KR100923217B1 - Composition for the prevention and treatment of neuropathic pain comprising an extract of loranthus yadoriki or active compounds isolated therefrom - Google Patents

Abstract

The present invention is mistletoe ( Loranthus) yadoriki ) Extract, Viscumneoside III (Viscumneoside III) or Homoflavoyadorin B (Homoflavoyadorin B) isolated from the composition for the prevention and treatment of neuropathic pain (neuropathic pain) containing . Since the composition according to the present invention exhibits an excellent neuropathic pain inhibitory effect, as well as a pharmaceutical composition for preventing or treating chronic neuropathic pain, which is an intractable disease caused by trauma or inflammation, ischemic damage, or nerve damage caused by metabolites. It can also be usefully used as a food additive.

Mistletoe, Neuropathic Pain, Biscum Neoside III, Homoflavoyadorin B, Inflammation, Ischemic Injury

Description

FIELD OF THE PREVENTION AND TREATMENT OF NEUROPATHIC PAIN COMPRISING AN EXTRACT OF LORANTHUS YADORIKI OR ACTIVE COMPOUNDS ISOLATED THEREFROM}

The present invention is mistletoe ( Loranthus) yadoriki ) Extract, Viscumneoside III (Viscumneoside III) or Homoflavoyadorin B (Homoflavoyadorin B) isolated from the composition for the prevention and treatment of neuropathic pain (neuropathic pain) containing .

Neuropathic pain is a refractory disease caused by trauma or inflammation, ischemic damage or nerve damage caused by metabolites and is characterized by symptoms such as spontaneous pain, burning pain, allodynia and hyperalgesia. Neuropathic pain inhibitors include anticonvulsants such as Gabapentin and Pregabalin that act on calcium channels; Anticonvulsants, such as Carbamazepine, which act on sodium channels; Serotonin Norepinephrine Reuptake Inhibitor (SNRI) family antidepressants; And four types of opioids recommended only in emergencies are being used clinically. These drugs are used in most cases, but the drugs of each class have similar side effects, and thus the side effects may be worsened in some patients due to the combination, but the therapeutic effect is low. Because neuropathic pain inhibitors are usually administered to patients in the long term, the most ideal of preventing and treating neuropathic pain should be less toxic and orally administrable. Therefore, there is a need for the development of drugs and functional foods with insufficient toxicity as effective inhibitors for neuropathic pain.

According to Heo Joon's consent (Agreement's National Board of Directors, Dong-Bo-Am Promoted by the Government, Namsandang, 1217, Seoul, 1988), Mistletoe (Mulletula Mistletoe) has a flat nature, taste and bitterness, and poison. It has been documented to be effective against back pain and arthritis. In addition, in the West, mistletoe is known to be effective enough to be called golden branches. Mistletoe is a parasitic plant, viscum album var. Coloratum (represented by the parasitic host), mistletoe (Loranthus yadoriki, organic) on oak, and mistletoe (Pseudicis ramulus, young) Parasitic) (Kim Jae-gil, Metabolism of Native Natural Medicines (Hakwon), 138, Namsan Party, Seoul, 1989).

Mistletoe extract in the 1920s in in vitro and in As the anti-cancer activity has been revealed in vivo , attention has been focused and many studies have since been reported (Taek Joon Yoon et al., International Immunopharmacology 1881-1889, 2001; Gorter RW et al., Alternative Therapies in Health & Medicine 5 (6): 37-44, 47-8, 1999; Hajto T et al, Forschende Komplementarmedizin 6 (4): 186-94, 1999; Vehmeyer K et al., European Journal of Haematology 60 (1): 16-20, 1998; Bruseth S et al., Tidsskrift for Den Norske Laegeforening 113 (9): 1058-60, 1993; Kast A et al., Schweizerische Rundschau fur Medizin Praxis 79 (11): 332-4, 1990; Stein GM et al., Anticancer Research 20 (3A): 1673-8, 2000; Kunze E et al., Journal of Cancer Research & Clinical Oncology 126 (3): 125-38, 2000; Stein GM et al., Anticancer Research 19 (4B): 2925-8, 1999 Jul-Aug .; Yoon TJ et al., Cancer Letters 97 (1): 83-91, 1995; Mannel DN et al., Immunotherapy 33 (3): 177-82, 1991; Bussing A, Anticancer Research 19 (1A): 23-8, 1999; And Weber K et al., Arzneimittel - Forschung 48 (5): 497-502, 1998).

Most of the anticancer active ingredients isolated from mistletoe extracts were lectins (Lectin) and some alkaloids were reported (Khwaja TA et al., Oncology 43 Suppl 1: 42-50, 1986), but their structure has not been identified yet. . Mistletoe diabetes (Gray AM and Flatt PR, Journal of Endocrinology 160 (3: 409-14, 1999), which is expected to be used in the private sector as a therapeutic agent, and this therapeutic effect is presumed to be due to an active ingredient other than lectin. Antithrombotic Effects of Mistletoe and Its Active Ingredients (Hyun Ok Yang et al., Natural Product Sciences 8 (4), 2002) and Cytotoxcity on Cancer Cells of Korean Mistletoe Water Extract (Park JH et al., Cancer Letters 126 (1): 43-8, 1998). Lectin in the mistletoe extract also induces apoptosis in tumor cells (Yoon TJ et al., Cancer Letters . 136 (1): 33-40, 1999), mistletoe itself has been reported to have a protective effect on tumor metastasis (Yoon TJ et al., International) Journal of Immunopharmacology 20 (4-5): 163-72, 1998). In addition to this, mistletoe has anticancer and immunomodulatory effects, and its active ingredient contains sugars and proteins, which are known to affect the immune system, lectins (1-14), flavonoids and phytosterols (Ohta). N, Agric . Biol . Chem . 34: 900-907, 1970; Sam S et al ., Thrombosis Research 98: 531-540, 2001).

However, no neuropathic pain inhibitory effect of mistletoe extract has been reported. The present inventors have found the neuropathic pain inhibitory ability of the mistletoe extract that has not been identified so far, and completed the present invention by separating and purifying the active ingredient exhibiting the neuropathic pain inhibitory ability therefrom.

Accordingly, an object of the present invention is to suppress neuropathic pain using mistletoe extract as an active ingredient, which is useful for the prevention and treatment of chronic neuropathic pain, which is an intractable disease caused by trauma or inflammation, ischemic damage or nerve damage caused by metabolites. It is to provide a composition.

It is also an object of the present invention to provide a food having a neuropathic pain inhibitory effect, including mistletoe extract.

In order to achieve the above object, the present invention includes biscum neoside III and homoflavoyadorin B as active ingredients, and mistletoe showing a neuropathic pain inhibitory effect ( Loranthus) yadoriki ) to give an extract.

The present invention also provides Viscumneoside III and its pharmaceutically acceptable salts, which are isolated and purified from the mistletoe extract and exhibit neuropathic pain inhibitory effects.

In addition, the present invention provides a homoflavoyadorin B (Homoflavoyadorin B) and its pharmaceutically acceptable salts that are isolated and purified from the mistletoe extract and exhibit neuropathic pain inhibitory effect.

In addition, the present invention comprises a mistletoe extract comprising a compound selected from the group consisting of biscum neoside III, homoflavoyadorin B, pharmaceutically acceptable salts of the compounds or mixtures thereof as an active ingredient. It provides a pharmaceutical composition for the prevention and treatment of neuropathic pain.

Finally, the present invention is a neuropathic pain containing a mistletoe extract containing a compound selected from the group consisting of biscum neoside III, homoflavoyadorin B, a pharmaceutically acceptable salt of the compounds or mixtures thereof. To provide a dietary supplement with the effect of inhibiting it.

The composition of the present invention is a chronic neuropathic disease that is a refractory disease caused by trauma or inflammation, ischemic damage or nerve damage caused by metabolites by containing an active ingredient of mistletoe extract having an neuropathic pain inhibitory ability or an active ingredient isolated therefrom. It can be usefully used to prevent or treat pain.

The present invention includes biscum neoside Ⅲ and homoflavoyadorin B as active ingredients and mistletoe ( Loranthus) showing neuropathic pain inhibitory effect yadoriki ) to give an extract.

Mistletoe extract of the present invention is added to the extraction solvent 1 to 20 times, preferably 3 to 10 times the volume of the dried mistletoe, and 1 to 10 days at 20 to 100 ℃, preferably 2 to Extraction may be carried out for 3 days, cooled, filtered, and then concentrated under reduced pressure. In this case, water, lower alcohol or a mixture thereof may be used as the extraction solvent, and preferably water, methanol, aqueous methanol solution, ethanol, ethanol aqueous solution, butanol, methylene chloride, ethyl acetate or a mixture thereof may be used. The extraction process may be performed by hot water extraction, ultrasonic extraction, reflux cooling extraction, cold soaking and the like.

Mistletoe extract may be prepared according to a conventional extraction method known in the art, in a preferred embodiment of the present invention 2 to 24 hours using 5 to 10 times the volume of 100% methanol in the mistletoe or its dry matter as the extraction solvent , Preferably extracted under reflux for 3 hours, filtered using filtrate, and the filtrate was concentrated under reduced pressure to prepare a methanol extract of mistletoe (see FIG. 1 ).

In another embodiment of the present invention, 1 to 20 times the volume of distilled water is added to mistletoe or its dry matter and heated and extracted at 70 to 100 ° C., preferably at 90 to 100 ° C. for 3 to 10 hours, preferably 6 hours. Filter under atmospheric pressure using and freeze-dried the filtrate to prepare a mistletoe water extract (see Figure 1 ).

In addition, the present invention isolates and purifies biscum neoside III and homoflavoyadorin B as active ingredients exhibiting neuropathic pain inhibitory effect from the mistletoe extract.

To this end, the mistletoe extract is fractionally extracted using a nonpolar solvent selected from the group consisting of ether, hexane, methylene chloride, ethyl acetate and mixtures thereof. In this case, as the nonpolar solvent, hexane, methylene chloride or ethyl acetate is preferable.

Specifically, methanol soluble water obtained by concentrating the methanol extract of mistletoe under reduced pressure was suspended in distilled water, extracted with hexane, and separated into hexane soluble fraction and distilled water fraction. Methylene chloride is added to the distilled water fraction and extracted to separate the methylene chloride soluble fraction and the distilled water fraction, ethyl acetate is added to the distilled water fraction separated therefrom and extracted to separate the ethyl acetate soluble fraction and the distilled water fraction. In order to isolate and purify the active ingredient exhibiting neuropathic pain inhibitory effect from the obtained ethyl acetate soluble fraction, column chromatography and reverse phase column chromatography using a stepwise gradient of an eluting solvent are performed (see FIG. 1 ). The column chromatography uses a filler selected from the group consisting of silica gel, Sephadex, RP-18, polyamide and XAD resin. Column chromatography uses a mixture of methylene chloride and methanol as the elution solvent, and reverse phase column chromatography uses a mixture of methanol and water as the elution solvent. In this case, gradient elution is used to sequentially increase the methanol concentration. Elution fractions).

By virtue of the above process, as an active ingredient exhibiting neuropathic pain inhibitory effect from the mistletoe extract, biscumneoside III of formula 1 and homoflavoyadorin B of formula 2 are separated and purified.

Biscumneoside III of Formula 1 and Homoflavoyadorin B compounds of Formula 2 according to the invention can be used in the form of pharmaceutically acceptable salts derived from inorganic or organic acids, or bases according to conventional methods in the art. Can be. The salt derived from the inorganic acid or organic acid is not particularly limited, but for example, inorganic acids such as hydrochloric acid, hydrobromic acid, phosphoric acid, sulfuric acid, acetic acid, trifluoro, and the like which are known in the art to the compounds of Formula 1 or 2 above. Salts obtained by imposing organic carboxylic acids such as acetic acid, citric acid, formic acid, maleic acid, hydroxyl, succinic acid, benzoic acid, tartaric acid, fumaric acid, mandelic acid, ascorbic acid or malic acid, methanesulfonic acid, or para-toluenesulfonic acid may be used. have. These acid addition salts can be prepared by conventional methods, and other acids, such as oxalic acid, which are not pharmaceutically acceptable on their own, can also be used to prepare pharmaceutically acceptable acid addition salts of the compounds according to the invention. Can be.

The compounds of the formulas (1) and ( 2) according to the invention can also be used in the form of pharmaceutically acceptable salts with bases, which include alkali metal hydroxides (e.g. sodium hydroxide, potassium hydroxide), alkali metal bicarbonates (e.g. Inorganic bases such as sodium bicarbonate, potassium bicarbonate), alkali metal carbonates (eg sodium carbonate, potassium carbonate, calcium carbonate) and organic bases such as primary, secondary and tertiary amine amino acids.

The compounds of the invention may also be in the form of solvates, especially hydrates. Hydration may occur during separation of the compound or may occur over time due to the hygroscopicity of the compound.

The neuropathic pain inhibitory effect of the mistletoe extract of the present invention and the active ingredients separated therefrom was investigated in the animal model in which the pain was induced to confirm that they exhibited excellent antiallodynic effect ( Tables 1 to 5 and FIGS. 2 to 2). 6 ). In addition, the biscum neoside III of the active ingredient isolated from the mistletoe extract of the present invention is a neuropathic pain statistically similar or better than the group administered 300 mg / kg of gabapentin even in a small amount of 10 mg / kg While showing an inhibitory effect (see Table 6 and Figure 7 ), it is confirmed that it does not exhibit toxicity without causing side effects such as sleepiness, motor dysfunction, which is a disadvantage of gabapentin. Therefore, the mistletoe extract of the present invention and the active ingredient separated therefrom can be usefully used as an oral therapeutic agent for the inhibition of neuropathic pain which does not affect daily life, as well as health foods and patch products. In particular, it can be prescribed more safely to patients with neuropathic pain due to metabolic diseases such as surgery, trauma, nerve damage, and diabetes, and can be used as an easy-to-use medicine.

Accordingly, the present invention provides a composition for inhibiting neuropathic pain, which contains mistletoe extract, biscum neoside III or homoflavoyadorin B purified therefrom as an active ingredient. The composition may be a pharmaceutical composition or a food composition.

Mistletoe extract contained in the pharmaceutical composition of the present invention is preferably purified through column chromatography, but may also include an extract obtained by simply solvent extraction. In addition, the biscum neoside III of Formula 1 and Homoflavoyadorin B of Formula 2 may be isolated and purified from mistletoe extract or chemically synthesized according to methods known in the art.

The pharmaceutical composition according to the invention is characterized in that it is used for the prevention or treatment of neuropathic pain. In the present invention, neuropathic pain is a peripheral or central nerve that transmits pain due to trauma or inflammation, ischemic damage, or nerve damage caused by metabolites, and sends a wrong signal to the pain control center, thereby causing no pain. It means an incurable disease that causes pain.

The pharmaceutical compositions according to the invention may be administered individually or in combination with other types of neuropathic pain inhibitors.

The pharmaceutical composition of the present invention may contain 0.1 to 20% by weight of the mistletoe extract or compound, and may further include appropriate carriers, excipients and diluents commonly used in the preparation of pharmaceutical compositions. Carriers, excipients and diluents that may be included in the pharmaceutical composition include lactose, dextrose, sucrose, sorbitol, mannitol, xylitol, erythritol, maltitol, starch, acacia rubber, alginate, gelatin, calcium phosphate, calcium silicate, cellulose, methyl Cellulose, microcrystalline cellulose, polyvinyl pyrrolidone, water, methylhydroxybenzoate, propylhydroxybenzoate, talc, magnesium stearate and mineral oil.

The pharmaceutical compositions of the present invention may be formulated using methods well known in the art to provide rapid, sustained or delayed release of the active ingredient after administration to a mammal. Such formulations may be in the form of powders, granules, tablets, capsules, suspensions, emulsions, syrups, aerosols and the like, oral formulations, suppositories, sterile injectables and the like. When formulated, it is formulated using diluents or excipients such as fillers, extenders, binders, wetting agents, disintegrating agents, surfactants, etc. conventional in the art.

Solid preparations for oral administration include tablets, pills, powders, granules, capsules and the like, which solid preparations comprise at least one excipient in the active ingredient, eg dextrin, starch, calcium carbonate. Prepared by mixing with sucrose, lactose and gelatin. In addition to simple excipients, lubricants such as magnesium stearate and talc are also used. Liquid preparations for oral administration include suspensions, contents, emulsions, and syrups.In addition to commonly used simple diluents such as water and liquid paraffin, various excipients such as wetting agents, sweeteners, fragrances, and preservatives can be used. May be included.

Formulations for parenteral administration include sterile aqueous solutions, non-aqueous solvents, suspensions, emulsions, lyophilized preparations, suppositories. As the non-aqueous solvent and suspending agent, propylene glycol, polyethylene glycol, vegetable oil such as olive oil, injectable ester such as ethyl oleate and the like can be used. As the base of the suppository, witepsol, macrogol, tween 61, cacao butter, laurin butter, glycerogelatin and the like can be used.

The pharmaceutical compositions of the invention can be administered via several routes including oral, transdermal, subcutaneous, intravenous or intramuscular. A preferred daily dosage of the pharmaceutical composition of the present invention containing at least one of mistletoe extract, biscum neoside III, homoflavoyadorin B, pharmaceutically acceptable salts of the compounds and mixtures thereof as an active ingredient is a patient. It should be appropriately selected by those skilled in the art to be determined in view of various related factors such as the condition and weight of the disease, the extent of the disease, the form of the drug, the route and duration of administration, the severity of the disease, and the like. However, in order to achieve a desirable neuropathic pain inhibitory effect, the pharmaceutical composition of the present invention is based on the active ingredient mistletoe extract of 0.01 to 10,000 mg / kg body weight, preferably 1 to 1,000 mg / kg body weight per day Range, biscumeoside III and homoflavoyadorin B, respectively, are preferably administered in the range of 0.01 to 1,000 mg / kg body weight, preferably 1 to 500 mg / kg body weight. Administration can be administered once a day or divided into several times.

In addition, the present invention comprises the mistletoe extract, biscum neoside III, homoflavoyadorin B, a pharmaceutically acceptable salt of the compounds or a mixture thereof, a health promoting food having a neuropathic pain inhibitory effect To provide a composition.

According to the present invention, foods to which mistletoe extract, biscum neoside III or homoflavoyadorin B may be added include, for example, various foods, beverages, gums, teas, vitamin complexes, health supplements, and the like. It is not limited.

When added to foods or beverages for the purpose of suppressing neuropathic pain, the amount of mistletoe extract may generally be added at 0.01 to 90% by weight, preferably 0.1 to 80% by weight of the total food weight. It may be added in a ratio of 0.01 to 90 g, preferably 0.1 to 50 g based on 100 ml.

The health beverage composition of the present invention does not have any particular limitation on the type and content of other liquid components except for containing the mistletoe extract in the indicated ratio as essential ingredients, and various flavors or natural carbohydrates are added as in general beverages. It may be contained as a component.

Examples of the natural carbohydrates include monosaccharides such as glucose and fructose; Disaccharides such as maltose and sucrose; And conventional sugars such as polysaccharides such as dextrin, cyclodextrin, and sugar alcohols such as xylitol, sorbitol, and erythritol. As flavoring agents other than those mentioned above, natural flavoring agents (tautin, stevia extract (rebaudioside A, glycyrzin, etc.), and synthetic flavoring agents (saccharin, aspartame, etc.) can be used. The proportion of cargo is generally 1 to 20 g, preferably 5 to 12 g per 100 ml of the health beverage composition of the present invention.

In addition to the above, the composition of the present invention includes various nutrients, vitamins, minerals (electrolytes), flavors such as synthetic flavors and natural flavors, coloring and neutralizing agents (such as cheese and chocolate), pectic acid and salts thereof, alginic acid and its Salts, organic acids, protective colloidal thickeners, pH adjusting agents, stabilizers, preservatives, glycerin, alcohols, carbonation agents used in carbonated drinks, and the like. The compositions of the present invention may also contain pulp for the production of natural fruit juices, fruit juice drinks and vegetable drinks. These components can be used independently or in combination.

Hereinafter, the present invention will be described in more detail by Examples, Experimental Examples and Preparation Examples. However, the following Examples, Experimental Examples and Preparation Examples are only for illustrating the present invention, and the scope of the present invention is not limited thereto.

< Example  1> Preparation of Mistletoe Extract

<1-1> Mistletoe Methanol Extract

In April 2004, 10 kg of 100% methanol was added to 2 kg of dried mistletoe, dried and dried at room temperature in Chiaksan, Gangwon-do, and extracted three times under reflux for about 3 hours. It was filtered. The filtrate was concentrated under reduced pressure to obtain 160 g of mistletoe methanol extract ( FIG. 1 ).

<1-2> Mistletoe Water Extract Preparation

In April 2004, 1 liter of distilled water was added to 200 kg of dried mistletoe, dried and dried at room temperature in Chiaksan, Gangwon-do, and extracted by heating twice at 90 to 100 ° C for 6 hours. The extract was filtered under normal pressure using a filter paper and lyophilized to obtain 6.73 g of mistletoe water extract ( FIG. 1 ).

< Example  2> Isolation, Purification and Identification of Active Ingredients

<2-1> Nonpolar Solvent Extraction

In order to separate the active ingredient from the mistletoe extract, 160 g of the mistletoe methanol extract obtained in Example 1 was suspended in 1 L of distilled water, and 1 l of n -hexane ( n -hexane) was added to the mixture, and the mixture was left to be separated. Extraction was performed three times with hexane to separate the hexane soluble fraction and the distilled water fraction. To the distilled water fraction, 1 L of methylene chloride (CH ₂ Cl ₂ ) was added and extracted, and the methylene chloride soluble fraction and the distilled water fraction were separated. 1 L of ethyl acetate (ethyl acetate, EtOAc) was added to the remaining distilled water fractions except for the methylene chloride soluble fraction, and the extract was concentrated under reduced pressure to obtain 5 g of an ethyl acetate soluble fraction ( FIG. 1 ).

<2-2> Biscum neoside  Isolation, Purification and Identification of III

Silica gel (70-230 mesh, 350 g) column (φ = 3.0 × 50 cm) chromatograph using 4.5 g of ethyl acetate soluble fraction obtained in Example <2-1> as an eluent as a methylene chloride / methanol mixed solvent Analysis by graphy. At this time, a concentration gradient elution method (20: 1 → 1: 1) was used to sequentially increase the concentration of methanol while flowing the elution solvent at a flow rate of 5 ml / min. This gave a total of five fractions of KW-E1 (1.05 g), KW-E2 (0.87 g), KW-E3 (0.56 g), KW-E4 (1.2 g) and KW-E5 (0.3 g). Among them, 1.0 g of fraction KW-E4, which exhibits neuropathic pain inhibition effect, was subjected to reverse phase silica gel (Reverse Phase, C18, 350 g) column (φ = 2.0 × 40 cm) chromatography using a methanol / water mixture as an elution solvent. Analyzed. At this time, a concentration gradient elution method (1: 1 → 9: 1) was used to sequentially increase the concentration of methanol while flowing the elution solvent at a flow rate of 1 ml / min. From this, a total of six fractions (KW-E4-A to KW-E4-F) were obtained, and 170 mg of Compound 1 was isolated from the fraction KW-E4-B showing neuropathic pain inhibitory effect among them ( FIG. 1 ). The compound 1 was analyzed using H-NMR, C-NMR, EI-MS, etc., and it was confirmed that the active ingredient exhibiting neuropathic pain inhibitory effect was biscum neoside III.

UV λ _max ^MeOH nm: 282, 222, 248, EI-MS (m / z): 596 C ₂₇ H ₃₂ O ₁₅ , m / e: 596.17 (100%), 597.18 (30.1%), 598.18 (7.5%) , 599.18 (1.3%)

¹ H-NMR (500 MHz, CDCl ₃ ): δ 2.71 (1H, dd, J = 3.0, 17.4 Hz, H-3), 3.15 (1H, dd, J = 8.5, 19.5 Hz, H-3), 3.32 (1H, m, H-4 "), 3.45 (1H, m, H-5"), 3.49 (2H, s, H-5 "), 3.62 (1H, m, H-2"), 3.64 (1H , H-4 "'), 3.75 (1H, d, J = 9.5 Hz, H-6"), 3.82 (1H, H-4 "'), 3.88 (3H, s, C-3'-OCH ₃ ) , 3.91 (1H, s, H-2 '"), 3.94 (1H, dd, J = 4.5, 9.5 Hz, H-6"), 5.01 (1H, d, J = 2.5 Hz, H-1 "), 5.37 (1H, m, H-1 '"), 5.38 (1H, d, J = 1.0 Hz, H-1'"), 6.12 (1H, d, J = 2.5 Hz, H-8), 6.15 (1H , d, J = 2.5 Hz, H-6), 6.78 (1H, d, J = 8.1 Hz, H-5 '), 6.88 (1H, d, J = 8.1 Hz, H-6'), 7.04 (1H , s, H-2 ')

¹³ C-NMR (125 MHz, CDCl ₃ ): δ 44.3 (C-3), 56.6 (C-3'-OCH ₃ ), 62.3 (C-6 "), 65.9 (C-5"), 71.2 (C -4 "), 75.4 (C-4"'), 78.1 (C-2 "'), 80.7 (C-3"'), 80.9 (C-2), 96.9 (C-6), 98.0 (C- 8), 99.8 (C-1 "), 105.0 (C-10), 110.9 (C-1"'), 111.4 (C-2'), 116.2 (C-5 '), 120.7 (C-6') , 130.5 (C-1 '), 148.2 (C-3'), 149.2 (C-4 '), 165.0 (2C, C-5, 9), 166.8 (C-7), 198.6 (C-4)

<2-3> Homoflavoyadorin  Isolation, Purification and Identification of B

Silica gel (70-230 mesh, 350 g) column (φ = 3.0 × 50 cm) chromatograph using 4.5 g of ethyl acetate soluble fraction obtained in Example <2-1> as an eluent as a methylene chloride / methanol mixed solvent Analysis by graphy. At this time, a concentration gradient elution method (20: 1 → 1: 1) was used to sequentially increase the concentration of methanol while flowing the elution solvent at a flow rate of 5 ml / min. This gave a total of five fractions of KW-E1 (1.05 g), KW-E2 (0.87 g), KW-E3 (0.56 g), KW-E4 (1.2 g) and KW-E5 (0.3 g). Among them, 1.0 g of fraction KW-E4, which exhibits neuropathic pain inhibition effect, was subjected to reverse phase silica gel (Reverse Phase, C18, 350 g) column (φ = 2.0 × 40 cm) chromatography using a methanol / water mixture as an elution solvent. Analyzed. At this time, a concentration gradient elution method (1: 1 → 9: 1) was used to sequentially increase the concentration of methanol while flowing the elution solvent at a flow rate of 1 ml / min. From this, a total of six fractions (KW-E4-A to KW-E4-F) were obtained, and 160 mg of Compound 2 was isolated from the fraction KW-E4-E, which shows neuropathic pain inhibitory effect. Analysis of the compound 2 using H-NMR, C-NMR, EI-MS, etc., it was confirmed that the active ingredient exhibiting neuropathic pain inhibitory effect homoflavoyadorin B.

UV λ _max ^MeOH nm: 343.5, 286.5, 269.0, 257.5, 250.5, 232.5, 214.5, EI-MS (m / z): 608 C ₂₈ H ₃₂ O ₁₅ , m / e: 59608.17 (100%), 609.18 (31.2 %), 610.18 (7.8%), 611.18 (1.4%)

¹ H-NMR (500 MHz, CDCl ₃ ): δ 3.12 (1H, m, H-4 ″), 3.28 (2H, br s, H-5 ″), 3.41 (1H, m, H-5 ″) , 3.44 (1H, m, H-6 ") 3.50 (1H, m, H-3"), 3.59 (1H, m, H-2 "), 3.59 (1H, d, J = 9.5 Hz, H-4 '"), 3.69 (1H, m, H-6"), 3.76 (1H, br s, H-2'"), 3.88 (3H, s, C-3'-OCH ₃ ), 3.90 (3H, s , C-3'-OCH ₃ ), 4.06 (1H, d, J = 9.5 Hz, H-4 '"), 5.14 (1H, d, J = 7.7 Hz, H-1"), 5.42 (1H, br s, H-1 '"), 6.40 (1H, d, J = 1.5 Hz, H-6), 6.86 (1H, d, J = 1.5 Hz, H-8), 7.08 (1H, s, H-3 ), 7.22 (1H, d, J = 8.6 Hz, H-5 '), 7.65 (1H, s, H-2'), 7.68 (1H, d, J = 8.6 Hz, H-6 ')

¹³ C-NMR (125 MHz, CDCl ₃ ): δ 55.9 (2C, C-3'-OCH _{3 ,} C-3 ', C-7), 64.3 (C-5 "), 69.8 (C-4" ), 73.9 (C-4 '"), 74.7 (C-2"), 76.0 (C-2'"), 76.1 (C-6"), 76.8 (C-5 "), 77.0 (C-3" ), 79.3 (C-3 '"), 93.1 (C-8), 97.9 (C-6), 98.1 (C-1"), 104.3 (C-3), 104.8 (C-10), 108.3 (C -1 "'), 110.0 (C-2'), 114.6 (C-5 '), 119.9 (C-6'), 124.1 (C-1 '), 148.9 (C-4'), 149.1 (C- 3 '), 157.2 (C-9), 161.4 (C-5), 163.4 (C-2), 165.1 (C-7), 182.1 (C-4)

< Experimental Example  1> Mistletoe Extract and Active Ingredients Isolated therefrom Neuropathic  Pain inhibitory effect

<1-1> Neuropathic  Pain Model Animal Experiment

Animal studies to investigate the neuropathic pain inhibitory effects of mistletoe extracts and the active ingredients isolated from them were conducted in accordance with the Ethics Regulations of the International Association for the Study of Pain.

The experimental animals used Sprague Dawley rats, 6 weeks old, weighing about 180 to 200 g at the time of surgery, and were adapted to the room atmosphere by arriving at the breeding room one week before the surgery. The breeding room was adapted to maintain a constant photoperiod (daytime: 6 am-6 pm) under sufficient supply of water and food, and was reared in four litter pails and maintained at a constant temperature (20-20) during the experiment. 24 ° C.) and constant humidity (40-60%) were maintained.

The construction and behavioral tests of neuropathic pain model mice were performed according to the methods described in Kim SH and Chung JM, Pain 50: 255-63, 1992. Three mechanical avoidance reactions were performed for one week prior to surgery to familiarize themselves with the experimental situation, and all three of these pain behavioral tests were performed on mice that did not exhibit pain avoidance. Surgery was performed by anesthetizing the experimental animals in a transparent anesthesia induction box with 100% oxygen and 4 vol% sevoflurane flowed at 3 l per minute, moving to the operating table, masking, and maintaining seboflurane at 4 vol%. Spontaneously breathing. Skin incisions from approximately L3 to S2 along the midline of experimental animals were exposed to the left L6-S1 interarticular projections. From this, the L6 transverse was removed, the left L4 and L5 spinal nerves were found at the base of the fascia, and the spinal nerves were carefully separated. The L5 spinal nerves were ligated with 5-0 silk thread. After irrigation with physiological saline and bleeding, pain response tests were performed on days 1, 4, 7, and 14, and the drug was administered to the rats in which the pain was apparent.

The mechanical avoidance reaction was performed by selecting a certain time period (9 am to 3 pm) to reduce the errors caused by photoperiodic. The pain avoidance response to mechanical stimulation was measured after the subject was placed in a transparent plastic box (8 × 8 × 18 cm) made of wire mesh and stabilized for 30 minutes. In order to quantify pain objectively, the steady-state control method quantifies the avoidance response to pain caused by pressure that does not cause pain. The von Frey filament (Semmes-Weinstein monofilament) has a constant pressure when bent. , USA). The filaments are eight in that the pressure is bent 0.41, 0.70, 1.20, 2.00, 3.63, 5.50, 8.50 and 15.14 g by applying a stimulus to the foot of the experimental animal. Among them, low pressure filaments were used sequentially to high filaments, and the stimulus was vertically applied for 6 seconds to bend the filament slightly from the bottom to the top of the center of the left foot of the animal exposed between the bottom wire mesh of the plastic box. At this time, the phenomenon that the test animal quickly avoided, lifted the soles, licked, whisked or escaped was considered as a positive avoidance reaction, and the pressure of the filament that caused the avoidance reaction was determined as the threshold value of the avoidance reaction.

The experiments were conducted blindly to compare the differences between the experimental and non-drug groups.

Statistical analysis was performed using the Sigma Stat (version 3.1) program, and a paired t-test was used for comparison with the reference values in the experimental and control groups. In addition, in order to verify the effect difference between the experimental group and the control group and to compare the effects over time, the One Way Repeated Measures Analysis of Variance was used and the Student-Newman-Keuls method was used. Post-validation was performed. All experimental results were expressed as mean ± standard deviation and considered significant only when P value was less than 0.05.

<1-2> Mistletoe Methanol Of extract Neuropathic  Pain inhibitory effect

Mistletoe Water Extract Neuropathic  Pain inhibitory effect

Inhibitory effect of neuropathic pain by administering 1 g / kg (n = 9) of methanol extract of mistletoe prepared in Example <1-1> to an experimental animal having pain according to the method of Experimental Example <1-1>. Was measured, and the results are shown in Table 1 and FIG. 2 . At this time, the same amount of solvent (0.5% CMC / water suspension, sodium carboxymethyl cellulose) was administered to the control group (n = 21).

Change in pain avoidance threshold (g) 0 min 30 minutes 60 minutes 90 minutes 120 minutes 150 minutes 180 minutes Control 1.5 ± 0.9 3.4 ± 3.4 4.4 ± 3.7 5.0 ± 3.4 4.0 ± 3.4 4.3 ± 3.6 3.4 ± 2.2 Methanol 1.9 ± 0.7 9.6 ± 5.7 11.9 ± 4.8 15.0 ± 0.0 10.0 ± 5.1 10.4 ± 4.8 9.6 ± 5.5

As shown in Table 1 and Figure 2 , the mistletoe methanol extract of the present invention was confirmed that the neuropathic pain inhibitory effect statistically significant (p <0.001) compared to the control group.

<1-3> Mistletoe Water Extract Neuropathic  Pain inhibitory effect

Inhibitory effect of neuropathic pain by administering 1 g / kg (n = 8) of the water extract of mistletoe prepared in Example <1-2> to the experimental animal having pain according to the method of Experimental Example <1-1> Was measured, and the results are shown in Table 2 and FIG. 3 . At this time, the same amount of solvent (0.5% CMC / water suspension, sodium carboxymethyl cellulose) was administered to the control group (n = 21).

Change in pain avoidance threshold (g) 0 min 30 minutes 60 minutes 90 minutes 120 minutes 150 minutes 180 minutes Control 1.5 ± 0.9 3.4 ± 3.4 4.4 ± 3.7 5.0 ± 3.4 4.0 ± 3.4 4.3 ± 3.6 3.4 ± 2.2 Experimental group 1.9 ± 0.5 8.9 ± 5.8 9.0 ± 5.9 9.3 ± 5.5 12.1 ± 4.6 15.0 ± 0 10.9 ± 5.7

As shown in Table 2 and Figure 3 , it was confirmed that the experimental group administered the mistletoe water extract of the present invention showed a neuropathic pain inhibitory effect statistically significant (p <0.001) compared to the control group.

<1-4> Mistletoe Ethyl Acetate Fraction Neuropathic  Pain inhibitory effect

Neuropathic pain by administering 1 g / kg (n = 8) of the ethyl acetate soluble fraction of mistletoe prepared in Example <2-1> to the experimental animal with pain according to the method of Experimental Example <1-1>. Inhibitory effect was measured, and the results are shown in Table 3 and FIG. 4 . At this time, the same amount of solvent (0.5% CMC / water suspension, sodium carboxymethyl cellulose) was administered to the control group (n = 21).

Change in pain avoidance threshold (g) 0 min 30 minutes 60 minutes 90 minutes 120 minutes 150 minutes 180 minutes Control 1.5 ± 0.9 3.4 ± 3.4 4.4 ± 3.7 5.0 ± 3.4 4.0 ± 3.4 4.3 ± 3.6 3.4 ± 2.2 Experimental group 1.6 ± 0.6 8.7 ± 5.7 11.6 ± 6.2 11.5 ± 6.0 12.0 ± 5.1 12.2 ± 5.3 10.9 ± 5.8

As shown in Table 3 and Figure 4 , the mistletoe of the present invention was confirmed that the neuropathic pain inhibitory effect statistically significant (p <0.001) in the experimental group administered the ethyl acetate fraction.

<1-5> Biscum neoside  Ⅲ Neuropathic  Pain inhibitory effect

Inhibition of neuropathic pain by administering 20 mg / kg (n = 19) of biscum neoside III isolated in Example <2-2> to the experimental animal with pain according to the method of Experimental Example <1-1>. The effect was measured and the results are shown in Table 4 and FIG. 5 . At this time, the same amount of solvent (0.5% CMC / water suspension, sodium carboxymethyl cellulose) was administered to the control group (n = 21).

Change in pain avoidance threshold (g) 0 min 30 minutes 60 minutes 90 minutes 120 minutes 150 minutes 180 minutes Control 1.5 ± 0.9 3.4 ± 3.4 4.4 ± 3.7 5.0 ± 3.4 4.0 ± 3.4 4.3 ± 3.6 3.4 ± 2.2 Experimental group 1.5 ± 0.7 6.7 ± 4.3 7.3 ± 5.2 9.1 ± 5.6 9.3 ± 4.8 8.9 ± 5.3 10.7 ± 5.0

As shown in Table 4 and Figure 5 , it was confirmed that in the experimental group administered the biscum neoside III of the present invention showed a neuropathic pain inhibitory effect statistically significant (p <0.001) compared to the control group.

<1-6> Homoflavoyadorin  B's Neuropathic  Pain inhibitory effect

Inhibition of neuropathic pain by administering 20 mg / kg (n = 11) of homoflavoyadorin B isolated in Example <2-3> to an experimental animal having pain according to the method of Experimental Example <1-1>. The effect was measured and the results are shown in Table 5 and FIG. 6 . At this time, the same amount of solvent (0.5% CMC / water suspension, sodium carboxymethyl cellulose) was administered to the control group (n = 21).

Change in pain avoidance threshold (g) 0 min 30 minutes 60 minutes 90 minutes 120 minutes 150 minutes 180 minutes Control 1.5 ± 0.9 3.4 ± 3.4 4.4 ± 3.7 5.0 ± 3.4 4.0 ± 3.4 4.3 ± 3.6 3.4 ± 2.2 Experimental group 1.8 ± 0.6 7.5 ± 5.4 7.8 ± 5.3 10.7 ± 5.5 9.4 ± 5.6 9.2 ± 5.0 8.9 ± 5.1

As shown in Table 5 and Figure 6 , it was confirmed that the neuropathic pain inhibitory effect statistically significant (p <0.001) in the experimental group administered homoflavoyadorin B of the present invention compared to the control group.

<1-7> Biscum neoside  Ⅲ and Gabapentin Neuropathic  Comparison of pain inhibitory effect

In order to compare the neuropathic pain inhibitory effect of biscum neoside III with gabapentin, which is currently used as a neuropathic pain inhibitor, biscum neoscis in the experimental animals suffering from pain according to the method of Experimental Example <1-1>. Cumneoside III 10 mg / kg (n = 12) and gabapentin 300 mg / kg (n = 11) were respectively administered to measure neuropathic pain inhibitory effects, and the results are shown in Table 6 and FIG. 7 . . At this time, the same amount of solvent (0.5% CMC / water suspension, sodium carboxymethyl cellulose) was administered to the control group (n = 21).

Change in pain avoidance threshold (g) 0 min 30 minutes 60 minutes 90 minutes 120 minutes 150 minutes 180 minutes Control 1.5 ± 9.9 3.4 ± 3.4 4.4 ± 3.7 5.0 ± 3.4 4.0 ± 3.4 4.3 ± 3.6 3.4 ± 2.2 Gabapentin 1.9 ± 1.0 3.9 ± 5.2 8.2 ± 6.8 9.0 ± 7.5 9.7 ± 6.6 9.2 ± 7.3 11.0 ± 6.8 Biscum neoside Ⅲ 2.3 ± 0.6 6.5 ± 5.9 13.1 ± 5.1 12.4 ± 4.5 13.3 ± 3.2 13.4 ± 4.3 11.8 ± 5.5

As shown in Table 6 and Figure 7 , all of the experimental groups administered the biscum neoside III and gabapentin of the present invention showed a neuropathic pain inhibitory effect statistically significant (p <0.05) compared to the control group. In addition, the biscum neoside III of the present invention shows a neuropathic pain inhibitory effect similar to or better than the gabapentin group, even though a small amount of 10 mg / kg was administered compared to the gabapentin to which 300 mg / kg was administered. .

< Experimental Example  2> Mistletoe Extract Acute Toxicity  Experiment

The acute toxicity (LD ₅₀ ) of mistletoe extract and the active ingredients isolated therefrom was investigated in the following manner.

As experimental animals, 6-week-old male mice (ICR mice) weighing about 18 to 22 g were used. The animals were allowed to arrive at the breeding room one week before the experiment. The breeding room was adapted to maintain a constant photoperiod (daytime: 6 am-6 pm) under sufficient water and feeding conditions, and was reared in four litter pails, and at a constant temperature (20 to 24 ° C). And constant humidity (40-60%). The experimental animals were fasted from the night before the start of the experiment to remove the experimental error due to food in advance. The experimental group was divided into eight groups per group, all samples were suspended orally in 0.5% CMC (Sodium Carboxy Methyl Cellulose) aqueous solution. Each sample was orally administered to the test animals at different doses (2, 1.0, 0.5, 0.25, and 0.125 g / kg), and death and abnormal behavior of the test animals were visually observed for 2 weeks. After 2 weeks, the animals were dissected to observe abnormalities of each organ.

The acute toxicity of Methanol extract of Mistletoe, Biscum neoside III and Homoflavoyadorin B isolated from the above-mentioned method was measured. There were no dead animals, and no toxic changes were observed in weight changes, blood tests, blood biochemistry tests, and autopsy findings.

Therefore, Mistletoe Methanol Extract, Biscum neoside III and Homoflavoyadorin B according to the present invention do not show toxic changes even up to 2 g / kg in mice, and the minimum lethal dose (LD50) of oral administration is at least 2 g / kg or more. It was considered safe.

Examples of preparations for the compositions of the present invention are illustrated below.

< Formulation example  1> Pilled  Produce

Biscum neoside III 120 mg

100 mg corn starch

Sterile distilled water

All of the above components were mixed, and refilled in a 0.3 cm diameter according to a conventional pill manufacturing method to prepare a pill.

< Formulation example  2> Preparation of Tablet

Homoflavoyadorin B 200 mg

Lactose 100 mg

Starch 100 mg

Magnesium stearate proper amount

All of the above components were mixed and tableted according to a conventional tablet manufacturing method to prepare a tablet.

< Formulation example  3> Liquid  Produce

Mistletoe Extract 1000 mg

20 g of sugar

20 g of isomerized sugar

Lemon flavor

According to the conventional liquid preparation method, all of the above components were mixed and purified water was added to adjust the total volume to 1000 ml, and then filled in a brown bottle and sterilized to prepare a liquid preparation.

< Formulation example  4> Patch  Produce

Mistletoe extract 15% by weight

Crodamol CAP 10% by weight

Eutanol GM 2% by weight

Polysorbate 80 4 wt%

1% by weight of oleic acid

Adhesive polymer 68% by weight

After mixing all the above components according to a conventional patch preparation method, the adhesive polymer solution dissolved in ethyl acetate was added and mixed for 10 minutes. After removing bubbles from the mixture, the nonwoven fabric was applied to a thickness of 1.3 mm, and then dried at 70 ° C. for 24 hours to remove all volatile solvents. Thereafter, the polyurethane film was adhered onto the nonwoven fabric and cut into 20 cm 2 to obtain a final product.

< Formulation example  5> Health drink  Produce

Mistletoe Extract 1000 mg

Citric acid 1000 mg

100 g oligosaccharides

Plum concentrate 2 g

1 g of taurine

According to the conventional method for preparing a healthy beverage, all of the above components were mixed and purified water was added to adjust the total volume to 900 ml, followed by heating with stirring at 85 ° C. for about 1 hour. As a result, the solution was filtered, sealed in sterilized 2 L containers, sterilized, and then refrigerated to prepare a healthy beverage. The composition ratio is a mixture of relatively suitable components for a preferred beverage in a preferred embodiment, and may be arbitrarily modified according to regional and ethnic preferences such as demand hierarchy, demand country, use.

1 is a schematic diagram showing a process of preparing mistletoe extract according to the present invention and separating the active ingredient from the extract,

Figure 2 is a graph showing the threshold change in pain avoidance response with time after administration of the mistletoe methanol extract of the present invention to the let,

Figure 3 is a graph showing the threshold change of pain avoidance response with time after administration of mistletoe water extract of the present invention to the let,

Figure 4 is a graph showing the threshold change of pain avoidance response with time after administration of the mistletoe ethyl acetate soluble fraction of the present invention to the let,

Figure 5 is a graph showing the change in threshold of pain avoidance response with time after administration of purified biscum neoside III separated from the mistletoe methanol extract of the present invention,

Figure 6 is a graph showing the change in threshold value of pain avoidance response with time after administration of purified homoflavoyadorin B isolated and purified from the mistletoe methanol extract of the present invention,

7 shows the present invention It is a graph showing the neuropathic pain inhibitory effect of biscum neoside III compared with gabapentin.

In the Fig. 2 to 7 * is a p <0.05 compared to the control values, * is a p <0.01 compared to the control value, # indicates a p <0.05 compared with the reference value (10 min.).

Claims (5)

Loranthus yadoriki extract comprising or a compound selected from the group consisting of Viscumneoside III, Homoflavoyadorin B, pharmaceutically acceptable salts of the compounds and mixtures thereof Pharmaceutical composition for the prevention or treatment of neuropathic pain containing as an active ingredient. The method of claim 1, wherein the mistletoe extract is characterized in that the mistletoe or its dried material is extracted with a solvent selected from the group consisting of water, methanol, aqueous methanol solution, ethanol, aqueous ethanol, butanol, methylene chloride, ethyl acetate and mixtures thereof. Pharmaceutical composition. delete Efficacy in inhibiting neuropathic pain, comprising a mistletoe extract comprising a compound selected from the group consisting of biscumneoside III, homoflavoyadorin B, pharmaceutically acceptable salts of the compounds and mixtures thereof Having health functional food. The health functional food according to claim 4, which is in the form of a beverage.

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