CN101619055A - Oxidated genistein and biotransformation preparation and application method thereof - Google Patents
Oxidated genistein and biotransformation preparation and application method thereof Download PDFInfo
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Abstract
The invention provides oxidated genistein and a biotransformation preparation and application method thereof. The oxidated genistein is an isoflavone chloride compound of beta-oxidated genistein and 3', 8-double oxidated genistein. The invention also provides a method of utilizing Actinoplanes sp. HBDN08 separated from soil to establish biotransformation and obtain the beta-oxidated genistein and the 3', 8-double oxidated genistein. The method facilitates the high-efficiency synthesis of a transformation product, has little environmental pollution and low cost and can be widely applied to large-scale preparation. The invention can directly add substrate genistein/genistin into a culture medium or jointly culture microorganisms and a culture medium of a carbon-nitrogen source containing the genistein/genistin to obtain the oxidated genistein. The invention also provides the application of the compound in anti-oxidation and anti-tumor aspects, and the transformation product thereof has more remarkable anti-oxidation activity and activity of restraining the propagation of cancer cells than genistein.
Description
(1) technical field
The present invention relates to oxidated genistein and Biotransfer process for preparing thereof and the application in anticancer, be specifically utilize one from soil isolating microorganism genistein carried out bio-transformation prepare oxidated genistein, and oxidated genistein is in the application of anti-oxidant and anti-tumor aspect.
(2) background technology
Isoflavones belongs to a kind of of flavonoid compound, owing to its tool different physiological roles receives much attention.Genistein is one of important composition composition of isoflavones, has female hormone and anti-female hormone character, and has antioxygenation, and genistein can also suppress the activity of tyrosine protein kinase and topoisomerase II simultaneously.Experiment shows that genistein also has the effect of bringing out programmed cell death, improving anticancer drug effect and inhibition vasculogenesis in cell in vitro and the animal body, it is a kind of very potential cancer chemopreventive agent, and its antitumous effect and mechanism are with a wide range of applications.
Halogenide extensively is present in occurring in nature, not only can obtain by synthetic, also can be synthesized into by the microorganism biological body.Up to the present, isolated more than 4000 kind of halogenation metabolite nearly, many have a biological activity preferably.Because halogenation can strengthen or reduce the biological activity of parent compound, make people constantly find and explore this new compounds.
The chlorination of genistein mainly occurs in 6 and 83 ' positions of encircling with B of A ring, and up to now, the oxidated genistein that is obtained comprises: 6-chlorine genistein, 8-chlorine genistein, 3 '-the chlorine genistein, 6, the two chlorine genisteins of 8-, 3 ', the two chlorine genisteins of 6-.Chlorization can change the biological effect of genistein, for example, compare with genistein, 3 '-the anti-LDL oxidation activity of chlorine genistein can improve about 4 times, 6, the two chlorine genisteins of 8-then have the female hormone activity that is different from genistein.
(3) summary of the invention
The object of the present invention is to provide a kind of the efficient synthetic of converted product that help, and it is low in the pollution of the environment, cost is low, can be widely used in mass preparation, and converted product has the oxidated genistein than tangible more anti-oxidant activity of genistein and anticancer proliferation activity.
Oxidated genistein provided by the invention: 8-chlorine genistein, 3 ', the two chlorine genisteins of 8-; Through structure identify and retrieve 3 ', the two chlorine genisteins of 8-are a new compound, and provide the preparation method of oxidated genistein, this method can directly add substratum and the microorganism that substrate genistein/Genistoside maybe will contain the carbon nitrogen source of genistein/Genistoside in substratum cultivates altogether, thereby obtains oxidated genistein; The present invention also provides the application method of this compound at anti-oxidant and anti-tumor aspect.
One of technical scheme of the present invention has following structural formula for a class oxidated genistein is provided:
8-chlorine genistein: R=H; 3 ', two chlorine genistein: the R=Cl of 8-
Technical scheme of the present invention two for the preparation method of above-mentioned oxidated genistein is provided, comprise step:
A) genistein/Genistoside directly being joined in the substratum substratum and the actinoplanes Actinoplanes sp.HBDN08 that maybe will contain the carbon nitrogen source material of genistein/Genistoside in cultivating altogether with actinoplanes Actinoplanes sp.HBDN08 as substrate cultivates altogether;
B) utilize high pressure liquid chromatography (HPLC) or extraction/silica gel column chromatography or extraction/silica gel/gel chromatography method separation and purification to obtain product.
Technical scheme of the present invention three for the application method of above-claimed cpd at anti-oxidant and anti-tumor aspect is provided, compare with genistein, 8-chlorine genistein and 3 ', the two chlorine genisteins of 8-have higher anti-oxidant activity; Anti-tumor experiment demonstration 8-chlorine genistein and 3 ', the two chlorine genisteins of 8-are aspect the growth of inhibition human breast carcinoma MDA-MB-231 cell, and effect is stronger.
The present invention relates to oxidated genistein and Biotransfer process for preparing thereof and the application in anticancer, be specifically utilize one from soil isolating microorganism genistein carried out bio-transformation prepare oxidated genistein, and oxidated genistein is in the application of anti-oxidant and anti-tumor aspect.
Beneficial effect of the present invention is:
1. utilize actinoplanes Actinoplanes sp.HBDN08 set up bio-transformation obtain 8-chlorine genistein and 3 ', the method for the two chlorine genisteins of 8-.
2. identify and retrieval by nuclear-magnetism, resulting oxidated genistein 3 ', the two chlorine genisteins of 8-are a new compound.
3.8-chlorine genistein and 3 ', the two chlorine genisteins of 8-have anti-oxidant and antineoplastic activity.
4. the improvement of bioconversion method such as the method for Microbial Breeding, gene clone and conversion, helps oxidated genistein more synthetic.
5. the biotransformation method cost is lower, can be widely used in mass preparation.
6. biotransformation method is a kind of method of safety and environmental protection, can not pollute environment in scale operation.
Among the present invention actinoplanes Actinoplanes sp.HBDN08 be preserved on 03 11st, 2009 " Chinese biological culture presevation management committee common micro-organisms " center "; preservation registration number is: CGMCC NO.2944; the bacterial strain of called after HBDN08 (Actinoplanes sp.), the dna sequence dna of actinomycetes Actinoplanes sp.HBDN 08 is:
gtggaaagtt?tttcggtctg?ggatgggctc?gcggcctatc?agcttgttgg?tggggtgatg 60
gcctaccaag?gcgacgacgg?gtagccggcc?tgagagggcg?accggccaca?ctgggactga 120
gacacggccc?agactcctac?gggaggcagc?agtggggaat?attgcacaat?gggcggaagc 180
ctgatgcagc?gacgccgcgt?gagggatgac?ggccttcggg?ttgtaaacct?ctttcagcag 240
ggacgaagcg?caagtgacgg?tacctgcaga?agaagcgccg?gccaactacg?tgccagcagc 300
cgcggtaaga?cgtagggcgc?gagcgttgtc?cggatttatt?gggcgtaaag?agctcgtagg 360
cggcttgtcg?cgtcgaccgt?gaaaactcgc?agctcaactg?cgagcctgcg?gtcgatacgg 420
gcaggctaga?gttcggtagg?ggagactgga?attcctggtg?tagcggtgaa?atgcgcagat 480
atcaggagga?acaccggtgg?cgaaggcggg?tctctgggcc?gatactgacg?ctgaggagcg 540
aaagcgtggg?gagcgaacag?gattagatac?cctggtagtc?cacgctgtaa?acgttgggcg 600
ctaggtgtgg?ggagcctctc?cggttctctg?tgccgcagct?aacgcattaa?gcgccccgcc 660
tggggagtac?ggccgcaagg?ctaaaactca?aaggaattga?cgggggcccg?cacaagcggc 720
ggagcatgcg?gattaattcg?atgcaacgcg?aagaacctta?cctgggtttg?acatggccgc 780
aaaactgtca?gagatggcag?gtccttcggg?ggcggtcaca?ggtggtgcat?ggctgtcgtc 840
agctcgtgtc?gtgagatgtt?gggttaagtc?ccgcaacgag?cgcaaccctc?gttcgatgtt 900
gccagcgcgt?tatggcgggg?actcatcgaa?gactgccggg?gtcaactcgg?aggaaggtgg 960
ggatgacgtc?aagtcatcat?gccccttatg?tccagggctt?cacgcatgct?acaatggccg?1020
gtacaaaggg?ttgcgatgcc?gtgaggtgga?gcgaatccca?aaaagccggt?ctcagttcgg?1080
atcggggtct?gcaactcgac?cccgtgaagt?cggagtcgct?agtaatcgca?gatcagcaac?1140
gctgcggtga?atacgttccc?gggccttgta?cacaccgccc?gtcacgtcac?gaaagtcggc?1200
aacacccgaa?gccggtggcc?taaccccgtt?aggggaggga?gccgtcgaag?gtggggctgg?1260
cgattgggac?gaagtcgtaa?caaggtagcc?gtaccgga 1298
Oxidated genistein also can obtain by microbial transformation except obtaining by the method for chemosynthesis.At present, realize that by bio-transformation genistein chlorating microorganism mainly belongs to streptomyces griseus, other microorganism with oxidated genistein yet there are no report.Actinomycetes Actinoplanes sp.HBDN08 separates from Taizhou, Zhejiang Province city soil.The homology comparative result of relevant bacterial strain shows among the DNA database GeneBank of the 16SrDNA sequence of this bacterial strain and NIH biotech research center, the whole nation (NCBI), the 16S rDNA sequence homology of it and known kind of small single-cell bacteria (Micromonospora sp.) and actinoplanes (Actinoplanes sp.) is up to more than 98%, registration number is FJ770217, and the evolutionary tree analysis shows that bacterial strain HBDN08 belongs to actinoplanes.Name sp.HBDN08 according to The above results bacterial strain HBDN08 into actinoplanes Actinoplanes.
(4) description of drawings
Fig. 1 transforms preceding HPLC collection of illustrative plates for genistein;
Fig. 2 is the HPLC collection of illustrative plates of genistein after transforming.
(5) embodiment
The present invention is further illustrated below in conjunction with specific embodiment, but should notice that scope of the present invention is not subjected to any restriction of these embodiment.
Embodiment one
Actinoplanes Actinoplanes sp.HBDN08 after 2 days is that 4% inoculum size inserts in the fermention medium with volume percent in seed culture, and the 250rpm shaking culture is 6 days under 28 ℃ of conditions, the concentration of sampling and measuring oxidated genistein after the fermentation ends.Its detailed step that relates to is as follows:
(1) slant culture: actinoplanes Actinoplanes sp.HBDN08 is inoculated on the solid slant culture base, under 28 ℃ of conditions, leaves standstill and cultivated 6 days;
(2) preparation seed culture fluid: with the resulting slant culture of step (1), be inoculated in the liquid seed culture medium, under 28 ℃ of conditions, 250rpm cultivated 2 days, made seed culture fluid;
(3) transform: use the seed culture fluid of step (2) gained, by volume the inoculum size of per-cent 4% inserts and transforms in the substratum, and under 28 ℃ of conditions, 250rpm cultivated 6 days;
(4) detection of oxidated genistein: transform and finish back centrifugal collection supernatant liquor of 4000r/min and cell precipitation, supernatant liquor is through ethyl acetate extraction, cell precipitation adds the methyl alcohol soaked overnight of 3 times of volumes, acetic acid ethyl acetate extract and methyl alcohol soak solution are merged, be dissolved in the ethanol after the vacuum-drying, through the content of rp-hplc mensuration oxidated genistein, calculating 8-chlorine genistein and 3 ', the transformation efficiency of the two chlorine genisteins of 8-is respectively 76.8% and 12.8%.
Transform in the method for producing oxidated genistein at above-mentioned this actinoplanes that utilizes, the solid slant culture based formulas is: Zulkovsky starch 10g/L, yeast extract paste 4g/L, caseinhydrolysate 3g/L, K
2HPO
43H
2O 0.5g/L, MgSO
47H
2O 0.5g/L, agar 20g/L, 7.2,120 ℃ of sterilizations of pH 20 minutes.
Transform in the method for producing oxidated genistein at above-mentioned this actinoplanes that utilizes, the liquid seed culture medium prescription is: W-Gum 24g/L, yeast extract paste 5g/L, extractum carnis 3g/L, peptone 5g/L, glucose 1g/L, lime carbonate 4g/L, pH 7.0, and liquid amount is the bottled liquid 25ml of 250ml triangle, sterilize 20 minutes for 120 ℃.
Transform in the method for producing oxidated genistein at above-mentioned this actinoplanes that utilizes, the conversion culture medium prescription is: Zulkovsky starch 50g/L, glucose 20g/L, soybean cake powder 30g/L, sodium-chlor 3g/L, lime carbonate 8g/L, pH 7.2, liquid amount is the bottled liquid 25ml of 250ml triangle, sterilizes 20 minutes for 120 ℃.Soybean cake powder in the substratum can be replaced by other carbon nitrogen source that contains genistein/Genistoside.
The RP-HPLC analysis condition:
Chromatographic column: Agilent TC-C18,5 μ m, 4.6mm * 250mm
Temperature: 25 ℃
Flow velocity: 1mL/min
Detect: 260nm
Elutriant: methanol (50: 50V/V)
The two chlorine genisteins of retention time: 8-are 22.5min, 3 ', the two chlorine genisteins of 8-are 36.9min
Embodiment two
Actinoplanes Actinoplanes sp.HBDN08 after 2 days is that 4% inoculum size inserts in the fermention medium with volume percent in seed culture, and the 250rpm shaking culture is 6 days under 28 ℃ of conditions, the concentration of sampling and measuring oxidated genistein after the fermentation ends.Its detailed step that relates to is as follows:
(1) slant culture: actinoplanes Actinoplanes sp.HBDN08 is inoculated on the solid slant culture base, under 28 ℃ of conditions, leaves standstill and cultivated 6 days;
(2) preparation seed culture fluid: with the resulting slant culture of step (1), be inoculated in the liquid seed culture medium, under 28 ℃ of conditions, 250rpm cultivated 2 days, made seed culture fluid;
(3) transform: use the seed culture fluid of step (2) gained, by volume the inoculum size of per-cent 4% inserts and transforms in the substratum, and under 28 ℃ of conditions, 250rpm cultivated 4 days, adds genistein or Genistoside by 1% volume ratio
Dimethyl formamide solution, the final concentration of substrate are 0.5mmol/L, and under 28 ℃ of conditions, 250rpm continues to cultivate 2 days;
(4) detection of oxidated genistein: transform and finish back centrifugal collection supernatant liquor of 4000r/min and cell precipitation, supernatant liquor is through ethyl acetate extraction, cell precipitation adds the methyl alcohol soaked overnight of 3 times of volumes, acetic acid ethyl acetate extract and methyl alcohol soak solution are merged, be dissolved in the ethanol after the vacuum-drying, through the content of rp-hplc mensuration oxidated genistein, calculating 8-chlorine genistein and 3 ', the transformation efficiency of the two chlorine genisteins of 8-is respectively 0.98% and 0.17%.
Transform in the method for producing oxidated genistein at above-mentioned this actinoplanes that utilizes, the solid slant culture based formulas is: Zulkovsky starch 8～12g/L, yeast extract paste 3～5g/L, caseinhydrolysate 2～4g/L, K
2HPO
43H
2O 0.5g/L, MgSO
47H
2O0.5g/L, agar 18～22g/L, 7.2,120 ℃ of sterilizations of pH 20 minutes.
Transform in the method for producing oxidated genistein at above-mentioned this actinoplanes that utilizes, the liquid seed culture medium prescription is: W-Gum 22～25g/L, yeast extract paste 4～6g/L, extractum carnis 2～4g/L, peptone 4～6g/L, glucose 0.8～1.2g/L, lime carbonate 3～5g/L, pH 7.0, and liquid amount is the bottled liquid 25ml of 250ml triangle, sterilize 20 minutes for 120 ℃.
Transform in the method for producing oxidated genistein at above-mentioned this actinoplanes that utilizes, the conversion culture medium prescription is: Zulkovsky starch 45～55g/L, glucose 18～22g/L, soybean cake powder 28～32g/L, sodium-chlor 2～4g/L, lime carbonate 6～10g/L, pH 7.2, liquid amount is the bottled liquid 25ml of 250ml triangle, sterilizes 20 minutes for 120 ℃.Soybean cake powder in the substratum can be replaced by other carbon nitrogen source that contains genistein/Genistoside.Substrate genistein or Genistoside be earlier with the dimethyl formamide dissolving, in fermentation joined in the 4th day transform in the substratum to final concentration be 0.5mmol/L.
The RP-HPLC analysis condition:
Chromatographic column: Agilent TC-C18,5 μ m, 4.6mm * 250mm
Temperature: 25 ℃
Flow velocity: 1mL/min
Detect: 260nm
Elutriant: methanol (50: 50V/V)
The two chlorine genisteins of retention time: 8-are 22.5min, 3 ', the two chlorine genisteins of 8-are 36.9min
Embodiment three
Press the method for embodiment one, gained conversion fluid 5000ml, conversion fluid is centrifugal in 4000r/min, collect supernatant liquor and cell precipitation, supernatant liquor is through ethyl acetate extraction, cell precipitation adds the methyl alcohol soaked overnight of 3 times of volumes, and acetic acid ethyl acetate extract and methyl alcohol soak solution are merged, and rotary evaporation in vacuo obtains the oily enriched material after concentrating.Gained oily enriched material is dissolved in a spot of chloroform methanol, mixes with silica gel, removes and to desolvate, and treats the silicagel column of packing into behind the complete drying, and the silica gel granularity is the 100-200 order.Elutriant is chloroform/methanol elutriant (99: 1～90: 10), and the Fractional Collections merging obtains 5 components.Component 3 is dissolved in ethanol after rotary evaporation in vacuo is done, joins then among the gel column Sephadex LH-20 and also use ethanol elution, obtain 4 components.The 4th component obtains compound 1 (15mg) and compound 2 (90mg) through half preparative liquid chromatography post.
Compound 1 and 2 is carried out structural characterization, comprise the evaluation of infrared, ultraviolet, mass spectrum and 1H and 13C NMR, experimental data shows, compound 1 is 3 ' and, the two chlorine genisteins of 8-are a new compound; Compound 2 is a 8-chlorine genistein.
Experimental data is as follows:
Compound 1:3 ', the two chlorine genisteins of 8-; The pale solid powder; UV (ethanol) λ
MaxNm (log ε): 267 (4.35); IR v
Max(KBr) cm
-1: 3271 (OH), 1655,1579,1503,1424,1369,1287,1248,1068,826;
1H NMR (acetone-d
6, 400MHz): δ 6.50 (1H, s, H-6), and 7.10 (1H, d, J=8.4Hz, H-5 '), 7.45 (1H, dd, J=8.4,2.0Hz, H-6 '), 7.68 (1H, d, J=2.0Hz, H-2 '), 8.41 (1H, s, H-2), 12.89 (1H, OH-5);
13C NMR (acetone-d
6, 100MHz): δ 98.5 (s, C-8), 100.2 (d, C-6), 106.9 (s, C-4a), 117.4 (d, C-5 '), 120.9 (s, C-3 '), 123.0 (s, C-3), 124.1 (s, C-1 '), 129.7 (d, C-6 '), 131.4 (d, C-2 '), 154.0 (s, C-8a), (154.1 s, C-4 '), 155.0 (d, C-2), 160.8 (s, C-7), 161.7 (s, C-5), 181.4 (s, C-4); : ESI-MS m/z 337[M-H]
+
HRESI-MS?m/z?336.9651([M-H]
+，calcd?for?C
15H
7Cl
2O
5，336.9676).
Compound 2:8-chlorine genistein; The pale solid powder;
1H NMR (acetone-d
6, 400MHz) δ 6.48 (1H, s, H-6), 6.92 (2H, d, J=8.4Hz, H-3 ', 5 '), 7.49 (2H, d, J=8.4Hz, H-2 ', 6 '), 8.32 (1H, s, H-2), 13.00 (1H, OH-5);
13C NMR (acetone-d
6, 100MHz) δ 98.6 (s, C-8), 100.2 (d, C-6), 106.8 (s, C-4a), 116.1 (d, C-3 ', 5 '), (122.6 s, C-1 '), 124.4 (s, C-3), 131.3 (d, C-2 ', 6 '), 154.2 (s, C-8a), 154.4 (d, C-2), 158.7 (s, C-4 '), 160.8 (s, C-7), 161.8 (s, C-5), 181.6 (s, C-4); ESI-MS m/z 303[M-H]
+.
Embodiment four
Lipid peroxidation suppresses research:
Rat is put to death the back and separates liver organization rapidly, wash repeatedly with the phosphate buffered saline buffer that contains Repone K (0.15mol/L) (0.1mol/L pH7.4), remove the blood ingredient in the tissue, add above-mentioned Repone K-phosphate buffered saline buffer by 1: 4 (W/V) at last, put into then and stir into liver homogenate in the tissue mashing machine.The centrifugal 15min of liver homogenate 9000r/min on ultracentrifuge with preparing gets supernatant liquor in the centrifugal 60min of 10000r/min, abandons supernatant liquor, obtains the pink precipitation, is hepatomicrosome.Hepatomicrosome is suspended in the Repone K-phosphate buffered saline buffer that contains 30% glycerine, in-20 ℃ of refrigerators, preserves standby.Above all operations all carries out under condition of ice bath.
Measure protein content in the hepatomicrosome with the Bradford method, and to be diluted to protein concentration with phosphate buffered saline buffer (80 μ mol/LpH 7.4) be 300-500 μ g/mL.The reaction system of anti-oxidant experiment comprises phosphate buffered saline buffer (80 μ mol/LpH 7.4), hepatomicrosome (300-500 μ g albumen/mL), vitamins C (200 μ mol/L), and the test agent of different concns (genistein, 8-chlorine genistein or 3 ', the two chlorine genisteins of 8-).(500 μ L) places 37 ℃ of water-baths to hatch 90min with reaction system, adds the trichoroacetic acid(TCA) termination reaction of 250 μ L20% then.Reaction solution in whizzer 10000g high speed centrifugation 3min, is got supernatant liquor 600 μ L, add the thiobarbituricacid of 250 μ L 0.67%, boil 20min, measure its light absorption value at the 535nm place after cooling, each concentration determination is three times in the experiment.With the sample that does not add testing compound is blank.
Genistein in the experiment, the two chlorine genisteins and 3 of 8-', the concentration gradient of the two chlorine genisteins of 8-is 5,10,20,30,40 μ mol/L.
The anti-oxidant activity of compound is with IC
50Expression, IC
50Be defined as the concentration that suppresses 50% testing compound when generating with the material of thiobarbituric acid reaction.
The calculation formula of inhibiting rate is:
Inhibiting rate (%)=(1-OD
Medication/ OD
Contrast) * 100%.
Experimental result shows (seeing Table 1), the two chlorine genisteins and 3 of 8-' and, the two chlorine genisteins of 8-have the anti-oxidant activity stronger than genistein.
Anti-oxidant experimental study (the IC of table 1 genistein and oxidated genistein
50).
Embodiment five
MTT colorimetric method for determining human breast carcinoma MDA-MB-231 cell proliferation inhibition rate:
The human breast carcinoma MDA-MB-231 cell of taking the logarithm vegetative period is with being made into individual cells suspension with the DMEM nutrient solution behind 0.25% tryptic digestion; With every hole 1.0 * 10
4Individual cell inoculation in 96 well culture plates, every pore volume 100 μ l.Culture plate is moved into CO
2In the incubator, at 37 ℃, 5%CO
2And under the saturated humidity condition, leave standstill and cultivated 24 hours, abandon stoste, the appropriate incubation liquid that every hole adds 100 μ l serum-frees continues to cultivate 24 hours synchronizing them, and abandons supernatant then.Treatment group adds the nutrient solution that contains 100 μ g/ml, 50 μ g/ml, 25 μ g/ml, 12.5 μ g/ml, 6.25 μ g/ml, 3.12 μ g/ml, 1.56 μ g/ml, 0.78 μ g/ml, 0.39 μ g/ml, 0.19 μ g/ml genistein or oxidated genistein respectively, control group is not for containing the nutrient solution of genistein or oxidated genistein, and each organizes 2 repeating holes.
Culture plate is moved into CO
2In the incubator, at 37 ℃, 5%CO
2And cultivated 72 hours under the saturated humidity condition.After cultivating end, every hole adds 20 μ l MTT solution (5g/L) in 96 well culture plates, and culture plate is moved into CO
2In the incubator, at 37 ℃, 5%CO
2And continue under the saturated humidity condition to cultivate 4 hours, abandon supernatant then, every hole adds 100 μ l dimethyl sulfoxide (DMSO) (DMSO), after allowing it fully dissolve, the 570nm place measures light absorption value on microplate reader, returns to zero with control wells, measure the OD value in each hole, calculate cell proliferation inhibition rate according to following formula:
Proliferation inhibition rate (%)=(1-OD
Medication/ OD
Contrast) * 100%
The antitumour activity of genistein and oxidated genistein is with IC
50Expression, promptly the growth inhibition ratio of cancer cells is the concentration of 50% o'clock testing sample, calculates with the LOGIT method.
Genistein and oxidated genistein see Table 2 to human breast carcinoma MDA-MB-231 cell inhibitory effect experimental result, the result shows, 8-chlorine genistein and 3 ', the two chlorine genisteins of 8-have the activity of the inhibition human breast carcinoma MDA-MB-231 cell proliferation stronger than genistein.
Table 2 genistein and oxidated genistein are to the inhibition (IC of human breast carcinoma MDA-MB-231 cell proliferation
50)
The sequence table sample
＜110〉Northeast Agricultural University
＜120〉oxidated genistein and bio-transformation methods for making and using same thereof
<160>1
<210>1
<211>1298
<212>DNA
<213>Actinoplanes?sp.
<400>
gtggaaagtt?tttcggtctg?ggatgggctc?gcggcctatc?agcttgttgg?tggggtgatg 60
gcctaccaag?gcgacgacgg?gtagccggcc?tgagagggcg?accggccaca?ctgggactga?120
gacacggccc?agactcctac?gggaggcagc?agtggggaat?attgcacaat?gggcggaagc?180
ctgatgcagc?gacgccgcgt?gagggatgac?ggccttcggg?ttgtaaacct?ctttcagcag?240
ggacgaagcg?caagtgacgg?tacctgcaga?agaagcgccg?gccaactacg?tgccagcagc?300
cgcggtaaga?cgtagggcgc?gagcgttgtc?cggatttatt?gggcgtaaag?agctcgtagg?360
cggcttgtcg?cgtcgaccgt?gaaaactcgc?agctcaactg?cgagcctgcg?gtcgatacgg?420
gcaggctaga?gttcggtagg?ggagactgga?attcctggtg?tagcggtgaa?atgcgcagat?480
atcaggagga?acaccggtgg?cgaaggcggg?tctctgggcc?gatactgacg?ctgaggagcg?540
aaagcgtggg?gagcgaacag?gattagatac?cctggtagtc?cacgctgtaa?acgttgggcg?600
ctaggtgtgg?ggagcctctc?cggttctctg?tgccgcagct?aacgcattaa?gcgccccgcc?660
tggggagtac?ggccgcaagg?ctaaaactca?aaggaattga?cgggggcccg?cacaagcggc?720
ggagcatgcg?gattaattcg?atgcaacgcg?aagaacctta?cctgggtttg?acatggccgc 780
aaaactgtca?gagatggcag?gtccttcggg?ggcggtcaca?ggtggtgcat?ggctgtcgtc 840
agctcgtgtc?gtgagatgtt?gggttaagtc?ccgcaacgag?cgcaaccctc?gttcgatgtt 900
gccagcgcgt?tatggcgggg?actcatcgaa?gactgccggg?gtcaactcgg?aggaaggtgg 960
ggatgacgtc?aagtcatcat?gccccttatg?tccagggctt?cacgcatgct?acaatggccg?1020
gtacaaaggg?ttgcgatgcc?gtgaggtgga?gcgaatccca?aaaagccggt?ctcagttcgg?1080
atcggggtct?gcaactcgac?cccgtgaagt?cggagtcgct?agtaatcgca?gatcagcaac?1140
gctgcggtga?atacgttccc?gggccttgta?cacaccgccc?gtcacgtcac?gaaagtcggc?1200
aacacccgaa?gccggtggcc?taaccccgtt?aggggaggga?gccgtcgaag?gtggggctgg?1260
cgattgggac?gaagtcgtaa?caaggtagcc?gtaccgga 1298
Claims (6)
1, a kind of oxidated genistein, it is characterized in that it be a kind of chlorination isoflavonoid 8-chlorine genistein and 3 ', the two chlorine genisteins of 8-, structural formula is:
8-chlorine genistein: R=II; 3 ', two chlorine genistein: the R=Cl of 8-.
2, a kind of Biotransfer process for preparing for preparing the described oxidated genistein of claim 1 is characterized in that it may further comprise the steps:
A) genistein/Genistoside directly being joined in the substratum substratum and the actinomycetes Actinoplanes sp.HBDN 08 that maybe will contain the carbon nitrogen source material of genistein/Genistoside in cultivating altogether with actinomycetes Actinoplanes sp.HBDN08 as substrate cultivates altogether;
B) utilize high pressure liquid chromatography (HPLC) or extraction/silica gel column chromatography or extraction/silica gel/gel chromatography method separation and purification to obtain product.
3, the Biotransfer process for preparing of oxidated genistein according to claim 2, it is characterized in that described actinomycetes Actinoplanes sp.HBDN 08 is what be separated to from soil, be to be preserved in " Chinese biological culture presevation management committee common micro-organisms " center "; preservation registration number is: CGMCC NO.2944, the bacterial strain of called after HBDN 08 (Actinoplanes sp.) on 03 11st, 2009.
4, the Biotransfer process for preparing of oxidated genistein according to claim 3 is characterized in that the 16S rDNA sequence of described actinomycetes Actinoplanes sp.HBDN 08 is:
gtggaaagtt?tttcggtctg?ggatgggctc?gcggcctatc?agcttgttgg?tggggtgatg?60
gcctaccaag?gcgacgacgg?gtagccggcc?tgagagggcg?accggccaca?ctgggactga?120
gacacggccc?agactcctac?gggaggcagc?agtggggaat?attgcacaat?gggcggaagc?180
ctgatgcagc?gacgccgcgt?gagggatgac?ggccttcggg?ttgtaaacct?ctttcagcag?240
ggacgaagcg?caagtgacgg?tacctgcaga?agaagcgccg?gccaactacg?tgccagcagc?300
cgcggtaaga?cgtagggcgc?gagcgttgtc?cggatttatt?gggcgtaaag?agctcgtagg?360
cggcttgtcg?cgtcgaccgt?gaaaactcgc?agctcaactg?cgagcctgcg?gtcgatacgg?420
gcaggctaga?gttcggtagg?ggagactgga?attcctggtg?tagcggtgaa?atgcgcagat?480
atcaggagga?acaccggtgg?cgaaggcggg?tctctgggcc?gatactgacg?ctgaggagcg?540
aaagcgtggg?gagcgaacag?gattagatac?cctggtagtc?cacgctgtaa?acgttgggcg?600
ctaggtgtgg?ggagcctctc?cggttctctg?tgccgcagct?aacgcattaa?gcgccccgcc?660
tggggagtac?ggccgcaagg?ctaaaactca?aaggaattga?cgggggcccg?cacaagcggc?720
ggagcatgcg?gattaattcg?atgcaacgcg?aagaacctta?cctgggtttg?acatggccgc?780
aaaactgtca?gagatggcag?gtccttcggg?ggcggtcaca?ggtggtgcat?ggctgtcgtc?840
agctcgtgtc?gtgagatgtt?gggttaagtc?ccgcaacgag?cgcaaccctc?gttcgatgtt?900
gccagcgcgt?tatggcgggg?actcatcgaa?gactgccggg?gtcaactcgg?aggaaggtgg?960
ggatgacgtc?aagtcatcat?gccccttatg?tccagggctt?cacgcatgct?acaatggccg?1020
gtacaaaggg?ttgcgatgcc?gtgaggtgga?gcgaatccca?aaaagccggt?ctcagttcgg?1080
atcggggtct?gcaactcgac?cccgtgaagt?cggagtcgct?agtaatcgca?gatcagcaac?1140
gctgcggtga?atacgttccc?gggccttgta?cacaccgccc?gtcacgtcac?gaaagtcggc?1200
aacacccgaa?gccggtggcc?taaccccgtt?aggggaggga?gccgtcgaag?gtggggctgg?1260
cgattgggac?gaagtcgtaa?caaggtagcc?gtaccgga?1298。
5, the Biotransfer process for preparing of oxidated genistein according to claim 2, it is characterized in that it may further comprise the steps: actinoplanes Actinoplanes sp.HBDN 08 after 2 days is that 4% inoculum size inserts in the fermention medium with volume percent in seed culture, 250 rpm shaking culture are 6 days under 28 ℃ of conditions, the concentration of sampling and measuring oxidated genistein after the fermentation ends, its detailed step that relates to is as follows:
(1) slant culture: actinoplanes Actinoplanes sp.HBDN 08 is inoculated on the solid slant culture base, under 28 ℃ of conditions, leaves standstill and cultivated 6 days;
(2) preparation seed culture fluid: with the resulting slant culture of step (1), be inoculated in the liquid seed culture medium, under 28 ℃ of conditions, 250 rpm cultivated 2 days, made seed culture fluid;
(3) transform: use the seed culture fluid of step (2) gained, by volume the inoculum size of per-cent 4% inserts and transforms in the substratum, and under 28 ℃ of conditions, 250 rpm cultivated 6 days;
(4) detection of oxidated genistein: transform and finish back centrifugal collection supernatant liquor of 4000 r/min and cell precipitation, supernatant liquor is through ethyl acetate extraction, cell precipitation adds the methyl alcohol soaked overnight of 3 times of volumes, acetic acid ethyl acetate extract and methyl alcohol soak solution are merged, be dissolved in the ethanol after the vacuum-drying, through the content of rp-hplc mensuration oxidated genistein, calculating 8-chlorine genistein and 3 ', the transformation efficiency of the two chlorine genisteins of 8-is respectively 76.8% and 12.8%; Transform in the method for producing oxidated genistein at above-mentioned this actinoplanes that utilizes, the solid slant culture based formulas is: Zulkovsky starch 8～12g/L, yeast extract paste 3～5g/L, caseinhydrolysate 2～4g/L, K
2HPO
43H
2O 0.5g/L, MgSO
47H
2O0.5g/L, agar 18～22g/L, 7.2,120 ℃ of sterilizations of pH 20 minutes;
Transform in the method for producing oxidated genistein at above-mentioned this actinoplanes that utilizes, the liquid seed culture medium prescription is: W-Gum 22～25g/L, yeast extract paste 4～6g/L, extractum carnis 2～4g/L, peptone 4～6g/L, glucose 0.8～1.2g/L, lime carbonate 3～5g/L, pH 7.0, and liquid amount is the bottled liquid 25ml of 250ml triangle, sterilize 20 minutes for 120 ℃;
Transform in the method for producing oxidated genistein at above-mentioned this actinoplanes that utilizes, the conversion culture medium prescription is: Zulkovsky starch 45～55g/L, glucose 18～22g/L, soybean cake powder 28～32g/L, sodium-chlor 2～4g/L, lime carbonate 6～10g/L, pH 7.2, liquid amount is the bottled liquid 25ml of 250ml triangle, sterilizes 20 minutes for 120 ℃.Soybean cake powder in the substratum can be replaced by other carbon nitrogen source that contains genistein/Genistoside.
6, a kind of application method of oxidated genistein according to claim 1 is characterized in that at it it being the application method of the anti-oxidant and anti-tumor aspect of oxidated genistein, may further comprise the steps:
(1). lipid peroxidation suppresses research:
Rat is put to death the back and separates liver organization rapidly, wash repeatedly with the phosphate buffered saline buffer 0.1mol/L pH 7.4 that contains Repone K 0.15mol/L, remove the blood ingredient in the tissue, add above-mentioned Repone K-phosphate buffered saline buffer by 1: 4 (W/V) at last, put into then and stir into liver homogenate in the tissue mashing machine; The centrifugal 15min of liver homogenate 9000r/min on ultracentrifuge with preparing gets supernatant liquor in the centrifugal 60min of 10000r/min, abandons supernatant liquor, obtains the pink precipitation, is hepatomicrosome.Hepatomicrosome is suspended in the Repone K-phosphate buffered saline buffer that contains 30% glycerine, in-20 ℃ of refrigerators, preserves standby; Above all operations all carries out under condition of ice bath;
Measure protein content in the hepatomicrosome with the Bradford method, and to be diluted to protein concentration with phosphate buffered saline buffer 80 μ mol/L pH 7.4 be 300-500 μ g/mL; The reaction system of anti-oxidant experiment comprises phosphate buffered saline buffer 80 μ mol/L pH 7.4, hepatomicrosome 300-500 μ g albumen/mL, vitamins C (200 μ mol/L), and the test agent genistein of different concns, 8-chlorine genistein or 3 ', the two chlorine genisteins of 8-; Place 37 ℃ of water-baths to hatch 90min reaction system 500 μ L, add the trichoroacetic acid(TCA) termination reaction of 250 μ L 20% then; Reaction solution in whizzer 10000g high speed centrifugation 3min, is got supernatant liquor 600 μ L, add the thiobarbituricacid of 250 μ L 0.67%, boil 20min, measure its light absorption value at the 535nm place after cooling, each concentration determination three times is a blank with the sample that does not add testing compound;
Wherein the two chlorine genisteins and 3 of genistein, 8-', the concentration gradient of the two chlorine genisteins of 8-is 5,10,20,30,40 μ mol/L;
The anti-oxidant activity of compound is with IC
50Expression, IC
50Be defined as the concentration that suppresses 50% testing compound when generating with the material of thiobarbituric acid reaction;
The calculation formula of inhibiting rate is:
Inhibiting rate (%)=(1-OD
Medication/ OD
Contrast) * 100%;
(2) .MTT colorimetric method for determining human breast carcinoma MDA-MB-231 cell proliferation inhibition rate:
The human breast carcinoma MDA-MB-231 cell of taking the logarithm vegetative period is with being made into individual cells suspension with the DMEM nutrient solution behind 0.25% tryptic digestion; With every hole 1.0 * 10
4Individual cell inoculation in 96 well culture plates, every pore volume 100 μ l; Culture plate is moved into CO
2In the incubator, at 37 ℃, 5%CO
2And under the saturated humidity condition, leave standstill and cultivated 24 hours, abandon stoste, the appropriate incubation liquid that every hole adds 100 μ l serum-frees continues to cultivate 24 hours synchronizing them, and abandons supernatant then; Treatment group adds the nutrient solution that contains 100 μ g/ml, 50 μ g/ml, 25 μ g/ml, 12.5 μ g/ml, 6.25 μ g/ml, 3.12 μ g/ml, 1.56 μ g/ml, 0.78 μ g/ml, 0.39 μ g/ml, 0.19 μ g/ml genistein or oxidated genistein respectively, control group is not for containing the nutrient solution of genistein or oxidated genistein, and each organizes 2 repeating holes;
Culture plate is moved into CO
2In the incubator, at 37 ℃, 5%CO
2And cultivated 72 hours under the saturated humidity condition, after cultivation finished, every hole added 20 μ l MTT solution 5g/L in 96 well culture plates, and culture plate is moved into CO
2In the incubator, at 37 ℃, 5%CO
2And continue under the saturated humidity condition to cultivate 4 hours, abandon supernatant then, every hole adds 100 μ l dimethyl sulfoxide (DMSO) DMSO, after allowing it fully dissolve, the 570nm place measures light absorption value on microplate reader, returns to zero with control wells, measure the OD value in each hole, calculate cell proliferation inhibition rate according to following formula:
Proliferation inhibition rate (%)=(1-OD
Medication/ OD
Contrast) * 100%
The antitumour activity of genistein and oxidated genistein is with IC
50Expression, promptly the growth inhibition ratio of cancer cells is the concentration of 50% o'clock testing sample, calculates with the LOGIT method;
Genistein, 8-oxidated genistein and 3 ', the two chlorine genisteins of 8-are to the inhibition IC of human breast carcinoma MDA-MB-231 cell proliferation
50Be respectively 27.94 μ g/ml, 8.26 μ g/ml, 3.15 μ g/ml.
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| CN111297807A (en) * | 2020-02-13 | 2020-06-19 | 东北农业大学 | Method for preparing bifunctional genistein-high polymer nano complex by ion crosslinking method and application |
| CN111795939A (en) * | 2020-07-21 | 2020-10-20 | 郑州安图生物工程股份有限公司 | Method for monitoring subpackaging uniformity of magnetic particle suspension |
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| CN111297807A (en) * | 2020-02-13 | 2020-06-19 | 东北农业大学 | Method for preparing bifunctional genistein-high polymer nano complex by ion crosslinking method and application |
| CN111795939A (en) * | 2020-07-21 | 2020-10-20 | 郑州安图生物工程股份有限公司 | Method for monitoring subpackaging uniformity of magnetic particle suspension |
| CN111795939B (en) * | 2020-07-21 | 2023-06-23 | 郑州安图生物工程股份有限公司 | Method for monitoring split charging uniformity of magnetic particle suspension |
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