Antihypertensive effect of passion fruit peel extract and its major bioactive components following acute supplementation in spontaneously hypertensive rats

Available online at www.sciencedirect.com

Journal of Nutritional Biochemistry 24 (2013) 1359 – 1366

Antihypertensive effect of passion fruit peel extract and its major bioactive components following acute supplementation in spontaneously hypertensive rats☆ Brandon J. Lewis a,⁎, Kelli A. Herrlinger a , Teresa A. Craig b, Cynthia E. Mehring-Franklin b, Zoraida DeFreitas a , Carmen Hinojosa-Laborde b b

a Kemin Foods, L.C. Research and Development, Des Moines, IA 50309 Department of Anesthesiology, University of Texas Health Science Center, San Antonio, TX

Received 14 September 2011; received in revised form 24 October 2012; accepted 2 November 2012

Abstract Extracts from leaves, peels or flowers of Passiflora are noted for their medicinal effects. Passiflora edulis peel extract (PFPE) has been proposed to lower blood pressure (BP); however, only indirect measurement techniques have been employed. To more accurately measure the effect of PFPE on hemodynamic parameters and determine the minimal effective dose, hemodynamic parameters were directly measured in spontaneously hypertensive rats (SHR) implanted with radiotelemeters. PFPE was given orally at 0, 2.5, 50 or 200 mg/kg body weight (BW) to determine the minimal effective dose. Once this dose was determined, the potential active components, edulilic acid (EA), anthocyanin fraction (AF) or γ-aminobutyric acid (GABA), were tested to determine which may contribute to the reductions in BP. The 50 mg PFPE/kg BW dose was the lowest dose that significantly reduced all hemodynamic parameters from baseline when compared to control. When the potential actives were provided at equivalent doses to those found in 50 mg PFPE/kg BW, the EA and AF significantly reduced all measured hemodynamic parameters from baseline when compared to control. GABA did not significantly affect any hemodynamic parameters compared to control and significantly increased heart rate. These direct measurements indicate that PFPE can decrease hemodynamic parameters in SHR and indicate that EA and AF are active compounds that contribute to the antihypertensive effects of PFPE supplementation. While these results are encouraging, detailed mechanistic studies are needed to determine the putative value of PFPE for blood pressure control in humans. © 2013 Elsevier Inc. All rights reserved. Keywords: Passion fruit; Spontaneously hypertensive rats; Blood pressure; Telemetry; Edulilic acid; Anthocyanins

1. Introduction The genus Passiflora consists of approximately 450–500 passion fruit plant species that are distributed in the warm temperate and tropical regions of North and South America, Asia, Australia and Africa [1,2]. The extracts from leaves, peel, flowers and roots of several species have been noted for their medicinal effects including treatments for alcoholism, anxiety, migraines, nervousness, insomnia, asthma, bronchitis, whooping cough, heart tonics, diuretics and urinary tract infections [3–5]. Recent studies have been published demonstrating the effectiveness of passion fruit (Passiflora edulis SIMS) peel extract (PFPE) against chloroform-induced liver toxicity, asthma, joint pain and hypertension in vitro, in rodent and in human studies [5–7]. Cardiovascular disease is the single largest cause of death in America, and hypertension is the primary risk factor [8]. Spontaneously hypertensive rats (SHR) have been extensively utilized and are acknowledged as an excellent animal model of human essential ☆

Funding: Funding for this research was provided by Kemin Foods, L.C. ⁎ Corresponding author. E-mail address: [email protected] (B.J. Lewis).

0955-2863/$ - see front matter © 2013 Elsevier Inc. All rights reserved. http://dx.doi.org/10.1016/j.jnutbio.2012.11.003

hypertension [9–11]. This breed is known to have continuous increases in all measurements of blood pressure (BP) and heart rate (HR) as the animal ages. Physiological and pharmacological studies of SHR provide strong evidence that the hypertension in SHR is similar in etiology to the most common form of hypertension found in humans [12]. Two studies have been published investigating the role of PFPE in SHR. Ichimura et al. fed 14-week-old male SHR single doses of a PFPE prepared by methanol extraction sourced from the Okinawa Prefecture [7]. Systolic blood pressure (SBP), as measured by tail cuff, was followed over 24 h, and a significant reduction in SBP was reported at various time points following dosing. Zibadi et al. supplemented 6-week-old female SHR for 8 weeks with PFPE (10 and 50 mg/kg) prepared by first extracting the peels with water followed by column chromatography and elution with methanol to remove sugars and pectins. Zibadi et al. found a significant reduction in SBP, as measured by tail cuff, in SHR when supplemented with 50 mg/kg dose of the water methanol extract of PFPE [6]. In these studies, only SBP data were reported using tail cuff technology, and other cardiovascular parameters were not measured continuously during the experimental protocol. The purpose of the current study was to determine the acute (6 h) and chronic (5 days)

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hemodynamic responses to PFPE and its active components in SHR by using radiotelemetry, a sensitive, less variable technology than the previously used tail cuff [13]. It was hypothesized that PFPE will not only lower SBP but will also lower diastolic blood pressure (DBP), mean arterial pressure (MAP) as well as HR in a dose-dependent manner. Following the determination of the minimum effective dose of PFPE, the second hypothesis tested was that specific active components [edulilic acid (EA), anthocyanin fraction (AF) or γaminobutyric acid (GABA)] in PFPE contributed to the hemodynamic effects. These hemodynamic parameters were measured directly and continuously utilizing radiotelemetry technology upon supplementation of PFPE or the hypothesized active components. 2. Materials and methods 2.1. Extract preparation and isolated compounds. Frozen passion fruit, sourced from New Zealand, was defrosted overnight, and the seeds, pulp and juice were removed. The peels were chopped using a food processor (Cutter model VCM 40, Hobart), minced using a Commitrol dicer (model 1700, Urschel Laboratories Inc.) and immersed in 6 vol (by weight) of deionized water for 16–24 h at ambient temperature. The solids were removed by centrifugation (model 20/24, Tolhurst), and the filtrate was further clarified (b0.2% solids) using a Sharples Centrifuge (model AS26VB, Alfa-Laval). Sugars and pectin were removed from the clarified filtrate by column filtration (12×48, model 7554-90, ACE) with SP70 polymeric resin (Sorbent, Mitsubishi Chemical Industries Ltd.) and washed with four column volumes of deionized water. The material adsorbed on the column was then eluted with ethanol (190 proof, ISP). The PFPE was concentrated using a thin film evaporator (model KDL-6, UIC GmbH) and then freeze dried (Lyophilizer SRC 100, model DL102A, VirTis). The EA and the AF were isolated from PFPE using semipreparative highperformance liquid chromatography. PFPE (4.5 g) was dissolved in 9 ml of 90% deionized water/9.8 % acetonitrile/0.2 % acetic acid. The solution was injected onto a Varian Dynamax Microsorb 100 column Å (5 μm particle size, 250×21.4 mm) and eluted with a gradient of 2% acetic acid in water (v/v) and 2% acetic acid in acetonitrile (v/v). EA was monitored at 280 nm and collected as it eluted from the column between 86 and 89 min. The AF was monitored at 520 nm and collected as it eluted from the column between the retention times of 99 and 103 min. GABA was purchased from Chromadex (Irvine, CA, USA). 2.2. Animals and implantation of radiotelemeters Male SHRs (Charles River Laboratories, Inc.) were purchased at 11 weeks of age. Upon arrival, all animals were housed in the Laboratory Animal Resources facility at the University of Texas Health Science Center at San Antonio under standard environmental conditions with a 10-h light/14-h dark cycle. Food (Harlan Teklad LM-485 mouse/rat diet) and water were provided ad libitum throughout the study. The study timeline is shown in Fig. 1. Three to four days after arrival, a catheter attached to a CA11PA-C40 radiotelemetry transmitter (Data Science, Inc.) was implanted into the abdominal aorta. Prior to surgery, all instruments and transmitters were sterilized by exposure to pressurized ethylene oxide gas. Using aseptic surgical techniques, an abdominal incision was made, and the catheter end of the transmitter was inserted into the abdominal aorta. The catheter was secured by skin adhesive (Vetbond). The transmitter/battery was secured to the abdominal muscle with sutures and remained in the abdominal cavity for the duration of the experiment. Beginning 4 h before surgery until 2 days after surgery, the animals were given drinking water (ad libitum) containing ibuprofen (2 mg/ml). SHR were allowed to recover from radiotelemeter implantation for 10 days prior to the initiation of monitoring of hemodynamic parameters. All study protocols were approved by the University of Texas Health Science Center at San Antonio, Institutional Animal Care and Use Committee. All animal

procedures and BP monitoring were performed at the University of Texas Health Science Center at San Antonio according to the Guide for the Care and Use of Laboratory Animals of the National Institutes of Health as well as the guidelines of the Animal Welfare Act. 2.3. Blood pressure analysis and supplementation 2.3.1. PFPE dose response Continuous measurement of BP and HR began for all SHR at 13 weeks of age (10 days postimplantation of the radiotelemeter, Fig. 1). Measurements of BP (MAP, SBP, DBP) and HR were recorded for 5 s at 10-min intervals beginning 5 days prior to supplementation (during study acclimatization). Hemodynamic parameters were recorded at 10-s intervals at the time of supplementation through 3 h postsupplementation. The recording rate was then returned to 10-min intervals for the 5 days following supplementation. The purpose of the shorter measurement intervals at the time of supplementation was to ensure capture of any potential rapid effects resulting from supplementation as well as to monitor the full recovery of hemodynamic parameters to baseline values. During the study acclimatization period, all SHR were given 0.25–0.30 g/day of the vehicle, Nutri-Cal (Vetoquinol, Fort Worth, TX, USA). Nutri-Cal is a high-calorie vitamin mixture routinely used as a nutritional supplement in animal diets. Nutri-Cal has no known vasoactive components and has no reported side effects. Since Nutri-Cal is very palatable and was not expected to affect blood pressure or HR in SHR, it was used as the vehicle for PFPE. Baseline parameters were assessed 24 h prior to supplementation. On the day of supplementation, SHR were randomly assigned to one of four groups (n=10/group): control (Nutri-Cal) or 2.5, 50 or 200 mg PFPE/kg body weight (BW). PFPE powder was mixed directly into 0.6 g Nutri-Cal. The Nutri-Cal mass was increased from 0.25–0.30 g during the study acclimatization period to 0.6 g at supplementation to maintain a soft consistency of Nutri-Cal after the addition of PFPE. Intake of the PFPE/Nutri-Cal mixture was monitored by weighing the container before and after consumption. In addition, the time required to consume the mixture was recorded. Animals were euthanized 5 days after supplementation by cardiac puncture. 2.3.2. Evaluation of PFPE components On the day of PFPE component supplementation, SHR were randomly assigned to one of five groups (n=8/group). For evaluation of the components, PFPE, EA (77% purity), AF (87% cyaniding 3-O-glucoside) and GABA (93% purity) were individually reconstituted in 0.5% w/v methyl cellulose in water at concentrations so that equal volumes of each were able to be added to the Nutri-Cal (approximately 1:20, test solution:Nutri-Cal ratio). At supplementation, each group received Nutri-Cal with either 0.5% w/v methylcellulose in water (control), 50 mg PFPE/kg BW, 0.42 mg GABA/ kg BW, 1.19 mg EA/kg BW or 2.39 mg AF/kg BW. The doses of GABA, EA and the AF were approximately equivalent to the amount of each component present in the 50 mg/kg BW PFPE dose, which was determined to be the minimal effective dose in the previous dose–response study. GABA was evaluated because it was postulated to be the active component responsible for lowering BP in the Ichimura et al. study [7]. EA is a novel compound uniquely present in purple passion fruit peel and thus was chosen as a compound that may also be responsible for reduction of BP parameters. Finally, the AF was evaluated due to the documented evidence of its ability to reduce BP [14,15]. The container of compound/Nutri-Cal mixture was weighed before and after placing it in the rat cage. The amount of mixture consumed and the time required to consume the mixture were recorded. Hemodynamic parameters were monitored prior, during and postsupplementation as described previously. Animals were euthanized 5 days after supplementation by cardiac puncture. 2.4. Statistics The short-term dose–response effects of PFPE on BP and HR were calculated as 10min averages during 6 h after supplementation. Repeated-measures analysis of variance (ANOVA) followed by a contrast analysis was performed by Summit Analytical, LLC., utilizing the Statistical Analysis Software Program for the evaluation

Fig. 1. Timeline of experimental procedures.

B.J. Lewis et al. / Journal of Nutritional Biochemistry 24 (2013) 1359–1366

of the change from baseline during this time to evaluate short-term observed differences following dosing. Differences between groups at baseline were analyzed by one-way ANOVA with a Dunnett's multiple-comparison test. The long-term dose–response effects of PFPE on BP and HR were calculated as 24-h averages for each of the 5 days after supplementation. A two-way ANOVA was performed using GraphPad Prism (Version 5.02) to determine the daily effect of PFPE dose over 5 days. The effects of PFPE components on BP and HR were calculated as 1-h averages, and a repeated-measures one-way ANOVA with a Tukey's multiple comparison posttest was performed using GraphPad Prism (Version 5.02) to determine the change from baseline in BP and HR measured over 5 days following administration of the PFPE components. Data are expressed as mean±S.E.M. Significance level was Pb.05.

3. Results 3.1. PFPE dose response All animals consumed 96%–99% of the target doses of PFPE administered within a 5-min period (data not shown) and had a sharp increase in all hemodynamic parameters at the first time point after dosing likely due to the excitatory response associated with feeding behavior. Baseline data for the 0, 2.5, 50, and 200 mg/kg BW groups are shown in Table 1. No significant differences were detected between groups for any parameter. Fig. 2 shows the real-time changes from baseline over the first 6 h for MAP, SBP, DBP and HR for all doses of PFPE administered. Repeated-measures ANOVA showed a treatment effect in the form of a treatment by time interaction for the 10-min measurements over the first 6 h postadministration of PFPE for MAP (Pb.05), SBP (Pb.05), DBP (Pb.01) and HR (Pb.05). Maximum reductions in MAP for any single 10-min time point were −8.9±3.0 mmHg, −13.0±2.5 mmHg and −14.0±2.7 mmHg after consuming 2.5, 50 and 200 mg PFPE/kg BW, respectively. Contrast analysis of the first 6 h postadministration of PFPE found that MAP trended (P≤0.1) or was significantly (Pb.05) less than control at 3% of the time points measured in the 2.5 mg PFPE/kg BW group, 33% of the time points for the 50 mg PFPE/kg BW group and 39% of time points for the 200 mg PFPE/kg BW group. The maximum reductions in SBP that occurred were −10.0±2.9 mmHg, −13.8±2.8 mmHg and −14.6±2.8 mmHg (Pb.05) after consuming 2.5, 50 and 200 mg PFPE/kg BW, respectively. Change in SBP from baseline over the first 6 h trended (P≤0.1) or was significantly (Pb.05) less than control at 0%, 28% or 39% of the time points (2.5, 50 and 200 mg PFPE/kg BW, respectively). The maximum reductions in DBP were −7.6±2.9 mmHg, −10.2± 2.2 mmHg and −12.4±2.3 mmHg after consuming 2.5, 50 and 200 mg PFPE/kg BW, respectively. Change in DBP from baseline trended (P≤0.1) or was significantly (Pb.05) less than control at 3%, 33% or 39% of the time points (2.5, 50 and 200 mg PFPE/kg BW, respectively) during the first 6 h following administration of dose. HR had a maximal reduction of −25.0±11.1 bpm, −46.4±9.7 bpm and −36.1±7.8 bpm at 2.5, 50 and 200 mg PFPE/kg BW, respectively. Change in HR during the first 6 h trended (P≤0.1) or was significantly (Pb.05) less than control at 3%, 11% or 17% of time points (2.5, 50 and 200 mg PFPE/kg BW, respectively). Fig. 3 shows the daily changes from baseline over 5 days for MAP, SBP, DBP and HR for all doses of PFPE administered. A two-way ANOVA evaluating the daily effect of PFPE dose for each of 5 days found a significant dose effect for MAP (P=.01), SBP (Pb.001), DBP (Pb.001) and HR (Pb.001). No significant time or dose/time interaction was found. A one-way ANOVA evaluating change from baseline over 5 days following acute dosing found a significant reduction (Pb.0001) in MAP, SBP and DBP for all doses of PFPE in comparison to control (Fig. 3). However, the change from baseline for HR over 5 days found a significant reduction (Pb.0001) in the 50 and 200 mg PFPE/kg BW

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doses of PFPE in comparison to control, while the 2.5 mg PFPE/kg BW dose was not significantly different than control (Fig. 4).

3.2. Evaluation of PFPE components To determine the potential compound(s) contributing to the reduction in BP, SHR were fed components of PFPE proportional to 50 mg PFPE/kg, which was the lowest dose eliciting the maximal response. All animals consumed 96%–99% of the PFPE components administered within a 5-min period (data not shown). Baseline data for the control, PFPE, AF, EA, and GABA groups are shown in Table 2. No significant differences were found between groups for all parameters with the exception of HR for which EA was significantly higher than control. Data indicate that a single dose of 50 mg/kg BW PFPE, 2.39 mg/kg BW AF or 1.19 mg/kg BW EA significantly decreased (Pb.001) MAP, SBP or DBP in SHR over the 5 days posttreatment in comparison to the control group (Fig. 4). Only the AF and EA significantly (Pb.001) decreased the average change from baseline in HR over the 5 days posttreatment in comparison to control. Administration of GABA had no effect on any of the hemodynamic measurements (Fig. 5).

4. Discussion This study demonstrates that PFPE can significantly reduce BP and HR both acutely and chronically in SHR (a model that is known to have continuous increases in all BP measurements and HR as the animal ages). The dose response of MAP, SBP and DBP to PFPE within 6 h of consumption revealed a significant acute dosing effect between the 2.5 and 50 mg/kg BW groups. There was no additional BP response between the 50 and 200 mg/kg BW groups, indicating that the maximal reduction in BP after a single dose of PFPE occurred between 2.5 and 50 mg/kg BW. All three doses of PFPE caused acute reductions in HR that were not dose dependent. Five days after the single doses of PFPE, the control group demonstrated the normal progression of hypertension with a 4–5 mmHg increase in blood pressure as would be expected in the SHR model [16,17]. Concurrently, 2.5 mg/kg PFPE blunted this elevation in MAP, SBP and DBP, while 50 and 200 mg/kg PFPE prohibited the elevation in MAP, SBP and DBP. The elevation in HR observed after 5 days in control group animals was also significantly reduced by 50 and 200 mg/kg PFPE. All parameters except HR were significantly decreased in the 2.5 mg/kg BW supplemental group 5 days following a single oral dose, indicating that a lower dose could potentially affect all parameters if ingested for an extended period. This hypothesis is supported by Zibadi et al. who supplemented female SHR with PFPE in the diet (50 mg/kg diet) for 8 weeks and demonstrated a significant reduction in SBP by the end of the study. Although no BWs were reported in this study, food intake was provided, allowing for the extrapolation of the dose consumed based upon the mg PFPE/kg BW/day. Utilizing the average BW of the SHR in this study, the dose of PFPE in the Zibadi et al. study was approximated to be 2 mg/kg BW/day. This was just below our acute dose of 2.5 mg/kg BW. Table 1 Average baseline values by dose for MAP, SBP, DBP and HR PFPE dose (mg/kg BW) 0

MAP SBP DBP HR

2.5

50

200

Mean

S.E.M.

Mean

S.E.M.

Mean

S.E.M.

Mean

S.E.M.

141.7 169.9 113.5 310.7

2.1 2.2 1.9 6.5

140.7 167.5 113.9 315.9

3.6 3.8 3.2 8.9

148.7 177.1 120.3 320.0

3.2 3.5 2.9 11.0

148.0 176.0 120.4 324.1

2.6 2.4 2.5 7.6

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A 24

B 25

20

12

15

8 4 0 20

40

60

80

100 120 140 160 180 200 220 240 260 280 300 320 340 360

-4

Change from Baseline, mmHg

16

10

5

0 20

40

60

80 100 120 140 160 180 200 220 240 260 280 300 320 340 360

-5 -8 -10

-12 Time, minutes

-16

0 mg/kg BW

C

Time, minutes

-15

2.5 mg/kg BW

50 mg/kg BW

D

20

200 mg/kg BW

60 50

15

30

10

5

0 20

40

60

80

100 120 140 160 180 200 220 240 260 280 300 320 340 360

-5

Change from Baseline, BPM

Change from Baseline, mmHg

40

20 10 0 20

40

60

80

100 120 140 160 180 200 220 240 260 280 300 320 340 360

-10 -20 -30

-10 -40 -15

Time, minutes

-50

Time, minutes

Fig. 2. The change in MAP (A), SBP (B), DBP (C) and HR (D) from baseline over the first 6 h following acute supplementation of SHR with 0, 2.5, 50 or 200 mg/kg BW of PFPE.

B.J. Lewis et al. / Journal of Nutritional Biochemistry 24 (2013) 1359–1366

Change from Baseline, mmHg

20

A8

B

4

2

0 1

2

3

4

5

Change from Baseline, mmHg

6

-2

2

0 1

Time, Days

3

4

5

3

4

5

Time, Days

-4

0 mg/kg BW

2.5 mg/kg BW

50 mg/kg BW

C8

D

6

200 mg/kg BW

25

2

0 1

2

3

-2

4

5

Change from Baseline, BPM

20

4

15

10

5

0 1

-4

2

-2

-4

Change from Baseline, mmHg

4

B.J. Lewis et al. / Journal of Nutritional Biochemistry 24 (2013) 1359–1366

Change from Baseline, mmHg

6

8

Time, Days

-5

2

Time, Days 1363

Fig. 3. The change in MAP (A), SBP (B), DBP (C) and HR (D) from baseline by day following acute supplementation of SHR with 0, 2.5, 50 or 200 mg/kg BW of PFPE. Two-way ANOVA demonstrated a significant dose effect (Pb.05) but no significant difference in time or dose/time interaction.

B.J. Lewis et al. / Journal of Nutritional Biochemistry 24 (2013) 1359–1366

B

c

c

0

c

0

20 0

5 2.

50

-2

Dose, mg/kg BW

15 10

c b

5 0

20 0

c

Heart Rate a

50

2

a

2. 5

b

20

0

a

Change from Baseline, mmHg

D

6

0

Change from Baseline, mmHg

Dose, mg/kg BW

Diastolic Blood Pressure

4

d

-2

Dose, mg/kg BW

C

c 0

20 0

50

2. 5

-2

2

20 0

2

b 4

50

b

4

a

2. 5

a

Systolic Blood Pressure 6

0

Mean Arterial Pressure 6

0

Change from Baseline, mmHg

A

Change from Baseline, mmHg

1364

Dose, mg/kg BW

Fig. 4. The change in MAP (A), SBP (B), DBP (C) and HR (D) from baseline through 5 days following acute supplementation of SHR with 0, 2.5, 50 or 200 mg/kg BW of PFPE. Values represent means±S.E.M. Columns marked with different letters are significantly different than each other (Pb.05).

These findings combined with the dramatic reduction (~30 mmHg) observed in hypertensive humans supplemented with 400 mg PFPE/day (7 mg/kg BW) for 4 weeks support the idea that chronic treatment with 2.5 mg PFPE/kg BW or less may significantly reduce BP and HR [6]. Further studies to test this hypothesis will provide useful insight into determining the lowest potential dose that can be chronically given to a human to obtain cardiovascular benefits. After demonstrating maximal decreases in BP and HR with the 50 mg/kg BW dose, the components of PFPE postulated to be responsible for these significant effects were evaluated. Three components found in PFPE were evaluated to determine their effect on hemodynamic parameters: EA, AF and GABA. EA is a novel compound initially identified at a concentration of approximately 3% in PFPE. Due to the significant presence in the extract, the hemodynamic effects of EA were evaluated in the present study. The AF was found to be approximately 5% of the extract. As the most abundant class of compounds in the extract and coupled with the evidence that these compounds have an effect on BP [14,15], anthocyanin was chosen for evaluation. Finally, GABA, a potent sympathoinhibitory neurotransmitter with BP-lowering effects [18], was included in the study

Table 2 Average baseline values by test compound for MAP, SBP, DBP and HR Group Control

MAP SBP DBP HR a

PFPE

AF

EA

GABA

Mean

S.E.M.

Mean

S.E.M.

Mean

S.E.M.

Mean

S.E.M.

Mean

S.E.M.

138.0 162.5 112.6 293.8

5.2 8.2 2.3 5.9

141.5 171.6 112.6 309.6

2.0 2.6 1.4 12.8

141.5 169.5 114.1 321.7

3.2 3.3 3.0 6.0

139.6 165.7 113.3 324.7 a

4.5 5.7 3.9 3.2

143.2 161.0 126.1 293.3

2.3 13.0 10.1 5.8

EA significantly higher than control.

because it has been hypothesized by Ichimura et al. as an active ingredient in PFPE. As observed in the PFPE dose–response study, the control group had progressively increasing BP and HR in the 5 days postsupplementation, while the 50 mg/kg PFPE dose blunted the progression of hypertension. Both EA and the AF caused further reductions in cardiovascular parameters compared to the dose of the whole extract. Since these components were provided at the approximate doses found in the whole extract at the 50 mg PFPE/kg BW dose, there appears to be either a competitive inhibition for absorption or an interference/inhibition that reduces the effectiveness of the extract when administered as a whole. SHR treated with GABA demonstrated similar BP and HR responses as control animals, indicating that GABA is not a major contributor of PFPE responsible for the reduction of cardiovascular parameters in this study. The current study was designed to identify the hemodynamic effects of various doses of PFPE and its components. Our observation that a single dose of PFPE had an effect on blood pressure and HR that lasted 5 days is intriguing. However, identifying the mechanism of this effect was beyond the scope of the study. Based on the etiology and suspected causes of essential hypertension which is modeled in the SHR, we speculate that PFPE is eliciting its hypotensive effect by diminishing activation of the sympathetic nervous system. While this mechanism seems to be the common pathway to essential hypertension, other potential targets for PFPE are the variables that can enhance sympathetic activation such as angiotensin II, inflammation and oxidative stress. Both the EA and the AF fractions of PFPE had a significant hypotensive effect in SHR, and we propose that these compounds are likely contributing to PFPE's cardiovascular effects. The absorption, bioavailability and bioactivity of anthocyanins and EA are not clearly understood [19], but may be responsible for the long-term effect of one dose of PFPE.

B.J. Lewis et al. / Journal of Nutritional Biochemistry 24 (2013) 1359–1366

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Fig. 5. The change in MAP (A), SBP (B), DBP (C) and HR (D) from baseline through 5 days following acute supplementation of SHR with either 0 mg/kg (control), 50 mg/kg BW of PFPE or components of PFPE. Components evaluated were AF, EA or GABA. These components were administered at levels equivalent to those present in the 50 mg/kg BW PFPE. Values represent means±S.E.M. Columns marked with different letters are significantly different than each other (Pb.05).

Anthocyanins have been identified as potent cardioprotective mediators. The mechanisms by which anthocyanins elicit their cardioprotective effects are the same pathways that inhibit the sympathetic nervous system [20]. Less is known about the EA mechanisms to elicit their cardioprotective effect; however, it is feasible that EA is also functioning through these pathways. This study confirms the ability of PFPE to reduce SBP and extends the current body of evidence that PFPE also decreases MAP, DBP and HR following acute administration. In addition, these studies indicate that the EA and AF of PFPE may be responsible for contributing to the cardiovascular benefits observed following administration. Further research will be needed to determine the minimum effective dose in humans and to elucidate the potential mechanisms of action. These results, obtained using a common animal model for hypertension, support the concept that certain phytochemicals present in PFPE can have potent vascular effects. Mechanistic studies aimed at evaluating the effects of these phytochemicals both alone and in combinations are warranted. Acute and chronic studies with human subjects are needed to determine the relevance of the experimental animal data with respect to the putative value of PFPE for blood pressure control in at-risk as well as healthy human subjects. Studies addressing the safety of PFPE represent another important area of future research.

Acknowledgments We would like to thank Prakash Bhosale, Ph.D., Tatania Emmick, Carrie Wray, John Warfield and Chengjian Liu, Adam (Billy) Quang and Renae Scroggins (Kemin Health) for their contributions in manufacturing the PFPE and isolating the fractions used for

evaluation in this study. We would also like to thank Jim Rogers and Mark Jaros from Summit Analytical for their contribution to the statistical analysis of the data in the PFPE dose–response study.

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