Assessment of Fatigue Endurance Potential of Blitum bonus-henricus and Spinacia oleracea Extracts in Mice

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.

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 strength ¹ and reduced risk of developing sarcopenia later in old age. ² By stimulating muscle protein synthesis, endurance training compensates muscle protein degradation ³ 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 leaves ¹⁵ ; 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

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. ¹⁸

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 K ³³ ; 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

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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