CN104211690B - Method for separating and purifying mangiferin from aquilaria sinensis leaves - Google Patents
Method for separating and purifying mangiferin from aquilaria sinensis leaves Download PDFInfo
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- CN104211690B CN104211690B CN201410428303.3A CN201410428303A CN104211690B CN 104211690 B CN104211690 B CN 104211690B CN 201410428303 A CN201410428303 A CN 201410428303A CN 104211690 B CN104211690 B CN 104211690B
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- C07D407/00—Heterocyclic compounds containing two or more hetero rings, at least one ring having oxygen atoms as the only ring hetero atoms, not provided for by group C07D405/00
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Abstract
The invention discloses a method for separating and purifying mangiferin from aquilaria sinensis leaves. The method is used for solving the problems such as low purity, low yield and complex operation of the mangiferin separated by virtue of the existing process. The method comprises the following step: quickly preparing the high-purity mangiferin by use of the aquilaria sinensis leaves used as raw materials by virtue of adopting a method of combining normal phase column chromatography with high-speed counter-current chromatography. The method has the advantages of great preparation amount, high efficiency, quickness, low cost, simplicity in operation, high product quality and no pollution; and moreover, a solvent can be recycled, the solvent cost and the solvent treatment cost can be lowered, and the method can be widely applied to industrial production.
Description
【Technical field】
The invention belongs to the extraction separation and purification field of native compound, and in particular to be combined with HSCCC using column chromatography
The method that technology quickly prepares high-purity compound.
【Background technology】
Lignum Aquilariae Resinatum (Aquilaria sinensis), also known as Aquilaria sinensis (Lour.) Spreng., agallochum, Aquilaria sinensis, tooth perfume, daughter's perfume (or spice) etc., is auspicious
Fragrant section eaglewood, aiphyllium is the peculiar Chinese Second Class Key Protected Plant of China, on Guangdong, Hainan, Guangxi, Fujian and other places
There is plantation, be the conventional rare medicinal herbss of the traditional Chinese medical science.Lignum Aquilariae Resinatum heartwood is pungent, bitter, tepor, with sending down the abnormal ascending QI adjust in, warming the kidney pain relieving work(
Effect, be usually used in chest and abdomen pain, uncomfortable in chest, vomiting singultuss, borborygmus have loose bowels, the disease such as dyspnea due to adverseness of QI.The resiniferous heartwood of Lignum Aquilariae Resinatum tree body
(being commonly called as Lignum Aquilariae Resinatum), can make perfume base, and to control gastropathy specific drug；Bark fiber is flexible, Se Bai and it is careful, fine paper can be made former
Material and synthetic cotton；Xylem can extract aromatic oil, flower can extractum processed, be a kind of higher plant of economic benefit.Its medicinal part
For grease-contained heart wood or resin (being commonly called as Edgeworthia chrysantha Lindl.).Under natural conditions, Edgeworthia chrysantha Lindl. needs just carry out under special circumstances, and the cycle is
10 years even longer, and artificial growth is employed new technology and makes Lignum Aquilariae Resinatum Edgeworthia chrysantha Lindl. in recent years, though shortening the Lignum Aquilariae Resinatum Edgeworthia chrysantha Lindl. cycle, also will
5～8 years.And resourceful Lignum Aquilariae Resinatum leaveves part, especially manually promote the tree body of Edgeworthia chrysantha Lindl. adopt the leaf after perfume (or spice) frequently as
Waste process, causes the wasting of resources.The present invention using Lignum Aquilariae Resinatum leaveves as raw material, using normal phase column chromatography and high-speed counter-current color
Spectrum multiple techniques, it is quick to prepare high-purity blood-sugar decreasing active chimonin, as the material of blood sugar lowering series of products.
Chimonin (mangiferin), also known as mangiferin, mangiferin, is tetrahydroxy pyrrone carbon glycoside, the double benzene pyrrones of category
Class compound.Chimonin is present in plant extensively, contains in Anacardiaceae plant Fructus Mangifera Indicae (Mangifera indica L.) blade
Measure as 0.33%～2.98%；In the liliaceous plant Rhizoma Anemarrhenae (Anemarrhenaasphodeloides Bunge) dried root
Content be 0.320%～0.325%；In Hypericaceae plant Herba Hyperici Sampsonii (Hypericum sampsonii Hance) herb
Content be 0.048%～0.66%；In gentianaceae plant Herba Swertiae bimaculatae (Swertia davidii Franch.) herb
Content is 0.094%～0.216%.Discovered in recent years is also distributed in Lignum Aquilariae Resinatum, and content is 2.76%～3.24% (handsome Europe
Deng. the extraction process [J] of chimonin in Lignum Aquilariae Resinatum leaf. forestry science and technology is developed, and 2013,27 (5):101-104.).
Chimonin has preferably physiologically active.In terms of hypoglycemic activity, S.Muruganandan etc. is sent out by research
Existing, chimonin has hyperglycemia, lipidemia, antiatherogenic effect, proposes that chimonin can be used for diabetes, tremulous pulse
Auxiliary treatment (S.Muruganandan, the et al.Effect of mangiferin of the related cardiovascular complication such as gruel type
on hyperglycemia and atherogenicity in streptozotocin diabetic rats.Journal
of Ethnopharmacology.97(2005):497-501.)；And Yao-Wu Liu, Prabhu Sukumaran Nair etc.
Reconfirm that chimonin has effect (Yao-Wu Liu, the et al.Up-regulation of of hyperglycemia, lipidemia
glyoxalase 1by mangiferin prevents diabetic nephropathy progression in
streptozotocin-induced diabetic rats.European Journal of Pharmacology(2013)1-
10.)；The studies above shows that chimonin has significantly hypoglycemic activity.
Additionally, chimonin radiation protection, reduce cause because of active oxygen natural death of cerebral cells, improved by antioxidative approach
Heart defense function, the poisoning of anti-nerves, antiinflammatory, antibacterial, bring down a fever, ease pain, anticancer, anti-Parkinson syndrome, anti-A Erzi it is extra large
The aspects such as Mo's disease show good physiologically active, are widely used in medicine, cosmetics, health product.But, due to
The reason such as separation preparation complex operation, long preparation period, the response rate be low, low in economic efficiency, with chimonin as it is main into
The Related product for dividing fails industrialization production so far.
Chimonin structure is：
It is larger to the demand of chimonin in terms of because of medicine and health product, occur in that some are isolated and purified from natural plants
Chimonin and the achievement in research and patent of chemosynthesis.It is entitled《Rhizoma Anemarrhenae extract and its production and use》(application number
For 03115509.X) patent in disclose one kind with the Rhizoma Anemarrhenae as raw material, it is size-reduced, alcohol extraction, macroporous resin adsorption eluting,
Precipitate with ethanol, after being dried, has obtained chimonin and the extract for diabetes control that Neomangiferin total content is 50%.It is entitled
《Fructus Mangifera Indicae general glycoside preparation and its production method》One kind is disclosed in the patent of (Application No. 03128247.4) with Folium mangiferae or flat
Folium Persicae is raw material, isolates and purifies the extract for obtaining that Fructus Mangifera Indicae general glycoside is up to 50% or more, for eliminating phlegm and stopping cough oral Chinese medicine system
The method of agent.It is entitled《The preparation method of high purity mangiferin》Disclose in the patent of (Application No. 200610079234.5)
One kind is decolourized with Folium mangiferae or myrica rubra leaf as raw material using resin method, the method for obtaining chimonin purity >=90% extract.Name
Referred to as《Extract a kind of method of chimonin》One kind is disclosed in the patent of (Application No. 200710066354.6) with Folium mangiferae
Or myrica rubra leaf is raw material, using the method for High Temperature High Pressure, in the method that water or alcohol-water extract chimonin as solvent.It is entitled《Awns
The preparation method of fruit glycosides》One kind is disclosed in the patent of (Application No. 200910175781.7) with Folium mangiferae as raw material, is adopted
With isolated chimonins of technique such as enzymatic treatment, Jing petroleum ether degreasings, 60%～80% alcohol extraction, centrifugations.It is entitled《A kind of awns
The preparation method of fruit glycosides》One kind disclosed in the patent of (Application No. 200910234065.1) is crushed with Folium mangiferae as raw material
Saturated limewater soak extraction is added afterwards, then by macroporous resin adsorption eluting, Fructus Mangifera Indicae glycoside product is recrystallized to give after concentration.Title
For《A kind of preparation method of chimonin》One kind is disclosed in the patent of (Application No. 201010543720.4) to contain Fructus Mangifera Indicae
The natural plants of glycosides are raw material, add aqueous alkali, boil extraction, and extracting solution acid is heavy, and Jing macroporous resin enrichments are decolourized, at activated carbon
After reason, the method for high purity mangiferin is concentrated to give.It is entitled《The method that chimonin is extracted from Folium mangiferae》(Application No.
One kind is disclosed in patent 200910194672.X) with Folium mangiferae as raw material, is carried using aqueous alkali temperature, be acidified, precipitate with ethanol centrifugation is used
After sephadex LH-20 enrichments, the method for high purity mangiferin is recrystallized to give.It is entitled《The full chemistry synthesis of mango aglycone
Method》The side that a kind of chemical synthesis process prepares mango aglycone is provided in the patent of (Application No. 201010529474.7)
Method.It is entitled《A kind of preparation method of chimonin》One kind is disclosed in the patent of (Application No. 201010292465.0) with awns
Tree Fruit, Folium mangiferae, mango bark and Asphodeloides Bge Rhizome are raw material, using alcohol water extraction, are extracted, prepared by the method such as recrystallization
High purity mangiferin.It is entitled《The high purity mangiferin prepared from Rhizoma Atractylodis macrocephalae Herba Pelargonii Graveolentiss and twig and its method》(Application No.
201110411308.1) one kind is disclosed in patent with Rhizoma Atractylodis macrocephalae Herba Pelargonii Graveolentiss and twig as raw material, using alcohol extraction, water precipitating, precipitate with ethanol, analysis
Crystalline substance, the method that recrystallization prepares high purity mangiferin.It is entitled《A kind of side that chimonin is isolated and purified from mango pericarp
Method》One kind is disclosed in the patent of (Application No. 201210053385.9) with mango pericarp dried powder as raw material, using liquid
Liquid is extracted, and macroporous resin isolates and purifies the method for obtaining high purity mangiferin with HSCCC multiple techniques.
In above-mentioned, existing disclosure prepares the problem that the method for chimonin is primarily present and has：
1st, the purity of the isolated chimonin of most of technique is relatively low, or does not have definite purity guarantee；
2nd, some processes complex operation, need to carry out eluting using substantial amounts of solvent, easily operator are damaged, and
Solvent cost is high；
3rd, the long preparation period of most of technique, the response rate are low, low in economic efficiency；
4th, chimonin is unstable under acid or alkaline conditions, is carried using acid or alkali carries, and most of chimonin may be
Preparation process deactivation；
5th, it is most of using Folium mangiferae as raw material, it is residual exceeded agriculture easily occur, dangerous；
【The content of the invention】
The invention is intended to solve the above problems, there is provided one kind quickly prepares high purity mangiferin from Rhizoma Atractylodis macrocephalae Herba Pelargonii Graveolentis extract
Method, by following steps realize：
A kind of method that chimonin is isolated and purified in leaveves from Lignum Aquilariae Resinatum, it is characterised in that the method is comprised the following steps：
(1) the blade after perfume (or spice) is adopted as raw material with the Lignum Aquilariae Resinatum tree body for manually promoting Edgeworthia chrysantha Lindl., drying carries out percolation after breaking into coarse powder
Or extraction, merging all percolates or lixiviating solution, concentration removes organic solvent；
(2) lipid and fat-soluble pigment are removed with petroleum ether extraction 2～4 times, phase of fetching water is extracted with n-butyl alcohol successively
After 2～4 times, merge n-butanol portion, the low pressure concentration at 40～60 DEG C obtains the main Rhizoma Atractylodis macrocephalae Herba Pelargonii Graveolentiss containing chimonin position and soak
Cream；
(3) gained Rhizoma Atractylodis macrocephalae Herba Pelargonii Graveolentiss extractum and purification on normal-phase silica gel are mixed after sample, using chloroform-methanol system, be respectively with volume ratio
9:2→7:3→6:4→1:1→0:1 stage carries out eluting, is connected in portion with 1 column volume, while the stream part that will be received is low
After pressure concentration, with chimonin reference substance point on same plate, volume ratio is adopted for (10:3:1) chloroform-methanol-aqueous solvent body
The expansion system of the lower phase of system carries out TLC detections, the main stream part containing chimonin is merged and reclaims molten after 35-45 DEG C of low pressure
Agent, that is, obtain the main normal phase column chromatography rough segmentation containing chimonin；
(4) the rough segmentation of gained normal phase column chromatography and purification on normal-phase silica gel are mixed after sample, go up normal phase column chromatography separation again, by main containing awns
After the stream part of fruit glycosides is merged, low pressure recycling design obtains the main yellow powder containing chimonin；
(5) acetate-methanol-aqueous solvent is prepared, after stratification, the above is mutually fixing phase, lower is mutually flowing
Phase；Upper HSCCC after gained yellow powder molten sample is separated, after the stream part containing single substance chimonin is merged, low pressure reclaims molten
Agent, obtains final product chimonin sterling.
The described method that chimonin is isolated and purified from Lignum Aquilariae Resinatum leaveves, it is characterised in that the percolation or extraction are
Percolation is carried out using 50%～80% aqueous acetone solution, or volume ratio is adopted for 1:20 solid-liquid ratio room temperature extraction.
The described method that chimonin is isolated and purified from Lignum Aquilariae Resinatum leaveves, it is characterised in that in the step (3) just
In phase column chromatography for separation, the extractum and purification on normal-phase silica gel in mass ratio 1:1 carries out mixing sample, and it is 1/3～1 cylinder that stream part receives volume
Product is received as portion.
The described method that chimonin is isolated and purified from Lignum Aquilariae Resinatum leaveves, it is characterised in that described to go up normal phase column again
The method of chromatography is to adopt chloroform-methanol volume ratio for (75:25) dicyandiamide solution carries out eluting, elution speed 5ml/
min；Portion is connected in 1/2 column volume, while after by the stream part low pressure concentration for receiving, with chimonin reference substance point in same plate
On, volume ratio is adopted for (10:3:1) under chloroform-methanol-aqueous solvent, the expansion system of phase carries out TLC detections, and master is contained
The stream part of chimonin merges the low pressure recycling design at 35 DEG C, obtains the main yellow powder containing chimonin.
The described method that chimonin is isolated and purified from Lignum Aquilariae Resinatum leaveves, it is characterised in that the step (1) middle concentration
The temperature of recovery is 40～60 DEG C.
The described method that chimonin is isolated and purified from Lignum Aquilariae Resinatum leaveves, it is characterised in that the step (1) middle concentration
The temperature of recovery is 45 DEG C, the temperature of the step (2) middle concentration and recovery is 50 DEG C.
The described method that chimonin is isolated and purified from Lignum Aquilariae Resinatum leaveves, it is characterised in that the detached side of the HSCCC
Method is that the volume ratio of the dicyandiamide solution acetate-methanol-water is (5.3～5.6):(0.4～0.6):(4.3～4.7),
It is 300～500rpm to adjust high-speed counter-current chromatograph and rotate forward rotating speed；Fixing phase retention is 78%～89%；Mobile phase with 8～
12ml/min passes through chromatograph；Detection wavelength：254nm；Separation temperature is：20～25 DEG C.
The described method that chimonin is isolated and purified from Lignum Aquilariae Resinatum leaveves, it is characterised in that in the normal phase column chromatography
Filler and sample ratio are 10～20 times.
The described method that chimonin is isolated and purified from Lignum Aquilariae Resinatum leaveves, it is characterised in that in the normal phase column chromatography
Filler and sample ratio are 15 times, and (3) middle stream part receives volume to the step is that 1 column volume is portion, the step (4) middle stream
It is that 1/2 column volume is portion that part receives volume.
The present invention has advantages below：
1st, discarded Rhizoma Atractylodis macrocephalae Herba Pelargonii Graveolentiss are adopted for raw material, low cost；
2nd, the equal recoverable of solvent used in above-mentioned technique, reduces cost；
3rd, the technological operation is simple, and feasibility is high, and short preparation period can shorten time cost；
4th, the technique is prepared using HSCCC, and isolated chimonin purity is high, pollution-free, stable, can be maximum
The physiologically active of the preservation chimonin of degree, and preparation amount is big, can through engineering approaches production.
【Description of the drawings】
Fig. 1 prepares high-purity hypoglycemic activity composition chimonin process from Rhizoma Atractylodis macrocephalae Herba Pelargonii Graveolentiss；
Fig. 2 chimonin HSCCC separating spectrums (dash area is chimonin chromatographic peak).
【Specific embodiment】
With reference to example and accompanying drawing, the present invention is described further.
The discovery of Jing research, the chimonin containing high level in Rhizoma Atractylodis macrocephalae Herba Pelargonii Graveolentiss.Compared with Folium mangiferae, there is no agriculture residual
Problem, it is safe, therefore smashed after Rhizoma Atractylodis macrocephalae Herba Pelargonii Graveolentiss are dried, you can oozed as raw material from Rhizoma Atractylodis macrocephalae Herba Pelargonii Graveolentiss in the present invention
Filter or extract.
High speed adverse current chromatogram (High-speed Counter-current Chromatography, abbreviation HSCCC) is one
Kind of LLC isolation technics, not by any solid packing in the case of, it is biphase as solid using immiscible liquid
Fixed phase and mobile phase, in the serpentine pipe of high speed rotation set up a kind of unipolarity fluid dynamic equilibrium, with wherein one are mutually
Fixing phase, another is mutually mobile phase, can retain a large amount of fixing phases, with material in biphase middle distribution during continuous eluting
Difference reaches separating effect.Compared with traditional separation means, the technology is not because adopting any solid packing to support and adsorb, it is to avoid
Because Irreversible Adsorption causes inactivation, degeneration of sample etc., can not only recovery sample completely, additionally it is possible to reflect that sample was originally special
Property, it is particularly suitable for natural bioactivity substance separation, is at present generally acknowledged to be in the world widely used in Separation of Natural Products
The effective ways of purification.Due to special environment in serpentine pipe, separate substance can be made mutually to be fully contacted with liquid up and down, made
Sample preparation amount is improved, and is a kind of ideal preparative separation technology.It has sample nondestructive lose, it is pollution-free, efficient, fast
Fast, easy to operate, preparation amount is big and low cost and other advantages, and in natural product, principal component or non-principal component are isolated and purified, chemistry
Synthetic is isolated and purified, Chinese medicine fingerprint and quality controling research, and marine bioactivity composition is isolated and purified, polypeptide and egg
The field such as the separation of the macromole such as white matter and chiral separation is widely used.
The concrete operations technique of the present invention is as shown in Figure 1.Comprise the steps：
1st, extract prepares：Take the artificial Lignum Aquilariae Resinatum tree body for promoting Edgeworthia chrysantha Lindl. and adopt the blade and blade after perfume (or spice), coarse powder, using 50%～
80% aqueous acetone solution (or 50%～80% ethanol water) percolation (or volume ratio is adopted for 1:Soak under 20 solid-liquid ratio room temperature
Carry), merge all percolates or lixiviating solution, after concentration removes organic solvent, using petroleum ether extraction 2～4 times, remove lipid and
Fat-soluble pigment, phase of fetching water are carried out with n-butyl alcohol after extraction 2～4 times, merge n-butanol portion, the low pressure concentration at 50 DEG C, i.e.,
Obtain Rhizoma Atractylodis macrocephalae's Herba Pelargonii Graveolentiss extractum.
2nd, normal phase column chromatography rough segmentation：By the extractum of step 1 and purification on normal-phase silica gel in mass ratio 1:After 1 carries out mixing sample, upper positive
Column chromatography for separation, using chloroform-methanol system, with stage (volume ratio 9:2→7:3→6:4→1:1→0:1) eluting is carried out,
Portion is connected in 1 column volume, while after by the stream part low pressure concentration for receiving, with chimonin reference substance point on same plate, adopting
Chloroform-methanol-water (volume ratio=10:3:1, remove phase) expansion system carry out TLC detections, the main stream part containing chimonin is entered
Row merges after 35 DEG C of low pressure recycling designs, obtains final product.
3rd, normal phase column chromatography is refined：The merging stream part that step 2 is obtained and purification on normal-phase silica gel in mass ratio 1:1 carries out mixing sample
Afterwards, upper normal phase column chromatography is separated, using chloroform-methanol=75:25 dicyandiamide solution carries out eluting, elution speed 5ml/min, with
1/2 column volume is connected in portion, while after by the stream part low pressure concentration for receiving, with chimonin reference substance point on same plate, adopting
With chloroform-methanol-water (volume ratio=10:3:1, remove phase) expansion system carry out TLC detections, will the main stream part containing chimonin
The low pressure recycling design at 35 DEG C is merged, the main yellow powder containing chimonin is obtained.
4th, HSCCC prepares chimonin：Prepare acetate-methanol-water (volume ratio 5.4:0.5:4.4) dicyandiamide solution with point
In liquid funnel, after fully shaking up, stratification mutually will separate up and down, and ultrasound degassing 10min, the above are mutually fixing phase, lower to be mutually
Mobile phase, fixing phase is pumped in high speed adverse current chromatogram post with the flow velocity of 30ml/min, is noted in treating high speed adverse current chromatogram post completely
During full fixing phase, it is 500rpm/min to adjust high-speed counter-current chromatograph and rotate forward rotating speed, and by mobile phase with the flow velocity of 10ml/min
High-speed counter-current chromatograph is pumped into, after chromatograph column equilibration is finished in high-speed counter-current chromatograph, the yellow powder that step 3 is obtained is matched somebody with somebody
Solution is set to, after pumping into chromatographic column, and mobile phase is pumped in current chromatographic column with 10ml/min flow velocitys, according to work station appearance
Peak type carries out stream part collection, every part of 5ml.Each stream part is carried out into purity detecting using HPLC, single substance chimonin will be contained
Low pressure recycling design after stream part merging, obtains final product chimonin sterling, the response rate >=87%, product purity >=98%.
In above-mentioned steps, in normal phase column chromatography, filler and sample ratio are 10～20 times, preferably 15 times.Stream part receives volume
Portion is received as 1/3～1 column volume, preferably 1 column volume of coarse powder is portion, preferably 1/2 column volume is refined for portion.
The chimonin Jing high performance liquid chromatography for preparing and nuclear-magnetism identification, it is consistent with document report.HSCCC is isolated
Stream part detected using HPLC that its separating spectrum refers to Fig. 2.
Embodiment 1：
Take Lignum Aquilariae Resinatum leaf coarse powder 30kg to be placed in percolation bucket, add 70% aqueous acetone solution to carry out percolation, receive percolate,
Merging is concentrated into 180L after 45 DEG C of low pressure.Petroleum ether, n-butanol extraction are respectively adopted, n-butanol portion extractum 165.2g is obtained,
Mix after sample with purification on normal-phase silica gel, upper normal phase column chromatography is separated, 1 column volume is connected in portion, while the stream part low pressure for receiving is concentrated
Afterwards, TLC detections are carried out.The main stream part containing chimonin is merged into the low pressure recycling design at 35 DEG C, obtains main containing Fructus Mangifera Indicae
Stream part 3.366g of glycosides.Stream part after merging and purification on normal-phase silica gel are mixed after sample, then upper normal phase column chromatography is separated, 1/2 column volume connects
For portion, while the stream part for receiving is carried out TLC detections after low pressure concentration at 35 DEG C.The main stream part containing chimonin is carried out
Merge the low pressure recycling design at 35 DEG C, obtain the main yellow powder 1.146g containing chimonin.After molten sample, upper HSCCC is separated,
Acetate-methanol-water volume ratio=5.5:0.4:4.5, wherein being mutually fixing phase above, lower is mutually mobile phase；Adjust at a high speed
It is 400rpm/min that counter-current chromatograph rotates forward rotating speed；Fixing phase retention is 79%；Mobile phase is with 8ml/min；Detection wavelength：
254nm；Separation temperature is：20～25 DEG C, prepare chimonin 0.9970g, purity >=98%, the response rate >=87%.
Embodiment 2：
Lignum Aquilariae Resinatum leaf coarse powder 60kg is taken in percolation bucket, is added 75% ethanol water to carry out percolation, is received percolate, close
And 300L is concentrated into after 50 DEG C of low pressure.Petroleum ether, n-butanol extraction are respectively adopted, n-butanol portion extractum 340.7g is taken, with
After purification on normal-phase silica gel mixes sample, upper normal phase column chromatography is separated, and 1 column volume is connected in portion, while after by the stream part low pressure concentration for receiving,
Carry out TLC detections.The main stream part containing chimonin is merged into the low pressure recycling design at 40 DEG C, master is obtained and is contained chimonin
Stream part 7.098g.Stream part after merging and purification on normal-phase silica gel are mixed after sample, then upper normal phase column chromatography is separated, 1/2 column volume is connected in one
Part, while after by the stream part low pressure concentration for receiving, carrying out TLC detections.The main stream part containing chimonin is merged after 40 DEG C
Lower low pressure recycling design, obtains the main yellow powder 2.389g containing chimonin.After molten sample, upper HSCCC is separated, ethyl acetate-first
Alcohol-water accumulates ratio=5.3:0.4:4.3, wherein being mutually fixing phase above, lower is mutually mobile phase；High-speed counter-current chromatograph is adjusted just
Speed walk around for 500rpm/min；Fixing phase retention is 85%；Mobile phase is with 12ml/min；Detection wavelength：254nm；Separate temperature
Spend and be：20～25 DEG C, prepare chimonin 2.008g, purity >=98%, the response rate >=89%.
Embodiment 3：
Lignum Aquilariae Resinatum leaf coarse powder 90kg is taken in percolation bucket, is added 75% aqueous acetone solution to carry out percolation, is received percolate, close
And 480L is concentrated into after 45 DEG C of low pressure.Petroleum ether, n-butanol extraction are respectively adopted, n-butanol portion extractum 478.2g is obtained, with
After purification on normal-phase silica gel mixes sample, upper normal phase column chromatography is separated, and 1 column volume is connected in portion, while after by the stream part low pressure concentration for receiving,
Carry out TLC detections.The main stream part containing chimonin is merged into the low pressure recycling design at 45 DEG C, master is obtained and is contained chimonin
Stream part 9.998g.Stream part after merging and purification on normal-phase silica gel are mixed after sample, then upper normal phase column chromatography is separated, 1/2 column volume is connected in one
Part, while after by the stream part low pressure concentration for receiving, carrying out TLC detections.The main stream part containing chimonin is merged after 40 DEG C
Lower low pressure recycling design, obtains the main yellow powder 3.1487g containing chimonin.After molten sample, upper HSCCC is separated, ethyl acetate-first
Alcohol-water accumulates ratio=5.6:0.5:4.6, wherein being mutually fixing phase above, lower is mutually mobile phase；High-speed counter-current chromatograph is adjusted just
Speed walk around for 400rpm/min；Fixing phase retention is 89%；Mobile phase is with 10ml/min；Detection wavelength：254nm；Separate temperature
Spend and be：20～25 DEG C, prepare chimonin 3.287g, purity >=98%, the response rate >=87%.
Embodiment 4：
Take Lignum Aquilariae Resinatum leaf coarse powder 30kg to be placed in percolation bucket, add 80% ethanol water to carry out percolation, receive percolate,
Merging is concentrated into 150L after 50 DEG C of low pressure, and petroleum ether is respectively adopted, and n-butanol extraction takes n-butanol portion extractum 170.4g,
Mix after sample with purification on normal-phase silica gel, upper normal phase column chromatography is separated, 1 column volume is connected in portion, while the stream part low pressure for receiving is concentrated
Afterwards, TLC detections are carried out.The main stream part containing chimonin is merged into the low pressure recycling design at 35 DEG C, obtains main containing Fructus Mangifera Indicae
Stream part 3.508g of glycosides.Stream part after merging and purification on normal-phase silica gel are mixed after sample, then upper normal phase column chromatography is separated, 1/2 column volume connects
For portion, while after by the stream part low pressure concentration for receiving, carrying out TLC detections.Will the main stream part containing chimonin merge after
Low pressure recycling design at 35 DEG C, obtains the main yellow powder 1.435g containing chimonin.After molten sample, upper HSCCC is separated, and ethyl acetate-
Methanol-water volume ratio=5.4:0.4:4.3, wherein being mutually fixing phase above, lower is mutually mobile phase；Adjust high-speed counter-current chromatograph
Rotating forward rotating speed is 500rpm/min；Fixing phase retention is 85%；Mobile phase is with 12ml/min；Detection wavelength：254nm；Separate
Temperature is：20～25 DEG C, prepare chimonin 1.087g, purity >=98%, the response rate >=88%.
Claims (9)
1. a kind of method that chimonin is isolated and purified in leaveves from Lignum Aquilariae Resinatum, it is characterised in that the method is comprised the following steps：
(1) the blade after perfume (or spice) is adopted as raw material with the Lignum Aquilariae Resinatum tree body for manually promoting Edgeworthia chrysantha Lindl., drying carries out percolation or leaching after breaking into coarse powder
Carry, merge all percolates or lixiviating solution, concentration removes organic solvent；
(2) lipid and fat-soluble pigment are removed with petroleum ether extraction 2～4 times, phase of fetching water carries out extraction 2～4 with n-butyl alcohol successively
After secondary, merge n-butanol portion, the low pressure concentration at 40～60 DEG C obtains main Rhizoma Atractylodis macrocephalae's Herba Pelargonii Graveolentiss extractum containing chimonin position；
(3) gained Rhizoma Atractylodis macrocephalae Herba Pelargonii Graveolentiss extractum and purification on normal-phase silica gel are mixed after sample, using chloroform-methanol system, 9 are respectively with volume ratio:2
→7:3→6:4→1:1→0:1 stage carries out eluting, is connected in portion with 1 column volume, while the stream part low-press thick that will be received
After contracting, with chimonin reference substance point on same plate, volume ratio is adopted for (10:3:1) under chloroform-methanol-aqueous solvent
The expansion system of phase carries out TLC detections, the main stream part containing chimonin is merged after 35-45 DEG C of low pressure recycling design, i.e.,
Obtain the main normal phase column chromatography rough segmentation containing chimonin；
(4) the rough segmentation of gained normal phase column chromatography and purification on normal-phase silica gel are mixed after sample, go up normal phase column chromatography separation again, by main containing chimonin
Stream part merge after, low pressure recycling design obtains the main yellow powder containing chimonin；
(5) acetate-methanol-aqueous solvent is prepared, after stratification, the above is mutually fixing phase, lower is mutually mobile phase；Will
After the molten sample of gained yellow powder, upper HSCCC is separated, and low pressure recycling design after the stream part containing single substance chimonin is merged is obtained final product
Chimonin sterling.
2. the method that chimonin is isolated and purified in the leaveves from Lignum Aquilariae Resinatum according to claim 1, it is characterised in that described to ooze
It is to carry out percolation using 50%～80% aqueous acetone solution to filter or extract, or adopts volume ratio for 1:20 solid-liquid ratio room temperature leaching
Carry.
3. the method that chimonin is isolated and purified in the leaveves from Lignum Aquilariae Resinatum according to claim 1, it is characterised in that described
During step normal phase column chromatography (3) is separated, the extractum and purification on normal-phase silica gel in mass ratio 1:1 carries out mixing sample, and stream part receives volume and is
1/3～1 column volume is received as portion.
4. the method that chimonin is isolated and purified in the leaveves from Lignum Aquilariae Resinatum according to claim 1, it is characterised in that it is described again
The detached method of secondary upper normal phase column chromatography is to adopt chloroform-methanol volume ratio for (75:25) dicyandiamide solution carries out eluting, washes
De- speed 5ml/min；Portion is connected in 1/2 column volume, while after by the stream part low pressure concentration for receiving, with chimonin reference substance
Point adopts volume ratio for (10 on same plate:3:1) under chloroform-methanol-aqueous solvent, the expansion system of phase carries out TLC
The main stream part containing chimonin is merged the low pressure recycling design at 35 DEG C by detection, obtains the main yellow powder containing chimonin
End.
5. the method that chimonin is isolated and purified in the leaveves from Lignum Aquilariae Resinatum according to claim 1, it is characterised in that the step
The temperature of rapid (1) middle concentration and recovery is 40～60 DEG C.
6. according to the method that chimonin is isolated and purified from Lignum Aquilariae Resinatum leaveves described in claim 5, it is characterised in that the step
The temperature of middle concentration and recovery be 45 DEG C, the temperature of the step (2) middle concentration and recovery be 50 DEG C.
7. the method that chimonin is isolated and purified in the leaveves from Lignum Aquilariae Resinatum according to claim 1, it is characterised in that described
The detached methods of HSCCC are that the volume ratio of the dicyandiamide solution acetate-methanol-water is (5.3～5.6):(0.4～
0.6):(4.3～4.7), it is 300～500rpm to adjust high-speed counter-current chromatograph and rotate forward rotating speed；Fixing phase retention be 78%～
89%；Mobile phase passes through chromatograph with 8～12ml/min；Detection wavelength：254nm；Separation temperature is：20～25 DEG C.
8. the method that chimonin is isolated and purified from Lignum Aquilariae Resinatum leaveves according to any claim in claim 1 to 7,
Characterized in that, filler and sample ratio are 10～20 times in the normal phase column chromatography.
9. the method that chimonin is isolated and purified in the leaveves from Lignum Aquilariae Resinatum according to claim 8, it is characterised in that it is described just
In phase column chromatography, filler and sample ratio are 15 times, and (3) middle stream part reception volume is that 1 column volume is portion to the step, described
(4) middle stream part reception volume is that 1/2 column volume is portion to step.
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| CN104644880A (en) * | 2014-12-31 | 2015-05-27 | 海南省农业科学院农产品加工设计研究所 | Extraction method of flavonol in agalloch leaves |
| CN104569236A (en) * | 2015-01-08 | 2015-04-29 | 东莞广州中医药大学中医药数理工程研究院 | A method for establishing the HPLC fingerprint of A. |
| CN106565693A (en) * | 2016-11-05 | 2017-04-19 | 林文练 | Method for extracting mangiferin |
| CN113429401A (en) * | 2021-07-06 | 2021-09-24 | 蒙汉富 | Mangiferin extraction and separation method |
| CN117434170B (en) * | 2023-08-23 | 2024-12-31 | 珠海莱森博萃生物科技有限公司 | A method for simultaneously determining benzyl acetone, adenosine and mangiferin in Aquilaria sinensis leaves |
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