| BackgroundPsoriasis, which is known as psora, is a very common skin disease. Due to the characteristics that its rash appears as red macula, papules or plaques, covered with multilevel argenteous scales, this pathognomonic representation is the name resource of psoriasis. And its pathological mechanisms represent that the cells are formed from cutins and proliferating excessively with parakeratosis, dermis angiotelectasis, and inflammatory cell infiltration. The pathogenesis of psoriasis is not completely known to the people, and it is very difficult to be cured, which has been classified as one of the stubborn disease by W. H.O. In recent years, a lot of researches and achievements have been achieved from the pathogenesis of psoriasis, however, the clinical manifestation of psoriasis is complex and changeable, and the cause of disease has not been completely discovered still. According to the existing research situation, psoriasis is connected with the inheritance, infection, dysbolism, disorder of immunologic function and endocrinopathy. In recent years, most of the scholars think that the attack of psoriasis is a kind of T cellular immunity mediated skin disease that is involved with polygenic inheritance and multi factors, and its pathogenesis is very complex. CD4+T Lymphocyte immune dysfunction is particularly important in attack. CD4+T Lymphocyte is divided into the major subgroups of Th1and Th2. Thl secretes cell factors such as IFN-γ and IL-12, the major characteristic is secreting IFN-γ, and Th2secretes the cell factors such as IL-4, IL-2, its major characteristic is secreting IL-4. Respectively, IFN-γ and IL-4are the symbolic cell factors of the subgroups of Th1and Th2. It is discovered from the research that the attack of psoriasis is in relation to the unbalance of Th1/Th2cell factors. A large number of CD4+T Lymphocytes are gathered in the cutaneous lesion of psoriasis, cell factors are formed from activated cutin that is secreted from cells, and they can act on T-Cells, and it is further facilitated to get mature. Cell factors are formed from T-Cells and cutin that are secreted from cells, and then forming the cytokine network, which results in the pathological damage of psoriasis.According to the existing development of antipsoriatic drugs, traditional drugs for external use have poor therapeutical effect. Although many oral drugs have the therapeutical effect for systematic treatment, meanwhile, the side effect is very serious. Although biotherapy is the development direction of antipsoriatic drugs, the drugs used for clinical treatment are very few, and the application is not mature enough, besides, the cost of treatment is very high. The traditional healing drugs for psoriasis in China have a very long history. Clinical traditional Chinese medicine for curing psoriasis has to not only follow the principle of diagnosis and treatment, it can also be selected after diagnosis and treatment, or the herbal extracts of active constituents can be used to cure the psoriasis.Zanthoxylum ungeanum is the dry and ripe fruit of Rutaceae that has a very long officinal history. It is recorded in the Compendium of Materia Medica that its taste is bitter, and it is warm in nature, slightly poisonous, anaesthesia, effective on spleen, stomach and kidney channel. Its major effects are for monsoon pathogenic factors, arthritis, letting out cold in lungs, xeransis in spleen, oedema dysentery. Its major chemical compositions are alkaloid, acid amides, lignans, coumarin, essential oil and fatty acid that have a strong pharmacological activity on cardiovascular system, digestive system, immune action, blood coagulation function, analgesia, composure, anti-inflammatory, bacteriostasis and killing worm, resisting the tumor, etc. As the in-depth study is being made to the pharmacological effects of Zanthoxylum ungeanum essential oil, it is found that it has an action on resisting tumor. Zang Linquan et al found that high-concentration (4-16mg/ml) Zanthoxylum ungeanum essential oil has the killing effect on A549cell strain of human lung cancer, and the low-concentration (lmg/ml) Zanthoxylum ungeanum essential oil has the effect of inducing apoptosis to A549. Yuan Taining et al found that Zanthoxylum ungeanum essential oil has the effect of restraining normal transformation on mice H22hepatocarcinoma, A549cell cycle of human lung cancer, restraining mitosis in vitro, resisting cell growth and stimulating procedural apoptosis.Li Pinyi et al found that Zanthoxylum ungeanum essential oil has the effect of directly restraining SGC-7901cell proliferation of gastric cancer and promoting apoptosis. It is reported in the overseas study that Zanthoxylum ungeanum essential oil is rich in terpenoids substance such as isoprene to have antitumor effect on blocking tumor cell cycle and inducing apoptosis. Dextrorotation limonene in isoprene has a strong activity on resisting tumor, and has a therapeutical effect on various tumors. Indeed, Zanthoxylum ungeanum essential oil can restrain various proliferation of tumor cells, and prevent its normal mitosis by means of resisting the cycle of tumor cells so as to reach the purpose of restraining cell growth and then induce the apoptosis, which can provide a theoretical basis to the Zanthoxylum ungeanum essential oil that is ameliorated by propranolol, and the local skin pathological changes of sample Guinea Pig psoriasis in the part of ear is resulted. In this connection, a pathological model for the sample Guinea Pig psoriasis is structured and used to observe the external curative effect of Zanthoxylum ungeanum essential oil so as to provide the original basis to the study of curing psoriasis with Zanthoxylum ungeanum essential oil. In China, so far, there is no relevant experiment or clinical report that the Zanthoxylum ungeanum essential oil is used for curing psoriasis.ObjectiveIn this experiment, the method of microwave-assisted extraction is used for extracting Zanthoxylum ungeanum essential oil from clovershrub. Besides, gas chromatography and mass spectrometry are applied to analyze the major ingredients in Zanthoxylum ungeanum essential oil so as to manufacture the DMSO solution of Zanthoxylum ungeanum essential oil. It is also the task to establish animal model for the sample psoriasis in the part of Guinea Pig ears through DMSO solution of5%Zanthoxylum ungeanum essential oil, DMSO solution of20%Zanthoxylum ungeanum essential oil and positive control medicine (Halcinonide emulsifiable paste) that is parallel to the medicine for external use so as to observe the grade of Zanthoxylum ungeanum essential oil to the histology of animal model of sample psoriasis, infiltration of inflammatory cell and the pathological effects of other major visceral organs such as heart, liver, lungs, kidney, etc. This is aimed at exploring the pathological feasibility of Zanthoxylum ungeanum essential oil that is for external use on improving the psoriasis in animal models. Meanwhile, the cutaneous lesions of sample psoriasis in the part of Guinea Pig ear are collected. After the homogenate is conducted, apply the enzyme-linked immuno sorbent assay (ELISA) to assay the concentration of IFN-γ and IL-4in cutaneous lesions, and the effect of expression on IFN-γ and IL-4in cutaneous lesion before molding and after curing with Zanthoxylum ungeanum essential oil. This is aimed at discussing the feasible mechanism of the solution of Zanthoxylum ungeanum essential oil on improving the animal model pathology of sample psoriasis.MethodThe method of microwave-assisted extraction is used to extract Zanthoxylum ungeanum essential oil, experiment condition:material ratio, Zanthoxylum ungeanum ungeanum (g):ethanol (ml),1:12; microwave power is390kw; time of microwave-assisted extraction is6min; initial extraction temperature is60℃.GC-MS experiment condition:Chromatographic conditions:AB-5MS capillary chromatographic column (30m×0.25mm×O.25μm); temperature programming:initial column temperature is60℃, keep for1min, rise to120℃from the condition of3℃/min, and then rise to220℃from the condition of8℃/min, keep for1min. Carrier gas is He, flow velocity is1.0ml/min; temperature in the injection port is230℃;1:100split-flow sample injection, capacity of sample injection is0.02μl; temperature in the connector is230℃. Mass spectrometer conditions:El-source electron energy is70eV, the temperature of ion source is230℃; mass scanning range:45～350amu; the time for dissolvent to delay is3min.Preparation of animal model of sample psoriasis:spread5%propranolol ointment on the outside ears of Guinea Pig, three times a day, three weeks continuously, the action that causes some typical psoriasis pathological changes such as hyperkeratosis in the ear skin, and the increasing inflammatory cell infiltration. Therefore, the model of sample psoriasis is established.Experimental animal packet processing:The healthy and grown-up Guinea Pig are randomly divided into seven groups. Group1:normal group, normal feeding×5W; group2:modeling group, after the model is established, cut the sample ear and the sample visceral organs; group3: modeled observation group, any processing is not allowed after the model is successfully established, feedingx2W; group4:dissolvent control group, spread40%DMSO solution on the ear outside after the model is successfully established, tid×2W; group5:positive drugs control group, spread halcinonide emulsifiable paste on the ear outside after the model is successfully established, tid×2W; group6: high-dosage treatment group, spread20%Zanthoxylum ungeanum essential oil DMSO solution on the ear outside after the model is successfully established, tidx2W; group7:low-dosage treatment group, spread5%Zanthoxylum ungeanum essential oil DMSO solution on the ear outside after the model is successfully established, tidx2W.Preparation of tissue samples:after each group of Guinea Pig reaches the scheduled time, put them to death in the method of cervical vertebra dislocation, adopt the ears and internal organs as the samples, keep them in the10%formaldehyde solution, paraffin embedding, cutting into slices, HE staining, observe the skin pathological changes through light microscopy.Preparation of tissue homogenate of Guinea Pig ear skin:cut the two sides of Guinea Pig ears in each group as the samples, wash them with the normal saline, swab the water away, cut1.0g of the ear skin respectively, weigh them accurately, put them into the test tubes that have1.0ml of normal saline, use the super-speed disperser to do10000rpm homogenate for two times, lOs/each time, make centrifugation for10min in the method of having3000rpm, adopt the supernatant liquid to make test.Evaluation of experimental effect:make three slices from both sides of each Guinea Pig’ears, randomly select5fields of vision under the optical microscope that has a10×20field of vision, use the computer image analysis software to do image analysis for the fields of vision selected, compare with the Baker method to do histological scores evaluation, and then count the inflammatory cells. Compare the statistical difference of the data in each group.Use the ELISA method to detect the concentration IFN-y and IL-4in cutaneous lesions:combine the specific antibody with solid-phase carriers to make solid-phase antibodies; wipe off the antibodies not combined and impurities. Add the censorable samples so as that they can have haptoreaction with solid-phase antibodies for a period of time; combine the antigen of samples to the antibodies of solid-phase carriers so as to make the complex of solid-phase antigen. Thoroughly wipe off the other substances not combined. Add enzyme-labeled antibody so as to make the antigen of solid-phase immune complex combined with enzyme-labeled antibody. Thoroughly wipe off the enzyme-labeled antibody not combined. At the moment, the quantity of enzyme carried on the solid-phase carries has a positive correlation to the censorable substances in the samples. Add substrate:the enzymatic substrate within sandwich complex becomes non-ferrous product. The shade and the IFN-y and IL-4of samples have a positive correlation. Use enzyme-labeling instrument to figure out the absorbance (OD value) under the circumstance when the wavelength is450nm, and then calculate the concentration of samples.Result1. Extraction and component analysis of Zanthoxylum ungeanum essential oilThe Zanthoxylum ungeanum essential oil extracted is bronzing, the yield is12.92%. The number of the major components of GC-MS analysis is20varieties.2. HE staining of Guinea Pig ear skinThe skin texture of group1(normal control group) under the optical microscope is intact, and there is no hyperkeratosis, parakeratosis, stratum granulosum insufficiency or acanthosis on stratum epidermis. Besides, the hyperaemia or edema resulted from the connective tissue hemangiectasis does not exist on dermis, dermis is internally scattered and infiltrated within the inflammatory cells. The grade of Baker is0.75±0.44, and the quantity of inflammatory cells infiltrated is22.15±2.82. An extensive hyperkeratosis in group2(modeling group) can be seen under the optical microscope, and the stratum spinosum is obviously thickened. Cutaneous nerve is lengthened, which rises and falls in the shape of wave. Small Munro abscesses can be seen in three cases; angiectasis hyperaemia, edema, mild and moderate infiltration of inflammatory cells exist within the dermis. The grade of Baker is6.48±0.83, and the quantity of inflammatory cells infiltrated is90.45±9.18. The analysis of variance indicated that the histological grade between normal group and modeling group, and the quantity of inflammatory cells infiltrated have a significant difference (P<0.001), it is prompted that the model is successfully established.The conditions of group3(observation group after the model is established) and group4(solvent control group) seen under the optical microscope is similar to it of group2. The grades of Baker are6.35±0.75and6.10±0.68respectively; the quantities of inflammatory cells infiltrated are88.65±7.60and85.85±8.05respectively. The analysis of variance indicated that the histological grades of group3and group4, and the quantity of inflammatory cells infiltrated have no significant difference compared to group2(P=1.000), from which it is prompted that the pathological changes can keep2w immovable under the circumstance of not accepting the stimulation of5%propranolol ointment. The ground substance (40%DMSO solution) of solution of Zanthoxylum ungeanum essential oil has no therapeutical effect.The skin texture that is intact in group5(positive drugs control group), group6(high-dosage treatment group) and group7(low-dosage treatment group) can be seen under the optical microscope. The degree of hyperkeratosis within stratum epidermis is alleviated, and the degree of thickening of stratum spinosum is lightened, and the degree of cutaneous nerve lengthened is alleviate. Small Munro abscesses cannot be seen; the cases of hemangiectasis and hyperaemia arise within dermis, and the degree of edema is alleviate, the quantity of inflammatory cells infiltrated is reduced obviously. The grades of Baker are2.45±0.71,3.13±0.56and4.60±0.45respectively. The histological grades of the groups in medicine and the quantity of inflammatory cells infiltrated have significant difference compared to group3and group4(P<0.001), from which it is prompted that the solution of Zanthoxylum ungeanum essential oil has inhibitory action on the pathological changes of sample psoriasis of Guinea Pig’ ears and the phenomenon of inflammation. In addition, the histological grades of group6and group7, and the quantity of inflammatory cells infiltrated have significant difference (P<0.001), it is prompted that the solution of20%Zanthoxylum ungeanum essential oil has an obvious therapeutical effect compared to the solution of5%Zanthoxylum ungeanum essential oil.3. The result of HE staining of Guinea Pig’visceral organs and tissuesThere is no pathological structural change to the heart, liver, lungs and kidney of the Guinea Pig in each group.4. The method of ELISA is used to determine the concentration of IFN-γ and IL-4for the skin tissue homogenate of Guinea Pig in each group.Compared to group1, the concentration of IFN-y of skin lesions in group2, group3and group4is increased obviously, and the difference is significant (P<0.001). There is no significant difference after the comparison among group2, group3and group4. It is prompted that the expression of IFN-γ of cutaneous lesion of Guinea Pig ears is increased after the5%propranolol ointment is for external use, and there will be no obvious decline after the model is established for2w and the drugs are given to the blank solvent substrate for2w.Compared to group3and group4, the concentration of IFN-y of skin lesions in group5, group6and group7is reduced obviously, and the difference is significant (P<0.001). There is significant difference (P=0.008) after the comparison between group6and group7. It is prompted that the expression of IFN-y of cutaneous lesion of Guinea Pig ears is decreased, and it has a positive correlation to the concentration of Zanthoxylum ungeanum essential oil.There is no significant difference (P=1.000) for the comparison of IL-4concentration in the ear tissues of Guinea Pig.Conclusion1. The condition of microwave-assisted extraction:the microwave power is390w, the time of microwave-assisted extraction is6min, the amount of dissolvent is1:12(g/ml), and the initial extraction temperature is60℃. The extraction efficiency of Zanthoxylum ungeanum essential oil is12.92%. GC-MS makes analysis on Zanthoxylum ungeanum essential oil, and the analysis is conducted through MSD chemistry workstation, and the spectral library is retrieved, the variety of components that has a compatibility of90%and over90%is20, which account for91%of the gross peak area, and the major ingredients are limonene, eucalyptol, pinene, etc.2. The DMSO solution of Zanthoxylum ungeanum essential oil for external use can help alleviate the local pathological changes of sample psoriasis of Guinea Pig, and it has a positive correlation to the concentration of Zanthoxylum ungeanum essential oil.3. The DMSO solution of Zanthoxylum ungeanum essential oil for external use can decrease the expression of IFN-y in the cutaneous lesions for the sample psoriasis of Guinea Pig, and it has a positive correlation to the concentration of Zanthoxylum ungeanum essential oil.4. The expression of IFN-y has no pathological changes in the cutaneous lesions for the sample psoriasis of Guinea Pig when the DMSO solution of Zanthoxylum ungeanum essential oil is for external use.5. There is no local stimulation or symptoms of allergy when the DMSO solution of Zanthoxylum ungeanum essential oil is for external use, and there are no pathological changes to the hearts, livers, lungs and kidneys of experimental animals. |
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