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WO2010036065A2 - Composition pharmaceutique servant à la prévention et au traitement de maladies inflammatoires et contenant une fraction acétate d'éthyle d'extrait sec de caulis trachelospermi comme principe actif, ainsi que procédé de production de cette fraction - Google Patents

Composition pharmaceutique servant à la prévention et au traitement de maladies inflammatoires et contenant une fraction acétate d'éthyle d'extrait sec de caulis trachelospermi comme principe actif, ainsi que procédé de production de cette fraction Download PDF

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Publication number
WO2010036065A2
WO2010036065A2 PCT/KR2009/005504 KR2009005504W WO2010036065A2 WO 2010036065 A2 WO2010036065 A2 WO 2010036065A2 KR 2009005504 W KR2009005504 W KR 2009005504W WO 2010036065 A2 WO2010036065 A2 WO 2010036065A2
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Prior art keywords
ethyl acetate
rockfall
fraction
extract
inflammatory diseases
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Ceased
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PCT/KR2009/005504
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English (en)
Korean (ko)
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WO2010036065A3 (fr
WO2010036065A9 (fr
Inventor
이정민
박지훈
유재훈
노경미
김세나
안은숙
이영석
이영준
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SHIN-IL PHARMACEUTICAL Co Ltd
Shin il Pharmaceutical Co Ltd
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SHIN-IL PHARMACEUTICAL Co Ltd
Shin il Pharmaceutical Co Ltd
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Publication date
Priority claimed from KR1020090090715A external-priority patent/KR101192409B1/ko
Application filed by SHIN-IL PHARMACEUTICAL Co Ltd, Shin il Pharmaceutical Co Ltd filed Critical SHIN-IL PHARMACEUTICAL Co Ltd
Priority to US13/120,943 priority Critical patent/US8945633B2/en
Priority to JP2011528943A priority patent/JP5514216B2/ja
Priority to CN2009801414976A priority patent/CN102186485B/zh
Publication of WO2010036065A2 publication Critical patent/WO2010036065A2/fr
Publication of WO2010036065A9 publication Critical patent/WO2010036065A9/fr
Publication of WO2010036065A3 publication Critical patent/WO2010036065A3/fr
Anticipated expiration legal-status Critical
Ceased legal-status Critical Current

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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K36/00—Medicinal preparations of undetermined constitution containing material from algae, lichens, fungi or plants, or derivatives thereof, e.g. traditional herbal medicines
    • A61K36/18—Magnoliophyta (angiosperms)
    • A61K36/185—Magnoliopsida (dicotyledons)
    • A61K36/24—Apocynaceae (Dogbane family), e.g. plumeria or periwinkle
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P29/00—Non-central analgesic, antipyretic or antiinflammatory agents, e.g. antirheumatic agents; Non-steroidal antiinflammatory drugs [NSAID]

Definitions

  • the present invention relates to a pharmaceutical composition for the prevention and treatment of inflammatory diseases containing an ethyl acetate fraction of a dry extract of rockfall, etc. as an active ingredient and a method for producing the fraction.
  • arthritis is a medical name collectively referred to as an inflammatory change in a joint caused by some cause such as bacteria or trauma. Arthritis is divided into acute and chronic depending on the duration of inflammation.
  • Serous arthritis is usually caused by trauma, with some unknown causes, usually occurring in only one joint.
  • Serous fibrotic arthritis occurs during acute joint rheumatism, which is characterized by turbid effusions in the joint cavity, and in general, fibrous gastric membranes that cause severe movement disorders even when inflammation subsides. .
  • Purulent arthritis is common in open windows of joints or infectious diseases such as gonorrhea, typhoid fever, sun red fever, and sepsis. Infants who are 1 to 2 months old are severely injured and cannot be treated. Caused by periosteal osteomyelitis in adults, the pus burst into the joints and often go into the joints, this is called secondary arthritis.
  • Chronic arthritis is classified as follows:
  • Specific inflammation refers to gouty arthritis caused by tuberculous syphilis toxicity or metabolic disorders of many uric acid in middle-aged men. Multiple arthritis is caused by chronic arthritis. ( ⁇ ) or in the course of tuberculosis, syphilis, gonorrhea may appear multiple, and one of the sepsis.
  • Still's disease In addition, it also includes arthritis called Still's disease.
  • Deformed osteoarthritis is caused by aging or trauma to bones or joints, and hemophilia ( ⁇ ⁇ ⁇ ⁇ ) arthritis is caused by bleeding in the joints when hemophilia.
  • the rockfall lamp (Trachelospermi caulis), also known as the bark line, is a evergreen vine of the oleander family. It grows up to 5 meters long. The petals are deeply divided into five, with a pinwheel-shaped white flower from May to June, and the fragrance is very good. The fruit is held from September to November. The two long fruits often represent human beings, and sometimes they make a round circle like a bracelet and admire them.
  • rockfall grows on the southern coast of Korea, in the foothills and fields, and in the wasteland, and usually grows on rocks, fences, and other trees and plants. In places where there are many rockfalls, there is a place where no other grass grows and all sides are covered with rockfalls.
  • Falling rocks are used for adding paraplegia, dew, etc. to limb paralysis pain, muscle cramps, and inability to flex. Since the blood cools, sore throat, boil, water, etc., take the month to take. The fruit of the rockfall is called the rockfall family ( ⁇ ⁇ ⁇ ), and ripe mature fruits are collected around July and used. Leaves and stems are mainly used, and they can be harvested at any time regardless of the season and dried in the sun to be chopped.
  • tracheloside Tracheloside
  • actin Arctiin
  • materesinoside Moatairesinoside
  • arctigenin notracheloside
  • an object of the present invention is to provide a method for preparing an ethyl acetate fraction of a dried rock extract, which has been purified and concentrated to have the best anti-inflammatory activity in the rock extract.
  • Another object is to provide a pharmaceutical composition for preventing and treating inflammatory diseases containing ethyl acetate fraction of the dry extract such as rockfall as an active ingredient.
  • the present invention is an ethyl acetate fraction of the dry extract of rockfall, etc., containing an extract of purified and concentrated so as to contain 0.05 to 12% by weight of actinogen, an indicator, as an active ingredient. It is characterized by a pharmaceutical composition for preventing and treating inflammatory diseases.
  • the present invention has developed an ethyl acetate fraction (hereinafter referred to as ethyl acetate fraction of rockfall) and the preparation method of the dried rockfall extract which significantly improved the anti-inflammatory effect of the conventional rock extract such as rockfall, and applied to the pharmaceutical composition It is to be done.
  • the ethyl acetate fraction of rockfall, etc., in the present invention refers to a material obtained by adding a solvent to a rockfall, etc. dry extract and then partitioning and concentrating.
  • the dry extract of rockfall lamp refers to the dry rock extract extracted with water or an organic solvent before fractionation.
  • water or alcohols selected from methanol, ethanol and butanol as the solvent used for extraction and fractionation of rockfall. This is because water and alcohol meet the pharmacopeia regulations.
  • chromatography is preferably performed. Specifically, it is preferable to use a method of separating the polar mobile phase and the nonpolar stationary phase or the nonpolar mobile phase and the polar stationary phase. More specifically, it is preferable to perform normal phase and reverse phase chromatography repeatedly to further increase the purity.
  • the normal phase chromatography refers to a silica gel base stationary phase
  • the reverse phase chromatography refers to an ODS (Octar-desil-silica) -based stationary phase, specifically, C-18 ODS, and the like. Solvents used in the art will be apparent to those skilled in the art, and detailed descriptions thereof will be omitted.
  • Actigenin in the present invention is a substance consisting of a molecular weight of 372 of the molecular formula C21H24O6, Dibenzylbutyroactone ligand (Dibenzylbutyroactone ligand), one of the substances that are actively studied on the biological activity in various plants, particularly Asteraceae, Actinin ) Is a substance consisting of molecular weight 534 of the molecular formula C27H34O11 and refers to a substance found in various plants and having a glycoside-bound form of actinogen.
  • the present invention is an ethyl acetate fraction of the dry extract of rockfall, etc., which should contain 0.05 to 12% by weight of the actinogen, indicator material, which is less than 0.05% by weight, the synergistic effect of the drug is reduced and exceeds 12% by weight. Since the lower surface process is added and the production cost is increased while the drug efficacy is not increased, it is preferable to apply the above-described content range of the indicator substance to the manufacturing process. In general, it is more preferable to standardize the content of actinizenin, which is an indicator, at 0.4 to 3.0% by weight in order to simplify the manufacturing cost and the process. In addition, it is more preferable that 6-20 wt% of actin is contained as another indicator of the fraction of the present invention.
  • Ethyl acetate fractions such as rockfall extracted and purified according to the present invention can be used as such, but are mixed with pharmaceutically acceptable carriers, excipients, diluents, powders, granules, capsules, Ointments, transdermal or injectables can be prepared.
  • fractions such as rockfall according to the present invention has been used for food and medicinal use since ancient times, and there is no particular restriction on the dosage, body absorption, weight, age, sex, health condition, diet, administration time, administration of the body. It may vary depending on the method, the rate of excretion, the severity of the disease and the like.
  • ethyl acetate fractions such as rockfall are preferably administered in an amount of 100 to 1000 mg / day for the adult (60 kg basis). Since the ethyl acetate fraction of rockfall according to the present invention shows an excellent effect of about 20 ⁇ 300% compared to the efficacy of the ordinary rockfall extract, it can be administered by setting a dose applied thereto.
  • the pharmaceutical composition comprising the active ingredient of the present invention is to be prepared in consideration of the effective amount range, the unit dosage form formulated in this way is the needs of the individual and the judgment of the expert to monitor or observe the administration of the drug as needed Depending on the specific dosage regimen, it may be administered several times at regular time intervals.
  • Inflammatory diseases in the present invention include acute, chronic inflammatory diseases, rheumatic inflammatory diseases caused by autoimmune reactions, degenerative inflammatory diseases and the like.
  • a dry extract of rockfall or the like is dissolved in water, and ethyl acetate (ethyl acetate) is added to the dissolved solution and mixed to separate the water layer and the ethyl acetate layer.
  • the solvent is removed from the separated ethyl acetate layer and concentrated to contain 0.05 to 12% by weight of the actinogen, which is an indicator, to obtain an ethyl acetate fraction having excellent anti-inflammatory effect.
  • rock extracts such as rockfall, are not limited to alcohols, and may be extracted using any solvent as long as it is a solvent that can obtain a lot of active ingredients and is contained in the pharmacopoeia or is safe for human body.
  • the second process and the third process may be selectively performed according to the efficacy or effective concentration of the drug, the physical properties of the extract and the like.
  • the ethyl acetate layer separated in the first step is chromatographed to obtain an active fraction.
  • various mobile phase or fixed phase solvents used in the normal phase or reverse phase chromatography will be apparent to those of ordinary skill in the art and will not be described in detail.
  • This process is carried out in the second process to obtain an active fraction with a higher degree of purification, and then dried or concentrated to obtain an extract purified.
  • the ethyl acetate fraction of rockfall according to the present invention exhibits a markedly improved anti-inflammatory effect compared to rockfall light extract obtained by the conventional method, and can be developed as a pharmaceutical composition or functional food for preventing and treating inflammatory diseases. It was made possible.
  • Example 1 is a photograph showing the results of TLC development pattern of rock extract and fractions according to the present invention [a: Example 2, b: Example 3, c: Example 1, d: Example 4],
  • Figure 2a and Figure 2b is a photograph showing the chromatogram of actin and actinigen of the rock extract and fractions (Examples 1 and 4) according to the present invention
  • FIG. 3 is a photograph showing column fractions of ethyl acetate fractions according to the present invention separated by normal phase chromatography, separated by TLC, and
  • FIG. 5 is a photograph showing the expression of MMP-1 and MMP-3 stimulated with TNF- ⁇ in arthritis synovial tissue cells (FLS) of Example 3 of rockfall, etc., according to the present invention.
  • Figure 6 is a photograph showing the arthritis inhibitory effect of the arthritis inflammation edema evaluation test using the arthritis-induced mouse model (Example 3),
  • Figure 7 is a photograph showing the results of H & E staining of the CIA mouse joint of the rockfall fraction (Example 3) according to the present invention.
  • Figure 8 is a photograph showing the inhibitory effect of IL-1 ⁇ , IL-6, RANTES, MMP-3 mRNA expression of the rockfall fraction (Example 3) in arthritis induced mouse joints,
  • FIG. 9 is a photograph showing the inhibitory effect of MMP-1, 3 protein expression in arthritis model animal joint tissue of the rockfall fraction (Example 3) according to the present invention.
  • Example 2 100 g of the 30% ethanol extract prepared in Example 2 was dissolved in about 0.8 L of water, dissolved, and then mixed with the same amount of ethyl acetate (330 mL X 5), followed by fractionation, to obtain an ethyl acetate soluble fraction.
  • An appropriate amount (about 125 g) of sodium thiosulfate was added to the ethyl acetate soluble fraction, the water molecules of the fraction were absorbed and filtered to purify the ethyl acetate soluble fraction.
  • the purified fractions were concentrated using a vacuum filter to obtain about 11 g of ethyl acetate fractions such as rockfall.
  • Example 1 100 g of the 95% ethanol extract prepared in Example 1 was dissolved in about 0.8 to 2 L of water, dissolved, and the same amount to 2 times of ethyl acetate were mixed and fractionated to obtain an ethyl acetate soluble fraction.
  • An appropriate amount (about 125 g) of sodium thiosulfate was added to the ethyl acetate soluble fraction, the water molecules of the fraction were absorbed and filtered to purify the ethyl acetate soluble fraction.
  • the purified fractions were concentrated using a vacuum filter to obtain about 16.7 g of ethyl acetate fractions such as rockfall.
  • the content of the indicator component Actin of the rockfall lamp extract of Example 2 and the rockfall lamp fraction of Example 3 was analyzed using a high performance liquid chromatography apparatus.
  • Example 2 the actin content was found to contain about 3% by weight, and the actin content in Example 3 was changed in content every fraction. That is, the largest amount of ethyl acetate fraction was detected in about 19% at one time, and the actin content was gradually decreased from 2 to 5 times, indicating the content of about 6%. The ethyl acetate fraction fractionated five times was analyzed for an average content of about 12-15%.
  • Example 1 the actin content was found to contain about 2% by weight and 0.5% by weight of actinigen, and the actin content in Example 4 was about 6% by weight, and about 1.2% by weight of actigenin when fractionated three times. This was measured.
  • Ethyl acetate fractions such as rockfall obtained in Example 3 were prepared using silica gel column chromatography using a step gradient solvent system composed of methylene chloride: methanol (1000: 5 to 95: 5). Separated.
  • the rock acetate ethyl acetate fraction is suspended about 12 times with silica gel in the ratio of methylene chloride 1000: methanol 5, which is a mobile phase, and finely stacked on a glass column.Then, the rock acetate ethyl acetate fraction is about 0.5 to 1 times.
  • Suspended in silica gel of and adsorbed by concentrated under reduced pressure was injected into a column formed.
  • the concentration gradient was changed stepwise to the ratio of methylene chloride 95: methanol 5 at about 5 times the capacity of the silica gel to receive the fractions in the capacity of the silica gel column (about 100 mL units), and each of the obtained fractions was obtained in Example 5 above. Separation was confirmed through silica gel TLC as in the method (see FIG. 3). In this case, 95% of methylene chloride, 5% of methanol or 80% of chloroform, 15% of methanol, and 5% of water were used.
  • RAW 264.7 (ATCC, # TIB-71) cells, mouse-derived macrophages, were treated with Dulbecco's Modified Eagle Medium (DMEM), 10% fetal bovine serum (FBS), penicillin (100 units / ml) and streptophore at 5% CO2 and 37 ° C. Cultures were made using mycin sulfate (100 g / ml). For each assay, RAW 264.7 cells were seeded at 96 ⁇ well plates at 2 ⁇ 10 4 cells / well and treated with concentrations of 300 ng / ml LPS and fractions after 24 hours.
  • DMEM Dulbecco's Modified Eagle Medium
  • FBS fetal bovine serum
  • penicillin 100 units / ml
  • streptophore streptophore at 5% CO2 and 37 ° C. Cultures were made using mycin sulfate (100 g / ml).
  • RAW 264.7 cells were seeded at 96
  • Cytotoxicity was incubated for 4 hours using TACS XTT Cell Proliferation / Viability Assay kit (R & D systems, Cat. 4891-025-K) and measured at OD490nm.
  • Table 1 is a result of converting the NO production inhibitory effect of the 30% ethanol extract (Example 2), such as rockfall and ethyl acetate extract (Example 3), converted to IC50. It was found that the effect was improved to 379 ⁇ g / ml while the range of concentration inhibiting 50% was purified from Example 1-3 at 1180 ⁇ g / ml in Example 2.
  • Example 4 the NO production inhibition rate of the extracts and fractions (Examples 1 and 4) of rockfall, etc. was analyzed in the same manner as described above. At the highest concentration of 320 ⁇ g / ml showing no cytotoxicity of Examples 1 and 4, the NO production inhibition rate of the ethanol extract of Example 1 showed a relatively high effect of 68%. In addition, it can be seen that Example 4, which is an ethyl acetate extract, showed a higher effect by showing a higher inhibition rate of 80% [Table 2].
  • the levels of IL-1 ⁇ and TNF- ⁇ in the culture were measured by ELISA using an IL-1 ⁇ assay kit (R & D systems, Cat. MLB00B) and TNF- ⁇ assay kit (R & D systems, Cat. MTA00). Absorbance was measured using a microplate reader at OD450nm UV. The results of the analysis were obtained in relative% based on the LPS-stimulated treatment group.
  • the treatment concentration of the sample in the cytokine analysis was performed based on a concentration range that does not exhibit cytotoxicity as a result of cytotoxicity experiments.
  • Example 3 Comparative analysis of the 30% ethanol rockfall extract (Example 2) and the rock acetate ethyl acetate fraction (Example 3) is shown in Table 3 below.
  • the inhibition rate of 38% and 42% was shown in Example 3, respectively, which showed higher inhibitory effect than that of Example 2 [Table 3].
  • This is the value analyzed in the highest toxicity range (Example 2: 640 [mu] g / ml, Example 3: 160 [mu] g / ml,) showing no effect on cytotoxicity.
  • the ethyl acetate fractions showed relatively superior inhibitory effects of TNF- ⁇ and IL- ⁇ , which are classified as inflammatory cytokines.
  • the active ingredients are indirectly more friendly to nonpolar solvents, and it can be seen that the ethyl acetate solubles of the active substances were purified.
  • Example 7 the purification result of the cytokine analysis of fractions 2, 5, and 6 fractions purified in Example 7 for further purification test is shown in FIG. 4.
  • IL-1 ⁇ , TNF- ⁇ inhibitory effect was shown to be a more purified form showing a high inhibitory effect at a lower concentration.
  • the extract purified according to the present invention is considered to have a very good anti-inflammatory effect in comparison with the conventional extract.
  • Synovial tissues from arthritis patients were cut with scissors to about 1 ⁇ 1 ⁇ 1 mm and incubated at 37 ° C. for 2 hours in HBSS buffer in which 1 mg / ml collagenase was dissolved. Only the supernatant was separated using a 70 ⁇ m nylon cell strainer (BD, NJ, USA). The isolated cells were washed by centrifugation at 1500 rpm for 2 minutes at 10 minutes. After removing the supernatant, add ⁇ -MEM medium (10% FBS, 1% L-Glutamine, 1% penicillin-streptomycin) to the pellet, transfer to T75-flask, 37 °C, 2 ⁇ 3 hours in CO2 incubator After incubation, washed twice and incubated. More than 95% FLS was obtained after 3-6 passages (> 95% surface markers for fibroblasts (CD90 +), ⁇ 1% CD3 +, ⁇ 1% CD11b, ⁇ 1% Fc ⁇ Receptor II positive). It was.
  • the FLS obtained in Test Example 3 was dispensed into 6 well plates at 2 ⁇ 10 5 / well. After incubation for 3 days, the culture medium was replaced with incomplete ⁇ -MEM (1% L-glutamin, 1% antibiotics), and after treatment of fractions such as rockfall (Example 3) at 10, 25, 50 ⁇ g / ml concentration, 30 Minutes later, TNF- ⁇ was treated with 20 ng / ml, and after 24 hours, only the culture medium was separated and extracted. The separated extracted culture was subjected to immunoblotting analysis using a 10% SDS-PAGE gel. Antibodies MMP-1 and MMP-3 were used to confirm the effects on MMPs secreted from FLS and are shown in FIG. 5. As shown in FIG. 5, significant decreases of MMP-1 and MMP-3 were confirmed depending on the concentrations of 10, 25, and 50 ⁇ g / ml of the rockfall fractions of Example 3.
  • Test Example 5 Collagen-induced arthritis (CIA) mouse model
  • mice were purchased 5 weeks old male DBA / 1J mice, purified for 2 weeks, and then tested at 7 weeks of age.
  • 100 ⁇ g bovine Type II collagen (in 0.05M acetic acid) was mixed with the same amount of Freund's complete adjuvant (Chondrex Inc., Redmond, Washington) and injected into the tail muscles (1st Immunization).
  • 50 ⁇ l (2 mg / ml) bovine type II collagen was injected again by intraperitoneal injection (i.p) (2nd Immunization).
  • i.p intraperitoneal injection
  • 2nd immunoassay distilled water (vehicle), IND (indomethacin), and rockfall fractions (Example 3) were injected orally every day as a suspension in 5% Arabian gum.
  • the arthritis score was evaluated based on the evaluation criteria of multiple joints. Evaluation was measured up to 18 days after 2nd immunization. 6 shows the results of visual evaluation of the CIA test.
  • Figure 7 shows the evaluation results of the tissue evaluation picture (H & E staining).
  • CIA animal model hind paws sacrificed 38 days after 1st immunization were extracted, immobilized CIA in fixative solution (4% paraform-alehyde in PBS), and then in demineralized solution (10% EDTA, 4% paraform-aldehyde in PBS). Weekly demineralization process was performed. Next, paraffin was treated and sectioned to 5 ⁇ m in thickness, followed by H & E (hematoxylin and eosin) staining for cartilage destruction, bone erosion, infiltration of immune cells, and degree of pannus formation. Measured.
  • H & E hematoxylin and eosin
  • RNA isolation kit (Intron, Korea). RNA was extracted and separated. After the RNA isolation lysis buffer was added, vortex was added for 30 minutes, and 200 ⁇ l of chloroform was added. After centrifugation at 13,000 rpm, 4 ° C. for 30 minutes, the supernatant was obtained, and total RNA was extracted using an RNA separation filter. The total extracted RNA was quantified using a spectrophotometer (Eppendorf, German).
  • Test Example 7 Reverse transcription-polymerase chain reaction (RT-PCR)
  • RNA extracted in Test Example 6 was carried out using a Superscript first strand synthesis system (Invitrogen, CA, USA) to perform RT-PCR. After 5 min reaction at 72 ° C. with OligodT 18mer (invitrogen, CA, USA) at 1 ug of total RNA, reaction buffer Mix (3 mM MgCl 2, 0.5 mM dNTP, 5x reaction buffer, 20U ribonuclase inhibitor, 1 ⁇ l reverse transcriptase ) was added, followed by reaction at 25 ° C-5 minutes, 42 ° C-1 hour, and 72 ° C-5 minutes. The template thus produced was used for the PCR reaction.
  • reaction buffer Mix 3 mM MgCl 2, 0.5 mM dNTP, 5x reaction buffer, 20U ribonuclase inhibitor, 1 ⁇ l reverse transcriptase
  • IL-1 ⁇ was 33 cycles (IL-6: 35 cycles) at 94 ° C-2 minutes, 94 ° C-20 seconds, 60 ° C-10 seconds (RANTES, MMP-3, GAPDH: 50 ° C) and 72 ° C-40 seconds. , RANTES, MMP-3: 35 cycles), and finally the experiment was carried out by reacting for 5 minutes at 72 °C.
  • the product thus obtained was electrophoresed on a 2% agarose gel, stained with ethidium bromide and confirmed in a UV-system as shown in FIG. 8.
  • Each PCR primer sequence is shown in Table 4 below.
  • the paw and hind leg tissues collected from CIA experimental animals were frozen in liquid nitrogen, and first crushed using a mortar and then lysis buffer (20 mM HEPES, pH 7.5, 150 mM). NaCl, 1% Nonidet p-40, 10% glycerol, 60 mM octyl ⁇ -glucoside, 10 mM NaF, 1 mM Na3VO4, 1 mM phenylmethylsulfonyl fluoride, 2.5 mM nitrophenylphosphate, 0.7 ⁇ g / ml pepstatin, and a protease-inhibitor cocktail tablet ) was added to extract the total protein. The separated and extracted proteins were quantified by Bradford assay, and immunoblot analysis was performed.
  • Tablets for oral administration were prepared using the wet granules method and the dry granules method using the ethyl acetate fraction of the present invention.
  • the ointment was prepared using the ethyl acetate fraction of the rockfall of the present invention with the following composition.
  • Injectables were prepared using the ethyl acetate fraction of the present invention, such as rockfall, in the following composition.
  • transdermal agent was prepared in the following composition.
  • ethyl acetate fractions such as rockfall, 1.3 g of sodium polyacrylate, 3.6 g of glycerin, 0.004 g of aluminum oxide, 0.2 g of methyl paraben, and 14 ml of acrylic adhesive solution.

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Abstract

L'invention concerne une composition pharmaceutique servant au traitement et à la prévention de maladies inflammatoires et contenant une fraction acétate d'éthyle d'extrait sec de Caulis trachelospermi en tant que principe actif, ainsi qu'un procédé de production correspondant. L'invention concerne plus particulièrement une composition servant à la prévention et au traitement de maladies inflammatoires et contenant une fraction acétate d'éthyle (séparation) d'extrait sec de Caulis trachelospermi en tant que principe actif, caractérisée en ce que l'extrait de Caulis trachelospermi est raffiné et concentré pour contenir 0,05 à 12% en poids d'arctigénine comme substance répertoriée.
PCT/KR2009/005504 2008-09-25 2009-09-25 Composition pharmaceutique servant à la prévention et au traitement de maladies inflammatoires et contenant une fraction acétate d'éthyle d'extrait sec de caulis trachelospermi comme principe actif, ainsi que procédé de production de cette fraction Ceased WO2010036065A2 (fr)

Priority Applications (3)

Application Number Priority Date Filing Date Title
US13/120,943 US8945633B2 (en) 2008-09-25 2009-09-25 Pharmaceutical composition for preventing and treating inflammatory diseases containing an ethyl acetate fraction of dried extract of Trachelospermi caulis as an active ingredient, and method for producing the fraction
JP2011528943A JP5514216B2 (ja) 2008-09-25 2009-09-25 絡石藤乾燥抽出物の酢酸エチル分画物を有効成分として含有する炎症性疾患の予防及び治療用薬剤学的組成物とその分画物の製造方法
CN2009801414976A CN102186485B (zh) 2008-09-25 2009-09-25 含有络石藤干燥提取物的乙酸乙酯级分作为有效成分的炎症性疾病预防及治疗用药物组合物与所述级分的制备方法

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KR10-2008-0094224 2008-09-25
KR20080094224 2008-09-25
KR10-2009-0090715 2009-09-24
KR1020090090715A KR101192409B1 (ko) 2008-09-25 2009-09-24 낙석등 건조 추출물의 에틸아세테이트 분획물을 유효성분으로 함유하는 염증성 질환 예방 및 치료용 약제학적 조성물과 상기 분획물의 제조방법

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WO2010036065A9 WO2010036065A9 (fr) 2010-06-10
WO2010036065A3 WO2010036065A3 (fr) 2010-07-22

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CN103327992A (zh) * 2010-12-03 2013-09-25 信一制药株式会社 用于预防或治疗炎性疾病的包含络石藤提取物和牡丹皮提取物的药物组合物以及制备该药物组合物的方法

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KR100847440B1 (ko) * 2007-01-24 2008-07-21 신일제약주식회사 염증성 질환 예방 및 치료용 복합 생약제

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* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN103327992A (zh) * 2010-12-03 2013-09-25 信一制药株式会社 用于预防或治疗炎性疾病的包含络石藤提取物和牡丹皮提取物的药物组合物以及制备该药物组合物的方法
CN103327992B (zh) * 2010-12-03 2016-10-12 信一制药株式会社 用于预防或治疗炎性疾病的包含络石藤提取物和牡丹皮提取物的药物组合物以及制备该药物组合物的方法
US9694045B2 (en) 2010-12-03 2017-07-04 Sinil Pharmaceutical Co., Ltd. Pharmaceutical composition for preventing or treating inflammatory diseases comprising trachelospermi caulis extract and paeonia suffruticosa andrews extract, and method for preparing the same

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