Studies And Utilization Of The Characteristic Medicinal Materials Of Dicranostigma Leptopodum(Maxim.) Fedde In Southeast Region Of Gansu Province

Dicranostigma leptopodum(Maxim.)Fedde,(DLF), also named “Tuchuangflower, Tuzi flower, and Lemahui” in Chinese, a biennial or perennial herbagebelonging to Dicranostigma in papaveraceae and growing in hills, braes, roadsides,fields, meadows, etc, in latitude of400～1300m, is characterized by resistance todrought and barren. DLF mainly distributes in Gansu, Shanxi, Henan, Shanxi,Qinghai, Ningxia, Sichuan and Tibet, particularly in the south and north of QinlingMountains in southeast region of Gansu province and Weihe River Basin.In the present study, DLF in the southeast of Gansu was used to investigate itsartificial and field standard cultivation, to extract and identify its active ingredientsand to determine the bioactivities in order to further exploitation and utilization.1. By means of investigation combining experiment, the indoor test combiningoutdoor observation, the experimental design combining statistical analysis in a3-year term study, the preliminary results of the botanical and biologicalcharacterization of DLF was obtained, including the monitoring of the variation ofalkaloids content in different periods of growth and the technology of artificial andstandard cultivation. The results showed that DLF did not need high standard growingcircumstances and its phonological performance and resistance in the artificialcultivated condition were generally consistent with that in the natural conditions. It issuitable to grow widely in an artificial cultivation condition in southeast region ofGansu province.2. The DLF dry powder was extracted with water and ethanol using the alkaloidsystematic extracts method. We found the alkaloid precipitation was most obvious.The thin-layer chromatographic analysis showed that the95%ethanol extract of DLFusing the ice acetic acid: water: strong aqua (15:0.5:2.5:0.5) as the developing solventhave a good repeat, strong speciality and stability. We built a special acid dyecolorimetry method to determine the total alkaloid content of DLF. In this method, thesample in pH4.0citric acid-sodium hydrogen phosphate buffer were stained by1.5mL0.1%bromcresol, then measured the optical density at415nm using isocorydine as the standard, the content of alkaloid is1.386%. Then the refinedflavonoids (rutin) were measured by HPLC. The HPLC condition arechromatographic column Eclipse×DB–C18(4.6mm×250mm), mobile phase ofmethanol:1%glacial acetic acid=60∶40(v/v); The UV wavelength at254nm;flowrate in1.0mL/min, injection volume at20μL; column oven temperature at30℃;HPLC result showed the content of alkaloid is0.106%.3. Firstly,1mg/mL alkaloid of DLF and360μL1%sodium molybdate-oil ofvitriol reagent was incubated at70℃water for20min, a light blue substancegenerated, the maximum absorbance was measured at310nm. The alkaloids averagecontent was18.65～18.75%. Comparing with the positive control of1mg/mLisocorydine, the0.493mg is little less than the actual value of0.50mg.4.The content of19trace metals in DLF including Cu, Zn, Fe, Mn, Co, Ni, Cr,Cd, As, K, Na, Ca, Mg, Li, Sr, Al, Pb, Se, Hg were determined by Flame AtomicAbsorption Spectrometry. The statistical analyze with SPSS17.0software showedthat the contents of K, Ca, Mg and Al were higher than others. The content of K fromdifferent tissue followed the order: flower> stem> root> leave> pod> herb> seed.3.16g volatile oils were extracted by supercritical CO2with0.63%oil yield rate.201chromatographic peaks were found in the GC-MS spectrum, then retrieve them withNIST02standard fragmentation in the warehouse,46compounds are recognized,which account for96.59%of total volatile oils. The alkaloids of DLF were furtherseparated and purified using silica gel column chromatographic and identified itsstructure by superconducting nuclear magnet resonance, isocorydine, corydine，protopine and chelidonine were obtained. Finally, the structure and stability offlavonoid (Rutin) extracted from DLF was tested by ultraviolet spectrum, infraredspectrum, and DTA-TG.5. All analyses were conducted by the single factor and L625(5) orthogonalmethod by using SPSS software. The optimal condition of extraction is65%ethanol,ultrasonic for20min, liquid to solid ratio:15:1ml/g, reflux2.5h, pH8extractingsolution, macerate4h. The alkaloids content in DLF extraction is0.928%under thisoptimized condition. Using response surface analysis the technique of alkaloids extraction from DLF, the result showed the main influencing factors are: Liquid tosolid ratio is15ml/g, the treatment time of ultrasonic is35min, reflux time is2.5h, andconcentration of ethanol is65%. Under this condition the alkaloids content is0.933%.The alkaloids of DLF were extracted by vacuum freeze drying, the two main alkaloids(isocorydine and protopine) content were determined by HPLC. The results showedthat the freezing rate of alkaloids is44.26%, RSD is0.21%. The isocorydine andprotopine showed good linear relation, the R2value of isocorydine is0.9998, the R2value of protopine is0.9996respectively. This method has a good specificity andcould provide reference standard for the monitoring and controlling the quality ofDLF drugs.6. The hydroxyl radical(·OH) and superoxide anion radical (O-2·), scavengingability of the extract of DLF using the optimization condition and the volatile oils ofDLF using supercritical CO2were determined.25.0mg/mL and1.8mg/mL alkaloidsof DLF showed the58.70%hydroxyl radical and49.26%superoxide anion radicalscavenging activity respectively, which IC50value is23.50mg/mLand1.75mg/mLrespectively. Comparing the hydroxyl radical scavenging activity of these extract at0.45ug/mL, they showed the following order: Vc> isocorydine> Rutin> extract ofDLF> volatile oils. When these extracts at0.50ug/mL, their hydroxyl radicalscavenging activity exhibited a different order: Vc> Rutin> volatile oils>isocorydine> extract of DLF.7. Anti-bacteria activity of alkaloids extraction of DLF was determined onEscherichia coli, Staphylococcus, Candida albicans and Pseudomonas aeruginosausing the bacteria-grow-curve test, slip method, MIC, MBC, IC50, the permeability ofEscherichia coli and morphology, transmission electron microscope observation. Theresults showed that the MIC of alkaloids extraction of DLF on Escherichia coli,Staphylococcus aureus, Candida albicans and Pseudomonas aeruginosa are0.28g/mL,0.20g/mL,0.20g/mL and0.30g/mL respectively, the IC50are0.2162g/mL,0.1805g/mL,0.1805g/mL and0.2604g/mL respectively. After treatment with thealkaloids extraction of DLF, the cells permeability of E.coli were obvious improved,making its morphological change.Finally, the thalli were splited.all these showed that this extract has a good anti-bacteria activity. The effect of flavonoid (Rutin) extractedfrom DLF on electrochemical behavior of lipid bilayer membranes was investigatedby Cyclic Voltammetry (CV) and SECM using s-BLM supported by Pt/C electrode asbio-membrane model, Fe(CN)3-/4-6as probe. We found that there are stronginteractions between s-BLM and rutin; meanwhile the flavone compounds have someextent effect on the electrochemical behavior of bilayer lecithin membrane. Theseresults provide some theory for further understanding the bioactivity of DLF and itsmechanism as drugs.8. The water-soluble external preparation of DLF exhibited the good effect onthe goat with orf. After treatment with different doses of alkaloids extraction, the heart,the liver, the spleen, the lung, the kidney and the muscular tissue in injection wereharvested at different periods, then stain all the tissue with HE, we found it presentedobvious acute inflammation and in injection part of muscular tissue, however thereare no obvious pathological changes in all the tissues. All these showed that theextract of DLF is safe for goat and mice and provideed some evidence for the clinicalpractice.All the results provided some important experimental and theoretical supports forfurther and deeper exploitation and utilization of DLF as the special medicinal plantin southeast region of Gansu province.