Main Research Findings

1) Results collected from in vitro bioassays emphasize the ability of 5-alpha-hydroxy-laxogenin to enhance androgenic potential and anabolic activity.

2) Application of laxogenin to tomato plants was shown to promote the germination of seeds as well as seedling growth in a dose-dependent manner

 

Selected Data

1) The research team of Beer et. Al examined the androgenic properties of 5-alpha-hydroxy-laxogenin through two in vitro bioassays: a yeast androgen screen and a reporter gene assay in a human prostate cell line. Dihydrotestosterone, bicalutamide, and hydroxyflutamide were all provided by Sigma-Aldrich in Munich Germany while 5-alpha-hydroxy-laxogenin was purchased from Biomol, located in Hamburg, Germany. DImethyl sulfoxide (DMSO) was provided by Carl Roth; all test compounds were dissolved in DMSO. DMEM, FBS, and penicillin/streptomycin were supplied by BioWest and luciferase assay systems were obtained by Promega. Human PC3(AR2) cells as well as the reporter plasmid encoding the luciferase gene in the mouse mammary tumor virus, were provided by Dr. Aria Baniahmad [2].

The first in vitro study performed was a yeast androgen screen that was performed using a Saccharomyces cerevisiae strain that was previously transfected with a human androgen receptor (AR) construct as well as a reporter plasmid carrying the beta-galactosidase encoding LacZ under control of androgen responsive elements. The yeast cells used in the study were treated with either DMSO as a solvent control, DHT as a positive control, or dilutions of 5-alpha-hydroxy-laxogenin. In order to examine antagonistic properties, 5-alpha-hydroxy-laxogenin was co-incubated with flutamide with beta-galactosidase activity was measured through the hydrolysis of chloro-phenol red-beta-D-galactopyranoside. All experimental treatments were performed independent from each other and Student’s t test was used to determine statistical significance.

Human PC3(AR)2 cells were maintained in DMEM/F12 and supplemented with 1% penicillin/streptomycin, 0.25 mg/mL G418, and 10% FBS. The cells were then cultivated in DMEM/F12 containing 1% penicillin/streptomycin, and 5% dextran-coated charcoal-treated FBS. 70,000 cells were seeded in a 24-well plate and transfected with the reporter plasmid mmTV-luc. PC3(AR)2 cells were treated with 0.1% DMSA as a solvent control, DHT as a positive control, or 5-alpha-hydroxy-laxogenin. The cells were then co-incubated with bicalutamide and 5-alpha-hydroxy-laxogenin in order to examine the antagonistic effects of the experimental compounds. Luciferase activity and quantified protein concentration was measured using the bicinchoninic acid assay with BSA as standard protein. Additionally, Relative Luminescence Units (RLU) were calculated by normalizing the luminescence with the protein concentrations. Three independent cell culture experiments were performed and one-way ANOVA followed by Bonferroni’s post hoc test were used to assess statistical significance for this portion of the study [2].

2) The research team of Deng et. Al evaluated how laxogenin affects metabolics mechanisms and lignin response in tomato plants. The study began by isolating and purifying laxogenin, followed by the extraction of Smilax scobinicaulis from dried powder through the use of 75% ethanol at reflux for 8 hours. The extract was concentrated under reduced pressure in order to yield a crude residue extracted from EtOAc and n-BuOH. Additionally, the n-butanol extracted fraction was separated on macroporous adsorbent resin, silica gel column, and purified on RP-18 column chromatography. The chemical structures of the resulting compounds were identified by comparing NMR and high-resolution MS data [3].

The next portion of the study included the placing of tomato seed into 10% H2O2 for 5 minutes, followed by washing in sterilized distilled water. The seeds were then put into clear water and doses of laxogenin varying from 1, 5, 10, 50, and 100 ug/L, and soaked 24 hours without light exposure. After washing the seeds in distilled water, 20 seeds of each treatment were sown in a petri dish, covered with filter paper, and transferred in a constant temperature incubator. All experiments were completed three times and the number of germinated seeds was counted daily. The seeds with at least 2.0 mm of radicle were considered to be germinated; germination percentage, energy, and index were calculated by the formula below:

Root and plant length as well as fresh and dry weight of the tomato seedlings were measured and recorded for 5 days after culturing. All seedlings were scanned on an Epson scanner while ImageJ software was used to quantify root and seedling length. All averages and deviations of germination energy and index and the plant weight were counted on the germinated seedlings in each culture dish [3].

Three groups were made by the research team: G1 was set as a blank group, G2 was the group treated with 5 ug/L of laxogenin, and the G3 group received 100 ug/L of laxogenin. The groups were developed to investigate the variation of tomato metabolites and each group had six biological replicates, each of which were collected with 1.0 grams of fresh seedlings following 5 days of culturing. The collections were frozen and ground into powder while the resulting samples were extracted with 5 mL MeCN and sonicated for 30 minutes. This was followed by 10 minutes of centrifugation; the supernatant was then filtered through a 0.22 um filter and stored prior to LC-MS analysis. 5 uL of each same was then injected on a UPLC-q-ESIMS system for analytical purposes. All metabolites were separated on a C18 column and eluted with MeCN over the course of 18 minutes. The full MS was scanned in positive mode and all MS/MS data was collected using the SWATH method [3].

In terms of data analysis and visualization, procedures began by converting the LC-MS WIFF raw data filed to ABF format. The ABF files were then imported into MS-Dial to explore the MS data which was then aligned and normalized according to the TIC and the internal standard, 6-bromo-4-hydroxycoumarin. Next the MS-DIAL metabolomics MSP Spectral kit was used for metabolite annotations, however, too many unrelated compounds, such as synthetic compounds and plasma components, identified within the MS-DIAL MSP database. That being said, the research team cleaned MSP data to exclude any compounds not included in the KEGG database in order to avoid the apparent wrong annotation of synthetic products. The final step of this portion of the study included LC-MS data analysis and visualization, performed according to previous methods. This procedure was followed by the exportation of the alignment data as a CSV file for further visualization purposes [3].

In order to verify the effect of laxogenin on tomato lignin, the research team cultured tomatoes and determined lignin content through the use of UV spectrophotometry. 0.5 grams of the fresh sample was ground into a homogenate using 95% ethanol and a 1:1 (v/v) of EtOH and n-hexane. The next step included the extraction of the dried plant material with 0.5 mL of 25% bromoacetyl in AcOH for 30 minutes. The reaction was terminated by adding 0.9 mL NaOH; this step was followed by the addition of 5 mL AcOH and 0.1 mL hydroxylamine. The mixture was then centrifuged for 5 minutes and an additional 3.0 mL of AcOh was added while the lignin concentration was quantified by the absorption at 280 nm.

 

Discussion

1) The study conducted by Beer et. Al performed two in vitro assays in order to examine the androgenic properties of 5-alpha-hydroxy-laxogenin. The SARM was shown to be able to trans-activate the androgen receptors in human prostate cells in a dose-dependent manner. Additionally, a biphasic response was observed with antagonist properties at low concentrations and agonist properties at high concentrations. The androgenic properties of the 5-alpha-hydroxy-laxogenin suggest that further research should be conducted regarding the anabolic properties of the compound [2].

The first in vitro examination that was performed was a yeast androgen screen that used a Saccharomyces cerevisiae strain transfected with a human AR construct and reporter plasmid carrying the beta-galactosidase encoding the LacZ gene. The results of the yeast androgen screen reported that the addition of DHT stimulated the reporter gene expression in a dose-dependent manner. However, the reporter gene beta-galactosidase was not activated by any of the concentrations of 5-alpha-hydroxy-laxogenin administered to the cells.

Figure 1: Androgenic dose response in the yeast androgen screen A) DHT as a positive control, B) 5-alpha-hydroxy-laxogenin

On the other hand, 5-alpha-hydroxy-laxogenin was able to induce luciferase expression in human PC3(AR)2 cells in a dose-dependent manner. The SARM elicited biphasic effects with lower doses acting as an antagonist and higher doses acting as an agonist. Bicalutamide, a non-steroidal AR antagonist, was co-incubated with 5-alpha-hydroxy-laxogenin resulting in the antagonism of luciferase activity. These results suggest that 5-alpha-hydroxy-laxogenin has the potential to bind to human AR and acts as an agonist in the PC3(AR)2 cells. The research team hypothesized that the discrepancy between the results of the two in vitro examinations could potentially be due to the different cofactor pattern in yeast and mammalian cells, as well as the additional yeast cell wall. That being said, the researchers thought it was important to note that false negative results of the yeast androgen screen are possible [2].

Figure 2: Androgen receptor transactivation in human PC3(AR)2 cells

2) Results of the study conducted by Deng et. Al examining the regulation of tomato seed germination and seedling growth reported that germination energy peaked when laxogenin was provided in concentrations of 5 ug/L. When laxogenin was administered in concentrations of 10 ug/L, seed germination was effectively enhanced. Additionally, the study found that the germination percentage of tomato seed demonstrated that low concentrations (1-10 ug/L) caused early seed germination as well as delayed seed germination as the concentration increased. The growth of the plants was evaluated by root length, plant length, and fresh and dry weight of the seedlings. Again, growth was shown to peak at a concentration of 10 ug/L before decreasing again [3].

Figure 3: Effects of laxogenin on A) germination energy, B) germination index, C) seedling root length D) plant length, E) fresh weight, and F) dry weight.

Three groups, G1, G2, and G3, were developed in order to observe changes in metabolites in response to increasing concentrations of laxogenin. All three groups detected a total of 33 ions and metabolites, followed by analysis of the effects of laxogenin on the overall metabolome of tomato seedlings. The PCA diagram developed by the research team indicated that laxogenin only had a minor effect on the overall metabolome with a difference of 24% in both the vertical and horizontal directions. Through the use of the sPLS-DA plot, the three groups were shown to occupy relatively independent spaces.

The metabolites were identified based on MS m/z and MS/MS spectra, the entries included in this portion of the study were washed and limited specifically to KEGG compounds that have important physiological roles. The differential metabolites (DMs) were found by comparing groups G2 vs. G1, G3 vs. G2, and G3 vs. G1. A total of 10 DMs were identified and out of the 10, two metabolites, L-phenylalanine and L-tryptophan were associated with the comparisons between G2 vs. G1 and G3 vs. G2, respectively. 3 more metabolites, nicotinamide, L-pyroglutamic acid, and glutamine, were related to the group comparisons between G2 vs. G1 and G3 vs. G1 [3].

Seven out of the ten identified metabolites were amino acids. When the G2 group was compared to G1, the most significant changes in expression were observed in phenylalanine, nicotinamide, tryptophan, and trans-2-hydroxycinnamate. These compounds had down-regulated more than 2-fold. When the G3 groups were compared to G2 expression levels of phenylalanine, valine, tryptophan, and isoleucine all significantly increased. The expression levels of L-phenylalanine increased the most when comparing groups G2 and G3, however, the expression of L-phenylalanine was also the most significantly decreased when comparing groups G3 and G1.

One of the most significant metabolites identified in the previously mentioned metabolic pathways was lignin, the final product of phenylpropanoid metabolism and a compound that plays a crucial role in plant growth.. Results of the study reported that expression of lignin significantly decreased in the G2 group but increased in the G3 group as the concentration of laxogenin increased. This same pattern was seen with the expression of phenylalanine, ferulate, and 2-hydroxycinnamate evaluated through metabolome analysis. The metabolomic analysis suggested that phenylalanine and ferulate were related to the phenylpropanoid metabolism, a component directly affected by the mediation of laxogenin [3].

The results reported by the research team indicate that high doses of laxogenin up-regulate phenylalanine metabolism as well as the phenylpropanoid pathway. In combination with increased expression of lignin the cell wall will solidify and root growth is inhibited. However, low concentrations of laxogenin were shown to down-regulate the metabolism of phenylalanine as well as the synthesis pathway of the lignan precursor, phenylpropanoid, that is capable of inhibiting the expression of lignans and promoting plant growth. Additionally, an overexpression of phenylalanine has been shown to promote plant growth by affecting the lignin content. This allowed the researchers to conclude that laxogenin can control lignin synthesis through the regulation of

Figure 4: expression of lignin, phenylalanine, 2-hydroxycinnamate, and ferulate, regulated by laxogenin

 

Disclaimer

**LAB USE ONLY**
*This information is for educational purposes only and does not constitute medical advice. THE PRODUCTS DESCRIBED HEREIN ARE FOR RESEARCH USE ONLY. All clinical research must be conducted with oversight from the appropriate Institutional Review Board (IRB). All preclinical research must be conducted with oversight from the appropriate Institutional Animal Care and Use Committee (IACUC) following the guidelines of the Animal Welfare Act (AWA).

 

Citations

[1] Tang, J., Han, Z. & Chai, J. Q&A: what are brassinosteroids and how do they act in plants?. BMC Biol 14, 113 (2016). https://doi.org/10.1186/s12915-016-0340-8

[2] Beer C, Keiler AM. Androgenic properties of the dietary supplement 5α-hydroxy-laxogenin. Arch Toxicol. 2022 Jul;96(7):2139-2142. doi: 10.1007/s00204-022-03283-5. Epub 2022 Mar 28. PMID: 35344071; PMCID: PMC9151512.

[3] Deng Y, Wang J, Zhang A, Zhu Z, Ren S, Zhang C, Zhang Q. Metabolomics Mechanism and Lignin Response to Laxogenin C, a Natural Regulator of Plants Growth. Int J Mol Sci. 2022 Mar 10;23(6):2990. doi: 10.3390/ijms23062990. PMID: 35328410; PMCID: PMC8951225.

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