Phytochemical profiling of Symplocos tanakana Nakai and S. sawafutagi Nagam. leaf and identification of their antioxidant and anti-diabetic potential.

Symplocos sp. contains various phytochemicals and is used as a folk remedy for treatment of diseases such as enteritis, malaria, and leprosy. In this study, we discovered that 70% ethanol extracts of Symplocos sawafutagi Nagam. and S. tanakana Nakai leaves have antioxidant and anti-diabetic effects. The components in the extracts were profiled using high-performance liquid chromatography coupled to electrospray ionization and quadrupole time-of-flight mass spectrometry; quercetin-3-O-(6''-O-galloyl)-ß-d-galactopyranoside (6) and tellimagrandin II (7) were the main phenolic compounds. They acted as strong antioxidants with excellent radical scavenging activity and as inhibitors of non-enzymatic advanced glycation end-products (AGEs) formation. Mass fragmentation analysis demonstrated that compounds 6 and 7 could form mono- or di-methylglyoxal adducts via reaction with methylglyoxal, which is a reactive carbonyl intermediate and an important precursor of AGEs. In addition, compound 7 effectively inhibited the binding between AGE2 and receptor for AGEs as well as the activity of α-glucosidase. Enzyme kinetic study revealed that compound 7 acts as a competitive inhibitor of α-glucosidase, through interaction with the active site of the enzyme. Therefore, compounds 6 and 7, the major constituents of S. sawafutagi and S. tanakana leaves, are promising for developing drugs for preventing or treating diseases caused by aging and excessive sugar consumption.

Phytoestrogen Coumestrol Selectively Inhibits Monoamine Oxidase-A and Amyloid ß Self-Aggregation.

Pueraria lobata leaves contain a variety of phytoestrogens, including flavonoids, isoflavonoids, and coumestan derivatives. In this study, we aimed to identify the active ingredients of P. lobata leaves and to elucidate their function in monoamine oxidase (MAO) activation and Aß self-aggregation using in vitro and in silico approaches. To the best of our knowledge, this is the first study to elucidate coumestrol as a selective and competitive MAO-A inhibitor. We identified that coumestrol, a coumestan-derivative, exhibited a selective inhibitory effect against MAO-A (IC50 = 1.99 ± 0.68 µM), a key target protein for depression. In a kinetics analysis with 0.5 µg MAO-A, 40-160 µM substrate, and 25 °C reaction conditions, coumestrol acts as a competitive MAO-A inhibitor with an inhibition constant of 1.32 µM. During an in silico molecular docking analysis, coumestrol formed hydrogen bonds with FAD and pi-pi bonds with hydrophobic residues at the active site of the enzyme. Moreover, based on thioflavin-T-based fluorometric assays, we elucidated that coumestrol effectively prevented self-aggregation of amyloid beta (Aß), which induces an inflammatory response in the central nervous system (CNS) and is a major cause of Alzheimer's disease (AD). Therefore, coumestrol could be used as a CNS drug to prevent diseases such as depression and AD by the inhibition of MAO-A and Aß self-aggregation.

Pygenic Acid A (PA) Sensitizes Metastatic Breast Cancer Cells to Anoikis and Inhibits Metastasis In Vivo.

Metastasis is the main cause of cancer-related deaths. Anoikis is a type of apoptosis caused by cell detachment, and cancer cells become anoikis resistant such that they survive during circulation and can successfully metastasize. Therefore, sensitization of cancer cells to anoikis could prevent metastasis. Here, by screening for anoikis sensitizer using natural compounds, we found that pygenic acid A (PA), a natural compound from Prunella vulgaris, not only induced apoptosis but also sensitized the metastatic triple-negative breast cancer cell lines, MDA-MB-231 cells (human) and 4T1 cells (mouse), to anoikis. Apoptosis protein array and immunoblotting analysis revealed that PA downregulated the pro-survival proteins, including cIAP1, cIAP2, and survivin, leading to cell death of both attached and suspended cells. Interestingly, PA decreased the levels of proteins associated with anoikis resistance, including p21, cyclin D1, p-STAT3, and HO-1. Ectopic expression of active STAT3 attenuated PA-induced anoikis sensitivity. Although PA activated ER stress and autophagy, as determined by increases in the levels of characteristic markers, such as IRE1α, p-elF2α, LC3B I, and LC3B II, PA treatment resulted in p62 accumulation, which could be due to PA-induced defects in autophagy flux. PA also decreased metastatic characteristics, such as cell invasion, migration, wound closure, and 3D growth. Finally, lung metastasis of luciferase-labeled 4T1 cells decreased following PA treatment in a syngeneic mouse model when compared with the control. These data suggest that PA sensitizes metastatic breast cancer cells to anoikis via multiple pathways, such as inhibition of pro-survival pathways and activation of ER stress and autophagy, leading to the inhibition of metastasis. These findings suggest that sensitization to anoikis by PA could be used as a new therapeutic strategy to control the metastasis of breast cancer.

Isoharringtonine Induces Apoptosis of Non-Small Cell Lung Cancer Cells in Tumorspheroids via the Intrinsic Pathway.

Lung cancer is the major cause of cancer-associated death worldwide, and development of new therapeutic drugs is needed to improve treatment outcomes. Three-dimensional (3D) tumorspheroids offer many advantages over conventional two-dimensional cell cultures due to the similarities to in vivo tumors. We found that isoharringtonine, a natural product purified from Cephalotaxus koreana Nakai, significantly inhibited the growth of tumorspheroids with NCI-H460 cells in a dose-dependent manner and induced apoptotic cell death in our 3D cell culture system. On the other hand, A549 tumorspheroids displayed low sensitivity to isoharringtonine-induced apoptosis. Nuclear receptor subfamily 4 group A member 1 (NR4A1) is an orphan nuclear receptor known to regulate proliferation and apoptosis of cancer cells. We observed that knockdown of NR4A1 dramatically increased isoharringtonine-induced cancer cell death in A549 tumorspheroids by activating the intrinsic apoptosis pathway. Furthermore, treatment with combined isoharringtonine and iNR4A1 significantly inhibited multivulva formation in a Caenorhabditis elegans model and tumor development in a xenograft mouse model. Taken together, our data suggest that isoharringtonine is a potential natural product for treatment of non-small cell lung cancers, and inhibition of NR4A1 sensitizes cancer cells to anti-cancer treatment.

Potent and selective inhibition of human monoamine oxidase-B by 4-dimethylaminochalcone and selected chalcone derivatives.

Six synthetic (1-6) and six natural (7-12) chalcones were tested for human monoamine oxidases (hMAOs) and acetylcholinesterase (AChE) inhibitory activities. Compounds 4-dimethylaminochalcone (2), 4'-chloro-4-dimethylaminochalcone (5), and 2,4'-dichloro-4-dimethylaminochalcone (1) potently inhibited hMAO-B with IC50 values of 0.029, 0.061, and 0.075 µM, respectively. 4-Nitrochalcone (4) and 4-chlorochalcone (3) also potently inhibited hMAO-B with IC50 values of 0.066 and 0.082 µM, respectively (2.3- and 2.6-fold less than compound 2). Compound 2 had a high selectivity index (113.1) for hMAO-B over hMAO-A (IC50 = 3.28 µM). Compounds 1 and 2,2'-dihydroxy-4',6'-dimethoxychalcone (12) potently inhibited hMAO-A with IC50 values of 0.18 and 0.39 µM, respectively. In addition, compounds 4 and 2 also effectively inhibited AChE with IC50 values of 1.25 and 6.07 µM, respectively, and thus, exhibited dual-targeting. Compound 2 reversibly and competitively inhibited hMAO-B with a Ki value of 0.0066 µM. Docking simulations showed binding affinities of compounds 1 to 5 for hMAO-B were higher than those for hMAO-A or AChE and suggested these five chalcones form hydrogen bonds with MAO-B at Cys172 but that they do not form hydrogen bonds with hMAO-A or AChE. These findings suggest compound 2 be considered a promising and dual-targeting lead compound for the treatment of Alzheimer's disease.

Potent inhibition of acetylcholinesterase by sargachromanol I from Sargassum siliquastrum and by selected natural compounds.

Six hundred forty natural compounds were tested for acetylcholinesterase (AChE) and butyrylcholinesterase (BChE) inhibitory activities. Of those, sargachromanol I (SCI) and G (SCG) isolated from the brown alga Sargassum siliquastrum, dihydroberberine (DB) isolated from Coptis chinensis, and macelignan (ML) isolated from Myristica fragrans, potently and effectively inhibited AChE with IC50 values of 0.79, 1.81, 1.18, and 4.16 µM, respectively. SCI, DB, and ML reversibly inhibited AChE and showed mixed, competitive, and noncompetitive inhibition, respectively, with Ki values of 0.63, 0.77, and 4.46 µM, respectively. Broussonin A most potently inhibited BChE (IC50 = 4.16 µM), followed by ML, SCG, and SCI (9.69, 10.79, and 13.69 µM, respectively). In dual-targeting experiments, ML effectively inhibited monoamine oxidase B with the greatest potency (IC50 = 7.42 µM). Molecular docking simulation suggested the binding affinity of SCI (-8.6 kcal/mol) with AChE was greater than those of SCG (-7.9 kcal/mol) and DB (-8.2 kcal/mol). Docking simulation indicated SCI interacts with AChE at Trp81, and that SCG interacts at Ser119. No hydrogen bond was predicted for the interaction between AChE and DB. This study suggests SCI, SCG, DB, and ML be viewed as new reversible AChE inhibitors and useful lead compounds for the development for the treatment of Alzheimer's disease.

Osthenol, a prenylated coumarin, as a monoamine oxidase A inhibitor with high selectivity.

Osthenol (6), a prenylated coumarin isolated from the dried roots of Angelica pubescens, potently and selectively inhibited recombinant human monoamine oxidase-A (hMAO-A) with an IC50 value of 0.74 µM and showed a high selectivity index (SI > 81.1) for hMAO-A versus hMAO-B. Compound 6 was a reversible competitive hMAO-A inhibitor (Ki = 0.26 µM) with a potency greater than toloxatone (IC50 = 0.93 µM), a marketed drug. Isopsoralen (3) and bakuchicin (1), furanocoumarin derivatives isolated from Psoralea corylifolia L., showed slightly higher IC50 values (0.88 and 1.78 µM, respectively) for hMAO-A than 6, but had low SI values (3.1 for both). Other coumarins tested did not effectively inhibit hMAO-A or hMAO-B. A structural comparison suggested that the 8-(3,3-dimethylallyl) group of 6 increased its inhibitory activity against hMAO-A compared with the 6-methoxy group of scopoletin (4). Molecular docking simulations revealed that the binding affinity of 6 for hMAO-A (-8.5 kcal/mol) was greater than that for hMAO-B (-5.6 kcal/mol) and that of 4 for hMAO-A (-7.3 kcal/mol). Docking simulations also implied that 6 interacted with hMAO-A at Phe208 and with hMAO-B at Ile199 by carbon hydrogen bondings. Our findings suggest that osthenol, derived from natural products, is a selective and potent reversible inhibitor of MAO-A, and can be regarded a potential lead compound for the design of novel reversible MAO-A inhibitors.

In vitro activity of collinin isolated from the leaves of Zanthoxylum schinifolium against multidrug- and extensively drug-resistant Mycobacterium tuberculosis.

BACKGROUND: Tuberculosis is a very serious infectious disease that threatens humanity, and the emergence of multidrug-resistant (MDR), extensively drug-resistant (XDR) strains resistant to drugs suggests that new drug development is urgent. In order to develop new tuberculosis drug, we have conducted in vitro anti-tubercular tests on thousands of plant-derived substances and finally found collinin extracted from the leaves of Zanthoxylum schinifolium, which has an excellent anti-tuberculosis effect. PURPOSE: To isolate an anti-tubercular bioactive compound from the leaves of Z. schinifolium and evaluate whether this agent demonstrates any potential in vitro characteristics suitable for the development of future anti-tubercular drugs to treat MDR and XDR Mycobacterium tuberculosis. METHODS: The methanolic extracts of the leaves of Z. schinifolium were subjected to bioassay-guided fractionation against M. tuberculosis using a microbial cell viability assay. In addition, following cell cytotoxicity assay, an intracellular anti-mycobacterial activity of the most active anti-tubercular compound was investigated after it was purified. RESULTS: The active compound with anti-tubercular activity isolated from leaves of Z. schinifolium was identified as a collinin. The extracted collinin showed anti-tubercular activity against both drug-susceptible and -resistant strains of M. tuberculosis at 50% minimum inhibitory concentrations (MIC50s) of 3.13-6.25 µg/ml in culture broth and MIC50s of 6.25-12.50 µg/ml inside Raw264.7 and A549 cells. Collinin had no cytotoxicity against human lung pneumocytes up to a concentration of 100 µg/ml (selectivity index > 16-32). CONCLUSIONS: Collinin extracted from the leaves of Z. schinifolium significantly inhibits the growth of MDR and XDR M. tuberculosis in the culture broth. In addition, it also inhibits the growth of intracellular drug-susceptible and drug-resistant tuberculosis in Raw264.7 and A549 cells. To our knowledge, this is the first report on the in vitro anti-tubercular activity of collinin, and our data suggest collinin as a potential drug to treat drug-resistant tuberculosis. Further studies are warranted to assess the in vivo efficacy and therapeutic potential of collinin.

Selective inhibition of monoamine oxidase A by chelerythrine, an isoquinoline alkaloid.

Chelerythrine, an isoquinoline alkaloid isolated from the herbaceous perennial Chelidonium majus, was found to potently and selectively inhibit an isoform of recombinant human monoamine oxidase-A (MAO-A) with an IC50 value of 0.55 µM. Chelerythrine was a reversible competitive MAO-A inhibitor (Ki = 0.22 µM) with a potency much greater than toloxatone (IC50 = 1.10 µM), a marketed drug. Other isoquinoline alkaloids tested did not effectively inhibit MAO-A or MAO-B. A structural comparison with corynoline suggested the 1- and/or 2-methoxy groups of chelerythrine increase its inhibitory activity against MAO-A. Molecular docking simulations revealed that the binding affinity of chelerythrine for MAO-A (-9.7 kcal/mol) was greater than that for MAO-B (-4.6 kcal/mol). Docking simulation implied that Cys323 and Tyr444 of MAO-A are key residues for hydrogen-bond interaction with chelerythrine. Our findings suggest chelerythrine is one of the most reversible selective and potent natural inhibitor of MAO-A, and that it be regarded a potential lead compound for the design of novel reversible MAO-A inhibitors.

Selective inhibition of monoamine oxidase A by hispidol.

Hispidol, an aurone, isolated from Glycine max Merrill, was found to potently and selectively inhibit an isoform of recombinant human monoamine oxidase-A (MAO-A), with an IC50 value of 0.26 µM, and to inhibit MAO-B, but with lower potency (IC50 = 2.45 µM). Hispidol reversibly and competitively inhibited MAO-A with a Ki value of 0.10 µM with a potency much greater than toloxatone (IC50 = 1.10 µM), a marketed drug. It also reversibly and competitively inhibited MAO-B (Ki = 0.51 µM). Sulfuretin, an analog of hispidol, effectively inhibited MAO-A (IC50 = 4.16 µM) but not MAO-B (IC50 > 80 µM). A comparison of their chemical structures showed that the 3'-hydroxyl group of sulfuretin might reduce its inhibitory activities against MAO-A and MAO-B. Flexible docking simulation revealed that the binding affinity of hispidol for MAO-A (-9.1 kcal/mol) was greater than its affinity for MAO-B (-8.7 kcal/mol). The docking simulation showed hispidol binds to the major pocket of MAO-A or MAO-B. The findings suggest hispidol is a potent, selective, reversible inhibitor of MAO-A, and that it be considered a novel lead compound for development of novel reversible inhibitors of MAO-A.

In vitro activity of alpha-viniferin isolated from the roots of Carex humilis against Mycobacterium tuberculosis.

This study explores the antitubercular activity of α-viniferin, a bioactive phytochemical compound obtained from Carex humilis. α-Viniferin was active against both drug-susceptible and -resistant strains of Mycobacterium tuberculosis at MIC50s of 4.6 µM in culture broth medium and MIC50s of 2.3-4.6 µM inside macrophages and pneumocytes. In combination with streptomycin and ethambutol, α-viniferin exhibited an additive effect and partial synergy, respectively, against M. tuberculosis H37Rv. α-Viniferin also did not show cytotoxicity in any of the cell lines tested up to a concentration of 147 µM, which gives this compound a selectivity index of >32. Moreover, α-viniferin was active against 3 Staphylococcus species, including methicillin-susceptible Staphylococcus aureus (MSSA), methicillin-resistant Staphylococcus aureus (MRSA), and methicillin-resistant Staphylococcus epidermidis (MRSE).

In vitro Antitubercular Activity of 3-Deoxysappanchalcone Isolated From the Heartwood of Caesalpinia sappan Linn.

Responsible for nearly 1.5 million deaths every year, the infectious disease tuberculosis remains one of the most serious challenges to global health. The emergence of multidrug-resistant tuberculosis and, more recently, extensively drug-resistant tuberculosis poses a significant threat in our effort to control this epidemic. New drugs are urgently needed to combat the growing threat of antimicrobial resistance. To achieve this goal, we screened approximately 500 species of medicinal plant methanol extracts and their solvent partitioned fractions for potential inhibitors of Mycobacterium tuberculosis growth. Using microdilution screening, the ethyl acetate solvent partitioned fraction from the heartwood of Caesalpinia sappan exhibited strong antitubercular activity. We isolated the active compound and identified it as 3-deoxysappanchalcone. The extracted 3-deoxysappanchalcone possessed activity against both drug-susceptible and drug-resistant strains of M. tuberculosis at MIC50 s of 3.125-12.5 µg/mL in culture broth and MIC50 s of 6.25-12.5 µg/mL inside macrophages and pneumocytes. 3-Deoxysappanchalcone was also found to act in partial synergy with streptomycin/ethambutol against M. tuberculosis H37Rv. 3-Deoxysappanchalcone had no cytotoxicity against the A549 cell line up to a concentration of 100 µg/mL (selectivity index > 8-32). Further studies are warranted to establish the in vivo effect and therapeutic potential of 3-deoxysappanchalcone. Copyright © 2017 John Wiley & Sons, Ltd.

The antioxidant activity and their major antioxidant compounds from Acanthopanax senticosus and A. koreanum.

The antioxidant activity and chlorogenic acid and caffeic acid contents were investigated from different parts of Acanthopanax senticosus and A. koreanum. Antioxidant activity was assessed by various in vitro assays such as DPPH, ABTS, FRAP, reducing power assays and ORAC, and the chlorogenic acid and caffeic acid were validated by HPLC chromatography. Among the various extracts, the fruit extracts of A. senticosus and A. koreanum exhibited strongest antioxidant activities including ABTS, FRAP, reducing power and ORAC, however, strongest DPPH radical scavenging activity was observed from the leaf extract of A. senticosus. In addition, the antioxidant activities of various extracts were correlated with total phenolic and proanthocyanidin contents. The major phenolic contents from various parts of these plants observed that leaf extract of A. senticosus expressed higher levels of chlorogenic acid (14.86 mg/dry weigh g) and caffeic acid (3.09 mg/dry weigh g) than other parts. Therefore, these results suggest that the leaf of A. senticosus may be an excellent natural source for functional foods and pharmaceutical agents, and the validated method was useful for the quality control of A. senticosus.

In vitro and in vivo immunomodulatory activity of sulfated polysaccharides from Enteromorpha prolifera.

Water-soluble sulfated polysaccharides extracted from Enteromorpha prolifera and fractionated using ion-exchange chromatography (crude, F(1), F(2) and F(3) fractions) were investigated to determine their in vitro and in vivo immunomodulatory activities. The sulfated polysaccharides, especially the F(1) and F(2) fractions, stimulated a macrophage cell line, Raw 264.7, inducing considerable nitric oxide (NO) and various cytokine production via up-regulated mRNA expression. The in vivo experiment results show that the sulfated polysaccharides (the crude and F(2) fractions) significantly increased Con A-induced splenocyte proliferation, revealing their potential comitogenic activity. In addition, IFN-γ and IL-2 secretions were considerably increased by the F(2) fraction without altering the release of IL-4 and IL-5. This implies that the F(2) fraction can activate T cells by up-regulating Th-1 response and that Th-1 cells might be the main target cells of the F(2) fraction. These in vitro and in vivo results suggest that the sulfated polysaccharides are strong immunostimulators.

Relationship between oversulfation and conformation of low and high molecular weight fucoidans and evaluation of their in vitro anticancer activity.

Low and high molecular weight fucoidans (F(5-30K) and F(>30K)) were chemically modified through the addition of sulfate groups, and the effect of oversulfation on the in vitro anticancer activity was investigated. After the addition of sulfate groups, a considerable increase of 35.5 to 56.8% was observed in the sulfate content of the F(5-30K) fraction, while the sulfate content of the F(>30K) fraction increased to a lesser extent (from 31.7 to 41.2%). Significant differences in anticancer activity were observed between the oversulfated F(5-30K) and F(>30K) fractions, with activities of 37.3-68.0% and 20.6-35.8%, respectively. This variation in the anticancer activity of oversulfated fucoidan derivatives was likely due to differences in their sulfate content. The results suggest that the molecular conformation of these molecules is closely related to the extent of sulfation in the fucan backbones and that the sulfates are preferably substituted when the fucoidan polymers are in a loose molecular conformation.