Presence of fatty acid synthase inhibitors in the rhizome of Alpinia officinarum hance

The galangal (the rhizome of Alpinia officinarum, Hance) is popular in Asia as a traditional herbal medicine. The present study reports that the galangal extract (GE) can potently inhibit fatty-acid synthase (FAS, E.C.2.3.1.85). The inhibition consists of both reversible inhibition with an IC50 value of 1.73 microg dried GE/ml, and biphasic slow-binding inactivation. Subsequently the reversible inhibition and slow-binding inactivation to FAS were further studied. The inhibition of FAS by galangin, quercetin and kaempferol, which are the main flavonoids existing in the galangal, showed that quercetin and kaempferol had potent reversible inhibitory activity, but all three flavonoids had no obvious slow-binding inactivation. Analysis of the kinetic results led to the conclusion that the inhibitory mechanism of GE is totally different from that of some other previously reported inhibitors of FAS, such as cerulenin, EGCG (epigallocatechin gallate) and C75.     

Differential effects of flavonoids on bovine kidney low molecular mass protein tyrosine phosphatase

Among the structurally related flavonoids tested on the bovine kidney low molecular weight protein tyrosine phosphatase (LMrPTP) activity, quercetin activated by about 2.6-fold the p-nitrophenyl-phosphate (p-NPP)-directed reaction, in contrast to morin that acted as a competitive inhibitor, with Ki values of 87, 73 and 50 μM for p-NPP, FMN, and tyrosine-phosphate, respectively. Other related flavonoids, such as rutin, kaempferol, catechin, narigin, phloretin and taxifolin did not significantly affect the LMrPTP activity.

The positions of the hydroxyl groups in the structures of the flavonoids were important for their distinct effects on LMrPTP activity. The hydroxyl groups at C3′ and C4′ and the presence of a double bond at C2 and C3 were essential for the activating effect of quercetin. The absence of the 3′-OH (kaempferol), absence of the double bond (taxifolin) and the presence of the sugar rutinose at the 3-OH (rutin) suppressed the effect of quercetin. The C2′- and C4′-hydroxyl groups, the presence of the double bond, and a C4-ketone group were important requirements for the inhibitory effects of morin.     

Influence of dietary flavonoids on the glycation of plasma proteins

It has been suggested that the increasing glycation in diabetes can influence the ability of plasma proteins to bind to small molecules. Herein, the influence of flavonoids on the glycation of plasma proteins was investigated. After being incubated with glucose at 37 °C, the levels of glycated albumin (HGA) were significantly improved in healthy human plasma proteins (HPP). The inhibitory effects of flavonoids against the formation of advanced glycation products (AGEs) in HPP were determined as: galangin > apigenin > kaempferol ≈ luteolin > myricetin > quercetin. After being combined with 20 μmol L?1 of quercetin for 11 days, the fresh plasma with δ-glucose caused 323.05-32.07% inhibition of HGA formation in type II diabetes plasma proteins (TPP). Luteolin showed weak inhibition of HGA formation in TPP. However, kaempferol, galangin and apigenin hardly inhibited the formation of HGA in TPP. These results showed that more hydroxyl groups on ring B of flavonoids will enhance the inhibitory effects on the HGA formation in TPP.     

Antifungal effects of the flavonoids kaempferol and quercetin: a possible alternative for the control of fungal biofilms

This study aimed to determine the minimum inhibitory concentration (MIC) of kaempferol and quercetin against planktonic and biofilm forms of the Candida parapsilosis complex. Initially, nine C. parapsilosis sensu stricto, nine C. orthopsilosis and nine C. metapsilosis strains were used. Planktonic susceptibility to kaempferol and quercetin was assessed. Growing and mature biofilms were then exposed to the flavonoids at MIC or 10xMIC, respectively, and theywere also analyzed by confocal laser scanning microscopy. The MIC ranges were 32-128 µg ml^?1 for kaempferol and 0.5-16 µg ml^?1 for quercetin. Kaempferol and quercetin decreased (P?<?0.05) the metabolic activity and biomass of growing biofilms of the C. parapsilosis complex. As for mature biofilms, the metabolic effects of the flavonoids varied, according to the cryptic species, but kaempferol caused an overall reduction in biofilm biomass. Microscopic analyses showed restructuring of biofilms after flavonoid exposure. These results highlight the potential use of these compounds as sustainable resources for the control of fungal biofilms.     

Evaluation of the inhibitory activities of aceraceous plants on fatty acid synthase

Fatty acid synthase (FAS) is a very significant lipogenic enzyme participating in energy metabolism in vivo and has been reported as a potential new therapeutic target for cancer treatment. The extracts from sixteen Aceraceae were prepared to assay their inhibitory activities against duck liver FAS and their correlated antitumor bioactivity. Their inhibition of FAS was composed of a reversible fast-binding inhibition, by which 0.41 μg/mL of the A. campestre extract inhibits 50% FAS activity, and an irreversible slow-binding inhibition with inactivation rate constants, k_obs, ranging between 1.5 × 10^{? 3} and 10.6 × 10^{? 3} min^{? 1}. Three Aceraceae extracts were selected from their smaller IC₅₀ values to study different type of inhibitions against the three substrates in the FAS overall reaction. As compared with other reported FAS inhibitors including EGCG with regard to inhibition constant and IC₅₀ value, the extracts appeared to be more efficient inhibitors, and exhibited a considerable inhibition against the growth of five types of cancer cells (China patent application number 200610088901.6), which may be related to the inhibition of lipogenesis in these cells.     

Evaluation of inhibition of fatty acid synthase by ursolic acid: Positive cooperation mechanism

The inhibitory effect of ursolic acid (UA) on fatty acid synthase (FAS, EC 2.3.1.85) was investigated. We found that UA potently inhibited the activity of FAS with a half-inhibitory concentration value (IC₅₀) of 6.0 μg/ml. The inhibition kinetic results showed that the inhibition of FAS by UA was competitive against acetyl-CoA and malonyl-CoA, but uncompetitive to NADPH. UA at low concentration slowly inactivated FAS, but FAS was fast inactivated by high concentration of UA in a positive cooperative manner. Moreover, NADPH significantly enhanced the inactivation of FAS by low concentration of UA, but NADPH slightly decreased the inactivation of FAS by high concentration of UA. Taken together, the results suggest that ursolic acid decreases the FAS activity through inactivation of acetyl/malonyl transferase. The combination of NADPH and KR domain promotes the inhibitory effect of UA on FAS.     

The extract of leaves of Acer truncatum Bunge: A natural inhibitor of fatty acid synthase with antitumor activity

Fatty acid synthase (FAS) has been identified as a potential antitumor target. The extract from the leaves of Acer truncatum Bunge (Extr) was prepared to assay its inhibitory activity against FAS, which was isolated from duck liver, and the correlated antitumor bioactivity. Its inhibition of FAS is composed of reversible fast-binding inhibition, IC₅₀ = 0.7 μg/ml, and irreversible slow-binding inhibition following saturation kinetics with a dissociation constant of 0.68 μg/ml and a limiting rate constant of 0.0288 min^{? 1}. The Extr exhibited different type of inhibitions against the three substrates in the FAS overall reaction. Compared with EGCG in inhibition constant and IC₅₀ value, the Extr appeared to be a more efficient inhibitor, and exhibited a considerable inhibition against the growth of four kinds of cancer cells (patent application number 200510068054.2). It was infered that the inhibitory activity is likely attributable to the co-operative effect of the components.     

Evaluation of the inhibitory activities of aceraceous plants on fatty acid synthase

Fatty acid synthase (FAS) is a very significant lipogenic enzyme participating in energy metabolism in vivo and has been reported as a potential new therapeutic target for cancer treatment. The extracts from sixteen Aceraceae were prepared to assay their inhibitory activities against duck liver FAS and their correlated antitumor bioactivity. Their inhibition of FAS was composed of a reversible fast-binding inhibition, by which 0.41 microg/mL of the A. campestre extract inhibits 50% FAS activity, and an irreversible slow-binding inhibition with inactivation rate constants, k(obs), ranging between 1.5 x 10(-3) and 10.6 x 10(-3) min(-1). Three Aceraceae extracts were selected from their smaller IC50 values to study different type of inhibitions against the three substrates in the FAS overall reaction. As compared with other reported FAS inhibitors including EGCG with regard to inhibition constant and IC50 value, the extracts appeared to be more efficient inhibitors, and exhibited a considerable inhibition against the growth of five types of cancer cells (China patent application number 200610088901.6), which may be related to the inhibition of lipogenesis in these cells.     

Screening and Inhibition Mechanism of Xanthine Oxidase Inhibitors in Ethanolic Extracts of Chimonanthus salicifolius Hu Leaves

This study aimed to evaluate the inhibition of the ethanol elutions of Chimonanthus salicifolius Hu leaves (CsHL) against xanthine oxidase (XO). The results of XO inhibition assay and enzymatic superoxide free radical scavenging assay in vitro showed that 70 % ethanol eluate (EE) had the best inhibitory effect and followed by 40 % EE. High performance liquid chromatograph analysis showed that quercetin and kaempferol were the potential active components of XO inhibition. The inhibition mechanism of quercetin and kaempferol on XO was investigated by kinetic analysis and fluorescence quenching titration assay. The molecular simulation further revealed that quercetin and kaempferol bind to XO mainly by hydrogen bonding and van der Waals, blocking the entry of substrates and leading to the inhibition of XO. In conclusion, the CsHL have inhibitory effects on XO activity, which provides a theoretical basis for relieving or preventing hyperuricemia and gout as a natural food or medicinal plant in the future.     

Elastase release by stimulated neutrophils inhibited by flavonoids: importance of the catechol group

Pathogenesis of chronic inflammatory diseases is associated with excessive elastase release through neutrophil degranulation. In the present study, inhibition of human neutrophil degranulation by four flavonoids (myricetin, quercetin, kaempferol, galangin) was evaluated by using released elastase as a biomarker. Inhibitory potency was observed in the following order: quercetin > myricetin > kaempferol = galangin. Quercetin, the most potent inhibitor of elastase release also had a weak inhibitory effect on the enzyme catalytic activity. Furthermore, the observed effects were highly dependent on the presence of a catechol group at the flavonoid B-ring. The results of the present study suggest that quercetin may be a promising therapeutic agent in the treatment of neutrophil-dependent inflammatory diseases.     

The extract of leaves of Acer truncatum Bunge: A natural inhibitor of fatty acid synthase with antitumor activity

Fatty acid synthase (FAS) has been identified as a potential antitumor target. The extract from the leaves of Acer truncatum Bunge (Extr) was prepared to assay its inhibitory activity against FAS, which was isolated from duck liver, and the correlated antitumor bioactivity. Its inhibition of FAS is composed of reversible fast-binding inhibition, IC50 = 0.7 microg/ml, and irreversible slow-binding inhibition following saturation kinetics with a dissociation constant of 0.68 microg/ml and a limiting rate constant of 0.0288 min(-1). The Extr exhibited different type of inhibitions against the three substrates in the FAS overall reaction. Compared with EGCG in inhibition constant and IC50 value, the Extr appeared to be a more efficient inhibitor, and exhibited a considerable inhibition against the growth of four kinds of cancer cells (patent application number 200510068054.2). It was infered that the inhibitory activity is likely attributable to the co-operative effect of the components.     

Induction of cancer cell apoptosis by flavonoids is associated with their ability to inhibit fatty acid synthase activity

The consumption of food products containing high amounts of flavonoids has been reported to lower the risk of various cancers. The mechanisms underlying the cancer-protective effects of these naturally occurring polyphenolic compounds, however, remain elusive. Based on our previous finding that the cytotoxic effect of the flavanol epigallocatechin-3-gallate on prostate cancer cells correlates with its ability to inhibit fatty acid synthase (FAS, a key lipogenic enzyme overexpressed in many human cancers), we examined the anti-lipogenic effects of a panel of 18 naturally occurring polyphenolic compounds. In addition to epigallocatechin-3-gallate, five other flavonoids, more particularly luteolin, quercetin, kaempferol, apigenin, and taxifolin, also markedly inhibited cancer cell lipogenesis. Interestingly, in both prostate and breast cancer cells, a remarkable dose-response parallelism was observed between flavonoid-induced inhibition of fatty acid synthesis, inhibition of cell growth, and induction of apoptosis. In support for a role of fatty acid synthesis in these effects, the addition of exogenous palmitate, the end product of FAS, markedly suppressed the cytotoxic effects of flavonoids. Taken together, these findings indicate that the potential of flavonoids to induce apoptosis in cancer cells is strongly associated with their FAS inhibitory properties, thereby providing a new mechanism by which polyphenolic compounds may exert their cancer-preventive and antineoplastic effects.     

In vitro inhibition of fatty acid synthase by 1,2,3,4,6-penta-O-galloyl-β-d-glucose plays a vital role in anti-tumour activity

1,2,3,4,6-Penta-O-galloyl-β-d-glucose (PGG) inhibits glioma cancer U251 cells, more strongly than MDA-MB-231 and U87 cells. In addition, PGG is transported across cancer cell membrane to further down-regulate FAS and activate caspase-3 in MDA-MB-231 cells. Compared with other FAS inhibitors, including catechin gallate and morin, PGG involves a higher reversible fast-binding inhibition with half-inhibitory concentration value (IC₅₀) of 1.16 μM and an irreversible slow-binding inhibition, i.e. saturation kinetics with a dissociation constant of 0.59 μM and a limiting rate constant of 0.16 min^−l. The major reacting site of PGG is on the β-ketoacyl reduction domain of FAS. PGG exhibits different types of inhibitions against the three substrates in the FAS overall reaction. The higher concentrations of PGG tested (higher than 20 μM) clearly altered the secondary structure of FAS by increasing the α-helix and induced a redshift in the FAS spectra. In addition, only PGG concentrations higher than 20 μM resulted in FAS precipitation.     

Complex I and cytochrome c are molecular targets of flavonoids that inhibit hydrogen peroxide production by mitochondria

Flavonoids can protect cells from different insults that lead to mitochondria-mediated cell death, and epidemiological data show that some of these compounds attenuate the progression of diseases associated with oxidative stress and mitochondrial dysfunction. In this work, a screening of 5 flavonoids representing major subclasses showed that they display different effects on H?O? production by mitochondria isolated from rat brain and heart. Quercetin, kaempferol and epicatechin are potent inhibitors of H?O? production by mitochondria from both tissues (IC?? approximately 1-2 μM), even when H?O? production rate was stimulated by the mitochondrial inhibitors rotenone and antimycin A. Although the rate of oxygen consumption was unaffected by concentrations up to 10 μM of these flavonoids, quercetin, kaempferol and apigenin inhibited complex I activity, while up to 100 μM epicatechin produced less than 20% inhibition. The extent of this inhibition was found to be dependent on the concentration of coenzyme Q in the medium, suggesting competition between the flavonoids and ubiquinone for close binding sites in the complex. In contrast, these flavonoids did not significantly inhibit the activity of complexes II and III, and did not affect the redox state of complex IV. However, we have found that epicatechin, quercetin and kaempferol are able to stoichiometrically reduce purified cytochrome c. Our results reveal that mitochondria are a plausible main target of flavonoids mediating, at least in part, their reported preventive actions against oxidative stress and mitochondrial dysfunction-associated pathologies.     

Differential effects of flavonoids on bovine kidney low molecular mass protein tyrosine phosphatase

Among the structurally related flavonoids tested on the bovine kidney low molecular weight protein tyrosine phosphatase (LMrPTP) activity, quercetin activated by about 2.6-fold the p-nitrophenyl-phosphate (p-NPP)-directed reaction, in contrast to morin that acted as a competitive inhibitor, with Ki values of 87, 73 and 50 microM for p-NPP, FMN, and tyrosine-phosphate, respectively. Other related flavonoids, such as rutin, kaempferol, catechin, narigin, phloretin and taxifolin did not significantly affect the LMrPTP activity. The positions of the hydroxyl groups in the structures of the flavonoids were important for their distinct effects on LMrPTP activity. The hydroxyl groups at C3' and C4' and the presence of a double bond at C2 and C3 were essential for the activating effect of quercetin. The absence of the 3'-OH (kaempferol), absence of the double bond (taxifolin) and the presence of the sugar rutinose at the 3-OH (rutin) suppressed the effect of quercetin. The C2'- and C4'-hydroxyl groups, the presence of the double bond, and a C4-ketone group were important requirements for the inhibitory effects of morin.     

Solid-phase extraction and gas chromatography–mass spectrometry determination of kaempferol and quercetin in human urine after consumption of Ginkgo biloba tablets

A method was developed for the quantification of the flavonoids quercetin and kaempferol in human urine using a solid-phase extraction procedure followed by gas chromatography–mass spectrometry. Deuterated internal standards of the analytes were spiked into the samples prior to extraction. The limit of detection of the method was ca. 10 pg on column and precision of the method for quantification in a sample of urine was ±9.40% for kaempferol and ±7.34% for quercetin (n=6). The levels of quercetin and kaempferol found in urine samples were only a small fraction of the amount ingested. The treatment of urine samples with β-glucuronidase markedly increased the levels of flavonoids detected, supporting the view that kaempferol and quercetin are eliminated in the urine as glucuronides.     

Structure-activity relationship of polyphenols that inhibit fatty acid synthase

Many flavone derivatives inhibit FAS, and their A and B rings play an important role, but is the C ring necessary for the inhibition of FAS? Here, using nordihydroguaiaretic acid (NDGA), with two phenyl rings connected by a four-carbon chain, as a representative, the structural basis for the inhibition of animal fatty acid synthase (FAS) by polyphenols was investigated. NDGA potently inhibits the overall reaction of FAS (IC(50) = 9.3 +/- 0.1 muM). The kinetic study indicated that NDGA inhibits FAS competitively with respect to acetyl-CoA, noncompetitively with respect to malonyl-CoA, and in a mixed manner with respect to NADPH. The inhibitory mechanism is the same as that of FAS flavonoid inhibitors. This suggests that the C ring of flavonoids is not essential for their FAS inhibitory effect. This conclusion was further confirmed by the results obtained for different polyphenols. A structure-activity relationship study indicated that a biphenyl core exists in all FAS polyphenol inhibitors. Thus, we propose a common model possibly shared by all FAS polyphenol inhibitors. The model includes two almost planar aromatic rings with their respective hydroxyl groups, and a proper ester linkage between the two rings that possibly causes the inhibition of FAS by irreversibly inhibiting the beta-ketoacyl reductase domain.     

Acylated flavonol tri- and tetraglycosides in the flavonoid metabolome of Cladrastis kentukea (Leguminosae)

The foliar metabolome of Cladrastis kentukea (Leguminosae) contains a complex mixture of flavonoids including acylated derivatives of the 3-O-rhamnosyl(1→2)[rhamnosyl(1→6)]-galactosides of kaempferol and quercetin and their 7-O-rhamnosides, together with an array of non-acylated kaempferol and quercetin di-, tri- and tetraglycosides. Thirteen of the acylated flavonoids, 12 of which had not been reported previously, were characterised by spectroscopic and chemical methods. Eight of these were the four isomers of kaempferol 3-O-α-l-rhamnopyranosyl(1→2)[α-l-rhamnopyranosyl(1→6)]-(3/4-O-E/Z-p-coumaroyl-β-d-galactopyranoside) and their 7-O-α-l-rhamnopyranosides, and three were isomers of quercetin 3-O-α-l-rhamnopyranosyl(1→2)[α-l-rhamnopyranosyl(1→6)]-(3/4-O-E/Z-p-coumaroyl-β-d-galactopyranoside) - the remaining 4Z isomer was identified by LC-UV-MS analysis of a crude extract. The final two acylated flavonoids characterised by NMR were the 3E and 4E isomers of kaempferol 3-O-α-l-rhamnopyranosyl(1→2)[α-l-rhamnopyranosyl(1→6)]-(3/4-O-E-feruloyl-β-d-galactopyranoside)-7-O-α-l-rhamnopyranoside while the 3Z and 4Z isomers were again detected by LC-UV-MS. Using the observed fragmentation behaviour of the isolated compounds following a variety of MS experiments, a further 18 acylated flavonoids were given tentative structures by LC-MS analysis of a crude extract. Acylated flavonoids were absent from the flowers of C. kentukea, which contained an array of non-acylated kaempferol and quercetin glycosides. Immature fruits contained kaempferol 3-O-α-rhamnopyranosyl(1→2)[α-rhamnopyranosyl(1→6)]-β-galactopyranoside and its 7-O-α-rhamnopyranoside as the major flavonoids with acylated flavonoids, different from those in the leaves, only present as minor constituents. The presence of acylated flavonoids distinguishes the foliar flavonoid metabolome of C. kentukea from that of a closely related legume, Styphnolobium japonicum, which contains a similar range of non-acylated flavonoids.     

Suppression of fatty acid synthase, differentiation and lipid accumulation in adipocytes by curcumin

Curcumin is a well-known component of the cook seasoning and traditional herb turmeric (Curcuma longa), which has been reported to prevent obesity. However, the mechanism still remains to be determined. In this study, curcumin is found to be an effective inhibitor of fatty acid synthase (FAS), and its effects on adipocytes are further evaluated. Curcumin shows both fast-binding and slow-binding inhibitions to FAS. Curcumin inhibits FAS with an IC?? value of 26.8 μM, noncompetitively with respect to NADPH, and partially competitively against both substrates acetyl-CoA and malonyl-CoA. This suggests that the malonyl/acetyl transferase domain of FAS possibly is the main target of curcumin. The time-dependent inactivation shows that curcumin inactivates FAS with two-step irreversible inhibition, a specific reversible binding followed by an irreversible modification by curcumin. Like other classic FAS inhibitors, curcumin prevents the differentiation of 3T3-L1 cells, and thus represses lipid accumulation. In the meantime, curcumin decreases the expression of FAS, down-regulates the mRNA level of PPARγ and CD36 during adipocyte differentiation. Curcumin is reported here as a novel FAS inhibitor, and it suppresses adipocyte differentiation and lipid accumulation, which is associated with its inhibition of FAS. Hence, curcumin is considered to be having potential application in the prevention of obesity.     

Purification and Identification of Flavonoids from the Yellow Green Cocoon Shell (Sasamayu) of the Silkworm,Bombyx mori

Three quercetin glycosides, quercetin 5-O-β-D-glucoside, quercetin 7-O-β-D-glucoside, and quercetin 4′-O-β-D-glucoside, and two kaempferol glycosides, kaempferol 5-O-β-D-glucoside and kaempferol 7-O-β-D-glucoside, along with their aglycones, quercetin and kaempferol, were isolated from an ethanolic extract of Sasamayu cocoon shells. The chemical structures were characterized by chemical and spectroscopic methods including UV spectrometry and HPLC-ESI-MS. The five flavonol glycosides of the shell are different structurally from those of the leaves of mulberry (Morus alba). It was suggested that potent antioxidative activity in the cocoon is mainly due to flavonoid compounds since free radical scavenging activity was found in the cocoon flavonoids identified here.