Main Research Findings

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

2) Brassinosteroids have the potential to enhance lean body mass and androgenic activity as well as significantly increase the amount of muscle fibers present.

 

Selected Data

1) 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 [2].

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 [2].

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 [2].

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.

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 [2].

2) As it was previously mentioned brassinosteroids are plant-derived polyhydroxylated derivatives of 5a-cholestane. The research team of Esposito et. Al examined how 28-Homobrassinolide (HB) has the potential to promote growth and protein synthesis while inhibiting protein degrading in L6 rat skeletal muscle cells. HB was initially purchased from Waterstone Technology. Phenylalanine was obtained from GE Healthcare while all reagents and enzymes used for quantitative PCR were collected from Stratagene and Applied Biosystems. Rat L6 skeletal muscle cell line CRL-1458 was obtained from the American Type Culture Collection.

In order to measure the rate of protein synthesis in cells treated with HB, differentiated myotubes were washed with serum-free DMEM and treated three times with either a vehicle composed of 0.1% ethanol, increasing concentrations of HB, or 6.5 nM of IGF-1 as a positive control. The experimental compounds were added to serum-free medium and incubated for 4 hours. Incubation was stopped by placing the cells on ice, discarding the medium, and washing the cells with PBS to remove the nonincorporated trace. The proteins were then precipitated with 5% trichloroacetic acid and dissolved in 0.5 N NaOH while specific radioactivity of protein-bound phenylalanine was quantified using the liquid scintillation counter. Samples were then normalized to milligrams of total protein and determined by BCA protein assay [3].

When investigating the effects of HB on protein degradation, the research team utilized differentiated myotubes, as previously described. The myotubes were incubated for 16 hours to allow labeling of cellular protein followed by washing with PBS to remove the nonincorporated trace. The cells were then treated for 4 hours with either a vehicle composed of 0.1% ethanol, increasing concentrations of HB, or 10 nM of insulin in serum-free medium. Incubation was stopped by placing the cells on ice, and 5% trichloroacetic acid was used to precipitate the protein in the medium.

All animal-based experiments were performed according to the procedures approved by the Rutgers Institutional Animal Care and Use Committee in an Association for Assessment and Accreditation of Laboratory Animal Care. The animals were housed in individual cages and maintained at a constant temperature and strict 12-hour light/12-hour dark schedule. The animals adapted for 7 days followed by random distribution into groups according to body weight prior to dosing [3].

The first protocol of the study included three groups of Wistar rats fed a normal diet containing 23.9% protein, 10.7% fat, 5.1% fiber, and 58.7% carbohydrates, resulting in 4.61 kcal/g energy value. The subjects were gavaged daily for 24 days with 1 ml of a vehicle composed of 5% DMSO in corn oil, or 20 or 60 mg/kg body weight of HB. Every 2 days the researchers recorded the body weight of each animal as well as the total amount of food consumed over the course of the experimental period. At the end of the experimental blood was collected via heart puncture after inhalation of CO2. Body composition of the test subjects was examined prior to necropsy; at necropsy the tissue weights were measured and recorded while tissue samples were collected by snap-freezing in liquid nitrogen for further examination. Protocol 2 included three groups of Wistar rats fed a high-protein diet containing 39.4% protein, 10.0% fat, 4.3% fiber, and 37.0% carbohydrates resulting in 3.93 kcal/g energy value. The subjects were gavaged daily for 24 days with 1 ml of a vehicle composed of 5% DMSO in corn oil, or 20 or 60 mg/kg body weight of HB. Further procedures and measurements followed the protocol 1 setup [3].

Protocol 3 included four-week-old sham-operated Wistar rats that were subjected to 10 day Hershberger assay. The experimental groups included: sham, ORX (vehicle), ORX (20 mg/kg of HB orally), ORX (60 mg/kg of HB orally), and ORX (0.4 mg/kg of testosterone propionate subcutaneously, acting as a positive control). Procedures and measurements followed the protocol 1 setup, however no body composition measurements were recorded. The control animals and the animals receiving 60 mg/kg of HB had limb grip strength measured using a digital force gauge. Three trials were performed on each animal and the significance of the results was determined using Student’s t test. Additionally, the gastrocnemius muscle, ventral prostate, seminal vesicles, bulbocavernosus/levator ani muscle complex, glans penis, and Cowper’s gland were extracted post-euthanasia and weighed. Finally, protocol 4 included four-week-old sham-operated or ORX Wistar rats that were subject to 10 day Hershberger assay. The experimental groups included sham, ORX (vehicle), ORX (0.4 mg/kg of HB subcutaneously), ORX (4 mg/kg of HB subcutaneously), and ORX (0.4 mg/kg of testosterone propionate subcutaneously to act as a positive control) [3].

Body composition of the test subjects was assessed through the use of DEXA scanning using Lunar Prodigy total body scanner that used Encore small animal body software. Quality control was performed on a daily basis and the coefficient of variation for total body, lean mass, and bone mineral content was measured in 5 rats, 3 times each. Furthermore, the muscle samples for the histochemical analysis were taken from the mixed-fiber gastrocnemius muscle. Serial transverse cryosections were prepared from each muscle and analyzed for myofibrillar adenosine triphosphate (mATPase) histochemistry, following alkaline preincubation. Fiber cross section area and enzyme activity levels were determined by examining digitized images of the muscle cross-sections.

 

Discussion

1) 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 [2].

Figure 1: 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 [2].

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 [2].

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

2) The results of the study conducted by Esposito et. Al initially reported that HB showed no toxicity to the fully differentiated L6 rat skeletal myotubes. In order to determine the effects of HB on protein synthesis, radiolabeled phenylalanine was incorporated and the response was assessed by the researchers. The cell samples were treated with different doses of HB for 4 hours; the lower concentration of 1 uM HB was shown to increase protein synthesis by 12.4 ± 2.3% above control levels. The response approached saturation when the cell samples were treated with concentrations of HB between 10 and 20 uM; protein synthesis increased by 34.9 ± 3.1 and 36.9 ± 2.9%, respectively. A 6.5 nM concentration of IGF-1 was used as a positive control in this portion of the study, resulting in a 42.5 ± 4.5% increase in protein synthesis. The cell samples treated with the higher concentrations of HB were shown to be less effective. A 1- to 24-hour study was performed next in order to investigate the time-dependent effects of HB on protein synthesis. The researchers observed a time-dependent increase in protein synthesis in response to HB. Protein synthesis induced by HB peaked at 3 hours and began to decrease at 4 hours; a similar kinetic pattern was seen in the cells treated with IGF-1 [3].

In terms of protein degradation, results of the study reported that IGF-1, used as a positive control in this study, has an anabolic effect on protein synthesis and an anticatabolic effect on protein degradation in muscle tissue, in a manner similar to insulin. Protein synthesis is more responsive to infusion with IGF-1 than to infusion with insulin, however, insulin is capable of inhibiting protein degradation. The researchers monitored degradation of the protein labels with phenylalanine through the release of acid-soluble radioactivity into the medium. When the cell samples were treated with concentrations of HB ranging from 0.3-20 uM, protein degradation was inhibited in a dose-dependent manner. Overall activity plateaued between 3 and 10 uM concentration of HB. A 1 uM concentration of HB was shown to decrease protein degradation by 8.2 ± 0.6% above control levels. At a higher concentration of 10 uM of HB, protein degradation was decreased by 9.5 ± 0.9% above control levels. 10 nM of insulin was used as a positive control and was shown to inhibit protein degradation by 13.0 ± 1.6%. A 1- to 4-hour study was performed next in order to investigate the time-dependent effects of HB on protein synthesis. The researchers observed a time-dependent increase in protein synthesis in response to both HB and insulin that reached a plateau after 3 hours [3].

Figure 3: Concentration- and time-dependent effects of HB on protein synthesis and degradation. A) cells incubated for 4 hours with phenylalanine, HB, or IGF-1, B) time-dependent effects of HB treatment on protein synthesis in cells treated with HB or IGF-1, C) dose-dependent effects on protein degradation, D) HB time-course study of protein degradation.

When examining the effects of HB on body composition in Wistar rats, the test subjects were administered daily doses of 20 and 60 mg/kg of HB (HB20 and HB60, respectively) over the course of 24 days. At the end of treatment the HB20 and HB60 treatment groups experienced an 18.3% and 26.6% respective increase in total body weight gain relative to initial body weight. When adjusted for body weight there were no significant changes in food intake among the treatment groups. This indicates that the increase in body weight in the HB groups was not caused by increased food intake. DEXA scans found that body composition of the test subjects reflected an increase in lean body mass; the HB20 and HB60 groups experienced a 7.0% and 14.2% increase in lean body mass as well as a 3.9% and 4.9% decrease in fat mass, respectively. These results allowed the researchers to conclude that the reported weight gain was due to a significant increase in lean body mass. Additionally, treatment with HB increased muscle mass of the gastrocnemius and slightly improved total body bone mineral content [3].

The anabolic effects of HB were examined in rats that were fed a high-protein diet rather than a standard diet. The diet of the test subjects consisted of approximately 40% protein, in addition to oral administration with 20 and 60 mg/kg of HB, daily for 24 days. In comparison, the control animals that were fed a high-protein diet ended up consuming less food and gaining less food than the control animals that were fed a standard diet. Furthermore, the high-protein diet was capable of enhancing the stimulatory effects of the 20 mg/kg dose of HB on body weight. However, there were no HB-related increases in food intake in either the control or experimental test subjects. Other than the recorded changes in body weight there were no additional changes in body composition or blood biochemistry that could be linked to HB treatment or a high-protein diet [3].

Figure 4: Effect of HB on body weight gain and food intake in rats fed (A,B) a normal diet or (C,D) a high-protein diet

The next portion of the study compared the ability of HB and testosterone propionate to restore androgen-dependent tissues after androgen deprivation was induced through surgical castration. Androgen deprivation led to a significant decrease in the size of the prostate, seminal vesicles, bulbocavernosus/levator ani muscle complex, glans penis, and Cowper’s gland; these organs decreased by 8.6, 6.5, 23.9, 54.6, and 40.5%, respectively, in comparison to the sham-operated animals. Testosterone propionate was injected in doses of 0.4 mg/kg resulting in a 3- to 8-fold increase in androgen sensitive tissues. However, testosterone propionate failed to restore the weight of the ventral prostate, seminal vesicles, and the bulbocavernosus/levator ani muscle complex.

In comparison to testosterone propionate, 10 days or oral administration with 20 and 60 mg/kg of HB did not prevent the loss of androgen sensitive tissue that was related to surgical castration. However, there was a dose-dependent increase in the glans penis following HB treatment. HB also increased the weight of the bulbocavernosus/levator ani muscle complex that is typically used as the skeletal muscle biomarker of anabolic activity. The two previously mentioned organs were the only androgen-sensitive tissues that displayed a significant change in weight in comparison to the control animals [3].

 

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] 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.

[3] Esposito D, Komarnytsky S, Shapses S, Raskin I. Anabolic effect of plant brassinosteroid. FASEB J. 2011 Oct;25(10):3708-19. doi: 10.1096/fj.11-181271. Epub 2011 Jul 11. PMID: 21746867; PMCID: PMC3177571.

 

5-Alpha-Hydroxy Laxogenin is sold for laboratory research use only. Terms of sale apply. Not for human consumption, nor medical, veterinary, or household uses. Please familiarize yourself with our Terms & Conditions prior to ordering.