The effect of dietary factors on strawberry anthocyanins oral bioavailability

Di Xiao a, Amandeep Sandhu a, Yancui Huang a, Eunyoung Park a, Indika Edirisinghe a and Britt M. Burton-Freeman *ab
aCenter for Nutrition Research, Institute for Food Safety and Health, Illinois Institute of Technology, IL, USA. E-mail: bburton@iit.edu; bbfreeman@ucdavis.edu
bDepartment of Nutrition, University of California, Davis, CA, USA

Received 15th June 2017 , Accepted 8th September 2017

First published on 5th October 2017


Strawberries are a dietary source of anthocyanins, particularly pelargonidin glycosides. Dietary anthocyanins have received increasing attention among researchers and consumers due to their health benefits. The oral bioavailability of anthocyanins is reported to be low and various dietary factors may influence their oral bioavailability further. Milk is suggested to reduce (poly)phenols’ oral bioavailability. However, the effect of milk on anthocyanin oral bioavailability remains uncertain. Likewise, mixed nutrient meals may influence the oral bioavailability of anthocyanins. Therefore, the purpose of this study was to assess the effect of milk on the oral bioavailability and other pharmacokinetic (PK) variables of strawberry anthocyanins consumed with and without a meal. Nine healthy participants consumed a strawberry beverage prepared in milk or water with a standard meal on two occasions. On two additional occasions, the beverages were given to a subset (n = 4) of participants to determine the impact of the meal on anthocyanin PK variables, including oral bioavailability. Independent of the meal, beverages prepared in milk significantly reduced the peak plasma concentrations (Cmax) of pelargonidin-3-O-glucoside (P-3-G), pelargonidin-glucuronide (PG) and pelargonidin-3-O-rutinoside (P-3-R), as well as the PG and P-3-R area under the curve (AUC) (p < 0.05) compared to beverages prepared in water. Milk did not influence the oral relative bioavailability of pelargonidin anthocyanins under meal conditions; however, the oral relative bioavailability of pelargonidin anthocyanins was reduced by ∼50% by milk under without meal conditions (p < 0.05). Consuming strawberry beverages made with milk and consuming those made with water with and without a meal influenced different aspects of strawberry anthocyanin PKs. The significance of this effect on clinical efficacy requires additional research.


Introduction

Strawberries (Fragaria spp.) are a rich source of polyphenols, primarily anthocyanins, which are responsible for their red colour. Epidemiological and clinical studies have shown health benefits associated with consumption of anthocyanins derived from strawberries, including reduced risk of chronic diseases such as type 2 diabetes and cardiovascular diseases.1–4 The health benefits of anthocyanins are suggested to be associated with their oral bioavailability and other kinetic behaviors, such as peak concentration and exposure of anthocyanins or their metabolites to peripheral tissues.5–8 The oral bioavailability of dietary anthocyanins that maintain their parent C6–C3–C6 structure is reported to be relatively low and several dietary factors may impact it further.9

Strawberries are one of the most commonly consumed berries. They may be eaten with meals, before or after meals as a snack or in formulated products such as yogurt, milk-based beverages and smoothies. When consumed with other foods or in formulated products, coexisting nutrients, such as proteins, fats and carbohydrates may interfere with the oral bioavailability of strawberry anthocyanins or alter their PK profile. Among the studies that have been conducted to test the effects of dietary factors on the oral bioavailability and PK profiles of polyphenols, alterations in these variables have been reported with milk. Mullen et al. (2009) reported that milk hinders the absorption and excretion of cocoa flavan-3-ols,10 and Serafini et al. found suppressed absorption of caffeic and ferulic acid when blueberries were consumed with milk compared to blueberries consumed with water.11 In a study with strawberries served with and without cream, which is almost exclusively fat, oral bioavailability was not significantly different, however, the peak in pelargonidin-3-O-glucuronide, a major strawberry anthocyanin metabolite, was delayed by more than 1 hour (h).12 In contrast, a recent in vitro digestion study by Cebaci et al. reported that whole or skim milk decreased the total phenolic content of blueberries as well as an oatmeal and blueberry mix, but does not impact the potential bioavailability measured by a co-digestion assay.13

Previous studies conducted by our research group have shown favorable health effects acutely when strawberry was consumed as part of a milk-based beverage (compared to the control beverage with no strawberry) with a high carbohydrate and high fat challenge meal.14–16 Moreover, the health effects were positively correlated with pelargonidin-3-O-glucoside and its conjugated metabolite (pelargonidin glucuronide) in plasma.17–20 However, anthocyanin content and antioxidant capacity were lower in the milk-based strawberry beverage than those in a water-based strawberry beverage.21 Therefore, we were interested in determining whether the reduction of detectable anthocyanin content in the milk-based beverage would result in altered PK variables when consumed with a meal compared to consuming the same amount of strawberry in a water-based beverage. Because the meal itself may influence PK outcomes, as suggested by one of our recent publications showing that the oral bioavailability of anthocyanins from strawberry (prepared in water only) was higher when consumed alone than that when consumed with a meal,22 we conducted a subset study with the milk-based and water-based beverages without the meal.

Materials and methods

Study design

All procedures in this study were approved by the Institutional Review Board at the Illinois Institute of Technology (Illinois Tech, Chicago, IL, USA). Written informed consent was obtained from all study participants before beginning any study procedure. This was a single-center, randomized, single blinded, cross over study. The first part of the study was to investigate the PK profile, including the oral bioavailability of strawberry anthocyanins when strawberry was consumed with milk or without milk along with a meal (Meal-STR-Milk and Meal-STR-Water, respectively). To assess the potential effect of the meal, a subset of participants were asked to participate in the second part of the study in which they consumed only the strawberry milk beverage (STR-Milk) or strawberry water beverage (without milk) (STR-Water) on two different occasions. A study diagram is given in Fig. 1. The study is registered with ClinicalTrial.gov, registration number, NCT01908634.
image file: c7fo00885f-f1.tif
Fig. 1 Study progress. Flow diagram of participants in the study served milk based strawberry drink (STR-Milk) and cross over to water based strawberry drink (STR-Water), with (Part 1 of the study) and without (Part 2 of the study) meal.

Study subjects

Relatively healthy individuals were screened and recruited for the study at the Clinical Nutrition Research Center (CNRC) at Illinois Tech. Inclusion criteria for the study were as follows: men and women between 18–78 years and in relatively good health. Subjects who met these inclusion criteria but were not eligible to participate were: those who were currently smoking or past smokers with abstinence <2 years; subjects who had allergies or intolerances to study foods; those who consumed more than 2 servings per day of berries; had a fasting blood glucose ≥126 mg dL−1; those who were taking medications or supplements that may interfere with the study procedures or endpoints; those who reported unusual dietary habits, e.g., pica; those who were actively losing weight or trying to lose weight (unstable body weight fluctuations of >5 lbs in a 60 day period); and those who were excessively exercising or people who are trained athletes or in training. In addition, subjects indicating addiction or suspected to be addicted to drugs and/or alcohol or who met criteria for severe obesity as defined by >39.9 kg m−2 Body Mass Index (BMI) or who were underweight for height (BMI <18.5 kg m−2) were also not eligible to participate in the study. A total of 10 subjects were enrolled and randomized and 9 completed the study. One subject dropped out due to schedule conflicts.

Study treatment

In Part 1 of the study, subjects received a strawberry beverage along with a standard breakfast meal typically used in our laboratory (Meal-STR-Milk or Meal-STR-Water). The strawberry beverage was prepared using 12 g of freeze-dried strawberry powder (equivalent to 1 cup fresh weight, provided by the California Strawberry Commission, Watsonville, CA). The ingredients used in the preparation of the beverages and meals are listed in Table 1. The Meal-STR-Water beverage was prepared by mixing the strawberry powder with water, whereas the Meal-STR-Milk beverage had additional ingredients that matched our recipes from previous studies.14 Both beverages made up a total quantity of 307 g. The breakfast meal served to the subjects was a high-carbohydrate and moderate-fat meal consisting of a croissant with butter and jelly, sausage links, and corn flakes and frosted-flakes cereal with whole milk. The nutritional composition of Meal-STR-Milk and Meal-STR-Water were matched in terms of total energy (1071.6 vs. 1077.4 kcal, respectively) (Table 2). Energy from total fat, carbohydrates, and protein are matched as well.
Table 1 Ingredients of STR-Water and STR-Milk beverage and meals served with STR-Milk and with STR-Water
Ingredienta Without meal With meal
STR-Water STR-Milk Meal-STR-Water Meal-STR-Milk
a Freeze-dried strawberry powder was provided by California Strawberry Commission, Watsonville, CA, USA. All other foods were purchased from a local store. General mills frozen croissants (Pillsbury Frozen Baked); butter (Sweet Salted Half Sticks, LAND O LAKES); apple jelly (Essential Everyday (Jewel) Supervalu Inc. Eden Prairie, MN, USA); Pork sausage links original (Bob Evans Farms, Inc., New Albany, OH, USA); Frosted flakes cereal (Kellogg Sales Co. Battle Creek, MI, USA); Corn flakes cereal (Kellogg Sales Co. Battle Creek, MI, USA); Whole milk (Dean's), sugar (Domino Foods, Yonkers, NY, USA); Nonfat dry milk (Essential Everyday, Supervalu Inc. Eden Prairie, MN, USA), Nesquik powder, strawberry flavored (Nestlé USA, Inc., Glendale, CA, USA).
Meal
Frozen baked croissant (g) 56 56
Butter (g) 4 2
Apple jelly (g) 26 12
Pork sausage links (g) 60 65
Frosted flakes cereal (g) 20 12
Corn flakes cereal (g) 35 38
Whole milk (g) 170 170
Sugar (g) 24 0
Non-fat dry milk (g) 22 0
Total quantity (g) 417 355
 
Beverage
Freeze-dried strawberry powder (g) 12 12 12 12
Nesquik powder, strawberry flavored (g) 0 34 0 34
Non-fat dry milk (g) 0 21 0 21
Sugar (g) 0 3 0 3
Water (g) 295 237 295 237
Total quantity (g) 307 307 307 307


Table 2 Nutritional composition of beverages (STR-Milk and STR-Water) and meal with beverages (Meal-STR-Milk and Meal-STR-Water)a
Treatment Calories (kcal) Protein (g) Carbohydrate (g) Fat (g)
a Nutrient analysis by Food Processor SQL Edition (version 10.6.0, ESHA Research, Salem, OR).
STR-Water 42.3 0.9 9.9 0.6
STR-Milk 261.9 9.3 56.1 0.6
Meal-STR-Water 1077.4 31 143.6 43.2
Meal-STR-Milk 1071.6 32.1 140.2 43.4


In Part 2 of the study, subjects received only the strawberry beverages (STR-Milk or STR-Water) prepared in the same way as the beverages served in the Part 1 of the study but without the meal.

Test day visit

Eligible participants followed a low polyphenol diet 3 days before each test day visit (3 days run-in), while maintaining their usual diet and physical activity patterns. The subjects were asked to have a similar dinner meal the night before each test day. The subjects were asked to keep food diaries for 3 consecutive days prior to each test day for confirmation of the subjects’ compliance with the low polyphenol diet and maintenance of consistency in dietary patterns, including the dinner meal the night before each test day. On each test day, participants came to the center after fasting overnight (∼10 h) on different occasions at least 3 days apart. After general health evaluation (anthropometrics measurements, vital signs and blood glucose check) and dietary compliance review, a catheter was placed in the subjects’ non-dominant arm followed by a baseline (fasting) blood draw (t = 0 h).

Thereafter, in Part 1 of the study, the subjects were provided with the standardized breakfast meal and one of two beverages (Meal-STR-Milk or Meal-STR-Water) based on a computer generated randomization sequence. The subjects were instructed to consume the entire beverage and breakfast in 15 minutes (min), at which time a second blood sample was collected. Then, blood samples were collected at 0, 0.25, 0.5, 1, 1.5, and 2 h, and hourly until 6 h.

A subset of subjects (n = 4) who participated in Part 2 of the study were provided only with the study beverages (STR-Milk or STR-Water) after the catheter placement and fasting blood draw. Study procedures were identical to Part 1 of the study except that no meal was served with the beverages and blood collection ended at 3 h post consumption of the beverages.

Blood samples were collected in vacutainers containing ethylenediaminetetraacetic acid and immediately placed on ice until centrifuged (within 1 h). After centrifugation at 453g for 15 min at 4 °C, plasma was aliquoted into individual cryovials and stored at −80 °C until analysis. The study was completed in Oct, 2013 and samples were analysed in Sep 2015.

Analysis of anthocyanins/metabolites in plasma

Analytical standards of cyanidin-3-O-glucoside (C-3-G), cyanidin-3-O-rutinoside (C-3-R), pelargonidin-3-O-glucoside (P-3-G), pelargonidin-3-O-rutinoside (P-3-R) and malvidin-3-O-glucoside (M-3-G) were purchased from Extrasynthese (Genay, France). All the solvents and reagents were High-Performance Liquid Chromatography (HPLC) grade. Acetonitrile, methanol, and formic acid were purchased from Fisher Scientific (Houston, TX). Bond Elut Plexa cartridges (3 ml) for solid-phase extraction (SPE) were from Agilent Technologies (Santa Clara, CA). Duplicate plasma samples were extracted using SPE methodology. Briefly, plasma samples (500 μL) were thawed on ice and mixed with an internal standard (M-3-G, 10 ng mL−1) to adjust for extraction losses. The samples were diluted with 1.5 mL of acidified water (1% formic acid), loaded on the cartridges pre-conditioned with 3 mL acidified methanol (1% formic acid) and washed with 3 mL of acidified water (1% formic acid). After loading the diluted sample, the SPE cartridges were washed with 1.5 mL of acidified water (1% formic acid). Anthocyanins were eluted with 1.5 mL of acidified methanol (1% formic acid). The eluted liquid was evaporated to dryness under nitrogen at room temperature (25 °C). The dried samples were dissolved in 5% acetonitrile (containing 1% formic acid), sonicated for 5 min and centrifuged at 18[thin space (1/6-em)]514g at 4 °C for 10 min. The supernatant was transferred to amber HPLC vials and injected for analysis. The samples were analyzed on an Agilent 1290 Infinity UHPLC system with a 6460 Triple Quadrupole Mass Spectrometer (Agilent Technologies, Santa Clara, CA). The system was equipped with a binary pump with an integrated vacuum degasser, an auto sampler with a thermostat, and a column compartment with a thermostat. A Poroshell 120 Stablebond C18 column (2.1 mm × 150 mm, 2.7 micron) was used for separation with a flow rate of 0.3 mL min−1 at a constant temperature of 30 °C. The mobile phase used for the separation of compounds was water with 1% formic acid (A) and acetonitrile (B). The injection volume was 5 μL. The gradient consisted of 5% B at the beginning (0 min), increasing to 15% B at 10 min; followed by 20% B at 15 min; 30% B at 18 min; and 90% B at 20 min, reaching back to the final condition of 5% B at 22 min. A post time of 5 min was used to re-equilibrate the column back to the initial conditions. The analysis was carried out using a full MS scan followed by a MS2 product ion scan with multiple reactions monitoring (MRM) by monitoring specific transitions of the parent and product ions. Analysis of anthocyanins utilized positive ion mode with capillary voltage of 4500 V and drying gas flow rate of 10 L min−1 at 250 °C.

Commercially available reference standards of C-3-G, C-3-R, P-3-G and P-3-R were prepared in blank plasma and optimized for collision energies and MRM transitions using a Mass Hunter Optimizer. The MRM transitions used for quantifying the compounds were: 433–271 for P-3-G and 579–271 for P-3-R. The MRM transition for pelargonidin glucuronide (PG) (447–271) was based on an analysis conducted in our previous study.23 Due to unavailability of a PG reference standard, PG was quantified using P-3-G.

Pharmacokinetic analysis of anthocyanins in plasma

Maximum plasma concentration of the metabolites was defined as Cmax, from 0 to 6 h post consumption of strawberry beverage for Part 1 (beverage with meal), and 0 to 3 h post consumption for Part 2 (beverage without meal), respectively. The time to reach maximum plasma concentration (Tmax) was defined as the time in hours at which Cmax was achieved. Total area under the concentration time curve (AUC) was calculated by the linear trapezoidal method using Microsoft Excel 2013 v.15.24 The oral relative bioavailability of anthocyanins was based on the parent compounds and metabolites maintaining the parent flavonoid structure. Therefore the oral relative bioavailability of PG was measured by intake of P-3-G in the beverage. Calculations of plasma mass were based on compound/metabolite AUC for 3 and 6 h (Part 1: AUC0–3 h and AUC0–6 h, Part 2: AUC0–3 h; nmol h L−1) corrected for individual plasma volume based on sex and body weight (kg) according to the methods described in the technical manual of American Association of Blood Banks (AABB)25 where plasma volume is estimated to be 36 mL kg−1 for women and 40 mL kg−1 for men. Oral relative bioavailability was calculated by dividing the plasma mass (nmol, measured by calculating the area under the concentration–time curve) by the mass of anthocyanin/metabolite per drink (nmol) and multiplying by 100.

Statistical analysis

Subject characteristics were analyzed using descriptive statistics and tabulated. The results of the statistical analysis are presented as mean ± standard error of the mean (SEM). Data in Part 1 (meal & beverage) were analyzed by repeated measures (RM) ANOVA using the mixed procedure of SAS 9.3 (SAS Institute Inc., Cary, NC) with treatment and time as the main effects and the subject as the blocking variable to test the main effects of treatment (Meal-STR-Water & Meal-STR-Milk), time (h) and treatment–time interaction. The Kenward–Roger correction and the method of restricted maximum likelihood were used in all mixed models.26–28 All efficacy variables were first examined for normality by Shapiro–Wilk's tests and data not conforming to normal distribution patterns were log transformed prior to analysis. To determine the difference between treatments on Cmax, Tmax and total AUC variables, paired data were analyzed using two-tailed Student's t tests and one-way ANOVA followed by a Tukey–Kramer adjusted t-test of SAS 9.3 (SAS Institute Inc., Cary, NC) for appropriate comparisons. Treatments in Part 2 (STR-Water & STR-Milk) were compared on Cmax, Tmax, AUC0–3 h and oral bioavailability by paired-two-tailed Student's t test with Microsoft Excel software 2013. A p < 0.05 was considered to be of statistical significance.

Result

Subject demographics

Nine subjects completed Part 1 (beverage with meal) and a subset of 4 completed Part 2 (beverages without meal). The subjects were relatively young individuals with a mean age and BMI of 24 ± 2 years (y) and 22 ± 2 kg m−2 (Part 1) and 25± 2 years and 22 ± 2 kg m−2 in Part 2. Baseline subject characteristics are listed in Table 3.
Table 3 Subject demographic characteristics at screening visit
Variable Part 1: Beverage with meal (n = 9) Part 2: Beverage without meal (n = 4)
Data are mean ± standard deviation, BMI = body mass index, His = Hispanic, Cau = Caucasian, AA = African American.
Age (years) 24 ± 2 25 ± 2
Weight (kg) 67 ± 9 68 ± 7
BMI (kg m−2) 22 ± 2 22 ± 2
Male[thin space (1/6-em)]:[thin space (1/6-em)]female 8[thin space (1/6-em)]:[thin space (1/6-em)]1 4[thin space (1/6-em)]:[thin space (1/6-em)]0
Ethnicity/race (Asian[thin space (1/6-em)]:[thin space (1/6-em)]His[thin space (1/6-em)]:[thin space (1/6-em)]Cau[thin space (1/6-em)]:[thin space (1/6-em)]AA) 6[thin space (1/6-em)]:[thin space (1/6-em)]1[thin space (1/6-em)]:[thin space (1/6-em)]1[thin space (1/6-em)]:[thin space (1/6-em)]1 4[thin space (1/6-em)]:[thin space (1/6-em)]0[thin space (1/6-em)]:[thin space (1/6-em)]0[thin space (1/6-em)]:[thin space (1/6-em)]0


Analysis of anthocyanins in test beverages

Table 4 shows the content of strawberry anthocyanins identified and quantified in STR-Water and STR-Milk beverages. Four anthocyanins were identified: P-3-G, P-3-R, C-3-G and C-3-R. The most abundant anthocyanin was P-3-G in both beverages, contributing 80.4% and 79.6% of the total anthocyanins content in STR-Water and STR-Milk respectively, followed by P-3-R (10.8%, 11.4%) and C-3-G (8.5%, 8.7%) and C-3-R (0.3%, 0%). The total concentration of pelargonidin-based anthocyanins was 130.9 ± 1.2 μmol in the STR-Water beverage, and 117.5 ± 2.8 μmol in the STR-Milk beverage.
Table 4 Anthocyanin contents of test beverages
Anthocyanins STR-Water (μmol per drink) STR-Milk (μmol per drink)
Data are mean ± standard deviation for three replicates.
Cyanidin-3-O-glucoside 12.2 ± 0.3 11.3 ± 0.2
Cyanidin-3-O-rutinoside 0.4 ± 0.0 0.4 ± 0.0
Pelargonidin-3-O-glucoside 115.5 ± 1.2 102.8 ± 2.6
Pelargonidin-3-O-rutinoside 15.5 ± 0.1 14.7 ± 0.2
Total anthocyanins 143.6 ± 1.6 129.2 ± 3.0


Part 1: Strawberry beverage served with meal – postprandial plasma anthocyanin concentrations

No anthocyanins were detected in baseline (t = 0 h) plasma samples. After consumption of the meal plus beverages (Meal-STR-Water and Meal-STR-Milk), four compounds were detected in the plasma samples, including three parent anthocyanins (C-3-G, P-3-G and P-3-R) and one conjugated metabolite, PG (Fig. 2). C-3-R was not detected in the plasma as it was below the detection level, C-3-G (limit of detection of 2.5 pg mL−1) was present in only trace amounts and could not be quantified accurately as it was below the quantification levels. As illustrated in Fig. 2, regardless of the treatment, peak plasma concentrations of PG, P-3-G and P-3-R were achieved between 2–4 hours after consumption of the meal. The concentrations of compounds declined with time, but remained detectable at 6 h. A significant treatment effect was observed for P-3-R, suggesting that consuming strawberry in milk with a meal reduced the plasma concentrations of P-3-R; however this was not the case for P-3-G (p = 0.7) or PG (p = 0.06). A significant time–treatment interaction was observed for postprandial plasma concentration of PG, P-3-G and P-3-R (p < 0.05).
image file: c7fo00885f-f2.tif
Fig. 2 Mean (±SE) concentration of (A) pelargonidin glucuronide, (B) pelargonidin-3-O-glucoside and (C) pelargonidin-3-O-rutinoside in plasma of subjects at each monitored time point (0–6 hours) after consumption of meal along with 307 mL of strawberry beverage (containing 12 g of freeze dried strawberry powder) prepared in water (Meal-STR-Water (- - -) or prepared in milk (Meal-STR-Milk (—), n = 9. Statistical analysis using the ANOVA/MIXED procedure of SAS 9.3 to test the main effects of the strawberry treatment (Meal-STR-Water and Meal-STR-Milk), time (h), and time–treatment interaction is shown on the right-hand side of each figure. NS: not significant. X, significant differences (p < 0.05).

The pharmacokinetic analyses of anthocyanins/metabolites are given in Table 5. The highest plasma concentration value (Cmax) was observed with PG while the lowest value was observed with P-3-R. The Cmax of PG, P-3-G and P-3-R were significantly lower (p < 0.05) after the Meal-STR-Milk conditions than those of the Meal-STR-Water conditions. No significant difference was observed on time to reach peak plasma concentration (Tmax) between treatments. The area under the curve (AUC) was calculated to provide information about exposure. The AUC0–6 h of PG and P-3-R were significantly reduced (p < 0.05) when strawberry was consumed in the milk-based beverage with a meal compared to the water-based beverage with a meal. No significant effect was observed in the AUC0–6 h of P-3-G.

Table 5 Pharmacokinetic parameters of pelargonidins/metabolites in plasma (0–6 hours) after healthy participants consumed test beverages with meal
Anthocyanins/metabolites C max (nmol L−1) T max (h) AUC0–6 h (nmol h L−1)
Meal-STR-Water Meal-STR-Milk Meal-STR-Water Meal-STR-Milk Meal-STR-Water Meal-STR-Milk
C max: maximum content in time period of 0–6 h; Tmax: time point when Cmax was achieved; AUC: area under curve derived by trapezoidal method. a, b[thin space (1/6-em)]Different letters in each row indicate significant differences in numbers in Cmax (p < 0.05). y, z[thin space (1/6-em)]Different letters in each row indicate significant differences in numbers in AUC0–6 h.
Pelargonidin glucuronide 11.3 ± 2.7a 7.6 ± 1.5b 3.8 ± 0.5 3.6 ± 0.6 44.4 ± 11.2y 30.3 ± 6.4z
Pelargonidin-3-O-glucoside 4.9 ± 0.5a 4.2 ± 0.6b 2.7 ± 0.2 2.2 ± 0.3 18.8 ± 1.9 17.6 ± 1.9
Pelargonidin-3-O-rutinoside 0.4 ± 0.0a 0.3 ± 0.0b 3.4 ± 0.4 3.8 ± 0.5 1.3 ± 0.2y 1.1 ± 0.2z


The oral relative bioavailability of pelargonidins and their conjugated metabolites maintaining the C6–C3–C6 structure was determined using both 3 and 6 h AUC, the former (AUC0–3 h) to compare with meal and without meal conditions. No differences in oral relative bioavailability were observed for P-3-G between treatments; however, the oral relative bioavailability of the P-3-R was significantly lower after the Meal-STR-Milk than that after the Meal-STR-Water (p < 0.05, Table 7, oral bioavailability (0–6 h)). The oral relative bioavailability for total pelargonidin based parent compounds and metabolites was ∼0.20% ± 0.04 (p > 0.05).

Table 6 Pharmacokinetic parameters of anthocyanins/metabolites in plasma (0–3 h) after healthy participants consumed strawberry beverages alone (no meal)
Anthocyanins/metabolites C max (nmol L−1) T max (h) AUC (nmol h L−1)
STR-Water STR-Milk STR-Water STR-Milk STR-Water STR-Milk
C max: maximum content in time period of 0–3 h; Tmax: time point when Cmax was achieved; AUC: area under curve derived by trapezoidal method. a, b[thin space (1/6-em)]Different letters in each row indicate significant differences in numbers in Cmax (p < 0.05). y, z[thin space (1/6-em)]Different letters in each row indicate significant differences in numbers in AUC0–3 h.
Pelargonidin glucuronide 22.1 ± 3.8a 8.7 ± 0.7b 1.0 ± 0.2 1.8 ± 0.3 35.8 ± 4.1y 16.0 ± 1.4z
Pelargonidin-3-O-glucoside 6.3 ± 0.9a 4.0 ± 0.6b 1.0 ± 0.2 1.4 ± 0.2 10.7 ± 1.3 7.7 ± 0.3
Pelargonidin-3-O-rutinoside 0.6 ± 0.1a 0.3 ± 0.0b 1.5 ± 0.5 1.6 ± 0.6 1.0 ± 0.2y 0.5 ± 0.0z


Table 7 Oral relative bioavailability of anthocyanins/metabolites in plasma under meal & beverage (0–6 h, and 0–3 h) and beverage only (0–3 h) conditions
  Part 1: Meal & beverage Part 2: Beverage only
% Bioavailability (0–6 h) % Bioavailability (0–3 h) % Bioavailability (0–3 h)
Meal-STR-Milk Meal-STR-Water Meal-STR-Milk Meal-STR-Water STR-Milk STR-Water
a, bDifferent letters in each row indicate significant differences in numbers in each variable p < 0.05.
Pelargonidin glucuronide 0.08 ± 0.02 0.11 ± 0.03 0.04 ± 0.01 0.04 ± 0.01 0.04 ± 0.01a 0.08 ± 0.00b
Pelargonidin-3-O-glucoside 0.05 ± 0.00 0.04 ± 0.00 0.02 ± 0.00a 0.03 ± 0.01b 0.02 ± 0.0 0.02 ± 0.0
Pelargonidin-3-O-rutinoside 0.02 ± 0.00a 0.02 ± 0.00b 0.01 ± 0.00 0.01 ± 0.00 0.01 ± 0.0 0.02 ± 0.0
Total pelargonidin 0.15 ± 0.02 0.17 ± 0.03 0.07 ± 0.01 0.07 ± 0.02 0.07 ± 0.0b 0.12 ± 0.0a


The oral relative bioavailability using AUC0–3 h data to compare with meal conditions and without meal conditions showed that the oral bioavailability of pelargonidins accounting for metabolism to PG were higher after the STR-Water conditions than that after the Meal-STR-Water conditions (p < 0.05).

Part 2: Strawberry beverage served without a meal – postprandial plasma anthocyanin concentrations

After the consumption of beverages only (STR-Water and STR-Milk), P-3-G, P-3-R and PG were detected and quantified in plasma samples (Fig. 3). The anthocyanins/metabolite concentrations increased faster and reached significantly higher Cmax after intake of STR-Water compared with the STR-Milk group (p < 0.05, Table 6). As shown in Table 6, the highest Cmax values were obtained for PG while the lowest values were observed for P-3-R. A trend of delayed Tmax of P-3-G in the STR-Milk group compared to the STR-Water group was observed, but due to subject-to-subject variation, the Tmax were not statistically significant (p = 0.06). The AUC0–3 h values of PG and P-3-R were significantly lower (p < 0.05) when strawberry was consumed with milk (STR-Milk) compared to water (STR-Water). No effect was observed in the AUC0–3 h of P-3-G. The relative bioavailability of pelargonidin anthocyanins as determined by their metabolism to PG was significantly reduced when strawberry was consumed with milk (PG: 0.04% ± 0.01), compared to strawberry intake with water (PG: 0.08% ± 0.00) (p < 0.05), which is consistent with calculations for total pelargonidin oral bioavailability of ∼0.08% vs. 0.13%, STR-Milk vs. STR-Water, respectively, p < 0.05.
image file: c7fo00885f-f3.tif
Fig. 3 Mean (±SE) concentration of (A) pelargonidin glucuronide, (B) pelargonidin-3-O-glucoside and (C) pelargonidin-3-O-rutinoside in plasma of subjects at each monitored time point after consumption of 307 mL of strawberry beverage (containing 12 g of freeze dried strawberry powder) prepared in water (STR-Water (- - -)) or prepared in milk (STR-Milk (—)).

Discussion

The purpose of this study was to assess whether the milk-based strawberry beverage would have an influence on PK variables when consumed with a meal compared to consuming the same amount of strawberry in a water-based beverage with a meal.

In this study, we quantified 4 anthocyanins including P-3-G, P-3-R, C-3-G and C-3-R in strawberry beverages. The total anthocyanins were about 10% higher in the water based beverage compared to the milk based beverage. This difference may have been due to small differences in the pH of the beverages (3.5 in water and 4.8 in milk) influencing anthocyanin stability/degradation or association with milk proteins rendering them undetectable. Several studies have reported that anthocyanin degradation was not significant at pH ≤ 5.0 in blueberry, black carrot and mixed anthocyanin solutions,29–31 suggesting complexation with milk proteins or other constituents may have been responsible for the 10% difference.

Plasma samples obtained after consumption of strawberry beverages contained P-3-G, P-3-R and C-3-G and PG, independent of meal intake. C-3-R was not detected in plasma samples. In addition, C-3-G in plasma samples was present in only trace amounts and could not be quantified accurately. The limited ability to quantify C-3-G may have been due to its minor contribution to total anthocyanins in strawberries (∼9% of total anthocyanins). Age, genetic factors and ethnicity may also affect the absorption and excretion profiles of anthocyanins in humans.32 Therefore, the anthocyanins discussed in this paper are pelargonidin-based anthocyanins, which were reported to be the most abundant form of anthocyanins present in strawberry.14 PG was the main anthocyanin metabolite detected in plasma after strawberry beverage consumption.23

Peak plasma concentrations (Cmax) of strawberry pelargonidin glycosides (P-3-G, P-3-R) and their primary conjugated metabolite (PG) were significantly reduced after ingestion of the milk-based strawberry drink compared to those after ingestion of the water-based strawberry drink under the “with meal” and “without meal” conditions. Further, milk reduced plasma exposure (expressed by AUC0–6 h) to PG and P-3-R, whether ingested with and without a meal. Several reports indicated that milk impairs the absorption of phenolics in humans. Serafini et al. (2009) reported reduced Cmax of caffeic and ferulic acids in plasma, as well as a significant reduction of caffeic acid absorption overall, when blueberries were consumed with whole milk.11 Additionally, peak concentrations of plasma flavan-3-ols were significantly lower after healthy male subjects consumed tea with milk than those after consuming black tea alone.33 The inhibitory effects of milk may be due to interactions among nutrients, and in the present study, the interaction of anthocyanins and milk proteins. Polyphenols can bind to proteins via hydrogen bonding involving polar groups and hydrophobic interactions involving non-polar aromatic rings of polyphenols and aromatic amino acids of proteins.34–41 Milk caseins34 and β-lactoglobulin42 are suggested to be interfering agents by creating a binding complex between milk protein and anthocyanins. Arroyo-Maya et al. (2016)43 indicated that hydrophobic interactions accounted for the binding of pelargonidin to β-lactoglobulin. In contrast, milk caseins bind to pelargonidin through hydrogen bonding at pH 3.0, and through hydrophobic interactions at pH 7.0. In addition, it has been reported that milk can alter the excretion profile of cocoa epicatechin metabolites in urine.44 Overall, milk appears to influence the pharmacokinetics of polyphenols in a variety of food/beverage products when co-ingested.

The oral bioavailability of strawberry anthocyanins estimated from plasma or urine is low, ranging between <0.1% and 2.4% when strawberries are consumed with a meal.45 Consistent with previous findings, the oral bioavailability in plasma was low in our study; however, delivery of strawberry in the milk-based beverage did not interfere remarkably with the oral bioavailability of pelargonidin anthocyanins when consumed with a meal. This may have been due to the complex nature of the meal, which had an over-riding effect on the results. To assess the meal effect, we conducted the sub-set study (Part 2), where only the beverages were consumed. In the no meal condition, the effect of the milk vs. water on strawberry oral bioavailability was apparent, showing approximately 50% reductions in oral bioavailability with the milk-based beverage. Similar results have been observed in studies of milk on the oral bioavailability of tea catechins46 and cocoa flavan-3-ol metabolites10 in plasma. However, semi-skimmed milk and whole milk were used in these studies, where the fat content may play a role influencing oral bioavailability.47 Limited in vivo research could be found using skim milk; however, an in vitro study showed that the addition of skimmed milk to coffee caused a 19–20% decrease in the phenolic acid bio-accessibility due to binding to proteins. Whereas analysis of fat concentration of beverages prepared with whole milk, semi-skim milk and skim milk revealed that the addition of fat in milk increased bioaccessibility.48 Cream was reported to delay the absorption of strawberry anthocyanins but does not significantly reduce oral bioavailability.12 In addition to proteins and fat, carbohydrates and other micronutrient may interact with polyphenols impacting oral bioavailability.49–51 Hence, the data comparing anthocyanins’ oral bioavailability between with meal and no meal conditions support the fact that various components in a meal are likely playing a role in the oral bioavailability of these compounds. This is the second study in our lab to show a meal effect on PK variables and the oral relative bioavailability of strawberry anthocyanins.45 Overall, the oral bioavailability of parent anthocyanins compounds and their metabolites is low when strawberries are consumed in a beverage with or without milk and with a meal or without a meal (<1%). When differences are observed in oral bioavailability (in this study or other studies), the question that remains to be answered is whether this translates to differences in their health benefits when eating strawberries. Huang et al. (2016)22 showed that consuming a water based strawberry slushy 2 h before a challenge meal reduced interleukin-6 (IL-6) concentrations, a marker of inflammation, over a 10 h period compared to a control no strawberry condition, whereas the strawberry provided with the meal did not statistically suppress IL-6 compared to the control condition. However, comparing IL-6 responses between the two conditions of strawberry with a meal and without a meal revealed no difference between the experimental conditions. Collectively, the data suggest that future research is warranted to better understand the mechanisms underlying the interactive effects of food components and matrix interactions on pharmacokinetic variance, including the oral bioavailability of anthocyanins and other polyphenols to optimally combine foods for maximal nutrition and delivery of health benefits.

Conflicts of interest

The authors declared no potential conflicts of interest associated with this study.

Acknowledgements

Funding and strawberry powder were provided by the California Strawberry Commission. SPE, analytical columns, and instruments were provided by Agilent Technologies.

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