The antityrosinase and antioxidant activities of flavonoids dominated by the number and location of phenolic hydroxyl groups.

BACKGROUND: Compounds with the ability to scavenge reactive oxygen species (ROS) and inhibit tyrosinase may be useful for the treatment and prevention from ROS-related diseases. The number and location of phenolic hydroxyl of the flavonoids will significantly influence the inhibition of tyrosinase activity. Phenolic hydroxyl is indispensable to the antioxidant activity of flavonoids. Isoeugenol, shikonin, baicalein, rosmarinic acid, and dihydromyricetin have respectively one, two, three, four, or five phenolic hydroxyls. The different molecular structures with the similar structure to l-3,4-dihydroxyphenylalanine (l-DOPA) were expected to the different antityrosinase and antioxidant activities. METHODS: This investigation tested the antityrosinase activity, the inhibition constant, and inhibition type of isoeugenol, shikonin, baicalein, rosmarinic acid, and dihydromyricetin. Molecular docking was examined by the Discovery Studio 2.5 (CDOCKER Dock, Dassault Systemes BIOVIA, USA). This experiment also examined the antioxidant effects of the five compounds on supercoiled pBR322 plasmid DNA, lipid peroxidation in rat liver mitochondria in vitro, and DPPH, ABTS, hydroxyl, or superoxide free radical scavenging activity in vitro. RESULTS: The compounds exhibited good antityrosinase activities. Molecular docking results implied that the compounds could interact with the amino acid residues in the active site center of antityrosinase. These compounds also exhibited antioxidant effects on DPPH, ABTS, hydroxyl, or superoxide free radical scavenging activity in vitro, lipid peroxidation in rat liver mitochondria induced by Fe2+/vitamin C system in vitro, and supercoiled pBR322 plasmid DNA. The activity order is isoeugenol < shikonin < baicalein < rosmarinic acid < dihydromyricetin. The results showed the compounds with more phenolic hydroxyls have more antioxidant and antityrosinase activities. CONCLUSION: This was the first study of molecular docking for modeling the antityrosinase activity of compounds. This was also the first study of the protective effects of compounds on supercoiled pBR322 plasmid DNA, the lipid peroxidation inhibition activity in liver mitochondria. These results suggest that the compounds exhibited antityrosinase and antioxidant activities may be useful in skin pigmentation and food additives.

Hepatoprotective effects and antioxidant, antityrosinase activities of phloretin and phloretin isonicotinyl hydrazone.

BACKGROUND: Acute liver damage is primarily induced by one of several causes, among them viral exposure, alcohol consumption, and drug and immune system issues. Agents with the ability to inhibit tyrosinase and protect against DNA damage caused by reactive oxygen species (ROS) may be therapeutically useful for the prevention or treatment of ROS-related diseases. METHODS: This investigation examined the hepatoprotective effects of phloretin and phloretin isonicotinyl hydrazone (PIH) on d-galactosamine (D-GalN)-induced acute liver damage in Kunming mice, as well as the possible mechanisms. The serum levels of alanine aminotransferase (ALT), aspartate aminotransferase (AST), γ-glutamyl transferase (γ-GT), alkaline phosphatase (ALP), and total bilirubin (TB) as well as the histopathological changes in mouse liver sections were determined. The antioxidant effects of phloretin, quercetin, and PIH on lipid peroxidation in rat liver mitochondria in vitro, 1,1-diphenyl-2-picrylhydrazyl (DPPH) or 2,2-azino-bis-(3-ethylbenzthiazoline-6-sulphonic acid) (ABTS) free radical scavenging activity in vitro, and supercoiled pBR322 plasmid DNA were confirmed. The experiment also examined the antityrosinase activity, inhibition type, and inhibition constant of phloretin and PIH. RESULTS: Phloretin, quercetin, or PIH significantly prevented the increase in serum ALT, AST, γ-GT, ALP, and TB in acute liver damage induced by D-GalN, and produced a marked reduction in the histopathological hepatic lesions. Phloretin, quercetin, or PIH also exhibited antioxidant effects on lipid peroxidation in rat liver mitochondria in vitro, DPPH or ABTS free radical scavenging activity in vitro, and supercoiled pBR322 plasmid DNA. Phloretin, quercetin, or PIH also exhibited good antityrosinase activity. CONCLUSION: To the best of our knowledge, this was the first study of the hepatoprotective effects of phloretin and PIH on D-GalN-induced acute liver damage in Kunming mice as well as the possible mechanisms. This was also the first study of the lipid peroxidation inhibition activity of phloretin and PIH in liver mitochondria induced by the Fe(2+)/vitamin C (Vc) system in vitro, the protective effects on supercoiled pBR322 plasmid DNA, and the antityrosinase activity of phloretin and PIH.

Synthesis, characterization and biological evaluation of paeonol thiosemicarbazone analogues as mushroom tyrosinase inhibitors.

A series of hydroxy- and methoxy-substituted paeonol thiosemicarbazone analogues were synthesized as potential tyrosinase inhibitors and their inhibitory effects on mushroom tyrosinase and inhibitory mechanism were evaluated. Paeonol thiosemicarbazone analogues have been found exhibiting more remarkable inhibition than their indexcompounds on mushroom tyrosinase. Among them, compound 2,4-dihydroxy acetophenone-4-phenyl-3-thiosemicarbazone (d1) had the most potent inhibition activity with the IC50 value of 0.006 ± 0.001 mM, displayed as a reversible competitive inhibitor. The inhibitory ability of o- or p-substituted acetophenone thiosemicarbazones was: di-substituted acetophenone thiosemicarbazones>mono-substituted acetophenone thiosemicarbazones>non-substituted acetophenone thiosemicarbazones. Copper ions chelation assay explained that compound d1 exhibited competitive inhibition by forming a chelate with the copper ions at the catalytic domain of tyrosinase as well as indicate a 1.5:1 binding ratio of compound d1 with copper ions. In the fluorescence spectrum study, compound d1 behaved stronger fluorescence quenching on tyrosinase towards d1-Cu(2+) complex, inhibiting tyrosinase mainly by means of chelating the two copper ions in the active site. The newly synthesized compounds may serve as structural templates for designing and developing novel tyrosinase inhibitors.

[Expressions and significances of CD147, OPN and MMP-2 in oral squamous cell carcinoma].

OBJECTIVE: To evaluate the expressions and significances of CD147, OPN and MMP-2 in oral squamous cell carcinoma (OSCC). METHODS: The expressions of CD147, OPN and MMP-2 were detected by immunohistochemical method (SP) in 35 cases of ()SCC. The relationships between the expressions of CD147, OPN and MMP-2 and clinic histopathologic parameters of OSCC were analyzed. RESULTS: The expression rates of CD147, OPN and MMP-2 in OSCC tissues were 65.71%, 71.43% and 68.57% respectively. The expressions of CD147, OPN and MMP-2 were positively correlated with tumor histopathologic type, clinical stage and lymph node metastasis (P < 0.05), but not correlated with age, gender and the location of tumor (P > 0.05). The positive correlations were found among the expressions of CD147, OPN and MMP-2 (MMP-2 and CD147, r = 0. 653; MMP-2 and OPN, r = 0.540; CD147 and OPN, r = 0.381; P < 0.05). CONCLUSION: The expressions of CD147, OPN and MMP-2 increase in OSCC and may be the potential predictor of the malignant degree of OSCC.

[Study on the interactions of genisten esterified derivatives with bovine serum albumin].

Under the imitated physiological conditions (pH = 7.4), the interactions of two novel genistein esterified derivatives, genistein 7-acetylferulic acid ester and genistein 7, 4'-di-acetylferulic acid ester (1 and 2), with bovine serum albumin (BSA) were investigated by the fluorescence and UV-Vis spectroscopy. It was observed that both of them can effectively quench the intrinsic fluorescence of BSA. The results suggested that the fluorescence quenching process of BSA at low concentrations of the compounds may be mainly governed by static quenching mechanisms. The binding constants (KA) and the number of binding sites (n) at different temperatures were calculated. From the thermodynamic parameters, it can be judged that the binding of 1 to BSA involved electrostatic interactions, whereas the binding of 2 to BSA involved hydrogen bonds and Van der Waals forces. The binding average distances r between BSA (donor) and the compounds (acceptor) were determined to be 2.63 nm and 2.92 nm respectively based on the Forster theory. Besides, the interactions of BSA with the compounds did not change the conformation of BSA and the binding of compounds to BSA is near tryptophan subunit via synchronous fluorescence spectrometry.

[Research on the interactions between genistein and its glucosides with DNA].

The interactions of genistein (GEN), genistein glucoside (GENG) and genistein 7,4'-di-O-beta-D- glucoside(GEND) with calf thymus DNA (ctDNA) in Tris (pH 7.2) buffer were investigated by UV spectra, fluorescence spectra and viscosity. From the absorption titration experiments, no obvious red shifts were found, but the notable hypochromicities were observed. The pi-->pi* transitions of GEN at 262 nm showed a 10% decrease in intensity at [GEN]/[DNA] = 2, and for the GENG and GEND, the decreases were 24.8% and 18% at 260 and 258 nm, respectively. These results indicated that there were intercalations between these compounds and ctDNA, involving a strong pi-stacking interacting. The hypochromism of the two glucosides was bigger than that of GEN, which suggested that the two glucosides intercalated deeply into the DNA base pairs. The emission intensity of DNA-EB system at 600 nm decreased remarkably with increasing the three compounds, indicating that these compounds could intercalate into DNA and replace EB from the DNA-EB system. And at 25 and 37 degrees C, the fluorescence quenching curves of these compounds with DNA-EB system were not linear curves. According to the classical Stern-Volmer equation, it was not single static or dynamic quenching model, so there would be hydrogen bonding besides intercalation. Viscosity experiments were carried out by an Ubbelodhe viscometer at (20.0 +/- 0.1) degrees C. The relative viscosity of ctDNA increased steadily with increasing these compounds. The results clearly showed that these compounds could intercalate between DNA base pairs, causing an extension of the helix, and thus increased the viscosity of DNA. And because of the greatest increase in viscosity of the DNA, the interaction of GENG with DNA was the strongest, followed by GEND, and then GEN. The results were consistent with the above spectral results. These results suggested that genistein and its glucosides could bind to ctDNA partly by intercalation and hydrogen bonding mode, and the binding ability to ctDNA followed the order of GENG > GEND > GEN from which, the authors speculated that 7 or 4'-O-glycosylation modification maybe an effective way to improve medicinal activity of genistein, and its glucoside modified derivatives may be a promising candidate for anticancer drug, which deserves further research.

[Research on the interaction of Cr(III) complex of genistein with DNA].

The interaction of the Cr(III) complex of genistein (GEN-Cr) with calf thymus DNA (ctDNA) in Tris (pH 7.2) buffer was investigated using UV spectra, DNA melting, fluorescence spectra and viscosity. From the absorption titration experiment, no obvious red shift was found, but the notable hypochromicities were observed. When C(DNA)/C(GEN-Cr) = 3, the pi-pi* transitions of the complex at 272 nm showed a decrease in intensity of 29.1%, which indicated that there was remarkable intercalation between complex and DNA base pairs, involving a strong pi-stacking interacting between them. The binding constant for the complex was K = 1.9 x 10(5) mol x L(-1). From the melting curves of ctDNA in the absence and presence of the complex, the melting temperature of ctDNA was found to increase by 5.5 degrees C from 74 to 79.5 degrees C, owing to the increased stability of the helix in the presence of the complex that was intercalated into the double helix. The complex could emit weak luminescence in Tris buffer. The emission intensity of the complex at 340 nm increased steadily with the addition of ctDNA. The result suggested that the complex got into a hydrophobic environment inside the DNA and avoided the effect of solvent water molecules. The strong interaction of the complex and ctDNA also resulted in greatly enhanced intensity of the resonance light scattering spectra. The emission intensity of DNA-EB system at 600 nm decreased remarkably with increasing the complex concentration, which indicated that the complex could be intercalated into DNA and replace EB from the DNA-EB system. According to the classical Stern-Volmer equation, the quenching plots at 25 and 37 degrees C both appeared approximately linear. These results showed that there was one predominant quenching style in this process. Viscosity experiments were carried out by an Ubbelodhe viscometer at 20.0 (+/- 0.1) degrees C. The relative viscosity of ctDNA increased steadily with the increased in the complex. The result clearly showed that the complex could be intercalated between DNA base pairs, causing an extension of the helix, and thus increased the viscosity of DNA. The results above indicated that there is a relatively strong interaction between the GEN-Cr complex and ctDNA, and the complex could bind ctDNA mainly by intercalation. The research suggested that the GEN-Cr complex may be a promising candidate for anticancer, which deserves further research.

Effect of polysaccharides of cassiae seeds on the intestinal microflora of piglets.

The objective of this study was to examine if polysaccharides from Cassiae Seeds (PCS) can be used as prebiotics to improve the intestinal microflora of piglets with an in vitro and an in vivo trial. The in vitro trial was conducted to study the dose-response effect of PCS on the growth of E. coli 09 and Lactobacillus with traditional plate count method. The gradient culture mediums, containing 3.2, 1.6, 0.8, 0.4, 0.2, 0.1, 0.05, 0.025 and 0% PCS, were inoculated with E. coli09, Lactobacillus and cecum content, respectively. PCS had no influence on the growth of E. coli09 from rejuvenation fluid, but inhibited the growth of E. coli09 from cecum content when the concentration of PCS was higher than 0.1%. Lactobacillus counts were significantly increased with 0.1% PCS or higher (p< 0.05); and the largest increase was found with 0.8% PCS. With the inoculum of cecum content in the medium, Lactobacillus counts increased when the concentration of PCS was 0.4% and 0.8%, whilst E. coli 09 counts decreased. The in vivo trial was carried out to investigate the effect of PCS on the growth of E. coli 09 and Lactobacillus in piglets. Thirty six barrows (average initial BW = 6.5 kg) were randomly divided into 3 groups with 6 each, fed diets supplemented without or with 0.4% or 0.8% PCS. After 14 days, 3 piglets were slaughtered from each group; digesta samples were collected from the ileum, cecum and colon for detection of E.coli 09 and Lactobacillus with plate count method. Samples of the tissue and content of the cecum were taken for detection of caecal microflora profiles with Denaturing Gradient Gel Electrophoresis (DGGE) technique. The dietary inclusion of PCS increased Lactobacillus counts, but reduced E. coli 09 counts in digesta of ileum, cecum and colon of piglets. The dietary inclusion of 0.8% PCS significantly increased the number of electrophoresis brands of caecal bacterial microflora in mucosa and content of the cecum (p< 0.05). These results confirmed the dynamic change in the intestinal microflora profile with the dietary inclusion of PCS in piglets. Thus, PCS can be used as prebiotics to improve the intestinal microflora.

[Study on the multicomponent quantitative analysis using near infrared spectroscopy based on building elman model].

The present paper introduces an application of near infrared spectroscopy (NIRS) multi-component quantitative analysis by building a kind of recurrent network (Elman) model. Elman prediction model for phenylalanine (Phe), lysine (Lys), tyrosine (Tyr) and cystine (Cys) in 45 feedstuff samples was established with good veracity. Twelve peak value data from 3 principal components straight forward compressed from the original data by PLS were taken as inputs of Elman, while 4 predictive targets as outputs. Forty seven nerve cells were taken as hidden nodes with the lowest error compared with taking 43 and 45 nerve cells. Its training iteration times was supposed to be 1000. Predictive correlation coefficients by the model are 0.960, 0.981, 0.979 and 0.952. The results show that Elman using in NIRS is a rapid, effective means for measuring Phe, Lys, Tyr and Cys in feedstuff powder, and can also be used in quantitative analysis of other samples.

[Determination of four contents of feedstuff powder using near infrared spectroscopy by PLS-BP model].

Partial least squares (PLS) and artificial neural networks (ANN) prediction model for four components of feedstuff has been established with good veracity and recurrence. The spectra put into the model should be processed by second derivative and standard normal variate (SNV). Ten principal components compressed from original data by PLS and two peak values were taken as the inputs of Back-Propagation Network (BP), while four predictive targets as outputs, according to Kolmogorov theorem and experiment, and twenty three nerve cells were taken as hidden nodes. Its training iteration times was supposed to be 10,000. Prediction deciding coefficient of four components by the model are 0.9950, 0.9980, 0.9990 and 0.9670, while the standard deviation of an unknown sample scanned parallelly are 0.02774, 0.04853, 0.03292 and 0.02204.

[Multicomponent quantitative analysis using near infrared spectroscopy by building PLS-GRNN model].

The present paper introduces an application of near infrared spectroscopy(NIRS) multi-component quantitative analysis by building partial least squares (PLS)-generalized regression neural networks (GRNN) model. The PLS-GRNN prediction model for chlorine, fibre and fat in 45 feedstuff samples was established with good veracity and recurrence. Eight peak values in principal components compressed from original data by PLS and four in original spectra were taken as inputs of GRNN while 4 predictive targets as outputs. 0.1 was chosen as smoothing factor for its good approximation and prediction with the lowest error compared with 0.2, 0.3, 0.4 and 0.5. Predictive correlation coefficient and Standard error of the estimate of three components by the model are 0.984 0, 0.987 0 and 0.983 0, and 0.015 89, 0.154 1 and 0.115 1, while the Standard deviations of an unknown sample scanned 8 times are 0.003 26, 0.065 5 and 0.031 4. The results show that PLS-GRNN used in NIRS is a rapid, effective means for measuring chlorine, fibre in the fat in feedstuff powder, and can also be used in quantitative analysis of other samples. A settlement in the high error of prediction of other samples with lower contents was also shown.