CN102988457A - Total flavone extract of lonicera macranthoides leaves, and preparation method and application thereof - Google Patents
Total flavone extract of lonicera macranthoides leaves, and preparation method and application thereof Download PDFInfo
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Abstract
The invention provides an extraction method and application of total flavone extract from lonicera macranthoides leaves. The total flavone of lonicera macranthoides leaves is furthest extracted by adopting a water-extraction and alcohol precipitation process; and the flavone compound is selectively separated through macroporous resin, and is concentrated and dried to obtain the flavone compound with high purity. The process is simple, and is suitable for large-scale industrial production, and the total flavone content of the total flavone extract prepared from the lonicera macranthoides leaves is higher than 50 percent. The total flavone extract has excellent activity in treating autoimmune hepatitis, hyperglycemia and hyperlipidemia syndromes.
Description
Technical field
The present invention relates to a kind of extracting method of Chinese crude drug active component, relate in particular a kind of extractive of general flavone, its preparation method and application thereof of grayer-hair honeysuckle leaf.
Technical background
Largeflower-like honeysuckle flower is the Caprifoliaceae woodbine, has heat-clearing and toxic substances removing, and the effect of wind-heat dissipating is usually used in the carbuncle furuncle, sore throat, erysipelas, toxic-heat and blood stasis, anemopyretic cold, epidemic febrile disease heating etc.Largeflower-like honeysuckle flower is not only conventional Chinese medicine, and is the green natural product of a kind of " integration of edible and medicinal herbs ", and its by-product-blade, but is regarded as dis-medicinal part always and does not obtain good utilization for a long time and discard.Existing document shows, flavones content is about 2.78 times of the medicinal gold Flos Lonicerae, 6.98 times of Caulis Lonicerae among the largeflower-like honeysuckle flower Lao Ye.Grayer-hair honeysuckle leaf may be medicinal part preferably, and the fungistatic effect of its crude flavonoid powder and antioxygenic activity all are higher than the chlorogenic acid crude product in the Flos Lonicerae.Flavone compound has the biological activity of the series such as obvious antiulcer, spasmolytic, antiinflammatory and blood fat reducing, and the preparation of its preparation has the value for the treatment of and long-term taking to have no side effect to diseases such as cardiovascular and cerebrovascular vessel, arteriosclerosis, hypertension.Also having broad application prospects aspect health care product and the cosmetic industry simultaneously.Be widely used at medicine and food, be this waste resource of rational exploitation and utilization, have very important significance so from grayer-hair honeysuckle leaf, extract chromocor compound.
The existing method of extracting total flavones in the purification grayer-hair honeysuckle leaf, " assay of total flavones in main product ground, the Chongqing largeflower-like honeysuckle flower branch and leaf " literary composition such as the 1st phase of the 31st volume " Southwestern University's journal " January in 2009, disclosed method is: the grayer-hair honeysuckle leaf after the drying and crushing or branch powder are as raw material, ethanol with 60% at room temperature soaks 12h. then behind 50 ℃ of supersound extraction 30min, filters to obtain flavone extractive.The major defect of the method is: 1. can not effectively remove the small molecular weight impurities such as inorganic salt, aminoacid, poor product quality; 2. can not further remove the ethanol organic solvent, purification is not thorough, " three waste discharge " contaminated environment; 3. the purity of product is not high, and color and luster is bad etc." the extraction process optimization of Flos Lonicerae total flavones and bacteriostasis research thereof " literary composition for another " Food Additives Used in China " the 4th phase in 2012, disclosed method is: adopt Soxhlet to extract, condition is: take the aqueous solution of ethanol volumetric concentration as 70% as extracting solution, m (Flos Lonicerae): m (extracting solution)=1:50, extract 220 ℃ of temperature, extraction time 2h, the extraction ratio of Flos Lonicerae total flavones are 8.43%.The major defect of the method is: 1. use alcoholic solution as extracting solution, the solution impurity after the extraction is many, is unfavorable for follow-up purification, directly causes the quality shade deviation of the low and product of the purity of product; 2. extract used excess Temperature, the quality of product is affected to some extent, to having relatively high expectations of producing instruments, improved production cost, not environmental protection wastes energy; 3. the solution after extracting is not done and is further purified, and product quality is lower.
Summary of the invention
The invention provides a kind of method and application thereof that total flavones extracts of from the leaf of largeflower-like honeysuckle flower, extracting, the present invention adopts aqueous extraction-alcohol precipitation technology, realization is to the extraction to large degree of the total flavones of grayer-hair honeysuckle leaf, again by the Selective Separation of macroporous resin realization to chromocor compound, and pass through the processing such as concentrated, dry, can obtain highly purified chromocor compound; Concrete processing step is:
A kind of preparation method of extractive of general flavone of grayer-hair honeysuckle leaf may further comprise the steps:
1) with grayer-hair honeysuckle leaf, adopt extraction process by water to extract, collect respectively water extraction liquid and medicine residue;
2) the described water extraction liquid of step 1) is concentrated, add the ethanol precipitation, filter, respectively collecting precipitation and filtrate;
Described adding ethanol is precipitated as and adds 95% alcoholic solution in the concentrated solution of collection, stirs, and carries out precipitating first time, until the ethanol content in the concentrated solution is 60%～85%(v/v), leaves standstill 24 hours.And then pump in the centrifuge, be under 3000～6000 rev/mins at rotating speed, carry out centrifugal 10～20 minutes of the first time, collect for the first time centrifugal supernatant.For the supernatant of collecting, carry out the precipitation second time, centrifugal with method, collect for the second time centrifugal supernatant.For the centrifugal supernatant second time of collecting, Recycled ethanol is until without till the ethanol flavor, collect concentrated solution and be used for next step processing; Difference collecting precipitation and filtrate.
3) with step 2) described filtrate adjust pH is to 4-6, upper D101 or AB-8 macroporous absorption post, eluent is the ethanol of 40%-80% concentration, effluent volume be column volume 3-10 doubly;
4) collect the described eluent of step 3), concentrate eluant, lyophilizing obtains extractive of general flavone.
The described extraction process by water of step 1) is extracted as: pulverize grayer-hair honeysuckle leaf, with the water reflux, extract, 30-50min of 3-8 times of weight of medical material.
The described upper D101 of step 3) or AB-8 macroporous resin column are: concentrated filtrate is to the 1/6-1/8 of original volume, and upper prop, the consumption of macroporous resin are 1-2.5 times of crude drug weight.
The flavone concentrated solution of the described lyophilizing of step 4) for collecting, first pre-freeze 5～6 hours in-18 ℃ refrigerator, and then place freezer dryer, be that-50～-60 ℃, vacuum are under the condition of 25～50Pa in temperature, dry 24～30 hours, prepare total flavones and be higher than 50% flavone lyophilized powder,, as extractive of general flavone.
A kind of extractive of general flavone of grayer-hair honeysuckle leaf, described extractive of general flavone prepares for adopting the described method of claim 1-4 to extract from the leaf of largeflower-like honeysuckle flower.
The leaf of described largeflower-like honeysuckle flower be largeflower-like honeysuckle flower upper end trophophase at 2 months with interior tender leaf.
The application of the extractive of general flavone of described grayer-hair honeysuckle leaf in preparation treatment autoimmune hepatitis medicine.
The application of the extractive of general flavone of described grayer-hair honeysuckle leaf in preparation treatment hyperglycemia medicine.
The application of the extractive of general flavone of described grayer-hair honeysuckle leaf in preparation treatment high blood cholesterol drug.
Useful technique effect of the present invention is: technique of the present invention is simple, be fit to large-scale industrial production, the extractive of general flavone general flavone content for preparing from grayer-hair honeysuckle leaf is higher than 50%, and the present invention obtains extractive of general flavone and have good activity in treatment autoimmune hepatitis, hyperglycemia and hyperlipidemia disease.
The specific embodiment
The preparation of extractive of general flavone in embodiment 1 grayer-hair honeysuckle leaf
Take by weighing and get grayer-hair honeysuckle leaf 10kg, oven dry is pulverized, and is the ratio reflux, extract, 30-50min of 1:3 with medical material and pure water weight ratio, the extracting solution vacuum filtration, and filtrate collection is for subsequent use, precipitates behind the interpolation medical material again extraction and application;
The filtrate water-bath of above-mentioned collection is concentrated into 1/3 of original volume, collects the concentrated solution I;
Ethanol to the concentration of alcohol in the concentrated solution of adding 95% is 60% in the concentrated solution I, left standstill 24 hours, then under 3000 rev/mins rotating speed, centrifugal 20 minutes, get the supernatant and carry out the precipitate with ethanol second time, centrifugal with method, collect for the second time centrifugal supernatant, Recycled ethanol is until without the alcohol flavor, obtain the concentrated solution II;
With the dissolved in purified water of concentrated solution II with 6 times of volumes, and then to regulate pH value with dilute hydrochloric acid be 4-6, obtains upper prop liquid;
With activating suitable D101 macroporous resin dress post, use first with the isopyknic pure water of macroporous resin column and recoil, discharge the bubble in the post, pump into again the upper prop liquid absorption that step obtains.
Pumping into concentration in the macroporous resin column with the absorption flavone is that the alcoholic solution of 40%-80% carries out eluting, and optimized concentration is 40%; Until in the eluent during without flavone till, the consumption of ethanol elution for for the 3-8 of macroporous resin column volume doubly, collect the effluent III, macroporous resin can reuse after with distilled water wash;
With the effluent III, be to be concentrated into without the alcohol flavor under 75 ℃ the condition in vacuum Wei – 0.9Mpa, temperature, for the ethanol that recovery obtains, can reuse after regulating concentration; For the flavone concentrated solution that obtains, first pre-freeze 5 hours in-18 ℃ refrigerator, and then place freezer dryer is under the condition of 25Pa in 60 ℃ of temperature Wei – 50～–, vacuum, and dry 24 hours, HPLC detected and determines that general flavone content surpasses 50%; Namely prepare purity and be 50% flavone lyophilized powder; Obtain extractive of general flavone.
Take by weighing the branch 10kg that gets largeflower-like honeysuckle flower, adopt said method to prepare the extraction matched group.
Extractive of general flavone is to the protective effect of immunological liver injury in mice in embodiment 2 grayer-hair honeysuckle leafs
70 of KM kind mices, male, weight 18 ~ 22g, be divided at random normal group, model group, largeflower-like honeysuckle flower leaf total flavone extract low dose group (100mg/kg), middle dosage group (200mg/kg), high dose group (400mg/kg) group, extract matched group (400mg/kg) and bifendate 150mg/kg, every group 10, all the other were respectively organized every mouse tail intravenous injection bacillus calmette-guerin vaccine (BCG) 12.5mg/mL and (contain approximately 5 * 10 except normal group in the 1st day in modeling
7Individual viable count), each treatment group ig gives relative medicine; Normal group and model group ig give the isometric(al) solvent, continuously 10d.The 10th day, except normal group iv gave the isometric(al) solvent, all the other respectively organized every mouse mainline bacteria lipopolysaccharide (LPS) 37.5 μ g/mL.Fasting 16h after the injection, from the blood sampling of mice posterior orbit venous plexus, the centrifugal 10min of 3000r/min surveys Serum ALT, AST, AKP value with full automatic biochemical apparatus.Put to death animal, the dissection liver is weighed, calculate organ coefficient, with the floating blood of cold saline flush away, take by weighing the 0.5g hepatic tissue, make 10% liver homogenate with cold saline, by the test kit operating instruction superoxide dismutase (SOD), malonaldehyde (MDA), glutathion peroxidase (GSH-Px) are measured.The results are shown in Table 1 and table 2.
Table 1 largeflower-like honeysuckle flower leaf flavonoids is on the impact of immunological liver injury in mice serum liver function zymetology
Annotate: * P＜0.05 of comparing with model group, * * P＜0.01
By as seen from Table 1: the largeflower-like honeysuckle flower leaf flavonoids is high, middle dosage group and extract matched group the ALT of immunologic liver injury and the rising of AST are had obvious reducing effect, with model group significant difference is arranged relatively; It has certain reducing effect trend to AKP, but not statistically significant.Prompting largeflower-like honeysuckle flower leaf flavonoids has certain protective effect to immunological liver injury.
Table 2 largeflower-like honeysuckle flower leaf flavonoids is on the impact of immunological liver injury in mice hepatic tissue SOD, MDA, GSH-Px
Annotate: * P＜0.05 of comparing with model group, * * P＜0.01
By as seen from Table 2: with normal group relatively, model group murine liver tissue MDA level all significantly raises, SOD, GSH-Px level significantly descend; Compare with model group, largeflower-like honeysuckle flower leaf flavonoids high dose group can significantly reduce liver coefficient and the MDA content of immunological liver injury in mice, and its SOD and GSH-Px content raise.The ability that prompting largeflower-like honeysuckle flower leaf total flavone extract improves body anti peroxidation of lipid and removing free radical shields to immunological liver injury in mice.
Extractive of general flavone is on the impact of high fat+high sugar+chain urine rhzomorph diabetes model rat blood sugar, blood fat in embodiment 3 grayer-hair honeysuckle leafs
10 in 70 rats are left intact, are normal group; All the other 70 rats give continuously high-sugar-fat-diet and fed one month, behind the fasting 24h, and lumbar injection chain urine rhzomorph (STZ) 25mg/kg.Survey blood glucose behind the 72h, get the rat random packet of blood glucose value 〉=11.0mmol/L, be model group, largeflower-like honeysuckle flower leaf total flavone extract low dose group (100mg/kg), middle dosage group (200mg/kg), high dose group (400mg/kg) group, extract matched group (400mg/kg) and metformin 800mg/kg, every group 10, be administered once every day, continuously January.Detect administration 10,20,30 days blood glucose and T-CHOL (TC), triglyceride (TG).The results are shown in Table 3 and table 4.
Table 3 largeflower-like honeysuckle flower leaf flavonoids is on the impact of STZ blood glucose in diabetic rats changing value
Annotate: * compares p ﹤ 0.05 with model group, and * * and matched group be p ﹤ 0.01 relatively.
Table 4 largeflower-like honeysuckle flower leaf flavonoids is on the impact of STZ diabetes rat TC, TG
Annotate: * compares p ﹤ 0.05 with model group, and * * and matched group be p ﹤ 0.01 relatively.
By table 3 and as seen from Table 4: largeflower-like honeysuckle flower leaf flavonoids high dose group in administration 10, all can obviously reduce the hyperglycemia that STZ causes in 20 and 30 days, wherein in the dosage group in administration obvious blood sugar lowering effect was also arranged in 30 days.The largeflower-like honeysuckle flower leaf flavonoids is high in addition, middle dosage group and extract T-CHOL (TC) and triglyceride (TG) rising that matched group causes STZ, in administration obvious reducing effect is arranged all in the time of 30 days.The hyperglycemia that prompting largeflower-like honeysuckle flower leaf flavonoids causes at reduction STZ has the effect of blood fat reducing simultaneously concurrently.
Extractive of general flavone is on the impact of serum lipids in rats in embodiment 4 grayer-hair honeysuckle leafs
70 rats conformed 5 days, the feed normal diet, and the tail venous blood sampling was surveyed TC in the 6th day.Be divided into 7 groups according to TC result, be normal group, model group, largeflower-like honeysuckle flower leaf total flavone extract low dose group (100mg/kg), middle dosage group (200mg/kg), high dose group (400mg/kg) group, extract matched group (400mg/kg) and Zhibituo Tabiet 226mg/kg, 10 every group.Except Normal group, all the other are respectively organized at administration while feed fat milk 1ml/100g, once a day, and continuous 14 days.1h after the last administration gets tail vein and surveys TC, TG, HDL-C.Begin to withdraw fat milk on the 15th day, continue administration, once a day, continuous 14 days.1h after the last administration, TC, TG, HDL-C are surveyed in the femoral artery blood-letting.The results are shown in Table 5 and table 6.
Table 5 largeflower-like honeysuckle flower leaf flavonoids is on the impact of the 15th day rat serum TC of administration, TG, HDL-C
Annotate: * P＜0.05 of comparing with model group, * * P＜0.01
Table 6 largeflower-like honeysuckle flower leaf flavonoids is on the impact of the 30th day rat serum TC of administration, TG, HDL-C
Annotate: * P＜0.05 of comparing with model group, * * P＜0.01
By table 5 and as seen from Table 6: the largeflower-like honeysuckle flower leaf flavonoids is high, middle dosage group more all has significance reduction to the serum TC of Hyperlipemia model rat and TG value and model group in administration after 15 days; Relatively there is not significant difference extraction matched group that serum TC, TG value and Serum HDL-C value and the model group of Hyperlipemia model rat are not relatively had significant difference to Serum HDL-C value and model group.Relatively have significance to reduce in administration 30 days serum TC value and model group to Hyperlipemia model rat, and the reducing effect to serum TC have certain dose-effect relationship between each dosage group; Serum TG, HDL-C value and model group more all there is not significant difference.Prompting largeflower-like honeysuckle flower leaf flavonoids has obvious effect for reducing blood fat to the hyperlipidemia model rat, and it is very unobvious that the extract that the preparation of largeflower-like honeysuckle flower branch is adopted in simultaneously prompting falls the rat fat effect to hyperlipidemia model.
Claims (9)
1. the preparation method of the extractive of general flavone of a grayer-hair honeysuckle leaf is characterised in that: may further comprise the steps:
1) with grayer-hair honeysuckle leaf, adopt extraction process by water to extract, collect respectively water extraction liquid and medicine residue;
2) the described water extraction liquid of step 1) is concentrated, add the ethanol precipitation, filter, respectively collecting precipitation and filtrate;
3) with step 2) described filtrate adjust pH is to 4-6, upper D101 or AB-8 macroporous absorption post, eluent is the ethanol of 40%-80% concentration, effluent volume be column volume 3-10 doubly;
4) collect the described eluent of step 3), concentrate eluant, lyophilizing obtains extractive of general flavone.
2. the preparation method of the extractive of general flavone of grayer-hair honeysuckle leaf according to claim 1, be characterised in that: the described extraction process by water of step 1) is extracted as: pulverize grayer-hair honeysuckle leaf, with the water reflux, extract, 30-50min of 3-8 times of weight of medical material.
3. the preparation method of the extractive of general flavone of grayer-hair honeysuckle leaf according to claim 1, be characterised in that: the described upper D101 of step 3) or AB-8 macroporous resin column are: concentrated filtrate is to the 1/6-1/8 of original volume, upper prop, the consumption of macroporous resin are 1-2.5 times of crude drug weight.
4. the preparation method of the extractive of general flavone of grayer-hair honeysuckle leaf according to claim 1, be characterised in that: the flavone concentrated solution of the described lyophilizing of step 4) for collecting, first pre-freeze 5～6 hours in-18 ℃ refrigerator, and then place freezer dryer, be under the condition of 25～50Pa in 60 ℃ of temperature Wei – 50～–, vacuum, dry 24～30 hours, prepare total flavones and be higher than 50% flavone lyophilized powder, as extractive of general flavone.
5. the extractive of general flavone of a grayer-hair honeysuckle leaf is characterised in that: described extractive of general flavone prepares for adopting the described method of claim 1-4 to extract from the leaf of largeflower-like honeysuckle flower.
6. the extractive of general flavone of a grayer-hair honeysuckle leaf is characterised in that: the leaf of described largeflower-like honeysuckle flower be largeflower-like honeysuckle flower upper end trophophase at 2 months with interior tender leaf.
7. the application of the extractive of general flavone of the described grayer-hair honeysuckle leaf of claim 1-6 in preparation treatment autoimmune hepatitis medicine.
8. the application of the extractive of general flavone of the described grayer-hair honeysuckle leaf of claim 1-6 in preparation treatment hyperglycemia medicine.
9. the application of the extractive of general flavone of the described grayer-hair honeysuckle leaf of claim 1-6 in preparation treatment high blood cholesterol drug.
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| CN103446027A (en) * | 2013-09-18 | 2013-12-18 | 重庆市中药研究院 | Preparation method for largeflower-like honeysuckle flower leaf gargle |
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| CN105663174A (en) * | 2016-01-05 | 2016-06-15 | 安徽宇宁生物科技有限公司 | Production technology for recovery of bioflavonoid from pectin extracted ethanol waste liquid |
| CN106858061A (en) * | 2017-03-24 | 2017-06-20 | 河南科高中标检测技术有限公司 | A kind of application for the composition of natural products of additive for farm animal feed and its in feed addictive |
| CN111088262A (en) * | 2020-01-19 | 2020-05-01 | 贵州大学 | Method for improving flavone content of safflower hairy roots by transferring CTMYB1 gene |
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| Publication number | Priority date | Publication date | Assignee | Title |
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| CN103446027A (en) * | 2013-09-18 | 2013-12-18 | 重庆市中药研究院 | Preparation method for largeflower-like honeysuckle flower leaf gargle |
| CN103450019A (en) * | 2013-09-18 | 2013-12-18 | 重庆市中药研究院 | Method for extracting and purifying active components from flower buds and leaves of largeflower-like honeysuckle flower |
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| CN105663174A (en) * | 2016-01-05 | 2016-06-15 | 安徽宇宁生物科技有限公司 | Production technology for recovery of bioflavonoid from pectin extracted ethanol waste liquid |
| CN106858061A (en) * | 2017-03-24 | 2017-06-20 | 河南科高中标检测技术有限公司 | A kind of application for the composition of natural products of additive for farm animal feed and its in feed addictive |
| CN111088262A (en) * | 2020-01-19 | 2020-05-01 | 贵州大学 | Method for improving flavone content of safflower hairy roots by transferring CTMYB1 gene |
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Application publication date: 20130327 |