A New Method For Quality Control Of Liuwei Dihuang Pill

Liuwei Dihuang Pill (LDP) is a classic Traditional Chinese Medicine formula, which is derived from the Shen Qi Pill by Qian Yi in Song dynasty. It is produced by a certain kind of procedure containing radix Radix Rehmannide Preparata, Rhizoma Dioscoreae, Fructus Corni, Cortex Moutan, Rhizoma Alismatis and Poria at the ratios of 8:4:4:3:3:3 and is applied for the treatment of various disorders such as backache, alopecia, menoxenia, sore waist and knees. Previous pharmacological studies demonstrated its efficacy for anti-aging, regulating the function of T lymphocytes and cytokines, treating type II diabetes disease and modulating neutrals system. In this study, we mainly focused on eight chemical components which belong to the class of glycosides, terpenes, phenolics, tetracyclic triterpenes and tannins, respectively. A new method for quality control and deepen research of LDP is established.60 batches of water-honeyed pill, concentrated pill and capsule of LDP, which are sold in local drug store, are mainly from 23 different manufacturers such as Beijing TongRen Tang Co. Ltd, Henan Wan Xi Co. Ltd. and Guangdong Fo Ci Co. Ltd., et al. In order to do the basic research and applied research of LDP, in this study, we conducted the chemical fingerpriting and quality control of LDP. We mainly focus on the reseach as follows.(1) Chemical fingerprinting of LDP samplesAn approach of chemical fingerprinting of LDP is established by using HPLC with ultraviolet detection (HPLC/UV) technique through graduate elution. The similarity of LDP was performed on the basis of the fingerprinting data. Total numbers of 15 batches LDP samples from the same manufacturer, Beijing TongRen Tang Co. Ltd., were chosen. Most of the similarity results are higher than 0.950 which reflected their difference was small. A number of 12 batches of samples, which were from different dosage forms and different manufacturers, have the similarity results among 0.657-0.950. It indicated that dosages form is the most influencing factor. From the result above, we may take a conclusion that capsule is with the best quality among three dosage forms compared with reference standard. Based on the chromatographic fingerprint data, partial least square (PLS) and discriminate analysis were utilized to visualize the quality information of 60 batches of LDP, and a partial least square-discriminate analysis (PLS-DA) model was constructed with acceptable predictive performance for the discrimination of various products. The proposed approach was expected to be developed as a simple and accurate method for the quality control of LDP.(2) Qualitative control of LDP by combined techniqies of HPLC-MS/MS and RRLC-QTOF/MS.Both the techniques of HPLC-MS/MS and RRLC-QTOF/MS had been used for the analysis of its chemical components. The HPLC-ESI-MS/MS method could separate the components well, and providing the molecular weight information by MS spectra and the fragment structural information from MS/MS spectrum to presume the structural information of 21 chemical components. A RRLC-Q-TOF/MS has characteristics of high sensitive, high resolution, low sample consumption, high accuracy, et al., thus can be applied for obtaining the accurate molecular weight of the target components. Finally, the accuate molecular weights of 11 chemical compounds were obtained. Both of the techniques was validated and could be applied for the quality analysis of the chemical components, which was the basis of the establishment of the quality standard and the illustration of effective constituents for LDP.(3) Simultaneous determination and quantitative control of multiple bioactive components in LDP.Multiple bioactive components were selected for quality control of LDP. For quality control purpose, an approach of simultaneous determination of its multiple bioactive components was established by using high performance liquid chromatography (HPLC) coupled with multiple detection techniques. HPLC with combined detections of diode array detector and evaporative light scattering detector (HPLC-DAD-ELSD) was performed to simultaneously determine the eight bioactive constituents, including gallic acid,5-hydroxymethyl furfural, morroniside, sweroside, loganin, paeoniflorin, paeonol and alisol B-23 acetate at the wavelength of 254 nm and 238 run. Samples were extracted with methanol for 60 min. A TSKgel C18 column,150 mm×4.6 mm i.d. and a mobile phase of 0.1% formic acid-acetonitrile containing 0.1% formic were used for the assay, a gradient program was used as follows (v/v):0-20 min, A 1%-12%; 20-30 min, A 12%-15%; 30-70 min, A 15%-50%; 70-80 min, A 50%-100% and hold at 100% for 10 min. The flow rate was 1.0 ml/min, and column temperature was set at 35℃. The lower limits of detection and lower limits of quantification were ranged in 0.11-1.93μg/mL and 0.38-3.85μg/mL, respectively. The intra-and inter-day precisions of the method were evaluated and were less than 3.87%, with accuracy from 95.3% to 103.4%. It is proved that this method is reliable, accurate and easy to operate, which is good for the quality control of LDP.