| Identification of efficient constituents, a core content of the modernization of traditional Chinesemedicine (TCM), is always considered as a difficult problem because of the limitation of techniques andmethods. To establish a new pattern that not only covered the theory of TCM but also interpreted theactive constituents with modern science languages, spectrum-effect relationships were applied as a newtool to investigate the identification of TMC in this study. Acalypha australis Linn. and its antibacterialeffect were chosen as the model of TCM and the pharmacodynamic evaluation criterion respectively,UPLC/UV technology and UPLC/MS technology were also applied to establish chemical fingerprints.The overall chemical information and pharmacodynamic data were acquired from the chemicalfingerprints and analysis of the inhibitory effects of various samples originated from different sources.Coupled with chemometric and mathematical statistical methods, the spectrum-effect relationships ofAcalypha australis Linn between chemical fingerprints and antibacterial effects were developed, andthen the characteristics peaks associated with pharmacodynamic effects in the fingerprints were located,and thus the active ingredients were speculated.To further certify its reliability, ingredients isolated and purified from Acalypha australis Linnusing semi-preparative chromatography were located the corresponding characteristic peaks in thechemical fingerprints and thus confirmed the putative active ingredient. Prior to the structures of theantibacterial ingredients were identified by UPLC-Q-TOF, the antibacterial experiments were performed.The main results are as follows:Since the pronounced effects of Acalypha australis Linn. on bacterial diarrhea,Aeromonashydrophila, Aeromonassobria, Vibrio parahaemolyticus, Vibrioanguillarum andStaphylococcusaureus were chosen to evaluate the antibacterial activities of Acalypha australis Linn..Crude extracts of14samples were produced using the optimum extraction process judged by theirantibacterial activity. The results from the antibacterial experiments showed that the magnitude ofantibacterial activity of the Acalypha australis Linn. samples was S8> S3> S4> S7> S12> S11> S1>S13> S5> S2> S10> S9> S6> S14.UPLC/UV fingerprint endowed with fourteen characteristic peaks and UPLC/MS fingerprintprovided with twenty ones were established respectively. UPLC/UV fingerprints and UPLC/MSfingerprints of Acalypha australis Linn. from fourteen samples were acquired to analyze similarityvalues between the reference fingerprint and the chromatograms of fourteen samples and to performhierarchical clustering analysis of samples. The results indicated that the samples could be divided intodifferent clusters according to similarity values and sample exhibiting greater similarity with referencefingerprint might produce stronger antibacterial activity.The differences, which mainly existed in the area of characteristic peaks in the fingerprints and thedegree of antibacterial effects, among the Acalypha australis Linn. samples, depended on species, origin,collection time and extraction methods. The crucial information of―spectrum‖and―effect‖weregathered from the chemical fingerprints and antibacterial effect of fourteen samples. The spectrum-effect relationships were established respectively with direct-viewing comparisons,correlational analysis method and multi-element linear regression (stepwise regression method andpartial least square method). Interestingly, the good consistency in predicting efficient constituentsanalyzed with different methods appeared.5,6,8and9on UPLC/UV fingerprint and Characteristicpeaks1’,2’,6’,7’,8’,9’,10’ and11’on UPLC/MS fingerprint were the putative efficient constituentsof Acalypha australis Linn..Nineteen fractions of Acalypha australis Linn.were separated and purified by semi-preparativeHPLC and then analyzed carefully to locate the corresponding position in the fingerprint. Eight ofthem were the efficient constituents in agreement with the results predicted by the spectrum-effectrelationship, which were belonged to B（4、1’）、C（5、2’）、D（6、6’）、E（7’）、F（8’）、H（8、9’）、K（10’）、L（9、11’）.After the antibacterial effects of these fractions prepared were performed,Fraction C, L were confirmed as the main antibacterial constituents of Acalypha australis Linn. whilefraction D, H and K were acted as the important auxiliary antibacterial constituents of Acalyphaaustralis Linn., but fraction B, E were low antibacterial effects. Either, besides the putative efficientconstituents, the role of fraction M and fraction Q in antibacterial effects played was only minorantibacterial constituents.The structures of nine antibacterial constituents of Acalypha australis Linn. were analyzed byUPLC-Q-TOF. Nicotinic acid, gallicacid, protocatechuicacid, p-hydroxybenzonic acid and quercetinwere confirmed by comparison with their standard substances. However, with the aid of data ofQ-TOF and without standard compound, one compound was inferred as copperleafelement, but themolecular formulas of the remaining three unknown constituents of Acalypha australis Linn. wediscovered for the first time were inferred as C13H8O8, C22H12O13and C27H22O18,respectively.In conclusion, eight antibacterial constituents of Acalypha australis Linn. were predicted byspectrum-effect relationship and their antibacterial effects of seven constituents have been testified. Itwas worth to notice that seven out of nine inhibitory bacterial ingredients obtained by semi-preparativechromatography were in accordance with the corresponding constituents predicted by spectrum-effectrelationship. The results from the analysis of structures demonstrated that6antibacterial constituentswere known compounds of Acalypha australis Linn. and three antibacterial constituents were unknowncompounds we discover first. Taken together, Acalypha australis Linn. was a good example as therepresentative TCM to evidence the feasibility of spectrum-effect relationship in identifying effectiveingredients from the complicated TCM. Our study would also provide the experimental basis for a newpattern that was beneficial to the recognition of efficient constituents of TCM. |
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