Quality Assessment of Camellia oleifera Oil Cultivated in Southwest China

: Camellia oleifera oil has attracted increasing attention due to its well-balanced composition. In this study, we evaluated the oil content and chemical composition of C. oleifera oil cultivated in southwest China. The results showed that the acid and peroxide values were in line with the optimal quality index of the national standard of China. Oleic acid was the most predominant and important fatty acid, which accounted for 80.34–86.18%. The α -tocopherol, polyphenols and squalene ranged from 112.36 to 410.46 mg/kg oil, 14.22 to 53.63 mg/kg oil and 14.80 to 52.49 mg/kg oil, respectively. Principal component analysis (PCA) results showed that the synthesis score of introduced cultivars (‘Changlin 3’, ‘Changlin 4’ and ‘Changlin 18’) was higher that the local cultivars (‘Chuanya 21’ and ‘Chuanlin 2’). This research demonstrated that the introduced C. oleifera could adapt to the environment and climate of southwest China and large-scale plant of these introduced cultivars. In addition, the C. oleifera oil rich in unsaturated fatty acid has enormous potential to become a kind of functional oil and possesses great prospects for pharmaceutical and industrial applications.


Introduction
Camellia oleifera Abel. is the evergreen shrub or small tree plant of the Camellia of Theaceae and has been primarily distributed in the south of China for approximately 2300 years cultivation. In addition, Vietnam, Myanmar, Thailand, Malaysia and Japan have a small distribution [1]. C. oleifera oil, a kind of healthy and high-quality edible oil, is extracted from C. oleifera seed.
C. oleifera oil has both effects in nutrition and medicine owing to its rich beneficial ingredients, such as unsaturated fatty acid (UFA), vitamin E, phytosterols, polyphenols, squalene and a number of trace elements [2]. It is also known as "eastern olive oil" because of the fatty acid composition similar to olive oil [3]. Interestingly, the level of UFA in C. oleifera oil reaches more than 90%, principally including oleic acid and linoleic acid, and higher than that of olive oil [4]. Oleic acid is beneficial to people health through reducing the levels of low-density lipoprotein cholesterol, total cholesterol and glycemic index in the blood [5,6].
Linoleic acid, an essential polyunsaturated fatty acid (PUFA), can decrease the risk of cardiovascular and coronary heart disease [7]. The vitamin E (tocopherols) mainly exists in C. oleifera oil in the form of α-tocopherol and the content is twice as much as that of olive oil [8]. Polyphenols of C. oleifera oil contribute to the health effect on people and also are very significant for the oxidative stability of the polyunsaturated fatty acids in oil [9]. Moreover, squalene has a particular benefit for vascular health [10]. The trace element concentration in vegetable oil is significant because of the toxicological and nutritional characteristics [11].

Oil Content Assay
The oil content of C. oleifera was determined according to the international standard (ISO 659-2009) [20]. In short, the soxhlet extraction method was used. Petroleum ether was the extraction solvent, the extraction temperature was 85 • C, the time was 10 h, and each sample was repeated three times.

Oil Extraction Assay
The oil extraction was performed as described previously with some modifications [21]. Briefly, the C. oleifera seeds were dried in an oven at 60 • C, and then the dried C. oleifera seed was ground to fine powder. The ratio of powder to the water was 1:4.5 (g/mL). Then, the mixture was shaken at 75 • C (160 rpm) for 150 min in a thermostat water bath. The mixture was centrifuged to separate the oil and stored in the dark at 4 • C for the following assays.

Determination of Fatty Acid Composition
The fatty acid composition was established by gas chromatography mass spectrometry (GC-MS) after transesterification. The fatty acids were methyl esterified (FAMEs) by potassium hydroxide/methanol according to previous method [25]. About 100 mg of oil was transferred into a ground glass stoppered test tube, treated with 2 mL 1 mol/L NaOH/methanol. The sample was mixed on vortex mixer, shook for 30 min at 40 • C. The methyl esters were extracted by 2 mL n-hexane, and the aqueous phase was discarded. The n-hexane extract was washed with distilled water and dried by anhydrous sodium sulfate, filtered for later analysis by GC-MS.
Then, the GC-MS analysis for the FAMEs were conducted by Agilent 7890A gas chromatograph and 5977C mass spectrometry (Agilent Technologies, Santa Clara, California, USA) and equipped with a capillary column HP-5MS (30 m × 0.25 mm; 0.25 µm). The oven temperature programmed from 60 • C hold of 2 min, increasing at 15 • C/min to 150 • C hold of 2 min, then 15 • C/min increasing to 280 • C hold of 3 min. The carrier gas was helium at a flow rate of 0.6 mL/min, the split ratio 100:1. The injector temperature was 240 • C, and the detector temperature was 260 • C. EI ion source, and mass scan ranged from 50 to 500 m/z, and the solvent was delayed 3 min. The FAMEs profiles were identified by comparing the database (National Institute of Standard Technology Library, NIST). The individual fatty acid content was expressed as a percentage of total fatty acids [26].

Determination of Tocopherols
Qualitative and quantitative analysis of tocopherols in C. oleifera oil were determined by high performance liquid chromatography (HPLC) [27]. The analysis was performed by an Agilent 1260 HPLC (Agilent Technologies, Santa Clara, California, USA) equipped with ZORBAX SB-C18 column (150 × 4.6 mm, 5.0 µm) and a fluorescence detector. The excitation wavelength was 295 nm, and the emission wavelength was 325 nm. Methanol was the mobile phase with a flow rate of 0.8 mL/min, and the column temperature was 35 • C.

Determination of Polyphenols
The polyphenol extraction was performed according to the previous method with some modifications [28]. The 0.5 g oil sample was added to 5 mL n-hexane. 2 mL aqueous methanol (80%, v/v) was applied to extract oil for three times. The extract was stored at room temperature overnight. The residual oil was removed by n-hexane. The extract was concentrated by rotary evaporator, and adjusted to 10 mL with methanol. Then, 0.1 mL polyphenols extract was added with 0.02 mL Folin-Ciocalteu and 0.08 mL 10% sodium carbonate solution for 5 min, and then we added 0.8 mL distilled water. The absorbance of mixture was read at 765 nm by Spectra Max M2 microplate reader (Molecular Devices Corp., Silicon Valley, California, USA) after incubation in dark for 1 h. The polyphenols quantities were given as milligram gallic acid equivalents per kilogram.

Determination of Squalene
The squalene was evaluated by HPLC. Firstly, the oil was saponified as previous method [29]. About 1.0 g of oil was transferred into 250 mL separatory funnel, treated with 50 mL of 2 mol/L potassium hydroxide/ethanol solution. The sample was mixed on vortex mixer, and saponified for 60 min at 80 • C in water bath. After cooling to room temperature, we added 50 mL of petroleum ether and extracted three times. The petroleum ether phase was washed to neutrality by distilled water and concentrated using a rotary evaporator at 40 • C and dissolved with acetonitrile (1 mL). Subsequently, the sample was analyzed by Agilent 1260 HPLC (Agilent Technologies, Santa Clara, California, USA). The detection conditions were as follow: ZORBAX SB-C18 column (150 × 4.6 mm, 5.0 µm) with a diode array detector. The wavelength was 325 nm, column temperature was 30 • C and mobile phase was methanol/acetonitrile (60/40, v/v) at a flow rate of 1.0 mL/min.

Determination of Trace Elements
The trace elements were analyzed by inductively coupled plasma mass spectrometry (ICP-MS) [30]. We placed 0.3 g oil sample into a digestion vessel, and then 5 mL HNO 3 (5%, v/v) and 1 mL H 2 O 2 solution were added and fully digested in a microwave digester (Anton Paar Co., Graz, Austria). Excess acid was removed under 120 • C, and the residual was diluted to 25 mL with ultrapure water. The trace elements were detected on a PerkinElmer NexION350D system (PerkinElmer Co., Waltham, Massachusetts, USA). The RF power was 1500 W, scanning mode was peak hopping, nebulizer flow rate was 0.8 L/min, coolant gas flow was 15 L/min, replicates were three times, sampling depth was 10 mm and isotopes were selected as 24 Mg + , 43 Ca + , 53 Cr + , 55 Mn + , 57 Fe + , 63Cu+, 66 Zn + , 75 As + , 111 Cd + and 208 Pb + .

Statistical Analysis
The results were expressed as the mean ± standard deviation (SD) and were carried out in triplicate. Analysis of variance (ANOVA), DUNCAN test and principal component analysis (PCA) were performed using the IBM SPSS Statistics version 20.0 (SSPS Inc., Chicago, IL, USA). P-values were reported based on 5% probability and 95% confidence level.

Oil Content
The oil content of the C. oleifera oil is presented in Figure 1, and the oil content of all introduced cultivars was higher than the local cultivars ('chuanya 21' and 'chuanlin 2'). In addition, the oil content of 'changlin 166' was the highest and reached 51.92%. The oil contents of 'changlin 3', 'changlin 4', 'changlin 18', 'changlin 23' and 'changlin 166' were higher than the excellent tree standard (45%) established by the People's Republic of China Forestry Standard (LY/T 1730.1-2008) [31]. The introduced cultivars were adapted to the local climate and had high oil contents.

Acid, Peroxide and Iodine Values
As shown in Table 1, the acid value, peroxide value and iodine value of C. oleifera oil were ranged from 1.23 to 1.92 mg/g, 0.52 to 1.73 mmol/kg and 83.80 to 94.69 g/100 g, respectively. The acid value of C. oleifera oil was less than 2.0 mg/g, and the peroxide value of C. oleifera oil was no more than 0.25 g/100g (9.85 mmol/kg). Thus, the acid and peroxide values were up to the optimal quality index of the China national standard (GB/T 11765-2018) [32].

Acid, Peroxide and Iodine Values
As shown in Table 1, the acid value, peroxide value and iodine value of C. oleifera oil were ranged from 1.23 to 1.92 mg/g, 0.52 to 1.73 mmol/kg and 83.80 to 94.69 g/100 g, respectively. The acid value of C. oleifera oil was less than 2.0 mg/g, and the peroxide value of C. oleifera oil was no more than 0.25 g/100g (9.85 mmol/kg). Thus, the acid and peroxide values were up to the optimal quality index of the China national standard (GB/T 11765-2018) [32].
The acid value reflects the amount of free fatty acid, and the peroxide value represents the degree of oxidation of oil and fatty acid [33]. In this study, the acid value and peroxide value was lower than that of previous works [34,35]. The iodine value of local cultivar was lower than introduced cultivars. The iodine value expresses the storage stability of oil. The iodine value was less than 100 g/100 g, indicating that they were nondrying oil [36]. The lower acid, peroxide value and moderate iodine value were related to the C. oleifera seed ripeness, processing method, storage condition and time of oil samples and also showed the excellent quality of the C. oleifera oil [37].   The acid value reflects the amount of free fatty acid, and the peroxide value represents the degree of oxidation of oil and fatty acid [33]. In this study, the acid value and peroxide value was lower than that of previous works [34,35]. The iodine value of local cultivar was lower than introduced cultivars. The iodine value expresses the storage stability of oil. The iodine value was less than 100 g/100 g, indicating that they were non-drying oil [36]. The lower acid, peroxide value and moderate iodine value were related to the C. oleifera seed ripeness, processing method, storage condition and time of oil samples and also showed the excellent quality of the C. oleifera oil [37].
The contents of oleic acid and UFA in C. oleifera oil from southwest China were higher than that east China could be attributed to the higher altitude [40,41]. The abundant UFA in C. oleifera oil is very important in developing and maintaining the nervous system as well as physiological function in body [19]. The content and composition fatty acid of C. oleifera oil at southwest China were consistent with origin that offer a manifestation that the C. oleifera has adapted to the southwest China environment and climate.
The previous research results were reported by Su and colleagues [26]. The contents of oleic acid were 74.20% (olive oil), 58.19% (canola oil), 25.90% (corn oil), 79.71% (sunflower oil), 39.46% (peanut oil), 22.40% (soybean oil) and 28.06% (pumpkin oil). In this study, the average content of oleic acid in C. oleifera oil was 83.57%. Therefore, the content of oleic acid in C. oleifera oil was higher than the common vegetable oils. The oleic acid is a monounsaturated fatty acid. The higher oleic acid of C. oleifera oil means it has high stability and digestibility and health-promoting effects that include lowering blood pressure, cholesterol and triglycerides [6].

Tocopherols Content
As shown in Table 3, the α-tocopherol content of C. oleifera oil ranged from 112.36 to 410.46 mg/kg oil. It should be noted that the β-tocopherol, γ-tocopherol and δ-tocopherol were not detected in C. oleifera oil. The α-tocopherol content was higher than that of other works in which the α-tocopherol contents were 97.80 mg/kg and 150.66 mg/kg (excepted for the local cultivars) [25,27].
These results suggested that the content of α-tocopherol was closely related to the cultivars and that the content of α-tocopherol from introduced cultivars was higher than the local cultivars. The α-tocopherol is the most bioactive structure in vitamin E [42]. Some previous studies demonstrated that α-tocopherol could facilitate fertility, lower oxidative stress, relief oxidative damage, reduce neuro-inflammation and delay aging [14,43]. In this study, the α-tocopherol content in the C. oleifera oil exhibited a higher level. Therefore, C. oleifera oil could be regarded as a natural supplement for obtaining α-tocopherol. Table 3. Content of tocopherols, polyphenols and squalene in C. oleifera oil of ten cultivars (mg/kg oil). Data are expressed as the mean ± standard deviations, n = 3. The "-" represents not detected in the identification process. Different letters in the same row indicate significantly different values, p < 0.05.

Polyphenol Content
Polyphenols are natural antioxidants that scavenging free radicals in vivo and have a clear effect on preventing tumorigenesis, preventing cardiovascular disease and lowering cholesterol [37]. The polyphenol content of C. oleifera oil is presented in Table 3. The polyphenol content of 'Changlin 26' was the highest and reached 53.63 mg/kg. The polyphenol contents of 'Changlin 26', 'Changlin 27' and 'Changlin 53' exceeded that of the local cultivars.
These results suggested that the content of polyphenols was related to the cultivars. Furthermore, previous studies indicated that the polyphenol content was affected by the planting soil, environment and processing technology of C. oleifera oil [44,45]. In addition, in C. oleifera oil, phenolic compounds contribute to C. oleifera oil's oxidative stability and can extend the shelf life of C. oleifera oil as well as the sensory characteristics, such as its bitter, astringent and pungent taste [46].

Squalene Content
Squalene has plenty of physiologic functions, such as enhancement of the immune responses and anti-aging, anti-fatigue and anti-tumor effects [47,48]. Thus, we measured the squalene content of C. oleifera oil. The squalene content of C. oleifera oil was varied with the cultivars (Table 3). In this study, the squalene content of C. oleifera oil was lower than that of previous studies, which may be related to the C. oleifera cultivars and the process technology of oil [49].

Trace Elements Concentration
The concentrations of trace elements in C. oleifera oil sample are presented in Table 4.   The presence of some trace elements, like Mg, Mn, Ca, Fe, Cu, Zn and Cr, could increase the oxidation rate of edible oil [52]. The elements of As, Cd, and Pb in edible oil could exhibit toxic effects on humans when the content exceeds certain boundaries [53]. In our study, the concentrations of harmful elements of Pb, Cd and As in all C. oleifera oil samples were lower than 0.02 µg/kg. Among them, As was not detected, and Pb was extremely lower than that the maximum limits of China national standard of GB 2762-2017 (0.1 mg/kg) in all oil samples [54]. These results indicated that the C. oleifera oil showed an excellent quality, and the soil of cultivation region (southwest China) did not suffer from contamination. Thus, this region is suitable to establish a standardized cultivation base of C. oleifera.

Principal Component Analysis
PCA is a statistical tool that can interpret most of the information of inter-correlated raw variables through several uncorrelated linear combinations [55]. We performed a comprehensive evaluation of the quality of C. oleifera oil by PCA based on oil content, acid value, peroxide value, iodine value, fatty acid composition, α-tocopherol, polyphenols, squalene and trace elements. The results were shown in Figures 2 and 3 and Table 5. The contribution rates of the first four principal components were 27.58, 23.52, 18.35 and 10.93%, and the cumulative contribution rates of first four principal components were 80.38%, which contained all of the information of C. oleifera oil and could fully reflect the overall oil quality. Therefore, the first four principal components were selected for analysis.

Principal Component Analysis
PCA is a statistical tool that can interpret most of the information of inter-correlated raw variables through several uncorrelated linear combinations [55]. We performed a comprehensive evaluation of the quality of C. oleifera oil by PCA based on oil content, acid value, peroxide value, iodine value, fatty acid composition, α-tocopherol, polyphenols, squalene and trace elements. The results were shown in Figures 2,3 and Table 5. The contribution rates of the first four principal components were 27.58, 23.52, 18.35 and 10.93%, and the cumulative contribution rates of first four principal components were 80.38%, which contained all of the information of C. oleifera oil and could fully reflect the overall oil quality. Therefore, the first four principal components were selected for analysis.  As shown in Figure 2, the distribution of the oil samples on different principal components was scattered; this indicated that the components of the oil samples from different  scores of 'Changlin 3', 'Changlin 4', 'Changlin 18' and 'Changlin 27' were higher than 'Chuanya 21' (local cultivar). 'Changlin 3', 'Changlin 4' and 'Changlin 18' were higher than 'Chuanlin 2' (local cultivar).
The higher the synthesis score, the better the quality of oil. Therefore, the oil quality of 'Changlin 3' was the highest, followed by 'Changlin 4' and 'Changlin 18'. In addition, the results suggested that the introduced cultivars of 'Changlin 3', 'Changlin 4' and 'Changlin 18' were fully adapted to the local climate and superior to the local cultivar.

Conclusions
According to the results in the study, the introduced cultivars of C. oleifera could adapt to the environment and climate of southwest China. The C. oleifera oil expressed unique qualitative characteristics. The chemical properties of C. oleifera oil were in accord with the national standard of China. The C. oleifera oil was rich in UFA, α-tocopherol, polyphenols and squalene and included some trace elements. However, the concentrations of harmful elements of Pb, Cd and As in C. oleifera oil samples were extremely low compared with that the maximum limits of the China national standard. The C. oleifera oil cultivated in southwest China demonstrated excellent quality, and the oil qualities of the introduced cultivars 'Changlin 3', 'Changlin 4' and 'Changlin 18' were superior to the local cultivars ('Chuanya 21' and 'Chuanlin 2'). This presents a promising option for the large-scale planting of these introduced cultivars.

Conflicts of Interest:
The authors declare that there is no conflict of interests to this work. As shown in Figure 2, the distribution of the oil samples on different principal components was scattered; this indicated that the components of the oil samples from different cultivars varied greatly. As illustrated in Figure 3, the peroxide value, myristoleic acid, palmitic acid, stearic acid, linoleic acid and eicosenoic acid had a higher loading on the first principal component. The second principal component included the oil content, iodine value, linoleic acid, squalene, Ca and Fe.
The higher the synthesis score, the better the quality of oil. Therefore, the oil quality of 'Changlin 3' was the highest, followed by 'Changlin 4' and 'Changlin 18'. In addition, the results suggested that the introduced cultivars of 'Changlin 3', 'Changlin 4' and 'Changlin 18' were fully adapted to the local climate and superior to the local cultivar.

Conclusions
According to the results in the study, the introduced cultivars of C. oleifera could adapt to the environment and climate of southwest China. The C. oleifera oil expressed unique qualitative characteristics. The chemical properties of C. oleifera oil were in accord with the national standard of China. The C. oleifera oil was rich in UFA, α-tocopherol, polyphenols and squalene and included some trace elements. However, the concentrations of harmful elements of Pb, Cd and As in C. oleifera oil samples were extremely low compared with that the maximum limits of the China national standard. The C. oleifera oil cultivated in southwest China demonstrated excellent quality, and the oil qualities of the introduced cultivars 'Changlin 3', 'Changlin 4' and 'Changlin 18' were superior to the local cultivars ('Chuanya 21' and 'Chuanlin 2'). This presents a promising option for the large-scale planting of these introduced cultivars.