Introduction

Focus on molecular structure

Chlorophyll is part of the human diet mainly in the form of fruits, vegetables and food coloring. The molecular structure of chlorophyll consists of a head (formed by a porphyrin ring) from which extends a long hydrophobic tail (phytol). At the center of the porphyrin ring there is a magnesium atom that gives stiffness to the structure avoiding dispersion of solar energy through heat. The porphyrin structure is very similar to the heme group of hemoglobin from which it differs for the presence of a magnesium atom instead of an iron atom [1].

Properties of chlorophyll

Several studies have demonstrated a potent anticancer action of chlorophyll and its derivatives due to its ability to form complex structures with some toxic substances, interfering with the gastrointestinal absorption of potential carcinogens and decreasing the amount of these substances in the susceptible tissues [2]. This anticancer activity has been observed especially in chlorophylline (CHL), a semi-synthetic derivative that differs from chlorophyll mainly for the substitution of the magnesium atom with a copper atom and for absence of the hydrophobic tail: precisely the absence of phytol could increase its effectiveness against carcinogenic and mutagenic agents [3].

The efficacy of chlorophyll and its derivatives has been demonstrated against dangerous substances for humans health such as: aflatoxins and mycotoxins [4]; polycyclic aromatic hydrocarbons formed as a result of incomplete combustion [5]; heterocyclic amines that are formed in high temperature cooked meat [6].

Chlorophyll is also a good source of antioxidants such as vitamin A, C and E, which help neutralize free radicals, that cause cellular damage [7, 8]. Many studies have shown antioxidant properties of chlorophyll and its derivatives [9] with a higher effect by pheophytin (chlorophyll in which the magnesium atom has been replaced by two hydrogen ions) [10]. The protective effect against oxidative damage by chlorophyll would be due to the stimulation of heme oxygenase-1 (HO-1), an enzyme that contributes to maintaining cell homeostasis and whose deficiency could make the organism more vulnerable to inflammatory stimuli and oxidative stress [11].

In addition, chlorophyll has been shown to be effective for the prevention of colorectal cancer [12], lung cancer [13], excretion of dioxins [14] and as antimicrobial [15].

Hypotheses of implication of physical activity

Although chlorophyll has deep been studied for its medicinal and therapeutic properties [16], there are currently no research that investigate its effects as sport supplement in physical activity or for its supposed ergogenic aid and anti-anemic properties.

The endurance training produces iron deficiency in post-exercise due to many factors such as hemolysis, hematuria and sweating. An additional factor is given by the role of hepcidin, a recently discovered hormone produced by liver that regulates iron homeostasis by inhibiting its cellular release and thus lowering blood levels [17]. The endurance activity produces an increase of hepcidin [18] in response to the inflammatory state caused by physical exercise, thereby it determines the condition (multifactorial and multi-causal) defined as “pseudo-anemia” by sports.

Objective of the study

The purpose of this study is to verify the effects of the protracted chlorophyll intake by athletes practicing endurance sports on blood count and serum iron values, to assess supposed ergogenic effects and contrasting “pseudo-anemia”.

Materials and methods

Subjects

Twenty-two endurance athletes (15 males, 7 females) signed the informed consent and authorization to process personal data before the start of treatment. Participants were randomly divided into an experimental group (EG) and a control group (CG) considering: age, gender, weight, height, weekly training amount and discipline practiced. National and regional level athletes with at least five years of experience in endurance-related activities were included in the study and they were randomly divided into experimental group (EG) and control group (CG) considering: age (EG: 32 ± 11 years; CG: 31 ± 13 years), body weight (EG: 64 ± 11 kg; CG: 61 ± 7 kg), height (EG: 174 ± 8 cm; CG: 174 ± 7 cm), BMI (EG: 20.9 ± 2.7 kg/m2; CG: 20.2 ± 2.1 kg/m2) and weekly training amount (EG: 9.5 ± 3.0 h/w; 10.0 ± 3.0 h/w) as shown in Table 1. All participants did not change the diet and the amount of habitual calorie consumption. Inclusion criteria also considered sporting experience, competitive level and the absence of food allergies and sports injuries.

Table 1 Mean ± SD of morpho-anthropometric characteristics of the two groups
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Experimental design

The experimental protocol was conducted using double-blind study design.

During the competitive period EG consumed daily an alcoholic extract of magnesiac chlorophyll (Herboristic laboratory Di Leo srl, Anzola dell’Emilia, BO, Italy) containing 86% water, alcohol (Vol. 34.4%) and 14% chlorophyll (extracted from festuca leaves) at a dosage of 1.6 drops × kg per day [19]; during the same period CG consumed a placebo containing 98% water, alcohol (Vol. 34.4%), 1.5% caramel , 0.4% yellow dye and 0.1% blue dye at the same dosage. The chlorophyll was guaranteed by the herboristic laboratory regarding the absence of contaminants that could have influenced the result.

Both the EG and the CG took the extract or placebo uninterruptedly for 120 days in the morning (from 7 am to 10 am) on an empty stomach and maintained their respective training program and usual diet. In addition, a daily diary was provided to monitor the time of consumption, training distance, duration of training, training type and fasting weight.

Three blood counts and serum iron tests were performed: before starting the intake (T0), after 30 days (T1) and after 120 days (T2).

For each examination 7 ml of blood was taken and analyzed (LARC, hematic laboratory in Turin) through the XE-2100D System (Sysmex, Kobe, Japan) to evaluate the following parameters: leukocytes (WBC), erythrocytes (RBC), hemoglobin (Hb), hematocrit (Ht), mean corpuscular volume (MCV), mean corpuscular hemoglobin (MCH), mean corpuscular hemoglobin concentration (MCHC), red blood cell distribution width (RDW-SD, RDW-CV), platelets (PLTS), platelet distribution width (PDW), mean platelet volume (MPV), platelet/large cell ratio (P-LCR), and serum iron (Fe).

The amount of unbound iron (Fe) was analyzed by the ARCHITECT system (c8000, Abbott Laboratories, Abbott Park, Illinois, USA).

Statistical analysis

Statistical analysis was done using the R software (version 3.0.1, R Core Team, Foundation for Statistical Computing, Vienna, Austria). Blinded randomization procedures were performed to minimize selection bias. The normality assumption of the data was analyzed by the Kolmogorov–Smirnov and Shapiro–Wilk tests; the homogeneity of the variables was analyzed by the Levene and Brown–Forsythe tests. Intra-group differences were investigated by the analysis of variance (ANOVA) for repeated measurements. The level of significance for all tests was set at p < 0.05. The Tukey test was used for multiple comparisons.

Power calculation

Threshold probability for rejecting the null hypothesis (Type I error rate) was set at α (two-tailed) = 0.05. Probability of failing to reject the null hypothesis under the alternative hypothesis (Type II error rate) was set at β = 0.20. Standard deviation of the change in the outcome is S(Δ) = 0.40. This study has 80.0% power to detect an effect size of effect size E = E/S(Δ) × S(Δ) = 0.375. R package ‘pwr’ was used to estimates the power of the study [20].

Results

The EG and the CG did not show significant differences at T0. In intra-group analysis (T2 vs T0), significant increases were seen only in the experimental group regarding PDW (0.83, 95% CI [0.41, 1.38], p < 0.01), MPV (0.41, 95% CI [0.19, 0.67], p < 0.05) and P-LCR (3.28, 95% CI [1.51, 5.25], p < 0.001). The control group did not show statistically significant differences during follow-up. The results are reported from Figs. 1, 2 and 3.

Fig. 1
figure 1

Platelet distribution width (PDW). Significant (p < 0.01) increase in the experimental group (EG): 12.48 ± 1.12 fL at T0, 13.36 ± 2.17 fL at T1 and 13.31 ± 1.23 fL at T2. No significant difference in the control group (CG)

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Fig. 2
figure 2

Mean platelet volume (MPV). Significant (p < 0.05) increase in the experimental group (EG): 10.79 ± 1.23 fL at T0, 11.07 ± 2.34 fL at T1 and 11.20 ± 8.10 fL at T2. No significant difference in the control group (CG)

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Fig. 3
figure 3

Platelet/large cell ratio (P-LCR). Significant (p < 0.001) increase in the experimental group (EG): 32.16 ± 3.13 fL at T0, 33.46 ± 4.59 fL at T1 and 35.44 ± 3.65 fL at T2. No significant difference in the control group (CG)

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Discussion

Blood count parameters, normally related to performance or as detecting elements of a pseudo-anemia state (in particular Ht, Hb, MCH, MCHC and Fe), showed no significant changes in intra-group analysis. In particular, hemoglobin (Hb) values remained stable in both groups; this parameter has a strong correlation with VO2Max [21] and performance in lactic acid tolerance [22]. About serum iron, it is important to point up how the iron turn-over is accelerated during exercise [23] and the deficiencies of this mineral can adversely affect performance [24], whereas iron homeostasis contributes to the production of free radicals, causing oxidative damage [25]; however no statistically significant difference was observed between the two groups or over time. The total number of platelets (PLTS) did not show significant intra-group variations, whereas there was a significant increase over T0 only in the experimental group about platelet distribution width (PDW), mean platelet volume (MPV), and platelet/large cell ratio (P-LCR); these parameters can be considered as a platelet activation index. Platelet-rich plasma has anti-inflammatory and anabolic effects [26] and several studies show its effectiveness in the healing process by muscle injury [27], tendon injury [28] and in the treatment of osteoarthritis [29]. A recent study shows a significant correlation between MPV and the running time in a half marathon [30], while in short-term performance at maximum intensity it appears no significant relationships between PLTS, MPV and PDW with VO2Max, resistance and running speed [31]. These results suggest that platelets may play a role in the performance of medium-long term by promoting the gradual release of growth factors and thereby relieving muscular pain and/or fatigue, or that MPV increase could be attributed to a greater turn-over of platelets that could reflect other chronic physical adaptation without necessarily having a direct ergogenic effect. In the present study, however, only the experimental group obtained a significant increase, indicating a role of chlorophyll in the modification of the above factor. It has been shown that exercise increases PLTS [32] due to adrenergic stimulation (due to epinephrine and norepinephrine activity) that induces splenic contractions resulting in PLTS release into blood circulation [33]; however, the PLTS and MPV values after exercises would return to basal levels after 3 h from the end of activity [34].

Conclusions

Protracted chlorophyll intake in endurance athletes has not shown significant changes about the hemochromocytometric and sideremic parameters. These results could indicate the absence of a direct ergogenic potential of chlorophyll in endurance sports. In the experimental group there was a significant increase in some platelet-related variables, which could affect sport performance and mitigate the pseudo-anemic effects of endurance sport practice.

Chlorophyll supplement can be considered in endurance sports for the proprieties previously described. However, the role of platelets in sports performance is not yet clear and the few findings currently available in the literature are conflicting. It should also be considered that MPV is used as a bio-marker of cardiovascular disease [35] and its eventually sudden increase, due to chlorophyll intake, could induce a false positive in the clinical monitoring.