CN105037479A

CN105037479A - Antimicrobial compound based on fungus transformation, and preparation method and application thereof

Info

Publication number: CN105037479A
Application number: CN201510316433.2A
Authority: CN
Inventors: 张立新; 何文妮; 刘雪婷; 罗厚蔚; 张小平; 韩建营; 李小林; 刘苗苗; 代焕琴
Original assignee: Institute of Microbiology of CAS
Current assignee: Institute of Microbiology of CAS
Priority date: 2015-06-10
Filing date: 2015-06-10
Publication date: 2015-11-11
Anticipated expiration: 2035-06-10
Also published as: CN105037479B

Abstract

The invention discloses an antimicrobial compound based on fungus transformation, and a preparation method and application thereof. The compound disclosed by the invention is disclosed as Formula I. The preparation comprises the following steps: by using Hypocrea sp. as a transformation strain and Tanshinone IIA as a substrate, carrying out microbial transformation to prepare a Diels-Alder reaction product, and separating to obtain the compound Tanshisorbicin. The detection by ultraviolet spectroscopy, mass spectroscopy, nuclear magnetic resonance spectroscopy and CD spectroscopy proves that the structure is correct. The compound disclosed by the invention has a brand-new structure, has favorable antimicrobial activity, and is suitable for researching antimicrobial-activity lead compounds or preparing antimicrobial drugs.

Description

Antimicrobial compounds of a kind of fungal transformation and preparation method thereof and application

Technical field

The invention belongs to biomedicine field, relate to antimicrobial compounds of a kind of fungal transformation and preparation method thereof and application.

Background technology

Gram-positive microorganism spread scope is extremely wide, and can cause multi-infection, be the modal pathogenic bacterium of hospital, its infection rate is also higher.In recent years, due to reasons such as antibiotic abuses, resistance phenomenon increases year by year, and this is the one of the main reasons causing infectious diseases mortality ratio high.Therefore, active development has efficiently, the overriding resistance medicine of low toxicity is significant.

Microbe transformation method utilizes microorganism system to carry out structural modification to compound, its essence is the catalysis of enzyme, be characterized in that there is solid highly, site and regioselectivity, the racemic derivant of chemosynthesis, precursor or latent chipal compounds can be changed into chiral product, selective structure modification can be carried out to the complicated natural compounds containing multiple functional group simultaneously.Therefore, the main active ingredient that microbial transformation can be utilized large to content in Chinese medicine carries out structural modification, obtains new active compound, not only safe and effective but also can reduce medicament research and development cost.

Diels-Alder reaction (Diels-Alder reaction) is a kind of cycloaddition reaction, and its process is exactly that conjugated dienes and substituted olefine (being commonly referred to as dienophile) react and generate substituted cyclohexene in simple terms.This reaction be nineteen twenty-eight by Germanization scholar Otto Deere this with his student Ku Erte Alder Late Cambrian.The little energy of Diels-Alder reaction just can form six-ring, is one of principal reaction of C―C bond formation.This reaction once generates two carbon-carbon bonds and maximum four adjacent chiral centres, can greatly reduce reactions steps simultaneously, improve the efficiency of synthesis, so paid attention to very much in organic synthesis.But occurring in nature find can promote that the natural enzyme albumen of [4+2] Diels-Alder cyclization is little, current report Diels-Alder katalaze enzyme only has 5 (Enzyme-catalysed [4+2] cycloadditionisakeystepinthebiosynthesisofspinosynA., Nature, 2011,473 (7345): 109-112; ThestructureofSpnF, astandaloneenzymethatcatalyzes [4+2] cycloaddition, NatureChemicalBiology, 2015,11 (4): 256-258), the structure of these enzymes is very novel, can efficiently, the reaction of stereoselectivity catalysis natural product, the research finding Diels-Alder enzyme and relevant mechanism thereof from occurring in nature attracts the interest of numerous investigator for a long time.

Summary of the invention

The object of this invention is to provide antimicrobial compounds of a kind of fungal transformation and preparation method thereof and application.

Compound provided by the present invention, shown in I:

The application of described compound or pharmaceutically acceptable salt thereof in preparation antibacterials also belongs to protection scope of the present invention.

Another object of the present invention is to provide a kind of antibacterials.

The activeconstituents of antibacterials provided by the present invention is described compound or pharmaceutically acceptable salt thereof.

In the present invention, described antibacterials are specially the medicine of resisting gram-positive bacterium.

Further, described gram positive bacterium can be at least one as follows: methicillin-resistant staphylococcus aureus, Bacillus subtilus, bacille Calmette-Guerin vaccine and streptococcus aureus.

In an embodiment of the present invention, described bacille Calmette-Guerin vaccine is specially mycobacterium bovis (Mycobacteriumbovis) attenuated strain BCG (Pasteur1173P2); Described streptococcus aureus is specially streptococcus aureus ATCC6538; Described methicillin-resistant staphylococcus aureus is specially the methicillin-resistant staphylococcus aureus be recorded in " Gosbell.Methicillin-resistantStaphylococcusaureus.Americ alJournalofClinicalDermatology.2004,5 (4): 239-259. " literary composition; Described Bacillus subtilus is specially Bacillus subtilus (Bacillussubtilis) ATCC6633.

In described antibacterials, the mass content of described compound can be 0.1-90%.

The medicine of described antibacterials imports body as muscle, intracutaneous, subcutaneous, vein, mucosal tissue by the method that injection, injection, collunarium, eye drip, infiltration, absorption, physics or chemistry mediate; Or to be mixed by other materials or to import body after wrapping up.

When needing, one or more pharmaceutically acceptable carriers can also be added in said medicine.Described carrier comprises the thinner, vehicle, weighting agent, tackiness agent, wetting agent, disintegrating agent, absorption enhancer, tensio-active agent, absorption carrier, lubricant etc. of pharmaceutical field routine.

The antibacterials being active fraction preparation with described compound can make the various ways such as injection liquid, tablet, pulvis, granule, capsule, oral liquid, paste, creme.The medicine of above-mentioned various formulation all can be prepared according to the ordinary method of pharmaceutical field.

Also object of the present invention is to provide the preparation method of described compound.

The preparation method of described compound, for transforming bacterial strain with meat seat bacterium (Hypocreasp.), microbial transformation is carried out to tanshinone IIA, make the own metabolism product sorbicillinol of described tanshinone IIA and described meat seat bacterium (Hypocreasp.) that Diels-Alder reaction occur, products therefrom is described compound.

Described meat seat bacterium (Hypocreasp.) is the teleomorph of wood mould (Trichoderma), sorbicillinol be also wood mould in common secondary metabolite.Preliminary proof of the present invention by experiment, this bacterium can produce enzyme and substrate sorbicillinol that Diels-Alder reaction occurs.

Described meat seat bacterium (Hypocreasp.) is specially meat seat bacterium (Hypocreasp.) FS4-3CGMCC3.17108 in the present invention.

Specifically, described preparation method comprises the steps: described meat seat bacterium (Hypocreasp.) FS4-3CGMCC3.17108 to be inoculated in fermention medium, cultivate under 25-30 DEG C (as 28 DEG C) 3-4 days (as 3 days), then described tanshinone IIA is added, cultivate under 25-30 DEG C (as 28 DEG C) 5-7 days (as 5 days), obtain fermented liquid; Described compound is obtained from described fermented liquid.

Wherein, described cultivation is shaking table rotating and culturing, and rotating speed is 220rpm.

Described fermention medium is composed as follows: containing 200g potato (peeling, is cut into small pieces and boils 20min, filtered through gauze), 20g glucose in often liter of described fermention medium, surplus is water; Nature pH.

In the process, described compound is obtained by realizing as follows: described filtering fermentation liquor (as adopted 4 layers of filtered through gauze) is collected filtrate afterwards from described fermented liquid, described filtrate is obtained enriched material (as concentrated enriched material as described in evaporate to dryness acquisition with Rotary Evaporators) with organic solvent extraction (as extracted 3 times) is concentrated afterwards, described enriched material is separated by silica gel column chromatography, RPLC successively, obtains described compound.

In the present invention, when carrying out described extraction, the consumption of described organic solvent is and described filtrate equal-volume.Described organic solvent is ethyl acetate.

In the present invention, when carrying out described silica gel column chromatography, wash-out is carried out successively, the equal wash-out of each elutriant 5 column volumes with 7 kinds of elutriants that the hexanaphthene and acetone that are respectively 100:1,100:2,100:5,100:10,100:20,100:50 and 100:100 by volume ratio mix; Collecting by volume ratio is the elution fraction that elutriant that the hexanaphthene of 100:5 and acetone mix is corresponding, obtains concentrated solution after concentrated (concentrating as adopted Rotary Evaporators); When carrying out described RPLC, described concentrated solution is carried out RPLC according to following condition: chromatographic column is C ₃reverse-phase chromatographic column, determined wavelength is 254nm, moving phase to be volumn concentration be 65% acetonitrile solution, isocratic elution, being collected in the retention time that 254nm wavelength place occurs is the elution peak of 31.0min, obtains described compound; The flow velocity of described isocratic elution specifically can be 2mL/min.

In addition, in the present invention, when carrying out described silica gel column chromatography, the silica gel granularity of employing is specially 200-300 order; Silicagel column is specially normal pressure post (normal pressure post), and specification is 50 × 400mm (glass column); Column temperature is room temperature (20-25 DEG C); Used silica gel amount is 15g; The applied sample amount of described enriched material is 1.35mg.

The present invention adopts meat seat bacterium (Hypocreasp.) FS4-3CGMCC3.17108 for transforming bacterial strain, take Tanshinone II A as substrate, Diels-Alder reaction product is prepared by microbial conversion process, separation obtains compound Tanshisorbicin, through UV spectrum, mass spectrum, nuclear magnetic resonance spectrum, CD spectrum, detect its structure correct.Experiment proves that compound structure provided by the invention is brand-new, has good anti-microbial activity, is suitable for research or the preparation antibacterials of anti-microbial activity lead compound.

Accompanying drawing explanation

Fig. 1 is the uv-spectrogram of compound Tanshisorbicin of the present invention.

Fig. 2 is the mass spectrum of compound Tanshisorbicin of the present invention.

Fig. 3 is that compound Tanshisorbicin of the present invention is dissolved in DMSO-d ₆in ¹h-NMR spectrogram.

Fig. 4 is that compound Tanshisorbicin of the present invention is dissolved in DMSO-d ₆in ¹³c-NMR spectrogram.

Fig. 5 is that compound Tanshisorbicin of the present invention is dissolved in DMSO-d ₆in ¹h- ¹hCOSY spectrogram

Fig. 6 is that compound Tanshisorbicin of the present invention is dissolved in DMSO-d ₆in hsqc spectrum figure.

Fig. 7 is that compound Tanshisorbicin of the present invention is dissolved in DMSO-d ₆in HMBC spectrogram.

Fig. 8 is that compound Tanshisorbicin of the present invention is dissolved in DMSO-d ₆in NOESY spectrogram.

Fig. 9 is circular dichroism spectrum (CD) figure that compound Tanshisorbicin of the present invention is dissolved in chloroform.

Figure 10 is the vitro enzyme experiment that meat seat bacterium (Hypocreasp.) FS4-3CGMCC3.17108 transforms tanshinone IIA.Wherein, 1: meat seat bacterium (Hypocreasp.) FS4-3CGMCC3.17108 cultivates the HPLC collection of illustrative plates of bacterium liquid after 3 days; 2: meat seat bacterium (Hypocreasp.) FS4-3CGMCC3.17108 cultivates bacterium liquid after 3 days heats the HPLC collection of illustrative plates after 5 minutes in boiling water; 3: after meat seat bacterium (Hypocreasp.) FS4-3CGMCC3.17108 cultivates 3 days, bacterium liquid places the HPLC collection of illustrative plates after 10 hours; 4: after meat seat bacterium (Hypocreasp.) FS4-3CGMCC3.17108 cultivates 3 days, bacterium liquid heats the HPLC collection of illustrative plates placed again for 5 minutes after 10 hours in boiling water; The HPLC collection of illustrative plates that tanshinone IIA reacts 10 hours is added in 5: meat seat bacterium (Hypocreasp.) FS4-3CGMCC3.17108 bacterium liquid; In boiling water, the HPLC collection of illustrative plates adding tanshinone IIA after 5 minutes and react 10 hours is heated in 6: meat seat bacterium (Hypocreasp.) FS4-3CGMCC3.17108 bacterium liquid; 7: the HPLC collection of illustrative plates of tanshinone IIA reference substance; The HPLC collection of illustrative plates of 8:Tanshisorbicin reference substance.

Embodiment

The experimental technique used in following embodiment if no special instructions, is ordinary method.

Material used in following embodiment, reagent etc., if no special instructions, all can obtain from commercial channels.

The potato that following embodiment uses is purchased from common market.

Glucose is purchased from Chemical Reagent Co., Ltd., Sinopharm Group, and catalog number is K491390.

Meat seat bacterium (Hypocreasp.) FS4-3: preserving number is CGMCC3.17108.

Tanshinone II A is Hao Xuan bio tech ltd, Xi'an product.

Mycobacterium tuberculosis var.bovis (Mycobacteriumbovis) attenuated strain BCG (Pasteur1173P2): with green fluorescent protein (GFP) expression vector, be recorded in " RolandBrosch, StephenV.Gordon, ThierryGarnier, etal.GenomeplasticityofBCGandimpactonvaccineefficacy.Pro ceedingsoftheNationalAcademyoftheSciencesoftheUnitedStat esofAmerica, 2007, 104 (13): 5596-5601. " in a literary composition, the public can obtain to repeat the present invention from applicant.

Streptococcus aureus (Staphylococcusaureus) ATCC6538: be recorded in " Gao Xuehui. the clone of streptococcus aureus (ATCC6538) protein A gene and applied research. Northeast Agricultural University, Master's thesis, 2010." literary composition, the public can obtain to repeat the present invention from applicant.

Methicillin-resistant staphylococcus aureus (methicillin-resistantStaphylococcusaureus): come from the clinical separation Resistant strain of Beijing Chaoyang Hospital Attached to Capital Medical Univ..Be recorded in " Gosbell.Methicillin-resistantStaphylococcusaureus.Americ alJournalofClinicalDermatology.2004; 5 (4): 239-259. " civilian, the public can obtain to repeat the present invention from applicant.

Bacillus subtilus (Bacillussubtilis) ATCC6633: be recorded in " Tong Kezhong; Li Mingfeng. the spontaneous mutation at Bacillus subtilus (Bacillussubtilis) Streptomycin sulphate seat; microorganism journal; 1965; 11 (04): 544-553. " in a literary composition, the public can obtain to repeat the present invention from applicant.

The Preparation and identification of embodiment 1, antimicrobial compounds

One, fermentation is for antimicrobial compounds

(1) microbial transformation tanshinone IIA fermentation is for antimicrobial compounds

1, seed culture

(1) by slant medium sterilizing 20min at 121 DEG C, bevel, in 28 DEG C of constant temperature culture 3 days.Slightly dry to surface-moisture, during without varied bacteria growing, by meat seat bacterium (Hypocreasp.) FS4-3CGMCC3.17108 bacterial classification spore inoculating in slant medium, cultivate 3 days in 28 DEG C, outward appearance is white hypha, upper attachment green spores, and mycelia is plentiful, can use be collected without during microbiological contamination, namely obtain slant strains.

Above-mentioned slant medium consists of the following composition: potato, glucose, agar and water;

Each composition concentration in described slant medium is respectively (g/L): potato (peeling, be cut into small pieces and boil 20min, filtered through gauze) 200g/L, glucose 20g/L, agar 20g/L, the pH value of described slant medium is nature pH, and solvent is water.

(2) in multiple 250mL vial, be respectively charged into 50mL seed culture medium, add sealed membrane, sterilizing 20 minutes at 121 DEG C, dig block by inclined-plane and inoculate, to connect bacterial classification be slant strains prepared by above-mentioned steps (1).In 28 DEG C rotary shaker rotating and culturing (rotating speed is 220rpm) 48 hours, obtain seed liquor.

Described seed culture medium consists of the following composition: potato, glucose and water;

Each composition concentration in described seed culture medium is respectively: potato 200g/L (peeling, is cut into small pieces and boils 20min, filtered through gauze), glucose 20g/L, the pH value of described seed culture medium is nature pH, and solvent is water.

2, fermentation culture

Preparation fermention medium.Packing 300mL fermention medium in the triangular flask of 1000mL, according to the inoculum size of 2% (volume percent), the seed liquor that above-mentioned steps 1 obtains is inoculated in fermention medium after sterilizing, in 28 DEG C of rotating and culturing (rotating speed is 220rpm) after 3 days, every bottle adds substrate tanshinone IIA 15mg, continue at 28 DEG C of rotating and culturing (rotating speed is 220rpm) 5 days, obtain fermented liquid.Fermented liquid is all substances in container.

Described fermention medium consists of the following composition: potato, glucose and water;

Each composition concentration in described fermention medium is respectively: potato 200g/L (peeling, is cut into small pieces and boils 20min, filtered through gauze), glucose 20g/L, the pH value of described fermention medium is nature pH, and solvent is water.

Three, from fermented liquid, be separated antimicrobial compounds and identify

1, separation and purification antimicrobial compounds

The filtering fermentation liquor (adopting 4 layers of filtered through gauze) step 2 obtained, collects filtrate.Filtrate is extracted 3 times by equal-volume ethyl acetate (15L), concentrates evaporate to dryness with Rotary Evaporators, weigh, obtain extract.Then extract silica gel chromatographic column (silica gel granularity is 200-300 order) is separated.Silica gel column chromatography condition is as follows: silicagel column is specially normal pressure post, and specification is 50 × 400mm (general glass column); Column temperature is room temperature; Used silica gel amount is 15g; The applied sample amount of described enriched material is 1.35mg; Elutriant and elution program are: employing volume ratio is the cyclohexane-acetone mixing solutions of 100:1,100:2,100:5,100:10,100:20,100:50 and 100:100 is successively elutriant, often kind of elution 5 column volumes.Collecting by volume ratio is the elution fraction that elutriant that the hexanaphthene of 100:5 and acetone mix is corresponding, is concentrated, obtain concentrated solution by Rotary Evaporators.

Described concentrated solution is separated through reversed-phased high performace liquid chromatographic, actual conditions is as follows: AgilentZORBAXSB-C3 reverse-phase chromatographic column, 9.4 × 250mm, determined wavelength 254nm, moving phase to be volumn concentration be 65% acetonitrile solution, isocratic elution (flow velocity is 2.0mL/min clock), the retention time being collected in the appearance of 254nm wavelength place is the elution peak component of 31.0min, obtains Tanshisorbicin compound after concentrating evaporate to dryness with Rotary Evaporators.

2, authenticating compound Tanshisorbicin

The compound Tanshisorbicin that above-mentioned steps 1 separation and purification obtains is identified:

(1) outward appearance: Tanshisorbicin is amorphous orange powder.

(2) solvability: be soluble in chloroform, is slightly soluble in acetone, methyl alcohol.

(3) UV spectrum: the UV spectrum of Tanshisorbicin chloroformic solution 365, there is maximum absorption band at 270nm place, sees Fig. 1.UV spectrum testing tool is MarinerSystem5304instrument.

(4) optical value: Tanshisorbicin, (c0.025, CHCl ₃); Optical rotational activity spectrum testing tool is NPerkin-ElmerModel343polarimeter, adopts sodium spectrum D line (589.3nm) to measure, measures length of tube 1dm.

(5) high resolution mass spectrum: Fig. 2 is the HRESIMS mass spectrum of Tanshisorbicin, shows its [M+H] ⁺peak is m/z543.2394, and provides most probable molecular formula to be C ₃₃h ₃₄o ₇.HRESIMS test adopts BrukerAPEX III 7.0Tspectrometer.Methyl alcohol is solvent.

(6) the NMR test of nuclear magnetic resonance spectrum: Tanshisorbicin adopt Bruker600MHz instrument ( ¹hNMR600MHz; ¹³cNMR125MHz), solvent is DMSO-d ₆(solvent peak corrects δ _h2.50/ δ _c39.5).Fig. 3 is Tanshisorbicin ¹h-NMR spectrogram.Fig. 4 is Tanshisorbicin ¹³c-NMR spectrogram.According to compound ¹h-NMR, ¹³c-NMR, ¹h- ¹hCOSY (Fig. 5), HSQC (Fig. 6), HMBC (Fig. 7) and NOESY (Fig. 8), is studied the nuclear magnetic resonance spectrum of compound and belongs to, in table 1 all hydrocarbon signals.

And finally determine that structure is such as formula I:

Table 1Tanshisorbicin's ¹h-NMR and ¹³c-NMR composes each peak ownership

Position	δ _H,mult(J)	δ _C	COSY	HMBC	NOE
						1		68.5
2		197.5
						3		105.7
4	3.90,d(3.7)	45.6	8	2,3,5,6,7,8,9	8,10
						5		73.4
6		209.1
						7		54.6
8	5.50,d(3.7)	91.1	4	1,3,28,31	4
						9		170.6
10	6.66,d(14.9)	119.1	11	9,12	4
						11	7.17,dd(11.0,14.8)	143.2	10,12	9,13
12	6.44,m	131.6	11,13	14
						13	6.26,td(14.8,6.7)	140.8	12,14	11,14
14	1.87,d(6.7)	19.3	13	12,13
						15		116.5
16		183.6
						17		175.4
18		128.7
						19		143.0
20	3.04,m	29.8	21	18,19,21,22,24
						21	1.70,m	19.2	20	22

22	1.59,br.d	37.8		20,23,29,30
					23		35.2
24		152.9
					25	7.82,d(8.2)	133.7	26	19,23,27
26	7.38,d(8.2)	123.0	25	18,24,28
					27		125.6
28		171.0
					29	1.24,s	32.1		22,23,24,30
30	1.27,s	31.9		22,23,24,29
					31	1.44,s	21.5		1,7,8,15
32	1.28,s	9.3		1,2,6,7
					33	1.17,s	24.2		4,5,6

(7) the CD spectrum signature of CD spectrum signature: Tanshisorbicin is shown in Fig. 9.Testing tool is JASCOJ-815Spectropolarimeter, CHCl ₃for solvent.

(8) absolute configuration is determined: calculated by the TDDFT rule of CD, compared with detection collection of illustrative plates, the absolute configuration of final deterministic compound is 1S, 4R, 5S, 7S and 8R.

(2) prove that meat seat bacterium (Hypocreasp.) FS4-3CGMCC3.17108 can produce the enzyme and substrate sorbicillinol that Diels-Alder reaction occurs

The preparation method of antimicrobial compounds of the present invention, for transforming bacterial strain with meat seat bacterium (Hypocreasp.) FS4-3CGMCC3.17108, microbial transformation is carried out to tanshinone IIA, make the own metabolism product sorbicillinol of described tanshinone IIA and described meat seat bacterium (Hypocreasp.) FS4-3CGMCC3.17108 that Diels-Alder reaction occur, products therefrom is described compound.

Described meat seat bacterium (Hypocreasp.) FS4-3CGMCC3.17108 is the teleomorph of wood mould (Trichoderma), sorbicillinol be also wood mould in common secondary metabolite.Preliminary proof of the present invention by experiment, this bacterium can produce enzyme and substrate sorbicillinol that Diels-Alder reaction occurs.

A. experimental technique

(1) meat seat bacterium (Hypocreasp.) FS4-3CGMCC3.17108 is inoculated into fermention medium (formula sees above) from slant medium (formula sees above), 250mL shaking flask contains 50mL substratum, cultivate 3 days, filter paper filtering, with distilled water wash, discard mycelia, get bacterium liquid, the further mistake of bacterium liquid 0.22 μm of filter membrane.

(2) get bacterium liquid 1mL, carry out HPLC detection (actual conditions is shown in following steps (6)), the retention time of sorbicillinol is at 4.4min.

(3) get bacterium liquid 1mL, be placed in closed EP pipe, in boiling water heating 5min, be cooled to room temperature, make enzyme deactivation, carry out HPLC detection (actual conditions is shown in following steps (6)), inspection sorbicillinol stability.

(4) bacterium liquid 10mL is got, add 0.3mg tanshinone IIA, react 10 hours, get bacterium liquid 1mL, add same volume extraction into ethyl acetate, ethyl acetate layer sample decompression and solvent recovery obtains solid, redissolves be prepared into sample liquid with 1mL methyl alcohol, carry out HPLC detection (actual conditions is shown in following steps (6)), detect Tanshisorbicin output.

(5) bacterium liquid 10mL is got, be placed in closed EP pipe, in boiling water heating 5min, be cooled to room temperature, then add tanshinone IIA 0.3mg, react 10 hours at 37 DEG C, get bacterium liquid 1mL, add same volume extraction into ethyl acetate, ethyl acetate layer sample decompression and solvent recovery obtains solid, redissolve with 1mL methyl alcohol and be prepared into sample liquid, carry out HPLC detection (actual conditions is shown in following steps (6)), detect Tanshisorbicin output.

(6) condition of high performance liquid chromatography is: AgilentZORBAXSB-C3 reverse-phase chromatographic column, 4.6 × 150mm, determined wavelength 370nm, flow velocity 1mL/min, moving phase is the acetonitrile-aqueous acid aqueous formic acid of 0.1% (sour water to be Volume fraction be), elution requirement: 0-10 minute volume ratio is the acetonitrile-sour water (volumn concentration of acetonitrile in moving phase at the uniform velocity increases to 70% by 40% in 0-10 minute) of 40%-70%, 10-15 minute volume ratio is the acetonitrile-sour water (volumn concentration of acetonitrile in moving phase is 70% in 10-15 minute) of 70%, 15-20 minute volume ratio is the acetonitrile-sour water (volumn concentration of acetonitrile in moving phase at the uniform velocity increases to 95% by 70% in 15-20 minute) of 70%-95%.

B. experimental result

Because sorbicillinol is variable, so need the method to removing proteolytic enzyme to investigate.The method not only can have been removed proteolytic enzyme but also not affect the stability of sorbicillinol.Known by preliminary experiment, the treatment process of heated and inactivated albumen can not make sorbicillinol change, and therefore we have selected the method and remove proteolytic enzyme, then the tanshinone IIA (0.3mg) of equivalent is added, react 10 hours, carry out HPLC analysis, as Figure 10.After removing proteolytic enzyme, the amount of product Tanshisorbicin obviously reduces.Before proteolytic enzyme inactivation, Tanshisorbicin productive rate can reach 53.6%, and after heating, proteolytic enzyme inactivation productive rate is reduced to 10.3% (calculation formula 1.3 × Tanshisorbicin peak area/1201514/0.55306).

Above result proves, meat seat bacterium (Hypocreasp.) FS4-3CGMCC3.17108 can produce enzyme and substrate sorbicillinol that Diels-Alder reaction occurs.Namely Tanshisorbicin compound of the present invention is for transforming bacterial strain with meat seat bacterium (Hypocreasp.) FS4-3CGMCC3.17108, microbial transformation is carried out to tanshinone IIA, makes the own metabolism product sorbicillinol of described tanshinone IIA and described meat seat bacterium (Hypocreasp.) FS4-3CGMCC3.17108 that the product of Diels-Alder reaction gained occur.

The Determination of Antibacterial Activity of embodiment 2, compound Tanshisorbicin

One, experimental technique

1, anti-BCG determination of activity

Bacille Calmette-Guerin vaccine (BCG) bacterial strain used is mycobacterium bovis (Mycobacteriumbovis) attenuated strain BCG (Pasteur1173P2) (being called for short BCG bacterium), and this bacterial strain is with green fluorescent protein (GFP) expression vector.When cell proliferation, can continuous expression GFP, under the exciting light of specific wavelength, produce specific fluorescent absorption value, thus quantization cell growing multiplication situation.By reading corresponding fluorescent absorption value, the BCG that can weigh testing compound is active.Concrete operations are as follows:

By mycobacterium bovis (Mycobacteriumbovis) attenuated strain BCG (Pasteur1173P2) access containing 40mL7H9 substratum (Shanghai Yu Chen Bioisystech Co., Ltd, U.S. BD271310) 250mL specification triangular flask in, within 3 days, BCG bacterium liquid is obtained afterwards in 37 DEG C of rotating and culturing (rotating speed is 60rpm), microplate reader is used to measure absorbance, controlling initial absorbance value is 0.05-0.055, excitation wavelength is 485nm, and wavelength of transmitted light is 535nm.Packing 40 μ L7H9 substratum is to each hole of 96 well culture plates; Transferase 12 μ L contains dimethyl sulfoxide (DMSO) (DMSO) solution of testing compound Tanshisorbicin, and (testing compound starting point concentration is that 40mmol/mL starts, carry out two times of gradient dilutions 7 times, the concentration obtaining 8 gradients is altogether followed successively by 40mmol/mL, 20mmol/mL, 10mmol/mL, 5mmol/mL, 2.50mmol/mL, 1.25mmol/mL, 0.625mmol/mL and 0.313mmol/mL; Corresponding final concentration is respectively 1000 μm of ol/mL, 500 μm of ol/mL, 250 μm of ol/mL, 125 μm of ol/mL, 62.5 μm of ol/mL, 31.25 μm of ol/mL, 15.6 μm of ol/mL, 7.8 μm of ol/mL) in 96 well culture plates; Join OD ₆₀₀be the BCG bacterium liquid of 0.025 concentration, add every hole 40 μ L in 96 well culture plates.96 well culture plates are placed in 37 DEG C of incubators and cultivate after 72 hours, and statistics testing compound is to the minimum inhibitory concentration (MIC value) of BCG bacterium.By observing the growing state (GFP expresses and produces green fluorescence) of BCG bacterium, the final compound concentration growing suppressed more than 90% is the minimum inhibitory concentration MIC value of this compound to BCG.Experiment is with vazadrine (Sigma, St.Louis, USA, I3377) be positive control (vazadrine final concentration of correspondence in system is respectively final concentration corresponding to vancomycin and is respectively 10 μm of ol/mL, 5 μm of ol/mL, 2.5 μm of ol/mL, 1.25 μm of ol/mL, 0.625 μm of ol/mL, 0.313 μm of ol/mL, 0.156 μm of ol/mL and 0.078 μm ol/mL), with DMSO for negative contrast), with DMSO for negative contrast.

Test in triplicate, results averaged.

2, anti-MRSA, BS, SA determination of activity

Methicillin-resistant staphylococcus aureus (methicillin-resistantStaphylococcusaureus) (being called for short MRSA bacterium) being activated by being inoculated in cryopreservation tube on LB Agar Plating, cultivating 20 hours in 37 DEG C; With aseptic inoculation ring picking 3 mono-bacterium colonies of MRSA in 3mLMHB substratum (Qingdao Hai Bo Bioisystech Co., Ltd, HB6231), using turbula shaker fully to mix becomes bacterium liquid mother liquor, uses blood counting chamber to detect bacterium dense; With MHB substratum, bacterium liquid mother liquor is diluted to 2 × 10 ⁴individual/mL, becomes stand-by bacterium liquid.Get aseptic 96 porocyte culture plates, pipette 40 μ LMHB substratum to each hole; Transferase 12 μ L vancomycin (Sigma, St.Louis, USA, V2002) DMSO solution (final concentration that vancomycin is corresponding is respectively 10 μm of ol/mL, 5 μm of ol/mL, 2.5 μm of ol/mL, 1.25 μm of ol/mL, 0.625 μm of ol/mL, 0.313 μm of ol/mL, 0.156 μm of ol/mL and 0.078 μm ol/mL), in 8 holes of 96 porocyte culture plate first rows, is positive controls; Draw 2 μ LDMSO, adding in 8 holes of 96 porocyte culture plates the 12 row, is negative control group; Transferase 12 μ L contains the DMSO solution of testing compound Tanshisorbicin, and (testing compound starting point concentration is that 40mmol/mL starts, carry out two times of gradient dilutions 7 times, the concentration obtaining 8 gradients is altogether followed successively by 40mmol/mL, 20mmol/mL, 10mmol/mL, 5mmol/mL, 2.50mmol/mL, 1.25mmol/mL, 0.625mmol/mL and 0.313mmol/mL; Corresponding final concentration is respectively 1000 μm of ol/mL, 500 μm of ol/mL, 250 μm of ol/mL, 125 μm of ol/mL, 62.5 μm of ol/mL, 31.25 μm of ol/mL, 15.6 μm of ol/mL, 7.8 μm of ol/mL) to 96 porocyte culture plate secondary series in the 11 each hole of row; Pipette the stand-by bacterium liquid of 40 μ LMRSA, add in the 96 each holes of porocyte culture plate; Above-mentioned 96 porocyte culture plates are placed in 37 DEG C of incubators, cultivate after 16 hours, observe the growing state of bacterium, statistics testing compound is to the minimum inhibitory concentration (MIC value) of MRSA bacterium.By observing the growing state of MRSA bacterium, visible MRSA grows the final compound concentration corresponding to hole be totally constrained and is the minimum inhibitory concentration MIC value of this compound to MRSA.

Same method measures the anti-microbial activity of compound Tanshisorbicin to streptococcus aureus (Staphylococcusaureus) ATCC6538 (being called for short SA bacterium) and Bacillus subtilus (Bacillussubtilis) ATCC6633 (being called for short BS bacterium).

Test in triplicate, results averaged.

Two, experimental result

Result shows, and in anti-BCG bacterium experiment, the MIC value of positive control vazadrine is 0.313 μM; In anti-MRSA bacterium experiment, the MIC value of positive control vancomycin is 0.625 μM; In anti-BS bacterium experiment, the MIC value of positive control vancomycin is 0.313 μM; In anti-SA bacterium experiment, the MIC value of positive control vancomycin is 0.625 μM.The Determination of Antibacterial Activity result of the compound Tanshisorbicin that embodiment 1 prepares to BCG bacterium, MRSA bacterium, BS bacterium and SA bacterium is specifically as shown in table 2, and itself MRSA with BS anti-microbial activity is significantly improved compared with substrate tanshinone IIA.

The anti-microbial activity of table 2 compound Tanshisorbicin and Tanshinone II A

Note: [a] represents vazadrine; [b] represents vancomycin.

Claims

1. a compound, shown in I:

2. the application of compound or pharmaceutically acceptable salt thereof described in claim 1 in preparation antibacterials.

3. antibacterials, its activeconstituents is compound or pharmaceutically acceptable salt thereof described in claim 1.

4. application according to claim 2 or antibacterials according to claim 3, is characterized in that: described antibacterials are the medicine of resisting gram-positive bacterium.

5. application according to claim 4 or antibacterials, is characterized in that: described gram positive bacterium be following at least one: methicillin-resistant staphylococcus aureus, Bacillus subtilus, bacille Calmette-Guerin vaccine and streptococcus aureus.

6. prepare the method for compound described in claim 1 for one kind, for transforming bacterial strain with meat seat bacterium (Hypocreasp.), microbial transformation is carried out to tanshinone IIA, make the own metabolism product sorbicillinol of described tanshinone IIA and described meat seat bacterium (Hypocreasp.) that Diels-Alder reaction occur, products therefrom is described compound.

7. method according to claim 6, it is characterized in that: described method comprises the steps: described meat seat bacterium (Hypocreasp.) CGMCC3.17108 to be inoculated in fermention medium, 3-4 days is cultivated at 25-30 DEG C, then described tanshinone IIA is added, at 25-30 DEG C, cultivate 5-7 days, obtain fermented liquid; Described compound is obtained from described fermented liquid.

8. method according to claim 7, is characterized in that: described cultivation is shaking culture, and rotating speed is 220rpm; And/or

Described fermention medium is composed as follows: containing the filtrate, the 20g glucose that are boiled filtration gained by 200g peeling potatoes in often liter of described fermention medium, surplus is water.

9. the method according to claim 7 or 8, it is characterized in that: in described method, described compound is obtained by realizing as follows: collect filtrate by after described filtering fermentation liquor from described fermented liquid, enriched material is obtained by concentrated after described filtrate organic solvent extraction, described enriched material is separated by silica gel column chromatography, RPLC successively, obtains described compound.

10. method according to claim 9, is characterized in that: described organic solvent is ethyl acetate; And/or

When carrying out described silica gel column chromatography, wash-out is carried out successively, the equal wash-out of each elutriant 5 column volumes with 7 kinds of elutriants that the hexanaphthene and acetone that are respectively 100:1,100:2,100:5,100:10,100:20,100:50 and 100:100 by volume ratio mix; Collecting by volume ratio is the elution fraction that elutriant that the hexanaphthene of 100:5 and acetone mix is corresponding, obtains concentrated solution after concentrated;

When carrying out described RPLC, described concentrated solution is carried out RPLC according to following condition: chromatographic column is C ₃reverse-phase chromatographic column, determined wavelength is 254nm, moving phase to be volumn concentration be 65% acetonitrile solution, isocratic elution, being collected in the retention time that 254nm wavelength place occurs is the elution peak of 31.0min, obtains described compound;

The flow velocity of described isocratic elution is specially 2mL/min.