Supercritical CO2 Fluid Extraction Research Articles

P 178 - Rosemary (Rosmarinus officinalis L) extract increases ROS and modulates Nrf2 pathway in human colon cancer cell lines

Carnosic acid, carnosol and betulinic acid are the main compounds present in rosemaryextract (RE) and have shown anti-proliferative effects on various tumor cell lines. The objective of this work was to study the anticancer effects of rosemary extract on different human colon cancer cell lines (HGUE-C-1, HT-29 and SW480). For this purpose, intracellular free radical generation and modulation of nuclear factor erythroid 2 [NF-E2]-related factor 2 (Nrf2) pathway was explored. Cells were treated with RE (20–40 µg/mL) for 24 h at 37 °C and cell viability was determined by MTT assay. Reactive oxygen species (ROS) production was measured with DCF-DA. In addition, cells were transfected with Nrf2-targeting siRNA to determine the potential role of this transcription factor. The inhibition of cell proliferation was correlated to an increase of intracellular ROS content by RE. Furthermore, Nrf2 silencing in siRNA experiments decreased cell viability in all cell lines, then the molecular mechanism seems to involve activation Nrf2/ARE pathway as a response for cell survival at least in this cell line. In conclusion, the antiproliferative effects of a rosemary extract obtained by CO2-supercritical fluid extraction may be related to their pro-oxidative capability by increasing the intracellular generation of ROS, which leads to the activation of Nrf2 as a response for cancer cell survival.

Multi-Dimensional Spectrum-Effect Relationship of the Impact of Chinese Herbal Formula Lichong Shengsui Yin on Ovarian Cancer.

Lichong Shengsui Yin (LCSSY) is an effective and classic compound prescription of Traditional Chinese Medicines (TCMs) used for the treatment of ovarian cancer. To investigate its pharmacodynamic basis for treating ovarian cancer, the multi-dimensional spectrum-effect relationship was determined. Four compositions (I to IV) were obtained by extracting LCSSY successively with supercritical CO2 fluid extraction, 75% ethanol reflux extraction, and the water extraction-ethanol precipitation method. Nine samples for pharmacological evaluation and fingerprint analysis were prepared by changing the content of the four compositions. The specific proportions of the four compositions were designed according to a four-factor, three-level L9(34) orthogonal test. The pharmacological evaluation included in vitro tumor inhibition experiments and the survival extension rate in tumor-bearing nude mice. The fingerprint analyzed by chromatographic condition I (high-performance liquid chromatography-photodiode array detec tor，HPLC-PDA) identified 19 common peaks. High-performance liquid chromatography-photodiode array detector-Evaporative Light-scattering Detector (HPLC-PDA-ELSD )hyphenated techniques were used to compensate for the use of a single detector, and the fingerprint analyzed by chromatographic condition II identified 28 common peaks in PDA and 23 common peaks in ELSD. Furthermore, multiple statistical analyses were utilized to calculate the relationships between the peaks and the pharmacological results. The union of the regression and the correlation analysis results were the peaks of X5, X9, X11, X12, X16, X18, Y5, Y8, Y12, Y14, Y20, Z4, Z5, Z6, and Z8. The intersection of the regression and the correlation analysis results were the peaks of X11, X12, X16, X18, Y5, Y12, and Z5. The correlated peaks were assigned by comparing the fingerprints with the negative control samples and reference standard samples, and identifying the structure using high-performance liquid chromatography-mass spectrometry detector(HPLC-MS). The results suggested that the pharmacodynamic basis of LCSSY on anti-ovarian cancer activities were germacrone, furandiene, β-elemene, calycosin-7-glucoside, ononin, epimedin B, icariin, ginsenoside Rc, astragaloside, ginsenoside Rd, astragaloside II, and some unknown components.

Chemical Constituents of Supercritical Extracts from Alpinia officinarum and the Feeding Deterrent Activity against Tribolium castaneum.

Alpinia officinarum has been confirmed to possess bioactivities against some pests. In this work, a sample was obtained from A. officinarum rhizomes by supercritical fluid CO2 extraction (SFE). According to GC-MS analysis, the main chemical components for SFE-sample included benzylacetone (26.77%), 1,7-diphenyl-5-hydroxy-3-heptanone (17.78%), guaiacylacetone (10.03%) and benzenepropanal (7.42%). The essential oil of A. officinarum rhizomes (LD50 = 20.71 μg/adult) exhibited more contact toxicity than SFE extract (LD50 = 82.72 μg/adult) against Tribolium castaneum. From SFE extracts, one new compound, 1-phenyl-4-(16,17-dimethyl-9,13-octadiene)-5-isopentenyl-7-(4”-methoxyl-3”-hydroxyl-phenyl)-3-heptanone (3), together with five known compounds identified as 5-hydroxy-1,7-diphenyl-3-heptanone (1), 1,7-diphenyl-4-hepten-3-one (2), galangin (4), galangin-3-methyl ether (5) and pinocembrin (6), were isolated and their feeding deterrent activities against T. castaneum adults were assessed. It was found that compounds 1–6 had feeding deterrent activities against T. castaneum with feeding deterrent indices of 18.21%, 18.94%, 19.79%, 26.99%, 20.34%, and 35.81%, respectively, at the concentration of 1500 ppm. Hence, the essential oil and SFE extracts/compounds of A. officinarum rhizomes represent promising alternatives in the control of T. castaneum adults.

Pilot scale production of a phospholipid-enriched dairy ingredient by means of an optimised integrated process employing enzymatic hydrolysis, ultrafiltration and super-critical fluid extraction

Abstract Pilot scale production of a dairy ingredient enriched in phospholipids (PLs) was generated from a buttermilk powder (BMP) substrate utilising a combined process of targeted enzymatic hydrolysis of the innate milk proteins followed by ultrafiltration with a 50 kDa membrane. An 8.5 fold increase in PL was achieved in the 50 kDa retentate (50 R) compared to the BMP, 11.05 ± 0.02% and 1.30 ± 0.00% total PL, respectively. Simultaneously, total lipid content in the retentate increased 8.7 fold with reference to the BMP, 60.07 ± 0.54% and 6.84 ± 0.17% total lipid respectively. Protein reduced to 10.58 ± 0.09% (50 R) from 31.40 ± 0.57% in BMP. Supercritical CO 2 fluid extraction (SFE) was employed to generate a purified lipid fraction. SFE with ethanol as a co-solvent yielded a purified lipid extract with enriched PLs level of 56.24 ± 0.07% on a dry matter basis. Industrial relevance PLs have many associated health and nutritional benefits including those related to cognitive development and repair. Generation of an ingredient enriched in dairy PLs would be advantageous from an industrial view to allow fortification of nutritionals, both infant and geriatric, in promoting brain health. The present work demonstrates a novel pilot scale process for the generation of a PL enriched ingredient from a BMP substrate. Utilising a combined process of targeted protein hydrolysis followed by selective removal by ultrafiltration of the smaller molecular weight peptide material, an ingredient with 8.5 fold increase in PL material was achieved. SFE technology was utilised to generate a purified lipid extract with greater PL levels for future applications in biological assays to determine these pathways. The need for investigate and further develop the knowledge relating to the modes of action of these bioactive compounds would be beneficial from a nutritional perspective.

GC-MS法分析沙蒿籽中挥发油与种子油的化学组成

本研究以沙蒿籽为原料，采用水中蒸馏法得到沙蒿籽挥发油，分别采用石油醚溶剂萃取和超临界二氧化碳萃取的方法，得到沙蒿籽种子油，并应用气质联用法(GC-MS)分析其化学组成。实验结果表明：沙蒿籽挥发性成分多为烯烃类或烯醇类化合物，其中含量最高的为β-罗勒烯以及它的同分异构体(相对含量为25.34%)；其次是β-没药烯(相对含量为10.318%)；D-柠檬烯含量也较高(相对含量为5.293%)。CO2超临界流体萃取所得沙蒿种子油中含有70.246%的亚油酸，13.433%的油酸和8.150%的棕榈酸。 In this study, the seed of Artemisia desterorum Spreng as the raw material, the volatile oil was extracted by water distillation, and the seed oil was extracted by petroleum ether solvent extraction and carbon dioxide supercritical fluid extraction, respectively. The chemical composition of volatile oil and seed oil was analyzed by GC-MS. The results showed that the volatile composition main consisted of olefins and enol, including β-basilene and its isomers (RA% = 25.34%), β-myrrhene (RA% = 10.318%), D-limonene (RA% = 5.293%). The chemical composition of seed oil extracted by CO2 supercritical fluid extraction was mainly linoleic acid (RA% = 70.246%), oleic acid (RA% = 13.433%) and palmitic acid (RA% = 8.150%).

GC-MS法分析沙葱籽中挥发油与种子油的化学组成

本研究以沙葱籽为原料，采用水中蒸馏法得到沙葱籽挥发油，采用超临界二氧化碳萃取法得到沙葱种子油，并应用气质联用法(GC-MS)分析其化学组成。实验结果表明：沙葱籽挥发性成分多为酯类或烯烃类化合物，其中含量最高的为二乙基十六酸二乙酯，以及它的同分异构体(相对含量为20.828%)；其次是角鲨烯(相对含量为7.383%)；联二苯酚类含量也较高(相对含量为5.798%)。CO2超临界流体萃取所得沙葱种子油中含有68.533%的亚油酸，16.640%的油酸和6.374%的棕榈酸，此外还含有2.257%花生酸和饱和脂肪酸(十九碳酸1.368%、二十碳酸0.760%、二十二碳酸0.244%)，还含有微量角鲨烯(相对含量为0.242%)。 In this study, the seed of Allium mongolicum Regel as the raw material, extracted volatile oil by water distillation; and seed oil by carbon dioxide supercritical fluid extraction, respectively. The chemical composition of volatile oil and seed oil was analyzed by GC-MS. The results showed that the volatile composition main consisted of ethers, olefins, and it also contains acids, alcohols, aldehydes, furan and pyrrole. The content of adipic acid bis (2-ethylhexyl) ester and its isomers was the highest (RA% = 20.828%), and then squalene (RA% = 7.383%), and 2,2'-Methylenebis (6-tert- butyl-4-methylphenol) (RA% = 5.798%). The chemical composition of seed oil extracted by CO2 supercritical fluid extraction was mainly linoleic acid (RA% = 68.533%), oleic acid (RA% = 16.640%), and palmitic acid (RA% = 6.374%), respectively. It also contained arachidic acid (RA% = 2.257%), and saturated fatty acids (19 carbonic acid RA% = 1.368%, 20 carbonic acid RA% = 0.760% and 22 carbonic acid RA% = 0.244%). The RA% of squalene in seed oil was 0.242%.

Thermodynamics and statistical correlation between supercritical-CO2 fluid extraction and bioactivity profile of locally available Mexican plants extracts

Supercritical-CO2 fluid extraction (SFE) is a green technology which is being used for the extraction of bioactive compounds such as pigments and fatty acids, in recent years. Naturally occurring plants or plant-based extracts are considered potential candidates with antioxidant and antimicrobial activities. Therefore, in this work, a range of locally available natural plants i.e. oregano, Mexican arnica also known as Heterotheca inuloides, lemon peel, and blackberry marc was analyzed for the extraction of high-value compounds. A multivariable linear regression model was used to analyze the output variables in relation with the inputs i.e. pressure (P), temperature (T), and co-solvent (CoS). Based on Arrhenius equation, the linear correlativity of the data with an activation energy Ea=6064.14kJ/mol and a pre-exponential factor of k0=0.388s−1 were recorded. The thermodynamic parameters of enthalpy, entropy, and Gibbs free energy were also evaluated, correlating the thermodynamics with the extraction yield of the process.

Some thermodynamic processes of anthracite–carbon dioxide mixture in supercritical fluid state

In the context of the development of the catalyst regeneration procedure via supercritical fluid CO2 extraction, some thermodynamic properties of the anthracene–carbon dioxide mixture in supercritical fluid state have been studied. Data on anthracene solubility in pure and modified (dimethyl sulfoxide, 5 wt %) supercritical carbon dioxide (SC–CO2), the heat capacity of anthracene and its mixtures with carbon dioxide, and the heat of solution of anthracene in SC–CO2 are presented. Anthracene solubility in SC–CO2 is described satisfactorily using the Peng–Robinson equation of state.

Chemical composition, antibacterial activity and related mechanism of the essential oil from the leaves of Juniperus rigida Sieb. et Zucc against Klebsiella pneumoniae

Ethnopharmacological relevanceJuniperus rigida is used as Tibetan and Mongolian medicine in China for the treatment of rheumatoid arthritis, nephritis, brucellosis and other various inflammatory diseases. Aim of the studyTo evaluate antibacterial potential of essential oils from J. rigida leaves against Klebsiella pneumoniae and to examine its possible related mechanisms. The study was undertaken in order to scientifically validate the traditional use of J. rigida. Materials and methodsThe essential oil was extracted from the leaves of J. rigida by supercritical CO2 fluid extraction technology. Chemical composition of essential oils was analyzed by gas chromatography-mass spectrometry (GC–MS). The antibacterial activity was evaluated against 10 bacteria by the paper disc diffusion method. The minimum inhibitory concentration (MIC) and minimal bactericidal concentration (MBC) values of the essential oil were estimated by agar dilution method. The antibacterial mechanism was evaluated by growth curve, the integrity of cell membrane, the SDS-PAGE of protein patterns and scanning electron microscope (SEM). Results61 components were identified from the essential oil. Caryophyllene (13.11%) and α-Caryophyllene (11.72%) were found to be the major components. The antibacterial activities of the essential oil were screened and compared against 10 bacteria. The essential oil showed good antibacterial activity against K. pneumoniae, with the biggest diameters of inhibition zones (DIZ) (16.00±0.25mm) and the lowest MIC and MBC values of 3.125mg/mL. The increase in proteins, 260nm absorbing materials of bacterial cells suspension indicated that the cytoplasmic membranes were broken by the essential oil. The SDS-PAGE of bacterial proteins demonstrated that the essential oil could damage bacterial cells through the destruction of cellular proteins. Scanning electron microscopy (SEM) showed that the essential oil damaged the morphology of cell wall and membrane. ConclusionsThe essential oil of J. rigida has potential antibacterial activities against K. pneumoniae. The antibacterial mechanism is the essential oil causing the irreversible damage to the cell wall and membrane, leading to the leakage of proteins and 260nm absorbing materials (DNA and RNA). Further phytochemical and pharmacological studies are required for proper scientific validation of the folk use of this plant species.

Characteristics, composition, and antioxidant activities in vitro and in vivo of Gynostemma pentaphyllum (Thunb.) Makino seed oil.

In order to further develop and utilise Gynostemma pentaphyllum (Thunb.) Makino seeds, a detailed analysis of the characteristics of G. pentaphyllum seed oil (GPSO), including its physico-chemical parameters, fatty acid composition and unsaponifiable matter constituents, has been investigated in this study. The antioxidant potential of GPSO was evaluated by radical-scavenging activity and ferric-reducing antioxidant power assay in vitro, and the antioxidant activity in vivo was examined by using an aged mice model. The main components of the seeds are lipids (485.54 g kg-1 ) and proteins (203.26 g kg-1 ). The GPSO obtained by supercritical CO2 fluid extraction was rich in polyunsaturated fatty acids (92.85%), especially conjugated linolenic acid (88.17%); and various useful compounds (squalene, tocopherol and phytosterols) were identified in the unsaponifiable matter. The overall antioxidant capacity of GPSO in vitro was shown to be comparable to that of Camellia seed oil as a positive control. GPSO could provide protection to the aged mice against oxidative stress and minimised the impact of ageing. All the results suggest that GPSO has direct and potent antioxidant activities; it could be utilised as a functional food to supplement or replace some conventional oils. © 2016 Society of Chemical Industry.

Chemical Composition, Anti‐oxidant, and Antimicrobial Activities of Four Saline‐Tolerant Plant Seed Oils Extracted by SFC

AbstractThe supercritical CO2 fluid extraction process was used to obtain seed oils. The compositional analyses of the seed oils of Amorpha fruticosa L., Caragana microphylla Lam and Elaeagnus angustifolia L. were performed by GC–MS, from which 18–22 fatty acid compositions were identified, and their main components were linoleic acid (49.10–66.39 %) and oleic acid (11.95–41.10 %). The seed oils were rich in unsaturated fatty acids, which accounted for 79.75–91.19 %. The in vitro anti‐oxidant activities of the seed oils were measured by the DPPH, ABTS and phosphomolybdenum complex assays. The IC50s of the seed oils were 6.5110–12.6599 mg/mL tested by DPPH assay, and the IC50s were in the range of 0.6872–1.9310 mg/mL tested by ABTS assay. The anti‐oxidant activities of seed oils decreased in the order of A. fruticosa > C. microphylla > E. angustifolia (A) > E. angustifolia (D). In vitro antimicrobial activities of seed oils against five bacteria were tested by microdilution method. The MIC values of the oils against Bacillus subtilis, E. coli, S. aureus, P. solanacearum and B. thuringiensis were in the range of 26–36, 30–38, 58, 56–58 and 56–58 mg/mL, respectively, while the MBC values were in the range of 30–40, 34–42, 58, 56–58 and 58 mg/mL, respectively. In vitro antifungal properties of the seed oils against four plant pathogenic fungi were tested by the mycelial growth rate method. The EC50s of seed oils against V. mali, C. gloeosporioides, F. graminearum and B. cinerea were in the range of 17.51–19.25, 15.58–19.12, 8.00–17.12 and 18.76–19.14 mg/mL, respectively. The seed oils showed moderate anti‐oxidant and antimicrobial activities.

Antioxidant and anti-inflammatory activities of Lonicera japonica Thunb. var. sempervillosa Hayata flower bud extracts prepared by water, ethanol and supercritical fluid extraction techniques

Lonicera japonica Thunberg (LJ) has long been used as an antipyretic, anti-inflammatory and anti-infectious agent in East Asia. The subspecies L. japonica Thunb. var. sempervillosa Hayata (LJv) is a variant that mainly grows in Taiwan. This study examined the antioxidant and anti-inflammatory activities of the extracts from the flower buds of these two species. The extracts were obtained by three extraction methods: water extraction, ethanol extraction, and supercritical-CO2 fluid extraction (SFE). The antioxidant activities of dry LJ (dLJ) extracts were superior to those of LJv extracts. Water extracts possessed higher activities than that prepared by ethanol or SFE. The total polyphenols content, total flavonoids content, and the amount of chlorogenic acid and luteolin-7-O-glucoside were all higher in the water extracts compared to the other two. The SFE extracts of these two species all exhibited excellent anti-inflammatory activities. Although the water and ethanol extracts of dLJ extracts had higher anti-inflammatory activity than that of LJv extracts, the SFE extracts prepared from fresh LJv flower buds (fLJv) exhibited the highest activity among all extracts. The SFE effectively isolates the bioactive components of L. japonica and can obtain the L. japonica extracts with high anti-inflammatory activity.

Chemical, physical, and in vitro characterization of research cigarettes containing denicotinized tobacco

The use of very low nicotine tobacco cigarettes is currently being investigated as a possible harm reduction strategy. Here, we report the smoke chemistry, toxicity, and physical characteristics of very low nicotine cigarettes that were made using blended tobacco processed through a supercritical CO2 fluid extraction, which resulted in elimination of 96% of nicotine content (denicotinized (denic) tobacco). Three types of test cigarettes (TCs) were manufactured with tobacco filler containing 100% denic tobacco (TC100), 50% denic tobacco and 50% unextracted tobacco (TC50/50), and 100% unextracted tobacco (TC0). Mainstream smoke (MS) was generated for measurement of 46 analytes and cytotoxicity and mutagenicity determination. Analysis of physical characteristics of TCs demonstrated they were well made with <5% variability among cigarettes for most parameters measured. We observed significant changes in the levels of smoke constituents, including decreases in formaldehyde, nitrosamines, and phenol, and increases in aliphatic hydrocarbons, aliphatic nitrogen compounds, aromatic amines, halogen compounds, and metals. Use of denic tobacco resulted in changes in the chemical composition of MS, but these changes did not modify biological activity as measured in the mutagenicity and cytotoxicity assays.

Regeneration of Aluminopalladium Catalyst for Hydrogenation Using Supercritical Fluid CO2-Extraction Process

An experimental setup was designed for studying the solubility of a catalyst-deactivating compound – polyisoprene – in pure supercritical carbon dioxide (SC-CO2) and in SC-CO2 with a co-solvent (3,5 wt % chloroform). In pure SC-CO2, the crossover-type variations in the solubility was observed in isotherms t = 70, 100 and 120 °C at the pressure region between 15 and 34 MPa, and the pressure of ca. 19 MPA determined in the top crossover point. Addition of chloroform to SC-CO2 was shown to result in an average 2 to 2,5 times increase in the solubility. The process of supercritical fluid CO2-extraction regeneration of the aluminopalladium catalyst LD-265 for hydrogenation was studied. Dimethylsulfoxide and ethanol were used as co-solvents for SC-CO2. The maximal efficiency of the regeneration was observed at the co-solvent concentration of 5,5–6,5 wt %, dimethylsulfoxide being more effective co-solvent than ethanol. With the catalysts subjected to the SC-CO2 regeneration, the resulting diene and bromine numbers, as well as conversions of styrene and methylcyclopentadiene, meet the standards on catalytic systems for selective hydrogenation of diene hydrocarbons to benzene-toluene-xylene fractions.

Molecular composition of oxygenated compounds in fast pyrolysis bio-oil and its supercritical fluid extracts

A three-step supercritical CO2 extraction was used for selective fractionation of fast pyrolysis bio-oil. Oxygen-containing compounds in bio-oil and its fractions were characterized using infrared spectroscopy (IR), 1H nuclear magnetic resonance spectroscopy (NMR), gas chromatography–mass spectrometry (GC–MS) and negative-ion electrospray ionization (ESI) Fourier transform ion cyclotron resonance mass spectrometry (FT-ICR MS). With appropriate optimization of extraction parameters, lipids, hemicellulose, lignin and condensed aromatics were found enriched in three different fractions. The molecular composition and compound classes were identified by combined analytical techniques, and the selectivity of the fractionation was proved. The compound-type-specific fractionation also facilitated the following characterization and provided insights into the chemical composition of bio-oil. Results indicated that CO2 supercritical fluid extraction was potential for bio-oil preprocessing.

Chemical Composition of Essential Oils from Zanthoxylum bungeanum Maxim. and Their Bioactivities against Lasioderma serricorne.

Two essential oil samples were obtained from the pericarp of Zanthoxylum bungeanum with the methods of hydrodistillation (HD) and supercritical fluid CO2 extraction (SFE), their chemical components were identified, and their bioactivities against Lasioderma serricorne adults were evaluated. In the process of testing, the two samples showed significant bioactivities against Lasioderma serricorne adults. For an example, the SFE-sample expressed relatively stronger fumigant toxicity on Lasioderma serricorne adults (LC50 = 3.99 μg/mL air) than that of the HD-sample (LC50 = 12.54 μg/mL air). According to GC-MS analysis, the chemical components between two samples were different. The major chemical components for HD included linalool (25.99%), limonene (19.34%), linalyl anthranilate (12.22%), 4-terpinenol (10.49%), eucalyptol (6.53%) and α-terpineol (5.02%), while for SFE included nonanoic acid (21.43%), γ-terpinene (14.51%), eucalyptol (13.45%), α-terpineol (5.83%) and caryophyllene oxide (5.48%). The results showed that different chemical components result in different bioactivities. This work provides theoretical basis for traditional Chinese concept of antagonistic storage, and it also provides important information for the development and comprehensive utilization of Zanthoxylum bungeanum.

Regeneration of an alumopalladium hydrogenation catalyst in the process of supercritical CO2 fluid extraction

Using a specially created experimental setup, the crossover character of a change in solubility is determined, and the approximate pressure at the upper crossover point is found (≈19 MPa). The modification of carbon dioxide with chloroform is shown to increase its solubility by 2–2.5 times on average within the above range of conditions. The supercritical CO2 fluid extraction (SCFE) process is studied as it applies to the regeneration of LD-265 alumopalladium hydrogenation catalyst. Dimethylsulfoxide and ethanol are selected as cosolvents (modifiers). Maximum regeneration efficiency is established at a 5.5–6.5 wt % concentration of cosolvents, and dimethylsulfoxide proves to be a more efficient cosolvent than ethanol. The diene and bromine numbers and styrene and methylcyclopentadiene conversions obtained on catalysts regenerated via SCFE satisfy the requirements for catalytic systems used in the selective hydrogenation of dienes in a benzene–toluene–xylene fraction.

Supercritical Fluid Extract of Spent Coffee Grounds Attenuates Melanogenesis through Downregulation of the PKA, PI3K/Akt, and MAPK Signaling Pathways.

The mode of action of spent coffee grounds supercritical fluid CO2 extract (SFE) in melanogenesis has never been reported. In the study, the spent coffee grounds were extracted by the supercritical fluid CO2 extraction method; the chemical constituents of the SFE were investigated by gas chromatography-mass spectrometry (GC-MS). The effects of the SFE and its major fatty acid components on melanogenesis were evaluated by mushroom tyrosinase activity assay and determination of intracellular tyrosinase activity and melanin content. The expression level of melanogenesis-related proteins was analyzed by western blotting assay. The results revealed that the SFE of spent coffee grounds (1–10 mg/mL) and its major fatty acids such as linoleic acid and oleic acid (6.25–50 μM) effectively suppressed melanogenesis in the B16F10 murine melanoma cells. Furthermore, the SFE decreased the expression of melanocortin 1 receptor (MC1R), microphthalmia-associated transcription factor (MITF), tyrosinase, tyrosinase-related protein-1 (TRP-1), and tyrosinase-related protein-2 (TRP-2). The SFE also decreased the protein expression levels of p-JNK, p-p38, p-ERK, and p-CREB. Our results revealed that the SFE of spent coffee grounds attenuated melanogenesis in B16F10 cells by downregulation of protein kinase A (PKA), phosphatidylinositol-3-kinase (PI3K/Akt), and mitogen-activated protein kinases (MAPK) signaling pathways, which may be due to linoleic acid and oleic acid.

Supercritical Fluid CO2-Extraction Regeneration of the Nickel-Molybdenum Catalyst for Hydrotreatment

Results of the studies of a supercritical fluid CO 2 -extraction regeneration of the industrial nickel-molybdenum catalyst DN-3531 for hydrotreatment at 323–383 K and pressure up to 30 MPa and of the basic extractant to be modified with polar compounds such as chloroform, methanol, ethanol, acetone and dimethylsulfoxide (DMSO) are reported. The said modifier sequence indicate an increase in the solubilizing ability of the modified supercritical carbon dioxide (SC-CO 2 ) with respect to the catalyst deactivating sediments. However, with DMSO as the most effective modifier, not only deactivating compounds but also nickel and molybdenum are removed that causes a considerable decrease in the final activity of the regenerated catalyst. The coke content in the catalyst decreases to one third while the specific surface area and pore volume increase during the extractive regeneration. After the catalyst deactivated during hydrodesulfurization of dibenzothiophene and hydrogenation of naphthaline was once subjected to the SC-CO 2 treatment, the catalyst activity increased several times to be as high as 2,5 times of that of the catalyst regenerated using the traditional oxidative method.

Biochemical and biophysical characterization of maize-derived HBsAg for the development of an oral vaccine

Although a vaccine against hepatitis B virus (HBV) has been available since 1982, it is estimated that 600,000 people die every year due to HBV. An affordable oral vaccine could help alleviate the disease burden and to this end the hepatitis B surface antigen (HBsAg) was expressed in maize. Orally delivered maize material induced the strongest immune response in mice when lipid was extracted by CO2 supercritical fluid extraction (SFE), compared to full fat and hexane-extracted material. The present study provides a biochemical and biophysical basis for these immunological differences by comparing the active ingredient in the differently treated maize material. Purified maize-derived HBsAg underwent biophysical characterization by gel filtration, transmission electron microscopy (TEM), dynamic light scattering (DLS), UV-CD, and fluorescence. Gel filtration showed that HBsAg forms higher-order oligomers and TEM demonstrated virus-like particle (VLP) formation. The VLPs obtained from SFE were more regular in shape and size compared to hexane or full fat material. In addition, SFE-derived HBsAg showed the greatest extent of α-helical structure by far UV-CD spectrum. Fluorescence experiments also revealed differences in protein conformation. This work establishes SFE-treated maize material as a viable oral vaccine candidate and advances the development of the first oral subunit vaccine.