Characterization of eight terpenoids from tissue cultures of the Chinese herbal plant, Tripterygium wilfordii, by high-performance liquid chromatography coupled with electrospray ionization tandem mass spectrometry.

Research article Received: 5 July 2013,

Revised: 14 December 2013,

Accepted: 28 December 2013

Published online in Wiley Online Library

(wileyonlinelibrary.com) DOI 10.1002/bmc.3140

Characterization of eight terpenoids from tissue cultures of the Chinese herbal plant, Tripterygium wilfordii, by high-performance liquid chromatography coupled with electrospray ionization tandem mass spectrometry Ping Sua,b, Qiqing Chenga,b, Xiujuan Wanga, Xiaoqing Chenga, Meng Zhanga, Yuru Tonga,b, Fei Lic, Wei Gaoa* and Luqi Huangb* ABSTRACT: In this study, a reliable method for analysis and identification of eight terpenoids in tissue cultures of Tripterygium wilfordii has been established using high-performance liquid chromatography coupled with electrospray ionization tandem mass spectrometry (HPLC-ESI-MS). Our study indicated that sterile seedlings, callus cultures and cell-suspension cultures can rapidly increase the amount of biological materials. HPLC-ESI-MS was used to identify terpenoids from the extracts of these tissue cultures. Triptolide, triptophenolide, celastrol and wilforlide A were unambiguously determined by comparing the retention times, UV spectral data, and mass fragmentation behaviors with those of the reference compounds. Another four compounds were tentatively identified as triptonoterpenol, triptonoterpene, 22β-hydroxy-3-oxoolean-12-en-29-oic acid and wilforlide B, based on their UV and mass spectrometry spectra. The quantitative analysis showed that all three materials contain triptolide, triptophenolide, celastrol, wilforlide A, and the contents of the four compounds in the cell-suspension cultures were 53.1, 240, 129 and 964 μg/g, respectively, which were at least 2.0-fold higher than these in the sterile seedlings and callus cultures. Considering the known pharmacological activity of triptolide and celastrol, we recommend the cellsuspension cultures as biological materials for future studies, such as clinical and toxicological studies. The developed method was validated by the evaluation of its precision, linearity, detection limits and recovery, and it was successfully used to identify and quantify the terpenoids in the tissue cultures. Copyright © 2014 John Wiley & Sons, Ltd. Keywords: tissue culture; HPLC-ESI-MS; terpenoid; Tripterygium wilfordii Hook. F.

Introduction Tripterygium wilfordii Hook. F. (TWHF), a perennial twining vine of the family Celastraceae, is one of the most commonly used traditional Chinese medicinal plants. TWHF mainly grows in southern China, including Zhejiang, Anhui, Yunnan, Fujian and Taiwan. The earliest record of its medical usage appears in Dian Nan Ben Cao, which was written by Mao Lan in 1476 (Tu, 2009). In ancient times, TWHF was used for the treatment of fever, chills, edema and carbuncle, and Chinese farmers used the powdered root to protect their crops from insects. Since the 1960s, this plant has attracted interest because its extracts were found to be effective for the treatment of autoimmune diseases and inflammatory dermatoses, including rheumatoid arthritis (Tao and Lipsky, 2000), systemic lupus erythematosus (Li et al., 2005), psoriasis (Xu et al., 1985) and erythema nodosum (Koo and Arain, 1998), and it was also found to have anti-inflammatory (Li et al., 2004), immunosuppressive (Li et al., 2004), antitumor (Chen et al., 2009), anti-HIV (Duan et al., 2000) and anti-Parkinsonian effects (Wang et al., 2008). Since the 1970s, domestic and foreign scholars have investigated tissue cultures of TWHF in pharmacological studies rather than the raw material of TWHF (Kutney et al., 1981, 1983, 1992, 1993; Li et al., 2008) because TWHF grows slowly and plant resources have been depleted.

Biomed. Chromatogr. 2014

Various diterpenoid compounds have been detected and accurately measured in TWHF tissue cultures (Kutney et al., 1993; Nakano et al., 1997), and terpenoids are the main bioactive components responsible for the pharmacological effects of TWHF. For example, triptolide, triptophenolide, celastrol and wilforlide

* Correspondence to: W. Gao, School of Traditional Chinese Medicine, Capital Medical University, Beijing 100069, People’s Republic of China. Email: [email protected] L. Huang, National Resource Center for Chinese Materia Medica, China Academy of Chinese Medical Sciences, Beijing 100700, People’s Republic of China. Email: [email protected] a

School of Traditional Chinese Medicine, Capital Medical University, Beijing 100069, People’s Republic of China

b

National Resource Center for Chinese Materia Medica, China Academy of Chinese Medical Sciences, Beijing 100700, People’s Republic of China

c

Laboratory of Metabolism, Center for Cancer Research, National Cancer Institute, National Institutes of Health, Bethesda MD, 20892, USA Abbreviations used: 2,4-D, 2,4-dichlorophenoxyacetic acid; 6-BA, 6-benzylaminopurine; IBA, indole-3-butytric acid; KT, kinetin; TWHF, Tripterygium wilfordii Hook. F.

Copyright © 2014 John Wiley & Sons, Ltd.

P. Su et al. A have potent antitumor (Chen et al., 2009), anti-inflammatory (Liu and Wang, 2005), immunosuppressive (Wang and Xie, 1999) and antifertility effects (Lee et al., 1999). However, most of the diterpenoids in TWHF have a narrow therapeutic index and can have serious side effects in humans and livestock (Zheng et al., 1981, 1991a, 1991b; Sun et al., 2001). Therefore, it is essential to accurately determine the concentration of terpenoids in TWHF medicinal plants as well as TWHF tissue cultures. Until now, many analytical methods have been published for measurement of the main bioactive components in TWHF, including thin-layer chromatography (Guo, 1986), micellar electrokinetic capillary chromatography (Song et al., 2003), high-performance liquid chromatography (HPLC) with a solid-phase extraction cleanup step (Brinker and Raskin, 2005), gas chromatography with derivatization (Rao et al., 2005), HPLC coupled with evaporative light scattering detection (Luo et al., 2007), and liquid chromatography–mass spectrometry (LC-MS; Shao et al., 2006; OuYang et al., 2007; Yao et al., 2006; Chen et al., 2008; Zhou et al., 2011). However, the reports about TWHF tissue cultures are limited. In this study, we mainly focused on the analysis and identification of the terpenoids in TWHF tissue cultures. HPLC coupled with electrospray ionization tandem MS (HPLCESI-MSn) is one of the most powerful analytical techniques available for the analysis of natural products (Stobiecki et al., 2006). The mass spectrometer is sensitive and selective, which allows the detection of minor or even trace amounts of constituents from a microscale sample. ESI, which produces only protonated or deprotonated molecules, is one of the most frequently used ion sources in HPLC/MS analysis. MS/MS, particularly multistage MS/ MS with Fourier-transform ion cyclotron resonance, can provide further information regarding the structures of the compounds. The important fragment ions can be used to determine the molecular mass and resolve subtle differences in molecular structure (Lee et al., 2005; Zhao et al., 2005). The tandem mass spectrometric fragmentation pattern of terpenoids has been investigated extensively, which allows the characterization of unknown compounds even when reference standards are not available (Kutney et al., 1992; Zhong and Li, 2002; Duan et al., 2001; Yang et al., 2006; Xue, 2011). The aim of this study was to obtain sterile seedlings, callus cultures and suspended-cell cultures of TWHF using tissue culture methods in the laboratory, and then develop an accurate and reliable HPLC-ESI-MSn method for simultaneous quantification of triptolide, triptophenolide, celastrol and wilforlide A. The other terpenoids also were identified in tissue cultures of TWHF.

was cultured in an artificial climate chamber. Chromatographically pure samples of triptolide, triptophenolide, celastrol and wilforlide A were purchased from Nanjing Zelang Medical Technology Co. Ltd (Jiangsu Province, China). Acetonitrile and methanol were of HPLC grade and purchased from Fisher Scientific (Pittsburgh, PA, USA). All chemicals, except where otherwise stated, were of analytical grade, and the water used in this assay was double-distilled. Murashige & Skoog basal medium with vitamins (MS medium) was purchased from PhytoTech Co. Ltd (Lenexa, KS, USA). 2,4-Dichlorophenoxyacetic acid (2,4-D), 6-benzylaminopurine (6-BA), kinetin (KT), and indole-3-butytric acid (IBA) were purchased from Sigma Co. Ltd (USA).

Tissue culture Tender stems with axillary buds were explanted from robustly growing TWHF plants and then subjected to a series of sterilization treatments: (1) a 20 s immersion in 70% alcohol followed by rinsing three times with sterile distilled water; (2) a 5 min disinfection in 1 g/L HgCl2 solution; and (3) rinsing three times with sterile distilled water. After that, the stems (0.5–1.0 cm) with axillary buds were cut and seeded in MS medium containing 0.1 mg/L IBA, 2.0 mg/L 6-BA, 30 g/L sucrose and 0.8% agar at room temperature under a 16 h light/8 h dark photoperiod in a growth chamber fitted with a cool white-fluorescent lamp providing a PPFD level of 25 μmol/m2 s per day. Approximately 10 days later, the explants gradually sprouted tender green buds, which elongated to 4–5 cm after another 30–35 days. The leaves of sterile seedlings were cut to 0.5 × 0.5 cm and cultured in MS medium containing 1.0 mg/L 2,4-D and 0.8% agar. The cultures were grown in the Intellus Control System (in the dark at 25 °C, Percival Scientific, Inc., USA). More than 20 days later, calli had grown out and were transferred routinely at 4-week intervals to MS medium containing 0.5 mg/L 2,4-D, 0.1 mg/L KT, 0.5 mg/L IBA, 30 g/L sucrose and 0.8% agar (pH = 5.8) in the dark at 25 °C. The calli obtained through growth on solid medium were used to initiate cell suspension cultures (supplemented with 0.5 mg/L 2,4-D, 0.1 mg/L KT, 0.5 mg/L IBA and 30 g/L sucrose in MS medium, pH = 5.8) by incubation in the dark at 25 °C on a rotary shaker (120 rpm) with regular subculturing at 20 day intervals. All these operations were completed on a super-clean bench (Fig. 1).

Preparation of the sample

Materials and methods Samples, reagents and plant hormones TWHF was obtained from the experimental fields of Fujian Agriculture and Forestry University (Fujian Province, China) and

Suspended-cell cultures were harvested by mild suction filtration followed by one rinse with distilled water, whereas callus cultures and sterile seedlings were removed from the MS medium directly. All samples were frozen at 20 °C before use. These samples were ground into small pieces using a mortar

Figure 1. Tissue culture flowchart of Tripterygium wilfordii Hook. F.

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Terpenoids from tissue cultures of Tripterygium wilfordii and placed in an ultralow-temperature refrigerator at 80 °C; 4 h later, the samples were freeze-dried for 8 h. Methanol was added in a ratio of 50 mL/g of dry weight of the sample, soaked overnight at room temperature, and the samples were extracted in an ultrasonic water bath for 30 min. After filtration, the residue was extracted in methanol for 30 min again. The combined filtrates were evaporated approximately to dryness under vacuum in a rotary evaporator with a thermostatic water bath at 45 °C (Shanghai Ailang Instrument Co. Ltd, China). The reside was dissolved in 3 mL of ethyl acetate (3 times). The supernatant was evaporated to dryness and transferred to a 2 mL volumetric flask, where it was diluted to the mark with methanol. All the sample solutions were filtered through a 0.45 μm membrane filter before injection into the HPLC system for analysis. Preparation of standard stock solutions Stock solutions of the standards triptolide, triptophenolide, celastrol and wilforlide A were accurately weighed, dissolved in methanol at a concentration of 1.0 mg/mL, and stored at 4 °C before use. Stock solutions were successively diluted with methanol in appropriate quantities to provide a series of working solutions, each of which was injected directly in triplicate, and the peak area was used for the calibration curve. HPLC conditions The analyses were conducted using a Agilent 1260 HPLC system (USA) equipped with an online degasser, quaternary pump, autosampler, column oven and diode array detector. The chromatographic separation was conducted using an Agilent Eclipse XDB-C18 analytical column (5 μm, 4.6 × 250 mm; Agilent, USA) protected by a precolumn and kept at 35 °C. The mobile phase consisting of a mixture of 0.05% (v/v) acetic acid in water (A) and acetonitrile (B) was set at a flow rate of 0.8 mL/min. The gradient program was as follows: 38% B at 0–12 min, 38–60% B at 12–25 min, 60–65% B at 25–30 min, 65% B at 30–35 min, 65–85% B at 35–48 min, 85% B at 48–52 min, 85-83% B at 52–54 min, and 83% B at 54–65 min. The detection wavelength was 210 nm, and UV spectra from 190 to 800 nm were also recorded. The injection volume was 10 μL. LC-ESI-MS detection conditions For HPLC/MS analysis, a Bruker FT-ICR-MS Solarix Maldi/EDI 9.4 T system was connected to the Agilent 1260 HPLC system through an ESI interface. The LC effluent was introduced into the ESI source in a post-column splitting ratio of 2:1. All mass spectra were acquired in the negative ion mode, and the parameters were as follows: dry gas, 4 L/min; dry gas temperature, 180 °C; use of high-purity nitrogen as the nebulizing gas; pressure, 1.0 bar; capillary voltage, 4.0 kV; and an end plate offset at 500 V. For full-scan MS analysis, the spectra were recorded in the range of m/z 100–1000. Data were analyzed on a computer equipped with a data acquisition software (ftmsControl 2.0, Bruker, Germany) and data processing software (dataAnalysis 4.1, Bruker, Germany).

Results and discussion Optimization of HPLC conditions Separation of the chemical constituents from tissue cultures of TWHF was difficult because of the vast number of chemical


components present in the tissue cultures. Four terpenoid compounds were used as standards for optimization of the separation of terpenoids using HPLC. In preliminary experiments, we tried to determine the optimum extraction solvent, extraction method, mobile phase and detection wavelength. Given the properties of the TWHF terpenoids, we selected methanol and ethyl acetate as the extraction solvents. We found that methanol can extract more terpenoids. We then verified that the efficiency of ultrasonic extraction (30 min, 2 times) was comparable to that of reflux extraction but that the former had the advantage of greater convenience. Therefore, we employed ultrasonic extraction in this study. At the same time, comparison of methanol and acetonitrile as the mobile phase showed that acetonitrile was more suitable for this study because of its shorter run time. Moreover, when we added 0.05% (v/v) acetic acid in water to the mobile phase, good separation was obtained and the peaks became more symmetrical. Four wavelengths (203, 210, 220 and 254 nm) were tested to determine the highest detection sensitivity. The detection wavelength of 210 nm gave the best results (Fig. 2). For MS analysis, ESI sources were investigated for the ionization of terpenoids in both positive and negative ion modes. The negative ion mode of ESI was selected because it yielded prominent [M H] ions as the base peak. In addition, collision-induced dissociation fragmentation of the [M H] ion provided extensive information for the characterization of terpenoids in tissue cultures of TWHF. Characterization of terpenoids in tissue cultures The common features of the ESI-MS-MS data for the [M H] ions observed in this study were the loss of H2O (18 Da), CO (28 Da) and CO2 (44 Da), and retro-Diels-Alder (RDA) cleavage of the C-ring. The compounds containing methyl or methoxyl groups gave an ion loss of CH3 (15 Da), which was consistent with that reported in the literature (Zhong and Li, 2002; Duan et al., 2001; Yang et al., 2006; Xue, 2011). In total, eight terpenoids (Fig. 3) were identified or tentatively characterized based on their LC retention behavior, UV spectra and MS fragmentation patterns obtained online (Table 1). Under the optimized LC/MS conditions, most of the terpenoid compounds gave sufficient [M H] ions for MSn analysis. Compound 2 showed an [M H] ion at m/z 359 and two adduct ions [M + HCOOH H] and [M + CH3COOH-H] at m/z 405 and 419 in the MS spectrum and had a molecular formula of C20H24O6 (high-resolution mass spectrometry). Its retention behavior was similar to that of the available reference standard (retention time, RT = 9.18 min). Thus, compound 2 was identified as triptolide. Compound 6 displayed a molecular ion at m/z 345 [M H] in the MS spectrum, with a molecular formula of C21H30O4 (Fig. 4). Its MS2 spectrum showed a base peak at m/z 300 that originated from the concurrent loss of ·CH3 and CH2OH. The fragment ion at m/z 191 suggested the loss of the entire A-ring. Thus, compound 6 was tentatively characterized as triptonoterpenol. Compound 7 showed [M H] , [2 M H] and [3 M H] ions at m/z 311, 623, and 935, respectively, in the MS spectrum with a molecular formula of C20H24O3 (Fig. 5). Its MS2 spectrum yielded a series of product ions at m/z 283 [M – H 28] , 265 [M – H 46] and 237 [M – H 74] , which were attributed to the successive loss of CO and H2O. Its retention behavior was similar to that of the available reference standard (RT = 31.76 min). Thus, compound 7 was identified as triptophenolide.



P. Su et al.

Figure 2. LC/MS chromatograms monitored at 210 nm: (a) negative ion LC-ESI-MS total ion current of suspended-cell culture; (b) LC-UV chromatogram of terpenoid standards; (c) LC-UV chromatogram of sterile seedling; (d) LC-UV chromatogram of callus culture; and (e) LC-UV chromatogram of suspended-cell culture.

Figure 3. Chemical structures of the eight identified terpenoids in tissue culture.

The MS spectrum of compound 8, with a molecular formula of C20H28O2, displayed a molecular ion at m/z 299 [M H] (Fig. 6). The fragment ion at m/z 283 [M H-16] in the MS2 spectrum suggested the loss of CH3 (C10) and formation of a double bond at C1–C10. The RDA fragmentation reaction of the A-ring produced the ion at m/z 213, and the ion at m/z 201 was also


derived from a cleavage of the A-ring, which was the same as that in ar-abietatriene (Zhong and Li, 2002). Thus, compound 8 was tentatively identified as triptonoterpene. The MS spectrum of compound 10, with a molecular formula of C30H46O4, displayed a molecular ion at m/z 469 [M H] and fragment ions at m/z 451 [M H-18] and 407 [M H-62] in



Terpenoids from tissue cultures of Tripterygium wilfordii Table 1. Characterization of eight terpenoids in tissue cultures Number

Retention time (min)

[M

H]-

MS2

UV (nm)

2 6 7 8 10

9.18 25.63 31.75 37.75 43.03

359 345 311 299 469

220 202 200 217 218

191 300, 283, 283, 451,

12

52.76

449

425

13 14

54.61 57.36

451 453

277 280

405, 390, 375, 361, 347, 253, 200 267, 163 301, 163

285, 265, 213, 423,

191, 150, 100 237, 201, 155 201 407, 391, 234

Compound

Reference

Triptolide Triptonoterpenol Triptophenolide Triptonoterpene 22β-hydroxy-3-oxoolean12-en-29-oic acid Celastrol

Xue et al. (2010)

Wilforlide B Wilforlide A

Figure 4. MS and MS/MS spectra of compound 6: (a) MS spectrum and (b) MS/MS spectrum from the [M

the MS2 spectrum (Fig. 7), which suggested the presence of -OH and -COOH groups. The ions at m/z 234 indicated RDA cleavage fragmentation of the C-ring, which provides evidence for an oleanene-type triterpene skeleton in compound 10. The ions at m/z 451, 423, 407, and 391 in the MS2 spectrum were similar to those in previously reported data (Kutney et al., 1992). Thus, compound 10 was tentatively identified as 22β-hydroxy-3oxoolean-12-en-29-oic acid. Compound 12, with a molecular formula of C29H38O4, displayed a molecular ion at m/z 449 [M H] in the MS spectrum and fragment ions at m/z 405 [M H-44] , 390 [M H-


H]¯ ion at m/z 345.

59] , 375 [M H-74] , 361 [M H-88] and 347 [M H-102] , presumably originating from the successive loss of CO2 and CH3 in the MS2 spectrum. The ions at m/z 200 and 253 indicated RDA cleavage fragmentation of the B-ring and C-ring, respectively (Xue, 2011). These findings, coupled with retention behavior similar to that of the reference standard (RT = 52.76 min), helped identify compound 12 as celastrol. Compound 14 exhibited an [M H] ion at m/z 453 and an adduct ion [M + HCOOH H] at m/z 489 and had a molecular formula of C30H46O3 in the MS spectrum. Compound 14 was similar to the reference standard (RT = 57.36 min) with regard



P. Su et al.








Terpenoids from tissue cultures of Tripterygium wilfordii


to retention behavior. This analysis helped identify compound 14 as wilforlide A. The MS spectrum of compound 13 showed a molecular ion at m/z 451 [M H] and fragment ions at m/z 267 and 163 in the MS2 spectrum, with a molecular formula of C30H44O3 (Fig. 8). The ions at m/z 163 indicated RDA cleavage fragmentation of the C-ring followed by cleavage of the A-ring (loss of ·C2H2O), which was similar to the pattern observed for compound 14. Thus, compound 13 was tentatively characterized as wilforlide B. Validation of HPLC assay Precision. The analytical precision from the data of the intraday (six times on one day) and inter-day (twice a day for three consecutive days) determinations is indicated by the RSD values, which were 0.9922) within the determination ranges. The detection limits (LOD) of triptolide, triptophenolide, celastrol and wilforlide A were defined in terms of the amount of the compounds that produced a peak area at least 3 times larger than the noise level (signal-to-noise ratio, S/N > 3). The limit of quantitative



determination (LOQ) was defined as the lowest concentration of a compound that was 10 times higher than the noise level (S/N > 10) Recovery. A recovery test was used to evaluate the accuracy of this method. The standards of triptolide, triptophenolide, celastrol and wilforlide A were added into the suspended-cell culture. The samples (n = 6) were extracted, and then injected directly into the HPLC system in accordance with the developed method. As shown in Table 4, the analytical method developed had good accuracy with overall recovery rates in the range of 92.1 ± 3.9 to 103.5 ± 3.3% for all four terpenoids (RSD < 4.51%). The RSD of the repeatability tests was

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