KR100901078B1 - SNARE complex formation inhibiting composition comprising natural extracts - Google Patents

Abstract

In the present invention, holly, schizandra, rhizome, worms, flies, babies, pheasant's amaranth, rhododendron, spines, rowan, persimmon, camellia, ginger, oyster, scab, chestnut, horse urine, seo, Flower extracts of Ryoyang, Negesvari, Raj briksha, Dhayaro, Rosemary and Enamel. The flower extract according to the present invention can inhibit the formation of the SNARE complex to regulate neurotransmitter release, and can provide a raw material at a lower cost than BoNT (Botulinum neurotoxin), and because it is a natural material, it is possible to be familiar to consumers and industrially useful.

SNARE (Soluble N-ethylmaleimide-sensitive factor attachment protein receptor) complex, neurotransmitter, extract, botox

Description

SNARE complex formation inhibiting composition comprising natural extracts}

The present invention relates to a composition for inhibiting the formation of the SNARE complex containing the flower extract as an active ingredient.

 The release of neurotransmitters is caused by the formation of a pathway between the two boundaries after the fusion of synaptic vesicles and presynaptic membranes located at the nerve endings containing the neurotransmitters. The vesicle protein VAMP (synaptobrevin) and the plasma membrane binding proteins Syntaxin 1a and SNAP-25 are three protein complexes, the SNARE protein, which provides a fundamental force in the fusion of synaptic vesicles and synaptic membranes. Herein, the neurotransmitter excretion pathway is opened by membrane fusion between synaptic vesicles and the presynaptic membrane, t-SNARE and vesicles, which are two complexes of syntaxin 1a protein and SNAP-25 protein attached to the target membrane. This is a result of the action of v-SNARE attached to the vesicle. When the membrane is fused, rearrangement of the lipid bilayer occurs. Since the biofilms push strongly against each other, these membranes are not spontaneously fused and have a strong force from outside to overcome the repulsion between the membranes, which is known to provide SNARE protein. As such, the formation of the SNARE complex is a key phenomenon of extracellular exocytosis, including the release of neurotransmitters.

 Botulinum neurotoxin, which is currently used as an injection for skin wrinkle improvement, is a protease that cleaves SNARE protein, a key protein involved in neurotransmitter release.It cuts SNARE protein to block neurotransmission and consequently invades Botox. Numb the muscle cells. While Botox, which is being used as an injection, blocks nerve transmission by irreversibly cutting the SNARE protein, the so-called "coating Botox" is a kind of "competitive inhibitor" that inhibits neurotransmission by inhibiting the formation of the SNARE complex. A key component of "Bortox" is a hexapeptide called Argireline and has the amino acid sequence of EEMQRR (Blanes-Mira et al., International Journal of Cosmetic Science. 24, 303-310, 2002). This is the site that matches the amino acid sequence of the N-terminal region of SNAP-25. In other words, a small hexapeptide is inserted instead of an intact SNAP-25 to bind to, thereby preventing the intact SNAP-25 from binding to other SNARE proteins, Syntaxin 1 and VAMP-2. do. However, argireline, the main component of the above-mentioned Botox product, has a disadvantage that the effect is uncertain, and the price is disadvantageous, and should be performed by a specialist due to skin side effects such as muscle paralysis.

Accordingly, the present inventors completed the present invention by finding competitive inhibitors on SNARE complex formation using flower extracts and confirming their inhibition of membrane fusion and inhibition of neurotransmitter release and inhibition of SNARE complex formation in neurons. It was.

The present invention has been made to solve the above problems, the present invention has an object to provide a natural composition that inhibits the formation of SNARE complex involved in neurotransmitter release. It is another object of the present invention to provide a neurotransmitter inhibitor composition comprising the flower extract as an active ingredient.

The natural composition according to the present invention has been studied to develop an excellent SNARE complex formation regulator for plants native to nature, as a result of the extract of tigers, Schisandra chinensis, yellow root, worms, flies, babies, pheasant, Extracts from rhododendrons, rhododendrons, rowan, persimmon, camellia, ginger, oyster, scab, chestnut, urinary, erhu, ryoyang, Negesvari, Raj briksha, Dhayaro, laurel and narcissus It was found to inhibit the formation of SNARE complexes involved in.

The natural composition according to the present invention can provide a raw material that is safer and significantly cheaper than skin aesthetics using conventional botox as an inhibitor of SNARE complex formation. In addition, the stability in the formulation of skin wrinkle improvers, which has been raised as a conventional problem, has been solved by using plant extracts.

In the present invention, 24 kinds of flower extracts having the most efficacy in regulating neurotransmitter emission among natural products (Table 1) which are the subject of efficacy test were selected as follows. Willows of the Salicaceae ( Salix hultenii ), Schisandra chinensis of the Magnoliaceae ( Schisandra chinensis ), Roots of the Malvaceae (Hibiscus hamabo), Typhaceae units (scientific name: Typha orientalis), Mesquite (Leguminosae) Hagi of (scientific name: Lespedeza bicolor), portions and (Typhaceae) Typha of (scientific name: Typha angustata), keunkkwong amaranth of stone Araliaceae (Crassulaceae) (Scientific name: Sedum spectabile ), Rhododendron mucronulatum (Ericaceae), the side of Polygonaceae (Scientific name: Persicaria tinctoria ), Rowanaceae genus ( Sorbus commixta ), Persimmon (Ebenaceae) Scientific names: Diospyros kaki ), Camellia japonica (Theaceae), Ginger ( La Lindera obtusiloba ), and Juphyraceae ( Plamycarya strobilacea ), Rosaceae (Scientific name: Potentilla chinensis ) in Rosaceae , Chestnut of the Fagaceae (Canadian name: Castanea crenata ), of the Staphyleaceae Horse urine when (scientific name: Euscaphis japonica), hornbeam trees betulaceae (Betulaceae) (scientific name: Carpinus laxiflora), charge quantification of the Ericaceae (Ericaceae) (scientific name: Rhododendron molle), wheel Araliaceae (Guttiferae) of Negesvari (scientific name: Mesua of Rosa chinensis), and the rose family (Rosaceae): ferrea), Mesquite (Leguminosae) Raj briksha (scientific name: Cassia fistula), lythraceae (Lythraceae) of Dhayaro (scientific name: Chinese rose in Woodfordia fruticosa), rose family (Rosaceae) (scientific name The flower area with the enchantment ( rosa rugosa ) Used.

Method for producing a natural product extract of the present invention is as follows. To the purchased natural product (100 g), C ₁ to C ₄ alcohol, preferably, anhydrous methanol or ethanol and 1000 ml of water were added and the extractor equipped with a reflux condenser (Model: HB 4 basic, manufacturer: IKA) for 1 hour at 60-90 ° C. The extract was warmed for 30 minutes to 2 hours 30 minutes. The extract was filtered through Whatman No. 2 filter paper, and the remaining residue was extracted one or more times in the same manner as above. The extracts were combined and concentrated under reduced pressure (model name: Rotavapor, manufacturer: Buchi, temperature: 40 ° C.), and then frozen. Drying yielded a dry extract. Among the flower extracts prepared in this way, holly willow, schisandra chinensis, rhubarb, worm, fern, babies, pheasant's amaranth, azalea, spinach, rowan, persimmon, camellia, ginger, oyster, scab, chestnut, horse urine At the same time, it was found that Rhizome, Ryoyang, Negesvari, Raj briksha, Dhayaro, Laurel and Lactobacillus extracts inhibited the formation of SNARE complex involved in the neurotransmitter pathway.

The natural product extract composition of the present invention contains ingredients commonly used in cosmetics and pharmaceuticals in addition to the natural product extracts described herein as active ingredients. Such ingredients include, but are not limited to, conventional auxiliaries and carriers such as, for example, antioxidants, stabilizers, solubilizers, vitamins, pigments, flavorings and the like.

The natural extract composition of the present invention may be prepared in any formulation conventionally prepared in the art, such as solutions, suspensions, emulsions, pastes, gels, creams, lotions, powders, soaps, surfactant-containing cleansing, oils, Powder foundations, emulsion foundations, wax foundations, sprays and the like can be formulated, but are not limited to these.

When the formulations of the present invention are pastes, creams and gels, animal oils, vegetable oils, waxes, paraffins, starches, trachants, cellulose derivatives, polyethylene glycols, silicones, bentonites, silicas, talc and zinc oxide may be used as carrier components. It is possible, but not limited to.

When the formulations of the present invention are powders and sprays, lactose, talc, silica, aluminum hydroxide, calcium silicate and polyamide powders can be used as carrier components, especially in the case of sprays additionally chlorofluorohydrocarbons, propane Propellants such as butane and dimethyl ether may be included, but are not limited thereto.

When the formulations of the present invention are solutions and emulsions, solvents, solubilizers and emulsions are used as carrier components, such as water, ethanol, isopropanol, ethyl carbonate, ethyl acetate, benzyl alcohol, benzyl benzoate, propylene glycol, 1 Fatty acid esters of, 3-butylglycol oil, glycerol aliphatic esters, polyethylene glycols and sorbitan.

When the formulation of the present invention is a suspension, liquid diluents such as water, ethanol and propylene glycol, suspending agents such as ethoxylated isostearyl alcohol, polyoxyethylene sorbitol ester and polyoxyethylene sorbitan ester, and microcrystals Castle cellulose, aluminum metahydroxy, bentonite, agar and tracant and the like can be used, but are not limited thereto.

When the formulation of the present invention is a surfactant-containing cleansing, the carrier component is an aliphatic alcohol sulfate, an aliphatic alcohol ether sulfate, a sulfosuccinic acid monoester, an isethionate, an imidazolinium derivative, a methyltaurate, a sarcosinate, a fatty acid amide. Ether sulfates, alkylamidoventins, fatty alcohols, fatty acid glycerides, fatty acid diethanolamides, vegetable oils, lanolin derivatives and ethoxylated glycerol fatty acid esters and the like can be used, but are not limited thereto.

Hereinafter, the present invention will be described in more detail based on the following examples. The following examples are intended to illustrate the invention, not to limit the invention. All documents disclosed in the present invention are incorporated by reference.

Experimental material

1-palmitoyl-1,2-dioleoyl-sn-glycero-3-phosphatidylcholine (POPC), 1,2-dioleoyl-sn-glycero-3-phosphatidylserine (DOPS), 1,2- Dioleoyl-sn-glycero-3-phosphoseline-N- (7-nitro-2-1,3-benzoxadiazol-4-yl) (NBD-PS) and 1,2-dioleoyl- sn-glycero-3-phosphoethanolamine-N- (rissamine rhodamine B sulfenyl) (rhodamine-PE) was purchased from Avanti Polar Lipid (Alabama, USA). RPMI 1640, 10% fetal calf serum, 5% fetal bovine serum was purchased from GIBCO-BRL (Grand Island, NY, USA).

Example 1 Preparation of Natural Product Extracts from Horners

Holly willow [ Salix hultenii , Salicaceae] Flower purchased from Korean plant extract bank, washed with 100 g of purified water, dried, and added with 1000 ml of anhydrous methane, ethanol and water (extractor with reflux cooler) Model: HB 4 basic, manufacturer: IKA) was extracted by heating for 2 hours at 80 ℃. The extract was filtered with Whatman No. 2 filter, and the remaining residue was extracted one or more times in the same way as above. The filtered extracts were concentrated under reduced pressure (model name: Rotavapor, manufacturer: Buchi, temperature: 40 ° C.). Lyophilization (model name: FD5518, manufacturer: Bonjaro, temperature: -45 ℃, pressure: 50 mTorr) to give a dry extract. Among the flower extracts prepared in this way, holly willow, schisandra chinensis, rhubarb, worm, fern, babies, pheasant's amaranth, azalea, spinach, rowan, persimmon, camellia, ginger, oyster, scab, chestnut, horse urine At the same time, it was found that Rhizome, Ryoyang, Negesvari, Raj briksha, Dhayaro, Laurel and Sesame extract inhibited the formation of SNARE complex involved in the neurotransmitter pathway.

Example 2: Preparation of Extracts from the remaining Natural Products

Extracted in the same manner as in Example 1, but extracted from the natural products shown in Table 1 below.

Table 1. Example  Natural product used for 2

Table 1. Number Scientific name F amily One Erysimum aurantiacum Cruciferae 2 Aster spathulifolius Compositae 3 Salix hultenii Salicaceae 4 Prunus padus Rosaceae 5 Spiraea prunifolia var. simpliciflora Rosaceae 6 Aruncus dioicus var. kamtschaticus Rosaceae 7 Schisandra chinensis Magnoliaceae 8 Hibiscus hamabo Malvaceae 9 Typha orientalis Typhaceae 10 Lespedeza bicolor Leguminosae 11 Pueraria thunbergiana Leguminosae 12 Synulus excelsus Compositae 13 Agastache rugosa Labiatae 14 Aster koraiensis Compositae 15 Eupatorium fortunei Compositae 16 Aster yomena Compositae 17 Typha angustata Typhaceae 18 Miscanthus sinensis var. purpurascens Gramineae 19 Aster scaber Compositae 20 Caryopteris incana Verbenaceae 21 Sedum spectabile Crassulaceae 22 Aristolochia manshuriensis Aristolochiaceae 23 Coreanomecon hylomeconoides Papaveraceae 24 Serratula coronata var . insularis Compositae 25 Rhododendron mucronulatum Ericaceae 26 Persicaria tinctoria Polygonaceae 27 Sorbus commixta Rosaceae 28 Robinia pseudo - accacia Leguminosae 29 Paulownia coreana Scrophulariaceae 30 Pseudosasa japonica Gramineae 31 Chionanthus retusa Oleaceae 32 Majanthemum dilatatum Liliaceae 33 Diospyros kaki Ebenaceae 34 Campsis grandiflora Bignoniaceae 35 Hydrangea paniculata Saxifragaceae 36 Helianthus tuberosus Compositae 37 Angelica fallax Umbelliferae 38 Cosmos bipinnatus Compositae 39 Farfugium japonicum Compositae 40 Alnus japonica Betulaceae 41 Camellia japonica Theaceae 42 Weigela subsessilis Caprifoliaceae 43 Prunus persica Rosaceae 44 Alnus firma Betulaceae 45 Morus bombycis Moraceae 46 Lindera obtusiloba Lauraceae 47 Platycarya strobilacea Juglandaceae 48 Potentilla chinensis Rosaceae 49 Castanea crenata Fagaceae 50 Euscaphis japonica Staphyleaceae 51 Spiraea blumei Rosaceae 52 Carpinus laxiflora Betulaceae 53 Lonicera japonica Caprifoliaceae  54 Inula helianthus - aquatilis Compositae 55 Rhododendron molle Ericaceae 56 Chamomilla recutita Compositae 57 Hibiscus sabdariffa Malvaceae 58 Buddleia officinalis Loganiaceae 59 Carthamus tinctorius Compositae 60 Mesua ferrea Guttiferae 61 Albizzia julibrissin Leguminosae 62 Celosia cristata Amaranthaceae 63 Gomphrena globosa Amaranthaceae 64 Sophora japonica Leguminosae 65 Butea minor Leguminosae 66 Cassia fistula Leguminosae 67 Matricaria chamomilla Compositae 68 Nyctanthes arbor - tristis Oleaceae 69 Woodfordia fruticosa Lythraceae 70 Rosa chinensis Rosaceae 71 Rosa rugosa Rosaceae

Example  3: SDS - PAGE

SNARE complexes form complexes that are strong enough to not be released by SDS. Using this property, SDS-PAGE was performed to confirm the formation of SNARE complex.

M)로 혼합하 여 형성되는 복합체에 대해, 천연물 추출물의 첨가에 의한 억제 여부를 확인하였다. 각각의 단백질 10 ㎕을 1 ml 튜브에 일정한 간격으로 점적한 후 천연물 추출물 2㎕을 넣고 보텍스를 사용하여 재빨리 혼합한 다음 실온에서 30분간 반응시켰다. 반응이 종료되면 12% SDS-PAGE상에서 전기영동하여 SNARE 복합체 형성여부를 확인하였다. 결과를 도 4에 도시하였다. 도 4의 하단 숫자는 표 1의 일련번호를 의마한다.The pGEX-2T vector (GE Healthcare, Inc.) with amino-terminal glutathione S-transferase (GST) -tag cleaved with thrombin was used for the protein of the invention, ie, full-length syntaxin 1a (FIG. 1). , SEQ ID NO: 1), SNAP-25 (FIG. 2, SEQ ID NO: 2), full length VAMP-2 (FIG. 3, SEQ ID NO: 3), soluble SNARE motif of syntaxin 1a (SynH3, SEQ ID NO: 4), and Soluble SNARE motif (VpS, SEQ ID NO: 5) of VAMP was synthesized. All recombinant proteins were expressed in E. coli (DE3, Novagen). All N-terminal GST tagged proteins were purified by affinity chromatography using glutathione agarose beads. Proteins were cleaved with thrombin in digestion buffer (50 mM Tris-HCl, 150 mM NaCl, pH8.0). Molarity of purified protein SNAP-25, SynH 3 and Vps 1: 1: 1

For the complex formed by mixing in M), it was confirmed whether the inhibition by the addition of natural product extract. 10 μl of each protein was dropped into the 1 ml tube at regular intervals, and then 2 μl of the natural product extract was added, mixed quickly using a vortex, and reacted at room temperature for 30 minutes. Upon completion of the reaction, electrophoresis on 12% SDS-PAGE confirmed the formation of SNARE complex. The results are shown in FIG. The lower digit of Figure 4 refers to the serial number of Table 1.

As a result, tigers (3), Schisandra chinensis (7), rhizome (8), buds (9), flies (10), babies (17), pheasant's larvae (21), rhododendrons (25), spines (26) , Rowan (27), persimmon (33), camellia (41), ginger tree (46), oysterwood (47), scab (48), chestnut (49), urinary tract (50) 52), extracts of ryoyang (55), Negesvari (60), Raj briksha (66), Dhayaro (69), laurel (70) and sesame (71) inhibited SNARE complex formation.

FIG. 5 suggests that Rhubarb 8, oyster tree 47, scab flower 48, chestnut tree 49, Westwood tree 52, Ryoyang 55, and Dhayaro 69 are most effectively inhibited.

Example 4 Membrane Inhibition Effect

In order to search for a membrane fusion inhibitor, the extract obtained in Examples 1 to 2 was tested for the membrane fusion inhibitory effect. SNARE protein was obtained by overexpressing the protein in transformed Escherichia coli, and selectively expressing the expressed protein using glutathione agarose beads. 1-palmitoyl-1,2-dioleoyl-sn-glycero-3-phosphatidylcholine (POPC, 62 mol%), 1,2-dioleoyl to prepare liposomes, which are microencapsulated particles labeled with the following fluorescent materials -sn-glycero-3-phosphatidylserine (DOPS, 35 mol%), 1,2-dioleoyl-sn-glycero-3-phosphoseline-N- (7-nitro-2-1,3-benz 10 mM liposomes (v-vesicle) were prepared by mixing oxadiazol-4-yl) (NBD-PS, 1.5 mol%) and fluorescent substance rhodamine (Rhodamin-PE, 1.5 mol%). Next, 50mM liposomes (t-vesicles) were prepared by mixing DOPS and POPC in a molar concentration of 35:65 to prepare liposomes that are not labeled with fluorescent materials. To make the complex of purified SNAP-25 and Syntaxin 1a, the mixture was mixed so that the molar concentration was 1: 1 and reacted at room temperature for 1 hour, followed by mixing the liposome with no fluorescent substance labeled with a molar ratio of 100: 1, and VAMP. -2 was bound to the liposome containing the fluorescent material with a molar ratio of 50: 1. Subsequently, after dialysis of two liposomes, the mixture was mixed at a ratio of 1: 9 (v-vesicle: t-vesicle), and the fluorescence intensity was measured using a fluorescence intensity measuring device (Model: SpectraMax M2, manufacturer: Molecular Device). Measured. The results are shown in FIGS. 6A and 6B. 6A and 6B, the fluorescence intensity means a membrane fusion phenomenon between SNARE proteins, and thus, lower fluorescence intensity shows an excellent membrane fusion inhibitory effect. The red circle is the path through which the control reaction proceeds. When methanol was added instead of the extract, only about 40% of the maximum fluorescence intensity proceeded, and most of the extracts followed a reaction path similar to that of the control. 6a and 6b show the results of experiments on some natural extracts in order to search for membrane fusion inhibitors by natural extracts. Figure 6a shows a case where the concentration of the flower extract used in the experiment is 100 µg / ml, Figure 6b is 10 µg / ml, respectively. The yellow root (8), oyster bark (47), scabflower (48), chestnut (49), seowal (52), lye yanghwa (55) and Dhayaro (69) used in the experiment showed the best membrane fusion inhibitory effect. Seemed. In addition, the worms, Schisandra chinensis, worms, flies, babies, large pheasant's amaranth, rhododendron, spines, rowan, persimmon, camellia, ginger, Negesvari, Raj briksha, laurel, and narcissus extracts inhibit membrane fusion Indicated. The results of measuring the membrane fusion inhibitory effect at the concentration of 100 ㎍ / ml of various extracts are summarized in Table 2 below. Based on the response of the control group, about 43%, and the extract showing 30 to 60% was considered to be insignificant, the hoverbirds (3), schisandra chinensis (7), yellow root (8), worms (9), Prunus (10), Baby Boys (17), Big Pheasant's Amaranth (21), Rhododendron (25), Spinach (26), Rowan (27), Persimmon (33), Camellia (41), Ginger (46), Oysterwood (47), Elderflower (48), Chestnut (49), Pee (50), West Tree (52), Ryoyang (55), Negesvari (60), Raj briksha (66), Dhayaro (69) ), Laurel (70) and sesame (71) extracts (24 species) were effective.

Table 2. Membrane Fusion  Measurement result of inhibitory effect

Table 2. Example Number Country Scientific name % of Maximum 0 Control MeOH 44 One Poker Erysimum aurantiacum 35 2 Sea country Aster spathulifolius 36 3 Tiger Salix hultenii 12 4 Oak tree Prunus padus 35 5 Maple Tree Spiraea prunifolia var. simpliciflora 32 6 Snow horseback riding Aruncus dioicus var. kamtschaticus 45 7 Schisandra Schisandra chinensis 21 8 Yellow root Hibiscus hamabo 6 9 reed mace Typha orientalis 16 10 Frugality Lespedeza bicolor 10 11 칡 Pueraria thunbergiana 48 12 Large repair Synulus excelsus 52 13 Pear Agastache rugosa 32 14 Hummingbird Aster koraiensis 29 15 Bee Sprouts Eupatorium fortunei 31 16 A worm Aster yomena 35 17 Babes Typha angustata 17 18 Silver grass Miscanthus sinensis var. purpurascens 44 19 Booze Aster scaber 39 20 Dogwood Caryopteris incana 45 21 Greater Pheasant Sedum spectabile 10 22 Back Aristolochia manshuriensis 44 23 Cicada flower Coreanomecon hylomeconoides 42 24 Ferret Serratula coronata var . insularis 35 25 Azalea Rhododendron mucronulatum 12 26 side Persicaria tinctoria 20 27 rowan Sorbus commixta 21 28 Holly tree Robinia pseudo - accacia 38 29 Paulownia Paulownia coreana 35 30 Idae Pseudosasa japonica 41 31 Popcorn Chionanthus retusa 35 32 Crane Majanthemum dilatatum 39 33 Persimmon tree Diospyros kaki 12 34 Jacaranda Campsis grandiflora 42 35 Tree hydrangea Hydrangea paniculata 49 36 Fat Helianthus tuberosus 45 37 Covenant Angelica fallax 41 38 Cosmos Cosmos bipinnatus 35 39 Fur head Farfugium japonicum 32 40 Alder Alnus japonica 30 41 Camellia Camellia japonica 7 42 Flower tree Weigela subsessilis 38 43 Copy wood Prunus persica 32 44 Ducks Alnus firma 31 45 Mountain mulberry Morus bombycis 42 46 Ginger Tree Lindera obtusiloba -12 47 Oyster tree Platycarya strobilacea 2 48 Scab Potentilla chinensis -5 49 chestnut Castanea crenata -10 50 When you pee Euscaphis japonica -5 51 Sanjo Pop Tree Spiraea blumei 45  52 West tree Carpinus laxiflora 4 53 Indong Lonicera japonica 53 54 Fish tank Inula helianthus - aquatilis 32 55 Ryoyang Rhododendron molle 12 56 Camomila Chamomilla recutita 45 57 Hibisco Hibiscus sabdariffa 44 58 Wheat dream Buddleia officinalis 39 59 Safflower Carthamus tinctorius 35 60 Negesvari Mesua ferrea 15 61 Ringback Albizzia julibrissin 45 62 Coronary Celosia cristata 38 63 Cheon Il Hong Gomphrena globosa 32 64 Lump Sophora japonica 33 65 Bhuletro ko phul Butea minor 38 66 Raj briksha Cassia fistula 21 67 Chandra gandha Matricaria chamomilla 44 68 Paarijaat Nyctanthes arbor - tristis 48 69 Dhayaro Woodfordia fruticosa 10 70 Chinese rose Rosa chinensis 20 71 Enchantment Rosa rugosa 17

Example  5: PC12  Inhibition of neurotransmitter release in cells

Ci/ml)를 첨가한 후 탄산가스 배양기에서 90분간 도입하였다. 반응 후 완충액을 제거한 후 PBS로 3회 세정한 후 새로운 배지와 각 추출물을 넣고 30분간 반응시켰다. 배지를 제거한 후 고농도의 K⁺ 완충액(115 mM NaCl, 30 mM KCl, 1.2 mM KH2PO4, 2.5 mM CaCl2, 1.2 mM MgSO4, 11 mM 글루코스, 15 mM HEPES-tris, pH 7.4)을 넣은 후 12분간 탄산가스 배양기에서 배양한 후 상등액을 회수하여 [³H]-노르에페네피린의 방출을 저해하는지를 신틸레이션 계수관로 측정하였다. 상기의 방법으로 본 발명의 꽃 추출물이 PC12 세포내에서 도파민 유도체인 노르에피네피린의 방출을 저해하는지를 확인하였으며, 그 결과를 도 7에 제시하였다. 그 결과는 도 5, 도 6a 및 도 6b의 실험결과와 일부 일치하지 않는 것도 있었으나, 황근(8), 굴피나무(47), 딱지꽃(48), 밤나무(49), 서어나무(52) 및 Dhayaro(69) 등의 꽃 추출물이 PC12 세포내에서 도파민 유도체인 노르에피네피린의 방출을 저해하는 효과가 상대적으로 높게 측정되었다. 도 7에서 CPM은 Count Per minute을 의미한다. PBS (Phosphate) after aspirating a medium containing 10% fetal calf serum, 5% fetal bovine serum and antibiotics to collagen-coated plates (60 mm dish) of PC12 cells (Korea Cell Line Bank) Buffered 2 ml of Saline) was added and pipetted to separate the cells from the dish wall, followed by centrifugation at 1,000 × g for 5 minutes to collect the cells. Next, after dispensing the cell pellet by pipetting with fresh RPMI1640 medium (GIBCO), it was placed in a new culture plate and subcultured in an incubator supplied with 37 ° C. and 5% CO ₂ gas. [ ³ H] -norepinephrine used in the experiment was purchased from Amersham. After inhaling the medium from the plate of PC12 cells, PBS was added and the cells were removed from the plate wall, and the cell number was measured by a hematocytometer and dispersed in fresh medium at a concentration of 2 × 10 ⁵ cells / ml and inoculated. After 24 hours norepinephrine assay buffer ([ ³ H] -NE, 1.5

Ci / ml) was added and then introduced in a carbon dioxide incubator for 90 minutes. After the reaction, the buffer solution was removed, and then washed three times with PBS. Then, fresh medium and each extract were added and reacted for 30 minutes. After removing the medium, high concentration of K ⁺ buffer (115 mM NaCl, 30 mM KCl, 1.2 mM KH2PO4, 2.5 mM CaCl2, 1.2 mM MgSO4, 11 mM glucose, 15 mM HEPES-tris, pH 7.4) After incubation in the incubator, the supernatant was recovered to determine whether it inhibited the release of [ ³ H] -norepinephrine by scintillation counter. It was confirmed whether the flower extract of the present invention by the above method inhibits the release of the dopamine derivative norepinephrine in PC12 cells, the results are shown in FIG. The results were inconsistent with the experimental results of FIGS. 5, 6A, and 6B, but the root roots (8), oyster tree (47), scab flower (48), chestnut tree (49), cypress (52), and Dhayaro (69) The effect of inhibiting the release of dopamine derivative norepinephrine in PC12 cells was relatively high. In FIG. 7, CPM means Count Per minute.

Example 6: Inhibition of SNARE Complex Formation in PC12 Cells

PC12 cells were cultured in collagen coated plates (60 mm dish) in RPMI 1640 medium containing 10% fetal calf serum, 5% fetal bovine serum and antibiotics. After aspirating the medium, add 2 ml of PBS and pipette to separate the cells from the dish wall, centrifuge at 1,000 x g for 5 minutes, and then disperse the cells with fresh medium and pipette to disperse the cell pellet. And subcultured in an incubator at 37 ° C. and 5% CO ₂ gas supplied. After inhaling the medium from the plate of PC12 cells, PBS was added, the cells were removed from the plate wall, and the cells were measured with a hematocytometer and dispersed in fresh medium at a concentration of 2 × 10 ⁶ cell / 100π. After 24 hours, each flower extract was added and reacted for 2 hours. After removing the medium, the high concentration of the K ⁺ buffer, and then incubated in a carbon dioxide gas incubator for 15 minutes, the supernatant was removed and the cell membrane was separated using a lysis buffer. The separated cell membrane proteins were subjected to electrophoresis using 5 to 21% SDS-PAGE gel. The electrotransfer was performed as follows. The NC membrane (Nitrocellulose membrane) was immersed in the transfer buffer for 5 minutes and placed on a cassette, and the gel to be transferred was slightly wetted with transfer buffer and placed on the membrane. Prepare a transfer by making a gel sandwich, transfer it at 100 V for 1 hour, disassemble the device, dye the transfer gel in Ponceau S solution (Sigma-Altrik) for 5 minutes, and dye with distilled water. The band was washed by washing. The dyed Ponceau S was completely decolorized with PBST and blocked by stirring for 1 hour (room temperature) in a blocking solution. After washing with PBST, the primary antibody (SNAP-25) was added and stirred at 4 ° C. overnight. After washing twice with PBST for 5 minutes, a secondary antibody (antibody isolated from affinity by binding to anti-antimouse IgG and peroxidase, No. A4416, Lot. 125K6059, Sigma) was recommended. : 2000) was diluted with a blocking solution and reacted with stirring at room temperature for about 1 hour. After the reaction was washed three times with PBST and then mixed in an enhanced chemiluminescense (ECL) kit and evenly reacted with the membrane was detected.

It was confirmed whether the flower extract inhibited SNARE complex formation in PC12 cells by the above method, and the results are shown in FIG. 8.

As a result, some of the results were inconsistent with the experimental results of FIGS. 5, 6A, and 6B, but the roots of 8, oyster tree 47, scab flower 48, chestnut tree 49, and Dhayaro 69, etc. The effect of the extract on the inhibition of SNARE complex formation in PC12 cells was relatively high. SNARE complex 1 is a SNARE complex consisting of one each of syntaxin 1a, SNAP-25 and VAMP-2, and SNARE complex 2 is a complex in which the SNARE complex is large through domain swapping. In addition, N.con means that before processing the high concentration of K ⁺ buffer to know the amount of SNARE complex that is always present, Con. By adding only methanol without the addition of other extracts after treatment of high concentration of K ⁺ buffer Show how induced the formation of the SNARE complex is. Therefore, it can be confirmed that a significant amount of SNARE complex formation was inhibited through the treatment of the extract.

Although the above has been described with reference to a preferred embodiment of the present invention, those skilled in the art will be able to variously modify and change the present invention without departing from the spirit and scope of the invention described in the claims below. It will be appreciated.

While the effects of argireline, the main ingredient of current Botox products, are uncertain, and due to the peculiarity of synthetic peptides, they are disadvantageous in causing skin side effects. The raw material can be provided at a lower cost than the botox to be suppressed, and since it is a natural material, a user-friendly approach is possible, so the industrial application is easy.

1 depicts the amino acid sequence of syntaxin 1a protein among the three proteins involved in SNARE complex formation.

Figure 2 shows the amino acid sequence of the SNAP-25 protein among the three proteins involved in SNARE complex formation.

Figure 3 shows the amino acid sequence of the VAMP-2 protein among the three proteins involved in SNARE complex formation.

Figure 4 shows the results of Sodium dodecyl sulfate-polyacryamide gel electrophoresis (SDS-PAGE). The numbers at the bottom of the gel photograph show the serial numbers in Table 1.

FIG. 5 shows SDS-PAGE analysis of flower extracts selected from the results of FIG. 4, showing flower extracts having the highest inhibitory effect of SNARE complex formation.

6a and 6b suggest that the flower extracts of the present invention, yellow root, oyster tree, scab, chestnut, seo, ryoyang and dhayaro flower extracts prevent membrane fusion by SNARE complex formation.

Figure 7 shows the results of inhibiting neurotransmitter release from PC12 cells of the root, oyster bark, scab, chestnut, cypress, ryoyang and dhayaro flower extracts of the present invention.

8 is a result of confirming by inhibiting the formation of SNARE complex in PC12 cells of the root, oyster, scab, chestnut and dhayaro flower extract of the present invention, by performing Western blot using the antibody SNAP-25.

<110> SUNGKYUNKWAN UNIVERSITY Foundation for Corporate Collaboration <120> SNARE complex formation inhibiting composition comprising natural          extracts <130> IPM-34421 <160> 5 <170> KopatentIn 1.71 <210> 1 <211> 288 <212> PRT <213> Artificial Sequence <220> <223> Syntaxin 1a <400> 1 Met Lys Asp Arg Thr Gln Glu Leu Arg Thr Ala Lys Asp Ser Asp Asp   1 5 10 15 Asp Asp Asp Val Thr Val Thr Val Asp Arg Asp Arg Phe Met Asp Glu              20 25 30 Phe Phe Glu Gln Val Glu Glu Ile Arg Gly Phe Ile Asp Lys Ile Ala          35 40 45 Glu Asn Val Glu Glu Val Lys Arg Lys His Ser Ala Ile Leu Ala Ser      50 55 60 Pro Asn Pro Asp Glu Lys Thr Lys Glu Glu Leu Glu Glu Leu Met Ser  65 70 75 80 Asp Ile Lys Lys Thr Ala Asn Lys Val Arg Ser Lys Leu Lys Ser Ile                  85 90 95 Glu Gln Ser Ile Glu Gln Glu Glu Gly Leu Asn Arg Ser Ser Ala Asp             100 105 110 Leu Arg Ile Arg Lys Thr Gln His Ser Thr Leu Ser Arg Lys Phe Val         115 120 125 Glu Val Met Ser Glu Tyr Asn Ala Thr Gln Ser Asp Tyr Arg Glu Arg     130 135 140 Cys Lys Gly Arg Ile Gln Arg Gln Leu Glu Ile Thr Gly Arg Thr Thr 145 150 155 160 Thr Ser Glu Glu Leu Glu Asp Met Leu Glu Ser Gly Asn Pro Ala Ile                 165 170 175 Phe Ala Ser Gly Ile Ile Met Asp Ser Ser Ile Ser Lys Gln Ala Leu             180 185 190 Ser Glu Ile Glu Thr Arg His Ser Glu Ile Ile Lys Leu Glu Asn Ser         195 200 205 Ile Arg Glu Leu His Asp Met Phe Met Asp Met Ala Met Leu Val Glu     210 215 220 Ser Gln Gly Glu Met Ile Asp Arg Ile Glu Tyr Asn Val Glu His Ala 225 230 235 240 Val Asp Tyr Val Glu Arg Ala Val Ser Asp Thr Lys Lys Ala Val Lys                 245 250 255 Tyr Gln Ser Lys Ala Arg Arg Lys Lys Ile Met Ile Ile Is Cys Cys             260 265 270 Val Ile Leu Gly Ile Ile Ile Ala Ser Thr Ile Gly Gly Ile Phe Gly         275 280 285 <210> 2 <211> 206 <212> PRT <213> Artificial Sequence <220> <223> SNAP-25 <400> 2 Met Ala Glu Asp Ala Asp Met Arg Asn Glu Leu Glu Glu Met Gln Arg   1 5 10 15 Arg Ala Asp Gln Leu Ala Asp Glu Ser Leu Glu Ser Thr Arg Arg Met              20 25 30 Leu Gln Leu Val Glu Glu Ser Lys Asp Ala Gly Ile Arg Thr Leu Val          35 40 45 Met Leu Asp Glu Gln Gly Glu Gln Leu Glu Arg Ile Glu Glu Gly Met      50 55 60 Asp Gln Ile Asn Lys Asp Met Lys Glu Ala Glu Lys Asn Leu Thr Asp  65 70 75 80 Leu Gly Lys Phe Cys Gly Leu Cys Val Cys Pro Cys Asn Lys Leu Lys                  85 90 95 Ser Ser Asp Ala Tyr Lys Lys Ala Trp Gly Asn Asn Gln Asp Gly Val             100 105 110 Val Ala Ser Gln Pro Ala Arg Val Val Asp Glu Arg Glu Gln Met Ala         115 120 125 Ile Ser Gly Gly Phe Ile Arg Arg Val Thr Asn Asp Ala Arg Glu Asn     130 135 140 Glu Met Asp Glu Asn Leu Glu Gln Val Ser Gly Ile Gly Asn Leu 145 150 155 160 Arg His Met Ala Leu Asp Met Gly Asn Glu Ile Asp Thr Gln Asn Arg                 165 170 175 Gln Ile Asp Arg Ile Met Glu Lys Ala Asp Ser Asn Lys Thr Arg Ile             180 185 190 Asp Glu Ala Asn Gln Arg Ala Thr Lys Met Leu Gly Ser Gly         195 200 205 <210> 3 <211> 116 <212> PRT <213> Artificial Sequence <220> <223> VAMP-2 <400> 3 Met Ser Ala Thr Ala Ala Thr Val Pro Pro Ala Ala Pro Ala Gly Glu   1 5 10 15 Gly Gly Pro Pro Ala Pro Pro Pro Asn Leu Thr Ser Asn Arg Arg Leu              20 25 30 Gln Gln Thr Gln Ala Gln Val Asp Glu Val Val Asp Ile Met Arg Val          35 40 45 Asn Val Asp Lys Val Leu Glu Arg Asp Gln Lys Leu Ser Glu Leu Asp      50 55 60 Asp Arg Ala Asp Ala Leu Gln Ala Gly Ala Ser Gln Phe Glu Thr Ser  65 70 75 80 Ala Ala Lys Leu Lys Arg Lys Tyr Trp Trp Lys Asn Leu Lys Met Met                  85 90 95 Ile Ile Leu Gly Val Ile Cys Ala Ile Ile Leu Ile Ile Ile Ile Val             100 105 110 Tyr Phe Ser Thr         115 <210> 4 <211> 76 <212> PRT <213> Artificial Sequence <220> <223> SyNH3 <400> 4 Ala Leu Ser Glu Ile Glu Thr Arg His Ser Glu Ile Ile Lys Leu Glu   1 5 10 15 Asn Ser Ile Arg Glu Leu His Asp Met Phe Met Asp Met Ala Met Leu              20 25 30 Val Glu Ser Gln Gly Glu Met Ile Asp Arg Ile Glu Tyr Asn Val Glu          35 40 45 His Ala Val Asp Tyr Val Glu Arg Ala Val Ser Asp Thr Lys Lys Ala      50 55 60 Val Lys Tyr Gln Ser Lys Ala Arg Arg Lys Lys Ile  65 70 75 <210> 5 <211> 96 <212> PRT <213> Artificial Sequence <220> <223> VpS <400> 5 Met Ser Ala Thr Ala Ala Thr Val Pro Pro Ala Ala Pro Ala Gly Glu   1 5 10 15 Gly Gly Pro Pro Ala Pro Pro Pro Asn Leu Thr Ser Asn Arg Arg Leu              20 25 30 Gln Gln Thr Gln Ala Gln Val Asp Glu Val Val Asp Ile Met Arg Val          35 40 45 Asn Val Asp Lys Val Leu Glu Arg Asp Gln Lys Leu Ser Glu Leu Asp      50 55 60 Asp Arg Ala Asp Ala Leu Gln Ala Gly Ala Ser Gln Phe Glu Thr Ser  65 70 75 80 Ala Ala Lys Leu Lys Arg Lys Tyr Trp Trp Lys Asn Leu Lys Met Met                  85 90 95  

Claims (4)

Cosmetic composition for improving skin wrinkles by inhibiting the formation of SNARE complexes and controlling the release of neurotransmitters, including the extract of the flower root as an active ingredient. According to claim 1, The flower extract is characterized in that extracted with water or C ₁ ~ C ₄ alcohol, cosmetic composition for improving skin wrinkles. The cosmetic composition for improving skin wrinkles according to claim 2, wherein the C ₁ to C ₄ alcohol is methanol or ethanol. The cosmetic composition according to any one of claims 1 to 3, wherein the cosmetic composition is a solution, a suspension, an emulsion, a paste, a gel, a cream, a lotion, a powder, a soap, a surfactant-containing cleansing oil, a powder foundation, an emulsion foundation Cosmetic composition for improving skin wrinkles having a formulation selected from the group consisting of, wax foundation and spray.

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