Screening of xanthine oxidase inhibitors in complex mixtures using online HPLC coupled with postcolumn fluorescence‐based biochemical detection

Xanthine oxidase (XO) catalyzes the metabolism of hypoxanthine and xanthine to uric acid, the overproduction and/or underexcretion of which could cause the incidence of hyperuricemia such as gout. Herein, the inhibition of XO is recognized as one of the therapeutic approaches to treat gout. In the present study, an off‐line fluorescence‐based microplate method was first developed for an XO assay in which the enzyme converted pterin to its fluorescent metabolite isoxanthopterin. Then, a postcolumn continuous XO assay as a means of bioactivity assessment was coupled to HPLC separation to establish the online HPLC with diode array detection, biochemical detection, and MS/MS system for the screening of XO inhibitors. The availability of the online system was first tested with a positive drug, allopurinol, a well‐known XO inhibitor, and subsequent analysis of Scutellaria baicalensis extract showed that two main bioactive compounds with XO inhibitory activities were observed, indicating that the developed online system was applicable to complex mixtures.     

Phloroglucinol Derivatives from Dryopteris crassirhizoma as Potent Xanthine Oxidase Inhibitors

Dryopteris crassirhizoma rhizomes are used as a traditional medicine in Asia. The EtOAc extract of these roots has shown potent xanthine oxidase (XO) inhibitory activity. However, the main phloroglucinols in D. crassirhizoma rhizomes have not been analyzed. Thus, we investigated the major constituents responsible for this effect. Bioassay-guided purification isolated four compounds: flavaspidic acid AP (1), flavaspidic acid AB (2), flavaspidic acid PB (3), and flavaspidic acid BB (4). Among these, 1 showed the most potent inhibitory activity with a half-maximal inhibitory concentration (IC₅₀) value of 6.3 µM, similar to that of allopurinol (IC₅₀ = 5.7 µM) and better than that of oxypurinol (IC₅₀ = 43.1 µM), which are XO inhibitors. A comparative activity screen indicated that the acetyl group at C3 and C3′ is crucial for XO inhibition. For example, 1 showed nearly 4-fold higher efficacy than 4 (IC₅₀ = 20.9 µM). Representative inhibitors (1–4) in the rhizomes of D. crassirhizoma showed reversible and noncompetitive inhibition toward XO. Furthermore, the potent inhibitors were shown to be present in high quantities in the rhizomes by a UPLC-QTOF-MS analysis. Therefore, the rhizomes of D. crassirhizoma could be used to develop nutraceuticals and medicines for the treatment of gout.     

Bioassay-Guided Isolation of Anti-Alzheimer Active Components from the Aerial Parts of Hedyotis diffusa and Simultaneous Analysis for Marker Compounds

Previous studies have reported that Hedyotis diffusa Willdenow extract shows various biological activities on cerebropathia, such as neuroprotection and short-term memory enhancement. However, there has been a lack of studies on the inhibitory activity on neurodegenerative diseases such as Alzheimer’s disease (AD) through enzyme assays of H. diffusa. Therefore, H. diffusa extract and fractions were evaluated for their inhibitory effects through assays of enzymes related to AD, including acetylcholinesterase (AChE) and butyrylcholinesterase (BChE), and β-site amyloid precursor protein cleaving enzyme 1 (BACE1), and on the formation of advanced glycation end-product (AGE). In this study, ten bioactive compounds, including nine iridoid glycosides 1–9 and one flavonol glycoside 10, were isolated from the ethyl acetate and n-butanol fractions of H. diffusa using a bioassay-guided approach. Compound 10 was the strongest inhibitor of cholinesterase, BACE1, and the formation of AGEs of all isolated compounds, while compound 5 had the lowest inhibitory activity. Compounds 3, 6, and 9 exhibited better inhibitory activity than other compounds on AChE, and two pairs of diastereomeric iridoid glycoside structures (compounds 4, 8, and 6, 7) showed higher inhibitory activity than others on BChE. In the BACE1 inhibitory assay, compounds 1–3 were good inhibitors, and compound 10 showed higher inhibitory activity than quercetin, the positive control. Moreover, compounds 1 and 3 were stronger inhibitors of the formation of AGE than aminoguanidine (AMG), the positive control. In conclusion, this study is significant since it demonstrated that the potential inhibitory activity of H. diffusa on enzymes related to AD and showed the potential use for further study as a natural medicine for AD treatment on the basis of the bioactive components isolated from H. diffusa.     

Potential Xanthine Oxidase Inhibitory Activity of Endophytic Lasiodiplodia pseudotheobromae

Xanthine oxidase is considered as a potential target for treatment of hyperuricemia. Hyperuricemia is predisposing factor for gout, chronic heart failure, atherosclerosis, tissue injury, and ischemia. To date, only two inhibitors of xanthine oxidase viz. allopurinol and febuxostat have been clinically approved for used as drugs. In the process of searching for new xanthine oxidase inhibitors, we screened culture filtrates of 42 endophytic fungi using in vitro qualitative and quantitative XO inhibitory assays. The qualitative assay exhibited potential XO inhibition by culture filtrates of four isolates viz. #1048 AMSTITYEL, #2CCSTITD, #6AMLWLS, and #96 CMSTITNEY. The XO inhibitory activity was present only in the chloroform extract of the culture filtrates. Chloroform extract of culture filtrate #1048 AMSTITYEL exhibited the highest inhibition of XO with an IC₅₀ value of 0.61 μg ml^?1 which was better than allopurinol exhibiting an IC₅₀ of 0.937 μg ml^?1 while febuxostat exhibited a much lower IC₅₀ of 0.076 μg ml^?1. Further, molecular phylogenetic tools and morphological studies were used to identify #1048 AMSTITYEL as Lasiodiplodia pseudotheobromae. This is the first report of an endophytic Lasiodiplodia pseudotheobromae from Aegle marmelos exhibiting potential XO Inhibitory activity.     

Fractionation of Lycopodiaceae Alkaloids and Evaluation of Their Anticholinesterase and Cytotoxic Activities

In view of the abundant evidence that Lycopodiaceae alkaloids, including the well-known huperzine A (HupA), are among the potent acetylcholinesterase (AChE) inhibitors, an attempt was made to search for new compounds responsible for this property. For this purpose, three plant species belonging to the Lycopodiaceae family, commonly found in the Euro-Asia region, were subjected to the isolation of bioactive compounds, their identification and subsequent evaluation of their anticholinesterase and cytotoxic activities. Methanolic extracts of two Lycopodium and one Hupezia species were obtained via optimized pressurized liquid extraction (PLE) and then pre-purified using innovative gradient vacuum liquid chromatography (gVLC). For the first time, three sorbents of different porosity packed in polypropylene cartridges and mobile phase systems of different polarity were used to elute the target compounds. This technique proved to be a rapid tool for the obtainment of alkaloid fractions and allowed one to select the appropriate process conditions to yield potent AChE inhibitors in each of the species studied. More than 100 collected fractions were analyzed via HPLC/ESI-QTOF-MS, which enabled one to detect more than 50 compounds, including several new ones previously unreported. Some of them were present in high purity fractions (60–90% of the established purity). TLC bioautography assays proved that the analyzed species are rich sources of AChE inhibitors, but H. selago showed the highest anti-AChE activity. Additionally, the modified silanized silica gel sorbent used allowed one to isolate L. clavatum alkaloids more efficiently using an aqueous reversed-phase solvent system. Furthermore, the tested extracts from the three plant extracts were found to be safe, as they did not exhibit cytotoxicity to skin fibroblasts.     

Combinatorial synthesis of novel and potent inhibitors of NADH:ubiquinone oxidoreductase

BACKGROUND: NADH:ubiquinone oxidoreductase (complex I) is the first of three large enzyme complexes located in the cell's inner mitochondrial membrane which form the electron transport chain that carries electrons from NADH to molecular oxygen during oxidative phosphorylation. There is significant interest in developing small molecule inhibitors of this enzyme for use as biological probes, insecticides and potential chemopreventive/chemotherapeutic agents. Herein we describe the application of novel natural product-like libraries to the discovery of a family of potent benzopyran-based inhibitors. RESULTS: Initially a combinatorial library of benzopyrans, modeled after natural products, was synthesized using a solid phase cycloloading strategy. Screening of this diversity oriented library for inhibitory potency against NADH:ubiquinone oxidoreductase activity in vitro using bovine heart electron transport particles provided several lead compounds which were further refined through a series of focused libraries. CONCLUSIONS: Using this combinatorial library approach, a family of potent 2,2-dimethylbenzopyran-based inhibitors was developed with IC(50) values in the range of 18-55 nM. Cell-based assays revealed that these inhibitors were rather non-cytotoxic in the MCF-7 cell line; however, they were quite cytostatic in a panel of cancer cell lines suggesting their potential as chemotherapeutic/chemopreventive candidates.     

基于拉伸分子动力学模拟的黄嘌呤氧化酶抑制剂解离途径的理论研究

采用拉伸分子动力学模拟的方法研究了黄嘌呤氧化酶(Xanthine oxidase, XO)抑制剂别嘌呤醇(Allopurinol)和葛根素(Puerarin)从XO离去通道解离的动态过程. 分子对接结果表明, 别嘌呤醇和葛根素均结合在XO的钼蝶呤中心(Molybdopterin, Mo-pt); 丙氨酸扫描的结果显示, Val789, Arg880, Phe911, Phe914和Val1081在XO与抑制剂的结合中起到非常重要的作用. 拉伸分子动力学模拟结果显示, 相比于葛根素, 别嘌呤醇需要更大的外力和更长的时间才能从XO中解离, 拉伸过程中Arg880, Phe1009, Thr1010, Val1011和Ala1079均对维持2种复合物的结构稳定起到重要作用, Phe649和Phe1013在抑制剂解离过程中起到门控的作用, His875起到阻碍抑制剂解离的作用.     

Isolation of Anti-Diabetic Active Compounds from Benincasae Exocarpium and Development of Simultaneous Analysis by HPLC-PDA

Diabetes is a chronic metabolic disease that is a constant problem. Previous studies have reported that Benincasa cerifera Savi. extracts are effective in treating diabetes and its complications. Benincasae Exocarpium (BE) is a fruit peel of B. cerifera that has been reported to be used for the prevention and treatment of metabolic diseases such as hyperglycemia, obesity, and hyperlipidemia. However, there are not enough studies on the compounds and bioassays to support the efficacy of BE. The inhibitory activity of the BE extracts and fractions against advanced glycation end-products (AGE) formation and α-glucosidase activity was evaluated. These assays are relevant for the treatment of type 2 diabetes and its complications. Based on these results, compounds 1–11 were isolated through bioassay-guided isolation. In addition, we developed a high-performance liquid chromatography (HPLC) method that can simultaneously analyze these 11 compounds. Activity evaluation of the compounds was also conducted, and eight compounds exhibited significant activity. Among these, flavonoid compounds showed strong activity. A quantitative evaluation of eight bioactive compounds (2, 5–11) was conducted. In conclusion, this study demonstrated the potential of BE for prevention and treatment of type 2 diabetes and its complications.     

Evaluation of xanthine oxidase inhibitory activity of flavonoids by an online capillary electrophoresis-based immobilized enzyme microreactor

Xanthine oxidase (XOD) is a key enzyme in the human body to produce uric acid, and its inhibitor can be used for the treatment of hyperuricemia and gout. In this study, an online CE-based XOD immobilized enzyme microreactor (IMER) was developed for the enzyme kinetics assays and inhibitor screening. After 30 consecutive runs, the XOD activity remained about 95.6% of the initial immobilized activity. The Michaelis–Menten constant (K_m) of the immobilized XOD was determined as 0.39 mM using xanthine as substrate. The half-maximal inhibitory concentration and inhibition constant of the known inhibitor 4-aminopyrazolo[3,4-d]pyrimidine on XOD were determined as 11.9 and 5.2 μM, respectively. Then, the developed method was applied to evaluate the XOD inhibitory activity of 10 flavonoids, which indicated that dihydroquercetin, quercetin, biochanin A, and epicatechin had significant inhibitory effect on XOD. In addition, molecular docking results verified that the binding energy of the flavonoids with enzyme were in line with their inhibitory activity determined by XOD–IMER. Therefore, the developed XOD–IMER is a potential tool for the primary screening of XOD inhibitors from natural products.     

In vitro antiperoxidative, free radical scavenging and xanthine oxidase inhibitory potentials of ethyl acetate fraction of Saraca ashoka flowers

Saraca ashoka is a widely used medicinal herb claimed to cure many diseases. This study investigated the antiperoxidative, free radical scavenging and xanthine oxidase (XO) inhibitory potential of the ethyl acetate fraction of S. ashoka flowers (SAF) and compared it with standard compounds like gallic acid, ascorbic acid, butylated hydroxyl toluene and allopurinol. The ethyl acetate fraction of SAF exhibited free radical scavenging activity against the 1,1-diphenyl-2-picrylhydrazyl radical and superoxide radical, along with hydroxyl radical scavenging activity. Lipid peroxidation inhibitory potential of SAF was studied using a linoleic acid emulsion system, which shows significant antioxidant potential. SAF also demonstrated significant XO (key enzyme linked to inflammation) inhibitory activity, which revealed its therapeutic potential as an antioxidant and XO inhibitor. HPLC profiling of the ethyl acetate fraction of SAF revealed that it contains ellagic acid as a major compound and thus the beneficial effects of this fraction may be due to the presence of this compound.     

Discovery of the first non-planar flavonoid that can strongly inhibit xanthine oxidase: protoapigenone 1′-O-propargyl ether

Xanthine oxidase (XO) is a key enzyme in purine metabolism with an important role in various pathologies. Several flavonoids have been reported for their capacity to inhibit this enzyme, and, for these compounds, the ability to adopt a planar 3D structure has been accepted as fundamental prerequisite for such activity. Here we report the in vitro investigation of a series of non-planar protoflavone derivatives as XO inhibitors, among which protoapigenone 1′-O-propargyl ether was found to be an efficient competitive inhibitor of the enzyme with an IC₅₀ value of 3.61 μM, significantly (p <0.001) stronger than the anti-gout drug allopurinol (IC₅₀ = 8.72 μM). Methoxy substitution at C-7, however, resulted in complete loss of activity. In silico docking supported the observed structure–activity relationships, based on which a ‘planar structure’ itself can no longer be considered as a criterion for flavonoid-type inhibitors of XO.     

Discovery of xanthine oxidase inhibitors from a complex mixture using an online,restricted-access material coupled with column-switching liquid chromatography with a diode-array detection system

To find potential lead compounds for antigout drug discovery, an automated online, restricted-access material coupled with column-switching liquid chromatography with a diode-array detection (RAM–LC–DAD) system was developed for screening of xanthine oxidase (XO) inhibitors and their affinity rankings in complex mixtures. The system was first evaluated by analyzing a mixture of six compounds with known inhibition of XO. Nonspecific binding to the denatured XO was investigated and used as the control for screening. Subsequently, the newly developed system was applied to screening of a natural product, Oroxylum indicum extract, and four compounds which could specifically interact with XO were found and identified as oroxin B, oroxin A, baicalin, and baicalein. The results were verified by a competitive binding test using the known competitive inhibitor allopurinol and were further validated by an inhibition assay in vitro. The online RAM–LC–DAD system developed was shown to be a simple and effective strategy for the rapid screening of bioactive compounds from a complex mixture.

Quick identification of xanthine oxidase inhibitor and antioxidant from Erycibe obtusifolia by a drug discovery platform composed of multiple mass spectrometric platforms and thin‐layer chromatography bioautography

As a final step of the purine metabolism process, xanthine oxidase catalyzes the oxidation of hypoxanthine and xanthine into uric acid. Our research has demonstrated that Erycibe obtusifolia has xanthine oxidase inhibitory properties. The purpose of this paper is to describe a new strategy based on a combination of multiple mass spectrometric platforms and thin‐layer chromatography bioautography for effectively screening the xanthine oxidase inhibitory and antioxidant properties of E. obtusifolia. This strategy was accomplished through the following steps. (i) Separate the extract of E. obtusifolia into fractions by an autopurification system controlled by liquid chromatography with mass spectrometry. (ii) Determine the active fractions of E. obtusifolia by thin‐layer chromatography bioautography. (iii) Identify the structure of the main active compounds with the information provided by direct analysis in real time mass spectrometry. (iv) Calculate the IC₅₀ value of each compound against xanthine oxidase using high‐performance liquid chromatography. Using the caulis of E. obtusifolia as the experimental material, seven target peaks were screened out as xanthine oxidase inhibitors or antioxidants. Our screening strategy allows for rapid analysis of small molecules with almost no sample preparation and can be completed within a week, making it a useful assay to identify unstable compounds and provide the empirical foundation for E. obtusifolia as a natural remedy for gout and oxidative‐stress‐related diseases.     

Synthesis and structure activity relationship studies of benzothieno[3,2-b]furan derivatives as a novel class of IKKbeta inhibitors

As a novel class of IKKbeta inhibitors, a series of tricyclic furan derivatives was designed and synthesized based on the structure of known thiophene IKKbeta inhibitors. Among the various fused furan derivatives synthesized, a benzothieno[3,2-b]furan derivative 13a displayed potent inhibitory activity towards IKKbeta in enzymatic and cellular assays. The potent inhibitory activity originates from an intramolecular non-bonded S...O interaction which was confirmed by the X-ray structure of JNK3 with 16k. The introduction of further substituents on the core structure led to the discovery of the 6-alkoxy derivatives, which possessed a comparable IKKbeta inhibitory activity to 13a and an improved metabolic stability. Among these, appropriately lipophilic compounds 16a, h, i, and 13g (log D>2) were found to possess good oral bioavailability.     

Thermochemiluminescence as a fast method to detect and characterize hydroperoxide moieties in novel 3-hydroperoxyisothiazole 1,1-dioxides

Hydroperoxy compounds are important for selective oxidation of organic precursors in the chemical industry. Moreover, novel 3-hydroperoxyisothiazole 1,1-dioxides (N-phthalimidyl sultams) have been shown to be potent and specific inhibitors of acetylcholinesterase (AChE) an important enzyme in the neurotransmitter process. The inhibitory potential of these novel compounds is clearly linked to the hydroperoxide moiety. Hydroperoxides are known for their chemi- and thermochemiluminescent reactivity. In this study we tested thermochemiluminescence (TCL) as a fast method to detect and characterize the hydroperoxide moiety in structurally different synthetic organic hydroperoxides.     

Rapid determination of the angiotensin I-converting enzyme inhibitory activity of peptide by HPLC method: A simulated gastrointestinal digestion study

Angiotensin I-converting enzyme (ACE) plays a vital role in blood pressure regulation. In vitro ACE inhibitory activity assays are commonly used for evaluating the activity of inhibitors in many researches. In the present study, a rapid, sensitive, simple, and accurate HPLC method for ACE inhibitory activity determination using chicken meat as a model was developed. Specific attention was paid to assess the ACE inhibitory activity of peptides released during simulated gastrointestinal digestion. Using hippuryl–histidyl–leucine as the substrate, ACE can catalyze it into hippuric acid, which is rapidly and sensitively detected by the HPLC at the retention time of 8–10?min. The ACE inhibitory activity of each digestion solution can be rapidly determined every 15?min or less. The model protein displayed a high ACE inhibitory activity during simulated gastrointestinal digestion, but the digestion solution in the stomach showed stronger activity than in the intestine.     

Design,synthesis and evaluation of 2-(4-(substituted benzoyl)-1,4-diazepan-1-yl)-N-phenylacetamide derivatives as a new class of falcipain-2 inhibitors

The cysteine protease, falcipain-2 is an important drug target in human malaria parasite Plasmodium falciparum. A new series of 2-(4-(substituted benzoyl)-1,4-diazepan-1-yl)-N-phenylacetamide derivatives 5(a–t) were designed as per pharmacophoric requirements of falcipain-2 inhibitors using ligand-based approach. The target compounds were synthesized from the key intermediate, 2-(1,4-Diazepan-1-yl)-N-phenylacetamide, by coupling it with appropriate carboxylic acids using carbodiimide chemistry. Structural features of target compounds were characterized by spectral data (¹H NMR, and mass) and elemental analyses. The purity of the final compounds was confirmed by HPLC. The compounds were tested for their in vitro falcipain-2 inhibitor activity on recombinant falcipain-2 enzyme. Five compounds 5b, 5g, 5h, 5j, 5k showed good inhibitory activity (>60%), against falcipain-2 at 10 μM concentration, and fifteen compounds showed weak to moderate inhibitor activity. Compound 5g, the most potent compound from this series showed 72% inhibition at 10 μM concentrations.     

Inhibitors of xylose reductase from the yeast pichia stipitis

Several compounds were examined for their inhibitory effects on xylose reductase from the yeastPichia stipitis NRC 2548. Mercuric chloride, cupric chloride, menadione sodium bisulfite, and sodium bisulfite inhibited enzyme activity in a sigmoidal dose-dependent manner, whereas quercetin and rutin were observed to have nonsigmoidal dose-response curves. Diphenylhydantoin, hydantoin, and valproic acid had no effect on xylose reductase activity. Mercuric chloride was the most potent inhibitor tested, with an IC₅₀ (the concentration that inhibited enzyme activity by 50%) of 4.7xl0^-6M. Three distinct inhibition patterns were observed amongst selected inhibitors. Mercuric chloride and quercetin were noncompetitive inhibitors of xylose reductase with respect to substrate and cofactor. Sodium bisulfite was an uncompetitive inhibitor with respect to substrate and cofactor, whereas menadione sodium bisulfite was a competitive inhibitor with respect to substrate, but noncompetitive to the cofactor.     

A rapid and simple HPLC-UV method for the determination of inhibition characteristics of farnesyl transferase inhibitors

Ras proteins play an important role in the development of cancer. Farnesyl transferase inhibitors (FTIs) block the first obligatory post-translational step for activation, prenylation, of Ras proteins. To find new potent FTIs, rapid enzyme activity assays are required to reduce FTI development time. Most assays to date are based on radioactive labelled substrates. We developed a new, in vitro, farnesyl transferase assay based on gradient chromatography coupled to UV detection. Unfarnesylated and farnesylated H-Ras proteins were resolved on a C18 wide-pore HPLC column and their concentrations were determined with use of a calibration curve of unfarnesylated H-Ras. The assay was used to investigate inhibition characteristics of FTIs. The IC50 values of the FTIs L778,123 and SCH66336 were 4.2 nm and 78 microm, respectively. This assay could support the screening and development of FTIs to obtain rapid insights into their inhibitory properties.