Department of Natural Medicine, School of Pharmacy, Fourth Military Medical University, 169 West Changle Road, Xi’an 710032, Shaanxi, China
Received date: December 29, 2014; Accepted date: March 03, 2015; Published date: March 10, 2015
Citation: Wang SW, Duan LR, Cao W, Xie YH, Yuan JN, et al. (2015) Simultaneous Quantitative Determination of Nine Bufadienolides in Traditional Chinese Medicinal Toad Skin from Different Regions of China by High-Performance Liquid Chromatography–Photodiode Array Detection.Pharm Anal Acta 6:345. doi: 10.4172/2153-2435.1000345
Copyright: © 2015 Wang SW, et al. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.
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Toad skin is a traditional Chinese medicine for the treatment of various tumors. The major active components in toad skin are bufadienolides. In this paper, a simple, accurate and reliable method for the simultaneous separation and determination of nine active components (gamabufotalin, arenobufagin, telocinobufagin, desacetylcinobufotalin, bufotalin, cinobufotalin, bufalin, cinobufagin and resibufogeni) in toad skin was developed using High-Performance Liquid Chromatography (HPLC) coupled with Photodiode Array Detection (PDA) detection. The chromatographic separation was performed on a SinoChrom ODS-BP C18 column with gradient elution using acetonitrile and 0.1% acetic acid-0.5% potassium dihydrogen phosphate aqueous solution at a flow rate of 0.8 mL min-1. All compounds showed good linearity in a wide concentration range with the values of r2 higher than 0.9994, and their limits of detection were at the range of 0.06-0.10 μg mL-1. The separation and identification of the nine bufadienolides in this paper may provide important experimental data for further research and application of toad skin.
HPLC-PDA; Bufadienolides; Toad skin; Traditional Chinese medicine; Quantitative determination
Toad skin is the dry epidermis of Bufo bufo gargarizans Cantor or Bufo melanostictus Schneider which is separated from internal organs, muscles and bones. The earliest record of toad skin appeared in Tang Dynasty . Thousands of years ago, ancient Chinese had found the way to extract secretions from toad skin; these secretions were denominated Venenum Bufonis, and were widely used in China as an anodyne, cardiotonic, antimicrobial, local anesthetic, and antineoplastic agent .
In recent years, more and more researchers have focused on the potential antitumor activity of toad skin [3-6]. Bufadienolides, such as bufalin, cinobufagin, resibufogenin and telocinobufagin are the major active compounds derived from the toad skin [7,8]. Numerous in vitro and in vivo data have suggested that these compounds exhibit a significant antitumor activity, including the inhibition of cell proliferation, induction of cell differentiation, induction of apoptosis, disruption of the cell cycle, inhibition of cancer angiogenesis, reversal of multi-drug resistance, and regulation of the immune response . Cinobufagin has been approved by the Chinese State Food and Drug Administration (SFDA) (ISO9002) and been widely used to treat patients with various cancers at oncology clinics in China . A total of 39 bufadienolides have been reported  and 11 have been isolated, namely, resibufogenin, cinobufagin, bufalin, telocinobufagin, bufotalin, desacetylcinobufotalin, hellebrigenin, arenobufagi, gamabufotalin, 11β-hydroxylresibufogenin and cinobufotalin .
Currently, only a few analytical methods have been reported to determine the active components in toad skin and these reports are simply confined to 3 bufadienolides at most [13,14]. Up to now, no studies on quantitative determination of multiple active components of toad skin in a single running have been reported. Although extensive research has been conducted on the active components of Venenum Bufonis [15-17], great differences in chemical constituents have been observed between Venenum Bufonis and toad skin. Compared with those in Venenum Bufonis, more types and less quantity of bufadienolides have been reported in toad skin . At the same time, the variation of environments and climates of different regions and the diverse ways of collection, processing and storage of the toad skin may result in different bufadienolides quantities separated from the toad skin from different regions. Therefore, it is meaningful to investigate the active components in toad skin from different regions.
To establish a standard and quality control for bufadienolides quantities separated from the toad skin from different regions, we collected and compared 10 samples of toad skin from different regions of China and an HPLC-UV method was developed for the simultaneous quantitative determination of 9 active components contained in toad skin (gamabufotalin, arenobufagin, telocinobufagin, desacetylcinobufotalin, bufotalin, cinobufotalin, bufalin, cinobufagin and resibufogenin). Our findings showed that this method is simple, accurate, reliable and particularly suitable for the routine analysis of toad skin and its quality control.
Chemicals and reagents
Standards of gamabufotalin, arenobufagin, telocinobufagin, desacetylcinobufotalin, bufotalin, cinobufotalin, bufalin, cinobufagin and resibufogenin were purchased from the National Institute for the Control of Pharmaceutical and Biological Products (NICPBP) (Beijing, China). The structures of the nine bufadienolides are presented in figure 1.
Acetonitrile (MeCN) (HPLC-grade) and methanol (MeOH) (HPLC-grade) were purchased from Honeywell (Muskegon, MI, USA). Acetic acid (HOAc) (HPLC-grade) was purchased from Mallinckrodt Baker (Phillipsburg, NJ, USA). The ultrapure water was prepared from Millipore water purification system (Milford, MA, USA) and filtered with a 0.22 μm membrane. Phosphoric acid (H3PO4) and potassium dihydrogen phosphate (KH2PO4) used in the mobile phase were of ARgrade.
Ten samples of toad skin were purchased from the herbal market or local pharmacy in the following different regions in China: Jinan, Shandong Province (1 batch, NO. S-1); Anguo, Hebei Province (2 batches, NO. S-2, S-3); Nanyang, Henan Province (2 batches, NO. S-4 S-5); Bozhou, Anhui Province (2 batches, NO. S-6 S-7) and Xian, Shaanxi Province (3 batches, NO. S-8 S-9 S-10).
Preparation of standard solutions
A standard stock solution containing the 9 components (gamabufotalin 200 μg ml−1, arenobufagin 100 μg ml−1, telocinobufagin 150 μg ml−1, desacetylcinobufotalin 150 μg ml−1, bufotalin 240 μg ml−1, cinobufotalin 180 μg ml−1, bufalin 300 μg ml−1 cinobufagin 400 μg ml−1 and resibufogenin 450 μg ml−1) was prepared in chromatographic pure methanol and stored away from light at 4°C. Working standard solutions containing the 9 compounds were prepared by appropriate dilution of the stock solution.
Preparation of sample solutions
1 g of each of the 10 powdered samples of toad skin was extracted with methanol (20 mL HPLC-grade) by recirculate for 1 h at 70°C, and then methanol was added to make up for a loss due to volatilization. The resulting extract was centrifuged for 15 min at 12,000×g and was filtered with a 0.45 μm microporous membrane prior to analysis. Aliquot (20 μL) of sample solution was injected into the HPLC system for analysis.
Apparatus and chromatographic conditions
The analyses were performed using a Shimadzu Prominence LC- 20A HPLC system (Shimadzu, Kyoto, Japan) equipped with a LC- 20AD quaternary pump, a SPD-M20A PDA detector, a SIL-20AC HT auto sampler, a CTO-20A thermostat compartment and a LC-solution software. The samples were separated on a SinoChrom ODS-BP C18 column (4.6 mm×250 mm 5 μm Yilite, Dalian, China) and a C18 guard column was used before the analytical column. The mobile phase consisted of MeCN and 0.1% HOAc-0.5% KH2PO4 aqueous solution (adjusted to pH 2.4 with H3PO4). A multistep gradient program was used as follows: 8% MeCN (0 min), 30% MeCN (20 min), 40% MeCN (45 min), 50% MeCN (70 min), 40% MeCN (75 min) and 8% MeCN (80 min). The flowrate was 0.8 mL min-1, column temperature was maintained at 30°C and detection wavelength was recorded at 296 nm.
Optimization of HPLC conditions
In order to achieve the best separation, various factors were examined, including chromatographic column, mobile phase, elution mode, and detection wavelength as well as flow rate and column temperature. Ultimate XB C18 column, Phenomenex Luna 5u C18 column, Yilite Hypersil BDS C18column and Yilite SinoChrom ODSBP C18 column were employed. Compared with methanol, acetonitrile combined with phosphoric acid and potassium dihydrogen phosphate buffer solution remarkably improved the separation of the major constituents in toad skin. In addition, the addition of acetic acid had substantial effects on the selectivity and efficiency of some compounds. Under different gradient elution modes, various kinds of pH, flow rate and column temperature were also compared. The detection wavelength was selected according to the characteristic UV profile, gamabufotalin, arenobufagin, telocinobufagin, desacetylcinobufotalin, bufotalin, cinobufotalin, bufalin, cinobufagin and resibufogenin at 300.1, 299.0, 296.6, 295.4, 295.4, 301.3, 295.4, 300.1 and 296.6 nm, respectively. The ultraviolet absorption spectrums are presented in figure 2. Nine bufadienolides compounds from toad skin were detected under the current HPLC condition and were compared with the bufadienolides standards. After many tests, SinoChrom ODS-BP C18 column with the acetonitrile and 0.1% acetic acid-0.5% potassium dihydrogen phosphate aqueous solution (adjusted to pH 2.4 with phosphoric acid) using gradient elution was selected for the simultaneous separation and determination. The representative HPLC chromatograms of the toad skin samples and authentic standards are presented in figure 3.
Calibration curves and the limit of detection: All calibration curves were plotted based on linear regression analysis of the integrated peak areas (y) versus concentrations (x, μg mL-1) of the 9 marker constituents in the standard solution at seven different concentrations. The regression equations, correlation coefficients and linear ranges for the analysis of the 9 marker constituents are shown in table 1.
|Constituent||Regression equationa||Correlation coefficient (r2)||Linearity range(μg mL-1)||LOD (μg mL-1)|
|gamabufotalin||y = 9213.8x + 14106||0.9994||3.13-200||0.08|
|arenobufagin||y = 10915x + 5079.1||0.9998||1.56-100||0.07|
|telocinobufagin||y = 12147x + 12169||0.9996||2.34-150||0.06|
|desacetylcinobufotalin||y = 11225x + 10293||0.9997||2.34-150||0.07|
|bufotalin||y = 9079.4x + 14123||0.9996||3.75-240||0.09|
|cinobufotalin||y = 10229x + 8819.8||0.9998||2.81-180||0.08|
|bufalin||y = 7979.2x + 13995||0.9996||4.69-300||0.10|
|cinobufagin||y = 10636x + 23504||0.9997||6.25-400||0.07|
|resibufogenin||y = 10860x + 28880||0.9998||7.03-450||0.07|
Table 1: Regression equation, linear range and LODs of the developed method.
The limit of detection value (LOD) was calculated as the amount of the injected sample which gave a signal-to-noise ratio of 3 (S/N= 3). The LOD values of the method for the 9 components are also listed (Table 1).
Precision and accuracy: The precision and the accuracy of the assay were evaluated with standard solutions at low, medium and high concentrations and measured by the Relative Standard Deviation (RSD). The intra- and inter-day precision was determined during a single day and on five consecutive days, respectively. Results are summarized (Table 2). The intra- and inter-day precision calculated as the RSD were less than 0.8%. The accuracies of nine bufadienolides were within the range of 99.67-100.69%.
|Intra-day (n =5)||Inter-day (n =5)|
|Accuracy (%)||RSD (%)||Mean±SD
|Accuracy (%)||RSD (%)|
|gamabufotalin||6.25||6.23 ±0.03||99.67||0.47||6.24 ±0.01||99.86||0.21|
Table 2: Precision and accuracy of the developed method.
Recovery: Three different concentrations (low, medium and high) of authentic standards were added into samples (NO. S-1). The resultant samples were processed and analyzed. The quantity of each analyte was subsequently obtained from the corresponding calibration curve. Recovery of all 9 tested bioactive constituents was within the range of 98.21-101.69%, with an RSD of between 0.03% and 1.23% (n=3). The corresponding values are shown in table 3.
Table 3: Recovery of the developed method.
Reproducibility and stability: For reproducibility test, five different samples from a same batch of toad skin (NO. S-1) was analyzed, and the RSD values of the peak area and retention times were no more than 2.06% and 0.15%, respectively. For stability, the same sample solution was analyzed every 12 h in 3 days at the room temperature. The RSD values of the peak area and retention times were no more than 3.94% and 0.21%, respectively. The results of reproducibility and stability test showed that the method manifested good reproducibility and the solution was considered to be stable within 72 h.
With the newly established method, 9 marker constituents in 10 different batches of toad skin were determined. As shown in figure 3, table 4 under the analytical conditions, the 9 marker constituents (gamabufotalin, arenobufagin, telocinobufagin, desacetylcinobufotalin, bufotalin, cinobufotalin, bufalin, cinobufagin and resibufogenin) in toad skin could be sufficiently resolved and separated. The analysis results notably indicate that the contents of the 9 bufadienolides components in toad skin from different regions of China are very different. The main difference of the main active component content may result from the variation of environments and climates of these regions, and from the varied ways of collection, processing and storage of the toad skin.
|Componentsa||Content (μg g-1)|
Table 4: Sample analysis.
In this study, 10 samples of toad skin from different regions of China were analyzed and 9 bufadienolides compounds were simultaneously identified in the methanolic extract of the different samples of toad skin. This is the first report of the simultaneous determination of 9 major active components in toad skin by high-performance liquid chromatography, coupled with photodiode array detection. The analytical method we used is sensitive, accurate and reliable, which is suitable for the routine analysis and quality control of toad skin form different regions of China.