CN102827220B - Method for separating rutin, hyperoside, isoquercitrin and quercetin from lotus leaves - Google Patents
Method for separating rutin, hyperoside, isoquercitrin and quercetin from lotus leaves Download PDFInfo
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- CN102827220B CN102827220B CN201210303085.1A CN201210303085A CN102827220B CN 102827220 B CN102827220 B CN 102827220B CN 201210303085 A CN201210303085 A CN 201210303085A CN 102827220 B CN102827220 B CN 102827220B
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Abstract
The invention relates to the technical field of traditional Chinese medicine, especially to a method for separating rutin, hyperoside, isoquercitrin and quercetin from lotus leaves. The method for separating the rutin, the hyperoside, the isoquercitrin and the quercetin from the lotus leaf comprises separating components in the lotus leaves powder by using a column chromatography method for a gradient elution for 65 to 75 minutes with a flow rate of 0.6 to 0.8 ml/min under a temperature of 28 to 32 DEG C and a pH value of 3.5 to 4.5, wherein a stationary phase of the column chromatography is C18, a mobile phase A is a phosphatic buffer with a pH value of 3.5 to 4.5, a mobile phase B is acetonitrile, and the outflow components are successively the rutin, the hyperoside, the isoquercitrin and the quercetin according to a time sequence; respectively recovering eluents containing the rutin, the hyperoside, the isoquercitrin and the quercetin; and finally removing solvents with reduced pressure distillation to obtain the rutin, the hyperoside, the isoquercitrin and the quercetin. The method not only can separate the rutin, the hyperoside, the isoquercitrin and the quercetin, but also is high is separation efficiency.
Description
Technical field
The present invention relates to technical field of traditional Chinese medicines, particularly relate to a kind of method being separated rutin, Quercetin 3-galactoside, isoquercitrin and Quercetin from lotus leaf.
Background technology
Lotus leaf is the dry leave of water lily plant lotus (Nelumbo nucifera Gaertn), have another name called lotus leaf, lotus root leaf, in China, most area has plantation more, is widely used in food and medicine, the kind that the second batch specified for the Ministry of Health " is food and medicine ".Traditional medicine is thought, lotus leaf bitter, flat, returns liver, spleen, stomach warp, there is effect of clearing away summer-heat and eliminating dampness, sending up the lucid YANG, cooling blood for hemostasis, record " lotus leaf takes it, makes us thin bad ", " hair tonic vigour, benefit helps taste; puckery essence is turbid, loose hemostasis, and detumescence pain, sends out variola " according to Compendium of Material Medica.Modern medicine study proves, containing a large amount of flavonoid compound in lotus leaf, has reducing blood-fat, treatment atherosclerosis, antimitotic, the effect such as antibacterial.
At present, in lotus leaf, the mensuration of flavones content has been reported, and the ultraviolet spectrometry range method that adopts measures the flavonoid content such as total flavones or high effective liquid chromatography for measuring Quercetin, rutin more.
The isoquercitrin of the certain content of tool in lotus leaf, but due to isoquercitrin and the flavonoid compound such as rutin, Quercetin structural similitude, ordinary method is difficult to effectively be separated.
CN100367976(2008-2-13) provide a kind of Herba Hyperici perforati extract and preparation method, wherein relate to the peak area of isoquercitrin and the fingerprint atlas detection method of Herba Hyperici perforati extract, however the method for from lotus leaf be separated and inapplicable.
" pharmaceutical analysis impurity " 2010,30 Yuan Li spring, Liu Bin, Shi Renbing discloses one " HPLC method measures the content of Quercetin 3-galactoside and isoquercitrin in different commercially available lotus leaf medicinal material ", but the method is routine analysis method of purification, separation efficiency is not high, and is difficult to Quercetin, rutin, Quercetin 3-galactoside and isoquercitrin be separated respectively.
Summary of the invention
The object of this invention is to provide the method that high a kind of of a kind of separation efficiency is separated rutin, Quercetin 3-galactoside, isoquercitrin and Quercetin from lotus leaf.
Above-mentioned technical purpose of the present invention is achieved by the following technical programs: a kind of method being separated rutin, Quercetin 3-galactoside, isoquercitrin and Quercetin from lotus leaf, comprise the component adopted in column chromatography method fractionation Lotus Leaf, and the stationary phase of column chromatography is C18, mobile phase A is the phosphate buffered saline buffer of pH value 3.5-4.5, Mobile phase B is acetonitrile, column temperature 28-32 DEG C, press the flow rate gradient 65-75min of 0.6-0.8ml/min under the condition of pH value 3.5-4.5; Described condition of gradient elution is: at 0-45min, and the volume ratio of described mobile phase A and Mobile phase B is (90-95)/(5-10); At 46-55min, the volume ratio of described mobile phase A and Mobile phase B is (80-85)/(15-20); At 56-65min, the volume ratio of described mobile phase A and Mobile phase B is (68-72)/(28-32); At 66-75min, the volume ratio of described mobile phase A and Mobile phase B is (90-95)/(5-10); Reclaim the elutriant containing rutin, Quercetin 3-galactoside, isoquercitrin, Quercetin respectively, after last underpressure distillation desolvation, obtain rutin, Quercetin 3-galactoside, isoquercitrin and Quercetin.
Rutin, Quercetin 3-galactoside, isoquercitrin, Quercetin differ a glucosyl respectively, fixed proportion wash-out is difficult to be separated, particularly rutin and isoquercitrin, retention time closely, easily cause mixing, by above-mentioned gradient separations method of the present invention, finally achieve effective separation, and the separation efficiency of rutin, Quercetin 3-galactoside, isoquercitrin, Quercetin reaches more than 98.5% respectively, and separation efficiency is higher.
As preferably, described phosphate buffered saline buffer is that the phosphoric acid solution of 0.15-0.25% and the sodium hydroxide solution of 8-12% are mixed to pH value 3.5-4.5 and are prepared from.
More preferably, the compound method of described phosphate buffered saline buffer is: the phosphoric acid solution of 0.2% is titrated to the sodium hydroxide solution of 10%, adds air in titration simultaneously by stirring, is mixed to pH value 4.0 and is prepared from.
As preferably, the pH value of phosphate buffered saline buffer is 4.0.
As preferably, at 0-45min, the volume ratio of described mobile phase A and Mobile phase B is 92/8.
As preferably, at 46-55min, the volume ratio of described mobile phase A and Mobile phase B is 82/18.
As preferably, at 56-65min, the volume ratio of described mobile phase A and Mobile phase B is 70/30.
As preferably, at 66-75min, the volume ratio of described mobile phase A and Mobile phase B is 92/8.
As preferably, described Lotus Leaf is first the dissolve with methanol solution of 75-85% and ultrasonic 28-35min with massfraction, and then filters after the dissolve with methanol solution adding 75-85%, gets the to be separated liquid of filtrate as described column chromatography.
More preferably, described Lotus Leaf first with massfraction be 80% dissolve with methanol solution and ultrasonic 30min, and then to filter after the dissolve with methanol solution adding 80%, get the to be separated liquid of filtrate as described column chromatography.
Accompanying drawing explanation
Fig. 1 is the color atlas of the embodiment of the present invention one;
Fig. 2 is the color atlas of control substance of Rutin;
Fig. 3 is the color atlas of Quercetin 3-galactoside reference substance;
Fig. 4 is the color atlas of isoquercitrin reference substance;
Fig. 5 is the color atlas of Quercetin reference substance;
In figure, I-rutin; II-Quercetin 3-galactoside; III-isoquercitrin; IV-Quercetin.
Embodiment
Embodiment one
1 instrument and reagent
Agilent 1100 type high performance liquid chromatograph; Isoquercitrin reference substance (lot number is 10391, and purity is 93.0%, German HWI ANALYTIK company); Quercetin reference substance (lot number is 100081-200406, Nat'l Pharmaceutical & Biological Products Control Institute); Control substance of Rutin (lot number is 100080-200707, Nat'l Pharmaceutical & Biological Products Control Institute); Acetonitrile (chromatographically pure, German Merck company); Water is ultrapure water; The former powder of lotus leaf (self-control, South Pacific lakeside is plucked, dry, pulverize, cross No. 5 sieves); All the other reagent are analytical pure.
2 methods and result
2.1 chromatographic condition
Chromatographic column: Agilent C18 post (250mm × 4.6mm, 5 μm) column temperature 30 DEG C.Mobile phase A: pH4.0 phosphate buffered saline buffer (0.2% phosphoric acid solution, by 10% sodium hydroxide solution adjust ph to 4.0); Mobile phase B: acetonitrile; Gradient elution refers to table 1.UV-detector detects, determined wavelength 356nm.Sample size: 5 μ l.Theoretical plate number is not less than 2500 in isoquercitrin.
The compound method of phosphate buffered saline buffer is: the phosphoric acid solution of 0.2% is titrated to the sodium hydroxide solution of 10%, adds air in titration simultaneously by stirring, is mixed to pH value 4.0 and is prepared from.
Column temperature 28-32 DEG C, press the flow rate gradient 65-75min of 0.6-0.8ml/min under the condition of pH value 3.5-4.5; The component flowed out is followed successively by rutin, Quercetin 3-galactoside, isoquercitrin, Quercetin according to time order and function, reclaim the elutriant containing rutin, Quercetin 3-galactoside, isoquercitrin, Quercetin respectively, after last underpressure distillation desolvation, obtain rutin, Quercetin 3-galactoside, isoquercitrin and Quercetin.
Rutin yield is 96.8%, and purity reaches 90%; Radix Hyperici Monogyni (Herba Hyperici Monogyni)
glycosidesyield is 97.6%, and purity reaches 88%; Isoquercitrin yield is 99.1%, and purity reaches 92%; Quercetin yield is 98.1%, and purity reaches 91%.
Table 1 embodiment one gradient elution table
2.2 solution preparations
Get the former powder 0.25g of lotus leaf, add 80% methyl alcohol 30ml, ultrasonic 30min, add 80% methyl alcohol to 50ml, shake up, filter, get filtrate as need testing solution (liquid to be separated).
Get isoquercitrin reference substance 0.00513g, add 80% methanol solution and be diluted to 50ml, mixing, makes recovery test liquid; Fetch yield experimental liquid 5.00ml and put 25ml measuring bottle, add 80% methyl alcohol to scale, mixing, product solution in contrast.
Get control substance of Rutin 0.00253g, add 80% methanol solution and be diluted to 100ml, mixing; Get Quercetin reference substance 0.00283g, add 80% methanol solution and be diluted to 100ml, mixing; As specificity experimental control liquid.
The same rutin of specificity experimental control liquid making method of Quercetin 3-galactoside, Quercetin.
2.3 methodological study
Specificity is tested: get reference substance solution, need testing solution, each 5 μ l of specificity experimental control liquid respectively, measure by above-mentioned chromatographic condition.The former powder collection of illustrative plates of lotus leaf refers to Fig. 1, and specificity test collection of illustrative plates refers to Fig. 2, Fig. 3, Fig. 4 and Fig. 5.
In result display need testing solution chromatogram, there is chromatographic peak in the place identical with reference substance solution chromatographic retention, and specificity experimental control liquid is effectively separated with reference substance solution chromatogram under this chromatographic condition.
Linear relationship is investigated: by above-mentioned chromatographic condition, reference substance solution sample size 2 μ l, 4 μ l, 8 μ l, 14 μ l, 20 μ l, measure peak area, typical curve is made with sample size (X) and peak area (Y), obtain regression equation Y=48.82X-1.119, r=0.9990(n=5), result shows, isoquercitrin sample size is good with peak area linear relationship in 0.0382ng-0.382ng scope.
Precision test: accurate absorption isoquercitrin reference substance solution 10 μ l, repeats sample introduction 6 times.The RSD=1.6%(n=6 of results peaks area).
Replica test: take lotus leaf 6 parts, former powder (about 0.25g), prepare need testing solution in accordance with the law, measure by above-mentioned chromatographic condition.The RSD=0.56% (n=6) of result isoquercitrin content.
Stability test: get same need testing solution, respectively 0.5,2,4,8,12h measures.Peak area RSD=1.7%(n=6), show that need testing solution is good at 12 hours internal stabilities.
Application of sample recovery test: the former powder of lotus leaf (content 3.727mg/g) getting known content, precision adds isoquercitrin recovery test liquid in right amount respectively, prepares and measure content by need testing solution preparation method, calculates the rate of recovery, the results are shown in Table 2.
Table 2 isoquercitrin application of sample recovery test result (n=6)
2.4 sample determination
Separately get 3 batches of former powder samples of lotus leaf, by need testing solution preparation method operation, by 2.1 lower chromatographic condition sample introductions, calculate content with external standard method.Result is: 3.567mg/g, 4.087mg/g, 2.908mg/g.
Embodiment two
With embodiment one, unlike in different time, the volume ratio of mobile phase A and Mobile phase B and condition of gradient elution are in table 3.Wherein phosphate buffered saline buffer is that the phosphoric acid solution of 0.15% and the sodium hydroxide solution of 8% are mixed to pH value 3.5 and are prepared from.Lotus Leaf first with massfraction be 75% dissolve with methanol solution and ultrasonic 28min, and then to filter after the dissolve with methanol solution adding 85%, get the to be separated liquid of filtrate as described column chromatography.
Recording rutin yield is 96.5%, and purity reaches 90.5%; Quercetin 3-galactoside yield is 97.3%, and purity reaches 89%; Isoquercitrin yield is 99%, and purity reaches 92.3%; Quercetin yield is 98.4%, and purity reaches 90.4%.
Table 3 embodiment two gradient elution table
Embodiment three
With embodiment one, unlike in different time, the volume ratio of mobile phase A and Mobile phase B and condition of gradient elution are in table 4.
Recording rutin yield is 96.2%, and purity reaches 89.9%; Quercetin 3-galactoside yield is 97.1%, and purity reaches 89.4%; Isoquercitrin yield is 98.9%, and purity reaches 91.8%; Quercetin yield is 97.8%, and purity reaches 90.1%.
Table 4 embodiment three gradient elution table
Comparative example one
With embodiment one.Unlike chromatographic condition: chromatographic column: C
18chromatographic column; Moving phase: acetonitrile-0.1% formic acid water (13:87); Flow velocity 1.0ml/min; Determined wavelength: 255nm; Column temperature: 34 DEG C.
The method only can be separated Quercetin 3-galactoside and isoquercitrin.Recording Quercetin 3-galactoside yield is 96.1%, purity 80%; Isoquercitrin yield is 98.8%, purity 75%.
Comparative example two
With embodiment two, under the condition of column temperature 20 DEG C, pH value 6, press 1.2ml/min gradient elution unlike pressing, elution requirement is in table 5.
Table 5 comparative example two gradient elution table
Recording rutin yield is 86.2%, and purity reaches 80.9%; Quercetin 3-galactoside yield is 92.5%, and purity reaches 85.4%; Isoquercitrin yield is 94.9%, and purity reaches 81.8%; Quercetin yield is 82.8%, and purity reaches 80%.
This specific embodiment is only explanation of the invention; it is not limitation of the present invention; those skilled in the art can make to the present embodiment the amendment not having creative contribution as required after reading this specification sheets, as long as but be all subject to the protection of patent law in right of the present invention.
Claims (2)
1. from lotus leaf, be separated the method for rutin, Quercetin 3-galactoside, isoquercitrin and Quercetin, comprise the component adopted in column chromatography method fractionation Lotus Leaf, it is characterized in that: described Lotus Leaf is first the dissolve with methanol solution of 75-85% and ultrasonic 28-35min with massfraction, and then filter after the dissolve with methanol solution adding 75-85wt%, get the to be separated liquid of filtrate as described column chromatography;
The stationary phase of described column chromatography is C18, mobile phase A is phosphate buffered saline buffer, Mobile phase B is acetonitrile, the compound method of described phosphate buffered saline buffer is: the phosphoric acid solution of 0.2% is titrated to the sodium hydroxide solution of 10%, add air by stirring in titration simultaneously, be mixed to pH value 4.0 and be prepared from; The pH value of described phosphate buffered saline buffer is 4.0; Column temperature 28-32 DEG C, press the flow rate gradient 65-75min of 0.6-0.8ml/min under the condition of pH value 3.5-4.5;
Described condition of gradient elution is:
At 0-45min, the volume ratio of described mobile phase A and Mobile phase B is 92/8;
At 46-55min, the volume ratio of described mobile phase A and Mobile phase B is 82/18;
At 56-65min, the volume ratio of described mobile phase A and Mobile phase B is 70/30;
At 66-75min, the volume ratio of described mobile phase A and Mobile phase B is 92/8;
Reclaim the elutriant containing rutin, Quercetin 3-galactoside, isoquercitrin, Quercetin respectively, after last underpressure distillation desolvation, obtain rutin, Quercetin 3-galactoside, isoquercitrin and Quercetin.
2. the method being separated rutin, Quercetin 3-galactoside, isoquercitrin and Quercetin from lotus leaf according to claim 1, it is characterized in that: described Lotus Leaf first with massfraction be 80% dissolve with methanol solution and ultrasonic 30min, and then filter after the dissolve with methanol solution adding 80wt%, get the to be separated liquid of filtrate as described column chromatography.
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| CN107522683A (en) * | 2017-07-17 | 2017-12-29 | 长沙爱扬医药科技有限公司 | A kind of method that OPC and Hyperoside are extracted from lotus pod |
| CN110658295B (en) * | 2019-10-23 | 2022-02-25 | 葵花药业集团（襄阳）隆中有限公司 | Method for measuring fingerprint spectrum of lotus leaf decoction pieces in Erdong decoction formula |
| CN113717139A (en) * | 2021-09-15 | 2021-11-30 | 耒阳市刘燕酿制生物科技有限公司 | Method for extracting quercetin from pomegranate peel |
| CN114235974B (en) * | 2021-11-05 | 2024-05-31 | 新疆维吾尔药业有限责任公司 | Method for detecting rutin, isoquercitrin and rosmarinic acid content in herba Hedyotidis Diffusae or extract thereof |
| CN114441687A (en) * | 2022-02-17 | 2022-05-06 | 山东福瑞达生物股份有限公司 | Fingerprint spectrum construction method and application of antioxidant lotus leaf extract |
| CN116577436A (en) * | 2023-05-31 | 2023-08-11 | 态创生物科技(广州)有限公司 | HPLC analysis method suitable for continuous measurement of hyperin and quercetin system |
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| CN101095724A (en) * | 2006-06-28 | 2008-01-02 | 扬州大学 | Technics for extracting lotus leaf flavone |
| CA2593623A1 (en) * | 2007-07-09 | 2009-01-09 | Mian Long | The applications of kidney secreted bone growth factor and pharmaceutical use of flavonol and flavonol glycosides for stimulating the secretion of kidney secreted bone growth factor |
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