CN101891722B - Method for preparing usnic acid - Google Patents
Method for preparing usnic acid Download PDFInfo
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- CN101891722B CN101891722B CN2010102368429A CN201010236842A CN101891722B CN 101891722 B CN101891722 B CN 101891722B CN 2010102368429 A CN2010102368429 A CN 2010102368429A CN 201010236842 A CN201010236842 A CN 201010236842A CN 101891722 B CN101891722 B CN 101891722B
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Abstract
The invention discloses a method for preparing usnic acid. The method comprises the following steps of: 1) crushing usnea longissima and blending the crushed usnea longissima with an organic solvent, and performing refluxing extraction to obtain usnic acid crude extracts; and 2) selecting a two-phase solvent system to separate the usnic acid crude extracts obtained by step 1) by high speed counter-current chromatography to obtain the usnic acid. The method adopts the high speed counter-current chromatography for separating the usnic acid; and compared with conventional experimental preparation method, the method has the obvious advantages of large sample volume at one time, high separation capability, short separation time, small sample loss and synchronous completion of separation and purification. The method provided by the invention is suitable for a high speed counter-current chromatograph with the column capacity of 325ml, and the usnic acid prepared by the method has a purity of up to 99 percent and a very important application value.
Description
Technical field
The present invention relates to the method for separating and preparing of natural antibiotics in the lichen, particularly the preparation method of usninic acid.
Background technology
Usninic acid is one of main bioactive ingredients of Usnea Longissima.Usnea Longissima (Usnea.longissima) is a kind of lichen of Usneaceae pine Polyalthia; Also claim rime, Herba Stellariae Alsines; Mainly grow in ground such as Xinjiang, the Inner Mongol, Yunnan, Gansu, Tibet, Heilungkiang, Jilin, Liaoning, have effects such as clearing heat and detoxicating, relieving cough and reducing sputum, anti-inflammatory.Usninic acid demonstrates stronger retarding effect to streptococcus aureus; Intestinal bacteria, mould, yeast and pathogenic bacteria also there are certain restraining effect, can be used as natural antiseptic agent and be used for food and feed and have effects such as clearing heat and detoxicating, relieving cough and reducing sputum, antiviral, anti-inflammatory, promotion wound healing.
Organic solvent extraction is generally adopted in the preparation of usninic acid, through the mode of recrystallization or purification by silica gel column chromatography repeatedly, obtains highly purified product again.The usninic acid generally content in Usnea Longissima is about 1.4%.Because repeatedly recrystallization and silica gel column chromatography can cause bigger loss to title product, make yield reduce greatly, its process is loaded down with trivial details tediously long simultaneously, and preparation amount is little.
Summary of the invention
The purpose of this invention is to provide a kind of method for preparing usninic acid.
The method for preparing usninic acid provided by the invention comprises the steps:
1) Usnea Longissima is pulverized back and organic solvent mixing, carry out refluxing extraction, obtain the usninic acid crude extract;
2) select two phase solvent system, adopt high-speed countercurrent chromatography, the usninic acid crude extract that said step 1) is obtained separates, and obtains said usninic acid.
In the step 1) of aforesaid method, Usnea Longissima can be bought by open commercial sources and obtain, and the order number of the Usnea Longissima after the said pulverizing is the 20-80 order; Preferred 60 orders; Said organic solvent is selected from any one in ether, ETHYLE ACETATE and the sherwood oil, preferred sherwood oil or ether, more preferably sherwood oil; Said sherwood oil is that boiling range is 60 ℃～90 ℃ a sherwood oil; The amount ratio of said organic solvent and said Usnea Longissima is the 4-10 milliliter: 1 gram, preferred 5-8 milliliter: 1 gram; In the said refluxing extraction step, the time of each refluxing extraction is 3-6 hour, is preferably 4-5 hour, and number of times is 2-4 time.
Said step 2) in; Said two phase solvent system is any one among the following two phase solvent system a-c; Said two phase solvent system a is for being that normal hexane, acetonitrile, ETHYLE ACETATE and the water of 8-13: 7-11: 5-8: 0.8-1.5 is formed by volume ratio; Said two phase solvent system b is for being that normal hexane, ethanol, ETHYLE ACETATE and the water of 8-13: 7-11: 5-8: 0.8-1.5 is formed by volume ratio; Said two phase solvent system c is for being that normal hexane, methyl alcohol, ETHYLE ACETATE and the water of 8-13: 7-11: 5-8: 0.g-1.5 is formed by volume ratio; Being preferably volume ratio is 8: 7: 6: 0.9 the two phase solvent system of being made up of normal hexane, acetonitrile, ETHYLE ACETATE and water, more preferably volume ratio is 8: 7: 5: 0.8 two phase solvent system, the volume ratio be made up of normal hexane, acetonitrile, ETHYLE ACETATE and water are 8: 7: 5: 0.8 two phase solvent system or the volume ratio be made up of normal hexane, ethanol, ETHYLE ACETATE and water are 8: 7: 5: 0.8 the two phase solvent system of being made up of normal hexane, methyl alcohol, ETHYLE ACETATE and water.The rotating speed of said high speed adverse current chromatogram separator column is 500rpm-1000rpm, is preferably 600rpm-800rpm.The TV of the high speed adverse current chromatogram separator column that said high-speed countercurrent chromatography is used is 325mL.
The sample that said high-speed countercurrent chromatography is used is that said step 1) gained usninic acid crude extract is dissolved in the solution that forms in the appearance solvent; Said molten appearance solvent is to be gone up mutually and the solvent of phase composite under the 5ml by the 5ml in the said two phase solvent system; The amount ratio of said usninic acid crude extract and said molten appearance solvent is 10mg-300mg: 10ml, specifically can be 10mg-50mg: 10ml, 10mg-25mg: 10ml, 25mg-300mg: 10ml, 25mg-50mg: 10ml or 50mg-300mg: 10ml.
In the used high speed adverse current chromatogram separator column of said high-speed countercurrent chromatography; Moving phase is the following phase in the said two phase solvent system; The flow velocity of said moving phase is 0.5-4ml/min; Be specially 1.5-2ml/min, preferred 1-3ml/min, the flow direction of moving phase pumps into for the head end by said high speed adverse current chromatogram separator column; Stationary phase is the last phase in the said two phase solvent system, and the flow velocity of said stationary phase is 6ml/min.
The separation temperature of said high speed adverse current chromatogram separator column is 20 ℃-30 ℃.The detection wavelength of said high speed adverse current chromatogram separator column is 254nm.
In addition, after said step 1), said step 2) before the separating step, in sepn process, have bubble to generate, also can carry out ultrasonic degas, the condition of this ultrasonic degas is the 100KHz degassing 10 minutes.
The present invention adopts high performance countercurrent chromatography (High-speed Counter-current Chromatography; HSCCC) carry out the separation and purification of usninic acid; With in the past test is that the technology of preparing means are compared and had tangible advantage: the single injected sampling amount is big, separating power is strong, disengaging time is short, sample loss is little, and separation and purifying can be accomplished synchronously.Method provided by the invention is suitable for the high-speed counter-current chromatograph of the high-speed counter-current chromatograph of column capacity 325ml, utilizes purity that this method prepares the gained usninic acid up to 99%, has important use and is worth.
Description of drawings
Fig. 1 is 600 rev/mins HSCCC spectrogram for embodiment 1 medium speed.
Fig. 2 is the liquid chromatogram of embodiment 1 preparation gained HSCCC discrete group swarming A (usninic acid).
Embodiment
Below in conjunction with specific embodiment the present invention is described further, but the present invention is not limited to following examples.Among the following embodiment, like no specified otherwise, be ordinary method, said percentage composition is the quality percentage composition like no specified otherwise.
Embodiment 1
1) the order number that takes by weighing after the pulverizing is 20 order Usnea Longissima 100g, adds the 600ml boiling range and be 60 ℃-90 ℃ sherwood oil, refluxing extraction 6h, suction filtration.Repeat above refluxing extraction step 3 time.Merging filtrate, 45 ℃ of vacuum (vacuum tightness is 0MPa) are concentrated into 15ml, and hold over night under the room temperature obtains the usninic acid crude extract.
2) be 8: 7: 5 with volume ratio: 0.8 the mixed solution of being made up of normal hexane, acetonitrile, ETHYLE ACETATE and water is as two phase solvent system; Adopt high-speed counter-current chromatograph, the usninic acid crude extract that step 1) is obtained is dissolved in and carries out separation and purification in this two phase solvent system.Concrete steps are following: behind this two phase solvent system thorough mixing, leave standstill phase-splitting, two 30min that under 100KHz UW condition, outgas successively mutually.Getting is stationary phase mutually, is moving phase mutually down.The flow velocity of stationary phase with 6ml/min pumped in the high speed adverse current chromatogram separator column that the separator column TV is 325ml, start the high speed adverse current chromatogram main frame, regulating the separator column rotating speed is 600rpm, and the flow velocity of moving phase with 1.5ml/min caudad pumped into by head end.The separator column temperature transfers to 25 ℃.The usninic acid crude extract that the 25mg step 1) is obtained is dissolved in 5ml and goes up the solution that obtains this usninic acid crude extract mutually and in the solvent of phase composite under the 5ml.Treat that the high speed adverse current chromatogram separator column reaches balance, with the solution injection injection annulus of this usninic acid crude extract, the detection wavelength is 254nm.Use the substep scoop to collect the target that contains usninic acid and flow out component.By the chromatographic working station on the connection detector is on the on-line data acquisition analyser, obtains HSCCC spectrogram shown in Figure 1, and wherein peak A is a usninic acid.The collection liquid that will contain this target components merges, and nitrogen dries up, and obtains product 11.2mg, and yield is 44.8%.
The nuclear-magnetism detected result of this product is following:
1H-NMR(300MHz，CDCL
3)：76(3H，s)，2.10(3H，s)，2.66(3H，s)，2.68(3H，s)，5.98(1H，s)，11.03(1H，s)，13.33(1H，s)；
13C-NMR(75MHz，CDCL
3)：197.80(s，C-1)，104.98(s，C-2)，205.10(s，2-OCCH
3)，27.58(q，2-OCCH
3)，191.44(s，C-3)，98.06(d，C-4)，179.10(s，C-4a)，101.27(s，C-6)，200.03(s，6-OCCH
3)，30.97(q，6-OCCH
3)，154.95(s，C-5a)，63.6(s，C-9)，103.69(s，C-7)，109.07(s，C-8)，7.25(q，8-CH3)，157.25(s，C-9)，103.69(s，C-9a)，58.81(s，C-9b)，31.84(q，9b-CH
3)。
In addition, according to following condition this peak A product being carried out performance liquid chromatography (HPLC) detects: chromatographic column: Agilent anti-phase C18 post (150 * 4.6mm, id 10 μ m; Moving phase: by volume ratio be 60: 40 be the mixed solution that 0.1% acetic acid aqueous solution is formed by acetonitrile and mass percentage concentration; Flow velocity: 1.0ml/min; Detect wavelength: 254nm; Detected temperatures: 30 ℃; Applied sample amount: 10 μ l.The gained result is as shown in Figure 2.
Can be known that by above-mentioned nucleus magnetic resonance and HPLC detected result peak A product structure is correct in this HSCCC spectrogram, is usninic acid, purity is 99%.
Embodiment 2
1) the order number that takes by weighing after the pulverizing is 60 purpose Usnea Longissima 100g, adds the 800ml boiling range and be 60-90 ℃ sherwood oil, refluxing extraction 4h, suction filtration.Repeat above refluxing extraction step 3 time.Merging filtrate, 45 ℃ of vacuum (vacuum tightness is 0.01MPa) are concentrated into 15ml, and hold over night under the room temperature obtains the usninic acid crude extract.
2) be 8: 7: 5 with volume ratio: 0.8 the mixed solution of being made up of normal hexane, acetonitrile, ETHYLE ACETATE and water is as two phase solvent system; Adopt high-speed counter-current chromatograph, the usninic acid crude extract that step 1) is obtained is dissolved in and carries out separation and purification in this two phase solvent system.Concrete steps are following: behind this two phase solvent system thorough mixing, leave standstill phase-splitting, two 30min that under 100KHz UW condition, outgas successively mutually.Getting is stationary phase mutually, is moving phase mutually down.The flow velocity of stationary phase with 6ml/min pumped in the high speed adverse current chromatogram separator column that the separator column TV is 325ml, start the high speed adverse current chromatogram main frame, regulating the separator column rotating speed is 800rpm, and the flow velocity of moving phase with 2ml/min caudad pumped into by head end.The separator column temperature transfers to 30 ℃.50mg usninic acid crude extract is dissolved in 5ml goes up the solution that obtains this usninic acid crude extract mutually and in the solvent of phase composite under the 5ml.Treat that the high speed adverse current chromatogram separator column reaches balance, with the solution injection injection annulus of this usninic acid crude extract, the detection wavelength is 254nm.Use the substep scoop to collect and flow out component.Collect the target components that contains usninic acid according to the chromatographic working station spectrogram.The collection liquid that will contain this target components merges, and nitrogen dries up, and obtains product 22.8mg, and yield is 45.4%.
Detect according to embodiment 1 identical testing conditions peak A product being carried out HPLC, the gained detected result is identical with embodiment 1.The nuclear-magnetism detected result of this product is also identical with embodiment 1.Can be known that by above-mentioned nucleus magnetic resonance and HPLC detected result peak A product structure is correct in this HSCCC spectrogram, is usninic acid, purity is 99%.
Embodiment 3
According to preparing usninic acid with embodiment 2 identical steps, only the order number with the Usnea Longissima in the step 1) changes 80 orders into, and the extraction solvent is an ether, consumption 1000mL/ time, and refluxing extraction 3 hours is extracted 2 times, and 35 ℃ of vacuum concentrated filtrates are to 10ml; Step 2) usninic acid crude extract amount changes 10mg in, and flow rate of mobile phase changes 4ml/min into, 20 ℃ of separator column temperature, and the separator column rotating speed changes 500rpm into.
Detect according to embodiment 1 identical testing conditions peak A product being carried out HPLC, the gained detected result is identical with embodiment 1.The nuclear-magnetism detected result of this product is also identical with embodiment 1.Can be known that by above-mentioned nucleus magnetic resonance and HPLC detected result peak A product structure is correct in this HSCCC spectrogram, is usninic acid, purity is 99.5%, and yield is 40.08%.
Embodiment 4
According to preparing usninic acid with embodiment 1 identical step, only change the extraction solvent in the step 1) into ETHYLE ACETATE, consumption 400mL/ time refluxed 6 hours, extracted 4 times, and 50 ℃ of vacuum concentrated filtrates are to 30ml; In the step 2) in two phase solvent system to change volume ratio into be 8: 7: 5: 0.8 the mixed solution of forming by normal hexane, methyl alcohol, ETHYLE ACETATE and water, usninic acid crude extract amount changes 300mg into, the separator column rotating speed changes 1000rpm into.
Detect according to embodiment 1 identical testing conditions peak A product being carried out HPLC, the gained detected result is identical with embodiment 1.The nuclear-magnetism detected result of this product is also identical with embodiment 1.Can be known that by above-mentioned nucleus magnetic resonance and HPLC detected result peak A product structure is correct in this HSCCC spectrogram, is usninic acid, purity is 99%, and yield is 43.6%.
Embodiment 5
According to preparing usninic acid, only with step 2 with embodiment 1 identical step) in two phase solvent system to change volume ratio into be 8: 7: 5: 0.8 the mixed solution of being made up of normal hexane, ethanol, ETHYLE ACETATE and water, flow rate of mobile phase changes 0.5ml/min into.
Detect according to embodiment 1 identical testing conditions peak A product being carried out HPLC, the gained detected result is identical with embodiment 1.The nuclear-magnetism detected result of this product is also identical with embodiment 1.Can be known that by above-mentioned nucleus magnetic resonance and HPLC detected result peak A product structure is correct in this HSCCC spectrogram, is usninic acid, purity is 99%, and yield is 41%.
Claims (1)
1. method for preparing usninic acid, step is following:
1) the order number that takes by weighing after the pulverizing is 80 purpose Usnea Longissima 100g, adds ether, refluxing extraction 3h, suction filtration; Extract 2 times, the ether consumption is 1000mL/ time, merging filtrate, and 35 ℃ of said filtratings of vacuum concentration to 10ml, hold over night under the room temperature obtains the usninic acid crude extract;
2) be 8: 7: 5 with volume ratio: 0.8 the mixed solution of being made up of normal hexane, acetonitrile, ETHYLE ACETATE and water is as two phase solvent system; Adopt high-speed counter-current chromatograph; The usninic acid crude extract that step 1) is obtained is dissolved in and carries out separation and purification in this two phase solvent system; Step is: behind this two phase solvent system thorough mixing, leave standstill phase-splitting, two 30min that under 100KHz UW condition, outgas successively mutually; Getting is stationary phase mutually, is moving phase mutually down; The flow velocity of said stationary phase with 6ml/min pumped in the high speed adverse current chromatogram separator column that the separator column TV is 325ml; Start the high speed adverse current chromatogram main frame; Regulating the separator column rotating speed is 500rpm, and the flow velocity of said moving phase with 4ml/min caudad pumped into by head end; The separator column temperature transfers to 20 ℃; The said usninic acid crude extract of 10mg is dissolved in the solution that obtains this usninic acid crude extract in the said solvent of going up mutually with the said following phase composite of 5ml of 5ml; Treat that the high speed adverse current chromatogram separator column reaches balance; The solution of this usninic acid crude extract is injected injection annulus, and the detection wavelength is 254nm, uses the substep scoop to collect and flows out component; Collect the target components that contains usninic acid according to the chromatographic working station spectrogram; The collection liquid that will contain this target components merges, and nitrogen dries up, and obtains usninic acid; Said usninic acid purity is 99.5%.
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| CN102973767A (en) * | 2012-12-02 | 2013-03-20 | 西北农林科技大学 | Chinese herbal medicine combination for effectively relieving cough and preparation method thereof |
| CN103044372B (en) * | 2012-12-13 | 2016-03-30 | 大兴安岭林格贝寒带生物科技股份有限公司 | A kind of production technique extracting usninic acid from Usnea Longissima |
| CN105734091A (en) * | 2016-04-19 | 2016-07-06 | 天津科技大学 | Method for producing high-purity monascine and ankafaflavin through monascus liquid-state fermentation |
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| 迟春艳.高速逆流色谱分离制备无花果叶和长松萝中主要活性成分的研究.《中国农业科学院学位论文》.2008,第26页第3.2.2节-第34页最后1段. * |
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