The antioxidant and antisenescence activities of physically refined rice bran oil surpass those of the combination of γ-oryzanol, α-tocopherol and sitosterol

ABSTRACT There are a variety of bioactive components in rice bran oil (RBO), among which the richer ones include γ-oryzanol, α-tocopherol and sitosterol. The activity of these ingredients is affected by the vehicle and formulation, and there is a significant synergy between these antioxidants. At present, the content of antioxidants in physically refined RBO has reached a high level. Therefore, comparing the activity of physically refined RBO with γ-oryzanol, α-tocopherol, and sitosterol as single components and the above three ingredients dissolved in the same base oil as RBO can provide information for better application of these antioxidants. The results showed that the antioxidant and anti-senescence capacities of physically refined RBO were significantly higher than those of dissolved γ-oryzanol, α-tocopherol, and sitosterol and better than those of the mixture. This study illustrates that the antioxidant and anti-senescence effects of highly active physically refined RBO are superior to those antioxidants derived from RBO.

At present, γ-oryzanol, α-tocopherol and sitosterol can be extracted and isolated from plant oils using different methods.Among them, γ-oryzanol has become a clinical drug, and αtocopherol and sitosterol are also being utilized in the development of clinical drugs (Fernandez & Vega-López, 2005;Pang & Chin, 2019;Ramazani et al., 2021).However, during the preparation of rice bran oil and the extraction and isolation of these natural products, many factors affect its biological activity (Aryusuk et al., 2008;Junyusen et al., 2022; R. J. Liu et al., 2019).Owing to several technical and nontechnical problems in oil refining, the actual annual production of RBO does not meet demand (Pestana-Bauer et al., 2012).The major difficulties in processing crude RBO for edible purposes are its high levels of FFA, waxes, gums, and pigments.However, the chemical and physical deacidification process causes the loss of bioactive compounds such as γ-oryzanol and α-tocopherol.
Based on the components content in RBO, it is possible that the antioxidant capacity and biological activity of highquality physically refined RBO is partially inferior to those of γ-oryzanol, α-tocopherol and sitosterol purified products at the same dose.Moreover, many studies have shown that the ratio between different antioxidants in RBO affects its bioactivity (R. R. Liu et al., 2020).When the ratio of α-tocopherol and β-carotene in liposomes was near 1:1, the synergistic effect was decreased, and when the ratio of α-tocopherol and myricetin was 1:1, the strongest synergistic effect was achieved (R. R. Liu et al., 2020).In addition, the total concentration of the mixture also affects the bioactivity.In this study, we compared the antioxidant and antisenescence activities of physically refined rice bran oil and purified γoryzanol, α-tocopherol, sitosterol, and a combination of these three active ingredients.

Materials and the preparation of samples
Physically refined RBO was obtained from HanKang Food Co., Ltd.(Jiangsu, China).γ-oryzanol (1479202, United States Pharmacopeia Reference Standard), α-tocopherol (258024, purity ≥ 95.5%) and sitosterol (1612947, United States Pharmacopeia Reference Standard) were purchased from Sigma-Aldrich (Bellefonte, PA, U.S.A).Palmitic acid (C16:0), stearic acid (C18:0), oleic acid (C18:1n9c), and linoleic acid (C18:2n6c) were purchased from J&K Scientific (China).The volume ratios of fatty acids and the concentration of α-tocopherol, γ-oryzanol and sitosterol were determined through analyzing of physically refined rice bran oil according to Chinese standard methods (GB/T 25,223-2010 for the determination of individual and total sterols contents, GB/T 26,635-2011 for the determination of tocopherol and tocotrienol contents, LS/T 6121.1-2017 for the determination of γ-oryzanol content, and GB 5009 168-2016 for the determination of fatty acids contents).The base oil was prepared by mixing different fatty acids in the following volume ratios: C16:0 19%, C18:0 4%, C18:1n9c 42%, and C18:2n6c 35%.The single ingredient sample was prepared by adding a single component to the base oil, and the final concentrations of the three minor constituents were as follows: α-tocopherol 228 mg/kg, γ-oryzanol 20,300 mg/kg, and sitosterol 10,490 mg/kg.The mixed sample was obtained by mixing the above three single components together and then adding them to the base oil (γ-oryzanol plus αtocopherol and sitosterol, OTS).The samples were mixed by mechanical vortexing and stored at 4°C.

Oxidative stability
The oxidative stability was determined using a 743 Rancimat apparatus (Metrohm Instruments, Herisau, Switzerland) (R. R. Liu et al., 2020), utilizing a sample of 2.50 g ± 0.01 g.A flow of air (20 L/h) was bubbled through the oil samples heated at 100°C, 110°C or 120°C.The test was applied in triplicate.The volatile organic acids from the oil sample were collected in cold water to increase the conductivity, and the conductivity was recorded continuously.The induction times [h] were printed automatically by the apparatus software with an accuracy of 0.005.The induction time is related to the antioxidant effect of the oil sample, and the longer the induction time, the stronger the antioxidant effect of the oil.

Scavenging capacity test
The scavenging capacity test were conducted as described previously (R. R. Liu et al., 2020).Briefly, a 0.1 mmol/L 2-Diphenyl-1-picrylhydrazyl (DPPH) solution was first prepared in ethyl acetate, and the samples were prepared at 25 mg/mL in ethyl acetate.A 2.0 mL sample solution was mixed with 2.0 mL DPPH solution, the mixtures were reacted in the dark at room temperature for 30 min, and the absorbance at 517 nm was recorded using a spectrophotometer.The experimental scavenging capacity (ESC) was calculated from the following equation: ESC = [1 -(Ai -Aj) A0] * 100%.where A0, Ai, and Aj are the absorbance values of 2.0 mL ethyl acetate + 2.0 mL DPPH solution, 2.0 mL sample solution + 2.0 mL DPPH solution and 2.0 mL sample solution + 2.0 mL ethyl acetate, respectively.The scavenging capacity is related to the antioxidant capacity of the sample, and the higher the scavenging capacity, the stronger the antioxidant capacity of the sample.

Analysis of the reducing capacity
The reducing capacity test were conducted as described previously (Arab et al., 2011). Different oil samples (0.375, 0.75, 1.5, 3.125, 6.25, 12.5, 25, 50 mg/ml) were mixed with phosphate buffer (2.5 ml, 2.0 M, pH 6.6), and then mixed with 2.5 ml of 1% potassium ferricyanide and incubated at 50°C for 20 min.2.5 ml of 10% trichloroacetic acid was added to the mixture.After centrifugation for 10 min, the upper layer of the solution (2.5 ml) was mixed with distilled water (2.5 ml) and 1% ferric chloride (0.5 ml), and the absorbance at 700 nm was recorded.The reducing capacity is related to the antioxidant capacity of the sample, and the higher the absorbance at 700 nm, the stronger the antioxidant capacity of the sample.

Determination of reactive oxygen species (ROS) in cells
The effect of the samples on excessive ROS generation was detected in 293T cells.293T cells were exposed to 2 mM H 2 O 2 for 30 min and incubated for 2 days in the presence of 0.1% (v/v) H 2 O 2 .The intracellular ROS were measured with a 2",7"-dichlorodihydrofluorescein diacetate (DCFDA) cellular ROS detection assay kit according to the manufacturer's instructions (Aranda et al., 2013).The intracellular fluorescence intensity is related to the amount of ROS in the cell, and the higher the DCFDA fluorescence intensity represents the more ROS in the sample.

Animals and treatments
Male C57BL/6J mice (18 g, 6-8 wk) and Sprague-Dawley rats (180-200 g) were obtained from Vital River Laboratory Animal Technology (Beijing, China).All animal protocols conformed to the Guidelines for the Care and Use of Laboratory Animals approved by the Animal Care and Use Committee of the Chinese Academy of Medical Sciences and Peking Union Medical College.Mice had free access to food and water and were housed in an air-conditioned room with a 12-hr/12-hr light -dark cycle.The mice were divided randomly into seven groups (n = 10 each): the blank control group, model group, γ-oryzanol group, α-tocopherol group, sitosterol group, combination group (γ-oryzanol plus αtocopherol and sitosterol, OTS), and the RBO group.Except for the blank control group, all groups were subcutaneously injected with D-galactose (D-gal) (120 mg kg −1 d −1 ) dissolved in normal saline solution (6%, w/v) for 8 weeks.All mice were orally gavaged with 2.5 ml/kg test samples.All animals were regularly monitored for weight on a weekly basis, and the dose was adjusted accordingly.

Morris water maze task
The Morris water maze equipment was used to test the learning and memory ability of mice in each group (Barnhart et al., 2015).The pool is 50 cm high, 120 cm in diameter, and 10 cm in diameter on the platform.The pool is divided into quadrants I, II, III, and IV.There is a video camera connected to the computer above the maze.The activity was tracked throughout the whole process, and the activity track of the experimental mice was displayed and analyzed through special computer software.The time required for each group of mice to successfully find the platform after the same learning training was recorded as the escape latency, and the number of times that each group of mice crossed the platform after the platform was removed was calculated.Learning and training: on the first day, the mice of each group were allowed to enter the water from different quadrants of the pool and swim freely for 2 min to familiarize themselves with the water maze environment.The swimming time was set to 2 min, and the mice were allowed to stand on the platform for 10 seconds to consolidate their memory each time they found the platform.Orientation navigation experiment: orientation navigation tests were carried out on the 2nd-6th day, and the result on the 6th day was used as the experimental data of this stage.At the same time, every day, the mice were put into the pool from 4 entry points facing the pool wall in turn, and the time required for them to successfully find the platform (escape latency) was calculated.Space exploration experiment: on the 7th day, the platform was removed, the mice were placed in the pool from the fourth quadrant, the movement of the mice was recorded within 2 min, and the number of times the mouse crossed the original platform was calculated.The escape latency time is related to the memory function and learning ability of the mouse, and longer escape latency time indicates worse memory function and learning ability.

Western blotting
Proteins were extracted from liver tissue using Radio-Immunoprecipitation Assay (RIPA) buffer containing 1% NP-40 and 1% sodium deoxycholate (Cell Signaling Technology, Danvers, MA).Protein concentrations were determined using a BCA Protein Assay Kit.SDS-PAGE and Western blotting were conducted as described previously (Lv et al., 2022).

Tissue index
Tissue index was determined by thymus (mg) or spleen (mg) versus body weight (g).

Statistics
Data are expressed as the mean ± standard error of the mean (SEM).Groups were compared by one-way ANOVA followed by a Tukey-Kramer's multiple comparisons test.Comparisons between two groups were performed by unpaired Student's t tests.A P value < .05 was considered to be significant.The Prism 9 was used for the statistical analysis in this study.

The antioxidant capacity of RBO and its active ingredients
Because the free radical scavenging ability of antioxidants is usually affected by solvents, the same amount of γ-oryzanol, α-tocopherol and sitosterol as rice bran oil were dissolved in a base oil that has almost the same fatty acid composition as rice bran oil.In addition, a mixed sample of γ-oryzanol, αtocopherol and sitosterol with the same content as RBO was prepared also.In this work, the Rancimat test and DPPH free radical scavenging method were performed to study the antioxidant capability of different samples.The antioxidant capacity in the Rancimat test of the samples could be generally ranked as follows: RBO > OTS > α-Tocopherol > γ-Oryzanol > Sitosterol at 100°C, 110°C and 120°C (Figure 1a and Table 1).The DPPH scavenging capacity and reducing power of RBO were significantly higher than those of γoryzanol and sitosterol (Figure 1b,c).The reducing power of RBO and other bioactive ingredients increased with increasing concentration (Figure 1d and Table 2).The highest values of absorption for reducing power were observed in the RBO group (Figure 1c,d).In addition, all the bioactive ingredients could decrease the level of ROS in 293T cells induced by H 2 O 2 (Figure 2a), and among them, RBO showed the best ROS-clearing function (Figure 2b).

RBO inhibits oxygen free radical damage in senescent mice
Eight weeks after D-gal stimulation, the model group had a significant decrease in body weight compared with the control group, and the administration of RBO prohibited D-gal-induced weight loss (Figure 3a).The thymus index and spleen index of the model group were also significantly decreased compared with those of the control group.However, the administration of RBO increased the thymus index and spleen index compared to the D-gal-induced aging group (Figure 3b,c).
The oxidative damage marker Malondialdehyde (MDA) levels were significantly increased in the serum of aging mice (Figure 3d), whereas the activities of antioxidative marker superoxide dismutase (SOD) and phospholipid hydroperoxide glutathione peroxidase (GSH-Px) were significantly decreased in D-gal mice compared with the control group (Figure 3e,f).After RBO treatment, the activities of GSH-Px and SOD were increased, and the MDA level was decreased, and RBO treatment restored these indices to values closest to the control group compared with the other active ingredient treatments (Figure 3d,f).

RBO improves immune status in aging mice
Immunosenescence-related inflammation in aging is mainly characterized by upregulation of proinflammatory cytokines or chemokines (such as TGF-β and IL-8) and downregulation of anti-inflammatory cytokines (such as IL-2 and IL-4).The levels of serum IL-2 and IL-4 were increased in RBO-treated mice compared with D-gal-exposed mice (Figure 4a,b).In contrast, the levels of serum TGF-β and IL-8 were reduced in RBO-treated mice compared with D-gal-treated mice (Figure 4c,d).Notably, there was no remarkable difference in the intervention effects on IL-2, IL-4 and TGF-β between the γ-oryzanol, α-tocopherol or sitosterol alone treatment groups and the model group (Figure 4a-c).In summary, these findings consistently demonstrated that RBO could effectively regulate the immune environment in mice exposed to D-gal.

Effect of RBO on spatial learning and memory
The Morris water maze (MWM) test was used to assess the spatial learning and memory functions of D-gal-treated senescent mice.The results suggested that during the 6-day training period, all mice showed a gradually shortened latency to locate the hidden platform.However, D-galexposed mice spent more time reaching the platform than control mice (Figure 5a), indicating that long-term exposure to D-gal significantly reduced the learning and memory abilities of mice.In addition, the number of target platform crossings in D-gal-exposed aging mice was dramatically reduced compared with that in the control group (Figure 5b), indicating a high possibility of impairment of learning and memory functions.Notably, impaired cognitive ability was effectively rehabilitated by RBO, as indicated by the escape latency and platform crossover number (Figure 5a,b and Table 3).However, γ-oryzanol or αand sitosterol alone did not effectively alleviate D-gal-induced learning and memory impairment (Figure 5a, b and Table 3).

RBO attenuates cellular senescence by inhibiting the p53/p21 pathway
The p53/p21 and p16/Rb pathways are involved in cell senescence.We speculated whether RBO could inhibit cellular senescence by inhibiting the p53/p21 or p16/Rb pathway.After stimulation with D-gal, the pp53, p21, and p16 levels were increased, and pRb was decreased in the liver tissue of mice (Figure 6a-d), which indicates that D-gal induces senescence.The results showed that RBO significantly decreased the expression of pp53, p53 and p21  (Figure 6a,b) but not p16 and pRb (Figure 6c,d).These data illustrate that RBO prevents cellular damage by blocking the p53/p21 pathway.

Discussion
Plant oil contains many antioxidant and anti-inflammatory components, especially RBO, which contains a high abundance of γ-oryzanol, α-tocopherol or sitosterol (Maszewska et al., 2018).These bioactive components have shown good antioxidant, anti-inflammatory, and hypolipidemic effects in both in vitro and in vivo studies.However, the antioxidative and antisenescence effects produced by these single components extracted and isolated from RBO are not as good as those produced by natural physically refined RBO.Even if γoryzanol, α-tocopherol and sitosterol are mixed and   redissolved in base oil according to the content of each component in RBO, their biological activity is still inferior to that of natural physically refined rice bran oil.It has been confirmed that γ-oryzanol in oil form would be more bioactive than γ-oryzanol dissolved in corn oil, followed by crystalline γ-oryzanol (Dai, 2004).The results showed that the bioavailability of crystalline γ-oryzanol was lower than that of crystalline γ-oryzanol dissolved in corn oil, and the solvents largely affected the biological activity of γoryzanol.Crystalline γ-oryzanol might be excreted in much more intact forms than the dissolved oil form.Thus, γoryzanol is best consumed after being dissolved in vegetable oil.In addition, the bioavailability of γ-oryzanol in RBO is higher than that of the dissolved form of crystalline γoryzanol in corn oil since the compatibility of γ-oryzanol in RBO is higher than that in corn oil (Dai, 2004).Moreover, a large number of studies have shown that there is a clear synergistic effect between γ-oryzanol, α-tocopherol and phytosterol (R. R. Liu et al., 2020Liu et al., , 2022)), so the combined use of γ-oryzanol, α-tocopherol and phytosterol in a certain proportion range can produce better antioxidant and antiinflammatory effects.In our study, base oils with the same fatty acid composition as rice bran oil was used to dissolve γoryzanol, α-tocopherol or sitosterol.At the same time, the three bioactive components were dissolved in the base oil in the same proportion and concentration as rice bran oil to observe their antioxidant and anti-senescence effects.Unexpectedly, the results showed that simply dissolving γoryzanol in the base oil was far less effective than physically refining RBO.Even the OTS group had slightly worse antioxidant effects than RBO.This illustrates that the process of preparing pure γ-oryzanol, α-tocopherol or sitosterol may lead to a loss of activity.At the same time, we cannot rule out the synergistic effect of other active ingredients in RBO.In addition to γ-oryzanol, α-tocopherol or sitosterol and unsaturated fatty acids, RBO is rich in 24methylenecholesterol, stigmasterol, γ-tocopherol, squalene, α-tocotrienol, and γ-tocotrienol, and these ingredients have certain antioxidant and anti-senescence effects also.Therefore, the antioxidant and anti-senescence effects of composition of ingredients are not as good as natural RBO.However, it is very difficult to study the synergy and mechanism between all the components in RBO.
ROS play important roles in tissue homeostasis, cellular signaling, differentiation, and survival (Schieber & Chandel, 2014).Overproduction of ROS is associated with the development of various human diseases.However, upregulation of antioxidant systems in cells can effectively scavenge ROS.MDA acts as a hallmark of oxidative damage in lipids.And the anti-oxidative system consists of reducing substances and anti-oxidative enzymes, such as, GSH-Px and SOD.RBO has a good antioxidant effect both in vitro and in vivo.In addition, the antioxidant effect of RBO in senescent mice was accompanied by weight gain and immune regulation.After RBO administration, the levels of IL-2 and IL-4 were significantly increased in senescent mice, while the levels of aging-inducing factors TGF-β and IL-8 were significantly decreased.This indicates that the anti-senescence effects of RBO not just dependent on antioxidation.More importantly, RBO has the effect of improving cognitive function, which fully shows that RBO is not only a nutrient, but also an active ingredient with clinical application prospects.Our results show that there is indeed a synergistic effect between these ingredients, but physically refined RBO which containing the same concentration of active ingredients has better antioxidant and anti-senescence effects than the manually combined active ingredients.

Disclosure statement
No potential conflict of interest was reported by the author(s).

Figure 5 .
Figure 5. RBO alleviates learning and memory impairment of D-gal exposed mice.(a) the escape latency of the mice during MWM training.(b) the number of platform crossing of the mice during MWM training.All data are presented as Mean ± SEM, (n = 8).*P < .05.RBO: rice bran oil, OTS: γ-oryzanol plus α-tocopherol and sitosterol.