Hye Sook Yun-Choi

Insulin-mimetic and insulin-sensitizing activities of a pentacyclic triterpenoid insulin receptor activator.

Five pentacyclic triterpenoids isolated from Campsis grandiflora were tested for insulin-mimetic and insulin-sensitizing activity. The compounds enhanced the activity of insulin on tyrosine phosphorylation of the IR (insulin receptor) beta-subunit in CHO/IR (Chinese-hamster ovary cells expressing human IR). Among the compounds tested, CG7 (ursolic acid) showed the greatest enhancement and CG11 (myrianthic acid) the least. We characterized the effect of CG7 further, and showed that it acted as an effective insulin-mimetic agent at doses above 50 mug/ml and as an insulin-sensitizer at doses as low as 1 mug/ml. Additional experiments showed that CG7 increased the number of IRs that were activated by insulin. This indicates that a major mechanism by which CG7 enhances total IR auto-phosphorylation is by promoting the tyrosine phosphorylation of additional IRs. CG7 not only potentiated insulin-mediated signalling (tyrosine phosphorylation of the IR beta-subunit, phosphorylation of Akt and glycogen synthase kinase-3beta), but also enhanced the effect of insulin on translocation of glucose transporter 4 in a classical insulin-sensitive cell line, 3T3-L1 adipocytes. The results of the present study demonstrate that a specific pentacyclic triterpenoid, CG7, exerts an insulin-sensitizing effect as an IR activator in CHO/IR cells and adipocytes. The enhancement of insulin activity by CG7 may be useful for developing a new class of specific IR activators for treatment of Type 1 and Type 2 diabetes.

Anti-platelet effect of the constituents isolated from the Barks and fruits of Magnolia obovata

In the course of our work on anti-platelet constituents from plants, five phenolic compounds, mag-nolol, honokiol, obovatol, methyl caffeate, and syringin, were isolated from the methanol extracts of the barks and fruits ofMagnolia obovata. The compounds were identified based on the spectro-scopic data. Methyl caffeate was isolated for the first time from the genusMagnolia and it showed 3 – 4-folds higher potency than ASA. The activities of obovatol and honokiol were comparable to ASA. Magnolol and syringin showed only very mild inhibitory effects to all the stimulators.

HO-1 and JAK-2/STAT-1 signals are involved in preferential inhibition of iNOS over COX-2 gene expression by newly synthesized tetrahydroisoquinoline alkaloid, CKD712, in cells activated with lipopolysacchride

We found that CKD712, an S enantiomer of YS49, strongly inhibited inducible nitric oxide synthase (iNOS) and NO induction but showed a weak inhibitory effect on cyclooxygenase-2 (COX-2) and PGE(2) induction in LPS-stimulated RAW 264.7 cells. We, therefore, investigated the molecular mechanism(s) responsible for this by using CKD712 in LPS-activated RAW264.7 cells. Treatment with either SP600125, a specific JNK inhibitor or TPCK, a NF-kappaB inhibitor, but neither ERK inhibitor PD98059 nor p38 inhibitor SB203580, significantly inhibited LPS-mediated iNOS and COX-2 induction. CKD712 inhibited NF-kappaB (p65) activity and translocation but failed to prevent JNK activation. However, AG490, a specific JAK-2/STAT-1 inhibitor, efficiently prevented LPS-mediated iNOS induction but not the induction of COX-2, and CKD712 completely blocked STAT-1 phosphorylation by LPS, suggesting that the NF-kappaB and JAK-2/STAT-1 pathways but not the JNK pathway are important for CKD712 action. Interestingly, CKD712 induced heme oxygenase 1 (HO-1) gene expression in LPS-treated cells. LPS-induced NF-kappaB and STAT-1 activation was partially prevented by HO-1 overexpression. Furthermore, HO-1 siRNA partly reversed not only the LPS-induced NF-kappaB activation and STAT-1 phosphorylation but also inhibition of these actions by CKD 712. Additionally, silencing HO-1 by siRNA prevented CKD712 from inhibiting iNOS expression but not COX-2. When examined plasma NO and PGE(2) levels and iNOS and COX-2 protein levels in lung tissues of mice injected with LPS (10 mg/kg), pretreatment with CKD712 greatly prevented NO and iNOS induction in a dose-dependent manner and slightly affected PGE(2) and COX-2 production as expected. Taken together, we conclude that inhibition of JAK-2/STAT-1 pathways by CKD 712 is critical for the differential inhibition of iNOS and COX-2 by LPS in vitro and in vivo where HO-1 induction also contributes to this by partially modulating JAK-2/STAT-1 pathways.

Platelet anti-aggregatory effects of coumarins from the roots ofAngelica genuflexa andA. gigas

Five coumarins, isoimperatorin (1), pabulenol (2), isooxypeucedanin (3), oxypeucedanin hydrate (4) and osthol (5) were isolated from the MeOH extract ofAngelica genuflexa in the course of searching for anti-platelet and anti-coagulant components from plants. Pabulenol (2) was isolated fromA. genuflexa for the first time. The five compounds isolated fromA. genuflexa, together with decursinol angelate (6), decursin (7) and nodakenin (8) fromA. gigas were evaluated for their effects on platelet aggregation and blood coagulation. Compounds 2, 5, 6 and 7 were observed to be either equally effective or 2~4 times more inhibitory than ASA in both arachidonic acid and U46619 (TXA2 mimetic) induced platelet aggregations.

Anti-platelet effects of flavonoids and flavonoid-glycosides from Sophora japonica

A methanol extract of Sophora japonica was subjected to anti-platelet activity guided fractionation affording the isolation of four flavonoids and six flavonoid-glycosides: biochanin A (1), irisolidone (2), genistein (3), sissotrin (4), sophorabioside (5), genistin (6), tectoridin (7), apigenin (8), quercitrin (9), and rutin (10). The structure of each compound was determined by a variety of spectroscopic methods. Among the compounds, 1, 3, and 7 showed ∼2.5–6.5 fold greater inhibitory effects on arachidonic acid (AA) and U46619 induced platelet aggregation (IC50: 19.9 and 99.8 μM; 20.3 and 53.8 μM; 25.9 and 123.4 μM, respectively) than acetylsalicylic acid (ASA, IC50: 63.0 and 350.0 μM). Compound 2 was an ∼22–40 fold stronger inhibitor than ASA on AA and U46619 induced aggregation (IC50: 1.6 and 15.6 μM, respectively).

Antiplatelet and Antithrombotic Effects of a Combination of Ticlopidine and Ginkgo biloba Ext (EGb 761)

The antiplatelet and antithrombotic effects of the oral combination treatment of ticlopidine and Ginkgo biloba extract (EGb 761) were studied in normal and thrombosis-induced rats. The ex vivo inhibitory effect on ADP-induced platelet aggregation of a small dose of ticlopidine (50 mg/kg/day) in combination with EGb 761 (40 mg/kg/day) was comparable to a larger dose of only ticlopidine (200 mg/kg/day). Bleeding time was also prolonged by 150%. Thrombus weight was also consistently decreased by a combination of ticlopidine and EGb 761 in an arterio-venous shunt model at two doses of ticlopidine (50 mg/kg) plus EGb 761 (20 mg/kg) and ticlopidine (50 mg/kg) plus EGb 761 (40 mg/kg). A combinatory treatment in acute thrombosis model in mice also showed a higher recovery than a single treatment.

Enantioselective synthesis of (R)-(+)- and (S)-(-)-higenamine and their analogues with effects on platelet aggregation and experimental animal model of disseminated intravascular coagulation.

Optically active tetrahydroisoquinoline alkaloids, (R)-(+)-higenamine (1R) and (S)-(-)-higenamine (1 S), and their optically active 1-naphthylmethyl analogues (2 and 3), were synthesized by enantioselective hydrogenation of the corresponding dihydroisoquinoline intermediates 7 as a key step. The evaluation of the platelet anti-aggregation effect demonstrated clearly that the (S)-(-)-enantiomers, 1S, 2S, and 3S, had higher inhibitory potency than the corresponding (R)-(+)-antipodes, 1R, 2R, and 3R, respectively, to platelet aggregation induced by epinephrine. 1S enantiomer was superior to the corresponding 1R enantiomer in attenuating all of the disseminated intravascular coagulation (DIC) and multiple organ failure (MOF) parameters tested, while the S enantiomers 2S and 3S ameliorated some of the DIC and MOF parameters more effectively than the corresponding antipodes 2R and 3R.

Phenolic and furan type compounds isolated from Gastrodia elata and their anti-platelet effects.

Nine phenolic (1–9) and two furan type (10,11) compounds, were isolated from the methan-olic extract of the tuber ofGastrodia elata Blume (Orchidaceae) in the course of continuing search for platelet anit-aggregating plant components. Compound1 was identified as 4,4′-dihy-droxybenzyl sulfone, a novel compound for the best of our knowledge. Compound10, 5-hydroxymethyl-2-furancarboxaldehyde, was isolated for the first time from this plant. Compound1 (IC50; 83 μM) was about four times more inhibitory to U46619 induced aggregation than ASA (IC50; 340 μM). Compound9, 4,4′-dihydroxy-dibenzylether, (IC50; 5 μM, 3 μM and 33uM, respectively) was 10–80 fold more potent than ASA (IC50; 420 μM, 53 μM and 340 μM respectively) to collagen, epinephrine and U46619 induced aggregation, although it is less active than ASA to AA induced aggregation.

Anticoagulating activities of low-molecular weight fuco-oligosaccharides prepared by enzymatic digestion of fucoidan from the sporophyll of Korean Undaria pinnatifida.

In spite of their potential as biologically active compounds, the high molecular mass and viscous natures of fucoidans have hampered their applications especially as a therapeutic agent. Herein the fucoidan-degrading enzyme activities were partially purified from the cultured cells of Sphingomonas paucimobilis PF-1 mainly by ammonium sulfate precipitation. This enzyme preparation degraded fucoidans from the Korean Undaria pinnatifida sporophyll into several low-molecular weight fuco-oligosaccharides (LMFOs) with less than 3,749 Da. The FTIR spectra of intact fucoidan and mixture of LMFOs (1,389∼3,749 Da) showed no significant structural difference except for about 10% reduced level of sulfate esters in LMFOs. The LMFOs have exerted strong anticoagulating activities at which the activated partial thromboplastin time (APTT) and thrombin time (TT) were significantly prolonged, although 3∼20 times weaker activities were observed than those of intact fucoidan. In addition, unlike intact fucoidan, LMFOs did not affect significantly to the prothrombin time (PT). These results suggest that the partially purified fucoidan-degrading enzyme preparation is valuable for the production of fuco-oligosaccharides having anticoagulating activities, and that the molecular weight and/or sulfate content of the fucoidan from the Korean Undaria pinnatifida sporophyll could be important factors for its anticoagulating activity.

Regulation of Lipopolysaccharide-Induced Inducible Nitric-Oxide Synthase Expression through the Nuclear Factor-κB Pathway and Interferon-β/Tyrosine Kinase 2/Janus Tyrosine Kinase 2-Signal Transducer and Activator of Transcription-1 Signaling Cascades by 2-Naphthylethyl-6,7-dihydroxy-1,2,3,4-tetrahydroisoquinoline (THI 53), a New Synthetic Isoquinoline Alkaloid

The effects of 2-naphthylethyl-6,7-dihydroxy-1,2,3,4-tetrahydroisoquinoline (THI 53), on nitric oxide (NO) production and inducible nitric-oxide synthase (iNOS) protein induction by lipopolysaccharide (LPS) were investigated in RAW 264.7 cells and mice. In cells, THI 53 concentration dependently reduced NO production and iNOS protein induction by LPS. In addition, THI 53 inhibited NO production and iNOS protein induction in LPS-treated mice. LPS-mediated iNOS protein induction was inhibited significantly by the specific tyrosine kinase inhibitor α-cyano-(3-hydroxy-4-nitro)cinnamonitrile (AG126) as well as by THI 53. In addition, a c-Jun NH2-terminal kinase (JNK) inhibitor anthra[1,9-cd]pyrazole-6 (2H)-one) (SP600125) but not an extracellular regulated kinase inhibitor [2-(2-amino-3-methoxyphenyl)-4H-1-benzopyran-4-one (PD98029)] or a p38 inhibitor [4-(4-fluorophenyl)-2-(4-methylsulfinylphenyl)-5-(4-pyridyl)1H-imidazole (SB230580)] reduced the iNOS protein level induced by LPS. Moreover, a Janus kinase 2 (JAK2) inhibitor α-cyano-(3,4-dihydroxy)-N-benzylcinnamide (AG490) dose-dependently prevented LPS-mediated iNOS protein induction. LPS activated phosphorylations of tyrosine kinases, especially tyrosine kinase 2 (Tyk2) and signal transducer and activator of transcription-1 (STAT-1); these were reduced by THI 53. LPS also phosphorylated the JNK pathway; however, this phosphorylation was unaffected by THI 53. Interestingly, a JNK inhibitor (SP600125) and another tyrosine kinase inhibitor (genistein) significantly inhibited STAT-1 phosphorylation, suggesting that the LPS-activated JNK pathway and a tyrosine kinase pathway (especially Tyk2) may link to the STAT-1 pathway, which is involved in iNOS induction. However, THI 53 regulates LPS-mediated iNOS protein induction by affecting the Tyk2/JAK2-STAT-1 pathway, not the JNK pathway. The inhibition by THI 53 of LPS-induced NO production was recovered by a tyrosine phosphatase inhibitor (Na3VO4), which supports the possibility that THI 53 inhibits the LPS-induced inflammatory response through regulation of tyrosine kinase pathways. THI 53 also inhibited LPS-mediated interferon (IFN)-β production and nuclear factor-κB (NF-κB) activation. Thus, THI 53 may regulate LPS-mediated inflammatory response through both the NF-κB and IFN-β/Tyk2/JAK2-STAT-1 pathways.