Ming-Ying Shang

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.

Chemical Constituents from the Rhizomes of Smilax glabra and Their Antimicrobial Activity

Six new phenolic compounds, named smiglabrone A (1), smiglabrone B (2), smilachromanone (3), smiglastilbene (4), smiglactone (5), smiglabrol (6), together with fifty-seven known ones 7–63were isolated from the rhizomes of Smilax glabra. Their structures were elucidated on the basis of extensive spectroscopic analyses, as well as by comparison with literature data. Twenty-seven of these compounds were obtained from and identified in the genus Smilax for the first time. The absolute configuration of (2S)-1,2-O-di-trans-p-coumaroylglycerol (43) was determined for the first time using the exciton-coupled circular dichroism (ECCD) method. Thirty isolated compounds were evaluated for their antimicrobial activity against three Gram-negative bacteria, three Gram-positive bacteria and one fungus, and the corresponding structure-activity relationships were also discussed. Eighteen compounds were found to be antimicrobial against the microorganisms tested and the minimum inhibitory concentrations (MIC) were in the range of 0.0794–3.09 mM. Among them, compound 1 showed antimicrobial activity against Canidia albicans with MIC value of 0.146 mM, which was stronger than cinchonain Ia with an MIC of 0.332 mM. Compounds 3 and 4 exhibited inhibitory activity against Staphylococcus aureus with MIC values of 0.303 and 0.205 mM, respectively. The results indicated that these antimicrobial constituents of this crude drug might be responsible for its clinical antimicrobial effect.

Astragaloside IV synergizes with ferulic acid to inhibit renal tubulointerstitial fibrosis in rats with obstructive nephropathy

BACKGROUND AND PURPOSE The combination of Chinese herbs, Astragali Radix and Angelicae Sinensis Radix, could alleviate renal interstitial fibrosis. Astragaloside IV (AS‐IV) and ferulic acid (FA) are the two major active constituents in this combination. In this study, we employed rats with unilateral ureteral obstruction to determine whether AS‐IV and FA have the same renoprotective effects and investigated the mechanisms of this action.

Trollioside, a new compound from the flowers of Trollius chinensis

A new compound, named trollioside, together with nine known compounds, proglobeflowery acid, isoswertisin, isoswertiajaponin, cirsimaritin, veratric acid, vitexin, orientin, β-sitosterol and daucosterol, were isolated from the ethanol extract of the dried flowers of Trollius chinensis Bunge. The isolation and structural determination of these compounds are discussed.

Alkaloids from the seeds of Sterculia lychnophora (Pangdahai)

Two alkaloids, named sterculinine I and sterculinine II, together with thirteen known compounds were isolated from the ethanol extract of a well-known Chinese traditional drug, Pangdahai (the seeds of Sterculia lychnophora Hance). Their structures were elucidated by NMR, UV, IR and MS spectroscopic analysis.

Exploring in vitro, in vivo metabolism of mogroside V and distribution of its metabolites in rats by HPLC-ESI-IT-TOF-MSn

Mogroside V, a cucurbitane-type saponin, is not only the major bioactive constituent of traditional Chinese medicine Siraitiae Fructus, but also a widely used sweetener. To clarify its biotransformation process and identify its effective forms in vivo, we studied its metabolism in a human intestinal bacteria incubation system, a rat hepatic 9000g supernatant (S9) incubation system, and rats. Meanwhile, the distribution of mogroside V and its metabolites was also reported firstly. Seventy-seven new metabolites, including 52 oxidation products formed by mono- to tetra- hydroxylation/dehydrogenation, were identified with the aid of HPLC in tandem with ESI ion trap (IT) TOF multistage mass spectrometry (HPLC-ESI-IT-TOF-MS(n)). Specifically, 14 metabolites were identified in human intestinal bacteria incubation system, 4 in hepatic S9 incubation system, 58 in faeces, 29 in urine, 14 in plasma, 34 in heart, 33 in liver, 39 in spleen, 39 in lungs, 42 in kidneys, 45 in stomach, and 51 in small intestine. The metabolic pathways of mogroside V were proposed and the identified metabolic reactions were deglycosylation, hydroxylation, dehydrogenation, isomerization, glucosylation, and methylation. Mogroside V and its metabolites were distributed unevenly in the organs of treated rats. Seven bioactive metabolites of mogroside V were identified, among which mogroside IIE was abundant in heart, liver, spleen and lung, suggesting that it may contribute to the bioactivities of mogroside V. Mogroside V was mainly excreted in urine, whereas its metabolites were mainly excreted in faeces. To our knowledge, this is the first report that a plant constituent can be biotransformed into more than 65 metabolites in vivo. These findings will improve understanding of the in vivo metabolism, distribution, and effective forms of mogroside V and congeneric molecules.

A-Type Proanthocyanidins from the Stems of Ephedra sinica (Ephedraceae) and Their Antimicrobial Activities

Phytochemical investigation of the n-BuOH-soluble fraction of the EtOH extract of the herbaceous stems of Ephedra sinica, which is known as Ephedrae Herba in Traditional Chinese Medicine, led to the isolation and identification of 12 A-type proanthocyanidins, containing five dimers, two trimers and five tetramers [i.e., (+)-epigallocatechin-(2α→O→7,4α→8)-(-)-catechin, named ephedrannin D1, a dimer; epigallocatechin-(2α→O→7,4α→8)-epigallocatechin-(4α→8)-catechin (ephedrannin Tr1), a trimer; and epigallocatechin-(2α→O→7,4α→8)-epigallocatechin-(4α→8)-epigallocatechin-(2α→O→7,4α→8)-gallocatechin, named ephedrannin Te1, a tetramer). Tetramers composed of gallocatechin are reported for the first time in Ephedraceae. Catechin, epicatechin, gallocatechin, epigallocatechin and four known dimers were also isolated. The structures were elucidated by extensive spectroscopic analysis. The absolute configurations of the 4α linkages, which were confirmed by NOESY and CD experiments, are the outstanding characteristic of most of these isolated A-type proanthocyanidins. The antimicrobial activities of these compounds were tested by measuring the minimum inhibitory concentrations (MIC) against bacteria (both Gram positive and Gram negative) and fungi, and were found to be in the range of 0.00515–1.38 mM. Compounds 6, 8, 10 and 11 exhibited moderate antimicrobial activities against Canidia albicans.

Altered Expression of Genes Profiles Modulated by a Combination of Astragali Radix and Angelicae Sinensis Radix in Obstructed Rat Kidney

The decoction of a combination of two Chinese herbs, Astragali Radix (the roots of Astragalus membranaceus var. mongholicus) and Angelicae Sinensis Radix (the roots of Angelica sinensis), here named as A&A, has been demonstrated to have renoprotective effects in several animal models and may be considered as a complementary therapeutic medicine for chronic kidney disease. In this study, genomic approaches were employed to identify expression signatures in the obstructed kidney, which may be linked to the molecular actions associated with anti-fibrotic effects of A&A. Ninety-six male Wistar rats were divided randomly into sham, SAA (sham + A&A), UUO (unilateral ureteral obstruction), and UAA (UUO + A&A) groups. The rats in the SAA and UAA groups were administered A&A (14 g/kg) by oral gavage once daily; the ones in the sham and UUO groups were given equal volumes of water. Eight rats from each group were sacrificed at days 3, 7, and 10 after the operation, respectively. Changes in gene expression in the kidneys were determined using Affymetrix RAE-230A GeneChips. The differential expression of known genes between UAA and UUO was confirmed by RT-PCR. The results revealed that 40, 65, and 104 genes were upregulated and 30, 36, and 40 genes downregulated in UUO compared with the sham group at days 3, 7, and 10, respectively. Compared to the UUO group, eight genes were upregulated and two genes were downregulated at day 3 in the UAA group, and two genes were upregulated at day 10. These genes included transient receptor protein 3 (TRP3), bone marrow stromal cell antigen 1 (BST-1), peroxisomal biogenesis factor 6 (PEX6), xanthine dehydrogenase (XDH), cytochrome P450 subfamily I member A1 (CYP1A1), serine/cysteine proteinase inhibitor clade E member1 (PAI-1), fibroblast growth factor 23 (FGF23), and five ESTs. Among these genes, differential expression of PAI-1, FGF23, and CYP1A1 were further confirmed by RT-PCR. These data provide the evidence that the anti-fibrotic effects of A&A are mediated through multiple pathways in obstructive nephropathy, and novel mechanisms may be involved in the increasing degeneration of ECM, decreasing ROS reaction, and regulation of the calcium-phosphate metabolism.

The profiling and identification of the metabolites of (+)-catechin and study on their distribution in rats by HPLC- DAD-ESI-IT-TOF-MS n technique

(+)-Catechin, a potential beneficial compound to human health, is widely distributed in plants and foods. A high-performance liquid chromatography with diode array detector and combined with electrospray ionization ion trap time-of-flight multistage mass spectrometry method was applied to profile and identify the metabolites of (+)-catechin in rats and to study the distribution of these metabolites in rat organs for the first time. In total, 51 phase II metabolites (44 new) and three phase I metabolites were tentatively identified, comprising 16 (+)-catechin conjugates, 14 diarylpropan-2-ol metabolites, 6 phenyl valerolactone metabolites and 18 aromatic acid metabolites. Further, 19 phase II metabolites were new compounds. The in vivo metabolic reactions of (+)-catechin in rats were found to be ring-cleavage, sulfation, glucuronidation, methylation, dehydroxylation and dehydrogenation. The numbers of detected metabolites in urine, plasma, small intestine, kidney, liver, lung, heart, brain and spleen were 53, 23, 27, 9, 7, 5, 3, 2 and 1, respectively. This indicated that small intestine, kidney and liver were the major organs for the distribution of (+)-catechin metabolites. In addition, eight metabolites were found to possess bioactivities according to literature. These results are very helpful for better comprehension of the in vivo metabolism of (+)-catechin and its pharmacological actions, and also can give strong indications on the effective forms of (+)-catechin in vivo.