Ecdysteroid Content and Therapeutic Activity in Elicited Spinach Accessions
Open accessAbstract
While spinach is an established nutritionally important crop, its medicinal value is not as well known. Spinach is rich in ecdysteroids, insect hormone analogs with a number of medicinal properties including anti-oxidative, anti-inflammatory and even anabolic activity. However, the potential of spinach as a medicinal plant has not yet been developed. In this study, the ecdysteroid content of spinach was optimized to increase its therapeutic value. Spinach seeds from various sources were grown under controlled hydroponic conditions and analyzed for ecdysteroid content and related anabolic activity. Variations in ecdysteroid content and the related anabolic activity were observed among spinach accessions. A selected variety, Spinacia oleracea cv. Turkey, was exposed to various physical and chemical elicitors to increase and stabilize ecdysteroid content. A number of elicitors, including methyl salicylate and mechanical damage, significantly increased ecdysteroid content and anabolic activity 24 h after exposure. The effect was transient and disappeared 48 h thereafter. Further work is needed to identify the most suitable germplasm and elicitation conditions for optimal ecdysteroid content.
Verified 2026-10-10 · 1 reference not checked yet
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Written from the paper’s full text.
TL;DR
Spinach accessions varied in 20-hydroxyecdysone (20HE) content and in anabolic activity measured as protein synthesis in skeletal muscle cells. Elicitation with methyl salicylate or mechanical wounding transiently increased both 20HE content and anabolic activity at 24 h, with effects largely gone by 72 h.
Key findings
- Among 7 spinach types grown under controlled conditions, 20HE content ranged widely: PI604787 averaged 17.3 µg/g and PI175312 averaged 16.8 µg/g, while PI169673 and NLS6085 were lowest at 10.27 µg/g and 9.3 µg/g; Turkey contained 12.3 µg/g.
- All spinach accessions significantly stimulated leucine incorporation in skeletal muscle cells compared with untreated cells (4662 ± 304 DPM), but anabolic activity only partially correlated with 20HE content; for example, Turkey produced a high increase despite lower 20HE than PI604787, and PI370602 increased protein synthesis significantly more than Genesis 8413 despite similar 20HE (11.9–11.5 µg/g).
- Methyl salicylate treatment increased 20HE by ~200% at 24 h, from 8.3 to 24.1–24.7 µg/g; methyl jasmonate increased 20HE by ~100% to 17.2 µg/g; root or leaf wounding also produced large increases. The effect was transient, and after 72 h 20HE dropped to about half of control levels. Chitosan did not significantly increase 20HE.
- Elicited plants showed parallel changes in anabolic activity: untreated spinach increased protein synthesis versus vehicle (6485 ± 476.6 DPM), and at 24 h leaf/root wounding and methyl salicylate produced the largest increases; methyl jasmonate gave a smaller significant increase, and activity reverted by 72 h. Chitosan-treated plants were similar to non-elicited plants.
Methods
- Seven spinach accessions were grown hydroponically in a climate-controlled greenhouse at 21–30 °C in one-quarter strength modified Hoagland solution, pH 5.7, with 5 plants per 14 L container.
- Elicitation used methyl salicylate (390 µg/L), methyl jasmonate (390 µg/L), chitosan (150 mg/L), leaf-wounding, or root-wounding; plants were randomized into five replicated containers of five plants per treatment (25 plants per treatment) and sampled at 24 h or 72 h.
- 20HE was extracted and quantified by HPLC against purified 20-hydroxyecdysone standard curves; anabolic activity was measured as [3H] leucine incorporation into protein in L6 skeletal myotubes, with one-way ANOVA and Tukey’s HSD for means comparisons. Membrane leakage, osmotic potential, chlorophyll, and carotenoids were also measured.
Limitations
- Stated: a larger survey is needed to evaluate 20HE variation among spinach worldwide; further work is needed to identify suitable germplasm and elicitation conditions; anabolic activity did not completely correlate with ecdysteroid content, and other non-ecdysteroid compounds may contribute to or inhibit activity.
- Stated: development of a spinach-based ecdysteroid nutraceutical requires additional biotechnological research and monitoring with sophisticated chemical analysis plus cell-based biologic assays.
- Evident from the text: only 7 accessions were screened, elicitation focused on a single variety (Turkey), and the elicitation effects were tested at only 24 h and 72 h in one greenhouse/hydroponic setting, so broader genotype, environment, dose, and field validation are not established.
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Claims the paper makes
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- Findingown data
The range of 20HE concentrations found in each spinach type varied.
“Interestingly, the range of concentrations of 20HE found in each spinach type also varied.”
Quote in the paperNot a substantial claim - Findingown data
The lowest concentrations of 20HE were found in PI169673 and NLS6085, containing 10.27 μg/g and 9.3 μg/g 20HE, respectively.
“The lowest concentrations of 20HE were found in PI169673 and NLS6085, containing 10.27-µg/g 20HE and 9.3-µg/g 20HE, respectively.”
Quote in the paperNot a substantial claim - Findingown data
PI604787, which contained comparable 20HE with PI75312, stimulated a significantly smaller increase in protein synthesis.
“Surprisingly, PI604787, which contained comparable 20HE with PI75312, stimulated a significantly smaller increase in protein synthesis.”
Quote in the paperNot a substantial claim - Findingown data
Chitosan treatment did not significantly increase 20HE content.
“Surprisingly, chitosan treatment did not significantly increase 20HE content.”
Quote in the paperNot a substantial claim - Findingown data
A smaller yet significant increase in protein synthesis was observed in MJ treated plants compared with non-elicited plants.
“A smaller yet significant increase in protein synthesis was observed in MJ treated plants compared with non-elicited plants.”
Quote in the paperNot a substantial claim
- Findingown data
Different spinach types produced varying increases in protein synthesis that at least partially correlated with 20HE concentration.
“The different spinach types produced varying increases in protein synthesis at least partially correlating with 20HE concentration (Figure 1B).”
- Also stated in: § 3. Discussion · plain
“The anabolic activity associated with ecdysteroids was confirmed with the different spinach types producing varying increases in protein synthesis.”
Quote in the paperSubstantial claim - Also stated in: § 3. Discussion · plain
- Findingown data
Leaves from untreated spinach significantly increased protein synthesis in skeletal muscle cells compared with cells which received the vehicle (6485 ± 476.6 DPM).
“Supporting our previous findings, leaves from untreated spinach leaves significantly increased protein synthesis in skeletal muscle cells compared with cells which received the vehicle (6485 ± 476.6 DPM).”
Quote in the paperSubstantial claim - Findingown data
All spinach accessions tested significantly stimulated leucine incorporation compared to untreated cells (4662 ± 304 DPM).
“All spinach accessions tested significantly stimulated leucine incorporation compared to untreated cells (4662 ± 304 Decays per minute (DPM)).”
Quote in the paperSubstantial claim - Findingown data
The largest increase in protein synthesis was produced by plants treated with wounding of leaf or roots as well as MS.
“The largest increase in protein synthesis was produced by plants treated with wounding of leaf or roots as well as MS.”
Quote in the paperSubstantial claim - Findingown data
The greatest increase in 20HE content was observed in plants treated with MS or root or leaf-wounding.
“The greatest increase in 20HE content was observed in plants treated with MS or root or leaf-wounding.”
Quote in the paperSubstantial claim - Findingown data
Twenty-four hours after elicitation, plants stimulated a significantly larger increase in protein synthesis than that produced by untreated plants.
“Furthermore, 24 h after elicitation, plants stimulated a significantly larger increase in protein synthesis than that produced by untreated plants.”
Quote in the paperSubstantial claim - Findingown data
Almost no changes in pigmentation were observed following elicitation treatments.
“Almost no changes in pigmentation were observed following elicitation treatments (Figure 4).”
Quote in the paperNot a substantial claim - Findingown data
The effects on protein synthesis could only partially be correlated to ecdysteroid content.
“Interestingly, the effects on protein synthesis could only partially be correlated to ecdysteroid content.”
- Also stated in: § 3. Discussion · plain
“Interestingly, the anabolic activity did not completely correlate with ecdysteroid content.”
Quote in the paperSubstantial claim - Also stated in: § 3. Discussion · plain
- Findingown data
In the case of root-wounding, after 72 h the stimulated protein synthesis was lower, although not statistically significant, than in non-elicited plants.
“In the case of root-wounding, after 72 h the stimulated protein synthesis was actually lower, although not statistically significant, than in non-elicited plants.”
Quote in the paperNot a substantial claim - Findingown data
Elicitation treatments did not affect osmotic potential of the tissue sap.
“The elicitation treatments did not affect membrane leakage from the leaf tissue (Figure 3A), nor the osmotic potential of the tissue sap (Figure 3B).”
Quote in the paperSubstantial claim - Findingown data
All elicitors studied produced a change in 20HE content.
“All elicitors studied produced a change in 20HE content (Figure 2A).”
- Also stated in: § 3. Discussion · plain
“The ability of elicitation to modulate 20HE content was also observed, supporting previous findings [22].”
Quote in the paperSubstantial claim0 of 1 sources checked- cites [22] Schmelz, E.A., Grebenok, R.J., Galbraith, D.W., and Bowers, W.S. (1998). Damage-induced accumulation of phytoecdysteroids in spinach: A rapid root response involving the octadecanoic acid pathway. J. Chem. Ecol.
Not checked in the free run.
- Also stated in: § 3. Discussion · plain
- Findingown data
Twenty-four hours after initiation of MS treatments, a ~200% increase in 20HE from 8.3 to 24.1-24.7 μg/g was observed.
“Twenty-four hours after initiation of the MS treatments, a ~200% increase in 20HE from 8.3 to 24.1–24.7 µg/g was observed.”
Quote in the paperSubstantial claim - Findingown data
The increase in protein synthesis from chitosan treated plants was similar to non-elicited plants.
“The increase in protein synthesis from chitosan treated plants was similar to non-elicited plants.”
Quote in the paperNot a substantial claim - Findingown data
The increase in protein synthesis in MJ treated plants dissipated after 72 h, reverting to activity similar to non-elicited plants.
“This increase dissipated after 72 h, reverting to activity similar to the protein synthesis observed in non-elicited plants.”
Quote in the paperNot a substantial claim - Findingown data
Elicitation treatments did not affect membrane leakage from the leaf tissue.
“The elicitation treatments did not affect membrane leakage from the leaf tissue (Figure 3A), nor the osmotic potential of the tissue sap (Figure 3B).”
Quote in the paperSubstantial claim - Findingown data
Changes in anabolic activity, both the increase after 24 h and the subsequent decrease after 72 h, parallel the previously observed increases in 20HE content.
“These changes in anabolic activity, both the increase after 24 h and the subsequent decrease after 72 h, parallel the previously observed increases in 20HE content.”
Quote in the paperSubstantial claim - Premisecitation
Ecdysteroid content in spinach is variable.
“However, the ecdysteroid content found in spinach is quite variable [19].”
- Also stated in: § 3. Discussion · boosted
“Overall, we have confirmed that the 20HE content in spinach varies greatly among different germplasm.”
Quote in the paperSubstantial claim0 of 1 sources checked- cites [19] Bakrim, A., Maria, A., Sayah, F., Lafont, R., and Takvorian, N. (2008). Ecdysteroids in spinach (Spinacia oleracea L.): Biosynthesis, transport and regulation of levels. Plant Physiol. Biochem. source
Not checked in the free run.
- Also stated in: § 3. Discussion · boosted
- Interpretationreasoning
The lack of change in membrane leakage or osmotic potential suggests that the elicitation treatments which increased ecdysteroid content after 24 h did not produce significant physiological stress in the plants.
“The lack of change in membrane leakage or osmotic potential suggest that the elicitation treatments which increased ecdysteroid content after 24 h did not produce significant physiological stress in the plants.”
Quote in the paperSubstantial claim - Premisecitation
Methyl salicylate and chitosan have been shown to be very effective at inducing the plant defense response.
“Methyl salicylate (MS) and chitosan have been shown to be very effective at inducing the plant defense response [23,24].”
Quote in the paperSubstantial claim0 of 2 sources checked- cites [23] Ament, K., Krasikov, V., Allmann, S., Rep, M., Takken, F.L.W., and Schuurink, R.C. (2010). Methyl salicylate production in tomato affects biotic interactions. Plant J. source
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- cites [24] Adam, 2010, Chitosan in plant protection, Mar. Drugs source
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- Interpretationown data
The level of variation in ecdysteroid content within a single population may vary depending on the germplasm.
“These findings suggest that the level of variation in ecdysteroid content within a single population may vary depending on the germplasm.”
Quote in the paperSubstantial claim - Premisecitation
The elicitors selected are known to stimulate the production of secondary metabolites in plants.
“The elicitors selected are known to stimulate the production of secondary metabolites in plants.”
Quote in the paperSubstantial claim0 of 3 sources checked- cites [23] Ament, K., Krasikov, V., Allmann, S., Rep, M., Takken, F.L.W., and Schuurink, R.C. (2010). Methyl salicylate production in tomato affects biotic interactions. Plant J. source
Not checked in the free run.
- cites [24] Adam, 2010, Chitosan in plant protection, Mar. Drugs source
Not checked in the free run.
- cites [25] Shah, J., Chaturvedi, R., Chowdhury, Z., Venables, B., and Petros, R.A. (2014). Signaling by small metabolites in systemic acquired resistance. Plant J. source
Not checked in the free run.
- Premisecitation
Mechanical damage to either aerial or root tissue is known to activate the plant defense response and stimulate the production of secondary metabolites.
“Mechanical damage to either aerial or root tissue is known to activate the plant defense response and stimulate the production of secondary metabolites [28].”
Quote in the paperSubstantial claim0 of 1 sources checked- cites [28] Jacobo-Velázquez, D.A., González-Aguëro, M., and Cisneros-Zevallos, L. (2015). Cross-talk between signaling pathways: The link between plant secondary metabolite production and wounding stress response. Sci. Rep. source
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- May combine two claims. 19
- The quote only partly states this claim. 1
- Classified as probably not a substantial claim. 26
May combine two claims.
Findingown dataSpinach leaves from the Turkey variety contained less 20HE (12.3 μg/g) than the highest accessions, although this decrease was not significant.
“Spinach leaves from the Turkey variety contained less 20HE (12.3 µg/g) although this decrease was not significant.”
Quote in the paperNot a substantial claimMay combine two claims.
Findingown dataSpinach types with higher 20HE content tended to have greater variation between plants, while types with lower concentrations had less variation in 20HE content.
“The spinach types with higher 20HE content tended to have greater variation between plants while the types with lower concentrations had less variation in 20HE content.”
- Also stated in: § 3. Discussion · plain
“A range of 20HE concentrations was observed in the various spinach material with sources with higher 20HE content possessing greater interplant variation.”
Quote in the paperNot a substantial claim- Also stated in: § 3. Discussion · plain
May combine two claims.
Findingown dataThe accessions with the highest 20HE concentrations were PI604787 and P175312, containing an average of 17.3 μg/g and 16.8 μg/g 20HE, respectively.
“The accessions with the highest concentrations were PI604787 and P175312, containing an average of 17.3-µg/g 20HE and 16.8-µg/g 20HE, respectively.”
Quote in the paperNot a substantial claimThe quote only partly states this claim.
Findingown dataThere was a range of 20HE concentrations found in leaf material from various spinach sources.
“Interestingly, there was a range of concentrations found in the leaf material from the various spinach sources.”
Quote in the paperNot a substantial claimMay combine two claims.
Findingown dataPI604787 and P175312 displayed large ranges of 20HE concentrations among plants: 10.1-25.8 μg/g and 8.2-27.0 μg/g, respectively.
“These accessions also displayed a very large range of concentrations among the different plants from 10.1–25.8-µg/g 20HE and 8.2–27.0-µg/g 20HE, respectively.”
Quote in the paperNot a substantial claimMay combine two claims.
Findingown dataThe highest increase in protein synthesis was produced by the Turkey variety, even though its 20HE content was lower than PI604787, which contained the highest content of 20HE.
“The highest increase in protein synthesis was produced by the Turkey variety, even though its 20HE content was lower than PI604787, which contained the highest content of 20HE.”
Quote in the paperNot a substantial claimMay combine two claims.
Findingown dataGenesis 8413 and PI370602 contained similar 20HE content (11.9-11.5 μg/g), but PI370602 increased protein synthesis significantly more than Genesis 8413.
“While Genesis 8413 and PI370602 contained similar 20HE content (11.9–11.5 µg/g) PI370602 increased protein synthesis significantly more than Genesis 8413.”
Quote in the paperSubstantial claimMay combine two claims.
Findingown dataMJ treatment increased 20HE content by ~100%, reaching 17.2 μg/g.
“MJ treatment increased 20HE content by ~100% reaching 17.2 µg/g.”
Quote in the paperSubstantial claimMay combine two claims.
Findingown dataPI175312, with 20HE content of 16.8 μg/g, produced the greatest increase in protein synthesis, while NLS6085, with 20HE content of 9.3 μg/g, stimulated the smallest increase.
“PI175312, with a 20HE content of 16.8 µg/g, produced the greatest increase in protein synthesis, while NLS6085, with a 20HE content of 9.3 µg/g stimulated the smallest increase.”
Quote in the paperNot a substantial claimMay combine two claims.
Findingown dataPI6973, which contained slightly less 20HE (10.3 μg/g), generated a slightly less increase in protein synthesis, although the difference was not statistically significant.
“PI6973, which contained slightly less 20HE (10.3 µg/g) generated a slightly less increase, although the difference was not statistically significant.”
Quote in the paperNot a substantial claimMay combine two claims.
Findingown dataThe observed increase in 20HE content was transient; after 72 h, the increase dissipated and dropped to about half of the content found in control untreated plants.
“The observed increase was transient. After 72 h, not only did the previously observed increase in 20HE content dissipate, but it even dropped to about half of the content found in the control untreated plants.”
Quote in the paperNot a substantial claimMay combine two claims.
Findingown dataOnly chitosan treatment significantly reduced both chlorophyll a and b while increasing carotenoid levels.
“Only chitosan treatment significantly reduced both chlorophyll a and b while increasing carotenoid levels.”
Quote in the paperNot a substantial claimMay combine two claims.
PremisecitationAmong major food crops, spinach contains the highest amounts of ecdysteroids, about 0.01% fresh weight.
“However, of the major food crops, spinach contains the highest amounts of ecdysteroids, about 0.01% fresh weight [4].”
Quote in the paperSubstantial claim0 of 1 sources checked- cites [4] Grebenok, R.J., Ripa, P.V., and Adler, J.H. (1991). Occurrence and levels of ecdysteroids in spinach. Lipids. source
Not checked in the free run.
May combine two claims.
PremisecitationSpinach contains ecdysteroids, primarily 20-hydroxyecdysone.
“Spinach (Spinacia oleracea) has been known to contain ecdysteroids, primarily 20HE, for some time [17]”
- Also stated in: § 1.1. Spinach · plain
“spinach contains another type of biologically active compound, ecdysteroids [4]”
Quote in the paperSubstantial claim0 of 2 sources checked- cites [17] Bathori, 1987, Phytoecdysteroids in some species of Caryophyllaceae and Chenopodiaceae, Acta Bot. Hung.
Not checked in the free run.
- cites [4] Grebenok, R.J., Ripa, P.V., and Adler, J.H. (1991). Occurrence and levels of ecdysteroids in spinach. Lipids. source
Not checked in the free run.
- Also stated in: § 1.1. Spinach · plain
Probably not a substantial claim: may not pass S3 (load-bearing). May combine two claims.
PremisecitationProduction of 20HE in spinach is elicited by both mechanical wounding and insect feeding.
“Production of 20HE in spinach is elicited by both mechanical wounding and insect feeding [21].”
Quote in the paperNot a substantial claim0 of 1 sources checked- cites [21] Schmelz, E.A., Grebenok, R.J., Ohnmeiss, T.E., and Bowers, W.S. (2002). Interactions between Spinacia oleracea and Bradysia impatiens: A role for phytoecdysteroids. Arch. Insect Biochem. Physiol. source
Not checked in the free run.
Classified as probably not a substantial claim.
PremisecitationMechanical damage to the roots or leaves elicited increased ecdysteroid content.
“Mechanical damage to the roots or leaves elicited increased ecdysteroid content [22].”
Quote in the paperSubstantial claim0 of 1 sources checked- cites [22] Schmelz, E.A., Grebenok, R.J., Galbraith, D.W., and Bowers, W.S. (1998). Damage-induced accumulation of phytoecdysteroids in spinach: A rapid root response involving the octadecanoic acid pathway. J. Chem. Ecol.
Not checked in the free run.
Classified as probably not a substantial claim.
PremisecitationPhytoecdysteroids possess a wide range of potentially therapeutic properties for humans, including anabolic, adaptogenic, hepatoprotective, spasmolytic and anti-inflammatory activity.
“Phytoecdysteroids are acknowledged today to possess a wide range of potentially therapeutic properties for humans, including anabolic [10], adaptogenic [11], hepatoprotective [12], spasmolytic [13] and anti-inflammatory activity [14].”
Quote in the paperSubstantial claim0 of 3 sources checked- cites [10] Gorelick-Feldman, J., MacLean, D., Ilic, N., Poulev, A., Lila, M.A., Cheng, D., and Raskin, I. (2008). Phytoecdysteroids increase protein synthesis in skeletal muscle cells. J. Agric. Food Chem. source
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- cites [11] Lafont, R., and Dinan, L. (2003). Practical uses for ecdysteroids in mammals including humans: And update. J. Insect Sci. source
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- cites [12] Bajguz, A., Bąkała, I., and Talarek, M. (2015). Ecdysteroids in plants and their pharmacological effects in vertebrates and humans. Studies in Natural Products Chemistry, Elsevier. source
Not checked in the free run.
Classified as probably not a substantial claim.
PremisecitationFifteen accessions of spinach grown under controlled conditions displayed significant differences in ecdysteroid content.
“Fifteen accessions of spinach grown under controlled conditions displayed significant differences in ecdysteroid content [20].”
Quote in the paperSubstantial claim0 of 1 sources checked- cites [20] Cheng, 2010, Variation in phytoecdysteroid accumulation in seeds and shoots of Spinacia oleracea L. accessions, HortScience source
Not checked in the free run.
Probably not a substantial claim: may not pass S3 (load-bearing). May combine two claims.
PremisecitationPhytoecdysteroids protect spinach from plant-parasitic nematodes and may confer a mechanism for nematode resistance.
“Phytoecdysteroids were found to protect spinach from plant-parasitic nematodes and may confer a mechanism for nematode resistance [7].”
Quote in the paperNot a substantial claim0 of 1 sources checked- cites [7] Soriano, I.R., Riley, I.T., Potter, M.J., and Bowers, W.S. (2004). Phytoecdysteroids: A novel defense against plant-parasitic nematodes. J. Chem. Ecol. source
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Probably not a substantial claim: may not pass S3 (load-bearing).
PremisecitationMost studies using chitosan report an increase in chlorophyll content.
“This is surprising as most studies using chitosan report an increase in chlorophyll content [29,30].”
Quote in the paperNot a substantial claim0 of 2 sources checked- cites [29] Choudhary, 2017, Cu-chitosan nanoparticle boost defense responses and plant growth in maize (Zea mays L.), Sci. Rep. source
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- cites [30] 2013, Effect of chitosan on common bean (Phaseolus vulgaris L.) plants grown under water stress conditions, Int. Res. J. Agric. Sci. Soil Sci.
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Probably not a substantial claim: may not pass S3 (load-bearing).
PremisecitationSalicylic acid has been observed to lower chlorophyll content.
“While other elicitors like salicylic acid have been observed to lower chlorophyll content [31]”
Quote in the paperNot a substantial claim0 of 1 sources checked- cites [31] Moharekar, S.T., Lokhande, S.D., Hara, T., Tanaka, R., Tanaka, A., and Chavan, P.D. (2003). Effect of salicylic acid on chlorophyll and carotenoid contents of wheat and moong seedlings. Photosynthetica. source
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Probably not a substantial claim: may not pass S3 (load-bearing).
PremisecitationSalicylic acid treatment has been shown to increase carotenoid content in plants.
“Surprisingly, while salicylic acid treatment has been shown to increase carotenoid content in plants [32]”
Quote in the paperNot a substantial claim0 of 1 sources checked- cites [32] Ahmed, F., Fanning, K., Netzel, M., and Schenk, P.M. (2015). Induced carotenoid accumulation in Dunaliella salina and Tetraselmis suecica by plant hormones and UV-C radiation. Appl. Microbiol. Biotechnol. source
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Classified as probably not a substantial claim.
Study design and conductnone givenStatistical significance was determined using one-way ANOVA and Tukey's HSD for comparison of means.
“Statistical significance was determined using one-way ANOVA and Tukey’s HSD for comparison of means.”
Quote in the paperSubstantial claimMay combine two claims.
PremisecitationTreatment with chitosan mimics an attack from a fungal pathogen which activates the plant defense response.
“Treatment with chitosan mimics an attack from a fungal pathogen which activates the plant defense response [26].”
Quote in the paperSubstantial claim0 of 1 sources checked- cites [26] Hadwiger, 2013, Plant science review: Multiple effects of chitosan on plant systems: Solid science or hype, Plant Sci. source
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Probably not a substantial claim: may not pass S3 (load-bearing).
Study design and conductown dataThe elicitation experiment used a random block design with five replicated hydroponic containers of five plants each per treatment.
“Spinach seedlings grown under the previously described hydroponic conditions were divided using a random block design into five replicated hydroponic containers of five plants each per treatment (25 plants per elicitation treatment).”
Quote in the paperNot a substantial claimProbably not a substantial claim: may not pass S3 (load-bearing).
InterpretationreasoningDifferent levels of genetic heterogeneity may exist among the different accessions studied.
“In addition. different levels of genetic heterogeneity may exist among the different accessions studied.”
Quote in the paperNot a substantial claimProbably not a substantial claim: may not pass S3 (load-bearing).
Study design and conductnone givenFive replicated leaves were averaged to generate the final results for membrane leakage.
“Five replicated leaves were averaged to generate the final results.”
Quote in the paperNot a substantial claimProbably not a substantial claim: may not pass S3 (load-bearing).
PremisecitationThe effects of ecdysteroids on mammalian cells were first documented in 1961.
“The effects of ecdysteroids on mammalian cells were first documented in 1961 [8]”
Quote in the paperNot a substantial claim0 of 1 sources checked- cites [8] Couderc, J.-L. (1987). Ecdysone: From Metabolism to Regulation of Gene Expression. Papers from the Seventh Ecdysone Workshop, Edinburgh, UK, March 31-April 3, 1985. Mary Bownes. Q. Rev. Biol. source
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Probably not a substantial claim: may not pass S3 (load-bearing).
Interpretationnone givenThere may also be compounds which inhibit anabolic activity.
“In addition. there may also be compounds which inhibit anabolic activity.”
Quote in the paperNot a substantial claimProbably not a substantial claim: may not pass S3 (load-bearing). May combine two claims.
PremisecitationMethyl salicylate is a natural plant derivative of salicylic acid implicated as an airborne signal involved with systemic acquired resistance.
“MS is a natural plant derivative of salicylic acid implicated as an airborne signal involved with systemic acquired resistance [25].”
Quote in the paperNot a substantial claim0 of 1 sources checked- cites [25] Shah, J., Chaturvedi, R., Chowdhury, Z., Venables, B., and Petros, R.A. (2014). Signaling by small metabolites in systemic acquired resistance. Plant J. source
Not checked in the free run.
Probably not a substantial claim: may not pass S3 (load-bearing). May combine two claims.
PremisecitationEcdysteroids are produced primarily in arthropods and plants, but are also present in fungi and even in marine sponges.
“Ecdysteroids, polyhydroxylated ketosteroids with long carbon side chains, are produced primarily in arthropods and plants, but are also present in fungi and even in marine sponges [5].”
Quote in the paperNot a substantial claim0 of 1 sources checked- cites [5] Lafont, R., and Koolman, J. (2009). Diversity of Ecdysteroids in Animal Species. Ecdysone: Structures and Functions, Springer.
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Probably not a substantial claim: may not pass S3 (load-bearing). May combine two claims.
PremisecitationIn insects, ecdysteroids are involved in molting; in plants there is evidence they are involved in defense response to insect herbivory.
“In insects, ecdysteroids are involved in the molting process (ecdysis), while in plants there is evidence that they are involved in the plant defense response to insect herbivory [7].”
Quote in the paperNot a substantial claim0 of 1 sources checked- cites [7] Soriano, I.R., Riley, I.T., Potter, M.J., and Bowers, W.S. (2004). Phytoecdysteroids: A novel defense against plant-parasitic nematodes. J. Chem. Ecol. source
Not checked in the free run.
Probably not a substantial claim: may not pass S3 (load-bearing).
PremisecitationOver 200 different ecdysteroids have been discovered in plants.
“While 20-hydroxyecdysone (20HE) is the primary ecdysteroid found in arthropods, over 200 different ecdysteroids have been discovered in plants [6].”
Quote in the paperNot a substantial claim0 of 1 sources checked- cites [6] Dinan, L., Savchenko, T., and Whiting, P. (2001). On the distribution of phytoecdysteroids in plants. Cell. Mol. Life Sci. source
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Probably not a substantial claim: may not pass S3 (load-bearing).
Study design and conductnone givenEach protein synthesis experiment was performed in triplicate.
“Each experiment was performed in triplicate.”
Quote in the paperNot a substantial claimProbably not a substantial claim: may not pass S3 (load-bearing).
PremisecitationIncreased chemical variability among different plant populations has been previously documented for a number of species.
“This increased chemical variability among different plant populations have been previous documented for a number of species [33,34,35].”
Quote in the paperNot a substantial claim0 of 3 sources checked- cites [33] Pedneault, 2002, Variations in concentration of active compounds in four hydroponically- and field-grown medicinal plant species, IV International ISHS Symposium on Artificial Lighting
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- cites [34] Lopes, 2007, Medicinal plants: Factors of influence on the content of secondary metabolites | Plantas medicinais: Fatores de influência no conteúdo de metabólitos secundários, Quim. Nova
Not checked in the free run.
- cites [35] Towler, 2015, Variations in key artemisinic and other metabolites throughout plant development in Artemisia annua L. for potential therapeutic use, Ind. Crops Prod. source
Not checked in the free run.
Probably not a substantial claim: may not pass S3 (load-bearing).
InterpretationcitationIt is quite plausible that other non-ecdysteroid compounds are present in spinach with anabolic activity.
“It is quite plausible that other non-ecdysteroid compounds are present in spinach with anabolic activity [2].”
Quote in the paperNot a substantial claim0 of 1 sources checked- cites [2] Lomnitski, L., Bergman, M., Nyska, A., Ben-Shaul, V., and Grossman, S. (2003). Composition, Efficacy, and Safety of Spinach Extracts. Nutr. Cancer. source
Not checked in the free run.
Probably not a substantial claim: may not pass S3 (load-bearing).
Study design and conductnone givenFive replicates were analyzed from each treatment for osmotic potential.
“Five replicates were analyzed from each treatment.”
Quote in the paperNot a substantial claimClassified as probably not a substantial claim.
Study design and conductown data20HE was quantified using HPLC with standard curves generated with >93% purified 20-hydroxyecdysone.
“Quantification was performed using standard curves generated with >93% purified 20-hydroxyecdysone (1–1000 ng) (Sigma-Aldrich, St. Louis, MO, USA).”
Quote in the paperSubstantial claimProbably not a substantial claim: may not pass S3 (load-bearing).
PremisecitationOver 100 different preparations containing ecdysteroids can be found on the market.
“Over 100 different preparations containing ecdysteroids can be found on the market containing either crude plant extracts or purified extracts with defined ecdysteroid content [15].”
Quote in the paperNot a substantial claim0 of 1 sources checked- cites [15] Hunyadi, A., Herke, I., Lengyel, K., Báthori, M., Kele, Z., Simon, A., Tóth, G., and Szendrei, K. (2016). Ecdysteroid-containing food supplements from Cyanotis arachnoidea on the European market: Evidence for spinach product counterfeiting. Sci. Rep. source
Not checked in the free run.
Probably not a substantial claim: may not pass S2 (checkable).
InterpretationreasoningSpinach is an ideal species to study the therapeutic activity of ecdysteroid enriched foods.
“Since spinach has a long history of use as a food crop and is generally recognized as safe, it is an ideal species to study the therapeutic activity of ecdysteroid enriched foods.”
Quote in the paperNot a substantial claim- Rests on an assumption: Spinach has a long history of use as a food crop and is generally recognized as safe.
Probably not a substantial claim: may not pass S3 (load-bearing).
PremisecitationOver time, accumulation of data strongly supports their therapeutic activity.
“and over time, the accumulation of data strongly supports their therapeutic activity [9].”
Quote in the paperNot a substantial claim0 of 1 sources checked- cites [9] Vanyolos, A., and Bathori, M. (2008). New Perspectives in the Analysis of Ecdysteroids: A Promising Group of Biologically Active Compounds. Curr. Pharm. Anal. source
Not checked in the free run.
The paper relies on this without support.
The response of spinach to different biotic or abiotic elicitors with regard to therapeutic activity has not been characterized.
“However, the response of spinach to different biotic or abiotic elicitors with regards to its therapeutic activity has not been characterized.”
The paper relies on this without support.
Spinach has a long history of use as a food crop and is generally recognized as safe.
“Since spinach has a long history of use as a food crop and is generally recognized as safe, it is an ideal species to study the therapeutic activity of ecdysteroid enriched foods.”
The paper relies on this without support.
No wide-scale selection has been undertaken for higher ecdysteroid producing lines.
“However, no wide-scale selection was undertaken for higher ecdysteroid producing lines.”
The paper relies on this without support.
Elicitors induce physiological responses in plant cells, which in turn often induce alterations to secondary metabolism.
“Elicitors induce physiological responses in the plant cells, which in turn often induce alterations to secondary metabolism.”
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?
Spinacia oleracea produces large amounts of phytoecdysteroids, with the major component being 20-hydroxyecdysone.
“Moreover, Spinacia oleracea produces large amounts of PEs, with the major component being 20HE [155,156].”
From Ecdysterone and Turkesterone—Compounds with Prominent Potential in Sport and Healthy Nutrition (2024) · cites this paper as [156]
Section 1.3 says spinach contains ecdysteroids, primarily 20-hydroxyecdysone, and has the highest ecdysteroid content among major food crops. It also says spinach contains considerably less than many other ecdysteroid-containing plants, so the unqualified description of its production as “large amounts” overstates the source. (LLM, conf=0.87)
Experimental — accuracy not yet measuredVariations exist regarding the levels of 20-hydroxyecdysone in spinach, with some reporting lower amounts such as 50 μg/g dry mass, 10.3 and 16.8 μg/g in different spinach accessions, and 0.44 mg% dry weight.
“However, variations exist regarding the levels of 20HE in spinach, with some reporting lower amounts, such as 50 μg/g dry mass, 10.3 and 16.8 μg/g in different spinach accessions, and 0.44 mg% dry weight [8,156,160].”
From Ecdysterone and Turkesterone—Compounds with Prominent Potential in Sport and Healthy Nutrition (2024) · cites this paper as [156]
Results §2.1 reports varying 20HE concentrations across spinach accessions, including 10.27 µg/g (approximately 10.3) and 16.8 µg/g. The cited paper does not report the sentence’s 50 µg/g dry-mass or 0.44 mg% dry-weight figures; those figures may be supported by the other citations, but they are not supported by this source. (LLM, conf=0.87)
Experimental — accuracy not yet measuredSpinach is associated with numerous health advantages, such as antioxidant characteristics, lipid-lowering effects, appetite regulation, tissue regeneration, and protection against oxidative stress-induced damage.
“These results show that spinach is associated with numerous health advantages, such as antioxidant characteristics, lipid-lowering effects, appetite regulation, tissue regeneration, and protection against oxidative stress-induced damage [156,162,164,165,166,167,168,169,170].”
From Ecdysterone and Turkesterone—Compounds with Prominent Potential in Sport and Healthy Nutrition (2024) · cites this paper as [156]
The Introduction (§1.1; page not available) says spinach has anti-oxidative properties, and the Results (§2.4; page not available) report increased protein synthesis in skeletal muscle cells exposed to spinach leaves. Our copy does not establish lipid-lowering effects, appetite regulation, tissue regeneration, or protection against oxidative stress-induced damage. (LLM, conf=0.88)
Experimental — accuracy not yet measuredThe content of 20E assayed in spinach was of the same order of magnitude as that given in the literature.
“The content of the 20E assayed in spinach was of the same order of magnitude as that given in the literature (Bajkacz et al., 2020;Bakrim et al., 2008;Gorelick et al., 2020).”
From Efficient Extraction and Sensitive HPLC-MS/MS Quantification of Selected Ecdysteroids in Plants (2022) · cites this paper as Gorelick et al., 2020
In spinach, various elicitors, such as methyl salicylate and mechanical damage, facilitate the production of PEs, which remarkably induce metabolic and anabolic activity in plants.
“In spinach, various elicitors, such as methyl salicylate and mechanical damage, facilitate the production of PEs, which remarkably induce metabolic and anabolic activity in plants [199].”
From Phytoecdysteroids: Distribution, Structural Diversity, Biosynthesis, Activity, and Crosstalk with Phytohormones (2022) · cites this paper as [199]
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].”
From Adaptogenic Properties of a Phytoecdysteroid-Rich Extract from the Leaves of Spinacia oleracea L. (2021) · cites this paper as [34]
In a cited study, a significant increase in protein synthesis in skeletal muscle cells was shown after treatment with spinach leaves.
“A significant increase in protein synthesis in skeletal muscle cells was shown after treatment with spinach leaves.”
From Adaptogenic Properties of a Phytoecdysteroid-Rich Extract from the Leaves of Spinacia oleracea L. (2021) · cites this paper as [34]
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.”
From Adaptogenic Properties of a Phytoecdysteroid-Rich Extract from the Leaves of Spinacia oleracea L. (2021) · cites this paper as [34]
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.
Two accessions are each said to produce the greatest increase in protein synthesis
MajorSection 2.2 identifies PI175312 as producing the greatest increase in protein synthesis, then identifies the Turkey variety as producing the highest increase. Both cannot rank first for the same outcome.
“PI175312, with a 20HE content of 16.8 µg/g, produced the greatest increase in protein synthesis”
“The highest increase in protein synthesis was produced by the Turkey variety”
Physiological-stress interpretation reverses within the same paragraph
MinorThe text says physiological stress does seem to explain the changes in this study, but the next sentence says the treatments did not produce significant physiological stress.
“this does seem to be the case in the present study.”
“The lack of change in membrane leakage or osmotic potential suggest that the elicitation treatments which increased ecdysteroid content after 24 h did not produce significant physiological stress in the plants.”
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Flagged first, then by citations
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“Similarly [19] However, variations exist regarding the levels of 20HE in spinach, with some reporting lower amounts, such as 50 µ g/g dry mass, 10.3 and 16.8 µ g/g in different spinach accessions, and 0.44 mg% dry weight [8,156,160].”
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“Studies on foods such as spinach or quinoa, which generally contain 20E, show that the concentration is highly dependent on various factors such as origin, genetics and environmental conditions [38,85–90].”
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Ecdysterone: A Component of Dietary Supplements with Ergogenic Potential?
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- Authors
- Gorelick, J., Iraqi, R. H., & Bernstein, N.
- Affiliations
- Eastern R&D Center, Kiryat Arba 90100, Israel
- Hebrew University of Jerusalem (IL)
- Agricultural Research Organization (IL)
- Corresponding author
- Gorelick, Jonathan
- Bernstein, Nirit
- Venue
- Plants
- Publisher
- MDPI AG
- Published
- 2020-06-09
- Volume
- 9
- Issue
- 6
- Pages
- 727
- ISSN
- 2223-7747 (ISSN-L)
- PMC
- PMC7356866
- OpenAlex
- W3033831922
- Cited by
- 19
- References
- 42
- Citations by year
- 2026: 3 · 2025: 2 · 2024: 6 · 2023: 3 · 2022: 2 · 2021: 3
- FWCI
- 0.65
- Type
- journal article
- Language
- en
- Open access
- gold
- dx.doi.org
- 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
- Ministry of Science and Technology, Israel (0000000)
- Ministry of Agriculture and Rural Development (000000)
- Topics
- Life Sciences › Biochemistry, Genetics and Molecular Biology › Molecular Biology › Phytochemical Studies and Bioactivities
- Life Sciences › Agricultural and Biological Sciences › Plant Science › Plant Growth Enhancement Techniques
- Life Sciences › Biochemistry, Genetics and Molecular Biology › Molecular Biology › Plant biochemistry and biosynthesis
- Keywords
- spinach, ecdysteroids, elicitation, methyl salicylate, mechanical damage, anabolic activity, medicinal plants, germplasm diversity, hydroponics
About these checks
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.
Statistics and arithmetic are recomputed, not judged.
Confidence bands are shown only for fields calibrated on our annotated test set. None is calibrated yet, so no band is shown.
What counts as a claim ›How we measure confidence ›How we check this ›
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