Study On The Quanlity Control Of Traditional Chinese Medicine Fructus Xanthii

BackgroundXanthii Fructus, originating from Fructus Xanthii, has been long used for rhinitis, rheumatic arthralgia and rubella in traditional Chinese medicine. It shows slight toxicity and may cause medicine poisoning in clinical application. The quality evaluation standard of this herb recorded in Chinese Pharmacopoeia is too simple to control its quality effectively. Just only a few identity tests include description, microscopic characteristics, thin-layer chromatographic analysis, moisture and total ash. Unfortunately, there is no appropriate quantitative method for this herb so far.Objectives1. To extract chemical compositions of interest from Fructus Xanthii, then enrich the compositions of interest for isolating reference compounds.2. To establish chemical fingerprint as the quality evaluation method of Fructus Xanthii using high-performance liquid chromatography (HPLC).3. To study MS/MS fragmentation pattern of organic acids contained in Fructus Xanthii using liquid chromatography-mass spectrometry-mass spectrometry (LC-MS/MS), and determine chemical compositions by Ultra-pressure Performance Liquid Chromatography (UPLC).Methods1. The powder of Fructus Xanthii was extracted by reflux both with alcohol twice for 1 h every time, the solid-to-solvent ratios were 1:8 and 1:6, respectively. Then the extract solution was filtered and concentrated under reduced pressure consecutively. The residue was parted into three fractions by liquid-to-liquid extraction, which involved petroleum ether fraction, ethyl acetate fraction, and water-saturated n-butanol fraction. The fraction containing components of interest was chromatographed using Agilent 1200 HPLC type preparation instrument. The impure component was further purified and crystallized.2. UV absorption spectra of most common peaks of HPLC fingerprint of the extract of Fructus Xanthii were on-line scanned using HPLC-DAD, then these common peaks could be assigned by comparing the UV spectra and retention times of these peaks with those of standard compounds. The HPLC fingerprints of 27 batches of Fructus Xanthii were developed and then analyzed using fingerprint software and principal component analysis in order to establish fingerprint common pattern of Fructus Xanthii as the standardized quality control protocol. 3. A quantitative method for determining protocatechuic acid, neochlorogenic acid, cryptochlorogenic acid and chlorogenic acid was established by UPLC-TUV.Results1. An pure reference compound (C13H15NO4) was prepared. 2. By comparing and analyzing HPLC fingerprints of 27 batches of Xanthii Fructus,23 peaks were characterized as common ones of HPLC fingerprint common pattern of the herbal drug. Batches of herbs collecting from different product areas showed their HPLC Fingerprints were very similar and consistent. The result indirectly indicated that the herbal drug originated from just only one species, i.e. Fructus Xanthii and source was stable. By analyzing these HPLC fingerprints using principal component analysis, the similarity indices of most samples were over 0.90 except samples 4,12 and 14. It showed the established HPLC fingerprint common pattern combined with principal component analysis was feasible and convenient for quality evaluation, species authentication of Xanthii Fructus.3. The MS fragmentation patterns of dominant organic acids of Fructus Xanthii were analyzed using LC-MS/MS. The contents of protocatechuic acid, neochlorogenic acid, cryptochlorogenic acid and chlorogenic acid contained in the herb were determined by UPLC-TUV. As a result, there is a good linear relationship between UV absorption and concentration of protocatechuic acid, neochlorogenic acid, cryptochlorogenic acid and chlorogenic acid over a large range of concentration, and the values of correlation coefficients R2were not less than 0.9999.Conclusions Our research on quality evaluation of Xanthii Fructus has established a feasible and effective method for the first time. This method transcends the outdated quality control mode used in ages. The developed HPLC fingerprint method together with chemometric analyses for this herb can be widely used for quality evaluation, species authentication of Xanthii Fructus. The investigation on MS/MS fragmentation pattern of dominant organic acids and their quantitative analysis also offered enormous information, which facilitates man to understand this toxic herbal drug and enhances its clinical safety.