COMPOSITION FOR INHIBITING RELEASE OF NEUROTRANSMITTERS

Abstract

The present disclosure relates to a composition for inhibiting neurotransmitter secretion by inhibiting membrane potential generation of the nerve. The composition of the present disclosure has a synergistic effect related to inhibition of SNARE complex formation, and thereby, it can prevent active secretion of neurotransmitters, and thus can reduce muscle contraction to improve skin wrinkles.

Claims

1-10. (canceled)

11. A method for inhibiting neurotransmitter secretion, comprising applying a composition comprising a combination of (i) a Zanthoxylum piperitum fruit extract and (ii) a SNARE formation inhibitor as an active ingredient to a subject in need thereof.

12. The method according to claim 11, wherein the Zanthoxylum piperitum fruit extract is one or more selected from the group consisting of a squeezed juice, an ultrasonic extract, a water extract, an extract of a lower alcohol aqueous solution having 1 to 4 carbon atoms and an extract of a lower alcohol having 1 to 4 carbon atoms of a Zanthoxylum piperitum fruit.

13. The method according to claim 11, wherein the Zanthoxylum piperitum fruit extract comprises 3 to 50 ppm of quercitrin.

14. The method according to claim 11, wherein the weight of the Zanthoxylum piperitum fruit extract comprised in the total composition is equal to or greater than the weight of the SNARE formation inhibitor.

15. The method according to claim 11, wherein the composition comprises (i) 100 to 500 ppm of Zanthoxylum piperitum fruit extract and (ii) 10 to 100 ppm of SNARE formation inhibitor, and the weight of the comprised Zanthoxylum piperitum extract is equal to or greater than the weight of the SNARE formation inhibitor.

16. The method according to claim 11, wherein the method improves skin wrinkles in the subject.

17. The method according to claim 11, wherein the composition is a cosmetic.

Description

BRIEF DESCRIPTION OF DRAWINGS

[0034] FIG. 1 shows the result of confirming the muscle contraction inhibition effect of each of hyperoside and quercitrin known to be contained in a Zanthoxylum piperitum fruit.

[0035] FIG. 2 shows the ability to inhibit electric signal transmission of the Z. piperitum fruit extract in a nerve cell.

[0036] FIG. 3 shows the effect of inhibiting neurotransmitter secretion of the Z. piperitum fruit extract.

[0037] FIG. 4 shows the evaluation of the cytotoxicity of the Z. piperitum fruit extract.

[0038] FIG. 5 shows the muscle paralysis efficacy of the Z. piperitum fruit extract.

[0039] FIG. 6 shows the synergistic effect of the Z. piperitum extract and argireline (acetyl hexapeptide 8).

[0040] FIG. 7 shows the muscle contraction inhibition effect upon single treatment of argireline.

[0041] FIG. 8 shows the synergistic effect of the Z. piperitum extract and BoNT/A LC that is a Botox effective substance.

MODE FOR INVENTION

[0042] Hereinafter, the present disclosure will be described in more detail by examples. These examples are only for describing the present disclosure in more detail, and it will be apparent to those skilled in the art to which the present disclosure pertains that the scope of the present disclosure is not limited by these examples.

EXAMPLE

[0043] 1. Preparation of Zanthoxylum piperitum Extract and Confirmation of Effective Substance (FIG. 1)

[0044] (1) Preparation of Zanthoxylum piperitum Extract

[0045] Zanthoxylum piperitum fruits were washed, dried and pulverized, and using them, a Z. piperitum ethanol extract was prepared. Specifically, the ethanol extract was prepared by a method of extracting at a room temperature for 2 hours by adding 70% ethanol as an extraction solvent, and then powdering the obtained extract by filtering and concentrating.

[0046] (2) Confirmation of Effective Substance

[0047] In order to test the muscle contraction inhibition effect of each of hyperoside and quercitrin known to be contained in the Z. piperitum fruit, a Caenorhabditis elegans system was introduced. As the Caenorhabditis elegans, a wild-type N2strain was used, which was provided by Caenorhabditis Genetics Center. The Caenorhabditis elegans at a synchronized young-adult stage was harvested with M9 (+0.01% PEG, 0.1% DMSO) buffer and washed and spun down twice, and then diluted with a proper amount of M9 buffer and 50 ul each was distributed to an e-tube. After each compound was added, the final reaction volume was adjusted to 1 ml. After each compound was treated in an incubator at 20° C. for 1 hour, all 1 ml was transferred to a 24-well plate for video recording. In the recorded video, the number of swims per minute was calculated by measuring the number of swims for 20 seconds of 10 animals, where one round trip from the right end to the left end and back to the right end was counted as “one swim”. The muscle paralysis effect was calculated by 100−(number of swims of experimental group)/(number of swims of control group)×100. The error bar was calculated by S.E.M (Standard Error of Mean) and statistical processing was performed by Student's t-test.

[0048] As the result of the experiment, the muscle contraction effect of hyperoside was incomplete, but the muscle contraction effect of quercitrin was confirmed (FIG. 1).

[0049] (3) Confirmation of Quercitrin Content

[0050] The content of quercitrin in the Z. piperitum ethanol extract prepared above and a Z. piperitum hot water extract and a Z. piperitum ultrasonic extract was measured. As the Z. piperitum hot water extract, ‘Zanthoxylum piperitum fruit extract’ of BIO FD&C was used, and as the Z. piperitum ultrasonic extract, Jeju Chophi (Z. piperitum fruit extract) of The Garden of Natural Solution was used. The content of quercitrin of the ultrasonic extracted Zanthoxylum piperitum extract was 4 ppm, and the content of quercitrin of the hot water extract was 3 ppm, and the content of quercitrin of the ethanol extract was measured as 9 ppm, and therefore, it was confirmed that the quercitrin content was significantly increased in ethanol extraction than other solvent extraction methods. On the other hand, in ‘Zanthalene’ of Indena, which is the Zanthoxylum bungeanum extract, quercitrin was not detected (less than 1 ppm).

[0051] 2. Inhibition Ability of Electric Signal Transmission in Nerve Cells (FIG. 2)

[0052] (1) Experimental Method

[0053] Using the Zanthoxylum piperitum fruit ethanol extract prepared above, the ability to inhibit electric signal transmission in nerve cells was measured. Specifically, to measure the change of the calcium ion inflow by KCl stimulation in a nerve cell line, NG108-15, FLIPR Calcium Assay Kit was used. The NG108-15 was aliquoted in a poly-L-lysine-coated 96-well plate so as to be 1×10.sup.4 cells/well. At the 3.sup.rd day after aliquoting, the Z. piperitum fruit extract was diluted in serum-free DMEM and treated for 1 hour. Then, a loading buffer was treated and it was cultured in a CO.sub.2 incubator for 2 hours at 37° C. The loading butter contains a fluorescence substance which reacts to the calcium ion, and due to the masking dye fluorescence results only from the calcium ion in the cells. After culturing, stimulation was provided with 70 mM KCl to the cells using FlexStation and fluorescence was measured at 485/525 nm.

[0054] (2) Experimental Result

[0055] When depolarization is induced with 70 mM KCl in nerve cells, Ca.sup.2+ flows into the cells. When the Z. piperitum fruit extract was treated, Ca.sup.2+ inflow by depolarization was significantly inhibited (FIG. 2). Through this, it could be seen that the Z. piperitum fruit extract inhibits transmission of electric signals in nerve cells.

[0056] 3. Neurotransmitter Secretion Inhibition by Z. piperitum Fruit Extract (FIG. 3)

[0057] (1) Experimental Method

[0058] In order to find out the change of neurotransmitter secretion under the co-culture condition of nerve cells and muscle cells, Neurotransmitter Transporter Uptake Assay Kit was used. At the 3.sup.rd day after co-culture, dye solution was treated for 30 minutes, and then the Z. piperitum fruit extract prepared above was diluted in HBSS buffer (HBSS+20 mM HEPES) and treated for 30 minutes. The dye solution contained a fluorescence substance imitating neurotransmitters and a masking dye. By the masking dye, when the fluorescence substance enters the cells, fluorescence is shown, and when it is released, fluorescence disappears. After treatment, using FlexStation, the cells were stimulated with 70 mM KCl, and fluorescence was measured at 485/525 nm.

[0059] (2) Experimental Result

[0060] Whether the Z. piperitum fruit extract inhibits transmission of electric signals and ultimately inhibits neurotransmitter secretion was confirmed. Neurotransmitters are released from a presynaptic neuron to a synapse gap when an action potential is generated in nerve cells. The released neurotransmitters bind to a receptor of a postsynaptic neuron present in muscle to produce an action potential in a motor neuron and then signals are converted into muscle contraction. Since the environment in which the neuromuscular junction is formed is an appropriate condition to study this process, NG108-15 and C2C12 were co-cultured to create an in vitro condition to form a neuromuscular junction.

[0061] Experimental results showed that KCl treatment reduces the fluorescence value, relatively to the control group, which means that KCl treatment promotes the secretion of neurotransmitters. When the Z. piperitum fruit extract was treated together thereto, the secretion of neurotransmitters promoted by KCl treatment reduced (FIG. 3). Through this, it was confirmed that the Z. piperitum fruit extract inhibited secretion of neurotransmitters.

[0062] 4. Evaluation of Cytotoxicity of Z. piperitum Fruit Extract (FIG. 4)

[0063] (1) Experimental Method

[0064] In order to confirm the cytotoxicity of the Z. piperitum fruit extract treatment concentration, cell viability was measured by MTT assay. The NG108-15 cell was aliquoted to a poly-L-lysine-coated 96-well plate so as to be 1×10 4 cells/well, and cultured for 24 hours and attached to the plate. Then, two substances were diluted by serum-free DMEM to each concentration and treated to the cell, and then cultured in a CO2 incubator for 4 hours. After removing the supernatant, 0.5 mg/ml MTT solution dissolved in serum-free DMEM was treated to the cell by 100 μL each and reacted for 3 hours. After the reaction, the MTT solution was removed and to dissolve a formazan crystal, isopropanol 100 μL was added and after stirring for 10 minutes absorbance was measured at 540 nm with a microplate reader.

[0065] (2) Experimental Result

[0066] It was confirmed that there was no cytotoxicity, as the cell viability of 100% or more was shown at all concentrations, when the Z piperitum fruit extract was treated by 12.5, 25, 50 ppm (FIG. 4).

[0067] 5. Muscle Paralysis Efficacy of Z. piperitum Fruit Extract (FIG. 5)

[0068] Using the Caenorhabditis elegans system described in the 1. (2), the muscle paralysis efficacy of the Z. piperitum fruit extract prepared above was tested.

[0069] As the experimental result, when the Z. piperitum fruit extract was treated to Caenorhabditis elegans, the muscle paralysis effect increased according to the concentration. In case of 0.01% Z. piperitum fruit extract, there was no muscle paralysis effect, but when 0.05% Z piperitum fruit extract was treated, 8.5% of muscle paralysis effect was shown, and when 0.1% Z. piperitum fruit extract was treated, 24.1% of muscle paralysis effect was shown (FIG. 5). Through this, it was confirmed that there was muscle paralysis efficacy at a subject level when the Z. piperitum fruit extract was treated. In addition, based on the previous cell experiment result, it can be inferred that the Z. piperitum fruit extract induces paralysis of muscle by inhibiting secretion of neurotransmitters in nerve cells similarly to the Botox mechanism.

[0070] 6. Synergistic Effect Test of Combination of Z. piperitum Fruit Extract and Argireline (FIGS. 6 and 7)

[0071] The synergistic effect between the Z. piperitum fruit extract and Botox efficacy materials was tested. First, a synergistic effect with argireline (acetyl hexapeptide-8) which inhibits SNARE formation by binding to SNAP-25 in nerve cells was tested.

[0072] As the experimental result, it was confirmed that the synergistic effect does not appear when the Z. piperitum extract was used less than argireline (Z. piperitum 50 ppm, argireline 100 ppm), and the synergistic effect appears when the Z. piperitum extract was used at least equal to or greater than argireline (FIG. 6). In addition, it was confirmed that the muscle contraction inhibition effect of argireline (100 ppm) increased about 15 times or more when used together with Z. piperitum extract 500 ppm (FIG. 6).

[0073] An experiment was performed in order to find out how much argireline is needed to achieve this amount of inhibition effect by itself. By gradually increasing the amount of the substance, it was confirmed that 100,000 ppm of argireline was needed to obtain the same level of effect as the combination of argireline 100 ppm and Z. piperitum extract 500 pp (FIG. 7).

[0074] 7. Synergistic Effect Test of Combination of Z. piperitum Extract and BoNT/A LC (FIG. 8)

[0075] Next, it was tested whether the Z. piperitum fruit extract shows a synergistic effect on the effective part of the botulinum toxin. Since it is difficult to use botulinum toxin in experiments due to its severe toxicity and strict regulation, the synergistic effect was measured using a light chain region (BoNT/A LC) which is a site with enzymatic activity in the botulinum toxin protein. 0.002% (20 ppm) Treatment with BoNT/A LC alone had a muscle paralysis effect of about 50%, like the muscle paralysis effect of botulinum toxin, and through this, it can be seen that BoNT/A LC also showed Botox efficacy like botulinum toxin. To test the synergistic effect, when the 0.01% Z. piperitum fruit extract was treated to BoNT/A LC together, the synergistic effect of 20% or more was shown (FIG. 8). Through this result, it could be seen that the Z. piperitum fruit extract showed the synergistic effect throughout the materials showing Botox efficacy.