Ecdysterone and Turkesterone—Compounds with Prominent Potential in Sport and Healthy Nutrition

The naturally occurring compounds ecdysterone and turkesterone, which are present in plants, including Rhaponticum carthamoides Willd. (Iljin), Spinacia oleracea L., Chenopodium quinoa Willd., and Ajuga turkestanica (Regel) Briq, are widely recognized due to their possible advantages for both general health and athletic performance. The current review investigates the beneficial biological effects of ecdysterone and turkesterone in nutrition, highlighting their roles not only in enhancing athletic performance but also in the management of various health problems. Plant-based diets, associated with various health benefits and environmental sustainability, often include sources rich in phytoecdysteroids. However, the therapeutic potential of phytoecdysteroid-rich extracts extends beyond sports nutrition, with promising applications in treating chronic fatigue, cardiovascular diseases, and neurodegenerative disorders.


Introduction
Ecdysteroids are a class of invertebrate steroid hormones, first found in insects, in which they regulate activities such as molting, development, and reproduction, including the critical metamorphic phases in arthropods [1,2].The first ecdysteroid, ecdysone, was isolated from silkworm pupae by Butenandt and Karlson in 1954, and its structure was presented in 1965 by Huber and Hoppe [3,4].Nowadays, over 550 ecdysteroids are known [5].They possess a tetrahydroxylated four-ring structure, a cyclopentanoperhydrophenanthrene skeleton consisting of 27-30 carbon atoms with a β-side chain at C17, originating from cholesterol or alternative sterols [1,6].
Ecdysteroids are classified into three main groups based on their natural origin, including phytoecdysteroids (PEs), zooecdysteroids, and mycoecdysteroids [1].Phytoecdysteroids are a class of bioactive molecules produced by plants as a defense against herbivorous insects [7].They are widely distributed through the plant kingdom, and research results suggest that only around 6% of plant species contain detectable levels of PEs [6,[8][9][10].Phytoecdysteroids were first reported in the mid-1960s and discovered in diverse plant sources, including the leaves of Podocarpus nakaii, the pinnae of Pteridium aquilinum, the bark of Podocarpus elatus, the roots of Achyranthes fauriei, etc. [11].Some ecdysteroids, including ecdysone, 20-hydroxyecdysone (20HE), makisterone A, and ajugasterone C, are found in both botanical and zoological environments [6,8].Phytoecdysteroids occur in a variety of plant families, including Asteraceae, Amaranthaceae, Commelinaceae, Liliaceae, Lamiaceae, Magnoliaceae, Podocarpaceae, Ranunculaceae, etc. Representative species of plant ecdysterone, turkesterone, and plants containing these PEs, were selected and included in the current review.This was carried out in accordance with the Preferred Reporting Items for Systematic Reviews and Meta-Analyses (PRISMA) criteria shown in Figure 1.
The role of 20-HE as a defensive chemical in plants has been well-established [18].Although R. carthamoides is considered as one of the main sources of the compound, many other plant species that are rich sources of 20HE are C. arachnoidea, Pfaffia (P.glomerata, P. iresinoides), and Serratula (S. centauroides, S. coronata, etc.) [31].It has been reported that Serratula coronata L. juice and the roots of R. carthamoides contain roughly 1.5% 20HE.The C. arachnoidea roots contained up to 4-5% 20HE.Ecdysteroids are also found in substantial amounts in agricultural products, such as spinach, sugar beets, and saltbush seeds.Ecdysterone is used as an ingredient in dietary supplements in the field of sports nutrition [12,17,26,34].
The precise mechanism of action of 20HE remains unclear, but it is hypothesized that it exerts its effects by enhancing protein synthesis in skeletal muscles and the heart [42],
The role of 20-HE as a defensive chemical in plants has been well-established [18].Although R. carthamoides is considered as one of the main sources of the compound, many other plant species that are rich sources of 20HE are C. arachnoidea, Pfaffia (P.glomerata, P. iresinoides), and Serratula (S. centauroides, S. coronata, etc.) [31].It has been reported that Serratula coronata L. juice and the roots of R. carthamoides contain roughly 1.5% 20HE.The C. arachnoidea roots contained up to 4-5% 20HE.Ecdysteroids are also found in substantial amounts in agricultural products, such as spinach, sugar beets, and saltbush seeds.Ecdysterone is used as an ingredient in dietary supplements in the field of sports nutrition [12,17,26,34].
The precise mechanism of action of 20HE remains unclear, but it is hypothesized that it exerts its effects by enhancing protein synthesis in skeletal muscles and the heart [42], boosting ATP synthesis in muscles [43], reducing hyperglycemia in diabetic animals [44], lowering plasma cholesterol levels [45], promoting the production of red blood cells, and decreasing the activity of triglyceride lipase [31].
In recent decades, significant research has been performed on the potential performanceenhancing benefits and therapeutic applications of ecdysterone [15,23,28].The World Anti-Doping Agency (WADA) [33] agreed to add ecdysterone to their monitoring program in 2020, and the inquiry has continued ever since.Moreover, ecdysterone is widely thought to be nontoxic to mammals.According to Ogawa et al., the LD 50 values for orally administrated 20HE in mice exceeded 9 g/kg, whereas intraperitoneally injected 20HE had an LD 50 value of 7.8 g/kg [46].Seidlova-Wuttke et al. conducted a study in which ovariectomized rats were fed dosages of up to 500 mg/kg daily for three months with no obvious adverse responses [47].In general, ecdysteroids are considered safe for mammals [46].Nowadays, numerous DSs containing ecdysteroids are available on the market.The focus of most of these products is sporting individuals [48].
Faster adaptation, the stimulation of protein synthesis [49], stress and anxiety reduction [50], antioxidant defense, the protection of joint cartilage [51], and neuroprotection [52] are some of the most important benefits that foods and plants rich in 20-hydroxyecdysterone might provide.Table 1 presents in vitro studies investigating the biological effects of 20HE.

Study Objectives Cell Lines Main Results
Ref.
Neuroprotective effects of 20HE in Parkinson's disease.
At a MIC of 2.5 mg/mL, the methanolic extract inhibited the growth of Acinetobacter sp., E. faecalis, K. oxytoca, P. agglomerans, P. rettgeri, P. aeruginosa, and S. aureus, while E. coli and K. pneumoniae were inhibited at a MIC of 1.25 mg/mL.The chloroform extract significantly suppressed the growth of cancer cells.Aqueous and butanol extracts have high antioxidant activity.

20-hydroxyecdysone decreased the anti-IgE-induced histamine production
by mast cells in a dose-dependent manner.

20-hydroxyecdysone exerts neuroprotective effects through different
and complementary pathways.It is a promising agent for the treatment of Alzheimer's disease. [55]

20-hydroxyecdysone inhibits inflammation via SIRT6-mediated NF-κB
signaling in endothelial cells.20-hydroxyecdysone could be a promising therapeutic avenue for addressing cardiovascular ailments.[57] Effect of glucocorticoids in osteoblasts and the role of 20HE in the pathogenesis of glucocorticoid-induced osteoporosis.
The protective effect of 20HE on dexamethasone-induced osteoporosis was also shown.It can induce osteogenic differentiation and autophagy.
[58] The isolated ecdysteroids and iridoid glucosides from A. turkestanica were found to lack antioxidant properties and did not exhibit significant cytotoxic or antimicrobial effects.Interestingly, the chloroform extract, containing more lipophilic compounds, demonstrated higher activity. [59] Investigation of the potential of ecdysterone to lower glucose levels in hepatocytes and whether it stimulates insulin secretion.
Ecdysterone may exert the glucose-lowering effect in hepatocytes that is insulin-independent and shows no effect on insulin release. [60] Effect of ecdysterone on human breast cancer cell lines.MCF7, MDA-MB-231, MDAMB-468, DF2, and WI-38-breast cancer cell lines.
Tumor suppressive effect of 20HE on a panel of breast cancer cell lines.It shows synergism with doxorubicin to induce cell death in breast cancer cell lines. [61] Examine the effect of 20HE on the susceptibility of Escherichia coli to antibiotics.
In E. coli, 20HE altered antibiotic bactericidal effect.[62] 20-hydroxyecdysone and its derivatives have the ability to sensitize multidrug-resistant (MDR) and non-MDR cancer cell lines to chemotherapeutic agents, and liposomal formulations.
MDR and non-MDR cancer cell.
20-hydroxyecdysone-diacetonide can sensitize both MDR and non-MDR cancer cell lines to chemotherapeutic agents, and liposomal formulations. [63] Effect of 20HE and ecdysteroid fraction isolated from Serratula coronata L. on human blood cells and neutrophils.
ecdysteroneantiproliferative and antioxidant effects.
The chloroform extract showed potent cytotoxic effects, while the water and n-butanol extracts demonstrated notable antioxidant activities. [65] Investigation of 20HE, ajugasterone C, and polypodine B isolated from Serratula coronata effects on breast cancer.
Human breast cancer cells.

20-hydroxyecdysone and ajugasterone C
demonstrated notable inhibition of viability in the triple-negative cell line.

Study Objectives Cell Lines Main Results
Ref.
Cytotoxic effects of the butanolic extract from the underground parts of Helleborus caucasicus and the purified compounds: furostanol derivative, 20HE, and deglucohellebrin, on human cancer cell lines.
The extract of H. caucasicus and isolated compounds decreased cell viability in vitro on a lung cancer cell line and exerted a strong cytotoxic effect.Moreover, 20-HE and deglucohellebrin possess pro-apoptotic activities. [68] Antiadipogenic activity of R. carthamoides extract, ecdysterone, turkesterone, and ponasterone A.
Chondrocytes isolated from knee joints of 3 to 4-week-old male Sprague-Dawley rats.
Ecdysterone exhibited anti-apoptosis and anti-inflammatory effects in rat chondrocytes induced by IL-1β, which may be related to the NF-κB signaling pathway. [70] Effects of ecdysterone on brain acetylcholinesterase (AChE).
Rat cerebral cortex slices from Wistar albino rats.
Ecdysterone caused an increase in AChE in rat brain slices.No effect was observed in an in vitro assay using purified AChE. [71] Effects of β-ecdysone on osteoblast viability by assessing apoptosis following treatment with excess glucocorticoids.
BMSCs were isolated from the femurs and tibiae of mice-male BALB/C mice aged 8 weeks (20 ± 2 g body weight).
Ecdysterone induced a significant increase in the thickness of joint cartilage and increased the whole epiphyseal growth plate and its proliferative and hypertrophic zones. [51] β-ecdysterone effects on osteoblast differentiation and bone regeneration in vitro and in vivo.
β-ecdysterone is identified as a regulator for bone regeneration, can stimulate the BMP-2/Smad/Runx2/Osterix pathway and MC3T3-E1 cell regeneration and initiate bone rebuilding. [74] The effect of ecdysterone on lipid metabolism.
Male Wistar rats.Ecdysterone was injected intraperitoneally in a dose of 0.5 mg/kg, injection volume was 0.2 mL.
Ecdysterone: reduces free fatty acids, diglycerides, triglyceride lipase activity, and specific activity of phosphatidylcholine, increases the specific activity of phosphatidylethanolamine and phosphatidylserine in the liver.[75] Evaluate the effect of high-intensity interval training exercise (HIT) and ecdysterone consumption synergistically after Alzheimer's disease.
Due to the free radical scavenging and neuroprotective qualities, the combination of HIT exercise and ecdysterone therapy may be a viable therapeutic strategy for Alzheimer's disease.
Neuroprotective effect and Removed glutamatergic excitotoxicity. [80] Ecdysterone effects on the cyclic AMP kinase system in mouse adipose tissue.
Male mice were injected intraperitoneally with 10 µg of ecdysterone or control mice received saline.
Decrease AMP-binding protein activity and cyclic AMP-dependent protein kinase. [81] Efficacy of 20-HE in ameliorating memory deficits within an animal model of type 1 diabetes mellitus.
Adult male Sprague-Dawley rats, divided into 2 control groups (n = 10) and animals (n = 70) in high-fat group were treated with a high-fat diet for 15 weeks.Other groups were received 20HE (1, 10, and 100 mg/kg/day) for 12 weeks.
20-hydroxyecdysterone has a protective role in memory deficits in rats with diabetes mellitus. [82] Effect of 20HE on the osteogenic differentiation ability of bone marrow mesenchymal stem cells.
β-ecdysterone may be beneficial for the recovery of osteonecrosis of bone marrow mesenchymal stem cells. [83] Effects of ecdysterone on cAMP and cGMP levels in mouse plasma.
Male mice, ecdysterone was given intraperitoneally at a dosage of 10 µg/animal.
The heterophilic effects of ecdysterone in mammals may be facilitated by modulation of the cyclic AMP system.[84] Effects of ecdysterone on cAMP and cGMP levels in mouse liver.
Male mice, ecdysterone was given intraperitoneally at a dosage of 10 µg/animal.
The cyclic AMP-protein kinase system is likely implicated in mediating the heterophilic effects of ecdysterone.[85] Investigation the underlying molecular mechanisms, in particular the role of estrogen receptor beta.
Ecdysterone led to an increase in muscle fiber size, accompanied by elevated serum insulin-like growth factor 1 (IGF-1) levels and decreased levels of corticosterone and 17β-estradiol (E2).Hypertrophy was induced by treatment with 20HE, dihydrotestosterone, IGF-1, and E2. [86] Ecdysterone lipid-lowering effects in obese Zucker rats.
The obese rats were allocated to two groups (n = 8/group): control and ecdysterone (0.5 g/kg/food).
Ecdysterone supplementation lacks lipid-lowering effects in liver and plasma of lean and obese Zucker rats. [87] Table 2. Cont.

Study Objectives Study Design Main Results
Ref.
Effects of 20HE on CYP11B1/2 levels and activity in UVB-induced photoaging and skin lesions in hairless mice.
20-hydroxyecdysone prevents UVB-induced skin aging by inhibiting aldosterone synthase.It is a promising candidate for anti-aging therapy. [88] Effect of β-ecdysterone on osteogenic differentiation of bone marrow mesenchymal stem cells and its dependents on the estrogen receptor.
In vitro, β-ecdysterone stimulated osteogenic differentiation of mesenchymal stem cells in an estrogen receptor-dependent way, reducing osteoporotic processes in a mouse model.[89] 20-hydroxyecdysone for improving skin conditions in postmenopausal women, investigation in ovariectomized rats.
Ecdysterone prevented the ovariectomy-induced decrease in subcutaneous musculature, contrasting with the effects of 17β-estradiol. [90] Effect of 20HE on oxidative stress and inflammation in a collagen-induced rheumatoid arthritis rat model.
Forty healthy male Sprague-Dawley rats, (4 gropus, n = 10).Rats given saline for 28 days.Groups I and II: collagen-induced arthritis-induced rats and groups III and IV: rats treated with 10 or 20 mg/kg body weight 20HE for 28 days.
Administering 20HE (20 mg/kg) to collagen-induced rheumatoid arthritis rat models successfully reduces inflammation and oxidative stress, resulting in anti-rheumatoid arthritis effects.[92] Ecdysterone as a modulator of cytostatic therapy.
Ecdysterone enhanced the chemotherapeutic impact of low doses of cytostatic. [93] Ethanol extract from Pfaffia glomerata and isolated ecdysterone hypnotic effect.
The lipophilic fraction derived from P. glomerata demonstrates a hypnotic effect, with ecdysterone identified as one of the compounds responsible for this central nervous system activity. [94] "Ecdysterone-80"-an extract of phytoecdysteroids from Serratula coronata L., dominated by 20HE (81-86%).
168 male rats.Ecdysterone-80 at a dosage of 20 mg/kg for 60 days beginning in the second month of the trial.
Rats were orally administered the extract at a repeated dose of 10 mg/kg.
The ecdysteroid-containing preparation derived from S. hissarica exhibited notable effectiveness in promoting wound healing activity. [96] Table 2. Cont.

Study Objectives Study Design Main Results
Ref.

Ecdysterone derived from
Achyranthes bidentate and Paeonol derived from Cortex Moutan.
Male Sprague-Dawley rats.10% ecdysterone and 5% paeonol.Rats in each group were given vehicle or compound treatments three times a day.

20-hydroxyecdysone isolated from Silene viridiflora influence on size of the different muscle fiber types
Twenty male Wistar rats (5 groups, n = 4/group): 0.9% NaCl, a daily subcutaneous injection of 5 mg/kg body weight of 20HE, snake venom (notexin) injections.From the 5th day post-injection: a daily subcutaneous injection of 5 mg/kg body mass weight of 20HE for 7 days or a daily subcutaneous injection of 0.5 mg/kg body mass weight of 20HE for 7 days.
20-hydroxyecdysone also augmented the myonuclear count within the fibres of both normal and regenerating muscles.
[98] Ecdysterone, a spinach component, has the potential to prevent metabolic syndrome.
It has been determined that this product can be utilized to treat and prevent the development of metabolic syndrome.
[99] The in vivo studies presented in Table 2, suggest that ecdysterone possesses effects on osteoblast differentiation and bone regeneration, joint morphology and osteoporosis, Alzheimer's disease, and lipid metabolism, as well as having anti-obesity, anti-diabetic, and neuroprotective effects [47,[49][50][51][74][75][76].Some of the in vivo studies confirm the in vitro studies about anti-obesity, anti-diabetic, neuroprotective, and cytotoxic effects, as well as the prevention of Alzheimer's disease.A controlled randomized study investigated the utilization of 20HE in metabolic syndrome [99].Ecdysterone has great potential for use in medications intended to cure a variety of illnesses.Furthermore, ecdysterone does not appear to possess any severe side effects [19,47,[49][50][51].These suggest that ecdysterone supplementation is safe.In a study involving 20-hydroxyecdysone in the dietary supplement "Peak Ecdysone", conducted on 46 men over a 10-week period, it was discovered that 20HE supplementation resulted in increases in body weight and muscle mass, as well as improvements in power and strength performance, without adverse effects or changes to the steroid profile [26].It is considered that lower doses showed no significant effects but reported that amounts more than 5 µg/kg body weight were considered effective [102].This shows that ecdysterone administration may be beneficial for improving athletic performance without compromising health.
We found multicenter randomized double-blind studies on ecdysterone and ecdysterone-rich extracts.Further research is needed to understand the mechanisms of action and possible long-term consequences of these supplements.To fully assess the benefits and possible future applications of ecdysterone, multicenter randomized double-blind trials are required.

Rhaponticum carthamoides
Rhaponticum carthamoides (Willd.)Iljin is a perennial herb of the Asteraceae family that is also known as maral root or Russian leuzea.It grows in the harsh conditions of South Siberia's Altai and Saian mountains.It is a semi-rosulate plant that may grow to be 150 cm tall [13,103,104].The use of R. carthamoides for medicinal purposes dates back to ancient times, and traditional Siberian medicine has long praised the plant for its ability to treat weariness and debility after sickness [13,103].In the history of Russian scientific investigation, R. carthamoides has received a lot of attention in the domain of physical performance improvement.Research over the last century has shown its muscle-and strength-building capabilities, resulting in widespread use among elite athletes in Soviet and Russian sports [13].In 1969, Brekhman and Dardymov classified R. carthamoides as an adaptogen, now widely used in herbal medicine to promote resistance to stress, such as trauma, anxiety, and fatigue [13,103,105].
Extracts from its roots and rhizomes are currently employed in a wide range of DSs and nutraceutical formulations.They are used to increase muscle growth, alleviate impotence, and reduce physical and mental fatigue [13].Furthermore, R. carthamoides and its derivatives are used in cosmetics and herbal teas [13,106].Rhaponticum carthamoides gained serious popularity in the last decade, especially after the introduction of numerous DSs containing leuzea extracts [120].
One of the most important benefits of leuzea supplementation is the potential to increase the working capacity of the skeletal muscles [42].However, for the athletes, the adaptogenic activity of leuzea seems to be of the greatest importance [103].Table 3 summarizes a wide range of in vivo investigations on the pharmacological activities of R. carthamoides.adaptogenic activity of leuzea seems to be of the greatest importance [103].Table 3 summarizes a wide range of in vivo investigations on the pharmacological activities of R. carthamoides.

Study Objectives Cell Lines Main Results
Ref.

SGBS human adipocytes.
Rhaponticum carthamoides extract, ecdysterone and turkesterone reduced lipid accumulation in human adipocytes and R.carthamoides and ecdysterone stimulated lipolysis. [69] Influence of R. carthamoides transformed roots extract on the growth of grade II and III human glioma cells.
Human primary glioma cell lines (grade II and III astrocytoma).
The extract from R. carthamoides transformed roots shows anticancer properties by impeding the proliferation of glioma cells and prompting apoptotic cell demise. [121] The cytotoxic effect and apoptotic potential of extracts derived from R. carthamoides transformed roots and roots of soil-grown plants evaluated in human glioma primary cells.
Human glioma cancer cells and normal human astrocytes.
Rhaponticum carthamoides root extracts reduce cell proliferation and promote apoptosis in human glioma cells. [122] Efficacy of R. carthamoides against myocardial injury.
A myocardial ischaemia in male SD rats and H9c2 cell lines.

Study Objectives Cell Lines Main Results
Ref.

SGBS human adipocytes.
Rhaponticum carthamoides extract, ecdysterone and turkesterone reduced lipid accumulation in human adipocytes and R. carthamoides and ecdysterone stimulated lipolysis.[69] Influence of R. carthamoides transformed roots extract on the growth of grade II and III human glioma cells.Human primary glioma cell lines (grade II and III astrocytoma).
The extract from R. carthamoides transformed roots shows anticancer properties by impeding the proliferation of glioma cells and prompting apoptotic cell demise.[121] The cytotoxic effect and apoptotic potential of extracts derived from R. carthamoides transformed roots and roots of soil-grown plants evaluated in human glioma primary cells.
Human glioma cancer cells and normal human astrocytes.
Rhaponticum carthamoides root extracts reduce cell proliferation and promote apoptosis in human glioma cells. [122] Efficacy of R. carthamoides against myocardial injury.
A myocardial ischaemia in male SD rats and H9c2 cell lines.
The extracts showed a significant modulation of the NF-κB inhibitory effect of dexamethasone. [125] Table 3. Cont.

Study Objectives Cell Lines Main Results
Ref.
Rhaponticum carthamoides root extract and 20HE, effects on human breast adenocarcinoma.
20-hydroxyecdysone does not affect cell proliferation, ERα protein, or 5α-reductase activity.The extract inhibited cell proliferation and affected AhR-agonistic activity. [126] Extracts from R. carthamoides transform roots and soil-grown plant roots' effect on oxidative stress.
Rhaponicum carthamoides possess antioxidant properties protecting CHO cells from oxidative stress. [127] Compare the composition and antioxidant activity of R. carthamoides leaf extracts taken from plants growing in Poland and Russia.

HL-60 cells (human leukaemia).
The extracts from the Polish material had better antioxidative and cytotoxic activity than those from the Russian. [128] The effect of R. carthamoides on the proliferation of a diverse population of rumen bacteria and their influence on the fermentation process of a feed mixture in an artificial rumen (Rusitec).

Rumen bacteria and artificial rumen (RUSITEC)
No significant influence of R. carthamoides on rumen microflora metabolism, nor any effect of stimulative chemicals found in the plant's above-ground parts. [129] The antibacterial activity of crude ethanol extracts from the aerial parts and roots of four leuzea species incl.

Study Objectives Study Design Main Results
Ref.
Ecdysterone did not affect blood glucose levels in non-diabetic animals but reduced blood glucose levels in alloxan-diabetic mice. [44] Rhaponticum carthamoides extract effects on rats' learning and memory processes.
Rats were orally administered R. carthamoides extract at doses of 0.25, 0.5, and 2.5 g/kg body weight for 10 days preceding the training sessions.
The extract increased learning and memory indices to varied degrees, depending on the amount used.

Study Objectives Study Design Main Results
Ref.
The central neurotropic activity of an aqueous-alcoholic extract from R. carthamoides cultivated in Bulgaria.
Male Wistar rats.The extract in doses of 200, 500, and 1000 mg/kg was injected subcutaneously 1 h prior to or immediately after training.Effect of extract on blood pressure conducted on male cats weighing 3.0 to 4.0 kg, administered intraduodenally in 1 mL/kg body weight.
The extract of R. carthamoides showed significant central stimulating effects, including enhanced ambulation increased central nervous system excitability, and a minor antinarcotic action and rearing behavior, improved learning, memory ability, and physical endurance in rats. [134] Rhaponticum carthamoides extract effects compared to those of Glycyrrhiza glabra and Punica granatum extracts.
Male Wistar Albino Glaxo rats incorporated with 300 mg/kg/day extracts.
Rhaponticum carthamoides extract powder supplementation significantly enhanced glucose and lipid metabolism compared to Glycyrrhiza glabra and Punica granatum extracts. [135] Investigate the impact of R. carthamoides, R. rosea, and their combined administration on resistance exercise and mechanical power.
Eleven [136] Ecdysterone from the roots of R. carthamoides, influence on the mitochondrial level.
White male mongrel rats.Ecdysterone was administered orally to experimental group for 10 days before the start of the experiment at a daily dose of 5 mg/kg.
Male mongrel white mice subjected to immobilization on their back for 6 h.Ecdysterone, turkesterone, and T-activin were administered intraperitoneally before immobilization.
Ecdysterone and turkesterone prevented and facilitated the consequences of stress and restored the immunological activity. [138] Genotoxic evaluation of β-ecdysone.
The results suggest cytogenotoxic activity of 2HE.[139] Effect of R. carthamoides extract on the lipid content of the erythrocyte membrane in rats with cerebral ischemia.
Rhaponticum carthamoides extract increased the total lipid and phospholipid contents. [140] The impact of different levels of R. carthamoides hay meal in rat diets.
The use of a diet containing 20% R. carthamoides has resulted in significant variability in teste weight.[141] Effect of an herbal supplement comprising a 70:30 combination of extracts R. carthamoides and R. rosea on performance fatigue.
Thirty healthy active men (age 22.3 ± 4.1 years): 350 mg dose of the DS, a 175 mg dose of the DS with 175 mg of maltodextrin, or a placebo-350 mg (maltodextrin).
No significant influence on performance fatigability during high-intensity, repetitive muscular activities. [142] The diverse range of bioactivities of R. carthamoides are presented in Table 4.These investigations include a wide range of activities, such as cognitive function effects, metabolic regulation, stress management, and cytotoxicity [44,[133][134][135][136][137][138][139][140][141][142].Mosharrof et al. investigated the effect of R. carthamoides extract on learning and memory processes in rats [133].Petkov et al. found that an aqueous-alcoholic extract of R. carthamoides exhibited significant stimulating effects on the central nervous system, including improved learning and memory ability in rats [134].Dushkin et al. investigated the therapeutic potential of R. carthamoides extract, as well as extracts from Glycyrrhiza glabra and Punica granatum.They observed that treatment with R. carthamoides extract significantly improved glucose and lipid metabolism compared to the other extracts [135].Furthermore, studies on the combination administration of R. carthamoides and Rhodiola rosea on resistance exercise, the combination of these extracts boosted muscle protein synthesis and average power performance in rats [136].These findings provide valuable insights into R. carthamoides' therapeutic potential for enhancing overall health and well-being.There are no observable indications or symptoms of toxicity, indicating a wide margin of safety [44,[133][134][135][136][137][138][139][140][141][142].Contrary to the randomized trial, which reported improved physical performance after the intake of 20HE, the investigation into the effects of R. carthamoides and R. rosea extracts suggests no potential effects on physical performance and fatigue [26,142].These made R. carthamoides a valuable resource in sports nutrition.Further research is needed to understand the mechanisms of action and possible long-term consequences of these supplements.Moreover, the multicenter randomized controlled trials are limited.

Spinacia oleracea
Spinach (Spinacia oleracea L.) is an annual plant belonging to the family Chenopodiaceae; it can be divided into two subspecies, ssp.glabra and ssp.spinosa [143,144].It originated in central Asia, specifically Persia, and its use dates back to ancient times [145,146].
Spinach is a great source of nutrition and phytochemical constituents [143,144,146,147].The nutrient composition can be divided into six major components, including carbohydrates (approximately 50%), proteins (approximately 14%), fats (approximately 23%), fiber, minerals, and vitamins (Figure 3) [146,[148][149][150][151][152].The high iron content makes it a valuable food for anemia [143].A disadvantage of spinach is its high nitrate content, which may cause methemoglobinemia [143].Spinach is a versatile plant that may be consumed fresh in salads and smoothies or cooked in recipes such as steamed vegetables, casseroles, and soups [143,144,146,147,149].Due to the wide variety of bioactive and phytochemical compounds, S. oleracea has a wide variety of potential functionalities, mainly antioxidant, antimicrobial, anticancer, antiobesity, hypoglycemic, and hypolipidemic [146,149,151].Spinach leaves are utilized for their, emollient, wholesome, antipyretic, diuretic, laxative, and anthelmintic substances, as well as their anti-inflammatory effects and joint pain relief (Figure 3) [151].Spinach, as a significant antioxidant and nitric oxide donor, is a valuable vegetable in athletes' diets [162].In recent years, the consumption of spinach has increased due to consumers' concerns about healthy eating [144].Spinach's traditional claim to increase muscle strength may have scientific support due to its ecdysteroid concentration.Because of its anabolic qualities, ecdysterone has gained popularity as a natural sports performance booster.As a result, ecdysterone-containing dietary supplements made from spinach and other plant extracts have become increasingly popular.Athletes are advised to take up to 1000 mg of ecdysterone daily; however, even high daily consumption of spinach (1 kg) rarely surpasses 100 mg.Studies on humans that have been given supplements containing ecdysterone have, over time, demonstrated increases in physical strength and muscular mass [34,163].In Table 5 are presents in vivo studies on the biological activities of S. oleracea.

Study Objectives Study Design Main Results
Ref.  Spinach is source of beneficial phytonutrient constituents, such as phenolic compounds (flavonoids, phenolic acids, stilbenes, and lignans) and carotenoids (lutein, zeaxanthin, and β-carotene) [146,149,153].Spinach also contains steroids, terpenes, tannins, and cardenolides [154].Moreover, Spinacia oleracea produces large amounts of PEs, with the major component being 20HE [155,156].Spinach has stimulated ecdysteroid accumulation in response to mechanical or insect injury, and PEs are metabolically stable in this plant species [11,157].The reported content of 20HE in spinach leaves ranges from 17.1 to 885 µg/g [158,159].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].Grucza et al. reported even lower levels of 20HE content in fresh spinach leaves at 10 µg/100 g [161].These discrepancies in 20HE and phytochemical content may be attributed to different processing methods applied to spinach leaves [149].

Mechanism of action of PEs
Spinach is a versatile plant that may be consumed fresh in salads and smoothies or cooked in recipes such as steamed vegetables, casseroles, and soups [143,144,146,147,149].Due to the wide variety of bioactive and phytochemical compounds, S. oleracea has a wide variety of potential functionalities, mainly antioxidant, antimicrobial, anticancer, anti-obesity, hypoglycemic, and hypolipidemic [146,149,151].Spinach leaves are utilized for their, emollient, wholesome, antipyretic, diuretic, laxative, and anthelmintic substances, as well as their anti-inflammatory effects and joint pain relief (Figure 3) [151].Spinach, as a significant antioxidant and nitric oxide donor, is a valuable vegetable in athletes' diets [162].In recent years, the consumption of spinach has increased due to consumers' concerns about healthy eating [144].Spinach's traditional claim to increase muscle strength may have scientific support due to its ecdysteroid concentration.Because of its anabolic qualities, ecdysterone has gained popularity as a natural sports performance booster.As a result, ecdysterone-containing dietary supplements made from spinach and other plant extracts have become increasingly popular.Athletes are advised to take up to 1000 mg of ecdysterone daily; however, even high daily consumption of spinach (1 kg) rarely surpasses 100 mg.Studies on humans that have been given supplements containing ecdysterone have, over time, demonstrated increases in physical strength and muscular mass [34,163].In Table 5 are presents in vivo studies on the biological activities of S. oleracea.S. oleracea extract demonstrated significantly reduced hyperlipidaemia.The combined therapy and aerobic exercise, underscored the importance of incorporating both exercise and antioxidant-rich dietary sources to managed lipid levels and obesity. [164] A flavonoid-rich extract from S. oleracea leaves effect on appetite in rats.
Spinach leaves extract shows promising appetite suppression effect. [165] Protective effect of spinach against radiation-induced oxidative stress.
Spinacia oleracea showed strong protective effects against radiation-induced oxidative stress in mouse livers, indicating its potential as a low-cost source of antioxidants. [166] Table 5. Cont.

Study Objectives Study Design Main Results
Ref.
Effect of Spinacia oleracea extracts on ulcer regeneration, particularly in diabetic conditions.
Then, they were exposed to diabetes and received 300 mg/kg S. oleracea alcoholic extract.Duration: 1 month.
Spinacia oleracea extracts may be effective in the regeneration of diabetic ulcers. [167] Anti-asthmatic activity of S. oleracea an aqueous extract.
In the ovalbumin-induced asthmatic mouse model, the aqueous spinach extract efficiently alleviates asthmatic symptoms. [168] Spinach-antioxidant and hyperlipidaemic effect.
HepG2 cells and male Sprague-Dawley rats (n = 24).Three groups of eight animals each.Normal diet, a high fat-cholesterol diet (HFCD), or a HFCD supplemented with 5% freeze-dried spinach powder.
Spinach led to improvements in lipid profiles, decreased oxidative stress markers in the liver and blood, and enhanced antioxidant enzyme activity. [169] Examined the preventive efficacy of spinach natural antioxidants.[170]

B16 melanoma cell line and female
Table 5 summarizes investigations into potential health benefits connected with S. oleracea.Gorelick-Feldman et al. investigated the effects of PEs and extracts from A. turkestanica and S. oleracea on protein synthesis and physical performance [19].Panda et.al. investigated whether spinach extract may reduce hyperlipidemia by reducing pancreatic lipase activity.This suggests the importance of consuming spinach in managing lipid levels and obesity [164].The spinach extract demonstrated promising appetite suppression effect which make it potential nutrient in appetite regulation [165].

Study Objectives Study Design Main Results
Ref.
The influence of chronic daily spinach supplementation on oxidative stress and muscle damage.
Twenty well-trained men, randomised in two groups: spinach (n = 10) and placebo (n = 10).The participants took spinach supplementation (1 g/kg body weight) or placebo daily for 14 days before running (21.1 km).
Persistent daily oral supplementation with spinach reduces indicators of oxidative stress and muscle injury after a half-marathon in well-trained healthy young men. [162] Effects of daily supplementation with a S. oleracea extract with a moderate-intensity exercise programmed on skeletal muscle.Training combined with daily intake of S. oleracea extract may increase muscle-related factors and muscle quality. [171] Benefits of spinach, in a therapeutic diet for obese and insulin-resistant patients.
Fourteen normal-weight and ten obese men, 20 to 35 years, consumed three test meals of bread, as a control, bread and butter, and bread and butter with boiled spinach.
Green leafy vegetable intake with a fat-rich meal may effectively supply postprandial α-tocopherol in obese subjects. [172] Supplementation with thylakoid membranes from spinach, combined with a hypocaloric diet, effect on lipopolysaccharide (LPS) levels, neurotrophic factors, and oxidative stress in polycystic ovary syndrome (PCOS).
Forty-eight obese women diagnosed with PCOS, aged 20-45 years: thylakoid (n = 21) and placebo groups (n = 23).Supplementation with a thylakoid-rich spinach extract (5 g/day) or a placebo (5 g cornstarch.Duration: 12 weeks.Supplementation with thylakoid from spinach, combined with a hypocaloric diet, resulted in reduced LPS levels, increased neurotrophic factor levels, and improved glycaemic and sex hormone profiles in PCOS patients.Potential benefits of combining HIFT with spinach-derived thylakoid supplementation in improving adipokine profiles and insulin resistance in males with obesity over a 12-week period. [174] The impact of taking a single dosage of concentrated extract of thylakoids from spinach on satiety, food intake, lipids, and glucose.
Sixty overweight and obese individuals aged 18-65 years.They consumed the spinach extract or placebo.Blood was drawn for assessments of lipids and glucose before a breakfast meal, followed 4 h later by a 5 g dose of the extract and a standard lunch.Two hours after lunch a second blood draw was conducted.
Consuming the spinach extract significantly reduced hunger.Postprandial plasma glucose concentrations were increased following.There were no differences in plasma lipids and energy intake at dinner.[175] Carotenoid-rich leafy vegetables-spinach and perilla effect on carotenoid concentration and oxidative stress in human blood plasma.
Twelve volunteers, aged 19-44 years.Examination after a 10-day consumption of perilla or spinach, after the washout phase (10 days), and after the following 10-day consumption.Duration: 10 days.
Significant increases in lutein content were observed in human blood plasma after consumption of both spinach and perilla preparations.Both perilla and spinach preparations influence lipid peroxidation. [176] Calcium possibility to affect bioavailability of carotenoids from a vegetable matrix-spinach.
Calcium did not affect the bioavailability of carotenoids from a spinach-based meal.

Study Objectives Study Design Main Results
Ref.
To characterize tissue lutein and β-carotene concentrations in various spinach cultigens, and to determine serum carotenoid and macular pigment optical density (MPOD) responses in human subjects consuming spinach cultigens.
Consumption of low-lutein spinach led to a 22% increase in average serum lutein concentrations, while consumption of high-lutein spinach resulted in a 33% increase, and consuming high-lutein spinach demonstrated significant increases in MPOD. [178] The table presents randomized controlled trials investigating the effects of spinach and its derivatives.In a study on daily supplementation with S. oleracea extract, combined with moderate-intensity exercise, in over 50-year-old individuals, Perez-Pinero et.al. reported that spinach extract increases muscle-related factors and muscle quality [171].Due to these results, spinach can be included in the nutritional diets of athletes, combined with physical exercises.Bohlooli et.al. reported that in well-trained men, daily oral supplementation with spinach reduced indicators of oxidative stress and muscle injury after training [162].In a randomized control trial, Maruyama et.al. reported that the consumption of spinach in obese and insulin-resistant patients supplies antioxidants and improves lipid profiles in patients [172].Also, a potential therapeutic application of spinach-derived thylakoid supplementation might be obesity management [174].Moreover, the consumption of spinach extract significantly reduced hunger and increased postprandial plasma glucose concentrations, indicating a potential role in appetite regulation [175].The findings from these studies collectively underscore the diverse health-promoting effects of spinach and its derivatives, ranging from muscle health and oxidative stress reduction to metabolic regulation [162,[171][172][173][174][175][176][177][178].Furthermore, the consumption of spinach is not associated with adverse effects [162,[171][172][173][174][175][176][177][178].These effects and the safety profile of spinach could correspond with its use in sports nutrition.Further double-blind multicenter randomized controlled trials are required to confirm the good therapeutic profile of spinach.

Chenopodium quinoa
Quinoa (Chenopodium quinoa Willd., Chenopodiaceae) is an annual herbaceous plant that is classified as a gluten-free pseudo-cereal [179,180].Quinoa originated in the Andes of South America, specifically Peru, Bolivia, Ecuador, Colombia, and Chile [179,181].The consumption of quinoa dates back 5000 years [182].It grows to a height of 3-7 feet, the woody stem can be branched or unbranched, and it comes in different colors (green, red, and purple) [179,181].Quinoa seeds are small, spherical, and flat, with colors that range from white to grey, black, yellow, and red [182,183].
sumption of 50 g of quinoa for 6 weeks is safe [198,199].Apart from being rich in nutrients, quinoa also exhibits health-promoting properties, including anti-inflammatory, antidiabetic, antioxidant, anti-obesity, and cardio-beneficial effects (Figure 4) [35,[191][192][193].Moreover, not only the presence of phytoecdysteroids but also the presence of amino acids could increase muscle performance and lead to an increase in lean mass [200].Table 7 presents in vitro studies involving quinoa.

Study Objectives Cell Lines Main Results
Ref.
Quinoa and its polysaccharides effects on the gut microbiota.
In vitro, including three different stages: simulated oral, gastric, and small intestine digestion.
Quinoa and its polysaccharides improved microbiota composition and short-chain fatty acid synthesis.They are potential prebiotics. [201] The effect of C. quinoa leaves phenolic compounds on cancer cells.
The phenolic compounds may exert a chemopreventive and anticarcinogenic effect on oxidative stress.
[202] Quinoa seeds have been utilized as flour, added to soups, and incorporated into bread recipes.The rise of new food products featuring ancient grains, including quinoa, is observed globally, offering new opportunities for these nutritious grains in the market [196,197].Quinoa seeds are considered a "functional food"; it is considered that the consumption of 50 g of quinoa for 6 weeks is safe [198,199].Apart from being rich in nutrients, quinoa also exhibits health-promoting properties, including anti-inflammatory, antidiabetic, antioxidant, anti-obesity, and cardio-beneficial effects (Figure 4) [35,[191][192][193].Moreover, not only the presence of phytoecdysteroids but also the presence of amino acids could increase muscle performance and lead to an increase in lean mass [200].Table 7 presents in vitro studies involving quinoa.

Study Objectives Cell Lines Main Results
Ref.
Quinoa and its polysaccharides effects on the gut microbiota.
In vitro, including three different stages: simulated oral, gastric, and small intestine digestion.
Quinoa and its polysaccharides improved microbiota composition and short-chain fatty acid synthesis.
They are potential prebiotics. [201] The effect of C. quinoa leaves phenolic compounds on cancer cells.
The phenolic compounds may exert a chemopreventive and anticarcinogenic effect on oxidative stress. [202] The polyphenol composition and antioxidant properties of methanolic extracts from amaranth, quinoa, buckwheat, and wheat.
Antioxidant capacity is determined by using the radical DPPH scavenging capacity assay.
Total phenol content and antioxidant activity were shown to rise after sprouting, but levels decreased after breadmaking. [203] The presence and bioactivity of lunasin in quinoa.RAW 264.7 macrophage cells The lunasin isolated and purified from quinoa inhibited the production of nitric oxide, tumor necrosis factor-α, and interleukin-6.[204] Table 7. Cont.

Study Objectives Cell Lines Main Results
Ref.
Antioxidant, anti-diabetic, and immunoregulatory activities of the isolated polysaccharides from quinoa seeds.
Dose-dependent antioxidant and antidiabetic activities of the polysaccharides isolated from quinoa seeds, along with immunoregulatory activity.[205] The inhibition of collagenase activity of quinoa extracted ecdysteroids.
The potential of ecdysteroids from quinoa in modulating collagenase activity.[206] In vitro antiplatelet activity of quinoa and lupin bean extracts.
Six healthy young male volunteers (ages 20-30) participated in the study.10 mL venous blood samples were obtained for platelets.
Quinoa extracts did not exert an anti-aggregatory effect, whereas lupin extracts showed a significant effect. [207] The in vitro studies presented in Table 7 reported quinoa's effects on gut microbiota and cancer cells, antioxidant and antidiabetic properties, platelet activity, and modulating collagenase activity [201][202][203][204][205][206][207].Quinoa's polysaccharides enhanced the synthesis of short-chain fatty acids and the composition of the microbiota.They might function as prebiotics [201].Gawlik-Dziki et al. reported that quinoa possesses possible chemopreventive and anticarcinogenic qualities [202].Furthermore, quinoa's potential as a rich source of antioxidants is highlighted by the study by Alvarez-Jubete et.al. examining the polyphenol content and antioxidant qualities of methanolic extracts from the grain [203].Overall, these studies highlight quinoa's broad bioactive qualities and promise as a functional food.More studies, including in vivo, are required to explain the mechanism of action and the potential use of quinoa in DSs.Table 8 presents in vivo studies involving quinoa.

Study Objectives Study Design Main Results
Ref.
Effects of hydrolyzed quinoa.
Wistar rats (n = 64): control groups, treated group with hydrolyzed quinoa 2.000 mg/kg and treated and exercised group for 30 days.
Hydrolyzed quinoa decreased body weight, blood triacylglycerol level, and fat deposition. [200] The ability of quinoa extract enriched in 20-HE to prevent obesity in mice.
Quinoa possesses anti-obesity activity; similar effect was observed with ecdysterone.[208] Anxiolytic and antioxidant effects of PEs (incl.20-HE) and polyphenols from C. quinoa.
Sixty male Wistar rats.Three groups (n = 12): CTRL, IMM, and IMM-FFI.The CTRL and IMM groups were treated with a standard half-synthetic diet for 36 days.The third group, IMM-FFI, was treated with a standard diet with the addition of FFIs in the dose of 0.055 ± 0.003%.
The nutritional support from C. quinoa effectively normalized malondialdehyde and catecholamine levels under induced oxidative/nitrosative stress, while maintaining high superoxide dismutase activity, improving behavioral reactions and cognitive functions. [209] Table 8.Cont.

Study Objectives Study Design Main Results
Ref.
The optimized quinoa leachate, including 0.86% 20-HE, 1.00% total Pes, 2.59% flavonoid glycosides, 11.9% oil, and 20.4% protein, notably reduced fasting blood glucose levels in obese, hyperglycaemic mice. [210] The impact of chronic intake of quinoa extract and 20HE on the regulation of energy homeostasis in mice.
Both quinoa extract and 20HE promoted a higher rate of glucose oxidation.Quinoa extract possesses an anti-obesity effect. [211] Investigate the potential weight management effects of hydroalcoholic extracts from quinoa seeds and chia (Salvia hispanica L., Lamiaceae), and assess the hepatoprotective, anti-inflammatory, and antioxidant activities.

Chia and quinoa seed extracts
showed hepatoprotective, anti-inflammatory, and antioxidant activity in obese rats given a high-fat diet.These extracts also influenced important metabolic indicators such as leptin, adiponectin, serum lipids, and glycaemic levels. [212] Effect of quinoa on intestinal permeability and inflammation.
Quinoa has the ability to enhance intestinal permeability and promote compartment-specific protein uptake, potentially through mechanisms distinct from those of cholera toxin and capsaicin. [213] Polyphenol composition of red and yellow quinoa seeds, effect of antioxidant bioactivity of both raw and germinated quinoa seeds in vitro and effect against carbon tetrachloride (CCl4)-induced oxidative stress in rats.
Markers of oxidative stress were evaluated in Wistar rats, which were categorized into five groups, each comprising six rats.Group I received an intraperitoneal injection of fresh olive oil (1.0 mL/kg/twice per week) along with 0.5 mL of distilled water orally per day.
Group II received an intraperitoneal injection of a fresh mixture of CCl4 and olive oil (1.0 mL/kg/twice per week).
Group III was administered an intramuscular injection of vitamin E and selenium (at a dosage of 30 mg/kg/twice per week) in combination with CCl4.
Group IV was given an oral dose of 30 mg/kg gallic acid-yellow quinoa extract for 4 weeks concurrently with CCl4.Lastly, group V received 30 mg/kg gallic acid-red quinoa extract for 4 weeks along with CCl4.
Both red quinoa sprouts and yellow quinoa sprouts contain naturally synthesized polyphenols with superior antioxidant activity.The ethanolic extracts of quinoa sprouts demonstrated promising effects in counteracting oxidative stress. [214] The studies presented in the table correspond with the potential effects of quinoa extract.Foucault et.al. conducted a study on the ability of quinoa extract enriched in 20HE to prevent obesity, and it was observed that quinoa and 20HE demonstrate antiobesity activity in rats [208].Moreover, Foucault et.al. reported that quinoa extract and 20HE regulated glucose and lipid metabolism [211].Sidorova et.al. revealed that quinoa effectively normalized oxidative stress markers and improved cognitive function [209].Meneguetti et al. reported that hydrolyzed quinoa decreased body weight, blood triacyl-glycerol level, and fat deposition [200].Supplementation with quinoa and chia seed extracts may regulate metabolic indicators and showed hepatoprotective, anti-inflammatory, and antioxidant activities in rats [212].Quinoa extracts showed anti-obesity, antioxidant, hypoglycemic, immunoregulatory, and collagenase-modulating effects in in vivo experiments with rats [208][209][210][211][212][213][214].No serious adverse effects were reported within the experiments, which correlated with the safety profile of quinoa extracts and their safe nutrition in sports.No double-blind multicenter randomized controlled trials were found, so further trials are recommended to be conducted.

Turkesterone and Ajuga turkestanica
Turkesterone is a PE, with 27 carbon atoms and seven OH-groups; it is considered that the OH groups at C-20 and C-11 are responsible for the anabolic effects [28,215].Turkesterone is considered to possess an anabolic effect, and it is used as DS by athletes, instead of anabolic steroids [28,30].It is found in some endemic plants, such as A. turkestanica, R. carthamoides, Triticum aestivum L., Vitex scabra, etc. [13,69,[216][217][218].The highest content of turkesterone is considered to be in Ajuga turkestanica [19].Moreover, in DSs, turkesterone is derived from A. turkestanica [28].
Ajuga turkestanica, a plant species of the Lamiaceae family, grows wild in the Boysun mountainous region of Central Asia, Uzbekistan.It is a perennial plant, that normally grows on clay, petrous, and rubbly slopes and rocks, with a height of 40-60 cm [14].Both the aerial parts and root sections are utilized in folk medicine to prevent obesity, hair loss, and gastrointestinal diseases [14,216].
Extracts from A. turkestanica are associated with wound-healing effects [223], as well as antiproliferative, anti-stress and immunostimulating, antimicrobial, and antioxidant effects [12,59].Most of these effects are likely due to the presence of Pes [224].Table 9 presents studies about the biological effects of turkesterone.Ajuga turkestanica and turkesterone products are commercially available not only online but also in bodybuilding centers [12].Table 9 presents studies on the biological effects of turkesterone, while Table 10 presents studies on A. turkestanica.

Study Objectives Study Design Main Results
Ref.
Male mongrel white mice subjected to immobilization on their back for 6 h.Ecdysterone, turkesterone, and T-activin were administered intraperitoneally.
Ecdysterone and turkesterone prevented and facilitated the consequences of stress and restored immunological activity. [138] Table 9. Cont.

Study Objectives Study Design Main Results
Ref.
Turkesterone isolated from the roots of A. turkestanica and its anabolic effect.
Sexually immature male rats weighing 60-80 g.Turkesterone was administered orally at a dose of 5 mg/kg.For comparison, nerobol (methandrostenolone) was used in a dose of 10 mg/kg.
Turkesterone exhibited pronounced anabolic, leading to significant increases in body weight, comparable in anabolic effect to nerobol.It did not exhibit androgenic effects. [219] The impact of turkesterone on the endocrine and exocrine function in alloxan-induced diabetic rats.

Study Objectives Study Design Main Results
Ref.
Mechanism of action of Pes (20HE, polypodine B, and ponesterone) and A. turkestanica and S. oleracea in mammalian tissue, focusing on protein synthesis and physical performance.
A The isolated ecdysteroids and iridoid glucosides from A. turkestanica were found to lack antioxidant properties and did not exhibit significant cytotoxic or antimicrobial effects.Interestingly, the chloroform extract, containing more lipophilic compounds, demonstrated higher activity. [59] Extract from A. turkestanica or 20HE to sedentary aging mice would activate the key control point of protein synthesis.
Treatment did not alter body, muscle, or organ mass; fiber cross-sectional area; or fiber type in the triceps brachii or plantaris muscles.Phytoecdysteroid treatment does not alter muscle mass or fibre type. [77] Phytoecdysteroid enriched extract from A. turkestanica affects Notch and Wnt signaling in aged skeletal muscle.
Ajuga turkestanica extract supplementation in aged mice increases Notch and Wnt signaling in triceps brachii muscle.[226] Total ecdysteroid extract from A. turkestanica (including 22-acetylcyasterone, cyasterone, ecdysterone, and turkesterone) and to assess the hypoglycemic activity of this extract on the model of alloxan-induced hyperglycemia and diabetes.
White mongrel male rats.The hyperglycemia model was induced by alloxan (150 mg/kg, s.c.).The PE preparation was introduced once per day in a dose of 5 mg/kg over a period of seven days.

Ref.
Screen of A. turkestanica extract, rich in ecdysteroids for efficacy and safety.
The extract showed no androgenic activity within the dose range used.The potential for an A. turkestanica to support muscle mass without androgenic side effects. [29] The studies presented in the tables reported the various effects of turkesterone and A. turkestanica.Turkesterone is associated with a reduction in lipid accumulation in human adipocytes [69].In a stress-induced mouse model, ecdysterone and turkesterone were found to prevent stress-related consequences and restore immunological activity [138].Additionally, turkesterone exhibited beneficial effects on the endocrine and exocrine function in alloxan-induced diabetic rats, suggesting its potential therapeutic application in diabetes management [225].Furthermore, PEs from A. turkestanica demonstrate hypoglycemic activity in models of hyperglycemia and diabetes, suggesting their potential therapeutic use in managing blood glucose levels [227].Turkesterone presents a safer option to conventional anabolic steroids, displaying significant anabolic effects without inducing androgenic side effects in in vivo animal studies [19,219].Moreover, A. turkestanica also enhances muscle regeneration and maintenance [29,226].These results suggest that turkesterone extracted from A. turkestanica possesses diverse physiological effects, including potential performance enhancement and antiadipogenic, immunomodulatory, and hypoglycemic activities, making it a promising candidate for sports nutrition.Further research is needed to fully understand the mechanisms of action and therapeutic potential of turkesterone and A. turkestanica [19,29,59,69,77,138,219,220,[225][226][227].

Conclusions
Phytoecdysteroids, widely distributed across various plant species, have been valued for their adaptogenic properties and potential to enhance muscle strength and performance.20-hydroxyecdysone and turkesterone are two of the most noteworthy PEs, which are found in plants such as R. carthamoides, S. oleracea, C. quinoa, and A. turkestanica.Nowadays, these plants are used not just in plant-based diets for their high nutritional value but also as major components of DSs.Since 2020, ecdysterone has garnered attention and has been included in the WADA's monitoring program due to its ability to enhance physical endurance.Studies on the benefits of 20HE, turkesterone, and extracts from these plants are limited.The preparations from these plants show promise not only as nutritional supplements but also as therapeutic agents for treating medicinal disorders, such as chronic tiredness, heart disorders, and neurodegenerative diseases.Future double-blind randomized multicenter trials are critical for fully assessing the effectiveness and safety of these preparations for cardiovascular disease, chronic tiredness, obesity, and osteoporosis.These studies will provide valuable insights into the therapeutic potential of phytoecdysteroid-rich extracts.

[ 91 ]
Ability of 20HE to activate mTORC1 signaling in both skeletal muscle and liver tissues.Male Sprague-Dawley rats.Dose-response study: Rats were separated into groups (n = 5-6) and administered doses of 0, 10, 50, or 200 mg/kg of 20HE via gavage.Time-course study: Rats were separated into groups and given either 200 mg/kg of 20HE or an excipient.Combination study: Rats were separated into groups and given either an excipient, 200 mg/kg of 20HE, or 200 mg/kg of 20HE plus 1.35 g/kg L-leucine by gavage.20-hydroxyecdysone does not quickly activate mTORC1 signaling in muscle or liver.

Over 50 -
year-old adults (Caucasian men and postmenopausal women aged between 50 and 75 years).Daily intake of a spinach extract for 12 weeks and a 12-week physical exercise resistance training program on skeletal muscle.

[ 173 ]
High-intensity functional training(HIFT) combined with spinach-derived thylakoid supplementation effect on selected adipokines and insulin resistance in males with obesity.Sixty-eight participants, mean age: 27.6 ± 8.4 years, four groups (n = 17): control group, supplement group, training group, and training with supplement group.The two training groups started the 12 weeks of exercise training program.The eligible participants received 5 g/day of thylakoid-rich spinach extract or matching placebo as 5 g/day of raw corn starch.Duration: 12 weeks.

Table 4 .
Rhaponticum carthamoides in vivo studies and trials.