A kind of method for preparing iridoid glycoside unit
Technical field
The present invention relates to biological technical field, is from the iridoid glycoside compounds, and high productivity prepares the method for its glucoside unit.
Background technology
Iridoid extensively is present in vegitabilia, with the most general in Rubiaceae, Pirolaceae, the Monotropaceae plant, the iridoid constituents exists mainly with the form of glucoside (Iridoid glycosides) greatly in plant, for example contain gardenoside (gardenoside, C in the madder wort cape jasmine
17H
24O
11), Geniposide (geniposide, C
17H
24O
10), genipin-1-β-D-gentiobioside (genipin-gentiobioside), Geniposidic acid (geniposidic acid, C
16H
22O
10), woodruff (another name: the rubichloric acid Tender Catchweed Bedstraw Herb) (asperuloside, C
18H
22O
11), the scandoside in the Herba Paederiae (paederoside, C
18H
22O
11S), rhinanthin (aucubin, the C in the psyllium
15H
22O
9) etc.
Iridoid glycoside (Iridoid glycosides) great majority are white crystals or powder, such glucoside has been proved to be facile hydrolysis, especially in the presence of β-D-Glucose glucoside enzyme, easily be hydrolyzed to the first iridoid (Iridoid) of corresponding glucoside, the first easy and amino acids substance reaction generation coloring matter of iridoid glycoside can prepare natural blue pigment, haematochrome and melanochrome or brown pigments.For example, gardenia blue pigment and gardenia red pigment, the Geniposide in the iridoid glycoside is as raw material exactly, the lower hydrolysis that exists at the enzyme with hydrolysis β-glycoside enzymic activity, the natural pigment that obtains with the amino acids substance reaction again.Proved that such pigment is safe, can be widely used in the painted of food, makeup and color matching.
In addition, studies show that in a large number, iridoid glycoside is sloughed glycosyl in enzymic hydrolysis, become iridoid glycoside unit after, the hemiacetal structure in its molecule, easily open loop becomes dialdehyde, be a kind of Biological cross-linker of excellent performance, compare with glutaraldehyde, have similar crosslinked action, the Side effect that does not but have glutaraldehyde, its biocompatibility is better; Therefore have been widely used at biomedicine field; For example people study more iridoid glycoside unit genipin, and investigators successfully are applied to make biomaterial, as enzyme fixedly the linking agent of usefulness (US 4983524), (US 6545042 to make biological callus material with it
),Make the wall material (US 5023024) of foodstuff glue (US 5037664), micro-capsule pill, utilize the characteristic of itself and amino acid reaction solution, with its developer that detects as amino acid, genipin also has many-sided physiologically active: can suppress platelet aggregation, possess anti-tumor activity (referring to suzuki, yasuhiro, kondo.Planta medica.2001,67 (9), the 807-810 page or leaf), can prevent and treat neurotoxicity (M.Yamazaki et, Biolagical﹠amp because amyloid protein causes; Pharmaceutical Bulletin, 2001,24(12), 1454-1455).In a word, the glucoside of iridoid glycoside unit in the Application Areas in all fields, has demonstrated good using value.
At present, for iridoid glycoside, had several different methods it is purified and to make with extra care, wherein adopting macroreticular resin absorbing method is a kind of economy and technique that can large-scale production.Yet, iridoid glycoside unit is a kind of hemiacetal, and character is comparatively active, meets acid, alkali, carbonyl compound, primary amino compound in the preparation process and all is easy to occur polymerization and degraded, variable color, therefore refining for the production of iridoid glycoside unit, still lack at present cost-effective method.CN 200710090208 discloses a kind of extracting method of glucoside unit genipin of Geniposide, at first Geniposide is carried out enzymolysis, and again by macroreticular resin absorbing method, fractionation by adsorption genipin from enzymolysis solution is again through wash-out, concentrated, freeze-drying acquisition genipin.But there are the following problems for the method: 1, productive rate is lower; 2, still can contain the Geniposide of 2-10% and the impurity such as dimer of genipin in the product of genipin.
Summary of the invention
The invention provides a kind of method for preparing its corresponding iridoid glycoside unit from the iridoid glycoside high yield.
The present invention is based on contriver's following discovery: iridoid glycoside is in the glucoside unit process at hydrolysis, the glucoside unit of the hemiacetal structure that generates can react with the enzyme with protein structure under identical condition immediately, hydrolyzed solution is darkened turn blue, this side reaction on the one hand passivation enzyme activity, make the hydrolysis of iridoid glycoside be difficult to thoroughly finish, the aspect has consumed hydrolysate iridoid glycoside unit on the other hand, and productive rate is reduced; Polymerization also can self occur in iridoid glycoside unit in the aqueous solution after generation, generate dimer, and these situations all can detect by high performance liquid chromatography and be confirmed.
For example, with prior art the Geniposide that originates from cape jasmine is hydrolyzed, in order reasonably to obtain high as far as possible transformation efficiency in the time, every gram Geniposide need to add the nearly β of 2000-3000 activity unit-D glucosaccharase, could obtain the transformation efficiency greater than 90%, yet the enzyme addition is more, and the hydrolysate genipin that consumes of being combined with enzyme is also more, and the productive rate of target product genipin significantly reduced when end reaction was finished; On the other hand, if reduce the consumption of enzyme, the reaction times will prolong, and the product genipin is remarkable to the passivation of enzyme, and the hydrolysis of Geniposide can not be carried out thoroughly, and transformation efficiency also reduces.
The problem that exists in order to overcome prior art, the inventor finds by research, utilize the deliquescent difference in organic solvent of hydrolysate glucoside unit and iridoid glycoside, select suitable organic solvent, in enzyme digestion reaction, in time extracting and separating goes out the glucoside unit that enzymolysis generates from reaction solution, reduces the passivation of enzyme and the polymerization of product, can significantly improve the productive rate of object iridoid glycoside unit.
Therefore, the present invention relates to a kind of optimization method from iridoid glycoside hydrolysis preparation iridoid glycoside unit.
Described iridoid glycoside comprises Geniposide (geniposide), genipic acid glucoside (geniposidic acid), genipin gentiobioside (genipin-gentiobioside), gardenoside (gardenoside), scandoside (paederoside), rubichloric acid (asperuloside), kind order soft-shelled turtle glucoside (loganin), rhinanthin (aucubin).
One of key point of the present invention is the method by liquid-liquid extraction, in the iridoid glycoside hydrolysis, from reaction system hydrolysate iridoid glycoside unit is separated in time.In order to reach the instantaneity of separation, the present invention preferably adopts continuous liquid-liquid extraction method, reaction product iridoid glycoside unit is implemented to separate, the preferred continuous liquid-liquid extraction method of the present invention is also based on the following fact: iridoid glycoside unit solubleness in water is larger, extracting process with routine, need repeatedly extraction it could be separated from the aqueous solution fully, therefore conventional extracting process does not possess good operability.Described continuous liquid-liquid extraction method, comprise CCD method, droplet countercurrent chromatography method, high-speed countercurrent chromatography, described continuous liquid-liquid extraction method is to extraction equipment and have not a particular requirement, as long as can realize smoothly the equipment of continuous extraction process, all be applicable to the present invention.For example, be applicable to the continuous liquid-liquid extraction tower of commercial scale production, can be used as and implement a preferred equipment of the present invention.This extraction tower can one or morely be connected, and internal structure can be packed tower or perforated plate tower, can also be the extraction tower in many extractions storehouse of band stirring.It also can be the tower of other structure.
The selection of organic solvent is another one key point of the present invention, although so long as the iridoid glycoside that optionally generates in solubilizing reaction system unit, and with water mutually not miscible organic solvent all can use.But the present invention is the organic solvent larger with the Relatively density contrast of water preferably, for example ethers, halo alkanes, concrete preferred ether, isopropyl ether, tetrahydrofuran (THF), methyl isopropyl ether, methyl tert-butyl ether, trichloromethane, methylene dichloride, ethylene dichloride are used solvent as extraction, can realize easily like this sharp separation of two-phase after the continuous extraction.
The selection of catalyzer, the present invention preferably contains the enzyme of β-D-Glucose enzyme, because enzyme usually is the mixture of a series of enzymes, its title is change also more, therefore so long as the glycoside bond of iridoid glycoside is had the material of catalytic hydrolysis activity, all can use.The for example mixing of one or more in amygdalase, pentosanase, polygalacturonase, the cellulase.Described enzyme can be powder or liquid form.Liquid enzymes is preferred as one, is more suitable for enforcement of the present invention.
The method according to this invention, the mixed aqueous solution of certain density enzyme and iridoid glycoside is added in the liquid-liquid continuous extraction tower, wherein the ratio of iridoid glycoside in solution can be at 1%-30%(w/w) between, preferred 10%-20%, the ratio of enzyme and iridoid glycoside can remain between the 200u/g-3000u/g.Mixed solution keeps suitable temperature and pH value after adding extraction tower, and described temperature can be between 20-60 ℃, and preferred 30-50 ℃, more preferably at 40-50 ℃, described PH can be between 2.0-6.0, between the preferred 3.0-5.0.After adding mixed solution, even can be with after hydrolysis 1-4 hour, (when organic phase density ratio water hour) adds organic phase continuously at the bottom of cat head (when organic phase density ratio water is large) or the tower, reaction solution is carried out continuous extraction, the time that adds organic phase can be adjusted according to the speed of reaction, and the velocity of flow of organic phase in tower can be adjusted according to partition ratio difference and two complexities that are separated of product iridoid glycoside unit in organic phase.At the bottom of the tower or the organic phase of overhead collection, by a device that anhydrous sodium sulphate or other siccative are housed, remove the moisture content that wherein contains, enter into again solvent recovery unit, Distillation recovery organic phase solvent, the organic phase that reclaims is in the process condenser condenses, after passing through again the water saturation device, turn back to again in the extraction agent, participate in continuous extraction, the residuum after the solvent recovered under vacuum carries out recrystallization with mixing solutions or other solvent of ether and methyl alcohol, filters, vacuum drying can obtain iridoid glycoside unit crystal at last.
The final the method that adopts, can obtain the first iridoid of its corresponding glucoside with the transformation efficiency more than 95% from iridoid glycoside, as extra results, adopt above-mentioned method, can make the consumption of enzyme be reduced to the 30%-40% of ordinary method, hydrolysis time shortens to 5-6 hour, simultaneously because the minimizing of side reaction, can obtain that HPLC purity surpasses 95%, colourless iridoid crystal.
HPLC measures the following condition that adopts:
Chromatographic column: Shim-pack VP-ODS C18 post, 150mm * 4.6mm, 5 μ m
Moving phase: methanol/water=3/7(V/V)
Flow velocity: 1ml/min
Detector: Shimadzu SPD-10AVP UV-vis detector
Measure wavelength: 238nm
The following examples are as the present invention is further illustrated, are not construed as limiting the invention.
Embodiment 1
Geniposide (geniposide) 10.0g(0.0252mol with content 98%), the amygdalase 1ml of 3000u/ml, adding water dissolves in right amount, regulate ph4.0 with citric acid/sodium citrate, constant volume is to 100ml, join in Φ 25mm * 250mm orifice-plate type glass extraction tower, pass into hot water in the chuck, insulation to 40 ℃ connects up and down the liquid in-out pipe, after 1 hour, by peristaltic pump or utilize the discrepancy in elevation that ether is squeezed into extraction tower from the bottom, keep flow velocity 5ml/min, the extraction that cat head flows out is light by a tube type drying strainer that anhydrous sodium sulphate is housed, and imports in the rotatory evaporator, rotation is concentrated, reclaim ether, the ether of recovery is incorporated in the extraction phase that flows after with water saturation again, so circulates 5 hours.Concentrated residuum in the vaporizer arrives 100ml with ether dissolution.From water and organic phase, get respectively 1ml, with methanol/water (3/7, v/v) dissolved dilution is 2000 times, with the residual volume of water Geniposide behind the shimadzu 10Avp HPLC chromatographic instrument assaying reaction, the output of product genipin in the organic phase, all purchase the company in sigma-aldrich as reference substance Geniposide (geniposide), genipin (genipin) that content is demarcated, measurement result is listed in the table 1.
Comparative example 1
Geniposide (geniposide) 10.0g(0.0252mol with content 98%), the amygdalase 1ml of 3000u/ml, adding water dissolves in right amount, regulate ph4.0 with citric acid/sodium citrate, constant volume is to 100ml, join the 200ml triangular flask, close plug, 50 ℃ of insulations, enzymic hydrolysis is respectively at the 1ml that takes a sample respectively after 12 hours and after 24 hours, with methanol/water (3/7, v/v) dilution is 2000 times, and with the output of the genipin (genipin) of aqueous phase and the residual volume of Geniposide behind the shimadzu 10Avp HPLC chromatographic instrument assaying reaction, measurement result is listed in the table 1.
Comparative example 2
Except the Vitamin B17 enzyme dosage is 3ml, identical with comparative example 1.Reaction solution is followed the tracks of detection, and the results are shown in Table 1.
Embodiment 2
Get the gardenia extract of removing behind the Gardenia Yellow and (contain Geniposide 0.203mol/L, contain genipin gentiobioside 0.0432mol/L) 100ml, regulate ph4.0 with citric acid/sodium citrate, add pectinase 1ml(3000u/ml), mixing solutions joins in the orifice-plate type glass extraction tower, pass into hot water in the chuck, insulation to 50 ℃, connect up and down the liquid in-out pipe, by peristaltic pump isopropyl ether is squeezed into extraction tower from the bottom, keep flow velocity 5ml/min, the extraction that cat head flows out is light by a tube type drying strainer that anhydrous sodium sulphate is housed, and imports in the rotatory evaporator, rotation is concentrated, reclaim isopropyl ether, the isopropyl ether of recovery is incorporated in the extraction phase that flows after with water saturation again, so circulates 5 hours.Concentrated residuum in the vaporizer arrives 100ml with ether dissolution, get 1ml, water-bath volatilizes solvent, residual quantity with reaction mixture Geniposidic, genipin gentiobioside before and after the shimadzu 10Avp HPLC chromatographic instrument difference assaying reaction, and reaction finish after content, the purity of genipin (genipin) in the extraction liquid, measurement result is listed in the following table 2.
Embodiment 3
Get dry woodruff 3000g, pulverize, with 60 ° of alcohol 15L, under 50 ℃, diafiltration is extracted, extracting solution concentration and recovery alcohol, and concentrated solution is diluted with water to 3000ml, add Chinese medicine finings KBT-ZTC, muddy thing is removed in clarification, and supernatant liquid filtering is crossed the resin column absorption that 500ml is equipped with nonpolar macroporous adsorption resin, after saturated, wash 2BV with water, use again 40 ° of alcohol wash-outs, collect elutriant, concentration and recovery alcohol, must contain the concentrated solution of rubichloric acid (asperuloside), regulate PH4.0 with citric acid-sodium citrate, get the 215ml concentrated solution, HPLC detects, and rubichloric acid content is 0.185mol/L.Get the 100ml concentrated solution, add 3000u/g pectinase 1g, mixing, join in the orifice-plate type glass continuous extraction tower, pass into hot water in the chuck, insulation to 50 ℃, connect up and down the liquid in-out pipe, by peristaltic pump isopropyl ether is squeezed into extraction tower from the bottom, keep flow velocity 5ml/min, the extraction that cat head flows out is light by a tube type drying strainer that anhydrous sodium sulphate is housed, import in the rotatory evaporator, rotation is concentrated, reclaims isopropyl ether, the isopropyl ether that reclaims is incorporated in the extraction phase that flows after with water saturation again, so circulates 5 hours.Concentrated residuum in the vaporizer is dissolved into 100ml with isopropyl ether, gets 1ml, and water-bath volatilizes solvent, dilute 1000 times, measure the peak area of rubichloric acid unit in the extraction liquid, other is the phase 1ml that fetches water, the residual survey of moving phase dilution 1000 times of mensuration rubichloric acid the results are shown in the following table 3 surely.
Comparative example 3
Get the PH4.0 rubichloric acid concentrated solution 100ml in the example, add 3000u/g pectinase 1g, 50 ℃ of insulations of water-bath enzymolysis 24 hours is got 1ml, with 1000 times of liquid phase flow phase dilutions, the peak area of rubichloric acid and rubichloric acid unit in the assaying reaction liquid.Measurement result is listed in the following table 3.
From the peak area of product, the rubichloric acid transformation efficiency of embodiment 3 and rubichloric acid unit yield all significantly improve than existing methods.
Description of drawings
Fig. 1 is the HPLC figure of ether phase after embodiment 1 reaction is finished; Fig. 2 is comparative example 1 reaction afterreaction liquid HPLC figure.
Accompanying drawing is embodiment 1 and comparative example 1(24h) the product of reaction after finishing with 2000 times of dilutions after the HPLC collection of illustrative plates, wherein among the embodiment 1, the output of genipin (peak height) and purity and comparative example 1(24h) compare all and be significantly increased.