Screening,separation, and evaluation of xanthine oxidase inhibitors from Paeonia lactiflora using chromatography combined with a multi‐mode microplate reader

Natural products have become one of the most important resources for discovering novel xanthine oxidase inhibitors, which are commonly employed in the treatment of hyperuricemia and gout. However, to date, few reports exist regarding the use of monoterpene glycosides as xanthine oxidase inhibitors. Thus, we herein report the use of ultrafiltration coupled with liquid chromatography in the screening of monoterpene glycoside xanthine oxidase inhibitors from the extract of Paeonia lactiflora (P. lactiflora ), and both high‐performance counter‐current chromatography and medium‐pressure liquid chromatography were employed to separate the main constituents. Furthermore, the xanthine oxidase inhibitory activities and the mechanisms of inhibition of the isolated compounds were evaluated using a multi‐mode microplate reader by Molecular Devices. As a result, three monoterpene glycosides were separated by combined high‐performance counter‐current chromatography and medium‐pressure liquid chromatography in purities of 90.4, 98.0, and 86.3%, as determined by liquid chromatography. These three compounds were identified as albiflorin, paeoniflorin, and 1‐O‐β‐ᴅ‐glucopyranosyl‐8‐O‐benzoylpaeonisuffrone by electrospray ionization tandem mass spectrometry, and albiflorin and paeoniflorin were screened as potential xanthine oxidase inhibitors by ultrafiltration with liquid chromatography. The evaluation results of xanthine oxidase inhibitory activity corresponded with the screening results, as only albiflorin and paeoniflorin exhibited xanthine oxidase inhibitory activity.     

Ultrafiltration liquid chromatography combined with high‐speed countercurrent chromatography for screening and isolating potential α‐glucosidase and xanthine oxidase inhibitors from Cortex Phellodendri

Cortex Phellodendri is a typical Chinese herb with a large number of alkaloids existing in all parts of it. The most common methods for screening and isolating alkaloids are mostly labor intensive and time consuming. In this study, a new assay based upon ultrafiltration liquid chromatography was developed for the rapid screening of ligands for α‐glucosidase and xanthine oxidase. The C. Phellodendri extract was found to contain two alkaloids with both α‐glucosidase‐ and xanthine oxidase binding activities and one lactone with α‐glucosidase‐binding activity. Subsequently, with the help of high‐speed countercurrent chromatography, the specific binding ligands including palmatine, berberine, and obaculactone with purities of 97.38, 96.12, and 96.08%, respectively, were successfully separated. An optimized low‐toxicity two‐phase solvent system composed of ethyl acetate/n‐butanol/ethanol/water (3.5:1.7:0.5:5, v/v/v/v) was used to isolate the three compounds mentioned above from C. Phellodendri. The targeted compounds were identified by liquid chromatography coupled with mass spectrometry and NMR spectroscopy. Therefore, ultrafiltration liquid chromatography combined with high‐speed countercurrent chromatography is not only a powerful tool for screening and isolating α‐glucosidase and xanthine oxidase inhibitors in complex samples but is also a useful platform for discovering bioactive compounds for the prevention and treatment of diabetes mellitus and gout.     

Development of a method to screen and isolate xanthine oxidase inhibitors from black bean in a single step: Hyphenation of semipreparative liquid chromatography and stepwise flow rate countercurrent chromatography

Black bean, in which isoflavones are the main active constituent, also contains saponins and monoterpenes. Soybean isoflavone is a secondary metabolite that is formed during the growth of soybean; it exhibits antioxidant and cardiovascular activities and traces estrogen-like effects. In this study, black bean isoflavones were extracted with n-butanol, and ultrafiltration–liquid chromatography–mass spectrometry was used to screen their activity. Subsequently, the inhibitors were isolated and purified using semipreparative liquid chromatography and stepwise flow rate countercurrent chromatography. Thereafter, five active compounds were identified using mass spectrometry and nuclear magnetic resonance experiments. Finally, the inhibition types of the xanthine oxidase inhibitors were determined using enzymatic kinetic studies. The IC₅₀ values of daidzin, glycitein-7-O-glucoside, genistin, daidzein, and genistein were determined to be 35.08, 56.22, 30.76, 68.79, and 95.37 μg/mL, respectively. Daidzin, genistin, and daidzein exhibited reversible inhibition, whereas glycitein-7-O-glucoside and genistein presented irreversible inhibition. This novel approach, which was based on ultrafiltration–liquid chromatography–mass spectrometry and stepwise flow rate countercurrent chromatography, is a powerful method for screening and isolating xanthine oxidase inhibitors from complex matrices. The study of enzyme inhibition types is helpful for understanding the underlying inhibition mechanism. Therefore, a beneficial platform was developed for the large-scale production of bioactive and nutraceutical ingredients.     

Practical method for the rapid screening of xanthine oxidase inhibitors in herbal extracts by high‐performance liquid chromatography based on on‐line precolumn enzymatic reaction

A high‐performance liquid chromatography method with on‐line precolumn enzymatic reaction for the screening of xanthine oxidase inhibitors in natural extracts was developed. In this method, the enzymatic reaction occurred at the capillary inlet during a predetermined waiting period, after which the reaction product, uric acid, was separated and detected by liquid chromatography using ultraviolet absorption at 295 nm. Enzyme inhibition can be read out directly from the reduced peak area of uric acid in comparison to a reference chromatogram obtained in the absence of any inhibitor. In the present study, the availability of on‐line precolumn enzymatic reaction with ultraviolet detection was firstly evaluated by determining the inhibitory mechanism and IC₅₀ values of allopurinol, a commercially available positive drug. Then, the newly developed method was applied to screening of ten natural extracts from traditional Chinese medicine and as a result, the extract of Epimedium sagittatum (Sieb. et Zucc.) Maxim was found to be most positive for xanthine oxidase inhibition. The results obtained were compared with those obtained by offline enzyme assay and the effectiveness of the present method was confirmed. A rapid, low‐cost, and fully automated method for xanthine oxidase inhibitor screening was proposed.     

Study on the spectrum‐effect relationship of the xanthine oxidase inhibitory activity of Ligustrum lucidum

To evaluate the xanthine oxidase inhibitory activity of the chemical constituents of Ligustrum lucidum in vitro, the spectrum‐effect relationship was investigated. The high‐performance liquid chromatography fingerprint was established by ultraviolet spectrophotometry, and the xanthine oxidase inhibitory activity was tested in vitro by a high‐throughput screening method. Cluster analysis, principal component analysis, gray correlation analysis, and partial least squares regression were used to explore the spectrum‐effect relationships. Sixty batches of Ligustrum lucidum were collected from 16 provinces for testing. The results revealed differences among the batches of medicinal materials, and the similarity score was between 0.635 and 0.968. Thirty‐three characteristic peaks (1–33) were calibrated by fingerprint evaluation software for traditional Chinese medicine. The spectrum‐effect relationship study further revealed that the contents of peaks 1, 2, 4, 5, 6, 7, 14, 17, 25, 28, 31, and 33, which are potentially critical ingredients for quality control of Ligustrum lucidum fruit, were highly correlated with the inhibition of xanthine oxidase activity.     

Bioassay-Guided Isolation and Identification of Antimicrobial Compounds from Thyme Essential Oil by Means of Overpressured Layer Chromatography,Bioautography and GC–MS

A simple method is described for efficient isolation of compounds having an antibacterial effect. Two thyme (Thymus vulgaris) essential oils, obtained from the market, were chosen as prospective materials likely to feature several bioactive components when examined by thin layer chromatography coupled with direct bioautography as a screening method. The newly developed infusion overpressured layer chromatographic separation method coupled with direct bioautography assured that only the active components were isolated by means of overrun overpressured layer chromatography (OPLC) with on-line detection and fractionation. Each of the 5 collected fractions represented one of the five antimicrobial essential oil components designated at the screening. The purity and the activity of the fractions were confirmed with chromatography coupled with various detection methods (UV, vanillin–sulphuric acid reagent, direct bioautography). The antibacterial components were identified with GC–MS as thymol, carvacrol, (−)-linalool, diethyl-phthalate, and α-terpineol. The oil component diethyl-phthalate is an artificial compound, used as a plasticizer or detergent base in the industry. Our results support that exploiting its flexibility and the possible hyphenations, OPLC is especially attractive for isolation of antimicrobial components from various matrixes.

     

A strategy for screening trypsin inhibitors from traditional Chinese medicine based on a monolithic capillary immobilized enzyme reactor coupled with offline liquid chromatography and mass spectrometry

A novel strategy was successfully developed for screening trypsin inhibitors in traditional Chinese medicines based on monolithic capillary immobilized enzyme reactors combined with liquid chromatography‐tandem mass spectrometry. Organic polymer based monolithic enzyme reactors were firstly prepared by covalently bonding trypsin to a poly(glycidyl methacrylate‐co‐poly (ethylene glycol) diacrylate) monolith by the ring‐opening reaction of epoxy groups. The activity and kinetic parameters of the obtained monolithic trypsin reactors were systematically evaluated using micro‐liquid chromatography. Fourier transform infrared spectroscopy and scanning electron microscopy were also used to characterize the monolithic trypsin reactors. The resulting functional and denatured monolithic trypsin reactors were applied as affinity solid‐phase extraction columns, and offline coupled with a liquid chromatography‐tandem mass spectrometry system to construct a binding affinity screening platform. Subsequently, the proposed platform was applied for screening trypsin binders in a Scutellaria baicalensis Georgi extract. Three compounds, namely scutellarin, baicalin, and wogonoside were identified, and their inhibitory activities were further confirmed via an in vitro enzymatic inhibition assay. Additionally, molecular docking was also performed to study the interactions between trypsin and these three compounds.     

Rapid screening and evaluation of XOD inhibitors and O2•− scavenger from total flavonoids of Ginkgo biloba leaves by LC–MS and multimode microplate reader

Superoxide anion radical scavenger and xanthine oxidase inhibitor play an important role in the treatment of several relevant human diseases. In the present study, ultrafiltration liquid chromatography–mass spectrometry coupled to microplate reader was applied to screen and identify superoxide anion radical scavengers and xanthine oxidase inhibitors from total flavonoids of Ginkgo biloba leaves. As a result, four compounds (quercetin, apigenin, kaempferol and isorhamnetin) were screened as xanthine oxidase inhibitors by ultrafiltration LC–MS, and the 50% scavenging concentration values of the screened flavonoids were lower than those for allopurinol. Lineweaver–Burk plot results indicated that kaempferol was a competitive xanthine oxidase inhibitor; the other flavonoids were all anticompetitive inhibitors. Four flavonoids—rutin, quercetin, kaempferol and isorhamnetin—were screened as superoxide anion radical scavengers by LC–MS. The results demonstrate that the method for screening and evaluation of superoxide anion radical scavenger and xanthine oxidase inhibitor from a complex mixture system is feasible and efficient.     

Preparative separation of the flavonoid fractions from Periploca forrestii Schltr. ethanol extracts using macroporous resin combined with HPLC analysis and evaluation of their biological activities

A preparative separation method using macroporous absorptive resin coupled with high‐performance liquid chromatography was developed for the separation of six fractions of the 80% ethanol extract of Periploca forrestii Schltr. The six ethanol fractions (5–95; A, B, C, D, E, and F) obtained were carefully analyzed to locate the corresponding peaks in the high‐performance liquid chromatography chromatogram of the total extract, which was established in a previous study. Furthermore, the biological activities, including antioxidant activities, acetyl cholinesterase inhibitory capacities, antihyaluronidase activities, and anti‐inflammatory effects, were evaluated in MH7A cells. The results demonstrated that fraction E could significantly prevent oxidation and inhibit hyaluronidase and acetyl cholinesterase. Finally, the main flavonoids in fractions A and E from P. forrestii Schltr. were purified, and the compounds were identified as chlorogenic acid, quercetin‐3‐O‐α‐L‐arabinopyranoside, and quercetin‐7‐O‐β‐D‐glucopyranoside. The chemical structures were confirmed by mass spectrometry and nuclear magnetic resonance spectroscopy. Furthermore, the inhibitory effects of these compounds against complete Freund's adjuvant‐induced secondary immune arthritis in rats were evaluated.     

Identification of flavonoids in Dendrobium huoshanense and comparison with those in allied species of Dendrobium by TLC,HPLC and HPLC coupled with electrospray ionization multi‐stage tandem MS analyses

Dendrobium huoshanense, a unique species in the genus Orchidaceae, is only found in China and is known as “mihu”. Due to the lack of quality control, the use of D. huoshanense in the herbal market has been limited. In this study, methods based on thin‐layer chromatography, high‐performance liquid chromatography and high‐performance liquid chromatography coupled with electrospray ionization multi‐stage tandem mass spectrometry were used to identify the flavonoids in D. huoshanense and distinguish this species from other Dendrobium species. Using thin‐layer chromatography, a characteristic band was observed for D. huoshanense, and this band was absent from the thin‐layer chromatography plates of other Dendrobium species. Then, using high‐performance liquid chromatography, nine peaks of flavonoids were observed in the chromatograms of ten batches of D. huoshanense. Ultimately, 22 flavonoids in D. huoshanense were identified by multi‐stage tandem mass spectrometry, and 11 of these compounds are being reported from D. huoshanense for the first time. In addition, two compounds both with molecular weights of 710, were identified as being unique to D. huoshanense; one of these compounds, apigenin‐6‐C‐α‐L‐rhamnosyl‐(1→2)‐β‐D‐glucoside‐8‐C‐α‐L‐arabinoside, was proven to be responsible for the characteristic thin‐layer chromatography band of D. huoshanense. These analysis methods can be applied for the identification and quality control of D. Huoshanense.     

Rapid screening,identification, and purification of neuraminidase inhibitors from Lithospermum erythrorhizon Sieb.et Zucc. by ultrafiltration with HPLC–ESI‐TOF‐MS combined with semipreparative HPLC

We put forward an efficient strategy based on bioassay guidance for the rapid screening, identification, and purification of the neuraminidase inhibitors from traditional Chinese medicines, and apply to the discovery of anti‐influenza components from Lithospermiun erythrorhizon Sieb.et Zucc. Ultrafiltration with high‐performance liquid chromatography and electrospray ionization time‐of‐flight mass spectrometry was employed for the rapid screening and preliminarily identification of anti‐influenza components from Zicao. Semipreparative high‐performance liquid chromatography was used for the rapid separation and purification of the target compounds. NMR spectroscopy, mass spectrometry, and UV spectroscopy were used for further structural identification, and the activity of the compounds was verified by in vitro assay. Five compounds were found to have neuraminidase inhibitory activity by this method. Subsequently, the five compounds were separated by semipreparative high‐performance liquid chromatography with the purity over 98% for all of them by high‐performance liquid chromatography test. Combined with the NMR spectroscopy, mass spectrometry, and UV spectroscopy data, they were identified as alkannin, acetylalkannin, isobutyrylalkannin, β,β‐dimethylacryloylalkannin and isovalerylalkannin. The in vitro assay showed that all five compounds had good neuraminidase inhibitory activities. These results suggested that the method is highly efficient, and it can provide platform and methodology supports for the rapid discovery of anti‐influenza active ingredients from complex Chinese herbal medicines.     

Establishing the chromatographic fingerprints of flavan‐3‐ols and proanthocyanidins from rose hip (Rosa sp.) species

The profile of flavan‐3‐ols and proanthocyanidins in five different Rosa species (R. canina, R. glutinosa, R. rubiginosa, R. multiflora, and R. spinosissima) was estimated on high performance thin layer chromatography cellulose plates. Differences in flavanol and proanthocyanidin profiles of the extracts were evident, among which Rosa spinosissima stood out with catechin as the only detected flavanol and red zones as indication of anthocyanins. Furthermore, the elution solvent for thin layer chromatography with mass spectrometry analyses of glycosylated flavan‐3‐ols and proanthocyanidins was optimized, enabling identification of catechin, (epi)catechin hexoside, proanthocyanidin dimer, and proanthocyanidin dimers and trimers hexosides. A total of 15 flavanols and their derivatives were identified using ultra‐high‐performance liquid chromatography with linear trap quadrupole‐Orbitrap mass analyzer and epicatechin, gallocatechin, and proanthocyanidin trimer were identified only using this technique. However, proanthocyanidin trimer trihexoside was identified only by thin‐layer chromatography with mass spectrometry. To establish the relationships between the flavanols and proanthocyanidins composition of rose hip and their origin, principal component analysis was performed on the entire set of liquid chromatography/mass spectrometry data. Both principal components’ scores plots showed that Rosa spinosissima could be considered as an outlier. Our study demonstrated that flavanol and proanthocyanidin profiles of different rose hips depend on the geographical origin rather than on the cultivar and genotype.     

Rapid methods for the separation of natural mixtures of beauverolides,cholesterol acyltransferase inhibitors,isolated from the fungus Isaria fumosorosea

Beauverolides (beauveriolides) are abundant, biologically active cyclodepsipeptides produced by many entomopathogenic fungi, including those that are used as biopesticides. Beauverolides act as cholesterol acyltransferase inhibitors in humans; thus, their mode of action has been the subject of pharmacological and clinical research. The cost‐effective analytical methods are needed for fast, routine laboratory analysis of beauverolides. We isolated beauverolides from the fungal strain Isaria fumosorosea PFR 97‐Apopka and opened the rings of the isolated beauverolides using a pyridine alkaline medium. We separated fractions of cyclic and linearized beauverolides by thin‐layer chromatography, and found the chloroform–acetate (9:1, v/v) and chloroform–acetonitrile–acetate (8:1:1, v/v/v) mobile phases, respectively, to be the most efficient. We examined all the fractions by liquid chromatography–mass spectrometry using ion trap and Orbitrap high resolution mass spectrometry. For rapid screening of the contents of cyclic, and, particularly, linearized beauverolides, we developed a novel analytical method that consisted of using capillary electrophoresis coupled with contactless conductivity detection. Furthermore, we improved the separation of the peptides by applying capillary micellar electrokinetic chromatography with the N‐cyclohexyl‐2‐aminoethanesulfonic acid:SDS:NaOH buffer, pH 9.8 as the background electrolyte. The described novel methods allow fast and cost‐effective separation of chemically related groups of beauverolides.     

Rapid screening and purification of potential alkaloid neuraminidase inhibitors from Toddalia asiatica (Linn.) Lam. roots via ultrafiltration liquid chromatography combined with stepwise flow rate counter‐current chromatography

Toddalia asiatica (Linn.) Lam. is a medical plant traditionally used to treat coughs, fevers, and various diseases. Alkaloids are the main active ingredients in Toddalia asiatica (Linn.) Lam., but traditional methods for screening and separation are complex and labor‐intensive. In this work, an efficient strategy was developed to rapidly screen, identify, and separate neuraminidase inhibitors from Toddalia asiatica (Linn.) Lam. Ultrafiltration, high performance liquid chromatography, and time‐of‐flight mass spectrometry were employed for rapid screening and identification of neuraminidase inhibitors. A two‐phase solvent system comprising n‐hexane/ethyl acetate/methanol/water (5:5:3:7, v/v) was then selected for separation by high‐speed counter‐current chromatography. A sample loading of 200 mg and a stepwise flow rate were achieved by increasing the flow rate from 2 to 4 mL/min after 4 h. Three main fluoroquinoline alkaloids (haplopine, skimmianine, and 5‐methoxydictamnine) along with two coumarins were obtained via one‐step separation and their structures were determined by mass spectrometry and nuclear magnetic resonance. In vitro assays revealed skimmianine with half‐maximal inhibitory concentration of 16.2 ± 0.7 µmol/L was selected as the potential highest neuraminidase inhibitor. The results suggest that ultrafiltration high performance liquid chromatography–mass spectrometry combined with high‐speed counter‐current chromatography is efficient for the screening and isolation of neuraminidase inhibitors from complex natural products.     

Screening inhibitors of xanthine oxidase from natural products using enzyme immobilized magnetic beads by high‐performance liquid chromatography coupled with tandem mass spectrometry

In this study, high‐performance liquid chromatography coupled with tandem mass spectrometry was used to assess the results of bioactive compound screening from natural products using immobilized enzyme magnetic beads. We compared three commercial magnetic beads with modified amino, carboxy, and N‐hydroxysuccinimide groups, respectively. Amino magnetic beads performed best for immobilization and were selected for further experiments. Xanthine oxidase was immobilized on amino magnetic beads and applied to screen potential inhibitors in fresh Zingiber officinale Roscoe, extracts of Scutellaria baicalensis Georgi, and Pueraria lobata Ohwi. In total, 12 potential xanthine oxidase ligands were identified from fresh Zingiber root and Scutellaria root extracts, of which eight were characterized and the concentration required for 50% inhibition was determined. Preliminary structure–function relationships were discussed based on these results. A convenient and effective method was therefore developed for the identification of active compounds from complex natural product mixtures.     

Application of characteristic fragment filtering with ultra high performance liquid chromatography coupled with high‐resolution mass spectrometry for comprehensive identification of components in Schisandrae chinensis Fructus

An integrated strategy of characteristic fragment filtering combined with target database screening based on ultra‐high‐performance liquid chromatography coupled with high‐resolution mass spectrometry was proposed for comprehensive profiling of components in Schisandrae chinensis Fructus. The strategy consisted of following five steps: (1) Representative standards were analyzed by ultra high performance liquid chromatography coupled with linear ion trap‐Orbitrap mass spectrometer for characteristic fragments and fragmentation rules of each structure type. (2) The raw data of 70% methanol extract was collected by ultra high performance liquid chromatography quadrupole time‐of‐flight tandem mass spectrometry. (3) The chemical components database that consisted of names, chemical formulas and structures of potential components in Schisandrae chinensis Fructus was established by summarizing previous literature to screen the collected liquid chromatography with mass spectrometry data and obtain matched compounds. (4) Characteristic fragments, literature, and reference standards were used to verify the matches. (5) Characteristic fragment filtering combined with online database querying was used to deduce potential new compounds. As a result, a total of 94 compounds were identified or characterized and 16 of them were potential new compounds. The study provided a reference for comprehensive characterization of ingredients in herbal medicine and formed the foundation for pharmacodynamic study of Schisandrae chinensis Fructus.     

Determination of chlorogenic acid,polyphenols and antioxidants in green coffee by thin‐layer chromatography,effect‐directed analysis and dot blot – comparison to HPLC and spectrophotometry methods

The great prevalence of thin‐layer chromatography over high‐performance liquid chromatography is connected with the possibility of analyzing many samples in parallel. Therefore, the method is often used in screening and/or effect directed analysis to compare composition and chemical/biological properties of many samples in one run. It was already proved, that high performance thin‐layer chromatography, in many cases, can replace high‐performance liquid chromatography for quantitative analysis. The main aim of the paper is to show that simple thin‐layer chromatography can also be used as a quantitative or at least as a semi‐quantitative method, even when it concerns effect directed analysis e.g. direct bioautography. Chlorogenic acid content was measured in four methanol extracts of various green coffees and in one extract of black coffee using thin‐layer chromatography with ultraviolet detection and thin‐layer chromatography with effect directed detection. High‐performance liquid chromatography was used as a reference method. Additionally, total contents of polyphenols and antioxidants were estimated using thin‐layer chromatography or dot‐blot on chromatography plates. These results were compared to spectrophotometric methods. It was proved that thin‐layer chromatography can be used as a quantitative (using densitometry) or semi‐quantitative method (using other detection methods including effect directed detection) as well as for estimating total antioxidants or polyphenols content.     

Discovery of cancer cell proliferation inhibitors from Salviae miltiorrhizae radix et rhizoma by a trace peak enrichment approach

Salviae miltiorrhizae radix et rhizoma is a traditional herbal medicine with anti‐cancer activities. In this work, a trace peak enrichment approach combined with a cell proliferation assay was applied for screening cancer cell proliferation inhibitors from the extract of S. miltiorrhiza. A set of 123 peak fractions were prepared, and by comprehensive screening, 21 tanshinones were screened out as cancer cell proliferation inhibitors and their structures were tentatively identified by liquid chromatography coupled with quadrupole time‐of‐flight tandem mass spectrometry analysis. The inhibitory activities of nine available screened tanshinones were validated, with their IC₅₀ values ranging from 0.63 to 28.40 μM, indicating their activities strongly inhibit the proliferation of cancer cells. This study presents tanshinones that are potential cancer cell proliferation inhibitors and may explain the anti‐cancer activity of S. miltiorrhiza.     

Characterization of in vivo antioxidant constituents and dual‐standard quality assessment of Danhong injection

Antioxidants and oxidative stress play a critical role in cardiovascular diseases. Danhong injection (DHI) is a well prescribed cardiovascular medication in China, but its detailed chemical basis and mechanisms of action remain unknown. To prove the antioxidant activity of DHI, its free radical scavenging capacity (RSC) was assessed by 2,2‐diphenyl‐1‐picrylhydrazyl (DPPH) spectrophotometric assay. The 50% radical scavenging activity value was 1:129.2 mL/mL, against 0.95 mm DPPH. To further identify the antioxidant compounds, modified thin‐layer chromatography combined with DPPH bioautography assay was used. Compared with vitamin C, 11 of 16 available compounds displayed strong antioxidant activity, which were also detected in rat serum after intravenous administration of DHI by ultra‐performance liquid chromatography–tandem mass spectrometry, except for hydroxysafflor yellow A. Therefore, 10 antioxidants remaining in the blood as key markers, and six other compounds as general markers, were employed to perform the quality control of DHI by ultra‐performance liquid chromatography–ultraviolet detection after systematic methodological validation. The analytical results indicate a high correlation (r = 0.9) between the total content of those antioxidants remaining in blood and RSC of DHI among 10 batches. Further, the antioxidant profiling and chemical marker quantification as dual‐standard quality assessment was successfully applied to evaluate Danshen and safflower injections. Copyright © 2012 John Wiley & Sons, Ltd.     

Screening,and identification of the binding position,of xanthine oxidase inhibitors in the roots of Lindera reflexa Hemsl using ultrafiltration LC–MS combined with enzyme blocking

A method based on enzyme blocking combined with ultrafiltration liquid chromatography–mass spectrometry (LC–MS) has been developed to identify xanthine oxidase (XOD) inhibitors in the roots of Lindera reflexa Hemsl (LR) and determine their binding positions. Allopurinol and febuxostat, known XOD inhibitors, which occupy different binding positions in XOD, were used as blockers and pre‐incubated with XOD. Then the LR extract was incubated without XOD, and with XOD, allopurinol‐blocked XOD and febuxostat‐blocked XOD before ultrafiltration LC–MS was performed. By comparing the chromatographic profiles of the incubation samples, not only the ligands, but also the binding position of these ligands with XOD could be determined. Finally, three compounds, pinosylvin, pinocembrin and methoxy‐5‐hydroxy‐trans‐stilbene, were identified as potential XOD inhibitors and the binding modes of these three compounds were shown to be similar to those of febuxostat. To verify the XOD inhibitory activity of the screened compounds, the microplate method and molecular docking in silico were used to evaluate the enzyme inhibitory activities and the binding positions with XOD. The results showed that the developed method could screen for XOD ligands in LR extracts and also determine the binding positions of the ligands. To our knowledge, this is the first report of the XOD inhibitory activity of these three compounds.