| Euodiae Fructus, known as ‘‘Wu-Zhu-Yu’’ in Chinese, is a traditional herbal medicine derived from the dried nearly mature fruits of three Euodia species, namely, Euodia rutaecarpa(Juss.) Benth., Euodia rutaecarpa(Juss.) Benth var. officinalis(Dode) Huang, and Euodia rutaecarpa(Juss.) Benth. var. bodinieri(Dode) Huang in Chinese Pharmacopoeia,which has been widely used to treat vomit, headache, diarrhea, stomachache and dysmenorrhea for thousands years.However,the three species had been combined in Flora of China(2008,edition). Hence,the Chinese Pharmacopoeia and Flora of China showed differences and contradictions in Euodia species,but the quality evaluation of Euodia rutaecarpa among three species had not been reported.Based on the former research of alkaloid, in this study, we investigated the resources of Euodiae Fructus in China, collecting samples and studied the watersoluble components.And then,conduct a comprehensive quality evaluation to clarify the species and origin specificity of Euodiae Fructus.Besides,the variation in essential oil composition of Euodiae Fructus during different growth stages were also observed.1. Resources investigation and sample collectionThrough the field investigation on twenty-one districts in nine provinces of China,we collected 49 batches,including E. rutaecarpa 14 batches, E. officinalis 13 batches and E. bodinieri 22 batches.Meanwhile, 19 batches during different growth satges were also collected. The resources investigation revealed the main producing areas of Euodiae Fructus are provinces of Jiangxi, Guizhou, Hubei, and Hunan and part of provinces and places of Guangxi, Anhui, Shanxi, and Chongqing. Commercial Euodiae Fructus mainly comes from private investors. The wild resources of Euodiae Fructus are few. The national annual output of cultivated Euodiae Fructus is around 3500 t. The production and marketing is in balance. The price for Euodiae Fructus is relatively stable.2. The study on water-soluble constituents in Euodiae Fructus23 compounds were isolated by various column chromatography, such as VLC, Silica gel, Sephadex LH-20, Flash and HPLC preparative chromatogram from aqueous extract of Euodia rutaecarpa. On basis of spectroscopy technology as well as physical and chemical property analysis, the structures of 22 compounds were identified as follows: limocitrin 3-O-β-D- xylopyranosyl(1→2)-β-D- glucopyranoside(LSS-1**), limocitrin 3-O-[2-O-β-D-xylopyranosyl 6-O-α-L-rhamnopyranosyl]-β-Dglucopyranoside(LSS-13**), limocitrin 3-O-β-D- glucopyranoside(LSS-6), isorhamnetin 3-O-β-D- glucopyranoside(LSS-7), quercetin 3-O-α-D- arabinopyranoside(LSS-8), quercetin 3-O-β-D- glucopyranoside(LSS-2), isorhamnetin 3-O-β-D- xylopyranosyl(1→2)-β-D- glucopyranoside(LSS-3), limocitrin 3-O-rutinoside(LSS-4), isorhamnetin 3-O-rutinoside(LSS-5), quercetin 3-O-β-D-xylopyranosyl(1→2)-β-D-glucopyranoside(LSS-10), isorhamnetin 3-O-[2-O-β-D-xylopyranosyl-6-O-α-L-rhamnopyranosyl]-β-D-glucopyranoside(LSS-11), 5-Ocaffeoylquinic acid(LSS-22), chlorogenic acid(LSS-23), 4-O-caffeoylquinic acid(LSS-25), 6,7-dihydroxy-3,7-dimethyl-octa-2(E),4(E)-dienoic acid(LSS-19), coniferin(LSS-15), caffeic acid(LSS-26), limonin diosphenol 17-β-Dglucopyranoside(LSS-24), dehydroevodiamine(LSS-14), quercetin(LSS-9), hyperin(LSS-27), rutin(LSS-12).Compoud LSS-1,13 were new compounds and LSS-2,3,8,10,11,15,19,22,25 were isolated from Euodia genus for the first time.3. Comparison of the chemical compositions and antimicrobial activities of essential oils among three speciesThe results of essential oil analysis in 27 accessions of Euodia rutaecarpa revealed the main components were β-cis-ocimene, β-Myrcene, limonene& β-Phellandrene, β-trans-ocimene, linalool, Caryophyllene oxide and Caryophyllene,of which limonene& β-Phellandrene, β-cis-ocimene and β-Myrcene varied greatly among these samples.There was no species and origin specificity among three species based on hierarchical clustering analysis(HCA) results.The essential oil of Euodia showed moderate antibacterial activities against Gram-positive bacteria and fungus, especially Bacillus subtilis and Staphylococcus aureus.The antimicrobial activities of selected samples varied according to the chemical components.4. The UPLC fingerprint analysis and assessment on the analgesia and anti- flammation pharmacodynamicsAs a result,17 common peaks were found, representing 59.91% to 80.57% of whole peaks and another 17 peaks including 10 common peaks were identified by comparing the retention time with reference subatance in reference spectrum.The similarity degrees of 49 batches of the samples was ranged 0.602 to 0.967, which indicated large differences among Euodia samples. All the samples were divided into three groups by HCA and PCA, which showed no species and origin specificity. Polar compounds showed major differences in these samples, of which chlorogenic acid was the biggest contributors.The water extract and alcohol extract of three Euodia species showed different levels of analgesic and anti-inflammatory pharmacodynamics, and water extract was more effective. The activities of selected samples varied according to the chemical components.Besides, the maximal dose, i.e. 10 g·kg-1 per day, was safe to mice in acute toxicity test.5. The variation in essential oil composition of Euodiae Fructus during different growth stagesResults suggested the content of monoterpene hydrocarbons, including β-cisocimene, β-Myrcene, limonene&β-Phellandrene, β-trans-ocimene, increased quickly, whereas sesquiterpene hydrocarbons and oxygenated sesquiterpenes were in the declining trend during the whole growth stage. All the components were fastestchanging when petals falling down or a week after that time.According to the above analysis, the chemical compositions among the three species were similar, but the contents varied greatly because of origins, collecting time, growing environment and other factors.The intraspecific differences were greater than or equal to the interspecific differences.The pharmacological activities varied in the light of chemical components, but there was no significant differences among the three species.Therefore,according to above results,that the three species were merged into one in Flora of China was scientific and the combination in Chinese Pharmacopoeia was suggested. |
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