Stauntonia hexaphylla leaf extract (YRA-1909) suppresses inflammation by modulating Akt/NF-κB signaling in lipopolysaccharide-activated peritoneal macrophages and rodent models of inflammation.

BACKGROUND: Inflammation is emerging as a key contributor to many vascular diseases and furthermore plays a major role in autoimmune diseases, arthritis, allergic reactions, and cancer. Lipopolysaccharide (LPS), which is a component constituting the outer membrane of Gram-negative bacteria, is commonly used for an inflammatory stimuli to mimic inflammatory diseases. Nuclear factor-kappa B (NF-κB) is a transcription factor and regulates gene expression particularly related to the inflammatory process. Stauntonia hexaphylla (Lardizabalaceae) is widely used as a traditional herbal medicine for rheumatism and osteoporosis and as an analgesic, sedative, and diuretic in Korea, Japan, and China. OBJECTIVE: The purpose of this study was to investigate the anti-inflammatory activity of YRA-1909, the leaf aqueous extract of Stauntonia hexaphylla using LPS-activated rat peritoneal macrophages and rodent inflammation models. RESULTS: YRA-1909 inhibited the LPS-induced nitric oxide (NO) and proinflammatory cytokine production in rat peritoneal macrophages without causing cytotoxicity and reduced inducible NO synthase and prostaglandin E2 levels without affecting the cyclooxygenase-2 expression. YRA-1909 also prevented the LPS-stimulated Akt and NF-κB phosphorylation and reduced the carrageenan-induced hind paw edema, xylene-induced ear edema, acetic acid-induced vascular permeation, and cotton pellet-induced granuloma formation in a dose-dependent manner in mice and rats. CONCLUSIONS: S. hexaphylla leaf extract YRA-1909 had anti-inflammatory activity in vitro and in vivo that involves modulation of Akt/NF-κB signaling. Thus, YRA-1909 is safe and effective for the treatment of inflammation.

Simultaneous Determination of Chlorogenic Acid Isomers and Metabolites in Rat Plasma Using LC-MS/MS and Its Application to A Pharmacokinetic Study Following Oral Administration of Stauntonia Hexaphylla Leaf Extract (YRA-1909) to Rats.

Stauntonia hexaphylla leaf extract (YRA-1909), which is widely used for the antirheumatic properties, has been under phase 2 clinical trials in patients with rheumatoid arthritis since April 2017. Liquid chromatography-tandem mass spectrometric method while using liquid⁻liquid extraction with ethyl acetate was validated for the simultaneous determination of the major active components of YRA-1909, including chlorogenic acid (CGA), neochlorogenic acid (NCGA), cryptochlorogenic acid (CCGA), and their metabolites (i.e., caffeic acid (CA), caffeic acid 3-O-glucuronide (CA-3-G), caffeic acid 4-O-glucuronide (CA-4-G), and ferulic acid (FA)) in rat plasma and applied to a pharmacokinetic study of YRA-1909 in rats. Seven analytes were separated on Halo C18 while using gradient elution of formic acid and methanol, and then quantified in selected reaction monitoring mode whle using negative electrospray ionization. Following oral administration of YRA-1909 at doses of 25, 50, and 100 mg/kg to male Sprague-Dawley rats, CGA, NCGA, and CCGA were rapidly absorbed and metabolized to CA, CA-3-G, and CA-4-G. The area under the plasma concentration-time curve (AUClast) of CGA, NCGA, CCGA, and three metabolites linearly increased as the YRA-1909 dose increased. Other pharmacokinetic parameters were comparable among three doses studied. AUClast values for CA, CA-3-G, and CA-4-G exceeded those for CGA, NCGA, and CCGA.

Practical Synthesis of Chalcone Derivatives and Their Biological Activities.

Practical synthesis and biological activities of 4-hydroxy-3-methoxy-2-propene derivatives are described. The novel chalcone derivatives were prepared by acid catalysed one-step condensation of 1,3- or 1,4-diacetylbenzene and 1,3,5-triacetylbenzene with 4-hydroxy-3-methoxybenzaldehyde. They were then evaluated for free radical scavenging activity, suppression of lipopolysaccharides (LPS)-induced NO generation, and anti-excitotoxicity in vitro. It was found that all compounds showed good effects for 2,2-diphenyl-1-picrylhydrazyl (DPPH) free radical scavenging, LPS-induced NO generation, and anti-neurotoxicity. Compounds 6 and 7 were potent suppressor of NO generation with the concentration range 10 µM and especially compound 8 showed very potent anti-inflammatory activity with 1 µM. In addition, the di- and tri-acetylbenzyl derivatives 6, 7, and 8 showed enhanced anti-neurotoxicity activity in cultured cortical neurons. Molecular modelling studies to investigate the chemical structural characteristics required for the enhanced biological activities interestingly revealed that compound 8 has the smallest highest occupied molecular orbital-lowest energy unoccupied molecular orbital (HOMO-LUMO) gap, which signifies easy electron and radical transfer between HOMO and LUMO in model studies.

Multiple UDP-Glucuronosyltransferase and Sulfotransferase Enzymes are Responsible for the Metabolism of Verproside in Human Liver Preparations.

Verproside, an active iridoid glycoside component of Veronica species, such as Pseudolysimachion rotundum var. subintegrum and Veronica anagallis-aquatica, possesses anti-asthma, anti-inflammatory, anti-nociceptive, antioxidant, and cytostatic activities. Verproside is metabolized into nine metabolites in human hepatocytes: verproside glucuronides (M1, M2) via glucuronidation, verproside sulfate (M3) via sulfation, picroside II (M4) and isovanilloylcatalpol (M5) via O-methylation, M4 glucuronide (M6) and M4 sulfate (M8) via further glucuronidation and sulfation of M4, and M5 glucuronide (M7) and M5 sulfate (M9) via further glucuronidation and sulfation of M5. Drug-metabolizing enzymes responsible for verproside metabolism, including sulfotransferase (SULT) and UDP-glucuronosyltransferase (UGT), were characterized. The formation of verproside glucuronides (M1, M2), isovanilloylcatalpol glucuronide (M7), and picroside II glucuronide (M6) was catalyzed by commonly expressed UGT1A1 and UGT1A9 and gastrointestinal-specific UGT1A7, UGT1A8, and UGT1A10, consistent with the higher intrinsic clearance values for the formation of M1, M2, M6, and M7 in human intestinal microsomes compared with those in liver microsomes. The formation of verproside sulfate (M3) and M5 sulfate (M9) from verproside and isovanilloylcatalpol (M5), respectively, was catalyzed by SULT1A1. Metabolism of picroside II (M4) into M4 sulfate (M8) was catalyzed by SULT1A1, SULT1E1, SULT1A2, SULT1A3, and SULT1C4. Based on these results, the pharmacokinetics of verproside may be affected by the co-administration of relevant UGT and SULT inhibitors or inducers.

Verproside inhibits TNF-α-induced MUC5AC expression through suppression of the TNF-α/NF-κB pathway in human airway epithelial cells.

Airway mucus secretion is an essential innate immune response for host protection. However, overproduction and hypersecretion of mucus, mainly composed of MUC5AC, are significant risk factors in asthma and chronic obstructive pulmonary disease (COPD) patients. Previously, we reported that verproside, a catalpol derivative iridoid glycoside isolated from Pseudolysimachion rotundum var. subintegrum, is a potent anti-asthmatic candidate drug in vivo. However, the molecular mechanisms underlying the pharmacological actions of verproside remain unknown. Here, we found that verproside significantly reduces the expression levels of tumor necrosis factor alpha (TNF-α)-induced MUC5AC mRNA and protein by inhibiting both nuclear factor kappa B (NF-κB) transcriptional activity and the phosphorylation of its upstream effectors such as IκB kinase (IKK)ß, IκBα, and TGF-ß-activated kinase 1 (TAK1) in NCI-H292 cells. Moreover, verproside attenuated TNF-α-induced MUC5AC transcription more effectively when combined with an IKK (BAY11-7082) or a TAK1 (5z-7-oxozeaenol) inhibitor than when administered alone. Importantly, we demonstrated that verproside negatively modulates the formation of the TNF-α-receptor (TNFR) 1 signaling complex [TNF-RSC; TNFR1-recruited TNFR1-associated death domain protein (TRADD), TNFR-associated factor 2 (TRAF2), receptor-interacting protein kinase 1 (RIP1), and TAK1], the most upstream signaling factor of NF-κB signaling. In silico molecular docking studies show that verproside binds between TRADD and TRAF2 subunits. Altogether, these results suggest that verproside could be a good therapeutic candidate for treatment of inflammatory airway diseases such as asthma and COPD by blocking the TNF-α/NF-κB signaling pathway.

Organic anion transporter 3- and organic anion transporting polypeptides 1B1- and 1B3-mediated transport of catalposide.

We investigated the in vitro transport characteristics of catalposide in HEK293 cells overexpressing organic anion transporter 1 (OAT1), OAT3, organic anion transporting polypeptide 1B1 (OATP1B1), OATP1B3, organic cation transporter 1 (OCT1), OCT2, P-glycoprotein (P-gp), and breast cancer resistance protein (BCRP). The transport mechanism of catalposide was investigated in HEK293 and LLC-PK1 cells overexpressing the relevant transporters. The uptake of catalposide was 319-, 13.6-, and 9.3-fold greater in HEK293 cells overexpressing OAT3, OATP1B1, and OATP1B3 transporters, respectively, than in HEK293 control cells. The increased uptake of catalposide via the OAT3, OATP1B1, and OATP1B3 transporters was decreased to basal levels in the presence of representative inhibitors such as probenecid, furosemide, and cimetidine (for OAT3) and cyclosporin A, gemfibrozil, and rifampin (for OATP1B1 and OATP1B3). The concentration-dependent OAT3-mediated uptake of catalposide revealed the following kinetic parameters: Michaelis constant (K m) =41.5 µM, maximum uptake rate (V max) =46.2 pmol/minute, and intrinsic clearance (CL int) =1.11 µL/minute. OATP1B1- and OATP1B3-mediated catalposide uptake also showed concentration dependency, with low CL int values of 0.035 and 0.034 µL/minute, respectively. However, the OCT1, OCT2, OAT1, P-gp, and BCRP transporters were apparently not involved in the uptake of catalposide into cells. In addition, catalposide inhibited the transport activities of OAT3, OATP1B1, and OATP1B3 with half-maximal inhibitory concentration values of 83, 200, and 235 µM, respectively. However, catalposide did not significantly inhibit the transport activities of OCT1, OCT2, OAT1, P-gp, or BCRP. In conclusion, OAT3, OATP1B1, and OATP1B3 are major transporters that may regulate the pharmacokinetic properties and may cause herb-drug interactions of catalposide, although their clinical relevance awaits further evaluation.

Quantitative structure-activity relationship study of aromatic inhibitors against rat lens aldose reductase activity using variable selections.

A quantitative structure-activity relationship (QSAR) study of aromatic inhibitors against aldose reductase (AR) activity was performed using variable selection from stepwise multiple linear regression (MLR) and genetic algorithm (GA)-MLR. As a result of variable selection, stepwise MLR and GA-MLR gave the same results with one, two, three and five descriptors and different results with four and six descriptors. GA-MLR produced higher values and was better in explanatory and predictive power than stepwise MLR in four variables. AR activity (pIC50) of aromatic derivatives was expressed with acceptable explanatory (74.6-81.2%) and predictive power (68.8-74.4%) in models 3 and 4. The resulting models with the given descriptors illustrate that hydrophobic and electrostatic interactions play a significant role in inhibition of AR activity. This study suggests that the QSAR models can be used as guidelines to predict improved aldose reductase inhibitory activity and to obtain reliable predictions in structurally diverse compounds.

In vitro and in vivo metabolism of verproside in rats.

Verproside, a catalpol derivative iridoid glycoside isolated from Pseudolysimachion rotundum var. subintegrum, is a biologically active compound with anti-inflammatory, antinociceptic, antioxidant, and anti-asthmatic properties. Twenty-one metabolites were identified in bile and urine samples obtained after intravenous administration of verproside in rats using liquid chromatography-quadrupole Orbitrap mass spectrometry. Verproside was metabolized by O-methylation, glucuronidation, sulfation, and hydrolysis to verproside glucuronides (M1 and M2), verproside sulfates (M3 and M4), picroside II (M5), M5 glucuronide (M7), M5 sulfate (M9), isovanilloylcatalpol (M6), M6 glucuronide (M8), M6 sulfate (M10), 3,4-dihydroxybenzoic acid (M11), M11 glucuronide (M12), M11 sulfates (M13 and M14), 3-methyoxy-4-hydroxybenzoic acid (M15), M15 glucuronides (M17 and M18), M15 sulfate (M20), 3-hydroxy-4-methoxybenzoic acid (M16), M16 glucuronide (M19), and M16 sulfate (M21). Incubation of verproside with rat hepatocytes resulted in thirteen metabolites (M1-M11, M13, and M14). Verproside sulfate, M4 was a major metabolite in rat hepatocytes. After intravenous administration of verproside, the drug was recovered in bile (0.77% of dose) and urine (4.48% of dose), and O-methylation of verproside to picroside II (M5) and isovanilloylcatalpol (M6) followed by glucuronidation and sulfation was identified as major metabolic pathways compared to glucuronidation and sulfation of verproside in rats.

Total synthesis of flocoumafen via knoevenagel condensation and intramolecular ring cyclization: general access to natural products.

The total synthesis and structure determination of cis- and trans-flocoumafen was described. The key synthetic steps involve Knoevenagel condensation with p-methoxybenzaldehyde, in situ decarboxylation and intramolecular ring cyclization to construct the tetralone skeleton. Stereospecific reduction of the O-alkylated ketone 13 afforded good yield of precusor alcohol 5. Final coupling of alcohol 5 with 4-hydroxy-coumarin yielded flocoumafen (1). Separation and structure determination of cis- and trans-flocoumafen through 2D NMR analyses-assisted computer simulation techniques for the evaluation of anticoagulant activities are reported for the first time. This method is useful for generating the core tetralone skeleton of 4-hydroxycoumarin derivatives and provides a generalized access to various warfarin type anticoagulants.

Design, synthesis, and biological evaluation of 3,4,5-trimethoxyphenyl acrylamides as antinarcotic agents.

The synthesis and biological evaluation of 3,4,5-trimethoxyphenyl acrylamides 1a-f as novel antinarcotic agents are described. The molecules were prepared by the Wittig reaction, followed by a coupling reaction between 3,4,5-trimethoxycinnamic acid (9) and aliphatic amines, which resulted in good yields. When tested for biological activity, compounds 1d-f exhibited strong inhibitory effects on the morphine withdrawal syndrome in mice due to their high binding affinities with serotonergic 5-HT1A receptors.

Synthesis and anticancer activity of novel amide derivatives of non-acetal deoxoartemisinin.

Novel amide derivatives of C-12 non-acetal deoxoartemisinin were synthesized. Some of the derivatives had potent in vitro anticancer activity against major human cancer cell lines. The deoxoartemisinin amide trimer had potent in vivo antiangiogenic activity, according to the mouse matrigel plug assay.

Total synthesis and anticancer activity of highly potent novel glycolipid derivatives.

The total synthesis and anticancer activity of several novel derivatives based on a dauer effect-inducing glycolipid are presented. A versatile and convergent synthesis was accomplished through stereospecific alpha-glycosylation, which produced di- and tri-rhamnoside daumone derivatives. Most of the synthetic derivatives possessed potent anticancer activity against human cancer cell lines. Daumone and deoxyrhamnose trisaccharides with amide side chains had the most potent anticancer activity among all other known glycolipids, with an effective concentration of 20 nM, which is comparable to that of doxorubicin. Conversely, acyclic and macrocyclic daumone derivatives had drastically decreased anticancer activity. Due to the high lipophilic nature of the novel glycolipid derivatives, we propose that the observed anticancer activity is due to their potential to inhibit cell differentiation and proliferation via interaction with the membranes of cancer cells.

Synthesis of novel trans-stilbene derivatives and evaluation of their potent antioxidant and neuroprotective effects.

A convenient synthesis and the biological properties of new amides, esters and other derivatives of trans-stilbene are described. The key synthetic strategies involve the Wittig-Horner reaction of a phosphonium salt 9 and an aldehyde 10 to generate (E)- or (Z)-olefins and a coupling reaction of an acid 12 and various amines 13a-n to give trans-stilbene derivatives 15a-n in high yields. A amide derivative 15g showed three times more in vitro free radical-scavenging activity than resveratrol, while another 15d exhibited strong inhibitory activity against lipopolysaccharide (LPS)(a)-induced NO generation. Allylamide analogue 15a showed the most potent neuroprotective activity in glutamate-induced primary cortical neuron cells.

Synthesis, structural characterization and biological evaluation of novel stilbene derivatives as potential antimalarial agents.

A series of benzamide-containing stilbene derivatives was synthesized through the incorporation of short basic side-chains in the B-ring hydroxy position of resveratrol. Their antiplasmodial activity was evaluated in vitro against the chloroquine resistant Plasmodium falciparum D10 strain, showing IC(50) values between 1.5 and 80 microM, while their cytotoxicity was assessed using an human myeloid leukemia (U-937) cell line. With a selectivity ratio of > 51.02, the most selective of these derivatives, 29, also had the most lowest cytotoxic activity of the series.

The neuroprotective effects of benzylideneacetophenone derivatives on excitotoxicity and inflammation via phosphorylated Janus tyrosine kinase 2/phosphorylated signal transducer and activator of transcription 3 and mitogen-activated protein K pathways.

To search for new neuroprotective compounds, novel benzylideneacetophenone compounds (JCI, (3E)-4-(4-hydroxy-3-methoxyphenyl)but-3-en-2-one; JC2, (1E)-1-(4-hydroxy-3-methoxyphenyl)hept-1-en-3-one; JC3, (2E)-3-(4-hydroxy-3-methoxyphenyl)phenylpro-2-en-l-one; JC4, (1E)-1-(4-hydroxy-3-methoxyphenyl)-5-phenylpent-1-en-3-one; JC5, (1E)-3-(4-hydroxy-3-methoxyphenyl)-6-phenylhex-1-en-3-one; JC6, (1E)-1-(4-hydroxy-3-methoxyphenyl]-7-phenylhept-1-en-3-one) were synthesized, and their potential to prevent neurotoxicities were evaluated. All compounds (JC1-JC6) showed considerable effect on free radical scavenging, the inhibition of glutamate-induced neurotoxicity in cortical cells, and the suppression of lipopolysaccharide (LPS)-induced nitric oxide (NO) generation in microglia. (2E)-3-(4-Hydroxy-3-methoxyphenyl)-phenylpro-2-en-1-one (JC3) exhibited the most potent neuroprotective effect in ischemia model using organotypic hippocampal culture and middle cerebral artery occlusion (MCAO). Based on the above-mentioned results, the mechanisms underlying the biological activity of JC3, which exhibited potent antiexcitotoxic and anti-inflammatory effects, were determined using cortical neuronal cells and microglia. Compound JC3 exerted a neuroprotective effect on oxygen-glucose deprivation- and hydrogen peroxide-induced cytotoxicity in cultured cortical cells. In addition, it suppressed the generation of NO, proinflammatory cytokines, and reactive oxygen species in LPS-treated microglial cells. It also suppressed the activation of phosphorylated Janus tyrosine kinase 2/phosphorylated signal transducer and activator of transcription 3 and mitogen-activated protein kinase (MAPK) in activated microglia and in cortex and striatum after 3 days of the MCAO in mice. These results demonstrated that JC3 might affect a set of intracellular signaling cascades, including the Janus tyrosine kinase/signal transducers and activators of transcription and MAPK pathways. This study suggests that benzylideneacetophenone derivative could be useful antineurotoxic agents.

The convenient synthesis and evaluation of the anticancer activities of new resveratrol derivatives.

In the present study we report the simple synthesis and antitumour activity of novel stilbene derivatives 13-22. The key synthetic strategies involved Wadsworth-Horner-Emmons condensation and coupling reactions in high yields. All compounds showed significant growth inhibition on human tumour cell lines, with the most potent compound (19) exhibiting an IC(50) of 5.7 microM-11.4 microM in vitro.

Efficient synthesis and neuroprotective effect of substituted 1,3-diphenyl-2-propen-1-ones.

An efficient synthesis involving a key aldol reaction and biological properties of 1,3-diphenyl-2-propen-1-ones 8- 20 is described. The in vitro activity for 2,2-diphenyl-1-picrylhydrazyl (DPPH) radical scavenging of 10 and 11 was 2 times higher than that for resveratrol. Compounds 9 and 11 were the strongest in suppression of in vitro nitric oxide (NO) generation and antiexcitotoxicity. Molecular modeling proposes an electron-donating group at the para position of acetophenones that leads to a dramatic increase in the suppression of NO production.

Design, synthesis, and discovery of stilbene derivatives based on lithospermic acid B as potent protein tyrosine phosphatase 1B inhibitors.

Dihydroxy stilbene derivatives were designed based on lithospermic acid B and were prepared from 4-(chloromethyl)benzoic acid. The inhibitory activities of the novel compounds against protein tyrosine phosphatase 1B (PTP1B) were evaluated. 3,4-Dihydroxy stilbene carbonyl compounds (7, 11b, 27b) inhibited PTP1B with IC50 values comparable to molybdate, while the conjugation-extended compound (15b) showed inhibition 3-fold better than preclinical RK682. The introduction of electron withdrawing groups or amides into the second phenyl ring, or extension of the conjugation into the stilbene molecule may increase stability of the generated radicals.

Quantitative structure-activity relationship (QSAR) of tacrine derivatives against acetylcholinesterase (AChE) activity using variable selections.

A diverse approach to the quantitative structure-activity relationship (QSAR) of tacrine derivatives against acetylcholinesterase (AChE) activity was studied using variable selections of stepwise multiple linear regression (MLR), genetic algorithm (GA)-MLR, and simulated annealing (SA)-MLR. AChE activity (logRA) of tacrine derivatives was expressed with acceptable explanation (95.5-95.9%) and good predictive power (94.5-95.2%), respectively, in the models. The best equation was obtained from simulated annealing (SA) MLR with greater explanatory capability and better prediction, with a smaller standard error than other methods. The resulting models with the given descriptors illustrate the significant roles of hydrophobic and electrostatic interaction on increasing AChE activity, but hydrophilic and topological feature of molecules were shown to decrease AChE activity.