CN104829664A - Anti-bacterial and anti-tumor compound, preparation method and application of same - Google Patents
Anti-bacterial and anti-tumor compound, preparation method and application of same Download PDFInfo
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- CN104829664A CN104829664A CN201510114000.9A CN201510114000A CN104829664A CN 104829664 A CN104829664 A CN 104829664A CN 201510114000 A CN201510114000 A CN 201510114000A CN 104829664 A CN104829664 A CN 104829664A
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- C07—ORGANIC CHEMISTRY
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- C07J73/00—Steroids in which the cyclopenta[a]hydrophenanthrene skeleton has been modified by substitution of one or two carbon atoms by hetero atoms
- C07J73/001—Steroids in which the cyclopenta[a]hydrophenanthrene skeleton has been modified by substitution of one or two carbon atoms by hetero atoms by one hetero atom
- C07J73/003—Steroids in which the cyclopenta[a]hydrophenanthrene skeleton has been modified by substitution of one or two carbon atoms by hetero atoms by one hetero atom by oxygen as hetero atom
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Abstract
The invention provides an anti-bacterial and anti-tumor compound, a preparation method and an application of the same, wherein the compound is a diterpene glucoside compound and is represented as the structure formula I or the structure formula II. The preparation method includes a step of with Mucor roxianus (accession number: CGMCC 3.3447) as a conversion strain, performing microorganism conversion to tanshinone I to obtain the diterpene glucoside compound, wherein the tanshinone I preferably is a nano preparation of the tanshinone I. A pharmacological experiment proves that the compound can inhibit growth of staphylococcus aureus and drug-resistant staphylococcus aureus and meanwhile inhibit proliferation of human gastric carcinoma cells and human breast cancer cells, so that the anti-bacterial and anti-tumor compound can be used for researching of an anti-bacterial and anti-tumor active lead compound or preparation of an anti-bacterial and anti-tumor drug.
Description
Technical field
The invention belongs to biomedicine field, be specifically related to antibacterial antineoplastic compound and preparation method thereof and application.
Background technology
Streptococcus aureus is a kind of gram-positive microorganism, spread scope is extremely wide, so far be found everywhere through the world from discovery, become burn, the main infection pathogenic bacteria of postoperative and children's, be the modal pathogenic bacterium of hospital, its infection rate is also higher, about has 100,000 examples to infect the report of this bacterium in worldwide.
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, wherein common with MRSA methicillin-resistant staphylococcus aureus.MRSA has more extensively, more serious Drug-fast case phenomenon, this means, all there is resistance phenomenon in other all beta-lactam with X-1497 same structure and cephalosporin analog antibiotic, vancomycin is as the unique selection of clinical treatment Drug-fast case MRSA, its resistance increasing pressure is large, now conventional clinically vancomycin has also engendered resistance phenomenon, and the research of microbiotic new drug has met with maximum bottleneck in 30-40 in the past, this cause the most at last without medicine can stage.Therefore, active development has efficiently, the medicine of the overriding resistance streptococcus aureus of low toxicity is significant.
Cancer is a kind of very ancient disease, and along with the history of whole human development, health and the life of the mankind in serious threat.The World Health Organization nearest one investigation display, cancer becomes the number one killer of global human health by surmounting heart trouble, the number of worldwide internal cause cancer and death is the situation of cumulative year after year.Within 2012, the whole world increases 1,400 ten thousand cases of cancers newly altogether and has 8,200,000 people dead.Wherein, newly-increased 3,070,000 cancer patientss of China also cause about 2,200,000 people dead, account for 21.9% and 26.8% of global total amount respectively.In 4 kinds of malignant tumours such as liver, esophagus, stomach and lung, Chinese new cases and death toll all occupy first place in the world.Therefore the treatment for cancer is faced with very stern challenge.Find and study that to have antitumor drug that is efficient, low toxicity most important.
Summary of the invention
An object of the present invention is to provide a kind of new Diterpene glucoside compounds.
Described Diterpene glucoside compounds is obtained by microbial transformation by the main component tanshinone compound in Salvia salviamiltiorrhizabung Salvia miltiorrhiza Bunge, is brand new type compound; Described Diterpene glucoside compounds can distinguish called after compound Tanshinoside A and compound Tanshinoside B by the difference on glycosylation position.
The structural formula of described compound Tanshinoside A is as shown in following formula I:
The structural formula of described compound Tanshinoside B is as shown in following formula II:
Diterpene glucoside compounds provided by the present invention prepares by the method comprised the steps: with Zygosaccharomyces rouxii (Mucor roxianus) CGMCC 3.3447 for transforming bacterial strain, microbial transformation is carried out to Tanshinone I, obtains Diterpene glucoside compounds.
Described microbial transformation is: cultivate 3 days-4 days under Zygosaccharomyces rouxii (Mucor roxianus) CGMCC 3.3447 seed liquor being inoculated in fermention medium room temperature, then add Tanshinone I room temperature bottom fermentation to cultivate 5 days-7 days (as 5 days), obtain fermented liquid.
Described Zygosaccharomyces rouxii (Mucor roxianus) CGMCC 3.3447 seed liquor is for be inoculated in slant medium by Zygosaccharomyces rouxii (Mucor roxianus) CGMCC 3.3447, and incubated at room temperature, obtains slant strains; Again described slant strains is inoculated into seed culture medium, Zygosaccharomyces rouxii (Mucor roxianus) CGMCC 3.3447 seed liquor obtained after incubated at room temperature.
In aforesaid method, described Tanshinone I is preferably Tanshinone I nanometer formulation.
Described Tanshinone I nanometer formulation is the eutectic pulvis be made up of the Macrogol 4000 of the Tanshinone I of massfraction 5.0-10% and massfraction 90-95% or polyethylene glycol 6000.
Described Tanshinone I nanometer formulation is that the method recorded in the Chinese patent of CN1391889 is prepared with reference to the patent No..Concrete preparation method is as follows: mix after Macrogol 4000 or polyethylene glycol 6000 heating and melting with Tanshinone I, form Tanshinone I-Macrogol 4000 or polyethylene glycol 6000 eutectic, after described eutectic cooling, 3 hours are ground with the rotating speed of 450 revs/min, cross 300 mesh sieves, obtain described Tanshinone I nanometer formulation.
Aforesaid method also comprises the step being separated from described fermented liquid and obtaining Tanshinoside A and Tanshinoside B.Concrete operations are as follows: collect filtrate by after described filtering fermentation liquor, described filtrate is obtained enriched material with concentrated after organic solvent (as ethyl acetate) extraction, described enriched material is separated by silica gel column chromatography, medium pressure column chromatography, reversed-phased high performace liquid chromatographic successively, obtains Tanshinoside A and Tanshinoside B compound.
Wherein, described silica gel column chromatography is: be that the chloroform-acetone mixing solutions of 100:0-100:50 carries out wash-out for elutriant with volume ratio, collect the elution fraction under each volume ratio, minimal inhibitory concentration (MIC) active testing is all carried out to each elution fraction, by the removal of solvents in elution fraction the highest for bacteriostatic activity, obtain concentrated solution.
Above-mentioned minimal inhibitory concentration (MIC) active testing carries out based on streptococcus aureus and methicillin-resistant staphylococcus aureus.
Particularly, described silica gel column chromatography is: employing volume ratio is the chloroform-acetone mixing solutions of 100:0,100:2-5,100:8-12,100:15-25 and 100:30-50 is successively that elutriant carries out wash-out, collected volume is than the elution fraction of the chloroform-acetone mixing solutions for 100:8-12, by the removal of solvents in described elution fraction, obtain concentrated solution.
More specifically, described silica gel column chromatography is: employing volume ratio is the chloroform-acetone mixing solutions of 100:0,100:2,100:3,100:10,100:20 and 100:50 is successively elutriant, collecting chloroform-acetone volume ratio is the component of 100:10, remove the solvent in described component, obtain concentrated solution.
Described medium pressure column chromatography is: the above-mentioned concentrated solution obtained through silica gel column chromatography is carried out medium pressure column chromatography separation, the acetonitrile solution being 10%-100% with the volumn concentration of acetonitrile carries out gradient elution for elutriant, collected volume percentage composition is the elution fraction of the acetonitrile solution of 40%-70%, remove the solvent in described component, concentrate and obtain concentrated solution, wherein, the Gradient program of gradient elution is that in acetonitrile solution, the volumn concentration of acetonitrile is from 10% to 100% from 0min to 90min.
Described reversed-phased high performace liquid chromatographic is: the above-mentioned concentrated solution obtained through medium pressure column chromatography is carried out reversed-phased high performace liquid chromatographic separation, wherein, the condition of described reversed-phased high performace liquid chromatographic is: Agilent ZORBAX – XDB reverse-phase chromatographic column, RP-C8,9.4*250mm, determined wavelength 254nm, moving phase to be volumn concentration be 42% acetonitrile solution, isocratic elution, be collected in elution fraction corresponding when absorption peak appears in 254nm wavelength place successively, obtain Tanshinoside A and Tanshinoside B compound.
Another object of the present invention is to provide the purposes of above-mentioned Diterpene glucoside compounds.
The application that the purposes of Diterpene glucoside compounds provided by the present invention is it in following:
1) application in preparation antibacterials;
2) application in preparation eukaryote tumor cell proliferation inhibitor;
3) application prevented and/or treated in tumour medicine is being prepared
Described antibacterials are mainly the medicine of anti-Staphylococcus aureus and/or resistant Staphylococcus aureus.
Described streptococcus aureus specifically can be streptococcus aureus (Staphylococcus aureus) ATCC 6538;
Described resistant Staphylococcus aureus specifically can be methicillin-resistant staphylococcus aureus (Methicillin-resistant Staphylococcus aureus).
Described eukaryote is Mammals; Described tumour cell is cancer cells; Described cancer cells can be stomach cancer cell and breast cancer cell.
Described stomach cancer cell specifically can be SGC-7901 cells; Described breast cancer cell specifically can be human breast cancer cell line Bcap-37.
Described tumour is cancer; Described cancer is cancer of the stomach or mammary cancer.
The antibacterials being active fraction preparation with above-mentioned Diterpene glucoside compounds, eukaryote tumor cell proliferation inhibitor, the medicine preventing and/or treating tumour also belong to protection scope of the present invention.
Described antibacterials, eukaryote tumor cell proliferation inhibitor or prevent and/or treat in the medicine of tumour, the mass content of described Diterpene glucoside compounds is 0.1-90%.
Described antibacterials, eukaryote tumor cell proliferation inhibitor or the medicine that prevents and/or treats tumour import body as muscle, intracutaneous, subcutaneous, vein, mucosal tissue by the method for injection, injection, collunarium, eye drip, infiltration, absorption, physics or chemistry mediation; 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 Diterpene glucoside compounds, eukaryote tumor cell proliferation inhibitor or the medicine preventing and/or treating tumour 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.
Compound structure provided by the invention is brand-new, has well antibacterial and anti-tumor activity, is suitable for the research of antibacterial and anti-tumor activity lead compound or prepares antibacterial and antitumor drug.
The present invention adopts Zygosaccharomyces rouxii (Mucor roxianus) AS 3.3447 for transforming bacterial strain, respectively with Tanshinone I, Tanshinone I nanometer formulation and Tanshinone II A are substrate, Diterpene glucoside compounds is prepared by microbial conversion process, respectively HPLC spectrum analysis is carried out to system after these three kinds reactions, find with Tanshinone I nanometer formulation for substrate time, the product amount obtained is the highest, then by easy extraction process, separation obtains compound Tanshinoside A and Tanshinoside B, through nucleus magnetic resonance, UV spectrum, mass spectrometric detection, its structure is correct.
Accompanying drawing explanation
Figure 1A is the HPLC spectrogram that Zygosaccharomyces rouxii of the present invention (Mucor roxianus) CGMCC 3.3447 pairs of Tanshinone Is transform; Figure 1B is the HPLC spectrogram that Zygosaccharomyces rouxii (Mucor roxianus) CGMCC 3.3447 pairs of Tanshinone I nanometer formulations transform; Fig. 1 C is the HPLC spectrogram that Zygosaccharomyces rouxii (Mucor roxianus) CGMCC 3.3447 pairs of Tanshinone II As transform; Fig. 1 D is the standard HPLC spectrogram of Tanshinoside A; Fig. 1 E is the standard HPLC spectrogram of Tanshinoside B.
Fig. 2 is the uv-spectrogram of compound Tanshinoside A of the present invention.
Fig. 3 is the uv-spectrogram of compound Tanshinoside B of the present invention.
Fig. 4 is the high resolution mass spectrum figure of compound Tanshinoside A of the present invention.
Fig. 5 is the high resolution mass spectrum figure of compound Tanshinoside B of the present invention.
Fig. 6 is that compound Tanshinoside A of the present invention is dissolved in DMSO-d
6in
1h-NMR spectrogram.
Fig. 7 is that compound Tanshinoside B of the present invention is dissolved in DMSO-d
6in
1h-NMR spectrogram.
Fig. 8 is that compound Tanshinoside A of the present invention is dissolved in DMSO-d
6in
13c-NMR spectrogram.
Fig. 9 is that compound Tanshinoside B of the present invention is dissolved in DMSO-d
6in
13c-NMR spectrogram.
Figure 10 is that compound Tanshinoside A of the present invention is dissolved in DMSO-d
6in
1h-
1hCOSY composes.
Figure 11 is that compound Tanshinoside B of the present invention is dissolved in DMSO-d
6in
1h-
1hCOSY composes.
Figure 12 is that compound Tanshinoside A of the present invention is dissolved in DMSO-d
6in hsqc spectrum figure.
Figure 13 is that compound Tanshinoside B of the present invention is dissolved in DMSO-d
6in hsqc spectrum figure.
Figure 14 is that compound Tanshinoside A of the present invention is dissolved in DMSO-d
6in HMBC spectrogram.
Figure 15 is that compound Tanshinoside B of the present invention is dissolved in DMSO-d
6in HMBC spectrogram.
Figure 16 is that compound Tanshinoside A of the present invention is dissolved in DMSO-d
6in ROESY spectrogram.
Figure 17 is that compound Tanshinoside B of the present invention is dissolved in DMSO-d
6in ROESY spectrogram.
Embodiment
Below by specific embodiment, the present invention will be described, but the present invention is not limited thereto.
The experimental technique used in following embodiment if no special instructions, is ordinary method; Reagent used in following embodiment, biomaterial 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.
Zygosaccharomyces rouxii (Mucor roxianus) preserving number is CGMCC 3.3447.
Tanshinone I and Tanshinone II A are purchased from Hao Xuan bio tech ltd, Xi'an.
Tanshinone I nanometer formulation is that the method recorded in the Chinese patent of CN1391889 is prepared with reference to the patent No..Concrete preparation method is as follows: mix after 95g Macrogol 4000 heating and melting with 5g Tanshinone I, form Tanshinone I-Macrogol 4000 eutectic, after described eutectic cooling, grind 3 hours with the rotating speed of 450 revs/min, cross 300 mesh sieves, obtain Tanshinone I nanometer formulation 70.5g.In described Tanshinone I nanometer formulation, the mass content of Tanshinone I is 6.65%.
Streptococcus aureus (Staphylococcus aureus) ATCC 6538: be recorded in " Gao Xuehui. the clone of streptococcus aureus (ATCC6538) protein A gene and applied research. Northeast Agricultural University; Master's thesis, 2010 ".
Methicillin-resistant staphylococcus aureus (Methicillin-resistant Staphylococcus aureus): be recorded in " IB Gosbell.Methicillin-resistant Staphylococcus aureus.Americal Journal of Clinical Dermatology.2004,5 (4): 239-259 ".
SGC-7901 cells, human liver cancer cell BEL-7402 and human breast cancer cell line Bcap-37 are all purchased from Shanghai cell bank.
The Preparation and identification of embodiment 1, antibacterial and antineoplastic compound.
One, substrate that is antibacterial and antineoplastic compound is prepared in screening
1, seed culture
(1) by slant medium sterilizing 20 minutes 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 Zygosaccharomyces rouxii (Mucor roxianus) CGMCC 3.3447 bacterial classification spore inoculating in slant medium, cultivate 3 days in 28 DEG C, outward appearance is white hypha, upper attachment black spore, 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;
Above composition concentration in described slant medium is respectively (g/L):
Potato 200g/L, glucose 20g/L, agar 20g/L, the pH value of described slant medium is nature pH.
(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;
Above composition concentration in described seed culture medium is respectively:
Potato 200g/L, glucose 20g/L, the pH value of described seed culture medium is nature pH.
2, substrate screening
Preparation screening is with fermention medium 9 bottles (Media Components: potato 200g/L, glucose 20g/L, the pH value of described fermention medium is nature pH, and solvent is water).Packing 50ml fermention medium in the triangular flask of 250ml, 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, add substrate Tanshinone I (3 bottles) respectively, Tanshinone I nanometer formulation (3 bottles), Tanshinone II A (3 bottles), wherein Tanshinone I and Tanshinone II A every bottle add 1.5 mg, Tanshinone I nanometer formulation every bottle adds 22mg (wherein Tanshinone I is 1.5mg), continues cultivation and obtains fermented liquid in 5 days.Fermented liquid is all substances in container.
By the filtering fermentation liquor obtained, collect filtrate.Filtrate is extracted 1 time by equal-volume ethyl acetate (50mL), concentrates evaporate to dryness with Rotary Evaporators, weigh, be dissolved in 1mL methyl alcohol, carry out analysed by reverse phase HPLC.The analysis condition of reversed-phase HPLC is: Agilent ZORBAX – XDB reverse-phase chromatographic column, RP-C8,4.5*150mm, determined wavelength 254nm, moving phase is volumn concentration is 5%-95% acetonitrile solution, gradient elution 35min (Gradient program of gradient elution is that in acetonitrile solution, the volumn concentration of acetonitrile is from 5% to 95% from 0min to 35min).
Figure 1A is the HPLC spectrogram that Zygosaccharomyces rouxii (Mucor roxianus) CGMCC 3.3447 pairs of Tanshinone Is transform; Figure 1B is the HPLC spectrogram that Zygosaccharomyces rouxii (Mucor roxianus) CGMCC 3.3447 pairs of Tanshinone I nanometer formulations transform; Fig. 1 C is the HPLC spectrogram that Zygosaccharomyces rouxii (Mucor roxianus) CGMCC 3.3447 pairs of Tanshinone II As transform; Fig. 1 D is the standard HPLC spectrogram of Tanshinoside A; Fig. 1 E is the standard HPLC spectrogram of Tanshinoside B.
As shown in Figure 1, the highest with the product amount that Tanshinone I nanometer formulation obtains for substrate, therefore select Tanshinone I nanometer formulation to be that substrate prepares described Diterpene glucoside compounds (Tanshinosides A and Tanshinoside B).
Two, fermentation is for antibacterial and antineoplastic compound
1, seed culture
Same (1)
2, fermentation culture
Then fermention medium (Media Components: potato 200g/L, glucose 20g/L, the pH value of described fermention medium is nature pH, and solvent is water) is prepared.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 I nanometer formulation 120mg and continues cultivation 5 days, obtains fermented liquid.Fermented liquid is all substances in container.
Three, be separated antibacterial and antineoplastic compound and identify
1, the antibacterial and antineoplastic compound of separation and purification
By the filtering fermentation liquor that above-mentioned experiment obtains, collect filtrate.Filtrate is extracted 3 times by equal-volume ethyl acetate (10L), concentrates evaporate to dryness with Rotary Evaporators, weigh.Then extract silica gel chromatographic column (200-300 order) is separated, employing volume ratio is the chloroform-acetone mixing solutions of 100:0,100:2,100:3,100:10,100:20 and 100:50 is successively elutriant, often kind of elution 5 column volumes, then after the material got off by the elution of different volumes ratio concentrates respectively, the testing sample solution being mixed with 4mg/ml is dissolved respectively, for minimal inhibitory concentration active testing with DMSO.
Active testing result shows, it is 100:10 chloroform-acetone elution fraction that active part concentrates on volume ratio, minimal inhibitory concentration and the MIC value of 100:10 part are respectively anti-Staphylococcus aureus 1.56 μ g/mL, methicillin-resistant staphylococcus aureus resistance 1.56 μ g/mL.
The component of chloroform-acetone 100:10 part is concentrated by Rotary Evaporators, concentrated solution is carried out ODS (5 μm) medium pressure column chromatography (purchased from Beijing Hui De Easytech Inc.), with acetonitrile-water system 10%-100% (volumn concentration of acetonitrile) gradient elution, (Gradient program is from 0min to 90min, in acetonitrile solution, the volumn concentration of acetonitrile is from 10% to 100%), collect the acetonitrile liquid (chromatographic peak is concentrated and appeared at this region) of 40%-70%, be separated through reversed-phased high performace liquid chromatographic again after concentrated, wherein, the condition of reversed-phase HPLC is: Agilent ZORBAX – XDB reverse-phase chromatographic column, RP-C8, 9.4*250mm, determined wavelength 254nm, moving phase to be volumn concentration be 42% acetonitrile solution, isocratic elution, be collected in elution fraction corresponding when absorption peak appears in 254nm wavelength place successively, obtain Tanshinoside A and Tanshinoside B compound.
2, authenticating compound Tanshinoside A and Tanshinoside B
The compound Tanshinoside A obtain above-mentioned steps 1 separation and purification and Tanshinoside B carries out following qualification:
(1) outward appearance: Tanshinoside A, Tanshinoside B is amorphous dark red powder.
(2) solvability: be soluble in methyl alcohol, is slightly soluble in acetone, is insoluble to the low polar organic solvents such as chloroform.
(3) UV spectrum: the UV spectrum of Tanshinoside A methanol solution has maximum absorption band at 196.0,273.0nm place.The UV spectrum of Tanshinoside B methanol solution has maximum absorption band at 194.0,273.0nm place.See Fig. 2 and Fig. 3 respectively.UV spectrum testing tool is Mariner System 5304instrument.
(4) optical value: Tanshinoside A,
(c 0.035, MeOH); Tanshinoside B,
(c 0.05, MeOH); Optical rotational activity spectrum testing tool is N Perkin-Elmer Model 343polarimeter, adopts sodium spectrum D line (589.3nm) to measure, measures length of tube 1dm.
(5) high resolution mass spectrum: Fig. 4 is the HRESIMS mass spectrum of Tanshinoside A, shows its [M+H]
+peak is m/z 441.1540, and provides most probable molecular formula to be C
24h
24o
8.Fig. 5 is the HRESIMS mass spectrum of Tanshinoside B, shows its [M+Na]
+peak is m/z 463.1353, and provides most probable molecular formula to be C
24h
24o
8.HRESIMS test adopts Bruker APEX III 7.0T spectrometer.Methyl alcohol is solvent.
(6) nuclear magnetic resonance spectrum: the NMR test of Tanshinoside A and Tanshinoside B adopt Bruker 600MHz instrument (
1h 500MHz;
13c 125MHz), solvent is DMSO-d
6(solvent peak corrects δ
h2.50/ δ
c39.5).Fig. 6 and Fig. 7 is Tanshinoside A and Tanshinoside B respectively
1h-NMR spectrogram.Fig. 8 and Fig. 9 is Tanshinoside A and Tanshinoside B respectively
13c-NMR spectrogram.According to compound
1h-NMR,
13c-NMR,
1h-
1h COSY (Figure 10 and Figure 11), HSQC (Figure 12 and 13), HMBC (Figure 14 and 15) and ROESY (Figure 16 and 17), is studied the nuclear magnetic resonance spectrum of two compounds and right
13c signal belongs to, in table 1.And finally determine that structure is as follows:
Table 1Tanshinoside A and Tanshinoside B
1h-NMR and
13c-NMR composes each peak ownership
The Determination of Antibacterial Activity of embodiment 2, compound Tanshinosides A and Tanshinoside B
One, minimal inhibitory concentration MIC activity test method
Methicillin-resistant staphylococcus aureus MRSA bacterial strain 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 3ml MHB (Qingdao Hai Bo Bioisystech Co., Ltd, HB6231) substratum, 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
4cell/mL, becomes stand-by bacterium liquid.
Get aseptic 96 porocyte culture plates, pipette 40 μ L MHB substratum to each hole;
Transferase 12 μ L vancomycin (Sigma, St.Louis, USA, V2002) (concentration is that 320 μ g/mL start to DMSO solution, carry out two times of gradient dilutions 7 times, concentration is followed successively by 160 μ g/mL, 80 μ g/mL, 40 μ g/mL, 20 μ g/mL, 10 μ g/mL, 5 μ g/mL and 2.5 μ g/mL) in 8 holes of 96 porocyte culture plate first rows, be positive controls.
Draw 2 μ L DMSO, adding in 8 holes of 96 porocyte culture plates the 12 row, is negative control group.
(concentration is that 4mg/mL starts to the DMSO solution of transferase 12 μ L testing compound Tanshinoside A and Tanshinoside B, carry out two times of gradient dilutions 8 times, concentration is followed successively by 2000 μ g/mL, 1000 μ g/mL, 500 μ g/mL, 250 μ g/mL, 125 μ g/mL, 62.5 μ g/mL and 31.25 μ g/mL) to 96 porocyte culture plate secondary series in the 3rd each hole of row, be experimental group to be measured.
(concentration is that 4mg/mL starts to the DMSO solution of transferase 12 μ L Tanshinone II A (Tan IIA), carry out two times of gradient dilutions 8 times, concentration is followed successively by 2000 μ g/mL, 1000 μ g/mL, 500 μ g/mL, 250 μ g/mL, 125 μ g/mL, 62.5 μ g/mL and 31.25 μ g/mL) arrange in each hole to 96 porocyte culture plates the 4th; Arrange in each hole with method transfer Tanshinone I (Tan I) and tsiklomitsin (Sigma, St.Louis, USA, T3383) to 96 porocyte culture plates the 5th row and the 6th; Above-mentioned three groups is contrast experiment's group.
Pipette the stand-by bacterium liquid of 38 μ L MRSA, 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, use microplate reader to read the light absorption value OD600 in each hole.
For each compound, 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 anti-Staphylococcus aureus SA (Staphylococcus aureus, ATCC 6538).Positive control is vancomycin, and MIC value is 1 μ g/mL.
Two, minimal inhibitory concentration active testing result
Compound Tanshinoside A, Tanshinoside B, Tanshinone II A (Tan IIA), Tanshinone I (Tan I), tsiklomitsin respectively to the MIC value test result of SA and MRSA in table 2.
The anti-microbial activity of table 2 compound
As shown in Table 2: the anti-microbial activity of Tanshinoside A and Tanshinoside B and tanshinone IIA are compared with Tanshinone I and be all significantly improved; And Tanshinoside A to the inhibit activities of streptococcus aureus (ATCC 6538) higher than the inhibit activities of vancomycin to streptococcus aureus (ATCC 6538), Tanshinoside A to the inhibit activities of methicillin-resistant staphylococcus aureus apparently higher than the inhibit activities of tsiklomitsin to methicillin-resistant staphylococcus aureus.
The antitumor cytolytic activity of embodiment 3, Tanshinoside B
One, IC
50the measuring method of value
1. cell culture condition
In cell cultivation process, all with the addition of microbiotic in substratum, usage quantity is: penicillin 100U/ml, Streptomycin sulphate 100U/ml; Cell is all incubated at 37 DEG C, 5%CO
2in the incubator of saturated humidity.
1.1 gastric carcinoma cells
SGC-7901 uses 1640 culture medium culturing containing 10% foetal calf serum.
1.2 human breast cancer cell
MCF-7 uses 1640 culture medium culturing containing 10% foetal calf serum.
1.3 the cultivation of human liver cancer cell
BEL-7402 uses the RPMI1640 culture medium culturing containing 10% foetal calf serum.
2. Tanshinone I, Tanshinoside B are to the inhibited proliferation of different people tumor cell line
Cell Counting Kit-8 (be called for short CCK-8) reagent can be used for easy and cell proliferation accurately and oxicity analysis.Its ultimate principle is: containing WST-8[chemical name: 2-(2-methoxyl group-4-nitrophenyl)-3-(4-nitrophenyl)-5-(2 in this reagent, 4-disulfonic acid benzene)-2H-tetrazolium monosodium salt], it is reduced to the yellow first a ceremonial jade-ladle, used in libation product (Formazan dye) with high water soluble under the effect of electron carrier 1-methoxyl group-5-toluphenazine methyl-sulfate (1-Methoxy PMS) by the desaturase in cell.The quantity of the first a ceremonial jade-ladle, used in libation thing generated is directly proportional to the quantity of viable cell.Therefore this characteristic can be utilized directly to carry out cell proliferation and oxicity analysis.
Get and be in one bottle, cell in good condition exponential phase of growth, add 0.05% tryptic digestive juice, digestion makes attached cell come off, counting 2 ~ 4 × 10
4individual/ml, makes cell suspension; Obtained cell suspension is inoculated on 96 orifice plates, and 180 μ l/ holes, put constant temperature CO
2cultivate 24 hours in incubator, cell inoculation requires as tested material 3-6 multiple hole, blank 3-6 multiple hole.Each hole adds the test medicine of different concns, 20 μ l/ holes, continues cultivation 72 hours; Add detection reagent (CCK-8) and hatch detection after 1-2 hour; With the light absorption value of enzyme-linked immunosorbent assay instrument in every hole, wavelength 450nm place, and by following formulae discovery cell inhibitory rate.After obtaining inhibiting rate, Origin software (8.0) is adopted to calculate IC
50.
Negative control group OD value=survey negative control group OD value-susceptibility zeroing group OD value
Administration group OD value=survey administration group group OD value-susceptibility zeroing group OD value
Two, anti-tumor activity test result
The IC of compound Tanshinoside B and Tanshinone I (Tan I)
50(nM) value test result is in table 3.
The anti-tumor activity of table 3 compound
As shown in Table 3: compared with Tanshinone I (Tan I), Tanshinoside B to the inhibit activities of SGC-7901 cells apparently higher than Tanshinone I (Tan I), Tanshinoside B is suitable with Tanshinone I (Tan I) to the inhibit activities of human breast cancer cell line Bcap-37, and Tanshinoside B is poor to human liver cancer cell BEL-7402 inhibit activities.
Claims (10)
1. a Diterpene glucoside compounds, its structural formula is such as formula shown in I:
2. a Diterpene glucoside compounds, its structural formula is such as formula shown in II:
3. prepare a method for Diterpene glucoside compounds described in claim 1 or 2, comprise the steps: to transform bacterial strain with Zygosaccharomyces rouxii CGMCC 3.3447, microbial transformation is carried out to Tanshinone I, obtains Diterpene glucoside compounds.
4. method according to claim 3, it is characterized in that: described microbial transformation is: cultivate 3 days-4 days under Zygosaccharomyces rouxii CGMCC 3.3447 seed liquor being inoculated in fermention medium room temperature, then add Tanshinone I room temperature bottom fermentation and cultivate 5 days-7 days, obtain fermented liquid.
5. the method according to any one of claim 3-4, is characterized in that: described Tanshinone I is Tanshinone I nanometer formulation;
Described Tanshinone I nanometer formulation is the eutectic pulvis be made up of the Macrogol 4000 of the Tanshinone I of massfraction 5.0-10% and massfraction 90-95% or polyethylene glycol 6000.
6. method according to claim 4, is characterized in that: described method also comprises the step being separated from described fermented liquid and obtaining Tanshinoside A and Tanshinoside B respectively;
Operate as follows: collect filtrate by after described filtering fermentation liquor, enriched material is obtained by concentrated after described filtrate organic solvent extraction, described enriched material is separated by silica gel column chromatography, medium pressure column chromatography, reversed-phased high performace liquid chromatographic successively, obtains Tanshinoside A and Tanshinoside B compound respectively.
7. method according to claim 6, it is characterized in that: described silica gel column chromatography is: be that the chloroform-acetone mixing solutions of 100:0-100:50 carries out wash-out for elutriant with volume ratio, collect the elution fraction under each volume ratio, all minimal inhibitory concentration active testing is carried out to each elution fraction, by the removal of solvents in elution fraction the highest for bacteriostatic activity, obtain concentrated solution;
Described medium pressure column chromatography is: the above-mentioned concentrated solution obtained through silica gel column chromatography is carried out medium pressure column chromatography separation, the acetonitrile solution being 10%-100% with the volumn concentration of acetonitrile carries out gradient elution for elutriant, collected volume percentage composition is the elution fraction of the acetonitrile solution of 40%-70%, remove the solvent in described component, concentrate and obtain concentrated solution, wherein, the Gradient program of gradient elution is that in acetonitrile solution, the volumn concentration of acetonitrile is from 10% to 100% from 0min to 90min;
Described reversed-phased high performace liquid chromatographic is: the above-mentioned concentrated solution obtained through medium pressure column chromatography is carried out reversed-phased high performace liquid chromatographic separation, wherein, the condition of described reversed-phased high performace liquid chromatographic is: Agilent ZORBAX – XDB reverse-phase chromatographic column, RP-C8,9.4*250mm, determined wavelength 254nm, moving phase to be volumn concentration be 42% acetonitrile solution, isocratic elution, be collected in elution fraction corresponding when absorption peak appears in 254nm wavelength place successively, obtain Tanshinoside A and Tanshinoside B compound.
8. the application of the compound described in claim 1 or 2 in following:
1) application in preparation antibacterials;
2) application in preparation eukaryote tumor cell proliferation inhibitor;
3) application prevented and/or treated in tumour medicine is being prepared.
9. application according to claim 8, is characterized in that:
Described antibacterials are the medicine of anti-Staphylococcus aureus and/or resistant Staphylococcus aureus;
Described streptococcus aureus is streptococcus aureus ATCC 6538;
Described resistant Staphylococcus aureus is methicillin-resistant staphylococcus aureus;
Described eukaryote is Mammals;
Described tumour cell is cancer cells;
Described cancer cells is stomach cancer cell and breast cancer cell;
Described tumour is cancer; Described cancer is cancer of the stomach or mammary cancer.
10. a product, its activeconstituents is the compound described in claim 1 or 2, and wherein, described product is: 1) antibacterials;
2) eukaryote tumor cell proliferation inhibitor;
3) tumour medicine is prevented and/or treated.
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| CN108102933A (en) * | 2018-02-28 | 2018-06-01 | 中国科学院微生物研究所 | One plant of white yellow black streptomycete bacterial strain and its application |
| US11547691B1 (en) | 2018-12-14 | 2023-01-10 | University Of South Florida | Dendrilla membranosa compounds, derivatives thereof, and uses thereof |
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| US4797495A (en) * | 1985-11-22 | 1989-01-10 | Burroughs Wellcome Co. | Benzo[c]carbazole propanediol compound and salts thereof |
| WO2012138297A1 (en) * | 2011-04-07 | 2012-10-11 | Nanyang Technological University | Acetyltanshinone iia (ata) as anticancer agent |
| CN103980341B (en) * | 2014-06-05 | 2016-01-20 | 上海朗萨医药科技有限公司 | One seed amino acid TANSHINONES phenol ester derivative and preparation method thereof |
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| XI CHENG，等: "Syntheses of diacyltanshinol derivatives and their suppressive effects on macrophage foam cell formation by reducing oxidized LDL uptake", 《BIOORGANIC CHEMISTRY 》 * |
| YASUAKI OKUMURA, 等: "The Structure of Tanshinone-II", 《BULLETIN OF THE CHEMICAL SOCIETY OF JAPAN》 * |
| 张朝晖: "丹参酮IIA衍生物的设计与合成研究", 《中国海洋大学硕士学位论文》 * |
Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
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| CN108102933A (en) * | 2018-02-28 | 2018-06-01 | 中国科学院微生物研究所 | One plant of white yellow black streptomycete bacterial strain and its application |
| US11547691B1 (en) | 2018-12-14 | 2023-01-10 | University Of South Florida | Dendrilla membranosa compounds, derivatives thereof, and uses thereof |
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