Natural products such as plant extracts, provide unlimited opportunities for new drug discoveries because of the unmatched chemical diversity. This has captured the interest of many researchers to explore local medicinal plants for valuable medicinal traits. Antioxidants are substances that protect against damaging oxidative processes in biological and environmental systems, inhibiting the harmful chain reactions initiated by free radicals and other oxidative processes(Halliwell et al., 1991). Naturally active antioxidants can play major roles in health promoting activities(Braga et al., 2003; Barjaktarovic et al., 2005; Carvalh et al., 2005; Hwang et al., 2005). Litsea cubeba contains a range of active substances with high therapeutic potential(Bighelli et al., 2008; Blois, 1958; Jiang et al., 2009)such as antimicrobial(Wang et al., 2010), anticancer(Ho et al., 2010), antioxidan and insecticidal activities(Park et al., 2007; Amer et al., 2006; Pumnuan et al., 2010; Feng et al., 2009). Because Litsea cubeba has biological functions and potential applications, it has become attractive to researchers. A number of reports have been previously published on the extraction of the essential oils from Litsea cubeba(Yang et al., 2010; Zhao et al., 2010; Wang et al., 1999). Previous researchers also reported the qualitative and quantitative analysis on essential oils composition from L. cubeba.

Although there are a considerable number of reports on L. cubeba extraction and biological activity, only a limited number of studies appear on the most efficient methods to obtain antioxidant fractions and the correlation between chemical composition and antioxidant activity of extracts. Biological antioxidant capacity of natural product extracts mainly depends on their composition and their combined action, various biological activities may be derived from different extraction methods(Huang et al., 2005). In this context, the objective of this work is to compare the chemical compositions derived from different extracted methods and identify a reliable and practical method for the liberation of desired natural antioxidants from L.cubeba.

The antioxidant extracts of L.cubeba were obtained using liquid-liquid extraction, SFE(CO₂ super-critical fluid extraction) and steam distillation extraction. The chemical compositions were determined by using gas chromatography-mass spectrometry(GC-MS). The antioxidant activities were measured by scavenging activity against 1, 1-diphenyl-2-picrylhydrazyl(Ho et al., 2010), The IC₅₀ value was calculated from the results.

Dry fruits of L.cubeba were purchased from Jianmin drugstore in Fuan city of Fujian Province.

An ISCO SFX^TM super-critical fluid extractor from Teledyne ISCO(Lincoln, NE, USA). UV and visible spectrum-photometer CARY100 bio-VARIAN EL05063009 was used to measure the absorbance for the DPPH test.

Dichloromethane was of AR grade supplied from Fisher Scientific, Atlanta, GA, USA. Citral(96%)mixture of cis and tran, 1, 1-diphenyl-2-picrylhydrazyl(DPPH) and 1, 4-Dibromobenzene were HPLC grade purchased from Sigma-Aldrich.(Sigma Aldrich, St. Louis, MO, USA). Butylated hydroxyanisol(artificially synthesized antioxidant, analytical st and ard)were produced by sigma-aldrich(Sigma, USA).

1)Liquid-liquid extraction with ultra-sonic assistance   Five grams of dry L.cubeba seeds were ground to a powder. The powder was taken up in 20 mLs of CH₂Cl₂ and 10 mL of saturated NaCl solution in a 100 mL breaker, employing an ultrasonic probe to disrupt the cell structures of the plant. The beaker containing the mixture was subject to ultrasonic treatment for 10 min. The resulting slurry was digested for 3 days in room temperature(20 ℃). Using a separating funnel, the lower organic solvent layer was extracted and condensed to get the crude oil.

2)Liquid-liquid extraction    Five grams of dry L.cubeba seeds were ground to a powder, 20 mL of CH₂Cl₂ was added followed by 10 mL of saturated NaCl solution in a 100 mL beaker. The resulting slurry was digested for 3 days in room temperature(20℃). Using a separating funnel the lower organic solvent layer was extracted and condensed to get the crude oil.

3)Liquid-liquid extraction with magnetic assistance    Five grams of dry Litsea cubeba seeds were ground to a powder, 20 mL of CH₂Cl₂ was added followed by 10 mL of saturated NaCl solution in a 100 mL beaker. The breaker was placed above a U-shape magnetization iron and allowed to digest inside the magnetic field for 3 days. Using a separating funnel the lower organic solvent layer was extracted and condensed to get the crude oil.

4)Steam distillation extraction    The 100 g powder of crushed seed of L.cubeba plant materials were placed in a flask(2 L)together with double distilled water(1.5 L). The mixture was boiled for 4 hours, followed by exhaustive extraction of the distillate with CH₂Cl₂, The extract was condensed in cooling vapor to collect the essential oil.

5)SFE    Five grams of dry L.cubeba seeds were ground to a powder and placed inside a supercritical fluid extractor vessel. The powder was extracted at a pressure of 31 MPa and a temperature of 100 ℃ for one hour to get the essential oil.

The overall yield of oil was calculated as Y(%): where Y=mass of extracts / mass of crushed seed ×100. All essential oils were stored at a temperature of -4 ℃ until used for the analysis

The dichloromethane solutions of the extracts were analyzed by GC-MS. GC-MS samples was prepared by taking a 100 μL aliquot of the oil. The oil was dried over anhydrous sodium sulphate prior to GC-MS analysis. To this was added 5 μL of 1, 4-Dibromobenzene(20 mg ·mL^-1)solution which served as an internal st and ard and 900 μL CH₂Cl₂(reagent grade)to make up 1mL GC-MS tested sample. GC column was a DB-5 length 30 m, diameter 0.25 mm, phase thickness 0.25 μm. GC Oven parameters were initial temperature of 40 ℃ and held for 1 min followed by a 10 ℃ ·min^-1 ramp to 300 ℃ and held for 1 min, inlet was maintained at 280 ℃. Mass spectrometer was electron impact at 70 eV in full scan mode from 50 to 550 amu.

Compounds were identified by comparison of their mass spectra with those of the NIST 98 mass spectral database. The components were analyzed and identified with GC-MS and relative index(RI value)in the same column compared with st and ard mixtures of authentic 1, 4-Dibromobenzene. For quantification, relative peak area percentages were used and normalized with the internal st and ard(accurate 1, 4-Dibromobenzene added and response peak)to obtain the relative content of major component without the use of correction factors. Peak area normalization was used to get the relative amount of main compounds in the respective extracts.

The extracted oils were diluted in pure methanol(reagent grade)giving a range of 100, 50, 25, 12.5, 6.5 mg ·mL^-1. 0.1 mL of each dilution was placed in a test tube in duplicate. The reaction was initiated by addition of 1.9 mL DPPH solution(51.54 mg ·L^-1 in methanol). Using a UV-Vis spectrophotometer, the absorbance was read at 517 nm until the reading reached a plateau. IC₅₀ value was determined from the plotted graph of scavenging activity versus the concentration of essential oils, which was defined as the total antioxidant necessary to decrease the initial DPPH radical concentration by 50%.

The DPPH radical scavenging activity was calculated with the following formula: DPPH radical scavenging activity(%)=[A₀-(A₁-A_S)]/A₀ × 100. Where A₀ is the absorbance of the control solution containing only DPPH after incubation; A₁ is the absorbance in the presence of plant extract in DPPH solution after incubation; and A_S is the absorbance of sample extract solution without DPPH for baseline correction arising from unequal color of the sample solutions(optical blank for A₁). The higher the DPPH radical scavenging activity, the more powerful the antioxidant activity of sample which results in a lower IC₅₀ value(Blois, 1958). For comparison a sample of Butylated hydroxyanisol(BHA)a man made antioxidant commonly added to food was also analyzed by DPPH to reveal its IC₅₀ value using this method.

Tab.1 shows the oil yields and DPPH radical scavenging rates based on the different extraction methods used. The yellow or coffee colored essential oil obtained in yields from 0.30 to 26.3%, The ultrasonic assisted liquid-liquid extraction exhibiting the highest oil yields of 26.34% with higher antioxidant activity of 32.16 mg ·mL^-1 IC₅₀ value.

Tab.1 The DPPH radical scavenging activity on rate of extraction with different method

Tab.1 The DPPH radical scavenging activity on rate of extraction with different method Extraction method Yield of crude oil (W/W)(%) Regression equation IC50/ (mg·mL-1) Extracts colour R2 Liquid-liquid extraction with ultra-sonic assisted 26.3 Y=0.79x + 24.51 32.16 Coffee 0.914 Liquid-liquid extraction 18.4 Y=0.78x + 21.33 36.95 Coffee 0.917 Liquid-liquid extraction with magnetic assisted 19.4 Y=0.74x + 25.82 31.22 Coffee 0.976 Steam distillation extraction 0.26 Y=0.75x + 1.35 64.95 Yellowish 0.975 SFE 0.69 Y=0.51x + 210 166 56.95 Light yellow 0.909 Standard 96% authentic Citral Y=0.64x + 2.25 74.33 Transparent 0.948 Butylated hydroxyanisol (BHA) Analytical standard Y=3.687 9x+45.628 1.185 4 Transparent 0.961 4

Two-fold dilutions of all crude essential oil extracts were made with methanol and tested from a starting dilution 100 to 6.25 mg ·mL^-1. There was a positive correlation between radical scavenging rate and the concentration of the essential oil. Tab.1 also shows the positive influence of ultrasonic or magnetic agitation extraction on antioxidant activity.

Tab.2 showed probable identity of substances and the relatives percents obtained in GC-MS analysis on extracts obtained using different extraction methods. The retention times of Citral(mixture of cis and trans)stayed are 11.06(trans) and 11.47(cis)minutes. Internal st and ard 1, 4 dibromobenzene had a retention time of 10.49 minutes. Retention times obtained were from 6.26-32.79 min, indicating more than 122 identified compounds including 12 kinds of fatty acids, 16 kinds of terpenes, 18 kinds of oxygenated terpenes, and other trace compounds including alkenes, alcohols, ketones, alkanes from these extracts. The observed variations in the profile and amounts of individual components have resulted from the variations in the extraction methods used.

Tab.2 Probable identity of substances and the relatives percents obtained in GC-MS analysis

Tab.2 Probable identity of substances and the relatives percents obtained in GC-MS analysis Substances Molecular Formula Molecular Weight Relative composition on extraction method (%) Similarity (%)≥ (1) (2) (3) (4) (5) 1 Dodecanoic acid C12H24O2 200.32 28.55 26.99 27.08 2.95 11.55 93 2 n-Decanoic acid C12H24O2 200.32 7.97 7.50 8.61 4.30 5.27 94 3 Tetradecanoic acid C14H28O2 228.36 0.48 0.64 7.80 0.65 0.24 98 4 N-Hexadecenoic acid C16H32O2 256.43 5.63 4.11 0.29 / / 95 5 Tridecanoic acid C13H26O2 214.34 0.43 / / / 0.24 93 6 Octadecadienoic acid C18H30O2 278.43 / / 0.53 / / 42 Total of saturated fatty acid 43.06 39.24 44.31 7.90 17.30 1 9-Octadecenoic acid C18H34O2 282.46 13.98 10.47 14.98 / / 98 2 9,12-Octadecadienoic acid C18H32O2 280.45 5.68 3.63 7.31 / / 97 3 9-Hexadecenoic acid C16H30O2 254.41 0.12 / / / / 42 4 Trans-2-undecenoic acid C11H20O2 184.28 / 0.17 0.01 / / 45 5 2-Myristynoic acid C14H24O2 224.34 / / / 0.20 / 52 6 10,12-Octadecadiynoic acid C18H28O2 276.41 / / / / 0.34 48 Total of unsaturated fatty acid 19.78 14.27 22.30 0.20 0.34 1 D-limonene C10H16 136.23 1.50 1.80 1.52 5.52 1.55 95 2 Camphene C10H16 136.23 0.10 0.14 0.11 0.33 0.06 94 3 β-Pinene C10H16 136.24 0.25 0.32 0.33 0.76 / 97 4 α-Pinene C10H16 136.24 0.33 0.39 / 0.25 / 97 5 Bicyclo[3.1.0] hexane C10H16 136.24 0.38 0.46 0.46 0.86 0.35 91 6 β-Myrcene C10H16 136.24 0.07 0.15 0.13 0.47 59 7 Bicyclo[3.1.1] heptane C10H16 136.24 / / / 0.28 / 49 8 1,4,9-Decatriene C10H16 136.23 0.52 / / / / 47 9 3-Carene C10H16 136.23 / / / / 1.20 43 10 Bicyclo[3.1.0] hex-2-ene C10H16 136.23 0.38 / / / 0.35 91 12 Copaene C15H24 204.35 0.12 0.06 0.14 / / 96 13 Ylangene C15H24 204.35 / 0.15 / / / 45 14 α-Caryophyllene C15H24 204.35 0.03 4.11 0.57 0.35 80 15 1,6,10-Dodecatriene C15H24 204.35 / / 0.13 / / 47 16 Bicyclo[7.2.0] undec-4-ene C15H24 204.35 0.32 0.09 0.31 / / 55 Total of terpene 4.00 7.67 3.70 8.47 3.86 1 Cis-citral C10H16O 152.24 0.13 0.08 0.09 1.74 2.29 99 2 Trans-Citral C10H16O 152.24 0.24 0.17 0.18 2.31 3.77 96 3 Caryophyllene oxide C15H24O 204.36 / 0.56 0.57 0.50 2.32 87 4 Camphor C10H16O 152.24 / / / 0.49 / 94 5 1,5,7-Octatrien-3-ol C10H16O 152.24 / / / 0.07 / 45 6 Bicyclo[3.1.1] hept-3-en-2-ol C10H16O 152.25 / / / 0.44 / 43 7 3-Cyclohexen-1-ol C10H18O 154.25 / / / 1.43 / 49 8 1,6-octadien-3-ol C10H18O 154.25 0.30 0.05 0.28 3.54 1.95 80 9 Cis-beta-terpineol C10H18O 154.25 0.11 0.01 0.12 / / 84 10 Eucalyptol C10H18O 154.25 0.46 0.52 0.41 4.56 0.48 98 11 Cis-beta-terpineol C10H18O 154.25 / 0.01 0.12 / / 49 12 Terpineol C10H18O 154.25 0.11 0.12 0.08 0.56 / 68 13 2,6-Octadien-1-ol C10H18O 154.25 1.07 0.48 0.84 / 1.07 72 14 1,6-Octadien-3-ol C10H18O 154.25 / / 0.28 3.54 1.95 80 15 2-Cyclohexen-1-ol C10H18O 154.25 0.17 0.08 0.03 / / 30 16 5,7-Octadien-3-ol C10H18O 154.25 / / / 0.59 / 38 17 Bicyclo[3.1.0] hexan-3-ol C10H18O 154.25 / / / 0.86 / 49 18 Isoborneol C10H18O 154.25 / / / 0.27 / 59 Total of oxygenated terpenes 2.49 2.08 3.00 20.90 13.83 Total of alcohols 1.37 1.00 2.40 2.93 1.67 Total of ketone 0.08 3.86 0.55 2.89 5.28 Total of others alkene 0 3.80 0.87 4.48 1.90 Total of alkane 3.73 1.86 5.99 0.83 6.78

As can be seen in Tab.2, the composition of the crude oil obtained using liquid-liquid extractions were primarily of fatty acids(53.51%-66.61%), including saturated fatty acids such as dodecanoic acid(26.99%-28.55%), monounsaturated fatty acids such as 9-octadecenoic acid(10.47%-14.98%), and 9, 12-octadecadienoic acid(3.63%-7.31%). Ultrasonic or magnetic agitation general extracted higher fatty acid yields up to 62.84% and 66.61% respectively. A higher yield of terpenes(8.47%) and oxygenated terpenes(20.90%)were obtained from steam distillation, namely D-limonene(5.52%), eucalyptol(4.56%), citral(4.05%) and 1, 6-octadien-3-ol(3.54%). Meanwhile, SFE extraction yields 17.30% of saturated fatty acids, 0.34% of monounsaturated fatty acids, 3.86% of terpenes and 13.83% of oxygenated terpenes.

As shown in 1,2,3, liquid-liquid extractions obtained a higher proportion of fatty acids in qualitative and quantitative composition, while more abundant odor-activevolatile compounds such as terpenes, alkenes and oxygenated terpenes were detected from the steam distillation(method 4) and SFE(method 5). Both steam distillation and SFE extraction were rich in terpene compounds which showed lower antioxidant activity(IC₅₀ values of 64.95 and 56.95 mg ·mL^-1 respectively)in comparison to the liquid-liquid extracts. Meanwhile shown in Tab.1, HPLC grade citral exhibited the lowest antioxidant activity of IC₅₀ values of 74.33 mg ·mL^-1. Thus, taken together, this study concludes that extraction of substances with the greatest antioxidant properties is enhanced by ultrasonic and magnetic mechanisms.

Tab.3 Relatives content of main composition obtained in GC-MS analysis

Tab.3 Relatives content of main composition obtained in GC-MS analysis Rt Main compound Relative content(mg·mL-1) to 1,4-Dibromobenzene Liquid-liquid extraction 1,2,3 4. Steam distillation 5. SFE 1.Ultrasonic treat 2. No assisted treat 3.Magnetic treat 6.26 α-Pinene 0.003 0.003 4 / 0.006 35 / 6.55 Camphene 0.000 91 0.001 3 0.001 7 0.008 4 0.000 21 7.02 β-Pinene 0.002 3 0.002 75 0.005 0 0.001 9 / 7.15 β-Myrcene 0.000 34 0.001 3 0.002 0 0.001 2 7.84 D-limonene 0.013 7 0.015 5 0.023 03 0.014 02 0.005 4 13.04 Copaene 0.001 1 0.000 52 0.002 1 / / 13.04 Ylangene / 0.001 3 / / / 13.65 Caryophyllene 0.000 27 0.035 3 0.008 6 / 0.00123 Total of main terpene 0.021 62 0.060 0 0.042 5 0.031 87 0.006 8 7.91 Eucalyptol 0.004 2 0.004 5 0.006 21 0.011 6 0.001 68 8.51 Terpineol 0.002 0 0.001 0 0.003 03 0.001 42 / 8.93 1,6-octadien-3-ol 0.002 7 0.000 43 0.004 242 0.009 0 0.006 8 9.77 Camphor / / / 0.001 24 / 10.02 Bicyclo[3.1.0] hexan-3-ol / / / 0.002 2 / 10.25 2-Cyclohexen-1-ol 0.001 5 0.000 7 0.000 45 / / 11.06 Cis-citral 0.001 2 0.000 69 0.001 4 0.004 42 0.008 03 11.47 Trans-Citral 0.002 2 0.001 46 0.002 7 0.005 9 0.013 23 12.86 5,7-Octadien-3-ol / / / 0.001 5 / 14.11 1,6-Octadien-3-ol / / 0.004 242 0.009 0 0.006 84 15.70 Caryophyllene oxide / 0.004 81 0.008 6 0.001 27 0.011 65 16.61 1,5,7-Octatrien-3-ol / / / 0.000 2 / 26.03 2,6-Octadien-1-ol 0.009 7 0.004 1 0.012 73 / 0.00375 Total of main oxygenated terpenes 0.023 5 0.017 7 0.045 43 0.047 75 0.051 98 12.75 N-decanoic acid / 0.064 4 0.130 4 / 0.0185 15.21 n-Dodecanoic acid 0.072 6 / / 0.011 / 15.27 Dodecanoic acid 0.260 1 0.230 9 0.410 3 0.007 5 0.040 5 16.11 z-7-Tetradecanoic acid` 0.000 5 0.002 5 / / / 17.49 Tridecanoic acid 0.003 9 / / / 0.000 84 19.47 Hexadecenoic acid / / 0.004 4 / / 19.73 N-Hexadecenoic acid 0.051 3 0.035 3 / / / 19.74 Tetradecanoic acid 0.003 9 0.003 0 0.118 2 0.001 7 0.008 4 23.06 Octadecadienoic acid / / 0.008 0 / / Total of main saturated fatty acids 0.396 8 0.336 1 0.671 3 0.010 3 0.068 24 7.21 2-Myristynoic acid / / / 0.000 51 / 10.34 10,12-Octadecadiynoic acid / / / / 0.001 2 15.09 9-Hexadecenoic acid 0.001 1 / / / / 22.44 9,12-Octadecadienoic acid 0.051 7 0.031 2 0.110 7 / / 22.57 9-Octadecenoic acid 0.127 4 0.089 9 0.226 9 / / Total of main unsaturated fatty acid 0.180 2 0.121 1 0.337 6 0.000 51 0.001 2

The results obtained in the present study showed that ultrasonic or magnetic assisted liquid-liquid extraction has advantages that include simplified equipment usage, higher extraction rates and antioxidant activity, with the antioxidant activity mainly related to the complex variety of fatty acids and terpenes within extracts.

Litsea cubeba seeds are known to contain monoterpenes(C₁₀H₁₆), sesquiterpenes(C₁₅H₂₄), oxygenated terpenes(such as citral), alkenes and unsaturated fatty acids(such as 9-Octadecenoic acid, 9, 12-Octadecadienoic acid), which are rich in with the majority of these possessing unsaturated double bonds of C=C which are the main reasons for their biological activity of natural extraction.

Steam distillation requires low-cost equipment, and as a result is more available in rural areas where L.cubeba is grown and harvested. Steam distillation with higher temperatures will increase the extraction rate of small volatile compounds such as mono-terpenes and oxygenated terpenes. However, long extraction times at elevated temperatures may destroy some thermo-sensitive compounds or others compound with high molecule weight such as fatty acids shown in Tab.3.

According to the principle of similarly dissolved solutions, the solubility rate of the non-polarity fraction will be enhanced in a similar non-polarity solvent. Carbon dioxide is an ideal solvent for the extraction of natural products which are normally non-polar. However, its non-polarity means that polar compounds may only be slightly soluble in CO₂, which is the main reason to explain the Supercritical carbon dioxide extraction showing weak antioxidant activity(Tab.1), due to poor extraction of the more polar antioxidant compounds such as unsaturated fatty acids. Despite a lengthy extraction time, the liquid-liquid extraction is advantageous to obtain both polar and non-polarity compounds. Ultrasonic assistance resulted in additional disruption of the plant cells that lead to higher overall oil yields and higher antioxidant activity of essential oil.

The results obtained in the present study revealed that liquid-liquid extraction with magnetic assisted extraction(method 3)made a significantly positive affect on the antioxidant activity and yield of the main active compounds, particularly unsaturated fatty acid. In addition, unsaturated fatty acids, such as 9-octadecenoic acid, are well-known as natural active components that show significant antioxidant activity. Thus, making a legitimate inference that antioxidant biological activity has been mainly attributed from fatty acid composition rather than terpenes or oxygenated terpenes.

The enhancement observed with magnetic assistance may be attributed to the magnetic field as it has been reported that an energy field can change the microstructure of biomembrane, thus enhancing the substances diffuse rate(Gribova et al., 2008; Cosio et al., 2006). This magnetic effect was in accordance with that previously published and states that natural antioxidants are most completely extracted by maceration in a constant electric field(Gribova et al., 2008)This finding indicates that magnetic-effect extraction is an acceptable technique for in-depth studies for improving the extraction of substances with antioxidant capacity from L.cubeba.

Some previous studies have reported that Litsea cubeba have significant antioxidant properties and that the essential oil from the fresh fruit contains 75% citral(Deng et al., 2011). The present study did not produce these results. However, this could be explained in terms of attributed to a significance factors the citral mainly come from fresh fruit peel, while fatty acid composition primarily from dry fruit kernel(Ho et al., 2010; Duan et al., 2010).

Acknowledgements

The work was supported by the Florida International University, Department of Chemistry and Biochemistry, special thanks to Dr. Kenneth G. Furton for use of his supercritical fluid extractor.