HPLC

    Five days after inoculation with bacterial suspension, leaf samples were harvested and 0.1 g of leaf tissue was extracted with 800 μL of 80% methanol for HPLC quantification. Among all test groups, the highest yield was observed in WT expressing BvAD6,BvADH,P19, with an L-DOPA yield of 2068.02 ng/20 μL, equivalent to 80.396 mg L-DOPA per 100 g of leaf tissue.

    In terms of plant synthesis, a yield of 80 mg per 1 g of leaf tissue is considered remarkably high, indicating a highly efficient production pathway for synthesizing compounds in other plants. Beyond the high yield, our production cycle is relatively short—requiring only one month of cultivation before bacterial inoculation, followed by just five days until harvest. This approach has the potential to offer a new method for high-yield, short-cycle plant-based drug synthesis in the future.

Neuroprotection Experiment

    L-DOPA Dose Test

    Zebrafish embryos were immersed in 0.5 mM and 1.0 mM L-DOPA bath treatments 24 hours post-fertilization (hpf), and their behavior was observed at 120 hpf using Danio Vision. Swimming distance was recorded and analyzed.

    Zebrafish embryos immersed in 0.5 mM and 1.0 mM L-DOPA from 24 to 120 hours post-fertilization (hpf) showed a significant decrease in swimming distance, with the reduction being more pronounced at 1.0 mM. When zebrafish embryos are healthy, immersion in L-DOPA at concentrations of 0.5 mM and 1.0 mM negatively impacts their motor abilities, with the 1.0 mM concentration causing more severe damage. Therefore, in protective assays, only 0.5 mM L-DOPA was used for testing.

    Control 0.5mM 1.0mM
    Distance Moved (cm) 47.151 10.239 5.498
    Death rate 1/16 1/16 1/16

    MPTP Dose Test

    MPTP is a drug used to establish a Parkinson’s disease (PD) model in zebrafish. We immersed zebrafish embryos in 50 μM, 100 μM, and 200 μM MPTP baths starting 48 hours post-fertilization (hpf), and observed their behavior at 120 hpf using Danio Vision. Swimming distance was recorded and analyzed.

    Embryos treated with 100 μM and 200 μM MPTP from 48 to 120 hpf exhibited a significant decrease in swimming distance. At 100 μM MPTP, zebrafish behavior was already impaired, indicating that this concentration is sufficient to cause damage. Therefore, 100 μM MPTP was selected as the concentration for establishing a PD model in protective assays.

    Control 0.5mM 1.0mM
    Distance Moved (cm) 47.151 10.239 5.498
    Death rate 1/16 1/16 1/16

    Neuroprotection Experiment

    At 24 hours post-fertilization (hpf), zebrafish embryos were immersed in a 2 mL bath of 0.5 mM L-DOPA. At 48 hpf, the solution was replaced with 2 mL of 0.5 mM L-DOPA + 100 μM MPTP, and their behavior was observed at 120 hpf using Danio Vision. Swimming distance was recorded and analyzed.

    The group treated with MPTP alone and the group treated with MPTP + L-DOPA showed a significant increase in swimming distance, suggesting that L-DOPA at a concentration of 0.5 mM provides a protective effect on the motor abilities of zebrafish embryos.

    Control MPTP MPTP L-DOPA
    Distance Moved (cm) 34.810 2.188 11.006
    Death rate 1/16 0/16 0/16

Recovery Experiment

    L-DOPA Dose Test

    At 72 hours post-fertilization (hpf), zebrafish embryos were immersed in 0.5 mM and 1.0 mM L-DOPA bath treatments, and their behavior was observed at 120 hpf using Danio Vision. Swimming distance was recorded and analyzed.

    Zebrafish embryos immersed in 0.5 mM and 1.0 mM L-DOPA from 72 to 120 hours post-fertilization (hpf) showed no significant difference in swimming distance, indicating that these concentrations do not impair zebrafish motor abilities. Therefore, both concentrations will be used in subsequent therapeutic tests for further experimentation.

    Control 0.5 mM 1.0 mM
    Distance Moved (cm) 15.489 17.500 16.445
    Death rate 1/16 0/16 0/16

    MPTP Dose Test

    Zebrafish embryos were immersed in 200 μM, 400 μM, and 600 μM MPTP baths from 48 to 72 hours post-fertilization (hpf), and their behavior was observed at 120 hpf using Danio Vision. Swimming distance was recorded and analyzed.

    Embryos treated with 400 μM and 600 μM MPTP from 48 to 72 hpf showed a significant decrease in swimming distance. At 400 μM MPTP, zebrafish behavior was already impaired, indicating that this concentration is sufficient to cause damage. Therefore, 400 μM MPTP was chosen as the concentration for establishing a Parkinson's disease (PD) model in therapeutic assays.

    Control 200 μM 400 μM 600 μM
    Distance Moved (cm) 33.672 41.320 19.757 15.681
    Death rate 0/16 0/16 0/16 0/16

    Recovery Experiment

    At 48 hours post-fertilization (hpf), zebrafish embryos were immersed in a 2 mL bath of 400 μM MPTP. At 72 hpf, the MPTP bath was replaced with L-DOPA, and their swimming distance was observed at 120 hpf using Danio Vision.

Immunohistochemical Staining

    control

    L-DOPA (0.5 mM)

    control

    MPTP (100 mM)

    MPTP (100 µM)+L-DOPA(0.5mM)

    MPTP (100 µM)+L-DOPA(1.0 mM)

    MPTP (200 µM)

    MPTP (400 µM)