Abstract:

Ziziphi Spinosae Semen is the dried seeds of Ziziphus jujuba Mill. var. spinosa (Bunge) Hu ex H. F. Chou, and its extract has broad application prospects in the development of sleep-aid functional foods. However, the quality parameters of Ziziphi Spinosae Semen extracts currently available in the market are not uniform and there is a lack of unified standards. Therefore, it is important to establish an accurate and comprehensive method for quality evaluation. In view of the problems that the UV responses of flavonoids and saponins in the Ziziphi Spinosae Semen extracts vary dramatically and the saponin content in Ziziphi Spinosae Semen water extract is very low, high performance liquid chromatography (HPLC) was used to establish the fingerprint and quantify spinosin. The separation was carried out on a Waters XSelect HSS C18 column (250 mm×4.6 mm, 5 μm), and the mobile phase was acetonitrile-0.1% (v/v) phosphoric acid aqueous solution for gradient elution. The eight common peaks in the fingerprint of the Ziziphi Spinosae Semen extracts, identified by HPLC-quadrupole time-of-flight mass spectrometry, were attributed to flavonoids by reference substance identification, literature comparison, and high-resolution mass spectrometry data analysis. Semi-quantitative analysis of seven flavonoids and quantitative analysis of spinosin were conducted using the established HPLC quantitative fingerprint. The contents of jujuboside A and jujuboside B were determined by ultra-high performance liquid chromatography-triple quadrupole mass spectrometry. Chromatographic separation was performed on a Waters ACQUITY UPLC BEH C18 column (50 mm×2.1 mm, 1.7 μm) by gradient elution using a mobile phase of acetonitrile-0.1%(v/v) formic acid aqueous solution. The target compounds were analyzed in multiple reaction monitoring mode with positive electrospray ionization.
The semi-quantitative and quantitative data of the above-mentioned 10 components are displayed in the form of radar. Using the above methods, three batches of Ziziphi Spinosae Semen water extracts prepared in the laboratory and 15 batches of extract samples obtained from 15 suppliers were analyzed and compared. The results showed that although the raw materials of three batches of Ziziphi Spinosae Semen water extracts prepared in the laboratory were from different enterprises, the overall difference was not significant. However, the component contents of the samples provided by different manufacturers were greatly different, suggesting that there are some problems associated with the different manufacturers, such as dilution of excipients, adulteration of Ziziphi Mauritianae Semen, alcohol extraction, purification, and enrichment. For example, the representative composition contents in the Ziziphi Spinosae Semen extracts obtained from manufacturers B, C, E, F, G, H, I, and O were low, which were approximately 1/10 of corresponding contents in the normal water extracts prepared in the laboratory. It is speculated that to reduce the unit price of the product, the manufacturer used fewer raw materials or a large number of auxiliary materials to dilute the Ziziphi Spinosae Semen extracts. The contents of some flavonoids in the Ziziphi Spinosae Semen extract from manufacturer N were slightly higher than that in the self-preparation Ziziphi Spinosae Semen water extract, but it did not contain jujuboside A; thus, it was speculated that the Ziziphi Mauritianae Semen might be used for extraction. The contents of 10 components in the Ziziphi Spinosae Semen extract obtained from manufacturer D were all higher than the corresponding ones in the self-preparation Ziziphi Spinosae Semen water extract. Combined with the quality label of total saponin content > 20% and poor water solubility, it was speculated that the product might be prepared by alcohol extraction or purified and enriched by using resin. These results provided the basis for the enterprise to establish internal control quality standards for Ziziphi Spinosae Semen extracts and to select qualified suppliers.

Key words: high performance liquid chromatography (HPLC), mass spectrometry (MS), quantitative fingerprint, Ziziphi Spinosae Semen extracts, radar-gram