| Qing-tian-kui(QTK),one of the genuses of Nervilia fordii(Hance)Schirt.,is a farmous ttaditional Chinese medicine and widely distributed in South China for treatment of pneumonia,bronchitis,acute laryngitis and so on.For the huge consumption,the wilding resources of QTK,nowadays,is dramatically exhausted due to the limit of distribution and the low reproduced factor.At present,it has been not comprehasively and systematically to stusdy of QTK for the better monitoring and regulating the market.Therefore,the aim of this work is to investigate the bioactivities and compounds,and the study of quality control. The other one of the purposes is to study the inflammatorymechanism of anti-SIRS-ALL of QTK.Methods:1.The extracted fractions were enriched by the various isolating methods including macroporous resin column,polyamide resin,ion exchange resin and so on.After the concentration under the vacuum,the dryness under the reducing pressure,the dryness under the refrigeration and the dryness under the spray,the bioactive compounds was determined by the HPLC.2.This study was to evaluate the bioactive fraction of the QTk based on the subjects of the QTK and it's extracts by the methods of the pharmacological models of anti-inflammatory and analgesic effects,antipyretic and immunopotentiation effects, relieve cough and asthma.The anti-inflammatory and analgesic effects were studied by mice ear swelling,rat paw edema,mouse writhing induced by acetic acid,and so on.The antipyretic activity was studied by fresh yeast method.The experiments of immunopotentiation effects included the models of rat carbo clearance and mensurate the weight of immune organ.The experiments of relieving cough and asthma included the models of guinea pig asthma and mice cough induced by ammonia water.3.It is to investigate the protective effect of QTK and its bioactive fractions,and study the the anti-inflamed mechanism of QTK by the model of acute lung injury causing by endotoxin(Lipopolysaccharides)in rats with the positive medicines of the injection of Qing-kai-ling or Shen-fu.4.According to the results of the pharmacological experiment of QTK and the aim to the bioactive fractions,the bioactive chemicals were isolated by the techniques of chromatogram and identified the chemical structure by the data of MS,UV,IR and MNR.5.The targets including moisture,the water-soluble extracts and the alcohol soluble extractive were determined based on the experimental method of Chinese Pharmacopoeia, and also to identify the compounds of amino acid and flavone by TLC.Besides,the levels of the flavone in the QTK were measured by HPLC.Results:1.The total flavone(about 1%of the total amount of QTK)of QTK was purified by the isolating method of macroporous resin column and polyamide resin.The majour extensive UV absorption was at 210nm,260nm and 350nm in the produces of the total flavone.The total amino acid(about 4%of the total amount of QTK)was enriched by the ion exchange resin and determined by the auto-analysis of amino acid.The results showed that there were 7 kinds of needed amino acid and some non-satisfied needed amino acid including histidine and arginine,as well as non-needed amino acid in human except for tryptophan.2.The results of animal experiment of anti-inflammation indicated that the bioactive fractions of QTK were likely to alleviate the rate of the mice ear swelling by dimethylbenzene(p＜0.05,or p＜0.01)and to inhibite the accentuation of capillary permeability of mouse abdomen induced by acetic acid(p＜0.05,or p＜0.01);and also to inhibite the rat's tumefaction by carrageen(p＜0.05,or p＜0.01).Those results suggested that a rather potent effect of anti-inflammation was found in the the bioactive fractions of QTK.The results of animal experiment of anti-pain indicated the times of writhing caused by acetic acid obviously decreased after the administration of the bioactive fractions of QTK in rats(p＜0.01)and also to reduce the accumulative time after subcutaneous injecting formaldehyde by toe.Those results demonstrated that a rather potent effect of anti-pain was found in the the bioactive fractions of QTK. The results of animal experiment of immunity indicated the expurgated exponential obviously increased in the the bioactive fractions of QTK,and also inhanced the weight coefficient of the immune organs such as thymus gland and spleen.Those results demonstrated that the effect of functions of regulative immunity was found in the the bioactive fractions of QTK.The results of antipyretic activity experiment suggested that these bioactive fractions could remarkablely contribute to decrease the rat's body temperature(p＜0.05,or p＜0.01), indicating that good antifebrile effect was found in the bioactive fractions of QTK.The results of anti-tussis's and anti-asthma's experiment suggested that these bioactive fractions could contribute to prolong the delitescence of cough in mice(p＜0.05,or p＜0.01) and possess some effect to inhibite tussis in mice(p＜0.05).Besides,the delitescence of dyspnoea could also be prolonged by acetylcholine and histamine in the certain degree in the guinea pig,showing that QTK possesses the effect in anti-tussis and anti-asthma.In addition,an interest phenominon could be found that some effect is likely to reinforce when the total flavone cooperates with the total amino acid in some effect.3.Rats were caused acute lung injury by via i.v LPS,then they were subjected to received QTK and its bioactive fractions.The results showed that the average arterial pressure obviously lowed comparing with the control after administration of water-extracted fractions of QTK(p＜0.05);the pressure of CO2 QTK and its bioactive fractions dramatically decreased comparing with control after administration of water-extracted fractions of QTK(p＜0.01)in the ananlysis of arterial pressure;the results of QTK could inhibite the decline of anoxic blood pressure causing by ALI based on the results of the remarkable rise of the saturation degree of blood oxygen saturation(O2Sat) and oxyhemoglobin(HbO2)(p＜0.01,p＜0.05).When comparing with the control,the level of TNF-αand IL-6 obviously declined(p＜0.01)after administration of QTK,whereas the level of IL-10 incresed(p＜0.01);The results of pathological slice by scanning electron microscope showed that the symptoms of ALI such as the increased thickness of pulmonary alveolar wall,the alveolar cavity decreased,and the effusion of inflammatory mediators could be avoided after after administration of QTK causing by LPS;and compared with model group,the results of pathological slice by transmission electron microscope of test groups showed that there was few secretion effused to the alveolar cavity,meanwhile,the structures of the endothelial cells and basilar membranes were not destroied.The exponent of pulmonary microvascular permeability obciously declined after administration of QTK(p＜0.01)and the pneumonic weight ratio of wetness/dryness also obciously declined(p＜0.01) 4.Two compounds of flavone were isolated from the total flavone of QTK by the isolating method of HSCCC,and then identified as rhamnazin-3,4'-di-O-glucoside and rhamnazin-3-β-D-glucoside based on the data of sperum5.The analyses were carried out the moisture,the water-soluble extracts and the alcohol soluble extractive,the results suggested that the content of moisture ranged from 6.82%～11.06%in the herba of QTK(The present data of was confined not to exceed to 12.0%);the content of water-soluble extracts ranged from 20.45%～30.55%,which was confined not to exceed to 20.0%in the herba of QTK;while the content of 95% ethanol-soIuble extracts ranged from 4.66%～5.74%,which was confined not to exceed to 4.0%in the herba of QTK,respecitively.6.Three amino acid(leucine,viline and alanine)were identified by the TLC with n-BuOH:acetic acid:water(4:1:1)as chromogenic reagent and with 5%ninhydrin ethanol solution as developing solvent.Whereas two spots from the herba of QTK were also identified as rhamnazin-3,4'-di-O-glucoside and rhamnazin-3-β-D-glucoside by the TLC in 365 nm with(Ac)2EtOH:formic acid:water:methanol(5:3:0.6:0.5:0.8)as developing solvent and with 5%AlCl3 solution as chromogenic reagent.7.A HPLC assay was developed for determination of rhamnazin-3,4'-di-O-glucoside and rhamnazin-3-β-D-glucoside in the herba of QTK.The chromatographic condition was described as follow:Separations of two compounds were achieved on a Kromasil C18 column(5-μm,4.6×250 mm).Mobile phase A was methanol,while mobile phase B was water(pH=2.5).Linear gradient elution was performed with in a 42min running time and its sequence was as follows:The beginning with 40%of mobile phase A and 60%of mobile phase B,which was maintained for 5 min,then the mobile phase A to B ratio was gradually change to 50:50 at 10 min with steps of increasing A on 1%/min,then the mobile phase A to B ratio was gradually change to 52:48 at 10 min with steps of increasing A on 0.2%/min,then the mobile phase A to B ratio was gradually change to 55: 45 at 10 min with steps of increasing A on 0.3%/min,followed by the mobile phase A to B ratio was gradually change to 90:10 at 2 min with steps of increasing A on 17.5%/min and maintained for 5 min.The flow rate was set to 1.0ml/min and the UV detector was set to 266 nm.The column temperature was set to 35℃.After the method validation,there were two good linearity over the range of 0.112～2.800μg and 0.098～2.450μg in two compounds with a correlation coefficient greater than 0.999,respectively.Good precision and stability were obtained in this method with RSD little than 2.0%,while the recovery ranged from 96%to 102%.This method was successfully applied for determination of QTK.When performing on determining multiple groups of QTK,the results suggested that the total amount of two compounds of flavone ranged from 0.36mg/g～0.52 mg/g in the QTK.The total amount of two compounds of flavone(rhamnazin-3,4'-di-O-glucoside and rhamnazin-3-β-D-glucoside)was confined no less than 0.3mg per gram of the dry herba of QTK.8.The preliminary draft of quality control of QTK was temporarily established according to the experimental results of 5,6 and 7.Conclusion:1.The preparation methods of amonio acid and flavone from QTK were simple.High purity and rich yield of amonio acid and flavone were available in this study.The methods could be applied for preparation technology of QTK as reference.2.The results of pharmacodynamics suggested that the good effects of anti-inflammatory,analgesia,strengthening immunity,antipyresis and relieving cough and asthma were found in QTK for the pharmacological experimental base of pneumonia, bronchitis,cough and asthma and fever in clinic therapy.Moreover,the above pharmacological effects of total amonio acid and flavone in the QTK were confirmed in this work.3.The total amonio acid and flavone and QTK possessed the protective effect of ALI in rats.The results showed that the mean arterial pressure obviously lowed comparing with the control after tested administrations,and maintained stable pressure.The results of blood gas analysis suggested that the above tested drugs could be used for inhibition of blood pressure decrease induced by hypoxia,and shock induced by acid-base disturbance.In summary,the total amonio acid and flavone and QTK possessed the effects of inhibiting the pulmonary microvascular permeability and the effusion of inflammatory mediators, alleviating the symptom of pulmonary edema,decreasing the level of proinflammation cytokines such as TNF-α,IL-6,ICAM-1,and increasing the level of anti-inflammation cytokines such as IL-10.4.The preliminary draft of quality control of QTK was temporarily established.The draft could reflect the essential features of the medicinal material of QTK,alsocould ensure the demands of "effective,stable,controllable",and could be used to regulate the market of QTK.Moreover,the draft was able to provide basic study for the promotion of rational expoitation and comprehensive utilization of QTK.Major innovation of this dissertation:1.Two flavone,isolated from QTK for the firs time,were identified by the isolating chromatographic method of TLC and then determined by assay method of HPLC. 2.The results of the total amino acid and flavone in the QTK and its bioactive fraction showed that good effects were found in this study and also possessed to cooperate with function for strengthening effects when carring out the study of anti-inflammatory, analgesic effects,antipyretic and immunopotentiation effects,relieving cough and asthma.. In this work,nti-inflammatory,analgesic effects of the study were firstly performed on QTK and it's amino acid and flavone and also investigated the model experiment of ALI in rat's and its mixed bioactive compounds.Meanwhile,the mechanism of QTK and its mixed bioactive compounds were also investiged in this work.3.The quality criterion of QTK was tentatively developed for the evaluation the study of quality standard in the QTK according to the results of TCL,HPLC assay and other examined programs.4.The selective Chinese medicine based on the authentic curative effect were subjected to moniotor by the advanced and credible assay methods according to the isolation of effective and bioactive monomer in the animal experimental model in vivo for the filtration and evaluation of pharmacological effect.This above pathway of the scientific research could contribute to explain the anti-inflammative effect and its substance base in the QTK and also develop a scientific method for evaluation of Chinese medicine.It will provide scientific basis for sustainable exploitation and utilization of chinese medicinal materials,and for new drug research and development of TCM.
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