Inhibition of neuraminidase activity by polyphenol compounds isolated from the roots of Glycyrrhiza uralensis

We isolated 18 polyphenols with neuraminidase inhibitory activity from methanol extracts of the roots of Glycyrrhiza uralensis. These polyphenols consisted of four chalcones (1–4), nine flavonoids (5–13), four coumarins (14–17), and one phenylbenzofuran (18). When we tested the effects of these individual compounds and analogs thereof on neuraminidase activation, we found that isoliquiritigenin (1, IC₅₀ = 9.0 μM) and glycyrol (14, IC₅₀ = 3.1 μM) had strong inhibitory activity. Structure–activity analysis showed that the furan rings of the polyphenols were essential for neuraminidase inhibitory activity, and that this activity was enhanced by the apioside group on the chalcone and flavanone backbone. In addition, the presence of a five-membered ring between C-4 and C-2′ in coumestan was critical for neuraminidase inhibition. All neuraminidase inhibitors screened were found to be reversible noncompetitive inhibitors.     

新疆石榴皮多酚的提取及其对酪氨酸酶的抑制作用

优化新疆石榴皮多酚的提取工艺,并研究石榴皮提取物对酪氨酸酶的抑制作用。采用超声波辅助法提取石榴皮多酚,通过单因素试验和正交试验确定其最佳提取工艺条件:丙酮体积分数70%、超声波功率200 W、提取温度40℃、料液比1∶35 g/m L、提取时间50 min、提取2次,在此条件下石榴皮多酚得率为23.77 mg/g。同时测定了石榴皮多酚提取物对蘑菇酪氨酸酶的抑制活性,石榴皮多酚提取物质量浓度在12 mg/m L时,对酪氨酸酶活力的相对抑制率为44.05%。该研究为从石榴皮中提取天然酪氨酸酶抑制剂提供了实验依据。     

Inhibition of tyrosinase by indole compounds and reaction products protection by albumin

Tyrosinase activity decreases as the reaction proceeds and is inhibited by L-3,4-dihydroxyphenylalanine oxidation products. Indole and tryptophan inhibit tyrosinase reaction and bovine albumin protects against end-products(s) inhibiton or inactivation. Since the same tyrosinase reaction products are indole compounds and some authors reported the binding of indole derivatives with albumin, it is here suggested that indole intermediates of melanin synthesis inhibit or inactivate tyrosinase.     

Competitive neutrophil elastase inhibitory isoflavones from the roots of Flemingia philippinensis

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 K_i^app = 0.1534 μM. The K_ik/K_iv 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 (K_SV).     

Bacterial neuraminidase inhibitory effects of prenylated isoflavones from roots of Flemingia philippinensis

Bacterial neuraminidase (NA) is one of the key enzymes involved in pathogenesis of inflammation during infection. The organic extract of the roots of Flemingia philippinensis showed high bacterial NA inhibitory activity with an IC₅₀ of around 5 μg/mL. Activity-guided separation of the methanol extract yielded nine prenylated isoflavones together with the novel species isoflavone (2) which was given the name flemingsin. Isolated prenylated isoflavones (1–9) were evaluated for NA inhibition and their IC₅₀ values were determined to range between 0.30 and 56.8 μM. The most potent inhibitor 4 (IC₅₀ = 300 nM, K_i = 130 nM) features a catechol motif in the B-ring and a furan in the A-ring. Structure–activity analysis also showed a 4-hydroxyl group within the B-ring was essential for NA inhibitory activity, because isoflavone (9) having protected 4-hydroxyl group was much less potent than its hydroxylated counterpart. All neuraminidase compounds screened were found to be reversible noncompetitive inhibitors. Furthermore, the most active NA inhibitors (1–9) were proven to be present in the native roots in high quantities by HPLC and LC-DAD-ESI/MS.     

Effect of p-aminophenols on tyrosinase activity

Tyrosinase is involved in the synthesis of melanin in the skin and hair as well as neuromelanin in the brain. This rate limiting enzyme catalyzes two critical steps (reactions) in melanogenesis; the hydroxylation of tyrosine to form DOPA and the subsequent oxidation of DOPA into dopaquinone. Several new aminophenol derivatives have been synthesized based on structure–activity relationship studies of N-(4-hydroxyphenyl)retinamide (1), a derivative of retinoic acid. In order to find new tyrosinase inhibitors, we investigated the effects of these p-aminophenols, including p-decylaminophenol (3), on the activity of mushroom tyrosinase. Compound 3 was the most potent agent, showing significant inhibition as compared with control. The inhibitory effects of 3 on tyrosinase activities were greater than seen with kojic acid, a well-known potent inhibitor of tyrosinase activity, which also causes adverse effects, including rash and dermatitis. A Lineweaver–Burk kinetic analysis of inhibition showed that 3 suppresses tyrosinase activity in a non-competitive fashion for both substrates, tyrosine and DOPA. These results suggest that 3 might be a useful alternative to kojic acid as a tyrosinase inhibitor.     

In vitro anti-rotavirus activity of polyphenol compounds isolated from the roots of Glycyrrhiza uralensis

We evaluated the ability of six polyphenols isolated from the roots of Glycyrrhiza uralensis to inactivate rotaviruses, specially G5P[7] and G8P[7]. Upon finding that all polyphenols possessed anti-rotavirus activity, we evaluated whether these properties were attributable to direct inhibition of the binding of rotavirus to cells and/or to inhibition of viral replication. Using the virucidal assay, we found that all six compounds directly inhibited rotavirus binding, with activity being dependent on the type of virus. The 50% effective inhibitory concentrations (EC₅₀) of the six compounds were 18.7–69.5 μM against G5P[7] and 14.7–88.1 μM against G8P[7], respectively. Five of the six compounds inhibited hemagglutination activity. Moreover, the CPE inhibition assay showed that five compounds inhibited viral replication with EC₅₀ values of 12.1–24.0 μM against G5P[7] and 12.0–42.0 μM against G8P[7], respectively. RT-PCR showed that the compounds suppressed viral RNA synthesis in TF-104 cells. Interestingly, the anti-rotavirus activities of four compounds were attributable to inhibition of both viral absorption and viral replication. These results suggest that compounds isolated from the roots of G. uralensis may be potent anti-rotavirus agents in vivo, acting by inhibiting both viral absorption and viral replication.     

Effects of hydroxystilbene derivatives on tyrosinase activity

Synthesis of melanin starts from the conversion of L-tyrosine to 3,4-dihydroxyphenylalanine (L-dopa) and then the oxidation of L-dopa yields dopaquinone by tyrosinase. Therefore, tyrosinase inhibitors have been established as important constituents of depigmentation agents. Recently, polyhydroxystilbene compounds, which are trans-resveratrol (3,4('),5-trihydroxy-trans-stilbene) analogs, have been demonstrated as potent tyrosinase inhibitors. However, their detailed inhibitory mechanisms are not clearly understood. In the present study, a variety of synthesized hydroxystilbene compounds were tested for their inhibitory effects against murine tyrosinase activity. The inhibitory potencies of the hydroxy-trans-stilbene compounds were remarkably elevated by increasing number of phenolic hydroxy substituents. Methylated hydroxy-trans-stilbene lost the inhibitory activity. Furthermore, hydrogenated hydroxystilbene or hydroxy-cis-stilbene exerted little or no inhibitory effect compared with hydroxy-trans-stilbene on tyrosinase activity. The structure-activity relationships demonstrated in the present study suggest that the phenolic hydroxy groups and trans-olefin structure of the parent stilbene skeleton contribute to the inhibitory potency of hydroxystilbene for tyrosinase activity.     

Polyamine derivatives from the bee pollen of Quercus mongolica with tyrosinase inhibitory activity

Eighteen constituents, including nine new compounds, were isolated from the bee pollen of Quercus mongolica. The structures of the new compounds were established on the basis of combined spectroscopic analysis. Structurally, the nine new compounds are polyamine derivatives with phenolic moieties which were assigned as one putrescine derivative, mogolicine A (2), seven spermidine derivatives, mongolidines A-G (3–5, 8, 12, 14, 17) and one spermine derivative, mogoline A (18). Evaluation of the biological activity of isolated compounds revealed that the polyamine derivatives with coumaroyl and caffeoyl moieties showed tyrosinase inhibition with IC₅₀ values of 19.5–85.8 μM; however, the addition of a methoxy group to phenolic derivatives reduced the inhibitory activity.     

Inhibition of tyrosinase reduces cell viability in catecholaminergic neuronal cells

The biosynthesis of dopamine (DA) in catecholaminergic neurons is regulated by tyrosine hydroxylase, which converts tyrosine into 3, 4-dihydroxyphenylalanine (L-DOPA). In melanocytes, tyrosinase catalyzes both the hydroxylation of tyrosine and the consequent oxidation of L-DOPA to form melanin. Although it has been demonstrated that tyrosinase is also expressed in the brain, the physiological role of tyrosinase in the brain is still obscure. In this study, to investigate the role of tyrosinase in catecholaminergic neuronal cells, we examined the effects of tyrosinase inhibition on the viability of CATH.a and SH-SY5Y cells using tyrosinase inhibitors-specifically, phenylthiourea (PTU) and 5-hydroxyindole (5-HI)-and the transfection of antisense tyrosinase cDNA. Both inhibitors significantly reduced the cell viability of CATH.a cells in a dose-dependent manner. PTU also specifically enhanced DA-induced cell death, but 5-HI did not. This discrepancy in cell death is probably due to the inhibitors' different mechanism of action: 5-HI inhibits the hydroxylation of tyrosine as a competitor for the substrate to induce cell death that may be due to depletion of DA, whereas PTU mainly inhibits the enzymatic oxidation of L-DOPA and DA rather than tyrosine hydroxylation to increase consequently autooxidation of DA. Indeed, the intracellular DA content in CATH.a cells was enhanced by PTU exposure. In contrast, PTU showed no enhancing effects on DA-induced cell death of SH-SY5Y cells, which express little tyrosinase. Furthermore, transfection with antisense tyrosinase cDNA into CATH.a cells dramatically reduced cell viability and significantly enhanced DA-induced cell death. These results suggest that tyrosinase controls the intracellular DA content by biosynthesis or enzymatic oxidation of DA, and the dysfunction of this activity induces cell death by elevation of intracellular DA level and consequent gradual autooxidation of DA to generate reactive oxygen species.     

Chemical synthesis and tyrosinase inhibitory activity of rhododendrol glycosides

The concise synthesis of rhododendrol glycosides 3–8, which are novel derivatives of (+)-epirhododendrin (1) and (−)-rhododendrin (2), has been achieved in six steps from benzaldehyde 9. The key reactions include aldol condensation and trichloroacetimidate glycosylation. From biological studies, it has been determined that synthetic derivatives of 1 and 2 possess potent tyrosinase inhibitory activity. Particularly, the inhibitory activity of cellobioside 8 (IC₅₀ = 1.51 μM) is six times higher than that of kojic acid. The R-epimers (4, 6, and 8) possessed more potent activity than the corresponding S-epimers (3, 5, and 7), indicating that tyrosinase inhibitory activity is significantly governed by stereochemistry of rhododendrol glycosides.     

Indirect oxidation of 6-tetrahydrobiopterin by tyrosinase

6-Tetrahydrobiopterin is known to bind to an allosteric site of tyrosinase to directly inhibit the enzyme. However, simultaneous measurements of ultraviolet-visible absorption spectra and oxygen consumption led us to conclude that the inhibition was due to oxidation of 6-tetrahydrobiopterin by dopaquinone. Immediately after addition of 6-tetrahydrobiopterin, tyrosinase stopped producing dopachrome from either tyrosine or dopa. Duration of inhibition was proportional to the concentration of added 6-tetrahydrobiopterin and the enzyme activity was fully restored after the inhibition. Surprisingly, there was a rapid consumption of oxygen during the inhibition period. In addition, absorption spectra indicated that the only reaction that occurred during the inhibition was oxidation of 6-tetrahydrobiopterin to 7,8-dihydrobiopterin. In the absence of tyrosine or dopa, tyrosinase did not oxidize 6-tetrahydrobiopterin, suggesting that a reaction intermediate between dopa and dopachrome was a target for the inhibition. We propose a new mechanism in which dopa is oxidized to dopaquinone and the latter, instead of producing dopachrome, is reduced back to dopa by 6-tetrahydrobiopterin.     

Synthesis and tyrosinase inhibition activity of trans-stilbene derivatives

Synthesis of a focussed library of trans-stilbene compounds through Wittig and other base catalysed condensation reactions is presented. The synthesized stilbenes were screened for their inhibitory potential against murine tyrosinase activity to explore the structure activity relationship (SAR). Presence of electron withdrawing group (–CN) at the double bond and hydroxyl group or halogen atom especially at para-position on the aromatic rings was found to significantly elevate the inhibitory activity. Among all the compounds screened, compounds 2, 6, 8, 10, 11, 15 and 21 were found to exhibit appreciable inhibitory activity. Compound 21 ((E)-2,3-bis(4-Hydroxyphenyl)acryonitrile) was found to be the most active with an IC₅₀ value of 5.06 μM which is less than half of the value 10.78 μM observed for resveratrol (common standard used in murine tyrosinase activity studies) under similar conditions. The results obtained from the present study reveal structural/functional group sensitivity for the tyrosinase inhibitory activity of stilbenoid moieties and are expected to be very helpful for the design and synthesis of novel, selective and effective tyrosinase inhibitors.     

pH-dependent inhibition of mushroom tyrosinase by N-substituted N-nitrosohydroxylamines

Several synthetic N-substituted N-nitrosohydroxylamines were found to inhibit mushroom tyrosinase in a pH-dependent manner regardless of the N-substituent. The inhibitory activity, or pI₅₀ ( ? log [IC₅₀, M]) value, linearly decreased as the pH of the media increased. The inhibitory activities of tested N-substituted N-nitrosohydroxylamines at pH 6.8 and 5.8 were found to be almost 10 times and 100 times greater than at pH 7.8, respectively. The types of inhibition were different at pH 6.8 and 5.8. These results suggest that the inhibitory effect of N-substituted N-nitrosohydroxylamines is caused by the non-ionized form of the inhibitor. Furthermore, the mechanism of inhibition depends on the interaction between the inhibitor and the active site of tyrosinase at different pH values.     

New tyrosinase inhibitory sesquiterpenes from Chloranthus henryi

Chemical investigation of the leaves and stems of Chloranthus henryi resulted in the isolation and characterization of two new eudesmane-type sesquiterpenes, and two new germacrane-type sesquiterpenes, together with two known compounds. The inhibitory activities against tyrosinase of all isolates were also evaluated.     

Chemical Composition of Roots Flemingia philippinensis and Their Inhibitory Kinetics on Aromatase

Aromatase is the key enzyme responsible for catalyzing the conversion of C₁₉ steroids to estrogens. Its inhibitors are widely used in breast cancer therapy. The CH₂Cl₂ partition of a crude ethanolic extract from the roots of Flemingia philippinensis showed potent inhibitory activity of aromatase. The constituents of the extract were analyzed and identified by liquid chromatography tandem mass spectrometry. Five purified prenylated isoflavones were evaluated for aromatase inhibition and their IC₅₀ values ranged between 2.98 and 58.08 μm . In kinetic studies, all tested compounds behaved as reversible competitive inhibitors and their K_i values were calculated by Dixon plots. The most potent inhibitor (6,8‐diprenylorobol) had a K_i value of 1.42 μm . Furthermore, using UPLC and LC/MS, 6,8‐diprenylorobol was proven to be present in the native roots in high quantities.     

Synthesis and tyrosinase inhibitory activity of novel N-hydroxybenzyl-N-nitrosohydroxylamines

Several novel N-substituted N-nitrosohydroxylamines were synthesized. They all inhibited mushroom tyrosinase, but the type of inhibition was different depending on the substituent. Some N-(mono- or dihydroxybenzyl)-N-nitrosohydroxylamines exhibited uncompetitive inhibition with respect to L-dopa. Among them, compound 6 was also a competitive inhibitor with respect to oxygen. This observation suggests that another interaction by the meta- or para-hydroxyl group might stabilize the binding of the inhibitor to the enzyme through the oxygen binding site.     

Proteolytic processing of polyphenol oxidase from plants and fungi

Polyphenol oxidase (PPO), a metalloenzyme containing a type-3 copper center, is produced by many species of plants, fungi, and bacteria. There is great variability in the subunit molecular mass reported for PPO, even from a single species. In some cases, experimental evidence (usually protein sequencing by Edman degradation) indicates that the variability in molecular mass for PPO from a given species is the result of proteolytic processing at the N and/or C-termini of the protein. In order to identify specific sequence regions where proteolysis occurs in PPO from most species, the experimentally established N and C-termini of these proteolyzed enzymes were compared to the protein sequences of other PPOs for which the N and C-termini have not been established by protein sequencing methods. In all cases the N-terminal proteolysis sites were located prior to a conserved arginine residue, and the C-terminal proteolysis sites were located following a conserved tyrosine motif. Based on the sites of proteolysis, molecular masses were calculated for the enzymes, and the calculated values were used to rationalize the varying molecular masses reported in the literature. To determine the structural implications of N and C-terminal proteolysis, the proteolysis sites were related to the two available PPO structures: Ipomoea batatas catechol oxidase and Streptomyces castaneoglobisporus tyrosinase. A structural “core” region that appears to be essential for structural stability and enzymatic activity was identified.     

Evaluation of new chalcone derivatives as polyphenol oxidase inhibitors

A newly series of 4-(phenylurenyl)chalcone (4a–j) and 4′-(phenylurenyl/thiourenyl)chalcone (9a–l) derivatives were synthesized and their inhibitory effects on the diphenolase activity of banana tyrosinase were evaluated. Tyrosinase has been purified from banana on an affinity gel comprised of Sepharose 4B-l-tyrosine-p-aminobenzoic acid. The result showed that 4a–j inhibited the PPO enzyme activity. Conversely, 9a–h and 9i–l showed activator effect on tyrosinase enzyme activity.