PURPOSE: This study examined the immunological activity and optimized the mixture conditions of Sargassum horneri (S. horneri) extracts in vitro and in vivo models.METHODS: S. horneri was extracted using three different methods: hot water extraction (HWE), 50% ethanol extraction (EE), and supercritical fluid extraction (SFE). Splenocyte proliferation and cytokine production (Interleukin-2 and Interferon-γ) were measured using a WST-1 assay and enzyme-linked immunosorbent assay, respectively. The levels of nitric oxide and T cell activation production were measured using a Griess assay and flow cytometry, respectively. The natural killer (NK) cell activity was determined using an EZ-LDH kit.RESULTS: Among the three different types of extracts, HWE showed the highest levels of splenocyte proliferation and cytokine production in vitro. In the animal model, three different types of extracts were administrated for 14 days (once/day) at 50 and 100 mg/kg body weight. HWE and SFE showed a high level of splenocyte proliferation and cytokine production in the with and without mitogen-treated groups, whereas EE administration did not induce the splenocyte activation. When RAW264.7 macrophage cells were treated with different mixtures (HWE with 5, 10, 15, 20% of SFE) to determine the optimal mixture ratio of HWE and SFE, the levels of nitric oxide and cytokine production increased strongly in the HWE with 5% and 10% of SFE containing group. In the animal model, HWE with 5% and 10% of SFE mixture administration increased the levels of splenocyte proliferation, cytokine production, and activated CD4⁺ cell population significantly, with the highest level observed in the HWE with 5% of SFE group. Moreover, the NK cell activity was increased significantly in the HWE with 5% of SFE mixture-treated group compared to the control group.CONCLUSION: The optimal mixture condition of S. horneri with immune-enhancing activity is the HWE with 5% of SFE mixture. These results confirmed that the extracts of S. horneri and its mixtures are potential candidate materials for immune enhancement.
Body Weight ; Chromatography, Supercritical Fluid ; Enzyme-Linked Immunosorbent Assay ; Ethanol ; Flow Cytometry ; In Vitro Techniques ; Killer Cells, Natural ; Macrophages ; Models, Animal ; Nitric Oxide ; Sargassum ; Water

PURPOSE: Sargassum horneri (S. horneri) is a species of brown macroalgae that is common along the coast of Japan and Korea. The present study investigated the immuno-modulatory effects of different types of S. horneri extracts in RAW264.7 macrophages. METHODS: S. horneri was extracted by three different methods, hot water extraction, 50% ethanol extraction, and supercritical fluid extraction. Cell viability was then measured by MTT assay, while the production levels of tumor necrosis factor-α (TNF-α), interleukin-6 (IL-6), and nitric oxide (NO) were measured by enzyme-linked immunosorbent assay and Griess assay, respectively. The expression and activation levels of inducible NO synthase (iNOS), mitogen-activated protein kinase (MAPK) and nuclear factor κB (NF-κB) were examined by western blot analysis. RESULTS: The three different S. horneri extracts were nontoxic against RAW 264.7 cells up to 50 µg/mL, among which treatment with hot water extract (HWE) of S. horneri significantly enhanced the production of TNF-α, IL-6, and NO in a dose-dependent manner. Hot water extract of S. horneri also increased the expression level of iNOS, suggesting that up-regulation of iNOS expression by HWE of S. horneri was responsible for the induction of NO production. In addition, treatment of RAW 264.7 macrophages with HWE of S. horneri increased the phosphorylation levels of ERK, p38 and JNK. Furthermore, the activation and subsequent nuclear translocation of NF-κB was enhanced upon treatment with HWE of S. horneri, indicating that HWE of S. horneri activates macrophages to secrete TNF-α, IL-6 and NO and induces iNOS expression via activation of the NF-κB and MAPKs signaling pathways. CONCLUSION: Taken together, these findings suggest that HWE of S. horneri possesses potential as a functional food with immunomodulatory activity.
Blotting, Western ; Cell Survival ; Chromatography, Supercritical Fluid ; Cytokines ; Enzyme-Linked Immunosorbent Assay ; Ethanol ; Functional Food ; Interleukin-6 ; Japan ; Korea ; Macrophages* ; Necrosis ; Nitric Oxide ; Nitric Oxide Synthase ; Phosphorylation ; Protein Kinases ; RAW 264.7 Cells ; Sargassum* ; Seaweed ; Up-Regulation ; Water

The volatile components of roots and stems of Zanthoxylum nitidum were investigated by supercritical fluid carbon dioxide extraction (SFE-CO2) and gas chromatography-mass spectrometry(GC-MS). Thirty-one and fifty-one compounds were identified in the supercritical extracts from roots and stems of Z. nitidum, respectively, and total twenty-seven compounds were the common constituents. Among them, the major constituents in root and stem supercritical extracts were spathulenol (18.49 and 26.18%), n-hexadecanoic acid (14.24% and 12.79%), ar-tumerone (6.95% and 8.88%), oleic acid (8.39% and 5.71%) and hexanoic acid (4.39% and 7.78%). The in-vitro MTT assay showed that the volatile components of roots and stems of Z. nitidum did not exhibited any cytotoxic activity against human cancer Huh-7 and normal IEC-6 cells. These results indicated the same nature of the volatile constituents in the root and stem of Z. nitidum. This investigation may provide further evidence for expansion of medicinal parts of Z. nitidum.
Animals ; Cell Line, Tumor ; Cell Survival ; drug effects ; Chromatography, Supercritical Fluid ; Drugs, Chinese Herbal ; chemistry ; isolation & purification ; toxicity ; Gas Chromatography-Mass Spectrometry ; methods ; Humans ; Mice ; Plant Roots ; chemistry ; Plant Stems ; chemistry ; Zanthoxylum ; chemistry

To determine the optimum conditions of supercritical CO2 extraction of Xiaoyaosan, and establish its fingerprint by gas chromatography-mass spectrometry (GC-MS), the yield of extract were investigated, an orthogonal test was used to quantify the effects of extraction temperature, pressure, CO2 flow rate and time, and fingerprint analysis of different batches of extracts were by GC-MS. The optimal extraction conditions were determined as follows: extraction pressure 20 MPa, extraction temperature 50 degrees C, CO2 flow rate 25 kg x h(-1), extraction time 3 h, and average yield 2.2%. The GC-MS fingerprint was established and 27 common peaks were found, whose contents add up to 81.89% of the total peak area. Among them, 21 compounds were identified, accounting for 53.20% of the total extract. The extraction process is reasonable and favorable for industrial production. The GC-MS method is accurate, reliable, reproducible, and can be used for quality control of supercritical CO2 extract from Xiaoyaosan.
Carbon Dioxide ; chemistry ; Chromatography, Supercritical Fluid ; methods ; Drugs, Chinese Herbal ; chemistry ; isolation & purification ; Gas Chromatography-Mass Spectrometry ; methods

With the yields of ferulic acid, coniferylferulate, Z-ligustilide, senkyunolide A, butylidenephthalide, butylphthalide, senkyunolide I, senkyunolide H, riligustilide, levistolide A, and total pharmacologically active ingredient as evaluation indexes, the extraction of Ligusticum chuanxiong by supercritical fluid technology was investigated through an orthogonal experiment L9 (3(4)). Four factors, namely temperature, pressure, flow rate of carbon dioxide, co-solvent concentration of the supercritical fluid, were investigated and optimized. Under the optimized conditions, namely 65 degrees C of temperature, 35 MPa of pressure, 1 L x min(-1) of CO2 flow rate, 8% of co-solvent concetration, supercritical fluid extraction could achieve a better yield than the conventional reflux extraction using methanol. And the supercritical fluid extraction process was validated to be stable and reliable.
Benzofurans ; analysis ; isolation & purification ; Chromatography, Supercritical Fluid ; instrumentation ; methods ; Coumaric Acids ; analysis ; isolation & purification ; Drugs, Chinese Herbal ; analysis ; isolation & purification ; Ligusticum ; chemistry

OBJECTIVETo compare the component difference of herb materials extracts of sesame oil fry and SFE-CO2 technique for compound ulcer oil.

METHODQualitative analysis of main component of dahuan, baizhi and chuangxiong in two extracts above was conducted by TLC. The contents of total anthraquinones, imperatorin and ferulic acid in two extracts were determined by UV and HPLC.

RESULTTLC experiment found that spots color of small Rf value component in oil extract were lighter than that in SFE-CO2 extract, but there was not obvious different between two extracts. Quantity analysis showed that SFE-CO2 extract owned much higher transfer rate of total anthraquinones, and it was 1.9 times of oil extract. Ferulic acid was similar in two extracts, and they were all below 10%. The contents of imperatorin in oil extracts were slight higher than that in SFE-CO2 extract.

CONCLUSIONThe components in the extracts of sesame oil fry for the herb materials of compound ulcer oil are the same as SFE-CO2 extract. Because SFE-CO2 extracts have no solvent limited for next preparation, it has more advantage.

Carbon Dioxide ; chemistry ; Chemical Fractionation ; instrumentation ; methods ; Chromatography, Supercritical Fluid ; instrumentation ; methods ; Chromatography, Thin Layer ; Drugs, Chinese Herbal ; analysis ; isolation & purification ; therapeutic use ; Hot Temperature ; Humans ; Plants, Medicinal ; chemistry ; Sesame Oil ; chemistry ; Ulcer ; drug therapy

OBJECTIVEThe optimum conditions of SFE-CO2 extraction of Plantaginis Semen oil (SPO), the composition of SPO and its antioxidant activities of SPO were all investigated in this paper.

METHODResponse surface method (RSM) was used to establish the mathematical model of SFE-CO2 extraction of SPO to obtain the optimum conditions based on Single factor experiments. Fatty acid compositions and contents of SPO were tested by GC-MS, and antioxidant activities of SPO were studied by DPPH and ABTS free radical elimination method.

RESULTThe optimum conditions obtained through RSM analysis were as follows: extraction tempreture 70 degrees C, extraction pressure 30 MPa, extraction time 120 min and flow rate 30 L x h(-1). Under the optimal condition, predicted value was 35.91%, while the experimental value was 35.07%. The experimental values agree with the predicted from the regression model with a relative error less than 5%. The main components of SPO were 9,12,15-octadecatrienoic acid, 8,11-octadecadienoic acid, octadecanoic acid and hexadecanoic acid. Most of the fatty acids were polyunsaturated fatty ones, whose quantities were obtained more than 88%. The IC50DPPH and IC50ABTS were 1.13, 3.57 g x L(-1) respectively in DPPH and ABTS assay.

CONCLUSIONOptimization of the extraction process by RSM of SPO is convenient and feasible. SPO has good antioxidant activity and is worth to develop for application.

Antioxidants ; chemistry ; isolation & purification ; Carbon Dioxide ; chemistry ; Chromatography, Supercritical Fluid ; methods ; Cupressaceae ; chemistry ; Gas Chromatography-Mass Spectrometry ; Plant Oils ; chemistry ; isolation & purification

OBJECTIVETo investigate USMM coupled techniques applied in active ingredient extraction and separation of Salvia.

METHODSupercritical fluid extraction (SFE) CO2 was used to extract and separate tanshinone liposoluble constituent, ultrasonic was used to extract danshen phenolic acids ternate, membrane separation and macroporous resin was used to purify water extraction from HDP. Transfer rate and purity of Danshen active ingredients were employed as the investigation indexes, the feasibility of USMM technology used in extraction and separation S. miltiorrhiza was investigated.

RESULTSFE-CO2 extraction process for S. miltiorrhiza was stable and feasible. Danshen phenolic acids extracted from slag of SFE-CO2 by ultrasound got a high yield. Macroporous resin purification technology could improve the purity of active ingredients of S. miltiorrhiza. Membrane separation and membrane separation coupled with macroporous resin technology applied to the purification process of S. miltiorrhiza phenolic acids still needed further research.

CONCLUSIONIt is feasible basically that USMM technology apply in extraction and separation of Salvia active ingredient.

Chromatography, Supercritical Fluid ; methods ; Salvia miltiorrhiza ; chemistry ; Technology, Pharmaceutical ; methods ; Ultrasonics

OBJECTIVETo extract the volatile components of water caltrop and kernel and to analyze them.

METHODThe volatiles were separated by supercritical fluid extraction (SFE) and determined by GC-MS.

RESULTThe extraction rates of water caltrop and kernel were 5.96% and 0.23%, respectively. The components determined by normalization method were mainly 9, 12-octadecadienoic acid (Z, Z), but the content was different.

CONCLUSIONThe researches showed that the components in the volatile components of water caltrop and kernel were mainly 12-octadecadienoic acid (Z, Z), and then palmitinic acid, with a higher extraction rate of caltrop.

Chromatography, Supercritical Fluid ; methods ; Fruit ; chemistry ; Gas Chromatography-Mass Spectrometry ; Linoleic Acid ; analysis ; isolation & purification ; Lythraceae ; chemistry ; Oils, Volatile ; chemistry ; isolation & purification ; Palmitic Acid ; analysis ; isolation & purification ; Plant Oils ; chemistry ; isolation & purification ; Plants, Medicinal ; chemistry ; Seeds ; chemistry

OBJECTIVETo analyze the chemical composition and compare acute hepatotoxicity of essential oils extracted from argy wormwood leaf in Guangdong by four different methods.

METHODFour extraction methods, including hydrodistillation extraction, supercritical fluid CO2 extraction, petroleum ether ultrasonic extraction and petroleum ether microwave extraction, were employed to prepare essential oil from argy wormwood leaf in Guangdong. The products were analyzed qualitatively and quantitatively using GC-MS and GC-FID. Sixty mice were divided into 5 groups according to different essential oils and took the same dose orally, then after 5 hours, hepatic functional parameters in serum were detected such as alanine aminotransferase (ALT) and so on, and morphologic change of hepatic tissues was observed.

RESULTThe extraction rate of the four methods and identified compounds was 1.02%, 80 (hydrodistillation extraction), 2.46%, 56 (supercritical fluid CO2 extraction), 3.17%, 45 (petroleum ether ultrasonic extraction) and 3.32%, 78 (petroleum ether microwave extraction) respectively. Totally 153 compounds were identified from those essential oils. Compared with that of the control group, some hepatic functional parameters of hydrodistillation and supercritical fluid CO2 extraction groups rose significantly (P < 0.01 or P < 0.05), and hepatic tissues of hydrodistillation group were damaged significantly.

CONCLUSIONThe essential oils extracted from argy wormwood leaf by different methods may have not only different chemical composition, but also different acute hepatotoxicity, and monoterpenes and benzenes in the essential oils might induce acute hepatotoxicity.

Alanine Transaminase ; metabolism ; Animals ; Artemisia ; chemistry ; Chemical Fractionation ; methods ; Chromatography, Supercritical Fluid ; methods ; Female ; Liver ; chemistry ; drug effects ; enzymology ; Male ; Mice ; Oils, Volatile ; analysis ; isolation & purification ; toxicity ; Plant Extracts ; analysis ; isolation & purification ; toxicity ; Plant Leaves ; chemistry ; Random Allocation