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Four new caged xanthones (1–4) and two known compounds (5, 6) were isolated from the roots of Cratoxylum cochinchinense, a polyphenol rich plant, collected in China. The structures of the isolated compounds (1–6) were characterized by obtaining their detailed spectroscopic data. In particular, compounds 1 and 6 were fully identified by X-ray crystallographic data. The isolated compounds (1–6) were evaluated against protein tyrosine phosphatase 1B (PTP1B), which plays an important role in diabetes, obesity, and cancer. Among these compounds, 3, 4, and 6 displayed significant inhibition with IC50 values of 76.3, 43.2, and 6.6 µM, respectively. A detailed kinetic study was conducted by determining Km, Vmax, and the ratio of Kik and Kiv, which revealed that all the compounds behaved as competitive inhibitors.

This study evaluated the changes in γ-aminobutyric acid (GABA) and isoflavone aglycone contents from soy powder yogurt (SPY) due to sprouting of soybean (1 cm) and fermentation with Lactobacillus brevis. The levels of GABA and the aglycone form increased, and the glutamate decarboxylase and β-glucosidase activities increased; however, the isoflavone glycoside and malonylglycoside contents decreased after fermentation for 72 h. In particular, after 60 h, the SPY presented the highest GABA content (120.38 mg/100 mL). The highest daidzein (179.93 µg/g), glycitein (44.10 µg/g), and genistein (126.24 µg/g) contents were present after 72 h of fermentation. In addition, the 2,2-diphenyl-1-picrylhydrazyl, 2,2-azinobis (3-ethylbenzothiazoline-6-sulfonic acid) diammonium salt, and hydroxyl radical scavenging activities increased from 69.65, 97.94, and 70.90% during this fermentation, respectively. This result suggests that SPY may be used for the preparation of high-protein soybean with high GABA and isoflavone aglycone contents, which can then be used as a natural ingredient of functional foods.

Human neutrophil elastase (HNE) has been linked to immune system functions, including inflammation, as well as the development of skin wrinkles, suggesting that HNE inhibitors have many practical applications. In this study, the methanol extract of Epimedium koreanum Nakai contained potent HNE inhibitory flavonoids (1–4) with IC 50 values of 6.06–125.80 μM. Prenyl groups within these flavonoids were critical for the inhibitory effects. For example, epimedokoreanin B (1) (IC 50 = 6.06 μM) showed 6-fold higher efficacy than that of its parent luteolin (IC 50 = 36.01 μM). A similar trend was observed for compound 2 (IC 50 = 6.28 μM) and its parent, apigenin (IC 50 = 37.94 μM). Lineweaver–Burk plots showed that the compounds exhibit mixed-type inhibition. In a detailed kinetic study, compound 1 was assigned to mixed type I and exhibited the greatest binding to the free enzyme (K I = 11.7 μM, K IS = 40.9 μM).

A series of flavonoids were isolated from Dodonaea Viscosa and tested for inhibition of human neutrophil elastase (HNE), enzyme involved in inflammatory disorders. Isolated compounds were identified as a novel flavonol ( 1 ) along with eight known flavonoids ( 2 – 9 ). Novel flavonol, visconata ( 1 ) has a very rare skeleton having odd numbered long chain (C19) fatty acid, which was completely identified by mass fragmentation and 2D NMR analysis. All compounds ( 1 – 9 ) inhibited HNE in dose dependent manner with IC 50 s ranging between 2.4 and 150 μM. Visconata ( 1 ) emerged to be the most potent compound with 2.4 μM of IC 50 . In kinetic studies, compound ( 1 ) was observed to be reversible, noncompetitive inhibitor having K i = 1.8 μM, whereas other flavonoids ( 2 – 9 ) displayed mixed type inhibition.

The α-glucosidase inhibitory potential of Tribulus terrestris extracts has been reported but as yet the active ingredients are unknown. This study attempted to isolate the responsible metabolites and elucidate their inhibition mechanism of α-glucosidase. By fractionating T. terristris extracts, three cinnamic acid amide derivatives (1–3) were ascertained to be active components against α-glucosidase. The lead structure, N-trans-coumaroyltyramine 1, showed significant inhibition of α-glucosidase (IC50 = 0.42 μM). Moreover, all active compounds displayed uncompetitive inhibition mechanisms that have rarely been reported for α-glucosidase inhibitors. This kinetic behavior was fully demonstrated by showing a decrease of both Km and Vmax, and Kik/Kiv ratio ranging between 1.029 and 1.053. We progressed to study how chemical modifications to the lead structure 1 may impact inhibition. An α, β-unsaturation carbonyl group and hydroxyl group in A-ring of cinnamic acid amide emerged to be critical functionalities for α-glucosidase inhibition. The molecular modeling study revealed that the inhibitory activities are tightly related to π-π interaction as well as hydrogen bond interaction between enzyme and inhibitors.

Protein tyrosine phosphatase 1B (PTP1B) is an important target to treat obesity and diabetes due to its key roles in insulin and leptin signaling. The MeOH extracts of the root bark of Flemingia philippinensis yielded eight inhibitory molecules (1-8) capable of targeting PTP1B. Three of them were identified to be novel compounds, philippin A (1), philippin B (2), and philippin C (3) which have a rare 3-phenylpropanoyl chromenedione skeleton. The other compounds (4-8) were known prenylated isoflavones. All compounds (1-8) inhibited PTP1B in a dose dependent manner with IC50s ranging between 2.4 and 29.4μM. The most potent compound emerged to be prenylated isoflavone 5 (IC50=2.4μM). In kinetic studies, chromenedione derivatives (1-3) emerged to be reversible, competitive inhibitors, whereas prenylated isoflavones (5-8) were noncompetitive inhibitors.

F. philippinensis Merr. et Rolfe has been cultivated on a large scale and is widely consumed by local inhabitants as an important nutraceutical, especially against rheumatism which has a deep connection with antioxidants. In this study, a total of 18 different phenolic metabolite compounds in F. philippinensis were isolated and identified, and evaluated for their antioxidant and DNA damage protection potential. The antioxidant activity of the 18 identified compounds was screened using DPPH, ORAC, hydroxyl and superoxide radical scavenging assays. The antioxidant potential of the compounds was found to differ by functionality and skeleton. However, most compounds showed a good antioxidant potential. In particular, seven of the identified compounds, namely, compounds 2, 3, 6, 10, 11, 15 and 16, showed significant protective effects on pBR322 plasmid DNA against the mutagenic and toxic effects of Fenton’s reaction. The most active compound, compound 2, displayed a dose-dependent DNA damage protection potential in the range of 7.5~60.0 μM. The DNA damage protective effect of the identified compounds was significantly correlated with the hydroxyl radical scavenging activity. Compounds that exhibited effective (IC50 = 5.4~12.5 μg/mL) hydroxyl radical scavenging activity were found to be the ones with higher DNA damage protection potential.

Flemingia philippinensis has been used throughout history to cure rheumatism associated with neutrophil elastase (NE). In this study, we isolated sixteen NE inhibitory flavonoids (1-16), including the most potent and abundant prenyl isoflavones (1-9), from the F. philippinensis plant. These prenyl isoflavones (2, 3, 5, 7, and 9) competitively inhibited NE, with IC50 values of 1.3-12.0 μM. In addition, they were reversible, simple, slow-binding inhibitors according to their respective parameters. Representative compound 3 had an IC50 = 1.3 μM, k3 = 0.04172 μM-1 min-1, k4 = 0.0064 min-1, and Kiapp = 0.1534 μM. The Kik/Kiv ratios (18.5 ∼ 24.6) for compound 3 were consistent with typical competitive inhibitors. The prenyl functionality of isoflavones significantly affected inhibitory potencies and mechanistic behavior by shifting the competitive mode to a noncompetitive one. The remaining flavonoids (10-16) were confirmed as mixed type I inhibitors that preferred to bind free enzyme rather than the enzyme-substrate complex. Fluorescence quenching analyses indicated that the inhibitory potency (IC50) closely followed the binding affinity (KSV).

Human neutrophil elastase (HNE) represents a goodtherapeutic target for the treatment of inflammatory diseases aswell as invasion of microorganism. The methanol extract of aaerial part of Chelidonium majus L. showed high activity againstthe neutrophil elastase with an IC 50 value of 100µg/mL. Due toits potency, subsequent bioactivity-guided fractionation ofmethanol extract led to six alkaloids (1-6), which were identifiedas dihydrosanguinarine (1), (s)-stylopine (2), arnottianamide (3),(+)-chelidonine (4), spallidamine (5), and N-trans-feruloyltyramine (6). Among of them, three alkaloids (2, 5, and 6)inhibited HNE in a dose-dependent manner with IC 50 rangingbetween 11.6 and 51.0µM. Lineweaver-Burk and Dixon plots,and their secondary replots showed that alkaloids (2, 5, and 6)were mixed inhibitors of HNE. The analysis of K I and K IS valueproved that all inhibitors (2, 5, and 6) had reversible mixed typeI mechanism.Keywords Chelidonium majus L. · human neutrophil elastase ·inflammation · isoquinoline alkaloidIntroductionThe regulation of the enzymatic activity of human neutrophilelastase (HNE) has been the attractive field because neutrophilsare the first cells recruited to inflammatory sites and from theearliest line of defense against the invasion of microorganism(Brice et al., 2010). The human neutrophil elastase (EC 3. 4. 21.37) is the family of serine proteases that possess the ability tohydrolyze the extracellular matrix protein (Siedle et al., 2007). Itis present in azurophil granules in the neutrophil cytoplasm. HNEis a proteolytic enzyme involved in pathogenesis of emphysema,adult respiratory distress syndrome and rheumatoid arthritis(Crocetti et al., 2013). The catalytic site of HNE molecule iscomposed of the triad His41-Asp99-Ser173, of which the oxygenof serine attacks carbonyl group on the target substrate (Bode etal., 1989). The activity of HNE is controlled by inhibitor namedα1-antitrypsin produced in the liver. However, their HNE affinityis strongly decreased by oxidative stress and by proteases releasedfrom leukocytes that are recruited to inflammation sites (Brice etal., 2010). Thus, the imbalance between HNE and its inhibitors isimplicated in many inflammation diseases as like pulmonaryemphysema.Chelidonium majus L. is a perennial herbaceous plant of thefamily Papaveraceae, which is widely distributed around theworld. This plant has been traditionally used as an herbal medicinefor treatment of gastric ulcer, oral infection and liver disease(Lenfield et al., 1981). These effects are mainly due to the alkaloidspresent in milk sap. The major bioactive components of this plantare isoquinoline alkaloids such as chelidonine, chelerythrine,sanguianarine, bebeerine and coptisine (Barreto et al., 2003). Itincluded flavonoids and various acids such as ferulic, cumaric,caffeic and chelidonic acids (Barens J et al., 2007). The constituentsof C. majus L. have been found to exhibit antitumor, antiviral, andanti-inflammatory activities (Colombo and Bosisio, 1996). Additionally,the potent and selective acetylcholinesterase inhibition wasobserved on 8-hydroxydihydrochelerythrine in C. majus L (Cho etal., 2006).During a screening procedure on higher plant to find neutrophilelastase inhibitors, methanol extract of C. majus L. was shown toexhibit considerable inhibitory activity. In this study, we isolatedsix alkaloids targeting to HNE from the methanol extract of aerial

Cratoxylum cochinchinense displayed significant inhibition against protein tyrosine phosphatase 1B (PTP1B) and α-glucosidase, both of which are key target enzymes to attenuate diabetes and obesity. The compounds responsible for both enzymes inhibition were identified as twelve xanthones (1–12) among which compounds 1 and 2 were found to be new ones. All of them simultaneously inhibited PTP1B with IC50s of (2.4–52.5 µM), and α-glucosidase with IC50 values of (1.7–72.7 µM), respectively. Cratoxanthone A (3) and γ-mangostin (7) were estimated to be most active inhibitors against both PTP1B (IC50 = 2.4 µM for 3, 2.8 µM for 7) and α-glucosidase (IC50 = 4.8 µM for 3, 1.7 µM for 7). In kinetic studies, all isolated xanthones emerged to be mixed inhibitors of α-glucosidase, whereas they behaved as competitive inhibitors of PTP1B. In time dependent experiments, compound 3 showed isomerization inhibitory behavior with following kinetic parameters: Kiapp = 2.4 µM; k5 = 0.05001 µM−1 S−1 and k6 = 0.02076 µM−1 S−1.

Protein tyrosine phosphatase 1B (PTP1B) plays important role in diabetes, obesity and cancer. The methanol extract of the gum resin of Garcinia hanburyi (G. hanburyi) showed potent PTP1B inhibition at 10µg/ml. The active compounds were identified as prenylated caged xanthones (1-9) which inhibited PTP1B in dose-dependent manner. Carboxybutenyl group within caged motif (A ring) was found to play a critical role in enzyme inhibition such as 1-6 (IC50s=0.47-4.69µM), whereas compounds having hydroxymethylbutenyl 7 (IC50=70.25µM) and methylbutenyl 8 (IC50>200µM) showed less activity. The most potent inhibitor, gambogic acid 1 (IC50=0.47µM) showed 30-fold more potency than ursolic acid (IC50=15.5µM), a positive control. In kinetic study, all isolated xanthones behaved as competitive inhibitors which were fully demonstrated with Km, Vmax and Kik/Kiv ratio. It was also proved that inhibitor 1 operated under the enzyme isomerization model having k5=0.0751µM-1S-1, k6=0.0249µM-1S-1 and Kiapp=0.499µM. To develop a pharmacophore model, we explored the binding sites of compound 1 and 7 in PTP1B. These modeling results were in agreement with our findings, which revealed that the inhibitory activities are tightly related to caged motif and prenyl group in A ring.

Tyrosinase inhibition may be a means to alleviate not only skin hyperpigmentation but also neurodegeneration associated with Parkinson's disease. In the course of metabolite analysis from tyrosinase inhibitory methanol extract (80% inhibition at 20 μg/ml) of Campylotropis hirtella, we isolated fourteen phenolic compounds, among which neorauflavane 3 emerged as a lead structure for tyrosinase inhibition. Neorauflavane 3 inhibited tyrosinase monophenolase activity with an IC50 of 30 nM. Thus this compound is 400-fold more active than kojic acid. It also inhibited diphenolase (IC50=500 nM), significantly. Another potent inhibitor 1 (IC50=2.9 μM) was found to be the most abundant metabolite in C. hirtella. In kinetic studies, compounds 3 showed competitive inhibitory behavior against both monophenolase and diphenolase. It manifested simple reversible slow-binding inhibition against monophenolase with the following kinetic parameters: Ki(app)=1.48 nM, k3=0.0033 nM(-1) min(-1) and k4=0.0049 min(-1). Neorauflavane 3 efficiently reduced melanin content in B16 melanoma cells with 12.95 μM of IC50. To develop a pharmacophore model, we explored the binding mode of neuroflavane 3 in the active site of tyrosinase. Docking results show that resorcinol motif of B-ring and methoxy group in A-ring play crucial roles in the binding the enzyme.