Bioavailability of polyphenols from peanut skin extract associated with plasma lipid lowering function

Food Chemistry 148 (2014) 24–29

Contents lists available at ScienceDirect

Food Chemistry journal homepage: www.elsevier.com/locate/foodchem

Bioavailability of polyphenols from peanut skin extract associated with plasma lipid lowering function Rishipal R. Bansode a,⇑, Priscilla Randolph a, Mohamed Ahmedna b, Steven Hurley c, Tracy Hanner c, Sarah A. Schwatrz Baxter d, Thomas A. Johnston d, Mingming Su d, Bryce M. Holmes e, Jianmei Yu e, Leonard L. Williams a a

Center for Excellence in Post-Harvest Technologies, North Carolina Agricultural and Technical State University, North Carolina Research Campus, Kannapolis, NC 28081, USA Department of Health Sciences, Qatar University, P.O. Box 2713, Doha, Qatar Department of Animal Sciences, North Carolina Agricultural and Technical State University, Web Hall, East Market Street, Greensboro, NC 27411, USA d David H. Murdoch Research Institute, North Carolina Research Campus, Kannapolis, NC 28081, USA e Department of Family and Consumer Sciences, North Carolina Agricultural and Technical State University, Carver Hall, Greensboro, NC 27411, USA b c

a r t i c l e

i n f o

Article history: Received 14 April 2013 Received in revised form 24 July 2013 Accepted 24 September 2013 Available online 1 October 2013 Keywords: Procyanidins Peanut skin Polyphenols Flavanoids Hypolipidemia

a b s t r a c t Peanut skin is a rich source of polyphenols including procyanidins and is shown to have hypolipidemic properties. This study investigated the bioavailability of peanut skin polyphenols using a rat model. First, the bioavailability of peanut skin polyphenols in rat plasma was evaluated. Our results showed procyanidin A2 levels in plasma peaked within 30 min of ingestion. The results of a second study show that peanut skin extract supplemented in addition to oil gavage resulted in significant decrease in plasma triglyceride and VLDL within 5 h. In the third study, rats were given a Western type diet for 5 weeks with peanut skin extract at a dose of 150 and 300 mg/kg body weight. The main effects observed were lowering of total blood lipid and reduction of the plasma fatty acids profile. Our results suggest that procyanidin A may impart a key role of hypolipidemic effect seen in peanut skin polyphenols. Ó 2013 Elsevier Ltd. All rights reserved.

1. Introduction Dyslipidemias are abnormal amounts of lipid and/or lipoprotein in the blood (Peterson & McBride, 2012). It is one of the major risk factors for cardiovascular diseases in diabetes mellitus, obese and overweight individuals (Mooradian, Haas, Wehmeier, & Wong, 2008; Weiss et al., 2004). It has been reported that 88% of U.S. adults have abnormalities of all three standard lipid parameters (LDL-C, HDL-C, TG) and are in need of therapy with lifestyle modification in an effort to relieve insulin resistance (Toth, Potter, & Ming, 2012). Similarly, approximately one-third of American children are overweight or obese and rates of pediatric dyslipedimia in the United States are rising (Kennedy, Jellerson, Snow, & Zacchetti, 2013; Weiss et al., 2004). In recent times, research has been focused on understanding the influence of diet on health and well-being (Serra et al., 2010). Foods enhanced with bioactive components, such as polyphenols, are attracting growing interest (Franck, 2006; Grassi et al., 2008; Schroeter et al., 2010; Shoji et al., 2006). Natural polyphenols range from simple molecules such as phenolic acids to highly polymerized compounds such as tannins and proanthocyanidins (Yang ⇑ Corresponding author. Tel.: +1 7042505707. E-mail address: [email protected] (R.R. Bansode). 0308-8146/$ - see front matter Ó 2013 Elsevier Ltd. All rights reserved. http://dx.doi.org/10.1016/j.foodchem.2013.09.129

et al., 2010). Proanthocyanidins comprises of the most abundant subclasses of phenolic compounds in our diet (Gu et al., 2004). The most common subgroup of proanthocyanidins are the procyanidins, which are oligomers of (epi)catechin units and their galloyl derivatives (Appeldoorn, Vincken, Gruppen, & Hollman, 2009). Procyanidins consists of two sub-types, namely A-type and B-type. Procyanidin B-type is common in most food sources, while A-type is additionally present in cranberries, peanuts, plums and spices like cinnamon (Gu et al., 2004). We have recently demonstrated that peanut skin extract prevents hepatic steatosis and showed hypolipidemic effect in rats on a Western type diet (Bansode, Randolph, Hurley, & Ahmedna, 2012). In the present study the bioavailability of peanut skin procyanidins and its derivatives in rat plasma is evaluated. This study also investigated the effect of peanut skin extract on the lipid and free fatty acid profile of rats on a Western type diet. 2. Materials and methods 2.1. Chemicals Ethanol, methanol, chloroform, isoflurane, tetrahydrofuran, Folin–Ciocalteu reagent, gallic acid, sodium chloride, sodium hydroxide, potassium hydroxide, acetonitrile, ammonium fluoride,

R.R. Bansode et al. / Food Chemistry 148 (2014) 24–29

ammonium actetate,formic acid, b-glucoronidase, catechin, epicatechin were purchased from Sigma–Aldrich (St. Louis, MO). transResveratrol, was purchased from Cayman Chemicals (Ann Arbor, MI). Procyanidin A2 and Procyanidin B1 were from Indofine Chemical Company (Hillsborough, NJ). Isooctane was purchased from Thermo Fisher Scientific Inc., (Waltham, MA). Heptadecanoic acid and fatty acid methyl ester was from Nu-Check Prep, Inc (Elysian, MN). All reagents were greater than or equal to 99% purity unless stated otherwise. 2.2. Extraction of polyphenols from peanut skin Polyphenol-rich extract was prepared from peanut skin as described previously by Bansode et al. (2012). Mildly roasted peanut skins were generously gifted by American Peanut Shellers Association (Albany, GA). Peanut skins were suspended in distilled water and boiled for 30 min as described by Shimizu-Ibuka et al. (2009). The residue was decanted and freeze dried. The lyophilized water-soluble fraction was further treated with 80% ethanol at 5 °C for 24 h and subsequently centrifuged at 10,000 rpm for 10 min. The supernatant was separated and concentrated by using a rotary evaporator in vacuo at 40 °C. The above-mentioned extraction and purification process were completed under dim light to minimize light-induced degradation/ oxidation of phenolics, which are generally light sensitive. The final concentrate of peanut skin extract (PSE) was stored at 30 °C until further use. 2.3. Determination of total phelonics assay Total phenolics in the peanut skin extract were evaluated by the Folin–Ciocalteu method as described by Yu, Ahmedna, and Goktepe (2005). Gallic acid was used as the standard and the phenolic concentrations in the PSE samples were calculated as gallic acid equivalent (mg GAE ml1). Samples were analyzed in triplicate. 2.4. Animal study and experimental design 2.4.1. Polyphenols bioavailability in rats receiving peanut skin extract gavage Male Wistar rats, approximately 6 weeks old, were used in this study. Each animal was individually housed and acclimated in laboratory conditions (18–23 °C, humidity 55–60%, 12 h light/dark cycles) for at least 1 week before the study. All rats were fed with a standard Purina 5001 chow diet (Purina Mills, Inc.) and given free access to water. After one week of acclimation, the rats were randomly divided into two groups (n = 6 per group). Food was withheld from the rats for 15 h prior to the experiments. Peanut skin polyphenols were dissolved in water at the concentration of 25 mg/ml and administered intragastrically by direct stomach intubation to six rats at a dose of 250 mg/kg of body weight. In a control group, six rats were administered 2 ml of saline solution. Blood (70–100 ll) was withdrawn from the intravenous catheter at 0, 10, 20, 30, 45, 60, 90, 150, 240, 300 and 360 min post administration. 2.4.2. Gastrointestinal absorption of lipids in rats receiving vegetable oil gavage with and without peanut skin extract The effect of peanut skin polyphenols on intestinal absorption of lipids after an acute lipid load was assessed in rats. Male wistar rats 8 weeks of age were individually housed at 22 °C with 12 h light/dark cycle and were fed with standard chow diet ad libitum. The animals were given a standard Purina chow diet and water ad libitum for a week. Food was withheld from the rats for 15 h prior to the experiment. On the experimental day, the rats were fed an oral gavage of peanut-skin extract (0, 100, 200, 300 and

25

400 mg/kg body weight) in 1 ml vegetable oil emulsion containing 0.1% lecithin as emulsifier or an oral gavage with vehicle (saline). The used procyanidin dose is one-fifth of the no-observed-adverse-effect level (NOAEL) described for grape seed procyanidin extract (GSPE), tested in male rats (Yamakoshi, Saito, Kataoka, & Kikuchi, 2002). Five hours after treatment, blood samples (200 ll) were collected by retro-orbital bleeding into heparinized tubes. Retro-orbital bleeding was conducted while the animals were under the influence of deep anesthesia attained using Isoflurane. Plasma was obtained by centrifugation and analyzed for lipids, glucose and liver enzymes (ALT and AST). 2.4.3. Evaluation of blood lipids in rats fed with a Western-type diet Male wistar rats were randomly divided into three groups (n = 6 per group). The rat groups were fed with one of the following diets: AIN93G based Western-type diet (WD) containing 30% of fat from lard, 30% from butterfat, 30% from Crisco (hydrogenated vegetable oil), and for EFA, 7% from soybean oil and 3% from corn oil. Cholesterol content was 0.15% and the approximate energy from fat was 39.9%, and 44.0% of energy from carbohydrate. WD with a gavage of peanut skin extract (PSE) at a low-dose of 150 mg/kg body weight/day (WD + PSE150) and WD with a high-dose of 300 mg/ kg body weight/day (WD + PSE300). Animals on WD received gavage of distilled water. Animals on WD + PSE150 and WD + PSE300 received gavage of peanut skin extract at the mentioned dosages five times a week for 5 weeks. Body weights of the animals and food intake were measured twice a week. The experiment was terminated after 5 weeks. Plasma samples were collected for further lipid profile and free fatty acid estimation. All animal-experimental protocols used in this study were approved by the Institutional Animal Care and Use Committee of North Carolina A&T State University, Greensboro, North Carolina. 2.5. Collection of plasma samples Rats were fasted overnight (approximately 15 h) in order to measure plasma biochemical parameters. Blood samples were collected by retro-orbital bleeding by putting animals under deep anesthesia using isoflurane. Plasma was separated from blood by centrifugation at 3000 g for 10 min and immediately analyzed for blood profile. Plasma aliquots were frozen immediately for free fatty acids analysis. 2.6. Determination of plasma profile Plasma was analyzed for lipid profile including total cholesterol (TC), triglyceride (TG), high-density-lipoprotein cholesterol (HDL), low-density-lipoprotein (LDL), and very-low-density-lipoprotein (VLDL) as well as glucose (GLU), alanine aminotransferase (ALT), aspartate amino- transferase (AST) using an Abaxis Piccolo express chemistry analyzer (Abaxis, CA). 2.7. Determination of plasma free fatty acids analysis by GC–MS For free fatty acids (FFA) analysis, lipids were extracted from plasma with chloroform–methanol (2:1, v/v). The extract was washed with a saline solution to remove proteins. The chloroform layer containing the lipids was transferred into an amber vial. The chloroform phase was concentrated under a stream of nitrogen and redissolved in 3 ml of chloroform–methanol. The free fatty acids extract was dried under N2 and resolubilized in 1 ml tetrahydrofuran at ambient temperature and 1 ml methanolic 1 M KOH and vortexed briefly. One ml BF3-Methanol (14%, w/w) was added and mixed thoroughly. The solution was subsequently heated for 15 min in a 100 °C water bath and cooled, then mixed with 0.5 ml of saturated NaCl. Fatty acid methyl esters (FAME) prepared

26

R.R. Bansode et al. / Food Chemistry 148 (2014) 24–29

by BF3 in methanol was mixed with 0.5 ml saturated NaCl. One ml of heptadecanoic acid (1 mg/ml in isooctane) was added followed by 1 ml isooctane. The solution was mixed thoroughly and allowed to separate. The upper layer was drawn off and a 1 ll quantity of sample injected into a Trace Ultra gas chromatography system (Thermo Scientific, West Palm Beach, FL) equipped with DB-23 (50% cyanopropyl/ 50% dimethylpolysiloxane) column (Agilent Technologies, Santa Clara, CA) with a 60 m  0.18 mm internal diameter, 0.14-lm film thicknesses with helium as the carrier gas. The temperature program was as follows: initial at 45 °C with a 2 min hold; ramp: 10 °C/min to 80 °C, hold: 80 °C for 2 min; ramp: 80 °C at 8 °C/min to 180 °C; hold: 180 °C for 2 min; ramp: 180 °C at 2 °C/min to 230 °C; hold: 230 °C for min. The injector and detector were set at 230 °C. Calibration was done with fatty acid methyl ester GLC-463 as a reference standard (Nu-Check Prep, Elysian, MN). 2.8. UPLC–qTOF MS 2.8.1. Sample preparation For the peanut skin extracts, three milliliters of peanut skin extract was centrifuged at 12,000 g for 10 min to remove particulates. The supernatant was separated and evaporated to dryness and reconstituted in 90:10 acetonitrile (ACN):H2O. This process was repeated and the dried sample was reconstituted in 500 lL 90:10 ACN: H2O and transferred to a UPLC vial for analysis. For the rat plasma samples, three selected plasma samples were pooled to generate enough volume for analysis (800 ll). The combined sample was divided into two samples of 300 ll each, the first as a test sample and the second as a spiked recovery sample (standard mix: trans-resveratrol, catechin, epicatechin, procyanidin A2, and procyanidin B1 at 1 lg/ml). To each sample was added 50 ll of b-glucuronidase solution (5000 Units/ml in 100 mM ammonium acetate buffer pH 5.0) and 25 ll of 10 mM ascorbic acid solution. The sample was allowed to shake for 2 h at 37 °C and 1200 rpm, and then 750 ll of acetonitrile (ACN) was added to precipitate proteins. The sample was allowed to shake for 30 min at 37 °C and 1200 rpm and subsequently was centrifuged for 20 min at 12,000 g and 4 °C. The supernatant was transferred into a glass vial and the pellet was reextracted with 750 ll of methanol. The methanol extract was shaken and centrifuged as described above. The supernatant was combined with the previous supernatant and dried under a gentle nitrogen flow. The sample was reconstituted in 50 ll methanol and 50 ll water, centrifuged and transferred to a UPLC vial for analysis. Prepared samples were analyzed on a Waters Acquity UPLC coupled with a Waters Synapt quadrupole-Time of Flight (q-TOF) mass spectrometer with an electrospray ionization source. The system was operated in V-mode and controlled by Masslynx 4.1. The samples were separated by reversed phase UPLC utilizing an Acquity BEH C18 column (2.1  100 mm, 1.8 lm) at 40 °C and a solvent flow rate of 0.3 ml/min. The mobile phase was comprised of a binary solvent system of 1 mM ammonium fluoride in water (A) and 100% acetonitrile (B) with the following gradient: 2% B for 0.4 min, 2–5% B over 0.4–2.8 min, 5–20% B over 2.8–5.0 min, 20–30% B over 5.0–9.0 min, 30–100% B over 9.0–12.0 min, the composition was held at 100% B for 1.5 min, then 100–2% B over 13.5–13.6 min, holding at 2% B over 13.9–15.0 min. The injection volume was 5.0 ll and the autosampler temperature was 4 °C. The mass spectrometric data was collected in both the positive and negative modes with a mass range of 50–1000 Da and a scan time of 0.3s in centroid mode. The analytes were noted to analyze well in the negative ion mode. The mass spectrometer was operated with a transfer CE at 4.0 eV and trap CE at 6.0 eV. The source cone and desolvation temperatures were 120 and 350 °C, respectively, with a desolvation gas flow of 700 L/h. The source operating

voltages for the capillary, source cone, and extraction cone were 3.2 kV, 35 eV, and 4.0 eV respectively. Calibration was performed using a sodium formate solution (1/1/8 (v/v) 10% formic acid/ 0.1 M NaOH/Acetonitrile) over mass range of 50–1200 (mass accuracy <5 ppm). Lockmass calibration was utilized during the analytical run with a 50-pg/ml solution of Leucine-Enkaphalin (m/ z = 554.2620 [MH]) in 1:1 ACN: H2O w/0.1% formic acid operated at 0.1 ml/min. 2.9. Statistical analysis Results are reported as mean ± standard error mean of six animals unless stated otherwise. The significance of differences was examined using analysis of variance (ANOVA), followed by Tukey’s HSD post hoc test (P < 0.05 or P < 0.01) using SAS 9.2 software (SAS Institute, Inc., Cary, NC). 3. Results 3.1. UPLC–qTOF analysis of peanut skin polyphenolic extract Twenty-two flavonoids and their derivatives were identified from PSE extract (Table 1). The retention times and transitions of the compounds were determined from the mass scan and used in identification of the compounds. The 22 compounds included 3 procyanidins (procyanidin A2, procyanidin B1, procyanidin B2), 4 anthocyanins (cyanidin, cyanidin 3-O-glucoside, petunidin 3-Oglucoside, peonidin 3-galactoside), 2 flavanols (catechin and epicatechin), 5 flavonols (isohamnetin, quercetin, kaempferol, fisetin, myricetin), 3 flavonol glycosides (rutin, kaempferol 3-O-glucoside, quercetin 4-glucoside), 1 isoflavone (genistein), 1 flavanone (hesperetin) and 1 flavone (apigenin). The remaining compound detected was (e and z) resveratrol. The theoretical fragment ions corresponded to the experimental mass spectral data and confirmed the structures. 3.2. Bioavailabity of procyanidin A2 in plasma of rats UPLC–MS analysis of procyanidin A2 in rat plasma. Utilizing UPLC–MS/MS analysis in MRM mode, five targeted polyphenolic compounds: catechin, epicatechin, procyanidin B1, procyanidin A2 and trans-resveratrol were quantitated (LOQ < 0.02 ng/ll). Other compounds of interest including gallic acid, chlorogenic acid and caffeic acid were not detected in plasma test samples. Plasma sample of PSE ingested rats were positive for catechin, epicatechin and procyanidin A. However, procyanidin B1 and tran-resveratrol were undetectable in the plasma samples at the dosage of PSE used for this experiment. The method was applied to the analysis of procyanidin A in rat plasma obtained after oral administration of peanut skin extract (250 mg/kg body weight). The mean plasma concentration–time curve of procyanidin A2 is shown in Fig 1. After PSE intake, the main metabolite quantified in plasma were procyanidin A2, followed by catechin and epicatechin. Procyanidin A2 reached the Cmax at 30 min. The procyanidin A2 was rapidly cleared from the plasma within 1 h after ingestion. Catechin and epicatechin were other metabolites determined in plasma after PSE intake, which followed similar trend at lower concentrations. 3.3. Lipid profile of plasma upon short-term exposure of rats receiving PSE with and without oil gavage The administration of PSE with the vegetable oil for 5 h produced a decrease in plasma TG at doses of 300 and 400 mg/kg body

27

R.R. Bansode et al. / Food Chemistry 148 (2014) 24–29 Table 1 Compounds identified from the 90:10 MeCN:H2O fraction of peanut skin extract as determined by UPLC–qTOF. Compound

Retention time (min)

Collision energy (eV)

Molecular ion [M]- (m/z)

Product ion (m/z)

Apigenin Catechin cis-Resveratrol Cyanidin Cyanidin 3-O-glucoside Cyanidin 3-sophoroside Epicatechin Fisetin Genistein Hesperetin Isohamnetin Kaempferol Kaempferol 3-O-glucoside Myricetin Peonidin 3-galactoside Petunidin 3-O-glucoside Procyanidin A2 Procyanidin B1 Procyanidin B2 Quercetin Quercetin 4-glucoside Rutin

10.13 5.05 8.54 9.25 8.04 6.86 3.83 9.25 10.13 10.32 10.56 9.25 8.04 8.30 8.56 7.77 6.38 3.26 3.49 9.32 8.32 6.86

6.0 6.0 6.0 6.0 6.0 6.0 6.0 6.0 6.0 6.0 6.0 6.0 6.0 6.0 6.0 6.0 6.0 6.0 6.0 6.0 6.0 6.0

269.0432 289.0680 227.0683 285.0389 447.0717 609.1462 289.0672 285.0389 269.0432 301.0684 315.0476 285.0375 447.0717 317.0639 461.1066 477.1023 575.1197 577.1345 577.1342 301.0323 463.1210 609.1462

116.9241 245.0791 184.8951 125.0216 284.0287 285.0391 245.0787 135.0435 133.0228 177.0181 300.0282 151.0019 285.0387 125.0230 299.0533 315.0490 285.0387 289.0686 289.0693 151.0015 301.0703 299.0259

VLDL levels in groups receiving 300 and 400 mg/kg dosage were significantly lower (P < 0.05). In addition, TC/H was reduced in all groups receiving PSE dose. The ranges of ALT and AST in plasma remained significantly unaltered implying minimal liver damage to groups receiving oil gavages.

0.012

Concentration (ng/µl)

0.01

0.008

3.4. Lipid content of plasma upon exposure of PSE in rats fed with a Western-type diet 0.006

0.004

0.002

0

0

50

100

150

200

Time (min)

250

300

350

400

Fig. 1. Plasma kinetics of procyanidin A2, catechin and epicatechin detected in rat plasma collected between 0 and 360 min after ingestion of peanut skin extract at a concentration of 250 mg/kg body weight.  procyanidin A2; h catechin; 4 epicatechin.

weight (P < 0.05 versus oil alone) to reach values within the range obtained in the control group receiving saline (Table 2). Likewise,

The effects of PSE administration on plasma lipids concentrations (overnight starved) for rats fed on a Western diet for five weeks are shown in Table 3. The control group receiving a Western diet increased its body weight by 73%, while the treatment groups WD + PSE150 and WD + PSE300 showed an increase of 59% and 57%, respectively. Plasma glucose levels did not show any significant difference between control and treatment groups. Rats receiving a daily dose of 150 and 300 mg/kg PSE showed reduced TG levels. The reduction in TG was significantly different in WD + PSE300 (P < 0.05) with as much as a 55% decrease. The levels of HDL were also slightly lower in treatment groups, although the decrease was not statistically significant. Total cholesterol, LDL and VLDL levels showed a dose-dependent decrease. However, VLDL levels were statistically significant (P < 0.05) in WD + PSE300 group. Also, the levels of ALT and AST were not significant in control and treatment groups.

Table 2 Plasma biochemistry of rats receiving PSE with and without oil gavage. Plasma profile

Triglyceride (mg DL1) Cholesterol (mg DL1) HDL (mg DL1) LDL (mg DL1) VLDL (mg DL1) Glucose (mmol L1) ALT (U L1) AST (U L1) TC/H a b

Saline

72.33 ± 13.09 74.00 ± 15.70 40.00 ± 11.79 19.67 ± 4.18 14.67 ± 2.73 112.67 ± 1.20 20.33 ± 3.84 73.00 ± 5.51 1.90 ± 0.15

Oil

205.67 ± 26.84 75.67 ± 6.74 34.33 ± 2.40 5.67 ± 2.85 41.33 ± 5.24a 118.33 ± 9.13 30.00 ± 3.21 88.00 ± 5.20 2.20 ± 0.20

Significantly different from saline treated sample (P < 0.05). Significantly different from oil treated sample (P < 0.05).

Oil + PSE 100 mg/kg

200 mg/kg

300 mg/kg

400 mg/kg

150.67 ± 51.52 91.67 ± 4.81 50.00 ± 5.69 11.33 ± 7.69 30.00 ± 10.15 103.67 ± 3.67 28.00 ± 2.31 80.33 ± 5.36 1.87 ± 0.17

154.00 ± 33.78 80.00 ± 11.06 40.00 ± 7.64 9.00 ± 2.31 31.00 ± 6.56 122.67 ± 3.93 29.33 ± 2.40 80.00 ± 2.31 1.37 ± 0.44

70.00 ± 5.00b 75.00 ± 2.52 48.33 ± 4.10 13.00 ± 1.73 31.00 ± 6.56b 124.00 ± 10.44 32.33 ± 3.18 80.33 ± 6.44 1.53 ± 0.09

60.67 ± 8.88b 70.67 ± 4.33 40.67 ± 0.88 18.00 ± 3.21 12.33 ± 1.67b 117.67 ± 2.67 30.33 ± 6.01 76.00 ± 4.51 1.73 ± 0.12

28

R.R. Bansode et al. / Food Chemistry 148 (2014) 24–29

Table 3 Plasma biochemistry of rats fed experimental diet for 5 weeks.

4. Discussion

Parameters

WD

WD + PSE150

WD + PSE300

Initial body weight (g) Final body weight (g) Triglycerides (mg DL1) Cholesterol (mg DL1) Glucose (mg DL1) HDL (mg DL1) LDL (mg DL1) VLDL (mg DL1) TC/H ALT (U/L1) AST (U/L1)

247.5 ± 2.9 427.5 ± 15.8 164.3 ± 30.0 84.3 ± 7.4 132.0 ± 0.49 41.2 ± 5.6 9.3 ± 0.7 29.8 ± 6.1 1.75 ± 0.11 26.7 ± 0.4 99.5 ± 0.8

243.7 ± 8.0 388.5 ± 24.2 105.3 ± 17.4 64.3 ± 7.1 128.83 ± 0.58 33.5 ± 2.6 6.3 ± 1.9 19.3 ± 3.8 1.82 ± 0.09 29.5 ± 0.4 92.7 ± 0.6

244.7 ± 1.9 384.7 ± 20.0 73.5 ± 5.6a 62.2 ± 5.5 130.83 ± 0.57 34.4 ± 1.5 6.5 ± 0.5 13.2 ± 1.2a 1.87 ± 0.11 27.7 ± 0.5 92.8 ± 1.0

WD, Western diet; WD + PSE150, Western diet with a daily infusion of peanut skin extract at 150 mg/kg body weight; WD + PSE300, Western diet with a daily infusion of peanut skin extract at 300 mg/kg body weight. Values represent the mean ± standard error mean of rats per group (n = 6). Tukey’s post-hoc test: a Significantly different from Western diet (WD)-fed group (P < 0.05).

3.5. Free fatty acid contents of plasma samples of rats receiving PSE with a Western-type diet As expected from the lipid profile data, the PSE supplemented group resulted in decreased saturated fatty acids (SFA), monounsaturated fatty acids (MUFA) and polyunsaturated fatty acids (PUFA) in blood plasma. The unsaturation index of the whole particle measured as PUFA/(SFA + MUFA) was slightly lower but not significantly different in PSE supplemented animal groups. SFAs represented a decrease of 30% and 40%, respectively; in the blood lipid fraction of PSE administered groups (Table 4). The proportion of MUFAs was significantly lower in PSEs than in control group (63% and 66%, respectively). PUFA represented a decrease of 46% and 53% in the PSE administered plasma fractions. PSE treated groups had a lower unsaturation index or PUFA/(SFA + MUFA) in both lipid fraction than the control group receiving Western diet only.

Table 4 Plasma lipid fatty acid composition in rats on a Western diet for 5 weeks with and without peanut skin extract infusiona. Fatty acid type 14:0 16:0 18:0 SFA 16:1 18:1 MUFA 18:2n6 18:3n6 18:3n3 20:3n6 20:4n6 22:5n6 22:6n3 PUFA PUFA/(SFA + MUFA)

WD 2.02 ± 0.05 48.37 ± 0.43 22.16 ± 0.23 72.55 ± 0.42 4.60 ± 0.12 35.50 ± 1.02 40.10 ± 1.09 37.25 ± 0.44 0.59 ± 0.03 2.14 ± 0.14 2.76 ± 0.20 20.17 ± 0.39 1.08 ± 0.10 2.30 ± 0.20 66.28 ± 0.74 0.59 ± 0.01

WD + PSE150 c

1.01 ± 0.05 29.23 ± 0.21c 20.40 ± 0.57b 50.64 ± 0.73c 0.57 ± 0.02c 14.13 ± 0.17c 14.71 ± 0.15c 17.67 ± 0.42c 0.32 ± 0.02c 0.78 ± 0.03c 0.36 ± 0.02c 14.89 ± 0.15c 0.53 ± 0.02c 1.36 ± 0.04c 35.92 ± 0.38c 0.55 ± 0.00b

WD + PSE300 1.04 ± 0.03c 26.24 ± 0.14c 16.40 ± 0.28c 43.68 ± 0.44c 0.33 ± 0.03c 13.19 ± 0.16c 13.52 ± 0.19c 16.11 ± 0.15c 0.21 ± 0.01c 0.61 ± 0.01c 0.65 ± 0.02c 11.82 ± 0.15c 0.64 ± 0.02c 1.35 ± 0.05c 31.39 ± 0.35c 0.55 ± 0.00b

a All values are expressed as microgram of fatty acid per milligram LDL protein represented as mean ± SEM. WD, Western diet alone; WD + PSE150, Western diet with peanut skin extract infusion of 150 mg/kg body weight; WD + PSE300, Western diet with peanut skin extract infusion of 300 mg/kg body weight; SFA, saturated fatty acids (14:0 + 16:0 + 18:0); MUFA, monounsaturated fatty acid (16:1 + 18:1); PUFA, polyunsaturated fatty acids (18:2n6 + 18:3n6 + 18:3n3 + 20:3n6 + 20:4n6 + 22:5n6 + 22:6n3). Tukey’s HSD post-hoc test: b Significantly different from control Western diet (WD), P < 0.05. c Significantly different from control Western diet (WD), P < 0.01.

The Western population is susceptible to health complications arising from metabolic syndrome (Del Bas et al., 2005). Our previous study has shown that oral administration of PSE triggered hypolipidemic effects in rats subjected to a Western type diet (Bansode et al., 2012). This study demonstrates the bioavailability of PSE in rat plasma, especially procyanidin A2, and shows the reduction in plasma lipid and fatty acid profiles from rats given a Western type diet for 5 weeks. Procyanidin profiles of blanched and roasted peanut skin extract has been previously reported (Yu, Ahmedna, Goktepe, & Dai, 2006). Our finding showed that peanut skin extract is a rich source of procyanidins, especially procyanidin type-A and type-B (Yu et al., 2006). In this study LC/MS was used to profile the peanut skin extract and confirmed the detection of procyanidins, flavonoids and catechins. However, further evaluation for bioavailability demonstrated limited absorption of procyanidins. In a targeted LC/MS analysis, only procyanidin type-A was observed and the level of procyanidin type-B was undetectable. This is in agreement with previous studies, which showed that procyanidin B2 is poorly absorbed, whereas, procyanidin B3 is not absorbed in rats (Baba, Osakabe, Natsume, & Terao, 2002; Donovan et al., 2002; Manach, Scalbert, Morand, Remesy, & Jimenez, 2004). Due to the polymeric nature and high molecular weight, procyanidins are not easily absorbed through the gut barrier (Manach et al., 2004). It is reported that procyanidins exert local activity in the gastrointestinal tract or activity mediated by phenolic acids produced through microbial degradation (Halliwell, Zhao, & Whiteman, 2000). Our results showed procyanidin A2 levels in plasma peaked within 30 min of PSE ingestion. Other targeted polyphenols such as catechin and epicatechin reached maximum levels in plasma at around 90 min from the administration of PSE. In a similar study on cocoa cream and hazelnut skin extract the catechin and epicatechin peak plasma levels were reported to be 2 h (Serra et al., 2013). In this study, the gastrointestinal absorption of vegetable oil in animals with and without PSE supplementation for 5 h was investigated. The results clearly showed a reduced plasma triglyceride upon PSE supplementation. Also, plasma VLDL-C levels were significantly lower than the group receiving oil and PSE at 300 and 400 mg/kg dosage. Similarly, supplementation of 300 mg/kg PSE in groups on a Western diet for five weeks also showed significantly lower triglyceride and VLDL-C levels. A reduction in HDL levels was also observed, signifying an overall reduction of lipids in rats receiving PSE. Interestingly, the study conducted by Zern, West, and Fernandez, 2003, showed that grape polyphenols reduced VLDL-C and TG by 50% and 39% compared to control. The reduction in TG and VLDL-C may be attributed to the decrease in apo E concentrations and therefore may result in an increase in lipoprotein lipase (LPL) activity and lipolysis (Zern & Fernandez, 2005). Our previous investigation indicated the essential role of PSE in regulating fatty acid synthesis gene and its subsequent effect on triglyceride levels in plasma (Bansode et al., 2012). Other reports have suggested that polyphenols regulate AMP-activated protein kinase (AMPK) signaling which then shuts down anabolic pathways and promotes catabolic activity through activation of enzymes such as fatty acid synthase and acyl coA carboxylase (Aoun et al., 2010; Hou et al., 2008; Kim et al., 2009; Lin, Huang, & Lin, 2007). Our recent study has shown that peanut skin polyphenols also effect peroxisome proliferator–activator receptor (PPAR)-regulated gene expression (Bansode et al., 2012). In conclusion, this study confirmed the bioavailability of procyanidin A2 in peanut skin polyphenols extract, administered orally

R.R. Bansode et al. / Food Chemistry 148 (2014) 24–29

in healthy, chow-fed rats in a postprandial situation. The absorption capacity of lipid in rat plasma was ascertained by subjecting the animals with oral administration of oil in presence or absence of peanut skin extract supplementation. The results showed reduction in total lipid levels upon peanut skin extract supplementation. A 5-week study, where animals on a Western type diet receiving a daily bolus of peanut skin extract orally, also exhibited hypolipidemic effects. The free fatty acid levels in the plasma sample of rats on five-week diet showed decreased SFA, MUFA and PUFA levels. These findings suggest that peanut skin polyphenols especially procyanidin A2 may impart hypolipidemic properties. Acknowledgements The authors would like to thank John Carver for analyzing plasma fatty acids. This research was supported by North Carolina State appropriation for the Center for Excellence in Post-Harvest Technologies at the North Carolina Research Campus, Kannapolis, NC. References Aoun, M., Michel, F., Fouret, G., Casas, F., Jullien, M., Wrutniak-Cabello, C., et al. (2010). A polyphenol extract modifies quantity but not quality of liver fatty acid content in high-fat-high-sucrose diet-fed rats: possible implication of the sirtuin pathway. British Journal of Nutrition, 104(12), 1760–1770. Appeldoorn, M. M., Vincken, J.-P., Gruppen, H., & Hollman, P. C. (2009). Procyanidin dimers A1, A2, and B2 are absorbed without conjugation or methylation from the small intestine of rats. Journal of Nutrition, 139(8), 1469–1473. Baba, S., Osakabe, N., Natsume, M., & Terao, J. (2002). Absorption and urinary excretion of procyanidin B2 [epicatechin-(4 beta-8)-epicatechin] in rats. Free Radical Biology and Medicine, 33(1), 142–148. Bansode, R. R., Randolph, P., Hurley, S., & Ahmedna, M. (2012). Evaluation of hypolipidemic effects of peanut skin-derived polyphenols in rats on Westerndiet. Food Chemistry, 135(3), 1659–1666. Del Bas, J. M., Fernández-Larrea, J., Blay, M., Ardèvol, A., Salvadó, M. J., Arola, L, et al. (2005). Grape seed procyanidins improve atherosclerotic risk index and induce liver CYP7A1 and SHP expression in healthy rats. The FASEB Journal, 19(3), 479–481. Donovan, J. L., Manach, C., Rios, L., Morand, C., Scalbert, A., & Remesy, C. (2002). Procyanidins are not bioavailable in rats fed a single meal containing a grapeseed extract or the procyanidin dimer B3. British Journal of Nutrition, 87(4), 299–306. Franck, A. (2006). Oligofructose-enriched inulin stimulates calcium absorption and bone mineralisation. Nutrition Bulletin, 31(4), 341–345. Grassi, D., Desideri, G., Necozione, S., Lippi, C., Casale, R., Properzi, G., et al. (2008). Blood pressure is reduced and insulin sensitivity increased in glucoseintolerant, hypertensive subjects after 15 days of consuming high-polyphenol dark chocolate. Journal of Nutrition, 138(9), 1671–1676. Gu, L., Kelm, M. A., Hammerstone, J. F., Beecher, G., Holden, J., Haytowitz, D., et al. (2004). Concentrations of proanthocyanidins in common foods and estimations of normal consumption. Journal of Nutrition, 134(3), 613–617. Halliwell, B., Zhao, K., & Whiteman, M. (2000). The gastrointestinal tract: a major site of antioxidant action? Free Radical Research, 33(6), 819–830. Hou, X., Xu, S., Maitland-Toolan, K. A., Sato, K., Jiang, B., Ido, Y., et al. (2008). SIRT1 regulates hepatocyte lipid metabolism through activating AMP-activated protein kinase. Journal of Biological Chemistry, 283(29), 20015–20026.

29

Kennedy, M. J., Jellerson, K. D., Snow, M. Z., & Zacchetti, M. L. (2013). Challenges in the pharmacologic management of obesity and secondary dyslipidemia in children and adolescents. Paediatr Drugs, 15(5), 335–342. Kim, W. S., Lee, Y. S., Cha, S. H., Jeong, H. W., Choe, S. S., Lee, M.-R., et al. (2009). Berberine improves lipid dysregulation in obesity by controlling central and peripheral AMPK activity. American Journal of Physiology-Endocrinology and Metabolism, 296(4), E812–E819. Lin, C.-L., Huang, H.-C., & Lin, J.-K. (2007). Theaflavins attenuate hepatic lipid accumulation through activating AMPK in human HepG2 cells. Journal of Lipid Research, 48(11), 2334–2343. Manach, C., Scalbert, A., Morand, C., Remesy, C., & Jimenez, L. (2004). Polyphenols: food sources and bioavailability. American Journal of Clinical Nutrition, 79(5), 727–747. Mooradian, A. D., Haas, M. J., Wehmeier, K. R., & Wong, N. C. (2008). Obesity-related changes in high-density lipoprotein metabolism. Obesity, 16(6), 1152–1160. Peterson, A. L., & McBride, P. E. (2012). A review of guidelines for dyslipidemia in children and adolescents. Wisconsin Medical Journal, 111(6), 274–281. Schroeter, H., Heiss, C., Spencer, J. P., Keen, C. L., Lupton, J. R., & Schmitz, H. H. (2010). Recommending flavanols and procyanidins for cardiovascular health: current knowledge and future needs. Molecular Aspects of Medicine, 31(6), 546– 557. Serra, A., Macià, A., Romero, M.-P., Valls, J., Bladé, C., Arola, L., et al. (2010). Bioavailability of procyanidin dimers and trimers and matrix food effects in in vitro and in vivo models. British Journal of Nutrition, 103(7), 944–952. Serra, A., Macià, A., Rubió, L., Anglès, N., Ortega, N., Morelló, J. R., et al. (2013). Distribution of procyanidins and their metabolites in rat plasma and tissues in relation to ingestion of procyanidin-enriched or procyanidin-rich cocoa creams. European Journal of Nutrition, 52(3), 1029–1038. Shimizu-Ibuka, A., Udagawa, H., Kobayashi-Hattori, K., Mura, K., Tokue, C., Takita, T., et al. (2009). Hypocholesterolemic effect of peanut skin and its fractions: a case record of rats fed on a high-cholesterol diet. Bioscience Biotechnology and Biochemistry, 73(1), 205–208. Shoji, T., Masumoto, S., Moriichi, N., Akiyama, H., Kanda, T., Ohtake, Y., et al. (2006). Apple procyanidin oligomers absorption in rats after oral administration: analysis of procyanidins in plasma using the porter method and highperformance liquid chromatography/tandem mass spectrometry. Journal of Agricultural and Food Chemistry, 54(3), 884–892. Toth, P. P., Potter, D., & Ming, E. E. (2012). Prevalence of lipid abnormalities in the United States: the national health and nutrition examination survey 2003– 2006. Journal of Clinical Lipidology, 6(4), 325–330. Weiss, R., Dziura, J., Burgert, T. S., Tamborlane, W. V., Taksali, S. E., Yeckel, C. W., et al. (2004). Obesity and the metabolic syndrome in children and adolescents. New England Journal of Medicine, 350(23), 2362–2374. Yamakoshi, J., Saito, M., Kataoka, S., & Kikuchi, M. (2002). Safety evaluation of proanthocyanidin-rich extract from grape seeds. Food and chemical toxicology: an international journal published for the British industrial biological research association, 40(5), 599–607. Yang, M.-Y., Peng, C.-H., Chan, K.-C., Yang, Y.-S., Huang, C.-N., & Wang, C.-J. (2010). The hypolipidemic effect of hibiscus sabdariffa polyphenols via inhibiting lipogenesis and promoting hepatic lipid clearance. Journal of Agricultural and Food Chemistry, 58(2), 850–859. Yu, J., Ahmedna, M., & Goktepe, I. (2005). Effects of processing methods and extraction solvents on concentration and antioxidant activity of peanut skin phenolics. Food Chemistry, 90(1–2), 199–206. Yu, J., Ahmedna, M., Goktepe, I., & Dai, J. A. (2006). Peanut skin procyanidins: composition and antioxidant activities as affected by processing. Journal of Food Composition and Analysis, 19(4), 364–371. Zern, T. L., & Fernandez, M. L. (2005). Cardioprotective effects of dietary polyphenols. Journal of Nutrition, 135(10), 2291–2294. Zern, T. L., West, K. L., & Fernandez, M. L. (2003). Grape polyphenols decrease plasma triglycerides and cholesterol accumulation in the aorta of ovariectomized guinea pigs. Journal of Nutrition, 133(7), 2268–2272.