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.     

Supercritical fluid extraction and hydrodistillation for the recovery of bioactive compounds from Lavandula viridis L’Hér

The chemical profiles of bioactive essential oil and extracts obtained by hydrodistillation (HD) and supercritical fluid extraction (SFE), respectively, from Lavandula viridis were compared. The SFE was performed at 40 °C and at extraction pressures of 12 or 18 MPa in two different separators. Evaluation of the essential oil and SFE extracts by GC–FID and GC–IT–MS revealed that oxygen-containing monoterpenes were the major constituents in both cases, but there were important differences between the chemical profiles produced by the different extraction techniques. More compounds were isolated by HD but higher yields were achieved by SFE. Camphor was the main component identified in the essential oil (31.59 ± 1.32%), and in extracts from the first (1.61 ± 0.34%) and second SFE separators (22.48 ± 1.49%) at 12 MPa. In contrast, the first separator SFE extract at 18 MPa (heavy compounds) was dominated by myrtenol (5.38 ± 2.04%) and camphor (4.81 ± 1.93%), whereas the second separator SFE extract (volatiles) was dominated by verbenone (13.97 ± 5.27%). The essential oil and heavy compound extracts from the first separator possessed antioxidant and anti-cholinesterase activities. Our data show that phytochemicals from the aerial parts of L. viridis could be developed as natural antioxidant and anti-cholinesterase drugs, with particular applications in the symptomatic treatment of Alzheimer’s disease.     

Preparative separation and purification of sulforaphene from radish seeds by high-speed countercurrent chromatography

Sulforaphene, a kind of isothiocyanates, derived from glucoraphenin which is the important ingredient of radish (Raphanus sativus L.) seeds, has shown significant pharmacological activities. In this paper, the separation and purification of sulforaphene from radish seeds, was achieved by high-speed countercurrent chromatography (HSCCC). A two-phase solvent system consisted of n-hexane–ethyl acetate–methanol–water (35:100:35:100, v/v/v/v) was applied. The revolution speed of the separation column, flow rate of the mobile phase and separation temperature were 800 rpm, 2 ml/min and 30 °C, respectively. From about 1000 mg amount of the crude plant extract, 249.4 mg of pure sulforaphene was obtained by one-step separation on a 280 ml HSCCC column. The purified sulforaphene was at a high purity of 96.9% and the mass recovery was more than 95%. The purity of sulforaphene was determined by HPLC analysis and its chemical structure was assessed by MS, ¹H NMR, ¹³C NMR and DEPT-135 NMR.     

Supercritical carbon dioxide fluid extraction of Hibiscus cannabinus L. seed oil: A potential solvent-free and high antioxidative edible oil

The supercritical fluid extraction (SFE) trends and antioxidant activities of Hibiscus cannabinus seed oils were studied. SFE results indicate that extraction pressure is the major factor determining the oil yield. In comparison, classic Soxhlet extraction (SOX/L) yielded higher oil content than SFE (P < 0.05). However, no significant differences in oil content were observed in SFE at 600 bars/80 °C, rapid Soxhlet extraction (SOX/S) and conventional ultra-sonic assisted solvent extraction (SONIC) (P > 0.05). Antioxidant activities of H. cannabinus seed oils were compared with 7 types of commercial edible oils. DPPH scavenging activity test indicated that H. cannabinus seed oil extracted by SFE at 200 bars/80 °C possessed the highest antiradical activity whereas beta-carotene bleaching (BCB) assay revealed that all H. cannabinus seed oils (except for SFE at 400 bars/80 °C and 600 bars/80 °C) exhibited higher antioxidant activity than all commercial edible oils (P < 0.05). Thus, SFE – H. cannabinus seed oil may serve as an excellent source of solvent-free edible oil with high antioxidant properties.     

Comparison of essential oil compositions of Salvia mirzayanii obtained by supercritical carbon dioxide extraction and hydrodistillation methods

Essential oil of Salvia mirzayanii cultivated in Iran was obtained by hydrodistillation and supercritical (carbon dioxide) extraction methods. The oil was analysed by capillary gas chromatography using flame ionization and mass spectrometric detections. The compounds were identified according to their retention indices and mass spectra (EI, 70 eV). The effects of different parameters such as pressure, temperature, modifier volume and extraction times (dynamic and static) on the supercritical fluid extraction (SFE) of S. mirzayanii oil were investigated. The results showed that, under a pressure of 35.5 MPa, temperature of 35 °C, 6% methanol, dynamic extraction time of 50 min and static extraction time of 30 min, extraction was more selective for the linalyl acetate. Thirty four compounds were identified in the hydrodistilled oil. The major components of S. mirzayanii were linalyl acetate (7.6%), 1,8-cineole (8.0%), linalool (9.0%) and 8-acetoxy linalool (11.0%). However, by using supercritical carbon dioxide in optimum conditions, only three components contain more than 63% of the oil. The yield of the obtained oil based on hydrodistillation was 2.20% (v/w). Extraction yield based on the SFE varied in the range of 1.50–9.67% (w/w) under different conditions. The results revealed that, in Iranian S. mirzayanii oil, linalyl acetate is a major component.     

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.     

Preparative separation and purification of gingerols from ginger (Zingiber officinale Roscoe) by high-speed counter-current chromatography

A novel method for purifying gingerols from ginger was developed using a high-speed counter-current chromatography (HSCCC). The two-phase solvent system such as light petroleum (bp 60–90 °C)–ethyl acetate–methanol–water (5:5:6.5:3.5, v/v/v/v) was applied to the separation and purification of 6-, 8- and 10-gingerol from a crude extract of ginger. The experiment yielded 30.2 mg of 6-gingerol, 40.5 mg of 8-gingerol, 50.5 mg of 10-gingerol from 200 mg of crude extract in one-step separation. And the purity of these compounds was 99.9%, 99.9% and 99.2%, respectively, as determined by high-performance liquid chromatography (HPLC). Their structures were identified by gas chromatography–mass spectrometry (GC/MS) and ¹H, ¹³C nuclear magnetic resonance (NMR).     

Supercriticial fluid extraction of flavors and fragrances from Hyssopus officinalis L. cultivated in Iran

Hyssopus officinalis L. (hyssop) as a food ingredient has its own importance in flavor industry and also in sauce formulations. Supercritical fluid extraction (SFE) of hyssop, cultivated in Iran, was performed at various pressures, temperatures, extraction (dynamic and static) times and modifier (methanol) concentrations using an orthogonal array design with an OA₂₅(5⁵) matrix conditions. Pressure, temperature and modifier in the SFE system influenced the extraction yield. Also, the composition of the extracted oils was greatly impacted by the operating conditions. Main components of the extracts under different SFE conditions were sabinene (4.2–17.1%, w/w), iso-pinocamphene (0.9–16.5%) and pinocamphene (0.7–13.6%). The extraction of sabinene, for example, was favored at 100 atm, 55 °C, 1.5% (v/v) methanol, 30 min dynamic time and 35 min static time. Use of SFE under different conditions can allow targeting the extraction of different constituents.     

Determination of tertiary butylhydroquinone in edible vegetable oil by liquid chromatography/ion trap mass spectrometry

A simple, sensitive and accurate analytical method for quantification of tertiary butylhydroquinone (TBHQ) in edible vegetable oil was established by liquid chromatography/ion trap mass spectrometry (LC/ITMS). After extraction, 5 μl of the extracts was directly injected into LC/ITMS for TBHQ determination. Ethanol was selected as the extraction solvent. The optimized fragmentation amplitude was 1.70 V and electrospray ionization (ESI) was more suitable than atmospheric pressure chemical ionization (APCI) for TBHQ detection. The calibration curve showed good linearity (R² = 0.9990) and recoveries from spiked samples ranged from 81.9% to 110.5%. Relative standard deviations of intra-day and inter-day were in the ranges 2.5–5.7% and 3.9–13.8%, respectively. The procedure allows the detection of 0.3 mg/kg TBHQ in edible vegetable oil. Typical edible vegetable oils in the market were detected for TBHQ by the proposed method. As results, TBHQ was detected in blend oil, soybean salad oil and camellia oil samples.     

Isolation and purification of phenylethanoid glycosides from Cistanche deserticola by high-speed counter-current chromatography

Five phenylethanoid glycosides (PhGs), echinacoside, cistanoside A, acteoside, isoacteoside and 2′-acetylacteoside, were isolated and purified from Cistanche deserticola for the first time by high-speed counter-current chromatography (HSCCC) using two biphasic systems, one consisting of ethyl acetate–ethanol–water (5:0.5:4.5, v/v/v) and another of ethyl acetate–n-butanol–ethanol–water (0.5:0.5:0.1:1, v/v/v/v). A total of 28.5 mg of echinacoside, 18.4 mg of cistanoside A, 14.6 mg of acteoside, 30.1 mg of isoacteoside and 25.2 mg of 2′-acetylacteoside were purified from 1412 mg of the n-butanol extract of C. deserticola, each at over 92.5% purity as determined by HPLC. The structures were identified by their retention time, UV, LC–ESI-MS in the negative ion mode, and confirmed by NMR experiments. The characteristic LC–ESI-MSⁿ fragmentation pattern of the five compounds is discussed, and found to be a very specific and useful tool for the structural identification of PhGs from this important medicinal plant.     

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 response surface methodology and central composite design for the optimisation of supercritical fluid extraction of essential oils from Myrtus communis L. leaves

Essential oils of Myrtus communis L. leaves were obtained using supercritical fluid extraction (SFE) and hydrodistillation methods. The experimental parameters of SFE such as pressure, temperature, modifier volume, static and dynamic extraction time were optimised using a central composite design after a 2ⁿ⁻¹ fractional factorial design. The chemical compositions of the SFE extract were identified by GC–MS and determined by GC–FID. The major components of essential oils obtained by hydrodistillation were α-pinene (31.8%), 1,8-cineole (24.6%), limonene (14.8%), linalool (8.3%) and α-terpinolene (4.8%). However, by using the supercritical carbon dioxide in optimum conditions, only three components represented more than 85% of the extract. Therefore, by using the proper SFE conditions, the supercritical extraction is more selective than the conventional hydrodistillation methods. The oil yields based on the hydrodistillation was 0.47% (v/w). Extraction yields based on the SFE varied in the range of 0.5–6.3% (w/w) under different conditions.     

α-Glucosidase inhibitors from Chinese Yam (Dioscoreaopposita Thunb.)

The inhibitory activities of crude extracts and purified constituents from the fresh tuberous rhizomes of Chinese Yam (Dioscorea opposita Thunb.), which is commonly called Huai Shan Yao in Chinese, were evaluated against yeast α-glucosidase in order to search for the active principals for treatment of diabetes. Bioassay-guiding isolation gave four compounds: trans-N-p-coumaroyltyramine (1) (IC₅₀ = 0.40 μM), 1,7-bis(4-hydroxyphenyl)heptane-3,5-diol (2) (IC₅₀ = 0.38 mM), 6-hydroxy-2,4,7-trimethoxyphenanthrene (3) (IC₅₀ = 0.77 mM) as α-glucosidase inhibitors, and cis-N-p-coumaroyltyramine (4), an isomer of compound 1, which showed no inhibitory activity against α-glucosidase. Furthermore, the separation and purification of compound 3 from Chinese Yam (Huai Shan Yao) was conducted by high-speed counter-current chromatography (HSCCC) using hexane–ethyl acetate–methanol–water (1:1:1:1, v/v/v/v). Compound 1, 2 and 4 were isolated from or detected in the Dioscoreaceae family for the first time.     

Determination of diquat and paraquat in olive oil by ion-pair liquid chromatography–electrospray ionization mass spectrometry (MRM)

For the first time a method for determination of herbicides diquat (DQ) and paraquat (PQ) in olive oil was developed utilising liquid chromatography–electrospray ionization mass spectrometry (MRM). n-Hexane/10 mM HFBA aqueous solution partitioning was used as the extraction method. Separation was carried out in an Xterra C8 column (100 × 21 mm, 3 μm), using the gradient mode. Solvent A was a HFBA aqueous solution (5 mM, pH 2) and solvent B acetonitrile/methanol 75/25 (v/v). Peaks used for quantification were m/z = 157 (diquat) and m/z = 158 (paraquat). Detection limit found for both diquat and paraquat was 4 μg kg⁻¹. The method can also be applied for determination of chlormequat (CQ, quantification peak m/z = 58), the detection limit being 0.3 μg kg⁻¹. Such limits are clearly lower than the MCLs commonly applied to olive oil as reference criteria (5 times MCLs in olives). Good reproducibilities (day to day and run to run) were obtained.     

The essential oil of Glossogyne tenuifolia

Glossogyne tenuifolia (Labill) Cass. (Compositae) is a traditional anti-pyretic and hepatoprotective herb in The Pescadores Islands. The essential oil of the dried herb, from four seasons, was isolated using a simultaneous steam-distillation and solvent-extraction (SDE) apparatus. The essential oil contents were in the range of 0.48–0.77 mg g⁻¹, with an average of 0.66 mg g⁻¹, and declined with the seasons. Generally, the essential oils from four seasons exhibited similar volatile profiles. A total of 62 different compounds were isolated by the SDE method and, among them, 30 compounds were identified, including 13 terpenes, 16 oxygen-containing compounds (eight alcohols, five aldehydes, one ester and two ketones) and one other compound. Terpenes were predominantly present in the essential oil and accounted for 61.3–76.0% of the essential oil with an average of 69.1%. The second most abundant class was alcohols, accounting for 12.4–15.9% of the essential oil, with an average of 14.1%. Consistently for four seasons, the most abundant eight compounds were in the descending order: p-cymene > β-pinene > β-phellandrene > limonene > cryptone > α-pinene > 4-terpineol + γ-muurolene. However, these eight compounds accounted for 71.5% of the average of the essential oil and, in combination, might give rise to the overall citrus-like aroma of the G. tenuifolia.     

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.     

Economical viability of SFE from peach almond, spearmint and marigold

Supercritical fluid extraction (SFE) applies near critical carbon dioxide (CO₂) for the extraction of aromatic and bioactive products such as peach (Prunus persica) almond oil, spearmint (Mentha spicata L.) essential oil and marigold (Calendula officinalis L., Asteraceae) oleoresin. Due to the cost intensive nature of SFE, the estimation of process cost is necessary to appraise operation viability. Therefore, this work evaluated the economical viability of SFE from peach almond, spearmint leaves and marigold flowers, as a function of process parameters, using extraction data from the literature. The costs were determined by the software Tecanalysis, considering the specific costs to be equal to the market value of the oils. The results show that SFE from peach almond for 30 min in a 2 × 400 L extraction unit and from spearmint at 300 bar are lucrative processes. SFE from marigold was not economically viable, probably because of the imprecise operational parameters assumed.     

Purification and anti-tumour activity of cyanidin-3-O-glucoside from Chinese bayberry fruit

Chinese bayberry (Myrica rubra Sieb. & Zucc.) is a rich source of cyanidin-3-O-glucoside (C3G). Purification of C3G was established by using high-speed counter-current chromatography (HSCCC) at 520 nm. The optimised flow rate was 2 ml/min and rotational speed was 850 rpm, and n-butanol-methyl tertiary butyl ether-acetonitrile-trifluoroacetic acid-water (30:20:40:1:100, v/v/v/v/v) solvent showed the highest partition coefficient (K = 1.50) to purify C3G. Different HSCCC fractions showed significant different anti-tumour effect on SGC7901, AGS, and BGC823 gastric cells, which were correlated with their C3G concentrations and DPPH^• scavenging activities. Cells treated with C3G resulted in reduced cell proliferation, decreased cell adherence, and abnormal morphological changes characteristics of apoptosis, all in dose-dependent manners. In addition, increased concentrations of C3G treatment resulted in increased inhibition of matrix metalloproteinase 2 (MMP-2) in SGC7901 cells, which may provide important information for the possible mechanism of C3G-induced anti-tumour activity against gastric adenocarcinoma cells.     

Extraction and characterisation of lipids from Antarctic krill (Euphausia superba)

There is significant commercial interest in oil extraction from krill because it is rich in omega-3 polyunsaturated fatty acids (n-3 PUFA) such as eicosapentaenoic (EPA, 20:5n3) and docosahexaenoic (DHA, 22:6n3) acids. The objectives were to determine oil extraction efficiency using different solvent systems and the composition of extracted oil and spent krill following extraction. Extraction efficiency was the highest (P < 0.05) for one-step extraction using freeze-dried krill with 1:12 or 1:30 krill:solvent ratio (w:v) compared to Folch, Soxhlet, and conventional two-step extraction. Extracted oils contained predominantly phospholipids (20–33%), polar non-phospholipids (64–77%), and minor triglycerides (1–3%). Triglycerides contained much less (P < 0.05) total n-3 (4.0%), DHA (1.1%), and EPA (2.3%), but more (P < 0.05) saturated FA (38.7%) than phospholipids (total n-3-47.4%, DHA-18.0%, EPA-28.2%, saturated FA-23.5%). Antioxidant capacity of krill oil extracted by one-step extraction (9.4–14.2 μmol Trolox Equivalents/ml oil) was generally similar to antioxidant capacity of krill oil extracted by ethanol (22.9), but greater (P < 0.05) than antioxidant capacity of krill oil extracted by acetone (1.2) and Folch method (1.5). The spent krill following oil extraction contained protein (72.9–75.8%, dry basis). Based on the extraction efficiency and composition of the extracted oil, the one-step extraction using 1:12 krill:solvent ratio is recommended.     

Chemical fingerprinting and bioactivity of Amazonian Ecuador Croton lechleri Müll. Arg. (Euphorbiaceae) stem bark essential oil: A new functional food ingredient?

Croton lechleri essential oil has been obtained by steam distillation of fresh stem bark from Amazonian Ecuador adult plants (yield: 0.61 ml/kg [0.061%]; density: 1.01 g/ml), and then chemically characterised by GC (Gas Chromatography) and GC–MS (gas chromatography–mass spectrometry). Seventy-four chemicals were detected and identified; the most abundant in descending order, were the sesquiterpenes sesquicineole (17.29%), α-calacorene (11.29%), 1,10-di-epi-cubenol (4.75%), β-calacorene (4.34%) and epi-cedrol (4.09%). Monoterpenes checked with a relative peak area higher than 2.0% were α-pinene (2.01%), p-cymene (2.61%), limonene (4.20%) and borneol (2.67%). The structure of the main chemicals were confirmed by GC–MS and ¹H NMR analyses. Spectrophotometric 1,1-diphenyl-2-picrylhydrazyl (DPPH) and DPPH-(high performance) thin layer chromatography (DPPH-(HP)TLC) bioautographic assays showed a lower radical scavenging capacity (IC₅₀) with respect to commercial thyme essential oil and BHA (butylated hydroxyl anisole), pointing out, however, that the C. lechleri essential oil fraction, characterised by α-calacorene, β-calacorene and δ-cadalene, was the most involved in the bioactivity. Similar results were obtained with β-carotene bleaching assay, where the IC₅₀ values were 0.291 ± 0.024 mg/ml for C. lechleri essential oil, 0.164 ± 0.013 and 1.34 × 10⁻⁴ ± 10⁻⁵ mg/ml for thyme essential oil and BHA, respectively. (HP)TLC-bioautographic assay performed with Gram positive and Gram negative bacteria revealed a minimum inhibitory concentration (MIC) values comprised between 0.10 mg/ml (Escherichia coli) and 10.10 mg/ml (for e.g. Pseudomonas aeruginosa), and the fraction mainly characterised by sesquicineole (97.38%) as the most involved in antibacterial capacity. Ames test employing Salmonella typhimurium TA98 and TA100 with and without a metabolic activation mixture (S9 mix) demonstrated the absence of mutagenicity of the C. lechleri essential oil between a concentration range of 10⁻² and 100 mg/plate. The same results were achieved by Saccharomyces cerevisiae D7 strain assay. An interesting mutagen-protective efficacy was evidenced by a 30% and 33% revertants reduction of TA98 strain treated with 2-aminoanthracene and nitrofluorene (2 μg/plate), suggesting, above all, the possibility to employ C. lechleri essential oil as a new flavouring protective ingredient for foods or dietary supplements against potential mutagens formed during cooking and/or processing in general.