CN1037539A - The manufacture method of new antitumor antibiotic C-1027 - Google Patents

Abstract

A kind of new large-molecular peptides antitumorigenic substance C-1027, be to become strain with the bacterial strain that can produce this material or its, under the suitable culture condition, cultivate (pH of substratum with neutrality for well, culture temperature is 20-37 ℃ of scopes, general 2-5 days of incubation time), its product C-1027 can adopt the method for general separation and Extraction tunning to carry out, as saltout, the hydrophobicity chromatography, ion exchange chromatography, adsorption chromatography, gel permeation chromatography etc., can be separately or the arbitrary combination mode carry out, centrifugal then, separate, filter, refining repeatedly, lyophilize, make pure product, this material has tangible anti-tumor activity, and curative effect is better than the Zorubicin and the ametycin of clinical use.

Description

The manufacture method of new antitumor antibiotic C-1027

The present invention relates to a kind of manufacture method of new large-molecular peptides antitumor antibiotics.

The C-1027 microbiotic is in the process of screening antitumor antibiotics, from the actinomycetic fermented liquid of nearly 2000 strains, detected with spermatogonium, not only have stronger anti-tumor activity, and also have the effect of resisting gram-positive bacterium and anti-gram negative bacterium, but there is not antimycotic effect.The C-1027 microbiotic that the present invention relates to after testing and consult existing technical information, all found no report, so can be defined as a new compound.

The objective of the invention is new antibiotic, with antitumor antibiotics kind deficiency or the not good enough problem of curative effect that solves present clinical use for providing one to have better antitumor action.

Main points of the present invention are to cultivate under proper condition by having the bacterial strain (call C-1027 in the following text and produce bacterium) that produces the C-1027 ability, and separation from nutrient solution, extraction obtain this material.

The generation bacterium that is used to make C-1027 is streptomyces bacterial strain C-1027, and available Streptomyces globisporus var.qianjiangensis n.var.C-1027 represents.This bacterial strain obtains from the separation of Qianjiang county, Chinese Hubei Province soil, (in the former Japanese patent specification mistake be written as " Chinese yunnan soil "), and be preserved in China Committee for Culture Collection of Microorganisms common micro-organisms center for No. 0135 with CGMCC NO.It is as follows that little rz thing of this bacterial strain is learned character:

Morphological specificity:

The fibrillae of spores of C-1027 bacterial strain is until ripple song (Fig. 1), 10 to 30 on every chain of sophisticated spore chain or more spore, spore cylindricality, smooth surface (Fig. 2).

Cultural characteristic:

The C-1027 bacterial strain is at various synthetic or organically form white to yellowish aerial hyphae on the substratum, often has little brown or light cinnamon color.

Substrate mycelium is general colourless or butterfat is yellowish, and pale yellow ash or ecru do not have specific color.

On the substratum of being tested, do not produce soluble pigment.The cultural characteristic of C-1027 sees table 1 for details.

The physiological characteristic of C-1027 sees Table 2.

The former utilization spectrum of the carbon of C-1027 sees Table 3.

The full cellular water hydrolysis products of C-1027 bacterial strain contains LL-diaminopimelic acid and glucose, ribose.

The cultural characteristic of table 1 C-1027 bacterial strain

Substratum growth aerial mycelium substrate mycelium soluble pigment

The luxuriant nude of ISP NO2 is to yellow ash or the yellow nothing of the yellow shallow moccasin of light olive moccasin or pale yellow

ISP NO3 appropriateness is pale yellow whitely not to be had to the yellow colourless or butterfat of light olive moccasin

ISP NO3 appropriateness is pale yellow whitely not to be had to the yellow colourless or butterfat of light olive moccasin

The yellow colourless or butterfat of the luxuriant pale yellow ash of ISP NO5 or light olive moccasin does not have

ISP NO6 is thin white to light yellow pale yellow nothing

The yellow colourless or ecru of the luxuriant pale yellow ash of ISP NO7 or light olive moccasin does not have

Czapek's agar (sucrose) is thin pale yellow white to the yellow colourless nothing of light olive moccasin

Czapek's agar (glucose) is luxuriant pale yellowly white not to be had to the yellow colourless or butterfat of light olive moccasin

The yellow colourless or pale yellow ash of luxuriant pale yellow ash of No. 1 agar of Gao Shi or light olive moccasin does not have

The yellow colourless nothing of glucose asparagine agar pale yellow ash of appropriateness or light olive moccasin

Pale yellow or the nothing of the white extremely pale yellow ash of Bennetts agar appropriateness

The luxuriant light olive moccasin of potato agar is yellow colourless to the yellow nothing of olive moccasin

The physiological property of table 2 C-1027 bacterial strain

The observation item feature

Melanochrome produces

ISP NO1 substratum feminine gender

ISP NO6 substratum feminine gender

ISP NO7 substratum feminine gender

H ₂The generation positive of S

The gelatine liquefication positive

Cow's milk: solidify the positive

Peptonize the positive

The nitrate reductase positive

The temperature (ISP NO2 substratum) of growth

28-32 ℃ is luxuriant, and growth is rapid,

37 ℃ of appropriateness, slow

Do not grow for 45 ℃

The utilization of carbon source spectrum of table 3 C-1027 bacterial strain

Carbon source for growth

L-arabinose ++ ++

The D-wood sugar +++

D-glucose +++

D-fructose ++ ++

Sucrose-

Inositol-

The L-rhamnosyl ++ ++

D-N.F,USP MANNITOL ++ ++

The D-semi-lactosi ++ ++

Lactose-

Mierocrystalline cellulose-

Melampyrin-

Saligenin+

Raffinose-

Result of study based on features such as form, cultivation, Physiology and biochemistry, cell wall analyses shows that the C-1027 bacterial strain should belong to the ball spore monoid of streptomyces.All very similar to styreptomyces globispotus strain Streptomyces globispous with west tang style streptomycete Streptomyces setonii.Show through comparative result under same culture condition, C-1027 bacterial strain and styreptomyces globispotus strain are more approaching but still have 3 differences, are decided to be the Streptomyces setonii C-1027 west streptomycete C-1027 of Tang so the C-1027 bacterial strain named to styreptomyces globispotus strain Qianjiang mutation Stretomyces globisporus var qianjiangensis n.var.(Japanese patent specification)

C-1027 material of the present invention can be cultivated, make under suitable condition with above-mentioned C-1027 bacterial strain, should vibrate under aerobic environment usually, ventilation, stir culture.

C-1027 produces the employed substratum of bacterium, and for well, various synthetic, semi-synthetic or natural mediums all can use with the substratum that contains the available nutritive substance of this bacterium.The carbon source that substratum is formed has that glucose, fructose, glycerine, lake essence, starch, sugar are close, corn steep liquor, organic acid etc., can use or be used in combination by single degree.Nitrogenous source has organic nitrogen sources such as peptone, meat extract, yeast extract paste, soyflour, casein food grade, amino acid, urea and SODIUMNITRATE, ammonium sulfate etc. inorganic nitrogen-sourced, can be used singly or in combination.Also can in substratum, look it and need suitably to add sodium salt, sylvite, magnesium salts, phosphoric acid salt and other heavy metallic salt class.

Produce foam in the culturing process, can add foam killers such as senior alcohols such as soybean oil, Semen Lini oil and stearyl alcohol, tetradecyl alcohol, heptadecanol or various silicides.

For well, culture temperature is 20-37 ℃ of scope, optimum temps 27-30 ℃ with neutrality for the PH of substratum.Incubation time is generally between 2-5 days.Cultivate by above-mentioned culture condition, can constantly put aside out the C-1027 material.Above-mentioned various culture condition, the kind of visual microorganism used therefor, characteristic and various external conditions appropriate change also can be adjusted and be selected optimum condition in above-mentioned scope.

By the C-1027 material that above-mentioned cultivation produces, its separation and Extraction can adopt the method for general extraction tunning to carry out, as saltout, hydrophobicity chromatography, ion exchange chromatography, adsorption chromatography, gel permeation chromatography etc., can be separately or the mode of arbitrary combination carry out.

Separating and extracting method is: will be present in the C-1027 material in the nutrient solution, at first filtration, centrifugal separates filtrate and mycelium.Get filtrate and add ammonium sulfate precipitation, will contain the throw out centrifugation of C-1027 then or add and help the filter powder to filter.In order to be beneficial to precipitation, can add precipitation agents such as ferric sulfate, iron trichloride, Tai-Ace S 150.As making solution PH that bigger variation take place because of adding precipitation agent, can add neutralizing agents such as yellow soda ash, salt of wormwood, SODIUM PHOSPHATE, MONOBASIC earlier, PH is remained between the 5-8, above-mentioned salting-out process can carry out repeatedly.

Above-described throw out, water-soluble or suitable damping fluid such as Tris-hydrochloride buffer, phosphoric acid buffer etc. are used the cellophane film again, and are after the desalinations such as the outer filtration membrane of limit, further refining.Strong basic ion exchange resins such as refining available Dowex.1 Amberlite IRA-400, weak-base ion-exchange resins such as Amberlite IR-45, DEAE-cellulose, help sorbent materials such as filter powder, Hydroxy phosphatic rock, with the C-1027 aqueous solution by adsorbing, and then wash-out, can remove impurity.Elutriant is through ion exchange resin Sephadex and Cellulose absorption, with wash-outs such as the Nacl aqueous solution, Tris-hydrochloride buffer, phosphoric acid buffers.After the filtering and concentrating, again through Sephadex G-50, the G-75 isogel filters refining this elutriant, preferably obtains pure product outside limitting, and contained low molecule salt can pass through Sephadex G-25 chromatography, and means such as the outer filtration of limit, dialysis are removed.Carry out repeatedly with above-mentioned various chromatography, will more help removing impurity.

After adopting above-mentioned various method for refining and making up the lyophilize of resulting C-1027 refined liquid, can obtain the C-1027 white powder.

The C-1027 of gained of the present invention is an one matter, show that by the SDS-polyacrylamide gel electrophoresis single band, Sephadex G-50 column chromatography show that symmetrical simple spike, TSK-gel G-2000SW high performance liquid chromatography are that simple spike, LKB iso-electric point electrophoresis are single band, all can be confirmed.

The physico-chemical property of C-1027 material is as follows:

Proterties: white powder

Solvability: soluble in water, be insoluble to organic solvents such as methyl alcohol, acetone, vinyl acetic monomer.

Ultra-violet absorption spectrum: in the result who measures among water, 0.01N Hcl, the 0.01N NaOH respectively shown in the curve among Fig. 31,2,3, from figure, can be observed, 270-275,350-360nm in neutrality, acidic aqueous solution, there is obtained the maximum absorption at 270-275,340-345nm place in basic solution.

Infrared absorption spectrum: with KBr sheet measurement result, as shown in Figure 4, mainly be absorbed in 3300,1640,1530cm ^-1The place.

Fusing point: no sharp melting point R decomposition point, very long browning, blackening in foaming, 260 ℃ of complete charings.

Color reaction: triketohydrindene hydrate, biuret, Folin-Lowry reacting positive, anthrone, aniline one phthalic acid reaction negative.

Iso-electric point: PH 3.5-3.7

Acid-basicity: acidity

Ultimate analysis: C45.22%, H6.65%, N14.03%

Molecular weight: utilize TSK gelG-2000SW high performance gel filtration chromatography and sds polyacrylamide gel electrophoresis method to calculate molecular weight and be about 15,000.

Amino acid is formed: 6NHCl, 110 ℃ of hydrolysis 20 hours are measured and amino acid containedly be the results are shown in Table 4.Detecting it amino acid containedly has: aspartic acid, Threonine, Serine, L-glutamic acid, proline(Pro), glycine, tyrosine, leucine, L-Ala, Gelucystine, Xie Ansuan, Isoleucine, phenylalanine, Histidine, Methionin, arginine and halfcystine is to form with S-sulfocysteine, tryptophane is detected after the NaOH hydrolysis.

-terminal amino acid: detecting-terminal amino acid according to the DNP method is L-Ala.

Table 4 C-1027 amino acid group

Amino acid weight % amino acid weight % amino acid weight %

Aspartic acid 6.16 Threonines 6.27 Serines 8.97

L-glutamic acid 4.74 proline-4 .96 glycine 6.41

L-Ala 9.36 Gelucystines 1.90 Xie Ansuans 6.85

Methionine(Met) 0 Isoleucine 0.76 leucine 3.76

Tryptophane 0 tyrosine 2.76 phenylalanines 4.78

Histidine 1.19 Methionins 1.10 arginine 1.14

Biological activity of the present invention shows: the C-1027 material has stronger restraining effect to the gram positive bacterium growth, and its antimicrobial spectrum is as shown in table 5.The C-1027 material also has tangible anti-tumor activity, and the in vitro tests effect is very strong.Generate the assay method inspection with the clone, to human hepatocellular carcinoma BEL-7402 cell's half casualty-producing concentrations (IC ₅₀) be 0.000013 μ m, its specific activity Zorubicin and ametycin are strong.(the IC of Zorubicin and ametycin ₅₀Be respectively 0.00084 μ M and 0.0059 μ M).The C-1027 material is to mouse L ₁₂₁₀And P ₃₈₈Leukemia has obvious restraining effect.Inoculation leukemia cell in the DBA mouse peritoneal, 24 hours venter posterior intracavitary administration C-1027 once, 60 days observationss.This material comprises ehrlich carcinoma, H to the mouse ascites cancer ₂₂Ascites liver cancer all has the obvious suppression effect.Kunming mouse intraperitoneal inoculation cancer cells, 24 hours venter posterior intracavitary administration C-1027 materials, 60 days observationss.Above curative effect all sees Table 6.

The C-1027 material comprises S to the mouse transplanted solid tumor ₁₈₀Interior knurl, U ₁₄Cervical cancer and Harding-passey melanoma all have the obvious suppression effect.The subcutaneous vaccination tumour, shot C-1027 material in 24 hours posterior veins sees Table 7 to the melanomatous inhibiting rate of Harding-passy.

The anti-microbial activity of table 5 C-1027 material

Test organisms minimum inhibitory concentration mcg/ml

Streptococcus aureus 209P 0.78

Bacillus subtilus 6,633 0.78

Sarcine 0.78

Bacillus proteus 0 * 19＞100

Intestinal bacteria B 3.13

Intestinal bacteria 0,111 50

Klebsiella pneumoniae 25

Dysentery bacterium 302＞100

Green pus liver bacterium 11＞100

Mycobacterium 607 (3.13)

Mycobacterium phlei 1.56

Candida albicans＞100

Penicillin 5404＞100

Spermatogonium 0.0039

Annotate: it is clear that () expression suppresses the circle blur margin.

Table 6 C-1027 material anti-tumor activity

Tumour title dosage (mg/kg) administration number of times lifetime (my god) increase in life span (%)

Leukemia 0.025 1＞60＞275

L ₁₂₁₀0.012 1 ＞60 ＞275

0.006 1 ＞60 ＞275

Contrast 16

Leukemia 0.025 1＞60＞275

P ₃₈₈， 0.012 1 ＞60 ＞275

0.006 1 22 37.5

Contrast 16

Ascites liver cancer 0.025 1 38.5 156

H ₂₂0.012 1 48 220

0.006 1 22 47

Contrast 15

Table 7 C-1027 material anti-tumor activity

The tumour title	Dosage	The animal dead number	Knurl heavy (gram)	Inhibiting rate (%)	The P value
						Harding-Passey melanoma	0.2 0.1 0.05 contrasts	1 0 0 0	0.20 0.56 0.75 2.98	93 81 75	<0.01 <0.01 <0.01

In addition, the C-1027 material is to KB cells in vitro fragmentation effect ED ₅₀(50% effective dose) is fine, and median effective dose has only 0.001-0.0001 μ g/ml.

Embodiment is as follows for C-1027 material manufacture method:

Glycerine 2%, dextrin 2%, Soytone1%, yeast extract paste 0.3%(NH ₄) ₂SO ₄0.2%, CaCO ₃0.2%, substratum PH6.6 adds the 100ml medium sterilization in the 500ml triangular flask, the C-1027 bacterium is gone in inoculation, and 27 ℃, 48 hours (180 rev/mins, amplitude 10cm) are cultivated in the rotation concussion.And then with glycerine 2%, dextrin 2.0%, fish meal 1%, (NH ₄) ₂SO ₄0.2%, CaCO ₂0.2%, adorns 100ml in the PH6.8 substratum 500ml triangular flask, sterilization.Above-mentioned seed plants with 5% amount, cultivates 96 hours for 27 ℃.

After having cultivated, centrifugal, the filtration of the nutrient solution 10l that gets PH8.0 obtains 7l filtrate, transfers PH4 with HCl, adds 3.5Kg(NH ₄) ₂SO ₄, 5 ℃ were stirred 3 hours, and the C-1027 precipitation and centrifugal separation of separating out (5 ℃, 3000 change, 30 minutes) obtains.This precipitation is dissolved in 400ml deionized water, dialysis, lyophilize and gets meal 5.3g.

Above-mentioned meal 2g is dissolved in the 400ml deionized water, centrifugal (5 ℃, 3000 change, 30 minutes), removes insolubles.Activator adsorbs through the DEAE-Cellulose post, after the washing of 400ml deionization, and 0.1MNacl wash-out, active component 112ml, dialysis, liquid freezing drying in the film.

The raw product 245mg that obtains like this, water-soluble, through Sephadex G-50 post deionized water chromatography, active component 80ml, lyophilize gets the 120mg white powder, and this thing is water-soluble again, through Sephadex G-75 column chromatography, collect the active component lyophilize, obtain 62mg C-1027 white powder highly finished product, its physico-chemical property and biological characteristics are as previously mentioned.

Description of drawings:

Fig. 1: the fibrillae of spores of C-1027 bacterial strain (300 *)

Fig. 2: the spore of C-1027 bacterial strain (15000 *)

The UV spectrum of Fig. 3: C-1027

The infrared spectra of Fig. 4: C-1027

Claims (6)

1, a kind of manufacture method that can extract large-molecular peptides antitumor antibiotics C-1027 from microorganisms cultures is characterized in that it is from streptomyces C-1027 bacterial strain, is prepared with the general method that extracts tunning.

2, manufacture method according to claim 1, the fibrillae of spores that it is characterized in that the C-1027 bacterial strain is until the ripple song, the spore cylindricality, smooth surface, aerial mycelium is that pale yellow white extremely light olive moccasin look often has shallow brown or shallow Chinese cassia tree color, colourless or the creamy of base silk, full cellular water hydrolysis products contains the LL-diaminopimelic acid.

3, manufacture method according to claim 2, it is characterized in that the C-1027 bacterial strain, can in various complete synthesis, semi-synthetic or natural mediums, cultivate, these substratum contain usually and are suitable for cultivating actinomycetic carbon source, nitrogenous source and an amount of inorganic salts, fermentation culture is generally carried out under vibration or aeration-agitation aerobic condition, culture temperature 27-30 ℃, pH value neutrality, incubation time 2-5 days.

4, manufacture method according to claim 1, it is characterized in that for from culturing filtrate, extracting the C-1027 antibiotics, available saltouing, the hydrophobicity chromatography, ion exchange chromatography, adsorption chromatography, gel permeation chromatographies etc. are handled individually or in combination, throw out is centrifugal, separate, filter, desalination, refining, wash-out, concentrate, lyophilize, finally obtain the pure product of C-1027 white powder, show single band by the SDS-polyacrylamide gel electrophoresis, Sephadex G-50 column chromatography shows simple spike, TSK-gel G.2000SW high pressure liquid chromatography is a simple spike, LKB iso-electric point electrophoresis is single band, proves that gained C-1027 is a monomer.

5, manufacture method according to claim 4 is characterized in that the antibiotic molecular weight of C-1027 that extracts preparation with this method is about 15000, and the-terminal amino acid during amino acid is formed is a L-Ala.

6, manufacture method according to claim 4, it is characterized in that compound with this method preparation, not only the gram positive bacterium growth there is stronger restraining effect, also has tangible anti-tumor activity, generate the assay method inspection with the clone, half casualty-producing concentrations to the human hepatocellular carcinoma BEL-7402 cell is stronger 64 times and 454 times respectively than Zorubicin and mitomycin, and the mouse transplanted solid tumor is comprised S ₁₈₀Sarcoma, U ₁₄Cervical cancer and Harding-passey melanoma all have obvious restraining effect, and therefore, the resulting C-1027 microbiotic of the present invention is useful as antitumor drug.

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