CN106589050A - Method for separating and preparing mastic monomer - Google Patents
Method for separating and preparing mastic monomer Download PDFInfo
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- CN106589050A CN106589050A CN201611131125.3A CN201611131125A CN106589050A CN 106589050 A CN106589050 A CN 106589050A CN 201611131125 A CN201611131125 A CN 201611131125A CN 106589050 A CN106589050 A CN 106589050A
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- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07J—STEROIDS
- C07J75/00—Processes for the preparation of steroids in general
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- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07J—STEROIDS
- C07J9/00—Normal steroids containing carbon, hydrogen, halogen or oxygen substituted in position 17 beta by a chain of more than two carbon atoms, e.g. cholane, cholestane, coprostane
- C07J9/005—Normal steroids containing carbon, hydrogen, halogen or oxygen substituted in position 17 beta by a chain of more than two carbon atoms, e.g. cholane, cholestane, coprostane containing a carboxylic function directly attached or attached by a chain containing only carbon atoms to the cyclopenta[a]hydrophenanthrene skeleton
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Abstract
The invention discloses a method for separating and preparing a mastic monomer. The method comprises the following steps: (1) preparing a coarse mastic extract: smashing a mastic medicine, heating and extracting the mastic medicine with an ethanol solution for multiple times, combining filtrate, and carrying out decompression rotary evaporation and freeze drying to obtain the coarse mastic extract; (2) dissolving the coarse mastic extract obtained in the step (1) with water, extracting the coarse mastic extract respectively with petroleum ether, ethyl acetate and n-butyl alcohol, filtering extract liquor, and carrying out decompression concentration to respectively obtain petroleum ether, ethyl acetate, n-butyl alcohol and water extraction sections; (3) separating a terpene monomer: separating and purifying the ethyl acetate extraction section in the step (2) by adopting a high-speed counter-current chromatography method to obtain a terpene compound. The method disclosed by the invention overcomes the shortcomings that the conventional prepration method has complicated operation, sample dead adsorption loss, low yield and the like. The method is high in efficiency, easy to operate, large in preparation amount and low in comprehensive cost, and has an extremely good popularization and use value.
Description
Technical field
The invention belongs to the efficient preparing technical field of Effective Component of Chinese Medicine, and in particular to a kind of high speed adverse current chromatogram is separated
The method of terpene monomer in purifying frankincense.
Background technology
Frankincense is Burseraceae Boswellia Cattell Boswellia carterii (Boswellia carterii Birdw.) and belongs to plant together
The resin that thing Bao is oozed out up to Boswellia carterii (Boswellia bhaw-dajiana Birdw.) bark, main product is in Somalia, Ethiopia Russia
Than Asia and other places.The pungent scattered temperature of frankincense is led to, and the effect of with promoting blood circulation, pain relieving, detumescence, myogenic, cures mainly qi and blood stagnation, trusted subordinate's pain, carbuncle
Epidemic disease pyogenic infections, traumatic injury, dysmenorrhoea, postpartum stasis etc..Research finds that frankincense has the medicine such as anti-inflammatory, antitumor, anti-oxidant, hypoglycemic
Reason is acted on.For preparation, domestic frankincense class medicine is mainly compound preparation, and the single preparations of ephedrine such as tablet, external application of frankincense is soft
Cream only sees overseas market.Clinic is used for dysmenorrhoea, amenorrhoea, gastral cavilty, pain, arthralgia due to wind-dampness, injuries from falls as well and sore, carbuncle and painful swelling, intestines
The diseases such as carbuncle, are a kind of autonomic drugs with extensive exploitation prospect.
The main chemical compositions being separated to from frankincense at present are the chemical combination such as pentacyclic triterpene, tetracyclic triterpene and macrocyclic diterpene
Thing, wherein boswellic acids and the macrocyclic diterpene constituents are the active ingredient of its anti-inflammatory, antitumor action.At present document report, newborn
The isolation and purification method of the active component is column chromatography, such as silica gel column chromatography in perfume (or spice).The method weak point is：Separate week
Phase is long, and the rate of recovery is low, and separating effect is undesirable, causes because long-time is contacted with silica filler when being separated using silica gel column chromatography
Such constituent structure is set to change.
High speed adverse current chromatogram (High-Speed Counter-Current Chromatography, HSCCC) is to utilize two
Phase solvent system sets up a kind of special one-way fluid dynamic equilibrium in the helix tube of high speed rotation, wherein one mutually makees
It is another to retain the separation method of a large amount of fixing phases during continuous wash-out as mobile phase for fixing phase, it is one kind
The liquid luquid partition chromatography isolation technics of continuous high-efficient.Its early CCD method (CCD) for originating from the 1950's, but by
In its equipment bulky complex, frangible, volume system easily emulsification etc. and by a kind of new liquid-liquid distribution method-" adverse current chromatogram "
Replace.70～eighties of 20th century, doctor Ito of U.S. National Institutes have developed the adverse current chromatogram instrument of early stage, including
The planetary centrifugation instrument (CPC) of helix tube, spiral reverse flow chromatograph, the planetary centrifugation instrument of circulation type helix tube, company
Continuous wash-out type centrifugation instrument etc..High speed adverse current chromatogram is the result that countercurrent chromatography has gone through development in nearly 60 years, without solid
Supporter makees fixing phase, it is to avoid the sample loss caused because of Irreversible Adsorption, inactivation, denaturation etc., not only can all reclaim
Sample, and the preparation amount of sample greatly improves, is a kind of suitable for Chinese medicine and the modern equipment of Natural products research, is had
Applied widely, preparation amount is big, simple to operate, it is efficiently quick the advantages of, in biochemistry, bioengineering, medicine, natural products
The fields such as, organic synthesis, environmental analysis, food, geology, material are widely used.
The content of the invention
It is an object of the invention to provide a kind of method that high speed adverse current chromatogram isolates and purifies terpene monomer in frankincense, so as to gram
Take the deficiencies in the prior art, there is provided a kind of simple to operate, product purity is high, the little efficient fast method of sample loss amount.
The technical solution used in the present invention is as follows：
A kind of method that high speed adverse current chromatogram isolates and purifies terpene monomer in frankincense, step is as follows：
(1) preparation of frankincense crude extract：Frankincense medicinal material is taken, is crushed, heating extraction for several times using ethanol solution, merge filter
Liquid, vacuum rotary steam, freeze-drying obtains frankincense crude extract；
(2) the frankincense crude extract that step (1) is obtained is adopted into water dissolves, respectively with petroleum ether, ethyl acetate, n-butanol extraction
Take, extract is filtered, reduced pressure concentration respectively obtains petroleum ether, ethyl acetate, n-butanol and water extraction section；
(3) separation of terpene monomer：The ethyl acetate extraction section in step (2) is carried out using high speed adverse current chromatogram method
Terpenoid is isolated and purified；Wherein, solvent system is consisted of：According to volume ratio, petroleum ether:Ethyl acetate:Methyl alcohol:Water=
1～1.2:0.6～0.9:1～1.2:0.5～0.8, rotating speed is 700～900rpm, and flow velocity is 1～3mL/min.
HSCCC separation is carried out to ethyl acetate extraction section, UV-detector on-line monitoring is collected respectively different fractions and subtracted
Press dry dry, obtain Boscartol A, 3, the 7- dicarbapentaborane-root of gansui -8,24- diene -21- carboxylic acids, 3 α-acetoxyl group -7- carbonyls-sweet
Then -8,24- diene -21- carboxylic acids, 11- keto-β-boswellic acids, acetyl -11- keto-β-boswellic acids, wherein 3,7- dicarbapentaborane-sweet
Then -8,24- diene -21- carboxylic acids and the 3 α-acetoxyl group -7- carbonyls-root of gansui -8,24- diene -21- carboxylic acids are noval chemical compound.
In step (1), the extraction of the pretreatment of frankincense to follow-up terpenoid separates important, and frankincense adopts ethanol
Heating extraction, compared to the extraction of other solvents, enable to terpene monomer in frankincense and farthest extract.
From for separating effect, it is preferred that the mass concentration of ethanol solution is 90～95%, using heating and refluxing extraction side
Method, solid-to-liquid ratio is 1：2～4, extraction time 2～3 times, the time is respectively 1～2h, merging filtrate, vacuum rotary steam, freeze-drying,
Obtain frankincense crude extract.
One of the invention is preferred embodiment, adopts mass concentration for 95% ethanol heating and refluxing extraction, Gu
Liquor ratio is 1:3, to extract three times, the time is respectively 2h, 1h, 1h, and merging filtrate, vacuum rotary steam, freeze-drying obtains frankincense and slightly carries
Thing.
In step (2), the solvent for selecting petroleum ether, ethyl acetate, n-butanol these three opposed polarities is extracted, and can be entered
Alcohol extract in step (1) is carried out polarity rough segmentation by one step, this 5 kinds of ter penoidses is enriched with, compared to other solvents
Extraction, can be such that this 5 kinds of compositions concentrate to greatest extent, and avoid intersection from being present in different extraction positions.The extractant is heavier
Will, it is to ensure that HSCCC can efficiently separate the precondition of 5 kinds of terpenoids.
In step (3), the configuration process of solvent system is：Solvent system is prepared by each solvent ratios, separatory funnel is placed in
In, rear stratification is shaken up, upper and lower two-phase is separated after ready to balance setting time, upper phase is fixing phase, and lower phase is mobile phase.
Ethyl acetate extraction section in step (2) is dissolved in into upper and lower phase standby, further, ethyl acetate extraction
Section is dissolved in volume ratio for 1：It is standby in the mixed liquor of 1 upper and lower phase；The use of the ethyl acetate extraction section and mixed liquor
Magnitude relation is：(0.3～0.5) g/10mL.
The selection of solvent system is very crucial for HSCCC is separated.Up to the present the selection of solvent system is not also filled
Divide, the more complete and theoretical system with directive significance, but selected according to the rich experiences of actual accumulation.The present invention
For the separation of the terpene monomer in frankincense, through substantial amounts of experimental analysis and checking, different solvent systems is selected,
Petroleum ether, ethyl acetate, the solvent system of first alcohol and water and the accurate ratio of each solvent have been obtained, petroleum ether has been obtained:
Ethyl acetate:Methyl alcohol:Volume ratio=1～1.2 of water:0.6～0.9:1～1.2:When 0.5～0.8, each terpene monomer point
It is preferable from effect.Further, the petroleum ether:Ethyl acetate:Methyl alcohol:Volume ratio=1 of water:0.8:1.1:When 0.6, obtain
Separating effect it is best.If using other solvent systems, such as petroleum ether is replaced with into n-hexane, or other proportional quantities
Solvent system can cause this five kinds of terpene monomers can not be completely separable.
Preferably, ultraviolet detection wavelength is 254nm.
Preferably, applied sample amount is 0.3～0.5g, further preferred for 0.3g.
Because high speed adverse current chromatogram is the liquid-liquid chromatography without the need for any solid carrier support, wherein, as the liquid of fixing phase
Body reserving degree in the chromatography column is highly important for the separation process of high speed chromatograph.Preferably, solid phase retention rate is
60～70%, it is further preferred that solid phase retention rate is 67%.
In terms of flow rate of mobile phase, flow rate of mobile phase is unfavorable for soon the reservation of fixing phase, and appearance time can cause soon very much
Peak is poor with peak-to-peak separating degree, although and low flow velocity can meet the requirement for improving fixing phase retention rate, separation wash-out
Time is oversize, and peak shape broadens, and expends substantial amounts of mobile phase, therefore selects suitable flow velocity extremely important to whole separation system.
Through substantial amounts of experimental analysis and checking, selective flow phase flow velocity is 1～3mL/min to the present invention.Preferably 2mL/min.
The rotating speed of splitter and the centrifugal force field of its generation have conclusive impact to the mixability of two-phase, this
It is bright to have selected higher rotating speed, it is 700～900rpm, so that mixing that can be violent between fixing phase and mobile phase, so as to promote
Enter distribution and reduce the resistance of particle transmission.In order to reach more preferable separating effect, preferred rotating speed is 800rpm.
Present invention determine that Optimum separation condition of the high speed adverse current chromatogram method to frankincense terpene monomer：Solvent system is stone
Oily ether:Ethyl acetate:Methyl alcohol:Volume ratio=1 of water:0.8:1.1:0.6, upper phase is fixing phase, and lower phase is mobile phase, at a high speed
Counter-current chromatograph column volume be 300mL, applied sample amount 0.3g, splitter rotating speed be 800rpm, flow rate of mobile phase 2.0mL/min, Gu
Determine phase retention rate 67%, ultraviolet detection wavelength 254nm.
The present invention also protects the compound as shown in formula I：
Wherein, R is carbonyl group or acetyloxy group, specifically as shown in formula II or formula III structure compound.
Compound chemical name shown in formula II and III is respectively：3,7- dicarbapentaborane-the root of gansui -8,24- diene -21- carboxylic acids,
3 α-acetoxyl group -7- carbonyls-the root of gansui -8,24- diene -21- carboxylic acids.The preparation of two kinds of compounds can adopt high-speed counter-current color
The method that spectrum isolates and purifies terpene monomer in frankincense, concrete preparation method is comprised the following steps：
(1) preparation of frankincense crude extract：Frankincense medicinal material is taken, is crushed, heating extraction for several times using ethanol solution, merge filter
Liquid, vacuum rotary steam, freeze-drying obtains frankincense crude extract；
(2) the frankincense crude extract that step (1) is obtained is broken up using water, respectively with petroleum ether, ethyl acetate, n-butanol extraction
Take, extract is filtered, reduced pressure concentration respectively obtains petroleum ether, ethyl acetate, n-butanol and water extraction section；
(3) preparation of compound shown in formula I：
Isolating and purifying for terpenoid is carried out to the ethyl acetate extraction section in step (2) using high speed adverse current chromatogram,
UV-detector is monitored on-line, and efflux corresponding with the 3rd peak and the 4th peak retention time is collected respectively, and is reduced pressure and done
It is dry, obtain compound shown in formula II and III；Wherein, solvent system is consisted of：According to volume ratio, petroleum ether:Ethyl acetate:First
Alcohol:Water=1～1.2:0.6～0.9:1～1.2:0.5～0.8, rotating speed is 700～900rpm, and flow velocity is 1～3mL/min；
The 3rd corresponding efflux of peak retention time is the efflux containing the compound of formula II, the 4th peak retention time pair
The efflux answered is the efflux containing the compound of formula III.
Structure based on two kinds of compounds has altogether the mother nucleus structure of terpenoid, so two kinds of compounds are also
The active ingredient of anti-inflammatory, antitumor action, for frankincense exploitation provide with further reference to.
Above-mentioned technical proposal has the advantages that：
(1) frankincense slightly takes each cut Jing HPLC after thing is separated and carries out purity detecting, and more than 97% sterling is obtained
The Boscartol A, 3,7- dicarbapentaborane-root of gansui -8,24- diene -21- carboxylic acids, the 3 α-acetoxyl group -7- carbonyls-root of gansui -8,24- two
Alkene -21- carboxylic acids, 11- keto-β-boswellic acids, acetyl -11- keto-β-boswellic acids, and 3, the 7- dicarbapentaborane-root of gansui -8,24- bis-
Alkene -21- carboxylic acids and the 3 α-acetoxyl group -7- carbonyls-root of gansui -8,24- diene -21- carboxylic acids are noval chemical compound, and the method is applied to
From frankincense one step purifying prepare the highly purified Boscartol A, 3,7- dicarbapentaborane-root of gansui -8,24- diene -21- carboxylic acids, 3
α-acetoxyl group -7- carbonyls-the root of gansui -8,24- diene -21- carboxylic acids, 11- keto-β-boswellic acids, acetyl -11- carbonyl-β-frankincense
Acid.
(2) in order to preferably separate frankincense terpenoid, screening and optimizing of the present invention obtains a set of for frankincense raw material point
From the technological parameter of terpenoid, using the technological parameter after screening and optimizing of the present invention, can isolated purity it is higher
Terpenoid.
(3) the present invention overcomes traditional preparation methods complex operation, the dead adsorption loss of sample, the low shortcoming of yield.
This method efficiency high, simple to operate, good stability, preparation amount is big, and integrated cost is low, has good value for applications.
Description of the drawings
Fig. 1 is the high-efficient liquid phase chromatogram of frankincense ethyl acetate extract；
Fig. 2 isolates and purifies the chromatogram of frankincense for high speed adverse current chromatogram；
Fig. 3 is to isolate and purify the high-efficient liquid phase chromatogram for obtaining Boscartol A；
Fig. 4 is to isolate and purify the high-efficient liquid phase chromatogram for obtaining the 3,7- dicarbapentaborane-root of gansui -8,24- diene -21- carboxylic acids；
Fig. 5 is to isolate and purify the efficient liquid phase for obtaining the 3 α-acetoxyl group -7- carbonyls-root of gansui -8,24- diene -21- carboxylic acids
Chromatogram；
Fig. 6 is to isolate and purify the high-efficient liquid phase chromatogram for obtaining 11- keto-β-boswellic acids；
Fig. 7 is to isolate and purify the high-efficient liquid phase chromatogram for obtaining acetyl -11- carbonyls-masticinic acid；
Fig. 8 is the structural formula of five kinds of terpene monomers.
Specific embodiment
With reference to embodiment, the present invention is further described.
Embodiment 1
A kind of method that high speed adverse current chromatogram isolates and purifies terpene monomer in frankincense, comprises the following steps：Frankincense medicinal material is taken,
It is crushed to 40-60 mesh, 95% ethanol heating and refluxing extraction, solid-to-liquid ratio is 1:3, to extract three times, the time is respectively 2h, 1h, 1h, closes
And filtrate, vacuum rotary steam, freeze-drying, obtain frankincense crude extract；Frankincense crude extract is broken up with water, respectively with petroleum ether, acetic acid
Ethyl ester, extracting n-butyl alcohol, extract is filtered, and reduced pressure concentration respectively obtains petroleum ether, ethyl acetate, n-butanol and water extraction section,
The HPLC analysis charts of Ethyl acetate fraction, as shown in Figure 1.
Terpenoid is carried out using high speed adverse current chromatogram to gained frankincense ethyl acetate extract to isolate and purify：
Using petroleum ether:Ethyl acetate:Methyl alcohol:Water=1:0.8:1.1:0.6 is solvent system, and upper phase is fixing phase, under
It is mutually mobile phase, high-speed counter-current chromatograph column volume is 300mL, applied sample amount 0.3g, splitter rotating speed is 800rpm, flow velocity
2.0mL/min, fixing phase retention rate 67%, Detection wavelength 254nm.HSCCC separation is carried out to frankincense ethyl acetate extraction section, it is purple
External detector is monitored on-line, and different fractions and drying under reduced pressure are collected respectively, corresponding high-purity compound is obtained, as shown in Fig. 2 a is
The corresponding efflux of second peak retention time, is Boscartol A；B is the corresponding efflux of first peak retention time：For
3,7- dicarbapentaborane-the root of gansui -8,24- diene -21- carboxylic acids；C is the 4th corresponding efflux of peak retention time, is 3 α-acetyl oxygen
Base -7- carbonyls-the root of gansui -8,24- diene -21- carboxylic acids；D be the 3rd corresponding efflux of peak retention time, be 11- carbonyl-β-
Masticinic acid；E is the 5th corresponding efflux of peak retention time, is acetyl -11- keto-β-boswellic acids.
Specifically operating procedure is：Solvent system is prepared by above-mentioned solvent ratios, in being placed in separatory funnel, is stood after shaking up
Layering, ready to balance for a period of time afterwards separates upper and lower two-phase, and upper phase is fixing phase, and lower phase is mobile phase, takes 0.3g frankincense acetic acid
Ethyl ester extraction section, is dissolved on 5mL mutually stand-by with phase under 5mL.It is inverse with the semi-preparative high speed that field company develops using Shanghai
Flow chromatography instrument, it is (by polyfluortetraethylene pipe multilayer by plunger displacement pump, sampling valve, Ultraviolet Detector, recorder and chromatography column
The spiral tube being wound, capacity is 300mL) etc. composition, first fixing phase pump is filled into chromatographic isolation with certain flow rate
Post, termination of pumping.Opening speed controller, rotates forward the chromatographic chromatography column of high velocity stream, during turn up 800rpm, arranges flowing
Phase flow velocity is 2.0mL/min, starts pump mobile phase, until biphase equilibrium, not termination of pumping, then make sampling valve in sample introduction state,
The sample for having dissolved is injected in the liquid storage tube of counter-current chromatograph sampling valve with syringe, rotation sampling valve makes to connect column state
Sample enters chromatography column.Then target component is received according to detector ultraviolet spectrogram (Fig. 2), obtains Boscartol A
(3.4mg, Fig. 3), 3, the 7- dicarbapentaborane-root of gansui -8,24- diene -21- carboxylic acids (8.5mg, Fig. 4), 3 α-acetoxyl group -7- carbonyls -
The root of gansui -8,24- diene -21- carboxylic acids (4.2mg, Fig. 5), 11- keto-β-boswellic acids (50.1mg, Fig. 6), acetyl -11- carbonyls -
Masticinic acid (60.4mg, Fig. 7), HPLC purity assays are more than 97%.
Using efficient liquid phase chromatographic analysis separator, liquid-phase condition：Kromasil 100-5C18Post (4.6 × 250mm) is purple
Outer Detection wavelength 254nm, column temperature：25 DEG C, flow velocity：1.0mL/min, sample size：10 μ L, mobile phase is using acetonitrile (A) and water-soluble
Liquid (B) gradient elution, gradient condition is as follows：0-10min, 40%-60%A；10-30min, 60%-80%A；30-40min,
100%A；40-50min, 100%A.
Structural Identification：To isolated monomer component application Agilent 5973N mass spectrographs and Burker 400MHz cores
Resonance spectrometer carries out respectively MS, and the measure of H NMR spectroscopy, the data obtained is as follows, and structural formula is shown in Fig. 8：
Boscartol A：ESI-MS,m/z 325.5[M+Na]+.1H-NMR(DMSO-d6,400MHz)δ:1.09(3H,s,
Me-14),1.11(3H,s,Me-11),1.73(3H,s,Me-20),4.01(2H,s,H-19),4.63(1H,s,Hb-12),
4.65(1H,s,Ha- 12), 5.24 (1H, d, J=15.2Hz, H-15), 5.75 (1H, d, J=11.2Hz, H-17), 6.21 (1H,
Dd, J=11.2,15.2Hz, H-16).13C-NMR(DMSO-d6,100MHz)δ:13.1(C-14),21.8(C-20),24.3(C-
13),26.4(C-8),26.5(C-11),26.9(C-2),28.7(C-7),31.8(C-6),38.6(C-9),41.8(C-3),
53.1(C-1),53.2(C-5),60.2(C-19),79.2(C-4),106.8(C-12),120.4(C-16),126.9(C-17),
135.2(C-18),143.7(C-15),153.4(C-10).
3,7- dicarbapentaborane-the root of gansui -8,24- diene -21- carboxylic acids：ESI-MS,m/z 467.3[M-H]-.1H-NMR(DMSO-
d6,400MHz)δ:0.70(3H,s,Me-18),0.89(3H,s,Me-30),0.97(3H,s,Me-28),1.03(3H,s,Me-
29), 1.21 (3H, s, Me-19), 1.53 (3H, s, Me-27), 1.64 (3H, s, Me-26), 5.08 (1H, t, J=6.8Hz, H-
24).13C-NMR(DMSO-d6,100MHz)δ:16.0(C-18),17.9(C-27),17.9(C-19),21.5(C-29),23.7
(C-11),24.6(C-26),24.7(C-23),25.9(C-28),26.0(C-16),27.3(C-12),28.0(C-15),31.5
(C-22),32.7(C-30),34.6(C-2),34.8(C-1),36.1(C-6),39.1(C-10),44.3(C-13),45.5(C-
17),47.1(C-4),47.3(C-14),47.7(C-20),48.8(C-5),124.3(C-24),131.6(C-25),138.2
(C-8),164.7(C-9),177.6(C-21),196.9(C-7),214.3(C-3).
3 α-acetoxyl group -7- carbonyls-the root of gansui -8,24- diene -21- carboxylic acids：ESI-MS,m/z 513.4[M+H]+.1H-
NMR(DMSO-d6,400MHz)δ:0.73(3H,s,Me-18),0.79(3H,s,Me-30),0.88(3H,s,Me-28),0.95
(3H,s,Me-29),1.02(3H,s,Me-19),1.54(3H,s,Me-27),1.64(3H,s,Me-26),4.56(1H,brs),
5.08 (1H, t, J=7.2Hz, H-24), 12.1 (1H, brs, COOH), 2.01 (3H, s,CH 3 CO-).13C-NMR(DMSO-d6,
100MHz)δ:16.1(C-18),17.9(C-27),18.3(C-19),21.5(C-11),22.9(C-2),23.2(C-26),
24.6(C-30),25.9(C-23),26.0(C-28),27.1(C-29),27.4(C-16),28.0(C-12),29.5(C-15),
31.5(C-22),32.7(C-1),35.4(C-6),36.7(C-4),39.2(C-10),43.6(C-13),44.3(C-14),
45.6(C-20),47.2(C-17),47.8(C-5),76.6(C-3),124.3(C-24),131.6(C-25),138.0(C-8),
166.1(C-9),177.6(C-21),197.0(C-7),21.4(CH 3 CO-),170.3(CH3 CO-).
11- keto-β-boswellic acids：ESI-MS,m/z 471.4[M+H]+.1H-NMR(CDCl3,400MHz)δ:0.81(3H,
D, J=6.0Hz, Me-29), 0.84 (3H, s, Me-30), 0.96 (3H, s, Me-28), 1.15 (3H, s, Me-25), 1.20 (3H,
s,Me-26),1.33(3H,s,Me-27),1.36(3H,s,Me-23),4.10(1H,brs,H-3),5.57(1H,brs,H-
12).13C-NMR(CDCl3,100MHz)δ:13.2(C-25),17.4(C-29),18.4(C-26),18.8(C-6),20.5(C-
27),21.1(C-28),24.3(C-23),26.2(C-2),27.2(C-16),27.5(C-21),28.9(C-30),30.9(C-
7),32.9(C-15),34.0(C-1),37.5(C-10),39.3(C-19),39.3(C-20),40.9(C-22),43.8(C-
8),45.1(C-14),47.3(C-4),48.8(C-5),59.0(C-18),60.4(C-9),70.5(C-3),130.5(C-12),
165.1(C-13),182.3(C-24),199.6(C-11).
Acetyl -11- carbonyls-masticinic acid：ESI-MS,m/z 471.4[M+H]+.1H-NMR(CDCl3,400MHz)δ:0.79
(3H, d, J=6.0Hz, Me-29), 0.82 (3H, s, Me-28), 0.94 (3H, s, Me-30), 1.14 (3H, s, Me-25), 1.19
(3H,s,Me-26),1.34(3H,s,Me-27),1.23(3H,s,Me-23),2.08(3H,s,CH 3 CO-),5.30(1H,brs,
H-3),5.55(1H,brs,H-12).13C-NMR(CDCl3,100MHz)δ:13.2(C-25),17.4(C-29),18.4(C-
26),18.8(C-6),20.5(C-30),21.1(C-27),23.5(C-2),23.8(C-23),27.2(C-16),27.4(C-
15),28.8(C-28),32.8(C-7),34.6(C-1),37.4(C-10),39.3(C-19),39.3(C-20),40.9(C-
22),43.8(C-8),45.1(C-14),46.5(C-21),46.5(C-4),50.4(C-5),59.0(C-18),60.3(C-9),
73.1(C-3),130.5(C-12),166.1(C-13),181.8(C-24),199.4(C-11),21.3(CH 3 CO-),170.3
(CH3 CO-).
Although the above-mentioned accompanying drawing that combines is described to the specific embodiment of the present invention, not to present invention protection model
The restriction enclosed, one of ordinary skill in the art should be understood that on the basis of technical scheme those skilled in the art are not
Need the various modifications made by paying creative work or deformation still within protection scope of the present invention.
Claims (10)
1. a kind of method that high speed adverse current chromatogram isolates and purifies terpene monomer in frankincense, is characterized in that, step is as follows：
(1) preparation of frankincense crude extract：Frankincense medicinal material is taken, is crushed, heating extraction for several times using ethanol solution, merging filtrate subtracts
Pressure revolving, freeze-drying obtains frankincense crude extract；
(2) the frankincense crude extract that step (1) is obtained is adopted into water dissolves, respectively with petroleum ether, ethyl acetate, extracting n-butyl alcohol,
Extract is filtered, and reduced pressure concentration respectively obtains petroleum ether, ethyl acetate, n-butanol and water extraction section；
(3) separation of terpene monomer：Terpene is carried out to the ethyl acetate extraction section in step (2) using high speed adverse current chromatogram method
Compound is isolated and purified；
Wherein, solvent system is consisted of：According to volume ratio, petroleum ether:Ethyl acetate:Methyl alcohol:Water=1～1.2:0.6～0.9:1
～1.2:0.5～0.8, rotating speed is 700～900rpm, and flow rate of mobile phase is 1～3mL/min.
2. the method for claim 1, is characterized in that：In step (1), the mass concentration of ethanol solution is 90～95%,
Using heating and refluxing extraction method, solid-to-liquid ratio is 1：2～4, extraction time 2～3 times, the time is respectively 1～2h, merging filtrate,
Vacuum rotary steam, freeze-drying obtains frankincense crude extract；
Preferably, the mass concentration of ethanol solution is 90～95%, and using heating and refluxing extraction method, solid-to-liquid ratio is 1：2～4,
Extraction time 2～3 times, the time is respectively 1～2h, and merging filtrate, vacuum rotary steam, freeze-drying obtains frankincense crude extract.
3. the method for claim 1, is characterized in that：In step (3), the configuration process of solvent system is：According to oil
Ether:Ethyl acetate:Methyl alcohol:Water=1～1.2:0.6～0.9:1～1.2:0.5～0.8 volume ratio prepares solvent system, puts
In separatory funnel, shake up rear stratification, separate upper and lower two-phase after ready to balance setting time, upper phase be fixing phase, lower phase
For mobile phase.
4. the method for claim 1, is characterized in that：In step (3), the petroleum ether:Ethyl acetate:Methyl alcohol:Water
Volume ratio=1:0.8:1.1:0.6.
5. the method for claim 1, is characterized in that：In step (3), ultraviolet detection wavelength is 254nm.
6. the method for claim 1, is characterized in that：In step (3), solid phase retention rate is 60～70%, it is preferred that Gu
Phase retention rate is 67%.
7. the method for claim 1, is characterized in that：In step (3), splitter rotating speed is 800rpm；Flow rate of mobile phase
For 2mL/min.
8. the method for claim 1, is characterized in that：In step (3), separation condition is set to：Solvent system is oil
Ether:Ethyl acetate:Methyl alcohol:Volume ratio=1 of water:0.8:1.1:0.6, upper phase is fixing phase, and lower phase is mobile phase, inverse at a high speed
Flow chromatography instrument column volume is 300mL, and applied sample amount 0.3g, splitter rotating speed is 800rpm, flow rate of mobile phase 2.0mL/min, is fixed
Phase retention rate 67%, ultraviolet detection wavelength 248nm.
9. the compound shown in formula I, is characterized in that：
Wherein, R is carbonyl group or acetyloxy group.
10. the preparation method of the compound shown in formula I, is characterized in that, comprise the following steps：
(1) preparation of frankincense crude extract：Frankincense medicinal material is taken, is crushed, heating extraction for several times using ethanol solution, merging filtrate subtracts
Pressure revolving, freeze-drying obtains frankincense crude extract；
(2) the frankincense crude extract that step (1) is obtained is broken up using water, respectively with petroleum ether, ethyl acetate, extracting n-butyl alcohol,
Extract is filtered, and reduced pressure concentration respectively obtains petroleum ether, ethyl acetate, n-butanol and water extraction section；
(3) preparation of compound shown in formula I：
Isolating and purifying for terpenoid is carried out to the ethyl acetate extraction section in step (2) using high speed adverse current chromatogram, it is ultraviolet
Detector is monitored on-line, and efflux corresponding with the 3rd peak and the 4th peak retention time, and drying under reduced pressure are collected respectively, is obtained
To compound shown in formula II and III；Wherein, solvent system is consisted of：According to volume ratio, petroleum ether:Ethyl acetate:Methyl alcohol:Water=
1～1.2:0.6～0.9:1～1.2:0.5～0.8, rotating speed is 700～900rpm, and flow velocity is 1～3mL/min.
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| CN108689969A (en) * | 2018-05-15 | 2018-10-23 | 山东省分析测试中心 | A method of isolating and purifying aromadendrane type diterpene-kind compound in frankincense |
| CN111196832A (en) * | 2018-11-20 | 2020-05-26 | 沈阳药科大学 | Kansui alkyl type triterpene compound and preparation method thereof |
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| CN111196832B (en) * | 2018-11-20 | 2021-03-23 | 沈阳药科大学 | Kansui alkyl type triterpene compound and preparation method thereof |
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