CN101559050A - Use of butin and derivatives thereof for preparing drugs having inhibitive effect on immune system - Google Patents

Abstract

The invention discloses use of butin and derivatives thereof for preparing drugs having inhibitive effect on an immune system. The use is characterized by preparing drugs having inhibitive effect on an immune system with the butin and the derivatives thereof as active ingredients and a pharmaceutically acceptable carrier.

Description

Butin and derivant thereof have the purposes that suppresses the immune system drugs with function in preparation

Invention field

The present invention relates to the new purposes of a kind of chemical compound and derivant thereof, specifically, is butin and derivant thereof the purposes at the medicine of preparation treatment metabolism of pigment disease such as vitiligo and poliosis.

Background technology

Vitiligo is a common a kind of limitation hypopigmentation dermatoses, and sickness rate is 0.5-2.0%, because pathogenesis and pathogeny are not clear and definite as yet at present, still is in the situation of easily examining refractory in the world so far.This disease is classified as one of difficult and complicated illness by world medical circle.Present leukodermic drug treatment has psoralen combined U VA treatment, 17-hydroxy-11-dehydrocorticosterone therapy, khellin's combined U VA therapy, metallic element to cooperate PUVA therapy, phenylalanine to cooperate UVA therapy and herbal treatment.Caulis Vernoniae andersonii is the achene of feverfew Caulis Vernoniae andersonii Vernoniaanthelmintica Willd.Extract during fruit maturation in the autumn in summer; Or extract ripe Fruit branch respectively, and lay fruit, be stored in shady and cool dry place.Originate in India, Pakistan, the existing cultivation history that is about 400 years in Chinese Xinjiang.Mainly be distributed in the Xinjiang Aksu Prefecture.Be that the Xinjiang uighur folk is treated leukodermic medicine, personnel are made into hospital preparation in the Xinjiang study of pharmacy seventies, are applied to the clinical treatment vitiligo, and cure rate is 7-20%, and effective percentage is greater than 90%, and do not see toxic and side effects.But so far the effective ingredient in the Caulis Vernoniae andersonii be it be unclear that.For this reason, the inventor has carried out further screening and separating work, has found that the chemical compound of other metabolism of pigment diseases such as Caulis Vernoniae andersonii treatment vitiligo and poliosis is a butin, thereby has finished the present invention.

Goal of the invention

The purpose of this invention is to provide butin and derivant thereof purposes at the medicine of preparation treatment metabolism of pigment disease such as vitiligo and poliosis.

Summary of the invention

Butin of the present invention is 3 "-4 '-7-trihydroxy flavanone, structural formula is:

The butin derivant is included in 5,6,7,8, the one group of chemical compound that 3 ', 4 ', 5 ', 6 ' quilt-OH replaces and the corresponding glucoside compound of generation; Or quilt-OR or-one group of chemical compound that R replaces, wherein R is C ₁-C ₈Alkyl.

It can extract acquisition from plant Caulis Vernoniae andersonii seed or other plant, also can obtain by chemosynthesis.Obtain if from the Caulis Vernoniae andersonii seed, extract, one of extracting method is: use the methanol reflux, obtain methanol extract liquid, reclaim under reduced pressure, concentrate, obtain concentrated solution,, get ethyl acetate layer with ethyl acetate and water dispenser, supernatant is passed through silica gel column chromatography, use the chloroform-methanol gradient elution, obtain eluent, again at silica gel column chromatography petroleum ether-acetone (4: 1-1: 1) eluting, with eluent, reuse polydextran gel LH20 50-80% methanol-eluted fractions obtains eluent, reuse octadecyl silane chromatography, use the 50-100% methanol-eluted fractions, obtain eluent, (10-25: 1) eluting obtains the recycle silicon plastic column chromatography with chloroform-methanol.Turn out to be butin through chromatography.

We carry out pharmacological evaluation and confirm, butin can make melanocyte propagation, the activity of tryrosinase improves, melanin content increases, can improve the melanin synthetic gene (tyrosinase cdna. tryrosinase related gene-1, tryrosinase related gene-2) expression, make melanosome type generation significant change, more trend towards the generation of pigment granule.Therefore, butin can be treated the metabolism of pigment disease.The immunity of butin pair cell, humoral immunization are inhibited.By to serum antibody, complement and T lymphocytic emiocytosis IL-2 activity analysis, further show that at molecular level butin is inhibited to immune system.Butin is to K-1735 SKMel-19, SKMel-23, and Mel-2A, MeWo, Bro. has shown inhibitory action.

Brief description

Fig. 1-----butin and derivant thereof are in external influence to melanin content.

Fig. 2-----butin external to melanocyte in the influence of tyrosinase activity.

Fig. 3-----butin is in external influence to melanocyte propagation.

Fig. 4-----butin influences the melanin synthetic gene expression external.

Fig. 5-----butin external to melanocyte in the influence of melanosome.

Fig. 6-----butin is in the influence to brown guinea pig skin melanocyte.

Fig. 7-----butin is in external influence to the mice spleen lymphocytes proliferation reaction.

Fig. 8-----butin is secreted the active influence of IL-12 external to mouse boosting cell.

Specific embodiment

The external pharmacodynamic study of experimental example 1 butin

Impaired because of the pigment cell and the function thereof in patients with vitiligo skin lesion district, we have studied the influence of butin to the human body melanocyte of In vitro culture.

1.1. experimental program

1.1.1 screening model: normal person's melanocyte;

1.1.2. source: the unnecessary foreskin of child's Wrapping annulus cuts operation, do not have and infect.

1.1.3. condition of culture: obtain melanocyte after getting the trypsinization of normal children foreskin, in the DMEM culture medium, add bFGF, CT, TPA, FCS, two various factors such as anti-, 5%CO ₂, to cultivate after 7 days under 37 ℃ of conditions and go down to posterity, passage cell is cultivated medicine and the contrast that adds variable concentrations after 48 hours under these conditions, measures the correlation analysis index.

1.1.4. assay method: cell increment mtt assay; Tyrosinase activity is measured and is used the DOPA staining; Melanin content alkali digestion method; The melanin synthetic gene (tyrosinase cdna. tryrosinase related gene-1, tryrosinase related gene-2) expression Northern Blotting; The electronic microscope photos scanning electron microscope.

1.2. result

1.2.1. butin and derivant thereof external to melanocyte in melanic influence

Butin and derivant thereof external to melanocyte in the content of synthesis of melanin the raising effect is arranged, see Fig. 1.Wherein chemical compound is:

Butin (Butin):

The butin derivative I:

Puerarta chalcone (Butein)

Butin derivative I I:

5,6,7,4 '-kaempferol

1.2.2. butin external to melanocyte in the influence of tyrosinase activity

Butin is seen Fig. 2 in the external activity that improves tryrosinase in the melanocyte.

1.2.3. butin external to the value-added influence of melanocyte

Butin can promote melanocyte propagation, sees Fig. 3.

1.2.4. butin is in external influence to the melanin synthetic gene expression

To its mechanism inquire into draw butin can improve the melanin synthetic gene (tyrosinase cdna. tryrosinase related gene-1, tryrosinase related gene-2) expression, see Fig. 4.

1.2.5. butin external to melanocyte in the influence of melanosome

Can see melanosome type generation significant change from electronic microscope photos, more trend towards the generation of pigment granule.See Fig. 5.

Pharmacodynamic study in the body of experimental example 2. butins

By gastric infusion observe butin to melanocyte in the brown Cavia porcellus body in the influence of the contained ratio of basal layer.

2.1. experimental program

Laboratory animal: the brown Cavia porcellus of Britain's undercoat, the Xinjiang Experimental Animal Center provides.

Be provided with by the reagent thing:

Butin:

High concentration group: 8.74mg/kg (calculating) by 1/25 of maximum tolerated dose

Middle concentration group: 4.37mg/kg (calculating) by 1/50 of maximum tolerated dose

Low concentration group: 2.19mg/kg (calculating) by 1/100 of maximum tolerated dose

Positive controls: 8-MOP (8 methoxypsoralens, Chinese pharmaceutical biological product is identified institute) negative control group: 2.5% hydroquinone blank group; Normal saline

2.2. the foundation of animal model

Elder generation external hydroquinone and Fructus Psoraleae (8-MOP) are observed the influence to coloured guinea pig skin basal layer melanocyte of hydroquinone, 8-MOP: behind the external hydroquinone, coloured guinea pig skin basal layer is thickened, melanin be generated reduce this result to conform to relevant document; After using 8-MOP, coloured guinea pig skin basal layer melanocyte is increased.Determine the model establishment.

2.3. result

Butin can improve the ratio of coloured guinea pig skin basal layer melanocyte.Coloured guinea pig skin basal layer melanocyte had significance to change (P＜0.05) before and after the butin of high, medium and low dosage can make medication; There was no significant difference between the butin group of high, medium and low dosage (P＞0.05), but numerically change.Wherein middle dosage group is bigger to the influence of coloured guinea pig skin basal layer melanocyte.See Fig. 6.

Experimental example 3. butins are to the influence of mouse immune system

3.1. experimental program

3.1.1. experimental model: cultivating Kunming is the newborn mice splenocyte.

3.1.2. animal origin: the Xinjiang Experimental Animal Center provides.

3.1.3. condition of culture: getting Kunming is to obtain splenocyte after the trypsinization of newborn mice spleen,

In the RPMI1640 culture medium, add FCS, two various factors such as anti-, 5%CO ₂, cultivate down under 37 ℃ of conditions and cultivate medicine and the contrast that adds variable concentrations after 48 hours, measure the correlation analysis index.

3.2. result

Support that leukodermic morbidity with immune system relevant because etiological study at present more, find to have in the patients with vitiligo body specific antibody to exist.Carrying out butin shows the result of study of mouse immune systematic influence: the immunity of butin pair cell, humoral immunization are inhibited.By to splenocyte and splenocyte secretion IL-2 activity analysis, further show that at molecular level butin is inhibited to immune system, see Fig. 7,8.

Experimental example 4. butins are in external influence to melanoma cell

Because butin can make melanocyte propagation, we have investigated it in external influence to melanoma cell.

4.1. screening model: K-1735 SKMel-19, SKMel-23, Mel-2A, MeWo, Bro;

4.2. source: Berlin, Germany Free University Benjamin Franklin medical research center.

4.3. condition of culture: in the DMEM culture medium, add FCS, two various factors such as anti-, 5%CO ₂, cultivate medicine and the contrast that adds variable concentrations after 24 hours under 37 ℃ of conditions, measure the correlation analysis index.

4.4. assay method: cell increment mtt assay

4.5. result

Butin can be to K-1735 SKMel-19, SKMel-23, and Mel-2A, MeWo, Bro have shown inhibitory action in various degree.

Claims (1)

1. butin and derivant thereof are in the purposes of preparation inhibition immune system drugs with function, and the structural formula of wherein said butin and derivant thereof is:

The butin derivant is included in 5,6,7,8, the one group of chemical compound that 3 ', 4 ', 5 ', 6 ' quilt-OH replaces and the corresponding glucoside compound of generation; Or quilt-OR or-one group of chemical compound that R replaces, wherein R is C ₁-C ₈Alkyl.

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