| Objective:To study the diversity, variation and distribution of the chemical constituents in Uygur medicine Ziziphora clinopodioides Lam. from different geographgic regions, collection times and plant parts. And investigated the effect on mushroom tyrosinase activity. To lay the foundation of quality standard, resource optimization and find new pharmacological activity for Z. clinopodioides. Methods:Using thin layer chromatographic (TLC) to qualitative analysis rosmarinic acid, caffeic acid, diosmin and linarin and simultaneous quantitative analysis for ursolic acid (UA) and oleanolic acid (OA) in Z. clinopodioides. Applied fourier transform infrared (FT-IR) Spectroscopy to study the herb material and oil to establish the IR fingerprint. Applied atomic absorption spectrometry (AAS) method to determine ten trace elements in Z. clinopodioides. Principal component analysis (PCA) and hierarchical cluster analysis (HCA) were applied according to its elements contents from different geograplgic samples. And also determined the samples of collection times and plant parts. Different extracts and identified chemical compounds in Z. clinopodioides were investigated to effect the activity on mushroom tyrosinase and mocleular docking with these compounds. Study the UV-Vis spectrum changing of extracts and compounds to chelate with different doses of Cu2+ to illustrated the interaction between samples and the active centre of this enzyme. Result: The optimized TLC condition is silica gel plate from Anhui Liangchen as the stationary phase and chloroform-methanol-water-formic acid-acetic acid (10:5:0.3:1:1) as the development system for qualitative analysis of rosmarinic acid, caffeic acid, diosmin and linarin. With this method, Z. clinopodioides samples from 18 different regions were analysed and found most compounds were the same, but different from the contents. TLC-DPPH method was used to screeming of anti-oxidation conpounds for the 18 samples. At least, five compounds were displayed as antioxidants, including caffeic acid and rosmarinic acid. The average contents of UA and OA in different regions were 1.84± 0.41 mg/g and 2.82±0.89 mg/g. Variation in 9 collection times for UA and OA were rising from late spring to the beginng of summer but decreasing in follow periods. The UA and OA contents in three plant parts were as follows:leaves>flowers>stems; According to the information of IR peak shape and peak position, the characteristic of spectum have not distinguished for different regions until the second derivative infrared spectra was used, no matter herb material or essential oil. The second derivative IR can enlarge the differences between samples. Three and five groups were classified according to cluster analysis for herb material and essential oil. The second derivative IR spectum for different collection times were found some regular changing on peak heights and shapes, so that the chemical constituents changing can be deduced by the functional groups of peaks; The decreasing sequence of trace elements levels was K> Ca> Mg> Fe> Cu> Zn> Na> Mn> Cd> Pb. PCA revealed 83.51% of the variance with the first four principal component (PC) variables. HCA indicated five groups from the 18 regions. Variation in the elements exhibited a decreasing trend, but of different types in the studied periods. Elemental contents in leaves were higher than those in flowers and stems; Only water extract and essential oil have weak inhibitiory effect on tyrosinase. Rosmarinic acid as one of the major compounds in Z. clinopodioides showed active effect at the dose of 500～62.5 μg/mL, but inhibit at 0.1～0.05 μg/mL. And showed a dose-dependent effect. In molecular docking study, the 15 known compounds, hyperoside, diosmin, quercetin, linarin, rosmarinic acid and oleanolic acid have better docking results that the docking energy were -8.6～7.8 kcal/mol. The scores were correspound with the actual result. In Cu2+ chelating assay, water extracts and high docking compounds react with different dose of Cu2+, most samples showed the same result with the kojic acid to rise the UV-Vis spectrum when Cu2+ add. But caffeic acid was opposed. Conclusion:TLC analysis laid the foundation of chemical analysis, resource optimization and quality standard for Z. clinopodioides. DPPH couple with the TLC fingerprint can be a referance for optimization with spectrum-activity relationship. The result of cluster analysis from IR and teace elements analysis was corresponded to the geographical distribution for the most regions. The variations from IR and trace elements can reflect the chemical constituents variations. The extracts have weak anti-tyrosinase activity. According to the emzyme effect, rosmarinic acid and Cu2+ chelating, the unsatisfactory result may be attributed to 1. Low contents of activity compounds in extracts.2. Both activator and inhibitor exist. |
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