Effect of Supplementation With Anthocyanins on Iron Metabolism in Female Swimmers

Effect of Supplementation With Anthocyanins Contained in Black Chokeberry (Aronia Melanocarpa) on Selected Indicators of Iron Metabolism in Female Swimmers

The study included 8-weeks supplementation of black chokeberry extract with 18% standardization of the anthocyanins content (dose 2×200 mg per day), or a placebo product that was made from chokeberry fiber.

Purpose:

The aim of the study is effectiveness check of black chokeberry intake as a potential factor which, due to its ability to reduce inflammation and chelate iron ions, will have an impact on minimizing undesirable changes in the red blood cell system.

Hypothesis:

Main hypothesis: Supplementation of black chokeberry (Aronia melanocarpa) has a positive effect on iron economy of female swimmers.

Auxiliary hypotheses:

1. Supplementation of black chokeberry enhances the antioxidant status and has a positive effect on the prooxidant-antioxidant balance.
2. Supplementation of black chokeberry reduce inflammation induced by intensive exercises, by reducing inflammatory markers.

Study plan:

Before first term of the study:

Control of Menstrual Cycle Phases since March 2023 (installing the application on the phone - Menstrual Period Tracker calendar).

First term of the study:

1. Menstrual cycle control survey.
2. Conduct a body composition analysis.
3. Collection of blood samples before the exercise test for biochemical determinations.
4. Exercise test in the swimming pool - crawl style intensive test (800 m + 200 m + 50 m).
5. Collection of blood immediately after exercise and 3 hours later.
6. Supplementation for nearly 56 days, depending on the length of the menstrual cycle.

Second term of the study:

Repeat the measurements from the 1st date of test.

Participants: The group of selective and homogeneous female swimmers, who train swimming intensively and attend the Sports Championship School Complex.

Study Overview

• Status: Completed
• Conditions: Inflammation; Oxidative Stress; Female Athlete Triad; Iron Deficiencies
• Intervention / Treatment: Drug: Black chokeberry extract in capsules with 18% standardization of the anthocyanins content; Drug: Chokeberry fiber in capsules

Detailed Description

This study included 8-weeks supplementation of black chokeberry extract with 18% standardization of the anthocyanins content (dose 2×200 mg per day), or a placebo product that was made from chokeberry fiber. The first date of research completed the group of twenty-four and homogeneous female swimmers, who train swimming intensively and attend the Sports Championship School Complex. The second date of research completed the group of twenty-two selective and homogeneous female swimmers, who train swimming intensively and attend the Sports Championship School Complex.

Purpose:

The aim of the study is effectiveness check of black chokeberry intake as a potential factor which, due to its ability to reduce inflammation and chelate iron ions, will have an impact on minimizing undesirable changes in the red blood cell system.

Hypothesis:

Main hypothesis: Supplementation of black chokeberry (Aronia melanocarpa) has a positive effect on iron economy of female swimmers.

Auxiliary hypotheses:

1. Supplementation of black chokeberry enhances the antioxidant status and has a positive effect on the prooxidant-antioxidant balance.
2. Supplementation of black chokeberry reduce inflammation induced by intensive exercises, by reducing inflammatory markers.

The research is going to be carried out as a medicinal experiment with tissue interference (approval of Research Ethics Committee: 538/22 of 06/23/2022, with changes: 221/23 of 03/09/2023). All swimmers qualified to study based on inclusion and exclusion criteria and were informed of the extract course of the study and gave written consent to participate.

EXPERIMENTAL PROCEDURE, WHICH WAS APPLIED (STUDY PLAN):

A. Before first term of the study:

Control of Menstrual Cycle Phases since March 2023 (installing the application on the phone - Menstrual Period Tracker calendar) and three surveys in three terms.

B. First term of the study:

1. Menstrual cycle control by application Menstrual Tracker Period Calendar and surveys, before swimming test.
2. Body composition analysis (TANITA MC-780 S MA).

3. Biochemical determinations - blood samples will be taken before and after exercise test as well as after 3-hour restitution, in order to determine:

   • morphological parameters,

   • value of indicators:

     1. iron status problems (iron, hepcidin, erythroferrone, total iron-binding capacity, unsaturated iron-binding capacity, ferritin, transferrin,
     2. oxidative stress (total antioxidant capacity),
     3. immunological indicators (interleukins: IL-6, IL-2, IL-10, C-reactive protein),
     4. hormonal balance (progesterone, estradiol, luteinizing hormone, follicle-stimulating hormone),
     5. other: lactic acid, myoglobin, hypoxanthine.

   Blood samples were collected from the elbow vein into two 4-ml tubes (to obtain serum) and centrifuged at 3,000 rpn and into 2,7-ml tubes to determine morphology. Morphology was determined on the same day the samples were collected, while serum was frozen and stored at -80 Celsius degrees until assays were performed. In addition, before and immediately after the exercise test, capillary blood was drawn from the earlobe to determine lactate levels.

   Lactate level were determined immediately after capillary blood sampling using a commercial available kit (Dr Lange, Germany). The concentration of lactate was determined in mmol/l. Other measurements were determined using ELISA kits (DRG Medtek).

4. Intensive exercise test in the swimming pool, before supplementation - crawl style (800 m + 200 m + 50 m) with 15-minute active rest between test sections.
5. Supplementation of black chokeberry - nearly 56-days supplementation of black chokeberry extract with 18% standardization of the anthocyanins content (connection with menstrual cycle phase).The swimmers were randomly divided into two groups: supplementation and placebo. The placebo contained chokeberry fiber. Whole products were manufactured by MLB BIOTRADE, Poznan. The swimmers took 2 capsules per day (before breakfast and dinner) and they sipped each capsule with glass of water.

C. Second term of the study:

1. Menstrual cycle control by application Menstrual Tracker Period Calendar and surveys, before swimming test.
2. Body composition analysis (TANITA MC-780 S MA).

3. Biochemical determinations - blood samples will be taken before and after exercise test as well as after 3-hour restitution, in order to determine:

   • morphological parameters (erythrocytes, leukocytes, thrombocytes),

   • value of indicators responsible for:

     1. iron status problems (iron, hepcidin, erythroferrone, total iron-binding capacity, unsaturated iron-binding capacity, ferritin, transferrin),
     2. oxidative stress (total antioxidant capacity),
     3. immunological indicators (interleukins: IL-6, IL-2, IL-10, C-reactive protein (CRP)),
     4. hormonal balance (progesterone, estradiol, luteinizing hormone (LH), follicle-stimulating hormone (FSH)),
     5. other: lactic acid, myoglobin, hypoxanthine.

   Blood samples were collected from the elbow vein into two 4-ml tubes (to obtain serum) and centrifuged at 3,000 rpn and into 2,7-ml tubes to determine morphology. Morphology wa determined on the same day the samples were collected, while serum was frozen and stored at -80 Celsius degrees until assays were performed. In addition, before and immediately after the exercise test, capillary blood was drawn from the earlobe to determine lactate levels.

   Lactate level were determined immediately after capillary blood sampling using a commercial available kit (Dr Lange, Germany). The concentration of lactate was determined in mmol/l. Other measurements were determined using ELISA kits (DRG Medtek).

4. Intensive exercise test in the swimming pool, after supplementation - crawl style (800 m + 200 m + 50 m) with 15-minute active rest between test sections.

Data collection, analysis in laboratory, statistical analysis and preparation articles to publication.

• Study Type: Interventional
• Enrollment (Actual): 22
• Phase: Phase 2; Phase 1

Contacts and Locations

Study Locations

• Poland
  • Poznań, Poland
    • University of Physical Education in Poznan

Participation Criteria

Eligibility Criteria

• Ages Eligible for Study: Child; Adult
• Accepts Healthy Volunteers: No

Description

Inclusion Criteria:

• Current medical examinations issued by a sports medicine doctor
• Training experience a minimum of three years
• Student of a sports championship school team
• Consent of the athlete/legal guardian to participate in the research
• Completion of the exercise test in the swimming pool - crawl style intensive test (800 m + 200 m + 50 m) on two test deadline

Exclusion Criteria:

• Taking: oral contraceptive pills, supplements containing anthocyanin compounds during the study or a one month earlier
• Health problems
• Antibiotic therapy

Study Plan

How is the study designed?

Design Details

• Primary Purpose: Basic Science
• Allocation: Randomized
• Interventional Model: Single Group Assignment
• Masking: Double

Number of Arms

2

Arms and Interventions

Participant Group / Arm	Intervention / Treatment
Experimental: 1. Supplemented group; The supplemented group (n=12) consumed capsules with highly concentrated 18% chokeberry extract (one capsule contained 200 mg). Each capsule also contained: chokeberry fiber, E460b magnesium salts of stearic acid (anti-caking agent), E551 silicon dioxide (anti-caking agent), hydrocypropyl methylcellulose (capsule shell) and E171 titanium dioxide (shell colour). The producer of all capsules is MLD Biotrade in Poznan. The swimmers took 2 capsules a day (before breakfast and dinner) for nearly 56 days (depending on length menstrual cycle phase). The swimmers sipped each capsule with glass of water.	Drug: Black chokeberry extract in capsules with 18% standardization of the anthocyanins content; Supplement group: supplement of diet - black chokeberry extract (capsules) with 18% standardization of the anthocyanins content (one capsule contained 200 mg). Each capsule also contained: chokeberry fiber, E460b magnesium salts of stearic acid (anti-caking agent), E551 silicon dioxide (anti-caking agent), hydrocypropyl methylcellulose (capsule shell) and E171 titanium dioxide (shell colour). The producer of all capsules is MLD Biotrade in Poznan.; Other Names: Aronia melanocarpa
Placebo Comparator: 2. Placebo group; The control group (n=10) consumed capsules that were made from chokeberry fiber. The producer of all capsules is MLD Biotrade in Poznan. The swimmers took 2 capsules a day (before breakfast and dinner) for nearly 56 days (depending on length menstrual cycle phase). The swimmers sipped each capsule with glass of water.	Drug: Chokeberry fiber in capsules; Placebo group: placebo product - chokeberry fiber (placebo capsules, which were made from chokeberry fiber). The producer of all capsules is MLD Biotrade in Poznan.; Other Names: Placebo product

What is the study measuring?

Primary Outcome Measures

Outcome Measure	Measure Description	Time Frame
Physiological parameters of blood morphology I	erythrocytes: RBC [10^6/mL], HGB [g/dL], HCT [%], MCV [mm^3], MCH [pg], MCHC [g/dL], RDW [%],	At each exercise test (up to 15 minute before exercise, up to 1 minute post exercise, 3 hours after)
Physiological parameters of blood morphology II	leukocytes: WBC [10^3/mL], LYM [10^3/mL], MON [10^3/mL], GRA [10^3/mL], LYM [%], MON [%], GRA [%],	At each exercise test (up to 15 minute before exercise, up to 1 minute post exercise, 3 hours after)
Physiological parameters of blood morphology III	thrombocytes: PLT [10^3/mL], MPV [mm^3], PCT [%], PDW [%]	At each exercise test (up to 15 minute before exercise, up to 1 minute post exercise, 3 hours after)
Physiological parameters of iron metabolism indicator I	iron [µg/dl]	At each exercise test (up to 15 minute before exercise, up to 1 minute post exercise, 3 hours after)
Physiological parameters of iron metabolism indicator II	hepcidin [ng/ml]	At each exercise test (up to 15 minute before exercise, up to 1 minute post exercise, 3 hours after)
Physiological parameters of iron metabolism indicator III	transferrin [mg/ml]	At each exercise test (up to 15 minute before exercise, up to 1 minute post exercise, 3 hours after)
Physiological parameters of iron metabolism indicator IV	total iron-binding capacity (TIBC) [µg/dl]	At each exercise test (up to 15 minute before exercise, up to 1 minute post exercise, 3 hours after)
Physiological parameters of iron metabolism indicator V	ferritin [ng/ml]	At each exercise test (up to 15 minute before exercise, up to 1 minute post exercise, 3 hours after)
Physiological parameters of iron metabolism indicator VI	erythroferrone [ng/mL]	At each exercise test (up to 15 minute before exercise, up to 1 minute post exercise, 3 hours after)
Physiological parameters of antioxidant-prooxidant indicator	total antioxidant capacity [mmol/l]	At each exercise test (up to 15 minute before exercise, up to 1 minute post exercise, 3 hours after)
Physiological parameters of immunological indicator I	interleukin 6 [ng/L]	At each exercise test (up to 15 minute before exercise, up to 1 minute post exercise, 3 hours after)
Physiological parameters of immunological indicator II	interleukin 2 [ng/L]	At each exercise test (up to 15 minute before exercise, up to 1 minute post exercise, 3 hours after)
Physiological parameters of immunological indicator III	interleukin 10 [ng/L]	At each exercise test (up to 15 minute before exercise, up to 1 minute post exercise, 3 hours after)
Physiological parameters of immunological indicator IV	C-reactive protein [mg/dL]	At each exercise test (up to 15 minute before exercise, up to 1 minute post exercise, 3 hours after)
Physiological parameter of hormonal indicator I	progesterone [ng/ml]	At each exercise test (up to 15 minute before exercise)
Physiological parameter of hormonal indicator II	estradiol [pg/ml]	At each exercise test (up to 15 minute before exercise)
Physiological parameter of hormonal indicator III	luteinizing hormone (LH) [mIU/ml]	At each exercise test (up to 15 minute before exercise)
Physiological parameter of hormonal indicator IV	follicle-stimulating hormone (FSH) [mIU/ml]	At each exercise test (up to 15 minute before exercise)
Other physiological parameter I	myoglobin [ng/ml]	At each exercise test (up to 15 minute before exercise, up to 1 minute post exercise, 3 hours after)
Other physiological parameter II	hypoxanthine [mmol/l]	At each exercise test (up to 15 minute before exercise, up to 1 minute post exercise, 3 hours after)
Other physiological parameter (with capillary blood - earlobe)	lactic acid [mmol/l]	At each exercise test (up to 15 minute before exercise, up to 1 minute post exercise, 3 hours after)

Secondary Outcome Measures

Outcome Measure	Measure Description	Time Frame
Physiological parameters of iron metabolism indicator VII	unsaturated iron-binding capacity (UIBC)	Calculated with blood samples at each exercise test (up to 15 minute before exercise, up to 1 minute post exercise, 3 hours after)

Collaborators and Investigators

• Sponsor: Poznan University of Physical Education
• Investigators: Principal Investigator: Marta Mydłowska, PhD student, Poznań University of Physical Education, Faculty of sport sciences in Gorzów Wielkopolski

Study record dates

Study Major Dates

• Study Start (Actual): March 1, 2023
• Primary Completion (Actual): December 28, 2023
• Study Completion (Actual): December 28, 2023

Study Registration Dates

• First Submitted: February 6, 2024
• First Submitted That Met QC Criteria: June 27, 2024
• First Posted (Actual): July 3, 2024

Study Record Updates

• Last Update Posted (Actual): July 3, 2024
• Last Update Submitted That Met QC Criteria: June 27, 2024
• Last Verified: January 1, 2024

More Information

Terms related to this study

• Additional Relevant MeSH Terms: Mental Disorders; Pathologic Processes; Metabolic Diseases; Feeding and Eating Disorders; Iron Metabolism Disorders; Relative Energy Deficiency in Sport; Inflammation; Female Athlete Triad Syndrome; Iron Deficiencies
• Other Study ID Numbers: MMydłowska_PhD

Plan for Individual participant data (IPD)

• Plan to Share Individual Participant Data (IPD)?: NO

Drug and device information, study documents

• Studies a U.S. FDA-regulated drug product: No
• Studies a U.S. FDA-regulated device product: No