CN102100721A - Method for producing total flavonoids of chrysanthemum - Google Patents
Method for producing total flavonoids of chrysanthemum Download PDFInfo
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Abstract
The invention relates to a method for producing total flavonoids of chrysanthemum. The method comprises the following steps of: extracting crude extracts from the chrysanthemum, and purifying the crude extracts to obtain the total flavonoids of the chrysanthemum. The method is characterized in that: the macroporous resin which is AB-8 type macroporous resin is used for purification. The content of the total flavonoids of the chrysanthemum, prepared by the method, is over 50 percent, nearly reaches 70 percent; the total flavonoids are high in purity and yield; moreover, the method is environmentally-friendly and applicable to industrial mass production.
Description
Technical field
The present invention discloses a kind of production method of BOJU total flavones.Belong to pharmaceutical field.
Background technology
Flos Chrysanthemi is the dry capitulum of feverfew Flos Chrysanthemi Dendranthema morifolium (Ramat.), is China's conventional Chinese medicine, sweet in the mouth, hardship.Return lung, Liver Channel.Included CHUJU on 2010 editions pharmacopeia, BOJU, Flos Chrysanthemi and Flos Chrysanthemi, wherein BOJU and CHUJU are Anhui Province's genuine medicinal materials.Flos Chrysanthemi is the cultivation history of China existing more than 1500 year at least, it is edible be used as medicine just on the books as far back as Shennong's Herbal: " obey sharp vim and vigour for a long time, make light of one's life by commiting suicide, anti-old, prolong life." essentials of Matea Medica cloud then: " and the Flos Chrysanthemi flavor sweetness and bitterness of holding concurrently, the property official report is gentle, enjoys four gas, experiences all kinds of frost and dew, the essence in Jinsui River, lung benefiting kidney two is dirty "." herd administration chat " said: " the food chrysanthemum prolongs age, and purple person goes into blood system, but but the medicine bait can make and can rest the head on, celestial through weight ".Say that all Flos Chrysanthemi has the effect of anti-senility, Glycerin
Flos Chrysanthemi uses very extensive clinically, is used for multiple treatment of diseases.Contained complex chemical composition has multiple biological activity, and volatile oil, flavone, polysaccharide, chlorogenic acid etc. are arranged.Wherein flavone compound extensively is present in the plant; have no adverse reaction; molecular weight is little; can be absorbed rapidly by human body; can pass through blood brain barrier; can enter fatty tissue; and then embody following function: allaying tiredness; the protection blood vessel; preventing arteriosclerosis; the expansion blood capillary; the mediation microcirculation; the oxidation of lipotropism fat; defying age; the function of activation brain and other cells of organs; both can be developed as the Natural antioxidant; as old functional food; the additive of food; simultaneously to coronary heart disease; angina pectoris; hypertension; cardiovascular disease all has better curative effect, has the wide development prospect, and research is active in natural medicine field.
The Flos Chrysanthemi preparation is very few in the market, because production technology limits, and impure more, taking dose is big, quality controllability is poor, directly influences product quality and clinical efficacy mostly.Along with a series of researchs that chemical constituent, extracting method and the pharmacological action etc. of Flos Chrysanthemi are carried out, the exploitation value and the economic worth of Flos Chrysanthemi will embody day by day.
For this reason, the present invention's theoretical method and technology of modern science, on the basis of existing research, choose that general flavone content is an index in the BOJU, the essential condition that influences the Flos Chrysanthemi flavones content is optimized, select optimum extraction process, and pass through repetition test, selected the method for the most suitable a kind of purification and purified BOJU total flavones, it has been carried out enriching and purifying, the BOJU general flavone content is reached more than 50%, thereby be suitable as effective ingredient in Chinese, be suitable for carrying out the crude drug of modern medicines preparation.
Summary of the invention
For achieving the above object, the invention provides a kind of production method of BOJU total flavones.Specifically be achieved through the following technical solutions:
A kind of production method of BOJU total flavones comprises the steps:
(1) the bo-chrysanthemum crude drug obtains crude extract through extraction;
(2) the purified process of the resulting crude extract of step (1) makes the BOJU total flavones;
It is characterized in that the purification described in the step (2) is to adopt purification by macroporous resin, and described macroporous resin is an AB-8 type macroporous resin.
The production method of above-mentioned described BOJU total flavones is characterized in that in the described step (2), and macroporous resin adopts wet method dress post, and adsorption capacity is 8.50mg/g, 10.41mg/g, 12.30mg/g, 7.95mg/g, 11.67mg/g; Wherein be preferably 12.30mg/g (being equivalent to 0.25g crude drug/g resin).
The production method of above-mentioned described BOJU total flavones is characterized in that in the described step (2), sample solution concentration is 4.61mg/ml, 1.54mg/ml, 0.92mg/ml, 0.66mg/ml; Wherein be preferably 4.61mg/ml.(be equivalent to 0.1g crude drug/ml)
The production method of above-mentioned described BOJU total flavones is characterized in that in the described step (2), the last sample rate of adsorption is 1BV/h, 2BV/h, and 3BV/h, 4BV/h, 5BV/h is preferably 1BV/h.
The production method of above-mentioned described BOJU total flavones is characterized in that in the described step (2), and water and ethanol gradient elution specifically are to wash with water earlier to remove decontamination, and reuse concentration is 50~90%, preferred 50~70% ethanol elutions, collects ethanol elution.
The production method of above-mentioned described BOJU total flavones is characterized in that in the described step (2), elution rate is 1BV/h, 2BV/h, and 3BV/h, 4BV/h, 5BV/h is preferably 2BV/h.
The production method of above-mentioned described BOJU total flavones, it is characterized in that the pretreatment with the following method of the used resin of step (2): resin is with 95% soak with ethanol 24h, wet method is adorned post after treating the abundant swelling of resin, mix no white casse phenomenon with 95% ethanol flow wash to effluent on post with water (1: 3), the reuse distilled water washes repeatedly to there not being the alcohol flavor, and is standby.
The production method of above-mentioned described BOJU total flavones, it is characterized in that the exhausted resin of step (2) Regeneration Treatment with the following method: soaked resin 2-4 hour with the 3%-5% hydrochloric acid solution, dash with the hydrochloric acid solution of concentration with 5-7BV and to drench resin bed, be washed till pH value with distilled water then and be neutral; The sodium hydroxide solution of reuse 3%-5% soaked resin 2-4 hour, dashed with the sodium hydroxide solution of concentration with 5-7BV and drenched resin bed, was washed till pH value with distilled water at last and was neutral.
The production method of above-mentioned described BOJU total flavones is characterized in that in the described step (1), adopts solvent refluxing to extract, and described solvent is water and/or ethanol, is preferably 70% ethanol.
As one of best mode for carrying out the invention, the invention provides a kind of production method of described BOJU total flavones, it is characterized in that:
(1) get bo-chrysanthemum, add 10 times of amount 70% ethanol, reflux, extract, 2 times, each 2h, merge extractive liquid, reclaims ethanol to there not being the alcohol flavor, and is centrifugal, gets supernatant, with distilled water be settled to concentration be 4.61mg/ml (be equivalent to 0.1g crude drug/ml), standby;
(2) solution with step (1) gained is splined on the AB-8 macroporous adsorptive resins, sample solution concentration is 4.61mg/ml (being equivalent to 0.25g crude drug/g resin), with sample absorption on the 1BV/h speed, earlier remove impurity with 10BV distilled water eluting, reuse 4BV/h50% ethanol carries out eluting with the flow velocity of 2BV/h, collects 50% ethanol elution, reclaims solvent, drying under reduced pressure obtains the BOJU total flavones.
The mensuration of general flavone content
(1) preparation of reference substance solution
Precision takes by weighing 120 ℃ of control substance of Rutin 10mg that are dried to constant weight, puts in the 50mL measuring bottle, and it is an amount of to add methanol, puts that slight fever makes dissolving in the water-bath, puts coldly, adds methanol to scale, shakes up, and promptly gets 0.2mg/mL rutin standard solution.
(2) preparation of need testing solution
Orthogonal test is extracted the gained solution concentration be settled to 100mL, precision is measured 50mL and is dried to constant weight through concentrating final vacuum, calculates yield of extract.Get among the 50ml in addition in right amount in the 25mL volumetric flask, operate, develop the color, measure absorbance, calculate general flavone content by the standard curve making method.
(3) selection of mensuration wavelength
Precision is measured a certain amount of reference substance solution and sample solution respectively, puts in the 25mL measuring bottle, and the sodium nitrite solution 1mL of accurate adding 5% shakes up, and places 6min; The solution 1mL that adds 10% aluminum nitrate, 6min is placed in jolting; Add 10% sodium hydroxide solution 10mL again, jolting also adds water to scale, places 15min.In addition precision is measured distilled water 1mL and is put in the 25mL measuring bottle, and accurate 5% the sodium nitrite solution 1mL of adding shakes up, and the same method operation is as reference solution.According to ultraviolet visible spectrophotometry, in the interscan of 400~600nm wave-length coverage, results sample solution and reference substance solution have at the 510nm place absorption maximum are all arranged, so select 510nm for measuring wavelength.
(4) preparation of standard curve
Precision is measured control substance of Rutin solution 0.0,1.0,2.0,3.0,4.0,5.0,6.0mL, places the 25mL volumetric flask respectively, and adding distil water is to 6mL, add 5% (g/v) sodium nitrite solution 1mL, mixing is placed 6min, add 10% (g/v) aluminum nitrate solution 1mL, shake up, place 6min, add 4% (g/v) sodium hydroxide solution 10mL, add water to scale, shake up, place 15min, with first part as blank reagent, measure each solution absorbance at the 510nm place.With the absorbance is vertical coordinate, and quality is an abscissa, the drawing standard curve.Getting regression equation is: y=0.5479x-0.0107, r=0.9996 is good linear relationship in the 0.2-1.2mg scope as can be known.
The method obtain the BOJU total flavones of from BOJU, producing of the present invention, technology is simple, environmental protection, the BOJU total flavones purity that obtains surpasses 50%, can be used as effective site, when crude drug is made preparation, thereby improves the preparation level and the clinical use of BOJU medical material.The inventive method also is easy to industrialization, is suitable for big production.
The specific embodiment
Below further specify technical scheme of the present invention by most preferred embodiment, but do not constitute restriction to claim protection domain of the present invention.
Embodiment 1: optimum extracting method
Selected respectively water is carried, each four factor of alcohol extraction, 3 levels of each factor.Getting bo-chrysanthemum 5g, test by L9 (34) orthogonal table, is evaluation index with total flavones extraction ratio in the medical material, the screening optimum extraction process.
Water is proposed the factor level arrangement and is seen Table 1, orthogonal experiments table 2, and The results of analysis of variance sees Table 3, table 4.
Table 1 water is carried the factor level table
Table 2 water is put forward orthogonal experiments
Table 3 water is put forward The results of analysis of variance (total flavones extraction ratio)
F0.01(1.2)=99;F0.05(1,2)=19
Table 4 water is put forward The results of analysis of variance (yield of extract)
F0.01(1.2)=99;F0.05(1,2)=19
The optimised process that is got the distilled water reflux, extract, by the table intuitive analysis is A
2B
2C
3D
3Be soak time 60 minutes, solid-liquid ratio 12, extraction time 2h, extraction time 3 times, get solid-liquid ratio by analysis of variance table 9, table 10, extraction time, extraction time has significant difference, and extraction time>extraction time>solid-liquid ratio, in addition from yield of extract, extraction time has significant difference, sees that from intuitive analysis soak time 40 minutes is not little with 60 minutes K value differences.For saving time, be soak time 40 minutes so choose the optimised process that water carries, solid-liquid ratio 12, extraction time 2h, extraction time 3 times.
The arrangement of alcohol extraction factor level sees Table 5, and orthogonal experiments sees Table 6, and The results of analysis of variance sees Table 7, table 8.
Table 5 alcohol extraction factor level table
Table 6 alcohol extraction orthogonal experiments
Table 7 alcohol extraction The results of analysis of variance (total flavones extraction ratio)
F0.01(1.2)=99;F0.05(1,2)=19
Table 8 alcohol extraction The results of analysis of variance (yield of extract)
F0.01(1.2)=99;F0.05(1,2)=19
The optimised process that is got the Different concentrations of alcohol reflux, extract, by the table intuitive analysis is A
2B
2C
3D
2Be that concentration of alcohol is 70%, solid-liquid ratio 10, extraction time 2h, extraction time 2 times, get concentration of alcohol, solid-liquid ratio by analysis of variance table 11,12, there is significant difference extraction time, and solid-liquid ratio>extraction time>concentration of alcohol, in addition from yield of extract, concentration of alcohol has significant difference, see that from intuitive analysis concentration of alcohol is 70%, solid-liquid ratio 10, extraction time 2h, extraction time are for 3 times best. considers from the time angle equally, the optimised process concentration of alcohol of choosing alcohol extraction is 70%, solid-liquid ratio 10, extraction time 2h, extraction time 2 times.
For investigating the stability of above-mentioned preferred extraction process, carry out replica test by these process conditions, measure its yield of extract and general flavone content respectively, get its meansigma methods.The results are shown in Table 9, this feasible process is described.
The preferred extraction process demonstration test result of table 9
By Biao Kede, the alcohol extraction general flavone content is that concentration of alcohol is 70% apparently higher than water extraction so choose optimised process, solid-liquid ratio 10, extraction time 2h, extraction time 2 times.
Embodiment 2: the optimal separation purification process
Relatively AB-8 type (low pole), D101 type (nonpolar), DA201 type (polarity), DM301 type (Semi-polarity), 5 kinds of opposed polarity macroporous resins of HPD600 type (polarity) are to the absorption property of BOJU total flavones.
Take by weighing good various each 30g of model resin of pretreatment respectively, the wet method upper prop (φ 1.5cm * 40cm), the accurate sample solution 80ml that draws (is equivalent to 0.1g crude drug/ml) upper prop absorption, preadsorption 1h crosses the post effluent and heavily adsorbs once, makes its absorption fully, collect effluent, be washed till no reducing sugar reaction with distilled water, reuse 10%, 30%.50%, 70%, 90% ethanol elution is collected eluent separately.Measure wherein the concentration of total flavones total saponins and calculate it respectively than adsorbance and compare elution amount.Parallel test 3 times is averaged.The results are shown in Table 10.
Table 10 different model macroporous resin is to the absorption of BOJU total flavones and the comparison of eluting power
By comparative test result as can be known, in 5 kinds of different common resins, AB-8 type macroporous resin is best to total flavones adsorptivity reconciliation absorption in the BOJU.
Embodiment 3:
Get bo-chrysanthemum 500g, add 10 times of amount 70% ethanol, reflux, extract, 2 times, each 2h, merge extractive liquid, reclaims ethanol to there not being the alcohol flavor, and is centrifugal, gets supernatant, with distilled water be settled to concentration be 4.61mg/ml (be equivalent to 0.1g crude drug/ml), standby;
The solution of step (1) gained is splined on the AB-8 macroporous adsorptive resins, sample solution concentration is 4.61mg/ml (being equivalent to 0.25g crude drug/g resin), with sample absorption on the 1BV/h speed, earlier remove impurity with 10BV distilled water eluting, reuse 4BV/h50% ethanol carries out eluting with the flow velocity of 2BV/h, collects 50% ethanol elution, reclaims solvent, drying under reduced pressure obtains the BOJU total flavones.
Test for parallel 3 parts.Measure general flavone content, dried cream amount, calculate the rate of transform, yield and refining degree.See Table 11.
Table 11 result of the test
Differentiate: hydrochloric acid-magnesium powder reaction, it is an amount of to get the sample that makes, and adds small amount of ethanol and makes dissolving, and a little drips with the salt acid number to add magnesium powder, jolting, solution shows redness to aubergine.
Sample thief is an amount of, adds methanol 10ml and makes dissolving as need testing solution.It is an amount of that other gets luteolin-7-O-β-D-glucoside, acacetin-7-O-β-D-glucoside, luteolin and acacetin reference substance, adds methanol and make dissolving and standardize solution, product solution in contrast.Draw each 5 μ l of above-mentioned solution, put respectively on same polyamide film with water-n-butyl alcohol-acetone-acetic acid=4: 2: 2: 1 launches, dry, aluminum chloride alcoholic solution colour developing with 2%, uviol lamp is observed fluorescence down, in the test sample chromatograph, with the corresponding position of reference substance chromatograph on, show the speckle of same color.
Claims (10)
1. the production method of a BOJU total flavones comprises the steps:
(1) the bo-chrysanthemum crude drug obtains crude extract through extraction;
(2) the purified process of the resulting crude extract of step (1) makes the BOJU total flavones;
It is characterized in that the purification described in the step (2) is to adopt purification by macroporous resin, and described macroporous resin is an AB-8 type macroporous resin.
2. the production method of BOJU total flavones according to claim 1 is characterized in that in the described step (2), and macroporous resin adopts wet method dress post, adsorption capacity is respectively 8.50mg/g, 10.41mg/g, 12.30mg/g, 7.95mg/g 11.67mg/g is preferably 12.30mg/g.
3. the production method of BOJU total flavones according to claim 1 is characterized in that in the described step (2), sample solution concentration is 4.61mg/ml, 1.54mg/ml, and 0.92mg/ml, 0.66mg/ml is preferably 4.61mg/ml.
4. the production method of BOJU total flavones according to claim 1 is characterized in that in the described step (2), the last sample rate of adsorption is 1BV/h, 2BV/h, and 3BV/h, 4BV/h, 5BV/h is preferably 1BV/h.
5. the production method of BOJU total flavones according to claim 1, it is characterized in that in the described step (2) that water and ethanol gradient elution specifically are to wash with water earlier to remove decontamination, reuse concentration is 50~90%, preferred 50~70% ethanol elutions, collects ethanol elution.
6. the production method of BOJU total flavones according to claim 1 is characterized in that in the described step (2), elution rate is 1BV/h, 2BV/h, and 3BV/h, 4BV/h, 5BV/h is preferably 2BV/h.
7. according to the production method of the described BOJU total flavones of claim 1-6, it is characterized in that the pretreatment with the following method of the used resin of step (2): resin is with 95% soak with ethanol 24h, wet method is adorned post after treating the abundant swelling of resin, mix no white casse phenomenon with 95% ethanol flow wash to effluent on post with water (1: 3), the reuse distilled water washes repeatedly to there not being the alcohol flavor, and is standby.
8. according to the production method of the described BOJU total flavones of claim 1-6, it is characterized in that the exhausted resin of step (2) Regeneration Treatment with the following method: soaked resin 2-4 hour with the 3%-5% hydrochloric acid solution, dash with the hydrochloric acid solution of concentration with 5-7BV and to drench resin bed, be washed till pH value with distilled water then and be neutral; The sodium hydroxide solution of reuse 3%-5% soaked resin 2-4 hour, dashed with the sodium hydroxide solution of concentration with 5-7BV and drenched resin bed, was washed till pH value with distilled water at last and was neutral.
9. according to the production method of the described BOJU total flavones of claim 1-6, it is characterized in that in the described step (1) that adopt solvent refluxing to extract, described solvent is water and/or ethanol, is preferably 70% ethanol.
10. according to the production method of the described BOJU total flavones of claim 1-6, it is characterized in that:
(1) get bo-chrysanthemum, add 10 times of amount 70% ethanol, reflux, extract, 2 times, each 2h, merge extractive liquid, reclaims ethanol to there not being the alcohol flavor, and is centrifugal, gets supernatant, uses the distilled water standardize solution.Concentration be 4.61mg/ml (be equivalent to 0.1g crude drug/ml), standby;
(2) solution with step (1) gained is splined on the AB-8 macroporous adsorptive resins, sample solution concentration is 4.61mg/ml (being equivalent to 0.25g crude drug/g resin), with sample absorption on the 1BV/h speed, earlier remove impurity with 10BV distilled water eluting, reuse 4BV/h50% ethanol carries out eluting with the flow velocity of 2BV/h, collects 50% ethanol elution, reclaims solvent, drying under reduced pressure obtains the BOJU total flavones.
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Cited By (3)
Publication number | Priority date | Publication date | Assignee | Title |
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CN102586021A (en) * | 2012-03-06 | 2012-07-18 | 烟台张裕集团有限公司 | Method for recovering aroma components from solution containing aroma components |
CN105030869A (en) * | 2015-06-29 | 2015-11-11 | 安徽瑞思威尔科技有限公司 | Bo-chrysanthemum total flavone extract preparation method based on response surface optimization |
CN105998126A (en) * | 2016-05-18 | 2016-10-12 | 安徽瑞思威尔科技有限公司 | Method for preparing Bozhou chrysanthemum general flavone extracting solution and concentrated solution by extraction and concentration machine |
Citations (1)
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CN1460678A (en) * | 2003-06-04 | 2003-12-10 | 浙江现代中药与天然药物研究院有限公司 | Chrysanthemum total flavone resin purification method |
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CN1460678A (en) * | 2003-06-04 | 2003-12-10 | 浙江现代中药与天然药物研究院有限公司 | Chrysanthemum total flavone resin purification method |
Non-Patent Citations (1)
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Cited By (4)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
CN102586021A (en) * | 2012-03-06 | 2012-07-18 | 烟台张裕集团有限公司 | Method for recovering aroma components from solution containing aroma components |
CN102586021B (en) * | 2012-03-06 | 2013-12-25 | 烟台张裕集团有限公司 | Method for recovering aroma components from solution containing aroma components |
CN105030869A (en) * | 2015-06-29 | 2015-11-11 | 安徽瑞思威尔科技有限公司 | Bo-chrysanthemum total flavone extract preparation method based on response surface optimization |
CN105998126A (en) * | 2016-05-18 | 2016-10-12 | 安徽瑞思威尔科技有限公司 | Method for preparing Bozhou chrysanthemum general flavone extracting solution and concentrated solution by extraction and concentration machine |
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Application publication date: 20110622 |