Structure-activity relationship of flavonoids as influenza virus neuraminidase inhibitors and their in vitro anti-viral activities

Flavonoids are polyphenolic compounds that widely exist in plant kingdom, and the structure-activity relationship (SAR) of 25 flavonoids was studied on neuraminidase (NA) activity of influenza virus. Three typical influenza virus strains A/PR/8/34 (H1N1), A/Jinan/15/90 (H3N2), and B/Jiangshu/10/2003 were used as the source of NAs, the average of IC(50)s of these compounds on these NAs was used in the SAR analysis. The order of potency for NA inhibition was as follows: aurones>flavon(ol)es>isoflavones>flavanon(ol)es and flavan(ol)es. The SAR analysis of flavonoids on influenza virus NAs revealed that for good inhibitory effect, the 4'-OH, 7-OH, C4O, and C2C3 functionalities were essential, and the presence of a glycosylation group greatly reduced NA inhibition. The in vitro anti-viral activities of eight flavonoids were evaluated using a cytopathic effect (CPE) reduction method, the assay results confirmed the SAR as influenza virus neuraminidase inhibitors. The findings of this study provide important information for the exploitation and utilization of flavonoids as NA inhibitors for influenza treatment.     

Effects of intrinsic fluorescence and quenching on fluorescence-based screening of natural products

To evaluate the effects of intrinsic (natural) fluorescence and quenching as confounding variables in fluorescence-based enzyme inhibition assays of natural products, we measured the fluorescence and quenching properties of 25 components of popular herbal products. The analyses were performed under conditions typically employed in drug-drug interaction studies that use c-DNA-derived P450 isoforms and surrogate fluorogenic substrates. Four of the 25 compounds tested (isorhamnetin, quercetin, vitexin, and yangonin) fluoresced or quenched sufficiently to interfere with these assays. Intrinsic fluorescence had a greater effect on these assays than quenching and for one compound, yangonin, was sufficient to mask inhibition and potentially produce a false negative result. Quenching had less of an effect on these assays, but was significant enough for one compound, quercetin, to mimic "weak" inhibition. Therefore, because intrinsic fluorescence or quenching could render some natural products unsuitable for testing in certain fluorometric assays, it would be prudent to include an evaluation of these properties in experimental protocols.     

Inhibition of a Putative Dihydropyrimidinase from Pseudomonas aeruginosa PAO1 by Flavonoids and Substrates of Cyclic Amidohydrolases

Dihydropyrimidinase is a member of the cyclic amidohydrolase family, which also includes allantoinase, dihydroorotase, hydantoinase, and imidase. These metalloenzymes possess very similar active sites and may use a similar mechanism for catalysis. However, whether the substrates and inhibitors of other cyclic amidohydrolases can inhibit dihydropyrimidinase remains unclear. This study investigated the inhibition of dihydropyrimidinase by flavonoids and substrates of other cyclic amidohydrolases. Allantoin, dihydroorotate, 5-hydantoin acetic acid, acetohydroxamate, orotic acid, and 3-amino-1,2,4-triazole could slightly inhibit dihydropyrimidinase, and the IC₅₀ values of these compounds were within the millimolar range. The inhibition of dihydropyrimidinase by flavonoids, such as myricetin, quercetin, kaempferol, galangin, dihydromyricetin, and myricitrin, was also investigated. Some of these compounds are known as inhibitors of allantoinase and dihydroorotase. Although the inhibitory effects of these flavonoids on dihydropyrimidinase were substrate-dependent, dihydromyricetin significantly inhibited dihydropyrimidinase with IC₅₀ values of 48 and 40 μM for the substrates dihydrouracil and 5-propyl-hydantoin, respectively. The results from the Lineweaver−Burk plot indicated that dihydromyricetin was a competitive inhibitor. Results from fluorescence quenching analysis indicated that dihydromyricetin could form a stable complex with dihydropyrimidinase with the K_d value of 22.6 μM. A structural study using PatchDock showed that dihydromyricetin was docked in the active site pocket of dihydropyrimidinase, which was consistent with the findings from kinetic and fluorescence studies. This study was the first to demonstrate that naturally occurring product dihydromyricetin inhibited dihydropyrimidinase, even more than the substrate analogs (>3 orders of magnitude). These flavonols, particularly myricetin, may serve as drug leads and dirty drugs (for multiple targets) for designing compounds that target several cyclic amidohydrolases.     

Isolation and characterization of a novel flavonoid possessing a 4,2'-glycosidic linkage from green mature acerola (Malpighia emarginata DC.) fruit

The novel flavonoid, leucocyanidin-3-O-beta-D-glucoside, possessing a 4,2'-glycosidic linkage was isolated from green mature acerola (Malpighia emarginata DC.) puree and given the trivial name "aceronidin." To examine the functions of aceronidin, its antioxidative activity and both its alpha-glucosidase and alpha-amylase inhibition activities, as a potential inhibitor of the sugar catabolic enzyme, were evaluated against those of taxifolin, catechin, isoquercitrin and quercitrin which each have a similar structure. The 1,1-diphenyl-2-picrylhydrazyl (DPPH) radical quenching activity of aceronidin was stronger than that of alpha-tocopherol and comparable to that of flavonoids. In the yeast alpha-glucosidase inhibitory assay, aceronidin showed significantly greater inhibition than the other flavonoids tested. In the human salivary alpha-amylase inhibitory assay, aceronidin showed inhibition activity. Taken together, these results indicate aceronidin to be a potent antioxidant that may be valuable as an inhibitor of sugar catabolic enzymes.     

Effects of synthetic and naturally occurring flavonoids on mitogen-induced proliferation of human peripheral-blood lymphocytes

Examination was made of the effects of 17 synthetic and naturally occurring flavonoids on human lymphocyte proliferation in the presence of concanavalin A as a mitogen. Twelve of the flavonoids examined were mono-hydroxy or methoxy derivatives. The mitogen-induced response of lymphocytes was evaluated from the extent of the incorporation of [3H]thymidine into cells in vitro. All the compounds showed inhibitory effects; 4.5-77.7% of [3H]thymidine incorporation was blocked by an 1.0 micrograms/ml concentration. The viability of lymphocytes before and after treatment, as assessed by a dye exclusion test, indicated no change, and thus the flavonoids may inhibit DNA synthesis. The flavonoids possessing 5-hydroxyl, 5-methoxyl and 6-methoxyl groups, and those with cyclohexyl instead of phenyl substituent (i.e. 2-cyclohexyl-benzopyran-4-one), showed the greatest inhibition. The inhibitory effect of any one of them was less than one half that of prednisolone, but essentially the same or somewhat exceeding that of bredinine or azathioprine. It would thus appear that the well-known anti-inflammatory effects of flavonoids may possibly arise in part from the inhibition of the proliferative response of lymphocytes.     

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.     

Inhibition of alpha-glucosidase and amylase by luteolin, a flavonoid

Twenty-one naturally occurring flavonoids were tested for inhibitory activities against alpha-glucosidase (EC 3.2.1.20) and alpha-amylase (EC 3.2.1.1). Luteolin, amentoflavone, luteolin 7-O-glucoside, and daidzein were the strongest inhibitors among the compounds tested. Luteolin inhibited alpha-glucosidase by 36% at the concentration of 0.5 mg/ml and was stronger than acarbose, the most widely prescribed drug, in inhibitory potency, suggesting that it has the possibility to effectively suppress postprandial hyperglycemia in patients with non-insulin dependent diabetes mellitus. Luteolin also inhibited alpha-amylase effectively although it was less potent than acarbose. The clinical value of luteolin needs to be further evaluated.     

A Mass Spectrometry-Based Assay for Improved Quantitative Measurements of Efflux Pump Inhibition

Bacterial efflux pumps are active transport proteins responsible for resistance to selected biocides and antibiotics. It has been shown that production of efflux pumps is up-regulated in a number of highly pathogenic bacteria, including methicillin resistant Staphylococcus aureus. Thus, the identification of new bacterial efflux pump inhibitors is a topic of great interest. Existing assays to evaluate efflux pump inhibitory activity rely on fluorescence by an efflux pump substrate. When employing these assays to evaluate efflux pump inhibitory activity of plant extracts and some purified compounds, we observed severe optical interference that gave rise to false negative results. To circumvent this problem, a new mass spectrometry-based method was developed for the quantitative measurement of bacterial efflux pump inhibition. The assay was employed to evaluate efflux pump inhibitory activity of a crude extract of the botanical Hydrastis Canadensis, and to compare the efflux pump inhibitory activity of several pure flavonoids. The flavonoid quercetin, which appeared to be completely inactive with a fluorescence-based method, showed an IC₅₀ value of 75 μg/mL with the new method. The other flavonoids evaluated (apigenin, kaempferol, rhamnetin, luteolin, myricetin), were also active, with IC₅₀ values ranging from 19 μg/mL to 75 μg/mL. The assay described herein could be useful in future screening efforts to identify efflux pump inhibitors, particularly in situations where optical interference precludes the application of methods that rely on fluorescence.     

Inhibition of Alpha-glucosidase and Amylase by Luteolin,a Flavonoid

Twenty-one naturally occurring flavonoids were tested for inhibitory activities against alpha-glucosidase (EC 3.2.1.20) and alpha-amylase (EC 3.2.1.1). Luteolin, amentoflavone, luteolin 7-O-glucoside, and daidzein were the strongest inhibitors among the compounds tested. Luteolin inhibited alpha-glucosidase by 36% at the concentration of 0.5 mg/ml and was stronger than acarbose, the most widely prescribed drug, in inhibitory potency, suggesting that it has the possibility to effectively suppress postprandial hyperglycemia in patients with non-insulin dependent diabetes mellitus. Luteolin also inhibited alpha-amylase effectively although it was less potent than acarbose. The clinical value of luteolin needs to be further evaluated.     

Synthesis and in vitro study of novel neuraminidase inhibitors against avian influenza virus

Evidences of oseltamivir resistant influenza patients raised the need of novel neuraminidase inhibitors. In this study, five oseltamivir analogs PMC-31-PMC-36, synthesised according to the outcomes of a rational design analysis aimed to investigate the effects of substitution at the 5-amino and 4-amido groups of oseltamivir on its antiviral activity, were screened for their inhibition against neuraminidase N1 and N3. The enzymes used as models were from the avian influenza A H7N1 and H7N3 viruses. The neuraminidase inhibition assay was carried out by using recombinant species obtained from a baculovirus expression system and the fluorogenic substrate MUNANA. The assay was validated by using oseltamivir carboxylate as a reference inhibitor. Among the tested compounds, PMC-36 showed the highest inhibition on N1 with an IC(50) of 14.6±3.0nM (oseltamivir 25±4nM), while PMC-35 showed a significant inhibitory effect on N3 with an IC(50) of 0.1±0.03nM (oseltamivir 0.2±0.02nM). The analysis of the inhibitory properties of this panel of compounds allowed a preliminary assessment of a structure-activity relationship for the modification of the 4-amido and 5-amino groups of oseltamivir carboxylate. The substitution of the acetamido group in the oseltamivir structure with a 2-butenylamido moiety reduced the observed activity, while the introduction of a propenylamido group was well tolerated. Substitution of the free 5-amino group of oseltamivir carboxylate with an azide, decreased the activity against both N1 and N3. When these structural changes were both introduced, a dramatic reduction of activity was observed for both N1 and N3. The alkylation of the free 5-amino group in oseltamivir carboxylate introducing an isopropyl group seemed to increase the inhibitory effect for both N1 and N3 neuraminidases, displaying a more pronounced effect against N1.     

Inhibitory effects of adenine nucleotides and related substances on UDP-glucuronosyltransferase: structure-effect relationships and evidence for an allosteric mechanism

The inhibitory effects of nucleotides and related substances on rat hepatic UDP-glucuronosyltransferase (UGT) were studied. ATP and NADP+ markedly reduced 4-methylumbelliferone (4-MU) UGT activity only when detergent-treated rat liver microsomes were used as the enzyme source. The IC50 values of adenine, ATP, NAD+ and NADP+ were estimated to be below 20 microM, whereas AMP had no inhibitory effect. From the kinetic behavior observed, these adenine-related compounds were assumed to inhibit UGT activity non-competitively without competing with either 4-MU or UDP-glucuronic acid. Among guanine, cytosine and their related nucleotides, only triphosphate nucleotides (CTP and GTP) exhibited potent UGT inhibition, although the effect of GTP was weak. Estradiol 3- and 17-glucuronidation were also inhibited by the inhibitors of 4-MU UGT. The only exception was that estradiol 17-glucuronidation activity was inhibited by AMP (IC50=31 microM). In addition, AMP antagonized the inhibitory effects of adenine, ATP, and NADP+ on 4-MU and estradiol 3- glucuronidation activities. These results suggest that (1) a number of cellular nucleotides present within the endoplasmic reticulum regulate UGT function; and (2) these substances bind to a common allosteric site on UGT to reduce catalytic function.     

Evaluation of fluorescent compound interference in 4 fluorescence polarization assays: 2 kinases, 1 protease,and 1 phosphatase

With the increasing use of fluorescence-based assays in high-throughput screening (HTS), the possibility of interference by fluorescent compounds needs to be considered. To investigate compound interference, a well-defined sample set of biologically active compounds, LOPAC, was evaluated using 4 fluorescein-based fluorescence polarization (FP) assays. Two kinase assays, a protease assay, and a phosphatase assay were studied. Fluorescent compound interference and light scattering were observed in both mixture- and single-compound testing under certain circumstances. In the kinase assays, which used low levels (1-3 nM) of fluorophore, an increase in total fluorescence, an abnormal decrease in mP readings, and negative inhibition values were attributed to compound fluorescence. Light scattering was observed by an increase in total fluorescence and minimal reduction in mP, leading to false positives. The protease and phosphatase assays, which used a higher concentration of fluorophore (20-1200 nM) than the kinase assays, showed minimal interference from fluorescent compounds, demonstrating that an increase in the concentration of the fluorophore minimized potential fluorescent compound interference. The data also suggests that mixtures containing fluorescent compounds can result in either false negatives that can mask a potential "hit" or false positives, depending on the assay format. Cy dyes (e.g., Cy3B and Cy5 ) excite and emit further into the red region than fluorescein and, when used in place of fluorescein in kinase 1, eliminate fluorescence interference and light scattering by LOPAC compounds. This work demonstrates that fluorescent compound and light scattering interferences can be overcome by increasing the fluorophore concentration in an assay or by using longer wavelength dyes.     

New functional assay of P-glycoprotein activity using Hoechst 33342

In this study we describe a simplified, HTS-capable functional assay for the multidrug resistance (MDR) transporter P-glycoprotein (P-gp) based on its substrate Hoechst 33342. The physicochemical properties of Hoechst 33342 and the enormous milieu dependency of its fluorescence intensity allowed performing the assay in a homogeneous manner. This new assay served as an effective tool to estimate the potency of 10 well recognized P-gp substrates and modulators. Further, the potency of these compounds was also estimated in the calcein AM assay. The Hoechst 33342 and calcein AM assays yielded significantly comparable results for all compounds tested. Principal component analysis (PCA) applied to literature data on inhibition of P-gp activity and our results obtained in the Hoechst 33342 and calcein AM assay indicated similarity of compared functional transport assays. However, no correlation could be detected between these functional assays and the ATPase activity assay.     

A prenylated flavonol,sophoflavescenol: a potent and selective inhibitor of cGMP phosphodiesterase 5

During the search for naturally occurring cyclic guanosine monophosphate (cGMP)-specific phosphodiesterase type 5 (PDE5) inhibitors, it was found that the extracts from Sophora flavescens exhibit potent inhibitory activity against cGMP PDE5 prepared from rat diaphragm. Therefore, the inhibitory activities of five flavonoids, kushenol H (1), kushenol K (2), kurarinol (3), sophoflavescenol (4) and kuraridine (5), isolated from S. flavescens were measured against cGMP PDE5 to identify potent cGMP PDE5 inhibitory constituents. Among tested compounds, sophoflavescenol (4), a C-8 prenylated flavonol, showed the most potent inhibitory activity (IC(50)=0.013 microM) against cGMP PDE5 with 31.5- and 196.2-fold selectivity over PDE3 and PDE4, respectively. Kinetic analysis revealed that sophoflavescenol was a mixed inhibitor of PDE5 with a K(i) value of 0.005 microM.     

Antioxidative activity of natural products from plants

A variety of flavonoids, lignans, an alkaloid, a bisbenzyl, coumarins and terpenes isolated from Chinese herbs was tested for antioxidant activity as reflected in the ability to inhibit lipid peroxidation in rat brain and kidney homogenates and rat erythrocyte hemolysis. The pro-oxidant activities of the aforementioned compounds were assessed by their effects on bleomycin-induced DNA damage. The flavonoids baicalin and luteolin-7-glucuronide-6'-methyl ester, the lignan 4'-demethyldeoxypodophyllotoxin, the alkaloid tetrahydropalmatine, the bisbenzyl erianin and the coumarin xanthotoxol exhibited potent antioxidative activity in both lipid peroxidation and hemolysis assays. The flavonoid rutin and the terpene tanshinone I manifested potent antioxidative activity in the lipid peroxidation assay but no inhibitory activity in the hemolysis assay. The lignan deoxypodophyllotoxin, the flavonoid naringin and the coumarins columbianetin, bergapten and angelicin slightly inhibited lipid peroxidation in brain and kidney homogenates. It is worth stressing that the compounds with antioxidant effects in this assay, with the exception of tetrahydropalmatin and tanshinone I, have at least one free aromatic hydroxyl group in structure. Obviously, the aromatic hydroxyl group is very important for antioxidative effects of the compounds. None of the compounds tested exerted an obvious pro-oxidant effect.     

Inhibition of Hyaluronic Acid Synthesis Suppresses Angiogenesis in Developing Endometriotic Lesions

Background

The development and long-term survival of endometriotic lesions is crucially dependent on an adequate vascularization. Hyaluronic acid (HA) through its receptor CD44 has been described to be involved in the process of angiogenesis.

Objective

To study the effect of HA synthesis inhibition using non-toxic doses of 4-methylumbelliferone (4-MU) on endometriosis-related angiogenesis.

Materials and Methods

The cytotoxicity of different in vitro doses of 4-MU on endothelial cells was firstly tested by means of a lactate dehydrogenase assay. The anti-angiogenic action of non-cytotoxic doses of 4-MU was then assessed by a rat aortic ring assay. In addition, endometriotic lesions were induced in dorsal skinfold chambers of female BALB/c mice, which were daily treated with an intraperitoneal injection of 0.9% NaCl (vehicle group; n = 6), 20mg/kg 4-MU (n = 8) or 80mg/kg 4-MU (n = 7) throughout an observation period of 14 days. The effect of 4-MU on their vascularization, survival and growth were studied by intravital fluorescence microscopy, histology and immunohistochemistry.

Main Results

Non-cytotoxic doses of 4-MU effectively inhibited vascular sprout formation in the rat aortic ring assay. Endometriotic lesions in dorsal skinfold chambers of 4-MU-treated mice dose-dependently exhibited a significantly smaller vascularized area and lower functional microvessel density when compared to vehicle-treated controls. Histological analyses revealed a downregulation of HA expression in 4-MU-treated lesions. This was associated with a reduced density of CD31-positive microvessels within the lesions. In contrast, numbers of PCNA-positive proliferating and cleaved caspase-3-positive apoptotic cells did not differ between 4-MU-treated and control lesions.

Conclusions

The present study demonstrates for the first time that targeting the synthesis of HA suppresses angiogenesis in developing endometriotic lesions. Further studies have to clarify now whether in the future this anti-angiogenic effect can be used beneficially for the treatment of endometriosis.     

Flavonoids as protectors against doxorubicin cardiotoxicity: role of iron chelation, antioxidant activity and inhibition of carbonyl reductase

Anthracycline antibiotics (e.g. doxorubicin and daunorubicin) are among the most effective and widely used anticancer drugs. Unfortunately, their clinical use is limited by the dose-dependent cardiotoxicity. Flavonoids represent a potentially attractive class of compounds to mitigate the anthracycline cardiotoxicity due to their iron-chelating, antioxidant and carbonyl reductase-inhibitory effects. The relative contribution of various characteristics of the flavonoids to their cardioprotective activity is, however, not known. A series of ten flavonoids including quercetin, quercitrin, 7-monohydroxyethylrutoside (monoHER) and seven original synthetic compounds were employed to examine the relationships between their inhibitory effects on carbonyl reduction, iron-chelation and antioxidant properties with respect to their protective potential against doxorubicin-induced cardiotoxicity. Cardioprotection was investigated in the neonatal rat ventricular cardiomyocytes whereas the H9c2 cardiomyoblast cells were used for cytotoxicity testing. Iron chelation was examined via the calcein assay and antioxidant effects and site-specific scavenging were quantified by means of inhibition of lipid peroxidation and hydroxyl radical scavenging activity, respectively. Inhibition of carbonyl reductases was assessed in cytosol from human liver. None of the flavonoids tested had better cardioprotective action than the reference cardioprotector, monoHER. However, a newly synthesized quaternary ammonium analog with comparable cardioprotective effects has been identified. No direct correlation between the iron-chelating and/or antioxidant effect and cardioprotective potential has been found. A major role of carbonyl reductase inhibition seems unlikely, as the best two cardioprotectors of the series are only weak reductase inhibitors.     

Pharmacological profiling of chemokine receptor-directed compounds using high-content screening

High-content screening, typically defined as automated fluorescence microscopy combined with image analysis, is now well established as a means to study test compound effects in cellular disease-modeling systems. In this work, the authors establish several high-content screening assays in the 384-well format to measure the activation of the CC-type chemokine receptors 2B and 3 (CCR2B, CCR3). As a cellular model system, the authors use Chinese hamster ovary cells, stably transfected with 1 of the respective receptors. They characterize receptor stimulation by human monocyte chemoattractant protein-1 for CCR2B and by human eotaxin-1 for CCR3: Receptor internalization and receptor-induced phosphorylation of ERK1/2 (pERK) were quantified using fluorescence imaging and image analysis. The 4 assay formats were robust, displayed little day-to-day variability, and delivered good Z' statistics for both CCRs. For each of the 2 receptors, the authors evaluated the potency of inhibitory compounds in the internalization format and the pERK assay and compared the results with those from other assays (ligand displacement binding, Ca(2+) mobilization, guanosine triphosphate exchange, chemotaxis). Both physiological agonists and test compounds differed significantly with respect to potencies and efficacies in the various profiling assays. The diverse assay formats delivered partially overlapping and partially complementary information, enabling the authors to reduce the probability of test compound-related technology artifacts and to specify the mode of action for individual test compounds. Transfer of the high-content screening format to a fully automated medium-throughput screening platform for CCR3 enabled the profiling of large compound numbers with respect to G protein signaling and possible tolerance-inducing liabilities.     

Biflavones from Ginkgo biloba as inhibitors of human thrombin

Ginkgo Biloba leaf extract has been widely used for the prevention and treatment of thrombosis and cardiovascular disease in both eastern and western countries, but the bioactive constituents and the underlying mechanism of anti-thrombosis have not been fully characterized. The purpose of this study was to investigate the inhibitory effects of major constituents in Ginkgo biloba on human thrombin, a key serine protease regulating the blood coagulation cascade and the processes of thrombosis. To this end, a fluorescence-based biochemical assay was used to assay the inhibitory effects of sixteen major constituents from Ginkgo biloba on human thrombin. Among all tested natural compounds, four biflavones (ginkgetin, isoginkgetin, bilobetin and amentoflavone), and five flavonoids (luteolin, apigenin, quercetin, kaempferol and isorhamnetin) were found with thrombin inhibition activity, with the IC₅₀ values ranging from 8.05 μM to 82.08 μM. Inhibition kinetic analyses demonstrated that four biflavones were mixed inhibitors against thrombin-mediated Z-GGRAMC acetate hydrolysis, with the K_i values ranging from 4.12 μM to 11.01 μM. Molecular docking method showed that the four biflavones could occupy the active cavity with strong interactions of salt bridges and hydrogen bonds. In addition, mass spectrometry-based lysine labeling reactivity assay suggested that the biflavones could bind on human thrombin at exosite I rather than exosite II. All these findings suggested that the biflavones in Ginkgo biloba were naturally occurring inhibitors of human thrombin, and these compounds could be used as lead compounds for the development of novel thrombin inhibitors with improved efficacy and high safety profiles.     

Potential cancer chemopreventive activity of simple isoquinolines, 1-benzylisoquinolines, and protoberberines

Seventeen simple isoquinolines, 15 1-benzylisoquinolines, and 19 protoberberines were tested for their inhibitory activities against Epstein-Barr virus early antigen (EBV-EA) activation induced by 12-O-tetradecanoylphorbol-13-acetate (TPA) in Raji cells. Among the tested alkaloids, the inhibitory activity of all 1-benzylisoquinolines and 11 protoberberines was higher than that of beta-carotene. The 1-benzylisoquinolines 19, 21, 22, 29, and 34 and protoberberines 41, 47-49, 51, 52, and 55 showed potent inhibitory effects on EBV-EA induction (96-100% inhibition at 1 x 10(3) mol ratio/TPA). These alkaloids were more active than the naturally occurring alkaloids, 23, 25, 33, 53, and 54. In addition, fifteen simple isoquinolines, eighteen 1-benzylisoquinolines and eight protoberberines were evaluated with respect to their ability to scavenge 1,1-diphenyl-2-picrylhydrazyl (DPPH) free radicals. Nine simple isoquinolines, ten 1-benzylisoquinolines, and four protoberberines were more potent than alpha-tocopherol, and four 1-benzylisoquinolines, 20 and 28-30, exhibited potent activities (SC50 4.5-5.8 microM). Their activities were higher than the naturally occurring alkaloids, 23, 25, and 33. Therefore, some of the isoquinoline alkaloids indicating the high activity on both assays may be potentially valuable cancer chemopreventive agents. Structure-activity relationships are discussed for both tests.