Investigation On The Quality Control Method Of Hebra Sarcandrae

Hebra Sarcandrae was the whole plant of Sarcandra glabra (Thunb.) Nakai, which was used as antibiotic, anti-inflammatory and anti-tumor traditional Chinese medicine (TCM).Up to now, there were 56 domestic manufactures for Hebra Sarcandrae products, with low quality standards and poor quality control methods which are independent of anti-imflammatory activity and whole process quality control. Thus a further intensive study was needed urgently to improve the quality control methods of Hebra Sarcandrae and its products.On the basis of our research on chemical constituents and their bioactivities in Hebra Sarcandrae water extract which was an important intermediate step in manufacturing process of its preparations, three parts of research were carried out in this thesis for improving the quality control of Hebra Sarcandrae water extract and its preparations as well as formulation of the national standard of Hebra Sarcandrae water extract.1. Study on HPLC fingerprint chromatogram of Hebra Sarcandrae water extract and its preparationsThis section mainly focused on establishing an HPLC fingerprint chromatogram of Hebra Sarcandrae water extract and its preparations with the main peaks being chemically identified. The method was developed on a C18 column (250 mm×4.6 mm i.d.,5μm) by gradient elution with acetonitrile-water (both containing 0.1% formic acid) as mobile phase at a flow rate of 1.00 mL/min with the wavelength at 330 nm. As a result the contrast HPLC fingerprint chromatogram was set up with good stability, precision and reproducibility by statistical and similarity analysis, then it was used as reference to evaluate the quality of the water extract and its preparations via similarity assay. The similarity of fingerprint chromatograms for 12 batches of Hebra Sarcandrae extract and 20 batches of its preparations ranged in 0.883～0.987 and 0.797～0.982 respectively. Furthermore, eight peaks in the chromatogram were identified as neochlorogenic acid, chlorogenic acid, cryptochlorogenic acid,β-hydroxypropiovanillone, caffeic acid, isofraxidin, rosmarinic acid-4-O-glucoside, rosmarinic acid, respectively. Our investigation on HPLC fingerprint chromatogram with anti-inflammatory and characteristic components included, which reflected the profile of multi-component in Hebra Sarcandrae water extract and its preparations, would contribute to its whole process and effective quality control.2. Assay of rosmarinic acid and isofraxidinin in Hebra Sarcandrae water extract and preparations by HPLC-UVDThis section mainly focused on developing an HPLC-UVD method for assay of multi-component including rosmarinic acid as anti-inflammatory component and isofraxidin as characteristic component in Sarcandra water extract and its preparations. The method was developed on a C18 column (250 mm×4.6 mm,5μm) by gradient elution with acetonitrile-water (both containing 0.1% formic acid) as mobile phase at a flow rate of 1.00 mL/min with the wavelength at 330 nm and column temperature at 35℃. The linearity was over the range of 0.04746～1.898μg for rosmarinic acid and 0.01018～0.4072μg for isofraxidin; The RSD was no more than 2.0% in precision test, reproducibility test and stability test, respectively; The average recovery of rosmarinic acid and isofraxidin in Hebra Sarcandrae extract were 99.8%(RSD=0.70%) and 99.2% (RSD=0.63%), while in paparations were 100.7% (RSD=1.6%) and 98.4% (RSD=0.44%), respectively. The twelve batches of Hebra Sarcandrae water extract sample shared an average content over the range of 1.479-6.447 mg/g for rosmarinic acid and 1.912-3.540 mg/g for isofraxidin, while 0.847-15.946 mg/g for rosmarinic acid and 2.032-4.093 mg/g for isofraxidin in its preparations. The method was simple, specific, precise and accurate and would help to improve the quality standard of Hebra Sarcandrae water extract and its preparations.3. Investigation on rapid recognition of caffeic acid derivatives in Hebra Sarcandrae water extract by HPLC-ESI-MS"This section mainly focused on rapid recognition of caffeic acid derivatives which were potential anti-inflammatory activity components in Hebra Sarcandrae water extract by HPLC-ESI-MSn. Based on the features of MS" fragmentation ions and DAD absorption spectra of caffeic acid derivatives in literature incorporated with those of reference chemical substances isolated from Hebra Sarcandrae water extract, a total of 19 caffeic acid derivatives out of 45 peaks in ESI(-)TIC MS chromatogram were rapidly recognized by HPLC-ESI-MSn,6 of them were identified compared with reference chemical substances, and the others were tentatively recognized according to the features of MSn fragmentation ions and DAD absorption spectra. This result would contribute to illuminate the relationship between anti-inflammatory activity and the related component in Hebra Sarcandrae.