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%.     

Enrichment and isolation of barbigerone from Millettia pachycarpa Benth. using high‐speed counter‐current chromatography and preparative HPLC

Enrichment of the anti‐tumor compound barbigerone along with a rotenoid derivative from Millettia pachycarpa Benth. was performed by a two‐step high‐speed counter‐current chromatography (HSCCC) separation process. In the first step, 155.8 mg of target fraction (Fra6) was obtained from 400 mg ethyl acetate extract of M. pachycarpa Benth. with an increase in barbigerone from 5.1 to 13% via HSCCC using a solvent system of n‐hexane–ethyl acetate–methanol–water (5:4:5:3, v/v) under normal phase head to tail elution. HSCCC was repeated to eliminate the major contaminant in this initial fraction 6. After a separation time of 65 min, 22.1 mg barbigerone of 87.7% purity was obtained from Fra6 with the ternary solvent system of n‐hexane–methanol–water (2:2:1, v/v) under normal phase elution. Finally, preparative HPLC was employed for the further isolation of barbigerone and the rotenoid derivative. The structures were confirmed by ESI‐MS, ¹H NMR and ¹³C NMR.     

Preparative isolation and purification of antioxidative stilbene oligomers from Vitis chunganeniss using high‐speed counter‐current chromatography in stepwise elution mode

Preparative high‐speed counter‐current chromatography (HSCCC) was successfully applied to the isolation and purification of three stilbene oligomers from Vitis chunganeniss using stepwise elution with a pair of two‐phase solvent systems composed of n‐hexane–ethyl acetate–methanol–water at (2:5:2:5, v/v) and (1:2:1:2, v/v). The preparative HSCCC separation was performed on 800 mg of crude sample yielding hopeaphenol (21.1 mg), amurensin G (37.2 mg) and vitisin A (95.6 mg) in a one‐step separation, with purities over 95% as determined by HPLC. The structures of these three compounds were identified by MS, ¹H NMR and ¹³C NMR. In addition, their antioxidant activities were screened by DPPH assay, where vitisin A showed strong antioxidant activity. Further EPR experiments with spin‐trapping technique demonstrated that vitisin A is a potent and selective singlet oxygen quencher, which may be used in singlet oxygen‐mediated diseases as a pharmacological agent.     

Preparative isolation of pseudolaric acids A and B,and their glucosides from the root bark of Pseudolarix kaempferi using high‐speed counter‐current chromatography

In order to provide the chemical markers for the quality control of herbal medicines, four diterpenoids, pseudolaric acids A and B (PAA and PAB), and their glucosides were isolated from the methanol extract of the Chinese herb Pseudolarix kaempferi using high‐speed counter‐current chromatography (HSCCC). The diphase solvent system was n‐hexane/EtOAc/MeOH/H₂O which was used at two ratios (5:5:5:5 and 1:9:4:6 by volume) in the separation of pseudolaric acids and their glycosides, respectively. As a result, PAA (14 mg), PAB (129 mg), PAA‐O‐β‐D ‐glucopyranoside (8 mg, PAAG), and PAB‐O‐β‐D ‐glucopyranoside (42 mg, PABG) were obtained from 0.5 g of the crude extract. Their purities were determined to be above 97% by HPLC analysis. Their chemical structures were confirmed by ¹H and ¹³C NMR analysis or HPLC comparison with the reference compounds.     

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.     

Preparative isolation of alkaloids from Corydalis bungeana Turcz. by high-speed counter-current chromatography using stepwise elution

High‐speed counter‐current chromatography (HSCCC) was successfully applied for the preparative separation and purification of alkaloids from Corydalis bungeana Turcz. (Kudiding in Chinese) for the first time. After the measurement of partition coefficient of seven target alkaloids in the nine two‐phase solvent systems composed of CHCl₃–MeOH–(0.1 M; 0.2 M; 0.3 M) HCl (4:1.5:2; 4:2:2; 4:3:2, v/v), CHCl₃–MeOH–0.2 M HCl (4:2:2, v/v) and CHCl₃–MeOH–0.3 M HCl (4:3:2, v/v) were finally selected for the HSCCC separation using the first upper phase as the stationary phase and the stepwise elution of the two lower mobile phases. Consequently, sanguinarine (10 mg), corynoline (25 mg), protopine (20 mg), corynoloxine (18 mg), and 12‐hydroxycorynoline (8 mg) were obtained from 200 mg of crude alkaloid extracts with purities of 94–99% as determined by HPLC. Their chemical structures were characterized on the basis of ¹H‐NMR, ¹³C‐NMR, and LC‐ESI‐Q‐TOF‐MS/MS analyses.     

A New HPLC Method for the Simultaneous Determination of Acetaminophen,Phenylephrine, Dextromethorphan and Chlorpheniramine in Pharmaceutical Formulations

Abstract

Acetaminophen, phenylephrine, dextromethorphan, and chlorpheniramine are frequently associated in pharmaceutical formulations against the common cold. A new high performance liquid chromatography (HPLC) method has been developed for the simultaneous determination of these active pharmaceutical ingredients in pharmaceutical formulations. The separation and quantitation were achieved on a 25 cm underivatized silica column using a mobile phase of methanol: water (containing 6.0 g of ammonium acetate and 10 ml of triethylamine per liter, pH adjusted to 5.0 with orthophosphoric acid), 95:5%(v/v). Detection was carried out using a variable wavelength UV-vis detector at 254 nm for acetaminophen, at 220 nm for phenylephrine, and at 227 nm for dextromethorphan and chlorpheniramine. The method showed linearity for the acetaminophen, phenylephrine, dextromethorphan, and chlorpheniramine in the 162.5–650, 2.5–10, 7.5–30, and 1–4 µg/ml ranges, respectively. The intraday and interday RSDs ranged from 0.92 to 1.52%, 1.00 to 1.76%, 1.21 to 1.74% and 1.26 to 1.80% for the acetaminophen, phenylephrine, dextromethorphan, and chlorpheniramine, respectively. Compounds were eluted in a run time of less than 12 min.     

New cadinane sesquiterpenoids from Mikania micrantha

Abstract

Chemical investigation of the aerial parts of Mikania micrantha led to the isolation of eight sesquiterpenoids and ten diterpenoids, including five cadinane sesquiterpenoids (1?5), three bisabolene sesquiterpenoids (6???8), nine ent-kaurane diterpenoids (9–17), and an abietane diterpenoid (18). Among them, 1???3 are new and feature a rare lactone or furan ring derived from C-6 isopropyl group side chain. Compound 18 was isolated from genus Mikania for the first time, and was also the first example of abietane-type diterpenoids from this plant. Their structures were elucidated on the basis of extensive spectroscopic analyses (1D and 2D NMR, HRESIMS, and ECD). All compounds were examined for their inhibitory effects on lipopolysaccharide (LPS)-induced nitric oxide (NO) production in RAW 264.7 macrophage cells, and compound 18 exhibited pronounced inhibition on NO production (IC₅₀ = 11.04?µM), being comparable to the positive control, quercetin (IC₅₀ = 11.15?µM).     

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.     

Isolation and purification of diterpenoids from the aerial parts of Isodon excisoides target-guided by UPLC-LTQ-Orbitrap-MS

Cytotoxic diterpenoids were enriched and orientation prepared from the aerial parts of Isodon excisoides target-guided by UPLC-LTQ-Orbitrap-MS. Four diterpenoids were obtained, including a novel compound: 1α-acetoxy-7α, 14β, 20α-trihydroxy-ent-kaur-16-en-15-one (1); together with three known compounds kamebakaurin (2), lasiokaurin (3), enmenol-1-β-glucoside (4). Their structures were elucidated on the basis of spectroscopic methods in conjunction with published data for their analogues. All compounds were tested for their cytotoxic effects against five human cancer cell lines HCT-116, HepG2, A2780, NCI-H1650 and BGC-823, respectively. Compounds 1 and 2 showed obviously cytotoxic activity against the five cancer cell lines with IC₅₀ ranging from 1.06 to 3.60 μM. Compounds 3 and 4 showed selective cytotoxic activity.     

Three new biflavonoids from the branches and leaves of Cephalotaxus oliveri and their antioxidant activity

Three new biflavonoids, named oliveriflavones A-C (1–3), together with two known flavonoids (quercetin (4) and rutin (5)), were isolated from the endangered plant Cephalotaxus oliveri. The chemical structures of these compounds were elucidated by comprehensive spectroscopic methods including NMR, HRESIMS, IR, UV, and CD spectra. Compounds 1–5 were first isolated from the genus Cephalotaxus. All the compounds were tested for their antioxidant activity. Compounds 4 and 5 showed excellent activity with IC₅₀ values of 0.03 ± 0.06 μM and 0.02 ± 0.10 μM, respectively.     

Diterpenoids from Blumea balsamifera and Their Anti-Inflammatory Activities

Six new diterpenoids, blusamiferoids A–F (1–6), including four pimarane-type diterpenoids, one rosane-type diterpenoid (3), and one rearranged abietane-type diterpenoid (6), were isolated from the dry aerial parts of Blumea balsamifera. Their structures were characterized by spectroscopic and computational methods. In particular, the structures of 1 and 4 were confirmed by X-ray crystallography. Compounds 5 and 6 were found to dose-dependently inhibit the production of TNF-α, IL-6, and nitrite oxide, and compound 5 also downregulated NF-κB phosphorylation in lipopolysaccharide (LPS)-induced RAW 264.7 cells.     

半制备型高速逆流色谱分离制备铁棒锤根中的一种咪唑生物碱

采用高速逆流色谱（HSCCC）技术从铁棒锤根氯仿提取物中分离制备了一种高纯度咪唑类生物碱1H-imidazole-2-carboxylic acid,butyl ester （ICABE）。采用高效液相色谱（HPLC）测定目标化合物在两相溶剂中的分配系数,优化HSCCC分离ICABE的溶剂体系,确定了以正己烷-氯仿-乙醇-水（10：1：13：2,v/v/v/v）为HSCCC的两相溶剂系统,以上相为固定相,下相为流动相,流动相流速为1.8 mL/min,主机转速850 r/min,检测波长为230 nm条件下进行分离制备,在350 min内从100 mg粗样品中一步分离得到7.5 mg ICABE,经HPLC检测其纯度达98%以上（峰面积归一化法）,结构由UV、¹H-NMR和¹³C-NMR得以鉴定。该方法简便、快速,所得产物纯度高,适合于铁棒锤中ICABE的制备分离。     

SEPARATION OF THE MINOR FLAVONOLS FROM FLOS GOSSYPII BY HIGH-SPEED COUNTERCURRENT CHROMATOGRAPHY

An effective high-speed countercurrent chromatography (HSCCC) method was established for further separation and purification of four minor flavonols in addition to five major flavonols which were reported by our previous study from extracts of Flos Gossypii. HSCCC was performed with three two-phase solvent systems composed of n-hexane-ethyl acetate-methanol-water (7.5:15:6:7, v/v), (2.5:15:2:7, v/v) and (0:1:0:1, v/v). The separation was repeated 3 times, and 3.8 mg of 8-methoxyl-kaempferol-7-O-β-D-rhamnoside (HPLC purity 98.27%), 6.7 mg of astragalin (HPLC purity 94.18%), 3.3 mg of 4'-methoxyl-quercetin-7-O-β-D-glucoside (HPLC purity 94.30%) and 8.2 mg of hyperoside (HPLC purity 93.48%) were separated from 150 mg of the crude sample. The chemical structures of the flavonols were confirmed by MS, (1)H NMR and (13)C NMR. Meanwhile, the results indicated that the target compound with smaller K value (<0.5) can be separated by increasing column length of HSCCC. And four separation rules of flavonols according to the present study and references were summarized, which can be used as a useful guide for separation of flavonols by HSCCC.     

An efficient strategy for the extraction and purification of lignans from Schisandra chinensis by a combination of supercritical fluid extraction and high‐speed counter‐current chromatography

An efficient strategy for extracting and separating five lignans from Schisandra chinensis (Turcz.) Baill has been developed using supercritical fluid extraction (SFE) and high‐speed counter‐current chromatography (HSCCC) in the present study. First, the extraction was performed by a preparative SFE system under 15 MPa of pressure at 36°C for 4 h. Then, the SFE extract was successfully separated and purified by HSCCC with a two‐phase solvent system composed of n‐hexane/ethyl acetate/methanol/water (6:4:5:5, 6:4:6:4, 6:4:8:2, v/v) in a stepwise elution mode. The fractions were analyzed by HPLC, and the chemical structures of the products were identified by ESI‐MS and ¹H NMR spectroscopy. As a result, a total of 12.5 mg of schisandrin at 98.0% purity, 7.1 mg of gomisin A at 98.1% purity, 1.8 mg of schisantherin B at 93.3% purity, 4.4 mg of deoxyschisandrin at 92.9% purity, and 6.8 mg of γ‐schisandrin at 89.1% purity were obtained from 300 mg crude extract in a one‐step purification.     

Rapid preparative separation of six bioactive compounds from Gentiana crassicaulis Duthie ex Burk. using microwave‐assisted extraction coupled with high‐speed counter‐current chromatography

A rapid method combining microwave‐assisted extraction (MAE) and high‐speed counter‐current chromatography (HSCCC) was applied for preparative separation of six bioactive compounds including loganic acid ( I ), isoorientin‐4′‐O‐glucoside ( II ), 6′‐O‐β‐d ‐glucopyranosyl gentiopicroside ( III ), swertiamarin ( IV ), gentiopicroside ( V ), sweroside ( VI ) from traditional Tibetan medicine Gentiana crassicaulis Duthie ex Burk. MAE parameters were predicted by central composite design response surface methodology. That is, 5.0 g dried roots of G. crassicaulis were extracted with 50 mL 57.5% aqueous ethanol under 630 W for 3.39 min. The extract (gentian total glycosides) was separated by HSCCC with n‐butanol/ethyl acetate/methanol/1% acetic acid water (7.5:0.5:0.5:3.5, v/v/v/v) using upper phase mobile in tail‐to‐head elution mode. 16.3, 8.8, 12., 25.1, 40.7, and 21.8 mg of compounds I–VI were obtained with high purities in one run from 500 mg of original sample. The purities and identities of separated components were confirmed using HPLC with photo diode array detection and quadrupole TOF‐MS and NMR spectroscopy. The study reveals that response surface methodology is convenient and highly predictive for optimizing extraction process, MAE coupled with HSCCC could be an expeditious method for extraction and separation of phytochemicals from ethnomedicine.     

Application of supercritical fluid extraction coupled with counter–current chromatography for extraction and online isolation of unstable chemical components from Rosa damascena

Supercritical fluid extraction (SFE) coupled with high‐speed counter‐current chromatography (HSCCC) was successfully used for the extraction and online isolation of the unstable compounds from Rosa damascene in a single extraction and separation operation in two stages. The solvent systems of SFE/HSCCC were optimized with the help of multiexponential function model. At the first stage, the upper phase of the solvent system of n‐butanol–tert‐butyl methyl ether–acetonitrile–0.1% aqueous TFA (1.7:1.0:0.8:4.0, v/v/v/v) was used as both the SFE entrainer and the HSCCC stationary phase, and the target compounds were eluted with the corresponding lower phase to separate the hydrophobic compounds. At the second stage, the upper phase of the solvent system of n‐hexane–ethyl acetate–methanol–water (3.2:1.0:2.8:2.6, v/v/v/v) was used as both the SFE entrainer and the HSCCC stationary phase, followed by elution with the corresponding lower phase to separate the moderate hydrophobic compounds. Six compounds including formononetin, delphinidin, cyaniding, 5,6,4′‐trihydroxy‐7,8‐dimethoxy flavone, 5,3′‐dihydroxy‐7,8‐dimethoxy flavone, and 5‐hydroxy‐6,7,8,3′,4′‐pentamethoxy flavone were successfully separated in one extraction–separation operation within 300 min. The targeted compounds were identified by MS and NMR spectroscopy. This research has opened up great prospects for industrial application of SFE/HSCCC to the extraction and separation of unstable compounds.     

Separation of polar antioxidants from Rhizoma Polygonatum Odorati by high-speed counter-current chromatography with a hydrophilic solvent system

Some highly polar compounds are quality control makers for medicinal herbs. However, investigation of them has been hampered because the existing fractionation steps are difficult and laborious to purify them. Similar situations happen to Rhizoma Polygonatum Odorati, a widely used food supplement and medicinal herb with strong antioxidant activity, and up to date, only ethyl acetate fraction of Rhizoma Polygonatum Odorati has been comprehensively investigated. Here, HSCCC using a hydrophilic solvent system composed of n-butanol–ethanol–2 M ammonium sulfate (1:1:2, v/v/v) was performed to isolate highly polar antioxidants in n–butanol fraction of Rhizoma Polygonatum Odorati, guided by DPPH–HPLC experiment. Afterward, Sephadex LH-20 column chromatography eluted by methanol was selected to eliminate ammonium sulfate and purify co-eluted compounds in HSCCC collected fractions. Finally, nine compounds, including four nucleosides, cytidine (1), uridine (4), guanosine (5), and adenosine (8); two nucleobases, guanine (3), and adenine (6); and three amino acids, tyrosine (2), phenylalanine (7), and tryptophan (9) with purities over 98% were achieved and identified by UV, MS, and ¹H NMR data. Notably, compounds 1–9 were first reported in genus Polygonatum. The results indicated that the proposed method was an efficient approach to isolate and purify highly polar compounds from complex extracts.     

High-Speed Counter-Current Chromatography Combined with Column Chromatography for Isolation of Methyllycaconitine from Delphinium pseudocyanthum

Preparative high-speed counter-current chromatography (HSCCC) combined with conventional column chromatography (CC) has been used for isolation and purification of methyllycaconitine from Delphinium pseudocyanthum. n-Hexane-ethyl acetate-methanol-water, 1:1:1:2 (v/v), was used as the solvent system for HSCCC. Separation of methyllycaconitine from an HSCCC fraction was successfully achieved by CC on silica gel using chloroform-methanol, 7:1 (v/v), as mobile phase. A total of 113.45 mg methyllycaconitine of purity >95% was obtained from 1.044 g extract of D. pseudocyanthum. Its structure was identified by MS and NMR.