RSC Advances View Article Online PAPER View Journal | View Issue Changes of physicochemical properties, oxidative stability and cellular anti-inflammatory potentials Cite this: RSC Adv., 2020, 10,36678 for sea-buckthorn pulp oils during refining Xiaofei Jiang,a Wei Li,ab Shengmin Zhou *a and Yuanrong Jianga The impact of the refining process on physicochemical properties, oxidative stability and cellular anti- inflammatory potentials of sea-buckthorn pulp oil (SBO) was investigated in this study. The results showed that acid and peroxide values of the tested SBOs decreased significantly after the refining process, while oxidative stability index (OSI) and anti-inflammatory potentials, measured as reduction in cellular inflammatory cytokine production, increased significantly. Interestingly, bleaching caused an unexpected increase in Received 18th August 2020 tocopherols as well as the greatest reduction in polycyclic aromatic hydrocarbons (PAHs). According to Accepted 23rd September 2020 correlation analyses, tocopherol concentrations were significantly and positively correlated with OSI values DOI: 10.1039/d0ra07095e and cellular anti-inflammatory potentials, while PHAs were negatively correlated with these factors. In general, Creative Commons Attribution-NonCommercial 3.0 Unported Licence. rsc.li/rsc-advances refining is an effective way to improve the oxidative stability and anti-inflammatory capacity of SBO. 1. Introduction micronutrient content of SBO.13 Compared with conventional (solvent, pressing and expelling) extraction methods, supercritical Sea buckthorn (Hippopha¨e rhamnoides L., SB) is a hardy, and subcritical extraction technologies can be considered as deciduous, fast-growing spiny shrub with orange or yellow e- alternative ways to enhance the quality and micronutrients of shy, juicy berries.1 It can grow in extreme conditions like SBO.2,14 However, few studies have focused on the changes of droughts, high salinity and low or high temperatures. There- physicochemical properties in SBO during the subsequent pro- This article is licensed under a fore, SB is cultivated in Asia and Northern Europe because of its cessing steps aer its extraction from the berry pulp. excellent abilities for water and soil conservation, and for land In fact, crude SBO also contains certain amounts of undesir- reclamation based on its nitrogen-xing root nodules.2 able minor components, i.e., free fatty acid (FFAs) and polycyclic Sea buckthorn pulp oil (SBO), a nutritive oil product extrac- aromatic hydrocarbons (PAHs), which can adversely affect oil Open Access Article. Published on 06 October 2020. Downloaded 9/24/2021 6:29:27 AM. ted from the pulp of SB berries, has been reported to contain quality and physicochemical properties. Rening is an effective a broad range of functional fatty acids such as palmitoleic acid way to eliminate these undesirable components in crude oils. (C16:1 u-7), oleic acid (C18:1 u-9), linoleic acid (C18:2 u-6) and However, micronutrients such as phytosterols and tocopherols linolenic acid (C18:3 u-3).3 More importantly, SBO is an edible can also be destroyed or removed due to the high temperature oil that is remarkedly rich in natural micronutrients such as and/or chemical reagents used in the rening process.15 tocopherols, phytosterols and carotenoids.4 Previous studies Considering the effect of rening on the quality and safety of have been reported that these micronutrients have anti- oil products, the present work is aimed (i) to make a compre- oxidative, antimicrobial, anti-aging, anti-cancer and anti- hensive comparison of physicochemical properties, oxidative inammatory properties beneting human health.5–7 In addi- stability and cellular anti-inammatory potentials of SBOs ob- tion, SBO or individual substances seperated from SB berries tained during the rening steps; (ii) to evaluate the relationship have also shown positive effects on symptoms and diseases between physicochemical properties, oxidative stability and such as acute alcohol intoxication,8 atherosclerosis,9 depres- anti-inammatory potentials of these different SBO samples. In sion,10 burn wounds,11 and dry eyes.12 brief, this study provides a quality assessment and comparison Due to these outstanding characteristics, SBO has attracted of important characteristics of SBOs obtained from different more and more attention in recent years. Most researchers focused steps in rening. It can provide the academic foundation for the on the effects of different oil extraction methods on the quality and usage of SBO as a food ingredient. aWilmar (Shanghai) Biotechnology Research & Development Center Co., Ltd, No. 118 2. Materials and methods Gaodong Road, Shanghai 200137, P. R. China. E-mail: zhoushengmin@cn. 2.1 Materials and reagents wilmar-intl.com; Fax: +86 21 58481079; Tel: +86 21 31153015 bUniversity of Shanghai for Science and Technology, School of Medical Instrument & Crude SBO was extracted from frozen dried sea-buckthorn pulp by Food Engineering, Shanghai 200093, P. R. China cold pressing. It was provided by Aikang Sea Buckthorn 36678 | RSC Adv.,2020,10, 36678–36685 This journal is © The Royal Society of Chemistry 2020 View Article Online Paper RSC Advances Pharmaceutical Co., Ltd (Xian, Shanxi, China). The neutralized, comparing with the corresponding standards and quantied bleached and deodorized SBOs were prepared in laboratory, and using the standard curves of corresponding tocopherols. the detailed procedures are listed in Section 2.2. The phytosterols contents were determined based on the Standards of 5a-cholestan-3b-ol, tocopherols (a-, b-, g-andd- method of Xie et al.15 The oil samples were added with internal À tocopherols), phytosterols (campesterol, b-sitosterol, and 7-stig- standard (1 mg mL 1 5a-cholestan-3b-ol in hexane). Then, 5 mL À masterol), and b-carotene were purchased from Sigma-Aldrich of KOH–ethanol (2 mol L 1) was added for saponication at (Shanghai, China). 3-Monochloropropane-1,2-diol (3-MCPD) and 60 C for 1 h. Aer that, 4 mL of H2O was added and then 5 mL 3-MCPD-d5 were purchased from CDN Isotopes Inc (Pointe-Claire, hexane was added to extract the unsaponiable matter for three Canada). A standard mixture of PAHs was purchased from AccuS- times. Aerwards, 1.5 g of sodium sulfate anhydrous was tandard (New Haven, USA). BSTFA + TMCS (99 : 1) for derivatization added. All the organic phases were combined and evaporated. was obtained from Sigma-Aldrich (Shanghai, China). The HPLC Aerwards, 0.2 mL (BSTFA + TMCS) was added and silylated at grade solvents, such as tetrahydrofuran, acetone, n-hexane, toluene, 105 C for 20 min, and the resulting product was redissolved in isopropanol, dichloromethane, acetonitrile, chloroform, were 1 mL hexane before analysis. The phytosterols were analyzed provided by CNW (Darmstadt, Germany). All other reagents and using a Thermo Scientic GC spectrometry (GC) equipped with solvents were obtained from Sinopharm Chemical Reagent Co. Ltd. a DB-5 capillary column (0.32 mm  30 m, 0.25 mm, Agilent (Shanghai, China). Corp.). The phytosterols (campesterol, b-sitosterol, and 7-stig- masterol) were identied with comparison of corresponding 2.2 Oil rening process standards, and quanti ed based on the internal standard. The squalene content was determined based on the method 2.2.1 Degumming & neutralization. The citric acid of Chiou et al.17 Aer the sample was saponied and derivat- degumming process was modied according to Jiang et al.16 ized. The squalene content was analyzed using a Thermo Crude SBO (1000 g) was heated to 70 C under stirring (500 Scientic GC spectrometry (GC) equipped with a HP-5 capillary rpm). Then 20 mL of citric acid solution (20% concentration)  m Creative Commons Attribution-NonCommercial 3.0 Unported Licence. column (0.32 mm 30 m, 0.25 m, Agilent Corp.). The squa- was added, and mixed vigorously (10 000 rpm) for 1 min. Aer lene was identi ed by comparing with the squalene standard that, the mixture was stirred (500 rpm) at 70 C for acid treat- (Merk, Life Science) and quantied using the standard curves of ment for 60 min. Then the mixture was centrifuged at squalene. 10 000 rpm for 10 min (HITACHI, model CR21G). The super- The b-carotene content in the oil samples was determined natant was collected and named degummed SBO. based on the AOCS Recommended Method Ce 9-01 using The degummed SBO was added with sodium hydroxide a Agilent high-performance liquid chromatography (HPLC) solution (15% concentration) and mixed at 88 C. A er being system with a UV detector (45 nm) and a C30 column (4.6 mm  stirred (250 rpm) at this temperature for 10 min, the mixture was 150 mm, 5 mm, YMC-Carotenoid, Japan). The oil is saponied This article is licensed under a centrifuged at 10 000 rpm for 10 min. Then the supernatant was and then extracted with petroleum ether. The extract is washed, collected, and it was washed three times with hot distilled water, concentrated, and then dissolved in methylene chloride for and dried at 105 C under vacuum to obtain neutralized SBO. HPLC analysis. The b-carotene was identied by comparing 2.2.2 Bleaching. The neutralized SBO was treated with 3% with the b-carotene standard and quantied using the standard Open Access Article. Published on 06 October 2020. Downloaded 9/24/2021 6:29:27 AM. acid-activated bleaching earth (w/w), 1% diatomaceous earth curves of b-carotene. (w/w) and 1% activated carbon (w/w). Aerward, it was bleached under vacuum at 105 C with vigorous stirring for 30 min. Then, 2.5 Determination of hazardous substances the oil was cooled to 60 C and ltered. ff 2.2.3 Deodorization. The bleached oil was placed in a three- Four di erent kinds of PAHs including benzo(a)anthracene necked ask, and stirred with nitrogen and deodorized at 250 C (B(a)A), chrysene (CHR), benzo(b) uoranthene (B(b)F) and for 3 h under 0.1 kPa vacuum (Edwards, model RV12).