CN103233055A - Preparation method and application of oleanolic 28-O-beta-D-glucopyranoside - Google Patents
Preparation method and application of oleanolic 28-O-beta-D-glucopyranoside Download PDFInfo
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- CN103233055A CN103233055A CN2013101656935A CN201310165693A CN103233055A CN 103233055 A CN103233055 A CN 103233055A CN 2013101656935 A CN2013101656935 A CN 2013101656935A CN 201310165693 A CN201310165693 A CN 201310165693A CN 103233055 A CN103233055 A CN 103233055A
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Abstract
The invention relates to the field of biological medicaments, in particular relates to oleanolic 28-O-beta-D-glucopyranoside (I) and discloses a preparation method of the oleanolic 28-O-beta-D-glucopyranoside. The oleanolic 28-O-beta-D-glucopyranoside is prepared by carrying out bioconversion on oleanolic acid by using a bacterial strain with the preservation number of NRRL1086. The invention further discloses an application of the oleanolic 28-O-beta-D-glucopyranoside in terms of anti-inflammation.
Description
Technical field
The present invention relates to the bio-pharmaceutical field, be specifically related to the preparation method of Oleanolic Acid 28-O-β-D-glucopyranoside, the invention also discloses the purposes of its anti-inflammatory aspect.
Background technology
Inflammation is modal a kind of pathologic process in the human diseases, with most of disease-relateds.Anti-inflammatory drug is the second largest class medicine that is only second to anti-infectives clinically, at present using more anti-inflammatory drug clinically mainly is non-steroidal anti-inflammatory medicine and adrenal cortex hormones drug, but these drug effects are many target spots, multipath, the dosage that uses and also have nothing in common with each other the course for the treatment of and various mechanism between also have complicated cyberrelationship, life-time service can bring a lot of untoward reactions.Along with constantly developing and be familiar with and for many years clinical and experiment confirm Chinese medicine, a lot of Chinese medicines have good anti-inflammatory action, little and the aboundresources of toxic side effect, so the exploitation of traditional Chinese medicine monomer and structure of modification and the focus that the research of its pharmacological mechanism become new drug development in the world today.Pentacyclic triterpenoid is distributed widely in each kind of plant, has biological activity widely, studies show that to have significant activity aspect anti-inflammatory.Have tangible anti-inflammatory action as Oleanolic Acid, but poorly water-soluble may influence the performance of its drug effect.Water-soluble in order to increase, keep simultaneously or improve its activity, it has been carried out structural modification, chemosynthesis a large amount of derivatives, wherein being no lack of has active good compound to find.But the microorganism of relevant Oleanolic Acid is glycosylation modified rarely in report.
Microbial transformation is to utilize the specific enzymes of microorganisms exogenous compounds to be carried out the specific biochemical reaction of structural modification.Microbial transformation has the three-dimensional arrangement selectivity of height, can the catalysis of single-minded ground specifically react, need not loaded down with trivial details protection and deprotection operation, has advantage efficient, environmental protection, is a kind of effective ways that the natural radioactivity compound carries out structural modification.If in containing the oleanane type pentacyclic triterpene aglycon compounds of carboxyl, introduce the problem that glycosyl can solve the bioavailability difference to a certain extent, keep its activity simultaneously.
Summary of the invention
The invention discloses compound in structural formula I:
Called after Oleanolic Acid 28-O-β-D-glucopyranoside.The present invention also comprises the solvate of compound in structural formula I simultaneously.
Table 1 Oleanolic Acid and Oleanolic Acid 28-O-β-D-glucopyranoside
13The C-NMR data are as follows:
Pharmacological testing proves that formula I compound of the present invention has tangible anti-inflammatory activity, and its solvate has same pharmacological effect.
Formula I compound of the present invention is that substrate transforms preparation with the Oleanolic Acid with bioconversion method preferably, and method is as follows:
In order to seek the suitable bacterial classification that Oleanolic Acid becomes Oleanolic Acid 28-O-β-D-glucopyranoside that transforms, the present invention has screened a plurality of bacterial classifications altogether, found that, preserving number is that the NRRL1086 bacterial classification has stronger conversion capability (seeing Table 2) to Oleanolic Acid.
Screen used liquid nutrient medium (PDA): 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, Vb
110.0mg 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 ofartenium B to artmisinn. natural product magazine (Jounal ofNatural 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: Oleanolic Acid is dissolved in ethanol, is made into the 10mg/mL solution for standby; Every bottle of bacterium liquid (30mL) through the activation of two steps adds the 0.5mL sample solution, makes every bottle of bacterium liquid contain Oleanolic Acid 5mg.Behind condition cultivation 120h, 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 extraction using alcohol 3 times, united extraction liquid reclaims ethanol, obtains extracting medicinal extract, and two kinds of medicinal extract are merged, and 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:
Thin layer plate: the silica gel G plate that 0.7% CMC-Na makes.
Developping agent: toluene-ethyl acetate-Glacial acetic acid (14: 4: 0.5).
Developer: 10% sulfuric acid ethanol
Operation:
Transform extract and blank extract and put in thin layer plate respectively, ascending method is fully launched back taking-up nature and is dried, and spray is with 10% sulfuric acid ethanol, 105 ℃ of heating colour developings.Conversion results sees Table 2.
Table 2 microbial transformation Oleanolic Acid The selection result
From The selection result, there are 2 kinds of bacterium to transform, NRRL1086, through measuring and calculating, transformation efficiency can reach 80%.And NRRL5646 is not the glycosylation conversion.Therefore, the present invention selects bacterial strain NRRL1086 for use, is that raw material prepares Oleanolic Acid 28-O-β-D-glucopyranoside with the Oleanolic Acid.
The invention discloses a kind of preparation method of preparation I compound, is that the NRRL1086 bacterial strain carries out the bio-transformation acquisition to Oleanolic Acid with preserving number.
Preferred method is: be substrate with the Oleanolic Acid, utilize preserving number to be the bacterial strain of NRRL1086, substrate and this bacterial strain were cultivated in substratum 3～8 days altogether, termination reaction use organic solvent extraction, concentrated extract, by the silica gel column chromatography separation namely.
Wherein organic solvent is selected from one or more in ethyl acetate, chloroform, methylene dichloride, ethyl formate, butylacetate, toluene, normal hexane, the propyl carbinol.More preferably ethyl acetate.
The silica gel column chromatography separation is carried out gradient elution with chloroform and methyl alcohol.
Substratum contains 10～30% potato decoction liquor, KH
2PO
4, MgSO
4, VITMAIN B1 and be selected from one or more carbon sources in glucose, sucrose, bud sugar, the starch, medium pH=5.0～7.0.Be weight percentage.
Wherein by 100 milliliters of 20% potato decoction liquor, the preferred content of each component is KH in the substratum
2PO
4Contain 0.1～0.5 gram; MgSO
4Contain 0.05～0.5 gram; Glucose or sucrose 0.5-2.5 gram.
Highly preferred preparation method is: the bacterial strain that with preserving number is NRRL1086 is inoculated in the liquid nutrient medium by the 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 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; Be elution system with the chloroform-methanol.Converted product obtains the white powder converted product through recrystallizing methanol.
Pharmacological testing proves that structural formula I compound of the present invention has significant anti-inflammatory activity, below is test method and result:
Oleanolic Acid 28-O-β-D-glucopyranoside anti-inflammatory activity research
1, the influence of Oleanolic Acid 28-O-β-D-glucopyranoside p-Xylol induced mice ear swelling model
Get 60 of normal mouses, male and female half and half are divided into 6 groups at random, are respectively control group, Oleanolic Acid group (100mg/kg), administration group (25,50,100mg/kg), positive drug group (Naproxen Base 45mg/kg).Each organizes continuous gastric infusion three days, and once a day, control group then gives the CMC-Na of respective volume.The last administration was only evenly smeared dimethylbenzene 20 μ L/ in the positive and negative two sides of left auricle after 40 minutes, 20 minutes post-tensioning necks are put to death, and cut ears along the auricle baseline, swell with the YLS-Q4 ear and make the ear device and sweep away auricle at the same position of left and right sides ear, weigh respectively, represent the swelling degree with the difference of self left and right sides auricle quality.
Inhibiting rate=[(control group swelling degree mean value-administration group swelling mean value)/control group swelling mean value] * 100%
The result is as follows:
This shows that can suppress dimethylbenzene induced mice auricle acute inflammation after Oleanolic Acid 28-O-β-D-glucopyranoside administration, middle and high dosage group effect is particularly evident, and effect is better than the positive drug group and with the Oleanolic Acid high dose group of concentration.
2, Oleanolic Acid 28-O-β-D-glucopyranoside on Carrageenan causes the influence of rat paw edema model
Get 60 of normal male rats, be divided into 6 groups at random, be respectively control group, Oleanolic Acid group (100mg/kg), administration (25,50,100mg/kg) group, positive drug group (Naproxen Base 30mg/kg).Each organizes continuous gastric infusion three days, and once a day, control group then gives the CMC-Na of respective volume.Measure right back sufficient volume with YLS-7B toes volume determination instrument before the last administration, as administration front volume V
0, administration is after 40 minutes, and injection 1% λ-carrageenin 0.1mL/ only causes inflammation between the right back whole sole of the foot stndon sheath of rat, and 1h, 3h, 5h, 7h measure right back sufficient volume V after causing inflammation respectively
S, so that scorching forebody-afterbody product moment (V
S-V
0) be the swelling degree.
The result is as follows:
Compare with control group
*P＜0.05,
*P＜0.01
This shows that on Carrageenan causes rat paw edema after Oleanolic Acid 28-O-β-D-glucopyranoside medication restraining effect, and has certain concentration dependent, and its high dose group effect is better than the Oleanolic Acid high dose group with concentration.
3, Oleanolic Acid 28-O-β-D-glucopyranoside is to the influence of rat AA model
Rat grouping, administration are the same, with same lot number 80 ℃ of water-bath 1h of bacille Calmette-Guerin vaccine deactivation, be made into the emulsion of 10g/L with sterilising liq paraffin, fully grinding mixing is Cheng Foshi Freund's complete adjuvant (FCA).It is standby to place 4 ℃ of refrigerators preservations, shakes up before the use.Cause the left and right back of the scorching preceding rat following volume of articulatio talocruralis (mL) with toes volumetric measurement instrument mensuration, cause inflammation in the left back toes intradermal injection of every mouse 0.1mL FCA then, normal control group injection 0.1mL PBS.Cause scorching preceding 3 days gastric infusions.Respectively at cause scorching before and cause scorching back 6,12,18 and 24h, detect with toes volumetric measurement instrument and cause scorching forward and backward rat and cause scorching parapodum swelling, with observation AA rat primary pathology.
The result is as follows:
Compare with control group
*P＜0.05,
*P＜0.01
Each is organized rats with left foot 6h after causing inflammation and begins swelling, and 18h peaks.The AA rat primary inflammation of after the medication FCA being induced has the obvious suppression effect, and has certain concentration dependent.
Oleanolic Acid 28-O-β of the present invention-D-glucopyranoside can mix with pharmaceutically acceptable carrier, for the preparation of the medicine for treatment compositions.Can be prepared into formulation general on the pharmaceutics, as parenteral dosage forms such as gastrointestinal administration formulations such as capsule, tablet, pill, oral liquid, granule, tincture, sustained release dosage and injection, transdermal patch, external preparations.
Embodiment
Embodiment 1
The preparation of Oleanolic Acid 28-O-β-D-glucopyranoside:
Substrate Oleanolic Acid 200mg is dissolved in 20mL ethanol; The glucose solution 100mL of preparation 0.5g/mL, sterilization (the same PDA of condition); Bacterial classification NRRL1086 is seeded to liquid PDA 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; VitaminB10 .01 restrains preparation, and the liquid nutrient medium 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 PDA substratum (substratum is formed the same), in shaking table, continues to cultivate 24 hours.Every bottle adds 1mL Oleanolic Acid ethanolic soln 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 309mg, take by weighing 100-200 order silica gel 0.8g and residue and evenly mix sample, column chromatography 200～300 order 20g dry column-packings, the chloroform-methanol gradient elution, in the eluting fraction of chloroform-methanol=95: 5, can get target converted product Oleanolic Acid 28-O-β-D-glucopyranoside, through recrystallizing methanol, obtain white powder 217mg.
Its carbon spectrum sees Table 1
Embodiment 2
Tablet
Get Oleanolic Acid 28-O-β-D-glucopyranoside 100g and starch 50g that embodiment 1 makes, dextrin 50g mixes, and makes wetting agent with an amount of 30% ethanol, makes softwood, and ordinary method is granulated, and adds an amount of Magnesium Stearate and mixes, and makes tablet.
Claims (8)
1. the preparation method of compound Oleanolic Acid 28-O-β-D-glucopyranoside (I) or its pharmaceutically acceptable solvate is characterized in that: be that the NRRL1086 bacterial strain carries out bio-transformation to Oleanolic Acid and obtains with preserving number.
2. the preparation method of claim 1, wherein bioconversion method is as follows: be substrate with the Oleanolic Acid, utilize preserving number to be the bacterial strain of NRRL1086, substrate and this bacterial strain were cultivated in substratum 4～7 days altogether, termination reaction, fermented liquid organic solvent extraction, concentrated extract, get medicinal extract, separate obtaining target compound by silica gel column chromatography.
3. the preparation method of claim 2, wherein organic solvent comprises: ethyl acetate, chloroform, methylene dichloride, ethyl formate, butylacetate, toluene, hexane, propyl carbinol etc.
4. the preparation method of claim 2, substratum contains 10～30% potato decoction liquor, KH
2PO
4, MgSO
4, VITMAIN B1 and be selected from glucose, sucrose, bud sugar, the starch one or more carbon source, medium pH=5.0～7.0.
5. the preparation method of claim 2 is wherein by 100 milliliters of 20% potato decoction liquor, KH
2PO
40.1～0.5 gram; MgSO
40.05～0.5 gram; Glucose 0.5-2.5 gram (or sucrose 0.5-2.5 gram).
6. the preparation method of claim 2, wherein bacterial classification activates earlier before cultivation, soak time 20～30h.
7. pharmaceutical composition wherein contains compound or its pharmaceutically acceptable solvate and the pharmaceutically acceptable carrier of claim 1.
8. the compound of claim 1 or its pharmaceutically acceptable solvate are for the preparation of the purposes of anti-inflammatory drug.
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|---|---|---|---|---|
| CN117379446A (en) * | 2023-12-12 | 2024-01-12 | 吉林大学 | Application of oleanolic acid-28-O-beta-D-glucopyranoside in preparation of anti-colitis drugs |
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2013
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Cited By (2)
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| CN117379446A (en) * | 2023-12-12 | 2024-01-12 | 吉林大学 | Application of oleanolic acid-28-O-beta-D-glucopyranoside in preparation of anti-colitis drugs |
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