CN102670696B - Eucommia ulmoids leaf extracts and preparation method and application thereof - Google Patents
Eucommia ulmoids leaf extracts and preparation method and application thereof Download PDFInfo
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- CN102670696B CN102670696B CN201210134193.0A CN201210134193A CN102670696B CN 102670696 B CN102670696 B CN 102670696B CN 201210134193 A CN201210134193 A CN 201210134193A CN 102670696 B CN102670696 B CN 102670696B
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- Prior art keywords
- folium eucommiae
- extract
- water
- eucommia
- ulmoids
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Landscapes
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Abstract
The invention discloses an eucommia ulmoids leaf water and/or low alcohol extract and preparation method and application thereof, which comprise crude extracts and refined eucommia ulmoids leaf extracts, wherein the crude extracts are extracted from folium cortex eucommiae through water or low alcohol, or are obtained by mixing and extracting the water and the low alcohol, and the refined eucommia ulmoids leaf extracts are obtained after the crude extracts are refined. The eucommia ulmoids leaf water and/or low alcohol extract has obvious effect in application of preparing drugs for promoting fracture healing. The eucommia ulmoids leaf water and/or low alcohol extract can obviously increase alkaline phosphatase, reduces blood calcium, increases serium inorganic phosphorus, increases bone mineral density, and can obviously promote healing of bones after fracturing. Furthermore, medicinal parts of eucommia ulmoids plants can be increased by developing application of extracts of the eucommia ulmoids leaf extracts, the application range of the eucommia ulmoids plants is enlarged, because the eucommia ulmoids leaf is easier to be obtained than eucommia ulmoids skins, the cost of eucommia ulmoids can be reduced.
Description
Technical field
The invention belongs to the field of Chinese medicines, be specifically related to a kind of Folium Eucommiae extract and its preparation method and application.
Background technology
The Cortex Eucommiae is the dry bark of the Eucommiaceae plant Cortex Eucommiae (Eucommia ulmoides Oliver), is Chinese famous and precious tonic herb.Tool invigorating the liver and kidney, bone and muscle strengthening, blood pressure lowering, many effects such as antiabortive.< < Sheng Nong's herbal classic > > classifies as top grade.Call its " cure mainly waist and knee, invigorating middle warmer, beneficial vital essence, hard muscles and bones, wet except itching under the moon, dribbling urination.Clothes for a long time, make light of one's life by commiting suicide resistance to old." Cortex Eucommiae is Chinese peculiar medical material, its medicinal history is long, clinical, has a wide range of applications.Find on earth so far nearly 14 kinds of Cortex Eucommiae platymisciums, they became extinct in succession in continent and Europe afterwards.Being present in the Chinese Cortex Eucommiae is that the Eucommiaceae Cortex Eucommiae belongs to the Relict Plant of only depositing, and it not only has very high economic worth.
In recent years, both at home and abroad expert has carried out repeatedly careful research to Cortex Eucommiae composition, finds to contain the necessary threonine of human body, methionine, isoleucine in the Cortex Eucommiae, 17 kinds of free amino acids such as lysine, and zinc, copper, magnesium, ferrum, calcium, phosphorus, 15 kinds of trace element such as potassium.The Cortex Eucommiae has invigorating the liver and kidney, and rubbish in body is removed in bone and muscle strengthening, strengthen human body cell substance metabolism, prevent that muscle skeleton is aging, balance human blood-pressure, decomposer inner cholesterol, reduce body fat, recover blood vessel elasticity, diuresis heat clearing away, broad-spectrum antiseptic, stimulating central nervous system, improves white blood cell count, strengthens the remarkable efficacy such as body immunity.
Existing Cortex Eucommiae medicinal part is Cortex Eucommiae, and the acquisition method of Cortex Eucommiae is: will 10 years above Cortexs Eucommiae of cultivation, process after stripping bark with half annular stripping method.Due to gutta-percha tree quantity rareness, and for guaranteeing that the quality of medical material needs Growth of Eucommia ulmoides to 10 year or the above age of tree could gather Cortex Eucommiae, the while is because the medical value of the Cortex Eucommiae is huge, so cause it expensive.
Summary of the invention
The object of the present invention is to provide a kind of Folium Eucommiae water and/or lower alcohol extraction thing, comprise Folium Eucommiae crude extract and Folium Eucommiae extract.
Another object of the present invention is to provide Folium Eucommiae water and/or the application of lower alcohol extraction thing in the anti-fracture medicine of preparation.
Object of the present invention can be achieved through the following technical solutions:
A kind of Folium Eucommiae water and/or lower alcohol extraction thing, it is that described Folium Eucommiae water and/or lower alcohol extraction thing comprise Folium Eucommiae water or lower alcohol extraction, or the crude extract that water and lower alcohol mixed extraction obtain, and the refining Folium Eucommiae extract that obtains after refining of described crude extract.
Water extraction Folium Eucommiae is prepared the method for Folium Eucommiae crude extract: after Folium Eucommiae medicinal material drying is pulverized, with water extraction three times, the quality of each institute's water is followed successively by 10 times, 10 times and 9 times of Folium Eucommiae quality of medicinal material, each return time extracting is followed successively by 2h, 1.5h and 1h, by after three extracting liquid filterings, merging filtrate is also concentrated into small size and obtains Folium Eucommiae crude extract.
Ethanol extraction Folium Eucommiae is prepared the method for Folium Eucommiae crude extract: after Folium Eucommiae medicinal material drying is pulverized, the alcoholic solution that is 45%～55% by concentration expressed in percentage by volume extracts three times, each alcoholic solution quality used is followed successively by 10 times, 8 times and 6 times of Folium Eucommiae quality of medicinal material, each return time extracting is followed successively by 2h, 1.5h and 1h, to after three extracting liquid filterings, merge, reclaim ethanol, obtain alcohol extract; Alcohol extract is concentrated into small size and obtains Folium Eucommiae crude extract.
Water and ethanol mixed extraction Folium Eucommiae are prepared the method for Folium Eucommiae crude extract: after Folium Eucommiae medicinal material drying is pulverized, the alcoholic solution that is 45%～55% by concentration expressed in percentage by volume extracts three times, each alcoholic solution quality used is followed successively by 10 times, 8 times and 6 times of Folium Eucommiae quality of medicinal material, each return time extracting is followed successively by 2h, 1.5h and 1h, to after three extracting liquid filterings, merge, reclaim ethanol, obtain alcohol extract; Medicinal residues after ethanol extraction add 10 times of water gagings and decoct 2h, obtain water extraction liquid; By being concentrated into small size after water extraction liquid and alcohol extract merging, obtain Folium Eucommiae crude extract.
When water and ethanol mixed extraction Folium Eucommiae, crude extract is carried out to refining method is: after first water extraction liquid being concentrated, by concentration expressed in percentage by volume, be not less than 90% the rear and described alcohol extract merging of ethanol precipitation, then through HPD100 macroporous resin enrichment and polyamide purification, obtain refining Folium Eucommiae extract successively.
Described HPD100 macroporous resin enrichment and polyamide purge process are: after the water extraction liquid after ethanol is precipitated and described alcohol extract merge, adding water adjusting concentration is 0.01g medical material/ml, regulate pH=4 simultaneously, obtain sample solution, upper HPD100 macroporous resin adsorption post, the ethanol elution that is 50～60% through concentration expressed in percentage by volume, ethanol in reclaim under reduced pressure eluent, to without alcohol taste, obtains HPD100 macroporous resin eluent;
It is 0.01g medical material/ml that HPD100 macroporous resin eluent is adjusted to concentration, the ethanol elution that upper 30-60 order polyamide resin column is 40%～50% through concentration expressed in percentage by volume obtains eluent, then after the ethanol in reclaim under reduced pressure eluent, continue decompression and steam aqueous solution, to small size, it is eluting concentrated solution; Eluting concentrated solution, through 65～70 ℃, is dried under negative pressure 0.08Mpa condition, obtains refining Folium Eucommiae extract.
In refining Folium Eucommiae extract, comprise 4 kinds of flavone compounds and chlorogenic acid, 4 kinds of described flavone compounds are: rutin, isoquercitrin, Herba Astragali Melilotoidis (Herba Astragali Sinici) glycosides, Quercetin-3-O-α-L-arabopyranose base-(1 → 2)-β-D-Glucose base glycosides, described refining after in Folium Eucommiae extract the quality percentage composition of 4 kinds of flavone compounds and chlorogenic acid be not less than 50%.
A kind of medicine that promotes union of fracture, it is to add that by the treatment Folium Eucommiae water of effective dose and/or lower alcohol extraction thing pharmaceutically acceptable adjuvant or complementary composition make preparation, and described preparation comprises pill, powder, capsule, tablet, granule, injection or compound preparation.
The application in the medicine of preparation promotion union of fracture of upper Folium Eucommiae water and/or lower alcohol extraction thing.
Beneficial effect of the present invention:
The invention provides Folium Eucommiae extract can significantly increase alkali phosphatase, reduces blood calcium, increases serium inorganic phosphorus, increases bone density, symphysis after obviously promoting to fracture.In addition, study the application of the extract of Folium Eucommiae, can increase the medicinal part of Cortex Eucommiae plant, expand its range of application, and because Folium Eucommiae is compared with Cortex Eucommiae, more easily obtain, can reduce the cost of the Cortex Eucommiae.
The specific embodiment
The preparation of embodiment 1 Folium Eucommiae extract
I. water extraction Folium Eucommiae is prepared Folium Eucommiae crude extract (being Folium Eucommiae aqueous extract)
Folium Eucommiae (collection of medical material plantation, Wuxi City Yixing City Zhang Zhu town) medicinal material drying, shred (shredding granularity 1～3mm2), with water extraction three times, the quality of each institute's water is followed successively by 10 times, 10 times and 9 times of Folium Eucommiae quality of medicinal material, each return time extracting is followed successively by 2h, 1.5h and 1h, by after three extracting liquid filterings, merging filtrate is also concentrated into 2.857g crude drug/ml, obtains Folium Eucommiae crude extract.
II. ethanol extraction Folium Eucommiae is prepared Folium Eucommiae crude extract (being Folium Eucommiae alcohol extract)
Folium Eucommiae (collection of medical material plantation, Wuxi City Yixing City Zhang Zhu town) medicinal material drying, shreds and (shreds granularity 1～3mm
2), by mass, adding the soak with ethanol 2h that 10 times of amount concentration expressed in percentage by volumes are 50%, backflow 2h, filters and obtains extracting solution for the first time; Adding 8 times of amount concentration expressed in percentage by volumes is 50% alcohol reflux 1.5h, filters and obtains extracting solution for the second time; Adding 6 times of amount concentration expressed in percentage by volumes is 50% alcohol reflux 1h, filters and obtains extracting solution for the third time; Merge three times extracting solution, reclaim ethanol, and be concentrated into 2.857g crude drug/ml, obtain Folium Eucommiae crude extract.
III. water and ethanol mixed extraction Folium Eucommiae are prepared Folium Eucommiae crude extract (being Cortex Eucommiae folic alcohol water mixed extract)
Folium Eucommiae (collection of medical material plantation, Wuxi City Yixing City Zhang Zhu town) medicinal material drying, shreds and (shreds granularity 1～3mm
2), by mass, adding 10 times of amount concentration expressed in percentage by volumes is 50% soak with ethanol 2h, backflow 2h, filters and obtains extracting solution for the first time; Adding 8 times of amount concentration expressed in percentage by volumes is 50% alcohol reflux 1.5h, filters and obtains extracting solution for the second time; Adding 6 times of amount concentration expressed in percentage by volumes is 50% alcohol reflux 1h, filters and obtains extracting solution for the third time; Merge three times extracting solution, reclaim ethanol, obtain alcohol extract.Medicinal residues after ethanol extraction add 10 times of water gagings and decoct 2h, obtain water extraction liquid.Merge alcohol extract and water extraction liquid, and be concentrated into 2.857g crude drug/ml, obtain Folium Eucommiae crude extract.
IV. the refining Folium Eucommiae extract of preparation
Folium Eucommiae medical material is after alcohol, water mixed extraction, and water extraction liquid merges with alcohol extract after second precipitation, then can prepare refining Folium Eucommiae extract after HPD100 macroporous resin enrichment and polyamide purifying.
1. medicinal material extract method
Folium Eucommiae (collection of medical material plantation, Wuxi City Yixing City Zhang Zhu town) medicinal material drying, shreds and (shreds granularity 1～3mm
2), adding by mass the soak with ethanol 2h that 10 times of amount concentration expressed in percentage by volumes are 50%, backflow 2h, filters and obtains extracting solution for the first time; Add the alcohol reflux 1.5h that 8 times of amount concentration expressed in percentage by volumes are 50%, filter and obtain extracting solution for the second time; Add the alcohol reflux 1h that 6 times of amount concentration expressed in percentage by volumes are 50%, filter and obtain extracting solution for the third time.Merge three times extracting solution, reclaim ethanol to without alcohol taste, obtain alcohol extract.
Medicinal residues after ethanol extraction add 10 times of water gagings and decoct 2h, obtain extracting solution.Water-bath is concentrated, take water, is settled to the water extraction liquid of concentration as 1g crude drug/ml.
2. the ethanol intermediate processing of water extraction liquid
Getting concentration is the water extraction liquid of 1g crude drug/ml, and adding concentration expressed in percentage by volume is 95% ethanol, makes concentration of alcohol in system reach 70%, and standing 2h filters, and obtains filtrate.The ethanol cyclic washing that precipitate is 50% by concentration expressed in percentage by volume, merges cleaning mixture among filtrate.Reclaim ethanol to without alcohol taste, obtain the water extraction liquid after ethanol precipitation.
3. the preparation method of the total extracting solution of Folium Eucommiae
Water extraction liquid after the alcohol extract of merging Folium Eucommiae and ethanol precipitation, obtains the total extracting solution of Folium Eucommiae.
4.HPD100 macroporous resin enrichment method
HPD100 macroporous resin preprocess method: by appropriate HPD100 macroporous resin, with 95% soak with ethanol 12h of 1.5 times of volumes, remove float, dress post, washes post with 95% ethanol, to effluent add water without muddiness till.Use distilled water flushing instead, be washed till without alcohol taste.
The blade diameter length ratio of pillar: 1: 9.
The preparation of sample solution: get the total extracting solution of Folium Eucommiae, adding water adjusting concentration is 0.01g medical material/ml, adds appropriate concentrated hydrochloric acid simultaneously, makes the pH=4 of system, and this liquid is HPD100 macroporous resin sample solution.
Applied sample amount: 1g medical material/1g dried resin, 1g dried resin=wet resin 4.4ml.Be that 100mlHPD100 macroporous resin sample solution needs 4.4ml wet resin.
Loading flow velocity: 6BV/h.
Remove impurity condition: use the aqueous hydrochloric acid solution remove impurity of the pH=4 of 2BV, rinse with 2BV/h flow velocity.
Ethanol elution condition: the ethanol elution that is 55% by concentration expressed in percentage by volume, speed 2BV/h, elution volume is 6BV.
The recovery of eluent: the ethanol in reclaim under reduced pressure eluent, to without alcohol taste, obtains HPD100 macroporous resin eluent.
Resin regeneration method: resin, can reuse to colourless through 95% alcohol flushing.
4. polyamide purification process
30-60 order polyamide preprocess method: adopt 95% soak with ethanol 12h of 1.5 times of amounts, remove float, dress post, 95% ethanol is washed post, adds water without muddiness to effluent.Use again distilled water flushing instead, to flowing out without ethanol.
The blade diameter length ratio of pillar: blade diameter length ratio is 1: 10.
The preparation of sample solution: get HPD100 macroporous resin eluent, adding water adjusting concentration is 0.01g medical material/ml, is 30-60 order polyamide sample solution.
Applied sample amount: applied sample amount is the dry polyamide of 0.5g medical material/1g, 1g polyamide dried resin=wet resin 4.4ml.Be that 100ml30-60 order polyamide sample solution needs the polyamide that 8.8ml is wet.
Loading flow velocity: loading flow velocity is 2BV/h.
Ethanol elution condition: the ethanol elution that is 45% by concentration expressed in percentage by volume, carries out the consumption 8BV of eluent with the flow velocity of 4BV/h.
The recovery of eluent: after the ethanol in reclaim under reduced pressure eluent, continuing decompression and steam aqueous solution, to small size, is eluting concentrated solution.
Dry: through 65～70 ℃, under negative pressure 0.08Mpa condition, to be dried, to obtain refining Folium Eucommiae extract.
Resin regeneration method: use 2-3%NaOH2BV soaked overnight, aqueous solution is washed till neutral colourless, then with 95% ethanol, be washed till colourlessly, can reuse.
The yield of refining Folium Eucommiae extract: be about 3.5%.
Each component content detection method in the refining Folium Eucommiae extract of embodiment 2
Adopt HPLC method, detect the content of 5 kinds of compositions in Folium Eucommiae extract simultaneously, they are respectively: chlorogenic acid, Quercetin-3-O-α-L-arabopyranose base-(1 → 2)-β-D-Glucose base glycosides (being designated hereinafter simply as: DY-1), rutin, isoquercitrin, Herba Astragali Melilotoidis (Herba Astragali Sinici) glycosides.Use one point external standard method to calculate each component content in Folium Eucommiae extract sample.
1. sample preparation
1.1 mix the preparation of reference substance
Precision takes chlorogenic acid reference substance 4.89mg, and the dissolve with ethanol that concentration expressed in percentage by volume is 50%, is settled to 10ml, and obtaining concentration is the chlorogenic acid reference substance solution of 489 μ g/ml;
Precision takes DY-1 reference substance 6.43mg, and the dissolve with ethanol that concentration expressed in percentage by volume is 50%, is settled to 10ml, and obtaining concentration is the DY-1 reference substance solution of 643 μ g/ml;
Precision takes control substance of Rutin 1.69mg, and the dissolve with ethanol that concentration expressed in percentage by volume is 50%, is settled to 10ml, and obtaining concentration is the control substance of Rutin solution of 169 μ g/ml;
Precision takes isoquercitrin reference substance 3.15mg, and the dissolve with ethanol that concentration expressed in percentage by volume is 50%, is settled to 10ml, and obtaining concentration is the isoquercitrin reference substance solution of 315 μ g/ml;
Precision takes Herba Astragali Melilotoidis (Herba Astragali Sinici) glycosides reference substance 1.23mg, and the dissolve with ethanol that concentration expressed in percentage by volume is 50%, is settled to 10ml, and obtaining concentration is the Herba Astragali Melilotoidis (Herba Astragali Sinici) glycosides reference substance solution of 123 μ g/ml;
Accurate chlorogenic acid reference substance solution 4.5ml, DY-1 reference substance solution 1ml, control substance of Rutin solution 1ml, isoquercitrin reference substance solution 1ml, Herba Astragali Melilotoidis (Herba Astragali Sinici) glycosides reference substance solution 1.5ml, Quercetin reference substance solution 0.3ml, kaempferol reference substance solution 0.2ml to the 10ml volumetric flask drawn respectively, be settled to scale with the ethanol that concentration expressed in percentage by volume is 50%, obtain mixing reference substance solution.Each reference substance concentration is respectively: chlorogenic acid 220.05 μ g/ml, DY-164.3 μ g/ml, rutin 16.9 μ g/ml, isoquercitrin 31.5 μ g/ml, Herba Astragali Melilotoidis (Herba Astragali Sinici) glycosides 18.45 μ g/ml.
The preparation of 1.2 samples
Precision takes refining Folium Eucommiae extract 20.1mg prepared by embodiment 1IV, and the dissolve with ethanol that is 50% by concentration expressed in percentage by volume, is settled to 50ml, and obtaining concentration is the refining Folium Eucommiae extract solution of 0.402mg/ml.
2.HPLC chromatographic condition
Chromatographic column: Hedera C18, mobile phase: solvent orange 2 A (0.1% formic acid acetonitrile, v/v) and solvent B (0.1% formic acid water, v/v) gradient elution, detect wavelength 270nm, sampling volume 20 μ L, column temperature is 30 ℃, flow velocity 1.0ml/min.The variation of mobile phase gradient sees the following form 1.
Table 1 mobile phase gradient
3. computing formula
Testing result is calculated according to formula:
Tie element peak area in tie element concentration × sample composition peak area/mixing reference substance in concentration=mixing reference substance of composition in refining Folium Eucommiae extract sample.
The volume (50ml) of concentration × Folium Eucommiae extract sample of composition in the weight of each composition in refining Folium Eucommiae extract sample=refining Folium Eucommiae extract sample.
The quality (0.0201g) × 100% of each component content sum in refining Folium Eucommiae extract content %=sample/refining Folium Eucommiae extract.
Refining Folium Eucommiae extract component content testing result is in Table 2:
Table 2 is refined Folium Eucommiae extract component content testing result
Each component content sum=55.48% in Folium Eucommiae extract.
Embodiment 3 Folium Eucommiae extracts promote the experimentation that affects of MC3T3-E1 osteoblastic proliferation rate
1. positive drug concentration screening
The selection of 1.1 positive control drugs
Positive drug is selected the clinical commonly used drug JIEGU QILI PIAN (Hunan Jinsa Pharmaceutical Co., Inc.'s production) for the treatment of fracture.
The preparation of 1.2 positive control drug sample solutions
Get JIEGU QILI PIAN, grind, take 0.3g, the ultrasonic 10min of ethanol that 20ml concentration expressed in percentage by volume is 50%, the centrifugal 10min of 3000rmin-1, obtains the extracting solution that concentration is 15mg/ml.The accurate 0.5ml supernatant of drawing, add the ethanol hydrotropy that 75 μ l concentration expressed in percentage by volumes are 95%, add 675 μ l DMEM high glucose mediums, mix, obtain the culture medium solution that concentration is the JIEGU QILI PIAN of 10mg/ml, through 0.25 μ m filtering with microporous membrane, obtaining concentration is the positive control drug solns of 10mg/ml.Draw the positive control drug solns that 100 μ l concentration are 10mg/ml, add 900 μ l DMEM high glucose mediums, obtaining concentration is the positive control drug solns of 1mg/ml.Draw the positive control drug solns that 100 μ l concentration are 1mg/ml, add 900 μ l DMEM high glucose mediums, obtaining concentration is the positive control drug solns of 0.1mg/ml.
2. cell kind plate
Get the osteoblast in culture bottle, use 0.25% trypsinization, with the DMEM culture fluid containing 10%FBS, repeatedly blow and beat, make cell suspension, blood counting chamber counting, adds culture fluid and is diluted to 1 × 10
4the cell concentration of cell/ml, 200 μ l/ holes are inoculated in 96 orifice plates, every hole inoculation 2 × 10
3cell.Put 37 ℃, 5%CO
2incubator is cultivated 24h.
3. the impact of on cell proliferation rate
Formula one: final volume=dosing volume+culture fluid volume=22 μ l+200 μ l=222 μ l
Formula two: ultimate density=liquor strength × dosing volume/final volume
Note: due to the volatilization of culture fluid in incubator, ultimate density may increase to some extent, eliminates concentration change by operation repetitive.
3. the impact of the Folium Eucommiae extract that prepared by distinct methods on osteoblastic proliferation rate
The preparation of 3.1 medicinal liquids
Refining Folium Eucommiae extract prepared by embodiment 1 IV is used 100 μ l 95% ethanol and DMEM high glucose medium 900 μ l to dissolve.Obtaining concentration is 1 × 10
-1the sample solution of mg/ml.Sample solution is through 0.22 μ m filtration sterilization.
3.2 cell fishplate bars
Get the osteoblast in culture bottle, with after 0.25% trypsinization, add the DMEM culture fluid containing 10%FBS of certain volume, make cell suspension, blood counting chamber counting, adds culture fluid and is diluted to 2 × 10
4the cell concentration of cell/ml, 200 μ l/ holes are inoculated in 96 orifice plates, every hole inoculation 4 × 10
3cell.Put 37 ℃, 5%CO
2in incubator, cultivate after 24h cell attachment, start dosing.
The various extracting solution of table 3 Folium Eucommiae, the impact of the Folium Eucommiae extract of different purity on osteoblastic proliferation rate
Note: the percentage composition summation that refining Folium Eucommiae extract (purity 55.48%) is 5 kinds of compositions in Folium Eucommiae extract is 55.48%.
Folium Eucommiae extract (purity 25%), for after alcohol, water mixed extraction, precipitates through ethanol, then the Folium Eucommiae extract preparing after polyamide purifying.This Folium Eucommiae extract is in preparation process, except identical with the method for embodiment 1IV without additive method HPD100 macroporous resin enrichment.In extract, the percentage composition detection method of each composition is identical with embodiment 2, and testing result shows that the percentage composition summation of 5 kinds of compositions in Folium Eucommiae extract is 25%.
Conclusion: the various extracting solution of Folium Eucommiae, the Folium Eucommiae extract of different purity all have the significance activity that promotes osteoblastic proliferation.But the Folium Eucommiae extract effect that purity is 56% is the most remarkable.
Embodiment 4 Folium Eucommiae extract antiinflammatories, the hematoma that disappears effect experiment
1 experiment purpose:
Whether investigation Folium Eucommiae extract has antiinflammatory, the hematoma that disappears effect.
2 experiment materials
2.1 experiment reagents
Refining Folium Eucommiae extract prepared by embodiment 1 IV, dimethylbenzene (AR, Nanjing Chemistry Reagent Co., Ltd.); Glacial acetic acid (AR, Nanjing chemical reagent one factory); Benzylpenicillin sodium for injection (Lukang Medical Co., Ltd., Shandong, lot number B110931); Anhydrous calcium chloride (AR, Guangdong Xilong Chemical Co., Ltd); Aspirin Enteric-coated Tablets (Baijingyu Pharmaceutical Co., Ltd., Nanjing, lot number 110503); Sodium carboxymethyl cellulose (AR, Ke Miou chemical reagent development centre, Tianjin)
2.2 laboratory animal
Kunming mouse, male 80, body weight 18-22g.
2.3 experimental apparatus and equipment
Analytical balance (BSA124S type, sartorius company), card punch, 50 μ l pipettors, 1ml syringe.
3 experimental techniques
3.1 grouping and dosages
3.1.1 model group
0.4%CMC-Na solution, 20ml/kg.
3.1.2 positive controls
Aspirin, mice dosage is 0.27g/kg, mice administration volume is 20ml/kg, the 0.4%CMC-Na solution that this solution concentration is 13.5mg/ml.
3.1.3 Folium Eucommiae extract high dose group
Refining Folium Eucommiae extract dosage prepared by embodiment 1 IV is 1g/kg, and mice administration volume is 20ml/kg, the 0.4%CMC-Na solution that this solution concentration is 0.05g/ml.
3.1.4 Folium Eucommiae extract low dose group
Refining Folium Eucommiae extract dosage prepared by embodiment 1 IV is 0.25g/kg, and mice administration volume is 20ml/kg, the 0.4%CMC-Na solution that this solution concentration is 0.0125g/ml.
The preparation of 3.2 sample liquid
3.2.1 the preparation of positive drug solution
In Aspirin Enteric-coated Tablets, the content of aspirin is 25mg/ sheet, gets 54 (containing aspirin 1350mg), grinds as fine powder, adds 0.4%CMC-Na solution and grinds, and is settled to 100ml, shakes up, and obtains 13.5mg/ml aspirin suspension.
3.2.2 the preparation of the each dosage solution of Folium Eucommiae extract
Take respectively refining Folium Eucommiae extract 1.25g, 5g prepared by embodiment 1IV, add 0.4%CMC-Na solution and grind, be settled to 100ml, shake up, obtain that Folium Eucommiae extract is low, high dose suspension.
3.3 administrations, modeling method and detection index
3.3.1 the antiinflammatory of mice auricle swelling model experiment
Get body weight 18-22g, 40 of male mices.Being divided at random 4 groups, being respectively model group, positive controls, Folium Eucommiae extract low dose group, Folium Eucommiae extract high dose group, is 4 groups altogether.Gastric infusion, after 4 days, is coated with 20 μ l dimethylbenzene at the auris dextra of every mice after last administration 1h continuously, after 1h, puts to death.The card punch that is 8mm with diameter, at the same position of mice left and right ear, is laid circular auricle, on electronic balance, weighs, and records experimental result, calculates swelling rate.
Swelling rate (%)=(auris dextra weight-left ear weight)/left ear heavy × 100%.
2.3.2 disappear hematoma experiment of mice
Get body weight and be 40 of 18～22g mices, male, be divided at random 4 groups, be respectively model group, positive controls, Folium Eucommiae extract low dose group, Folium Eucommiae extract high dose group, be 4 groups altogether.Before experiment, first cause self hematoma model: with glass capillary in the mice eyeground vein clump 0.05ml that quantitatively takes a blood sample, add the 0.1mol/LCaCl containing penicillin 100,000 units
2solution 0.1ml mixes, and injects immediately mice self nape portion subcutaneous, causes hematoma model.Cause after model successive administration 4 days, mice dislocation of cervical vertebra was put to death in the 5th day, residual hematoma piece subcutaneous mice nape place is separated, with scales/electronic balance weighing, by the residual hematoma piece weight of more different groups, judge drug effect.
4 experimental results
4.1 mice auricle swelling experiments
Mice auricle swelling degree experimental result t inspection statistics through between group is analyzed, show that the each dosage group of Folium Eucommiae extract and model group relatively have utmost point significant difference (P < 0.01), show that the each dosage group of Folium Eucommiae extract all has good antiinflammatory action.Mice auricle swelling experimental result is in Table 4.
Table 4 mice auricle swelling experimental result
Note: with model group comparison,
*p < 0.01,
* *p < 0.001.
The disappear hematoma experiment of 4.2 mices
The mice hematoma experimental result t inspection statistics analysis through between group that disappears, Folium Eucommiae extract low dose group, high dose group and model group relatively have utmost point significant difference (P < 0.01), show that Folium Eucommiae extract has the hematoma effect that well disappears.Mice disappears hematoma experimental result in Table 5.
The table 5 mice hematoma experimental result that disappears
Note: with model group comparison,
*p < 0.05,
*p < 0.01.
4.3 conclusions:
The demonstration of this experimental result, the each dosage group of Folium Eucommiae extract all has obvious antiinflammatory and the hematoma effect that disappears.
The impact of embodiment 5 Folium Eucommiae extracts on rat tibia fracture
Observe the impact of Folium Eucommiae extract on rat tibia fracture.Get 40 SD rats, male and female half and half, by literature method, preparation rat tibia fracture model.Be divided at random 5 groups: Folium Eucommiae extract high dose group (360mg/kg), Folium Eucommiae extract low dose group (90mg/kg), GUZHE CUOSHANG JIAONANG group (470mg/kg), model group and sham operated rats (0.5%CMC-Na).Every group 8, gastric infusion, administration volume 1mL/100g body weight, continuous 30 days.By serum alkaline phosphatase, blood calcium and serium inorganic phosphorus, tibia X sheet, the biomechanics experiment of bone density and skeleton is observed the curative effect of each medicine.Result shows, each group and model group comparison, when 20 days and 30 days, the serum alkaline phosphatase of Folium Eucommiae extract high dose group, Folium Eucommiae extract low dose group, blood calcium, serium inorganic phosphorus raise, bone density significantly rises compared with model group, with normal group there was no significant difference, X sheet shows with model group significant difference.But the demonstration of the biomechanical analysis of each group, respectively organizing fracture resistence force experiment all has significant difference with model group.Result of the test explanation: in dosage range used, Folium Eucommiae extract has the significance effect that promotes union of fracture.
1. pharmaceutical agent compound method
Folium Eucommiae extract high dose preparation: get refining Folium Eucommiae extract prepared by 3.6g embodiment 1IV and be dissolved in 100mL0.5%CMC-Na solution, Folium Eucommiae extract high dose concentration is 36mg/mL.
Folium Eucommiae extract low dosage preparation: get refining Folium Eucommiae extract high dose solution 0.5%CMCNa solution standardize solution prepared by 20mL embodiment 1IV to 80mL, concentration is 9mg/mL.
2. experimental technique
The foundation of 2.1 fracture models
Get SD rat, male and female half and half, totally 80, body weight 180-220g.Adaptability is fed one week, standard Mus grain, and drinking-water is raised.Be divided into 5 groups, be respectively sham operated rats, model group, positive controls, Folium Eucommiae extract high dose group and low dose group.The processing of sham operated rats animal: use 3% chloral hydrate, intraperitoneal injection of anesthesia, anaesthesia dosage is: 10ml/kg.Dorsal position is fixed, and wound circumference sterilization, with sterile operating theater instruments incision skin, then layer-by-layer suture.After stitching, do not fix, the gentamycin of intramuscular injection 1mL 40,000 units, and in wound, sprinkle the anti-infection of levofloxacin.The next day operation group every intramuscular injection gentamycin 1mL, continuous two days.The animal of model group, positive controls, Folium Eucommiae extract high dose group and low dose group all needs modeling, the preparation method of tibia standard fracture model: use 3% chloral hydrate, and intraperitoneal injection of anesthesia, anaesthesia dosage is: 10ml/kg.Dorsal position is fixed, and wound circumference sterilization is cut skin by sterile operating theater instruments, finds tibia, at lower 1/3 place, with hacksaw, cut 3mm long, be equivalent to 2/3 of tibia diameter and cut, leave side seam cortex and fibula and play fixed supporting role, irrigate with 3% hydrogen peroxide, layer-by-layer suture.After stitching, do not fix, the gentamycin of intramuscular injection 1mL 40,000 units, and in wound, sprinkle the anti-infection of levofloxacin.The next day operation group every intramuscular injection gentamycin 1mL, continuous two days.
2.2 dosages and method
Each group laboratory animal starts gastric infusion for the 3rd day after operation.Administration volume 1mL/100g body weight.Once a day, continuous 30 days.Claim weekly body weight one time, according to body weight, adjust dosage.Dosage is: positive controls gives GUZHE CUOSHANG JIAONANG 470mg/kg, and Folium Eucommiae extract high dose group gives 360mg/kg, Folium Eucommiae extract low dose group gives 90mg/kg, model group and sham operated rats and gives isopyknic 0.5%CMC-Na aqueous solution.After administration the 20th day and the 30th day, gets blood from laboratory animal eye socket respectively, measures serum alkaline phosphatase, blood calcium and serium inorganic phosphorus.And put to death after animal in 30 days, take off rat tibia, measure the fracture resistence force of tibia, bone density, X sheet detects symphysis degree, by relatively drawing the impact of each group of medicine on fracture healing in rats with model group.
3. experimental technique and result
The measurement result of 3.1 serum alkaline phosphatases
In 20 days and 30 days, eye socket is got the about 600uL of blood left and right respectively, is placed in clean 1mL centrifuge tube, and 3500rpm frozen centrifugation 10min, draws upper serum, and-20 ℃ of storages, wait until serum alkaline phosphatase, the detection of blood calcium, serium inorganic phosphorus.Testing result is in Table 6.
Result shows: with model group comparison, when 20 days and 30 days, Folium Eucommiae extract low dosage (90mg/kg) group and high dose (360mg/kg) group have significant difference (p < 0.05, p < 0.01), can obviously increase serum alkaline phosphatase.
Table 620 day and 30 days each group serum alkaline phosphatase (ALP) comparison (
unit: IU/L)
Note: * and model group comparison, * P < 0.05, * * P < 0.01.
The measurement result of 3.2 serum blood calcium
With model group comparison, when 20 days and 30 days, Folium Eucommiae extract low dosage (90mg/kg) group and high dose (360mg/kg) group have significant difference (p < 0.05), can significantly reduce blood calcium.Testing result is in Table 7.
The comparison of table 720 day and 30 days each group blood calcium (
unit: mmol/L)
Note: * and model group comparison, * P < 0.05, * * P < 0.01.
The measurement result of 3.3 serum serium inorganic phosphorus
With model group comparison, in the time of 20 days, Folium Eucommiae extract low dosage (90mg/kg) group and high dose (360mg/kg) group have significant difference (p < 0.05), can significantly increase serium inorganic phosphorus; In the time of 30 days, Folium Eucommiae extract low dosage (90mg/kg) group and high dose (360mg/kg) group have significant difference (p < 0.05), can significantly increase serium inorganic phosphorus.Testing result is in Table 8.
The comparison of table 820 day and 30 days each group serium inorganic phosphorus (
unit: mmol/L)
Note: * and model group comparison, * P < 0.05, * * P < 0.01.
3.4 tibia X sheets detect
In administration the 30th day, put to death animal after getting blood, take off rat tibia, at 50KV, 50mA, takes X-ray film under 0.125S condition, by the variation of X-ray film, by following standard, mark: 1. dislocation: 1 point of damaged tibia dislocation-free (comprising dislocation < 0.1cm); Have dislocation (comprising that fracture site dislocation does not exceed the slight angulation of 1/2 or fracture end of its broken ends of fractured bone) 0 point.2. fracture seam: damaged tibial bone crease part is fuzzy or disappear 1 point; There is 0 point of fracture seam.3. connect: the damaged fracture of tibia broken ends of fractured bone is connected to 1 point of an entirety; Do not connect 0 point.4. fibula: connect 1 point after fibula does not rupture or ruptures; 0 point of fibula fracture.5. healing: damaged tibia (the remaining unchanged without interior external callus or fracture seam) 0 point of not healing; 1 point of union of fracture (have inside and outside slight callus to change, fracture seam shoals or be fuzzy simultaneously).Testing result is in Table 9.
Result demonstration, positive group fracture line disappears substantially, and fracture end has a large amount of connectivity callus shades, and some visible pulp cavity is logical again, model group fracture end edge blurry, a small amount of growth of spur.With model group comparison, Folium Eucommiae extract low dosage (90mg/kg) group has significant difference (p < 0.05), high dose (360mg/kg) has utmost point significant difference (p < 0.01), fracture line is tending towards disappearing, and callus generates and increases.
Table 9 is fractured and within 30 days, is respectively organized the impact of Chinese medicine on X sheet score value
Note: * and model group comparison, * P < 0.05, * * P < 0.01.
3.5 Bone mineral density
Get the rat tibia of having clapped after X sheet, reject surperficial soft tissue, detect after healing bone biomechanical, measure respectively the bone density of each group of healing bone.With model group comparison, Folium Eucommiae extract low dosage (90mg/kg) group and high dose (360mg/kg) group have significant difference (p < 0.05), can significantly increase bone density.
Table 10 fracture within 30 days, respectively organize afterwards bone density comparison (n=4,
)
Note: * and model group comparison, * P < 0.05, * * P < 0.01.
The biomechanics of 3.6 rat tibia detects
Get the rat tibia of having clapped after X sheet, reject surperficial soft tissue, by healing skeleton two slidably, on parallel, the support of height in same level, keep two support distances suitably, centered by fracture, hang a cord, underneath one pallet, to the counterweight of putting known weight in pallet, until the bone of this healing fractures, calculate the gross weight of hung weight as the index of tibia fracture resistence force.With model group comparison, Folium Eucommiae extract low dosage (90mg/kg) group and high dose (360mg/kg) group have significant difference (p < 0.05), can significantly increase the fracture resistence force of bone.Testing result is in Table 11.
Table 11 is fractured and within 30 days, is respectively organized afterwards the comparison of healing bone fracture resistence force
Note: * and model group comparison, * P < 0.05, * * P < 0.01.
4. conclusion (of pressure testing)
By with model group comparison, when 20 days and 30 days, Folium Eucommiae extract high dose group, Folium Eucommiae extract low dose group can significantly increase alkali phosphatase, reduce blood calcium, increase serium inorganic phosphorus, increase bone density, X sheet also shows that healing result and the model group of this medication group have significant difference, obviously promotes the rear symphysis of fracture.
Embodiment 6 prepares granule
Take refining Folium Eucommiae extract prepared by embodiment 1IV, add the adjuvant dextrin of 2 times of quality of extract, mix homogeneously, crosses 65 mesh sieves, with appropriate 95% ethanol be binding agent, make moist wood, through 20 mesh sieves extruding, granulate, after 65 ℃ are dried, obtain granule.
Embodiment 5 prepares tablet
Take refining Folium Eucommiae extract prepared by embodiment 1IV, add appropriate supplementary product starch and magnesium stearate, mix, beat sheet, obtain tablet.
Claims (4)
1. a Folium Eucommiae water and/or lower alcohol extraction thing, Folium Eucommiae water described in it is characterized in that and/or lower alcohol extraction thing are adopted preparation with the following method: after Folium Eucommiae medicinal material drying is pulverized, the alcoholic solution that is 45% ~ 55% by concentration expressed in percentage by volume extracts three times, each alcoholic solution quality used is followed successively by 10 times, 8 times and 6 times of Folium Eucommiae quality of medicinal material, each return time extracting is followed successively by 2h, 1.5 h and 1h, to after three extracting liquid filterings, merge, reclaim ethanol, obtain alcohol extract; Medicinal residues after ethanol extraction add 10 times of water gagings and decoct 2h, obtain water extraction liquid;
After first water extraction liquid being concentrated, by concentration expressed in percentage by volume, be not less than 90% the rear and described alcohol extract merging of ethanol precipitation, then through HPD100 macroporous resin enrichment and polyamide purification, obtain refining Folium Eucommiae extract successively;
Described HPD100 macroporous resin enrichment and polyamide purge process are: after the water extraction liquid after ethanol is precipitated and described alcohol extract merge, adding water adjusting concentration is 0.01 g medical material/ml, regulate pH=4 simultaneously, obtain sample solution, upper HPD100 macroporous resin adsorption post, the ethanol elution that is 50 ~ 60% through concentration expressed in percentage by volume, ethanol in reclaim under reduced pressure eluent, to without alcohol taste, obtains HPD100 macroporous resin eluent;
It is 0.01 g medical material/ml that HPD100 macroporous resin eluent is adjusted to concentration, the ethanol elution that upper 30-60 order polyamide resin column is 40% ~ 50% through concentration expressed in percentage by volume obtains eluent, then after the ethanol in reclaim under reduced pressure eluent, continue decompression and steam aqueous solution, to small size, it is eluting concentrated solution; Eluting concentrated solution, through 65 ~ 70 ℃, is dried under negative pressure 0.08MPa condition, obtains refining Folium Eucommiae extract.
2. Folium Eucommiae water according to claim 1 and/or lower alcohol extraction thing, it is characterized in that comprising 4 kinds of flavone compounds and chlorogenic acid in described refining rear Folium Eucommiae extract, 4 kinds of described flavone compounds are: rutin, isoquercitrin, Herba Astragali Melilotoidis (Herba Astragali Sinici) glycosides, Quercetin-3-O-α-L-arabopyranose base-(1 → 2)-β-D-Glucose base glycosides, described refining after in Folium Eucommiae extract the quality percentage composition of 4 kinds of flavone compounds and chlorogenic acid be not less than 50%.
3. one kind promotes the medicine of union of fracture, it is characterized in that adding that by the Folium Eucommiae water described in any one and/or lower alcohol extraction thing in the claim 1 ~ 2 for the treatment of effective dose pharmaceutically acceptable adjuvant makes preparation, described preparation is selected from pill, powder, capsule, tablet, granule, injection.
4. any one Folium Eucommiae water described in claim 1 ~ 2 and/or the lower alcohol extraction thing application in the medicine of preparation promotion union of fracture.
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| CN103570662A (en) * | 2013-11-19 | 2014-02-12 | 东南大学 | Preparation method of high-purity rutin |
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