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This work describes a sensitive and specific LC-ESI-MSn methodology for identification of the major constituents in Zhimu-Huangqi herb-pair extract and their metabolites in rats after oral administration. A total of 30 compounds were characterized from the extract. 13 were unambiguously identified by comparing the retention times and mass spectra with those of reference standards, and 17 were tentatively identified on the basis of their MSn fragmentation behaviors and molecular weight information from literatures. In addition, the metabolites in vivo were also identified. The Zhimu-Huangqi herb-pair was actively metabolized in rats, including 3 prototypes and 9 metabolites in serum, while 7 prototypes and 23 metabolites in urine. This study proposed a serious of potential bioactive components of this herb-pair in traditional Chinese medicine (TCM) and could facilitate further pharmacokinetic studies of the Zhimu-Huangqi herb-pair.
Keywords: LC-ESI-MSn; Zhimu-Huangqi; herb-pair; major constituents; metabolites
Compatibility of Chinese medicinal herbs refers to the combination of two or more herbs with purpose in the light of the clinical requirement and medicinal properties and actions . Unique combinations of the traditionally defined herbal properties of traditional Chinese medicine (TCM) are frequently used for achieving mutual reinforcement, mutual assistance, mutual restraint, and mutual detoxication [2, 3]. The Zhimu-Huangqi herb-pair is a combination of Rhizoma Anemarrhenae (RA) and Astragalus Membranaceus (AM). Nowadays, as a famous Chinese medicine formula, numerous studies have demonstrated the Zhimu-Huangqi herb-pair have attractive pharmacological activities, including tonic, diuretic, immunostimulant, antioxidant, hepatoprotective, anti-diabetic, anti-cancer and expectorant effects [4-7].
Various analytical techniques currently used for identification of crude drugs of RA or AM include thin layer chromatography , high-performance liquid chromatography coupled with evaporative light scattering detection (HPLC-ELSD) , and HPLC-diode array detection (DAD) . However, the saponins in Zhimu-Huangqi herb-pair with poor UV absorption (at short wavelengths 200-210 nm) are difficult to detect using DAD. Although it is a universal detection suitable for the analysis of non-chromophoric compounds, ELSD only provides information about retention time for purposes of identification, leading to poor accuracy.
Recently, the development of high performance liquid chromatography coupled with electrospray ionization multistage tandem mass spectrometry (LC-ESI-MSn) has demonstrated its value in analysis of the complex mixtures like the Traditional Chinese medicine (TCM) and their metabolites in vivo [11-13]. The compounds in a mixture can be efficiently separated by high-performance liquid chromatography (HPLC), and then be characterized by MS. Due to its high sensitivity and functional versatility, MS detection can provide more valuable structural information than conventional detectors like UV, and allows for fast and specific structural elucidation of unknown compounds at fairly low levels, even when standard samples are not available .
Here, we report a simple and robust LC-ESI-MSn method for the identification of the major constituents and their metabolites in rat urine after oral administration of decoction of Zhimu-Huangqi herb-pair. A total of 30 compounds were identified or tentatively characterized which mainly included xanthones, isoflavonoids and saponins. In addition, the metabolites in vivo were also identified, including 3 prototypes and 9 metabolites in serum while 7 prototypes and 23 metabolites in urine. Our results will facilitate the comprehensive research on the Zhimu-Huangqi herb-pair and assist in the better understanding the mechanism of action of this TCM.
Chemicals and Reagents
Reference standards (Timosaponin A-III, Timosaponin B-III, Timosaponin B-II, Calycosin-7-O-Î²-glucoside, Calycosin and Formononetin-7-O-Î²-glucoside) were prepared in our laboratory and their chemical structures were unambiguously identified by comparison of their 1H-NMR, 13C-NMR and MS data with the literature data [15-18]. The Mangiferin (NO.111607-200301), the Neomangiferin (NO.111597-200308), the Astragaloside IV (NO.110781-200613) and the Formononetin (NO.111703-200501) were purchased from the National Institutes for Food and Drug Control (Beijing, China). The Astragaloside I, Astragaloside II and Astragaloside III were purchased from DELTA information Centre for Nature Organic Compounds (Anhui, China). The purities of all ingredients were above 98% according to HPLC analysis.
RA and AM were purchased from Shanghai Kangqiao Traditional Chinese Medicine Co. Ltd., Shanghai, China and identified by Professor C. G. Huang of the Shanghai Institute of Meteria Medica, Chinese Academy of Sciences.
Acetonitrile and formic acid of HPLC grade purchased from Dikma Company (Dikma, USA). All other analytical chemical reagents of analytical grade purchased from Sinopharm Chemical Reagent Co. Ltd., Shanghai, China. Deionized water was purified using a Milli-Q system (Millipore, Billerica, MA, USA), Double-distilled water was used for all the preparations.
Chromatographic System and Mass Spectrometry Conditions
The chromatographic separations were performed using a reversed-phase column (Inertsil ODS-3, 4.6Ã-250 mm i.d., 5 Î¼m, GL Science, Tokyo, Japan) connected to an Easy-Guard Kit C18 (4Ã-2 mm, Grace, USA) guard column, with the column temperature set at 25°C. The mobile phase consisted of linear gradients of 0.2% (v/v) formic acid (A) and acetonitrile (B): 0-10 min, 10-27% B (v/v); 10-18 min, 27-29% B; 18-40 min, 29-95% B; 40-55 min, held at 95% B. The mobile phase flow rate was 1.0mL/min and the analysis time was 55 min.
The LC-MSn analysis was performed on the 6300 Series Ion Trap LC/MS (Agilent technology, Pala Alto, CA, USA). The hardware including an Agilent 1200 Series LC, the ion trap mass spectrometer and the data system. The software (version 6.1) included the Trap Control Program for trap control, data acquisition, data analysis, quantitative analysis and the LC control. MSn analyses were conducted in negative-ion mode and the instrument was operated under the following optimized conditions: collision gas, ultrahigh-purity Helium (He); nebulizing gas, high-purity nitrogen (N2); capillary voltage, 3.5 kV; end plate offset, 500 V; nebulizer, 30 psi; drying gas flow rate, 10 LÂ·min-1; drying gas temperature, 350 °C. For full-scan MS analysis, the spectra were recorded in the range of 50-1500 m/z.
To 50 g of RA and 150 g AM crude herbs were added 3000 mL of H2O. The mixture was refluxed at 65 °C twice for 1.5 h each. The sample was immediately cooled to room temperature, and was then centrifuged at 12,000 rpm for 10 min. The supernatant was concentrated under reduced pressure to afford 400 mL residue, 2 mL of the residue was filtered through a 0.22 Î¼m micro-porous membrane before use. Samples of individual herbs were prepared with the same procedure to make 0.5 gÂ·mL-1 extracts. An aliquot of 10 Î¼L of each sample was injected into the LC/MS instrument for analysis.
Animals and drug administration
All animal procedures were conducted in accordance with the guidelines from the Review Committee of Animal Care and Use at the Shanghai Institute of Materia Medica (Shanghai, China). 18 male Sprague-Dawley rats (200-220 g; Shanghai SLAC Laboratory Animal Co., Shanghai, China) were kept in an environmentally controlled breeding room for one week and fasted 12 h (but with access to water) before starting the experiments. These animals were randomly divided into three groups: a dosed urine collection group (group A, n=6); a dosed plasma collection group (group B, n=6) and a control group (group C, n=6).
After the oral administration, heparinized blood samples were collected at 0.5, 1.0, 2.0, 4.0, 6.0 and 8.0 h (n=6) from the abdominal aorta of the rats, shaken up and then centrifuged at 12,000 g for 10 min to obtain the plasma. Urine samples were collected during the time period 0-24h (n=6). All samples were stored at -80°C until analysis.
All the preparation procedures of plasma and urine samples were according to Liu et al  and Chen et al . Then, a 600 Î¼L aliquot of urine and plasma samples was loaded onto an SPE cartridge, which was preconditioned with 2 mL methanol and 2 mL water. Then the cartridge was washed with 1 mL of water and the analyte was eluted with 1 mL of methanol. The eluted solution was evaporated to dryness in a water bath at 37°C under a gentle stream of nitrogen and the residue was reconstituted in 200 Î¼L methanol. The resulting solution was centrifuged at 12,000 g for 10 min at 4°C and 10 Î¼L of supernatant was injected into the chromatographic system for LC-MS/MS analysis. Blank samples as controls were prepared with the same method as the drug-containing samples.
Results and discussion
Structure elucidation of the reference compounds by LC-MSn
In terms of the chemical diversity of nature products, the strategy we recently proposed was used with minor modifications to identify the compounds in Zhimu-Huangqi herb-pair by LC/MS. When a pure standard was available, the compound was identified by comparing its HPLC retention time and mass spectra with those of the standards. When no standard was available, the structures were proposed mainly based on the mass spectra. Analysis of retention time and MS spectral data of the reference compounds and conclusion of the rules will be valuable for subsequent on-line elucidation of structurally related compounds. Thus, the first step of this work was to characterize the chromatographic and mass spectral properties of the reference compounds. These data will provide a scientific basis for identification of other bioactive compounds in plant extracts. A typical MS total ion current chromatogram of 13 reference compounds studied in the experiment is in shown in Fig. 1.
Among the 13 reference compounds analyzed, there were 2 xanthones, 3 timosaponins, 4 astragaloside and 4 isoflavonoids. The MSn spectra for S1 (m/z 583), S3 (m/z 491), S4 (m/z 919) and S10 (m/z 871) in negative ion mode were shown in Fig. 2. Negative ESI analysis of S1 gave the [M-H]ï¼ ion at m/z 583. The MS2 experiment of the m/z 583 ion yielded three prominent ions at m/z 565, 493 and 421 through neutral losses of H2O (18 Da), C3H6O3 (90 Da) and C6H11O5 (162 Da), respectively. The ion at m/z 421 was subjected to MS3 analysis to afford four ions at m/z 403, 331, 301 and 259 by losses of H2O (18 Da), C3H6O3 (90 Da), C4H8O4 (120 Da) and C6H11O5 (162 Da), respectively. Based on these data, the structure of S1 could be identified as neomangiferin. The mechanistic pathway for fragments formed may be explained according to Fig. 3(A).
Compound S3 exhibited a [M+Cl-H]ï¼ and [M+HCOOH-H]ï¼ ion at m/z 481 and 491. Its MS/MS spectra produced a prominent ion at m/z 283, originating from loss of branch glycoside chain (162 Da). In the MS3 of m/z 283, fragments of m/z 268 was generated owning to loss of one water molecule. Thus compound S4 was tentatively identified as Calycosin-7-O-Î²-glucoside. The proposed fragmentation pathway for compound S3 is shown in Fig. 3(B).
Compound S4 displayed a typical [M-H]ï¼ at m/z 919. In addition, three ions at m/z 757 [M-H-Glu]ï¼, 577 [M-H-Glu-Gal]ï¼ and 433 [M-H-2Glu-Gal]ï¼ could be detected in the MS/MS spectrum of the ion at m/z 919, originating from loss of branch glycoside chain (162 Da). The above fragmentations were consistent with those of Timosaponin B-II. The mechanistic pathway for fragments formed may be explained according to Fig. 3(C).
Compound S10 showed a typical [M-H+HCOOH]ï¼ and [M-H]ï¼ at m/z 871 and 825. Moreover, three ions at m/z 765 [M-H-Ac-H2O]ï¼, 633 [M-H-H2O-Acetylated Xyl]ï¼ and 489 [M-H-Glu-Acetylated Xyl]ï¼ could be detected in the MS/MS spectrum of the ion at m/z 825. The above fragmentations were consistent with those of Astragaloside II. The proposed fragmentation pathway for compound S10 is shown in Fig. 3(D).
Compound S8 and compound S9 displayed the same quasi-molecular ion at m/z 829. Additionally, the characteristic fragment ions in compounds S8 and S9 are the same, i.e., m/z 651 [M-H-Xyl]ï¼, 621 [M-H-Glu]ï¼ and 489 [M-H-Xyl-Glu]ï¼. Similar to Astragaloside II, these characteristic fragment ions are yielded by the loss of branch glycoside chain. However, compounds S8 and S9 can be differentiated by their retention times since Astragaloside IV was eluted earlier than the compound Astragaloside III on different chromatographic columns. Thus, compounds S8 and S9 were identified as Astragaloside IV and Astragaloside III, respectively.
Taking the same identification method as for S1, S3, S4 and S10 described above, the other 7 reference compounds, S2, S5, S6, S7, S11, S12 and S13, were unambiguously identified as Mangiferin, Ononin, Calycosin, Timosaponin B-III, Formononetin, Astragaloside I and Timaosaponin A-III. The retention time, ESI-MSn data and fragmentations of 13 standard compounds are summarized in Table 1. The ESI-MSn data of these compounds shared some common features, such as the neutral loss of H2O (18 Da), branch glycoside chain (162 Da) in timosaponins and astragalosides or C3H6O3 (90 Da) and C4H8O4 (120 Da) in xanthones. These specific fragmentation rules discussed above can be reliably and effectively used for rapid screening and identification of other bioactive xanthones, timosaponins, isoflavonoids and astragalosides in Zhimu-Huangqi herb-pair extract and their metabolites in vivo in the following sections.
Identification of the major bioactivity constituents in Zhimu-Huangqi herb-pair extract
In the total ion current (TIC) chromatograms of Zhimu-Huangqi herb-pair extract, a total of 30 compounds were identified on the basis of their retention time and mass spectrometry fragmentation patterns. The LC-MS total ion chromatograms for Zhimu-Huangqi herb-pair extract are shown in Fig. 4 and their chemical structures are presented in Fig. 5. Among them, peaks 1, 3, 6, 10, 11, 16, 17, 20, 21, 24, 27, 28 and 31 could be unambiguously identified as Neomangiferin, Mangiferin, Calycosin-7-O-Î²-glycoside, Timosaponin B-II, Ononin, Calycosin, Timosaponin B-III, Astragaloside IV, Astragaloside III, Astragaloside III, Formononetin, Astragaloside I and Timaosaponin A-III by comparing their retention times, molecular weights and fragment ions with those of standards S1-S13. From the ESI-MSn studies on standards S1-S3, we can conclude that the characteristic fragmentation pathways for the bioactivity constituents of Zhimu-Huangqi herb-pair extract were the characteristic neutral losses of 18 Da (H2O), 90 Da (C3H6O3), 120 Da (C4H8O4) and the branch glycoside chain (162 Da), which could be used for speculating as to the existence of xanthones and saponins. These characteristic neutral losses and the characteristic cleavage of branch glycoside chain provided a sound basis for elucidation of other 17 compounds in Zhimu-Huangqi herb-pair extract.
Peak 2 exhibited a [M+HCOOH-H]ï¼ ion at m/z 521. The prominent ions at m/z 313 resulted from the loss of branch glycoside chain C6H11O5 (162 Da), which was similar to Calycosin-7-O-Î²-glycoside and Ononin. Compared with the known compound reported in the literature [21, 22], peak 2 was tentatively identified as Odoratin-7-O-Î²-glycoside. Similarly, peaks 13, 14 and 15 were identified as 9,10-dimethoxypteracarpan-3-O-Î²-D-glycoside, Afrormosin-7-O-Î²-glycoside and 2'-hydroxy-3',4'-dimethoxyisoflavan-7-O-D-glycoside as reported in the literatures [22-24].
Peak 4 gave a [M-H]ï¼ ion at m/z 421, indicating that it was the isomer of Mangiferin. Its MS2 spectrum yielded a typical fragmentation ion at m/z 283, corresponding to the loss of branchglycoside chain (162 Da). The ion represented a similar pathway to Mangiferin. By searching the known compound reported in the literature , peak 4 was tentatively identified as Isomangiferin.
Peak 5 displayed a [M-H]ï¼ ion at m/z 901, suggesting that it was a pair of isomer with Timosaponin B-III. The prominent ions at m/z 739, 577, 415 resulted from the loss of branch glycoside chains, which was the similar to Timosaponin B-III. These characteristic ions of peak 5 manifested the same pathway as peak 17. The above fragmentations were consistent with known Timosaponin C, thus, peak 5 was tentatively identified as Timosaponin C. Similarly, peaks 9, 12, 22, 23 and 26 were Timosaponin D, Timosaponin B-I, Timosaponin F, Anemarrhenasaponin I and Timosaponin G as reported in the literatures [16, 25-27].
Peak 7 and 8 in the MSn spectra gave a quasi-molecular ion at m/z 931, including that these two compounds were isomers. Moreover, five ions at m/z 773 [M-H-Glu]ï¼, 755 [M-H-H2O-Glu]ï¼, 611 [M-H-Glu-Gal]ï¼ and 449 [M-H-2Glu-Gal]ï¼, 593 [M-H-H2O-Glu-Gal]ï¼ could be detected in MSn spectrum of the ion at m/z 935. Based on these data, the structure of peak 7 and 8 could be identified as timosaponin N or timosaponin E1. Owning to lack of reference compounds and more characteristic signals in ESI-MSn, we couldn't distinguish them.
Peaks 18-21, 24, 25, 28, 29 displayed common characteristic ions of m/z 143, 401, 419, 437, 455, and 473, which were the same as the Astragaloside IV. All the major peaks in the MSn spectra of these compounds were in accordance with [M+HCOOH-H]ï¼ions showed that all of these 8 saponins, because of the inevitable presence of formic acid during the sample separation process, MS/MS spectra of these [M-H+HCOOH] - were 9,19-cyclolanostane aglycone (m/z 489) and some sugar moieties. Based on the comparison of the retention time and MS/MS data with the standard, peak 20 was identified as Astragaloside IV and peak 21 was identified as Astragaloside III. Peak 19 gave the same MS and MS/MS data as those of standard, and was thus ascribed to isoastragaloside IV.
Peaks 24 and 25 are a pair of isomers. Both of them gave an adduct ion [M-H+HCOOH]ï¼ at m/z 871 and significant [M-H]ï¼ signal at m/z 825, and m/z 871 produced similar fragment ions to those of standard, except for the ions containing an acetoxy group. Additional fragmentation peaks in the MSn spectrum at m/z 489 [M-H-(Ac-H2O)-Glu-Xyl]ï¼, 603 [M-H-(Ac-H2O)-180]ï¼ and 633 [M-H-(Ac-H2O)-180]ï¼, indicated that the acetyl group was linked to the glucose in the sugar portion. By examining the known Astragalosides in AM, it was found that there were two saponins, named Astragaloside II and isoastragaloside II, consisted with the above data. According to the content difference and the retention behavior in previous reports [28, 29], peaks 24 and 25 were tentatively assigned to Astragaloside II and isoastragaloside II, repectively. Similarly, peaks 28 and 29 containing [M+HCOOH-H]ï¼ ion (m/z 913) and [M-H]ï¼ ion (m/z 867) were tentatively assigned as Astragaloside I and isoastragaloside I, respectively. Additionally, peak 18 was ascribed to acetylastragaloside I or its isomer based on their MS and MS/MS spectra and by comparison with the literature data .
These 30 identified compounds represented the major components of the Zhimu-Huangqi herb-pair extract. The majority of them had been reported as the main bioactive constituents of the individual herbs.
Identification of the metabolism compounds in rat serum and urine samples
By comparing the accurate masses of peaks appearing in the chromatograms of drug-containing serum and urine with those previously identified in Zhimu-Huangqi herb-pair extract (Fig. 6), 3 peaks and 7 peaks were detected as prototype components of Zhimu-Huangqi herb-pair in serum and urine, respectively. Beyond the prototype compounds, more than 23 peaks were tentatively predicted to be metabolites of Zhimu-Huangqi herb-pair and could be generally divided into two groups: isoflavonoid-related and xanthone-related metabolites. As shown in Table 3, most of the metabolites were identified respectively by comparison of their retention times and MSn data with those of the prototypes and the literatures reported before [19, 20, 24].
Based on these metabolites, the possible metabolic pathways of Mangiferin, Calycosin and Formononetin in Zhimu-Huangqi herb-pair were proposed and summarized in Fig. 7. The results revealed that glucuronidation and sulfation were the main metabolic pathways of isoflavonoids in Zhimu-Huangqi herb-pair. In addition, it showed that some Phase I reactions, such as hydrolysis, demethylation, methylation and hydroxylation, also occurred in the metabolism of isoflavonoids in Zhimu-Huangqi herb-pair.
Saponins also play an important role in Zhimu-Huangqi herb-pair. However, only very low amounts of Astragaloside II (P4), Astragaloside IV (P5), Isoastragaloside II (P6) and Timosaponin A-III (P8) were detected in the drug-containing serum and urine. This can be possibly attributed to the fact that saponins were poorly absorbed directly, and could be mainly excreted through feces or bile.
In the present study, a LC-ESI-MSn method was developed for the analysis of the major constituents of the Zhimu-Huangqi herb-pair extract and their metabolites in rat serum and urine. A total of 30 compounds were identified or tentatively characterized in the decoction based on their mass spectra determined in negative ion mode. The Zhimu-Huangqi herb-pair was actively metabolized in rats, including 3 prototypes and 9 metabolites in serum, while 7 prototypes and 23 metabolites in urine. The developed method was simple, reliable and sensitive, which revealed it to be appropriate for rapid screening and structural characterization of the major constituents and their metabolites of Zhimu-Huangqi herb-pair. These results could facilitate the clarification of the metabolic process of the Zhimu-Huangqi herb-pair in vivo and better understand its action mechanism.
We would like to appreciate the State Key Program of National Natural Science Foundation of China (no. 81030065) and the National Science & Technology Major Project "Key New Drug Creation and Manufacturing Program", China (no. 2012ZX09301001-001 and 2013ZX09102027) for ï¬nancial support of this work.