Profiling of the charged metabolites of traditional herbal medicines using capillary electrophoresis time-of-flight mass spectrometry
The quantification of a small number of bioactive components in herbal medicines is often inadequate when attempting to elucidate a medicine’s biological effects. Despite rapid advances in analytical technologies, obtaining comprehensive metabolomic profiles of herbal medicines remains difficult, due to the complexity of natural product mixtures. Toki-Shakuyaku-San is a Chinese medicine used widely to treat gynecological and obstetric disorders, such as infertility, dysmenorrhea, toxemia during pregnancy and neural dysfunction. It consists of Angelica acutiloba Radix (Toki), Cnidium officinale Rhizoma (Senkyu), Paeonia lactiflora Radix (Shakuyaku), Atractylodes lancea Rhizoma (Sojutsu), Alisma orientale Rhizoma (Takusha) and Poria cocos Hoelen (Bukuryo). To elucidate the composition of these herbal medicines individually, we conducted non-targeted profiling analyses of extracts of these herbs using capillary electrophoresis time-of-flight mass spectrometry (CE-TOFMS), which allows the simultaneous quantification of hundreds of charged metabolites. In total, 737 ± 183.1 (average ± SD) metabolite-derived features were observed, and of these, 119 metabolites were identified. Score plots of principal component analysis (PCA) showed a clear cluster including Shakuyaku, Bukuryo, and Sojutsu, while the other three herbs were distributed over PCA spaces. Loading plots revealed that amino acids and shikimate-derived alkaloids were the predominant metabolite constituents. Hierarchical clustering analysis revealed that few clusters overlapped in the herbal medicines tested. This report is the first demonstration of the characterization of a herbal medicine using large-scale metabolomic analysis, which is complementary to traditional quality control methods.
KeywordsCapillary electrophoresis time-of-flight mass spectrometry Herbal medicine Charged metabolite Metabolomic profiling
Herbal medicines have been used as therapeutic agents for several thousands of years in Asian countries (Wang and Ren 2002; Wang et al. 2009). The evaluation of a herbal medicine’s safety and efficacy is required before its clinical use as a complementary and alternative medicine (CAM) in western countries (Miller et al. 2004; Murray and Rubel 1992; Calixto 2000; Zhang et al. 2010). The conventional pharmacological approach to characterize herbal medicines focuses on the identification and quantification of a single or several bioactive components (Murray and Rubel 1992; Liu et al. 2008), however, the complex properties of herbal medicines often render such an approach inadequate (Wang and Ren 2002; Lao et al. 2009; Liang et al. 2009; Chan 2003). The quantitative analysis of the complete metabolite profile of herbal medicines would therefore be of significant value (Liu et al. 2008).
Recent advances in the development of mass spectrometry (MS)-based profiling techniques have made a significant contribution to the study and quantification of the metabolome, the complete range of low molecular weight compounds of a natural sample, which is a field known as metabolomics. MS is typically used in combination with a separation system, such as liquid chromatography (LC), gas chromatography (GC) or capillary electrophoresis (CE). The selection of the separation system to be used is based on the chemical properties of the target compounds, since there is no single analytical methodology currently available capable of profiling complete metabolome. GC–MS and LC–MS are used routinely for targeted analysis (Liang et al. 2009) and are considered to be mature techniques (Weng and Jin 2002). GC–MS is limited to profiling volatile molecules, such as essential oils (Li et al. 2003; Gong et al. 2001), while LC–MS is suitable for separating and detecting more diverse molecules, such as flavonoids, glycosides, organic acids, saponins and lipids (Lao et al. 2009; Ma et al. 2007; Ren et al. 2008). However, the separation conditions used for LC should be optimized for individual molecular classes, which limits the number of metabolites simultaneously detected. CE coupled MS, in particular CE time-of-flight MS (CE-TOFMS), has been demonstrated to have high separation capability and sensitivity for the profiling of charged metabolites, including primary metabolites such as amino acids, amines, organic acids and nucleic acids (Soga et al. 2003, 2006; Monton and Soga 2007). CE alone has been used for the targeted analysis of alkaloids and flavonoids (Ganzera 2008; Lao et al. 2009; Hurtado-Fernandez et al. 2010) and used for quality control (QC) of herbal medicines (Ganzera 2008). LC-MS-based profiling showed variance attributed to harvesting region and processing protocol (Xie et al. 2008; Chan et al. 2007). Our CE–MS-based profiling method is also important for QC of herbal medicines, since charged metabolites are the dominant species dissolved in water extracts (Lao et al. 2009).
Toki-Shakuyaku-San, or Danggui-Shaoyao-San, is a Chinese herbal medicine used widely for the therapy of hematopoiesis and menstrual disorders, and has several functions, including smooth muscle relaxation and biofluid control (Akase et al. 2004). Toki-Shakuyaku-San consists of six individual herbal medicines, with each providing different pharmacological effects. These herbal medicines are Toki (Angelica acutiloba Radix), Senkyu (Cnidium officinale Rhizoma), Shakuyaku (Paeonia lactiflora Radix), Sojutsu (Atractylodes lancea Rhizoma), Takusha (Alisma orientale Rhizoma), and Bukuryo (Poria cocos Hoelen). Toki and Senkyu are used for the treatment of gynecological diseases (Lu et al. 2005; Kim et al. 2006; Yi et al. 2007; Bohrmann et al. 1967), while Takusha, Bukuryo and Sojutsu are used to improve water metabolism (Wang et al. 2004, 2008; Kitajima et al. 2003; Zhao et al. 2008), and Shakuyaku provides an anti-inflammatory effect (He et al. 2010; Ohta et al. 1993). While some of the active components, such as l-ornithine, gallic acid, paeoniflorin, ferulic acid and benzoic acid in this mixture, and their effects, have been well studied, the relationships between the complete compound profile and the pharmacological effects is not yet well understood (Hatip-Al-Khatib et al. 2004; Chen et al. 2009).
This study aimed to reveal the variance of the charged metabolites found in the six herbal medicines that constitute Toki-Shakuyaku-San, using CE-TOFMS. We conducted multivariate analysis to examine variations in the primary metabolites of the herbal medicine and to understand the relationships between the charged metabolites and the pharmacological effects. Most studies focus on the importance of secondary metabolites to the pharmacological effects of a herbal medicine, however we show that the profile of charged metabolites also varies among herbal medicines.
2 Materials and methods
2.1 Sample preparation
Six herbal medicines, Toki, Bukuryo, Sojutsu, Takusha, Shakuyaku and Senkyu, were obtained from Yatsume Pharmaceutical Co., Ltd. (Tokyo, Japan). Each herb was homogenized without solvent using a multi-bead shocker (Yasui Kikai Co., Osaka, Japan) at 2,500 rpm for 300 s. The crushed material (1.00 g) was dissolved in 3 ml of 50% MeOH containing 2 internal standards (200 μM each of methionine sulfone and 3-aminopyrrolidine). This solution was centrifuged at 3,000 rpm for 10 min at 4°C, and 0.2 ml of the supernatant fluid was transferred to a Millipore 5 kDa cutoff filter tube for centrifugal centrifugation (10,000 rpm × 2 h at 4°C). The filtrate was lyophilized and dissolved in 100 μl of Milli-Q (Millipore, Bedford, MA, USA) water prior to CE-TOFMS analysis.
2.2 Standard chemical compounds
Cysteine-glutathione disulphide was purchased from Apollo Scientific Ltd (Tokyo, Japan). 5-Methyl-2′-deoxycytidine was purchased from MP Biomedicals LLC. (Tokyo, Japan). γ-Glu-2AB and cystathionine were purchased from Toray Research Center (Tokyo, Japan). N-α-Dimethylhistidine was purchased from Bachem AG (Bubendorf, Switzerland). Isobutylamine and azetidine-2-carboxylate were purchased from Chem Service Inc. (West Chester, PA, USA). N8-Acetylspermidine, 5-aminovalerate, O-acetylcarnitine, betaine, glucosaminate, 7-methylguanine, 5-methoxy-3-indoleacetate and pyridoxamine were purchased from Fluka (Buchs, Switzerland). Isonicotinamide was purchased from Tokyo Chemical Industry (Tokyo, Japan). All other compounds were purchased from Sigma–Aldrich (St. Louis, MO, USA) or Wako (Osaka, Japan). All chemical standards were dissolved in Milli-Q water, 0.1 M HCl or 0.1 M NaOH to give 10 mM or 100 mM stock solutions. A standard mixture was prepared by diluting the stock solutions with Milli-Q water just prior to injection into the CE-TOFMS. The chemicals used were of analytical or reagent grade.
2.3 Instrument parameters
The instrumentation and measurement conditions used for CE-TOFMS were as described elsewhere (Sugimoto et al. 2010a). All CE-TOFMS experiments were performed using an Agilent CE capillary electrophoresis system (Agilent Technologies, Waldbronn, Germany), an Agilent G3250AA LS/MSD TOF system (Agilent Technologies, Palo Alto, CA), an Agilent 1100 series binary HPLC pump which delivers sheath liquid, a G1603A Agilent CE–MS adapter kit, and a G1607A Agilent CE–ESI–MS sprayer kit (Agilent Technologies, Waldbronn, Germany). The CE–MS adapter kit used includes a capillary cassette that facilitates thermostatting of the capillary. The CE–ESI–MS sprayer kit simplifies the coupling of the CE system with the MS system, which was equipped with an electrospray ionization source. For system control and data acquisition, G2201AA Agilent ChemStation software for CE and Analyst QS software for TOFMS were used.
2.4 Measurement conditions for CE-TOFMS
Samples were separated in fused silica capillaries (50 μm i.d. × 100 cm total length) filled with 1 M formic acid (pH 1.8) as the background electrolyte. The sample solutions were injected at 50 mbar for 3 s and a voltage of 30 kV was applied. The capillary temperature was maintained at 20°C and the temperature of the sample tray was maintained below 5°C using the external coolant system. The sheath liquid, comprising of methanol/water (50% v/v) and 0.1 μM hexakis-(2,2-difluorothoxy)-phosphazene (Hexakis), was delivered at 10 μl/min. ESI–TOFMS was conducted in positive ion mode. The capillary voltage was set at 4 kV and the nitrogen gas flow rate (heater temperature 300°C) was 10 psig. In TOFMS, the fragmentor, skimmer and OCT RFV voltage were set at 75, 50 and 125 V, respectively. Automatic recalibration of each acquired spectrum was performed using the reference masses of reference standards (13C isotopic ion of protonated methanol dimer [2MeOH + H]+, m/z 66.06371, and [Hexakis + H]+, m/z 622.02896). Mass spectra were acquired at a rate of 1.5 cycles/s over a 50–1,000 m/z range.
2.5 Data processing for CE-TOFMS
Raw data were analyzed with our proprietary software, MasterHands (Sugimoto et al. 2010b). Initially noise-filtering, baseline correction, peak detection and peak area integration from sliced electropherograms (m/z 0.02 width) were carried out. Subsequently, the accurate m/z value for each peak detected was calculated with Gaussian curve-fitting. A migration time normalization function was established using dynamic programming and the simplex optimization method (Soga et al. 2006). Peaks with small differences in m/z value (<20 ppm) and normalized migration time (<1.0 min) were treated as features. Subsequently, neutral compounds, salt ions of Na+ and K+, and redundant features such as fragments, adducts, isotopes, dimers and trimers were eliminated on the basis of established m/z differences (Brown et al. 2009). For the remaining features, metabolite identities were assigned by matching their m/z values and migration times with those of standard compounds. To quantify the detected metabolites, the injected volume for CE and sensitivity of MS were corrected using internal standards, then all annotated metabolites were further corrected with the same chemicals in a standard mixture to overcome different ionization patterns.
2.6 Statistical analysis
The concentration of each metabolite was divided by its average to scale the data, clustered on the basis of Euclidean distance, and visualized as a heat map representation using Mev TM4 software, version 4.6 (Dana-Farber Cancer Institute, Boston, MA) (Saeed et al. 2006). We conducted principal component analysis (PCA) using JMP software (version 8.0.2, SAS, Cary, NC). Metabolite data were mapped on the pathway map using Pathway Projector (Kono et al. 2009), a web-based pathway map application using KEGG Atlas (Kanehisa et al. 2010).
3.1 PCA analysis of charged metabolites in herbal medicines and pharmacological effects
In the obtained profiles, 39 metabolites showed higher loading score values at the first PC (>0.8). Of these, 20 metabolites were amino acids and amino acid derived metabolites; threonine, valine, serine, tyrosine, glycine, phenylalanine, isoleucine, alanine, lysine, asparagine, arginine, citrulline, homoserine, N-ε-acetyllysine, N-acetylvaline, octopine, ornithine, N-acetylornithine, N-γ-ethylglutamine and N-methylalanine. Along the second PC axis, seven metabolites, 5-aminovalerate, acetylcholine, isoamylamine, O-acetyl carnitine, tryptamine, tyramine and anthranilate, showed high loading score values. Along the third PC axis, 2′-deoxyguanosine, 5′-deoxyadenosine, 5-methyl-2′-deoxycytidine, 5-methylthioadenosine, adenosine, ethanolamine phosphate, N6-methyl-2′-deoxyadenosine and phosphorylcholine showed high loading score values. Of these, four compounds, 5′-deoxyadenosine, 5-methylthioadenosine, adenosine and N6-methyl-2′-deoxyadenosine, were adenosine derived metabolites. In short, these three PCs characterized amino acids, amines and adenosine-derived metabolites, respectively. The pharmacological effects of the herbal medicines used in this study are summarized in Supplementary Information Table 1. To evaluate the relationships between these efficacies and the observed metabolite profiles of the herbal medicines, the efficacies shared by multiple herbal medicines were mapped on the score plot of PCA results (Fig. 1c). One distinct result found was the shared diuretic effect of Bukuryo and Sojutsu, which were closely located on the score plot, indicating a correlation between the observed metabolite profiles and this effect (Fig. 1c). Both Bukuryo and Sojutsu showed minus values in the first PC on score plots (Fig. 1a). The metabolites located at large minus values at the first PC on loading plots, such as β-alanine, isobutylamine, cytosine, cystathionine, nicotine and nicotinamide, may be characteristic of the PC scores of Bukuryo and Sojutsu (Supplementary Information Fig. 1). Senkyu and Toki are used for purposes including tranquilization, alleviation of pain, menstruation problems, removing blood stasis, as a tonic and for anemia, while they are located at a significant distance in PC score plots (Fig. 1c). Although, the PC values of Senkyu and Toki were not similar, the concentrations of several individual metabolites were clearly higher than in the other herbs; 3-methylhistidine, spermidine, adenosine, pipecolate, tryptophan, thiamine and uridine were high in Senkyu and Toki, while agmatine was high in Takusha, Bukuryo and Sojutsu (Supplementary Information Fig. 2).
3.2 Profiling of charged metabolites in herbal medicines
3.3 Standard amino acid concentration
4.1 Overview of charged metabolite profiles and pharmacological effects
CE-TOFMS-based metabolomics has been used to successfully annotate mainly primary metabolites in herbal medicines, while many studies based on LC–MS or GC–MS give profiles of the secondary metabolites contained. The observed metabolite profiles of six herbs studied here showed large variances. PCA revealed that there was a particularly large variance in the herbs Toki, Takusha and Senkyu when compared with the other herbs tested (Fig. 1a). Several metabolites observed in this study have known pharmaceutical efficacies. Tryptophan is present at higher concentrations in Toki and Senkyu (Supplementary Information Fig. 2a, which have several common activities, including the improvement of blood stasis and anemia. It is a precursor of kynurenine, which plays a role in arterial vessel relaxation (Wang et al. 2010). Agmatine was present at high concentrations in Takusha, Bukuryo and Sojutsu, and is known to have a diuretic effect (Smyth and Penner 1995). Bukuryo, Sojutsu and Shakuyaku showed negative scores at the first PC and were closely plotted on the score plot (Fig. 1a) and β-alanine, isobutylamine, cytosine, cystathionine, nicotine and nicotinamide showed higher negative loading score values at the first PC (Supplementary Information Fig. 1). β-Alanine is the rate-limiting precursor of carnosine, which has an anti-inflammatory effect (Zhu et al. 2007), and single oral doses of isobutylamine have a sedative effect (Cheever et al. 1982). These metabolites therefore correlate with the clinical application of these herbs.
Clustering results, shown using a heat map, indicated that there was little similarity among the herbs tested (Fig. 2). Thus, the concentrations of many metabolites vary widely between the herbs. Izzettin Hatip-Al-Khatib et al. have shown previously that only ornithine in Toki extract is responsible for improving memory impairment (Hatip-Al-Khatib et al. 2004). Our profile revealed high concentrations of ornithine in Toki, which was consistent with their study, and in addition, other amines were also found to be present at high concentrations in Takusha. Such findings were obtained using the non-targeted profiling analysis demonstrated. Here, we discuss prominent features observed for amino acids, shikimate derived metabolites, and xanthine analogs.
4.2 Profile of amino acids
Amino acids have been reported to be the main components of Takusha extracts (Namba and Tsuda 1998), and our results are consistent with these findings; 74% of the total annotated metabolite composition was made up of standard amino acids in Takusha (Fig. 3a). However, particularly high amino acid content was not restricted to Takusha, with amino acids making up 64–85% of the composition of the other herbs tested. The only exception to this trend was Bukuryo, whose extract was composed of only 34% standard amino acids (Fig. 3a). Arginine was found to be the most dominant amino acid measured in all of the herbs tested (21–42%), again, with the exception of Bukuryo (9.4%) (Fig. 3b). Proline was found to be abundant in Toki (30%) and Sojutsu (38%), and alanine was abundant in Bukuryo (38%) (Fig. 3b). Indeed, PCA results showed high loading scores for all standard amino acids except for glutamine, asparagine and tyrosine, indicating that these amino acids may be a prominent factor useful for the characterization of these herbs (Fig. 1b).
4.3 Profile of shikimate derived metabolites
The shikimate-derived metabolites observed in our profiles are known to be bioactive, for example dopamine and kynurenine act as signal molecules in the central and peripheral nervous systems. Metabolites contained in the Toki extract are known to bind to a receptor of the central nervous system (Liao et al. 1995) and a liquid extract of Toki containing alkaloids improved retention memory in rats (Hatip-Al-Khatib et al. 2004). Our profiles showed the presence of seven shikimate-derived metabolites and three precursor amino acids (tryptophan, tyrosine and phenylalanine). The total concentration of these three amino acids was higher in Toki, Senkyu and Takusha (Fig. 3a). The concentrations of the shikimate-derived metabolites detected, including kynurenine, harman, tryptamine, tyramine and anthranilate, were also high in Toki, Senkyu and Takusha (Supplementary Information Fig. 3b). These results suggest a correlation between the concentrations of shikimate-derived metabolites and those of the precursor amino acids.
Toki and Senkyu have been reported to contain ferulic acid, which is derived from tyrosine or phenylalanine (Lu et al. 2005; Yi et al. 2007). Although, ferulic acid was not observed in our profiles, the high concentrations of the precursor amino acids of ferulic acid suggest that ferulic acid may be abundant in Toki and Senkyu. In conclusion, we have found some degree of quantitative interaction between bioactive compounds and their precursor amino acids, though there were some shikimate-derived metabolites, such as dopamine, 3-hydroxyanthranilate, which did not follow this trend.
4.4 Profiles of xanthine analogs
Xanthine analogs are known to have a wide range of biological targets. Examples include the adenosine receptor, where xanthines act as antagonists, and calcium release ryanodine-sensitive channels, where they act to sensitize the channels to calcium activation (Daly 2007). Xanthines are known to have several therapeutic benefits, including analgesic and diuretic effects (Daly 2007). Takusha was found to contain the xanthine analogs guanine (EC 188.8.131.52), hypoxanthine (EC 184.108.40.206) and xanthosine (EC 220.127.116.11 or EC 18.104.22.168) at levels more than ten, four and two times higher than the other herbs tested, respectively. Xanthine analogs may be expected to participate in the analgesic and diuretic effects of Takusha.
4.5 Limitation of this study
In the profiles we obtained, the total concentration of all annotated metabolites was higher in Toki, Takusha, and Senkyu than in the other herbs tested (Fig. 3a). The complex metabolome of these three herbal medicines, including shikimate-derived metabolites and xanthine analogs, may be responsible for the therapeutic functions associated with the herbal medicines, such as analgesia and sedation, since structurally similar compounds are known to have similar functions by receptor binding (Wambach and Casals-Stenzel 1983; Monteith et al. 1996). Further analyses are necessary to compare metabolite extraction methods, with methanol useful to achieve high recovery rates of metabolites (Ren et al. 2008; Sato et al. 2004) and water useful when considering dosage (Liang et al. 2009). More intensive studies on the individual metabolites may give insight into the effect of chemical structure on these therapeutic effects. Further study is needed to elucidate the relationship between the many polar low molecular weight compounds found in these herbal medicines and the herb’s therapeutic functions, particularly their interactions with the nervous system. The quantified profiles obtained should also be validated using different samples of these herbal medicines, for example samples grown at different production sites, harvested at different times, and different batches, in order to establish the natural variability of the metabolites. Profiles of negatively charged metabolites and those obtained using other “omics” platforms should also be integrated to allow for more comprehensive analyses.
5 Concluding remarks
In this study, we have conducted a non-targeted analysis of the polar metabolite profiles of the six herbal medicines which compose Toki-Shakuyaku-San. Overall, there were few common characteristics observed among the herbs tested, and while the total amino acids content was consistently high among the herbs, individual amino acid content showed large variation. Notably, high concentrations of shikimate-derived metabolites were observed in Toki, Takusha and Senkyu, which helps to explain the herb’s biological effects, such as sedation, analgesia and diuretic effects. Multivariable analysis revealed a high correlation between the concentrations of shikimate-derived metabolites and the concentrations of their precursor amino acids, tryptophan, tyrosine and phenylalanine, in the herbs tested. The xanthine analogs guanine, hypoxanthine and xanthosine also showed a high correlation. Analysis of the multiple pharmacological effects of these herbal medicines and comparison with the quantitative compound datasets described here would be valuable in the evaluation of the quality and efficacy of herbal medicines.
This work was supported by research funds from the Yamagata Prefectural Government and the city of Tsuruoka. We thank Dr. Kazuko Otomo for technical assistance, and Wanjun Kong and Guo Jing for fruitful discussions.
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