Chemical Characterization of Chinese Perilla Seed Oil

: Physicochemical properties and chemical composition of Chinese perilla seed oil has been characterized in this study. The result showed that both the cold press oil and the solvent extracted oil possessed low acid value and peroxide value. The fatty acid composition result showed that the oil has high content of linolenic acid (C18:3) up to 66.4 g/100 g, followed by linoleic acid (C18:2) of 15.3 g/100 g. The total triacylglycerol (TAG) profiles results showed that the oil contained 20 TAGs including 17 regioisomers, including LnLnLn (35.8 g/100 g), LLnLn (20.2 g/100 g), LLLn (17.7 g/100 g) and PLnLn (14.9 g/100 g) (Ln, linolenic acid; L, linoleic acid; P, palmitic acid). With content of only 0.57 g/100 g oil, the unsaponifiable matters were mainly composed of phytosterols, squalene, tocopherol, alcohols and hydrocarbons. The total phytosterols content was 0.39 g/100 g oil, in which β -sitosterol has high content of 0.31 g/100 g oil.


Perilla seed oil extraction
Two methods were selected for oil extraction: solvent extraction and cold pressing. The perilla seed powder of 100 g was extracted by 600 mL n-hexane at 40 . The extraction was carried out for 8 h. The solvent was collected, evaporated and centrifuged, after which the perilla seed oil was obtained. Cold pressing was carried out using a screw oil press CA-59-G model, TBG, Germany without heat treatment. The obtained oil was stand for 30 min, and then centrifuged at 3500 r/min for 10 min to separate oil from sediments 11 . Both oil samples were stored at 20 in amber bottles to avoid oxidization.

Analysis of the perilla seed oil 2.3.1 Physicochemical parameters
Determination of acid value AV , peroxide value PV , saponification value, and unsaponifiable matter content of the oil was carried out according to AOCS methods 12 .
The tocopherol α -, β -, γ -and δ -isomers level of the oil were determined as described by 13 using an Agilent 1200 HPLC system Agilent, USA equipped with Supelcosil LC-Si column 250 4.6 mm, 5 μm Supelco, USA and fluorescence detector. The emission and excitation wavelength were set at 330 and 290 nm, respectively. The column temperature was held at 20 . The oil samples were dissolved in hexane at 100 mg/mL. A 5 μL volume of the loaded sample on the column was eluted with n-hexane/isopropyl ether 93/7, v/v isocratically at 1.5 mL/min. The absolute contents of tocopherols were determined by comparison with the calibrated standard curves.

Fatty acid composition
Derivatisation reaction of the oil was carried out by using the literature method 14 . Analysis of the obtained fatty acid methyl esters was carried out on Agilent 7890B gas chromatograph system equipped with BPX70 capillary column 30 m 320 μm 0.5 μm Agilent, USA and flame ionization detector FID . The column, injector, and detector temperatures were set at 210, 230, and 300 , respectively. The flow rate of carrier gas N 2 with split ratio of 1:50 was set at 50 mL/min. The fatty acids were identified with reference to the retention times of standard fatty acid methyl ester performed at the same conditions.

TAG profile analysis
Overall TAG profile analysis was carried out using offline 2D HPLC system coupled with atmosphere pressure chemical ionisation APCI MS according to the literature 15 . The oil sample was first separated by non-aqueous reversed-phase NARP using Agilent 1200 HPLC system equipped with an Agilent ZORBAX SB-C18 column 250 4.6 mm, 5 μm . Each fraction of the NARP eluent was collected following ten injections. Same fractions were combined and condensed by nitrogen dryer. Fractions collected in NARP dimension were solubilized in hexane, and then injected into the silver ion HPLC Ag-HPLC system, using Agilent 1200 HPLC system equipped with Agilent Chrom-Spher 5 Lipids column 250 4.6 mm, 5 μm . LC-MS detection was achieved through Agilent 6300 Mass Spectrometer Detector ion trap Agilent, USA equipped with APCI.

Unsaponifiable matters of the oil
Saponification reaction was performed at present of internal standards α -colestanol and eicosanol according to the literature 15 . The obtained unsaponifiable matters was then boiled with BSTFA TMCS 99:1 for derivatisation reaction.
Derivated unsaponifiable matters were analyzed by Agilent GC-7890B GC-MS with an HP-5MS column 30 m 250 μm 0.25 μm, Agilent, USA ; carrier gas: helium; flow rate: 1 mL/min; injection temperature: 280 ; detection temperature: 280 . The temperature was firstly kept at 50 for 1 min, increased from 50 to 150 at 8 /min and kept at 150 for 1 min, then increased from 150 to 300 at 5 /min, and kept at 300 for 10 min at last. MS parameters were as follows: electronic ionization voltage, 70 eV; ion source temperature, 250 ; scan range, m/z 50-550 16 . Unsaponifiable matters identification was performed by comparison of the retention times and mass spectra of eluting compounds to those of the Wiley library WILEY 275, NIST 98; Wiley, West Sussex, UK .

Statistical analysis
All experiments were repeated in triplication, and results were expressed as mean standard deviation. Table 1 shows the physicochemical properties of the perilla seed oil. The oil yield of the solvent extracted method was up to 39.61 g/100 g, while that of the cold press method was only 34.40 g/100 g. Both of them were higher than the aqueous enzymatic extracted method 31.28 g/100 g 17 . Recently, the oil yield has been reported to be 78.81 g/100 g by superheated steam treatment method 14 and 78.81 g/100 g by freeze-thaw pretreatment method 18 . Both oil presented pleasant golden yellow color. Low acid value 0.57 and 0.62 mg KOH/g oil and peroxide value 1.60 and 1.78 meq O 2 /kg oil indicated good quality of the oil. But the aqueous enzymatic extracted oil possessed relatively higher acid value 3.06 mg KOH/g oil and peroxide value 3.20 meq O 2 /kg oil 17 . High iodine value indicated high unsaturated fatty acids content of the two oils. Total content of tocopherols in the solvent extracted oil was up to 743.3 mg/kg oil, higher than the peanut oil 345.0 mg/kg oil , peanut oil 455.0 mg/kg oil , and most grape seed oil 397.8-755.8 mg/kg 19 21 . It has been reported that the tocopherol content of polyunsaturated fatty acid PUFA rich oils was higher than that of the oleic acid-rich oils 22 . With good antioxidant, tocopherol in the perilla oil could reduce free radical, therefore protecting the unsaturated fatty acids of the oil 1 . However, the unsaponifiable matter of this oil was only 0.57 g/100 g, relatively lower than oliver oil 1.23 g/100 g , peanut oil 0.94 g/100 g and sunflower oil 0.81 g/100 g 23 .

Fatty acid composition
Fatty acid composition of the cold press perilla seed oil was shown in Table 2. The majority fatty acid of the perilla seed oil was α-linolenic acid C18:3, ALA at concentration of 66.4 g/100 g, which has high nutrition and medicinal value. This was comparable to that of the aqueous enzymatic extracted oil 64.1 g/100 g and ultrasound-assisted hexane extracted oil 61.9 g/100 g . It has been reported that after a series of metabolic activities in the body, ALA could finally produce eicosapentaenoic acid EPA and docosahexaenoic acid DHA , which are especially beneficial to human health 24 . The common commercial oils contain relatively lower content of ALA, such as soybean oil 5.0-11.0 g/100 g , rapeseed oil 5.0-13.0 g/100 g and peanut oil 0.3 g/100 g 25 . The ALA content of the perilla seed oil Results are presented as mean value±standard deviation (n=3). PS-CHI, perilla seed samples harvested from Sichuan Province of China; N.D., not detected; N.G., not given. Table 2 Fatty acid composition of the cold press Chinese perilla seed oil (g/100 g).
was higher than that other ALA rich oils, such as linseed oil 52.1 g/100 g , sacha inchi oil 50.7 g/100 g , and chia oil 63.5 g/100 g 26,27 . The second one was linoleic acid C18:2 with concentration of 15.3 g/100 g, while the third one was oleic acid C18:1 at 11.3 g/100 g. The unsaturated fatty acids content was 93.0 g/100 g, among which polyunsaturated fatty acids amounted up to 81.7 g/100 g. The polyunsaturated/saturated fatty acids PUFA/SFA ratio reached up to 11.6 g/100 g. High PUFA/SFA ratio has been reported to be relative with reduction of cholesterol, therefore decreasing cardiovascular and atherosclerosis disease 28 .

TAG composition
The oil sample was separated into six TAGs fractions in NARP dimension, but baseline separation was not achieved in peak 3, 4 and 5 Fig. 1 . Since the separation of TAGs in NARP was dependent on the equivalent carbon number ECN , overall separation of TAGs with the same ECN could not be resolved by NARP. Especially positional isomers were always co-eluted in NARP-mode 29 . For instance, PLnLn and LLLn with same ECN value of 40 are co-eluted in fraction 3, LLnP and OLnL with same ECN value of 42 are co-eluted in fraction 4, while PLL, POLn, LLO and OOLn with same ECN value of 44 are co-eluted in fraction 5. The separation of TAG in Ag-HPLC depends on the interaction of fatty acid double bonds and silver ions. Resolution of TAGs with the same ECN could be achieved by Ag-HPLC. Therefore, it is necessary to combine the NARP with the Ag-HPLC separation for overall TAGs profiles analysis. Figure 2 shows the Ag-HPLC-MS chromatogram of fraction 1. As LnLnLn has no positional isomers, there is only one peak in Ag-HPLC mode.
As shown in Fig. 3, fraction 2 is separated into two regioisomers in Ag-HPLC mode: sn-LLnLn and sn-LnLLn. With both linolenic acids located in sn-1/3 position, sn-LnLLn is more retained than sn-LLnLn.
Fraction 3 is separated into sn-PLnLn, sn-LnPLn, sn-LLnL and sn-LLLn in Ag-HPLC Fig. 4 . With a higher unsaturation degree, the LLnL group are more retained than the PLnLn group. As shown in Table 4, LLLn has 7 double bonds, while PLnLn has 6 double bonds. sn-LnPLn is more retained than sn-PLnLn due to the linolenic acids position. Similarly, sn-LLLn is more retained than sn-LLnL.
As shown in Fig. 5, fraction 4 is separated into 5 species: sn-LnLP, sn-LnPL, sn-LLnO, sn-LnLO and sn-LOLn. LnLO group are more retained because LnLO has 6 double bonds, more than LnLP 5 . For the LnLP group, the relative abundance of LLn 597.3 in the first peak is relatively higher the other peak, as well as that of PLn   573.3 in the second peak. sn-LnPL is more retained due to location of linoleic acid and oleic acid in sn-1/3 position. Therefore, the first peak is presumed to be sn-LnLP, and the second one is sn-LnPL. For the OLnL group, the relative abundance of LO 601.4 in the first peak is the lowest of the three peaks, as well as LnO 599.4 in the second peak and LLn 597.6 in the third peak. Also, the retaining sequence is sn-LLnO, sn-LnLO and sn-LOLn. Therefore, the first peak is presumed to be sn-LLnO, the second one is sn-LnLO, and the third one is sn-LOLn.
As shown in Fig. 6, fraction 5 is separated into 7 species in Ag-HPLC. Having more double bonds 5 , LLO and LnOO groups are more retained than LPL and POLn groups 4 . With higher relative abundance of PL 575.3 , the first peak is presumed to be sn-LPL. In the PLnO group, with lower relative abundance of PLn 573.4 and stronger retaining, the third peak is presumed to be sn-POLn. With lower relative abundance of PO 577.4 , the second peak is sn-PLnO. For the LLO group, the relative abundance of LL 599.4 in sn-LLO is higher than sn-LOL, and sn-LOL is more retained than sn-LLO. So the fourth peak is sn-LLO, and the fifth one is sn-LOL. In the sixth peak, the relative abundance of OLn 599.4 is higher than that of OO 603.5 , so this one is sn-OLnO.
As shown in Fig. 7, fraction 6 is separated into 2 species: sn-OLnS S, stearic acid and sn-OSLn. The relative abundance of OS 605.4 in the first peak and OLn 599.4 in the second peak are relatively lower. Also, the retaining of sn-OSLn is stronger than sn-OLnS. So the first and second peak is sn-OLnS and sn-OSLn, respectively.
The TAG profiles of Chinese perilla seed oil analysis results were listed in Table 3. With content of 35.8 g/100 g, LnLnLn is the dominant TAG of this oil. Principal TAGs include LLnLn 20.2 g/100 g , LLLn 17.7 g/100 g and PLnLn 14.9 g/100 g . Other TAGs are minor TAGs, such as    J. Oleo Sci. 6 LLnO 4.2 g/100 g , LnPL 2.5 g/100 g , OOLn 2.0 g/100 g , and so on. Relative abundance of TAG regioisomers was listed in Table 4. LLnO has three isomers, while other 7 TAGs all have two isomers. The total TAG profiles results showed that this oil contained 20 TAGs including 17 regioisomers.

Unsaponi able matters composition
As shown in Tables 5-7, the unsaponifiable matters of Chinese perilla seed oil were composed of sterols, squalene, tocopherol, alcohols and hydrocarbons. Total hydrocarbons content was 44.02 mg/kg.
As shown in Table 6, total alcohols content was 299.3 mg/kg, including phytol and farnesol with contents of 53.20 mg/kg and 30.93 mg/kg, respectively. Phytol and farnesol has good anti-carcinogenic, anti-inflammatory and antimetabolic-syndrome properties 30,31 .
As shown in Table 7, the total phytosterols were mainly   Results are presented as mean value±standard deviation (n=3).
composed of campesterol, stigmasterol, β-sitosterol, isofucosterol and lanosterol. The total phytosterols content of the perilla seed oil 0.39 g/100 g oil was higher than that of the grape seed oil 0.23-0.34 g/100 g oil 20 and the peanut oil 0.28 g/100 g oil . Phytosterols have been reported to have important antioxidant, analgesic, anti-inflammatory and anticancer properties 32,33 . The content of β-sitosterol was up to 0.31 g/100 g oil. Other important sterols include campesterol 273.3 mg/kg , stigmasterol 237.1 mg/kg and lanosterol 201.7 mg/kg . Campesterol and β -sitosterol have been proved to be effective in inhibiting intestinal cholesterol absorption in humans 34,35 . The squalene content of the oil was tested to be 275.4 mg/kg. It has been certified that squalene has several beneficial health effects, such as antioxidant activity, decreasing cancer, and reducing the side effects of chemotherapy 36 . δ -Tocopherol and γ -tocopherol were also found with contents of 2.87 mg/kg and 151.5 mg/kg, respectively. Previous studies reported the health benefits of tocopherols, including neuroprotective, cardioprotective and anti-inflammatory activities 37 .

Conclusions
The results showed that the oil yield of the solvent extracted method was up to 39.61 g/100 g, higher than that of the cold press method 34.40 g/100 g . Both oil possessed low acid value and peroxide value. Total content of tocopherols in the solvent extracted oil was up to 743.3 mg/kg oil. The majority fatty acid of the Chinese perilla seed oil was linolenic acid 66.4 g/100 g , followed by linoleic acid 15.3 g/100 g . The overall TAG profile showed that this oil contained 20 TAGs including 17 regioisomers, in which LnLnLn 35.8 g/100 g was dominant. The unsaponifiable matters of this oil possessed high content of phytosterols 0.39 g/100 g oil , among which β-sitosterol was the most predominant 0.31 g/100 g oil . Therefore, the Chinese perilla seed oil is a good edible oil. And it also can be a good source of biological activity compounds, especially linolenic acid and β-sitosterol. Results are presented as mean value±standard deviation (n=3). Total sterols 3914±33 Results are presented as mean value±standard deviation (n=3).