Anoectochilus burmannicus Extract Rescues Aging-Related Phenotypes in Drosophila Susceptible to Oxidative Stress-Induced Senescence

Abstract

Aging is a significant risk factor for various conditions, including neurodegeneration, cardiovascular disease, and type 2 diabetes. The accumulation of reactive oxygen species (ROS) and a decline in antioxidant defense are mechanisms that are widely acknowledged as causing the acceleration of both aging and the onset of age-related diseases. To promote longevity and reduce the risk of the development of aging-related disorders, it is essential to prevent or minimize oxidative stress and enhance antioxidant defense. It has been shown that Anoectochilus burmannicus (AB), a jewel orchid rich in phenolic compounds, can impact various biological activities associated with aging prevention. These activities include antioxidant, anti-inflammation, anti-insulin resistance, and anti-obesity effects. The aim of this study was to explore whether AB extract (ABE) could serve as an anti-aging agent using a Sod1-deficient Drosophila model, which accelerates the process of aging through ROS production. The results demonstrated that ABE, at a concentration of 2.5 mg/mL, significantly extended the lifespan of the flies and helped maintain their locomotor activity as they aged. ABE also reduced the age-related accumulation of damaged proteins in the muscle of the flies by inhibiting the expression of Gstd1, a genetic marker for oxidative stress. This finding agrees with those from in vitro experiments, which have shown the potential for ABE to reduce the production of ROS induced by H₂O₂ in myoblasts. ABE has been shown to attenuate insulin resistance, an age-related disorder, by inhibiting the pro-inflammatory cytokine TNF-α, which in turn increased insulin-stimulated glucose uptake in adipocytes. These findings suggest a promising role of ABE as an ingredient in functional foods or nutraceuticals aimed at promoting health, preventing oxidative stress, and potentially managing age-associated diseases.

Keywords: age-related disorder; functional food; locomotor activity; longevity promotion; medicinal plants; orchid; oxidative stress.

Figure 1

The effect of ABE consumption on adult life span (A) and locomotor activity (B) in a Drosophila aging-accelerated model. (A) Survival curve plotted by Kaplan–Meier survival analysis of Sod1ⁿ¹ mutant flies fed with or without ABE (control, n = 103; 0.5 mg/mL ABE, n = 114; 2.5 mg/mL ABE, n = 106). ** p < 0.05, *** p < 0.001 compared to control, Log-rank test. (B) The effect of ABE (2.5 mg/mL) on locomotor activity of muscle-specific Sod1 depletion in Drosophila adults. The climbing score over time is shown as relative to the day 5 value. Data are represented as mean ± SD (n = 100). One-way ANOVA with Tukey’s multiple comparisons test, *** p < 0.001 compared to day 5 value (control group), ## p < 0.05, ### p < 0.001 compared to day 5 value (ABE group). $$ p < 0.05, $$$ p < 0.001 control group compared to ABE-treated group.

Figure 2

The effect of ABE on protein aggregation in indirect flight muscle (IFM) (A,B) and expression of the oxidative response gene, GstD1, (C) in Drosophila adults. (A) Immunostaining of the indirect flight muscles from the model flies with a muscle-specific depletion of Sod1 (Mef2 > Sod1RNAi) using an anti-ubiquitin-conjugated antibody (green) and phalloidin staining for F-actin (red). (A) Flies were fed with a control diet or with an ABE-supplemented diet (ABE 2.5 mg/mL) for 12 days after eclosion. (B) The average number of protein aggregates containing polyubiquitinated proteins in each confocal optic field. The area of ubiquitinated protein aggregates (green) was analyzed using ImageJ software (version 1.54g) and converted into the number of protein aggregates as a unit/4 × 10⁻² mm². Circles and squares represent the number of protein aggregates in each muscle of the control group (n = 25) and the ABE feeding group (n = 25), respectively. (C) Quantitation of mRNA of GstD1, a marker gene used to monitor the oxidative stress accumulation in the flies (Mef2 > Sod1RNAi) fed with control diet or ABE-containing diet. Data are represented as mean ± SD from two independent experiments (n = 10, total n = 20). Student’s t-test ** p < 0.01.

Figure 3

Cytotoxicity of ABE on (A) C2C12 myoblasts and (B) mature 3T3-L1 adipocytes. The cells were treated with various doses of ABE (12.5–200 µg/mL) for 48 h. Cell viability was determined by sulforhodamine B (SRB) assay. The data are shown as mean ± SD of three independent experiments.

Figure 4

The effect of ABE against H₂O₂-generated intracellular ROS in C2C12 myoblasts. (A) Fluorescent microscopy images of the cells, a bright-field image showing the general morphology of the cells (left panel), a fluorescent image showing the DCF fluorescence (middle panel), and an overlay image combining bright-field and fluorescent images to illustrate the spatial relationship between cellular structures and fluorescent signals (right panel). (B) Relative DCF fluorescence pixel intensity as analyzed by the Zeiss ZEN Pro software (version 3.9). Data are represented as mean ± SD of triplicate experiments and analyzed using a one-way ANOVA with Tukey’s multiple comparisons test. *** p < 0.001 compared to H₂O₂-treated controls.

Figure 5

The effect of ABE on insulin-induced cellular glucose uptake in TNF-α-treated 3T3-L1 adipocytes. (A) Fluorescent microscopic images of the cell, the bright-field image showing the general morphology of the cells (left panel), a fluorescent image showing the 2-NBDG fluorescence (middle panel), and overlay image combining bright-field and fluorescent images to illustrate the spatial relationship between cellular structures and fluorescent signals (right panel). (B) Cellular uptake of 2-NBDG measured by a fluorescence microplate reader. Data presented as mean ± SD of triplicate experiments and analyzed with a one-way ANOVA with Tukey’s multiple comparisons test. ** p < 0.01 compared to untreated controls, # p <0.05 ### p < 0.001 compared to TNF-α-treated controls.