Nematode growth medium (NGM) plates were used to maintained the Bristol N2 (wild type) strains of the worms with Escherichia coli strain OP50 as a food sources, at 20°C±1.5ºC 27.

Wheat (Triticum, Family: Poaceae) HI1605 variety seeds were procured by ICAR-Indian Agricultural Research Institute, Regional station, Indore. Prior to sowing, the seeds were soaked in clean water for several hours. We used small plastic container with many holes at the bottom side. The soaked seeds are sprinkled densely over growing media i.e. coco peat of about 2.5 inches. Then again, a thin layer of growing media is covered over the seed layer. Now the prepared tray is sprinkled over with water. Microgreen was grown in plant culture room at the temperature of 24-27ºC with relative 70% humidity and alternate day watering. We have not used any sort of chemical such as pesticides, fungicide or fertilizers as it is an organic nutrient rich food so any nutrient should not be compromised. Wheat microgreen were harvested after 7 days of sowing using a sharp pair of scissors and then washed under running water 28 (Figure 1).

Freshly harvested wheat microgreens were used to prepare extract. For that, 3 grams of microgreen were crushed in motor pastel then chloroform and methanol were added in 1:1 ratio. The crushed leaves were further homogenized and sonicated to ensure that majority of the cells breaks and releases most of their phytocompounds. The samples were centrifuged at 4000 rpm for 10 min. The supernatant was collected in separate tube and lyophilized. Lyophilized samples were dissolved in DMSO to prepare a stock concentration of 20 mg/ml of WME.

L1 stage worms were exposed to the different concentrations (2, 4, 6, 8 and 10 µM) of rotenone every day for 48 h and observed under stereomicroscope for survival and growth.

In order to choose concentrations for microgreen extract on C. elegans, we have taken 2 parameters in consideration 1st visual observation and 2nd behavior assay. We have exposed worms to the 0.5, 1 and 2 mg/ml concentration of microgreen extract along with the 4 µM of rotenone for 48 h and then observe worms under the stereomicroscope for clear vision and behavior changes.

Behavior assays were performed as reported in 29. Briefly, after 48 h treatment worms were washed, harvested and transferred individually on an unseeded NGM plate. After 1 min recovery period individual worm was manually scored under the microscope, 1 min for head trashes and 20 sec for body bends. Change in the direction of bending at the midbody is defined as head thrash that is useful to observe the effect of compounds on motor neuron. A body bend is referred as a change in the direction of the part of the nematodes corresponding to the posterior bulb of the pharynx along the Y-axis, assuming that the nematode was traveling along the X-axis. Both the assays have been performed in triplicate with 30 replicates (Randomly selected) for each group.

Level of ROS was determined as reported in 30. Briefly, after the treatment worms were washed with 1XPBS and about 1000 worms from each group were separately transferred into the each well of black 96-well plate with transparent bottom in triplicates. 2',7'-dichlorodihydrofluorescein diacetate (H2DCFDA) (Sigma, U.S.A) dye (0.05 mM working concentration) was added individually and incubated for 30 min on an orbital shaker at 20ºC. Inside the cell nonfluorescent dye oxidized into highly fluorescent 2′,7′-dichlorofluorescein and was measured at 485/535 nm in Spectrophotometer Spectramax (Molecular Devices, UK).

Tetrazolium MTT dye [3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide] is used to assess cellular metabolic activity. MTT is a cell-membrane permeable dye and intracellularly reduced by NAD(P)H-dependent cellular oxido-reductase enzyme to an insoluble purple colored formazan product. This product is soluble in organic solvent and thus, measured calorimetrically. Hermaphrodite worms (~1000 worms) in triplicate were transferred to each well of transparent 96-well microtitre plate along with 50 µl of MTT (10 mg/ml) dye and incubated at 20°C for 3 h. the plate was centrifuged at 2000 rpm for 10 min, and the supernatant was aspirated. Formazan formed in worms was solubilized in 100 μl DMSO and measured absorbance at 595 nm in Spectrophotometer (Spectramax, Molecular Devices, UK). MTT gives a measure of viable cells and is shown in results section as fold change in comparison to control.

A nonanol repulsive assay is an established and indirect method to estimation of dopamine level in C. elegans. Level of dopamine is related to the recognition, motivation, memory, adaptation and motor activity in worms. Nonanol assay has been performed as reported in 31. Briefly, worm after the treatment were washed 3 times with 1X PBS buffer and left tabletop to settle down. A drop of worm suspension was placed at 90 mm NGM plates and let it dry. An eye lash attached with a toothpick dipped in 1- nonanol (Sigma Aldrich; 131210; 0.01% 1-nonanol dissolved in DMSO) and was placed near the head of worm. Time taken by worm to exhibit the repulsive behavior was calculated using a stopwatch.

We observed delayed development in the worms those were exposed to the 6, 8 and 10 µM of rotenone (Figure 2). Thus, these concentrations were excluded since these are very high concentration to analyze the rotenone induced neurodegeneration in C. elegans. 4 µM concentration of rotenone was chosen to work with which was also supported by literature 32, 33.

We found that 0.5 mg/ml microgreen extract were not able to recover from rotenone (4 µM) induced significant changes in the behavior of worms compared to control (data not shown). While plates seeded with 2 mg/ml microgreen extract were more greenish and harder to observe in microscope. Worms exposed to the 1 mg/ml microgreen extract along with rotenone were easy to observe in the stereomicroscope as well as ameliorate rotenone induce behaviors changes (Figure 3). So, we have chosen 1 mg/ml of microgreens concentration to work further.

It was observed that rotenone induces significant (p<0.05) change in the body bend (Mean value 6.4±0.3) and head trashes (Mean value 60±2.5) behavior of C. elegans at 4 µM concentration (Figure 4 a, b). While, when WME was exposed along with the rotenone treatment worms were observed to have normal body bend and head trashes behaviour with the mean value 18.25±0.35 and 143.35±0.85 respectively, comparable to control (Mean value 17.4±0.2 and 135.85±1.15) (Figure 4 a, b). So, wheat microgreens extract can ameliorate rotenone induced behavior changes in worm. 61-68% recovery was observed in behaviour of WME+rotenone treated worms compared to rotenone exposed worms.

ROS production in organisms exposed to rotenone significantly increased in comparison to control. 4-fold increase in ROS generation was observed after 48 h exposures of rotenone at 4 µM concentration, respectively (Figure 5). Microgreen treatment at 1 mg/ml concentration along with rotenone was found to completely attenuate ROS generation in case of rotenone treatment (Figure 5; Control=1).

Mitochondrial activity reduces significantly (p<0.05) in rotenone exposed organisms compared to controls. We observed 33% reduction in mitochondrial activity of the organism exposed to rotenone, respectively. When WME was given along with rotenone mitochondrial activity was found comparable to the control (Figure 6; Control=1).

Effect of the dopamine level on the behavior of worms was assayed through 1- nonanol assay. 1- nonanol responsive movement of C. elegans is associated with the dopamine levels, where higher repulsive time related with lower the dopamine level and vice versa. Control was used to normalize the repulsive time. Compared to control, a significant increase (p<0.05) in the repulsive time was observed in the worm treated with rotenone. While, in comparison to control when worms were exposed to rotenone along with WME no significant change was observed (Figure 7; Control=1).

Wheat microgreen extract: WME; Neurodegeneration: ND; Caenorhabditis elegans: C. elegans

Both the authors are thankful to Babasaheb Bhimrao Ambedkar University for providing necessary infrastructure. We also acknowledge research fellowships from University Grant commission to MS. The authors declare that no funds, grants or other support were received during the preparation of the manuscript.

MS conceptualization, Methodology, Investigation, data analysis, writing work; SS Investigation, Conceptualization. Both the authors have seen and approved the content of written work.