Simultaneous Determination of 11 High-Polarity Components from Fructus Corni: A Quantitative LC–MS/MS Method for Improved Quality Control

Simultaneous Determination of 11 High-Polarity Components from Fructus Corni: A Quantitative... Abstract Fructus Corni, the dried ripe sarcocarp of Cornus officinalis Sieb.et Zucc (Cornaceae), is widely used in traditional medicine. Pharmacological studies to date have attributed many biological effects to the high-polarity components. However, current quality control methods focus on only several iridoid glycoside components, and, of note, there is no comprehensive method available to simultaneously quantify the high-polarity components in Fructus Corni. Here, a simple, sensitive and robust liquid chromatography-electrospray ionization-tandem mass spectrometry method was developed to simultaneously determine 11 high-polarity constituents (5-hydroxymethyl-2-furfural, gallic acid, sweroside, cornin, loganin, morroniside, 7α-O-methylmorroniside, 7β-O-methylmorroniside, 7α-O-ethylmorroniside and 7β-O-ethylmorroniside, cornuside) of Fructus Corni. This method showed good specificity, linearity (r2 ≥ 0.9907), repeatability (RSD < 5.98%) and recovery (93.24 ~ 112.92%, RSD < 9.06%). This validated method was successfully employed to assess the component variation of crude Fructus Corni of three regional origins as well as after processing. In particular, the iridoid isomers were, for the first time, included as the quality markers for Fructus Corni. We propose that this method may provide a new and powerful tool for achieving comprehensive quality control of Fructus Corni. Introduction Fructus Corni, a well-known traditional Chinese medicine widely used in China for thousands of years, is derived from the dried ripe sarcocarp of Cornus officinalis Sieb.et Zucc (Cornaceae). Due to its biological and pharmacological activities such as anti-inflammation, anti-diabetes and anti-oxidation (1–4), Fructus Corni has received intensive attention as one of the most popular and cherished clinical herbal medicines in the world (1, 5–7). Chemical constituents from Fructus Corni that have been reported mainly include iridoids, organic acids and carbohydrates (8–10). To date, the pharmacological activity of high-polarity components such as the iridoid glycosides from Fructus Corni has received huge attention. For example, loganin has been shown to exhibit immune regulation, anti-inflammatory and inhibition of aldose reductase (11); morroniside was reported to contribute to the effect of Fructus Corni in protecting neurons and preventing diabetic angiopathies (12–16); and 7-O-methylmorroniside, a methylation product of morroniside, was also proposed as a potential bioactive iridoid constituent, which was shown to possess comparable anti-inflammatory effects as morroniside (17–18). Given the high medicinal value of Fructus Corni, a reliable, sensitive and comprehensive analytical method is indispensible for the quality control. To date, only a few analytical methods have been reported for the determination of several active components in crude or processed Fructus Corni (10, 19–23). However, it is notable that, current methods largely fail to focus on the high-polarity components in a comprehensive manner. Moreover, although several isomeric iridoids, such as 7-O-ethylmorroniside and 7-O-methylmorroniside, are reported to exist in Fructus Corni, they are not covered in these conventional methods (19, 22, 24–27). These limitations highlight the need to efficiently separate and determine these newly emerging active components for achieving a comprehensive quality assessment of the Fructus Corni. Therefore, in this study, we aimed to improve the current method for the quality control of Fructus Corni, focusing on developing a high-throughput and time-efficient detection and quantification of the major high-polarity components from Fructus Corni. By employing the high sensitivity and wide coverage of liquid chromatography tandem mass spectrometry (LC–MS/MS), we successfully developed a simple and robust LC–MS/MS method to simultaneously identify and quantify 11 bioactive constituents in Fructus Corni. All the target components, including two pairs of iridoid isomers, could be well separated and detected within 18 min. This method was then validated and successfully applied to examine the component changes of several batches of crude Fructus Corni from three different origins as well as after procession. The results suggested that the present method could provide a new and feasible method that can be employed for improving the quality control of Fructus Corni. Experimental Chemicals, reagents and materials Gallic acid, loganin and 5-hydroxymethyl-2-furfural (5-HMF) were purchased from the National Institute for Food and Drug Control (Beijing, China). Morroniside, cornuside, cornin and sweroside were obtained from Nanjing Ningqi Biotechnology Co. Ltd. (Nanjing, China). The purity for each reference compound was greater than 98% as validated by HPLC analysis. 7α-O-methylmorroniside, 7β-O-methylmorroniside and 7α-O-ethylmorroniside, 7β-O-ethylmorroniside were prepared by our lab, and structurally characterized based on 1H-NMR, 13C-NMR spectra. Their contents were 99.7% and 99.6% as determined by HPLC analysis, respectively. The structures of these 11 compounds are shown in Figure 1. Acetonitrile used was of chromatographical grade and purchased from Merck (Darmstadt, Germany). A Milli-Q water (Millipore Inc. USA) purification system was used to obtain purified water for the HPLC analysis. Figure 1. View largeDownload slide The chemical structures of the 11 bioactive compounds in Fructus Corni. Figure 1. View largeDownload slide The chemical structures of the 11 bioactive compounds in Fructus Corni. Crude Fructus Corni were purchased from Henan, Zhejiang and Hubei suppliers (Batch No.: HN-1, HN-2, HN-3; ZJ-1, ZJ-2, ZJ-3, HB-1, HB-2, HB-3), and the processed product (HN-1), named Jiu zheng pin, were collected from Henan (steaming with yellow wine according to the procedures in Pharmacopoeia of the People’s Republic of China (2015) (28)). All the Crude Fructus Corni identified by Qinmei Zhou, a Professor worked at Department of Pharmacy, Nanjing University of Chinese Medicine Affiliated Hospital. Preparation of sample solutions The developed optimized method was used for the quantification of the 11 bioactive compounds in three crude Fructus Corni from different origins and their processed product, respectively. The powder of Fructus Corni samples was precisely weighed (0.100 g) and immersed in 25 mL of methanol. Additional methanol was added to make up the loss after ultrasonic extraction for 30 min. All solutions were diluted to proper concentrations and filtered through 0.45 μm filter membranes (ASDlab, Nanjing, China) before being injected into the HPLC system. All the samples were extracted and analyzed by LC–MS/MS in triplicate. Preparation of standard solutions Standard solutions of 5-HMF (10.01 mg), gallic acid (10.10 mg), sweroside (10.16 mg), cornin (10.06 mg), loganin (10.05 mg), morroniside (10.04 mg), 7α-O-methylmorroniside (10.05 mg), 7β-O-methylmorroniside (10.19 mg), and 7α-O-ethylmorroniside (10.08 mg), 7β-O-ethylmorroniside (10.06 mg) and cornuside (10.05 mg) were prepared in 10 mL of methanol, respectively. Each standard solution after dilution with methanol was mixed and then further diluted with methanol to give eight different appropriate concentrations for the establishment of calibration curves. All stock and working standard solutions were stored in brown bottles at 4°C until used for analysis. LC–MS/MS analysis The chromatographic analysis was performed on a Diamonsil C18 column (200 × 4.6 mm, i.d. 5 μm) for separation. The column was maintained at 30°C, and the injection volume of reference solution or sample solution was 10 μL. The mobile phase consisted of aqueous solution containing 0.1% formic acid (A) and acetonitrile (B). The column was eluted at a flow rate of 1.0 mL/min using a gradient as follows: 20% (B) at 0–6 min, 20–22% (B) at 6–7 min, 22% (B) at 7–10 min, 22–80% (B) at 10–13 min, 80% (B) at 13–16 min, 80–20% (B) at 16–17 min, 20% (B) at 17–18 min. The mobile phase was diverted before MS analysis. Detection was carried out on a Thermo Fisher TSQ Quantum Access MAX Triple Quad LC/MS (Thermo Scientific) equipped with electrospray ionization (ESI). The parameters for the ionization source conditions were as follows: spray voltage of 4,000 V(+)/3,000 V(−), capillary temperature of 270°C and vaporizer temperature of 200°C. Nitrogen was used as Aux gas and Sheath Gas pressure were set at 5 and 20 psi. Quantification was operated at multiple reaction monitoring (MRM) modes. The compounds were ionized in the positive or negative ion polarity mode, and the mass detection parameters are summarized in Table I. Data acquisition and processing were performed by Xcalibar 1.4 Workstation. Table I. MS/MS Detection Parameters for the 11 Bioactive Compounds and Internal Standard in Fructus Corni Compound  Rt (min)  Precursor ion (m/z)  Product ion (m/z)  Collision energy (eV)  Tube lens  5-HMF  3.59  127.1  109.2  9 (+)  58  Gallic acid  3.11  170.1  126.4  16 (−)  39  Sweroside  4.64  403  126.4  25(−)  57  Cornin  4.47  433.3  226.1  17 (−)  61  Loganin  3.82  435  228.1  19 (−)  51  Morroniside  3.24  451.2  243.9  19 (−)  44  7α-O-methylmorroniside  6.87  465.1  258  21 (−)  59  7β-O-methylmorroniside  7.33  465.1  258  21 (−)  59  7α-O-ethylmorroniside  11.48  479.2  271.9  17 (−)  54  7β-O-ethylmorroniside  12.29  479.2  271.9  17 (−)  54  Cornuside  13.29  541.1  170.1  35 (−)  66  Diazepam  16.09  285  154  27 (+)  81  Compound  Rt (min)  Precursor ion (m/z)  Product ion (m/z)  Collision energy (eV)  Tube lens  5-HMF  3.59  127.1  109.2  9 (+)  58  Gallic acid  3.11  170.1  126.4  16 (−)  39  Sweroside  4.64  403  126.4  25(−)  57  Cornin  4.47  433.3  226.1  17 (−)  61  Loganin  3.82  435  228.1  19 (−)  51  Morroniside  3.24  451.2  243.9  19 (−)  44  7α-O-methylmorroniside  6.87  465.1  258  21 (−)  59  7β-O-methylmorroniside  7.33  465.1  258  21 (−)  59  7α-O-ethylmorroniside  11.48  479.2  271.9  17 (−)  54  7β-O-ethylmorroniside  12.29  479.2  271.9  17 (−)  54  Cornuside  13.29  541.1  170.1  35 (−)  66  Diazepam  16.09  285  154  27 (+)  81  Method validation Linearity A series of standard solutions with eight different concentrations were analyzed by an established method in triplicate. Every calibration curve was plotted based on linear regression analysis of the peak areas ratio (Y) versus concentrations (X, μg/mL). LOD and LOQ The limits of detection (LOD) and quantification (LOQ) under the chromatographic conditions were determined at a signal-to-noise (S/N) ratio of 3 and 10, respectively. The stock solutions containing 11 reference compounds were diluted to a series of appropriate concentrations, and injected for analysis under the present chromatographic conditions. Precision, repeatability and accuracy Intra- and inter-day variations were chosen to determine the precision of the developed method. The intra-day variation was determined by analyzing the same mixed standard solution for six times within a single day. Six replicate mixed standard solutions were determined each day over three consecutive days to determine inter-day precision. The analytic repeatability was examined by the injection of six different samples (HN-2), which were prepared with the same preparation procedure. To investigate the recovery, accurate amounts of reference compounds were added to Fructus Corni samples, and then extracted and analyzed as described (n = 6). Recovery and stability The accuracy was achieved on recovery determination. A recovery test was performed with the method of standard addition. The standard compounds were added to Fructus Corni samples exactly, and were then extracted and analyzed as described (n = 6). The stability was appraised by analyzing the same sample at 0, 2, 4, 6, 12 and 24 h at room temperature. Statistical analysis All data are presented as means ± standard deviation (S.D.). Statistical differences were evaluated by one-way ANOVA with Tukey’s multiple comparison test. For all of the analyses, a value of P < 0.05 was considered statistically significant. Results and Discussion Optimization of the LC–MS/MS analysis The optimal mass detection parameter for each component was firstly investigated. To this end, ESI both in negative and positive modes were tried and the results showed that ESI in the negative ion mode was more sensitive for gallic acid, sweroside, cornin, loganin, morroniside, 7α-O-methylmorroniside, 7β-O-methylmorroniside, 7α-O-ethylmorroniside, 7β-O-ethylmorroniside and cornuside, while ESI in the positive ion mode was more sensitive for 5-HMF and diazepam. In this study, chromatographic conditions of the mobile phase and gradient elution system were then optimized, with the special goal to effectively separate the four iridoid isomers. Firstly, several mobile phase systems, including acetonitrile–water, acetonitrile–aqueous formic acid (0.1%, v/v) and acetonitrile–aqueous formic acid (0.1%, v/v) with CH3COONH4 (5 μM) were tried to optimize the mobile phase, with the aim to obtain good resolution and symmetrical peak shapes of the 11 target compounds, especially for the isomers. It was found that the combination of acetonitrile–aqueous formic acid (0.1%, v/v) with CH3COONH4 (5 μM) and acetonitrile–aqueous formic acid (0.1%, v/v) gave similar performance in the separation of the 11 bioactive compounds, and other methods cannot separate the isomers well or showed bad peaks. Therefore, acetonitrile–water with 0.1% formic acid was finally chosen as the mobile phase system. Under these chromatographic and mass detection conditions, all the 11 target components and the internal standard could be well separated within 18 min (Figure 2). As shown in Figure 2, both 7-O-methylmorroniside and 7-O-ethylmorroniside produced very close peaks in their elution, which are in line with the existence of isomers. The results therefore indicated that the present method could achieve good resolution of the 11 active compounds from Fructus Corni especially the isomers. The quantitative analysis was performed by means of the internal standard method. Figure 2. View largeDownload slide Typical LC–MS/MS chromatograms of the 11 targeted compounds and internal standard after injection of standard mix solutions.Note: 1. 5-HMF; 2. gallic acid; 3. sweroside; 4. cornin; 5. loganin; 6. morroniside; 7. 7α-O-methylmorroniside; 8. 7β-O-methylmorroniside; 9. 7α-O-ethylmorroniside; 10. 7β-O-ethylmorroniside; 11. cornuside; 12. internal standard. Figure 2. View largeDownload slide Typical LC–MS/MS chromatograms of the 11 targeted compounds and internal standard after injection of standard mix solutions.Note: 1. 5-HMF; 2. gallic acid; 3. sweroside; 4. cornin; 5. loganin; 6. morroniside; 7. 7α-O-methylmorroniside; 8. 7β-O-methylmorroniside; 9. 7α-O-ethylmorroniside; 10. 7β-O-ethylmorroniside; 11. cornuside; 12. internal standard. Method validation of the 11 compounds After successful development of the LC–MS/MS method, we then sought to validate this method in terms of its linearity, sensitivity, precision, repeatability, recovery and stability. The calibration curves, correlation coefficients and linearity ranges of the 11 analytes were given in Table II. The calibration results indicated that the calibration curves were linear with correlation coefficients (r2 ≥ 0.9907) for all the 11 analytes. The LOD and LOQ under the chromatographic conditions were shown in Table II. These data suggested that the developed method had adequate sensitivity and dynamic ranges for the determination of target compounds. Table II. The Calibration Curve, LOD, LOQ for the LC–MS/MS Determination of 11 Bioactive Compounds in Fructus Corni Compound  Calibration curve  r2  Linear range (μg/mL)  LOD (μg/mL)  LOQ (μg/mL)  5-HMF  Y = 1.2451X + 1.7258  0.9993  1.564 ~ 200.2  0.1954  0.3911  Gallic acid  Y = 0.1760X + 0.001  0.9989  0.158 ~ 20.20  0.0395  0.1102  Sweroside  Y = 0.0058X − 0.0010  0.9996  0.635 ~ 81.28  0.2024  0.4827  Cornin  Y = 0.0882X + 0.0378  0.9980  0.314 ~ 40.24  0.0157  0.0689  Loganin  Y = 0.0138X + 0.1503  0.9907  2.433 ~ 311.4  0.1518  0.5782  Morroniside  Y = 0.0618X + 0.6278  0.9914  2.412 ~ 308.7  0.1507  0.4725  7α-O-methylmorroniside  Y = 0.0288X − 0.0032  0.9991  0.118 ~ 15.08  0.0257  0.0838  7β-O-methylmorroniside  Y = 0.0264X − 0.0053  0.9995  0.119 ~ 15.29  0.0392  0.0931  7α-O-ethylmorroniside  Y = 0.0420X − 0.0056  0.9991  0.118 ~ 15.12  0.0139  0.0627  7β-O-ethylmorroniside  Y = 0.0434X − 0.0044  0.9994  0.059 ~ 7.545  0.0146  0.0457  Cornuside  Y = 0.4889X + 0.71  0.9929  0.589 ~ 75.37  0.0588  0.1178  Compound  Calibration curve  r2  Linear range (μg/mL)  LOD (μg/mL)  LOQ (μg/mL)  5-HMF  Y = 1.2451X + 1.7258  0.9993  1.564 ~ 200.2  0.1954  0.3911  Gallic acid  Y = 0.1760X + 0.001  0.9989  0.158 ~ 20.20  0.0395  0.1102  Sweroside  Y = 0.0058X − 0.0010  0.9996  0.635 ~ 81.28  0.2024  0.4827  Cornin  Y = 0.0882X + 0.0378  0.9980  0.314 ~ 40.24  0.0157  0.0689  Loganin  Y = 0.0138X + 0.1503  0.9907  2.433 ~ 311.4  0.1518  0.5782  Morroniside  Y = 0.0618X + 0.6278  0.9914  2.412 ~ 308.7  0.1507  0.4725  7α-O-methylmorroniside  Y = 0.0288X − 0.0032  0.9991  0.118 ~ 15.08  0.0257  0.0838  7β-O-methylmorroniside  Y = 0.0264X − 0.0053  0.9995  0.119 ~ 15.29  0.0392  0.0931  7α-O-ethylmorroniside  Y = 0.0420X − 0.0056  0.9991  0.118 ~ 15.12  0.0139  0.0627  7β-O-ethylmorroniside  Y = 0.0434X − 0.0044  0.9994  0.059 ~ 7.545  0.0146  0.0457  Cornuside  Y = 0.4889X + 0.71  0.9929  0.589 ~ 75.37  0.0588  0.1178  Intra- and inter-day variations were further investigated to determine the precision of the developed method. RSD values for both intra- and inter-day precision were below 9.33% (Table III). The repeatability of the solution was less than 5.98% and the results showed good average recovery from 93.24% to 112.92% for the 11 compounds with RSD < 9.06% (Table III). And the RSD values for stability of these 111 components were less than 13.99%. Taken together, these results validate that this method was precise and accurate enough for achieving a simultaneous quantitative evaluation of the 11 compounds from Fructus Corni. Table III. Precision, Repeatability, Recovery and Stability of 11 Compounds Compound  Precision (RSD, %, n = 6)  Repeatability (RSD, %, n = 6)  Recovery (%, n = 6)  Stability (RSD,%, n = 6)  Intra-day  Inter-day  Mean  RSD  5-HMF  1.89  2.86  N.D.  93.24  1.90  9.27  Gallic acid  2.71  4.06  3.78  112.92  3.62  11.64  Sweroside  7.32  5.17  4.65  109.72  6.99  13.91  Cornin  3.49  5.20  4.47  95.20  8.22  13.22  Loganin  3.27  4.27  3.48  95.40  9.06  10.12  Morroniside  1.55  3.39  5.31  96.30  6.37  4.68  7α-O-methylmorroniside  2.29  9.33  4.50  97.68  8.45  10.16  7β-O-methylmorroniside  3.59  5.74  5.98  110.21  7.59  5.78  7α-O-ethylmorroniside  3.62  5.69  N.D.  103.22  1.56  13.99  7β-O-ethylmorroniside  4.22  6.44  2.92  93.61  2.13  11.86  Cornuside  2.50  3.54  1.02  103.19  5.54  7.29  Compound  Precision (RSD, %, n = 6)  Repeatability (RSD, %, n = 6)  Recovery (%, n = 6)  Stability (RSD,%, n = 6)  Intra-day  Inter-day  Mean  RSD  5-HMF  1.89  2.86  N.D.  93.24  1.90  9.27  Gallic acid  2.71  4.06  3.78  112.92  3.62  11.64  Sweroside  7.32  5.17  4.65  109.72  6.99  13.91  Cornin  3.49  5.20  4.47  95.20  8.22  13.22  Loganin  3.27  4.27  3.48  95.40  9.06  10.12  Morroniside  1.55  3.39  5.31  96.30  6.37  4.68  7α-O-methylmorroniside  2.29  9.33  4.50  97.68  8.45  10.16  7β-O-methylmorroniside  3.59  5.74  5.98  110.21  7.59  5.78  7α-O-ethylmorroniside  3.62  5.69  N.D.  103.22  1.56  13.99  7β-O-ethylmorroniside  4.22  6.44  2.92  93.61  2.13  11.86  Cornuside  2.50  3.54  1.02  103.19  5.54  7.29  N.D.: not detected. Sample analysis The optimized and validated method was then used for the quantification of the 11 bioactive compounds in crude Fructus Corni from three different origins and a processed product. All the samples were extracted and analyzed in triplicate. The 11 bioactive compounds in crude Fructus Corni and its processed product were successfully identified by the LC–MS/MS method (Figures 3 and 4). The contents of the 11 compounds in Fructus Corni samples analyzed are listed in Table IV. Table IV. The Mean Contents of the 11 Bioactive Compounds in Crude and Processed Fructus Corni Samples (mg/g + SD, n = 3) Origins  Henan  Hubei  Zhejiang  Processed product  Compounds  5-HMF  N.D.  N.D.  N.D.  0.41 ± 0.14  Gallic acid  0.26 ± 0.04  0.28 ± 0.03  0.18 ± 0.03  0.45 ± 0.03  Sweroside  0.53 ± 0.12  0.46 ± 0.12  0.46 ± 0.19  0.47 ± 0.10  Cornin  0.32 ± 0.10  0.37 ± 0.13  0.40 ± 0.12  0.31 ± 0.13  Loganin  6.75 ± 1.13  6.74 ± 0.71  6.03 ± 0.97  6.39 ± 0.51  Morroniside  7.61 ± 1.33  8.65 ± 1.23  9.43 ± 1.03  7.45 ± 0.49  7α-O-methylmorroniside  0.05 ± 0.01  0.05 ± 0.02  0.05 ± 0.01  0.04 ± 0.005  7β-O-methylmorroniside  0.08 ± 0.01  0.08 ± 0.01  0.07 ± 0.01  0.06 ± 0.02  7α-O-ethylmorroniside  N.D.  N.D.  N.D.  0.08 ± 0.03  7β-O-ethylmorroniside  0.011 ± 0.002  0.008 ± 0.001  N.D.  0.06 ± 0.02  Cornuside  2.28 ± 0.51  2.53 ± 0.60  2.78 ± 0.44  2.22 ± 0.52  Origins  Henan  Hubei  Zhejiang  Processed product  Compounds  5-HMF  N.D.  N.D.  N.D.  0.41 ± 0.14  Gallic acid  0.26 ± 0.04  0.28 ± 0.03  0.18 ± 0.03  0.45 ± 0.03  Sweroside  0.53 ± 0.12  0.46 ± 0.12  0.46 ± 0.19  0.47 ± 0.10  Cornin  0.32 ± 0.10  0.37 ± 0.13  0.40 ± 0.12  0.31 ± 0.13  Loganin  6.75 ± 1.13  6.74 ± 0.71  6.03 ± 0.97  6.39 ± 0.51  Morroniside  7.61 ± 1.33  8.65 ± 1.23  9.43 ± 1.03  7.45 ± 0.49  7α-O-methylmorroniside  0.05 ± 0.01  0.05 ± 0.02  0.05 ± 0.01  0.04 ± 0.005  7β-O-methylmorroniside  0.08 ± 0.01  0.08 ± 0.01  0.07 ± 0.01  0.06 ± 0.02  7α-O-ethylmorroniside  N.D.  N.D.  N.D.  0.08 ± 0.03  7β-O-ethylmorroniside  0.011 ± 0.002  0.008 ± 0.001  N.D.  0.06 ± 0.02  Cornuside  2.28 ± 0.51  2.53 ± 0.60  2.78 ± 0.44  2.22 ± 0.52  N.D.: not detected. Figure 3. View largeDownload slide Representative TIC chromatogram of the 11 targeted compounds from the whole extract of Fructus Coni. Figure 3. View largeDownload slide Representative TIC chromatogram of the 11 targeted compounds from the whole extract of Fructus Coni. Figure 4. View largeDownload slide Typical LC–MS/MS chromatograms of the 11 targeted compounds analyzed in Fructus Coni extract.Note: 1. 5-HMF; 2. gallic acid; 3. sweroside; 4. cornin; 5. loganin; 6. morroniside; 7. 7α-O-methylmorroniside; 8. 7β-O-methylmorroniside; 9. 7α-O-ethylmorroniside; 10. 7β-O-ethylmorroniside; 11. cornuside; 12. internal standard. Figure 4. View largeDownload slide Typical LC–MS/MS chromatograms of the 11 targeted compounds analyzed in Fructus Coni extract.Note: 1. 5-HMF; 2. gallic acid; 3. sweroside; 4. cornin; 5. loganin; 6. morroniside; 7. 7α-O-methylmorroniside; 8. 7β-O-methylmorroniside; 9. 7α-O-ethylmorroniside; 10. 7β-O-ethylmorroniside; 11. cornuside; 12. internal standard. The contents of 11 constituents in Fructus Corni from different origins and one processed product were determined and showed in Figure 5. It is clear that the contents of gallic acid and 7β-O-ethylmorroniside showed significant batch differences, while other nine compounds did not. The quantitative results for these components in crude Fructus Corni from different origins display various degrees of difference, as shown in Figure 6. For instance, contents of gallic acid in Henan and Hubei supplier (0.26 mg/g, 0.28 mg/g) were higher than those provided by Zhejiang supplier (0.17 mg/g). However, the content of morroniside from Henan (7.6 mg/g) was less than that in Zhejiang (9.4 mg/g). In addition, compared to the processed product, some chemical constituents in crude Fructus Corni varied significantly (P < 0.05). For example, the content of gallic acid and 7β-O-ethylmorroniside from crude products were lower than that in the processed product (0.45 and 0.056 mg/g), while other compounds such as 7α-methylmorroniside and 7β-O-methylmorroniside did not show significant changes after procession, as shown in Figure 6. It was previously reported that processing or heating could drastically increase the content of 5-HMF and tannin in Fructus Corni, which was hydrolyzed to generate gallic acid because of high temperature (19). In our study, we observed the most dramatic changes for 7α-O-ethylmorroniside, 5-HMF and gallic acid in crude and processed Fructus Corni, which supported the capability of our method to monitor the content variations of Fructus Corni. Figure 5. View largeDownload slide Batch differences of the 11 compounds in crude Fructu Corni and the effect of processing. Henan, Zhejiang and Hubei suppliers, and the processed product, n = 3. *P < 0.05, **P < 0.01, ***P < 0.001, compared with corresponding processed product.Note: HN, ZJ and HB refer to Henan, Zhejiang and Hubei suppliers. 1, 2, 3 refer to the different batches No. from the same suppliers. Figure 5. View largeDownload slide Batch differences of the 11 compounds in crude Fructu Corni and the effect of processing. Henan, Zhejiang and Hubei suppliers, and the processed product, n = 3. *P < 0.05, **P < 0.01, ***P < 0.001, compared with corresponding processed product.Note: HN, ZJ and HB refer to Henan, Zhejiang and Hubei suppliers. 1, 2, 3 refer to the different batches No. from the same suppliers. Figure 6. View largeDownload slide Comparison of the contents of the 11 compounds in crude Fructu Corni from different origins and a processed product. Each column represents the mean content of 3 samples in the same origin, *P < 0.05, ***P < 0.001. Note: HN, ZJ and HB refer to Henan, Zhejiang and Hubei suppliers. Figure 6. View largeDownload slide Comparison of the contents of the 11 compounds in crude Fructu Corni from different origins and a processed product. Each column represents the mean content of 3 samples in the same origin, *P < 0.05, ***P < 0.001. Note: HN, ZJ and HB refer to Henan, Zhejiang and Hubei suppliers. Although previously isomers have been reported for the methyl and ethylmorroniside, these isomers were not differentiated in previous quantitative studies (20, 22, 27, 29). In our experiment, with the developed method, 7α-O-methylmorroniside, 7β-O-methylmorroniside and 7β-O-ethylmorroniside were all detected in the crude and processed Fructus Corni, while 7α-O-ethylmorroniside was only found in processed product. This finding suggested that 7α-O-ethylmorroniside was an artificial product generated during ethanol extraction. Previously, we have also reported a HPLC-based quantitative method for the simultaneous determination of polar active components in Fructus Corni (30). This largely features a multi-components by single-marker (QAMS) strategy that depends on the calculation of relative correction factors for each target component compared to the internal standard, thereby dose not necessitate the preparation of all standards. Despite the advantages of the method, time-efficient separation of the numerous target components of Fructus Corni, especially concerning the existence of isomers, by HPLC-UV proved challenging. Also, this method may have limitations when applied to the analysis of biological samples. In light of this caveat, the current study makes an important complementary progress by integrating HPLC separation with tandem mass spectrometry, which only takes 18 min for a single run and confers much better sensitivity. Conclusion In this study, a highly selective method for simultaneous identification and determination of the 11 compounds in Fructus Corni using LC–MS/MS was established and validated. With this developed method, we performed a comparative quantitative study of 11 bioactive components in crude Fructus Corni of three different origins as well as a processed product. We contributed to the establishment of a simple and comprehensive method for efficiently integrating the iridoid isomers (7α-O-methylmorroniside, 7β-O-methylmorroniside, and 7α-O-ethylmorroniside, 7β-O-ethylmorroniside) in the quality control of Fructus Corni. This method is expected to find a wide and routine application in the quantitative analysis of crude and processed Fructus Corni. Funding This work was supported by the Project Phase II of Priority Academic Program Development of Jiangsu Higher Education Institutions; Jiangsu Project of Chinese Medicine (No.: FY201503) and Natural Science Foundation of China (No.: 81202580). References 1 Lau, C.H., Chan, C.M., Chan, Y.W., Lau, K.M., Lau, T.W., Lam, F.C., et al.  .; In vitro antidiabetic activities of five medicinal herbs used in Chinese medicinal formulae; Phytotherapy Research , ( 2008); 22: 1384– 1388. Google Scholar CrossRef Search ADS PubMed  2 Wu, Y., Wang, X., Shen, B., Kang, L., Fan, E.; Extraction, structure and bioactivities of the polysaccharides from Fructus corni; Recent Patents on Food, Nutrition and Agriculture , ( 2013); 5: 57– 61. Google Scholar CrossRef Search ADS   3 Park, C.H., Xu, F.H., Roh, S.S., Song, Y.O., Uebaba, K., Noh, J.S., et al.  .; Astaxanthin and Corni Fructus protect against diabetes-induced oxidative stress, inflammation, and advanced glycation end product in livers of streptozotocin-induced diabetic rats; Journal of Medicinal Food , ( 2015); 18: 337– 344. Google Scholar CrossRef Search ADS PubMed  4 Park, C.H., Noh, J.S., Park, J.C., Yokozawa, T.; Beneficial Effect of 7-O-Galloyl-D-sedoheptulose, a polyphenol isolated from Corni Fructus, against diabetes-induced alterations in kidney and adipose tissue of Type 2 diabetic db/db mice; Evidence-based Complementary and Alternative Medicine , ( 2013); 2013: 736856. Google Scholar PubMed  5 Zhou, H.Y., Yang, P.P., Cong, X.D., Zhang, C.R., Cai, B.C.; Comparative study on decoction and dissolution of crude and processed Corni Fructus; Zhongguo Zhong Yao Za Zhi , ( 2013); 38: 3888– 3892. Google Scholar PubMed  6 Telang, N.T., Li, G., Sepkovic, D.W., Bradlow, H.L., Wong, G.Y.; Anti-proliferative effects of Chinese herb Cornus officinalis in a cell culture model for estrogen receptor-positive clinical breast cancer; Molecular Medicine Reports , ( 2012); 5: 22– 28. Google Scholar PubMed  7 Cho, S., Won, C.H., Lee, D.H., Lee, M.J., Lee, S., So, S.H., et al.  .; Red ginseng root extract mixed with Torilus fructus and Corni Fructus improves facial wrinkles and increases type I procollagen synthesis in human skin: a randomized, double-blind, placebo-controlled study; Journal of Medicinal Food , ( 2009); 12: 1252– 1259. Google Scholar CrossRef Search ADS PubMed  8 Sun, H., Li, L., Zhang, A., Zhang, N., Lv, H., Sun, W., et al.  .; Protective effects of sweroside on human MG-63 cells and rat osteoblasts; Fitoterapia , ( 2013); 84: 174– 179. Google Scholar CrossRef Search ADS PubMed  9 Yamabe, N., Noh, J.S., Park, C.H., Kang, K.S., Shibahara, N., Tanaka, T., et al.  .; Evaluation of loganin, iridoid glycoside from Corni Fructus, on hepatic and renal glucolipotoxicity and inflammation in type 2 diabetic db/db mice; European Journal of Pharmacology , ( 2010); 648: 179– 187. Google Scholar CrossRef Search ADS PubMed  10 Wang, S.F., Chen, X.G., Hu, Z.D., Ju, Y.; Analysis of three effective components in Fructus Corni and its preparations by micellar electrokinetic capillary chromatography; Biomedical Chromatography , ( 2003); 17: 306– 311. Google Scholar CrossRef Search ADS PubMed  11 Lee, C.M., Jung, H.A., Oh, S.H., Park, C.H., Tanaka, T., Yokozawa, T., et al.  .; Kinetic and molecular docking studies of loganin and 7-O-galloyl-D-sedoheptulose from Corni Fructus as therapeutic agents for diabetic complications through inhibition of aldose reductase; Archives of Pharmacal Research , ( 2015); 38: 1090– 1098. Google Scholar CrossRef Search ADS PubMed  12 Yokozawa, T., Kang, K.S., Park, C.H., Noh, J.S., Yamabe, N., Shibahara, N., et al.  .; Bioactive constituents of Corni Fructus: the therapeutic use of morroniside, loganin, and 7-O-galloyl-D-sedoheptulose as renoprotective agents in type 2 diabetes; Drug Discovery and Therapeutics , ( 2010); 4: 223– 234. 13 Park, C.H., Noh, J.S., Tanaka, T., Yokozawa, T.; Effects of morroniside isolated from Corni Fructus on renal lipids and inflammation in type 2 diabetic mice; Journal of Pharmacy and Pharmacology , ( 2010); 62: 374– 380. Google Scholar CrossRef Search ADS PubMed  14 Wang, W., Sun, F., An, Y., Ai, H., Zhang, L., Huang, W., et al.  .; Morroniside protects human neuroblastoma SH-SY5Y cells against hydrogen peroxide-induced cytotoxicity; European Journal of Pharmacology , ( 2009); 613: 19– 23. Google Scholar CrossRef Search ADS PubMed  15 Yao, R.Q., Zhang, L., Wang, W., Li, L.; Cornel iridoid glycoside promotes neurogenesis and angiogenesis and improves neurological function after focal cerebral ischemia in rats; Brain Research Bulletin , ( 2009); 79: 69– 76. Google Scholar CrossRef Search ADS PubMed  16 Liang, J., He, J., Zhu, S., Zhao, W., Zhang, Y., Ito, Y., et al.  .; Preparative isolation and purification of iridoid glycosides from Fructus Corni by high-speed countercurrent chromatography; Journal of Liquid Chromatography & Related Technologies , ( 2013); 36: 983– 999. Google Scholar PubMed  17 Sunghwa, F., Sakurai, H., Saiki, I., Koketsu, M.; Iodine-catalyzed etherification of morroniside; Chemical and Pharmaceutical Bulletin , ( 2009); 57: 112– 115. Google Scholar CrossRef Search ADS PubMed  18 Takeda, Y., Tanigawa, N., Sunghwa, F., Ninomiya, M., Hagiwara, M., Matsushita, K., et al.  .; Morroniside cinnamic acid conjugate as an anti-inflammatory agent; Bioorganic & Medicinal Chemistry Letters , ( 2010); 16: 4855– 4857. Google Scholar CrossRef Search ADS   19 Cai, H., Cao, G., Cai, B.; Rapid simultaneous identification and determination of the multiple compounds in crude Fructus Corni and its processed products by HPLC-MS/MS with multiple reaction monitoring mode; Pharmaceutical Biology , ( 2013); 51: 273– 278. Google Scholar CrossRef Search ADS PubMed  20 Cao, G., Zhang, C., Zhang, Y., Cong, X., Cai, H., Cai, B., et al.  .; Global detection and identification of components from crude and processed traditional Chinese medicine by liquid chromatography connected with hybrid ion trap and time-of-flight-mass spectrometry; Journal of Separation Science , ( 2011); 34: 1845– 1852. Google Scholar CrossRef Search ADS PubMed  21 Du, W., Cai, H., Wang, M., Ding, X., Yang, H., Cai, B.; Simultaneous determination of six active components in crude and processed Fructus Corni by high performance liquid chromatography; Journal of Pharmaceutical and Biomedical Analysis , ( 2008); 48: 194– 197. Google Scholar CrossRef Search ADS PubMed  22 Liu, Z., Zhu, Z., Zhang, H., Tan, G., Chen, X., Chai, Y.; Qualitative and quantitative analysis of Fructus Corni using ultrasound assisted microwave extraction and high performance liquid chromatography coupled with diode array UV detection and time-of-flight mass spectrometry; Journal of Pharmaceutical and Biomedical Analysis , ( 2011); 55: 557– 562. Google Scholar CrossRef Search ADS PubMed  23 Ouyang, E., Zhang, C., Li, X.; Determination of 5-hydroxymethyl-2-furaldehyde of crude and processed Fructus Corni in freely moving rats using in vivo microdialysis sampling and liquid chromatography; Pharmacognosy Maganize , ( 2011); 7: 271– 276. Google Scholar CrossRef Search ADS   24 Choi, W.H., Chu, J.P., Jiang, M.H., Baek, S.H., Park, H.D.; Effects of fraction obtained from Korean Corni Fructus extracts causing anti-proliferation and p53-dependent apoptosis in A549 lung cancer cells; Nutrition and Cancer-an International Journal , ( 2011); 63: 121– 129. 25 Wang, X.Y., Sun, L., Qiao, S.Y.; Chemical constituents of glucoside fraction from Liuwei Dihuang Gantang; Zhongguo Zhong Yao Za Zhi , ( 2012); 37: 2576– 2580. Google Scholar PubMed  26 Cao, G., Zhang, C., Zhang, Y., Cong, X., Cai, H., Cai, B.; Screening and identification of potential active components in crude Fructus Corni using solid-phase extraction and LC-LTQ-linear ion trap mass spectrometry; Le Pharmacien Biologiste , ( 2012); 50: 278– 283. 27 Shi, T., Yao, Z., Qin, Z., Ding, B., Dai, Y., Yao, X.; Identification of absorbed constituents and metabolites in rat plasma after oral administration of Shen-Song-Yang-Xin using ultra-performance liquid chromatography combined with quadrupole time-of-flight mass spectrometry; Biomedical Chromatography , ( 2015); 29: 1440– 1452. Google Scholar CrossRef Search ADS PubMed  28 Chinese Pharmacopoeia Commission.; Pharmacopoeia of the People’s Republic of China . Chemical Industry Press, Beijing, ( 2015); p. 27. 29 Cao, G., Cai, H., Zhang, Y., Cong, X., Zhang, C., Cai, B.; Identification of metabolites of crude and processed Fructus Corni in rats by microdialysis sampling coupled with electrospray ionization linear quadrupole ion trap mass spectrometry; Journal of Pharmaceutical and Biomedical Analysis , ( 2011); 56: 118– 125. Google Scholar CrossRef Search ADS PubMed  30 Jiang, Y., Chen, H., Wang, L., Zou, J., Zheng, X., Liu, Z.; Quality evaluation of polar and active components in crude and processed Fructus Corni by quantitative analysis of multicomponents with single marker; Journal of Analytical Methods in Chemistry , ( 2016); 2016: 6496840. Google Scholar CrossRef Search ADS PubMed  © The Author 2017. Published by Oxford University Press. All rights reserved. For Permissions, please email: journals.permissions@oup.com http://www.deepdyve.com/assets/images/DeepDyve-Logo-lg.png Journal of Chromatographic Science Oxford University Press

Simultaneous Determination of 11 High-Polarity Components from Fructus Corni: A Quantitative LC–MS/MS Method for Improved Quality Control

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Oxford University Press
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© The Author 2017. Published by Oxford University Press. All rights reserved. For Permissions, please email: journals.permissions@oup.com
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0021-9665
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1945-239X
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10.1093/chromsci/bmx083
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Abstract

Abstract Fructus Corni, the dried ripe sarcocarp of Cornus officinalis Sieb.et Zucc (Cornaceae), is widely used in traditional medicine. Pharmacological studies to date have attributed many biological effects to the high-polarity components. However, current quality control methods focus on only several iridoid glycoside components, and, of note, there is no comprehensive method available to simultaneously quantify the high-polarity components in Fructus Corni. Here, a simple, sensitive and robust liquid chromatography-electrospray ionization-tandem mass spectrometry method was developed to simultaneously determine 11 high-polarity constituents (5-hydroxymethyl-2-furfural, gallic acid, sweroside, cornin, loganin, morroniside, 7α-O-methylmorroniside, 7β-O-methylmorroniside, 7α-O-ethylmorroniside and 7β-O-ethylmorroniside, cornuside) of Fructus Corni. This method showed good specificity, linearity (r2 ≥ 0.9907), repeatability (RSD < 5.98%) and recovery (93.24 ~ 112.92%, RSD < 9.06%). This validated method was successfully employed to assess the component variation of crude Fructus Corni of three regional origins as well as after processing. In particular, the iridoid isomers were, for the first time, included as the quality markers for Fructus Corni. We propose that this method may provide a new and powerful tool for achieving comprehensive quality control of Fructus Corni. Introduction Fructus Corni, a well-known traditional Chinese medicine widely used in China for thousands of years, is derived from the dried ripe sarcocarp of Cornus officinalis Sieb.et Zucc (Cornaceae). Due to its biological and pharmacological activities such as anti-inflammation, anti-diabetes and anti-oxidation (1–4), Fructus Corni has received intensive attention as one of the most popular and cherished clinical herbal medicines in the world (1, 5–7). Chemical constituents from Fructus Corni that have been reported mainly include iridoids, organic acids and carbohydrates (8–10). To date, the pharmacological activity of high-polarity components such as the iridoid glycosides from Fructus Corni has received huge attention. For example, loganin has been shown to exhibit immune regulation, anti-inflammatory and inhibition of aldose reductase (11); morroniside was reported to contribute to the effect of Fructus Corni in protecting neurons and preventing diabetic angiopathies (12–16); and 7-O-methylmorroniside, a methylation product of morroniside, was also proposed as a potential bioactive iridoid constituent, which was shown to possess comparable anti-inflammatory effects as morroniside (17–18). Given the high medicinal value of Fructus Corni, a reliable, sensitive and comprehensive analytical method is indispensible for the quality control. To date, only a few analytical methods have been reported for the determination of several active components in crude or processed Fructus Corni (10, 19–23). However, it is notable that, current methods largely fail to focus on the high-polarity components in a comprehensive manner. Moreover, although several isomeric iridoids, such as 7-O-ethylmorroniside and 7-O-methylmorroniside, are reported to exist in Fructus Corni, they are not covered in these conventional methods (19, 22, 24–27). These limitations highlight the need to efficiently separate and determine these newly emerging active components for achieving a comprehensive quality assessment of the Fructus Corni. Therefore, in this study, we aimed to improve the current method for the quality control of Fructus Corni, focusing on developing a high-throughput and time-efficient detection and quantification of the major high-polarity components from Fructus Corni. By employing the high sensitivity and wide coverage of liquid chromatography tandem mass spectrometry (LC–MS/MS), we successfully developed a simple and robust LC–MS/MS method to simultaneously identify and quantify 11 bioactive constituents in Fructus Corni. All the target components, including two pairs of iridoid isomers, could be well separated and detected within 18 min. This method was then validated and successfully applied to examine the component changes of several batches of crude Fructus Corni from three different origins as well as after procession. The results suggested that the present method could provide a new and feasible method that can be employed for improving the quality control of Fructus Corni. Experimental Chemicals, reagents and materials Gallic acid, loganin and 5-hydroxymethyl-2-furfural (5-HMF) were purchased from the National Institute for Food and Drug Control (Beijing, China). Morroniside, cornuside, cornin and sweroside were obtained from Nanjing Ningqi Biotechnology Co. Ltd. (Nanjing, China). The purity for each reference compound was greater than 98% as validated by HPLC analysis. 7α-O-methylmorroniside, 7β-O-methylmorroniside and 7α-O-ethylmorroniside, 7β-O-ethylmorroniside were prepared by our lab, and structurally characterized based on 1H-NMR, 13C-NMR spectra. Their contents were 99.7% and 99.6% as determined by HPLC analysis, respectively. The structures of these 11 compounds are shown in Figure 1. Acetonitrile used was of chromatographical grade and purchased from Merck (Darmstadt, Germany). A Milli-Q water (Millipore Inc. USA) purification system was used to obtain purified water for the HPLC analysis. Figure 1. View largeDownload slide The chemical structures of the 11 bioactive compounds in Fructus Corni. Figure 1. View largeDownload slide The chemical structures of the 11 bioactive compounds in Fructus Corni. Crude Fructus Corni were purchased from Henan, Zhejiang and Hubei suppliers (Batch No.: HN-1, HN-2, HN-3; ZJ-1, ZJ-2, ZJ-3, HB-1, HB-2, HB-3), and the processed product (HN-1), named Jiu zheng pin, were collected from Henan (steaming with yellow wine according to the procedures in Pharmacopoeia of the People’s Republic of China (2015) (28)). All the Crude Fructus Corni identified by Qinmei Zhou, a Professor worked at Department of Pharmacy, Nanjing University of Chinese Medicine Affiliated Hospital. Preparation of sample solutions The developed optimized method was used for the quantification of the 11 bioactive compounds in three crude Fructus Corni from different origins and their processed product, respectively. The powder of Fructus Corni samples was precisely weighed (0.100 g) and immersed in 25 mL of methanol. Additional methanol was added to make up the loss after ultrasonic extraction for 30 min. All solutions were diluted to proper concentrations and filtered through 0.45 μm filter membranes (ASDlab, Nanjing, China) before being injected into the HPLC system. All the samples were extracted and analyzed by LC–MS/MS in triplicate. Preparation of standard solutions Standard solutions of 5-HMF (10.01 mg), gallic acid (10.10 mg), sweroside (10.16 mg), cornin (10.06 mg), loganin (10.05 mg), morroniside (10.04 mg), 7α-O-methylmorroniside (10.05 mg), 7β-O-methylmorroniside (10.19 mg), and 7α-O-ethylmorroniside (10.08 mg), 7β-O-ethylmorroniside (10.06 mg) and cornuside (10.05 mg) were prepared in 10 mL of methanol, respectively. Each standard solution after dilution with methanol was mixed and then further diluted with methanol to give eight different appropriate concentrations for the establishment of calibration curves. All stock and working standard solutions were stored in brown bottles at 4°C until used for analysis. LC–MS/MS analysis The chromatographic analysis was performed on a Diamonsil C18 column (200 × 4.6 mm, i.d. 5 μm) for separation. The column was maintained at 30°C, and the injection volume of reference solution or sample solution was 10 μL. The mobile phase consisted of aqueous solution containing 0.1% formic acid (A) and acetonitrile (B). The column was eluted at a flow rate of 1.0 mL/min using a gradient as follows: 20% (B) at 0–6 min, 20–22% (B) at 6–7 min, 22% (B) at 7–10 min, 22–80% (B) at 10–13 min, 80% (B) at 13–16 min, 80–20% (B) at 16–17 min, 20% (B) at 17–18 min. The mobile phase was diverted before MS analysis. Detection was carried out on a Thermo Fisher TSQ Quantum Access MAX Triple Quad LC/MS (Thermo Scientific) equipped with electrospray ionization (ESI). The parameters for the ionization source conditions were as follows: spray voltage of 4,000 V(+)/3,000 V(−), capillary temperature of 270°C and vaporizer temperature of 200°C. Nitrogen was used as Aux gas and Sheath Gas pressure were set at 5 and 20 psi. Quantification was operated at multiple reaction monitoring (MRM) modes. The compounds were ionized in the positive or negative ion polarity mode, and the mass detection parameters are summarized in Table I. Data acquisition and processing were performed by Xcalibar 1.4 Workstation. Table I. MS/MS Detection Parameters for the 11 Bioactive Compounds and Internal Standard in Fructus Corni Compound  Rt (min)  Precursor ion (m/z)  Product ion (m/z)  Collision energy (eV)  Tube lens  5-HMF  3.59  127.1  109.2  9 (+)  58  Gallic acid  3.11  170.1  126.4  16 (−)  39  Sweroside  4.64  403  126.4  25(−)  57  Cornin  4.47  433.3  226.1  17 (−)  61  Loganin  3.82  435  228.1  19 (−)  51  Morroniside  3.24  451.2  243.9  19 (−)  44  7α-O-methylmorroniside  6.87  465.1  258  21 (−)  59  7β-O-methylmorroniside  7.33  465.1  258  21 (−)  59  7α-O-ethylmorroniside  11.48  479.2  271.9  17 (−)  54  7β-O-ethylmorroniside  12.29  479.2  271.9  17 (−)  54  Cornuside  13.29  541.1  170.1  35 (−)  66  Diazepam  16.09  285  154  27 (+)  81  Compound  Rt (min)  Precursor ion (m/z)  Product ion (m/z)  Collision energy (eV)  Tube lens  5-HMF  3.59  127.1  109.2  9 (+)  58  Gallic acid  3.11  170.1  126.4  16 (−)  39  Sweroside  4.64  403  126.4  25(−)  57  Cornin  4.47  433.3  226.1  17 (−)  61  Loganin  3.82  435  228.1  19 (−)  51  Morroniside  3.24  451.2  243.9  19 (−)  44  7α-O-methylmorroniside  6.87  465.1  258  21 (−)  59  7β-O-methylmorroniside  7.33  465.1  258  21 (−)  59  7α-O-ethylmorroniside  11.48  479.2  271.9  17 (−)  54  7β-O-ethylmorroniside  12.29  479.2  271.9  17 (−)  54  Cornuside  13.29  541.1  170.1  35 (−)  66  Diazepam  16.09  285  154  27 (+)  81  Method validation Linearity A series of standard solutions with eight different concentrations were analyzed by an established method in triplicate. Every calibration curve was plotted based on linear regression analysis of the peak areas ratio (Y) versus concentrations (X, μg/mL). LOD and LOQ The limits of detection (LOD) and quantification (LOQ) under the chromatographic conditions were determined at a signal-to-noise (S/N) ratio of 3 and 10, respectively. The stock solutions containing 11 reference compounds were diluted to a series of appropriate concentrations, and injected for analysis under the present chromatographic conditions. Precision, repeatability and accuracy Intra- and inter-day variations were chosen to determine the precision of the developed method. The intra-day variation was determined by analyzing the same mixed standard solution for six times within a single day. Six replicate mixed standard solutions were determined each day over three consecutive days to determine inter-day precision. The analytic repeatability was examined by the injection of six different samples (HN-2), which were prepared with the same preparation procedure. To investigate the recovery, accurate amounts of reference compounds were added to Fructus Corni samples, and then extracted and analyzed as described (n = 6). Recovery and stability The accuracy was achieved on recovery determination. A recovery test was performed with the method of standard addition. The standard compounds were added to Fructus Corni samples exactly, and were then extracted and analyzed as described (n = 6). The stability was appraised by analyzing the same sample at 0, 2, 4, 6, 12 and 24 h at room temperature. Statistical analysis All data are presented as means ± standard deviation (S.D.). Statistical differences were evaluated by one-way ANOVA with Tukey’s multiple comparison test. For all of the analyses, a value of P < 0.05 was considered statistically significant. Results and Discussion Optimization of the LC–MS/MS analysis The optimal mass detection parameter for each component was firstly investigated. To this end, ESI both in negative and positive modes were tried and the results showed that ESI in the negative ion mode was more sensitive for gallic acid, sweroside, cornin, loganin, morroniside, 7α-O-methylmorroniside, 7β-O-methylmorroniside, 7α-O-ethylmorroniside, 7β-O-ethylmorroniside and cornuside, while ESI in the positive ion mode was more sensitive for 5-HMF and diazepam. In this study, chromatographic conditions of the mobile phase and gradient elution system were then optimized, with the special goal to effectively separate the four iridoid isomers. Firstly, several mobile phase systems, including acetonitrile–water, acetonitrile–aqueous formic acid (0.1%, v/v) and acetonitrile–aqueous formic acid (0.1%, v/v) with CH3COONH4 (5 μM) were tried to optimize the mobile phase, with the aim to obtain good resolution and symmetrical peak shapes of the 11 target compounds, especially for the isomers. It was found that the combination of acetonitrile–aqueous formic acid (0.1%, v/v) with CH3COONH4 (5 μM) and acetonitrile–aqueous formic acid (0.1%, v/v) gave similar performance in the separation of the 11 bioactive compounds, and other methods cannot separate the isomers well or showed bad peaks. Therefore, acetonitrile–water with 0.1% formic acid was finally chosen as the mobile phase system. Under these chromatographic and mass detection conditions, all the 11 target components and the internal standard could be well separated within 18 min (Figure 2). As shown in Figure 2, both 7-O-methylmorroniside and 7-O-ethylmorroniside produced very close peaks in their elution, which are in line with the existence of isomers. The results therefore indicated that the present method could achieve good resolution of the 11 active compounds from Fructus Corni especially the isomers. The quantitative analysis was performed by means of the internal standard method. Figure 2. View largeDownload slide Typical LC–MS/MS chromatograms of the 11 targeted compounds and internal standard after injection of standard mix solutions.Note: 1. 5-HMF; 2. gallic acid; 3. sweroside; 4. cornin; 5. loganin; 6. morroniside; 7. 7α-O-methylmorroniside; 8. 7β-O-methylmorroniside; 9. 7α-O-ethylmorroniside; 10. 7β-O-ethylmorroniside; 11. cornuside; 12. internal standard. Figure 2. View largeDownload slide Typical LC–MS/MS chromatograms of the 11 targeted compounds and internal standard after injection of standard mix solutions.Note: 1. 5-HMF; 2. gallic acid; 3. sweroside; 4. cornin; 5. loganin; 6. morroniside; 7. 7α-O-methylmorroniside; 8. 7β-O-methylmorroniside; 9. 7α-O-ethylmorroniside; 10. 7β-O-ethylmorroniside; 11. cornuside; 12. internal standard. Method validation of the 11 compounds After successful development of the LC–MS/MS method, we then sought to validate this method in terms of its linearity, sensitivity, precision, repeatability, recovery and stability. The calibration curves, correlation coefficients and linearity ranges of the 11 analytes were given in Table II. The calibration results indicated that the calibration curves were linear with correlation coefficients (r2 ≥ 0.9907) for all the 11 analytes. The LOD and LOQ under the chromatographic conditions were shown in Table II. These data suggested that the developed method had adequate sensitivity and dynamic ranges for the determination of target compounds. Table II. The Calibration Curve, LOD, LOQ for the LC–MS/MS Determination of 11 Bioactive Compounds in Fructus Corni Compound  Calibration curve  r2  Linear range (μg/mL)  LOD (μg/mL)  LOQ (μg/mL)  5-HMF  Y = 1.2451X + 1.7258  0.9993  1.564 ~ 200.2  0.1954  0.3911  Gallic acid  Y = 0.1760X + 0.001  0.9989  0.158 ~ 20.20  0.0395  0.1102  Sweroside  Y = 0.0058X − 0.0010  0.9996  0.635 ~ 81.28  0.2024  0.4827  Cornin  Y = 0.0882X + 0.0378  0.9980  0.314 ~ 40.24  0.0157  0.0689  Loganin  Y = 0.0138X + 0.1503  0.9907  2.433 ~ 311.4  0.1518  0.5782  Morroniside  Y = 0.0618X + 0.6278  0.9914  2.412 ~ 308.7  0.1507  0.4725  7α-O-methylmorroniside  Y = 0.0288X − 0.0032  0.9991  0.118 ~ 15.08  0.0257  0.0838  7β-O-methylmorroniside  Y = 0.0264X − 0.0053  0.9995  0.119 ~ 15.29  0.0392  0.0931  7α-O-ethylmorroniside  Y = 0.0420X − 0.0056  0.9991  0.118 ~ 15.12  0.0139  0.0627  7β-O-ethylmorroniside  Y = 0.0434X − 0.0044  0.9994  0.059 ~ 7.545  0.0146  0.0457  Cornuside  Y = 0.4889X + 0.71  0.9929  0.589 ~ 75.37  0.0588  0.1178  Compound  Calibration curve  r2  Linear range (μg/mL)  LOD (μg/mL)  LOQ (μg/mL)  5-HMF  Y = 1.2451X + 1.7258  0.9993  1.564 ~ 200.2  0.1954  0.3911  Gallic acid  Y = 0.1760X + 0.001  0.9989  0.158 ~ 20.20  0.0395  0.1102  Sweroside  Y = 0.0058X − 0.0010  0.9996  0.635 ~ 81.28  0.2024  0.4827  Cornin  Y = 0.0882X + 0.0378  0.9980  0.314 ~ 40.24  0.0157  0.0689  Loganin  Y = 0.0138X + 0.1503  0.9907  2.433 ~ 311.4  0.1518  0.5782  Morroniside  Y = 0.0618X + 0.6278  0.9914  2.412 ~ 308.7  0.1507  0.4725  7α-O-methylmorroniside  Y = 0.0288X − 0.0032  0.9991  0.118 ~ 15.08  0.0257  0.0838  7β-O-methylmorroniside  Y = 0.0264X − 0.0053  0.9995  0.119 ~ 15.29  0.0392  0.0931  7α-O-ethylmorroniside  Y = 0.0420X − 0.0056  0.9991  0.118 ~ 15.12  0.0139  0.0627  7β-O-ethylmorroniside  Y = 0.0434X − 0.0044  0.9994  0.059 ~ 7.545  0.0146  0.0457  Cornuside  Y = 0.4889X + 0.71  0.9929  0.589 ~ 75.37  0.0588  0.1178  Intra- and inter-day variations were further investigated to determine the precision of the developed method. RSD values for both intra- and inter-day precision were below 9.33% (Table III). The repeatability of the solution was less than 5.98% and the results showed good average recovery from 93.24% to 112.92% for the 11 compounds with RSD < 9.06% (Table III). And the RSD values for stability of these 111 components were less than 13.99%. Taken together, these results validate that this method was precise and accurate enough for achieving a simultaneous quantitative evaluation of the 11 compounds from Fructus Corni. Table III. Precision, Repeatability, Recovery and Stability of 11 Compounds Compound  Precision (RSD, %, n = 6)  Repeatability (RSD, %, n = 6)  Recovery (%, n = 6)  Stability (RSD,%, n = 6)  Intra-day  Inter-day  Mean  RSD  5-HMF  1.89  2.86  N.D.  93.24  1.90  9.27  Gallic acid  2.71  4.06  3.78  112.92  3.62  11.64  Sweroside  7.32  5.17  4.65  109.72  6.99  13.91  Cornin  3.49  5.20  4.47  95.20  8.22  13.22  Loganin  3.27  4.27  3.48  95.40  9.06  10.12  Morroniside  1.55  3.39  5.31  96.30  6.37  4.68  7α-O-methylmorroniside  2.29  9.33  4.50  97.68  8.45  10.16  7β-O-methylmorroniside  3.59  5.74  5.98  110.21  7.59  5.78  7α-O-ethylmorroniside  3.62  5.69  N.D.  103.22  1.56  13.99  7β-O-ethylmorroniside  4.22  6.44  2.92  93.61  2.13  11.86  Cornuside  2.50  3.54  1.02  103.19  5.54  7.29  Compound  Precision (RSD, %, n = 6)  Repeatability (RSD, %, n = 6)  Recovery (%, n = 6)  Stability (RSD,%, n = 6)  Intra-day  Inter-day  Mean  RSD  5-HMF  1.89  2.86  N.D.  93.24  1.90  9.27  Gallic acid  2.71  4.06  3.78  112.92  3.62  11.64  Sweroside  7.32  5.17  4.65  109.72  6.99  13.91  Cornin  3.49  5.20  4.47  95.20  8.22  13.22  Loganin  3.27  4.27  3.48  95.40  9.06  10.12  Morroniside  1.55  3.39  5.31  96.30  6.37  4.68  7α-O-methylmorroniside  2.29  9.33  4.50  97.68  8.45  10.16  7β-O-methylmorroniside  3.59  5.74  5.98  110.21  7.59  5.78  7α-O-ethylmorroniside  3.62  5.69  N.D.  103.22  1.56  13.99  7β-O-ethylmorroniside  4.22  6.44  2.92  93.61  2.13  11.86  Cornuside  2.50  3.54  1.02  103.19  5.54  7.29  N.D.: not detected. Sample analysis The optimized and validated method was then used for the quantification of the 11 bioactive compounds in crude Fructus Corni from three different origins and a processed product. All the samples were extracted and analyzed in triplicate. The 11 bioactive compounds in crude Fructus Corni and its processed product were successfully identified by the LC–MS/MS method (Figures 3 and 4). The contents of the 11 compounds in Fructus Corni samples analyzed are listed in Table IV. Table IV. The Mean Contents of the 11 Bioactive Compounds in Crude and Processed Fructus Corni Samples (mg/g + SD, n = 3) Origins  Henan  Hubei  Zhejiang  Processed product  Compounds  5-HMF  N.D.  N.D.  N.D.  0.41 ± 0.14  Gallic acid  0.26 ± 0.04  0.28 ± 0.03  0.18 ± 0.03  0.45 ± 0.03  Sweroside  0.53 ± 0.12  0.46 ± 0.12  0.46 ± 0.19  0.47 ± 0.10  Cornin  0.32 ± 0.10  0.37 ± 0.13  0.40 ± 0.12  0.31 ± 0.13  Loganin  6.75 ± 1.13  6.74 ± 0.71  6.03 ± 0.97  6.39 ± 0.51  Morroniside  7.61 ± 1.33  8.65 ± 1.23  9.43 ± 1.03  7.45 ± 0.49  7α-O-methylmorroniside  0.05 ± 0.01  0.05 ± 0.02  0.05 ± 0.01  0.04 ± 0.005  7β-O-methylmorroniside  0.08 ± 0.01  0.08 ± 0.01  0.07 ± 0.01  0.06 ± 0.02  7α-O-ethylmorroniside  N.D.  N.D.  N.D.  0.08 ± 0.03  7β-O-ethylmorroniside  0.011 ± 0.002  0.008 ± 0.001  N.D.  0.06 ± 0.02  Cornuside  2.28 ± 0.51  2.53 ± 0.60  2.78 ± 0.44  2.22 ± 0.52  Origins  Henan  Hubei  Zhejiang  Processed product  Compounds  5-HMF  N.D.  N.D.  N.D.  0.41 ± 0.14  Gallic acid  0.26 ± 0.04  0.28 ± 0.03  0.18 ± 0.03  0.45 ± 0.03  Sweroside  0.53 ± 0.12  0.46 ± 0.12  0.46 ± 0.19  0.47 ± 0.10  Cornin  0.32 ± 0.10  0.37 ± 0.13  0.40 ± 0.12  0.31 ± 0.13  Loganin  6.75 ± 1.13  6.74 ± 0.71  6.03 ± 0.97  6.39 ± 0.51  Morroniside  7.61 ± 1.33  8.65 ± 1.23  9.43 ± 1.03  7.45 ± 0.49  7α-O-methylmorroniside  0.05 ± 0.01  0.05 ± 0.02  0.05 ± 0.01  0.04 ± 0.005  7β-O-methylmorroniside  0.08 ± 0.01  0.08 ± 0.01  0.07 ± 0.01  0.06 ± 0.02  7α-O-ethylmorroniside  N.D.  N.D.  N.D.  0.08 ± 0.03  7β-O-ethylmorroniside  0.011 ± 0.002  0.008 ± 0.001  N.D.  0.06 ± 0.02  Cornuside  2.28 ± 0.51  2.53 ± 0.60  2.78 ± 0.44  2.22 ± 0.52  N.D.: not detected. Figure 3. View largeDownload slide Representative TIC chromatogram of the 11 targeted compounds from the whole extract of Fructus Coni. Figure 3. View largeDownload slide Representative TIC chromatogram of the 11 targeted compounds from the whole extract of Fructus Coni. Figure 4. View largeDownload slide Typical LC–MS/MS chromatograms of the 11 targeted compounds analyzed in Fructus Coni extract.Note: 1. 5-HMF; 2. gallic acid; 3. sweroside; 4. cornin; 5. loganin; 6. morroniside; 7. 7α-O-methylmorroniside; 8. 7β-O-methylmorroniside; 9. 7α-O-ethylmorroniside; 10. 7β-O-ethylmorroniside; 11. cornuside; 12. internal standard. Figure 4. View largeDownload slide Typical LC–MS/MS chromatograms of the 11 targeted compounds analyzed in Fructus Coni extract.Note: 1. 5-HMF; 2. gallic acid; 3. sweroside; 4. cornin; 5. loganin; 6. morroniside; 7. 7α-O-methylmorroniside; 8. 7β-O-methylmorroniside; 9. 7α-O-ethylmorroniside; 10. 7β-O-ethylmorroniside; 11. cornuside; 12. internal standard. The contents of 11 constituents in Fructus Corni from different origins and one processed product were determined and showed in Figure 5. It is clear that the contents of gallic acid and 7β-O-ethylmorroniside showed significant batch differences, while other nine compounds did not. The quantitative results for these components in crude Fructus Corni from different origins display various degrees of difference, as shown in Figure 6. For instance, contents of gallic acid in Henan and Hubei supplier (0.26 mg/g, 0.28 mg/g) were higher than those provided by Zhejiang supplier (0.17 mg/g). However, the content of morroniside from Henan (7.6 mg/g) was less than that in Zhejiang (9.4 mg/g). In addition, compared to the processed product, some chemical constituents in crude Fructus Corni varied significantly (P < 0.05). For example, the content of gallic acid and 7β-O-ethylmorroniside from crude products were lower than that in the processed product (0.45 and 0.056 mg/g), while other compounds such as 7α-methylmorroniside and 7β-O-methylmorroniside did not show significant changes after procession, as shown in Figure 6. It was previously reported that processing or heating could drastically increase the content of 5-HMF and tannin in Fructus Corni, which was hydrolyzed to generate gallic acid because of high temperature (19). In our study, we observed the most dramatic changes for 7α-O-ethylmorroniside, 5-HMF and gallic acid in crude and processed Fructus Corni, which supported the capability of our method to monitor the content variations of Fructus Corni. Figure 5. View largeDownload slide Batch differences of the 11 compounds in crude Fructu Corni and the effect of processing. Henan, Zhejiang and Hubei suppliers, and the processed product, n = 3. *P < 0.05, **P < 0.01, ***P < 0.001, compared with corresponding processed product.Note: HN, ZJ and HB refer to Henan, Zhejiang and Hubei suppliers. 1, 2, 3 refer to the different batches No. from the same suppliers. Figure 5. View largeDownload slide Batch differences of the 11 compounds in crude Fructu Corni and the effect of processing. Henan, Zhejiang and Hubei suppliers, and the processed product, n = 3. *P < 0.05, **P < 0.01, ***P < 0.001, compared with corresponding processed product.Note: HN, ZJ and HB refer to Henan, Zhejiang and Hubei suppliers. 1, 2, 3 refer to the different batches No. from the same suppliers. Figure 6. View largeDownload slide Comparison of the contents of the 11 compounds in crude Fructu Corni from different origins and a processed product. Each column represents the mean content of 3 samples in the same origin, *P < 0.05, ***P < 0.001. Note: HN, ZJ and HB refer to Henan, Zhejiang and Hubei suppliers. Figure 6. View largeDownload slide Comparison of the contents of the 11 compounds in crude Fructu Corni from different origins and a processed product. Each column represents the mean content of 3 samples in the same origin, *P < 0.05, ***P < 0.001. Note: HN, ZJ and HB refer to Henan, Zhejiang and Hubei suppliers. Although previously isomers have been reported for the methyl and ethylmorroniside, these isomers were not differentiated in previous quantitative studies (20, 22, 27, 29). In our experiment, with the developed method, 7α-O-methylmorroniside, 7β-O-methylmorroniside and 7β-O-ethylmorroniside were all detected in the crude and processed Fructus Corni, while 7α-O-ethylmorroniside was only found in processed product. This finding suggested that 7α-O-ethylmorroniside was an artificial product generated during ethanol extraction. Previously, we have also reported a HPLC-based quantitative method for the simultaneous determination of polar active components in Fructus Corni (30). This largely features a multi-components by single-marker (QAMS) strategy that depends on the calculation of relative correction factors for each target component compared to the internal standard, thereby dose not necessitate the preparation of all standards. Despite the advantages of the method, time-efficient separation of the numerous target components of Fructus Corni, especially concerning the existence of isomers, by HPLC-UV proved challenging. Also, this method may have limitations when applied to the analysis of biological samples. In light of this caveat, the current study makes an important complementary progress by integrating HPLC separation with tandem mass spectrometry, which only takes 18 min for a single run and confers much better sensitivity. Conclusion In this study, a highly selective method for simultaneous identification and determination of the 11 compounds in Fructus Corni using LC–MS/MS was established and validated. With this developed method, we performed a comparative quantitative study of 11 bioactive components in crude Fructus Corni of three different origins as well as a processed product. We contributed to the establishment of a simple and comprehensive method for efficiently integrating the iridoid isomers (7α-O-methylmorroniside, 7β-O-methylmorroniside, and 7α-O-ethylmorroniside, 7β-O-ethylmorroniside) in the quality control of Fructus Corni. This method is expected to find a wide and routine application in the quantitative analysis of crude and processed Fructus Corni. Funding This work was supported by the Project Phase II of Priority Academic Program Development of Jiangsu Higher Education Institutions; Jiangsu Project of Chinese Medicine (No.: FY201503) and Natural Science Foundation of China (No.: 81202580). References 1 Lau, C.H., Chan, C.M., Chan, Y.W., Lau, K.M., Lau, T.W., Lam, F.C., et al.  .; In vitro antidiabetic activities of five medicinal herbs used in Chinese medicinal formulae; Phytotherapy Research , ( 2008); 22: 1384– 1388. Google Scholar CrossRef Search ADS PubMed  2 Wu, Y., Wang, X., Shen, B., Kang, L., Fan, E.; Extraction, structure and bioactivities of the polysaccharides from Fructus corni; Recent Patents on Food, Nutrition and Agriculture , ( 2013); 5: 57– 61. Google Scholar CrossRef Search ADS   3 Park, C.H., Xu, F.H., Roh, S.S., Song, Y.O., Uebaba, K., Noh, J.S., et al.  .; Astaxanthin and Corni Fructus protect against diabetes-induced oxidative stress, inflammation, and advanced glycation end product in livers of streptozotocin-induced diabetic rats; Journal of Medicinal Food , ( 2015); 18: 337– 344. Google Scholar CrossRef Search ADS PubMed  4 Park, C.H., Noh, J.S., Park, J.C., Yokozawa, T.; Beneficial Effect of 7-O-Galloyl-D-sedoheptulose, a polyphenol isolated from Corni Fructus, against diabetes-induced alterations in kidney and adipose tissue of Type 2 diabetic db/db mice; Evidence-based Complementary and Alternative Medicine , ( 2013); 2013: 736856. Google Scholar PubMed  5 Zhou, H.Y., Yang, P.P., Cong, X.D., Zhang, C.R., Cai, B.C.; Comparative study on decoction and dissolution of crude and processed Corni Fructus; Zhongguo Zhong Yao Za Zhi , ( 2013); 38: 3888– 3892. Google Scholar PubMed  6 Telang, N.T., Li, G., Sepkovic, D.W., Bradlow, H.L., Wong, G.Y.; Anti-proliferative effects of Chinese herb Cornus officinalis in a cell culture model for estrogen receptor-positive clinical breast cancer; Molecular Medicine Reports , ( 2012); 5: 22– 28. Google Scholar PubMed  7 Cho, S., Won, C.H., Lee, D.H., Lee, M.J., Lee, S., So, S.H., et al.  .; Red ginseng root extract mixed with Torilus fructus and Corni Fructus improves facial wrinkles and increases type I procollagen synthesis in human skin: a randomized, double-blind, placebo-controlled study; Journal of Medicinal Food , ( 2009); 12: 1252– 1259. Google Scholar CrossRef Search ADS PubMed  8 Sun, H., Li, L., Zhang, A., Zhang, N., Lv, H., Sun, W., et al.  .; Protective effects of sweroside on human MG-63 cells and rat osteoblasts; Fitoterapia , ( 2013); 84: 174– 179. Google Scholar CrossRef Search ADS PubMed  9 Yamabe, N., Noh, J.S., Park, C.H., Kang, K.S., Shibahara, N., Tanaka, T., et al.  .; Evaluation of loganin, iridoid glycoside from Corni Fructus, on hepatic and renal glucolipotoxicity and inflammation in type 2 diabetic db/db mice; European Journal of Pharmacology , ( 2010); 648: 179– 187. Google Scholar CrossRef Search ADS PubMed  10 Wang, S.F., Chen, X.G., Hu, Z.D., Ju, Y.; Analysis of three effective components in Fructus Corni and its preparations by micellar electrokinetic capillary chromatography; Biomedical Chromatography , ( 2003); 17: 306– 311. Google Scholar CrossRef Search ADS PubMed  11 Lee, C.M., Jung, H.A., Oh, S.H., Park, C.H., Tanaka, T., Yokozawa, T., et al.  .; Kinetic and molecular docking studies of loganin and 7-O-galloyl-D-sedoheptulose from Corni Fructus as therapeutic agents for diabetic complications through inhibition of aldose reductase; Archives of Pharmacal Research , ( 2015); 38: 1090– 1098. Google Scholar CrossRef Search ADS PubMed  12 Yokozawa, T., Kang, K.S., Park, C.H., Noh, J.S., Yamabe, N., Shibahara, N., et al.  .; Bioactive constituents of Corni Fructus: the therapeutic use of morroniside, loganin, and 7-O-galloyl-D-sedoheptulose as renoprotective agents in type 2 diabetes; Drug Discovery and Therapeutics , ( 2010); 4: 223– 234. 13 Park, C.H., Noh, J.S., Tanaka, T., Yokozawa, T.; Effects of morroniside isolated from Corni Fructus on renal lipids and inflammation in type 2 diabetic mice; Journal of Pharmacy and Pharmacology , ( 2010); 62: 374– 380. Google Scholar CrossRef Search ADS PubMed  14 Wang, W., Sun, F., An, Y., Ai, H., Zhang, L., Huang, W., et al.  .; Morroniside protects human neuroblastoma SH-SY5Y cells against hydrogen peroxide-induced cytotoxicity; European Journal of Pharmacology , ( 2009); 613: 19– 23. Google Scholar CrossRef Search ADS PubMed  15 Yao, R.Q., Zhang, L., Wang, W., Li, L.; Cornel iridoid glycoside promotes neurogenesis and angiogenesis and improves neurological function after focal cerebral ischemia in rats; Brain Research Bulletin , ( 2009); 79: 69– 76. Google Scholar CrossRef Search ADS PubMed  16 Liang, J., He, J., Zhu, S., Zhao, W., Zhang, Y., Ito, Y., et al.  .; Preparative isolation and purification of iridoid glycosides from Fructus Corni by high-speed countercurrent chromatography; Journal of Liquid Chromatography & Related Technologies , ( 2013); 36: 983– 999. Google Scholar PubMed  17 Sunghwa, F., Sakurai, H., Saiki, I., Koketsu, M.; Iodine-catalyzed etherification of morroniside; Chemical and Pharmaceutical Bulletin , ( 2009); 57: 112– 115. Google Scholar CrossRef Search ADS PubMed  18 Takeda, Y., Tanigawa, N., Sunghwa, F., Ninomiya, M., Hagiwara, M., Matsushita, K., et al.  .; Morroniside cinnamic acid conjugate as an anti-inflammatory agent; Bioorganic & Medicinal Chemistry Letters , ( 2010); 16: 4855– 4857. Google Scholar CrossRef Search ADS   19 Cai, H., Cao, G., Cai, B.; Rapid simultaneous identification and determination of the multiple compounds in crude Fructus Corni and its processed products by HPLC-MS/MS with multiple reaction monitoring mode; Pharmaceutical Biology , ( 2013); 51: 273– 278. Google Scholar CrossRef Search ADS PubMed  20 Cao, G., Zhang, C., Zhang, Y., Cong, X., Cai, H., Cai, B., et al.  .; Global detection and identification of components from crude and processed traditional Chinese medicine by liquid chromatography connected with hybrid ion trap and time-of-flight-mass spectrometry; Journal of Separation Science , ( 2011); 34: 1845– 1852. Google Scholar CrossRef Search ADS PubMed  21 Du, W., Cai, H., Wang, M., Ding, X., Yang, H., Cai, B.; Simultaneous determination of six active components in crude and processed Fructus Corni by high performance liquid chromatography; Journal of Pharmaceutical and Biomedical Analysis , ( 2008); 48: 194– 197. Google Scholar CrossRef Search ADS PubMed  22 Liu, Z., Zhu, Z., Zhang, H., Tan, G., Chen, X., Chai, Y.; Qualitative and quantitative analysis of Fructus Corni using ultrasound assisted microwave extraction and high performance liquid chromatography coupled with diode array UV detection and time-of-flight mass spectrometry; Journal of Pharmaceutical and Biomedical Analysis , ( 2011); 55: 557– 562. Google Scholar CrossRef Search ADS PubMed  23 Ouyang, E., Zhang, C., Li, X.; Determination of 5-hydroxymethyl-2-furaldehyde of crude and processed Fructus Corni in freely moving rats using in vivo microdialysis sampling and liquid chromatography; Pharmacognosy Maganize , ( 2011); 7: 271– 276. Google Scholar CrossRef Search ADS   24 Choi, W.H., Chu, J.P., Jiang, M.H., Baek, S.H., Park, H.D.; Effects of fraction obtained from Korean Corni Fructus extracts causing anti-proliferation and p53-dependent apoptosis in A549 lung cancer cells; Nutrition and Cancer-an International Journal , ( 2011); 63: 121– 129. 25 Wang, X.Y., Sun, L., Qiao, S.Y.; Chemical constituents of glucoside fraction from Liuwei Dihuang Gantang; Zhongguo Zhong Yao Za Zhi , ( 2012); 37: 2576– 2580. Google Scholar PubMed  26 Cao, G., Zhang, C., Zhang, Y., Cong, X., Cai, H., Cai, B.; Screening and identification of potential active components in crude Fructus Corni using solid-phase extraction and LC-LTQ-linear ion trap mass spectrometry; Le Pharmacien Biologiste , ( 2012); 50: 278– 283. 27 Shi, T., Yao, Z., Qin, Z., Ding, B., Dai, Y., Yao, X.; Identification of absorbed constituents and metabolites in rat plasma after oral administration of Shen-Song-Yang-Xin using ultra-performance liquid chromatography combined with quadrupole time-of-flight mass spectrometry; Biomedical Chromatography , ( 2015); 29: 1440– 1452. Google Scholar CrossRef Search ADS PubMed  28 Chinese Pharmacopoeia Commission.; Pharmacopoeia of the People’s Republic of China . Chemical Industry Press, Beijing, ( 2015); p. 27. 29 Cao, G., Cai, H., Zhang, Y., Cong, X., Zhang, C., Cai, B.; Identification of metabolites of crude and processed Fructus Corni in rats by microdialysis sampling coupled with electrospray ionization linear quadrupole ion trap mass spectrometry; Journal of Pharmaceutical and Biomedical Analysis , ( 2011); 56: 118– 125. Google Scholar CrossRef Search ADS PubMed  30 Jiang, Y., Chen, H., Wang, L., Zou, J., Zheng, X., Liu, Z.; Quality evaluation of polar and active components in crude and processed Fructus Corni by quantitative analysis of multicomponents with single marker; Journal of Analytical Methods in Chemistry , ( 2016); 2016: 6496840. Google Scholar CrossRef Search ADS PubMed  © The Author 2017. Published by Oxford University Press. All rights reserved. For Permissions, please email: journals.permissions@oup.com

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