Testing the anabolic activity, potency and mechanisms of action of ecdysterone and diosgenin combinations

Code: 21C19EI

Our research hypothesis is that combined intake of diosgenin and ecdysterone is an effective strategy to induce anabolic responses resulting in the enhancement of performance, in particular strength and speed. Therefore, we want to test the dose-dependent anabolic activity of different combinations of ecdysterone and diosgenin in our well-established in vitro test system for anabolic activity. Diosgenin and ecdysterone will be tested dose-dependently as single compounds and in combinations in comparison to other anabolic substances (testosterone, estradiol and IGF1). 

Mechanistic studies will provide more evidence which molecular pathways are involved in its anabolic activity. Comparison with the recently characterised plant-derived anabolic substance ecdysterone in our in vitro test system will provide first information regarding its potential anabolic potency if abused orally in athletes. Ecdysterone was first identified to be anabolic in this test system, which was later confirmed in a human training study [10]. Taken together, the results of our investigations will be part in the pharmacological characterisation of these substances. This knowledge will be helpful to verify and justify the already existing limits of the combinations of ecdysterone and diosgenin for positive testing, to get additional information about side effects and to improve already existing doping tests for these substances. 

Study 1: Binding affinity of ecdysterone and diosgenin combination to the androgen and estrogen receptor.
In our yeast transactivation system, we will examine the binding affinity of ecdysterone and diosgenin combination to the androgen and estrogen receptor in comparison to testosterone, ecdysterone and diosgenin separately. We will determine the respective Michaelis constant (MC) of the combined substances.

Study 2: Anabolic potency of ecdysterone and diosgenin combination
In our C2C12 cell culture system, we will examine dose-dependent anabolic effects of ecdysterone and diosgenin combination in comparison to testosterone, IGF1 and ecdysterone and diosgenin. This could allow to estimate the anabolic potency of a combination of phytosteroids in comparison to the other anabolic substances.

Study 3: Mechanisms of action
In coincubation experiments with anti-estrogens, antiandrogens, glucocorticoids and antagonists to the IGF-1 receptor (like Losartan), we will determine signal transduction pathways mainly involved in the anabolic activity of this combinations. 

Main Findings

For decades, plant-derived phytosteroids like ecdysterone (Ecdy) have been used to enhance physical performance, especially in terms of strength, due to their anabolic properties. Recent findings indicate that Ecdy is increasingly being used in combination with other phytosteroids such as diosgenin (Dio). However, there is limited data available on the combinatorial effects of phytosteroids. Our study aimed to investigate the anabolic effects and underlying molecular mechanisms of various combinations of Dio and Ecdy in C2C12 myotubes in a dose-dependent manner.

In differentiated C2C12 cells, dose-dependent effects of Dio and Ecdy and combinations (ranging from 2 µM to 0.1 nM) on myotube size were investigated. In addition, the mRNA expression of genes associated with muscle hypertrophy, such as IGF-1 and PIK3R1, was analyzed by RT PCR. To delve into the molecular mechanisms, we co-incubated Dio with inhibitors of the estrogen receptor (ER) and androgen receptor (AR), along with dexamethasone (DEX), and evaluated myotube diameter. Moreover, the interaction of Dio with the AR and the ER was examined in a yeast transactivation assay.

Different combinations of Dio and Ecdy had positive effects on the myotube diameter. Combinations of Dio and Ecdy also affected mRNA expression of IGF-1 and PIK3R1. Contrary to the ER inhibitor ZK 191703, the AR inhibitor Flutamide, and DEX treatment effectively counteracted Dio induced myotube diameter growth. Dio did not exhibit agonistic activity in the AR and ER yeast transactivation assays, but it displayed antagonistic effects on the AR rather than the ER. The full mechanism affecting myotube diameter needs to be further elucidated.

Our in vitro findings provide evidence that combinations of Dio and Ecdy display anabolic activity in C2C12 cells in an additive manner. Dio has anti-androgenic activity in the yeast cell model, while Ecdy has anti-oestrogenic activity. Moreover, our data suggest that activation of the PI3K/Akt/mTOR pathway is essential for Dio effects on myotubule diameter. Considering that these results were obtained in vitro in myoblast cells (C2C12) from rodents, future studies are needed to investigate these effects in human training interventions to conclude on any anabolic effect in human.