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Chem. Pharm. Bull. 60(6) 712–721 (2012)
Regular Article
Vol. 60, No. 6
Characterization Using LC/MS of the Absorption Compounds and Metabolites in Rat Plasma after Oral Administration of a Single or Mixed Decoction of Shaoyao and Gancao Lan Shen,*,a,b Wen-Juan Cong,a Xiao Lin,a,b Yan-Long Hong,a Rong-Wan Hu,a,b Yi Feng,a,b De-Sheng Xu,c and Ke-Feng Ruana a
Engineering Research Center of Modern Preparation Technology of Traditional Chinese Medicine, Ministry of Education, Shanghai University of Traditional Chinese Medicine; b College of Chinese Material Medica, Shanghai University of Traditional Chinese Medicine; Shanghai 201203, P. R. China: and c Shuguang Hospital, Affiliated to Shanghai University of Traditional Chinese Medicine; Shanghai 200021, P. R. China. Received November 22, 2011; accepted March 12, 2012; published online March 14, 2012 Shaoyao–Gancao decoction (SGD), a traditional Chinese formulation containing Paeoniae Radix (SY) and Glycyrrhizae Radix (GC), is commonly used to relieve abdominal pain. However, the absorption and metabolites of the characteristic constituents of the two herbs in vivo have been reported rarely. The purpose of this study was to investigate the compatibility rationality and the mechanism of the enhanced efficiency of SGD. A single or a mixed decoction (SG and S+G, respectively) was orally administered to rats. Blood samples were collected at different intervals following treatment and analyzed by liquid LC/MS. A total of fifteen ingredients (denoted as M1 to M15) were found in both rat plasma after treatment with the two decoctions. Furthermore, the proposed structures of the remained twelve ingredients were obtained except M9, M10 and M15. The quality of the ingredients in the rat plasma showed no significant difference between the two decoctions. However, the quantity of twelve ingredients differed greatly, indicating that the absorption of SG was greater than that of S+G except M7, M12 and M15. As the compositions associated with the efficacy of SG and S+G were inconsistent, the degree of the absorption of the 15 ingredients by the gastrointestinal tract were different, which caused a significantly enhanced efficacy of certain ingredients. This study presents an exploration of the mechanism behind the improved efficacy of individual components in traditional Chinese medicine therapies through combination with other components. Key words
Shaoyao–Gancao decoction; absorption; metabolite; LC/MS
Traditional Chinese medicine (TCM) formulations are often composed of more than two herbs and are used for a wide range of treatments. The efficacy of a formulation cannot be calculated simply as the sum of the efficacies of the individual components. In combination, components may either yield additive therapeutic effects or diminish possible adverse effects. Furthermore, classical research methods for active constituents of herbal or medical formulas in vitro cannot replace the study of absorption characteristics in vivo. The effective constituents of Chinese herbal formulas can potentially be ascertained by analyzing the compounds absorbed in the blood after oral administration, since the compounds absorbed in the blood are likely to be potent. Changes in efficacy in vivo include both quantitative changes, such as significantly improving the absorption efficacy of related substances in combination with each other,1–5) and qualitative changes. Combination formulations can mean that some substances may disappear or new active metabolites may emerge.6,7) The qualitative or quantitative changes in combination could reflect the mechanism of the enhanced efficiency or reduced toxicity of the TCM, and may potentially provide the theoretical basis for the the effects of combining substances in TCM. The in vivo study of a single compound or ingredient cannot fully explain the combination mechanisms based on multiple ingredients, targets and/or mechanisms. In addition, traditional technologies such as HPLC often focus on the changes of pharmacokinetic parameters. The exact structures of the absorbed components or potential metabolites in rat plasma after administration have not yet been identified. However, it is supposed that powerful hyphenated instruments, such as LC/MS, are useful tools for * To whom correspondence should be addressed.
the identification of component structure. Shaoyao–Gancao decoction (SGD), a traditional Chinese formulation composed of Paeoniae Radix (SY) and Glycyrrhizae Radix (GC), is frequently used in clinical treatment. Modern pharmacological studies have revealed that SGD has a variety of effects, including analgesia,8) anti-inflammatory effects9) and anti-allergy/anti-asthma effects.10) It has been reported that triterpenes, flavonoids and pinanes have been extracted from SGD.11) Human intestinal bacterial studies on the metabolites of SY and GC have been carried out in vitro.12) However, there are few in vivo studies on the absorption and metabolites of the characteristic constituents of the two herbs. In previous studies, an obvious analgesic effect in the mixed decoction of SY and GC (SG) has been observed, compared to the single decoction (S+G). Additionally, paeoniflorin, glycyrrhizic acid and liquiritin have been found in the three kinds of effective plant extracts in the analgesic mouse model.13) On this basis, we found that certain peaks originated from either SY or GC. We also investigated changes in the pharmacokinetic behavior of the characteristic peaks to elucidate the nature of the absorption and metabolites of the characteristic constituents of the two herbs. In the present study, rat plasma were analyzed using LC/MS after the oral administration of SG or S+G. This study aimed to explore the mechanism of enhanced efficiency of the mixed decoction of Shaoyao and Gancao from the absorbed compounds and their potential metabolites, and to clarify the compatibility of this decoction mixture. The outcome of the study is a comprehensive explanation of the compatibility of SGD, in a qualitative and quantitative way. Furthermore, the synergistic mechanism
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© 2012 The Pharmaceutical Society of Japan
June 2012
of SGD is elucidated on the basis of metabolite variations.
Experimental
Chemicals and Materials Paeoniflorin, liquiritin, glycyrrhizic acid and glycyrrhetic acid standards were purchased from the National Institute for the Control of Pharmaceutical and Biological Products (Beijing, China). Liquiritigenin and isoliquiritigenin standards were purchased from Yuan Ye Biotechnology Co., Ltd. (Shanghai, China). Distilled water and methanol were used to extract and prepare the samples. HPLC-grade acetonitrile and methanol (Sigma-Aldrich, U.S.A.) and Milli-Q system (Millipore, Bedford, MA, U.S.A.) were used for the HPLC analysis. All other chemicals were of analytical grade and were obtained from Sinopharm Chemical Reagent Co., Ltd. (Shanghai, China). SY and GC were purchased from Kang Qiao Chinese Cut Crude Drug Co., Ltd. (Shanghai, China). Animals Healthy, male Sprague-Dawley rats, weighing 200–220 g (Certificate No. SCXK 2008-0016), were provided by the Animal Center of Shanghai University of TCM. Animals were kept in a standard breeding room (25°C, 65% relative humidity, 12 h dark–light cycle) for one week prior to the start of the experiments. The animals were fed standard laboratory chow with water ad libitum but were fasted overnight before the experiments. The animal facilities and protocols were approved by the Institutional Animal Care and Use Committee, Shanghai University of TCM. All procedures were conducted in accordance with the Guide for the Care and Use of Laboratory Animals (National Academies Press, revised edition 2010). Sample Preparation. Preparation of Reference Substance Solutions Paeoniflorin, liquiritin, glycyrrhizic acid, glycyrrhetic acid, liquiritigenin and isoliquiritigenin were weighed accurately (5 mg), placed into 10 mL volumetric flasks, and diluted with methanol to volume. The reference substance solutions were then obtainde by futher dilution of stock solution with methanol, through which the concentrations were 0.5 µg/ mL. These served as reference solutions for the analysis. Preparation of Decoctions SGD was obtained by decocting a mixture of P. Radix and G. Radix (1 : 1, w/w) twice, by boiling in water (1 : 8, w/v) for 45 min each time. The solution obtained was filtered and concentrated under reduced pressure at 60°C to 4.24 g crude material per mL (2.12 g SY, 2.12 g GC per mL). The same procedure was followed for the Shaoyao decoction (SYD), containing SY only, and the Gancao decoction (GCD), containing GC only, (4.24 g crude material per mL in each). The two decoctions were mixed together to obtain a single decoction of SY and GC (S+G), containing 2.12 g SY per mL and 2.12 g GC per mL. Plasma Sample Preparation SG, S+G or distilled water was orally administered (20 mL·kg−1) to rats twice daily and blood samples (3 mL) by abdominal aorta blood sampling were collected in heparinized tubes 30 min after administration of the dose on the second day. Blood samples were centrifuged (3000 rpm, 4°C, 15 min) to obtain the plasma samples. Each plasma sample (0.5 mL) was transferred to a 5.0 mL polypropylene tube and 2.5 mL of methanol was added. The tube was mixed by vortexing for 30 s. The precipitated protein was removed by centrifugation at 6000 rpm for 15 min. The supernatant was then transferred to a fresh tube, ready for analysis.
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Instrumentation and Conditions. Instruments A Shimadzu LC-20A liquid chromatographic system equipped with a DGL-20A vacuum degasser, a dual pump, and a SIL-20A autosampler (Shimadzu, Kyoto, Japan) was used. Detection was performed on an API 4000 QTRAP mass spectrometer equipped with TurboIonSpray (ESI) Interface (Applied Biosystems, Concord, Ontario, Canada). Analyst 1.5 software packages (Applied Biosystems) were used to control the LC/MS system, as well as for data acquisition and processing. Chromatographic and Mass Spectrometric Conditions The analytical column used was a Thermo kromasil (Switzerland) C18 (5 µm, 250×4.6 mm). A post-column diverter valve was used to direct HPLC column elute to a waste container for the first 3.5 min of the chromatographic run and then to the ionization source. The chromatographic conditions were as follows: mobile phase gradient 85% A (5 mM NH4Ac); 15% B (0.1% formic acid in a mixture of acetonitrile/methanol [50/50]) to 70% A; 30% B for 0–8 min to 50% A; 50% B for 8–17 min to 10% A; 90% B for 17–25 min to 10% A; 90% B for 25–35 min to 85% A; 15% B for 35–36 min to 85% A; 15% B for 36–45 min, flow rate 0.8 mL·min−1, column temperature 25°C, detection wavelength 232 nm and injection volume 20 µL. The product ions, enhanced mass spectrometry (EMS) and multiple reaction monitoring with enhanced product ions (MRM-EPI) scan modes were selected for analysis of the standard reference solutions and plasma samples. The ESI conditions were as follows: 5500 V spray voltage, source temperature 500°C, sheath gas nitrogen gas flow 50 psi. A 20 µL sample of the solution was injected into the LC/MS system for analysis. LC/MS Assay Validation Validation was performed by establishing inter- and intra-batch precision and repeatability for all characteristic compounds. Full scan spectra of blank plasma, plasma samples obtained 30 min after oral administration of SG and S+G in rats using the chromatographic conditions described in Sect. Instrumentation and Conditions. Chromatographic and Mass Spectrometric Conditions (Fig. 3). The samples were determined five times in a single day (intrabatch) and once per day over 5 consecutive days (inter-batch) for precision. Plasma were kept at 25°C for 24 h, 4°C for 24 h and −20°C for 15 d to determine stability. Statistical Analysis Each sample was parallel tested five times. Results are expressed as the mean±S.D. Differences of relative responses between SG and S+G was calculated by ratio. Times of the ratio of relative responses was considered increasing or deceaseing trend of absorption.
Results
Validation of LC/MS Method The LC/MS method was validated (inter- and intra-batch precision and repeatability) for all characteristic compounds (Table 1). The validation data indicated that the analytical method was specific and sensitive, and could be used for the qualitative research and comparison on the absorption of all characteristic compounds in rat plasma. Plasma were kept at 25°C for 24 h, 4°C for 24 h and −20°C for 15 d to determine the stability. There was no significant change in the retention time and relative response in any of these samples, indicating that the samples were stable under the conditions tested.
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Table 1. Inter-batch and Intra-batch Precision, Repeatability (n=5) Retention time (% R.S.D.) SG
Compounds
Intra-batch Inter-batch precision precision M1 M2 M3 M4 M5 M6 M7 M8 M9 M10 M11 M12 M13 M14 M15
0.23 0.16 0.18 0.67 0.19 0.14 0.17 0.23 0.13 0.08 0.15 0.11 0.25 0.28 0.23
0.49 0.15 0.21 0.74 0.36 0.18 0.40 0.29 0.10 0.10 0.15 0.13 0.36 0.30 0.32
Relative responses (% R.S.D.) S+G
Repeatability 0.32 0.10 0.17 0.48 0.29 0.20 0.39 0.30 0.18 0.10 0.22 0.16 0.41 0.37 0.39
Intra-batch Inter-batch precision precision 0.28 0.21 0.15 0.54 0.20 0.12 0.18 0.30 0.12 0.10 0.19 0.15 0.30 0.30 0.28
SG Repeatability
0.44 0.24 0.16 0.60 0.31 0.15 0.24 0.35 0.15 0.09 0.23 0.20 0.40 0.31 0.27
Analysis of Reference Substance Solutions To identify the metabolites, the MS fragmentation and chromatography characteristics of reference standards (paeoniflorin, liquiritin, glycyrrhizic acid, glycyrrhetic acid, liquiritigenin and isoliquiritigenin) were first identified. Under the chromatographic conditions described previously, the retention times of paeoniflorin, liquiritin, glycyrrhizic acid, glycyrrhetic acid, liquiritigenin and isoliquiritigenin were found to be 12.7, 14.2, 25.5, 34.5, 20.1 and 24.0 min, respectively (Fig. 1). In positive scan mode, the reference compound of paeoniflorin formed predominately ammonium adduct ions [M+NH4]+ at m/z 498.2, and no protonated molecules [M+H]+ were observed. Liquiritin, glycyrrhizic acid and glycyrrhetic acid formed predominately protonated molecules [M+H]+ at m/z 419.3, 823.8, and 471.2 respectively. The full scan of liquiritigenin and isoliquiritigenin showed identical protonated molecules at m/z 257. The mass spectra of the Enhanced Productions Ion (EPI) scan for the reference standards are shown in Figs. 2A–F. The major fragment ions existed at m/z 179, 151, 135 and 133 for paeoniflorin; at m/z 257, 239, 211, 147, 137 and 119 for liquiritin; at m/z 647, 471, 453, 435, 407, 343, 299, 241, 189 and 119 for glycyrrhizic acid; at m/z 436, 408, 317, 235,199, 189, 149 and 119 for glycyrrhetic acid; and at m/z 239, 147, 137 and 119 for liquiritigenin and isoliquiritigenin. Analysis of Plasma Samples. Identification of Absorption Compounds and Metabolites Full scan chromatograms for blank and dosed plasma samples, following oral administration of SG or S+G, are shown in Figs. 3A–C. Compared with blank samples, fifteen peaks were found in both the SG and S+G dosed samples. Because of a low response and strong interference, the selected ion scan was used to identify the absorption and metabolite in rat plasma after oral administration of SG or S+G. The retention times for the peaks, denoted as M1 (m/z 471.2), M2 (m/z 823.8), M3 (m/z 498.2), M4 and M5 (m/z 250), M6 and M7 (m/z 257), M8 (m/z 503), M9 and M10 (m/z 455.5), M11 (m/z 339), M12 (m/z 247.5), M13 and M14 (m/z 249.4), M15 (m/z 188), were 35.3, 26.0, 13.4, 6.4, 7.5, 11.5, 12.4, 12.2, 3.2, 4.5, 14.8, 5.6, 3.3, 5.2 and 7.6 min, respectively.
Intra-batch Inter-batch precision precision
0.37 0.19 0.17 0.52 0.30 0.13 0.33 0.33 0.16 0.11 0.29 0.23 0.44 0.40 0.37
Fig. 1.
10.23 7.66 5.57 11.48 10.78 6.23 13.52 11.23 8.10 4.66 5.80 10.77 15.03 12.24 6.05
11.25 6.98 6.23 12.31 11.33 6.14 14.20 11.59 7.63 5.27 6.03 11.82 14.58 12.09 7.11
S+G Repeatability 13.80 9.82 7.92 12.08 11.02 8.05 14.21 13.65 10.99 5.47 7.59 13.13 15.22 14.80 7.19
Intra-batch Inter-batch precision precision 10.08 6.98 7.72 11.59 10.44 5.37 11.42 11.00 9.96 3.27 6.30 10.18 12.03 13.14 4.30
10.83 7.21 7.29 12.16 10.23 7.00 13.21 11.31 10.08 3.45 5.77 11.41 13.19 12.79 5.00
Repeatability 12.07 9.00 8.80 13.01 11.14 8.71 14.69 13.96 12.09 5.43 7.12 12.33 14.63 14.47 6.17
EPI Chromatograms of Standard Reference Solutions
Based on comparison of the retention times and MRM spectra for the identified peaks and those of the standard solutions, M1, M2 and M3 were characterized as glycyrrhetinic acid, glycyrrhizic acid and paeoniflorin. MS2 scans of the remaining twelve compounds ions were performed, and part of the product ions were obtained. Based on analysis of the product ion spectra, the fragment ions for M4 were observed at m/z 166, 148, 142 and 124. The serial dehydration peaks formed were consistent with those of paeoniflorin. The fragment ions for M5 were observed at m/z 166, 135, 132. Therefore, M4 and M5 were predicted to be the isomeric metabolites of paeoniflorin. Since paeniflorin was found to form ammonium adduct ion [M+NH4]+, and no protonated molecules [M+H]+ were observed, the peak at m/z 250 is likely to be the ammonium adduct ion [M+NH4]+ for M4 and M5. The molecular weight was predicted to be 232 Da and to be a ring oxidation and cleavage product of paeoniflorin (Fig. 4). The exact structure requires confirmation by further investigation, using powerful hyphenated instruments, such as HPLC-MSn, HPLC-NMR and high resolution-MS (HR-MS). The molecular weight of M6 and M7 were same for
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Fig. 2. EPI MS Spectra of Standard Reference Solutions (A) Paeoniflorin, (B) Liquiritin, (C) Glycyrrhizic Acid, (D) Glycyrrhetic Acid, (E), Liquiritigenin and (F) Isoliquiritigenin
liquiritigenin and isoliquiritigenin (Fig. 5), but the corresponding retention times differed greatly. Therefore the possibility of M6 and M7 being liquiritigenin and isoliquiritigenin was excluded. M6 and M7 were found to have identical product ions, liquiritigenin and isoliquiritigenin, which indicated that M6 and M7 were possibly progenitor ions, or phase II metabolites of liquiritigenin and isoliquiritigenin. A selected ion monitoring (SIM) scan of possible glucuronide conjugates at m/z 433 showed identical chromatographic peaks with M6 and M7 (Fig. 6). This meant that M6 and M7 (m/z 257, tR=11.5, 12.4 min) could be tentatively identified as the glucuronide conjugates of liquiritigenin and isoliquiritigenin respectively. According to the literatures,7,14) the binding sites of glucuronic acid of isoliquiritigenin and liquiritigenin may be isoliquiritigenin 4′-O-glucuronide, isoliquiritigenin 2′-O-glucuronide and liquiritigenin 4′-O-glucoside. The mass spectra of product ions for M8 could not be obtained due to a relatively low peak response. However, information from the study by Akao et al.15) indicates that M8 might related to the 22α- and 24-hydroxylation product of glycyrrhetic acid.
The extracted ion chromatogram of the ion at m/z 455.5 from the full LC-MS scan showed clear chromatographic peaks at 3.2 min (M9) and 4.5 min (M10). However, corresponding chromatographic peaks in the MRM-EPI scan could not be found. As a result, the source of these peaks warrants further exploration that is outside the scope of this study. The ion at m/z 339 (M11), 80 Da higher than that at m/z 259, and its MRM-EPI scan showed a predominant product ion at m/z 153 and m/z 107, similar to that of liquiritigenin and isoliquiritigenin (Fig. 7). This suggested that M11 could be the sulfate conjugate of deoxidized liquiritigenin or isoliquiritigenin. Information from the study by Lu et al.,16) it was easy for the glycyrrhizin 7-OH and 4-OH with sulfuric acid or glucuronidation. By MS2 analysis, the existence of m/z 219 fragment peaks may arise from glycyrrhizin-7-O-sulfate Retro-Diels–Alder (RDA) cleavage fragments generated by A ring, so there is speculation of the glycyrrhizin-7-O-sulfuric acid ester. Based on similar molecular weights and retention times (3.2, 5.2, 5.6 min, respectively), it was proposed that M13 (m/z 249.4) and M14 (m/z 249.4) were the reduction products
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Fig. 3.
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Full Scan Spectra of (A) Blank Plasma Samples, (B) Plasma Sample Dosed with SG and (C) Plasma Sample Dosed with S+G
of M12 (m/z 247.5). The MRM-EPI spectra of M12 and paeoniflorin showed identical fragment ions at m/z 179, 160, 150 and 122 (Fig. 8). Therefore it is speculated that M12, M13
and M14 were metabolites of paeoniflorin. The existence of a product ion at m/z 179 suggested that these metabolites had the characteristic glucosyl moiety. Further investigation is
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Fig. 4.
MRM-EPI MS Spectrum of M4 and M5 (A: M4; B: M5)
required to determine the detailed structure of the metabolites. The extracted ion chromatogram of M15 at m/z 188 from the full LC-MS scan showed significant chromatographic peak at 7.6 min. The corresponding chromatographic peak could not be found in the MRM-EPI scan. Similarly as for M9 and M10, further work is necessary to determine the source of the peak in M15. The specific information for each compound observed in the blood plasma of the rats is presented in Table 2. The proposed structures of the absorption compounds and metabolites in rat plasma after oral administration of SG or S+G are shown in Fig. 9. Comparison of Absorption Compounds and Metabolites Each sample was parallel tested five times. The relative responses of the ionized compounds are shown in Table 3, to compare the quantity of the above mentioned metabolites in rat plasma after oral administration of SG and S+G. Significant differences were observed in the quantity of certain metabolites in plasma samples that were treated with doses of the two decoction mixtures, which indicated that the absorption of SG was greater than that of S+G. Of the fifteen ingredients, six of them had higher concentrations in the plasma samples that were more than 1-fold higher in SG than in S+G (M2, 2.04-fold; M4, 2.00-fold; M8, 2.86-fold; M11, 2.50-fold; M13, 2.32-fold and M14, 2.16-fold). Meanwhile, the contents of another six also showed an increasing trend (M1, 1.60-fold; M3, 1.85-fold; M5, 1.35-fold; M6, 1.82-fold; M9, 1.36-fold and M10, 1.32-fold). However, this was not the case for the M7, M12 and M15 components of the misture (M7, 0.88-fold; M12, 0.81-fold and M15, 0.79-fold).
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Fig. 5.
MRM-EPI MS Spectrum of M6 and M7 (A: M6; B: M7)
Discussion
Generally, TCM or herbal medicines are a complex mixture of a variety components. As discussed previously, conventional compatibility rationality studies, used in the assesment of in vivo effects of the prescription, are often based on the pharmacokinetic parameters of one component. However, in reality, due to the existence of multiple constituents, this cannot give a complete picture of the true activity of a formulation. For example, it has been reported that when SGD is used as an antispasmodic analgesic, the therapeutic effect arises mainly from SY.17) However, another study has showed that the efficacy of SY could be improved by co-administration with GC, even though it has been shown that GC alone has almost no analgesic effect.18,19) Some studies have focused on enhanced efficacy through the combination of SY and GC. For instance, paeoniflorin, glycyrrhizic acid and glycyrrhetinic acid, were considered as the biologically active components in SGD and it has been found that the absorption of paeoniflorin increased significantly after co-administration with GC.20) The Cmax and bioavailability of glycyrrhizic acid and glycyrrhetinic acid were also found to increase when SY and GC were co-administered.21) It was observed that there were more significant anti-inflammation and analgesic effects for SGD than GC or SY.22) Generally, however, these reports focused only on the compatibility rationality of SY or GC. There has been no comparison study between the single or mixed decoction. Therefore, the purpose of this paper was to explore the absorbed compounds of SGD and the compatibility rationality of a mixed decoction of SGD, on the basis of known absorbed compounds or metabolites. Actually, due to the existence of multiple constituents, it
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Fig. 6. SIM Scan Chromatograms of M6 and M7 at m/z 433 (A: Blank Plasma Sample; B: Plasma Sample Dosed with SG; C: Plasma Sample Dosed with S+G)
Fig. 7. MRM-EPI Chromatogram and MS Spectrum of M11 at m/z 339 (A: MRM-EPI Chromatogram; B: MS Spectrum)
Fig. 8. MRM-EPI Chromatogram and MS Spectrum of M12 at m/z 247.5 (A: MRM-EPI Chromatogram; B: MS Spectrum)
could not give a complete picture of the activity of a formulation. The multiple constituents may work “synergistically,” or it may be that the individual consitituents cannot be separated. Qualitative or quantitative changes after compatability was assessed could reflect the mechanism of the enhanced efficiency or reduced toxicity of the TCM.23,24) The enhanced efficiency after the formulation is produced could not only be
investigated in vitro but also in vivo. For instance, it has been previously observed that the active ingredients increased 25,26) and that new active ingredients were formed.27) Meanwhile, it has also been shown that new ingredients or quantification changes developed for some metabolites.28–31) In the previous study, the analgesic effect of SG was found to be greater than S+G, and there were obvious component
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Table 2. The Retention Time (tR), the Fragment Ions, and m/z of the Compounds Absorbed in Rat Blood Plasma, Following Oral Administration of SG and S+G Mass charge ratio [M+H]+
Compounds
tR/min
M1
35.3
Glycyrrhetinic acid
SG/S+G
471.2
M2
26.0
Glycyrrhizic acid
SG/S+G
823.8
M3 M4
13.4 6.4
SG/S+G SG/S+G
498.2 250
M5
7.5
SG/S+G
250
166, 135, 132
M6
11.5
M7
12.4
M8
12.2
M9 M10 M11
3.2 4.5 14.8
M12 M13 M14 M15
5.6 3.2 5.2 7.6
Paeoniflorin The ring oxidation and cleavage product of paeoniflorin The ring oxidation and cleavage product of paeoniflorin The glucuronide conjugates of liquiritigenin The glucuronide conjugates of isoliquiritigenin The hydroxylation product of glycyrrhetic acid Unknown Unknown The sulfate conjugate of deoxidized liquiritigenin or isoliquiritigenin Metabolites of paeoniflorin Metabolites of paeoniflorin Metabolites of paeoniflorin Unknown
454, 436, 408, 327, 317, 189, 149, 135, 123, 119 647, 471, 453, 435, 407, 343, 299, 241, 189, 119 179, 161, 151, 135, 133 166, 148, 142, 124
Table 3.
Identitified compounds
Origin
The fragment ions
SG/S+G
433 (257)
147, 137, 119
SG/S+G
433 (257)
239, 165, 147, 137, 119
SG/S+G
503
SG/S+G SG/S+G SG/S+G
455.5 455.5 339
SG/S+G SG/S+G SG/S+G SG/S+G
247.5 249.4 249.4 188
219, 153, 107 179, 160, 150, 122
Relative Responses of the Ionized Metabolites (Mean±S.D., n=5)
Compounds M1 M2 M3 M4 M5 M6 M7 M8 M9 M10 M11 M12 M13 M14 M15
Mass charge ratio [M+H]+ 471.2 823.8 498.2 250 250 433 (257) 433 (257) 503 455.5 455.5 339 247.5 249.4 249.4 188
Retention time/min 35.3 26.0 13.4 6.4 7.5 11.5 12.4 12.2 3.2 4.5 14.8 5.6 3.3 5.2 7.6
changes for the two decoction mixtures in vitro.32) The ingredients of rat plasma dosed with SG and S+G were determined in this work, and according to our research, fifteen ingredients were discovered in the rat plasma after administration of both decoctions. This finding indicated that the quality of the absorbed compounds and potential metabolites did not change. Therefore, we compared the quantity of the above mentioned absorbed compounds and their potential metabolites in rat plasma samples after the oral administration of either SG or
Ratio of relative responses
Relative responses SG
S+G
2.40E+06±3.32E+05 1.10E+06±1.08E+05 3.70E+05±2.93E+05 1.20E+06±1.45E+05 2.70E+05±2.97E+04 4.00E+05±3.22E+04 2.20E+05±3.08E+04 2.00E+05±2.73E+04 3.13E+06±3.44E+05 3.04E+06±1.52E+05 1.20E+06±9.11E+05 2.20E+06±2.86E+05 1.30E+06±1.95E+05 4.10E+06±6.07E+05 1.90E+06±1.33E+05
1.50E+06±1.81E+05 5.40E+05±4.86E+05 2.00E+05±1.76E+05 6.00E+05±7.85E+05 2.00E+05±2.22E+04 2.20E+05±1.76E+04 2.50E+05±3.65E+04 7.00E+04±9.77E+04 2.30E+06±2.76E+05 2.30E+06±1.25E+05 4.80E+05±3.36E+04 2.70E+06±3.33E+05 5.60E+05±8.19E+05 1.90E+06±2.75E+05 2.40E+06±1.48E+05
SG/(S+G) 1.60 2.04 1.85 2.00 1.35 1.82 0.88 2.86 1.36 1.32 2.50 0.81 2.32 2.16 0.79
S+G. Significant differences were observed in the quantity of some metabolites in plasma samples treated with the two decoctions, which showed that the absorption of SG was better than that of S+G, with the exception of the M7, M12 and M15 components. The results of comprehensive qualitative and quantitative studies showed that the behaviors of components in vivo after the administration of SG was better than those in S+G, which was consistent with previous experimental results which demonstrated that the analgesic effect of SG
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Fig. 9. Proposed Structure of Some Absorption and Metabolite Compounds
was better than S+G. We have also investigated the spectrum and efficiency of such compounds, which indicated that the characteristic constituents of Shaoyao, and some of those from Gancao, were positively correlated with the efficacy of the compound.30) In addition, the pharmacokinetics of characteristic peaks associated with efficacy were studied; the results showed that the AUC was increased and T1/2 was extended after their coadministration with GC.20) Previous reports that investigated the relevance of fingerprinting and efficacy of the Ginkgo biloba extract33,34) showed that changes in the chemical composition ratio may alter the “synergistic” behaviour of active ingredients, which leads to changes in efficacy. Therefore, the efficacy of a formulation cannot be simply calculated as the sum of the efficacies of all the components. Furthermore, most TCMs can play a local or systemic role after oral administration, where the TCMs are absorbed by the digestive tract through the fat-soluble substance absorption mechanisms.35) As the compositions associated with the efficacy of SG and S+G were inconsistent, the degree of the absorption of the 15
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ingredients by the gastrointestinal tract were different, which caused a significantly enhanced efficacy of certain ingredients. This might be one of the mechanisms that causes the enhanced efficacy of SY or GC. Moreover, drug metabolism is classified into phase I and phase II reactions. Phase I metabolism usually does not result in a large change in molecular weight or water solubility of the substrate, but is of great importance because oxidative reactions add or expose sites where phase II metabolism can subsequently occur. In contrast, phase II conjugation typically results in an appreciable increase in molecular weight and water solubility which includes the three most relevant phase II drug conjugation reactions, sulfation, glucuronidation, and glutathione conjugation.36) The combination reaction of Oglucuronides happens more common in flavonoid compounds, which binding site exist sselectivity. Due to the biological activity of flavonoids depends mainly on the hydroxyl groups, especially the free hydroxyl number and position, so the study on flavonoid metabolism site selectivity has important significance.37–40) According to eht literature information,7,14) the phase II transformation of isoliquiritigenin by human hepatocytes and pooled human liver microsomes (HLMs) was investigated using liquid chromatography/tandem mass spectrometry and UV absorbance. Five glucuronides were detected corresponding to monoglucuronides of isoliquiritigenin and liquiritigenin. UGT1A1 and UGT1A9 were the major enzymes responsible for the formation of the most abundant conjugate, isoliquiritigenin 4′-O-glucuronide, at the same time UGT1A1 and UGT1A10 converted isoliquiritigenin to the next most abundant phase II metabolite, isoliquiritigenin 2′-Oglucuronide. Thereout, we speculate that the binding sites of glucuronic acid of isoliquiritigenin and liquiritigenin may be like that. In the majority of drugs and endogenous compounds, can happen glucuronidation also can be sulfated,36,41) so it can happen glucuronidation flavonoid compounds, two-phase metabolism also occurred in the sulfation reaction.16) Ultra-high performance liquid chromatography-quadrupole time-of-flight mass spectrometry (UPLC-Q-TOF/MS) was applied to identify the metabolites of Sinisan extract in rat plasma, urine, feces and bile after intragastric administration. Using MSE and mass defect filter techniques, 41 metabolites of 10 parent compounds (naringin, naringenin, hesperidin, neohesperidin, liquiritin, liquiritigenin, glycyrrhizic acid, glycyrrhetinic acid, saikosaponin a and saikosaponin d) were detected and tentatively identified. It was shown by our results that these compounds was metabolized to the forms of hydroxylation, glucuronidation, sulfation, glucuronidation with sulfation and glucuronidation with hydroxylation in vivo. Among them, it was easy for the glycyrrhizin 7-OH and 4-OH with sulfuric acid or glucuronidation. By MS2 analysis, the existence of m/z 215 fragment peaks may arise from glycyrrhizin- 7-Osulfate RDA cleavage fragments generated by A ring, so there is speculation of the glycyrrhizin-7-O-sulfuric acid ester. It was shown in our reslut that the ion at m/z 339 (M11), 80 Da higher than that at m/z 259 could be the sulfate conjugate of deoxidized liquiritigenin or isoliquiritigenin. In addition, the intestinal metabolism studies also show that flavonoids intestinal metabolism is also to glucuronic acid, sulfuric acid as the main.42)
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Conclusion
In conclusion, the results from this study demonstrate the enhanced absorption mechanism behind the “synergistic” effect of TCM combination therapies, which thus improved the efficacy of individual components. Acknowledgements The work was supported by Grants from the National Natural Science Foundation of China (Grant No. 30801548), the Shanghai Municipal Education Committee (Grant No. 12ZZ124, 11zz111), the Shanghai Science and Technology Committee (Grant No. 11ZR1434500) and the Shanghai Education Commission Leading Academic Discipline Project (Grant No. J50302). Declaration of Interest have no conflict of interest.
References
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