KR20070068689A - Composition for immunosuppression comprising an extract of saururus chinensis or active compounds isolated therefrom - Google Patents

Abstract

A composition for immunosuppression comprising the extract of Saururus chinensis or active compounds isolated therefrom is provided to inhibit abnormal proliferation of B cell and T cell, so that the composition is useful for prevention and treatment of diseases caused by hypersensitive immune responses. The composition for immunosuppression comprises the extract of Saururus chinensis or an active compound isolated from the Saururus chinensis extract selected from (-)-saucerneol, saucerneol C, manassantin A, manassantin B and a mixture thereof as effective ingredient, wherein the Saururus chinensis extract is prepared by using an organic solvent selected from methanol, methanol solution, ethanol, ethanol solution, butanol, dichloromethane, ethylacetate and a mixture thereof. The composition is a pharmaceutical or health food composition. The pharmaceutical composition prevents or treats diseases caused by hypersensitive immune responses including organ transplant rejection, autoimmune disease including lupous erythematosus and rheumatoid arthritis, and allergy disease including rhinitis, asthma and atopic dermatitis, and the health food composition is formulated as drink.

Description

COMPOSITION FOR IMMUNOSUPPRESSION COMPRISING AN EXTRACT OF SAURURUS CHINENSIS OR ACTIVE COMPOUNDS ISOLATED THEREFROM}

Figure 1 shows the inhibitory effect on the LPS (lipopolysaccharide) induced B cell proliferation of the compounds isolated and purified from three hundred s. Extract according to the present invention,

Figure 2 shows the inhibitory effect on Con A-induced T cell proliferation of the compounds isolated and purified from three hundred sec extract according to the present invention,

Figure 3 shows the inhibitory effect on the mixed leukocyte response of the compounds isolated and purified from three hundred seconds extract according to the present invention.

The present invention provides an active ingredient containing Saururus chinensis extract, (-)-sorneol [(-)-saucerneol], soserenol C (saucerneol C), manasantin A or manasantin B as an active ingredient. It relates to a composition for inhibition.

Although diseases due to immune hypersensitivity reactions are increasing all over the world, the root cause for the occurrence of these diseases is insufficient. Current treatments for immune diseases include the use of two or more immunosuppressive agents in combination to alleviate or reduce various symptoms caused by the disease. Herein, immunosuppressants refer to a variety of substances used to reduce or block the host's ability to produce antibodies (humoral immune response) or to generate a cellular immune response to the action of an antigen.

Currently, cyclosporin A and FK506 are mainly used as immunosuppressive agents. These are compounds derived from natural products with complex chemical structures, which are uneconomical due to high cost in terms of supply and demand of raw materials, and may cause various side effects due to long-term administration. have. Therefore, there is an urgent need for the development of new immunosuppressive agents capable of economical production with low toxicity and induction of immune tolerance. Such immunosuppressive agents may be useful for autoimmune diseases such as lupus and rheumatoid arthritis, as well as for skin hypersensitivity reactions such as atopy and allergy.

Saururus chinensis is a perennial herb belonging to the genus Saururaceae of the three hundred (Saururaceae), which has long been used as a folk medicine in China. Chinese herbal medicine dictionary, three hundred seconds have the effect of deciduous fever, small seed detoxification, species, each, jaundice, sediment, lobster, Ongjong, it is effective to read. In the private sector, outposts, roots and leaves have been used for the treatment of wind poison, diuresis, gonorrhea, gonorrhea, hepatitis, pneumonia, venom and hypertension.

The study of the components of the three hundred seconds has been conducted for flavonoids (alkaloids), alkaloids (amino acids), fatty acids (fatty acids), quinones (essence) and essential oils. The main components of the outpost are methyl n-nonyl ketone, the stem contains hydrolyzable tannins, and the leaves contain quercetin, isoquercetrin and abiculin. It has been reported to contain hyperin, rutin and hydrolyzable tannins.

Meanwhile, it is known that sesquiterpene-based compounds exist in extracts of the same species, Saururus cernuus , which is widely distributed in the wetlands of North America. Rao et al., Tetrahedron Lett . 24 (45): 4947-4950, 1983 have isolated compounds called SC-8, SC-9, manasanthin A, manasanthin B, and soserenol from extracts of the US Triticale. These structures were identified, and it was confirmed that the double manaxanthin A had a neurocentral inhibitory effect. In Korea, dimethyl terephthalate and quercetin were isolated and identified to confirm their pharmacological and antimicrobial activity (Kwak Jae Wook, Ph.D. dissertation, Kyung Hee University, 1988). In addition, a patent has recently been filed for anticancer activity (Korean Patent No. 10-0248942) and anti-inflammatory activity (Korean Patent Publication No. 10-2004-0075135) of three hundred seconds.

However, there has been no report on the immunosuppressive ability of the extract of 300 seconds. Therefore, the present inventors have identified the immunosuppressive ability of the extract of three hundred seconds, which has not been identified so far, and isolates five single substances having immunosuppressive effect from the three hundred seconds extract, which are useful for the prevention and treatment of diseases caused by immune hypersensitivity reactions. The present invention was completed by confirming that it can.

Accordingly, it is an object of the present invention to provide an immunosuppressive composition comprising as an active ingredient three hundred seconds extract useful for the prevention and treatment of diseases caused by immune hypersensitivity reactions.

In addition, it is an object of the present invention to provide a food having an immunosuppressive effect of the three hundred seconds extract as an active ingredient.

In order to achieve the above object, the present invention comprises an effective amount of an extract of three hundred seconds, (-) soserol, soserolol C, manasanthin A or manasanthin B as an active ingredient with a pharmaceutically acceptable carrier, immunosuppressive effect It provides a composition showing.

Hereinafter, the present invention will be described in detail.

The composition according to the present invention is characterized in that it contains three hundred sec extract showing an immunosuppressive effect, (-) soserenol, soserenol C, manasanthin A or manasanthin B separated and purified therefrom as an active ingredient.

First, the three hundred seconds extract according to the present invention can be prepared by solvent extraction of three hundred seconds or its dried product.

Three hundred seconds extract of the present invention is added to the organic solvent of 2 to 200 times, preferably 10 to 30 times with respect to the volume of the dried three hundred seconds, dried, 1 to 20 days, preferably 3 to 7 days at 10 to 30 ℃ Extraction, cooling, filtration and concentration under reduced pressure. At this time, the extraction solvent may be used methanol, methanol aqueous solution, ethanol, ethanol aqueous solution, butanol, dichloromethane, ethyl acetate or a mixture thereof, 80 to 100% aqueous methanol solution is preferred. The three hundred sec extract extracted with methanol may be re-extracted with a solvent such as butanol, dichloromethane or ethyl acetate. Preferably re-extracted with ethyl acetate.

Specifically, in a preferred embodiment of the present invention, three hundred seconds or its dried product is cooled for 7 days using 100% aqueous methanol solution, filtered, and the filtrate is concentrated under reduced pressure to obtain a methanol extract, the methanol extract is suspended in distilled water and then the same amount The extracted water layer was extracted with dichloromethane and extracted with the same amount of ethyl acetate and concentrated under reduced pressure to obtain a fraction of 300 seconds. The dichloromethane and ethyl acetate fractions in the methanol extract of 300 seconds show the immunosuppressive effect, of which the immunosuppressive effect of the dichloromethane fraction of 300 seconds is the best (see Table 1 ).

In the present invention, chromatography is performed to separate and purify the active ingredient from the dichloromethane fraction of 300 seconds which exhibits immunosuppressive effect. The chromatography is preferably carried out 1-2 times using a silica gel column. The mixed solution of dichloromethane and methanol can be used as the in-phase. At this time, the eluted fraction by gradient elution to sequentially increase the methanol concentration to 0, 20, 40, 60, 80 and 100%, and to obtain the active fraction by measuring the immunosuppressive effect of the collected fractions Can be.

The active fraction showing the immunosuppressive effect was further purified by silica gel column chromatography to obtain (-) soserenol of formula (1 ), soserenol (C) of formula 2 , manasanthin A of formula (3 ) and manasanthin B of formula (4 ).

In order to confirm the immunosuppressive effects of the compounds, sacchinone, soseneol C, manasanthin A, isolated and purified from extract of 300 sec in a culture medium in which abnormal cell proliferation of B cells or T cells was induced by LPS or Con A. As a result of treatment with manasanthin B or (-)-soseneol by concentration, it was confirmed that B- or T-cell proliferation was suppressed in a concentration-dependent manner in the experimental group treated with four compounds except sacchinone ( FIGS. 1 and 2). , See Tables 2 and 3 ). In addition, in the growth of mouse splenocytes through the mouse mixed leukocyte reaction, Socerolol C, Manastanthin A, Manastanthin B, and (-)-Soseneol compounds except Saucinin effectively inhibited cell proliferation ( FIG. 3 and See Table 4 ). Succinone has been reported to inhibit the activity of NF-κB, which is important in immune response, as a major active ingredient contained in three hundred sec extracts (Hwang et al., Plant Med. 69: 1096-1101, 2003). In the present invention, it can be seen that other components such as (-) socerol, soserolol C, manasanthin A, and manasanthin B act as an active ingredient of immunosuppressive effect rather than sacchinone.

Immunosuppressive composition containing (300) extract, (-) soserenol, soserenol C, manasanthin A or manasanthin B as an active ingredient isolated from the three hundred sec extract according to the present invention may be a pharmaceutical composition or food composition. .

The three hundred sec extract included in the pharmaceutical composition of the present invention is preferably purified by column chromatography, but may also include an extract obtained by simply solvent extraction. Further, the formula (1) to 4 (-) borneol, O, O-Borneol C, Mana A xanthine and xanthine Mana B can be isolated and purified or synthesized chemically from the Houttuynia cordata extract.

The pharmaceutical composition according to the invention is characterized in that it is used for the prevention or treatment of diseases caused by immune hypersensitivity reactions. Diseases caused by immune hypersensitivity reactions mean that the immune function is abnormally activated to reach a pathological state, for example, but not limited to, rejection in organ transplantation; Autoimmune diseases such as lupus and rheumatoid arthritis; Skin hypersensitivity reactions such as rhinitis, asthma and allergic diseases such as atopic dermatitis. In addition, the pharmaceutical compositions according to the invention may be administered individually or in combination with other types of immunosuppressive agents.

Extract of trichophytium, (-) socerenol, soserenol C, manasanthin A or manasanthin B isolated and purified therefrom may be mixed with a suitable pharmaceutically acceptable carrier or excipient or diluted with a diluent according to conventional methods. Pharmaceutical compositions having the function can be prepared. Examples of suitable carriers, excipients and diluents include lactose, dextrose, sucrose, sorbitol, mannitol, xylitol, erythritol, malditol, starch, acacia rubber, alginate, gelatin, calcium phosphate, calcium silicate, cellulose, methyl cellulose, undecided Vaginal cellulose, polyvinylpyrrolidone, water, methylhydroxybenzoate, propylhydroxybenzoate, talc, magnesium stearate and mineral oil. The pharmaceutical composition may further include fillers, anti-coagulants, lubricants, wetting agents, fragrances, emulsifiers, preservatives and the like. The pharmaceutical compositions of the invention can be formulated using methods well known in the art to provide rapid, sustained or delayed release of the active ingredient after administration to a mammal. The formulations may be in the form of tablets, pills, powders, sachets, elixirs, suspensions, emulsions, solutions, syrups, aerosols, soft or hard gelatin capsules, sterile injectable solutions, sterile powders and the like.

The pharmaceutical compositions of the invention can be administered via several routes including oral, transdermal, subcutaneous, intravenous or intramuscular. Typical daily dosages of the pharmaceutical compositions of the present invention, based on the active ingredient, the three hundred sec extract is in the range of 0.1 to 500 mg / kg body weight, preferably 10 to 100 mg / kg body weight, (-) soserol , Soseneol C, manasanthin A and manasanthin B each ranges from 0.1 to 500 mg / kg body weight, preferably 5 to 100 mg / kg body weight, and may be administered once or in several divided doses. However, it should be understood that the actual dosage of the active ingredient should be determined in light of several relevant factors such as the route of administration, the age, sex and weight of the patient, and the severity of the disease, and therefore, the dosage may be determined in any respect to the invention It does not limit the scope of.

In addition, the present invention can prevent or alleviate diseases caused by immune hypersensitivity reactions, including an effective amount of the extract of 300 seconds, (-) soserolol, soserolol C, manasanthin A or manasanthin B separated and purified therefrom It provides a health promoting food composition. As the food to which the trichophytium extract of the present invention, (-) soserolol, socerolol C, manasanthol A or manasanthin B separated and purified therefrom, can be added, for example, various foods, beverages, snacks, confectionery, Chewing gums, ice creams, tea bags, instant teas, granules, flavorings, vitamin complexes, and other health supplements, but are not limited thereto.

Extract of the three hundred seconds of the present invention, (-) soserolol, soserolol C, mansaanthin A, or manasanthin B isolated and purified therefrom to the raw material during food preparation or properly mixed in the cooked food for the above-mentioned health promotion Food or beverages may be prepared, in which case the content of trichoola extract, (-) soserenol, soserenol C, manasanthin A or manasanthin B, respectively, separated and purified from the final food or beverage is 0.01 To 30% by weight.

The pharmaceutical composition or health promoting food or beverage of the present invention may further include other medicinal herbs or extracts thereof that are pharmaceutically acceptable to enhance or supplement the desired effect.

Hereinafter, the present invention will be described in more detail by the following Preparation Examples and Examples.

The following Preparation Examples and Examples are only for illustrating the present invention, but the scope of the present invention is not limited thereto.

Example 1: Preparation of Extract of Trichophyte and Isolation and Purification of Active Ingredients

15 kg of dried three hundred seconds was chilled with 200 L of methanol for one week, filtered, and the filtrate was concentrated under reduced pressure to obtain 1.7 kg of methanol extract. The methanol extract was suspended in 20 L of distilled water, extracted with the same amount of dichloromethane (CH ₂ Cl ₂ ), and the extract was concentrated under reduced pressure to obtain 922 g of dichloromethane extract. The remaining aqueous layer was extracted again with the same amount of ethyl acetate (ethylacetate, EtOAc), and the extract obtained therefrom was concentrated under reduced pressure to obtain 18 g of ethyl acetate (EtOAc) fraction, and the remaining aqueous layer was freeze-dried.

430 g of the dichloromethane (CH ₂ Cl ₂ ) fraction was mixed with dichloromethane / methanol (100: 1) as a mobile phase and flown at a flow rate of 3 ml / min, and the concentration of methanol was sequentially changed from 0% to 100%. It was analyzed by silica gel column chromatography in gradient gradient elution. This gave a total of four fractions (fractions 1 to 4). Among them, fraction 2 showing immunosuppressive effect was analyzed by a concentration gradient elution method of silica gel column chromatography using hexane / ethyl acetate (10: 1 to 1: 1) mixed solution as a mobile phase at a flow rate of 3 ml / min. Fractions (fractions 21 to 25) were obtained. Among them, fraction 25 showing an immunosuppressive effect was purified by repeating silica gel column chromatography (mobile phase: hexane / ethyl acetate = 10: 1 to 1: 1), and as a result, 1.5 g of (-)-soseneol, 28 Mg mg of sorcerol C, 202 mg manasanthin A, and 630 mg manasanthin B were isolated. On the other hand, fraction 23 was crystallized using hexane (Hx) / ethyl acetate (EA) (8: 1) to obtain 4.0 g of sacchinone.

Immunosuppressive effect of the extract from the three hundred seconds and fractions separated therefrom is shown in Table 1 by measuring the inhibitory effect on the growth of mouse splenocytes.

(-)-Socerol

¹ H-NMR (CD ₃ OD, 500 MHz) δ 7.00-6.77 (9H, H-2 ′, 2 '', 5,2,6 ', 6,6'',5', 5 ''), 5.46 (2H, d, J = 7.0 Hz, H-7,7 '), 4.72 (1H, d, J = 6.3 Hz, H-7``), 4.42 (1H, d, J = 6.6 Hz, H-8 ''), 3.89 (3H, s, -OCH <sub>3</sub> ), 3.86 (3H, s, -OCH <sub>3</sub> ), 3.82 (3H, s, -OCH <sub>3</sub> ), 3.81 (3H, s, -OCH <sub>3</sub> ), 2.30 ( 2H, m, H-8, 8 '), 1.10 (3H, d, J = 6.2 Hz, H-9``), 0.69 (6H, d, J = 6.3 Hz, H-9,9')

¹³ C-NMR (CD ₃ OD, 125 MHz) δ 151.5, 150.3, 150.1, 148.7, 147.8, 146.7, 136.8, 135.2, 134.1, 120.9, 120.2, 120.1, 117.8, 115.8, 112.5, 112.1, 111.3, 85.7, 85.4 , 81.6,77.9, 56.6, 56.5, 56.4, 44.7, 44.6, 16.5, 14.8, 14.7

Sorcerol C

¹ H-NMR (CD ₃ OD, 500 MHz) δ 7.07 (1H, d, J = 1.7 Hz, H-2 ′), 7.00 (1H, d, J = 1.8 Hz, H-2``), 6.99 ( 1H, d, J = 8.1 Hz, H-5), 6.98 (1H, d, J = 1.7 Hz, H-2), 6.95 (1H, dd, J = 1.7, 8.1 Hz, H-6 '), 6.88 (1H, dd, J = 1.7, 8.1 Hz, H-6), 6.84 (1H, dd, J = 1.8, 8.3 Hz, H-6``), 6.83 (1H, d, J = 8.1 Hz, H- 5 '), 6.76 (1H, d, J = 8.3 Hz, H-5``), 5.11 (1H, d, J = 8.3 Hz, H-7), 4.64 (1H, d, J = 6.6 Hz, H -7 ''), 4.42 (1H, q, J = 6.6 Hz, H-8``), 4.36 (1H, d, J = 9.5 Hz, H-7 '), 3.87 (3H, s, -OCH ₃ ), 3.83 (3H, s, -OCH ₃ ), 3.84 (3H, s, -OCH ₃ ), 2.25 (1H, m, H-8), 1.79 (1H, m, H-8 '), 1.07 (3H , d, J = 6.1 Hz, H-9``), 1.01 (3H, d, J = 6.6 Hz, H-9 '), 0.65 (3H, d, J = 6.8 Hz, H-9)

¹³ C-NMR (CD ₃ OD, 125 MHz) δ 151.4 (C-3), 149.1 (C-3 '), 148.3 (C-3''), 147.9 (C-4), 147.6 (C-4' ), 147.3 (C-4``), 136.3 (C-1), 133.7 (C-1 ''), 132.9 (C-1 '), 121.4 (C-6''), 120.9 (C-6) , 120.7 (C-6 '), 117.7 (C-5), 116.2 (C-5'), 115.8 (C-5``), 112.7 (C-2), 111.8 (C-2 ''), 111.7 (C-2 '), 89.1 (C-7'), 84.4 (C-7), 81.7 (C-8 ''), 78.3 (C-7 ''), 56.6 (-OCH3), 56.4 (-OCH3 ), 56.3 (-OCH3), 49.5 (C-8 '), 47.0 (C-8), 16.5 (C-9``), 15.2 (C-9), 14.8 (C-9')

Manasanthin A

¹ H-NMR (CDCl ₃ , 500 MHz) δ 6.99 (1H, d, J = 8.1 Hz, H-2 ′), 6.94 (2H, d, J = 8.1 Hz, H-2,3 ′), 6.92 ( 1H, s, H-6 '), 6.84 (1H, d, J = 8.1 Hz, H-3), 6.84 (1H, s, H-6), 5.47 (2H, d, J = 5.8 Hz, H- 7 ', 7''), 4.65 (1H, d, J = 8.3 Hz, H-7), 4.15 (1H, m, H-8), 3.89 (6 x -OCH ₃ ), 2.31 (2H, m, H-8 ', 8''), 1.17 (3H, d, J = 6.2 Hz, H-9), 0.73 (6H, d, J = 6.3 Hz, H-9', 9 '')

¹³ C-NMR (CDCl ₃ , 125 MHz) δ 150.6 (C-5 '), 148.9 (C-5), 148.8 (C-4), 146.5 (C-4'), 136.5 (C-1 '), 132.5 (C-1), 120.0 (C-3 '), 118.7 (C-2'), 118.7 (C-6), 110.8 (C-3), 110.1 (C-6 '), 109.9 (C-2 ), 84.1 (C-8), 83.4 (C-7 ', 7''), 78.4 (C-7), 55.9 (6x-OCH3), 44.2 (C-8,8''), 17.1 (C- 9), 14.9 (C-9 ', 9'')

Manasanthin B

¹ H-NMR (CDCl ₃ , 500 MHz) δ 7.02-6.79 (12H, H-6``, 6 ', 5,6,5', 5 '', 6 ''',2''', 2, 2 ', 2'',5'''), 5.95 (2H, s, -OCH ₂ O-), 5.46 (2H, d, J = 5.7 Hz, H-7 ', 7''), 4.67 (1H , d, J = 8.2 Hz, H-7), 4.63 (1H, d, J = 8.2 Hz, H-7 '''), 4.12 (1H, m, H-8), 4.12 (1H, m, H -8 '''), 3.90 (4 x -OCH ₃ ), 2.32 (2H, m, H-8', 8 ''), 1.20 (3H, d, J = 4.6 Hz, H-9 ''') , 1.18 (3H, d, J = 4.6 Hz, H-9), 0.73 (6H, d, J = 5.7 Hz, H-9 ', 9'')

¹³ C-NMR (CDCl ₃ , 125 MHz) δ 150.6 (C-4 ', C-4``), 149.0 (C-3'''), 148.8 (C-4 '''), 147.8 (C- 3), 147.4 (C-3``), 146.5 (C-3 '), 146.3 (C-4), 136.6 (C-1''), 136.5 (C-1'), 134.0 (C-1 '''), 132.6 (C-1), 121.1 (C-6), 120.0 (C-6 '''), 118.9 (C-5'), 118.7 (C-6``, C-5 '', C-6 '), 118.7 (C-5), 110.8 (C-2``), 110.1 (C-2'), 110.0 (C-2), 108.1 (C-5 '''), 107.6 (C -2 '''), 101.0 (-OCH ₂ O-), 84.1 (C-8'''), 84.0 (C-8), 83.4 (C-7 ', C-7''), 78.4 (C -7 '''), 78.4 (C-7), 56.0 (-OCH3), 44.2 (C-8', C-8 ''), 17.1 (C-9 '''), 17.0 (C-9) , 15.0 (C-9 ', C-9'')

Sauuccinon

¹ H-NMR (CDCl ₃ , 500 MHz) δ 6.38 (1H, s, H-3), 6.82 (1H, s, H-6), 3.03 (1H, d, J = 5.4, H-7), 2.44 (1H, m, H-8), 1.22 (3H, d, J = 7.3, H-9), 2.52 (1H, td, J = 11.9, 3.5, H-1 '), 5.57 (1H, s, H -3 '), 2.48 (1H, d, J = 5.4, H-6'), 1.74 (1H, m, H-7'eq), 1.92 (1H, m, H-7'ax), 1.88 (1H) , m, H-8 '), 0.71 (3H, d, J = 7.4, H-9'), 5.90, 5.87, 5.65, 5.60 (4H, s, OCH ₂ O)

¹³ C-NMR (CDCl ₃ , 125 MHz) δ 115.73 (C-1), 145.70 (C-2), 99.50 (C-3), 143.26 (C-4), 146.72 (C-5), 105.54 (C -6), 35.06 (C-7), 34.82 (C-8), 21.29 (C-9), 37.55 (C-1 '), 194.69 (C-2'), 101.34 (C-3 '), 168.66 (C-4 '), 100.42 (C-5'), 37.60 (C-6 '), 25.27 (C-7'eq, ax), 33.45 (C-8'), 20.92 (C-9 '), 101.24 (OCH ₂ O), 98.19 (OCH ₂ O)

Example 2: Inhibitory Effect of Three hundred Second Extracts on Proliferation of Mouse Spleen Cells Induced by LPS or Con A

The immunosuppressive effects of the three hundred sec extract according to the present invention, sachinone, (-) soserol, soserenol C, manasanthin A and manasanthin B isolated and purified from the extract were investigated as follows.

Spleens were extracted by disinfection with 70% isopropyl alcohol from untreated BALB / c mice (Orien Bio Co., Ltd.) and soaked in EBSS (Earl's Balanced Salt Solution, Sigma). Using a syringe plunger, the spleen was crushed into single cells, placed in a 15 ml tube and left at room temperature for 5 minutes. When the dregs settled down, the Pasteur pipette was used to transfer only the upper layer to a new tube. The supernatant was discarded by centrifugation at 1,200 rpm for 10 minutes to separate only splenocyte pellets, which were RPMI 1640 supplemented with 10% FBS, 2 mM L-glutamine, 100 units / ml penicillin and 100 µg / ml streptomycin. Suspended in the medium. At this time, the concentration of the cell suspension was 1 × 10 ⁶ cells per ml.

Dispense the cell suspension into a 96-well flat bottom microplate (Falcon) at 100 μl per well, and then add Con A or LPS (lipopolysaccharide) as a mitogen to a final concentration of 1 μg / ml or 20 μg / ml, respectively. Treated as possible. The test substance to be tested was diluted to concentrations of 0, 0.1, 1 and 10 μg / ml and added to each well, and then the total culture volume in the well was adjusted to 200 μl. After addition of the constituents to each well, the microplates were incubated for 72 hours in a 37 ° C., 5% CO ₂ incubator.

After 72 hours, 20 μl of the reagent in the cell counting kit (Cell Counting Kit, DOJIDO Laboratories) was added to each well and incubated for another 2 to 4 hours. The extent of reagent development in the 96-well microplate was checked and the absorbance was measured at 450 nm using an ELISA reader.

The absorbance values of the wells treated with the test substance were limited to the blank value of the well in which only the test substance was added to the culture medium without cells, and then compared to the absorbance value of the control group in which the same amount of the medium was used instead of the test substance. It was converted according to the following equation (1 ). In addition, based on the cell proliferation rate of the sample group measured at each concentration, using a data regression of the SigmaPlot program, the sample inhibits the proliferation of immune cells by 50% (ED ₅₀ ). Calculated.

[(T-Tz) / (C-Cz)] × 100

Wherein T is the absorbance of the cell culture solution treated with the test substance; Tz is the absorbance of the culture medium without test cells added; C is the absorbance of the cell culture without treatment of the test substance; Cz is the absorbance of the culture without both test substance and cells added.

As described above, the concentrations of Saucinone, Soserenol C, Manastanthin A, Manastanthin B, and (-)-Soseneol, which were isolated and purified from the extract of 300 sec, in mouse splenocyte culture medium pretreated with 20 µg / ml LPS As a result, as shown in FIG . 1 , B cell proliferation was inhibited in a concentration-dependent manner in the experimental group treated with four compounds except sacchinone, as shown in FIG . 1 . Table 2 below shows the 50% cell growth inhibition concentration (ED ₅₀ ) of the sample compound, while the sacchinone showed an ED ₅₀ value of 10 μg / ml or more, whereas soserenol C, manasanthin A, manasanthin B, and ( -)-Soserenol showed a very low ED ₅₀ value, it was confirmed that the immunosuppressive effect is excellent.

In addition, Succinone, Soserenol C, Manasanthin A, Manasanthin B, and (-) isolated from Pur 300 extracts in mouse spleen cell cultures treated with Con A, a T cell proliferative substance, at a concentration of 1 μg / ml. As a result of treating each of the) -soseneol by concentration, as shown in Figure 2 , the proliferation of T cells was significantly inhibited in the experimental group treated with four compounds except sacchinone. In addition, the 50% cell proliferation inhibitory concentration (ED ₅₀ ) of each sample compound is shown in Table 3 below , where the ED ₅₀ values of socerenol C, manasanthin A, manasanthin B, and (-)-soseneol are very low. Able to know.

Example 3: Inhibitory Effect of Three hundred Second Extract on Proliferation of Mouse Spleen Cells through Mixed Leukocyte Reaction

In order to determine the effect of the compounds isolated and purified from the Triticale extract on cells extracted from DBA mice and Balb / C mice, which are not genetically identical, the mixed leukocyte reaction was performed as follows. At this time, cells extracted from DBA mice were treated with mitomycin C to stop DNA synthesis, and only proliferation of splenocytes derived from Balb / C was possible.

Specifically, the spleen extracted aseptically with 70% isopropyl alcohol from an untreated DBA mouse (Orient Bio Co., Ltd.) was immersed in EBSS. After crushing the spleen into a single cell using a syringe plunger, mitomycin C was added to a tube containing cells at a concentration of 50 μg / ml and incubated for 45 minutes in a 37 ° C., 5% CO ₂ incubator. After incubation, cells were washed three times with EBSS and then suspended in RPMI 1640 medium supplemented with 10% FBS, 2 mM L-glutamine, 100 units / ml penicillin and 100 μg / ml streptomycin. At this time, the concentration of the cell suspension was 4 × 10 ⁶ cells per ml.

On the other hand, splenocytes in the form of single cells prepared in the same manner as above from untreated BALB / c mice were placed in a 15 ml tube and allowed to stand at room temperature for 5 minutes. When the dregs settled down, the Pasteur pipette was used to transfer only the upper layer to a new tube. The supernatant was discarded by centrifugation at 1,200 rpm for 10 minutes, and only splenocyte pellets were separated and suspended in RPMI 1640 medium supplemented with 10% FBS, 2 mM L-glutamine, 100 units / ml penicillin, and 100 µg / ml streptomycin. At this time, the concentration of the cell suspension was 1 × 10 ⁶ cells per ml.

The two kinds of cell suspensions prepared above were added to the 96-well U-type bottom microplate (Falcon) by 90 μl per well, and each test substance was diluted to 0,1, 10 and 100 μg / ml concentrations. 20 μl each was added. After adding all the components to each well was incubated for 5 days at 37 ℃, 5% CO ₂ incubator.

After the incubation, 20 μl of each reagent in a cell counting kit (Cell Counting Kit, DOJIDO Laboratories) was added to check the proliferation capacity of the splenocytes and incubated for another 2 to 4 hours. The extent of reagent development in the 96-well microplate was checked and the absorbance was measured at 450 nm using an ELISA reader.

The absorbance values of the wells treated with the test substance were limited to the blank value of the well in which only the test substance was added to the culture medium without cells, and then compared to the absorbance value of the control group in which the same amount of the medium was used instead of the test substance. Converted according to Equation 1 above. Based on the cell proliferation rate of the sample group measured at each concentration, using the data regression analysis of the sigma plot program was calculated the concentration (ED ₅₀ ) that the sample inhibits the proliferation of immune cells by 50%.

As a result, as shown in Figure 3 , except for sacchinone, Soserenol C, Manasanthin A, Manasanthin B and (-)-Soseranol showed the effect of inhibiting the proliferation of B cells, the mouse of each compound ED ₅₀ of the inhibitory effect on the mixed leukocyte response was calculated as shown in Table 4 below.

The extract of the present invention or the active ingredient separated and purified therefrom may be in the form of preparations such as powders, tablets, capsules, injections, liquids and the like according to the methods commonly used pharmaceutically or in combination with excipients used alone or pharmaceutically. Can be formulated and used.

Formulation examples are illustrated below.

Production Example 1 Powder

3 g dry extract or 2 g of active ingredient isolated and purified from

1 g lactose

The above ingredients are mixed and filled in an airtight cloth to prepare a powder.

Preparation Example 2 Tablet

100 mg dry extract or active ingredient isolated and purified from it

Corn starch 100 mg

Lactose 100 mg

2 mg magnesium stearate

After mixing the above components and tableting according to a conventional tablet production method to produce a tablet.

Preparation Example 3 Capsule

100 mg dry extract or active ingredient isolated and purified from it

Corn starch 100 mg

Lactose 100 mg

2 mg magnesium stearate

After mixing the above components to fill a gelatin capsule in accordance with the conventional capsule preparation method to prepare a capsule.

Production Example 4 Injection

100 mg dry extract or active ingredient isolated and purified from it

Suitable amount of distilled water for injection

pH adjuster

According to a conventional injection preparation method, the active ingredient is dissolved in distilled water for injection, the pH is adjusted to about 7.5, and the whole is filled with 2 ml of ampoule with injection distilled water and sterilized to prepare an injection.

In addition, health foods and alcoholic beverages are prepared in the following manner.

<Manufacture example 5> Wire type

Brown rice, barley, glutinous rice, and yulmu were alphad by a known method, and then dried and roasted to make a powder having a particle size of 60 mesh. Black beans, black sesame seeds, and perilla were also steamed and dried in a known manner, and then ground to a powder having a particle size of 60 mesh.

The extract of the present invention was concentrated under reduced pressure and concentrated in a vacuum concentrator, and dried by spraying and drying with a hot air dryer. The dried product was pulverized with a particle size of 60 mesh to obtain an extract dry powder.

Granules were prepared by combining the dry powders of the grains, seeds and triticale extract prepared above in the following proportions.

Cereals: Brown rice 30% by weight, barley 15% by weight, barley 20% by weight, glutinous rice 9% by weight,

Seeds: perilla 7% by weight, black beans 8% by weight, black sesame 7% by weight,

Three hundred percent extract dry powder 3% by weight, ganoderma lucidum 0.5% by weight, sulfuric acid 0.5% by weight

Preparation Example 6 Chewing Gum

Chewing gum was prepared in a conventional manner by combining 20% by weight of gum base, 76.9% by weight of sugar, 1% by weight of perfume, and 2% by weight of water, and 0.1% by weight of Trichophyt extract of the present invention.

Production Example 7 Candy

Candy was prepared by the conventional method by combining 60% by weight of sugar, 39.8% by weight of starch syrup, 0.1% by weight of fragrance, and 0.1% by weight of three hundred vine extract of the present invention.

Production Example 8 Biscuits

Force 1st class 25.59 wt%, 1st class gravity 22.22 wt%, white sugar 4.80 wt%, salt 0.73 wt%, glucose 0.78 wt%, palm shortening 11.78 wt%, ammonium 1.54 wt%, sodium bicarbonate 0.17 wt%, sodium bisulfite 0.16 wt %, Rice flour 1.45 wt%, Vitamin B₁0.0001 wt%, Vitamin B20.0001 wt%, Milk flavor 0.04 wt%, Water 20.6998 wt%, Whole milk powder 1.16 wt%, Substitute milk powder 0.29 wt%, Calcium phosphate 0.03 wt% , Biscuits were prepared in a conventional manner by combining 0.29% by weight of spraying salt and 7.27% by weight of spray oil with 1% by weight of three hundred vine extract of the present invention.

<Manufacture example 9> Healthy drink

0.26% by weight of honey, 0.0002% by weight of thioctoamide, 0.0004% by weight of nicotinic acid, 0.0001% by weight of sodium riboflavinate, 0.0001% by weight of pyridoxine hydrochloride, 0.001% by weight of inositol, 0.002% by weight of orthoic acid and 98.7362% by weight of water 1300% by weight of 300 seconds extract of was prepared by the conventional method for the health beverage.

Preparation Example 10 Health Supplement

55% by weight of spirulina, 10% by weight of guar gum enzyme, 0.01% by weight of vitamin B ₁ hydrochloride, 0.01% by weight of vitamin B6 hydrochloride, 0.23% by weight of DL-methionine, 0.7% by weight of magnesium stearate, 22.2% by weight of lactose and 1.85% by weight of corn starch And formulated with tablets health supplements in a conventional manner by combining 10% by weight of three hundred seconds extract of the present invention.

As described above, the composition containing the three hundred sec extract according to the present invention, (-) soserolol, soserolol C, manasanthin A or manasanthin B isolated and purified from the B cell and T cells Since it effectively inhibits abnormal proliferation and shows an excellent immunosuppressive effect, it can be usefully used for the prevention and treatment of diseases caused by immune hypersensitivity reactions.

Claims (7)

Compound selected from the group consisting of (-)-soseneol [(-)-saucerneol], sosserol C, manasantin A, manasanthin B, and mixtures thereof; chinensis) Pharmaceutical composition for immunosuppression containing the extract as an active ingredient. The method of claim 1, Pharmaceutical composition, characterized in that the extract extracts three hundred seconds or its dried matter with an organic solvent selected from the group consisting of methanol, aqueous methanol solution, ethanol, aqueous ethanol solution, butanol, dichloromethane, ethyl acetate and mixtures thereof. The method of claim 1, Organ transplant rejection reactions caused by the immunological hypersensitivity reaction of the pharmaceutical composition; Autoimmune diseases of lupus or rheumatoid arthritis; Pharmaceutical composition, characterized in that used for the prevention or treatment of allergic diseases of rhinitis, asthma, atopy. A health promoting food that suppresses immune hypersensitivity reactions containing an active ingredient selected from the group consisting of (-)-soseneol, soseneol C, manasanthin A, manasanthin B, and mixtures thereof, or three hundred sec extract comprising the same. The method of claim 4, wherein Health foods, characterized in that the food is in the form of a drink. 300 seconds extract obtained by extraction with an organic solvent selected from the group consisting of methanol, methanol aqueous solution, ethanol, ethanol aqueous solution, butanol, dichloromethane, ethyl acetate and mixtures thereof, and exhibits an immunosuppressive effect. The method of claim 6, The extract is a three hundred seconds extract, characterized in that after extracting three hundred seconds with methanol, the obtained extract is suspended in distilled water, extracted with dichloromethane, and then the obtained water layer is extracted with ethyl acetate.

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