KR101836406B1 - The pharmaceutical compositions for prevention or treatment of inflammatory spine disease containing Scolopendra subspinipes and Peony as an active ingredient - Google Patents

Abstract

The present invention relates to a pharmaceutical composition for the prevention and treatment of inflammatory spinal diseases, comprising a peony and a pit extract as an active ingredient, wherein the peony and pit mixed extracts exhibit a concentration-dependent inhibitory effect on nitric oxide production and cytokine production, And it was confirmed that ear edema was alleviated in a concentration dependent manner by the mixed extract in ear edema-induced mouse model. Further, it was confirmed that ear edema was alleviated by the mixed extract in ear edema-induced mouse model, It was confirmed that the mixed extract of the present invention had the best inhibitory effect on inflammation. As a result, it was confirmed that the pain was alleviated and the disk was improved by administering it as a herb to a patient with herniated disc herniation. The composition is useful for the prevention and treatment of inflammatory spinal diseases It can be used and may be useful to use acupuncture for the prevention and treatment of inflammatory spinal disease.

Description

BACKGROUND OF THE INVENTION 1. Field of the Invention The present invention relates to a pharmaceutical composition for preventing and treating inflammatory spinal diseases,

The present invention relates to a pharmaceutical composition for the prevention and treatment of inflammatory spinal diseases, comprising a peony and a pore as an active ingredient, and a pharmaceutical preparation using the same.

It is known that there are various bone disease patients in Korea and the number of patients is also increasing every year. Bone disease is mainly inflammatory bone disease, and typical herniated disc herniation is classified as bulging disc, protruded disc, extruded disc, and sequestrated disc depending on the type of disc herniation. do. A bulging disc is a fibrous ring that is displaced symmetrically more than 3 mm outward from the normal range of the intervertebral disc. The protruded disc is in a state in which the nucleus pulposus protrudes through the inner fiber gap of the fibrous ring, but the outer fibrous ring is normal. The extruded disc ruptures to the outer fibrous ring so that the nucleus protrudes through the entire layer of the fibrous ring but is connected to the central nucleus. The sequestrated disc is the nucleus of the extruded nucleus, State. It is presumed that the typical symptoms such as lower limb pain, sensory abnormality, weakness of muscle strength, paralysis are caused by inflammation or autoimmune reaction due to chemical stimulation caused by escape of the nucleus pulposus or fibrous ring into the epididymis rather than mechanical compression caused by the herniated disc.

Arthritis, a typical inflammatory bone disease, is a skeletal inflammatory disease caused by aging. It is known that about 20% of the total population in Korea suffer from arthritis. Although there are about 100 kinds of arthritis, osteoarthritis accounts for most of the arthritis.

Osteoarthritis (osteoarthritis) of the joints are worn out of the cartilage of the joint bones to cause the pain caused by the muscles around the joints do not use the power gradually falls out of the disease. Osteoarthritis has been known in the past as a degenerative joint disease and can occur without a joint anomaly or damage, or without joint damage.

Chemotherapy and surgery for the treatment of bone diseases have not yet achieved perfect results. For example, osteoarthritis has been treated with a corticosteroid-type anti-inflammatory substance, such as hydrocortisone and betamethasone, which have a prostaglandin synthesis inhibitory function. However, administration of such a steroid hormone Is a temporary effect, but due to serious side effects, continuous attention should be observed. Also, there is a limitation that intra-articular injection is the principle rather than oral administration.

Therefore, in view of the fact that despite the increasing tendency of inflammatory bone disease, there is no proper treatment plan, it is urgently required to develop new materials and new treatment methods that can be used generally in clinical fields of inflammatory bone diseases .

Peony is a perennial herbaceous plant belonging to Ranunculaceae and is also called Paeonia lactiflora . It is the most widely used medicinal crop after ginseng and Angelica gigas in Korea, and it is widely used as a raw material for medicinal herbs such as Ssanghwang and Sangmul. The roots of peonies are mainly prescribed for therapeutic purposes such as amenorrhea control, trauma treatment, non-hemorrhage, inflammation, boils and wound treatment, pain relief at the chest and rib sites for the purpose of inflammation, pain and swelling come. The physiologically active components of peanut roots were separated from monoterpene glucoside compounds including paeoniflorin and albiflorin and several pheonolic compounds and its chemical structure was reported (Kim Se-jong et al., Isolation and Identification of Physiologically Active Substances in Peony Leaf and Stem, Korean Medicinal Crop Science Society 15 (1), 6-11, 2007).

Ogong is an extermination of Wangzine ( Scolopendra morsitans L.) and Zine ( Scolopendra subspinipes mitilans L) which belong to Wangjingen family. It is also called Chongryong, It is called. It is collected in spring and it removes poisonous head, tail and legs and it is put in boiling water and it is dried in the sun or it is used, and it is used for sake washing or powder, and the mood is god, It has the efficacy of releasing the poison of the snake, which is gentle and bloody. It is applied to symptoms such as seizures, pincers, imprisonment, and headache due to bruising, breeze, etc. due to the efficacy of bruxism. In addition, the detoxification effect was remarkable, and it was used as a remedy for bruxism, lethargy, and vomiting, and was used as a remedy for bruises caused by vipers (Shin Min-gyo, primary color clinical herbal medicine, Yeonglim, p665, 1991).

Cibotium belongs to the Dicksoniaceae family and uses cibotium barometz J Smith in the tropics. Based on the herbal literature, it has been recorded in the private sector that it has been shown to be effective in strengthening muscular strength, and is said to be transmitted to the public. Examples of the components of the gold molybdate include onitin, onitin 4-O- beta-di- onitin 4-O-beta-D-allopyranoside, onitin 4-O-beta-Dglucopyranoside, pterosin R 4-deoxy 4 -chloro-onitin is known, and onitin has been found to have a smooth muscle relaxation effect (Yang, Meei-Shieu, Studies on the Tween fork medicine VI. Studies on onitin, Planta Medica, p25, 1986).

Ostericum Koreanum is a perennial herbaceous perennial herbaceous plant of the dicotyledonous plant, and is distributed in valley valleys of Korea (Gyeongbuk, Gangwon, Gyeonggi, Pyeongbuk, and North Hamgyong) and northeastern China. Korean ancestry, angelikoreanol and iso-imperatorin are known as ganghwam. In the private sector, the paralysis is solved, the bones are tangled and sore, Headache, neuralgia, rheumatism, arthritis and paralysis.

Eucommia ulmoides is a deciduous arboreous tree belonging to Eucommiaceae . It is a rare tree belonging to 1, 1 and 1 species. The pharmacological actions of the two persimmons are known as strengthening of blood pressure, strengthening of blood cholesterol and soothing, analgesic action, and diuretic action.

The name Achyranthes refers to the herbaceous plants or their roots of the perennial plant belonging to the Byrimaceae family. It is a name given to the fact that the grasshopper grass is swollen like a cow's knee. The general efficacy of the wax is that it works to smooth the blood circulation and remove the eosinophilia, and it is used for the treatment of physiological impurity, postpartum abdominal pain, and also works well in organs such as liver and kidney.

Acanthopanax ( acanthopanax ) is a plant called acanthopanax . It is a medicinal plant containing a lot of saponin like ginseng. In Oriental medicine, it helps to improve liver function, detoxification, immune function, It is known that it regulates bad blood cleanly, strengthens muscles and bones, shows Oro (七 勞) and Seven Wounds (七 傷), and is effective for enhancing energetic power.

Saposhnikovia divaricata is a perennial herbaceous perennial plant of the dicotyledonous plant. It is distributed in grassland or many mountainous areas such as Korea, China (Northeast, North China), Mongolia, Siberia and is cultivated as a medicinal crop. The windmill is known as coumarin-based materials such as imperatorin, psoralen, and bergapten, and it is known that windmills are produced by extracting roots of spiders from which spring flowers do not come out and drying them, . It is used for the purpose of sweating in one room, fever, pain, and sight.

Aralia contientalis is a perennial plant belonging to the Araliaceae . It is mainly grown in the mountains. It is used for the treatment of migraine by drying the roots and roots in the spring and fall, drying the sun. Until now, the ingredients of chewing gum have been known as vitamin B12, folic acid, limonen, sabinene, α-pinene, γ-terpinene, myrcene, and α-caryophyllene have been most widely known, and effects such as diuretic action, tonic action, hypotensive effect, anticonvulsant, and anti-ulcer have been reported.

Accordingly, the inventors of the present invention have completed the present invention by confirming that the extract of Paeonia japonica and Pseudomonas aeruginosa exhibits the therapeutic effect of inflammatory spinal diseases while trying to find a composition capable of treating inflammatory spinal diseases.

An object of the present invention is to provide a pharmaceutical composition for the prevention and treatment of inflammatory spinal diseases, which comprises peony and pore as an active ingredient.

The present invention provides a pharmaceutical composition for the prevention and treatment of inflammatory spinal diseases, which comprises a peony root extract and an extract as an active ingredient.

The present invention also provides a medicine using the pharmaceutical composition for the prevention and treatment of inflammatory spinal diseases of the present invention.

The present invention relates to a pharmaceutical composition for the prevention and treatment of inflammatory spinal diseases, which comprises a peony root extract and a perennial herb extract as an active ingredient, wherein the peony and pung mixture extract exhibits a nitric oxide (NO) production and a cytokine production inhibitory effect in a concentration- And it was confirmed that ear edema was alleviated by the mixed extract in a concentration dependent manner in the mouse model induced by ear swelling. In addition, it was confirmed that ear edema was alleviated by the mixed extract, It was confirmed that the combined extracts of wind, wind and hunting mixed extracts showed the best inhibitory effect on inflammation, and it was confirmed that the pain was alleviated and the disk was improved by administering it as a herb to the herniated disc herniation. The pharmaceutical compositions containing are useful for the prevention and / Advantageously it may be used for.

FIG. 1 is a diagram illustrating a method of manufacturing a mixed extract. FIG.
FIG. 2 is a diagram illustrating a method of manufacturing a medicine using a mixed extract. FIG.
FIG. 3 is a graph showing the inhibitory effect of the mixed extract on nitric oxide (NO) activity.
FIG. 4 is a graph showing the inhibitory effect of the mixed extract on cell proliferation.
FIG. 5 is a view showing the inhibitory effect of the inflammatory cytokine TNF-a of the mixed extract.
6 is a graph showing the inhibitory effect of the combined extract on the inflammatory cytokine IL-1 ?.
FIG. 7 is a graph showing the effect of suppressing ear edema by treating the mixed extract with ear edema-induced mice.
FIG. 8 is a graph showing sex, age, and pathological characteristics of the herniated disc herniation participated in the clinical experiment.
FIG. 9 is a graph showing the degree of pain before and after administration of the mixed extract by the NRS (Numeric Rating Scale) method.
FIG. 10 is a graph showing the improvement of the disk of the herniated disc herniated with mixed extract. FIG.
In Figures 3, 4, 5, 6 and 7, (a) is a peony, (b) is a peony, a peony, a peony, a peony, a peony, a peony, a peony, a peony, , (g), (g), (g), (g), and (g) (K), (g), (g), (g), (g), and (g) (M), (g), (g), (g), (g), (g), and (g) n) is peony, pung, kookchik, ganghwam, mungwoo, wuss, ogapi, windshield, and simpleness.

Hereinafter, the present invention will be described in detail.

The present invention provides a pharmaceutical composition for the prevention and treatment of inflammatory spinal diseases, which comprises a peony root extract and an extract as an active ingredient.

In the present invention, the extract is preferably a peony root extract, a peony root extract, and a mixture of peony root, pore and globe. Preferably, the extract is an extract obtained by mixing peony root, It is preferable that the extract is an extract extracted from a mixture of peony and pear, and it is preferably an extract extracted from a mixture of peony, pear, pear, korean, and bamboo. It is preferable that the extract is an extract obtained by mixing peony root, pore, claw root, ganghwam and ogapi. It is preferable that the extract is an extract obtained by mixing peony, pung, It is preferable that the extract is a mixture obtained by mixing two kinds of insecticides and acacia, and extracts obtained by mixing peonies, It is preferable that the extract is an extract obtained by mixing peony, pore, claw, ganghwam, mugwort, mugwort, and ocher. The extract is preferably an extract obtained by mixing peony, It is preferable that the extract is an extract obtained by mixing peony root, loquat, cormorant, hardy, bumblebee, oyster, ocher and chewing gum. Most preferred is an extract.

In the present invention, the peony and the pore are mixed preferably in the range of 1: 1 to 3: 1, and most preferably 2: 1. In addition, it is preferable to mix with the same amount as the pore, the hardy, the mite, the wax, the oak, the wind, and the virgin.

In the present invention, the extract may be extracted with water, C ₁ to C ₂ lower alcohol or a mixture thereof. According to a preferred embodiment of the present invention, extraction with 70% ethanol is most preferred.

As the extraction method, shaking extraction, Soxhlet extraction, or reflux extraction may be used. The extraction solvent may be extracted by adding 1 to 40 times the amount of dried peony and pore, and the extraction temperature may be 20 to 50 ° C, The extraction time may be 10 to 100 hours, the extraction frequency may be 1 to 5 times, and the extract may be filtered and concentrated under reduced pressure using a vacuum decompression concentrator or a vacuum rotary evaporator. Drying may be carried out under reduced pressure, , Boiling drying, spray drying or lyophilization.

In the present invention, it is preferable that the inflammatory spinal disease is any one selected from the group consisting of herniated disc herniation, degenerative arthritis, and discitis.

In the present invention, the composition may be a formulation for oral or parenteral administration, but it is most preferably a formulation for injection.

In a specific example of the present invention, the present inventors produced extracts of peony and pomace mixed extracts (see Fig. 1), and the peony and pomaceous mixture extracts showed inhibitory effects on NO production and cytokine production in a concentration-dependent manner, (See FIG. 3, FIG. 5, and FIG. 6), it was confirmed that ear edema was alleviated in a concentration-dependent manner by the mixed extract in the ear edematization-induced mouse model (see FIG. 7) , Extracts obtained by mixing peony extracts, pore extracts and extracts, peony extracts, extracts obtained by mixing peony pits, pore extracts and strong extracts, extracts obtained by mixing peony extracts, pore extracts, , Extracts made by mixing peony, pungpu, pungchul, ganghwam, and mulberry extract, peony, pungpu Mixture of extracts extracted from a mixture of extracts, peonies, pomopsis, moxibustion, moxibustion, moxibustion, and moxibustion extract, peony moxibustion, Extracts extracted from a mixture of extracts, peonies, pungs, mushrooms, mushrooms, mushrooms, mushrooms and mushrooms, extracts obtained by mixing mushrooms, mushrooms, mushrooms, mushrooms, mushrooms, mushrooms, It was confirmed that the extracts obtained by extracting mixed extracts of ganghwam, mugwort, oak, and chewy gum, extracts of peony, It was confirmed that the extract inhibited the inflammatory reaction in the extracts obtained by mixing with the extracts of Pseudomonas aeruginosa, 9 and 10), the pharmaceutical composition containing the peony extract and the extract of the present invention is useful for the prevention and treatment of inflammatory spinal diseases .

The composition of the present invention may further comprise a pharmaceutically acceptable carrier. The term " pharmaceutically acceptable "as used herein means that the composition is free of toxicity to cells or humans exposed to the composition. Compositions comprising a pharmaceutically acceptable carrier can be of various oral or parenteral formulations. In the case of formulation, it can be prepared using diluents or excipients such as fillers, extenders, binders, humectants, disintegrants, surfactants and the like which are usually used. The carriers, excipients and diluents include lactose, dextrose, sucrose, sorbitol, mannitol, xylitol, erythritol, maltitol, starch, acacia rubber, alginate, gelatin, calcium phosphate, calcium silicate, cellulose, methylcellulose, microcrystalline cellulose, But may be at least one selected from the group consisting of polyvinylpyrrolidone, physiological saline, methylhydroxybenzoate, propylhydroxybenzoate, talc, magnesium stearate and mineral oil, dextrin, calcium carbonate, propylene glycol and liquid paraffin, But are not limited to, ordinary carriers, excipients or diluents. These components can be added to the active ingredient, peony root extract and extract, independently or in combination.

Solid formulations for oral administration may include tablet pills, powders, granules, capsules and the like, which may contain one or more excipients, such as starch, calcium carbonate, sucrose or lactose, lactose, gelatin, and the like. In addition to simple excipients, lubricants such as magnesium stearate, talc, and the like may also be used. Liquid preparations for oral administration include suspensions, solutions, emulsions, syrups and the like. Various excipients such as wetting agents, sweeteners, fragrances, preservatives and the like may be included in addition to water and liquid paraffin, which are simple diluents commonly used. have. Formulations for parenteral administration may include sterile aqueous solutions, non-aqueous solutions, suspensions, emulsions, freeze-dried preparations, and suppositories. Examples of the non-aqueous solvent and the suspending agent include propylene glycol, polyethylene glycol, vegetable oil such as olive oil, and injectable ester such as ethyl oleate. Examples of the suppository base include witepsol, macrogol, tween 61, cacao paper, laurin, glycerogelatin and the like.

The pharmaceutical composition of the present invention may also be in the form of tablets, pills, powders, granules, capsules, suspensions, solutions, emulsions, syrups, sterile aqueous solutions, non-aqueous solutions, suspensions, emulsions, Or a pharmaceutically acceptable salt thereof.

The pharmaceutical composition of the present invention can be administered in a pharmaceutically effective amount. There is no particular restriction on the dosage, and it may vary depending on the body's absorption, body weight, age, sex, health condition, diet, administration time, administration method, excretion rate, severity of disease and the like. The pharmaceutical composition of the present invention is prepared in consideration of an effective dose range, and the unit dosage formulations thus formulated are classified according to the judgment of the expert who monitors or observes the administration of the drug, if necessary, Or may be administered several times at a predetermined time interval. Preferably, the composition of the present invention may be administered at a dose of 0.5 to 5000 mg / kg, preferably 50 to 500 mg / kg, more preferably 50 mg / kg, per day, based on the amount of peony and perilla extract The above administration may be administered once a day, or may be administered several times.

The present invention also provides a pharmaceutical preparation comprising the pharmaceutical composition for the prevention and treatment of inflammatory spinal diseases of the present invention.

In the present invention, the pharmaceutical composition for the prevention and treatment of inflammatory spinal diseases is sterilized and / or used as a preservative, a stabilizer, a wetting agent or an emulsifying accelerator, an adjuvant such as a salt and / or a buffer for controlling osmotic pressure and other therapeutically useful substances And may be formulated according to a conventional method.

The pharmaceutical composition for the prevention and treatment of inflammatory spinal diseases of the present invention can be formulated for the preparation of a medicament. The present invention also provides a pharmaceutical composition for the prevention and treatment of inflammatory spinal diseases, which comprises a pharmaceutical composition for the prevention and treatment of inflammatory spinal diseases according to the present invention. It is a method of direct injection of purified liquid medicine (药液) to a specific site such as acupuncture points or tender point. It injects the drug extract prepared in the form of injection into acupuncture points or the mucus using a syringe, It is a new herbal medicine treatment that combines the pharmacological effects of drugs. In general, the one-handed saliva is related to hemorrhagic type according to the type, and there are therapies for directly stimulating the skin or stabbing the affected area with a needle, a blood transfusion or a needle stitch, and various other therapies are being performed. Herbal medicine therapy is widely used because it stimulates the acupuncture point which is a herbal medicine treatment point and at the same time shows the effect of skin penetration of herbal medicine extracts having inherent pharmacological effect. In addition, the herbal medicine therapy can be applied easily to patients or emergency patients who are not able to take the medicine unlike the injection of the chemical into the muscles and blood vessels.

Hereinafter, the present invention will be described in detail with reference to the following Examples, Production Examples and Experimental Examples.

However, the following Examples, Preparation Examples and Experimental Examples are merely illustrative of the present invention, and the content of the present invention is not limited by the following Examples, Production Examples and Experimental Examples.

Example 1 Preparation of Peony Pomace and Ouong Mixed Extract

The mixture was mixed with 2: 1 ratio of peanut and pore, and the mixture was extracted with 70% ethanol. The mixture was filtered with a 1 ㎛ filter paper, concentrated under reduced pressure, and sequentially treated with 80% ethanol and 90 & After filtration, the mixture was filtered through a 0.6 mu m filter paper, followed by filtering with a 0.2 mu m capsule filter. Then, the mixture was concentrated under reduced pressure, lyophilized at -80 ° C, powdered, and stored in a freezer.

Example 2 Preparation of Peony, Pore and Crown Mixed Extracts

Peanut, lozenge, and lozenge were weighed at a ratio of 2: 1: 1, and extracted and powdered in the same manner as in Example 1,

<Example 3> Preparation of a mixture of peanut, pore, and hard mixture

The extracts were weighed in the ratio of 2: 1: 1, and extracted and powdered in the same manner as in Example 1,

<Example 4> Preparation of mixed extracts,

The extracts were weighed in the ratio of 2: 1: 1: 1, and extracted and powdered in the same manner as in Example 1,

<Example 5> Preparation of mixed extracts of Peony root, Pseudomonas sp., Pseudomonas sp.

The extracts were weighed in the ratio of 2: 1: 1: 1: 1, extracted in the same manner as in Example 1, powdered, and stored frozen.

&Lt; Example 6 > &gt;

The mixture was weighed in the ratio of 2: 1: 1: 1: 1, and extracted and powdered in the same manner as in Example 1,

<Example 7> Preparation of mixed extracts of peanut, pomace, gochu, ganghwam and ogapi

The extracts were extracted and powdered in the same manner as in <Example 1> after being weighed at a ratio of 2: 1: 1: 1: 1,

<Example 8> Preparation of mixed extracts of peony root, loquat, chrysanthemum, mandarin, quadriplegia and mist

The mixture was weighed in a ratio of 2: 1: 1: 1: 1: 1, and then extracted and powdered in the same manner as in Example 1,

<Example 9> Preparation of mixed extracts of peony root, loquat, chrysanthemum, mandarin, quadriceps, and acerola

1: 1: 1, and extracted and pulverized in the same manner as in Example 1, and then stored in a freezer. Then,

<Example 10> Preparation of mixed extracts of peony root, loam, chrysanthemum, mandarin,

The extracts were extracted and powdered in the same manner as in Example 1 after being weighed at a ratio of 2: 1: 1: 1: 1: 1,

<Example 11> Preparation of mixed extracts of peanut, pomace, moxa, ganghwam, mugwort, moxa and ogumi

1: 1: 1: 1: 1, followed by extraction and pulverization in the same manner as in Example 1, followed by freezing &Lt; / RTI &

<Example 12> Preparation of mixed extracts of peony root, pseudo-pomaceus, pseudomonas,

Weighed in the ratio of 2: 1: 1: 1: 1: 1: 1: 1, and then extracted in the same manner as in <Example 1> Powdered and frozen,

<Example 13> Preparation of mixed extracts of peony root, loquat, chrysanthemum, ganghwam, quadriceps, horses,

1: 1: 1: 1: 1: 1: 1, and then extracted in the same manner as in <Example 1> Powdered and frozen,

<Example 14> Preparation of mixed extracts of peony root, loquat, chrysanthemum, ganghwam, mugwort, hwal,

1: 1: 1: 1: 1: 1: 1, and the same amount as in Example 1 was obtained after weighing in a ratio of 2: 1: 1: 1: And then stored in a freezer.

&Lt; Preparation Example 1 >

In order to prepare the extract prepared in Examples 1 to 14 as a medicinal herb, the above-mentioned filtered extract was filled in a vial sterilized at 250 ° C for 120 minutes, and then sterilized cap ), And the medicament was sterilized at 121 DEG C for 25 minutes. The sterilized medicinal herb was packed and finished after inspection of the foreign objects and the finished product such as properties, pH, specific gravity and microorganism.

Example 15 Cell culture

To evaluate the immunological activity of the mixed extracts, Raw264.7 cells, macrophages, were cultured and tested.

Specifically, Raw264.7 cells were obtained from the American Type Culture Collection (ATCC) and cultured in Dulbecco's Modified Eagle Medium (DMEM, Gibco) supplemented with 10% FBS (fetal bovine serum, Gibco-Invitrogen) and 1% penicillin- streptomycin -Invitrogen) medium at 37 ° C and 5% CO ₂ for 2 days.

<Experimental Example 1> Confirmation of inhibitory effect of nitrogen oxides (NO) activity by mixed extracts

The inhibitory effect of the mixed extract prepared in Example 1 to Example 14 of the present invention was confirmed.

Specifically, the formation of NO (NO) was confirmed using Raw264.7 cells cultured in Example 15 above. Cells were seeded at a density of 2 × 10 ⁵ cells / 1 ml in a 96-well plate and cultured in a CO ₂ cell incubator at 37 ° C. for 24 hours. The cultured cells were treated with the mixed extracts prepared in Examples 1 to 14 at 100, 400 and 800 占 퐂 / ml and cultured for 6 hours. For induction of inflammation, LPS (lipopolysaccharide) was added to each well at 1 ㎍ / ㎖ and cultured for 18 hours. Then, the supernatant was collected, treated with the same amount of Griess reagent, and NO production was measured at 540 nm on a microplate reader (TECAN Austria, Australia).

As a result, as shown in FIG. 3, it was found that NO production was decreased in a concentration-dependent manner in the mixed extract. In addition, all of the mixed extracts containing peanut and pomace were significantly reduced in NO production as compared with the control, and the mixed extracts prepared in Example 14 were excellent (FIG. 3).

<Experimental Example 2> Identification of cell toxicity by mixed extracts

The cytotoxicity of the mixed extract prepared in Example 1 to Example 14 of the present invention was confirmed.

Specifically, the formation of NO (NO) was confirmed using Raw264.7 cells cultured in Example 15 above. Cells were seeded at a density of 2 × 10 ⁵ cells / 1 ml in a 96-well plate, and cultured in a CO ₂ cell incubator at 37 ° C. for 24 hours. The cultured cells were treated with the mixed extracts prepared in Examples 1 to 14 at the concentrations of 100, 400 and 800 / / ml and cultured for 6 hours. For induction of inflammation, LPS was added to each well at 1 ㎍ / ㎖ and cultured for 18 hours. 20 μl of MTT reagent (10 mg / ml) diluted with DPBS was added to each well and incubated for 4 hours. After removing the supernatant, 50 μl of DMSO was added and slightly shaken. After 30 minutes, the absorbance at 570 nm was measured using an ELISA reader (TECAN Austria, Australia). The cytotoxicity (%) was calculated as the ratio of the absorbance of the extract addition well to the absorbance of the control well without addition of the extract (%).

As a result, as shown in Fig. 4, the cytotoxicity was not confirmed at all concentrations of the mixed extract (Fig. 4).

<Experimental Example 3> Identification of inflammatory cytokine inhibitory effect by mixed extracts

The inhibitory effect of the mixed extracts prepared in Examples 1 to 14 of the present invention on inflammatory cytokines was confirmed.

Specifically, the Raw 264.7 cells cultured in Example 15 were divided into 96-well plates at a concentration of 1 × 10 ⁵ cells / ml and cultured for 24 hours. Then, the cells were cultured in the same manner as in Example 1 to Example 14 Were cultured at 100, 400 and 800 ㎍ / ㎖ for 24 hours. Then, only the supernatant was taken and the cytokine content was measured by an ELISA kit (product name, manufacturer) using Enzyme-linked immunosorbent assay (ELISA). 100 μl of the supernatant sample was added to each well plate coated with an antibody of cytokine (TNF-α and IL-1β) (Abcam, UK), allowed to react at room temperature for 2 hours, 20 was washed with washing buffer 5 times or more. Detection antibody solution was added to react with antibody. Then, Horseradish peroxidase (HRP) enzyme conjugated with Avidin was added and reacted at room temperature for 15 minutes. After stopping the reaction between the HRP enzyme and the TMB substrate by adding a stop solution (H ₂ SO ₄ ), the absorbance was measured at 450 nm using a microplate reader (TECAN Austria, Australia).

As a result, as shown in FIG. 5 and FIG. 6, the cytokine inhibitory effect was confirmed in a concentration-dependent manner in the mixed extract treatment group. In particular, TNF-α and IL-1β inhibitory effect was remarkable at a concentration of 800 μg / (Figs. 5 and 6). In addition, it was confirmed that the mixed extract of Example 14 had the best cytokine inhibitory effect (FIGS. 5 and 6).

<Experimental Example 4> Confirmation of the effect of mixed extracts on mouse edema in mice

The effect of inhibiting ear edema of the mixed extracts prepared in Examples 1 to 14 of the present invention was confirmed in mice.

Specifically, ICR mice at 7 weeks of age were purchased from Orient Bio (Korea). After the mice were separated from each experimental group, the mixed extracts of Examples 1 to 14 were injected at 100, 400, and 800 / / kg, respectively, for 30 minutes after intramuscular injection. TPA (12-O-tetradecanoylphorbol-13 -acetate) was dissolved in acetone (1 μg / 20 μl) to induce edema in the ear. As a control, the left ear was treated with acetone only. When inducing edema, the experimenter fixed the specimen completely from the back, and then the second experimenter stimulated the edematous substance in the ear using a micropipette. Four hours later, both ears of the mouse were punched and weighed to determine the degree of edema.

As a result, as shown in FIG. 7, ear edema was remarkably alleviated at a concentration of 800 μg / kg of the mixed extract, and ear edema was relieved depending on the mixed extract concentration (FIG. 7). In addition, among the mixed extracts, the extract extract prepared in Example 14 showed the best effect of alleviating edema of the ear (FIG. 7).

<Experimental Example 5> Confirmatory effect of mixed extract on herniated disc prolapse

Confirming the efficacy of the mixed extract of the present invention in clinical patients.

Specifically, a mixed extract prepared in Example 14 was applied to a 26-gauge needle (4 cm) of 23 back and lower back pain patients who were diagnosed with disc herniation by visiting a spinal hospital in the spine specialist hospital in 2014, And acupuncture points close to the disk and the acetabular joints. Pain levels and adverse reactions were assessed and confirmed by MRI.

The average age of the participants was 49 ± 14, 14 (61%) were males, 10 (44%) had previously suffered back pain, and 3 had muscle weakness or sensory loss. Three patients experienced only lower back pain and the rest had lower back pain, and 16 (70%) of them experienced pain under the knee. Currently, general medical treatment (14 analgesics, 8 received 1.9 ± 1.5 nerve block sessions) and herbal treatment, the composition of the invention was administered twice weekly for 3 weeks. The pain level was assessed by the Lumbar disc herniation Numerical Rating Scale (NRS) method. NRS is a method of measuring the intensity of back pain that a patient feels consciously. It consists of a series of numbers from 0 to 10, where 0 is no pain. 10 is the most severe pain that has been felt so far. The intensity of the most consistent pain at that moment was selected and converted to a score.

As a result, as shown in FIG. 9, NRS was significantly reduced from 7.9 ± 1.7 to 3.4 ± 1.6, and there was no report of side effects (FIG. 9).

Claims (6)

Peony 2 parts by weight, Goku 1 part by weight, nine 1 part by weight, ganghwal 1 part by weight, Eucommia 1 part by weight, wooseul 1 part by weight of Acanthopanax 1 part by weight, wind-1 parts by weight of Aralia cordata 1 part by weight of a mixture of water, C ₁ To C ₂ lower alcohol, or a mixture thereof, as an active ingredient. The pharmaceutical composition for the prevention and treatment of inflammatory spinal diseases according to claim 1,
The pharmaceutical composition according to claim 1, wherein the inflammatory spinal disease is any one selected from the group consisting of herniated disc herniation, degenerative arthritis and discitis.
The pharmaceutical composition for the prevention and treatment of inflammatory spinal diseases according to claim 1, wherein the composition is in a form of injection.
A pharmaceutical preparation comprising a pharmaceutical composition for the prophylaxis and treatment of inflammatory spinal diseases according to any one of claims 1 to 3.
Peony 2 parts by weight, Goku 1 part by weight, nine 1 part by weight, ganghwal 1 part by weight, Eucommia 1 part by weight, wooseul 1 part by weight of Acanthopanax 1 part by weight, wind-1 parts by weight of Aralia cordata 1 part by weight of a mixture of water, C ₁ To C ₂ lower alcohol, or a mixture thereof, as an active ingredient, as an effective ingredient for the prevention and / or amelioration of an inflammatory spinal disease.
[Claim 7] The health functional food according to claim 5, wherein the inflammatory spinal disease is any one selected from the group consisting of herniated disc herniation, degenerative arthritis and discitis.

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