Oxidation of low-density lipoprotein (LDL) is crucial in plaque formation and the onset of atherosclerosis. The LDL is subject to oxidative modification to form oxidized LDL (ox-LDL) due to its high content of unsaturated fatty acyl groups [1
]. Ox-LDL can cause macrophage growth inhibition, induction of monocyte differentiation into macrophages, promotion of its uptake by macrophages through scavenger receptors, and cytotoxicity of macrophages [2
]. Consequently, macrophage apoptosis plays an important role in the development of atherosclerotic lesion. Occurrence of macrophage apoptosis in human atheroma has been demonstrated [3
]. Much evidence has implicated that atherosclerosis has a close relationship with ox-LDL, which is clearly a main risk factor for coronary heart disease. In contrast, much evidence shows that dietary antioxidants (e.g., polyphenol and flavonoid) can potentially protect against LDL oxidation [5
]. Epidemiological studies have repeatedly shown that diets rich in fruit and vegetables containing antioxidants are well associated with lower risks of cardiovascular diseases [6
]. Thus, increased intakes of dietary antioxidants may be useful to prevent LDL oxidation and the atherosclerotic progression.
Pine (Pinus morrisonicola
Hay, PM), also called Taiwanese short-leaf pine, belongs to the Pinaceae family [8
]. PM needles have been used as tonic drinks and a folk medicine for antihypertension in Asia [8
]. PM needles extracted with various solvents (water, ethanol, ethyl acetate) exhibit in vitro
biological functions, including antioxidant activity, anti-mutagenicity, anti-inflammatory actions, inhibition of the growth of human leukemic cell line U937, and antitumor effects [9
]. In addition, the ethyl acetate extract of PM needle was shown to inhibit Cu2+
-induced LDL oxidation and attenuate excessive NO generation in mouse RAW 264.7 macrophages treated with lipopolysaccharide (LPS) [14
The solvent lipid extraction methods for plant essential oils are being superseded by supercritical fluid extraction (SFE) methods, as the latter are rapid, automatable, selective, and can avoid the use of large amounts of solvent [15
]. Among different supercritical fluids, CO2
is considered environmentally friendly, safe, non-toxic, non-carcinogenic, non-flammable, inexpensive, and to have modest critical conditions. The selectivity of the supercritical CO2
) can be adjusted by varying temperature and pressure to obtain fractions consisting of desirable compounds [16
]. It has been shown that the essential oils can be obtained from plants by SF-CO2
) methods [17
] and that the essential oil has hypolipidemic effects in vivo
] and can potentially be used as a natural antioxidant and antimicrobial agent in food processing [19
]. Besides, the majority of the essential oils are classified as Generally Recognized As Safe (GRAS) ingredients [21
]. These features of SFE are ideal for extraction of essential oil from plant materials. However, to our knowledge, no reports have been available on the use of SFE for PM needle essential oil. Therefore, the present study aimed to determine the optimal conditions of SFE for PM needles and whether the PM extracts (PME) exert antioxidant activities in RAW 264.7 macrophages treated with ox-LDL. Furthermore, we analyzed the possible bioactive compounds in PME using GC/MS.
The aim of this study was to evaluate optimal extraction conditions of SFE for PM (i.e.
, temperature, pressure, time, and adjuvant volume) on antioxidant activity and to identify the active fractions and compounds in PM. The SFE conditions for extracting essential oil from PM reported in the present study may be applicable to the extraction of essential oil or pigment in various plants. The results reveal that the optimal SF-CO2
conditions for the antioxidant activity of PM were 25 MPa for 15 min at 40 °C. Under these conditions, the yield of SF extracts from PM increased with increasing ethanol volume. As indicated by Lin et al.
], the SF-CO2
system has low polarity at high pressure, so only traces of polar compounds were extracted and often only the low polarity compounds, such as alkanes, alkoxy, aliphatic aldehydes and fat, were obtained. Consequently, SF with carbon dioxide requires the addition of small amounts of more polar solvents (such as ethanol) or modifiers to increase the solvent strength of the mobile phase, deactivate active sites on the surface of the packing material, and allow the elution of polar compounds with high efficiency [23
]. It has been shown that plant extracts that have higher polarity are less cytotoxic [24
]. We found that the inhibitory effects of PME on cell viability decreased with increasing polarity (i.e.
, no ethanol added) and that PME2 and PME5 had the strongest cell cytotoxicity (cell viability decreased to 40% and 50%, respectively, at the concentration of 100 μg/mL). However, PME fractions 1, 3, 4, and 6, which had higher polarity (75%–80%) displayed less cytotoxicity than the other PME fractions. In addition, the extracts obtained by higher extraction temperature had higher cytotoxic effects (i.e.
, PME fractions 7, 8, and 9). These results indicate that the lower polarity or higher temperature in the extraction conditions of SF-CO2
results in higher cytotoxicity in PME fractions.
Among the several SFE fractions of PM needles, PME3 was not cytotoxic and was the only fraction that effectively inhibited lipid peroxidation in RAW 264.7 macrophages. We then analyzed the components in PME3 and found that PME3 contained polyphenols, flavonoids, and p
-coumaric acid (14,500, 960, and 460 μg/g) (data not shown). Yen et al.
] indicated that the ethyl acetate extracts of PM may contribute to its anti-atherosclerotic effects by inhibition of LDL oxidation and that the main bioactive compounds in the ethyl acetate extracts of PM are epicatechin and p
-coumaric acid. Fang et al.
] also indicated that the acetone extract of heartwood from PM contains various flavonoids and stilbenes.
We further fractionated PME3 by column and thin-layer chromatography to obtain PME3-1 through PME3-4, and we found that the inhibitory effect of PME3-1 on conjugated diene and foam cell formation was about two times higher than those of PME3. Furthermore, we analyzed the components in PME3-1 by GC/MS, and our data suggest that the essential oil compounds, such as 1-docosene, neophytadiene, and methyl abietate, could be the major active components in PME3-1. This study represents the first to explore the appropriate condition to obtain SFE-CO2 extracts from PM and to identify the possible active compounds responsible for their antioxidant activities.
Regarding the active compounds in PM needle essential oil, a new abietic acid-type diterpene glucoside and terpinolene were found to exhibit remarkable protection against LDL-oxidation [26
]. In addition, 3-methylene-7,11,15-trimethylhexadec-1-ene (neophytadiene) was found in the hexane extracts of Bursera simaruba
(L.) Sarg. leaves which displays anti-inflammatory activities on carrageenan-induced inflammation in rats, and the effect observed at the dose of 1.50 g/kg·bw was comparable to that of phenylbutazone at the dose of 80 mg/kg·bw [28
]. The same study also identified a hydrocarbon compound, cedrane-8,13-diol, a kind of sesquiterpenoid, which exhibits hypolipidemic and antioxidant activities [18
]. In the present study, four major constituents were identified in PM needle essential oil by GC/MS in PME3-1, namely 1-docosene, cedrane-8,13-diol, neophytadiene, and methyl abietate, and their contents were 5.2-, 4.2-, 1.7- and 4.3-fold, respectively, higher than those of PME3. However, 1-(ethenyloxy)octadecane detected in PME3 was not found in PME3-1. Though we did not prove that cedrane-8, 13-diol, neophytadiene and methyl abietate were the active components in PME3-1. Evidence shows that cedrane-8,13-diol, neophytadiene and methyl abietate have hypolipidemic and antioxidant activities [18
]. Thus, we speculate that cedrane-8,13-diol, neophytadiene and methyl abietate may participate in the antioxidant activity of PM3-1.
In conclusion, the present results demonstrate that the optimal operation conditions of SFE-CO2 for antioxidant activity of PM are 25 MPa for 15 min at 40 °C. The PME3 fraction of the PM needle is able to inhibit lipid peroxidation and foam cell formation in RAW 264.7 cells. We further showed that PME3-1 may contribute to the biological function of PME3 and that cedrane-8,13-diol may be the major active compound in PME3. Further studies are needed to ascertain the hypolipidemic effect of PME3-1 in vivo and the major active compounds in PME3.
4. Experimental Section
4.1. Chemical and Reagents
Sodium nitrite, albumin, bovine, potassium chloride, sodium bicarbonate, lipopolysaccharide (LPS), Oil Red O (ORO), 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) were purchased from Sigma Co. Ltd. (St Louis, MO, USA). Copper sulfate pentahydrate, sodium chloride, trichloroacetic acid, copper sulfate pentahydrate, and dimethyl sulfoxide (DMSO) were obtained from Merck (Darmstadt, Germany). Pine (P. morrisonicola Hay) needles were kindly supplied by the KARA Legend Biotechnology Co., Ltd. (Pingchen, Taoyuan County, Taiwan). All other chemicals were reagent grade.
4.2. Sample Preparation
Fresh pine (P. morrisonicola) needles were freeze-dried, ground to powder, passed through a 100 mesh sieve and stored at −20 °C until use.
4.3. Supercritical Fluid Extraction
The model 260D system for supercritical fluid extraction was purchased from Teledyne ISCO (Lincoln, NE, USA). Twenty grams of powdered sample of pine needles were placed in the 30-mL extraction vessel and set at 40, 50, 60 °C. The pressure was adjusted at 15, 20, and 25 MPa, respectively. Ethanol was used as an adjuvant solvent, and the volume was 1, 2, and 3 mL, respectively. The flow rates for CO2
were fixed at 6 mL/min. Once the set temperature and pressure were achieved after turning on the injection valve and the system was in equilibrium, the extraction was carried out for 15 min in each experimental run. The final extract was collected in a flask connected to the back pressure regulator. The solvent was evaporated by drying under vacuum using rotary evaporator; the extract was weighed to obtain the yield (calculated as percentage of 20-g pine needles converted into extract by SF) and stored at −20 °C before further analysis of bioactive compounds. Conditions and of the yields of supercritical fluid extraction for PME are shown in Table 2
. Fractions of PME1 to 9 were obtained by different supercritical extraction conditions with 1%–4% yields, of which PME1 had the lowest yield (1.1%) and PME3 had the highest (3.9%).
Conditions and the yields of supercritical fluid extraction for Pinus morrisonicola extract (PME).
Conditions and the yields of supercritical fluid extraction for Pinus morrisonicola extract (PME).
|Extract||Temperature (°C)||Pressure (MPa)||Time (min)||Adjuvant (mL)||Yield (%)|
|PME 1||40||15||5||1||1.1 ± 0.04 *|
|PME 2||40||20||10||2||3.2 ± 0.52|
|PME 3||40||25||15||3||3.9 ± 1.07|
|PME 4||50||15||20||3||3.7 ± 0.90|
|PME 5||50||20||25||1||3.4 ± 0.02|
|PME 6||50||25||15||2||2.7 ± 0.27|
|PME 7||60||15||25||2||2.8 ± 0.50|
|PME 8||60||20||15||3||3.7 ± 0.05|
|PME 9||60||25||20||1||2.5 ± 0.06|
4.4. Separation and Characterization of PME3
Fractions of PME3 are readily separated by silica gel column chromatography using an elution system of n-hexane–ethyl acetate = 1:0.2 (200 mL/bottle). The overall recovery of PME3 isolates from the column is 90%–95% at a loading capacity of 150 mg of PME per 10 g of silica gel. The complete separation of four PME3 subfractions (PME3-1, PME3-2, PME3-3 and PME3-4) was also achieved by one-dimensional silica gel thin-layer chromatography with the solvent system n-hexane–ethyl acetate = 1:0.2.
4.5. GC/MS Analysis of Chemical Components in PME3 and PME3-1
A HP6890 series GC coupled with a 5973 Network Mass Selective Detector (Agilent Technologies, Santa Clara, CA, USA) was used for quantification and determination of the chemical components in the PME3 and PME3-1 of the pine extract. The SF-CO2
extraction for PM in our study primarily fractionated the compounds with low polarity and low boiling point. To some extent, increasing the initial temperature of column will increase the polarity or boiling point of compounds [18
]. Thus, we used low temperature and a high retention time to identification of the compounds. A capillary column type DB-1 (i.d. 0.25 mm × 60 m; membrane thickness, 0.25 μm) was used. Helium was used as the carrier gas and operated at a flow rate of 1 mL/min. The ionization potential used was 70 eV. The temperature of the ion source was set at 230 °C. The flux ratio was set at 50:1. Initially, the temperature was set at 40 °C for 10 min, then programmed at 1 °C/min up to 150 °C, than programmed at 5 °C/min up to 200 °C, then programmed at 11 °C/min up to 250 °C, and held at this temperature for 10 min. Quantification of PME3 and PME3-1. Aliquots (1.0 μL) of the extracts were, respectively, measured with a GC microsyringe from fractionates of pine extract and analyzed with GC/MS. Quantification of each constituent in PME3 and PME3-1 were calculated from the integrated diagrams obtained by Equation (1):
where Q = the quantity of each volatile constituent in extracts PME3 and PME3-1, A = the percent peak area in the gas chromatograms occupied by each constituent, and Y = the recovery yield of extracts.
The molecular weight and chemical structure splitting pattern of each peak in the GC chromatogram of PME3 and PME3-1 were compared with known compounds in the databases such as the Wiley MS Chemstation Libraries, NBS Computer Data [29
], and reference mixture of n
-alkanes (C5–C25) to calculate the retention indices (RI) from the retention time for each component. A compound is identified, when the similarity of the peak with the library compound is above 90%.
4.6. Lipoprotein Separation
Human plasma was obtained from the Taichung Blood Bank (Taichung, Taiwan), and LDL was isolated using sequential ultracentrifugation (p
= 1.019–1.063 g/mL) in KBr solution containing 30 mM EDTA, stored at 4 °C in a sterile, dark environment, and used within 3 days as previously described [30
]. Immediately before the oxidation tests, LDL was separated from EDTA and from diffusible low molecular mass compounds by gel filtration on PD-10 Sephadex G-25 M gel (Amersham Pharmacia Biotech Inc., Piscataway, NJ, USA) in 0.01 mol/L phosphate-buffered saline (136.9 mmol/L NaCl, 2.68 mmol/L KCl, LDL (100 μg of protein/mL) to obtain the native-LDL (n-LDL) for various tests in a cell-free LDL oxidation system and in macrophages.
4.7. Cell Culture and Treatment with Oxidized LDL
RAW 264.7 cells (BCRC 60001), the murine macrophage cell line, were obtained from the Bioresources Collection and Research Center, Food Industry Research and Development Institute (Hsinchu, Taiwan). Cells were cultured in endotoxin-free Dulbecco’s modified Eagle’s medium, supplemented with 10% fetal bovine serum and penicillin (100 units/mL) plus streptomycin (100 mg/mL) and maintained at 37 °C in 5% CO2. To induce lipid peroxidation and foam cell formation, RAW 264.7 macrophages were treated with oxidized-LDL (ox-LDL) which was obtained by incubating n-LDL with CuSO4 (50 μM) at 37 °C for 24 h. To determine the protective effects of PME extracts, the macrophages were pretreated with PME extracts for 2 h before incubation with ox-LDL or n-LDL.
4.8. Determination of Cell Viability
A portion (0–100 μg/mL) of PME fractions 1 to 9 was incubated with RAW 264.7 macrophages at 37 °C for 24 h. Cell viability was measured using the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide (MTT) assay according to the method of Denizot et al.
]. Briefly, cells (1 × 104
) were dispensed into 96-well plates and sample (PME1–9) was added then incubation at 37 °C in humidified atmosphere containing 5% CO2
for 24 h. Then, culture solutions were removed and replaced by 90 μL culture medium. Ten microliters of sterile filtered MTT solution (5 mg/mL) in PBS was added to each well, reaching a final concentration of 0.5 mg MTT/mL. Un-reacted dye was removed after 5 h. The insoluble formazan crystals were dissolved in 200 mL/well dimethyl sulfoxide (DMSO) and measured spectrophotometrically at 570 nm. The cell viability was expressed as the percentage of cell growth compared to control non-treated cells (at which growth is considered 100%) and it was calculated by A570 nm (sample)/A570 nm (control) × 100.
4.9. Determination of Thiobarbituric Acid-Reactive Substances (TBARS) in Macrophages Treated with Oxidized LDL
Lipid peroxidation in RAW 264.7 macrophages was determined as TBARS using the TBA assay [32
]. Briefly, a portion (0–100 μg/mL) of PME 1, 3, 4, and 6 was incubated with macrophages for 2 h before incubation with ox-LDL (100 μg/mL) at 37 °C for 24 h. Butylated hydroxytoluene (10 μL, 50 mM) was added to terminate peroxidation reaction, and the mixture was then added with 1 mL of 7.5% (w
) cold trichloroacetic acid. After centrifugation, the supernatant was allowed to react with 1 mL of 0.8% (w
) TBA in a boiling water bath for 45 min. After cooling, levels of TBARS were determined spectrofluorimetrically (at 515 nm excitation and 555 nm emission) using 1,1,3,3-tetraethoxypropane as the standard. Results are expressed as pmoles of TBARS content per milligram protein.
4.10. Determination of Foam cell Formation in Macrophages Treated with Ox-LDL
The foam cell formation assay was carried out by the method of Halvorsen et al.
]. RAW 264.7 cells were incubated with ox-LDL (100 μg/mL) or native LDL (n-LDL) in the absence or presence of PME3 and PME3-1 (25–100 μg/mL) for 24 h. After incubation, the cells were washed once in ice-cold PBS (phosphate buffered saline, pH 7.4), followed by formaldehyde fixation for 30 min at room temperature (25 °C). Neutral lipids were stained using 0.5% oil-red-O in isopropanol for 60 min. The ox-LDL uptake was using microscopy.
4.11. Determination of Conjugated Diene Formation in Cell-Free LDL Oxidized by Cu2+
Native LDL (n-LDL, 100 μg/mL) was pre-incubated with PME1, 3, 4, and 6 (100 μg/mL) at room temp for 1 h followed by addition of CuSO4
(50 μM) at 37 °C for 150 min. Conjugated diene formation was monitored by the method described by Yen et al.
]. The UV spectrum (OD234nm
) was recorded, and the increase in conjugated diene expressed in relative unit was obtained from the absorbance at 234 nm.
4.12. Statistical Analysis
All statistical analyses were performed using SPSS for Windows, version 17 (SPSS, Inc., Chicago, IL, USA). Data are expressed as means ± SD and analyzed using one-way ANOVA followed by Duncan’s multiple range test. p < 0.05 is considered statistically significant.