Effects of Rosa roxburghii and kiwifruit composite supplements on improving exercise performance and anti-fatigue in mice

Abstract

Rosa roxburghii Tratt (RR) and Actinidia chinensis Planch (AC, kiwifruit) are nutrient-dense plant foods with diverse bioactive components. This study aimed to evaluate the effect of supplementation with Rosa roxburghii and Kiwifruit (RRAC)on improving the exercise performance-enhancing and anti-fatigue effects of a RR-AC composite supplement (RRAC) in experimental murine models. Fifty male institute of Cancer Research (ICR) mice were randomly allocated to five groups (n = 10/group): (1) the blank control group (Rest, normal saline), (2) the fatigue model control group (Con, normal saline), (3) RRAC-L (130 mg/kg/d), (4) RRAC-M (260 mg/kg/d), (5) RRAC-H (520 mg/kg/d). Following 28 consecutive days intervention, the mice in all groups except the Rest group underwent a swimming exhaustion test. Then, mice were subsequently euthanized for plasma biochemical markers and analysis and tissue collection. We found that supplementation with 28-day RRAC could significantly enhance mice’s exercise endurance performance, elevated hepatic and muscle glycogen content (all p < 0.05), and significantly reduced post-exercise fatigue biochemical parameters, including blood urea nitrogen (BUN), L-lactic acid (LAC), lactate dehydrogenase (LDH), creatine kinase (CK) concentration. Moreover, RRAC supplementation reduced the oxidative stress indicators malondialdehyde (MDA) activity, and increased the superoxide dismutase (SOD) , glutathione peroxidase (GSH-Px) (p < 0.05). In summary, supplementation with RRAC for 28 days could significantly enhance the exercise tolerance in mice during the swimming exhaustion test via glycogen accumulation and oxidative stress modulation, with no detectable adverse effects on organ structure or function.

Introduction

Fatigue is a complex physiological state characterized by the body’s reduced capacity to maintain normal physiological function or sustain adequate exercise intensity¹. Chronic fatigue, defined as fatigue persisting for six months or longer, affects approximately one-third of the global population², and represents a significant burden on both individual quality of life and broader public health systems. Beyond impairment of daily activities and work performance, prolonged fatigue has been identified as a contributing risk factor in the development of several chronic diseases, including multiple sclerosis, Parkinson’s disease, and depression^3,4. As a result, consumer interest in anti-fatigue functional products has grown substantially in recent years⁵, with these products typically marketed as dietary supplements whose composition differs considerably from that of ordinary foodstuffs⁶. The category encompasses a range of formulations, including sports foods, medical nutritional supplements, ergogenic aids, and functional foods, with bioactive ingredients—such as glucosamine, curcumin, L-carnitine, and lycopene—sourced from both animal and plant origins⁷. Among these, plant-based supplementation has received particular attention as a means of improving athletic performance, given that plant-derived foods are abundant in polyphenols, vitamins, phytosterols, biogenic amines, and bioactive proteins, several of which have demonstrated anti-fatigue properties in both in vitro and in vivo studies^8–10.

Rosa roxburghii Tratt (RR, CiLi) is a functional fruit native to southwestern China and is recognized for its high content of biologically active constituents, including vitamins, superoxide dismutase (SOD), minerals, polysaccharides, phenolic compounds, triterpenoids, and organic acids^11,12. Despite its nutritional profile, the commercial application of RR as a ready-to-drink beverage has been limited due to its astringent and sour sensory characteristics, which are largely attributable to its high tannin content¹³. To address this issue, combining RR with other complementary fruits has been suggested as a practical approach for improving palatability while retaining its functional benefits.

Actinidia chinensis Planch (AC, kiwifruit) is a nutrient-rich fruit also native to China, and is considered a suitable candidate for such a combination. Kiwifruit contains substantial amounts of natural sugars, essential minerals, ascorbic acid (vitamin C), dietary fiber, and various bioactive compounds, including flavonoids and proteolytic enzymes^14,15. A number of studies have reported the medicinal properties of kiwifruit, including antioxidative, antiproliferative, anti-inflammatory, antimicrobial, antihypertensive, antihypercholesterolemic, and neuroprotective effects, as well as a beneficial role in promoting gut health¹⁶. Many of these effects have been attributed to its high phenolic content and associated antioxidant activity, which is also of relevance to exercise recovery and fatigue management.

Several of the antioxidant compounds found in both RR and kiwifruit are thought to exert ergogenic effects during exercise, possibly by improving cellular redox balance and reducing the production of free radicals, thereby supporting muscle contractile function, delaying the onset of fatigue, and prolonging exercise performance¹⁷. For example, Chen et al.¹⁸ reported that supplementation with resveratrol, administered as an antioxidant beverage, significantly improved endurance performance, as measured by forelimb grip strength, in trained SAMP8 mice. Similarly, dietary polyphenols appear to play a plausible role in modulating various exercise-related physiological responses and recovery processes, including the promotion of mitochondrial adaptation and the enhancement of blood flow, thus improving overall endurance capacity. Furthermore, polyphenol intake has been associated with reduced age-related pathological changes and increased longevity in both animal and human populations^19,20.

Although evidence supporting the independent antioxidant and medicinal properties of RR and kiwifruit continues to accumulate, few in vivo investigations have been conducted to determine whether a composite formulation of the two fruits (RRAC) provides synergistic benefits for exercise performance and fatigue mitigation. Furthermore, the effects of RRAC on fatigue-related blood biochemical markers have not been adequately addressed in the existing literature. The present scoping investigation was therefore designed to examine the effects of 28 consecutive days of RRAC supplementation on exercise endurance capacity and fatigue-related hematological parameters in a murine model. In addition, organ indices and histopathological assessments were performed to evaluate the physiological safety of this supplementation regimen across the experimental period.

Materials and methods

RRAC preparation

The Rosa roxburghii Tratt and Actinidia chinensis Planch (kiwifruit) used in the present study were obtained from Guizhou Province in southwestern China, a region well known for cultivating high-quality Rosa roxburghii and kiwifruit. Mature fruits of both species were randomly selected and processed using a fruit extractor to separate the juice from the pulp. Rosa roxburghii and kiwifruit were then mixed at a volume ratio of 1:3 (Rosa roxburghii : kiwifruit) on the basis of preliminary sensory evaluations. The extracted juice was double-filtered through a clean filter cloth to obtain a clear liquid, after which carrageenan was added as a gelling agent and the mixture was heated at moderate temperature with continuous stirring until fully homogenized. The mixture was subsequently cooled at room temperature (22–28 °C) for approximately 30 min and stored at 4 °C until use. The preparation protocol was adapted from a previously described method for kiwifruit jelly development²¹, with appropriate modifications to accommodate the unique sensory and physicochemical characteristics of Rosa roxburghii, since no standardized recipe for a Rosa roxburghii–kiwifruit composite supplement (RRAC) was available at the time of this study. To ensure standardization and batch-to-batch consistency, all fruits were harvested from the same crop, and all RRAC batches were prepared by a single trained operator under identical conditions (Fig. 1).

Fig. 1.

Preparation of RRAC. (A) Raw materials of Rosa roxburghii fruit. (B) Raw materials of Kiwifruit. (C) RRAC sample.

Analysis of RRAC constituents

The basic nutritional composition of RRAC was determined by the Guizhou Testing Technology Research and Application Center (Guizhou, China). On a per 100 g basis, RRAC provided approximately 703 kcal of energy, primarily derived from carbohydrate (33.4 g), with smaller contributions from fat (2.90 g) and protein (1.65 g). The detailed nutritional composition, including vitamins, minerals, and amino acids, is presented in Tables 1, 2 and 3, indicating that RRAC is particularly rich in vitamin C, potassium, and several essential amino acids. The polyphenolic profile of RRAC was further characterized by high-performance liquid chromatography (HPLC) using an Agilent 1260 Infinity III liquid chromatography system (Agilent Technologies, Santa Clara, CA, USA), in accordance with a previously reported method²². Chromatographic separation was performed on an Eclipse C18 column (4.6 mm × 250 mm i.d., 5 μm). The mobile phase consisted of 0.1% phosphoric acid in water (solvent A) and HPLC-grade methanol (solvent B), at a constant flow rate of 1 mL/min. The gradient elution program was set as follows: 0 min, 20% A / 80% B; 0–10 min, 30% A / 70% B; 10–20 min, 40% A / 60% B; 20–30 min, 50% A / 50% B; 30–40 min, 60% A / 40% B; and 40–50 min, 70% A / 30% B. Detection was performed at 270 nm using a multi-wavelength detector, with an injection volume of 10 µL and a column temperature maintained at 25 °C. Individual polyphenolic compounds were identified by retention time matching and co-injection with authentic standards.

Table 1.

Nutritional content of the RRAC composite supplement.

Nutrition facts	/100 g
Total calories	703 kcal
Fat	2.9
Protein	1.65
Carbohydrate	33.4
Dietary fiber	1.04
SOD	1.88 × 103 (U/g)

Table 2.

Vitamin and element of the RRAC composite supplement.

Vitamin and element	mg/100 g
Vitamin E	1.27
Vitamin B1	0.245
Vitamin B2	0.245
Vitamin B6	0.375
Vitamin C	671
Na	166
Ca	351
K	3.27 × 103

Table 3.

Amino acids content of the RRAC composite supplement.

Amino acids	g/100 g
Arginine	0.11
Lysine	0.052
Histidine	0.014
Phenylalanine	0.026
Leucine	0.041
Isoleucine	0.024
Methionine	0.016
Valine	0.033
Alanine	0.11
Glycine	0.25
Proline	0.12
Glutamic acid	0.17
Serine	0.039
Threonine	0.035
Aspartic acid	0.097

Animals

In this experimental, a total of Fifty healthy independent ventilation cages (IVC) grade ICR male mice, 6-8weeks old with an initial weight of 28–32 g, were purchased from the SPF Biotechnology Co., Ltd. (Beijing, China). The animals were housed in Guizhou Foster Biotechnology Co., Ltd. (Guizhou, China), and was carried out according to the standard guidelines. Standard food and water were freely provided to the animals and cared for under standard conditions with exposure to 12 light/dark cycles during the experiment period. All experimental procedures were strictly performed in accordance with the Guide for the Care and Use of Laboratory Animals published by the U.S. National Institutes of Health, and the study protocol was approved by the Animal Research Ethics Committee of Guizhou Foster Biotechnology Co., Ltd. (IACUC No. FST/LL-03).

Experimental design

Rosa roxburghii and kiwifruit were recognized as rich in vitamin C, and the vitamin C content of RRAC detected in this study is 671 mg per 100 g of dry weight (Table 2). Based on “The Chinese Dietary Guidelines”, which recommends a daily Vitamin C intake of 100–200 mg, conversion calculation shows that only 14.9 g of RRAC can meet the daily vitamin C requirement of healthy adults. Therefore, the mouse dosage was converted from a human equivalent dose (HED) based on body surface area by the following formula from the US Food and Drug Administration (available from http://www.fda.gov/downloads/Drugs/GuidanceComplianceRegulatoryInformation/Guidances/ ucm078932.pdf): ): assuming a human weight of 60 kg, the human equivalent dose for 14.9 g /60 kg (0.2483 g/kg) = 0.2483 × 12.3 = a mouse dose of 3.05 g/kg (305 mg/kg); the conversion coefficient 12.3 was used to account for differences in body surface area between a mouse and a human. To determine the optimal dose of RRAC, we found through preliminary experiments that the maximum gavage dose that can be used to prepare RRAC is 520 mg/kg. Therefore, three experimental doses (130, 260, and 520 mg/kg/d) were designed to systematically evaluate the anti-fatigue efficacy and dose response relationship of RRAC.

After one week of adaptation, the animals were randomly divided into five groups according to their body weight (n = 10 in each group), and they were administered by gavage once a day for 28 days: (1) the blank control group (Rest, saline), (2) the fatigue model control group (Con, saline), (3) the RRAC-L group (130 mg/kg/d), (4) the RRAC-M group (260 mg/kg/d), (5) the RRAC-H group (520 mg/kg/d). The RRAC was dissolved in physiological saline and administered at a volume of 10 ml/kg body weight via oral gavage. In the control group, an equal volume of normal saline was administered by gavage according to the body weight to match that of the experimental group. All mouse body weights were recorded every 7 days for inter group comparisons.

Exercise performance test

In order to understand the effect of RRAC on improving exercise endurance performance, we conducted a swimming exhaustion test. The tail of mice were attached with 5% body weight, and the mice in all group except the Rest group were subjected to swim individually in a plastic pool (50 × 50 × 40 cm) filled with water (25 ± 1℃) to a depth of 30 cm, and the duration of such swimming was recorded. The test mice were then forced to swim until they lost coordinated movement or could not return to the surface within 10s, as described previously²³.

Determination of fatigue-associated biochemical variables

In order to understand the effect of RRAC on fatigue-related indicators and physiological adaptation after exercise, after swimming exhaustion and resting for 20 min, all the mice were euthanized by 95% CO2 after the last treatment, and blood was immediately collected and mix thoroughly with anticoagulant (heparin). Blood collected by cardiac puncture was centrifuged at 2000 rpm for 15 min at 4 ℃ and then plasma was collected for − 80 ℃ storage. Biochemical variables were estimated by commercial assay kits according to the procedures provided in the kits. Levels of blood urea nitrogen (BUN), L-lactic acid (LAC), lactate dehydrogenase (LDH), creatine kinase (CK), glycogen(GLU), superoxide dismutase (SOD), malondialdehyde (MDA) and glutathione peroxidase (GSH-Px) were measured.

Body composition, glycogen content, and histopathology

After the mice were euthanized, the heart, lung, liver, kidney, spleen, adipose, and skeletal muscle were accurately exercise and weighed to calculate the organ indexes according to the formula as follows:

The organs were carefully removed, chopped, and fixed in 10% formalin. The tissue was embedded in paraffin and cut into 2 μm-thick sections for morphological and pathological evaluation. The sections were prepared by deparaffinization, stained with hematoxylin and eosin (H&E), dehydrated through a series of graded alcohols (100%, 95%, and 75%), and rinsed twice in xylene. Photomicrographs were obtained by using a Motic digital pathology slide scanner. Parts of the liver and gastrocnemius muscle tissues were stored in liquid nitrogen for glycogen content analysis, as previously described.

Statistical analysis

All data are expressed as mean ± SD. The statistical analysis was performed in SPSS 24.0 software (IBM Corporation, Armonk, NY, USA). Using Tukey’s post hoc test, multiple group comparisons were analyzed by one-way analysis of variance (ANOVA). Statistical significance was set at p < 0.05.

Results

Polyphenolic compounds in RRAC

The polyphenol of RRAC was determined by a high-performance liquid chromatography (HPLC) method, as shown in Fig. 2, polyphenolic compounds including gallic acid, catechins, and chlorogenic acid are present in RRAC.

Fig. 2.

HPLC chromatogram of polyphenols extracted from RRAC, detected at 270 nm. (A) The HPLC chromatogram of RRAC. (B) The HPLC chromatogram of mixed standards. (a.Gallic acid; b.Catechin; c.Chlorogenic acid)

General characteristics of mice with RRAC for 28 days

As shown in Fig. 3, after 28 days of continuous gavage intervention, the weight of each group of mice showed stable growth and ; there was no significant difference between the groups (p > 0.05). Also, there were no significant differences in the organ indexes of the lungs, kidney, liver, spleen, and testis.

Fig. 3.

The effect of RRAC on mice various parameters. (A) The effect RRAC intervention on body weight in ICR mice. (B) The effect of RRAC intervention on the organ index in ICR mice. Data are expressed as mean ± SD for n = 10 mice per group. Different superscript letters (a, b) mean significant difference at p<0.05 and # indicates that there is no significant difference between the group (p>0.05).

Effect of RRAC on exercise performance

In swimming exhaustion test, the time for the mice to resist to fatigue is normally noted to assess the anti-fatigue activity of several compounds or extracts. The swimming time of mice in the Con, RRAC-L, RRAC-M, and RRAC-H groups were 369.90 ± 81.635, 459.60 ± 85.73, 721.10 ± 160.874, and 597.40 ± 180.22 s, respectively. Compared with the Con group, the RRAC-L, RRAC-M and RRAC-H groups were significantly increased relative swimming exhaustion time by 1.24-fold (p = 0.145), 1.95-fold (p<0.001), 1.62-fold (p<0.001).

Effect of RRAC on the plasma biochemical parameters of ICR mice

To confirm the anti-fatigue effect exerted by RRAC, the plasma levels were evaluated for BUN, CK, LAC, LDH, GLU content and MDA, SOD, GSH-Px activity after 20 min rest following the swimming exhaustion test.

Effect of RRAC on liver and muscle glycogen levels of ICR mice

To confirm the effect of RRAC on liver and muscle glycogen, the levels of glycogen were measured using liver/muscle glycogen assay kits. The liver glycogen levels of the mice in the Rest, Con, RRAC-L, RRAC-L, RRAC-M and RRAC-H groups were 6.10 ± 0.88, 6.48 ± 0.90, 8.15 ± 0.64, 11.92 ± 2.37 and 10.46 ± 1.70 mg/g liver, respectively (Fig. 6A). Compared with the Rest group, the Con, RRAC-L, RRAC-L, RRAC-M and RRAC-H groups were significantly improved by 1.06-fold (p = 0.570), 1.34-fold (p = 0.003), 1.95-fold (p < 0.001), and 1.71-fold (p < 0.001), respectively. Muscle glycogen levels in the Rest group, the Con, RRAC-L, RRAC-L, RRAC-M and RRAC-H groups were 1.38 ± 0.04, 1.26 ± 0.03, 1.32 ± 0.03, 1.34 ± 0.03 and 1.32 ± 0.05 mg/g muscle, respectively (Fig. 6B). The RRAC-L, RRAC-M, and RRAC-H groups were significantly increased by 1.05-fold (p=0.001), 1.06-fold (p < 0.001), and 1.05-fold (p༝0.004), respectively, as compared to the Con group.

Fig. 6.

The effect of RRAC on liver and muscle glycogen levels. (A) The liver glycogen levels; (B) The muscle glycogen levels. Data are expressed as mean ± SD for n = 10 mice per group. Different superscript letters (a, b, c, d) mean significant difference at p < 0.05.

Effect of RRAC on tissue histology

At the end of the study, we conducted histological analysis of the heart, lungs, liver, kidney, spleen, testis, and muscle, were performed to understand the morphological damage in tissues. These results indicate that RRAC has no adverse effects on organs and tissues at the doses tested in this study.

Discussion

Fatigue is an unavoidable physiological state in modern life, and the prevention or alleviation of fatigue has long been a challenging issue in the field of nutrition and functional food development. Regular exercise, balanced nutrition, and complementary and alternative medicine have been reported to effectively relieve fatigue by providing energy substrates and supporting recovery²⁴. Apart from conventional nutritional supplements such as vitamins, β-alanine, and creatine, in recent years a growing number of studies have focused on identifying novel natural anti-fatigue components as alternatives to synthetic compounds²⁵. Not only traditional herbal plants but also several common plant-based foods have been found to exhibit considerable potential, owing to their abundant bioactive constituents, including polyphenols, vitamins, phytosterols, biogenic amines, and biologically active proteins^26–28. Rosa roxburghii Tratt is particularly rich in vitamin C, SOD, polysaccharides, and phenolic compounds, while kiwifruit contributes ascorbic acid, flavonoids, and dietary fiber; the two fruits therefore appear to be nutritionally complementary rather than redundant. In order to improve the portability of the composite supplement and to mitigate the astringency associated with Rosa roxburghii, we formulated a jelly-like product (RRAC) by mixing Rosa roxburghii and kiwifruit at a 1:3 volume ratio, with gelatin as a gelling agent. The 1:3 ratio was selected on the basis of preliminary sensory evaluations indicating that this proportion provided acceptable palatability while retaining the bioactive characteristics of Rosa roxburghii. The jelly matrix could also be expected to slow gastric emptying and prolong the contact time of polyphenols with the intestinal mucosa, which may favor their absorption.

The swimming exhaustion test has been widely used in animal models to evaluate the anti-fatigue efficacy of drugs or natural compounds²⁹. In our study, supplementation with different doses of RRAC for 28 consecutive days significantly prolonged the swimming time of mice in a non-linear, dose-dependent manner (Fig. 4). The swimming time of the RRAC-M group (721.10 ± 160.87 s) was 1.95-fold longer than that of the Con group (369.90 ± 81.64 s, p < 0.001), and was also longer than that of the RRAC-H group (597.40 ± 180.22 s, 1.62-fold of Con, p < 0.001). This inverted-U dose response is consistent with the well-documented hormetic behavior of dietary antioxidants, in which moderate doses optimize redox balance, while excessive intakes may impair endogenous adaptive signaling and even exert mild pro-oxidant effects. A similar pattern has also been observed in previous animal studies of polyphenol-rich and polysaccharide-based supplements, in which moderate doses produced the most pronounced anti-fatigue effects, while higher doses provided no additional benefit²⁵.

Fig. 4.

The effect of RRAC on swimming exhaustion test in ICR mice. Data are expressed as mean ± SD for n = 10 mice per group. Different superscript letters (a, b, c) mean significant difference at p<0.05.

Blood biochemical parameters have been commonly used as markers for fatigue. Previous studies have shown that when the organism does not acquire energy replenishment in time after intense exercise, proteins undergo deamination to produce pyruvate and a large amount of ammonia, which must be metabolized into urea through the urea cycle in the liver and subsequently excreted via the kidneys through the blood circulation system. At the same time, glycolysis is accelerated, generating large amounts of lactic acid and contributing to muscle fatigue. When muscle is damaged, creatine kinase (CK) is released into the blood, while lactate dehydrogenase (LDH) in skeletal muscle also penetrates into the circulation, where LDH catalyzes the conversion of pyruvate to lactic acid (LAC), thereby reducing exercise tolerance. Prolonged or high-intensity exercise therefore disrupts the balance of energy metabolism, and elevated levels of blood urea nitrogen (BUN), LAC, LDH, and CK are typically observed^30–33. Our results are consistent with these previous reports: gavage with RRAC at certain doses significantly reduced post-exercise BUN, LAC, LDH, and CK levels in mice (Fig. 5A-D). The magnitude of LAC and BUN reduction observed in the RRAC-M group is of a similar order to that previously reported for other natural bioactive supplements administered in the same exhaustive swimming model^29,31, suggesting that polyphenol-based composite supplementation could achieve effects comparable to those of single bioactive compounds or extracts.

Fig. 5.

The effect of RRAC on biochemical parameters. (A) Blood urea nitrogen, BUN (B) Creatine kinase, CK (C) L-lactic acid, LAC (D) lactate dehydrogenase, LDH (E) Glycogen (GLU), (F) malondialdehyde, MDA, (G) superoxide dismutase, SOD, (H) glutathione peroxidase, GSH-Px. Data are expressed as mean ± SD for n = 10 mice per group. Different superscript letters (a, b, c, d) mean significant difference at p<0.05.

In addition, blood glucose is also a crucial fatigue-related biochemical indicator. Long-term or high-intensity exercise stimulates the activity of glucose transporters on the membrane of muscle fibers, leading to a decrease in blood glucose concentration. However, glycogen is an essential energy source for the organism, and it could increase the glucose uptake of cells for glycolysis to maintain blood glucose concentration and supply energy^34,35. In the present study, the blood glucose (GLU) level of mice in the RRAC group was higher than that of the control group (Fig. 5E). After prolonged or high-intensity exercise, muscle glycogen generates ATP to meet energy demand, while hepatic glycogen releases glucose for other tissues to maintain physiological balance, which would result in the depletion of muscle and liver glycogen contents in the body^36,37. In our study, hepatic glycogen in the RRAC-M group reached 11.92 ± 2.37 mg/g, 1.95-fold higher than that of the Rest group (p < 0.001), while muscle glycogen in the RRAC-L, RRAC-M, and RRAC-H groups was significantly increased by 1.05- to 1.06-fold compared with the Con group (Fig. 6). Liver glycogen appeared more responsive to RRAC supplementation than muscle glycogen, which may reflect the preferential delivery of orally absorbed polyphenols and monosaccharides to the liver via the portal circulation, and is in line with the central role of glycogen metabolism in maintaining systemic energy balance during exercise³⁸.

Previous studies have shown that the anti-fatigue and antioxidant effects are generally closely related in their functional metabolic pathways. The excessive oxygen free radicals generated during prolonged and high-intensity exercise could cause oxidative damage to tissues or organs, ultimately leading to muscle fatigue³⁹. Based on this rationale, we hypothesized that ingestion of the Rosa roxburghii and kiwifruit composite supplement (RRAC) would enhance endurance performance by improving antioxidant status. The results support this hypothesis, demonstrating that RRAC significantly improved endurance performance, and that the upregulated antioxidant activity may underlie the performance-enhancing effect of RRAC (Fig. 5F–H).

SOD and GSH-Px, as important antioxidant enzymes, are commonly used as biomarkers for evaluating antioxidant capacity and oxidative damage, owing to their capability of scavenging oxygen free radicals in the body, and the levels of SOD and GSH-Px could indirectly reflect the extent of tissue damage^33,40. MDA, on the other hand, is a product of lipid peroxidation and serves as an indicator of oxidative stress in cells and tissues³⁸. These markers are recognized contributors to premature muscle fatigue during sustained contractions and exercise. The accumulation of reactive oxygen species (ROS) within active muscle fibers impairs muscle function and promotes fatigue³⁹. Accordingly, exogenous antioxidant supplementation has been proposed as a strategy to delay fatigue and enhance endurance performance⁴⁰. The benefits of antioxidant supplementation are thought to arise from improvements in cellular redox balance and reductions in oxidative damage to DNA, lipids, and proteins. Although the antioxidant properties of Rosa roxburghii and kiwifruit have been well established when studied separately, their potential in a composite supplement has remained largely unexplored.

Nutritionally, Rosa roxburghii and kiwifruit are rich sources of polyphenols, organic acids, vitamin C, and other bioactive compounds. HPLC analysis of RRAC in the present study identified several polyphenols, including gallic acid, catechins, and chlorogenic acid (Fig. 2). Polyphenolic compounds have been shown to activate the Nrf2/Keap1 signaling pathway, which subsequently upregulates the expression of phase II antioxidant enzymes such as SOD and GSH-Px and attenuates exercise-induced oxidative damage; a similar mechanism has previously been demonstrated for curcumin supplementation in an exhaustive swimming mouse model²³. The increased SOD and GSH-Px activities together with the reduced MDA levels observed in the RRAC-M group are therefore in agreement with this antioxidant property of dietary polyphenols. In addition, gallic acid, catechins, and chlorogenic acid have each been individually associated with improvements in mitochondrial function and energy metabolism in previous studies, and the co-occurrence of these three polyphenols in RRAC could provide a broader phenolic spectrum than either Rosa roxburghii or kiwifruit alone. The observed benefits are therefore likely the result of additive or synergistic actions of multiple bioactive constituents rather than the contribution of any single compound.

Moreover, the effects of RRAC on exercise-induced oxidative stress and physical performance appear to be influenced by dosage. In the present study, the RRAC-M group showed the best performance among all indicators tested, while the higher dose (RRAC-H) did not produce additional benefit and in some indicators showed a slight regression toward the Con group. This further supports a hormetic dose–response relationship for polyphenol supplementation in exercise contexts, in which moderate intake appears to be more favorable than high-dose supplementation for optimizing endogenous antioxidant defenses²⁵.

In terms of safety, no significant differences in body weight or organ indices (lung, kidney, liver, spleen, and testis) were observed among groups during the 28-day intervention (Fig. 3), and histopathological examination of the heart, lung, liver, kidney, spleen, testis, and skeletal muscle revealed no morphological abnormalities in any of the RRAC-supplemented groups (Fig. 7). These results indicate that daily oral administration of RRAC at the tested doses is well tolerated and does not cause detectable organ-level toxicity under the present experimental conditions.

Fig. 7.

The effect of RRAC on histology in various organs: (A) heart, (B) lung, (C) liver, (D) kidney, (E) spleen, (F) testis, (G) muscle. (H&E stain, magnification: 100×, bar, 80 μm).

Several limitations of this study should be acknowledged. First, only ICR mice of a single sex were used, and sex-specific responses to RRAC supplementation remain to be examined. Second, although the swimming exhaustion test is a well-established model for evaluating anti-fatigue activity, it does not fully recapitulate the complexity of human exercise performance, and human trials are required to validate the present findings. Third, the underlying molecular mechanisms—particularly the involvement of the Nrf2/Keap1 antioxidant pathway—were inferred from the literature rather than directly verified at the gene and protein expression level in our experimental samples, and further mechanistic studies are required. Fourth, although HPLC analysis confirmed the presence of gallic acid, catechins, and chlorogenic acid in RRAC, the individual contribution of each polyphenol to the observed anti-fatigue effects was not separately evaluated. Finally, single-ingredient comparison groups (Rosa roxburghii alone and kiwifruit alone) were not included in the present study, and future work should incorporate such groups to formally confirm the synergistic nature of the composite formulation.

Future studies should therefore focus on the following directions: (i) verification of the anti-fatigue and antioxidant effects of RRAC in human subjects through randomized controlled trials, including measurement of post-exercise blood biochemistry and perceived fatigue; (ii) elucidation of the molecular mechanisms underlying RRAC supplementation, with particular attention to the Nrf2/Keap1 and related antioxidant signaling pathways; (iii) quantitative determination of individual polyphenols in RRAC and pharmacokinetic studies on their bioavailability from the jelly matrix; and (iv) evaluation of long-term supplementation effects, including potential influences on gut microbiota composition and inflammatory cytokine profiles.

In conclusion, the present study demonstrates that 28 days of oral RRAC supplementation could significantly prolong swimming endurance, reduce post-exercise BUN, LAC, LDH, and CK levels, increase hepatic and muscle glycogen storage, and enhance antioxidant enzyme activity in ICR mice, without producing detectable adverse effects on body weight, organ indices, or tissue histology. Among the three doses tested, the moderate dose (RRAC-M) showed the most pronounced effects, consistent with a hormetic dose–response pattern commonly reported for dietary polyphenols. Therefore, we believe that RRAC could be used as a functional supplement to improve exercise performance and to exert anti-fatigue bioactivity, and it provides a practical example of how the combination of two phenolic-rich fruits in a jelly form can address both palatability and functionality in the development of anti-fatigue nutritional products.

Conclusion

In summary, our results provide evidence that supplementation with RRAC for 28 consecutive days could significantly increase exercise endurance performance by increasing glycogen storage. In addition, supplementation significantly reduced post-exercise biochemical parameters of fatigue, such as BUN, LAC, LDH concentration and CK activity. Meanwhile, the activities of SOD and GSH-Px were increased to varying degrees, and the contents of MDA were reduced significantly, while the supplementation with the proper amount of RRAC would not cause damage to various physiological organs. Collectively, these results indicate that RRAC supplementation can be used to mitigate fatigue during exercise and increase performance.

Author contributions

W.Y.Y. performed the experiments and wrote the main manuscript text; L.L.P. and X.X.L provided the idea and wrote the main manuscript text; X.X.L. andL.Y. performed the experiments and processed the data; H.H.Z. supervised the project. All authors have read and agreed to the published version of the manuscrip.

Funding

This work was supported by grants from the High-level innovative talent project in Guizhou Province (NO. GCC [2023]073), and Guizhou Provincial Science and Technology Plan Project (NO. [2024]019), Guizhou, China.

Data availability

The datasets used and/or analysed during the current study are available from the corresponding author on reasonable request.

Declarations

Competing interests

The authors declare no competing interests.

Institutional review board statement

The animal experiment was approved on 25 September 2025 by the Animal Ethics Committee of Guizhou Foster Biotechnology Co., Ltd, Guizhou, China (Approval No. FST/LL−03).