RESUMO
Meconopsis integrifolia (Maxim.) Franch. is used extensively in traditional Tibetan medicine for its potent anti-inflammatory properties. In this study, six cyclooxygenase-2 (COX-2) inhibitors were purified from M. integrifolia using high-speed counter-current chromatography guided by ultrafiltration liquid chromatography (ultrafiltration-LC). First, ultrafiltration-LC was performed to profile the COX-2 inhibitors in M. integrifolia. The reflux extraction conditions were further optimized using response surface methodology, and the results showed that the targeted COX-2 inhibitors could be well enriched under the optimized extraction conditions. Then the six target COX-2 inhibitors were separated by high-speed countercurrent chromatography with a solvent system composed of ethyl acetate/n-butanol/water (4:1:4, v/v/v. Finally, the six COX-2 inhibitors, including 21.2 mg of 8-hydroxyluteolin 7-sophoroside, 29.6 mg of 8-hydroxyluteolin 7-[6'''-acetylallosyl-(1â2)-glucoside], 42.5 mg of Sinocrassoside D3, 54.1 mg of Hypolaetin 7-[6'''-acetylallosyll-(lâ2)-3''-acetylglucoside, 30.6 mg of Hypolaetin 7-[6'''-acetylallosyll-(lâ2)-6''-acetylglucoside and 17.8 mg of Hypolaetin were obtained from 500 mg of sample. Their structures were elucidated by 1 H-NMR spectroscopy. This study reveals that ultrafiltration-LC combined with high-speed counter-current chromatography is a robust and efficient strategy for target-guided isolation and purification of bioactive molecules. It also enhances the scientific understanding of the anti-inflammatory properties of M. integrifolia but also paves the way for its further medicinal applications.
Assuntos
Distribuição Contracorrente , Inibidores de Ciclo-Oxigenase 2 , Papaveraceae , Distribuição Contracorrente/métodos , Inibidores de Ciclo-Oxigenase 2/farmacologia , Ultrafiltração/métodos , Cromatografia Líquida de Alta Pressão/métodos , Cromatografia LíquidaRESUMO
This study presents an efficient strategy for large-scale preparation of low polarity gingerols directly from ginger crude extract by high-speed countercurrent chromatography with different rotation mode. The ultrasonic-assisted extraction conditions were optimized by response surface methodology and the results showed the major low polarity gingerols could be well enriched under the optimized extraction conditions. Then the crude extract without any pretreatment was directly separated by high-speed countercurrent chromatography with different rotation mode using n-hexane/ethyl acetate/methanol/water (6:4:6:4, v/v/v/v) as the solvent system. In about 400 min, five major gingerols including 150 mg of [6]-gingerol, 50 mg of [8]-gingerol, 20 mg of [6]-shogaol, 43 mg of [6]-dehydrogingerdione, and 40 mg of [10]-gingerol were obtained from 1.2 g of crude extract in a single run with repeated injection. Their structures were identified by 1 H-NMR spectroscopy.
Assuntos
Distribuição Contracorrente , Zingiber officinale , Distribuição Contracorrente/métodos , Zingiber officinale/química , Rotação , Extratos Vegetais/química , Álcoois Graxos/químicaRESUMO
Partition coefficient is a key parameter for counter-current chromatography separation. High-performance liquid chromatography (HPLC) is the most commonly used tool for the screening of partition coefficients. However, HPLC technology is not applicable to the compounds present in the same chromatographic peak. Nuclear magnetic resonance (NMR) technology could easily distinguish compounds according to their characteristic absorption even if they exist in the same HPLC peak. In this study, two flavonoids present in the same HPLC peak were successfully purified by counter-current chromatography with a solvent system screened by NMR to show the great potential of NMR technology in the screening of the partition coefficient of co-efflux compounds. Through NMR screening, an optimized ethyl acetate/n-buthanol/water (7:3:10, v/v/v) system was applied in this study. As a result, two flavonoids, including 4.8 mg of 3'-methoxyl-6'''-O-feruloylsaponarin and 9.8 mg of 6'''-O-feruloylsaponarin were separated from 15 mg of the mixture. There is only one methoxy group difference between the two flavonoids. This study provides a new strategy for the screening of counter-current chromatography solvent systems and broadens the application scope of counter-current chromatography.
Assuntos
Distribuição Contracorrente , Hordeum , Solventes/química , Cromatografia Líquida de Alta Pressão/métodos , Distribuição Contracorrente/métodos , Plântula/química , Flavonoides/análise , Extratos Vegetais/química , Espectroscopia de Ressonância MagnéticaRESUMO
Urtica laetevirens Maxim. is used extensively in traditional Chinese medicine (TCM) for its potent antioxidative properties. In this study, three antioxidants were purified from U. laetevirens. using HSCCC guided by online DPPH-HPLC analysis. Firstly, the online DPPH-HPLC analysis was performed to profile out the antioxidant active molecules in U. laetevirens. The ultrasonic-assisted extraction conditions were optimized by response surface methodology and the results showed the targeted antioxidant active molecules could be well enriched under the optimized extraction conditions. Then, the antioxidant active molecules were separated by high-speed countercurrent chromatography ethyl acetate/n-butanol/water (2:3:5, v/v/v) as the solvent system. Finally, the three targets including 16.8 mg of Isovitexin, 9.8 mg of Isoorientin, and 26.7 mg of Apigenin-6,8-di-C-ß-d-glucopyranoside were obtained from 100 mg of sample. Their structures were identified by 1H NMR spectroscopy.
Assuntos
Antioxidantes , Urticaceae , Antioxidantes/química , Cromatografia Líquida de Alta Pressão/métodos , Extratos Vegetais/química , Espectroscopia de Ressonância Magnética , Distribuição Contracorrente/métodosRESUMO
In high-throughput scenarios of targeted metabolomics, it is a significant challenge to process complex NMR spectra with severely overlapping signals produced by metabolites with similar chemical structures. Traditional frequency-domain NMR is ineffective to some degree due to the low sensitivity and poor resolution, and the precision of quantitation is usually affected by poorly or inconsistently phased and baselined spectra. Here, we established a strategy based on time-domain NMR focusing on methyl protons for targeted metabolomics. The targeted metabolomics focusing on bufadienolides for varietal discrimination of three toad venoms was conducted to demonstrate the practicability of time-domain-based methyl proton NMR. It revealed that the signals could be precisely identified and quantitated with an signal-to-noise ratio (SNR) of about 10 and a resolution of about 1.0 Hz. The sensitivity and resolution improvement make it particularly applicable in targeted metabolomics. The precise and absolute quantitation ability confirmed by triple-quadrupole mass spectrometry (QqQ-MS) could further extend its application range. Importantly, the methyl group is common in metabolites with a relatively wide chemical shift range. Time-domain analysis could be conducted in common NMR software. This technique is very easy and convenient for general researchers when employed as a complementary alternative to traditional frequency-domain NMR, especially in the field of targeted metabolomics.
Assuntos
Metabolômica , Prótons , Imageamento por Ressonância Magnética , Espectroscopia de Ressonância Magnética/métodos , Espectrometria de Massas/métodos , Metabolômica/métodosRESUMO
Biosynthesis is a promising way to manufacture desired products, however, the purification of its final products is a tough work due to the huge amount of reaction matrix. Liquid stationary phase of high-speed counter-current chromatography could easily avoid the commonly disadvantages that occurred in traditional column chromatography in the field of biosynthesized products purification. This characteristic makes high-speed counter-current chromatography particularly applicable for final products separation in biosynthesis. In this study, the glycosylation products of Silybin B by one-pot glycosylation were successfully purified by high-speed counter-current chromatography to show the applicability of high-speed counter-current chromatography for preparative separation of biosynthesis products. An optimized n-hexane/ethyl acetate/methanol/water (2:5:2:3, v/v/v/v) system was applied in this study. As a result, four Silybin B glycosylation products, including 7 mg of Silybin B-5-O-ß-D-glucoside (SG-1), 12 mg of Silybin B-3-O-ß-D-glucoside (SG-2), 10 mg of Silybin B-7-O-ß-D-glucoside (SG-3), and 24 mg of Silybin B-20-O-ß-D-glucoside (SG-4), were simultaneously separated from 200 mg of glycosylation crude products, with the purity of 89.3, 95.2, 96.4, and 97.5%, respectively. Their structures were identified by spectroscopic analysis.
Assuntos
Distribuição Contracorrente/métodos , Acetatos/química , Glicosilação , Hexanos/química , Metanol/químicaRESUMO
This study presents an efficient strategy based on liquid-liquid extraction and pH-zone-refining counter-current chromatography for selective enrichment, separation, and purification of alkaloids and organic acids from natural products. First, an acid or base modified two-phase solvent system with maximum or minimum partition coefficient was developed for the liquid-liquid extraction of the crude extract. As a result, alkaloids or organic acids could be selectively enriched in the upper or lower phase. Then pH-zone-refining counter-current chromatography was employed to separate and purify the selectively enriched alkaloids or organic acids efficiently. The selective enrichment and separation of five bufadienolide from toad venom of Bufo marinus was used as an example to show the advantage of this strategy. As a result, 759 mg of selectively enriched bufadienolide was obtained from 2 g of crude extract and the total content of five targets was increased from 14.64 to 83%. A total of 31 mg of marinobufagin-3-adipoyl-l-arginine, 42 mg of telocinobufagin-3-pimeloyl-l-arginine, 51 mg of telocinobufagin-3-suberoyl-l-arginine, 132 mg of marinobufagin-3-suberoyl-l-arginine, and 57 mg of bufalin-3-suberoyl-l-arginine were all simultaneously separated from 500 mg of selectively enriched sample, with the purity of 92.4, 97.5, 90.3, 92.1, and 92.8%, respectively.
Assuntos
Alcaloides/isolamento & purificação , Produtos Biológicos/isolamento & purificação , Distribuição Contracorrente , Extração Líquido-Líquido , Alcaloides/química , Animais , Produtos Biológicos/química , Bufo marinus , Concentração de Íons de HidrogênioRESUMO
This study presents an efficient strategy for separation of three phenolic compounds with high molecular weight from the crude extract of Terminalia chebula Retz. by ultrasound-assisted extraction and high-speed counter-current chromatography. The ultrasound-assisted extraction conditions were optimized by response surface methodology and the results showed the target compounds could be well enriched under the optimized extraction conditions. Then the crude extract was directly separated by high-speed counter-current chromatography without any pretreatment using n-hexane/ethyl acetate/methanol/water (1:7:0.5:3, v/v/v/v) as the solvent system. In 180 min, 13 mg of A, 18 mg of B, and 9 mg of C were obtained from 200 mg of crude sample. Their structures were identified as Chebulagic acid (A, 954 Da), Chebulinic acid (B, 956 Da), and Ellagic acid (C) by (1) H NMR spectroscopy.
Assuntos
Fenóis/química , Fenóis/isolamento & purificação , Extratos Vegetais/química , Terminalia/química , Distribuição Contracorrente , Peso Molecular , UltrassomRESUMO
Anthraquinone glycosides, such as chrysophanol 1-O-ß-d-glucoside, chrysophanol 8-O-ß-d-glucoside, and physion 8-O-ß-d-glucoside, are the accepted important active components of Rheum tanguticum Maxim. ex Balf. due to their pharmacological properties: antifungal, antimicrobial, cytotoxic, and antioxidant activities. However, an effective method for the separation of the above-mentioned anthraquinone glycosides from this herb is not currently available. Especially, greater difficulty existed in the separation of the two isomers chrysophanol 1-O-ß-d-glucoside and chrysophanol 8-O-ß-d-glucoside. This study demonstrated an efficient strategy based on preparative high-performance liquid chromatography and high-speed countercurrent chromatography for the separation of the above-mentioned anthraquinone glycosides from Rheum tanguticum Maxim.ex Balf.
Assuntos
Antraquinonas/isolamento & purificação , Cromatografia Líquida de Alta Pressão/métodos , Distribuição Contracorrente/métodos , Medicamentos de Ervas Chinesas/isolamento & purificação , Glucosídeos/isolamento & purificação , Rheum/química , Antraquinonas/química , Glucosídeos/química , IsomerismoRESUMO
Barley seedlings are rich in flavones that can have positive effects on people with antihypoxia and antifatigue. Lutonarin and saponarin are two major flavonoid glycosides that have unique structures in barley seedlings. This study presents a new approach for the preparation of lutonarin and saponarin from barely seedlings by membrane separation technology and preparative high-performance liquid chromatography. Preparative conditions of these two flavonoid glycosides by membrane separation technology were studied using response surface methodology. Under the optimized conditions, the total contents of these two flavonoid glycosides amounts to 17.0%.
Assuntos
Apigenina/síntese química , Apigenina/isolamento & purificação , Flavonoides/isolamento & purificação , Glucosídeos/síntese química , Glucosídeos/isolamento & purificação , Glicosídeos/isolamento & purificação , Hordeum/química , Plântula/química , Apigenina/química , Cromatografia Líquida de Alta Pressão , Flavonoides/síntese química , Flavonoides/química , Glucosídeos/química , Glicosídeos/síntese química , Glicosídeos/química , Estrutura MolecularRESUMO
This study presents an efficient strategy based on liquid-liquid extraction, high-speed counter-current chromatography, and preparative HPLC for the rapid enrichment, separation, and purification of four anthraquinones from Rheum tanguticum. A new solvent system composed of petroleum ether/ethyl acetate/water (4:2:1, v/v/v) was developed for the liquid-liquid extraction of the crude extract from R. tanguticum. As a result, emodin, aloe-emodin, physcion, and chrysophanol were greatly enriched in the organic layer. In addition, an efficient method was successfully established to separate and purify the above anthraquinones by high-speed counter-current chromatography and preparative HPLC. This study supplies a new alternative method for the rapid enrichment, separation, and purification of emodin, aloe-emodin, physcione, and chrysophanol.
Assuntos
Antraquinonas/isolamento & purificação , Cromatografia Líquida de Alta Pressão/métodos , Distribuição Contracorrente/métodos , Extração Líquido-Líquido/métodos , Extratos Vegetais/isolamento & purificação , Rheum/química , Antraquinonas/química , Extratos Vegetais/químicaRESUMO
One new amine 2-dimethyl-Penidilamine (1), together with seventeen known compounds (2-18) were isolated from the 95% ethanol extract of Urtica thunbergiana Siebold & Zucc. Their structures were characterised by extensive spectroscopic analysis including NMR, mass spectra and single X-ray crystallography. Among them, compound 1 is a new compound, and compounds 3, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 17 and 18 were isolated from Urtica thunbergiana Siebold & Zucc for the first time. Among them, compound 1, 10, 15, 17 and 18 exhibited significant α-glucosidase inhibitory activity with an IC50 value of 65.12 µM, 7.42 µM, 26.24 µM, 71.31 µM and 72.55 µM, respectively. Our study provided the scientific report for the medicinal value of Urtica thunbergiana Siebold & Zucc.
RESUMO
Ginger has been used as consumed food spice and folk medicine in daily life for thousands of years in various regions of the world. Considerable antioxidation is one of the major activities for Ginger to exhibit health-promoting effects. In this study, a bioinformatic workflow was developed to generate activity labelled molecular networking (ALMN) to fuel the antioxidation active molecules profile of Ginger. In ALMN, antioxidation activity data, which was defined as correlation (r and p value) between the relative abundance of a molecule in fractions and the activity level of each fraction, was labelled to feature-based molecular network to profile out antioxidation active molecules visually. Fragmentation tree was further computed as a complementary way to conduct high confidence structure annotations of antioxidation active molecules. Consequently, 48 molecules were prioritized as antioxidation active molecules from 11,720 metabolite molecules of Ginger in a systematical way.
Assuntos
Antioxidantes , Zingiber officinale , Extratos Vegetais/química , Zingiber officinale/químicaRESUMO
In this study, a strategy based on COSMO-RS (Conductor-like Screening Model for Real Solvents) with a constrained optimization calculation was developed for ab initio calculation based solvent system selection in silico for counter-current chromatography. The separation of resibufogenin glycosylation products was selected as an example to show its practicability. The selected solvent system in silico gave the K values consistent with the experimentally measured data (RMSD=0.2861) and the glycosylation products, namely Resibufogenin-3-O-ß-D-glucoside (R-G) and Resibufogenin-3-O-ß-D-glucosyl (1â2)-ß-D-glucoside (R-2G), were successfully separated by HSCCC.
Assuntos
Distribuição Contracorrente , Glucosídeos , Solventes , GlicosilaçãoRESUMO
Bufadienolide, an essential member of the C-24 steroid family, is characterized by an α-pyrone positioned at C-17. As the predominantly active constituent in traditional Chinese medicine of Chansu, bufadienolide has been prescribed in the treatment of numerous ailments. It is a specifically potent inhibitor of Na+/K+ ATPase with excellent anti-inflammatory activity. However, the severe side effects triggered by unbiased inhibition of the whole-body cells distributed α1-subtype of Na+/K+ ATPase, restrict its future applicability. Thus, researchers have paved the road for the structural alteration of desirable bufadienolide derivatives with minimal adverse effects via biotransformation. In this review, we give priority to the present evidence for structural diversity, MS fragmentation principles, anti-inflammatory efficacy, and structure modification of bufadienolides derived from toads to offer a scientific foundation for future in-depth investigations and views.
RESUMO
Biosynthesis is a research hot-spot in recent years, however, the purification of its final products is a tough work. Liquid stationary phase and large-scale separation ability of PZRCCC could easily avoid the commonly disadvantages occurred in traditional column chromatography. These characteristics makes PZRCCC particularly applicable for final products separation in biosynthesis. In this study, the glycosylation products of ellagic acid by one-pot glycosylation were successfully purified by PZRCCC to show the applicability of PZRCCC for preparative separation of biosynthesis products. An optimized ethyl acetate/n-buthanol/water (3:3:5, v/v/v) system was applied in this study, where 5 mM trifluoroacetic acid (TFA) as the retainer and 30 mM triethylamine (TEA) as the eluter were added. As a result, four ellagic acid glycosylation products, including 51 mg of ellagic acid-4, 3'-O-ß-D-diglucoside (EG-1), 24 mg of ellagic acid-4, 4'-O-ß-D-diglucoside (EG-2), 11 mg of ellagic acid-4-O-ß-D-glucosyl (1â2)-ß-D-glucoside (EG-3) and 64 mg of ellagic acid-4-O-ß-D-glucoside (EG-4) were simultaneously separated from 500 mg of glycosylation crude products, with the purity of 93.3%, 91.2%, 89.4% and 95.5%, respectively. Their structures were identified by spectroscopic analysis.
Assuntos
Distribuição Contracorrente , Extratos Vegetais , Glicosilação , Concentração de Íons de HidrogênioRESUMO
Traditional Tibetan medicine (TTM) is a valuable source of novel therapeutic lead molecules inspired by natural products (NPs). The health benefits of Saxifraga atrata are well documented in TTM, but reports on its chemical composition are limited, most likely due to the complicated purification process. Herein, target separation and identification of 4 main radical scavenging compounds from the methanolic extract of S. atrata was were performed using medium- and high-pressure liquid chromatography coupled with online HPLC-DPPH detection. The sample was pretreated using medium pressure liquid chromatography with MCI GELâ CHP20P styrene-divinylbenzene beads as a stationary phase, yielding 1.4 g of the target DPPH inhibitors (Fr4, 11.9% recovery). The compounds were further purified and isolated using HPLC on RP-C18 (ReproSil-Pur C18 AQ) followed by HILIC (Click XIon) column separation, resulting in 2.8 mg of fraction Fr4-1-1, 6.8 mg of fraction Fr4-2, 244.9 mg of the Fr4-3-1 sample, and 38.3 mg of Fr4-4-1. The structure and purity of the target compounds were determined, and four compounds (ethyl gallate, 11-O-galloylbergenin, rutin and isoquercitrin) were isolated with >95% purity. The developed methodology is efficient for targeted isolation of high-purity radical scavengers from NP extracts and could be used for rapid identification and isolation of DPPH inhibitors from various NPs.
Assuntos
Compostos de Bifenilo/análise , Técnicas de Química Analítica/métodos , Cromatografia Líquida de Alta Pressão , Picratos/análise , Extratos Vegetais/isolamento & purificação , Saxifragaceae/química , Antioxidantes/análise , Compostos de Bifenilo/antagonistas & inibidores , Picratos/antagonistas & inibidores , Extratos Vegetais/químicaRESUMO
This study presents an efficient strategy based on pH-zone-refining counter-current chromatography with a hydrophilic organic/salt-containing two-phase system composed of acetonitrile, sodium chloride and water for preparative separation of polar alkaloids from natural products. Acetonitrile-sodium chloride-water system provides a wider range of polarity for polar alkaloids than classical aqueous two-phase systems. It gets rid of the effect of free hydrogen ion, strong ionic strength, hold low viscosity and the sharp retainer border could be formed easily. So acetonitrile-sodium chloride-water system showed great advantages to pH-zone-refining counter-current chromatography for polar alkaloids. The separation of polar indole alkaloids from toad venom was selected as an example to show the advantage and practicability of this strategy. An optimized acetonitrile-sodium chloride-water (54%:5%:41%, w%) system was applied in this study, where 10â¯mM triethylamine (TEA) as the retainer and 15â¯mM hydrochloric acid (HCl) as the eluter were added. As a result, three polar indole alkaloids, including 19â¯mg of serotonin, 45â¯mg of 5-Hydroxy-N'-methyl tryptamine, 33â¯mg of bufotenine were simultaneously separated from 500â¯mg of 5% ethanol elution fraction of toad venom on macroporous resin chromatography, with the purity of 91.3%, 97.5% and 89.4%, respectively. Their structures were identified by spectroscopic analysis.
Assuntos
Produtos Biológicos/química , Distribuição Contracorrente/métodos , Alcaloides Indólicos/isolamento & purificação , Acetonitrilas/química , Venenos de Anfíbios/química , Concentração de Íons de Hidrogênio , Alcaloides Indólicos/química , Cloreto de Sódio/química , Solventes/químicaRESUMO
This study presents an efficient strategy based on liquid-liquid extraction with three-phase solvent system and high speed counter-current chromatography for rapid enrichment and separation of epimers of minor bufadienolide from toad meat. The reflux extraction conditions were optimized by response surface methodology first, and a novel three-phase solvent system composed of n-hexane/methyl acetate/acetonitrile/water (3:6:5:5, v/v) was developed for liquid-liquid extraction of the crude extract. This integrative extraction process could enrich minor bufadienolide from complex matrix efficiently and minimize the loss of minor targets induced by repeated extraction with different kinds of organic solvents occurring in the classical liquid two-phase extraction. As a result, four epimers of minor bufadienolide were greatly enriched in the middle phase and total content of these epimers of minor bufadienolide was increased from 3.25% to 46.23%. Then, the enriched four epimers were separated by HSCCC with a two-phase solvent system composed of chloroform/methanol/water (4:2:2, v/v) successfully. Furthermore, we tested Na+,K+-ATPase (NKA) inhibitory effect of the four epimers. 3ß-Isomers of bufadienolide showed stronger (>8-fold) inhibitory activity than 3α-isomers. The characterization of minor bufadienolide in toad meat and their significant difference of inhibitory effect on NKA would promote the further quantitative analysis and safety evaluation of toad meat as a food source.
Assuntos
Bufanolídeos/química , Bufanolídeos/isolamento & purificação , Bufonidae , Distribuição Contracorrente/métodos , Extração Líquido-Líquido/métodos , Carne/análise , Animais , Bufanolídeos/farmacologia , Inibidores Enzimáticos , Isomerismo , ATPase Trocadora de Sódio-Potássio/antagonistas & inibidores , SolventesRESUMO
The glycosyltransferase OleD variant as a catalyst for the glycosylation of four pairs of epimers of cardiotonic steroids (CTS) are assessed. The results of this study demonstrated that the OleD-catalyze glycosylation of CTS is significantly influenced by the configuration at C-3 and the A/B fusion mode. 3ß-OH and A/B ring cis fusion are favoured by OleD (ASP). An epoxide ring at C-14 and C-15 further increases the bioconversion rate; while an acetyl group at C-16 and lactone ring type at C-17 did not influence the biotransformation. A high conversion rate corresponded to a low K m value. A molecular docking simulation showed that filling of hydrophobic pocket II and interaction with residue Tyr115 may play an important role in the glycosylation reactions catalyzed by OleD glycosyltransferases. Furthermore, the glycosylation products showed a stronger inhibitory activity for Na+, K+-ATPase than the corresponding aglycones. This study provides the first stereoselective properties for OleD (ASP) catalyzed glycosylation.