Study On The Guiding Action Of Platycodi Radix In Shengxian Decotion And Its Chemical Constituents

A classical Chinese Formula is a combination of compatible Chinese herbs in fixeddosages according to classical or well-known texts of Traditional Chinese Medicine(TCM), which including one of TCM pharmacological theories---principal (Jun),adjuvant (Chen), assistant (Zuo) and guide (Shi). It has existed for over two thousandyears, and plays an important role in the medical system used in health care and treatmentof diseases. A well-designed formula tailored to the pathophysiologic state of the patient.In a formula, the "guiding drug" which could change other drug's direction or/and location,and then made them concentrated in specially appointed direction or/and location. It ismeaningful to study the "guiding action" of the drug, which has contributed tounderstanding the TCM formula compatibility, new drug exploitation, and rational use ofdrugs in clinical practice.It is a TCM theory that Platycodi Radix acts as a "guiding drug" which assists indelivering the main herb to the organ or meridian. Platycodi Radix plays an important andspecific role in Shengxian Decoction, a famous TCM formula, which was composed ofAstragali Radix, Asphodeloides Anemarrhena, Platycodi Radix, Cimicifugae Rhizoma, andBupleuri Radix. This study established systematic method for identification of mainconstituents and determination of main active constituents in Shengxian Decoction in ratserum, and provided a scientific explanation of "guiding action" for Platycodi Radix. Thecontent includes the following three main parts:The first, myocardial ischemisa was created in rats by ligating the left anteriordescending brach of the coronary artery (LAD), resulting in chronic cardiac failure in thismodel; a metabonomics-based approach to study the anti-chronic cardiac failure activitiesbetween Shengxian Decoction and Shengxian Decoction that removed Platycodi Radix:In a rat model of chronic cardiac failure, metabonomics-based approaches were used to study the function of Shengxian Decoction (SXT), decreased Shengxian Decoction(Platycodi Radix removed, SXT-PG) and Platycodi Radix extraction. In this study, aUPLC-Q-TOF/MS-based metabolomics platform was developed for the qualitativeprofiling of urinary and serum metabonomics in rats, which were randomly assigned infive groups: Sham Surgery group, Model group, positive control group (Betaloc), PGgroup, SXT-PG group, and SXT group. Our method was successful in discriminating thedifferentially processed rats. Both the unsupervised principal component analysis (PCA)and the supervised partial least square-data analysis (PLS-DA) demonstrated strongclassification and clear trajectory patterns with regard to different groups. The PLS-DAresults (37days after oral administration) showed that most of the raw and differentiallyprocessed samples were clearly clustered in the score plot, and SXT group and ShamSurgery group had similar scores clustering together, while SXT-PG group didn't act.Besides, this time-dependent metabolic profiling of SXT group achieved its desiredactivity and clustered near Sham Surgery group in the PLS-DA model.Echocardiography results (35days after oral administration) showed that ejectionfraction (EF) value and shortening fraction (FS) value of ventriculus sinister in SXT-PGgroup and SXT group were larger than those in Model group, which showed no significantdifference from those in PG group. In addition, EF value in SXT group were larger thanthose in Model group (P<0.01), while EF value in SXT-PG group showed no significantdifference from those in Model group and PG group (P>0.05).Serum activities of LDH and CK were determined according to the instructions ofbiochemical reagent. The velocity study results showed that LDH and CK contents inModel group were larger than those in Sham Surgery group (P<0.01), while LDH and CKcontents in SXT group and Betaloc group were smaller than those in Sham Surgery group(P<0.05). Besides, LDH and CK contents in PG group showed no significant differencefrom those in SXT-PG group.The above study verified that Shengxian Decoction (SXT group) exhibited potentanti-chronic cardiac failure activity, but the Shengxian Decoction that removed Platycodi Radix (SXT-PG group) not, which revealed the "guiding action" of the Platycodi Radix. Potential biomarkers related with chronic cardiac failure were identified, and theirmetabolic pathways were also discussed.The second, an UPLC-Q-TOF/MS method was established and validated to analyzethe chemical profiles of Shengxian Decoction and Shengxian Decoction that removedPlatycodi Radix:A rapid UPLC-Q-TOF/MS based chemical profiling method was established andvalidated to qualitative evaluation of Shengxian Decoction. Based on m/z, mass spectrumcomparison and retention time data,36compounds were unambiguously identified asneomangiferin, caffeic acid, mangiferin, isomangiferin, rutin, calycosin-7-O-β-D-glucoside,fukinolic acid, ferulic acid, isoferulic acid, timosaponin E1, timosaponin O (or timosaponinP), timosaponin N, timosaponin B-II, ononin,2-feruloyl-piscidic acid (or2-isoferuloyl-piscidic acid), platycodin D,3"-O-acetylplatycodin D,2,6,4′-trihydroxy-4-methoxybenzophenone,2"-O-acetylplatycodin D, calycosin,timosaponin B, astragaloside IV, anemarrhenasaponin I (or anemarrhenasaponin II),formononetin, saikosaponin C, saikosaponin A,10-hydroxy-3,9-dimethoxy-pterocarpan (or3-hydroxy-9,10-dimethoxy-pterocarpan), timosaponin G (or anemarrhenasaponin III),24-O-acetylhydroshengmanol-3-O-xyloside,1α-hydroxycimigenol-3-O-β-D-galactopyranoside, astragaloside I (or isoastragaloside I),1α-hydroxycimigenol-3-O-β-D-galactopyranoside,7,8-didehydrocimigenol-3-O-α-L-arabinopyranoside, timosaponin A-III,24-O-acetylhydroshengmanol-3-O-xyloside,7,8-didehydrocimigenol. This study indicatedthat astragalosides, isoflavonoids that from Radix Astragali, timosaponins, xanthones thatfrom Anemarrhena Asphodeloides, saikosaponins, platycodins, flavonoids, caffeic acidderivatives were the main compositions of Shengxian Decoction, which was a powerfultool to reveal the chemical profile of Shengxian Decoction. Besides, comparison of thedefference of chemical fingerprints between Shengxian Decoction and ShengxianDecoction that removed Platycodi Radix truly reflected the influence in the presentation ofthe chemical components of other four TCM in Shengxian Decoction by Platycodi Radix. The third, establishement of a sensitive and validated method for determination ofnine active compounds, and a comparetive study of the pharmacokinetics in rat plasmabetween SXT and SXT-PG groups:A sensitive and reliable high performance liquid chromatography-electrosprayionization-tandem mass spectrometry (HPLC-MS/MS) has been developed and validatedfor determination of9main active compounds (formononetin,calycosin-7-O-β-D-glucoside, ononin, caffeic acid, isoferulic acid, mangiferin,timosaponin E1, timosaponin B-II and timosaponin B) of Shengxian Decotion. Plasmasamples were pretreated by protein precipitation with acetonitrile containing formic acid(0.1%, v/v). Ginsenoside Re and puerarin were used as the internal standards. Then treatedsamples were devided into two parts. Two LC separation methods were established foranalyzing each part, respectly. The detection was carried out by a triple-quadrupole tandemmass spectrometer in negative ionization mode. This method was successfully applied topharmacokinetic study of the main active compounds in rats.A comparative pharmacokinetic study was carried out for the main active ingredientsusing the established method between SXT and SXT-PG. The results showed that theprocesses in vivo of mangiferin, timosaponin E1, timosaponin B-II and timosaponin Bwere changed significantly when removed PG from SXT. The shape of concentration-timecurves has a greater difference between the two groups. This may be due to thecompositions fail to distributing to the tissues timely when missing PG in SXT. Theoutcome reflected the corrigent function of Platycodi Radix in SXT at some extent.The last, the chemical investigation of Platycodi Radix:A total of46compounds including3new ones were isolated from the aqueousexaction of Platycodi Radix through various separation techniques and structurallyidentified on the basis of diverse spectroscopic methods including UV, IR, ESI-MS,1Dand2D NMR spectra.35triterpenoids and triterpenoid glycosides as well as11compoundsin other types were obtained. All compounds were listed as follows:1-pentacosanol(Rpg-1), α-spinasterol (Rpg-2),7-stigmasterol (Rpg-3), α-spinasteryl-3-O-β-D-glucoside (Rpg-4), ergosterol peroxide (Rpg-5), platycodonoid A (Rpg-6), β-amyrin (Rpg-7),Δ7-stigmastenol-3-O-β-D-glucoside (Rpg-8), platycodoniod B (Rpg-9),3-O-β-D-glucopyranosyl-polygalacic acid (Rpg-10),3-O-β-D-glucopyranosyl-platycodigenin (Rpg-11),3-O-β-D-laminaribiosyl-polygalacic acid (Rpg-12),hexyl-β-D-glucopyranosyl-(1→2)-β-D-glucopyranoside (Rpg-13), platycoside K (Rpg-14),platycoside L (Rpg-15),2"-O-acetylpolygalacin D (Rpg-16), platycodinA(2"-O-acetylplatycodin D, Rpg-17),2"-O-acetylplatycodin D2(Rpg-18),3"-O-acetylpolygalacin D (Rpg-19), platycoside B (Rpg-20), polygalacin D (Rpg-21),platycoside C (Rpg-22), platycodin C(3"-O-acetylplatycodin D, Rpg-23), polygalacin D2(Rpg-24),3"-O-acetylplatycodin D2(Rpg-25), hexadecanoic acid (Rpg-26), platycosideH(deapio-polygalacin D3, Rpg-27), platycodin D (Rpg-28), platycodin D2(Rpg-29),16-oxo-platycodin D (Rpg-30), platyconic acid A (Rpg-31), platycoside G3(Rpg-32),deapio platycodin D (Rpg-33),3-O-β-D-glucopyranosyl platycodigenin methyl ester(Rpg-34), platycoside P (Rpg-35), deapio-platyconic acid A lactone (Rpg-36), platycosideN (Rpg-37),2"-O-acetylplatycodin D3(Rpg-38), platycodin D3(Rpg-39),3"-O-acetylplatycodin D3(Rpg-40), deapio-platycodin D3(Rpg-41), platycoside D(Rpg-42),6-O-β-D-glucopyranosyl-(1→6)-O-β-D-glucopyranosyl-(1→6)-O-β-D-glucopyranosyl-platycodigenin (Rpg-43), hexitol (Rpg-44), platycoside E (Rpg-45),deapio-platycoside E (Rpg-46). Among these constituents, Rpg-6, Rpg-9, and Rpg-35were identified as new compounds, and Rpg-1, Rpg-5, Rpg-13, and Rpg-44were reportedfrom Platycodi Radix for the first time. The platycodins yielded in this research detailedthe chemical profile of Platycodi Radix, provided standard substance for quality control ofthis herb, and made contribution for the scientific explanation of "guiding action" for thisherb.