Yun-Cheol Na

Liquid chromatography/mass spectrometry-based structural analysis of new platycoside metabolites transformed by human intestinal bacteria.

Platycosides, the main active constituents of Platycodi Radix, have been thoroughly studied for the characterization of their potent biological activities. However, metabolism of platycosides has not yet been characterized. A HPLC electrospray ionization-tandem mass spectrometry (LC/ESI-MS(n)) approach was applied to new complex platycoside metabolites transformed by human intestinal bacteria to identify their structures and determine metabolic pathway. The molecular weights of metabolites were identified by LC/ESI-MS analysis in both positive and negative modes. Structures for the platycoside metabolites were proposed by the molecular weights and the expected enzymatic activity of intestinal microbes on platycoside. In the second step, successive LC-MS(n) analysis was used to demonstrate the proposed structures. Under ESI tandem mass conditions, the sequential fragmentation patterns of [M+Na](+) ions exclusively showed signals, consistent with the cleavage of glycoside bonds, rearrangement and some cross-ring cleavage, thus allowing the rapid identification of platycoside metabolites. The metabolites identified in the time-dependent metabolism experiments enable us to propose several microbial pathways for platycosides. Even though the metabolites of some platycosides may have unknown structures and low levels, the analytical tools presented in this study made it possible to obtain a rapid and complete characterization of new metabolites and their metabolism pathway in human intestinal bacteria.

Development and optimization of a method for the separation of platycosides in Platycodi Radix by comprehensive two-dimensional liquid chromatography with mass spectrometric detection

Comprehensive two-dimensional chromatography (LCxLC) using combinations of two columns (C(18) x CN and C(18) x NH(2)) was employed with electrospray (ESI) mass spectrometry to analyze platycosides from root extract. Based on the capability of the C(18), CN and NH(2) columns to separate the platycosides, the orthogonality in two-dimensional space according to each combination of columns was predicted from the correlation coefficients between the retention times of the 17 compounds separated by the independent CN and C(18) columns, and NH(2) and C(18) columns. The expected distribution of the peaks was also compared with the two-dimensional plots obtained by practical separation in an LCxLC system. The increased peak capacities using C(18) x NH(2) allowed three minor components and five isomers of the platycosides to be newly separated, which were not identified with 1D-LC using the individual C(18) column, whereas the combination of C(18) x CN did not result in any improvement of the separation performance.

The aglycone of ginsenoside Rg3 enables glucagon-like peptide-1 secretion in enteroendocrine cells and alleviates hyperglycemia in type 2 diabetic mice

Ginsenosides can be classified on the basis of the skeleton of their aglycones. Here, we hypothesized that the sugar moieties attached to the dammarane backbone enable binding of the ginsenosides to the sweet taste receptor, eliciting glucagon-like peptide-1 (GLP-1) secretion in the enteroendocrine L cells. Using the human enteroendocrine NCI-H716 cells, we demonstrated that 15 ginsenosides stimulate GLP-1 secretion according to the position of their sugar moieties. Through a pharmacological approach and RNA interference technique to inhibit the cellular signal cascade and using the Gαgust−/− mice, we elucidated that GLP-1 secreting effect of Rg3 mediated by the sweet taste receptor mediated the signaling pathway. Rg3, a ginsenoside metabolite that transformed the structure through a steaming process, showed the strongest GLP-1 secreting effects in NCI-H716 cells and also showed an anti-hyperglycemic effect on a type 2 diabetic mouse model through increased plasma GLP-1 and plasma insulin levels during an oral glucose tolerance test. Our study reveals a novel mechanism where the sugar moieties of ginsenosides Rg3 stimulates GLP-1 secretion in enteroendocrine L cells through a sweet taste receptor-mediated signal transduction pathway and thus has an anti-hyperglycemic effect on the type 2 diabetic mouse model.

A bitter herbal medicine Gentiana scabra root extract stimulates glucagon-like peptide-1 secretion and regulates blood glucose in db/db mouse.

ETHNOPHARMACOLOGICAL RELEVANCE Gentiana scabra root extract (GS) is frequently prescribed as an internal remedy in traditional Korean medicine for treatment of diabetes mellitus. GS contains bitter iridoid glycosides including loganic acid, gentiopicrin, trifloroside, and rindoside. We previously reported that the intestinal bitter taste sensation stimulates GLP-1 secretion, and thereupon hypothesized that the blood glucose regulatory effect of GS is due to its GLP-1 secreting effect in enteroendocrine L cells. MATERIALS AND METHOD We studied GLP-1 secreting effect of GS treatment and its cellular downstream mechanism in human enteroendocrine NCI-H716 cells using the G protein-coupled receptor (GPCR) pathway inhibitors. Intracellular calcium assay also demonstrated the signal transduction pathway stimulated by the GS treatment. Using db/db mice, we performed oral glucose tolerance test (OGTT) to examine the blood glucose lowering effect of GS administration. We also collected the mouse plasma during the OGTT to measure the GLP-1 and insulin levels. RESULT We demonstrated dose-dependent GLP-1 secreting effect of GS on the NCI-H716 cells. The GLP-1 secreting effect of GS is mediated by the G protein βγ-subunit and inositol triphosphate. Using db/db mice, we found that the effect of GS on lowering blood glucose is due to its GLP-1 secretion, and consequential insulinotropic effect. The chemical fingerprint of GS was obtained through a direct analysis in realtime mass spectrometry (DART-MS) and high-performance liquid chromatography (HPLC)/MS. Through the GLP-1 secretion study, we found that loganic acid, an iridoid glycoside, contributes to the GLP-1 secreting effect of GS. CONCLUSION The findings of this study highlight the potential of exploiting the antidiabetic effect of GS on type 2 diabetes mellitus patients.

Effects of β-sitosterol derived from Artemisia capillaris on the activated human hepatic stellate cells and dimethylnitrosamine-induced mouse liver fibrosis

Backgroundβ-sitosterol is a cholesterol-like phytosterol, which widely distributed in the plant kingdom. Here, anti-fibrotic effect of the β-sitosterol was studied using the activated human hepatic stellate cell (HSC) model and dimethylnitrosamine (DMN)-induced mouse hepatic fibrosis model.MethodHSCs were activated by transforming growth factor-β (TGF-β) and the collagen-1 and α-smooth muscle actin (α-SMA) expressions were measured at the mRNA and protein level. We also studied the effect β-sitosterol using DMN-induced mouse hepatic fibrosis model. We then measured the collagen-1 and α-SMA expression levels in vivo to investigate anti-hepatofibrotic effect of β-sitosterol, at both of the mRNA and protein level.Resultsβ-sitosterol down regulated the mRNA and protein expression levels of collagen-1 and α-SMA in activated HSC. Oral administration of the β-sitosterol successfully alleviated the DMN-induced mouse liver damage and prevented collagen accumulation. The mRNA and protein expression levels of collagen-1 and α-SMA were also down regulated in β-sitosterol treated mouse group.ConclusionsThis study shows the effect of β-sitosterol on the TGF-β -or DMN-induced hepatofibrosis. Hence, we demonstrate the β-sitosterol as a potential therapeutic agent for the hepatofibrosis.

Effects of the inhaled treatment of liriope radix on an asthmatic mouse model.

As a treatment for allergic asthma, inhaled treatments such as bronchodilators that contain β2-agonists have an immediate effect, which attenuates airway obstructions and decreases airway hypersensitivity. However, bronchodilators only perform on a one off basis, but not consistently. Asthma is defined as a chronic inflammatory disease of the airways accompanying the overproduction of mucus, airway wall remodeling, bronchial hyperreactivity and airway obstruction. Liriope platyphylla radix extract (LPP), a traditional Korean medicine, has been thoroughly studied and found to be an effective anti-inflammatory medicine. Here, we demonstrate that an inhaled treatment of LPP can attenuate airway hyperresponsiveness (AHR) in an ovalbumin-induced asthmatic mouse model, compared to the saline-treated group (p < 0.01). Moreover, LPP decreases inflammatory cytokine levels, such as eotaxin (p < 0.05), IL-5 (p < 0.05), IL-13 (p < 0.001), RANTES (p < 0.01), and TNF-α (p < 0.05) in the bronchoalveolar lavage (BAL) fluid of asthmatic mice. A histopathological study was carried out to determine the effects of LPP inhalation on mice lung tissue. We performed UPLC/ESI-QTOF-MS, LC/MS, and GC/MS analyses to analyze the chemical constituents of LPP, finding that these are ophiopogonin D, spicatoside A, spicatoside B, benzyl alcohol, and 5-hydroxymethylfurfural. This study demonstrates the effect of an inhaled LPP treatment both on airway AHR and on the inflammatory response in an asthmatic mouse model. Hence, LPP holds significant promise as a nasal inhalant for the treatment of asthmatic airway disease.

Antiproliferative Effects of Dangyuja (Citrus grandis Osbeck) Leaves Through Suppression of Constitutive Signal Transducer and Activator of Transcription 3 Activation in Human Prostate Carcinoma DU145 Cells

Although Dangyuja (Citrus grandis Osbeck) exhibits anti-inflammatory and anticancer activities, its molecular targets and pathways, especially in human prostate cancer cells, are not fully understood. In this study, the antiproliferative effect of Dangyuja leaves through the signal transducer and activator of transcription (STAT) 3 signaling pathway was investigated in human prostate carcinoma DU145 cells. The solvent fractions (n-hexane, chloroform, ethyl acetate, and n-butanol) were obtained from a crude extract (80% methanol extract) of Dangyuja leaves. We first found that the chloroform fraction of Dangyuja leaves (DCF) was the most cytotoxic against DU145 cells. DCF inhibited constitutive STAT3 activation through blocking upstream Janus-like kinase 2 and c-Src. Consistent with STAT3 inactivation, DCF down-regulated the expression of STAT3 target genes, including bcl-2, bcl-xl, and cyclin D1; this correlated with the suppression of proliferation, the accumulation of cell cycle at the sub-G(1) phase, and the induction of apoptosis. Furthermore, DCF exerted a relatively minor effect on the growth of human prostate noncancerous RWPE-1 cells. Nobiletin, a major active constituent of DCF, could induce apoptosis via the suppression of constitutive STAT3 activation. Overall, our results indicate that the anti-inflammatory and anticancer activities previously assigned to DCF may be mediated partially through the suppression of the STAT3 signaling.

Glycosylated platycosides: identification by enzymatic hydrolysis and structural determination by LC-MS/MS.

In this study, enzymatic hydrolysis and chemometric methods were utilized to discriminate glycosylated platycosides in the extract of Platycodi Radix by LC-MS. Laminarinase, whose enzymatic activity was evaluated using gentiobiose and laminaritriose, was a suitable enzyme to identify the glycosylated platycosides. The laminarinase produced deapi-platycodin D and platycodin D from the isolated deapi-platycoside E and platycoside E through the loss of two glucose units by enzymatic reaction, respectively. After hydrolyzing a crude extract by laminarinase, the reconstructed total ion chromatogram generated by a chemometric technique sorted peaks of deglycosylated platycosides easily. Structural information of the glycosylated isomers was revealed through fragment ions generated by the sodiated C0β ion corresponding to reduced disaccharides in the positive MS(4) spectra. Characteristic fragment ions of Glc-(1→6)-Glc moieties were observed through ring cleavages of (0,2)A0β, (0,3)A0β, and (0,4)A0β, whereas Glc-(1→3)-Glc moieties produced only (0,3)A0β ions. Lithium-adducted platycosides allowed more detailed structural analysis of glycosidic bond cleavage corresponding to Y1β and B1β in addition to ring cleavage.

Metabolic Profiling of Liver Tissue in Diabetic Mice Treated with Artemisia Capillaris and Alisma Rhizome Using LC-MS and CE-MS.

Artemisia Capillaris (AC) and Alisma Rhizome (AR) are natural products for the treatment of liver disorders in oriental medicine clinics. Here, we report metabolomic changes in the evaluation of the treatment effects of AC and AR on fatty livers in diabetic mice, along with a proposition of the underlying metabolic pathway. Hydrophobic and hydrophilic metabolites extracted from mouse livers were analyzed using HPLC-QTOF and CE-QTOF, respectively, to generate metabolic profiles. Statistical analysis of the metabolites by PLS-DA and OPLA-DA fairly discriminated between the diabetic, and the AC- and AR-treated mice groups. Various PEs mostly contributed to the discrimination of the diabetic mice from the normal mice, and besides, DG (18:1/16:0), TG (16:1/16:1/20:1), PE (21:0/20:5), and PA (18:0/21:0) were also associated with discrimination by s-plot. Nevertheless, the effects of AC and AR treatment were indistinct with respect to lipid metabolites. Of the 97 polar metabolites extracted from the CE-MS data, 40 compounds related to amino acid, central carbon, lipid, purine, and pyrimidine metabolism, with [Formula: see text] values less than 0.05, were shown to contribute to liver dysregulation. Following treatment with AC and AR, the metabolites belonging to purine metabolism preferentially recovered to the metabolic state of the normal mice. The AMP/ATP ratio of cellular energy homeostasis in AR-treated mice was more apparently increased ([Formula: see text]) than that of AC-treated mice. On the other hand, amino acids, which showed the main alterations in diabetic mice, did not return to the normal levels upon treatment with AR or AC. In terms of metabolomics, AR was a more effective natural product in the treatment of liver dysfunction than AC. These results may provide putative biomarkers for the prognosis of fatty liver disorder following treatment with AC and AR extracts.

Antihyperglycemic and Antiobesity Effects of JAL2 on db/db Mice

Lonicera japonica Thunb. (LJT) and Rehmannia glutinosa Libosch. (RGL) have been used traditionally as a herbal medicine in Korean medicine. Using LC/Q-TOF was performed to profile the two herbal medicines and the mixture of LJR and RGL (JAL2, ratio 1 : 1). We performed oral glucose tolerance test (OGTT) and plasma GLP-1 and insulin secretion by multiplex assays to investigate antidiabetic effects of LJT, RGL, and JAL2 in db/db mice, the mice model of type 2 diabetes mellitus (T2DM). Also, the antiobesity-related factors such as plasma peptide YY (PYY), triglyceride, total cholesterol, HDL, LDL, and weight of liver, epididymal, and retroperitoneal fat tissue were investigated. Through the multiplex assay, it was found that JAL2 treatment more efficiently attenuated high levels of blood glucose by stimulating GLP-1 secretion and reduced LDL concentration and weight of liver and retroperitoneal fat tissue compared to LJT or RGL treated separately. These results suggest that the JAL2 has antidiabetes and antiobesity effects in T2DM mice model.