CN103694120B - A kind of method extracting high-purity chlorogenic acid from clover - Google Patents
A kind of method extracting high-purity chlorogenic acid from clover Download PDFInfo
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- CN103694120B CN103694120B CN201410008928.4A CN201410008928A CN103694120B CN 103694120 B CN103694120 B CN 103694120B CN 201410008928 A CN201410008928 A CN 201410008928A CN 103694120 B CN103694120 B CN 103694120B
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Abstract
From clover, extract a method for high-purity chlorogenic acid, comprise the following steps: by clover cleaning, dry, pulverizing grinding; Add water high-pressure homogenization, and centrifuging and taking supernatant liquor obtains high density crude extract, and the ultrafiltration of high density crude extract obtains filtrate; Macroporous resin column is adsorbed, ethanol elution, after collecting elutriant, repeats described absorption and wash-out; Elutriant is vacuum-drying at low temperatures; Add acetone solution again to filter, get filtrate and reclaim acetone, vacuum-drying.Cheaper starting materials of the present invention is easy to get, for the leguminous plants that domestic and international cultivated area is maximum, can in enormous quantities for the extraction of chlorogenic acid, production cost is low, simple to operate, method environmental protection, without hazardous solvent, also can be widely used in the raw material chlorogenic acid extractings such as Japanese Honeysuckle, Folium Eucommiae or coffee berry, mild condition, does not heat without high light, effectively prevent the decomposition of chlorogenic acid, improve the purity of chlorogenic acid, use high-pressure homogenization, repeatedly centrifugal and ultrafiltration membrance filter, substantially increase extraction yield and the purity of chlorogenic acid.
Description
Technical field
The invention belongs to technical field of chlorogenic acid extraction, relate to the method for chlorogenic acid extracting from plant, particularly from clover, high efficiency separation extracts the method for high-purity chlorogenic acid.
Background technology
Clover is pulse family clover, and its flower for purple, because grass yield is high, rich in proteins, strong adaptability are called as " King of Pasture ", is one of China's Dominant Species of Forage Grass, is the maximum leguminous forage of domestic and international cultivated area.Clover is also rich in the valuable bioactive ingredients of chlorogenic acid, but does not have the correlative study of separation and Extraction chlorogenic acid from clover at present.Chlorogenic acid in clover has biological activity widely, as anti-oxidant, mutation inhibiting and antitumor, obviously can promote the effects such as enterogastric peristalsis, gastric secretion and cholagogic, scavenging free radicals and anti peroxidation of lipid, its hydrolysate caffeine also has the effect of cholagogic, leukocyte increasing.
Chlorogenic acid is mainly distributed in the plants such as Caprifoliaceae, the Rosaceae, composite family, Rubiaceae and Eucommiaceae, that the traditional Chinese medicine such as the bark of eucommia, Japanese Honeysuckle is clearing heat and detoxicating, the main component of antiphlogistic antibacterial, be plant a kind of phenylpropanoids through shikimic acid pathway synthesis in aerobic repiration process, molecular formula is C
16h
18o
9, molecular weight is 354.90, facile hydrolysis under alkalescence or hot conditions.Its effective biological activity is paid much attention to, and becomes internationally recognized " plant gold ".Highly purified chlorogenic acid has high economic worth and market outlook.
There is the preparation technology of a lot of chlorogenic acid at present, chlorogenic acid itself has unstable, high temperature, long-time heating and strong illumination to be avoided during extraction, existing decocting cooking method, water extraction and alcohol precipitation method, supercritical fluid extraction etc., supercritical fluid extraction productive rate is high but cost is also high, can't be applied to suitability for industrialized production at present; Alkaline precipitation easily makes chlorogenic acid be hydrolyzed; Although microwave extraction and supersound extraction extraction time, energy consumption etc. reduce greatly, exist noise large, be difficult to the deficiencies such as large-scale production; It is large to there is facility investment in supercritical fluid extraction, and cost is high, and the material that, polarity high to relative molecular mass is strong extracts the problems such as difficulty.All there is larger problem in these methods, some purity is low, and some costs are high, the too very complicated had, and some product waste liquor contamination environment, what have is not suitable for large-scale commercial production etc.Extracting method as described in Chinese patent 01144170.4 can only extract the chlorogenic acid that purity is 84%.Along with on market, chlorogenic acid demand is constantly increased, how easy, rapidly obtain from raw material steady quality, purity is high, yield is high, safe and reliable chlorogenic acid Study on Extraction Method necessary.
Summary of the invention
The present invention is directed to the weak point of existing chlorogenic acid technology of preparing, propose a kind of method extracting high-purity chlorogenic acid from clover simply, fast, easily.Comprise the following steps:
(1) by clover cleaning, dry, pulverizing grinding;
(2) add water high-pressure homogenization, and centrifuging and taking supernatant liquor obtains high density crude extract, and the ultrafiltration of high density crude extract obtains filtrate;
(3) macroporous resin column absorption, ethanol elution, after collecting elutriant, repeats described absorption and wash-out;
(4) elutriant vacuum-drying at low temperatures; Add acetone solution again to filter, get filtrate and reclaim acetone, vacuum-drying.
Further, described high-pressure homogenization temperature maintains 15 ~ 20 DEG C, avoids chlorogenic acid to decompose, and amount of water is 10 ~ 15 times of raw material.
Further, describedly centrifugally to carry out at twice, rotating speed is respectively 1500r/h and 2000r/h.
Further, the film hole diameter of ultra-filtration membrane that uses of described ultrafiltration is below 0.4 micron.
Further, described macroporous resin column adopts NKA-2 macroporous resin, and its polarity is good, maximum can adsorb chlorogenic acid, thus improve the extraction yield of chlorogenic acid.
Further, described wash-out adopts the ethanol of 20% of 5 times of resin column volumes to carry out wash-out, and flow velocity is 0.5/h, can at utmost be eluted by chlorogenic acid.
Further, the described clover diameter through grinding, at below 1mm, increases the surface-area of clover, is conducive to the chlorogenic acid extracting high density.
Further, the product that (4) (3) (2) (1) described method obtain through step, adopts Thin-layer chromatography-ultraviolet spectrophotometry to record chlorogenic acid purity more than 95%.
Thin-layer chromatography-ultraviolet spectrophotometry of the present invention is, point sample on silica-gel plate, adopts developing agent to launch, scraping spot under ultraviolet lamp, through dissolve with ethanol, then by the content of ultraviolet spectrophotometry detection by quantitative chlorogenic acid.
Developing agent of the present invention selects volume ratio to be the mixed solution of the ethyl acetate of 10:2:3, acetic acid and water; Uv-spectrophotometric detects and adopts TU-1800SPC type ultraviolet spectrophotometer to detect absorbancy in 330nm place.
Further, described method chooses the clover that leaf weight reaches more than 50% of complete stool.Wherein preferred, take the fresh pollution-free fresh and tender clover being in bud stage and initial bloom stage, it contains abundant chlorogenic acid, is not relate in current chlorogenic acid extraction field both at home and abroad.
The invention has the beneficial effects as follows:
1. the cheaper starting materials of the inventive method is easy to get, and is the leguminous plants that domestic and international cultivated area is maximum, can in enormous quantities for the extraction of chlorogenic acid, and production cost is low, simple to operate, method environmental protection.The method, except available clover is as except raw material, also can be widely used in Japanese Honeysuckle, Folium Eucommiae or coffee berry equipotential raw material chlorogenic acid extracting.
2. in whole operating process, mild condition, does not heat without high light, effectively prevent the decomposition of chlorogenic acid, improves the purity of chlorogenic acid.
3. use high-pressure homogenization, repeatedly centrifugal and ultrafiltration membrance filter, substantially increase extraction yield and the purity of chlorogenic acid.
4. method of the present invention have technique advanced person, easy and simple to handle, filter easily, cost is low, high without hazardous solvent, purity, rate high, be applicable to the advantages such as industrialized production.
Embodiment
Following non-limiting example can make the present invention of those of ordinary skill in the art's comprehend, but does not limit the present invention in any way.
Embodiment 1
1. get Herba Medicaginis leaf 50kg, removal of impurities clean dry, pulverize and grind to form 10-20 object powder, add 10 times to the distilled water of powder, suspension liquid is put into high pressure homogenizer and pulls an oar, pulverize raw material further, increase surface-area;
2. above-mentioned mixed solution is centrifugal in supercentrifuge, and centrifugal rotational speed is 1000r/h for the first time, abandons precipitation and gets supernatant liquor recentrifuge, and second time centrifugal rotational speed is 1500r/h;
3. by said extracted liquid again through the polymer substance such as ultrafiltration membrance filter leaf protein, polysaccharide of less than 0.4 micron;
4. by said extracted liquid after macroporous resin NKA-2 enrichment, 5 times of column volume 20% ethanol repeatedly wash-out, collect elutriant in vacuum-drying, use acetone solution again, collect filtrate and reclaim acetone spraying dry, adopt the absorbancy at thin-layer chromatography-UV spectrophotometer measuring 330nm place, find that chlorogenic acid has good linear relationship (y=absorbancy, x=chlorogenic acid mass concentration within the scope of 1.2 ~ 12.6 μ g/ml, regression equation is y=0.023x+0.499, r
2=0.9463), measurement result display chlorogenic acid sample recovery rate is 88.2%.
Embodiment 2
1. get Herba Medicaginis leaf 80kg, removal of impurities clean dry, pulverize and grind to form 10-20 object powder, add 10 times to the distilled water of powder, suspension liquid is put into high pressure homogenizer and pulls an oar, pulverize raw material further, increase surface-area;
2. above-mentioned mixed solution is centrifugal in supercentrifuge, and centrifugal rotational speed is 1000r/h, abandons precipitation and gets supernatant liquor recentrifuge, and rotating speed is 1500r/h;
3. by said extracted liquid again through the polymer substance such as ultrafiltration membrance filter leaf protein, polysaccharide of less than 0.4 micron;
4. by said extracted liquid after macroporous resin NKA-2,5 times of column volume 20% ethanol repeatedly wash-out, collect the powder that elutriant is dry under vacuo, use acetone solution again, collect filtrate and reclaim acetone spraying dry, adopt the absorbancy at thin-layer chromatography-UV spectrophotometer measuring 330nm place, find that chlorogenic acid has good linear relationship (y=absorbancy, x=chlorogenic acid mass concentration within the scope of 1.2 ~ 12.6 μ g/ml, regression equation is y=0.021x+0.529, r
2=0.9690), measurement result display chlorogenic acid sample recovery rate is 90%.
Embodiment 3
1. get Herba Medicaginis leaf 100kg, removal of impurities clean dry, pulverize and grind to form 10-20 object powder, add the distilled water decupling powder, suspension liquid is put into high pressure homogenizer and pulls an oar, pulverize raw material further, increase surface-area;
2. above-mentioned mixed solution is centrifugal in supercentrifuge, and centrifugal rotational speed is 1000r/h for the first time, abandons precipitation and gets supernatant liquor recentrifuge, and second time centrifugal rotational speed is 1500r/h;
3. by said extracted liquid after macroporous resin NKA-2 enrichment, 5 times of column volume 20% ethanol repeatedly wash-out, collect the powder that elutriant is dry under vacuo, use acetone solution again, collect filtrate and reclaim acetone spraying dry, adopt the absorbancy at thin-layer chromatography-UV spectrophotometer measuring 330nm place, find that chlorogenic acid has good linear relationship (y=absorbancy, x=chlorogenic acid mass concentration within the scope of 1.2 ~ 12.6 μ g/ml, regression equation is y=0.021x+0.533, r
2=0.9840), measurement result display chlorogenic acid sample recovery rate is 93.4%.
Embodiment 4
1. get Herba Medicaginis leaf 100kg, removal of impurities clean dry, pulverize and grind to form 10-20 object powder, add 10 times to the distilled water of powder, suspension liquid is put into high pressure homogenizer and pulls an oar, pulverize raw material further, increase surface-area;
2. above-mentioned mixed solution is centrifugal in supercentrifuge, and centrifugal rotational speed is 1500r/h for the first time, abandons precipitation and gets supernatant liquor recentrifuge, and second time centrifugal rotational speed is 2000r/h;
3. by said extracted liquid again through the polymer substance such as ultrafiltration membrance filter leaf protein, polysaccharide of less than 0.4 micron;
4. said extracted liquid is used after macroporous resin NKA-2 enrichment, 5 times of column volume 20% ethanol repeatedly wash-out, collect the powder that elutriant is dry under vacuo, use acetone solution again, collect filtrate and reclaim acetone spraying dry, detect the content of chlorogenic acid in 330nm place with thin-layer chromatography-TU-1800SPC type uv-spectrophotometric, thin-layer chromatography developing agent proportioning is, ethyl acetate: acetic acid: water (10:2:3, V/V).First point sample on silica-gel plate, employing developing agent launches, scraping spot under ultraviolet lamp, through dissolve with ethanol, then by the absorbancy at UV spectrophotometer measuring 330nm place, find that chlorogenic acid has good linear relationship (y=absorbancy, x=chlorogenic acid mass concentration within the scope of 1.2 ~ 12.6 μ g/ml, regression equation is y=0.019x+0.543, r
2=0.9996), measurement result display chlorogenic acid sample recovery rate is 95.7%.
Embodiment 5
1. get Herba Medicaginis leaf 100kg, removal of impurities clean dry, pulverize and grind to form 10-20 object powder, add 15 times to the distilled water of powder, suspension liquid is put into high pressure homogenizer and pulls an oar, pulverize raw material further, increase surface-area;
2. above-mentioned mixed solution is centrifugal in supercentrifuge, and centrifugal rotational speed is 1500r/h for the first time, abandons precipitation and gets supernatant liquor recentrifuge, and second time centrifugal rotational speed is 2000r/h;
3. by said extracted liquid again through the polymer substance such as ultrafiltration membrance filter leaf protein, polysaccharide of less than 0.4 micron;
4. by said extracted liquid after macroporous resin NKA-2 enrichment, 5 times of column volume 20% ethanol repeatedly wash-out, collect the powder that elutriant is dry under vacuo, use acetone solution again, collect filtrate and reclaim acetone spraying dry, adopt the absorbancy at thin-layer chromatography-UV spectrophotometer measuring 330nm place, find that chlorogenic acid has good linear relationship (y=absorbancy, x=chlorogenic acid mass concentration within the scope of 1.2 ~ 12.6 μ g/ml, regression equation is y=0.021x+0.537, r
2=0.9993), measurement result display chlorogenic acid sample recovery rate is 94.1%.
Claims (7)
1. from clover, extract a method for high-purity chlorogenic acid, it is characterized in that comprising the following steps:
(1) by clover cleaning, dry, pulverizing grinding;
(2) add water high-pressure homogenization, and centrifuging and taking supernatant liquor obtains high density crude extract, and the ultrafiltration of high density crude extract obtains filtrate;
(3) macroporous resin column absorption, ethanol elution, after collecting elutriant, repeats described absorption and wash-out;
(4) elutriant vacuum-drying at low temperatures; Add acetone solution again to filter, get filtrate and reclaim acetone, vacuum-drying;
Described macroporous resin column adopts NKA-2 macroporous resin;
Described wash-out adopts the ethanol of 20% of 5 times of resin column volumes to carry out wash-out, and flow velocity is 0.5/h.
2. method according to claim 1, when it is characterized in that described high-pressure homogenization, temperature maintains 15 ~ 20 DEG C, and amount of water is 10 ~ 15 times of raw material.
3. method according to claim 1, it is characterized in that described centrifugally to carry out at twice, rotating speed is respectively 1500r/h and 2000r/h.
4. method according to claim 1, is characterized in that the film hole diameter of the ultra-filtration membrane that described ultrafiltration uses is below 0.4 micron.
5. method according to claim 1, is characterized in that the described clover diameter through grinding is at below 1mm.
6. the method according to claim 1-5 any one, is characterized in that the product that (1), (2), (3), (4) described method obtains through step, adopts Thin-layer chromatography-ultraviolet spectrophotometry to record chlorogenic acid purity more than 95%.
7. method according to claim 1, is characterized in that described method chooses the clover that leaf weight reaches more than 50% of complete stool.
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| CN102648932A (en) * | 2011-02-25 | 2012-08-29 | 苏州宝泽堂医药科技有限公司 | Method for extracting alfalfa saponin |
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| Publication number | Priority date | Publication date | Assignee | Title |
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| CN102648932A (en) * | 2011-02-25 | 2012-08-29 | 苏州宝泽堂医药科技有限公司 | Method for extracting alfalfa saponin |
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| Title |
|---|
| 反相高效液相色谱法测定苜蓿中绿原酸的含量;古丽巴哈尔•达吾提 等;《中国民族医药杂志》;20130831(第8期);第61-62页 * |
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