Comparison of different extraction methods for the extraction of major bioactive flavonoid compounds from spearmint (Mentha spicata L.) leaves

Different bioactive flavonoid compounds including catechin, epicatechin, rutin, myricetin, luteolin, apigenin and naringenin were obtained from spearmint (Mentha spicata L.) leaves by using conventional soxhlet extraction (CSE) and supercritical carbon dioxide (SC-CO₂) extraction at different extraction schemes and parameters. The effect of different parameters such as temperature (40, 50 and 60 °C), pressure (100, 200 and 300 bar) and dynamic extraction time (30, 60 and 90 min) on the supercritical carbon dioxide (SC-CO₂) extraction of spearmint flavonoids was investigated using full factorial arrangement in a completely randomized design (CRD). The extracts of spearmint leaves obtained by CSE and optimal SC-CO₂ extraction conditions were further analyzed by high performance liquid chromatography (HPLC) to identify and quantify major bioactive flavonoid compounds profile. Comparable results were obtained by optimum SC-CO₂ extraction condition (60 °C, 200 bar, 60 min) and 70% ethanol soxhlet extraction. As revealed by the results, soxhlet extraction had a higher crude extract yield (257.67 mg/g) comparing to the SC-CO₂ extraction (60.57 mg/g). Supercritical carbon dioxide extract (optimum condition) was found to have more main flavonoid compounds (seven bioactive flavonoids) with high concentration comparing to the 70% ethanol soxhlet extraction (five bioactive flavonoids). Therefore, SC-CO₂ extraction is considered as an alternative process compared to the CSE for obtaining the bioactive flavonoid compounds with high concentration from spearmint leaves.     

Biodiesel production from supercritical carbon dioxide extracted Jatropha oil using subcritical hydrolysis and supercritical methylation

This study investigates supercritical carbon dioxide (SC-CO₂) extraction of triglycerides from powdered Jatropha curcas kernels followed by subcritical hydrolysis and supercritical methylation of the extracted SC-CO₂ oil to obtain a 98.5% purity level of biodiesel. Effects of the reaction temperature, the reaction time and the solvent to feed ratio on free fatty acids in the hydrolyzed oil and fatty acid esters in the methylated oil via two experimental designs were also examined. Supercritical methylation of the hydrolyzed oil following subcritical hydrolysis of the SC-CO₂ extract yielded a methylation reaction conversion of 99%. The activation energy of hydrolysis and trans-esterified reactions were 68.5 and 45.2 kJ/mole, respectively. This study demonstrates that supercritical methylation preceded by subcritical hydrolysis of the SC-CO₂ oil is a feasible two-step process in producing biodiesel from powdered Jatropha kernels.     

Supercritical carbon dioxide and solvent extraction of 2-acetyl-1-pyrroline from Pandan leaf: The effect of pre-treatment

Pandan (Pandanus amaryllifolius Roxb.) leaf is a source of natural flavoring widely used in South-east Asia. The major compound contributing to the characteristic flavor of Pandan is 2-acetyl-1-pyrroline (2AP). This highly volatile compound also contributes significantly to the flavor of aromatic rice such as basmati and jasmine rice. As the consumer requirement for use of natural flavors, extraction of components from natural sources has been sought. In this study, supercritical carbon dioxide (SC-CO₂) and solvent extraction of components from Pandan leaves were performed. Experimental parameters included particle size and drying method (oven and freeze drying). Results indicated that the initial value of moisture content and particle size of Pandan leaves had the greatest effect on the total yield and 2AP concentration of the extracts. Almost 80% of water in Pandan leaves can be removed by drying. Yields of supercritical extracts were 10 times lower when compared to the hexane extracts. The total yield of extracts was increased up to 50% with decreasing particle size of Pandan leaves. Extraction of coarsely ground freeze-dried Pandan leaves by SC-CO₂ obtained the highest yield (0.88 ± 0.06%) followed by oven dried (0.38 ± 0.09%) and fresh leaves (0.34 ± 0.01%). The 2AP was identified by GC-MS and analyzed by GC-FID. Supercritical and hexane extracts of pre-treated Pandan leaves were found to have a small quantity of 2AP ranging between 0.04 ± 0.01 and 0.45 ± 0.01 ppm. Grinding pre-treatment was the best method for both SC-CO₂ and hexane extractions while the freeze drying method was the best for SC-CO₂.     

Preparation of PEGylated liposomes of docetaxel using supercritical fluid technology

The aims of this investigation were to develop a procedure to prepare and characterize docetaxel encapsulated PEGylated liposomes using supercritical carbon dioxide as antisolvent and optimize the process and formulation variables using response surface methodology. The process and formulation variables were optimized by “Box Behnken Design (BBD)” of response surface methodology (RSM) with temperature, pressure and CO₂ flow rate as independent variables and particle size and yield as dependent variables for process variables while the amount of hydrogenated soya phosphotidylcholine (HSPC), soya phosphotidylcholine (SPC) and cholesterol as independent variables and % entrapment efficiency as dependent variables for optimization of formulation variables. The optimized liposomal formulation was characterized for particle size, shape, morphology, crystallinity, zeta potential, % entrapment efficiency, residual solvent content and in vitro drug release. The process and formulation variables were optimized to achieve maximum drug entrapment efficiency and yield. Results for particle size, zeta potential, % entrapment efficiency and in vitro drug release of PEGylated liposomes were found to be 269.2 ± 2.8 nm, −27.2 ± 1.8 mV, 79.2 ± 4.4 %w/w and 37.5 ± 4.5% in 24 h, respectively. The liposomes were found to be small, unilamellar and spherical with smooth surface as seen in photomicrographs of scanning electron microscopy and transmission electron microscopy. Differential scanning calorimetry and X-ray diffraction results indicated a molecular dispersion of docetaxel in the liposomes. In this study, supercritical fluid technology was successfully used to prepare small, spherical and unilamellar liposomes of docetaxel with high entrapment. It was observed that supercritical fluid technique can be an excellent technique for preparation of docetaxel liposomes which otherwise is very difficult to prepare as a solvent free and stable liposomes.     

Guava (Psidium guajava L.) seed oil obtained with a homemade supercritical fluid extraction system using supercritical CO2 and co-solvent

In this work we designed and built a homemade supercritical fluid extraction (HM-SFE) system, in which pure CO₂ and CO₂ with co-solvents were used. The HM-SFE was made by means of thermal dilatation-contraction (TDC). This HM-SFE system was used for obtaining guava (Psidium guajava L.) seed oil, using supercritical CO₂ adding ethanol as co-solvent (CO₂ SC/EtOH), extractions were performed at 313 K and different pressures (10, 20 and 30 MPa), each one in four stages of 30 min, the extract with higher yield was subjected to transesterification and high-resolution gas chromatography (HRGC) analysis. The highest extraction yield was obtained at 30 MPa (17.30% w/w), this yield was higher than one observed in a previous work using SC-CO₂, and near to the one obtained by Soxhlet extraction (20.2% w/w). HRGC enabled the identification of components of the derivatized extract as methyl esters of palmitic, oleic, linoleic, and stearic fatty acids. The results obtained with HM-SFE system was compared with a commercial SFE system, obtained very similar results. In this work was possible to construct a low cost and simple manner HM-SFE system which was employed for obtaining guava seed oil, using CO₂ SC/EtOH.     

Supercritical fluids extraction and anti-solvent purification of carotenoids from microalgae and associated bioactivity

This study investigated co-solvent modified supercritical carbon dioxide extraction of lipids and carotenoids from the microalgal species of Nannochloropsis oculata. Supercritical carbon dioxide (SCCO₂) anti-solvent precipitation of carotenoids from the extracts following purification of Zeaxanthin was also examined. Continuous modification by ethanol of supercritical carbon dioxide extractions showed that the addition ratio was important for extraction efficiency of lipids and carotenoids. SCCO₂ extraction at 350 bar, 323 K and 16.7 wt% of ethanol addition yielded 239.7 mg of triglycerides and 7.61 mg of carotenoids per gram extract with a total yield of 15.5%. SCCO₂ anti-solvent experiments showed that the content of Zeaxanthin in the precipitate was greater than that in the fraction of normal phase column chromatography. The purest Zeaxanthin (93.8%) was then successfully isolated from the purified fraction by using a reverse-phase HPLC column chromatography. Rat macrophages treated by ultra-sonicated water extracts of the SCCO₂ defatted algae showed a positive phagocytotic activity.     

Supercritical antisolvent precipitation of andrographolide from Andrographis paniculata extracts: Effect of pressure, temperature and CO2 flow rate

Andrographis paniculata extracts were precipitated using the so-called supercritical antisolvent (SAS) technique. Ethanol was used as the solvent and compressed CO₂ as the antisolvent. The effects of process operating conditions (pressure: 5-24 MPa, temperature: 308-328 K and CO₂ flow rate: 0.5-1.5 g/min) on particle size and morphology of precipitated andrographolide were evaluated. X-ray diffraction (XRD) patterns showed significant changes in andrographolide morphology depending on process operating conditions; both column-like and slice-like crystals were observed depending on operating conditions. Crystals with mean diameters of 3.30-228.35 μm were produced, smaller crystals were obtained at high pressure, low temperature and high CO₂ flow rate and vice versa for large crystals. In addition, SAS process also produced high precipitation yields, since solubility of andrographolide is small in the supercritical CO₂ plus ethanol. When operating under subcritical conditions, amorphous particles were produced.     

Ethanol modified supercritical carbon dioxide extraction of flavonoids from Momordica charantia L. and its antioxidant activity

Ethanol modified supercritical carbon dioxide (SC-CO₂) extraction of flavonoids from Momordica charantia L. fruits and its antioxidant activity were performed. The influences of parameters such as temperature, extraction time and pressure on the yield of flavonoids were investigated. The antioxidant activities of flavonoids were assessed by means of 2,2-diphenyl-1-picrylhydrazyl (DPPH) radical-scavenging assay and β-carotene bleaching test. The experimental data obtained indicated that pressure, temperature and time had significant effect on the extraction yield. The optimum extraction conditions, determined by the 3D response surface and contour plots derived from the mathematical models, were as follows: extraction temperature 46 °C, pressure 33.4 MPa, and extraction time 53.2 min. Under these conditions, the experimental value was 15.47 mg/g, which was well matched with value predicted by the model. The antioxidant activity of flavonoids obtained by ethanol modified SC-CO₂ extraction method had higher antioxidant activity than the flavonoids extracted by conventional solvent extraction (CSE) method. The DPPH radical-scavenging ability of flavonoids obtained by ethanol modified SC-CO₂ extraction method reached to 96.14 ± 1.02%, equivalent to the clearance rate of ascorbic acid at 1.2 mg/mL. Results indicated that ethanol modified SC-CO₂ extraction was a suitable approach for the selective extraction of flavonoids from M. charantia L.     

Extraction of biologically active compounds from Sideritis ssp. L.

In this study extraction of polyphenols and flavonoids from cultivated hybrid Sideritis scardica × Sideritis syriaca, known for its rich content of phenolics and flavonoids with antioxidant activity, was investigated. Extractions have been done by ethanol and water-ethanol, respectively. High equilibrium values of the extracted species were obtained—17.55 mg/(g solid) total phenolics and 5.7 mg/(g solid) total flavonoids with ethanol as solvent. The influence of the solvent on the total yield and the content of biologically active compounds were studied. Maximum polyphenolics and flavonoids extraction was observed for water-ethanol solvent ratio 20/80. Increase of the content of ethanol in the solvents led to lower total yield of extracts but higher percentage of polyphenolics. The extraction kinetics showed that 90% of the phenolic compounds were extracted during the first 2.5 h. The experimental kinetics was described by a constant effective diffusion coefficient D_e = 1.5 × 10⁻¹² m²/s in the solid, accounting for the actual particle size distribution.     

Micronization and characterization of drug substances by RESS with supercritical CO2

A RESS (rapid expansion of supercritical solution) process for the preparation of ultra-fine drug particles with no organic solvent has been developed with supercritical CO₂. Three drug substances with different solubility in supercritical CO₂ were used, and orifice disks and capillary tubes were adapted as an expansion device. The solubilities of drug substances in supercritical CO₂ and the effects of various operating parameters on the characteristics of the particles prepared by RESS process were experimentally investigated. The solubility of the drug substance in supercritical CO₂ had a major effect on the average diameter of the particle prepared by RESS process, and the particle diameter decreased with the solubility for all the drugs and operating conditions. The particle diameter also decreased with preexpansion temperature and increased with the hole diameter of the orifice nozzle and aspect ratio (L/D) of the capillary tube.     

Supercritical anti-solvent micronization of marigold-derived lutein dissolved in dichloromethane and ethanol

This work aims to study supercritical anti-solvent (SAS) micronization of lutein derived from marigold flowers. Lutein solution in dichloromethane (DCM) or ethanol was atomized into the stream of supercritical carbon dioxide (SC-CO₂) through a concentric nozzle in a pressurized vessel. The effects of pressure and SC-CO₂ flow rate on morphology, mean particle size (MPS) and particle size distribution (PSD) were investigated. The reduction in lutein MPS from 202.3 μm of unprocessed lutein to 1.58 μm and 902 nm could be achieved by SAS micronization using DCM and ethanol, respectively. In both solvent systems, no significant effects of pressure and SC-CO₂ flow rate on particle morphology were observed. However, pressure was found to have a significant effect on MPS and PSDs of lutein particles.     

Extraction of the essential oil of abajeru (Chrysobalanus icaco) using supercritical CO2

Abajeru (Chrysobalanus icaco) is a plant that has hypoglycemic properties and is often used in Brazilian popular medicine. In order to identify the flavoring and hypoglycemic substances present in this plant, this work has an objective for the extraction of the essential oil presented in the leaves of abajeru using the supercritical fluid extraction (SFE). The supercritical solvent used is CO₂, because of its moderate critical temperature and pressure, atoxicity, low cost and volatility. The experiments were conducted using dried leaves in an apparatus containing a high-pressure pump, a stainless steel extractor of 42 mL of volume and a micrometric valve for sampling. Different operational conditions were tested, varying mainly the temperature (313.15-353.15 K) and the pressure (10.5-20 kPa) in order to investigate the efficiency of the process. The results showed that the best operational condition was at 20 kPa and 353.15 K. To compare the supercritical carbon dioxide results, the essential oil was also extracted by hydrodistillation and soxhlet, using ethanol as solvent. The chromatographic analysis showed that the different technologies studied extracted the same classes of compounds but the SFE obtained the extract with potential hypoglycemic activity with the presence of lupenol.     

Reactivity of α-pinene epoxide in supercritical solvents

Thermal transformations of α-pinene epoxide in composite supercritical solvents that contain CO₂, lower alcohols (ethanol, isopropyl alcohol) and water were studied in the temperature range of 387-575 K at pressure 13.5-21.5 MPa. Campholenic aldehyde and carveol were shown to be the main products of α-pinene epoxide reactions in supercritical solvents containing water. In the absence of water, thermolysis of α-pinene epoxide in supercritical solvent yields campholenic aldehyde and pinocamphone, with their total amount in the reaction mixture attaining 80%. Suggestions were made on the mechanism of α-pinene epoxide thermal isomerization depending on acidity of supercritical solvent.     

Supercritical impregnation of lavandin (Lavandula hybrida) essential oil in modified starch

Lavandin (Lavandula hybrida) essential oil contains components with biocide properties that can be used as substitutes of synthetic drugs in livestock. This application requires an appropriate formulation of the essential oil. In this work, supercritical impregnation of lavandin oil has been proposed as a possible formulation process, due to the high solubility of lavandin essential oil in supercritical carbon dioxide. The polymer used in this work as carrier material was starch modified with the n-octenil succinate (OSA) group, in the form of powder with a particle size of 30 μm. The effects of operational pressure (10-12 MPa), temperature (313-323 K) and lavandin oil to starch mass ratio (0.2-1) were studied. Impregnation loads ranging from 25 to 150 mg lavandin oil/g OSA-starch were obtained. The distribution coefficient of essential oil between the starch and the supercritical phase as well as the essential oil load depended on the density of CO₂.     

Preparation of submicron-sized RDX particles by rapid expansion of solution using compressed liquid dimethyl ether

Cyclotrimethylenetrinitramine (RDX) was precipitated to submicron-sized particles with spherical morphology by the rapid expansion from supercritical solution (RESS). Compressed liquid dimethyl ether (DME) was used as a solvent for the RDX. This study examined the influence of extraction temperature (293-333 K), extraction pressure (8-20 MPa) and size of orifice nozzle (50, 100, 200, and 250 μm) on the size and morphology of the RDX particles in the RESS process. The precipitated RDX particles were characterized by using the following instruments: field emission scanning electron microscope (FE-SEM), image analyzer, powder X-ray diffraction (PXRD), Fourier transform infrared (FT-IR) spectroscopy, and differential scanning calorimetry (DSC). The precipitated RDX particles showed granular and spherical morphologies, submicron-sized particles, and narrow particle size distributions. The mean particle size of the precipitated RDX ranged from 2.48 to 0.36 μm, and the crystallinity of the precipitated RDX decreased. The enthalpy change for the exothermic decomposition of the precipitated RDX (ΔH = 714.4 J/g) was much higher than that of the original RDX (ΔH = 381.5 J/g).     

Supercritical carbon dioxide extraction of cottonseed with co-solvents

Extraction of cottonseed lipids with supercritical carbon dioxide (SC-CO₂) was conducted with and without a cosolvent, ethanol or 2-propanol (IPA). At 7000 psi and 80°C, the reduced pressure, temperature and density of SC-CO₂ was at 6.5, 1.17 and 1.85, respectively; the specific gravity was 0.87. Under these conditions, CO₂ is denser than most liquid extraction agents such as hexane, ethanol and IPA. The extraction of cottonseed with SC-CO₂ gave a yield of more than 30% (moisture-free basis). This is comparable to yields obtained by the more commonly used solvent, hexane. The crude cottonseed oil extracted by SC-CO₂ was visually lighter than refined cottonseed oil. This was substantiated by colorimetric measurements. No gossypol was detected in the crude oil. However, crude oil extracted by SC-CO₂, to which less than 5% of ethanol or IPA as co-solvent was added, containedca. 200 ppm of gossypol, resulting in the typical dark color of cottonseed crude oil with gossypol. CO₂ extracted a small amount of cottonseed phosphatides, about one-third of that extracted by pure ethanol, IPA or hexane. A second extraction with 100% ethanol or IPA after the initial SC-CO₂ extraction produced a water-soluble lipid fraction that contained a significant amount of gossypol, ranging between 1500 and 5000 ppm. Because pure gossypol is practically insoluble in water, this fraction is believed to be made up of gossypol complexed with polysaccharides and phosphatides. Partially presented at the AOCS 1993 Annual Meeting & Expo in Anaheim, California.     

Study on supercritical extraction of lipids and enrichment of DHA from oil-rich microalgae

The present study aims to isolate the lipids from microalgae by supercritical CO₂ (SC-CO₂) extraction followed by a further enrichment of crude lipids to produce high-purity docosahexenoic acid (DHA) by an urea complexation method. Our systematic approach indicates the optimum conditions of supercritical CO₂ extraction were obtained as follows: 35 MPa, 40 °C, ethanol (95%, v/v) as the co-solvent, and the mass ratio of material to co-solvent 1:1. Under these conditions, 33.9% of lipid yield and 27.5% of DHA content were achieved. Despite the relatively low lipid yield, supercritical CO₂ extraction has exhibited many advantages over the Soxhlet extraction for the DHA enrichment such as high DHA purity and superb product quality. Furthermore, urea complexation method on DHA enrichment considerably increased the DHA purity from 29.7% to 60.4% with an enrichment ratio of 60.6%, under the optimum complexation conditions of urea/fatty acid 2:1, complexation time 8 h, and the complexation temperature of −10 °C.     

Formation of retinyl palmitate-loaded poly(l-lactide) nanoparticles using rapid expansion of supercritical solutions into liquid solvents (RESOLV)

Poly(l-lactide) (PLLA) nanoparticles loaded with retinyl palmitate (RP) were successfully prepared by rapid expansion of a supercritical carbon dioxide (CO₂) solution into an aqueous receiving solution containing a stabilizing agent (RESOLV). Three stabilizing agents, Pluronic F127, Pluronic F68, and sodium dodecyl sulfate (SDS) have been employed and the Pluronic F127 was found to be more effective for stabilizing PLLA/RP nanoparticles than Pluronic F68 and SDS, as RESOLV into a 0.1 wt% Pluronic F127 solution produced a stable nanosuspension consisting mainly of well-dispersed, individual nanoparticles. The effect of rapid expansion processing conditions (i.e., degree of saturation (S), pre-expansion temperature (T_pre), and concentrations of PLLA and RP (C_PLLA, C_RP)) on the particle size, form, and RP loading was systematically investigated. It was found that spherical PLLA/RP nanoparticles with an average size range of ∼40-110 nm and RP loadings of 0.9-6.2 wt% were consistently produced by RESOLV. The size of PLLA/RP nanoparticles increased from ∼30-80 to ∼30-160 nm as the solution degree of saturation changed from S < 1 to S > 1, independent of T_pre, C_PLLA, and C_RP. The entrapment capacity of RP in PLLA nanoparticles was predominantly determined by T_pre and C_RP. Increasing the T_pre from 70 to 100 °C and the C_RP from 0.05 to 0.15 wt% increased the encapsulated RP content at least twofold. Our results show that the technique with benign supercritical CO₂ should be generally applicable to nanoparticle fabrications of other important active ingredients, especially in liquid form, in polymeric nanoparticles.     

Quality of residual oil from palm-pressed mesocarp fiber (Elaeis guineensis) using supercritical CO2 with and without ethanol

The qualities of oils extracted from fresh and dried palm-pressed mesocarp fiber were evaluated. The means of extraction included conventional solvent extraction and supercritical carbon dioxide (SC-CO₂) extraction with and without addition of ethanol. Extraction efficiency using pure SC-CO₂ and the effect of moisture content on efficiency were studied. Minor components, such as vitamin F, carotenoids, squalene and phytosterols, obtained by different methods were compared. The quality of oil recovered from fresh palm-pressed fiber is generally better than that of oil recovered from dried fiber. The SC-CO₂ extraction rate was lower for fresh fiber than for dried fiber. The incorporation of ethanol with SC-CO₂ resulted in oil with higher oxidative stability than did SC-CO₂ alone. Concentrations of minor components and the acylglycerol compositions of the oils extracted from both types of fibers were similar.     

Polymeric microparticles prepared by supercritical antisolvent precipitation

This work has as main objective the development of a model process to obtain microparticles of an acrylate-methacrylate copolymer (Eudragit L100^® and Eudragit EPO^®) using supercritical carbon dioxide (SC-CO₂) as antisolvent (GAS). After studying the behaviour of the copolymers in SC-CO₂ at different operation conditions (pressure, temperature and presence of ethanol (EtOH)), efforts were invested in the optimization of Eudragit EPO^® precipitation from an organic solution using carbon dioxide as antisolvent in batch mode. After loading the precipitation vessel with a fixed quantity of the copolymer dissolution, the SC-CO₂ has been added until the pressure of operation has been reached. Three process parameters, namely, solution nature, presence of surfactants and organic solvent removal step, have been evaluated. Microparticles with mean diameter from about 2 to 12 μm are obtained.