Studies On The Effective Components Of Fufangfulingtang And The Pharmacologic Activities Of The Major Components

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 resultsThere 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.