Effect of sample handling on alkaloid and mineral content of aqueous extracts of greater celandine (Chelidonium majus L.)

The authors examined the extraction of alkaloids from the greater celandine (Chelidonium majus L.) by different methods (traditional pressing and tea making, microwave and supercritical fluid extraction). The extractants were water and propylene glycol. For comparison of the extraction methods, the yield was evaluated according to total alkaloid content measured by spectroscopy. The highest alkaloid yield was obtained by microwave extraction and by making tea. Distribution of the components was studied by thin-layer chromatography and densitometry. The concentration and the ratio of alkaloid components in extracts are significantly different depending on the extraction method. The solution obtained by supercritical fluid extraction contains coptisine and chelidonine, while berberine could be obtained by microwave extraction only. Extracts with high coptisine content were obtained by supercritical fluid extraction, followed by pressing and microwave extraction. Mineral element content of the drug and extracts was also determined by inductively coupled plasma atomic emission spectrometry. Element content (Na, Ca, Fe) was found to be highest in microwave extracts.     

GC-MS Identification and GC-FID Quantitation of Terpenoids in Ononidis spinosae Radix

A GC-MS method has been developed for the qualitative analysis of sterol and triterpene (terpenoid) constituents of Ononis spinosa L. (spiny restharrow) root without derivatization. β-Sitosterol, campesterol, stigmasterol, stigmastan-3,5-diene sterol compounds and the triterpene derivatives β-amyrin and α-onocerin were identified. A validated GC-FID quantitative method was developed for measuring β-sitosterol, the main sterol component, in various extracts of this plant, obtained with organic solvents and by supercritical fluid extraction. The extracts were cleaned by saponifying and then the β-sitosterol was quantified in the non-saponifiable fractions by GC-FID with an internal standard. In addition, the relative concentrations of the other terpenoids were also determined. The β-sitosterol content in the non-saponifiable solvent extraction fractions was 0.19–5.5%, that of the supercritical fractions were 4.8–9.2%, depending on the experimental conditions. The hexane and the pilot scale SFE extracts were considered as main sources of terpenoids (71.8%; 93.3%, respectively).     

Supercritical extraction of thyme (Thymus vulgaris L.)

Summary Chromatographic methods for the qualitative and quantitative analysis of thyme (Thymus vulgaris L.) extracts (essential oil obtained by steam distillation and extracts obtained by carbon dioxide supercritical fluid extraction and methylene chloride) are described. The composition of extracts obtained at different pressures (from 80 bar to 400 bar) and constant temperature (40°C) is discussed. The extraction system thyme— supercritical carbon dioxide was modelled by empirical equations defining the dependence of the total extract (TE) solubility and thymol solubility in CO₂ on the density of carbon dioxide.     

Comparison of various extraction techniques for isolation and determination of isoflavonoids in plants

In the present paper, the following extraction techniques have been used for extracting isoflavonoids from the species Matricaria recutita, Rosmarinus officinalis, Foeniculum vulgare, and Agrimonia eupatoria L.: supercritical fluid extraction (SFE), pressurized fluid extraction, matrix solid phase dispersion, ultrasonic extraction in an ultrasonic bath (USE) and by means of an ultrasonic homogeniser (HOM), extraction by means of Soxhlet apparatus (SOX), and solid phase extraction. Experimental optimization of all techniques has been carried out using a soybean flour. Subsequent analyses of the extracts were carried out by liquid chromatography with UV detection. The maximum yields of daidzein and genistein were obtained by extraction with the SOX, USE, and HOM techniques. The maximum yields of apigenin and biochanin A from herb samples were obtained by SFE.     

Modeling of supercritical fluid extraction of flavonoids from Calycopteris floribunda leaves

The aim of this study was to obtain flavonoids extracts from Calycopteris floribunda leaves using supercritical fluid extraction (SFE) with CO₂ and a co-solvent. Pachypodol, a potential anticancer drug lead compound separated from the extracts, was examined. Classical organic solvent extraction (CE) with ethanol was performed to evaluate the high pressure method. HPLC analysis was introduced to interpret the differences between SFE and CE extracts in terms of antioxidant activity and the concentration of pachypodol. SFE kinetics and mathematical modeling of the overall extraction curves (OEC) were investigated. Evaluation of the models against experimental data showed that the Sovová model performs the best. The supercritical fluid extraction process was optimized using a central composite design (CCD), where temperature and pressure were adjusted. The optimal conditions of SFE were: pressure of 30 MPa and temperature of 35°C.     

SFE of rosemary oil: Assessment of the influence of process variables and extract characterization

The effects of temperature and solvent density on the characteristics of the extracts obtained from supercritical fluid extraction (SFE) of the volatile compounds from Brazilian rosemary (Rosamarinus officianalis L., Labiatae) using carbon dioxide (CO₂) as solvent were investigated. The experiments were performed in a semi-batch laboratory scale home-made unit at two temperatures, viz. 310.15 and 320.15 K, over the pressure range of 100-160 bar. This study allowed to determine the crossover point and the maximum solubility of the oil. The products were analyzed by HRGC-MS, and compounds grouped in three different classes according to their molecular mass distribution in order to evaluate the influence of the variables studied on the characteristics of the extracts. The model proposed by Sovová was adopted in an attempt to interpret the mass transfer phenomena in the extraction process.     

Green processes for the extraction of bioactives from Rosemary: Chemical and functional characterization via ultra-performance liquid chromatography-tandem mass spectrometry and in-vitro assays

In this contribution, the performance of three different extraction procedures towards the extraction of antioxidants from rosemary (Rosmarinus officinalis) is presented. Namely, pressurized liquid extraction (PLE), using water and ethanol as solvents, supercritical fluid extraction (SFE), using neat CO₂ and supercritical CO₂ modified with ethanol, as well as a novel extraction process called Water Extraction and Particle formation On-line (WEPO) are directly compared. Different extraction conditions including temperatures, times and pressures have been studied. The produced extracts have been characterized in terms of extraction yield, antioxidant activity (using the DPPH radical scavenging method) and total phenols (using the Folin method). Besides, all the extracts have been chemically characterized using a new quantitative UPLC-MS/MS method. This method allowed the determination of the main antioxidants present in rosemary, including, among others, rosmarinic acid, carnosic acid and carnosol, attaining detection limits as low as 2 ng/mL. The results obtained in this study show that PLE using ethanol at high temperatures (200 °C) was able to produce extracts with high antioxidant activity (EC₅₀ 8.8 μg/mL) and high yield (ca. 40%) while efficiently extracting antioxidants of diverse polarity, among them, carnosic and rosmarinic acids, regarded as the most important antioxidants present in rosemary. Nevertheless, in this work, the ability of the three studied environmentally friendly extraction techniques to obtain bioactives from natural sources is demonstrated.     

Comparison of techniques for the extraction of the hypotensive drugs geniposidic acid and geniposide from Eucommia ulmoides

The extraction of geniposidic acid and geniposide from Eucommia ulmoides was carried out by Soxhlet extraction, enzyme-assisted aqueous extraction, semi-bionic extraction and supercritical fluid extraction techniques and the extracts were analyzed by high performance liquid chromatography. The results showed that the best methods for extraction of geniposidic acid and geniposide are enzyme-assisted aqueous extraction and semi-bionic extraction. Compared with various extraction methods, the semi-bionic extraction was more efficient, yielding the highest amount of geniposidic acid and geniposide. In semi-bionic extraction, no organic solvent was used. Semi-bionic extraction was performed at the same pH as that of the human body. The results indicated that semi-bionic extraction was the best method for large-scale efficient extraction of geniposidic acid and geniposide from plant materials. The supercritical fluid extraction process required more time than the other methods, and gave the lowest yields of geniposidic acid and geniposide.     

Bixin extraction from defatted annatto seeds

Bixin is the major carotenoid in the seed of the Annatto plant (Bixa orellana L.). The aim of this study was to obtain extracts containing bixin from seeds that had been partially defatted by supercritical fluid extraction. Pressurized liquid extraction (PLE) and low-pressure solvent extraction (LPSE) methods were used, and the effects of the solvent, temperature, pressure, solvent mass to feed mass (S/F) ratio and ultrasonication were evaluated for the global yield (X₀(%)) and the bixin yield (BY(%)). Extraction conditions producing high yields of bixin were established for both the PLE and LPSE methods. Analysis of variance was used to examine the influence of the individual extraction variables in LPSE and PLE. For LPSE; significant effects were found for solvent, temperature, and the interactions of temperature with solvent and temperature with S/F. Solvent was the only variable that significantly affected X₀(%) and BY(%), for PLE. While ultrasonication did not significantly affect X₀(%) or BY(%), scanning electron microscopy analysis revealed structural changes in the vegetal matrix following this treatment.     

Supercritical Fluid Extraction of Phenolic Compounds from Mango (Mangifera indica L.) Seed Kernels and Their Application as an Antioxidant in an Edible Oil

Phenolic compounds from mango (M. indica) seed kernels (MSK) var. Sugar were obtained using supercritical CO₂ and EtOH as an extraction solvent. For this purpose, a central composite design was carried out to evaluate the effect of extraction pressure (11–21 MPa), temperature (40–60 °C), and co-solvent contribution (5–15% w/w EtOH) on (i) extraction yield, (ii) oxidative stability (OS) of sunflower edible oil (SEO) with added extract using the Rancimat method, (iii) total phenolics content, (iv) total flavonoids content, and (v) DPPH radical assay. The most influential variable of the supercritical fluid extraction (SFE) process was the concentration of the co-solvent. The best OS of SEO was reached with the extract obtained at 21.0 MPa, 60 °C and 15% EtOH. Under these conditions, the extract increased the OS of SEO by up to 6.1 ± 0.2 h (OS of SEO without antioxidant, Control, was 3.5 h). The composition of the extract influenced the oxidative stability of the sunflower edible oil. By SFE it was possible to obtain extracts from mango seed kernels (MSK) var. Sugar that transfer OS to the SEO. These promissory extracts could be applied to foods and other products.     

Comparison of supercritical fluid and ultrasound-assisted extraction of carotenoids and chlorophyll a from Dunaliella salina

In the work described here the extraction processes of carotenoids and chlorophylls were analysed using two extraction techniques, namely ultrasound-assisted extraction and supercritical fluid extraction, and the results are compared. The solvents used for the ultrasound-assisted extraction were N,N′-dimethylformamide and methanol and for the supercritical fluid extraction, carbon dioxide. The raw material studied was Dunaliella salina, a microalgae characterized by the high levels of carotenoids present in its cellular structure. The results indicate that the supercritical fluid extraction process is comparable to the ultrasound-assisted extraction when methanol is used as solvent. In addition, the supercritical extraction process is more selective for the recovery of carotenoids than the conventional technique since it leads to higher values for the ratio carotenoids/chlorophylls. Finally, the effects of pressure and temperature on the extraction yields of the supercritical fluid extraction process were studied.     

Effect of CO2 Flow Rate on the Extraction of Astaxanthin and Fatty Acids from Haematococcus pluvialis Using Supercritical Fluid Technology

Haematococcus pluvialis is the largest producer of natural astaxanthin in the world. Astaxanthin is a bioactive compound used in food, feed, nutraceutics, and cosmetics. In this study, astaxanthin extraction from H. pluvialis by supercritical fluid extraction was evaluated. The effects of temperature (40 and 50 °C), pressure (40 and 50 MPa), and CO₂ flow rate (2 and 4 L/min) were investigated. The results showed that the highest astaxanthin recovery was obtained at 50 °C/50 MPa and the CO₂ flow rates evaluated had no significant effect. It was possible to achieve astaxanthin recoveries of 95% after 175 min for a CO₂ flow rate of 2 L/min, and 95 min for CO₂ flow rate of 4 L/min. The ω-6/ω-3 ratios obtained were similar in all conditions, reaching 0.87, demonstrating that the extracts from H. pluvialis by SFE are rich in unsaturated fatty acids (UFA) which increases their positive effects when used as a functional ingredient in food.     

Molecular Distillation of Lavender Supercritical Extracts: Physicochemical and Antimicrobial Characterization of Feedstocks and Assessment of Distillates Enriched with Oxygenated Fragrance Components

Lavandula angustifolia is one of the most widely cultivated non-food crops used in the production of essential oil; it is used in perfumery, aromatherapy, pharmaceutical preparations, and food ingredients. In this study, supercritical fluid extraction (SFE) and molecular distillation (MD) were combined, primarily to enrich scCO₂ extracts with lavender oxygenated monoterpenes, avoiding thermal degradation, hydrolysis, and solvent contamination, and maintaining the natural characteristics of the obtained oils. Molecular distillation was developed for the first time for the extraction of crucial lavender fragrance ingredients, i.e., from two scCO₂ extracts obtained from dry flower stems of lavender cultivated in Poland and Bulgaria. The best results for high-quality distillates were obtained at 85 °C (EVT) and confirmed that linalyl acetate content increased from 51.54 mg/g (initial Bulgarian lavender extract, L-Bg-E) and 89.53 mg/g (initial Polish lavender extract, L-Pl-E) to 118.41 and 185.42 mg/g, respectively, corresponding to increases of 2.3 and 2.1 times in both distillate streams, respectively. The distillates, light oils, and extracts from lavender were also evaluated for their antimicrobial properties by determining the minimum inhibitory concentration (MIC) by the broth microdilution method. Generally, Gram-positive bacteria and Candida spp. were more sensitive to all distilled fractions and extracts than Escherichia coli (Gram-negative bacteria).     

Isolation and determination of saponin hydrolysis products from Medicago sativa using supercritical fluid extraction,solid‐phase extraction and liquid chromatography with evaporative light scattering detection

Saponins are widespread secondary metabolites with various beneficial properties: fungicidal, antibacterial, antiviral, and anticancer. Alfalfa saponin molecules contain mainly: medicagenic acid, hederagenin, bayogenin, and soyasapogenol B. Structural diversity of saponins makes their determination in Medicago sativa extracts very difficult. The most popular determination technique is high‐performance liquid chromatography applied with evaporative light scattering detection. Qualitative and quantitative analysis of sapogenins from Medicago sativa by high‐performance liquid chromatography with evaporative light scattering detection required hydrolysis and purification of extracts obtained by supercritical fluid extraction. Hydrolysis of saponins with concentrated hydrochloric acid provided high concentration of medicagenic acid. In the purification process, satisfactory results were obtained for solid‐phase extraction using octadecyl. Recoveries were from 71 to 99% with a standard deviation from 2 to 8. Hydrolysis with concentrated hydrochloric acid was the only method that allowed identification of all four analyzed sapogenins. Moreover, it is characterized by a short time of preparation, simplicity of execution, a small amount of the sample and solvents. The hydrolysis and purification methods coupled with high‐performance liquid chromatography and evaporative light scattering detection can be successfully used for qualitative and quantitative analysis of the main saponins present in Medicago sativa plant extracts obtained by supercritical fluid extraction.     

Study on the stability of supercritical fluid extracted rosemary (<Emphasis Type="Italic">Rosmarinus offcinalis</Emphasis> L.) essential oil

The aim of this study was to examine the influence of storage conditions and duration on composition and antioxidant activity of supercritical fluid (SCF) extracted essential oil of rosemary (Rosmarinus officinalis L.). Supercritical extraction was carried out sequentially by using SCF carbondioxide in the first two steps and with 5% ethanol as entrainer in the third step. The compositions of the extracts were determined by gas chromatography/mass spectrometry. The total phenolics were analyzed using Folin-Ciocalteau assay. Antioxidant activities of the extracts were tested by β-carotene-linoleic acid bleaching method.     

Preparative isolation and purification of hainanmurpanin,meranzin, and phebalosin from leaves of Murraya exotica L. using supercritical fluid extraction combined with consecutive high‐speed countercurrent chromatography

The objective of this study was to develop a consecutive preparation method for the isolation and purification of hainanmurpanin, meranzin, and phebalosin from leaves of Murraya exotica L. The process involved supercritical fluid extraction with CO₂, solvent extraction, and two‐step high‐speed countercurrent chromatography. Pressure, temperature, and the volume of entrainer were optimized as 27 MPa, 52°C, and 60 mL by response surface methodology in supercritical fluid extraction with CO₂, and the yield of the crude extracts was 7.91 g from 100 g of leaves. Subsequently, 80% methanol/water was used to extract and condense the three compounds from the crude extracts, and 4.23 g of methanol/water extracts was obtained. Then, a two‐step high‐speed countercurrent chromatography procedure was developed for the isolation of the three target compounds from methanol/water extracts, including conventional high‐speed countercurrent chromatography for further enrichment and consecutive high‐speed countercurrent chromatography for purification. The yield of concentrates from high‐speed countercurrent chromatography was 2.50 g from 4.23 g of methanol/water extracts. Finally, the consecutive high‐speed countercurrent chromatography produced 103.2 mg of hainanmurpanin, 244.7 mg of meranzin, and 255.4 mg of phebalosin with purities up to 97.66, 99.36, and 98.64%, respectively, from 900 mg of high‐speed countercurrent chromatography concentrates in one run of three consecutive sample loadings without exchanging a solvent system.     

GC–MS Studies of Thiophenes in the Supercritical Fluid CO2 and Solvent Extracts of Tagetes patula L.

The intact plant parts and genetically modified hairy root clone #TpA6 of Tagetes patula were extracted with supercritical fluid CO₂ extraction (SFE) and a conventional solvent extraction. SFE optimization included the variation of fluid CO₂ pressure, dynamic time, and the addition of methanol modifier co-solvent. The four characteristic thiophene metabolites, 5-(3-buten-1-ynyl)-2,2′-bithienyl (BBT), 2,2′:5′,2″-terthiophene (α-T), 5-(4-acetoxy-1-butynyl)-2,2′-bithienyl (BBTOAc), and 5-(3,4-diacetoxy-1-butynyl)-2,2′-bithienyl [BBT(OAc)₂], were analysed by GC–MS. The proposed SFE method allowed the selective extraction of thiophenes in 60 min dynamic time with supercritical CO₂ without modifier co-solvent, at 30 MPa and 40 °C. The SFE and the reference solvent extraction yielded similar results. The SFE of intact roots and flowers yielded 717 ± 31.3 and 480 ± 26.6 μg g^?1 α-T, respectively, while the leaves did not contain considerable amounts of thiophenes. Remarkable amounts of BBT, BBTOAc, and BBT(OAc)₂ were characteristic of the SFE of hairy root cultures.     

Assessment of Conventional Solvent Extraction vs. Supercritical Fluid Extraction of Khella (Ammi visnaga L.) Furanochromones and Their Cytotoxicity

Background: Khella (Ammi visnaga Lam.) fruits (Apiaceae) are rich in furanochromones, mainly khellin and visnagin, and are thus incorporated in several pharmaceutical products used mainly for treatment of renal stones. Methods: The objective of this study was to compare the yield of khellin and visnagin obtained using different conventional solvents and supercritical fluid extraction (SCFE) with carbon dioxide (containing 5% methanol as co-solvent). Water, acetone and ethanol (30% and 95%) were selected as conventional solvents. Results: Highest extract yield was obtained from 30% ethanol (15.44%), while SCFE gave the lowest yield (4.50%). However, the percentage of furanochromones were highest in SCFE (30.1%), and lowest in boiling water extract (5.95%). HPLC analysis of conventional solvent extracts showed other coumarins that did not appear in supercritical fluid extraction chromatogram due to non-selectivity of solvent extraction. Ammi visnaga extracts as well as standard khellin and visnagin were tested for their cytotoxic activity using sulforhodamine B assay on breast cancer (MCF-7) and hepatocellular carcinoma (Hep G2) cell lines. Results revealed a strong cytotoxic activity (IC₅₀ < 20 µg/mL) for the SCFE and standard compounds (khellin and visnagin) (IC₅₀ ranging between 12.54 ± 0.57 and 17.53 ± 1.03 µg/mL). However, ethanol and acetone extracts had moderate cytotoxic activity (IC₅₀ 20–90 µg/mL) and aqueous extract had a weak activity (IC₅₀ > 90 µg/mL). Conclusions: Thus, supercritical fluid extraction is an efficient, relatively safe, and cheap technique that yielded a more selective purified extract with better cytotoxic activity.     

Comparison of supercritical fluid and Soxhlet extractions for the quantification of hydrocarbons from Euphorbia macroclada

This study compares conventional Soxhlet extraction and analytical scale supercritical fluid extraction (SFE) for their yields in extracting of hydrocarbons from arid-land plant Euphorbia macroclada. The plant material was firstly sequentially extracted with supercritical carbon dioxide, modified with 10% methanol (v/v) in the optimum conditions that is a pressure of 400 atm and a temperature of 50 °C and then it was sonicated in methylene chloride for an additional 4 h. E. macroclada was secondly extracted by using a Soxhlet apparatus at 30 °C for 8 h in methylene chloride. The validated SFE was then compared to the extraction yield of E. macroclada with a Soxhlet extraction by using the Student’s t-test at the 95% confidence level. All of extracts were fractionated with silica-gel in a glass column to get better hydrocarbon yields. Thus, the highest hydrocarbons yield from E. macroclada was achieved with SFE (5.8%) when it compared with Soxhlet extractions (1.1%). Gas chromatography (GC) analysis was performed to determine the quantitative hydrocarbons from plant material. The greatest quantitative hydrocarbon recovery from GC was obtained by supercritical carbon dioxide extract (0.6 mg g⁻¹).     

Extraction of volatile oil from aromatic plants with supercritical carbon dioxide: experiments and modeling

An overview of the studies carried out in our laboratories on supercritical fluid extraction (SFE) of volatile oils from seven aromatic plants: pennyroyal (Mentha pulegium L.), fennel seeds (Foeniculum vulgare Mill.), coriander (Coriandrum sativum L.), savory (Satureja fruticosa Béguinot), winter savory (Satureja montana L.), cotton lavender (Santolina chamaecyparisus) and thyme (Thymus vulgaris), is presented. A flow apparatus with a 1 L extractor and two 0.27 L separators was built to perform studies at temperatures ranging from 298 to 353 K and pressures up to 30.0 MPa. The best compromise between yield and composition compared with hydrodistillation (HD) was achieved selecting the optimum experimental conditions of extraction and fractionation. The major differences between HD and SFE oils is the presence of a small percentage of cuticular waxes and the relative amount of thymoquinone, an oxygenated monoterpene with important biological properties, which is present in the oils from thyme and winter savory. On the other hand, the modeling of our data on supercritical extraction of volatile oil from pennyroyal is discussed using Sovová's models. These models have been applied successfully to the other volatile oil extractions. Furthermore, other experimental studies involving supercritical CO₂ carried out in our laboratories are also mentioned.