WO2018008973A1 - Composition comprising forsythiae fructus extract as effective ingredient for preventing, ameliorating or treating peripheral neuropathy - Google Patents

Composition comprising forsythiae fructus extract as effective ingredient for preventing, ameliorating or treating peripheral neuropathy Download PDF

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WO2018008973A1
WO2018008973A1 PCT/KR2017/007168 KR2017007168W WO2018008973A1 WO 2018008973 A1 WO2018008973 A1 WO 2018008973A1 KR 2017007168 W KR2017007168 W KR 2017007168W WO 2018008973 A1 WO2018008973 A1 WO 2018008973A1
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peripheral neuropathy
extract
preventing
pharmaceutical composition
anticancer
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PCT/KR2017/007168
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French (fr)
Korean (ko)
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방옥선
김노수
김진희
이진무
이유진
김영아
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한국 한의학 연구원
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    • A—HUMAN NECESSITIES
    • A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
    • A23L—FOODS, FOODSTUFFS, OR NON-ALCOHOLIC BEVERAGES, NOT COVERED BY SUBCLASSES A21D OR A23B-A23J; THEIR PREPARATION OR TREATMENT, e.g. COOKING, MODIFICATION OF NUTRITIVE QUALITIES, PHYSICAL TREATMENT; PRESERVATION OF FOODS OR FOODSTUFFS, IN GENERAL
    • A23L33/00—Modifying nutritive qualities of foods; Dietetic products; Preparation or treatment thereof
    • A23L33/10—Modifying nutritive qualities of foods; Dietetic products; Preparation or treatment thereof using additives
    • A23L33/105—Plant extracts, their artificial duplicates or their derivatives
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/28—Compounds containing heavy metals
    • A61K31/282—Platinum compounds
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/33—Heterocyclic compounds
    • A61K31/335—Heterocyclic compounds having oxygen as the only ring hetero atom, e.g. fungichromin
    • A61K31/337—Heterocyclic compounds having oxygen as the only ring hetero atom, e.g. fungichromin having four-membered rings, e.g. taxol
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/69—Boron compounds
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K36/00—Medicinal preparations of undetermined constitution containing material from algae, lichens, fungi or plants, or derivatives thereof, e.g. traditional herbal medicines
    • A61K36/18—Magnoliophyta (angiosperms)
    • A61K36/185—Magnoliopsida (dicotyledons)
    • A61K36/63—Oleaceae (Olive family), e.g. jasmine, lilac or ash tree
    • A61K36/634—Forsythia
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K45/00—Medicinal preparations containing active ingredients not provided for in groups A61K31/00 - A61K41/00
    • A61K45/06—Mixtures of active ingredients without chemical characterisation, e.g. antiphlogistics and cardiaca

Definitions

  • the present invention relates to a composition for the prevention, improvement or treatment of peripheral neuropathy (peripheral neuropathy) containing a Forsythiae Fructus extract as an active ingredient.
  • Peripheral neuropathy is caused by a disease that directly damages or affects nerve tissue. Depending on the type of nerve tissue affected, sensory neuropathy and motor neuropathy neuropathy) and autonomic neuropathy. In sensory neuropathy, sensation of touch and tremor, sensation of temperature change, tingling or burning pain, and allodynia in the skin are experienced. Motor neuropathy is accompanied by loss of balance and muscle weakness. In autonomic neuropathy, the ability to control the bladder is weakened depending on the organs affected by the nerve, resulting in incontinence or abnormal blood pressure and heart rate. Induced (RA Hughes, 2002, BMJ, v324, pp466-469; JM Torpy et al., 2010, JAMA, v303, p1556).
  • CIPN chemotherapy-induced peripheral neuropathy
  • CIPN-related symptoms include numbness, tingling, burning, cold and pain in the fingers and toes, and loss of feel and strength (T. Armstrong et al., 2005) , Oncol Nurs Forum, v32, pp305-311; C. Visovsky et al., 2008, Clin J Oncol Nurs, v12, pp243-247).
  • Anticancer agents known to cause CIPN include platinum, taxane, vinca alkaloids, bortezomib or thalidomide, and the incidence of CIPN depends on the type and dose of anticancer agent used. And 20-75%, depending on the duration of administration (G. Cavaletti et al., 2011, Curr Treat Options Neurol, v13, pp180-190).
  • the mechanism of neurotoxicity caused by anticancer drugs is not exactly known. However, it is assumed that the mechanisms by which anticancer drugs act on general cancer cells and the mechanisms on peripheral nervous system cells are similar to each other. It is known that anticancer agents administered to patients accumulate not only in cancer tissues but also in the peripheral nervous system, and the accumulation of such anticancer agents eventually causes neurotoxicity and cause CIPN.
  • Forsythia Fructus refers to the dried fruit of the ash of Forsythia viridissima Lindley or Forsythia suspensa Vahl, which is used as a medicinal herb in Japan, but in China It is known to use only dried fruit of Dangari forsythia without using.
  • Yeon-kyo is the name that fruit is formed in a room similar to lotus, and it rises long among various grasses. Or, if a seed is split, a piece and a piece are similar to feathers, so it is known that it is called a bridge using a kite that means 'to touch' and a bridge that means 'feather'. This drug has a peculiar smell, its bitter taste, and a little cold.
  • the drug has the effect of lowering and detoxifying the fever, eliminating wind and fever, and causing boils and cuts to swell, swell, or loosen the swelling. Therefore, the fever can be lowered early in the fever to reduce the fever and mental confusion, or can be used to boil, lymphadenitis, and sore throat. In ideology, it is a medicine that can be used by Soyangin. As a pharmacological action, antibacterial action, anti-inflammatory action, blood pressure drop, hemostatic action, liver treatment action, antipyretic effect, clay, diuretic action have been reported.
  • the main chemicals include forsythol, sterol compounds, saponins, and oleanolic acids.
  • Appearance is oval or long oval granules, with pointed ends and fruit stems at the base.
  • the outer surface is light brown or dark brown with scattered light gray small ridges and two vertical grooves.
  • the well-done splits along the longitudinal groove and the end is twisted.
  • the inside of the split rind is yellowish brown with a septum in the middle. Seeds have elongated oval wings.
  • Korean Patent Publication No. 2016-0062303 discloses a composition for the prophylaxis or treatment of an immune disease containing a duct extract, and Korean Patent Publication No. 2009-0047501 contributes to the treatment of neuropathy induced by chemotherapy. It is disclosed with respect to the method and pharmaceutical preparations, Korean Patent Publication No. 2014-0123444 discloses a pharmaceutical composition and health functional food for the prevention or treatment of cancer comprising a natural product extract as an active ingredient, There is no disclosure regarding a composition for preventing, improving or treating peripheral neuropathy containing the extract as an active ingredient.
  • the present invention is derived from the above requirements, the present invention provides a composition for preventing, ameliorating or treating peripheral neuropathy containing a duct extract as an active ingredient.
  • the present invention reduces pain response by administering the duct extract to an animal model in which peripheral neuropathy was induced by the administration of oxaliplatin, and inhibited neuronal toxicity and neurite growth by oxaliplatin as the duct extract was treated in nerve cells.
  • the present invention was completed by confirming that the mitigatory extract did not affect the cytotoxicity of lung cancer and colorectal cancer cell lines by oxaliplatin.
  • the present invention provides a pharmaceutical composition for the prevention or treatment of peripheral neuropathy (peripheral neuropathy) containing a duct extract as an active ingredient.
  • the present invention provides an anticancer adjuvant containing the duct extract as an active ingredient.
  • the present invention provides a health functional food composition for the prevention or improvement of peripheral neuropathy (peripheral neuropathy) containing a duct extract as an active ingredient.
  • peripheral neuropathy peripheral neuropathy
  • the present invention relates to a composition for the prevention, improvement or treatment of peripheral neuropathy (peripheral neuropathy), in particular, chemotherapy-induced peripheral neuropathy caused by an anticancer agent, containing as a active ingredient extract, specifically Yeongyo extract of the present invention does not affect the intrinsic cytotoxic effects of anticancer agents in human cancer cell lines, and at the same time mitigates neuronal cytotoxicity and neurite growth inhibition by anticancer agents, and increased pain in animal models of anticancer drug induced peripheral neuropathy. The effect is to effectively reduce the sensitivity to stimulation.
  • peripheral neuropathy peripheral neuropathy
  • chemotherapy-induced peripheral neuropathy caused by an anticancer agent containing as a active ingredient extract, specifically Yeongyo extract of the present invention does not affect the intrinsic cytotoxic effects of anticancer agents in human cancer cell lines, and at the same time mitigates neuronal cytotoxicity and neurite growth inhibition by anticancer agents, and increased pain in animal models of anticancer drug induced peripheral neuropathy.
  • the effect is to effectively reduce the sensitivity to stimulation.
  • 1 is a ratio of cells forming neurites when treated with increasing concentrations of GF extracts of the present invention (WEFF) sequentially in PC-12 cells induced with nerve growth factor (NGF) (A ) And a graph showing the sum of the lengths of the grown neurites (B) and the effect of the neurites growing under a microscope (C).
  • WEFF GF extracts of the present invention
  • Figure 2 shows that after the induction of neuronal differentiation of PC-12 cells using nerve growth factors, the treatment of oxaliplatin induced the toxicity of neurons and the duct extract of the present invention protects neurons. ** indicates that the toxicity of neurons was statistically significant by treatment with oxaliplatin as compared to PC-12 cells ((-) Oxal + (-) WEFF) not treated with oxaliplatin, p ⁇ 0.01 , ## means that when treated with ductile water extract of the present invention compared to the control treated with oxaliplatin and vehicle (PBS), there is a statistically significant neuroprotective effect of reducing the toxicity of neurons, p ⁇ 0.01.
  • Figure 3 is injected once a week, 10 times / total (20 mg / kg) intraperitoneal injection of 10 mg / kg oxaliplatin to induce peripheral neuropathic pain in the mouse model (C57BL / 6), pontoon water extract (WEFF) is a graph showing the weight change measured by daily oral administration.
  • WEFF pontoon water extract
  • ** and *** indicate a statistically significant increase in pain response in animal models administered oxaliplatin compared to normal, ** is p ⁇ 0.01, and *** is p ⁇ 0.001.
  • the test was performed with von Frey filament with 0.4g (A) and 0.16g (B) bending force.
  • ##, ### indicates that the oxaliplatin administered group compared to the oxaliplatin + vehicle (0.5% CMC solution) administration group, and the response to pain in the group administered the ductal water extract of the present invention statistically significantly reduced ## is p ⁇ 0.01 and ### means p ⁇ 0.001.
  • Figure 5 is a result confirming the relative viability of the lung cancer cell lines (A549, A) and colon cancer cell lines (HCT116, B) treated with the ductile water extract of the present invention in combination with oxaliplatin.
  • the present invention relates to a pharmaceutical composition for the prevention or treatment of peripheral neuropathy (peripheral neuropathy) containing a duct extract as an active ingredient.
  • the duct bridge extract is preferably extracted with water, a lower alcohol of C 1 ⁇ C 4 or a mixture thereof as a solvent, the lower alcohol is preferably methanol or ethanol.
  • the duct bridge extract is preferably prepared by a manufacturing method including the following steps, but is not limited thereto.
  • step 3 drying the filtered extract of step 2) under reduced pressure.
  • the bridge of step 1) can be used without limitation, such as grown or commercially available.
  • the extraction method of the duct bridge extract of step 1) conventional methods in the art such as filtration, hot water extraction, dipping extraction, reflux cooling extraction, and ultrasonic extraction may be used.
  • the extraction solvent is preferably extracted by adding 2 to 40 times the volume based on the weight of the dried duct bridge. Extraction temperature is preferably 20 ⁇ 100 °C but is not limited thereto.
  • the extraction time is preferably 0.5 to 10 hours, more preferably 1 to 4 hours, most preferably 2 hours is not limited thereto.
  • the extraction is preferably repeated 2-3 times, but is not limited thereto.
  • the decompression concentration in step 3) preferably uses a vacuum decompression concentrator or a vacuum rotary evaporator, but is not limited thereto.
  • the drying is preferably reduced pressure drying, vacuum drying, boiling drying, spray drying or freeze drying, but is not limited thereto.
  • the peripheral neuropathy is preferably a chemotherapy-induced peripheral neuropathy induced by an anticancer agent
  • the anticancer agent is a platinum-based, taxane-based, vinca alkaloids, bortezomib ( bortezomib) or thalidomide, including, but not limited to, all anticancer agents that are clinically, pharmacologically and biomedically available.
  • the platinum-based anticancer agent is preferably at least one selected from cisplatin, carboplatin, and oxaliplatin, and the taxane-based anticancer agent is preferably at least one selected from paclitaxel and docetaxel. It is not limited to this.
  • compositions of the present invention may be in various oral or parenteral formulations.
  • diluents or excipients such as fillers, extenders, binders, wetting agents, disintegrating agents, and surfactants are usually used.
  • Solid form preparations for oral administration include tablets, pills, powders, granules, capsules, and the like, which form at least one excipient such as starch, calcium carbonate, sucrose or lactose (at least one compound). lactose) and gelatin. In addition to simple excipients, lubricants such as magnesium stearate, talc and the like are also used.
  • Liquid preparations for oral administration include suspensions, solutions, emulsions or syrups, and include various excipients such as wetting agents, sweeteners, fragrances or preservatives, in addition to the commonly used simple diluents such as water and liquid paraffin. Can be.
  • Formulations for parenteral administration include sterile aqueous solutions, non-aqueous solvents, suspensions, emulsions, freeze-dried preparations or suppositories.
  • non-aqueous solvent and the suspension solvent propylene glycol, polyethylene glycol, vegetable oil such as olive oil, injectable ester such as ethyl oleate, and the like can be used.
  • injectable ester such as ethyl oleate, and the like
  • suppositories witepsol, macrogol, tween 61, cacao butter, laurin butter, glycerol, gelatin, and the like may be used.
  • the pharmaceutical composition of the present invention may be administered orally or parenterally, and it is preferable to select an external skin or intraperitoneal, rectal, intravenous, intramuscular, subcutaneous, intrauterine dural or cerebrovascular injection method for parenteral administration. I never do that.
  • the pharmaceutical composition of the present invention is administered in a pharmaceutically effective amount.
  • the pharmaceutically effective amount means an amount sufficient to treat the disease at a reasonable benefit / risk ratio applicable to the medical treatment, and the effective dose level refers to the type, severity, drug activity, and drug of the patient. Sensitivity, time of administration, route of administration and rate of release, duration of treatment, factors including concurrent use of drugs, and other factors well known in the medical arts.
  • the compositions of the present invention may be administered as individual therapeutic agents or in combination with other therapeutic agents, may be administered sequentially or simultaneously with conventional therapeutic agents, and may be single or multiple doses. Taking all of the above factors into consideration, it is important to administer an amount that can achieve the maximum effect with a minimum amount without side effects, which can be readily determined by one skilled in the art.
  • the dosage of the composition of the present invention varies depending on the patient's weight, age, sex, health status, diet, time of administration, administration method, excretion rate and severity of the disease, the daily dosage is the amount of duct extract It is 0.01-1000 mg / kg, Preferably it is 30-500 mg / kg, More preferably, it is 50-300 mg / kg, It can be administered 1-6 times a day. However, the dosage may be increased or decreased depending on the route of administration, the severity of obesity, sex, weight, age, etc., and the above dosage does not limit the scope of the present invention in any way.
  • the pharmaceutical composition of the present invention is preferably used in combination with an anticancer agent, but may be used alone or in combination with methods using surgery, radiation therapy, hormonal therapy, and biological response modifiers.
  • the present invention relates to an anticancer adjuvant containing duct extract as an active ingredient.
  • the anticancer adjuvant is preferably administered in combination with an anticancer agent including platinum, taxane, vinca alkaloids, bortezomib or thalidomide, and is caused by an anticancer agent.
  • an anticancer agent including platinum, taxane, vinca alkaloids, bortezomib or thalidomide
  • the present invention also relates to a nutraceutical composition for the prevention or improvement of peripheral neuropathy (peripheral neuropathy) containing a duct extract as an active ingredient.
  • peripheral neuropathy peripheral neuropathy
  • the duct bridge extract is preferably extracted with water, a lower alcohol of C 1 ⁇ C 4 or a mixture thereof as a solvent, the lower alcohol is preferably methanol or ethanol.
  • the duct bridge extract is preferably prepared by a manufacturing method including the following steps, but is not limited thereto.
  • step 3 drying the filtered extract of step 2) under reduced pressure.
  • the bridge of step 1) can be used without limitation, such as grown or commercially available.
  • the extraction method of the duct bridge extract of step 1) conventional methods in the art such as filtration, hot water extraction, dipping extraction, reflux cooling extraction, and ultrasonic extraction may be used.
  • the extraction solvent is preferably extracted by adding 2 to 40 times the volume based on the weight of the dried duct bridge. Extraction temperature is preferably 20 ⁇ 100 °C but is not limited thereto.
  • the extraction time is preferably 0.5 to 10 hours, more preferably 1 to 4 hours, most preferably 2 hours is not limited thereto.
  • the extraction is preferably repeated 2-3 times, but is not limited thereto.
  • the decompression concentration in step 3) preferably uses a vacuum decompression concentrator or a vacuum rotary evaporator, but is not limited thereto.
  • the drying is preferably reduced pressure drying, vacuum drying, boiling drying, spray drying or freeze drying, but is not limited thereto.
  • the peripheral neuropathy is preferably a chemotherapy-induced peripheral neuropathy induced by an anticancer agent
  • the anticancer agent is a platinum-based, taxane-based, vinca alkaloids, bortezomib ( bortezomib) or thalidomide, including, but not limited to, all anticancer agents that are clinically, pharmacologically and biomedically available.
  • the platinum-based anticancer agent is preferably at least one selected from cisplatin, carboplatin and oxaliplatin, and the taxane-based anticancer agent is preferably at least one selected from paclitaxel and docetaxel. It is not limited to this.
  • the health functional food composition may be manufactured and processed in the form of tablets, capsules, powders, granules, liquids, pills, and the like, but is not limited thereto and may be manufactured and processed in any form according to the law.
  • the composition containing the duct extract of the present invention as an active ingredient may be added as it is to food or used together with other food or food ingredients, and may be appropriately used according to a conventional method.
  • the mixing amount of the active ingredient can be suitably determined according to the purpose of use (prevention or improvement).
  • the amount of the extract in the health food can be added to 0.1 to 90 parts by weight of the total food weight.
  • the amount may be below the above range, and the active ingredient may be used in an amount above the above range because there is no problem in terms of safety.
  • the health functional food composition of the present invention When used as a beverage, there is no particular limitation on other ingredients except for containing the extract as an essential ingredient in the indicated ratio, and various flavors or natural carbohydrates are added as in the general beverage. It may contain as a component.
  • the natural carbohydrates include monosaccharides such as glucose, fructose and the like; Disaccharides such as maltose, sucrose and the like; And conventional sugars such as polysaccharides such as dextrin, cyclodextrin, and sugar alcohols such as xylitol, sorbitol, and erythritol.
  • natural flavoring agents tautin, stevia extract (e.g., Rebaudioside A, glycyrginine, etc.)
  • synthetic flavoring agents sacharin, aspartame, etc.
  • the duct extract of the present invention is a variety of nutrients, vitamins, minerals (electrolytes), flavors such as synthetic and natural flavors, coloring and neutralizing agents (such as cheese, chocolate), pectic acid and salts thereof, alginic acid and Salts, organic acids, protective colloid thickeners, pH adjusting agents, stabilizers, preservatives, glycerin, alcohols, carbonation agents used in carbonated drinks, and the like.
  • the duct extract of the present invention may contain natural fruit juice and fruit flesh for the production of fruit juice drinks and vegetable drinks. These components can be used independently or in combination. The ratio of such additives is not so important, but is generally selected in the range of 0.1 to 20 parts by weight per 100 parts by weight of duct extract.
  • the duct bridge extract of the present invention 100 g of the dried duct bridge purchased from Gwangmyeongdang Pharmaceutical Co., Ltd. (Ulsan, South Korea) was ground to prepare a sample in powder form. 100 g of ground dry sample and 2 l of water Put into a round flask and mix. A water bath connected to a reflux extractor with a cooling tube was heated and extracted twice, once every two hours. The extracted extract was filtered under reduced pressure using a paper filter paper (Whatman No. 2) and a vacuum pump (GAST). The filtered liquid extract was concentrated under reduced pressure using a rotary evaporator (YEYELA). The concentrated extract was lyophilized and then homogenized with a mortar and pestle to obtain soft water extract (WEFF). The resulting soft water extract was placed in a sealed plastic container and stored at 4 ° C. cold storage until the experiment.
  • GEEYELA rotary evaporator
  • PC-12 cells to be used in the Examples for confirming the effect of the duct extract of the present invention is a rat-derived Chromocytocytoma (Pheochromocytoma), when the neuronal growth factor (NGF) is treated to finally differentiate into neurons It is a cell line that is useful as a cell model of neurological research because of its characteristics.
  • PC-12 cells were treated with 5% (v / v) non-heat inactivated fetal bovine serum (FBS), 10% (v / v) heat inactivated horse serum (HS), Coating with collagen type I in an incubator maintained at 37 ° C., 5% (v / v) CO 2 using DMEM growth medium containing 100 units / ml penicillin and 100 ⁇ g / ml streptomycin Cultured in a 100 mm cell culture dish.
  • FBS non-heat inactivated fetal bovine serum
  • HS heat inactivated horse serum
  • Human lung cancer cells (A549) and human colon cancer cells (HCT116) were treated at 37 ° C. using RPMI1640 growth medium containing 10% (v / v) heat inactivated FBS, 100 units / ml penicillin, 100 ⁇ g / ml streptomycin.
  • the cells were cultured in 100 mm cell culture dishes in an incubator maintained at 5% (v / v) CO 2 and used for later in vitro experiments.
  • CIPN chemotherapy-induced peripheral neuropathy
  • results obtained in the examples of the present invention were expressed as mean ⁇ standard deviation (mean ⁇ SD) or mean ⁇ standard error (mean ⁇ SE), and the statistical mean comparison between groups was T-test and ANOVA / Tukey post-hoc. The test was conducted. p values were expressed as * ⁇ 0.05, ** ⁇ 0.01, *** ⁇ 0.001.
  • Example 1-1 Determination of Neuronal Growth Inhibitory Effect of Yeon-kyo Water Extract
  • the neurite outgrowth inhibitory effect of the water extract of Fructus of the present invention was confirmed. Specifically, the cultured PC-12 cells were uniformly inoculated in a 24 well plate coated with collagen type IV at a concentration of 1 ⁇ 10 4 cells / well, and then cultured in DMEM growth medium to adhere to the bottom surface. After 24 hours, the cells were replaced with DMEM medium containing 100 ng / ml neuronal growth factor (NGF) without serum to induce the production of neurites, and treated with 200 nM oxaliplatin to induce neurotoxicity.
  • NGF neuronal growth factor
  • the prepared soft water extract (WEFF) was treated at concentrations of 12.5, 25, 50, and 100 ⁇ g / ml, respectively, and after 3 days, observed with an optical microscope to analyze the ratio of neurites-produced cells and the length of neurites. It was.
  • oxaliplatin oxaliplatin
  • the cultured PC-12 cells were uniformly inoculated into a 96 well plate coated with collagen type IV at a concentration of 2 ⁇ 10 3 cells / well and then cultured in DMEM growth medium to adhere to the bottom surface. After 24 hours, differentiation into neurons was induced by replacing with DMEM medium containing 100 ng / ml of neuronal growth factor (NGF) without serum to induce neurites. Four days later, 200 nM of oxaliplatin was treated to induce cytotoxicity to neurons.
  • the prepared soft water extract (WEFF) was treated at concentrations of 25, 50, and 100 ⁇ g / ml, respectively, and after 2 days, viability of each well was measured using Ez-cytox.
  • the experimental animal group administered oxaliplatin decreased body weight compared to the normal group, and after induction of CIPN for 2 weeks, the group administered oral extract (WEFF) orally was 0.5 as vehicle. Compared with the group administered the% CMC solution, no side effects such as weight loss were seen, but rather recovered faster than the vehicle administration group (FIG. 3).
  • CMC carboxymethyl cellulose
  • WEFF soft water extract
  • each cell was divided into 5 x 10 3 cells per well in a 96 well culture dish. After 24 hours, oxaliplatin (0-100 ⁇ g / ml) and soft water extract (100 ⁇ g / ml) diluted sequentially were treated simultaneously. The negative control group was treated with oxaliplatin and used a cell group treated with PBS as a vehicle instead of the soft water extract. After 72 hours of treatment, cell viability was measured using an Ez-Cytox cell viability measurement kit.

Abstract

The present invention relates to a composition comprising a Forsythiae Fructus extract as an effective ingredient for preventing, ameliorating or treating peripheral neuropathy, particularly chemotherapy-induced peripheral neuropathy (CIPN). In detail, a Forsythiae Fructus extract of the present invention relieves the neurotoxicity and neurite growth suppression induced by anticancer agents, and effectively reduces sensitivity to increased pain stimuli in animal models with chemotherapy-induced peripheral neuropathy while not affecting the inherent cytotoxicity of anticancer agents in human cancer cell lines.

Description

연교 추출물을 유효성분으로 함유하는 말초신경병증 예방, 개선 또는 치료용 조성물Composition for the prevention, improvement or treatment of peripheral neuropathy containing poncho extract as an active ingredient
본 발명은 연교(Forsythiae Fructus) 추출물을 유효성분으로 함유하는 말초신경병증(peripheral neuropathy)의 예방, 개선 또는 치료용 조성물에 관한 것이다.The present invention relates to a composition for the prevention, improvement or treatment of peripheral neuropathy (peripheral neuropathy) containing a Forsythiae Fructus extract as an active ingredient.
말초신경병증(peripheral neuropathy)은 신경조직에 직접 손상을 주거나 혹은 신경조직에 영향을 주는 질병에 의해 발병하는데, 영향을 받는 신경조직의 종류에 따라 감각신경병증(sensory neuropathy), 운동신경병증(motor neuropathy), 자율신경증(autonomic neuropathy)으로 구별할 수 있다. 감각신경병증의 경우 촉각 및 떨림 현상, 온도 변화에 대한 감각이 저하되거나, 따끔거리는 느낌 혹은 타는 듯한 통증을 느끼게 되고, 피부에서 이질통 등을 경험하게 된다. 운동신경병증의 경우에는 균형감각의 손실 및 근력 약화 등이 동반되며, 자율신경병증의 경우에는 해당 신경이 미치는 기관에 따라 방광을 조절하는 기능이 약화되어 요실금을 경험하거나, 비정상적인 혈압 및 심장박동을 유발한다(R.A. Hughes, 2002, BMJ, v324, pp466-469; J.M. Torpy et al., 2010, JAMA, v303, p1556). Peripheral neuropathy is caused by a disease that directly damages or affects nerve tissue. Depending on the type of nerve tissue affected, sensory neuropathy and motor neuropathy neuropathy) and autonomic neuropathy. In sensory neuropathy, sensation of touch and tremor, sensation of temperature change, tingling or burning pain, and allodynia in the skin are experienced. Motor neuropathy is accompanied by loss of balance and muscle weakness.In autonomic neuropathy, the ability to control the bladder is weakened depending on the organs affected by the nerve, resulting in incontinence or abnormal blood pressure and heart rate. Induced (RA Hughes, 2002, BMJ, v324, pp466-469; JM Torpy et al., 2010, JAMA, v303, p1556).
말초신경병증의 원인으로 다양한 요인들이 제시되었는데, 유전적 질환, 대사 및 내분비계통의 질환, 염증성 질환, 비타민 등 영양결핍, 암 치료 과정에서 투여되는 항암제 등이 발병 원인으로 알려져 있다. 이 중 항암제에 의해 유발된 말초신경병증(chemotherapy-induced peripheral neuropathy, CIPN)은 증상의 경도에 따라 환자에게 투여되는 항암제의 용량을 줄이거나 심한 경우 항암치료 자체를 중단해야 하기 때문에 CIPN은 항암 치료에 매우 부정적인 영향을 주는 부작용으로 작용한다. 항암치료를 받은 환자의 약 3분의 1 정도가 CIPN을 경험하며, 또 CIPN을 경험하는 환자의 3분의 1은 영구적으로 신경 손상을 받는다고 알려져 있다(A. Bhagra, R.D. Rao, 2007, Curr Oncol Rep, v9, pp290-299). CIPN 관련 증상으로는 손가락 및 발가락에 저리고(numbness), 따끔거리고(tingling), 타는 듯한(burning) 느낌이 오거나 냉감 및 통증을 느끼게 되며, 촉감과 근력이 저하된다(T. Armstrong et al., 2005, Oncol Nurs Forum, v32, pp305-311; C. Visovsky et al., 2008, Clin J Oncol Nurs, v12, pp243-247). Various factors have been suggested as a cause of peripheral neuropathy. Genetic disorders, metabolic and endocrine system diseases, inflammatory diseases, vitamin deficiency, and anticancer drugs administered during cancer treatment are known to cause the onset. Among these, chemotherapy-induced peripheral neuropathy (CIPN) caused by chemotherapy should reduce the dose of chemotherapy given to the patient depending on the severity of symptoms or, in severe cases, stop chemotherapy itself. It has a very negative side effect. It is known that about one third of patients receiving chemotherapy experience CIPN, and one third of patients experiencing CIPN are permanently damaged (A. Bhagra, RD Rao, 2007, Curr). Oncol Rep, v9, pp 290-299). CIPN-related symptoms include numbness, tingling, burning, cold and pain in the fingers and toes, and loss of feel and strength (T. Armstrong et al., 2005) , Oncol Nurs Forum, v32, pp305-311; C. Visovsky et al., 2008, Clin J Oncol Nurs, v12, pp243-247).
CIPN을 일으키는 것으로 알려진 항암제로는 플래티넘(platinum) 계열, 탁산(taxane) 계열, 빈카알카로이드(vinca alkaloids), 보르테조밉(bortezomib) 또는 탈리도마이드(thalidomide) 등이 있으며, CIPN 발병률은 사용한 항암제의 종류, 용량 및 투여 기간에 따라 20-75%에 이른다고 알려져 있다(G. Cavaletti et al., 2011, Curr Treat Options Neurol, v13, pp180-190). 현재 항암제에 의한 신경독성의 기전은 정확히 알려져 있지 않으며, 다만 항암제가 일반 암세포에 작용하는 세포독성 기전과 말초신경계 세포에 작용하는 기전이 서로 비슷할 것이라고 추측될 뿐이다. 환자에게 투여된 항암제는 암 조직뿐만 아니라 말초신경계에 축적되고, 이러한 항암제의 축적이 결국 신경 독성의 원인으로 작용하여 CIPN을 유발하는 것으로 알려져 있다.Anticancer agents known to cause CIPN include platinum, taxane, vinca alkaloids, bortezomib or thalidomide, and the incidence of CIPN depends on the type and dose of anticancer agent used. And 20-75%, depending on the duration of administration (G. Cavaletti et al., 2011, Curr Treat Options Neurol, v13, pp180-190). Currently, the mechanism of neurotoxicity caused by anticancer drugs is not exactly known. However, it is assumed that the mechanisms by which anticancer drugs act on general cancer cells and the mechanisms on peripheral nervous system cells are similar to each other. It is known that anticancer agents administered to patients accumulate not only in cancer tissues but also in the peripheral nervous system, and the accumulation of such anticancer agents eventually causes neurotoxicity and cause CIPN.
현재까지 CIPN 치료를 목적으로 미국 FDA에서 승인된 표준치료제는 없다. 임상적으로 CIPN의 증상 완화를 위해 가바펜틴(Gabapentin)과 같은 항경련제, 혹은 아미트리프틸린(Amitriptyline)과 같은 항우울제 등을 사용하고 있으나, CIPN에 대한 대규모 임상시험에서 그 효과가 입증되지 못하였다. 또한, 이러한 약물들은 어지럼증, 졸림 등의 부작용을 내재하고 있으며, 낮은 안전역(safety margin)으로 인하여 고용량 투여가 불가능하다는 단점이 있다. 따라서 이러한 CIPN의 의학적 미충족으로 인하여 보다 안전하고 효과가 입증된 CIPN 치료제 및 예방을 위한 새로운 소재 개발이 절실한 상황이다. CIPN은 복합적인 기전을 갖는 질병이기 때문에 다중표적 기반의 소재 개발이 적절하다고 판단되며, 이에 전통적으로 사용되어 온 약용식물 혹은 천연물 소재는 이러한 소재개발전략 관점에서 중요한 출발 물질로 다뤄질 수 있을 것이다. To date, there is no standard treatment approved by the US FDA for the treatment of CIPN. Clinically, anticonvulsants such as gabapentin or antidepressants such as amitriptyline have been used to alleviate the symptoms of CIPN. However, large-scale clinical trials of CIPN have not been proven effective. In addition, these drugs have side effects such as dizziness and drowsiness, and have a disadvantage in that high dose administration is impossible due to a low safety margin. Therefore, due to the lack of medical care of CIPN, there is an urgent need to develop new materials for safer and more effective CIPN treatment and prevention. Since CIPN is a disease with multiple mechanisms, it is considered appropriate to develop a multi-target-based material, and medicinal plants or natural materials, which have been used traditionally, may be treated as important starting materials from the viewpoint of this material development strategy.
한편, 연교(Forsythia Fructus)는 물푸레나무과의 의성개나리(Forsythia viridissima Lindley) 또는 당개나리(Forsythia suspensa Vahl)의 건조시킨 열매를 말하며, 일본에서는 우리나라와 동일한 기원식물을 사용해 약재로 쓰지만, 중국에서는 의성개나리는 사용하지 않고, 당개나리의 열매만을 건조시켜 사용하는 것으로 알려져 있다. 연교는 열매가 연꽃과 비슷하게 방(房)을 형성하고 있고, 여러 풀 중에 길게 솟아나와 있어 얻은 이름이다. 또는 씨앗을 쪼개면 한 조각, 한 조각이 깃털과 비슷하기 때문에 '잇닿다'라는 의미의 연과 '깃털'이라는 의미의 교를 써서 연교라고 부른다고도 알려져 있다. 이 약은 특이한 냄새가 있으며 그 맛은 쓰고, 기운은 약간 차다.Meanwhile, Forsythia Fructus refers to the dried fruit of the ash of Forsythia viridissima Lindley or Forsythia suspensa Vahl, which is used as a medicinal herb in Japan, but in China It is known to use only dried fruit of Dangari forsythia without using. Yeon-kyo is the name that fruit is formed in a room similar to lotus, and it rises long among various grasses. Or, if a seed is split, a piece and a piece are similar to feathers, so it is known that it is called a bridge using a kite that means 'to touch' and a bridge that means 'feather'. This drug has a peculiar smell, its bitter taste, and a little cold.
약효는 열을 내리고 해독하며, 풍열(風熱)을 없애고, 종기나 상처가 부은 것을 삭도록 하거나 뭉치거나 몰린 것을 풀어주는 소종산결(消腫散結)하는 효능을 갖는다. 따라서 온열병 초기에 열을 내려서 고열과 정신이 혼몽한 것을 가라앉히거나 종기, 림프절염, 인후염 등에 활용할 수 있다. 사상의학에서는 소양인에게 활용할 수 있는 약재이다. 약리작용으로 항균작용, 항염증작용, 혈압강하, 지혈작용, 간치료작용, 해열, 진토, 이뇨작용이 보고되었다. 주요 화학성분으로는 포르시톨(forsythol), 스테롤(sterol) 화합물, 사포닌(saponin), 올레아놀산(oleanolic acid) 등이 있다. 생김새는 난원형 또는 긴 난원형의 삭과로, 양 끝이 뾰족하고 기부에 열매의 꼭지가 남아있는 것도 있다. 바깥 면은 엷은 갈색이나 어두운 갈색을 띠고 여기에 엷은 회색의 작은 융기점이 흩어져 있으며 2개의 세로 홈이 있다. 잘 여문 것은 세로 홈에 따라 갈라지고 끝 쪽은 뒤틀려 있다. 갈라진 과피의 안쪽은 황갈색으로 가운데에 격벽(隔壁)이 있다. 씨에는 가늘고 긴 타원형의 날개가 있다.The drug has the effect of lowering and detoxifying the fever, eliminating wind and fever, and causing boils and cuts to swell, swell, or loosen the swelling. Therefore, the fever can be lowered early in the fever to reduce the fever and mental confusion, or can be used to boil, lymphadenitis, and sore throat. In ideology, it is a medicine that can be used by Soyangin. As a pharmacological action, antibacterial action, anti-inflammatory action, blood pressure drop, hemostatic action, liver treatment action, antipyretic effect, clay, diuretic action have been reported. The main chemicals include forsythol, sterol compounds, saponins, and oleanolic acids. Appearance is oval or long oval granules, with pointed ends and fruit stems at the base. The outer surface is light brown or dark brown with scattered light gray small ridges and two vertical grooves. The well-done splits along the longitudinal groove and the end is twisted. The inside of the split rind is yellowish brown with a septum in the middle. Seeds have elongated oval wings.
한국공개특허 제2016-0062303호에 연교 추출물을 함유하는 면역 질환의 예방 또는 치료용 조성물에 관하여 개시되어 있고, 한국공개특허 제2009-0047501호에 화학요법에 의해 유발된 신경병증의 치료에 기여하는 방법 및 약학 제제에 관하여 개시되어 있으며, 한국공개특허 제2014-0123444호에 천연물 추출물을 유효성분으로 포함하는 암의 예방 또는 치료용 약학적 조성물 및 건강기능식품에 관하여 개시되어 있으나, 본 발명의 연교 추출물을 유효성분으로 함유하는 말초신경병증 예방, 개선 또는 치료용 조성물에 관한 기술이 개시된 바 없다. Korean Patent Publication No. 2016-0062303 discloses a composition for the prophylaxis or treatment of an immune disease containing a duct extract, and Korean Patent Publication No. 2009-0047501 contributes to the treatment of neuropathy induced by chemotherapy. It is disclosed with respect to the method and pharmaceutical preparations, Korean Patent Publication No. 2014-0123444 discloses a pharmaceutical composition and health functional food for the prevention or treatment of cancer comprising a natural product extract as an active ingredient, There is no disclosure regarding a composition for preventing, improving or treating peripheral neuropathy containing the extract as an active ingredient.
본 발명은 상기와 같은 요구에 의해 도출된 것으로서, 본 발명은 연교 추출물을 유효성분으로 함유하는 말초신경병증 예방, 개선 또는 치료용 조성물을 제공한다. 본 발명자들은 옥살리플라틴의 투여에 의해 말초신경병증이 유발된 동물 모델에 상기 연교 추출물을 투여함으로써 통증반응을 감소시키는 것과, 신경세포에서 연교 추출물을 처리함에 따라 옥살리플라틴에 의한 신경세포 독성 및 신경돌기 성장 억제에 완화 효과가 있다는 것을 확인하였고, 옥살리플라틴에 의한 폐암 및 대장암 세포주의 세포 독성에 연교 추출물이 영향을 미치지 않는다는 것을 확인함으로써, 본 발명을 완성하였다.The present invention is derived from the above requirements, the present invention provides a composition for preventing, ameliorating or treating peripheral neuropathy containing a duct extract as an active ingredient. The present invention reduces pain response by administering the duct extract to an animal model in which peripheral neuropathy was induced by the administration of oxaliplatin, and inhibited neuronal toxicity and neurite growth by oxaliplatin as the duct extract was treated in nerve cells. The present invention was completed by confirming that the mitigatory extract did not affect the cytotoxicity of lung cancer and colorectal cancer cell lines by oxaliplatin.
상기 목적을 달성하기 위하여, 본 발명은 연교 추출물을 유효성분으로 함유하는 말초신경병증(peripheral neuropathy)의 예방 또는 치료용 약학 조성물을 제공한다.In order to achieve the above object, the present invention provides a pharmaceutical composition for the prevention or treatment of peripheral neuropathy (peripheral neuropathy) containing a duct extract as an active ingredient.
또한, 본 발명은 연교 추출물을 유효성분으로 함유하는 항암 보조제를 제공한다.In addition, the present invention provides an anticancer adjuvant containing the duct extract as an active ingredient.
또한, 본 발명은 연교 추출물을 유효성분으로 함유하는 말초신경병증(peripheral neuropathy)의 예방 또는 개선용 건강기능식품 조성물을 제공한다.In addition, the present invention provides a health functional food composition for the prevention or improvement of peripheral neuropathy (peripheral neuropathy) containing a duct extract as an active ingredient.
본 발명은 연교 추출물을 유효성분으로 함유하는 말초신경병증(peripheral neuropathy), 특히 항암제에 의해 유발된 말초신경병증(chemotherapy-induced peripheral neuropathy)의 예방, 개선 또는 치료용 조성물에 관한 것으로, 구체적으로 본 발명의 연교 추출물은 인간 암세포주에서 항암제의 고유한 세포독성 효력에 영향을 주지 않으면서 동시에, 항암제에 의한 신경세포 독성과 신경돌기 성장 억제를 완화하며, 항암제 유발 말초신경병증 동물모델에서 증가된 통증 자극에 대한 민감도를 효과적으로 감소시키는 효과가 있는 것이다.The present invention relates to a composition for the prevention, improvement or treatment of peripheral neuropathy (peripheral neuropathy), in particular, chemotherapy-induced peripheral neuropathy caused by an anticancer agent, containing as a active ingredient extract, specifically Yeongyo extract of the present invention does not affect the intrinsic cytotoxic effects of anticancer agents in human cancer cell lines, and at the same time mitigates neuronal cytotoxicity and neurite growth inhibition by anticancer agents, and increased pain in animal models of anticancer drug induced peripheral neuropathy. The effect is to effectively reduce the sensitivity to stimulation.
도 1은 신경성장인자(nerve growth factor, NGF)로 유도된 PC-12 세포에서 본 발명의 연교 추출물(WEFF)을 순차적으로 농도를 증가시키면서 처리하였을 때, 신경돌기를 형성한 세포의 비율(A) 및 성장한 신경돌기의 길이의 합(B)을 상대적으로 나타낸 그래프와 신경돌기 성장 효과를 현미경으로 확인한 것이다(C). 1 is a ratio of cells forming neurites when treated with increasing concentrations of GF extracts of the present invention (WEFF) sequentially in PC-12 cells induced with nerve growth factor (NGF) (A ) And a graph showing the sum of the lengths of the grown neurites (B) and the effect of the neurites growing under a microscope (C).
a: (+)NGF, 신경성장인자(100ng/㎖)를 처리한 PC-12 세포;a: PC-12 cells treated with (+) NGF, nerve growth factor (100 ng / ml);
b: (+)NGF+Oxal(200nM)+V, 신경성장인자(100ng/㎖), 옥살리플라틴(200nM) 및 vehicle로서 PBS를 처리한 PC-12 세포;b: PC-12 cells treated with (+) NGF + Oxal (200nM) + V, nerve growth factor (100ng / ml), oxaliplatin (200nM) and PBS as vehicle;
c: (+)NGF+Oxal+WEFF(12.5㎍/㎖), 신경성장인자(100ng/㎖), 옥살리플라틴(200nM) 및 연교 물 추출물(12.5㎍/㎖)을 처리한 PC-12 세포;c: PC-12 cells treated with (+) NGF + Oxal + WEFF (12.5 μg / ml), nerve growth factor (100 ng / ml), oxaliplatin (200 nM) and soft water extract (12.5 μg / ml);
d: (+)NGF+Oxal+WEFF(25㎍/㎖), 신경성장인자(100ng/㎖), 옥살리플라틴(200nM) 및 연교 물 추출물(25㎍/㎖)을 처리한 PC-12 세포;d: PC-12 cells treated with (+) NGF + Oxal + WEFF (25 μg / ml), nerve growth factor (100 ng / ml), oxaliplatin (200 nM) and soft water extract (25 μg / ml);
e: (+)NGF+Oxal+WEFF(50㎍/㎖), 신경성장인자(100ng/㎖), 옥살리플라틴(200nM) 및 연교 물 추출물(50㎍/㎖)을 처리한 PC-12 세포; 및e: PC-12 cells treated with (+) NGF + Oxal + WEFF (50 μg / ml), nerve growth factor (100 ng / ml), oxaliplatin (200 nM) and soft water extract (50 μg / ml); And
f: (+)NGF+Oxal+WEFF(100㎍/㎖), 신경성장인자(100ng/㎖), 옥살리플라틴(200nM) 및 연교 물 추출물(100㎍/㎖)을 처리한 PC-12 세포이고,f: PC-12 cells treated with (+) NGF + Oxal + WEFF (100 μg / ml), nerve growth factor (100 ng / ml), oxaliplatin (200 nM), and soft water extract (100 μg / ml),
***는 (+)NGF 처리군(a) 대비 통계적으로 유의성 있게 차이(p < 0.001)가 있다는 것을 의미하고, #, ## 또는 ###는 NGF+Oxal+V 처리군(b) 대비 유의성 있게 차이(#, p < 0.05; ##, p < 0.01; ###, p < 0.001)가 있다는 것을 의미한다.*** means that there is a statistically significant difference ( p <0.001) compared to the (+) NGF treatment group (a), and #, ## or ### compared to the NGF + Oxal + V treatment group (b) This means that there is a significant difference (#, p <0.05;##, p <0.01;###, p <0.001).
도 2는 신경성장인자를 이용하여 PC-12세포의 신경세포 분화 유도 후, 옥살리플라틴을 처리하여 신경세포의 독성이 유발되는 것과 본 발명의 연교 물 추출물이 신경세포를 보호하는 것을 확인한 결과이다. **는 옥살리플라틴을 처리하지 않는 PC-12 세포((-)Oxal + (-)WEFF)에 대비하여 옥살리플라틴의 처리에 의해 신경세포의 독성이 통계적으로 유의하게 발생하였다는 것을 나타내는 것으로, p<0.01이고, ##는 옥살리플라틴과 vehicle(PBS)를 처리한 대조군에 비해 본 발명의 연교 물 추출물을 처리한 경우, 통계적으로 유의하게 신경세포의 독성이 감소하는 신경보호 효과가 있다는 것을 의미하며, p<0.01이다.Figure 2 shows that after the induction of neuronal differentiation of PC-12 cells using nerve growth factors, the treatment of oxaliplatin induced the toxicity of neurons and the duct extract of the present invention protects neurons. ** indicates that the toxicity of neurons was statistically significant by treatment with oxaliplatin as compared to PC-12 cells ((-) Oxal + (-) WEFF) not treated with oxaliplatin, p <0.01 , ## means that when treated with ductile water extract of the present invention compared to the control treated with oxaliplatin and vehicle (PBS), there is a statistically significant neuroprotective effect of reducing the toxicity of neurons, p < 0.01.
도 3은 동물 모델인 마우스(C57BL/6)의 말초신경병증 통증 유발을 위해 10㎎/㎏의 옥살리플라틴을 주 1회, 총 2번(총 20㎎/㎏) 복강에 주사한 후, 연교 물 추출물(WEFF)을 매일 경구 투여하면서 측정한 체중변화를 나타낸 그래프이다.Figure 3 is injected once a week, 10 times / total (20 mg / kg) intraperitoneal injection of 10 mg / kg oxaliplatin to induce peripheral neuropathic pain in the mouse model (C57BL / 6), pontoon water extract (WEFF) is a graph showing the weight change measured by daily oral administration.
도 4는 C57BL/6 마우스에 옥살리플라틴을 투여하여 말초신경병증을 유발하고, 본 발명의 연교 물 추출물 투여에 따른 통증반응율의 변화를 확인한 결과이다.4 is a result of confirming the change in the pain response rate according to administration of oxaliplatin to C57BL / 6 mice to induce peripheral neuropathy and administration of ductal water extract of the present invention.
**, ***는 정상대조군(Normal)에 대비하여 옥살리플라틴을 투여한 동물모델에서 통증에 대한 반응이 통계적으로 유의하게 증가하였다는 것을 나타내는 것이며, **는 p<0.01이고, ***는 p<0.001임을 의미한다. 실험에서는 0.4g(A) 과 0.16g(B)의 bending force를 가진 von Frey filament를 가지고 test하였다.** and *** indicate a statistically significant increase in pain response in animal models administered oxaliplatin compared to normal, ** is p <0.01, and *** is p <0.001. In the experiment, the test was performed with von Frey filament with 0.4g (A) and 0.16g (B) bending force.
##, ###은 옥살리플라틴+vehicle(0.5% CMC 용액) 투여군에 대비하여 옥살리플라틴 투여 후, 본 발명의 연교 물 추출물을 투여한 군에서의 통증에 대한 반응이 통계적으로 유의하게 감소하였다는 것을 나타내는 것이며, ##는 p<0.01이고, ###는 p<0.001임을 의미한다.##, ### indicates that the oxaliplatin administered group compared to the oxaliplatin + vehicle (0.5% CMC solution) administration group, and the response to pain in the group administered the ductal water extract of the present invention statistically significantly reduced ## is p <0.01 and ### means p <0.001.
도 5는 본 발명의 연교 물 추출물을 옥살리플라틴과 병용 처리한 폐암 세포주(A549, A) 및 대장암 세포주(HCT116, B)의 상대적인 세포 생존률(viability)을 확인한 결과이다.Figure 5 is a result confirming the relative viability of the lung cancer cell lines (A549, A) and colon cancer cell lines (HCT116, B) treated with the ductile water extract of the present invention in combination with oxaliplatin.
본 발명은 연교 추출물을 유효성분으로 함유하는 말초신경병증(peripheral neuropathy)의 예방 또는 치료용 약학 조성물에 관한 것이다.The present invention relates to a pharmaceutical composition for the prevention or treatment of peripheral neuropathy (peripheral neuropathy) containing a duct extract as an active ingredient.
상기 연교 추출물은 물, C1~C4의 저급 알코올 또는 이들의 혼합물을 용매로 하여 추출하는 것이 바람직하며, 상기 저급 알코올은 메탄올 또는 에탄올인 것이 바람직하다. 상기 연교 추출물은 하기 단계들을 포함하는 제조방법에 의해 제조된 것이 바람직하지만 이에 한정하는 것은 아니다.The duct bridge extract is preferably extracted with water, a lower alcohol of C 1 ~ C 4 or a mixture thereof as a solvent, the lower alcohol is preferably methanol or ethanol. The duct bridge extract is preferably prepared by a manufacturing method including the following steps, but is not limited thereto.
1) 연교에 추출용매를 가하여 추출하는 단계;1) extracting by adding an extraction solvent to the bridge;
2) 단계 1)의 추출물을 여과하는 단계; 및2) filtering the extract of step 1); And
3) 단계 2)의 여과한 추출물을 감압 농축한 후 건조하는 단계.3) drying the filtered extract of step 2) under reduced pressure.
상기 방법에 있어서, 단계 1)의 연교는 재배한 것 또는 시판되는 것 등 제한 없이 사용할 수 있다.In the above method, the bridge of step 1) can be used without limitation, such as grown or commercially available.
상기 방법에 있어서, 단계 1)의 연교 추출물의 추출 방법으로는 여과법, 열수 추출, 침지 추출, 환류냉각 추출 및 초음파 추출 등 당업계의 통상적인 방법을 이용할 수 있다. 상기 추출용매는 건조된 연교의 중량을 기준으로 2~40배 부피가 되도록 첨가하여 추출하는 것이 바람직하다. 추출온도는 20~100℃인 것이 바람직하나 이에 한정하지 않는다. 또한, 추출시간은 0.5~10시간인 것이 바람직하며, 1~4시간이 더욱 바람직하고, 2시간이 가장 바람직하나 이에 한정하지 않는다. 또한, 추출은 2~3회 반복하는 것이 바람직하나 이에 한정하지 않는다.In the above method, as the extraction method of the duct bridge extract of step 1), conventional methods in the art such as filtration, hot water extraction, dipping extraction, reflux cooling extraction, and ultrasonic extraction may be used. The extraction solvent is preferably extracted by adding 2 to 40 times the volume based on the weight of the dried duct bridge. Extraction temperature is preferably 20 ~ 100 ℃ but is not limited thereto. In addition, the extraction time is preferably 0.5 to 10 hours, more preferably 1 to 4 hours, most preferably 2 hours is not limited thereto. In addition, the extraction is preferably repeated 2-3 times, but is not limited thereto.
상기 방법에 있어서, 단계 3)의 감압농축은 진공감압농축기 또는 진공회전증발기를 이용하는 것이 바람직하나 이에 한정하지 않는다. 또한, 건조는 감압건조, 진공건조, 비등건조, 분무건조 또는 동결건조하는 것이 바람직하나 이에 한정하지 않는다.In the above method, the decompression concentration in step 3) preferably uses a vacuum decompression concentrator or a vacuum rotary evaporator, but is not limited thereto. In addition, the drying is preferably reduced pressure drying, vacuum drying, boiling drying, spray drying or freeze drying, but is not limited thereto.
상기 말초신경병증은 항암제에 의해 유발되는 말초신경병증(chemotherapy-induced peripheral neuropathy)인 것이 바람직하고, 상기 항암제는 플래티넘(platinum) 계열, 탁산(taxane) 계열, 빈카알카로이드(vinca alkaloids), 보르테조밉(bortezomib) 또는 탈리도마이드(thalidomide)를 포함하지만, 이에 한정되지 않으며 임상, 약학 및 생의학적으로 사용 가능한 모든 항암제를 포함한다. The peripheral neuropathy is preferably a chemotherapy-induced peripheral neuropathy induced by an anticancer agent, and the anticancer agent is a platinum-based, taxane-based, vinca alkaloids, bortezomib ( bortezomib) or thalidomide, including, but not limited to, all anticancer agents that are clinically, pharmacologically and biomedically available.
상기 플래티넘 계열 항암제는 시스플라틴(cisplatin), 카르보플라틴(carboplatin) 및 옥살리플라틴(oxaliplatin) 중에서 선택된 하나 이상인 것이 바람직하며, 상기 탁산 계열 항암제는 파클리탁셀(paclitaxel) 및 도세탁셀(docetaxel) 중에서 선택된 하나 이상인 것이 바람직하지만 이에 한정하는 것은 아니다. The platinum-based anticancer agent is preferably at least one selected from cisplatin, carboplatin, and oxaliplatin, and the taxane-based anticancer agent is preferably at least one selected from paclitaxel and docetaxel. It is not limited to this.
본 발명의 약학 조성물은 경구 또는 비경구의 여러 가지 제형일 수 있다. 상기 조성물을 제제화할 경우에는 보통 사용하는 충진제, 증량제, 결합제, 습윤제, 붕해제, 계면활성제 등의 희석제 또는 부형제를 사용하여 조제된다.The pharmaceutical compositions of the present invention may be in various oral or parenteral formulations. In formulating the composition, diluents or excipients such as fillers, extenders, binders, wetting agents, disintegrating agents, and surfactants are usually used.
경구투여를 위한 고형제제에는 정제, 환제, 산제, 과립제, 캡슐제 등이 포함되며, 이러한 고형제제는 하나 이상의 화합물에 적어도 하나 이상의 부형제 예를 들면, 전분, 탄산칼슘, 수크로오스(sucrose) 또는 락토오스(lactose), 젤라틴 등을 섞어 조제된다. 또한, 단순한 부형제 이외에 스테아린산 마그네슘, 탈크 등과 같은 윤활제들도 사용된다. 경구투여를 위한 액상 제제로는 현탁제, 내용액제, 유제 또는 시럽제 등이 해당되는데, 흔히 사용되는 단순 희석제인 물, 리퀴드 파라핀 이외에 여러 가지 부형제, 예를 들면 습윤제, 감미제, 방향제 또는 보존제 등이 포함될 수 있다.Solid form preparations for oral administration include tablets, pills, powders, granules, capsules, and the like, which form at least one excipient such as starch, calcium carbonate, sucrose or lactose (at least one compound). lactose) and gelatin. In addition to simple excipients, lubricants such as magnesium stearate, talc and the like are also used. Liquid preparations for oral administration include suspensions, solutions, emulsions or syrups, and include various excipients such as wetting agents, sweeteners, fragrances or preservatives, in addition to the commonly used simple diluents such as water and liquid paraffin. Can be.
비경구 투여를 위한 제제에는 멸균된 수용액, 비수성용제, 현탁용제, 유제,동결건조제제 또는 좌제 등이 포함된다. 비수성용제 및 현탁용제로는 프로필렌글리콜(propylene glycol), 폴리에틸렌 글리콜, 올리브 오일과 같은 식물성 기름, 에틸올레이트와 같은 주사 가능한 에스테르 등이 사용될 수 있다. 좌제의 기제(base)로는 위텝솔(witepsol), 마크로골, 트윈(tween) 61, 카카오지, 라우린지, 글리세롤, 젤라틴 등이 사용될 수 있다.Formulations for parenteral administration include sterile aqueous solutions, non-aqueous solvents, suspensions, emulsions, freeze-dried preparations or suppositories. As the non-aqueous solvent and the suspension solvent, propylene glycol, polyethylene glycol, vegetable oil such as olive oil, injectable ester such as ethyl oleate, and the like can be used. As a base of suppositories, witepsol, macrogol, tween 61, cacao butter, laurin butter, glycerol, gelatin, and the like may be used.
본 발명의 약학 조성물은 경구 또는 비경구로 투여될 수 있으며, 비경구 투여시 피부외용 또는 복강 내, 직장, 정맥, 근육, 피하, 자궁 내 경막 또는 뇌혈관 내 주사 방식을 선택하는 것이 바람직하지만 이에 제한하지 않는다.The pharmaceutical composition of the present invention may be administered orally or parenterally, and it is preferable to select an external skin or intraperitoneal, rectal, intravenous, intramuscular, subcutaneous, intrauterine dural or cerebrovascular injection method for parenteral administration. I never do that.
본 발명의 약학 조성물은 약학적으로 유효한 양으로 투여한다. 본 발명에 있어서, 약학적으로 유효한 양은 의학적 치료에 적용 가능한 합리적인 수혜/위험 비율로 질환을 치료하기에 충분한 양을 의미하며, 유효용량 수준은 환자의 질환의 종류, 중증도, 약물의 활성, 약물에 대한 민감도, 투여 시간, 투여 경로 및 배출 비율, 치료기간, 동시 사용되는 약물을 포함한 요소 및 기타 의학 분야에 잘 알려진 요소에 따라 결정될 수 있다. 본 발명의 조성물은 개별 치료제로 투여하거나 다른 치료제와 병용하여 투여될 수 있고 종래의 치료제와는 순차적 또는 동시에 투여될 수 있으며, 단일 또는 다중 투여될 수 있다. 상기한 요소들을 모두 고려하여 부작용없이 최소한의 양으로 최대 효과를 얻을 수 있는 양을 투여하는 것이 중요하며, 이는 당업자에 의해 용이하게 결정될 수 있다.The pharmaceutical composition of the present invention is administered in a pharmaceutically effective amount. In the present invention, the pharmaceutically effective amount means an amount sufficient to treat the disease at a reasonable benefit / risk ratio applicable to the medical treatment, and the effective dose level refers to the type, severity, drug activity, and drug of the patient. Sensitivity, time of administration, route of administration and rate of release, duration of treatment, factors including concurrent use of drugs, and other factors well known in the medical arts. The compositions of the present invention may be administered as individual therapeutic agents or in combination with other therapeutic agents, may be administered sequentially or simultaneously with conventional therapeutic agents, and may be single or multiple doses. Taking all of the above factors into consideration, it is important to administer an amount that can achieve the maximum effect with a minimum amount without side effects, which can be readily determined by one skilled in the art.
본 발명의 조성물의 투여량은 환자의 체중, 연령, 성별, 건강상태, 식이, 투여시간, 투여방법, 배설율 및 질환의 중증도에 따라 그 범위가 다양하며, 일일 투여량은 연교 추출물의 양을 기준으로 0.01~1000㎎/㎏이고, 바람직하게는 30~500㎎/㎏이고, 더욱 바람직하게는 50~300㎎/㎏이며, 하루 1~6 회 투여될 수 있다. 그러나 투여 경로, 비만의 중증도, 성별, 체중, 연령 등에 따라서 증감될 수 있으므로 상기 투여량이 어떠한 방법으로도 본 발명의 범위를 한정하는 것은 아니다.The dosage of the composition of the present invention varies depending on the patient's weight, age, sex, health status, diet, time of administration, administration method, excretion rate and severity of the disease, the daily dosage is the amount of duct extract It is 0.01-1000 mg / kg, Preferably it is 30-500 mg / kg, More preferably, it is 50-300 mg / kg, It can be administered 1-6 times a day. However, the dosage may be increased or decreased depending on the route of administration, the severity of obesity, sex, weight, age, etc., and the above dosage does not limit the scope of the present invention in any way.
본 발명의 약학 조성물은 항암제와 함께 병용하여 사용하는 것이 바람직하나, 단독으로, 또는 수술, 방사선 치료, 호르몬 치료, 및 생물학적 반응 조절제를 사용하는 방법들과 병용하여 사용할 수 있다.The pharmaceutical composition of the present invention is preferably used in combination with an anticancer agent, but may be used alone or in combination with methods using surgery, radiation therapy, hormonal therapy, and biological response modifiers.
또한, 본 발명은 연교 추출물을 유효성분으로 함유하는 항암 보조제에 관한 것이다. 상기 항암 보조제는 플래티넘(platinum) 계열, 탁산(taxane) 계열, 빈카알카로이드(vinca alkaloids), 보르테조밉(bortezomib) 또는 탈리도마이드(thalidomide)를 포함하는 항암제와 병용투여하는 것이 바람직하고, 항암제에 의해 유발되는 말초신경병증 예방 또는 치료에 효과가 있는 특징이 있다.In addition, the present invention relates to an anticancer adjuvant containing duct extract as an active ingredient. The anticancer adjuvant is preferably administered in combination with an anticancer agent including platinum, taxane, vinca alkaloids, bortezomib or thalidomide, and is caused by an anticancer agent. There is a characteristic that is effective in preventing or treating peripheral neuropathy.
또한, 본 발명은 연교 추출물을 유효성분으로 함유하는 말초신경병증(peripheral neuropathy)의 예방 또는 개선용 건강기능식품 조성물에 관한 것이다. The present invention also relates to a nutraceutical composition for the prevention or improvement of peripheral neuropathy (peripheral neuropathy) containing a duct extract as an active ingredient.
상기 연교 추출물은 물, C1~C4의 저급 알코올 또는 이들의 혼합물을 용매로 하여 추출하는 것이 바람직하며, 상기 저급 알코올은 메탄올 또는 에탄올인 것이 바람직하다. 상기 연교 추출물은 하기 단계들을 포함하는 제조방법에 의해 제조된 것이 바람직하지만 이에 한정하는 것은 아니다.The duct bridge extract is preferably extracted with water, a lower alcohol of C 1 ~ C 4 or a mixture thereof as a solvent, the lower alcohol is preferably methanol or ethanol. The duct bridge extract is preferably prepared by a manufacturing method including the following steps, but is not limited thereto.
1) 연교에 추출용매를 가하여 추출하는 단계;1) extracting by adding an extraction solvent to the bridge;
2) 단계 1)의 추출물을 여과하는 단계; 및2) filtering the extract of step 1); And
3) 단계 2)의 여과한 추출물을 감압 농축한 후 건조하는 단계.3) drying the filtered extract of step 2) under reduced pressure.
상기 방법에 있어서, 단계 1)의 연교는 재배한 것 또는 시판되는 것 등 제한 없이 사용할 수 있다.In the above method, the bridge of step 1) can be used without limitation, such as grown or commercially available.
상기 방법에 있어서, 단계 1)의 연교 추출물의 추출 방법으로는 여과법, 열수 추출, 침지 추출, 환류냉각 추출 및 초음파 추출 등 당업계의 통상적인 방법을 이용할 수 있다. 상기 추출용매는 건조된 연교의 중량을 기준으로 2~40배 부피가 되도록 첨가하여 추출하는 것이 바람직하다. 추출온도는 20~100℃인 것이 바람직하나 이에 한정하지 않는다. 또한, 추출시간은 0.5~10시간인 것이 바람직하며, 1~4시간이 더욱 바람직하고, 2시간이 가장 바람직하나 이에 한정하지 않는다. 또한, 추출은 2~3회 반복하는 것이 바람직하나 이에 한정하지 않는다.In the above method, as the extraction method of the duct bridge extract of step 1), conventional methods in the art such as filtration, hot water extraction, dipping extraction, reflux cooling extraction, and ultrasonic extraction may be used. The extraction solvent is preferably extracted by adding 2 to 40 times the volume based on the weight of the dried duct bridge. Extraction temperature is preferably 20 ~ 100 ℃ but is not limited thereto. In addition, the extraction time is preferably 0.5 to 10 hours, more preferably 1 to 4 hours, most preferably 2 hours is not limited thereto. In addition, the extraction is preferably repeated 2-3 times, but is not limited thereto.
상기 방법에 있어서, 단계 3)의 감압농축은 진공감압농축기 또는 진공회전증발기를 이용하는 것이 바람직하나 이에 한정하지 않는다. 또한, 건조는 감압건조, 진공건조, 비등건조, 분무건조 또는 동결건조하는 것이 바람직하나 이에 한정하지 않는다.In the above method, the decompression concentration in step 3) preferably uses a vacuum decompression concentrator or a vacuum rotary evaporator, but is not limited thereto. In addition, the drying is preferably reduced pressure drying, vacuum drying, boiling drying, spray drying or freeze drying, but is not limited thereto.
상기 말초신경병증은 항암제에 의해 유발되는 말초신경병증(chemotherapy-induced peripheral neuropathy)인 것이 바람직하고, 상기 항암제는 플래티넘(platinum) 계열, 탁산(taxane) 계열, 빈카알카로이드(vinca alkaloids), 보르테조밉(bortezomib) 또는 탈리도마이드(thalidomide)를 포함하지만, 이에 한정되지 않으며 임상, 약학 및 생의학적으로 사용 가능한 모든 항암제를 포함한다.The peripheral neuropathy is preferably a chemotherapy-induced peripheral neuropathy induced by an anticancer agent, and the anticancer agent is a platinum-based, taxane-based, vinca alkaloids, bortezomib ( bortezomib) or thalidomide, including, but not limited to, all anticancer agents that are clinically, pharmacologically and biomedically available.
상기 플래티넘 계열 항암제는 시스플라틴(cisplatin), 카르보플라틴(carboplatin) 및 옥살리플라틴(oxaliplatin) 중에서 선택된 하나 이상인 것이 바람직하며, 상기 탁산 계열 항암제는 파클리탁셀(paclitaxel) 및 도세탁셀(docetaxel) 중에서 선택된 하나 이상인 것이 바람직하지만 이에 한정하는 것은 아니다.The platinum-based anticancer agent is preferably at least one selected from cisplatin, carboplatin and oxaliplatin, and the taxane-based anticancer agent is preferably at least one selected from paclitaxel and docetaxel. It is not limited to this.
상기 건강기능식품 조성물은 정제, 캡슐, 분말, 과립, 액상, 환 등의 형태로 제조, 가공될 수 있으나, 이에 한정되지 않으며 법률에 따라 어떤 형태로든지 제조, 가공될 수 있다.The health functional food composition may be manufactured and processed in the form of tablets, capsules, powders, granules, liquids, pills, and the like, but is not limited thereto and may be manufactured and processed in any form according to the law.
본 발명의 연교 추출물을 유효성분으로 함유하는 조성물은 식품에 그대로 첨가하거나 다른 식품 또는 식품 성분과 함께 사용될 수 있고, 통상적인 방법에 따라 적절하게 사용될 수 있다. 유효 성분의 혼합량은 그의 사용 목적(예방 또는 개선용)에 따라 적합하게 결정될 수 있다. 일반적으로, 건강식품 중의 상기 추출물의 양은 전체 식품 중량의 0.1~90중량부로 가할 수 있다. 그러나 건강 및 위생을 목적으로 하거나 또는 건강 조절을 목적으로 하는 장기간의 섭취의 경우에는 상기 양은 상기 범위 이하일 수 있으며, 안전성 면에서 아무런 문제가 없기 때문에 유효성분은 상기 범위 이상의 양으로도 사용될 수 있다.The composition containing the duct extract of the present invention as an active ingredient may be added as it is to food or used together with other food or food ingredients, and may be appropriately used according to a conventional method. The mixing amount of the active ingredient can be suitably determined according to the purpose of use (prevention or improvement). In general, the amount of the extract in the health food can be added to 0.1 to 90 parts by weight of the total food weight. However, in the case of long-term intake for health and hygiene or health control purposes, the amount may be below the above range, and the active ingredient may be used in an amount above the above range because there is no problem in terms of safety.
본 발명의 건강기능식품 조성물을 음료로 사용하는 경우, 지시된 비율로 필수 성분으로서 상기 추출물을 함유하는 것 외에는 다른 성분에는 특별한 제한이 없으며 통상의 음료와 같이 여러 가지 향미제 또는 천연 탄수화물 등을 추가 성분으로서 함유할 수 있다. 상기 천연 탄수화물의 일례로는 모노사카라이드, 예를 들어, 포도당, 과당 등; 디사카라이드, 예를 들어 말토스, 슈크로스 등; 및 폴리사카라이드, 예를 들어 덱스트린, 시클로덱스트린 등과 같은 통상적인 당, 및 자일리톨, 소르비톨, 에리트리톨 등의 당알콜이 있다. 또한 상기한 것 이외의 향미제로서 천연 향미제(타우마틴, 스테비아 추출물(예를 들어 레바우디오시드 A, 글리시르히진 등)) 및 합성 향미제(사카린, 아스파르탐 등)를 유리하게 사용할 수 있다.When the health functional food composition of the present invention is used as a beverage, there is no particular limitation on other ingredients except for containing the extract as an essential ingredient in the indicated ratio, and various flavors or natural carbohydrates are added as in the general beverage. It may contain as a component. Examples of the natural carbohydrates include monosaccharides such as glucose, fructose and the like; Disaccharides such as maltose, sucrose and the like; 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 (e.g., Rebaudioside A, glycyrginine, etc.)) and synthetic flavoring agents (saccharin, aspartame, etc.) can be advantageously used. Can be.
또한, 본 발명의 연교 추출물은 여러 가지 영양제, 비타민, 광물(전해질), 합성 풍미제 및 천연 풍미제 등의 풍미제, 착색제 및 중진제(치즈, 초콜릿 등), 펙트산 및 그의 염, 알긴산 및 그의 염, 유기산, 보호성 콜로이드 증점제, pH 조절제, 안정화제, 방부제, 글리세린, 알코올, 탄산음료에 사용되는 탄산화제 등을 함유할 수 있다. 그 밖에 본 발명의 연교 추출물은 천연 과일 쥬스 및 과일 쥬스 음료 및 야채 음료의 제조를 위한 과육을 함유할 수 있다. 이러한 성분은 독립적으로 또는 조합하여 사용할 수 있다. 이러한 첨가제의 비율은 그렇게 중요하진 않지만 본 발명의 연교 추출물 100 중량부 당 0.1~20 중량부의 범위에서 선택되는 것이 일반적이다.In addition, the duct extract of the present invention is a variety of nutrients, vitamins, minerals (electrolytes), flavors such as synthetic and natural flavors, coloring and neutralizing agents (such as cheese, chocolate), pectic acid and salts thereof, alginic acid and Salts, organic acids, protective colloid thickeners, pH adjusting agents, stabilizers, preservatives, glycerin, alcohols, carbonation agents used in carbonated drinks, and the like. In addition, the duct extract of the present invention may contain natural fruit juice and fruit flesh for the production of fruit juice drinks and vegetable drinks. These components can be used independently or in combination. The ratio of such additives is not so important, but is generally selected in the range of 0.1 to 20 parts by weight per 100 parts by weight of duct extract.
이하, 실시예를 이용하여 본 발명을 더욱 상세하게 설명하고자 한다. 이들 실시예는 오로지 본 발명을 보다 구체적으로 설명하기 위한 것으로 본 발명의 범위가 이들에 의해 제한되지 않는다는 것은 당해 기술분야에서 통상의 지식을 가진 자에게 있어 자명한 것이다. Hereinafter, the present invention will be described in more detail with reference to Examples. These examples are only for explaining the present invention in more detail, it is obvious to those skilled in the art that the scope of the present invention is not limited by them.
1. 연교(Forsythiae Fructus) 물 추출물(WEFF)의 제조1. Preparation of Forsythiae Fructus Water Extract (WEFF)
본 발명의 연교 추출물을 제조하기 위하여, 광명당제약(울산, 대한민국)으로부터 구입한 건조된 연교 100g을 분쇄하여 가루 형태의 시료를 준비하였다. 분쇄된 건조 시료 100g과 물 2ℓ를 둥근 플라스크에 넣어 혼합하였다. 냉각관이 부착된 환류추출장치에 연결된 항온수조(water bath)를 가열하여 1회 2시간씩, 총 2회 반복 추출하였다. 추출된 추출물을 종이여과지(Whatman No.2)와 진공펌프(vacuum pump)(GAST)를 사용하여 감압 여과하였다. 회전농축기(Rotary Evaporator, EYELA) 기기를 이용하여 여과된 액상 추출물을 감압 농축하였다. 농축된 추출물을 동결건조시킨 후, 막자 사발로 균질화하여 연교 물 추출물(WEFF)을 수득하였다. 수득된 연교 물 추출물은 실험 전까지 밀봉 플라스틱 용기에 담아 4℃ 저온 저장소에서 보관하였다.In order to prepare the duct bridge extract of the present invention, 100 g of the dried duct bridge purchased from Gwangmyeongdang Pharmaceutical Co., Ltd. (Ulsan, South Korea) was ground to prepare a sample in powder form. 100 g of ground dry sample and 2 l of water Put into a round flask and mix. A water bath connected to a reflux extractor with a cooling tube was heated and extracted twice, once every two hours. The extracted extract was filtered under reduced pressure using a paper filter paper (Whatman No. 2) and a vacuum pump (GAST). The filtered liquid extract was concentrated under reduced pressure using a rotary evaporator (YEYELA). The concentrated extract was lyophilized and then homogenized with a mortar and pestle to obtain soft water extract (WEFF). The resulting soft water extract was placed in a sealed plastic container and stored at 4 ° C. cold storage until the experiment.
2. 세포의 배양2. Culture of Cells
1) PC-12 세포의 배양1) Culture of PC-12 Cells
본 발명의 연교 추출물의 효과를 확인하기 위한 실시예에 사용될 PC-12 세포는 쥐(Rat)에서 유래된 크롬친화세포종(Pheochromocytoma)으로, 신경성장인자(NGF)를 처리하면 신경세포로 최종 분화하는 특성을 가지도 있어 신경관련분야 연구의 세포모델로 유용하게 사용되는 세포주이다. PC-12 세포는 5%(v/v)의 비가열 비활성화 소태아혈청(non-heat inactivated fetal bovine serum, FBS), 10%(v/v)의 가열 비활성화 말 혈청(Horse serum, HS), 100unit/㎖의 페니실린(penicillin), 100㎍/㎖의 스트렙토마이신(streptomycin)이 함유된 DMEM 성장 배지를 사용하여 37℃, 5%(v/v) CO2로 유지되는 배양기에서 콜라겐타입 Ⅰ으로 코팅된 100㎜ 세포 배양 디쉬에 배양하였다.PC-12 cells to be used in the Examples for confirming the effect of the duct extract of the present invention is a rat-derived Chromocytocytoma (Pheochromocytoma), when the neuronal growth factor (NGF) is treated to finally differentiate into neurons It is a cell line that is useful as a cell model of neurological research because of its characteristics. PC-12 cells were treated with 5% (v / v) non-heat inactivated fetal bovine serum (FBS), 10% (v / v) heat inactivated horse serum (HS), Coating with collagen type I in an incubator maintained at 37 ° C., 5% (v / v) CO 2 using DMEM growth medium containing 100 units / ml penicillin and 100 μg / ml streptomycin Cultured in a 100 mm cell culture dish.
2) 인간폐암세포(A549) 및 인간대장암세포(HCT116)의 배양2) Culture of Human Lung Cancer Cell (A549) and Human Colon Cancer Cell (HCT116)
인간폐암세포(A549) 및 인간대장암세포(HCT116)는 10%(v/v) 가열 비활성화 FBS, 100unit/㎖의 페니실린, 100㎍/㎖의 스트렙토마이신이 함유된 RPMI1640 성장 배지를 사용하여 37℃, 5%(v/v) CO2로 유지되는 배양기에서 100㎜ 세포 배양 디쉬에 배양하여 추후 생체 외(in vitro) 실험에 사용하였다.Human lung cancer cells (A549) and human colon cancer cells (HCT116) were treated at 37 ° C. using RPMI1640 growth medium containing 10% (v / v) heat inactivated FBS, 100 units / ml penicillin, 100 μg / ml streptomycin. The cells were cultured in 100 mm cell culture dishes in an incubator maintained at 5% (v / v) CO 2 and used for later in vitro experiments.
3. 항암제 유발 말초신경병증(chemotherapy-induced peripheral neuropathy, CIPN) 동물모델의 준비3. Preparation of Chemotherapy-induced Peripheral Neuropathy (CIPN) Animal Model
본 발명의 실시예를 수행하기 위하여, 옥살리플라틴(oxaliplatin)으로 유발된 말초신경병증(chemotherapy-induced peripheral neuropathy, CIPN) 동물모델을 준비하였다.In order to carry out an embodiment of the present invention, a animal model of chemotherapy-induced peripheral neuropathy (CIPN) induced with oxaliplatin was prepared.
구체적으로, 8~10주령의 C57BL/6 수컷 마우스를 동물 실험실 내에 케이지에서 순화 기간을 거친 후, 3회에 걸쳐 기준선(base Line, BL)을 잡고, 군 분리(N=6 마우스/그룹)를 실시하였다. 이 후, 말초신경병증 통증 유발을 위해 10㎎/㎏의 옥살리플라틴을 2주에 걸쳐 총 20㎎/㎏을 복강에 주사하였다.Specifically, 8-10 weeks old C57BL / 6 male mice were subjected to a purification period in a cage in an animal laboratory, followed by three baseline (BL), and group separation (N = 6 mice / group). Was carried out. Subsequently, 10 mg / kg of oxaliplatin was injected into the abdominal cavity for a total of 20 mg / kg over 2 weeks to induce peripheral neuropathic pain.
4. 통계처리4. Statistical Processing
본 발명의 실시예에서 획득된 결과는 평균±표준편차(mean±S.D.) 또는 평균±표준오차(mean±S.E.)로 표기하였고, 그룹간의 통계학적 평균 비교는 T-test와 ANOVA/Tukey post-hoc테스트를 실시하였다. p값은 * <0.05, ** <0.01, *** <0.001로 표기하였다.The results obtained in the examples of the present invention were expressed as mean ± standard deviation (mean ± SD) or mean ± standard error (mean ± SE), and the statistical mean comparison between groups was T-test and ANOVA / Tukey post-hoc. The test was conducted. p values were expressed as * <0.05, ** <0.01, *** <0.001.
실시예 1. 생체 외(in vitro)에서 연교 물 추출물의 CIPN 억제 효과 확인Example 1 Confirmation of CIPN Inhibitory Effect of Pyrophyllum Water Extract in Vitro
실시예 1-1. 연교 물 추출물의 신경돌기 성장 억제 완화 효과 확인Example 1-1. Determination of Neuronal Growth Inhibitory Effect of Yeon-kyo Water Extract
본 발명의 연교 물 추출물의 신경돌기 성장 억제 효과를 확인하였다. 구체적으로, 상기 배양한 PC-12 세포를 1×104 세포/웰의 농도로 콜라겐 타입 Ⅳ로 코팅된 24 웰 플레이트에 균일하게 접종한 후, 바닥면에 부착되도록 DMEM 성장배지에 배양하였다. 24시간 후 신경돌기의 생성을 유도하기 위하여 혈청 없이 100ng/㎖ 신경성장인자(NGF)가 함유된 DMEM 배지로 교체하고, 동시에 신경독성을 유도하기 위하여 200nM 옥살리플라틴을 처리하였다. The neurite outgrowth inhibitory effect of the water extract of Fructus of the present invention was confirmed. Specifically, the cultured PC-12 cells were uniformly inoculated in a 24 well plate coated with collagen type IV at a concentration of 1 × 10 4 cells / well, and then cultured in DMEM growth medium to adhere to the bottom surface. After 24 hours, the cells were replaced with DMEM medium containing 100 ng / ml neuronal growth factor (NGF) without serum to induce the production of neurites, and treated with 200 nM oxaliplatin to induce neurotoxicity.
상기 제조한 연교 물 추출물(WEFF)은 각각 12.5, 25, 50 및 100㎍/㎖의 농도로 처리하였으며, 3일 후 광학현미경으로 관찰하여 신경돌기가 생성된 세포의 비율과 신경돌기의 길이를 분석하였다. The prepared soft water extract (WEFF) was treated at concentrations of 12.5, 25, 50, and 100 ㎍ / ml, respectively, and after 3 days, observed with an optical microscope to analyze the ratio of neurites-produced cells and the length of neurites. It was.
그 결과 도 1에 나타낸 바와 같이, 신경성장인자(NGF)에 의해 유도된 신경돌기 성장은 옥살리플라틴 처리(NGF+Oxal)에 의해 감소 되며, 신경돌기를 형성한 세포의 비율(A)과 성장한 신경돌기의 길이의 합(B)이 연교 물 추출물(WEFF)에 의해 회복되는 것을 확인하였고, 연교 물 추출물(WEFF)에 의해 옥살리플라틴에 의한 신경돌기 성장 억제가 완화(C)되는 것을 확인하였다.As a result, as shown in Figure 1, the neurites induced by the neuronal growth factor (NGF) is reduced by oxaliplatin treatment (NGF + Oxal), the ratio of cells forming the neurites (A) and the grown neurites It was confirmed that the sum of the lengths (B) was recovered by the duct water extract (WEFF), and the inhibition of neurites growth inhibition by oxaliplatin was relieved by the duct water extract (WEFF).
실시예 1-2. 연교 물 추출물의 세포독성 보호 효과 확인Example 1-2. Confirmation of Cytotoxic Protective Effects of Yeonkyo Water Extract
본 발명의 연교 물 추출물의 옥살리플라틴(Oxaliplatin)에 의한 세포독성에 대한 보호 효과를 확인하였다. 구체적으로, 상기 배양한 PC-12 세포를 2×103 세포/웰의 농도로 콜라겐 타입 Ⅳ로 코팅된 96웰 플레이트에 균일하게 접종한 후 바닥면에 부착되도록 DMEM 성장배지에 배양하였다. 24시간 후 신경돌기의 생성을 유도하기 위하여 혈청 없이 100ng/㎖의 신경성장인자(NGF)가 함유된 DMEM 배지로 교체하여 신경세포로의 분화를 유도하였다. 4일 후 신경세포에 세포독성을 유도하기 위하여 200nM의 옥살리플라틴을 처리하였다. 상기 제조한 연교 물 추출물(WEFF)은 각각 25, 50, 100㎍/㎖의 농도로 처리하였으며, 2일 후 Ez-cytox를 이용하여 각 웰의 viability를 측정하였다.The protective effect of oxaliplatin (Oxaliplatin) on the cytotoxicity of the water extract of the present invention was confirmed. Specifically, the cultured PC-12 cells were uniformly inoculated into a 96 well plate coated with collagen type IV at a concentration of 2 × 10 3 cells / well and then cultured in DMEM growth medium to adhere to the bottom surface. After 24 hours, differentiation into neurons was induced by replacing with DMEM medium containing 100 ng / ml of neuronal growth factor (NGF) without serum to induce neurites. Four days later, 200 nM of oxaliplatin was treated to induce cytotoxicity to neurons. The prepared soft water extract (WEFF) was treated at concentrations of 25, 50, and 100 µg / ml, respectively, and after 2 days, viability of each well was measured using Ez-cytox.
그 결과 도 2에 나타낸 바와 같이, 신경성장인자(NGF)에 의해 신경세포로 분화 유도된 PC12세포는 옥살리플라틴 처리(NGF+Oxal)에 의해 세포 생존률(cell viability)이 감소되었으며, 연교 물 추출물(WEFF)에 의해 옥살리플라틴에 의한 신경세포 독성이 완화되는 것을 확인하였다.As a result, as shown in Figure 2, PC12 cells induced differentiation into neurons by nerve growth factor (NGF) was reduced cell viability by oxaliplatin treatment (NGF + Oxal), duct water extract (WEFF) It was confirmed that neuronal toxicity caused by oxaliplatin is alleviated by).
실시예 2. 항암제 유발 말초신경병증(chemotherapy-induced peripheral neuropathy, CIPN) 동물모델의 체중변화 확인Example 2 Identification of Weight Changes in Animal Models of Chemotherapy-induced Peripheral Neuropathy (CIPN)
상기 준비한 동물모델의 체중변화를 확인한 결과, 옥살리플라틴을 투여한 실험 동물 그룹은 정상그룹에 대비하여 체중이 감소하였으며, 2주간의 CIPN 유발 후, 연교 추출물(WEFF)을 경구 투여한 군은 vehicle로 0.5%의 CMC 용액을 투여한 그룹과 비교하여 체중 감소와 같은 부작용은 보이지 않았고, 오히려 vehicle 투여그룹보다 빠르게 체중을 회복하였다(도 3).As a result of confirming the weight change of the prepared animal model, the experimental animal group administered oxaliplatin decreased body weight compared to the normal group, and after induction of CIPN for 2 weeks, the group administered oral extract (WEFF) orally was 0.5 as vehicle. Compared with the group administered the% CMC solution, no side effects such as weight loss were seen, but rather recovered faster than the vehicle administration group (FIG. 3).
실시예 3. 생체 내(in vivo)에서 연교 물 추출물의 CIPN(chmotherapy-induced peripheral neuropathy) 완화 효과 확인Example 3 Confirmation of ChIPtherapy-induced Peripheral Neuropathy (CIPN) Mitigation Effect of Fructus Water Extract in Vivo
옥살리플라틴으로 유발된 말초신경병증 동물모델을 이용하여 기계적 통각과민(mechanical hyperalgesia)을 측정함으로써, 본 발명의 연교 물 추출물의 CIPN 억제 효과를 확인하였다.By measuring mechanical hyperalgesia using an oxaliplatin-induced peripheral neuropathy animal model, the CIPN inhibitory effect of the soft water extract of the present invention was confirmed.
구체적으로, 상기한 말초신경병증 동물모델에 통증이 유발된 2주 후부터 상기 250㎎/㎏ 연교 물 추출물(WEFF) 및 음성대조군으로 0.5%(v/v) 카르복시메틸셀룰로스(carboxymethyl cellulose, CMC) 용액을 각각 주 7회씩, 매일 경구투여하였다. 통증이 유발된 2주 후부터 약물투여 기간 동안 주 1회 마우스의 뒷발바닥(hind paw)에 0.16g과 0.4g의 동일한 자극을 주는 von Frey filament 테스트를 시행하였다. 자극에 대한 민감도는 자극에 반응하는 동물 개체의 빈도(%)로 표시하였다.Specifically, 0.5% (v / v) carboxymethyl cellulose (CMC) solution as the 250 mg / kg soft water extract (WEFF) and negative control group after two weeks of pain induced in the peripheral neuropathy animal model Were administered orally 7 times a week each day. Two weeks after the pain-induced von Frey filament test, the same stimulation of 0.16 g and 0.4 g was applied to the hind paw of the mouse once a week during the administration period. Sensitivity to stimuli is expressed as the percentage of animal subjects responding to the stimulus.
그 결과, 도 4에 개시한 바와 같이, 연교 물 추출물 투여한 한 후, 통증에 대한 반응율이 감소하는 것을 확인할 수 있었다. As a result, as shown in FIG. 4, after administration of the soft water extract, it was confirmed that the response rate to pain was decreased.
실시예 4. 생체 외(in vitro) 인간종양세포에서 연교 물 추출물이 옥살리플라틴의 세포독성에 미치는 영향 확인Example 4. Confirmation of the effect of soft water extract on the cytotoxicity of oxaliplatin in human tumor cells in vitro
종양 임상에서 암환자에게 항암제와 연교 물 추출물을 동시에, 혹은 시간적인 간격을 두고 투여하였을 때, 연교 물 추출물이 항암제를 이용한 항암치료 결과에 부정적인 영향을 줄 수 있는 가능성을 배제하기 위하여 생체 외 인간종양세포에서 연교 물 추출물이 항암제의 세포독성에 영향을 주는지 확인하였다.In tumor clinical trials, in vitro human tumors were excluded in order to rule out the possibility that chemotherapeutic water extracts could negatively affect chemotherapy outcomes with chemotherapy when the anticancer agent and ductile water extract were administered simultaneously or at intervals. In the cells, it was confirmed that the duct extracts affect the cytotoxicity of anticancer drugs.
구체적으로, 상기한 바와 같이, 인간폐암세포(A549)와 인간대장암세포(HCT116)를 배양한 후, 각각의 세포를 96웰 배양 디쉬에 웰당 5×103개의 세포로 분주하였다. 24시간 후에 순차적으로 희석된 옥살리플라틴(0~100㎍/㎖)과 연교 물 추출물(100㎍/㎖)을 동시에 처리하였다. 음성대조군은 옥살리플라틴을 처리하고, 연교 물 추출물 대신 vehicle로서 PBS를 처리한 세포군을 사용하였다. 처리 72시간 후에 Ez-Cytox 세포생존률 측정키트를 사용하여 세포생존률을 측정하였다.Specifically, as described above, after culturing human lung cancer cells (A549) and human colon cancer cells (HCT116), each cell was divided into 5 x 10 3 cells per well in a 96 well culture dish. After 24 hours, oxaliplatin (0-100 µg / ml) and soft water extract (100 µg / ml) diluted sequentially were treated simultaneously. The negative control group was treated with oxaliplatin and used a cell group treated with PBS as a vehicle instead of the soft water extract. After 72 hours of treatment, cell viability was measured using an Ez-Cytox cell viability measurement kit.
그 결과 도 5에 나타난 것과 같이, 옥살리플라틴을 단독으로 처리하였을 때, 농도의존적으로 A549 인간폐암세포와 HCT116 인간대장암세포의 성장을 억제하였고, 연교 물 추출물을 100㎍/㎖의 농도로 옥살리플라틴과 동시에 처리하여도 연교 물 추출물이 옥살리플라틴의 암세포성장 억제 효과에 부정적 영향을 주지 않음을 확인하였다. 따라서, 상기 결과로 연교 물 추출물은 임상적으로 옥살리플라틴과 함께 암환자에게 병용 투여하였을 때, 옥살리플라틴의 항암 효과에 영향을 주지 않는 것을 확인하였다.As a result, as shown in Figure 5, when treated with oxaliplatin alone, concentration-dependently inhibited the growth of A549 human lung cancer cells and HCT116 human colon cancer cells, and treated with oxaliplatin at the concentration of 100 ㎍ / ㎖ Even if it was confirmed that the water extract did not have a negative effect on the cancer cell growth inhibitory effect of oxaliplatin. Therefore, as a result, it was confirmed that the soft water extract did not affect the anticancer effect of oxaliplatin when used in combination with oxaliplatin to cancer patients.

Claims (13)

  1. 연교 추출물을 유효성분으로 함유하는 말초신경병증(peripheral neuropathy)의 예방 또는 치료용 약학 조성물.Pharmaceutical composition for the prevention or treatment of peripheral neuropathy (peripheral neuropathy) containing a cheongyo extract as an active ingredient.
  2. 제1항에 있어서, 상기 연교 추출물은 물, C1~C4의 저급 알코올 또는 이들의 혼합물을 용매로 하여 추출하는 것을 특징으로 하는 말초신경병증 예방 또는 치료용 약학 조성물.The pharmaceutical composition for preventing or treating peripheral neuropathy according to claim 1, wherein the duct extract is extracted with water, lower alcohols of C 1 to C 4 or a mixture thereof as a solvent.
  3. 제2항에 있어서, 상기 저급 알코올은 메탄올 또는 에탄올인 것을 특징으로 하는 말초신경병증 예방 또는 치료용 약학 조성물.The pharmaceutical composition for preventing or treating peripheral neuropathy according to claim 2, wherein the lower alcohol is methanol or ethanol.
  4. 제1항에 있어서, 상기 말초신경병증은 항암제에 의해 유발되는 말초신경병증(chemotherapy-induced peripheral neuropathy)인 것을 특징으로 하는 말초신경병증 예방 또는 치료용 약학 조성물.The pharmaceutical composition for preventing or treating peripheral neuropathy according to claim 1, wherein the peripheral neuropathy is a chemotherapy-induced peripheral neuropathy induced by an anticancer agent.
  5. 제4항에 있어서, 상기 항암제는 플래티넘(platinum) 계열, 탁산(taxane) 계열, 빈카알카로이드(vinca alkaloids), 보르테조밉(bortezomib) 또는 탈리도마이드(thalidomide)인 것을 특징으로 하는 말초신경병증 예방 또는 치료용 약학 조성물.The method of claim 4, wherein the anticancer agent is platinum-based, taxane-based, vinca alkaloids, bortezomib, or thalidomide, which is used for preventing or treating peripheral neuropathy. Pharmaceutical composition.
  6. 제5항에 있어서, 상기 플래티넘 계열 항암제는 시스플라틴(cisplatin), 카르보플라틴(carboplatin) 및 옥살리플라틴(oxaliplatin) 중에서 선택된 하나 이상인 것을 특징으로 하는 말초신경병증 예방 또는 치료용 약학 조성물.The pharmaceutical composition for preventing or treating peripheral neuropathy according to claim 5, wherein the platinum-based anticancer agent is at least one selected from cisplatin, carboplatin, and oxaliplatin.
  7. 제5항에 있어서, 상기 탁산 계열 항암제는 파클리탁셀(paclitaxel) 및 도세탁셀(docetaxel) 중에서 선택된 하나 이상인 것을 특징으로 하는 말초신경병증 예방 또는 치료용 약학 조성물.The pharmaceutical composition for preventing or treating peripheral neuropathy according to claim 5, wherein the taxane-based anticancer agent is at least one selected from paclitaxel and docetaxel.
  8. 제1항에 있어서, 상기 유효성분 이외에 추가로 부형제 또는 희석제를 포함하는 것을 특징으로 하는 말초신경병증 예방 또는 치료용 약학 조성물.The pharmaceutical composition for preventing or treating peripheral neuropathy according to claim 1, further comprising an excipient or a diluent in addition to the active ingredient.
  9. 연교 추출물을 유효성분으로 함유하는 항암 보조제.Anticancer adjuvant containing Yeongyo extract as an active ingredient.
  10. 제9항에 있어서, 상기 항암 보조제는 플래티넘(platinum) 계열, 탁산(taxane) 계열, 빈카알카로이드(vinca alkaloids), 보르테조밉(bortezomib) 또는 탈리도마이드(thalidomide)를 포함하는 항암제와 병용투여하는 것을 특징으로 하는 항암 보조제.The method of claim 9, wherein the anticancer adjuvant is used in combination with an anticancer agent including platinum, taxane, vinca alkaloids, bortezomib, or thalidomide. Anticancer supplements.
  11. 제9항에 있어서, 항암제에 의해 유발되는 말초신경병증의 예방 또는 치료에 효과가 있는 것을 특징으로 하는 항암 보조제.10. The anticancer adjuvant according to claim 9, which is effective for preventing or treating peripheral neuropathy induced by an anticancer agent.
  12. 연교 추출물을 유효성분으로 함유하는 말초신경병증(peripheral neuropathy)의 예방 또는 개선용 건강기능식품 조성물.Health functional food composition for the prevention or improvement of peripheral neuropathy (peripheral neuropathy) containing Yeongyo extract as an active ingredient.
  13. 제11항에 있어서, 상기 유효 성분 이외에 추가로, 영양제, 비타민, 전해질, 풍미제, 착색제, 증진제, 펙트산 및 그의 염, 알킨산 및 그의 염, 유기산, 보호성 콜로이드 증점제, pH 조절제, 안정화제, 방부제, 글리세린, 알코올 및 탄산음료에 사용되는 탄산화제 중에서 선택된 하나 이상을 더 함유하는 것을 특징으로 하는 말초신경병증(peripheral neuropathy)의 예방 또는 개선용 건강기능식품 조성물.The method according to claim 11, wherein, in addition to the active ingredient, a nutrient, a vitamin, an electrolyte, a flavoring agent, a coloring agent, an enhancer, a pectic acid and a salt thereof, an alkanoic acid and a salt thereof, an organic acid, a protective colloidal thickener, a pH adjuster, a stabilizer Functional food composition for the prevention or improvement of peripheral neuropathy (peripheral neuropathy), characterized in that it further comprises at least one selected from preservatives, glycerin, alcohol and carbonation agent used in carbonated drinks.
PCT/KR2017/007168 2016-07-06 2017-07-05 Composition comprising forsythiae fructus extract as effective ingredient for preventing, ameliorating or treating peripheral neuropathy WO2018008973A1 (en)

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