KR20070037041A - A process for purifying adenosine derivatives derived from silkworm and a composition for treating vasculogenic impotence comprising the adenosine derivatives - Google Patents

Abstract

The present invention relates to a method for extracting and purifying adenosine derivative compounds from silkworm pupae and to a composition for treating erectile dysfunction comprising the extracted adenosine derivative compounds. Specifically, the present invention is a method for extracting and purifying silkworm pupae with an organic solvent, followed by anion exchange gel filtration chromatography, selective desalting and optionally reverse phase HPLC to extract and purify adenosine derivative compounds from silkworm pupae. And it relates to a composition for treating erectile dysfunction comprising the same.

 Silkworm pupa, adenosine derivative compound, erectile dysfunction

Description

Process for purifying adenosine derivatives derived from silkworm and a composition for treating vasculogenic impotence comprising the adenosine derivatives}

1 is a graph showing the effect of alcohol extracts of the silkworm pupae on the NO production in the mouse brain endothelial cells (bEND3), where SNP means sodium nitrofurside used as a positive control.

FIG. 2 is a graph showing the effect of alcohol extracts at each time of silkworm pupae on cell growth rate in mouse brain endothelial cells (bEND3), where SNP means sodium nitrofurside used as a positive control.

Figure 3 is a graph showing the effect of alcohol extract by the time period of silkworm pupae on cell growth rate in macrophage (RAW264.7) cells.

Figure 4 is a graph showing the effect of alcohol extracts according to the silkworm pupa phase on NO production in macrophage (RAW264.7) cells.

Figure 5 is a graph showing the activity of the secretory phospholipase A ₂ according to the alcohol extract according to the silkworm (male) pupa phase in macrophage (RAW264.7) cells.

6a and 6b are graphs showing cGMP and its ED ₅₀ (cGMP 50% effective concentration) when three days of silkworm pupae alcohol extracts were administered to mice.

Figure 7 is a graph showing the mTNF-α production of alcohol extracts of the silkworm pupae in the spleen cells.

8 is a schematic diagram of organic fractions for silkworm pupa.

9 is a graph showing NO production in small blood vessel endothelial cells (CPAE) for each organic fraction.

Figure 10 is a graph showing the DPPH activity for the fraction subjected to anion exchange gel filtration chromatography with a solvent-specific extract (400mg) for silkworm pupa.

FIG. 11 is a graph showing NO activity in small blood vessel endothelial cells for a fraction subjected to anion exchange gel filtration chromatography with silkworm pupal extract (2 g).

FIG. 12 is a graph showing NO activity in small blood vessel endothelial cells of an extract of each silkworm pupa by anion exchange gel filtration chromatography and desalting (Biogel P10). The x coordinate means the number of each fraction.

FIG. 13 is a graph showing the results of cytotoxicity in small blood vessel endothelial cells of an extract subjected to anion gel filtration chromatography and desalting of the extract of each silkworm pupa. The x coordinate means the number of each fraction.

14 is a graph showing the peaks for the fractions subjected to reverse phase high performance liquid chromatography (RP-HPLC, also called RPC), and II, III, and IV peaks are RPC-II fraction, RPC-III fraction, and RPC-IV fraction, respectively. Indicates.

15A and 15B show MALDI-TOF MS spectra of the RPC-II fraction and MALDI-TOF MS spectra of the RPC-IV fraction, respectively.

16A and 16B show infrared absorption spectra of the RPC-II fraction and infrared absorption spectra of the RPC-IV fraction, respectively.

17A, 17B, and 17C show ultraviolet absorbance spectra of the RPC-II fraction, ultraviolet absorbance analysis of the RPC-IV fraction, and RPC-VI (18 minute fractions), respectively.

18A, 18B and 18C show the ¹ H-NMR spectrum of the RPC-II fraction, the ¹³ C-NMR spectrum of the RPC-II fraction, and the two dimensions of the RPC-II fraction, respectively. Hydrogen-Hydrogen Bond-High Resolution Nuclear Magnetic Resonance Spectroscopy ( ¹ H- ¹ H COSY NMR) spectrum.

19A, 19B, and 19C show ¹ H-NMR of the RPC-IV fraction, ¹³ C-NMR of the RPC-IV fraction, and ¹ H- ¹ H COSY of RPC-IV, respectively. NMR is shown.

20A and 20B are electro-ion molecular weight absorption spectra (EI Mass) of RPC-II fractions measured by varying the range of the x-axis, and FIGS. 20C and 20D are electro-ion molecular weight absorption spectra (EI Mass) for RPC-IV fractions. ) Spectrum.

Figure 21 shows the PDE [ ³ H] cAMP SPA analysis data according to the concentration change of the anion exchange gel filtration chromatography purified fraction (DX fraction) and reverse phase HPLC purified fraction (RPC fraction, 14.5-16 minutes).

22 shows the inhibition of PDE type IV and PDE type V when anion exchange gel filtration chromatography purified fraction (DX fraction) and conventional commercialized PDE inhibitors (I, V, IV, III) were administered in combination. Graph showing activity.

      FIG. 23 is an HPLC diagram showing the residence time per addition by HPLC-ECD detector after hydrolysis of the fractions subjected to anion gel filtration chromatography and desalting with 0.1 N HCl.

The present invention relates to a method for extracting and purifying adenosine derivative compounds from silkworm pupae and to a composition for treating erectile dysfunction comprising the extracted adenosine derivative compounds.

The causes of erectile dysfunction can be largely divided into psychogenic (mental cause) and temperament (physical cause). The organic causes include abnormalities of the nervous system, the vascular system or the endocrine system, and specifically include central nervous system diseases or peripheral neurological diseases. Representative diseases causing erectile dysfunction include diabetes, hypertension, and kidney disease. As a method for treating such erectile dysfunction, there are conventional psychological therapies, non-surgical methods, surgical methods, and non-surgical treatments have been introduced, such as drug use, erectile prostheses in the corpus cavernosum, vacuum penis inhalers, and the like.

Neurotransmitters involved in penile erection are known to be acetylcholine, ATP, VIP, NO (nitrogen oxide), and NO is known to be the most important substance before erection (Palmer et al., 1987). NO, which acts on the penile erection mechanism, is produced by two different NO synthases, which are endothelial NOS (eNOS) and neuronal NOS (nNOS). It is known that the decrease in NO production that occurs as aging progresses as a major cause of erectile dysfunction.

When the erection and contraction mechanism of the penis is subjected to psychological and physical stimulation that causes sexual excitement, NO expands the blood vessels, and 5-6 times more blood flows into the corpus cavernosum, causing both penile cavernous bodies to expand. do. At this time, the expanded cavernous body compresses the blood vessels and prevents blood from flowing back through the veins, and the penis becomes firm and enlarged. When the erection of the penis ejaculates or stops sexual stimulation, the closed vein opens and the blood escapes, returning to a relaxed state by the action of phosphodiesterase-V (PDE-V). An erection is normally done when the endocrine, vascular, and nervous systems involved in the erection are working properly and there is no abnormality in the penis itself.

Currently, the most effective erectile dysfunction treatment agent in the world is Viagra, which uses a mechanism that inhibits the activity of cGMP hydrolase, PDE-V, to increase cGMP concentration in the NO-cGMP pathway. Linast material is known to induce erections to a lesser extent than Viagra (Trigo-Rocha F, et al., Am J Physiol 264, H419-422, 1993; Taher A, et al., World J Urol 12 , 289-291, 1994). Viagra has been evaluated for efficacy and safety that has been proven over the past five years, Lily's Cialis has been recognized for up to 36 hours of drug efficacy. It is evaluated.

However, Viagra, which has an excellent erectile dysfunction effect, is known to cause serious side effects in patients with myocardial infarction, stroke, and cardiovascular disease because Viagra has a low blood pressure in heart patients and can lead to death due to heart attack. Although the duration has been extended to 36 hours with sugars, many more unexpected side effects have been reported than natural products.

Also on July 14, 2005, the pharmacist order issued a warning on July 8, 2005, for the US erectile dysfunction Viagra, Cialis and Levitra, to warn that the package could lose sight. There is an urgent need for the development of natural product-derived erectile dysfunction without these side effects.

In order to solve the above problems, the present invention is not only an excellent therapeutic effect against male sexual dysfunction, but also a method for extracting and purifying an active substance from a natural extract that can minimize the side effects of conventional drugs and the active substance It is an object to provide a pharmaceutical composition for treating erectile dysfunction comprising.

One aspect of the present invention relates to a method for extracting and purifying the compounds of Formula 1 and Formula 2 from silkworm pupa,

(a) The organic fraction of (i) silkworm pupa with hexane: water volume ratio of 1: 50-50: 1 was subjected to organic fractionation to obtain water fraction a, and (ii) water fraction a. The organic fraction was purified by chloroform to obtain a water fraction b. (Iii) The organic fraction was fractionated with ethyl acetate for the water fraction b to obtain a water fraction c. (Iv) Butanol: water for the water fraction c. Performing organic fractionation with an aqueous butanol solution having a volume ratio of 1:50 to 50: 1 to obtain a water fraction d,

(b) performing anion exchange gel filtration chromatography on the water fraction d to select an active fraction a having an ultraviolet absorbance of 540 nm or greater and a cell survival rate of 80% or greater at 540 nm,

(c) selectively desalting the active fraction a to select an active fraction b having an ultraviolet absorbance of 0.9 or more at 540 nm and a cell survival rate of 85% or more for measuring NO production; and

(d) optionally performing reversed phase HPLC on the active fraction a or active fraction b to select the active fraction c having an ultraviolet absorbance of 540 nm or more and a cell survival rate of 20% or more at 540 nm.

It relates to a method for extracting adenosine compound derivative represented by the formula (1) or (2) or a pharmaceutically acceptable solvate thereof from silkworm pupa comprising.

The compound of Formula 1 or Formula 2 contains an asymmetric carbon so that optical isomers exist, and the compounds according to the present invention include both such individual optical isomers and racemic mixtures of such optical isomers. In addition, the compound according to the present invention also includes a hydrate of the formula (3) is formed by the addition of · OH ₂ as a hydrate to the compound of formula (2) or · O is added to the compound of formula (1).

·OH₂

OH ₂

In the present invention, the active substance is very unstable due to the strong reactivity associated with oxidation, and for this reason, it is very difficult to separate, purify, and identify the structure, but the extraction method developed by the present inventors through numerous trials and errors and a series of studies. The active substance can be extracted and separated by

The dimethyl adenosine derivative is an acidic compound containing ribose (5 saccharides) and nucleic acid, which is bound to an anion exchange resin in an aqueous buffer solution and is separable according to the concentration gradient of salts. It is an important step in extracting, and cation exchange gel filtration chromatography and cation exchange chromatography are not effective because they are not separated from the acidic compound, which is a dimethyl adenosine derivative and a resin, are not separated and purified.

In addition, the (c) desalting process and (d) reverse phase HPLC process may be omitted as a natural pharmaceutical preparation as a natural extract containing silkworm pupae-derived active substance having an effect of treating erectile dysfunction. However, when the purpose of obtaining the pure pharmacological effect of a single substance, such as to obtain a pure dimethyl adenosine derivative compound, it is preferable to perform the steps (c) and (d).

In addition, NO production amount is measured by general ultraviolet spectroscopy, and it is preferable to use Griess reagent. The absorbance of at least 0.9 corresponds to a NO production of at least about 25 μM (or mg / mL).

On the other hand, in the present invention, it is preferable to use silkworm pupae as silkworm pupae, and the term "upper" used in the present invention means to raise the silkworm to silkworm.

Another aspect of the present invention relates to a composition for treating erectile dysfunction comprising an extract comprising Formula 1 or Formula 2 extracted from silkworm pupa by the above method, and specifically, may be used as a pharmaceutical composition or a food composition.

Instead of separating and purifying these extracts into pure compounds of Formula 1 or Formula 2, the intermediate extracts having physiological activity can be used directly, thereby avoiding the process of separation or purification.

In addition, it is possible to select a fraction having a physiological activity by performing only the (a) organic fractionation step and (b) anion gel filtration chromatography step and confirming NO production and cell viability at each step. There is an advantage that can greatly contribute to the simplification of the process.

Another aspect of the present invention relates to a composition for treating erectile dysfunction comprising adenosine derivative compound extracted and purified from silkworm pupa, and specifically, an adenosine compound derivative represented by Formula 1 or Formula 2, a pharmaceutically acceptable salt thereof Or it relates to a composition for treating erectile dysfunction comprising a solvate thereof as an active ingredient, and may be specifically used as a pharmaceutical composition or a food composition.

The pharmaceutical composition according to the present invention exhibits the effect of erectile dysfunction by increasing the concentration of cGMP by selectively inhibiting the activity of PDE-V, a cGMP hydrolase, without any side effects even when used in combination with conventional PDE inhibitors. Synergistic or at least an additive effect of the treatment of erectile dysfunction can be seen, there is an advantage that can be administered or formulated in combination with existing PDE inhibitors.

The compound can be administered orally or parenterally during clinical administration and can be administered in various formulations of a general pharmaceutical formulation. At this time, it is prepared using diluents or excipients, such as fillers, weights, binders, wetting agents, disintegrating agents, surfactants commonly used in the formulation step.

It includes formulations of dosage units of the invention. The formulations are in individual dosage forms, i.e. tablets, coated tablets, capsules, pills, suppositories, and ampoules, the content of active compound in the drug being determined by the fraction of individual dosage (e.g. 1/2, 1/3. 1/4). .) Or multiples (e.g. 2, 3, 4 times). The dosage of the present invention varies depending on the weight, age, sex, health status, diet, time of administration, method of administration, excretion rate and severity of the disease of the patient, the adult daily dosage is 10-100 mg / kg It is preferable to divide the amount of once into several times.

The pharmaceutical compositions of the present invention may comprise non-toxic and inert pharmaceutical excipients, wherein the non-toxic and inert pharmaceutically suitable excipients are solid, semisolid or liquid diluents, fillers and formulation aids of all types. Preferred formulations include tablets, coated tablets, capsules, pills, granules, suppositories, solutions, suspensions and emulsions, pastes, ointments, gels, creams, lotions, powders and sprays.

Tablets, coated tablets, capsules, pills, and granules are conventional excipients, such as (a) fillers and weighting agents (e.g. starch, lactose, sucrose, glucose, mannitol and silicic acid) (b) binders (e.g. carboxy) Methyl cellulose, alginate, gelatin and polyvinylpyrrolidone) (c) hygroscopic agents (e.g. glycerol), (d) disintegrants (e.g. agar, calcium carbonate and sodium carbonate), (e) dissolution retardants (e.g. paraffin) (f) absorption accelerators (e.g. quaternary ammonium compounds), (g) wetting agents (e.g. cetyl alcohol and glycerol monostearate), (h) sorbents (e.g. kaolin and bentonite), and (i) lubricants ( Examples: talc, calcium stearate, magnesium stearate, magnesium starrate talc and solid polyethylene glycol), or a mixture of the substances described in (a) to (i) above, may contain an active compound or compounds. Tablets, capsules, pills, and granules can be encapsulated, can release only active compounds to the intestinal tract, and can be microencapsulated that polymers or adjuvants compositions can be formulated to sustained release if necessary. .

Formulations for parenteral administration include injections, suspensions, emulsions, lyophilizers, suppositories, and the like. Suppositories are conventional water-soluble or water-insoluble excipients, for example polyethylene glycol, such as cacao fat fat, higher esters in addition to the emulsion compounds (for example: C ₁₆ - C ₁₄ having a fatty acid-alcohol), above tepsol, macrogol, tween 61 And a mixture of laurin and glycerol gelatin materials.

Ointments, pastes, creams and gels may contain, in addition to the active compounds, conventional excipients, lactose, talc, silicic acid, aluminum hydroxide, calcium silicate and polyamide powder, or mixtures of these materials. The nebulizer may further contain a conventional foaming agent, for example, chlorofulurohydrocarbon, and may also be formulated as a nasal spray containing PEG-4000 and glycerin.

Liquids and emulsions, in addition to the active compounds, include conventional excipients such as solvents, solubilizing agents and emulsifiers, for example water, ethyl alcohol, isopropyl alcohol, ethyl carbonate, ethyl acetate, benzyl alcohol, benzyl benzoate, propylene glycol , 1,3-butylene glycol, dimethylformromamide oil, especially cottonseed oil, peanut oil, corn seed oil, olive oil, castor oil and sesame oil, glycerol, glycerol form alcohol, tethyrahydrofurfuryl alcohol, polyethylene glycol and sorb Non-fatty acid esters, or mixtures of these materials. Parenteral solutions and emulsions may be formulated in sterile form, isotonic with blood.

Suspending agents, in addition to the active compounds, include conventional excipients, for example liquid diluents such as water, ethyl alcohol, propylene glycol, polyethylene glycol, and suspending agents such as ethoxylated isostearyl alcohol, polyoxyethylene sorbitol and sorbitan esters. ), Microcrystalline cellulose, aluminum metahydroxy, bentonite, agar and injectable esters such as tragacanth, ethyl oleate or mixtures of these materials.

The formulations may also contain coloring agents, preservatives and additives that improve odor or taste, such as peppermint oil and eucalyptus oil and sweeteners such as saccharin, and may contain other pharmaceutically effective compounds in addition to the compounds according to the invention. Can be.

The formulations are prepared in a known manner, for example in a conventional manner in which the active compound is mixed with excipients. The therapeutically effective compound should be contained in an amount of about 0.1-99.5% by weight, preferably 0.5-95% by weight of the total mixture in the pharmaceutical formulation, and the extract containing the compound corresponding to this amount is directly It may be included in the pharmaceutical composition of the present invention, it will be apparent to those skilled in the art that such an effective amount can be easily determined.

In addition, it may be further formulated preferably according to each disease or component using a method disclosed in the art or a method disclosed in Remington's Pharmaceutical Science (Mack Publishing Company, Easton PA) and the like.

In addition, the food composition according to the present invention may include a food-acceptable food supplement additive in addition to the extract or compound according to the present invention, the preferred formulation of such a food composition is powder, granules, pills, solid, flaky, Capsules, tablets and liquid solutions, beverages, and the like.

In this case, the amount of the extract in the food or beverage is generally preferably 0.01-50% by weight based on the total food, the compound of the formula (1) or formula (2) or salts thereof in an amount corresponding to the amount of the compound included therein Cargo can also be added directly.

The health functional beverage composition of the present invention is not particularly limited in the liquid component except for containing the extract as an essential ingredient in the indicated ratio, and may contain various flavors or natural carbohydrates, etc. as additional ingredients, as in general beverages. . Examples of the above-mentioned natural carbohydrates include monosaccharides such as glucose, fructose and the like; Disaccharides such as maltose, sucrose and the like; Polysaccharides such as conventional sugars such as dextrin, cyclodextrin and the like and sugar alcohols such as xylitol, sorbitol, erythritol and the like. The proportion of such natural carbohydrates is generally about 1 to 20 g, preferably about 5 to 12 g per 100 ml of the composition of the present invention.

As other flavors, natural flavors (tauumatin, stevia extracts (e.g., rebaudioside A, glycyrzin, etc.) and synthetic flavors (saccharin, aspartame, etc.) can be advantageously used. The composition of the present invention is a variety of nutrients, vitamins, minerals (electrolytes), flavors such as synthetic flavors and natural flavors, coloring and neutralizing agents (such as cheese, chocolate), pectic acid and salts thereof, alginic acid and salts thereof, Organic acids, protective colloidal thickeners, pH adjusters, stabilizers, preservatives, glycerin, alcohols, carbonation agents used in carbonated beverages, and the like.

The compositions of the present invention may also contain pulp for the production of natural fruit juices and fruit juice beverages and vegetable beverages. These components can be used independently or in combination. The proportion of such additives is not critical but is usually selected in the range of 0-20 parts by weight per 100 parts by weight of the composition of the present invention.

Example

The following examples are provided to more specifically understand the present invention, but the present invention is not limited thereto.

Example 1: Confirmation of the sedative effect and other activity of silkworm pupa alcohol extract in endothelial cells or blood

end. NO production, cytotoxicity, secreted phospholipase A ₂ (s-PLA2) Activity Study

8, 9, 10, 11, 12, 14, and 15 days after extraction, silkworm pupa was extracted using 80% ethanol, and ethanol was evaporated to sample by concentration (1 mg / mL or 3 mg / mL). After preparation, it was administered to endothelial cells (mouse brain endothelial cells (bEND3) and macrophage cells (RAW 264.7 cells) and incubated for 12, 24, 48 hours in a CO ₂ incubator.

Investigation of NO production is carried out by Nims et al ., Nims, et al ., Nims, RW, Darbyshire, JF, Saavedra, JE, 1995.Colorimetric methods for the determination of nitric oxide concemtration in neutral aqueous solutions, Methods 7, 48 -54. Experiment using Griess reagent according to the method of 1995).

Roche's XTT assay (Geldof, AA, Mastbergen, SC, Henrar, RE and Faircloth, GT, Cytotoxicity and neurocytotoxicity of new marine anticancer agents evaluated using in vitro assays. Cancer Chemother.Pharmacol., 44 , 312-318, 1999) Was examined.

In addition, s-PLA ₂ activity was measured by Reynolds et al. (Reynolds, LJ, Hughes, LL, and Dennis, EA Analysis of human synovial fluid phospholipase A ₂ on short chain phosphatidylcholine-mixed micelles: development of a spectrophotometric using Cayman kit). assay suitable for a microtiterplate reader , Anal.Biochem . 204, 190-197, 1992).

As a result, as shown in FIG. 1, the NO production was highest in the pupa 11 days after the incubation in endothelial cells bEND3. In addition, as shown in Figure 2, the endothelial cells (bEND3) in the pupa, silkworms, male moth cell survival rate of each stage was examined by MTT assay, there is no cytotoxicity from 11 to 15 days after injured, especially at 15 days Very high cell growth rate was confirmed.

In addition, the results of NO production, cell viability, and s-PLA ₂ irradiation in macrophage cells (RAW 264.7 cells) are shown in FIGS. 3 to 5 and Table 1 below. The highest NO production was observed at 14 and 15 days, and the survival rate of macrophages increased in all stages of the pupal stage, indicating that cell growth was increased. Secretion-type phospholipase A ₂ was significantly lower than the positive control bee venom. The secretion was confirmed to be independent of the inflammatory response.

Post processing time 12 hours 24 hours 48 hours density 1 mg / mL 3 mg / mL 1 mg / mL 3 mg / mL 3 mg / mL Control  1.77 ± 0.09  1.15 ± 0.09 12.24 ± 0.24 SNP  8.71 ± 0.06 12.84 ± 0.26 63.56 ± 1.39 8 days after foot 6.29 ± 0.06 5.18 ± 0.24 8.98 ± 0.28 9 days after foot 5.44 ± 0.10 5.70 ± 0.25 4.74 ± 0.16 10 days after foot 4.55 ± 0.04 12.01 ± 0.21 3.98 ± 0.06 18.50 ± 0.54 15.93 ± 0.41 11 days after foot 3.53 ± 0.04 6.75 ± 0.10 4.10 ± 0.12 6.45 ± 0.23 32.66 ± 0.33 12 days after foot 3.78 ± 0.10 8.34 ± 0.15 2.03 ± 0.07 9.41 ± 0.43 9.30 ± 0.16 14 days after foot 8.92 ± 0.26 9.69 ± 0.42 8.16 ± 0.29 15 days after foot 8.75 ± 0.04 7.57 ± 0.27 7.13 ± 0.17 silkworm 3.87 ± 0.06 9.05 ± 0.07 2.27 ± 0.09 8.25 ± 0.19 10.39 ± 0.21 Male moth 5.18 ± 0.15 4.86 ± 0.07 6.21 ± 0.16

* 100 μM SNP is used, NO production amount (x100 μM)

I. Cyclic GMP Measurement

ICR 40 ± 1 g male mice were acclimated for 10 days, followed by intraperitoneal administration of 80 mg / kg of pupal ethanol extract for 3 days after incubation, and then blood was collected from the orbital vein of the mouse to isolate serum and replace it with nitrogen, acetyl Cyclic GMP was prepared using the Cayman kit by Pradelles et al. (Pradelles, P. and Grassi, J. Enzyme Immunoassays of Adenosine cyclic 3 ', 5'-monophosphate using acethylcholineesterase.Anal. Chem., 61, 447, 1989). And analyzed.

The results are shown in Figure 6, the amount of cyclic GMP per unit protein until day 15 after the silkworm pupae showed a relatively increased even value (Fig. 6a) yielded a 50% effective concentration of Cyclic GMP (Fig. 6b).

All. rat In splenocytes mTNF -α activity investigation

BALB / c, ethanol extract (8mg / 20ul) per silkworm pupal stage, per 200 uL of splenocyte suspension isolated from 6-week-old female rats, were inoculated into 96well for 16 hours and cultured in 5% CO2 incubator for mTNF-α activity ( Vilcek J. and TH Lee, J. biol. Chem. 266, 7313, 1991) are shown in Figure 7 the results of the R & D kit.

In contrast, a mouse necrosis factor (mTNF-α) was used as a control. As a result, the cancer necrosis factor, a type of cytokine secreted by the activation of splenocytes, which are immune cells, was slightly increased in silkworm pupa on the 15th day. It showed only a slight increase in cell-immunoactivation at the concentration of use, and confirmed that there is no problem of side effects due to severe cell-immune action.

Example 2 Identification of Chrysalis Gangster Material

end. Obtaining Gangjeong Fractions and Their Biological Activities

Using organic fractionation, Gangjeong fraction was obtained to have the therapeutic effect of erectile dysfunction by improving the sexual function by causing expansion of vascular endothelial cells of penis with relatively low cytotoxicity. same.

As shown in the schematic diagram of silkworm pupae fraction extraction of FIG. Fractions were then performed in aqueous butanol solution, and each fraction was concentrated.

The amount of NO produced was investigated for each of the obtained fractions. Specifically, 100 μL of the CPAE cell line was cultured into 96 wells, and each fraction was diluted in multiples of 10 (10 mg / mL, 20). μL), and incubated in a 5% CO ₂ incubator for one day, NO production was measured by Nims et al.

The result is shown in FIG. 9, and it was confirmed that the total amount of nitric acid was 156.87 μM in the water (water) fraction. In Figure 9, the first group is the water that dissolved the sample as a control, the second group is chloroform extract, the third group is the water-soluble water fraction at the bottom in the schematic diagram, the fourth group is butanol extract, the fifth group is hexane first extract, and the sixth group is hexane Residue, group 7 means ethyl acetate extract 10mg / mL, group 8 means ethyl acetate extract 50mg / mL, group 9 means chloroform middle emulsion layer extract, group 10 means hexane middle emulsion layer extract, group 11 means saturated butanol extract .

In general, NO production has the advantage of vasodilation, but in some cases, superoxide may cause cell damage, resulting in maximum NO activity, which is an indicator of vascular endothelial cell expansion capacity, and minimal cytotoxicity. It is important to find the fraction. As a result, experiments were conducted to obtain the desired fractions. The water-soluble fractions were found to be non-toxic, showing a 50% cytotoxicity concentration of 362.3 mg / mL (Table 2).

Cytotoxicity in Bovine Endothelial Cells (CPAE) Organic-fraction 1 (control) 2 3 4 5 6 7 8 9 10 11 IC ₅₀ (mg / mL) > 100 > 100 362.3 2.31 1.35 3.73 > 100 > 100 > 100 6.33 > 100

In Table 2, each group name is identical to the group name of FIG. 9, and each sample was added 10 mg / mL, 20 μL, and the cells used were CPAE cells, and were cultured in a 5% carbon dioxide gas incubator for 24 hours.

I. Active fraction, maximum active fraction obtained and investigation of its activity

(1) Adopt NO assay in endothelial cells instead of DPPH activity assay

Organic-fraction 3, the final water fraction obtained above, was purified by anion chromatography and subjected to DPPH (1,1-diphenyl-2-picrylhydrazyl) color reaction with a large number of peaks detected (Fig. 10), the tablets were purified based on NO activity indices in endothelial cells.

(2) Obtaining Active Fractions and Identification thereof

The organic-fraction 3 obtained above was subjected to anion exchange gel filtration chromatography and desalting to obtain an active fraction, the specific method of obtaining and identifying the method as follows.

A. Perform anion exchange gel filtration chromatography and investigate NO activity

Anion gel filtration chromatography was performed on organo-fraction 3, specifically 1.6 × 70 cm column of DEAE sepadex A25 resin with increasing salt (0, 0.1, 0.5, 1, 2.5M NaCl) for 2 g of organo-fraction 3 Purification was carried out with 10 mL / 5 min per fraction in a solvent of 50 mM phosphate buffer (pH 7.4), and the NO activity of each was measured and shown in FIG. 11. DEAE-fraction 52-59 was taken.

B. Desalting and Separation by Molecular Weight (Optional Step)

After desalting and separating by molecular weight, Biogel P10 (BMS) (1.5 x 80 cm) was charged, and the fractions taken in the previous step were concentrated and poured. The fractions obtained by flowing tertiary distilled water into the solvent were again absorbed by UV absorbance and NO activity. Irradiation gave a desalted fraction 14-18, an active fraction showing the maximum NO production (Fig. 12). However, in some cases, such a desalting step may be omitted, or the desalting column may be concentrated in distilled water using a desalting column and a dialysis membrane molecular weight cut off, and then concentrated.

C. Cytotoxicity Study on Active Fraction

As a result of examining the cytotoxicity of the active fraction obtained above, as shown in FIG. 13, it was confirmed that the 490 nm absorbance was 0.4 or more and there was no cytotoxicity (based on 0.35 in the non-treated cells).

(3) Obtaining the maximum active fraction through reverse phase HPLC and purification of the tablet

For the active fraction obtained above, a Luna 5μ C ₁₈ column (25 cm X 1.0 cm, ID, Phenomex, using HPLC-RPC (Thermo Spectra Products, San Jose, USA, P4000, Spectrasystem UV 3000 photodiode UV-Vis detector) USA) with 70% solvent for methanol and 1.0 mL / min flow rate at 254 nm using a UV absorbance spectrometer. The amount and cytotoxicity were examined by XTT assay. The results are shown in Table 3. Among them, 14.5 minutes, 15 minutes, and 16 minutes, which are excellent fractions in terms of NO production and cytotoxicity, were analyzed for the subsequent structural identification.

Also, in some cases, RPC (Source 15RPC, amersham pharmacia biotech) resin is filled with polar ethyl alcohol or methanol in the range of 0-100% of gradient or isocratic 60% or more of dimethyladenosine. Elution is possible. A suitable flow rate is 0.7-3.0 mL / min.

As shown in Table 3 below, 15 minutes (RPC-III fraction) and 16 minutes (RPC-IV fraction) release the maximum amount of NO production in vascular endothelial cells to relax, expand blood vessels and induce erection, cytotoxicity Was a non-treatment contrasting level, indicating no toxicity.

Measurement of NO Production and Cytotoxicity by Chrysalis Fraction in CPAE Cells Fraction 13 minutes (control) (RPC-I) 14.5 minutes (RPC-II) 15 minutes (RPC-III) 16 minutes (RPC-IV) 17 minutes (RPC-V) 18 minutes (RPC-VI) NO production (540 nm) 0.079 1.225 1.412 1.426 0.878 0.853 Cell viability 82.4 23.3 93.6 86.9 107.0 93.9

Example 3 Confirmation of Structure of Steel Crystal

end. Molecular Weight Measurement

Molecular weights were measured using MALDI-TOF MS (Voyager DE-STR, Applied Biosystems, USA). As a result, the molecular weights of the active substances within 14.5 minutes (II), 15 minutes (III), and 16 minutes (IV) of the active liquid separation were 451, 451, and 432, respectively.

I. IR spectrum

IR was measured for each fraction, and as a result, the following characteristic peaks were obtained as shown in FIGS. 16A and 16B.

(1) 14.5 min (II) and 15 min (III): v _max cm ^-1 (KBr); 3522 (OH), 3441 (NH), 2955 (CH-CH ₂ ), 2361 (CH ₃ CO), 1572 (C = O), 1141 (glycosidic CO)

(2) 16 min (IV): v _max cm ^-1 (KBr); 3495 (OH), 3227, 2874 (CH-CH ₂ ), 2361 (CH ₃ CO), 1949, 1657 (C = O), 1146 (glycosidic CO)

All. Melting Point Measurement

Melting points were measured for each fraction, and the results are shown in Table 4 below.

14.5 minutes (RPC-II fraction) 16 minutes (RPC-IV fraction) Start riding 188.5 172 Shot temperature 189 173

la. UV maximum absorbance measurement

Ultraviolet maximal absorbance was measured, and as a result, as shown in FIGS. 17A and 17B, there was no 280 nm peak in 14.5 fractions (II) and 16 fractions (IV) of active liquid, but NO activity was 16 in 18 minutes of FIG. The protein (peptide) wavelength is found at 60% lower than the minute, but appears to be a small portion of the biodistribution absorption with the potential for secondary role.

hemp. Various qualitative and TLC color reactions

Fehling, ρ-Anisaldehyde, Ehrlich's reagent, Dragendorff's reagent, Serium oxide, Somogi method, and silver staining were performed.

White powder, TLC Ehrilich Reagent R f 0.6 (CH ₃ Cl ₃ : MeOH: H ₂ O) = 5: 4: 1

Yellow powder, TLC Ehrilich Reagent R f 0.6 (CH ₃ Cl ₃ : MeOH: H ₂ O) = 5: 4: 1

bar. NMR analysis

For fraction II, ¹ H-NMR, ¹³ C-NMR, and ¹ H- ¹ H COSY were performed, and the results are shown in FIGS. 18A, 18B, and 18C, respectively. Fraction IV was also performed and shown in FIGS. 19A, 19B and 19C, respectively.

In particular, the NMR results for fraction IV are as follows:

¹ H-NMR (600 MHz, D ₂ O) δ: 2.26 (1H, dd, J = 10.3, 15.3 Hz, H-2`a), 2.30 (6H, s, 2 x NC H <sub>3</sub> ), 2.57 (1H , dd, J = 2.9, 5.4 Hz, H-2`b), 3.91 (1H, br t, J = 3.5 Hz, H-5`a), 4.20 (1H, dd, J = 2.9, 10.3 Hz, H -3`), 4.27 (1H, br s, H-4`), 4.41 (1H, dd, J = 3.5, 4.9 Hz, H-5`b), 6.04 (1H, d, J = 5.95 Hz, H -1`), 8.16 (1H, s, H-8), 8.51 (1H, s, H-2)

¹³ C-NMR (150 MHz, D ₂ O) δ: 34.9 (N- C H ₃ ), 43.4 (C-2`), 63.2 (C-5`), 71.1 (C-3`), 85.4 (C -1`), 87.5 (C-4`), 116.6 (C-5), 141.0 (C-8), 151.6 (C-4), 153.6 (C-2), 156.4 (C-6)

From this, when [· OH ₂ ] is added to the structure of fraction II it can be confirmed that the fraction IV.

four. Electric ion molecular weight absorbance analysis

As a result of measuring the molecular weight of falling ions using an electro ion molecular weight absorber (High resolution Tandem Mass spectrometer, Autospec OA-TOF, Micromass, UK, EI-mode, Electron 70eVDIP mode, Direct Inlet Probe) Reference).

EI-MS m / z (Rel. Int.,%) [M] ⁺ 182, 183 [M + H] ⁺ , 267 (84), 317 (50), 432 (19)

Through the structural identification of the above material, it was confirmed that the active material of the pupa extract was a dioxydimethyladenosine compound as follows.

<Formula 1>

The compound of Formula 1 is 5- {6- (dimethylamino) -9H-purin-9-yl} -tetrahydro-4 '-(hydromethyl) furan-3'-ol according to the IUPAC nomenclature. The compound may be present in the form of a OH group (hydroxy adenosine form) bonded to the carbon position 2 of the ribose pentose instead of hydrogen.

Monosaccharide can be attached to the compound of Formula 1, in particular, RPC-IV fraction with 0.1 N HCl or endoglycosidase F (Endogycosidase F, Sigma) after enzymatic treatment culture culture supernatant is injected to the sugar structure by HPLC-AU ECD (Dionex submmit HPLC, ED50, carbo pak PA1 column, sodium hydroxide solvent) detector confirmed that L (+)-Arabinose, and sometimes D (+)-xylose, peaks This is consistent with the retention time of 16.6 minutes (see Figure 23).

That is, the active substance of the RPC-IV fraction was confirmed to have the structure of formula (2) to which L (+)-arabinose is bound (molecular weight 432), and according to the IUPAC nomenclature, 5- {6- (dimethylamino ) -9H-purin-9-yl} -tetrahydro-4'-furan-3'-ol-5'-o-tetrafyro-2H-pyran-3 '', 4 '', 5 ' '-Triol.

<Formula 2>

A hydrate of OH ₂ may be added to the compound of Formula 2, an RPC-II fraction corresponds to this (molecular weight 451), and may be formed by addition of · O to the compound of Formula 1.

<Formula 3>

·OH₂

OH ₂

Example 4: Confirmation of the reinforcing effect

end. Investigation of phosphodiesterase inhibitory activity

The phosphodiesterase inhibitory activity according to each purification fraction was investigated as follows, and the results are shown in Table 5 below.

The phosphodiesterase I used was performed according to the manual of PDE type IV (Sigma) 5 mg / mL, 10 μL, Phosphodiestrase [ ³ H] cAMP SPA Enzyme Assay from Crotalus atrox (Amersham Biosciences), Inhibitors were used at concentrations of 0, 2, 10, and 20 μg according to the concentration of the purified fraction (FIG. 21). The kit used is that 3'5 '-[ ³ H] cAMP is converted into' 5 '-[ ³ H] cAMP by PDE, and is absorbed by scintillation proximity assay (SPA) beads to measure tritium radiation. If PDE inhibitor is present, it is the principle that '5'-[ ³ H] cAMP is not generated and SPA count is not generated. RPC-II and RPC-IV fractions produced less radioactivity with increasing treatment concentrations, indicating a dose-dependent inhibition of phosphodiesterase.

Purified fraction IC ₅₀ (μg / mL) Anion Exchange Gel Filtration Chromatography DX fraction 2.8 x 10 ^-3 Reverse Phase HPLC RPC-II Fraction Capacity independent RPC-III fraction 8.63 RPC-IV Fraction 0.88

I. Comparison of the Inhibitors of PDE Type V in Conventional Inhibitors and Aliquots According to the Invention

Sandwich relativity comparing phosphodiesterase inhibitory effects when combined with existing inhibitors by adding the same amount of inhibitor to the product and changing the dose of DX fraction, a purified fraction only by anion exchange gel filtration chromatography, to 20μg, 10μg, 4μg Comparison was made. The enzyme used was also compared with 10 μL of phosphodiesterase V (cGMP-characteristics, bovine, compared to Crotalus atrox ) -derived phosphodiesterase (PDE) type IV (Sigma, 5 mg / mL, 10 μL). PDE Type V inhibition was also examined using recombinant, Spodera frugiperda (Calbiochem).

As shown in FIG. 21, DX fractions showed an excellent inhibitory effect on Cyclic GMP selective phosphodiesterase type V and showed superior effects to conventional phosphodiesterase I, V, IV, and III inhibitors. .

All. Confirmation of the possibility of coadministration of the aliquot according to the present invention and the existing PDE inhibitor

In addition, as a control, 8-Methoymethyl-IBMX (PDE type I inhibitor) 15μM, 10μl, Trequinsin, hydrochloride (PDE type III inhibitor) 10 mM, 10μl, Rolipiram (PDE type IV inhibitor) 10 mM, 10μl, 4- Trequinsin, hydrochloride (PDE type III inhibitor) as analyzed using 10 mM, 10 μl of {[3 ', 4'-(methylenedioxy) benzyl] amino} -6-methoxyquinazolin (PDE type V inhibitor) In co-administration with PDE inhibitors, there was a synergistic effect, and any combination of PDE inhibitors with PDE inhibitors showed dose-dependent inhibition of PDEs.

In addition, the DX purified fraction of the Cyclic GMP selective phosphodiesterase type V enzyme was confirmed that the PDE inhibitory activity is comparable or even better than the conventional PDE inhibitors.

As described above, the silkworm-derived (dimethyl) adenosine compounds and derivatives thereof according to the present invention produce nitric oxide (NO) in sildenafil-level superior vascular endothelial cells by the mechanism of inhibition of phosphodiesterase-5 enzyme. In addition to inducing erection, the extraction and purification method according to the present invention was able to isolate and identify these substances. These silkworm-derived (dimethyl) adenosine compounds and derivatives thereof are useful as pharmaceutical materials by purely separating them into substances directly related to erectile dysfunction or masculinity.

Claims (10)

Adenosine compound derivative represented by the following formula (1), that is, a composition for treating erectile dysfunction comprising a pharmaceutically acceptable salt or solvate thereof as an active ingredient. <Formula 1>

A composition for treating erectile dysfunction comprising an adenosine compound represented by Formula 1 or a pharmaceutically acceptable salt thereof or solvate thereof as an active ingredient. <Formula 2>

In the composition for treating erectile dysfunction, (i) organic fractionation was performed with (i) ethanol extract of silkworm pupa with hexane: water volume ratio of 1: 100-100: 1 to obtain water fraction a, and (ii) water fraction a. The organic fraction was purified by chloroform to obtain a water fraction b. (Iii) The organic fraction was fractionated with ethyl acetate for the water fraction b to obtain a water fraction c. (Iv) Butanol: water for the water fraction c. Performing an organic fraction with an aqueous butanol solution having a volume ratio of 1: 100 to 100: 1 to obtain a water fraction d, (b) performing anion exchange gel filtration chromatography on the water fraction d to select an active fraction a having an ultraviolet absorbance of 0.9 or more at 540 nm and a cell survival rate of 80% or more for measuring NO production, and (c) selectively desalting the active fraction a to select an active fraction b having an ultraviolet absorbance of 0.9 or more at 540 nm and a cell survival rate of 85% or more for measuring NO production An erectile dysfunction treatment composition comprising an active fraction a or active fraction b extracted by a method comprising an active ingredient. In the composition for treating erectile dysfunction, (i) organic fractionation was performed with (i) ethanol extract of silkworm pupa with hexane: water volume ratio of 1: 100-100: 1 to obtain water fraction a, and (ii) water fraction a. The organic fraction was purified by chloroform to obtain a water fraction b. (Iii) The organic fraction was fractionated with ethyl acetate for the water fraction b to obtain a water fraction c. (Iv) Butanol: water for the water fraction c. Performing an organic fraction with an aqueous butanol solution having a volume ratio of 1: 100 to 100: 1 to obtain a water fraction d, (b) performing anion exchange gel filtration chromatography on the water fraction d to select an active fraction a having an ultraviolet absorbance of 540 nm or greater and a cell survival rate of 80% or greater at 540 nm, (c) selectively desalting the active fraction a to select an active fraction b having an ultraviolet absorbance of 0.9 or more at 540 nm and a cell survival rate of 85% or more for measuring NO production; and (d) performing reverse phase HPLC on the active fraction a or active fraction b to select an active fraction c having an ultraviolet absorbance of 0.9 or more and a cell survival rate of 20% or more for measuring NO production An erectile dysfunction treatment composition comprising the active fraction c obtained by the method comprising as an active ingredient. The composition for treating erectile dysfunction according to any one of claims 1 to 4, further comprising an existing PDE inhibitor. (a) (i) performing organic fractionation with an aqueous hexane solution of hexane: water volume ratio of 1: 100-100: 1 with respect to the ethanol extract of silkworm pupa to obtain a water fraction a, (ii) the water fraction a Performing organic fractionation with chloroform to obtain water fraction b, (iii) performing organic fractionation with ethyl acetate on water fraction b to obtain water fraction c, and (iv) water fraction c with respect to water fraction c. Performing organic fractionation with an aqueous butanol solution having a butanol: water volume ratio of 1: 100 to 100: 1 to obtain a water fraction d, (b) performing anion exchange gel filtration chromatography on the water fraction d to select an active fraction a having an ultraviolet absorbance of 540 nm or greater and a cell survival rate of 80% or greater at 540 nm, (c) selectively desalting the active fraction a to select an active fraction b having an ultraviolet absorbance of 0.9 or more at 540 nm and a cell survival rate of 85% or more for measuring NO production Method for extracting adenosine compound derivative represented by the following formula (1) or a pharmaceutically acceptable solvate thereof from silkworm pupa comprising: <Formula 1>

(a) (i) performing organic fractionation with an aqueous hexane solution of hexane: water volume ratio of 1: 100-100: 1 with respect to the ethanol extract of silkworm pupa to obtain a water fraction a, (ii) the water fraction a Performing organic fractionation with chloroform to obtain water fraction b, (iii) performing organic fractionation with ethyl acetate on water fraction b to obtain water fraction c, and (iv) water fraction c with respect to water fraction c. Performing organic fractionation with an aqueous butanol solution having a butanol: water volume ratio of 1: 100 to 100: 1 to obtain a water fraction d, (b) performing anion exchange gel filtration chromatography on the water fraction d to select an active fraction a having an ultraviolet absorbance of 540 nm or greater and a cell survival rate of 80% or greater at 540 nm, (c) selectively desalting the active fraction a to select an active fraction b having an ultraviolet absorbance of 0.9 or more at 540 nm and a cell survival rate of 85% or more for measuring NO production A method for extracting adenosine compound derivative represented by the following formula (2) or a pharmaceutically acceptable solvate thereof from silkworm pupa comprising: <Formula 2>

(a) (i) performing organic fractionation with an aqueous hexane solution of hexane: water volume ratio of 1: 100-100: 1 with respect to the ethanol extract of silkworm pupa to obtain a water fraction a, (ii) the water fraction a Performing organic fractionation with chloroform to obtain water fraction b, (iii) performing organic fractionation with ethyl acetate on water fraction b to obtain water fraction c, and (iv) water fraction c with respect to water fraction c. Performing organic fractionation with an aqueous butanol solution having a butanol: water volume ratio of 1: 100 to 100: 1 to obtain a water fraction d, (b) performing anion exchange gel filtration chromatography on the water fraction d to select an active fraction a having an ultraviolet absorbance of 540 nm or greater and a cell survival rate of 80% or greater at 540 nm, (c) optionally performing a desalting process on the active fraction a to select an active fraction b having an ultraviolet absorbance of 0.9 or more at 540 nm and a cell survival rate of 85% or more for measuring NO production, and (d) an active fraction having an ultraviolet absorbance of 0.9 or more at 540 nm and a cell survival rate of 20% or more for the determination of NO production by activating the active fraction a or the active fraction b and performing reverse phase HPLC. step to select c Method for extracting adenosine compound derivative represented by the following formula (1) or a pharmaceutically acceptable solvate thereof from silkworm pupa comprising: <Formula 1>

(a) (i) performing organic fractionation with an aqueous hexane solution of hexane: water volume ratio of 1: 100-100: 1 with respect to the ethanol extract of silkworm pupa to obtain a water fraction a, (ii) the water fraction a Performing an organic fraction with chloroform to obtain a water fraction b, (iii) performing an organic fraction with ethyl acetate on the water fraction b to obtain a water fraction c, and (iv) obtaining the water fraction c with respect to the water fraction c Performing organic fractionation with an aqueous butanol solution having a butanol: water volume ratio of 1: 100 to 100: 1 to obtain a water fraction d, (b) performing anion exchange gel filtration chromatography on the water fraction d to select an active fraction a having an ultraviolet absorbance of 540 nm or greater and a cell survival rate of 80% or greater at 540 nm, (c) selectively desalting the active fraction a to select an active fraction b having an ultraviolet absorbance of 0.9 or more at 540 nm and a cell survival rate of 85% or more for measuring NO production; and (d) an active fraction having an ultraviolet absorbance of 0.9 or more at 540 nm and a cell survival rate of 20% or more for the determination of NO production by activating the active fraction a or the active fraction b and performing reverse phase HPLC. step to select c A method for extracting adenosine compound derivative represented by the following formula (2) or a pharmaceutically acceptable solvate thereof from silkworm pupa comprising: <Formula 2>

10. The extracting method according to any one of claims 6 to 9, wherein the silkworm pupa is silkworm pupa.

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