Effect of tangeretin supplements on simulated precompetition weight loss in female combat sports athletes

Abstract

Background

Weight reduction before competition has long been a major challenge for combat-sports athletes. Unscientific weight-loss practices not only increase the risk of hormonal imbalance, electrolyte disturbance, and impaired immune function but also compromise athletic capacities such as aerobic and anaerobic performance and explosive power. This study investigated the potential benefits of tangeretin, a natural citrus extract, on athletic performance. These benefits include the regulation of hormone secretion, the optimization of carbohydrate and lipid metabolism, and the promotion of muscle synthesis and lipid utilization, which may help athletes reach their target weight.

Objective

To examine the effects of tangeretin supplementation on body-composition and sports-performance indicators in female combat-sports athletes, and to evaluate its efficacy for simulated precompetition weight loss.

Methods

Thirty female combat-sports athletes (4 wrestlers, 3 boxers, 9 sanda athletes, 3 judokas, and 11 taekwondo athletes) were recruited. A paired, randomized, double-blind design was used. All 30 participants were first paired according to sport and competitive level, then randomly allocated to either a tangeretin group or a placebo group (15 per group). The 30-day intervention comprised a double-blind nutritional supplement, a dietary questionnaire, blood sampling and analysis, body-composition assessment, isokinetic muscle-strength testing, and aerobic-capacity testing, to evaluate the effects of tangeretin (400 mg/day) on performance and health-related outcomes. These outcomes included body-composition indicators (e.g. body-fat percentage, fat mass, lean-body mass), fat-distribution indicators (e.g. limb and regional fat mass, gynoid fat mass), glucose and lipid-metabolism indicators (e.g. LDL-C, total cholesterol, free fatty acids), hormone indicators (e.g. cortisol, testosterone), muscle-strength indicators (e.g. peak torque of hip, knee, and ankle flexors and extensors), and maximal-oxygen-uptake-derived indicators (e.g. VO₂max heart rate, time to exhaustion). Statistical significance was assessed using two-way repeated-measures analysis of variance (ANOVA).

Results

(1) Body weight: 80.0% of athletes in the tangeretin group lost weight to varying degrees, and 33.3% (5 athletes) reached their competition weight class. (2) Body fat: body-fat percentage in the tangeretin group decreased from 23.92 ± 0.87% to 22.92 ± 0.89%; trunk, upper-limb, lower-limb, and android-region fat mass decreased by 6.77%, 8.77%, 4.43%, and 8.09%, respectively. (3) Glucose and lipid metabolism: HDL-C increased by 8.86% (P = 0.002), whereas triglycerides decreased by 25.00% (P = 0.048). (4) Sports performance: peak torque during hip, knee, and ankle flexion and extension increased significantly in the tangeretin group. VO₂max also improved from 42.80 ± 1.13 to 43.46 ± 1.32 ml/kg/min.

Conclusion

Tangeretin supplementation effectively reduced body weight and fat mass (both overall and regionally) in female combat-sports athletes. Among the participants, 80.0% lost weight to varying degrees, and 33.3% reached their competition weight class, indicating a strong effect on precompetition weight loss. Moreover, tangeretin improved aerobic capacity and lower-limb joint muscle strength, thereby enhancing competitive performance.

1. Introduction

The problem of precompetition weight loss has always been a major challenge for athletes in combat sports (e.g. wrestling, boxing, and judo) [1]. Studies have shown that 85%–90% of combat sports athletes need to lose weight before competitions, among whom 43.6% lose 5.0%–15% of their body weight, and approximately 3% miss competitions because of failed weight loss [2,3]. At the 2024 Paris Olympics, the Indian wrestler Vinesh Phogat was disqualified for being 100 g overweight, which highlights the strictness of the weight classification system in combat sports and the importance of choosing appropriate precompetition weight-loss strategies.

Currently, commonly used precompetition weight loss methods for athletes include gradual body weight reduction (GBR) and rapid weight loss (RWL). GBR relies mainly on low-calorie dietary intervention (cycle: 1–12 weeks, caloric range: 80–135 kJ/kg/day), whereas RWL uses high-temperature sauna dehydration or weight-control clothing shortly before competition (2–20 h) [4,5].

Multiple studies have confirmed that these weight loss methods can cause problems such as hormonal secretion disorders, fluid–electrolyte imbalance, and decreased immune function, and lead to declines in various athletic capacities, such as aerobic capacity, anaerobic capacity, and explosive power, thereby negatively affecting athletic performance and competitive readiness [1,5–8]. Webster et al. [9] reported that after 36 h of rapid dehydration, wrestlers’ maximal oxygen uptake (VO₂max) and time to exhaustion (TLim) decreased by 6.7% and 13.4%, respectively. Hammer et al. [10] reported that during RWL interventions, for every 1% decrease in body weight, the risk of sports injuries increased by 11%. In addition, long-term low-calorie diets and rapid dehydration with salt restriction can cause serious problems, such as depression, irritability, and suppressed immunity in athletes [11]. Moreover, some combat sports athletes resort to banned substances because they cannot effectively manage their precompetition weight. According to data from the China Anti-Doping Agency [12], from January 2020 to December 2024, 16 cases of illegal doping use were recorded among domestic combat sports athletes, all involving S5 diuretics (e.g. furosemide and hydrochlorothiazide). These cases accounted for 27.3% of all doping violations in China during the same period, causing widespread public concern and seriously damaging the fairness and integrity of competitive sports.

Addressing the challenge of precompetition weight loss while minimizing the reduction of fat-free mass (FFM) and mitigating the decline in precompetition athletic performance has emerged as a central focus of research within the domain of sports science [1,10]. In preliminary work, the team used tangeretin, a natural citrus extract, to intervene in athletes across different events and showed that tangeretin can regulate hormone secretion (e.g. testosterone and cortisol), optimize glucose and lipid metabolism (e.g. fatty acids and glucose), and accelerate muscle synthesis and lipid consumption [13–15]. One study reported that after 28 days of tangeretin intervention (200 mg/day), the body-fat percentage of track and field athletes decreased from 13.8 ± 3.5% to 12.7 ± 3.9%, and the muscle mass increased from 29.2 ± 5.7 to 31.2 ± 4.3 kg [14]. A separate study demonstrated that tangeretin supplementation significantly enhances the secretion of adiponectin, an insulin-sensitizing factor, by 3T3-L1 adipocytes, while simultaneously reducing the secretion of monocyte chemoattractant protein-1, a factor associated with insulin resistance [16]. Tangeretin can also inhibit the further accumulation of triglycerides [16]. These studies suggest that tangeretin may have potential in weight control, including fat reduction and muscle gain, in athletes. However, whether it can be applied to solve athletes’ precompetition weight loss problems needs further research. Building on these findings, the present study seeks to investigate the effects of tangeretin supplementation on precompetition weight loss in female athletes participating in combat sports, utilizing a simulated precompetition weight reduction protocol. If the research results meet or partially meet expectations, they may provide a new solution to the long-standing issue of precompetition weight loss for combat sports athletes.

2. Materials and methods

2.1. Participants

In mid-October 2023, this study recruited female combat athletes from Shanghai professional teams with reference to the participant selection criteria of previous studies [14]. The inclusion criteria were as follows: national first-class athletes or above, training days ≥ 200 days in the past year, and weekly training time ≥ 15 h. The exclusion criteria were as follows: taking other supplements or drugs, having major injuries, or participating in other intervention trials in the past three months. G*Power 3.1 was utilized for a priori sample size estimation, employing a two-way repeated measures experimental design. Based on the findings from prior studies (η ² = 0.590), the statistical effect size was determined to be 1.20, with a significance level set at α = 0.05 and a desired power of 1-β = 0.80, indicating a minimum requirement of 21 participants. To mitigate potential sample attrition due to non-research-related factors, such as sports injuries and modifications to training regimens, we increased the target sample size accordingly. Following the screening process, a total of 30 female athletes were recruited for participation in this study (4 wrestlers, 3 boxers, 9 sanda athletes, 3 judokas, and 11 taekwondo athletes). A paired, randomized, double-blind experimental design was adopted. All 30 participants were paired individually according to their sport and competition levels. They were subsequently randomly allocated to either the tangeretin group or the placebo group, with each group comprising 15 participants, as illustrated in Figure 1A. The tangeretin group was administered the research team’s self-developed product, the “Qingguo Ren” tangeretin supplement (production license number: SC10611310–113704, doping test certification number: 2023FD233), at a daily dosage of 30 g. This supplement consisted of 29.6 g of whey protein isolate (purity: 95%, Cansure Inc., USA) and 400 mg of tangeretin (purity: 99.79%). Based on prior animal studies [17,18] and relevant toxicological reports on tangeretin [19], it was determined that daily supplementation with tangeretin at a dosage of ≤10  mg/kg was safe. Consequently, in order to enhance the intervention effects of tangeretin, the dosage administered in this study was increased from 200 mg/day (as utilized in previous research) to 400 mg/day. The placebo group received 30 g whey protein isolate (95% purity, Cansure Inc., USA; doping test number: 2023FD221) per day. Furthermore, there were no significant differences in taste or color between the tangeretin and placebo supplements, both of which were dispensed in brown, opaque drinking cups. Except for the authors, other personnel were unaware of the specific grouping, numbering, and supplement distribution of the participants. Before the study began, the researchers explained its content, process, and potential risks to all the participants in detail, and they signed informed consent forms. The team purchased corresponding commercial insurance for all participants to protect their rights and interests. This study was conducted in accordance with the principles set forth in the Declaration of Helsinki and obtained approval from the Academic and Ethics Committee of Shanghai University of Sport (document number: 102772020RT082).

Figure 1.

Participants grouping (A), overall study design (B), and trial procedure (C).

2.2. Supplement intake, blood sample collection, and analysis

The study was carried out during the early winter training phase of 2023, with tangeretin or placebo supplementation administered over a 30-day period (from November 6 to December 5). The participants reported to the training center restaurant daily between 7:30 and 8:30 a.m., where they received and consumed the prepared supplement or placebo under the supervision of the research team. Two researchers collected fasting blood samples the day before the baseline (E1) and the day after the end of the trial (E2) from 7:00 to 8:00 (Figure 1B). Three milliliters of venous blood were drawn each time, and the serum samples were separated via a high-speed centrifuge (model: TG16, Shuke, China; 3000 r/min, 15 min) and stored in a medical refrigerator (model: BDF-86V158, Haier, China) at low temperature (−80 °C). After the trial, the researchers uniformly analyzed all the serum samples (SAL-6000 immune pipeline model, Mindray, China) to record data on indicators such as blood glucose (Glu), high-density lipoprotein cholesterol (HDL-C), low-density lipoprotein cholesterol (LDL-C), total cholesterol (TCHO), triglyceride (TG), free fatty acid (FFA), testosterone (TE), and cortisol (COR) levels. During the entire trial, the daily diet of each participant was strictly controlled and recorded in detail. The training center restaurant was responsible for food preparation and distribution. One researcher provided guidance and supervision on food selection and cooking to exclude foods and seasonings that might affect the research results. Moreover, to reduce the potential influence of confounding factors, all participants were instructed to refrain from using any additional supplements or traditional Chinese herbal medicines for a period of 2 weeks prior to the initiation of the study and throughout its duration, amounting to a total of 44 days. No participants experienced discomfort or adverse reactions during the trial.

2.3. Dietary intake

One week before the start of the study, the researchers used the Nutritionist Pro™ software (version 1.3, First Databank Inc., USA) to record and analyze the total daily caloric and macronutrient contents of all the participants (Table 1). The participants’ weight-loss program did not include controlled calorie restriction or restrictions on exercise intensity. During the main study period (November 6 to December 5), the participants’ daily calorie intake remained consistent with their pre-intervention levels, and their macronutrient ratios remained unchanged. All three meals were uniformly prepared by two registered dietitians and four research assistants and eaten under the supervision of the researchers. The nuts, fruits, and other post-meal foods received by athletes each day were included in the daily dietary caloric intake statistics. In addition, the training base was closed; athletes could not obtain outside food. During the study, athletes were prohibited from purchasing extra food via online retailers or supermarkets. Researchers and coaches conducted regular inspections of the athletes' dormitories to mitigate the influence of external factors on the study.

Table 1.

The information of the participants, average daily energy, and macronutrient intakes during the study.

Study indicators	Tangeretin group (n = 15)	Placebo group (n = 15)
Age (yrs)	18.53 ± 1.19	19.27 ± 1.58
Height (cm)	168.00 ± 7.78	170.67 ± 8.53
Body weight (kg)	61.58 ± 1.90	60.96 ± 1.90
Professional training years (yrs)	4.33 ± 1.29	4.33 ± 0.72
Training time per week (h)	27.73 ± 2.34	28.80 ± 3.30
Energy intake (kj/d)	11984.80 ± 2701.91	12879.60 ± 2678.67
Protein (%)	28.57% ± 2.28%	27.78% ± 5.03%
Fat (%)	20.12% ± 3. 13%	21.26% ± 4.48%
Carbohydrate (%)	50.26% ± 4. 13%	51.37% ± 4.24%

2.4. Body composition testing

All participants entered the laboratory in a sequential manner between 9:00 and 11:30 on testing days E1 and E2. Initially, researchers performed preliminary assessments of the participants' morphological indicators, followed by the evaluation of body composition utilizing dual-energy X-ray absorptiometry (DXA). The participants were prohibited from eating and drinking for 1 h before the test and were advised to avoid strenuous exercise. They were also asked to urinate as much as possible 30 min before the test. Three minutes before the test, the participants wore shorts and a short-sleeved top, then lay flat on the GE Healthcare dual-energy X-ray bone densitometer (model: Lunar, USA; software version enCORE version 14.10) according to the test requirements. The top of the head was positioned  ≥3  cm from the marking line on the head side of the examination bed, arms were placed vertically on both sides of the body and within the edge line of the mattress, palms facing the foot side, legs were naturally parallel (angle approximately 15°), and the feet were touching each other with slight internal rotation (Figure 2). Researchers used two nylon straps to secure the participants' knees and ankles and attempted to keep their bodies still and breathing normally. Researchers with DXA operation qualifications subsequently performed a full-body scan, which lasted approximately 5 min. During the test, the operator began by selecting “Quality Assurance” (QA) on the main interface, then accurately positioned the QA phantom with the copper sheet facing downward and the long axis aligned parallel to the scanning arm. The laser was aligned with the cross-center of the phantom before proceeding with the scan by clicking “OK”. After the scan, upper limb area, lower limb area, trunk area, abdominal area (above the pelvic tangent, approximately 20% of the area between the pelvic tangent and the neck tangent), gynoid hip area (the upper boundary parallel to the pelvic tangent, and the lower boundary 3.5 times the height of the female area below the pelvic tangent), and the whole-body area were subsequently scanned. This process generated data on indicators such as body weight (BW), fat mass (FM), lean tissue mass (LBM), FFM, bone mineral content (BMC), and body-fat percentage (BF%). After the scan, the instrument performed automatic analysis, and the researchers printed and saved the data reports.

Figure 2.

DXA scanning of the full body to assess body composition partitioning.

2.5. Muscle strength testing

This study used an isokinetic muscle strength testing system (model: ISOMED 2000, Germany) to test the flexion and extension muscle strength of the hip, knee, and ankle joints of the dominant leg of the participants in both groups [20]. Fifteen minutes before the test, the researchers explained the test content, process, and action requirements in detail and conducted demonstrations. Formal tests were conducted after all the athletes had become familiar with the process and mastered the actions. Five minutes before the test, the participants performed warm-up activities (including joint flexion and extension activities and stretching) under the guidance of the researchers. During the test, the participants assumed a seated position and were secured and tested in strict accordance with the methods specified in the operation manual. The knee joint was fixed in the test position, keeping the hip and knee flexed, and the knee joint was in a neutral position. The lower-limb joints were secured with straps, with hands naturally grasping the handles on both sides. The rotational axis of the power head was oriented to align with the anatomical joint axis, thereby ensuring compatibility with the power arm of the instrument. The test action selected in this study involved flexion and extension in the “concentric-concentric mode”. The hip and knee joints were tested at isokinetic velocities of 60°/s (slow) and 300°/s (fast), whereas the ankle joint was tested at velocities of 60°/s and 180°/s. The participants practiced applying force three times at each joint and speed before testing. Each participant performed five flexion–extension repetitions per set and rested for 1 min between sets. The peak torque (PT) was recorded, and the average of the five test results was used for statistical analysis.

2.6. Aerobic capacity testing

All participants entered the laboratory in a sequential manner between 14:30 and 18:30 on testing days E1 and E2, where each individual underwent an assessment of aerobic capacity. With reference to relevant research reports [21], this study adopted the direct testing method. The specific operation was as follows: participants ran on a treadmill (model: T150, COSMED, Italy) at a speed of 8 km/h for 5 min, after which the speed was linearly increased by an average of 1 km/h per minute until exhaustion. According to the report by Oyama et al. [22], participants were considered to have reached exercise exhaustion if they met two or more of the following conditions: (1) The athlete was unable to sustain the prescribed exercise intensity. (2) There was no further increase in VO₂, indicating the onset of a plateau. (3) The exercise heart rate surpassed the maximum estimated heart rate, calculated as [(220 − age) − 10 bpm]. (4) The respiratory quotient exceeded 1.10. (5) The post-exercise blood lactate concentration was greater than 8 mmol/L.

During the entire aerobic capacity test, a cardiopulmonary exercise testing system (Cortex, Germany) and a heart-rate monitor (model: M430, Polar, Finland) were used to continuously measure exhaled components, heart rate, and other indicators. With reference to previous studies, the maximum value of VO₂ in this study was defined as VO₂max, and the heart rate corresponding to the occurrence of VO₂max during exhaustive exercise was considered the VO₂max heart rate. During the test, researchers recorded indicators such as the VO₂max, VO₂max heart rate, and time to exhaustion (TLim).

2.7. Statistical analysis

Statistical analyses were performed utilizing SPSS software version 23.0. The results are presented as the means  ±  standard deviations. The Shapiro‒Wilk test was applied to assess data normality, and Levene’s test was used to evaluate the homogeneity of variance. Two-way repeated-measures ANOVA (different groups ×  different time points) was used to compare distribution indicators (e.g. trunk fat mass, limb fat mass, android region fat mass, and gynoid regional fat mass), muscle strength indicators (e.g. peak torque of flexors and extensors of the hip, knee, and ankle joints at 60°/s and 300°/s for the hip and knee joints and 60°/s and 180°/s for the ankle joints), glucose and lipid metabolism indicators (e.g. LDL-C, TCHO, and FFA), hormone indicators (e.g. COR and TE), and maximal oxygen uptake and its derived indicators (e.g. VO₂max heart rate and TLim) between the tangeretin group and the placebo group. In the case of significant differences, pairwise comparisons were performed on the basis of the LSD standard. The threshold for statistical significance was set at P < 0.05, and that for high significance was set at P < 0.01.

3. Results

3.1. Basic information of the participants, average daily energy, and macronutrient intakes

Table 1 presents the basic information of the participants obtained from the questionnaire survey. The study cohort had a mean (±SD) age of 18.90 ± 1.43 years, a height of 169.33 ± 8.14  cm, and a weight of 61.27 ± 7.26 kg. Under the matched-pairs design, no significant differences were detected between the two groups in terms of height, weight, years of professional training, daily caloric intake, and macronutrient proportions (Table 1).

3.2. Changes in body composition indicators

Table 2 and Figure 3 show the changes in the body weights of the participants in the tangeretin group and the placebo group before and after the intervention experiment. After 30 days of intervention, the body weight of the tangeretin group decreased from 61.58 ± 1.90 to 60.86 ± 1.92 kg (a reduction of 1.17%, P = 0.018), whereas the body weight of the placebo group increased from 60.96 ± 1.90 to 61.26 ± 1.92 kg (a gain of 0.49%, P = 0.300), with a significant group × time interaction effect (P = 0.018; η _p ² = 0.185). Among the 15 participants in the tangeretin group, 12 (80.0%) experienced varying degrees of weight loss, of whom five reached their competition weight class (33.3%). During the same period, six participants in the placebo group experienced slight weight loss (40.0%), one participant had stable weight (No. 3, 6.6%), and eight participants experienced weight gain (53.3%). Among them, Athlete No. 3 (weight class, 52 kg; 52.40 kg before intervention vs. 51.80 kg after intervention) and Athlete No. 13 (weight class, 78 kg; 78.50 kg before intervention vs. 77.20 kg after intervention) reached their competition weight class.

Table 2.

Effects of tangeretin intervention on body composition.

Group	Time point	Body weight (kg)	body-fat percentage (%)	Total fat mass (g)	Total muscle mass (g)	Bone mineral content (g)	Lean body mass (g)
Placebo group (n = 15)	E1	60.96 ± 1.90	24.36 ± 0.81	13898.00 ± 862.79	43419.26 ± 1001.65	2515.40 ± 79.32	52116.80 ± 1278.46
	E2	61.26 ± 1.92	24.44 ± 0.83	14669.86 ± 856.22 #	43362.60 ± 987.54	2531.53 ± 84.71	52071.80 ± 1289.16
Tangeretin group (n = 15)	E1	61.58 ± 1.90	23.92 ± 0.87	13899.42 ± 893.07	45992.46 ± 1001.65	2752.20 ± 79.33	55741.66 ± 1278.46
	E2	60.86 ± 1.92 #	22.92 ± 0.89 #	13359.42 ± 886.27 #	46691.66 ± 1001.86 # *	2801.00 ± 84.71 # *	56352.66 ± 1289.16 #
Main effect – Time		P = 0.312;ηp 2 = 0.036	P = 0.020;ηp 2 = 0.190	P = 0.507;ηp 2 = 0.016	P = 0.066;ηp 2 = 0.116	P = 0.029;ηp 2 = 0.159	P = 0.069;ηp 2 = 0.113
Main effect – group		P = 0.969;ηp 2 = 0.000	P = 0.417;ηp 2 = 0.025	P = 0.597;ηp 2 = 0.010	P = 0.013;ηp 2 = 0.153	P = 0.036;ηp 2 = 0.147	P = 0.038;ηp 2 = 0.145
Interaction – Time × group		P = 0.018;ηp 2 = 0.185	P = 0.008;ηp 2 = 0.244	P = 0.001;ηp 2 = 0.348	P = 0.032;ηp 2 = 0.153	P = 0.257;ηp 2 = 0.046	P = 0.036;ηp 2 = 0.148

Note: Compared with E1.

^# P < 0.05, compared with the placebo group.

^* P < 0.05.

Figure 3.

Weight changes of all participants before and after intervention.

Table 2 presents the changes in other body composition indicators of the participants. Prior to the intervention trial, there were no significant differences noted in parameters such as body-fat percentage, fat mass, lean body mass, or bone mineral content between the two groups of participants. Following the intervention trial, significant interaction effects were identified between the tangeretin group and the placebo group with respect to body weight (P = 0.018; η _p ² = 0.185), body-fat percentage (P = 0.008; η _p ² = 0.244), total fat mass (P = 0.001; η _p ² = 0.348), and lean body mass (P = 0.036; η _p ² = 0.148). In contrast, no significant interaction effects were detected regarding bone mineral content (P = 0.257; η _p ² = 0.046). Compared with baseline (E1), the body-fat percentage (P = 0.001) and total fat mass (P = 0.039) significantly decreased, whereas the lean body mass (P = 0.007) and bone mineral content (P = 0.021) significantly increased. In the placebo group, total fat mass demonstrated a significant increase (P = 0.003). Additionally, body-fat percentage (P = 0.755) and bone mineral content (P = 0.426) demonstrated slight increases, whereas lean body mass (P = 0.825) exhibited a slight decrease; however, these changes were not statistically significant.

3.3. Changes in fat mass and lean body mass indicators

Figure 4 illustrates the changes in the limb fat mass of the participants. Significant interaction effects between the two groups were observed for regional fat mass distribution, including total upper limb fat mass (P = 0.025, η _p ² = 0.192), right upper limb fat mass (P = 0.037, η _p ² = 0.162), total lower limb fat mass (P = 0.008, η _p ² = 0.138), and left lower limb fat mass (P = 0.035, η _p ² = 0.172). It is worth noting that two groups exhibited divergent patterns of change in limb fat mass. Compared with baseline (E1), the total upper limb fat mass (P = 0.001), right upper limb fat mass (P = 0.003), left upper limb fat mass (P = 0.001), total lower limb fat mass (P = 0.039), and left lower limb fat mass (P = 0.009) in the tangeretin group decreased significantly after the intervention. Conversely, those indicators in the placebo group exhibited varying degrees of increases, albeit with non-significant differences both between and within groups.

Figure 4.

Effect of tangeretin intervention on trunk, upper, and lower limb fat (x ± SD, n = 30). A = Total trunk fat mass; A-1 = Right trunk fat mass; A-2 = Left trunk fat mass; B = Total arm fat mass; B-1 = Right arm fat mass; B-2 = Left arm fat mass; C = Total leg fat mass; C-1 = Right leg fat mass; C-2 = Left leg fat mass; D = Android fat mass; E = Gynoid fat mass.

Figure 4 also shows the changes in trunk fat mass. Notable interaction effects between the tangeretin group and the placebo group were noted for total trunk fat mass (P = 0.039; η _p ² = 0.166) and android fat mass (P = 0.048; η _p ² = 0.153). The results indicated that the two groups exhibited different trends over time. Compared with baseline (E1), the left trunk fat mass (P = 0.199), right trunk fat mass (P = 0.103), and gynoid fat mass (P = 0.162) in the tangeretin group decreased to varying degrees. Among these factors, the decreases in total trunk fat mass (P = 0.046) and android fat mass (P = 0.044) reached statistical significance. Within the same time frame, the same indicators in the placebo group increased to varying degrees, but without significant differences between or within groups.

Table 3 outlines the changes in lean body mass across various anatomical regions. No significant differences were observed in upper limb lean body mass, trunk lean body mass, or regional lean body mass in the tangeretin group, nor in trunk lean body mass or gynoid regional lean body mass in the placebo group, when compared with baseline (E1).

Table 3.

Effect of tangeretin intervention on the LBM.

Group	Time point	Arm lean body mass (g)	Leg lean body mass (g)	Trunk lean body mass (g)	Android lean body mass (g)	Gynoid lean body mass (g)
Placebo Group (n = 15)	E1	4789.20 ± 187.92	16762.93 ± 385.18	20836.40 ± 469.69	2845.28 ± 90.92	7308.00 ± 195.72
	E2	4706.26 ± 181.61	16798.86 ± 381.57	20955.13 ± 543.56	2850.92 ± 93.54	7378.86 ± 216.05
Tangeretin Group (n=15)	E1	5263.13 ± 187.92	17863.33 ± 385.18	22089.73 ± 469.69	3004.33 ± 98.21	7814.66 ± 195.72
	E2	5285.20 ± 181.61	17795.26 ± 384.81	22217.53 ± 543.56	3044.08 ± 101.04	7804.93 ± 216.05
Main effect – Time		P = 0.528;ηp 2 = 0.014	P = 0.836;ηp 2 = 0.002	P = 0.308;ηp 2 = 0.037	P = 0.214;ηp 2 = 0.063	P = 0.482;ηp 2 = 0.018
Main effect – Group		P = 0.050;ηp 2 = 0.130	P = 0.062;ηp 2 = 0.119	P = 0.087;ηp 2 = 0.101	P = 0.203;ηp 2 = 0.067	P = 0.117;ηp 2 = 0.085
Interaction – Time × Group		P = 0.279;ηp 2 = 0.042	P = 0.504;ηp 2 = 0.016	P = 0.970;ηp 2 = 0.000	P = 0.347;ηp 2 = 0.037	P = 0.356;ηp 2 = 0.030

3.4. Changes in glucose, lipid metabolism, and hormone secretion indicators

Table 4 summarizes the changes in glucose and lipid metabolism and hormone indicators of the participants. A notable interaction was identified between the two groups of participants for HDL-C (P = 0.021, η _p ² = 0.176). No significant interaction effects were found for LDL-C (P = 0.213, η _p ² = 0.055), TG (P = 0.545, η _p ² = 0.013), TCHO (P = 0.447, η _p ² = 0.021), FFA (P = 0.167, η _p ² = 0.078), or Glu (P = 0.383, η _p ² = 0.027). In the tangeretin group, HDL-C increased significantly compared with baseline (E1) (P = 0.002), whereas TG significantly decreased (P = 0.048). In addition, LDL-C, TCHO, and FFA in the tangeretin group slightly decreased (P = 0.152, P = 0.196, and P = 0.317, respectively), but no significant differences were noted compared with baseline (E1). Compared with baseline (E1), HDL-C (P = 0.300), TG (P = 0.240), TCHO (P = 0.818), and Glu (P = 0.197) in the placebo group slightly decreased, but LDL-C (P = 0.745) and FFA (P = 0.081) slightly increased. However, these variations were not statistically significant. This study also found no significant interaction effects between the two groups for serum cortisol (P = 0.164, η _p ² = 0.111) and testosterone levels (P = 0.077, η _p ² = 0.280). However, the trends in both groups were opposite: the serum cortisol levels decreased by 8.27% in the tangeretin group, while they increased by 5.39% in the placebo group. At the same time, serum testosterone levels in the placebo group decreased significantly compared to pre-intervention levels (P = 0.003); although testosterone levels in the tangeretin group also decreased, there was no statistically significant difference compared to pre-intervention levels.

Table 4.

Effects of tangeretin intake on glycolipid metabolism and hormonal indicators (x ± SD, n = 30).

Group	Time point	HDL-C (mmol/L)	LDL-C (mmol/L)	TG (mmol/L)	TCHO (mmol/L)	FFA (mmol/L)	GLU (mmol/L)	COR (nmol/L)	TE (ng/ml)
Placebo Group (n=15)	E1	1.70 ± 0.05	2.30 ± 0.17	0.87 ± 0.08	4.46 ± 0.22	0.40 ± 0.06	4.82 ± 0.12	267.78 ± 46.82	0.37 ± 0.11
	E2	1.64 ± 0.06	2.36 ± 0.14	0.77 ± 0.04	4.42 ± 0.18	1.26 ± 0.47	4.62 ± 0.14	282.20 ± 79.00	0.16 ± 0.09 ##
Tangeretin Group (n=15)	E1	1.58 ± 0.05	2.23 ± 0.17	0.68 ± 0.08	4.37 ± 0.22	0.30 ± 0.06	4.76 ± 0.12	272.07 ± 65.47	0.32 ± 0.13
	E2	1.72 ± 0.06 #	1.99 ± 0.17	0.51 ± 0.45 #	4.12 ± 0.18	0.21 ± 0.47	4.75 ± 0.11	249.57 ± 33.83	0.26 ± 0.13
Main effect - Time		P = 0.348; ηp 2 = 0.032	P = 0.426; ηp 2 = 0.023	P = 0.029; ηp 2 = 0.160	P = 0.281; ηp 2 = 0.041	P = 0.262; ηp 2 = 0.052	P = 0.334; ηp 2 = 0.033	P = 0.811; ηp 2 = 0.003	P = 0.005; ηp 2 = 0.560
Main effect - Group		P = 0.836; ηp 2 = 0.002	P = 0.256; ηp 2 = 0.044	P = 0.010; ηp 2 = 0.215	P = 0.452; ηp 2 = 0.020	P = 0.105; ηp 2 = 0.106	P = 0.764; ηp 2 = 0.003	P = 0.564; ηp 2 = 0.020	P = 0.718; ηp 2 = 0.014
Interaction - Time × Group		P = 0.021; ηp 2 = 0.176	P = 0.213; ηp 2 = 0.055	P = 0.545; ηp 2 = 0.013	P = 0.447; ηp 2 = 0.021	P = 0.167; ηp 2 = 0.078	P = 0.383; ηp 2 = 0.027	P = 0.164; ηp 2 = 0.111	P = 0.077; ηp 2 = 0.280

Note: Compared with E1.

^# P < 0.05.

^## P < 0.01.

3.5. Changes in lower limb joint muscle strength and aerobic capacity-related indicators

Tables 5 and 6 present the changes in the lower limb joint muscle strength and aerobic capacity-related indicators. At baseline, the two groups did not significantly differ on any of these indicators. After the intervention, the lower limb joint muscle strength of the tangeretin group changed positively. In terms of the hip joint, the peak torque of the flexor and the extensor at 60°/s angular velocity (P = 0.034 and P = 0.004, respectively) in the tangeretin group significantly increased compared with those before the intervention. Furthermore, the peak torque of the extensor at an angular velocity of 60°/s (P = 0.015) was significantly elevated compared to the values recorded in the placebo group during the same period. With regard to the knee joint, following the intervention, there were no significant alterations observed in the peak torque of either the flexor or extensor at angular velocities of 60°/s and 300°/s within the tangeretin group. However, the peak torque of the extensor at 60°/s in the placebo group demonstrated a significant reduction when compared to the levels recorded prior to the intervention (P = 0.037). In addition, the peak flexor torque of the knee joint in the tangeretin group at an angular velocity of 60°/s (P = 0.141, η _p ² = 0.076) and peak extensor torque at 300°/s (P = 0.664, η _p ² = 0.007) did not show a significant interaction effect. However, both values were significantly higher than those in the placebo group (P = 0.009, η _p ² = 0.221 and P = 0.008, η _p ² = 0.228, respectively). Regarding the ankle joint, although no significant interaction effect was observed for peak torque in the flexor muscles of the tangeretin group at an angular velocity of 180°/s (P = 0.223, η _p ² = 0.053), it is worth noting that it increased significantly compared with the pre-intervention value (P = 0.028). Positive changes were also observed in the aerobic capacity-related indicators of the tangeretin group. After the intervention, the first ventilatory threshold heart rate (P = 0.049), second ventilatory threshold heart rate (P = 0.017), and maximum heart rate (HRmax) (P = 0.031) in the tangeretin group significantly decreased, whereas the TLim demonstrated a significant increase in comparison to those before the intervention (P = 0.049). However, no statistically significant differences were detected when compared to the placebo group during the same timeframe. Additionally, no interaction effects were observed between the tangeretin group and the placebo group concerning the first ventilatory threshold, second ventilatory threshold, oxygen uptake, heart rate at exhaustion, or duration until exhaustion.

Table 5.

Effects of tangeretin intake on the flexion and extension strength of hip, knee, and ankle joints (x ± SD, n = 30).

Group	Time point	Hip flexor (Nm)		Hip extensor (Nm)		Knee flexor (Nm)		Knee extensor (Nm)		Ankle flexor (Nm)		Ankle extensor (Nm)
		60°/s	300°/s	60°/s	300°/s	60°/s	300°/s	60°/s	300°/s	60°/s	180°/s	60°/s	180°/s
Placebo Group (n = 15)	E1	88.26 ± 4.42	49.20 ± 2.69	173.93 ± 13.63	116.66 ± 12.85	75.53 ± 3.77	55.80 ± 2.65	137.00 ± 5.90	89.53 ± 4.75	64.00 ± 4.04	44.73 ± 2.16	19.26 ± 1.46	15.13 ± 0.78
	E2	91.66 ± 4.84	50.33 ± 3.36	176.20 ± 13.56	117.93 ± 11.70	73.26 ± 3.79	56.13 ± 2.71	127.33 ± 5.78*	82.86 ± 4.62	66.13 ± 4.57	46.33 ± 3.51	18.86 ± 1.33	15.20 ± 0.99
Tangeretin Group (n = 15)	E1	96.60 ± 4.42	55.60 ± 2.69	210.06 ± 13.63	144.26 ± 12.85	83.13 ± 3.77	61.26 ± 2.65	153.46 ± 5.90	100.67 ± 4.75	72.93 ± 4.04	41.80 ± 2.16	22.93 ± 1.46	17.26 ± 0.78
	E2	105.46 ± 4.84 #	59.13 ± 3.36	225.86 ± 13.56 # *	149.93 ± 11.70	90.26 ± 3.79*	63.86 ± 2.71	154.86 ± 5.78	96.46 ± 4.62*	80.40 ± 4.57	48.46 ± 3.51*	21.60 ± 1.33	16.60 ± 0.99
Main effect – Time		P = 0.038;	P = 0.143	P = 0.017;	P = 0.581;	P = 0.439;	P = 0.463;	P = 0.195;	P = 0.063;	P = 0.114;	P = 0.052;	P = 0.123;	P = 0.478;
		ηp 2 = 0.145	ηp 2 = 0.075	ηp 2 = 0.187	ηp 2 = 0.011	ηp 2 = 0.022	ηp 2 = 0.019	ηp 2 = 0.059	ηp 2 = 0.188	ηp 2 = 0.087	ηp 2 = 0.129	ηp 2 = 0.083	ηp 2 = 0.018
		P = 0.073;	P = 0.069;	P = 0.031;	P = 0.077;	P = 0.009;	P = 0.051;	P = 0.008;	P = 0.049;	P = 0.039;	P = 0.912;	P = 0.104;	P = 0.151;
Main effect – Group		ηp2 = 0.111 P = 0.340;	ηp2 = 0.113 P = 0.445	ηp2 = 0.155 P = 0.067;	ηp2 = 0.107 P = 0.726;	ηp2 = 0.221 P = 0.141;	ηp2 = 0.129 P = 0.570;	ηp2 = 0.228 P = 0.087;	ηp2 = 0.131 P = 0.664;	ηp2 = 0.144 P = 0.372;	ηp2 = 0.000 P = 0.223;	ηp2 = 0.092 P = 0.399;	ηp2 = 0.072 P = 0.387;
Interaction –- Time × Group		ηp 2 = 0.033	ηp 2 = 0.021	ηp 2 = 0.114	ηp 2 = 0.004	ηp 2 = 0.076	ηp 2 = 0.012	ηp 2 = 0.101	ηp 2 = 0.007	ηp 2 = 0.029	ηp 2 = 0.053	ηp 2 = 0.026	ηp 2 = 0.027

Note: Compared with E1.

^# P < 0.05, Compared with placebo group.

^* P  < 0.05.

Table 6.

Effects of tangeretin intervention on oxygen uptake and heart rate during VT1, VT2, and exhaustion (x ± SD, n=30).

Group	Time point	VT1_VO2	VT1_HR (bpm)	VT2_VO2	VT2_HR (bpm)	VO2max (ml/min/kg)	VO2max_HR	TLim (min)
Placebo Group (n = 15)	E1	27.20 ± 0.68	143.26 ± 1.85	41.00 ± 0.95	179.86 ± 2.02	45.60 ± 1.13	190.86 ± 1.92	11.52 ± 1.11
	E2	27.66 ± 1.13	139.60 ± 3.11	38.40 ± 1.36	177.53 ± 2.15	44.93 ± 1.32	187.93 ± 2.40	11.33 ± 1.12
Tangeretin Group (n = 15)	E1	26.66 ± 0.68	144.26 ± 1.85	38.26 ± 0.95	178.00 ± 2.09	42.80 ± 1.13	190.84 ± 2.06	11.26 ± 1.65
	E2	25.26 ± 1.13	137.40 ± 3.11 #	38.86 ± 1.36	173.42 ± 2.22 #	43.46 ± 1.32	185.61 ± 2.58 #	12.18 ± 1.37 #
Main effect - Time		P = 0.569; ηp 2 = 0.012	P = 0.034; ηp 2 = 0.150	P = 0.313; ηp 2 = 0.036	P = 0.010; ηp 2 = 0.220	P = 1.000; ηp 2 = 0.000	P = 0.009; ηp 2 = 0.235	P = 0.039; ηp 2 = 0.143
Main effect - Group		P = 0.175; ηp 2 = 0.065	P = 0.829; ηp 2 = 0.002	P = 0.111; ηp 2 = 0.088	P = 0.284; ηp 2 = 0.012	P = 0.170; ηp 2 = 0.066	P = 0.574; ηp 2 = 0.012	P = 0.567; ηp 2 = 0.012
Interaction - Time × Group		P = 0.258; ηp 2 = 0.045	P = 0.504; ηp 2 = 0.016	P = 0.111; ηp 2 = 0.088	P = 0.379; ηp 2 = 0.029	P = 0.445; ηp 2 = 0.021	P = 0.613; ηp 2 = 0.010	P = 0.166; ηp 2 = 0.061

Note: Compared with E1.

^# P < 0.05.

4. Discussion

This study simulated and observed the intervention effect of a 30-day tangeretin supplementation (400 mg/day) on precompetition weight loss in female combat athletes and its regulatory effect on physical function. The results revealed that the tangeretin group had advantages in weight reduction, body composition optimization, glucose and lipid metabolism regulation, and sports performance improvement.

• 1)Weight reduction: The majority of the participants in the tangeretin group (80.00%) experienced varying degrees of weight loss. Of these, five participants reduced their weight to the competition weight class (33.33%).
• 2)Body composition optimization: The body-fat percentage of athletes in the tangeretin group and their regional fat mass significantly decreased. The fat mass in the upper limbs (↓8.77%), lower limbs (↓4.43%), trunk (↓6.77%), and android region (↓8.09%) significantly decreased. By contrast, the placebo group exhibited the opposite trend.
• 3)Lean body mass improvement: The lean body mass of athletes in the tangeretin group significantly increased (↑1.09%). Additionally, the regional lean body mass of these athletes also demonstrated an increase, and their overall performance exceeded that of the placebo group during the same timeframe.
• 4)Glucose and lipid metabolism regulation: The HDL-C level in the tangeretin group increased by 8.86% (P = 0.002), whereas the TG level decreased by 25.00% (P = 0.048).

4.1. Optimization of body composition by tangeretin supplements

Tangeretin is a polymethoxyflavonoid unique to citrus fruits. It does not contain any doping substances. Its excellent glucose and lipid metabolism regulatory properties have been verified by multiple studies [23,24].

At the molecular level, in vitro experiments have reported that at concentrations of 32–64 μM, tangeretin can activate the protein kinase A signaling pathway, promote the phosphorylation of adipose triglyceride lipase, and effectively inhibit the hypertrophy and proliferation of 3T3-L1 preadipocytes [18]. At the animal level, an 8-week tangeretin intervention (25  mg/kg body weight) in mice significantly reduced their LDL-C and TG levels, which may be due to the key role of tangeretin in regulating hepatic lipid metabolism [25]. In a hypercholesterolemic rat model, a 7-week tangeretin intervention (50  mg/kg body weight) not only markedly diminished the TCHO and LDL-C levels but also inhibited the activity of hepatic 3-hydroxy-3-methylglutaryl-coenzyme A reductase [26]. This finding suggests that tangeretin has a regulatory effect on cholesterol synthesis. At the clinical trial level, the team’s previous study revealed that after 4 weeks of tangeretin supplementation (200 mg/day), football athletes’ body-fat percentage decreased from 13.3 ± 4% to 12.9 ± 3.7% (↓3.0%) [14]. In this study, increasing tangeretin from 200 mg/day to 400 mg/day produced a corresponding enhancement in fat reduction, supporting a dose-dependent effect. Compared with the 200 mg/day regimen, the 400 mg/day regimen yielded statistically and practically significant improvements in fat loss. Given that previous studies examined only a single low dose, this finding represents a critical advancement for clinical and exercise-sport applications. The 400  mg/day dose falls within an effective range and is well tolerated, laying a foundation for dose optimization. Future studies incorporating multi-dose designs are warranted to confirm this dose‒response relationship and to assess long-term safety.

Together, the regulatory effects of tangeretin on glucose metabolism and lipid metabolism constitute the core mechanism for improving the body’s metabolic homeostasis [27]. At the molecular level, Kang et al. [25] confirmed through C2C12 myoblast experiments that 100  μM tangeretin can activate the AMP-activated protein kinase (AMPK) signaling pathway, induce AS160 protein phosphorylation, and promote GLUT4 translocation from the cytoplasm to the cell membrane, thereby significantly improving glucose uptake efficiency. This mechanism has also been investigated and validated in a mouse model of diabetes induced by a high-fat diet. A previous study revealed that 200  mg/kg tangeretin can activate the skeletal muscle AMPK pathway and improve glucose intolerance, effects closely associated with the recovery of the activity of key liver glucose-metabolism enzymes (e.g. glucokinase and pyruvate kinase) [28]. Sundaram et al. [29] further reported in a streptozotocin-induced diabetic rat model that tangeretin (100 mg/kg/day × 30 days) can reduce not only blood glucose and glycosylated hemoglobin levels but also endogenous glucose output by inhibiting hepatic glucose-6-phosphatase activity (↓37%), suggesting a regulatory effect on gluconeogenesis. However, the team’s previous human trial showed that after 4 weeks of supplementation with 200 mg/day tangeretin in athletes, although the serum cortisol level significantly decreased (P = 0.017), the blood glucose level immediately after exercise was not affected [13]. This phenomenon may be attributed to the metabolic compensation effect of tangeretin through multiple targets (e.g. AMPK activation and gluconeogenesis inhibition), but its dynamic regulatory mechanism under exercise stress conditions still requires in-depth analysis.

This study further verified the bidirectional regulatory effect of tangeretin on glucose and lipid metabolism and revealed its comprehensive effect on improving metabolic homeostasis through quantitative analysis of body fat components. The results revealed that after tangeretin intervention, the participants presented a significant increase in HDL-C levels (P = 0.002) and a simultaneous notable reduction in TG levels (P = 0.048), which exhibited a high degree of consistency with the findings from prior animal models [19,29] and human trials [14]. This finding suggests that tangeretin may synergistically improve lipid metabolism disorders by activating cholesterol reverse transport-related proteins (e.g. ATP-binding cassette transporter A1) and accelerating adipose tissue lipolysis (e.g. via lipase phosphorylation mechanisms). More importantly, the significant decreases in body-fat percentage (P = 0.001) and total regional fat mass in the tangeretin group (e.g. trunk fat mass ↓6.77% vs. placebo group ↑2.69%) directly corresponded to the findings in the team’s previous athlete trial [14]. Owing to the higher dose used in this study (twice that of the previous study), the effect magnitude was further enhanced, confirming the dose-dependent characteristics of tangeretin intervention. This study further demonstrated that the stability of glucose levels aligned with the results of earlier research [30] (e.g. no significant change in postprandial blood glucose), which may be due to the bidirectional regulatory mechanism of tangeretin on glucose metabolism. On the one hand, tangeretin enhances skeletal muscle glucose uptake through the AMPK–GLUT4 axis (e.g. the 42% increase in uptake efficiency reported by Kang et al. [25]). On the other hand, it reduces endogenous glucose output by inhibiting hepatic gluconeogenesis (e.g. the 37% decrease in glucose-6-phosphatase activity reported by Sundaram et al. [29]), thereby maintaining blood glucose homeostasis under non-stress conditions. However, the nonsignificant decreases in LDL-C and TCHO in this study (P > 0.05) differ from the cholesterol synthesis inhibition observed in animal models [26], suggesting that the metabolic response of humans to tangeretin may be influenced by species-specific differences in the HMG-CoA reductase regulatory pathway or baseline metabolic status. In addition, the more pronounced reduction observed in FFA levels in the tangeretin group (↓30%) and the improved regional fat distribution (e.g. 8.09% decrease in android fat mass) may be related to the multitarget effect of tangeretin in inhibiting preadipocyte differentiation (via lipase phosphorylation) and promoting fatty acid β-oxidation (via AMPK pathway activation), as indirectly supported by diabetic mouse models. In brief, tangeretin achieves metabolic optimization from the molecular level to the overall level by regulating multiple targets in lipid synthesis/decomposition balance and glucose metabolism networks. However, the dose–response relationship in humans, tissue-specific effects, and potential synergistic mechanisms with exercise intervention still require further clarification through long-term multicenter studies to promote its precise application in metabolic disease prevention and sports performance improvement. The ability of tangeretin to optimize body composition is reflected not only in fat metabolism but also in its unique role in promoting lean body mass growth, which has gradually shown biological importance. The team’s previous randomized controlled trial on football athletes (a 4-week regimen of 200 mg/day tangeretin in conjunction with whey protein) revealed that muscle mass in the experimental group slightly increased (from 30.5 ± 4.3 kg to 30.8 ± 4.3 kg), and this alteration exhibited a significant correlation with a reduction in cortisol levels (P = 0.017) [14]. This finding provides direct evidence for the hypothesis that tangeretin promotes muscle synthesis through endocrine regulation. Cortisol, a key regulatory factor of catabolism, when reduced, may inhibit the activity of the muscle protein hydrolase system and synergistically enhance the anabolic effect of testosterone, jointly creating a metabolic microenvironment conducive to maintaining or increasing lean body mass. In this study, although the serum testosterone levels in the tangeretin group and the placebo group decreased (possibly due to increased training intensity and volume during winter training), the reduction in the tangeretin group (↓18.75%) was markedly less substantial compared to that in the placebo group (↓56.76%). The observed improvement trend in lean body mass in the upper limbs, lower limbs, and regional areas (e.g. a 1.30% increase in android regional lean body mass vs. 0.19% in the placebo group) corresponds with the observed hormonal regulation pattern. This gain in lean body mass may also be closely related to the metabolic homeostasis regulation of tangeretin. It may increase the supply of energy substrates to muscle cells through enhanced glucose uptake via the AMPK pathway and inhibit adipose tissue lipolysis, thereby reducing the interference of FFAs in the insulin signaling pathway. This dual mechanism may indirectly promote protein synthesis by improving skeletal muscle insulin sensitivity.

However, a gap remains in current evidence regarding hormone regulatory mechanisms. Although animal experiments have confirmed that tangeretin can regulate cortisol secretion via the hypothalamic–pituitary–adrenal axis [31,32], the molecular pathways directly related to lean body mass in human studies have yet to be analyzed. Therefore, future research should combine dynamic hormone detection with muscle tissue-specific gene expression analysis to reveal the multilevel regulatory network by which tangeretin affects body composition. Such an endeavor would have important implications for the precise application of tangeretin in synergistic intervention strategies for increasing muscle and reducing fat content.

4.2. Promotion of sports performance by tangeretin supplements

A previous animal study revealed that 4 weeks of treatment with tangeretin (50 mg/kg/day) substantially elevated antioxidant enzyme activity in Kunming mice, prolonged TLim time (↑53%), and alleviated oxidative stress damage to the myocardium and skeletal muscle. Tangeretin supplementation also positively affected the aerobic capacity of athletes with exercise-induced bronchoconstriction (EIB) [21]. The data showed that after 4 weeks of tangeretin intervention (200 mg/day), the aerobic capacity-related indicators, such as VO₂max, TLim, and VO₂max running speed, of EIB athletes increased from 52.77 ± 6.82 ml/kg/min, 12.27 ± 1.79 min, and 15.55 ± 1.73 km/h to 54.54 ± 5.18 ml/kg/min (P = 0.042), 13.03 ± 1.35 min (P = 0.050), and 15.97 ± 1.37 km/h, respectively. At the same time, tangeretin also improved red blood cell indicators, optimized body composition and glucose and lipid metabolism, and alleviated the cortisol stress response and oxidative stress damage caused by strenuous exercise. In the current investigation, the VO₂max of athletes in the tangeretin group exhibited a slight increase following the intervention (42.80 ± 1.13 ml/kg/min before vs. 43.46 ± 1.32 ml/kg/min after). In the placebo group, VO₂max exhibited a decline, but without statistical significance. Additionally, the first ventilatory threshold heart rate (144.26 ± 1.85 bpm before vs. 137.40 ± 3.11 bpm after, P = 0.049) and the second ventilatory threshold heart rate (178.00 ± 2.09 bpm before vs. 173.42 ± 2.22 bpm after, P = 0.017) in the tangeretin group notably reduced, whereas the TLim value increased significantly (11.26 ± 1.65 min before vs. 12.18 ± 1.37 min after, P = 0.049). These results suggest that 30-day tangeretin supplementation (400 mg/day) effectively improved the aerobic capacity of female combat athletes, thereby positively affecting their sports performance.

In the oxygen transport system, the synergistic effect of hemoglobin and red blood cells is key to determining the body's aerobic capacity [33]. Multiple studies have confirmed that tangeretin can enhance this link by improving red blood cell indicators. Sundaram et al. [29] reported that 30 days of tangeretin intervention (100 mg/kg body weight) significantly improved insulin and hemoglobin levels in diabetic rats. After the intervention, the hemoglobin level in the “tangeretin + dietary control group” was 61.4% higher than that in the “dietary control group.” Yuan et al. [34] reported similar findings. In human clinical research, the team previously reported (2019) that after 5 weeks of tangeretin supplementation (200 mg/day), the hemoglobin and serum ferritin levels in weightlifters increased by 8.6% and 4.2%, respectively. They concluded that tangeretin was beneficial for improving red blood cell indices. In another clinical study, supplementation with tangeretin at a dosage of 200 mg/day resulted in reductions of 11.7% in glycosylated hemoglobin levels and 7.5% in fasting blood glucose levels among patients with type 2 diabetes. Another clinical study by the team on EIB athletes verified the above conclusion [21]. Following a 30-day intervention with tangeretin at a dosage of 200 mg/day, the red blood cell count in the tangeretin group increased by 1.96%, whereas a decrease of 6.76% was observed in the placebo group. Hemoglobin levels rose by 3.89% in the tangeretin group, compared to an increase of 1.34% in the placebo group. The serum iron levels decreased slightly in the tangeretin group (↓3.8%) but dropped significantly in the placebo group (↓38.4%). These results highlight the positive effect of tangeretin on oxygen transport. Although red blood cell indicators were not tested in this study because of experimental constraints, the improvements in aerobic capacity-related indicators such as VO₂max and TLim observed in the tangeretin group aligned with the team’s earlier findings and the broader literature. This outcome suggests that tangeretin may enhance aerobic capacity through its effects on hemoglobin and serum iron.

Skeletal muscle is the main site of oxygen utilization, and increases in muscle mass can improve the body’s oxygen demand and work efficiency, thereby increasing VO₂max [35]. A study by Posnakidis et al. [36] revealed that 8 weeks of high-intensity functional training combined with a daily 20  ml nutritional supplement (a combination of ω-3 and ω-6 polyunsaturated fatty acids, along with vitamin A, vitamin E, and γ-tocopherol) increased participants’ muscle mass (24.8 kg before vs. 25.2 kg after), decreased fat mass (21.0% before vs. 19.1% after), and significantly improved aerobic capacity (VO₂max: 46.95 ± 5.24 ml/kg/min vs. 50.29 ± 4.83 ml/kg/min; maximum heart rate: 187 ± 10 bpm vs. 186 ± 6 bpm). The enhancement in VO₂max was more pronounced compared to that observed in the placebo group (7.1% vs. 5.5%), clearly demonstrating the importance of muscle mass for aerobic capacity. However, long-term and repeated precompetition weight control can stimulate cortisol secretion [37,38]. As a catabolic hormone, chronically elevated cortisol levels can inhibit muscle protein synthesis and promote fat synthesis and storage [39]. Prior research has established that tangeretin is capable of modulating cortisol secretion [13,21]. In this study, during high-intensity winter training, the serum cortisol level in the placebo group increased (by 5.39%), whereas that in the tangeretin group decreased from 272.07 ± 65.47 to 249.57 ± 33.83 nmol/L, indicating the positive regulatory effect of tangeretin on cortisol. In addition, the overall lean body mass of the tangeretin group, as well as the lean mass in the upper limbs, lower limbs, trunk, android, and gynoid regions, increased more than in the placebo group during the same period. This improvement likely contributed to the observed increases in VO₂max and aerobic capacity in the tangeretin group.

At the energy metabolism level, the optimization of glucose and lipid metabolism can enhance energy supply efficiency during high-intensity exercise, thereby improving aerobic capacity [40]. Tangeretin has exhibited biological activity in the regulation of glucose and lipid metabolism and has yielded encouraging outcomes in preclinical animal studies [41]. Its regulatory effects involve the modulation of the AMPK pathway, the insulin signaling pathway, along with the facilitation of GLUT4 translocation from the cytoplasm to the plasma membrane to enhance glucose uptake. Kang et al. [25] found that after intervention with a citrus active extract (with tangeretin as the main component), TCHO and TG levels in obese mice significantly decreased. Tangeretin also increased intracellular protein kinase A activity and promoted the phosphorylation of adipose triglyceride lipase, thereby inhibiting the hypertrophy and proliferation of 3T3-L1 adipocytes. In addition, it significantly reduced the activity of hepatic 3-hydroxy-3-methylglutaryl-coenzyme A reductase. Together, these effects positively contribute to glucose and lipid metabolism [26]. This study further verified the ability of tangeretin to optimize glucose and lipid metabolism in humans. After 30 days of intervention, LDL-C levels increased in the placebo group, and HDL-C levels decreased. By contrast, in the tangeretin group, TG and LDL-C levels remained stable, and HDL-C and Glu levels increased steadily. These results suggest that tangeretin has promising regulatory effects and potential for improving metabolic health.

Muscle strength serves as a critical metric for assessing performance adaptations in combat sports athletes. Supplementation with relevant nutritional compounds is an effective strategy to promote muscle synthesis and improve muscle strength [42]. Smith et al. [43] reported that resistance training combined with supplementation rich in n-3 polyunsaturated fatty acids can effectively activate the mammalian target of rapamycin growth signaling pathway, thereby increasing lean body mass, muscle mass, muscle strength, and the rate of muscle protein synthesis. Their study also revealed that increases in muscle mass can enhance the rapid power output of skeletal muscle during high-intensity exercise. Church et al. [44] reported that after three months of intervention with a mixture of tangeretin and branched-chain amino acids (15 g/day), the lower limb muscle mass and 30-second Wingate test output power of elite cyclists significantly increased by 7.4% and 16%, respectively. Their study revealed that the mixed intervention of tangeretin and branched-chain amino acids contributed to the improvements in muscle mass and explosive power. Although the present study measured the lean body mass of athletes, it did not conduct a detailed assessment of muscle mass. However, the team’s previous study demonstrated that after 30 days of supplementation with tangeretin (200 mg/day) and whey protein (15 g/day), the athletes’ muscle mass increased from 29.2 ± 4.3 to 31.2 ± 4.3 kg, representing a 6.12% increase. Explosive performance, as measured by the medicine ball throw, also significantly improved compared with that before intervention (5.59 ± 1.82  m/s vs. 6.86 ± 1.59  m/s, P = 0.015). Other indicators reflecting explosive muscle power, such as pre-swing vertical jumps, non-swing vertical jumps, left foot jumps, right foot jumps, and depth jump touch-down times, also improved to varying degrees. In this study, after 30 days of tangeretin supplementation, the lower limb joint muscle strength of the athletes showed changes similar to those in the team’s previous research. In terms of the hip joint, the peak torque of the flexor and extensor at 60°/s angular velocity increased by 9.17% and 7.52%, respectively, and the peak torque of the flexor at 300°/s angular velocity increased by 6.34%, exceeding the corresponding value in the placebo group by 63.88% during the same timeframe.

In terms of the knee joint, the peak torque of the flexors at an angular velocity of 60°/s and the peak torque of the extensors at an angular velocity of 300°/s in the tangeretin group were significantly higher than those recorded in the placebo group, suggesting a positive effect of tangeretin on the flexion and extension strength of the knee joint. This outcome may be attributed to either muscle hypertrophy around the knee or improved neuromuscular activation, which enhances contraction efficiency.

In terms of the ankle joint, the peak torque of the flexor at 180°/s angular velocity increased significantly compared with the pre-intervention value, whereas other indicators showed no significant differences. This positive change suggests that tangeretin may improve ankle flexor function, potentially by enhancing the local blood supply or promoting muscle contractility, thereby increasing torque at specific angular velocities. Therefore, tangeretin intervention had a significant positive effect on the lower limb joint muscle strength of the athletes, improving the strength at different joints and angular velocities. Some strength indicators were significantly better than those in the placebo group. These findings provide theoretical support for the practical and reasonable use of tangeretin by athletes. However, while the effects of metabolism and body composition may be universal, even though all combat sports require weight cutting, sport-specific improvements in muscle strength (such as in the knee joint) may still be influenced by differences between disciplines. Future studies can further explore long-term effects, optimal dosing strategies, and sport-specific outcomes to provide more comprehensive guidance for use in the field of sports science.

5. Conclusion

This study is the first attempt to apply a natural citrus extract to address precompetition weight control in female combat sports athletes. The results show that under the premise of maintaining normal dietary intake and fluid supplementation, a 30-day tangeretin intervention (400 mg/day) effectively reduces total body and regional fat mass, increases lean body mass, and significantly improves body composition. Among the athletes, 80.0% experienced varying degrees of weight loss, and 33.3% achieved weight reduction to their competition weight class, achieving a favorable precompetition weight control effect. Tangeretin supplementation also improved aerobic capacity and markedly enhanced lower limb joint muscle strength. These improvements had a positive impact on the athletes’ precompetition sports performance and competitive readiness, directly increasing their winning probability in high-difficulty, high-intensity competitive events.

Funding Statement

This research was funded by the Shanghai Key Laboratory of Human Performance at Shanghai University of Sport.

Disclosure statement

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

Data availability statement

The original contributions presented in this study are included in the article. Further inquiries can be directed to the corresponding author.

Institutional review board statement

The study was conducted in accordance with the Declaration of Helsinki, and ethical approval was obtained from Shanghai University of Sport.

Informed consent statement

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