WO2006012779A1

WO2006012779A1 - Wikstroemia indeca extract their production and use in the production of anti-inflammatory pharmaceutical

Info

Publication number: WO2006012779A1
Application number: PCT/CN2004/000907
Authority: WO
Inventors: Susumu Kitanaka; Liyan Wang
Original assignee: Nihon University
Priority date: 2004-08-06
Filing date: 2004-08-06
Publication date: 2006-02-09
Also published as: WO2006012779A8; CN101014560A; CN101014560B

Abstract

The invention relates to extracts from wilstroemia indeca C.A.Mey, the preparation method and pharmaceutical uses thereof, the formula (1) of the extracted active compound.

Description

Gewang extract, preparation method thereof and application thereof in preparing anti-inflammatory drugs

The present invention relates to a genus extract, a preparation method thereof and an application thereof in the preparation of a medicament, and in particular to a compound having an anti-inflammatory effect extracted from a plant. Background technique

Daily inflammatory diseases that plague people include joint inflammation such as osteoarthritis, rheumatoid arthritis, rheumatoid arthritis, gout arthritis, etc.; inflammatory skin diseases such as eczema, psoriasis, dermatitis, etc.; inflammatory eye diseases such as Uveitis, conjunctivitis, etc.; pulmonary diseases such as asthma, bronchitis, acute respiratory distress syndrome, etc.; bacteremia, toxemia, aphthous ulcer, gingivitis, pancreatitis, etc.; gastrointestinal diseases such as festivals Segmental ileitis, atrophic gastritis, ulcerative colitis, abdominal inflammation, peptic ulcer, irritable bowel syndrome such as mucosal inflammation caused by H. pylori infection or caused by non-steroidal anti-inflammatory drugs Gastrointestinal diseases and so on.

The mechanism of various inflammatory pathogenic effects is: It is known that nitric oxide (NO) is a toxic substance that mediates cellular immunity and inflammation. Its precursor is L-arginine (L-arg), and L-arg produces NO under the action of NO synthase (NOS). Three different types of NOS have been isolated _: including endothelial NO synthase (eNOS), neuronal NO synthase (nNOS) and inducible NO synthase (iN0S). It is currently known that macrophages, hepatocytes, smooth muscle cells, adenocarcinoma cells, and epithelial cells all express iN0S. Certain inflammatory cytokines and microbial products such as lipopolysaccharide (LPS) induce iNOS expression. iNOS is highly active when it is induced, producing a large amount of NO, which in turn leads to cell damage, which causes inflammation to worsen and even causes cancer.

Therefore, for a long time, people have been searching for inhibitors of NO synthase to treat inflammatory diseases associated with them. However, most of these inhibitors are limited to chemically synthesized substances with large side effects.

Since various inflammations have long plagued humans' healthy lives, scientists have been sparing no effort to develop new types of anti-inflammatory active compounds with definite curative effects, few side effects, and low cost of development. Deepening the study of Chinese herbal medicines may be a good vehicle to achieve this desire.

Wiks troemia indica CA Mey is distributed in Guangdong, Guangxi, China. Rutaceae plants in Taiwan and other places. It has been used for swelling, pain relief, rheumatism, and tumors very early. The clinical effect of this plant in the treatment of chronic bronchitis and cancer has been affirmed. In the Dictionary of Traditional Chinese Medicine

(Shanghai People's Publishing House, published in 1977, 5, 53 pages) records: The king was born in the foothills and the damp shrubs on the slopes of the plant. Stems, leaves, roots and fruits can be used as medicines, which have the effects of clearing away heat and detoxifying, reducing swelling and dispersing, and relieving pain, treating bloating, rheumatism and bruises.

The method for extracting the active substance in the king of the king is described in the patent application No. 9811403. 9 of the "Anti-AIDS New Drugs for the Preparation of the King's Extract". However, the chemical structure and physical and chemical properties of the active substance group have not been confirmed. The parameters, so that the performance of the material in question cannot be clearly understood, and further research is not possible. Summary of the invention

One of the objects of the present invention is to find an effective active compound which can be used for the preparation of an anti-inflammatory drug by analyzing the active ingredient extracted from the king.

Another object of the present invention is to provide a method for extracting an active ingredient from a phylum.

The invention also provides a compound extracted from the king of the king for preparation: ^

The drug use of the disease.

In the present invention, there is provided a compound (1) of the formula (1), which is extracted from the king, and the method for extracting the compound of the active formula (1) from the king of the invention comprises the following steps:

1) The extract obtained by extracting the saponin from the king is concentrated under reduced pressure and dried to obtain an extract;

2) extracting the extract sequentially with n-ethane, ethyl acetate and n-butanol;

3) The ethyl acetate extract is eluted with methanol through a chromatography column;

4) The eluate collected in step 3) is further separated and purified by high pressure liquid chromatography to obtain a compound of the formula (1).

In the above extraction method, concentration under reduced pressure is preferably carried out at 40 °C.

In the above extraction method, the mobile phase of the high pressure liquid chromatography is methanol: water = 30:70.

Another aspect of the invention provides a compound of formula (1), or a pharmaceutically acceptable salt thereof, Use of an enantiomer, racemate or tautomer in the manufacture of a medicament for the treatment or prevention of a disease in which inhibition of NO production activity is beneficial.

Another aspect of the invention provides a compound of formula (1), or a pharmaceutically acceptable salt, enantiomer, racemate, tautomer or physiologically functional derivative thereof, for use in the treatment or prevention of an inflammatory disease Use in the preparation of a drug.

Suitable salts include those formed with organic and inorganic acids or bases; pharmaceutically acceptable salts include those formed with hydrochloric acid, hydrobromic acid, sulfuric acid, citric acid, tartaric acid, phosphoric acid, lactic acid, pyruvic acid, acetic acid, Fluoroacetic acid, succinic acid, oxalic acid, fumaric acid, maleic acid, butanone diacid, methanesulfonic acid, ethanesulfonic acid, p-toluenesulfonic acid, benzenesulfonic acid, isethionate; pharmaceutically acceptable basic formula Salts include ammonium salts, alkali metal salts such as sodium and potassium salts, alkaline earth metal salts such as calcium and magnesium salts, and salts with organic bases such as dicyclohexylamine and N-mercapto-D-glucosamine.

Examples of physiologically functional derivatives include esters, amides, carbamates, preferably esters and amides. The compounds of the invention may also be advantageously administered in combination with a second pharmaceutically active substance, in particular a selective inhibitor of an inducible cyclooxygenase (COX-2) isoenzyme, specifically a compound provided by the invention or a pharmaceutical thereof Use of an acceptable salt, enantiomer, racemate or tautomer in combination with a COX-2 inhibitor for the treatment of inflammation, inflammatory diseases, and diseases associated with inflammation.

Inflammatory diseases and clinical conditions include joint diseases, especially arthritis (such as rheumatoid arthritis, osteoarthritis), or gastrointestinal diseases (such as ulcerative colitis, gastritis, and other infections caused by mucosal inflammation, by Intestinal diseases caused by non-steroidal anti-inflammatory drugs), lung diseases (such as adult respiratory distress, asthma, sputum fibrosis, chronic obstructive pulmonary disease), heart disease (such as myocarditis), neurological diseases (such as multiple Sclerosis), pancreatic diseases (such as diabetes and complications), kidney diseases (such as glomerulonephritis), skin diseases (such as dermatitis, psoriasis, eczema, urticaria), eye diseases (such as glaucoma), transplant disease (eg rejection), multiple organ diseases (eg systemic lupus erythematosus) and inflammatory sequelae following viral and bacterial infections.

In addition, the compounds provided by the present invention can help prevent or treat lymphocyte loss associated with HIV infection, increase the radiosensitivity of tumor cells during radiotherapy, P tumor growth, tumor development, angiogenesis, and tumor metastasis. .

The compounds provided by the present invention can also be used in the preparation of health foods, beverages, or feeds and the like. The method for extracting the active compound in the king of the present invention is simple and practical, and the extracted active compound has a clear structure and has an exact NO inhibitory activity, and has an anti-inflammatory effect without cytotoxicity. detailed description

Example 1: Preparation of extract and determination of compound structure

2.3 kg of the king's diameter or whole grass was pulverized, added 80% methanol (melanol: water = 8:2), 10 liters, extracted three times, and concentrated and dried under reduced pressure at 40 ° C to obtain 286 g of an extract. The whole extract was dissolved in 2 liters of water, and extracted with 1 liter of each of n-hexane, ethyl acetate and n-butanol in three portions. These extracts were concentrated and dried under reduced pressure at 40 ° C to obtain an n-hexane extract. ^6. ⁵ g, ethyl acetate extract 62.0g, n-butanol extract 25.0g. The NO production inhibition activity test was carried out on various extracts (refer to Example 2), and the hexane extract (76, 1%) and ethyl acetate extract were obtained under the conditions of a test sample concentration of 100 g/ml. 86.9%), butanol extract ⁽⁶ ^45%), N0 production inhibition rate affirmative;. all extracts were found not to poison fine packet '1' born in the MTT cell toxicity in acetic acid to take the most active. Ester extract 62g, separated by Sephadex LH-20 column (6.5 X 35cm), eluted with 30% methanol, 70% sterol, decyl alcohol, concentrated and concentrated under reduced pressure at 40 ° C, four Fractions eluted: fr.1 (6.9g) fr.2 (14.7g), fr.3 (12. lg), fr.4 (2.7g). The NO production inhibition test was performed on these extracts again, and N0 production inhibition was performed. The rate is fr.1 (22.6%), fr.2 (89.7%), fr.3 (58.6%) fr.4 (89·7%). Extract extract fr.2, using silica column (wako gel) C-300, 2.2 χ 22cm) Separation, elution with chloroform:methanol (100: 0-0: 100), concentration and drying under reduced pressure at 40 ° C, yielding 11 (fr.2-1 to fr.2- 11) Elution of the fraction. Take fr.2 - 3 (1. lg), using CHP-20 column (4x 48cra) Separation, followed by Elution with 30% methanol, 50% methanol, 70% methanol, methanol gave four fractions: fr. 2-3- A (0.12 g), fr. 2-3-B (0. 65 g), Fr. 2-3-C (0. 03 g ), fr. 2 - 3-D (0. 07 g ). Purification of fr. 2- 3-C by reversed-phase HPLC, chromatography column YMC Guard park ODS-AL, mobile phase H ₂ 0 -methanol (30:70), flow rate 4 ml/min, measurement wavelength 254 nm, collection retention The peak of the time of 6.4 minutes was concentrated and dried under reduced pressure at 40 ° C to give Compound (1) [3.5 mg]. After the structure was determined, it was found that no literature was recorded. Example 2: Determining physical and chemical parameters and structure of compounds

The properties of the compound of the formula (1) are shown in Table 1, and the data of ¹³ C-NMR are shown in Table 2. The structural formula of the compound was finally determined from the above data.

Table 1 :

¹ H and ¹³ C-awake R-spectrum data of compound (1) (500 MHz, CDC1 ₃ )

s:—heavy line, d: double line, t: triple line, m: multiple line (1) The structural formula of the compound is determined as follows:

(1)

Example 3: Activity test for inhibiting NO production

The inhibitory effect of macrophages on NO production by the stimulation of stems and lipopolysaccharide (the effect of inhibiting NO production) was obtained by the following real face method. Further, the suppression effect of NO generation was evaluated based on IC50 (urn) which hindered the effect.

Materials used:

RAW 264.7 cells (Greater Japan Pharmaceuticals)

Naphthylethylenediamine hydrochloride (lg and pure medicine)

Sulfonamide (500g and pure medicine)

Ham's F12 Medium (SIGMA N488 500mL)

IFN- γ (Geneyme/Techne lOO g)

Lipopolysaccharide (LPS, 055: B5 lOmg, Sigma)

Phosphoric acid ( ⁵⁰⁰ ml and pure medicine)

DMS0 (500ml and pure medicine)

96 holes ^: titration plate (50 / box Sumitomo Bakelite, trade name [8096R])

Test method for NO production inhibition activity:

Place RAW264.7 cells (2 plates) in a 50ml Falcon hose. The cells were pelleted by a centrifuge (100 rpm, 3 min, 4 ° C) and the supernatant was removed with a pipette. 10 ml of fresh medium was added to suspend it. The concentration was adjusted to 1, 5 X 10 ⁵ / ml, and injected into a 96-well plate (Sumitomo Electric 8096R) at a rate of 20 (^L per well), and the cells were cultured in a C0 ₂ incubator for 1-2 hours. LPS ( lO g/Ml, Sigma, 055: B5) 2 L, mouse INF-γ (33 ng/raL, Genzyme) 2 μΙ, sample 0.4 L. Incubate for 16 hours in a CO ₂ incubator. Final concentration is INF- γ 0.33 Ng/mK LPS100ng/ml. The sample is dissolved in DMS0, the content of DMS0 relative to the medium is adjusted to 0, 2%. Take the culture supernatant 100, add 0.1% naphthalene diamine solution 50 L, p-aminobenzenesulfonate Amide solution 5 (^L, placed in the dark for 10 minutes at room temperature. The absorbance OD of 57 Onm (control 655mn) was measured with a spectrophotometer. STD was treated with sodium nitrite solution (100, 50, 20, 10, 5, 2, 1 , 0 um ). For cell survival rate, microscopic observation and MTT assay were performed.

Cytotoxicity was determined by MTT assay and microscopic examination. The MTT method is a known conventional method. That is, in a 96-well ^: titration plate, the cell concentration of 200 μ 1 per well is 1.0 χ 10 ⁵ cells/ml, and different concentrations of extract or monomer components are added, the cells are cultured for 16 hours, and MTT reagent is added. Incubate for another 4 hours. The supernatant was discarded, 150 L DMSO was added, and the resulting formazan (f ormazane) was completely dissolved, and the absorbance at 570 nm was measured.

Activity evaluation

Calculate the amount of N0 ₂ — and substitute the following formula to find the suppression effect.

Inhibition effect ( % ) = {1- ( X-Y ) / ( Z-Y ) } X 100

X: the amount of N0 ₂ - induced by IFN-γ and LPS in the presence of the test compound, Y; induced N0 ₂ - in the absence of real-face compound, IFN-γ and LPS the amount,

Z: The amount of N0 ₂ - induced by IFN-γ and LPS.

IC _{5 of the} inhibitory effect of the compound (1). As follows:

Example 4: Preparation of a pharmaceutical dosage form

When the compound (1) or a pharmaceutically acceptable salt, enantiomer, racemate, tautomer or physiological functional derivative thereof is administered alone, it is preferably provided in a pharmaceutical preparation, and the preparation includes a compound (1) or a pharmaceutically acceptable salt, enantiomer, racemate, tautomer or physiologically functional derivative thereof, a pharmaceutically acceptable carrier or excipient thereof, and optionally one or A variety of other therapeutic ingredients.

The preparation includes an orally administered preparation, a parenteral preparation (including intradermal injection, intramuscular injection, intravenous injection, and intra-articular injection), an inhalation preparation (including a pressurized aerosol device, a sprayer, and a different dose). Microparticle powders or aerosols produced in insufflators, rectal and topical formulations (including dermal, buccal, sublingual, intraocular). Most suitable administration The route depends on the condition and disease of the patient being administered. The formulations are usually provided in unit dosage form and may be prepared by any methods known in the art of pharmacy. All methods include the step of mixing the active ingredient with a carrier which is comprised of one or more accessory ingredients. Usually, the active ingredient is uniformly and tightly bound to a liquid or a fine solid carrier or both, and then the product is formulated as needed; the preparation of the present invention suitable for oral administration can be in a separate unit such as a capsule, a flat An elixirs or tablets are provided, and each unit contains a predetermined amount of the active ingredient; a powder or granule; a water-soluble liquid or suspension; or an oil-in-water emulsion or a water-in-oil emulsion; or a granule or a syrup Agent or paste.

Tablets may be prepared by tableting or molding, optionally with one or more accessory ingredients; by compressing free-flowing forms such as powders or granules on a suitable machine (optional with binders, lubricants, inert diluents) The active component of the surfactant, or dispersion of the dispersing agent can be used to prepare compressed tablets; the tablets may optionally be coated or indented and formulated to provide sustained or controlled release of the active ingredient.

Formulations for parenteral administration include aqueous and non-aqueous sterile injectable solutions, which may contain antioxidants, buffers, antibacterial agents, and solutes, aqueous and non-aqueous sterile suspensions which render the formulation invigorating with the patient's blood. The suspension may contain a suspending agent and a thickening agent; the formulation may be packaged in unit or multi-dose, for example, in a sealed ampoule and vial; and may be stored under lyophilization conditions, prior to use It is only necessary to provide a sterile liquid carrier such as saline or water for injection; the injectable solutions and suspensions may be prepared in the form of powders, granules and various the various compositions described above.

Formulations for rectal administration may be presented as a suppository in a conventional carrier such as cocoa butter or polyethylene glycol.

Formulations for topical administration to the oral cavity (e.g., buccal or sublingual agents), including lozenges of the active ingredient in a flavoring base such as sucrose, arabinose.

It will be understood that in addition to the ingredients specifically mentioned above, the formulations of the present invention also include other conventional ingredients of the type of preparations known in the art, such as flavoring agents suitable for oral administration and the like.

1 10 g of the compound (1) extracted from the king of the king is mixed with 40 g of corn starch, added with water to make a soft material, granulated by a 12-mesh sieve, and dried to obtain a granule containing a compound per 500 mg in the granule (1) ) 100mg. 2 40 g of the compound (1) extracted from the king was mixed with 100 g of lactose and 10 g of magnesium stearate, and the intestinal sol was filled every 600 mg. In the intestinal sol preparation, each capsule contained 160 mg of the compound (1).

3 25 g of the compound (1) extracted from the king was dissolved in an ordinary injection preparation method, and dissolved in 1000 ml of distilled water for injection to 60 ° C. The isotonic solution was adjusted by NaCL and sealed in a vial. This injection contains 10 mg of compound (1) in 10 ml.

Example 5, preparing food

The compound (1) provided by the present invention may also be provided in the form of a food. Preferred food forms include powders, granules, pastes, gelatins and the like, and the granule form may be added with a sugar such as lactose to increase the sweetness; In the form of a beverage; in addition to the extract of Shixian peach, these foods or beverages may also contain vitamins, inorganic elements such as calcium, alcohols, deodorants, such as polyphenols. These foods include special health foods, medical foods, and the like.

Claims

Rights request

1. A compound of the formula (1) or a salt, enantiomer, racemate, tautomer or physiological functional derivative thereof, extracted from the king

( 1 ).

2. Use of a compound according to claim 1 or a salt, enantiomer, racemate, tautomer or physiologically functional derivative thereof for the manufacture of a medicament for the treatment or prevention of an inflammatory disease.

3. Use according to claim 2, characterized in that the compound or a salt, enantiomer, racemate, tautomer or physiologically functional derivative thereof can be used in combination with a second pharmaceutically active substance For the preparation of a medicament for treating or preventing an inflammatory disease.

4. Use according to claim 2 or 3, characterized in that the inflammatory diseases and clinical conditions include joint diseases, in particular arthritis (e.g. rheumatoid arthritis, osteoarthritis), or gastrointestinal diseases (e.g. Ulcerative colitis, gastritis, and other mucosal inflammation caused by infection, intestinal diseases caused by non-anti-inflammatory drugs), lung diseases (such as adult respiratory distress, asthma, sputum fibrosis, chronic obstructive pulmonary disease) , heart disease (such as myocarditis), nervous tissue diseases (such as multiple sclerosis), pancreatic diseases (such as diabetes and complications), kidney disease (such as glomerulonephritis), skin diseases (such as dermatitis, psoriasis, eczema, Urticaria), eye diseases (such as glaucoma), transplant organ diseases (such as rejection), multiple organ diseases (such as systemic lupus erythematosus), and inflammatory sequelae after viral and bacterial infections.

5. A compound according to claim 1 or a salt, enantiomer, racemate, tautomer or physiologically functional derivative thereof, characterized in that a suitable salt comprises an organic or inorganic acid or base. A pharmaceutically acceptable salt includes the following acids: hydrochloric acid, hydrobromic acid, sulfuric acid, citric acid, tartaric acid, phosphoric acid, lactic acid, pyruvic acid, acetic acid, trifluoroacetic acid, succinic acid, oxalic acid, fumaric acid, horse Toluene, butanone diacid, methanesulfonic acid, ethanesulfonic acid, p-toluenesulfonic acid, benzenesulfonic acid, isethionethane; pharmaceutically acceptable basic salts include ammonium salts, alkali metal salts, such as sodium salts And potassium salts, alkaline earth metal salts such as calcium salts and magnesium salts, and salts with organic bases such as dicyclohexylamine and N-mercapto-D-glucosamine.

6. A compound according to claim 1 or a salt, enantiomer, racemate, tautomer or physiologically functional derivative thereof, characterized in that the physiological function comprises an ester, an amide or a carbamate. Ester.

7. Use of a compound according to claim 1 or a salt, enantiomer, racemate, tautomer or physiologically functional derivative thereof for the preparation of a health food, beverage, or feed.

8. A method of extracting an active compound from a king, comprising the steps of:

1) The extract obtained by methanol extraction from the king is concentrated under reduced pressure and dried to obtain an extract;

4) The eluate collected in step 3) is further separated and purified by high pressure liquid chromatography to obtain an active compound.

9. A method according to claim 8 wherein the active compound structure obtained

For:

10. Method according to claim 8, characterized in that the concentration under reduced pressure is carried out at 4G degrees Celsius.

11. Process according to claim 8, characterized in that the mobile phase of the high pressure liquid chromatography is methanol: water = 30:70.