Isolation and Purification of Isoquercitrin and Quercitrin from sf Hypericum Japonicum Thunb.ex Murray by Counter-Current Chromatography

Isoquercitrin and quercitrin were successfully isolated and purified from Hypericum japonicum Thunb.ex Murray by counter-current chromatography with a solvent system of n-hexane-ethyl acetate-methanol-water (1:7:1:7, v/v/v/v) in one step. From 100 mg of the extract of Hypericum japonicum Thunb.ex Murray, 9.8 mg of isoquercitrin and 12 mg of quercitrin were obtained with the purities of 95.9% and 99.1%, respectively, as determined by HPLC. Their structures were identified by UV, MS, and NMR analysis. In this study, a rapid method for isolation and purification of the two major compounds from Hypericum japonicum Thunb.ex Murray crude extract was established.     

Separation and Purification of Rosmarinic Acid and Rutin from Glechoma hederacea L. using High-Speed Counter-Current Chromatography

Rosmarinic acid and rutin were successfully separated from Glechoma hederaceaL. using high-speed counter-current chromatography for the first time. Eleven milligrams of rosmarinic acid (chromatographic purity 97.2 %) and 10 mg of rutin (chromatographic purity 98.1 %) were obtained from 100 mg ethyl acetate extract and 100 mg n -butanol extract of Glechoma hederacea L., respectively, with the separation procedure less than 2 h. Their structures were characterized by UV, MS, and NMR. The established methods were simple, fast, and convenient, which can be applied to the preparation of reference substances of rosmarinic acid and rutin.     

One Step Purification of Piperine Directly from Piper nigrum L. by High Performance Centrifugal Partition Chromatography

Abstract

In this study, a preparative high performance centrifugal partition chromatography (HPCPC) method for isolation and purification of the bioactive component piperine directly from the ethanol extract of Piper nigrum L. was successfully established by using n-hexane-ethyl acetate-methanol-water as the two-phase solvent system. The upper phase of n-hexane-ethyl acetate-methanol-water (6:5:6:5, v/v) was used as the stationary phase of CPC. Under the optimum conditions, 40 mg of piperine at 98.5% purity, as determined by HPLC, was yielded from 300 mg of the crude extract in a single CPC separation. The peak fraction of CPC was identified by ¹H NMR and ¹³C NMR.     

Optimization of conditions for single-step purification of recombinant hepatitis B surface antigen produced in Pichia pastoris using ion exchange chromatography

ABSTRACT

The adsorption and release of rHBsAg extracted from the final dosage form on various ion exchange resins and under different pH conditions were investigated after its peptide map and isoelectric point (PI) determination. Efficient antigen adsorption to the anion exchange resins occurred when the pH value of the protein buffer was adjusted to 5.0. In purification of rHBsAg derived from the yeast crude extract using Q Sepharose FF column, with adjusting the pH value of the crude extract to 5.0 (i.e., near to the target protein PI) and using 2M NaCl, rHBsAg with high purity (up to >95%) was obtained.

Abbreviations: rHBsAg, recombinant hepatitis B surface antigen; Alhydrogel, aluminum hydroxide; IEF, isoelectric focusing; PI, isoelectric point; SDS-PAGE, sodium dodecyl sulfate-polyacrylamide gel electrophoresis; RP-HPLC, reversed-phase high-performance liquid chromatography; SE-HPLC, size-exclusion high-performance liquid chromatography; PBS, phosphate-buffered saline     

Impacts of spray drying conditions on stability of isoflavones in microencapsulated soybean extract

Abstract

Soybean extract rich in isoflavones has attracted widespread attention for dietary supplement and pharmaceutical purposes. However, it has poor solubility and low stability. Encapsulation using spray drying is a good alternative for overcoming these problems in soybean extract. Isoflavones profiles in soybean extract are altered during encapsulation and storage. The objective of this work was to investigate the effect of spray drying conditions on the isoflavones profiles and the various properties of microencapsulated soybean extract. The studied parameters comprised the type of wall material (maltodextrin [MD], gum arabic [GA], and β-cyclodextrin [βCD]), inlet air temperature (130–170?°C) and storage time (0–6?months), while the investigated properties included moisture content, particle size, hygroscopicity, morphology, isoflavones content, encapsulation properties, and Fourier transform infrared analysis. Type of wall material had a more significant impact on the properties of microencapsulated soybean extract than inlet air temperature. The degradation of total isoflavones during storage mainly depended on the inter-conversion level of isoflavones during encapsulation, hygroscopicity and heating history of microencapsulated soybean extract. The use of βCD as wall material could preserve total isoflavones after encapsulation and storage at 0.1–1.3 and 2.4–3.1 times that in the case of MD and 1.1–1.3 and 1.5–1.8 times that in the case of GA, respectively. Abbreviations AI aglycone isoflavones (daidzein and genistein)

AGI acetyl β-glucoside isoflavones (6″-O-acetyldaidzin and 6″-O-acetylgenistin)

βCD β-cyclodextrin

GA gum arabic

GI β-glucoside isoflavones (daidzin and genistin)

MD maltodextrin

MGI malonyl β-glucoside isoflavones (6″-O-malonyldaidzin and 6″-O-malonylgenistin)

SE soybean extract

SE-βCD microencapsulated soybean extract using β-cyclodextrin as wall material

SE-GA microencapsulated soybean extract using gum arabic as wall material

SE-MD microencapsulated soybean extract using maltodextrin as wall material

TI total isoflavones (sum of MGI, AGI, GI and AI)

     

One Step Large-Scale Separation and Purification of Rutin from Boenninghausenia sessilicarpa by Countercurrent Chromatography

In this study, a preparative countercurrent chromatography (CCC) method for isolation and purification of the bioactive component rutin directly from the ethanol extract of Boenninghausenia sessilicarpa was successfully established by using n-butanol-ethyl acetate-water as the two-phase solvent system. The upper phase of n-butanol-ethyl acetate-water (4:1:5, v/v) was used as the stationary phase of CCC. Under the optimum conditions, 112 mg of rutin at 98.6% purity was obtained from 2.0 g of the crude extract in a single CCC separation. The peak fraction of CCC was identified by negative ESI, ¹H NMR, and ¹³C NMR.     

Application of high-speed counter-current chromatography coupled with high performance liquid chromatography for the separation and purification of Quercetin-3-O-sambubioside from the leaves of Nelumbo nucifera

Quercetin-3-O-sambubioside [Quercetin-3-O-β-D-xylopyranosyl (1→2)-β-D-glucopyranoside] was separated and purified by semi-preparative high-speed counter-current chromatography (HSCCC) with a two-phase-solvent system composed of ethyl acetate-n-butanol-water (4:1:5, v/v) from the leaves of Nelumbo nucifera (Lotus). A total of 5.0 mg of the targeted compound with a purity of 98.6% as determined by high performance liquid chromatography (HPLC) was obtained from 100 mg of the crude extract cleaned up by AB-8 macroporous resin in a one-step separation. Quercetin-3-O-sambubioside was a novel flavonoid glycoside from the leaves of Nelumbo nucifera, and its chemical structure was identified by means of ESI-MS, 1D NMR and 2D NMR.     

The determination of the protium-deuterium electrolytic separation factor by infrared-cryogenic gas chromatography method

A method IR-CGC (infrared-cryogenic gas chromatography) was established directly through the combination of infrared spectrum (IR) and cryogenic gas chromatography (CGC) to measure the electrolytic separation factor (α) between protium and deuterium. The n_1,liq(D)/n_2,liq(H) ratio of alkalic aqueous solution after electrolysis was tested quantitatively by the IR method, and the n_1,gas(D)/n_2,gas(H) ratio of mixed gases from electrolysis was determined by CGC. Then α of different electrode materials were obtained according to the formula of electrolytic separation factor. As a result, it provides an accurate, simple, feasible, suitable approach for evaluating the electrolytic protium-deuterium separation ability of different electrode materials, which is helpful for searching excellent hydrogen isotopes separation materials.     

Critical analysis of phospholipid hydrolyzing activities in ripening tomato fruits. Study by spectrofluorimetry and high-performance liquid chromatography

Using the spectrofluorimetric method described by Wittenaueret al. [Wittenauer, L.A., Shirai, K., Jackson, R.L., and Johnson, J.D. (1984)Biochem. Biophys. Res. Commun. 118, 894–901] for phospholipase A₂ (PLA₂) measurement, we have detected a phospholipase activity in Ailsa Craig and in mutantrin tomatoes at their normal harvest time (mature green stage). This activity in Ailsa Craig tomatoes increased at the beginning of fruit ripening (green-orange stage) and then decreased slowly. The decrease in activity, however, was greater when ripening occurred after tomato picking at normal harvest time than when ripening occurred on tomato plants. This phospholipase activity was always higher inrin tomatoes than in normal ones. Thin-layer chromatography of compounds obtained after incubation of tomato extract demonstrated a decrease in the substrate 1-acyl-2-{6[(7-nitro-2,1,3, benzoxadiazol-4-yl)amino]-caproyl}-sn-glycero-3-phosphocholine (C₆-NBD-PC), and an increase in one product (NBD-aminohexanoic acid), but failed to detect the second product (1-acyl-sn-glycero-3-phosphocholine). We, therefore, developed a new one-step method for separation and quantification of a mixture of phospholipids and other lipids, using straight-phase-high-performance liquid chromatography with light-scattering detection. This method detected another fatty acid-releasing activity in enzyme extract from green-orange tomatoes. This lipolytic enzyme (or family of enzymes) slowly produced free fatty acids when 1-oleoyl-sn-glycero-3-phosphocholine was added as substrate. The production of fatty acids was stoichiometric and more rapid when 1-oleoyl-sn-glycero-3-phosphate and 1-oleoyl-sn-glycerol were used as substrates. On the other hand, the same tomato extract was unable to hydrolyze 1,2-dioleoyl-sn-glycero-3-phosphate and 1,2-dioleoyl-sn-glycerol. Crude tomato extract exhibited lipid acyl hydrolase activity according to the definition of Galliard [Galliard, T. (1979), inAdvances in the Biochemistry and Physiology of Plant Lipids (Appelqvist, L.A., and Liljenberg, C. eds.), pp. 121–132, Elsevier, Amsterdam]. But in order to demonstrate whether tomato extract contains PLA₂ activity and/or lysophospholipase activity, further work on purified tomato extract will be necessary.     

Combination of supercritical fluid extraction with high-speed countercurrent chromatography for extraction and isolation of ethyl p-methoxycinnamate and ethyl cinnamate from Kaempferia galanga L.

Supercritical fluid extraction (SFE) with high-speed countercurrent chromatography (HSCCC) was successfully used for the extraction and isolation of ethyl p-methoxycinnamate (EPMC) and ethyl cinnamate (EC) from Kaempferia galanga L. The SFE parameters including extraction temperature, extraction pressure and entrainer volume were optimized by central composite design (CCD). Then the crude extract was separated by HSCCC with a two-phase solvent system composed of n-hexane:ethyl acetate:methanol:water (7:3:8:2, v/v/v/v) in one-step within 60 min. As a result, 13 mg of EPMC and 2 mg of EC were isolated from 100 mg of crude extract with purities of 98.4% and 98.1%, as determined by HPLC. The structural identification was carried out by UV, MS and NMR spectra.     

用大孔树脂从大豆乳清中纯化大豆异黄酮

引言 大豆异黄酮是大豆生长中形成的一类次生代谢产物.大豆异黄酮由于具有弱雌激素活性、抗氧化活性、抗溶血活性,能有效地预防和治疗癌症、骨质疏松、妇女更年期综合征等多种疾病,因此在保健食品和医药中有广泛的应用[1-4].大孔吸附树脂是一类新型的高分子分离材料,具有化学性质稳定、选择性吸附、再生简便等许多优点,在天然产物的分离纯化方面其应用日趋广泛[5-6].     

Using High-Performance Counter-Current Chromatography Combined with Preparative High Performance Liquid Chromatogramphy for the Separation of Bioactive Compounds from the Water Extract of Gentiana macrophylla Pall

In this paper, a combined high performance counter-current chromatography (HPCCC) and preparative high-performance liquid chromatography (HPLC) method was employed for rapid separation and enrichment of bioactive constituents from a water extract of Gentiana macrophylla Pall. With a two phase solvent system composed of ethyl acetate-n-butanol-water-acetic acid (2: 3: 5: 0.6, v/v), the water extract of G. macrophylla Pall was fractionated into six fractions with three targets isolated and four others highly concentrated, which were then further purified by preparative-HPLC. As a result, 37 mg deglucoserrulatoside, 22.4 mg loganic acid, 3.9 mg isoorientin, 22.4 mg swertiamarin, 52.3 mg gentiopicroside, 27.5 mg sweroside, and 7.9 mg macrophylloside D with the purity of 95.3%, 90.2%, 98%, 98%, 99.2%, 98.8%, and 98.4%, respectively, were isolated from the water extract of Gentiana macrophylla Pall. The structures were confirmed by UV spectra, MS, as well as NMR measurements.     

Gel permeation chromatography of vegetable-oil-based printing ink vehicles

The apparent weight-average molecular weights (M_w ) of ink vehicles made from soybean, safflower, sunflower, cottonseed, and canola oils were compared by gel permeation chromatography (GPC), and the correlation between viscosity and M_w of these vehicles was established. Apparent M_w of vegetable oil gels that were used in vehicle preparation were also obtained by GPC. © 1992 John Wiley & Sons, Inc.     

Rapid screening and separating two radical scavengers in Lycium barbarum L. by DPPH-HPLC analysis-combined dual-mode high-speed countercurrent chromatography

A target-guidance separation strategy composed of activity screening process with high-performance liquid chromatography and separating process with high-speed countercurrent chromatography (HSCCC) has been developed and used to screen and separate radical scavengers from Lycium barbarum extract. Two compounds were found to be radical scavengers and dual-mode HSCCC was used to separate these two defined compounds due to the big gap in polarity between them. Rutin and quercetin were separated with purities of 96.5% and 95.0%. Their structures were identified by nuclear magnetic resonance and both of them exhibit potent radical scavenging activity with the IC₅₀ values being 20.07 ± 0.10 and 3.11 ± 0.03 μg/mL, respectively.     

Analysis of oxylipins by high-performance liquid chromatography with evaporative light-scattering detection and particle beam-mass spectrometry

The metabolism of 13 S-hydroperoxy-9Z,11E,15Z-octadecatrienoic acid was investigated in a crude enzyme extract from mung bean seedlings (Phaseolus radiatus L.). Hydroperoxide-metabolizing activity was mainly due to a hydroperoxide lyase and, to a lesser extent, to an allene oxide synthase and a peroxygenase. Oxylipins originating from hydrolysis and cyclization of the allene oxide synthase product 12,13-epoxy-9Z,11,15Z-octadecatrienoic acid and from peroxygenase catalysis were identified by high-performance liquid chromatography (HPLC) particle beam-mass spectrometry (PB-MS) and quantified by normal-phase HPLC with an evaporative light-scattering detector (ELSD). An advantage of this methodology was the possibility to avoid extensive derivatization procedures commonly used for the gas chromatographic analysis of oxylipins. Owing to a comparable sample inlet system, the ELSD served an important analytical pilot function for the PB-MS: Qualitatively identical chromatographic patterns were obtained with both detection systems. The HPLC system enabled the separation of methyl 12-oxo-phytodienoate, methyl 11-hydroxy-12-oxo-9Z,15Z-octadecadienoate, methyl 12-oxo-13-hydroxy-9Z,15Z-octadecadienoate, methyl 9-hydroxy-12-oxo-10E,15Z-octadecadienoate, methyl 13-hydroxy-9Z,11E,15Z-octadecatrienoate, methyl 15,16-epoxy-13-hydroxy-9Z,11E,15Z-octadecatrienoate, and methyl 13-hydroperoxy-9Z,11E,15Z-octadecatrienoate on a Lichrospher DIOL column within 33 min. Compared with a diode array detector, the ELSD proved to be more sensitive, in the case of methyl 12-oxo-13-hydroxy-9Z, 15Z-octadecadienoate by a factor of about 15. In addition, volatile metabolites were analyzed by capillary gas chromatography. The yield of the hydroperoxide lyase product 2E-hexenal was 49%, whereas the sum of oxylipins reached about 15%.     

Separation of squalene from palm fatty acid distillate using adsorption chromatography

A method to separate squalene from palm fatty acid distillate (PFAD) using neutralization‐hydrolysis‐neutralization before employing adsorption column chromatography was developed. Extraneous matters, especially free fatty acids (83.8%) and acylglycerols (12.7%), were first neutralized and removed before being subjected to hydrolysis by using commercially available, immobilized Candida antartica lipase, at 65 °C for 8 h. Neutralization followed by hydrolysis and repeated neutralization successfully concentrated squalene from an initial amount of 3.76% to 27.5%. Oil extracted from neutralized‐hydrolyzed‐neutralized PFAD was then passed through a Diaion HP‐20 column using reverse‐phase adsorption chromatography. Squalene was desorbed by hexane, with a recovery of 93%.     

A highly efficient method for concentrating DNA from river water by combined coagulation and foam separation

ABSTRACT

Both Escherichia coli and Enterococci were collected in foam within 7 min from 500 mL of bacteria-spiked water by coagulation and foam separation using ferric chloride and milk casein. These bacterial DNA isolated in the 100 µL of extract from the foam more than 87.5% recovery using the DNeasy PowerWater® Kit. To test this method with water from three natural rivers, 0.67–2.70 µg of DNA were concentrated in 100 µL of extract from 1,000 mL of river water. When the DNA extract was subjected to 16S rRNA gene sequencing analysis, information on the bacterial flora could be obtained.     

Oviposition stimulants inBarbarea vulgaris forPieris rapae andP. napi oleracea: isolation,identification and differential activity

The closely related butterflies,Pieris rapae andP. napi oleracea, readily laid eggs onBarbarea vulgaris in greenhouse cages. When offered a choice between cabbage andB. vulgaris, P. rapae showed no preference, butP. napi oleracea preferredB. vulgaris. Bioassays of extracts ofB. vulgaris foliage revealed the presence of oviposition deterrent(s) in l-butanol extracts as well as stimulants in the postbutanol water extracts. However, the deterrent effect was apparently outweighed by the strong stimulatory effect in the whole plants. The postbutanol water extract was preferred over an equivalent cabbage extract by both species, but more significantly in the case ofP. napi oleracea. The stimulants were isolated by open column chromatography and HPLC, and the activity was associated with three glucosinolates.P. napi oleracea was more sensitive thanP. rapae to the natural concentration of compounds1 and3, whereas both species were strongly stimulated to oviposit by natural concentrations of compound2. Compounds1 and2 were identified as (2R)-glucobarbarin and (2S)-glucobarbarin, respectively, and3 was identified as glucobrassicin, on the basis of their UV, mass, and NMR spectra. When the pure compounds were tested at the same concentrations applied to bean plants, the (2R)-glucobarbarin at 0.2 mg/plant was preferred over a standard cabbage extract by both butterfly species. However, at a dose of 0.02 mg/plant,P. rapae preferred the cabbage extract whereasP. napi oleracea still preferred the (2R)-glucobarbarin. No such difference in response of the two species to the same two concentrations of (2S)-glucobarbarin was obtained. The results indicate a distinct difference in sensitivity of these butterflies to the epimers of glucobarbarin, and the differences in behavioral responses of the two butterfly species depend to a large extent on the concentration of stimulant present.     

Separation and purification of six biosurfactant rhamnolipids by high-speed countercurrent chromatography utilizing novel solvent selection method

In this study, an original solvent selection method was developed for the separation of biosurfactant rhamnolipids from fermentation broth by high-speed countercurrent chromatography. For this method, calculations were based on solubility and functional group parameters that enabled the rapid selection of suitable solvent systems at no more than 10% of average time by traditional method. Based on selection results, a three-phase solvent system composed of n-hexane-methyl acetate-acetonitrile-water (2:2:2:5, v/v) was efficiently used for one-step separation of a mixture of six rhamnolipids. Thereby, RhaC₁₀C₁₀ (0.73 mg), RhaC₁₀ (0.51 mg), Rha₂C₁₀ (0.12 mg), RhaC₁₀C₁₂ (1.26 mg), Rha₂C₁₀C₁₀ (1.03 mg), and Rha₂C₁₀C₁₂ (0.87 mg) were extracted from prepared Pseudomonas aeruginosa (100.0 mg) with purities of 96.37, 95.20, 91.25, 84.41, 89.8, and 90.26%, respectively. The purified compounds were identified by high-performance liquid chromatography/mass spectroscopy, plus ¹H and ¹³C nuclear magnetic resonance studies. This study provides a new perspective on concepts involving efficient solvent system selection.     

Optimization of extraction conditions for active ingredients of Suye Huanglian Decoction and its anti-influenza effect in vivo

ABSTRACT

Uniform design method (UD) was performed to define the levels of impact factors. The contents of anti-influenza active ingredients, extraction yield, and the influenza virus inhibition ratio were measured to evaluate the Suye Huanglian Decoction (SHD) aqueous extraction processes. The best optimal extraction process was optimized by genetic algorithm (GA). Further SHD extract was obtained under the optimal extraction process (add 28-fold water, extract for 108 min at 97°C for triple re-extraction). And the pneumonia mouse model established was used to investigate the anti-influenza effect of SHD. The results in vivo showed the optimized SHD exerted an excellent anti-influenza agent.