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Cited by 11Medicinal Plants and Bioactive CompoundsPhytochemical Studies and BioactivitiesPhytochemicals and Medicinal Plants

Adaptogenic Properties of a Phytoecdysteroid-Rich Extract from the Leaves of Spinacia oleracea L.

Open access
Sidorova, Yuliya S., Shipelin, Vladimir A., Petrov, Nikita A., Zorin, Sergey N., Mazo, Vladimir K.·2021·Plants
DOI 10.3390/plants10122555PMC PMC8704498
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Abstract

Increasing the ability of the human body to adapt in conditions of physical or emotional stress is promising from the standpoint of the use of preventive nutrition containing functional food ingredients (FFI) with proven effectiveness in complex physiological in vivo studies. In this work, we developed FFI from spinach leaves (Spinacia oleracea L.) with a high content of polyphenols and adaptogens—phytoecdysteroids. Using in vivo models of increased physical activity and immobilization-induced emotional stress, we evaluated the nonspecific resistance of rats in response to the addition of the developed FFI to the diet. In the acute toxicity experiment, we found no signs of FFI toxicity up to 5000 mg/kg body weight. As a result of the daily 26-day consumption of FFI, we observed an anxiolytic effect in physiological studies. FFI prevented an increase in the content of biogenic amines in the blood, the main markers of the stress system, and had a positive effect on the lipid metabolism of the rats. The obtained results demonstrate a “smoothing” effect on the body’s reaction in response to induced stress conditions.

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Written from the paper’s full text.

TL;DR

A phytoecdysteroid-rich spinach-leaf extract (FFI) was non-toxic up to 5000 mg/kg in mice and, in 26-day rat studies, showed anxiolytic-like, hypolipidemic, catecholamine-normalizing, and possible anabolic/performance effects under immobilization stress and exhausting treadmill exercise. The authors conclude it is a promising adaptogenic ingredient for specialized foods, while noting some effects were trend-level and further dose/body-composition studies are needed.

Key findings

  • FFI composition: total polyphenols were 41.9 ± 4.1 mg-eq gallic acid/g and 20-hydroxyecdysone (20E) was 14.7 ± 1.5 mg/g; in acute oral toxicity testing, no mice died at 5000 mg/kg, LD50 exceeded 5000 mg/kg, and FFI was classified as low-hazard (class V).
  • In immobilization-induced emotional stress, FFI intake was 84.0 ± 6.8 mg FFI/kg/day (1.4 ± 0.1 mg 20E/kg/day; 199.4 ± 11.5 mg total polyphenols GAE/kg/day); it prevented the immobilization-associated feed-intake and body-weight decline, produced anxiolytic-like EPM effects (less time in closed arms and more time in open arms; discussion reports three times longer open-arm stay vs IMM), lowered triglycerides versus CTRL and IMM (p < 0.05), normalized bilirubin/globulins, and prevented depletion of norepinephrine and dopamine (p < 0.05).
  • In increased physical energy expenditure, FFI intake was 91.1 ± 12.9 mg FFI/kg/day (1.6 ± 0.1 mg 20E/kg/day); distance traveled and electric shocks did not differ, but shock time was more than two times higher in RUN controls than in FFI-fed rats (p < 0.05), triglycerides were reduced almost two times versus RUN, and all urinary catecholamines were significantly higher in RUN than RUN-FFI (p < 0.05).
  • FFI did not affect learning or memory in the CRPA test, and in the exercise experiment it did not affect EPM anxiety-like indices.

Methods

  • FFI was produced from freeze-dried Spinacia oleracea L. leaves by 20% aqueous ethanol extraction, centrifugation, 10 kD ultrafiltration, reverse-osmosis concentration, freeze-drying, and oxalic acid removal; it was characterized by HPLC-MS for 20E, Folin–Ciocalteu for total polyphenols, and HPLC-DAD for flavonoids.
  • Acute oral toxicity used 16 male and 16 female ICR mice given FFI intragastrically at 5000 mg/kg under a modified OECD 420 fixed-dose procedure, with 14-day observation.
  • Two 26-day rat experiments used male Wistar rats phenotyped by EPM and fed FFI at 600 mg/100 g diet: Experiment 1 used daily 40-min immobilization plus 3-h exhausting immobilization (CTRL n = 20, IMM n = 10, IMM-FFI n = 10); Experiment 2 used treadmill training and a 45-min exhausting treadmill test (CTRL n = 20, RUN n = 10, RUN-FFI n = 10). Outcomes included EPM, CRPA, blood biochemistry, and urinary catecholamines by HPLC, analyzed with nonparametric tests at p < 0.05.

Limitations

  • Stated: the CRPA anxiolytic-like result in the immobilization experiment was only at the trend level; the authors also call for further studies with a wide range of FFI dosages under physical exercise, using noninvasive NMR relaxometry for body-weight composition and specific markers to clarify anabolic properties.
  • Evident: the two main adaptogenic stress experiments used only male Wistar rats, with no female rats in those models; only one FFI dose (600 mg/100 g diet) was tested, so no dose–response can be assessed; the studies were short (26 days) and preclinical, with no human clinical data reported.

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Integrity signals

Registry

Crossref · 10.3390/plants10122555

Cites

Statistics

Nothing to recompute.

Claims the paper makes

0 of 32 cited sources checked · 0 supported · 0 flagged

  • Findingown data

    Necropsy on the 14th day after FFI administration at 5000 mg/kg did not reveal specific pathological changes in the internal organs of mice.

    “Necropsy examination on the 14th day did not reveal any specific pathological changes in the internal organs.”
    Quote in the paperSubstantial claim
  • Findingown data

    The total polyphenol content of the developed FFI, determined spectrophotometrically using the Folin-Ciocalteu method, was 41.9 ± 4.1 mg-eq. of gallic acid/g.

    “The total polyphenol content, determined spectrophotometrically using the Folin–Ciocalteu method, was 41.9 ± 4.1 mg-eq. of gallic acid/g.”
    Quote in the paperNot a substantial claim
  • Findingown data

    Animals in the IMM group subjected to daily immobilization consumed significantly less feed compared to animals in the CTRL group (p < 0.05).

    “animals in the IMM group subjected to daily immobilization consumed significantly less feed compared to animals in the CTRL group (p < 0.05, Mann–Whitney test)”
    Quote in the paperSubstantial claim
  • Findingown data

    A single oral administration of FFI at a dose of 5000 mg/kg body weight caused no death in any mice, male or female.

    “Following a single oral administration of FFI at a dose of 5000 mg/kg of body weight, we did not observe the death of any mice, either male or female.”
    Quote in the paperSubstantial claim
  • Findingown data

    The phytoecdysteroid content (20-hydroxyecdysone, 20E) of the developed FFI, determined by HPLC-MS, was 14.7 ± 1.5 mg/g.

    “Phytoecdysteroid content (20-hydroxyecdysone, 20E) determined by HPLC-MS was 14.7 ± 1.5 mg/g.”
    Quote in the paperSubstantial claim
  • May combine two claims. 29
  • Classified as probably not a substantial claim. 18

  • May combine two claims.

    Findingown data

    After FFI administration at 5000 mg/kg, all mice had normal appearance, stool, appetite, mobility, and equal body weight gain for 13 days.

    “In the first six hours after the administration of FFI, and subsequently for 13 days, all animals had normal appearance, stool, and appetite, were mobile, and gained body weight equally.”
    Quote in the paperSubstantial claim

  • May combine two claims.

    Findingown data

    After FFI administration at 5000 mg/kg, no negative changes were detected in body weight dynamics, liver mass, liver lipid profiles, or corticosterone and prostaglandin E2 levels in blood plasma compared with control animals.

    “When assessing integral indicators (body weight dynamics, liver mass) as well as liver lipid profiles and corticosterone and prostaglandin E2 levels in the blood plasma, no negative changes were detected compared with the control animals”
    Quote in the paperSubstantial claim

  • May combine two claims.

    Findingown data

    At the end of treadmill testing, there were no significant differences in distance traveled and number of electric shocks received between the compared groups.

    “At the end of testing, there were no significant differences in indicators such as distance traveled and number of electric shocks received between the compared groups.”
    Quote in the paperNot a substantial claim

  • May combine two claims.

    Findingown data

    In RUN-FFI animals, triglycerides were reduced by almost two times compared with the RUN group, and total cholesterol levels were increased, together with HDL and LDL fractions.

    “In these animals, triglycerides were reduced by almost two times in comparison with the RUN group, and total cholesterol levels were increased, together with HDL and LDL fractions.”
    Quote in the paperSubstantial claim

  • Probably not a substantial claim: may not pass S3 (load-bearing).

    Findingown data

    During the first CRPA formation test, the animals of all groups entered the dark compartment of a chamber (100% reflex).

    “During the first CRPA formation test, the animals of all groups entered the dark compartment of a chamber (100% reflex).”
    Quote in the paperNot a substantial claim

  • May combine two claims.

    Findingown data

    The animals in the RUN group showed significant increases in total cholesterol level, AsAT activity, and phosphorus and protein content in the blood after a single exhausting physical activity.

    “The animals in the RUN group showed significant increases in total cholesterol level, AsAT activity, and phosphorus and protein content in the blood due to an increase in both fractions after a single exhausting physical activity.”
    Quote in the paperSubstantial claim

  • May combine two claims.

    Findingown data

    In the second test, animals of the IMM-FFI group spent significantly less time in closed arms and more time in open arms compared to the animals of the IMM control group.

    “They also spent significantly less time in closed arms (Figure 2c) in the second test and, accordingly, spent more time in open arms compared to the animals of the IMM control group (Figure 2d).”
    Quote in the paperSubstantial claim

  • May combine two claims.

    Findingown data

    In the RUN-FFI animals, protein metabolism indicators remained unchanged and phosphorus levels were below the level of the control group.

    “It is noteworthy that in the RUN-FFI animals, protein metabolism indicators remained unchanged and phosphorus levels were below the level of the control group.”
    Quote in the paperNot a substantial claim

  • May combine two claims.

    Findingown data

    According to the latency of entrance to the dark compartment, the animals of the IMM-FFI group were closer to the control group, characterized by fewer anxiety-like functions, but this result was at the trend level.

    “According to the latency of the entrance to the dark compartment, the animals of the IMM-FFI group were closer to the control group, i.e., they were characterized by fewer anxiety-like functions. However, this result was at the trend level.”
    Quote in the paperNot a substantial claim

  • May combine two claims.

    Findingown data

    The FFI intake of the RUN-FFI experimental group animals was 91.1 ± 12.9 mg FFI per kg body weight per day, corresponding to 1.6 ± 0.1 mg 20E per kg body weight per day.

    “The FFI intake of the RUN-FFI experimental group animals was 91.1 ± 12.9 mg FFI per kg body weight per day, corresponding to 1.6 ± 0.1 mg 20E per kg body weight per day.”
    Quote in the paperNot a substantial claim

  • May combine two claims.

    Findingown data

    Daily immobilization of the IMM group reduced the pool of globulins in the blood of rats compared with intact control animals, and consumption of FFI returned this indicator to the values of the control group (p < 0.05).

    “Daily immobilization of the IMM group reduced the pool of globulins in the blood of rats compared with intact animals of the control group; consumption of FFI positively affected this indicator, returning it to the values of the control group (p < 0.05, Mann-Whitney test).”
    Quote in the paperSubstantial claim

  • May combine two claims.

    Findingown data

    The consumption of FFI by animals in the IMM-FFI group was 84.0 ± 6.8 mg FFI per kg body weight/day, corresponding to 1.4 ± 0.1 mg 20E per kg body weight/day and 199.4 ± 11.5 mg of total polyphenols in gallic acid equivalent/kg body weight.

    “The consumption of FFI by animals in the experimental group IMM-FFI was 84.0 ± 6.8 mg FFI per kg body weight/day, which corresponds to 1.4 ± 0.1 mg 20E per kg body weight/day and 199.4 ± 11.5 mg of total polyphenols in gallic acid equivalent/kg body weight.”
    Quote in the paperNot a substantial claim

  • May combine two claims.

    Findingown data

    Daily immobilization reduced appetite in the rats of the IMM group, which led to a lag in their growth starting from the 10th day of the experiment.

    “Daily immobilization reduced appetite in the rats of the IMM group, which led to a lag in their growth starting from the 10th day of the experiment.”
    Quote in the paperSubstantial claim

  • May combine two claims.

    Findingown data

    Despite the hypolipidemic effect of FFI, the animals were characterized by normal body weight, and in the experiment with daily immobilization they even normalized it compared to the IMM-FFI group.

    “It should be noted that despite the hypolipidemic effect of FFI, the animals were characterized by normal body weight, and in the experiment with daily immobilization they even normalized it compared to the IMM-FFI group.”
    Quote in the paperSubstantial claim

  • Probably not a substantial claim: may not pass S4 (based).

    Findingnone given

    FFI lacks oxalates.

    “Due to the lack of oxalates in FFI”
    Quote in the paperNot a substantial claim

  • May combine two claims.

    Interpretationown data

    The FFI developed from spinach leaves has a high content of polyphenols and phytoecdysteroids.

    “we developed FFI from spinach leaves (Spinacia oleracea L.) with a high content of polyphenols and adaptogens—phytoecdysteroids”
    Quote in the paperSubstantial claim

  • May combine two claims.

    Interpretationown data

    Consumption of FFI by rats positively affected the balance of the stress activators studied and prevented depletion of the pool of biogenic amines in stressed animals.

    “Consumption of FFI by rats positively affected the balance of the stress activators studied, and prevented depletion of the pool of biogenic amines in stressed animals.”
    Quote in the paperSubstantial claim

  • May combine two claims.

    Interpretationown data

    Consumption of FFI contributed to the improvement of physical performance and reaction rate in animals under simulated conditions.

    “Thus, the consumption of FFI contributed to the improvement of physical performance and reaction rate in animals under simulated conditions.”
    Quote in the paperSubstantial claim

  • May combine two claims.

    Interpretationreasoning

    The lag in growth of animals in the IMM group throughout the experiment may be due to both lower feed consumption and daily stressing immobilization.

    “The lag in the growth of animals in control group IMM is shown throughout the experiment, and may be due to both lower feed consumption and daily stressing immobilization.”
    Quote in the paperNot a substantial claim

  • Probably not a substantial claim: may not pass S3 (load-bearing).

    Premisecitation

    Quantification of 20E under controlled spinach cultivation conditions can range from 50 to 800 μg per g fresh tissue, depending on the growth rate.

    “Quantification of 20E under controlled spinach cultivation conditions can range from 50 to 800 µg per g fresh tissue, depending on the growth rate [6].”
    Quote in the paperNot a substantial claim0 of 1 sources checked
    • cites [6] Das, 2020, The phytochemical, biological, and medicinal attributes of phytoecdysteroids: An updated review, Acta Pharm. Sin. B source

      Not checked in the free run.

  • Probably not a substantial claim: may not pass S3 (load-bearing).

    Premisecitation

    The average rate of 20E is about 300 μg per g of fresh tissue.

    “The average rate of 20E is about 300 µg per g of fresh tissue [33].”
    Quote in the paperNot a substantial claim0 of 1 sources checked
    • cites [33] Grebenok, 1991, Occurrence and levels of ecdysteroids in spinach, Lipids source

      Not checked in the free run.

  • Probably not a substantial claim: may not pass S3 (load-bearing).

    Premisecitation

    In most spinach species, the average amount of 20E ranges from 8.2 to 27 μg per g fresh tissue.

    “In most species, the average amount of 20E ranges from 8.2 to 27 µg per g fresh tissue [34].”
    Quote in the paperNot a substantial claim0 of 1 sources checked
    • cites [34] Gorelick, J., Iraqi, R.H., and Bernstein, N. (2020). Ecdysteroid Content and Therapeutic Activity in Elicited Spinach Accessions. Plants, 9. source

      Not checked in the free run.

  • Probably not a substantial claim: may not pass S3 (load-bearing).

    Premisecitation

    High concentrations of some plant polyphenols may exhibit toxicological characteristics.

    “Despite the apparent safety of a plant such as spinach, high concentrations of some plant polyphenols may exhibit toxicological characteristics [35,36].”
    Quote in the paperNot a substantial claim0 of 2 sources checked
    • cites [35] Rojo, M.Á., Garrosa, M., Jiménez, P., Girbés, T., Garcia-Recio, V., Cordoba-Diaz, M., and Cordoba-Diaz, D. (2020). Unexpected Toxicity of Green Tea Polyphenols in Combination with the Sambucus RIL Ebulin. Toxins, 12. source

      Not checked in the free run.

    • cites [36] Mennen, 2005, Risks and safety of polyphenol consumption, Am. J. Clin. Nutr. source

      Not checked in the free run.

  • May combine two claims.

    Interpretationcitation

    The profile of polyphenols contained in extracts of Spinacia oleracea L. has low toxicity and is classified as low-hazard substances.

    “Taken together, it can be concluded that the profile of polyphenols contained in extracts of Spinacia oleracea L. have low toxicity and are classified as low-hazard substances.”
    Quote in the paperSubstantial claim

  • May combine two claims.

    Interpretationown data

    Consumption of FFI had a positive effect on animal feed intake and maintained body weight despite these animals being characterized by a reduced level of triglycerides.

    “Consumption of FFI had a positive effect on animal feed intake and, maintained body weight despite these animals being characterized by a reduced level of triglycerides.”
    Quote in the paperSubstantial claim

  • May combine two claims.

    Interpretationown data

    Consumption of FFI reduced the reaction time of animals by more than two times when touching the electrode, which against the background of the same distance traveled characterizes rats with greater adaptability to the treadmill.

    “Consumption of FFI reduced the reaction time of animals by more than two times when touching the electrode, which against the background of the same distance traveled characterizes rats with greater adaptability to the treadmill.”
    Quote in the paperSubstantial claim

  • May combine two claims.

    Premisecitation

    In a cited study, aging female rats of the Long-Evans line consumed dry spinach leaves at 8 g per kg body weight and demonstrated improved cognitive function and memory during spatial learning in the Morris water maze test.

    “In [45], aging female rats of the Long–Evans line consumed dry spinach leaves at the rate of 8 g per kg of body weight. The animals demonstrated improved cognitive function and memory during spatial learning in the “Morris water maze” test.”
    Quote in the paperNot a substantial claim0 of 1 sources checked
    • cites [45] Millin, P.M., and Rickert, G.T. (2018). Effect of a Strawberry and Spinach Dietary Supplement on Spatial Learning in Early and Late Middle-Aged Female Rats. Antioxidants, 8. source

      Not checked in the free run.

  • May combine two claims.

    Premisecitation

    In the cited study, expression of IL-1β and TNF-α was increased in the hippocampus of rats exposed to stress, and as a result of consuming the extract, the authors observed a decrease in neuroinflammation in the hippocampus.

    “The results showed that the expression of IL-1β and TNF-α was increased in the hippocampus of rats exposed to stress. As a result of consuming the extract, the authors observed a decrease in neuroinflammation in the hippocampus.”
    Quote in the paperNot a substantial claim0 of 1 sources checked
    • cites [12] Mahmazi, 2020, Spinacia oleracea L. extract attenuates hippocampal expression of TNF-α and IL-1β in rats exposed to chronic restraint stress, Med. J. Islam. Repub. Iran

      Not checked in the free run.

  • Probably not a substantial claim: may not pass S3 (load-bearing).

    Premisecitation

    The treadmill technique allows assessment of the degree of fatigue in rodents by registering the distance traveled, the number of electric shocks received, and the total time during which the electric shock occurred.

    “The technique allows assessment of the degree of fatigue in rodents by registering the distance traveled, the number of electric shocks received, and the total time during which the electric shock occurred [43,44].”
    Quote in the paperNot a substantial claim0 of 2 sources checked
    • cites [43] Poole, 2020, Guidelines for animal exercise and training protocols for cardiovascular studies, Am. J. Physiol. Circ. Physiol. source

      Not checked in the free run.

    • cites [44] Augusto, 2020, Ultra-Endurance Associated With Moderate Exercise in Rats Induces Cerebellar Oxidative Stress and Impairs Reactive GFAP Isoform Profile, Front. Mol. Neurosci. source

      Not checked in the free run.

  • Probably not a substantial claim: may not pass S3 (load-bearing). May combine two claims.

    Premisecitation

    The authors of the cited study linked the results of anabolic activity with the presence of phytoecdysteroids in spinach, but also concluded that spinach contains other non-ecdysteroid compounds with anabolic activity.

    “The authors linked the results of the anabolic activity with the presence of phytoecdysteroids in spinach. However, the authors also concluded that spinach contains other non-ecdysteroid compounds with anabolic activity.”
    Quote in the paperNot a substantial claim0 of 1 sources checked
    • cites [34] Gorelick, J., Iraqi, R.H., and Bernstein, N. (2020). Ecdysteroid Content and Therapeutic Activity in Elicited Spinach Accessions. Plants, 9. source

      Not checked in the free run.

  • May combine two claims.

    Interpretationown data

    The 25-day consumption of FFI had a positive effect not only on anxiety-like functions but also on the reaction rate in the Treadmill test, as well as indicators of search and locomotor activity in the EPM test.

    “We can conclude that the 25-day consumption of FFI had a positive effect not only on anxiety-like functions but also on the reaction rate in the “Treadmill test”, as well as indicators of search and locomotor activity in the EPM test.”
    Quote in the paperSubstantial claim
    • Rests on an assumption: The adaptogenic effects observed in rats under immobilization-induced emotional stress and increased physical activity can be extrapolated to humans.
    • Rests on an assumption: The observed effects of the developed FFI are attributable to the combined action of its phytoecdysteroids and other minor biologically active substances.

  • Probably not a substantial claim: may not pass S3 (load-bearing). May combine two claims.

    Premisecitation

    In a cited study, the antihyperlipidemic effects of spinach extract were evaluated using a model of aerobic exercise (swimming) in rats fed a high-fat diet, and the authors concluded that spinach extracts inhibit pancreatic lipase in vivo in rats, preventing intestinal absorption of food fat and thereby reducing weight gain-induced weight.

    “However, this is most consistent with [13], where the antihyperlipidemic effects of spinach extract were evaluated using a model of aerobic exercise (swimming) in rats fed a high-fat diet. The authors concluded that spinach extracts inhibit pancreatic lipase in vivo in rats, preventing the intestinal absorption of food fat and thereby reducing weight gain-induced weight.”
    Quote in the paperNot a substantial claim0 of 1 sources checked
    • cites [13] Panda, 2020, Consumption of Spinacia oleracea (spinach) and aerobic exercise controls obesity in rats by an inhibitory action on pancreatic lipase, Arch. Physiol. Biochem. source

      Not checked in the free run.

  • May combine two claims.

    Interpretationcitation

    The hypolipidemic effect of FFI is in some part probably due to increased activity of lipoprotein lipase, which increases under the influence of physical exertion, leading to a decrease in blood triglyceride levels.

    “In some part, this is probably due to the increased activity of lipoprotein lipase, which increases under the influence of physical exertion, leading to a decrease in blood triglyceride levels [46].”
    Quote in the paperNot a substantial claim0 of 1 sources checked
    • cites [46] Harrison, 2012, Lipoprotein particle distribution and skeletal muscle lipoprotein lipase activity after acute exercise, Lipids Health Dis. source

      Not checked in the free run.

  • May combine two claims.

    Interpretationcitation

    The anxiolytic effect observed during EPM testing in animals that consumed FFI was manifested in a longer (three times compared to IMM) stay of rats in open arms, indicating a decrease in their anxiety-like functions.

    “The anxiolytic effect observed during EPM testing in animals that consumed FFI was manifested in a longer (three times compared to IMM) stay of rats in open arms, which indicates a decrease in their anxiety-like functions [41].”
    Quote in the paperSubstantial claim0 of 1 sources checked
    • cites [41] Mzhelskaya, 2020, Effects of quercetin on the neuromotor function and behavioral responses of Wistar and Zucker rats fed a high-fat and high-carbohydrate diet, Behav. Brain Res. source

      Not checked in the free run.

  • Probably not a substantial claim: may not pass S3 (load-bearing). May combine two claims.

    Study design and conductnone given

    The studies were conducted according to the guidelines of the Declaration of Helsinki and approved by the Ethics Committee of the Federal Research Centre of Nutrition and Biotechnology (protocol code No. 02-19, 6 October 2019).

    “The studies were conducted according to the guidelines of the Declaration of Helsinki and approved by the Ethics Committee of the Federal Research Centre of Nutrition and Biotechnology (protocol code No. 02-19, 6 October 2019).”
    Quote in the paperNot a substantial claim

  • Probably not a substantial claim: may not pass S3 (load-bearing).

    Study design and conductcitation

    Biochemical indices were determined in blood plasma using an automatic biochemical analyzer according to standard methods [42].

    “The protein metabolism (total protein, albumin, globulins), lipid metabolism (total cholesterol, HDL cholesterol, LDL cholesterol, triglycerides), mineral metabolism (phosphorus), and liver functional state (total bilirubin, AlAT, AsAT, AsAT/AlAT) were determined in blood plasma using an automatic biochemical analyzer “Konelab 20i” (Thermo Fisher Scientific, Waltham, MA, USA) according to standard”
    Quote in the paperNot a substantial claim0 of 1 sources checked
    • cites [42] Apryatin, 2019, Comparative analysis of the influence of a high-fat/high-carbohydrate diet on the level of anxiety and neuromotor and cognitive functions in Wistar and DAT-KO rats, Physiol. Rep. source

      Not checked in the free run.

  • Classified as probably not a substantial claim.

    Study design and conductnone given

    In Experiment No. 2, 40 animals were randomly divided into three groups according to their body weight, EPM test results, and treadmill training: CTRL (n = 20), RUN (n = 10), and RUN-FFI (n = 10).

    “Afterwards, 40 animals were randomly divided into three groups according to their body weight, EPM test results, and treadmill training: CTRL (n = 20), RUN (n = 10), and RUN-FFI (n = 10).”
    Quote in the paperSubstantial claim

  • Probably not a substantial claim: may not pass S3 (load-bearing).

    Study design and conductcitation

    The acute oral toxicity study of FFI was carried out according to the OECD 420 guideline with some modifications.

    “The study of FFI acute toxicity was carried out according to the “420 OECD guideline for testing of chemicals” [31], with some modifications.”
    Quote in the paperNot a substantial claim0 of 1 sources checked
    • cites [31] OECD (2008). Test No. 425: Acute Oral Toxicity: Up-and-Down Procedure, OECD Publishing.

      Not checked in the free run.

  • Probably not a substantial claim: may not pass S3 (load-bearing).

    Study design and conductcitation

    Preliminary separation of animals into groups similar in behavior increases the verifiability of the obtained results.

    “Preliminary separation of animals into groups similar in behavior increases the verifiability of the obtained results [39,40].”
    Quote in the paperNot a substantial claim0 of 2 sources checked
    • cites [39] Sharanova, 2016, Effect of Acute Emotional Stress on Proteomic Profile of Selected Brain Areas and Lysosomal Proteolysis in Rats with Different Behavioral Activity, Bull. Exp. Biol. Med. source

      Not checked in the free run.

    • cites [40] Pertsov, 2016, Effects of Melatonin on Lipid Peroxidation in Blood in Rats with Different Behavioral Characteristics in Acute Emotional Stress, Neurosci. Behav. Physiol. source

      Not checked in the free run.

  • May combine two claims.

    Interpretationreasoning

    The greater muscle mass advantage allowed the animals to overcome exhausting physical activity without much effort, which is reflected in a slight increase in urinary excretion of catecholamines.

    “This advantage allowed them to overcome the exhausting physical activity without much effort, which is also reflected in a slight increase in urinary excretion of catecholamines, the main activators of the stress system [47].”
    Quote in the paperSubstantial claim0 of 1 sources checked
    • cites [47] Salvi, S., and Casadio, V. (2021). Urinary Catecholamines as Markers in Overtraining Syndrome BT—Urinary Biomarkers: Methods and Proto-Cols, Springer US. source

      Not checked in the free run.

  • Classified as probably not a substantial claim.

    Study design and conductnone given

    Animals in Experiment No. 1 were randomly divided by body weight and EPM test results into three groups: CTRL (n = 20), IMM (n = 10), and IMM-FFI (n = 10).

    “Animals were randomly divided by body weight and EPM test results into three groups: CTRL (n = 20), IMM (n = 10), and IMM-FFI (n = 10).”
    Quote in the paperSubstantial claim

  • Classified as probably not a substantial claim.

    Study design and conductcitation

    The assessment of behavior and short-term memory of animals was carried out using the CRPA test according to methodology and equipment described earlier [41].

    “The assessment of the behavior and short-term memory of animals was carried out on the 15th day of the experiment using the “Conditioned reflex of passive Avoidance” (CRPA) test according to the methodology and on equipment described earlier [41].”
    Quote in the paperSubstantial claim0 of 1 sources checked
    • cites [41] Mzhelskaya, 2020, Effects of quercetin on the neuromotor function and behavioral responses of Wistar and Zucker rats fed a high-fat and high-carbohydrate diet, Behav. Brain Res. source

      Not checked in the free run.

  • Probably not a substantial claim: may not pass S3 (load-bearing).

    Study design and conductcitation

    The work with animals adhered to the standard principles described in [49].

    “The work with animals adhered to the standard principles described in [49].”
    Quote in the paperNot a substantial claim0 of 1 sources checked
    • cites [49] Committee for the Update of the Guide for the Care and Use of Laboratory Animals (2011). Guide Laboratory for the Care and Use of Animals, National Academies Press (US).

      Not checked in the free run.

  • Probably not a substantial claim: may not pass S3 (load-bearing).

    Study design and conductnone given

    The chromatographic column used for catecholamine determination was pre-calibrated according to an internal standard.

    “The resulting eluent was injected into a chromatographic column (Nucleodur C18, 5 microns, 250 × 5 mm), pre-calibrated according to an internal standard (“Sigma-Aldrich”, Burlington, MA, USA).”
    Quote in the paperNot a substantial claim
44 of 44

  • The paper relies on this without support.

    The adaptogenic effects observed in rats under immobilization-induced emotional stress and increased physical activity can be extrapolated to humans.

    “Increasing the ability of the human body to adapt in conditions of physical or emotional stress is promising from the standpoint of the use of preventive nutrition containing functional food ingredients (FFI) with proven effectiveness in complex physiological in vivo studies.”
    “Nowadays, the daily impact of psychoemotional stress, the development of chronic fatigue syndrome, and increased physical and mental stress makes it necessary to use specialized food products (SFP) with proven adaptogenic effects in human nutrition [1].”

  • The paper relies on this without support.

    The observed effects of the developed FFI are attributable to the combined action of its phytoecdysteroids and other minor biologically active substances.

    “The present study aimed to evaluate the non-specific resistance of rats in light of the manifestation of pharmacological effects caused by the combined effect of phytoecdysteroids and other minor BAS included in FFI developed from spinach, under conditions of increased physical energy expenditure and immobilization-induced emotional stress.”

  • The paper relies on this without support.

    The rat immobilization and treadmill models used in this study are valid for assessing adaptogenic properties relevant to human psychoemotional stress and increased physical activity.

    “In the conditions of the reproduced models, FFI demonstrated an anxiolytic effect, positively influenced lipogenesis, normalized the excretion of catecholamines in urine, and had a pronounced anabolic effect.”
    “Thus, the studied safety, adaptogenic and anabolic properties of the FFI make it a promising component of specialized food products for conducting clinical trials of products in certain categories of humans subjected to psychoemotional stress and increased physical activity.”

  • The paper relies on this without support.

    The absence of oxalates in FFI makes it suitable for people suffering from or prone to kidney stones, rheumatism, and gout.

    “Due to the lack of oxalates in FFI, such products will be able to find their use in the future, including in people suffering from or prone to kidney stones, rheumatism and gout.”
4 of 4

Claims other papers make about this one

Each is checked against this paper’s own text: does it say what the citing paper says it does?

  • Immobilization has a strong stressful impact on male Wistar rats, leads to increased excretion of catecholamines and circulating corticosterone levels, reduced energy consumption, and a lag in body weight gain.

    “We have previously shown that immobilization has a strong stressful impact on male Wistar rats, leads to the increased excretion of catecholamines and circulating corticosterone levels, reduced energy consumption, and, as a consequence, a lag in body weight gain [33].”

    From Anxiolytic and Antioxidant Effect of Phytoecdysteroids and Polyphenols from Chenopodium quinoa on an In Vivo Restraint Stress Model (2022) · cites this paper as [33]

  • Restraint stress in rats leads to a decrease in memory function, the development of anxiety-like functions, and an increase in motor activity, changing the functional state of antioxidant enzymes in the brain, especially in the poststress period.

    “Restraint stress in rats leads to a decrease in memory function, the development of anxiety-like functions, and an increase in motor activity, changing the functional state of antioxidant enzymes in the brain, especially in the poststress period [33,34].”

    From Anxiolytic and Antioxidant Effect of Phytoecdysteroids and Polyphenols from Chenopodium quinoa on an In Vivo Restraint Stress Model (2022) · cites this paper as [33]

Self-check

Percentages, sums and reported differences re-computed from the paper’s own numbers.

Each contradiction between two passages listed here was confirmed by an independent check.

  • Acute-toxicity guideline number conflicts with its citation

    Minor

    The methods identify the cited OECD guideline as number 420, whereas reference 31 identifies it as Test No. 425.

1 of 1

The citation lists haven’t been fetched yet

References 44 · Cited by 11

Research works printed but not linked: 4

  1. Kaur, 2016, Comparative evaluation of anxiolytic effects of various extracts of oats (Avena Sativa), rice bran (Oryza Sativa) and spinach (Spinacia oleracea) in experimental animals, Int. J. Pharm. Sci. Res., 7, 4110
  2. Zorin, 2021, Development of a method for producing purified spinach extract with a high content of 20-hydroxyecdysone and polyphenols, Int. J. Pharm. Qual. Assur., 12, 1
  3. Watson, R.R., and Preedy, V.R. (2010). Chapter 26—Spinach and Health: Anticancer Effect, Academic Press.
  4. Committee for the Update of the Guide for the Care and Use of Laboratory Animals (2011). Guide Laboratory for the Care and Use of Animals, National Academies Press (US).

Authors
Sidorova, Y. S., Shipelin, V. A., Petrov, N. A., Zorin, S. N., & Mazo, V. K.
Affiliations
  • Federal Research Centre of Nutrition and Biotechnology, 109240 Moscow, Russia
  • Federal Research Centre of Nutrition and Biotechnology (RU)
ORCID
Venue
Plants
Publisher
MDPI AG
Published
2021-11-23
Volume
10
Issue
12
Pages
2555
ISSN
2223-7747 (ISSN-L)
DOI
10.3390/plants10122555
PMC
PMC8704498
OpenAlex
W3216269459
Cited by
11
References
44
Citations by year
2026: 4 · 2025: 1 · 2024: 1 · 2023: 2 · 2022: 3
FWCI
0.47
Type
journal article
Language
en
Open access
Licence
Locations
  • Plants (publishedVersion, cc-by)
  • PubMed Central (publishedVersion, cc-by)
  • PubMed (publishedVersion)
  • DOAJ (DOAJ: Directory of Open Access Journals) (submittedVersion, cc-by-sa)
Funders
Russian Science Foundation (19-16-00107)
Topics
  • Life Sciences › Biochemistry, Genetics and Molecular Biology › Molecular Biology › Medicinal Plants and Bioactive Compounds
  • Life Sciences › Biochemistry, Genetics and Molecular Biology › Molecular Biology › Phytochemical Studies and Bioactivities
  • Health Sciences › Medicine › Complementary and alternative medicine › Phytochemicals and Medicinal Plants
Keywords
phytoecdysteroids, spinach, spinach leaves, adaptogens, functional food ingredients, stress tolerance, anxiolytic effects, biogenic amines, lipid metabolism, acute toxicity, polyphenols, non-specific immunity

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Registry, retraction, Cites and statistics results are computed, not judged, with one exception: a printed reference that no index lists is read once by a model before it counts as a citation error. Claims are extracted by a model and kept only when their quote appears verbatim in the text; each cited source is then checked, with a model’s help, for whether it says what the claim relies on. Those verdicts can be wrong — read the claim’s quote and the cited source before relying on one.

Claims read by deepseek-v4.1-flash under definition version 3 (extract-v3) on 2026-10-10.

Classified by jev-1.13-20260917 (gold-guideline-v0.2) on 2026-10-10.

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