The sample used for the study was used to oil extracted from a sancho (Zanthoxylum schinifolium Siebold & Zucc.) tree farm located in Korea. Sancho seed-pods were collected on 5^th October, 2013 and then the oil was immediately extracted from them. The extraction of sansho oil was performed by the compression method. After picking the sancho seeds, they were extracted with a extraction machine (Gaebangagan, Oscarelectronic Co., Ltd., Gimhae, Korea). The oil was cooled at room temperature and centrifuged at 2,500 rpm for 5 minutes using a centrifuge. The supernatant was collected and stored at 5℃ and used as a sample.

We added 10㎖ phosphoric acid and acetic acid that concentration each of 0.05 - 0.3% into 100 g of sancho oil, and then the efficiency was measured according to the degumming effect. Degummed sancho oil was analyzed based on the method described below, to obtain the acid value, polyphenol content, antioxidant activity and fatty acid content.

We prepared 5 N H₂SO₄, 0.01 M potassium antimonyl tartrate, 4% ammonium molybdate, and 1M ascorbic acid by mixing reagents. These reagents (50㎖ of 5 N sulfuric acid, 5㎖ potassium antimonyl tartarate, 15㎖ ammonium molybdate, and 30㎖ ascorbic acid) were mixed and adjusted to a total volume of 100㎖, and we then mixed 5㎖ sancho oil with 0.8㎖ of the mixed reagents. These mixtures were allowed to stand for 30 minutes, and then 2㎖ of 95% EtOH was added to each mixture vortexing again, then the absorbance was measured using 880 ㎚ UV/Vis Spectrophotometer (LibraS22, Biochrom, Cambridge, England). 0.8㎖ KH₂PO₄ was added into each reagent mixture and kept for 30 minutes in order to examine the standard calibration curve of phosphate. We added 2㎖ of 95% EtOH into the reagent mixtures and vortexed again, and then measured the absorbance using 880㎚ VIS Spectrophotometer. Total phosphorus content was calculated using the calibration curve.

In order to calculate the yield and acid value of oil, we added 1 - 3 N sodium hydroxide (NaOH) into 100 g of sancho oil, and then examined the mixture after the addition of an optimal concentration of NaOH. The mixture of sancho oil and NaOH was reacted in a water bath with temperature of 80℃, and then stirred for 10 min using a glass rod and the sample was further reacted for 30 min. The deoxidized oil was analyzed based on the method described below, to obtain the acid value, polyphenol content, antioxidant activity and fatty acid content.

After the input of 0.3% Chinese pepper oil, 1㎖ ether was collected for dilution of the sancho oil. Sancho oil was diluted with 1㎖ 95% ethanol, 5㎖ of distilled water and 0.5㎖ 1 N Folin-Ciocalteau (Sigma-Aldrich Co., St. Louis, MO, USA), and then vortexed and reacted for 1 hour under dark condition. The total polyphenol content of the sample was calculated by using tannic acid as standard, and then measured with a 725㎚ Vis Spectrophotometer (Vernier Software and Technology, Beaverton, OR, USA).

Analysis of fatty acid (FA) content in refined sancho oil was conducted by slightly modified method of Bae et al. (2011). For the preparation of FAME, the AOAC method (2001) was modified as follows: we weighed 0.5 g of the oil sample accurately in a flask (30㎖) and 5㎖ of 0.5M methanolic sodium hydroxide (Sigma-Aldrich Co., St. Louis, MO, USA) solution was added for the saponification. Reflux condenser (15㎝, in length) was attached to the flask and heated at 140 - 160℃ for 5 min. on a heating mantle. 5㎖ of boron trifluoride-methanol (Sigma-Aldrich Co., St. Louis, MO, USA) was then added from a pipet through a condenser and we continued heating at 140 - 160℃ for 5 min.

The flask was removed from the heating mantle and refluxed, and the solution allowed at room temperature. 5㎖ of n-hexane (Sigma-Aldrich Co., St. Louis, MO, USA) and about 10㎖ of saturated sodium chloride (Sigma-Aldrich Co., St. Louis, MO, USA) solution were added, and then the content was agitated thoroughly. An aliquot of the supernatant hexane layer was transferred into a Pasteur pipette (5 3/4 inch) packed with anhydrous sodium sulfate (about 1 g) to remove water. Following filtration, 0.1㎕ of filtrate was analyzed using gas chromatography (GC).

The purified samples were analyzed using GC (Agilent GC 6890, Agilent Technologies Inc., Santa Clara, CA, USA). Analysis column was used for DV-WAX (60 m × 0.25㎜ id, 0.25㎛). The mobile phase used was a nitrogen gas, and the moving speed was set at 1.3㎖/min. The amount injected was 0.1㎕ and the split ratio was 1 : 60. Temperature settings were as follows: injector 230℃; FID 230℃. The oven temperature was maintained at 160℃ for 5 min, and then programmed to 210℃ at 4℃/min, and maintained for 6 min at 210℃. GC peak areas were integrated with a Chromato integrator (D-2500, Hitachi Ltd., Tokyo, Japan).

The peak areas were normalized using unity response factors. The total amounts of palmitic (C_{16 : 0}), stearic (C_{18 : 0}), oleic (C_{18 : 1}), linoleic (C_{18 : 2}), and linolenic (C_{18 : 3}) acids were calculated as hundredth of a percent. All traces of FA less than 0.1% or a specific FA found in limited amounts in the oil were excluded in this study for the simple comparison.

We measured the antioxidant activity of refined oil by using the DPPH method (Kiers et al., 1976). The measurement wavelength of DPPH was 517㎚ in UV/ VIS-spectrophotometer (Uvikon XL, BIO-TEK, Winooski, VT, USA).

Change of acid value in sancho oil treat by degumminf and deoxygenation, and polyphenol content, saturated and unsaturated fatty acid content according to the purification process were analyzed by One-way ANOVA using the IBM SPSS statistical package, and significant differences among treatments were tested at the 5% significance level, Duncan's Multiple Range Test (DMRT).

The results showed that the density of sancho oil is 0.903㎏/㎥ (Table 1). In general, the density value of the edible oil in Korea differed from the 0.92 - 0.94㎏/㎥ value (Korea Food Sanitation Act Food Sanitation Act. Act No. 10787). The extracted sancho (Zanthoxylum schinifolium Siebold & Zucc.) oil was 0.906㎏/㎥, similar to other edible oil. Also, crude fatty acid content of sancho oil was 61.8%.

Degumming efficiency differed depending on the phosphoric acid and acetic acid treatments (Fig. 1). The acetic acid treatment had a better phosphorous removal effect than the phosphoric acid treatment. Degumming efficiency of sancho oil increased depending on the concentration of the two acids treatments. Degumming efficiency using 0.05% acetic acid was about 80% higher compared to the non-treatment. Also, the degumming efficiency of the 0.15% phosphoric acid treatment was dramatically improved. However, the reason for this improvement is unknown. The use of acetic acid showed an efficiency of about double that of phosphoric acid.

Fig. 1  Phosphorus content of sancho oil in accordance with the phosphoric acid and acetic acid treatment.

Values are reported as the means of six replicates ± standard deviation, and the alphabetical words in the Figure show the results of the DMRT analysis (p < 0.05).

Degumming process was not give to significant change for the acid value of sancho oil. (p-value : 0.392) (Fig. 2). Although mean of acid value was higher acetic acid than phosphoric acid, there was not shown the significant different. (p-value : 0.123) In degumming process, slightly increase of acid value be likely to degumming with only use water (data not shown).

Fig. 2  Change of acid value in sancho oil treat by acetic acid and phosphoric acid.

Acetic acid and phosphoric acid concentration; 0.15%. Values are reported as the means of three replicates ± standard deviation, and the alphabetical words in the figure show the results of the DMRT analysis (p < 0.05).

The precipitate amount in sancho oil increased with increase in NaOH concentration (Fig. 3). The formation of precipitates was not significantly influenced by the concentration of NaOH.

Fig. 3  Change of the precipitate and acid value in sancho oil treated with different concentrations of NaOH.

Values are reported as the means of three replicates ± standard deviation, and the alphabetical words in the Figure show the results of the DMRT analysis (p < 0.05).

Acid value also differed depending on the NaOH concentration, as it decreased with increase in NaOH concentration (Fig. 3A). When treated with 1 N NaOH, acid value of oil somewhat decreased. The amount of precipitate increased rapidly with NaOH treatment (Fig. 3B). The amount of precipitate increased as the concentration of NaOH increased. Oil yield decreased with the amount of NaOH treated (Fig. 3C). Oil yield decreased with increasing NaOH concentration.

The total polyphenol content was dramatically decreased through the purification process (Fig. 4). Polyphenol content of untreated sancho oil was 667 ppm. Polyphenol content in degummed and deoxidated oil was reduced to 44.5% and 46.4%, respectively. The content of total polyphenol in sancho oil after completing all the purification processes was reduced by approximately 9% compared to the raw material.

Fig. 4  The variation of polyphenol content in sancho oil in accordance with the purification process.

Values are reported as the means of three replicates±standard deviation, and the alphabetical words in the Figure show the results of the DMRT analysis (p < 0.05).

Antioxidant activity of the sancho oil was different for each refining process (Fig. 5). Antioxidant activity was decreased in the degumming process and then increased again in the deoxygenation process.

Fig. 5  The antioxidant activity of Sancho oil in accordance with the purification process.

The saturated fatty acid content varied according to the concentration of NaOH (Fig. 6). Myristic acid (C_{14 : 0}) and stearic acid (C_{18 : 0}), which are saturated fatty acids contained in the sancho oil, decreased with increased concentration of NaOH. The amount of stearic acid (C_{18 : 0}) was less than that of myristic acid (C_{14 : 0}) in the 50 ppm NaOH concentration.

Fig. 6  The content of saturated fatty acid in sancho oil in accordance with the NaOH treatment.

Values are reported as the means of three replicates±standard deviation, and the alphabetical words in the Figure show the results of the DMRT analysis (p < 0.05).

For the unsaturated fatty acids, the contents of palmitic acid (C_{14 : 1}) and palmitoleic acid (C_{16 : 1}) slightly decreased with increase in the concentration of NaOH used in the deoxygenation process (Fig. 7). However, the contents of unsaturated fatty acids were not significantly influenced by the concentration of NaOH. The contents of unsaturated fatty acids were much decreased after treatment with 10 ppm NaOH concentration. The palmitic acid content of 100 g/ℓ decreased by 44.6% compared to the control.

Fig. 7  The content of the unsaturated fatty acids in sancho oil in accordance with the NaOH treatment.

Values are reported as the mean of three replicates ± standard deviation, and the alphabetical words in the Figure show the results of the DMRT analysis (p < 0.05).

In 10 ppm NaOH concentration, palmitoleic acid content of 195 g/ℓ was reduced by 43.5% compared to the control. Oleic acid (C_{18 : 1}), linoleic acid (C_{18 : 2}), and linolenic acid (C_{18 : 3}) showed the greatest decrease rate when treated with 10 ppm NaOH concentration. The contents of oleic acid, linoleic acid and linolenic acid decreased by 52.5%, 59.4% and 56.8%, respectively, whereas comparatively, the contents of palmitic acid (C_{14 : 1}) and palmitoleic acid (C_{16 : 1}) decreased by about 10%. However, the content of oleic acid (C_{18 : 1}) experienced an increase even though it was lower compared to that of the control experiment (p value < 0.05).

The content of saturated fatty acids in the sancho oil reduced while passing through the purification process (Fig. 8). The content of myristic acid (C_{14 : 0}) was higher than that of stearic acid (C_{18 : 0}) in the degumming process, but it was similar in the deoxidation process. The degree of reduction varied according to the type of saturated fatty acids (Fig. 8A). The decline of myristic acid (C_{14 : 0}) content was twice more than that of stearic acid (C_{18 : 0}). Therefore, the NaOH used in the deoxygenation step was more effective in the removal of lower fatty acids than the higher fatty acids.

Fig. 8  Changes in the saturated fatty acids contents in sancho oil according to the purification process.

Values are reported as the mean of three replicates±standard deviation. and the alphabetical words in the Figure show the results of the DMRT analysis (p< 0.05).

The content of the unsaturated fatty acids; palmitic acid (C_{14 : 1}) and palmitoleic acid (C_{16 : 1}) decreased depending on the purification process, with the reduction rate similar to that of the saturated fatty acids in the degumming process (Fig. 8B).

The content of Palmitic acid (C_{14 : 1}) was reduced by about 2.4% in the degumming process, and about 23.7% in the deoxidation process. The Palmitoleic acid (C_{16 : 1}) content was also reduced by 1.7% in the degumming process, and 17% in the deoxidation process. This decline in the degumming process was 10 times higher than in the deoxidation process.

The contents of major unsaturated fatty acids such as oleic acid (C_{18 : 1}) and linolenic acid (C_{18 : 3}) decreased in the degumming process (Fig. 9). Also, the contents of oleic acid (C_{18 : 1}) and linolenic acid (C_{18 : 3}) decreased in the degumming process.

Fig. 9  Changes in the unsaturated fatty acids contents in sancho oil according to the purification process.

Values are reported as the mean of three replicates±standard deviation. and the alphabetical words in the Figure show the results of the DMRT analysis (p < 0.05).

When treated with NaOH, the decomposition rate of high fatty acids in sancho oil was greater than that of low fatty acids. The linoleic acid (C_{18 : 2}) content showed a large reduction of about 39.3% in the degumming process, whereas its content in the deoxidation process was increased by about 7%. This means that the unsaturated fatty acid, linoleic acid (C_{18 : 2}) is easily decomposed by an acid. In addition, the increase in the contents of linolenic acid (C_{18 : 3}) and linoleic acid (C_{18 : 2}) in the NaOH treatment appears to be due to decomposition.