复方茯苓汤有效成分及其主要成分单体的药理活性研究
|
摘要
目的：
湿疹-皮炎是一种瘙痒性、复发性的皮肤病,对患者生活有严重影响。目前湿疹-皮炎的治疗还是个难题,传统的皮质类固醇激素和免疫抑制剂均有不可忽视的副作用,且不能长期应用,开发研究中药成为湿疹-皮炎治疗的发展趋势。
中医中药是中华民族历史文化和现代文明的重要组成部分,中药治疗湿疹-皮炎在中医医籍上早有记载,为后人进一步研究和开发中药提供了丰富的空间和资源。中药复方茯苓汤(Fu Fang Fu Ling Tang, FFFLT)由茯苓、赤芍、栀子、泽泻、黄柏、当归、浮萍、甘草组成,前期的实验研究及临床应用证明,复方茯苓汤对小鼠变应性接触性皮炎(allergic contact dermatitis, ACD)有显著抑制作用,临床也可有效治疗湿疹-皮炎。但复方茯苓汤的化学成分及其有效抗炎成分尚未研究,使其临床应用、质量控制、产业化都受到限制。
近十年来,基于液相色谱的技术被广泛应用于中药的质量控制,尤其是LC的联用技术液相色谱-质谱(liquid chromatography-mass spectra, LC-MS)和液相色谱-二极管阵列检测器(liquid chromatography-photodiode array detector, LC-DAD),分别作为首选技术用于复杂中药样品中目标化合物的定性和定量分析。目前,超高效液相色谱(ultra performance liquid chromatography, UPLC)和超高速液相色谱(ultra fast liquid chromatography, UFLC)由于其色谱柱选用微粒填料,能获得更高的柱效,节省分析时间,提高分析通量,适于快速分析植物样品及天然产物,同时也推动了中药药理活性作用的研究。
肝脏是药物主要的和重要的代谢器官,是药物生物转化的主要场所,大多数药物的Ⅰ相和Ⅱ相代谢反应都是在肝药酶系统的参与下发生的,因此药物的体外代谢模型主要是以肝脏为基础的,并以其特有的优势和特点在药物代谢的研究中得到广泛的应用。目前,有关药物代谢研究的热点主要是以肝微粒体和肝细胞为基础,通过分离培养的肝细胞及重组单酶进行药物的体外代谢研究,了解药物的代谢途径、代谢稳定性及经代谢酶生物转化的代谢产物,也可考察药物经代谢后对细胞的毒副作用。
湿疹-皮炎的免疫发病机制与T淋巴细胞密切相关。Thl和Th2型细胞亚群的平衡保持着机体正常免疫功能,而Th1／Th2免疫模式的确定是以Thl和Th2型细胞因子水平为主要依据。表皮海绵肿是湿疹-皮炎特征性病理变化,其机制是T细胞诱导的角质形成细胞凋亡。湿疹-皮炎的活化CD4+T细胞分泌IFN-γ,是引发皮肤病理的主要细胞因子之一。
本研究以中药复方茯苓汤为研究对象,先对复方茯苓汤在植物活性成分分离平台上用超高速液相色谱-二极管阵列检测器-电喷雾-质谱(UFLC-DAD-ESI-MS)等方法进行主要化学成分分离,分析和鉴定；再结合中药分析鉴定结果,选用2,4-二硝基氟苯(DNFB)诱导的小鼠变应性接触性皮炎(ACD)模型为药效学实验模型,进一步确定复方茯苓汤在活体内的抗炎有效成分；再选取有效成分单体及其体内脱糖代谢产物进行体外药物代谢研究,了解其代谢途径、鉴定其代谢产物；最后,从有效成分单体中选定目标化合物,研究其对人外周血T淋巴细胞增殖的抑制、诱导凋亡,细胞周期的变化及对Thl.Th2型细胞因子的影响,从分子水平和免疫调节机制方面探讨FFFLT中的目标化合物对湿疹-皮炎发病机制的影响。通过上述实验,为复方茯苓汤的临床应用提供理论依据。
方法和结果：
全文共分四部分。第一部分是采用UFLC-DAD-ESI-MS法分析复方茯苓汤的主要化学成分与质量控制。色谱条件：分析柱为Shim-pack XR-ODS(75 mm×2.0 mm,2.2μm)和Shim-pack XR-ODS(100 mm×2.0 mm,2.2μm),以乙腈(A)及0.1%甲酸-水(B)为流动相,采用梯度程序洗脱,流速为0.4 mL／min,柱温设定在40℃。质谱条件：在正负离子模式下分别扫描,检测器电压分别为+1.55kV与-1.55kV；扫描范围为m/z100-500；曲线脱溶剂管和加热块温度均为250℃,接口电压为4 kV,CDL电压为40 V；雾化气(氮气)流速设定在1.5 L／min,干燥气压力设定为0.06 MPa。结果：复方茯苓汤中共分析出14种成分,其中主要化学成分为京尼平龙胆双糖苷,京尼平苷,芍药苷及甘草苷,其最大吸收波长分别为239 nm,239 nm,230 nm及276 nm。在本研究方法下,京尼平龙胆双糖苷,京尼平苷,芍药苷及甘草苷的保留时间分别为4.30min,4.84min,5.46min及6.11 min,四种成分的定量线性范围在1.0 ng-200 ng之间,线性相关系数R2介于0.9998-1.0之间,日内差及日间差测定显示4个目标物的保留时间及峰面积相对标准偏差均小于1.7%；平均加样回收率介于95%-104%之间。
第二部分是研究复方茯苓汤作用于小鼠皮炎的有效组分。用2,4-二硝基氟苯(2,4-Dinitrofluorobenzene,DNFB)诱发的小鼠变应性接触性皮炎为动物模型,比较灌饲不同中药组合(Ⅰ-Ⅳ)及阳性对照氢化可的松和阴性对照生理盐水的药效学指标：Ⅰ组为茯苓,泽泻,黄柏,栀子,赤芍,浮萍,当归,甘草8味中药(复方茯苓汤)的水提物,Ⅱ组为栀子,赤芍,甘草3味中药的水提物,Ⅲ组为茯苓,当归2味中药的水提物,Ⅳ组为茯苓,栀子,赤芍,当归,甘草5味中药的水提物。观察中药对小鼠耳重量、耳厚度、耳真皮炎性细胞浸润以及血清细胞因子的影响。结果：与生理盐水组比较,不同中药组和氢化可的松组对诱发皮炎前后小鼠的耳重量差、耳厚度差及真皮炎症细胞浸润的影响均有显著差异(P<0.05),其中,8味中药组、5味中药组和氢化可的松组更优(P<0.01)。与生理盐水组比较,氢化可的松组和中药组小鼠血清IFN-γ水平均有显著降低(P<0.05),8味组和氢化可的松组尤为明显(P<0.01)；而各组IL-4,IL-10水平无显著性差异(P>0.05)。
第三部分是对该复方中的有效成分单体及其体内脱糖代谢产物进行体外代谢研究。采用体外肝微粒体孵育体系及肝细胞培养体系结合超快速液相色谱-质谱(UFLC-MS)技术,通过特异性细胞色素酶(CYP)抑制剂,重组单酶及相关性分析,对以上成分进行代谢酶研究及主要代谢产物分析。结果：芍药苷、京尼平苷、京尼平、甘草苷都不经肝脏Ⅰ相酶代谢；甘草苷的脱糖产物甘草素与人肝微粒体(HLMs)和烟酰胺腺嘌呤二核苷酸(NADPH)-孵育体系孵育30 min后,产生一个代谢产物,为4’,5,7-三羟基黄酮。采用9种主要CYP的选择性抑制剂来测定HLMs中产生4’,5,7-三羟基黄酮的CYP亚型,确定CYP1A2为生成该代谢产物的主要代谢酶。
第四部分是研究芍药苷抑制人外周血T淋巴细胞增殖、诱导凋亡及其对Th1及Th2型细胞因子的影响。采用SRB法检测CD3+CD28共刺激下的T淋巴细胞增殖及芍药苷对其增殖的抑制,流式细胞术检测芍药苷诱导T淋巴细胞凋亡及其细胞周期的变化,ELISA法分析芍药苷对T淋巴细胞上清中细胞因子的影响。结果：不同浓度芍药苷加药组与CD3+CD28组相比较,可呈浓度依赖性抑制CD3+CD28共刺激下的T淋巴细胞增殖(P<0.01)；不同浓度芍药苷加药组诱导活化的T淋巴细胞凋亡与CD3+CD28组比较,有显著性差异(P<0.01)。与CD3+CD28组相比较,不同浓度芍药苷加药组均能显著降低T淋巴细胞上清液中IFN-γ的水平(P<0.01),而对T淋巴细胞上清液中IL-4水平的影响无显著性差异(P>0.01)。
结论：
1.本研究采用UFLC-DAD-ESI-MS法首次对复方茯苓汤的化学成分进行分析,共分析出14种成分,其中主要化学成分是京尼平龙胆双糖苷,京尼平苷,芍药苷及甘草苷。
2.基于UFLC-DAD技术开发了复方茯苓汤中京尼平龙胆双糖苷,京尼平苷,芍药苷及甘草苷4种主要化学成分的快速定量分析方法。该方法快速、灵敏,稳定可靠,适于分析复方茯苓汤提取物中的活性成分,可用于复方茯苓汤原材料的质量控制。
3.复方茯苓汤的主要药效成分多来源于栀子,赤芍,甘草,茯苓和当归5味中药。复方茯苓汤抑制DNFB诱发的小鼠ACD的作用机制可能与降低IFN-γ水平有关。
4.复方茯苓汤中甘草苷的脱糖产物甘草素可被肝脏Ⅰ相酶代谢,代谢产物是4’,5,7-三羟基黄酮,CYP1A2为生成该代谢产物的主要代谢酶。澄清甘草素的代谢产物和代谢酶有利于对代谢产物的药理和毒理学活性进行深入研究,也可指导含甘草素药物的临床用药。
5.复方茯苓汤中芍药苷可抑制CD3+CD28共刺激的人外周血T淋巴细胞增殖,诱导T淋巴细胞凋亡,芍药苷可呈浓度依赖性显著抑制培养的人T淋巴细胞上清中IFN-γ水平,其抗炎机制可能是通过诱导T淋巴细胞凋亡、降低IFN-γ水平来起作用。
Objective:
Eczema-dermatitis is a kind of pruritic, recurrent skin disorder which has serious impact on patients'lives and is usually refractory. The treatment of this disease still needs to be developed. Conventional modalities of treatment such as corticosteroids and immunosuppressive agents and antihistaminics can not be used for long period because of their side effects. Therefore, it is imperative to develop a new recipe to treat this disease. Traditional Chinese Medicine (TCM) is supposed to be a promising and expectable method.
TCM is one of the important constitutes of Chinese ancient culture. Many records of TCM treating eczema-dermatitis have been kept in traditional Chinese books, which may provide abundant resources for us to study and develop TCM. Fu Fang Fu Ling Tang (FFFLT) was composed of Poria cocos, Paeonia lactiflora Pall, Gardenia jasminoides, Glycyrrhiza uralensis, Alisma orientalis, Phellodendron amurense, Angelica sinensis and Spirodela polyrrhiza. It has been proved via previous research and clinical experience that FFFLT could not only inhibit DNFB-induced allergic contact dermatitis (ACD) in mice, but also treat eczema effectively in clinical practice. However, the chemical constitutes and effective anti-inflammatory components of this herbal formula have not been studied, which limited its clinical application, quality control and industrialization.
Over recent ten years, the liquid chromatography (LC)-based methods have been widely used in quality control of TCMs and herbal medicines. Its hyphenated techniques, liquid chromatography-mass spectra(LC-MS) and liquid chromatography-photodiode array detector(LC-DAD) have been first choice of qualitative and quantitative analysis of target compounds in complex TCM samples. More recently, ultra performance liquid chromatography (UPLC) and ultra fast liquid chromatography (UFLC) become more and more popular in rapid profiling and target analysis of crude biological or botanical samples because of saving analytical time, enhancing separation performance, improving sensitivity, facilitating the rapid screening. Given these advantages, UPLC and UFLC are utilized for quality control and quantification of trace constitutes and also promote further investigation on pharmacologic activity of TCM.
Liver is the main organ of drug metabolism and biotransformation, whereⅠphase andⅡphase metabolism reaction of most drugs occur under the control of liver drug enzymes. The drug metabolism models in vitro based on liver microsomes and hepatic cells are widely used in the study of drug metabolism. Recently, research of drug metabolism which mainly focus on isolated hepatic cells and recombinant monoenzymes may contribute to understanding drug metabolism pathway and stability, as well as metabolites by biotransformation of metabolic enzymes and their toxic and side effects on cells.
The pathogenesis of eczema-dermatitis is closely related to T lymph-ocytes. The balance of Th1 and Th2 cell subsets maintain normal function of human immune system, and the Th1/Th2 immune reaction mainly depends on the level of Th1 and Th2 cytokines. Abnormal regulation of T lymphocytes cytokine network involves in the pathogenesis of eczema-dermatitis. Epidermal spongiosis is the characteristic pathologic changes of eczeima-dermatitis, the mechanism of which is T cell induced apoptosis of keratinocytes. The activated CD4+T cells in eczema-dermatitis secrete IFN-γ, which is one of the major cytokines promoting the pathologic changes in the skin.
This study aims mainly to analyze the effective components of FFFLT and explore the implication of these components in the pathogenesis of eczema-dermatitis. The principal chemical components of FFFLT were firstly isolated, analysed and identified by UFLC-DAD-ESI-MS. In order to determine the effective anti-inflammatory constitutes of FFFLT in vivo, mouse model of 2,4-dinitrofluorobenzene (DNFB) induced allergic contact dermatitis (ACD) were used to test the effects of these chemical compo-nents. Then the effective constitutes and their deglycosylation products were chosen in vitro so as to study the drug metabolites and drug metabolic pathway. In the last part, the target chemical substance was cocultured with the human peripheral blood T lymphocytes which were costimulated by CD3 plus CD28. The inhibition effects of the chemical substance on T lymphocytes proliferation were observed. The induction of apoptosis and transition of cell cycle were analysed and the Thl and Th2 type cytokines level in the culture supernatants were detected to investigate the role of the target chemical substance in the pathogenesis of eczema-dermatitis. On the whole, the studies are supposed to provide theoretical foundation for clinical application of FFFLT.
Methods and results
There were 4 parts in this study.
The first part:rapid analysis and quality control of principal compo-nents in the FFFLT by ultra fast liquid chromatography. Shim-pack XR-ODS column(75 mmx2.0 mm,2.2μm) and Shim-pack XR-ODS column(100 mm×2.0 mm,2.2μm) were used as solid phase, and the mobile phase consisted of acetonitrile(A) and water-formic acid (B,100:0.1,v/v). The gradient profiles were as follows:0-4 min,4%A; 4-6 min,20% A; 6-7.5 min,22% A; 7.5-10 min,90% A; 10-13.00 min,4% A. The flow rate was 0.4 mL/min, and the temperature was set at 40℃. Results:Using this method, most chemicals in FFFLT samples were baseline separated within 7 minutes. Fourteen constitutes were rapidly identified based on their product ions by UFLC-ESI-MS and the most abundant constitutes from FFFLT were genipingentiobioside, geniposide, paeoniflorin and liquiritin, by compa-rison of retention times, UV and MS spectra with authentic standards. For quantification analysis, the detection wavelengths were set at the maximal absorption wavelengths of genipingentiobioside, geniposide, paeoniflorin and liquiritin, which were 239 nm,239 nm,230 nm and 276 nm, respectively. Under this circumstance, the retention time of genipin, gentiobioside, geniposide, paeoniflorin and liquiritin were 4.30 min,4.84 min,5.46 min and 6.11 min respectively. The linear range of these four components ranged from 1.0 ng to 200ng with good correlation coefficient (0.9998 to 1.0). The inter-day and intra-day reproducibility were evaluated and the results showed that the relative standard deviations (RSDs) of both retention times and peak areas were less than 1.7%. The recovery was also evaluated and the results showed acceptable values between 95% to 104%.
The second part:the effective components of Fu Fang Fu Ling Tang on mouse dermatitis. Sixty mice with DNFB induced allergic contact dermatitis (ACD) were randomly divided into 6 groups, includingⅠ-Ⅳherb groups, positive control group (hydrocortisone, HC) and negative control group (physiological saline, PS). Herb groupⅠwas composed of Poria cocos, Paeonia lactiflora Pall, Gardenia jasminoides, Glycyrrhiza uralensis, Alisma orientalis, Phellodendron amurense, Angelica sinensis and Spirodela polyrrhiza; groupⅡwas composed of Paeonia lactiflora Pall, Gardenia jasminoides, Glycyrrhiza uralensis; groupⅢwas composed of Poria cocos and Angelica sinensis; and group IV was composed of Poria cocos, Paeonia lactiflora Pall, Gardenia jasminoides, Glycyrrhiza uralensis and Angelica sinensis. The thickness and weight of mouse ears, the pathological changes of ears tissue and the serum levels of IFN-γ, IL-4 and IL-10 were observed. Compared with PS, significant decrease in ear swelling and dermal inflammatory infiltration were seen in all herb groups and the HC group (P<0.05). Furthermore,ⅠandⅣgroup and the hydrocor-tisone group showed better effects (P<0.01). Compared with PS, significant decrease in the levels of the mouse serum IFN-γwere seen in all herb groups and the HC group (P<0.05), with the herb groupⅠand the HC group showing better effects (P<0.01); while the levels of IL-4 and IL-10 in all the groups showed no significant changes (P>0.05).
The third part:the metabolism of target chemical substances and their deglycosylation products of FFFLT in vitro. Chemical selective inhibitor, cDNA-expressed human CYPs, correlation assay and kinetics study were used to determine the metabolic enzyme of the chemicals with the liver microsomes incubation system and hepatic cell culture system by UFLC-MS. The results showed that paeoniflorin, geniposide, genipin and liquiritin were not metabolized by I phase enzyme. It was also found that liquiritigenin, the deglycosylation product of liquiritin, after incubated in the HLMs and NADPH incubation system for 30 minutes, produced a metabolite, which was identified as naringenin (4',5,7-trihydroxyflavanone) by comparing the tandem mass spectra and the chromatographic retention time with that of the standard compound. CYP1A2 was the specific isozyme responsible for the C5 hydroxylation of liquiritigenin in human liver microsomes.
The fourth part:the impacts of paeoniflorin on human peripheral blood T lymphocytes proliferation, apoptosis, cell cycle and the Thl and Th2 type cytokine level in supernatant. The inhibition of paeoniflorin on T lymphocytes proliferation after costimulated by CD3 plus CD28 was measured by Sulforhodamine B (SRB) method. Apoptosis and transition of cell cycle of T lymphocytes were analysed by flow cytometry, and the Th1、Th2 type cytokine level in supernatant were measured by ELISA. Different doses of paeoniflorin inhibited proliferation and induced apoptosis of these cells after costimulated by CD3 plus CD28 in dose-dependent manner (P<0.01). Paeoniflorin lowered IFN-y level in T lymphocytes supernatant (P<0.01), but had no effects on IL-4 level (P>0.01).
Conclusions:
1. Fourteen chemical components in FFFLT were identified firstly by ultra fast liquid chromatography-photodiode array detector-electrospray ionization-mass spectra (UFLC-DAD-ESI-MS). Genipingentiobioside, geniposide, paeoniflorin and liquiritin were four major components in FFFLT.
2. Simultaneous quantitative determination of abovementioned four major bioactive compounds were developed based on rapid method incorporating of UFLC-DAD. Distinctive advantages were shown that the UFLC-based method was rapid, sensitive, valid and applicable for the analysis of active components in FFFLT. It may also be used for quality control of FFFLT.
3. The effective components of FFFLT in treating eczema-dermatitis mostly came from Paeonia lactiflora Pall, Gardenia jasminoides, Glycyrrhiza uralensis, Poria cocos and Angelica sinensis. FFFLT may inhibite DNFB-induced ACD in mice by decreasing IFN-y level.
4. Liquiritigenin, the deglycosylation product of liquiritin from FFFLT, could be metabolized by liver I phase enzyme, and the metabolite was 4',5,7-trihydroxyflavanone. CYP1A2 was major metabolic enzyme catalyzing liquiritigenin hydroxylation in HLMs. Identification of metabolite of liquiritigenin and the significance of CYP1A2 as metabolic enzyme of liquiritigenin will intrigue further study of pharmacological and toxicology of 4',5,7-trihydroxyflavanone, and guide future investigations on individual differences of CYP1A2 in liquiritigenin metabolism.
5. Paeoniflorin of FFFLT inhibited proliferation and induced apoptosis of human peripheral blood T lymphocytes after costimulated by CD3 plus CD28 in dose-dependent manner. Paeoniflorin decreased IFN-y level in T lymphocytes supernatant in dose-dependent manner. The anti-inflammation mechanism may be related to apoptosis of T lymphocytes and decrease in IFN-y level.
湿疹-皮炎是一种瘙痒性、复发性的皮肤病,对患者生活有严重影响。目前湿疹-皮炎的治疗还是个难题,传统的皮质类固醇激素和免疫抑制剂均有不可忽视的副作用,且不能长期应用,开发研究中药成为湿疹-皮炎治疗的发展趋势。
中医中药是中华民族历史文化和现代文明的重要组成部分,中药治疗湿疹-皮炎在中医医籍上早有记载,为后人进一步研究和开发中药提供了丰富的空间和资源。中药复方茯苓汤(Fu Fang Fu Ling Tang, FFFLT)由茯苓、赤芍、栀子、泽泻、黄柏、当归、浮萍、甘草组成,前期的实验研究及临床应用证明,复方茯苓汤对小鼠变应性接触性皮炎(allergic contact dermatitis, ACD)有显著抑制作用,临床也可有效治疗湿疹-皮炎。但复方茯苓汤的化学成分及其有效抗炎成分尚未研究,使其临床应用、质量控制、产业化都受到限制。
近十年来,基于液相色谱的技术被广泛应用于中药的质量控制,尤其是LC的联用技术液相色谱-质谱(liquid chromatography-mass spectra, LC-MS)和液相色谱-二极管阵列检测器(liquid chromatography-photodiode array detector, LC-DAD),分别作为首选技术用于复杂中药样品中目标化合物的定性和定量分析。目前,超高效液相色谱(ultra performance liquid chromatography, UPLC)和超高速液相色谱(ultra fast liquid chromatography, UFLC)由于其色谱柱选用微粒填料,能获得更高的柱效,节省分析时间,提高分析通量,适于快速分析植物样品及天然产物,同时也推动了中药药理活性作用的研究。
肝脏是药物主要的和重要的代谢器官,是药物生物转化的主要场所,大多数药物的Ⅰ相和Ⅱ相代谢反应都是在肝药酶系统的参与下发生的,因此药物的体外代谢模型主要是以肝脏为基础的,并以其特有的优势和特点在药物代谢的研究中得到广泛的应用。目前,有关药物代谢研究的热点主要是以肝微粒体和肝细胞为基础,通过分离培养的肝细胞及重组单酶进行药物的体外代谢研究,了解药物的代谢途径、代谢稳定性及经代谢酶生物转化的代谢产物,也可考察药物经代谢后对细胞的毒副作用。
湿疹-皮炎的免疫发病机制与T淋巴细胞密切相关。Thl和Th2型细胞亚群的平衡保持着机体正常免疫功能,而Th1／Th2免疫模式的确定是以Thl和Th2型细胞因子水平为主要依据。表皮海绵肿是湿疹-皮炎特征性病理变化,其机制是T细胞诱导的角质形成细胞凋亡。湿疹-皮炎的活化CD4+T细胞分泌IFN-γ,是引发皮肤病理的主要细胞因子之一。
本研究以中药复方茯苓汤为研究对象,先对复方茯苓汤在植物活性成分分离平台上用超高速液相色谱-二极管阵列检测器-电喷雾-质谱(UFLC-DAD-ESI-MS)等方法进行主要化学成分分离,分析和鉴定；再结合中药分析鉴定结果,选用2,4-二硝基氟苯(DNFB)诱导的小鼠变应性接触性皮炎(ACD)模型为药效学实验模型,进一步确定复方茯苓汤在活体内的抗炎有效成分；再选取有效成分单体及其体内脱糖代谢产物进行体外药物代谢研究,了解其代谢途径、鉴定其代谢产物；最后,从有效成分单体中选定目标化合物,研究其对人外周血T淋巴细胞增殖的抑制、诱导凋亡,细胞周期的变化及对Thl.Th2型细胞因子的影响,从分子水平和免疫调节机制方面探讨FFFLT中的目标化合物对湿疹-皮炎发病机制的影响。通过上述实验,为复方茯苓汤的临床应用提供理论依据。
方法和结果：
全文共分四部分。第一部分是采用UFLC-DAD-ESI-MS法分析复方茯苓汤的主要化学成分与质量控制。色谱条件：分析柱为Shim-pack XR-ODS(75 mm×2.0 mm,2.2μm)和Shim-pack XR-ODS(100 mm×2.0 mm,2.2μm),以乙腈(A)及0.1%甲酸-水(B)为流动相,采用梯度程序洗脱,流速为0.4 mL／min,柱温设定在40℃。质谱条件：在正负离子模式下分别扫描,检测器电压分别为+1.55kV与-1.55kV；扫描范围为m/z100-500；曲线脱溶剂管和加热块温度均为250℃,接口电压为4 kV,CDL电压为40 V；雾化气(氮气)流速设定在1.5 L／min,干燥气压力设定为0.06 MPa。结果：复方茯苓汤中共分析出14种成分,其中主要化学成分为京尼平龙胆双糖苷,京尼平苷,芍药苷及甘草苷,其最大吸收波长分别为239 nm,239 nm,230 nm及276 nm。在本研究方法下,京尼平龙胆双糖苷,京尼平苷,芍药苷及甘草苷的保留时间分别为4.30min,4.84min,5.46min及6.11 min,四种成分的定量线性范围在1.0 ng-200 ng之间,线性相关系数R2介于0.9998-1.0之间,日内差及日间差测定显示4个目标物的保留时间及峰面积相对标准偏差均小于1.7%；平均加样回收率介于95%-104%之间。
第二部分是研究复方茯苓汤作用于小鼠皮炎的有效组分。用2,4-二硝基氟苯(2,4-Dinitrofluorobenzene,DNFB)诱发的小鼠变应性接触性皮炎为动物模型,比较灌饲不同中药组合(Ⅰ-Ⅳ)及阳性对照氢化可的松和阴性对照生理盐水的药效学指标：Ⅰ组为茯苓,泽泻,黄柏,栀子,赤芍,浮萍,当归,甘草8味中药(复方茯苓汤)的水提物,Ⅱ组为栀子,赤芍,甘草3味中药的水提物,Ⅲ组为茯苓,当归2味中药的水提物,Ⅳ组为茯苓,栀子,赤芍,当归,甘草5味中药的水提物。观察中药对小鼠耳重量、耳厚度、耳真皮炎性细胞浸润以及血清细胞因子的影响。结果：与生理盐水组比较,不同中药组和氢化可的松组对诱发皮炎前后小鼠的耳重量差、耳厚度差及真皮炎症细胞浸润的影响均有显著差异(P<0.05),其中,8味中药组、5味中药组和氢化可的松组更优(P<0.01)。与生理盐水组比较,氢化可的松组和中药组小鼠血清IFN-γ水平均有显著降低(P<0.05),8味组和氢化可的松组尤为明显(P<0.01)；而各组IL-4,IL-10水平无显著性差异(P>0.05)。
第三部分是对该复方中的有效成分单体及其体内脱糖代谢产物进行体外代谢研究。采用体外肝微粒体孵育体系及肝细胞培养体系结合超快速液相色谱-质谱(UFLC-MS)技术,通过特异性细胞色素酶(CYP)抑制剂,重组单酶及相关性分析,对以上成分进行代谢酶研究及主要代谢产物分析。结果：芍药苷、京尼平苷、京尼平、甘草苷都不经肝脏Ⅰ相酶代谢；甘草苷的脱糖产物甘草素与人肝微粒体(HLMs)和烟酰胺腺嘌呤二核苷酸(NADPH)-孵育体系孵育30 min后,产生一个代谢产物,为4’,5,7-三羟基黄酮。采用9种主要CYP的选择性抑制剂来测定HLMs中产生4’,5,7-三羟基黄酮的CYP亚型,确定CYP1A2为生成该代谢产物的主要代谢酶。
第四部分是研究芍药苷抑制人外周血T淋巴细胞增殖、诱导凋亡及其对Th1及Th2型细胞因子的影响。采用SRB法检测CD3+CD28共刺激下的T淋巴细胞增殖及芍药苷对其增殖的抑制,流式细胞术检测芍药苷诱导T淋巴细胞凋亡及其细胞周期的变化,ELISA法分析芍药苷对T淋巴细胞上清中细胞因子的影响。结果：不同浓度芍药苷加药组与CD3+CD28组相比较,可呈浓度依赖性抑制CD3+CD28共刺激下的T淋巴细胞增殖(P<0.01)；不同浓度芍药苷加药组诱导活化的T淋巴细胞凋亡与CD3+CD28组比较,有显著性差异(P<0.01)。与CD3+CD28组相比较,不同浓度芍药苷加药组均能显著降低T淋巴细胞上清液中IFN-γ的水平(P<0.01),而对T淋巴细胞上清液中IL-4水平的影响无显著性差异(P>0.01)。
结论：
1.本研究采用UFLC-DAD-ESI-MS法首次对复方茯苓汤的化学成分进行分析,共分析出14种成分,其中主要化学成分是京尼平龙胆双糖苷,京尼平苷,芍药苷及甘草苷。
2.基于UFLC-DAD技术开发了复方茯苓汤中京尼平龙胆双糖苷,京尼平苷,芍药苷及甘草苷4种主要化学成分的快速定量分析方法。该方法快速、灵敏,稳定可靠,适于分析复方茯苓汤提取物中的活性成分,可用于复方茯苓汤原材料的质量控制。
3.复方茯苓汤的主要药效成分多来源于栀子,赤芍,甘草,茯苓和当归5味中药。复方茯苓汤抑制DNFB诱发的小鼠ACD的作用机制可能与降低IFN-γ水平有关。
4.复方茯苓汤中甘草苷的脱糖产物甘草素可被肝脏Ⅰ相酶代谢,代谢产物是4’,5,7-三羟基黄酮,CYP1A2为生成该代谢产物的主要代谢酶。澄清甘草素的代谢产物和代谢酶有利于对代谢产物的药理和毒理学活性进行深入研究,也可指导含甘草素药物的临床用药。
5.复方茯苓汤中芍药苷可抑制CD3+CD28共刺激的人外周血T淋巴细胞增殖,诱导T淋巴细胞凋亡,芍药苷可呈浓度依赖性显著抑制培养的人T淋巴细胞上清中IFN-γ水平,其抗炎机制可能是通过诱导T淋巴细胞凋亡、降低IFN-γ水平来起作用。
Objective:
Eczema-dermatitis is a kind of pruritic, recurrent skin disorder which has serious impact on patients'lives and is usually refractory. The treatment of this disease still needs to be developed. Conventional modalities of treatment such as corticosteroids and immunosuppressive agents and antihistaminics can not be used for long period because of their side effects. Therefore, it is imperative to develop a new recipe to treat this disease. Traditional Chinese Medicine (TCM) is supposed to be a promising and expectable method.
TCM is one of the important constitutes of Chinese ancient culture. Many records of TCM treating eczema-dermatitis have been kept in traditional Chinese books, which may provide abundant resources for us to study and develop TCM. Fu Fang Fu Ling Tang (FFFLT) was composed of Poria cocos, Paeonia lactiflora Pall, Gardenia jasminoides, Glycyrrhiza uralensis, Alisma orientalis, Phellodendron amurense, Angelica sinensis and Spirodela polyrrhiza. It has been proved via previous research and clinical experience that FFFLT could not only inhibit DNFB-induced allergic contact dermatitis (ACD) in mice, but also treat eczema effectively in clinical practice. However, the chemical constitutes and effective anti-inflammatory components of this herbal formula have not been studied, which limited its clinical application, quality control and industrialization.
Over recent ten years, the liquid chromatography (LC)-based methods have been widely used in quality control of TCMs and herbal medicines. Its hyphenated techniques, liquid chromatography-mass spectra(LC-MS) and liquid chromatography-photodiode array detector(LC-DAD) have been first choice of qualitative and quantitative analysis of target compounds in complex TCM samples. More recently, ultra performance liquid chromatography (UPLC) and ultra fast liquid chromatography (UFLC) become more and more popular in rapid profiling and target analysis of crude biological or botanical samples because of saving analytical time, enhancing separation performance, improving sensitivity, facilitating the rapid screening. Given these advantages, UPLC and UFLC are utilized for quality control and quantification of trace constitutes and also promote further investigation on pharmacologic activity of TCM.
Liver is the main organ of drug metabolism and biotransformation, whereⅠphase andⅡphase metabolism reaction of most drugs occur under the control of liver drug enzymes. The drug metabolism models in vitro based on liver microsomes and hepatic cells are widely used in the study of drug metabolism. Recently, research of drug metabolism which mainly focus on isolated hepatic cells and recombinant monoenzymes may contribute to understanding drug metabolism pathway and stability, as well as metabolites by biotransformation of metabolic enzymes and their toxic and side effects on cells.
The pathogenesis of eczema-dermatitis is closely related to T lymph-ocytes. The balance of Th1 and Th2 cell subsets maintain normal function of human immune system, and the Th1/Th2 immune reaction mainly depends on the level of Th1 and Th2 cytokines. Abnormal regulation of T lymphocytes cytokine network involves in the pathogenesis of eczema-dermatitis. Epidermal spongiosis is the characteristic pathologic changes of eczeima-dermatitis, the mechanism of which is T cell induced apoptosis of keratinocytes. The activated CD4+T cells in eczema-dermatitis secrete IFN-γ, which is one of the major cytokines promoting the pathologic changes in the skin.
This study aims mainly to analyze the effective components of FFFLT and explore the implication of these components in the pathogenesis of eczema-dermatitis. The principal chemical components of FFFLT were firstly isolated, analysed and identified by UFLC-DAD-ESI-MS. In order to determine the effective anti-inflammatory constitutes of FFFLT in vivo, mouse model of 2,4-dinitrofluorobenzene (DNFB) induced allergic contact dermatitis (ACD) were used to test the effects of these chemical compo-nents. Then the effective constitutes and their deglycosylation products were chosen in vitro so as to study the drug metabolites and drug metabolic pathway. In the last part, the target chemical substance was cocultured with the human peripheral blood T lymphocytes which were costimulated by CD3 plus CD28. The inhibition effects of the chemical substance on T lymphocytes proliferation were observed. The induction of apoptosis and transition of cell cycle were analysed and the Thl and Th2 type cytokines level in the culture supernatants were detected to investigate the role of the target chemical substance in the pathogenesis of eczema-dermatitis. On the whole, the studies are supposed to provide theoretical foundation for clinical application of FFFLT.
Methods and results
There were 4 parts in this study.
The first part:rapid analysis and quality control of principal compo-nents in the FFFLT by ultra fast liquid chromatography. Shim-pack XR-ODS column(75 mmx2.0 mm,2.2μm) and Shim-pack XR-ODS column(100 mm×2.0 mm,2.2μm) were used as solid phase, and the mobile phase consisted of acetonitrile(A) and water-formic acid (B,100:0.1,v/v). The gradient profiles were as follows:0-4 min,4%A; 4-6 min,20% A; 6-7.5 min,22% A; 7.5-10 min,90% A; 10-13.00 min,4% A. The flow rate was 0.4 mL/min, and the temperature was set at 40℃. Results:Using this method, most chemicals in FFFLT samples were baseline separated within 7 minutes. Fourteen constitutes were rapidly identified based on their product ions by UFLC-ESI-MS and the most abundant constitutes from FFFLT were genipingentiobioside, geniposide, paeoniflorin and liquiritin, by compa-rison of retention times, UV and MS spectra with authentic standards. For quantification analysis, the detection wavelengths were set at the maximal absorption wavelengths of genipingentiobioside, geniposide, paeoniflorin and liquiritin, which were 239 nm,239 nm,230 nm and 276 nm, respectively. Under this circumstance, the retention time of genipin, gentiobioside, geniposide, paeoniflorin and liquiritin were 4.30 min,4.84 min,5.46 min and 6.11 min respectively. The linear range of these four components ranged from 1.0 ng to 200ng with good correlation coefficient (0.9998 to 1.0). The inter-day and intra-day reproducibility were evaluated and the results showed that the relative standard deviations (RSDs) of both retention times and peak areas were less than 1.7%. The recovery was also evaluated and the results showed acceptable values between 95% to 104%.
The second part:the effective components of Fu Fang Fu Ling Tang on mouse dermatitis. Sixty mice with DNFB induced allergic contact dermatitis (ACD) were randomly divided into 6 groups, includingⅠ-Ⅳherb groups, positive control group (hydrocortisone, HC) and negative control group (physiological saline, PS). Herb groupⅠwas composed of Poria cocos, Paeonia lactiflora Pall, Gardenia jasminoides, Glycyrrhiza uralensis, Alisma orientalis, Phellodendron amurense, Angelica sinensis and Spirodela polyrrhiza; groupⅡwas composed of Paeonia lactiflora Pall, Gardenia jasminoides, Glycyrrhiza uralensis; groupⅢwas composed of Poria cocos and Angelica sinensis; and group IV was composed of Poria cocos, Paeonia lactiflora Pall, Gardenia jasminoides, Glycyrrhiza uralensis and Angelica sinensis. The thickness and weight of mouse ears, the pathological changes of ears tissue and the serum levels of IFN-γ, IL-4 and IL-10 were observed. Compared with PS, significant decrease in ear swelling and dermal inflammatory infiltration were seen in all herb groups and the HC group (P<0.05). Furthermore,ⅠandⅣgroup and the hydrocor-tisone group showed better effects (P<0.01). Compared with PS, significant decrease in the levels of the mouse serum IFN-γwere seen in all herb groups and the HC group (P<0.05), with the herb groupⅠand the HC group showing better effects (P<0.01); while the levels of IL-4 and IL-10 in all the groups showed no significant changes (P>0.05).
The third part:the metabolism of target chemical substances and their deglycosylation products of FFFLT in vitro. Chemical selective inhibitor, cDNA-expressed human CYPs, correlation assay and kinetics study were used to determine the metabolic enzyme of the chemicals with the liver microsomes incubation system and hepatic cell culture system by UFLC-MS. The results showed that paeoniflorin, geniposide, genipin and liquiritin were not metabolized by I phase enzyme. It was also found that liquiritigenin, the deglycosylation product of liquiritin, after incubated in the HLMs and NADPH incubation system for 30 minutes, produced a metabolite, which was identified as naringenin (4',5,7-trihydroxyflavanone) by comparing the tandem mass spectra and the chromatographic retention time with that of the standard compound. CYP1A2 was the specific isozyme responsible for the C5 hydroxylation of liquiritigenin in human liver microsomes.
The fourth part:the impacts of paeoniflorin on human peripheral blood T lymphocytes proliferation, apoptosis, cell cycle and the Thl and Th2 type cytokine level in supernatant. The inhibition of paeoniflorin on T lymphocytes proliferation after costimulated by CD3 plus CD28 was measured by Sulforhodamine B (SRB) method. Apoptosis and transition of cell cycle of T lymphocytes were analysed by flow cytometry, and the Th1、Th2 type cytokine level in supernatant were measured by ELISA. Different doses of paeoniflorin inhibited proliferation and induced apoptosis of these cells after costimulated by CD3 plus CD28 in dose-dependent manner (P<0.01). Paeoniflorin lowered IFN-y level in T lymphocytes supernatant (P<0.01), but had no effects on IL-4 level (P>0.01).
Conclusions:
1. Fourteen chemical components in FFFLT were identified firstly by ultra fast liquid chromatography-photodiode array detector-electrospray ionization-mass spectra (UFLC-DAD-ESI-MS). Genipingentiobioside, geniposide, paeoniflorin and liquiritin were four major components in FFFLT.
2. Simultaneous quantitative determination of abovementioned four major bioactive compounds were developed based on rapid method incorporating of UFLC-DAD. Distinctive advantages were shown that the UFLC-based method was rapid, sensitive, valid and applicable for the analysis of active components in FFFLT. It may also be used for quality control of FFFLT.
3. The effective components of FFFLT in treating eczema-dermatitis mostly came from Paeonia lactiflora Pall, Gardenia jasminoides, Glycyrrhiza uralensis, Poria cocos and Angelica sinensis. FFFLT may inhibite DNFB-induced ACD in mice by decreasing IFN-y level.
4. Liquiritigenin, the deglycosylation product of liquiritin from FFFLT, could be metabolized by liver I phase enzyme, and the metabolite was 4',5,7-trihydroxyflavanone. CYP1A2 was major metabolic enzyme catalyzing liquiritigenin hydroxylation in HLMs. Identification of metabolite of liquiritigenin and the significance of CYP1A2 as metabolic enzyme of liquiritigenin will intrigue further study of pharmacological and toxicology of 4',5,7-trihydroxyflavanone, and guide future investigations on individual differences of CYP1A2 in liquiritigenin metabolism.
5. Paeoniflorin of FFFLT inhibited proliferation and induced apoptosis of human peripheral blood T lymphocytes after costimulated by CD3 plus CD28 in dose-dependent manner. Paeoniflorin decreased IFN-y level in T lymphocytes supernatant in dose-dependent manner. The anti-inflammation mechanism may be related to apoptosis of T lymphocytes and decrease in IFN-y level.
引文
1. 刘辅仁.实用皮肤科学.第二版.北京：人民卫生出版社.1997,244.
2. 黑玉英,马双弟.中医中药治疗慢性湿疹的体会.内蒙古中医药.2006；(6)：23-24.
3. 郭爱群,马万里.滋阴除湿汤加味治疗慢性湿疹.湖北中医杂志.2001；23(6)：37.
4. 王丽娟,毕湘杰,张秀云.中药治疗湿疹100例临床疗效观察.厂矿医药卫生.2000；16(3)：211.
5. 王惠玲.急性湿疹的中药治疗.首都医药.1999；6(9)：34.
6. 徐金鹏.中药治疗慢性湿疹89例.吉林中医药.2004；24(5)：33.
7. 黄为群.加减除湿汤治疗湿疹样皮炎疗效观察.亚太传统医药.2009；5(8)：80-81.
8. 戴溱,毛舒和.中药治疗泛发性湿疹126例疗效分析.天津中医.1999；16(1)：17.
9. 涂彩霞,张新军,张蕴颖,等.复方甘草酸苷治疗特应性皮炎的临床及实验研究.中国麻风皮肤病杂志.2007；23(5)：393-395.
10.卢艳红.雷公藤多甙治疗泛发性湿疹疗效观察.包头医学院学报.2007；23(4)：388-389.
11.杨春梅,刘晓明,涂彩霞,等.丹皮酚治疗湿疹的研究.中国皮肤性病学杂志.1997,11(1)：25.
12.王建湘.中医辨证加熏洗治疗小腿湿疹临床疗效分析.中国临床药理学与治疗学.2003；8(1)：104-106.
13.王兆梓,张立平,王波.中药治疗湿疹128例.中医研究.2002；15(3)：43.
14.徐关冰,闫景东,周妍妍.中药治疗婴儿湿疹76例临床观察.中医药信息.2006；23(4)：37.
15.王小萍,秦亮甫.秦亮甫教授治疗湿疹150例.上海中医药杂志.2001；10(7)：31.
16.方晴,朱永先,吴兰芬.中药治疗湿疹及皮炎类皮肤病临床疗效观察.北京中医药大学学报.2003；26(4)：83-84.
17.王光亚,朱奎.中药治疗湿疹临床观察.当代医学.2009；15(34)：157.
18.朱正君.中药治疗婴儿湿疹的临床研究.中医儿科杂志.2008；4(2)：36-39.
19.李建华.中药治疗肛门湿疹68例临床观察.中国麻风皮肤病杂志.2005；21(2)：153.
20.付宏伟.中药治疗婴儿期异位性皮炎160例.中国中医药信息杂志.2000；7(2)：66.
21.胡宝春.湿疹的中药治疗及护理.时珍国医国药.2002；13(2)：125.
22.胡春菊.急性湿疹的中药治疗及护理.辽宁中医药大学学报.2008；10(4)：118.
23.刘明学.除湿止痒熏洗剂治疗肛门湿疹127例.陕西中医.2000；21(7)：303.
24.兰绍波,宋修亭.如意金黄散外治湿疹132例.辽宁中医杂志.2006；33(4)：482.
25.宋岚,武静.苦参等中药治疗皮炎湿疹类皮肤病临床疗效观察.职业与健康.2002；18(5)：140-141.
26.刘金燕.针灸治疗湿疹52例疗效观察.实用医技杂志.2006；13(1)：148-149.
27.王丽娟,周丽波,沈仲秋,等.梅花针与中药治疗湿疹100例.针灸临床杂志.2001；17(5)：15.
28.陈淑琴.针药并用治疗湿疹.中国针灸.2003；23(4)：221.
29.宫振甲,周立东.中药保留灌肠治疗婴幼儿湿疹96例临床观察.中国中西医结合皮肤性病学杂志.2007；6(2)：103-104.
30.涂彩霞,林熙然,谷玲.四种中药复方对小鼠实验性皮炎作用的研究.中华皮肤科杂志.1998；31(4)：244-246.
31.林熙然,涂彩霞,杨春梅,等.抗Ⅳ型变态反应中药治疗湿疹的研究.中国中西医结合杂志.2000；20(4)：258-260.
32.涂彩霞,刘芳,谷玲,等.复方茯苓汤对小鼠接触性过敏反应的影响.中华皮肤科杂志.2001；34(6)：452.
33.涂彩霞,刘芳,李敬,等.复方茯苓汤治疗湿疹582例临床观察及实验研究.中国中西医结合皮肤性病学杂志.2002；1(1)：13-15.
34. Lin XR, Tu CX, Meng XM, et al. Studies on treating eczema by Chinese herbal medicine with anti-type IV allergic activity. Chin J Integr Med.2001;7(1):7-11.
35. Kim MJ, Im KR, Yoon KS.et al. Anti-inflammatory effects of YeongyoSeungma-tang. J Ethnopharmacol.2009; 126(3):377-381.
36. Chan BC, Hon KL, Leung PC, et al. Traditional Chinese medicine for atopic eczema:PentaHerbs formula suppresses inflammatory mediators release from mast cells. J Ethnopharmacol.2008; 120(1):85-91.
37. Kim DY, Jung JA, Kim TH, et al. Oral administration of Uncariae rhynchophylla inhibits the development of DNFB-induced atopic dermatitis-like skin lesions via IFN-gamma down-regulation in NC/Nga mice. J Ethnopharmacol. 2009;122(3):567-572.
38. Choi JJ, Park B, Kim DH, et al. Blockade of atopic dermatitis-like skin lesions by DA-9102, a natural medicine isolated from Actinidia arguta, in the Mg-deficiency induced dermatitis model of hairless rats. Exp Biol Med. 2008;233(8):1026-1034.
39. Leung TF, Wong KY, Wong CK, et al. In vitro and clinical immunomodulatory effects of a novel Pentaherbs concoction for atopic dermatitis. Br J Dermatol. 2008;158(6):1216-1223.
40. Hon KL, Lee VW, Leung TF, et al. Corticosteroids are not present in a traditional Chinese medicine formulation for atopic dermatitis in children. Ann Acad Med Singapore.2006;35(11):759-763.
41. Makino T, Hamanaka M, Yamashita H, et al. Effect of bakumijiogan, an herbal formula in traditional Chinese medicine, on atopic dermatitis-like skin lesions induced by mite antigen in NC/Jic mice. Biol Pharm Bull. 2008;31(11):2108-2113.
42. Choi MS, Kim EC, Lee HS, et al. Inhibitory effects of Saururus chinensis (LOUR.) BAILL on the development of atopic dermatitis-like skin lesions in NC/Nga mice. Biol Pharm Bull.2008;31 (1):51-56.
43. Nakatsukasa H, Tago F, Okamoto T, et al. Therapeutic Effects of Gyokuheifusan on NC/Nga Mouse Model of Allergic Dermatitis. J Health Sci.2009;55(4): 516-524.
44. Kobayashi H, Mizuno N, Teramae H, et al. Diet and Japanese herbal medicine for recalcitrant atopic dermatitis:Efficacy and safety. Drugs Exp Clin Res.2004;30 (5-6):197-202.
45. Kobayashi H, Mizuno N, Teramae H,et al. The effects of Hochu-ekki-to in patients with atopic dermatitis resistant to conventional treatment. Int J Tissue React.2004;26(3-4):113-117.
46. Kobayashi H, Mizuno N, Kutsuna H, et al. Hochu-ekki-to suppresses development of dermatitis and elevation of serum IgE level in NC/NGA mice. Drugs Exp Clin Res.2003;29(2):81-84.
47. Nakada T, Watanabe K, Matsumoto T, et al. Effect of orally administered Hochu-ekki-to, a Japanese herbal medicine, on contact hypersensitivity caused by repeated application of antigen. Int Immunopharmacol.2002;2(7):901-911.
48. Andoh T, Honma Y, Kawaharada S, et al. Inhibitory effect of the repeated treatment with Unsei-in on substance P-induced itch-associated responses through the downregulation of the expression of NK1 tachykinin receptor in mice. Biol Pharm Bull.2003;26(6):896-898.
49. Novak N, Haberstok J, Kraft S, et al. Standardized extracts from Chinese herbs induce IL-10 production in human monocyte-derived dendritic cells and alter their differentiation in vitro. J Allergy Clin Immunol.2001; 108(4):588-593.
50. Xu XJ, Banerjee P, Rustin MH, et al. Modulation by Chinese herbal therapy of immune mechanisms in the skin of patients with atopic eczema. Br J Dermatol. 1997;136(1):54-59.
51. Tahara E, Satoh T, Toriizuka K,et al. Effect of Shimotsu-to (a Kampo medicine, Si-Wu-Tang) and its constituents on triphasic skin reaction in passively sensitized mice. J Ethnopharmacol.1999;68(1-3):219-228.
52. Shichijo, Kosaburo; Saito, Hirohisa; Ikura, Yoji.Inhibitory effect of the Sho-saiko-to, Shofu-san, and Makyo-kanseki-to on histamine release from murine bone marrow-derived cultured mast cells. Jikeikai Medical Journal.1995; 42(3):183-194.
53. Lee B, Shin YW, Bae EA, et al. Antiallergic effect of the root of Paeonia lactiflora and its constituents paeoniflorin and paeonol. Arch Pharm Res. 2008;31(4):445-450.
54. Yamahara J, Yamada T, Kimura H, et al. Biologically active principles of crude drugs. Ⅱ. Anti-allergic principles in "Shoseiryu-To" anti-inflammatory pro-perties of paeoniflorin and its derivatives. J Pharmacobiodyn.1982;5(11): 921-929.
55. Sun Y, Dong Y, Jiang HJ, et al. Dissection of the role of paeoniflorin in the traditional Chinese medicinal formula Si-Ni-San against contact dermatitis in mice. Life Sci.2009;84(11-12):337-344.
1. 涂彩霞,刘芳,谷玲,等.复方茯苓汤对小鼠接触性过敏反应的影响.中华皮肤科杂志.2001；34(6)：452.
2. 涂彩霞,刘芳,李敬,等.复方茯苓汤治疗湿疹582例临床观察及实验研究.中国中西医结合皮肤性病学杂志.2002；1(1)：13-15.
3. 涂彩霞,林熙然,谷玲.四种中药复方对小鼠实验性皮炎作用的研究.中华皮肤科杂志.1998,3 1(4)：244-246.
4. 林熙然,涂彩霞,杨春梅,等.抗Ⅳ型变态反应中药治疗湿疹的研究.中国中西医结合杂志.2000,20(4)：258-260.
5. Meng J, Leung KS, Dong XP, Zhou YS, Jiang ZH, Zhao ZZ. Simultaneous quantification of eight bioactive components of Houttuynia cordata and related Saururaceae medicinal plants by on-line high performance liquid chromato-graphy-diode array detector-electrospray mass spectrometry.Fitoterapia.2009; 80(8):468-474.
6. Ni LJ, Zhang LG, Hou J, Shi WZ, Guo ML. A strategy for evaluating antipyretic efficacy of Chinese herbal medicines based on UV spectra fingerprints. J Ethnopharmacol.2009; 124(1):79-86.
7. Liu AH, Lin YH, Yang M, Guo H, Guan SH, Sun JH, Guo DA.Development of the fingerprints for the quality of the roots of Salvia miltiorrhiza and its related preparations by HPLC-DAD and LC-MS(n). J Chromatogr B Analyt Technol Biomed Life Sci.2007;846(1-2):32-41.
8. Liang XM, Jin Y, Wang YP, Jin GW, Fu Q, Xiao YS.Qualitative and quantitative analysis in quality control of traditional Chinese medicines. J Chromatogr A. 2009;1216(11):2033-2044.
9. Kong WJ, Zhao YL, Xiao XH, Jin C, Li ZL.Quantitative and chemical fingerprint analysis for quality control of rhizoma Coptidischinensis based on UPLC-PAD combined with chemometrics methods.Phytomedicine.2009;16(10):950-959.
10. Min JZ, Shimizu Y, Toyo'oka T, Inagaki S, Kikura-Hanajiri R, Goda Y.Simul-taneous determination of 11 designated hallucinogenic phenethylamines by ultra-fast liquid chromatography with fluorescence detection. J Chromatogr B Analyt Technol Biomed Life Sci.2008;873(2):187-194.
11. Ge GB, Luan HW, Zhang YY, He YQ, Liu XB, Yang L, Wang ZT, Yang L.Profiling of yew hair roots from various species using ultra-performance liquid
chromatography/electrospray ionization mass spectrometry. Rapid Commun Mass Spectrom.2008;22(15):2315-2323.
12. Li R, Wang X, Zhou Y, Cai M, Ding L. Analysis of sodium adduct paeoniflorin, albiflorin and their derivatives by (+)ESI-MSn, DFT calculations and computer-aided mass spectrometry analysis program.J Mass Spectrom.2007;42(3): 335-345.
13. Xu SJ, Yang L, Zeng X, Zhang M, Wang ZT.Characterization of compounds in the Chinese herbal drug Mu-Dan-Pi by liquid chromatography coupled to electrospray ionization mass spectrometry. Rapid Commun Mass Spectrom. 2006;20(22):3275-3288.
14. Moiteiro C, Gaspar H, Rodrigues AI, Lopes JF, Carnide V.HPLC quantification of dye flavonoids in Reseda luteola L. from Portugal. J Sep Sci.2008;31(21): 3683-3687.
15. Svehlikova V, Bennett RN, Mellon FA, Needs PW, Piacente S, Kroon PA, Bao Y.Isolation, identification and stability of acylated derivatives of apigenin 7-O-glucoside from chamomile (Chamomilla recutita [L.] Rauschert). Phytochemistry.2004;65(16):2323-2332.
16. Hu YM, Su GH, Sze SC, Ye W, Tong Y. Quality assessment of Cortex Phellodendri by high-performance liquid chromatography coupled with electrospray ionization mass spectrometry. Biomed Chromatogr.2010;24(4): 438-453.
17. Hatano T, Takagi M, Ito H, Yoshida T. Acylated flavonoid glycosides and accompanying phenolics from licorice.Phytochemistry.1998;47(2):287-293.
18. Cao J, Xu Y, Zhang Y, Wang Y, Luo G. Chin J Anal Chem.2004;32(4):875.
19. Chen F, Lu HT, Jiang Y. Purification of paeoniflorin from Paeonia lactiflora Pall, by high-speed counter-current chromatography. J Chromatogr A. 2004;1040(2):205-208.
20. Rauchensteiner F, Matsumura Y, Yamamoto Y, Yamaji S, Tani T. Analysis and comparison of Radix Glycyrrhizae (licorice) from Europe and China by capillary-zone electrophoresis (CZE). J Pharm Biomed Anal.2005;38(4): 594-600.
21. Lee KY, You HJ, Jeong HG, Kang JS, Kim HM, Rhee SD, Jeon YJ. Polysac-charide isolated from Poria cocos sclerotium induces NF-kappaB/Rel activation and iNOS expression through the activation of p38 kinase in murine macrophages. Int Immunopharmacol.2004;4(8):1029-1038.
22. Cuellar MJ, Giner RM, Recio MC, Just MJ, Manez S, Rios JL.Effect of the basidiomycete Poria cocos on experimental dermatitis and other inflammatory conditions. Chem Pharm Bull (Tokyo).1997;45(3):492-494.
23. Wong VK, Yu L, Cho CH.Protective effect of polysaccharides from Angelica sinensis on ulcerative colitis in rats.Inflammopharmacology.2008; 16(4): 162-167.
24. Sun Y, Tang J, Gu X, Li D. Water-soluble polysaccharides from Angelica sinensis (Oliv.) Diels:Preparation, characterization and bioactivity. Int J Biol Macromol. 2005;36(5):283-289.
25. Zhang C, Zhou A, Zhang M. Chemical constituents of Alisma orientalis and their immunosuppressive function. Zhongguo Zhong Yao Za Zhi.2009;34(8):994-998.
26. Koo HJ, Lim KH, Jung HJ, Park EH. Anti-inflammatory evaluation of gardenia extract, geniposide and genipin. J Ethnopharmacol.2006;103(3):496-500.
27. Sun Y, Dong Y, Jiang HJ, Cai TT, Chen L, Zhou X, Chen T, Xu Q.Dissection of the role of paeoniflorin in the traditional Chinese medicinal formula Si-Ni-San against contact dermatitis in mice.Life Sci.2009;84(11-12):337-344.
28. Yoon T, Cheon MS, Kim SJ, Choo BK, Moon BC, Lee AY. Evaluation of the anti-inflammatory efficacy of Glycyrrhiza uralensis according to extracting solvents [J]. Planta Medica.2009;75:1050-1051.
29. Chen Y, Zhang H, Li YX, Cai L, Huang J, Zhao C, Jia L, Buchanan R, Yang T, Jiang LJ. Crocin and geniposide profiles and radical scavenging activity of gardenia fruits (Gardenia jasminoides Ellis) from different cultivars and at the various stages of maturation. Fitoterapia.2010;81(4):269-273.
1. 涂彩霞,刘芳,谷玲,等.复方茯苓汤对小鼠接触性过敏反应的影响.中华皮肤科杂志.2001；34(6)：452.
2. 涂彩霞,刘芳,李敬,等.复方茯苓汤治疗湿疹582例临床观察及实验研究.中国中西医结合皮肤性病学杂志.2002；1(1)：13-15.
3. Lin IH, Lee MC, Chuang WC, et al. Application of LC/MS and ICP/MS for establishing the fingerprint spectrum of the traditional Chinese medicinal preparation Gan-Lu-Yin. J Sep Sci.2006;29 (1):172-179.
4. Wang H, Feng F. Identification of components in Zhi-Zi-Da-Huang decoction by HPLC coupled with electrospray ionization tandem mass spectrometry, photodiode array and fluorescence detectors. Pharm Biomed Anal.2009;49(5): 1157-1165.
5. Bedi MK, Shenefelt PD. Herbal therapy in dermatology. Arch Dermatol. 2002;138(2):232-242.
6. Leung TF, Wong KY, Wong CK, et al. In vitro and clinical immunomodulatory effects of a novel Pentaherbs concoction for atopic dermatitis. Br J Dermatol. 2008;158(6):1216-1223.
7. Rowe A, Bunker CB. Interleukin-4 and the interleukin-4 receptor in allergic contact dermatitis. Contact Dermatitis.1998;38(1):36-39.
8. Neis MM, Peters B, Dreuw A, et al. Enhanced expression levels of IL-31 correlate with IL-4 and IL-13 in atopic and allergic contact dermatitis. J Allergy Clin Immunol.2006; 118(4):930-937.
9. 许宗严,吴铁,吴志华.鼠皮炎湿疹模型建立的研究进展.中国临床药理学与治疗学.2005；10(7)：730-733.
10. Kim DY, Jung JA, Kim TH, et al. Oral administration of Uncariae rhynchophylla inhibits the development of DNFB-induced atopic dermatitis-like skin lesions via IFN-gamma down-regulation in NC/Nga mice. J Ethnopharmacol. 2009;122(3):567-572.
11. Hopkins J E, Naisbitt DJ, Kitteringham NR, et al. Selective haptenation of cellular or extracellular protein by chemical allergens:association with cytokine polarization.Chem Res Toxicol.2005;18(4):375-381.
12. Ge GB, Zhang YY, Hao DC, et al. Chemotaxonomic study of medicinal Taxus species with fingerprint and multivariate analysis.Planta Med.2008; 74(7):
773-779.
13. Chan EC, Yap SL, Lau AJ, et al. Ultra-performance liquid chromatography/-time-of-flight mass pectrometry based metabolomics of raw and steamed Panax notoginseng. Rapid Commun Mass Spectrom.2007;21(4):519-528.
14. Apollonio LG, Pianca DJ, Whittall IR, et al. A demonstration of the use of ultra-performance liquid chromatography-mass spectrometry [UPLC/MS] in the determination of amphetamine-type substances and ketamine for forensic and toxicological analysis. Chromatogr B Analyt Technol Biomed Life Sci.2006;836(1-2):111-115.
15. Lee B, Shin YW, Bae EA, et al. Antiallergic effect of the root of Paeonia lactiflora and its constituents paeoniflorin and paeonol. Arch Pharm Res.2008;31(4):445-450.
16. Wu M, Gu Z. Screening of bioactive compounds from moutan cortex and their anti-inflammatory activities in rat synoviocytes. Evid Based Complement Alternat Med.2009;6(1):57-63.
17. Sun Y, Dong Y, Jiang HJ, et al. Dissection of the role of paeoniflorin in the traditional Chinese medicinal formula Si-Ni-San against contact dermatitis in mice. Life Sci.2009;84(11-12):337-344.
18. Jiang WL, Chen XG, Zhu HB, et al. Paeoniflorin inhibits systemic inflammation and improves survival in experimental sepsis. Basic Clin Pharmacol Toxicol. 2009;105(1):64-71.
19. Chang WL, Wang HY, Shi LS, et al. Immunosuppressive iridoids from the fruits of Gardenia jasminoides. J Nat Prod.2005; 68(11):1683-1685.
20. Koo HJ, Song YS, Kim HJ, et al. Antiinflammatory effects of genipin, an active principle of gardenia. Eur J Pharmacol.2004;495(2-3):201-208.
21. Koo HJ, Lim KH, Jung HJ, et al. Anti-inflammatory evaluation of gardenia extract, geniposide and genipin. J Ethnopharmacol.2006; 103(3):496-500.
22. Yoon T, Cheon MS, Kim SJ, et al. Evaluation of the anti-inflammatory efficacy of Glycyrrhiza uralensis according to extracting solvents. Planta Medica.2009; 75:1050-1051.
23. Wang Y, Zhang M, Ruan D, et al. Chemical components and molecular mass of six polysaccharides isolated from the sclerotium of Poria cocos. Carbohydr Res. 2004;339(2):327-334.
24. Sun Y, Tang J, Gu X, et al. Water-soluble polysaccharides from Angelica sinensis (Oliv.) Diels:Preparation, characterization and bioactivity. Int J Biol Macromol. 2005;36(5):283-289.
25. Wong VK, Yu L, Cho CH. et al. Protective effect of polysaccharides from Angelica sinensis on ulcerative colitis in rats. Inflammopharmacology.2008; 16(4):162-167.
26. Yang TH, Jia M, Mei QB,et al. Effect of Angelica sinensis polysaccharide fraction AP-3 on IL-2 and IFN-gamma induction.Yao Xue Xue Bao.2006;41(1):54-57.
27. Yang X, Zhao Y, Wang H, et al. Macrophage activation by an acidic polysaccharide isolated from Angelica sinensis (Oliv.) Diels. J Biochem Mol Biol.2007; 40(5):636-643.
28. Lee KY, You HJ, Jeong HG, et al. Polysaccharide isolated from Poria cocos sclerotium induces NF-kappaB/Rel activation and iNOS expression through the activation of p38 kinase in murine macrophages. Int Immunopharmacol. 2004;4(8):1029-1038.
29. Cuellar MJ, Giner RM, Recio MC, et al. Effect of the basidiomycete Poria cocos on experimental dermatitis and other inflammatory conditions. Chem Pharm Bull. 1997;45(3):492-494.
30. Choy YM, Leung KN, Cho CS, et al. Immunopharmacological studies of low molecular weight polysaccharide from Angelica sinensis. Am J Chin Med. 1994;22(2):137-145.
31. Kondo S, McKenzie RC, Sauder DN.Interleukin-10 inhibits the elicitation phase of allergic contact hypersensitivity. J Invest Dermatol.1994; 103(6):811-814.
1. Kanaze FI, Bounartzi MI, Georgarakis M, et al.Pharmacokinetics of the citrus flavanone aglycones hesperetin and naringenin after single oral administration in human subjects. Eur J Clin Nutr.2007; 61:472-477.
2. Liu ZQ, Jiang ZH, Liu L, et al. Mechanisms responsible for poor oral bioavailability of paeoniflorin:Role of intestinal disposition and interactions with sinomenine. Pharm Res.2006; 23:2768-2780.
3. 徐萌萌,王建芳,徐春,等.微生物转化苷类中药的机理及应用.世界科学技术-中医药现代化.2006；8：24-27.
4. Nemeth K, Plumb GW, Berrin JG, et al. Deglycosylation by small intestinal epithelial cell beta-glucosidases is a critical step in the absorption and meta-bolism of dietary flavonoid glycosides in humans. Eur J Nutr.2003; 42:29-42.
5. Akao T, Kobashi K, Aburada M.Enzymic studies on the animal and intestinal bacterial metabolism of geniposide. Biol Pharm Bull.1994;17(12):1573-1576.
6. He JX, Goto E, Akao T, et al. Interaction between Shaoyao-Gancao-Tang and a laxative with respect to alteration of paeoniflorin metabolism by intestinal bacteria in rats. Phytomedicine 2007; 14(7-8):452-459.
7. Liu ZQ, Jiang ZH. Liu L, et al. Mechanisms responsible for poor oral bioavai-lability of paeoniflorin:Role of intestinal disposition and interactions with sinomenine. Pharm Res.2006;23(12):2768-2780.
8. Asano T, Ishihara K, Morota T,et al. Permeability of the flavonoids liquiritigenin and its glycosides in licorice roots and davidigenin, a hydrogenated metabolite of liquiritigenin. using human intestinal cell line Caco-2. J Ethnopharmacol. 2003;89(2-3):285-289.
9. 中国药典1一部[S].2005：59.
10. Wang ZY, Nixon DW. Licorice and cancer. Nutr Cancer.2001;39(1):1-11.
11. Yokozawa T, Cho EJ, Rhyu DY, et al. Glycyrrhizae Radix attenuates peroxy-nitrite-induced renal oxidative damage through inhibition of protein nitration. Free Radic Res.2005;39(2):203-211.
12. Mori H, Niwa K, Zheng Q, et al.Cell proliferation in cancer prevention; effects of preventive agents on estrogen-related endometrial carcinogenesis model and on an in vitro model in human colorectal cells. Mutat Res.2001;480-481: 201-207.
13. Tsukamoto S, Aburatani M, Yoshida T, et al.CYP3A4 inhibitors isolated from Licorice. Biol Pharm Bull.2005;28(10):2000-2002.
14. Hatano T, Yasuhara T, Miyamoto K, et al. Anti-human immunodeficiency virus phenolics from licorice. Chem Pharm Bull.1988;36(6):2286-2288.
15. Hatano T, Yasuhara T, Fukuda T, et al.Phenolic constituents of licorice. Ⅱ. Structures of licopyranocoumarin, licoarylcoumarin and glisoflavone, and inhibitory effects of licorice phenolics on xanthine oxidase. Chem Pharm Bull. 1989;37(11):3005-3009.
16. Gao HX, Shao S H,Wang GQ, et al.Research progress of Glycyrrhiza uralensis Fisch. Jinganshan Medical College.2004;11(5):8-11.
17. Falcao MJ, Pouliquem YB, Lima MA, et al. Cytotoxic flavonoids from Platymiscium floribundum. J Nat Prod.2005;68(3):423-426.
18. Sanderink GJ, Bournique B, Stevens J,et al. Involvement of human CYP1A isoenzymes in the metabolism and drug interactions of riluzole in vitro. J Pharmacol Exp Ther.1997;282(3):1465-1472.
19. Lowry OH, Rosebrough NJ, Farr AL,et al. Protein measurement with the Folin phenol reagent. J Biol Chem.1951;193(1):265-275.
20. Omura T, Sato R.The carbon monoxide-binding pigment of liver microsomes. I. evidence for its hemoprotein nature. J Biol Chem.1964;239:2370-2378.
21. Chotnopparatpattara P, Taneepanichskul S. Use of depot medroxyprogesterone acetate in Thai adolescents. Contraception.2000; 62 (3):137-140.
22. Ortiz A, Hirol M, Stanczyk FZ, et al. Serum medroxyprogesterone acetate (MPA)concentrations and ovarian function following intramuscular injection of depo-MPA. J Clin Endocrinol Metab.1977; 44 (1):32-38.
23. Kobayashi K, Mimura N, Fujii H, et al. Role of human cytochrome P450 3A4 in metabolism of medroxyprogesterone acetate. Clin Cancer Res.2000; 6 (8): 3297-3303.
24. Mimura N, Kobayashi K, Nakamura Y, et al. Metabolism of medroxyprogesterone acetate (MPA) via CYP enzymes in vitro and effect of MPA on bleeding time in female rats in dependence on CYP activity in vivo. Life Sci.2003; 73 (25): 3201-3212.
25. Tucker GT, Houston JB, Huang SM. Optimizing drug development:strategies to assess drug metabolism/transporter interaction potential--towards a consensus. Br J Clin Pharmacol.2001; 52 (1):107-117.
26. Fukushima DK, levin J, Liang JS, et al. Isolation and partial synthesis of a new metabolite of medroxyrogesterone acetate. Steroids.1979; 34 (1):57-72.
27. Bjornsson TD, Callaghan JT, Einolf HJ, et al. The conduct of in vitro and in vivo drug-drug interaction studies:a PhRMA perspective. J Clin Pharmacol.2003; 43 (5):443-469.
28. Bachmann KA, Lewis JD. Predicting inhibitory drug-drug interactions and evaluating drug interaction reports using inhibition constants. Ann Pharmacother. 2005; 39 (6):1064-1072.
29. Ito K, Iwatsubo T, Kanamitsu S, et al. Prediction of pharmacokinetic alterations caused by drug-drug interactions:metabolic interaction in the liver. Pharmacol Rev.1998; 50 (3):387-412.
30. Sturm G, Haberlein H. Bauer T, et al. Mass spectrometric and high-performance liquid chromatographic studies of medroxyprogesterone acetate metabolites in human plasma. J Chromatogr.1991; 562 (1-2):351-362.
31. Vecht CJ, Wagner GL, Wilms EB. Interactions between antiepileptic and chemotherapeutic drugs. Lancet Neurol.2003; 2 (7):404-409.
32. Bjornsson TD, Callaghan JT, Einolf HJ, et al. The conduct of in vitro and in vivo drug-drug interaction studies:a Pharmaceutical Research and Manufacturers of America (PhRMA) perspective. Drug Metab Dispos.2003;31(7):815-832.
33. Liu HX, Hu Y, Liu Y,et al.Hydroxylation of tanshinone Ⅱa in human liver microsomes is specifically catalysed by cytochrome P4502A6. Xenobiotica. 2009;39(5):382-390.
34. Liu HX, Liu Y, Zhang JW,et al. UDP-glucuronosyltransferase 1A6 is the major isozyme responsible for protocatechuic aldehyde glucuronidation in human liver microsomes. Drug Metab Dispos.2008.36(8):1562-1569.
35. Eaton DL, Gallagher EP, Bammler TK, et al. Role of cytochrome P4501A2 in chemical carcinogenesis:implication for human variability in expression and enzyme activity. Pharmacogenetics.1995;5(5):259-274.
36. Aklillu E, Carrillo JA, Makonnen E, et al. Genetic polymorphism of CYP1A2 in Ethiopians affecting induction and expression:characterization of novel haplotypes with single-nucleotide polymorphisms in intron 1. Molecular Pharmacology.2003:64(3):659-669.
37. Shimada T, Yamazaki H. Mimura M, et al. Interindividual variations in human liver cytochrome P-450 enzymes involved in the oxidation of drugs, carcinogens and toxic chemicals:Studies with liver microsomes of 30 Japanese and 30 Gaucasians. J Pharmacol Exp Ther.1994;270(1):414-423.
38. Corchero J, Pimprale S, Kimura S, et al.Organization of the CYPIA cluster on human chromosome 15:implications for gene regulation. Pharmacogenetics. 2001;11(1):1-6.
39. Boke O, Gunes S, Kara N, et al. Association of serotonin 2A receptor and lack of association of CYP1A2 gene polymorphism with tardive dyskinesia in a Turkish population. DNA and Cell Biology.2007;26(8):527-531.
40. Kalow W, Tang B K. Caffeine as a metabolic probe:exploration of the enzyme-inducing effect of cigarette smoking. Clin Pharmacol Ther. 1991;49(I):44-48.
1. Steinman L. Immune therapy for autoimmune diseases. Science.2004; 305(5681): 212-216.
2. Mamessier E, Milhe F, Guillot C, et al.T-cell activation in occupational asthma and rhinitis. Allergy.2007;62(2):162-169.
3. Werfel T, Wittmann M. Regulatory role of T lymphocytes in atopic dermatit-is. Chem Immunol Allergy.2008;94:101-111.
4. Tsuboi H, Hossain K, Akhand AA, et al. Paeoniflorin induces apoptosis of lymphocytes through a redox-linked mechanism. J Cell Biochem.2004;93(1): 162-172.
5. Mosmann TR, Cherwinski H, Bond MW. Two types of murine helper T cell clone. I. Definition according to profiles of Iymphokine activities and secreted proteins. J Immunol.1986; 136(7):2348-2357.
6. Liu DZ, Xie KQ, Ji XQ, et al. Neuroprotective effect of paeoniflorin on cerebral ischemic rat by activating adenosine A1 receptor in a manner different from its classical agonist. Br J Pharmacol.2005; 146(4):604-611.
7. Chu DY, Li CL, Wu Q, et al. Paeoniflorin prevents hepatic fibrosis of Schistoso-miasis japonica by inhibiting TGF-b1 production from macrophages in mice. Front Med China.2008;2(2):154-165.
8. Liu DF, Wei W, Song LH, et al. Protective effect of paeoniflorin on immuno-logical liver injury induced by bacillus calmette-guerin plus lipopolysac-charide:modulation of tumour necrosis factor-a and interleukin-6 mRNA.Clinical and Experimental Pharmacology and Physiology.2006; 33:332-339.
9. Liu DZ, Zhao FL, Liu J. et al. Potentiation of adenosine Al receptor agonist CPA-induced antinociception by paeoniflorin in mice. Biol Pharm Bull. 2006;29(8):1630-1633.
10. Zheng YQ, Wei W, Zhu L, et al. Effects and mechanisms of Paeoniflorin, a bioactive glucoside from paeony root, on adjuvant arthritis in rats. Inflamm Res. 2007;56(5):182-188.
11. Chen Y, Wei W, Wu H, et al. Effects of paeoniflorin on the level of antibodies and cAMP produced by splenocytes in rats with adjuvant arthritis.Yao Xue Xue Bao.2007;42(11):1147-1151.
12. Sun Y, Dong Y, Jiang HJ, et al. Dissection of the role of paeoniflorin in the
traditional Chinese medicinal formula Si-Ni-San against contact dermatitis in mice. Life Sci.2009;84(11-12):337-344.
13. Lee B, Shin YW, Bae EA, et al. Antiallergic effect of the root of Paeonia lactiflora and its constituents paeoniflorin and paeonol. Arch Pharm Res. 2008;31(4):445-450.
14. Wu M, Gu Z. Screening of bioactive compounds from moutan cortex and their anti-inflammatory activities in rat synoviocytes. Evid Based Complement Alternat Med.2009;6(1):57-63.
15. Nori M, Iwata S, Munakata Y,et al. Ebastine inhibits T cell migration, production of Th2-type cytokines and proinflammatory cytokines. Clin Exp Allergy. 2003;33(11):1544-1554.
16.骆群,吕明,于鸣,等.抗CD28抗体协同刺激增强抗CD3抗体体外激活T淋巴细胞并降低TGF-β的表达.中国实验血液学杂志.2006；14(3)：547-551.
17. Kubota T, Takahara T, Nagata M, et al. Colorimetric chemosensitivity testing using sulforhodamine B.J Surg Oncol.1993;52(2):83-88.
18. Verant P, Ricard C, Serduc R, et al. In vivo staining of neocortical astrocytes via the cerebral microcirculation using sulforhodamine B. J Biomed Opt. 2008;13(6):064028.
19. Lei S, Chien PY, Sheikh S, et al. Enhanced therapeutic efficacy of a novel liposome-based formulation of SN-38 against human tumor models in SCID mice. Anticancer Drugs.2004;15(8):773-778.
20.周思朗,屈艳妮,张健等.SRB法与MTT法细胞计数应用比较.中国现代医学杂志.2005；15(17)：2615-2616.
21. Liu HQ, Zhang WY, Luo XT, et al. Paeoniflorin attenuates neuroinflammation and dopaminergic neurodegeneration in the MPTP model of Parkinson's disease by activation of adenosine A1 receptor.Br J Pharmacol.2006; 148(3):314-325.
22. Egwuagu CE. STAT3 in CD4+T helper cell differentiation and inflammatory diseases. Cytokine.2009;47(3):149-156.
23. Aguilar Angeles D, Serrano Miranda E, Rojo Gutierrez MI, et al. Th1 and Th2 lymphocytes in perennial allergic rhinitis.Rev Alerg Mex.2006;53(3):85-88.
24. Parnia S, Frew AJ. Chemokines and atopic dermatitis.J Allergy Clin Immunol. 2000; 105(5):892-894.
25. Tomimori Y, Tanaka Y, Goto M, et al. Repeated topical challenge with chemical antigen elicits sustained dermatitis in NC/Nga mice in specific-pathogen-free condition. J Invest Dermatol.2005;124(1):119-124.
26. Trautmann A, Akdis M, Kleemann D, et al. T cell-mediated Fas-induced keratinocyte apoptosis plays a key pathogenetic role in eczematous dermatitis. J Clin Invest.2000; 106(1):25-35.
27. Armbruster N, Trautmann A, Brocker EB, et al. Suprabasal spongiosis in acute eczematous dermatitis:cFLIP maintains resistance of basal keratinocytes to T-cell-mediated apoptosis. J Invest Dermatol.2009; 129(7):1696-1702.
2. 黑玉英,马双弟.中医中药治疗慢性湿疹的体会.内蒙古中医药.2006；(6)：23-24.
3. 郭爱群,马万里.滋阴除湿汤加味治疗慢性湿疹.湖北中医杂志.2001；23(6)：37.
4. 王丽娟,毕湘杰,张秀云.中药治疗湿疹100例临床疗效观察.厂矿医药卫生.2000；16(3)：211.
5. 王惠玲.急性湿疹的中药治疗.首都医药.1999；6(9)：34.
6. 徐金鹏.中药治疗慢性湿疹89例.吉林中医药.2004；24(5)：33.
7. 黄为群.加减除湿汤治疗湿疹样皮炎疗效观察.亚太传统医药.2009；5(8)：80-81.
8. 戴溱,毛舒和.中药治疗泛发性湿疹126例疗效分析.天津中医.1999；16(1)：17.
9. 涂彩霞,张新军,张蕴颖,等.复方甘草酸苷治疗特应性皮炎的临床及实验研究.中国麻风皮肤病杂志.2007；23(5)：393-395.
10.卢艳红.雷公藤多甙治疗泛发性湿疹疗效观察.包头医学院学报.2007；23(4)：388-389.
11.杨春梅,刘晓明,涂彩霞,等.丹皮酚治疗湿疹的研究.中国皮肤性病学杂志.1997,11(1)：25.
12.王建湘.中医辨证加熏洗治疗小腿湿疹临床疗效分析.中国临床药理学与治疗学.2003；8(1)：104-106.
13.王兆梓,张立平,王波.中药治疗湿疹128例.中医研究.2002；15(3)：43.
14.徐关冰,闫景东,周妍妍.中药治疗婴儿湿疹76例临床观察.中医药信息.2006；23(4)：37.
15.王小萍,秦亮甫.秦亮甫教授治疗湿疹150例.上海中医药杂志.2001；10(7)：31.
16.方晴,朱永先,吴兰芬.中药治疗湿疹及皮炎类皮肤病临床疗效观察.北京中医药大学学报.2003；26(4)：83-84.
17.王光亚,朱奎.中药治疗湿疹临床观察.当代医学.2009；15(34)：157.
18.朱正君.中药治疗婴儿湿疹的临床研究.中医儿科杂志.2008；4(2)：36-39.
19.李建华.中药治疗肛门湿疹68例临床观察.中国麻风皮肤病杂志.2005；21(2)：153.
20.付宏伟.中药治疗婴儿期异位性皮炎160例.中国中医药信息杂志.2000；7(2)：66.
21.胡宝春.湿疹的中药治疗及护理.时珍国医国药.2002；13(2)：125.
22.胡春菊.急性湿疹的中药治疗及护理.辽宁中医药大学学报.2008；10(4)：118.
23.刘明学.除湿止痒熏洗剂治疗肛门湿疹127例.陕西中医.2000；21(7)：303.
24.兰绍波,宋修亭.如意金黄散外治湿疹132例.辽宁中医杂志.2006；33(4)：482.
25.宋岚,武静.苦参等中药治疗皮炎湿疹类皮肤病临床疗效观察.职业与健康.2002；18(5)：140-141.
26.刘金燕.针灸治疗湿疹52例疗效观察.实用医技杂志.2006；13(1)：148-149.
27.王丽娟,周丽波,沈仲秋,等.梅花针与中药治疗湿疹100例.针灸临床杂志.2001；17(5)：15.
28.陈淑琴.针药并用治疗湿疹.中国针灸.2003；23(4)：221.
29.宫振甲,周立东.中药保留灌肠治疗婴幼儿湿疹96例临床观察.中国中西医结合皮肤性病学杂志.2007；6(2)：103-104.
30.涂彩霞,林熙然,谷玲.四种中药复方对小鼠实验性皮炎作用的研究.中华皮肤科杂志.1998；31(4)：244-246.
31.林熙然,涂彩霞,杨春梅,等.抗Ⅳ型变态反应中药治疗湿疹的研究.中国中西医结合杂志.2000；20(4)：258-260.
32.涂彩霞,刘芳,谷玲,等.复方茯苓汤对小鼠接触性过敏反应的影响.中华皮肤科杂志.2001；34(6)：452.
33.涂彩霞,刘芳,李敬,等.复方茯苓汤治疗湿疹582例临床观察及实验研究.中国中西医结合皮肤性病学杂志.2002；1(1)：13-15.
34. Lin XR, Tu CX, Meng XM, et al. Studies on treating eczema by Chinese herbal medicine with anti-type IV allergic activity. Chin J Integr Med.2001;7(1):7-11.
35. Kim MJ, Im KR, Yoon KS.et al. Anti-inflammatory effects of YeongyoSeungma-tang. J Ethnopharmacol.2009; 126(3):377-381.
36. Chan BC, Hon KL, Leung PC, et al. Traditional Chinese medicine for atopic eczema:PentaHerbs formula suppresses inflammatory mediators release from mast cells. J Ethnopharmacol.2008; 120(1):85-91.
37. Kim DY, Jung JA, Kim TH, et al. Oral administration of Uncariae rhynchophylla inhibits the development of DNFB-induced atopic dermatitis-like skin lesions via IFN-gamma down-regulation in NC/Nga mice. J Ethnopharmacol. 2009;122(3):567-572.
38. Choi JJ, Park B, Kim DH, et al. Blockade of atopic dermatitis-like skin lesions by DA-9102, a natural medicine isolated from Actinidia arguta, in the Mg-deficiency induced dermatitis model of hairless rats. Exp Biol Med. 2008;233(8):1026-1034.
39. Leung TF, Wong KY, Wong CK, et al. In vitro and clinical immunomodulatory effects of a novel Pentaherbs concoction for atopic dermatitis. Br J Dermatol. 2008;158(6):1216-1223.
40. Hon KL, Lee VW, Leung TF, et al. Corticosteroids are not present in a traditional Chinese medicine formulation for atopic dermatitis in children. Ann Acad Med Singapore.2006;35(11):759-763.
41. Makino T, Hamanaka M, Yamashita H, et al. Effect of bakumijiogan, an herbal formula in traditional Chinese medicine, on atopic dermatitis-like skin lesions induced by mite antigen in NC/Jic mice. Biol Pharm Bull. 2008;31(11):2108-2113.
42. Choi MS, Kim EC, Lee HS, et al. Inhibitory effects of Saururus chinensis (LOUR.) BAILL on the development of atopic dermatitis-like skin lesions in NC/Nga mice. Biol Pharm Bull.2008;31 (1):51-56.
43. Nakatsukasa H, Tago F, Okamoto T, et al. Therapeutic Effects of Gyokuheifusan on NC/Nga Mouse Model of Allergic Dermatitis. J Health Sci.2009;55(4): 516-524.
44. Kobayashi H, Mizuno N, Teramae H, et al. Diet and Japanese herbal medicine for recalcitrant atopic dermatitis:Efficacy and safety. Drugs Exp Clin Res.2004;30 (5-6):197-202.
45. Kobayashi H, Mizuno N, Teramae H,et al. The effects of Hochu-ekki-to in patients with atopic dermatitis resistant to conventional treatment. Int J Tissue React.2004;26(3-4):113-117.
46. Kobayashi H, Mizuno N, Kutsuna H, et al. Hochu-ekki-to suppresses development of dermatitis and elevation of serum IgE level in NC/NGA mice. Drugs Exp Clin Res.2003;29(2):81-84.
47. Nakada T, Watanabe K, Matsumoto T, et al. Effect of orally administered Hochu-ekki-to, a Japanese herbal medicine, on contact hypersensitivity caused by repeated application of antigen. Int Immunopharmacol.2002;2(7):901-911.
48. Andoh T, Honma Y, Kawaharada S, et al. Inhibitory effect of the repeated treatment with Unsei-in on substance P-induced itch-associated responses through the downregulation of the expression of NK1 tachykinin receptor in mice. Biol Pharm Bull.2003;26(6):896-898.
49. Novak N, Haberstok J, Kraft S, et al. Standardized extracts from Chinese herbs induce IL-10 production in human monocyte-derived dendritic cells and alter their differentiation in vitro. J Allergy Clin Immunol.2001; 108(4):588-593.
50. Xu XJ, Banerjee P, Rustin MH, et al. Modulation by Chinese herbal therapy of immune mechanisms in the skin of patients with atopic eczema. Br J Dermatol. 1997;136(1):54-59.
51. Tahara E, Satoh T, Toriizuka K,et al. Effect of Shimotsu-to (a Kampo medicine, Si-Wu-Tang) and its constituents on triphasic skin reaction in passively sensitized mice. J Ethnopharmacol.1999;68(1-3):219-228.
52. Shichijo, Kosaburo; Saito, Hirohisa; Ikura, Yoji.Inhibitory effect of the Sho-saiko-to, Shofu-san, and Makyo-kanseki-to on histamine release from murine bone marrow-derived cultured mast cells. Jikeikai Medical Journal.1995; 42(3):183-194.
53. Lee B, Shin YW, Bae EA, et al. Antiallergic effect of the root of Paeonia lactiflora and its constituents paeoniflorin and paeonol. Arch Pharm Res. 2008;31(4):445-450.
54. Yamahara J, Yamada T, Kimura H, et al. Biologically active principles of crude drugs. Ⅱ. Anti-allergic principles in "Shoseiryu-To" anti-inflammatory pro-perties of paeoniflorin and its derivatives. J Pharmacobiodyn.1982;5(11): 921-929.
55. Sun Y, Dong Y, Jiang HJ, et al. Dissection of the role of paeoniflorin in the traditional Chinese medicinal formula Si-Ni-San against contact dermatitis in mice. Life Sci.2009;84(11-12):337-344.
1. 涂彩霞,刘芳,谷玲,等.复方茯苓汤对小鼠接触性过敏反应的影响.中华皮肤科杂志.2001；34(6)：452.
2. 涂彩霞,刘芳,李敬,等.复方茯苓汤治疗湿疹582例临床观察及实验研究.中国中西医结合皮肤性病学杂志.2002；1(1)：13-15.
3. 涂彩霞,林熙然,谷玲.四种中药复方对小鼠实验性皮炎作用的研究.中华皮肤科杂志.1998,3 1(4)：244-246.
4. 林熙然,涂彩霞,杨春梅,等.抗Ⅳ型变态反应中药治疗湿疹的研究.中国中西医结合杂志.2000,20(4)：258-260.
5. Meng J, Leung KS, Dong XP, Zhou YS, Jiang ZH, Zhao ZZ. Simultaneous quantification of eight bioactive components of Houttuynia cordata and related Saururaceae medicinal plants by on-line high performance liquid chromato-graphy-diode array detector-electrospray mass spectrometry.Fitoterapia.2009; 80(8):468-474.
6. Ni LJ, Zhang LG, Hou J, Shi WZ, Guo ML. A strategy for evaluating antipyretic efficacy of Chinese herbal medicines based on UV spectra fingerprints. J Ethnopharmacol.2009; 124(1):79-86.
7. Liu AH, Lin YH, Yang M, Guo H, Guan SH, Sun JH, Guo DA.Development of the fingerprints for the quality of the roots of Salvia miltiorrhiza and its related preparations by HPLC-DAD and LC-MS(n). J Chromatogr B Analyt Technol Biomed Life Sci.2007;846(1-2):32-41.
8. Liang XM, Jin Y, Wang YP, Jin GW, Fu Q, Xiao YS.Qualitative and quantitative analysis in quality control of traditional Chinese medicines. J Chromatogr A. 2009;1216(11):2033-2044.
9. Kong WJ, Zhao YL, Xiao XH, Jin C, Li ZL.Quantitative and chemical fingerprint analysis for quality control of rhizoma Coptidischinensis based on UPLC-PAD combined with chemometrics methods.Phytomedicine.2009;16(10):950-959.
10. Min JZ, Shimizu Y, Toyo'oka T, Inagaki S, Kikura-Hanajiri R, Goda Y.Simul-taneous determination of 11 designated hallucinogenic phenethylamines by ultra-fast liquid chromatography with fluorescence detection. J Chromatogr B Analyt Technol Biomed Life Sci.2008;873(2):187-194.
11. Ge GB, Luan HW, Zhang YY, He YQ, Liu XB, Yang L, Wang ZT, Yang L.Profiling of yew hair roots from various species using ultra-performance liquid
chromatography/electrospray ionization mass spectrometry. Rapid Commun Mass Spectrom.2008;22(15):2315-2323.
12. Li R, Wang X, Zhou Y, Cai M, Ding L. Analysis of sodium adduct paeoniflorin, albiflorin and their derivatives by (+)ESI-MSn, DFT calculations and computer-aided mass spectrometry analysis program.J Mass Spectrom.2007;42(3): 335-345.
13. Xu SJ, Yang L, Zeng X, Zhang M, Wang ZT.Characterization of compounds in the Chinese herbal drug Mu-Dan-Pi by liquid chromatography coupled to electrospray ionization mass spectrometry. Rapid Commun Mass Spectrom. 2006;20(22):3275-3288.
14. Moiteiro C, Gaspar H, Rodrigues AI, Lopes JF, Carnide V.HPLC quantification of dye flavonoids in Reseda luteola L. from Portugal. J Sep Sci.2008;31(21): 3683-3687.
15. Svehlikova V, Bennett RN, Mellon FA, Needs PW, Piacente S, Kroon PA, Bao Y.Isolation, identification and stability of acylated derivatives of apigenin 7-O-glucoside from chamomile (Chamomilla recutita [L.] Rauschert). Phytochemistry.2004;65(16):2323-2332.
16. Hu YM, Su GH, Sze SC, Ye W, Tong Y. Quality assessment of Cortex Phellodendri by high-performance liquid chromatography coupled with electrospray ionization mass spectrometry. Biomed Chromatogr.2010;24(4): 438-453.
17. Hatano T, Takagi M, Ito H, Yoshida T. Acylated flavonoid glycosides and accompanying phenolics from licorice.Phytochemistry.1998;47(2):287-293.
18. Cao J, Xu Y, Zhang Y, Wang Y, Luo G. Chin J Anal Chem.2004;32(4):875.
19. Chen F, Lu HT, Jiang Y. Purification of paeoniflorin from Paeonia lactiflora Pall, by high-speed counter-current chromatography. J Chromatogr A. 2004;1040(2):205-208.
20. Rauchensteiner F, Matsumura Y, Yamamoto Y, Yamaji S, Tani T. Analysis and comparison of Radix Glycyrrhizae (licorice) from Europe and China by capillary-zone electrophoresis (CZE). J Pharm Biomed Anal.2005;38(4): 594-600.
21. Lee KY, You HJ, Jeong HG, Kang JS, Kim HM, Rhee SD, Jeon YJ. Polysac-charide isolated from Poria cocos sclerotium induces NF-kappaB/Rel activation and iNOS expression through the activation of p38 kinase in murine macrophages. Int Immunopharmacol.2004;4(8):1029-1038.
22. Cuellar MJ, Giner RM, Recio MC, Just MJ, Manez S, Rios JL.Effect of the basidiomycete Poria cocos on experimental dermatitis and other inflammatory conditions. Chem Pharm Bull (Tokyo).1997;45(3):492-494.
23. Wong VK, Yu L, Cho CH.Protective effect of polysaccharides from Angelica sinensis on ulcerative colitis in rats.Inflammopharmacology.2008; 16(4): 162-167.
24. Sun Y, Tang J, Gu X, Li D. Water-soluble polysaccharides from Angelica sinensis (Oliv.) Diels:Preparation, characterization and bioactivity. Int J Biol Macromol. 2005;36(5):283-289.
25. Zhang C, Zhou A, Zhang M. Chemical constituents of Alisma orientalis and their immunosuppressive function. Zhongguo Zhong Yao Za Zhi.2009;34(8):994-998.
26. Koo HJ, Lim KH, Jung HJ, Park EH. Anti-inflammatory evaluation of gardenia extract, geniposide and genipin. J Ethnopharmacol.2006;103(3):496-500.
27. Sun Y, Dong Y, Jiang HJ, Cai TT, Chen L, Zhou X, Chen T, Xu Q.Dissection of the role of paeoniflorin in the traditional Chinese medicinal formula Si-Ni-San against contact dermatitis in mice.Life Sci.2009;84(11-12):337-344.
28. Yoon T, Cheon MS, Kim SJ, Choo BK, Moon BC, Lee AY. Evaluation of the anti-inflammatory efficacy of Glycyrrhiza uralensis according to extracting solvents [J]. Planta Medica.2009;75:1050-1051.
29. Chen Y, Zhang H, Li YX, Cai L, Huang J, Zhao C, Jia L, Buchanan R, Yang T, Jiang LJ. Crocin and geniposide profiles and radical scavenging activity of gardenia fruits (Gardenia jasminoides Ellis) from different cultivars and at the various stages of maturation. Fitoterapia.2010;81(4):269-273.
1. 涂彩霞,刘芳,谷玲,等.复方茯苓汤对小鼠接触性过敏反应的影响.中华皮肤科杂志.2001；34(6)：452.
2. 涂彩霞,刘芳,李敬,等.复方茯苓汤治疗湿疹582例临床观察及实验研究.中国中西医结合皮肤性病学杂志.2002；1(1)：13-15.
3. Lin IH, Lee MC, Chuang WC, et al. Application of LC/MS and ICP/MS for establishing the fingerprint spectrum of the traditional Chinese medicinal preparation Gan-Lu-Yin. J Sep Sci.2006;29 (1):172-179.
4. Wang H, Feng F. Identification of components in Zhi-Zi-Da-Huang decoction by HPLC coupled with electrospray ionization tandem mass spectrometry, photodiode array and fluorescence detectors. Pharm Biomed Anal.2009;49(5): 1157-1165.
5. Bedi MK, Shenefelt PD. Herbal therapy in dermatology. Arch Dermatol. 2002;138(2):232-242.
6. Leung TF, Wong KY, Wong CK, et al. In vitro and clinical immunomodulatory effects of a novel Pentaherbs concoction for atopic dermatitis. Br J Dermatol. 2008;158(6):1216-1223.
7. Rowe A, Bunker CB. Interleukin-4 and the interleukin-4 receptor in allergic contact dermatitis. Contact Dermatitis.1998;38(1):36-39.
8. Neis MM, Peters B, Dreuw A, et al. Enhanced expression levels of IL-31 correlate with IL-4 and IL-13 in atopic and allergic contact dermatitis. J Allergy Clin Immunol.2006; 118(4):930-937.
9. 许宗严,吴铁,吴志华.鼠皮炎湿疹模型建立的研究进展.中国临床药理学与治疗学.2005；10(7)：730-733.
10. Kim DY, Jung JA, Kim TH, et al. Oral administration of Uncariae rhynchophylla inhibits the development of DNFB-induced atopic dermatitis-like skin lesions via IFN-gamma down-regulation in NC/Nga mice. J Ethnopharmacol. 2009;122(3):567-572.
11. Hopkins J E, Naisbitt DJ, Kitteringham NR, et al. Selective haptenation of cellular or extracellular protein by chemical allergens:association with cytokine polarization.Chem Res Toxicol.2005;18(4):375-381.
12. Ge GB, Zhang YY, Hao DC, et al. Chemotaxonomic study of medicinal Taxus species with fingerprint and multivariate analysis.Planta Med.2008; 74(7):
773-779.
13. Chan EC, Yap SL, Lau AJ, et al. Ultra-performance liquid chromatography/-time-of-flight mass pectrometry based metabolomics of raw and steamed Panax notoginseng. Rapid Commun Mass Spectrom.2007;21(4):519-528.
14. Apollonio LG, Pianca DJ, Whittall IR, et al. A demonstration of the use of ultra-performance liquid chromatography-mass spectrometry [UPLC/MS] in the determination of amphetamine-type substances and ketamine for forensic and toxicological analysis. Chromatogr B Analyt Technol Biomed Life Sci.2006;836(1-2):111-115.
15. Lee B, Shin YW, Bae EA, et al. Antiallergic effect of the root of Paeonia lactiflora and its constituents paeoniflorin and paeonol. Arch Pharm Res.2008;31(4):445-450.
16. Wu M, Gu Z. Screening of bioactive compounds from moutan cortex and their anti-inflammatory activities in rat synoviocytes. Evid Based Complement Alternat Med.2009;6(1):57-63.
17. Sun Y, Dong Y, Jiang HJ, et al. Dissection of the role of paeoniflorin in the traditional Chinese medicinal formula Si-Ni-San against contact dermatitis in mice. Life Sci.2009;84(11-12):337-344.
18. Jiang WL, Chen XG, Zhu HB, et al. Paeoniflorin inhibits systemic inflammation and improves survival in experimental sepsis. Basic Clin Pharmacol Toxicol. 2009;105(1):64-71.
19. Chang WL, Wang HY, Shi LS, et al. Immunosuppressive iridoids from the fruits of Gardenia jasminoides. J Nat Prod.2005; 68(11):1683-1685.
20. Koo HJ, Song YS, Kim HJ, et al. Antiinflammatory effects of genipin, an active principle of gardenia. Eur J Pharmacol.2004;495(2-3):201-208.
21. Koo HJ, Lim KH, Jung HJ, et al. Anti-inflammatory evaluation of gardenia extract, geniposide and genipin. J Ethnopharmacol.2006; 103(3):496-500.
22. Yoon T, Cheon MS, Kim SJ, et al. Evaluation of the anti-inflammatory efficacy of Glycyrrhiza uralensis according to extracting solvents. Planta Medica.2009; 75:1050-1051.
23. Wang Y, Zhang M, Ruan D, et al. Chemical components and molecular mass of six polysaccharides isolated from the sclerotium of Poria cocos. Carbohydr Res. 2004;339(2):327-334.
24. Sun Y, Tang J, Gu X, et al. Water-soluble polysaccharides from Angelica sinensis (Oliv.) Diels:Preparation, characterization and bioactivity. Int J Biol Macromol. 2005;36(5):283-289.
25. Wong VK, Yu L, Cho CH. et al. Protective effect of polysaccharides from Angelica sinensis on ulcerative colitis in rats. Inflammopharmacology.2008; 16(4):162-167.
26. Yang TH, Jia M, Mei QB,et al. Effect of Angelica sinensis polysaccharide fraction AP-3 on IL-2 and IFN-gamma induction.Yao Xue Xue Bao.2006;41(1):54-57.
27. Yang X, Zhao Y, Wang H, et al. Macrophage activation by an acidic polysaccharide isolated from Angelica sinensis (Oliv.) Diels. J Biochem Mol Biol.2007; 40(5):636-643.
28. Lee KY, You HJ, Jeong HG, et al. Polysaccharide isolated from Poria cocos sclerotium induces NF-kappaB/Rel activation and iNOS expression through the activation of p38 kinase in murine macrophages. Int Immunopharmacol. 2004;4(8):1029-1038.
29. Cuellar MJ, Giner RM, Recio MC, et al. Effect of the basidiomycete Poria cocos on experimental dermatitis and other inflammatory conditions. Chem Pharm Bull. 1997;45(3):492-494.
30. Choy YM, Leung KN, Cho CS, et al. Immunopharmacological studies of low molecular weight polysaccharide from Angelica sinensis. Am J Chin Med. 1994;22(2):137-145.
31. Kondo S, McKenzie RC, Sauder DN.Interleukin-10 inhibits the elicitation phase of allergic contact hypersensitivity. J Invest Dermatol.1994; 103(6):811-814.
1. Kanaze FI, Bounartzi MI, Georgarakis M, et al.Pharmacokinetics of the citrus flavanone aglycones hesperetin and naringenin after single oral administration in human subjects. Eur J Clin Nutr.2007; 61:472-477.
2. Liu ZQ, Jiang ZH, Liu L, et al. Mechanisms responsible for poor oral bioavailability of paeoniflorin:Role of intestinal disposition and interactions with sinomenine. Pharm Res.2006; 23:2768-2780.
3. 徐萌萌,王建芳,徐春,等.微生物转化苷类中药的机理及应用.世界科学技术-中医药现代化.2006；8：24-27.
4. Nemeth K, Plumb GW, Berrin JG, et al. Deglycosylation by small intestinal epithelial cell beta-glucosidases is a critical step in the absorption and meta-bolism of dietary flavonoid glycosides in humans. Eur J Nutr.2003; 42:29-42.
5. Akao T, Kobashi K, Aburada M.Enzymic studies on the animal and intestinal bacterial metabolism of geniposide. Biol Pharm Bull.1994;17(12):1573-1576.
6. He JX, Goto E, Akao T, et al. Interaction between Shaoyao-Gancao-Tang and a laxative with respect to alteration of paeoniflorin metabolism by intestinal bacteria in rats. Phytomedicine 2007; 14(7-8):452-459.
7. Liu ZQ, Jiang ZH. Liu L, et al. Mechanisms responsible for poor oral bioavai-lability of paeoniflorin:Role of intestinal disposition and interactions with sinomenine. Pharm Res.2006;23(12):2768-2780.
8. Asano T, Ishihara K, Morota T,et al. Permeability of the flavonoids liquiritigenin and its glycosides in licorice roots and davidigenin, a hydrogenated metabolite of liquiritigenin. using human intestinal cell line Caco-2. J Ethnopharmacol. 2003;89(2-3):285-289.
9. 中国药典1一部[S].2005：59.
10. Wang ZY, Nixon DW. Licorice and cancer. Nutr Cancer.2001;39(1):1-11.
11. Yokozawa T, Cho EJ, Rhyu DY, et al. Glycyrrhizae Radix attenuates peroxy-nitrite-induced renal oxidative damage through inhibition of protein nitration. Free Radic Res.2005;39(2):203-211.
12. Mori H, Niwa K, Zheng Q, et al.Cell proliferation in cancer prevention; effects of preventive agents on estrogen-related endometrial carcinogenesis model and on an in vitro model in human colorectal cells. Mutat Res.2001;480-481: 201-207.
13. Tsukamoto S, Aburatani M, Yoshida T, et al.CYP3A4 inhibitors isolated from Licorice. Biol Pharm Bull.2005;28(10):2000-2002.
14. Hatano T, Yasuhara T, Miyamoto K, et al. Anti-human immunodeficiency virus phenolics from licorice. Chem Pharm Bull.1988;36(6):2286-2288.
15. Hatano T, Yasuhara T, Fukuda T, et al.Phenolic constituents of licorice. Ⅱ. Structures of licopyranocoumarin, licoarylcoumarin and glisoflavone, and inhibitory effects of licorice phenolics on xanthine oxidase. Chem Pharm Bull. 1989;37(11):3005-3009.
16. Gao HX, Shao S H,Wang GQ, et al.Research progress of Glycyrrhiza uralensis Fisch. Jinganshan Medical College.2004;11(5):8-11.
17. Falcao MJ, Pouliquem YB, Lima MA, et al. Cytotoxic flavonoids from Platymiscium floribundum. J Nat Prod.2005;68(3):423-426.
18. Sanderink GJ, Bournique B, Stevens J,et al. Involvement of human CYP1A isoenzymes in the metabolism and drug interactions of riluzole in vitro. J Pharmacol Exp Ther.1997;282(3):1465-1472.
19. Lowry OH, Rosebrough NJ, Farr AL,et al. Protein measurement with the Folin phenol reagent. J Biol Chem.1951;193(1):265-275.
20. Omura T, Sato R.The carbon monoxide-binding pigment of liver microsomes. I. evidence for its hemoprotein nature. J Biol Chem.1964;239:2370-2378.
21. Chotnopparatpattara P, Taneepanichskul S. Use of depot medroxyprogesterone acetate in Thai adolescents. Contraception.2000; 62 (3):137-140.
22. Ortiz A, Hirol M, Stanczyk FZ, et al. Serum medroxyprogesterone acetate (MPA)concentrations and ovarian function following intramuscular injection of depo-MPA. J Clin Endocrinol Metab.1977; 44 (1):32-38.
23. Kobayashi K, Mimura N, Fujii H, et al. Role of human cytochrome P450 3A4 in metabolism of medroxyprogesterone acetate. Clin Cancer Res.2000; 6 (8): 3297-3303.
24. Mimura N, Kobayashi K, Nakamura Y, et al. Metabolism of medroxyprogesterone acetate (MPA) via CYP enzymes in vitro and effect of MPA on bleeding time in female rats in dependence on CYP activity in vivo. Life Sci.2003; 73 (25): 3201-3212.
25. Tucker GT, Houston JB, Huang SM. Optimizing drug development:strategies to assess drug metabolism/transporter interaction potential--towards a consensus. Br J Clin Pharmacol.2001; 52 (1):107-117.
26. Fukushima DK, levin J, Liang JS, et al. Isolation and partial synthesis of a new metabolite of medroxyrogesterone acetate. Steroids.1979; 34 (1):57-72.
27. Bjornsson TD, Callaghan JT, Einolf HJ, et al. The conduct of in vitro and in vivo drug-drug interaction studies:a PhRMA perspective. J Clin Pharmacol.2003; 43 (5):443-469.
28. Bachmann KA, Lewis JD. Predicting inhibitory drug-drug interactions and evaluating drug interaction reports using inhibition constants. Ann Pharmacother. 2005; 39 (6):1064-1072.
29. Ito K, Iwatsubo T, Kanamitsu S, et al. Prediction of pharmacokinetic alterations caused by drug-drug interactions:metabolic interaction in the liver. Pharmacol Rev.1998; 50 (3):387-412.
30. Sturm G, Haberlein H. Bauer T, et al. Mass spectrometric and high-performance liquid chromatographic studies of medroxyprogesterone acetate metabolites in human plasma. J Chromatogr.1991; 562 (1-2):351-362.
31. Vecht CJ, Wagner GL, Wilms EB. Interactions between antiepileptic and chemotherapeutic drugs. Lancet Neurol.2003; 2 (7):404-409.
32. Bjornsson TD, Callaghan JT, Einolf HJ, et al. The conduct of in vitro and in vivo drug-drug interaction studies:a Pharmaceutical Research and Manufacturers of America (PhRMA) perspective. Drug Metab Dispos.2003;31(7):815-832.
33. Liu HX, Hu Y, Liu Y,et al.Hydroxylation of tanshinone Ⅱa in human liver microsomes is specifically catalysed by cytochrome P4502A6. Xenobiotica. 2009;39(5):382-390.
34. Liu HX, Liu Y, Zhang JW,et al. UDP-glucuronosyltransferase 1A6 is the major isozyme responsible for protocatechuic aldehyde glucuronidation in human liver microsomes. Drug Metab Dispos.2008.36(8):1562-1569.
35. Eaton DL, Gallagher EP, Bammler TK, et al. Role of cytochrome P4501A2 in chemical carcinogenesis:implication for human variability in expression and enzyme activity. Pharmacogenetics.1995;5(5):259-274.
36. Aklillu E, Carrillo JA, Makonnen E, et al. Genetic polymorphism of CYP1A2 in Ethiopians affecting induction and expression:characterization of novel haplotypes with single-nucleotide polymorphisms in intron 1. Molecular Pharmacology.2003:64(3):659-669.
37. Shimada T, Yamazaki H. Mimura M, et al. Interindividual variations in human liver cytochrome P-450 enzymes involved in the oxidation of drugs, carcinogens and toxic chemicals:Studies with liver microsomes of 30 Japanese and 30 Gaucasians. J Pharmacol Exp Ther.1994;270(1):414-423.
38. Corchero J, Pimprale S, Kimura S, et al.Organization of the CYPIA cluster on human chromosome 15:implications for gene regulation. Pharmacogenetics. 2001;11(1):1-6.
39. Boke O, Gunes S, Kara N, et al. Association of serotonin 2A receptor and lack of association of CYP1A2 gene polymorphism with tardive dyskinesia in a Turkish population. DNA and Cell Biology.2007;26(8):527-531.
40. Kalow W, Tang B K. Caffeine as a metabolic probe:exploration of the enzyme-inducing effect of cigarette smoking. Clin Pharmacol Ther. 1991;49(I):44-48.
1. Steinman L. Immune therapy for autoimmune diseases. Science.2004; 305(5681): 212-216.
2. Mamessier E, Milhe F, Guillot C, et al.T-cell activation in occupational asthma and rhinitis. Allergy.2007;62(2):162-169.
3. Werfel T, Wittmann M. Regulatory role of T lymphocytes in atopic dermatit-is. Chem Immunol Allergy.2008;94:101-111.
4. Tsuboi H, Hossain K, Akhand AA, et al. Paeoniflorin induces apoptosis of lymphocytes through a redox-linked mechanism. J Cell Biochem.2004;93(1): 162-172.
5. Mosmann TR, Cherwinski H, Bond MW. Two types of murine helper T cell clone. I. Definition according to profiles of Iymphokine activities and secreted proteins. J Immunol.1986; 136(7):2348-2357.
6. Liu DZ, Xie KQ, Ji XQ, et al. Neuroprotective effect of paeoniflorin on cerebral ischemic rat by activating adenosine A1 receptor in a manner different from its classical agonist. Br J Pharmacol.2005; 146(4):604-611.
7. Chu DY, Li CL, Wu Q, et al. Paeoniflorin prevents hepatic fibrosis of Schistoso-miasis japonica by inhibiting TGF-b1 production from macrophages in mice. Front Med China.2008;2(2):154-165.
8. Liu DF, Wei W, Song LH, et al. Protective effect of paeoniflorin on immuno-logical liver injury induced by bacillus calmette-guerin plus lipopolysac-charide:modulation of tumour necrosis factor-a and interleukin-6 mRNA.Clinical and Experimental Pharmacology and Physiology.2006; 33:332-339.
9. Liu DZ, Zhao FL, Liu J. et al. Potentiation of adenosine Al receptor agonist CPA-induced antinociception by paeoniflorin in mice. Biol Pharm Bull. 2006;29(8):1630-1633.
10. Zheng YQ, Wei W, Zhu L, et al. Effects and mechanisms of Paeoniflorin, a bioactive glucoside from paeony root, on adjuvant arthritis in rats. Inflamm Res. 2007;56(5):182-188.
11. Chen Y, Wei W, Wu H, et al. Effects of paeoniflorin on the level of antibodies and cAMP produced by splenocytes in rats with adjuvant arthritis.Yao Xue Xue Bao.2007;42(11):1147-1151.
12. Sun Y, Dong Y, Jiang HJ, et al. Dissection of the role of paeoniflorin in the
traditional Chinese medicinal formula Si-Ni-San against contact dermatitis in mice. Life Sci.2009;84(11-12):337-344.
13. Lee B, Shin YW, Bae EA, et al. Antiallergic effect of the root of Paeonia lactiflora and its constituents paeoniflorin and paeonol. Arch Pharm Res. 2008;31(4):445-450.
14. Wu M, Gu Z. Screening of bioactive compounds from moutan cortex and their anti-inflammatory activities in rat synoviocytes. Evid Based Complement Alternat Med.2009;6(1):57-63.
15. Nori M, Iwata S, Munakata Y,et al. Ebastine inhibits T cell migration, production of Th2-type cytokines and proinflammatory cytokines. Clin Exp Allergy. 2003;33(11):1544-1554.
16.骆群,吕明,于鸣,等.抗CD28抗体协同刺激增强抗CD3抗体体外激活T淋巴细胞并降低TGF-β的表达.中国实验血液学杂志.2006；14(3)：547-551.
17. Kubota T, Takahara T, Nagata M, et al. Colorimetric chemosensitivity testing using sulforhodamine B.J Surg Oncol.1993;52(2):83-88.
18. Verant P, Ricard C, Serduc R, et al. In vivo staining of neocortical astrocytes via the cerebral microcirculation using sulforhodamine B. J Biomed Opt. 2008;13(6):064028.
19. Lei S, Chien PY, Sheikh S, et al. Enhanced therapeutic efficacy of a novel liposome-based formulation of SN-38 against human tumor models in SCID mice. Anticancer Drugs.2004;15(8):773-778.
20.周思朗,屈艳妮,张健等.SRB法与MTT法细胞计数应用比较.中国现代医学杂志.2005；15(17)：2615-2616.
21. Liu HQ, Zhang WY, Luo XT, et al. Paeoniflorin attenuates neuroinflammation and dopaminergic neurodegeneration in the MPTP model of Parkinson's disease by activation of adenosine A1 receptor.Br J Pharmacol.2006; 148(3):314-325.
22. Egwuagu CE. STAT3 in CD4+T helper cell differentiation and inflammatory diseases. Cytokine.2009;47(3):149-156.
23. Aguilar Angeles D, Serrano Miranda E, Rojo Gutierrez MI, et al. Th1 and Th2 lymphocytes in perennial allergic rhinitis.Rev Alerg Mex.2006;53(3):85-88.
24. Parnia S, Frew AJ. Chemokines and atopic dermatitis.J Allergy Clin Immunol. 2000; 105(5):892-894.
25. Tomimori Y, Tanaka Y, Goto M, et al. Repeated topical challenge with chemical antigen elicits sustained dermatitis in NC/Nga mice in specific-pathogen-free condition. J Invest Dermatol.2005;124(1):119-124.
26. Trautmann A, Akdis M, Kleemann D, et al. T cell-mediated Fas-induced keratinocyte apoptosis plays a key pathogenetic role in eczematous dermatitis. J Clin Invest.2000; 106(1):25-35.
27. Armbruster N, Trautmann A, Brocker EB, et al. Suprabasal spongiosis in acute eczematous dermatitis:cFLIP maintains resistance of basal keratinocytes to T-cell-mediated apoptosis. J Invest Dermatol.2009; 129(7):1696-1702.