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. 2018 Feb 15;26(3):1113–1121. doi: 10.1016/j.jfda.2018.01.011

Quantitative and qualitative analyses of cytotoxic triterpenoids in the rhizomes of Anemone raddeana using HPLC and HPLC-ESI-Q/TOF-MS

Ying Zhao 1, Xin Zhang 1, Chongning Lv 1, Yang Yu 1, Yu Zhang 1, Jincai Lu 1,*
PMCID: PMC9303036  PMID: 29976404

Abstract

Anemone raddeana Regel, a Traditional Chinese Medicine, has been demonstrated to possess cytotoxicity and anti-inflammatory activities. The purpose of this study is to establish analytical methods to identify and quantify the major active constituents in Anemone raddeana. A high-performance liquid chromatography coupled with electrospray ionization quadrupole time-of-flight tandem mass spectrometry (HPLC-ESI-Q/TOF-MS) was used to identify the components in the title plant material. To quantify the major components, a HPLC-UV method was developed and validated. The results showed that 37 compounds were identified based on the MS data and retention times. The contents of eight main bioactive compounds were determined by HPLC simultaneously. These methods could be used to effectively evaluate the quality of A. raddeana and provide a valuable reference for further study. In addition, the cytotoxicity activity of the different fractions of A. raddeana was determined. Hederacolchiside A1 (f) showed promising activity against ten human cancer cell lines with IC50 values from 0.29 to 3.48 μM.

Keywords: Anemone raddeana Regel, Quantification, Cytotoxicity

1. Introduction

Anemone raddeana Regel is a well-known Traditional Chinese Medicine (TCM) recorded in the Chinese Pharmacopoeia (2015 edition) for the treatment of rheumatism and pain [1]. This herbal is the major components in a few TCM formulas such as Huo-Luo-Wan and Zai-Zao-Wan [2]. Pharmacological and clinical results showed that the total triterpenoid saponins prepared from A. raddeana had cytotoxic and anti-inflammation activities [3]. Furthermore, chemical analysis demonstrated that triterpenoids were the major constituents in A. raddeana [4]. Some of the pure compounds have been tested using cell line or animal models. For example, raddeanoside A displayed significant cytotoxicity by inhibiting VEGFR2 signaling [5]. In the previous studies, we found that some triterpenoid isolated from A. raddeana exhibited promising effects on superoxide generation in human neutrophil, which was associated with anti-inflammation activity [6–9].

TCM plays an important role in public health due to its effectiveness [10]. However, quality control is challenge for TCM. A few methods, such as HPLC-UV, LC-MS/MS, and LC-NMR, have been developed and tested for the quality control of TCM [11–14]. For A. raddeana, a few analytic methods using HPLC have been reported. However, most of them only measured raddeanin A or raddeanin D [15–17]. The concern is that other components may also be active. To globally control the quality of the title plant material, we developed a HPLC-ESI-Q/TOF-MS method to identify the constituents in A. raddeana. In addition, we developed and validated an HPLC method to quantify eight bioactive triterpenoids. To provide scientific evidence to justify the traditional usage, we tested the activity of the major components in A. raddeana against several cancer cell lines.

2. Materials and methods

2.1. Chemicals and reagents

HPLC-grade methanol and acetonitrile were purchased from Yuwang Group Co., Ltd. (Shandong, China). The standard compounds, 3-O-α-l-rhamnopyranosyl (1 → 2)[β-d-glucopyr-anosyl (1 → 4)]-α-l-arabinopyranosyl-27-hydroxy-oleanolic acid 28-O-α-l-rhamnopyranosyl (1 → 4)-β-d-glucopyranosyl (1 → 6)-β-d-glucopyranoside (a), hederacholichiside E (b), raddeanoside R19 (c), hederacholichiside B (d), raddeanoside R20 (e), hederacolchiside A1 (f), eleutheroside K (g), and betulinic acid (h) were purified from the rhizome of A. raddeana. The purity of these standards was all above 98.0% by HPLC analysis. Their structures (Fig. 1) were elucidated by spectroscopic analysis (1D, 2D NMR) [4,18–22]. Other chemicals were analytical grade.

Fig. 1.

Fig. 1

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Chemical structures of the eight compounds.

2.2. Plants materials

Nineteen batches of A. raddeana (Sample 1–19) were collected from different regions in northeast of China in May 2014. These samples were authenticated by Professor Lu, (Department of Pharmacognosy, Shenyang Pharmaceutical University). The information was listed in Table 1.

Table 1.

Samples information of A. raddeana.

Sample No. Growth location Sample No. Growth location
1 Zuojia, Jilin 11 Yabuli, Heilongjiang
2 Huadian, Jilin 12 Heilongjiang
3 Tiangang, Jilin 13 Heilongjiang
4 Jingyu, Jilin 14 Qingyuan, Liaoning
5 Tonghua, Jilin 15 Benxi, Liaoning
6 Meihekou, Jilin 16 Fengcheng, Liaoning
7 Jilinshi, Jilin 17 Qianshan, Liaoning
8 Wuchang, Heilongjiang 18 Kuandian, Liaoning
9 Acheng, Heilongjiang 19 Aiyang, Liaoning
10 Yimianpo, Heilongjiang
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2.3. Preparation of sample solutions

2.3.1. Preparation of sample solutions for quantification

Nineteen batches of A. raddeana were grounded into powder. The powder (5.0 g) of each batch was extracted by reflux with 75% ethanol (3 × 50 mL, each 1 h). The combined extract was filtered and the solvent was removed using rotary vaporization under vacuum. The residue was dissolved in water and loaded to the HPD400 macroporous resin column (10.0 g), which was eluted with water till the eluate showed negative response to Molish reaction, followed by 70% ethanol till void of saponin. The saponin containing fractions were evaporated to remove ethanol, followed by in vacuo-drying at 40 °C to obtain the total saponin. An accurately weighed total saponin was dissolved in methanol and filtrated through 0.45 μm micropore membrane for analysis.

2.3.2. Preparation of sample solutions for cytotoxic activity assay

Air-dried and powdered A. raddeana was refluxed with 75% ethanol. The dry extract was suspended in water and then successively partitioned with petroleum ether (PE), dichloromethane (CH2Cl2), ethyl acetate (EtOAc), and n-butanol (BuOH). Remove solvent under vacuum to afford PE, CH2Cl2, EtOAc, and BuOH fractions. These fractions were dissolved in DMSO to afford stock solutions used in MTT assay. Doxorubicin was used as the positive control.

2.4. Preparation of standard solutions

A standard solution of initial concentration was prepared by accurately weighing each standard sample (compound a–h) and dissolved with methanol, which was stored in a 10 mL volumetric flask. Then the standard solution was diluted to a series of different concentrations. Each standard working solution was filtered by 0.45 μm micropore membrane.

2.5. HPLC-ESI-Q/TOF-MS analysis

The experiment was performed on quadrupole time-of-flight tandem mass spectrometer coupled with an electrospray ionization source in the negative ion mode from m/z 50 to 1500 (Bruker Co., Karlsruhe, Germany). The instrumental parameters for the mass spectrometric analysis were set as follows: end plate offset, −500 V; capillary voltage, 3800 V; nebulizer gas pressure, 1.2 bar; temperature 180 °C and the flow rate was 8.0 L/min. All data were acquired and processed using Bruker Daltonics DataAnalysis 3.4. Software (Bruker Co., Germany).

2.6. HPLC analysis

2.6.1. HPLC conditions

HPLC analysis was operated on a Lab Alliance-Series III (SSI, USA). A C18 analytical column (250 mm × 4.6 mm, 5 μm, Alltech Associates Co., USA) was used. The separation was achieved by a linear gradient elution program of the two combined eluents: A (acetonitrile) and B [0.1% phosphoric acid (v/v) in water]. The detailed gradient elution was as follows: 0–28 min, 23%–36% A; 28–42 min, 36%–56% A; 42–52 min, 56%–90% A. The flow rate of the mobile phase was 1.0 mL/min. The wavelength was set at 203 nm. The column temperature was 30 °C. The injection volume was 10 μL.

2.6.2. HPLC method validation

The developed method was validated for its linearity, LODs (limit of detection), LOQs (limit of quantification), precision, repeatability, and sample stability. All calibration curves were plotted based on linear regression analysis of the integrated peak areas (y) versus concentrations (x) of identified constituents in the standard solutions to obtain a linear equation y = ax + b. The standard solution containing eight reference compounds was diluted to six different concentrations with methanol for HPLC injection. The experiment was performed in triplicate.

The LODs and LOQs were determined at signal-to-noise ratios (S/N) of 3 and 10, respectively.

Intra-day and inter-day variations were utilized to evaluate the precision. The intra-day variation was determined by successive analysis of the same sample solution six times within one day and inter-day variation was determined on three consecutive days. For testing stability, the same sample solution was analyzed after prepared for 0 h, 2 h, 4 h, 8 h, 12 h, and 24 h. To confirm the repeatability, six different working solutions from the same sample were injected. The recovery was determined by spiking the extracts with exact amount of each reference compound. Eight triterpenoids were spiked into the samples (the same as the known amounts), and then extracted, processed and quantified in accordance with the established method. The precision, repeatability and the stability were analyzed and variations expressed by RSD. The percent recovery rates for the analytes were presented as mean ± SD.

2.7. Cell culture

All of the cell lines, including Lung cancer (A549), human hepatocarcinoma (Hep-G2), human breast adenocarcinoma cell lines (MCF-7), human pancreatic cancer (CFPAC-1), human hepatocarcinoma (Hep 3B), human colon cancer (HT-29), human oral epidermoid carcinoma cells (KB), human esophageal cancer cell line (Eca-109), Lung cancer (SPC-A-1), gastric cancer cell line (SGC-7901), bladder cancer cell (5637), acute myeloid leukemia cells (HL-60), and Human glioma cells (U251), were cultured in Dulbecco’s Modified Eagle’s medium (DMEM) supplemented with 10% fetal bovine serum and maintained at 37 °C with 5% CO2.

2.8. Viability assay

The cells were treated with the test compounds at different concentrations for 72 h. Then 10 μL MTT solution was added and the mixture was incubated for another 4 h at 37 °C. After the removal of the culture medium, 100 μL dimethyl sulfoxide (DMSO) was added. The absorbance was measured at 570 nm on Varioskan Flash Microplate Reader (Thermo Scientific, USA).

2.9. Statistical analysis

All values were represented by mean ± SD. The IC50 values were calculated by the SPSS 19.0 software.

3. Results and discussion

3.1. Identification of main chemical constituents of A. raddeana

To identify the main chemical constituents in A. raddeana, a HPLC-ESI-Q/TOF-MS method was used in negative scan mode. A representative chromatogram was shown in Fig. 2. Based on the retention times, molecular formula, and the MS/MS data, 37 compounds were identified (Table 2).

Fig. 2.

Fig. 2

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Representative total ion chromatograms of A. raddeana obtained by HPLC-ESI-Q/TOF-MS in negative scan mode.

Table 2.

The compounds identified by HPLC-ESI-Q/TOF-MS.

Peak tR (min) Formula M [M–H]− Fragmentation Structurea Reference
Measured mass Calcd mass R1 R2 R3 R4
1 11.371 C65H106O31 1381.6645 1381.6645 911.5011 [M-H-rha-glc-glc] rha1 → 2 [glc (1 →4)]ara rha1 → 4glc1 →6glc CH2OH CH3 [23,24]
2 12.509 C59H96O27 1235.6066 1235.6138 765.4435 [M-H-rha-glc-glc] glc1 → 4ara rha1 → 4glc1 →6glc CH2OH CH3 [23]
3 12.86 C65H106O31 1381.6645 1381.665 911.4989 [M-H-rha-glc-glc] rha1 → 2 [glc (1 →4)]ara rha1 → 4glc1 →6glc CH3 CH2OH [25]
4 14.200 C65H106O31 1381.6645 1381.6698 911.4986 [M-H-rha-glc-glc] rha1 → 2glc1 → 2ara rha1 → 4glc1 →6glc CH3 CH2OH [20]
5 14.568 C59H96O26 1219.6117 1219.6134 749.4490 [M-H-rha-glc-glc] rha1 → 2ara rha1 → 4glc1 →6glc CH2OH CH3 [24,26]
6 15.455 C53H86O22 1073.5538 1073.5523 603.3893 [M-H-rha-glc-glc] ara rha1 → 4glc1 →6glc CH2OH CH3 [23,24]
7 16.041 C59H96O27 1235.6066 1235.6083 765.4467 [M-H-rha-glc-glc] glc1 → 2ara rha1 → 4glc1 →6glc CH2OH CH3 [27]
8 17.933 C59H96O27 1235.6066 1235.6141 – glc1 → 4ara rha1 → 4glc1 →6glc CH3 CH2OH [25]
9 19.322 C59H96O26 1219.6117 1219.6166 749.4540 [M-H-rha-glc-glc] rha1 → 2ara rha1 → 4glc1 →6glc CH3 CH2OH [21]
10 21.013 C65H106O31 1381.6645 1381.6715 911.5004 [M-H-rha-glc-glc] rha1 → 2glc1 → 2ara rha1 → 4glc1 →6glc CH3 CH2OH [25]
11 21.465 C70H114O34 1497.7119 1497.7165 1027.549 [M-H-rha-glc-glc] ara1 → 3rha1 →2 [glc (1 → 4)]ara rha1 → 4glc1 →6glc CH3 CH3 [25]
12 24.027 C65H106O30 1365.6696 1365.6686 895.5039 [M-H-rha-glc-glc] rha1 → 2glc1 → 2ara rha1 → 4glc1 →6glc CH3 CH3 [25,26]
13 24.579 C65H106O30 1365.6696 1365.6678 – rha1 → 2 [glc (1 →4)]ara rha1 → 4glc1 →6glc CH3 CH3 [28]
14 25.266 C59H96O26 1219.6117 1219.6166 749.4502 [M-H-rha-glc-glc] glc1 → 4ara rha1 → 4glc1 →6glc CH3 CH3 [19]
15 25.701 C59H96O26 1219.6117 1219.6155 749.4504 [M-H-rha-glc-glc] glc1 → 2ara rha1 → 4glc1 →6glc CH3 CH3 [29]
16 26.572 C64H104O29 1335.6591 1335.6655 865.4963 [M-H-rha-glc-glc] ara1 → 3rha1 →2ara rha1 → 4glc1 →6glc CH3 CH3 [25]
17 27.559 C65H106O31 1381.6645 1381.6715 911.5011 [M-H-rha-glc-glc] glc1 → 2 [glc (1 →4)]ara rha1 → 4glc1 →6glc CH3 CH3 [25]
18 27.76 C59H96O25 1203.6168 1203.627 733.4583 [M-H-rha-glc-glc] rha1 → 2ara rha1 → 4glc1 →6glc CH3 CH3 [25,30]
19 30.573 C53H86O21 1057.5589 1057.5684 587.4005 [M-H-rha-glc-glc] ara rha1 → 4glc1 →6glc CH3 CH3 [23,26]
20 33.603 C47H76O18 927.4959 927.5085 – glc1 → 2 [glc (1 →4)]ara H CH2OH CH3 [27]
21 34.205 C52H84O21 1043.5432 1043.5538 – ara1 → 3rha1 →2 [glc (1 → 4)]ara H CH2OH CH3 [26]
22 36.767 C47H76O17 911.5010 911.5055 – rha1 → 2 [glc (1 →4)]ara H CH2OH CH3 [21,31]
23 37.503 C47H76O17 911.5010 911.5126 – rha1 → 2 [glc (1 →4)]ara H CH3 CH2OH [25]
24 38.457 C48H78O17 925.5166 925.5286 455.3650 [M-H-rha-glc-glc] H rha1 → 4glc1 →6glc CH3 CH3 [32]
25 38.809 C41H66O12 749.4482 749.4586 – rha1 → 2ara H CH2OH CH3 [24]
26 39.077 C41H66O13 765.4431 765.4547 603.3992 [M-H-glc] glc1 → 4ara H CH2OH CH3 [23,24]
27 39.646 C36H56O9 631.3852 631.3931 455.2545 [M-H-glcA] glcA H CH3 CH3 [33]
28 40.784 C53H86O21 1057.5589 1057.5677 – glc1 → 2ara rha1 → 4glc CH3 CH3 [25,34]
29 40.868 C47H76O17 911.5010 911.5102 455.2526 [M-H-glc-glc-ara] glc1 → 2 [glc (1 →4)]ara H CH3 CH3 [28]
30 41.571 C41H66O12 749.4482 749.4543 – rha1 → 2ara H CH3 CH2OH [22,25]
31 42.459 C47H76O16 895.5061 895.5156 – rha1 → 2 [glc (1 →4)]ara H CH3 CH3 [22,24]
32 43.296 C35H55O8 603.3891 603.3922 471.3511 [M-H-ara] ara H CH2OH CH3 [26]
33 44.518 C47H76O16 895.5061 895.5102 – rha1 → 2glc1 → 2ara H CH3 CH3 [35]
34 45.187 C41H66O12 749.4482 749.4516 587.3973 [M-H-glc] glc1 → 2ara H CH3 CH3 [25,35]
35 45.639 C46H74O15 865.4955 865.4979 – ara1 → 3rha1 →2ara H CH3 CH3 [18]
36 47.849 C41H66O11 733.4521 733.4602 587.4005 [M-H-rha] rha1 → 2ara H CH3 CH3 [22,26]
37 52.152 C35H56O7 587.3953 587.3992 – ara H CH3 CH3 [25]
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a

The basic skeleton is the same as depicted in Fig. 1 for compounds a–g.

3.2. Optimization of HPLC method

The RP-HPLC condition for analyzing these triterpenoid glycosides was optimized. Acetonitrile and 0.1% phosphoric acid system as eluent using a C18 reversed-phase column and detection at 203 nm showed the best separation and peak shape.

3.3. Calibration and validation

The method was validated in terms of linearity, LODs, LOQs, precision, stability, repeatability and recovery test [36]. All calibration curves showed good linear regression (R2 > 0.999). The LODs and the LOQs for the analytes were less than 1.21 and 4.06 μg/mL (Table 3). The RSD values of precision, stability, and repeatability were less than 2.74%. The average recoveries of the analytes were 94.55%–103.57% and RSD values were less than 3.55% (Table 4). Therefore, the HPLC method was precise, accurate and sensitive enough for simultaneously quantitating eight compounds (a–h) in the extracts of A. raddeana.

Table 3.

Results of calibration curves, LOD and LOQ.

Compound Calibration curve R2 Linear range (μg/mL) LOD (μg/mL) LOQ (μg/mL)
a y = 1.006 × 106x + 371 381 0.9990 34–270 1.05 4.06
b y = 1.174 × 106x + 4753 0.9991 86–688 0.33 0.79
c y = 6.916 × 105x + 92951 0.9997 42–338 1.21 3.86
d y = 1.088 × 106x + 1528223 0.9998 27–213 0.48 1.28
e y = 1.155 × 106x + 67640 0.9999 34–270 0.52 1.37
f y = 1.880 × 106x + 195490 0.9997 44–354 0.24 0.80
g y = 1.458 × 106x + 261874 0.9990 46–371 0.21 0.69
h y = 4.394 × 105x + 367245 0.9995 27–216 0.41 1.62
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Table 4.

Precision, Stability, Reproducibility and Recovery (n = 6) in quantitation.

Compound Precision (RSD, %) Stability Repeatability Recovery
Intra-day Inter-day (RSD, %) (RSD, %) Average (%) RSD (%)
a 1.12 2.59 1.45 1.45 98.3 ± 1.78 1.81
b 1.64 1.68 2.45 2.20 101.03 ± 2.92 2.89
c 1.02 2.05 1.10 1.10 95.92 ± 3.41 3.55
d 2.22 2.20 1.60 0.88 94.55 ± 2.95 3.12
e 1.81 3.18 1.81 1.81 96.67 ± 3.25 3.36
f 2.49 2.74 1.80 1.88 99.13 ± 2.57 2.59
g 1.07 1.21 0.70 2.74 103.57 ± 2.45 1.90
h 1.02 1.97 2.05 2.44 96.77 ± 2.91 3.01
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3.4. Quality evaluation of the eight compounds

The proposed method was applied to simultaneously determine the eight compounds in 19 batches of A. raddeana samples. A representative HPLC chromatogram was shown in Fig. 3A. The content of eight analytes in 19 samples were listed in Table 5 and that of hederacholichiside B (d) (0.68–5.94 mg/g) showed the most remarkable difference and that of hederacholichiside E (b) (8.20–14.12 mg/g) had the highest amount. Based on this result, the content of each compound varied to a great extent among different sources. The relationship between the bioactivity and the content of these major glycosides should be further studied.

Fig. 3.

Fig. 3

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HPLC chromatogram of standard chemicals (A) and sample extract (B).

Table 5.

Contents of the eight components in different batches of A. raddeana samples (mg/g, n = 3).

Samples.NO. compound a compound b compound c compound d compound e compound f compound g compound h
1 3.48 ± 0.080 10.65 ± 0.269 1.25 ± 0.030 2.16 ± 0.042 0.61 ± 0.008 1.66 ± 0.037 – 0.18 ± 0.004
2 3.06 ± 0.080 10.73 ± 0.178 1.96 ± 0.036 2.19 ± 0.039 0.58 ± 0.007 2.18 ± 0.039 0.46 ± 0.012 –
3 3.59 ± 0.090 12.25 ± 0.239 1.98 ± 0.041 1.86 ± 0.022 0.81 ± 0.015 2.16 ± 0.029 0.35 ± 0.004 0.17 ± 0.005
4 3.63 ± 0.100 12.13 ± 0.232 1.40 ± 0.036 0.68 ± 0.009 0.42 ± 0.006 1.16 ± 0.023 0.19 ± 0.004 –
5 4.17 ± 0.060 12.58 ± 0.308 1.92 ± 0.021 1.59 ± 0.016 0.32 ± 0.008 0.74 ± 0.014 0.22 ± 0.006 0.10 ± 0.001
6 4.21 ± 0.090 11.71 ± 0.253 1.27 ± 0.014 1.87 ± 0.028 0.82 ± 0.018 2.33 ± 0.046 0.52 ± 0.009 –
7 2.95 ± 0.050 10.88 ± 0.185 1.46 ± 0.038 1.99 ± 0.044 0.49 ± 0.006 1.50 ± 0.017 0.36 ± 0.006 0.10 ± 0.001
8 3.06 ± 0.060 10.73 ± 0.145 1.96 ± 0.030 2.19 ± 0.037 0.58 ± 0.016 2.18 ± 0.054 0.46 ± 0.011 –
9 2.39 ± 0.040 8.70 ± 0.114 1.20 ± 0.020 3.41 ± 0.080 0.64 ± 0.010 2.45 ± 0.026 0.62 ± 0.017 0.31 ± 0.003
10 3.93 ± 0.070 9.01 ± 0.105 1.64 ± 0.028 2.13 ± 0.052 0.78 ± 0.020 1.92 ± 0.020 0.47 ± 0.013 0.10 ± 0.002
11 3.60 ± 0.070 10.38 ± 0.274 2.78 ± 0.064 2.14 ± 0.037 0.48 ± 0.006 1.58 ± 0.037 0.25 ± 0.004 0.10 ± 0.001
12 3.93 ± 0.100 9.29 ± 0.252 2.09 ± 0.058 4.32 ± 0.055 0.72 ± 0.011 2.41 ± 0.045 0.88 ± 0.020 0.13 ± 0.002
13 1.96 ± 0.030 12.14 ± 0.243 1.25 ± 0.032 1.88 ± 0.040 0.86 ± 0.015 2.87 ± 0.068 0.42 ± 0.008 0.18 ± 0.002
14 2.66 ± 0.050 8.20 ± 0.216 2.39 ± 0.057 1.39 ± 0.024 0.73 ± 0.010 2.58 ± 0.050 0.27 ± 0.006 0.27 ± 0.004
15 2.94 ± 0.070 11.65 ± 0.308 0.48 ± 0.009 2.18 ± 0.047 0.69 ± 0.016 2.29 ± 0.062 0.56 ± 0.010 0.60 ± 0.011
16 2.73 ± 0.030 10.20 ± 0.141 1.11 ± 0.026 5.94 ± 0.163 0.68 ± 0.009 1.86 ± 0.042 0.84 ± 0.016 0.53 ± 0.006
17 4.06 ± 0.110 14.12 ± 0.387 2.85 ± 0.048 2.03 ± 0.049 0.55 ± 0.011 1.55 ± 0.018 0.16 ± 0.004 –
18 3.31 ± 0.050 13.93 ± 0.299 1.61 ± 0.042 1.83 ± 0.027 0.46 ± 0.012 1.30 ± 0.028 0.14 ± 0.003 0.17 ± 0.002
19 3.69 ± 0.050 12.25 ± 0.272 1.70 ± 0.026 2.09 ± 0.037 0.66 ± 0.016 1.81 ± 0.034 0.26 ± 0.006 0.27 ± 0.005
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3.5. MTT cytotoxicity assay

The results of different fraction of A. raddeana were shown in Fig. 4A. Petroleum ether and ethyl acetate fraction showed the potential cytotoxicity. The anti-proliferation activity of hederacolchiside A1 (f) against ten cancer cells lines was evaluated. As compound f exhibited a strong cytotoxicity with IC50 values from 0.29 to 3.48 μM (Fig. 4B) and was isolated from the ethyl acetate fraction, this fraction contained the cytotoxic ingredients of A. raddeana.

Fig. 4.

Fig. 4

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Cytotoxic activity of different part of extract (A) and hederacolchiside A1 (B).

4. Conclusions

Thirty seven compounds were identified from A. raddeana primarily by HPLC-ESI-Q/TOF-MS for the first time. Eight tri-terpenoid glycosides were simultaneously quantified by HPLC for the quality control. The cytotoxic activity of the extract of A. raddeana was tested. The result indicated that the ethyl acetate soluble fraction was the most potent. Further separation from this fraction yielded hederacolchiside A1, a tri-terpenoid glycoside, which showed good inhibitory activity against ten human cancer cells lines, providing scientific evidence for the potential of A. raddeana as an anti-cancer traditional medicine.

Acknowledgments

This work was supported by National Natural Science Foundation of China [No.81373900]; the Special Fund for TCM supported by State Administration of Traditional Chinese Medicine of China [No. 201407002]; and National Natural Science Foundation of China [No. U15082201009120].

Funding Statement

This work was supported by National Natural Science Foundation of China [No.81373900]; the Special Fund for TCM supported by State Administration of Traditional Chinese Medicine of China [No. 201407002]; and National Natural Science Foundation of China [No. U15082201009120].

Footnotes

Conflicts of interest statement

The authors declare that there is no conflict of interest.

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