Multi-step infrared macro-fingerprint features of ethanol extracts from different Cistanche species in China combined with HPLC fingerprint

• Multi-steps IR show different features on ethanol extracts of four Cistanche species.
• Analysis results of IR macro-fingerprint were verified by HPLC fingerprint method.
• Relationships of four species of Cistanche were inferred by IR and HPLC fingerprint.
• Main active ingredients of four Cistanche were compared and estimated by IR spectra.
• 2D-correlation analysis provided additional further information for differentiation.

The genus Cistanche generally has four species in China, including C. deserticola (CD), C. tubulosa (CT), C. salsa (CS) and C. sinensis (CSN), among which CD and CT are official herbal sources of Cistanche Herba (CH). To clarify the sources of CH and ensure the clinical efficacy and safety, a multi-step IR macro-fingerprint method was developed to analyze and evaluate the ethanol extracts of the four species. Through this method, the four species were distinctively distinguished, and the main active components phenylethanoid glycosides (PhGs) were estimated rapidly according to the fingerprint features in the original IR spectra, second derivative spectra, correlation coefficients and 2D-IR correlation spectra. The exclusive IR fingerprints in the spectra including the positions, shapes and numbers of peaks indicated that constitutes of CD were the most abundant, and CT had the highest level of PhGs. The results deduced by some macroscopic features in IR fingerprint were in agreement with the HPLC fingerprint of PhGs from the four species, but it should be noted that the IR provided more chemical information than HPLC. In conclusion, with the advantages of high resolution, cost effective and speediness, the macroscopic IR fingerprint method should be a promising analytical technique for discriminating extremely similar herbal medicine, monitoring and tracing the constituents of different extracts and even for quality control of the complex systems such as TCM.