. The structures of the 6 indole alkaloids were shown in Fig. 4b.

Bufogenins were widely investigated in recent years because of their pharmacological activities. In our present study, a total of 40 bufogenins were identified and 16 of them including 23, 25, 26, 28, 36, 41, 43, 50, 57, 63, 66, 67, 75, 81, 85, and 86 were confirmed as hellebrigenol, 16-desacetyl-19-oxo-cinobufotalin, Ψ-bufarenogin, gamabufotalin, arenobufagin, hellebrigenin, desacetylcinobufotalin, bufotalinin, argentinogenin, telocinobufagin, bufotalin, desacetylcinobufagin, cinobufotalin, bufalin, cinobufagin, and resibufogenin, respectively by standard substances (Fig. 2). For example, 85 was eluted at tR = 28.0 min with [M + H]+ ion at m/z 443.2420 (C26H35O6, Cal. 443.2428, error − 1.77 ppm) in the full mass spectrum. In its MS/MS spectrum, the common neutral losses of [M + H-C2H2O]+, [M + H-C2H4O2-H2O]+, and [M + H-C2H4O2-2H2O]+ fragmentations at m/z 401.2333, 365.2122, and 347.2015 were observed (Fig. 3c). The tR and HR-MSn data of 85 were the same as those of standard substance cinobufagin. Thus, 85 was confirmed as cinobufagin. Among the 40 bufogenins detected, compounds 82, 89, and 90 were detected in toad venom for the first time. Compound 82 was diluted at tR = 26.8 min with [M + H]+ ion at m/z 473.2531 (C27H37O7, Cal. 473.2534, error − 0.51 ppm) in the full mass spectrum. The molecular weight of 82 was 14 Da (CH2) more than that of 64 (12β-hydroxylcinobufagin), indicating that 82 might be a methylation product of 12β-hydroxylcinobufagin. In its MS/MS spectrum, the product ions of 82 at m/z 417.2283, 399.2174, 381.2068, 363.1964, and 345.1858 (ESM Fig. S2C) were the same as those of 12β-hydroxylcinobufagin (ESM Fig. S2D). By comparison with the proposed fragmentation pathway of 12β-hydroxylcinobufagin, the methylation site of 82 was inferred to be on the ester methyl at C16 (ESM Fig. S3A). Compound 89 (tR = 28.8 min) was detected with [M + H]+ ion at m/z 457.2582 (C27H37O6, Cal. 457.2585, error − 0.69 ppm), which was 14 Da (CH2) higher than that of cinobufagin at m/z 443.2420, suggesting that 89 might be a methylation product of cinobufagin. Further, the MS/MS fragments of 89 at m/z 401.2332, 383.2227, 365.2122, 347.2014, and 319.2061 (ESM Fig. S2E) were identical to those of cinobufagin (Fig. 3c), indicating that the methylation site occurred on the unique ester methyl at C16 (ESM Fig. S3B). Compound 90 (tR = 28.9 min) was observed with [M + H]+ ion at m/z 423.2165 (C26H31O5, Cal. 423.2166, error − 0.33 ppm), which was 2 Da (2H) less than that of 93 (dehydro-cinobufagin), demonstrating that 90 might be a dehydrogenation product of dehydrated-cinobufagin. The MS2 fragments of 90 at m/z 405.2069, 381.2071, 363.1965, and 345.1858 (ESM Fig. S2F) were all 2 Da (2H) less than the corresponding fragments of 93 (dehydrated-cinobufagin) at m/z 407.2226, 383.2228, 365.2122, and 347.2015 (ESM Fig. S2G), which supported the deduction that 90 was a dehydrogenation product of dehydrated-cinobufagin. Similarly, the other 21 bufogenins were identified based on their tR, HR-MSn data, and by comparison with the reported literature data (ESM Table S1). For example, compounds 52, 67, 74, and 84 were detected with the same [M + H]+ ion at m/z 401.2319 (C24H33O5, Cal. 423.2323, error − 0.80 ppm). Among them, 67 was confirmed as desacetylcinobufagin by standard substance and compounds 52, 74, and 84 were identified as marinobufagin, 19-oxo-bufalin, and resibufaginol, respectively, based on their tR and HR-MSn data. Among the 40 bufogenins, 20 bufogenins belong to 14-hydroxy bufogenins (Fig. 4c, e) while the other 20 bufogenins belong to 14,15-epoxy bufogenins (Fig. 4d, e).

On the analysis of amino acid-conjugated bufogenins (bufotoxins) in toad venom, a total of 34 bufotoxins were detected in this paper. Compound 80 displayed [M + H]+ ion at m/z 755.4240 (C40H59O10N4, Cal. 755.4246; error − 0.76 ppm) in the positive ion mode. In its MS/MS spectrum, the characteristic product ion at m/z 331.1982 by the loss of 424 Da was detected (Fig. 3d), demonstrating the existence of one suberoyl arginine in 80. Accurate mass measurement showed that the formula of the lossed 424 Da was C26H32O5, which was 18 Da (H2O) less than the molecular weight of cinobufagin. Thus, 80 was tentatively inferred as 3-(N-suberoyl argininyl) cinobufagin. Further, the [M + H-C2H4O2]+ and [M + H-C2H4O2-H2O]+ fragments at m/z 695.4021 and 677.3911, which were similar to the fragmentation pattern of cinobufagin (Fig. 3c) and the same as those of the published literatures [[32] , [33] , [41] ], assured its structure as 3-(N-suberoyl argininyl) cinobufagin (Cinobufotoxin). Analogously, the other bufotoxins, were identified based on their HR-MSn data and confirmed by comparison with the literatures. In the fragmentation pattern analysis of bufotoxins, the characteristic product ions (acid residues) attributed to the loss of bufogenins were observed, which could help identification of bufotoxins in toad venom. For example, compounds 30, 40, 55, and 60 were succinic acid conjugates of bufogenins as the succinic acid residue (m/z 275.1357) was detected in their MS/MS fragments. Similarly, 2 glutaric acid conjugates (49 and 62), 6 adipic acids conjugates (22, 32, 42, 56, 61, and 72), 5 pimelic acids conjugates (27, 38, 68, 77, and 78), 14 suberic acids conjugates (33-35, 39, 45, 46, 51, 58, 59, 65, 70, 76, 79, and 80), and 1 azelic acid conjugate of bufogenins (83) were identified based on their high-resolution full MS data and characteristic product ions. Among the 34 bufotoxins detected, the structures of 33 bufotoxins were confirmed and 16 of them belong to 14-hydroxy bufotoxins (Fig. 4f) while the other 17 bufotoxins belong to 14,15-epoxy bufotoxins (Fig. 4g).

Additionally, three compounds (2, 14, and 19) were detected in toad venom and 14 and 19 were detected in toad venom for the first time. Compounds 14 (tR = 7.5 min) and 19 (tR = 9.0 min) were detected with [M + H]+ ion at m/z 340.1855 (C16H26O5N3, Cal. 340.1867; error − 3.46 ppm) and m/z 211.1438 (C11H19O2N2, Cal. 211.1441; error − 1.39 ppm), respectively. MS/MS spectra of 14 generated the [M + H-H2O]+, [M + H-CO2]+, and [M + H-H2O-CO2]+ product ions at m/z 322.1770, 296.1980, and 278.1875 (ESM Fig. S4A), demonstrating the existence of one hydroxyl group and one carbanyl group. The product ions of 19 at m/z 194.1183, 183.1498, and 155.1546 by the neutral loss of NH3, CO, and 2CO (ESM Fig. S4B), indicating the existence of one amidogen group and two carbanyl groups in 19.

Though HR-MS has the advantages of high accuracy for the identification of chemicals, its quantitative or semi-quantitative sensitivity is limited. As reported, UPLC-Qtrap-MS in MRM mode shows high sensitivity, specificity, and selectivity in the quantitation of trace compounds in complex matrices [[16] ]. In the present study, a compound database of toad venom containing 93 chemicals with high sensitivity and selectivity was successfully established for the first time using UPLC-Qtrap-MS in MRM mode on the basis of the identified constituents in toad venom. The MRM ion pairs and corresponding DP and CE of each constituent were optimized and presented in Table S2 (see ESM).

To obtain reliable screening results, the reliability of the target cell-based screening method was evaluated using a mixed standard solution composed of captopril (CA), nifedipine (NI), paclitaxel (PA), and sorafini (SO). CA is an angiotensin-converting enzyme (ACE) antagonist and NI is a calcium channel receptor (CCR) antagonist. They act selectively at cell membranes expressing abundant ACE [[42] ] and CCR [[43] ]. PA and SO are two wide-spectrum anticancer drugs with experimental and clinical pharmacology towards a variety of solid tumors such as hepatocellular carcinoma and breast cancers [[44] ]. In theory, when incubated with cancer cells, PA and SO would interact with cancer cells tightly while CA and NI would not. Thus, CA and NI were selected as negative controls (N) while PA and SO as positive controls (P) to validate the cancer cell-based screening method.

UPLC-Qtrap-MS/MS parameters for quantification of CA, NI, PA, and SO were optimized based on the standards (ESM Table S3). The typical TIC of the mixed standards was presented in Fig. 5a. To test the reliability of the cell-based screening method, the mixed standard solutions at various concentrations were incubated with HepG2 cells and MCF-7 cells individually for 4 h, and after extraction, the supernatants were detected by UPLC-Qtrap MS/MS. The TIC of the extract of HepG2 cells incubated with a mixed standard solution was shown in Fig. 5b. Compared with the control, PA and SO at tR = 28.9 and 29.6 min respectively, were detected in the extract of HepG2 cells, whereas CA and NI were not detected in the screening procedure. Similarly, PA and SO were detected in the extract of MCF-7 cells while absence of CA and NI (ESM Fig. S5). The results clearly demonstrated specific binding of PA and SO to cancer cells. Further, cancer cells incubated with various concentrations of mixed standards were investigated. As shown in Fig. S6 (see ESM), PA and SO were detected after incubation with cancer cells even at the lower concentration (20 ng/mL of PA and 1 ng/mL of SO), demonstrating high sensitivity and selectivity of the UPLC-Qtrap-MS in MRM mode to screen for trace bioactive compounds in complex mixtures. The cell binding degree (CBD) of PA and SO was calculated using the following formula:CBD=AiPa/PisPb/Pis×100, where Pa and Pb are the peak area of compounds after and before incubation with target cancer cells, respectively, and Pis is the peak area of NO (IS). It showed that the CBD of 200 ng/mL of PA to HepG2 cells and MCF-7 cells is 1.2 ± 0.3 and 13.5 ± 2.1%, respectively. And the CBD of 10 ng/mL of SO to HepG2 cells and MCF-7 cells is 8.1 ± 1.1 and 10.2 ± 2.3%, respectively.Typical TICs of the five standards using UPLC-Qtrap-MS. a Mixed standards of NO, CA, NI, PA, and SO (black line). b The extract of HepG2 cells incubated with 100 ng/mL of CA, 20 ng/mL of NI, 200 ng/mL of PA, and 10 ng/mL of SO (red line), the extract of HepG2 cells incubated without four standards (cyan line) and the fifth eluate (blue line)

In the screening experiment, factors that influencing the drug-cell interaction such as drug concentration, cell quantity, incubation time, washing times, and extraction method were investigated carefully. The optimized conditions were as follows: concentration of toad venom, 1 mg/mL; incubation time, 4 h; five washes with PBS buffer; extraction with methanol.

After compound database construction of toad venom and successful validation of the target cell-based screening method using the positive and negative controls, target cell-based screening of potential bioactive compounds in toad venom was performed as described in “UPLC-HR-MS analysis” section using UPLC-Qtrap-MS in MRM mode. The total ion chromatography (TIC) of the compound database of toad venom containing 93 chemicals was shown in Fig. S7A. The typical TIC of the extract of HepG2 cells and MCF-7 cells incubated with toad venom extract are shown in Figs. S7B and S7C, respectively (see ESM). In general, when cells are incubated with drugs, the bioactive molecules may selectively bind with the cells or be transported into the cells [[35] , [36] ]. As can be seen from Fig. S7, some relatively high abundance compounds (tR eluated from 6 to 20 min) were not detected in the target-cell extract samples. However, some components with low content are detected after incubation with cancer cells, indicating that the interaction between cancer cells and drugs is selective and the proposed compound database has suitable sensitivity to screen for trace bioactive compounds. Totally, 17 compounds including 3 alkaloids (A1-A3), 1 amino acid (A4), 6 bufogenins, and 7 bufotoxins (B1-B13) in toad venom were discovered to interact with the target cancer cells by comparing the chromatograms between target cell-extract groups (red line in ESM Figs. S7B and S7C) with the control samples (cyan line in ESM Figs. S7B and S7C). The peak area of each compound was obtained to calculate the cell binding degree (CBD) of these compounds. Extracted ion chromatograms of B1-B13 using UPLC-Qtrap-MS were shown in Fig. S8 (see ESM). Based on the compound database of toad venom and standards, the 17 peaks were identified (ESM Table S4) and their structures are provided in Fig. 6.The chemical structures of the 17 potential bioactive constituents identified from the toad venom extract

Further, an MTT assay was carried out to evaluate the anti-cancer activity of the identified compounds including 1 alkaloid (serotonin) and 3 bufogenins (arenobufagin, cinobufagin, and resibufogenin). As shown in Fig. 7a and ESM Fig. S9A, serotonin (A1) showed no cytotoxicity toward MCF-7 and HepG-2 cancer cells even at the concentration of 5000 ng/mL. As we know, serotonin was an endogenous substance; it could interact with the serotonin receptors such as 5-HT1 and 5-HT2 receptors [[45] ] in the cancer cells, resulting in the high CBD of serotonin in cancer cells. Minor suberoyl arginine (A4) was detected in the extract of cancer cells, but the CBD of suberoyl arginine (A4) was very low among the 17 compounds. Thus, we deduce that serotonin (A1), N-methyl serotonin (A2), bufotenidine (A3), and suberoyl arginine (A4) in toad venom may not be the main active compounds for the treatment of cancers.The cytotoxicity of serotonin (a), arenobufagin (b), cinobufagin (c), and resibufogenin (d) toward MCF-7 cancer cells

Bufogenins have been increasingly regarded as a source of promising anticancer agents due to their potent pharmacological activities [[46] ]. In our present study, 6 bufogenins including arenobufagin (B1), bufotalin (B3), cinobufotalin (B5), bufalin (B8), cinobufagin (B12), and resibufogenin (B13) were detected to bind with cancer cells. In the MTT assay, arenobufagin (B1) exhibited the most cytotoxicity toward MCF-7 cells with the IC50 value of 2.78 ± 0.18 ng/mL, followed by cinobufagin (B12, IC50 = 15.19 ± 3.01 ng/mL) and resibufogenin (B13, IC50 = 106.04 ± 16.80 ng/mL) (Fig. 7b-d). To HepG-2 cells, arenobufagin (B1) also showed the most cytotoxicity with the IC50 value of 3.14 ± 1.04 ng/mL, followed by cinobufagin (B12, IC50 = 22.51 ± 3.11 ng/mL) and resibufogenin (B13, IC50 = 141.18 ± 0.30 ng/mL) (ESM Fig. S9B-D). In our previous report, the IC50 values of bufotalin (B3) and bufalin (B8) against HepG2 cell lines were 154.53 and 78.68 ng/mL, respectively [[47] ]. The results above demonstrated that the bufogenins, especially arenobufagin (B1), exhibited potent cytotoxicity toward cancer cells.

Though the biological affinity of ten bufogenins in toad venom with Hela cells [[48] ] and eight bufogenins in toad venom with MGC-803 cells [[49] ] have been performed, full-scale screening of the potential active compounds in toad venom was lacking, especially the interaction of bufotoxins with the cancer cells. In this paper, 7 bufotoxins including telocinobufatoxin (B2), bufalin-3-pimelate-arginine ester (B4), bufalitoxin (B6), resibufogenin-3-pimelate-arginine ester (B7), resibufotoxin (B9), cinobufotoxin (B10), and resibufogenin-3-azelate-arginine ester (B11) were discovered to interact compactly with cancer cells and their CBD ranked relatively higher than those of some bufogenins. However, owing to the lacking of corresponding standards, the validation of their pharmalogical activities against cancer cells was hindered. Qiong Meng et al. [[32] ] revealed that the IC50 values of bufalitoxin (B6) toward HepG2 and MCF-7 cancer cells were 18.1 and 30.8 ng/mL, respectively, exhibiting potent cytotoxicity. And the arginine part was reported to play an important role in decreasing the toxicity of toad venom secretions [[50] ], which might decrease the poisonous side effects of toad venom. Our results demonstrated that the 7 bufotoxins identified in our present study should be taken into consideration for exploring anticancer agents with low cardiotoxic effect. In the future research, due attention would be paid to the isolation of single bufotoxins in toad venom to evaluate their pharmalogical activities.

Amphibians present pharmacologically active aliphatic, aromatic and heterocyclic molecules in their skin as defense against microorganisms, predators and infections, such as steroids, alkaloids, biogenic amines, and so on [[31] ]. These constituents possess a great potential to search for new compounds with a wide range of biomedical applications [[30] ]. Many reports have focused on the analysis of the indole alkaloids and free bufogenins in the toad venom, and the number of bufotoxins reported in the literature was limited [[27] , [28] , [33] , [51] ]. In our present study, a total of 34 bufotoxins were detected, representing a full-scale analysis of bufotoxins in toad venom. As the existence of many isomers in toad venom, it is difficult to accurately identify all the constituents in toad venom. However, with the isolation and identification of pure substances from toad skin in our previous work, many bufogenins were confirmed with standards in this paper. Taking the identification of the five compounds with the same [M + H]+ ion at m/z 417.2272 for example, the HR-MS data showed their formula was C24H33O6 (Cal. 417.2271, error 0.01 ppm). In their MS/MS fragments, some fragment ions were the same, making it difficult to distinguish them. Fortunately, with the isolation of pure substances from toad skin in our previous work, four of the five constituents including 26, 36, 41, and 43 were confirmed as Ψ-Bufarenogin, arenobufagin, hellebrigenin, and desacetylcinobufotalin, respectively, by standards. However, it would be hard to unambiguously confirm all the constituents solely by mass spectrometry and further NMR study is needed.

In summary, in the present study, the chemical profiling of toad venom was comprehensively investigated using UPLC-HR-MS and the constructed compound database using UPLC-Qtrap-MS has many advantages including comprehensive covering of the constituents in toad venom, high sensitivity and selectivity in the quantitation of trace compounds, which avoided the ignorance of bioactive chemicals with low content. In contrast to traditional procedures of active compound screening, the target cell-based screening method avoids the need for systematic purification of a large number of different molecules and provides a rapid and efficient technique for screening of active components in TCMs. To our best knowledge, few analyses have been performed to rapidly screen and identify the potential bioactive constituents of toad venom using cell-based screening method except the identification of 2 anti-proliferation compounds in cinobufacini by bioassay-guided isolation [[52] ] and the present research may shed some new lights on the targeted cell-based screening of potential active agents in TCMs.

In this study, a total of 93 compounds including 9 amino acids, 7 indole alkaloids, 40 bufogenins, 34 bufotoxins, and 3 other compounds were characterized in toad venom with the proposed UPLC-HR-MS. Among the 93 compounds detected, 17 components were confirmed by standard substances and 8 trace constituents were detected in toad venom for the first time. Further, a compound database of toad venom containing 93 chemicals with full-scale covering, high sensitivity and selectivity in the quantitation of trace compounds was successfully established using UPLC-Qtrap-MS in MRM mode. After successful validation of the cell-based screening method using negative and positive controls, the developed method was applied to the predication of potential candidates in toad venom with HepG2 and MCF7 cells as target cells, and 17 compounds were detected to interact with cancer cells. Together with the MTT assay, the results indicated that 6 bufogenins and 7 bufotoxins in toad venom represented a promising resource to search for anticancer agents with low cardiotoxic effect, which should be further investigated. The target cell-based screening method coupled with the compound database construction of toad venom provide us a new strategy to quickly screen for the potential trace bioactive components in TCMs interacting with the target cells.

The online version of this article (10.1007/s00216-018-1097-4) contains supplementary material, which is available to authorized users.

This work was supported by National Natural Science Foundation of China (Grant No. 21575146, 21635008, and 21621062), the Fundamental Research Funds for the Central public welfare research institutes (Grant No. ZZ10-007), and National Standardlization Project of Chinese Medicine (Grant No. ZYBZH-C-AH-01).

The authors declare that they have no conflict of interest.

Animal experiments were conducted with the formal approval of the ethics committee of the China Academy of Chinese Medical Sciences (Beijing, China).

1 Hsiao WL, Liu L, The role of traditional Chinese herbal medicines in cancer therapy-from TCM theory to mechanistic insights, Planta Med, 2010, 76, 11, 1118, 1131, 10.1055/s-0030-1250186

• 2 Wang CY, Bai XY, Wang CH, Traditional Chinese medicine: a treasured natural resource of anticancer drug research and development, Am J Chin Med, 2014, 42, 3, 543, 559, 10.1142/S0192415X14500359
• 3 Konkimalla VB, Efferth T, Anti-cancer natural product library from traditional Chinese medicine, Comb Chem High Throughput Screen, 2008, 11, 1, 7, 15, 10.2174/138620708783398368
• 4 Otvos RA, Nierop P, Niessen WM, Kini RM, Somsen GW, Smit AB, Development of an online cell-based bioactivity screening method by coupling liquid chromatography to flow cytometry with parallel mass spectrometry, Anal Chem, 2016, 88, 9, 4825, 4832, 10.1021/acs.analchem.6b00455
• 5 Wang X, Zhang R, Gu L, Zhang Y, Zhao X, Bi K, Cell-based screening identifies the active ingredients from traditional Chinese medicine formula Shixiao san as the inhibitors of atherosclerotic endothelial dysfunction, PLoS One, 2015, 10, 2, e0116601, 10.1371/journal.pone.0116601
• 6 Yuan J, Chen Y, Liang J, Wang CZ, Liu X, Yan Z, Component analysis and target cell-based neuroactivity screening of Panax ginseng by ultra-performance liquid chromatography coupled with quadrupole-time-of-flight mass spectrometry, J Chromatogr B, 2016, 1038, 1, 11, 10.1016/j.jchromb.2016.10.014
• 7 Sun M, Huang L, Zhu J, Bu W, Sun J, Fang Z, Screening nephroprotective compounds from cortex Moutan by mesangial cell extraction and UPLC, Arch Pharm Res, 2015, 38, 6, 1044, 1053, 10.1007/s12272-014-0469-3
• 8 Mou ZL, Qi XN, Liu RL, Zhang J, Zhang ZQ, Three-dimensional cell bioreactor coupled with high performance liquid chromatography-mass spectrometry for the affinity screening of bioactive components from herb medicine, J Chromatogr A, 2012, 1243, 33, 38, 10.1016/j.chroma.2012.04.041
• 9 Li Y, Wang P, Xiao W, Zhao L, Wang Z, Yu L, Screening and analyzing the potential bioactive components from reduning injection, using macrophage cell extraction and ultra-high performance liquid chromatography coupled with mass spectrometry, Am J Chin Med, 2013, 41, 1, 221, 229, 10.1142/S0192415X1350016X
• 10 Li SL, Li P, Sheng LH, Li RY, Qi LW, Zhang LY, Live cell extraction and HPLC-MS analysis for predicting bioactive components of traditional Chinese medicines, J Pharm Biomed Anal, 2006, 41, 2, 576, 581, 10.1016/j.jpba.2006.01.014
• 11 Cheng Z, Huang M, Chen G, Yang G, Zhou X, Chen C, Cell-based assays in combination with ultra-high performance liquid chromatography-quadrupole time of flight tandem mass spectrometry for screening bioactive capilliposide C metabolites generated by rat intestinal microflora, J Pharm Biomed Anal, 2016, 119, 130, 138, 10.1016/j.jpba.2015.11.029
• 12 Qiu JY, Chen X, Zheng XX, Jiang XL, Yang DZ, Yu YY, Target cell extraction coupled with LC-MS/MS analysis for screening potential bioactive components in Ginkgo biloba extract with preventive effect against diabetic nephropathy, Biomed Chromatogr, 2015, 29, 2, 226, 232, 10.1002/bmc.3264
• 13 Liao SG, Li YT, Zhang LJ, Wang Z, Chen TX, Huang Y, UPLC-PDA-ESI-MS/MS analysis of compounds extracted by cardiac h9c2 cell from Polygonum orientale, Phytochem Anal, 2013, 24, 1, 25, 35, 10.1002/pca.2374
• 14 Ren W, Xin SK, Han LY, Zuo R, Li Y, Gong MX, Comparative metabolism of four limonoids in human liver microsomes using ultra-high-performance liquid chromatography coupled with high-resolution LTQ-Orbitrap mass spectrometry, Rapid Commun Mass Spectrom, 2015, 29, 21, 2045, 2056, 10.1002/rcm.7365
• 15 Yang H, Yao W, Wang Y, Shi L, Su R, Wan D, et al. High-throughput screening of triplex DNA binders from complicated samples by 96-well pate format in conjunction with peak area-fading UPLC-Orbitrap MS. Analyst. 2017. 10.1039/c6an01974a.
• 16 Guan M, Dai D, Li L, Wei J, Yang H, Li S, Comprehensive qualification and quantification of triacylglycerols with specific fatty acid chain composition in horse adipose tissue, human plasma and liver tissue, Talanta, 2017, 172, 206, 214, 10.1016/j.talanta.2017.05.042
• 17 Wang S, Wu X, Tan M, Gong J, Tan W, Bian B, Fighting fire with fire: poisonous Chinese herbal medicine for cancer therapy, J Ethnopharmacol, 2012, 140, 1, 33, 45, 10.1016/j.jep.2011.12.041
• 18 Man S, Gao W, Wei C, Liu C, Anticancer drugs from traditional toxic Chinese medicines, Phytother Res, 2012, 26, 10, 1449, 1465
• 19 Xu H, Zhao X, Liu X, Xu P, Zhang K, Lin X, Antitumor effects of traditional Chinese medicine targeting the cellular apoptotic pathway, Drug Des Devel Ther, 2015, 9, 2735, 2744
• 20 Lee S, Lee Y, Choi YJ, Han KS, Chung HW, Cyto−/genotoxic effects of the ethanol extract of Chan Su, a traditional Chinese medicine, in human cancer cell lines, J Ethnopharmacol, 2014, 152, 2, 372, 376, 10.1016/j.jep.2014.01.023
• 21 Li C, Hashimi SM, Cao S, Qi J, Good D, Duan W, Chansu inhibits the expression of cortactin in colon cancer cell lines in vitro and in vivo, BMC Complement Altern Med, 2015, 15, 207, 10.1186/s12906-015-0723-3
• 22 Li C, Hashimi SM, Cao S, Mellick AS, Duan W, Good D, The mechanisms of chansu in inducing efficient apoptosis in colon cancer cells, Evid Based Complement Alternat Med, 2013, 2013, 849054
• 23 Meng Z, Yang P, Shen Y, Bei W, Zhang Y, Ge Y, Pilot study of huachansu in patients with hepatocellular carcinoma, nonsmall-cell lung cancer, or pancreatic cancer, Cancer, 2009, 115, 22, 5309, 5318, 10.1002/cncr.24602
• 24 Wu T, Sun R, Wang Z, Yang W, Shen S, Zhao Z, A meta-analysis of Cinobufacini combined with transcatheterarterial chemoembolization in the treatment of advanced hepatocellular carcinoma, J Cancer Res Ther, 2014, 10, Suppl 1, 60, 64
• 25 Dong J, Zhai X, Chen Z, Liu Q, Ye H, Chen W, Treatment of huge hepatocellular carcinoma using cinobufacini injection in transarterial chemoembolization: a retrospective study, Evid Based Complement Alternat Med, 2016, 2016, 2754542
• 26 Chen Z, Chen HY, Lang QB, Li B, Zhai XF, Guo YY, Preventive effects of jiedu granules combined with cinobufacini injection versus transcatheter arterial chemoembolization in post-surgical patients with hepatocellular carcinoma: a case-control trial, Chin J Integr Med, 2012, 18, 5, 339, 344, 10.1007/s11655-012-1083-1
• 27 Li X, Liu Y, Shen A, Wang C, Yan J, Zhao W, Efficient purification of active bufadienolides by a class separation method based on hydrophilic solid-phase extraction and reversed-phase high performance liquid chromatography, J Pharm Biomed Anal, 2014, 97, 54, 64, 10.1016/j.jpba.2014.04.015
• 28 Li XL, Guo ZM, Wang CR, Shen AJ, Liu YF, Zhang XL, Purification of bufadienolides from the skin of Bufo bufo gargarizans Cantor with positively charged C18 column, J Pharm Biomed Anal, 2014, 92, 105, 113, 10.1016/j.jpba.2014.01.002
• 29 Wang YM, Li ZY, Wang JJ, Wu XY, Gao HM, Wang ZM, Bufadienolides and polyhydroxycholestane derivatives from Bufo bufo gargarizans, J Asian Nat Prod Res, 2015, 17, 4, 364, 376, 10.1080/10286020.2014.995174
• 30 Rodriguez C, Rollins-Smith L, Ibanez R, Durant-Archibold AA, Gutierrez M, Toxins and pharmacologically active compounds from species of the family Bufonidae (Amphibia, Anura), J Ethnopharmacol, 2017, 198, 235, 254, 10.1016/j.jep.2016.12.021
• 31 Sousa LQ, Machado KD, Oliveira SF, Araujo LD, Moncao-Filho ED, Melo-Cavalcante AA, Bufadienolides from amphibians: a promising source of anticancer prototypes for radical innovation, apoptosis triggering and Na, +/K, +-ATPase inhibition, Toxicon, 2017, 127, 63, 76, 10.1016/j.toxicon.2017.01.004
• 32 Meng Q, Yau LF, Lu JG, Wu ZZ, Zhang BX, Wang JR, Chemical profiling and cytotoxicity assay of bufadienolides in toad venom and toad skin, J Ethnopharmacol, 2016, 187, 74, 82, 10.1016/j.jep.2016.03.062
• 33 Zhou J, Gong Y, Ma H, Wang H, Qian D, Wen H, Effect of drying methods on the free and conjugated bufadienolide content in toad venom determined by ultra-performance liquid chromatography-triple quadrupole mass spectrometry coupled with a pattern recognition approach, J Pharm Biomed Anal, 2015, 114, 482, 487, 10.1016/j.jpba.2015.05.032
• 34 Hu YM, Yu ZL, Yang ZJ, Zhu GY, Fong WF, Comprehensive chemical analysis of Venenum Bufonis by using liquid chromatography/electrospray ionization tandem mass spectrometry, J Pharm Biomed Anal, 2011, 56, 2, 210, 220, 10.1016/j.jpba.2011.05.014
• 35 Zhang HY, Hu CX, Liu CP, Li HF, Wang JS, Yuan KL, Screening and analysis of bioactive compounds in traditional Chinese medicines using cell extract and gas chromatography-mass spectrometry, J Pharm Biomed Anal, 2007, 43, 1, 151, 157, 10.1016/j.jpba.2006.06.033
• 36 Hong M, Wang XZ, Wang L, Hua YQ, Wen HM, Duan JA, Screening of immunomodulatory components in Yu-ping-feng-san using splenocyte binding and HPLC, J Pharm Biomed Anal, 2011, 54, 1, 87, 93, 10.1016/j.jpba.2010.08.016
• 37 Chen X, Deng Y, Xue Y, Liang J, Screening of bioactive compounds in Radix Salviae Miltiorrhizae with liposomes and cell membranes using HPLC, J Pharm Biomed Anal, 2012, 70, 194, 201, 10.1016/j.jpba.2012.06.030
• 38 Zhang P, Cui Z, Liu Y, Wang D, Liu N, Yoshikawa M, Quality evaluation of traditional Chinese drug toad venom from different origins through a simultaneous determination of bufogenins and indole alkaloids by HPLC, Chem Pharm Bull (Tokyo), 2005, 53, 12, 1582, 1586, 10.1248/cpb.53.1582
• 39 Schmeda-Hirschmann G, Quispe C, Arana GV, Theoduloz C, Urra FA, Cardenas C, Antiproliferative activity and chemical composition of the venom from the Amazonian toad Rhinella marina (Anura: Bufonidae), Toxicon, 2016, 121, 119, 129, 10.1016/j.toxicon.2016.09.004
• 40 Zulfiker AH, Sohrabi M, Qi J, Matthews B, Wei MQ, Grice ID, Multi-constituent identification in Australian cane toad skin extracts using high-performance liquid chromatography high-resolution tandem mass spectrometry, J Pharm Biomed Anal, 2016, 129, 260, 272, 10.1016/j.jpba.2016.06.031
• 41 Schmeda-Hirschmann G, Gomez CV, Rojas de Arias A, Burgos-Edwards A, Alfonso J, Rolon M, The Paraguayan Rhinella toad venom: implications in the traditional medicine and proliferation of breast cancer cells, J Ethnopharmacol, 2017, 199, 106, 118, 10.1016/j.jep.2017.01.047
• 42 Coates D, The angiotensin converting enzyme (ACE), Int J Biochem Cell Biol, 2003, 35, 6, 769, 773, 10.1016/S1357-2725(02)00309-6
• 43 Hou X, Zhou M, Jiang Q, Wang S, He L, A vascular smooth muscle/cell membrane chromatography-offline-gas chromatography/mass spectrometry method for recognition, separation and identification of active components from traditional Chinese medicines, J Chromatogr A, 2009, 1216, 42, 7081, 7087, 10.1016/j.chroma.2009.08.062
• 44 Dal Lago L, D'Hondt V, Awada A, Selected combination therapy with sorafenib: a review of clinical data and perspectives in advanced solid tumors, Oncologist, 2008, 13, 8, 845, 858, 10.1634/theoncologist.2007-0233
• 45 Sarrouilhe D, Clarhaut J, Defamie N, Mesnil M, Serotonin and cancer: what is the link?, Curr Mol Med, 2015, 15, 1, 62, 77, 10.2174/1566524015666150114113411
• 46 Gao H, Popescu R, Kopp B, Wang Z, Bufadienolides and their antitumor activity, Nat Prod Rep, 2011, 28, 5, 953, 969, 10.1039/c0np00032a
• 47 Han L, Wang H, Si N, Ren W, Gao B, Li Y, Metabolites profiling of 10 bufadienolides in human liver microsomes and their cytotoxicity variation in HepG2 cell, Anal Bioanal Chem, 2016, 408, 10, 2485, 2495, 10.1007/s00216-016-9345-y
• 48 Zhou Q, Wang J, Ma HY, Ding AW, Shang EX, Zhan Z, Cell extraction-UPLC-QTOF determination of the biological affinity of bufadienolides with Hela cells and their correlation with properties computed from structure, Chin Pharmacol Bull, 2012, 8, 1079, 1083
• 49 Jiang JJ, You FQ, Ma HY, Zhou Q, Zhang JF, Zhan Z, Cell continuous extraction-HPLC determination biological affinity of 8 bufadienolides on MGC-803 cells and their correlation with antitumor activities, Chin J Chin Mater Med, 2011, 2, 205, 208
• 50 Dai YH, Shen B, Xia MY, Wang AD, Chen YL, Liu DC, A new indole alkaloid from the toad venom of Bufo bufo gargarizans, Molecules, 2016, 21, 3, 349, 10.3390/molecules21030349
• 51 Ye M, Guo DA, Analysis of bufadienolides in the Chinese drug ChanSu by high-performance liquid chromatography with atmospheric pressure chemical ionization tandem mass spectrometry, Rapid Commun Mass Spectrom, 2005, 19, 13, 1881, 1892, 10.1002/rcm.1989
• 52 Wang DL, Qi FH, Xu HL, Inagaki Y, Orihara Y, Sekimizu K, Apoptosis-inducing activity of compounds screened and characterized from cinobufacini by bioassay-guided isolation, Mol Med Rep, 2010, 3, 4, 717, 722

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