Anti-Fatigue Efficacy and Mechanism of Perilla Seed Peptides

Fatigue is a non-specific and multi-dimensional symptom arising from complex interactions among biological, psychological, social and behavioural processes[1]. Physiological fatigue is classified into central fatigue and peripheral fatigue. Peripheral fatigue, also known as chronic fatigue, is mainly triggered by intrinsic muscular factors such as insufficient energy supply and accumulation of metabolic metabolites[2]. With technological advances and accelerating work and living rhythms, an increasing number of people suffer from fatigue. Modern nutritional studies have demonstrated that bioactive peptides are readily digested and absorbed by the human body, which can effectively improve cellular metabolism, restore physiological homeostasis, and thereby delay fatigue onset[3].

In recent years, nutritional intervention for fatigue relief has attracted extensive research attention[4]. The commonly adopted evaluation protocol involves intragastric administration followed by detection of exercise endurance and related biochemical indicators in mice[5]. Among these assessment models, the weight-loaded swimming test in mice is most widely applied, where the prolongation of exhaustive swimming time is used to evaluate the anti-fatigue capacity of bioactive substances[6]. Based on the energy depletion theory and metabolite accumulation hypothesis, key fatigue-related biochemical markers include muscle/hepatic glycogen contents, blood lactic acid (BLA), blood urea nitrogen (BUN) and lactate dehydrogenase (LDH) activity[7]. Existing literature has reported the anti-fatigue functions of various bioactive peptides. For instance, grass carp-derived anti-fatigue peptides can elevate bodily glycogen reserves and reduce metabolite accumulation[8]. Liu et al.[9] verified that peanut peptides enhance exercise endurance in mice by regulating glucose metabolism and inhibiting lactic acid accumulation.

Perilla frutescens (L.) Britt., an annual herb of the Lamiaceae family, is predominantly cultivated across Asian countries including China, India, Japan, Vietnam and Thailand[10]. Perilla has a cultivation history of over 2,000 years in China[11]. Its seeds, rhizomes and leaves are rich in diverse nutrients and bioactive compounds. Perilla seed oil features a high fatty acid content, with oleic acid, linoleic acid and linolenic acid accounting for approximately 93% of total fatty acids[12]. Perilla seeds contain roughly 25% protein with abundant and well-balanced amino acid profiles[13]. Although perilla seed oil has been extensively developed and commercialised, research and industrial utilisation of perilla seed isolated protein and perilla peptides remain insufficient. In this study, perilla seed peptides were prepared via double enzymatic hydrolysis. Their anti-fatigue activity and underlying molecular mechanisms were investigated to provide theoretical support for the high-value utilisation of perilla seed peptides.

Abstract

Perilla seeds are abundant in protein with comprehensive amino acid composition, and their derived bioactive peptides possess great development potential. In this research, perilla seed peptides were prepared by enzymatic hydrolysis. Male mice were subjected to continuous intragastric administration of low, medium and high doses of perilla seed peptides for 28 days. The exhaustive swimming time of mice under load was recorded, together with the detection of muscle/hepatic glycogen contents, serum BLA, BUN and LDH levels. Compared with the blank control group, the weight-loaded swimming time was prolonged by 10.61%, 22.76% and 56.14% in the low-, medium- and high-dose peptide groups, respectively. Perilla seed peptides significantly (P<0.05) promoted muscle and hepatic glycogen deposition, elevated serum LDH activity, and accelerated the catabolism of BLA and BUN. These results confirm that perilla seed peptides exhibit prominent anti-fatigue effects.

Results and Analysis

2.1 Determination of Main Components of Perilla Seed Peptides

The compositional parameters of prepared perilla seed peptides are summarised in Table 1. The total protein content was 87.70%, ash content 6.21%, total free amino acid content 5.87%, small peptide content 78.05%, and the proportion of short peptides with molecular weight below 1000 u reached 80.12%.

2.2 Effects of Perilla Seed Peptides on Body Weight of Mice

As shown in Table 2, the body weight variation trends across all perilla seed peptide dose groups were similar to those of the blank control and positive control groups, with no statistically significant differences (P＞0.05). The findings indicate that dietary supplementation with perilla seed peptides supports normal mouse growth and does not induce obesity.

2.3 Effects of Perilla Seed Peptides on Weight-Loaded Swimming Endurance in Mice

Experimental results are presented in Figure 1. After 28 days of intragastric administration, the exhaustive swimming time of mice in low-, medium- and high-dose groups increased by 10.61%, 22.76% and 56.14% relative to the blank control group, respectively. The high-dose group achieved a swimming endurance of 116.40 min, which was significantly longer than the 104.19 min recorded in the positive control group (P<0.05). A clear dose-effect relationship was observed: higher peptide dosage corresponded to longer exhaustive swimming time, verifying the dose-dependent anti-fatigue activity of perilla seed peptides.

2.4 Effects of Perilla Seed Peptides on Serum BUN Levels in Mice

As illustrated in Figure 2, serum BUN concentrations in all three peptide dose groups were markedly lower (P<0.05) than those in the blank control group following 28 days of treatment. The medium- and high-dose groups recorded BUN levels of (11.40±0.16) mmol/L and (11.12±0.31) mmol/L, both significantly lower than the positive control value of (12.56±0.26) mmol/L (P<0.05). Perilla seed peptides effectively accelerate the catabolism of blood urea nitrogen, demonstrating superior efficacy compared with whey protein.

2.5 Effects of Perilla Seed Peptides on Muscle and Hepatic Glycogen Contents in Mice

After 28 consecutive days of intragastric gavage, the high-dose group showed muscle glycogen and hepatic glycogen levels of (1.63±0.01) mg/g and (13.11±0.36) mg/g, significantly higher than the positive control group [(1.56±0.05) mg/g and (9.07±0.56) mg/g, P<0.05] (Figure 3). Relative to the blank control group, muscle glycogen and hepatic glycogen contents in the high-dose group rose by 117.33% and 89.73%, respectively. Perilla seed peptides effectively enhance glycogen accumulation in muscle and liver tissues, thereby boosting bodily energy supply during exercise.

2.6 Effects of Perilla Seed Peptides on Serum BLA and LDH Activities in Mice

Figure 4 shows that LDH activity was elevated in all peptide treatment groups compared with the blank control group. Serum BLA concentrations in the medium- and high-dose groups were (5.89±0.31) mmol/L and (5.73±0.20) mmol/L, both significantly reduced relative to the blank control group (8.56±0.15 mmol/L, P<0.05). Perilla seed peptides can upregulate LDH activity and inhibit lactic acid accumulation, thus delivering potent anti-fatigue benefits.

Discussion and Conclusion

Four-week intragastric administration of low, medium and high doses of perilla seed peptides prolonged mice weight-loaded swimming time by 10.61%, 22.76% and 56.14% respectively in a concentration-dependent manner, which markedly improved exercise capacity. In terms of anti-fatigue mechanisms, muscle glycogen is preferentially consumed during physical activity, followed by hepatic glycogen to sustain energy expenditure; accordingly, glycogen consumption is closely correlated with fatigue severity[18]. The high-dose perilla seed peptide group increased muscle and hepatic glycogen contents by 117.33% and 89.73% versus the blank control, facilitating greater energy reserve for physical exercise.

Under oxygen-deficient conditions, muscular aerobic metabolism shifts to anaerobic glycolysis, leading to lactic acid accumulation[19]. Excessive lactic acid impairs circulatory and skeletal muscle function and reduces motor performance[20-21]. Lactate dehydrogenase catalyses the conversion of excess muscle lactic acid into pyruvate, mitigating lactic acid build-up and delaying fatigue progression[22]. All three tested doses of perilla seed peptides significantly enhanced systemic LDH activity, accelerated the metabolic clearance of circulating BLA and BUN during exercise, and outperformed the whey protein positive control in retarding the decline of muscular exercise capacity[23,24].

In conclusion, enzymatically prepared perilla seed peptides improve exercise performance and exert favourable anti-fatigue effects in mice via promoting glycogen deposition, elevating LDH activity and accelerating the elimination of exercise-induced metabolic waste products.

References

[1] HSIAO C Y, HSU Y J, TUNG Y T, et al. Effects of Antrodia camphorata and Panax ginseng supplementation on anti-fatigue properties in mice [J]. Journal of Veterinary Medical Science, 2018, 80 (2):284-291.

[2] HAO J M, LIU H Y. Research progress on the essence of fatigue from integrated traditional Chinese and western medicine perspectives [J]. Journal of Changchun University of Chinese Medicine, 2017, 33 (5):853-854.

[3] WANG P X, CHEN Y Y, WANG R, et al. Research advances in anti-fatigue peptides [J]. Journal of Fujian Agriculture and Forestry University (Natural Science Edition), 2019, 48 (3):273-284.

[4] HU M, DU J, DU L D, et al. Anti-fatigue activity of purified anthocyanins prepared from purple passion fruit (P. edulis Sim) epicarp in mice [J]. Journal of Functional Foods, 2020, 65:103725.

[5] CHEN X F. Physiological activity analysis and application of anti-fatigue properties of maca and kelp [D]. Xiamen: Jimei University, 2015.

[6] CHI A, LI H, KANG C, et al. Anti-fatigue activity of a novel polysaccharide conjugates from Ziyang green tea [J]. International Journal of Biological Macromolecules, 2015, 80:566-572.

[7] ZHAO Y Q, XIONG H J, SU Y C, et al. Research progress on natural marine bioactive substances with anti-fatigue effects [J]. Journal of Food Safety & Quality, 2019, 10 (1):158-164.

[8] REN J, ZHAO M, WANG H, et al. Effects of supplementation with grass carp protein versus peptide on swimming endurance in mice [J]. Nutrition, 2011, 27 (7):789-795.

[9] LIU J, CHEN L W, JI K M, et al. An endurance-enhancing effect of peanut meal protein hydrolysate in mice: Possible involvement of a specific peanut peptide [J]. Journal of Animal Physiology and Animal Nutrition, 2014, 98 (5):830-837.

[10] JUN H I, KIM B T, SONG G S, et al. Structural characterization of phenolic antioxidants from purple perilla (Perilla frutescens var. acuta) leaves [J]. Food Chemistry, 2014, 148:367-372.

[11] DAI C H, XU Z J, SHEN X K, et al. Nutritional composition analysis of different perilla seed varieties [J]. Journal of the Chinese Cereals and Oils Association, 2015, 30 (3):55-58.

[12] LI H Z, ZHANG Z J, HOU T Y, et al. Optimization of ultrasound-assisted hexane extraction of perilla oil using response surface methodology [J]. Industrial Crops and Products, 2015, 76:18-24.

[13] SHENG C H. Development of perilla protein products [D]. Wuhan: Wuhan Polytechnic University, 2011.

[14] National Health and Family Planning Commission of the People’s Republic of China. Determination of moisture in food: GB 5009.3-2016 [S]. Beijing: China Standards Press, 2016:1-6.

[15] National Health and Family Planning Commission of the People’s Republic of China. Determination of ash in food: GB 5009.4-2016 [S]. Beijing: China Standards Press, 2016:1-7.

[16] National Health and Family Planning Commission, China Food and Drug Administration. Determination of protein in food: GB/T 5009.5-2016 [S]. Beijing: China Standards Press, 2016:1-7.

[17] National Health and Family Planning Commission, China Food and Drug Administration. Determination of amino acids in food: GB/T 5009.124-2016 [S]. Beijing: China Standards Press, 2016:1-6.

[18] WEI L, WEN Y T, LEE M C, et al. Effects of isolated soy protein and strength exercise training on exercise performance and biochemical profile in postpartum mice [J]. Metabolism, 2019, 94:18-27.

[19] VETHARANIAM I, THOMSON R A, DEVINE C E, et al. Modelling muscle energy metabolism in anaerobic muscle [J]. Meat Science, 2010, 85 (1):134-148.

[20] CHI A P, KANG C Z, GUO H H, et al. Composition, anti-fatigue and anti-hypoxia activities of Patrinia scabiosaefolia polysaccharides [J]. Food Science, 2014, 35 (21):212-215.

[21] ZHANG Z R, MAO D R, GAO H, et al. Anti-fatigue and in vivo antioxidant effects of blueberry anthocyanins in mice [J]. Food Science, 2017, 38 (21):207-211.

[22] DENG S H, SONG L, DUAN X J, et al. Correlation analysis between HPLC fingerprint and anti-fatigue activity of Astragalus membranaceus extracts [J]. Chinese Materia Medica, 2013, 36 (2):260-264.

[23] LIN Y, LIU H L, FANG J, et al. Anti-fatigue and vasoprotective effects of quercetin-3-O-gentiobiose on oxidative stress and vascular endothelial dysfunction induced by endurance swimming in rats [J]. Food and Chemical Toxicology, 2014, 68:290-296.

[24] YOU L, ZHAO M, REGENSTEIN J M, et al. In vitro antioxidant activity and in vivo anti-fatigue effect of loach (Misgurnus anguillicaudatus) peptides prepared by papain digestion [J]. Food Chemistry, 2011, 124 (1):188-194.