CN101693913A - Process for preparing rusco monoglycosides high-efficiently - Google Patents
Process for preparing rusco monoglycosides high-efficiently Download PDFInfo
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- CN101693913A CN101693913A CN200910233989A CN200910233989A CN101693913A CN 101693913 A CN101693913 A CN 101693913A CN 200910233989 A CN200910233989 A CN 200910233989A CN 200910233989 A CN200910233989 A CN 200910233989A CN 101693913 A CN101693913 A CN 101693913A
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Abstract
The invention relates to a process for preparing rusco monoglycosides, which utilizes 25(R/S) rusco as a substrate and utilizes bacterial strain with storage number of NRRL1086. The substrate and the bacterial strain are cultivated for 4-7 days in a culture medium and then stop reacting, fermentation liquor is extracted by 3-5 times via organic solvent, extractum is obtained by concentrating extract liquor, and an objective compound is obtained by separation through chromatographic separation of silicagel columns. Further, the process optimizes cultivated conditions and screens glycosylated accelerators so as to increase convert ratio. The process can high-efficiently prepare the rusco monoglycosides with the convert ratio higher than 70%.
Description
Technical field
The present invention relates to 25 (R, S) 1-O-β-D-glucosyl Shandong this can efficiently, preparation method fast, belong to field of biological pharmacy.
Background technology
Steroidal saponin (steroidal saponins) is the important natural glucosides of a class, is distributed widely in higher plant and some marine organisms, and be the important drug resource of a class.Experimentation on animals proves: natural or synthetic saponin(e can suppress the absorption of cholesterol and reduce content of cholesterol in the serum; Platelet aggregation and thrombosis are the major reasons of ischemia heart and brain damage, and steroidal saponin can anticoagulant.Structure activity study shows that the pharmaceutical activity of steroidal body saponin(e is not only relevant with aglycon, and with sugar chain closer the contact is arranged partly.Configuration of monose composition, mode of connection, glycosidic link etc. all directly influences the activity of steroidal saponin in the sugar chain, and steroidal saponin has stronger activity than its aglycon.Multinomially studies show that the variation of the sugar chain with cell recognition ability can regulate and control the biologic activity of steroidal saponin.
The acquisition of steroidal saponin mainly separates from natural product, but is difficult to a large amount of a certain specific saponin(es of acquisition.Can obtain a certain specific saponin(e in a large number by chemosynthesis.But which kind of approach chemosynthesis takes all exist a common shortcoming in actually operating: loaded down with trivial details protection and deprotection work and repeatedly intermediate purification step and productive rate are very low; thereby cause synthetic cost higher, be unfavorable for suitability for industrialized production.Adopt glycosylation strategy synthesizing steroid saponin(e at random,, shortcomings such as the wayward and product separation and purification difficulty of level of glycosylation are arranged also though save loaded down with trivial details protection and deprotection work.
How to overcome the shortcoming of chemosynthesis, efficient, environmental protection, the cheap a large amount of steroidal saponin of acquisition be the steroid saponin drug development and produce in need one of problem of solution badly.Adopting microbial transformation that exogenous compounds is carried out structure repaiies to obtain a certain specific, the more difficult synthetic target compound of chemosynthesis; three-dimensional arrangement selectivity with height; can single-minded ground catalysis specific reaction, need not loaded down with trivial details protection and deprotection is operated; advantages such as tool is efficient, environmental protection; being a kind of effective ways that the natural radioactivity compound carries out structural modification, is the important channel that lead compound is found.
Ruscogenin (Ruscogenin), have another name called the ruscus aculeatus aglycon, be from the ruscus aculeatus (Ruscus aculeatusL.) that is grown in Mediterranean Zone, to separate a kind of steroid sapogenin that obtains the earliest, also be the main sapogenin of enriching yin Chinese medicine in the tuber of dwarf lilyturf commonly used, externally can obviously suppress sticking of Buddhist ripple ester inductive ECV304 cell and HL260 cell.But the water-insoluble of ruscogenin has influenced the performance of its drug effect.
But the introducing glycosyl is converted into its corresponding glucosides with ruscogenin and can obtains water-soluble better, active higher this class anti-inflammatory of Shandong, the lead compound of anti-cardiovascular disease on the side chain of ruscogenin.
Adopt above-mentioned two-steps tissue culture method, the yield that obtains rusco monoglycosides is at 10%-75%.
Summary of the invention
The present invention wants the technical solution problem to be: overcome the low deficiency of existing preparation rusco monoglycosides method transformation efficiency, a kind of method of preparing rusco monoglycosides high-efficiently is provided, its ruscogenin is a substrate, utilize Gliocladium deliquescens NRRL1086 to be the conversion instrument, and culture condition is optimized, screens glycosylated promotor to improve transformation efficiency.
In order to solve above technical problem, the method for a kind of preparing rusco monoglycosides high-efficiently of the present invention, it is that the bacterial strain of NRRL1086 carries out bio-transformation to ruscogenin and obtains rusco monoglycosides with preserving number, the steps in sequence of described bio-transformation comprises:
A, obtaining liq substratum;
B, with the NRRL1086 inoculation to liquid nutrient medium, in shaking table, cultivate (12～48 hours) and obtain seed liquor;
C, seed liquor sent out be forwarded to liquid nutrient medium and continue cultivate, obtain fermented liquid;
But d, add in the fermented liquid and be dissolved with Lu Si acetone soln and carbon source, and cultivated altogether 4～7 days, described carbon source be in glucose, sucrose, maltose, the starch one or more;
E, fermented liquid be with organic solvent A extraction 3～6 times, and described organic solvent A is a kind of in ethyl acetate, chloroform, methylene dichloride, ethyl formate, butylacetate, toluene, hexane, the propyl carbinol;
F, concentrated extract get medicinal extract, separate by silica gel column chromatography, obtain target compound--rusco monoglycosides.
Described liquid nutrient medium contains 10%～30% potato decoction liquor, KH
2PO
4, MgSO
4, vitamins B
1, NaCl, KNO
3And FeSO
4, the pH=5.0 of liquid nutrient medium～7.0;
The present invention also optimizes liquid nutrient medium, in described liquid nutrient medium, in 1000 milliliters of potato decoction liquor, contains 0.1～10 gram KH
2PO
4, 0.1～5 the gram MgSO
4, 0.01～1 the gram vitamins B
1, 0.1～10 the gram NaCl, 0.1～10 the gram KNO
3, 0.1～7 the gram FeSO
4
By 1000 milliliters of 20% potato decoction liquor, KH
2PO
40.1～0.5 gram; MgSO
40.05～0.5 gram; NaCl 0.5～0.8g, KNO
32.0～4.0g, FeSO
40.005～0.03g; Carbon source 0.5～2.5 gram.
Bacterial classification activates earlier before cultivation, and soak time 20～30h with the activity of raising bacterial classification, and then improves transformation efficiency.
Utilize the inventive method efficiently to prepare rusco monoglycosides fast, its transformation efficiency can reach more than 70%.
Description of drawings
The present invention is further illustrated below in conjunction with accompanying drawing.
Fig. 1 is application of sample time and conversion rate curve figure.
Fig. 2 is fermentation time and conversion rate curve figure.
Fig. 3 is for adding the influence curve figure of glucose to transforming.
Fig. 4 measures the glucose curve figure that adds different mass for HPLC-ELSD.
Fig. 5 is for adding the influence curve figure of sucrose to transforming.
Fig. 6 HPLC-ELSD measures the glucose curve figure that adds different mass.
Fig. 7 enzyme inhibitors SDS and imidazoles are to the influence curve figure of transformation efficiency.
Embodiment
But this can become 1-O-β-this bacterial classification of D-glucosyl group Shandong in order to seek suitable conversion Shandong, and the present invention has screened a plurality of bacterial classifications altogether, found that, preserving number is that the NRRL1086 bacterial classification can have stronger conversion capability (seeing Table .1) to Lu Si.
Screen used liquid culture: the fresh peeling potato of 200 grams adds boiling water 500ml and boils the 15min after-filtration, and filtrate adds glucose 20g, KH
2PO
43.0g, MgSO
40.75g, Vb110.0mg, NaCl 0.5g, KNO
33.0g, FeSO
4The 10mg adding distil water is settled to 1.0L, adds NaOH or HCl adjust pH to 6.0, every bottle of packing 30ml of 150ml Erlenmeyer flask liquid nutrient medium, 121 ℃, the 0.15MPa 20min that sterilizes.
During strain screening, preserve the used slant medium of bacterial classification and be and add 1.5% agar at the aforesaid liquid substratum, make behind the every pipe 5ml of 15ml tool plug test tube, 121 ℃, 0.15MPa sterilization 20min cooling.
The preservation of bacterial classification and activation: actication of culture adopts two step activation methods, referring to: Nair, M.S.R., and Basile, D.V.Bioconversion of artenium B toartmisinn. natural product magazine (Jounal of Natural Products) 1993,56 (9): 1559.Elder generation in liquid nutrient medium, behind 28 ℃ of 180r/min rotating and culturing 24～48h, transfers bacterial classification inoculation in another liquid nutrient medium, and inoculum size 1-5% (V/V) is with application of sample behind the condition cultivation 24h.
The preparation of sample and application of sample: 25 (R/S) Lu Si dissolves in acetone, is made into the 5.0mg/ml solution for standby; Every bottle through two the step activatory bacterium liquid (30ml) add the 1.0ml sample solution, but make every bottle of bacterium liquid contain Lu Si 5mg.Behind condition cultivation 120～144h, stopped reaction is filtered thalline, the ethyl acetate extraction of fermented liquid usefulness equivalent 3～5 times, combining extraction liquid, recovery ethyl acetate; Obtain extracting medicinal extract, thalline is with extraction using alcohol 3 times, and united extraction liquid reclaims ethanol, obtains extracting medicinal extract, with two kinds of medicinal extract and also, adds a little acetic acid ethyl dissolution reserve thin layer and identifies usefulness.
Blank: blank is established positive control (substrate+substratum), negative control (substratum+bacterium), solvent control (substratum+bacterium+ethanol), and cultivation and extraction conditions are the same.
The thin layer of converted product is identified:
The thin layer condition:
1. thin layer plate: the silica gel G plate that 0.7% CMC-Na makes.
2. developping agent: chloroform: methyl alcohol (8: 1) ascending method is launched.
3. developer: 10% ethanol solution of sulfuric acid.
Operation:
Conversion extract and blank extract are put respectively in thin layer plate, fully launch back taking-up nature and dry, and spray the sulfuric acid ethanol with 10%, hair dryer heating colour developing.Conversion results sees Table 1.
But this The selection result of table 1 microbial transformation Shandong
From The selection result, G.deliquescens.NRRL-1086, A.coerulea and N.sp.NRRL.5646 can have activity of conversion to Lu Si, wherein, G.deliquescens, through measuring and calculating, transformation efficiency can reach 20%; Though A.coerulea is glycosylation conversion but transformation efficiency is lower.And N.sp.NRRL.5646 is not the glycosylation conversion.Therefore, the present invention selects bacterial strain G.deliquescens NRRL.1086 for use, can be raw material with 25 (R/S) Lu Si and prepares 25 (R/S) 1-O-β-D-glucosyl group Shandong this can.
The G.deliquescens bacterial strain is inoculated in the liquid nutrient medium by the inclined-plane, puts 28 ℃, rotating speed is to cultivate in the shaking table of 180r/m to become seed liquor in 24 hours, and seed liquor continues to cultivate 24 hours after being forwarded to fermented liquid, adds conversion of substrate; Continue to cultivate after 144 hours and stop catalyzed reaction, filter thalline, fermented liquid is with the ethyl acetate extraction of equivalent 5 times, combining extraction liquid, reclaims ethyl acetate, obtains extracting medicinal extract, and medicinal extract is column chromatography for separation behind silica gel mixed sample; With the chloroform-methanol is elution system.Converted product obtains the Powdered converted product of pale through recrystallizing methanol.
Pharmacological testing proves that structural formula of the present invention is that I and II compound have excellent anti-inflammatory, anti-cardiovascular disease effect, below is test method and result: this can study 25 (R/S) 1-O-β-D-glucosyl group Shandong in anti-mouse cardiac muscle Hypoxia and ischemia effect
Method: adopt Racemic isoproterenol to cause chmice acute myocardial anoxia model and investigate 25 (R/S) 1-O-β-D-glucosyl group Shandong this can be to the influence of mouse survival time, and the preliminary observation amount, imitate time-effect relationship; Adopting Pituitrin to cause the chmice acute myocardial infarction and ischemia model investigates the two and share influence to related biochemical indicator.
Result: 25 (R/S) but 1-O-β-this single of D-glucosyl group Shandong give, can obviously prolong the myocardial anoxia mouse hypoxia tolerance survival time, remarkable drug effect is promptly arranged about 1h, onset is more slow, the effect hold time longer; And can improve myocardial ischemia mouse related biochemical indicator, comprise reduction serum mda (MDA) content and creatine kinase (CK), serum lactic dehydrogenase (LDH) vigor, reduce MDA, Ca2+, NO content in the heart tissue, rising superoxide dismutase (SOD) vigor (seeing Table 2).
Conclusion: this can have obvious provide protection to acute myocardial anoxia ischemic 1-O-β-D-glucosyl group Shandong, and it is more slow to have an onset, acts on persistent characteristics.
This can cause the influence of myocardial ischemia mouse cardiac muscle biochemical indicator to Pi t table 2 25 (R/S) 1-O-β-D-glucosyl group Shandong
(.x±s，n＝10)
The preliminary study of 25 (R/S) 1-O-β-this Anhydroalkannin analgesic activities of D-glucosyl group Shandong
Method: employing acetic acid causes mouse peritoneal capillary permeability elevation model and carrageenin causes mouse pedal swelling model, investigates its anti-inflammatory action; Employing acetic acid causes the mouse writhing model and thermal stimulus causes pain model, investigates its analgesic activities, and compare with the former side of LIUSHEN WAN.
The result: 25 (R/S) 1-O-β-D-glucosyl group Shandong this can 16,32, three dosage groups of 64mg/kg single gastric infusion all can suppress the rising that acetic acid causes the mouse peritoneal capillary permeability significantly; Wherein, middle and high dosage group can obviously suppress the mouse pedal swelling that carrageenin causes, injection back 3h reaches the effect peak, and can continue until more than the 5h.Three dosage groups all significantly reduce the mouse writhing number of times of acetic-acid induced; 0.5h behind the middle and high dosage group single-dose gets final product the mouse threshold of pain that the significant prolongation thermal stimulus causes, acts on more than the sustainable 3h.
Conclusion: this can have significant anti-inflammatory, analgesic activities 25 (R/S) 1-O-β-D-glucosyl group Shandong.
Determining of application of sample time and fermentation time
Liquid nutrient medium substratum, every bottle of packing 30ml of 150ml Erlenmeyer flask liquid nutrient medium, 121 ℃, the 0.15MPa 20min that sterilizes.Bacterial classification NRRL.1086 is seeded to liquid nutrient medium by 4 ℃ solid inclined-plane.Put 28 ℃, rotating speed is to cultivate in the shaking table of 180r/m to become seed liquor in 24 hours, and seed liquor is forwarded to continue to cultivate after 1,2,3,4,5,6,7 days after the fermented liquid and adds 1ml 5mg/ml 25 (R/S) but the Lu Si acetone soln; Continue to cultivate after 144 hours and stop catalyzed reaction, fermented liquid is with the ethyl acetate extraction of equivalent 5 times, combining extraction liquid, reclaims ethyl acetate, obtains extracting medicinal extract, and HPLC-ELSD measures, and conversion results is seen Fig. 1.
With above-mentioned operation, but add this acetone soln of 1ml 5mg/ml Shandong behind the switching 24h, cultivate 1,2,3,4,5,6,7,8 respectively, sampling after 9 days, ethyl acetate extraction, HPLC-ELSD measures the conversion results (see figure 2)
As seen NRRL.1086 transforms this back 24h application of sample of can transferring of Shandong, and fermentation 144～168h sampling transformation efficiency is higher.
Determining of glycosylation promotor
Glucose
Operation is with " determining of application of sample time and fermentation time " part, but in application of sample, adding 0.5,1.5,2.5,4.0 gram glucose the results are shown in Figure 3.Add glucose and can significantly improve transformation efficiency, but addition is when reaching 1.5 gram/bottles, transformation efficiency can reach 67.64%, be when not adding glucose more than three times.But when glucose addition during greater than 1.5 gram/bottles, transformation efficiency sharply descends.
Among Fig. 4,1 refers to the substratum blank; 2 refer to substratum+5mg Shandong, and this can; 3 refer to substratum+5mg Shandongs this can+0.5g glucose; 4 refer to substratum+5mg Shandongs this can+1.5g glucose; 5 refer to substratum+5mg Shandongs this can+2.5g glucose; 6 refer to substratum+5mg Shandongs this can+4.0g glucose; A refers to the substrate Shandong, and this can; B refers to glycation product 1-O-β-D-glucosyl group Shandong, and this can.
Sucrose
Operation is with " determining of application of sample time and fermentation time " part, but in application of sample, adding 0.5,1.5,2.5,4.0 gram sucrose the results are shown in Figure 5.Add sucrose and can improve transformation efficiency, but addition is when reaching 1.5 gram/bottles, transformation efficiency can reach 67.64%, be when not adding glucose more than three times.But when glucose addition during greater than 1.5 gram/bottles, transformation efficiency sharply descends, and when the sucrose addition reaches 4.0 gram/bottles, transforms and almost stops.
Among Fig. 6,1 refers to the substratum blank; 2 refer to substratum+5mg Shandong, and this can; 3 refer to substratum+5mg Shandongs this can+0.5g sucrose; 4 refer to substratum+5mg Shandongs this can+1.5g sucrose; 5 refer to substratum+5mg Shandongs this can+2.5g sucrose; 6 refer to substratum+5mg Shandongs this can+4.0g sucrose; A refers to the substrate Shandong, and this can; B refers to glycation product 1-O-β-D-glucosyl group Shandong, and this can.Enzyme inhibitors
Operation is " determining of application of sample time and fermentation time " part together, but in application of sample, adds an amount of glycosyl transferase inhibitor imidazoles and glycosidase inhibitor sodium lauryl sulphate (SDS), and HPLC-ELSD detects conversion results (Fig. 7).This can have stronger restraining effect to imidazoles to NRRL.1086 glycosylation Shandong, every bottle add 4.5mM imidazoles 1ml almost can stop fully 1086 couples of Lu Si can conversion; The effect of SDS is opposite, and to transforming not only unrestraint effect, situation transforms on the contrary, every bottle of SDS that adds 1ml 17.5mM, and visible transformation efficiency is increased to more than 70%.
Among Fig. 7,1 refers to the substratum blank; 2 refer to substratum+5mg Shandong, and this can; 3 refer to substratum+imidazoles; 4 refer to substratum+5mg Shandongs this can+imidazoles; 5 refer to substratum+SDS; 6 refer to substratum+5mg Shandongs this can+SDS.A refers to the substrate Shandong, and this can; B refers to glycation product 1-O-β-D-glucosyl group Shandong, and this can.
Rusco monoglycosides 1-O-β-D-glucosyl group Shandong this can preparation
But this 150mg of substrate Shandong is dissolved in 30mL acetone; The glucose solution 100ml of preparation 0.5g/ml, sterilization (the same substratum of condition); Bacterial classification NRRL1086 is seeded to the liquid nutrient medium substratum by 4 ℃ solid inclined-plane, and (substratum is by containing KH in 100 milliliter of 20% potato decoction liquor
2PO
40.3 gram; MgSO
40.75 gram; Glucose 10 grams; Microorganism B
10.01 gram; NaCl 0.5g, KNO
33.0g, FeSO
40.01g obtaining liq substratum branch is filled to the triangular flask of 150mL, every bottled liquid 30mL), to shaking table, cultivate 24 hours (28 ℃ 180r/m), are seed liquor; Seed liquor is transferred under aseptic condition in the liquid nutrient medium substratum (substratum is formed the same), in shaking table, continues to cultivate 24 hours.But every bottle adds this acetone soln of 1ml Shandong and 3ml sucrose solution, cultivates termination reaction after 144 hours.The ethyl acetate extraction of fermented liquid usefulness equivalent 6 times, combining extraction liquid, recovery ethyl acetate; Obtain extracting medicinal extract 0.65g, take by weighing 100-200 order silica gel 0.9g and residue and evenly mix sample, column chromatography 200～300 order 15g dry column-packings, the chloroform-methanol gradient elution, in the eluting fraction of chloroform-methanol=1: 10, can get target converted product rusco monoglycosides 1-O-β-D-glucosyl group Shandong this can, through recrystallizing methanol, obtain white toner end 100mg.
In addition to the implementation, the present invention can also have other embodiments.All employings are equal to the technical scheme of replacement or equivalent transformation formation, all drop on the protection domain of requirement of the present invention.
Claims (6)
1. the method for a preparing rusco monoglycosides high-efficiently, it is that the bacterial strain of NRRL1086 carries out bio-transformation to ruscogenin and obtains rusco monoglycosides with preserving number, the steps in sequence of described bio-transformation comprises:
A, obtaining liq substratum;
B, with the NRRL1086 inoculation to liquid nutrient medium, in shaking table, cultivate and obtained seed liquor in 10～48 hours;
C, seed liquor sent out be forwarded to liquid nutrient medium and continue cultivate, obtain fermented liquid;
But d, add in the fermented liquid and be dissolved with Lu Si acetone soln and carbon source, and cultivated altogether 4～7 days, described carbon source be in glucose, sucrose, maltose, the starch one or more;
E, fermented liquid be with organic solvent A extraction 3～6 times, and described organic solvent A is a kind of in ethyl acetate, chloroform, methylene dichloride, ethyl formate, butylacetate, toluene, hexane, the propyl carbinol;
F, concentrated extract get medicinal extract, separate by silica gel column chromatography, obtain target compound---rusco monoglycosides.
2. the method for preparing rusco monoglycosides high-efficiently according to claim 1 is characterized in that described liquid nutrient medium contains 10%～30% potato decoction liquor, KH
2PO
4, MgSO
4, vitamins B
1, NaCl, KNO
3And FeSO
4
3. the method for preparing rusco monoglycosides high-efficiently according to claim 2 is characterized in that in the described liquid nutrient medium, in 1000 milliliters of potato decoction liquor, contains 0.1～10 gram KH
2PO
4, 0.1～5 the gram MgSO
4, 0.01～1 the gram vitamins B
1, 0.1～10 the gram NaCl, 0.1～10 the gram KNO
3, 0.1～7 the gram FeSO
4
4. the method for preparing rusco monoglycosides high-efficiently according to claim 3 is characterized in that in the described liquid nutrient medium, by 1000 milliliters of 20% potato decoction liquor, KH
2PO
40.1～0.5 gram; MgSO
40.05～0.5 gram; NaCl 0.5～0.8g, KNO
32.0～4.0g, FeSO
40.005～0.03g; Carbon source 0.5～2.5 gram.
5. the method for preparing rusco monoglycosides high-efficiently according to claim 4 is characterized in that pH=5.0～7.0 of described liquid nutrient medium.
6. the method for preparing rusco monoglycosides high-efficiently according to claim 5 is characterized in that bacterial classification activates earlier, soak time 20～30h before cultivation.
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Cited By (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN103215336A (en) * | 2013-05-08 | 2013-07-24 | 中国药科大学 | Preparation method of betulinic acid 28-O-beta-D-glucopyranoside and use of betulinic acid 28-O-beta-D-glucopyranoside |
| CN103233055A (en) * | 2013-05-08 | 2013-08-07 | 中国药科大学 | Preparation method and application of oleanolic 28-O-beta-D-glucopyranoside |
| CN105906676A (en) * | 2016-05-03 | 2016-08-31 | 中国药科大学 | Preparation method of isoquercitrin and application of isoquercitrin to aspect of resisting asthma |
-
2009
- 2009-10-22 CN CN200910233989A patent/CN101693913A/en active Pending
Cited By (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN103215336A (en) * | 2013-05-08 | 2013-07-24 | 中国药科大学 | Preparation method of betulinic acid 28-O-beta-D-glucopyranoside and use of betulinic acid 28-O-beta-D-glucopyranoside |
| CN103233055A (en) * | 2013-05-08 | 2013-08-07 | 中国药科大学 | Preparation method and application of oleanolic 28-O-beta-D-glucopyranoside |
| CN105906676A (en) * | 2016-05-03 | 2016-08-31 | 中国药科大学 | Preparation method of isoquercitrin and application of isoquercitrin to aspect of resisting asthma |
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Open date: 20100414 |