Studies On Active Components Of The Medicinal Plants Erycibe Schmidtii And Lonicera Japonica And The Discrimination Of Their Adulterants

The medicinal plants Erycibe schmidtii Craib and Lonicera japonica Thunb.are widely used as traditional Chinese medicine.E.schmidtii,a perennial vine of Erycibe in the family Convolvulaceae,is mainly used to rheumarthritis,rheumatoid arthritis,sciatica and other inflammatory diseases.Recently,with reduction of wild E.schmidtii sources,at least nine adulterants have emerged in herbal markets.However,the studies on the compositions and anti-inflammatory activities of E.schmidtii and its adulterants are still not in-depth.Moreover,the resource of E.schmidtii is scarce,so a substitute is needed to alleviate this shortage.Which of these adulterants can replace E.schmidtii,their anti-inflammatory indicators and anti-inflammatory mechanism are all not clear.L.japonica is a perennial vine of Lonicerae in Caprifoliaceae,also known as honeysuckle,and widely used in antibacterial,antioxidant,anti-inflammatory and anti-cancer.Since 2010,the Chinese Pharmacopoeia has classified the dried flower buds of L.macranthoides,L.hypoglauca,L.confusa,and L.fulvotomentosa as“Shanyinhua”,and dried buds of L.japonica individually as“Jinyinhua”.Before that,however,all the above species were called as“Jinyinhua”.The studies on Jinyinhua and Shanyinhua are mostly focused on the comparison of the characteristic compounds,and the research on other differential metabolites is rare.Moreover,L.japonica is also an important ornamental plant because its flower color transits from the initial green to silvery and finally golden,and the silvery and golden flowers set each other off beautifully.However,little is known about the metabolic and molecular information of the change of its decors.The purpose of this study is?1?to screen out effective substitutes of E.schmidtii through determining and comparing the components,antioxidant and anti-inflammatory activities of E.schmidtii and its adulterants,and establishing their HPLC fingerprints.?2?to identify anti-inflammatory indicators of substitutes through the separation and purification of the crude extract of the substitute by macroporous resin,as well as the determination of material composition,antioxidant and anti-inflammatory activity.?3?to determine the different metabolites of L.japonica and L.macranthoides flowers by UPLC–Q Exactive Orbitrap–MS/MS and GC-MS.?4?to uncover the molecular and material basis of pigmentation in L.japonica flowers at different developmental stages by transcriptome and target metabolome analysis.The main results of this study are as follows:?1?Compared with E.schmidtii?S1?,Porana sinensis?S2?,Porana sinensis var.delavayi?S3?,Celastrus hindsii?S4?,and Morinda umbellata?S5?exhibited similar or higher total phenols,flavonoids and tannins along with similar or greater capacities scavenging DPPH^?,ABTS^+?and AAPH^?,contained similar or higher total amounts of the three marker compounds,and possessed higher similarities in HPLC profiles.The other five adulterants?S6–S10,i.e.,Illigera parviflora,Morinda parvifolia,Piper puberulum,Piper kadsura and Iodes seguini in sequence?were identified as absolute fakes,thus were excluded from alternatives of S1.Further anti-inflammatory experiments with LPS-induced RAW264.7 macrophages cells on NO release and transcriptions of inflammatory factors i NOS,COX-2,IL-6 and IL-1b showed that S2 and S3 possessed higher anti-inflammation activities than and similar mechanisms to those of S1.Taken together,S2 and S3 could be the best potentially alternatives of E.schmidtii among the nine adulterants.?2?P.sinensis var.delavayi is a variety of P.sinensis,and the main adulterants of E.schmidtii is also P.sinensis in the market.Therefore,we have made a further study on P.sinensis.Twenty-three phenolics were identified by UPLC-QTOF-MS/MS from crude extract?CE?prepared optimally with 80%methanol.Further fractionalization using D101 macroporous resin resulted in predominant enrichment of total phenols and flavonoids into Fr.II.Correspondingly,the bioactive components-enriched Fr.II exhibited the lowest IC50 for scavenging DPPH and ABTS and the highest oxygen radical absorbance capacity or ORAC followed by Fractions Fr.I+Fr.II,CE and Fr.I,implying that certain phenolics possessing lower antioxidant activity completely remained in CE.Anti-inflammatory tests with LPS-stimulated RAW264.7 cells showed that CE possessed the highest inhibition of NO-production followed by Fr.II and Fr.I,meaning that CE might contain compounds that expressed higher anti-inflammatory but lower antioxidant activities or possessed synergistic interactions but were not fractionated together.Quantitative determination of nine major phenolics revealed that caffeic acid and 3-,4-and 5-caffeoylquinic acids were concentrated into Fr.I,whereas scopolin,scopoletin and 3,5-,3,4-and 4,5-dicaffeoylquinic acids were enriched into Fr.II.Further experiments with three selected major phenolics reduced the proposed synergistic interactions.Anti-inflammatory tests of the nine major phenolics evidenced that caffeic acid and the six caffeoylquinic acids produced higher,and the three dicaffeoylquinic acids at 140??showed even more significant activities in suppressing NO production and m RNA expression of i NOS,TNF-?,COX-2,and IL-6,suggesting that these three dicaffeoylquinic acids could be indicators of the anti-inflammatory potential of P.sinensis stem.?3?The methanol extracts and volatile oils of L.japonica and L.macranthoides flowers were obtained by ultrasonic assisted extraction and supercritical CO₂ extraction,respectively.The results showed that the contents of total phenols,flavonoids,and antioxidant activities of DPPH,FRAP,Fe²⁺chelating ability of the methanol extract of L.macranthoides flowers were significantly higher than those of L.japonica flowers,while the phenolic contents and antioxidant activities of the essential oil of L.macranthoides flowers were significantly lower than those of L.japonica flowers.Fifty-four compounds were identified in the two methanol extracts using UPLC–Q Exactive Orbitrap–MS.And the results of OPLS-DA analysis showed that macranthoidin B,macranthoidin A,macranthoside B,asperosaponin VI,secologanic acid and secologanin had the largest difference between L.japonica and L.macranthoides flowers,and were recommended to distinguish them.Sixty-four volatile compounds were identified by GC-MS from oils of L.japonica and L.macranthoides flowers.And the results of OPLS-DA analysis showed that dioctyl phthalate,2-phenylethyl benzoate,1-monolinoleoylglycerol trimethylsilyl ether,tocopherol acetate,isofucosterol and 3-phenylbutyric acid could be used to distinguish them.?4?To explore the molecular and metabolic bases of pigmentation in L.japonica flowers at different developmental stages,we profiled the transcriptome at six developmental stages?S1–S6?and constructed regulatory networks of anthocyanin biosynthesis,chlorophyll metabolism,and carotenoid biosynthesis by weighted gene co-expression network analysis?WGCNA?.The transcriptomics results demonstrated that the early flower colors were caused by anthocyanins and chlorophylls,and the late golden flower?S6?by carotenoids rather than anthocyanins.To identify the carotenoids responsible for the golden flowers,targeted metabolomics profiling of the flowers at S1,S4,and S6 was performed.The results showed that the golden flowers contained the highest contents of 11 of the 14 detectable carotenoids,and the total content of?-carotene,?-carotene,and zeaxanthin accounted for 79.6%of the content of the 13 carotenoids detected at S6,indicating that they are major contributors to the golden flowers.