Journal of Oleo Science Copyright ©2018 by Japan Oil Chemists’ Society J-STAGE Advance Publication date : November 15, 2018 doi : 10.5650/jos.ess18064 J. Oleo Sci. Role of Seed Oil and Fish Oil with or without Turmeric and Alpha- Tocopherol in Prevention of and Fatty Liver in Rats Sahar Y. Al-Okbi1＊ , Salah M. El-qousy2, Samah El-Ghlban3, and Hosam F. Moawad4 1 Nutrition and Food Sciences Department, National Research Centre, Cairo, EGYPT 2 Chemistry Department, Faculty of Science El Menoufeia University, Shebin El Kom, EGYPT 3 Chemistry Department, Biochemistry Division, Faculty of Science El Menoufeia University, Shebin El Kom, EGYPT 4 Medical Research Center, Faculty of Medicine, Ain Shams University, Cairo, EGYPT

Abstract: The aim of the present research was to Study the prevention of dyslipidemia, oxidative stress, and fatty liver as risk factors for cardiovascular disease via intervention by borage oil (B) and fish oil (F) with or without turmeric (T) and alpha-tocopherols (TC). Fatty acids were assessed in both oils while curcuminoids were determined in turmeric. Rats were divided into; first group fed on balanced diet and designated as normal control (NC), second fed on dyslipidemic and steatohepatitis (DS) inducer diet which represented the DS control group and groups 3-6 fed on DS inducer diet with daily oral administration of B, B+T+TC, F and F+T+TC; respectively for 5 weeks. Liver fat and plasma lipid profile, oxidative stress and inflammatory biomarker and liver and heart histopathology were assessed. Results showed gamma linolenic to be 21.01% in B. F contained eicosapentaenoic as 22.768% and docosahexaenoic acid as 13.574%.Total curcuminoids were 4.63 mg/g turmeric. The DS control group showed significant dyslipidemia, elevated malondialdehyde (MDA), tumor necrosis factor-alpha and liver fat with significant reduction in total antioxidant capacity (TAC) compared to NC. The different treatments produced significant improvement in all the parameters and histopathology. F was superior to B in ameliorating liver histopathological changes while B was more efficient in elevating TAC. B was more promising in improving lipid profile and liver fat compared to B + T + TC, while the latter was superior in improving MDA and liver histopathology. Fish oil was more efficient than F+TC+T except for TAC and high density lipoprotein cholesterol which were more improved on addition of TC and T. Conclusion: Borage and fish oil with or without antioxidants protect from cardiovascular and fatty liver diseases with variable degrees.

Key words: borage oil, fish oil, turmeric, alpha-tocopherol, cardiovascular disease, fatty liver

1 Introduction omega3 and omega6 could have different efficiency Cardiovascular diseases（CVDs）are considered as the towards dyslipidemia and CVDs. Omega3 fatty acids like al- major cause of morbidity and death worldwide in both de- pha-linolenic, eicosapentaenoic（EPA）and docosahexaenoic veloped and developing countries1）. Dyslipidemia, oxidative （DHA）possess both hypolipidemic and anti-inflammatory stress and inflammation are among the reported risk activity but there is a confliction in literature concerning factors for cardiovascular diseases2）. Recently fatty liver their effect on oxidative stress4）. Fish oil is rich in long with inflammation（steatohepatitis）, which is increasing chain polyunsaturated fatty acids（EPA and DHA）and was worldwide, is accused as being major player in induction of reported to possess the aforementioned health effects as cardiovascular diseases3）. Generally, the condition includ- well as hepatoprotective effect5）. On the other hand little is ing dyslipidemia, inflammation, steatohepatitis, oxidative known about the effect of omega6 fatty acid like linoleic stress and impaired glucose tolerance is called metabolic acid on metabolic syndrome. Although linoleic was report- syndrome. ed to have hypolipidemic effect but claimed to induce some Two series of polyunsaturated fatty acids represented by sorts of pro-inflammation. However; this claim was not

＊Correspondence to: Sahar Y. Al-Okbi, Nutrition and Food Sciences Department, National Research Centre, Cairo, EGYPT E-mail: [email protected] Accepted August 20, 2018 (received for review April 8, 2018) Journal of Oleo Science ISSN 1345-8957 print / ISSN 1347-3352 online http://www.jstage.jst.go.jp/browse/jos/ http://mc.manusriptcentral.com/jjocs

1 S. Y. Al-Okbi, S. M. El-qousy, S. E.-Ghlban et al.

supported by recent studies6, 7）. Linoleic acid was reported 2.2 Animals by Maruyama et al.8）to be a potent protector against lipo- Male Sprague Dawley rats weighing 140-150 g. were toxicity induced inflammation. Other omega6 fatty acid like used in the present study. Animals were obtained from gamma linolenic（GLA）was reported to have anti-inflamma- Animal house of National Research Centre, Cairo, Egypt. tory and antioxidant effect9, 10）. The ratio of n-6/n-3 polyun- Rats were kept individually in stainless steel cages; water saturated fatty acids is a better index of health benefit; un- and food were given adlibtium. Animal experiment was favourable ratio was reported to be associated by greater carried out according to the Medical Research Ethics Com- systemic inflammation11）. mittee for institutional and national guide for the care and Borago officinalis is native to the Mediterranean region, use of laboratory animals, National Research Centre, Cairo, North Africa and Middle East and is known as starflower or Egypt. borage. In traditional medicine borage is used in gastroin- testinal, respiratory, cardiovascular and urinary disor- 2.3 Methods ders12）. It is also used for pre and post-menopausal symp- 2.3.1 Assessment of fatty acids in borage and fish oil toms13）. Commercially, the plant is cultivated for borage Methyl esters were prepared via trans-esterification of seed oil which is used for different medical and nutritional fatty acids using methanol sulfuric acid and chloroform benefits. The seed contain 26-38％ oil which is the richest mixture according to Indarti et al.18）. Twenty mg of the oil source of gamma linolenic acid（GLA）that accounts for was weighed into clean, 10 mL screw-cap glass tubes, to 17-28％ of the oil14）. which 4 mL fresh solution of a mixture of freshly prepared Aside from being a popular condiment, turmeric has long methanol, concentrated sulfuric acid and chloroform history in medicinal use in Ayurvedic medicine for wound （1.7/0.3/2 v/v/v）was added. The tubes were covered; trans- healing, hepatic diseases, joint pains, gastrointestinal disor- esterification was run at 100℃ for 30 min. On completion der and cancer prevention. Curcuminoids and phenolic of the reaction, the tubes were brought to room tempera- compounds as well as the essential oil were reported to be ture. Then, 3 mL distilled water was added into the mixture the major bioactive constituents in turmeric and to which and thoroughly mixed by vortex for 1 min. After the forma- the bioactivity of turmeric were ascribed. The health bene- tion of two phases, the lower phase containing the fatty fits of such constituents are attributed mainly to their anti- methyl esters was transferred to clean glass tube and dried 15） oxidant and anti-inflammatory effect . A meta analysis with anhydrous Na2SO4. Fatty acids were analyzed by GC study showed curcuminoids therapy to reduce triglycerides after addition of a known volume of chloroform. and increase HDL-Ch16）. Alpha-tocopherol an antioxidant Assessment of the methyl ester was carried out by in- oil soluble vitamin was reported recently to possess anti- jecting 2 μL into a Hewlett Packard HP-system 6890 gas inflammatory, hepatoprotective and cardioprotective chromatograph equipped with FID. HP-5 capillary column effect17）. So it is hypothesized that combination of either （30 m×0.32 mm i.d.; 0.25 μm film thickness）was used to fish oil or borage seed oil with both turmeric and alpha to- separate the different methyl esters. The chromatographic copherol could impart an efficient cardiovascular protec- analysis conditions were; initial temperature 70℃ with a tive effect in addition to prevention of steatohepatitis. hold for 1 min, then raised to 120℃ at a rate of 40℃/min The objective of the present research was to set a com- with 2 min hold then the temperature was finally raised to parative study between the healthy effect of borage and 220℃ at a rate of 4℃/min with another 20 min hold. The fish oil in rat model of metabolic syndrome（dyslipidemia injector and detector temperatures were 250℃ and 280℃; and steatohepatitis）when administered alone or as a blend respectively. Identification of the fatty acid methyl esters with alpha tocopherol and turmeric. The aim included the were carried out by direct comparison of retention times of analysis of fatty acids contents of the oils and assessment each of the separated compounds with standards of the of curcuminoids content of turmeric. fatty acid methyl esters analyzed under the same condi- tions. Quantization was based on peak area integration. 2.3.2 Curcuminoids determination in turmeric Curcuminoids were determined according to Jayapraka- 2 Experimental sha et al.19）. Methanolic stock solutions of curcumin, deme- 2.1 Materials thoxycurcumin, and bisdemethoxycurcumin were prepared Borage oil（Borago officinalis, family Boraginaceae）and separately at a concentration of 0.5 mg/mL. Turmeric fish oil were obtained from Iherb, 1795 World Wide Blvd, powder sample（1.0 g）was extracted with hexane（50 mL） USA. Turmeric powder was purchased from herbal store, by using a Soxhlet extractor for 30min. The hexane extract local market, Cairo, Egypt. Alpha tocopherol was obtained was discarded, and the powder was re-extracted with 50 from local pharmacy, Cairo, Egypt; the manufacturer is mL of methanol for 2 h. One milliliter of this solution was Pharco Pharmaceuticals Company, Alexandria, Egypt. transferred to a 10 mL volumetric flask, and the volume was adjusted to 10 mL with methanol.

2 J. Oleo Sci. Edible Oils with or without Antioxidants for Healthy Heart and Liver

Chromatographic conditions: The elution was carried out stomach tube specially when mixed with the other constit- with gradient solvent systems with a flow rate of 1.0 mL/ uents while the second reason is the presence of another min at ambient temperature. The mobile phase consisted antioxidant represented by α-tocopherol in the nutraceuti- of methanol（A）, 2％ acetic acid（B）, and acetonitrile（C）. cals. So in the present study the previously mentioned Quantitative levels of curcuminoids were determined using doses were provided through administration of 0.75 mL of the above solvents programmed linearly from 45 to 65％ the nutraceuticals 2 and 4. acetonitrile in B for 0-15 min. The gradient then went from 2.3.5 Design of the animal experiment 65 to 45％ acetonitrile in B for 15-20 min, with a constant Thirty six male rats were divided into 6 groups; each of 6 of 5％ A. The compounds were quantified using standard rats. The first was the normal control（NC）group where the curcumin, demethoxycurcumin, and bisdemethoxycur- rats received a balanced diet throughout the study period cumin. （5 weeks）, all other remaining groups were fed on DS 2.3.3 Preparation of Diets inducer diet for 5 weeks. One served as a DS control group, A balanced diet containing 12％ casein, 10％ Sun-flower whereas the other 4 groups were fed on DS inducer diet oil, 68.5％ corn starch, 5％ cellulose, 3.5％ mineral mixture along with an oral administration of a daily oral dose of 0.75 and 1％ vitamin mixture was prepared. Metabolic mL from nutraceutical 1, 2, 3 and 4, respectively. The NC syndrome（Dyslipidemia and steatohepatitis）inducer diet and the DS control groups were given daily oral dose of （DS）composed of 12％ casein, 20％ coconut oil, 62.25％ 0.75 mL from the vehicle. During the experiment, body fructose, 3.5％, mineral mixture, 1％ vitamin mixture, 1％ weight and food intake were recorded once a week. At the cholesterol and 0.25％ sodium cholate was prepared utiliz- end of the study which extended for 5 weeks, total food ing a combination of two previous methods20, 21）with some intake and body weight gain were calculated. Blood modifications. samples were collected from anaesthetized fasted animals. 2.3.4 Preparation of different nutraceuticals Plasma was separated from heparinized blood by centrifu- Different mixtures were freshly prepared every three gation for the determination of different lipid parameters days to be given to rats. Borage oil（810 mg）was emulsified represented by total lipids（T.L）, total cholesterol（T.Ch）, by tween 80 in distilled water（final volume 15 mL）. Tween triglycerides（T.G）, low density lipoprotein-cholesterol 80 was used as 10％ from the oil. The mixture was mixed （LDL-Ch）and high density lipoprotein-cholesterol well by vortex and designated as nutraceutical 1. Borage （HDL-Ch）by colorimetric methods24－28）. Plasma malondial- oil（810 mg）＋ α-toccopherol（1.08 g）＋ turmeric（1.6 g） dehyde（MDA）was assessed by the colorimetric methods of were emulsified by tween 80 and completed to 15 mL by Satoh29）as oxidative stress biomarker. Plasma total antioxi- distilled water and mixed by vortex till became homoge- dant capacity（TAC）as an antioxidant biomarker was deter- neous（Nutraceutical 2）. Tween 80 was used as 10％. Fish mined using colorimetric method as reported by Koracevic oil（810 mg）was prepared as in case of nutraceutical 1 et al.30）. Plasma tumor necrosis factor-alpha（TNF-α）was where borage oil was exchanged by fish oil and marked as estimated as anti-inflammatory biomarker adopting en- nutraceutical 3. Nutraceutical 4 was prepared as nutraceu- zyme-linked immunosorbent assay（ELISA）according to tical 2 with replacing borage oil by fish oil. A vehicle was the method of Stepaniak et al.31）. T.Ch/HDL-Ch was calcu- prepared containing the same amount of tween 80 in dis- lated as indicator of cardiovascular disease risk. Rats were tilled water as in the nutraceuticals to be given to rats of dissected and the livers and hearts were separated. A part the control groups. from each rat liver was kept in deep freeze till analyzed for Turmeric and tocopherol were added together because total lipids according to Cequier-Sànchez et al.32）by extrac- mixing two types of antioxidant were reported to be more tion and gravimetric measure. The other part of each liver beneficial to health than using only one. Also; tocopherol together with the heart was preserved in 10％ formalin for was added as antioxidant while turmeric possesses both the histopathology examination. antioxidant and anti-inflammatory effect so the prepared The different analyzed plasma biochemical parameters nutraceuticals could afford an antioxidant effect due to were assessed using commercial kits. The reagents kits for two antioxidants from two different sources with simulta- T.Ch, T.G and HDL-Ch were obtained from Salucea, Neth- neous anti-inflammatory activity. erlands. Reagents for LDL-Ch were purchased from Cen- Alpha-Tocopherol was used previously in a dose of tronic GmbH, Germany. T.L, TAC and MDA reagents kits 360mg/kg rat body weight22）i.e 54 mg/150g rat body weight were supplemented from Biodiagnostic, Egypt. Rat TNF-α which was applied in the current study. Turmeric was used ELISA kit was obtained from KOMABIOTEH, Seoul, Korea. as 1100 mg/kg rat body weight according to Rajashekhara et al.23）i.e 165 mg/ 150 g rat body weight. About half the 2.4 Statistical analysis dose of turmeric（80 mg/150 rat body weight）was used in The results of animal experiments were expressed as the the present study for two reasons; first due to its high vis- mean±SE and they were analyzed statistically using the cosity that could be not easy to be given to rats by the one-way analysis of variance ANOVA followed by Tukey

3 J. Oleo Sci. S. Y. Al-Okbi, S. M. El-qousy, S. E.-Ghlban et al.

test. In all cases p＜0.05 was used as the criterion of statis- Table 1 Fatty acids’ content of borage and fish oil（as tical significance. percentage of total fatty acids）.

Fatty acids Borage oil Fish oil Myristic acid, C14:0 － 7.452 3 Results Palmitic, C16 : 0 10.603 18.474 3.1 Fatty acid composition of borage and sh oil Palmitoleic, C16:1 － 9.349 The fatty acid composition of borage and fish oil is Stearic acid, C18:0 4.689 5.243 present in Table 1. It could be noticed that linoleic acid Oleic, C18:1 20.163 12.771 was the major fatty acid（36.482％）followed by γ-Linolenic Linoleic, C18 : （2 ω6） 36.482 2.379 （21.01％）in borage oil. Oleic acid was the only monounsat- urated acid detected（20.163％）. Alpha linolenic acid was γ-Linolenic, C18 : （3 ω6） 21.010 － present as 4.559％. The major saturated fatty acid was pal- α- Linolenic, C18 : （3 ω3） 4.559 3.368 mitic followed by stearic and arachidic（10.603, 4.689 and Arachidic acid, C20:0 2.493 － 2.493, respectively）. The predominant fatty acid in fish oil EPA, C20:5（ω3） － 22.768 was eicosapentaenoic acid（22.768％）and decosahexanoic DHA, C22:6（ω3） － 13.574 acid（13.574％）. The rest of the unsaturated identified fatty Total saturated fatty acids 17.785 31.169 acids in fish oil were oleic, palmitoleic, α- Linolenic, and linoleic（12.771, 9.349, 3.368 and 2.379％, respectively） Total unsaturated fatty acids 62.051 64.209 while saturated fatty acids were palmitic, myristic and Total monounsaturated fatty acids 20.163 22.12 stearic（18.474, 7.452 and 5.243％, respectively）. n-6: n-3 fatty acids 12.66 0.06

3.2 Curcuminoids content of turmeric 3.3 Results of the animal experiment The major curcuminoid compound in turmeric was cur- Table 2 showed that DS control group demonstrated sig- cumin which present as 2.55 mg/g followed by deme- nificant increase in plasma total lipids reflected in the sig- thoxycurcumin（1.13 mg/g）. Bisdemethoxycurcumin nificant elevation of total cholesterol, triglycerides, and showed the least content（0.95 mg/g）. LDL-Ch compared to NC. Plasma HDL-Ch decreased sig- nificantly along with significant increase of T.Ch/HDL-Ch

Table 2 Biochemical parameters of different experimental groups. Groups DS＋Borage oil DS＋Fish oil Normal control Control DS DS＋Borage oil ＋turmeric＋ DS＋Fish oil ＋turmeric＋ Parameters vitamin E vitamin E Plasma T.L（mg/dl） 280.2a±10.329 658.2b±17.696 326.3a±28.272 350.5a±18.45 312.0a±24.59 319.8a±40.842 T.Ch（mg/dl） 81.5a±2.437 177.8d±5.075 82.9a±1.452 99.6b±5.35 83.3a±5.304 115.3c±1.067 T.G（mg/dl） 92.6a±0.217 140.2c±0.214 107.3e±1.323 114.3d±0.201 110.1de±0.469 105.6e±3.51 HDL-Ch（mg/dl） 46.5a±1.349 31.6c±1.389 41.8bd±1.216 40.4b±0.585 44.5ad±1.044 40.4b±0.792 LDL-Ch （mg/dl） 16.5a±1.138 118.1c±5.669 19.7a±1.092 36.4d±2.88 17.3a±0.98 53.1b±1.323 T.Ch/HDL-Ch 1.758a±0.068 5.687c±0.344 1.985a±0.068 2.473b±0.163 1.87a±0.112 2.692b±0.129 TAC（mM/L） 6.08a±0.178 3.27c±0.247 5.57a±0.251 6.04a±0.184 4.12b±0.251 5.71a±0.254 MDA（nmol/mL） 3.980a±0.606 11.505c±0.767 4.327a±0.824 2.012b±0.421 4.783a±0.773 4.843a±0.143 TNF-α（Pg/mL） 51.8a±1.415 85.3c±0.86 64.7d±1.506 63.8bd±0.495 59.0bd±2.245 58.5b±1.011 Liver ％Total fat 4.2a±0.616 7.2c±0.290 3.8a±0.479 6.1b±0.135 3.8a±0.222 5.3b±0.306 Values are means±SE; where n＝6. DS: Dyslipidemic and steatohepatitis inducer diet, T.L: total lipids, T.Ch: total cholesterol, T.G: Triglycerides, HDL-Ch: High density lipoprotein cholesterol, TAC: Total antioxidant capacity, MDA: Malondialdehyde, TNF-α: Tumor necrosis factor-alpha In the same row: Similar superscript letters mean insignificant difference, while different letters mean significant difference between groups at p < 0.05.

4 J. Oleo Sci. Edible Oils with or without Antioxidants for Healthy Heart and Liver

and liver percentage of total lipids in the DS control com- was no significant change in liver/ body weight％ when the pared to NC. It was noticed that plasma MDA and TNF-al- different treated groups were compared with each other. pha were significantly elevated along with significant re- Histopathological examination of livers and hearts of the duction of total antioxidant capacity in DS control group different groups are shown in Figs. 1 and 2. It could be compared to NC. Administration of any of the studied nu- noticed from Fig. 1 that liver of NC group showed normal traceuticals produced significant improvement of all liver structure, the hepatic cords radiating from central plasma parameters and liver lipids with variable degrees. vein separated by sinusoid（Fig. 1A）. Liver of control DS Fish and borage oil were almost equal in improving all bio- group demonstrated focal area of hepatic necrosis infiltrat- chemical parameters except for TAC which was more effi- ed with inflammatory cells and most of hepatocyte showing ciently improved by borage oil. Borage oil was more prom- fatty degeneration（Fig. 1B1）. Liver of control DS group ising in improving lipid profile and liver fat compared to B also showed fatty degeneration with the hepatocytes taking ＋T＋TC, while the latter was superior in reducing MDA. signet ring appearance（Fig.1B2）. Livers of the group Fish oil was more efficient in improving T.Ch, HDL-Ch, treated by borage seed oil contain multi focal areas of fatty LDL-Ch, T.Ch/HDL-Ch, and liver fat compared to F＋T＋ degeneration and focal aggregation of inflammatory cells TC; while the latter was more efficient in improving TAC. （Fig. 1C1）. Liver of another rat from the group that re- Compared to normal control; plasmaT.Ch, LDL-Ch, T.Ch/ ceived borage oil showed proliferation of fibers connective HDL-Ch and MDA as well as liver fats in the groups treated tissue in portal area with inflammatory cells infiltration and by fish and borage oil showed insignificant change. Treat- fatty degeneration of hepatocytes（Fig. 1C2）. Liver of all ment by fish oil restored the plasma HDL-Ch to normal rats treated by B＋T＋TC showed marked improvement of level as in NC group. When rats were treated by F＋T＋ histopathological picture（Figs. 1D1 and 1D2）. Liver from TC; the plasma level of MDA was normalized. Plasma TAC the majority of rats treated by fish oil showed marked im- of borage oil, B＋T＋TC and F＋T＋TC treated rats provement in the histopathological changes（Fig. 1E2）. showed insignificant change compared to NC group. Rats Only one rat from those given fish oil demonstrated mild treated by B＋T＋TC showed promising reduction of MDA vacuolar degenerative changes while most of cells are which was even significantly lower than NC group. intact（Fig. 1E1）. Liver of rats received F＋T＋TC revealed Different nutritional parameters and percentage organ marked improvement in histopathological picture（Figs. weight to body weight of different groups are illustrated in 1F1 and 1F2）while only one rat from this group（Fig. 1F3） Table 3. It was noticed that total food intake was signifi- showed focal area of vacuolar degeneration. cantly reduced in both DS control group and the treated The improvement of liver histopathological changes groups compared to NC group without change in body could be arranged from the best to the least according to weight gain. There was no significant change in heart/ body the following sequence; borage combined with T and TC＞ weight ％ among the different groups while liver/ body fish oil＝fish oil combined with T and TC＞borage oil. weight％ was increased significantly in DS control group Heart histopathology of different groups（Fig. 2）showed compared to NC. Treatment with either borage oil or F＋T heart of control normal rat with normal histological struc- ＋TC produced significant reduction in liver/ body ture（Fig. 2A）. Heart of control DS group contains focal weight％ compared to DS control group while the other areas of regenerative changes with loss of cardiac myofi- treated two groups only showed minor reduction. There bres striation and congestion of cardiac blood vessels（Fig.

Table 3 Nutritional parameters, liver/body weight％ and heart/body weight％ of different experimental groups. Groups DS＋Borage oil DS＋Fish oil Normal control Control DS DS＋Borage oil ＋turmeric＋ DS＋Fish oil ＋turmeric＋ Parameters vitamin E vitamin E Initial body weight（g） 147a±4.96 147a±4.54 147a±6.8 147a±4.19 147a±6.28 147a±5.85 Final body weight（g） 217.5a±6.407 209.3a±10.032 210.0a±9.007 210.5a±6.076 214.2a±2.182 215.3a±6.422 Body weight gain（g） 68.7a±10.026 62.2a±6.358 62.8a±7.472 63.7a±4.145 67.2a±4.785 68.2a±8.424 Total food intake（g） 622.5a±18.786 455.3bc±17.443 452.3bc±27.04 462.0bc±18.085 470.0bc±33.45 446.3c±47.8 Liver/body weight％ 2.5a±0.062 4.5c±0.259 3.9b±0.100 4.1bc±0.108 4.1bc±0.201 4.0b±0.143 Heart/body weight％ 0.29a±0.118 0.32a±0.011 0.33a±0.012 0.32a±0.023 0.31a±0.004 0.32a±0.020 Values are means ± SE; where n＝6. DS: Dyslipidemic and steatohepatitis inducer diet In the same row: The similar superscript letters mean insignificant difference, while different letters mean significant difference between groups at p＜0.05. 5 J. Oleo Sci. S. Y. Al-Okbi, S. M. El-qousy, S. E.-Ghlban et al.

Fig. 1 Liver histopathology of different groups. A）Liver of control normal rat showing normal liver structure, the hepatic cords radiating from central vein separated by sinusoid（H&EX100）. B-1）Liver of control DS group showing focal area of hepatic necrosis infiltrated with inflammatory cells and most of hepatocyte showing fatty degeneration（H& E X 200）. B-2）Liver of control DS group demonstrated fatty degeneration, the hepatocytes taking signet ring appearance（H& E X 400）. C-1）Liver of B group showed multi focal areas of fatty degeneration and focal aggregation of inflammatory cells（H&E X 100）. C-2）Liver of B group showed proliferation of fibers connective tissue in portal area with inflammatory cells infiltration and fatty degeneration of hepatocytes（H&E X 200）. D-1）Liver of B＋T＋TC group showing marked improvement of histopathological picture（H&E X 200）D-2）Liver of B＋T＋TC group showing marked improvement of histopathological picture（H&E X 400）. E-1）Liver of fish oil group showing mild vacuolar degenerative changes while most of cells are intact（H& E X 100）. E-2）Liver of fish oil group showing marked improvement in the histopathological changes（H& E X 400）.F-1）Liver of F＋T＋TC group showing marked improvement in histopathological picture（H& E X 100）. F-2）Liver of F＋T＋TC group showing marked improvement in histopathological picture（H& E X 400）. F-3）Liver of F＋T＋TC group showing focal area of vacuolar degeneration （H& E X 200）. B: Borage oil; F: Fish oil; T: Turmeric; TC: Alpha-tocopherol, DS: Dyslipidemic and steatohepatitis

2B1）. Heart of another rat from control DS group showed hearts from different test groups were improved without pericardial edema with separation of myofibers and inflam- distinct variations among them. matory cells infiltration（Fig. 2B2）. Generally hearts from the group treated by borage oil demonstrated improvement in histopathological changes（Fig. 2C2）while heart from only one rat from such group showed focal area of myocar- 4 Discussion dial edema with inflammatory cells infiltration（Fig. 2C1）. It is well documented that both cardiovascular diseases Hearts of rats given borage oil combined with T and TC and steatohepatitis involved high oxidative stress, inflam- showed marked improvement in the histopathological matory condition and lipid imbalance3, 33）. So, reduction of picture（Fig. 2D1 and 2D2）while heart from only one rat in oxidative stress, inflammation and improved lipids’ profile the same group still showed loss of striation of cardiac could play an important role in prevention and reduction of muscles（Fig. 2D3）. Hearts of fish oil treated group demon- the progression of such diseases. The nutraceuticals tested strated marked improvement of myocardial fibers（Fig. in the present study contained antioxidants, anti-inflamma- 2E2）while heart from one rat from such group showed tory and lipid lowering bioactive constituents. mild myocardial degenerative changes（Fig. 2E1）. Hearts The rat model used in the present study showed elevated from rats treated by fish oil with T and TC group illustrated oxidative stress represented by the increased MDA and the marked improvement in the histopathological picture（Fig. reduced total antioxidant which might be a biomarker of 2F2）while mild focal area of myocardial degeneration were tissue destruction and inflammation in both liver and heart. noticed in the heat from one rat of such group（Fig. 2F1）. An elevated inflammatory biomarker（TNF-alpha）was also It could be noticed that the histopathological picture of the noticed in the current results. In such model significant

6 J. Oleo Sci. Edible Oils with or without Antioxidants for Healthy Heart and Liver

Fig. 2 Heart histopathology of different groups. A）Heart of control normal rat showed normal histological structure（H&EX 400）. B1）Heart of control DS group demonstrated focal areas of regenerative changes with loss of cardiac myofibres striation and congestion of cardiac blood vessels（H& E X 400）. B2）Heart of control DS group exhibited pericardial edema and separation of myofibers and inflammatory cells infiltration（H& E X 200）. C1）Heart from B group showed focal area of myocardial edema with inflammatory cells infiltration（H& E X 200）. C2）Heart from B group demonstrated improvement in histopathological changes（H& E X 200）. D1）Heart from B＋T＋TC group showing marked improvement in the histopathological picture（H& E X 200）. D2）Heart from B＋T＋TC group showing marked improvement in the histopathological picture（H& E X 400）. D3）Heart from B＋T＋TC group still showed loss of striation of cardiac muscles（H& E X 400）. E1）Heart from fish oil group exhibited mild myocardial degenerative changes（H&EX200）. E2）Heart from fish oil group showed marked improvement of myocardial fibers （H&E X400）. F1）Heart from F＋T＋TC group showing focal area of myocardial degenerative（mild）（ H& E X 200）. F2）Heart from F＋T＋TC group showing marked improvement in the histopathological picture（H& E X 200）. B: Borage oil; F: Fish oil; T: Turmeric; TC: Alpha-tocopherol, DS: Dyslipidemic and steatohepatitis dyslipidemia along with elevated T.Ch/HDL-Ch emphasized fibers in the diet could promote induction of dyslipidemia, the induction of cardiovascular disease according to Yang cardiovascular disease and steatohepatitis. et al.34）. Also high hepatic fat with increased inflammatory Borage seed oil, besides containing 17-28％ gamma lino- biomarker confirm the induction of steatohepatitis. The lenic, it contains palmitic（10-11％）, stearic（3.5-4.5％）, biochemical changes in such model were supported by his- oleic（16-20％）, linoleic（35-38）as reported previously14） topathological changes in liver and heart. Induction of this which coincided with the results of the current study. In model was implemented by feeding high fructose-high sat- addition borage oil showed the presence of alpha- linolenic urated fat diet supplemented by cholesterol and sodium （4.559）. The main bioactive fatty acids in fish oil identified cholate and deficient in fibers. Fructose stimulates hepatic from GC analysis were EPA（22.768）and DHA（13.574） lipid synthesis and block fat oxidation35）thereby increase which were similar in concentration to that reported previ- hepatic deposition of fat. Plasma triglycerides were report- ously39）. Alpha-linolenic and linoleic acid in fish oil were ed to increase due to fructose moiety of sweetened bever- 3.368％ and 2.379％, respectively which were slightly ages36）. High intake of fructose and saturated fat produced higher than that identified in the aforementioned study39） insulin resistence, dyslipidemia, high oxidative stress, fatty which could be ascribed to the different origin of fish oil. liver and inflammatory state3, 37）. The presence of cholic Generally, all nutraceuticals used in the present study acid and cholesterol in the diet increased cholesterol ab- produced improvement of both biochemical and histopath- sorption with consequent hypercholesterolemia. The ological changes. Treatment by either fish or borage oil changes in endothelial function via saturated fat intake, alone restored the normal levels of plasma T.Ch, LDL-Ch, oxidative stress and inflammation promote initiation and T.Ch/HDL-Ch and liver fat. HDL-Ch was only normalized on induction of cardiovascular diseases38）. Also absence of treatment with fish oil. Plasma TAC was normalized on ad-

7 J. Oleo Sci. S. Y. Al-Okbi, S. M. El-qousy, S. E.-Ghlban et al.

ministration of any nutraceutical except for fish oil. MDA tective effect55, 56）. Omega3 polyunsaturated fatty acids ac- level matches that of normal control on treatment with the tivate the peroxisome proliferator receptor（PPAR）alpha in different nutraceuticals while borage oil supplemented by liver thereby stimulate fatty acid oxidation with simultane- turmeric and tocopherol showed lower level than normal ous activation of PPAR gamma that lead to increases pointed to high antioxidant effect of such nutraceutical insulin sensitivity, reduction of hepatic lipogenesis and that could be ascribed not only to the antioxidant activity reduced reactive oxygen species with consequent reduc- of T and TC but also to that of gamma-linolenic. Liver total tion of hepatic fat and slowing down the progression to fat was significantly reduced on treatment of the different steatohepatitis57）. This could reflect the significant reduc- nutraceuticals; while it was only normalized on treatment tion of liver fat in the present work on treatment with fish with fish oil and borage oil. oil. Increased insulin sensitivity on administration of fish oil Gamma linolenic acid from borage oil is converted into is an important issue because insulin resistance is one of the body to dihomo gamma linolenic acid, a precursor of the major proposed mechanisms involved in all stages of 1-series and 3- series , thereby non alcoholic fatty liver disease. Elevated cytokines, as in- inhibit leukotriene synthesis resulting in anti-inflammatory flammatory biomarker, have strong link to steatohepatitis, and antithrombotic effect. Borage oil holds great promise so reduction of them by omega3 fatty acids54）is essential in for modulating inflammatory disorders40）. So, it can prevent the management of such disease. the incidence of cardiovascular disease. Borage oil possess- It was confirmed that alpha and gamma-linolenic acids es antioxidant activity which is superior compared to gam- increased fatty acid oxidation in the liver while they differ ma-linolenic itself41）. Borage oil reduced progression of considerably in how they affect individual fatty acid oxida- cardiac remodeling after myocardial infarction and conges- tion enzyme58）. Gamma-linolenic acid enhanced the activity tive heart failure in rats through attenuation of inflamma- of carnitine palmitoyl transferase and peroxisomal beta oxi- tory infiltration and fibrosis in the myocardium42）as could dation that demonstrated reduction of body fat including be seen from histopathology examination of the heart and that of liver due to fatty acid beta-oxidation59）. the reduction in TNF- alpha in the present study. Gamma- Combination of antioxidants（turmeric and tocopherol） linolenic acid afforded protection from ventricular fibrilla- with either borage or fish oil in the present study showed tion in aged rats43）. marked reduction of the effect of both oils on liver fat Fish oil and borage oil were reported previously to without complete abolishing. This effect is difficult to be reduce T.Ch, T.Gs and hepatic lipids in mice44）which agreed explained however it might be related to some sorts of in- with the current research. Oleic acid a monounsaturated teraction or an effect on biotransformation enzyme in the fatty acid which is present as 20.163 and 12.771％ in liver that might have an impact on the metabolism of both borage and fish oil, respectively were reported to have car- gamma-linolenic and omega-3 polyunsaturated fatty acids. dioprotective effect via improving vascular endothelial On the other hand, curcumin was demonstrated to combat function45）. The cardiac and hepatic ameliorating effect of liver fatty acid oxidation; thereby might antagonize the re- fish oil seen in the present study can be justified based on ported effect of both fish oil and borage oil as enhancer of its content of the anti-inflammatory ω3 fatty acids DHA fatty acid beta-oxidation60）. and EPA via an effect on cytokines46, 47）. Despite the high Inclusion of turmeric and vitamin E within nutraceuticals unsaturation of fish oil fatty acids however it showed anti- did not add any beneficial effect concerning biochemical oxidant effect in the present study which agreed with the parameters except in few cases like the significant increase previous works48, 49）. EPA was reported as potent antioxi- in TAC in case of fish oil containing turmeric and vitamin E dant due to its distinct lipophilic and electron stabilization compared to fish oil as well as the significant reduction in property50, 51）. EPA also improves HDL function represent- MDA and more improvement in liver histopathology on ad- ed by enhancing cholesterol efflux, anti-inflammatory and ministration of borage oil combined with turmeric and antioxidant effect52）. Both DHA and EPA possess beneficial vitamin E compared to borage oil. The elevation of TAC effect towards cardiovascular health, promoting vascular and reduction of MDA are expected due to the antioxidant smooth muscle relaxation and vasodilatation53）. The low activity of both vitamin E and turmeric. Vitamin E was re- n-6: n-3 fatty acids ratio in fish oil compared to borage oil ported to stabilize mitochondrial function and protect the might add to the health benefit of fish oil in the present liver against oxidative damage with consequent anti-in- study. flammatory activity thereby could prevent progression of Monounsaturated fatty acids（20.163％ in borage oil and fatty liver to steatohepatitis54, 61, 62）. 22.12％ in fish oil）were demonstrated to reduce hepatic The health benefit of turmeric is certainly attributed to deposition of fat due to their insulin sensitizing effect54）. curcuminoids contents especially curcumin determined in Fish oil was reported to stimulate gene expression related the present study. Curcumin is metabolized in the body to to fatty acids beta oxidation with simultaneous reduction tetrahydrocurcumin that possess antioxidant property and of those induce lipogenesis in liver and possess hepatopro- improved cardiac hypertrophy in rats63）. Curcumin was re-

8 J. Oleo Sci. Edible Oils with or without Antioxidants for Healthy Heart and Liver

ported to alter serum peroxidation as could be seen in the Conflict of interest statement present study54）. Also turmeric was demonstrated to The authors declare that they have no conflict of interest contain bioactive essential oil and polyphenol of reported remedial anti-inflammatory effect. Vascular endothelial function which is a potential risk of cardiovascular disease was improved by curcumin supplementation via anti-in- Reference: flammatory and antioxidant mechanism. Curcumin im- 1） Lozano, R.; Naghavi, M.; Foreman, K.; Lim S.; Shibuya, proved dyslipidemia and liver dysfunction in high fat high K.; Aboyans, V. et al. Global and regional mortality sucrose fed rats15）. Curcumin has the ability to alleviate from 235 causes of death for 20 age groups in 1990 myocardial injury with inhibition of pro-inflammatory cyto- and 2010: a systematic analysis for the Global Burden kine in rats with coronary artery ligated and in isolated of Disease Study 2010. Lancet 380, 2095-128（2013）. heart64）which was likely demonstrated in the present study. 2） Al-Okbi, S.Y.; Mohamed, D.A.; Hamed, T.E.; Edris, A.E. Modulation of post-translational modifications（like protein Potential protective effect of Nigella sativa crude oils acetylation）is considered as one of the most recent strate- towards fatty liver in rats. Eur. J. Lipid Sci. Technol. gy to combat cardiovascular disease; curcumin and DHA 115, 774-782（2013）. are emerging agents that possess this activity mediated 3） Al-Okbi, S.Y.; Mohamed, D.A.; Hamed, T.E.; Esmail, through modulation of sirtuin65）. This is because protein R.S.H. Rice bran oil and pumpkin seed oil alleviate oxi- hyperacetylation is associated with different cardiovascular dative injury and fatty liver in rats fed high fructose diseases. diet. Polish Journal of Food and Nutrition Sciences The non significant change in body weight gain among 64, 127-133（2014）. the different experimental groups with simultaneous re- 4） Ulven, S.M.; Holven, K.B. Comparison of bioavailability duction in total food intake in rats fed on DS（control and of krill oil versus fish oil and health effect. Vasc. test groups）compared to NC fed on balanced diet could be Health Risk Manag. 11, 511-24（2015）. explained on the basis of the high Caloric contents of DS 5） Al-Okbi, S.Y.; Mohamed, D.A.; Hamed, T.E.; Edris, A.E.; compared to balanced diet due to high fat percentage. The Karem Fouda. Hepatic Regeneration and Reno-Protec- increase in liver/body weight％ in DC compared to NC tion by Fish oil, Nigella sativa Oil and Combined Fish might be attributed to the increased hepatic fat deposition Oil/Nigella sativa Volatiles in CCl4 Treated Rats. J. while the reduction in liver/body weight％ on administra- Oleo Sci. 67, 345-353（2018）. tion of different nutraceuticals is related to reduction of 6） Fritsche, K.L. The science of fatty acids and inflamma- liver fat. tion. Adv. Nutr. 6, 293S-301S（2015）. 7） Virtanen, J.K.; Mursu, J.; Voutilainen, S.; Tuomainen, T.P. The associations of serum n-6 polyunsaturated fatty acids with serum C-reactive protein in men: the 5 Conclusion Kuopio Ischaemic Heart Disease Risk Factor Study. Borage and fish oil possess equal protective effect Eur. J. Clin. Nutr. 72, 342-348（2018）. towards cardiovascular and fatty liver diseases except for 8） Maruyama, H.; Takahashi, M.; Sekimoto, T.; Shimada, liver histopathological changes which was more efficiently T.; Yokosuka, O. Linoleate appears to protect against improved by fish oil and the superiority in improving TAC palmitate-induced inflammation in Huh7 cells. Lipids in case of borage oil treatment. Health Dis. 13, 78（2014）. doi: 10.1186/1476-511X-13- Inclusion of turmeric and vitamin E within nutraceuticals 78. did not add any beneficial effect concerning biochemical 9） Al- Okbi, S.Y.; Ammar, N.M.; Soroor, Kh. A.; Moham- parameters except in few cases like the significant increase med, D.A. Impact of natural oils supplements on dis- in TAC in case of fish oil containing turmeric and vitamin E ease activity and antioxidant state of Egyptian pa- compared to fish oil. Another exception is the significant tients with rheumatiod . Med. J. Islamic reduction in MDA and more improvement in liver histopa- Acad. Sci. 13, 161-171（2000）. thology on administration of borage oil combined with tur- 10） Tortosa-Caparrós, E.; Navas-Carrillo, D.; Marín, F.; meric and vitamin E compared to borage oil. Orenes-Piñero, E. Anti-inflammatory effects of omega 3 and omega 6 polyunsaturated fatty acids in cardio- vascular disease and metabolic syndrome. Crit. Rev. Food Sci. Nutr. 57, 3421-3429（2017）. Acknowledgements 11） Poreba, M.; Rostoff, P.; Siniarski, A.; Mostowik, M.; This research did not receive any specific grant from Golebiowska-Wiatrak, R.; Nessler, J.; Undas, A.; Gajos, funding agencies in the public, commercial, or not-for-prof- G. Relationship between polyunsaturated fatty acid it sectors. composition in serum phospholipids, systemic low-

9 J. Oleo Sci. S. Y. Al-Okbi, S. M. El-qousy, S. E.-Ghlban et al.

grade inflammation, and glycemic control in patients kar, B; Evaluation of acute toxicity and anti-ulcerogen- with type 2 and atherosclerotic cardiovascu- ic study of rhizome starch of two source plants of lar disease. Cardiovasc. Diabetol. 17, 29（2018）. Tugaksheeree（Curcuma angustifolia Roxb. and Ma- 12） Gilani, A.H. Focused Conference Group: P16 - Natural ranta arundinacea Linn.）. Ayu. 35, 433-437（2014）. products: Past and future? Pharmacological use of 24） Zollner, N.; Kirsch, K. Colorimetric method for deter- borago officinalis”, Basic and Clinical Pharmacolo- mination of total lipids. Z. Ges. Exp. Med. 135, 545- gy and Toxicology. Conference: 16th World Congress 550（1962）. of Basic and Clinical Pharmacology. WorldPharma 25） Richmond, W. Preparation and properties of a choles- 2010 Copenhagen Denmark. Publication:（var. pag- terol oxidase from Nocardia sp. and its application to ings）. 107（pp 301）, 2010. Date of Publication: July the enzymatic assay of total cholesterol in serum. 2010. Clin. Chem. 19, 1350-1356（1973）. 13） Gupta, M.; Singh, S. Borago officinalis Linn. An im- 26） Fossati, P.; Prencipe, L. Serum triglycerides deter- portant medicinal plant of Mediterranean region: Re- mined colorimetrically with an enzyme that produces view. Int. J. Pharm. Sci. Rev. Res. 5, 27-34（2010）. hydrogen peroxide. Clin. Chem. 28, 2077-2080 14） National Non-Food Crops Centre. NNFCC Crop Fact- （1982）. sheet: Borage, Retrieved on 16 Feb 2011. 27） Wieland, H.; Seidel, D. A simple specific method for 15） Tsai, I.J.; Chen, C.W.; Tsai, S.Y.; Wang, P.Y.; Owaga, E.; precipitation of low density lipoproteins. J. Lipid Res. Hsieh, R.H. Curcumin supplementation ameliorated 24, 904-909（1983）. vascular dysfunction and antioxidant status in high su- 28） Burstein, M.; Scholnick, H.R.; Morfin, R. Rapid method crose, high fat fed rats. Appl. Physiol. Nutr. Metab. for the isolation of lipoproteins from human serum by 43, 669-676（2018）. precipitation with polyanions. J. Lipid Res. 11, 583- 16） Simental-Mendía, L.E.; Pirro, M.; Gotto, A.M. Jr.; Ban- 595（1970）. ach, M.; Atkin, S.L.; Majeed, M.; Sahebkar, A. Lipid- 29） Satoh, K. Serum lipid peroxide in cerebrovascular dis- modifying activity of curcuminoids: A systematic re- orders determined by a new colorimetric method. view and meta-analysis of randomized controlled Clin. Chim. Acta 20, 37-43（1987）. trials. Crit. Rev. Food Sci. Nutr. 29, 1-10（2017）. 30） Koracevic, D.; Koracevic, G.; Djordjevic, V.; Andre- 17） Azzi, A. Many tocopherols, one vitamin E. Mol. As- jevic, S.; Cosic, V. Method for the measurement of an- pects Med. 61, 92-103（2018）. tioxidant activity in human fluids. J. Clinic. Pathol. 18） Indarti, E.; Majid, M.I.A.; Hashim, R.; Chong, A. Direct 54, 356-361（2001）. FAME synthesis for rapid total lipid analysis from fish 31） Stepaniak, J.A.; Gould, K.E.; Sun, D.; Swanborg, R.H. oil and cod liver oil. J. Food Compost. Anal. 18, 161- A comparative study of experimental autoimmune en- 170（2005）. cephalomyelitis in Lewis and DA rats. J. Immunol. 19） Jayaprakasha, G.K.; Rao L.J.M.; Sakariah, K.K. Im- 155, 2762-2769（1995）. proved HPLC Method for the Determination of Cur- 32） Cequier- Sànchez, E.; Rodr´ıguez, C.; Ravelo, G.; Zárate, cumin, Demethoxycurcumin, and Bisdemethoxycur- R. Dichloromethane as a solvent for lipid extraction cumin. J. Agric. Food Chem. 50, 3668-3672（2002）. and assessment of lipid classes and fatty acids from 20） Zulet, M.A.; Macarulla, M.T.; Portillo, M.P.; Noel-Suber- samples of different natures. J. Agric. Food Chem. ville, C.; Higueret, P.; Martínez, J.A. Lipid and glucose 56, 4297-4303（2008）. utilization in hypercholesterolemic rats fed a diet con- 33） Mohamed, D.A.; Hamed, T.E.; Al-Okbi, S.Y. Reduction taining heated chickpea（Cicer aretinum L.）: a poten- in hypercholesterolemia and risk of cardiovascular dis- tial functional food. Int. J. Vitam. Nutr. Res. 69, 403- eases by mixtures of plant food extracts: a study on 411（1999）. plasma lipid profile, oxidative stress and testosterone 21） Kawasaki, T.; Igarashi, K.; Koeda, T.; Sugimoto, K.; Na- in rats. Grasas y Aceites 61, 378-389（2010）. kagawa, K.; Hayashi, S.; Yamaji, R.; Inui, H.; Fukusato, 34） Yang, R.; Le, G.; Li, A.; Zheng, J.; Shi, Y. Effect of anti- T.; Yamanouchi, T. Rats fed fructose-enriched diets oxidant capacity on blood lipid metabolism and lipo- have characteristics of nonalcoholic hepatic Steatosis. protein lipase capacity of rats feed a high-fat diet. J. Nutr. 139, 2067- 2071（2009）. Nutr. 22, 1185-1191（2006）. 22） Abedi, S.M.; Yarmand, F.; Motallebnejad, M.; Seyedma- 35） Ackerman, Z.; Oron-Herman, M.; Grozovski, M.; Rosen- jidi, M.; Moslemi, D.; Ashrafpour, M.; Bijani, A.; Mogha- thal, T.; Poppo, O. Link G, Sela BA, Fructose-induced damnia, A.; Mardanshahi, A.; Hosseinimehr, S.J. Vita- fatty liver disease: hepatic effects of blood pressure min E protects salivary glands dysfunction induced by and plasma triglyceride reduction. Hypertension 45, ionizing radiation in rats. Arch. Oral Biol. 60, 1403- 1012-1018（2005）. 1409（2015）. 36） Bray, G.A. Fructose and risk of cardiometabolic dis- 23） Rajashekhara, N.; Ashok, B.K.; Sharma, P.P.; Ravishan- ease. Curr. Atheroscler. Rep. 14, 570-578（2012）.

10 J. Oleo Sci. Edible Oils with or without Antioxidants for Healthy Heart and Liver

37） Jaya, C.; Anuradha, C.V. Cissus quadrangularis stem sential fatty acids against formaldehyde-induced cere- alleviates insulin resistance, oxidative injury and fatty bellar damage in rats. Toxicol. Ind. Health 27, 489- liver disease in rats fed high fat plus fructose diet. 495（2011）. Food Chem. Toxicol. 48, 2021-2029（2010）. 49） Meydan, S.; Altas, M.; Nacar, A.; Ozturk, H.; Tas, U.; 38） Thorand, B.; Baumert, J.; Chambless, L.; Meisinger, C.; Zararsiz, I.; Sarsilmaz, M. The protective effects of Kobb, H.; Doring, A. For the MONICA/KORA study omega-3 fatty acid against toluene-induced neurotox- group. Elevated markers of endothelial dysfunction icity in prefrontal cortex of rats. Hum. Exp. Toxicol. predict type 2 diabetes mellitus in middle-aged men 31, 1179-1185（2012）. and women from the general population. Arterioscler. 50） Mason, R.P.; Jacob, R.F., Eicosapentaenoic acid inhib- Thromb. Vasc. Biol. 26, 398-405（2006）. its glucose-induced membrane cholesterol crystalline 39） Al-Okbi, S.Y.; Mohamed, D.A., Hamed, T.E.; Edris, A.E. domain formation through a potent antioxidant mech- Evaluation of the therapeutic effect of Nigella sativa anism. Biochim. Biophys. Acta 1848, 502-509（2015）. crude oil and its blend with omega-3 fatty acid-rich 51） Mason, R.P.; Sherratt, S.C.R.; Jacob, R.F. Eicosapen- oils in a modified hepatorenal syndrome model in rats. taenoic acid inhibits oxidation of ApoB-containing li- Grasas y Aceites 66, e103（2015）. poprotein particles of different size in vitro when ad- 40） Chilton, F.H.; Rudel, L.L.; Parks J.S.; Arm, J.P. Seeds ministered alone or in combination with atorvastatin MC Mechanisms by which botanical lipids affect in- active metabolite compared with other triglyceride- flammatory disorders. Am. J. Clin. Nutr. 87, 498S- lowering agents. J. Cardiovasc. Pharmacol. 68, 503S（2008）. 33-40（2016）. 41） Tasset-Cuevas, I.; Fernández-Bedmar, Z.; Lozano-Bae- 52） Tanaka, N.; Ishida, T.; Nagao, M.; Mori, T.; Monguchi, T.; na, M.D.; Campos-Sánchez, J.; de Haro-Bailón A.; Mu- Sasaki, M.; Mori, K.; Kondo, K.; Nakajima, H.; Honjo, T.; ñoz-Serrano A.; Alonso-Moraga, A. Protective effect of Irino, Y.; Toh, R.; Shinohara, M.; Hirata, K. Administra- borage seed oil and gamma linolenic acid on DNA: in tion of high dose eicosapentaenoic acid enhances anti- vivo and in vitro studies. PLoS One 8（2）, e56986 inflammatory properties of high-density lipoprotein in （2013）. Japanese patients with dyslipidemia. Atherosclerosis 42） Maldonado-Menetti, J.; dos, S.; Vitor, T.; Edelmuth, 237, 577-583（2014）. R.C.; Ferrante, F.A.; Souza, P.R.; Koike, M.K. Borage 53） Limbu, R.; Cottrell, G.S.; McNeish, A.J. Characterisa- oil attenuates progression of cardiac remodeling in tion of the vasodilation effects of DHA and EPA, n-3 rats after myocardial infarction. Acta Cir. Bras. 31, PUFAs（fish oils）in rat aorta and mesenteric resistance 190-197（2016）. arteries. PLoS One 13（2）, e0192484（2018）. 43） Charnock, J.S. Gamma-linolenic acid provides addi- 54） Al-Okbi, S.Y. Role of nutraceuticals in prevention of tional protection against ventricular fibrillation in aged non-alcoholic fatty liver. in Plant- and marine- based rats fed linoleic acid rich diets. Prostaglandins Leu- phytochemicals for human health: Attributes, po- kot. Essent. Fatty Acids 62, 129-134（2000）. tential, and use（Goyal, M.R.; Chauhan, D.N. eds.）, 44） Shewale, S.V.; Brown, A.L.; Bi, X.; Boudyguina, E.; Apple Academic Press, Taylor and Francis（2018）, in Sawyer, J.K.; Alexander-Miller, M.A.; Parks J.S. In vivo press. activation of leukocyte GPR120/FFAR4 by PUFAs has 55） de Castro, G.; Deminice, R.; Cordeiro Simões-Ambro- minimal impact on atherosclerosis in LDL receptor sio, L.; Calder, P.; Jordão, A.; Vannucchi, H. Dietary knockout mice. J. Lipid Res. 58, 236-246（2017）. docosahexaenoic acid and eicosapentaenoic acid influ- 45） Christon, R.A. Mechanisms of action of dietary fatty ence liver triacylglycerol and insulin resistance in rats acids in regulating the activation of vascular endothe- fed a high-fructose diet. Mar. Drugs 13, 1864-1881 lial cells during atherogenesis. Nutr. Rev. 61, 272-279 （2015）. （2003）. 56） Al-Okbi, S.Y.; Mohamed, D.A.; Hamed, T.E.; Edris, A.E.; 46） Chen, Y.; Lin, Y.; Pan, Y.; Liao, L.; Chuang, H.; Yen, J.; Fouda, K. Hepatic regeneration and reno-protection Lin, Y.; Chen, J. Beneficial effect of docosahexaenoic by fish oil, Nigella sativa oil and combined fish oil/Ni- acid on cholestatic liver injury in rats. J. Nutr. Bio- gella sativa volatiles in CCl4 treated rats. J. Oleo Sci. chem. 23, 252-264（2012）. 67, 345-353（2018）. 47） Yilmaz, R.; Songur, A.; Ozyurt, B.; Zararsiz, I.; Sar- 57） Lu, W.; Li, S.; Li, J.,; Wang, J.; Zhang, R.; Zhou, Y.; Yin, silmaz, M. The effects of n-3 polyunsaturated fatty ac- Q.; Zheng,Y.,; Wang, F.; Xia, Y.; Chen, K.; Liu, T.; Lu, J.; ids by gavage on some metabolic enzymes of rat liver. Zhou,Y.; Guo, C. Effects of omega-3 fatty acid in nonal- Prostagladins Leukot. Essent. Fatty Acids 71, 131- coholic fatty liver disease: A meta-analysis. Gastroen- 135（2004）. terol. Res. Pract. 1459790（2016）. 48） Zararsiz, I.; Meydan, S.; Sarsilmaz, M.; Songur, A.; 58） Kumamoto, T.; Ide T. Comparative effects of alpha- Ozen, A.; Sogut, S. Protective effects of omega-3 es- and gamma-linolenic acids on rat liver fatty acid oxida-

11 J. Oleo Sci. S. Y. Al-Okbi, S. M. El-qousy, S. E.-Ghlban et al.

tion. Lipids 33, 647-654（1998）. coholic steatohepatitis? Expert Rev. Gastroenterol. 59） Takada, R.; Saitoh, M.; Mori, T. Dietary gamma-linole- Hepatol. 5, 147-150（2011）. nic acid-enriched oil reduces body fat content and in- 63） Lau, W.L.; Khazaeli, M.; Savoj, J.; Manekia, K.; Ban- duces liver enzyme activities relating to fatty acid be- gash, M.; Thakurta, R.G.; Dang, A.; Vaziri, N.D.; Singh, ta-oxidation in rats. J. Nutr. 124, 469-474（1994）. B. Dietary tetrahydrocurcumin reduces renal fibrosis 60） She, L.; Xu, D.; Wang, Z.; Zhang,Y.; Wei, Q.; Aa, J. and cardiac hypertrophy in 5/6 nephrectomized rats. Wang, G.; Liu, B.; Xie, Y. Curcumin inhibits hepatic Pharmacol. Res. Perspect. 6（2）, e00385（2018）. stellate cell activation via suppression of succinate-as- 64） Liu, K.; Chen, H.; You, Q.S.; Ye, Q.; Wang, F.; Wang, S.; sociated HIF-1α induction. Mol. Cell. Endocrinol. Zhang, S.L.; Yu, K.J.; Lu, Q. Curcumin attenuates myo- 476, 129-138（2018）. cardial ischemia-reperfusion injury. Oncotarget 8, 61） Ohta, Y.; Yashiro, K.; Ohashi, K.; Horikoshi, Y.; Kusu- 112051-112059（2017）. moto, C.; Matsura, T.; Fukuzawa, K. Effect of dietary 65） Treviño-Saldaña, N.; García-Rivas, G. Regulation of sir- vitamin E supplementation on liver oxidative damage tuin-mediated protein deacetylation by cardioprotec- in rats with water-immersion restraint stress. J. Nutr. tive phytochemicals. Oxid. Med. Cell. Longev. Sci. Vitaminol.（Tokyo）61, 113-122（2015）. 1750306（2017）. 62） Adinolfi, L.E.; Restivo, L. Does vitamin E cure nonal-

12 J. Oleo Sci.