CN104829664B - Antibacterial antitumoral compounds and preparation method and application - Google Patents
Antibacterial antitumoral compounds and preparation method and application Download PDFInfo
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Abstract
The present invention provides antibacterial antitumoral compounds and preparation method and application.The compound is Diterpene glucoside class compound, and its structural formula is shown in Formulas I or formula II.The compound is prepared by following methods：It is conversion bacterial strain with Zygosaccharomyces rouxii (Mucor roxianus) CGMCC 3.3447, microorganism conversion is carried out to Tanshinone I, the Diterpene glucoside class compound is obtained.The Tanshinone I is preferably Tanshinone I nanometer formulation.Effect experiment shows：The compound can suppress the growth of staphylococcus aureus and resistant Staphylococcus aureus, while can suppress the propagation of gastric carcinoma cells and human breast cancer cell.Therefore available for antibacterial and the research of antitumor activity lead compound or preparation antibacterial and antineoplastic.
Description
Technical field
The invention belongs to biomedicine field, and in particular to antibacterial antitumoral compounds and preparation method and application.
Background technology
Staphylococcus aureus is a kind of gram-positive bacteria, and spread scope is extremely wide, has spread all over full generation so far from discovery
Boundary, as burn, the main infection pathogen of postoperative and children, is the most common pathogenic bacteria of hospital, its infection rate is also higher, entirely
100,000 reports for infecting the bacterium are there are about in world wide.
In recent years, due to reasons such as the abuses of antibiotic, resistance phenomenon increases year by year, and this is to cause infectious diseases dead
The high one of the main reasons of rate, wherein most commonly seen with MRSA methicillin-resistant staphylococcus aureus.MRSA has
More extensively, more serious multi-drug resistant phenomenon, it means that, it is other all with the mutually isostructural beta-lactam in methicillin and head
Spore class antibiotic occurs in that resistance phenomenon, and vancomycin is used as the multi-drug resistant MRSA of clinical treatment unique selection, its resistance
Pressure is increasing, and the vancomycin clinically commonly used now has also engendered resistance phenomenon, and the research of antibiotic new drug exists
The bottleneck of maximum is met with past 30-40, this most causes the available stage of no medicine at last.Therefore, active development has height
Effect, the overriding resistance staphylococcus aureus of low toxicity medicine it is significant.
Cancer is a kind of very ancient disease, along with the history of whole human development, seriously threatens the strong of the mankind
Health and life.A nearest investigation display of the World Health Organization, cancer will surmount head of the heart disease as global human health
The number dead because of cancer is in the situation of cumulative year after year in number killer, worldwide.The whole world in 2012 increases 1400 newly altogether
Ten thousand cases of cancers simultaneously have 8,200,000 people dead.Wherein, China increases 3,070,000 cancer patients newly and causes about 2,200,000 people dead, respectively
Account for the 21.9% and 26.8% of global total amount.In 4 kinds of malignant tumours such as liver, esophagus, stomach and lung, Chinese new cases and death
Number occupies first place in the world.Therefore very stern challenge is faced with for the treatment of cancer.It was found that and studying with height
Effect, low toxicity antineoplastic it is most important.
The content of the invention
An object of the present invention is to provide a kind of new Diterpene glucoside class compound.
The Diterpene glucoside class compound is by the master in Salvia salviamiltiorrhizabung Salvia miltiorrhiza Bunge
Want composition tanshinone compound to be obtained by microorganism conversion, be brand new type compound；The Diterpene glucoside class
Compound can be respectively designated as compound Tanshinoside A and compound by the difference on glycosylation position
Tanshinoside B。
The structural formula of the compound Tanshinoside A is as shown in following Formulas I：
The structural formula of the compound Tanshinoside B is as shown in following formula II：
Diterpene glucoside class compound provided by the present invention can be prepared by the method comprised the steps：With Lu Shi
Mucor (Mucor roxianus) CGMCC 3.3447 is conversion bacterial strain, and microorganism conversion is carried out to Tanshinone I, obtains diterpene sugar
Glycosides compound.
The microorganism conversion is：The seed liquors of Zygosaccharomyces rouxii (Mucor roxianus) CGMCC 3.3447 are inoculated in hair
Ferment culture medium is cultivated -4 days 3 days at room temperature, is then added Tanshinone I fermented and cultured -7 days 5 days (such as 5 days) at room temperature, is sent out
Zymotic fluid.
The seed liquors of Zygosaccharomyces rouxii (Mucor roxianus) CGMCC 3.3447 are by Zygosaccharomyces rouxii (Mucor
Roxianus) CGMCC 3.3447 is inoculated in slant medium, and incubated at room temperature obtains slant strains；Again by the slant strains
It is inoculated into the seed liquors of Zygosaccharomyces rouxii (Mucor roxianus) CGMCC 3.3447 obtained after seed culture medium, incubated at room temperature.
In the above method, the Tanshinone I is preferably Tanshinone I nanometer formulation.
The Tanshinone I nanometer formulation is by mass fraction 5.0-10% Tanshinone I and gathering for mass fraction 90-95%
The congruent melting medicinal powder of ethylene glycol 4000 or Macrogol 6000 composition.
Method system of the Tanshinone I nanometer formulation with reference to described in Patent No. CN1391889 Chinese patent
It is standby.Specific preparation method is as follows：It will mix, formed with Tanshinone I after Macrogol 4000 or Macrogol 6000 heating and melting
Tanshinone I-Macrogol 4000 or Macrogol 6000 eutectic, after the eutectic is cooled down, with 450 revs/min of rotating speed
Grinding 3 hours, crosses 300 mesh sieves, produces the Tanshinone I nanometer formulation.
The above method also includes isolated Tanshinoside A and Tanshinoside B from the zymotic fluid
Step.Concrete operations are as follows：Filtrate will be collected after the filtering fermentation liquor, by the filtrate with organic solvent (such as ethyl acetate)
Concentrate is concentrated to give after extraction, the concentrate is passed sequentially through into silica gel column chromatography, medium pressure column chromatography, RP-HPLC color
Spectrometry is separated, and obtains Tanshinoside A and Tanshinoside B compounds.
Wherein, the silica gel column chromatography is：Using volume ratio as 100:0-100:50 chloroform-acetone mixed solution is elution
Liquid is eluted, and collects the elution fraction under each volume ratio, minimal inhibitory concentration (MIC) is carried out to each elution fraction living
Property test, by bacteriostatic activity highest elution fraction solvent remove, obtain concentrate.
Above-mentioned minimal inhibitory concentration (MIC) active testing is to be based on staphylococcus aureus and methicillin-resistant staphylococcus Portugal
What grape coccus was carried out.
Specifically, the silica gel column chromatography is：Volume ratio is used successively for 100:0、100:2-5、100:8-12、100:
15-25 and 100:30-50 chloroform-acetone mixed solution is that eluent is eluted, and collected volume ratio is 100:8-12 chlorine
The elution fraction of imitative-acetone mixed solution, the solvent in the elution fraction is removed, concentrate is obtained.
More specifically, the silica gel column chromatography is：Volume ratio is used successively for 100:0、100:2、100:3、100:10、
100:20 and 100:50 chloroform-acetone mixed solution is eluent, and it is 100 to collect chloroform-acetone volume ratio:10 component,
The solvent in the component is removed, concentrate is obtained.
Medium pressure column chromatography is：The above-mentioned concentrate obtained through silica gel column chromatography is subjected to medium pressure column chromatography separation, with
The acetonitrile solution that the volumn concentration of acetonitrile is 10%-100% is that eluent carries out gradient elution, and collected volume percentage contains
The elution fraction of the acetonitrile solution for 40%-70% is measured, the solvent in the component is removed, is concentrated to give concentrate, wherein,
The Gradient program of gradient elution be from 0min to 90min, in acetonitrile solution the volumn concentration of acetonitrile from 10% to
100%.
The reversed-phased high performace liquid chromatographic is：The above-mentioned concentrate obtained through medium pressure column chromatography is subjected to reversed phase high performance liquid
Phase chromatography is separated, wherein, the condition of the reversed-phased high performace liquid chromatographic is：Agilent ZORBAX-XDB reverse-phase chromatographies
Post, RP-C8,9.4*250mm, Detection wavelength 254nm, mobile phase is the acetonitrile solution that volumn concentration is 42%, isocratic
Elution, corresponding elution fraction when there is absworption peak at 254nm wavelength is collected successively, obtain Tanshinoside A and
Tanshinoside B compounds.
It is a further object to provide the purposes of above-mentioned Diterpene glucoside class compound.
The purposes of Diterpene glucoside class compound provided by the present invention is its application at following aspects：
1) application in antibacterials are prepared；
2) application in eucaryote tumor cell proliferation inhibitor is prepared；
3) application in prevention and/or tumor is prepared
The antibacterials are mainly the medicine of anti-Staphylococcus aureus and/or resistant Staphylococcus aureus.
The staphylococcus aureus concretely staphylococcus aureus (Staphylococcus aureus) ATCC
6538；
The resistant Staphylococcus aureus concretely methicillin-resistant staphylococcus aureus (Methicillin-
resistant Staphylococcus aureus)。
The eucaryote is mammal；The tumour cell is cancer cell；The cancer cell can for stomach cancer cell and
Breast cancer cell.
The stomach cancer cell concretely SGC-7901 cells；The breast cancer cell concretely human breast carcinoma
Cell MCF-7.
The tumour is cancer；The cancer is stomach cancer or breast cancer.
The antibacterials that are prepared using above-mentioned Diterpene glucoside class compound as active component, the suppression of eucaryote tumor cell proliferation
The medicine of preparation, prevention and/or treatment tumour falls within protection scope of the present invention.
In the medicine of the antibacterials, eucaryote tumor cell proliferation inhibitor or prevention and/or treatment tumour, institute
The mass content for stating Diterpene glucoside class compound is 0.1-90%.
The medicine of the antibacterials, eucaryote tumor cell proliferation inhibitor or prevention and/or treatment tumour can lead to
Cross injection, injection, collunarium, eye drip, infiltration, absorption, the method that physically or chemically mediates import body for example muscle, it is intracutaneous, subcutaneous,
Vein, mucosal tissue；Or imported body after other material mixings or parcel.
When needs, one or more pharmaceutically acceptable carriers can also be added in said medicine.It is described to carry
Body includes the conventional diluent of pharmaceutical field, excipient, filler, adhesive, wetting agent, disintegrant, sorbefacient, surface
Activating agent, absorption carrier, lubricant etc..
The antibacterials that are prepared using the Diterpene glucoside class compound as active component, the suppression of eucaryote tumor cell proliferation
Parenteral solution, tablet, pulvis, granule, capsule, oral liquid, cream can be made in the medicine of preparation or prevention and/or treatment tumour
The diversified forms such as agent, creme.The medicine of above-mentioned various formulations can be prepared according to the conventional method of pharmaceutical field.
The compound structure that the present invention is provided is brand-new, with good antibacterial and antitumor activity, is suitable for antibacterial and anti-
The research of tumor promotion lead compound prepares antibacterial and antineoplastic.
The present invention uses Zygosaccharomyces rouxii (Mucor roxianus) AS 3.3447 for conversion bacterial strain, respectively with Tanshinone I,
Tanshinone I nanometer formulation and tanshinone IIA are substrate, and Diterpene glucoside class compound is prepared by microbial conversion process, respectively
HPLC spectrum analysis are carried out to system after these three reactions, when finding using Tanshinone I nanometer formulation as substrate, obtained product amount
Highest, then by easy extraction process, isolated compound Tanshinoside A and Tanshinoside B, warp
Nuclear magnetic resonance, ultraviolet spectra, Mass Spectrometer Method, its structure are correct.
Brief description of the drawings
Fig. 1 is what Zygosaccharomyces rouxii of the present invention (Mucor roxianus) CGMCC 3.3447 was converted to Tanshinone I
HPLC spectrograms (A)；The HPLC spectrums that Zygosaccharomyces rouxii (Mucor roxianus) CGMCC 3.3447 is converted to Tanshinone I nanometer formulation
Scheme (B)；The HPLC spectrograms (C) that Zygosaccharomyces rouxii (Mucor roxianus) CGMCC 3.3447 is converted to tanshinone IIA；
Tanshinoside A standard HPLC spectrograms (D)；Tanshinoside B standard HPLC spectrograms (E).
Fig. 2 is compound Tanshinoside A of the present invention uv-spectrogram.
Fig. 3 is compound Tanshinoside B of the present invention uv-spectrogram.
Fig. 4 is compound Tanshinoside A of the present invention high resolution mass spectrum figure.
Fig. 5 is compound Tanshinoside B of the present invention high resolution mass spectrum figure.
Fig. 6 is that compound Tanshinoside A of the present invention are dissolved in DMSO-d6In1H-NMR spectrum.
Fig. 7 is that compound Tanshinoside B of the present invention are dissolved in DMSO-d6In1H-NMR spectrum.
Fig. 8 is that compound Tanshinoside A of the present invention are dissolved in DMSO-d6In13C-NMR spectrograms.
Fig. 9 is that compound Tanshinoside B of the present invention are dissolved in DMSO-d6In13C-NMR spectrograms.
Figure 10 is that compound Tanshinoside A of the present invention are dissolved in DMSO-d6In1H-1HCOSY is composed.
Figure 11 is that compound Tanshinoside B of the present invention are dissolved in DMSO-d6In1H-1HCOSY is composed.
Figure 12 is that compound Tanshinoside A of the present invention are dissolved in DMSO-d6In hsqc spectrum figure.
Figure 13 is that compound Tanshinoside B of the present invention are dissolved in DMSO-d6In hsqc spectrum figure.
Figure 14 is that compound Tanshinoside A of the present invention are dissolved in DMSO-d6In HMBC spectrograms.
Figure 15 is that compound Tanshinoside B of the present invention are dissolved in DMSO-d6In HMBC spectrograms.
Figure 16 is that compound Tanshinoside A of the present invention are dissolved in DMSO-d6In ROESY spectrograms.
Figure 17 is that compound Tanshinoside B of the present invention are dissolved in DMSO-d6In ROESY spectrograms.
Embodiment
Below by specific embodiment, the present invention will be described, but the invention is not limited in this.
Experimental method used in following embodiments is conventional method unless otherwise specified；Institute in following embodiments
Reagent, biomaterial etc., unless otherwise specified, are commercially obtained.
The potato that following embodiments are used 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 IIA are purchased from Xi'an Hao Xuan bio tech ltd.
It is prepared by method of the Tanshinone I nanometer formulation with reference to described in Patent No. CN1391889 Chinese patent.Tool
Preparation is as follows：It will be mixed after 95g Macrogol 4000 heating and meltings with 5g Tanshinone Is, form Tanshinone I-poly- second two
The eutectic of alcohol 4000, after the eutectic is cooled down, is ground 3 hours with 450 revs/min of rotating speed, crosses 300 mesh sieves, obtain the red sage root
Ketone I nanometer formulations 70.5g.The mass content of Tanshinone I is 6.65% in the Tanshinone I nanometer formulation.
Staphylococcus aureus (Staphylococcus aureus) ATCC 6538：It is recorded in " the golden yellow Portugals of the intelligent of height
The clone of grape coccus (ATCC6538) protein A gene and application study Northeast Agricultural Universities, Master's thesis, 2010 ".
Methicillin-resistant staphylococcus aureus (Methicillin-resistant Staphylococcus
aureus)：It is recorded in " IB Gosbell.Methicillin-resistant Staphylococcus
aureus.Americal Journal of Clinical Dermatology.2004,5(4):239-259”。
It is thin that SGC-7901 cells, human liver cancer cell BEL-7402 and human breast cancer cell line Bcap-37 are purchased from Shanghai
Born of the same parents storehouse.
The preparation and identification of embodiment 1, antibacterial and antitumoral compounds.
First, screening prepares the substrate of antibacterial and antitumoral compounds
1st, seed culture
(1) slant medium is sterilized 20 minutes at 121 DEG C, bevel is incubated 3 days in 28 DEG C.To surface
Moisture is slightly dry, during no varied bacteria growing, by the strain spore inoculatings of Zygosaccharomyces rouxii (Mucor roxianus) CGMCC 3.3447 in oblique
Face culture medium, is cultivated 3 days, outward appearance is white hypha, and upper attachment black spore, mycelia is plentiful, can be received during no microbiological contamination in 28 DEG C
Take and use, that is, obtain slant strains.
Above-mentioned slant medium is consisted of the following composition：
Potato, glucose, agar and water；
Above composition concentration in the slant medium is respectively (g/L)：
Potato 200g/L, glucose 20g/L, agar 20g/L, the pH value of the slant medium is nature pH.
(2) 50ml seed culture mediums are respectively charged into multiple 250ml vials, seal up membrana oralis, in sterilizing 20 at 121 DEG C
Minute, block inoculation is dug by inclined-plane, it is slant strains prepared by above-mentioned steps (1) to connect strain.Rotated in 28 DEG C in rotary shaker
Cultivate (rotating speed is 220rpm) 48 hours, obtain seed liquor.
The seed culture medium is consisted of the following composition：
Potato, glucose and water；
Above composition concentration in the seed culture medium is respectively：
Potato 200g/L, glucose 20g/L, the pH value of the seed culture medium is nature pH.
2nd, substrate is screened
Prepare screening 9 bottles of (Media Components of fermentation medium：Potato 200g/L, glucose 20g/L, the fermentation
The pH value of culture medium is nature pH, and solvent is water).In 250ml triangular flask dispense 50ml fermentation mediums, after sterilizing according to
The seed liquor that above-mentioned steps 1 are obtained is inoculated in fermentation medium by the inoculum concentration of 2% (percent by volume), in 28 DEG C of rotating and culturings
After (rotating speed is 220rpm) 3 days, substrate Tanshinone I (3 bottles), Tanshinone I nanometer formulation (3 bottles), tanshinone IIA (3 are separately added into
Bottle), wherein Tanshinone I and every bottle of tanshinone IIA addition 1.5mg, every bottle of Tanshinone I nanometer formulation add 22mg (the wherein reds sage root
Ketone I is 1.5mg), continue culture and obtain zymotic fluid in 5 days.Zymotic fluid is all substances in container.
By obtained filtering fermentation liquor, filtrate is collected.Filtrate is extracted 1 time with isometric ethyl acetate (50mL), with rotation
Turn evaporimeter concentration to be evaporated, weigh, be dissolved in 1mL methanol, carry out analysed by reverse phase HPLC.The analysis condition of reversed-phase HPLC is：
Agilent ZORBAX-XDB reverse-phase chromatographic columns, RP-C8,4.5*150mm, Detection wavelength 254nm, mobile phase contains for volume basis
Measure as 5%-95% acetonitrile solutions, (Gradient program of gradient elution is the acetonitrile water from 0min to 35min to gradient elution 35min
The volumn concentration of acetonitrile is from 5% to 95% in solution).
Figure 1A is the HPLC spectrograms that Zygosaccharomyces rouxii (Mucor roxianus) CGMCC 3.3447 is converted to Tanshinone I；Figure
1B is the HPLC spectrograms that Zygosaccharomyces rouxii (Mucor roxianus) CGMCC 3.3447 is converted to Tanshinone I nanometer formulation；Fig. 1 C
The HPLC spectrograms converted for Zygosaccharomyces rouxii (Mucor roxianus) CGMCC 3.3447 to tanshinone IIA；Fig. 1 D are
Tanshinoside A standard HPLC spectrograms；Fig. 1 E are Tanshinoside B standard HPLC spectrograms.
As shown in Figure 1, using Tanshinone I nanometer formulation as the product amount highest obtained by substrate, therefore selection Tanshinone I nanometer
Preparation is that substrate prepares the Diterpene glucoside class compound (Tanshinosides A and Tanshinoside B).
2nd, fermentation prepares antibacterial and antitumoral compounds
1st, seed culture
Same (1)
2nd, fermented and cultured
Then fermentation medium (Media Components are prepared：Potato 200g/L, glucose 20g/L, the fermentation medium
PH value be nature pH, solvent is water).300ml fermentation mediums are dispensed in 1000ml triangular flask, according to 2% after sterilizing
The seed liquor that above-mentioned steps 1 are obtained is inoculated in fermentation medium by the inoculum concentration of (percent by volume), (is turned in 28 DEG C of rotating and culturings
Speed is 220rpm) after 3 days, every bottle plus substrate Tanshinone I nanometer formulation 120mg is continued to cultivate 5 days, obtains zymotic fluid.Zymotic fluid
For all substances in container.
3rd, antibacterial and antitumoral compounds are separated and are identified
1st, antibacterial and antitumoral compounds are isolated and purified
The filtering fermentation liquor that above-mentioned experiment is obtained, collects filtrate.Filtrate is extracted 3 with isometric ethyl acetate (10L)
It is secondary, it is evaporated, is weighed with Rotary Evaporators concentration.Then extract is separated with silica gel chromatographic column (200-300 mesh), is adopted successively
It is 100 with volume ratio:0、100:2、100:3、100:10、100:20 and 100:50 chloroform-acetone mixed solution is eluent,
Every kind of 5 column volumes of elution, then by different volumes than the material that gets off of elution concentrate respectively after, respectively
The testing sample solution for being configured to 4mg/ml is dissolved with DMSO, for minimal inhibitory concentration active testing.
Active testing result shows that it is 100 that active part, which concentrates on volume ratio,:10 chloroforms-acetone elution fraction, 100:10
Partial minimal inhibitory concentration is that MIC value is respectively the μ g/mL of anti-Staphylococcus aureus 1.56, anti-methicillin-resistant staphylococcus Portugal
The μ g/mL of grape coccus 1.56.
By chloroform-acetone 100:The component of 10 parts is concentrated by Rotary Evaporators, and concentrate is carried out in ODS (5 μm)
Pressure column chromatography (is purchased from Beijing Hui De Easytech Inc.), and with acetonitrile-water system 10%-100%, (volume basis of acetonitrile contains
Amount) gradient elution (Gradient program be from 0min to 90min, in acetonitrile solution the volumn concentration of acetonitrile from 10% to
100%) 40%-70% acetonitrile eluent (chromatographic peak concentrate occur in this region), is collected, inverted efficient liquid again after concentration
Phase chromatography is separated, wherein, the condition of reversed-phase HPLC is：Agilent ZORBAX-XDB reverse-phase chromatographic columns, RP-C8,9.4*
250mm, Detection wavelength 254nm, mobile phase are the acetonitrile solution that volumn concentration is 42%, and isocratic elution is collected successively
Corresponding elution fraction when there is absworption peak at 254nm wavelength, obtains Tanshinoside A and Tanshinoside Bization
Compound.
2nd, authenticating compound Tanshinoside A and Tanshinoside B
Above-mentioned steps 1 are isolated and purified to obtained compound Tanshinoside A and Tanshinoside B progress following
Identification：
(1) outward appearance：Tanshinoside A, Tanshinoside B are amorphous dark red powder.
(2) dissolubility：Methanol is soluble in, acetone is slightly soluble in, insoluble in low polar organic solvents such as chloroforms.
(3) ultraviolet spectra：The ultraviolet spectra of Tanshinoside A methanol solutions has maximum suction at 196.0,273.0nm
Receive peak.The ultraviolet spectra of Tanshinoside B methanol solutions has maximum absorption band at 194.0,273.0nm.Fig. 2 is seen respectively
And Fig. 3.Ultraviolet spectra tester is Mariner System 5304instrument.
(4) optical value：Tanshinoside A,Tanshinoside B,Optical rotational activity spectrum tester is N Perkin-Elmer Model 343
Polarimeter, is determined using sodium spectrum D lines (589.3nm), determines length of tube 1dm.
(5) high resolution mass spectrum：Fig. 4 is Tanshinoside A HRESIMS mass spectrograms, is shown its [M+H]+Peak is m/z
441.1540, and most probable molecular formula is provided for C24H24O8.Fig. 5 is Tanshinoside B HRESIMS mass spectrograms, shows it
[M+Na]+Peak is m/z 463.1353, and provides most probable molecular formula for C24H24O8.HRESIMS tests are using Bruker APEX
Ⅲ 7.0 T spectrometer.Methanol is solvent.
(6) nuclear magnetic resoance spectrum：Tanshinoside A and Tanshinoside B NMR tests use Bruker
600MHz instruments (1H 500MHz；13C 125MHz), solvent is DMSO-d6(solvent peak corrects δH 2.50/δC39.5).Fig. 6 and
Fig. 7 is Tanshinoside A and Tanshinoside B respectively1H-NMR spectrum.Fig. 8 is to be respectively with Fig. 9
Tanshinoside A and Tanshinoside B's13C-NMR spectrograms.According to compound1H-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), to two compounds
Nuclear magnetic resoance spectrum has carried out research and right13C signal is belonged to, and is shown in Table 1.And finally determine that structure is as follows：
Table 1 Tanshinoside A and Tanshinoside B1H-NMR and13C-NMR composes each peak ownership
The Determination of Antibacterial Activity of embodiment 2, compound Tanshinosides A and Tanshinoside B
First, minimal inhibitory concentration MIC activity test methods
By methicillin-resistant staphylococcus aureus MRSA bacterial strains by being inoculated in cryopreservation tube on LB Agar Platings
Activated, cultivated 20 hours in 37 DEG C；3ml MHB (the rich biologies in Qingdao sea are fallen within 3 MRSA single bacteriums of aseptic inoculation ring picking
Technology Co., Ltd., HB6231) in culture medium, fully mixed as bacterium solution mother liquor using turbula shaker, use blood count
Plate detection bacterium is dense；Bacterium solution mother liquor is diluted to 2 × 10 with MHB culture mediums4Cell/mL, as stand-by bacterium solution.
Sterile 96 porocyte culture plates are taken, 40 μ L MHB culture mediums are pipetted to each hole；
(concentration is that 320 μ g/mL are opened to the DMSO solution of transferase 12 μ L vancomycins (Sigma, St.Louis, USA, V2002)
Begin, two times of progress gradient dilution 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) into 8 holes of 96 porocyte culture plates first rows, it is positive controls.
2 μ L DMSO are drawn, are negative control group in 8 holes for adding 96 porocyte culture plates the 12nd row.
(concentration is 4mg/ to transferase 12 μ L testing compound Tanshinoside A and Tanshinoside B DMSO solution
ML starts, and carries out two times of gradient dilutions 8 times, concentration be 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 plates secondary series into the 3rd each hole of row, be reality to be measured
Test group.
(concentration starts the DMSO solution of transferase 12 μ L tanshinone IIAs (Tan IIA) for 4mg/mL, carries 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) arranged to 96 porocyte culture plates the 4th in each hole；With method transfer Tanshinone I (Tan I) and tetracycline (Sigma,
St.Louis, USA, T3383) into 96 porocyte culture plates the 5th row and the 6th each hole of row；Above-mentioned three groups are contrast experiment's group.
The 38 stand-by bacterium solutions of μ L MRSA are pipetted, are added in each hole of 96 porocyte culture plates；Above-mentioned 96 porocyte culture plates are put
In 37 DEG C of incubators, after cultivating 16 hours, the light absorption value OD600 in each hole is read using ELIASA.
For each compound, it is seen that the final compound concentration corresponding to hole that MRSA growths are totally constrained is the change
Minimum inhibitory concentration MIC value of the compound to MRSA.Same method determines anti-Staphylococcus aureus SA (Staphylococcus
aureus,ATCC 6538).Positive control is vancomycin, and MIC value is 1 μ g/mL.
2nd, minimal inhibitory concentration active testing result
Compound Tanshinoside A, Tanshinoside B, tanshinone IIA (Tan IIA), Tanshinone I (Tan
I), MIC value test result of the tetracycline respectively to SA and MRSA is shown in Table 2.
The antibacterial activity of the compound of table 2
As shown in Table 2：Tanshinoside A and Tanshinoside B antibacterial activity and tanshinone IIA and tanshinone
I is compared and is significantly improved；And Tanshinoside A are higher than ten thousand to the inhibitory activity of staphylococcus aureus (ATCC 6538)
Ancient mycin is to staphylococcus aureus (ATCC 6538) inhibitory activity, and Tanshinoside A are to methicillin-resistant staphylococcus
Staphylococcic inhibitory activity is apparently higher than inhibitory activity of the tetracycline to methicillin-resistant staphylococcus aureus.
The antitumor cytolytic activity of embodiment 3, Tanshinoside B
First, IC50The assay method of value
1. cell culture condition
In cell cultivation process, antibiotic is with the addition of in culture medium, usage amount is：Penicillin 100U/ml, streptomysin
100U/ml；Cell is incubated at 37 DEG C, 5%CO2In the incubator of saturated humidity.
1.1 gastric carcinoma cells
SGC-7901 uses 1640 medium cultures containing 10% hyclone.
1.2 human breast cancer cell
MCF-7 uses 1640 medium cultures containing 10% hyclone.
The culture of 1.3 human liver cancer cells
BEL-7402 uses the RPMI1640 medium cultures containing 10% hyclone.
2. Tanshinone I, Tanshinoside B are to the inhibited proliferation of different human tumor cell lines
Cell Counting Kit-8 (abbreviation CCK-8) reagent can be used for easy and accurately cell is bred and toxicity point
Analysis.Its general principle is：Contain WST-8 in the reagent【Chemical name：2- (2- methoxyl group -4- nitrobenzophenones) -3- (4- nitrobenzene
Base) -5- (2,4- disulfonic acid benzene) -2H- tetrazolium monosodium salts】, it is in electron carrier 1- methoxyl group -5- toluphenazine dimethyl sulfates
The yellow first a ceremonial jade-ladle, used in libation product with high water soluble is reduced in the presence of ester (1-Methoxy PMS) by the dehydrogenase in cell
(Formazan dye).The quantity of the first a ceremonial jade-ladle, used in libation thing of generation is directly proportional to the quantity of living cells.Therefore can be direct using this characteristic
Carry out cell propagation and oxicity analysis.
Take in exponential phase of growth one bottle of cell in good condition, add 0.05% tryptic digestive juice, digestion makes patch
Parietal cell comes off, and counts 2~4 × 104Individual/ml, is made cell suspension；Cell suspension inoculation is taken on 96 orifice plates, 180 μ l/ holes,
Put constant temperature CO2Cultivated 24 hours in incubator, cell inoculation requires to be 3-6 multiple holes of tested material, 3-6 multiple holes of blank control.
Each hole adds the test medicine of various concentrations, and 20 μ l/ holes continue to cultivate 72 hours；Add detection reagent (CCK-8) and be incubated 1-2
Detected after hour；With enzyme-linked immunosorbent assay instrument at wavelength 450nm per hole light absorption value, and by following equation calculate Carbazole alkaloid
Rate.Obtain after inhibiting rate, IC is calculated using Origin softwares (8.0)50。
Negative control group OD values=survey negative control group OD values-susceptibility zeroing group OD values
Administration group OD values=survey administration group group OD values-susceptibility zeroing group OD values
2nd, antitumor activity test result
Compound Tanshinoside B and Tanshinone I (Tan I) IC50(nM) value test result is shown in Table 3.
The antitumor activity of the compound of table 3
As shown in Table 3：Compared with Tanshinone I (TanI), suppressions of the Tanshinoside B to SGC-7901 cells
System is active apparently higher than Tanshinone I (TanI), inhibitory activity and the red sage root of the Tanshinoside B to human breast cancer cell line Bcap-37
Quite, and Tanshinoside B are poor to human liver cancer cell BEL-7402 inhibitory activity by ketone I (TanI).
Claims (16)
1. a kind of Diterpene glucoside class compound, its structural formula is shown in formula I：
2. a kind of Diterpene glucoside class compound, its structural formula is as shown in formula II：
3. a kind of method for preparing Diterpene glucoside class compound described in claim 1 or 2, comprises the steps：With Zygosaccharomyces rouxii
CGMCC 3.3447 is conversion bacterial strain, carries out microorganism conversion to Tanshinone I, obtains Diterpene glucoside class compound.
4. method according to claim 3, it is characterised in that：The microorganism conversion is：By Zygosaccharomyces rouxii CGMCC
3.3447 seed liquors are inoculated in fermentation medium and cultivated at room temperature -4 days 3 days, then add Tanshinone I fermented and cultured 5 at room temperature
My god -7 days, obtain zymotic fluid.
5. the method according to any one of claim 3-4, it is characterised in that：The Tanshinone I is Tanshinone I nanometer system
Agent；
The Tanshinone I nanometer formulation is the Tanshinone I and mass fraction 90-95% poly- second two by mass fraction 5.0-10%
The congruent melting medicinal powder of alcohol 4000 or Macrogol 6000 composition.
6. method according to claim 4, it is characterised in that：Methods described also includes separating difference from the zymotic fluid
The step of obtaining Tanshinoside A and Tanshinoside B；
Operation is as follows：Filtrate will be collected after the filtering fermentation liquor, be concentrated to give after the filtrate is extracted with organic solvent dense
Contracting thing, passes sequentially through silica gel column chromatography, medium pressure column chromatography, reversed-phased high performace liquid chromatographic by the concentrate and is separated, point
Tanshinoside A and Tanshinoside B compounds are not obtained.
7. method according to claim 6, it is characterised in that：The silica gel column chromatography is：Using volume ratio as 100:0-
100:50 chloroform-acetone mixed solution is that eluent is eluted, and collects the elution fraction under each volume ratio, is washed to each
De- component carries out minimal inhibitory concentration active testing, and the solvent in bacteriostatic activity highest elution fraction is removed, obtains dense
Contracting liquid；
Medium pressure column chromatography is：The above-mentioned concentrate obtained through silica gel column chromatography is subjected to medium pressure column chromatography separation, with acetonitrile
Volumn concentration be 10%-100% acetonitrile solution be that eluent carries out gradient elution, collected volume percentage composition is
The elution fraction of 40%-70% acetonitrile solution, removes the solvent in the component, is concentrated to give concentrate, wherein, gradient
The Gradient program of elution is that from 0min to 90min, the volumn concentration of acetonitrile is from 10% to 100% in acetonitrile solution；
The reversed-phased high performace liquid chromatographic is：The above-mentioned concentrate obtained through medium pressure column chromatography is subjected to RP-HPLC color
Spectrometry is separated, wherein, the condition of the reversed-phased high performace liquid chromatographic is：Agilent ZORBAX-XDB reverse-phase chromatographic columns, RP-
C8,9.4*250mm, Detection wavelength 254nm, mobile phase is the acetonitrile solution that volumn concentration is 42%, isocratic elution, according to
Secondary collection corresponding elution fraction when there is absworption peak at 254nm wavelength, obtain Tanshinoside A and
Tanshinoside B compounds.
8. compound described in claim 1 or 2 is in the application of following aspects：
1) application in antibacterials are prepared；
2) application in eucaryote tumor cell proliferation inhibitor is prepared；
3) application in prevention and/or tumor is prepared.
9. application according to claim 8, it is characterised in that：The antibacterials be anti-Staphylococcus aureus and/or
The medicine of resistant Staphylococcus aureus.
10. application according to claim 9, it is characterised in that：The staphylococcus aureus is staphylococcus aureus
ATCC 6538；
The resistant Staphylococcus aureus is methicillin-resistant staphylococcus aureus.
11. application according to claim 8, it is characterised in that：The eucaryote is mammal.
12. application according to claim 8, it is characterised in that：The tumour cell is cancer cell.
13. application according to claim 12, it is characterised in that：The cancer cell is stomach cancer cell and breast cancer cell.
14. application according to claim 8, it is characterised in that：The tumour is cancer.
15. application according to claim 14, it is characterised in that：The cancer is stomach cancer or breast cancer.
16. a kind of product, its active component is the compound described in claim 1 or 2, wherein, the product is：1) antimicrobial
Thing；
2) eucaryote tumor cell proliferation inhibitor；
3) prevention and/or tumor.
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Citations (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US4797495A (en) * | 1985-11-22 | 1989-01-10 | Burroughs Wellcome Co. | Benzo[c]carbazole propanediol compound and salts thereof |
| CN103732586A (en) * | 2011-04-07 | 2014-04-16 | 南洋理工大学 | Acetyltanshinone IIA (ATA) as anticancer agent |
| CN103980341A (en) * | 2014-06-05 | 2014-08-13 | 上海朗萨医药科技有限公司 | Amino acid tanshinone phenolic ester derivatives and preparation method thereof |
-
2015
- 2015-03-16 CN CN201510114000.9A patent/CN104829664B/en active Active
Patent Citations (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US4797495A (en) * | 1985-11-22 | 1989-01-10 | Burroughs Wellcome Co. | Benzo[c]carbazole propanediol compound and salts thereof |
| CN103732586A (en) * | 2011-04-07 | 2014-04-16 | 南洋理工大学 | Acetyltanshinone IIA (ATA) as anticancer agent |
| CN103980341A (en) * | 2014-06-05 | 2014-08-13 | 上海朗萨医药科技有限公司 | Amino acid tanshinone phenolic ester derivatives and preparation method thereof |
Non-Patent Citations (3)
| Title |
|---|
| Syntheses of diacyltanshinol derivatives and their suppressive effects on macrophage foam cell formation by reducing oxidized LDL uptake;Xi Cheng，等;《Bioorganic Chemistry 》;20140228;第52卷;第24-30页 * |
| The Structure of Tanshinone-II;Yasuaki OKUMURA, 等;《Bulletin of the Chemical Society of Japan》;19611231;第34卷(第7期);第895-897页 * |
| 丹参酮IIA衍生物的设计与合成研究;张朝晖;《中国海洋大学硕士学位论文》;20150115;第28-30页 * |
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