The impact of tangeretin combined with whey protein on exercise-induced bronchoconstriction in professional athletes: a placebo-controlled trial

ABSTRACT

Background

Exercise-induced bronchoconstriction (EIB) is highly prevalent in athletes. The objective of this study was to assess the therapeutic efficacy of daily tangeretin combined with whey protein supplementation over a period of 4 weeks in professional athletes with EIB.

Methods

Using a placebo-controlled, double-blind, paired, randomized trial design, a cohort of 30 professional athletes with EIB, consisting of 14 females and 16 males, was divided into two groups: the tangeretin combined with whey protein intervention group (TIG), and the placebo control group (PCG). Both the TIG and PCG underwent exercise challenge tests (ECT) and VO_2max tests before (ECT₁, V1) and after (ECT₂, V2) the intervention. Blood (eosinophils, neutrophils, and basophils) and serum (interleukin-5, IL-5; interleukin-8, IL-8; Clara cell secretory protein-16, CC16; immunoglobulin E, IgE) levels were measured early in the morning of ECT₁ and ECT₂, respectively. Lung function was assessed immediately before and post-ECT immediately.

Results

Tangeretin combined with whey protein use for 4 weeks attenuated the decrease in forced expiratory volume in 1 s (FEV₁) post trials (∆FEV₁(ECT1-ECT2): mean (SD) TIG −7.51(6.9)% vs. PCG −2.33(11.49)%, p = 0.013). Tangeretin also substantially attenuated IL-5 concentration (∆IL-5(T₁-T₅): Tangeretin −19.4% vs Placebo + 8.37%, p = 0.022); IL-8 concentration (∆IL-8(T₁-T₅): Tangeretin −17.28% vs Placebo + 6.1%, p = 0.012); CC16 concentration (∆CC16(T₁-T₅): Tangeretin −11.77% vs Placebo + 24.19%); and IgE concentration in the serum (∆IgE(T₁-T₅): Tangeretin −24.1% vs Placebo −3.9%), and significantly decreased neutrophil count (∆N(T₁-T₅): Tangeretin −11.34% vs Placebo + 0.3%) and eosinophil count in blood (∆N(T₁-T₅): Tangeretin −38.5% vs Placebo + 4.35%). Compared with V1, VO_2max (p = 0.042) and TLim (p = 0.05) of V2 were significantly increased in the TIG, and there was no significant change in the PCG. Meanwhile, six athletes in the TIG and 0 athletes in the PCG became EIB-negative at ECT₂; the overall negative conversion rate of EIB was 40.00% in TCG. Additionally, the number of cough symptoms decreased from 9 to 3 and dyspnea from 4 to 2 in the TIG.

Conclusion

After high-intensity exercise, athletes with EIB achieved significant improvements in lung function and blood inflammatory factors by combining tangeretin and whey protein supplementation. EIB athletes also showed longer exercise endurance and VO_2max at 4 weeks after TI. In addition, some patient symptoms disappeared after combination supplementation. The effect of this treatment on professional athletes with EIB was beneficial.

1. Introduction

Exercise-induced bronchoconstriction (EIB) is characterized by temporary constriction of the lower airways during or after physical activity. Tests for EIB diagnosis include EVH (eucapnic voluntary hyperpnea), exercise, methacholine, dry powder mannitol, hypertonic saline, and histamine. The criteria for EIB positivity typically involves a reduction of at least 10% in forced expiratory volume in 1 s (FEV₁) posttest [1,2]. Symptoms of EIB include coughing, wheezing, chest tightness, dyspnea, and increased mucus production [3]. EIB has been shown to decrease maximal oxygen uptake, increase respiratory muscles’ work and oxygen costs, and lead to ventilation-perfusion mismatch, ultimately affecting athletic performance [4–7]. EIB is a significant contributor to sudden non-cardiac deaths during exercise, with numerous cases reported among professional athletes in the United States and Italy [8,9]. Research indicates that elite athletes (30–70%) face a higher EIB risk than the general population (5–20%) [10,11]. EIB prevalence in endurance athletes (>30%) was higher than that in other sports, especially in endurance athletes in winter or swimming pools (approximately 39–54.8%) [11]. This finding underscores the importance of addressing this issue within the athletic community.

Traditionally, beta-2 adrenergic agonists (β2-agonists) have been commonly prescribed for EIB and associated medical conditions. However, the use of β2-agonists among athletes has raised concerns regarding potential doping violations, leading to the inclusion of various β2-agonists such as salbutamol, fenoterol, vilanterol, higenamine, terbutaline, and clenbuterol on the Prohibited List by the World Anti-Doping Agency (WADA) in 2022. Improper dosages of β2-agonists may also have adverse effects on both physical and mental well-being [12], as they can stimulate the excretion of uric acid and cortisol, potentially leading to symptoms such as insomnia and anxiety [2,13–15]. In addition to inducing Cushing’s syndrome and coronary heart disease, long-term use of β2-Agonists may also pose a risk to patient safety [16,17].

Tangeretin (Figure 1), a citrus flavonoid extracted from citrus fruits, has shown promising anti-inflammatory properties in various in vitro, animal, and in vivo studies [18]. Tangeretin can donate hydrogen atoms, thereby facilitating the delocalization of free radicals and mitigating the endogenous production of reactive oxygen species. Tangeretin activates the Nrf2/Keap1 signaling pathway, reducing cellular lipid peroxides and inflammatory cytokines while concurrently enhancing the levels of both enzymatic and non-enzymatic antioxidants. Additionally, tangeretin inhibits intracellular signaling cascades, including those mediated by p38 MAPK, JNK, and PI3K, as well as the downstream activation of nuclear factor κB (NFκB). It resulted in suppression of COX-2 expression, a reduction in the pro-inflammatory cytokine TNF-α levels, an elevation of the anti-inflammatory cytokine IL-1α level, and a decrease in histamine-induced vascular permeability [19]. Notably, pharmacokinetic data indicate that the lungs are the second largest perfusion site for tangeretin [20]. Findings of mice suggest that tangeretin has potential therapeutic applications in the treatment of asthma, pharyngitis, tonsillitis, and other respiratory conditions [21,22]. Furthermore, tangeretin has effectively reduced airway hyperresponsiveness and bronchial inflammation triggered by strenuous exercise [21,22]. Jang et al. [22] demonstrated that tangeretin exhibited anti-inflammatory effects comparable to those of azelastine, a commonly used antihistamine for asthma, by inhibiting histamine-induced inflammation. This suggests that tangeretin may serve as a potential alternative to β2-agonists and related medications for the treatment of EIB. In recent preliminary studies [23,24] involving weightlifters, supplementation with tangeretin at a dosage of 200 mg/day for a period of 5 weeks, the two athletes with asthma had effectively reduced respiratory discomfort [23,24]. But EIB is not the only cause of respiratory discomfort in athletes. The effects of tangeretin on respiratory inflammation and pulmonary ventilation in athletes with EIB remain unclear.

Figure 1.

Tangeret in chemical structure and 3D chemical structure [28].

Therefore, this study aimed to investigate the impact of tangeretin supplementation on respiratory tract inflammation and pulmonary ventilation in athletes with EIB and to assess the therapeutic efficacy of a 28-day tangeretin intervention (200 mg/day) in professional athletes with EIB.

2. Materials and methods

2.1. Participants

The participants were selected from our study published in 2022, all of whom were diagnosed with EIB [25]. The participants are national elite athlete of track endurance (long distance running/race walking), from the provincial team of ChongQing. The inclusion criteria were 1) EIB diagnosis received by the authoritative institute (First Affiliated Hospital of Chongqing Medical University) within 3 months prior to participating in the study; 2) professional athletes from the provincial team; and 3) willingness to participate in this study. In accordance with the guidelines established by Graham et al., [26] participants were excluded if they met any of the following conditions: 1) presence of chronic obstructive pulmonary disease, pulmonary heart disease, depression, or other significant medical conditions; 2) recent chest surgery; 3) lung infection within 3 weeks before the examination; 4) training cessation for two or more weeks before the examination; 5) moderate or severe cold or respiratory infection within the week preceding the examination; 6) individuals who experienced chest pain during spirometry; and 7) who were unable to understand or unwilling to follow instructions during spirometry were excluded from the study. Finally, 30 professional athletes (14 females and 16 males) were recruited (Figure 2). Their mean (± standard deviation, SD) age, height, and body mass were, respectively, 24.2 ± 3.3 years, 181.9 ± 7.6 cm, and 68.7 ± 12.1 kg. They have received professional exercise training for 7.6 ± 1.8 years. None of the participants were taking any medications.

Figure 2.

The flow diagram of study.

All participants were paired according to their sex, athletic ability, anthropometric, and exercise training characteristics. And each pair of two participants was randomly divided into either tangeretin combined with whey protein intervention group (TIG) or placebo control group (PCG). The TIG received tangeretin supplementation, and the PCG received a placebo for 4 weeks. Throughout the study, both groups received regular training with similar training programs (frequency, duration, and intensity) and schedules (training and resting days) under their coach’s supervision. All participants were informed of the study design, experimental procedure, risks, and benefits and provided signed informed consent. This study was approved by the Institutional Academic and Human Rights Ethics Committee (No.102772020RT082) and conducted in accordance with the Declaration of Helsinki.

A priori sample size was estimated through G*Power 3.1 using the effect size data (Cohen’s d = 0.85), which was calculated based on the FEV₁ decline rate (16.12 ± 4.97% vs. 9.92 ± 9.03%) before and after 4-week intervention [24]. Considering 20% attrition, 8 participants were deemed sufficient to obtain a desired power of 80% at α = 0.05.

2.2. Methods

2.2.1. Experimental design

A placebo-controlled, double-blind, randomized trial was conducted. The TIG received a four-week tangeretin supplement intervention, while the PCG received a placebo. Both groups were instructed to perform the exercise program before and after the intervention. Throughout the study, the grouping details and assignment of tangeretin vs. placebo were blinded to the participants and investigators.

2.2.2. Tangeretin supplement intervention

Throughout the 4-week intervention, all participants were required to enter the laboratory and take a bottle of supplement drink (200 mL) assigned by a lab assistant early in the morning (7:30–8:30 a.m.) prior to their daily exercise training. The composition of the drink was unknown to both the participants and the lab assistant because it was prepared daily by another lab assistant in advance.

For TIG, the supplement drink was made using a dose of 20 g powdery mixture of whey protein isolate powder (CanSure, Vancouver, BC, Canada; China Anti-Doping Agency report NO: 2019FD279; ≥95% purity; 19.8 g) and tangeretin supplement powder (Qinguoren®, Chongqing, China; China Anti-Doping Agency report NO.: 2019FD234; 99.79% purity; 200 mg). The Qingguoren® tangeretin supplement powder was extracted and purified from citrus fruits using our self-developed techniques and has been developed into a commercial supplement product. It is yet to be massively produced and could only be purchased from the Southwest Institute of Fruits Nutrition (No. 73, Hongguang Avenue, Banan District, Chongqing, China). According to in vitro, animal, and in vivo studies [23,24,27–32], tangeretin supplementation at 200 mg/day has been proven safe for adults. For PCG, the supplement drink had identical aesthetics, weight, and flavor but contained only 19.8 g of whey protein isolate powder (≥95% purity).

During the study period, diets of all participants were strictly controlled by the Chongqing Competitive Sports Training Center to minimize the effects of food and condiments [33]. A registered dietician recorded each participant’s daily dietary intake in detail and submitted it to a laboratory assistant. The last assistant analyzed the diet record for macro/micronutrient content and energy using the “The Mint Nutritionist” APP (version 2.9.1, Mint Information Technology Co., Ltd, Shanghai, China). Aside from Gatorade sports drinks, athletes were also prohibited from other supplements, such as traditional Chinese medicine, fish oil, curcumin, polyphenols, and vitamins C and D. No participants reported signs or symptoms of discomfort during the study period.

2.2.3. Blood sample collection

To monitor the changes in each participant, blood samples were collected from the brachial venous vein by two experienced nurses on the first day of each week (T₁, T₂, T₃, and T₄) and the day after the intervention (T₅), as shown in Figure 3b. A total of 4 ml blood samples were collected as an aliquot in two different tubes (Tube A, 1 ml; Tube B, 3 ml). Tube A was analyzed using a hematology analyzer (Mindray BC-5150, China) to obtain leukocyte count (WBC), basophilia count (BAS), eosinophil count (EOSIN) and neutrophil (NEUT) within 10 min after collection. Tube B was centrifuged (TG16, Shuke, China) at 2,000 rpm for 15 min within 30 min after collection, and the isolated serum samples were stored at −80°C in a medical freezer (BDF-86V158, Haier, China). The serum samples were analyzed (SAL-6000 Immunoproduct line, Mindray, China; BioTek-Epoch Fluorescence Photometer, USA; Multiskan microplate, China) for obtaining the level of interleukin-5 (IL-5), interleukin-8 (IL-8), Clara cell protein 16 (CC16) and immunoglobulin E (IgE).

Figure 3.

Participants grouping (a), intervention and training protocol (b), and bronchial provocation tests (c).

2.2.4. Exercise challenge test

The exercise challenge test (ECT) is the most intuitive method for EIB diagnosis and therapy efficacy assessment of prophylactic drugs [34]. In this study, all participants underwent ECT the day before (T₁) and after (T₅) the intervention. ECT was conducted following the guidelines of the European Respiratory Society (ERS) and the American Thoracic Society (ATS) for ECT [35,35]. Each participant ran on a motorized treadmill (T150, Cosmed, Italy) with a portable heart rate monitor (M 430, Polar, Finland) and a nose clip. He/she was required to reach 80% of his/her maximum heart rate (HRmax) within 4 min and then maintain the speed for 6 min (80–90% HRmax), which was determined based on his/her HRmax (220-age) [35]. The entire procedure lasted no more than 10 min. The heart rate was monitored every 5 s. A nose clip was used to simulate mouth breathing to prevent the inhaled air from being conditioned by the nasal mucosa during running. All participants could only perform simple static muscle stretching and were informed of the target HR range of heart rate in advance. To minimize the effects of other factors, all participants were instructed to avoid caffeine, pulmonary medications, and strenuous physical activity within 24 h. The laboratory temperature and humidity were controlled and maintained under a standard state (20–22℃, <10 mg H₂O/L) [36].

2.2.5. Pulmonary function tests

Before and after ECT, all participants were instructed to undergo pulmonary function tests (Chest HI-101 Pulmonary Function Instrument, Japan) according to the ATS guidelines. Pulmonary function tests were performed before ECT (PRE) and at the 3rd min, 5th min, 10th min, 15th min, 20th min, and 30th min after ECT. For each test, the forced expiratory volume in 1 s (FEV₁), maximal voluntary ventilation (MVV), forced vital capacity (FVC), peak expiratory flow (PEF), maximal mid-expiratory flow curve (MMF), and FEV₁/FVC ratio were determined. To diagnose EIB, the decline rate (R) of FEV₁ was calculated based on the FEV₁ values before and after ECT using the following equation:

$$\mathit{R}=\frac{FE{V_1}_{\mathit{pre}}-\mathit{FE}{V_1}_{\mathit{post}}}{\mathit{FE}{V_1}_{\mathit{pre}}}\times 100$$

where $\mathit{FE}{V_1}_{\mathit{pre}}$ is the FEV₁ value before ECT (PRE), and $\mathit{FE}{V_1}_{\mathit{post}}$ is the FEV₁ value after ECT (at 3rd min, 5th min, 10th min, 15th min, 20th min, and 30th min). Accordingly, the participant was diagnosed as EIB-positive with R ≥ 10% [35,35].

EIB is usually accompanied by respiratory discomfort, such as cough, wheezing, chest tightness, and shortness of breath [36]. In this study, respiratory symptoms were recorded for each participant when they performed ECT on the first day of the tangeretin supplement intervention (ECT₁) and the first day after the intervention (ECT₂).

2.2.6. VO_2max test

In the first 24 h of the VO_2max test, all athletes were required to avoid high-intensity exercise. The direct test method was adopted in this study, with specific operations as follows (Figure 3d): Subjects exercised on a treadmill (model: T150, COSMED, Italy) at a speed of 8 km/h for 5 min and then at an average speed of 1 km/h per minute in a linear manner until exhaustion. In this study, according to the report of Oyama et al. [37], if two or more of the following conditions are met: 1) the athlete is unable to maintain the prescribed exercise speed; 2) VO₂ no longer increases and a platform appears; 3) exercise heart rate greater than the maximum calculated heart rate, that is, [(220-age) −10 bmp]; 4) respiratory quotient greater than 1.10; and 5) blood lactate level greater than 8 mmol/L after exercise. All subjects entered the laboratory from 14:00 to 17:00 on V1 and V2 and conducted the corresponding tests sequentially. The cardiopulmonary exercise assessment system (Model: Cortex, Germany; Model: M430, Polar, Finland) continuously measured the expiratory components and heart rate. Referring to relevant studies^[A4], the maximum value of VO₂ was set as VO_2max in this study, and the corresponding speed and heart rate when VO_2max appeared during exhaustive exercise were identified as the VO_2max speed and VO_2max heart rate, respectively. During the tests, VO_2max, VO_2max heart rate, VO_2max speed, Oxygen Pulse rate (VO₂/HR), and time to exhaustion (TLim) were recorded.

2.3. Statistical analysis

Statistical analyses were conducted using SPSS version 25.0. The results were shown as mean ± SD. An independent sample t-test was used to compare anthropometric differences between the groups. Two-way (two groups × different time points) repeated-measures analysis of variance was used to determine the differences in respiratory inflammation indicators and pulmonary ventilation function indicators. If a significant difference was indicated, pairwise comparisons were performed using the least significant difference (LSD). The chi-square test was used to compare differences between the two groups regarding the proportion of participants with and without respiratory symptoms. The significance level was set at p < 0.05.

3. Results

3.1. Participants general information and respiratory symptoms

Table 1 shows the general information of all participants. Before the intervention, there were no statistically significant differences in age, height, weight, weekly training hours, history of allergies, smoking history, or other general information between the TIG and the PCG. In addition, there was no significant difference between TIG and PCG, respectively, for daily energy intakes (9767 ± 2155 kJ vs. 10456 ± 2399 kJ) and the percent energy from protein (27% ± 5% vs 28% ± 2%), fat (21% ± 4% vs 20% ± 3%) and carbohydrate (51% ± 4% vs 50% ± 4%). The dietary records of all the participants revealed that they had normal eating patterns and no fad diets, signs, or eating disorders.

Table 1.

The general information of TIG and PCG.

	TIG (n = 15)	PCG (n = 15)	t	P
Age (yrs)	24.08 ± 3.49	24.19 ± 2.89	0.934	0.797
Height (cm)	180.11 ± 9.59	182.11 ± 10.99	0.001	0.912
Weight (kg)	67.78 ± 9.18	69.78 ± 10.41	1.053	0.688
Weekly training hours (h)	30.18 ± 7.15	31.26 ± 7.27	0.131	0.898
Training years (yrs)	5.61 ± 1.21	5.72 ± 1.33	0.032	0.858
History of allergy n (%)	3(20.0%)	5(33.3%)	0.396	0.411
Smoking history n (%)	1(6.7%)	1(6.7%)	0.000	0.758
Smoking history ≥3 yrs (%)	0(0%)	1(6.7%)	0.969	0.516
Smoking history <3 yrs (%)	1(6.7%)	0(0%)	0.969	0.516
Family history of asthma n (%)	1(6.7%)	2(13.3%)	0.303	0.523
β2-agonist therapy n (%)	1(6.7%)	1(6.7%)	0.000	0.758
Therapeutic Use Exemption n (%)	1(6.7%)	0(0%)	0.969	0.516

Before the 4-week tangeretin intervention (7 days before T1 day, from Nov.1st to Nov.7th), there was no significant difference in respiratory symptoms between the TIG and PCG groups, as shown in Figure 4. Seven days before the end of the intervention (between the period of T₄ and T₅, from Nov 29th to Dec 5th), the number of participants with “dyspnea” in TIG decreased from 4 to 2 (p = 0.386), while the number did not change in PCG. The number of participants with “wheeze” increased from 1 to 2 in TIG, while PCG still had no change. The number of cases of “chest tightness” increased from 1 to 2 in the TIG and from 0 to 2 in the PCG. For the symptom of “cough,” the cases decreased from 9 to 3 (p = 0.128) in TIG and decreased from 11 to 9 (p = 0.798) in PCG. The two groups had no significant differences (p = 0.128).

Figure 4.

Effects of tangeret in intervetion on respiratory symptoms of TIG and PCG.

3.2. Blood inflammatory parameters

The changes in inflammatory parameters in routine blood are shown in Table 2. During the intervention, eosinophil count in the TIG showed a downward trend, significantly lower than the initial value (T1) at T5 (p = 0.049), whereas no change was observed in the PCG. The changes in basophil counts were significant within and between the two groups. Neutrophils in the TIG showed a fluctuating downward trend significantly lower than the initial value (T1) at T5 (p = 0.039). In the PCG, the number of neutrophils remained stable. In terms of differences between groups, the number of neutrophils in the TIG was significantly lower than that in the PCG at T5 (p = 0.033).

Table 2.

Effects of tangeretin intervetion on leukocyte, eosinophils, Basophil, and neutrophil.

	Leukocyte (109/L)		Eosinophils (109/L)		Basophil (109/L)		Neutrophil (109/L)
	TIG (n = 15)	PCG (n = 15)	TIG (n = 15)	PCG (n = 15)	TIG (n = 15)	PCG (n = 15)	TIG (n = 15)	PCG (n = 15)
T1	6.45 ± 2.18	7.01 ± 2.46	0.26 ± 0.22	0.23 ± 0.17	0.038 ± 0.016	0.039 ± 0.015	3.88 ± 1.46	3.91 ± 1.29
T2	6.11 ± 1.52	5.49 ± 0.99	0.21 ± 0.09	0.21 ± 0.17	0.041 ± 0.023	0.036 ± 0.015	4.01 ± 2.01	3.93 ± 1.87
T3	6.09 ± 1.43	5.65 ± 0.77	0.23 ± 0.11	0.27 ± 0.26	0.047 ± 0.026	0.044 ± 0.021	3.66 ± 1.33	3.98 ± 1.52
T4	6.14 ± 1.63	6.43 ± 1.51	0.24 ± 0.16	0.27 ± 0.17	0.045 ± 0.019	0.046 ± 0.021	3.71 ± 1.45	3.84 ± 1.22
T5	6.13 ± 1.27	5.87 ± 1.62	0.16 ± 0.11 #	0.24 ± 0.19	0.042 ± 0.018	0.047 ± 0.034	3.44 ± 1.12 #*	3.92 ± 1.62
Intraclass Correlation Coefficient	0.558	0.344	0.443	0.848	0.179	0.303	0.154	0.779
Coefficient of Variation	26 %	24.2 %	63.5 %	80.8 %	48.1 %	52.3 %	39.4 %	33.7 %
Cohen’s d	0.18	0.55	0.56	−0.03	−0.36	−0.31	0.34	−0.01
Main effect – Time	F = 1.78; p = 0.157; η2 = 0.182		F = 1.581; p = 0.202; η2 = 0.124		F = 0.508; p = 0.454; η2 = 0.085		F=4.83; p = 0.009; η2 = 0.821
Main effect – Group	F = 0.359; p = 0.565; η2 = 0.043		F = 0.037; p = 0.809; η2 = 0.006		F = 0.835; p = 0.866; η2 = 0.003		F=4.09; p = 0.012; η2 = 0.484
Interaction – Time × Group	F = 1.49; p = 0.228; η2 = 0.157		F = 1.604; p = 0.278; η2 = 0.107		F = 0.726; p = 0.768; η2 = 0.044		F=1.249; p = 0.373; η2 = 0.471

TIG: tangeretin intervention group, PCG: placebo group. # p < 0.05 vs T1 (TIG), *p < 0.05 vs PCG.

Changes in serum inflammatory parameters in the TIG and PCG groups are shown in Table 3. The level of IL-5 in the TIG remained stable at T2 (p = 0.538) and T3 (p = 0.600) and then decreased gradually to T5, which was significantly lower than the initial value (T1) (p = 0.037). In terms of differences between the groups, the IL-5 levels in the TIG were significantly lower than those in the PCG at T5 (p = 0.022). IL-8 levels of IL-8 in the TIG decreased gradually and were substantially lower than the initial values (T1) at T4 (p = 0.033) and T5 (p = 0.003). The IL-8 levels in the PCG increased steadily and were significantly higher than the initial value (T1) at T3 (p = 0.042). For the difference between groups, the level of IL-8 at T4 (p = 0.050) and T5 (p = 0.012) in TIG was significantly higher than that in PCG. The level of CC16 in the TIG decreased gradually, but there was no statistical difference compared with the initial value (T1). CC16 levels in the PCG showed an upward fluctuating trend. Between the groups, the CC16 levels in the TIG were lower than those in the PCG at T2, T3, T4, and T5, but no statistical difference was observed. IgE levels in the TIG showed a fluctuating descent, which was significantly lower than the initial value (T1) at T2 (p = 0.031), T3 (p = 0.032), T4 (p = 0.036), and T5 (p = 0.011). In contrast, there was no significant change in IgE levels in the PCG or between the two groups.

Table 3.

Effects of tangeretin intervetion on sreum IL-5, IL-8, CC16, and IgE.

	IL-5 (pg/mL)		IL-8 (ng/mL)		CC16 (ng/ml)		IgE (μg/L)
	TIG (n = 15)	PCG (n = 15)	TIG (n = 15)	PCG (n = 15)	TIG (n = 15)	PCG (n = 15)	TIG (n = 15)	PCG (n = 15)
T1	25.57 ± 4.89	24.26 ± 5.37	47.73 ± 7.45	43.03 ± 5.63	28.13 ± 5.37	27.66 ± 5.03	3203.57 ± 883.19	3052.73 ± 1027.82
T2	24.64 ± 4.32	25.56 ± 4.68	46.55 ± 6.46	43.68 ± 5.37	27.10 ± 4.75	27.03 ± 4.49	2814.28 ± 765.41 #	3004.55 ± 1131.22
T3	26.60 ± 4.89	27.37 ± 7.66	45.13 ± 4.55	46.62 ± 5.35 &	29.26 ± 5.38	37.49 ± 17.09	2533.57 ± 907.11 #	2890.00 ± 1055.23
T4	23.42 ± 4.95	24.80 ± 6.29	41.32 ± 4.06 #*	45.13 ± 7.55	25.76 ± 5.44	31.68 ± 10.93	2678.57 ± 710.23 #	3120.91 ± 1128.21
T5	20.61 ± 5.51 #*	26.29 ± 6.31	39.48 ± 4.49 #*	45.64 ± 6.32	24.82 ± 4.05	34.35 ± 12.87	2432.14 ± 737.85 #	2933.64 ± 1198.01
Intraclass correlation coefficient	0.18	−0.029	0.209	0.174	0.521	0.907	0.116	0.36
Coefficient of Variation	20.3 %	23.6 %	12.6 %	10.8 %	18.5 %	36.3 %	29.3 %	36.9 %
Cohen’s d	0.95	−0.35	1.29	−0.57	0.69	−0.46	0.95	0.11
Main effect – Time	F = 1.214; p = 0.282; η2 = 0.098		F = 2.381; p = 0.068; η2 = 0.192		F = 2.257; p = 0.077; η2 = 0.523		F = 3.049; p = 0.028; η2 = 0.612
Main effect – Group	F = 1.762; p = 0.507; η2 = 0.037		F = 0.739; p = 0.410; η2 = 0.069		F = 3.055; p = 0.106; η2 = 0.203		F = 0.556; p = 0.473; η2 = 0.053
Interaction – Time × Group	F = 2.039; p = 0.078; η2 = 0.158		F = 5.129; p = 0.002; η2 = 0.339		F = 2.516; p = 0.054; η2 = 0.231		F = 2.248; p = 0.081; η2 = 0.340

TIG: tangeretin intervention group, PCG: placebo group. # p < 0.05 vs T1 (TIG), & p < 0.05 vs T1 (PCG), * p < 0.05 vs PCG.

3.3. Pulmonary ventilation parameters

The changes in the FEV₁ decline rate (R) at the first and second ECT sessions in the TIG and PCG are shown in Figure 5. At ECT1, there was no significant difference in the rate of decline between FEV₁ of the two groups. At ECT2, the overall FEV₁ decline rate in TIG significantly decreased from 16.12 ± 4.97% (ECT1) to 9.92 ± 9.03% (p = 0.013), Figure 5a. While the overall FEV₁ decline rate in PCG slightly fell from 17.60 ± 8.36% (ECT1) to 15.26 ± 5.54%, there was no statistical difference (p = 0.444), Figure 5b. The overall FEV₁ decrease rate in the TIG was lower than in the PCG during ECT2, but the difference was not statistically significant (p = 0.061) (Figure 5c). In addition, during the second ECT (ECT2), the change in the rate of decline of FEV₁ in the TIG was significantly lower than that in the PCG at 5^th min (p = 0.024) and 10^th min (p = 0.015), Figure 5d.

Figure 5.

Effects of tangeret in intervetion on the FEV1 declinerate(%).

At ECT2, the rate of decline of FEV₁ of six TIG participants (male, No. 1, 3, 5, 7; female, No. 8 and 12) dropped to less than 10% (the rate of decline of FEV₁ ≥10% was diagnosed as EIB-positive), and the overall negative conversion rate of EIB in TIG was 40.00% (6/15), Figure 6a. One female participant (no. 7) in PCG became EIB-negative (FEV₁ decrease rate = 9.84%), and the overall EIB negative rate was 6.66% (1/15), as shown in Figure 6b. In addition, there was no significant difference in the negative conversion rate of EIB between the two groups (p = 0.096).

Figure 6.

The FEV1 declinerate(%) at ECT1 and ECT2 of each participant.

At ECT1, there were no significant differences in FEV₁, FVC, or MVV between the two groups. During ECT2, the FEV₁ value of the TIG significantly increased. It was considerably higher in 3rd min (p < 0.001), 5th min (p < 0.001), 10th min (p = 0.001), 15th min (p = 0.016) and 20th min (p = 0.028) than those in corresponding time points of ECT1; Between groups, the FEV₁ value of TIG was significantly higher than that of the PCG at the 5th min (p = 0.044) and 10th min (p = 0.041), Figure 7a. At ECT2, the FVC value of TIG was significantly higher than that of ECT1 at 3rd min (p = 0.061), 5th min (p = 0.006), 10th min (p < 0.001), 15th min (p = 0.012) and 20th min (p = 0.057); The FVC value of PCG also increased to varying degrees, but no statistical difference was observed; Between groups, the FVC value of TIG was significantly higher than that of PCG at time point of 5th min (p = 0.046), 10th min (p = 0.031) and 15th min (p = 0.038), Figure 7b. At ECT2, the MVV values in both groups increased but without statistical significance (Figure 7c).

Figure 7.

Comparison of the FEV1, FVC and MVV at ECT1 and ECT2 in TIG and PCG.

At ECT1, there were no significant differences in PEF, FEV₁/FVC ratio, or MMF between TIG and PCG. At ECT2, the PEF and MFF values at 5th min in the TIG were significantly higher than those at the corresponding time points of ECT1 (p = 0.047 and p = 0.039, respectively), while there were no significant changes in the PCG (Figure 8a,c). The FEV₁/FVC ratio at the 5th min and 10th min in the TIG were significantly higher than those at the corresponding time points in the ECT1 (p = 0.021, p = 0.017), while there were no significant changes in PCG; In addition, the FEV₁ value of the TIG was significantly higher than that of the PCG at the 10th min (p = 0.046) (Figure 8b).

Figure 8.

Comparison of the PEF, FEV1/FVC and MMF at ECT1 and ECT2 in TIG and PCG.

3.4. VO_2max test result

Table 4 shows the changes in maximal oxygen uptake and its derived indices between the TIG and PCG groups. Compared to V1, there were no significant changes in VO_2max, VO_2max HR, VO₂/HR, VO2max speed, or TLim in the PCG at V2. At the same time, VO_2max (p = 0.042) and TLim (p = 0.050) significantly increased in the TIG. VO₂/HR (p = 0.147) and VO_2max speed (p = 0.304) were slightly improved.

Table 4.

Effects of Tangeretin Intervetion on the VO₂max, VO₂max HR, VO₂/HR, VO₂max speed and TLim.

	VO2(mL/min/kg)		VO2max HR(bpm)		VO2/HR(ml)		VO2max Speed(km/h)		TLim(min)
	TIG (n = 15)	PCG (n = 15)	TIG (n = 15)	PCG (n = 15)	TIG (n = 15)	PCG (n = 15)	TIG (n = 15)	PCG (n = 15)	TIG (n = 15)	PCG (n = 15)
ECT 1	52.77 ± 6.82	56.31 ± 4.80	188.92 ± 6.85	195.00 ± 6.38	17.31 ± 2.90	17.54 ± 3.45	15.55 ± 1.73	15.77 ± 1.18	12.27 ± 1.79	12.67 ± 1.12
ECT 2	54.54 ± 5.18#	54.06 ± 4.85	188.08 ± 9.00	189.32 ± 7.43	17.72 ± 2.47	17.38 ± 2.81	15.97 ± 1.37	15.68 ± 1.19	13.03 ± 1.35#	12.36 ± 1.11
Intraclass Correlation Coefficient	0.881	0.821	0.433	0.46	0.93	0.979	0.597	0.827	0.613	0.708
Coefficient of Variation	11.9 %	8.8 %	3.6 %	2.7 %	15.1 %	18.9 %	8.6 %	7.2 %	11.5 %	8.3 %
Cohen’s d	−0.27	0.25	0.11	0.8	−0.15	0.05	−0.27	0.08	−0.48	0.28
Main effect – Time	F = 0.147; p = 0.708		F = 2.787; p = 0.121		F = 0.005; p = 0.942		F = 0.012; p = 0.914		F = 0.187; p = 0.673
Main effect – Group	F = 0.744; p = 0.405		F = 1.724; p = 0.214		F = 0.558; p = 0.469		F = 0.93; p = 0.354		F = 1.308; p = 0.275
Interaction – Time × Group	F = 18; p = 0.001		F = 2.737; p = 0.124		F = 3.356; p = 0.092		F = 0.96; p = 0.347		F = 5.84; p = 0.033

Note: Compared with the initial value (E1), # P<0.05

3.5. Result of simple effect analysis

All the dependent variables with significant group × time interaction effects are analyzed with simple effects, and the results are as follows.

FEV₁: The mean of the PCG group at each time point following exercise in both ECT1 and ECT2 was significantly different from the baseline measurements taken before exercise (p < 0.002). Similarly, the mean of the TIG group at each time point following exercise in ECT1 was significantly different from baseline (p < 0.039), mirroring the pattern observed in the PCG group. However, in the TIG group during ECT2, a significant difference was observed only at the 5-min post-exercise mark compared with the baseline (p = 0.014), with no significant differences detected at other time points. FEV₁/FVC: The results of ECT2 measurements post-intervention indicated significant differences between the TIG and PCG groups both prior to exercise and at the 10th and 15th minutes post-exercise (p ≤ 0.003). Within the PCG group, significant differences were observed at the 3rd, 5th, and 7th minutes post-exercise compared to pre-exercise levels (p ≤ 0.035). In the ECT1 of TIG, a significant difference was noted at the 5th-minute post-exercise compared to pre-exercise levels (p = 0.028); However, in ECT2 of TIG, no significant differences were found at any post-exercise time points compared to pre-exercise levels.

IL-8: Significant differences were observed between the TIG and PCG groups at weeks 3 (p = 0.021) and 4 (p = 0.01). Within the TIG group, significant differences were noted between T1 and T5 (p = 0.03); however, no significant differences were found within the PCG group.

VO_2max: The TIG group exhibited a statistically significant difference between pre- and post-intervention (p = 0.042), whereas the PCG group did not demonstrate a statistically significant change. TLim: The TIG group showed a statistically significant difference between the pre- and post-measurements (p = 0.05), whereas the PCG group did not exhibit a statistically significant difference.

4. Discussion

This study aimed to evaluate the therapeutic effect of a 28-day tangeretin (200 mg/d) combined with whey protein (19.8 g/d) intervention in professional athletes with EIB. The evaluated parameters included changes in lung ventilation function, serum inflammatory cytokine levels, and airway epithelial injury biomarkers before and after the intervention. The results indicated that the 28-day tangeretin combined with whey protein intervention effectively improved EIB in professional athletes, which was attained within the framework of the athletes’ routine implementation of the training program. Tangeretin, a nonpharmacological intervention devoid of banned substances, has practical significance because it effectively mitigates the potential risks associated with drug-related side effects and illicit doping practices among athletes.

EIB is characterized by temporary constriction of the lower respiratory tract, leading to a decrease in lung ventilation indicators such as FEV₁ and FVC due to airflow limitation [10]. The diagnostic criteria for EIB include a ≥10% decrease in FEV₁ after bronchial provocation, such as exercise challenge testing (ECT) [10]. In this study, all athletes exhibited an FEV₁ decline rate of ≥10% within 30 min after ECT, meeting the diagnostic criteria for positive EIB. The findings indicate that following a 28-day intervention, the rate of decline in FEV₁ in the tangeretin combined with whey protein intervention group (TIG) decreased significantly from 16.12 ± 4.97% to 9.92 ± 9.03%, placebo (whey protein) control group (PCG) decreased from 17.6 ± 8.36% to 15.26 ± 5.54%. Specifically, the FEV₁ decline rate of six athletes in the TIG group dropped to below 10%, resulting in an overall EIB protection rate of 40.00%. In contrast, there was no notable change in the FEV₁ decline rate in the PCG, with a protection rate of 0%. The findings of this study indicate that a 28-day intervention with tangeretin combined with whey protein led to a significant improvement in the rate of decline of expiratory flow rate indicators, including FEV₁ and FEV₁/FVC, in athletes with EIB. But placebo did not lead to significant change. Furthermore, athletes in the tangeretin intervention group exhibited notable improvements in dyspnea and cough symptoms, whereas those in the placebo group showed only a slight improvement in cough symptoms.

The primary pathogenesis of EIB involves airway inflammation triggered by osmotic and cold stimuli and is exacerbated by repeated dehydration of the respiratory mucosa during intense physical activity [38]. Inflammation is characterized by elevated levels of inflammatory cytokines in the blood. Santos et al. [39] demonstrated that athletes with EIB (EIB+) exhibited significantly greater increases in blood eosinophils, neutrophils, and serum IL-5 levels following bronchial provocation tests than athletes without EIB (EIB-). Prior epidemiological studies involving 411 participants demonstrated that EIB+ athletes exhibited significantly elevated levels of blood eosinophils, neutrophils, and serum IL-8 compared to EIB athletes [25]. Alterations in blood inflammatory cytokine levels may serve as a supplementary measure for assessing the efficacy of EIB treatment. For instance, Das et al. [40] observed a significant reduction in serum IgE and IL-5 levels in the alveolar fluid of BALB/c mice treated with the natural flavonoid luteolin, leading to the effective inhibition of respiratory inflammation. Another study discovered that inhaled corticosteroids, a traditional pharmacological therapy for EIB, inhibited airway hypertonicity, cooling, and rewarming-induced production of IL-8 and chemokines [41]. The results of our study indicate that after 28 days of tangeretin (200 mg/d) combined with whey protein (19.8 g/d) intervention, resting blood eosinophils, neutrophils, serum IL-5, IL-8, and IgE levels reduced in EIB athletes. However, after 28 days of placebo (whey protein, 19.8 g/d), resting blood eosinophils, basophils, serum IL-5 and IL-8 levels were increased in EIB athletes. It should be noted that IL-8 was significant between groups, while eosinophils, basophils, IL-5 and IgE were significant only within TIG. This suggests that tangeretin combined with whey protein treatment may effectively alleviate airway inflammation in EIB, but placebo may not.

During intense physical activity, hyperventilation can damage the airway epithelial barrier, heightened risk of airway inflammation, and the subsequent development of EIB. Those engaging in prolonged and rigorous exercise are particularly susceptible to airway epithelial injury due to increased ventilation flow, contributing to the elevated prevalence of EIB within this population [42]. CC16 serves as a biomarker of airway epithelial injury [2]. Serum CC16 levels increased significantly due to the disruption of tight junctions in the airway epithelial barrier [42]. Anderson et al. highlighted that certain athletes reported symptoms of discomfort, including respiratory pain, following high-intensity exercise with a concomitant rise in serum CC16 levels [43]. Additionally, Liu’s epidemiological study revealed that serum CC16 levels were notably elevated in athletes with EIB compared to those without, even during periods of rest in the morning >24 h post-exercise [25]. Airway epithelial injury biomarkers can serve as additional indicators for evaluating the risk of EIB in athletes. Our study demonstrated that a 28-day tangeretin combined with whey protein intervention significantly decreased the serum CC16 levels in EIB+ athletes, whereas no significant changes were observed in the placebo group over the same period. This finding suggests that the observed decrease may be attributed to the inhibition of airway inflammation and the subsequent promotion of airway epithelial barrier repair.

Tangeretin, a polymethoxy flavonoid derived from citrus peels, exhibits anti-inflammatory properties. In vitro, experiments have demonstrated that when tangeretin is combined with luteolin at a concentration of 30 μM, a synergistic anti-inflammatory effect is observed in the lipopolysaccharide (LPS)-induced inflammation model. This combination inhibits the secretion of inflammatory cytokines, including NO, IL-1β, IL-5, and PGE2, by 44%, 44%, 55%, and 80%, respectively [44]. Additionally, Xu et al. found that tangeretin suppresses the phosphorylation of Extracellular Regulated Protein Kinases (ERK), IκB-α, IKK-β, and P38 induced by LPS [45]. Tangeretin has been shown to effectively inhibit the signaling pathways of various inflammatory mediators by reducing the phosphorylation of p38 MAPK, a crucial intracellular signal transduction molecule. The activation of the p38 MAPK pathway is central to the development of airway inflammation and hyperresponsiveness [46,47]. Additionally, tangeretin enhances mitochondrial membrane potential [47]. Furthermore, Liu et al. discovered that administering tangeretin at a dose of 50 mg/kg/day for 5 days resulted in a significant reduction in Th2, Th17, and IgE levels in P12 mice, leading to expedited elimination of airway hyperresponsiveness (AHR) [48]. The AHR is a crucial characteristic of EIB. These findings offer a plausible rationale for the mechanism by which tangeretin ameliorates airway inflammation in EIB. Our findings validate the effectiveness of tangeretin intervention in the prevention and treatment of EIB.

Finally, we assessed VO_2max in both groups before and after the intervention. Our results demonstrated that following tangeretin intervention, the VO_2max of athletes with EIB significantly increased compared to the pre-intervention levels, whereas no significant change was observed in the placebo group. This pattern was consistent with the observed changes in FEV₁, FVC, and FEV₁/FVC ratio before and after the intervention. We hypothesized that effective management of EIB in the TIG group was the primary factor contributing to the observed increase in VO_2max. Because research has indicated that the impairment or reduction in pulmonary ventilation function parameters (e.g. FEV₁, FVC, FEV₁/FVC) [49] is a critical factor in limiting $\dot{V}$O_2max. Conversely, the slight decrease in VO_2max in the PCG suggests inadequate control of the EIB, which aligns with the findings of the Sonna study [50]. Sonna et al. [50] implemented a two-month regular medium–high intensity aerobic training regimen for American soldiers. They observed a significant increase in VO_2max among healthy soldiers, from 50.6 ± 0.9 mL/min/kg pre-training to 52.2 ± 1.8 mL/min/kg post-training. Conversely, soldiers with EIB exhibited a slight decrease in VO_2max, from 53.5 ± 3.2 mL/min/kg before training to 52.2 ± 1.8 mL/min/kg after training. Furthermore, the TLim in the TIG group was significantly extended post-intervention, whereas it exhibited a slight reduction in the PCG. These findings indicate that the enhancement in physical performance observed in the TIG group was associated with the effective control of EIB. In contrast, the decline in physical performance in the PCG group was attributable to poor EIB management.

The advantage of our study is that it is the first practical case of tangeretin being applied to the treatment of EIB + in professional athletes, which completes the gap in human experimental data from previous studies. In terms of study control, randomized paired double-blind placebo-controlled trials ensured the objectivity of the results. In terms of the results of the study, 28-day (200 mg/d) tangeretin intervention improved about 40% of athletes from EIB+ to EIB-. Considering that tangeretin is a nonpharmacologic measure, but the therapeutic effect is close to traditional drugs of EIB (leukotriene antagonist protection rate is about 50%; short-acting/long-acting β2 agonists protection rate is approximately 68%) [51], which shows considerable promise for the treatment of EIB with tangeretin. This study had certain limitations. The dose of, 200 mg/kg is still far from the upper limit of the safe dose range(<5 mg/kg) [31]. However, we did not administer tangeretin at mg/kg/d to each subject; instead, we administered the intervention at a uniform dose of 200 mg/d, which may have slightly contributed to individual differences in the therapeutic effect. Furthermore, no significant group × time interaction terms were found for white blood cells. Only the group × time interaction of IL-8 was substantial. So, it is worth noting that the “differences” we report may only be within-group changes. But the other inflammatory cytokines/proteins (0.054–0.081) were very close to the criteria of significance. And the Cohen’s d values in the TIG group were moderate to highly effective, while the d values in the PCG group were low effect sizes.

5. Conclusion

This study demonstrated the novel use of tangeretin, a natural fruit extract nutrient, for treating EIB in professional elite athletes. Results indicated that a 4-week supplementation of 200 mg tangeretin combined with 19.8 g whey protein isolate powder per day alleviated respiratory symptoms, effectively suppressed inflammatory factors and airway injury markers in the blood of EIB+ athletes, and significantly improved pulmonary ventilation indicators such as FEV₁ and FVC following exercise challenge testing. Additionally, in the treatment intervention group, six out of 15 EIB+ athletes converted to EIB status post-intervention, resulting in an overall protection rate of 40.00%, demonstrating a favorable therapeutic outcome.

Funding Statement

The study was supported by Research and innovation Grant for Graduate Students, Shanghai University of Sport [Project No. YJSCX-2023-037].

Disclosure statement

No potential conflict of interest was reported by the author(s).

Data availability statement

The data presented in this study are available on request from the corresponding author. The data are not publicly available due to ethical, legal, and privacy issues.

Informed consent statement

Informed consent was obtained from all subjects involved in the study.

Institutional review board Statement

The study was conducted in accordance with the Declaration of Helsinki and approved by the Ethics Committee of Shanghai University of Sport. (Reference No.: 102772020RT082; 23 June 2020).