4 China Academy of Chinese Medical Sciences Institute of Chinese Materia Medica, Beijing,100011, China,
* Corresponding author, email: czlu@fjirsm.ac.cn, Tel: +86-592-3576166
Abstract : The Podocarpus Nagi kernel oil was prepared and its chemical constituents were confirmed accordingly in this work. The constituents and the contents of the obtained oil were identified as: total flavonoids (4.2%, calculated according to the rutin), (9Z ,12Z )-9,12-octadecadienoic acid(38.2%), arachidic acid(0.11%), cis -11-eicosenoic acid(1.40%),cis -11,14-eicosadienoic acid(8.04%), ꞷ-3(0.2%), ꞷ-6(48.0%), ꞷ-9(21.0%), vitamin E(2.11mg/100g). The microelements in this oil was also tested using ICP-MS and the results exhibited that this oil contains numbers of microelements beneficial for human health: such as V(0.006μg/g), Cr(0.024μg/g), Mn(40.109μg/g), Fe(2.292μg/g), Co(0.007μg/g), Zn(4.316μg/g), As(0.009μg/g), Se(0.240μg/g), Sr(0.453μg/g). Then, the oil was evaluated for its in vitroantioxidant effect against DPPH⸱, which exhibited noteworthy scavenging ability against DPPH⸱. In addition, the oil was also evaluated for its preliminary in vitro anticancer activity against four cancer cell lines: the results showed that it exhibited the highest inhibition against gastric cancer, breast cancer (MCF-7), lung cancer(A549) and Hela cell lines with the highest inhibitions of 64.30%±2.80, 52.87%±2.57, 93.21%±2.24 and 88.33%±2.08 at the concentrations of 25mg/mL, 25mg/mL, 50mg/mL and 50mg/mL, and with the IC50s of 48.47mg/mL, 763.14mg/mL, 11.47mg/mL and 23.77mg/mL respectively. These findings demonstrated that this oil can be regarded as the functional edible oil by comparing the contents of edible oils used in the field of food.
Key words : Podocarpus Nagi kernel oil; Preparation; Chemical constituents; Antioxidant activity; Anticancer activity
Introduction
According to the statistics provided by the National Grain and Oil Information Center, since 1996, China has imported more edible oils than the total domestic production, and become the largest importer of edible oils and oilseeds in the world. The self-sufficiency rate of edible oils in China is currently less than 40%. In China, the oilseeds mainly rely on rapeseeds, peanuts, flaxseeds etc., which makes it still difficult to ensure the safe supply of the oilseeds and edible oils. In 2011, the consumption of edible oils reached 25.15 million tons, while industrial and other consumption reached 2.5 million tons, totaling 27.65 million tons, and the annual consumption per person has reached 20.5 kg in China. Among the edible oils, the import dependence of high-grade woody (herbaceous) edible oil has reached 95%, and the external dependence of ordinary edible oils has also reached 68%. Thus, it is urgent to explore new approaches and new raw oil materials to develop the edible oils for long-term development.
Podocarpus Nagi (P. nagi , named Zhubai in Chinese), belonging to the Podocarpaceae family. This plant contains different kinds of biological components (such as volatile oil, flavonoids, steroids, sugar and glycosides, lactones, etc.) and it exhibits a wide spectrum of biological activities: such as hemostasis, bone setting, anti-bacterial, anti-tumor, antiviral, antioxidant and detumescence activities 1-4. The peel and fruit of P. nagiare rich in volatile components and oil, and especially the kernel ofP. nagi has an oil content up to about 30%. P. nagi has many kinds of active components such as abundant unsaturated fatty acids, flavonoids, β-vanillin, vitamin E and so on 5. Therefore, the P. nagi kernel oil should be a high-quality functional edible oil in theory. And according to the folk records of the Yao Nationality, P. nagi kernel oil has ever been used as an edible oil 6. However, up to now, P. nagikernel oil has not been used as an edible oil in the field of food yet. Considering the lack of edible oils in our native state and large scale of P. nagi kernel is produced ever year, it is very necessary to study the chemical constituents and relevant biological activities ofP. nagi kernel oil to develop it to the fields of food and nutrition. In this work, the preparation process, chemical constituents and relevant biological activities (antioxidant and anticancer activities) of P. nagi kernel oil were studied.
2. Experimental
2.1 Material and instruments
The P. nagi kernel were collected in September of 2018 from the Yangli town of Fujian province and which were air-dried and powdered; other chemicals used for chemical extractions, chemical constituents analysis and biological evaluations are analytical reagents and commercially available.
Instruments. ICP-MS was carried out on the Agilent Technologies 7800(USA); Microwave digestion instrument (Mars 6 Classic, China); Full-wavelength multifunctional microplate reader (Multiskan GO, USA); Energy-saving water bath steamer (RQ-ZBL-12, China); Yifeng extruding machine (6Y1.0, China).
2.2 General process of oil extraction
The oil was obtained according to the below processes. Briefly, 50kg powdered P. nagi kernel was placed on a steamer with the mechanical stirring for 20min. Then, the steamed powder was transferred to a fixed mold with the high pressure device, and squeezed through pressure-programmed route from 0MPa to 30MPa for 30min to obtain the light yellow oil, 10kg, yield:20%.
2.3 Chemical components analysis of the obtained oil
2.3.1 Organic chemical components analysis
The organic components of the obtained oil were analyzed accordingly. The total flavonoids were determined in accordance with the《Implementation Manual of Technical Specifications for Health Food Inspection and Evaluation》(2003 Edition) P1082 [Determination of Total Flavonoids (Spectrophotometry)]; the fatty acids were determined according to the method of GB 5009.168-2016; and vitamin E was determined according to the method of GB 5009.82-2016.
2.3.2 Trace elements analysis
2.3.2.1 Prepare the testing solution
0.5g oil together with 10 mL of 65% nitric acid was added into the polytetrafluoroethylene (PTFE) digestion tank and the tank was sealed. The mixture was digested according to the following working conditions (see to table 1 ; while the conditions of the ICP-MS instrument see to table 2 ). After completion of the digestion, the tank was cooled naturally to room temperature and the digestion solution was transferred to a 50 mL volumetric flask, and the tank was washed with a small amount of deionized water for three times and the washing solutions were also added to the volumetric flask. The volumetric flask was shaken well after filling to the constant volume with the deionized water to obtain the testing solution. The blank solution was prepared according to the above method.
Table 1. Conditions of microwave digestion