RESUMO
In the present work, comprehensive two-dimensional reversed-phase countercurrent chromatography × reversed-phase liquid chromatography combined (2D RPCCC × RPLC) with 2D microfraction bioactive evaluation was employed to screen and isolate α-glucosidase inhibitors from Rheum palmatum L. Countercurrent chromatography was employed to improve 2D analysis and preparative separation. A selected biphasic solvent system composed of petroleum ether/ethyl acetate/methanol/water with gradient elution mode was used for the first dimension RPCCC separation (1D RPCCC). Solid-phase extraction was applied to eliminate interfering polar compounds before the second dimension analysis (2D RPLC). 76 components were shown in 2D contour plot in UV 280 nm. 11 Candidates were separated by a scaled-up CCC and identified by 1H NMR and 13C NMR, including anthraquinones, flavonoids, stilbenes, phenols, and glucoside derivatives. In addition, it was found that two components, resveratrol-4'-O-(6â³-galloyl)glucoside (36) and lyciumaside (43) were identified as natural α-glucosidase inhibitors in Rheum palmatum L. for the first time.
Assuntos
Inibidores de Glicosídeo Hidrolases , Rheum , Inibidores de Glicosídeo Hidrolases/farmacologia , Inibidores de Glicosídeo Hidrolases/química , Rheum/química , Distribuição Contracorrente/métodos , Cromatografia Líquida de Alta Pressão/métodos , Cromatografia de Fase Reversa , Solventes/química , Extratos Vegetais/farmacologia , Extratos Vegetais/química , GlucosídeosRESUMO
In the present study, twelve compounds from Dioscorea spongiosa were successfully purified by an efficient technique combined bioassay-guided fractionation macroporous resin column chromatography (MRCC) pretreatment and high-speed counter-current chromatography (HSCCC) separation for the first time. Then, D101 MRCC was used to fractionate the crude extract into five parts, which further applied the bioassay-guided fractionation strategy to screen the active fractions of 2 and 4. As for the separation, 200 mg Fr.2 was purified by HSCCC using EtOAc/n-BuOH/H2 O (2:2:3, v/v), leading to annulatomarin (1), dioscoresides C (2), diosniponol C (3), methyl protodioscin (4), pseudoprotodioscin (5), protogracillin (6), as well as 200 mg Fr.4 yielding montroumarin (7), dioscorone A (8), diosniponol D (9), protodioscin (10), gracillin (11), and dioscin (12) using CH2 Cl2 /MeOH/H2 O (3:3:2, v/v) with the purities over 95.0%. Finally, the isolates were assayed for their anti-inflammatory, urico-lowering, and anti-diabetic activities in vitro, which indicated that the steroidal saponins of 5, 6, and 11 showed all these three activities.
Assuntos
Distribuição Contracorrente , Dioscorea , Distribuição Contracorrente/métodos , Dioscorea/química , Extratos Vegetais/farmacologia , Extratos Vegetais/química , Bioensaio , Cromatografia Líquida de Alta Pressão/métodosRESUMO
German chamomile is one of the most effective herbal elements used in anti-allergic products and as an antioxidant. Herein, the antioxidant activity of different extract fractions of German chamomile was initially evaluated using an off-line 2,2-diphenyl-1-picrylhydrazyl spectrophotometric assay. The ethyl acetate extract demonstrated the highest efficacy in scavenging free radicals. Based on this, a rapid screening and separation method using ultra-high-performance liquid chromatography combined with the 2,2-diphenyl-1-picrylhydrazyl assay was implemented to identify antioxidants in the ethyl acetate fraction of German chamomile flowers. Ten potential radical scavengers were tentatively screened from German chamomile using a target-guided isolating approach with off-line two-dimensional high-speed countercurrent chromatography and the structures of the compounds were analyzed and identified. Ultimately, 10 radical scavengers were obtained from the ethyl acetate extract with a purity quotient exceeding 90%. The results demonstrated the effectiveness and reproducibility of this method for isolating potential antioxidants from complex mixtures in a targeted manner. This strategy can be applied to the target-guided isolation of complex mixtures of natural products with broad K-values and similar structures.
Assuntos
Acetatos , Compostos de Bifenilo , Distribuição Contracorrente , Matricaria , Picratos , Distribuição Contracorrente/métodos , Extratos Vegetais/química , Antioxidantes/análise , Espectrometria de Massa com Cromatografia Líquida , Reprodutibilidade dos Testes , Misturas Complexas , Cromatografia Líquida de Alta Pressão/métodosRESUMO
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
The traditional method for isolation and purification of polysaccharides is time-consuming. It often involves toxic solvents that destroy the function and structure of the polysaccharides, thus limiting in-depth research on the essential active ingredient of Lycium barbarum L. Therefore, in this study, high-speed countercurrent chromatography (HSCCC) and aqueous two-phase system (ATPS) were combined for the separation of crude polysaccharides of Lycium barbarum L. (LBPs). Under the optimized HSCCC conditions of PEG1000-K2HPO4-KH2PO4-H2O (12:10:10:68, w/w), 1.0 g of LBPs-ILs was successfully divided into three fractions (126.0 mg of LBPs-ILs-1, 109.9 mg of LBPs-ILs-2, and 65.4 mg of LBPs-ILs-3). Moreover, ATPS was confirmed as an efficient alternative method of pigment removal for LBPs purification, with significantly better decolorization (97.1 %) than the traditional H2O2 method (88.5 %). Then, the different partitioning behavior of LBPs-ILs in the two-phase system of HSCCC was preliminarily explored, which may be related to the difference in monosaccharide composition of polysaccharides. LBPs-ILs-1 exhibited better hypoglycemic activities than LBPs-ILs-2 and LBPs-ILs-3 in vitro. Therefore, HSCCC, combined with aqueous two-phase system, was an efficient separation and purification method with great potential for separating and purifying active polysaccharides in biological samples.
Assuntos
Medicamentos de Ervas Chinesas , Lycium , Lycium/química , Distribuição Contracorrente/métodos , Peróxido de Hidrogênio , Solventes/química , Medicamentos de Ervas Chinesas/química , Polissacarídeos/químicaRESUMO
INTRODUCTION: Many secondary metabolites isolated from plants have been described in the literature owing to their important biological properties and possible pharmacological applications. However, the identification of compounds present in complex plant extracts has remained a great scientific challenge, is often laborious, and requires a long research time with high financial cost. OBJECTIVES: The aim of this study was to develop a method that allows the identification of secondary metabolites in plant extracts with a high degree of confidence in a short period of time. MATERIAL AND METHODS: In this study, an ethanolic extract of Coffea arabica leaves was used to validate the proposed method. Countercurrent chromatography was chosen as the initial step for extraction fractionation using gradient elution. Resulting fractions presented a variation of compounds concentrations, allowing for statistical total correlation spectroscopy (STOCSY) calculations between liquid chromatography coupled with high-resolution tandem mass spectrometry (LC-HRMS/MS) and NMR across fractions. RESULTS: The proposed method allowed the identification of 57 compounds. Of the annotated compounds, 20 were previously described in the literature for the species and 37 were reported for the first time. Among the inedited compounds, we identified flavonoids, alkaloids, phenolic acids, coumarins, and terpenes. CONCLUSION: The proposed method presents itself as a valid alternative for the study of complex extracts in an effective, fast, and reliable way that can be reproduced in the study of other extracts.
Assuntos
Coffea , Distribuição Contracorrente , Distribuição Contracorrente/métodos , Espectrometria de Massas por Ionização por Electrospray/métodos , Coffea/química , Extratos Vegetais/química , Espectroscopia de Ressonância Magnética , Cromatografia Líquida de Alta Pressão/métodosRESUMO
Separation and purification of naturally occurring isomers from herbs are still challenging. High-speed counter-current chromatography (HSCCC) has been applied to isolate natural products. In this study, an off-line multi-dimensional high-speed counter-current chromatography (multi-D HSCCC) strategy was developed utilizing the in situ concentration technique with online storage recycling elution to rapidly separate bioactive isomeric neolignans from chloroform-partitioned samples of the plant Piper betle L. In the procedure, the crude sample (105 mg) was implemented using the online storage recycling technique in a two-phase solvent system composed of petroleum ether-ethyl acetate-methanol-water (7: 5: 12: 3), which first simply afforded a neolignane kadsurenone (1, 5.3 mg) and its epimer (-)-denudatin B (2, 6.4 mg). Then, the remains fr a was subjected to the second-dimensional HSCCC elution using the in situ concentration technique with online storage recycling technique in another solvent system of petroleum ether-ethyl acetate-methanol-water (5: 5: 11, 15). As a result, kadsurenin I (3, 0.6 mg) and its regioisomer pibeneolignan C (4, 5.0 mg), together with the fractional remaining fr b and fr c, were obtained. Thirdly, the fr c was reloaded to allow the HSCCC for recycling elution with the former solvent system employing the in situ concentration strategy and yielded a pair of epimers, (7R,8S,1'S)-1'-allyl-5-methoxy-8-methyl-7-piperonyl-7,8,3,6-tetrahydro-2-oxobenzofuran (5, 10.2 mg), and 3-epi-(-)-burchullin (6, 2.6 mg). Finally, the three pairs of less amount and the structurally similar isomers 1-6 were isolated from the crude fraction of P. betle with a high HPLC purity of over 95.0 % for compound 2, 4-6 and 92.5 % for compound 1, 91.0 % for 3, while the purity of 1 and 3 in 1H NMR were 89.9 % and 91.1 %, respectively. The whole isolation process was quick and efficient. Compounds 1, 2, 4 and 5 showed significantly synergistic activities combining several antibiotics against five drug-resistant Staphylococcus aureus with FICIs from 0.156 to 0.375. This novel off-line multi-dimensional HSCCC strategy could be broadened to application for the rapid separation of complex natural products.
Assuntos
Acetatos , Alcanos , Lignanas , Staphylococcus aureus Resistente à Meticilina , Piper betle , Distribuição Contracorrente/métodos , Metanol , Extratos Vegetais/química , Lignanas/análise , Cromatografia Líquida de Alta Pressão/métodos , Solventes , ÁguaRESUMO
The chemical constituents of volatile oils used in traditional Chinese medicine are highly complex. Thus, achieving the complete separation of volatile oils by one-dimensional chromatography is difficult owing to the low peak capacity of the technique. Although comprehensive two-dimensional gas chromatography provides an efficient means for separating volatile oils, it cannot be used to screen bioactive components because of its limitations. Therefore, developing a new method to separate volatile oils based on liquid chromatography is of great significance in efforts to obtain new approaches to screen bioactive components in volatile oil. The objectives of the present study are to establish an efficient method for separating the chemical constituents of Curcuma volatile oil using off-line comprehensive two-dimensional countercurrent chromatography-liquid chromatography (CCC-LC) and to investigate the two-dimensional peak capacity, orthogonality, and spatial coverage of this method. Both CCC and LC conditions were optimized. A biphasic n-hexane-methanol-water solvent system was selected via the colorimetric method, and the lower phase was used as the mobile phase in gradient-elution mode: 0-55 min, n-hexane-methanol-water (5â¶2â¶3 v/v/v); 55-170 min, n-hexane-methanol-water (5â¶3â¶2, v/v/v); 170-290 min, n-hexane-methanol-water (5â¶4â¶1, v/v/v). After gradient elution, elution-extrusion elution mode was applied within 290-375 min. Good resolution was achieved by the CCC separation process. The HPLC separation process was carried out with gradient elution using a mobile phase composed of acetonitrile (A)-water (B): 0-10 min, 50%A-65%A; 10-14 min, 65%A; 14-21 min, 65%A-85%A; 21-25 min, 85%A-95%A; 25-30 min, 95%A-55%A; 30-40 min, 55%A. Curcuma volatile oil was separated under the above optimized two-dimensional separation conditions, and the data obtained were drawn into a two-dimensional contour map using Matlab software. The calculated total peak capacity exceeded 954, which was 10 times more than that of one-dimensional chromatography. High orthogonality (r=0.17) and spatial coverage factor (68.1%) were also obtained. Our research provides a new methodology for separating volatile oils used in traditional Chinese medicine as well as an approach for evaluating the quality of traditional Chinese medicinal herbs using two-dimensional chromatographic fingerprints.
Assuntos
Distribuição Contracorrente , Óleos Voláteis , Distribuição Contracorrente/métodos , Metanol , Curcuma/química , Cromatografia Líquida , Cromatografia Líquida de Alta Pressão , ÁguaRESUMO
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
This research investigated the effectiveness of an integrated method for the extraction and separation of naphthoquinones and diarylheptanes from exocarp of Juglands mandshurica Maxim. (namely, green walnut husks). The target compounds were obtained by ultra-turrax homogenization (UTH) coupled with ultrasound-assisted extraction (UAE) technology followed by high-speed countercurrent chromatography (HSCCC). The UTH-UAE extraction method achieved higher efficiency with 2.49- and 2.36-fold to those by UAE, and 1.39- and 1.34-fold to those by UTH in a short time. HSCCC was adopted for further separation and purification; six target compounds, namely, regiolone (RE), juglone (JU), myricatomento-genin (MG), galleon (GA), 2-oxatrycyclo[13.2.2.13,7]eicosa-3,5,7(20),15,17,18-hexaen-10-16-diol (OE), and juglanin A (JA), were separated with more than 95.37% purities and more than 84.71% final recovery rates, respectively. In this study, the integrated strategy of extraction and separation could get high purity compounds quickly, which would provide time and solvent saved method for the natural products separation from plants.
Assuntos
Juglans , Naftoquinonas , Distribuição Contracorrente/métodos , Extratos Vegetais/química , Nozes , Juglans/química , Cromatografia Líquida de Alta PressãoRESUMO
In this work, eight compounds from Phellodendron chinense were separated and purified by pH-zone refining counter-current chromatography and traditional counter-current chromatography coupled with online-storage inner-recycling counter-current chromatography (IRCCC). The pH-zone-refining mode was adopted for separating 2.0 g of crude extract with the solvent system of chloroform-methanol-water (4:3:3, v/v), in which 10 mM hydrochloric acid and 10 mM triethylamine were added in the stationary and mobile phases, respectively. Meanwhile, traditional counter-current chromatography coupled with online-storage IRCCC separation was performed by the solvent system of n-hexane-ethyl acetate-methanol-water (5:5:2:8, v/v). Finally, eight compounds, including six alkaloids as 6-methylpiperidin-2-one(1), isoplatydesmine(4), berlambine(5), epiberberine(6), palmatine(7), berberine(8) and two phenolic acids as ferulic acid(2), isoferulic acid(3), were successfully obtained using these three different CCC modes with the purities over 95.0%.
Assuntos
Alcaloides , Phellodendron , Extratos Vegetais/química , Metanol , Distribuição Contracorrente/métodos , Alcaloides/análise , Solventes/química , Água , Concentração de Íons de Hidrogênio , Cromatografia Líquida de Alta Pressão/métodosRESUMO
Bergenia ciliata (haw.) Sternb, the renowned pharmaceutical plant in Jammu and Kashmir of Pakistan, is widely applied in treating different illnesses including diabetes, diarrhea, and vomiting. This work employed an efficient one-step inner-recycling counter-current chromatography for preparative separating and purifying compounds with similar partition coefficients from the rhizome of Bergenia ciliate (haw.). Five compounds, including quercetin rhamnodiglucoside (1), quercetin-3-O-rutinoside (2), bergenine (3), kaempferol (4), and palmatic acid (5), were successfully separated using the optimized biphasic solvent system that contained ter-butylmetylether/n-butanol/acetonitrile/water (2:2:1:5, v/v) with the purities over 98%. Mass spectrometry and nuclear magnetic resonance were conducted for structural identification. As a result, our proposed strategy might be applied in separating compounds with similar partition coefficients, which was advantageous with regard to the less solvent and time consumption, and the increased number of theoretical plates.
Assuntos
Distribuição Contracorrente , Plantas Medicinais , Distribuição Contracorrente/métodos , Extratos Vegetais/análise , Rizoma/química , Solventes/análise , Cromatografia Líquida de Alta PressãoRESUMO
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
Peanut hulls (Arachis hypogaea, Leguminosae), which are a side stream of global peanut processing, are rich in bioactive flavonoids such as luteolin, eriodictyol, and 5,7-dihydroxychromone. This study aimed to isolate these flavonoid derivatives by liquid-liquid chromatography with as few steps as possible. To this end, luteolin, eriodictyol and 5,7-dihydroxychromone were isolated from peanut hulls using two different techniques, high-performance countercurrent chromatography (HPCCC) and fast-centrifugal partition chromatography (FCPC). The suitability of the biphasic solvent system composed of n-hexane/ethyl acetate/methanol/water (1.0/1.0/1.0/1.5; v/v/v/v) was determined by the Conductor like Screening Model for Real Solvents (COSMO-RS), which allowed the partition ratio KD-values of the three main flavonoids to be calculated. After a one-step HPCCC separation of ~1000 mg of an ethanolic peanut hull extract, 15 mg of luteolin and 8 mg of eriodictyol were isolated with purities over 96%. Furthermore, 3 mg of 5,7-dihydroxychromone could be isolated after purification by semi-preparative reversed-phase liquid chromatography (semi-prep. HPLC) in purity of over 99%. The compounds were identified by electrospray ionization mass spectrometry (ESI-MS) and nuclear magnetic resonance spectroscopy (NMR).
Assuntos
Distribuição Contracorrente , Flavonoides , Distribuição Contracorrente/métodos , Solventes/química , Flavonoides/análise , Arachis , Luteolina/análise , Extratos Vegetais/química , Cromatografia Líquida , Cromatografia Líquida de Alta Pressão/métodosRESUMO
In this study, a multi-dimensional chromatography system was developed by integrating normal-phase flash chromatography and counter-current chromatography to isolate flavonoids, phenylpropanoids, and thymol from the aerial parts of Thymus quinquecostatus Celak. In the online multi-dimensional switching system, a normal-phase flash chromatograph packed with 1.2 g of dry homogeneous silica gel mixture (containing 600 mg of methanol extract) was connected to counter-current chromatography via a six-port valve. Two two-dimensional separations were performed using n-heptane-ethyl acetate-methanol-water (6:4:6:4, v/v) and ethyl acetate-water solvent systems sequentially to separate the constituents of Thymus quinquecostatus Celak. The upper phase of the former solvent system was utilized as both elution solvent for flash chromatography and the stationary phase for counter-current chromatography, while the lower phase of the latter solvent system containing 10 mM trifluoroacetic acid was employed as elution solvent for flash chromatography and one mobile phase in pH gradient counter-current chromatography. Thymol (7) and xanthomicrol (8), two hydrophobic ingredients, were purified in the initial two-dimensional separation. The subsequent two-dimensional separation yielded six hydrophilic compounds, namely dihydrokaempferol-7-O-D-glucopyranoside (1), lithospermic acid (2), luteolin-7-O-glucuronide (3), rosmarinic acid (4), messerschmidin (5) and apigenin-7-O-D-glucuronide (6). This study represents the first documented use of online multi-dimensional normal-phase flash chromatography coupled to counter-current chromatography for separating constituents from Thymus quinquecostatus Celak.
Assuntos
Distribuição Contracorrente , Metanol , Metanol/química , Distribuição Contracorrente/métodos , Timol , Solventes/química , Extratos Vegetais , Água/química , Cromatografia Líquida de Alta Pressão/métodosRESUMO
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
In this work, the preparative separation of quinolyridine alkaloids from seeds of T. lanceolata by conventional and pH-zone-refining counter-current chromatography. Traditional counter-current chromatography separation was performed by a flow-rate changing strategy with a solvent system of ethyl acetate-n-butanol-water (1:9:10, v/v) and 200 mg sample loading. Meanwhile, the pH-zone-refining mode was adopted for separating 2.0 g crude alkaloid extracts with the chloroform-methanol-water (4:3:3, v/v) solvent system using the stationary and mobile phases of 40 mM hydrochloric acid and 10 mM triethylamine. Finally, six compounds, including N-formylcytisine (two conformers) (1), N-acetycytisine (two conformers) (2), (-)-cytisine (3), 13-ß-hydroxylthermopsine (4), N-methylcytisine (5), and thermopsine (6) were successfully obtained in the two counter-current chromatography modes with the purities over 96.5%. Moreover, we adopted nuclear magnetic resonance and mass spectrometry for structural characterization. Based on the obtained findings, the pH-zone-refining mode was the efficient method to separate quinolyridine alkaloids relative to the traditional mode.
Assuntos
Alcaloides , Extratos Vegetais , Extratos Vegetais/química , Distribuição Contracorrente/métodos , Concentração de Íons de Hidrogênio , Cromatografia Líquida de Alta Pressão/métodos , Alcaloides/análise , Solventes/química , Água , Sementes/químicaRESUMO
Alkaloids, widespread in plants, have a series of pharmacological activities and have been widely used to treat various diseases. Because alkaloids are usually presented in multicomponent mixtures and are deeply low in content, they are very difficult to extract and separate by traditional methods. High-speed counter current chromatography(HSCCC) is a kind of liquid-liquid chromatography without solid support phase, which has the advantages of large injection volume, low cost, and no irreversible adsorption. Compared with the traditional methods of extraction and separation of alkaloids, HSCCC can ensure the separation of many different alkaloids at one time, with a high recovery and large amount. In this paper, the advantages and disadvantages of HSCCC compared with traditional separation methods were discussed and the solvent system and elution mode of HSCCC used to separate alkaloids in recent years were summarized by referring to the relevant literature to provide some references for the separation of alkaloids by HSCCC.
Assuntos
Alcaloides , Produtos Biológicos , Distribuição Contracorrente/métodos , Cromatografia Líquida de Alta Pressão/métodos , Alcaloides/análise , Solventes/químicaRESUMO
Flavonoids are major active small-molecule compounds in bamboo leaves, which can be easily obtained from the bamboo leaves extraction residues (BLER) after the polysaccharides extraction. Six macroporous resins with different properties were screened to prepare and enrich isoorientin (IOR), orientin (OR), vitexin (VI), and isovitexin (IVI) from BLER, and the XAD-7HP resin with the best adsorption and desorption performance was selected for further evaluation. Based on the static adsorption experiments, the experimental results showed that the adsorption isotherm fitted well with the Langmuir isotherm model, and the adsorption process was better explained by the pseudo-second-order kinetic model. After the dynamic trial of resin column chromatography, 20 bed volume (BV) of upload sample and 60% ethanol as eluting solvent was used in a lab scale-up separation, and the results demonstrated that the content of four flavonoids could be increased by 4.5-fold, with recoveries between 72.86 and 88.21%. In addition, chlorogenic acid (CA) with purity of 95.1% was obtained in water-eluted parts during dynamic resin separation and further purified by high-speed countercurrent chromatography (HSCCC). In conclusion, this rapid and efficient method can provide a reference to utilize BLER to produce high-value-added food and pharmaceutical products.
Assuntos
Ácido Clorogênico , Distribuição Contracorrente , Distribuição Contracorrente/métodos , Extratos Vegetais/química , Flavonoides/química , Folhas de Planta/química , Adsorção , Resinas Vegetais/análise , Resinas Sintéticas/química , Cromatografia Líquida de Alta PressãoRESUMO
Premna fulva Craib, rich in iridoid glycosides, is widely used to treat periarthritis, osteoproliferation, pain, and other diseases. However, no studies have reported effective purification methods for obtaining iridoid glycosides as active materials. This paper describes an efficient strategy for separating iridoid glycosides from Premna fulva leaves using high-speed counter-current chromatography and preparative high-performance liquid chromatography. A two-phase solvent system, ethyl acetate/n-butanol/water (7.5:2.5:10, v/v), was selected for high-speed counter-current chromatography separation. The proposed method effectively separated and purified four iridoid glycosides and four lignans, including three new iridoid glycosides (4-6) and five known compounds (1-3, 7, 8), from Premna fulva leaves, indicating that high-speed counter-current chromatography combined with prep-HPLC can efficiently isolate catalpol derivatives from the genus Premna. Additionally, the in vitro anti-inflammatory activities of all isolated compounds were analyzed using lipopolysaccharide-stimulated RAW 264.7 cells, and the results indicated that six compounds (1 and 3-7) exhibited potential anti-inflammatory activities.