Dong-Hui Yang

The profiling and identification of the absorbed constituents and metabolites of Paeoniae Radix Rubra decoction in rat plasma and urine by the HPLC–DAD–ESI-IT-TOF-MSn technique: A novel strategy for the systematic screening and identification of absorbed constituents and metabolites from traditional Chinese medicines

Paeoniae Radix Rubra (PRR, the dried roots of Paeonia lactiflora) is a commonly used traditional Chinese medicine (TCM). A clear understanding of the absorption and metabolism of TCMs is very important in their rational clinical use and pharmacological research. To find more of the absorbed constituents and metabolites of TCMs, a novel strategy was proposed. This strategy was characterized by the following: the establishment and utilization of the databases of parent compounds, known metabolites and characteristic neutral losses; the comparison of base peak chromatograms and ClogPs; and the use of the HPLC-DAD-ESI-IT-TOF-MS(n) technique. This strategy was first applied to screen and identify the absorbed constituents and metabolites of PRR decoction and paeoniflorin in rats. In total, 13 new absorbed constituents and 90 new metabolites of PRR decoction were detected. Among these metabolites, the structures of 70 metabolites were identified, and the conjugation types and structure skeletons of the other 20 metabolites were preliminarily determined. Moreover, 35 new metabolites of some constituents of PRR, i.e., 22 new metabolites of paeoniflorin, 10 new metabolites of gallic acid-related compounds, 1 new metabolite of (epi)catechin-related compounds, and 2 new metabolites of other compounds, were reported for the first time. The results also indicated that (epi)catechin-related compounds, gallic acid-related compounds and paeoniflorin were the main precursors of these metabolites. Phase I reactions (dehydroxylation, decarboxylation, dehydrogenation) and phase II reactions (sulfation, glucuronidation and methylation) were observed as the main metabolic pathways of PRR. According to the literature, the 11 absorbed constituents and 11 metabolites have various bioactivities. This study is the first to explore the absorption and metabolism of PRR decoction, and the result also is a notable improvement in the discovery of paeoniflorin metabolites in vivo. These findings enhance our understanding of the metabolism and Effective forms (the truly active structures) of PRR decoction and paeoniflorin.

ABSORPTION AND METABOLISM OF ASTRAGALI RADIX DECOCTION: IN SILICO, IN VITRO, AND A CASE STUDY IN VIVO

To profile absorption of Astragali Radix decoction and identify its orally absorbable constituents and their metabolites, four complementary in silico, in vitro, and in vivo methods, i.e., a computational chemistry prediction method, a Caco-2 cell monolayer model experiment, an improved rat everted gut sac experiment, and a healthy human volunteer experiment, were used. According to the in silico computation result, 26 compounds of Astragali Radix could be regarded as orally available compounds, including 12 flavonoids. In the in vitro and in vivo experiments, 21 compounds were tentatively identified by high-performance liquid chromatography-diode array detection-electrospray ion trap tandem mass spectrometry data, which involved calycosin, formononetin, (6aR,11aR)-3-hydroxy-9,10-dimethoxypterocarpan, 7,2′-dihydroxy-3′,4′-dimethoxyisoflavan, calycosin-7-O-β-d-glucoside, formononetin-7-O-β-d-glucoside, 7,2′-dihydroxy-3′,4′-dimethoxyisoflavan-7-O-β-d-glucoside-6″-O-malonate, (6aR,11aR)-3-hydroxy-9,10-dimethoxypterocarpan-3-O-β-d-glucoside, and phase II metabolites calycosin-7-O-β-d-glucuronide, formononetin-7-O-β-d-glucuronide, (6aR,11aR)-3-hydroxy-9,10-dimethoxypterocarpan-3-O-β-d-glucuronide, 7,2′-dihydroxy-3′,4′-dimethoxyisoflavan-7-O-β-d-glucuronide, and calycosin sulfate. Calycosin and formononetin were proved absorbable by four methods; (6aR,11aR)-3-hydroxy-9,10-dimethoxypterocarpan and 7,2′-dihydroxy-3′,4′-dimethoxyisoflavan were proved absorbable by three methods; formononetin-7-O-β-d-glucoside and (6aR,11aR)-3-hydroxy-9,10-dimethoxypterocarpan-3-O-β-d-glucoside were proved absorbable by two methods. The existence of calycosin-7-O-β-d-glucuronide, formononetin-7-O-β-d-glucuronide, (6aR,11aR)-3-hydroxy-9,10-dimethoxypterocarpan-3-O-β-d-glucuronide, 7,2′-dihydroxy-3′,4′-dimethoxyisoflavan-7-O-β-d-glucuronide, and calycosin sulfate was proved by two or three methods. We found that besides isoflavones, pterocarpans and isoflavans also could be metabolized by the intestine during absorption, and the major metabolites were glucuronides. In conclusion, the present study demonstrated that the flavonoids in Astragali Radix decoction, including isoflavones, pterocarpans, and isoflavans, could be absorbed and metabolized by the intestine. These absorbable compounds, which were reported to have various bioactivities related to the curative effects of Astragali Radix decoction, could be regarded as an important component of the effective constituents of Astragali Radix decoction.

Production of enterodiol from defatted flaxseeds through biotransformation by human intestinal bacteria

BackgroundThe effects of enterolignans, e.g., enterodiol (END) and particularly its oxidation product, enterolactone (ENL), on prevention of hormone-dependent diseases, such as osteoporosis, cardiovascular diseases, hyperlipemia, breast cancer, colon cancer, prostate cancer and menopausal syndrome, have attracted much attention. To date, the main way to obtain END and ENL is chemical synthesis, which is expensive and inevitably leads to environmental pollution. To explore a more economic and eco-friendly production method, we explored biotransformation of enterolignans from precursors contained in defatted flaxseeds by human intestinal bacteria.ResultsWe cultured fecal specimens from healthy young adults in media containing defatted flaxseeds and detected END from the culture supernatant. Following selection through successive subcultures of the fecal microbiota with defatted flaxseeds as the only carbon source, we obtained a bacterial consortium, designated as END-49, which contained the smallest number of bacterial types still capable of metabolizing defatted flaxseeds to produce END. Based on analysis with pulsed field gel electrophoresis, END-49 was found to consist of five genomically distinct bacterial lineages, designated Group I-V, with Group I strains dominating the culture. None of the individual Group I-V strains produced END, demonstrating that the biotransformation of substrates in defatted flaxseeds into END is a joint work by different members of the END-49 bacterial consortium. Interestingly, Group I strains produced secoisolariciresinol, an important intermediate of END production; 16S rRNA analysis of one Group I strain established its close relatedness with Klebsiella. Genomic analysis is under way to identify all members in END-49 involved in the biotransformation and the actual pathway leading to END-production.ConclusionBiotransformation is a very economic, efficient and environmentally friendly way of mass-producing enterodiol from defatted flaxseeds.

A NEW ALKALOID FROM DYSOXYLUM BINECTARIFERUM

Two compounds (1 and 2) have been isolated from the stem bark of Dysoxylum binectariferum (Roxb.) Hook. f. ex Bedd. Compound 1 is rohitukine {5,7-dihydroxy-2-methyl-8-[4-(3-hydroxy-1-methyl)-piperidinyl]-4H-1-benzopyran-4-one}, a known compound that has anti-inflammatory and immunomodulatory activities. Compound 2 is a new alkaloid, named rohitukine N-oxide, the structure of which was elucidated on the basis of spectral evidences and comparison with the 1H NMR data of rohitukine.

Identification of metabolites in WZS‐miniature pig urine after oral administration of Danshen decoction by HPLC coupled with diode array detection with electrospray ionization tandem ion trap and time‐of‐flight mass spectrometry

Danshen (DS) is a widely used traditional Chinese medicine for treating cardiovascular and cerebrovascular diseases. A simple, rapid and sensitive method was developed for identification of the in vivo metabolites in urine of WZS-miniature pigs after oral administration of DS decoction by HPLC coupled with diode array detection with electrospray ionization tandem ion trap and time-of-flight mass spectrometry. This method has been successfully applied to simultaneous identification of 50 compounds (including 11 new ones) in pig urine. In addition, one new compound, (3-hydroxyphenyl) crylic acid glycine methyl ester (C1), along with eight known ones were first isolated by column chromatography and identified by spectroscopic means, including 1D/2DNMR and mass spectrometry, as reference substances. Ten phenolic compounds (protocatechuic aldehyde, protocatechuic acid, caffeic acid, danshensu, ferulic acid, isoferulic acid, rosmarinic acid and salvianolic acid A/B/D) were found to be the main absorbed original constituents of DS decoction, which underwent the metabolic reactions of glucuronidation, sulfation, methylation, hydrogenation and glycine conjugation in vivo. In conclusion, the developed method is applicable to the analysis and identification of constituents in biological matrices after administration of DS decoction.

The in vivo absorbed constituents and metabolites of Danshen decoction in rats identified by HPLC with electrospray ionization tandem ion trap and time-of-flight mass spectrometry

Danshen, the dried root and rhizome of Salvia miltiorrhiza Bunge, is widely used for the treatment of cardiovascular and cerebrovascular diseases. This research focuses on the in vivo metabolism of Danshen decoction (DSD) in rats. After oral administration of DSD, the absorptive constituents and their metabolites in urine and plasma were analyzed by HPLC coupled with a photodiode array detector and electrospray ionization hybrid ion trap and time-of-flight mass spectrometry. Samples were separated on a C18 column by gradient elution using 0.1% (v/v) aqueous formic acid and acetonitrile. As a result, 93 compounds from urine and 38 compounds from plasma were identified. Among them, lipo-soluble diterpenoids (24 in urine and 15 in plasma) were reported for the first time as in vivo metabolites of DSD. According to the quantities and contents of the identified compounds, tanshinone IIA, cryptotanshinone and tanshinone I were deduced to be the major absorptive diterpenoids of DSD. Moreover, nine water-soluble phenolics (caffeic acid, ferulic acid, danshensu, etc.) were proved to be the major absorptive constituents as reported. Most of the absorbed constituents underwent sulfation, glucuronidation, hydrogenation and hydroxylation in vivo. This investigation provided scientific evidence to obtain a more comprehensive metabolic profile of DSD.

Identification of the absorptive constituents and their metabolites in vivo of Puerariae Lobatae Radix decoction orally administered in WZS-miniature pigs by HPLC-ESI-Q-TOFMS

In this study, the technique of high-performance liquid chromatography coupled with electrospray ionization quadrupole time-of-flight mass spectrometry (HPLC-ESI-Q-TOFMS) was used to analyze and identify the absorptive constituents and their metabolites in drug-containing urine of Wuzhishan (WZS)-miniature pigs administered with Puerariae Lobatae Radix (PLR) decoction. With the accurate mass measurements (<5 ppm) and effective MS(2) fragment ions, 96 compounds, including eight original constituents and 88 metabolites, were identified from the drug-containing urine. Among these, 64 metabolites were new ones and their structures can be categorized into five types: isoflavones, puerols, O-desmethylangolensins, equols and isoflavanones. In particular, puerol-type constituents in PLR were first proved to be absorptive in vivo. Meanwhile, the metabolic pathways of PLR in vivo were investigated. On the basis of relative content of the identified compounds, 13 major metabolites accounting for approximately 50% of the contents, as well as their corresponding 12 prototype compounds, were determined as the major original absorptive constituents and metabolites of PLR in vivo. The HPLC-ESI-Q-TOFMS technique proved to be powerful for characterizing the chemical constituents from the complicated traditional Chinese medicine matrices in this research.

Metabolism of Chuanxiong Rhizoma decoction: Identification of the metabolites in WZS-miniature pig urine

Chuanxiong Rhizoma (CR), a well-known traditional Chinese medicine originated from the rhizome of Ligusticum chuanxiong Hort., was effective for treating various vascular diseases. To identify the metabolites of CR in vivo, the drug-containing urine samples of WZS-miniature pigs after orally administrated CR decoction were collected, after sequential column chromatography 17 metabolites (M1-M17) were isolated from the methanol extract of the urine samples. Their structures, including nine phthalides (M1-M9) and eight phenolic acids (M10-M17), were identified by spectroscopic means. Among them, 8 were new ones (M1-M6, M11-M12). On the basis of the structures of identified metabolites, seven original constituents, including 2 phthalides (senkyunolideI/H) and 5 phenolic acids (ferulic acid, isoferulic acid, caffeic acid, 3-hydroxycinnamoyl acid and 4-hydroxybenzonic acid) were deduced to be the major absorbed original constituents of CR in vivo. This is the first study on the metabolites of CR decoction in non-rodent animal (WZS-miniature pig), the results will give an insight into the metabolism profiles of phthalides and phenolic acids in CR decoction in vivo.