Sage Journals: Discover world-class research

Abstract

Background & Objectives

Phytoecdysteroids contained in common spinach (Spinacia oleracea L.) and wild spinach (Blitum bonus-henricus (L.) Rchb.) have been reported to produce anabolic effects, including increase in size and number of muscle fibers, increase in muscle strength, and improvement of physical performance. Based on this premise, the present study investigated whether treatment with hydroalcoholic raw extracts of Spinacia oleracea and Blitum bonus-henricus improved physical performance in mice exposed to an exhaustion running test.

Methods

Adult male mice were treated repeatedly and intragastrically with either 0, 1000, and 2000 mg/kg Spinacia oleracea extract or 0, 1000, and 2000 mg/kg Blitum bonus-henricus extract, and exposed to 20 daily running sessions at Rota-Rod.

Results

Treatment with Blitum bonus-henricus extract resulted in a dose-related, progressive increase in exhaustion time (defined as the third fall from the revolving drum). Over the entire second half of the 20-session period, mice treated with 2000 mg/kg Blitum bonus-henricus extract displayed an exhaustion time at least double than that recorded in vehicle-treated mice. Conversely, treatment with Spinacia oleracea extract was totally ineffective.

Conclusions

These results suggest that products derived from Blitum bonus-henricus may represent novel, promising anti-fatigue agents.

Keywords

phytoecdysteroids ecdysterone animal models of physical fatigue and endurance Rota-Rod

Introduction

Vigorous physical activity, including endurance training, has repeatedly been reported to result in a number of beneficial health effects, including – among several others – preservation of muscle mass and strength¹ and reduced risk of developing sarcopenia later in old age.² By stimulating muscle protein synthesis, endurance training compensates muscle protein degradation³ and reduces age-related loss of muscle mass, a phenomenon known to start already in young adulthood.^4,5

Proper nutritional interventions can effectively sustain muscle mass and strength.⁶ Specifically, diets rich in proteins, vegetables, fruits, and vitamin D may delay loss of muscle mass and strength.⁷ Combination of endurance training and specific nutritional interventions may therefore constitute an effective approach to counteract physiological decline in muscle mass and strength.

Spinach (Spinacia oleracea L.; common spinach) is often referred as “super food” or “power food” because of its high content of phytochemicals like carotenoids, flavonoids, phenolic compounds, vitamins, and minerals.^8,9 More specifically, phytoecdysteroids contained in spinach leaves have been reported to activate protein biosynthesis in murine and human myotubes,^10,11 increase size and number of muscle fibers in mice and rats,^12–14 improve physical performance (eg, forced swimming) and muscle strength (eg, increase in grip strength) in rats,¹¹ and enhance sports performance in humans.¹⁴

As a further contribution to this research field, the present study was aimed at investigating the effect of an hydroalcoholic raw extract of Spinacia oleracea on endurance in a highly challenging physical activity such as exhaustive running on a Rota-Rod in mice. Spinacia oleracea extract was titrated in 0.066% ecdysterone (20-hydroxyecdysone), ie the major phytoecdysteroid contained in spinach leaves¹⁵; it also contained 2.20% flavonoids. As an additional objective, the present study investigated the effect of an hydroalcoholic raw extract of Blitum bonus-henricus (L.) Rchb. on the same exhaustion test in mice. Blitum bonus-henricus (common names, among several others: wild spinach, good King Henry, and poor man's asparagus) belongs to the same Chenopodiaceae family of Spinacia oleracea (Cronquist system). Blitum bonus-henricus is widespread in Europe, Western Asia, and North America, where is used – since ancient times – because of its disparate medicinal and nutritional properties.^16,17 The Blitum bonus-henricus extract tested in the present study was titrated in 0.11% ecdysterone and contained 2.08% flavonoids.

Materials and Methods

Animals

Male CD1 mice [Charles River Laboratories, Calco (LC), Italy] were used. Mice were 8 weeks old at the start of each experiment. Mice were housed 10/cage in standard plastic cages with wood chip bedding. The animal facility was under a reversed 12:12 h light-dark cycle (lights on at 7:00 pm), at a constant temperature of 22 ± 2 °C and relative humidity of approximately 60%. Rota-rod sessions (see below) were conducted during the first 6 h of the dark phase of the light/dark cycle. Food pellets [standard chow for adult rodents; Mucedola, Settimo Milanese (MI), Italy] and tap water were always available (24 h/day). Mice were extensively habituated to handling and intragastric infusion.

Each experiment used an independent set of mice. Sample size was calculated using G*Power 3.1.9.7 software.¹⁹

Botanical Material

Extracts of Spinacia oleracea and Blitum bonus-henricus [donated by Indena, Milan (MI), Italy] were prepared starting from aerial parts of different biomasses. More specifically, aerial parts of Spinacia oleracea were collected in spring 2020, supplied by a food-grade company [Agrieuropa Società Cooperativa Agricola, Terracina (LT), Italy], and certified as food for human use; aerial parts of Blitum bonus-henricus, of spontaneous origin, were collected in summer 2018 around Lecco (LC, Italy) and summer 2019 near Parma (PR, Italy) and certified with taxonomic identification no. 5549 dated Jan. 26, 2018. Both extracts were identified botanically^20,21 and free of any contaminant (Indena; certificates of analysis available upon request).

Spinacia oleracea dry extract was prepared from 350 g of dried leaves, ground through a 10 mm grid, which were uploaded in a percolator and repeatedly extracted with 70% ethanol at room temperature, carrying out 5 extractions: 1 × 2.8L + 4 × 1.75L, contact time 2 h. The leachates were combined and vacuum concentrated. The concentrate was centrifuged to separate the insoluble residue from the solution. After decantation, a pitchy mass (dry residue) was separated and discarded. The clarified solution was concentrated to dryness and dried under vacuum for 24 h, yielding 86.2 g of dry extract.

Blitum bonus-henricus extract was prepared from 8200 g of dried leaves, ground through a 10 mm grid, were uploaded in two 20 L percolators and repeatedly extracted with 70% ethanol at room temperature, carrying out 5 extractions: 1 × 20L + 4 × 10L (for each percolator), contact time 2 h. The leachates were combined and vacuum concentrated. The concentrate was centrifuged to separate the insoluble residue from the solution. After decantation, a pitchy mass (dry residue) was separated and discarded. The clarified solution was concentrated to dryness and dried under vacuum for 24 h, yielding 1.5 kg of dry extract.

In both extracts, 20-hydroxyecdysone and flavonoid content was quantified by HPLC with an Ascentis^® Express C18 column and using water (A) and acetonitrile (B), both acidified with 0.1% formic acid, as eluents. The flow rate was set at 0.4 ml/min at a column temperature of 30 °C. In Spinacia oleracea extract the content of 20-hydroxyecdysone was 0.066%, while the flavonoid content was 2.20%. In Blitum bonus-henricus extract was titrated in 0.11% ecdysterone and contained 2.08% flavonoids.

Experimental Procedure

The experimental procedure was adapted from those of recent studies investigating fatigue endurance in mice.^22–24

Four identical Rota-Rod [accelerating Rota-Rod Treadmills for mice [Ugo Basile, Comerio (VA), Italy)] were used.

In each experiment, mice (n = 50) were initially trained to run on the revolving drum of the Rota-Rod for 5 consecutive daily sessions (Figure 1a). Each daily training session lasted 10 min and comprised (i) an initial phase of 5 min, during which the rotation speed was kept constant at 6 RPM, and (ii) a second phase of 5 min, during which the rotation speed was progressively accelerated from 7 to 60 RPM. Mice falling where immediately repositioned on the revolving drum.

Figure 1.

Panel a: Schematic representation of the study design. Panel b: Schematic representation of Rota-Rod sessions of the test period (Rota-Rod sessions of the training period comprised only the first two 5-min phases).

At the end of the 5-day training period, mice were divided into 3 groups of n = 16–17, matched for their body weight and performance at Rota-Rod over the last 3 sessions of the training period. The test period comprised 20 daily sessions at Rota-Rod (Monday to Friday for 4 consecutive weeks) (Figure 1a). Sixty min before exposure to Rota-Rod, mice were treated intragastrically with either 0, 1000, and 2000 mg/kg Spinacia oleracea extract (Experiment 1) or 0, 1000, and 2000 mg/kg Blitum bonus-henricus extract (Experiment 2). Both extracts were suspended in distilled water with 2% (w/v) Tween 80 (infusion volume: 10 ml/kg). Control mice (0 mg/kg Spinacia oleracea extract; 0 mg/kg Blitum bonus-henricus extract) received an equal volume of distilled water with 2% (w/v) Tween 80. Extract administration was performed by means of a flexible, plastic feeding tube (Instech Laboratories, Plymouth Meeting, PA, USA). Extracts were administered also on intervening Saturdays and Sundays, resulting in a total number of 26 consecutive days of treatment (Figure 1a).

In both experiments, Rota-Rod sessions of the test period lasted up to 60 min (the 60-min time limit corresponded to cut-off, when those mice still running on the revolving drum were removed by the operator) and comprised 3 different phases: (i) an initial phase of 5 min, during which the rotation speed was kept constant at 6 RPM; (ii) a second phase of 5 min, during which the rotation speed was progressively accelerated from 7 to 60 RPM; (iii) a third phase of variable duration (maximum 50 min), during which the rotation speed was kept constant at 60 RPM (Figure 1b). The measured variable was the time (expressed in min and defined as “exhaustion time”) each mouse managed to remain on the revolving drum between the beginning of the acceleration phase and the third fall from the revolving drum (mice not reaching the exhaustion time were assigned the value “60 min”).

Mouse body weight (expressed in g) was recorded twice weekly (on Monday and Friday).

Both in vivo experiments lasted 33 consecutive days (Figure 1a), were performed in March and April 2023, and were conducted at the animal facility of the Section of Cagliari, Neuroscience Institute, National Research Council of Italy, Monserrato (CA), Italy.

Statistical Analysis

In both experiments, data on body weight as well as on exhaustion time at the Rota-Rod test were analyzed by 2-way (treatment; time) ANOVA for repeated measures on the factor “time”, followed by the Tukey's test for post hoc analysis.

Results

Experiment 1 – Spinacia oleracea Extract

ANOVA indicated no effect of treatment with Spinacia oleracea extract [F(2,47) = 0.08, P > 0.05], a highly significant effect of time [F(19,893) = 7.26, P < 0.0001], and no “treatment x time” interaction [F(38,893) = 0.12, P > 0.05] on exhaustion time. Specifically, exhaustion time increased progressively over the 20 daily sessions with comparable magnitude in the 3 mouse groups (Figure 2).

Figure 2.

Effect of repeated, daily administration of two different doses of Spinacia oleracea extract on exhaustion time (expressed in min) in male CD1 mice exposed to daily sessions at the Rota-Rod test. Exhaustion time was defined as the time elapsed between the beginning of the acceleration phase and the third fall from the revolving drum (mice that did not fall 3 times over the session were assigned the value 60 min). Spinacia oleracea extract was administered intragastrically 60 min before the start of the Rota-Rod test (Monday to Friday) as well as on intervening Saturdays and Sundays (with a total of 26 daily treatments). Each point is the mean ± SEM of n = 16-17 mice.

ANOVA indicated no effect of treatment with Spinacia oleracea extract [F(2,47) = 0.54, P > 0.05], a highly significant effect of time [F(7,329) = 111.50, P < 0.0001, and no “treatment x time” interaction [F(14,329) = 1.54, P > 0.05] on mouse body weight (data not shown).

Experiment 2 – Blitum Bonus-Henricus Extract

ANOVA indicated a significant effect of treatment with Blitum bonus-henricus extract [F(2,47) = 4.93, P < 0.05], a highly significant effect of time [F(19,893) = 7.08, P < 0.0001], and a significant “treatment x time” interaction [F(38,893) = 1.44, P < 0.05] on exhaustion time. Specifically, exhaustion time increased progressively, in a dose-related fashion and over the 20 daily sessions, in the mouse groups treated with Blitum bonus-henricus extract (Figure 3). Post hoc analysis indicated that, over the last 12 daily sessions, exhaustion time in the mouse group treated with 2000 mg/kg Blitum bonus-henricus extract was significantly higher than that recorded in the vehicle-treated mouse group (with the sole exception of Session 10, P = 0.054); in these sessions, exhaustion time in the mouse group treated with 2000 mg/kg Blitum bonus-henricus extract was often more than double that recorded in the vehicle-treated mouse group (Figure 3).

Figure 3.

Effect of repeated, daily administration of two different doses of Blitum bonus-henricus extract on exhaustion time (expressed in min) in male CD1 mice exposed to daily sessions at the Rota-Rod test. Exhaustion time was defined as the time elapsed between the beginning of the acceleration phase and the third fall from the revolving drum (mice that did not fall 3 times over the session were assigned the value 60 min). Blitum bonus-henricus extract was administered intragastrically 60 min before the start of the Rota-Rod test (Monday to Friday) as well as on intervening Saturdays and Sundays (with a total of 26 daily treatments). Each point is the mean ± SEM of n = 16-17 mice. ×: P < 0.05 in comparison to vehicle-treated mouse group in the same daily session (Tukey's test).

ANOVA indicated no effect of treatment with Blitum bonus-henricus extract [F(2,47) = 0.36, P > 0.05], a highly significant effect of time [F(7,329) = 170.00, P < 0.0001, and no “treatment x time” interaction [F(14,329) = 0.42, P > 0.05] on mouse body weight (data not shown).

Discussion

With the intent of modeling intense endurance activity and physical fatigue, the present study set up a procedure in which adult healthy mice were exposed to 20 daily running sessions on the revolving drum of an accelerated Rota-Rod. Exhaustion time – defined as the third fall from the drum – was taken as index of the mouse performance at each running session. The procedure used in the present study was highly similar to those employed in multiple studies focusing on anti-fatigue drugs, including natural products, in rodent models of physical endurance.^22–24

The results of the present study indicate that an hydroalcoholic raw extract of Blitum bonus-henricus markedly improved the mouse performance. Notably, the effect of Blitum bonus-henricus extract was dose-related, progressed over time, and was of remarkable magnitude. With regard to the latter point, exhaustion time in mice treated with 2000 mg/kg Blitum bonus-henricus extract was steadily double than that recorded in vehicle-treated mice over the entire second half of the 20-session period; in other words, mice treated with 2000 mg/kg Blitum bonus-henricus extract ran on the revolving drum for at least twice as long compared to vehicle-treated mice. To our knowledge, these data constitute the very first line of experimental evidence on the anti-fatigue properties of a preparation derived from Blitum bonus-henricus. Blitum bonus-henricus-based preparations can thus be added to the list of natural products (eg, Panax ginseng, Rhodiola rosea) known to improve physical performance and counteract fatigue development in laboratory animals and humans.^22,25–30

Conversely, the hydroalcoholic raw extract of Spinacia oleracea, at least under the experimental conditions used in the present study, was totally ineffective on mouse performance. The limited improvement in mouse performance, recorded over time in all 3 mouse groups (including that treated with vehicle), was likely due to the continuous practice at the Rota-Rod task. At present, we cannot rule out that higher doses of the extract or different experimental procedures of physical endurance will disclose any possible anti-fatigue effect of this Spinacia oleracea extract.

Comparison of data from Experiments 1 and 2 is somewhat helpful in advancing some hypothesis on the active constituent(s) responsible for the observed anti-fatigue effects of Blitum bonus-henricus extract. The initial hypothesis is based on ecdysterone, the phytoecdysteroid known to exert clear anabolic effects, including increase in muscle size and strength.^11–13 Both extracts had relatively low contents of ecdysterone (0.11% and 0.066% in Blitum bonus-henricus and Spinacia oleracea extract, respectively), resulting in administered amounts of ecdysterone equal to approximately 1.1 and 2.2 mg/kg at the doses of 1000 and 2000 mg/kg Blitum bonus-henricus extract, respectively, and approximately 0.66 and 1.32 mg/kg at the doses of 1000 and 2000 mg/kg Spinacia oleracea extract, respectively. These ecdysterone dose-ranges were too similar to justify the opposite effects recorded in Experiments 1 and 2 (ie: remarkable improvement of running activity after treatment with Blitum bonus-henricus extract; complete absence of any effect after treatment with Spinacia oleracea extract). Additionally, these doses of ecdysterone were much lower than that reported to potentiate grip strength in rat forelimbs in the only fatigue-related in vivo study on ecdysterone of which we are aware (50 mg/kg administered intragastrically for 28 consecutive days).¹¹ It is therefore reasonable to hypothesize that other active constituents of Blitum bonus-henricus extract had a role in the extract effects on mouse performance, either being responsible per se for the observed pharmacological effects or potentiating ecdysterone action via a botanical synergy-like mechanism.^31,32

The nutritional content of Blitum bonus-henricus may provide additional hints on the possible active ingredient(s) responsible for its anti-fatigue effect. Blitum bonus-henricus is indeed rich in iron, calcium, vitamin C, and vitamin K³³; high vitamin and mineral contents are expected to support the immune system and improve recovery after exercise.³⁴ Additionally, Blitum bonus-henricus is a source of polyphenols and antioxidants,¹⁶ known to modulate oxidative stress and improve overall well-being.³⁵ Finally, recent studies indicated that nitrate supplementation enhanced muscle fatigue endurance^36,37; notably, remarkable nitrate concentrations have been detected in spinach.³⁸

Additional important research questions, deriving from the results of the present study, require to be addressed by future studies. These research questions – also intended to fill the gaps and limitations of the present, initial study – include: (i) Does the ability of this Blitum bonus-henricus extract to increase physical endurance extend to ageing-associated decline in muscle force and performance or pathological conditions of exacerbated muscle fatigue (ie, cancer cachexia, neurological and psychiatric disorders)? (ii) Is treatment with Blitum bonus-henricus extract associated with skeletal muscle hypertrophy (ie, increase in number and size of muscular fibers)? (iii) Does treatment with Blitum bonus-henricus extract also alter the mouse basal metabolism? (iv) Do the observed results extend to other rodent models of fatigue endurance? (v) Is the protective effect of Blitum bonus-henricus extract associated with changes in biomarkers of exercise-induced muscle fatigue?³⁹ (vi) Is Blitum bonus-henricus extract differently effective in female and male mice? (vii) Do different extract dose-ranges, extracts with higher ecdysterone content, or experimental procedures of physical endurance may unravel the anti-fatigue properties of the Spinacia oleracea extract?

Conclusions

In summary, the results of the present study indicate that repeated administration of an extract of Blitum bonus-henricus effectively improved the physical performance of mice exposed to a severe endurance test. Conversely, and somewhat contrary to our initial expectations, the anti-fatigue properties of Blitum bonus-henricus extract were not replicated by repeated treatment with an extract of Spinacia oleracea. The results of the present study lead to hypothesize that preparations based on Blitum bonus-henricus may have therapeutic potential as anti-fatigue agents.

Supplemental Material

sj-jpg-1-npx-10.1177_1934578X261434480 - Supplemental material for Assessment of Fatigue Endurance Potential of Blitum bonus-henricus and Spinacia oleracea Extracts in Mice

Supplemental material, sj-jpg-1-npx-10.1177_1934578X261434480 for Assessment of Fatigue Endurance Potential of Blitum bonus-henricus and Spinacia oleracea Extracts in Mice by Carla Lobina, Mauro AM Carai and Giancarlo Colombo in Natural Product Communications

Supplemental Material

sj-jpg-2-npx-10.1177_1934578X261434480 - Supplemental material for Assessment of Fatigue Endurance Potential of Blitum bonus-henricus and Spinacia oleracea Extracts in Mice

Supplemental material, sj-jpg-2-npx-10.1177_1934578X261434480 for Assessment of Fatigue Endurance Potential of Blitum bonus-henricus and Spinacia oleracea Extracts in Mice by Carla Lobina, Mauro AM Carai and Giancarlo Colombo in Natural Product Communications

Supplemental Material

sj-jpg-3-npx-10.1177_1934578X261434480 - Supplemental material for Assessment of Fatigue Endurance Potential of Blitum bonus-henricus and Spinacia oleracea Extracts in Mice

Supplemental material, sj-jpg-3-npx-10.1177_1934578X261434480 for Assessment of Fatigue Endurance Potential of Blitum bonus-henricus and Spinacia oleracea Extracts in Mice by Carla Lobina, Mauro AM Carai and Giancarlo Colombo in Natural Product Communications

Supplemental Material

sj-docx-4-npx-10.1177_1934578X261434480 - Supplemental material for Assessment of Fatigue Endurance Potential of Blitum bonus-henricus and Spinacia oleracea Extracts in Mice

Supplemental material, sj-docx-4-npx-10.1177_1934578X261434480 for Assessment of Fatigue Endurance Potential of Blitum bonus-henricus and Spinacia oleracea Extracts in Mice by Carla Lobina, Mauro AM Carai and Giancarlo Colombo in Natural Product Communications

Footnotes

Acknowledgements

The authors are grateful to Mrs. Carla Acciaro for animal care, Mr Alessandro Capra for equipment maintenance, and Ms. Anne Farmer for language editing of the manuscript.

ORCID iDs

Carla Lobina

Mauro A.M. Carai

Giancarlo Colombo

Ethical Statement

All experimental procedures employed in the present study fully complied with European Directive no. 2010/63/EU and subsequent Italian Legislative Decree no. 26, March 4, 2014, on the “Protection of animals used for scientific purposes”. The reporting of the present study fully conforms with ARRIVE 2.0 Guidelines [Percie du Sert et al, Br. J. Pharmacol. 177(16):3617–3624, 2020]. The current study was approved by the Italian Ministry of Health (Authorization no. 36/2023-BA8CE.12, released on January 23, 2023).

Consent to Participate

There are no human subjects in this article and consent to participate is not applicable.

Consent for Publication

There are no human subjects in this article and consent for publication is not applicable.

Author Contributions

Conceptualization: G.C. and M.AMC.; Methodology: C.L.; Formal analysis: C.L.; Investigation: C.L.; Data curation: C.L.; Writing—original draft preparation: G.C.; Writing—review and editing: C.L. and M.AMC.; Supervision: G.C.; Funding acquisition: G.C. All authors have read and agreed to the published version of the manuscript.

Funding

The authors received no financial support for the research, authorship, and/or publication of this article.

Declaration of Conflicting Interests

The authors declared no potential conflicts of interest with respect to the research, authorship, and/or publication of this article.

Data Availability

The datasets used and analyzed during the current study are available from the corresponding author on request.

Supplemental Material

Supplemental material for this article is available online.

References

Atherton

Smith

. Muscle protein synthesis in response to nutrition and exercise. J Physiol. 2012;590(5):1049‐1057. 10.1113/jphysiol.2011.225003

Akune

Muraki

Oka

, et al. Exercise habits during middle age are associated with lower prevalence of sarcopenia: the ROAD study. Osteoporos Int. 2014;25(3):1081‐1088. 10.1007/s00198-013-2550-z

Damas

Phillips

Vechin

Ugrinowitsch

. A review of resistance training-induced changes in skeletal muscle protein synthesis and their contribution to hypertrophy. Sports Med. 2015;45(6):801‐807. 10.1007/s40279-015-0320-0

Janssen

Heymsfield

Wang

Ross

. Skeletal muscle mass and distribution in 468 men and women aged 18-88yr. J Appl Physiol. 1985;89(1):81‐88. 10.1152/jappl.2000.89.1.81

Cruz-Jentoft

Bahat

Bauer

, et al. Sarcopenia: revised European consensus on definition and diagnosis. Age Ageing. 2019;48(1):16‐31. 10.1093/ageing/afy169

Cruz-Jentoft

Dawson Hughes

Scott

Sanders

Rizzoli

. Nutritional strategies for maintaining muscle mass and strength from middle age to later life: a narrative review. Maturitas. 2020;132:57‐64. 10.1016/j.maturitas.2019.11.007

Millward

. Nutrition and sarcopenia: evidence for an interaction. Proc Nutr Soc. 2012;71(4):566‐575. 10.1017/S0029665112000201

Jiraungkoorskul

. Review of neuro-nutrition used as anti-Alzheimer plant, spinach, Spinacia oleracea. Pharmacogn Rev. 2016;10(20):105‐108. 10.4103/0973-7847.194040

Gutierrez

RMP

Velazquez

Carrera

SPP

. Spinacia oleracea Linn considered as one of the most perfect foods: a pharmacological and phytochemical review. Mini Rev Med Chem. 2019;19(20):1666‐1680. 10.2174/1389557519666190603090347

10.

Syrov

. Comparative experimental investigation of the anabolic activity of phytoecdysteroids and steranabols. Pharm Chem J. 2000;34(4):193‐197. 10.1007/BF02524596

11.

Gorelick-Feldman

Maclean

Ilic

, et al. Phytoecdysteroids increase protein synthesis in skeletal muscle cells. J Agric Food Chem. 2008;56(10):3532‐3537. 10.1021/jf073059z

12.

Tóth

Szabó

Kacsala

Héger

Zádor

. 20-Hydroxyecdysone Increases fiber size in a muscle-specific fashion in rat. Phytomedicine. 2008;15(9):691‐698. 10.1016/j.phymed.2008.04.015

13.

Parr

Zhao

Haupt

, et al. Estrogen receptor beta is involved in skeletal muscle hypertrophy induced by the phytoecdysteroid ecdysterone. Mol Nutr Food Res. 2014;58(9):1861‐1872. 10.1002/mnfr.201300806

14.

Isenmann

Ambrosio

Joseph

, et al. Ecdysteroids as non-conventional anabolic agent: performance enhancement by ecdysterone supplementation in humans. Arch Toxicol. 2019;93(7):1807‐1816. 10.1007/s00204-019-02490-x

15.

Bakrim

Maria

Sayah

Lafont

Takvorian

. Ecdysteroids in spinach (Spinacia oleracea L.): biosynthesis, transport and regulation of levels. Plant Physiol Biochem. 2008;46(10):844‐854. 10.1016/j.plaphy.2008.06.002

16.

Kokanova-Nedialkova

Nedialkov

Kondeva-Burdina

Simeonova

Tzankova

Aluani

. Chenopodium bonus-henricus L. - A source of hepatoprotective flavonoids. Fitoterapia. 2017;118:13‐20. 10.1016/j.fitote.2017.02.001

17.

Kokanova-Nedialkova

Nedialkov

Momekov

. Saponins from the roots of Chenopodium bonus-henricus L. Nat Prod Res. 2019;33(14):2024‐2031. 10.1080/14786419.2018.1483928

18.

Percie du Sert

Hurst

Ahluwalia

, et al. The ARRIVE guidelines 2.0: updated guidelines for reporting animal research. Br J Pharmacol. 2020;177(16):3617‐3624. doi:10.1111/bph.15193

19.

Faul

Erdfelder

Lang

Buchner

. G*Power 3: a flexible statistical power analysis program for the social, behavioral, and biomedical sciences. Behav Res Methods. 2007;39(2):175‐191. 10.3758/bf03193146

20.

Patel

Shukla

. Pharmacognostical studies and evaluation of quality parameters of Spinacia oleracea. Int J Pharmacogn Phytochem Res. 2017;9(6):760‐765.

21.

Flora Europaea, Vol. 1. Cambridge University Press, 2010, 92–95.

22.

Kim

Choi

Kim

. Anti-fatigue effects of 20(S)-protopanaxadiol and 20(S)-protopanaxatriol in mice. Biol Pharm Bull. 2015;38(9):1415‐1419. 10.1248/bpb.b15-00230

23.

Cheng

Hwee

Kim

, et al. Fast skeletal muscle troponin activator CK-2066260 increases fatigue resistance by reducing the energetic cost of muscle contraction. J Physiol. 2019;597(17):4615‐4625. 10.1113/JP278235

24.

Huang

Sun

Lin

, et al. Camellia oil exhibits anti-fatigue property by modulating antioxidant capacity, muscle fiber, and gut microbial composition in mice. J Food Sci. 2024;89(4):2465‐2481. 10.1111/1750-3841.16983

25.

Huang

Lee

Kuo

Yang

Chien

. Attenuation of long-term Rhodiola rosea supplementation on exhaustive swimming-evoked oxidative stress in the rat. Chin J Physiol. 2009;52(5):316‐324. 10.4077/cjp.2009.amh029

26.

Shikov

Lazukina

Pozharitskaya

, et al. Pharmacological evaluation of Potentilla alba L. in mice: adaptogenic and central nervous system effects. Pharm Biol. 2011;49(10):1023‐1028. 10.3109/13880209.2011.560162

27.

Liu

Zhang

, et al. Anti-fatigue effects of proteins isolated from Panax quinquefolium. J Ethnopharmacol. 2014;153(2):430‐434. 10.1016/j.jep.2014.02.045

28.

Luo

Wei

, et al. Natural medicines for the treatment of fatigue: bioactive components, pharmacology, and mechanisms. Pharmacol Res. 2019;148:104409. 10.1016/j.phrs.2019.104409

29.

Todorova

Ivanov

Delattre

Nalbantova

Karcheva-Bahchevanska

Ivanova

. Plant adaptogens-history and future perspectives. Nutrients. 2021;3(8):2861. 10.3390/nu13082861

30.

Todorova

Ivanova

Chakarov

Kraev

Ivanov

. Ecdysterone and turkesterone-compounds with prominent potential in sport and healthy nutrition. Nutrients. 2024;16(9):1382. 10.3390/nu16091382

31.

Russo

. The case for the entourage effect and conventional breeding of clinical Cannabis: no “Strain,” No Gain. Front Plant Sci. 2019;9:1969. 10.3389/fpls.2018.01969

32.

Anand

Pacchetti

Anand

Sodergren

. Cannabis-based medicines and pain: a review of potential synergistic and entourage effects. Pain Manag. 2021;11(4):395‐403. 10.2217/pmt-2020-0110

33.

Paniagua-Zambrana

Bussmann

Kikvidze

. Chenopodium album L. Chenopodium bonus-henricus L. Chenopodium polyspermum L.Chenopodium urbicum L. Amaranthaceae. In: Bussmann

Paniagua-Zambrana

Kikvidze

, eds. Ethnobotany of the Mountain Regions of Eastern Europe. Ethnobotany of Mountain Regions. Springer; 2024:1‐16. 10.1007/978-3-030-98744-2_78-1

34.

Abd El-Hack

Allam

Aldhalmi

, et al. Nutritional value and therapeutic applications of Chenopodium album for human and livestock health: a comprehensive review. Food Biosci. 2025;68:106624. 10.1016/j.fbio.2025.106624

35.

Zhang

Tsao

. Dietary polyphenols, oxidative stress and antioxidant and anti-inflammatory effects. Curr Opin Food Sci. 2016;8:33‐42. 10.1016/j.cofs.2016.02.002

36.

Bailey

Gandra

Jones

Hogan

Nogueira

. Incubation with sodium nitrite attenuates fatigue development in intact single mouse fibres at physiological PO2. J Physiol. 2019;597(22):5429‐5443. 10.1113/JP278494

37.

Mosely

Landberg

. The Popeye mythology may be true: eating spinach can make you fatigue resistant!. J Physiol. 2020;598(4):627‐629. 10.1113/JP279264

38.

Chang

Yang

Riskowski

. Ascorbic acid, nitrate, and nitrite concentration relationship to the 24hour light/dark cycle for spinach grown in different conditions. Food Chem. 2013;138(1):382‐388. 10.1016/j.foodchem.2012.10.036

39.

Finsterer

. Biomarkers of peripheral muscle fatigue during exercise. BMC Musculoskelet Disord. 2012;13:218. doi:10.1186/1471-2474-13-218

Supplementary Material

Please find the following supplemental material available below.

For Open Access articles published under a Creative Commons License, all supplemental material carries the same license as the article it is associated with.

For non-Open Access articles published, all supplemental material carries a non-exclusive license, and permission requests for re-use of supplemental material or any part of supplemental material shall be sent directly to the copyright owner as specified in the copyright notice associated with the article.

0.00 MB

0.02 MB

0.36 MB

0.83 MB

0.59 MB