WO2019225845A1 - Composition for alleviating meningioma by using salidroside and betulin - Google Patents

Abstract

Disclosed in the present invention is a composition for alleviating meningioma, containing, as an active ingredient, salidroside and/or betulin, both of which are cytotoxic to HBL-52 cells, HBL-52 cells being human-derived meningioma cell lines.

Description

Composition for improving meningioma using salidroside or betulin

The present invention relates to a composition for improving meningioma, comprising salidroside of Formula 1 below or betulin of Formula 2 as an active ingredient.

Cancer is a life-threatening disease in which abnormal cells multiply indefinitely and spread throughout the body (LifeSciencePublishing Co. 2008. p3-49). Cancer accounts for about 20% of deaths worldwide (World HealthOrganization.Geneva. 2012. Switzerland.p80-81) and is the number one cause of death from 1983 to 2011 in Korea (National Statistical Office 2012). For this reason, various studies on cancer prevention have been conducted (PNAS. 1995. 92 (12): 5258-5265; JAMA. 1996. 276 (24): 1957-1963; NEJM.1996. 334 (18): Food Science and Industry 1150-1155. 1997. 30 (1): 59-63).

To this end, researches for effective anticancer drugs have been conducted, but since anticancer drugs are applied to cancer cells as well as normal cells, damage to normal tissues, ie toxicity and side effects, is inevitable when the anticancer drugs are administered.

In order to search for efficacy for developing new anticancer drugs or to examine the sensitivity of previously developed anticancer drugs, it is necessary to go through the process of examining the tumor cell inhibitory ability of drugs in vitro before applying them to living organisms such as animal experiments. The most direct and ideal way is to treat the cells with trypan blue, and then count the living cells using a microscope and hemocytometer. Were developed. At present, MTT (tetrazonlium-based colorimetric search) can be used to read many samples simply and quickly and with high objectivity, and is widely used with SRB search (sulforhodamin B protein search) as a cytotoxicity and cell proliferation search method.

The MTT assay is a method that utilizes the characteristic that living cells reduce MTT using mitochondrial dehydrogenase to form blue formazan crystals that can be measured spectrophotometrically. It was designed to analyze the degree of proliferation by mitogen, but recently it has been modified and used as a method for assessing the sensitivity of anticancer drugs to tumor cell lines (Cancer Res. 1987 Nov 15. 47 (22): 5875-9; Cancer Res. 1987 Feb 15. 47 (4): 943-6).

Brain tumors are on the rise in Korea, and meningiomas, in particular, have a relatively high incidence in women (J Korean Med Sci. 1989. 4: 77-90). Advances in imaging and surgical treatment have improved early detection and treatment of brain tumors, but meningiomas are found after tumor growth, even with early detection, with varying histologic subtypes of tumors, and the same differentiation. Even grades of tumors differ in the rate of tumor recurrence, and in many patients, despite the advancement of surgical procedures, they may become malignant and have a poor prognosis. Although meningioma has been identified as a cause of tumor biology as well as molecular biology factors (J Neurooncol. 2004. 70: 183-202), literature reports have been reported at home and abroad compared to cancers of other organs and other brain tumors. Rarely, tumor progenitor lesions or high-risk groups are less established than tumors of other organs.

Recently, due to the side effects of synthetic anticancer drugs used for cancer treatment, efforts to search for and develop cancer prevention substances targeting natural products have been actively conducted in order to develop effective natural anticancer drugs while reducing these side effects.

Salidroside is a substance in which glucose is an ester bond of hydroxy group of tyrosol and is also called rhodioloside. It is known as a therapeutic agent for degenerative nervous system diseases by preventing cardiovascular disease, protecting brain cell damage, protecting and regenerating nerve cells, and regulating neurotransmitters (Phytother Res. 2012. 26 (6): 878-83) . In addition, the compound has been studied to inhibit the irradiation of bone cells by acting on the formation and development of bone cells (PLoS One. 2013. 8 (2): e57251).

Betulin (Betulin) has been published in the literature has been widely used as a raw material used in the organic synthesis of betulinic acid (betulinic acid), betulinic acid (betulonic acid) or derivatives thereof useful as a therapeutic agent (therapeutic agent). Throughout the literature, betulinic acid has anticancer activity against melanoma (e.g., MEL-2, MEL-2 and MEL-4) (Nat Med. 1995 Oct; 1 (10): 1046-51) and betulinic acid in humans. The results show that H9 cells, which are lymphoma cells, have anti-HIV activity (J Nat Prod. 1994. 57 (2): 243-7). In addition, betulin was used in combination with acyclovir, and antiviral effects were reported for herpessimplex viruses (Antiviral Res. 2004. 64 (2): 127-30). In addition, clinical studies show that betulin can induce cell death in some types of tumor cells and delay the growth of several types of tumor cells (Eur J Pharm Sci. 2006. 29 (1): 1-13).

Accordingly, the present invention discloses the activity of meningioma using salidroside and / or betulin having cytotoxicity against HBL-52 cells, which are human-derived meningioma cell lines.

An object of the present invention is to provide a composition for improving meningioma using salidroside (Salidroside) and / or betulin (Betulin).

Other objects and advantages of the present invention will become apparent from the following detailed description, claims and drawings.

The present invention is a concentration-dependent cell proliferation when Salidroside (Salidroside) and / or Betulin (Betulin) is treated by concentration to HBL-52 cells, which are human-derived meningioma cells, as confirmed in the Examples below IC ₅₀ , a concentration that inhibits cell proliferation by 50%, was found to be 25.2, 8.8 and 2.3 μg / mL, respectively, and it was confirmed that the effect of inhibiting cell proliferation was higher than that of a single treatment group when mixed with salidroside and betulin. In view of the low IC ₅₀ value, it was confirmed that there is a combination effect than a single material.

In consideration of the experimental results described above, the present invention provides a compound selected from the group consisting of (i) Salidroside of Formula 1 below, an isomer thereof, a prodrug thereof, a hydrate thereof, and a solvate thereof, (ii) a compound selected from the group consisting of Betulin, an isomer thereof, a prodrug thereof, a hydrate thereof, and a solvate of Formula 2, below; It can be grasped as a composition for improving meningioma using a mixture of compounds as an active ingredient.

<Formula 1>

<Formula 2>

As used herein, "isomers" are not only essentially pure diastereomers, including optical isomers, but also conformational isomers, position isomers (especially tautomers or geometric isomers) or geometric isomers. geometric isomers (eg, cis-trans isomers).

In addition, in the present specification, "prodrug" refers to a drug that does not show physiological activity by itself but can exert its effect by changing to the original drug by chemical or enzymatic action in the body after administration. Manufactured for the purpose of controlling physical and chemical properties. In some cases prodrugs may be used to make administration easier. For example, some drugs do not ferment the drug by oral administration, but the prodrug may ferment the drug by oral administration. Prodrugs may also be prepared to increase the solubility of the drug. For example, prodrugs may be in the form of esters that facilitate the passage of cell membranes, where the water solubility is detrimental to mobility but once hydrolyzed is hydrolyzed to the active carboxylic acid by metabolism. Another example of a prodrug may be a short peptide that is bound to an acid group that is converted by metabolism to reveal the active site.

In addition, in the present specification, "hydrate" refers to a compound to which water is bound, and includes a compound containing no chemical bonding force between water and the compound. The hydrate may contain 1 equivalent or more, preferably 1 to 5 equivalents of water. Such hydrates can be prepared by crystallizing the compounds of the present invention, their pharmaceutically acceptable salts, or isomers thereof from water or a solvent containing water.

In addition, in this specification, "solvate" means the compound which generate | occur | produced between the molecule | numerator or ion of a solute, and the molecule | numerator or ion of a solvent. Such solvates may include stoichiometric or nonstoichiometric amounts of solvent that are bound by non-covalent intermolecular forces. Preferred solvents may be ethanol, methanol, propanol, methylene chloride, and the like, which are non-volatile and do not exhibit more toxic to humans.

In addition, in the present specification, the "active ingredient" means a component that can exhibit the desired activity alone or in combination with a carrier which is itself inactive.

In addition, in the present specification, "meningio meningioma improvement" is meant to include induction of death of meningioma cells or inhibition of their proliferation, thereby suppressing recurrence of meningiomas.

The composition of the present invention may include the active ingredient in any amount (effective amount) as long as it can exhibit the effect of treating and improving meningioma disease intended to be treated according to the use, formulation, formulation purpose, etc. It will be determined within the range of 0.001% to 15% by weight based on the total weight of the composition. The term “effective amount” herein refers to a mammal, preferably to a human subject, when a composition of the present invention is administered to a human being by the suggestion of a medical professional or the like for a disease involving muscle growth effect, exercise ability improvement effect, and muscle loss. It refers to the amount of the active ingredient included in the composition of the present invention, which can exhibit the intended medical and pharmacological effects such as an improvement effect. Such effective amounts can be determined experimentally within the range of ordinary skill in the art.

The present invention can be understood as a pharmaceutical composition in specific embodiments.

The pharmaceutical compositions of the present invention may be prepared in oral or parenteral formulations according to the route of administration by conventional methods known in the art, including pharmaceutically acceptable carriers in addition to the active ingredient. The route of administration here can be any suitable route, including the topical route, the oral route, the intravenous route, the intramuscular route, and direct absorption through mucosal tissue, and may be used in combination of two or more routes. An example of a combination of two or more routes is where a drug of two or more formulations according to the route of administration is combined, for example when one drug is administered first by the intravenous route and the other by the local route.

Pharmaceutically acceptable carriers are well known in the art depending on the route of administration or formulation, and specific reference may be made to the pharmacopoeia of each country, including "Korea Pharmacopoeia."

When the pharmaceutical composition of the present invention is prepared in an oral dosage form, powders, granules, tablets, pills, dragees, capsules, solutions, gels, syrups, suspensions, wafers according to methods known in the art with suitable carriers It may be prepared in a formulation such as. Examples of suitable carriers include lactose, glucose, sucrose, dextrose, sorbitol, sugars such as mannitol, xylitol, starch such as corn starch, potato starch, wheat starch, cellulose, methyl cellulose, ethyl cellulose, sodium carboxymethyl cellulose, Celluloses such as hydroxypropylmethylcellulose, polyvinyl pyrrolidone, water, methylhydroxybenzoate, propylhydroxybenzoate, magnesium stearate, mineral oil, malt, gelatin, talc, polyol, vegetable oil, ethanol, and Cerrol etc. are mentioned. If formulated, appropriate binders, lubricants, disintegrants, colorants, diluents and the like may be included as needed. Suitable binders include starch, magnesium aluminum silicate, starch ferrite, gelatin, methylcellulose, sodium carboxymethylcellulose, polyvinylpyrrolidone, glucose, corn sweeteners, sodium alginate, polyethylene glycol, waxes, and the like. Sodium, sodium stearate, magnesium stearate, sodium benzoate, sodium acetate, sodium chloride, silica, talcum, stearic acid, magnesium salts thereof, calcium salts, polydetylene glycol and the like, and the disintegrating agents include starch, methyl cellulose And agar, bentonite, xanthan gum, starch, alginic acid or its sodium salt and the like. Moreover, lactose, dextrose, sucrose, mannitol, sorbitol, cellulose, glycine etc. are mentioned as a diluent.

When the pharmaceutical compositions of the present invention are prepared in parenteral formulations, they may be formulated in the form of injections, transdermal administrations, nasal inhalants and suppositories with suitable carriers according to methods known in the art. When formulated as an injection, a suitable carrier may be an aqueous isotonic solution or suspension. Specifically, an isotonic solution such as phosphate buffered saline (PBS) containing triethanol amine, sterile water for injection, or 5% dextrose may be used. . When formulated as a transdermal administration, it may be formulated in the form of an ointment, cream, lotion, gel, external solution, pasta, linen, aerosol, and the like. Nasal inhalants can be formulated in the form of aerosol sprays using suitable propellants, such as dichlorofluoromethane, trichlorofluoromethane, dichlorotetrafluoroethane, carbon dioxide, etc. witepsol), tween 61, polyethylene glycols, cacao butter, laurin paper, polyoxyethylene sorbitan fatty acid esters, polyoxyethylene stearate, sorbitan fatty acid esters and the like.

In addition, the compositions of the present invention may be formulated into suitable formulations with acceptable pharmaceutical carriers and administered by a variety of routes. "Administration" means introducing a predetermined substance into a patient in any suitable way and the route of administration of the composition can be administered via any general route as long as it can reach the desired tissue. Intraperitoneal administration, intravenous administration, intramuscular administration, subcutaneous administration, intradermal administration, oral administration, topical administration, nasal administration, pulmonary administration, rectal administration, but is not limited thereto. However, upon oral administration, since the peptide is digested, it is desirable to formulate the oral composition to coat the active agent or to protect it from degradation in the stomach. In addition, the pharmaceutical composition may be administered by any device that allows the active substance to migrate to the target cell. Preferred modes of administration and preparations are intravenous, subcutaneous, intradermal, intramuscular, injectable and the like. Injectables include non-aqueous solvents such as aqueous solvents such as physiological saline solution and ring gel solution, vegetable oils, higher fatty acid esters (e.g., oleic acid, etc.), and alcohols (e.g., ethanol, benzyl alcohol, propylene glycol, glycerin, etc.). Stabilizers (e.g. ascorbic acid, sodium bisulfite, sodium pyrosulfite, BHA, tocopherol, EDTA, etc.), emulsifiers, buffers for pH control, to prevent microbial development Preservatives such as mercury nitrate, chimerosal, benzalkonium chloride, phenol, cresol, benzyl alcohol, and the like.

Specific formulations of pharmaceutical compositions are known in the art and can be found, for example, in Remington's Pharmaceutical Sciences (19th ed., 1995). The document is considered part of this specification.

Preferred dosages of the pharmaceutical compositions of the present invention are the type of disease, age, weight, health, sex of the patient, sensitivity to the drug of the patient, route of administration, method of administration, frequency of administration, duration of treatment, combination or drug used concurrently. It can be easily determined by those skilled in the art according to factors well known in the medical field. In general, the active substance can be administered at a dose of 0.001 mg / kg to 10 g / kg per day, preferably 0.001 mg / kg to 1 g / kg. Administration can be done once a day or divided into several times. Such dosage should not be construed as limiting the scope of the invention in any aspect.

In another specific aspect, this invention can be understood as a food composition.

The food composition of the present invention may be prepared in any form, for example, beverages such as tea, juice, carbonated beverages, ionic beverages, processed oils such as milk, yogurt, gums, rice cakes, sweets, bread, sweets, noodles, and the like. It can be prepared as a dietary supplement, such as foods, tablets, capsules, pills, granules, liquid, powder, flaky, paste, syrup, gel, jelly, bar. In addition, the food composition of the present invention can distinguish any product as long as it conforms to the enforcement regulations at the time of manufacture and distribution in the legal and functional divisions. For example, it is a health functional food according to the Act on Health Functional Foods, or confectionary, soybeans, soy milk, fermented beverages, special purpose foods according to each food type in the Food Code of the Food Sanitation Act (KFDA Notification, Food Standards and Standards). In particular, the weight control preparation) and the like.

The food composition of the present invention may include food additives in addition to the active ingredient. Food additives are generally understood as substances that are added to foods, mixed or infiltrated in the manufacture, processing or preservation of foods, and because they are taken daily with food for a long time, their safety should be ensured. The Food Additives Code of the Food Sanitation Act (KFDA Notification, Food Additives Standards and Standards) defines the food additives, which are guaranteed to be safe, divided into chemical synthetics, natural additives, and mixed preparations.

These food additives may be divided into sweeteners, flavors, preservatives, emulsifiers, acidulants, thickeners, and the like in terms of their functionalities.

Sweeteners are used to impart a proper sweetness to foods, and may be natural or synthesized. Preferably, a natural sweetener is used. Examples of the natural sweetener include sugar sweeteners such as corn syrup solids, honey, sucrose, fructose, lactose and maltose.

Flavoring agents can be used to enhance the taste or aroma, both natural and synthetic. It is the case of using a natural thing preferably. In addition to flavors, the use of natural ones can be combined with nutritional purposes. The natural flavor may be obtained from apples, lemons, citrus fruits, grapes, strawberries, peaches, and the like, or may be obtained from green tea leaves, round leaves, jujube leaves, cinnamon, chrysanthemum leaves, jasmine and the like. In addition, ginseng (red ginseng), bamboo shoots, aloe vera, ginkgo and the like can be used. Natural flavors can be liquid concentrates or solid extracts. In some cases, synthetic flavoring agents may be used, and synthetic flavoring agents may include esters, alcohols, aldehydes, terpenes, and the like.

Sodium sorbate, sodium sorbate, potassium sorbate, calcium benzoate, sodium benzoate, potassium benzoate, EDTA (ethylenediaminetetraacetic acid), etc. may be used as a preservative, and as an emulsifier, acacia gum, carboxymethylcellulose, xanthan gum, Pectin etc. can be mentioned, As acidic acid, acid, malic acid, fumaric acid, adipic acid, phosphoric acid, gluconic acid, tartaric acid, ascorbic acid, acetic acid, phosphoric acid, etc. can be used. The acidulant may be added so that the food composition is at an appropriate acidity for the purpose of inhibiting the growth of microorganisms in addition to the purpose of enhancing the taste.

As the thickener, suspending implementers, sedimenting agents, gel formers, swelling agents and the like can be used.

In addition to the food additives described above, the food composition of the present invention may include a bioactive substance or minerals known in the art for the purpose of supplementing and reinforcing the functionality and nutritional properties and ensuring the stability as a food additive.

Examples of such physiologically active substances include catechins, vitamin B1, vitamin C, vitamin E, vitamin B12 and the like, tocopherol, dibenzoylthiamine, and the like contained in green tea. Examples of the minerals include calcium preparations such as calcium citrate and magnesium stearate. Magnesium preparations such as iron, iron preparations such as iron citrate, chromium chloride, potassium iodine, selenium, germanium, vanadium, zinc and the like.

In the food composition of the present invention, the food additive as described above may be included in an amount that can achieve the purpose of addition according to the product type.

Regarding other food additives that may be included in the food composition of the present invention, reference may be made to food or food additives.

As described above, according to the present invention, it is possible to provide a composition for improving meningioma, including salidroside and / or betulin as an active ingredient. The composition of the present invention uses salidroside and / or betulin having cytotoxicity against HBL-52 cells, which are human-derived meningioma cell lines, as an active ingredient, and may be commercialized as drugs or foods (particularly health functional foods).

1 shows the results of evaluation of cell proliferation inhibition against meningioma cells of salidroside.

Fig. 2 shows the results of cell proliferation inhibition of betulin's meningioma cells.

Figure 3 shows the results of cell proliferation inhibition evaluation of the meningioma cells of the salidroside and betulin mixture.

One. Example

1.1 Preparation of Sample

Salidroside (CAS No. 10338-51-9) and betulin (CAS No. 473-98-3) were purchased from ChemFaces (China).

1.2 Cell Line Culture and Preparation

HBL-52, a human meningioma cell line, was suspended in a complete medium (DMEM medium containing 10% FBS, 1% Penicillin-Streptomycin) and placed in a flask for cell culture at 37 ° C and 5% CO ₂ incubator (MCO-20AIC, Sanyo, Japan Incubated). After the incubation, the cells were collected, placed in a centrifuge tube, and centrifuged (1,000 rpm, 5 minutes). After centrifugation, the supernatant was removed and a new complete medium was added to the pellet to make cell suspension. The cells were dispensed 200 μL into each well of a 96 well plate at a concentration of 5 × 10 ⁴ cells / 200 μL and incubated for 24 hours in a 37 ° C., 5% CO ₂ incubator.

1.3 Group Composition and Treatment Concentration

The experimental group consisted of a single salidroside (test substance 1), a single betulin (test substance 2), and a mixed salidroside and betulin (test substance 3, the mixing ratio is weight ratio). Group composition and specific treatment concentrations are shown in Table 1.

Test substance	Test substance concentration (Μg / ml)	Processing liquid volume (Ml / well)
Test substance 1	100, 50, 25, 12.5, 6.25, 3.125, 1.56, 0.78, 0.39, 0	0.2
Test substance 2	100, 50, 25, 12.5, 6.25, 3.125, 1.56, 0.78, 0.39, 0	0.2
Test substance 3	50/50, 25/25, 12.5 / 12.5, 6.25 / 6.25, 3.125 / 3.125, 1.56 / 1.56, 0.78 / 0.78, 0.39 / 0.39, 0.19 / 0.19, 0	0.2

1.4 MMT  assay

After removing the 96-well culture medium incubated at a concentration of 5 × 10 ⁴ cells / 200 μL / well, 200 μL of each concentration of each test substance was treated. Staining solution (MTT solution (3- (4,5-dimethylthiazol-2yl) -2,5-diphenyl-2H-tetrazolium bromide, 5 mg / mL) was added to each well by 50 μL, and then 37 ° C. and 5% CO ₂ incubator The negative control material was treated with excipient water for injection or dimethyl sulfoxide (DMSO) After completion of the culture, the culture solution of each well was removed, and 150 μL of DMSO (dimethyl sulfoxide) solution was added to each well. Cells were lysed Optical density (OD) was measured at 570 nm using an ELISA reader.

Based on the absorbance of the negative control group, the cell survival rate was calculated by substituting the absorbance of the wells treated with the test substance for each concentration into the following formula. When cell proliferation inhibition was observed, IC ₅₀ , a concentration that inhibits cell proliferation by 50%, was evaluated using regression analysis.

Cell viability (%) = [1-(B-A) / B] × 100

A: Average absorbance value of the group by concentration of each test substance

B: Absorbance average value of negative control group

2. Results (cell proliferation Inhibitory  evaluation)

2.1 Salidroside

When the degree of inhibition of cell proliferation when treated with a negative control substance was 0.0%, the degree of inhibition of cell proliferation by concentration of each test substance 1 (salidroside) is shown in FIG. 1. Referring to FIG. 1, as a result of treating Experiment 1 with concentrations of 0, 0.39, 0.78, 1.56, 3.125, 6.25, 12.5, 25, 50, and 100 μg / mL, the degree of inhibition of cell proliferation was 0.0, -3.2, 0.2 , 2.5, 4.5, 3.9, 29.7, 48.9, 73.5, and 90.2%, with an IC ₅₀ of 25.2 μg / mL.

2.2 Betulin

When the degree of inhibition of cell proliferation in the treatment of negative controls was 0.0%, the degree of inhibition of cell proliferation by concentration of each test substance 2 (betulin) is shown in FIG. 2. Referring to FIG. 2, as a result of treating Experiment 2 at concentrations of 0, 0.39, 0.78, 1.56, 3.125, 6.25, 12.5, 25, 50, and 100 μg / mL, the degree of inhibition of cell proliferation was 0.0, 3.7, 7.1, 8.9, 33.4, 48.5, 51.4, 66.9, 92.0 and 93.8%, with an IC ₅₀ of 8.8 μg / mL.

2.3 Salidroside Betulin  mixture

When the degree of inhibition of cell proliferation in the treatment of negative controls was 0.0%, the degree of inhibition of cell proliferation by concentration of each test substance 3 (salidroside and betulin weight ratio mixture) is shown in FIG. 3. Referring to Figure 3, the test material 3 is 0, 0.19 / 0.19, 0.39 / 0.39, 0.78 / 0.78, 1.56 / 1.56, 3.125 / 3.125, 6.25 / 6.25, 12.5 / 12.5, 25/25, 50/50 μg / mL Treatment with each concentration resulted in inhibition of cell proliferation of 0.0, 11.5, 13.6, 37.3, 60.0, 83.1, 89.2, 90.6, 91.6 and 92.1%, and IC ₅₀ was calculated to be 2.3 μg / mL.

3. Conclusion

In conclusion, cell proliferation was evaluated in the experiments using HBL-52, a human-derived meningioma cell line, to evaluate the cytotoxicity of test substance 1, salivaside, test substance 2, betulin, test substance 3, salidroside, and betulin. IC ₅₀ the concentration that inhibit 50% respectively 25.2, 8.8, appears to 2.3μg / mL appear to have cell proliferation inhibitory effect IC ₅₀ of the test substance 3 is combined in a single material than when viewed at a lower point than the single treatment groups The result shows that it works.

Claims (4)

1. A composition for improving meningioma, comprising a compound according to (i) below, a compound according to (ii) below, or a mixture thereof as an active ingredient:

   (i) salidoxide, a hydrate thereof, or a solvate thereof;

   (ii) Betulin, its hydrate, or solvate thereof.
2. The method of claim 1,

   The mixture of the salidroside and betulin is a composition for improving meningioma, characterized in that to use a mixture of salidroside and betulin in a weight ratio of 1: 1.
3. The method according to claim 1 or 2,

   The composition is a composition for improving meningioma, characterized in that the pharmaceutical composition.
4. The method according to claim 1 or 2,

   The composition is a composition for improving meningioma, characterized in that the food composition.

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