Additional file 1 of PRECIS-2 analysis of pragmatic acupuncture trials: a systematic review

Supplementary Material 1

Schematic representation of the effects of elderberry extract and the monosaccharide-amino acids derived from elderberry extract in the improvement of testosterone-deficiency syndrome.

EB: elderberry extract, FL: Fructose–leucine, E∙R: endoplasmic reticulum, ROS: reactive oxygen species.</p

Antibody information.

With aging, men develop testosterone-deficiency syndrome (TDS). The development is closely associated with age-related mitochondrial dysfunction of Leydig cell and oxidative stress-induced reactive oxygen species (ROS). Testosterone-replacement therapy (TRT) is used to improve the symptoms of TDS. However, due to its various side effects, research on functional ingredients derived from natural products that do not have side effects is urgently needed. In this study, using the mitochondrial dysfunction TM3 (mouse Leydig) cells, in which testosterone biosynthesis is reduced by H2O2, we evaluated the effects of elderberry extract and monosaccharide-amino acid (fructose–leucine; FL) on mRNA and protein levels related to steroidogenesis-related enzymes steroidogenic acute regulatory protein (StAR), cytochrome P450 11A1(CYP11A1, cytochrome P450 17A1(CYP17A1), cytochrome P450 19A1(CYP19A1, aromatase), 3β-hydroxysteroid dehydrogenase (3β-HSD), and 17β-hydroxysteroid dehydrogenase(17β-HSD). We analyzed elderberry extract and extract-derived FL for changes in ROS scavenging activity and testosterone secretion. Elderberry extract and FL significantly reduced H2O2-induced intracellular ROS levels, improved testosterone secretion, and increased the mRNA and protein expression levels of steroidogenesis-related enzymes (StAR, 3b-HSD, 17b-HSD, CYP11A1, CYp17A1). However, the conversion of testosterone to estradiol was inhibited by elderberry extract and extract-derived FL, which reduced the mRNA and protein expression of CYP19A1. In conclusion, elderberry extract and FL are predicted to have value as novel functional ingredients that may contribute to the prevention of TDS by ameliorating reduced steroidogenesis.</div

Effects of elderberry extract and elderberry extract-derived FL on inhibition of H₂O₂ treatment-induced reactive oxygen species (ROS) production and improvement of testosterone secretion in TM3 cells.

TM3 cells were treated with EB and FL combined with H2O2 (600 μM) for 24h and analyzed for intracellular ROS (A, B) and testosterone secretion (C, D). Data are expressed as mean ± SD (n = 3). #p ###p p p p 2O2 group, analyzed via one-way ANOVA. CTL: control, EB: elderberry extract, FL: fructose–leucine.</p

Effect of elderberry extract and extract-derived FL compound.

TM3 cells were treated with different concentrations of EB (A) and FL (B) for 24 and 48 h before an MTS assay was performed. Data are expressed as mean ± SD (n = 3). Student’s t-test was used to analyze differences between groups at *pp<0.01. EB; elderberry extract, FL; fructose–leucine.</p

Safety analysis of elderberry extract powder.

With aging, men develop testosterone-deficiency syndrome (TDS). The development is closely associated with age-related mitochondrial dysfunction of Leydig cell and oxidative stress-induced reactive oxygen species (ROS). Testosterone-replacement therapy (TRT) is used to improve the symptoms of TDS. However, due to its various side effects, research on functional ingredients derived from natural products that do not have side effects is urgently needed. In this study, using the mitochondrial dysfunction TM3 (mouse Leydig) cells, in which testosterone biosynthesis is reduced by H2O2, we evaluated the effects of elderberry extract and monosaccharide-amino acid (fructose–leucine; FL) on mRNA and protein levels related to steroidogenesis-related enzymes steroidogenic acute regulatory protein (StAR), cytochrome P450 11A1(CYP11A1, cytochrome P450 17A1(CYP17A1), cytochrome P450 19A1(CYP19A1, aromatase), 3β-hydroxysteroid dehydrogenase (3β-HSD), and 17β-hydroxysteroid dehydrogenase(17β-HSD). We analyzed elderberry extract and extract-derived FL for changes in ROS scavenging activity and testosterone secretion. Elderberry extract and FL significantly reduced H2O2-induced intracellular ROS levels, improved testosterone secretion, and increased the mRNA and protein expression levels of steroidogenesis-related enzymes (StAR, 3b-HSD, 17b-HSD, CYP11A1, CYp17A1). However, the conversion of testosterone to estradiol was inhibited by elderberry extract and extract-derived FL, which reduced the mRNA and protein expression of CYP19A1. In conclusion, elderberry extract and FL are predicted to have value as novel functional ingredients that may contribute to the prevention of TDS by ameliorating reduced steroidogenesis.</div

Effect of FL derived from elderberry extract on the expression of genes encoding enzymes involved in steroidogenesis in TM3 cells treated with H₂O₂.

The mRNA expression of these genes was analyzed after TM3 cells were treated with FL and H2O2 (600 μM) for 24 h. The expression was determined by quantitative real-time reverse-transcription polymerase chain reaction. Data are presented as mean ± SD (n = 3). Data are expressed as mean ± SD (n = 3). ##p ###p p p p 2O2 group, analyzed via one-way ANOVA. CTL: control, EB: elderberry extract, FL: fructose–leucine.</p

Primer sequences for quantitative reverse-transcription polymerase chain reaction (RT-PCR) analysis.

Primer sequences for quantitative reverse-transcription polymerase chain reaction (RT-PCR) analysis.</p

Effect of elderberry extract and elderberry extract-derived FL on the increase in protein expression of steroidogenesis-related enzymes in TM3 cells treated with H₂O₂.

The protein expression of the enzymes involved in steroidogenesis in TM3 cells treated with EB and FL combined with H2O2 (600 μM) for 24 h was determined by western blotting. Data are presented as mean ± SD (n = 3). Data are expressed as mean ± SD (n = 3). #p ###p p p p 2O2 group, analyzed via one-way ANOVA. CTL: control, EB: elderberry extract, FL: fructose–leucine.</p

Effect of elderberry extract on expression of genes encoding enzymes involved in steroidogenesis in TM3 cells treated with H₂O₂.

The mRNA expression of genes encoding enzymes involved in steroidogenesis was analyzed after these cells were treated with EB and H2O2 (600 μM) for 24 h. The expression was determined by quantitative real-time reverse -transcription polymerase chain reaction. Data are presented as mean ± SD (n = 3). Data are expressed as mean ± SD (n = 3). #p ##p ###p p p p 2O2 group, analyzed via one-way ANOVA. CTL: control, EB: elderberry extract, FL: fructose–leucine.</p