CN101385793A - High-purity extraction method of total flavone from cherokee rose and use thereof in preparing medicine for treating cardio-cerebrovascular disease - Google Patents
High-purity extraction method of total flavone from cherokee rose and use thereof in preparing medicine for treating cardio-cerebrovascular disease Download PDFInfo
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Abstract
Steps for extracting high-purity total flavone of cherokee rose-hip are as follows: a. firstly, a medical plant cherokee rose-hip is extracted by 30%-60% medicinal ethanol to obtain coarse powder of the pharmaceutical plant cherokee rose-hip, and the coarse powder of the medical plant cherokee rose-hip is extracted by 6-8 folds of the medicinal ethanol for 2-3 times with each time lasting 1-2h to obtain extract, and then the extracts from each time are mixed, decompressed and condensed to an extractum; b. the extractum from the step a is separated and purified by macroporous absorption resin, and added to the processed macroporous absorption resin, and the consumption ratio of the resin to the extractum is 1-2g:1ml; in macroporous resin column chromatography, the extractum is first eluted by water until the extractum is close to colorless, and then eluted by 40%-60% medicinal ethanol which is 5-8 times of the weight of resin bed, the fractions eluted by the ethanol are received, condensed, decompressed and dried to be made into the high-purity flavone of the cherokee rose-hip. The high-purity flavone of the cherokee rose-hip is applied to preparing drugs for treating cardiovascular and cerebrovascular diseases.
Description
Technical field
The present invention relates to a kind of application of method for extraction and purification and extract of Chinese medicine active component, particularly from the medicinal plants Fructus Rosae Laevigatae, prepare the method and the application of high-purity total flavones in the preparation heart, cerebrovascular drug of high-purity total flavones fast.
Background technology
The heart, cerebrovascular disease are one of harm humans three big diseases, and mortality rate tops the list.Wherein coronary heart disease, hypertension are commonly encountered diseases, frequently-occurring disease, serious harm people's life and health and life quality.The ischemic heart, cerebrovascular disease account for 56.6～80.8% of the heart, cerebrovascular disease, existent also decline greatly of quality of life, and about self care ability that loses more than 50% has caused very big misery for patient and family thereof, also brings utmost point heavy burden to society.The medicine of the treatment heart, cerebrovascular disease comprises a large amount of Western medicine at present, and its effect is unsatisfactory, and has toxic and side effects, has also caused other injury in the treatment heart, cerebrovascular disease to health.As everyone knows, Chinese medicine is compared with Western medicine has significantly characteristics, and toxic and side effects is little, and cost is low, and some is also had curative effect preferably with the disease that Western medicine is difficult to cure.
Fructus Rosae Laevigatae is the dry mature fruit of rosaceous plant Fructus Rosae Laevigatae (Rosa laevigate Michx), is distributed in ground such as Guizhou, ground such as south China, Central China, East China and southwest of China and Yunnan, has effects such as controlling nocturnal emission with astringent drugs, astringing intestine to stop diarrhea, reducing urination, antidiarrheal.Extract at present the method more complicated of total flavones in Fructus Rosae Laevigatae, speed is slow, the cost height, and the total flavones purity of extracting is lower than 50%, and existing this Fructus Rosae Laevigatae total flavones is not applied to treat the heart, cerebrovascular disease yet.
Summary of the invention
The purpose of this invention is to provide the extracting method and the application of Fructus Rosae Laevigatae high-purity total flavones in the preparation heart, cerebrovascular drug of the Fructus Rosae Laevigatae high-purity total flavones that a kind of speed is fast, cost is low, purity is high, overcome the deficiencies in the prior art.
The extracting method of Fructus Rosae Laevigatae high-purity total flavones of the present invention, it is characterized in that: it comprises the steps:
A, at first utilize medicinal alcohol that the medicinal plants Fructus Rosae Laevigatae is extracted, the concentration of medicinal alcohol is selected between 30%～60%, the coarse powder of the medicinal plants Fructus Rosae Laevigatae medicinal alcohol with 6～8 times of amounts is extracted 2～3 times, each 1～2 hour, be evaporated to extractum after merging each time extracting solution;
B, utilize macroporous adsorbent resin that the extractum that a step produces is carried out separation and purification treatment, extractum is added on the macroporous adsorbent resin of having handled well, the amount ratio of resin and extractum is 1～2 gram resin: 1 milliliter of extractum; First water is eluted to effluent near colourless in the macroporous resin chromatography, then with 40%～60% medicinal alcohol eluting of 5～8 times of resin beds, receives ethanol elution stream part, concentrates the back drying under reduced pressure and gets Fructus Rosae Laevigatae high-purity total flavones.
Described macroporous adsorbent resin is low pole macroporous adsorbent resins such as D101, AB-8.
Medicinal alcoholic acid dense crossing is selected between 40%～50% in the described a step, and the coarse powder of described medicinal plants Fructus Rosae Laevigatae extracts each 1.5 hours 2 times or 3 times with the medicinal alcohol of 7 times of amounts; The amount ratio of resin and extractum is 1.5 gram resins in the described b step: 1 milliliter of extractum, described water are eluted to effluent near after colourless, follow 50% medicinal alcohol eluting with 6～7 times of resin beds.
The application of the high-purity total flavones of the extracting method gained of Fructus Rosae Laevigatae high-purity total flavones of the present invention in the preparation heart, cerebrovascular drug.
The extracting method of Fructus Rosae Laevigatae high-purity total flavones of the present invention compared with prior art has that extraction rate is fast, cost is low, the product purity advantages of higher; The application of Fructus Rosae Laevigatae high-purity total flavones of the present invention in the preparation heart, cerebrovascular drug can alleviate the heart, cerebrovascular patient's toxic and side effects, and effect is obvious.
The specific embodiment
The fast preparation method of Fructus Rosae Laevigatae high-purity total flavones:
Embodiment 1:
1, get Fructus Rosae Laevigatae dry mature fruit 100g, utilize commonsense method to be ground into coarse powder, its granularity is 10 orders or 20 orders or 30 orders or 40 orders, promptly all can between the 10-40 order.With 30% or 40% or 50% or 60% medicinal alcohol reflux, extract, of 6 times of amounts or 7 times of amounts or 8 times of amounts 2 times or 3 times, each 1 hour or 2 hours, filter, merging filtrate is evaporated to the about 100ml of extractum.
2, get 100g or 150g or 200gD101 or AB-8 macroporous adsorbent resin, after 95% soak with ethanol 12-24 hour, be added to after being soaked in water again 12-24 hour on the chromatographic column (4.0 * 60cm), wash repeatedly with big water gaging.100ml extractum is added to the resin upper end, opens piston, flow velocity is 10ml/min, after the completion of the sample, closure piston, the about 2h of static absorption, then extremely colourless with distilled water flushing, then with 40% or 50% or 60% medicinal alcohol eluting of 5 times or 6 times or 7 times or 8 times resin beds, 200ml effluent before discarding merges all the other ethanol elution stream parts, decompression recycling ethanol final vacuum drying, get the bronzing powder, 3 parts of operation repetitives, average yield is 5.37--5.54%.
3, use state-promulgated pharmacopoeia version in 2000 and measure flavones content method mensuration general flavone content, average content is between 76.04%--79.86%.
Embodiment 2:
The antioxidant activity of Fructus Rosae Laevigatae total flavones:
Antioxidant activity in the Fructus Rosae Laevigatae total flavones body:
The KM mice is divided into 2 groups after adapting to a week at random, is respectively: matched group and administration group, 10 every group, male and female half and half.Matched group is irritated stomach NG and organize 0.9% normal saline every day, administration group 100mg/kg/d, administration 10d altogether.Experimental session all gives normal diet, and freely ingests and drink water.Testing the 11d post processing mice that spends the night on an empty stomach draws materials.The eye socket blood sampling, and the low-temperature centrifugation separation of serum (4 ℃, 3000r/min, 10min)-20 ℃ preservation is to be measured.The mice cervical vertebra dislocation of getting behind the blood is put to death, and gets liver, kidney, and with the floating blood of ice normal saline flush away, filter paper blots, and liquid nitrogen cools off rapidly, weighs.With the PBS buffer of pre-cooling (0.2mol/l, pH7.4) do the homogenate medium under ice bath with each organ-tissue machinery homogenate (1.0g wet tissue adds the 9.0ml buffer), 10% tissue homogenate.In 4 ℃, the centrifugal 10min of 6000r/min separates supernatant, and-20 ℃ of preservations are to be measured.Build up the institute's test kit that provides explanation according to Nanjing and measure and respectively organize serum and tissue T-AOC, the value of MDA and SOD, and with SPSS11.5 software data are added up.Get following table 1:
Table 1 is respectively organized the different sample T-AOC of mice, and MDA and SOD value (Mean ± S.D., n=10)
Annotate: administration group and matched group compare,
*P＜0.05,
*P＜0.01
The oxidation resistance of body defense system is strong and weak to exist close ties with health degree, and the reduction of defense system function often causes the generation of various diseases, thereby the T-AOC of the body fluid of measurement human body, tissue etc. is significant.As known from Table 1, the T-AOC of the serum of administration group and each tissue thereof and matched group relatively exist significance or utmost point significant difference.
MDA is a sensitive indicator of weighing the body Radical Metabolism as the representative product of lipid peroxidation, and its content can reflect objectively that body produces the level of free radical.After giving Fructus Rosae Laevigatae total flavones solution, mice serum and organize the MDA level all significantly or extremely significantly to reduce, illustrate that oral Fructus Rosae Laevigatae total flavones solution can reduce the formation of interior free yl, increase the drainage of cylinder super-oxidation lipid, show tangible interior antioxidation action.
SOD is an enzyme important in the body antioxidant system, and as known from Table 1, relatively there are significant difference in the SOD of the serum of administration group and each tissue thereof and matched group.
The analysis experimental result finds that the Fructus Rosae Laevigatae total flavones can reduce MDA level in the body significantly, improves T-AOC and SOD, shows that it has good interior antioxidation action.
The antioxidation in vitro experiment of Fructus Rosae Laevigatae total flavones:
The DPPH free radical scavenging activity is measured:
Operate according to pertinent literature, can get, the Fructus Rosae Laevigatae total flavones is removed the IC of DPPH
50Be 0.01mg/ml, maximal clearance is 88.3%; Vitamin C is removed the IC of DPPH
50Be 0.008mg/ml, maximal clearance is 96.5%.
Removing the ultra-oxygen anion free radical ability measures:
Fructus Rosae Laevigatae total flavones powder is mixed with the solution of mass concentration 5mg/ml with deionized water, and is diluted to different Concentraton gradient, the related kit description is seen in concrete operations.Can get, the Fructus Rosae Laevigatae total flavones is removed the IC of ultra-oxygen anion free radical
50Be 0.08mg/ml, maximal clearance is 50.3%; Vitamin C is removed the IC of ultra-oxygen anion free radical
50Be 0.01mg/ml, maximal clearance is 97.23%.
Removing hydroxyl radical free radical OH ability measures:
The Fructus Rosae Laevigatae total flavones is mixed with the solution of mass concentration 5mg/ml with deionized water, and is diluted to different Concentraton gradient, the related kit description is seen in concrete operations.Can get, the Fructus Rosae Laevigatae total flavones is removed the IC of hydroxyl radical free radical
50Be 0.041mg/ml, maximal clearance is 94.03%; Vitamin C is removed the IC of hydroxyl radical free radical
50Be 0.005mg/ml, maximal clearance is 96.67%.
The mensuration of reducing power:
Operate according to pertinent literature, can get, in 0～0.05mg/ml scope, Fructus Rosae Laevigatae total flavones maximum absorbance is 1.17; The vitamin C maximum absorbance is 1.45.
The hydrogen peroxide clearance rate is measured:
Operate according to pertinent literature, can get, Fructus Rosae Laevigatae total flavones and vitamin C all constantly increase along with the increase of concentration the removing of hydrogen peroxide, and when to finite concentration, clearance rate begins to descend.The maximal clearance of Fructus Rosae Laevigatae total flavones is 31.4%; Ascorbic maximal clearance is 96.67%.
The erythrocyte hemolysis suppression ratio is measured:
Operate according to pertinent literature, can get, the Fructus Rosae Laevigatae total flavones suppresses the IC of erythrocyte hemolysis
50Be 0.35mg/ml, maximal percentage inhibition is 71.62%; Vitamin C suppresses the IC of erythrocyte hemolysis
50Be 0.15mg/ml, maximal percentage inhibition is 94.63%.
The lipid peroxidation suppression ratio is measured:
Operate according to pertinent literature, can get, in 0～2.00mg/ml scope, Fructus Rosae Laevigatae total flavones maximal percentage inhibition is 48.45%; The vitamin C maximal percentage inhibition is 50.14%.
Embodiment 3:
The Fructus Rosae Laevigatae total flavones is to the influence of mice hyperlipemia and hypercholesterolemia:
The KM mice is divided into 5 groups adapt to a week under experimental situation after at random, and 10 every group, male and female half and half.Respectively group (removing blank group) is all giving to carry out following filling stomach (i.g.) processing under high lipid food (normal feedstuff 97.6%, cholesterol 2%, the cholate 0.4%) prerequisite.The blank group, model control group gives 0.9% normal saline; The positive drug group gives 100mg/kg/d zhibituo (production of Chengdu buchu pharmaceutical factory); High dose group gives 200mg/kg/d; Low dose group gives 100mg/kg/d, 9:00 administration every day, and in continuous 4 weeks, the experimental session animal freely drinks water and ingests.Adopt in last week and pluck the blood sampling of eyeball blood taking method.Blood sample is in 4 ℃, the centrifugal 20min of 6000r/min separate serum.Measure TC according to the test kit description, TG and HDL-C, and by formula LDL-C (mmol/l)=TC-(TG * 0.456+HDL-C) calculates low density lipoprotein, LDL (LDL-C).The results are shown in following table 2.
Table 2. respectively organize serum TC, TG, HDL-C, LDL-C value (Mean ± S.D., n=10)
Annotate: TC in the model group, TG, HDL-C and LDL-C value compare with blank group,
#P＜0.05,
##P＜0.001; Administration group desired value and model group compare,
*P＜0.05,
*P＜0.001
As known from Table 2, model group cholesterol, triglyceride and blank group relatively have significant difference, and hyperlipemia mice modeling success is described.Positive group, Fructus Rosae Laevigatae total flavones high dose group and low dose group and model group more all exist significance or utmost point significant difference.And the high dose group lipid-lowering effect is more a little better than positive controls as can be known.Illustrate that the Fructus Rosae Laevigatae total flavones has the biological activity of significant treatment cardiovascular disease.
Embodiment 4:
The Fructus Rosae Laevigatae total flavones is to the influence of serum lipids in rats and hypercholesterolemia
The SD rat is divided into 4 groups adapt to a week under experimental situation after at random, and is 10 every group, female.Each group is made hyperlipemia model under the condition that gives high lipid food (normal feedstuff 93.6%, Adeps Sus domestica 4%, cholesterol 2%, cholate 0.4%), detect its blood lipid level weekly, after two weeks, and the modeling success.According to different blood lipid levels, random packet, each group continue to give to carry out under the high lipid food prerequisite following filling stomach (i.g.) and handle.Model control group gives 0.9% normal saline; The positive drug group gives 40mg/kg/d fenofibrate capsules (French Li Bofuni drugmaker); High dose group gives 200mg/kg/d; Low dose group gives 100mg/kg/d, 14:00 administration every day, and in continuous 4 weeks, the experimental session animal freely drinks water and ingests.Its blood lipid level and each index of hemorheology are surveyed in last week blood sampling.Measure TC according to the test kit description, TG and HDL-C, and by formula LDL-C (mmol/l)=TC-(TG * 0.456+HDL-C) calculates low-density lipoprotein (LDL-C).Whole blood contrast viscosity, get anticoagulation, with the different shear rates of specific viscosity instrumentation (low cutting: 20s
-1, height is cut: 200s
-1) under specific viscosity; RBC hematocrit (%): select for use the about 0.8mm of internal diameter to be about the uniform and smooth glass capillary of 75mm, suck anticoagulation 30ul, account for 3/4 place of pipe range.Seal end mouth, change the centrifugal 5min of (12000r/min) dull and stereotyped centrifuge with height.Measure blood total length and erythrocyte length, calculate the shared percentage rate of erythrocyte, be packed cell volume; Plasma viscosity: anticoagulation, the centrifugal 5min of 1000r/min, separated plasma, fixed with the specific viscosity instrumentation; The serum specific viscosity: get non-anticoagulation, the centrifugal 10min of 3000r/min gets serum and surveys specific viscosity.And calculate: whole blood reduction ratio viscosity=whole blood contrast viscosity/RBC hematocrit; Fiber protein content (mg/ml)=(plasma viscosity-serum specific viscosity) * 5/0.002; RBC aggregate index=whole blood is low to be cut specific viscosity/whole blood height and cuts specific viscosity.The results are shown in Table 3, table 4 and table 5.
Table 3. is respectively organized serum TC, TG, the value of HDL-C and LDL-C (Mean ± S.D., n=10)
Annotate: administration group desired value and model group compare,
*P＜0.05,
*P＜0.001
Table 4. Fructus Rosae Laevigatae total flavones to each organize hemorheological influence (Mean ± S.D., n=10)
Annotate: administration group desired value and model group compare,
*P＜0.05,
*P＜0.001
Table 5. Fructus Rosae Laevigatae total flavones to each organize hemorheological influence (Mean ± S.D., n=10)
Annotate: administration group desired value and model group compare,
*P＜0.05,
*P＜0.001
As known from Table 3, positive group, Fructus Rosae Laevigatae total flavones high dose group and low dose group and model group more all exist significance or utmost point significant difference.And the high dose group lipid-lowering effect is more a little better than positive controls as can be known.Compare with model group, the Fructus Rosae Laevigatae total flavones has the biological activity of significant treatment cardiovascular disease.
Atherosclerosis (As) is the main pathological basis of cardiovascular and cerebrovascular disease.As pathogenesis complexity relates to lipoprotein infiltration, arterial endothelium (EC) damage, platelet adhesion reaction gathering, thrombosis, smooth muscle cell (SMC) propagation etc.Hyperlipemia and AS all have the unusual performance of hemorheology, Fibrinogen has the greatest impact to plasma viscosity, but and coup injury EC, promote the LDL-C retention, SMC propagation, monocyte chemotactics etc. participate in the formation of AS, Fructus Rosae Laevigatae total flavones heavy dose can obviously reduce fibrinogen content, reduce RBC aggregate index and hematocrit, show that it is dense, sticking to high fat rat blood, cohesion has antagonism preferably, help improving the ischemia symptom that AS causes.
In a word, the Fructus Rosae Laevigatae total flavones can have tangible action effect to hyperlipidemia rats blood lipid level and hemorheology, has tangible blood fat reducing activity.
Claims (4)
1, a kind of extracting method of Fructus Rosae Laevigatae high-purity total flavones, it is characterized in that: it comprises the steps:
A, at first utilize medicinal alcohol that the medicinal plants Fructus Rosae Laevigatae is extracted, the concentration of medicinal alcohol is selected between 30%～60%, the coarse powder of the medicinal plants Fructus Rosae Laevigatae medicinal alcohol with 6～8 times of amounts is extracted 2～3 times, each 1～2 hour, be evaporated to extractum after merging each time extracting solution;
B, utilize macroporous adsorbent resin that the extractum that a step produces is carried out separation and purification treatment, extractum is added on the macroporous adsorbent resin of having handled well, the amount ratio of resin and extractum is 1～2 gram resin: 1 milliliter of extractum; First water is eluted to effluent near colourless in the macroporous resin chromatography, then with 40%～60% medicinal alcohol eluting of 5～8 times of resin beds, receives ethanol elution stream part, concentrates the back drying under reduced pressure and gets Fructus Rosae Laevigatae high-purity total flavones.
2, the extracting method of Fructus Rosae Laevigatae high-purity total flavones according to claim 1 is characterized in that: described macroporous adsorbent resin is low pole macroporous adsorbent resins such as D101, AB-8.
3, the extracting method of Fructus Rosae Laevigatae high-purity total flavones according to claim 2, it is characterized in that: medicinal concentration of ethanol is selected between 40%～50% in the described a step, the coarse powder of described medicinal plants Fructus Rosae Laevigatae extracts each 1.5 hours 2 times or 3 times with the medicinal alcohol of 7 times of amounts; The amount ratio of resin and extractum is 1.5 gram resins in the described b step: 1 milliliter of extractum, described water are eluted to effluent near after colourless, follow 50% medicinal alcohol eluting with 6～7 times of resin beds.
4, a kind of application of high-purity total flavones in the preparation heart, cerebrovascular drug according to the extracting method gained of any described Fructus Rosae Laevigatae high-purity total flavones in the claim 1 to 3.
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Cited By (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN101829214A (en) * | 2010-05-21 | 2010-09-15 | 中国人民解放军第二军医大学 | Cherokee rose leaf extract and application thereof in preparing medicine capable of curing burn and scald |
| CN102240067A (en) * | 2011-07-01 | 2011-11-16 | 深圳烟草工业有限责任公司 | Method for preparing fructus fosae laevigatae extracts, application thereof in cigarette and cigarette |
| CN103385950A (en) * | 2013-07-22 | 2013-11-13 | 沈阳化工大学 | A preparation method for total rose laevigata michx flavones |
| CN105434591A (en) * | 2015-12-28 | 2016-03-30 | 青岛市市立医院 | Traditional Chinese medicine preparation capable of promoting blood circulation to remove meridian obstruction and preparation method of traditional Chinese medicine preparation |
-
2008
- 2008-10-22 CN CN 200810228214 patent/CN101385793B/en not_active Expired - Fee Related
Cited By (6)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN101829214A (en) * | 2010-05-21 | 2010-09-15 | 中国人民解放军第二军医大学 | Cherokee rose leaf extract and application thereof in preparing medicine capable of curing burn and scald |
| CN102240067A (en) * | 2011-07-01 | 2011-11-16 | 深圳烟草工业有限责任公司 | Method for preparing fructus fosae laevigatae extracts, application thereof in cigarette and cigarette |
| CN102240067B (en) * | 2011-07-01 | 2013-06-26 | 深圳烟草工业有限责任公司 | Method for preparing fructus fosae laevigatae extracts, application thereof in cigarette and cigarette |
| CN103385950A (en) * | 2013-07-22 | 2013-11-13 | 沈阳化工大学 | A preparation method for total rose laevigata michx flavones |
| CN103385950B (en) * | 2013-07-22 | 2015-08-26 | 沈阳化工大学 | A kind of preparation method of Fructus Rosae Laevigatae total flavones |
| CN105434591A (en) * | 2015-12-28 | 2016-03-30 | 青岛市市立医院 | Traditional Chinese medicine preparation capable of promoting blood circulation to remove meridian obstruction and preparation method of traditional Chinese medicine preparation |
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