KR20090026838A - 3-O-[beta-D-GLUCOPYRANOSYL(1->4)-alpha-L-ARABINOPYRANOSYL] -HEDERAGENIN INCREASING TRANSDUCING ACTIVITY OF CELL TRANSDUCING FUSION PROTEIN - Google Patents

Abstract

A 3-O-(beta-D-glucopyranosyl(1->4)-alpha-L-arabinopyranosyl)-hederagenin is provided to be applied to aiding agent, medicine, cosmetic material increasing infiltration efficiency of the cell permeability fusion protein including Tat-SOD etc. and various disease treating field. A 3-O-(beta-D-glucopyranosyl(1->4)-alpha-L-arabinopyranosyl)-hederagenin improves cell permeation rate of cell permeability fusion protein. The cell permeability fusion protein is formed by covalent bonding HIV-Tat(49-57) consisting of 6~15 amino acids, oligolysine, oligoarginine, oligo(lysine and arginine), protein transport domain selected from PEP-1 and cell non-porous protein. The cell permeability fusion protein is HIV-Tat-Cu,Zn superoxide dismutase. The 3-O- (beta-D-glucopyranosyl(1->4)-alpha-L-arabinopyranosyl)-hederagenin is separated from Fatsia japonica.

Description

3-O-[[beta] -D-glucopyranosyl (1 → 4)-[alpha] -L-arabinofyranosyl] -hederagenin {3-O-[[beta] -D- for increasing cell permeation fusion protein cell introduction activity Glucopyranosyl (1 → 4) -α-L-arabinopyranosyl] -hederagenin increasing transducing activity of cell transducing fusion protein}

The present invention relates to a compound for improving cell permeability of cell permeable fusion proteins.

Skin is constantly exposed to harmful environments such as ultraviolet (UV) light and air pollution. Reactive Oxygen Species (ROS) are known to be involved in inflammatory skin diseases, skin cancer, skin autoimmune diseases, phototoxicity, photosensitivity, and skin aging [Cross, C.C., Halliwell, B. and Borish, E.T. (1987) Ann. Intern. Med., Davis Conference 107, 526-545.].

Free radicals are inevitably produced as by-products of many intracellular metabolic processes in all living things that use oxygen for energy. These free radicals damage biopolymers such as proteins, nucleic acids and fats in cells. It is deeply involved in the progress of various diseases in the human body. In particular, it is involved in carcinogenesis, stroke, arthritis, arteriosclerosis, radiation damage, and inflammatory reactions, and plays an important role in promoting aging in normal aging [Floyd, R.A. (1990) FASEB J. 4, 2587-2597. Anderson, W. F. (1998) Nature 392, 25-30, Halliwell B. and Gutteridge J.M.C. (1999) Free radicals in biology and medicine, Oxford University Press, Oxford.

Instead of treating human diseases with traditional drugs, gene therapy or protein therapy is the key to gene therapy or protein therapy. Recently, as it is known that various human diseases are caused by abnormal activity of cellular proteins, the development of drugs that can treat fatal human diseases by regulating the activity of these proteins is of interest worldwide.

In vivo polymers, in particular the skin, are always exposed to the harmful effects of these inflammations and free radicals, so there is a great interest in using superoxide dismutase for therapeutic purposes in various skin diseases. At the moment the most attention is on gene therapy. However, gene therapy is not easy to carry genes, low expression in target cells, short duration of protein expression in cells, and very difficult to artificially control the amount of protein expressed in target cells. And several problems [Del Zoppo, GJ, Wagner, S. and Tagaya, M. (1997) Drugs 54, 9-38.

Thus, the present inventors have continued to study a fusion protein capable of penetrating into the cell through the cell membrane by binding a protein transducing domain consisting of 5 to 12 amino acids to the protein. It was confirmed in experiments using HeLa cells that the HIV-TAt protein transport domain was fused to human Cu, Zn-superoxide dismutase so that the fusion protein could be effectively permeated into cells [Kwon et al. FEBS letters 485, 163-167], Korean Patent No. 445186 added copper ions to Tat-Cu, Zn-superoxide dismutase to increase cell permeability of superoxide dismutase fusion protein having cell permeability.

Fatsia japonica is a unique evergreen plant that exists in Korea and Japan, with 6 to 8 broad leaves, mostly grown as shrubs. The arm does not have falcarinol, an atopic inducer of other plants of the same family. It has also long been used in the private sector to treat rheumatism and expectorants [Oka, K., F. Saito, T. Yasuhara, and A. Sugimoto. (1999) Contact Dermatitis 40, 209-2013, Boll. PM and L. Hansen. (1987) Phytochem. 26, 2955-2956.

An object of the present invention is to improve the cell permeability of cell-permeable fusion proteins.

It is also an object of the present invention to ensure that cell-permeable fusion proteins efficiently permeated into cells or tissues exhibit normal activity.

In addition, an object of the present invention is to use a compound derived from natural products in order to achieve the above object.

In order to achieve the above object, the present inventors extracted a compound showing activity from the palm of the hand, and identified its structure, and mixed it with HIV-Tat (hereinafter referred to as "Tat" in the detailed description, claims and drawings) of the protein. The peptide is mixed with an external protein, human Cu, Zn- superoxide dismutase ("SOD", "Cu, Zn-SOD", "superoxide dismutase" in the following detailed description, claims and drawings). Fusion protein (combined with "Tat-SOD" in the following description, claims and drawings) was overexpressed in Escherichia coli and purified by metal chelating affinity chromatography. Purified fusion protein was cultured for intracellular penetration and activity by compound 3-O- [β-D-glucopyranosyl (1 → 4) -α-L-arabinofyranosyl] -hederazinine Skin cell and skin tissue experiments were confirmed.

The present invention provides 3-O-[[beta] -D-glucopyranosyl (1 → 4)-[alpha] -L-arabinofyranosyl] -hederazenine {3-O-[[beta] -D through structural identification of human arms. -Glucopyranosyl (1 → 4) -α-L-arabinopyranosyl] -hederagenin; In the following detailed description, claims and drawings, "combined with" OGAH "has been shown to effectively increase the penetration efficiency of Tat-SOD into cells and tissues, through which superoxide dismutes free radicals and various diseases associated therewith. By using OGAH which effectively penetrates Izu, it can be effectively used for protein treatment for various free radical-related diseases.

OGAH increases the penetration efficiency and activity of the fusion protein, and thus can be widely used in the functional cosmetics industry more effectively in addition to drugs and supplements for the treatment of various diseases.

Several researchers have found that protein transport domains, such as the Tat protein of human immunodeficiency virus (HIV-1), can be used as carriers to transport heterologous proteins into cells. In the present invention, a study was conducted to find a compound or method for improving the cellular permeation efficiency of the cell permeable fusion protein in which the protein transport domain is bound to the heterologous protein.

The present invention relates to a compound for improving the cell permeability of the cell-permeable fusion protein, more specifically the compound 3-O- [β-D-glucopyranosyl (1 → 4) -α-L-arabino isolated from the arm Pyranosyl] -hederazenin {3-O- [β-D-Glucopyranosyl (1 → 4) -α-L-arabinopyranosyl] -hederagenin} (hereinafter referred to as "OGAH" in the description, claims and drawings of the invention). Mixed) increased the penetration efficiency of Tat-SOD fusion protein into cells and tissues.

The present inventors first purely separated 3-O-[[beta] -D-glucopyranosyl (1 → 4)-[alpha] -L-arabinofyranosyl] -hederagenin from the palmar arm in various ways. When the isolated material was treated with the purified Tat-SOD fusion protein, it was confirmed that the fusion protein was more efficiently transported to the cells in a concentration- and time-dependent manner than the control treated with Tat-SOD only. OGAH effectively infiltrated Tat-SOD fusion protein not only into cultured cells but also into skin tissues of mice, and it was found that the activity of fusion protein infiltrated into cells and tissues was significantly increased. These results suggest that OGAH could be used as an adjuvant to increase the penetration efficiency of Tat-SOD fusion proteins and in various disease treatment fields.

In a specific embodiment of the present invention, "Tat-SOD fusion protein" is mentioned as an example of the cell-induced fusion protein, but it is clearly understood that the scope of the present invention is not limited to the fusion protein. The method for preparing the Tat-SOD fusion protein was performed by the conventional method developed by the present inventors as described in the following Examples. Tat-SOD fusion protein prepared as described above is useful for the prevention and treatment / improvement of various immunological and inflammatory diseases including asthma, atopic dermatitis and dermatitis. It can be applied in various fields such as health functional food.

In the present invention, 3-O-[[beta] -D-glucopyranosyl (1 → 4)-[alpha] -L-arabinofyranosyl] -hederazenin, a compound that enhances the cell transduction activity of the cytotoxic fusion protein, is a drug. It can be used as a supplement, health functional food, cosmetics, and when used as a pharmaceutically acceptable carrier can be added.

Pharmaceutical compositions containing 3-O- [β-D-glucopyranosyl (1 → 4) -α-L-arabinofyranosyl] -hederazenin as an active ingredient are carriers commonly accepted in the pharmaceutical art. It can be combined with and formulated in oral or injection form by conventional methods. Oral compositions include, for example, tablets and gelatin capsules, which, in addition to the active ingredient, may contain diluents (e.g. lactose, dextrose, sucrose, mannitol, sorbitol, cellulose and / or glycine), lubricants (e.g. silica, talc) , Stearic acid and its magnesium or calcium salts and / or polyethylene glycols, the tablets also contain binders (e.g. magnesium aluminum silicate, starch paste, gelatin, methylcellulose, sodium carboxymethylcellulose and / or polyvinylpyrrolidone). ), And optionally a disintegrant (eg starch, agar, alginic acid or its sodium salt) or boiling mixture and / or absorbent, colorant, flavor and sweetener. Injectable compositions are preferably aqueous isotonic solutions or suspensions, and the compositions mentioned are sterile and / or contain auxiliaries such as preservatives, stabilizers, wetting or emulsifier solution promoters, salts or buffers for controlling osmotic pressure. They may also contain other therapeutically valuable substances.

The pharmaceutical preparations thus prepared may be administered orally as desired, or parenteral, that is, intravenous, subcutaneous, intraperitoneal, or topically applied, and when applied to asthma, etc. in the art to which the present invention pertains. As is well known to those skilled in the art, it may be formulated into inhaled or sprayed formulations. The dose may be administered by dividing the daily dose of 0.001-100 mg / kg in one to several times. Dosage levels for a particular patient may vary depending on the patient's weight, age, sex, health condition, time of administration, method of administration, rate of excretion, severity of the disease, and the like.

Furthermore, the present invention is a pharmaceutical composition comprising the 3-O- [β-D-glucopyranosyl (1 → 4) -α-L-arabinofyranosyl] -hederazenin and Tat-SOD fusion protein as an active ingredient. It provides a pharmaceutical composition useful for the prevention and treatment of various immune diseases and inflammatory diseases, including asthma, atopic dermatitis, dermatitis, characterized in that it comprises an acceptable carrier.

The present invention also provides a health functional food composition comprising the above 3-O- [β-D-glucopyranosyl (1 → 4) -α-L-arabinopyranosyl] -hederagenin as an active ingredient.

The present invention also provides a cosmetic composition comprising the 3-O- [β-D-glucopyranosyl (1 → 4) -α-L-arabinopyranosyl] -hederazenin and a cell-induced fusion protein as an active ingredient. It provides a composition for external application to the skin. The coating agent which uses the fusion protein of this invention as an active ingredient can be easily manufactured in any form according to the conventional manufacturing method. As an example, in preparing a cream coating agent, the Tat-SOD fusion protein of the present invention and 3-O- [β-D-glucopyranosil in a water-based (O / W) or water-in-oil (W / O) cream base may be used. (1 → 4) -α-L-arabinofyranosyl] -hederagenin, which is used as a fragrance, chelating agent, pigment, antioxidant, preservative, etc. as necessary, and for the purpose of improving physical properties. Synthetic or natural materials such as proteins, minerals and vitamins can be used in combination.

In addition, the fusion protein of the present invention can be used as a cosmetic, by adding a pH adjuster, fragrance, emulsifier, preservatives, etc. as needed, in the usual cosmetic preparation method, lotion, gel, water-soluble powder, fat-soluble powder, water-soluble liquid, cream Or an essence or the like.

The present invention also provides a method for efficient delivery of a cytotoxic fusion protein into a cell, including a Tat-SOD fusion protein. Intracellular delivery of a fusion protein according to the invention is a cell or tissue fusion protein in the form of covalently linked protein transport domains, including HIV-1 Tat, oligolysine, oligoarginine, oligos (lysine, arginine), Pep-1 peptide In the treatment, the treatment is performed before or concurrently with the fusion protein. An example of the transport domain of the present invention is an HIV Tat peptide consisting of nine amino acids of aa 49-57 and consisting of an amino acid sequence such as SEQ ID NO: 1. However, the protein transport domain of the present invention is not limited to the Tat peptide of SEQ ID NO: 1, and the production of a peptide having a function similar to the Tat peptide by partial substitution, addition or lack of the amino acid sequence of Tat in the field of the present invention belongs. Since it is easy for a person skilled in the art, a protein transport domain composed of 7 to 25 amino acids, including 5 or more lysine or arginine, and a protein transport function that performs the same or similar protein transport function by substituting partial amino acids therefrom. It will be apparent that the fusion protein using the domain belongs to the scope of the present invention.

Definitions of main terms used in the detailed description of the present invention are as follows.

"Fused protein" means a covalent complex comprising a protein transport domain and a target protein, formed by genetic fusion or chemical bonding of the transport domain and the target protein (which is referred to as "SOD" in one embodiment of the invention). .

A “target protein” is a molecule that is not originally a transport domain or fragment thereof that cannot enter the target cell or is not able to enter the target cell at its original useful rate, and is a molecule of the transport domain or of the transport domain-target protein complex prior to fusion with the transport domain. Refers to the target protein portion. Target proteins include polypeptides, proteins, and peptides. In one embodiment of the present invention, SOD is exemplified.

"Fused protein" means a complex comprising a transport domain and one or more target protein moieties and formed by genetic fusion or chemical bonding of the transport domain and the target protein.

In addition, the "genetic fusion" means a linear, covalent linkage formed through the genetic expression of the DNA sequence encoding the protein.

In addition, "target cell" means a cell to which a target protein is delivered by a transport domain, and a target cell refers to a cell in or outside the body. That is, a target cell is meant to include cells in the body, that is, microorganisms found in cells or living animals or humans constituting organs or tissues of a living animal or human. In addition, target cells are meant to include extracellular cells, ie cultured animal cells, human cells or microorganisms.

In the present invention, the "transport domain" may be covalently bonded to a polymer organic compound such as oligonucleotide, peptide, protein, oligosaccharide or polysaccharide to introduce the organic compound into cells without requiring a separate receptor, carrier, or energy. Say what it is.

In addition, in the present specification, expressions of "transport", "penetration", "transport", "transfer", "transmission", "pass" for "introducing" a protein, a peptide, etc. into a cell were mixed.

Hereinafter, the configuration of the present invention in more detail through the embodiment. However, the scope of the present invention is not limited only to the description of the examples.

< Example  1: material>

Restriction enzymes and T4 DNA ligase were purchased from Promega (USA) and Pfu polymerase was purchased from stratagene (USA). Tat oligonucleotides were synthesized in Gibco BRL custom primer (USA). IPTG was purchased from Duchefa (Netherland). pET-15b and BL21 (DE3) plasmids were purchased from Novagen (USA) and Ni-nitrilotriactic acid sepharose superflow was purchased from Qiagen (Germany). Human Cu, Zn-superoxide dismutase cDNA was isolated from human placental cDNA library by PCR method [Kang, JH, Choi, BJ and Kim, SM (1997) J. Biochem . Mol . Biol . 30, 60-65}. All other reagents were used as express products.

< Example  2: Tat - SOD  Preparation and Transformation of Fusion Protein Expression Vectors transformation )>

To produce a Tat-SOD fusion protein, first, a pET-Tat expression vector containing a basic domain of HIV-1 Tat (amino acids 49-57) was made. Two types of oligonucleotides (top chain, 5'-TAGGAAGAAGCGGAGACAGCGACGAAGAC-3 '; lower chain, 5'-TCGAGTCTTCGTCGCTGTCTCCGCTTCTTCC-3') corresponding to the Tat basic domain were ligated into pET15b cut with NdeI - XhoI restriction enzymes. It was. Then, based on the cDNA sequence of human Cu, Zn-SOD Two kinds of oligonucleotides were synthesized. The forward primer has a Xho I restriction site of 5'-CTCGAGGCGACGAAGGCCGTGTGCGTG-3 'and the reverse primer sequence has a BamH I restriction site of 5'-GGATCCTTATTG-GGCGATCCCAATTAC-3'.

Polymerase chain reaction (PCR) was performed in a thermal cycle reactor (Perkin-Elmer, model 9600), and the reaction mixture was placed in a 50 µl silicon tube (siliconized reaction tube) and heated at 94 ° C for 5 minutes. PCR reactions induced 30 minutes of denaturation at 94 ° C., annealing at 55 ° C. for 1 minute, and final extension at 74 ° C. for 1 minute 30 seconds, and 72 ° C. for 5 minutes. After PCR, the reactants were separated by agarose gel electrophoresis, which was then linked to a TA cloning vector (Invitrogen, Sandiego, USA). This vector was then transformed into competent cells and the plasmid was isolated from the transformed bacteria by alkaline lysis method. TA vectors containing human Cu, Zn-SOD cDNA were synthesized with Xho I and Cleavage with BamH I and insertion into the HIV-1 Tat expression vector. E. coli BL21 (DE3) transformed with Tat-SOD fusion protein was selected, then colonies were inoculated in 100 ml LB medium and IPTG (0.5 mM) was added to the medium to induce overexpression of recombinant Tat-SOD. . Overexpressed Tat-SOD was confirmed by SDS-PAGE and Western blot analysis.

< Example  3: Tat - SOD  Purification of Fusion Proteins>

Transformed E. coli BL21 was put in LB medium containing ampicillin (ampicillin) and incubated with stirring at 200 rpm at 37 ℃. When the bacterial concentration in the culture medium showed OD ₆₀₀ = 0.5-1.0, IPTG was added to the medium to bring the final concentration to 0.5 mM, followed by further incubation for 3 hours. The cultured cells were collected by centrifugation and sonicated by adding 6M urea to 5ml binding buffer (5mM imidazole, 0.5M NaCl, 20mM Tris-HCl, pH 7.9).

Centrifugation and the supernatant was immediately 2.5㎖ Ni ^{2 +} - nitro roteuri ethyl Sepharose load to the column, washed with buffer, binding buffer and 6 times the volume of 10-fold volume (60mM imidazole, 0.5M NaCl, 20mM Tris-HCl , pH 7.9 ) And the fusion protein was eluted with elution buffer (1 M imidazole, 0.5 M NaCl, 20 mM Tris-HCl, pH 7.9). Subsequently, the fractions containing the fusion protein were collected and PD10 chromatography was performed to remove salts contained in the fractions. Protein concentration was measured by Bradford method using bovine serum albumin as a standard.

< Example  4: Determination of enzyme activity>

The activity of Cu, Zn-SOD was determined by spectrophotometric observation of the degree of inhibition of ferricytochrome c reduction by xanthine / xanthine oxidase reaction according to McCord and Fridovich's method (1969). It was measured [McCord, JM and Fridovich, I. (1969) J. Biol. Chem. 244, 6049-6055.

Standard assays were performed in 50 mM potassium phosphate buffer (pH 7.8) containing 2 ml of 0.1 mM EDTA at 25 ° C. The reaction mixture contained 10 μM pericytochrome c, 50 μM xanthine and a sufficient amount of xanthine oxidase and the reduction of pericytochrome c was measured at 550 nm. Under the above conditions, the amount of SOD that decreases pericytochrome c by 50% was defined as 1 unit.

< Example  4: Cell Culture and Recombination Tat - Annexin  Intracellular Permeation of Fusion Proteins>

Fibroblasts, skin cells, provide 95% air and 5% CO ₂ at 37 ° C, 10% fetal bovine serum ("FBS") and antibiotics (100 µg / ml streptomycin, 100 U / ml penicillin). Cultured in DMEM (Dulbecco's Modified Eagle's Medium) medium.

To observe intracellular penetration of Tat-SOD, cells were grown for 4-6 hours in 6-well plates, replaced with 1 ml fresh culture medium without FBS, and various concentrations of Tat-SOD fusion protein were cultured. Treated within. After one hour, cells were treated with trypsin-EDTA (Gibco BRL) and washed sufficiently with PBS (phosphate buffered saline). After crushing the cells, the amount of Tat-SOD fusion protein permeated into the cells was compared by Western blot analysis.

< Example  5: Weston Blot  Analysis>

Western blotting was performed to confirm intracellular penetration of the Tat-SOD fusion protein. Tat-SOD fusion protein was treated to the prepared cells by concentration and time, and then cells were collected and western blotting was performed. Proteins in the cell mill were separated by 12% SDS polyacrylamide gel electrophoresis, and the proteins in the gels were then electrophoresed to a nitrocellulose membrane (Amersham, UK). The protein-transferred nitrocellulose membrane was blocked with Tris Buffered Saline with Tween (TBST) buffer containing 5% skim milk powder at room temperature for two hours. The membrane was then treated with a rabbit anti-histidine polyclonal antibody (Santacruze, USA, 1: 1,000) for 1 hour. The primary antibody was washed with TBST buffer and then reacted with horseradish peroxidase-linked anti-mouse IgG antibody (1: 10,000 dilution) for one hour. Finally, ECL kit (ECL; Amersham) was used to identify the protein bands that respond to SOD monoantibodies.

< Example  6: immunohistostaining ( Immunohistochemistry )>

Experimental animals are anesthetized with 3% isoflurance containing a mixture of nitrogen and oxygen, followed by a perfusion wash. After perfusion, the organs were fixed with 0.1 M PBS containing paraformaldehyde (pH 7.4) and the fixed tissues were detached and soaked in 4% paraformaldehyde solution. Tissues were embedded in embedding medium (Reichert-Jung, Germany) and sectioned using cryostat (Reichert-Jung, Germany) and used in the present invention.

Prior to immunohistochemical staining, the frozen tissue sections were dried at room temperature for 30 minutes and then completely dried at 37 ° C. for at least 2 hours before starting the experiment. Tissue sections were buffered with 0.01 M PBS for 10 minutes and washed again with distilled water for 10 minutes. After washing twice with PBS for 7 minutes each, it was reacted for 20 minutes in a PBS solution containing 0.5% hydrogen peroxide to remove endogenous peroxidase (peroxidase) in the tissue. After washing twice with PBS for 7 minutes each, the tissues were reacted with 5% normal goat serum in PBS for 30 minutes to prevent non-specific reactions. Subsequently, the immunohistochemical reaction was performed using a conventional streptavidin-biotin peroxidase method.

The primary antibody used was rabbit anti-mouse tyrosine hydroxylase (Santa cruz, USA). All antibodies used for immunohistochemistry were diluted in 0.1M PBS (pH 7.4) containing 2% normal goat serum and 0.1% triton X-100. Brain tissue was diluted at 1: 200 primary antibody and reacted at 4 ° C. for 48 hours. The biotin-conjugated rabbit anti-mouse IgG (Vector, USA) was then diluted at 1: 200 and reacted at room temperature for 2 hours, followed by a 1: 200 peroxidase-linked streptavidin (peroxidase). -conjugated streptavidin) (Vector, USA) was reacted at room temperature for 2 hours. After the reaction of each step was washed 4-5 times with PBS solution.

Antigen-reacted tissues were developed for 1-5 minutes in a solution of Tris buffer (pH 7.4) containing 0.03% hydrogen peroxide and 0.05% DAB (3,3'-diaminobenzidine tetrachloride; Sigma, USA). After completion of the color reaction, the tissue was encapsulated using a water-soluble encapsulant crystal mount (Biomeda, USA) and then photographed after a microscope observation (Axioscope microscope; Carl Zeiss, Germany).

< Example  7: Arms  Fractionation and Separation>

After extracting, the dry limbs were extracted by refluxing 1 kg of tree trunks into 3 l of 95% ethanol and condensing all of them to obtain 67.3 g of ethanol extract. 1.2 liters of distilled water was added and suspended, followed by solvent fractions in the order of n-hexane, chloroform, and ethylacetate. 6.9 g), ethyl acetate soluble fraction (12.9 g) and the remaining four fractions (40.2 g) were obtained. Of the four fractions, water-soluble fractions (40.0 g) with excellent activity were subjected to column chromatography with Diaion HP-20 as a filler to obtain 100% water (fraction 1, 7.9 g), 25% methanol (fraction 2, 3.2 g). ), 6 fractions, 40% methanol (fraction 3, 5.9 g), 65% methanol (fraction 4, 6.9 g), 80% methanol (fraction 5, 3.1 g) and 100% methanol (fraction 6, 10.5 g) Got. Among these six fractions, fraction 5, which had excellent activity, was subjected to ODS column chromatography (JAI, recycle HPLC system, Japan), 30% methanol under isocratic conditions to obtain a final product (36.4 mg). Melting point, ¹ H, ¹³ C NMR and the like of the extracted material were measured and 3-O- [β-D-glucopyranosyl (1 → 4) -α-L-arabinofyranosyl]- It was confirmed to be hederagenin.

Example  Result

1) recombination Tat - SOD Fusion protein  Overexpression, refining and Intracellular  Penetration

The preparation, overexpression, purification and intracellular penetration of Tat-SOD fusion proteins have already been confirmed in cells and tissues by the present researchers [8].

2) Arms  Fractions and their fractions Tat - SOD Efficiency analysis for cell infiltration

Suspended by adding distilled water to the ethanol extract, followed by solvent fractionation in the order of n-hexane, chloroform, ethylacetate, n-hexane soluble fraction (H), chloroform soluble fraction (C), ethylacetate soluble fraction (E) and the remaining water fraction. Four fractions, such as phosphorus water soluble fraction (W), were obtained. The water soluble fraction was the best as a result of comparing the infiltration efficiency of Tat-SOD with four obtained fractions.

In addition, 6 fractions were obtained using column chromatography with Diaion HP-20 as a filler for water soluble fraction (40.0 g). Six fractions were used to confirm the infiltration efficiency of Tat-SOD cells.

3) Arms  Structure identification

Among the six fractions, PS-1 was obtained by using an active fraction with ODS column chromatography (JAI, recycle HPLC system, Japan) and 30% methanol isocratic conditions. Melting point, ¹ H, ¹³ C nmr, etc. of the extracted material were measured to determine that PS-1 is 3-O- [β-D-Glucopyranosyl (1 → 4) -α-L-arabinopyranosyl] -hederagenin. It was confirmed that (OGAH).

Colorless needles (MeOH); mp 246-248 ° [α] ¹⁶ D + 44.6 ° (c = 0.79, pyridine); ¹ H nmr (pyridine- d ₅ ) δ 0.84, 0.95, 0.99, 1.00, 1.04, 1.23 (each 3H, s, tert- Me), 3.30 (1H, dd, J = 4.0 and 13.5 Hz, H-18), 3.65-3.80 (3H, m, H-3 and sugar-H), 4.03-4.30 (6H, m, H ₂ -23 and sugar-H), 4.36 (1H-dd, J = 4.0 and 12.0 Hz glycosyl H- 6), 4.45 (1H, dd, J = 2.5 and 12.0 Hz, glucosyl H-6 '), 4.56 (1H, t, J = 6.5 Hz, arabinosyl H-2), 5.20 (1H, d, J = 6.5 Hz , arabinosyl H-1), 5.21 (1H, d, J = 7.5 Hz, glycosyl H-1), 5.49 (1H, brs, H-12); ¹³ C nmr (pyridine- d ₅ ) δ 13.5 (C-24), 16.1 (C-25), 7.5 (C-26), 18.3 (C-6), 23.7 (C-11), 23.8 (C-30 ), 23.9 (C-16), 26.0 (C-2), 28.4 (C-15), 31.0 (c-20), 33.0 (C-7), 33.3 (C-22), 33.3 (C-29) , 34.3 (C-21), 37.0 (C-10), 38.8 (C-1), 39.8 (C-18), 42.0 (C-18), 42.2 (C-14), 43.6 (C-4), 46.5 (C-19), 46.7 (C-17), 48.0 (C-5), 48.2 (C-9), 62.6 (glycosyl C-6), 64.9 (C-23), 65.0 (arabinosyl C-5) , 68.3 (arabinosyl C-3), 71.5 (glycosyl C-4), 73.7 (arabinosyl C-2), 76.3 (glycosyl C-2), 78.3 (glycosyl C-5), 81.5 (arabinosyl C-4), 82.3 (C-3), 104.0 (arabinosyl C-1), 105.0 (glycosyl C-1), 122.6 (C012), 144.9 (C-13), 180.2 (C-28).

4) 3-O- [β-D- Glucopyranosyl (1 → 4) -α-L- arabinopyranosyl ]- hederagenin  By (OGAH) Tat - SOD Intracellular Permeation

In order to observe the intracellular penetration of Tat-SOD by OGAH, the purified Tat-SOD was administered to the cultured cells by concentration (1-5 μM) and hourly (10-60 min), followed by Western blotting. As a result, it was confirmed that Tat-SOD is permeated into cells by time and concentration.

In addition, as a result of measuring the activity of SOD after intracellular permeation, the activity of enzyme was significantly increased by concentration and time.

5) skin Within the organization Tat - SOD Permeation and Activity of

OGAH and Tat-SOD were applied to mice to determine whether TGA was permeated by OGAH into mouse skin tissues, and confirmed by immunohistostaining and enzyme activity analysis. As shown in the figure, OGAH confirmed that Tat-SOD increased the penetration efficiency into mouse skin tissue and significantly increased the enzyme activity.

Figure 1 shows the Tat-SOD penetration efficiency according to the solvent fraction. After 300 μg of each fraction was first pretreated with cells for 12 hours, 3 μM of Tat-SOD fusion protein was treated for 1 hour and then confirmed by Western blotting.

NC: Negative Control (Nothing Treated Cells)

PC: positive control (cells treated with Tat-SOD only)

H: n-hexane soluble fraction

C: chloroform soluble fraction

W: water soluble fraction

E: ethyl acetate soluble fraction

Figure 2 shows the Tat-SOD penetration efficiency according to the fractions. After 300 μg of each fraction was first pretreated with cells for 12 hours, 3 μM of Tat-SOD fusion protein was treated for 1 hour and then confirmed by Western blotting.

NC: negative control, PC: positive control,

1: 100% water, 2: 25% methanol,

3: 40% methanol, 4: 65% methanol,

5: 80% methanol, 6: 100% methanol.

3 is a chemical formula showing the structure of 3-O- [β-D-glucopyranosyl (1 → 4) -α-L-arabinofyranosyl] -hederagenin found through physical property analysis such as NMR.

Figure 4 shows the results of investigation of the intracellular penetration of Tat-SOD fusion protein by concentration, time by OGAH.

By concentration: After treatment for 12 hours OGAH to 50 ㎍ / ㎖ cells, Tat-SOD fusion protein treatment by concentration (1-5uM) for 1 hour was confirmed by Western blotting.

Hourly: After treatment for 12 hours to 50μg / ㎖ OGAH cells, the data was confirmed by Western blotting by treating the Tat-SOD fusion protein hourly (10-60 minutes).

5 is a result of examining the SOD activity of the Tat-SOD fusion protein infiltrated into cells by concentration and time by OGAH.

6 is a result of skin tissue penetration of Tat-SOD by OGAH. 100 μg of OGAH was applied to the skin of the mouse 6 hours in advance, and 50 μg of the fusion protein was treated for 1 hour, followed by tissue staining and activity analysis.

<110> Industry Academic Cooperation Foundation, Hallym University          Chuncheon Bioindustry Foundation <120> 3-O- [B-D-glucopyranosyl (1> 4) -a-L-arabinopyranosyl] -hederagenin          increasing transducing activity of cell transducing fusion          protein <130> hallymU-OGAH <160> 7 <170> KopatentIn 1.71 <210> 1 <211> 29 <212> DNA <213> Human immunodeficiency virus type 1 <400> 1 taggaagaag cggagacagc gacgaagac 29 <210> 2 <211> 31 <212> DNA <213> Human immunodeficiency virus type 1 <400> 2 tcgagtcttc gtcgctgtct ccgcttcttc c 31 <210> 3 <211> 27 <212> DNA <213> Artificial Sequence <220> <223> primer <400> 3 ctcgaggcga cgaaggccgt gtgcgtg 27 <210> 4 <211> 27 <212> DNA <213> Artificial Sequence <220> <223> primer <400> 4 ggatccttat tgggcgatcc caattac 27 <210> 5 <211> 9 <212> PRT <213> Human immunodeficiency virus type 1 <400> 5 Arg Lys Lys Arg Arg Gln Arg Arg Arg   1 5 <210> 6 <211> 492 <212> DNA <213> Artificial Sequence <220> <223> artificial sequence for Tat-superoxide dismutase fusion protein <220> <221> CDS (222) (1) .. (489) <400> 6 agg aag aag cgg aga cag cga cga aga atg gcg acg aag gcc gtg tgc 48 Arg Lys Lys Arg Arg Gln Arg Arg Arg Met Ala Thr Lys Ala Val Cys   1 5 10 15 gtg ctg aag ggc gac ggc cca gtg cag ggc atc atc aat ttc gag cag 96 Val Leu Lys Gly Asp Gly Pro Val Gln Gly Ile Ile Asn Phe Glu Gln              20 25 30 aag gaa agt aat gga cca gtg aag gtg tgg gga agc att aaa gga ctg 144 Lys Glu Ser Asn Gly Pro Val Lys Val Trp Gly Ser Ile Lys Gly Leu          35 40 45 act gaa ggc ctg cat gga ttc cat gtt cat gag ttt gga gat aat aca 192 Thr Glu Gly Leu His Gly Phe His Val His Glu Phe Gly Asp Asn Thr      50 55 60 gca ggc tgt acc agt gca ggt cct cac ttt aat cct cta tcc aga aaa 240 Ala Gly Cys Thr Ser Ala Gly Pro His Phe Asn Pro Leu Ser Arg Lys  65 70 75 80 cac ggt ggg cca aag gat gaa gag agg cat gtt gga gac ttg ggc aat 288 His Gly Gly Pro Lys Asp Glu Glu Arg His Val Gly Asp Leu Gly Asn                  85 90 95 gtg act gct gac aaa gat ggt gtg gcc gat gtg tct att gaa gat tct 336 Val Thr Ala Asp Lys Asp Gly Val Ala Asp Val Ser Ile Glu Asp Ser             100 105 110 gtg atc tca ctc tca gga gac cat tgc atc att ggc cgc aca ctg gtg 384 Val Ile Ser Leu Ser Gly Asp His Cys Ile Ile Gly Arg Thr Leu Val         115 120 125 gtc cat gaa aaa gca gat gac ttg ggc aaa ggt gga aat gaa gaa agt 432 Val His Glu Lys Ala Asp Asp Leu Gly Lys Gly Gly Asn Glu Glu Ser     130 135 140 aca aag aca gga aac gct gga agt cgt ttg gct tgt ggt gta att ggg 480 Thr Lys Thr Gly Asn Ala Gly Ser Arg Leu Ala Cys Gly Val Ile Gly 145 150 155 160 atc gcc caa t aa 492 Ile Ala Gln <210> 7 <211> 163 <212> PRT <213> Artificial Sequence <400> 7 Arg Lys Lys Arg Arg Gln Arg Arg Arg Met Ala Thr Lys Ala Val Cys   1 5 10 15 Val Leu Lys Gly Asp Gly Pro Val Gln Gly Ile Ile Asn Phe Glu Gln              20 25 30 Lys Glu Ser Asn Gly Pro Val Lys Val Trp Gly Ser Ile Lys Gly Leu          35 40 45 Thr Glu Gly Leu His Gly Phe His Val His Glu Phe Gly Asp Asn Thr      50 55 60 Ala Gly Cys Thr Ser Ala Gly Pro His Phe Asn Pro Leu Ser Arg Lys  65 70 75 80 His Gly Gly Pro Lys Asp Glu Glu Arg His Val Gly Asp Leu Gly Asn                  85 90 95 Val Thr Ala Asp Lys Asp Gly Val Ala Asp Val Ser Ile Glu Asp Ser             100 105 110 Val Ile Ser Leu Ser Gly Asp His Cys Ile Ile Gly Arg Thr Leu Val         115 120 125 Val His Glu Lys Ala Asp Asp Leu Gly Lys Gly Gly Asn Glu Glu Ser     130 135 140 Thr Lys Thr Gly Asn Ala Gly Ser Arg Leu Ala Cys Gly Val Ile Gly 145 150 155 160 Ile Ala Gln              

Claims (6)

Cell-permeable covalently linked to a protein non-invasive with a protein transport domain selected from HIV-Tat (49-57), oligolysine, oligoarginine, oligos (lysine, arginine) and PEP-1 consisting of 6-15 amino acids 3-O- [β-D-glucopyranosyl (1 → 4) -α-L-arabinofyranosyl] -hederagenin {3-O- [β-D-Glucopyranosyl to enhance cell permeability of the fusion protein (1 → 4) -α-L-arabinopyranosyl] -hederagenin} The method of claim 1, The cell-permeable fusion protein is 3-O- [β-D-glucopyranosyl (1 → 4), which enhances cell permeability of the cell-permeable fusion protein, characterized in that HIV-Tat-Cu, Zn superoxide dismutase. -α-L-arabinofyranosyl] -hederazinin The method according to claim 1 or 2, The 3-O- [β-D-glucopyranosyl (1 → 4) -α-L-arabinofyranosyl] -hederagenin is isolated from Fatsia japonica and is a cell permeable fusion protein. 3-O- [β-D-glucopyranosyl (1 → 4) -α-L-arabinofyranosyl] -hederazinin to enhance cell permeability Cell-permeable covalently linked to a protein non-invasive with a protein transport domain selected from HIV-Tat (49-57), oligolysine, oligoarginine, oligos (lysine, arginine) and PEP-1 consisting of 6-15 amino acids 3-O- [β-D-Glucopyyranosyl (1 → 4) -α-L-Arabinofyranosyl] -hederazenin {3-O- [β-D-Glucopyranosyl (1 → 4) -α-L-arabinopyranosyl] -hederagenin} to improve cell permeability of cell-permeable fusion proteins The method of claim 4, wherein The cell permeable fusion protein is a method for improving cell permeability of cell permeable fusion protein, characterized in that the HIV-Tat-Cu, Zn superoxide dismutase The method according to claim 4 or 5, The 3-O- [β-D-glucopyranosyl (1 → 4) -α-L-arabinofyranosyl] -hederagenin is isolated from Fatsia japonica and is a cell permeable fusion protein. How to improve cell permeability

Images