CN101407558B - Preparation of Dendrobium nobile polysaccharide extract and use there - Google Patents
Preparation of Dendrobium nobile polysaccharide extract and use there Download PDFInfo
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Abstract
The invention provides a preparation method of dendrobium nobile polysaccharide extracts. The dendrobium nobile polysaccharide extracts can be realized through steps of degreasing, extracting, alcohol precipitation, deproteinization, removing monosaccharide and oligosaccharide, gel column purification etc. The content of polysaccharide in the polysaccharide extracts is above 90 percent. The polysaccharide extracts are provided with obvious antioxidant activity and can be applied to the preparation of medicaments or medicament complex used for treating and preventing cardiovascular disease after combined with medicament excipients or carriers allowed by the preparation. The preparation method of the extracts has the advantages of reasonable design and easy operation.
Description
Technical field
The invention belongs to Chinese medical extract, be specifically related to a kind of Dendrobium nobile polysaccharide extract preparation method and application thereof.
Background technology
The stem of noble dendrobium is a rare traditional Chinese medicine commonly used, is the general designation of the fresh or dry stem of the orchid family (Orchidaceae) Dendrobium (Dendrobium Sw.) various plants.Dendrobium is bigger genus in the orchid, about 1100 kinds of the whole world, and China has an appointment 76 kinds.Stem of noble dendrobium beginning is stated from Shennong's Herbal, be listed in top grade, effect such as have nourishing Yin and clearing heat, reinforcing stomach reg fluid, moisten the lung and relieve the cough." Chinese pharmacopoeia version in 2005 has been recorded three kinds of dendrobium stem (D.nobileLindl.), Herba Dendrobii (D.officinale Kimura et Migo) and stem of Eyeshaped Dendrobium (D.fimbriatum Hook.).
The modern chemistry pharmacological research shows that dendrobium stem mainly contains polyose, alkaloids, and phenols isoreactivity material, wherein the polyose composition has the inhibition tumour, strengthens or the recovery immunologic function effect such as hypoglycemic grade.Drawbacks such as the research of dendrobium polysaccharide at present exists basic substance indeterminate, and quality is uncontrollable, and separation purifying technique is coarse lack active component content height, specific chemical components, significant document of drug activity and patent report.And, do not see appearance on the market about the medicine or the pharmaceutical composition of Dendrobium nobile polysaccharide yet.
Summary of the invention
The preparation method who the purpose of this invention is to provide a kind of Dendrobium nobile polysaccharide extract mainly contains polysaccharide in the described Dendrobium nobile polysaccharide extract, wherein, by weight percentage, the content of polysaccharide is more than 90%.The content of preferred polysaccharide is 93%-98%.Realize by following steps:
(1) degreasing: get the dendrobium stem medicine materical crude slice, doubly measure organic solvent with 8-15 and soak back backflow 1-3 time, filter, volatilize organic solvent and get the dregs of a decoction;
(2) extract: after the above-mentioned dregs of a decoction were soaked, water-bath was extracted 1-3 time, and each 1-3 hour, filter, concentrated filtrate extremely every ml is equivalent to contain crude drug 0.5-2.0g;
(3) alcohol precipitation: add an amount of ethanol in above-mentioned concentrated solution, ice bath is placed, and filters, and gets polyoses extract crude product I after the precipitation lyophilize;
(4) remove albumen: it is soluble in water in right amount to get above-mentioned polyoses extract crude product I, and adding volume ratio is the chloroform of 5:1: propyl carbinol, stir the back layering, after upper strata repetitive operation 4-8 time, add an amount of ethanol, ice bath is placed, filter, the precipitation lyophilize gets polyoses extract crude product II;
(5) remove monose and oligosaccharides: it is soluble in water in right amount to get above-mentioned polyoses extract crude product II, (end molecular weight: 8000-14000) dialysis was taken out the interior liquid of dialysis tubing and is added an amount of ethanol after 2 days, and ice bath is placed with dialysis membrane, filter, get polyoses extract crude product III after the precipitation lyophilize;
(6) gel column purifying: with above-mentioned polyoses extract crude product III gel chromatography column purification, follow the tracks of cut with the sulfuric acid phynol method, after the collection main peak merges, add an amount of ethanol, ice bath is placed, and filters, and the precipitation lyophilize gets white Dendrobium nobile polysaccharide extract.
The described organic solvent of step (1) is a sherwood oil, ethyl acetate, ethanol or the high concentration ethanol aqueous solution.
The described dregs of a decoction of step (2) are soaked, and wherein amount of water is that the 4-10 of the dregs of a decoction doubly measures, and preferred 8-10 doubly measures; Extracting temperature is 30-80 ℃, preferably 40-60 ℃.
Behind the adding ethanol described in step (3)-(6), the alcoholic acid volumn concentration is 50%-80%, and preferred alcoholic acid volumn concentration is 70%-80%.
Be more than 12 hours the ice bath storage period described in step (4)-(6), and preferred ice bath was placed 24 hours.
Another object of the present invention provides the application of described Dendrobium nobile polysaccharide extract in preparation treatment and preventing cardiovascular disease medicine.
Drug prepared also contains preparation allowable pharmaceutical excipients or carrier.
Usefulness of the present invention is the quality controllable of the dendrobium polysaccharide that extracts, the active component content height of extraction, and specific chemical components, drug activity is remarkable.The separation purifying technique of preparation method of extract of the present invention is reasonable in design, and is easy and simple to handle.The appearance of medicine provided by the invention or pharmaceutical composition is for Dendrobium nobile polysaccharide provides medicinal use widely.
Embodiment
Further specify flesh and blood of the present invention and beneficial effect below in conjunction with embodiment, this embodiment only is used to the present invention is described but not limitation of the present invention.
The preparation of embodiment one Dendrobium nobile polysaccharide extract
(1) degreasing: get the dendrobium stem medicine materical crude slice,, filter, volatilize organic solvent and get the dregs of a decoction with refluxing 2 times after 8 times of amount alcohol immersion; (2) extraction: after the above-mentioned dregs of a decoction added 10 times of amount immersions of water, water-bath was extracted 3 times, and each 1 hour, filter, concentrated filtrate extremely every ml is equivalent to contain crude drug 1.0g; (3) alcohol precipitation: adding an amount of ethanol to ethanol content in above-mentioned concentrated solution is 80%, and ice bath was placed 12 hours, filters, and gets polyoses extract crude product I after the precipitation lyophilize; (4) remove albumen: it is soluble in water in right amount to get above-mentioned polyoses extract crude product I, adds an amount of chloroform and propyl carbinol, stirs the back layering, after the upper strata repetitive operation 4 times, adding an amount of ethanol to ethanol content is 80%, and ice bath was placed 12 hours, filter, the precipitation lyophilize gets polyoses extract crude product II; (5) remove monose and oligosaccharides: it is soluble in water in right amount to get above-mentioned polyoses extract crude product II, (end molecular weight: 8000-14000) dialysis is after 2 days with dialysis membrane, it is 70% that the interior liquid of taking-up dialysis tubing adds an amount of ethanol to ethanol content, ice bath was placed 12 hours, filter, get polyoses extract crude product III after the precipitation lyophilize; (6) gel column purifying: with above-mentioned polyoses extract crude product III gel chromatography column purification, follow the tracks of cut, after the collection main peak merges with the sulfuric acid phynol method, adding an amount of ethanol to ethanol content is 70%, and ice bath is placed, and filters, the precipitation lyophilize gets white Dendrobium nobile polysaccharide extract.It is 91% that phenol sulfuric acid process mensuration polysaccharide gets content, and the bradford method is measured albumen and is respectively 1.0%.
The preparation of embodiment two Dendrobium nobile polysaccharide extracts
(1) degreasing: get the dendrobium stem medicine materical crude slice,, filter, volatilize organic solvent and get the dregs of a decoction with refluxing 2 times after 10 times of amount 80% alcohol immersion; (2) extraction: after the above-mentioned dregs of a decoction added 8 times of amount immersions of water, water-bath was extracted 2 times, and each 1 hour, filter, concentrated filtrate extremely every ml is equivalent to contain crude drug 1.5g; (3) alcohol precipitation: adding an amount of ethanol to ethanol content in above-mentioned concentrated solution is 80%, and ice bath was placed 24 hours, filters, and gets polyoses extract crude product I after the precipitation lyophilize; (4) remove albumen: it is soluble in water in right amount to get above-mentioned polyoses extract crude product I, adds an amount of chloroform and propyl carbinol, stirs the back layering, after the upper strata repetitive operation 8 times, adding an amount of ethanol to ethanol content is 80%, and ice bath was placed 24 hours, filter, the precipitation lyophilize gets polyoses extract crude product II; (5) remove monose and oligosaccharides: it is soluble in water in right amount to get above-mentioned polyoses extract crude product II, (end molecular weight: 8000-14000) dialysis is after 2 days with dialysis membrane, it is 80% that the interior liquid of taking-up dialysis tubing adds an amount of ethanol to ethanol content, ice bath was placed 24 hours, filter, get polyoses extract crude product III after the precipitation lyophilize; (6) gel column purifying: with above-mentioned polyoses extract crude product III gel chromatography column purification, follow the tracks of cut, after the collection main peak merges with the sulfuric acid phynol method, adding an amount of ethanol to ethanol content is 80%, and ice bath was placed 24 hours, filters, the precipitation lyophilize gets white polysaccharide CP50 elaboration.It is 93% that phenol sulfuric acid process mensuration polysaccharide gets content, and the bradford method is measured albumen and is respectively 1.0%.
The preparation of embodiment three Dendrobium nobile polysaccharide extracts
(1) degreasing: take by weighing stem of noble dendrobium medicinal material 4kg (dry product, block medicine materical crude slice), add 8 times of amounts of 80% ethanol and soak 3 hours (W/V), 85 ℃ were refluxed 2 times 5 hours.Filter, the dregs of a decoction are divided, and volatilize ethanol; (2) extract: the above-mentioned dregs of a decoction are waved to there not being the alcohol flavor, add 4 times of amount distilled water (W/V), soak 12 hours, and 50 ℃ of water-baths were extracted 2 times 2 hours.Four layers of gauze B suction filtration are collected filtrate, merge, and are concentrated into 3000ml in 60 ℃ of rotary evaporations; (3) alcohol precipitation: add about 9000ml dehydrated alcohol to above-mentioned concentrated solution, be adjusted to 80% ethanol content with alcoholometer, ice bath was placed 24 hours, flocks occurred, and with four layers of gauze B suction filtration, the precipitation lyophilize gets polyoses extract crude product I; (4) remove albumen: take by weighing above-mentioned polyoses extract crude product I5.0g, be dissolved in the 500ml distilled water, add CHCl3100ml, propyl carbinol 20ml, electronic stirring 30 minutes, separating funnel left standstill 10 minutes, and isolated for disposal subnatant, upper strata liquid repeat deproteinated operation 6 times.Afterwards, upper strata liquid adds dehydrated alcohol to 80%, and ice bath was placed 24 hours, and is centrifugal, and the precipitation lyophilize gets polyoses extract crude product II; (5) remove monose and oligosaccharides: get above-mentioned polyoses extract crude product II and be dissolved in right amount in the 300ml distilled water, (end molecular weight: 8000～14000) dialysis, dialysis tubing external application magnetic stir bar at the uniform velocity stirs, and changes water 1 time in per 2 hours, dialyses 2 days with dialysis membrane.Liquid takes out and adds dehydrated alcohol to 80% in the dialysis tubing, and ice bath 24 hours is centrifugal, gets gray precipitate, and lyophilize gets polyoses extract crude product III; (6) gel column purifying: above-mentioned polyoses extract crude product III with Deae sepharose FF gel chromatography column purification, is followed the tracks of cut with the sulfuric acid phynol method, after the collection main peak merges, add an amount of ethanol to 80%, ice bath is placed, and filters, the precipitation lyophilize gets white polysaccharide CP50 elaboration.It is 95% that phenol sulfuric acid process mensuration polysaccharide gets content, and the bradford method is measured albumen and is respectively 0.9%.
Embodiment four neonatal rat myocardial cell H
2O
2Damage model
Is 50mg/mL with Dendrobium nobile polysaccharide extract with the pure DMSO melt into concentration of top grade, and jolting is centrifugal.Store down at-20 ℃, standby.At first carry out cell cultures.The quantity of safeguarding cell can supply screening used to guarantee it, but cell count can not be too many, otherwise can influence the growth of cell, regularly change liquid to it and go down to posterity.H
9C
2Be attached cell, change liquid and only need inhale the nutrient solution supernatant, wash one time, add fresh medium again and get final product with PBS.Go down to posterity to inhale and remove the nutrient solution supernatant, wash one time with PBS, use trysinization, add nutrient solution again and stop digestion, centrifugal going down to posterity get final product (nutrient solution is that DMEM (high sugared)+10%FBS (G)+1% non-essential amino acid+0.1% pair resists).Reach one regularly Deng cell quantity, just can plant plate, for dosing is prepared.Blow and beat cell dispersion in culturing bottle, mixed solution is transferred in the centrifuge tube, draw a little and expect blue method of exclusion counting with platform on hematimeter, centrifugal 10 minutes remaining (900rpm) inhales and removes supernatant.Add an amount of nutrient solution again, piping and druming disperses the cell mixing gently, draws an amount of nutrient solution and joins in the cell groove, makes cell density be about 1 * 10
4Individual/0.15mL.Cell suspension is added on the 96 porocyte culture plates uniformly every hole 0.15mL with the octal road volley of rifle fire.After planting plate, be put in the constant temperature cell culture incubator and cultivate after 24 hours, it is carried out H
2O
2The manufacturing of damage model.Get plate, draw supernatant, wash one time, remove supernatant with PBS.Dosing is with H is arranged
2O
2Nutrient solution dilutes medicine, makes that the component drug final concentration is 50 μ g/mL, and the positive drug final concentration is 50 μ mol/mL, H
2O
2Final concentration be 200 μ mol/mL.With 96 orifice plates put into incubator (37 ℃, 5%CO
2, 21%O
2), get plate after 24 hours, mtt assay is surveyed cell activity.With the positive medicine of the VC of 50 μ mol/mL.Treatment rate=(each dosing group A value-DMSO group A value)/(DMSO group A value-blank group A value) * 100% the results are shown in Table 1.
Table 1 neonatal rat myocardial cell H
2O
2Damage model
Embodiment five neonatal rat myocardial cell anoxic reoxygenation models
Is 50mg/mL with Dendrobium nobile polysaccharide extract with the pure DMSO melt into concentration of top grade, and jolting is centrifugal.Store down at-20 ℃, standby.At first carry out cell cultures.The quantity of safeguarding cell can supply screening used to guarantee it, but cell count can not be too many, otherwise can influence the growth of cell, regularly change liquid to it and go down to posterity.H
9C
2Be attached cell, change liquid and only need inhale the nutrient solution supernatant, wash one time, add fresh medium again and get final product with PBS.Go down to posterity to inhale and remove the nutrient solution supernatant, wash one time with PBS, use trysinization, add nutrient solution again and stop digestion, centrifugal going down to posterity get final product (nutrient solution is that DMEM (high sugared)+10%FBS (G)+1% non-essential amino acid+0.1% pair resists).Reach one regularly Deng cell quantity, just can plant plate, for dosing is prepared.Blow and beat cell dispersion in culturing bottle, mixed solution is transferred in the centrifuge tube, draw a little and expect blue method of exclusion counting with platform on hematimeter, centrifugal 10 minutes remaining (900rpm) inhales and removes supernatant.Add an amount of nutrient solution again, piping and druming disperses the cell mixing gently, draws an amount of nutrient solution and joins in the cell groove, makes cell density be about 1 * 10
4Individual/0.15mL.Cell suspension is added on the 96 porocyte culture plates uniformly every hole 0.15mL with the octal road volley of rifle fire.After planting plate, be put in the constant temperature cell culture incubator and cultivate after 24 hours, it is carried out the manufacturing of anoxia model.Get some sugar-free Hank ' s, logical N
210 minutes, get plate, draw supernatant, use sugar-free Hank ' s to wash one time, remove supernatant.Dosing uses sugar-free Hank ' s that medicine is diluted, and makes that the component drug final concentration is 50 μ g/mL, and the positive drug final concentration is 50 μ mol/mL.96 orifice plates are put into closed environment, vacuumize, logical N
2, make its inner hypoxia that guarantees, put into three gas incubators (37 ℃, 5%CO
2, 5%O
2).Anoxic 6 hours.After 6 hours, get plate, supernatant 10 μ L are got in every hole, survey LDH.Upper plate draws unnecessary supernatant after taking out supernatant 10 μ L, add nutrient solution (low sugar DMEM+FBS), and dosing is supplied again, and making every hole contain amount of liquid is 100 μ L, and makes that the component drug final concentration is 50 μ g/mL, and the positive drug final concentration is 50 μ mol/mL.Put into constant incubator (37 ℃, 5%CO2).Hatched 4 hours.After 4 hours, get plate, supernatant 10 μ L are got in every hole, survey LDH.Get a cell groove, R1 and R2 in the LDH test kit are mixed with 2:1, mixed solution is joined in 96 orifice plates of need measuring with the octal road volley of rifle fire, every hole 300 μ L are hatched under 37 ℃.Set a series of time points, measure the LDH value at the 340nm place.With the positive medicine of the VC of 50 μ mol/mL.Press treatment rate=(DMSO group A value-each dosing group A value)/DMSO group A value * 100%, the results are shown in Table 2.
Table 2 neonatal rat myocardial cell anoxic reoxygenation model experiment result
The preparation of embodiment six pills
Get Dendrobium nobile polysaccharide extract 0.5g and 10.5g polyoxyethylene glycol-6000 mixes, heating and melting moves in the dripping pill drip irrigation after changing material, and in ℃ whiteruss of medicine liquid droplet to 6～8, oil removing makes 300 of dripping pills.
The preparation of embodiment seven lyophilized injectable powders
Get Lignum Dalbergiae Odoriferae oil 1.5g, join in the saturated hydroxypropyl of 13ml, stirring and dissolving filters, and the filtrate cryodrying gets the inclusion compound powder of Lignum Dalbergiae Odoriferae oil and hydroxypropyl.Except that above-mentioned Lignum Dalbergiae Odoriferae oil closes the inclusion compound powder of hydroxypropyl, get Dendrobium nobile polysaccharide extract 0.5g, N.F,USP MANNITOL 5.5g, Calcium Disodium Edetate 0.9g and distilled water 2ml again, behind the said components mixing, lyophilize, 350 of packing, promptly.
The preparation of embodiment eight tablets
Get that Dendrobium nobile polysaccharide extract is an amount of to mix with Microcrystalline Cellulose, add 3% polyvidone ethanolic soln system softwood, cross 18 mesh sieve system particles, 60 ℃ of dryings 1 hour, whole, the adding talcum powder is an amount of, mixing, compressing tablet, promptly.
The preparation of embodiment nine capsules
Get Dendrobium nobile polysaccharide extract and peanut oil and stir in the filling in stainless steel in the ratio of 2:18 is water-soluble, add an amount of gelatin and glycerine simultaneously and mix, emit and use the colloidal mill defibrination again, the mixed solution that grinds stirs, and makes soup; Under 40-50 ℃ of temperature, stir glycerine and distilled water miscible, again with the glycerin liquid temperature to 80-90 ℃, get gelatin and add wherein to mix and stir, become glue until off-bottom, glue was left standstill 4 hours, make offset plate with laminator and use for the pill operation; Above-mentioned soup of making and offset plate are sent into the pellet press pill, did cylinder typing in 4 hours at 22-25 ℃ of temperature katabatic wind then, again dry 16-20 hour of 25-30 ℃ of temperature katabatic wind, the qualified capsule and pill of pick, clean with 95% ethanol, dry 4 hours of 25-30 ℃ of katabatic wind, promptly.
The preparation of embodiment ten injections
Get Dendrobium nobile polysaccharide extract in right amount with 40 ℃ of water for injection dissolvings, add an amount of pharmaceutical carrier isotonic agent, the pH value of regulator solution is 7.0-8.0, adds to the full amount of water for injection, remove thermal source with the ultra-fine filter ultrafiltration, after the mensuration pH value, use membrane filtration, after the packing, autoclaving, check, packing, promptly.
Claims (7)
1. the preparation method of a Dendrobium nobile polysaccharide extract mainly contains polysaccharide in the described Dendrobium nobile polysaccharide extract, and wherein, by weight percentage, the content of polysaccharide is 93%-98%, realizes by following steps:
(1) degreasing: get the dendrobium stem medicine materical crude slice, doubly measure organic solvent with 8-15 and soak back backflow 1-3 time, filter, volatilize organic solvent and get the dregs of a decoction;
(2) extract: after the above-mentioned dregs of a decoction were soaked, water-bath was extracted 1-3 time, and each 1-3 hour, filter, concentrated filtrate is equivalent to contain crude drug 0.5-2.0g to every milliliter, and wherein amount of water is that the 4-10 of the dregs of a decoction doubly measures, and the extraction temperature is 30-80 ℃;
(3) alcohol precipitation: add ethanol in above-mentioned concentrated solution, ice bath is placed, and filters, and gets polyoses extract crude product I after the precipitation lyophilize;
(4) remove albumen: it is soluble in water to get above-mentioned polyoses extract crude product I, adds volume ratio and be 5: 1 chloroform: propyl carbinol, stir the back layering, after upper strata repetitive operation 4-8 time, add ethanol, ice bath is placed, filter, the precipitation lyophilize gets polyoses extract crude product II;
(5) remove monose and oligosaccharides: it is soluble in water to get above-mentioned polyoses extract crude product II, after dialysing 2 days by the dialysis membrane of molecular weight: 8000-14000, take out liquid adding ethanol in the dialysis tubing, ice bath is placed, filter, get polyoses extract crude product III after the precipitation lyophilize;
(6) gel column purifying: with above-mentioned polyoses extract crude product III gel chromatography column purification, follow the tracks of cut with the sulfuric acid phynol method, after the merging of collection main peak, add ethanol, ice bath is placed, and filters, and precipitates lyophilize, gets white Dendrobium nobile polysaccharide extract;
Adding ethanol described in step (3)-(6), wherein the alcoholic acid volumn concentration is 50%-80%.
2. the preparation method of a kind of Dendrobium nobile polysaccharide extract according to claim 1 is characterized in that: the used organic solvent of step (1) is selected a kind of in sherwood oil, ethyl acetate, ethanol or the high concentration ethanol aqueous solution for use.
3. the preparation method of a kind of Dendrobium nobile polysaccharide extract according to claim 1, it is characterized in that: the described dregs of a decoction of step (2) are soaked, and wherein amount of water is that the 8-10 of the dregs of a decoction doubly measures, and extracting temperature is 40-60 ℃.
4. the preparation method of a kind of Dendrobium nobile polysaccharide extract according to claim 1, it is characterized in that: the adding ethanol described in step (3)-(6), wherein the alcoholic acid volumn concentration is 70%-80%.
5. the preparation method of a kind of Dendrobium nobile polysaccharide extract according to claim 1 is characterized in that: be more than 12 hours the ice bath storage period described in step (3)-(6).
6. the application of the Dendrobium nobile polysaccharide extract of the preparation method of a kind of Dendrobium nobile polysaccharide extract according to claim 1 acquisition in preparation treatment and preventing cardiovascular disease medicine.
7. the application of a kind of Dendrobium nobile polysaccharide extract according to claim 6 is characterized in that: drug prepared also contains preparation allowable pharmaceutical excipients or carrier.
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| CN102274138A (en) * | 2010-06-11 | 2011-12-14 | 上海家化联合股份有限公司 | Dendrobe extract and preparation method and application thereof |
| CN102391385B (en) * | 2011-10-26 | 2013-06-26 | 安徽圣农生物科技股份有限公司 | Extraction and purification method of bioactive polysaccharides in dendrobium huoshanense |
| CN106220480B (en) * | 2016-06-24 | 2017-10-27 | 江西九草铁皮石斛科技协同创新有限公司 | The technique that tank assembling dynamic countercurrent extracts dendrophnol in dendrobium candidum |
| CN110274972A (en) * | 2019-06-21 | 2019-09-24 | 海南大学 | A method of series connection gel chromatography Dendrobium nobile polysaccharide molecular weight distribution |
| CN111196864B (en) * | 2020-03-06 | 2021-09-14 | 遵义医科大学 | Dendrobium nobile polysaccharide with moisturizing effect and preparation method and application thereof |
| CN113480675B (en) * | 2021-08-19 | 2023-03-17 | 遵义医科大学 | Extraction method and application of dendrobium nobile polysaccharide |
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