CN102670696A - 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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- CN102670696A CN102670696A CN2012101341930A CN201210134193A CN102670696A CN 102670696 A CN102670696 A CN 102670696A CN 2012101341930 A CN2012101341930 A CN 2012101341930A CN 201210134193 A CN201210134193 A CN 201210134193A CN 102670696 A CN102670696 A CN 102670696A
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- Prior art keywords
- folium eucommiae
- extract
- water
- ulmoids
- eucommia
- Prior art date
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Landscapes
- Medicines Containing Plant Substances (AREA)
- Coloring Foods And Improving Nutritive Qualities (AREA)
- Pharmaceuticals Containing Other Organic And Inorganic Compounds (AREA)
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.
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.Shennong's Herbal is classified as top grade.Call its " cure mainly waist and knee, invigorating middle warmer, beneficial vital essence, hard muscles and bones, remove itch under cloudy wet, dribbling urination.Obey for a long time, it is anti-old to make light of one's life by commiting suicide." Cortex Eucommiae is Chinese peculiar medical material, its medicinal history is long, has a wide range of applications clinical.Find nearly 14 kinds of Cortex Eucommiae platymisciums so far on earth, they became extinct in continent and Europe in succession afterwards.The Cortex Eucommiae that is present in China 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, the expert has carried out careful repeatedly research to Cortex Eucommiae composition both at home and abroad, finds to contain necessary for human body threonine, 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 the body is removed in bone and muscle strengthening, strengthens the human body cell substance metabolism; Prevent that muscle skeleton is aging, the balance human blood-pressure, the decomposer inner cholesterol reduces body fat; Recover blood vessel elasticity, diuresis heat clearing away, broad-spectrum antiseptic; Stimulating central nervous system improves white blood cell count, remarkable efficacy such as enhancing human immune.
Existing Cortex Eucommiae medicinal part is a Cortex Eucommiae, and the acquisition method of Cortex Eucommiae is: will cultivate the Cortex Eucommiae more than 10 years, and process after stripping bark with the semi-ring strip.Because gutta-percha tree quantity is rare, and for the quality that guarantees medical material needs the Cortex Eucommiae to grow to 10 years or the above age of tree could be gathered Cortex Eucommiae, the while is owing to the medical value of the Cortex Eucommiae is huge, so cause it to cost an arm and a leg.
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 purpose of the present invention is to provide Folium Eucommiae water and/or the application of lower alcohol extraction thing in the anti-fracture of preparation medicine.
The object of the invention can be realized through following technical scheme:
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; The refining Folium Eucommiae extract that obtains after the perhaps crude extract that obtains of water and lower alcohol mixed extraction, and said crude extract is refining.
The water extraction Folium Eucommiae prepares the method for Folium Eucommiae crude extract: after the Folium Eucommiae medicinal material drying is pulverized; With water extraction three times; The quality of each time institute water is followed successively by 10 times, 10 times and 9 times of Folium Eucommiae quality of medicinal material; Each return time that extracts is followed successively by 2h, 1.5h and 1h, and with behind three extracting liquid filterings, merging filtrate also is concentrated into small size and obtains the Folium Eucommiae crude extract.
The ethanol extraction Folium Eucommiae prepares the method for Folium Eucommiae crude extract: after the Folium Eucommiae medicinal material drying is pulverized; Using concentration expressed in percentage by volume is that 45%～55% alcoholic solution extracts three times; The used alcoholic solution quality of each time is followed successively by 10 times, 8 times and 6 times of Folium Eucommiae quality of medicinal material, and each return time that extracts is followed successively by 2h, 1.5h and 1h, with merging behind three extracting liquid filterings; Reclaim ethanol, get alcohol extract; Alcohol extract is concentrated into small size obtains the Folium Eucommiae crude extract.
Water and ethanol mixed extraction Folium Eucommiae prepare the method for Folium Eucommiae crude extract: after the Folium Eucommiae medicinal material drying is pulverized; Using concentration expressed in percentage by volume is that 45%～55% alcoholic solution extracts three times; The used alcoholic solution quality of each time is followed successively by 10 times, 8 times and 6 times of Folium Eucommiae quality of medicinal material, and each return time that extracts is followed successively by 2h, 1.5h and 1h, with merging behind three extracting liquid filterings; Reclaim ethanol, get alcohol extract; Medicinal residues behind the ethanol extraction add 10 times of water gagings and decoct 2h, get the water extract; Obtain the Folium Eucommiae crude extract with being concentrated into small size after water extract and the alcohol extract merging.
When water and ethanol mixed extraction Folium Eucommiae; Crude extract is carried out purified method is: the water extract is concentrated merge with described alcohol extract the back is not less than 90% ethanol precipitation with concentration expressed in percentage by volume after earlier, obtain making with extra care Folium Eucommiae extract through HPD100 macroporous resin enrichment and polyamide purification successively then.
Described HPD100 macroporous resin enrichment and polyamide purge process are: with adding water adjusting concentration after water extract behind the ethanol precipitation and the merging of described alcohol extract is 0.01g medical material/ml; Regulate pH=4 simultaneously; Obtain sample solution, last HPD100 macroporous resin adsorption post is 50～60% ethanol elution through concentration expressed in percentage by volume; Ethanol in the reclaim under reduced pressure eluent gets HPD100 macroporous resin eluent to there not being the alcohol flavor;
It is 0.01g medical material/ml that HPD100 macroporous resin eluent is adjusted to concentration; Last 30-60 order polyamide resin column is that 40%～50% ethanol elution gets eluent through concentration expressed in percentage by volume; Then behind the ethanol in the reclaim under reduced pressure eluent; Continue decompression and steam aqueous solution,, be the eluting concentrated solution to small size; The eluting concentrated solution through 65～70 ℃, is carried out drying under negative pressure 0.08Mpa condition, must make with extra care Folium Eucommiae extract.
Comprise 4 kinds of flavone compounds and chlorogenic acid in the purified Folium Eucommiae extract; Described 4 kinds of flavone compounds are: rutin, isoquercitrin, Herba Astragali Melilotoidis (Herba Astragali Sinici) glycosides, Quercetin-3-O-α-L-arabopyranose base-(1 → 2)-β-D-glucosyl group glycosides, the quality percentage composition of 4 kinds of flavone compounds and chlorogenic acid is not less than 50% in the Folium Eucommiae extract of described refining back.
A kind of medicine that promotes union of fracture; It is that Folium Eucommiae water and/or lower alcohol extraction thing by the treatment effective dose adds that acceptable accessories or complementary composition process 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 last Folium Eucommiae water and/or lower alcohol extraction thing.
Beneficial effect of the present invention:
The present invention provides Folium Eucommiae extract can significantly increase alkali phosphatase, reduces blood calcium, increases serium inorganic phosphorus, bone density improving, and the back symphysis can obviously promote to fracture.In addition, study the application of the extract of Folium Eucommiae, can increase the medicinal part of Cortex Eucommiae plant, enlarge its range of application, and, more be prone to obtain, can reduce the cost of the Cortex Eucommiae because Folium Eucommiae is compared with Cortex Eucommiae.
The specific embodiment
The preparation of embodiment 1 Folium Eucommiae extract
I. the water extraction Folium Eucommiae prepares Folium Eucommiae crude extract (being the Folium Eucommiae aqueous extract)
Folium Eucommiae (collection of medical material plantation, Wuxi City Yixing City Zhang Zhu town) medicinal material drying; Shred that (shred granularity 1～3mm2), with water extraction three times, the quality of each time institute water is followed successively by 10 times, 10 times and 9 times of Folium Eucommiae quality of medicinal material; Each return time that extracts is followed successively by 2h, 1.5h and 1h; With behind three extracting liquid filterings, merging filtrate also is concentrated into 2.857g crude drug/ml, obtains the Folium Eucommiae crude extract.
II. the ethanol extraction Folium Eucommiae prepares Folium Eucommiae crude extract (being the 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 quality, 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 to obtain 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 the Folium Eucommiae crude extract.
III. water and ethanol mixed extraction Folium Eucommiae prepare 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 quality, 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 to obtain 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, get alcohol extract.Medicinal residues behind the ethanol extraction add 10 times of water gagings and decoct 2h, get the water extract.Merge alcohol extract and water extract, and be concentrated into 2.857g crude drug/ml, obtain the Folium Eucommiae crude extract.
IV. Folium Eucommiae extract is made with extra care in preparation
The Folium Eucommiae medical material is after alcohol, water mixed extraction, and the water extract merges through second post precipitation and alcohol extract, after HPD100 macroporous resin enrichment and polyamide purifying, can prepare refining Folium Eucommiae extract again.
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 10 times of amount concentration expressed in percentage by volumes by quality 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 to obtain 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, recovery ethanol gets alcohol extract to there not being the alcohol flavor.
Medicinal residues behind the ethanol extraction add 10 times of water gagings and decoct 2h, get extracting solution.Water-bath concentrates, and is settled to the water extract that concentration is 1g crude drug/ml with water.
2. the ethanol precipitation method of water extract
Getting concentration is the water extract of 1g crude drug/ml, adds concentration expressed in percentage by volume and be 95% ethanol liquid, makes that concentration of alcohol reaches 70% in the system, leaves standstill 2h, filters, and must filtrate.Precipitate use concentration expressed in percentage by volume is 50% ethanol liquid cyclic washing, merges cleaning mixture among filtrating.Reclaim ethanol to there not being the alcohol flavor, the water extract behind the ethanol precipitation.
3. the method for preparing of the total extracting solution of Folium Eucommiae
Merge the alcohol extract of Folium Eucommiae and the water extract behind the ethanol precipitation, get the total extracting solution of Folium Eucommiae.
4.HPD100 macroporous resin enrichment method
HPD100 macroporous resin preprocess method: the HPD100 macroporous resin is an amount of, and 95% soak with ethanol 12h with 1.5 times of volumes removes float, and the dress post is washed post with 95% ethanol, till adding water and do not have a muddiness to effluent.Use distilled water flushing instead, being washed till does not have the alcohol flavor.
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 an amount of 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 the 4.4ml wet resin.
Last appearance flow velocity: 6BV/h.
Remove impurity condition: use the aqueous hydrochloric acid solution remove impurity of the pH=4 of 2BV, wash with the 2BV/h flow velocity.
The ethanol elution condition: the use concentration expressed in percentage by volume is 55% ethanol elution, and speed 2BV/h, elution volume are 6BV.
The recovery of eluent: the ethanol in the reclaim under reduced pressure eluent gets HPD100 macroporous resin eluent to there not being the alcohol flavor.
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, the dress post, 95% ethanol is washed post, and adding water to effluent does not have muddiness.Use distilled water flushing again instead, do not flow out to there being 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 dried 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 wet polyamide of 8.8ml.
Last appearance flow velocity: going up the appearance flow velocity is 2BV/h.
The ethanol elution condition: using concentration expressed in percentage by volume is 45% ethanol elution, carries out the consumption 8BV of eluent with the flow velocity of 4BV/h.
The recovery of eluent: behind the ethanol in the reclaim under reduced pressure eluent, continue decompression and steam aqueous solution,, be the eluting concentrated solution to small size.
Dry: as through 65～70 ℃, under negative pressure 0.08Mpa condition, to carry out drying, must make with extra care Folium Eucommiae extract.
Resin regeneration method: use the 2-3%NaOH2BV soaked overnight, aqueous solution is washed till neutral colourless, and reuse 95% ethanol is washed till colourless, can reuse.
The yield of refining Folium Eucommiae extract: be about 3.5%.
Each component content detection method in the embodiment 2 refining Folium Eucommiae extracts
Adopt the HPLC method, detect the content of 5 kinds of compositions in the Folium Eucommiae extract simultaneously, they are respectively: chlorogenic acid; Quercetin-3-O-α-L-arabopyranose base-(1 → 2)-β-D-glucosyl group glycosides (is 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 the Folium Eucommiae extract sample.
1. sample preparation
1.1 mix the preparation of reference substance
Precision takes by weighing chlorogenic acid reference substance 4.89mg, and concentration expressed in percentage by volume is 50% dissolve with ethanol, is settled to 10ml, and obtaining concentration is the chlorogenic acid reference substance solution of 489 μ g/ml;
Precision takes by weighing DY-1 reference substance 6.43mg, and concentration expressed in percentage by volume is 50% dissolve with ethanol, is settled to 10ml, and obtaining concentration is the DY-1 reference substance solution of 643 μ g/ml;
Precision takes by weighing control substance of Rutin 1.69mg, and concentration expressed in percentage by volume is 50% dissolve with ethanol, is settled to 10ml, and obtaining concentration is the control substance of Rutin solution of 169 μ g/ml;
Precision takes by weighing isoquercitrin reference substance 3.15mg, and concentration expressed in percentage by volume is 50% dissolve with ethanol, is settled to 10ml, and obtaining concentration is the isoquercitrin reference substance solution of 315 μ g/ml;
Precision takes by weighing Herba Astragali Melilotoidis (Herba Astragali Sinici) glycosides reference substance 1.23mg, and concentration expressed in percentage by volume is 50% dissolve with ethanol, 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 respectively 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; Using concentration expressed in percentage by volume is that 50% ethanol is settled to scale, promptly gets and mixes 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.
1.2 the preparation of sample
Precision takes by weighing the refining Folium Eucommiae extract 20.1mg of embodiment 1IV preparation, and the use concentration expressed in percentage by volume is 50% dissolve with ethanol, 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) with 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 the concentration=mixing reference substance of composition in the refining Folium Eucommiae extract sample.
The volume (50ml) of the concentration * Folium Eucommiae extract sample of composition in the weight of each composition in the refining Folium Eucommiae extract sample=refining Folium Eucommiae extract sample.
The quality (0.0201g) * 100% of each component content sum in the refining Folium Eucommiae extract content %=sample/refining Folium Eucommiae extract.
Refining Folium Eucommiae extract component content testing result is seen table 2:
Table 2 is made with extra care Folium Eucommiae extract component content testing result
Each component content sum=55.48% in the Folium Eucommiae extract.
Embodiment 3 Folium Eucommiae extracts promote the experimentation that influences of MC3T3-E1 osteoblastic proliferation rate
1. positive drug concentration screening
1.1 the selection of positive control drug
Positive drug is selected the clinical commonly used drug JIEGU QILI PIAN (Hunan Jinsa Pharmaceutical Co., Inc.'s production) of treatment fracture for use.
1.2 the preparation of positive control drug sample solution
Get JIEGU QILI PIAN, grind, take by weighing 0.3g, the 20ml concentration expressed in percentage by volume is 50% the ultrasonic 10min of ethanol, and the centrifugal 10min of 3000rmin-1 obtains the extracting solution that concentration is 15mg/ml.The accurate 0.5ml supernatant of drawing; Add 75 μ l concentration expressed in percentage by volumes and be 95% ethanol hydrotropy; Add 675 μ l DMEM high glucose mediums, mixing obtains the culture medium solution that concentration is the JIEGU QILI PIAN of 10mg/ml; Through 0.25 μ m filtering with microporous membrane, promptly getting concentration is the positive control drug solns of 10mg/ml.Drawing 100 μ l concentration is the positive control drug solns of 10mg/ml, adds 900 μ l DMEM high glucose mediums, and promptly obtaining concentration is the positive control drug solns of 1mg/ml.Drawing 100 μ l concentration is the positive control drug solns of 1mg/ml, adds 900 μ l DMEM high glucose mediums, and promptly obtaining concentration is the positive control drug solns of 0.1mg/ml.
2. cell kind plate
Get the osteoblast in the culture bottle, use 0.25% trypsinization, blow and beat repeatedly with the DMEM culture fluid that contains 10%FBS, process cell suspension, the 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 influence 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
Annotate: because the volatilization of culture fluid in incubator, ultimate density may increase to some extent, eliminates concentration change through operation repetitive.
3. the Folium Eucommiae extract of distinct methods preparation is to the influence of osteoblastic proliferation rate
3.1 the preparation of medicinal liquid
The refining Folium Eucommiae extract of embodiment 1 IV preparation uses 100 μ l, 95% ethanol and DMEM high glucose medium 900 μ l dissolving.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 bar
Get the osteoblast in the culture bottle, behind 0.25% trypsinization, add the DMEM culture fluid that contains 10%FBS of certain volume, process cell suspension, the 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 the incubator, behind the cultivation 24h cell attachment, the beginning dosing.
The various extracting solution of table 3 Folium Eucommiae, the Folium Eucommiae extract of different purity are to the influence of osteoblastic proliferation rate
Annotate: the percentage composition summation that refining Folium Eucommiae extract (purity 55.48%) is 5 kinds of compositions in the Folium Eucommiae extract is 55.48%.
Folium Eucommiae extract (purity 25%) is with after alcohol, the water mixed extraction, through ethanol precipitation, and the Folium Eucommiae extract that after polyamide purifying, prepares again.This Folium Eucommiae extract is in the preparation process, except that identical with the method for embodiment 1IV without additive method the HPD100 macroporous resin enrichment.The percentage composition detection method of each composition is identical with embodiment 2 in the extract, and testing result shows that the percentage composition summation of 5 kinds of compositions in the Folium Eucommiae extract is 25%.
Conclusion: it is active that the various extracting solution of Folium Eucommiae, the Folium Eucommiae extract of different purity all have the significance that promotes osteoblastic proliferation.But it is the most remarkable that purity is 56% Folium Eucommiae extract effect.
Embodiment 4 Folium Eucommiae extract antiinflammatories, the hematoma that disappears effect experiment
1 experiment purpose:
Whether the investigation Folium Eucommiae extract has antiinflammatory, the hematoma that disappears effect.
2 experiment materials
2.1 experiment reagent
The refining Folium Eucommiae extract of embodiment 1 IV preparation, xylene (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, Tianjin section close europeanized reagent development centre)
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 divide into groups and dosage
3.1.1 model group
0.4%CMC-Na solution, 20ml/kg.
3.1.2 positive controls
Aspirin, mice dosage are 0.27g/kg, and mice administration volume is 20ml/kg, and then this solution concentration is the 0.4%CMC-Na solution of 13.5mg/ml.
3.1.3 Folium Eucommiae extract high dose group
The refining Folium Eucommiae extract dosage of embodiment 1 IV preparation is 1g/kg, and mice administration volume is 20ml/kg, and then this solution concentration is the 0.4%CMC-Na solution of 0.05g/ml.
3.1.4 Folium Eucommiae extract low dose group
The refining Folium Eucommiae extract dosage of embodiment 1 IV preparation is 0.25g/kg, and mice administration volume is 20ml/kg, and then this solution concentration is the 0.4%CMC-Na solution of 0.0125g/ml.
3.2 the preparation of sample liquid
3.2.1 the preparation of positive drug solution
The content of aspirin is the 25mg/ sheet in the Aspirin Enteric-coated Tablets, gets 54 (containing aspirin 1350mg), grinds to be fine powder, adds 0.4%CMC-Na solution and grinds, and is settled to 100ml, shakes up, and promptly gets 13.5mg/ml aspirin suspension.
3.2.2 the preparation of each dosage solution of Folium Eucommiae extract
Take by weighing refining Folium Eucommiae extract 1.25g, the 5g of embodiment 1IV preparation respectively, add 0.4%CMC-Na solution and grind, be settled to 100ml, shake up, get promptly that Folium Eucommiae extract is low, the high dose suspension.
3.3 administration, 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 into 4 groups at random, being respectively model group, positive controls, Folium Eucommiae extract low dose group, Folium Eucommiae extract high dose group, is 4 groups altogether.Gastric infusion is after 4 days continuously, and the auris dextra every mice behind last administration 1h is coated with 20 μ l xylene, puts to death behind the 1h.Use the same position of diameter as card punch ear in the mice left and right sides of 8mm, lay circular auricle, on electronic balance, weigh, the record experimental result is calculated the swelling rate.
Swelling rate (%)=(auris dextra weight-left ear is heavy)/left ear heavy * 100%.
The hematoma experiment 2.3.2 mice disappears
Get body weight and be 40 of 18～22g mices, male, be divided into 4 groups at random, be respectively model group, positive controls, Folium Eucommiae extract low dose group, Folium Eucommiae extract high dose group, be 4 groups altogether.Cause self hematoma model before the experiment earlier: promptly with glass capillary in the mice eyeground vein clump 0.05ml that quantitatively takes a blood sample, add the 0.1mol/LCaCl that contains penicillin 100,000 units
2Solution 0.1ml mixing, it is subcutaneous to inject mice self nape portion immediately, causes the hematoma model.Caused behind the model successive administration 4 days, and the mice dislocation of cervical vertebra put to death in the 5th day, the residual hematoma piece that mice nape place is subcutaneous is separated, and uses scales/electronic balance weighing, judges drug effect through the residual hematoma piece weight of more different groups.
4 experimental results
4.1 mice auricle swelling experiment
Mice auricle swelling degree experimental result t inspection statistics through between group is analyzed, and shows that each dose groups of Folium Eucommiae extract and model group relatively have utmost point significant difference (P＜0.01), shows that each dose groups of Folium Eucommiae extract all has good antiinflammatory action.The mice auricle swelling experimental result is seen table 4.
Table 4 mice auricle swelling experimental result
Annotate: compare with model group,
*P＜0.01,
* *P＜0.001.
The hematoma experiment 4.2 mice disappears
Mice disappear the hematoma experimental result through the group between the analysis of t inspection statistics, Folium Eucommiae extract low dose group, high dose group and model group relatively have utmost point significant difference (P＜0.01), the demonstration Folium Eucommiae extract hematoma effect that well disappears is arranged.The mice hematoma experimental result that disappears is seen table 5.
The table 5 mice hematoma experimental result that disappears
Annotate: compare with model group,
*P＜0.05,
*P＜0.01.
4.3 conclusion:
This experimental result shows that each dose groups of Folium Eucommiae extract all has tangible antiinflammatory and the hematoma effect that disappears.
Embodiment 5 Folium Eucommiae extracts are to the influence of rat tibia fracture
Observe the influence of Folium Eucommiae extract to the rat tibia fracture.Get 40 SD rats, male and female half and half are pressed literature method, preparation rat tibia fracture model.Be divided into 5 groups at random: 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.Through 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.The result shows; Each group compares with model group; 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, and bone density significantly rises than model group; With the normal group there was no significant difference, the X sheet shows with model group has significant difference.But the biomechanical analysis of each group shows that each organizes the fracture resistence force experiment all has significant difference with model group.The result of the test explanation: in used dosage range, Folium Eucommiae extract has the significance effect that promotes union of fracture.
1. pharmaceutical agent compound method
The preparation of Folium Eucommiae extract high dose: the refining Folium Eucommiae extract of getting 3.6g embodiment 1IV preparation is dissolved in the 100mL0.5%CMC-Na solution, and promptly Folium Eucommiae extract high dose concentration is 36mg/mL.
Folium Eucommiae extract low dosage preparation: to 80mL, promptly concentration is 9mg/mL to the refining Folium Eucommiae extract high dose solution of getting 20mL embodiment 1IV preparation with 0.5%CMCNa solution standardize solution.
2. experimental technique
2.1 the foundation of fracture model
Get the SD rat, male and female half and half, totally 80, body weight 180-220g.Adaptability is fed a 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 the wound circumference sterilization is cut skin, layer-by-layer suture then with sterile operating theater instruments.After the stitching, do not fix, the gentamycin of intramuscular injection 1mL 40,000 units, and sprinkle in the wound that levofloxacin is anti-to be infected.The next day surgical groups 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, tibia standard fracture Preparation of model method: use 3% chloral hydrate, and intraperitoneal injection of anesthesia, anaesthesia dosage is: 10ml/kg.Dorsal position is fixed, and the wound circumference sterilization is cut skin with sterile operating theater instruments, finds tibia; Infra 1/3 place, it is long to cut 3mm with hacksaw, is equivalent to 2/3 of tibia diameter and is cut; Stay side seam cortex and fibula and play fixed supporting role, the hydrogen peroxide with 3% irrigates, layer-by-layer suture.After the stitching, do not fix, the gentamycin of intramuscular injection 1mL 40,000 units, and sprinkle in the wound that levofloxacin is anti-to be infected.The next day surgical groups every intramuscular injection gentamycin 1mL, continuous two days.
2.2 dosage and method
Each organizes laboratory animal in operation back beginning in the 3rd day gastric infusion.Administration volume 1mL/100g body weight.Once a day, continuous 30 days.Claim body weight weekly one time, according to body weight adjustment dosage.Dosage is: positive controls gives GUZHE CUOSHANG JIAONANG 470mg/kg, and the Folium Eucommiae extract high dose group gives 360mg/kg, the Folium Eucommiae extract low dose group gives 90mg/kg, model group and sham operated rats and gives isopyknic 0.5%CMC-Na aqueous solution.Blood is got from the laboratory animal eye socket respectively in after administration the 20th day and the 30th day, measures serum alkaline phosphatase, blood calcium and serium inorganic phosphorus.And after putting to death animal in 30 days, take off rat tibia, and measure the fracture resistence force of tibia, bone density, the X sheet detects the symphysis degree, respectively organizes the influence of medicine to fracture healing in rats through relatively drawing with model group.
3. experimental technique and result
3.1 the mensuration result of serum alkaline phosphatase
Eye socket is got about the about 600uL of blood respectively in 20 days and 30 days, places clean 1mL centrifuge tube, and 3500rpm frozen centrifugation 10min draws upper serum, and serum alkaline phosphatase is waited until in-20 ℃ of storages, the detection of blood calcium, serium inorganic phosphorus.Testing result is seen table 6.
The result shows: with model group relatively, when 20 days and 30 days, Folium Eucommiae extract low dosage (90mg/kg) group is organized with high dose (360mg/kg) has significant difference (p＜0.05, p＜0.01), can obviously increase serum alkaline phosphatase.
Table 620 day and respectively organized comparison (
unit: IU/L) of serum alkaline phosphatase (ALP) in 30 days
Annotate: * and model group compare, * P＜0.05, * * P＜0.01.
3.2 the mensuration result of serum blood calcium
With model group relatively, when 20 days and 30 days, Folium Eucommiae extract low dosage (90mg/kg) group is organized with high dose (360mg/kg) has significant difference (p＜0.05), can significantly reduce blood calcium.Testing result is seen table 7.
Table 720 day and respectively organized comparison (
unit: mmol/L) of blood calcium in 30 days
Annotate: * and model group compare, * P＜0.05, * * P＜0.01.
3.3 the mensuration result of serum serium inorganic phosphorus
Compare with model group, 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 seen table 8.
Table 820 day and respectively organized comparison (
unit: mmol/L) of serium inorganic phosphorus in 30 days
Annotate: * and model group compare, * P＜0.05, * * P＜0.01.
3.4 tibia X sheet detects
In administration the 30th day, put to death animal after getting blood, take off rat tibia, at 50KV, 50mA takes X-ray film under the 0.125S condition, and through the variation of X-ray film, by following standard scoring: 1. dislocation: damaged tibia dislocation-free (comprises dislocation＜0.1cm) 1 minute; Dislocation (comprising that the fracture site dislocation slightly becomes the angle above 1/2 or fracture end of its broken ends of fractured bone) 0 minute is arranged.2. fracture is stitched: damaged tibial bone crease partly blurs or disappeared 1 fen; Fracture seam 0 minute is arranged.3. connect: the damaged fracture of tibia broken ends of fractured bone was connected to an integral body 1 minute; Do not connect 0 fen.4. fibula: the fibula back of rupturing or rupture connects 1 fen; Fibula fracture 0 minute.5. healing: damaged tibia do not heal (external callus or fracture seam are not still in not having) 0 minute; Union of fracture (have inside and outside slight callus to change, the fracture seam shoals or be fuzzy simultaneously) 1 minute.Testing result is seen table 9.
The result shows that positive group fracture line disappears basically, 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.Compare with model group, Folium Eucommiae extract low dosage (90mg/kg) group has significant difference (p＜0.05), and high dose (360mg/kg) has utmost point significant difference (p＜0.01), and fracture line is tending towards disappearing, and callus generates and increases.
Table 9 fracture was respectively organized the influence
of Chinese medicine to X sheet score value in 30 days
Annotate: * and model group compare, * P＜0.05, * * P＜0.01.
3.5 bone density detects
Get the rat tibia of having clapped behind the X sheet, reject surperficial soft tissue, detect the healing bone biomechanical after, measure the bone density of each group healing bone respectively.Compare with model group, Folium Eucommiae extract low dosage (90mg/kg) group and high dose (360mg/kg) group have significant difference (p＜0.05), significantly bone density improving.
Table 10 fracture is respectively organized the comparison (n=4,
) of bone density after 30 days
Annotate: * and model group compare, * P＜0.05, * * P＜0.01.
3.6 the biomechanics of rat tibia detects
Get the rat tibia of having clapped behind the X sheet, reject surperficial soft tissue, with the healing skeleton two slidably, parallel, height is on the support on the same horizontal plane; Keep two support distances suitably; With the fracture is that the center hangs a cord, and underneath one pallet is put the counterweight of known weight in pallet; Bone up to this healing fractures, and calculates the index of the gross weight of the weight of hanging as the tibia fracture resistence force.Compare with model group, 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 seen table 11.
The comparison
of table 11 fracture each group healing bone fracture resistence force after 30 days
Annotate: * and model group compare, * P＜0.05, * * P＜0.01.
4. conclusion (of pressure testing)
Through comparing with model group; Folium Eucommiae extract high dose group, Folium Eucommiae extract low dose group can significantly increase alkali phosphatase when 20 days and 30 days; Reduce blood calcium, increase serium inorganic phosphorus, bone density improving; The X sheet shows that also the healing result and the model group of this medication group have significant difference, obviously promotes fracture back symphysis.
Embodiment 6 preparation granules
Take by weighing the refining Folium Eucommiae extract of embodiment 1IV preparation, the adjuvant dextrin of 2 times of quality of adding extract, mix homogeneously is crossed 65 mesh sieves, uses an amount of 95% ethanol to be binding agent, processes moist wood, granulates through the extruding of 20 mesh sieves, after 65 ℃ of dryings, gets granule.
Embodiment 5 preparation tablets
Take by weighing the refining Folium Eucommiae extract of embodiment 1IV preparation, add an amount of supplementary product starch and magnesium stearate, mixing is beaten sheet, promptly gets tablet.
Claims (9)
1. Folium Eucommiae water and/or lower alcohol extraction thing; It is characterized in that described Folium Eucommiae water and/or lower alcohol extraction thing comprise Folium Eucommiae water or lower alcohol extraction; The refining Folium Eucommiae extract that obtains after the perhaps crude extract that obtains of water and lower alcohol mixed extraction, and said crude extract is refining.
2. Folium Eucommiae water according to claim 1 and/or lower alcohol extraction thing; It is characterized in that described Folium Eucommiae water and/or lower alcohol extraction thing adopt following method preparation: after the Folium Eucommiae medicinal material drying is pulverized; With water extraction three times, the quality of each time institute water is followed successively by 10 times, 10 times and 9 times of Folium Eucommiae quality of medicinal material, and each return time that extracts is followed successively by 2h, 1.5h and 1h; With behind three extracting liquid filterings, merging filtrate also is concentrated into small size and obtains the Folium Eucommiae crude extract.
3. Folium Eucommiae water according to claim 1 and/or lower alcohol extraction thing; It is characterized in that described Folium Eucommiae water and/or lower alcohol extraction thing adopt following method preparation: after the Folium Eucommiae medicinal material drying is pulverized; Using concentration expressed in percentage by volume is that 45%～55% alcoholic solution extracts three times; The used alcoholic solution quality of each time is followed successively by 10 times, 8 times and 6 times of Folium Eucommiae quality of medicinal material, and each return time that extracts is followed successively by 2h, 1.5h and 1h, with merging behind three extracting liquid filterings; Reclaim ethanol, get alcohol extract; Alcohol extract is concentrated into small size obtains the Folium Eucommiae crude extract.
4. Folium Eucommiae water according to claim 1 and/or lower alcohol extraction thing; It is characterized in that described Folium Eucommiae water and/or lower alcohol extraction thing adopt following method preparation: after the Folium Eucommiae medicinal material drying is pulverized; Using concentration expressed in percentage by volume is that 45%～55% alcoholic solution extracts three times; The used alcoholic solution quality of each time is followed successively by 10 times, 8 times and 6 times of Folium Eucommiae quality of medicinal material, and each return time that extracts is followed successively by 2h, 1.5h and 1h, with merging behind three extracting liquid filterings; Reclaim ethanol, get alcohol extract; Medicinal residues behind the ethanol extraction add 10 times of water gagings and decoct 2h, get the water extract; Obtain the Folium Eucommiae crude extract with being concentrated into small size after water extract and the alcohol extract merging.
5. Folium Eucommiae water according to claim 4 and/or lower alcohol extraction thing; It is characterized in that described Folium Eucommiae crude extract adopts following method to make with extra care: the water extract is concentrated merge with described alcohol extract the back is not less than 90% ethanol precipitation with concentration expressed in percentage by volume after earlier, obtain making with extra care Folium Eucommiae extract through HPD100 macroporous resin enrichment and polyamide purification successively then.
6. Folium Eucommiae water according to claim 5 and/or lower alcohol extraction thing; It is characterized in that described HPD100 macroporous resin enrichment and polyamide purge process are: with adding water adjusting concentration after water extract behind the ethanol precipitation and the merging of described alcohol extract is 0.01g medical material/ml; Regulate pH=4 simultaneously; Obtain sample solution, last HPD100 macroporous resin adsorption post is 50～60% ethanol elution through concentration expressed in percentage by volume; Ethanol in the reclaim under reduced pressure eluent gets HPD100 macroporous resin eluent to there not being the alcohol flavor;
It is 0.01g medical material/ml that HPD100 macroporous resin eluent is adjusted to concentration; Last 30-60 order polyamide resin column is that 40%～50% ethanol elution gets eluent through concentration expressed in percentage by volume; Then behind the ethanol in the reclaim under reduced pressure eluent; Continue decompression and steam aqueous solution,, be the eluting concentrated solution to small size; The eluting concentrated solution through 65～70 ℃, is carried out drying under negative pressure 0.08Mpa condition, must make with extra care Folium Eucommiae extract.
7. Folium Eucommiae water according to claim 6 and/or lower alcohol extraction thing; It is characterized in that comprising 4 kinds of flavone compounds and chlorogenic acid in the Folium Eucommiae extract of described refining back; Described 4 kinds of flavone compounds are: rutin, isoquercitrin, Herba Astragali Melilotoidis (Herba Astragali Sinici) glycosides, Quercetin-3-O-α-L-arabopyranose base-(1 → 2)-β-D-glucosyl group glycosides, the quality percentage composition of 4 kinds of flavone compounds and chlorogenic acid is not less than 50% in the Folium Eucommiae extract of described refining back.
8. medicine that promotes union of fracture; It is characterized in that adding that by any described Folium Eucommiae water and/or lower alcohol extraction thing in the claim 1～7 of treatment effective dose acceptable accessories or complementary composition process preparation, described preparation comprises pill, powder, capsule, tablet, granule, injection or compound preparation.
9. any Folium Eucommiae water described in the claim 1～7 and/or the lower alcohol extraction thing application in the medicine of preparation promotion union of fracture.
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| CN104614480A (en) * | 2015-02-04 | 2015-05-13 | 东莞广州中医药大学中医药数理工程研究院 | Water-soluble general flavones of rabdosia lophanthide and fingerprint chromatographic detection method of water-soluble general flavones of rabdosia lophanthide |
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