Preparative separation of gingerols from Zingiber officinale by high-speed counter-current chromatography using stepwise elution

Following an initial clean-up step on silica column, high-speed counter-current chromatography (HSCCC) was used to purify gingerols from an extract of the dried rhizome of Zingiber officinale. The sample was separated with petroleum ether–ethyl acetate–methanol–water (1:0.2:0.5:0.7, v/v) and petroleum ether–ethyl acetate–methanol–water (1:0.2:0.7:0.5, v/v) in a stepwise elution and yielded 132 mg of 6-gingerol, 31 mg of 8-gingerol and 61 mg of 10-gingerol from 360 mg of pre-purified sample. The purity of each compound was over 98% as determined by HPLC. The structures of the three compounds have been identified by LC-ESI-MS, ¹H NMR and ¹³C NMR.     

Application of preparative high-speed counter-current chromatography for separation and purification of lignans from Taraxacum mongolicum

Apart from being used as a pharmaceutical, the inflorescences, leaves and roots of Taraxacum mongolicum are processed into different food products. However, only few phytochemical investigations on this plant have been performed. In the present study, a preparative high-speed counter-current chromatography (HSCCC) for the separation and purification of bioactive compounds from T. mongolicum was developed. Two lignans, mongolicumin A and rufescidride, were obtained. The target compounds were finally isolated and purified with a solvent system composed of ethyl acetate–n-butanol–water (2:5:7, v/v/v). The lower phase was used as the mobile phase in the head to tail elution mode. By injecting 500 mg of the enriched extract of T. mongolicum, one-step HSCCC procedure yielded 36.7 mg of mongolicumin A and 43.9 mg of rufescidride with the purity of 98.7% and 98.5%, respectively, as determined by high-performance liquid chromatography (HPLC). The chemical structures of the two lignans were confirmed by UV, IR, HRESIMS, 1D and 2D NMR. Among them, mongolicumin A is a new compound, and rufescidride was obtained from genus Taraxacum for the first time. Furthermore, lignans were first isolated and identified from genus Taraxacum.     

Preparative isolation and purification of xanthohumol from hops (Humulus lupulus L.) by high-speed counter-current chromatography

Xanthohumol (XN) and related prenylflavonoids are the main bioactive components of hops (Humulus lupulus L.). The current work is to investigate the use of high-speed counter-current chromatography (HSCCC) in search for high isolation of xanthohumol from hops. A solvent system consisted of n-hexane-ethyl acetate-methanol-water at a volume ratio of 5:5:4:3 was employed. The results demonstrated that the constructed method could be well applied for the isolation of xanthohumol from hops extract. After HSCCC isolation procedure, the purity of xanthohumol was over 95% assayed by HPLC and the yield of extraction was 93.60%. The chemical structure identification of xanthohumol was carried out by UV, ¹H NMR and ¹³C NMR. The present results demonstrated that xanthohumol could be efficiently obtained using a single HSCCC step from H. lupulus L. extract.     

Simultaneous determination of five phenylethanoid glycosides in small-leaved Kudingcha from the Ligustrum genus by UPLC/PDA

A quantitative method using ultra performance liquid chromatography with a photodiode array detector (UPLC/PDA) was established for the analysis of the five main phenylethanoid glycosides in small-leaved Kudingcha from the genus Ligustrum and was applied as a quality control for small-leaved Kudingcha. Acteoside (1), ligupurpuroside A (2), isoacteoside (3), lipedoside A-I (4), and ligupurpuroside B (5) were separated using a mobile phase of water/phosphoric acid and acetonitrile with a Waters Acquity BEH C₁₈ column. The method was validated according to regulatory guidelines with respect to precision, accuracy and linearity. The five phenylethanoid glycosides in 38 batches of small-leaved Kudingcha were determined by this method. As a result, Ligustrum robustum (syn. Ligustrum purpurascens), which contains a large amount of phenylethanoid glycosides, was tentatively determined to be the correct species for the small-leaved Kudingcha.     

Preparative separation of phenylpropenoid glycerides from the bulbs of Lilium lancifolium by high-speed counter-current chromatography and evaluation of their antioxidant activities

Preparative separation of seven phenylpropenoid glycerides from the bulbs of Liliumlancifolium (lily), was conducted by high-speed counter-current chromatography (HSCCC) with a two-phase solvent system composed of n-hexane-ethyl acetate-methanol-0.05% aqueous trifluoroacetic acid (TFA) (3:5:3:5, v/v/v/v). These phenylpropenoid glycerides were identified as 1-O-feruloylglycerol (1), 1-O-p-coumaroylglycerol (2), 1-O-caffeoyl-3-O-p-coumaroylglycerol (3), 1,2-O-diferuloylglycerol (4), 1,3-O-diferuloylglycerol (5), 1-O-feruloyl-3-O-p-coumaroylglycerol (6), and 1,3-O-di-p-coumaroylglycerol (7), respectively, by ESI-MS, ¹H NMR and ¹³C NMR. Their antioxidant activities were evaluated by DPPH radical scavenging activity, ABTS radical cation scavenging activity, and ferric-reducing antioxidant power (FRAP). The trend in antioxidant capacity was similar in all the three assays, with 3 > 1, 4, 5, 6 > 2, 7. This is the first report on simultaneous separation of seven antioxidantive phenylpropenoid glycerides from L. lancifolium by HSCCC.     

Antioxidant activity of phenolic and phenylethanoid glycosides from Teucrium polium L

In the present study, the chemical composition and antioxidant activities of Teucrium polium L. (Lamiaceae) were assessed. Fractionation of methanol extract obtained from the aerial parts of T. polium yielded one new phenylethanoid glycoside, named poliumoside B, together with four known flavonoids, two iridoid glycosides and a known poliumoside. The structures of all of these compounds were elucidated on the basis of spectroscopic data from 1D and 2D NMR experiments and MS spectral analyses. The antioxidant activities of the crude extracts and of the isolated compounds were evaluated through tests such as DPPH radical-scavenging capability, reducing power, xanthine oxidase inhibitory effect and inhibition of linoleic acid peroxidation. The highest antioxidant activity was found in the n-butanol extract, which also contained active polyphenols, thus suggesting that this plant could be used as a source of natural molecules, to provide safe antioxidant additives and nutraceuticals. The structure–activity relationships of the isolated compounds are also discussed.     

Separation of anti-ulcer flavonoids from Artemisia extracts by high-speed countercurrent chromatography

High-speed countercurrent chromatography (HSCCC) was applied to separate two anti-ulcer flavonoids, eupatilin (5,7-dihydroxy-6,3′,4′-trimethoxyflavone, 1) and jaceosidin (5,7,4′-trihydroxy-6,3′-dimethoxyflavone, 2), isolated from the ethanol, ethyl acetate and semi-purified ethanol extract of dietary wormwood (Artemisia princeps). The two phase solvent system comprising n-hexane–chloroform–methanol–water (2:5:5:2, v/v/v/v) was employed to HSCCC and the flow rate of mobile phase was optimised at 1.3 ml/min. Utilising this method, the recovery rate and purity of eupatilin (1) and jaceosidin (2) separated from three Artemisia extracts were evaluated to be over 95% and 90%, respectively. These results suggest that countercurrent separation method can be applied to isolate two anti-ulcer flavonoids, eupatilin (1) and jaceosidin (2), selectively in one step, and to produce anti-ulcer flavonoid-enriched preparations.     

Isolation and purification of nootkatone from the essential oil of fruits of Alpinia oxyphylla Miquel by high-speed counter-current chromatography

HSCCC technique in a semi-preparative scale was successfully applied in isolation and purification of nootkatone from the essential oil of fruits of Alpinia oxyphylla Miquel. Twelve kinds of two-phase solvent systems, consisting of seven non-aqueous and five organic-aqueous solvent systems, were selected with not only suitable partition coefficients of nootkatone but also suitable separation factors between nootkatone and valencene, the dominant impurity in the essential oil. Further on HSCCC, n-hexane–chloroform–acetonitrile (10:1:10, v/v) amongst the non-aqueous solvent systems and n-hexane–methanol–water (5:4:1, v/v) amongst the organic-aqueous solvent systems were separately screened out. However, n-hexane–methanol–water (5:4:1, v/v) was thought optimal due to quite shorter elution time and better HSCCC peak form. By eluting the lower phase of this solvent system in head–tail mode, 3.1 mg of nootkatone was obtained at a purity of 92.30% by GC–MS from 80 mg of crude essential oil in one step operation in less than 4 h. The chemical structure of nootkatone fraction was confirmed by EI-MS and ¹H NMR.     

Isolation of ursolic acid from apple peels by high speed counter-current chromatography

Cuticular waxes of four varieties of Malus domestica were investigated regarding their content of ursolic acid. Peels from Fuji, Gala, Smith and Granny Smith apples were extracted with chloroform, ethyl acetate and/or ethanol. The crude extracts were purified by high speed counter-current chromatography (HSCCC), by using mobile and stationary phases derived from the two-phase solvent system composed by n-hexane:ethyl acetate:methanol:water in the proportion of 10:5:2.5:1. The phase proportions and the relative distribution of ursolic acid between the two-phases were optimized by TLC and optical densitometry, by comparison with an authentic sample of ursolic acid. The amount of ursolic acid present in the extracts as well as the characterization of the isolated compound were made by high resolution gas chromatography coupled to mass spectrometry (GC–MS), ¹³C nuclear magnetic resonance (¹³C NMR), Infrared; and by comparing thin layer chromatography and flame ionization detection gas chromatography (GC–FID) patterns with the commercial sample. The average content of ursolic acid of 0.8 mg/cm² in the peel (around 50 mg per medium sized fruit with a surface area of 50–70 cm²) was found in the Fuji and Smith varieties, whereas 0.5 mg/cm² and 0.2 mg/cm² were the amounts calculated for Granny Smith and Gala, respectively. The HSCCC technique was shown to be a good method to purify free ursolic acid from apple peels and could represent a new technological tool to be developed to exploit industrially this source of product.     

肉苁蓉保鲜贮藏及加工研究进展

肉苁蓉是中国传统的名贵中药材之一,可鲜食,但由于产地区域限制以及新鲜肉苁蓉采收后仍具有非常旺盛的生命活动,其在运输和贮藏过程中极易发生褐变、软化甚至霉变等情况。通过检索查阅相关的文献资料,文章总结了近年来新采收鲜肉苁蓉的保鲜方法及贮藏技术,剖析了不同加工方法的优缺点差异,并对肉苁蓉加工技术的发展方向进行了展望。     

Preparative separation of flavonoids in Adinandranitida leaves by high-speed counter-current chromatography and their effects on human epidermal carcinoma cancer cells

Preparative separation of camellianins A and B in Adinandra nitida leaves, which is a flavonoid-rich plant source with great practical prospects, was conducted by high-speed counter-current chromatography (HSCCC) with a solvent system composed of ethyl acetate–ethanol–water (5:1:5, v/v). Apigenin (their aglycone) was prepared by hydrolysing a water extract and recrystallising. The purity and identity of these flavonoids was confirmed by HPLC-ESI/MS, and ¹H and ¹³C NMR. These flavones were used in cultures of the A431 cell line. Their inhibitory effect was evaluated by using the MTT assay. The results showed that three flavones could significantly inhibit the growth of human epidermal carcinoma cancer cells in a concentration-dependent manner. After 48 h of treatment, IC₅₀ values of camellianin A, camellianin B and apigenin against A431 tumour cells were 9.09 μM, 12.5 μM, 21.0 μM, respectively.     

Preparative isolation of triterpenoids from Ganoderma lucidum by counter-current chromatography combined with pH-zone-refining

Ganoderma lucidum is a famous fungus. The triterpenoids are the main bioactive components and exhibit various pharmacological activities. However, the scarcity of the pure triterpenoid has greatly restrained the modern research of G. lucidum. The present paper first describes a convenient method to separate the main triterpenoids from G. lucidum using counter-current chromatography (CCC) technique. Ganoderic acid C6 (38 mg), E (76 mg), F (32 mg) were successfully separated from the crude extract in 1 day with the HPLC purity of above 90% using stepwise CCC. Ganoderic acid G (36 mg), A (64 mg), B (25 mg), D (28 mg) and ganoderenic acid D (11 mg) were separated in 2 days with the HPLC purity of above 90% using a combination of stepwise CCC and pH-zone-refining CCC. The method presented in the paper is much quicker and easier than the previous methods.     

Phenylpropanoid glycosides from the seeds of Michelia hedyosperma

The seed of Michelia hedyosperma Law (Magnoliaceae) is a commonly used spice by the local people living in the south of Yunnan province, China. From which, six new phenylpropanoid glycosides, michehedyosides A–F (1–6) were obtained, in addition to six known compounds, eugenol, eugenol 4-O-β-d-glucopyranoside, martynoside, alaschanioside C, 3,4-methylenedioxycinnamyl alcohol, and (+)-pinoresinol 4-O-β-d-glucoside. Their structures were elucidated on the basis of detailed spectroscopic analyses and chemical methods.     

高速逆流色谱法用于纵条纹炭角菌中过氧麦角甾醇的分离纯化

过氧麦角甾醇生物活性多样,具有较大开发价值,但传统柱色谱分离手段步骤多效率低,本文建立了高速逆流色谱从纵条纹炭角菌中高效快速制备过氧麦角甾醇的方法。通过高效薄层色谱和高效液相色谱测定分配系数,再利用高速逆流色谱考查了多个溶剂体系的分离效果,确定最佳溶剂体系为:正己烷-乙酸乙酯-乙醇-水（体积比为3∶1∶2∶0.8）。以上相为固定相,下相为流动相,转速为800 r/min（正转）,流速为2 m L/min,检测波长为220 nm。制备所得过氧麦角甾醇经13C-NMR和1H-NMR鉴定,并经高效液相色谱分析纯度达到96.05%（峰面积归一化法）。经DPPH体外抗氧化活性测试,发现该化合物在0.2 mg/m L时,对DPPH自由基清除为28.36%,远低于阳性对照抗坏血酸（93.41%）,且量价关系也不明显,表明其DPPH自由基清除能力较弱。该方法制备过氧麦角甾醇简便、快速,所得产物纯度高,可用于纵条纹炭角菌中该化合物的分离。      

Spirostane and cholestane glycosides from the bulbs of Allium nigrum L

A phytochemical investigation of the fresh bulbs of Allium nigrum L. led to the isolation of new spirostane-type glycosides as two inseparable isomer mixtures, nigrosides A1/A2 (1a/1b) and nigrosides B1/B2 (2a/2b), two new cholestane-type glycosides, nigrosides C and D (3 and 4), together with the known compounds, 25(R,S)-5α-spirostan-2α,3β,6β-trio1-3-O-β-d-glucopyranosyl-(1 → 2)-O-[β-d-xylopyranosyl-(1 → 3)]-O-β-d-glucopyranosyl-(1 → 4)-β-d-galactopyranoside (5a/5b) and 25(R,S)-5α-spirostan-2α,3β,6β-trio1 3-O-β-d-glucopyranosyl-(1 → 2)-O-[4-O-(3S)-3-hydroxy-3-methylglutaryl-β-d-xylopyranosyl-(1 → 3)]-O-β-d-glucopyranosyl-(1 → 4)-β-d-galactopyranoside (6a/6b), isolated from this plant for the first time. All structures were elucidated mainly by spectroscopic analysis (1D and 2D NMR experiments, FABMS, HRESIMS) and by comparison with literature data. Cytotoxicity of the isolated compounds was assessed against human colon carcinoma (HT-29 and HCT 116) cell lines. Compounds 5a/5b and 6a/6b were found to be the most active with IC₅₀ values 1.09 and 2.82 μM against HT-29 and 1.59 and 3.45 μM against HCT 116, respectively.     

Supercritical fluid extraction of Coriandrum sativum and subsequent separation of isocoumarins by high-speed counter-current chromatography

A novel supercritical fluid extraction (SFE) method has been developed for extracting essential oil from the aerial part of Coriandrum sativum. Various experimental conditions were investigated to optimise the SFE. It suggested that the extraction of 35 °C, 8 MPa, 2 h might be the optimisation to get the maximum of coriander essential oil. Then, high-speed counter-current chromatography (HSCCC) was successfully used in one step for the isolation and purification of coriandrone B, coriandrin, dihydrocoriandrin and coriandrone A from the coriander oil with a two-phase system composed of n-hexane–ethyl acetate–methanol–water (3:7:5:5, v/v/v/v) for the first time. High-performance liquid chromatography (HPLC) was employed to analyse the HSCCC fractions and revealed that the purities of the isocoumarins were all above 90%, and the chemical structures were identified by spectroscopic analysis including ESIMS, ¹H NMR and UV.     

Chemical properties of the cultivated Sideritis raeseri Boiss. & Heldr. subsp. raeseri

Phytochemical analyses of the cultivated Sideritis raeseri subsp. raeseri in four different stages of flower development were performed. Traditionally used infusion and decoction were also prepared from aerial parts in full flowering stage, and analyses of active compounds and radical scavenging capacity were performed. The highest yield of the essential oil, obtained by hydrodistillation, was noticed in the full flowering phase (0.11%), with sesquiterpene bicyclogermacrene as the main constituent (42.5%). All examined extracts contained phenolic compounds and their amounts varied from 15.3 to 34.1 mg GAE/g DW. The amounts of total phenolics in infusion and decoction were similar (46.5 and 43.9 mg GAE/100 ml, respectively). LC–ESI-MS analyses of all samples allowed the characterisation of 22 phenolic compounds. Two dominant flavone glycosides, 4′-O-methylhypolaetin-7-O-[6?-O-acetyl-β-d-allopyranosyl (1 → 2)-β-d-glucopyranoside (17) and 4′-O-methylisoscutellarein-7-O-[6?-O-acetyl-β-d-allopyranosyl-(1 → 2)]-β-d-glucopyranoside (19) were quantified using HPLC. Moreover, the mineral content and the percent of transportation were investigated.     

Isolation, purification and characterization of a new gum from Acanthophyllum bracteatum roots

A new gum was isolated from the roots of Acanthophyllum bracteatum (ABG) by warm-water extraction. Purification was carried out by barium complexing to give a yield of 12.4% of pure air-dried or 5.8% of freeze-dried gum. The ABG contained 13.2% moisture, 84.3% carbohydrate, 0.9% protein and 1.5% ash. Its mineral content was comparable to commercial hydrocolloids. Monosaccharide analysis by HPLC showed the presence of galactose, glucose, arabinose, rhamnose and uronic acids in the ratio 16.0:7.2:3.0:1.0:3.1 respectively. The viscosity and pH value of 1% ABG solution at 25 °C were 51.5 mPa s and 6.85 respectively. ABG solutions (5-30 wt%) showed shear-thinning flow behavior at shear rates < 10 s⁻¹. The viscosity decreased as temperature increased, and was highest at the neutral state. ABG had low surface and emulsification properties but moderate foaming capacity and relatively high foaming stability, which suggests that ABG could potentially be used in food systems to improve foaming properties.     

Two unusual minor 18,19-seco-ursane glycosides from leaves of Ilex cornuta

The leaves of Ilex cornuta is traditionally used as a functional tea in China. Two new minor 18,19-seco-ursane glycosides, named cornutaoside A (1) and B (2), were isolated from leaves of I. cornuta, along with two known compounds (3 and 4). Their structures were elucidated as (3β,12β)-3-[β-d-glucopyranosyl-(1 → 2)-α-l-arabinopyranosyl]-12,21-dihydroxy-19-oxo-18,19-secours-13(18)-en-28-oic acid (1) and (3β,12β)-3-[β-d-glucopyranosyl-(1 → 2)-α-l-arabinopyranosyl]-12,19,21-trihydroxy-18,19-secours-13(18)-en-28-oic acid (2), by chemical methods, 1D and 2D NMR experiments, and by comparison with known analogues. This is the first report of E-seco triterpenoids and diterpene skeletons (4) from this plant. In a preliminary cytotoxic test against U937, L1210, and B16 cell lines, 1 and 2 had no significant activities as compared to controls, with concentrations up to 443.61 and 346.25 μM/plate, for 1 and 2, respectively.