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 [[
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 [[
Among the TCMs, poisonous Chinese herbal medicine toad venom gained increasing attention for cancer therapy [[
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 [[
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 [[
HepG2 cells and MCF-7 cells in the logarithmic growth phase were seeded into cell culture flasks at a density of 1.0 × 10
For cytotoxicity assay, MCF-7 cells and HepG2 cells in logarithmic growth were plated in 96-well plates at a density of 6.0 × 10
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 MS
In total, nine amino acids were detected and/or tentatively identified based on their HR-MS
Among the 93 compounds detected, 7 indole alkaloids (
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 t
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]
Additionally, three compounds (
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 [[
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 [[
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 t
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 [[
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-HT
Bufogenins have been increasingly regarded as a source of promising anticancer agents due to their potent pharmacological activities [[
Though the biological affinity of ten bufogenins in toad venom with Hela cells [[
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 [[
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 [[
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).
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By Wei Ren; Lingyu Han; Mengyi Luo; Baolin Bian; Ming Guan; Hui Yang; Chao Han; Na Li; Tuo Li; Shilei Li; Yangyang Zhang; Zhenwen Zhao and Haiyu Zhao