Patent ID: 12227747

MODE FOR INVENTION

Hereinafter, the present invention will be described in more detail by the following examples. However, the following examples are described with the intention of illustrating the present invention, and the scope of the present invention is not to be construed as being limited by the following examples.

Example 1: DNA Aptamer Selection and Sequence Analysis

Ascorbic Acid SELEX:

9 rounds of SELEX for ascorbic acid were performed using a DNA library (BasePair Biotechnologies) consisting of ˜1015unique oligonucleotides. The buffer composition used was as follows: 50 mM Sodium Acetate pH 5.5 (Sigma), 1 mM MgCl2(Sigma), 0.05% Tween 20 (Sigma), 1% BSA (Sigma) and 1 mM glutathione (Sigma). The stringency of SELEX was changed by reducing the binding time of the aptamer to the target, changing the buffer composition, and reducing the concentration of the target in free molecule elution. Negative selection for DHA was performed to remove aptamers that bind to oxidized form of Ascorbic acid from the enriched library (FIG.2).

Bioinformation analysis of the rich library produced by the SELEX method obtained candidate aptamers, and the ability to protect AA from oxidation from these top 20 was screened. The aptamer of SEQ ID NO: 1 showed the best effect.

Example 2: Fluorescence Analysis of Ascorbic Acid Oxidation Products

The oxidation of ascorbic acid was measured in reverse with detecting the oxidized product dehydroascorbic acid (DHA) with modifying a method described in Vislisel et al. (Vislisel, J. M. Schafer, F. O. and Buettner, G. R. (2007) Analytical biochemistry, 365, 31-39).

Briefly, aptamers were incubated with AA (10.3 μM) at 4× concentration for 30 minutes at room temperature before addition of 25 μM H2O2(Sigma). Before the addition of OPDA dye (Sigma), the oxidizing agent sample was added and incubated at room temperature for 10 minutes. Immediately after addition of the dye (954.6 μM) sample, and read at excitation 345 nm; emission: 425 nm with a SpectraMax® plate reader (Molecular Devices) for 45 minutes at intervals of 60 seconds until the control converges. To confirm that the screening data shows AA protection and that there is no interference of oxidation products (DHA) or analytical dyes (OPDA), fluorescence analysis was repeated with DHA (10.3 μM) (Sigma) with selected aptamers cultured in place of AA. All analyzes were performed by calibrating with 50 mM sodium acetate (Sigma), 1% BSA (Sigma), 0.05% Tween 20 (Sigma), and 1 mM MgCl2(Sigma) pH 5.5. All fluorescence assays were performed in black 384-well plates (greiner bio-one). Each sample was repeated three times (FIG.4).

Example 3: Increased Storage of Vitamin C by Aptamer

The aptamin C of the present invention was maintained at room temperature for 8 weeks and the reducing activity of ascorbic acid was measured using DCPIP (2,6-Dichlorophenolindophenol). As can be seen fromFIG.5, the aptamine C of the present invention inhibits the oxidation of ascorbic acid and maintains the reducing power, thereby increasing the shelf life by 4 times or more compared to the control group in which ascorbic acid alone exists.

Example 4: Aptamer Titration for AA

It was titrated against AA (10.3 μM) to determine the effective concentration of the optimal aptamer (A). The relative concentrations of aptamers for AA were 10×, 5×, 2×, 1×, 0.5×, 0.25× and 0.1×. All aptamer/AA mixtures were incubated at room temperature for 30 minutes before the addition of 10.3 μM CuSO4, and the samples were incubated at room temperature for another 10 minutes before addition of 954.6 μM OPDA. Plate ex: 345 nm; em: Read plate at ex: 345 nm; em: 425 nm for 45 minutes, and data were collected every 60 s. Each sample was run in triplicate. As can be seen inFIG.7, the binding affinity between the aptamer and ascorbic acid of the present invention is 0.9 μM,

Example 5: Effect of Complex of Aptamin C and Vitamin C on MPP+-Induced Cytotoxicity

SH-SY5Y cells (human neuroblastoma) were treated with MPP (1-methyl-4-phenylpyridinium) at the indicated concentration, and then cell viability was measured, and the cells were pre-treated with NXP031 (aptamer and vitamin C complex of the present invention) for 1 hour, and then exposed with 5 mM MPP for 24 hours, and then the cell viability was measured. The cell viability was measured by MTT assay.

Example 6: Effects on MPTP-Induced Parkinson's Disease Mouse Model

1-1. Parkinson's Disease Mouse Model

Parkinson's disease was acutely induced with intraperitoneal injection of 20 mg/kg of MPTP (1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine) at 2 hour intervals for a total of 4 consecutive days to 8-week-old C57BL/6 mice weighing 25 g or more.

1-2. Preparation and Administration of the Composition

A composition was prepared by mixing 200 mg/kg body weight of vitamin C and 4 mg of an aptamer binding to the vitamin C (GTGGA GGCGG TGGCC AGTCT CGCGG TGGCG GC; SEQ ID NO: 1)/kg body weight. 1 hour after the end of the last MPTP administration to the mouse inducing Parkinson's disease by the above method, the composition was diluted to a final dose of 200 mg of vitamin C/kg body weight and 4 mg of aptamin C/kg body weight and 50 μl of the resulting diluent was administered orally or intraperitoneally in the mouse. The single administration of vitamin C and aptamin C was administered intraperitoneally or orally with 50 μl at the same concentration as in the above composition. The administration of composition and vitamin C and aptamin C were performed once a day for 4 consecutive days.

1-3. Neurobehavioral Evaluation

In order to evaluate the neurobehavioral effect induced by the MPTP and composition administered by the above method, a pole test and a rotarod test were performed 4 days after Parkinson's disease was induced by the method described above.

A pole test was conducted using a 55 cm high pole. The mouse was placed above the pole to measure the time to come down to the floor.

Rota rod test starts with a speed of 2.5 rpm with the mouse on the rota rod treadmill, and then gradually increases the speed so that the maximum speed is 25 rpm (3.5 to 35 rpm in the case of high speed), and when the treadmill rotates, the time (seconds) until the mouse loses its balance and falls to the floor was measured.

1-4. Brain Tissue Immunohistochemical Staining

A section of the brain tissue obtained by the above method was reacted with 1% hydrogen peroxide for 15 minutes to remove the activity of endogenous peroxidase. Next, tyrosine hydroxylase antibody diluted to an appropriate concentration was added and stained overnight at 4° C. After washing and removing the unbound primary antibody, stain with biotinylated secondary antibody for 90 minutes at room temperature. After washing and removing unbound secondary antibody, it is stained with ABC solution for 1 hour at room temperature. After color reaction with 3,3-diaminobenzidine, it was observed under a microscope.

As can be seen fromFIGS.10to14, in the experiment result, the MPTP-induced Parkinson's disease mouse model test, it was confirmed that the deficit of exercise capacity was behaviorally reduced by the treatment of the composition (vitamin C and aptamin C) of the present invention. In addition, as a result of confirming the dopaminergic neurons in the tissue through immunohistochemical staining for tyrosine hydroxylase in the brain tissue sections obtained from the mice to which the sample was administered, it was confirmed that the apoptosis of dopaminergic neurons was significantly reduced in the composition-administered group.