Characterization of Linum usitatissimum L. oil obtained from different extraction technique and in vitro antioxidant potential of supercritical fluid extract
Abstract
Aim:
Present investigation was aimed to characterize the fixed oil of Linum usitatissimum L. using five different extraction methods: Supercritical fluid extraction (SFE), ultrasound-assistance, soxhlet extraction, solvent extraction, and three phase partitioning method.
Materials and Methods:
The SFE conditions (temperature, pressure, and volume of CO2) were optimized prior for better yield. The extracted oils were analyzed and compared for their physiochemical parameters, high performance thin layer chromatography (HPTLC), gas chromatography-mass spectrometry (GC-MS), and Fourier-transformed infrared spectroscopy (FT-IR) fingerprinting. Antioxidant activity was also determined using 1,1-diphenyl-2-picrylhydrazyl and superoxide scavenging method.
Result:
The main fatty acids were α-linolenic acid, linoleic acid, palmitic acid, and stearic acid as obtained by GC-MS. HPTLC analysis revealed the presence of similar major components in chromatograms. Similarly, the pattern of peaks, as obtained in FT-IR and GC-MS spectra of same oils by different extraction methods, were superimposable.
Conclusion:
Analysis reported that the fixed oil of L. usitatissimum L. is a good source of n-3 fatty acid with the significant antioxidant activity of oil obtained from SFE extraction method.
Nowadays, the interest in the use of flaxseed (Linum usitatissimum L.) has been increasing in order to improve the health status. The flaxseed contains fiber, lignans, proteins, and fixed oil, etc. The fixed oil of flaxseed is a good source of n-3 fatty acid. The beneficial effect of n-3 essential fatty acid on human health is well recognized.[12] The fixed oil of flaxseed also contains a significant amount of n-6 fatty acid that is also one of the essential fatty acids. The determination of the total oil content in the seeds is of paramount importance in the oil industry as the price of the raw material is a function of its richness in the final, commercial product. Traditionally, soxhlet extraction (SOXH), and solvent extraction (SOL) methods are used for the extraction of fixed oil. These methods mostly use hexane as an extracting solvent in a very large quantity, but hexane is a hazardous chemical. Furthermore, these conventional methods are very time-consuming and less efficient. Thus, there is a need for efficient methods to be made available for extracting the fixed oils. Over the last few years, there has been an increased interest in the development of solid, liquid extraction technique such as ultrasound-assisted (US) extraction, microwave assisted extraction, and supercritical fluid extraction (SFE). These techniques have the possibility of working at elevated pressure and/or temperatures and greatly decreasing the time of extraction, hydrolysis, and oxidation. Due to the environmental concerns and potential health hazards of organic solvents, nonorganic solvents have become popular. SFE technique mostly uses supercritical CO2. The supercritical fluid has characteristics of both gases and liquids.
In this study, we characterize the fixed oil of flaxseeds extracted by SFE and other conventional methods. We also studied the antioxidant potential of the extracted fixed oil of flaxseeds.
Materials and Methods
Plant materials
Flaxseeds were collected from the local market and the same were identified and authenticated by a pharmacognosist and voucher specimen deposited in the Department of Pharmacognosy and Phytochemistry, Jamia Hamdard, New Delhi (Specimen No. BNPL/JH/RCMPH: 01/2012). The seeds were cleaned, dried, and grinded and passed through a sieve (40 mesh).
Extraction of fixed oil of flaxseed
SOXH and US extraction was done using hexane (ratio 1:3) as a solvent, for 4 h at boiling point in SOXH whereas 1 h at 54°C ± 2°C with occasional stirring in US.[3] Fixed oil was obtained by three phase partitioning (TPP) method as per the reported method.[4] SFE extraction was performed at a pressure of 350 bar and temperature of 50°C for 60 min at CO2 flow rate 20 g/min[5] which was optimized before the extraction.
Physiochemical parameters
Physiochemical parameters such as acid value (AV), peroxide value (PV), iodine value (IV), ester value, saponification value, and unsaponification value were analyzed according to the Indian Pharmacopoeia 2007[6] and matched with the reported values.
High performance thin layer chromatography profiling
All the five samples obtained through the five different extraction procedures (50 times diluted) were applied to duplicate (5.0 µL each) on precoated silica gel 60 F254 plates (E. Merck, 0.20 mm thickness) using Linomat V. The chromatograms were scanned at 300 nm after the development. The plate was also scanned at 370 nm after spraying with ethanolic sulfuric acid, 10% v/v and drying at 100°C for 5 min in the oven.
Gas chromatography-mass spectrometry analysis of fatty acids
The fixed oils extracted using different extraction methods were analyzed for their fatty acid composition by preparing FAME (Khan et al.).[7]
Agilent 7890A series, GC-MS System (Agilent, USA) was used for the analysis attached with CTC-PAL autosampler (CTC Analytics AG, Switzerland), Apolar HP 5MS (5% phenyl polymethylsiloxane) capillary Column (30 m×0.25 mm i.d. and 0.25 µm film thickness) and mass detector. The splitless mode at 250°C inlet temperature, 0.1 ml injection volume with helium gas at 1 ml/min and 70-202°C of oven temperature was used for analysis.
Fourier-transformed infrared spectroscopy analysis
A Fourier-transformed infrared spectroscopy (FT-IR) spectrophotometer (IR Affinity, Shimatzu) equipped with a deuterated triglycerine sulfate detector with a resolution of 4 cm−1. The data interval provided by the instrument for a resolution of 4 cm−1 is 1.93 cm−1. All spectra were recorded from 4000 to 400 cm−1. A thin film of the fixed oil was created between the two polished KBr disks.[8]
In vitro antioxidant activity
The free radical scavenging activity was determined by using 1,1-diphenyl-2-picrylhydrazyl assay previously reported by Hasan et al.[9] and Liu et al.[10] The method described by Liu et al.[11] was used for the determination of anion scavenging activity.
Results and Discussion
Evaluation of extraction methods
The maximum yield of oil was found in soxhlet method that may be attributed to the combined effect of temperature and hexane (nonpolar). The yield in solvent, soxhlet, and US methods was 35.73, 36.26, and 33.05%; respectively whereas by SFE it was 32.16% that the approaches yields of above procedures with the use of solvent and it also a less time consuming (1 h). The higher value of yield in solvent methods may be because of some small amount of remaining solvents; however, SFE method is solvent less.
Physiochemical parameter
The physiochemical parameter of extracted oil was compared [Table 1]. The lower AV for oil recovered from TPP method showed 1.60 ± 0.7 mg KOH/g indicates that it contains less free fatty acid. The PV of SFE oil is slightly higher than the soxhlet and SOL extraction methods indicating the presence of some peroxides in plant material which have been extracted due to high pressure. Although, there was no significant difference in the parameters among the oils extracted through five different extraction methods and within the limit as mentioned in codex standard for named vegetable oils.
High performance thin layer chromatography profiling
The solvent system hexane:diethyl ether:acetic acid (7:4:1, v/v) was found best for the separation of the constituents of the extracted oils sample. The comparative high performance thin layer chromatography (HPTLC) finger printing of oils extracted was found matching with the presence of different common compounds at matching Rf [Figure 1]. The maximum component was observed in TPP method. The oils by SOXH and US extraction methods showed a similar pattern of separation. SOL extraction and SFE technique showed maximum components when the developed HPTLC was scanned at 370 nm. The substance at Rf0.58 comprised the highest % area.
Gas chromatography-mass spectrometry analysis
The fatty acid compositions of extracted oil of flaxseed are shown in Table 2. Alpha-linolenic acid and linoleic acid were found to be major fatty acids in the five extracted samples. The sharp peak around 23.09 min was identified as linolenic acid methyl ester [Figure 2] and the peak around 22.90 min. was identified as the linoleic acid methyl ester. SFE extract contained 70.16% of the n-3 fatty acid and 14.50% n-6 fatty acid. The total PUFA was the highest in TPP extraction method.
Fourier-transformed infrared spectroscopy fingerprinting of extracted fixed oil of flaxseeds
The FT-IR spectrum showed the characteristics bands associated with 2924 cm−1 (CH2 - asym stretching), 2856 cm−1 (CH2 - symmetric stretching). The band at 3008 cm−1 assigned to the C-H stretching vibration of the cis- double (=CH). The strong peak demonstrated at 1743 cm−1 is attributed to ester carbonyl functional group of triglyceride [Figure 3].
In vitro antioxidant activity of supercritical fluid extraction extracted fixed oil
The oil of flaxseed showed a good antioxidant activity, IC50 of standard ascorbic acid was 30.09 μg/mL whereas that of oil was 60.57 μg/mL. In super oxide anionic scavenging method, the fixed oil was less active with IC50 241.60 µg/mL as compared with the standard (ascorbic acid, IC50 217.40 μg/mL). These results can be attributed to the higher level of unsaturated fatty acids.
Conclusion
The result of gas chromatography-mass spectrometry analysis, FT-IR spectroscopy, and HPTLC showed that all the major peaks are present in each sample; indicated by the superimposed spectra. It means that all the oils obtained using different extraction methods are chemically similar and contains all major components. Considering the factors such as time, economy, and also the environment, SFE was found the best. The finished product is of good quality as it was solvent free.
Financial support and sponsorship
Nil.
Conflicts of interest
There are no conflicts of interest.
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