Studies On Chemical Constituents And Pharmacokinetics Of Zhimu Baihe Herb-pair

RHIZOMA ANEMARRHENAE and BULBUS LILII are TCM commonly used for clinlic treatment, which can clear away heat and purge fire, moisten the lung and nourish kidney, clear away heart-fire and relieve restlessness. Zhimu-Baihe herb-pair (ZMBHHP), composed of Zhimu [Anemarrhena asphodeloides, the rhizome of Anemarrhena asphodeloides Bge. (Fam. Liliaceae)] and Baihe [Lilium brownii F.E.Brown var.viridulum Baker. (Fam. Liliaceae)], is an important multiherb remedy in TCM and was first described by the famous Chinese physician Zhang Zhongjing (150 to 219 A. D. in the Chinese Eastern Han Dynasty) in his"Jingui Yaolue", a medical classic. In clinical applications, ZMBHHP has proved effective in stopping dry coughing due to yin deficiency and moisturizing the lung, clearing away the heat evil in the pericardium and calming the nerves , treating diabetes due to internal heat and constipation, and strengthening the immunity, etc. In this research, based on modern analytical techniques, from the two Chinese medicinal herbs of Zhimu and Baihe, we developed several methods and strategies of characterization of chemical components in Chinese medicinal herbs and preliminary screening of pharmaceutical active components, and apply these method and related analytical techniques in whole quality control of Chinese medicinal herbs and multiple components pharmacokinetics. In consideration of complex chemical components and ambiguous chromatographic peaks, we developed a combinative method and stragetry by using high performance liquid chromatography with time of flight mass spectrometry (HPLC-TOF/MS) according to the feature of multiple analytical techniques, which could be applied in non-target screening and identification of multi-component in vivo and in vitro of Chinese medicinal herbs.一,Studies on chemical constituents of ZhimuFirstly, chemical constituents of Zhimu were characterized and elucidated by HPLC-TOF/MS. Twenty-one components were precisely characterized by searching against the formula database of Zhimu, including eighteen saponins and three xanthones. The main differences of the chemical structures of various timosaponins are that the types and numbers of connected groups on the parent structures are different. Most timosaponins have the connected groups as -O-nG,-CH3, -OH, -OCH3,etc, so their CID spectra are easy to form various fragmentors as [M+H-n*162]~+, [M+H-15]~+, [M+H-18]~+, [M+H-31]~+ etc. All saponins comprise one steroid sapogenin and O-glycosides and their CID spectra also manifest similar characteristic fragmentation behaviors of steroid sapogenin. The major fragmentations of saponins corresponded to the losses of branched glycoside chains, side chains and small molecules such as H2O, CH3 and CH3OH. The fragmentation behaviors of saponins demonstrated that the main fragmentation was the loss of the side chain if there was no substituent group in C23 of the steroid sapogenin, otherwise, fragmentation was produced fromα-cleavage of the bond between C22 and C23 due to the existence of the C23-hydroxyl.Secondly, many different batches of RHIZOMA ANEMARRHENAE were collected and the pretreatment method as well as chromatographic method was investigated. The method was firstly established to assay the seven compounds and the pretreatment method was easy and effective. The method provided good accuracy and good precision and was successfully applied to quantitative determination of seven compounds in Zhimu.Thirdly, single-quadrupole mass spectrometry was applied to do pharmacokinetic studies of seven active ingredients in Zhimu simultaneously. And plasma sample was pretreated by liquid-liquid extraction method. Column was Agilent Zorbax XDB-C18 (2.1×50 mm,1.8μm). The mobile phase consisted of 0.1% formic acid in water and acetonitrile. A gradient program was used. SIM in the positive ion mode. The pharmacokinetics of the seven major components in the SD rats after oral administration of Zhimu extract was studied by using this method. The results showed that the pharmacokinetic behavior of timosaponins and xanthones was fitted to the one-compartment model after a singe administration of Zhimu extract, however, timosaponins have lag time during the absorption in vivo. The parameters of timosaponin E1 and timosaponin E like T1/2 and Cmax and Tmax are lower than those of the others. This implies that the rates of absorption and metabolism of timosaponin E1 and E are faster than those of the other compounds. Timosaponins showed double peak in concentration-time curve, which may be related to enterohepatic circulation. Pharmacokinetic parameters of timosaponin A_Ⅲshowed that its Tmax was obviously higher than others, which meant that its absorption and metabolism was slower than others. All in all, the results of pharmacokinetic parameters showed that mixed administration of herbal extracts extended pharmacodynamic effect, which showed that the compatibility of Chinese medicine could have synergistic effect.二,Studies on chemical constituents of Baihe and Zhimu-Baihe herb-pairFirstly, chemical constituents of Baihe were characterized and elucidated by HPLC-TOF/MS. Five components were precisely characterized by searching against the formula database of Baihe, including two saponins, one anthraquinone and two alkaloids. The main differences of the chemical structures of Browniosides are that the parent structures are different. Brownioside 1 has a steroid nucleus of tigogenin, while Brownioside 2 has a steroid nucleus of diosgenin. So their CID spectra are easy to form different fragmentors.Secondly, chemical constituents of Zhimu-Baihe herb-pair were characterized and elucidated by HPLC-TOF/MS. Thirty components were precisely characterized by searching against the formula database of Zhimu and Baihe, including twenty four saponins, three xanthones, one anthraquinone and two alkaloids.Thirdly, single-quadrupole mass spectrometry was applied to do pharmacokinetic studies of seven active ingredients in Zhimu-Baihe herb-pair simultaneously. The pharmacokinetics of the seven major components in the SD rats after oral administration of Zhimu-Baihe herb-pair extract was studied by using this method. The results showed that the pharmacokinetic behavior of timosaponins and xanthones was still fitted to the one-compartment model after a singe administration of Zhimu-Baihe herb-pair extract, however, timosaponins have no lag time during the absorption in vivo. The parameters of Zhimu-Baihe herb-pair like T1/2 and Tmax and Cmax and AUC are lower than those of Zhimu extract, while CL/F is higher than that of Zhimu extract. This implies that the rates of absorption and metabolism of these seven chemical constituents from Zhimu-Baihe herb-pair are faster than those of same seven chemical constituents from Zhimu extract. Timosaponins showed double peak in concentration-time curve, which may be related to enterohepatic circulation. There are many pharmacokinetic studies about Zhimu and Baihe, however, there is no research paper on pharmacokinetic study of Zhimu-Baihe herb-pair. Our study shows that compatibility of Zhimu and Baihe has changed some pharmacokinetic parameters of those seven active compounds. Adding Baihe to Zhimu extract might promote the absorption of saponins, so as to affect the pharmacokinetics of Zhimu. The fact that compatibility of Zhimu-Baihe herb-pair has synergistic effect needs further study.