Study On Fingerprint And Histochemical Lication Of Lomatogonium Rotatum In Mongolian

Mongolian medicinal stigma is the dried whole herb of Lomatogonium rotatum（L.）Fries ex Nym.The main active ingredients include iridoid terpenoids,flavonoids and so on.It has obvious clinical effects and is commonly used in Mongolian medicine.In recent years,more adulterants and substitutes of costal stigma have been introduced into clinical practice,which affects the clinical efficacy and drug safety.Objective:To establish the fingerprint,content determination method and microscopic histochemical method of Mongolian medicine stigma costatum by HPLC,and to provide basis for the identification and quality control of stigma costatum.Methods:（1）The fingerprints of 15 batches of Lomatogonium rotatum samples collected from different places in Inner Mongolia were studied by HPLC.（2）Simultaneous determination of four active ingredients in whole herb and different medicinal parts under this chromatographic condition.（3）The content of Total Iridoid terpenoids in 15 batches of Lomatogonium rotatum stigma costatum was determined by Ultraviolet Spectrophotometry with swertiamarin as control.（4）The active constituents of stigma costatum were localized by histochemical method.Results:（1）Establish the fingerprint of Lomatogonium rotatum by HPLC.The optimum chromatographic conditions are as follows:YMC C18 column（4.6 mm x250 mm,5 m）;mobile phase:acetonitrile（A）-0.4%phosphoric acid（B）-methanol（C）,gradient elution,acetonitrile is always 10%,0-10 min,90%-85%B;10-20 min,85%-80%B;20-24%-78%-78%B;27-37%B;75%-47%B;40 min.⁷0%B;40-45 min,70%-65%B;45-47 min,65%-60%B;47-48 min,60%-58%B;48-65 min,58%-50%B;65-77 min,50%-50%B.The flow rate was 0.8 mL min-1,the column temperature was 30 C,the injection volume was 10 L,and the detection wavelength was 254 nm.Fifteen common peaks were identified.Five common peaks were identified as swertiamarin,isohirudin,flavin,apigenin and luteolin.The similarity of fingerprints between 15 batches of stigma costatum and the control ranged from 0.881 to 0.997.（2）The contents of swertiamarin,isohigenin,luteolin and luteolin were simultaneously determined by using the above-mentioned chromatographic conditions of the fingerprint of Lomatogonium rotatum.Swertia picroside,isourea glycoside,flavin and luteolin showed good linear relationship in the range of 0.08⁰.48ug（r=0.9998）、0.02⁰.15ug（r=0.9999）、0.02⁰.12ug（r=0.9999）、0.03⁰.12ug（r=0.9998）,respectively.（3）Ultraviolet spectrophotometry was used to determine the content of total iridoids in 15 batches of Lomatogonium rotatum from different habitats in Inner Mongolia.The regression equation of swertiamarin was Y=3.4214X-0.0068 and R²=0.9995 in the range of0.04-0.14mg/ml^-1.There was a good linear relationship between absorbance and concentration.（4）Histochemical localization technique was used to determine that1%magnesium acetate and methanol showed fluorescent green on the epidermal cell wall,epidermal cuticle,xylem and upper and lower epidermal cell walls and xylem of Lomatogonium rotatum under fluorescence microscope.After 5%NaoH staining,the upper and lower epidermis cells of Lomatogonium rotatum showed yellow color,and the xylem did not change significantly.After staining with concentrated hydrochloric acid-aniline-perchloric acid,the xylem cells of the stem of Lomatogonium rotatum were yellow to brown.Conclusion:（1）The fingerprint of Lomatogonium rotatum and its four active ingredients were established for the first time.The method has good repeatability,high precision and stability.It can be used for qualitative and quantitative determination simultaneously,and can be used as a quality control method for Stylosanthes costatum.（2）The content determination of different parts provides a basis for the selection of Lomatogonium rotatum parts and the study of microscopic and histochemical localization of Lomatogonium rotatum.（3）The method of determination of Total Iridoid Glycosides in stigma costatum of Lomatogonium rotatum is simple,rapid and can be used as the basis for quality evaluation of this medicine by combining with the method of determination of Total Iridoid Glycosides in stigma costatum of Lomatogonium rotatum by high performance liquid chromatography.（4）Establish a method of histochemical localization of Lomatogonium rotatum,so as to lay a foundation for future research on accumulation and dynamic transfer of active ingredients and biosynthetic pathways.