Separation and purification of six compounds from the flowers of Cerasus yedoensis (Matsum.) by high-speed countercurrent chromatography in stepwise elution mode

Six compounds from the flower of Cerasus yedoensis (Matsum.) were successfully isolated by high-speed countercurrent chromatography (HSCCC) and preparative high performance liquid chromatography using stepwise elution with a pair of two-phase solvent systems composed of ethyl acetate–n-butanol–formic acid–water at volume ratio of 4:1.5:0.15:5 and ethyl acetate–ethanol–formic acid–water at volume ratio of 4:1:0.15:5 for the first time. This separation process produced (a) 141 mg of 1-O-caffeoyl-β-D-glucopyranoside, (b) 28 mg of p-coumaric acid glucoside, (c) 13 mg of chlorogenic acid, (d) 21 mg of quercetin-3-O-β-D-glucopyranoside, (e) 19 mg of kaempferol 3-O-β-D-glucopyranoside, and (f) 25 mg of caffeic acid from 400 mg of crude sample with the purities of 96.51, 98.82, 94.96, 99.01, 82.51, and 82.45%, respectively. MS, ¹H NMR, and ¹³C NMR analyses were used for the chemical structure identification.     

Isolation of Chlorogenic Acid from <Emphasis Type="Italic">Flaveria bidentis</Emphasis> (L.) Kuntze by CCC and Synthesis of Chlorogenic Acid-Intercalated Layered Double Hydroxide

Preparative counter-current chromatography (CCC) was successfully used for isolation and purification of chlorogenic acid from Flaveria bidentis (L.) Kuntze with a solvent system composed of ethyl acetate–methanol–water at a volume ratio of 50:1:50, v/v. Using a preparative unit of the CCC centrifuge, about 800 mg of the crude extract was separated, yielding 3.2 mg of chlorogenic acid at a purity of 92.0%. The blood pressure lowering and antivirus chlorogenic acid (C₁₆H₁₈O₉) was intercalated into magnesium–aluminum–layered double hydroxides, which was used as host materials for drug-LDH host-guest supermolecular structures by anion exchange under a nitrogen atmosphere. Chlorogenic acid–LDH is a functional and effective drug. The product chlorogenic acid–LDH has been characterized by powder X-ray diffraction (XRD), Fourier transform infrared (FT-IR), thermogravimetric analysis (TGA) and scanning electron micrographs (SEM). The X-ray diffraction patterns of NO₃ ^? form of LDH and chlorogenic acid–LDH were compared, and the basal d spacing value of NO₃ ^?-LDH layer was 8.75 Å (2θ = 10.100°); however, the basal reflection (003) of chlorogenic acid–LDH shifts to lower 2θ (for 003 reflection: 2θ = 5.119°) that is expanded to 17.25 Å, indicating the intercalation of chlorogenic acid into the interlayer of Mg–Al-LDH. Thermogravimetric analysis showed that chlorogenic acid stability had improved, and scanning electron micrographs showed that the morphology of the chlorogenic acid–LDH was irregular masses of distinctly thicker flakes, which was similar to the morphology of NO₃ ^? form of LDH.     

Separation and Purification of Three Flavonoids from Helichrysum arenarium (L.) Moench by HSCCC

Following an initial clean-up step on a Sephadex LH-20 column, high-speed countercurrent chromatography was successfully applied to the isolation and purification of three flavonoids from a crude sample of Helichrysum arenarium (L.) Moench. HSCCC was performed with a two-phase solvent system composed of ethyl acetate–water (1:1, v/v). Naringenin-7-O-β-d-glycoside (2.3 mg), isoquercitrin (3.5 mg), and astragalin (6.7 mg), with purities of 96.05%, 93.63%, 95.23%, respectively, were separated from 160 mg of crude sample in a one-step separation. The structure identification was by ¹H NMR and ¹³C NMR.     

Identification of Anthocyanin Compounds in Butterfly Pea Flowers (Clitoria ternatea L.) by Ultra Performance Liquid Chromatography/Ultraviolet Coupled to Mass Spectrometry

Butterfly pea flower have great sensory attraction, but they have not yet been used widely in Vietnam. Extracts of butterfly pea flowers can be used conveniently as a natural blue colorant for food products. In this study, the identification of anthocyanin compounds in butterfly pea flowers was performed by UPLC coupled with a UV and Mass spectrometer instrument. Positive and negative ion electrospray MS/MS chromatograms and spectra of the anthocyanin compounds were determined. By analyzing the chromatograms and spectra for each ion, five anthocyanins were identified in the butterfly pea flower extract; these were delphinidin-3-(6″-p-coumaroyl)-rutinoside, cyanidin 3-(6″-p-coumaroyl)-rutinoside, delphinidin-3-(p-coumaroyl) glucose in both cis- and trans- isomers, cyanidin-3-(p-coumaroyl-glucoside) and delphinidin-3-pyranoside. Additionally, based on their intensity, it was determined that cyanidin-3-(p-coumaroyl-glucoside) was the most abundant anthocyanin, followed by cyanidin 3-(6″-p-coumaroyl)-rutinoside, delphinidin-3-(p-coumaroyl-glucoside), delphinidin-3-(6″-p-coumaroyl)-rutinoside and delphinidin-3-pyranoside. In this study, cyanidin derivatives were discovered in butterfly pea flower extract, where these compounds had not been detected in previous studies.     

Simultaneous Isolation and Purification of Mollugin and Two Anthraquinones from Rubia cordifolia by HSCCC

A preparative high-speed countercurrent chromatography (HSCCC) method for isolation and purification of three bioactive components, tectoquinone; 1-hydroxy-2-methylanthraquinone and mollugin from the Chinese medicinal plant Rubia cordifolia was successfully established. With a two-phase solvent system composed of light petroleum/ethanol/diethyl ether/water (5:4:3:1, v/v), 10 mg tectoquinone, 19 mg 1-hydroxy-2-methylanthraquinone and 16 mg mollugin were obtained from 100 mg of the crude petroleum extract in a one-step separation at a purity of 98.8, 95.8 and 98.3%, respectively. The structures of mollugin and anthraquinones were identified by ¹H NMR and ¹³C NMR.     

Co-Ingestion of Natal Plums (Carissa macrocarpa) and Marula Nuts (Sclerocarya birrea) in a Snack Bar and Its Effect on Phenolic Compounds and Bioactivities

This study investigated the effect of co-ingesting Natal plums (Carissa macrocarpa) and Marula nuts (Sclerocarya birrea) on the bioaccessibility and uptake of anthocyanins, antioxidant capacity, and the ability to inhibit α-glucosidase. A Natal plum–Marula nut bar was made by mixing the raw nuts and the fruit pulp in a ratio 1:1 (v/v). The cyanidin-3-O-sambubioside (Cy-3-Sa) and cyanidin-3-O-glucoside content (Cy-3-G) were quantified using the ultra-high performance liquid chromatography-quadrupole time-of-flight mass spectrometry (UHPLC/Q-TOF-MS). Inclusion of Natal plum in the Marula nut bar increased the Cy-3-Sa, Cy-3-G content, antioxidants capacity and α-glucosidase inhibition compared to ingesting Marula nut separately at the internal phase. Adding Natal plum to the Marula nut bar increased bioaccessibility of Cy-3-Sa, Cy-3-G, quercetin, coumaric acid, syringic acid and ferulic acid to 80.2% and 71.9%, 98.7%, 95.2%, 51.9% and 89.3%, respectively, compared to ingesting the Natal plum fruit or nut separately.     

Ultrasound extracted flavonoids from four varieties of Portuguese red grape skins determined by reverse-phase high-performance liquid chromatography with electrochemical detection

Several flavonoids present in red grape skins from four varieties of Portuguese grapes were determined by reverse-phase high-performance liquid chromatography (RP-HPLC) with electrochemical detection (ECD). Extraction of flavonoids from red grape skins was performed by ultrasonication, and hydrochloric acid in methanol was used as extraction solvent. The developed RP-HPLC method used combined isocratic and gradient elution with amperometric detection with a glassy carbon-working electrode. Good peak resolution was obtained following direct injection of a sample of red grape extract in a pH 2.20 mobile phase. Eleven different flavonoids: cyanidin-3-O-glucoside (kuromanin), delphinidin-3-O-glucoside (myrtillin), petunidin-3-O-glucoside, peonidin-3-O-glucoside, malvidin-3-O-glucoside (oenin), (+)-catechin, rutin, fisetin, myricetin, morin and quercetin, can be separated in a single run by direct injection of sample solution. The limit of detection obtained for these compounds by ECD was 20-90 pg/L, 1000 times lower when compared with photodiode array (PDA) limit of detection of 12-55 ng/L. RP-HPLC-ECD was characterized by an excellent sensitivity and selectivity, and appropriate for the simultaneous determination of these electroactive phenolic compounds present in red grape skins.     

Preparative Isolation and Purification of Two Phenylpropanoids from Daphne giraldii Nitsche by HSCCC

Preparative high-speed counter-current chromatography (HSCCC) was successfully applied to purify phenylpropanoids from the stem and root bark of Daphne giraldii Nitsche, a traditional Chinese medicine. Their structures were identified on the basis of ¹H NMR and ¹³C NMR technology. The two-phase solvent system composed of n -hexane–ethyl acetate–methanol–water (2: 3: 0.5: 4, v/v/v/v) was selected for HSCCC. A total of 8.0 mg woonenoside XI (1) and 18.0 mg daphnetin (2) were obtained in one-step separation from 200 mg of the crude extract with purity of 96.0 and 99.1%, respectively, as determined by LC. And the major compound (2) showed antithrombotic activity in vitro.     

Selective extraction, separation, and identification of anthocyanins from Hibiscus sabdariffa L. using solid phase extraction-capillary electrophoresis-mass spectrometry (time-of-flight /ion trap)

A method for selective extraction using SPE, electrophoretic separation at basic condition and the identification by using exact masses and fragmentation patterns has been developed in order to know the anthocyanins in dried calyces of Hibiscus sabdariffa L. A detailed and comparative study of several extraction procedures has been carried out to obtain the maximum number of anthocyanidins from the calyces and then a CE-TOF-MS method in positive mode using ESI has been developed for the separation and rapid identification of anthocyanins in H. sabdariffa L. Delphinidin-3-sambubioside, cyanidin-3-sambubioside have been detected as main components and cyanidin-3-O-rutinoside, delphinidin-3-O-glucoside and cyanidin-3,5-diglucoside, and chlorogenic acid as minor constituents. The confirmation of the anthocyanidins and chlorogenic acid was carried out using fragmentation ions with the IT-mass spectrometer (IT-MS).     

An Efficient Method for Extraction, Separation and Purification of Naphthoquinone Pigments from Lithospermum erythrorhizon Sieb. et Zucc. by SFE and HSCCC

Supercritical fluid extraction was used to extract naphthoquinone pigments from Lithospermum erythrorhizon Sieb. et Zucc. The crude extracts were separated and purified by high-speed counter-current chromatography with light petroleum–ethyl acetate–methanol–water (5:5:8:2, v/v) as the two-phase solvent system. Three kinds of naphthoquinone pigments including 17.6 mg of β-hydroxyisovalerylshikonin (I), 17.6 mg of acetylshikonin (II), and 19.7 mg of isobutyrylshikonin (III) were obtained from 150 mg crude sample. The purity of these compounds was 96.7, 99.3 and 95.5%, respectively, as determined by liquid chromatograph. Their structures were identified by ¹H NMR and ¹³C NMR.     

Use of pH-Zone Refining Countercurrent Chromatography for Preparative Separation of Fangchinoline and Tetrandrine from Stephania tetrandra

pH-Zone-refining countercurrent chromatography with a multilayer coil planet centrifuge has been successfully used for separation of fangchinoline and tetrandrine from crude extracts of Stephania tetrandra. The two target compounds were completely resolved by use of the two-phase solvent system petroleum ether (60–90 °C)–ethyl acetate–methanol–water 5:5:1:9 (v/v), with 10 mm triethylamine in the organic stationary phase and 5 mm hydrochloric acid in the aqueous mobile phase. Separation of 3.5 g sample yielded 126 mg fangchinoline (LC purity >93%) and 249 mg tetrandrine (LC purity >95%). The structures of the compounds were confirmed by use of electrospray ionization mass spectrometry and ¹H NMR.     

Efficient Protocol for Large-Scale Purification of Naringin with High Recovery from Fructus aurantii by Macroporous Resin Column Chromatography and HSCCC

Several kinds of resins were investigated in the first step and D101 macroporous resin was selected for cleaning-up naringin (NAR), a major flavonoid glycoside from Fructus aurantii. In the subsequent column chromatography, 10% aqueous ethanol was first used to elute the column to remove the undesired constituents and 70% aqueous ethanol was used to elute the target. The content of NAR was 57.1% with 95.7% recovery in this process. In the second step, the obtained crude sample was directly isolated by high-speed counter-current chromatography (HSCCC) with a two-phase solvent system composed of ethyl acetate–n-butanol–water at a volume ratio of 2: 0.8: 3.2 (v/v/v), and 331 mg NAR with 98.3% purity was obtained from 600 mg crude extract in only one run. The recovery of the compound in this step was 95.0%. Thus, the total recovery of NAR was 90.9% after the two step purification. The established protocol for large-scale isolation and separation of NAR with high purity and recovery from F. aurantii was simple, efficient, and suitable for pharmace- utical and commercial use.     

A simple method to optimize the HSCCC two-phase solvent system by predicting the partition coefficient for target compound

A simple method was developed to optimize the solvent ratio of the two-phase solvent system used in the high-speed counter-current chromatography (HSCCC) separation. Some mathematic equations, such as the exponential and the power equations, were established to describe the relationship between the solvent ratio and the partition coefficient. Using this new method, the two-phase solvent system was easily optimized to obtain a proper partition coefficient for the CCC separation of the target compound. Furthermore, this method was satisfactorily applied in determining the two-phase solvent system for the HSCCC preparation of pseudolaric acid B from the Chinese herb Pseudolarix kaempferi Gordon (Pinaceae). The two-phase solvent system of n-hexane/EtOAc/MeOH/H(2)O (5:5:5:5 by volume) was used with a good partition coefficient K = 1.08. As a result, 232.05 mg of pseudolaric acid B was yielded from 0.5 g of the crude extract with a purity of 97.26% by HPLC analysis.     

Preparative Isolation and Purification of Flavonoids and Protocatechuic Acid from Sea Buckthorn Juice Concentrate (Hippophaë rhamnoides L. ssp. rhamnoides) by High-Speed Counter-Current Chromatography

High-speed counter-current chromatography (HSCCC)—a support free all liquid–liquid chromatography technique—has been successfully used for the preparative isolation of isorhamnetin 3-O-β-d-glucoside, isorhamnetin 3-O-β-rutinoside, quercetin 3-O-β-d-glucoside, syringetin 3-O-β-d-glucoside and protocatechuic acid from sea buckthorn juice concentrate (Hippophaë rhamnoides L. ssp. rhamnoides, Elaeagnaceae). The preparative HSCCC instrument was a multilayer coil planet centrifuge equipped with three preparative coils. Separation was performed with a two phase solvent system (n-hexane–n-butanol–water, 1:1:2 v/v/v) in ‘head-to-tail’ mode. Each injection of 4.1 g crude ethyl acetate extract yielded isorhamnetin 3-O-β-d-glucoside (95 mg), isorhamnetin 3-O-β-rutinoside (10 mg), quercetin 3-O-β-d-glucoside (5 mg), and protocatechuic acid (34 mg) with purities >98%. The flavonoid syringetin 3-O-β-d-glucoside (2 mg) was a novel compound for H. rhamnoides. Chemical structures of all compounds were determined by HPLC–ESI–MS–MS, 1D-NMR (¹H, ¹³C, DEPT 135) spectroscopy and for elucidation of glycosidic linkages 2D-NMR (HMBC) spectroscopy was used.     

Separation and Identification of Ergosta-4,6,8(14),22-tetraen-3-one from Ganoderma atrum by High-Speed Counter-Current Chromatography and Spectroscopic Methods

High speed counter-current chromatography in semi-preparative scale was used to separate and purify ergosta-4,6,8(14),22-tetraen-3-one from Ganoderma atrum, a famous traditional Chinese medicine. A two-phase solvent system composed of a mixture of n-hexane–ethanol–water (6: 5: 1, v/v/v) was used and the separation conditions were optimized. In a typical run in less than 400 min, 100 mg of samples can be separated to yield 14 mg of ergosta-4,6,8(14),22-tetraen-3-one with 99.1% purity. The structure of this compound was elucidated by UV, EI-MS, ¹H NMR and ¹³C NMR.     

Preparative Isolation of Imperatorin, Oxypeucedanin and Isoimperatorin from a Traditional Chinese Herb Using a HSCCC Strategy Based on Optimization of Rapid Flow Rate

Preparative high-speed counter-current chromatography has been used successfully for the isolation and purification of imperatorin, oxypeucedanin and isoimperatorin from traditional Chinese herb “bai zhi”—Angelica dahurica (Fisch. ex Hoffm) Benth. et Hook using high-speed counter-current chromatography (HSCCC). This was achieved in two stages. The first stage used a high flow HSCCC protocol with a two-phase solvent system composed of n-hexane–ethyl acetate–methanol–water (HEMW) with volume ratios of 5:5:5:5, v/v which isolated isoimperatorin but co-eluted imperatorin and oxypeucedanin. The second stage used HEMW 5:5:4:6, v/v at low flow rate to resolve the co-eluted components from the first stage. The flow rate was optimized by preparative HSCCC. 300 mg of the crude extract was separated, yielding 18.5 mg of imperatorin, 8.3 mg of oxypeucedanin and 9.8 mg of isoimperatorin all at a high purity of over 98%.     

Anthocyanin Recovery from Grape by-Products by Combining Ohmic Heating with Food-Grade Solvents: Phenolic Composition,Antioxidant, and Antimicrobial Properties

Usually, wine-making by-products are discarded, presenting a significant environmental impact. However, they can be used as a source of bioactive compounds. Moreover, consumers’ increasing demand for naturally nutritious and healthy products requires new formulations and food product improvement, together with sustainable, environmentally friendly extraction methods. Thus, this work aimed to compare ohmic heating (OH) with conventional methodology (CONV), using food-grade solvents, mainly water, compared to standard methanol extraction of anthocyanins. No significant differences were found between the CONV and OH for total phenolic compounds, which were 2.84 ± 0.037 and 3.28 ± 0.46 mg/g DW gallic acid equivalent, respectively. The same tendency was found for antioxidant capacity, where CONV and OH presented values of 2.02 ± 0.007 g/100 g and 2.34 ± 0.066 g/100 g ascorbic acid equivalent, respectively. The major anthocyanins identified were malvidin-3-O-acetylglucoside, delphinidin-3-O-glucoside, petunidine-3-O-glucoside, cyanidin-3-O-glucoside, and peonidine-3-O-glucoside. These extracts displayed antimicrobial potential against microorganisms such as Yersinia enterocolitica, Pseudomonas aeruginosa, Salmonella enteritidis, methicillin-sensitive Staphylococcus aureus, a methicillin-resistant Staph. aureus (MRSA), and Bacillus cereus. In conclusion, OH provides similar recovery yields with reduced treatment times, less energy consumption, and no need for organic solvents (green extraction routes). Thus, OH combined with water and citric acid allows a safe anthocyanin extraction from grape by-products, thus avoiding the use of toxic solvents such as methanol, and with high biological potential, including antimicrobial and antioxidant activity.     

Isolation and purification of oridonin from Rabdosia rubescens using upright counter-current chromatography

A preparative counter-current chromatography (CCC) method for isolation and purification of oridonin, a new cancer chemoprevention agent, from the Chinese medicinal plant Rabdosia rubescens was successfully established. The crude oridonin was obtained by elution with a light petroleum/acetone solvent mixture from ethanol extracts of R. rubescens using column chromatography on silica gel. With a two-phase solvent system composed of n-hexane/ethyl acetate/methanol/water (1:2:1:2, v/v), 120 mg of oridonin at the purity of 97.8% was obtained from 200 mg of the crude sample in a single-step CCC separation. The structure of oridonin was identified by ESI-MS, 1H NMR, and 13C NMR.     

Preparative isolation and purification of anthocyanins from purple sweet potato by high-speed counter-current chromatography

High-speed counter-current chromatography (HSCCC) was applied to the preparative isolation and purification of peonidin 3-O-(6-O-(E)-caffeoyl-2-O-β-D -glucopyranosyl-β-D -glucopyranoside)-5-O-β-D -glucoside ( 1 ), cyanidin 3-O-(6-O-p-coumaroyl)-β-D -glucopyranoside ( 2 ), peonidin 3-O-(2-O-(6-O-(E)-caffeoyl-β-D -glucopyranosyl)-6-O-(E)-caffeoyl-β-D -glucopyranoside)-5-O-β-D -glucopyranoside ( 3 ), peonidin 3-O-(2-O-(6-O-(E)-feruloyl-β-D -glucopyranosyl)-6-O-(E)-caffeoyl-β-D -glucopyranoside)-5-O-β-D -glucopyranoside ( 4 ) from purple sweet potato. Separation of crude extracts (200 mg) from the roots of purple sweet potato using methyl tert-butyl ether/n-butanol/acetonitrile/water/trifluoroacetic acid (1:4:1:5:0.01, v/v) as the two-phase solvent system yielded 1 (15 mg), 2 (7 mg), 3 (10 mg), and 4 (12 mg). The purities of 1 – 4 were 95.5%, 95.0%, 97.8%, and 96.3%, respectively, as determined by HPLC. Compound 2 was isolated from purple sweet potato for the first time. The chemical structures of these components were identified by ¹H NMR, ¹³C NMR and ESI-MSⁿ.     

Separation and Purification of Baishouwubenzophenone, 4-Hydroxyacetophenone and 2,4-Dihydroxyacetophenone from Cynanchum auriculatum Royle ex Wight by HSCCC

A preparative high-speed counter-current chromatography method for isolation and purification of three acetophenones, baishouwubenzophenone, 4-hydroxyacetophenone and 2,4-dihydroxyacetophenone from the Chinese medicinal plant Cynanchum auriculatum Royle ex Wight was successfully established. With a two-phase solvent system composed of light petroleum (b.p. 60–90 °C)–ethyl acetate–methanol–water (4:9:6:6, v/v), about 20.2 mg compound 1, 35.0 mg compound 2 and 8.3 mg compound 3, each at over 95% purity as determined by LC, were obtained in one-step elution from 400 mg of the ethanol extract. The structures of these compounds were identified by UV, IR, ESI-MS, ¹H NMR and ¹³C NMR spectroscopy. Among them, compounds 2,4-dihydroxyacetophenone and 4-hydroxyacetophenone were obtained from C. auriculatum for the first time.