Part 2 Chemical And Genetic Discrimination Of Cistanches Herba Based On UPLC-QTOF/MS And DNA Barcoding

Contact: Audrey Hu Whatsapp/hp: 0086 13880143964 Email: audrey.hu@wecistanche.com

OPLS-DA and marker identification To identify potential chemical markers for discrimination of the two species, the S-plot of OPLS-DA was generated (Fig. 3). In the S-plot, each point represents one tR–m/z ion pair. The X and Y axes represent the contribution and confidence of the ion, respectively; the farther the distance the ion tR–m/z pair points from zero, the larger the contribution/confidence of this icon is to the difference between the two groups. Thus, the tR–m/z ion pointing to the two ends of the ‘S’ represents the characteristic markers with the highest confidence in each group. The OPLS-DA results showed that UPLC-QTOF/MS could be used to distinguish Cistanche deserticola from Cistanche tubulosa (Fig. 3). A total of six credible and significant markers were determined to facilitate the discrimination of these groups (Table 3). The identities of three potential markers were tentatively assigned. The components correlated with these three ions were tentatively identified as isomers of campneoside II, cistanoside C, and cistanoside A. The marker compounds a, b and c could be used to distinguish the two plant species, as the ion intensities of a and b in Cistanche deserticola was higher than in Cistanche tubulosa (Fig. 4A, 4B), and marker c could be detected in Cistanche tubulosa, but not in Cistanche deserticola (Fig. 4C).

DNA barcoding: sequence information and identification efficiency

Sequence information was shown in Table 4. The average genetic distance of psbA-trnH (0.1732) was larger than the other two regions (0.0740, 0.1197) significantly. The average GC content of psbA-trnH (20.64%) was smaller than the other two regions (55.00%, 55.00%). Though the success rate of ITS and ITS2 was not obtained in this study, the psbA-trnH region performed well in PCR amplification and sequencing (100%, 87.23%). Identification efficiency was achieved by BLAST1 analysis and the nearest distance method, and mainly reflected the success rate of the barcodes. The psbA-trnH region was clearly higher than the other two barcodes in identification efficiency based on two methods. The shortage of sequences is most likely the reason that the ITS region exhibited 100% identification efficiency based on the BLAST1 method, and 0 based on the nearest-distance method.

Analysis of genetic divergence using six parameters

Six parameters were used to analyze intra-specific variation and interspecific divergence using three barcodes (Table 5). The significant difference between inter-and intra-specific variations was indicative of the utility of the DNA barcodes. Here, the minimum interspecific distance of three barcodes was all higher than the maximum intraspecific distance. Moreover, the psbA-trnH region had a larger maximum intraspecific distance and average interspecific distance than the other two barcodes indicating that psbA-trnH region performed well in discrimination of two origins of Cistanches Herba.

Analysis of barcoding gap to identify

Cistanche deserticola and Cistanche tubulosa The barcoding gap presents the remarkable variation of inter and intra-species and demonstrates separate, non-overlapping distributions between intra- and inter-specific samples. In this study (Fig. 5), the distance range was set to 0–0.45, because the greatest K2P distance of psbA-trnH between Cistanche deserticola and Cistanche tubulosa was close to 0.45. The three barcodes exhibited distinct gaps in the distributions of intra- and inter-specific variation. Furthermore, the gap of psbA-trnH was significantly larger than the other two barcodes. Therefore, the psbA-trnH region could be an ideal barcode for discriminating two origins of Cistanches Herba.

Neighbor-joining (NJ) tree

An NJ tree illustrates the relationship among species and facilitates the determination of their clustering. In this study, NJ tree of three barcodes was built based on K2P model (Fig. 6). The results demonstrated that two origins of Cistanches Herba clustered into two clades separately. Thus, the NJ tree clearly distinguished between Cistanche deserticola and Cistanche tubulosa.

Discussion and Conclusions

Cistanches Herba is an important medicinal material commonly used to nourish the Asian community [38]. However, the two origins of Cistanches Herba, Cistanche deserticola and Cistanche tubulosa, have different chemical compositions and pharmacological activities respectively. Concurrently, the two origins differ in clinical application and the commodity market. The classification of Cistanche is confused and massive substitutes and adulterants flood the market due to the shortage of resources and special growing environment for Cistanches Herba. Genus of Cistanche is accepted to include four species and one variant: Cistanche deserticola, Cistanche tubulosa, Cistanche sinensis, Cistanche salsa, and Cistanche salsa var. albiflora [39]. Researchers in Japan considered the origin of Cistanches Herba as Cistanche salsa [40–43], while it was identified as Cistanche deserticola by Tu [44–46]. Therefore, it is confused in the classification of Cistanche, and it is hard to discriminate the two origins of Cistanches Herba. Traditional methods for quality control of Cistanches Herba are morphological identification [47,48], microscopic identification [49] and TLC (Thin-Layer Chromatography) [50,51], FTIR (Fourier Transform Infrared Spectroscopy) [14], HPLC (High Performance Liquid Chromatography) [52,53]. The morphological and microscopic method can easily differentiate species from different genera or families that possess big differences in morphological and microscopic characteristics, while it is hard to distinguish sibling species. TLC and FTIR can clearly discriminate species that possess different kinds of chemical compositions, whereas it is difficult to determine the chemical component and content. HPLC is mainly used for differentiating species with different chemical elements contents, nevertheless, the time of analysis is longer and the sensitivity is relatively lower compared to UPLC. Correspondingly, UPLC-QTOF/MS technology was faster and more accurate in determining chemical composition than other chemical methods. Molecular identification methods exhibit well in discrimination based on the genetic variation, such as SDS-PAGE (Sodium Dodecyl Sulfate-Polyacrylamide Gel Electrophoresis) [54], AFLP (Amplified Fragment Length Polymorphism) [55,56]. However, these molecular methods are not easy to operate and are not universal. Correspondingly, DNA barcoding could discriminate species more universally, quickly, and accurately than other molecular methods. For the species from the same genus and close genetic relationship, those methods alone may not perform well in identification.

Here, we combined UPLC-QTOF/MS and DNA barcoding in identifying Cistanche deserticola and Cistanche tubulosa and evaluated the chemical and molecular markers that would allow them to be discriminated against. 23 qualified mass peaks were detected and 16 were identified by using UPLC–QTOF/ MS, and three potential marker compounds were firstly found to facilitate the discrimination of two origins by PCA and OPLS-DA analysis. Furthermore, four indicators were assessed by DNA barcoding technology in terms of their ability to differentiate two origins: Identification efficiency, genetic efficiency, barcoding gap, and NJ tree analysis. The psbA-trnH region was supported as a suitable DNA barcode for discriminating Cistanche deserticola and Cistanche tubulosa. In conclusion, we firstly established a new molecular and chemical analysis-combined method for discriminating and quality control in two origins of Cistanches Herba. DNA barcoding can discriminate two origins in genetic variation and authenticate species universally and accurately; UPLC-QTOF/MS technology can analyze chemical composition to evaluate the quality of medicinal materials rapidly and accurately. The combined method of DNA barcoding and UPLC-QTOF/MS technology guarantee the identification in multiple sources of medicinal materials more accurately and scientifically and may serve as a method for identifying other confusing species or genera in classification.

Author Contributions

Conceived and designed the experiments: LFH SHZ LBW. Performed the experiments: SHZ LBW. Analyzed the data: SHZ LBW. Contributed reagents/materials/analysis tools: LFH. Wrote the paper: SHZ XJ LBW. Check the manuscript: ZHW.

Cistanche deserticola has many effects, click here to know more

References

1. Tu PF, Lou ZC, Li SC, Wang CS, Jiang WY, et al. (1996) Comparison on the kidney nourishing and yang strengthening the efficacy of three different species of herba cistanches. Journal of Chinese medicinal materials 19: 420–421.

2. Zhang Y, Wu H, Wang SN, Zheng HC (1994) Comparison on the kidney nourishing and yang strengthening functions of three different species of herba cistanches. China Journal of Chinese Materia Medica 19: 169–171.

3. Lei L, Song ZH, Tu PF (2003) Advances in research of chemical constituents in plants of Cistanche Huffing. et Link. Chinese Traditional and Herbal Drugs 34: 473–476.

4. Yoshikawa M, Matsuda H, Morikawa T, Xie H, Nakamura S, et al. (2006) Phenylethanoid aminoglycosides and acylated oligosugars with vasorelaxant activity from Cistanche tubulosa. Bioorg Med Chem 14: 7468–7475.

5. Shimoda H, Tanaka J, Takahara Y, Takemoto K, Shan SJ, et al. (2009) The hypocholesterolemic effects of Cistanche tubulosa extract, a Chinese traditional crude medicine, in mice. Am J Chin Med 37: 1125–1138.

6. Yamada P, Iijima R, Han J, Shigemori H, Yokota S, et al. (2010) Inhibitory effect of acteoside isolated from Cistanche tubulosa on chemical mediator release and inflammatory cytokine production by RBL-2H3 and KU812 cells. Planta Med, 1512–1518.

7. Morikawa T, Pan Y, Ninomiya K, Imura K, Matsuda H, et al. (2010) Acylated phenylethanoid aminoglycosides with hepatoprotective activity from the desert plant Cistanche tubulosa. Bioorg Med Chem 18: 1882–1890.

8. Morikawa T, Pan Y, Ninomiya K, Imura K, Yuan D, et al. (2010) Iridoid and acyclic monoterpene glycosides, kankanosides L, M, N, O, and P from Cistanche tubulosa. Chem Pharm Bull (Tokyo) 58: 1403–1407.

9. He YP, Yin ZZ, Tu PF, Lou ZC (1997) Identification and survey of commercial crude drug of herba cistanche. Journal of Chinese medicinal materials 20: 117– 122.

10. Zhu SY (2001) Pharmacognostical identification of Cistanche tubulosa a confused species of Cistanche deserticola. Journal of Chinese medicinal materials 24: 24–25.

11. Xu XQ, Ni H, Wei HY, Jia XG, Zhang BG, et al. (2013) Microscopic identification of three herba cistanches in Xin Jiang. Shizhen Medicine and Materia Research 24: 881–883.

12. Zhang M, Fu XL, Wang SY (2004) Microscopic identification of commercial Herba Cistanches by digital imaging technique. Journal of Chinese medicinal materials 27: 400–402.

13. Li JS, Yao ZQ, Yu MX (2000) Study on identification of herba cistanches and adulterants. Shizhen Medicine and Materia Research 11: 317–318.

14. Xu R, Sun SQ, Liu YG, Chen J, Liu TN, et al. (2010) FTIR and 2D-IR spectroscopic studies on different sources of herba cistanches. Guang Pu Xue Yu Guang Pu Fen Xi 30: 897–900.

15. Chen J, Sun SQ, Xu R, Liu YG, Yu J, et al. (2009) Identification of Cistanche deserticola from Boschniakla rossica and Cynomorium solarium using FTIR and two-dimensional correlation IR spectroscopy. Guang Pu Xue Yu Guang Pu Fen Xi 29: 1502–1507.

16. Yang HC, Xia PX, Huang JX, Yuan HZ (2008) Study on HPLC fingerprint of Cistanche deserticola. Pharm Care & Res 8: 255–257.

17. Xie JN, Zhao MB, Wu FW, Tu PF (2005) Chromatographic fingerprint of Cistanche deserticola by HPLC. Chinese Traditional and Herbal Drugs 36: 268–271.

18. Han JP, Song JY, Liu C, Chen J, Qian J, et al. (2010) Identification of Cistanche species (Orobanchaceae) based on sequences of the plastid psbA-trnH intergenic region. Acta Pharmaceutica Sinica 45: 126–130.

19. Shi HM, Wang J, Wang MY, Tu PF, Li XB (2009) Identification of Cistanche Species by Chemical and Inter-simple Sequence Repeat Fingerprinting. Biol. Pharm. Bull., 32: 142–146.

20. Han LF, Yiadom MB, Liu EW, Zhang Y, Li W, et al. (2012) Structural Characterisation and Identification of Phenylethanoid Glycosides from Cistanches deserticola Y.C. Ma by UHPLC/ESI-QTOF- MS/MS. Phytochemical analysis 23: 668–676.

21. Jiang X, Huang LF, Wu LB, Wang ZH, Chen SL (2012) UPLC-QTOF/MS Analysis of Alkaloids in Traditional Processed Coptis chinensis Franch. Evid Based Complement Alternat Med 2012: 942384.

22. Wu LB, Jiang X, Huang LF, Chen SL (2013) Processing Technology Investigation of Loquat (Eriobotrya japonica) Leaf by Ultra-Performance Liquid Chromatography- Quadrupole Time-of-Flight Mass Spectrometry Combined with Chemometrics. PLoS ONE 8: e64178.

23. Hebert PD, Ratnasingham S, Waard JR (2003) Barcoding animal life: cytochrome c oxidase subunit 1 divergences among closely related species. Proc. R. Soc. Lond. B., 270 Suppl 1: S96–99.

24. Hebert PD, Cywinska A, Ball SL, Waard JR (2003) Biological identifications through DNA barcodes. Proc Biol Sci., 270: 313:321.

25. Kress WJ, Wurdack KJ, Zimmer EA, Weigt LA, Janzen DH (2005) Use of DNA barcodes to identify flowering plants. Proc Natl Acad Sci., 102: 8369–8374.

26. Chen S, Yao H, Han J, Liu C, Song J, et al. (2010) Validation of the ITS2 region as a novel DNA barcode for identifying medicinal plant species. PLoS One 5: e8613.

27. Tamura K, Peterson D, Peterson N, Stecher G, Nei M, et al. (2011) MEGA5: Molecular Evolutionary Genetics Analysis Using Maximum Likelihood, Evolutionary Distance, and Maximum Parsimony Methods. Mol Biol Evol 28: 2731–2739.

28. Meyer CP, Paulay G (2005) DNA barcoding: error rates based on comprehensive sampling. PLoS Biol 3: e422.

29. Kobayashi H, Karasawa H, Miyase T, Fukushima S (1984) Studies on the Constituents Cistanche Herba.IV. Isolation and Structures of Two New Phenylpropanoid Glycosides, Cistanosides C and D. Chem Pharm Bull 32: 3880–3885.

30. Kobayashi H, Karasawa H, Miyase T, Fukushima S (1985) Studies on the Constituents of Cistanche Herba. VI. Isolation and Structures of a New Iridoid Glycoside, 6-Deoxyeatalpol. Chem Pharm Bull 33: 3645–3650.

31. Jiang Y, Li SP, Wang YT, Chen XJ, Tu PF (2009) Differentiation of Herba Cistanches by fingerprint with high-performance liquid chromatography–diode array detection–mass spectrometry. Journal of Chromatography A 1216: 2156– 2162.

32. Han LF, Boakye YM, Liu E, Zhang Y, Li W, et al. (2012) Structural Characterisation and Identificationof Phenylethanoid Glycosides from Cistanches deserticola Y.C. Ma by UHPLC/ESI–QTOF–MS/MS. Phytochem Anal 23: 668–676.

33. Li L, Tsao R, Yang R, Liu CM, Young JC, et al. (2008) Isolation and purification of phenylethanoid glycosides from Cistanche deserticola by high- speed counter-current chromatography. Food Chemistry 108: 702–710.

34. Lu DY, Zhang JY, Yang ZY, Liu HM, Li S, et al. (2013) 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 26: 1945–1952.

35. Kobayashi H, Karasawa H, Miyase T, Fukushima S (1985) Studies on the Constiuents Cistanche Herba. V. Isolation and Structures of Two New Phenylpropanoid Glycosides, Cistanoside E and F. Chem Pharm Bull 33: 1452– 1457.

36. Jiang Y, Tu PF (2009) Analysis of chemical constituents in Cistanche species. J Chromatogr A 1216:1970–1979.

37. Masssart DL, Vandeginste BGM, Deming SN, Michotte Y, Kaufman L (1988) Data handling in science and technology. Chemometrics: a textbook.

38. Huang LF, Zheng SH, Wu LB, Jiang X, Chen SL (2014) Ecotypes of Cistanche deserticola based on chemical component and molecular traits. SCIENTIA SINICA Vitae 44: 318–328.

39. Tu PF, Jiang Y, Guo YH (2011) research and industry development of herba cistanches. Journal of Chinese pharmaceutical 46: 882–887.

40. Kobayashi H, Oguchi H, Takizawa, Miyase T, Ueno A, et al. (1987) New phenylethanoid glycosides from Cistanche tubulosa (SCHRENK) Hook. f. I. Chem Pharm Bull 35: 3309–3314.