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Dose-dependent targeted knockout methodology combined with deep structure elucidation strategies for Chinese licorice chemical profiling
Journal of Pharmaceutical and Biomedical Analysis 115 (2015) 130–137
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Dose-dependent targeted knockout methodology combined with deep structure elucidation strategies for Chinese licorice chemical profiling Zhenzuo Jiang a,b,1 , Yuefei Wang a,b,∗,1 , Yan Zhu a,b , Lei Zhang a,b , Xin Chai a,b , Miaomiao Jiang a,b,∗ , Lihua Shan a a b
Tianjin State Key Laboratory of Modern Chinese Medicine, Tianjin University of Traditional Chinese Medicine, Tianjin 300193, PR China Research and Development Center of TCM, Tianjin International Joint Academy of Biotechnology and Medicine, Tianjin 300457, PR China
a r t i c l e
i n f o
Article history: Received 28 January 2015 Received in revised form 8 June 2015 Accepted 14 June 2015 Available online 19 June 2015 Keywords: Licorice Dose-dependent targeted knockout technique UPLC-Q/TOF MS MS–NMR combination spectroscopy Flavonoid glycoside alkaloids Organic acid alkaloids
a b s t r a c t One of the limitations with regards to the chemical profiling of Chinese herbs is that low-level compounds are masked by high-level structures. Here, we established a novel methodology based on a dosedependent targeted knockout (DDTK) technique combined with deep structure elucidation strategies to allow the chemical profiling of Chinese licorice. We employed ultra-performance liquid chromatography coupled with quadrupole-time-of-flight mass spectrometry (UPLC-Q/TOF MS) incorporated with the DDTK technique to identify the compounds in different concentration samples and found that the compounds at the high- or medium-level were detected readily in the sample at a low concentration; subsequently, minor or trace-level constituents were identified in the sample at a high concentration by rejecting high-level constituents detected in the sample at a low concentration based on a heartcutting technique during analysis. In this study, among the 232 compounds detected, 27 compounds were unequivocally identified and 165 compounds, including 29 new compounds and two new natural products, were tentatively characterized. The novel methodology established in this work paves the way the further identification of compounds from complicated mixtures, especially traditional Chinese medicines. © 2015 Published by Elsevier B.V.
1. Introduction For thousands of years, traditional Chinese medicine (TCM) has been widely used in China for clinical therapy based on the theories of Chinese medical science. Nowadays, TCM is attracting increasing attention worldwide due to its distinguished efficiency and minimal or negligible toxicity [1–3]. However, the disadvantages, including poor quality control, ambiguous pharmacodynamics and indefinable therapeutic mechanisms, limit the worldwide spread of TCM [4]. Thus, the investigation of holistic chemical profiling of TCM contributes to clarifying its chemical material basis; how-
∗ Corresponding authors at: Tianjin State Key Laboratory of Modern Chinese Medicine, Tianjin University of Traditional Chinese Medicine, Tianjin 300193, PR China. Fax: +86 022 27386453. E-mail addresses: wangyuefei [email protected] (Y. Wang), [email protected] (M. Jiang). 1 These authors contributed equally to this work. http://dx.doi.org/10.1016/j.jpba.2015.06.020 0731-7085/© 2015 Published by Elsevier B.V.
ever, researchers have been challenged by the masking of low-level compounds by high-level constituents during analysis [5]. In order to characterize the chemical profiling of TCM, hyphenated techniques, including LC–MS [6–8], GC–MS [9–11], CE–DAD and/or MS [12–14], and LC–NMR [15], have been widely employed. Among these hyphenated techniques, ultra-performance liquid chromatography coupled with quadrupole-time-of-flight MS (UPLC-Q/TOF MS) is the most feasible method for rapid chemical profiling in TCM [16]. However, even with its excellent performance [5,7,17–20], the detection of minor- and trace-level compounds remains challenging with UPLC-Q/TOF MS, owning to the restricted dynamic range and suppression of ion signals in the presence of high-level constituents [21]. Furthermore, the technique is insufficient to confirm the structures of unknown compounds [22] and isomers using MS and MS/MS data [23]. To overcome these issues, we developed a novel methodology based on a dose-dependent targeted knockout (DDTK) technique (shown in Fig. 1), which facilitated the preparation of samples at multiple concentrations, including low, medium and high con-
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Fig. 1. Summary diagram of the integrated analytical strategy.
centrations. Subsequently, we detected high- and medium-level compounds in the sample at a low concentration and identified minor- and even trace-level constituents in the sample at a high concentration by specifically rejecting the high-level constituents that were detected in the sample at a low concentration based on a heart-cutting technique. In this work, Chinese licorice (GanCao; Glycyrrhiza uralensis), which is a member of the Glycyrrhiza species and Leguminosae family, was chosen as a challenging sample to evaluate via the DDTK method. As a renowned tonifying and harmonizing herb, licorice is a popular clinical treatment that presents in around 60% of all TCM prescriptions [24]. Through this new approach, the number of detected peaks was expanded from 50 to 232 compounds [25–27]. Furthermore, 192 compounds were identified by employing deep structure elucidation strategies, including 31 that were tentatively characterized as new compounds or natural products (five organic acid alkaloids, 23 flavonoid glycoside alkaloids and three flavonoid glycosides).
2. Materials and methods 2.1. Chemicals and materials HPLC grade acetonitrile (ACN) and methanol were purchased from Merck (Darmstadt, Germany). Formic acid with a purity of 96% was obtained from MREDA (Meridian Medical Technologies, USA). Ultra-pure water was purified by a Milli-Q system (Millipore, USA). Deuterated dimethylsulfoxide (DMSO-d6 ) was purchased from Cambridge Isotope Laboratories Inc. (CIL Inc., Andover, USA). Reference compounds, including schaftoside, neoliquiritin, liquiritin, liquiritin apioside, ononin, isoliquiritin apioside, isoliquiritin, neoisoliquiritin, liquiritigenin, calycosin, isoliquiritigenin, 18-glycyrrhizic acid, licochalcone A and 18-glycyrrhetinic acid, were purchased from National Institute for Food and Drug Control (Beijing, China), Tianjin ZhongXin Pharmaceutical Group Co., Ltd. (Tianjin, China) and Victory Biotechnology Co., Ltd. (Szechwan, China). Kumatakenin B, uralsaponin C, licoricesaponin A3, uralsaponin F, 24-hydroxy-licorice-saponin E2, 22-acetoxylglycyrrhizin, licorice-saponin E2, 22-acetoxylglycyrrhaldehyde, licorice-saponin G2, licoricone, licoisoflavone B, neoglycyrol and licoflavonol were supplied by Yunfeng Zheng, an associate professor at Pharmacy College, Nanjing University of Chinese Medicine. These reference compounds [28,29] were isolated and identified (1 H NMR, 13 C NMR, MS and UV spectra) from Glycyrrhiza uralensis by Professor Zheng. The purities of the standards were above 96%. Chinese licorice (Glycyrrhiza uralensis) was obtained from Chinese Traditional Medicine Pharmacy JianMinDaYaoFang (Tianjin, China).
2.2. Sample preparation 2.2.1. Sample preparation for DDTK analysis The decoction pieces of licorice were pulverized to a fine powder. The accurately weighed powder (0.5 g) was immersed in 10 mL 50% (v/v) methanol aqueous solution, ultrasonically extracted for 30 min and then cooled at room temperature. The extracted solution was centrifuged at 14,000 rpm for 10 min. Finally, the high concentration sample was acquired by filtering through a 0.22 m filter, and then serially diluted three and nine fold with 50% (v/v) methanol aqueous solution to obtain samples with medium and low concentrations for UPLC-Q/TOF MS analysis.
2.2.2. Sample preparation for MS–NMR combination spectroscopy (COSY) analysis The licorice powder (150 g) was macerated in 1500 mL 50% (v/v) methanol aqueous solution for 12 h and ultrasonically extracted for 30 min. The extracted solution was filtered through a funnel and concentrated to a solution with 2 g crude drug per mL by rotary evaporation under reduced pressure. Polysaccharides in the concentrated solution were precipitated and removed by adding an equal volume of methanol, and finally the solution was centrifuged for 10 min at 14,000 rpm for p-HPLC analysis. The effluent fractions, which were collected in chronological order, were concentrated and lyophilized to a powder. The accurately weighed powder (5 mg) was dissolved in 10 mL 50% (v/v) methanol aqueous solution for subsequent UPLC-Q/TOF MS analysis (a representative p-HPLC chromatogram of the licorice extract and corresponding extract max plot chromatograms of 21 effluent fractions are shown in Fig. S1). Powder (30 mg) was dissolved in 0.5 mL DMSO-d6 for subsequent NMR analysis.
2.3. DDTK analytical conditions In the low concentration sample, the high- or medium-level compounds appeared as obvious peaks. By knocking out these peaks using a heart-cutting technique during analysis, the minorand even trace-level constituents were identified in the high concentration sample. The details of this novel technique were as follows: (1) samples at multiple concentrations, including low, medium and high concentrations, were prepared; (2) high-level constituents in the sample at a low concentration were detected; (3) medium-level constituents in the sample at a medium concentration were detected by rejecting high-level constituents (abundance ≥50% base peak intensity); (4) minor- and trace-level constituents were detected in the high concentration sample by rejecting high- and medium-level constituents (Fig. S2).
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2.3.1. Chromatographic conditions Chromatographic analysis was performed on an ACQUITYTM UPLC system (Waters, Milford, USA) equipped with a binary solvent manager, sample manager and column oven. The system was controlled by Masslynx V4.1 software. Chromatographic separation was carried on an ACQUITYTM UPLC BEH Shield RP18 column (2.1 × 100 mm, 1.7 m) held at 40 ◦ C. The flow rate was 0.4 mL/min. The optimal mobile phase was composed of 0.1% formic acid aqueous solution (A) and acetonitrile (B) operating under a gradient elution program as follows: 0–10 min, 2–18% B; 10–15 min, 18–28% B; 15–22 min, 28–40% B; and 22–32 min, 40–67% B. The injection volume of the sample was 2 L. 2.3.2. MS conditions MS was performed by employing a Waters ACQUITY SYNAPTTM G2 high definition mass spectrometer system (Waters, Milford, USA) equipped with an electrospray ion (ESI) source operating in positive and negative ion modes. The optimal conditions of analysis were as follows: capillary voltage at 3.0 and 2.5 kV in positive and negative ion mode, respectively; sampling cone voltage at 30 V; extraction cone voltage at 3.0 V; source temperature at 120 ◦ C; desolvation temperature at 400 ◦ C; cone gas flow at 50 L/h; and desolvation gas flow at 700 L/h. Data were acquired in centroid mode from 50 to 1500 Da. For accurate mass to charge ratio acquisition, the MS was corrected during data acquisition using a lock mass of leucine–enkephalin (LE) at a concentration of 200 pg/mL via a LockSprayTM interface at a flow rate of 10 L/min, monitoring a reference ion for the positive ion mode ([M + H]+ = 556.2771) and negative ion mode ([M − H]– = 554.2615) to ensure accuracy during MS analysis. All the data were acquired and processed by Masslynx V4.1 software. 2.3.3. Heart-cutting conditions The DDTK technique was based on heart-cutting apparatus. In this work, a six-port valve equipped on the MS was employed to reject high-level constituents in the sample at a high concentration based on the designated valve-switching time, which referred to the retention time of the high-level constituents in the based peak intensity (BPI) chromatograms. The details of the valve-switching time were as follows: (1) to analyze the low concentration sample, high-level constituents in both the positive and negative ion modes were detected and recorded in the BPI chromatogram; (2) to analyze the medium concentration sample, peaks of the high-level constituents were rejected as waste during 0.00–1.15, 9.83–10.32 and 21.77–22.21 min (peaks 45–47 and 162) in the negative ion mode, and 9.81–10.03 and 21.75–22.26 min (peaks 45 and 161–162) in the positive ion mode; (3) to analyze the high concentration sample, peaks in 0.00–1.15, 4.15–4.65, 7.78–8.28, 9.83–10.32, 14.23–14.70, 18.96–19.15, 20.63–20.85, 21.77–22.21, 23.05–25.35, 26.70–28.40 and 29.05–29.62 min (peaks 6, 20–28, 45–47, 86–88, 138, 151, 162, 169–171, 173–185, 197–211 and 214–217) were set as waste in the negative ion mode, and 0.00–0.95, 8.62–9.28, 9.81–10.32, 17.28–17.52, 20.60–20.86, 21.75–22.26 and 23.05–23.40 min (peaks 35–37, 39, 45–47, 116–117, 151–152, 161–162 and 169–171) were set as waste in the positive ion mode. The superior reproducibility and stability of UPLC was a basic guarantee for the achievement of the DDTK technique.
powders; (3) to dissolve the powders for UPLC-Q/TOF MS and NMR assays; (4) to confirm the structures of compounds through m/z-ı spectra and assign them to the chromatographic peaks (Fig. S3). 2.4.1. p-HPLC conditions Effluent fractions were prepared by an Agilent 1260 Infinity p-HPLC system (Agilent Technologies, CA, USA) equipped with a preparative pump and multiple wavelength detector (MWD). Chromatographic separation was performed on a SymmetryPrepTM C18 column (19 × 150 mm, 7 m) held at ambient temperature and the flow rate was 10 mL/min. The optimal mobile phase was composed of 0.1% formic acid aqueous solution (A) and methanol (B). The gradient program was employed as follows: 0–5 min, 2% B; 5–60 min, 2–90% B; 60–65 min, 90–95% B. The detection wavelength was set at 254 nm. The injection volume was 5 mL. Effluent collected in the first 5 min under 2% B and labeled as fraction 0 was used to flush the strong polar compounds, such as monosaccharides, polysaccharides and amino acids, to reduce interference in the MS and NMR signals. The remaining 20 fractions (1–20) were collected from the p-HPLC every 3 min. Data were processed by ChemStation B.04.03 software. 2.4.2. NMR conditions 1 H NMR spectra were acquired at 298 K on a Bruker AVIII 600 MHz NMR spectrometer (Bruker, Zurich, Switzerland) using a PABBO probe. For spectral resonance assignment purposes, 1 H–1 H correlation spectroscopy (COSY), 1 H–1 H total correlation spectroscopy (TOCSY), 1 H–13 C heteronuclear single quantum correlation (HSQC) and heteronuclear multiple-bond correlation (HMBC) 2D-NMR spectra were acquired for interesting samples. The detailed 2D-NMR spectral conditions were as follows: COSY experiments were performed using a cosygpmfqf sequence, 9 kHz spectral width, 2 k data points, 32 scans per increment, 128 increments; TOCSY was acquired using a mlevphpp sequence, 100 ms mixing time, 9 kHz spectral width, 2 k data points, 64 scans per increment, 128 increments; HSQC spectra were acquired using an echo-antiecho phase sensitive standard pulse sequence (hsqcetgpsisp2.2) and 1.7 ms evolution time, 9 kHz spectral width in f2 , 36 kHz spectral width in f1 , 2 k data points, 32 scans per increment, 256 increments; HMBC spectra were recorded using a hmbcgpndqf sequence and 9 kHz spectral width in f2 , 36 kHz spectral width in f1 , 2 k data points, 128 scans per increment, 128 increments. These data were zero-filled to 2 k data points in the evolution dimension prior to appropriate apodization and Fourier transform (FT) with forward linear prediction. 3. Results and discussion 3.1. Holistic chemical profiling of licorice The study of holistic chemical profiling of licorice in both the positive and negative ion modes was performed by UPLC-Q/TOF MS incorporated with DDTK using the optimal conditions described above. High-, medium-, and minor- and trace-level constituents of BPI chromatograms in the negative and positive ion modes are shown in Fig. 2A and E, B and F, and C and G, respectively. In total, 232 compounds in licorice were detected, of which 192 compounds shown in Table S1 were tentatively characterized.
2.4. MS–NMR COSY analytical conditions
3.2. Deep structure elucidation
Fractions from licorice were separated by p-HPLC and verified by UPLC-Q/TOF MS and NMR. We combined the acquired data from MS and NMR, and set up a new structure elucidation strategy. The details were: (1) to collect the elutions in a time-dependent manner by p-HPLC; (2) to concentrate and lyophilize all fractions to
To achieve the delicate structure elucidation of the compounds from DDTK, a strategy of structure elucidation was applied in this study and was based on (1) authentic standards, (2) MS–NMR COSY, (3) polarity, (4) mathematics, (5) structure assembly and (6) the literature.
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3.2.1. Structure elucidation based on authentic standards Retention time data, and accurate MS and MS/MS spectra were compared for the samples with those of authentic standards, allowing 27 compounds, labeled ‘a’ in Table S1, to be unequivocally identified, including 16 flavones, 10 saponins and one coumarin. Representative BPI chromatograms of the authentic standards in the negative ion mode and positive ion mode are shown in Fig. 2D and H, respectively.
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3.2.2. Structure elucidation based on MS–NMR COSY Integrated chemical shift values (ı) acquired by NMR with tR m/z (retention time–mass to charge ratio) were generated from Q/TOF MS of the compounds in the same fraction and the structures of compounds were deduced accurately and assigned to chromatographic peaks. Taking compound 6 as an example, a detailed procedure of structure elucidation of MS–NMR COSY can be described.
Fig. 2. Representative BPI chromatograms of sample solution of licorice at (A) low conc., (B) medium conc., (C) high conc. and (D) mixed solution of 27 authentic standards in negative ion mode; sample solution of licorice at (E) low conc., (F) medium conc., (G) high conc. and (H) 27 authentic standards in positive ion mode.
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Fig. 2. (Continued).
Compound 6 presented in fractions 0–5. As shown in the 1 H NMR spectrum, the intensities of two aromatic proton signals at ı 6.99 (2H, d, J = 8.3 Hz, H-2, 6) and 6.64 (2H, d, J = 8.3 Hz, H-3, 5), one methine signal at ı 3.39 (1H, t, J = 7.5 Hz, H-7) and one methylene signal at ı 2.92 (2H, t, J = 7.5 Hz, H2 -8) were in proportion, suggesting these signals were born from the same compound with a 1,4-disubstituted benzene ring and >CH CH2 moiety in the structural skeleton. Moreover, in the HMBC spectrum shown in Fig. 3A, the proton signal of H-7 had a 3 JCH correlation with the carbon signal at ı 129.9 (C-2, 6), which showed the quaternary carbon (C-1) of a benzene ring substituted by a >CH CH2
moiety. The carbon signal at ı 156.3 (C-4) indicated that the other substituent of the benzene ring was a hydroxyl. In ESI negative ion mode, compound 6 showed a quasi-molecular ion at m/z 209.0477, indicating the molecular formula of C10 H10 O5 . The ESI–MS/MS spectrum (Fig. 3B) exhibited major fragment ions at m/z 165.0576 [M − H − CO2 ]– , 121.0673 [M − H – CO2 ]– and 93.0349 [M − H – 2CO2 – C2 H4 ]– , suggesting the presence of two carboxyl groups and one >CH CH2 moiety. Accordingly, the HMBC correlations between H-7/H2 -8 and the carbonyl carbon signal at ı 171.3 also confirmed the presence of two carboxylic groups at both sides of the >CH CH2 moiety. Consequently, compound 6 was iden-
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Fig. 3. Structure elucidation of compound 6 based on MS–NMR COSY strategy.
tified as 2-(4-hydroxyphenyl) succinic acid (Fig. 3D) and its MS fragmentation patterns are shown in Fig. 3E. By this way, 29 compounds, labeled ‘b’ in Table S1, were unequivocally identified or tentatively characterized based on MS–NMR COSY, including new compounds, natural products and isomers. 3.2.3. Structure elucidation based on polarity (chromatographic retention behaviors) Liquiritigenin and isoliquiritigenin are the basic skeletons of flavanones and chalcones in licorice, whose characteristic fragment ions appear at m/z 137.0233 in the positive mode, and m/z 135.0088 and 119.0502 in the negative mode. When liquiritigenin and isoliquiritigenin are connected to glucose (Glc), apiose (Api) or a combination of them (Glc, Glc–Api, Glc–Glc, etc.), glycosides may be achieved. Three characteristic eluotropic regulars were summarized according to the chromatographic behaviors of the authentic standards (neoliquiritin, liquiritin, liquiritin apioside, isoliquiritin apioside, isoliquiritin, neoisoliquiritin, liquiritigenin and isoliquiritigenin) as follows: (1) liquiritigenin and its glycosides were prior to isoliquiritigenin and its corresponding glycosides; (2) for the glycoside derivatives of liquiritigenin, 7-O-glucoside-substituted glycosides preceded 4 -O-glucoside, as well as glucosyl-substituted glycosides, which were prior to apiosyl–glucosyl-substituted glycosides that were substituted at same position; (3) for the glycoside derivatives of isoliquiritigenin, an inverse rule was displayed and 4-O-glucoside-substituted glycosides preceded 4 -O-glucoside, and apiosyl-glucosyl-substituted glycosides were prior to glucosyl-substituted glycosides that were substituted at same position. For example, in the ESI negative ion mode, compounds 15, 16, 17 and 18 generated quasi-molecular ions at m/z 499.0917, 499.0917, 631.1337 and 631.1349, respectively. In the (−) ESI–MS/MS mode, compounds 15 and 16 yielded the same fragment ions at m/z 417.1179/417.1172 [M – H – H2 SO3 ]– , 255.0656/255.0653 [M – H – H2 SO3 – Glc]– and 135.0094/135.0081 [M – H – H2 SO3 – Glc – VP]– , which were consistent with the structures of neoliquiritin sulfite and liquiritin sulfite. Compounds 17 and 18 gen-
erated the same fragment ions at m/z 549.1591/549.1591 [M – H – H2 SO3 ]– , 255.0657/255.0653 [M – H – H2 SO3 – Api – Glc]– and 135.0093/135.0076 [M – H – H2 SO3 – Api – Glc – VP]– , which were consistent with the structures of neoliquiritin apioside sulfite and liquiritin apioside sulfite. Moreover, by comparing the chromatographic behaviors of compounds 38, 40, 45 and 47, compounds 15, 16, 17 and 18 were identified as neoliquiritin sulfite, liquiritin sulfite, neoliquiritin apioside sulfite and liquiritin apioside sulfite. As a result, 27 compounds, labeled ‘c’ in Table S1, were unequivocally identified or tentatively characterized based on chromatographic retention behaviors (Fig. 4). 3.2.4. Structure elucidation based on mathematics (MS data calculation) In this study, 28 new alkaloids were observed that have rarely been reported in the literature. Thus, mathematics were proposed and introduced for structure elucidation of these alkaloids. For instance, in the (+) ESI–MS/MS spectrum of compound 4 (Fig. 5A), fragment ions at m/z 250.1433, 232.1313, 204.1370, 170.0764, 107.0495, 98.0965 and 84.0806 were observed. By mathematics (Fig. 5B), the ion at m/z 84.0806 was generated by loss of C10 H10 O5 (the same elemental composition as compound 6) from the precursor ion directly, which was consistent with the elemental composition of C5 H10 N+ (calc. 84.0808). Therefore, it was deduced to be 2,3,4,5-tetrahydropyridinium via ChemSpider based on the biosynthesis approaches of natural products [30,31] (see chapters 2 and 3 in Medicinal Natural Products). Thus, there were four possible combinations by assembling the two moieties (Fig. 5C). Moreover, ions at m/z 250.1433 [M + H – CO2 ]+ and 204.1370 [M + H – CO2 – HCOOH]+ indicated the presence of two free carboxyl groups. Furthermore, the ion at m/z 98.0965 was consistent with the elemental composition of C6 H12 N+ (calc. 98.0964), which indicated the nitrogen atom of 2,3,4,5-tetrahydropyridinium was substituted by CH2 with a double bond. Consequently, compound 4 was identified as structure (a), shown in Fig. 5C. Product ions of compound 4 were consistent with the MS cleavage patterns of structure (a) (Fig. 5D). Based on the mathematics strategy, 31
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Fig. 4. Structure elucidation based on polarity strategy.
alkaloids, labeled ‘d’ in Table S1, including 26 new compounds and one new natural product, were unequivocally identified or tentatively characterized. 3.2.5. Structure elucidation based on structure assembly (sapogenin–saccharide assembly) Triterpene saponins are composed of sapogenin and saccharide moieties. By carefully analyzing the structural features of all the triterpene saponins from licorice, 29 sapogenins (Fig. S4) and three main types of saccharide (glucuronic acid, glucose and rhamnose) were reported. In addition, for the known saponins in licorice, three possible substituted positions of saccharide chain were observed: (1) two glucuronic acid units (GluA–GluA) substituted at 3-OH of the sapogenin, which exhibited characteristic fragment ions at m/z 351.0564 [M – H – sapogenin]– and 193.0348
[M – H – sapogenin – GluAr]– in the (–) ESI–MS/MS mode; (2) two glucuronic acid units (GluA–GluA) substituted at 3-OH of the sapogenin, and one glucose substituted at 30-COOH of the sapogenin, which showed the same fragment ions as (1) in the (–) ESI–MS/MS; (3) two glucuronic acid units connected with a rhamnose (GluA–GluA–Rha) substituted at 3-OH of the sapogenin, which generated characteristic fragment ions at m/z 497.1143 [M – H – sapogenin]– , 339.0927 [M – H – sapogenin – GluA]– , 321.0822 [M – H – sapogenin – GluA]– and 175.0243 [M – H – sapogenin – GluA – Rha]– in the (–) ESI–MS/MS. In the (+) ESI–MS/MS, the predominant characteristic cleavages of the saponins were consecutive losses of saccharides and H2 O. Based on the above studies, 53 saponins, labeled ‘e’ in Table S1, were unequivocally identified or tentatively characterized.
Fig. 5. Structure elucidation of compound 4 based on mathematics strategy.
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3.2.6. Structure elucidation based on the literature Another 51 constituents, labeled ‘f’ in Table S1, which could not be identified based on the strategies mentioned above, were tentatively characterized by comparing the accurate MS and MS/MS spectra, characteristic fragmentation patterns and related botanical biogenesis with the literature. For example, compound 191, according to the quasi-molecular ions at m/z 341.1381 [M + H]+ and 339.1237 [M – H]– , had a molecular formula of C20 H20 O5 . Ten compounds (licoflavanone, 5 -prenyllicodione, licocoumarone, cyclolicocoumarone, 4 -hydroxyglabridin, (2R,3R)3,4 ,7-trihydroxy-3 -prenylflavanone, dehydroglyasperins C, glepidotin B, 5 -prenylbutein and morachalcone (A) matched reports in the relevant literature. Furthermore, in the (–) ESI–MS/MS, characteristic fragment ions at m/z 187.1116 and 151.0045 were produced by RDA cleavage patterns. Consequently, compound 191 was tentatively identified as licoflavanone (Fig. S5). 4. Conclusions In this work, a novel methodology based on a DDTK technique combined with deep structure elucidation strategies was developed for the holistic chemical profiling of Chinese licorice. Through the established method, 232 chemical compounds were detected. Furthermore, the structure elucidation strategies established in this study permitted extensive data analysis, which identified 192 compounds. What’s more, new compounds and natural products, including five organic acid alkaloids, 23 flavonoid glycoside alkaloids and three flavonoid glycosides, were identified by the proposed methodology. UPLC-Q/TOF MS incorporated with the DDTK technique allowed the characterization of micro and even trace constituents, which has not been previously possible with previously reported analytical methods. Acknowledgements This work was supported by grants from National Science and Technology Major Projects for “Major New Drugs Innovation and Development” (2015ZX09J15102-004-004, 2014ZX09304307001-005) and National Natural Science Foundation of China (81202877). The authors would like to thank Yunfeng Zheng associate professor (Pharmacy College, Nanjing University of Chinese Medicine) for providing 13 reference compounds. Appendix A. Supplementary data Supplementary data associated with this article can be found, in the online version, at http://dx.doi.org/10.1016/j.jpba.2015.06.020
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Journal of Pharmaceutical and Biomedical Analysis journal homepage: www.elsevier.com/locate/jpba
Dose-dependent targeted knockout methodology combined with deep structure elucidation strategies for Chinese licorice chemical profiling Zhenzuo Jiang a,b,1 , Yuefei Wang a,b,∗,1 , Yan Zhu a,b , Lei Zhang a,b , Xin Chai a,b , Miaomiao Jiang a,b,∗ , Lihua Shan a a b
Tianjin State Key Laboratory of Modern Chinese Medicine, Tianjin University of Traditional Chinese Medicine, Tianjin 300193, PR China Research and Development Center of TCM, Tianjin International Joint Academy of Biotechnology and Medicine, Tianjin 300457, PR China
a r t i c l e
i n f o
Article history: Received 28 January 2015 Received in revised form 8 June 2015 Accepted 14 June 2015 Available online 19 June 2015 Keywords: Licorice Dose-dependent targeted knockout technique UPLC-Q/TOF MS MS–NMR combination spectroscopy Flavonoid glycoside alkaloids Organic acid alkaloids
a b s t r a c t One of the limitations with regards to the chemical profiling of Chinese herbs is that low-level compounds are masked by high-level structures. Here, we established a novel methodology based on a dosedependent targeted knockout (DDTK) technique combined with deep structure elucidation strategies to allow the chemical profiling of Chinese licorice. We employed ultra-performance liquid chromatography coupled with quadrupole-time-of-flight mass spectrometry (UPLC-Q/TOF MS) incorporated with the DDTK technique to identify the compounds in different concentration samples and found that the compounds at the high- or medium-level were detected readily in the sample at a low concentration; subsequently, minor or trace-level constituents were identified in the sample at a high concentration by rejecting high-level constituents detected in the sample at a low concentration based on a heartcutting technique during analysis. In this study, among the 232 compounds detected, 27 compounds were unequivocally identified and 165 compounds, including 29 new compounds and two new natural products, were tentatively characterized. The novel methodology established in this work paves the way the further identification of compounds from complicated mixtures, especially traditional Chinese medicines. © 2015 Published by Elsevier B.V.
1. Introduction For thousands of years, traditional Chinese medicine (TCM) has been widely used in China for clinical therapy based on the theories of Chinese medical science. Nowadays, TCM is attracting increasing attention worldwide due to its distinguished efficiency and minimal or negligible toxicity [1–3]. However, the disadvantages, including poor quality control, ambiguous pharmacodynamics and indefinable therapeutic mechanisms, limit the worldwide spread of TCM [4]. Thus, the investigation of holistic chemical profiling of TCM contributes to clarifying its chemical material basis; how-
∗ Corresponding authors at: Tianjin State Key Laboratory of Modern Chinese Medicine, Tianjin University of Traditional Chinese Medicine, Tianjin 300193, PR China. Fax: +86 022 27386453. E-mail addresses: wangyuefei [email protected] (Y. Wang), [email protected] (M. Jiang). 1 These authors contributed equally to this work. http://dx.doi.org/10.1016/j.jpba.2015.06.020 0731-7085/© 2015 Published by Elsevier B.V.
ever, researchers have been challenged by the masking of low-level compounds by high-level constituents during analysis [5]. In order to characterize the chemical profiling of TCM, hyphenated techniques, including LC–MS [6–8], GC–MS [9–11], CE–DAD and/or MS [12–14], and LC–NMR [15], have been widely employed. Among these hyphenated techniques, ultra-performance liquid chromatography coupled with quadrupole-time-of-flight MS (UPLC-Q/TOF MS) is the most feasible method for rapid chemical profiling in TCM [16]. However, even with its excellent performance [5,7,17–20], the detection of minor- and trace-level compounds remains challenging with UPLC-Q/TOF MS, owning to the restricted dynamic range and suppression of ion signals in the presence of high-level constituents [21]. Furthermore, the technique is insufficient to confirm the structures of unknown compounds [22] and isomers using MS and MS/MS data [23]. To overcome these issues, we developed a novel methodology based on a dose-dependent targeted knockout (DDTK) technique (shown in Fig. 1), which facilitated the preparation of samples at multiple concentrations, including low, medium and high con-
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Fig. 1. Summary diagram of the integrated analytical strategy.
centrations. Subsequently, we detected high- and medium-level compounds in the sample at a low concentration and identified minor- and even trace-level constituents in the sample at a high concentration by specifically rejecting the high-level constituents that were detected in the sample at a low concentration based on a heart-cutting technique. In this work, Chinese licorice (GanCao; Glycyrrhiza uralensis), which is a member of the Glycyrrhiza species and Leguminosae family, was chosen as a challenging sample to evaluate via the DDTK method. As a renowned tonifying and harmonizing herb, licorice is a popular clinical treatment that presents in around 60% of all TCM prescriptions [24]. Through this new approach, the number of detected peaks was expanded from 50 to 232 compounds [25–27]. Furthermore, 192 compounds were identified by employing deep structure elucidation strategies, including 31 that were tentatively characterized as new compounds or natural products (five organic acid alkaloids, 23 flavonoid glycoside alkaloids and three flavonoid glycosides).
2. Materials and methods 2.1. Chemicals and materials HPLC grade acetonitrile (ACN) and methanol were purchased from Merck (Darmstadt, Germany). Formic acid with a purity of 96% was obtained from MREDA (Meridian Medical Technologies, USA). Ultra-pure water was purified by a Milli-Q system (Millipore, USA). Deuterated dimethylsulfoxide (DMSO-d6 ) was purchased from Cambridge Isotope Laboratories Inc. (CIL Inc., Andover, USA). Reference compounds, including schaftoside, neoliquiritin, liquiritin, liquiritin apioside, ononin, isoliquiritin apioside, isoliquiritin, neoisoliquiritin, liquiritigenin, calycosin, isoliquiritigenin, 18-glycyrrhizic acid, licochalcone A and 18-glycyrrhetinic acid, were purchased from National Institute for Food and Drug Control (Beijing, China), Tianjin ZhongXin Pharmaceutical Group Co., Ltd. (Tianjin, China) and Victory Biotechnology Co., Ltd. (Szechwan, China). Kumatakenin B, uralsaponin C, licoricesaponin A3, uralsaponin F, 24-hydroxy-licorice-saponin E2, 22-acetoxylglycyrrhizin, licorice-saponin E2, 22-acetoxylglycyrrhaldehyde, licorice-saponin G2, licoricone, licoisoflavone B, neoglycyrol and licoflavonol were supplied by Yunfeng Zheng, an associate professor at Pharmacy College, Nanjing University of Chinese Medicine. These reference compounds [28,29] were isolated and identified (1 H NMR, 13 C NMR, MS and UV spectra) from Glycyrrhiza uralensis by Professor Zheng. The purities of the standards were above 96%. Chinese licorice (Glycyrrhiza uralensis) was obtained from Chinese Traditional Medicine Pharmacy JianMinDaYaoFang (Tianjin, China).
2.2. Sample preparation 2.2.1. Sample preparation for DDTK analysis The decoction pieces of licorice were pulverized to a fine powder. The accurately weighed powder (0.5 g) was immersed in 10 mL 50% (v/v) methanol aqueous solution, ultrasonically extracted for 30 min and then cooled at room temperature. The extracted solution was centrifuged at 14,000 rpm for 10 min. Finally, the high concentration sample was acquired by filtering through a 0.22 m filter, and then serially diluted three and nine fold with 50% (v/v) methanol aqueous solution to obtain samples with medium and low concentrations for UPLC-Q/TOF MS analysis.
2.2.2. Sample preparation for MS–NMR combination spectroscopy (COSY) analysis The licorice powder (150 g) was macerated in 1500 mL 50% (v/v) methanol aqueous solution for 12 h and ultrasonically extracted for 30 min. The extracted solution was filtered through a funnel and concentrated to a solution with 2 g crude drug per mL by rotary evaporation under reduced pressure. Polysaccharides in the concentrated solution were precipitated and removed by adding an equal volume of methanol, and finally the solution was centrifuged for 10 min at 14,000 rpm for p-HPLC analysis. The effluent fractions, which were collected in chronological order, were concentrated and lyophilized to a powder. The accurately weighed powder (5 mg) was dissolved in 10 mL 50% (v/v) methanol aqueous solution for subsequent UPLC-Q/TOF MS analysis (a representative p-HPLC chromatogram of the licorice extract and corresponding extract max plot chromatograms of 21 effluent fractions are shown in Fig. S1). Powder (30 mg) was dissolved in 0.5 mL DMSO-d6 for subsequent NMR analysis.
2.3. DDTK analytical conditions In the low concentration sample, the high- or medium-level compounds appeared as obvious peaks. By knocking out these peaks using a heart-cutting technique during analysis, the minorand even trace-level constituents were identified in the high concentration sample. The details of this novel technique were as follows: (1) samples at multiple concentrations, including low, medium and high concentrations, were prepared; (2) high-level constituents in the sample at a low concentration were detected; (3) medium-level constituents in the sample at a medium concentration were detected by rejecting high-level constituents (abundance ≥50% base peak intensity); (4) minor- and trace-level constituents were detected in the high concentration sample by rejecting high- and medium-level constituents (Fig. S2).
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2.3.1. Chromatographic conditions Chromatographic analysis was performed on an ACQUITYTM UPLC system (Waters, Milford, USA) equipped with a binary solvent manager, sample manager and column oven. The system was controlled by Masslynx V4.1 software. Chromatographic separation was carried on an ACQUITYTM UPLC BEH Shield RP18 column (2.1 × 100 mm, 1.7 m) held at 40 ◦ C. The flow rate was 0.4 mL/min. The optimal mobile phase was composed of 0.1% formic acid aqueous solution (A) and acetonitrile (B) operating under a gradient elution program as follows: 0–10 min, 2–18% B; 10–15 min, 18–28% B; 15–22 min, 28–40% B; and 22–32 min, 40–67% B. The injection volume of the sample was 2 L. 2.3.2. MS conditions MS was performed by employing a Waters ACQUITY SYNAPTTM G2 high definition mass spectrometer system (Waters, Milford, USA) equipped with an electrospray ion (ESI) source operating in positive and negative ion modes. The optimal conditions of analysis were as follows: capillary voltage at 3.0 and 2.5 kV in positive and negative ion mode, respectively; sampling cone voltage at 30 V; extraction cone voltage at 3.0 V; source temperature at 120 ◦ C; desolvation temperature at 400 ◦ C; cone gas flow at 50 L/h; and desolvation gas flow at 700 L/h. Data were acquired in centroid mode from 50 to 1500 Da. For accurate mass to charge ratio acquisition, the MS was corrected during data acquisition using a lock mass of leucine–enkephalin (LE) at a concentration of 200 pg/mL via a LockSprayTM interface at a flow rate of 10 L/min, monitoring a reference ion for the positive ion mode ([M + H]+ = 556.2771) and negative ion mode ([M − H]– = 554.2615) to ensure accuracy during MS analysis. All the data were acquired and processed by Masslynx V4.1 software. 2.3.3. Heart-cutting conditions The DDTK technique was based on heart-cutting apparatus. In this work, a six-port valve equipped on the MS was employed to reject high-level constituents in the sample at a high concentration based on the designated valve-switching time, which referred to the retention time of the high-level constituents in the based peak intensity (BPI) chromatograms. The details of the valve-switching time were as follows: (1) to analyze the low concentration sample, high-level constituents in both the positive and negative ion modes were detected and recorded in the BPI chromatogram; (2) to analyze the medium concentration sample, peaks of the high-level constituents were rejected as waste during 0.00–1.15, 9.83–10.32 and 21.77–22.21 min (peaks 45–47 and 162) in the negative ion mode, and 9.81–10.03 and 21.75–22.26 min (peaks 45 and 161–162) in the positive ion mode; (3) to analyze the high concentration sample, peaks in 0.00–1.15, 4.15–4.65, 7.78–8.28, 9.83–10.32, 14.23–14.70, 18.96–19.15, 20.63–20.85, 21.77–22.21, 23.05–25.35, 26.70–28.40 and 29.05–29.62 min (peaks 6, 20–28, 45–47, 86–88, 138, 151, 162, 169–171, 173–185, 197–211 and 214–217) were set as waste in the negative ion mode, and 0.00–0.95, 8.62–9.28, 9.81–10.32, 17.28–17.52, 20.60–20.86, 21.75–22.26 and 23.05–23.40 min (peaks 35–37, 39, 45–47, 116–117, 151–152, 161–162 and 169–171) were set as waste in the positive ion mode. The superior reproducibility and stability of UPLC was a basic guarantee for the achievement of the DDTK technique.
powders; (3) to dissolve the powders for UPLC-Q/TOF MS and NMR assays; (4) to confirm the structures of compounds through m/z-ı spectra and assign them to the chromatographic peaks (Fig. S3). 2.4.1. p-HPLC conditions Effluent fractions were prepared by an Agilent 1260 Infinity p-HPLC system (Agilent Technologies, CA, USA) equipped with a preparative pump and multiple wavelength detector (MWD). Chromatographic separation was performed on a SymmetryPrepTM C18 column (19 × 150 mm, 7 m) held at ambient temperature and the flow rate was 10 mL/min. The optimal mobile phase was composed of 0.1% formic acid aqueous solution (A) and methanol (B). The gradient program was employed as follows: 0–5 min, 2% B; 5–60 min, 2–90% B; 60–65 min, 90–95% B. The detection wavelength was set at 254 nm. The injection volume was 5 mL. Effluent collected in the first 5 min under 2% B and labeled as fraction 0 was used to flush the strong polar compounds, such as monosaccharides, polysaccharides and amino acids, to reduce interference in the MS and NMR signals. The remaining 20 fractions (1–20) were collected from the p-HPLC every 3 min. Data were processed by ChemStation B.04.03 software. 2.4.2. NMR conditions 1 H NMR spectra were acquired at 298 K on a Bruker AVIII 600 MHz NMR spectrometer (Bruker, Zurich, Switzerland) using a PABBO probe. For spectral resonance assignment purposes, 1 H–1 H correlation spectroscopy (COSY), 1 H–1 H total correlation spectroscopy (TOCSY), 1 H–13 C heteronuclear single quantum correlation (HSQC) and heteronuclear multiple-bond correlation (HMBC) 2D-NMR spectra were acquired for interesting samples. The detailed 2D-NMR spectral conditions were as follows: COSY experiments were performed using a cosygpmfqf sequence, 9 kHz spectral width, 2 k data points, 32 scans per increment, 128 increments; TOCSY was acquired using a mlevphpp sequence, 100 ms mixing time, 9 kHz spectral width, 2 k data points, 64 scans per increment, 128 increments; HSQC spectra were acquired using an echo-antiecho phase sensitive standard pulse sequence (hsqcetgpsisp2.2) and 1.7 ms evolution time, 9 kHz spectral width in f2 , 36 kHz spectral width in f1 , 2 k data points, 32 scans per increment, 256 increments; HMBC spectra were recorded using a hmbcgpndqf sequence and 9 kHz spectral width in f2 , 36 kHz spectral width in f1 , 2 k data points, 128 scans per increment, 128 increments. These data were zero-filled to 2 k data points in the evolution dimension prior to appropriate apodization and Fourier transform (FT) with forward linear prediction. 3. Results and discussion 3.1. Holistic chemical profiling of licorice The study of holistic chemical profiling of licorice in both the positive and negative ion modes was performed by UPLC-Q/TOF MS incorporated with DDTK using the optimal conditions described above. High-, medium-, and minor- and trace-level constituents of BPI chromatograms in the negative and positive ion modes are shown in Fig. 2A and E, B and F, and C and G, respectively. In total, 232 compounds in licorice were detected, of which 192 compounds shown in Table S1 were tentatively characterized.
2.4. MS–NMR COSY analytical conditions
3.2. Deep structure elucidation
Fractions from licorice were separated by p-HPLC and verified by UPLC-Q/TOF MS and NMR. We combined the acquired data from MS and NMR, and set up a new structure elucidation strategy. The details were: (1) to collect the elutions in a time-dependent manner by p-HPLC; (2) to concentrate and lyophilize all fractions to
To achieve the delicate structure elucidation of the compounds from DDTK, a strategy of structure elucidation was applied in this study and was based on (1) authentic standards, (2) MS–NMR COSY, (3) polarity, (4) mathematics, (5) structure assembly and (6) the literature.
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3.2.1. Structure elucidation based on authentic standards Retention time data, and accurate MS and MS/MS spectra were compared for the samples with those of authentic standards, allowing 27 compounds, labeled ‘a’ in Table S1, to be unequivocally identified, including 16 flavones, 10 saponins and one coumarin. Representative BPI chromatograms of the authentic standards in the negative ion mode and positive ion mode are shown in Fig. 2D and H, respectively.
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3.2.2. Structure elucidation based on MS–NMR COSY Integrated chemical shift values (ı) acquired by NMR with tR m/z (retention time–mass to charge ratio) were generated from Q/TOF MS of the compounds in the same fraction and the structures of compounds were deduced accurately and assigned to chromatographic peaks. Taking compound 6 as an example, a detailed procedure of structure elucidation of MS–NMR COSY can be described.
Fig. 2. Representative BPI chromatograms of sample solution of licorice at (A) low conc., (B) medium conc., (C) high conc. and (D) mixed solution of 27 authentic standards in negative ion mode; sample solution of licorice at (E) low conc., (F) medium conc., (G) high conc. and (H) 27 authentic standards in positive ion mode.
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Fig. 2. (Continued).
Compound 6 presented in fractions 0–5. As shown in the 1 H NMR spectrum, the intensities of two aromatic proton signals at ı 6.99 (2H, d, J = 8.3 Hz, H-2, 6) and 6.64 (2H, d, J = 8.3 Hz, H-3, 5), one methine signal at ı 3.39 (1H, t, J = 7.5 Hz, H-7) and one methylene signal at ı 2.92 (2H, t, J = 7.5 Hz, H2 -8) were in proportion, suggesting these signals were born from the same compound with a 1,4-disubstituted benzene ring and >CH CH2 moiety in the structural skeleton. Moreover, in the HMBC spectrum shown in Fig. 3A, the proton signal of H-7 had a 3 JCH correlation with the carbon signal at ı 129.9 (C-2, 6), which showed the quaternary carbon (C-1) of a benzene ring substituted by a >CH CH2
moiety. The carbon signal at ı 156.3 (C-4) indicated that the other substituent of the benzene ring was a hydroxyl. In ESI negative ion mode, compound 6 showed a quasi-molecular ion at m/z 209.0477, indicating the molecular formula of C10 H10 O5 . The ESI–MS/MS spectrum (Fig. 3B) exhibited major fragment ions at m/z 165.0576 [M − H − CO2 ]– , 121.0673 [M − H – CO2 ]– and 93.0349 [M − H – 2CO2 – C2 H4 ]– , suggesting the presence of two carboxyl groups and one >CH CH2 moiety. Accordingly, the HMBC correlations between H-7/H2 -8 and the carbonyl carbon signal at ı 171.3 also confirmed the presence of two carboxylic groups at both sides of the >CH CH2 moiety. Consequently, compound 6 was iden-
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Fig. 3. Structure elucidation of compound 6 based on MS–NMR COSY strategy.
tified as 2-(4-hydroxyphenyl) succinic acid (Fig. 3D) and its MS fragmentation patterns are shown in Fig. 3E. By this way, 29 compounds, labeled ‘b’ in Table S1, were unequivocally identified or tentatively characterized based on MS–NMR COSY, including new compounds, natural products and isomers. 3.2.3. Structure elucidation based on polarity (chromatographic retention behaviors) Liquiritigenin and isoliquiritigenin are the basic skeletons of flavanones and chalcones in licorice, whose characteristic fragment ions appear at m/z 137.0233 in the positive mode, and m/z 135.0088 and 119.0502 in the negative mode. When liquiritigenin and isoliquiritigenin are connected to glucose (Glc), apiose (Api) or a combination of them (Glc, Glc–Api, Glc–Glc, etc.), glycosides may be achieved. Three characteristic eluotropic regulars were summarized according to the chromatographic behaviors of the authentic standards (neoliquiritin, liquiritin, liquiritin apioside, isoliquiritin apioside, isoliquiritin, neoisoliquiritin, liquiritigenin and isoliquiritigenin) as follows: (1) liquiritigenin and its glycosides were prior to isoliquiritigenin and its corresponding glycosides; (2) for the glycoside derivatives of liquiritigenin, 7-O-glucoside-substituted glycosides preceded 4 -O-glucoside, as well as glucosyl-substituted glycosides, which were prior to apiosyl–glucosyl-substituted glycosides that were substituted at same position; (3) for the glycoside derivatives of isoliquiritigenin, an inverse rule was displayed and 4-O-glucoside-substituted glycosides preceded 4 -O-glucoside, and apiosyl-glucosyl-substituted glycosides were prior to glucosyl-substituted glycosides that were substituted at same position. For example, in the ESI negative ion mode, compounds 15, 16, 17 and 18 generated quasi-molecular ions at m/z 499.0917, 499.0917, 631.1337 and 631.1349, respectively. In the (−) ESI–MS/MS mode, compounds 15 and 16 yielded the same fragment ions at m/z 417.1179/417.1172 [M – H – H2 SO3 ]– , 255.0656/255.0653 [M – H – H2 SO3 – Glc]– and 135.0094/135.0081 [M – H – H2 SO3 – Glc – VP]– , which were consistent with the structures of neoliquiritin sulfite and liquiritin sulfite. Compounds 17 and 18 gen-
erated the same fragment ions at m/z 549.1591/549.1591 [M – H – H2 SO3 ]– , 255.0657/255.0653 [M – H – H2 SO3 – Api – Glc]– and 135.0093/135.0076 [M – H – H2 SO3 – Api – Glc – VP]– , which were consistent with the structures of neoliquiritin apioside sulfite and liquiritin apioside sulfite. Moreover, by comparing the chromatographic behaviors of compounds 38, 40, 45 and 47, compounds 15, 16, 17 and 18 were identified as neoliquiritin sulfite, liquiritin sulfite, neoliquiritin apioside sulfite and liquiritin apioside sulfite. As a result, 27 compounds, labeled ‘c’ in Table S1, were unequivocally identified or tentatively characterized based on chromatographic retention behaviors (Fig. 4). 3.2.4. Structure elucidation based on mathematics (MS data calculation) In this study, 28 new alkaloids were observed that have rarely been reported in the literature. Thus, mathematics were proposed and introduced for structure elucidation of these alkaloids. For instance, in the (+) ESI–MS/MS spectrum of compound 4 (Fig. 5A), fragment ions at m/z 250.1433, 232.1313, 204.1370, 170.0764, 107.0495, 98.0965 and 84.0806 were observed. By mathematics (Fig. 5B), the ion at m/z 84.0806 was generated by loss of C10 H10 O5 (the same elemental composition as compound 6) from the precursor ion directly, which was consistent with the elemental composition of C5 H10 N+ (calc. 84.0808). Therefore, it was deduced to be 2,3,4,5-tetrahydropyridinium via ChemSpider based on the biosynthesis approaches of natural products [30,31] (see chapters 2 and 3 in Medicinal Natural Products). Thus, there were four possible combinations by assembling the two moieties (Fig. 5C). Moreover, ions at m/z 250.1433 [M + H – CO2 ]+ and 204.1370 [M + H – CO2 – HCOOH]+ indicated the presence of two free carboxyl groups. Furthermore, the ion at m/z 98.0965 was consistent with the elemental composition of C6 H12 N+ (calc. 98.0964), which indicated the nitrogen atom of 2,3,4,5-tetrahydropyridinium was substituted by CH2 with a double bond. Consequently, compound 4 was identified as structure (a), shown in Fig. 5C. Product ions of compound 4 were consistent with the MS cleavage patterns of structure (a) (Fig. 5D). Based on the mathematics strategy, 31
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Fig. 4. Structure elucidation based on polarity strategy.
alkaloids, labeled ‘d’ in Table S1, including 26 new compounds and one new natural product, were unequivocally identified or tentatively characterized. 3.2.5. Structure elucidation based on structure assembly (sapogenin–saccharide assembly) Triterpene saponins are composed of sapogenin and saccharide moieties. By carefully analyzing the structural features of all the triterpene saponins from licorice, 29 sapogenins (Fig. S4) and three main types of saccharide (glucuronic acid, glucose and rhamnose) were reported. In addition, for the known saponins in licorice, three possible substituted positions of saccharide chain were observed: (1) two glucuronic acid units (GluA–GluA) substituted at 3-OH of the sapogenin, which exhibited characteristic fragment ions at m/z 351.0564 [M – H – sapogenin]– and 193.0348
[M – H – sapogenin – GluAr]– in the (–) ESI–MS/MS mode; (2) two glucuronic acid units (GluA–GluA) substituted at 3-OH of the sapogenin, and one glucose substituted at 30-COOH of the sapogenin, which showed the same fragment ions as (1) in the (–) ESI–MS/MS; (3) two glucuronic acid units connected with a rhamnose (GluA–GluA–Rha) substituted at 3-OH of the sapogenin, which generated characteristic fragment ions at m/z 497.1143 [M – H – sapogenin]– , 339.0927 [M – H – sapogenin – GluA]– , 321.0822 [M – H – sapogenin – GluA]– and 175.0243 [M – H – sapogenin – GluA – Rha]– in the (–) ESI–MS/MS. In the (+) ESI–MS/MS, the predominant characteristic cleavages of the saponins were consecutive losses of saccharides and H2 O. Based on the above studies, 53 saponins, labeled ‘e’ in Table S1, were unequivocally identified or tentatively characterized.
Fig. 5. Structure elucidation of compound 4 based on mathematics strategy.
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3.2.6. Structure elucidation based on the literature Another 51 constituents, labeled ‘f’ in Table S1, which could not be identified based on the strategies mentioned above, were tentatively characterized by comparing the accurate MS and MS/MS spectra, characteristic fragmentation patterns and related botanical biogenesis with the literature. For example, compound 191, according to the quasi-molecular ions at m/z 341.1381 [M + H]+ and 339.1237 [M – H]– , had a molecular formula of C20 H20 O5 . Ten compounds (licoflavanone, 5 -prenyllicodione, licocoumarone, cyclolicocoumarone, 4 -hydroxyglabridin, (2R,3R)3,4 ,7-trihydroxy-3 -prenylflavanone, dehydroglyasperins C, glepidotin B, 5 -prenylbutein and morachalcone (A) matched reports in the relevant literature. Furthermore, in the (–) ESI–MS/MS, characteristic fragment ions at m/z 187.1116 and 151.0045 were produced by RDA cleavage patterns. Consequently, compound 191 was tentatively identified as licoflavanone (Fig. S5). 4. Conclusions In this work, a novel methodology based on a DDTK technique combined with deep structure elucidation strategies was developed for the holistic chemical profiling of Chinese licorice. Through the established method, 232 chemical compounds were detected. Furthermore, the structure elucidation strategies established in this study permitted extensive data analysis, which identified 192 compounds. What’s more, new compounds and natural products, including five organic acid alkaloids, 23 flavonoid glycoside alkaloids and three flavonoid glycosides, were identified by the proposed methodology. UPLC-Q/TOF MS incorporated with the DDTK technique allowed the characterization of micro and even trace constituents, which has not been previously possible with previously reported analytical methods. Acknowledgements This work was supported by grants from National Science and Technology Major Projects for “Major New Drugs Innovation and Development” (2015ZX09J15102-004-004, 2014ZX09304307001-005) and National Natural Science Foundation of China (81202877). The authors would like to thank Yunfeng Zheng associate professor (Pharmacy College, Nanjing University of Chinese Medicine) for providing 13 reference compounds. Appendix A. Supplementary data Supplementary data associated with this article can be found, in the online version, at http://dx.doi.org/10.1016/j.jpba.2015.06.020
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