CN102337308A - Method for converting bergenin into special nitrogenous derivative by using penicillium - Google Patents

Method for converting bergenin into special nitrogenous derivative by using penicillium Download PDF

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CN102337308A
CN102337308A CN2011103084787A CN201110308478A CN102337308A CN 102337308 A CN102337308 A CN 102337308A CN 2011103084787 A CN2011103084787 A CN 2011103084787A CN 201110308478 A CN201110308478 A CN 201110308478A CN 102337308 A CN102337308 A CN 102337308A
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bergeninum
converted product
penicillium
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solution
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崔桂友
朱志雄
陆广念
冉昇
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Yangzhou University
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Yangzhou University
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Abstract

The invention relates to a method for converting bergenin into a special nitrogenous derivative by using penicillium. The method comprises the following steps of: inoculating a microbial strain which has bergenin structure modified special enzyme activity into a culture solution, and performing flask shaking fermentation culture; at the middle stage of fermentation, adding a bergenin-containing substrate conversion liquor into a fermentation liquor; after the conversion is finished, removing mycelia, extracting supernatant liquor by using an organic solvent, concentrating an extract liquor, and recrystallizing to obtain a converted product crystal; or concentrating the extract liquor, and performing silica gel chromatographic separation, Sephadex purification or high performance liquid chromatography (HPLC) to obtain a solution containing a converted product; and concentrating or crystallizing the solution or performing low-temperature drying treatment on the solution to obtain converted product crystals or powder. The product has 2,2-diphenyl-1-picrylhydrazyl (DPPH) eliminating capacity and oxidation resistance which are higher than those of the bergenin and has a restraint effect on lipid peroxidation.

Description

Utilizing penicillium spp to transform Bergeninum is a kind of method of special nitrogen containing derivative
Technical field
The present invention relates to utilize the microbial strains that has the specific enzymes vigor in the penicillium, with Bergeninum (bergenin) through the method for microbial transformation carry out structural modification for ( E)-7-((6-acetyl-2-(hydroxyimino)-4-methyl-1,2-dihydropyridin-3-yl) methyl) 3,4,8,10-tetrahydroxy-2-(hydroxymethyl)-9-methoxy-2,3,4,4a-tetrahydropyrano [3,2- c] iso chromen-6 (10b H)-one, promptly ( E)-7-((6-ethanoyl-2-(oximido)-4-methyl isophthalic acid, 2-dihydropyridine-3-alkyl) methyl)-3,4,8,10-tetrahydroxy-2-(methylol)-9-methoxyl group-2,3,4, the 4a-tetrahydrochysene [3,2- c] method of different chromene-6 (10b H)-ketone.Converted product be ( E)-7-((6-ethanoyl-2-(oximido)-4-methyl isophthalic acid, 2-dihydropyridine-3-alkyl) methyl)-3,4,8,10-tetrahydroxy-2-(methylol)-9-methoxyl group-2,3,4, the 4a-tetrahydrochysene [3,2- c] different chromene-6 (10b H)-ketone.
Technical background
Bergeninum (bergenin) is a kind of naturally occurring aromatic series carbon glycosides, is the dihydro different coumarin derivative of glucopyranosyl gallic acid, contains the cyclic lactone structure in the molecule.Having found to be present in 30 various plants, is the natural constituent that a kind of high yield is easy to get.Particularly belong to the Rhizome or herb of Purple Bergenia of Saxifragaceae ( Bergenia), the Ardisa of Myrsinacea ( Ardisia), the mallotus of Euphorbiaceae ( Mallotus) content is abundant in the plant.The Rhizoma Seu Herba Bergeniae of Saxifragaceae ( Bergenia crassifolia), Rhizome or herb of Purple Bergenia ( B. purpurascens) in the Root of Crispateleaf Ardisia of Myrsinacea ( Ardisia crispa) wait in the plant, yield is up to 4.5%.The Bergeninum content of other a lot of plants is more than 1%.Structure called after 3,4,8,10-tetrahydroxy-2-(hydroxymethyl)-9-methoxy-3,4,4a, 10b-tetrahydropyrano [3,2-c] isochromen-6 (2H)-one.
In the Pharmacopoeia of the People's Republic of China, it has been recorded and be to be used for chronic bronchitis clinically by antisussive and expectorant agent.Its advantage is only to coughing centre restraining effect selectively, and toxic side effect is little, and untoward reaction is few, and use does not produce resistance continuously.Except this function; Also have and much to have reported but be not used for clinical biological activity and pharmacological action as yet, for example arthritis, antiulcer agent, protect the liver, anti-hepatitis c virus, anti HIV-1 virus, anti-mellitus, anti-heart disorder, arrestin tyrosine phosphatase 1B (FTP1B), arrhythmia, promotion burn wound healing, promote multiple effects such as pathological tissues recovery, trypanocidia, anticoagulation, anti-oxidant, enhancing immunity, reducing blood-fat.
Bergeninum distributes in plant extensively, and content is high, and extraction separation is cheap easily.But the Bergeninum poorly water-soluble, a lot of activity are not really remarkable, are necessary very much to carry out structural modification.Through structure of modification, find new Bergeninum converted product, be expected to solve the problem that its oral absorption is poor, bioavailability is low, biological activity is not high.Also enrich simultaneously the compound library of screening active ingredients, expanded new biological activity and pharmacological function.Therefore, Bergeninum is ideal natural product material and bio-transformation substrate.Up to the present, less relatively to the microbial transformation research of Bergeninum, rarely seen mistake 1 example report, 2005, Wang etc. utilized fungi Pleurotus ostreatusBergeninum is converted into dimer.
Summary of the invention
The object of the invention is to provide a kind of microbial conversion process of Bergeninum specific enzymes structural modification: but screening obtain catalysis Bergeninum (bergenin) carry out the specific enzymes structural modification become ( E)-7-((6-acetyl-2-(hydroxyimino)-4-methyl-1,2-dihydropyridin-3-yl) methyl) 3,4,8,10-tetrahydroxy-2-(hydroxymethyl)-9-methoxy-2,3,4,4a-tetrahydropyrano [3,2- c] iso chromen-6 (10b H)-one, promptly ( E)-7-((6-ethanoyl-2-(oximido)-4-methyl isophthalic acid, 2-dihydropyridine-3-alkyl) methyl)-3,4,8,10-tetrahydroxy-2-(methylol)-9-methoxyl group-2,3,4, the 4a-tetrahydrochysene [3,2- c] microorganism strains of different chromene-6 (10b H)-ketone so that adopt the method for microbial transformation, obtain Bergeninum special converted product ( E)-7-((6-acetyl-2-(hydroxyimino)-4-methyl-1,2-dihydropyridin-3-yl) methyl) 3,4,8,10-tetrahydroxy-2-(hydroxymethyl)-9-methoxy-2,3,4,4a-tetrahydropyrano [3,2- c] iso chromen-6 (10b H)-one.
The molecular structural formula of Bergeninum (bergenin) and converted product thereof is seen Fig. 1.
The present invention adopts the microbial strains with Bergeninum structural modification specific enzymes vigor to be inoculated in the nutrient solution, carries out shake flask fermentation and cultivates; Ferment middle adds the substrate conversion liquid contain Bergeninum (bergenin) in fermented liquid, make Bergeninum (bergenin) through the method for microbial transformation carry out special enzyme structural modification become ( E)-7-((6-acetyl-2-(hydroxyimino)-4-methyl-1,2-dihydropyridin-3-yl) methyl) 3,4,8,10-tetrahydroxy-2-(hydroxymethyl)-9-methoxy-2,3,4,4a-tetrahydropyrano [3,2- c] iso chromen-6 (10b H)-one, promptly ( E)-7-((6-ethanoyl-2-(oximido)-4-methyl isophthalic acid, 2-dihydropyridine-3-alkyl) methyl)-3,4,8,10-tetrahydroxy-2-(methylol)-9-methoxyl group-2,3,4, the 4a-tetrahydrochysene [3,2- c] method of different chromene-6 (10b H)-ketone; After transforming end; Remove mycelium; Supernatant is used organic solvent extraction, extraction liquid through concentrate, silica gel column chromatography separates, Sephadex purifying or again through the HPLC preparation, can obtain containing the solution of this converted product; Again with this solution through concentrating or after cryodrying handles available converted product crystal or powder.Utilize penicillium ( PenicilliumSpp.) bacterial strain conversion Bergeninum is seen Fig. 1 for the converted product synoptic diagram.
Above-mentioned penicillium with Bergeninum structural modification specific enzymes vigor ( PenicilliumSpp.) bacterial classification, available from Chinese common micro-organisms preservation administrative center, for penicillium crustaceum ( Penicillium crustosum, CGMCC numbering 3.7142).
The above-mentioned nutritive substance that is used for the nutrient solution of fermentation culture has no particular limits, and can be any nutrient media that is suitable for this microorganism growth, like the fungi fermentation nutrient solution.
Above-mentioned microbial fermentation culture condition with Bergeninum structural modification specific enzymes vigor is: 28 ℃, 180r/min shakes bottle vibration or ventilation or stirs ventilation or pipeline ventilation oxygen-supplying, cultivates 48 hours.
The substrate solution of the above-mentioned microbial transformation that is used for Bergeninum specific enzymes structural modification can be prepared in organic phase, and optional organic solvent is a DMSO 99.8MIN., or other organic solvents that pair cell toxicity is less, solubleness is bigger.
Above-mentioned transformation fermentation liquid separates with mycelium, adopts the method for filtering separation or spinning.
The microbial transformation condition of above-mentioned Bergeninum specific enzymes structural modification: invert point is 28 ℃; 180r/min shakes bottle vibration ventilation or stirs ventilation or pipeline ventilation oxygen-supplying; 96 ~ 120 hours reaction times; After transforming end, conversion fluid and mycelium are filtered or centrifugal separating, can obtain to contain the conversion fluid of converted product.
But the organic solvent that in the above-mentioned solution that contains this Bergeninum converted product, adds ETHYLE ACETATE or any other extraction product, vibration, extraction, leave standstill or centrifugal layering after, extraction liquid after concentrating, process recrystallization, available converted product crystal; Perhaps extraction liquid is after concentrating; Through silica gel column chromatography separate, Sephadex purifying or prepare through HPLC again; Can obtain containing the solution of the plain converted product of Rhizome or herb of Purple Bergenia; The solution that will contain converted product again is through concentrating or after recrystallization or cryodrying handle the powder of available converted product or crystal (purity >=90%).
The fermented liquid HPLC of the HPLC figure of Bergeninum, the HPLC figure that does not add the blank fermented liquid of substrate Bergeninum, interpolation Bergeninum schemes, and sees Fig. 2, Fig. 3 and Fig. 4 respectively.
The Bergeninum converted product each item spectrum analysis result who makes is following:
The quasi-molecule negative ions peak that ESI MS provides the Bergeninum converted product is respectively m/ z507 ([M+H] + ) and 505 ([M-H] -), can know that its molecular weight is 506.The quasi-molecular ion peak that HRESI MS provides does m/ z[507.1398 M+H] +, m/ z[529.1432 M+Na] +With m/ z[1035.3194 2M+Na] +, the ESI MS spectrogram of Bergeninum converted product and HRESI MS spectrogram are seen Fig. 5 and Fig. 6 respectively.
At the Bergeninum converted product 1In the H NMR spectrum, δ H3.70 (3H, the spectral line of s) locating can be thought OCH 3On H-12; Two proton signal δ appear in low place H5.39 (1H, d, J=6.0Hz) and δ H5.68 (1H, d, J=5.4Hz) can think respectively that 3-OH and 4-OH go up the hydrogen proton signal.δ H4.88 (1H, t, J=4.8 Hz), 4.70 (1H, d, J=15.0 Hz), 3.92 (1H, d, J=15.0 Hz), 3.59 (1H, m), 3.21 (1H, m), 3.65 (1H, m), 3.85 (1H, t, J=9.6 Hz), 4.91 (1H, d, J=10.2 Hz), 3.40 (1H, m) with 3.83 (1H, d, J=9.6Hz) be distinctive signal or the hydrogen signal on the newly-increased fragment on the Bergeninum carbon glycosides.δ H2.08 (3H, (3H s) is the CH on the newly-increased fragment s) with 2.48 3Signal.δ H8.45 (1H is s) with 7.4 (1H s) possibly be the reactive hydrogen that is connected on the phenyl ring.
From the Bergeninum converted product 13C NMR spectrum, 135 ° of spectrums of 90 ° of DEPT and DEPT confirm in the compound five CH peak (δ on the carbon glycosides are arranged c70.4,72.5,73.6,79.6,81.4) and a CH 2Peak (δ c61.9), an OCH 3(δ c59.9), a CH peak (δ c130.4), a CH 2Peak (δ c19.3), two CH 3(δ c16.7,26.6).12 quaternary carbon peak (δ c111.6,113.7,114.9,116.3,118.6,122.3,138.8,145.0,149.7,161.3,164.1,202.3), δ wherein c202.3 be the carbon peak on the carbonyl.
In conjunction with the Bergeninum converted product 1H NMR with 13The data of hydrogen and carbon among the C NMR confirm that tentatively converted product is the verivate of Bergeninum, and its elementary composition and molecular formula are C 23H 26O 11N 2The Bergeninum converted product 1H NMR, 1390 ° of C NMR and DEPT, 135 ° of collection of illustrative plates of DEPT are seen Fig. 7, Fig. 8 and Fig. 9, Figure 10 respectively.
The Bergeninum converted product 1H- 1H COSY spectrum shows δ H5.39 → δ H3.21, δ H5.68 → δ H3.65 locate coupled relation, δ H3.21 be respectively H-3 and H-4 with the spectral line at 3.65 places; By δ H3.21 → δ H3.65 → δ H3.85 coupled relation, δ H3.85 the spectral line of locating is H-4a; By δ H3.65 → δ H3.85 → δ H4.91 coupled relation, δ H3.85 the spectral line of locating is H-10b.By δ H3.65 → δ H3.21 → δ H3.59 coupled relation can be known δ H3.59 the spectral line of locating is H-2; By δ H3.42 → δ H3.59 and δ H3.85 → δ H3.59 coupled relation, δ H3.42 and the spectral line at 3.85 places is H-11.Converted product 1H- 1H COSY collection of illustrative plates is seen Figure 11.
In the hsqc spectrum of Bergeninum converted product, H-2, H-3, H-4, H-4a, H-10b, the relevant carbon peak of H-11 is δ c81.4,70.4,73.6,79.6,72.5,61.9, can confirm as C-2, C-3, C-4, C-4a, C10b, C-11.Converted product HSQC collection of illustrative plates is seen Figure 12.
In conjunction with 1H NMR, 13C NMR, DEPT 90, the characteristic of DEPT 135 spectrum and 1H- 1Correlationship among H COSY and the HSQC can be confirmed δ H3.59 (δ c81.4), δ H3.21 (δ c70.4), δ H3.65 (δ c73.6), δ H3.85 (δ c79.6), δ H4.91 (δ c72.5), δ H3.92,4.70 (δ c19.3), δ H3. 70 (δ c59.9) be respectively C-2, C-3, C-4, C-4a, C10b, C-11, the H that the C-12 position is connected.
In the HMBC spectrum, quaternary carbon peak δ c138.8 relevant with H-12, ownership δ c138.8 be C-9; δ H8.45 with C-9, quaternary carbon peak δ c145.1 114.9 is relevant, can belong to δ H8.45 be the H on the 10-OH, δ c145.1 be C-10, δ c114.9 be C-10a; There is C-2 at the carbon peak relevant with H-10b, C-4, C-4a, C-9, C-10, C-10a, δ c118.6,122.3,149.7,164.1, can belong to quaternary carbon peak δ c118.6 be C-6, δ c122.3 be C-7, δ c149.7 be C-8.Converted product section H MBC collection of illustrative plates A and B see Figure 13, Figure 14 and Figure 15 respectively.
Carrying out comprehensively 1H NMR with 13On the basis that C NMR detects, through DEPT, 1H- 12D NMR technology such as H COSY, HSQC and HMBC are all to this compound 1H with 13C NMR signal carries out detailed ownership (seeing table 1).
The NMR data of table 1 converted product and spectrum peak ownership (DMSO-d 6, 600 MHz)
Figure 2011103084787100002DEST_PATH_IMAGE001
The present invention use microbial conversion process to obtain the Bergeninum converted product and combine utilization MS, 1D NMR and 2D NMR technology ( 1H- 1H COSY, HSQC HMBC) has belonged to it to the Bergeninum converted product 1H with 13C NMR signal.And called after:
( E)-7-((6-acetyl-2-(hydroxyimino)-4-methyl-1,2-dihydropyridin-3-yl)methyl)3,4,8,10-tetrahydroxy-2-(hydroxymethyl)-9-methoxy-2,?3,?4,?4a-tetrahydropyrano?[3,2- c]?isochromen-6?(10b?H)-one。
That is: ( E)-7-((6-ethanoyl-2-(oximido)-4-methyl isophthalic acid, 2-dihydropyridine-3-alkyl) methyl)-3,4,8,10-tetrahydroxy-2-(methylol)-9-methoxyl group-2,3,4, the 4a-tetrahydrochysene [3,2- c] different chromene-6 (10b H)-ketone.
This converted product has following characteristics: 1. be a new compound; 2. introduce 2 external source N atoms, on side chain, formed a pyridine heterocycle that contains oximido and ethanoyl; 3. except a fragrant hydrogen of Bergeninum was substituted, rest part comprised structure parent nucleus, all side-chain radicals, chirality etc., does not all change.
The anti-oxidant activity of converted product of the present invention:
Product of the present invention ( E)-7-((6-ethanoyl-2-(oximido)-4-methyl isophthalic acid, 2-dihydropyridine-3-alkyl) methyl)-3,4,8,10-tetrahydroxy-2-(methylol)-9-methoxyl group-2,3,4, the 4a-tetrahydrochysene [3,2- c] different chromene-6 (10b H)-ketone removing the ability of DPPH, the IC of Bergeninum 50Be 2.65 mg/ml, the IC of converted product 50Be 1.10 mg/ml.The TAC of converted product is 7.28 U/mg, and Bergeninum is 1.60 U/mg.
Aspect removing ultra-oxygen anion free radical activity, the result shows that both all do not have this activity.The result shows aspect tyrosinase inhibitory activity; Under three different concentration, 10 μ g/ml, 25 μ g/ml, 50 μ g/ml; The inhibiting rate of Bergeninum is respectively 61.58%, 64.75%, 68.62%, all than converted product 44.12%, 56.32%, the last 57.89%.Adopt Fe 2+-H 2O 2Inductive mouse liver even slurry model of oxidation investigation Bergeninum and converted product are to the inhibition of lipid peroxidation; Under three different concentration 0.01 mg/ml, 0.1 mg/ml, 1 mg/ml; The converted product all inhibiting rate than Bergeninum is high, but effect and not obvious.Other pharmacologically actives all have improvement in various degree, and technology is simple, and product purity is high, and water-soluble than the substrate raising, have good application prospects.
Description of drawings
Fig. 1 for utilize penicillium ( PenicilliumSpp.) bacterial strain conversion Bergeninum is the converted product synoptic diagram.
Fig. 2 is the HPLC figure (T of Bergeninum R=3.897min).
Fig. 3 is not for adding the blank fermented liquid HPLC figure of substrate Bergeninum, Bergeninum T R=3.808min, converted product T R=21.110min.
Fig. 4 is for adding the fermented liquid HPLC figure of Bergeninum.
Fig. 5 is a converted product ESI MS spectrogram.
Fig. 6 is the HR ESI MS spectrogram of converted product.
Fig. 7 is a converted product 1H NMR spectrogram.
Fig. 8 is a converted product 13C NMR spectrogram.
Fig. 9 is 90 ° of spectrograms of DEPT of converted product.
Figure 10 is 135 ° of spectrograms of DEPT of converted product.
Figure 11 is a converted product 1H- 1H COSY spectrogram.
Figure 12 is the hsqc spectrum figure of converted product.
Figure 13 is the section H MBC spectrogram A of converted product.
Figure 14 is the section H MBC spectrogram B of converted product.
Figure 15 is a converted product HMBC correlogram.
Embodiment
Employed in the present invention term only if other explanation is arranged, generally has the implication of those of ordinary skills' common sense.
Below in conjunction with concrete embodiment, and comparable data is described the present invention in further detail.Should be understood that these embodiment just in order to demonstrate the invention, but not limit scope of the present invention by any way.
In following embodiment, various processes and the method do not described in detail are ordinary methods as known in the art.The source of agents useful for same, trade(brand)name and be necessary to list its moity person show when occurring first that all used thereafter identical reagent is like no specified otherwise, and is all identical with the content of indicating first.
Embodiment 1:
Fungi fermentation substratum: glucose 20 g, yeast extract paste 5 g, peptone 5 g, NaCl 5 g, K 2HPO 45 g, zero(ppm) water 1000 mL, pH 6.5.
Pour 50mL fungi fermentation substratum into the 250mL triangular flask, behind 121 ℃ of high pressure steam sterilizations, the access penicillium crustaceum ( Penicillium crustosum, CGMCC numbering 3.7142) and bacterial strain, 28 ℃; The 180r/min shake flask fermentation was cultivated after 48 hours, stopped fermentation, and the adding massfraction is 2% Bergeninum DMSO solution in substratum; 28 ℃, after 96 hours, stop conversion reaction; Filter to remove mycelium, contain in the supernatant Bergeninum converted product ( E)-7-((6-ethanoyl-2-(oximido)-4-methyl isophthalic acid, 2-dihydropyridine-3-alkyl) methyl)-3,4,8,10-tetrahydroxy-2-(methylol)-9-methoxyl group-2,3,4, the 4a-tetrahydrochysene [3,2- c] different chromene-6 (10b H)-ketone.Supernatant with 50mL ethyl acetate extraction three times after, again through silica gel column chromatography (chloroform: behind methyl alcohol=10:1) and the Sephadex purifying, obtain purity up to 90% Bergeninum converted product ( E)-7-((6-ethanoyl-2-(oximido)-4-methyl isophthalic acid, 2-dihydropyridine-3-alkyl) methyl)-3,4,8,10-tetrahydroxy-2-(methylol)-9-methoxyl group-2,3,4, the 4a-tetrahydrochysene [3,2- c] different chromene-6 (10b H)-ketone.
Embodiment 2:
Fungi fermentation substratum: glucose 20 g, yeast extract paste 5 g, peptone 5 g, NaCl 5 g, K 2HPO 45 g, zero(ppm) water 1000 mL, pH 6.5.
Pour 50mL fungi fermentation substratum into the 250mL triangular flask, behind 121 ℃ of high pressure steam sterilizations, the access penicillium crustaceum ( Penicillium crustosum, CGMCC numbering 3.7142) and bacterial strain, 28 ℃; The 180r/min shake flask fermentation was cultivated after 48 hours, stopped fermentation, and the adding massfraction is 2% Bergeninum DMSO solution in substratum; 28 ℃, after 120 hours, stop conversion reaction; Filter to remove mycelium, contain in the supernatant Bergeninum converted product ( E)-7-((6-ethanoyl-2-(oximido)-4-methyl isophthalic acid, 2-dihydropyridine-3-alkyl) methyl)-3,4,8,10-tetrahydroxy-2-(methylol)-9-methoxyl group-2,3,4, the 4a-tetrahydrochysene [3,2- c] different chromene-6 (10b H)-ketone.Supernatant with 50mL ethyl acetate extraction three times after, again through silica gel column chromatography (chloroform: behind methyl alcohol=10:1) and the Sephadex purifying, obtain purity up to 90% Bergeninum converted product ( E)-7-((6-ethanoyl-2-(oximido)-4-methyl isophthalic acid, 2-dihydropyridine-3-alkyl) methyl)-3,4,8,10-tetrahydroxy-2-(methylol)-9-methoxyl group-2,3,4, the 4a-tetrahydrochysene [3,2- c] different chromene-6 (10b H)-ketone.
Embodiment 3:
Fungi fermentation substratum: glucose 20 g, yeast extract paste 5 g, peptone 5 g, NaCl 5 g, K 2HPO 45 g, zero(ppm) water 1000 mL, pH 6.5.
Pour 50mL fungi fermentation substratum into the 250mL triangular flask, behind 121 ℃ of high pressure steam sterilizations, the access penicillium crustaceum ( Penicillium crustosum, CGMCC numbering 3.7142) and bacterial strain, 28 ℃; The 180r/min shake flask fermentation was cultivated after 48 hours, stopped fermentation, and the adding massfraction is 2% daidzein DMSO solution in substratum; 28 ℃, after 96 hours, stop conversion reaction; Filter to remove mycelium, contain in the supernatant Bergeninum converted product ( E)-7-((6-ethanoyl-2-(oximido)-4-methyl isophthalic acid, 2-dihydropyridine-3-alkyl) methyl)-3,4,8,10-tetrahydroxy-2-(methylol)-9-methoxyl group-2,3,4, the 4a-tetrahydrochysene [3,2- c] different chromene-6 (10b H)-ketone.Supernatant with 50mL ethyl acetate extraction three times after, again after HPLC preparative column preparation, obtain purity up to 90% Bergeninum converted product ( E)-7-((6-ethanoyl-2-(oximido)-4-methyl isophthalic acid, 2-dihydropyridine-3-alkyl) methyl)-3,4,8,10-tetrahydroxy-2-(methylol)-9-methoxyl group-2,3,4, the 4a-tetrahydrochysene [3,2- c] different chromene-6 (10b H)-ketone.
The condition that partly prepares for the first time liquid phase: chromatographic column: Zorbax Eclipse XDB-C18 (4.6 mm * 150 mm, 5 μ m), detect wavelength: 275 nm; Sample size: 20 μ L; Moving phase: methyl alcohol: water=20:80, flow velocity: 1.0 mL/min, column temperature: 25 ℃; Time: 30 min, back operation: 5 min.The condition that partly prepares for the second time liquid phase is identical with the condition that partly prepares for the first time liquid phase.
Embodiment 4:
Fungi fermentation substratum: glucose 20 g, yeast extract paste 5 g, peptone 5 g, NaCl 5 g, K 2HPO 45 g, zero(ppm) water 1000 mL, pH 6.5.
Pour 50mL fungi fermentation substratum into the 250mL triangular flask, behind 121 ℃ of high pressure steam sterilizations, the access penicillium crustaceum ( Penicillium crustosum, CGMCC numbering 3.7142) and bacterial strain, 28 ℃; The 180r/min shake flask fermentation was cultivated after 48 hours, stopped fermentation, and the adding massfraction is 2% daidzein DMSO solution in substratum; 28 ℃, after 120 hours, stop conversion reaction; Filter to remove mycelium, contain in the supernatant Bergeninum converted product ( E)-7-((6-ethanoyl-2-(oximido)-4-methyl isophthalic acid, 2-dihydropyridine-3-alkyl) methyl)-3,4,8,10-tetrahydroxy-2-(methylol)-9-methoxyl group-2,3,4, the 4a-tetrahydrochysene [3,2- c] different chromene-6 (10b H)-ketone.Supernatant with 50mL ethyl acetate extraction three times after, again after HPLC preparative column preparation, obtain purity up to 90% Bergeninum converted product ( E)-7-((6-ethanoyl-2-(oximido)-4-methyl isophthalic acid, 2-dihydropyridine-3-alkyl) methyl)-3,4,8,10-tetrahydroxy-2-(methylol)-9-methoxyl group-2,3,4, the 4a-tetrahydrochysene [3,2- c] different chromene-6 (10b H)-ketone.
The condition that partly prepares for the first time liquid phase: chromatographic column: Zorbax Eclipse XDB-C18 (4.6 mm * 150 mm, 5 μ m), detect wavelength: 275 nm; Sample size: 20 μ L; Moving phase: methyl alcohol: water=20:80, flow velocity: 1.0 mL/min, column temperature: 25 ℃; Time: 30 min, back operation: 5 min.The condition that partly prepares for the second time liquid phase is identical with the condition that partly prepares for the first time liquid phase.
Embodiment 5:
Fungi fermentation substratum: glucose 20 g, yeast extract paste 5 g, peptone 5 g, NaCl 5 g, K 2HPO 45 g, zero(ppm) water 1000 mL, pH 6.5.
Pour 50mL fungi fermentation substratum into the 250mL triangular flask, behind 121 ℃ of high pressure steam sterilizations, the access penicillium crustaceum ( Penicillium crustosum, CGMCC numbering 3.7142) and bacterial strain, 28 ℃; The 180r/min shake flask fermentation was cultivated after 48 hours, stopped fermentation, and the adding massfraction is 2% Bergeninum DMSO solution in substratum; 28 ℃, after 96 hours, stop conversion reaction; Filter to remove mycelium, contain in the supernatant Bergeninum converted product ( E)-7-((6-ethanoyl-2-(oximido)-4-methyl isophthalic acid, 2-dihydropyridine-3-alkyl) methyl)-3,4,8,10-tetrahydroxy-2-(methylol)-9-methoxyl group-2,3,4, the 4a-tetrahydrochysene [3,2- c] different chromene-6 (10b H)-ketone.Supernatant with 50mL ethyl acetate extraction three times after, cultivate through crystal under the room temperature, obtain purity up to 95% Bergeninum converted product ( E)-7-((6-ethanoyl-2-(oximido)-4-methyl isophthalic acid, 2-dihydropyridine-3-alkyl) methyl)-3,4,8,10-tetrahydroxy-2-(methylol)-9-methoxyl group-2,3,4, the 4a-tetrahydrochysene [3,2- c] powder or the crystal of different chromene-6 (10b H)-ketone.
Embodiment 6:
Fungi fermentation substratum: glucose 20 g, yeast extract paste 5 g, peptone 5 g, NaCl 5 g, K 2HPO 45 g, zero(ppm) water 1000 mL, pH 6.5.
Pour 50mL fungi fermentation substratum into the 250mL triangular flask, behind 121 ℃ of high pressure steam sterilizations, the access penicillium crustaceum ( Penicillium crustosum, CGMCC numbering 3.7142) and bacterial strain, 28 ℃; The 180r/min shake flask fermentation was cultivated after 48 hours, stopped fermentation, and the adding massfraction is 2% Bergeninum DMSO solution in substratum; 28 ℃, after 120 hours, stop conversion reaction; Filter to remove mycelium, contain in the supernatant Bergeninum converted product ( E)-7-((6-ethanoyl-2-(oximido)-4-methyl isophthalic acid, 2-dihydropyridine-3-alkyl) methyl)-3,4,8,10-tetrahydroxy-2-(methylol)-9-methoxyl group-2,3,4, the 4a-tetrahydrochysene [3,2- c] different chromene-6 (10b H)-ketone.Supernatant with 50mL ethyl acetate extraction three times after, cultivate through crystal under the room temperature, obtain purity up to 95% Bergeninum converted product ( E)-7-((6-ethanoyl-2-(oximido)-4-methyl isophthalic acid, 2-dihydropyridine-3-alkyl) methyl)-3,4,8,10-tetrahydroxy-2-(methylol)-9-methoxyl group-2,3,4, the 4a-tetrahydrochysene [3,2- c] powder or the crystal of different chromene-6 (10b H)-ketone.
Product of the present invention ( E)-7-((6-ethanoyl-2-(oximido)-4-methyl isophthalic acid, 2-dihydropyridine-3-alkyl) methyl)-3,4,8,10-tetrahydroxy-2-(methylol)-9-methoxyl group-2,3,4, the 4a-tetrahydrochysene [3,2- c] different chromene-6 (10b H)-ketone have the ability, resistance of oxidation of the removing DPPH stronger than Bergeninum and to the inhibition effect of lipid peroxidation, be potential anti-inflammatory, anti-HIV, arrhythmia, neuroprotective, protect the liver the medicine with aspects such as multiple biological activitys such as immunomodulatory and pharmacological actions.
The invention is not restricted to these disclosed embodiments, the present invention will cover the scope described in the patent book, and the various modification of claim scope change with equivalence.

Claims (9)

1. utilizing penicillium spp to transform Bergeninum is a kind of method of special nitrogen containing derivative, and described method is:
The microbial strains that will have Bergeninum structural modification specific enzymes vigor is inoculated in the nutrient solution, carries out shake flask fermentation and cultivates; Ferment middle adds the substrate conversion liquid contain Bergeninum in fermented liquid, make Bergeninum through the method for microbial transformation carry out special enzyme structural modification become ( E)-7-((6-ethanoyl-2-(oximido)-4-methyl isophthalic acid, 2-dihydropyridine-3-alkyl) methyl)-3,4,8,10-tetrahydroxy-2-(methylol)-9-methoxyl group-2,3,4, the 4a-tetrahydrochysene [3,2- c] different chromene-6 (10b H)-ketone; After transform finishing, remove mycelium, supernatant use organic solvent extraction, extraction liquid after concentrated, process recrystallization, available converted product crystal; Perhaps extraction liquid is after concentrating; Through silica gel column chromatography separate, Sephadex purifying or prepare through HPLC again; Can obtain containing the solution of this converted product, again with this solution through concentrating or after crystallization or cryodrying handle available converted product crystal or powder.
It is 2. according to claim 1 that to utilize penicillium spp to transform Bergeninum be a kind of method of special nitrogen containing derivative, it is characterized in that described microbial strains with Bergeninum structural modification specific enzymes vigor be penicillium ( PenicilliumSpp.) fungi, or any mikrobe or mixing microorganisms that makes Bergeninum carry out the specific enzymes structural modification.
3. said to utilize penicillium spp to transform Bergeninum be a kind of method of special nitrogen containing derivative according to claim 2, it is characterized in that described penicillium ( PenicilliumSpp.) fungi, for the bacterial strain penicillium crustaceum ( Penicillium crustosum) CGMCC numbering 3.7142.
4. said to utilize penicillium spp to transform Bergeninum be a kind of method of special nitrogen containing derivative according to claim 1; It is characterized in that the described nutrient solution that is used for fermentation culture, is that fungi fermentation nutrient solution or other any suitable penicillium fungies or other can make Bergeninum carry out the nutrient media of the mikrobe of specific enzymes structural modification.
It is 5. according to claim 1 that to utilize penicillium spp to transform Bergeninum be a kind of method of special nitrogen containing derivative; It is characterized in that the described conversion fluid that contains Bergeninum that in fermented liquid, adds; Being used for the substrate solution of the microbial transformation of Bergeninum specific enzymes structural modification, is in organic phase, to prepare; Organic phase is a DMSO 99.8MIN., or other organic solvents that pair cell toxicity is less, solubleness is bigger.
It is 6. according to claim 1 that to utilize penicillium spp to transform Bergeninum be a kind of method of special nitrogen containing derivative; It is characterized in that said transformation fermentation reaction conditions is: the invert point scope is at 28 ~ 30 ℃; 160 ~ 180 r/min shake bottle vibration ventilation or stir ventilation or pipeline ventilation oxygen-supplying, and 96 ~ 120 hours reaction times is after conversion finishes; Conversion fluid and mycelium are filtered or centrifugal separating, can obtain to contain the conversion fluid of Bergeninum converted product.
7. according to claim 6ly utilize penicillium spp to transform Bergeninum to be a kind of method of special nitrogen containing derivative, to it is characterized in that said fermentation conversion fluid separates with mycelium, adopt the method for filtering separation or spinning; Supernatant is used organic solvent extraction.
It is 8. according to claim 7 that to utilize penicillium spp to transform Bergeninum be a kind of method of special nitrogen containing derivative; It is characterized in that the organic solvent that described extraction contains the conversion fluid of Bergeninum converted product is an ETHYLE ACETATE, but or the organic solvent of any other extraction product.
9. according to claim 8ly utilize penicillium spp to transform Bergeninum to be a kind of method of special nitrogen containing derivative, to it is characterized in that described extraction liquid, after concentrating, pass through recrystallization, available converted product crystal; Perhaps extraction liquid separates the Sephadex purifying through silica gel column chromatography after concentrating; Or prepare through HPLC again; Can obtain containing the solution of the plain converted product of Rhizome or herb of Purple Bergenia, again with this solution through concentrate or crystallization or cryodrying processing after, can obtain the powder or the crystal of converted product.
CN2011103084787A 2011-10-12 2011-10-12 Method for converting bergenin into special nitrogenous derivative by using penicillium Pending CN102337308A (en)

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* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2014188440A1 (en) * 2013-05-24 2014-11-27 Council Of Scientific & Industrial Research Tetrahydro-2h-pyrano[3,2-c]isochromene-6-ones and analogs for the treatment of inflammatory disorders
CN106084461A (en) * 2016-06-27 2016-11-09 高大元 A kind of method that high-polarity polypropylene resin material is prepared in Rhizoma Seu Herba Bergeniae modification
CN113200989A (en) * 2021-05-18 2021-08-03 云南民族大学 Preparation method and application of chromone alkaloid compound

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Publication number Priority date Publication date Assignee Title
WO2014188440A1 (en) * 2013-05-24 2014-11-27 Council Of Scientific & Industrial Research Tetrahydro-2h-pyrano[3,2-c]isochromene-6-ones and analogs for the treatment of inflammatory disorders
CN106084461A (en) * 2016-06-27 2016-11-09 高大元 A kind of method that high-polarity polypropylene resin material is prepared in Rhizoma Seu Herba Bergeniae modification
CN113200989A (en) * 2021-05-18 2021-08-03 云南民族大学 Preparation method and application of chromone alkaloid compound

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