Next Article in Journal
Structure-Activity Relationship of Cannabis Derived Compounds for the Treatment of Neuronal Activity-Related Diseases
Previous Article in Journal
Facile Preparation of Metal-Organic Framework (MIL-125)/Chitosan Beads for Adsorption of Pb(II) from Aqueous Solutions
 
 
Font Type:
Arial Georgia Verdana
Font Size:
Aa Aa Aa
Line Spacing:
Column Width:
Background:
Article

Discovery of the Potential Biomarkers for Discrimination between Hedyotis diffusa and Hedyotis corymbosa by UPLC-QTOF/MS Metabolome Analysis

1
School of Pharmaceutical Sciences, Jilin University, Fujin Road 1266, Changchun 130021, China
2
College of Chinese Medicinal Materials, Jilin Agriculture University, Xincheng Street 2888, Changchun 130118, China
*
Author to whom correspondence should be addressed.
Molecules 2018, 23(7), 1525; https://doi.org/10.3390/molecules23071525
Submission received: 11 May 2018 / Revised: 21 June 2018 / Accepted: 22 June 2018 / Published: 25 June 2018
(This article belongs to the Section Analytical Chemistry)

Abstract

:
Hedyotis diffuse Willd. (HD) and Hedyotis corymbosa (L.) Lam. (HC), two closely related species of the same genus, are both used for health benefits and disease prevention in China. HC is also indiscriminately sold as HD in the wholesale chain and food markets. This confusion has led to a growing concern about their identification and quality evaluation. In order to further understand the molecular diversification between them, we focus on the screening of chemical components and the analysis of non-targeted metabolites. In this study, UPLC-QTOF-MSE, UNIFI platform and multivariate statistical analyses were used to profile them. Firstly, a total of 113 compounds, including 80 shared chemical constituents of the two plants, were identified from HC and HD by using the UNIFI platform. Secondly, the differences between two herbs were highlighted with the comparative analysis. As a result, a total of 33 robust biomarkers enabling the differentiation were discovered by using multivariate statistical analyses. For HC, there were 18 potential biomarkers (either the contents were much greater than in HD or being detected only in HC) including three iridoids, eight flavonoids, two tannins, two ketones, one alcohol and two monoterpenes. For HD, there were15 potential biomarkers (either the contents were much greater than in HC or being detected only in HD) including two iridoids, eight flavonoids, one tannin, one ketone, and three anthraquinones. With a comprehensive consideration of the contents or the MS responses of the chemical composition, Hedycoryside A and B, detected only in HC, could be used for rapid identification of HC. The compounds 1,3-dihydroxy-2-methylanthraquinone and 2-hydroxy-3-methylanthraquinone, detected only in HD, could be used for rapid identification of that plant. The systematic comparison of similarities and differences between two confusing Chinese herbs will provide reliable characterization profiles to clarify the pharmacological fundamental substances. HC should not be used as the substitute of HD.

1. Introduction

Hedyotis diffuse Willd. (HD) is a well-known Chinese folk-medicine with a spectrum of pharmacological activities, including anti-cancer, antioxidant, anti-inflammatory, anti-fibroblast, immunomodulatory and neuroprotective effects, especially the anti-cancer effect in practice [1]. Almost 200 compounds have been identified in HD, including iridoids, flavonoids, anthraquinones, phenylpropanoids, phenolics and their derivatives, sphingolipids, volatile oils and miscellaneous compounds [1,2,3].
Hedyotis corymbosa (L.) Lam. (HC), another species of the same genus, is also used interchangeably in China as a health supplement and for disease prevention. It is reported to possess antioxidant [4,5], anti-inflammatory [6], hepatoprotective [7,8], antitumor [9,10], antimalarial [11] and anti-nociceptive [12] activities. Iridoids, carboxylic acids, flavonoids, phenolics and their derivatives, triterpenes, anthranquinones and coumarins were isolated from HC [13,14,15]. Iridoid glycosides were reported as the main constituents [16]. Oleanolic acid and ursolic acid were also considered as biologically active ingredients [17,18].
HD and HC are closely related species of the Rubiaceae family. Due to their similar morphology, they are often mixed up. Recently, a systematic survey on confusable Chinese herbal medicines has revealed that HC is indiscriminately sold as HD in wholesale markets or food markets [19]. This confusion in the market has led to a growing concern about the identification and quality evaluation of HD and HC.
Several methods using various techniques have been established to distinguish between these two species, such as loop-mediated isothermal amplification technique (LAMP) [20], fluorescence microscopy [21], thin layer chromatography (TLC) [22], DNA sequencing of the complete internal transcribed spacer region and chemical analysis [23], phylogenetic utility of nuclear ribosomal DNA (nrDNA) internal transcribed spacers (ITS) [24], high-performance liquid chromatography (HPLC) [25], etc. As a result, markers such as hedyotiscone A [22], scandoside methyl ester [25], (9R,10S,7E)-6,9,10-trihydroxyoctadec-7-enoic acid [26] for HC, 6-O-(E)-p-coumaroyl scandoside methyl ester [23,25], (10S)-hydroxypheophytin a [23], 6-O-(E)-p-coumaroyl scandoside methyl ester-10-methyl ether and 6-O-p-feruloyl scandoside methyl ester [25] for HD have been found. The UPLC-UV (detection wavelength at 254 nm) fingerprint of HC was also established to distinguish it from HD [27]. The contents of oleanolic acid and ursolic acid were significantly different [28].
Untargeted metabolomics, with the ability to profile diverse classes of metabolites, is primarily used to compare the overall small-molecule metabolites of different samples [29]. It is mainly applied in metabolites identification through mass-based search strategy followed by manual or automated verification. The combination of ultra-high performance liquid chromatography (UPLC) separation, quadrupole time-of-flight tandem mass spectrometry (Q/TOF-MS) detection and the automated data processing software UNIFI with a scientific library is frequently applied in the characterization of chemical constituents of herbal medicines [30,31,32,33] and traditional Chinese medicine injection recently [34]. High-resolution tandem mass spectrum can provide an accurate and specific mass when the coeluting components possess different m/z values. UNIFI, a high throughput, comprehensive, simple and efficient platform, offers the approach to integrate data acquisition, data mining, library searching and report generation. The Traditional Medicine Library within the platform contains more than 6000 compounds from 600 herbs.
The aim of the study was search for potential biomarkers in order to systematically screen chemical components and the non-targeted metabolomic analysis of the two species, and in turn providing the basis for establishment of HC and HD quality criterion in the future. UPLC-QTOF-MSE, UNIFI platform and multivariate statistical analyses, such as principal component analysis (PCA) and orthogonal partial least squares discriminant analysis (OPLS-DA) were used to profile these two herbs. The established method could enable us to find the similarities and differences between them, and provide data for the establishment of HC and HD quality criterion in the future. This comprehensive and unique phytochemical profile study revealed the structural diversity of secondary metabolites and the different patterns in HC and HD. The method developed in this study can be used as a standard protocol for identifying and discriminating species of HC and HD.

2. Experimental

2.1. Materials and Reagents

HC and HD were purchased from herbal markets or collected from their respective cultivation areas in China (Table 1). The corresponding voucher specimens had been deposited in the Research Center of Natural Drug, School of Pharmaceutical Sciences, Jilin University, China. All the HC and HD samples were identified with the macroscopic and microscopic characters according to the Standard of Chinese Medicinal Materials in Guangdong Province (2004 Edition) and the Standard of Chinese Medicinal Materials in Shaanxi Province (2015 Edition). In these Standards, the identified methods only focus on the different macroscopic and microscopic characters. As the chemical constitutes are concerned, both oleanolic acid and ursolic acid are used to quality control. That is to say, there are no biomarkers to distinguish HC from HD.
Acetonitrile and methanol were UPLC-MS pure grade (Fisher Chemical Company, Geel, Belgium). Formic acid for UPLC was purchased from Sigma-Aldrich Company (St. Louis, MO, USA). Deionized water was purified using a Millipore water purification system (Millipore, Billerica, MA, USA). All other chemicals were of analytical grade. For reference substance, ursolic acid (110742-201622), citric acid (111679-201602), chlorogenic-acid (110753-201716), geniposide (110749-201718), luteolin 7-O-β-d-glucopyranoside (111968-201602), rutin (100080-201409), quercetin (100081-201610), kaempferol (110861-201611), hesperidin (110721-201617) were purchased from the National Institutes for Food and Drug Control (Beijing, China). Scandoside (20170503), alizarin 1-methyl ether (20170608) were purchased from Nanjing DASF Biotechnology Co., Ltd. (Nanjing, China). Scandoside methyl ester (20171001), 5,6,7,4′-tetramethoxyflavone (20171011), geniposidic acid (20171024) were purchased from Sichuan Weikeqi Biotechnology Co., Ltd. (Chengdu, China). 6-Methoxy-8-methylcoumarin (16018), sanlengdiphenyllactone (15025) were provided by the Research Center of Natural Drugs, School of Pharmaceutical Sciences, Jilin University, China.

2.2. Sample Preparation and Extraction

All the whole plants, including HC (HC1~HC10) and HD (HD1~HD10), were air-dried, grinded and sieved (40 mesh) to get the homogeneous powder respectively. Then, the powder of 20 samples (200 mg per sample) were extracted respectively with 80% methanol (2L × 3) at 80 °C for three times (3 h each time) with the reflux method. The extraction procedure is repeated until the extracted solution is colorless. After filteration, the extracts of each sample were combined, concentrated and evaporate to dryness. As a result, 20 desiccated extract powders were obtained. Each powder was dissolved in 1.0 mL of 80% methanol. Subsequently, each methanolic solution was filtered and injected directly into the UPLC system. The volume injected of each sample was 2 μL for each run. Furthermore, the methanol blank were run with the same gradient program between two samples during the whole sample list. The wash volume between injections was enough for avoiding carry over. Meanwhile, 20-μL aliquots of each HD and HC sample were mixed to obtain a quality control (QC) sample, which contained all of the components in the analysis. The QC sample was run every five samples to monitor the stability of the system.

2.3. Ultra-High Performance Liquid Chromatography with Quadrupole Time-of-Flight Tandem Mass Spectrometry (UPLC-QTOF-MS)

The separation and MS detection of components were performed on a Waters Xevo G2-XS QTOF mass spectrometer (Waters Co., Milford, MA, USA) connected to the UPLC system through an electrospray ionization (ESI) interface. UV wavelength did not trigger the MS detection of components. The column used was an ACQUITY UPLC BEH C18 (100 mm × 2.1 mm, 1.7 μm) from Waters Corporation (Milford, MA, USA). The mobile phases consisted of eluent A (0.1% formic acid in water, v/v) and eluent B (0.1% formic acid in acetonitrile, v/v) with a flow rate of 0.4 mL/min following a liner gradient program: 10% B from 0 to 2 min, 10–90% B from 2 to 25 min, 90% B from 25 to 26 min and 90–10% B from 26 to 26.1 min. The temperature of the UPLC column and sample was set at 30 °C and 15 °C. Mixtures of 10/90 and 90/10 water/acetonitrile were used as the strong wash and the weak wash solvent respectively. The optimized instrumental parameters were as follows: capillary voltage floating at 2.6 kV (ESI+) or 2.2 kV (ESI), cone voltage at 40 V, source temperature at 150 °C, desolvation temperature at 400 °C, cone gas flow at 50 L/h and desolvation gas flow at 800 L/h. In MSE mode, collision energy of low energy function was set to 6 V, while ramp collision energy of high energy function was set to 20–40 V. Each sample was analyzed by UPLC-QTOF-MSE mode; data acquisition was performed via the mass spectrometer by rapidly switching from a low-collision energy (CE) scan to a high-CE scan during a single LC run. The low-CE experiment provides information about the intact molecular ion, e.g., [M+H]+, while the high-CE scan generates fragment ion information. Alignment of the low-CE and high-CE data is automatically performed by the software. To ensure mass accuracy and reproducibility, the mass spectrometer was calibrated over a range of 100–1200 Da with sodium formate. Leucine enkephalin was used as external reference of Lock Spray™ infused at a constant flow of 10 μL/min. In addition, MassLynx data were recorded in continuous mode during acquisition.

2.4. Chemical Information Database for the Components of HC and HD

In addition to the Waters Traditional Medicine Library in the UNIFI software, a systematic investigation of chemical constituents was conducted. A self-built database of compounds isolated from HC and HD was established by searching online databases such as China Journals of Full-Text Database (CNKI), PubMed, Medline, Web of Science and ChemSpider. The name, molecular formula and structure of components from HC and HD were obtained in the database.

2.5. Data Analysis by UNIFI Platform

Data analysis was performed on UNIFI 1.7.0 software (Waters, Manchester, UK). Emphasis was put on analyzing structural characteristics and MS fragmentation behaviors, especially for characteristic fragments. Minimum peak area of 200 was set for 2D peak detection. The peak intensity of high energy over 200 counts and the peak intensity of low energy over 1000 counts were the selected parameters in 3D peak detection. A margin of error up to 5 ppm for identified compounds was allowed. We selected positive adducts containing +H and +Na and negative adducts including +COOH and −H. For exact mass accuracy, with leucine enkaplin as the reference compound, [M+H]+ 556.2766 was used for positive ion and [M−H] 554.2620 was used for negative ion in the UNIFI platform.
The MS raw data were processed using the streamlined workflow of UNIFI software to quickly identify the chemical components that met the match criteria with the Traditional Medicine Library. Firstly, an in-house scientific library was created including the information of chemical components from the target herbs based on the literature, saved as Mol file format, and then, the newly built library was imported into the analysis method, in virtue of some compounds being missing in the Traditional Medicine Library. Secondly, the raw data was compressed by Waters Compression and Archival Tool v1.10 and imported into the software. Thirdly, automated screening and identification were performed by the UNIFI platform instead of manually extracting each individual chromatographic peak, calculating the elementary composition and then analyzing MS fragmentation behaviors. Fourthly, we set up a filter to refine results, being mass error between −5 and 5 ppm, and additionally, response value greater than 6000. Finally, further verification of compounds by comparison with retention time of reference substances and characteristic MS fragmentation patterns reported in literature was carried out. After processing and filtering of the data by UNIFI, all selected components were listed for further verification, including information such as compound name, chemical structure, mass error, adducts, response, extracting ion chromatograms and spectra of low energy and high energy. The components were listed by descending response order and confirmed by reference substances or comparison with literatures.

2.6. Metabonomics Analysis

MarkerLynx XS V4.1 software (Waters, Manchester, UK) was used to process the raw data for alignment, deconvolution, data reduction, etc. As a result, the list of mass and retention time pairs with corresponding intensities for all the detected peaks from each data file. The main parameters were as follows: retention time range 0–26 min, mass range 100–1200 Da, mass tolerance 0.10, minimum intensity 5%, marker intensity threshold 2000 counts, mass window 0.10, retention time window 0.20, and noise elimination level 6. Furthermore, also with the MarkerLynx XS V4.1 software, principle component analysis (PCA) and orthogonal projections to latent structures discriminant analysis (OPLS-DA) were applied to analyze the above resulting data. Whether these two species are different would depend on the separation between HD and HC groups. The obvious separation in PCA score plots means they are differentiated. The supervised pattern recognition approach OPLS-DA can visualize and depict general metabolic variation between two groups. To identify the metabolites contributing to the discrimination, S-plots and VIP-plots were obtained via OPLS-DA analysis to find potential biomarkers that significantly contributed to the difference among HC and HD. Each spot in S-plots represents a variance. The importance of each variance to classification is determined by the value of variable importance in the projection (VIP) and metabolites with VIP value above 2.0 were considered as potential markers.

3. Results

3.1. Identification of Components from HC and HD

A total of 113 compounds were identified or tentatively characterized in both positive and negative mode from HC and HD (Table 2), the base peak intensity (BPI) chromatograms are shown in Figure 1, and their chemical structures are shown in Figure 2. In HC and HD 109 and 104 compounds were characterized, respectively. Both herbs are rich in natural components with various structural patterns, including iridoids, flavonoids, organic acids and organic acid esters, tannins, alcohols, ketones, coumarins, anthraquinones, monoterpenes, triterpenoids, etc. Some of these compounds have isomers may be distinguished based on characteristic MS fragmentation patterns reported in literature, or comparison of retention times to reference substances.
80 common constituents were identified from HC and HD. Among them, there were eleven iridoids (compounds 6, 8, 11, 14, 18, 20, 29, 51, 53, 58 and 59), thirteen flavonoids (compounds 7, 17, 25, 26, 27, 31, 36, 37, 38, 39, 43, 56 and 61), one monoterpene (compound 10), one anthraquinone (compound 68), two ketones (compounds 34 and 67), three tannins (compounds 4, 73 and 60), five alcohols (compounds 13, 80, 82, 98 and 99), and the rest are organic acids and organic acid esters, triterpenoids, coumarins, alkaloid, phenol, amide and glycoside. The contents of above components were similar in these two herbs.

3.2. Biomarker Discovery for HD and HC

PCA, a classic unsupervised lowering-dimension pattern recognition model, can be used to select distinct variables and to find potential biomarkers. It was firstly established based on the spectra of HD and HC samples to discern the presence of inherent similarities in mass spectral profiles as displayed in Figure 3. Two parameters, R2 (cum) and Q2 (cum), are commonly used to assess the quality of the PCA model, with values close to 1.0 indicative of good fitness and predictive ability. In the present study, R2X (cum) and Q2 (cum) were 0.6909 and 0.6257, respectively, indicating good fitness and prediction of the constructed PCA model.
Based on the obtained PCA score plots (Figure 3), the 20 samples were obviously divided into two main groups according to different species (HD and HC). The HD samples were noticeably overlapping, which indicates good similarity among them, and this result was also observed for HC samples. Meanwhile, the HD group and the HC group were completely separated, indicating that these two species herbs could be differentiated. The QC samples were between the two species, which came from the fact that they were mixed volumetrically in 50%.
In order to distinguish HD from HC, OPLS-DA models were built in both positive and negative modes. OPLS-DA score plot, S-plot, variable trend and VIP (variable importance in the projection) values were obtained to understand which variables are responsible for separation [109].
As shown in Figure 4, OPLS-DA models were constructed to discriminate the difference under the already established separation between different groups based on the PCA results. Each model has 2 score components (HD and HC). These scores are weighted averages of the original ones, hence providing a good summary. In addition, these scores display the separation of the groups in both ESI+ and ESI modes. The scores t[1] (x-axis) and to[1] (y-axis) are the two most important new variables in summarizing and separating the data. Each point in the plot corresponds to an observation. The groups are shown in different shapes and the separation of the groups is easily visible in t[1]. The to[1] score values show the variation within each class. This variation can either be caused by biological variation or by systematic changes in the experimental setup.
Figure 5 displays the variable importance (VIP) versus the PLS-regression coefficients. Important X-variables have large positive VIP values and large positive or negative coefficient values. The covariance p[1] and correlation p(corr)[1] loadings from a two class OPLS-DA model were shown here in S-Plot format (Figure 6). The points are Exact Mass/Retention Time pairs (EMRTs). The upper right quadrant of the S-plot shows those components which are elevated in HC, the control group, while the lower left quadrant shows EMRTs elevated in HD, the treated group. The farther along the x-axis the greater the contribution to the variance between the groups, while the farther the y-axis the higher the reliability of the analytical result. Based on VIP values (VIP > 4) (Figure 5) and p values (p < 0.05) [110] from univariate analysis, and the identification of components from HC and HD (Table 2), 33 robust known biomarkers enabling the differentiation between HD and HC were discovered and marked in S-plots (Figure 6). In order to systematically evaluate the biomarkers, a heatmap was generated from these biomarkers (shown in Figure 7), which shows distinct segregation between two species.

4. Discussion

There are 109 and 104 compounds characterized from HC and HD respectively. Sixty compounds were identified in ESI mode and 53 compounds were identified in ESI+ mode. According to the BPI chromatograms of HC and HD, it seems that ESI ionization mode is better than ESI+ based on the quantity and the responses of the identified compounds, but it is still necessary to run the ESI+ mode because some compounds showed better respond than in ESI mode.
It was revealed that HD and HC differed in their chemical composition according to the HPLC analysis [19]. It was also indicated that 6-O-(E)-p-coumaroyl scandoside methyl ester and 6-O-(E)-p-coumaroyl scandoside methyl ester-10-O-methyl ether were the main components of HD. In 2007, Liang et al. reported that HD and its substitutes could be identified based on HPLC chemical fingerprints and mass spectrometric analysis [25]. MS combined with UV spectra and literature values was used to obtain the chemical information. As a result, four compounds, asperuloside, 6-O-(E)-p-coumaroyl scandoside methyl ester, 6-O-(E)-p-coumaroyl scandoside methyl ester-10-methyl ester and 6-O-p-feruloyl scandoside methyl ester were recommended to be used as chemical markers for quality evaluation and chemical authentication of HD and its substitutes. In addition, scandoside methyl ester detected in the chromatograms of HC can be used as the characteristic peaks [25]. Furthermore, a previous report found that hedyotiscone A could be used to differentiate HC from HD using TLC method [22]. In our study, asperuloside, 6-O-(E)-p-coumaroyl scandoside methyl ester-10-methyl ester, scandoside methyl ester, 6-O-p-feruloyl scandoside methyl ester and hedyotiscone A were shared in HC and HD, but the reported result concerning 6-O-(E)-p-coumaroyl scandoside methyl ester was consistent with our findings.
In the other record, another marker compound, 10(S)-hydroxypheophytin a, isolated with a yield of 22 mg from 600 g of HC, was identified exclusively in HD [23]. It is a pity that it was not be detected under our experimental conditions. Similarly, (9R,10S,7E)-6,9,10-trihydroxyoctadec-7-enoic acid, isolated with a yield of 47.9 mg from 20 kg of HC, was reported to be used to differentiate HC from HD [26]. It was not be detected under our experimental conditions either.
In this study, 33 known compounds enabling the robust differentiation between HC and HD were detected. For HC, there were 18 potential biomarkers, including three iridoids (23, 55, 66), eight flavonoids (30, 35, 40, 42, 47, 71, 75, 81), two tannins (19, 45), two ketones (22, 91), one alcohol (92), two monoterpenes (89, 90). Among these potential biomarkers, the contents of nine components (19, 22, 23, 30, 35, 40, 45, 66, 92) in HC were much greater than in HD. Compounds 42, 47, 55, 71, 75, 81, 89, 90 and 91 could be detected only in HC. It’s worth mentioning that two iridoids, compounds 55 (hedycoryside B) and 66 (hedycoryside A), with high responses in UPLC-MS might be used for rapid identification of HC. For HD, there were 15 potential biomarkers including two iridoids (52, 50), eight flavonoids (41, 44, 49, 54, 57, 62, 79, 84), one tannin (46), one ketone (70), and three anthraquinones (69, 77, 78). Among them, the contents of eleven components (41, 44, 46, 49, 52, 57, 62, 70, 77, 78, 79) in HD were much higher than those in HC. Compounds 50, 54, 69 and 84 were detected only in HD. In addition, two anthraquinones, compounds 69 (1,3-dihydroxy-2-methylanthraquinone) and 78 (2-hydroxy-3-methylanthraquinone) with high responses in UPLC-MS might be used for rapid identification of HD.
However, there are still some unresolved issues. Firstly, the pharmaceutical effects associated with these identified compounds should be screened in the future. Secondly, as shown in BPI chromatograms, though 113 compounds were identified, there are still some unidentified components. Further research should be carried out based on the formula of these unknown compounds. Thirdly, source material is not seasonable as it was collected during summer time. Fourthly, collecting HC and HD in the same area may be the better way for comparison. But in this study, Haikou City for HC and Fuzhou City for HD were visited. To some extent, the collection of these samples might be used as negative controls for another species because it could eliminate the influence of the region on the analysis of the sample. But unfortunately, the regional factor should not be considered as there should be more samples per region.

5. Conclusions

Under the optimized conditions, a total of 109 chemical compounds with different structural types were identified from HC and 104 from HD. The similarities and differences between these two herbs were also highlighted in the paper. Various structural patterns including iridoids, flavonoids, organic acids and organic acid esters, tannins, alcohols, ketones, coumarins, anthraquinones, monoterpenes, triterpenoids were presenting in these two herbs, of which there were 80 shared compounds in HC and HD. There is quite a difference in the parent structures types between HC and HD. A total of 33 robust biomarkers enabling the differentiation between HC and HD were discovered. For HC and HD, 18 and 15 potential biomarkers, respectively, were identified in this paper. Two iridoids, hedycoryside B (compound 55) and hedycoryside A (66) might be used for rapid identification of HC, and two anthraquinones, 1,3-Dihydroxy-2-methylanthraquinone (compound 69) and 2-Hydroxy-3-methylanthraquinone (78) might be used for rapid identification of HD based on their presence and content. Actually, these solid biomarkers are recommended for further use in the recognition and distinction between HC and HD. The results provided reliable characterization profiles to identify these two herbs and to clarify the fundamental pharmacological substances. Different chemical compositions will inevitably lead to different biological effects of HC and HD in clinical application. HC should not be used as substitute of HD. The results provided data on the chemical constituents of HC and provide a reference for the quality control of HD in the aspect of quantitative determination.

Author Contributions

J.L. conceived and designed the experiments; Y.W., C.W. and H.L. performed the experiments; Y.W., Y.L. (Yunhe Liu), Y.L. (Yameng Li) and Y.Z. were responsible for data analysis. J.L. wrote the paper. J.L. and P.L. assisted paper revision.

Funding

This research was supported by the Biomedicine Special Foundation for Government-Univeristy Cooperation Project of Jilin Province [No. SXGJSF2017-1-1-(02)].

Conflicts of Interest

The authors declare that they have no conflicts of interest concerning this article.

References

  1. Chen, R.; He, J.; Tong, X.; Tang, L.; Liu, M. The Hedyotis diffusa Willd. (Rubiaceae): A Review on Phytochemistry, Pharmacology, Quality Control and Pharmacokinetics. Molecules 2016, 21, 710. [Google Scholar] [CrossRef] [PubMed]
  2. Wang, C.; Xin, P.; Wang, Y.; Zhou, X.; Wei, D.; Deng, C.; Sun, S. Iridoids and sfingolipids from Hedyotis diffusa. Fitoterapia 2017, 124, 152–159. [Google Scholar] [CrossRef] [PubMed]
  3. Li, C.; Zhao, Y.; Guo, Z.; Xue, X.; Liang, X. Effective 2D-RPLC/RPLC enrichment and separation of micro-components from Hedyotis diffusa Willd. and characterization by using ultra-performance liquid chromatography/quadrupole time-of-flight mass spectrometry. J. Pharm. Biomed. Anal. 2014, 99, 35–44. [Google Scholar] [CrossRef] [PubMed]
  4. Sasikumar, J.M.; Maheshu, V.; Aseervatham, G.S.; Darsini, D.T. In vitro antioxidant activity of Hedyotis corymbosa (L.) Lam. aerial parts. Indian J. Biochem. Biophys. 2010, 47, 49–52. [Google Scholar] [PubMed]
  5. Endrini, S. Antioxidant activity and anticarcinogenic properties of “rumput mutiara” {Hedyotis corymbosa (L.) Lam.} and “pohpohan” {Pilea trinervia (Roxb.) Wight}. J. Med. Plant Res. 2011, 5, 3715–3718. [Google Scholar]
  6. Lin, C.C.; Ng, L.T.; Yang, J.J.; Hsu, Y.F. Anti-inflammatory and hepatoprotective activity of peh-hue-juwa-chi-cao in male rats. Am. J. Chin. Med. 2002, 30, 225–234. [Google Scholar] [CrossRef] [PubMed]
  7. Sadasivan, S.; Latha, P.G.; Sasikumar, J.M.; Rajashekaran, S.; Shyamal, S.; Shine, V.J. Hepatoprotective studies on Hedyotis corymbosa, (L.) Lam. J. Ethnopharmacol. 2006, 106, 245–249. [Google Scholar] [CrossRef] [PubMed]
  8. Chimkode, R.; Patil, M.B.; Jalalpure, S.; Pasha, T.Y.; sarkar, S. A Study of hepatoprotective activity of Hedyotis corymbosa. Linn, in albino rats. Anc. Sci. Life 2009, 28, 32–35. [Google Scholar] [PubMed]
  9. Yue, G.G.; Kin-Ming, L.J.; Cheng, L.; Chung-Lap, C.B.; Jiang, L.; Fung, K.P.; Leung, P.C.; Bik-San Lau, C. Reversal of P-glycoprotein-mediated multidrug resistance in human hepatoma cells by hedyotiscone A, a compound isolated from Hedyotis corymbosa. Xenobiotica 2012, 42, 562–570. [Google Scholar] [CrossRef] [PubMed]
  10. You, B.J.; Wu, Y.C.; Wu, C.Y.; Bao, B.Y.; Chen, M.Y.; Chang, Y.H.; Lee, H.Z. Proteomics displays cytoskeletal proteins and chaperones involvement in Hedyotis corymbosa-induced photokilling in skin cancer cells. Exp. Dermatol. 2011, 20, 653–658. [Google Scholar] [CrossRef] [PubMed]
  11. Mishra, K.; Dash, A.P.; Swain, B.K.; Dey, N. Anti-malarial activities of Andrographis paniculata, and Hedyotis corymbosa, extracts and their combination with curcumin. Malar. J. 2009, 8, 26. [Google Scholar] [CrossRef] [PubMed]
  12. Moniruzzaman, M.; Ferdous, A.; Irin, S. Evaluation of antinociceptive effect of ethanol extract of Hedyotis corymbosa Linn. whole plant in mice. J. Ethnopharmacol. 2015, 161, 82–85. [Google Scholar] [CrossRef] [PubMed]
  13. Wei, J.; Kuang, L.; Hou, A.; Qian, M.; Li, J.Z. Iridoid Glycosides from Hedyotis corymbosa. Helv. Chim. Acta 2010, 90, 1296–1301. [Google Scholar]
  14. Takagi, S.; Yamaki, M.; Masuda, K.; Nishihama, Y.; Sakina, K. Studies on the herb medical materials used for some tumors. II. On the constituents of Hedyotis corymbosa Lam (author’s transl). Yakugaku Zasshi J. Pharm. Soc. Jpn. 1981, 101, 657–659. [Google Scholar] [CrossRef]
  15. Noiarsa, P.; Ruchirawat, S.; Otsuka, H.; Kanchanapoom, T. Chemical constituents from Oldenlandia corymbosa L. of Thai origin. J. Nat. Med. 2008, 62, 249–250. [Google Scholar] [CrossRef] [PubMed]
  16. Otsuka, H.; Yoshimura, K.; Yamasaki, K.; Cantoria, M.C. Isolation of 10-O-acyl iridoid glucosides from a Philippine medicinal plant, Oldenlandia corymbosa L. (Rubiaceae). Chem. Pharm. Bull. 1991, 39, 2049–2052. [Google Scholar] [CrossRef]
  17. Modi, K.; Shah, M.B. Determination of oleanolic acid, ursolic acid, lupeol, and stigmasterol by high-performance thin-layer chromatographic method in Oldenlandia Corymbosa Linn. J. Planar Chromatogr. Mod. TLC 2017, 30, 32–35. [Google Scholar] [CrossRef]
  18. Wei, M.C.; Hong, S.J.; Yang, Y.C. Isolation of triterpenic acid-rich extracts from Hedyotis corymbosa, using ultrasound-assisted supercritical carbon dioxide extraction and determination of their fictitious solubilities. J. Ind. Eng. Chem. 2017, 48, 202–211. [Google Scholar] [CrossRef]
  19. Liang, Z.; He, M.; Fong, W.; Jiang, Z.; Zhao, Z. A comparable, chemical and pharmacological analysis of the traditional Chinese medicinal herbs Oldenlandia diffusa and O. corymbosa and a new valuation of their biological potential. Phytomedicine 2008, 15, 259–267. [Google Scholar]
  20. Li, M.; Wong, Y.L.; Jiang, L.L.; Wong, K.L.; Wong, Y.T.; Lau, C.S.; Shaw, P.C. Application of novel loop-mediated isothermal amplification (LAMP) for rapid authentication of the herbal tea ingredient Hedyotis diffusa Willd. Food Chem. 2013, 141, 2522–2525. [Google Scholar] [CrossRef] [PubMed]
  21. Liang, Z.T.; Jiang, Z.H.; Leung, K.S.; Peng, Y.; Zhao, Z.Z. Distinguishing the medicinal herb Oldenlandia diffusa from similar species of the same genus using fluorescence microscopy. Microsc. Res. Tech. 2006, 69, 277–282. [Google Scholar] [CrossRef] [PubMed]
  22. Lau, C.B.; Cheng, L.; Cheng, B.W.; Yue, G.G.; Wong, E.C.; Lau, C.P.; Leung, P.C.; Fung, K.P. Development of a simple chromatographic method for distinguishing between two easily confused species, Hedyotis diffusa and Hedyotis corymbosa. Former. Nat. Prod. Lett. 2011, 26, 1446–1450. [Google Scholar] [CrossRef] [PubMed]
  23. Li, M.; Jiang, R.W.; Hon, P.M.; Cheng, L.; Li, L.L.; Zhou, J.R.; Shaw, P.C.; Paul, P.H. Authentication of the anti-tumor herb Baihuasheshecao with bioactive marker compounds and molecular sequences. Food Chem. 2010, 119, 1239–1245. [Google Scholar] [CrossRef]
  24. Sun, Y.L.; Wang, D.; Yeom, M.H.; Kim, D.H.; Kim, H.G.; Hong, S.K. Molecular identification of medicinal herbs, Oldenlandia diffusa and Oldenlandia corymbosa based on nrDNA ITS region sequence. J. Plant Biotechnol. 2011, 38, 301–307. [Google Scholar] [CrossRef]
  25. Liang, Z.; Jiang, Z.; Ho, H.; Zhao, Z. Comparative analysis of Oldenlandia diffusa and its substitutes by high performance liquid chromatographic fingerprint and mass spectrometric analysis. Planta Med. 2007, 73, 1502–1508. [Google Scholar] [CrossRef] [PubMed]
  26. Li, H.; Li, C.; Xia, B.; Zhou, Y.; Lin, L.; Liao, D. A chemotaxonomic study of phytochemicals in Hedyotis corymbosa. Biochem. Syst. Ecol. 2015, 62, 173–177. [Google Scholar] [CrossRef]
  27. Li, H.Q.; Cao, Y.; Bai, Y.B.; Xia, B.H.; Lin, L.M.; Liao, D.F. UPLC Fingerprint of Oldenlandia corymbosa. J. Chin. Med. Mater. 2015, 38, 735–738. [Google Scholar]
  28. Yang, Y.C.; Wei, M.C.; Chiu, H.F.; Huang, T.C. Development and validation of a modified ultrasound-assisted extraction method and a HPLC method for the quantitative determination of two triterpenic acids in Hedyotis diffusa. Nat. Prod. Commun. 2013, 8, 1683–1686. [Google Scholar] [PubMed]
  29. Wang, J.R.; Yau, L.F.; Gao, W.N.; Liu, Y.; Yick, P.W.; Liu, L.; Jiang, Z.H. Quantitative comparison and metabolite profiling of saponins in different parts of the root of Panax notoginseng. J. Agric. Food Chem. 2014, 62, 9024–9034. [Google Scholar] [CrossRef] [PubMed]
  30. Wang, C.; Zhang, N.; Wang, Z.; Qi, Z.; Zhu, H.; Zheng, B.; Li, P.; Liu, J. Nontargeted Metabolomic Analysis of Four Different Parts of Platycodon grandiflorum Grown in Northeast China. Molecules 2017, 22, 1280. [Google Scholar] [CrossRef] [PubMed]
  31. Wang, C.; Zhang, N.; Wang, Z.; Qi, Z.; Zhu, H.; Zheng, B.; Li, P.; Liu, J. Rapid characterization of chemical constituents of Platycodon grandiflorum and its adulterant Adenophora stricta by UPLC-QTOF-MS/MS. J. Mass Spectrom. 2017, 52, 643–656. [Google Scholar] [CrossRef] [PubMed]
  32. Zhang, F.X.; Li, M.; Qiao, L.R.; Yao, Z.H.; Li, C.; Shen, X.Y.; Wang, Y.; Yu, K.; Yao, X.S.; Dai, Y. Rapid characterization of Ziziphi Spinosae Semen by UPLC/Q-tof MS with novel informatics platform and its application in evaluation of two seeds from Ziziphus species. J. Pharm. Biomed. Anal. 2016, 122, 59–80. [Google Scholar] [CrossRef] [PubMed]
  33. Deng, L.; Shi, A.M.; Liu, H.Z.; Meruva, N.; Liu, L.; Hu, H.; Yang, Y.; Huang, C.; Li, P.; Wang, Q. Identification of chemical ingredients of peanut stems and leaves extracts using UPLC-QTOF-MS coupled with novel informatics UNIFI platform. J. Mass Spectrom. 2016, 51, 1157–1167. [Google Scholar] [CrossRef] [PubMed]
  34. Tang, J.; Li, W.; Tan, X.; Li, P.; Xiao, X.; Wang, J.; Zhu, M.; Li, X.; Meng, F. A novel and improved UHPLC-QTOF/MS method for the rapid analysis of the chemical constituents of Danhong Injection. Anal. Methods 2016, 8, 2904–2914. [Google Scholar] [CrossRef]
  35. Piacente, S.; Carbone, V.; Plaza, A.; Zampelli, A.; Pizza, C. Investigation of the Tuber Constituents of Maca (Lepidium meyenii Walp.). J. Agric. Food Chem. 2002, 50, 5621–5625. [Google Scholar] [CrossRef] [PubMed]
  36. Shoji, N.; Umeyana, A.; Iuchi, A.; Saito, N.; Arihara, S.; Nomoto, K.; Ohizumi, Y. Two Novel Alkaloids from Evodia rutaecarpa. J. Nat. Prod. 1989, 52, 1160–1162. [Google Scholar] [CrossRef]
  37. Li, C.; Xue, X.; Zhou, D.; Zhang, F.; Xu, Q.; Ren, L.L.; Liang, X.M. Analysis of iridoid glucosides in Hedyotis diffusa, by high-performance liquid chromatography/electrospray ionization tandem mass spectrometry. J. Pharm. Biomed. Anal. 2008, 48, 205–211. [Google Scholar] [CrossRef] [PubMed]
  38. Montoro, P.; Maldini, M.; Russo, M.; Postorino, S.; Piacente, S.; Pizza, C. Metabolic profiling of roots of liquorice (Glycyrrhiza glabra) from different geographical areas by ESI/MS/MS and determination of major metabolites by LC-ESI/MS and LC-ESI/MS/MS. J. Pharm. Biomed. Anal. 2011, 54, 535–544. [Google Scholar] [CrossRef] [PubMed]
  39. Guo, X.; Chen, X.; Li, L.; Shen, Z.; Wang, X.; Zheng, P.; Duan, F.; Ma, Y.; Bi, K. LC-MS determination and pharmacokinetic study of six phenolic components in rat plasma after taking traditional Chinese medicinal-preparation: Guanxinning lyophilized powder for injection. J. Chromatogr. B 2008, 873, 51–58. [Google Scholar] [CrossRef] [PubMed]
  40. Guy, P.A.; Renouf, M.; Barron, D.; Cavin, C.; Dionisi, F.; Kochhar, S.; Rezzi, S.; Williamson, G.; Steiling, H. Quantitative analysis of plasma caffeic and ferulic acid equivalents by liquid chromatography tandem mass spectrometry. J. Chromatogr. B 2009, 877, 3965–3974. [Google Scholar] [CrossRef] [PubMed]
  41. Yang, X.; Yang, L.; Xiong, A.; Li, D.; Wang, Z.T. Authentication of Senecio scandens, and S. vulgaris, based on the comprehensive secondary metabolic patterns gained by UPLC-DAD/ESI-MS. J. Pharm. Biomed. Anal. 2011, 56, 165–172. [Google Scholar] [CrossRef] [PubMed]
  42. Raiskila, S.; Fagerstedt, K.; Laakso, T.; Saranpää, P.; Löija, M.; Paajanen, L. Polymerisation of added coniferyl alcohol by inherent xylem peroxidases and its effect on fungal decay resistance of Norway spruce. Wood Sci. Technol. 2006, 40, 697–707. [Google Scholar] [CrossRef]
  43. Kim, D.H.; Lee, H.J.; Oh, Y.J.; Kim, M.J.; Kim, S.H. Iridoid glycosides isolated from Oldenlandia diffusa, inhibit LDL-oxidation. Arch. Pharm. Res. 2005, 28, 1156–1160. [Google Scholar] [CrossRef] [PubMed]
  44. Kim, J.E.; Jung, M.J.; Jung, H.A.; Woo, J.J.; Cheigh, H.S.; Chung, H.Y.; Choi, J.S. A new kaempferol 7-O-triglucoside from the leaves of Brassica juncea, L. Arch. Pharm. Res. 2002, 25, 621–624. [Google Scholar] [CrossRef] [PubMed]
  45. Hiltunen, E.; Pakkanen, T.T.; Alvila, L. Phenolic extractives from wood of birch (Betula pendula). Holzforschung 2004, 58, 326–329. [Google Scholar] [CrossRef]
  46. Liang, Z.T.; Jiang, Z.H.; Leung, K.S. Determination of iridoid glucosides for quality assessment of Herba Oldenlandiae by high-performance liquid chromatography. Chem. Pharm. Bull. 2006, 54, 1131–1137. [Google Scholar] [CrossRef] [PubMed]
  47. Bandyopadhyay, A.; Bagchi, B.; Podder, G.; Moitra, S.K. A new route for synthesis of aromatic Keto acid. Indian Chem. Soc. 1989, 66, 239–240. [Google Scholar]
  48. Huang, S.; Liao, X.; Nie, Q.; Ding, L.; Peng, S. Phenyl and phenylethyl glycosides from Picrorhiza scrophulariiflora. Helv. Chim. Acta 2004, 87, 598–604. [Google Scholar] [CrossRef]
  49. Wang, S.P.; Liu, L.; Wang, L.L.; Wang, S.P.; Liu, L.; Wang, L.L.; Jiang, P.; Zhang, J.Q.; Zhang, W.D.; Liu, R.H. Screening and analysis of the multiple absorbed bioactive components and metabolites in rat plasma after oral administration of Jitai tablets by high-performance liquid chromatography/diode-array detection coupled with electrospray ionization tandem ma. Rapid Commun. Mass Spectrom. 2010, 24, 1641–1652. [Google Scholar] [CrossRef] [PubMed]
  50. Luthria, D.L.; Lin, L.Z.; Robbins, R.J.; Finley, J.W.; Banuelos, G.S.; Harnly, J.M. Discriminating between cultivars and treatments of broccoli using mass spectral fingerprinting and analysis of variance-principal component analysis. J. Agric. Food Chem. 2008, 56, 9819–9827. [Google Scholar] [CrossRef] [PubMed]
  51. Regos, I.; Urbanella, A.; Treutter, D. Identification and quantification of phenolic compounds from the forage legume sainfoin (Onobrychis viciifolia). J. Agric. Food Chem. 2009, 57, 5843–5852. [Google Scholar] [CrossRef] [PubMed]
  52. Guvenalp, Z.; Kilic, N.; Kazaz, C.; Kaya, Y.; Demirezer, L.O. Chemical constituents of Galium tortumense. Turk. J. Chem. 2006, 30, 515–523. [Google Scholar]
  53. Cuyckens, F.; Shahat, A.A.; Pieters, L.; Claeys, M. Direct stereochemical assignment of hexose and pentose residues in flavonoid O-glycosides by fast atom bombardment and electrospray ionization mass spectrometry. J. Mass Spectrom. 2002, 37, 1272–1279. [Google Scholar] [CrossRef] [PubMed]
  54. Gao, X.; Pujosguillot, E.; Martin, J.F.; Galan, P.; Juste, C.; Jia, W.; Sebedio, J.L. Metabolite analysis of human fecal water by gas chromatography/mass spectrometry with ethyl chloroformate derivatization. Anal. Biochem. 2009, 393, 163–175. [Google Scholar] [CrossRef] [PubMed]
  55. Jerezano, A.; Jimenez, F.; Cruz, M.D.; Montiel, L.E.; Delgado, F.; Tamariz, J. New Approach for the Construction of the Coumarin Frame and Application in the Total Synthesis of Natural Products. Cheminform 2015, 94, 185–198. [Google Scholar] [CrossRef]
  56. Kuo, Y.H.; Lo, J.M.; Chan, Y.F. Cytotoxic Components from the Leaves of Schefflera Taiwaniana. J. Chin. Chem. Soc. 2002, 49, 427–431. [Google Scholar] [CrossRef]
  57. Shi, S.; Zhao, Y.; Zhou, H.G.; Zhang, Y.P.; Jiang, X.Y.; Huang, K.L. Identification of antioxidants from Taraxacum mongolicum by high-performance liquid chromatography-diode array detection-radical-scavenging detection-electrospray ionization mass spectrometry and nuclear magnetic resonance experiments. J. Chromatogr. 2008, 1209, 145–152. [Google Scholar] [CrossRef] [PubMed]
  58. Lee, E.H.; Kim, H.J.; Song, Y.S.; Jin, C.B.; Lee, K.T.; Cho, J.S.; Lee, Y.S. Constituents of the stems and fruits of Opuntia ficus-indica var. saboten. Arch. Pharm. Res. 2003, 26, 1018–1023. [Google Scholar] [CrossRef] [PubMed]
  59. Machida, K.; Osawa, K. On the Flavonoid constituents from the Peels of Citrus hassaku HORT. ex TANAKA. Chem. Pharm. Bull. 1989, 37, 1092–1094. [Google Scholar] [CrossRef]
  60. Luo, Y.D.; Wu, S.S.; Li, X.Y.; Li, P. LC-ESI-MS-MS determination of rat plasma protein binding of major flavonoids of Flos Lonicerae Japonicae by centrifugal ultrafiltration. Chromatographia 2010, 72, 71–77. [Google Scholar] [CrossRef]
  61. Li, Y.L.; Li, J.; Wang, N.L.; Yao, X.S. Flavonoids and a new polyacetylene from Bidens parviflora Willd. Molecules 2008, 13, 1931–1941. [Google Scholar] [CrossRef] [PubMed]
  62. Woo, K.W.; Moon, E.; Park, S.Y.; Kim, S.Y.; Lee, K.R. ChemInform Abstract: Flavonoid Glycosides from the Leaves of Allium victorialis var. Platyphyllum and Their Antineuroinflammatory Effects. Cheminform 2013, 44, 7465–7470. [Google Scholar] [CrossRef]
  63. Rösch, D.; Krumbein, A.; Mugge, C.; Kroh, L.W. Structural investigations of flavonol glycosides from sea buckthorn (Hippophaë rhamnoides) pomace by NMR spectroscopy and HPLC-ESI-MS(n). J. Agric. Food Chem. 2004, 52, 4039–4046. [Google Scholar] [CrossRef] [PubMed]
  64. Böttcher, C.; Roepenacklahaye, E.V.; Schmidt, J.; Schmotz, C.; Neumann, S.; Scheel, D.; Clemens, S. Metabolome Analysis of Biosynthetic Mutants Reveals a Diversity of Metabolic Changes and Allows Identification of a Large Number of New Compounds in Arabidopsis. Plant Physiol. 2008, 147, 2107–2120. [Google Scholar] [CrossRef] [PubMed] [Green Version]
  65. Mo, S.; Wang, S.; Zhou, G.; Yang, Y.C.; Li, Y.; Chen, X.G.; Shi, J.G. Phelligridins C-F: Cytotoxic pyrano [4,3-c] benzopyran-1,6-dione and furo[3,2-c]pyran-4-one derivatives from the fungus Phellinus igniarius. J. Nat. Prod. 2004, 67, 823–828. [Google Scholar] [CrossRef] [PubMed]
  66. Dunggun, L.; Jin, Q.L.; Jin, H.G.; Shin, J.F.; Choi, E.J.; Woo, E.R. Isolation of virus-cell fusion inhibitory components from the stem bark of Styrax japonica S. et Z. Arch. Pharm. Res. 2010, 33, 863–866. [Google Scholar]
  67. Palter, R.; Haddon, W.F.; Lundin, R.E. The complete structure of matairesinol monoglucoside. Phytochemistry 1971, 10, 1587–1589. [Google Scholar] [CrossRef]
  68. Sebok, A.; Vasanitszsigrai, A.; Helenkar, A.; Zaray, G.; Molnar-Perl, I. Multiresidue analysis of pollutants as their trimethylsilyl derivatives, by gas chromatography-mass spectrometry. J. Chromatogr. 2009, 1216, 2288–2301. [Google Scholar] [CrossRef] [PubMed]
  69. Dugo, P.; Presti, M.L.; Ohman, M.; Fazio, A.; Dugo, G.; Mondello, L. Determination of flavonoids in citrus juices by micro-HPLC-ESI/MS. J. Sep. Sci. 2005, 28, 1149–1156. [Google Scholar] [CrossRef] [PubMed]
  70. Wu, H.; Tao, X.; Chen, Q.; Lao, X. Iridoids from Hedyotis diffusa. J. Nat. Prod. 2004, 54, 254–256. [Google Scholar] [CrossRef]
  71. Jianyong, S.; Dihua, C.; Ruile, P. Study on the glycosides in Hedyotis diffusa. Chin. Tradit. Herb. Drugs 2008, 39, 507–509. [Google Scholar]
  72. Xu, G.H.; Younghee, K.; Chi, S.W.; Choo, S.J.; Ryoo, I.J.; Ahn, J.S.; Yoo, I.D. Evaluation of human neutrophil elastase inhibitory effect of iridoid glycosides from Hedyotis diffusa. Bioorg. Med. Chem. Lett. 2010, 20, 513–515. [Google Scholar] [CrossRef] [PubMed]
  73. Ding, Y.; Xiong, Y.; Zhou, B. Separation and identification of flavonoids from the oyster shell. Chin. J. Chin. Mater. Med. 2015, 40, 2352–2356. [Google Scholar]
  74. Kuang, L.S. 1. Isolation and Purification of Active Constituents from Chinese Herbal Medicine against Colon Cancer and Study on Its Mechanism, 2. Study on Fatty Acid Binding Protein of Haemonchus Contortus; East China Normal University: Shanghai, China, 2010. [Google Scholar]
  75. Sun, H.Y.; Xiao, C.F.; Cai, Y.C.; Chen, Y.; Wei, W.; Liu, X.K.; Lv, Z.L.; Zou, Y. Efficient synthesis of natural polyphenolic stilbenes: Resveratrol, piceatannol and oxyresveratrol. Chem. Pharm. Bull. 2010, 58, 1492–1496. [Google Scholar] [CrossRef] [PubMed]
  76. Bai, N.S.; He, K.; Zhu, Z.; Lai, C.S.; Zhang, L.; Quan, Z.; Shao, X.; Pan, M.H.; Ho, C.T. Flavonoids from Rabdosia rubescens exert anti-inflammatory and growth inhibitory effect against human leukemia HL-60 cells. Food Chem. 2010, 122, 831–835. [Google Scholar] [CrossRef]
  77. Li, W.; Fitzloff, J.F. HPLC-PDA determination of bioactive diterpenoids from plant materials and commercial products of Andrographis paniculata. J. Liq. Chromatogr. Relat. Technol. 2004, 27, 2407–2420. [Google Scholar] [CrossRef]
  78. Bhuyan, R.; Saikia, C. Isolation of colour components from native dye-bearing plants in northeastern India. Bioresour. Technol. 2005, 96, 363–372. [Google Scholar] [CrossRef] [PubMed]
  79. Nagase, H.; Omae, N.; Omori, A.; Nakagawasai, O.; Tadano, T.; Yokosuka, A.; Sashida, Y.; Mimaki, Y.; Yamakuni, T.; Ohizumi, Y. Nobiletin and its related flavonoids with CRE-dependent transcription-stimulating and neuritegenic activities. Biochem. Biophys. Res. Commun. 2005, 337, 1330–1336. [Google Scholar] [CrossRef] [PubMed]
  80. Wang, Q.; Yang, Y.; Li, Y.; Yu, W.; Hou, Z.J. An efficient method for the synthesis of lignans. Tetrahedron 2006, 62, 6107–6112. [Google Scholar] [CrossRef]
  81. Chen, Z. Comparative Analysis of Chemical Constituents of Hedyotis diffusa and Waterlilies; Liaoning Normal University: Dalian, China, 2011. [Google Scholar]
  82. Júnior, J.C.; Lemos, R.P.; Conserva, L.M. Chemical constituents from Spermacoce verticillata, (Rubiaceae). Biochem. Syst. Ecol. 2012, 44, 208–211. [Google Scholar] [CrossRef]
  83. Hasan, A.; Sadiq, A.; Abbas, A.; Mughal, E.; Khan, K.M.; Ali, M. Isolation and synthesis of flavonols and comparison of their antioxidant activity. Nat. Prod. Res. 2010, 24, 995–1003. [Google Scholar] [CrossRef] [PubMed]
  84. Dat, N.T.; Cai, X.F.; Shen, Q.; Lee, I.S.; Lee, E.J.; Park, Y.K.; Bae, K.; Kim, Y.H. Gymnasterkoreayne G, a new inhibitory polyacetylene against NFAT transcription factor from Gymnaster koraiensis. Cheminform 2005, 37, 1194–1196. [Google Scholar] [CrossRef]
  85. Han, S.; Kim, H.M.; Lee, J.M.; Mok, S.Y.; Lee, S. Isolation and Identification of Polymethoxyflavones from the Hybrid citrus, Hallabong. J. Agric. Food Chem. 2010, 58, 9488–9491. [Google Scholar] [CrossRef] [PubMed]
  86. Lechner, D.; Stavri, M.; Oluwatuyi, M.; Pereda-Miranda, R.; Gibbons, S. The anti-staphylococcal activity of Angelica dahurica, (Bai Zhi). Phytochemistry 2004, 65, 331–335. [Google Scholar] [CrossRef] [PubMed]
  87. Lee, T.; Juang, S.; Hsu, F.; Wu, C.; Cheng, Y.W. Triterpene Acids from the Leaves of Planchonella duclitan (Blanco) Bakhuizan. J. Chin. Chem. Soc. 2005, 52, 1275–1280. [Google Scholar] [CrossRef]
  88. Lai, J.P.; Lim, Y.H.; Su, J.; Shen, H.M.; Ong, C.N. Identification and characterization of major flavonoids and caffeoylquinic acids in three Compositae plants by LC/DAD-APCI/MS. J. Chromatogr. B 2007, 848, 215–225. [Google Scholar] [CrossRef] [PubMed]
  89. Catalan, C.A.; de Heluani, C.S.; Kotowicz, C.; Gedris, T.E.; Herz, W. A linear sesterterpene, two squalene derivatives and two peptide derivatives from Croton hieronymi. Phytochemistry 2003, 64, 625–629. [Google Scholar] [CrossRef]
  90. Alamsjah, M.A.; Hirao, S.; Ishibashi, F.; Fujita, Y. Isolation and Structure Determination of Algicidal Compounds from Ulva fasciata. J. Agric. Chem. Soc. Jpn. 2005, 69, 2186–2192. [Google Scholar]
  91. Yang, J.R.; An, Z.; Li, Z.H.; Jing, S.; Qina, H.L. Sesquiterpene coumarins from the roots of Ferula sinkiangensis and Ferula teterrima. Chem. Pharm. Bull. 2006, 54, 1595–1598. [Google Scholar] [CrossRef] [PubMed]
  92. Suebsasana, S.; Pongnaratorn, P.; Sattayasai, J.; Arkaravichien, T.; Tiamkao, S.; Aromdee, C. Analgesic, antipyretic, anti-inflammatory and toxic effects of andrographolide derivatives in experimental animals. Arch. Pharm. Res. 2009, 32, 1191–1200. [Google Scholar] [CrossRef] [PubMed]
  93. Shi, S.Y.; Zhou, C.X.; Xu, Y.; Tao, Q.F.; Bai, H.; Lu, F.S.; Lin, W.Y.; Chen, H.Y.; Zheng, W.; Wang, L.W. Studies on chemical constituents from herbs of Taraxacum mongolicum. Zhongguo Zhong Yao Za Zhi 2008, 33, 1147–1157. [Google Scholar] [PubMed]
  94. Yang, X.W.; Zhang, P.; Tao, H.Y.; Jiang, S.Y.; Zhou, Y. GC-MS Analysis of Essential Oil Constituents from Rhizome and Root of Notopterygium incisum. J. Chin. Pharm. Sci. 2006, 15, 172–176. [Google Scholar]
  95. Perret, D.; Gentili, A.; Marchese, S.; Serg, M.; Caporossi, L. Determination of free fatty acids in chocolate by liquid chromatography with tandem mass spectrometry. Rapid Commun. Mass Spectrom. 2004, 18, 1989–1994. [Google Scholar] [CrossRef] [PubMed]
  96. Liu, Y.Z.; Bing, Y.U.; Ding, G.; Wu, Y.J. Terminalic Acid, a New Tannin from the Fruit of Terminalia chebula. Chin. Chem. Lett. 1998, 9, 827–828. [Google Scholar]
  97. Chen, F.; Li, H.L.; Tan, Y.F.; Li, Y.H.; Lai, W.Y.; Guan, W.W.; Zhang, J.Q.; Zhao, Y.S.; Qin, Z.M. Identification of known chemicals and their metabolites from Alpinia oxyphylla, fruit extract in rat plasma using liquid chromatography/tandem mass spectrometry (LC–MS/MS) with selected reaction monitoring. J. Pharm. Biomed. Anal. 2014, 97, 166–177. [Google Scholar] [CrossRef] [PubMed]
  98. Zhang, Q.; Luo, S.; Wang, H.; Zhang, J.H. Study on the Chemical Constituents of Chinese Herbs, Zhiyiren. Chin. Tradit. Herb. Drugs 1997, 28, 131–133. [Google Scholar]
  99. Knothe, G.; Phoo, Z.W.; de Castro, M.E.; Razon, L.F. Fatty acid profile of Albizia lebbeck and Albizia saman seed oils: Presence of coronaric acid. Eur. J. Lipid Sci. Technol. 2015, 117, 567–574. [Google Scholar] [CrossRef]
  100. Igual, M.O.; Martucci, M.E.; Da Costa, F.B.; Gobbo-Neto, L. Sesquiterpene lactones, chlorogenic acids and flavonoids from leaves of Vernonia polyanthes, Less (Asteraceae). Biochem. Syst. Ecol. 2013, 51, 94–97. [Google Scholar] [CrossRef]
  101. Bankefors, J.; Nord, L.I.; Kenne, L. Multidimensional profiling of components in complex mixtures of natural products for metabolic analysis, proof of concept: Application to Quillaja saponins. J. Chromatogr. B 2010, 878, 471–476. [Google Scholar] [CrossRef] [PubMed]
  102. Bang, Y.H.; Lee, J.H.; Nam, J.B.; Hang, S.K.; Young, S.H.; Jung, J.L. Two New Furanoditerpenes from Saururus chinenesis and Their Effects on the Activation of Peroxisome Proliferator-Activated Receptor. J. Nat. Prod. 2002, 65, 616–617. [Google Scholar]
  103. Jalaliheravi, M.; Vosough, M. Characterization and determination of fatty acids in fish oil using gas chromatography-mass spectrometry coupled with chemometric resolution techniques. J. Chromatogr. 2004, 1024, 165–176. [Google Scholar] [CrossRef]
  104. Xiang, L.; Peng, G.; Gjetvaj, B.; Westcott, B.; Gruber, M.Y. Analysis of the metabolome and transcriptome of Brassica carinata seedlings after lithium chloride exposure. Plant Sci. 2009, 177, 68–80. [Google Scholar]
  105. Berkov, S.; Pavlov, A.; Georgiev, V.; Weber, J.; Bley, T.; Viladomat, F.; Bastida, J.; Codina, C. Changes in apolar metabolites during in vitro organogenesis of Pancratium maritimum. Plant. Physiol. Biochem. 2010, 48, 827–835. [Google Scholar] [CrossRef] [PubMed]
  106. Slivniak, R.; Domb, A.J. Macrolactones and polyesters from ricinoleic acid. Biomacromolecules 2005, 6, 1679–1688. [Google Scholar] [CrossRef] [PubMed]
  107. Kulyal, P. Section B: Organic Chemistry Including Medicinal Chemistry. Indian J. Chem. 2010, 49B, 356–359. [Google Scholar]
  108. Mei, H.N.; Choo, Y.M.; Ma, A.N. Separation of vitamin E (tocopherol, tocotrienol, and tocomonoenol) in palm oil. Lipids 2004, 39, 1031–1035. [Google Scholar]
  109. Ferreira, A.C.; Monforte, A.R.; Teixeira, C.S.; Martins, R.; Fairbairn, S.; Bauer, F.F. Monitoring alcoholic fermentation: An untargeted approach. J. Agric. Food Chem. 2014, 62, 6784–6793. [Google Scholar] [CrossRef] [PubMed]
  110. Zou, Z.J.; Liu, Z.H.; Gong, M.J.; Han, B.; Wang, S.M.; Liang, S.W. Intervention effects of puerarin on blood stasis in rats revealed by a 1H NMR-based metabonomic approach. Phytomedicine 2015, 22, 333–343. [Google Scholar] [CrossRef] [PubMed]
Sample Availability: Samples of the compounds 6-Methoxy-8-methyl coumarin and Sanlengdiphenyllactone are available from the authors.
Figure 1. The representative base peak intensity (BPI) chromatograms of HD and HC in positive mode (ESI+) and negative mode (ESI). (The character “,” represent the meaning of “and”).
Figure 1. The representative base peak intensity (BPI) chromatograms of HD and HC in positive mode (ESI+) and negative mode (ESI). (The character “,” represent the meaning of “and”).
Molecules 23 01525 g001
Figure 2. Chemical structures of compounds identified in HD and HC.
Figure 2. Chemical structures of compounds identified in HD and HC.
Molecules 23 01525 g002aMolecules 23 01525 g002b
Figure 3. The PCA of HC and HD in positive mode (ESI+) and negative mode (ESI). HD: Hedyotis diffuse Willd. HC: Hedyotis corymbosa (L.) Lam. QC: Quality Control.
Figure 3. The PCA of HC and HD in positive mode (ESI+) and negative mode (ESI). HD: Hedyotis diffuse Willd. HC: Hedyotis corymbosa (L.) Lam. QC: Quality Control.
Molecules 23 01525 g003
Figure 4. The OPLS-DA of HC and HD in positive mode (ESI+) and negative mode (ESI).
Figure 4. The OPLS-DA of HC and HD in positive mode (ESI+) and negative mode (ESI).
Molecules 23 01525 g004
Figure 5. The OPLS-DA/Coefficients vs. VIP of HC and HD in positive (ESI+) and negative mode (ESI).
Figure 5. The OPLS-DA/Coefficients vs. VIP of HC and HD in positive (ESI+) and negative mode (ESI).
Molecules 23 01525 g005
Figure 6. The OPLS-DA/S-Plot of HC and HD in positive mode (ESI+) and negative mode (ESI).
Figure 6. The OPLS-DA/S-Plot of HC and HD in positive mode (ESI+) and negative mode (ESI).
Molecules 23 01525 g006
Figure 7. Heatmap visualizing the intensities of potential biomarkers.
Figure 7. Heatmap visualizing the intensities of potential biomarkers.
Molecules 23 01525 g007
Table 1. The list of the tested samples from China.
Table 1. The list of the tested samples from China.
Sample No.SourceCollection Time
HC 1Guangzhou City, Guangdong Province, China; market15 September 2016
HC 226 August 2017
HC 3Haikou City, Hainan Province, China; market11 August 2017
HC 4Nanning City, Guangxi Province, China; field8 July 2016
HC 55 July 2017
HC 6Kunming City, Yunnan Province, China; market13 August 2016
HC 71 September 2017
HC 8Shenzhen City, Guangdong Province, China; cultivated28 September 2017
HC 9Luoding County, Guangdong Province, China; market24 August 2016
HC 1012 July 2017
HD 1Nanning City, Guangxi Province, China; field12 July 2016
HD 220 July 2016
HD 3Luoding County, Guangdong Province, China; market13 August 2016
HD 415 July 2017
HD 5Guangzhou City, Guangdong Province, China; market13 July 2017
HD 6Shenzhen City, Guangdong Province, China; cultivated21 September 2016
HD 721 August 2017
HD 8Kunming City, Yunnan Province, China; market8 August 2016
HD 9Fuzhou City, Fujian Province, China; field22 August 2017
HD 1013 September 2017
Table 2. Compounds identified from HD and HC by UPLC-QTOF-MSE.
Table 2. Compounds identified from HD and HC by UPLC-QTOF-MSE.
No.tR (min)FormulaCalculated Mass (Da)Theoretical Mass (Da)Mass Error (ppm)MSE FragmentationIdentificationSourceRef.
10.61C7H12O6192.0633192.0634−0.4191.0560 [M−H], 129.0190 [M−C2H2O2], 127.0407 [M−OH−COOH]Quinic acidHC, HDs
20.69C4H6O5134.0216134.02150.9133.0144 [M−H], 115.0037 [M−OH], 71.0150 [M-OH-COOH]2-hydroxy-succinic acidHC, HD[35]
30.75C6H8O7192.0270192.02700.0191.0197 [M−H], 173.0085 [M−OH], 117.0193 [M−OH−CH2COOH], 111.0089 [M−2 × OH−COOH], 101.0247 [M−2 × COOH], 89.0250 [M−CH2COOH−COOH]Citric acidHC, HDs
40.80C9H10O4182.0582182.05791.2227.0564 [M+HCOO], 165.0558 [M-OH], 153.0555 [M-CHO], 137.0611 [M−OH−CHO], 125.0244 [M−2CH3−CHO]SyringaldehydeHC, HDa
50.93C19H19N3O305.1505305.15287328.1397 [M+Na]+, 132.0812 [M−C10H9N2O]+, 117.0626 [M−C11H12N2O]+, 107.0503 [M−C11H10N2-NCH3]+Wuchuyuamide IHC, HD[36]
60.94C16H20O10372.1051372.1057−1.4371.0978 [M−H], 315.0723 [M−C3HO], 167.0712 [M−Glu−COOH], 153.0192 [M−Glu−C3HO], 123.0451 [M−Glu−C3HO3]Deacetyl asperulosideHC, HD[37]
71.11C22H18O10442.0866442.0900−7.8443.0938 [M+H]+, 319.0772 [M−C6H5O3]+, 145.0255 [M−C13H10O7]+(+)-Epicatechol 3-gallateHC, HD[38]
81.20C17H24O11404.1318404.1319−0.2449.1300 [M+HCOO], 353.0872 [M−OH−OCH3], 247.1184 [M−OH−C6H5O3], 241.0720 [M−Glu], 211.0610 [M−OCH3−Glu]Scandoside methyl esterHC, HDs
91.34C11H10O5222.0519222.0528−4.0223.0592 [M+H]+, 209.0418 [M−CH3]+, 191.0318 [M−OH−CH3]+, 181.0501 [M−C2H3O]+, 179.0680 [M−COOH]+, 163.0364 [M−OH−C2H3O]+4-O-acetyl-caffeic acidHC, HD[39]
101.34C10H10O4194.0568194.0579−5.8195.0641 [M+H]+, 181.0501 [M−CH3]+, 179.0680 [M−OH]+, 163.0364 [M-CH3−OH]+, 149.0581 [M−COOH]+, 145.0256 [M−OH−OCH3]+3-Hydroxy-4-methoxycinnamic acidHC, HD[40]
111.44C16H22O10374.1207374.1213−0.6419.1189 [M+HCOO], 357.1190 [M−OH], 343.0975 [M−CH2OH], 313.0909 [M−2 × CH2OH], 257.0671 [M−C5H4O3]Geniposidic acid HC, HDs
121.45C8H8O3152.0490152.04739.2175.0382 [M+Na]+, 136.0598 [M−OH]+, 119.0494 [M−2 × OH]+, 91.0561 [M−OH−COOH]+4-Hydroxybenzeneacetic acidHC, HD[41]
131.46C10H14O5214.0840214.0841−0.4213.0768 [M−H], 195.0657 [M−OH], 181.0498 [M−CH2OH], 177.0554 [M−2 × OH], 163.0395 [M−OH−CH2OH], 151.0397 [M−C2H5O2], 149.0593 [M−3 × OH−CH3]Guaiacyl glycerolHC, HD[42]
141.56C18H24O12432.1260432.1268−1.8431.1187 [M−H], 269.0663 [M−Glu], 165.0552 [M−Glu−OH−C3H5O2]Asperulosidic acidHC, HD[43]
151.83C16H18O9354.0939354.0951−3.4355.1011 [M+H]+, 163.0383 [M−quinic acid]+, 145.0264 [M−quinic acid−OH]+Chlorogenic acidHC, HDs
161.90C7H12O6192.0632192.0634−0.7191.0632 [M−H], 173.0445 [M−OH], 137.0239 [M−2 × OH], 121.0291 [M−4 × OH]1,3,4,5-Tetrahydroxycyclohexanecarboxylic acidHC, HDa
172.02C33H40O21772.2067772.20620.6817.2049 [M+HCOO], 609.1443 [M−Glu]Kaempferol-3-O-sophoroside-7-O-β-d-glucopyranosideHC, HD[44]
182.09C16H22O11390.1152390.1162−2.7389.1079 [M−H], 209.0454 [M−Glu], 165.0549 [M−Glu−OH−CH2OH], 121.0658 [M−Glu−OH−CH2OH−COOH]ScandosideHC, HDs
19 #2.39C20H30O13478.1677478.1686−1.8523.1659 [M+HCOO], 293.0873 [M−C9H11O4], 151.0395 [M−furanosyl−Glu]3,4,5-Trimethoxyphenyl−6-O-d-apio-β-d-furanosyl-β-d-glucopyranosideHC, HD
(HC>>HD)
[45]
202.50C18H22O11414.1157414.1162−1.0459.1139 [M+HCOO], 367.1029 [M−OH−CH2OH], 251.0555 [M−Glu], 191.0352 [M−Glu−CH3COOH], 177.0190 [M−Glu−CH2COOCH3]AsperulosideHC, HD[46]
212.65C9H8O4180.0423180.04230.2179.0350 [M−H], 165.0192 [M−CH3], 135.0451 [M−COOH](4-Methoxyphenyl)-oxoacetic acidHC, HD[47]
22 #3.07C15H20O8282.1108282.11031.3327.1090 [M−H], 165.0556, 147.0452 [M−Glu], 121.0294 [M−Glu−CH3−CO]AndrosinHC, HD
(HC>>HD)
[48]
23 #3.27C17H24O10388.1370388.13700.1433.1352 [M+HCOO], 355 [M−OCH3], 353.0876 [M−OH−CH3], 337.0932 [M−OH−OCH3], 225.0770 [M−Glu], 193.0506 [M−Glu−OCH3]Geniposide HC, HD
(HC>>HD)
s
243.82C14H17NO6295.1052295.1056−1.2340.1034 [M+HCOO], 167.0346 [M−N−C6H5−2OH], 166.0508 [M−3 × OH−C6H5]Prunasin HC, HD[49]
254.01C15H10O7302.0414302.0426−4.3303.0486 [M+H]+, 153.0171 [M−C8H6O3]+, 127.0389 [M−C9H6O4]+Moric acidHC, HDa
264.12C27H30O17626.1493626.14831.6625.1420 [M−H], 609.1424 [M−OH], 595.1373 [M−CH2OH], 400.0883 [M−OH−CH2OH−Glu], 300.0282 [M−Glu]Quercetin−3-sophorosideHC, HD[50]
274.14C43H48O25964.2508964.24852.4963.2435 [M−H], 903.2227 [M−CH2OH], 757.1849 [M−C11H11O4], 625.1419 [M−C11H11O4−Glu]Quercetin-3-O-(6-O-feruloyl-β-d-glucopyranosyl)-(1→2)-β-d-galactopyranosyl-(1→2)-β-d-glucopyranosideHC, HDa
284.15C15H18O8326.1002326.10020.0371.0984 [M+HCOO], 163.0403 [M−Glu], 119.0504 [M−Glu−COOH]trans-p-Coumaric acid-4-O-glucosideHC, HD[51]
294.19C19H26O12446.1421446.1424−0.7491.1403 [M+HCOO], 371.0986 [M−OCH3−C2H3O], 283.0824 [M−Glu], 163.0403 [M−Glu−OCH3−C3H5O2], 119.0504 [M−Glu−OH−C5H8O4]DaphyllosideHC, HD[52]
30 #4.24C21H20O11448.1000448.1006−1.3449.1072 [M+H]+,415.1006 [M−2 × OH]+, 397.0920 [M−3 × OH]+, 287.0490 [M−Glu]+, 137.0587 [M−Glu−C7H3O3]+Luteolin 7-O-β-d-glucopyranosideHC, HD
(HC>>HD)
s
314.39C26H28O14564.1486564.14791.3563.1414 [M−H], 403.1260 [M−OH−C9H6O2], 275.0578 [M−OH−CH2OH−C6H5O−apiofuranosyl]ApiinHC, HD[53]
324.48C9H10O3166.0631166.06300.7165.0558 [M−H], 147.0451 [M-OH], 119.0501 [M−COOH], 103.0556 [M−OH−COOH]Phloretic acidHC, HD[54]
334.59C10H8O4192.0413192.0423−4.8193.0486 [M+H]+, 178.0247 [M−CH3]+, 122.0350 [M−C3H2O2]+ScopoletinHC, HD[55]
344.75C11H16O3196.1097196.1099−0.2197.117 [M+H]+, 179.1057 [M−OH]+, 167.0688 [M−2× CH3]+, 147.0436 [M−2 × CH3-OH]+LoliolideHC, HD[56]
35 #4.96C26H28O16596.1375596.1377−0.4595.1302 [M−H], 300.0280 [M−Glu−Xyl]Isoetin-7-O-β-d-glucopyranosyl-2′-O-β-d-xyloypyranosideHC, HD
(HC>>HD)
[57]
364.98C15H12O7304.0573304.0583−2.8349.0555 [M+HCOO], 195.0294 [M−C6H5O2], 179.0323 [M−OH−C6H5O2], 151.0036 [M−C8H7O3]DihydroquercetinHC, HD[58]
375.04C15H10O7302.0424302.0427−0.3303.0496 [M+H]+, 287.0541, 127.0395 [M−C9H6O4]+5,7,8,3′,4′-pentamethoxy FlavonoidsHC, HD[59]
385.04C27H30O16610.1538610.15340.7611.1611 [M+H]+, 465.1016 [M−Rha]+, 303.0493 [M−Glu−Rha]+RutinHC, HDs
395.34C21H20O12464.0948464.0955−1.4463.0876 [M−H], 301.0353 [M−Glu]Quercetin-3-O-glucopyranosideHC, HD[60]
40 #5.34C22H22O10446.1212446.1213−0.2447.1285 [M+H]+, 429.1118 [M−OH]+, 175.0383 [M−Glu−C6H3O]+, 163.0388 [M−Glu−C6H5−OCH3]+, 131.0489 [M−Glu−C7H3O3]+Acacetin 7-O-β-d-glucopyranosideHC, HD
(HC>>HD)
[61]
41 *5.71C37H38O19786.2011786.20070.5831.1993 [M+HCOO], 565.1556 [M−CH2OH−C10H9O3], 379.0657 [M−Glu−CH2OH−C6H5O−C7H7O2]Allivictoside FHC, HD
(HD>>HC)
[62]
42 #5.75C20H18O11434.0848434.0849−0.2433.0775 [M−H], 300.0280 [M−Ara], 163.0401 [M−Ara−C6H4O3], 147.0450 [M−H−Ara−C6H4O3−OH]Quercetin-3-O-β-ArabinopyranoseHC[63]
435.79C27H30O15594.1588594.15850.6593.1515 [M−H], 285.0403 [M−Glu−Rha]Kaempferol 3-glucoside-7-rhamnosideHC, HD[64]
44 *5.89C28H32O16624.1682624.1690−1.3625.1755 [M+H]+, 501.1583 [M−C6H4O2]+, 479.1155 [M−Rha]+, 465.0997 [M−Rha−CH3]+, 317.0637 [M−Rha−Glu]+Isorhamnetin-3-rutinosideHC, HD
(HD>>HC)
[65]
45 #5.93C20H12O8380.0560380.05326.5425.0542 [M+HCOO],163.0399 [M−CO−C11H5O5]Phelligrindins d-9HC, HD
(HC>>HD)
[66]
46 *6.10C26H32O11520.1941520.1945−0.6565.1923 [M+HCOO], 501.1766 [M−OH], 489.1748 [M−CH2OH], 339.1233 [M−Glu]Matairesinol monoglucosideHC, HD
(HD>>HC)
[67]
47 #6.17C36H36O19772.1864772.18511.7771.1791 [M−H], 565.1548 [M−p-Hydroxy-cinnamic acid−CH2OH]Allivictoside GHC[62]
486.38C8H14O2187.1049187.09770.4187.0977 [M−H], 169.0871 [M−OH], 125.0973 [M−OH−COOH], 97.0663 [M−OH−C3H5O2]Azelaic acidHC, HD[68]
49 *6.52C27H32O15596.1749596.17411.3595.1676 [M−H], 549.1621 [M−OH−CH2OH], 387.1073 [M−OH−CH2OH−Rha], 369.0977 [M−2 × OH−CH2OH−Rha], 163.0400 [M−C18H24O12]NeoeriocitrinHC, HD
(HD>>HC)
[69]
50 *6.82C27H32O14580.1808580.17922.5625.1790 [M+HCOO], 529.1359 [M−OH−OCH3], 517.1356 [M−OCH3−CH2OH], 417.1204 [M−Glu], 193.0510 [M−C17H23O10], 147.0449 [M−OCH3−Glu−C10H9O4]6-O-Z-p-feruloyl scandoside methyl esterHD[70]
516.89C26H30O13550.1683550.1686−0.6595.1665 [M+HCOO], 433.14811 [M−OH−C4H4O3], 403.13121 [M−C9H7O2], 387.1093 [M−Glu], 355.0823 [M−Glu−OCH3]10-O-E-p-courmaroyl scandoside methyl esterHC, HD[71]
52 *7.07C26H30O13550.1683550.1686−0.6549.1610 [M−H], 595.1663 [M+HCOO], 387.1086 [M−Glu], 370.0789 [M−Glu−CH3], 193.0503 [M−Glu−OCH3−C9H7O3]6-O-p-coumaroyl scandoside methyl esterHC, HD
(HD>>HC)
[16]
537.13C27H32O13564.1843564.18430609.1825 [M+HCOO], 549.1613 [M−CH3], 387.1086 [M−CH3−Glu], 387.1086 [M−CH3−C10H9O2], 370.0789 [M−2 × CH3−C10H9O2], 337.1070 [M−OCH3−OH−Glu]6-O-(E)-p-coumaroyl scandoside methyl ester-10-methyl esterHC, HD[72]
54 *7.18C27H32O14580.1800580.17921.3579.1727 [M−H], 399.1051 [M−Glu], 223.0604 [M−Glu−C10H12O4]Nobiletin-3-O-β-d-glucosideHD[73]
55 #7.58C24H28O12508.1581508.15810.1553.1563 [M+HCOO], 345.0977 [M−Glu], 223.0602 [M−Glu−C7H4O2]+Hedycoryside BHC[74]
567.88C15H10O7302.0438302.04263.9303.0511 [M+H]+, 287.0549 [M−OH]+, 153.0181 [M−C8H6O3]+, 152.0565 [M−C7H4O4]+QuercetinHC, HDs
57 *7.91C15H10O6286.0489286.0477−3.7287.0540 [M+H]+,163.0361 [M−C6H4O3]+,149.0589 [M−C6H4O3−OH]+, 131.0487 [M−C6H4O3−2 × OH]+KaempferolHC, HD
(HD>>HC)
s
587.94C23H26O11478.1471478.1475−0.9477.1398 [M−H], 355.1035 [M−benzoic acid], 315.0879 [M−Glu], 285.0406 [M−Glu−C2H3], 241.1076 [M−OH−benzoic acid−C3H2O3]Hedycoryside CHC, HD[13]
598.12C24H28O12508.1585508.15810.7553.1567 [M+HCOO], 345.0976 [M−Glu], 207.0655 [M−Glu−benzoic acid], 137.0245 [M−Glu−benzoic acid−C4H4O2]10-O-benzoyl scandoside methyl esterHC, HD[43]
608.88C14H12O4244.0738244.07360.9245.0811 [M+H]+, 227.0693 [M−OH]+, 135.0429 [M−C6H3O2]+, 119.0493 [M−C6H3O2−OH]+, 95.0512 [M−C8H5O2]+PiceatannolHC, HD[75]
619.26C17H14O7330.0750330.07403.2331.0823 [M+H]+, 315.0485 [M−CH3]+, 301.0679 [M−OCH3]+, 207.0647 [M−OH−C6H5O2]+5,3′,4′-Trihydroxy-6,7-dimethoxy flavonoidsHC, HD[76]
62 *9.23C28H34O15610.1909610.18981.8609.1836 [M−H], 401.1232 [M−OH−OCH3−Rha], 193.0513 [M−2 × Rha−C6H3O], 177.0557 [M−2 × Rha−C6H3O2]HesperidinHC, HD
(HD>>HC)
s
639.62C20H30O5350.2078350.2093−4.2351.2151 [M+H]+, 293.2123 [M−C2H4O2]+, 275.1999 [M−C2H4O2−OH]+, 257.1917 [M−C2H4O2−OH−OH]+, 105.0713 [M−C6H6O3−C6H12O2]+14-AndrographolideHC, HD[77]
649.62C16H28O2252.2113252.20898.7275.2006 [M+Na]+, 195.1389 [M−C4H8]+, 155.1050 [M−C7H14]+, 151.1110 [M−C6H12O]+7-Hexadecenoic acid-16-hydroxy-O-lactoneHC, HDa
659.98C11H10O3190.0625190.0630−2.8191.0697 [M+H]+, 177.0533 [M−CH3]+, 159.0427 [M−CH3−OH]+, 105.0348 [M−C6H5O]+6-Methoxy-8-methyl coumarinHC, HDs
66 #10.00C24H28O11492.1634492.1632−0.4537.1616 [M+HCOO], 329.1028 [M−Glu], 207.0622 [M−Glu−C7H5O], 195.0664 [M−Glu−OCH3−C7H5O], 163.0397 [M−Glu−OCH3−C8H7O2]Hedycoryside AHC, HD
(HC>>HD)
[13]
6710.08C11H16O2180.1145180.1150−2.9181.1218 [M+H]+, 163.1114 [M−O]+, 121.1022 [M−C2HO2]+5,6,7,7α-Tetrahydro-4,4,7α-trimethyl-2(4H)-benzofuranoneHC, HD[77]
6810.31C15H10O4254.0589254.05793.7255.0661 [M+H]+, 240.0411 [M−CH3]+, 224.0466 [M−OCH3]+Alizarin 1-methyl etherHC, HDs
69 *10.64C15H10O4254.0579254.05790253.0506 [M−H], 224.0477 [M−CH2OH]1,3-Dihydroxy-2-methylanthraquinoneHD[78]
70 *11.03C15H8O4252.0424252.04230.7251.0352 [M−H], 223.0399 [M−O−CH], 207.0449 [M−COO]SanlengdiphenyllactoneHC, HD
(HD>>HC)
s
71 #11.65C21H22O8402.1311402.1315−0.8403.1384 [M+H]+, 387.1084 [M−CH3]+, 373.0905 [M−2 × CH3]+, 359.1092 [M−CH3−OCH3]+Chuan NecteinHC[79]
7211.82C15H16O4260.1065260.10496.3261.1138 [M+H]+, 205.0499 [M-C4H7]+, 190.0262 [M-C5H9]+, 177.0543 [M-C5H9O]+, 162.0316 [M-OCH3-C5H9]+5-Prenyloxy-7-methoxycoumarinHC, HDa
7311.85C20H26O4330.1805330.1831−7.8331.1878 [M+H]+, 149.0953 [M-OH-C10H13O2]+, 131.0489 [M-OH-CH3-C10H13O2]+, 135.0803 [M-OCH3-C10H12O2]+, 121.0646 [M-OCH3-C12H17O2]+Dihydroguaiac acidHC, HD[80]
7411.87C15H14O4258.0889258.0892−1.3259.0962 [M+H]+, 244.0707 [M-CH3]+, 229.0480 [M-2 × CH3]+, 227.0684 [M-OCH3]+, 217.0474 [M-C3H5]+, 212.0444 [M-CH3-OCH3]+Hedyotiscone AHC, HD[81]
75 #12.11C19H18O6342.1103342.1103−0.2343.1175 [M+H]+, 327.08434 [M−CH3]+, 313.06864 [M−2 × CH3]+, 299.08954 [M−CH3−OCH3]+, 285.07454 [M−2 × CH3−OCH3]+5,6,7,4′-TetramethoxyflavoneHCs
7612.11C16H28O3268.2056268.20386.2291.1949 [M+Na]+, 217.1566 [M−CH3−OH]+, 132.0863 [M−OH−C2H5−C4H7O2]+13-Hydroxy-9,11-Hexadecandienoic acidHC, HDb
77 *12.41C16H12O4286.0731268.0736−0.4269.0804 [M+H]+, 254.0557 [M−CH3]+, 251.06537 [M−OH]+, 239.0689 [M−OCH3]+, 225.0540 [M−OCH3−CH3]+Methylisotropine-1-methyletherHC, HD
(HD>>HC)
a
78 *12.44C15H10O3238.0630238.06300.2237.0558 [M−H], 224.0471 [M−CH3], 208.0518 [M−OH−CH3]2-Hydroxy-3-methylanthraquinoneHC, HD
(HD>>HC)
[82]
79 *12.49C15H10O5270.0524270.0528−1.6269.0451 [M−H], 237.0555 [M−2 × OH]5-Dehydroxykaempferol HC, HD
(HD>>HC)
[83]
8012.51C17H24O3276.1730276.17251.8277.1803 [M+H]+, 259.1608 [M−OH]+, 231.1774 [M−CH3−2 × OH]+, 213.1633 [M−CH3−3 × OH]+, 203.1776 [M−3 × OH−C2H3]+, 201.1612 [M−3 × OH−C2H5]+(10E)1,10-Heptadeca-diene-4,6-diyne-3,8,9-triolHC, HD[84]
81 #12.74C22H24O9432.1411432.1420−2.0433.1484 [M+H]+, 418.1231 [M−CH3]+, 403.0998 [M−2 × CH3]+, 388.0763 [M−3 × CH3]+, 385.0857 [M−CH3−OCH3]+, 372.1131 [M−2 × OCH3]+, 357.0934 [M−CH3−2 × OCH3]+3′,4′,5′,5,6,7,8-Seven-methoxyflavoneHC[85]
8212.80C17H24O2260.1774260.1776−0.8305.1756 [M+HCOO], 135.0813 [M−C3H7−C5H5O], 125.0969 [M−C2H5−C7H5O], 121.0656 [M−C4H9−C5H5O]FakalinediolHC, HD[86]
8313.35C30H48O5488.3497488.3502−0.9533.3479 [M+HCOO], 291.1956 [M−C12H20O2], 195.1029 [M−C19H29O2], 171.1025 [M−C21H33O2]3β,19α,23-Trihydroxyurs-12-en-28-oic acidHC, HD[87]
84 *13.36C17H14O6314.0793314.07900.8315.0866 [M+H]+, 300.0618 [M−CH3]+, 282.04958 [M−OCH3]+, 111.04458 [M−CH3−C10H6O4]+5,3′-Dihydroxy-7,4′-dimethoxyflavoneHD[88]
8513.96C27H28N2O4444.2060444.20492.5445.2133 [M+H]+, 385.1887 [M−C2H3O2]+, 224.1062 [M−C12H13NO3]+, 194.1172 [M−C16H13NO2]+, 134.0970 [M−C2H3O2−C16H13NO2]+Gold Amide Alcohol EsterHC, HD[89]
8614.44C17H32O2268.2398268.2402−1.4313.2380 [M+HCOO], 251.2019 [M−CH3], 183.1388 [M−C6H13], 129.0918 [M−C10H17]Methyl cis-9-hexadecenoateHC, HDa
8714.81C18H34O4314.2460314.24570.9337.2352 [M+Na]+, 139.1118 [M−C9H18O3]+, 125.09614 [M−C10H20O3]+Dibutyl sebacateHC, HDa
8814.81C18H30O2278.2244278.2246−0.8279.2316 [M+H]+, 249.1834 [M−C2H5]+, 217.1935 [M−CH3−COO]+, 191.1801 [M−C4H6O2]+, 163.1483 [M−C6H10O2]+9,12,15-Octadecatrienoic acidHC, HD[90]
89 #15.25C26H32O6440.2193440.2199−1.4441.2266 [M+H]+, 389.2315 [M−C3H2O]+, 340.1657 [M−C3H2O−C2H3O]+, 147.0437 [M−C17H25O4]+IsofeterinHC[91]
90 #15.25C20H28O4332.2016332.19887.9355.1908 [M+Na]+, 241.1946 [M−OH−CH2OH−COO]+, 217.1189 [M−OH−CH2OH−CH3−C4H8]+, 161.1320 [M−OH−CH2OH−CH3−C6H5O2]+14-Deoxy-11,12-dihydroandrographolideHC[92]
91 #15.69C15H22O218.1659218.1671−5.5219.1731 [M+H]+, 163.1106 [M−C4H7]+, 161.0935 [M−CH3−C3H6]+α-TurmeroneHCa
92 #15.87C15H28O2240.2090240.2089 285.2072 [M+HCOO], 223.2068 [M−OH]Isodonsesquitin AHC, HD
(HC>>HD)
[93]
9316.02C16H30O2254.2251254.22461.8277.2143 [M+Na]+, 137.1316 [M−C4H9−CH2COOH]+, 109.1012 [M−C4H9−C3H6COOH]+Z-11-Hexadecenoic acidHC, HD[94]
9416.02C20H28O3316.2025316.2038−4.1317.2098 [M+H]+, 289.1787 [M−C2H4]+, 277.2151 [M−C2H2O]+, 251.1930 [M−C4H4O]+, 235.1667 [M−C5H7O]+, 221.1503 [M−CH3−C5H7O]+7β-Senecioyloxyoplopa-3(14)Z,8(10)-dien-2-oneHC, HDa
9516.03C34H58O4530.4316530.4335−3.4553.4208 [M+Na]+, 483.3400 [M−3CH3]+, 317.2060 [M−OCH3−C13H27]+, 315.1595 [M−2 × CH3−C13H27]+, 313.1703 [M−OH−CH3−C13H27]+Ferulic acid esters lignocericHC, HDa
9616.23C16H30O2254.2258254.22464.6277.2151 [M+Na]+, 137.1329 [M−C2H5−C4H6O2]+, 123.1168 [M−C2H5−C5H8O2]+, 111.1171 [M−C8H14O2]+Palmitoleic acidHC, HD[95]
9716.23C20H28O3316.2021316.2038−5.5317.2094 [M+H]+, 301.2068 [M−OH]+, 277.2147 [M−C2H2O]+, 259.2029 [M−CH3−COOH]+, 215.1763 [M−C2H2O−COOH]+, 141.0911 [M−C11H15]+Terminalic acid HC, HD[96]
9816.61C20H26O3314.1859314.1882−7.2315.1932 [M+H]+, 159.1158 [M−OH−C8H9O2]+, 133.1005 [M−C10H13O3]+, 147.1165 [M−OH−C9H11O2]+OxyphyllacinolHC, HD[97]
9916.91C20H26O3314.1854314.1882−8.9315.1927 [M+H]+, 191.1040 [M−OH−C8H9]+, 173.1307 [M−OH−C7H7O2]+, 135.0799 [M−OH−OCH3−C10H13]+NeonootkatolHC, HD[98]
10017.08C17H30O2266.2646266.26460.1311.2228 [M+HCOO], 183.1387 [M−C6H12], 249.2224 [M−OH]7,10-Dienylhexadecanoic acid methyl esterHC, HDa
10117.37C18H32O3296.2355296.23511.3295.2283 [M−H], 277.2176 [M−OH], 233.2262 [M−O−COOH], 183.1024 [M−CH3−5×CH2−2×CH],125.0968 [M−OH−C10H17O], 123.1180 [M−O−CH2COOH−C7H13]Coronaric acidHC, HD[99]
10217.37C18H32O3296.2355296.23511.3295.2283 [M−H], 277.2176 [M−OH], 233.2262 [M−O−COOH], 125.0968 [M−C10H17O2], 123.1180 [M−COOH−C8H15O]Vernonia acidHC, HD[100]
10317.39C30H46O4470.3398470.33960.3471.347 [M+H]+, 455.3448 [M−OH]+, 437.3382 [M−2 × OH]+, 425.3421 [M−COO]+, 420.2712 [M−2 × CH3−OH]+, 409.3449 [M−OH−COO]+, 383.3309 [M−CH3−CO−COO]+CaryophyllosideHC, HD[101]
10417.87C20H28O3316.2019316.2038−6.2317.2092 [M+H]+, 235.1672 [M−C5H6O]+, 189.1622 [M−C5H6O−COOH]+, 179.1418 [M−OH−CH3−C7H8O]+Saurufuran BHC, HD[102]
10517.87C18H28O2276.2088276.2089−0.3277.2161 [M+H]+, 235.1672 [M−C3H6]+, 217.1967 [M−CH2COOH]+, 207.1729 [M−OH−C3H6]+, 189.1623 [M−C3H6COOH]+Stearidonic acidHC, HD[103]
10615.99C18H30O3294.2197294.21950.7293.2124 [M−H], 275.2016 [M−OH], 211.1340 [M−C6H12], 185.1180 [M−C8H14], 182.1305 [M−OH−C7H13](E,E)-9-Oxooctadeca-10,12-dienoic acidHC, HD[104]
10718.61C20H28O3316.2021316.2038−7.0317.2089 [M+H]+, 283.1680 [M−OH−CH3]+, 259.2034 [M−CH3−COOH]+, 235.1680 [M−C5H5O]+Saurufuran AHC, HD[103]
10818.61C16H30O2254.2270254.22468.7277.2162 [M+Na]+, 179.1405 [M−OH−C4H9]+, 165.1260 [M−OH−C5H11]+, 151.1111 [M−OH−C6H13]+, 125.0963 [M−OH−C8H15]+Hexadecenoic acidHC, HD[105]
10918.68C18H34O3298.2511298.25081.1297.2438 [M−H], 279.2332 [M−OH], 155.1076 [M−C9H18O]Ricinolic acidHC, HD[106]
11019.51C20H30O3318.2174318.2195−6.6319.2247 [M+H]+, 239.1776 [M−COOH−CH2OH]+, 233.193 [M−C4H3O2]+, 189.1630 [M−OH−C6H7O2]+AndrograpaninHC, HD[107]
11121.64C30H48O3456.3579456.3604−4.8501.3561 [M+HCOO], 340.2808 [M−2 × OH−C6H12], 277.2159 [M−C12H20O], 223.2062 [M−COOH−C14H19]Ursolic acidHC, HDs
11221.72C28H48O2416.3678416.36545.3439.357 [M+Na]+, 342.3004 [M−OH−C4H9]+, 327.2377 [M−2CH3−C4H9]+, 277.2119 [M−C10H21]+, 249.1820 [M−CH3−C11H23]+γ-TocopherolHC, HD[108]
11323.33C19H38O4330.2776330.27701.6353.2668 [M + Na]+, 313.2733 [M−OH]+, 283.2593 [M−2 × OH−CH3]+, 269.2161 [M−OH−C3H7]+, 239.2376 [M−C3H5O3]+PalmitinHC, HDa
* Characteritic component in HD; # Characteritic component in HC; s: Identified with reference substance. a: Compared with spectral data obtained from Wiley Subscription Services, Inc. (USA); b: Compared with NIST Chemistry WebBook; HD: Hedyotis diffuse Willd.; HC: Hedyotis corymbosa (L.) Lam.

Share and Cite

MDPI and ACS Style

Wang, Y.; Wang, C.; Lin, H.; Liu, Y.; Li, Y.; Zhao, Y.; Li, P.; Liu, J. Discovery of the Potential Biomarkers for Discrimination between Hedyotis diffusa and Hedyotis corymbosa by UPLC-QTOF/MS Metabolome Analysis. Molecules 2018, 23, 1525. https://doi.org/10.3390/molecules23071525

AMA Style

Wang Y, Wang C, Lin H, Liu Y, Li Y, Zhao Y, Li P, Liu J. Discovery of the Potential Biomarkers for Discrimination between Hedyotis diffusa and Hedyotis corymbosa by UPLC-QTOF/MS Metabolome Analysis. Molecules. 2018; 23(7):1525. https://doi.org/10.3390/molecules23071525

Chicago/Turabian Style

Wang, Yaru, Cuizhu Wang, Hongqiang Lin, Yunhe Liu, Yameng Li, Yan Zhao, Pingya Li, and Jinping Liu. 2018. "Discovery of the Potential Biomarkers for Discrimination between Hedyotis diffusa and Hedyotis corymbosa by UPLC-QTOF/MS Metabolome Analysis" Molecules 23, no. 7: 1525. https://doi.org/10.3390/molecules23071525

Article Metrics

Back to TopTop