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Journal of Exercise Physiologyonline

April 2018 Volume 21 Number 2

Editor-in-Chief Tommy Boone, PhD, MBA Review Board Todd Astorino, PhD Julien Baker, PhD Steve Brock, PhD Lance Dalleck, PhD Eric Goulet, PhD Robert Gotshall, PhD Alexander Hutchison, PhD M. Knight-Maloney, PhD Len Kravitz, PhD James Laskin, PhD Yit Aun Lim, PhD Lonnie Lowery, PhD Derek Marks, PhD Cristine Mermier, PhD Robert Robergs, PhD Chantal Vella, PhD Dale Wagner, PhD Frank Wyatt, PhD Ben Zhou, PhD Official Research Journal of the American Society of

Exercise Physiologists

ISSN 1097-9751

Official Research Journal of the American Society of Exercise Physiologists

ISSN 1097-9751

JEPonline

Effect of Black Rice Bran Extract Supplementation on Circulating Leukocyte Counts after Moderate-

Intensity Exercise in Dyslipidemic Subjects Nantaya Krasuaythong1,2, Panakaporn Wannanon3, Juntanee Uriyapongson4, Yupaporn Kanpetta1,2, Naruemon Leelayuwat2,3 1Graduate School, Khon Kaen University, Khon Kaen, Thailand, 2Exercise and Sport Sciences Development and Research Group, Khon Kaen University, Khon Kaen, Thailand, 3Faculty of Medicine, Khon Kaen University, Khon Kaen, Thailand, 4Faculty of Food Technology, Khon Kaen University, Khon Kaen, Thailand

ABSTRACT

Krasuaythong N, Wannanon P, Uriyapongson J, Kanpetta Y, Leelayuwat N. Effect of Black Rice Bran Extract Supplementation on Circulating Leukocyte Counts to Moderate-Intensity Exercise in Dyslipidemic Subjects. JEPonline 2018; 21(2):70-83. This study investigated the effects of black rice bran extract supplementation (BRE) on leukocyte counts at rest and after exercise in dyslipidemic individuals. Fifteen subjects performed two 5-wk phases with daily ingestion of either 100 mL BRE or placebo with a 5-wk washout. One day pre- (acute effect) and post- (prolonged effect), subjects randomly ingested the drink before a 20-min moderate-intensity exercise followed by a 30 min rest. Comparing with PLA, acute effect of BRE showed lower monocyte count at recovery. The prolonged effect showed higher neutrophils count immediately after exercise. Findings indicate that the acute effect of BRE seems to be anti-inflammatory induced by moderate-intensity exercise, while the prolonged effect is unclear whether this represents an immune activation or pro-inflammatory response to exercise in the dyslipidemic subjects.

Key Words: Activity, Anthocyanin, Lipid Profile, White Blood Cell

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INTRODUCTION Regular moderate-intensity exercise is recommended to improve blood lipid profiles and physical fitness as well as reduce cardiovascular risk in dyslipidemic patients (8,9). A single bout of aerobic exercise at this intensity promotes an increase in leukocytosis and neutrophil counts within 30 to 60 min of exercise (4,23,29). This condition induces oxidative stress and muscle inflammation that represent biological markers of inflammation in blood circulation, such as circulating total leukocytes (LE) and the differential leukocyte counts including neutrophils (NE), lymphocytes (LY), and monocytes (MO) (3). Neutrophils are the primary leukocyte subpopulation to arrive at damaged or stressed tissue sites. Therefore, the likely mechanisms responsible for this increase are multifactorial and may include an increase in the release of various biochemical products such as catecholamine or alternatively enhanced cell signalling or blood flow (6). Furthermore, the elevation of differential leukocyte counts was associated with serum lipid levels and increase risk of cardiovascular disease, morbidity, and mortality (1,12,18,28). Nieman and co-worker reported that NE counts increased from 3.99 ± 0.21 × 109 cells·L−1 at baseline to 5.36 ± 0.41 × 109 cells·L−1 immediately post moderate-intensity exercise (23). The relative increase in NE counts from baseline to post-exercise was 51%. Thammawong and co-workers (30) also found increases in LE and differential leukocyte counts after moderate-intensity exercise in trained subjects. Thus, the resting and exercise-induced alterations in LE were independent of gender and subject fitness level (21). The phenolic compound in food supplement can improve antioxidant status and attenuate LE and NE counts after a single session of moderate-intensity exercise (25). However, there are few reported effects of food supplement with anthocyanin on the circulating leukocyte counts after acute and prolonged ingestion at rest and in response to exercise in dyslipidemia (7,31). There are several studies that indicate Black rice (Oryza saltiva L.) contains active compounds such as dietary fiber (5) and phenolic compound especially anthocyanin (14), which were demonstrated to improve metabolic response (10,26), anti-oxidant, and anti-inflammatory effects (19). Recently, vivo studies showed that black rice bran extract can reduce free radical and improve antioxidant enzyme activities in HepG2 cells (15). However, there appears to be no studies that have investigated the effect of dietary fiber and phenolic compounds in black rice bran extract on the circulating total and differential leukocyte counts in dyslipidemic individuals. Therefore, we investigated the acute and prolonged effects of beverages containing dietary fiber plus anthocyanin (cyanidin-3-glucoside: Cy-3-G) extracted from Thai black sticky rice bran (BRE) on the circulating total and differential leukocyte counts at rest and after moderate-intensity exercise (50% VO2 peak) in dyslipidemic subjects. We hypothesized that BRE reduces the number of the circulating total and differential leukocyte counts at rest and in response to moderate-intensity exercise (50% VO2 peak) in dyslipidemic subjects.

METHODS Subjects Fifteen dyslipidemia subjects (12 women and 3 men, aged 49 ± 4.3 yrs) participated in this study. They were screened by physical examination, health questionnaires, blood chemistry, and electrocardiogram (ECG). Blood samples were collected after 12 hrs of overnight fasting

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to measure blood chemistry. Subjects were recruited if they had abnormal lipid profiles by inclusion criteria: (a) total cholesterol (TC) 200-239 mg·dL-1; (b) triglyceride (TG) 150-199 mg·dL-1; (c) low-density lipoprotein cholesterol (LDL-c) 130-159 mg·dL-1; and (d) high-density lipoprotein cholesterol (HDL-c) <40 mg·dL-1. However, if they had a history of CVD, respiratory disease, thyroid disease, kidney disease, liver disease or other severe chronic disease or use of any drug or food supplement known to affect blood lipid metabolism, they were excluded. Before starting the experiment, body composition was measured by Dual-energy X-ray absorptiometry (DEXA) (Lunar Prodigy whole body scanner, GE Medical System, USA). Each subject’s blood pressure was measured in a sitting position without a back support by an automatic sphygmomanometer (Data scope ACCUTORR#1A, Japan). This study was approved by the Ethics Committee of Khon Kaen University according to the guidelines detailed in the Declaration of Helsinki (HE 561298). All subjects provided informed consent to participate in the study after receiving both verbal and written explanations. Study Design This study was a randomized crossover (double-blinded) design. Subjects and researcher were blinded as to the composition of the BRE and PLA. Procedures Acute Effects (Pre- Supplementation) (First Day of Both Supplementations) All subjects randomly received either BRE or placebo drink (PLA) and rested for 20 min, followed by 20 min of moderate-intensity exercise. After exercise, the subjects rested for 30 min. Blood samples were collected before (T0) and 20-min after ingestion (T20), immediately after (T40), and 30 min after (T70) the 20-min moderate-intensity exercise.

Prolonged Effects (Post-Supplementation) (Last Day of Both Supplementations) Prolonged effects were determined on the 1st and the 35th day of each supplementation. On both days, all subjects randomly ingested a beverage containing either BRE or PLA within 10 min and rested for 30 min. Then, they performed a single bout of moderate-intensity exercise for 20 min following by resting for 30 min. Blood samples, heart rate, and blood pressure were collected before exercise, immediately and 30 min after the 20-min moderate-intensity exercise. One week before starting the study, the subjects were asked to record their physical activity and dietary records for 3 d (i.e., 2 weekdays and 1 weekend day). INMUCAL software (Mahidol University, Thailand) was used to analyze the dietary records. Serum glutamic-pyruvic transaminase (SGPT) was assessed for 2 d of both supplementations; the first and the second day at the second and the last week of the ingestion, respectively. Peak oxygen Consumption Test One week before each supplementation, peak oxygen consumption (VO2 peak) was determined at 85% of maximum heart rate (HR max) by cycling ergometer (Corival, Lode B.V., Groningen, The Netherlands) at a room temperature of 25 ± 0.5C, humidity 50 to 55% (Chanavirut et al. 2015). Throughout the test, expired gas was recorded and analyzed by a gas analysis system (Power lab 8/30, AD instruments, Australia). The HR and VO2 relationship was used to calculate VO2 peak and workload, and VO2 relationship was used to set work load at 50% VO2 peak. The workload was used for the exercise periods to confirm

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exercise intensity. On the day prior to the test, subjects were asked to restrict their intake of alcohol and caffeine-containing drinks. They were recommended to get at least 8 hrs of sleep and to not eat or drink at least 2 hrs before testing. Preparation of BRE The beverages containing BRE and PLA were prepared under reliable manufacturing practices (GMP) at the Faculty of Food Technology, Khon Kean University, Thailand. Contaminant level was measured for safety by the Central Lab, Thailand. The powder of BRE and PLA were packed in aluminum foil in packs of 10 gm per pack. The BRE contained spray-dried black rice extract at 41.8% (198 mg of anthocyanins, insoluble fiber 21 gm and soluble fiber 4 gm), sucralose 0.58% (0.058 gm), maltitol 57.6% (5.8 gm), salt 0.01% (0.001 gm). PLA was dye-coloured powder with water base type plus sucralose that consisted of sucralose 0.08%, moltodextrin (DE 10) 89.8% (8.98 gm), salt 0.15% (0.015 gm), synthetic colors such as carmoisine, amaranth and brilliant blue 0.02% (0.002 gm). The BRE and PLA powder had red color and were kept in aluminum foil envelopes. Then, the envelopes were labeled with expiration dates and the method required for drink preparation. Both BRE and PLA packages were stored at 4°C in a refrigerator. Before ingestion, the powder was put in 100 mL of warm water (50C). All subjects were explained the preparation of the beverage before taking home. Measurements of Main Outcomes Two ml blood sample was analyzed for routine complete blood count including total LE count and differential LE subfractions (Pentra 60, ABX Diagnostics, France). The quantity of each LE subfraction (LYM, MO, NE; cells·mL-1) was calculated by multiplying the measured fraction of each by the total LE count. All blood parameters were analyzed in the chemistry laboratory of Srinagarind hospital, Faculty of Medicine, Khon Kaen University, Thailand (30). Statistical Analyses and Power Calculation The study design employed simple random sampling, double-blinded, and cross over. There were fifteen subjects in this study, according to statistical calculation using a comparison of mean to hypothesize values with the WINPEPI program (WINPEPI Program COMPARE2 manual, Version 1.50:online 2012 Cite 2012 September 24. According to a study by Kestin and coworkers in 1990 (Kestin et al., 1990), the effects of rice bran can reduce LDL-c from 4.55 ± 0.79 mmol·L-1 to 4.49 ± 0.57 mmol·L-1 (5% difference). The data were significant at a P-value of <0.05, an -error = 0.05 with drop out 20% (N = 12) and Power = 90. The percentage changes of the data were calculated as follows: [(value after - value at before) / value at before x 100. RESULTS Baseline Anthropometrics and Physiological Characteristics Fifteen dyslipidemic subjects aged 49 ± 4.3 yrs participated in this study. They were overweight, high W/H circumference ratio, and a high percentage of body fat. Fasting blood glucose and fasting serum insulin were normal. They were classified as Type IIb dyslipidemia because of their high LDL-c, TG, and low HDL-c (Table 1). Furthermore, they had normal

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serum creatinine and SGPT (Table 1). They had normal blood hematology as well as normal hsCRP (Table 2). Before and during ingestion of BRE and PLA supplementations, the total energy intake and total energy expenditure were not different between supplementations (Table 4). The work rate and peak oxygen consumption were not different between supplementations (Table 3). Table 1. Baseline Data of Anthropometric, Physiological Characteristics, and Blood Chemistry in Dyslipidemic Subjects.

Conditions Mean ± SD

Body Mass (kg) 65.4 ± 8.40 BMI (kg·m-2) 27.2 ± 3.50 Waist Circumference (cm) 88.4 ± 8.90 Hip Circumference (cm) 100.1 ± 8.30 W/H Circumference Ratio 0.80 ± 0.04 Body Fat (%) 39.0 ± 8.05 Fat Mass (kg) 24.9 ± 6.80 Lean Body Mass (kg) 37.0 ± 4.30 HR (beats·min-1) 70 ± 6.7 SBP (mmHg) 121.8 ± 9.80 DBP (mmHg) 79.2 ± 8.50 MAP (mmHg) 93.4 ± 7.90 BG (mg·dL-1) 90.6 ± 19.5 TG (mg·dL-1) 175.9 ± 78.0 TC (mg·dL-1) 200.5 ± 29.6 LDL-c (mg·dL-1) 126.4 ± 28.2 HDL-c (mg·dL-1) 50.4 ± 16.2 Insulin (IU/mL) 15.3 ± 10.6 Creatinine (mg·dL-1) 0.8 ± 0.2 SGPT (U/L) 21.3 ± 8.3

The data are expressed as mean ± SD; N = 15 (12 females and 3 males). BMI = Body Mass Index; W/H ratio = Waist Hip Ratio; HR = Heart Rate; SBP = Systolic Blood Pressure; DBP = Diastolic Blood Pressure; MAP = Mean Arterial Pressure; BG = Blood Glucose; TG = Triglyceride; TC = Total Cholesterol; LDL-c = Low Density Lipoprotein-Cholesterol; HDL-c = High Density Lipoprotein-Cholesterol; SGPT = Serum Glutamic-Pyruvic Transaminase

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Table 2. Baseline Data of Hematology and hsCRP in Dyslipidemic Subjects.

Conditions Mean ± SD

Red Blood Cell (x106/L) 4.7 ± 0.6 Hemoglobin (g·dL-1) 12.8 ± 1.4 Hematocrit (%) 38.6 ± 3.6 Platelet Count ( cell/mm3) 278.9 ± 80.6 Leukocyte ( x103/L) 7.2 ± 1.3 Neutrophils ( x103/L) 3.9 ± 0.9 Lymphocytes ( x103/L) 2.5 ± 0.4 Monocytes ( x103/L) 0.5 ± 0.2 Eosinophils ( x103/L) 0.3 ± 0.2 Basophils (x103/L) 0.05 ± 0.02 hsCRP ( mg·dL-1) 2.4 ± 2.2

The data are expressed as mean ± SD; N =15 (12 females and 3 males); hsCRP = High-Sensitivity C-Reactive Protein Table 3. The Major Variables in VO2 peak Test Before Start the Beverages.

Conditions Mean ± SD P-value

BRE PLA

VO2 peak (mL·kg -1·min-1) 22.7 ± 7.7 25.7 ± 10.5 0.34 (NS) Work Rate at VO2 peak (watts) 92.5 ± 14.9 91.7 ± 15.8 0.46 (NS)

50% VO2 peak (mL·kg -1·min-1) 11.4 ± 3.8 12.9 ± 5.2 0.32 (NS)

Work Rate at 50% VO2 peak

(watts) 46.5 ± 9.4 45.9 ± 7.6 0.36 (NS)

The data are expressed as mean ± SD; N = 15 (12 females and 3 males)

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Table 4. Means of Daily Total Energy Intake and Energy Expenditures Before and During the Ingestion of The Beverage in Dyslipidemic Subjects.

Mean ± SD

Before

During

Conditions BRE PLA BRE PLA

CHO (g·d-1) 228.4 ± 36.7 172.7 ± 61.3 228.1 ± 23.7 175.6 ± 48.6

% CHO (%TEI) 59.6 ± 10.2 53.9 ± 21.2 57.5 ± 8.5 56.3 ± 17.0

Fat (g·d-1) 44.1 ± 29.5 73.6 ± 44.8 47.1 ± 21.1 81.6 ± 48.3

% Fat (%TEI) 22.9 ± 6.7 26.8 ± 13.4 25.1 ± 5.6 27.2 ± 14.5

Protein (g·d-1) 73.5 ± 41.9 73.6 ± 31.1 73.4 ± 32.2 82.1 ± 32.4

% Protein (%TEI) 17.4 ± 3.6 15.4 ± 2.9 14.4 ± 3.5 16.5 ± 3.7

Vitamin A (mg) 447.0 ± 287.6 216.0 ± 183.2 290.1 ± 85.9 194.2 ± 115.7

Thiamin (mg) 1.0 ± 0.4 1.1 ± 0.5 0.9 ± 0.3 1.1 ± 0.5

Riboflavin (mg) 0.8 ± 0.3 0.8 ± 0.3 0.8 ± 0.3 0.7 ± 0.3

Vitamin B6 (mg) 0.5 ± 0.3 0.4 ± 0.2 0.4 ± 0.2 0.9 ± 0.5

Vitamin B12 (g) 2.2 ± 1.4 1.4 ± 1.3 1.6 ± 1.1 2.9 ± 1.8

Vitamin C (mg) 59.9 ± 50.0 67.7 ± 23.5 46.1 ± 34.3 39.3 ± 18.9

Vitamin E (g) 1.5 ± 1.3 2.1 ± 0.9 1.5 ± 1.0 1.9 ± 0.8

Crude Fiber (g) 0.6 ± 0.4 0.4 ± 0.3 0.5 ± 0.2 0.3 ± 0.2

Dietary Fiber (g) 8.6 ± 2.5 6.0 ± 3.7 7.2 ± 3.0 4.4 ± 3.2

Total EI (kcal) 1,604.0 ± 525.9 1,648.0 ± 536.3 1,629.7 ± 364.3 1,765.0 ± 571.2

Total EE (kcal) 1,677.0 ± 130.5 1,685.7 ± 501.1 1,682.3 ± 205.5 1,794.7 ± 555.4

The data are expressed as mean ± SD; N = 15, (12 females and 3 males); CHO = Carbohydrate; TEI = Total Energy Intake; EE = Energy Intake; EE = Energy Expenditure

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The Acute Effect of BRE In both supplementations, there were significant increases (P<0.05) from baseline in total LE, LYM, and NE counts immediately after exercise and returned to baseline at the end of the recovery (P<0.05) without any difference between supplementations (Figures 1 and 2). Similarly, in BRE supplementation, there was significant increase (P<0.05) from baseline in MO count immediately after exercise and return to baseline at the end of the recovery (P<0.05). However, in PLA supplementation, MO count immediately after exercise was not different from baseline, but it increased to greater than baseline at the end of the recovery (P<0.05) when the lower value was in BRE supplementation (P<0.05) (Figures 1 and 2). The Prolonged Effect of BRE The prolonged effect of BRE supplementation shows a significant increase from baseline of NE counts at immediately after exercise when compared with that of PLA supplementation (P<0.05) (Figure 2). In both supplementations, total LE, LYM, and MO counts were increased immediately after exercise when compared with baseline without any difference between supplementations (Figure 2). At this time, there was a greater increase in MO count in BRE than PLA supplementation (P<0.05). In addition, at the end of recovery, LE, LYM, and NE counts were decreased in both supplementations without any difference between the supplementations (Figure 2).

Figure 1. The Acute Effects of BRE on the Percent Changes of Circulating Leukocyte Counts at Rest and to Moderate-Intensity Exercise in Dyslipidemic Subjects. The data are expressed as mean ± SE. BRE = Black Rice Bran Extract; N =15 (12 women and 3 men); PLA = placebo; N =14 (11 females and 3 males) ; T0 = Before Ingestion, T20 = 20 Min After Ingestion, T40 = Immediately After 20-min Moderate-Intensity Exercise, T70 = 30 Min After Exercise. *Significantly different from the percent change from baseline at immediate after exercise within group (P<0.05 ). @Significantly different between groups at the same time (P<0.05 )

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Figure 2. Acute and Prolonged Effects of BRE and PLA on Percent Changes of Circulating Leukocyte Counts during Moderate-Intensity Exercise in Dyslipidemic Subjects. The data are expressed as mean ± SE. BRE = Black Rice Bran Extract; N = 15 (12 women and 3 men); PLA = Placebo; N = 14 (11 females and 3 males). *Significantly different from the percent change from baseline at immediate after exercise within group (P<0.05). @Significantly different between groups at the same time (P<0.05) DISCUSSION The present study determined the acute and prolonged effects of BRE ingestion on the circulating total and differential LE counts at rest and during moderate-intensity exercise (50% VO2 peak) in dyslipidemic subjects. The results of this study provide new information that the acute effect of BRE supplementation was a reduction in the MO count at the end of recovery and the prolonged effect showed higher NE count immediately after exercise. However, there was no effect of BRE at rest condition. This shows that the acute effect of BRE supplementation was an anti-inflammatory effect 30 min after the single bout of 20-min moderate-intensity exercise at 50% VO2 peak. This may explain the beneficial effect of reducing cardiovascular risk of BRE on moderate-intensity exercise training. Moderate-intensity exercise not only yields beneficial effects on blood metabolism, but it also produces reactive oxygen and nitrogen species, resulting in oxidative stress through mitochondrial electron transport chain pathways in the muscle cells (27). Free radicals attack the lipid molecules of the membrane, causing lipid peroxidation induced inflammation. In the

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present study, BRE is the natural product that contains a phenolic compound known as anthocyanin that has been shown to attenuate inflammatory and oxidative responses to exercise-induced muscle damage, which leads to faster recovery after exercise bouts (7). However, BRE in this study only decreased MO count induced by the exercise. The greater dose or longer time of BRE supplementation should be further investigated. It may provide more significant results on other circulating total and differential LE counts. Furthermore, the prolonged effect of BRE supplementation that contributed to the higher NE count immediately after the exercise is currently unclear whether this represents an immune activation or pro-inflammatory response. This is a limitation of this study since NE function and inflammatory markers such as IL-6, IL-1-α, IL-8, IFN-, CRP, and TNF-alpha were not measured. Therefore, it is important to consider repeating the present study and measuring these variables. A previous study showed that the increase in body mass index, white blood cell count, TC, LDL-c, and TG and the decrease in HDL-c were associated with impaired blood rheology in healthy subjects (28). The circulating total LE and differential LE counts are associated with the presence of metabolic syndrome including dyslipidemia (13). Thus, the anti-inflammatory effect of BRE supplementation on monocytes at exercise recovery may be important in improving cardiovascular function in dyslipidemic individuals. However, the subjects’ blood rheology (i.e., hematocrit, plasma viscosity, and RBC aggregation) in the present study was in the normal range, which means that it would be rare to find the significant changes in these parameters as a function of the BRE supplementation. The recent systemic reviews and meta analysis demonstrated that anthocyanin supplementation significantly reduced serum TC, TG, LDL-c, and inflammatory response in patients with dyslipidemia (11,16,17,22,24,32,33). In contrast, our study did not find changes in lipid profiles after the ingestion of BRE. A possible explanation of this discrepancy may be the different dose and duration of the BRE supplementation. Unfortunately, we did not measure the blood concentration of anthocyanin and antioxidant status of the subjects. It would be interesting to exam a higher dose and longer duration of the BRE supplementation on WBC counts and functions, antioxidant activity, on cardiovascular risk factors at rest and during recovery after exercise in the dyslipidemic subjects. CONCLUSIONS The findings indicate that BRE supplementation, which is anthocyanin- and dietary fiber-riched food, had both an acute effect and a prolonged effect. The acute effect of BRE supplementation seems to be anti-inflammatory. Regarding the prolonged effect, it is unclear whether it represents an immune activation or a pro-inflammatory response to exercise in the dyslipidemic subjects.

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ACKNOWLEDGMENTS This study was partially supported by the Exercise and Sport Sciences Development and Research Group, the Graduate School, Khon Kaen University, the Agricultural Research Development Agency (Public organization) ARDA, Thailand and the College of Medicine and Public Health, UbonRatchathani University. We thank the subjects for their kind cooperation. Address for correspondence: Associate Professor Naruemon Leelayuwat, PhD, Department of Physiology, University of Khon Kaen, Nai Muang, Muang Khon Kaen, Khon Kaen 40002,Thailand, Tel: +66-043-363263, +66-043-363185, Fax: +66-043-348394, Email: naruemon.Leelayuwat@gmail.com REFERENCES

1. Babio N, Ibarrola-Jurado N, Bulló M, Martínez-González MÁ, Wärnberg J, Salaverría I, Ortega-Calvo M, Estruch R, Serra-Majem L, Covas MI, Sorli JV, Salas-Salvadó J. White blood cell counts as risk markers of developing metabolic syndrome and its components in the PREDIMED study. PLoS One. 2013;8(3):e58354.

2. Barriga C, Pedrera MI, Maynar M, Maynar J, Ortega E. Effect of submaximal physical exercise performed by sedentary men and women on some parameters of the immune system. Rev Esp Fisiol. 1993;49:79-85.

3. Bessa AL, Oliveira VN, Agostini GG, Oliveira RJ, Oliveira AC, White GE, et al.

Exercise intensity and recovery: Biomarkers of injury, inflammation, and oxidative stress. J Strength Cond Res. 2016;30(2):311-319.

4. Brown WM, Davison GW, McClean CM, Murphy MH. A systematic review of

the acute effects of exercise on immune and inflammatory indices in untrained adults. Sports Med Open. 2015;1(1):35.

5. Bruno B and Laura C. Therapeutic properties of rice constituents and derivatives

(Oryza sativa L): A review update. Trends in Food Sci Technol. 2014;40:82-98.

6. Butterfield TA, Best TM, Merrick MA. The dual roles of neutrophils and macrophages in inflammation: A critical balance between tissue damage and repair. J Athl Train. 2006;41(4):457-465.

7. Coelho Rabello Lima L, Oliveira Assumpção C, Prestes J, Sérgio Denadai B. Consumption of cherries as a strategy to attenuate exercise-induce muscle damage and inflammation in humans. Nutr Hosp. 2015;32(5):1885-1893.

8. Cordero A, Masiá MD, Galve E. Physical exercise and health. Rev Esp Cardiol (Engl

Ed). 2014;67(9):748-753.

81

9. Duscha BD, Slentz CA, Johnson JL, Houmard JA, Bensimhon DR, Knetzger KJ, Kraus WE. Effects of exercise training amount and intensity on peak oxygen consumption in middle-age men and women at risk for cardiovascular disease. Chest. 2005;128(4): 2788-2793.

10. Guo H, Ling W, Wang Q, Liu C, Hu Y, Xia M, Feng X, Xia X. Effect of anthocyanin-rich

extract from Black Rice (Oryza sativa L. Indic) on hyperlipidemia and insulin resistance in fructose-fed rats. Plant Food Hum Nutr. 2007;62:1-6.

11. Hassellund SS, Flaa A, Kjeldsen SE, Seljeflot I, Karlsen A, Erlund I, et al. Effects of

anthocyanins on cardiovascular risk factors and inflammation in pre-hypertensive men: A double-blind randomized placebo-controlled crossover study. J Hum Hypert. 2013; 27(2):100-106.

12. Huang ZS, Chien KL, Yang CY, Tsai KS, et al. Peripheral differential leukocyte counts in humans vary with hyperlipidemia, smoking, and body mass index. Lipids. 2001; 36(3):237-245.

13. Kim DJ, Noh JH, Lee BW, Choi YH, Chung JH, Min YK, Lee MS, Lee MK, Kim KW.

The associations of total and differential white blood cell counts with obesity, hypertension,dyslipidemia and glucose intolerance in a Korean population. J Korean Med Sci. 2008;23(2):193-198.

14. Laokuldilok T, Shoemaker CF, Jongkaewwattana S and Tulyathan V. Antioxidants and

antioxidant activity of several pigmented rice brans. J Agric Food Chem. 2011;59(1): 193-199.

15. Lee SM, Choi Y, Sung J, Kim Y, Jeong HS and Lee J. Protective effects of black rice extracts on oxidative stress induced by tert-butyl hydroperoxide in HepG2 cells. Prev Nutr Food Sci. 2014;19(4):348-352.

16. Li D, Zhang Y, Liu Y, Sun R, Xia M. Purified anthocyanin supplementation reduces dyslipidemia, enhances antioxidant capacity, and prevents insulin resistance in diabetic patients. J Nutri. 2015;145(4):742-748.

17. Liu C,Sun J, Lu Y, Bo Y.Effects of anthocyaninon serum lipids in dyslipidemia patients: A systematic review and meta-analysis. PLoS One. 2016;11(9):e0162089.

18. Liu Y, Kong X, Wang W, et al. Association of peripheral differential leukocyte counts with dyslipidemia risk in Chinese patients with hypertension: Insight from the China Stroke Primary Prevention Trial. J Lipid Res. 2017;58(1):256-266.

19. Maria JK, Neil D, Kathryn HM, Sandrine L. Proanthocyanins, anthocyanin and

cardiovascular disease. Food Res Intern. 2014;59:41-52.

20. Markovitch D, Tyrrell RM, Thompson D. Acute moderate-intensity exercise in middle-aged men has neither an anti-nor proinflammatory effect. J Appl Physiol. 2008;105 (1):260-265.

82

21. Moyna NM, Acker GR, Weber KM, Fulton JR, Goss FL, Robertson RJ, Rabin BS. The

effects of incremental submaximal exercise on circulating leukocytes in physically active and sedentary males and females. Eur J Appl Physiol Occup Physiol. 1996; 74(3):211-218.

22. Mu H, Qu Q, Liu J, Qin Y, Ling W, Ma J. Effect of anthocyanins on oxidative stress in subjects with hyperlipidemia. Acta Nutrimenta Sinica. 2010;32(6):551-555.

23. Nieman DC, Nehlsen-Cannarella SL, Donohue KM, Chritton DB, Haddock BL, Stout RW, et al. The effects of acute moderate exercise on leukocyte and lymphocyte subpopulations. Med Sci Sports Exerc.1991;23(5):578-585.

24. Qin Y, Xia M, Ma J, Hao Y, Liu J, Mou H, et al. Anthocyanin supplementation improves

serum LDL and HDL-cholesterol concentrations associated with the inhibition of cholesteryl ester transfer protein in dyslipidemic subjects. Am J Clin Nutri. 2009;90 (3):485-492.

25. Roengrit T, Wannanon P, Prasertsri P, et al. Antioxidant effect of Phyllanthus amarus after moderate-intensity exercise in sedentary males: A randomized crossover (double-blind) study. J Phys Ther Sci. 2015;27(4):1181-1186.

26. Sariano Sancho RA, Maria Pastore G. Evaluation of the effects of anthocyanins in type

2 diabetes. Food Res Intern. 2012;46:378-386.

27. Scott K. Powers, W. Bradley Nelson, Matthew B. Hudson. Exercise-induced oxidative stress in humans: Cause and consequences. Free Rad Biol Med. 2011;51:942-950.

28. Seki K, Sumino H, Nara M, Ishiyama N, Nishino M, Murakami M. Relationships

between blood rheology and age, body mass index, blood cell count, fibrinogen, and lipids in healthy subjects. Clin Hemorheol Microcirc. 2006;34(3):401-410.

29. Simpson RJ, Kunz H, Agha N, Graff R. Exercise and the regulation of immune

functions. Prog Mol Biol Transl Sci. 2015;135:355-380.

30. Thammawong S, Krasuaythong N, Kanpettha Y, Tunkamnerdthai O, Leelayuwat N. Effects of sex and intensity of exercise on circulating leukocyte counts after exercise in trained subjects. JEPonline. 2017;20(4):11-23.

31. Thompson K, Hosking H, Pederick W, Singh I, Santhakumar AB. The effect of anthocyanin supplementation in modulating platelet function in sedentary population: A randomised, double-blind, placebo-controlled, cross-over trial. Br J Nutr. 2017;118(5): 368-374.

32. Yu QIN, Wenhua L. Effect of anthocyanin-rich exact from black rice on patients with hyperlipidemia. Food Sci. 2008;29(10):540-542.

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33. Zhu Y, Ling W, Guo H, Song F, Ye Q, Zou T, et al. Anti-inflammatory effect of purified dietary anthocyanin in adults with hypercholesterolemia: A randomized controlled trial. Nutri Metab Cardiovas Dis. 2013;23(9):843-849.

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