CN105030853A - Method for separating and purifying kandelia candel leaf total flavonoids - Google Patents
Method for separating and purifying kandelia candel leaf total flavonoids Download PDFInfo
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- CN105030853A CN105030853A CN201510479849.6A CN201510479849A CN105030853A CN 105030853 A CN105030853 A CN 105030853A CN 201510479849 A CN201510479849 A CN 201510479849A CN 105030853 A CN105030853 A CN 105030853A
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- autumn
- total flavones
- solani melongenae
- folium solani
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Abstract
The invention discloses a method for separating and purifying kandelia candel leaf total flavonoids. The method comprises the steps that 1, kandelia candel leaves are taken and smashed into coarse powder, extraction is conducted on the coarse powder for two times through a 65%-75% ethanol solution which accounts for 15 times of the coarse powder, the extraction time is 1.0-2.0 h each time, and extracting solutions are mixed; 2, concentrating is conducted on the extracting solution obtained in the first step to make the concentration of the extracting solution range from 4 mg/ml to 5 mg/ml, centrifugation is conducted, supernatant liquid is taken and purified through nonpolar macroporous absorption resin, absorption is conducted for 30 min, water washing is conducted for removing impurities, and elution is conducted through 40%-70% ethyl alcohol; 3, eluant obtained in the second step is collected, decompression recycling of the solvent is conducted, drying and smashing are conducted, and a final product is obtained. The content of the kandelia candel leaf total flavonoids is at least 60%. The separation and purification method of the kandelia candel leaves is simple, convenient, efficient, environmentally friendly and strong in pertinence, and the kandelia candel leaf total flavonoids which are high in purity and whitening and freckle-removing activity can be obtained.
Description
Technical field
The invention belongs to technical field of bioseparation, specifically refer to a kind of isolation and purification method of autumn Folium Solani Melongenae total flavones.
Background technology
Autumn eggplant (Kandeliacandel) is Rhizophoraceae autumn nightshade, is seeds the most cold-resistant in China's mangrove, and in China's mangrove areal area from south orientation north, mangrove kind reduces gradually, only has the distribution in addition of autumn eggplant, reaches 28o25'N northernmost.Chemical composition in autumn eggplant mainly contains flavonoid, tannin class, sterols, fatty acid and polysaccharide etc.Many scholars are to the various chemical constitution studies in autumn eggplant in recent years, find that some possess the function of drug development value, such as scholar Li Baocai reports in " Rhizophora apiculata Blume autumn Folium Solani Melongenae chemistry of lipids and active matter Quality Research " literary composition: autumn Folium Solani Melongenae total flavones, content is 29.3%, Wheat Protein, obviously can reduce high lipid peroxide mice serum MDA value caused by alloxan.In addition, find after deliberation, autumn Folium Solani Melongenae total flavones has the generation of good check melanin, restraint of tyrosinase activity, antioxidation and uvioresistant effect, and the power of effect has certain associating with the purity of autumn Folium Solani Melongenae total flavones.
The preparation method of existing autumn Folium Solani Melongenae total flavones, mainly contain: one, adopt autumn Folium Solani Melongenae powder 1kg 5L60% alcohol dipping 4 times, centrifuging and taking supernatant after concentrated removing ethanol, again by AB-8 macroporous ion exchange resin, with deionized water rinsing pillar to limpid, then use 75% alcohol flushing, collect ethanol, reclaim ethanol, drying, obtains total flavones 45.3g, and the content of total flavones is 17.8%; Or adopt 1kg autumn Folium Solani Melongenae powder 5L60% alcohol dipping 4 times, centrifuging and taking supernatant after concentrated removing ethanol, again by DA201 macroporous ion exchange resin, with deionized water rinsing pillar to limpid, then use 80% alcohol flushing, collect ethanol, reclaim ethanol, drying, obtains crude flavonoid powder 50.2g, and the content of total flavones is 18.9%.By above-mentioned crude flavonoid powder 45g, pulverize, with 500ml ethanol/chloroform (3:17) mixed solvent, extract 2 times, centrifuging and taking supernatant.Residue 500ml ethanol/chloroform (1:1) extracts 2 times, centrifugal, gets supernatant.Merge all centrifugal liquid, recycling design, dry, obtain Refined flavonoid powder 9.6g, flavones content is 29.3%.Above two kinds of purification process are exemplified, the major defect of above-mentioned two kinds of methods is: the total flavones purity obtained is low, after having crossed macroporous resin, purity is all less than 20%, namely after using a large amount of toxic organic solvents extractions, general flavone content, also not more than 30%, affects drug development effect, and adopts toxic organic solvents to extract the hidden danger of also easily environmental pollution.
Based on this research and a kind of isolation and purification method of autumn Folium Solani Melongenae total flavones of development and Design.
Summary of the invention
The object of the invention is to: the isolation and purification method that a kind of autumn Folium Solani Melongenae total flavones is provided.
The present invention is achieved through the following technical solutions: a kind of isolation and purification method of autumn Folium Solani Melongenae total flavones, comprises the following steps:
(1) get autumn Folium Solani Melongenae and be ground into coarse powder, extract 2 times with the alcoholic solution of 15 times 65 ~ 75%, each extraction time is 1.0 ~ 2.0h, merge extractive liquid;
(2) extracting solution of concentration step (1) gained makes its concentration be 4 ~ 5mg/ml, centrifugal, gets supernatant, purifies with nonpolar macroporous adsorption resin, absorption 30min, ion, then the ethanol elution using 40 ~ 70%;
(3) collect the eluent of step (2) gained, decompression and solvent recovery, dry, pulverize, and obtains this product, and the content of described this product Folium Solani Melongenae in mid-autumn total flavones is at least 60%.
Further, in order to better realize the present invention, also comprise following technical characteristic, nonpolar macroporous adsorption resin described in described step (1) is any one in HPD722 type, HPD100 type, HPD826 type adsorbent resin.
Further, in order to better realize the present invention, also comprise following technical characteristic, in described step (2), nonpolar macroporous adsorption resin is HPD722 type.
Further, in order to better realize the present invention, also comprise following technical characteristic, the extraction time 1.0h of described step (1), Extracting temperature 60 ~ 80 DEG C.
Further, in order to better realize the present invention, also comprise following technical characteristic, described step (2) loading flow velocity is 2 ~ 3BV/h, and applied sample amount is 4.5 ~ 6.5BV.
Further, in order to better realize the present invention, also comprise following technical characteristic, described step (2) adopts the ethanol elution of 70%.
Further, in order to better realize the present invention, also comprise following technical characteristic, elution flow rate during described step (2) ethanol elution is 3 ~ 6BV/h, and eluent consumption is 3 ~ 5BV.
Further, in order to better realize the present invention, also comprise following technical characteristic, elution flow rate during described step (2) ethanol elution is 5BV/h, and eluent consumption is 4BV.
The present invention compared with prior art, has the following advantages and beneficial effect:
The isolation and purification method of a kind of autumn Folium Solani Melongenae total flavones provided by the invention, method is easy, efficient, environmental protection, and with strong points, can obtain highly purified, the active strong autumn Folium Solani Melongenae total flavones of whitening and speckle dispelling.
Detailed description of the invention
Below in conjunction with embodiment, the present invention is described in further detail, but embodiments of the present invention are not limited thereto.
1, autumn Folium Solani Melongenae determination of total flavonoids adopts NaNO
2-Al (NO
3)
3-NaOH method, concrete operations are: with 60% ethanol dilution and standardize solution sample, add 0.6mL5%NaNO
20.6mL10%Al (NO is added immediately after solution shakes up
3)
3solution, shakes up rear placement 6min, measures absorbance after adding 6ml1mol/LNaOH solution at wavelength 500nm place.
2, static adsorption-desorption experimental technique is specially: get the different model wet resin that pretreatment is good respectively, every part of 1.0mL, put in 250mL ground triangular flask, add 2.63mg/mL extracting solution sample 80mL, to place in constant-temperature table 25 DEG C, leached by resin after 100r/min constant temperature oscillation 12h, filtrate standardize solution, in 100mL volumetric flask, measures total flavones concentration in filtrate.Put into conical flask by after the resin distilled water wash of adsorption equilibrium, add 95% ethanol 80mL, 25 DEG C, 100r/min vibration 12h carries out eluting, and then leached by resin, filtrate standardize solution is in 100mL volumetric flask, measure total flavones concentration in eluent, calculate total flavones adsorbance, desorption efficiency.
Adsorbance and desorption efficiency press formulae discovery below:
Q=(C1 × V1-C2V2) × V0R=C3 × V3Q × V1 wherein, Q-adsorbance (mg/mL); R-desorption efficiency (%); The concentration (mg/mL) of total flavones in the front solution of C1-absorption; The concentration (mg/mL) of total flavones in the rear sample liquid of C2-absorption; Total flavones concentration (mg/mL) in C3-eluent; V0-resin volume (mL); V1-sample liquid volume (mL); V2-adsorption liquid volume (mL) V3-effluent volume (mL).
Dynamic adsorption-desorption experimental technique is specially: get the macroporous resin dress post that pretreatment is good, resin column volume 20mL, fixing blade diameter length ratio 1:6, the extracting solution adding 2.88mg/mL is positive to chromogenic reaction in effluent, leave standstill 30min, with distillation washing post to colourless, collect distilled water and survey volume and absorbance.Carry out being eluted to chromogenic reaction feminine gender with 80% ethanol, collect eluent and survey volume and absorbance, calculate adsorbance, desorption efficiency.
Adsorbance (mg/mL)=(C
onv
on-C
residualv
residual-C
washingv
washing)/V
Desorption efficiency (%)=C
alcoholv
alcohol/ (C
onv
on-C
residualv
residual-C
washingv
washing) × 100%
Wherein, C
onfor flavone concentration in sample solution, V
onfor sample solution cumulative volume, C
residualfor crossing flavone mean concentration in post effluent, V
residualfor crossing post effluent cumulative volume, C
washingfor the flavone mean concentration that distilled water elutes, V
washingfor the distilled water volume of eluting, C
alcoholfor the flavone mean concentration that ethanol elution gets off, V
alcoholfor the total ethanol volume of eluting, V is the volume of resin,
Embodiment 1:
The present embodiment provides a kind of autumn Folium Solani Melongenae total flavones to obtain isolation and purification method, specifically comprises the following steps:
(1) extract: get autumn Folium Solani Melongenae and be ground into coarse powder, get 1000g, with the ethanol of volume fraction 65% for extractant, solid-liquid ratio 1:15(quality and volume ratio), Extracting temperature 80 DEG C, extraction time 1.0h, extracts 2 times;
(2) separation and purification: purification is carried out to the autumn Folium Solani Melongenae total flavones that step (1) obtains with HPD722 macroporous adsorbent resin, concrete purifying process is: loading total flavones concentration is 5.0mg/ml, loading flow velocity is 3BV/h, applied sample amount is 6.5BV resin column volume, adsorption time 30min, remove impurity is rinsed with 3.5 times of water, eluant is the ethanol of volume fraction 50%, elution flow rate is 3BV/h, eluting agent is 4BV resin column volume, collect ethanol, decompression and solvent recovery, dry, pulverize, obtain yellowish-brown autumn Folium Solani Melongenae total flavones powder 94.51g, general flavone content is 77.58%.
Embodiment 2:
The present embodiment provides a kind of autumn Folium Solani Melongenae total flavones to obtain isolation and purification method, specifically comprises the following steps:
(1) extract: get autumn Folium Solani Melongenae and be ground into coarse powder, get 1000g, with the ethanol of volume fraction 70% for extractant, solid-liquid ratio 1:15, Extracting temperature 60 DEG C, extraction time 1.5h, extracts 2 times;
(2) separation and purification: purification is carried out to the autumn Folium Solani Melongenae total flavones that step (1) obtains with HPD722 macroporous adsorbent resin, concrete purifying process is: loading total flavones concentration is 5.0mg/mL, loading flow velocity is 2.5BV/h, applied sample amount is 6BV resin column volume, adsorption time 30min, remove impurity is rinsed with 3.5 times of water, eluant is the ethanol of volume fraction 35%, elution flow rate is 4BV/h, eluting agent is 4BV resin column volume, collect ethanol, decompression and solvent recovery, dry, pulverize, obtain yellowish-brown autumn Folium Solani Melongenae total flavones powder 93.23g, general flavone content is 75.11%.
Embodiment 3:
The present embodiment provides a kind of autumn Folium Solani Melongenae total flavones to obtain isolation and purification method, specifically comprises the following steps:
(1) extract: get autumn Folium Solani Melongenae and be ground into coarse powder, get 1000g, with the ethanol of volume fraction 70% for extractant, solid-liquid ratio 1:15, solid-liquid ratio is quality and volume ratio, Extracting temperature 80 DEG C, extraction time 2.0h, extracts 2 times;
(2) separation and purification: purification is carried out to the autumn Folium Solani Melongenae total flavones that step (1) obtains with HPD100 macroporous adsorbent resin, concrete purifying process is: loading total flavones concentration is 4.5mg/mL, loading flow velocity is 3BV/h, applied sample amount is 5.5BV resin column volume, adsorption time 30min, remove impurity is rinsed with 3.5 times of water, eluant is the ethanol of volume fraction 50%, elution flow rate is 3BV/h, eluting agent is 4BV resin column volume, collect ethanol, decompression and solvent recovery, dry, pulverize, obtain yellowish-brown autumn Folium Solani Melongenae total flavones powder 85.51g, general flavone content is 65.21%.
Embodiment 4:
The present embodiment provides a kind of autumn Folium Solani Melongenae total flavones to obtain isolation and purification method, specifically comprises the following steps:
(1) extract: get autumn Folium Solani Melongenae and be ground into coarse powder, get 1000g, with the ethanol of volume fraction 75% for extractant, solid-liquid ratio 1:15(quality and volume ratio), Extracting temperature 60 DEG C, extraction time 1.2h, extracts 2 times;
(2) separation and purification: purification is carried out to the autumn Folium Solani Melongenae total flavones that step (1) obtains with HPD826 macroporous adsorbent resin, concrete purifying process is: loading total flavones concentration is 5.0mg/mL, loading flow velocity is 2BV/h, applied sample amount is 4.5BV resin column volume, adsorption time 30min, remove impurity is rinsed with 3.5 times of water, eluant is the ethanol of volume fraction 50%, elution flow rate is 5BV/h, eluting agent is 4BV resin column volume, collect ethanol, decompression and solvent recovery, dry, pulverize, obtain yellowish-brown autumn Folium Solani Melongenae total flavones powder 84.25g, general flavone content is more than 64.51%.
Described, be only preferred embodiment of the present invention, not do any pro forma restriction to the present invention, every any simple modification, equivalent variations done above embodiment according to technical spirit of the present invention, all falls within protection scope of the present invention.
Claims (8)
1. an isolation and purification method for autumn Folium Solani Melongenae total flavones, comprises the steps:
(1) get autumn Folium Solani Melongenae and be ground into coarse powder, extract 2 times with the alcoholic solution of 15 times 65 ~ 75%, each extraction time is 1.0 ~ 2.0h, merge extractive liquid;
(2) extracting solution of concentration step (1) gained makes its concentration be 4-5mg/ml, centrifugal, gets supernatant, purifies with nonpolar macroporous adsorption resin, absorption 30min, ion, then uses the ethanol elution of 40 ~ 70% to obtain eluent;
(3) collect the eluent of step (2) gained, decompression and solvent recovery, dry, pulverize, and obtains this product, and the content of described this product Folium Solani Melongenae in mid-autumn total flavones is at least 60%.
2. the isolation and purification method of a kind of autumn Folium Solani Melongenae total flavones according to claim 1, is characterized in that: nonpolar macroporous adsorption resin described in described step (1) is any one in HPD722 type, HPD100 type, HPD826 type adsorbent resin.
3. the isolation and purification method of a kind of autumn Folium Solani Melongenae total flavones according to claim 2, is characterized in that:
In described step (2), nonpolar macroporous adsorption resin is HPD722 type.
4. the isolation and purification method of a kind of autumn Folium Solani Melongenae total flavones according to claim 3, is characterized in that: the extraction time 1.0h of described step (1), Extracting temperature 60 ~ 80 DEG C.
5. the isolation and purification method of a kind of autumn Folium Solani Melongenae total flavones according to claim 4, it is characterized in that: described step (2) loading flow velocity is 2 ~ 3BV/h, applied sample amount is 4.5 ~ 6.5BV.
6. the isolation and purification method of a kind of autumn Folium Solani Melongenae total flavones according to claim 5, is characterized in that: described step (2) adopts the ethanol elution of 70%.
7. the isolation and purification method of a kind of autumn Folium Solani Melongenae total flavones according to claim 6, it is characterized in that: elution flow rate during described step (2) ethanol elution is 3 ~ 6BV/h, eluent consumption is 3 ~ 5BV.
8. the isolation and purification method of a kind of autumn Folium Solani Melongenae total flavones according to claim 7, it is characterized in that: elution flow rate during described step (2) ethanol elution is 5BV/h, eluent consumption is 4BV.
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Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| JP2007084515A (en) * | 2005-09-26 | 2007-04-05 | Tohoku Univ | Medicine for ameliorating anorexia of patient in low nutritional state |
| CN104473981A (en) * | 2014-12-04 | 2015-04-01 | 浙江树人大学 | Method for separating and purifying high-purity general flavone in kandelia candel leaves |
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2015
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Patent Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| JP2007084515A (en) * | 2005-09-26 | 2007-04-05 | Tohoku Univ | Medicine for ameliorating anorexia of patient in low nutritional state |
| CN104473981A (en) * | 2014-12-04 | 2015-04-01 | 浙江树人大学 | Method for separating and purifying high-purity general flavone in kandelia candel leaves |
Non-Patent Citations (1)
| Title |
|---|
| 纪丽丽等: "红树植物秋茄叶黄酮的提取及抑菌活性实验", 《天然产物研究与开发》 * |
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