Abstract
BackgroundQuantitation analysis and chromatographic fingerprint of multi-components are frequently used to evaluate quality of herbal medicines but fail to reveal activity of the components. It is necessary to develop a rational approach of chromatography coupled with activity detection for quality assessment of herbal medicines.MethodsAn on-line HPLC-ultraviolet detection-2,2′-azino-bis(3-ethylbenzothiazoline-6-sulfonic acid) free radical scavenging (HPLC-UV-ABTS) method was developed to obtain the chromatographic fingerprints and ABTS+• inhibition profiles (active fingerprints) of Rehmanniae Radix (Dihuang) and Rehmannia Radix Praeparata (Shu Dihuang). Eighteen compounds showing ABTS+• inhibition activity were identified by HPLC-fourier-transform mass spectrometry (HPLC-FTMS). Verbascoside was used as a positive control to evaluate the total activities of the samples and the contribution rate of each compound. The similarities of the chromatographic and active fingerprints were estimated by the vectorial angle cosine method.ResultsThe results showed that the HPLC-UV-ABTS method could efficiently detect antioxidant activity of the herbal medicine samples. The antioxidants were different between the two herbs and several new antioxidants were identified in Shu Dihuang. A function equation was generated in terms of the negative peak area (x) and the concentrations of verbascoside (y, μg/mL), y = 2E-07 × 4 - 8E-05 × 3 + 0.0079 × 2 + 0.5755x + 1.4754, R2 = 1. Iridoid glycosides were identified as main antioxidants and showed their higher contributions to the total activity of the samples. The total contributions of the three main active components in the Dihuang and Shu Dihuang samples to the total activity, such as echinacoside, verbascoside and an unknown compound, were 39.2–58.1% and 55.9–69.4%, respectively. The potencies of the main active components in the Shu Dihuang samples were two to ten times those in the Dihuang samples. Similarity values for S12 in the chromatographic fingerprints and S03, S12 and P03 in the active fingerprints were less than 0.9. The three batches of samples might show their different quality with the other samples.ConclusionsThe results suggested that the combination of “quantity-effect” research strategy and the HPLC-UV-ABTS analysis method could comprehensively evaluate the active components and quality of Dihuang and Shu Dhuang.
Highlights
Quantitation analysis and chromatographic fingerprint of multi-components are frequently used to evaluate quality of herbal medicines but fail to reveal activity of the components
P), catalpol and verbascoside were quantified by high performance liquid chromatographic (HPLC) methods to control the quality of Dihuang and Shu Dihuang, and 1,1-Diphenyl-2-picrylhydrazyl (DPPH) scavenging method was applied to distinguish verbascoside through thin layer chromatography (TLC) [13]
Four batches of Dihuang samples from the four manufacturers in Anhui province were labeled as S07, S08, S09 and S13, while S10 and S11 were collected from the manufacturers of Guangzhou and Hangzhou, respectively
Summary
Materials and reagents The information of the Dihuang and Shu Dihuang samples was listed in Additional file 1. Verbascoside (7.78 mg and 3.81 mg) was weighed accurately and dissolved in 50 mL by methanol as the stock solutions. The on-line instrumentation consisted of a Waters HPLC for chromatographic fingerprint analysis at 334 nm or 250 nm and an Ultimate 3000 UV detector for ABTS+ scavenging analysis at 734 nm. ABTS solution was delivered with an injection pump of Pickering PCX Della (Pichering Laboratories Inc., USA) at the flow rate of 0.5 mL/min after PDA detector, and the elution was mixed with ABTS solution after PDA detector through a reaction coil with 1.4 mL volume. An aliquot of 20 μL sample solution was injected for HPLC-UV-ABTS analysis. The chromatographic and active fingerprints from HPLC-UV-ABTS method were represented mathematically by a vector of their chromatographic peak areas and inhibiting peak areas. The two vectors are more similar when the cosine values are near 1
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