MALDI-TOF-MS-Based Fingerprinting Using Cysteine-rich Peptides as Markers to Distinguish between Roots of Astragalus and Hedysarum

Reviewed: Huang J, Wong KH, Tay SV, How A, Tam JP. Cystein-rich peptide fingerprinting as a general method for herbal analysis to differentiate radix Astralagi and radix Hedysarum. Front Plant Sci. 2019;10:973.

Keywords: Adulteration, Astragalus membranaceus, Astragalus membranaceus var. mongholicus, cysteine-rich peptides, Hedysarum polybotrys, MALDI-TOF-MS

The roots of astragalus (Astragalus membranaceus or A. membranaceus var. mongholicus, Fabaceae) are popular as herbal medicine for patients suffering from “qi” deficiencies. They are regarded as immune tonics in the West, and in Chinese medicine to strengthen the lung and the digestive system. Known as huang qi in traditional Chinese medicine, astragalus roots are sometimes substituted with hong qi, the roots of hedysarum (Hedysarum polybotris, Fabaceae). Hedysarum is used for similar health issues as astragalus, and it is not clear if substitution is due to the similar medicinal uses for both plants, or due to mistaken identity.¹

Most reported methods to authenticate astragalus by chemical means use calycosin-7-O-glucoside and other isoflavones as markers, even if some of these compounds occur in hedysarum as well.² Not much is known about cysteine-rich peptides (CRPs) in astragalus roots. These proteins can bind to membrane receptors and thus induce plant growth, plant defense, and plant reproduction mechanisms. The amino acid composition of cysteine-rich peptides varies substantially across different plant species but they all share three common features: (1) small size (less than 160 amino acid residues), (2) a conserved N-terminal region which includes a secretion peptide signal, and (3) a C-terminal cysteine-rich domain usually containing 4–16 cysteine residues.^3,4

Samples of astragalus roots (51) and hedysarum roots (40) collected from herbal pharmacies in China and Singapore were extracted with water or 50% ethanol. After centrifugation, supernatants were loaded onto a micro-elution 96 well plate and the peptide fraction eluted with 80% acetonitrile. CRP fingerprints were obtained by matrix assisted laser desorption/ionization–time of flight (MALDI-TOF) mass spectrometry. Mass spectral data were then submitted to chemometric evaluation using a number of multivariate statistical tools. Similarly, fingerprints obtained by ultra-high-performance liquid chromatography with UV detection (UHPLC-UV) at 230 nm were also submitted to statistical analysis, and the ability to distinguish between astragalus and hedysarum compared.

Of the statistical models, the K-nearest neighbors (KNN) algorithm performed best with regards to predictability and interpretability. UHPLC-UV correctly predicted the classification for all samples, whereas MALDI-TOF showed prediction accuracies of 89% or greater in all models. The authors conclude that relying on CRP fingerprints to distinguish among the two species is reliable, and point out that the MALDI-TOF analysis is “500 fold faster” than conventional UHPLC-UV. However, the shorter analysis time does not make up for the lengthier and more cumbersome sample preparation required to perform the MALDI-TOF analysis. Calycosin-7-O-glucoside was proposed as a marker compound for astragalus, while medicarpin was determined to be present only in hedysarum.

Comment: The use of CRPs to differentiate among plant species represents an interesting approach for the identification of crude raw materials (whole, cut, or powdered plant), from which these peptides can be extracted. The comparison of MALDI-TOF fingerprints of 100 plant species, including Astragalus membranaceus and Hedysarum polybotris supports the notion that CRP-based plant identification is reliable. However, the results also show that analysis of the isoflavones, especially calycosin-7-O-glucoside which has been reported in trace amounts from hedysarum,⁵ provides equal or better prediction of the species. Another marker compound used to distinguish the species is the triterpene glycoside astragaloside IV.^6,7 In light of the relative ease to use the isoflavones or triterpenoids as marker compounds to distinguish between the two species, and the lack of MALDI-TOF instruments in most quality control laboratories, the use of HPLC- or UHPLC-based methods seems to make the most sense.

References

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