Part Ⅱ Comparison Of The Chemical Profiles And Antioxidant Activities Of Different Parts Of Cultivated Cistanche Deserticola Using Ultra Performance Liquid Chromatography-Quadrupole Time-of-Flight Mass Spectrometry And A 1,1-Diphenyl-2-picrylhydrazyl-Based Assay

For more information: emily.li@wecistanche.com

Click here to get more information about Cistanche

Detailed mass spectral data and the proposed fragmentation patterns were given in Figure 7. The structures of the six PhGs identified above are divided into three moieties: I-caffeic acid (CA), II-aglycone, and III-sugar (Glc and Rha). In the MS/MS spectra, the mass defects corresponding to the neutral cleavages of a caffeoyl group and Glc and Rha residues dominate the fragmentation pathways of PhGs and generate the diagnostic fragment ions (Figure 8). Additional MS/MS characteristic fragment ions at m/z 179, 161, and 135 further suggested that a caffeoyl substituent exists in their structures. Using these fragmentation features, we could confidently propose fragmentation pathways as follows. Acteoside (Figure 7c) produced a fragment at m/z 461 via the loss of a CA moiety and then produced an ion at m/z 315 via a further loss of Rha. The CA moiety was found at m/z 179, and its fragment ions found at m/z 161 and m/z 135 were produced by the loss of H2O and CO2, respectively. Isoacteoside (Figure 7d) produced the same ions as acteoside at m/z 461, 315, 179, 161, and 135. 20 -Acetylacteoside (Figure 7e) produced a fragment at m/z 623 via the loss of acetyl and then generated ions at m/z 461, 315, 179, 161, and 135, which was identical to the fragmentation pattern of acteoside. Meanwhile, 20 -acetylacteoside also produced a fragment at m/z 503 by losing its CA moiety and subsequently lost its Ac group to produce the ion at m/z 461. Tubuloside B (Figure 6f) displayed a similar fragmentation pattern to 20 -acetylacteoside. Echinacoside (Figure 7a) produced a fragment ion at m/z 623 via loss of its CA moiety, and then sequential losses of Rha and Glc moieties yielded the fragment ions at m/z 477 and 315. The fragment ion at m/z 461 was generated by losing the CA and Glc moieties from the precursor ion. The characteristic CA ions at m/z 179, 161, and 135 were also found. Cistanoside A (Figure 7b) produced a fragment ion at m/z 637 via loss of its CA moiety and then generated ions at m/z 491 and 475 by the further loss of Rha and Glc moieties, respectively. Therefore, the use of UPLC-PDA-QTOF/MS in combination could make a major contribution towards precisely elucidating the chemical structures of compounds.

2.7. Correlation between Antioxidants and A8ntioxidant Properties. 

Correlation analysis between the total contents of antioxidants and total antioxidant activities has been described in many studies [33–38]. To the best of our knowledge, there is a lack of studies on comparing the antioxidant properties and PhG contents of cultivated C. deserticola parts using such a concise characterization method. The preliminary results of the six PhG contents described in the “Quantitative analysis” section are summarized in Table 4, and antioxidant activity (IC50) determined using the DPPH assay as described in the “Antioxidant capacity” section are listed in Table 5. First, the total antioxidant contents and activities of each parts are presented in Figure 9. Clearly, there are conspicuous differences between the different parts. In fact, the obtained results indicated that the antioxidant activity of cultivated C. deserticola stems was comparable with that of the stems available on the market. These results suggested that the antioxidant activity of the stem extract may be correlated to its PhG content. Therefore, the phytochemicals in the stem extract of cultivated C. deserticola are proposed to play an important role in its DPPH radical scavenging activity. Overall, our results indicated a strong correlation between total PhG content and antioxidant activity: r = 0.92, ** p < 0.01.

In addition, certain samples with higher total antioxidant contents did not show high antioxidant activities, such as the axis and inflorescence. Meanwhile, the corolla, which had the highest activity of the six parts, had a lower total antioxidant content than the axis and inflorescence. Figure 4, as described in the “Quantitative analysis” section, shows the relative amounts of the PhG compounds to give a preliminary understanding of their individual contributions. In this study, Partial Least Squares (PLS) regression analysis [39–41] was also carried out to compare the correlation between the abundance of each antioxidant and the antioxidant activity in vitro. The PLS model was constructed using the contents of each antioxidant (as the descriptor matrix X) in the fingerprint chromatograms at 330 nm and the antioxidant activities (as the response matrix Y) of all the parts. The coefficient plots and variable importance in projection (VIP) values obtained using SIMCA-P+ software (Version 13.0, Umetrics, Umea, Sweden) are shown in Figure 10. Notably, the inverse of the IC50 values (1/IC50) was selected as the Y variable to establish the models because lower IC50 values represent stronger antioxidant activity. According to the obtained regression coefficient plot in Figure 10A, the linear regression models show that all the antioxidants except acteoside were positively correlated with 1/IC50. The obtained calibration model of PLS is expressed by the regression equation: Y = 0.50X1 + 0.57X2 − 0.11X3 + 0.32X4 + 0.58X5 + 0.24X6. VIP values reflect the importance of variables in the model. the larger the VIP, the more relevant for sample classification. As our results showed, 20 -acetylacteoside was an important contributor to the antioxidant activity (Figure 10B). This finding was in agreement with a study by Yang et al [20].

3 Materials and Methods

3.1 Plant Materials. Cultivated C. deserticola was generously provided by the Bencao Congrong planting base in Yongning County, Ningxia Province, and was authenticated by Prof. Jun Chen of the Institute of Medicinal Plant Development, Chinese Academy of Medical Sciences, Beijing, China. The six different parts of cultivated C. deserticola include the succulent stem (R1), inflorescence axis (R2), inflorescence (R3), flower (without seed) (R4), inflorescence stalk (R5), and corolla (R6). The detailed sample information is shown in Figure 11. Each part was dried separately in a dryer, crushed into powder, passed through a 40-mesh sieve, and then loaded into airtight containers and stored in a desiccator. Voucher specimens of these samples were deposited at the Institute of Medicinal Plant Development, Chinese Academy of Medical Sciences, Beijing, China.

3.2. Chemicals and Reagents. Isoacteoside (Lot 130809), acteoside (Lot 130421), and echinacoside (Lot 121027) were purchased from Chengdu Pure-Chem Standard Co. Ltd. (Beijing, China), Cistanoside A, 20 -Acetylacteoside and tubuloside B were isolated and purified from cultivated C. deserticola in our laboratory, with more than 98% purity (HPLC) and their structure was also confirmed based on HR-MS, 1H-NMR, and 13C-NMR and compared with previous literature. Their structures are displayed in Table 7. DPPH, L-ascorbic acid, and Trolox were purchased from Sigma-Aldrich (St. Louis, MO, USA). The solvents, acetonitrile, and methanol were of HPLC grade from Merck (Darmstadt, Germany), and formic acid with a purity of 96% was of HPLC grade (Tedia, Fairfield, OH, USA). Deionized water (18 MΩ) was prepared by distilled water through a Milli-Q system (Millipore, Milford, MA, USA). Other reagents and chemicals were of analytical grade.

3.3. Preparation of Solutions

3.3.1. Standard Solutions.A mixed standard stock solution containing echinacoside (501 µg/mL), cistanoside A (64 µg/mL),acteoside (239 µg/mL), isoacteoside (22.6 µg/mL), 20 -acetylacteoside (278 µg/mL) and tubuloside B(46.2 µg/mL) was prepared in methanol. Stock solutions were diluted with methanol to obtain a series of appropriate concentrations that were used as working solutions to develop a calibration curve and were stored at 4 ◦C prior to use.

3.3.2. Sample Solutions. An accurately weighed powder (40 mesh, 1 g) of six different parts of cultivated C. deserticola was added with 50 mL methanol, respectively. After being immersed for 30 min at room temperature, the mixtures were weighed and then extracted in an ultrasonic water bath (40 kHz, 250 W, Kunshan, China) at room temperature for 40 min. After cooling, additional methanol was added to compensate for the lost weight. All solutions were filtered through 0.22 µm membrane filters prior to injection. An aliquot (3 µL) of the supernatant solution was injected into the UPLC for analysis. Each extract was injected in triplicate.

3.3.3. DPPH Solutions. The DPPH radical stock solution (5 × 10−3 mol/L) was prepared by dissolving an accurately weighed DPPH sample in methanol immediately before the experiments and was protected from light. Then, the stock solution was freshly diluted with methanol to obtain a standard solution (1 × 10−4 mol/L).

3.4. UPLC-PDA Analysis Condition. UPLC analysis was conducted on a Waters Acquity UPLC system (Waters, Milford, MA, USA)comprising a column heater, a sample manager, a binary solvent manager, and a photodiode array detector. Chromatographic separation was performed on an Acquity UPLC BEH C18 column (1.7 µm,2.1 mm × 100 mm; Waters) by fixing the column heater at 30 ◦C. The mobile phase consisted of acetonitrile (A) and water containing 0.2% formic acid (B) at a flow rate of 0.4 mL/min. A gradient elution programme was employed as follows: 5% A at 0–2 min, 5–15% A at 2–4 min, 15% A at 4–6 min,15–20% A at 6–10 min, 20–35% A at 10–15 min, 35% A at 15–18 min, and 35–5% A at 18–18.1 min. The composition was then held at 5% A for an additional 10 min for re-equilibration. The detection wavelength was set at 330 nm.

3.5. UPLC-ESI-Q/TOF-MS Analysis. Condition The UPLC conditions were the same as those described in Section 3.4. MS analysis was performed on a Waters Xevo G2-XS QTOF mass spectrometer (Waters MS Technologies, Manchester, UK) equipped with an electrospray interface (ESI) in negative-ion mode with a full scan MS spectrum over the m/z range of 50–1200. In the source, the capillary voltage was 2000 V, the source temperature was 120 ◦C, the desolvation temperature was 450 ◦C, the cone voltage was 40 V, the cone gas was 30 L/h and the desolvation gas flow rate was 600 L/h. MS/MS experiments were conducted using MSE, and the collision energy was 20–40 V. All MS data were collected using the Lock Spray system to ensure mass accuracy and reproducibility. The [M − H]− ion of leucine-enkephalin at m/z 554.2615 was used as the lock mass in negative ESI mode. Data were acquired, analyzed, and processed using Waters Mass Lynx 4.1 software (Waters MS Technologies, Manchester, UK).

3.6. Offlfline DPPH Radical Scavenging Assay. The total antioxidant activity of the plant extracts was assessed using an offline DPPH radical-scavenging assay. Sample solutions (2 mL) at various concentrations were mixed with 2 mL of 1 × 10−4 mol/L DPPH solution (in methanol). All samples were shaken and allowed to stand in the dark at room temperature for 60 min. The reduction in DPPH free radicals was measured by reading the absorbance at 517 nm against a blank (methanol without sample) on an ELISA reader (TECAN, Growing, Austria). Methanol (2 mL) alone was used as the control of this experiment. Trolox and L-ascorbic acid were used as the positive controls. The radical scavenging activity (% inhibition) of the tested samples, which was expressed as the DPPH scavenging percentage, was calculated using the following formula: % inhibition = [(A control − A sample)/Acontrol] × 100. The total antioxidant activity was calculated by plotting the percent inhibition against the sample concentration and was represented as the sample concentration required to scavenge 50% of the DPPH radicals (IC50). All tests were carried out in triplicate, and the IC50 values were reported as the means ± SD.

3.7. UPLC-PDA Coupled with Pre-Column DPPH Assay. In this study, after 2 mL of DPPH solution (3 × 10−3 mol/L) and 1 mL of sample solutions (12 mg/mL) of different parts of cultivated C. deserticola were mixed, UPLC-PDA was employed to further screen the antioxidant profiles and identify the antioxidant components by detecting changes in the characteristic peaks of the sample solutions before and after the DPPH free radical scavenging reaction. For each different plant part, 10 µL aliquots of the sample solution before and after the DPPH assay were separately injected into the UPLC system under the same conditions as those described in Section 3.4. After the sample solutions were mixed with DPPH solution, changes in the characteristic peaks of the sample solutions were detected at 330 nm.

3.8. Data Analysis. Analytical data are expressed as the mean ± standard deviation (SD) of triplicate independent measurements. Bivariate correlation analysis was performed to study the relationship between the total PhG content and the antioxidant activity by determining the Pearson correlation coefficient (r)using IBM SPPS Statistics (Version 19; International Business Machines Corp., New York, NY, USA). The IC50 values were also calculated by probit analyses using SPPS Statistics 19. The DPPH free-radical scavenging rate and total PhG content were plotted using OriginPro 8.5.1 (Origin Lab Corp., Northampton, MA, USA). Partial least squares (PLS) regressions were determined using SIMCA-P+software (Version 13.0, Umetrics, Umea, Sweden).

4. Conclusions

Different parts of cultivated C. deserticola, a widely used herbal medicine, were investigated for their qualities. First, characteristic fingerprints of six different parts of the cultivated Cistanche deserticola samples were generated and evaluated using SES and simultaneous analysis of the contents of six marker compounds. Second, quantifying the six major compounds using UPLC demonstrated the distribution characteristics of these compounds in the different parts. However, the results could not be directly related to the activity of herbal medicine. Therefore, a better method for simultaneously determining the quality and activity of cultivated C. deserticola samples was necessary. First, we used an offline DPPH radical scavenging assay to evaluate the antioxidant activities of cultivated Cistanche deserticola extracts and found the highest activity in the stem extract. Furthermore, UPLC-PDA coupled with the pre-column DPPH assay was applied to screen and evaluate the antioxidants in the cultivated Cistanche deserticola stem extracts. This method not only provided more chemical information but also afforded some antioxidant information that could be used to identify and assess herbal medicines. Second, the active components in the stem extracts were separated and identified using UPLC-PAD-QTOF/MSto confirm the number and position of substituent groups and to improve the accuracy of the structural analysis. Finally, a relationship between the chemical components and in vitro antioxidant activity was established and validated using the PLS model.

In conclusion, for the first time, we have compared the chemical composition profiles and antioxidant activities of different parts of cultivated C. deserticola samples. These findings were of special interest since the distribution of PhG compounds, PhG content and their contribution to the antioxidant of different parts of cultivated C. deserticola samples have not been reported until the present study. Furthermore, PLS analysis was first established to give a preliminary understanding of six PhG’s individual contributions to the antioxidant activity. This approach may give us some new insights into the comprehensive quality assessment of cultivated C. deserticola. Finally, this work represents the first report of a strong correlation between the DPPH radical scavenging activity and total PhG content of the different parts from cultivated C. deserticola (r = 0.92). Among these different parts, the stems were found to be rich in PhGs and to have strong antioxidant activity. In particular, the discardedaerial parts of cultivated C. deserticola also contained some bioactive compounds and might be used as an alternative dietary supplement. We hope that the results will provide useful information for future utilization of cultivated C. deserticola. To the best of our knowledge, this is the first report on the rapid identification and quantification of natural antioxidants in different parts of cultivated Cistanche deserticola using UPLC-PAD-QTOF/MS, offline DPPH method and the UPLC-PAD-pre-columnDPPH method. According to our results, the method developed here could provide a powerful and meaningful tool for comprehensive quality control of complex herbal medicines. In our country, under the guidance of the “One Belt, One Road” initiative, this traditional crop with health benefits could be used to design new products with worldwide applications.

Acknowledgments

The authors gratefully acknowledge the financial supports of the Chinese Academy of Medical Sciences (CAMS) Innovation Fund for Medical Sciences (CIFMS) (No. 2016-I2M-1-012) and the NationalNatural Science Foundation of China (No. 31570344).

Author Contributions

Yue Shi and Xiaoming Wang participated in the research design. Xiaoming Wang, Jinfang Wang, Huanyu Guan, Rong Xu, Xiaomei Luo, Meifeng Su, Xiaoyan Chang, Wenting Tan, and Jun Chen were responsible for performing the experiments. Yue Shi, Xiaoming Wang, and Jinfang Wang performed data analysis. Yue Shi and Xiaoming Wang contributed to the writing and editing of the manuscript. Conflicts of Interest: The authors declare no conflicts of interest.

References

Wang, T.; Zhang, X.; Xie, W. Cistanche deserticola Y. C. Ma, “Desert Ginseng”: A Review. Am. J. Chin. Med. 2012, 40, 1123–1141. [CrossRef] [PubMed]

2. Li, Z.; Lin, H.; Gu, L.; Gao, J.; Tzeng, C. Herba Cistanche (Rou Cong-Rong): One of the Best Pharmaceutical Gifts of Traditional Chinese Medicine. Front. Pharmacol. 2016, 7, 41. [CrossRef] [PubMed]

3. Gu, C.; Yang, X.; Huang, L. Cistanches Herba: A Neuropharmacology Review. Front. Pharmacol. 2016, 7, 289. [CrossRef] [PubMed]

4. Xu, X.; Zhang, Z.; Wang, W.; Yao, H.; Ma, X. Therapeutic Effect of Cistanoside A on Bone Metabolism of Ovariectomized Mice. Molecules 2017, 22, 197. [CrossRef] [PubMed]

5. Wang, D.; Wang, H.; Gu, L. The Antidepressant and Cognitive Improvement Activities of the Traditional Chinese Herb Cistanche. Evid.-Based Complement. Altern. Med. 2017, 2017, 1–9. [CrossRef] [PubMed]

6. Xue, Z.; Yang, B. Phenylethanoid Glycosides: Research Advances in Their Phytochemistry, Pharmacological Activity and Pharmacokinetics. Molecules 2016, 21, 991. [CrossRef] [PubMed]

7. Jiang, Y.; Tu, P. Analysis of chemical constituents in Cistanche species. J. Chromatogr. A 2009, 1216, 1970–1979. [CrossRef] [PubMed]

8. Xiong, Q.; Kadota, S.; Tani, T. Antioxidative effects of phenylethanoids from Cistanche deserticola. Biol. Pharm. Bull. 1996, 19, 1580–1585. [CrossRef] [PubMed]

9. Ma, Z.; Yang, Z.; Li, P.; Li, C. Simultaneous Determination of Eight Phenylethanoid Glycosides in Different Species of the Genus Cistanche by High-Performance Liquid Chromatography. J. Liq. Chromatogr. Relat. Technol. 2008, 31, 2838–2850. [CrossRef]

10. Jiang, Y.; Li, S.P.; Wang, Y.T.; Chen, X.J.; Tu, P.F. Differentiation of Herba Cistanches by fingerprint with high-performance liquid chromatography-diode array detection-mass spectrometry. J. Chromatogr. 2009, 1216, 2156–2162. [CrossRef] [PubMed]

11. Lu, D.; Zhang, J.; Yang, Z.; Liu, H.; Li, S.; Wu, B.; Ma, Z. Quantitative analysis of Cistanches Herba using high-performance liquid chromatography coupled with diode array detection and high-resolution mass spectrometry combined with chemometric methods. J. Sep. Sci. 2013, 36, 1945–1952. [CrossRef] [PubMed]

12. Song, Q.; Li, J.; Liu, X.; Zhang, Y.; Guo, L.; Jiang, Y.; Song, Y.; Tu, P. Home-made online hyphenation of pressurized liquid extraction, turbulent flow chromatography, and high-performance liquid chromatography, Cistanche deserticola as a case study. J. Chromatogr. 2016, 1438, 189–197. [CrossRef] [PubMed]

13. Han, L.; Boakye-yiadom, M.; Liu, E.; Zhang, Y.; Li, W. Structural Characterisation and Identififi cation of Phenylethanoid Glycosides from Cistanches deserticola Y. C. Ma by UHPLC/ESI–QTOF–MS/MS. Phytochem. Anal. 2012, 23, 668–676. [CrossRef] [PubMed]

14. Zheng, S.; Jiang, X.; Wu, L.; Wang, Z.; Huang, L. Chemical and Genetic Discrimination of Cistanches Herba Based on UPLC-QTOF/MS and DNA Barcoding. PLoS ONE 2014, 9, 1–11. [CrossRef] [PubMed]

15. Song, Y.; Song, Q.; Li, J.; Zhang, N.; Zhao, Y.; Liu, X.; Jiang, Y.; Tu, P. An integrated strategy to quantitatively differentiate chamomile between Cistanche deserticola and C. tubulosa using high-performance liquid chromatography-hybrid triple quadrupole-linear ion trap mass spectrometry. J. Chromatogr. 2016, 1429, 238–247. [CrossRef] [PubMed]

16. Niu, Y.; Yin, L.; Luo, S.; Dong, J.; Wang, H.; Hashi, Y.; Chen, S. Identification of the Anti-oxidants in Flos Chrysanthemi by HPLC-DAD-ESI/MSN and HPLC Coupled with a Post-column Derivatisation System. Phytochem. Anal. 2013, 24, 59–68. [CrossRef] [PubMed]

17. Chen, L.X.; Hu, D.J.; Lam, S.C.; Ge, L.; Wu, D.; Zhao, J.; Long, Z.R.; Yang, W.J.; Fan, B.; Li, S.P. Comparison of antioxidant activities of different parts from snow chrysanthemum (Coreopsis tinctoria Nutt.) and identification of their natural antioxidants using high-performance liquid chromatography coupled with diode array detection and mass spectrometry and 2,20 -azinobis(3-ethylbenzthiazoline-sulfonic acid) diammonium salt-based assay. J. Chromatogr. 2016, 1428, 134–142. [PubMed]

18. Fu, M.; Xu, Y.; Chen, Y.; Wu, J.; Yu, Y.; Zou, B.; An, K.; Xiao, G. Evaluation of bioactive flavonoids and antioxidant activity in Pericarpium Citri Reticulate (Citrus reticulata “Chachi”) during storage. Food Chem. 2017, 230, 649–656. [CrossRef] [PubMed]

19. Zhang, X.; Xu, M.; Zhang, J.; Wu, L.; Liu, J.; Si, J. Identification and evaluation of antioxidant components in the flowers of five Chimonanthus species. Ind. Crop. Prod. 2017, 102, 164–172. [CrossRef]

20. Yang, J.; Hu, J.; Rena, K.; Ka, S.; Du, N. Structure-activity relationships of phenylethanoid glycosides in plants of Cistanche salsa on antioxidative activity. J. Chin. Med. Mater. 2009, 32, 1067–1069.

21. He, W.; Fang, T.; Tu, P. Research progress on pharmacological activities of echinacoside. China J. Chin. Mater. Med. 2009, 34, 476–479.

22. Li, Y.; Chen, J.; Li, Y.; Li, P. Identification and quantification of free radical scavengers in the flower buds of Lonicera species by online HPLC-DPPH assay coupled with electrospray ionization quadrupole time-of-flight tandem mass spectrometry. Biomed. Chromatogr. 2012, 26, 449–457. [CrossRef] [PubMed]

23. Yao, H.; Chen, Y.; Shi, P.; Hu, J.; Li, S.; Huang, L.; Lin, J.; Lin, X. Screening and quantitative analysis of antioxidants in the fruits of Livistona Chinensis R. Br using HPLC-DAD-ESI MS coupled with pre-column DPPH assay. Food Chem. 2012, 135, 2802–2807. [CrossRef] [PubMed]

24. Wang, Y.; Hao, H.; Wang, G.; Tu, P.; Jiang, Y.; Liang, Y.; Dai, L.; Yang, H.; Lai, L.; Zheng, C.; et al. An approach to identifying sequential metabolites of a typical phenylethanoid glycoside, echinacoside, based on liquid chromatography-ion trap-time of flight mass spectrometry analysis. Talanta 2009, 80, 572–580. [CrossRef] [PubMed]

25. Qi, M.; Xiong, A.; Li, P.; Yang, Q.; Yang, L.; Wang, Z. Identification of acteoside and its major metabolites in rat urine by ultra-performance liquid chromatography combined with electrospray ionization quadrupole time-of-flight tandem mass spectrometry. J. Chromatogr. B 2013, 940, 77–85. [CrossRef] [PubMed]

26. Li, W.; Sun, X.; Song, H.; Ding, J.; Bai, J.; Chen, Q. HPLC/Q-TOF-MS-Based Identification of Absorbed Constituents and Their Metabolites in Rat Serum and Urine after Oral Administration of Cistanche deserticola Extract. J. Food Sci. 2015, 80, H2079–H2087. [CrossRef] [PubMed]

27. Li, Y.; Zhou, G.; Xing, S.; Xing, S.; Tu, P.; Li, X. Identification of Echinacoside Metabolites Produced by Human Intestinal Bacteria Using Ultraperformance Liquid Chromatography-Quadrupole Time-of-Flight Mass Spectrometry. J. Agric. Food. Chem. 2015, 63, 6764–6771. [CrossRef] [PubMed]