CN104211690A - Method for separating and purifying mangiferin from aquilaria sinensis leaves - Google Patents
Method for separating and purifying mangiferin from aquilaria sinensis leaves Download PDFInfo
- Publication number
- CN104211690A CN104211690A CN201410428303.3A CN201410428303A CN104211690A CN 104211690 A CN104211690 A CN 104211690A CN 201410428303 A CN201410428303 A CN 201410428303A CN 104211690 A CN104211690 A CN 104211690A
- Authority
- CN
- China
- Prior art keywords
- mangiferin
- purification
- separation
- stream part
- column chromatography
- Prior art date
- Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
- Granted
Links
Classifications
-
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07D—HETEROCYCLIC COMPOUNDS
- 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
- C07D407/02—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 containing two hetero rings
- C07D407/04—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 containing two hetero rings directly linked by a ring-member-to-ring-member bond
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 natural compounds, be specifically related to adopt column chromatography and HSCCC coupling technique to prepare the method for high-purity compound fast.
[background technology]
Lignum Aquilariae Resinatum (Aquilaria sinensis); also known as aquilaria sinensis, agallochum, tabernaemontanus bulrush is fragrant, tooth is fragrant, daughter is fragrant; for thymelaeceae eaglewood; aiphyllium; for the peculiar Chinese Second Class Key Protected Plant of China, in Guangdong, Hainan; all there is plantation on the ground such as Guangxi, Fujian, is the rare medicinal herbs that the traditional Chinese medical science is commonly used.Lignum Aquilariae Resinatum heartwood is pungent, bitter, tepor, have sending down abnormally ascending adjust in, warm kidney analgesic efficacy, be usually used in chest abdomen pain, the disease such as uncomfortable in chest, vomiting hiccup, borborygmus are had loose bowels, dyspnea due to adverseness of QI.The Lignum Aquilariae Resinatum tree resiniferous heartwood of body (being commonly called as agalloch eaglewood), can make perfume base, and for controlling stomach trouble specifics; Bark fiber is pliable and tough, Se Bai and careful, can do fine paper raw material and artificial cotton; Xylem can extract perfume oil, and flower can medicinal extract processed, is the plant that a kind of economic benefit is higher.Its medicinal part is grease-contained heart wood or resin (being commonly called as Edgeworthia chrysantha).Under natural condition, Edgeworthia chrysantha needs just can carry out under special circumstances, the cycle be 10 years even longer, and artificial growth is employed new technology and is made Lignum Aquilariae Resinatum Edgeworthia chrysantha in recent years, though shorten the agalloch eaglewood Edgeworthia chrysantha cycle, also takes 5-8.And resourceful Lignum Aquilariae Resinatum leaf part, the leaf Chang Zuowei refuse process after especially manually promoting the tree body of Edgeworthia chrysantha to adopt perfume (or spice), causes the wasting of resources.The present invention, using Lignum Aquilariae Resinatum leaf as starting material, adopts normal phase column chromatography and high speed adverse current chromatograph joint used technology, fast preparation high purity blood-sugar decreasing active Mangiferin, as the material of hypoglycemic series product.
Mangiferin (mangiferin), also known as mangiferin, mangiferin, is tetrahydroxy pyrrone carbon glycoside, belongs to two benzene pyrrones compound.Mangiferin exists extensively in plant, and in Anacardiaceae plant mango (Mangifera indica L.) blade, content is 0.33%-2.98%; Content in the liliaceous plant wind-weed (Anemarrhenaasphodeloides Bunge) dried root is 0.320%-0.325%; Be 0.048%-0.66% at the content of Hypericaceae plant Sampson St.John's wort Herb (Hypericum sampsonii Hance) herb; Be 0.094%-0.216% at the content of gentianaceae plant Herba Swertiae bimaculatae (Swertia davidii Franch.) herb.Discovered in recent years also has distribution in Lignum Aquilariae Resinatum, content be 2.76%-3.24% (handsome Europe etc. the extraction process [J] of Mangiferin in eaglewood leaf. forestry science and technology develop, 2013,27 (5): 101-104.).
Mangiferin has preferably physiologically active.In hypoglycemic activity, S.Muruganandan etc. are found by research, Mangiferin has hyperglycemia, lipidemia, antiatherogenic effect, the assisting therapy (S.Muruganandan, et al.Effect of mangiferin on hyperglycemia and atherogenicity in streptozotocin diabetic rats.Journal of Ethnopharmacology.97 (2005): 497-501.) that Mangiferin can be used for the related cardiovascular such as diabetes, atheroma complication is proposed; And Yao-Wu Liu, Prabhu Sukumaran Nair etc. reconfirms that Mangiferin has the effect (Yao-Wu Liu, et al.Up-regulation of glyoxalase 1by mangiferin prevents diabetic nephropathy progression in streptozotocin-induced diabetic rats.European Journal of Pharmacology (2013) 1-10.) of hyperglycemia, lipidemia; Above-mentioned research all shows that Mangiferin has hypoglycemic activity significantly.
In addition, Mangiferin in radio-protective, reduce natural death of cerebral cells because active oxygen causes, improved heart defense function by oxidation resistant approach, anti-nervosa is poisoning, anti-inflammatory, antibacterial, bring down a fever, ease pain, all show good physiologically active in anticancer, anti-Parkinson syndromes, anti-alzheimer's disease etc., be widely used in medicine, makeup, healthcare products.But due to separation preparation complicated operation, preparation cycle is long, the rate of recovery is low, the reason such as low in economic efficiency, be that the related products of main component is failed industrialization so far and produced with Mangiferin.
Mangiferin structure is:
Because of medicine and healthcare products aspect comparatively large to the demand of Mangiferin, there is achievement in research and the patent of some separation and purification Mangiferin and chemosynthesis from natural phant.Name is called in the patent of " Rhizoma Anemarrhenae extract and its production and use " (application number is 03115509.X) that disclosing a kind of is starting material with the wind-weed, through pulverizing, alcohol lixiviate, macroporous resin adsorption wash-out, alcohol precipitation, after drying, obtain the extract for diabetes control that Mangiferin and Neomangiferin total content are 50%.Name be called in the patent of " mango general glycoside preparation and production method thereof " (application number is 03128247.4) disclose a kind of with Folium mangiferae or almond leaf for starting material, separation and purification obtains the extract that mango general glycoside reaches 50% or more, for the method for cough-relieving apophlegmatic oral traditional Chinese medicine preparation.Name be called in the patent of " preparation method of high purity mangiferin " (application number is 200610079234.5) disclose a kind of with Folium mangiferae or almond leaf for raw material, adopt resin method decolouring, obtain the method for Mangiferin purity >=90% extract.Name be called in the patent of a kind of method of Mangiferin " extract " (application number is 200710066354.6) disclose a kind of with Folium mangiferae or almond leaf for raw material, adopting the method for High Temperature High Pressure, is the method that solvent extracts Mangiferin with water or alcohol-water.Name is called in the patent of " preparation method of Mangiferin " (application number is 200910175781.7) that disclosing a kind of is starting material with Folium mangiferae, adopt enzymatic treatment, through petroleum ether degreasing, 60%-80% alcohol extracting, the technique such as centrifugal is separated and obtains Mangiferin.Name is called that in the patent of " a kind of preparation method of Mangiferin " (application number is 200910234065.1), open one is starting material with Folium mangiferae, saturated limewater soak extraction is added after pulverizing, again by macroporous resin adsorption wash-out, after concentrated, recrystallization obtains Mangiferin product.Name be called in the patent of " a kind of preparation method of Mangiferin " (application number is 201010543720.4) disclose a kind of with the natural phant containing Mangiferin for starting material, add buck, boil extraction, extracting solution acid is heavy, decolour through macroporous resin enrichment, after activated carbon treatment, the concentrated method obtaining high purity mangiferin.Name is called in the patent of " method extracting Mangiferin from Folium mangiferae " (application number is 200910194672.X) that disclosing a kind of is raw material with Folium mangiferae, buck temperature is adopted to carry, acidifying, alcohol precipitation is centrifugal, after sephadex LH-20 enrichment, recrystallization obtains the method for high purity mangiferin.Name is called in the patent of " the full chemical synthesis process of mango aglycone " (application number is 201010529474.7) provides a kind of chemical synthesis process to prepare the method for mango aglycone.Name be called in the patent of " a kind of preparation method of Mangiferin " (application number is 201010292465.0) disclose a kind of with mango fruit, Folium mangiferae, mango bark and Asphodeloides Bge Rhizome for raw material, adopt alcohol water extraction, extraction, the methods such as recrystallization, prepare high purity mangiferin.Name be called in the patent of " high purity mangiferin prepared from whitewood spiceleaf and spray and method thereof " (application number is 201110411308.1) disclose a kind of with whitewood spiceleaf and spray for raw material, adopt alcohol extracting, depositing in water, alcohol precipitation, crystallization, recrystallization prepares the method for high purity mangiferin.Name be called in the patent of " a kind of method of separation and purification Mangiferin from mango pericarp " (application number is 201210053385.9) disclose a kind of with mango pericarp dried powder for raw material, adopt liquid-liquid extraction, macroporous resin and the separation and purification of HSCCC coupling technique obtain the method for high purity mangiferin.
In above-mentioned, the existing main Problems existing of method openly preparing Mangiferin has:
1, to be separated the purity obtaining Mangiferin lower for most of technique, or do not have definite purity guarantee;
2, some processes complicated operation, need adopt a large amount of solvents to carry out wash-out, easily damage operator, and solvent cost be high;
3, the preparation cycle of most of technique is long, and the rate of recovery is low, low in economic efficiency;
4, Mangiferin is unstable under acid or alkaline conditions, and employing acid carries or alkali is carried, and most of Mangiferin may at preparation process deactivation;
5, major part adopts Folium mangiferae as starting material, easily occurs that agriculture is residual and exceeds standard, dangerous;
[summary of the invention]
The invention is intended to solve the problem, a kind of method preparing high purity mangiferin from Lignum Aquilariae Resinatum leaf extract be fast provided, realized by following steps:
From Lignum Aquilariae Resinatum leaf, a method for separation and purification Mangiferin, is characterized in that, the method comprises the following steps:
(1) manually to promote that the Lignum Aquilariae Resinatum of Edgeworthia chrysantha is set body and adopted the blade after perfume (or spice) for raw material, dry after breaking into meal and carry out diacolation or lixiviate, merge all percolates or vat liquor, concentrated removing organic solvent;
(2) use petroleum ether extraction 2-4 time successively, removing lipid and fat-soluble pigment, water intaking phase, after carrying out extraction 2-4 time with propyl carbinol, merge n-butanol portion, at 40-60 DEG C, low pressure concentrates, and namely obtains the whitewood spiceleaf medicinal extract that master contains Mangiferin position;
(3) after gained whitewood spiceleaf medicinal extract n-butanol portion and purification on normal-phase silica gel being mixed sample, adopt normal phase column chromatography to be separated, in 35-45 DEG C of low pressure recycling design after main stream part containing Mangiferin being merged, namely obtain the main normal phase column chromatography rough segmentation containing Mangiferin;
(4) after sample being mixed in the rough segmentation of gained normal phase column chromatography and purification on normal-phase silica gel, again go up normal phase column chromatography and be separated, after main stream part containing Mangiferin being merged, low pressure recycling design, obtains the main yellow powder containing Mangiferin;
(5) prepare acetate-methanol-aqueous solvent, after stratification, above is stationary phase mutually, and lower is moving phase mutually; Be separated by HSCCC upper after molten for gained yellow powder sample, low pressure recycling design after the stream part containing one matter Mangiferin being merged, obtains Mangiferin sterling.
The method of described separation and purification Mangiferin from Lignum Aquilariae Resinatum leaf, is characterized in that, described diacolation or lixiviate adopt 50%-80% aqueous acetone solution or 50%-80% aqueous ethanolic solution to carry out diacolation, or employing volume ratio is the solid-liquid ratio room temperature lixiviate of 1:20.
The method of described separation and purification Mangiferin from Lignum Aquilariae Resinatum leaf, it is characterized in that, in described step normal phase column chromatography (2), is (3) separated, described medicinal extract or normal phase column chromatography rough segmentation and purification on normal-phase silica gel by volume 1:1 carry out mixing sample, and stream part reception volume is that 1/3-1 column volume is received as portion.
The method of described separation and purification Mangiferin from Lignum Aquilariae Resinatum leaf, it is characterized in that, the method obtaining described normal phase column chromatography rough segmentation is, adopt chloroform-methanol system, wash-out is carried out with stage (volume ratio is respectively 9:2 → 7:3 → 6:4 → 1:1 → 0:1), connect as portion with 1 column volume, after the stream part low pressure received being concentrated simultaneously, with Mangiferin reference substance point on same plate, volume ratio is adopted to be that under the chloroform-methanol-aqueous solvent of (10:3:1), the expansion system of phase carries out TLC detection, in 35 DEG C of low pressure recycling design after main stream part containing Mangiferin is merged, namely winner is containing Mangiferin stream part.
The method of described separation and purification Mangiferin from Lignum Aquilariae Resinatum leaf, is characterized in that, the described method again going up normal phase column chromatography separation is, employing chloroform-methanol volume ratio is that the solvent system of (75:25) carries out wash-out, elution speed 5ml/min; Connect as portion with 1/2 column volume, after the stream part low pressure received being concentrated simultaneously, with Mangiferin reference substance point on same plate, volume ratio is adopted to be that under the chloroform-methanol-aqueous solvent of (10:3:1), the expansion system of phase carries out TLC detection, bend down pressure recycling design in 35 DEG C after main stream part containing Mangiferin being merged, obtain the main yellow powder containing Mangiferin.
The method of described separation and purification Mangiferin from Lignum Aquilariae Resinatum leaf, is characterized in that, described step (1) middle recovered temperature is 40-60 DEG C.
The method of described separation and purification Mangiferin from Lignum Aquilariae Resinatum leaf, is characterized in that, described step (1) in recovered temperature be 45 DEG C, described step (2) in recovered temperature be 50 DEG C.
The method of described separation and purification Mangiferin from Lignum Aquilariae Resinatum leaf, it is characterized in that, the method that described HSCCC is separated is, the volume ratio of described solvent system acetate-methanol-water is (5.3-5.6): (0.4-0.6): (4.3-4.7), and regulating high-speed counter-current chromatograph to rotate forward rotating speed is 300-500rpm/min; Stationary phase retention value is 78%-89%; Moving phase is with 8-12ml/min; Determined wavelength: 254nm; Separation temperature is: 20-25 DEG C.
The method of described separation and purification Mangiferin from Lignum Aquilariae Resinatum leaf, is characterized in that, in described normal phase column chromatography, filler and sample ratio are 10-20 times.
The method of described separation and purification Mangiferin from Lignum Aquilariae Resinatum leaf, it is characterized in that, in described normal phase column chromatography, filler and sample ratio are 15 times, described step (3) in stream part to receive volume be 1 column volume for a, described step (4) in stream part to receive volume be that 1/2 column volume is for a.
The present invention has the following advantages:
1, adopt discarded whitewood spiceleaf to be raw material, cost is low;
2, the equal recoverable of solvent used in above-mentioned technique, reduces costs;
3, this technological operation is simple, and feasibility is high, and preparation cycle is short, can shorten time cost;
4, this technique adopts HSCCC to be prepared, and is separated the Mangiferin purity obtained high, pollution-free, stable, farthest can preserve the physiologically active of Mangiferin, and preparation amount is large, can through engineering approaches produce.
[accompanying drawing explanation]
Fig. 1 prepares high purity hypoglycemic activity composition Mangiferin process from whitewood spiceleaf;
Fig. 2 Mangiferin HSCCC separating spectrum (dash area is Mangiferin chromatographic peak).
[embodiment]
Below in conjunction with example and accompanying drawing, the present invention is described further.
Investigate discovery after deliberation, the Mangiferin containing high level in whitewood spiceleaf.Compared with Folium mangiferae, there is not the residual problem of agriculture, security is high, therefore selects whitewood spiceleaf as raw material in the present invention, smashes, can carry out diacolation or lixiviate after being dried by whitewood spiceleaf.
High speed adverse current chromatogram (High-speed Counter-current Chromatography, be called for short HSCCC) be a kind of liquid-liquid chromatograph isolation technique, when not by any solid packing, using immiscible liquid two-phase as stationary phase and moving phase, a kind of one-way fluid dynamic equilibrium is set up in the spiral tube of high speed rotating, with wherein one for stationary phase, another is moving phase mutually, can a large amount of stationary phase be retained in the process of continuous wash-out, in two-phase, distribute difference with material and reach separating effect.Compared with traditional separation means, this technology is not because adopting any solid packing to support and absorption, avoid because irreversible adsorption causes the inactivation, sex change etc. of sample, can not only recovery sample completely, the original characteristic of sample can also be reflected, being particularly suitable for natural bioactivity substance to be separated, is the effective ways being widely used in Separation of Natural Products purifying of generally acknowledging in the world at present.Due to environment special in spiral tube, separate substance can be made fully to contact mutually up and down with liquid, sample preparation amount is improved, be a kind of ideal preparative separation technology.It has, and sample nondestructive loses, pollution-free, efficient, quick, easy to operate, preparation amount large and low cost and other advantages, at natural product principal component or unknown component separating purifying, the separation and purification of chemosynthesis material, traditional Chinese medicine fingerprint and quality controling research, marine bioactivity component separating purifying, the fields such as macromole separation and chiral separation such as peptide and protein are all widely used.
Concrete operations technique of the present invention as shown in Figure 1.Comprise the steps:
1, extract prepares: get the blade and blade after manually promoting the Lignum Aquilariae Resinatum of Edgeworthia chrysantha tree body to adopt perfume (or spice), meal, adopt 50%-80% aqueous acetone solution (or 50%-80% aqueous ethanolic solution) diacolation (or adopting volume ratio to be lixiviate under the solid-liquid ratio room temperature of 1:20), merge all percolates or vat liquor, after concentrated removing organic solvent, adopt petroleum ether extraction 2-4 time, removing lipid and fat-soluble pigment, water intaking phase, after carrying out extraction 2-4 time with propyl carbinol, merge n-butanol portion, at 50 DEG C, low pressure concentrates, and namely obtains whitewood spiceleaf medicinal extract.
2, normal phase column chromatography rough segmentation: after 1:1 carries out mixing sample by volume by the medicinal extract of step 1 and purification on normal-phase silica gel, upper normal phase column chromatography is separated, adopt chloroform-methanol system, wash-out is carried out with stage (volume ratio 9:2 → 7:3 → 6:4 → 1:1 → 0:1), connect as portion with 1 column volume, after the stream part low pressure received being concentrated simultaneously, with Mangiferin reference substance point on same plate, adopt chloroform-methanol-water (volume ratio=10:3:1, take off phase) expansion system carry out TLC detection, in 35 DEG C of low pressure recycling design after main stream part containing Mangiferin is merged, obtain.
3, normal phase column chromatography is refined: after merging step 2 obtained is flowed part and purification on normal-phase silica gel 1:1 is carried out mixing sample by volume, upper normal phase column chromatography is separated, the solvent system of chloroform-methanol=75:25 is adopted to carry out wash-out, elution speed 5ml/min, connect as portion with 1/2 column volume, after the stream part low pressure received being concentrated simultaneously, with Mangiferin reference substance point on same plate, adopt chloroform-methanol-water (volume ratio=10:3:1, take off phase) expansion system carry out TLC detection, pressure recycling design is bent down in 35 DEG C after main stream part containing Mangiferin being merged, obtain the main yellow powder containing Mangiferin.
4, HSCCC prepares Mangiferin: prepare in acetate-methanol-water (volume ratio 5.4:0.5:4.4) solvent system and separating funnel, after fully shaking up, stratification, to separate mutually up and down, ultrasonic degas 10min, below be stationary phase mutually, lower is moving phase mutually, stationary phase is pumped in high speed adverse current chromatogram post with the flow velocity of 30ml/min, when filling stationary phase completely in high speed adverse current chromatogram post, regulating high-speed counter-current chromatograph to rotate forward rotating speed is 500rpm/min, and moving phase is pumped into high-speed counter-current chromatograph with the flow velocity of 10ml/min, after treating that in high-speed counter-current chromatograph, chromatographic column balances, yellow powder step 3 obtained is configured to solution, after pumping into chromatographic column, and moving phase is pumped in current chromatographic column with 10ml/min flow velocity, go out peak-to-peak type according to workstation and carry out the collection of stream part, every part of 5ml.
Adopt HPLC to carry out purity detecting each stream part, low pressure recycling design after the stream part containing one matter Mangiferin being merged, obtains Mangiferin sterling, the rate of recovery >=87%, product purity >=98%.
In above-mentioned steps, in normal phase column chromatography, filler and sample ratio are 10-20 times, preferably 15 times.It is that 1/3-1 column volume is received as portion that stream part receives volume, and meal preferably 1 column volume is a, and refining preferably 1/2 column volume is a.
The Mangiferin prepared is through high performance liquid chromatography and nuclear-magnetism qualification, consistent with bibliographical information.HSCCC is separated the stream part obtained and all adopts HPLC to detect, and its separating spectrum refers to Fig. 2.
Embodiment 1:
Get whitewood spiceleaf meal 30kg and be placed in diacolation bucket, add 70% aqueous acetone solution and carry out diacolation, receive percolate, after merging, be reduced to 180L in 45 DEG C of low-press thick.Adopt sherwood oil, n-butanol extraction respectively, obtain n-butanol portion medicinal extract 165.2g, after mixing sample with purification on normal-phase silica gel, upper normal phase column chromatography is separated, and 1 column volume connects as portion, after the stream part low pressure received being concentrated, carries out TLC detection simultaneously.Bend down pressure recycling design in 35 DEG C after main stream part containing Mangiferin being merged, obtain main stream part 3.366g containing Mangiferin.After merging rear stream part and purification on normal-phase silica gel are mixed sample, upper normal phase column chromatography is separated, and 1/2 column volume connects as portion, after low pressure concentrates at 35 DEG C by stream part of reception simultaneously, carries out TLC detection.Bend down pressure recycling design in 35 DEG C after main stream part containing Mangiferin being merged, obtain the main yellow powder 1.146g containing Mangiferin.After molten sample, upper HSCCC is separated, acetate-methanol-water volume ratio=5.5:0.4:4.5, and wherein above is stationary phase mutually, and lower is moving phase mutually; Regulating high-speed counter-current chromatograph to rotate forward rotating speed is 400rpm/min; Stationary phase retention value is 79%; Moving phase is with 8ml/min; Determined wavelength: 254nm; Separation temperature is: 20-25 DEG C, prepares Mangiferin 0.9970g, purity >=98%, the rate of recovery >=87%.
Embodiment 2:
Get whitewood spiceleaf meal 60kg in diacolation bucket, add 75% aqueous ethanolic solution and carry out diacolation, receive percolate, after merging, be reduced to 300L in 50 DEG C of low-press thick.Adopt sherwood oil, n-butanol extraction respectively, get n-butanol portion medicinal extract 340.7g, after mixing sample with purification on normal-phase silica gel, upper normal phase column chromatography is separated, and 1 column volume connects as portion, after the stream part low pressure received being concentrated, carries out TLC detection simultaneously.Bend down pressure recycling design in 40 DEG C after main stream part containing Mangiferin being merged, obtain main stream part 7.098g containing Mangiferin.After merging rear stream part and purification on normal-phase silica gel are mixed sample, upper normal phase column chromatography is separated, and 1/2 column volume connects as portion, after the stream part low pressure received being concentrated, carries out TLC detection simultaneously.Bend down pressure recycling design in 40 DEG C after main stream part containing Mangiferin being merged, obtain the main yellow powder 2.389g containing Mangiferin.After molten sample, upper HSCCC is separated, acetate-methanol-water volume ratio=5.3:0.4:4.3, and wherein above is stationary phase mutually, and lower is moving phase mutually; Regulating high-speed counter-current chromatograph to rotate forward rotating speed is 500rpm/min; Stationary phase retention value is 85%; Moving phase is with 12ml/min; Determined wavelength: 254nm; Separation temperature is: 20-25 DEG C, prepares Mangiferin 2.008g, purity >=98%, the rate of recovery >=89%.
Embodiment 3:
Get whitewood spiceleaf meal 90kg in diacolation bucket, add 75% aqueous acetone solution and carry out diacolation, receive percolate, after merging, be reduced to 480L in 45 DEG C of low-press thick.Adopt sherwood oil, n-butanol extraction respectively, obtain n-butanol portion medicinal extract 478.2g, after mixing sample with purification on normal-phase silica gel, upper normal phase column chromatography is separated, and 1 column volume connects as portion, after the stream part low pressure received being concentrated, carries out TLC detection simultaneously.Bend down pressure recycling design in 45 DEG C after main stream part containing Mangiferin being merged, obtain main stream part 9.998g containing Mangiferin.After merging rear stream part and purification on normal-phase silica gel are mixed sample, upper normal phase column chromatography is separated, and 1/2 column volume connects as portion, after the stream part low pressure received being concentrated, carries out TLC detection simultaneously.Bend down pressure recycling design in 40 DEG C after main stream part containing Mangiferin being merged, obtain the main yellow powder 3.1487g containing Mangiferin.After molten sample, upper HSCCC is separated, acetate-methanol-water volume ratio=5.6:0.5:4.6, and wherein above is stationary phase mutually, and lower is moving phase mutually; Regulating high-speed counter-current chromatograph to rotate forward rotating speed is 400rpm/min; Stationary phase retention value is 89%; Moving phase is with 10ml/min; Determined wavelength: 254nm; Separation temperature is: 20-25 DEG C, prepares Mangiferin 3.287g, purity >=98%, the rate of recovery >=87%.
Embodiment 4:
Get whitewood spiceleaf meal 30kg and be placed in diacolation bucket, add 80% aqueous ethanolic solution and carry out diacolation, receive percolate, after merging, be reduced to 150L in 50 DEG C of low-press thick, adopt sherwood oil respectively, n-butanol extraction, gets n-butanol portion medicinal extract 170.4g, after mixing sample with purification on normal-phase silica gel, upper normal phase column chromatography is separated, 1 column volume connects as portion, after the stream part low pressure received being concentrated, carries out TLC detection simultaneously.Bend down pressure recycling design in 35 DEG C after main stream part containing Mangiferin being merged, obtain main stream part 3.508g containing Mangiferin.After merging rear stream part and purification on normal-phase silica gel are mixed sample, upper normal phase column chromatography is separated, and 1/2 column volume connects as portion, after the stream part low pressure received being concentrated, carries out TLC detection simultaneously.Bend down pressure recycling design in 35 DEG C after main stream part containing Mangiferin being merged, obtain the main yellow powder 1.435g containing Mangiferin.After molten sample, upper HSCCC is separated, acetate-methanol-water volume ratio=5.4:0.4:4.3, and wherein above is stationary phase mutually, and lower is moving phase mutually; Regulating high-speed counter-current chromatograph to rotate forward rotating speed is 500rpm/min; Stationary phase retention value is 85%; Moving phase is with 12ml/min; Determined wavelength: 254nm; Separation temperature is: 20--5 DEG C, prepares Mangiferin 1.087g, purity >=98%, the rate of recovery >=88%.
Claims (10)
1. the method for separation and purification Mangiferin from Lignum Aquilariae Resinatum leaf, it is characterized in that, the method comprises the following steps:
(1) manually to promote that the Lignum Aquilariae Resinatum of Edgeworthia chrysantha is set body and adopted the blade after perfume (or spice) for raw material, dry after breaking into meal and carry out diacolation or lixiviate, merge all percolates or vat liquor, concentrated removing organic solvent;
(2) use petroleum ether extraction 2-4 time successively, removing lipid and fat-soluble pigment, water intaking phase, after carrying out extraction 2-4 time with propyl carbinol, merge n-butanol portion, at 40-60 DEG C, low pressure concentrates, and namely obtains the whitewood spiceleaf medicinal extract that master contains Mangiferin position;
(3) after gained whitewood spiceleaf medicinal extract n-butanol portion and purification on normal-phase silica gel being mixed sample, adopt normal phase column chromatography to be separated, in 35-45 DEG C of low pressure recycling design after main stream part containing Mangiferin being merged, namely obtain the main normal phase column chromatography rough segmentation containing Mangiferin;
(4) after sample being mixed in the rough segmentation of gained normal phase column chromatography and purification on normal-phase silica gel, again go up normal phase column chromatography and be separated, after main stream part containing Mangiferin being merged, low pressure recycling design, obtains the main yellow powder containing Mangiferin;
(5) prepare acetate-methanol-aqueous solvent, after stratification, above is stationary phase mutually, and lower is moving phase mutually; Be separated by HSCCC upper after molten for gained yellow powder sample, low pressure recycling design after the stream part containing one matter Mangiferin being merged, obtains Mangiferin sterling.
2. the method for separation and purification Mangiferin from Lignum Aquilariae Resinatum leaf according to claim 1, it is characterized in that, described diacolation or lixiviate adopt 50%-80% aqueous acetone solution or 50%-80% aqueous ethanolic solution to carry out diacolation, or employing volume ratio is the solid-liquid ratio room temperature lixiviate of 1:20.
3. the method for separation and purification Mangiferin from Lignum Aquilariae Resinatum leaf according to claim 1, it is characterized in that, in described step normal phase column chromatography (2), is (3) separated, described medicinal extract or normal phase column chromatography rough segmentation and purification on normal-phase silica gel by volume 1:1 carry out mixing sample, and it is that 1/3-1 column volume is received as portion that stream part receives volume.
4. the method for separation and purification Mangiferin from Lignum Aquilariae Resinatum leaf according to claim 1, it is characterized in that, the method obtaining described normal phase column chromatography rough segmentation is, adopt chloroform-methanol system, wash-out is carried out with stage (volume ratio is respectively 9:2 → 7:3 → 6:4 → 1:1 → 0:1), connect as portion with 1 column volume, after the stream part low pressure received being concentrated simultaneously, with Mangiferin reference substance point on same plate, volume ratio is adopted to be that under the chloroform-methanol-aqueous solvent of (10:3:1), the expansion system of phase carries out TLC detection, in 35 DEG C of low pressure recycling design after main stream part containing Mangiferin is merged, namely winner is containing Mangiferin stream part.
5. the method for separation and purification Mangiferin from Lignum Aquilariae Resinatum leaf according to claim 1, it is characterized in that, the described method again going up normal phase column chromatography separation is, employing chloroform-methanol volume ratio is that the solvent system of (75:25) carries out wash-out, elution speed 5ml/min; Connect as portion with 1/2 column volume, after the stream part low pressure received being concentrated simultaneously, with Mangiferin reference substance point on same plate, volume ratio is adopted to be that under the chloroform-methanol-aqueous solvent of (10:3:1), the expansion system of phase carries out TLC detection, bend down pressure recycling design in 35 DEG C after main stream part containing Mangiferin being merged, obtain the main yellow powder containing Mangiferin.
6. the method for separation and purification Mangiferin from Lignum Aquilariae Resinatum leaf according to claim 1, is characterized in that, described step (1) middle recovered temperature is 40-60 DEG C.
7., according to the method for separation and purification Mangiferin from Lignum Aquilariae Resinatum leaf according to claim 6, it is characterized in that, described step (1) in recovered temperature be 45 DEG C, described step (2) in recovered temperature be 50 DEG C.
8. the method for separation and purification Mangiferin from Lignum Aquilariae Resinatum leaf according to claim 1, it is characterized in that, the method that described HSCCC is separated is, the volume ratio of described solvent system acetate-methanol-water is (5.3-5.6): (0.4-0.6): (4.3-4.7), and regulating high-speed counter-current chromatograph to rotate forward rotating speed is 300-500rpm/min; Stationary phase retention value is 78%-89%; Moving phase is with 8-12ml/min; Determined wavelength: 254nm; Separation temperature is: 20-25 DEG C.
9. the method for separation and purification Mangiferin from Lignum Aquilariae Resinatum leaf according to claim arbitrary in claim 1 to 8, is characterized in that, in described normal phase column chromatography filler and sample ratio be 10-20 doubly.
10. the method for separation and purification Mangiferin from Lignum Aquilariae Resinatum leaf according to claim 9, it is characterized in that, in described normal phase column chromatography, filler and sample ratio are 15 times, described step (3) in stream part to receive volume be 1 column volume for a, described step (4) in stream part to receive volume be that 1/2 column volume is for a.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201410428303.3A CN104211690B (en) | 2014-08-27 | 2014-08-27 | Method for separating and purifying mangiferin from aquilaria sinensis leaves |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201410428303.3A CN104211690B (en) | 2014-08-27 | 2014-08-27 | Method for separating and purifying mangiferin from aquilaria sinensis leaves |
Publications (2)
| Publication Number | Publication Date |
|---|---|
| CN104211690A true CN104211690A (en) | 2014-12-17 |
| CN104211690B CN104211690B (en) | 2017-05-03 |
Family
ID=52093635
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN201410428303.3A Active CN104211690B (en) | 2014-08-27 | 2014-08-27 | Method for separating and purifying mangiferin from aquilaria sinensis leaves |
Country Status (1)
| Country | Link |
|---|---|
| CN (1) | CN104211690B (en) |
Cited By (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| 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 | Establishment method of Aquilaria sinensis leaf high performance liquid chromatography (HPLC) fingerprint spectrum |
| 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 |
| 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 |
Citations (6)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| WO1996016632A1 (en) * | 1994-11-25 | 1996-06-06 | Laboratoires De Biologie Vegetale Yves Rocher | Cosmetic or pharmaceutical compositions containing mangiferin or derivatives thereof |
| CN101993437A (en) * | 2009-08-27 | 2011-03-30 | 上海新康制药厂 | Method for extracting mangiferin from mango leaf |
| WO2011044751A1 (en) * | 2009-10-16 | 2011-04-21 | 天津中医药大学 | Foliamangiferosides, preparation method and use thereof |
| CN102424678A (en) * | 2011-12-09 | 2012-04-25 | 林励 | High-purity mangiferin prepared from leaves and twigs of aquilaria sinensis and preparation method thereof |
| CN102617669A (en) * | 2012-03-03 | 2012-08-01 | 浙江大学 | Method for separating and purifying mangiferin from mango pericarp |
| CN103408602A (en) * | 2013-07-22 | 2013-11-27 | 中国科学院西北高原生物研究所 | Separation and preparation method for four glycoside chemical reference substances in Tibetan capillary artemisia |
-
2014
- 2014-08-27 CN CN201410428303.3A patent/CN104211690B/en active Active
Patent Citations (6)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| WO1996016632A1 (en) * | 1994-11-25 | 1996-06-06 | Laboratoires De Biologie Vegetale Yves Rocher | Cosmetic or pharmaceutical compositions containing mangiferin or derivatives thereof |
| CN101993437A (en) * | 2009-08-27 | 2011-03-30 | 上海新康制药厂 | Method for extracting mangiferin from mango leaf |
| WO2011044751A1 (en) * | 2009-10-16 | 2011-04-21 | 天津中医药大学 | Foliamangiferosides, preparation method and use thereof |
| CN102424678A (en) * | 2011-12-09 | 2012-04-25 | 林励 | High-purity mangiferin prepared from leaves and twigs of aquilaria sinensis and preparation method thereof |
| CN102617669A (en) * | 2012-03-03 | 2012-08-01 | 浙江大学 | Method for separating and purifying mangiferin from mango pericarp |
| CN103408602A (en) * | 2013-07-22 | 2013-11-27 | 中国科学院西北高原生物研究所 | Separation and preparation method for four glycoside chemical reference substances in Tibetan capillary artemisia |
Non-Patent Citations (1)
| Title |
|---|
| 胡彦君，等: "芒果叶的化学成分研究", 《亚太传统医药》 * |
Cited By (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| 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 | Establishment method of Aquilaria sinensis leaf high performance liquid chromatography (HPLC) fingerprint spectrum |
| 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 |
Also Published As
| Publication number | Publication date |
|---|---|
| CN104211690B (en) | 2017-05-03 |
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| CN102976909B (en) | Method for extracting and purifying 6-gingerol from ginger | |
| CN101190258A (en) | Total sesquiterpene lactone extract containing rich parthenolide and preparation method and application thereof | |
| CN105399656A (en) | Isobenzazole alkaloid compound, and preparation method and applications thereof | |
| CN102746362A (en) | Method for extracting refined astragaloside from astragaliradix | |
| CN101691330A (en) | Separation and purification methods of highly purified antiviral active components in artichoke | |
| CN102850416A (en) | Method and apparatus used for preparing olive leaf extract | |
| CN102351824A (en) | Method for preparing lactuca indica and lactucin | |
| CN104211690A (en) | Method for separating and purifying mangiferin from aquilaria sinensis leaves | |
| CN105348192A (en) | Antiviral-activity isoquinoline alkaloid compound in Cassia alata L. and preparation method of antiviral-activity isoquinoline alkaloid compound | |
| CN104940280A (en) | Method for extracting total flavones from radix puerariae employing enzyme preparation | |
| CN102731592A (en) | Method for extracting cleupin and amentoflavone from olive leaf | |
| CN102898347A (en) | Method for extracting caragana microphylla from hypaphorine | |
| US10111917B2 (en) | Method for preparing purified extracts of harpagophytum procumbens | |
| CN106831909B (en) | The extracting method of double benzene pyrrones compounds in rhizoma anemarrhenae fibrous root | |
| CN103467262B (en) | Method for preparing 9-oxonerolidol from camphor tree plants | |
| CN102391350A (en) | Method for purifying polygalasaponin F | |
| CN107325147A (en) | The screening technique of fulvoushair honeysuckle flower moderate resistance hypertensin conversion enzyme activity composition | |
| CN101880301B (en) | Novel method for preparing paeoniflorin extract | |
| CN102863489A (en) | Method for extracting catalpol from rehmannia stem | |
| CN102432419B (en) | Method for extracting and separating beta-elemene from Eupatorium adenophorum | |
| CN102432420B (en) | Method for extracting and separating beta-elemene from Lantana camara | |
| CN101973975B (en) | Method for fast extracting luteolin from Lonicera macranthoides | |
| CN101513448B (en) | Preparation and use of Ziziphora general lavone | |
| CN103819518A (en) | Preparation method for standardized extracts of eucommia ulmoides pinoresinol diglucoside | |
| CN104592322A (en) | Method for extracting and separating curculigoside from curculigo gaertn plants |
Legal Events
| Date | Code | Title | Description |
|---|---|---|---|
| C06 | Publication | ||
| PB01 | Publication | ||
| C10 | Entry into substantive examination | ||
| SE01 | Entry into force of request for substantive examination | ||
| GR01 | Patent grant | ||
| GR01 | Patent grant | ||
| TR01 | Transfer of patent right | ||
| TR01 | Transfer of patent right |
Effective date of registration: 20171120 Address after: 528403 room D, house D, No. 328, No. 328, Changjiang North Road, Eastern District, East District of Guangdong Province Co-patentee after: Yang Maoxun Patentee after: Zhongshan sunshine biological science and Technology Development Co.,Ltd. Co-patentee after: Liang Yaoguang Address before: 528403 room D, house D, No. 328, No. 328, Changjiang North Road, Eastern District, East District of Guangdong Province Co-patentee before: Yang Maoxun Patentee before: ZHONGSHAN GEHAO BIOLOGICAL SCIENCE & TECHNOLOGY DEVELOPMENT Co.,Ltd. Co-patentee before: Liang Yaoguang |
|
| TR01 | Transfer of patent right | ||
| TR01 | Transfer of patent right |
Effective date of registration: 20190628 Address after: Room 201H on the second floor of No. 4 workshop of South China Modern Chinese Medicine City Science Park, Nanlang Town, Zhongshan City, Guangdong Province Co-patentee after: Yang Maoxun Patentee after: Zhongshan Yixiang Biotechnology Co.,Ltd. Co-patentee after: Liang Yaoguang Address before: Room 421, Building D, Junxianju, 328 Changjiang North Road, East District, Zhongshan City, Guangdong Province Co-patentee before: Yang Maoxun Patentee before: Zhongshan sunshine biological science and Technology Development Co.,Ltd. Co-patentee before: Liang Yaoguang |
|
| TR01 | Transfer of patent right | ||
| TR01 | Transfer of patent right |
Effective date of registration: 20220919 Address after: Room 501-2333, Office Building, Development Zone, No. 8, Xingsheng South Road, Economic Development Zone, Miyun District, Beijing 100055 (Central Office Area of Economic Development Zone) Patentee after: BEIJING LIKEDE TECHNOLOGY Co.,Ltd. Address before: Room 201H on the second floor of No. 4 workshop of South China Modern Chinese Medicine City Science Park, Nanlang Town, Zhongshan City, Guangdong Province Patentee before: Zhongshan Yixiang Biotechnology Co.,Ltd. Patentee before: Yang Maoxun Patentee before: Liang Yaoguang |