Analysis Of The Active Ingredients Of TCM By Graphene-based Electrochemical Sensor Technology And UPLC-MS/MS Techniques

Analysis of the active ingredients of TCM is a hot and difficult in the field of traditional Chinese medicine research in recent years. On one hand, due to the high selectivity and sensitivity, low cost and many other advantages, electrochemical sensor analysis technique has attracted wide attention in the analysis of the active component of TCM. On the other hand, due to strong separation capacity, high sensitivity, high selectivity and can provide relative molecular mass and rich structural information, LC-MSn technique is widely used in the analysis of the active component and pharmacokinetic studies of TCM. Based on the above two aspects, the contents of this paper include the analysis of active ingredients of TCM based on electrochemical sensor technology and the pharmacokinetic studies and compatibility law of TCM based on LC-MSn technique. The specific studies are as follows:(一) The analysis of active ingredients of TCM based on electrochemical sensor technology1. An electrochemical sensor based on PDDA functionalized graphene sheets was developed for the sensitive detection of shikonin in Arnebiae Radix.The electrochemical behaviors of shikonin at a poly(diallyldimethylammonium chloride) functionalized graphene sheets modified glass carbon electrode (PDDA-GS/GCE) have been investigated. Due to the high shikon in-loading capacity on the electrode surface and the upstanding electric conductivity of graphene, the electrochemical response of the fabricated sensor was greatly enhanced and displayed excellent analytical performance for shikonin detection from 9.472×10-8 to 3.789×10-6 mol·L-1 with a low detection limit of 3.157×10-8 mol·L-1.In addition, the proposed electrochemical sensor also exhibited good stability, selectivity and acceptable reproducibility and it could be used for the detection of shikonin in real samples.2. Synthesis of a biocompatible gelatin functionalized graphene nanosheets and its application for drug delivery.A simple and environmentally friendly synthetic route for the preparation of gelatin functionalized graphene nanosheets (gelatin-GNS) was reported by using gelatin as a reducing agent. Because of its one-atom layer and large surface area, the obtained biocompatible gelatin-GNS attaching methotrexate (MTX) via strong n-n stacking interaction, exhibited a high drug loading capacity of MTX and excellent ability for cellular imaging and drug delivery. From the cytotoxicity assay, we can see that the MTX@gelatin-GNS showed remarkable toxicity while the gelatin-GNS showed nontoxic at appropriate concentration, both of them might be taken up by A549 cells through a nonspecific endocytosis process. The pH-dependent release behavior of MTX might experience a gelatin-mediated sustained release process. Therefore, the gelatin-GNS with good stability and biocompatibility can be selected as an ideal drug carrier to be applied in drug delivery and controlled release.(二) The pharmacokinetic studies and compatibility law of TCM based on LC-MSn technique.Qing’e fang is a classical prescription that can benefit kidney and strength bones. It consists of the monarch herb Eucommia ulmoides Oliv., the minister herb Psoralea corylifolia L., adjuvant Juglans regia L. and conductant drug Allium sativum L.. Although the compatibility law studies of Qing’e prescription have been reported, however, the compatibility law studies based on pharmacokinetic have not been reported. The details are as follows:1.The medicine constituents that can be detected in rat plasma after oral administration of Eucommia ulmoides Oliv. extract.To establish the chromatographic and mass conditions of 18 active ingredients in Eucommia ulmoides Oliv., including lignan ingredients pinoresinol di-O-β-D-glucopyranoside, pinoresinol 4’-O-β-D-glucopyranoside, syringaresinol, Lirioresinol-A, pinoresinol, epipinoresinol, medioresinol; iridoid ingredients geniposide, geniposidic acid, aucubin; phenolic ingredients chlorogenic acid, sinspslfrhyde, coniferyl aldehyde; and other ingredients 5-hydroxymethyl furfural by UPLC-MS/MS techniques. Simultaneously, to detect the medicine constituents in SD rats plasma after oral administration of Eucommia ulmoides Oliv. extracts. The medicine constituents that can be detected in rat plasma include:pinoresinol di-O-β-D-glucopyranoside, pinoresinol 4’-O-β-D-glucopyranoside, epipinoresinol, syringaresinol, aucubin, geniposide, geniposidic acid, chlorogenic acid and 5-hydroxymethyl furfural.2. Pharmacokinetic studies of the main active ingredient from monarch herb of Qing’e prescription:Eucommia ulmoides Oliv.Based on the medicine constituents that can be detected in rat plasma, an efficient, sensitive and selective ultra performance liquid chromatography/tandem mass spectrometry (UPLC-MS/MS) method for the simultaneous determination of pinoresinol di-O-p-d-glucopyranoside, geniposide, geniposidic acid, aucubin and chlorogenic acid of Eucommia ulmoides Oliv. extact in rat plasma has been developed and validated. The validated method has been successfully applied to the pharmacokinetic studies in male and female rats after a single oral administration of Eucommia ulmoides Oliv. extract.Pinoresinol di-O-β-d-glucopyranoside, geniposide, geniposidic acid, aucubin and chlorogenic acid have exhibited good absorption, distribution and elimination process both in male and female rats. And further research indicated that significant difference in pharmacokinetic parameters and basically consistent plasma concentration-time curve trend of the analytes was observed between the male and female rats, while absorptions of these analytes in male group were all significantly higher than those in female group.3. The pharmacokinetic and tissue distribution studies in normal mice and ovariectomized (OVX) mice after oral administration of Eucommia ulmoides Oliv. extract.To investigate the pharmacokinetics and tissue distribution differences of pinoresinol di-O-β-d-glucopyranoside, geniposide, geniposidic acid, aucubin and chlorogenic acid between the normal mice and the OVX mice after oral administration of Eucommia ulmoides Oliv. extracts by using the optimized UPLC-MS/MS method. The differences in plasma pharmacokinetics mainly as follows:the pharmacokinetic parameters AUC0-t, AUC0-∞, Cmax of pinoresinol di-O-∞-d-glucopyranoside, geniposide, geniposidic acid in OVX mice were higher than normal mice, while the parameters Vd and CL in OVX mice were lower than normal mice. On the contrary, the absorption of chlorogenic acid in normal mice plasma was significantly higher than in OVX mice plasma. Aucubin had been detected in normal mice plasma, while it had not been detected in OVX mice. The major differences in tissue distribution are as follows:Aucubin showed good distribution in normal mice liver, while it had not been detected in OVX mice liver. The distribution of pinoresinol di-O-β-d-glucopyranoside, geniposide, geniposidic acid in OVX mice liver, spleen and kidney was significantly higher than in normal mice. The distribution of chlorogenic acid in OVX mice spleen was lower than in normal mice, but the distribution of chlorogenic acid in OVX mice liver and kidney was still higher than in normal mice. The results indicate that the five active ingredients of Eucommia ulmoides Oliv. extract showed good and relatively fast distribution in the mice liver, spleen, kidney and uterus, and they showed a higher bioavailability in OVX mice. In addition, the five index components with good and rapid distribution showed quadratic distribution within 4 h in OVX mice uterus, which suggested the estrogen-like effects on the uterus.4. Study on the compound compatibility law of Qing’e prescription based on the pharmacokineticsTo investigate the pharmacokinetics of pinoresinol di-O-β-d-glucopyranoside, geniposide, geniposidic acid, aucubin and chlorogenic acid in rats plasma after oral administration of five kinds of prescription extracts related to Qing’e Fang (Du Zhong, Du Zhong+Bu Gu Zhi, Du Zhong+He Tao Ren, Du Zhong+Da Suan, Du Zhong+Bu Gu Zhi+He Tao Ren+Da Suan). The results indicated that the five index components exhibited good absorption, distribution and elimination process and basically consistent concentration-time curve trend in five Qing’e prescription groups of rats. However, the pharmacokinetic parameters of the five active ingredients showed significant difference among the five compatibility groups. By comparing the pharmacokinetic parameters AUCo_t, AUC0-∞ Cmax of the index components among the five compatibility groups, we found that the absorption of pinoresinol di-O-β-d-glucopyranoside, geniposide and geniposidic acid in (Du Zhong+Bu Gu Zhi) group rats plasma was significantly increased, in addition, Bu Gu Zhi can delay the plasma concentration time to peak; the absorption of aucubin and chlorogenic acid in (Du Zhong+He Tao Ren) group rats plasma was significantly decreased; the absorption of geniposide, geniposidic acid and aucubin in (Du Zhong+Da Suan) group rats plasma was significantly decreased; the absorption of chlorogenic acid was significantly increased, while the absorption of pinoresinol di-O-β-d-glucopyranoside and aucubin was significantly decreased in (Du Zhong+Bu Gu Zhi+He Tao Ren+Da Suan) group rats plasma. This experiment verified the rationality of Qing’e prescription compatibility from the perspective of the pharmacokinetics.