CN103880912A

CN103880912A - Method for extracting tripterine from plants

Info

Publication number: CN103880912A
Application number: CN201410116127.XA
Authority: CN
Inventors: 张梅; 彭学东; 赵金召; 邱洁
Original assignee: Jiangsu Swithin Biological Medicine Engineering Research Center Co Ltd
Current assignee: Jiangsu Swithin Biological Medicine Engineering Research Center Co Ltd
Priority date: 2014-03-27
Filing date: 2014-03-27
Publication date: 2014-06-25

Abstract

The invention discloses a method for extracting tripterine from root barks or rootstocks of tripterygium and celastrus orbiculatus plants. According to the method, the root barks or rootstocks of the tripterygium and celastrus orbiculatus plants are taken as a raw material and subjected to treatment steps including enzymolysis, ultrasonic extraction, separation, purification, recrystallization and the like. The extraction comprises the steps of firstly, using enzyme for enzymolysis and then performing ultrasonic extraction, concentrating an extracting solution to obtain a paste-like crude extract, subsequently suspending the crude extract in water, extracting by use of ethyl acetate and concentrating an extracting agent to obtain a thick paste-like extract, wherein the purification comprises the steps of stirring and mixing the crude extract with diatomite or silica gel, performing chromatographic separation and purification by use of a silica gel column, collecting an eluent, concentrating and drying to obtain powdery coarse crystal, and finally performing the recrystallization by use of a methanol-water or ethanol-water mixed solvent. The method is simple in the separation and purification process and can be used for preparing the tripterine of which the purity is as high as 98%, and the extraction yield is higher than 90%.

Description

A kind of method of extracting Tripterine from plant

Technical field

The invention belongs to medical technical field, be specifically related to a kind of method of extracting Tripterine from plant.

Background technology

Tripterine (celastrol), have another name called celastrin, be a kind of from Celastraceae Thunder God Calamus and celastraceae plants isolated pentacyclic triterpene material, the outward appearance acicular crystals that takes on a red color, has anti-inflammatory, immunosuppression, the multiple pharmacologically active such as antiviral and antitumor.The discovery of the antineoplastic new mechanism of Tripterine, has started again the new upsurge of studying its antitumour activity, and its economic worth is also high gradually simultaneously.

According to the solvability of Tripterine, utilize organic solvent to extract Tripterine from the higher plant of trypterygine cellulose content, be to produce a kind of method that Tripterine generally adopts.So far, the Tripterine extraction process of bibliographical information only has organic solvent extraction and SCF-CO 2.Organic solvent extraction method is low for equipment requirements, simple to operate, with low cost because of it, is current a kind of the most frequently used method.

But vegetable cell is made up of cell walls and protoplastis, cell walls is the dense structure being made up of materials such as Mierocrystalline cellulose, hemicellulose, pectin substance, xylogen.In leaching process, the Tripterine that is arranged in cell protoplast when extracting Medium Diffusion, must overcome the dual resistance of cell walls and intercellular substance, causes extraction time to extend, and extraction efficiency reduces.

Summary of the invention

The present invention extracts the lower problem of Tripterine yield for overcoming in existing method, invents a kind of method newly, improved extraction Tripterine.

The invention provides a kind of method of extracting Tripterine, wherein, the method comprises the plant material that contains Tripterine be dry, pulverize, and after enzymolysis, organic solvent carries out supersound extraction to it, extraction after extracting solution is concentrated, column chromatography for separation, recrystallization.Concrete steps comprise:

(1) raw material drying, pulverizing, cross 20 ~ 60 mesh sieves, by solid-liquid ratio 1:5 ~ 20(kg/L) add deionized water, pH is in 6 ~ 9 scopes in adjustment, adds enzyme enzymolysis, enzyme amount is raw materials quality 0.1 ~ 10%, 25 ~ 60 ℃ of hydrolysis temperatures, enzymolysis time 0.5 ~ 2 h.Described enzyme is a kind of in cellulase, polygalacturonase or both mixtures;

(2) reacting liquid filtering, collects filter residue, by solid-liquid ratio 1:5 ~ 15(kg/L) add methyl alcohol or ethanol, ultrasonic extraction 10 ~ 45 min in 20 ~ 45 ℃, filter, and repeat to extract 2 ~ 3 times, and united extraction liquid, is evaporated to without cut and steams;

(3) by concentrated extract with 2 ~ 5 times of volume aqueous suspensions after, add equal-volume ethyl acetate extraction, re-extract 3 times, combining extraction liquid, concentrating under reduced pressure;

(4) product that extraction liquid obtains after concentrating, with diatomite or silica gel mixed sample dry, is placed in silicagel column by the product obtaining and separates.Wherein, be 5 ~ 10% of the concentrated rear products therefrom weight of extraction liquid for mixing the diatomite of sample or the consumption of silica gel, eluent is petroleum ether-ethyl acetate, gradient elution adopts tlc to monitor in elution process;

(5) collect containing the elutriant of Tripterine, be concentrated into after dry and carry out recrystallization dry, gained is Tripterine.

Advantage of the present invention is: utilize enzyme to carry out effectively decomposing and destroying to the cell wall constituent of raw material, thereby reduce the resistance to mass transfer in leaching process, accelerate Tripterine stripping cell speed, shorten extraction time, improve extraction efficiency; In former processing condition, only increased an operating unit, reaction conditions is gentle easily to be obtained, lower to the requirement of conversion unit, simple to operate simultaneously.

Accompanying drawing explanation

Fig. 1 is the structural formula of Tripterine.

Fig. 2 is the extraction schema of Tripterine.

Embodiment

Specific implementation method described herein only, for description and interpretation the present invention, is not limited to the present invention.

Embodiment 1

Take the dry root skin of 1kg celastraceae plants celastrus angulatus, be crushed to 40 orders, 3% cellulase and the polygalacturonase recombiner of 10L deionized water and 20g, enzymolysis 2h under the state of 40 ℃ of constant temperature and pH value 6.0, filter, in filter residue, add methyl alcohol 10L to carry out ultrasonic extraction, extraction time 30min, filter, repeat to extract 2 times, merging filtrate, concentrating under reduced pressure, after aqueous suspension by the extract after concentrated with 4 times of volumes, add isopyknic ethyl acetate extraction, coextraction 3 times, combining extraction liquid concentrating under reduced pressure, product after concentrated is mixed sample dry with the diatomite of its quality 5%, upper 200-300 object silicagel column, gradient elution, eluent is petroleum ether-ethyl acetate, gradient is 1:0(2V), 5:1(2V), 1:1(10V) (V is column volume), collect the elutriant containing Tripterine, concentrating under reduced pressure is also dry, obtain red powder shape extract, this extract is dissolved into after saturated solution with hot ethanol, slowly add water, there is red crystals to separate out, after dry after dry, obtain the Tripterine that 7.37g purity is 98.1%, yield is 90.3%.

Embodiment 2

Take the root of 1kg trypterygine, be crushed to 60 orders, 3% cellulase and the polygalacturonase recombiner of 15L deionized water and 250g, enzymolysis 2h under the state of 40 ℃ of constant temperature and pH value 6.0, filter, in filter residue, add ethanol 15L to carry out ultrasonic extraction, extraction time 1h, filter, repeat to extract 2 times, merging filtrate, concentrating under reduced pressure, after aqueous suspension by the extract after concentrated with 4 times of volumes, add isopyknic ethyl acetate extraction, coextraction 3 times, combining extraction liquid concentrating under reduced pressure, product after concentrated is also dry with the silica gel mixed sample of its quality 5%, upper 200-300 object silicagel column, gradient elution, eluent is petroleum ether-ethyl acetate, gradient is 1:0(2V), 5:1(2V), 1:1(10V) (V is column volume), collect the elutriant containing Tripterine, concentrating under reduced pressure is also dry, obtain red powder shape extract, this extract is dissolved into after saturated solution with hot ethanol, slowly add cold water, there is red crystals to separate out, after dry, obtain the Tripterine that 6.19g purity is 98.3%, yield is 90.2%.

Claims

1. the extraction process of a Tripterine, it is characterized in that with roots of plants skin or rhizome raw material drying containing Tripterine, pulverize and sieve, the enzyme-added enzymolysis that carries out after adding appropriate amount of deionized water and adjusting PH, filter and in backward filter residue, add methyl alcohol or ethanol ultrasonic extraction 3 ~ 4 times, after extracting solution is concentrated, adds aqueous suspension and extract with ethyl acetate, after extraction liquid is concentrated with silica gel column chromatogram separating purification, collect elutriant, concentrate drying obtains Powdered coarse crystal, with methanol-water or ethanol-water mixed solvent recrystallization.

2. the method for claim 1, wherein enzyme used is the single or mixture in cellulase, polygalacturonase, and add-on is raw materials quality 0.1 ~ 10%, and temperature is 25 ~ 60 ℃, enzymolysis time 0.5 ~ 2 h.

3. the method for claim 1, adopts organic solvent methyl alcohol or ethanol ultrasonic extraction, and extraction time 10 ~ 45min, repeats 2 ~ 3 times.

4. the method for claim 1, adopts column chromatographic isolation and purification, and wherein eluent is petroleum ether-ethyl acetate, then with methanol-water or ethanol-water mixed solvent recrystallization elutriant, final gained Tripterine purity reaches 98%, and extract yield is higher than 90%.