CN101906091B - Method for preparing and separating tectorigenin from iris in high-speed countercurrent chromatography by one step - Google Patents
Method for preparing and separating tectorigenin from iris in high-speed countercurrent chromatography by one step Download PDFInfo
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- CN101906091B CN101906091B CN201010249254A CN201010249254A CN101906091B CN 101906091 B CN101906091 B CN 101906091B CN 201010249254 A CN201010249254 A CN 201010249254A CN 201010249254 A CN201010249254 A CN 201010249254A CN 101906091 B CN101906091 B CN 101906091B
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Abstract
The invention relates to a method for separating and purifying monomeric compound of tectorigenin from iris, in particular to a method for separating and purifying tectorigenin from iris. The method comprises the following steps: dipping with methanol solution to extract and prepare iris coarse extracts, adding a little of water in the coarse extracts to dilute, extracting with ethyl acetate solution and removing water soluble foreign matters, concentrating the ethyl acetate into an extract, separating and purifying an ethyl acetate sample in a high-speed countercurrent chromatography by one step to prepare the tectorigenin with purity higher than 95%. The method has simple operation, high separation efficiency and high purity of products and is suitable for the industrial production.
Description
Technical field
The present invention relates to medical technical field, be specifically related to utilize the method for high-speed countercurrent chromatography separation and purification iris aglycone from iris.
Background technology
Iris (Rhizoma Belamcandae) is is the dry rhizome of Iridaceae iris iris Iris tectorum Maxim; Has effect clearing heat and detoxicating, the dissolving phlegm relieve sore throat; Be mainly used in diseases such as swelling and pain in the throat, productive cough asthma; For Chinese Pharmacopoeia one one of version in 2005 record kind (Chinese Pharmacopoeia Commission. Pharmacopoeia of People's Republic of China [S], one one, Beijing: the .2005:28-29. of Chemical Industry Press).Main product in Sichuan, province such as Chongqing, Guizhou, Yunnan, Guangxi.Modern study is found, contains abundant osajin composition in the iris, wherein iris aglycone be in the iris main active ingredient (reward logistics, Qin Minjian, Wu Jinrong. the chemical ingredients of iris [J]. Chinese natural drug, 2007,5 (4): 312-314.).
(high speed counter-current chromatography HSCCC) is a kind of novel LLC separation method to high-speed countercurrent chromatography.It is different from traditional liquid phase chromatography; Can realize high efficiency separation and analysis at short notice; Having does not need solid carrier; Avoided sample separation and solid carrier surface to produce chemical reaction and advantages such as sex change and irreversible adsorption, and low to the The pretreatment requirement, be applicable to the separation of crude extract.In recent years, HSCCC widely uses in fields such as bio-science, medicine and chemical industry, and is more outstanding in the separation preparation of natural plant activeconstituents, and the amount of preparation and preparation purity all are greatly improved.
Iris aglycone in the traditional method separation and purification irises such as domestic and international at present many employing column chromatographies (Yuan Chongjun, Wang Jia, Chen Shuai, etc. iris The Chemical Constituents [J]. research and development of natural products, 2008,20 (9): 444-446,449; Qiu Qinghao, Zhang Zhi state, Wang Jianhua; Deng. osajin chemical constitution study [J] in the iris, Chinese medicinal materials, 2009; 32 (9): 1392-1394.), these class methods deleterious organic solvents such as a large amount of chloroforms, methyl alcohol that use can cause environmental pollution more; And the weak point of column chromatography is that separation cycle is long, and the recovery is low, and separating effect is undesirable.Recently, (Fan Guorong, Zhou Tingting, Feng Chao such as Fan Guorong; Xie Ying, Wen Jun, Cui Lijun, Chen Danxia; Li Ji, Xie Rui, Wei Hua opens crane. and the secondary high speed adverse current chromatogram separates the method for osajin monomeric compound in the blackberry lily; Publication number: CN101357933A, open day: on February 4th, 2009) utilize HSCCC from the equal not congener of iris blackberry lily (Belamcandachinensis (L.) DC.), to separate and obtained iris aglycone, method therefor was loaded down with trivial details, length consuming time.In the existing document, do not see that HSCCC is used for the report that a step prepares separation iris iris aglycone fast.Therefore, the invention discloses HSCCC one step from iris prepares the method for separating iris aglycone.
Summary of the invention
The object of the present invention is to provide a kind of fast, the novel method of iris aglycone in the high efficiency separation iris, promptly use high-speed countercurrent chromatography one step from iris to separate the method for preparing iris aglycone.The purity that adopts the performance liquid chromatography area normalization method to measure iris aglycone in the finished product reaches more than 95%.This method separation condition is gentle, and separation efficiency is high, and disengaging time is short, can obtain highly purified iris aglycone.
The present invention provide a kind of from iris the method for separation and purification iris aglycone, comprise the steps:
Step (1)
Get the iris medicinal material, sample is pulverized through kibbler, crosses 40 mesh sieves; Take by weighing exsiccant iris powder, add methyl alcohol, soaking at room temperature is extracted, and filters; Filter residue is repeated above-mentioned steps, extract 2 times again, merging filtrate, concentrating under reduced pressure become the medicinal extract shape, get the extract of iris;
Step (2)
After the iris extract that above-mentioned steps is made adds a spot of water-dispersion, use ethyl acetate extraction, the concentrating under reduced pressure ethyl acetate solution promptly gets ETHYLE ACETATE medicinal extract;
Step (3)
Preparation two-phase solvent system in separating funnel, hold over night at room temperature behind the shake well; Get last phase (as the stationary phase of HSCCC) and following (as the moving phase of HSCCC) mutually, ultrasonic degas in the separating funnel before the use respectively; Get ETHYLE ACETATE medicinal extract in the step (2) with the upper and lower phase sample dissolution of equal volume;
Step (4)
With injecting the high-speed counter-current chromatograph separator tube on the solvent systems mutually; Adjust engine speed after treating to be full of whole pipeline mutually, pump into down phase, treat that moving phase flows out from column outlet; Two in separator tube, reach running balance after, by the sample solution in the sampling valve implantation step (3); Under the 280nm wavelength, detect, the record color atlas confirms that according to color atlas collection obtains the monomeric compound iris aglycone.
The methyl alcohol weight ratio of iris powder and adding is 1: 20 described in the step (1).
Use methyl alcohol soaking and extracting 24h in the step (1), extract 3 times.
After the iris extract adds a spot of water-dispersion in the step (2), with ethyl acetate extraction more than 5 times.
Solvent systems described in the step (3) is sherwood oil (boiling range is 60-90 ℃): ETHYLE ACETATE: methyl alcohol: and water (1: 6: 4: 3, v/v).
The engine speed of high-speed counter-current chromatograph is 900rpm/min described in the step (4).
The flow rate of mobile phase of high-speed counter-current chromatograph is 2.0mL/min described in the step (4).
The present invention has adopted rational solvent systems, has controlled the conditions such as engine speed, flow rate of mobile phase and detector wavelength of high-speed counter-current chromatograph, can obtain the higher iris aglycone of purity with the inventive method.This separation condition is controlled easily, and disengaging time is short.Because do not introduce soda acid, and separate at normal temperatures, target compound does not change, and has farthest kept the stability of compound.
Embodiment
Embodiment
(1) preparation iris extract:
Get the iris medicinal material, sample is pulverized through kibbler, crosses 40 mesh sieves.Take by weighing exsiccant iris powder 20g, add in the methyl alcohol of 20 times of weight, soaking at room temperature is extracted 24h, filters; Filter residue is repeated above-mentioned steps, extract 2 times again, merging filtrate is evaporated to the medicinal extract shape, gets the iris crude extract.
(2) being further purified of extract:
After the iris extract that above-mentioned steps is made adds the suitable quantity of water suspendible, with ethyl acetate extraction 5 times, remove water-soluble impurity, concentrating under reduced pressure ethyl acetate solution to medicinal extract shape obtains ETHYLE ACETATE medicinal extract 2.9g.
(3) high speed adverse current chromatogram separates iris aglycone:
A. the preparation of solvent systems and sample solution
In separating funnel, prepare sherwood oil (boiling range 60-90 ℃): ETHYLE ACETATE: methyl alcohol: water (1: 6: 4: 3, v/v) two phase solvent system, hold over night at room temperature behind the shake well.Get last phase (as the stationary phase of HSCCC) and following (as the moving phase of HSCCC) mutually, ultrasonic degas 30min in the separating funnel before the use respectively.Get step (2) ETHYLE ACETATE medicinal extract 2.0g, go up phased soln sample under phase and the 1mL with 1mL, subsequent use.
B. high speed adverse current chromatogram separates the preparation process
With pumping into mutually in the high-speed counter-current chromatograph separator tube on the solvent systems, treat to be full of mutually adjustment engine speed 900rpm/min behind the whole pipeline with Peak Flow Rate (9.99mL/min), pump into down phase with the 2.0mL/min flow velocity again; Treat that moving phase flows out from column outlet; Two in separator tube, reach running balance after, inject the sample solution of a by sampling valve, in the 280nm wavelength to effluent UV-detector continuous detecting; The record color atlas is collected the flow point I according to color atlas.
C. the analysis of iris aglycone and evaluation
Use the performance liquid chromatography area normalization method to carry out purity detecting: to use Symmetry C to separating the flow point I that obtains among the b
18Chromatographic column (25mm * 4.6mm i.d., 5 μ m), acetonitrile-water is a moving phase, gradient elution, 0 to 20min, the acetonitrile ratio is 40%, and 20 to 30min, the acetonitrile ratio is raised to 50% by 40%, and the detection wavelength is 280nm, and the mensuration temperature is a room temperature.Recording the flow point I is simple spike, and purity is 95.2%.The flow point I is volatilized, get chemical compounds I white needle 15.7mg, use
1H NMR with
13C NMR carries out the structure evaluation to I and contrasts with data in literature, confirms that chemical compounds I is an iris aglycone.
Selection about solvent systems of the present invention
The selection of solvent systems is a key issue of using effective constituent in the HSCCC separation and purification natural drug.Different solvent systemss has dissimilar polarity, viscosity, density; The ratio of the upper and lower phase that the same solvent system is different also can produce different dissolving and distribution capability to identical composition; Form the difference of partition ratio, thereby separating effect is produced certain influence.Suitable two-phase solvent system generally should satisfy following pacing items: separation time is shorter than 30s; Two partition ratios in mutually of target compound are answered 0.5＜K＜2 (OuyangX K, Jin M C, He C H.Sep.Purif.Technol., 2007,56 (3): 319～324.).
The mensuration of partition ratio: get the 5mg crude extract in the 5mL test tube, add upper and lower phase solvent each 2mL of system that reaches partition equilibrium in advance respectively, standing demix behind the concuss 1min, the HPLC method detects target compound distributes upper and lower mutually, and last phase peak area is A
1, following phase peak area is A
2, partition ratio K=A
1/ A
2
The partition ratio (see table) of this measuring target compound in ETHYLE ACETATE-water, ETHYLE ACETATE-methanol-water, n-butanol-water, petroleum ether-ethyl acetate-methanol-water equal solvent system.The result shows: adopt ETHYLE ACETATE-water (1: 1, v/v) ETHYLE ACETATE-methanol-water (3: 1: 3, v/v) n-butanol-water is (1: 1; V/v) petroleum ether-ethyl acetate-methanol-water (1: 5: 2: 4, v/v), partition ratio is bigger; The chromatographic peak broadening, and disengaging time is long; Employing petroleum ether-ethyl acetate-methanol-water (1: 6: 4: 3, in the time of v/v), partition ratio is reasonable, and the distribution time is short, can realize the separation of target compound.Therefore, and experiment employing sherwood oil (60-90 ℃)-ETHYLE ACETATE-methanol-water (1: 6: 4: 3, v/v) system carries out the HSCCC separation and purification.
The partition ratio of table iris aglycone in the different solvents system
Compound
1H NMR with
13The appraising datum of C NMR:
1H NMR (DMSO-d
6) δ 13.06 (1H, s, 5-OH), 10.78 (1H, s, 7-OH), 9.59 (1H, s, 4 '-OH), 8.34 (1H; S, H-2), 7.37 (2H, d, J=8.4Hz, H-2 ', H-6 '), 6.82 (2H, d; J=8.4Hz, H-3 ', H-5 '), 6.50 (1H, s, H-8), 3.75 (3H, s, 6-OCH
3).
13C?NMR(DMSO-d
6)δ180.5(C-4)，157.4(C-5)，157.3(C-4′)，154.0(C-7)，153.2(C-9)，152.6(C-2)，131.3(C-6)，130.1(C-2′，6′)，121.7(C-1′)，121.1(C-3)，115.0(C-3′，5′)，104.7(C-10)，60.0(6-OCH
3)。Be accredited as 5,7 through above datumization compound, 4 '-trihydroxy--6-methoxyl group NOVASOY 400, i.e. iris aglycone (tectorigenin).
Claims (8)
1. high-speed countercurrent chromatography by one step prepares the method for separating iris aglycone in the iris, it is characterized in that comprising the steps:
Step (1)
Get the iris medicinal material, sample is pulverized through kibbler, crosses 40 mesh sieves; Take by weighing exsiccant iris powder, add methyl alcohol, soaking at room temperature is extracted, and filters; Filter residue is repeated above-mentioned steps, extract 2 times again, merging filtrate, concentrating under reduced pressure become the medicinal extract shape, get the extract of iris;
Step (2)
After the iris extract that above-mentioned steps is made adds a spot of water-dispersion, use ethyl acetate extraction, the concentrating under reduced pressure ethyl acetate solution promptly gets ETHYLE ACETATE medicinal extract;
Step (3)
Preparation two-phase solvent system in separating funnel, hold over night at room temperature behind the shake well; Get the upper and lower phase in the separating funnel before the use respectively, ultrasonic degas; Get ETHYLE ACETATE medicinal extract in the step (2) with the upper and lower phase sample dissolution of equal volume; Said solvent systems is that boiling range is 60-90 ℃ sherwood oil, ETHYLE ACETATE, methyl alcohol, a water, and its amount ratio is 1: 6: 4: 3, and v/v;
Step (4)
With injecting the high-speed counter-current chromatograph separator tube on the solvent systems mutually; Adjust engine speed after treating to be full of whole pipeline mutually, pump into down phase, treat that moving phase flows out from column outlet; Two in separator tube, reach running balance after, by the sample solution in the sampling valve implantation step (3); Under the 280nm wavelength, detect, the record color atlas confirms that according to color atlas collection obtains the monomeric compound iris aglycone.
2. high-speed countercurrent chromatography by one step according to claim 1 prepares the method for separating iris aglycone in the iris, it is characterized in that the methyl alcohol weight ratio of iris powder described in the step (1) and adding is 1: 20.
3. high-speed countercurrent chromatography by one step according to claim 1 and 2 prepares the method for separating iris aglycone in the iris, it is characterized in that using methyl alcohol soaking and extracting 24h in the step (1), extracts 3 times.
4. high-speed countercurrent chromatography by one step according to claim 1 and 2 prepares the method for separating iris aglycone in the iris, it is characterized in that the iris extract adds a spot of water-dispersion in the step (2) after, with ethyl acetate extraction more than 5 times.
5. high-speed countercurrent chromatography by one step according to claim 1 and 2 prepares the method for separating iris aglycone in the iris, and the engine speed that it is characterized in that high-speed counter-current chromatograph described in the step (4) is 900rpm.
6. high-speed countercurrent chromatography by one step according to claim 3 prepares the method for separating iris aglycone in the iris, and the engine speed that it is characterized in that high-speed counter-current chromatograph described in the step (4) is 900rpm.
7. high-speed countercurrent chromatography by one step according to claim 1 and 2 prepares the method for separating iris aglycone in the iris, and the flow rate of mobile phase that it is characterized in that high-speed counter-current chromatograph described in the step (4) is 2.0mL/min.
8. high-speed countercurrent chromatography by one step according to claim 3 prepares the method for separating iris aglycone in the iris, and the flow rate of mobile phase that it is characterized in that high-speed counter-current chromatograph described in the step (4) is 2.0mL/min.
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|---|---|---|---|---|
| CN101357933A (en) * | 2008-09-09 | 2009-02-04 | 中国人民解放军第二军医大学 | Method for separating isoflavones monomeric compound in blackberry lily by high speed countercurrent chromatography |
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| CN101357933A (en) * | 2008-09-09 | 2009-02-04 | 中国人民解放军第二军医大学 | Method for separating isoflavones monomeric compound in blackberry lily by high speed countercurrent chromatography |
Non-Patent Citations (2)
| Title |
|---|
| 袁崇均等.川射干化学成分的研究.《天然产物研究与开发》.2008,第20卷444-446,449. * |
| 邱庆浩等.川射干中异黄酮类化学成分研究.《中药材》.2009,第32卷(第9期),1392-1394. * |
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