WO2019098568A2 - Composition for alleviating, preventing or treating pain comprising camellia japonica extract - Google Patents

Abstract

Provided is a composition for alleviating, preventing or treating pain comprising a Camellia japonica extract as an active ingredient. The composition of the present invention exhibits the effects of not only alleviating and treating generated pain but also preventing pain when administered to a subject before pain occurs. The composition of the present invention exhibits a remarkable pain relieving effect not only by intravenous injection or application to the skin but also by oral administration. The composition of the present invention having such characteristics can be applied to food.

Description

Composition for alleviating, preventing or treating pain including camellia extract

The present invention was made by the task number E0164502-02 under the support of the Ministry of Science, Technology & Information and Communications of the Republic of Korea. The research institute of the above subject is Korea Food Research Institute, the research project name is "Korea Food Research Institute major project" The research period is 2016. 01. ~ 2021. December 31, the contribution rate is 1/1.

This patent application claims priority to Korean Patent Application No. 10-2017-0151554 filed on November 14, 2017, the Korean Intellectual Property Office, the disclosure of which is incorporated herein by reference.

The present invention relates to a composition for alleviating, preventing or treating pain comprising a camellia extract.

The camellia ( Camellia japonica L.) is an evergreen tree belonging to Camelliae (Camelliae). It is mainly distributed in southern coasts and islands, Of which Jeonnam area occupies about 67% of the total planted area.

It has been reported that many compounds such as camellin, pipecolic acid, eugenol, camelliagenin A and B, tsubaki saponin, triterpene, tannin, benzenoid, steroid, flavonoid and phenylpropanoid are present in the leaves, seeds and flowers of camellia trees.

In Japan, dried camellia buds were used as a hematopoietic agent in the private sector, as a hemostatic agent for red blood cells and as a powder of red flowers as a powder, along with urine, ginger juice, and alcohol. Antigenic and antipruritic effects, Inhibition, skin whitening, and other physiological activities have been reported. In addition, recent studies have reported effects of antihypertensive agents, anti-neurotoxicity, and antioxidant effects in rodent models of Parkinson's disease.

The inventors of the present invention have made efforts to develop a safe material for human body which can relieve pain effectively, particularly plant-derived materials. As a result, it has been found out that the extract of Camelliaceae is very effective for alleviating, Thereby completing the invention.

Accordingly, it is an object of the present invention to provide a pharmaceutical composition for alleviating, preventing or treating pain including camellia extract.

It is another object of the present invention to provide a food composition for alleviating or preventing pain comprising a camellia extract.

Another object of the present invention is to provide a method for alleviating, preventing or treating pain, comprising the step of administering to a subject in need thereof an effective amount of alleviating, preventing or treating pain of the camellia extract.

The present invention relates to a pharmaceutical composition for relieving, preventing or treating pain comprising Camellia japonica L. extract. The camellia extract according to the present invention comprises a solvent extract of camellia and a solvent fraction thereof, Camellia extracts exhibit relief, preventive and therapeutic effects against pain.

The present inventors have made extensive efforts to develop a safe material for humans, particularly plant-derived materials, which can relieve pain effectively, and as a result, it has been found that the extract of Camelliaceae is very effective for alleviating, preventing or treating pain.

Camellia japonica L. belongs to the genus Camellia. It is an arboreous tree with evergreen evergreen trees. It blooms from winter to early spring. Seeds are harvested at the end of October and contain 3-5 seeds per plant. Leaves are alternate oval or long oval with wavy sawtooth on edge. The surface is dark green with shine and the back side is yellowish green with no hairs and thick. It is reported that about 200 species are distributed in Asian region. One of them ( Camellia japonica L.) is native to Korea, and Camellia species, which are distributed in relatively cold places, are emphasized as genetic resources.

Camellia has been mainly used as a source of horticultural material since the past, and its main ingredients are saturated fatty acids (9.1-11.5%), oleic acid (85.6-89.4%), linoleic acid (1.3-2.9%) and glycerides with high oleic acid It is a clear, non-drying oil. Its freezing point is low at -25 ℃. In addition, the extract of camellia japonica leaf is known to be very useful as a natural preservative.

One aspect of the present invention relates to a pharmaceutical composition for alleviating, preventing or treating pain comprising a camellia extract.

The camellia may be at least one selected from the group consisting of fruits, roots, stems and leaves of a camellia, and may be, for example, a camellia leaf.

The camellia extract may be an extract obtained by extracting a camellia tree with a polar solvent, a non-polar solvent or a mixed solvent thereof.

The polar solvent may be water, alcohol, acetic acid, dimethylformamide (DMFO), dimethyl sulfoxide (DMSO), or a mixture thereof, but is not limited thereto.

The alcohol may be a linear or branched alcohol having 1 to 4 carbon atoms such as methanol, ethanol, propanol, butanol, n-propanol, iso-propanol, n-butanol, 1-pentanol, 2-butoxyethanol, Glycols, or mixtures thereof, but is not limited thereto.

When a mixture of water and alcohol is used as a solvent used in the production of the camellia extract, the solvent is used in an amount of 10% or more to less than 100% (v / v), 20% to less than 100% (v / (V / v), less than 100% (v / v), less than 100% (v / v) Or a linear or branched alcohol aqueous solution having from 1 to 4 carbon atoms and less than 100% (v / v), and may be, for example, an aqueous 70% (v / v) alcohol solution.

The nonpolar solvent may be selected from the group consisting of acetone, acetonitrile, ethyl acetate, methyl acetate, fluoroalkane, pentane, hexane, 2,2,4-trimethylpentane, decane, cyclohexane, cyclopentane, diisobutylene, Chlorobenzene, benzene, diethyl ether, diethylsulfide, chloroform, dichloromethane, dichloromethane, dichloromethane, chloroform, Diethylamine, ether, carbon tetrachloride, tetrahydrofuran or a mixture thereof, preferably acetone, ethyl acetate, chloroform, butyl acetate, 1,3-butylene glycol , Hexane, diethyl ether, or a mixture thereof, but is not limited thereto.

According to one embodiment of the present invention, the camellia extract is a camellia tree extract obtained by using ethanol as a solvent.

According to another embodiment of the present invention, the camellia extract is a camellia leaf extract obtained by extracting ethanol with a solvent.

The 'extract' includes a solvent crude extract, a specific solvent soluble extract (solvent fraction) and a solvent fraction of a solvent crude extract.

Specifically, the term " extract " as used herein means a crude extract in the art as described above, but broadly includes fractions obtained by further fractionating the extract.

Namely, the Camellia sinensis extract is obtained not only by using the above-mentioned extraction solvent but also by additionally applying a purification process thereto. For example, a fraction obtained by passing the above extract through an ultrafiltration membrane having a constant molecular weight cut-off value, and a separation by various chromatography (manufactured for separation according to size, charge, hydrophobicity or affinity) The fraction obtained through the purification method is also included in the camellia extract of the present invention.

The camellia extract used in the present invention can be prepared in a concentrate and / or powder state by an additional process such as vacuum distillation and freeze drying or spray drying.

Since the present invention includes an extract extracted from a camellia tree which is a natural plant material, there is no adverse effect on the human body even when administered in an excessive amount. Therefore, the quantitative upper limit of the camellia extract contained in the composition of the present invention can be selected by a person skilled in the art have.

The composition of the present invention is very effective for alleviating, preventing or treating pain.

The composition of the present invention exhibits remarkable pain relieving effect not only by intravenous injection or skin application but also by oral administration. The composition of the present invention having such characteristics can be applied to foods well.

The composition of the present invention not only alleviates and treats the pain generated, but also has an effect of preventing pain when administered to a subject before pain occurs.

The term " pain " is used in the present invention in its broadest sense and includes acute and chronic pain such as pain ache and pain, such as somatic pain and visceral pain; Pain, pain, inflammatory pain, dysfunctional pain, idiopathic pain, superficial pain, deep pain, itching, neuropathic pain such as centrally generated pain and peripherally generated pain, migraine, and cancer pain.

The term " nociceptive pain " refers to any pain caused by a noxious stimulus that is potentially damaging or actually damaging to body tissues. The painful water-soluble pain includes, without limitation, pain caused by ben, cut, bruise, bone fracture, crush injury, burn and similar wounds. Pain receptors for tissue damage (nociceptors) are mostly located in the skin, musculoskeletal or internal organs.

Acupuncture pain occurs in response to activation of nociceptors by a specific subset of peripheral sensory neurons, that is, intense or harmful stimuli. Acute water-soluble pain is a generally sensitive, self-limiting, and provides a biological function of protection by acting as a warning of potential or ongoing tissue damage.

Pain Acceptable pain is typically very limited to locality. Examples of painful painful pain include, but are not limited to, traumatic or surgical pain, labor pain, sprains, bone fractures, burns, bumps, but are not limited to, bruises, injections, dental procedures, skin biopsies and obstructions.

The term " somatic pain " refers to pain occurring in bones, joints, muscles, skin or connective tissue. This type of pain is typically very localized.

The term " visceral pain " is used herein to refer to any or all of the organs such as respiratory, gastrointestinal tract and pancreas, urinary tract and reproductive organs, Used to indicate pain that occurs. Embolic pain includes pain induced by tumor involvement of the organ capsule. Other types of visceral pain are typically caused by occlusion of the hollow viscus, characterized by intermittent cramping and severe local pain. Embolic pain may be associated with inflammation, as in the case of cystitis or reflux esophagitis.

The term " inflammatory pain " refers to pain associated with active inflammation, which may be caused by trauma, surgery, infection, and autoimmune disease.

Inflammatory pain is associated with joints such as postoperative, post-traumatic pain, arthritis (rheumatoid or osteoarthritis) pain, and axial low back pain. ), Pain associated with damage to muscles and tendons, or pain when there is a tissue injury or inflammation.

The term " superficial pain " refers to a pain sensed by a skin segment in which a dorsal root nerve is distributed, and refers to a direct pain sensation at the point where stimulation is felt.

The term " deep pain " refers to pain originating from deep organs, which varies in character and degree depending on the nature of the tissue. Particularly sensitive areas of pain are tendons, perineum, ligaments, joints, periosteum, blood vessels and nerves. In general, deep pain is dull, spreads around, and feels wide. Pain in the deep or intestine is complicated in its mechanism, making it difficult to locate the pain rather than the surface pain, and problems such as nausea, sweating, and elevated blood pressure also appear.

The term " neuropathic pain " as used herein refers to pain resulting from the sensory input of abnormal processing by the peripheral or central nervous system resulting from disturbance of the peripheral or central nervous system.

Pain In contrast to water-soluble pain, neuropathic pain is depicted as actual "burning," "electric," "tingling," or "shooting" do. Neuropathic pain is often defined as chronic allodynia (defined as pain due to stimuli that do not cause a normal pain response, such as a mild touch) and hyperalgesia (a high susceptibility to normal pain stimuli ). It can last for months or years after apparent healing of any damaged tissue.

The term " procedural pain " refers to pain that occurs in internal medicine, dentistry, or surgery. The procedure is usually scheduled or associated with acute trauma.

The term " itch " is used herein in its broadest sense and refers to all types of itchy, stinging sensations of acute intermittency and persistence that can be generally described by limiting to local. The itch may be due to idiopathic, allergic, metabolic, infectious, drug-induced, liver, kidney disease or by cancer. "Pruritus" is severe itching.

As used herein, the term " palliative " means any action in which pain is suppressed or delayed by administration of the composition.

As used herein, the term " prevention " means any action in which pain is suppressed or delayed due to administration of the composition in advance of the possibility of pain occurring.

As used herein, the term " treatment " means any action in which the development of pain is suppressed, pain relieved, or pain relieved by the administration of the composition.

According to one embodiment of the present invention, the composition of the present invention has the effect of alleviating, preventing or treating pain in various models of painful pain.

In a specific embodiment of the invention, the painful animal model is selected from the group consisting of a skin incision model, a neural branch ligation injury model, a complete Freund's adjuvant induced inflammatory pain model, a chemotherapy induced peripheral neuropathic pain model, Pain models, and the like.

According to a particular embodiment of the present invention, the nerve ligated in the neural branch ligation injury model is a total peroneal nerve and / or tibial nerve.

According to another specific embodiment of the present invention, the chemical agent to be administered in the preparation of the chemotherapy-induced peripheral neuropathic pain model is an anticancer agent, specifically, but not limited to, vincristine, paclitaxel, and the like.

In certain embodiments of the present invention, the pain sensed by the animal pain model may be quantified by von frey filament test or ultrasound vocalization calls measurements.

According to another embodiment of the present invention, the composition of the present invention has pain relief, prophylactic or therapeutic effects in an in vitro pain cell model.

In certain embodiments of the present invention, the in vitro pain cell model is a cell model expressing CCR2 (chemokine receptor type 2). Specifically, the cells may be HEK cells (Human embryonic kidney cells 293), but are not limited thereto.

The measurement of pain in the in vitro cell model can be carried out by treating a Camellia sinensis leaf extract, which is an effective ingredient of the composition of the present invention, with CCR2-expressing cells, followed by treatment with CCR2's monocyte chemoattractant protein 1 (MCP-1) or CCL2 CC motif chemokine ligand 2) and measuring changes in intracellular calcium concentration.

The content of the camellia extract in the composition according to the present invention can be appropriately adjusted depending on the mode and purpose of use, patient condition, symptom type, and the like, and is 0.1 to 99.9% by weight, 0.1 to 99.0% by weight, 0.1 To 90.0% by weight, 0.1 to 80.0% by weight, 0.1 to 70.0% by weight, 0.1 to 60.0% by weight, preferably 0.1 to 50% by weight.

The compositions according to the present invention can be administered to mammals, including humans, in a variety of routes. The mode of administration may be any conventional manner and may be administered, for example, by oral, skin, intravenous, intramuscular, subcutaneous, and the like routes, preferably orally.

The composition of the present invention may be formulated into oral formulations such as powders, granules, tablets, capsules, ointments, suspensions, emulsions, syrups and aerosols, or parenteral formulations such as transdermal preparations, suppositories, And the like.

The composition of the present invention may contain pharmaceutically acceptable and physiologically acceptable carriers, excipients and diluents as well as adjuvants such as the camellia extract.

Examples of carriers, excipients and diluents that can be included in the composition of the present invention include lactose, dextrose, sucrose, sorbitol, mannitol, xylitol, erythritol, maltitol, starch, acacia rubber, alginate, gelatin, calcium phosphate, calcium silicate , Cellulose, methylcellulose, microcrystalline cellulose, polyvinylpyrrolidone, water, methylhydroxybenzoate, propylhydroxybenzoate, talc, magnesium stearate and mineral oil.

In the case of formulation, diluents or excipients such as fillers, extenders, binders, humectants, disintegrants, surfactants and the like which are usually used can be used. Solid formulations for oral administration include tablets, pills, powders, granules, capsules and the like, which may contain at least one excipient such as starch, calcium carbonate, sucrose sucrose), lactose, gelatin, and the like. In addition to simple excipients, lubricants such as magnesium stearate talc may also be used.

Examples of the agent for oral administration include suspensions, solutions, emulsions, syrups, ointments and the like. Various excipients such as wetting agents, sweeteners, fragrances and preservatives are included in addition to water and liquid paraffin which are commonly used simple diluents .

Formulations for parenteral administration include sterilized aqueous solutions, non-aqueous solutions, suspensions, emulsions, freeze-dried preparations, suppositories, transdermal preparations and the like. Examples of the suspending agent include propylene glycol, polyethylene glycol, vegetable oil such as olive oil, injectable ester such as ethyl oleate, and the like.

As the suppository preparation, witepsol, macrogol, tween 61, cacao paper, laurin, glycerogelatin and the like can be used.

In an embodiment where the composition of the present invention is applied to humans, the composition comprising the camellia extract of the present invention may be administered alone, but is generally selected in consideration of the mode of administration and standard phamaceutical practice May be administered in admixture with a pharmaceutical carrier.

For example, a composition containing the camellia extract of the present invention may be in the form of tablets containing starch or lactose, in the form of capsules containing the active ingredient alone or as an excipient, or in the form of capsules containing flavoring or coloring chemicals Orally, sublingually or sublingually in the form of tablets, dragees, troches, Such liquid preparations may contain suspending agents, for example, a mixture of semisynthetic glycerides such as methylcellulose, withexol, or apricot kernel oil with a PEG-6 ester, or a mixture of PEG-8 and caprylic / capric glyceride ≪ / RTI > such as a mixture of glycerides, such as a mixture.

The dose of the composition containing the camellia extract of the present invention may be varied depending on the age, weight, sex, dosage form, health condition and disease level of the patient. It may be administered in divided doses.

For example, a daily dosage of 100 to 1000 mg / kg, 100 to 900 mg / kg, 100 to 800 mg / kg, 100 to 700 mg / kg, 100 to 600 mg / kg, 100 to 500 mg / kg, 150 to 1000 mg / kg, 150 to 900 mg / kg, 150 to 800 mg / kg, 150 to 700 mg / kg, 150 to 600 mg / kg, 150 to 500 mg / kg, 150 to 400 mg / kg or 150 to 350 mg / kg, but is not limited thereto. The above-mentioned dosage is exemplified as an average case, and the dose may be increased or decreased depending on individual differences.

Another aspect of the present invention relates to a food composition for alleviating or preventing pain comprising a camellia extract.

The food composition can be variously used for foods for relieving or preventing pain.

Examples of the food to which the extract of the present invention can be added include various foods such as beverage, gum, tea, vitamin complex, leavening tea and health food, and health food such as powder, granule, tablet and capsule Can be used.

The content of the camellia extract contained in the food composition is not particularly limited depending on the form of the food, the intended use, etc., and may be, for example, 0.01 to 15% by weight of the total food, May be added at a ratio of 0.02 to 10 g, preferably 0.3 to 1 g based on 100 mL.

The food composition of the present invention contains the above-mentioned Camellia sinensis extract as an essential ingredient in the indicated ratio, and there is no particular limitation on the liquid ingredient. The food composition may contain various flavors or natural carbohydrates, have.

Examples of such natural carbohydrates include monosaccharides such as disaccharides such as glucose and fructose such as maltose, sucrose and the like and polysaccharides such as dextrin, cyclodextrin and the like And sugar alcohols such as xylitol, sorbitol and erythritol. Natural flavors (tau martin, stevia extracts (e.g., rebaudioside A, glycyrrhizin, etc.) and synthetic flavors (saccharin, aspartame, etc.) can be advantageously used as flavors other than those described above .

In addition to the above, the food composition of the present invention can be used as a flavoring agent such as a variety of nutrients, vitamins, minerals (electrolytes), synthetic flavors and natural flavors, coloring agents and thickening agents (cheese, chocolate etc.), pectic acid and its salts, A salt thereof, an organic acid, a protective colloid thickener, a pH adjusting agent, a stabilizer, a preservative, a glycerin, an alcohol, a carbonating agent used in a carbonated drink, and the like. In addition, the compositions of the present invention may contain flesh for the production of natural fruit juices and fruit juice drinks and vegetable drinks. These components may be used independently or in combination. The ratio of such additives is not critical, but is generally selected in the range of 0 to about 20 parts by weight per 100 parts by weight of the composition of the present invention.

For example, when the food composition of the present invention is prepared as a drink, it may further contain citric acid, liquid fructose, sugar, glucose, acetic acid, malic acid, juice, mulberry extract, jujube extract, licorice extract, Can be included.

Another aspect of the present invention relates to a method for alleviating, preventing or treating pain comprising administering to a subject in need thereof an effective amount of alleviating, preventing or treating pain of a camellia extract.

Since the method of the present invention utilizes the camellia extract, the common content between the two is omitted in order to avoid the excessive complexity of the present specification.

The present invention provides a composition for alleviating, preventing or treating pain comprising a camellia extract. The composition of the present invention not only alleviates and treats the pain generated, but also has an effect of preventing pain when administered to a subject before pain occurs. The composition of the present invention exhibits remarkable pain relieving effect not only by intravenous injection or skin application but also by oral administration, and the composition of the present invention having such characteristics can be applied to foods.

FIG. 1 shows the results of a von Frey filament test for the pain sensitivity of the surgical site at 5 and 24 hours after oral administration of Camellia sinica leaf extract to an experimental animal.

Control group (n = 10) vs. Camellia sinensis leaf extract 300 mg / kg (n = 8), ** p < 0.01

Fig. 2 shows the result of measuring the number of times of generating ultrasonic waves of the 22-27 kHz lunar power band after oral administration of the camellia leaf extract to the experimental animal and then performing the foot incision surgery at 6 and 24 hours.

Control group (n = 10) vs. Camellia leaf extract 300 mg / kg (n = 8), * p < 0.05, ** p < 0.01

FIG. 3 shows the results of evaluation of pain sensitivity of the surgical site at 3 days, 6 days, 9 days, 12 days, and 15 days after the daily oral administration of Camellia sinensis leaf extract, .

Control group (n = 10) vs. (N = 8), * p < 0.05, ** p < 0.01, *** p < 0.001

FIG. 4 is a graph showing that the pain sensitivity of the surgical site was measured at 4 hours, 6 hours, 24 hours, 48 hours, and 72 hours after the daily oral administration of Camellia sinensis leaf extract by inducing inflammatory pain by administering CFA to the experimental animals. As shown in Fig.

Control group (n = 5) vs. Camellia sinensis leaf extract 300 mg / kg (n = 5), * p < 0.05

FIG. 5 shows the results obtained by administering vincristine to an experimental animal to induce peripheral neuropathic pain and administering a daily oral administration of the extract of Camelliaceae to the mice at 6 days, 7 days, 8 days, 11 days, 14 days, and 17 days The pain sensitivity at the postoperative site was measured by mechanical allodynia evaluation.

Control group (n = 5) vs. Camellia sinensis leaf extract 300 mg / kg (n = 5), * p < 0.05, ** p < 0.01

FIG. 6 shows the results of oral administration of the extract of Camellia yambenius L. on a daily basis from day 7 to day 13 after induction of peripheral neuropathic pain by administering paclitaxel to the experimental animals. Then, on days 6, 7, 8, 11 Day and 13 days after surgery, using mechanical allodynia evaluation.

Control group (n = 5) vs. Camellia sinensis leaf extract 300 mg / kg (n = 5), * p < 0.05, ** p < 0.01

FIG. 7 shows the results of measurement of mechanical sensitivity of the surgical site after 0 day and 12 days after the daily oral administration of Camellia sinensis leaf extract and diabetic induction neuropathic pain by administering STZ to experimental animals, to be.

Control group (n = 5) vs. Camelliaceae leaf extract 200 mg / kg (n = 5), * p < 0.05

Fig. 8 shows the result of measuring the intracellular calcium concentration after treating CCR2 expressing cell line with Camellia sinensis leaf extract.

Control vs. Camellia sinensis leaf extract 10 μg / ml, ** p <0.01

Hereinafter, the present invention will be described in more detail with reference to Examples. It is to be understood by those skilled in the art that these embodiments are only for describing the present invention in more detail and that the scope of the present invention is not limited by these embodiments in accordance with the gist of the present invention .

Example

Experimental Method

1. Camellia leaf extract manufacturing

The sample of Camellia sinensis leaf was dried by hot air, and 70 vol% of grain alcohol was added 10 times (v / v) per 100 g of raw material. After extraction at 80 ℃ for 4 hours, the filtrate and residue extract were combined and filtered and concentrated under reduced pressure. All the extracts were filtered and concentrated under reduced pressure, lyophilized and powdered for use in the experiment.

2. Experimental animals

Sprague-Dawley (SD) rats (200-250 g, male) and C57Bl / 6 mice (21-26 g, male) were purchased from Samtako Co., Respectively. The animals were fed under the conditions of temperature 22 ± 1 ℃, humidity 55 ± 5%, day and night cycle (12 hours day / 12 hours night), illumination 300 lux, feed and water free. All animals were managed by the KFRI-IACUC (National Food Research Institute, Laboratory Animal Care and Use Committee) guidelines.

3. Sample Preparation and Administration

Body weights were measured for the experimental animals, and the experimental group was randomly selected.

The 70% ethanol extract of Camellia sinensis was prepared by dissolving and suspending the extract in the second distilled water at an appropriate concentration. The prepared samples were orally administered at a dose of 300 mg / kg / 5 ml for rats and orally at a dose of 200 mg / kg / 10 ml for mice. The control group was orally administered the same amount of secondary distilled water.

4. Pain modeling

4-1. Skin incision model

A rat skin incision model was proposed by Brennan [Brennan et al. Pain 64: 493-501 (1996). Generally, postoperative pain is thought to be a form of acute pain, and the incision model of the rat is thought to be similar to the postoperative pain state in humans.

In order to produce a rat skin incision model, SD male rats (200-250 g) were anesthetized first with an isoflurane solution inhalation anesthesia equipment. After disinfection of the left footpad with 10% povidone solution, the skin and fascia 1 cm long in the longitudinal direction, starting from a point 0.5 cm away from the heel tip, were incised with the 11th surgical knife. The plantar muscle (plantar muscle) of the incision was lifted with a forceps to separate a length of 1 cm. At both ends of the vertical direction, the foot muscles were lifted carefully to avoid falling, and the bleeding was performed by gently pressing the incision. The fascia and skin of the bleeding incision site were closed with nylon 4-0 suture and disinfected with 10% povidone solution. After surgery, antibiotic ointment was applied to prevent infection and transferred to a cage for restoration.

Animals that developed infections during the observation period, or sutures and edema were excluded from the study.

4-2. Spinal nerve injury (SNI)

The nerve branch ligation impaired animal model was proposed by Decosterd and Woolf [Decosterd & Woolf Pain 87: 149-158 (2000)].

In order to construct an animal model of damage to the nerve branch ligation, SD male rats (200-250 g) were first anesthetized using isoflurane aspiration anesthesia equipment. Under general anesthesia, the lateral skin of the left thigh of the rat was shaved and incised. After removing the biceps femoris, the three distal branches of the sciatic nerve were exposed. The sciatic nerve consists of three nerves, the sural nerve, the common peroneal nerve, and the tibial nerve. The double sympathetic nerve should remain intact without damage, branching to the right when viewed from the left leg reference, and the smallest nerve. The total peroneal nerve and tibial nerve branches along the muscles in the direction of the leg. They were separated and ligated firmly using nylon 4-0 suture, and then cut by 2-4 mm length using forceps and scissors . After finishing the nerve cutting, the muscle layer was sutured with a suture, and the skin incision site was closed again, and the experimental animals were transferred to a breeding cage to recover.

4-3. Complete Freund's Adjuvant (CFA) Induced Inflammatory Pain Model

CFA is an antigen solution which is inactivated by heat treatment and then emulsified in dried oil / saline (1: 1) at a concentration of 0.5 mg / kg. The CFA-induced inflammatory pain model cited Sotocinal's method [D. De Rantere et al. Eur J Pain 20 (3): 417-426 (2016)].

CFA treatment was performed after general anesthesia using an inhalation anesthesia device with isoflurane solution. CFA (Sigma-Aldrich Korea) solution was diluted 50% with physiological saline and subcutaneously injected 150 μl under the skin 6 mm below the left footpad of the rat. The injected CFA solution was pressed to prevent counting, and when awakened from anesthesia, it was transferred to a breeding cage to recover.

4-4. Chemotherapy-induced peripheral neuropathy (CIPN)

The CIPN induction method using the anticancer drug vincristine, paclitaxel is described by Authier et al. NeuroReport 10: 965-968 (1999)] and Ghirardi [Ghirardi O et al. In Vivo 19 (3): 631-637 (2005)].

Cyclophosphamide was induced by intraperitoneal injection of vincristine 150 μg / kg, paclitaxel 2 mg / kg once daily for 7 days, and von Frey test was used.

4-5. Diabetic induction neuropathic pain model

The production of an animal model of diabetes induced by streptozotocin (STZ) proceeded according to Moura's experimental method [Moura LI. et al. Biochim Biophys Acta. 1842 (1): 32-43 (2014)].

The C57B1 / 6 mice fasted for 4 hours were allowed to induce STZ sodium citrate solution to 7.5 mg / ml, followed by intraperitoneal administration for 5 consecutive days at a dose of 50 mg / kg. Diabetic animal models were identified by measuring fasting blood glucose after fasting for 12 hours. The average fasting blood glucose level of the animal model of diabetes mellitus was 450 mg / dl, which was significantly higher than the mean fasting blood glucose of 95 mg / dl of the mouse without diabetes induction.

4-6. Calcium imaging

HEK cells expressing CCR2 (chemokine receptor type 2) were seeded in a black 96 well plate at a concentration of 1 × 10 ⁴ cells / mL and cultured overnight. Cells were washed once with HEPES (4- (2-Hydroxyethyl) piperazine-1-ethanesulfonic acid) buffer and treated with Fura-2 am 5 μM for 30 min. After washing three times with HEPES buffer, Camellia sinica leaf extract was treated at a concentration of 10 μg / ml and then cultured for 30 minutes. The 96-well plate was transferred to a fluorescence microscope and treated with MCP-1 (monocyte chemoattractant protein 1), a ligand for CCR2 at 15 seconds, and the intracellular calcium concentration was measured for 2 minutes 30 seconds. The ratio of the change when the MCP-1 was treated to the control group containing the HEPES buffer was set to 1.

5. Evaluation Method

5-1. Von frey filament test

A method for measuring the degree of mechanical allodynia after animal pain modeling has been described by Chaplan et al. [Chaplan et al. J Neurosci Methods 53: 55-63 (1994)].

The rats were placed in an acrylic box mounted on a wire mesh test bench with a mesh size of 2 x 2 mm and adapted for at least 15 minutes. When the movement of the mice was quiet, the pain threshold value (g) was evaluated by using a continuous fleece filament (Stoelting, USA). The filament is contacted vertically on the left footpad and maintained for 5-6 seconds, and the rat is regarded as showing a positive avoidance reaction or showing a positive response if it immediately flaps or licks the foot as the hairs are released. When stimulated from the main prefilament in the central part, it was stimulated with a weak filament if it showed a positive reaction, and stimulated with a strong filament if there was no positive reaction. The minimum stimulation size for positive reaction was the threshold value, and the upper limit was not applied when there was no reaction even at 15 g or more. CFA (Complete Freund`s Adjuvant) Induced Inflammatory Pain Model and Chemotherapy In the induced peripheral neuropathic pain model, the pressure was applied to the central part of the hindpaw of the experimental animal.

5-2. Ultrasound vocalization calls

Rats produce ultrasound when they are in pain, pain, atrophy, and stress, and their ultrasound range is reported to be 22-27 KHz (Portfors CV J Am Assoc Lab Anim. Sci. 46 (1): 28-34 )).

To assess the degree of pain at 6 hours and 24 hours postoperatively, the rats were exposed to ultrasound (22-27) using a USV measurement system (Sonotrack®, ver 1.5.0, Metris, Netherlands) KHz) were measured. The rats were placed in an acrylic box capable of measuring ultrasound, stabilized for 15 minutes, and ultrasonic measurement was conducted for 10 minutes.

Experiment result

1. Pain relief effect of Camellia sinica leaf extract on pain model after surgery

30 minutes before skin incision, 300 mg / kg of Camellia sinensis leaf extract was orally administered and mechanical allodynia was evaluated 5 hours after the skin incision operation to measure pain sensitivity at the surgical site.

As shown in FIG. 1, the pain threshold (g) of the control group was 0.700 ± 0.068 (g), while that of the camellia leaf extract 300 mg / kg was 2.175 ± 0.425 (g) Was significantly (p <0.01) increased. In addition, the results of postoperative Day 1 (POD1) pain threshold were 0.680 ± 0.090 (g) in the control group and 2.825 ± 0.688 (g) in the group treated with 300 mg / kg of camphor leaf extract Was significantly (p <0.01) increased.

2. Determination of pain relieving effect of Camellia sinica leaf extract by ultrasonic sound measurement

30 minutes before the skin incision surgery, the ultrasound lunar eclampsia (22-24 weeks) induced when the animals were given pain 300 mg / kg orally, and 6 hours and 24 hours (POD1) 27 kHz) was measured, and the number of times was quantified to confirm the pain relieving effect of Camellia sinica leaf extract.

As can be seen in FIG. 2, the control group was 18.8 ± 4.273 (calls) at 6 hours after surgery, whereas the control group was recorded at 5.125 ± 1.231 (calls) at 300 mg / kg of camphor tree leaf extract (P <0.01), respectively. After 24 hours (POD1), the control group showed 20.1 ± 5.786 (calls), whereas the group treated with 300 mg / kg of camellia japonica leaf extract had a significant (p <0.05) It was confirmed that ultrasonic measurement records were reduced.

3. Evaluation of pain relief effect of camellia leaves through evaluation of mechanical allodynia

Nerve branching ligation injury The daily doses of Camellia sinica leaf extract 300 mg / kg were orally administered for 15 days after surgery and mechanical allodynia was evaluated to determine pain sensitivity.

As can be seen in FIG. 3, the pain threshold (g) of the control group was 0.975 ± 0.179 (g), 0.49 ± 0.14 (g), 0.415 (G), 0.13 ± 0.436 (g), 1.743 ± 0.121 (g), and 1.629 ± 0.182 (g) in the group treated with Camellia sinica leaf extract at the concentration of 300 mg / kg g), 2.0 ± 0.338 (g), and 1.371 ± 0.211 (g), respectively. The results showed that Camellia sinensis leaf extract exhibited pain relief in long - term pain model as well as short - term pain model.

Nerve branch ligation injury To assess the baseline pain between the two groups, mechanical allodynia evaluation was performed. As a result, it was confirmed that the preoperative pain threshold value (g) was the same between the two experimental groups.

4. Identification of analgesic effects of camellia leaves in inflammatory pain animal models

CFA-induced inflammatory pain model, 300 mg / kg of Camellia sinica leaf extract was orally administered daily for 3 days. After 4, 6, 24, 48, and 72 hours of induction, .

As can be seen in FIG. 4, the pain threshold value (g) of the control group was 7.2 ± 1.02 (g), 5.2 ± 1.2 (g), 3.6 ± 1.13 (g), 4.52 ± 1.382 (g), 6.8 ± 0.8 (g), 8.4 ± 0.748 (g) and 12.2 ± 1.715 (g) in the group treated with 300 mg / kg of Camellia sinensis leaf extract (P <0.05), and the intensity of the sensation of pain increased significantly (p <0.05). As a result, the pain relief effect of Camellia sinica leaf extract was confirmed in the inflammatory pain model. CFA-induced inflammatory pain model A mechanical allodynia evaluation was performed to determine baseline pain between the two groups. As a result, it was confirmed that the preoperative pain threshold value (g) was the same between the two experimental groups.

5. Confirmation of pain relief effect of camellia leaves in an animal model of chemotherapy-induced pain

The chemotherapy induced peripheral neuropathic pain (CIPN) model induced by administration of the anticancer agent vincristine 150 μg / kg was orally administered at a dose of 300 mg / kg of Camellia sinensis leaf extract daily for 17 days. The von Frey filament test on the 7th day of induction The pain was confirmed. The von Frey filament test was used to observe the pain relief effect until the 17th day.

As shown in FIG. 5, the pain threshold value (g) of the control group was 4.5 ± 1.258 (g) at the last day of vincristine administration and 5.85 ± 1.938 (g) Of the total pain threshold value.

On the 11th day, 14th day and 17th day, the pain threshold (g) of the control group was 3.58 ± 0.943 (g), 4.0 ± 0.816 (g) and 7.5 ± 0.957 (P <0.05), the strength of the pain was increased to 8.0 ± 0.816 (g), 10.75 ± 1.493 (g) and 16.5 ± 3.379 (g) As a result, the pain relief effect of Camellia sinica leaf extract was confirmed in the chemotherapy induced peripheral neuropathic pain (CIPN) model.

Chemotherapy Induced Peripheral Neuropathic Pain (CIPN) Model Mechanical allodynia was evaluated to identify baseline pain between the two groups. As a result, it was confirmed that the preoperative pain threshold value (g) was the same between the two experimental groups.

6. Confirmation of the pain relief effect of camellia leaves in pain model animals treated with anticancer drugs

Chemotherapy Induced Peripheral Neuropathic Pain (CIPN) model induced by administration of 2 mg / kg of paclitaxel, an anticancer agent, was confirmed by von Frey filament test on the 7th day after induction of pain. From day 7 to day 13, 300 mg / kg of Camellia sinensis leaf extract was orally administered daily and von Frey filament test was used to observe the pain relief effect.

As shown in FIG. 6, the pain threshold value (g) of the control group was 3.0 ± 1.0 (g) on the last day of administration of paclitaxel, and 2.5 ± 1.5 (g) It was confirmed that there was no difference in the pain threshold values between the groups.

On the 11th day, on the 13th day, the pain threshold (g) of the control group was 1.7 ± 0.3 (g) and 1.2 ± 0.2 (g) 0.1 (g), the intensity of the pain felt was significantly increased (p <0.05). As a result, the pain relief effect of Camellia sinica leaf extract was confirmed in the chemotherapy induced peripheral neuropathic pain (CIPN) model.

Chemotherapy Induced Peripheral Neuropathic Pain (CIPN) Model A mechanical allodynia evaluation was performed to determine baseline pain between the two groups. As a result, it was confirmed that the preoperative pain threshold value (g) was the same between the two experimental groups.

7. Confirm the pain relief effect of camellia leaves in an animal model inducing diabetes

To determine whether diabetic pain was induced, the mean mechanical withdrawal threshold was measured using a von Frey filament test at 0 days before administration of Camellia sinensis leaf extract.

As shown in FIG. 7, the mean value of the mice not inducing diabetes was 2.25 ± 0.445 (g), and the mean pain threshold value of diabetic mice was 0.67 ± 0.16 (g). In the diabetic model, the pain threshold value was decreased. Camellia sinensis leaf extract was administered once daily for 12 days at a dose of 200 mg / kg. After 12 days, the pain threshold value was 2.26 ± 0.554 (g), which was significantly increased (p <0.05) Respectively. In addition, the mean fasting blood glucose was measured at the end of the experiment. As a result, it was confirmed that the model of diabetes mellitus was maintained at 440 mg / dl.

8. Determination of CCR2 antagonist efficacy of Camellia wood by measuring intracellular calcium concentration

CCL2 (CC motif chemokine ligand 2) and CCR2 receptor are known to be associated with various types of pain. It is known that CCL2 is involved in the mechanical hypersensitivity generated after induction of the skin incision model, which is the pain model used in the present invention [Abbadie, C. , et al. Proceedings of the National Academy of Sciences, 100 (13), 7947-7952 (2003)].

Like the nerve branch ligation injury model, the sciatic nerve injury model that induced pain by nerve injury induced pain. Pain was reduced in mice knocked out of CCR2 differently from wildtype. Also, pain was reduced in CCR2 knockout mice in CFA induced inflammatory pain models [Peters & Eisenach, Anesthesiology: The Journal of the American Society of Anesthesiologists, 112 (5), 1250-1258 (2010)].

Chemotherapy Induced peripheral neuropathic pain models have also been shown to relieve pain when inhibiting CCL2 / CCR2 signaling [Zhang, H. , et al. The Journal of Pain, 14 (10), 1031-1044. (2013)].

Through these documents, it was confirmed that the CCR2 antagonist has an effect on various kinds of pain relief, and in the present invention, it was confirmed that the camellia extract was effective as a CCR2 antagonist to alleviate the pain.

As can be seen from FIG. 8, when the CCR2 antagonist INCB3344 was pretreated, it was confirmed that the calcium concentration was completely reduced compared to the control. In addition, it was confirmed that the calcium concentration in the pretreated cells of Camellia sinensis leaf extract 10 μg / ml was significantly lower than that of the control. It was confirmed that Camellia sinica leaf extract had a CCR2 antagonistic effect.

conclusion

The Camellia japonica L. leaf extract of the present invention can be used as a pain model animal model, a nerve branch ligation injury model, a CFA induced inflammatory pain model, a chemotherapy induced peripheral neuropathic pain model, a diabetic induction neuropathic pain model , And also confirmed the effect of decreasing calcium concentration, which is a function of CCR2 antagonist, in intracellular experiments. Accordingly, the composition of the present invention containing the extract of Camellia sinensis as an active ingredient exhibits the effect of preventing, alleviating or treating pain.

Claims (12)

1. A pharmaceutical composition for alleviating, preventing or treating pain comprising camellia japonica L. extract.
2. The pharmaceutical composition according to claim 1, wherein said camellias are camellia leaves.
3. The pharmaceutical composition according to claim 1, wherein the camellia extract is an extract of at least one solvent selected from the group consisting of water, methanol and ethanol.
4. The method of claim 1, wherein the pain is selected from the group consisting of somatic pain, visceral pain, inflammatory pain, dysfunctional pain, idiopathic pain, neuropathic pain, superficial pain, deep pain, itching, migraine and cancer pain. &lt; Desc / Clms Page number 17 &gt;
5. The pharmaceutical composition according to claim 1, wherein the camellia extract increases the threshold value of the force to feel pain.
6. A food composition for alleviating or preventing pain comprising camellia japonica L. extract.
7. 7. The food composition of claim 6, wherein the camellias are camellia leaves.
8. A method for alleviating, preventing or treating pain, comprising administering to a subject in need thereof an alleviating, preventive or therapeutic effective amount of pain of a camellia extract.
9. 9. The method according to claim 8, wherein said camellias are camellia leaves.
10. 9. The method according to claim 8, wherein the camellia extract is an extract of at least one solvent selected from the group consisting of water, methanol and ethanol.
11. 10. The method of claim 8, wherein the pain is selected from the group consisting of somatic pain, visceral pain, inflammatory pain, dysfunctional pain, idiopathic pain, neuropathic pain, superficial pain, deep pain, itching, migraine, and cancer pain.
12. 9. The method of claim 8, wherein the camellia extract increases the threshold value of the force of pain.

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