Twenty-two samples of C. oleifera Abel seeds were collected from South Henan (China), harvested on November 2016. Each sample consisted of about 1.5 kg. These samples were divided into four groups based on the cultivation districts: (1) Guangshan (Gs, n=7; YuGuang variety), (2) Shangcheng (Sc, n=5; YuShang variety), (3) Xinxian (Xx, n=5; YuXin variety), (4) Luoshan (Ls, n=5; YuLuo variety). The detailed information is shown in Figure 1, which describes the places of origin. The standard fatty acid methyl esters (palmitic, stearic, oleic, linoleic, linolenic, and arachidic) and tocopherols (α-, β-, δ-, and γ), pancreatic lipase for the sn-2 position analysis were purchased from Sigma-Aldrich (Shanghai) Trading Co. Ltd (Shanghai, China). Silica gel used for TLC plate preparation was obtained from Qingdao Ocean Chemical Factory (Qingdao, China). The hexane for the fatty acid composition analysis was chromatographic grade, and the other reagents and solvents from local sources were of analytical grade and purified before use.

After removal of the hull, the seed was ground into a fine paste using a laboratory grinder. The paste was subjected to Soxhlet extraction with petroleum ether (boiling point, 60-90 °C) or ethyl ester for 8h. Briefly, ground seed (about 50g) was packed in a cellulose thimble, and the open end of the thimble was plugged with cotton to avoid spillage of the seed paste into the miscella. After extraction, a clear miscella was obtained and hence the crude C. oleifera oil was obtained after the removal of solvent from the miscella under vacuum.

The lipid content of C. oleifera seed was analyzed according to the IUPAC method (1.122) (Paquot and Hauntfenne, 1987). The important physicochemical properties of the crude C. oleifera oils concerning acid value (AV), saponification value (SV), iodine value (IV), and refractive index were characterized according to the IUPAC Methods 2.201, 2.202, 2.205, and 2.102, respectively (Paquot and Hauntfenne, 1987).

The lipid contents of these 22 seeds in our research were in the range of 28.39-56.94% with an average value of 41.05% on a moisture free basis (Table 1). The lipid contents of the seeds cultivated in Shangcheng were 41.73-56.94% with a mean value of 49.66% (Table 1), and significantly higher than those of the seeds cultivated in GuangShan, Xinxian and Luoshan. The values in our study are comparable to the report (Yao et al., 2011) on the lipid content (46%) of the C. oleifera seed from Shangcheng (Henan, China), higher than those (27.21-37.37% with a mean value of 31.31%) of the C. oleifera seeds (Xianglin variety cultivated in Henan) as reported by Wang et al. (2008). As a comparison, the oil contents of C. sasanqua seeds (Gunstone et al., 1994), C. sinensis seed (Sahari et al., 2004) and C. reticulate Lindl seeds (Li et al., 2013) were 56-70%, 30-33% and 49.4-51.4%, respectively.

The acid values (AVs) of the C. oleifera oils in our study ranged from 0.22 to 5.17 mg KOH/g with an average value of 1.24±1.17 mg KOH/g (Table 1). The AVs of C. oleifera oils from the seeds harvested from Guangshan were 2.01±0.57 mg KOH/g, and similar to those of the oils from the seeds harvested from Xinxian (1.71±0.97 mg KOH/g), however higher than those of the oils from the seeds harvested from Shangcheng (0.33±0.10 mg KOH/g) and Luoshan (0.62±0.13 mg KOH/g) (Table 1).

The saponification values (SVs) of the C. oleifera oils in our study ranged from 178.90 to 196.91 mg KOH/mg with an average value of 190.91±4.06 mg KOH/g (Table 1). The SVs of C. oleifera oils from the seeds obtained from Guangshan were in the range of 189.69 to 196.91 with an average of 193.44±2.52 mg KOH/g, similar to those of the oils from the seeds harvested from Luoshan (in the range of 191.53 to 194.72 with an average of 192.60±1.28 mg KOH/g), however higher than those of the oils from the seeds harvested from Xinxian (in the range of 183.17 to 192.39 with an average of 188.88±3.85 mg KOH/g) and Shangcheng (in the range of 178.90-192.81 with an average of 187.68±5.33) (Table 1).

The iodine values (IVs) of the C. oleifera oils in our study ranged from 82.0 to 87.1 g I₂/100g with an average value of 84.3±1.5 g I₂/100g (Table 1). The IVs of C. oleifera oils among the seeds from Guangshang, Shangcheng, Xinxian, and Luoshan showed no significant differences (Table 1). The refraction index (n ²⁵) of C. oleifera oils in our study ranged from 1.4667 to 1.4720 with an average of 1.4705±0.0011 (Table 1).

The SVs and IVs of the C. oleifera oils in our research are similar to those the C. oleifera oils from other province of China. The SV and IV of C. oleifera Abel. oil from the seed cultivated in Hunan (China) were 180.5 mg KOH/g and 81.5 g I₂/100g, respectively (Yun et al., 2011). The IV and SV of the C. oleifera oil from the seed cultivated in Fujian (China) were 85±2 g I₂/100g and 195±1 mg KOH/g, respectively (Yu et al., 2013).

As a comparison, the refractive index (n ²⁵), SV, and IV of Camellia sasanqua oil were 1.467-1.469, 193-196 mg KOH/g, and 83-89 g I₂/100g, respectively (Gunstone et al., 1994). The IV and SV of the oils from tea seeds (Lahijan variety of Iranian tea seed, Southern Indian tea seed, and Turkish tea seed) were in the range of 85-91 g I₂/100g and 192-194 mg KOH/g, respectively (Sahari et al., 2004). The refractive index (n ⁴⁰) of Camellia reticulate f. simpex seed oil was 1.4711-1.4736 (Huang et al., 2010). The SV and IV of Camellia sinensis O.Ktze oil were 189.7 mg KOH/g and 88.5 g I₂/100g, respectively (Yun et al., 2011). The IV, SV and refractive index (n ⁴⁰) of C. reticulate Lindl seed oil were 72.1-79.8 g I₂/100g, 174.1-185.0 mg KOH/g and 1.4727, respectively (Li et al., 2013).

The fatty acid composition of the C. oleifera oils in our research was 7.07-9.52% palmitic, 0.09-0.22% palmitoleic, 1.45-2.94% stearic, 77.02-84.33% oleic, 5.19-11.23% linoleic 0.53-0.70% linolinic, and 0.22-0.70% arachidic acid (Table 2). The proportion of unsaturated fatty acids (UFAs) and saturated fatty acids (SFAs) were 87.80-90.30% and 9.69-12.19%, respectively. And the ratios of UFAs/SFAs were in the range of 7.20-9.32.

The palmitic contents of the oils from C. oleifera seeds obtained from Guangshan and Luoshang were higher than those of the oils from C. oleifera seeds cultivated in Shangcheng and Xinxian (Table 2). The oleic contents of the oils from C. oleifera seeds obtained from Guangshan, Xinxian and Luoshang were similar, and lower than that of the C. oleifera seeds obtained from Shangcheng (Table 2). Linoleic contents of the oils from C. oleifera seeds obtained from Guangshan, Xinxian and Luoshang were similar, and higher than that of the C. oleifera seeds obtained from Shangcheng (Table 2). The geographic differences among the seeds from different districts could be attributed to the differences in the fatty acid composition of the oils.

The fatty acid composition of the C. oleifera oils in our study was comparable to those of the oils from the C. oleifera seeds cultivated in the other provinces of China reported by previous researchers (p<0.05) (Cao et al., 2017; Cheng et al., 2010; Jiang et al., 2010; Xi et al., 2002; Yang, 2010; Yao et al., 2011; Yu et al., 2013; Yuan et al., 2013). The oils from the C. oleifera seeds cultivated in Jinhua (Zhejiang, China) consisted of 8.10% palmitic, 0.10% palmitoleic, 1.90% stearic, 80.90% oleic, 8.10% linoleic acid, 0.30% linolenic, 0.60% eicosenoic acid, and UFAs were 90.00% (Cao et al., 2017). The fatty acid composition of the oil from C. oleifera seeds cultivated in Hangzhou (Zhejiang, China) was 8.60% palmitic, 0.10% palmitoleic, 1.80% stearic, 78.55% oleic, 9.90% linoleic acid, 0.30% linolenic, 0.70% eicosenoic acid, and UFAs were 89.55% (Cao et al., 2017). The fatty acid composition of the oils from the C. oleifera seeds cultivated in Hubei were 6.61-11.01% palmitic, 0.72-2.00% stearic acid, 65.70-84.48% oleic, and 6.14-13.37% linoleic (Cheng et al., 2010). The fatty acid composition of the oils from 35 C. oleifera seeds cultivated in Guangxi was 6.68-12.23% palmitic, 1.24-3.21% stearic, 74.49-86.16% oleic, and 1.94-12.97% linoleic acid (Jiang et al., 2010). The oils from C. oleifera seeds cultivated in the Jiangxi province consisted of 6.70-1.57% palmitic, 0.49-0.65% stearic, 72.1-90.1% oleic, 2.1-16.6% linoleic acid, and the UFAs were about 90% (Xi et al., 2002). Yang (Yang, 2012) analyzed the fatty acid composition of the oils from 38 C. oleifera seeds cultivated in the Jiangxi province in the 2011 season, and the oils consisted of 7.19-9.17% palmitic, 1.50-3.46% stearic, 78.34-82.79% oleic, 6.37-10.05% linoleic acid, and the UFAs were over 88.76%. Yao et al. (2011), analyzed the fatty acid composition of the C. oleifera oils from several provinces (such as Guangxi, Jiangxi, Hunan, Hubei, Guangdong, Zhengjiang, Anhui, Fujian, and Guizhou) of China. These oils consisted of 7.16-8.87% palmitic, 1.42-2.78% stearic, 76.72-83.18% oleic, 6.59-9.71% linoleic, 0-1.18% linolenic, and 0-0.79% arachidic (Yao et al., 2011). The fatty acid composition of the oil from the C. oleifera seeds cultivated in Fujian (China) was 10.20% palmitic, 1.70% stearic, 77.84% oleic, 8.30% linoleic, 0.37% linolenic, 0.59% arachidic (Yu et al., 2013). Yuan et al. (2013) evaluated the fatty acid composition of 132 C. oleifera oils from the seeds collected from several provinces of China in 2009. The fatty acid composition of these oils was 7.03-13.85% palmitic, 1.35-5.49% stearic, 70.33-86.21% oleic, and 3.25-17.18% linoleic acid.

The fatty acid composition of the C. oleifera oils in our study was different from the results of Chen et al. (1996) and Ma et al. (2011). Chen et al. (1996) reported the major fatty acid composition of the oils from the C. oleifera seeds cultivated in Hunan province to be 11.32-16.40% palmitic, 63.51-75.08 oleic, and 9.91-19.76% linoleic acid. The study of Ma et al. (2011) showed that the fatty acid composition of the oil from C. oleifera obtained from Guangxi was 68-77% monounsaturated fatty acids, 7-14% polyunsaturated acids, and 16-18% saturated fatty acids, including 4.4-4.7% nonadecanoic acid (C19:0). The differences in the fatty acid compositions might be attributed to geographic and varietal differences.

The chemical, physical and biological characteristics of lipids are largely dependent on the amount and positional distribution of fatty acids on the glycerol backbone, and therefore, the stereospecific analysis of fatty acids in the triacylglycerol is considered very important when using the lipids for dietary and industrial purposes (Hunter, 2001). The major fatty acids in the sn-2 position of the triacylglycerols in the C. oleifera oils in our study were oleic (84.36-91.33% with an average value of 86.74±2.06%), followed by linoleic (7.14-14.22% with an average value of 12.01±2.03%) (Table 3). The palmitic, stearic and arachidic acid contents were lower than 1%, while palmitoleic and linolenic acids were not detected (Table 3). Oleic in the sn-2 position of the oils from the C. oleifera seeds obtained from Shangcheng were in the range of 94.65-91.33% with an average of 88.56±2.88%, similar to those of the oils from C. oleifera seeds obtained from Xinxian, and higher than those of the oils from C. oleifera seeds obtained from Guangshan and Luoshan. Linoleic in the sn-2 position of the oils from the C. oleifera seeds obtained from Luoshan were in the range of 13.27-13.85% with an average of 13.62±0.24%, about the same as those of the oils from the C. oleifera seeds obtained from Guangshan and Xinxian, and higher than those of the oils from the C. oleifera seeds obtained from Shangcheng. The SFAs content in the sn-2 position of C. oleifera oils in our research was in the range of 0.89-1.96% with an average of 1.25±0.25%, and UFA content in the sn-2 position of these oils was in the range of 98.04-99.11% with an average of 98.75±0.26% (Table 3). These differences might be caused by geographic differences.

In compliance with the general law for the fatty acid distribution of natural triacylglycerols in vegetable oils, UFAs occupied over 98% of the sn-2 position of the glycerol backbone, in which only a very small amount of thermodynamically unfavorable palmitic, stearic and arachidic acid were detected. As a comparison, the oleic acid in the sn-1, 2, and 3 positions of C. japonica L. oil were 66.0%, 93.6% and 94.7%, respectively (Noh and Yoon, 2012). For other high-oleic acid vegetable oils, such as Chufa tuber oil, the major fatty acid composition of the oil was 15.4% palmitic, 65.5% oleic, and 16.2% linoleic, while the major fatty acid composition on its sn-2 position was 75.1% oleic and 23.1% linoleic (Kim et al., 2007). For olive oil, the major fatty acid composition of the oil was 12.1-16.3% palmitic and 74.3-77.5% oleic, while the major fatty acid composition on its sn-2 position was 82.2-88.4% oleic (Vichi et al., 2007).

The total tocopherol contents in the C. oleifera oils extracted by petroleum ether were in the range of 39.55-75.94 mg/100g with an average of 58.21±10.54 mg/100g, and the α-tocopherol and γ-tocopherol in these oils were 33.98-67.82 mg/100g with an average of 51.66±10.19 mg/100g and 5.57-8.27 mg/100g with an average of 6.55-8.27 mg/100g with an average of 6.55±0.76 mg/100g, respectively (Table 4). However, the total tocopherol contents in the C. oleifera oils extracted by ethyl ether were 14.91-44.36 mg/100g with an average of 30.35±7.77 mg/100g, the α-tocopherol and the γ-tocopherol contents in these oils were 9.42-37.08 mg/100g with an average of 23.74±7.44 mg/100g and 5.50-7.33 mg/100g with an average of 6.60±0.53 mg/100g, respectively (Table 4). β-tocopherol and δ-tocopherol were not detected in the C. oleifera oils extracted by petroleum ether or ethyl ether, which is consistent with the report of Li et al. (2009). The α-tocopherol contents in the oils extracted with petroleum ether (33.98-67.82 mg/100g with an average of 51.66±10.19 mg/100g were dramatically higher than those in the oils extracted with ethyl ether (9.42-37.08 mg/100g with an average of 23.74±7.44 mg/100g) (p<0.05) (Table 4). The γ-tocopherol contents in the oils from C. oleifera seeds obtained from different locations extracted by the same solvent showed no difference. Moreover, there was no difference between the γ-tocopherol contents in the oils obtained from the same seed extracted with different solvents (petroleum ether and ethyl ether) (Table 4).

Li et al. (2009) reported that the α- and γ-tocopherols in the C. oleifera oil were 1.5-187.7mg/100g and 0.66-1.32mg/100g, respectively. The study by Zhu et al., (2011) showed that α-, γ-, and δ-tocopherols in tea seed (seed of C. sinensis O. Ktze) oil were 24.91-55.31mg/100g, 1.68-3.55 mg/100g, and 0.08-1.32 mg/100g, respectively, and β-tocopherol was not detected. In the report of Zhu et al. (Zhu et al., 2013), total tocopherol was 29.78-58.63 mg/100g. In tea seed oils (seeds of C. sinensis O. Ktze), total tocopherols were 23.2-47.0 mg/100g, with the the major component being α-tocopherol with 22.6-45.0 mg/100g; and β-, γ-, and δ-tocopherol were lower than 2 mg/100g. In the research of Dong et al. (Dong et al., 2012), the tocopherol content of tea seed oils (C. sinensis L. seed) extracted by press was 24.71 mg/100g with 24.15 mg/100g of α-tocopherol in the oils extracted by the soxhlet method with petroleum ether, the α-tocopherol content was 21.80 mg/100g, and β-, γ- and δ-tocopherols were not detected. In the oils extracted by supercritical CO₂ (15MPa, 35°C), the tocopherol content was 70.11 mg/100g, including 68.90 mg/100g α-tocopherol. According to the three methods, supercritical CO₂ extraction might be the best one. Fazel et al. (Fazel et al., 2008) evaluated the β-carotene, vitamin E (8 compounds) and polyphenols (8 compounds) in oils obtained from C. sinensis seeds, and the results showed that the total content of tocopherols and tocotrienols were 376.0 ± 3.0 mg/kg and 13.4 ± 0.34 mg/kg, respectively, and in vitamin E; the α-tocopherol content (210.0 ± 2.3 mg/kg) was higher than the others.

In conclusion, this work has presented the general properties of the oils from C. oleifera seeds cultivated in south Henan of China along with their fatty acid profile. It turns out that physiochemical properties of the oils from the C. oleifera seeds cultivated in south Henan of China are similar to those of the oils from the C. oleifera seeds cultivated in other districts of China, and could be a good source of natural oil rich in oleic acid and tocopherols.