KR101584279B1 - Compositions for Alleviating, Preventing or Treating Pain Comprising Ecklonia cava Extracts as Active Ingredients - Google Patents

Abstract

The present invention provides a composition for alleviating, preventing or treating pain comprising a gangue extract as an active ingredient. 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 is highly applicable to foods.

Description

TECHNICAL FIELD The present invention relates to a composition for alleviating, preventing or treating pain comprising an extract of Ganoderma lucidum as an active ingredient. [0002] Compositions for Alleviating, Preventing or Treating Pain Comprising Ecklonia cava Extracts as Active Ingredients [

The present invention relates to a composition for alleviating, preventing or treating pain comprising a gangrene extract as an active ingredient.

Ecklonia cava is a seaweed of the perennial brown algae of the Laminariales seaweed (Alariaceae). It grows in the deep sea and is 1 to 2 meters long. The stem is cylindrical and the base is roots. It is an important algae plant that constitutes seaweed, and it mainly feeds abalone and turtle. It is the main raw material for making alginic acid, iodine, and potassium, and it is edible. It is distributed in Korea (south coast and Jeju island) and Japan (Korea Science and Technology Society, 2012).

In recent years, research on the components of brown algae including mites has been actively conducted, and studies on anticancer, antibacterial, antiviral, antibacterial, antioxidant and anti-inflammatory effects have been actively conducted domestically (Smit AJ 2004; Blunt JW et al Et al., 2007; Mayer AM et al., 2007; Mayer AM et al., 2009; Shin HC et al., 2006; Choi JS et al., 2011).

Undaria pinnatifida is brown algae with seaweed in the sea. It has been inhabited mainly in temperate coastal areas of relatively low temperatures in Korea, Japan, and China. However, during the recent decades in the South Pacific, during the North Atlantic, it has been found in the Mediterranean and Australian southern regions and is rapidly penetrating. Seaweed contains 87.6% of water, 3.0% of protein and 0.3% of fat per 100g of edible portion. The amino acid composition of the protein is high, the content of essential amino acid, and the calories of food is 35 kcal. The content of inorganic trace elements is high in calcium and phosphorus. It is used as main food of abalone style with kelp, and it is mainly used for food in Korea, Japan, and China. It is rich in dietary fiber, potassium, calcium, and iodine, making it metabolically active, and has been used early on for postpartum cooking, constipation and prevention of obesity, iron and calcium supplementation. Seaweed in one room is called seaweed, 甘 藿, itself (紫 菜), sea band.

Studies on the components of brown algae including seaweed have been actively conducted and researches on anti-cancer, antibacterial, antiviral, antibacterial, antioxidant, anti-inflammatory and anti-edema effects have been actively conducted domestically (Smit AJ. 2004; Blunt JW Mayer et al., 2009; Choi, JS et al., 2011; Khan et al., 2009).

Numerous papers and patent documents are referenced and cited throughout this specification. The disclosures of the cited papers and patent documents are incorporated herein by reference in their entirety to better understand the state of the art to which the present invention pertains and the content of the present invention.

The inventors of the present invention have sought to develop a safe material, particularly a plant-derived material, capable of effectively relieving pain, and as a result, it has been found out that the extracts of Ganoderma lucidum are very effective for alleviating, preventing or treating pain, .

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

Another object of the present invention is to provide a food composition for alleviating, preventing or improving pain.

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

According to one aspect of the present invention, there is provided a composition for alleviating, preventing or treating pain comprising Ecklonia cava extract as an active ingredient.

The inventors of the present invention have sought to develop a safe material, particularly a plant-derived material, capable of effectively relieving the pain, and as a result, it has been found that the extract of Ganoderma lucidum is very effective for alleviating, preventing or treating pain.

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

As demonstrated in the following examples, when the compositions of the present invention were administered to an animal prior to skin incision surgery, pain sensitivity at the surgical site was reduced. In addition, the phytotoxic extract of the present invention showed remarkable pain relieving effect as compared with the seaweed of the same seaweed and plant.

Ecklonia cava is a seaweed species of the seaweeds of the seaweeds. It is a seaweed species native to the depths of 10 m from the coast of Jeju. It contains nutrients such as alginic acid, iodine and potassium. Since it contains a component called phlorotannin, it has been known that it exhibits anti-cancer, anti-cancer, anti-aging and anti-hypertensive effects.

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.

Various extracting solvents can be used when the phytotoxic extract used in the composition of the present invention is obtained by treating the extractant with an extractant. According to the present invention, a polar solvent or a non-polar solvent can be used. Suitable polar solvents are (i) water, (ii) alcohols (preferably methanol, ethanol, propanol, butanol, n-propanol, iso-propanol, n-butanol, 1-pentanol, Or ethylene glycol), (iii) acetic acid, (iv) dimethyl-formamide (DMFO) and (v) dimethyl sulfoxide (DMSO). Suitable nonpolar solvents are acetone, acetonitrile, ethyl acetate, methyl acetate, fluoroalkane, pentane, hexane, 2,2,4-trimethylpentane, decane, cyclohexane, cyclopentane, diisobutylene, 1- But are not limited to, pentane, 1-chlorobutane, 1-chloropentane, o -xylene, diisopropyl ether, 2- chloropropane, toluene, 1- chloropropane, chlorobenzene, benzene, diethyl ether, diethylsulfide, Methane, 1,2-dichloroethane, aniline, diethylamine, ether, carbon tetrachloride, and THF.

According to an embodiment of the present invention, the extraction solvent used in the present invention may be selected from the group consisting of (a) water, (b) an anhydrous or hydrated lower alcohol having 1 to 4 carbon atoms (methanol, ethanol, propanol, butanol, (E) ethyl acetate, (f) chloroform, (g) butyl acetate, (h) 1,3-butylene glycol, (i) hexane, and (j) Diethyl ether. In another embodiment of the present invention, the extract of the present invention is obtained by treatment with water, ethanol or a combination thereof. According to a particular embodiment of the present invention, the extract of the present invention is obtained by treating 60 to 80 vol% of grain alcohol with menthol.

The phlegm extract used in the present invention can be prepared in powder form by an additional process such as vacuum distillation and freeze drying or spray drying.

As used herein, the term " extract " means that it is used in the art as a crude extract as described above, but broadly includes fractions obtained by further fractionating the extract. That is, the phlegm extract includes not only the extract obtained by using the above-mentioned extraction solvent, but also those obtained 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 by the purification method is also included in the phlegm extract of the present invention.

As used herein, the term " comprising as an active ingredient " is meant to include an amount sufficient to achieve efficacy or activity of the following gut extract. Since the composition of the present invention contains the extract obtained from the natural plant material, the extract has no adverse effect on the human body even when it is administered in an excessive amount. Therefore, the quantitative upper limit of the extract of the phytotoxic extract contained in the composition of the present invention is selected by a person skilled in the art .

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 " is used to include all pain caused by noxious stimuli that may or may damage nervous tissues and may include ben, cut, bruise, fracture, bone pain, bone fracture, crush injury, burn, and similar wounds. Pain receptors for tissue damage (nociceptors) are mostly located in the skin, musculoskeletal or internal organs.

The term " somatic pain " is used to indicate pain that occurs 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 " includes pain associated with active inflammation, which may be caused by trauma, surgery, infection, and autoimmune diseases.

The term " superficial pain " refers to the pain sensed by the skin segments of the nerves of the dorsal root, and is a direct pain to feel the pain at the point of stimulation.

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, pericardium, 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 " is used herein to denote pain resulting from sensory input of abnormal processing by the peripheral or central nervous system resulting from disturbance of the peripheral or central nervous system.

The term " procedural pain " refers to pain that occurs in internal medicine, dentistry, or surgery, and 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.

According to one embodiment of the present invention, the composition of the present invention exhibits mitigation, prophylactic or therapeutic effects on neuropathic pain.

Neuropathic, inflammatory, and nociceptive pain are different in etiology, pathophysiology, diagnosis and treatment. 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.

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.

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 ) And may last for months or years after apparent healing of any damaged tissue.

The composition of the present invention may be provided as a pharmaceutical composition.

When the composition of the present invention is manufactured from a pharmaceutical composition, it includes pharmacologically acceptable carriers as well as a phlegm extract as an active ingredient. The pharmaceutically acceptable carriers to be contained in the pharmaceutical composition of the present invention are those conventionally used in the present invention and include lactose, dextrose, sucrose, sorbitol, mannitol, starch, acacia rubber, calcium phosphate, alginate, gelatin, But are not limited to, calcium silicate, microcrystalline cellulose, polyvinylpyrrolidone, cellulose, water, syrups, methylcellulose, methylhydroxybenzoate, propylhydroxybenzoate, talc, magnesium stearate and mineral oil. It is not. The pharmaceutical composition of the present invention may further contain a lubricant, a wetting agent, a sweetening agent, a flavoring agent, an emulsifying agent, a suspending agent, a preservative, etc. in addition to the above components. Suitable pharmaceutically acceptable carriers and formulations are described in detail in Remington ' s Pharmaceutical Sciences (19th ed., 1995).

The pharmaceutical composition of the present invention can be administered orally or parenterally. In the case of parenteral administration, the composition can be administered by intravenous injection, subcutaneous injection, muscle injection, intraperitoneal injection, transdermal administration, or the like.

The appropriate dosage of the pharmaceutical composition of the present invention varies depending on factors such as the formulation method, administration method, age, body weight, sex, pathological condition, food, administration time, administration route, excretion rate and responsiveness of the patient, Usually, a skilled physician can readily determine and prescribe dosages effective for the desired treatment or prophylaxis. According to a preferred embodiment of the present invention, the daily dosage of the pharmaceutical composition of the present invention is 0.001-100 mg / kg.

The pharmaceutical composition of the present invention may be formulated into a unit dose form by formulating it using a pharmaceutically acceptable carrier and / or excipient according to a method which can be easily carried out by a person having ordinary skill in the art to which the present invention belongs. Or by intrusion into a multi-dose container. The formulations may be in the form of solutions, suspensions or emulsions in oils or aqueous media, or in the form of excipients, powders, granules, tablets or capsules, and may additionally contain dispersing or stabilizing agents.

According to another aspect of the present invention, there is provided a food composition for alleviating or ameliorating pain comprising a gangue extract as an active ingredient.

According to one embodiment of the invention, the pain relieved or prevented by the food composition of the present invention is selected from the group consisting of somatic pain, visceral pain, inflammatory pain, dysfunctional pain, idiopathic pain, Pain, superficial pain, deep pain, itching, migraine or cancer pain.

According to another embodiment of the present invention, the pain relieved or improved by the food composition of the present invention is neuropathic pain.

When the composition of the present invention is prepared with a food composition, it contains not only the gentle extract as an active ingredient, but also components that are ordinarily added in the manufacture of food, for example, proteins, carbohydrates, fats, nutrients, . Examples of the above-mentioned carbohydrates are monosaccharides such as glucose, fructose, and the like; Disaccharides such as maltose, sucrose, oligosaccharides 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 extract (e.g., rebaudioside A, glycyrrhizin, etc.) and synthetic flavors (saccharin, aspartame, etc.) can be used as flavorings.

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

The features and advantages of the present invention are summarized as follows:

(a) The present invention provides a composition for alleviating, preventing or treating pain comprising a gangrene extract as an active ingredient.

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

(c) 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 is highly applicable to food.

FIG. 1 shows the results of a von Frey filament test for the pain sensitivity of the surgical site 5 hours after performing the plantar incision surgery and then orally administering the Ganoderma sp.
Control group (n = 8) vs. (N = 8), * p < 0.05, ** p < 0.01
FIG. 2 shows the result of measuring the pain sensitivity of the surgical site after 24 hours after performing the plantar incision surgery, and the mechanical allodynia evaluation.
Control group (n = 8) vs. Mite, seaweed 300 mg / kg (n = 8), * p <0.05, *** p <0.001
FIG. 3 shows the results of measurement of the number of times of generating ultrasonic waves of the 22-27 kHz lunar band after 6 hours from performing the plantar incision operation after orally administering the extracts of Ganoderma lucidum or Ganoderma lucidum to the experimental animals.
Control group (n = 8) vs. (N = 8), * p < 0.05, ** p < 0.01
FIG. 4 shows the results of measurement of the number of times the ultrasonic waves of the 22-27 kHz lunar band were generated 24 hours after performing the plantar incision operation after orally administering the extracts of Ganoderma lucidum or the sea mustard to the experimental animals.
Control group (n = 8) vs. (N = 8), * p < 0.05, ** p < 0.01
FIG. 5 is a result of performing a hot plate test after 24 hours after performing a plantar incision operation after orally administering an extract of Ganoderma Lucidum or Ganoderma lucidum to an experimental animal.
Control group (n = 8) vs. Seaweed, Seaweed 300 mg / kg (n = 8), * p < 0.05
FIG. 6 is a result of cold plate test after 24 hours after performing plantar incision surgery after orally administering ganoderma lucidum or sea mustard extract to an experimental animal.
Control group (n = 8) vs. (N = 8), * p < 0.05, ** p < 0.01
FIG. 7 shows the results of measurement of pain sensitivity at the surgical site from 3 days to 15 days after the operation of spinal nerve injury (SNI) through mechanical allodynia evaluation.
Control group (n = 8) vs. (N = 8), * p < 0.05, ** p < 0.01
FIG. 8 shows the results of analysis of IL-1β expression in DRG tissue 24 hours after SNI surgery.
Control group (n = 6) vs. Seaweed, Seaweed 300 mg / kg (n = 6), * p < 0.05
FIG. 9 shows the results of analysis of IL-2 expression in DRG tissue 24 hours after SNI surgery.
Control group (n = 6) vs. Seaweed, Seaweed 300 mg / kg (n = 6), * p < 0.05
FIG. 10 shows the results of analysis of IL-6 expression in DRG tissue 24 hours after SNI surgery.
Control group (n = 6) vs. Seaweed, Seaweed 300 mg / kg (n = 6), * p < 0.05
FIG. 11 shows the results of analysis of IL-12 expression in DRG tissue 24 hours after SNI surgery.
Control group (n = 6) vs. Seaweed, Seaweed 300 mg / kg (n = 6), * p < 0.05

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. Seed extraction

The natural extracts were purchased from Jeodo Hadori Fisheries Market and purchased as a raw material. 70 vol% alcohol was added 10 times per 100 g of the raw guttae and the mixture was extracted at 80 ℃ for 4 hours. The filtrate and residue extracts were combined, 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. Seaweed extract

The samples of natural extracts were purchased from seaweeds from Hadori Fisheries Market in Jeju Island and extracted by cutting. After adding 70 vol% alcohol per 100 g of seaweed raw material, it was added 10 times and extracted at 80 ℃ for 4 hours. The filtrate and residue extract were combined, filtered and concentrated under reduced pressure. All the extracts were filtered and concentrated under reduced pressure, lyophilized and powdered for use in the experiment.

3. Experimental animals

Sprague-Dawley (SD) rats (200-250 g, male) were purchased from Samtako Co., Ltd. and used for experiments for one week in an experimental animal breeding box. 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.

4. Sample Preparation and Administration

The body weights were measured and the test group was randomly selected. The individual identification of the experimental animals was carried out using the pigment coloring method. And 70% of seaweed extracts were prepared by dissolving and suspending the extracts in an appropriate concentration in the second distilled water. The samples were orally administered at a dose of 300 mg / kg / 5 ml 30 min before surgery, and the same amount of the second distilled water was orally administered to the control group 30 min before the operation.

5. Pain modeling

Skin incision model

A rat skin incision model was proposed by Brennan (Brennan, 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. SD male rats (180-230 g) were anesthetized with pentobarbital and the left footpad was disinfected with 10% povidone solution and then started at a distance of 0.5 cm from the tip of the heel. The skin and fascia Were incised with a No. 11 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 fever during the observation period, or sutures and edema were excluded from the study.

Spinal nerve injury (SNI)

The nerve branch ligation impaired animal model was proposed by Decosterd and Woolf (Decosterd & Woolf, 2000). SD male rats (180-230 g) were anesthetized using pentobarbital. Under general anesthesia, the lateral skin of the left thigh of the rat was shaved, incised, and the biceps femoris separated and exposed to the three terminal branches of the sciatic nerve. The sciatic nerve consists of three nerves of 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.

6. Evaluation Method

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., 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 rats was quiet, the continuous whitening prefilament (Stoelting, USA) (g) were evaluated. 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. Stimulate with weak filaments when stimulating positive filaments from central filaments, stimulating with weak filaments, and stimulating with strong filaments. The minimum stimulation size for positive reactions should be considered as a threshold, and the upper limit should not be applied when there is no reaction even at 15 g or more.

Ultrasonic vocalization calls

Rats produce ultrasound when they are in pain, distress, atrophy or stress, and their ultrasound range is reported to be 22-27 KHz (Portfors, 2007). To determine the degree of pain at 6 and 24 hours postoperatively, we used ultrasonic waves (22-27 KHz) when we felt pain using USV measurement system (Sonotrack, ver 1.5.0, Metris, ) 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.

Thermal hyperalgesia

The method used by Bennett et al. (1988), designed by Bennett et al. (1988), was applied to measure pain hyperactivity after pain modeling. The chamber bottom temperature of the hot / cold plate (IITC life science, USA) apparatus was adjusted to 51 ° C for the hot plate test and 0 ° C for the cold plate test, then the experimental animals were placed in the chamber and observed for behavior. The time taken to jump in response to temperature or lift the hind leg was measured, and the average value was used for the data in three rounds. An experimental time limit of 30 seconds was set for the hot plate test and 60 seconds for the cold plate test to prevent animal tissue damage.

Cytokine analysis &lt; RTI ID = 0.0 &gt;

IL-1β, IL-2, IL-4, IL-10, IL-12p70, IFN-γ, To determine the changes of TNF-α, DRG tissues (L4-6) were isolated at 1 day and 3 days after SNI surgery, respectively. In addition, blood samples were collected on the 3rd postoperative day. The cytokine assay was performed using the Q-Plex assay of Quansys. After the extraction of DRG tissue, the tissue weight was measured. 300 μl of PBS containing protease inhibitor cocktail (Sigma) was added to the DRG tissue and the supernatant was obtained through sample pretreatment using micropestles Respectively. The supernatant of the tissue was used as the undiluted solution and the plasma sample was diluted 1: 1 with the sample diluent in the kit. IL-1β, IL-2, IL-4, IL-6, IL-10, IL-12p70, IFN-γ and TNF in the Q-Plex ™ rat cytokine- -α) kit was used to confirm the cytokine content of each sample. The Q-Plex ™ kit was run twice by ELISA. The reagents contained in the kit were prepared, and the Q-View ™ Imager and software were set up and then tested. Diluted 8-point standards were prepared for each series, and treated with 50 μl of each standard and sample, At 500 rpm for 3 hours. The plate was then washed three times and then the detection mixture was added to each well in 50 [mu] l increments. The mixture was stirred at room temperature for 1 hour at 500 rpm. The plates were then washed three times and then streptavidin-HRP 1x was added to each well in an amount of 50 μl, followed by stirring at 500 rpm for 15 minutes at room temperature. While repeating the plate washing for 6 times, mix the substrates A and B with care to avoid exposure to light, store at room temperature, add 50 μl of substrate to each well, and then take an image of the plate through Q-View ™ Imager The 4PL and 5PL data were obtained from Q-View ™ software and the results were statistically analyzed using 5PL data. The results were expressed in pg / mg protein.

Experiment result

1. Assessment of pain relief through von Frey filament test of sorghum, seaweed 300 mg / kg

After 5 hours of skin incision, mechanical allodynia was evaluated to determine the pain sensitivity at the surgical site. As a result, the mechanical withdrawal threshold (g) of the control group was 0.529 ± 0.122 (g), compared with 4.629 ± 1.080 (g) in the 300 mg / kg group and 2.629 ± 0.665 ) And the magnitude of the force feeling the pain showed statistical significance and increased (Fig. 1). In addition, the pain threshold value (POD1) of the postoperative day 1 (POD1) was 3.665 ± 0.913 (g) in the control group, 0.486 ± 0.071 ± 0.737 (g), indicating a statistically significant increase in the magnitude of the force (Figure 2).

2. Determination of pain relieving effect by measuring ultrasonic vocalization calls of 300 mg / kg of seaweed and seaweed

The ultrasonic lunar lunar band (22-27 kHz), which is induced when the animals feel pain at the time of 6 hours and 24 hours (POD1) after the skin incision operation, was measured and the number of times was quantified to confirm the pain relieving effect . At 6 hours after surgery, the ultrasound spoken sounds measured 14.500 ± 2.283 (calls) in the control group, 6.429 ± 1.771 (calls) in the spontaneous 300 mg / kg group and 8.571 ± 1.784 (calls) in the seaweed 300 mg / (Fig. 3). At the 24 hour postoperative POD1 ultrasonographic sound measurement, the control group was recorded at 17.375 ± 4.355 (calls), compared with 3.714 ± 1.340 (calls) at the spastic 300 mg / kg group and 8.286 ± 1.899 Indicating statistical significance and increased (Fig. 4).

3. Determination of pain relief effect by thermal hyperalgesia test with 300 mg / kg of seaweed and seaweed

The hot / cold plate test was performed to measure thermal pain hyperactivity in the experimental animals 24 hours after the skin incision operation (POD1). In the hot plate test, the time for the experimental animals to react to pain was measured at 51 ° C. The escape time (s) of the surgical control (sham) was 17.778 ± 0.444 (s) and the control group was 8.175 ± 0.575 (s). (Fig. 5). In the case of the seaweed 300 mg / kg group, 10.108 ± 0.561 (s) and seaweed 300 mg / kg group were 9.997 ± 0.864 (s).

For the cold plate test, the time for the experimental animals to react to pain was measured at 0 ° C. The escape time (s) of the control group was 48.222 ± 2.350 (s) and the control group was 23.834 ± 1.861 (s). (30 mg / kg body weight, 300 mg / kg body weight, 35.776 ± 3.156 s), and seaweed 300 mg / kg body weight (30.998 ± 2.331 s), respectively.

4. Assessment of pain relief through mechanical allodynia evaluation of sputum, seaweed 300 mg / kg (long-term model)

During the 15-day period after SNI surgery, long-term seaweed, 300 mg / kg of seaweed was orally administered, and mechanical allodynia was evaluated at 3-day intervals to measure pain sensitivity. As a result, the mechanical evasion threshold value (g) of POD3 and control group was 4.233 ± 1.244 (g) in the sputum 300 mg / kg group and 2.233 ± 0.578 (g) in the seaweed 300 mg / kg group compared with 0.467 ± 0.042 It was confirmed that the intensity of the pain felt was significantly increased. POD15 was 0.933 ± 0.256 (g) in the 300 mg / kg group and 0.513 ± 0.198 (g) in the seaweed 300 mg / kg group compared with the mechanical avoidance threshold value (g) of the control group of 0.066 ± 0.022 And the pain relieving effect was maintained from POD3 to POD15 (Fig. 7). As a result, the pain relieving effect of the seaweed and seaweed was confirmed not only in the short - term model of the sole incision model but also in the long - term model SNI model. In addition, mechanical allodynia evaluation was performed to confirm that the baseline pain level was the same between pre-SNI groups, and it was confirmed that pre-operative mechanical avoidance threshold (g) values were the same (Fig. 7).

5. Identification of anti-inflammatory effects in SNI model of mackerel and seaweed 300 mg / kg

The expression of IL-1β, one of the inflammatory cytokine factors, was analyzed after isolation of L4, L5 and L6 by DRG at 24 hours (POD1) after SNI surgery. As a result, the expression level of the control group was increased by 154.76% compared to that of the control group, and that of the control group was 61.34% and 42.03% compared with the control group of 300 mg / kg and 300 mg / kg of seaweed, respectively ) (FIG. 8).

The expression of IL-2, one of the inflammatory cytokine factors, was analyzed after isolation of L4, L5, and L6 by DRG at 24 hours (POD1) after SNI surgery. As a result, the expression level of the control group was increased by 75.43% compared to that of the control group. In the 300 mg / kg group and 300 mg / kg of seaweed group, 46.78% and 34.70% ) (FIG. 9).

The expression of IL-6, one of the inflammatory cytokine factors, was analyzed by DRG separation of L4, L5, and L6 at 24 hours (POD1) after SNI surgery. As a result, the expression level of the control group was increased by 66.33% compared to that of the control group. In the 300 mg / kg group and 300 mg / kg of seaweed group, the expression level of the control group was 39.77% and 28.84% ) (FIG. 10).

The expression of IL-12, an inflammatory cytokine, was analyzed by DRG separation of L4, L5 and L6 at 24 hours (POD1) after SNI surgery. As a result, the expression level of the control group was increased by 88.69% as compared with the control group. The expression level of IL-12 increased by SNI surgery was 47.65% and 32.40%, respectively (p <0.05), compared with the IL-12 expression level of control group in 300 mg / kg of seaweed and 300 mg / kg of seaweed (Fig. 11). In addition, the expression of IL-12 was increased in the control group as a result of IL-12 analysis in plasma samples after 3 days of SNI surgery (POD3) at the sacrifice of the experimental animals. Plasma 300 mg / Lt; RTI ID = 0.0 &gt; IL-12 &lt; / RTI &gt;

conclusion

The extract of Ecklonia cava of the present invention confirmed the pain relieving effect both in the pain model animal, the skin incision model and the nerve branch ligation damage model. In particular, the short-term effect as well as the long-term effect could be confirmed. In addition, the anti - inflammatory effect of the moth extract was confirmed in the neuronal branching injury model. The phytotoxic extract of the present invention showed remarkable pain relief as compared with the seaweed extract belonging to the same seaweed.

While the present invention has been particularly shown and described with reference to exemplary embodiments thereof, it is to be understood that the same is by way of illustration and example only and is not to be construed as limiting the scope of the present invention. Accordingly, the actual scope of the present invention will be defined by the appended claims and their equivalents.

references

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Claims (8)

A pharmaceutical composition for alleviating, preventing or treating neuropathic pain or traumatic pain comprising Ecklonia cava extract as an active ingredient.
2. The composition according to claim 1, wherein the ghatti extract is obtained by treating gherkin with water, methanol, ethanol or a combination thereof.
delete delete delete A food composition for alleviating or ameliorating neuropathic pain or traumatic pain comprising a ghatti extract as an active ingredient. delete delete

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