High-resolution monophenolase/diphenolase/radical scavenging profiling for rapid screening of natural whitening candidates from Rosa rugosa cv. ‘Plena’

Antioxidants and tyrosinase inhibitory components were successfully screened and separated from Rosa rugosa cv. ‘Plena’ by high-performance liquid chromatography microfractionation bioactive screening combined with several separation and purification methods. Ethyl acetate extract of Rosa rugosa cv. ‘Plena’ showed high antioxidant activity and tyrosinase inhibitory activity. High-speed countercurrent chromatography, silica gel column chromatography, and semi-preparative high-performance liquid chromatography were used for the preparative separation of four bioactive components from ethyl acetate extract. Two tyrosinase-inhibiting active substances, flavogallonic acid, and N¹-N⁵-N¹⁰-tri-4-p-coumaroylspermidine, were isolated from Rosa rugosa cv. ‘Plena’, and they showed great monophenolase inhibition activity (half-maximal inhibitory concentration: 664.60 and 23.77 μg/ml, respectively) and excellent diphenolase inhibition activity (half-maximal inhibitory concentration: 23 614.61 and 16.80 μg/ml, respectively). Meanwhile, gallic acid, flavogallonic acid, and ellagic acid were shown to have excellent 1,1-diphenyl-2-picryl-hydrazyl antioxidant activity (half maximal inhibitory concentration: 6.66, 20.17, and 13.45 μg/ml), and 2,2′-azino-bis(3-ethylbenzothiazoline-6-sulfonic acid) antioxidant activity (half maximal inhibitory concentration: 3.53, 3.83, and 2.78 μg/ml). Molecular docking revealed that flavogallonic acid and N¹-N⁵-N¹⁰-tri-4-p-coumaroylspermidine had a strong binding affinity (–9.3 and –10 kcal/mol, respectively) to tyrosinase through hydrogen bonding and hydrophobic interactions.     

Antioxidant,antibacterial and cytotoxic potential of the ripe fruits of Solanum lycocarpum A. St. Hil. (Solanaceae)

Ethanol extract (EE) and fractions obtained from the ripe fruits of Solanum lycocarpum were examined in order to determine their phenolic composition, antioxidant capacity, antibacterial activities and cytotoxic potential. High-performance liquid chromatography coupled with DAD analysis indicated that caffeic and chlorogenic acids were the main phenolic compounds present in the EE, dichloromethane (DCM) and ethyl acetate (Ac) fractions. The antioxidant activity assessed by the scavenging ability on 1,1-diphenyl-2-picrylhydrazyl radical was significantly more pronounced for DCM and Ac fractions than that of the commercial antioxidant 2,6-di-tert-butyl-4-methylphenol (BHT). EE and fractions exhibited selective antibacterial activity against Gram-positive bacteria, especially the hexane (Hex) and DCM fractions. EE and fractions exhibited low toxicity towards the LLC-MK2 cell line, especially the Hex, DCM and Ac fractions. This work provides the knowledge of phenolic composition in the extract and fractions from the ripe fruits of S. lycocarpum and their antioxidant, antibacterial and cytotoxic activities.     

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.     

Efficient strategies for preparative separation of iridoid glycosides and flavonoid glycosides from Hedyotis diffusa

Efficient strategies for the preparative separation of iridoid glycosides and flavonoid glycosides from Hedyotis diffusa using preparative high-performance liquid chromatography combined with appropriate pretreatment technologies were developed. Four fractions (Fr.1-1, Fr.1-2, Fr.1-3, and Fr.2-1) were firstly isolated from the crude extract of Hedyotis diffusa by column chromatography with C18, resin, and silica gel materials, respectively. Then, corresponding separation strategies were developed according to the polarity and chemical constituents. High-polar compounds of Fr.1-1 were purified by hydrophilic reversed-phase liquid chromatography and hydrophilic interaction liquid chromatography mode. The combination of C18 and phenyl columns realized the complementary separation of iridoid glycosides in Fr.1-2. Meanwhile, the improved selectivity caused by the change of organic solvent in the mobile phase was utilized to realize the purification of flavonoid glycosides in Fr.1-3 and Fr. 2-1. Finally, 27 compounds (purity > 95%) mainly involving nine iridoid glycosides and five flavonoid glycosides were obtained. A complete strategy was established for the separation of a complex sample with a wide polarity range, to jointly solve the problems of enrichment of target components and separation of structural analogs.     

Structures of novel norstilbene dimer, longusone A, and three new stilbene dimers, longusols A, B, and C, with antiallergic and radical scavenging activities from Egyptian natural medicine Cyperus longus

The methanolic extract of the whole plant of Cyperus longus originating in Egypt was found to show antiallergic effect on ear passive cutaneous anaphylaxis reactions in mice. By bioassay-guided separation, 11 stilbenes and stilbene dimers including a novel norstilbene dimer, longusone A, and three new stilbene dimers, longusols A, B, and C, were isolated. Their structures were elucidated on the basis of chemical and physicochemical evidence. Among the isolates, longusol B (IC(50)=96 μM), luteolin (3.0 μM), resveratrol (17 μM), piceatannol (24 μM), and cassigarols E (84 μM) and G (84 μM) were found to inhibit the release of β-hexosaminidase, as a marker of antigen-induced degranulations, in rat basophilic leukemia (RBL-2H3) cells. In addition, the methanolic extract and the constituents showed 1,1-diphenyl-2-picrylhydrazyl (DPPH) radical scavenging activity (SC(50)=22 μg/ml and 2.8-29 μM, respectively).     

Preparative High-Performance Liquid Chromatography Isolation of the True Proazulenes from Artemisia Arborescens L

Abstract

The use of the preparative medium-pressure liquid chromatography (prep-MPLC) and the preparative high-pressure liquid chromatography (prep-HPLC) techniques for the separation of the main true proazulenes from a purified extract of Artemisia arborescens L. are reported and discussed.     

Comparative phytochemical study of methanolic and ethanolic extracts of Thymus linearis and their antibacterial and antioxidant potential

Thymus linearis (Thyme) is a medicinal plant widely distributed throughout Asia. Various parts of thyme are utilized for diverse medicinal purposes, including its use as a tonic and diuretic, for cough relief, as a flavoring agent, in treating dysentery, and for alleviating stomach disorders. Numerous studies have been conducted to explore the unexploited potential of thyme. Thyme was collected from the northern region of Pakistan, and sun-mediated extraction was conducted. Phytochemical analysis, utilizing GC–MS, revealed numerous bioactive phytochemical constituents with disease-preventing roles, including detoxifying agents, antioxidants, anticancer compounds, dietary fiber, neuropharmacological agents, and immunity-potentiating agents, in the methanolic and ethanolic (14 days) extracts of the flower, leaf, and stem. The 1,1-diphenyl-2-picrylhydrazyl (DPPH) assay results indicated that the ethanolic and methanolic extracts of the stem exhibited the highest antioxidant activity, reaching up to 67.34% and 62.73%, respectively, while the values for the flower and leaf extracts (both methanol and ethanol) were around 60%. The IC₅₀ (half maximal inhibitory concentration) values were also calculated for all the samples, ranging between 7 and 9 μg/mL. Positive antibacterial and antifungal effects against Bacillus subtilis and Escherichia coli, as well as Aspergillus niger (fungi), were observed only in the extracts of the flower (both methanol and ethanol). The sun-mediated technique was used for extraction for the first time in this study. Therefore, this study introduces a novel approach to the extraction of bioactive compounds from medicinal plants, ultimately contributing to the development of herbal drugs with more convenient and cost-effective methods.     

Microcolumn liquid chromatography with thermionic detection of the enantiomers of O-ethyl S-2-diisopropylaminoethyl methylphosphonothioate (VX)

An improved interface for the on-line coupling of microcolumn liquid chromatography (micro-LC) with thermionic detection (TID) is described. Modifications have been made to enable separate adjustment of the eluent introduction and the detector flame temperature in order to improve the sensitivity and ease of use of the system. The micro-LC-TID was used for the chiral separation of the nerve agent O-ethyl S-2-diisopropylaminoethyl methylphosphonothioate (VX). Baseline separation for the enantiomers of VX was obtained on Chiralcel OD using 1% isopropanol in hexane as the eluent. The detection limit of VX using 60 nl injections is ca. 5 μg/ml (ppm range). However, when using large-volume injections (10 μl) the detection limit is ca. 25 ng/ml (ppb range).     

A rapid assay to screen adenosine deaminase inhibitors from Ligustri Lucidi Fructus against metabolism of cordycepin utilizing ultra-high-performance liquid chromatography–tandem mass spectrometry

Cordycepin has recently received increased attention owing to its extensive pharmacological activity. Adenosine deaminase (ADA) is widely distributed in mammalian blood and tissues; as a result, cordycepin is quickly metabolized upon entering into the body and converted into the inactive metabolite 3′-deoxyinosine, thus limiting its activity when administered alone. We herein present a novel ultra-high-performance liquid chromatography–tandem mass spectrometry (UHPLC–MS/MS) method for screening ADA inhibitors against the metabolism of cordycepin. Cordycepin and 3′-deoxyinosine were chosen as substrate and product, respectively. A proper separation was achieved for all analytes within 3 min. 3′-Deoxyinosine was quantified in the presence or absence of potential ADA inhibitors to evaluate ADA activity. The assay can simultaneously determine substrate and product, with the endogenous substance and ADA inhibitors added not interfering in its activity. After optimizing the enzymatic incubation and UHPLC–MS/MS conditions, K_m and V_max values for ADA deamination of cordycepin were 95.18 ± 7.85 μm and 363.90 ± 12.16 μmol/min/unit, respectively. Oleanolic acid and ursolic acid from Ligustri Lucidi Fructus were chosen as ADA inhibitors with half maximal inhibitory concentration values of 21.82 ± 0.39 and 18.41 ± 0.14 μm , respectively. A non-competitive inhibition model was constructed and this assay can be used to screen other potential ADA inhibitors quickly and accurately.     

Halioxepine, a new meroditerpene from an Indonesian sponge Haliclona sp

Chemical investigations on a sponge Haliclona sp. found a meroditerpene 1 having a new carbon skeleton. By analyzing spectroscopic data, the structure was elucidated to comprise a substituted hydroquinone, a tetrahydrooxepine, and a cyclohexene, and these components were united with C1 and C2 units. Compound 1 showed moderate cytotoxicity against NBT-T2 cells with IC50 4.8 μg/ml and also antioxidant activity against 1,1-diphenyl-2-picrylhydrazyl (DPPH) with IC50 3.2 μg/ml.     

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.     

Preparative isolation and analysis of alcohol dehydrogenase inhibitors from Glycyrrhiza uralensis root using ultrafiltration combined with high‐performance liquid chromatography and high‐speed countercurrent chromatography

A simple, rapid, and effective assay based on ultrafiltration combined with high‐performance liquid chromatography and high‐speed countercurrent chromatography was developed for screening and purifying alcohol dehydrogenase inhibitors from Glycyrrhiza uralensis root extract. Experiments were carried out to optimize binding conditions including alcohol dehydrogenase concentration, incubation time, temperature, and pH. By comparing the chromatograms, three compounds were found possessing alcohol dehydrogenase binding activity in Glycyrrhiza uralensis root. Under the target‐guidance of ultrafiltration combined with the high‐performance liquid chromatography experiment, liquiritin ( 1 ), isoliquiritin ( 2 ), and liquiritigenin ( 3 ) were separated by high‐speed countercurrent chromatography using ethyl acetate/methanol/water (5:1:4) as the solvent system. The alcohol dehydrogenase inhibitory activities of these three isolated compounds were assessed; compound 2 showed strongest inhibitory activity with an IC₅₀ of 8.95 μM. The results of the present study indicated that the combinative method using ultrafiltration, high‐performance liquid chromatography and high‐speed countercurrent chromatography could be widely applied for the rapid screening and isolation of enzyme inhibitors from complex mixtures.     

Isolation and Identification of Antioxidative Peptides from Frog (Hylarana guentheri) Protein Hydrolysate by Consecutive Chromatography and Electrospray Ionization Mass Spectrometry

Frog (Hylarana guentheri) proteins were hydrolyzed by papain and Flavourzyme to obtain antioxidative peptides. The antioxidant activities of the frog protein hydrolysates (FPHs) were measured, including 1,1-diphenyl-2-picrylhydrazyl (DPPH) radical scavenging activity (IC₅₀?=?9.94?±?0.13 mg/mL), reducing power (0.39?±?0.01 at 5.0 mg/mL), and oxygen radical absorbance capacity (ORAC) value (789.15?±?75.10 μmol Trolox equivalents/g). The hydrolysates were purified by ultrafiltration, ion exchange chromatography, gel filtration chromatography, and reverse-phase high-performance liquid chromatography (RP-HPLC). Through analysis of ESI-MS/MS, two dipeptides were identified as Leu/Ile-Lys (259.1607 Da) and Phe-Lys (293.1446 Da), respectively.     

Major flavonoid components of heartsease (<Emphasis Type="Italic">Viola tricolor</Emphasis> L.) and their antioxidant activities

Sephadex LH-20 column chromatography was used to separate flavonoid components in a heartsease methanol extract. One of the main components was identified by NMR as violanthin (6-C-glucosyl-8-C-rhamnosylapigenin). As a first approximation, the other main flavonoid component was considered to be rutin (3-O-rhamnoglucosylquercetin), based on comprehensive comparison of retention times and UV spectra of reference molecules, as well as molecular mass and fragmentation patterns obtained by mass spectrometry. The minor flavonoids were separated by polyamide column and analyzed by LC-MS. The antioxidant capacity of different flavonoid fractions was determined using both Trolox equivalent antioxidant capacity (TEAC) and 2,2-diphenyl-1-picrylhydrazyl (DPPH) in vitro antioxidant assays. The highest electron-donor capacity was found for the major flavonoid component (rutin), whereas one minor component-rich flavonoid fraction exhibited the highest hydrogen-donor activity.     

Identification and quantification of anthocyanins,flavonoids, and phenolic acids in flowers of Rhododendron arboreum and evaluation of their antioxidant potential

The current study aimed to investigate the anthocyanins, non-anthocyanins (flavonoids and phenolic acids), and free radicals scavenging potential in the flowers of Rhododendron arboreum using ultra high performance liquid chromatography with ion mobility quadrupole time of flight tandem mass spectrometry. A total of 25 constituents including nine anthocyanins, six phenolic acids, and ten flavonoids were identified in the flower extract. The major anthocyanins identified were cyanidin-3-O-β-galactoside ( 1 ), cyanidin-3-O-α-arabinoside ( 4 ), and cyanidin-3-O-rhamnoside ( 8 ), while quercetin glycosides were the main identified flavonoids in R. arboreum flowers. Additionally, ultra high performance liquid chromatography methods were developed and validated for the quantification of nine compounds (anthocyanins, flavonoid glycosides, and phenolic acids); five of them were quantified using internal standards. The extracts were analyzed for total phenolics (123.6 mg GAE/g), anthocyanin content (1.76% w/w), and evaluated for antioxidant properties against 2,2-diphenyl-1-picrylhydrazyl radical (IC₅₀: 102.06 and 96.92 μg/mL) and 2,2′-azino-bis (3-ethylbenzothiazoline-6-sulfonic acid) radical cation (112.25 and 45.59 μM TE/g) assays. The profiling of R. arboreum for anthocyanins is reported for the first time. The findings suggest that the flowers are a promising source of bioactive constituents and could be used as functional food, antioxidants, and nutraceuticals.     

Preparative separation of liquiritigenin and glycyrrhetic acid from Glycyrrhiza uralensis Fisch using hydrolytic extraction combined with high-speed countercurrent chromatography

The objective of this paper was to develop a preparative method for the isolation and purification of liquiritigenin and glycyrrhetic acid from Glycyrrhiza uralensis Fisch using hydrolytic extraction combined with high-speed countercurrent chromatography (HSCCC). Liquiritigenin and glycyrrhetic acid were well hydrolyzed from liquiritin and glycyrrhizic acid by hydrochloric acid, respectively. The optimal extraction conditions were obtained by single-factor and orthogonal experiments, which were 100% ethanol, 1.5 mol/L hydrochloric acid, 1:25 ratio of solid to liquid, and extracted 2 h for one time. Using the two-phase solvent system of n-hexane-ethyl acetate-methanol–water (4:5:4:5, v/v), 2.1 mg liquiritigenin (the purity was 96.5% with a recovery of 87.6%) and 12.3 mg glycyrrhetic acid (the purity was 97.1% with a recovery of 74.4%) were obtained from 315-mg crude extraction by HSCCC. The retention ratio of stationary phase was 47.2%. Their structures were identified by HPLC, melting points, UV, Fourier-transform infrared, Electrospray ionization-MS, ¹H nuclear magnetic resonance (NMR), and ¹³C NMR spectra. According to the antioxidant activity assays, liquiritigenin and glycyrrhetic acid had some scavenging abilities on 1,1-diphenyl-2-picrylhydrazyl free radicals; liquiritigenin had stronger scavenging ability on hydroxyl radicals.     

PREPARATIVE ISOLATION AND PURIFICATION OF CHEMICAL CONSTITUENTS OF BELAMCANDA BY MPLC, HSCCC AND PREP-HPLC

Combined with medium-pressure liquid chromatography (MPLC) and preparative high-pressure liquid chromatography (Prep-HPLC), high-speed countercurrent chromatography (HSCCC) was successfully applied for separation and purification of isoflavonoids from the extract of belamcanda. HSCCC separation was performed on a two-phase solvent system composed of methyl tert-butyl ether -ethyl acetate - n-butyl alcohol - acetonitrile -0.1% aqueous trifluoroacetic acid at a volume radio of 1:2:1:1:5. Semi-purified peak fractions from HSCCC separation were further purified by Prep-HPLC. Nine well-separated fractions were analyzed by HPLC-UV absorption spectrometry to determine their purities and characterized with ESI-MS(n). Except for peaksland VII (unknown) seven compounds were identified as apocynin (peak II), mangiferin (peak III), 7-O-methylmangiferin (peak IV), hispidulin (peak V), 3'-hydroxyltectoridin (peak VI), iristectorin B (peak VII), isoiridin (peak IX).     

Rotation planar extraction and rotation planar chromatography of oak (Quercus robur L.) bark

The versatile novel instrument for rotation planar extraction and rotation planar chromatography was exploited for the investigation of oak bark (Quercus robur L.). The same instrument enabled extraction of the bark, analytical proof of (+)-catechin directly in the crude extract and also its fractionation. Additionally, epimeric flavan-3-ols, (+)-catechin and (-)-epicatechin were separated by analytical ultra-micro rotation planar chromatography on cellulose plates with pure water as developing solvent. A comparison of the extraction of oak bark with 80% aqueous methanol by rotation planar extraction and medium pressure solid-liquid extraction was carried out and both techniques were shown to be suitable for the efficient extraction of oak bark. The raw extracts and fractions on thin-layer chromatography showed many compounds that possessed antioxidant activity after spraying with 1,1-diphenyl-2-picrylhydrazyl. Rotation planar fractionation of 840 mg of crude oak bark extract on silica gel gave 6.7 mg of pure (+)-catechin in one run.     

The generally useful estimate of solvent systems method facilitates off-line two-dimensional countercurrent chromatography for isolating compositions from Siraitia grosvenorii roots

Solvent system selection is a crucial and the most time-consuming step for successful countercurrent chromatography separation. A thin-layer chromatography-based generally useful estimate of solvent systems method has been developed to simplify the solvent system selection. We herein utilized the method to select a solvent system for off-line two-dimensional countercurrent chromatography to separate chemical compositions from a complex fraction of the Siraitia grosvenorii root extract. The first-dimensional countercurrent separation using chloroform/methanol/water (10:5.5:4.5, v/v/v) yielded four compounds with high purity and three mixture fractions (Fr I, III, and VII). The second-dimensional countercurrent separation conducted on Fr I, III, and VII using the hexane/ethyl acetate/methanol/water (4:6:6:4, 3:7:3:7, v/v/v) and chloroform/methanol/water (10:9:6, v/v/v) solvent systems, respectively, produced another four compounds. Four triterpenoids and four lignans were finally isolated, including two novel compounds. Hence, the generally useful estimate of solvent systems method is a feasible and efficient approach for selecting an applicable solvent system for separating complex samples. In addition, the off-line two-dimensional countercurrent chromatography method can improve both the peak resolution and the capacity of countercurrent chromatography.     

Tracking antiangiogenic components from Glycyrrhiza uralensis Fisch. based on zebrafish assays using high-speed countercurrent chromatography

Natural products are some of the most important sources of lead compounds for drug discovery. The advanced isolation technique of lead compounds of natural origin using therapeutically relevant bioassays is capable of enhancing work efficiency from complex multiconstituent extracts. In the present study, a bioassay-guided isolation strategy combined with bioactivity screening was used to identify novel angiogenesis inhibitors from licorice (Glycyrrhiza uralensis Fisch.) based on the zebrafish model and rapid preparative separation by high-speed countercurrent chromatography. Zebrafish embryos at 24 h postfertilization were chosen as the angiogenesis inhibition model for bioactivity screening. A solvent system (n-hexane-ethyl acetate-methanol-water) with different ratios was optimized and applied in the high-speed countercurrent chromatography separation of two fractions, Fr5 and Fr6, from the ethyl acetate extract of licorice. Blood circulation and vascular outgrowth in intersegmental vessels were found to be simultaneously inhibited by isoliquiritigenin and isolicoflavonol in a dose-dependent manner. Thus, these two compounds were identified and considered as active inhibitors against angiogenesis. These experimental results indicate that zebrafish bioassays combined with high-speed countercurrent chromatography may provide an alternative pathway for the rapid isolation of bioactive natural products.