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H: Health, Nutrition, & Food Hypolipidemic Activity in Sprague–Dawley Rats and Constituents of a Novel Natural Vegetable Oil from Cornus Wilsoniana Fruits Jie Fu, Xue-Wei Zhang, Kai Liu, Qing-Shan Li, Li-Rong Zhang, Xian-Hui Yang, Zhi-Ming Zhang, Chang-Zhu Li, Yin Luo, Zhen-Xiang He, and Hai-Liang Zhu Abstract: Cornus wilsoniana Wanger is a woody oil plant distributed in the south region of the Yellow River, China. Its oil has been taken as edible oil for over 100 y, and consumption of such oil is believed to prevent hyperlipidemia in Chinese folk recipe. This study has investigated the hypolipidemic effect of Cornus wilsoniana oil (CWO) in Sprague–Dawley rats. The results demonstrated that CWO could significantly decrease total cholesterol (TC), total triacylglycerol (TG), and low-density lipoprotein cholesterol (HDL-C) in serum, liver weight, hepatic TC, and TG. After analyzing the chemical constituents of CWO, we found that the content of unsaturated fatty acids (UFA) was very high (69.12%). Specially, the n-6 polyunsaturated fatty acids (PUFA), including linoleic acid, γ -linolenic acid, and 11,14-eicosadienoic acid, accounted very great proportion (38.86%). The high hypolipidemic activity of CWO might be attributed to the lipid-lowering functions of these polyunsaturated fatty acids. Molecular docking was further performed to study the binding model of fatty acids (FA) from CWO to a possible hypolipidemic target, peroxisome proliferator-activated receptor δ (PPARδ). The results showed that linoleic acid and γ -linolenic acid could bind PPARδ very well. Keywords: Cornus wilsoniana oil, docking, fatty acids, hypolipidemic activity Practical Application: Cornus wilsoniana oil could be used as equilibrated dietary oil, not only having hypolipidemic function, but also helping to overcome essential fatty acids deficiency. Introduction Hyperlipidemia is the condition of abnormally elevated levels of any or all lipids and/or lipoproteins in the blood. It is the most common form of dyslipidemia. Hyperlipidemia is common in the general population, and is regarded as a modifiable risk factor for cardiovascular disease due to its influence on atheroscle- rosis. A number of industrial and pharmaceutical agents have lipid-lowering effects in animals and humans (Rozman and Monostory 2010). These hypolipidemic agents act via variety of MS 20110896 Submitted 7/26/2011, Accepted 5/1/2012. Authors Fu, X-W Zhang, Liu, Q-S Li, L-R Zhang, Yang, Z-M Zhang, Luo, He, Zhu are with State Key Lab. of Pharmaceutical Biotechnology, School of Life Sciences, Nanjing Univ., Nan- jing 210093, China. Author C-Z Li is with Hu’nan Academy of Forestry, Changsha 410004, China. Direct inquiries to author Zhu (E-mail address: [email protected]). Author disclosures: The material is original and has not been submitted for publication elsewhere. There is no conflict of interest in the manuscript. The main research in animal experiment, constituent analysis, docking study, data analysis, and article writing were finished by Jie Fu (1st author). Xue- Wei Zhang (2nd author) contributed in constituent analysis. Kai Liu (3rd author) and Qing-Shan Li (4th author) contributed in docking study. Li- Rong Zhang (5th author), Xian-Hui Yang (6th author) and Zhi-Ming Zhang (7th author) participated in animal experiment and constituent analysis. Yin Luo (9th author) contributed to the revision of the manuscript. Chang-Zhu Li (8th author) and Zhen-Xiang He (co-corresponding author) provided the oil sample, experimental conditions, and financial support. Hai-Liang Zhu (corresponding author) designed and directed the experiments. mechanisms ranging from the enhancement of cholesterol flux through the bile acid pathway to the inhibition of hepatic choles- terol biosynthesis and elevation of low-density lipoprotein (LDL) receptor activity (Gibson and others 1995). However, many of these synthesized drugs could cause side effects that limited their clinical application. For example, there is a risk of sever muscle damage with statins, which are particularly well-suited for low- ering LDL (Kobayashi and others 2008). Niacin, a good drug for lowering triglycerides, may cause hyperglycemia and may also cause liver damage (Guyton and Bays 2007). Recently, many natural vegetable oils have been reported to show hypolipidemic effect due to their high content of unsatu- rated fatty acids (UFA), such as linoleic acid (Chandrashekar and others 2010). As reported, UFA and some other hypolipidemic drugs can improve the peroxisome level by regulating peroxisome proliferator-activated receptors (PPAR) mediated gene expression via liver fatty acid binding protein (Wolfrum and others 2001). Cornus wilsoniana Wanger is a woody oil plant with high-yield and high-oiliness characters in China. It is widely distributes in the south region of the Yellow River, and concentrated in the limestone mountains at an elevation below 1000 m. Cornus wilso- niana oil (CWO) has been taken as edible oil for over 100 y. Its physicochemical properties (acid value, 3.75 mg KOH/g; iodine value, 102 g I/100 g; saponification value, 198 mg KOH/g) are similar with other commonly used edible oils, such as soybean oil, peanut oil, and rapeseed oil. Long-term consumption of such oil is believed to reduce cholesterol and prevent hyperlipidemia in C 2012 Institute of Food Technologists R doi: 10.1111/j.1750-3841.2012.02786.x Vol. 00, Nr. 0, 2012 Journal of Food Science H1 Further reproduction without permission is prohibited

Hypolipidemic Activity in Sprague–Dawley Rats and Constituents of a Novel Natural Vegetable Oil from Cornus Wilsoniana Fruits

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Page 1: Hypolipidemic Activity in Sprague–Dawley Rats and Constituents of a Novel Natural Vegetable Oil from Cornus Wilsoniana Fruits

H:He

alth,

Nutrit

ion,&

Food

Hypolipidemic Activity in Sprague–Dawley Ratsand Constituents of a Novel Natural VegetableOil from Cornus Wilsoniana FruitsJie Fu, Xue-Wei Zhang, Kai Liu, Qing-Shan Li, Li-Rong Zhang, Xian-Hui Yang, Zhi-Ming Zhang, Chang-Zhu Li, Yin Luo,Zhen-Xiang He, and Hai-Liang Zhu

Abstract: Cornus wilsoniana Wanger is a woody oil plant distributed in the south region of the Yellow River, China. Its oilhas been taken as edible oil for over 100 y, and consumption of such oil is believed to prevent hyperlipidemia in Chinesefolk recipe. This study has investigated the hypolipidemic effect of Cornus wilsoniana oil (CWO) in Sprague–Dawley rats.The results demonstrated that CWO could significantly decrease total cholesterol (TC), total triacylglycerol (TG), andlow-density lipoprotein cholesterol (HDL-C) in serum, liver weight, hepatic TC, and TG. After analyzing the chemicalconstituents of CWO, we found that the content of unsaturated fatty acids (UFA) was very high (69.12%). Specially, then-6 polyunsaturated fatty acids (PUFA), including linoleic acid, γ -linolenic acid, and 11,14-eicosadienoic acid, accountedvery great proportion (38.86%). The high hypolipidemic activity of CWO might be attributed to the lipid-loweringfunctions of these polyunsaturated fatty acids. Molecular docking was further performed to study the binding model offatty acids (FA) from CWO to a possible hypolipidemic target, peroxisome proliferator-activated receptor δ (PPARδ).The results showed that linoleic acid and γ -linolenic acid could bind PPARδ very well.

Keywords: Cornus wilsoniana oil, docking, fatty acids, hypolipidemic activity

Practical Application: Cornus wilsoniana oil could be used as equilibrated dietary oil, not only having hypolipidemicfunction, but also helping to overcome essential fatty acids deficiency.

IntroductionHyperlipidemia is the condition of abnormally elevated levels

of any or all lipids and/or lipoproteins in the blood. It is themost common form of dyslipidemia. Hyperlipidemia is commonin the general population, and is regarded as a modifiable riskfactor for cardiovascular disease due to its influence on atheroscle-rosis. A number of industrial and pharmaceutical agents havelipid-lowering effects in animals and humans (Rozman andMonostory 2010). These hypolipidemic agents act via variety of

MS 20110896 Submitted 7/26/2011, Accepted 5/1/2012. Authors Fu, X-WZhang, Liu, Q-S Li, L-R Zhang, Yang, Z-M Zhang, Luo, He, Zhu are with StateKey Lab. of Pharmaceutical Biotechnology, School of Life Sciences, Nanjing Univ., Nan-jing 210093, China. Author C-Z Li is with Hu’nan Academy of Forestry, Changsha410004, China. Direct inquiries to author Zhu (E-mail address: [email protected]).

Author disclosures: The material is original and has not been submittedfor publication elsewhere. There is no conflict of interest in the manuscript.The main research in animal experiment, constituent analysis, docking study,data analysis, and article writing were finished by Jie Fu (1st author). Xue-Wei Zhang (2nd author) contributed in constituent analysis. Kai Liu (3rdauthor) and Qing-Shan Li (4th author) contributed in docking study. Li-Rong Zhang (5th author), Xian-Hui Yang (6th author) and Zhi-Ming Zhang(7th author) participated in animal experiment and constituent analysis. YinLuo (9th author) contributed to the revision of the manuscript. Chang-ZhuLi (8th author) and Zhen-Xiang He (co-corresponding author) provided theoil sample, experimental conditions, and financial support. Hai-Liang Zhu(corresponding author) designed and directed the experiments.

mechanisms ranging from the enhancement of cholesterol fluxthrough the bile acid pathway to the inhibition of hepatic choles-terol biosynthesis and elevation of low-density lipoprotein (LDL)receptor activity (Gibson and others 1995). However, many ofthese synthesized drugs could cause side effects that limited theirclinical application. For example, there is a risk of sever muscledamage with statins, which are particularly well-suited for low-ering LDL (Kobayashi and others 2008). Niacin, a good drugfor lowering triglycerides, may cause hyperglycemia and may alsocause liver damage (Guyton and Bays 2007).

Recently, many natural vegetable oils have been reported toshow hypolipidemic effect due to their high content of unsatu-rated fatty acids (UFA), such as linoleic acid (Chandrashekar andothers 2010). As reported, UFA and some other hypolipidemicdrugs can improve the peroxisome level by regulating peroxisomeproliferator-activated receptors (PPAR) mediated gene expressionvia liver fatty acid binding protein (Wolfrum and others 2001).

Cornus wilsoniana Wanger is a woody oil plant with high-yieldand high-oiliness characters in China. It is widely distributes inthe south region of the Yellow River, and concentrated in thelimestone mountains at an elevation below 1000 m. Cornus wilso-niana oil (CWO) has been taken as edible oil for over 100 y. Itsphysicochemical properties (acid value, 3.75 mg KOH/g; iodinevalue, 102 g I/100 g; saponification value, 198 mg KOH/g) aresimilar with other commonly used edible oils, such as soybeanoil, peanut oil, and rapeseed oil. Long-term consumption of suchoil is believed to reduce cholesterol and prevent hyperlipidemia in

C© 2012 Institute of Food Technologists R©doi: 10.1111/j.1750-3841.2012.02786.x Vol. 00, Nr. 0, 2012 � Journal of Food Science H1Further reproduction without permission is prohibited

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Hypolipidemic activity of an oil. . .

Chinese folk recipe. However, to the best of our knowledge, thereis no report on the hypolipidemic activity or chemical composi-tions of CWO. Therefore, in this study, the hypolipidemic effecton rat serum and hepatic tissue lipids of feeding oil from Cornuswilsoniana fruits were examined. The messenger RNA (mRNA)levels of PPAR target genes in the rat liver were analyzed byNorthern blot. And the chemical compositions of CWO wereanalyzed by gas chromatography-mass spectrometry (GC-MS). Inaddition, molecular docking was further performed to study thebinding model of fatty acids (FA) from CWO to a possible hy-polipidemic target, PPARδ. The object of this article is to disclosethe hypolipidemic activity and chemical compositions of CWO.We hope our results could provide some beneficial informationon the study of CWO.

Materials and Methods

Oil and chemicalsCWO, supplied by Hu’nan Academy of Forestry (Changsha,

Hu’nan Province, China), was obtained by pressing the driedfruits (collected in 2009) with a KOMET cold-pressing expeller(IBG Monforts, Monchengladbach, Germany) in August 2010. Allchemicals were purchased from Sigma Chemical Company (SaintLouis, Miss., U.S.A.). All solvents used were of analytical grade.

Experimental animals and dietsTotal of 50 adult male Sprague-Dawley rats, weighing 150 to

170 g each, were supplied by Yangzhou Univ. (Yangzhou, JiangsuProvince, China) and divided into 5 groups. Each group had 10rats housed in 1 cage and fed in the animal house of Nanjing Univ.(Nanjing, Jiangsu Province, China). The common feed was pre-pared according to state standard (GB14924—1994). The high-fatdiet (HFD) was composed of 78.8% common feed, 1% choles-terol, 10% yolk powder, 10% lard, and 0.2% cholate (Lu and others2010). Total of 40 rats were fed an HFD for 4 wk to establish thehyperlipidemia model and then the rats were randomly dividedinto 4 groups. The suspension of CWO was prepared in purifiedwater with 1% carboxymethyl cellulose (CMC) by stirring. Therats were continuously fed an HFD for 6 wk and administeredby oral gavage with 10 mL 1% CMC solution (high-fat controlgroup, HFCG), 100 mg CWO (low-dose group, LDG), 150 mgCWO (medium-dose group, MDG), and 300 mg CWO (high-dose group, HDG) per kilogram body weight (b.w.), respectively.The other 10 rats were given common feed as the normal controlgroup (NCG) and administered by oral gavage with 10 mL 1%CMC solution during the experimental period.

Animal experiment and sample collectionDuring the 6-wk experimental period, rats were given free ac-

cess to experimental diets and tap water. Tap water was refreshedevery 3 d, and the experimental diets were refreshed every day.Food consumption and body weights were recorded once a week.At the termination of animal experiment, rats were placed on anovernight fast, then weighed and anesthetized prior to being sac-rificed by carbon dioxide (CO2) inhalation. The blood sampleswas drawn from the abdominal vein and collected in tubes. Thetubes were placed in a 37 ◦C water bath for 1.5 h to coagulatethe blood, and then kept at 4 ◦C in a refrigerator for 1 h. Serumwas separated from the blood by centrifugation (365 g) for 10 minat 4 ◦C, then stored at –20 ◦C for further measurement. Af-ter exsanguination, liver was excised, washed in ice-cold saline,

weighed, frozen in liquid nitrogen, and then stored at –70 ◦C forliver lipids analysis.

Analysis of lipid parametersThe concentrations of total cholesterol (TC), total triacylglyc-

erol (TG), low-density lipoprotein cholesterol (LDL-C), and high-density lipoprotein cholesterol (HDL-C) in serum were directlymeasured using commercial kits (EHSY Co., Shanghai, China).TC and TG levels in liver were measured after extracting fromthe hepatic tissue according to the reported method (Tzang andothers 2009). Briefly, the hepatic lipid extraction was prepared asthe following procedure: extraction of hepatic tissue with chlo-roform and methanol (2 : 1, v/v), drying extract under nitrogen(N2), resuspending and dissolving in isopropanol with the help ofultrasound.

Analysis of mRNA levelsThe mRNA levels of PPAR target genes, acyl-CoA oxidase

(ACO) and carnitine palmitoyltransferase I (CPT1), in the ratliver were analyzed according to the reported method (Dyroy andothers 2007). Total RNA was extracted from rat liver by the acidguanidium thiocyanate-phenol-chloroform method. Polyadeny-lated mRNA was run on a denaturing gels and Northern blottedwith with ACO and CPT1, taking glyceraldehyde-3-phosphatedehydrogenase (GAPDH) as internal reference. The mRNA lev-els were quantified with a phosphoImager.

Risk testingWe have carried out the risk testing to evaluate the glycemic

effect and the possible liver damage of CWO. Total of 24 maleS-D rats of 8 wk were divided into 4 groups (n = 6). Animalsin different treatment groups were fed a normal diet for 2 wkand administered by oral gavage with 10 mL 1% CMC solution,100 mg CWO, 150 mg CWO, and 300 mg CWO per kilogramb.w., respectively. Blood glucose concentrations were measuredon Days 0, 7, and 14. Blood samples were collected from thetail into heparinized capillaries. Blood glucose was measured infresh whole blood using a glucometer. Then, rats were sacrificedby carbon dioxide inhalation. Livers were removed and fixed ina buffer solution of 10% formalin. Fixed tissues were processedroutinely for paraffin embedding, and 4 μm sections were preparedand dyed with hematoxylin-eosin. Stained areas were viewed usingan optical microscope with a magnifying power of ×400.

Preparation of methylated products of CWOThe preparation of methylated products of CWO was accord-

ing to the standard method (GB/T1736–2008). Briefly, CWO wassaponified with 0.5 M sodium hydroxide (NaOH) methanol solu-tion and methylated with 14% boron trifluoride (BF3) methanolsolution under N2. The methylated products were obtained by ex-traction using isooctane and expulsion of water using anhydroussodium sulfate (Na2SO4).

Analysis of methylated products of CWOAnalysis of methylated products of CWO was carried out on a

HP6890-HP5975 GC-MS apparatus (Agilent Technologies Co.,Santa Clara, Calif., U.S.A.). A HP-5 fused silica capillary column(30 m, 0.25 mm, 0.25 μm) was used for separation with helium asthe carrier gas at a constant flow rate of 0.8 mL/min. Oven tem-perature was programmed as follows: 100 ◦C hold for 0.5 min,then raised at 10 ◦C/min to 300 ◦C (held for 30 min). The in-jection was set on a split mode at 280 ◦C. A 1 μL sample wasinjected with a 1 min solvent delay. Detection was conducted by

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a mass selective detector (MSD) with electron impact ionization(EI). The mass scanning ranged from m/z 20 to 650. The com-ponents were identified by co-injection with standards (whereverpossible), confirmed with Natl. Inst. of Standards and Technology(NIST) mass library. The relative concentration of each compoundin was quantified based on the peak area integrated by the anal-ysis program. The total ion chromatogram (TIC) and identifiedcompounds of the methylated products of CWO are shown inFigure A1 and Table A1, respectively.

Docking studyThe analysis of 3-dimensional (3D) X-ray structure of PPARδ

(PDB code: 2baw) was conducted with Molsoft ICM 3.5–0a soft-ware. Molecular docking of FA in the CWO into the pocket ofPPARδ was carried out using the AutoDock software package(version 4.0) as implemented through the graphical user interfaceAutoDockTools (ADT 1.4.6). The 3D X-ray structure of PPARδ

was shown in Figure A2.

Statistical analysisThe results of animal experiment were analyzed by analysis of

variance (ANOVA) using SPSS 13.0 software. Data were expressedas means ± S.D. ANOVA was employed to evaluate the differencesbetween the groups. A difference of P < 0.05 was considered tobe significant. Principal component analysis (PCA) was carriedout using SPSS 13.0 for Windows.

Results

Food consumption and body weightsThroughout the 6-wk animal experiment, no adverse clini-

cal signs or effects on survival were found in rats. The groups’mean weekly body weights and food consumption compared withtime are presented in Figure 1. As shown in Figure 1A, therewas a significant difference (P < 0.05) in daily food consump-tion between the HFCG and NCG. Compared with the HFCG,food consumption decreased in the MDG (P < 0.05) and HDG(P < 0.01), but no significant difference (P > 0.05) was shownin the LMG. Hence, the administration of high-dose CWO couldaffect food consumption.

As shown in Figure 1B, a significant difference (P < 0.05) inbody weight was found between the HFCG and NCG. Com-pared with the HFC group, the final body weights were signif-icantly different (P < 0.05) in the MDG and extremely signifi-cantly different (P < 0.01) in the HDG. No significant difference(P > 0.05) was shown in the LDG. This suggested that high-doseof CWO might inhibit increases in body weight gain.

Hypolipidemic effect of CWOFigure 2 shows the changes of rat serum lipids, lipoprotein,

liver weights, and hepatic lipids profiles after 6 wk of feeding. Asshown in Figure 2A, rat serum TC and TG of the NCG were 2.98and 1.02 mmol/L, respectively. Serum TC and TG in the HFCGwere significantly raised to 5.48 and 2.69 mmol/L (P < 0.01),respectively. Compared with the HFCG, serum TC levels of MDG(P < 0.05) and HDG were significantly suppressed (P < 0.01),and showed a dose-dependent manner. Serum TC was reducedby 13.2%, 22.1%, and 29.8% LDG, MDG, and HDG, respectively.When comparing serum TG, HDG exhibited significantly lower(P < 0.05) value than the HFCG, by 51.6%. However, it was notsignificantly different (P > 0.05) in LDG and MDG.

Rat serum LDL-C and HDL-C of the NCG were 1.33 and2.08 mmol/L, respectively (Figure 2B). Serum LDL-C was signif-

icantly raised (P < 0.01) to 4.64 mmol/L, and HDL-C was signif-icantly lowered (P < 0.01) to 1.53 mmol/L in the HFCG. Com-pared with the HFCG, LDL-C was significantly lowered in LDG(P < 0.05), MDG (P < 0.01), and HDG (P < 0.01) by 26.7%,36.9%, and 39.7%, respectively. However, for HDL-C levels, noneof the LDG, MDG, and HDG showed significant differences com-paring with HFCG.

Figure 2C shows the changes of rat liver weight. There wasa significant increase (P < 0.01) in relative liver weight in theHFCG, compared with the NCG. Administrating 300 mg/kg b.w.CWO could lower relative liver weight significantly (P < 0.05)compared with the HFCG (the relative liver weight decreasedby 20.7%).

Figure 2D indicated that hepatic TC and TG concentrationswere significantly (P < 0.01) increased in the HFCG comparedwith the NCG. Rats in the MDG and HDG had significantlylower (P < 0.05 or P < 0.01) hepatic TC concentrations (de-creased by 24.6% and 39.3%, respectively), and rats in the HDGhad significantly lower (P < 0.05) TG (decreased by 16.2%) con-centrations compared with the HFCG.

The mRNA levels of PPAR target genes, ACO and CPT1, inthe rat liver were also analyzed, and the results were shown inFigure 3. Both the gene expressions of ACO and CPT1 increasedwith increasing CWO dose, which were favorable for the fattyacids metabolism.

Taken together, our data suggests consuming CWO might pro-vide benefits in the prevention and treatment of hyperlipidemia.

Chemical compositions of CWOThe main components of CWO were various FA, accounting

for 99.54%. Table 1 shows the contents of FA in the CWO. UFA

Figure 1–Changes of daily food consumption and body weights during the6-wk experimental period: (A) daily food consumption and (B) body weights.

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Hypolipidemic activity of an oil. . .

and saturated fatty acids (SFA) accounted for 69.12% and 30.42%,respectively. The most abundant UFA were linoleic acid (38.81%)and oleic acid (23.32%). Palmitic acid was the most SFA, account-ing for 26.05%. Besides the three fatty acids, palmitoleic acid

(3.47%), vaccenic acid (2.80%), and stearic acid (3.3%) also hadhigh contents. Considering the chemical structure, C18 FA werethe most, and C16 FA took the second place. The high content ofvarious UFA may contribute to the main health function of CWO.

Figure 2–Hypolipidemic effect of CWO on rats: (A) serum TC and TG concentrations in rats, (B) serum LDL-C and HDL-C concentrations in rats, (C)relative liver weights of rats, and (D) hepatic TC and TG concentrations in rats. Each value is shown as the mean ± S.D. (n = 10). ∗ P < 0.05, ∗∗ P < 0.01significantly different when compared with HFCG group; ## P < 0.01 significantly different when compared with NCG.

Figure 3–Gene expressions of ACO and CPT1 inrat liver. Target mRNA levels were normalizedby GAPDH and gene expressions obtained fromthe mRNA levels ratio relative to HFCG group.Each value is shown as the mean ± S.D. (n = 3).∗∗ P < 0.01 significantly different whencompared with HFCG group.

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In addition, there were some odorous compounds (0.36%),alkanes (0.075%) and glucoside (0.028%) existing in CWO(Table A1). The odorous compounds included some esters, alka-nols, and acetals. The chain lengths of alkanes were between C11-C18. The bad taste of CWO is possibly related with these odorouscompounds and alkanes. In addition, sterol compounds, reportedactive components, were not detected in this observation. This isprobably because of the low abundance in the CWO.

Docking studyThe PPARs are nuclear receptors for various natural FA, which

regulate lipid homeostasis. The only reported crystal structure ofa PPAR with bound FA is that of the ligand-binding domain(LBD) of PPARδ (Xu and others 1999). Figure 4A shows the

Table 1–Contents of FA in the CWO.

Content ContentCompound (%) Compound (%)

UFA 69.12 SFA 30.42Linoleic acid 38.81 Palmitic acid 26.05Oleic acid 23.32 Stearic acid 3.3Palmitoleic acid 3.47 Arachic acid 0.43Vaccenic acid 2.801 Lignoceric acid 0.27Gondoic acid 0.28 Margarinic acid 0.129,10-Methyle-

nehexadecanoic acid0.17 Behenic acid 0.073

Hypogeic acid 0.09 Myristic acid 0.068Linolenic acid 0.076 Tricosanoic acid 0.03γ -Linolenic acid 0.043 Ceratinic acid 0.03Conjucated linoleic acid 0.037 Heneicosanoic acid 0.0218,11-Octadecadienoic

acid0.021 Pentacosanoic acid 0.018

11,14-Eicosadienoicacid

0.0044 Octacosanoic acid 0.01

9,15-Octadecadienoicacid

0.0034 15-Methylpalmitic acid 0.0032

Table 2–Binding energy of FA in the CWO to PPARδ.

Binding Bindingenergy energy

UFA (kcal/mol) SFA (kcal/mol)

Hypogeic acid (C16:1,�7)

−5.91 Myristic acid(C14:0)

−4.26

Palmitoleic acid(C16:1, �9)

−3.17 Palmitic acid(C16:0)

−4.21

Oleic acid (C18:1, �9) −3.45 Margarinic acid(C17:0)

−2.25

Vaccenic acid (C18:1,�11)

−4.49 Stearic acid(C18:0)

−4.03

8,11-Octadecadienoicacid (C18:2, �8,11)

−4.25 Arachic acid(C20:0)

−3.02

Conjucated linoleicacid (C18:2, �9,11)

−2.89 Heneicosanoicacid (C21:0)

−1.71

Linoleic acid (C18:2,�9,12)

−4.64 Behenic Acid(C22:0)

−3.21

9,15-Octadecadienoicacid (C18:2, �9,15)

−3.19 Tricosanoic acid(C23:0)

−0.54

Linolenic acid (C18:3,�9,12,15)

−2.11 Lignoceric acid(C24:0)

−0.35

γ -Linolenic acid(C18:3, �6,9,12)

−4.15 Pentacosanoic acid(C25:0)

−0.14

Gondoic acid (C20:1,�11)

−0.89 Ceratinic acid(C26:0)

0.18

11,14-Eicosadienoicacid (C20:2, �11,14)

−2.48 Octacosanoic acid(C28:0)

\

9,10-Methyle-nehexadecanoicacid

−4.07 15-Methylpalmiticacid

−3.76

ligand-binding pocket of PPARδ, which is a very large cavitywithin the protein with a total volume of approximately 1300 A3.Molecular docking was performed on the binding model basedon the crystal structure of PPARδ and natural FA from CWOusing the AutoDock 4.0 software. Table 2 summaries the bindingenergies of these FA with PPARδ. Hypogeic acid (C16:1, �7)showed the lowest biding energy with PPARδ (–5.91 kcal/mol).The binding energies of long chain fatty acids (C > 20) were muchhigher, suggesting that these fatty acids could not fit into the pocketvery well. The binding mode of hypogeic acid with PPARδ wasshown in Figure 4B. The binding mode was stabilized througha combination of hydrogen bond and hydrophobic interactions.The acid group of hypogeic acid was oriented and held in placethrough a hydrogen bond with the side chain of residue Gln451,and hydrophobic interactions with Ala450, Val446 and Ile472.

Risk testingWe have carried out the risk testing to evaluate the glycemic ef-

fect and the possible liver damage of CWO. As shown in Figure 5A,there were no significant differences in the blood glucose level ofrats in different treatment groups, suggesting that appropriate doesof CWO might not change the blood glucose level. Also, nomorphological changes were found in hepaocytes (Figure 5B), in-dicating that administering CWO would not pose liver damage.

Discussion

Health functionThere are many reports that disclose the hypolipidemic effect

of various natural oils using different animal models (Table 3).Except for fish oil, the hypolipidemic oils were mainly from veg-etable sources. Because of the differences between experimentalmethods, it is difficult to compare the effect of various oils.

Although many oils could lower serum lipids, the rising effectson HDL-C were usually little (Berrougui and others 2003; Lu andothers 2010). Only Mountain celery seed essential oil was reportedthe HDL-C elevating capacity, which was probably relative withthe high content of oleamide in oil (Cheng and others 2008).Generally, vegetable oils are very rich in UFA, which are difficultto be oxidized and involved in the fluidity of lipoproteins andas a consequence in generation of HDL (Sola and others 1990).Therefore, usually the HDL-C concentration remained unaltered.However, we could evaluate the ratio of LDL-C/HDL-C, whichis an important index of atherogenicity. In our study, we noticeda significant increase (P < 0.01) of the LDL-C/HDL-C ratio,in the HFCG compared with the NCG (Figure A3). Comparedwith the HFCG, there was a significant decrease (P < 0.05 orP < 0.01) of LDL-C/HDL-C ratio in the groups administeredwith 150 and 300 mg/kg.b.w. CWO, indicating that consuminghigh dose of CWO might reduce the risks of coronary arterydisease (CAD).

Active compositionThe effective compositions of various natural oils with hypolipi-

demic effect are different (Table 3). For example, bamboo shoot oilhas high content of phytosterols (Lu and others 2010). Oleamideis the main active component of mountain celery seed essentialoil (Cheng and others 2008). The hypolipidemic effect of fishoil is attributed to the presences of eicosapentaenoic acid (EPA)and docosahexaenoic acid (DHA) (Saraswathi and others 2009).Rice bran oil has large quantities of tocotrienols (Minhajuddinand others 2005). Palm oil, diacylglycerol oil, and argan oil

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Figure 4–Docking study: (A) crystal structure of PPARδ with ligand-binding pocket, and (B) binding mode of hypogeic acid with PPARδ.

Figure 5–Risk testing: (a) blood glucose comparedwith time, each value is shown as the mean ± S.D.(n = 6); (b) H-E staining of liver tissue in S–D rats (×400).

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Table 3–Hypolipidemic effect of various natural oils.

Oil SourceMain

composition Animal Does Effect Mechanism Reference

Bamboo shootoil

Phyllostachyspubescens

FA and phytosterol S–D rats Oral gavage, 250to 1000 mg/kgb.w.

TC, TG, LDL-C inserum, and TC, TGin liver significantlydecreased

High content ofphytosterol inhibitcholesterolabsorption andincrease cholesterolexcretion

Lu and others(2010)

Mountaincelery oil

Cryptotaeniajaponica

Oleamide Hamster Fed on 0.01 to0.05 g/rat

Exhibited bothsignificanthypolipidemic andHDL-C elevatingcapabilities

Oleamide is the mainhypolipidemiccomponet

Cheng and others(2008)

Fish oil Menhaden oil EPA, DHA LDLR-/-mice

Fed on 6% (b.w.) Lipid-lowering effects Fish oil hashypolipidemiceffects in lean miceand insulinsensitizing effects inobese mice

Saraswathi andothers (2009)

Palm oil Okitipupa oilmill,Nigeria

16:0 and 18:1 FA Wistar rats Fed on 5% (b.w.) Lowering TC and TGin serum and liver

The tocotrienols andthe peculiarisomeric position ofits FA may playcrucial roles

Ojieh and others(2009)

Diacylglyceroloil

Commerciallyavailable

18:1 and 18:2 FA S-D rats Fed on 5% (b.w.) The hepatic TG levelwas significantlylowered

The activity wasattributable to anincreased fatty acidoxidation enzymeactivity and areduced fatty acidsynthesis enzymeactivity

Kim and others(2007)

Rice bran oilfraction

Rice bran oil Tocotrienol Albino rats Oral gavage, 4–50mg/kg b.w.

Plasma TC, TG andLDL-C weresignificantlylowered

Atocopherol playedthe crucial role

Minhajuddin andothers (2005)

Argan oil Argania spinosaL.

Oleic acid andLinoleic acid

Meriones shawirats

Gastric intubation,1 ml/100 g b.w.

Serum TC, TG,LDL-C and bodyweight significantlydecreased

This effect will berelated with thePUFA and otherconstituents ofstudied oil

Berrougui andothers (2003)

contain abundant UFA (Berrougui and others 2003; Kim andothers 2007; Ojieh and others 2009). The high content of UFA(69.12%) may be the main effective components in CWO. Earlystudies showed that dietary intake of n-6 polyunsaturated fattyacids (PUFA), which are abundant in vegetable oils, are inverselyrelated to the incidence of cardiovascular disease. Dietary linoleicacid (C18: 2n-6) serves as a precursor for biosynthesis of arachi-donic acid (C20: 4n-6), the substrate for eicosanoid synthesis.It has long been accepted as having hypocholesterolemic effects(Keys and others 1957; Hegsted and others 1965). More recently,acid derivatives, particularly γ -linolenic acid, were found to beeven more potent in reducing blood cholesterol in humans andrats (Takada and others 1994). In the CWO, the n-6 PUFA ac-counted for 38.86%, including linoleic acid (38.81%), γ -linolenicacid (0.043%), and 11,14-eicosadienoic acid (0.0044%). These n-6PUFA were possibly attributed to the strong hypolipidemic effectof CWO.

To increase content of the UFA, some oil full of SFA wasblended with other oils full of UFA, such as mixed coconut oilblended with rice bran oil or sesame oil (Chandrashekar and oth-ers 2010), and blend of coconut oil with rice bran oil or sesameoil (Reena and Lokesh 2007). These blended oils showed goodhypolipidemic effect. To explain the hypolipidemic effect of theseblended oils, it is interesting to consider its richness in PUFA witha ratio of PUFA/SFA. Studies examining the effects of PUFA/SFA

ratios of dietary fats on the regulation of lipid metabolism in ratshave shown that a diet containing lipids with a PUFA/SFA ratioabove 1.5 ameliorates risk factors for coronary heart disease (Oliver1987; Lee and others 1989). Studies using coconut oil blendswith rice bran oil or sesame oil, in which PUFA/SFA ratio wasmaintained at 0.8 to 1, reduced serum lipids significantly as com-pared with rats given coconut oil alone (Reena and Lokesh 2007).In the present study, it was observed that PUFA/SFA ratio was1.28. That is high enough to explain the hypolipidemic effect ofCWO. In addition, recommended dietary fat should have 1 : 1 : 1ratio for SFA/MUFA/PUFA and that the diet should provide 20%to 25% energy from fat. In the CWO, SFA/MUFA/PUFA was1 : 1.28 : 0.99. Moreover, essential fatty acids (EFA), which arenot made by the body, must be obtained through food. Total of2 EFA, linoleic acid and linolenic acid, are essential for normalbody function. These 2 EFA are found principally in seed oils,such as soybean oil and sunflower oil (Lance-Gould 2000). In thecomposition of CWO, the content of linoleic acid and linolenicacid is very high. From the previously mentioned discussion, wecan see that CWO is very suitable for dietary, not only having hy-polipidemic effect, but also helping to overcome EFA deficiency.

Possible targetThe PPAR are a group of nuclear receptor proteins that func-

tion as transcription factors regulating the expression of genes

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(Michalik and others 2006). PPAR play essential roles in the reg-ulation of lipid homeostasis. In our observation, when rats werereceived CWO, the hepatic mRNA levels of the PPAR targetgenes ACO and CPT1 were significantly increased, suggestingthat CWO might promote the activation of PPAR.

Various natural FA and hypolipidemic drugs serve as ligands forthe PPAR subtypes. Therefore, PPAR might serve as the targetfor the active FA in CWO, linking lipid level. Many previousstudies have conducted binding analyses between fatty acids andPPAR (Gani and Sylte 2009). However, they focused on PPARγ

or PPARα. The research on molecular recognition of fatty acidsby PPARδ has been rarely reported. It has been proposed thatPPARδ regulates FA oxidation and could play a critical role inlipid metabolism and obesity (Oliver and others 2001; Wang andothers 2003), making this PPAR subtype an especially interestingreceptor. The results of docking study suggested that the longchain FA (C ≥ 20) could not bind PPARδ well. This is becausethat the long chains of these FA exceed the capacity of bindingpocket. The ligand-binding pocket of PPARδ assumes roughly a“Y” shape with each of the 3 arms approximately 12 A in length(Xu and others 1999). Therefore, the diameter of this pocket islower than 24 A. However, the lengths of long chain FA (C ≥ 20)are usually longer than 24 A, though they can bend. Therefore,they could not insert this pocket completely.

Among the FA with the same number of carbons, the bindingenergies are different. This is the result of fold of double bonds.The two n-6 PUFA, linoleic acid and γ -linolenic acid, showedgood binding capability to PPARδ, which is possibly related withtheir potent hypolipidemic effects. However, the regulation of lipidmetabolism is complex, involving three PPAR subtypes. Hence,we could just say that the high hypolipidemic effects of linoleicacid and γ -linolenic acid were possible related to their high affinityto PPARδ.

In addition, we have carried out PCA on the properties of FA inthe CWO (Figure A4). The properties included content, molec-ular weight (MW), carbon number (C), degree of unsaturation(�), ratio of carbon and hydrogen (C/H), binding energy (E) andinhibitory constant (K). It can be seen that 2 principal compo-nents (eigenvalue > 1) were extracted, which explains 79% of thetotal variation. The first component accounts for 55% of the totalvariance and captures the molecular size (C, MW) and affinityto PPARδ (E, K). The 2nd component accounts for 24% of thetotal variance and is significantly related to the unsaturation (�and C/H). The results showed that the affinity of FA to PPARδ

is related with the molecular size. The content of FA with highaffinity to PPARδ is usually low. Saturation poses the impact onthe hypolipidemic effect in a different way.

ConclusionsCWO has strong hypolipidemic effect, which is related with

its high content of PUFA, especially linoleic acid and γ -linolenicacid. The activities of the two n-6 PUFA were possibly relatedto their high affinity to PPARδ. In addition, SFA/MUFA/PUFAratio and EPA content are both good in the CWO. Therefore,CWO could be used as equilibrated dietary oil, not only hav-ing hypolipidemic function, but also helping to overcome EFAdeficiency.

AcknowledgmentThe study was financed by the Natl. Natural Science Foun-

dation of China (nr 30772627), Jiangsu Natl. Science Founda-

tion (nr BK2009239), and Natl. Science & Technology Project(nr 201004071). The authors are very grateful to the editor andreviewers for their invaluable comments and suggestions.

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Figure A1–TIC of the methylated products of CWO.

Figure A2–3D X-ray structure of PPARδ.

Figure A3–LDL-C/HDL-C ratios of differentgroups. Each value is shown as the mean ± S.D.(n = 10). ∗ P < 0.05, ∗∗ P < 0.01 significantlydifferent when compared with HFCG group;## P <0.01 significantly different when compared withNCG.

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Figure A4–Changes of daily food consumption and bodyweights during the 6-wk experimental period: (A) dailyfood consumption and (B) body weights.

Table A1–Identified compounds of the methylated products of CWO.No. Name MW Formula RT (min) Area CAS Ref Identification Remarks

1 Hexanal dimethyl acetal 146 C8H18O2 1.371 396380 001599–47-9 21173 NIST,85, ST acetals, odorous compound2 Sulfuric acid, diethyl ester 154 C4H10O4S 1.497 4883241 000064–67-5 25927 NIST, 91 smelly compound3 Undecane 156 C11H24 1.734 230722 001120–21-4 27119 NIST, 90 alkane4 Silane, cyclohexyldimethoxymethyl- 188 C9H20O2Si 2.419 123426 017865–32-6 48442 NIST, 74, atificial

comparationloss from the column

5 2,6-Dimethyldecane 170 C12H26 3.218 34919 013150–81-7 36163 NIST, 88 alkane6 Decane, 2,3,6-trimethyl- 184 C13H28 3.305 94888 062238–12-4 45597 NIST, 82, atificial

comparationalkane

7 Tridecane 184 C13H28 3.427 330207 000629–50-5 45540 NIST, 90 alkane8 Undecane, 3-methyl- 198 C14H30 3.894 85857 000629–59-4 55007 NIST, 91 alkane9 Phenol, 2,5-bis(1,1-dimethylethyl)- 206 C14H22O 5.957 419974 005875–45-6 60201 NIST, 91 odorous compound10 Pentadecane 212 C15H32 6.228 103700 000629–62-9 64571 NIST, 92 alkane11 Decanal dimethyl acetal 202 C12H26O2 6.999 505371 007779–41-1 57286 NIST, 86, ST acetals, odorous compound12 Hexadecane 226 C16H34 8.025 52527 000544–76-3 73963 NIST, 93 alkane13 Heptadecane 240 C17H36 8.065 102266 000629–78-7 82606 NIST, 94 alkane14 Methyl tetradecanoate 242 C15H30O2 8.375 1294774 000124–10-7 83692 NIST, 97 methyl esterifical product of fatty acid15 Heptadecane, 2-methyl- 254 C18H38 8.538 92890 001560–89-0 91045 NIST, 95, atificial

comparationalkane

16 Hexadecanoic acid, 15-methyl-, methyl ester 284 C18H36O2 9.449 61210 006929–04-0 108888 NIST, 86, ST methyl esterifical product of fatty acid17 Undecanal dimethyl acetal 216 C13H28O2 10.002 57142 052517–67-6 66880 NIST, 82, ST acetals, odorous compound18 9-Hexadecenoic acid, methyl ester, (Z)- 268 C17H32O2 10.229 66603966 001120–25-8 99349 NIST, 99 methyl esterifical product of fatty acid19 7-Hexadecenoic acid, methyl ester, (Z)- 268 C17H36O2 10.323 1728425 056875–67-3 99348 NIST, 91 methyl esterifical product of fatty acid20 Hexadecanoic acid, methyl ester 270 C17H34O2 10.517 498393387 000112–39-0 100704 NIST, 98 methyl esterifical product of fatty acid21 Hexadecanoic acid, ethyl ester 284 C18H36O2 11.11 1141051 000628–97-7 108865 NIST, 98 ethyl esterifical product of fatty acid22 Cyclopropaneoctanoic acid, 2-hexyl-, methyl ester 282 C18H34O2 11.187 3184590 010152–61-1 107579 NIST, 90 methyl esterifical product of fatty acid23 Heptadecanoic acid, methyl ester 284 C18H36O2 11.425 2361032 001731–92-6 108875 NIST, 99 methyl esterifical product of fatty acid24 2(3H)-Furanone, 5-butyldihydro- 142 C8H14O2 11.761 251280 000104–50-7 18957 NIST, 90, ST spicy compound25 9,12-Octadecadienoic acid, methyl ester 294 C19H34O2 12.164 742272790 002462–85-3 114374 NIST, 99 methyl esterifical product of fatty acid26 9-Octadecenoic acid (Z)-, methyl ester 296 C19H36O2 12.222 445712634 000112–62-9 115454 NIST, 99 methyl esterifical product of fatty acid27 11-Octadecenoic acid, methyl ester, (Z)- 296 C19H36O2 12.269 53220559 001937–63-9 115462 NIST, 99 methyl esterifical product of fatty acid28 11-Octadecenoic acid, methyl ester 296 C19H36O2 12.304 406165 052380–33-3 115447 NIST, 99 methyl esterifical product of fatty acid29 Octadecanoic acid, methyl ester 298 C19H38O2 12.388 63314050 000112–61-8 116666 NIST, 99 methyl esterifical product of fatty acid30 8,11-Octadecadienoic acid, methyl ester 294 C19H34O2 12.545 411089 056599–58-7 114373 NIST, 99 methyl esterifical product of fatty acid31 Linoleic acid ethyl ester 308 C20H36O2 12.668 1803435 000544–35-4 121902 NIST, 99 ethyl esterifical product of fatty acid32 Ethyl Oleate 220 C14H20O2 12.727 1512982 000719–22-2 69382 NIST, 99 ethyl esterifical product of fatty acid33 9,11-Octadecadienoic acid, methyl ester, (E,E)- 294 C19H34O2 12.951 703036 013038–47-6 114391 NIST, 91 methyl esterifical product of fatty acid34 9,15-Octadecadienoic acid, methyl ester, (Z,Z)- 294 C19H34O2 13.015 65116 017309–05-6 114395 NIST, 95 methyl esterifical product of fatty acid35 9,12-Octadecadienoic acid (Z,Z)-, methyl ester 294 C19H34O2 13.051 77639 000112–63-0 114388 NIST, 95 methyl esterifical product of fatty acid36 9,12,15-Octadecatrienoic acid, methyl ester, (Z,Z,Z)- 292 C19H32O2 13.561 1450290 000301–00-8 113307 NIST, 81, ST methyl esterifical product of fatty acid37 6,9,12-Octadecatrienoic acid, methyl ester 292 C19H32O2 13.794 823845 002676–41-7 113294 NIST, 89, ST methyl esterifical product of fatty acid38 11,14-Eicosadienoic acid, methyl ester 322 C21H38O2 13.847 85531 002463–02-7 129033 NIST, 95 methyl esterifical product of fatty acid39 11-Eicosenoic acid, methyl ester 324 C21H40O2 13.895 5437712 003946–08-5 129957 NIST, 99 methyl esterifical product of fatty acid40 Eicosanoic acid, methyl ester 326 C21H42O2 14.123 8183964 001120–28-1 130937 NIST, 99 methyl esterifical product of fatty acid41 Phytol 296 C20H40O 14.453 273555 000150–86-7 115541 NIST, 88, ST smelly compound42 .alpha.-D-Glucopyranoside, methyl-4-O-octyl- 306 C15H30O6 14.723 528796 100015–74-7 120531 NIST, 92 glucoside43 Heneicosanoic acid, methyl ester 340 C22H44O2 14.967 396209 006064–90-0 137339 NIST, 93 methyl esterifical product of fatty acid44 1-Hexacosanol 382 C26H54O 15.567 73891 000506–52-5 152038 NIST, 85, ST smelly compound45 Docosanoic acid, methyl ester 354 C23H46O2 15.767 1404090 000929–77-1 142870 NIST, 99 methyl esterifical product of fatty acid46 Tricosanoic acid, methyl ester 368 C24H48O2 16.542 570547 002433–97-8 147736 NIST, 99 methyl esterifical product of fatty acid47 Tetracosanoic acid, methyl ester 382 C25H50O2 17.283 5244453 002442–49-1 152018 NIST, 99 methyl esterifical product of fatty acid48 Pentacosanoic acid, methyl ester 396 C26H52O2 18.02 347675 055373–89-2 155611 NIST, 68, ST methyl esterifical product of fatty acid49 Tridecanol, 2-ethyl-2-methyl- 242 C16H34O 18.479 125883 100011–56-6 83812 NIST, 76, ST smelly compound50 Hexacosanoic acid, methyl ester 410 C27H54O2 18.71 573331 005802–82-4 158462 NIST, 90 methyl esterifical product of fatty acid51 Cyclotrisiloxane, hexamethyl- 222 C6H18O3Si3 19.684 77895 000541–05-9 71175 NIST, 32, atificial

comparationloss from the column

52 Octacosanoic acid, methyl ester 438 C29H58O2 20.036 202608 055682–92-3 162993 NIST, 46, atificialcomparation

methyl esterifical product of fatty acid

53 Silicic acid, diethyl bis(trimethylsilyl) ester 296 C10H28O4Si3 21.534 244825 003555–45-1 114916 NIST, 38, atificialcomparation

loss from the column

54 Cyclotrisiloxane, hexamethyl- 222 C6H18O3Si3 21.775 104711 000541–05-9 71175 NIST, 47, atificialcomparation

loss from the column

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