Chemical Analysis And Vasodilative Effect Of Rosemary Leaves

Rosmarinus officinalis L(rosemary)is a folk medicine with a long history in Europe.In Wei period,rosemary was imported into China from the western regions,and it was recorded in some herbal books,such as "Oversea Materia Medica" and "A Supplement to Materia Medica".At present,there are many rosemary planting bases in Yunnan,Guangxi,Guizhou and Hainan provinces.Modern research shows that rosemary has antioxidant,analgesic,antibacterial,anti-inflammatory,anti-depressive effects and so on and it has been applied in medicine and other industries.According to market survey,the commercial rosemary is composed of domestic and imported sample with big difference in morphology.Because rosemary has not been recorded in Chinese Pharmacopoeia,there is no national standard for its quality estimation.The differences on quality of commercial rosemary will inevitably induce some security risks to consumer's health.In this paper,the chemical composition and vasodilative effect on 31 batches of rosemary,including both domestic and imported samples,were investigated with multidisciplinary techniques and methods.1.To analyze the chemical composition of rosemaryThe fingerprint of rosemary oil was established by GC/MS,and 63 volatile compounds,such as(+)-a-Pinene,Camphene,o-Cymene,Eucalyptol,(+)-Camphor,endo-Borneol,(-)-Verbenone,Bornyl acetate,Caryophyllene and etc.,were tentatively identified by comparising with data in Standard Mass Spectrometry Library of the National Institute of Standards and Technology(NIST)on an Xcalibur workstation.At the same time,the fingerprint of nonvolatile ingredients of rosemary was established by UPLC,and 39 nonvolatile components were tentatively identified using UPLC-Q-TOF-MS,including Rosmarinic acid,Rosmarinic acid-3-O-glucoside,Homoplantaginin,Carmosol,Camosic acid,and Eucommin A.2.To compare the chemical differences between the imported and the domestic samplesMultivariate data analysis was applied to analyze the GC/MS data and UPLC data of rosemary,respectively.The imported and domestic rosemary samples were classified into two groups by principal components analysis(PCA),which indicated the chemical differences between the imported and domestic commercial rosemary samples.Moreover,14 components including Eucalyptol,(+)-a-Pinene,D-Limonene,endo-Bomeol,o-Cymene,Bornyl acetate,and Carnosic acid,were found to be the discrimination markers between the imported and domestic rosemary sample by partial least squares discriminant analysis(PLS-DA),and the contents of Eucalyptol and(+)-Camphor were higher in the imported samples,while,the contents of Camosic acid and other discrimination markers were higher in the domestic samples.In addition,the essential oil of the imported rosemary samples belongs to Tunisian/Morocco type and the one of domestic samples belongs to Spanish type.3.To study the vasodilative effect of rosemaryThe vasodilative activities of 31 batches of rosemary oil were performed in isolated thoracic aorta of rat.The results showed that the rosemary oil had good vasodilative activity(0.10-1.60 mg/mL)with dose-dependent,and the maximum vasodilation of the sample reached to 65%.Moreover,no significant difference was found between the imported and domestic rosemary oil in terms of its vasodilative activity.As for non-volatile extract,the vasodilative activity of 80%ethanol extract(48%)was found to be stronger than that of water extract(9%).Accordingly,the 80%ethanol extract was purified by different solvents and by macroporous resin,respectively,and 8 kinds of extract were obtained.The vasodilative activities of the 8 extracts were decreased in the order as follows:petroleum ether fraction>macroporous resin fraction ?>macroporous resin fraction ??chloroform fraction>ethyl acetate fraction>butanol fraction>macroporous resin fraction ?>water fraction,which suggested that the lipid-soluble components were the active components of vasodilation.The contents of nitric oxide(NO),endothelin(ET)and angiotensin ?(AT-?)in blood vessels and K-H solution were determined by the Griess reagent method and Radioimmunoassay.The results showed that rosemary oil could promote the release of NO,and the petroleum ether extract can not only promote the release of NO but also reduce the amount of AT-? in endothelial cells.4.To discover efficacy-associated markers by fingerprint-activity relationship modelingThe "fingerprint-activity" relationship modeling between the vasodilative activity and chemical compound of rosemary was carried out by Grey relational analysis,Partial least squares regression and Lasso regression.According to results,(+)-Camphor,Eucalyptol,endo-Bomeol,a-Myrcene,a-Terpineol,Terpinen-4-ol,Homoplantaginin,Camosol,and Carnosic acid could be the active constituents of vasodilative activity,and Camphor,Eucalyptol,and Carnosic acid were proved to be the active compounds by verifying test.In summary,the volatile and nonvolatile ingredients of rosemary were analyzed by GC/MS and UPLC/MS combined with multivariate data analysis.Meanwhile,the vasodilative activity of rosemary was studied and the "fingerprint-activity"relationship was analyzed to discover the active ingredients.