CN104844723B - A kind of preparation method and application of Herba Dendrobii extract - Google Patents
A kind of preparation method and application of Herba Dendrobii extract Download PDFInfo
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Abstract
The invention discloses the preparation method of a kind of Herba Dendrobii extract, the method is with the fresh bar of Herba Dendrobii as raw material, neutral protein enzyme fermentation is used after squeezing the juice, fermentation liquid first passes through after removing the impurity such as starch after quick-freezing is thawed naturally, use storng-acid cation exchange resin and weak-base anion-exchange resin to process the solution removing starch, finally use titanium rod filter concentrations, precipitate with ethanol, centrifugal, collection supernatant lyophilization to prepare Herba Dendrobii extract；This technological operation is simple, low cost, and industrialization level is high；Being used for extract of the present invention preparing treatment wound healing medicine, compare with matched group, rabbit postoperative 10d wound surface size, healing express delivery and the average healing all have significant difference, and Herba Dendrobii extract can accelerate the healing of wound model rabbit wound.
Description
Technical field
The present invention relates to the preparation method and application of a kind of Herba Dendrobii extract, belong to active substance of plant and extract separation
Field.
Background technology
Herba Dendrobii (formal name used at school: Dendrobium officinale Kimura et Migo) is the orchid family Dendrobium Sw,
It is the perennial herbaceous plant that grows nonparasitically upon another plant.
Herba Dendrobii is classified as top grade in Shennong's Herbal, is described as first of " China's nine big Herba mesonae chinensis "；Iron sheet Radix Stephaniae Tetrandrae is lived
Originate on the south China Qinling Mountains and the Yangtze river basin, be distributed mainly on Yunnan, Guangxi, zhejiang and other places be grown on hot and humid area.
Dendrobium officinale chemical composition is mainly dendrobium polysaccharide, dendrobine, bibenzyl, luxuriant and rich with fragrance class, flavonoid, amino-acid trace element
Deng.Artificial organ cultivates Herba Dendrobii mainly rich in effective ingredient such as polysaccharide, and dendrobine, bibenzyl, luxuriant and rich with fragrance constituents content are extremely low；
Polysaccharide is topmost effective ingredient in Herba Dendrobii；Molecular weight about 400,000, configuration is based on α-Isosorbide-5-Nitrae-D-Gic straight-chain polysaccharide.
Pharmaceutical research shows that Herba Dendrobii has nourishing YIN and clearing away heat, the stomach reinforcing that promotes the production of body fluid, nourishing the lung to arrest cough, the kidney invigorating benefit power, benefit of prolonging life
The effects such as longevity, in vivo test finds, to laryngopharyngeal diseases, gastroenteropathy, cataract, diabetes, antitumor, defying age, to strengthen human body
Immunity has significant curative effect.
Method report that dendrobium polysaccharide extracts is more mainly has the dry pulverizing medicinal materials of employing, defat, water to carry, ethanol precipitation,
Deproteinization, it is dried to obtain total polysaccharides.Gained Herba Dendrobii total polysaccharides composition is mainly high physiologically active polysaccharide, starch-polysaccharides, really
Glue polysaccharide, protein etc., the high activity polyoses content wherein with drug action is not the highest.These report methods more but
Still it is weak, still needs to improve in terms of extraction ratio and purity.It is that raw material extracts that the present invention extracts the new fresh Herba Dendrobii of employing, improves and receives
Rate.Drying dendrobium polysaccharide in Herba Dendrobii to be heated generation intermolecular condensation, par-tial polysaccharide component damage water-soluble reduces, and extraction ratio drops
Low.
Summary of the invention
It is an object of the invention to provide the preparation method of a kind of Herba Dendrobii extract, the method is with the fresh bar of Herba Dendrobii
For raw material, juice uses neutral protein enzyme fermentation, and fermentation liquid first passes through after removing the impurity such as starch after quick-freezing is thawed naturally, uses
Storng-acid cation exchange resin and weak-base anion-exchange resin process the solution removing starch, finally use titanium rod to filter
Device concentration, precipitate with ethanol, centrifugal, collect supernatant lyophilization and prepare Herba Dendrobii extract.
The concrete operations of said method are as follows:
(1) in the fresh bar of Herba Dendrobii after cleaning add pure water smash squeezing to pieces, amount of water be fresh bar quality 10～
12 times, after squeezing the juice, filter to obtain the fresh juice of Herba Dendrobii；
(2) in step (1) cross filtering residue, add filtration slag amount 3～the water of 5 times and extract 1～2 time at 90～95 DEG C,
Filter merging filtrate after extraction time 40～60 minutes, abandon filtering residue；
(3) the fresh juice of combining step (1) Herba Dendrobii and step (2) filtrate, in amalgamation liquid add amalgamation liquid quality 0.03～
The neutral protease of 0.05% is incubated 1～2 hour in 45～60 DEG C, then room temperature standing for fermentation 12～16 hours；
(4) will the most naturally thaw after step (3) fermentation liquid quick freeze, make age of starch precipitation in extract；In collection
Clear liquid, remainder is centrifuged, and collects centrifugal liquid and is incorporated in supernatant；
(5) step (4) liquid is crossed strong acid cation exchange resin column, collect trickle；
(6) step (5) effluent is crossed weak-base anion-exchange resin post, collect colourless effluent；
(7) step (6) effluent uses titanium rod filter to concentrate, till being concentrated to concentrate front liquid volume 1/3～1/4；
(8) in step (7) concentrated solution, concentrated solution volume 4～the ethanol that mass percent concentration is 95% of 5 times are added
Carry out precipitate with ethanol overnight, centrifugal, collect supernatant, supernatant lyophilization prepares Herba Dendrobii extract.
Described titanium rod filter element aperture 0.2～3 microns.
The present invention another object is that and the Herba Dendrobii extract that said method prepares is applied to preparation treatment wound healing
In medicine.
Application of the present invention be with above-mentioned extract for active component preparation treatment wound healing medicine in.
Can also add one or more pharmaceutically acceptable adjuvants in application of the present invention, described adjuvant includes medicine
Filler, diluent, binding agent, excipient, absorption enhancer, filler, surfactant and the stabilizer that field is conventional
Deng, the most also can add flavouring agent, pigment and sweeting agent etc..
Application of the present invention is in addition to making capsule, it is also possible to make pill, powder, tablet, granule, oral liquid and injection
The various ways such as liquid.
The application crude extract use enzyme fermentation overnight, freeze thawing is macro-molecular protein, pectin, phlegmatic temperament, starch-polysaccharides
Little molecule class impurity centrifugal segregation is become etc. impurities ingredient degradation；Cation seperation column is used to slough aminoacid in centrifugal liquid, part fat
Dissolubility pigment, ash；Anion exchange resin is used to remove water colo(u)r and the small protein impurity of residual in Herba Dendrobii,
Substitute traditional organic solvent SAVAGE method deproteinization technique and there is more preferable industrial operation；Titanium candle filter is used to replace
Concentrate and expensive membrane separation plant for traditional heat, reduce production cost retentive activity polysaccharide simultaneously and do not have heat concentration
Destroyed phenomenon；The extract obtained protein after testing of precipitate with ethanol lyophilization, starch residual is extremely low, and this technological operation is simple,
Low cost, industrialization level is high.
The present invention is extract obtained preferable for treating wound healing effect, and in extract, protein, starch remove relatively
Spread on wound surface for thorough external and breed bacteria will not become inflammatory mediator；Experimental result shows extract of the present invention and Matrix controls
Group compares, and rabbit postoperative 10d wound surface size, healing express delivery and the average healing all have significant difference, Herba Dendrobii extract energy
Accelerate the healing of wound model rabbit wound.
Detailed description of the invention
Below by embodiment, the present invention is described in further detail, but protection scope of the present invention is not limited to described
Content.
Embodiment 1: the extraction separation method step of this Herba Dendrobii extract is as follows:
(1) adding pure water in the fresh bar of Herba Dendrobii after cleaning and smash squeezing to pieces, amount of water is 10 times of fresh bar quality,
The fresh juice of Herba Dendrobii is filtered to obtain after squeezing the juice；
(2) in step (1) crosses filtering residue, add the water of filtration slag amount 3 times and extract 1 time at 93 DEG C, extraction time 40
Filter merging filtrate after minute, abandon filtering residue；
(3) the fresh juice of combining step (1) Herba Dendrobii and step (2) filtrate, adds amalgamation liquid quality 0.03% in amalgamation liquid
Neutral protease 45 DEG C is incubated 2 hours, then room temperature standing for fermentation 12 hours；
(4) will the most naturally thaw after step (3) fermented liquid quick freeze, make age of starch precipitation in extract；Collect
Supernatant, remainder is centrifuged, and collects centrifugal liquid and is incorporated in supernatant；
(5) step (4) liquid is crossed strong acid cation exchange resin column (model D001), collect trickle；
(6) step (5) effluent is crossed weak-base anion-exchange resin post (model D941), collect colourless effluent；
(7) step (6) effluent uses titanium rod filter to concentrate, (titanium rod mistake till being concentrated to concentrate front liquid volume 1/3
2 microns of filter insert aperture)；
(8) ethanol that mass percent concentration is 95% adding concentrated solution volume 4 times in step (7) concentrated solution enters
Row precipitate with ethanol overnight, centrifugal, collect supernatant, supernatant lyophilization prepares Herba Dendrobii extract.
Use following experiment that the present embodiment extract carries out therapeutic wound healing:
1, laboratory animal: Japan large ear rabbit, 1.8 ± 0.2kg, male and female half and half, experimental animal is supported by Beijing KeYu animal
Grow center to provide, the quality certification number: SCXK(capital) 2012-0004；
2, the preparation of medicine: take Herba Dendrobii extract freeze-drying powder end 5g, with water for injection by Herba Dendrobii extract lyophilized powder
Stirring and evenly mixing becomes paste standby；
3, experimental technique: family's rabbit back unhairing, at back rostral 1/3, at the other 1.5cm of spinal column, symmetrical design 3cm
The rectangle wound surface of × 4cm size, major axis and spine parallel；After 1% procaine local anaesthesia, by design complete resection skin, superficial fascia
Tissue, until deep fascia superficial surface.By the above-mentioned rabbit 30 making wound model, it is randomly divided into 5 groups, i.e. blank group, substrate (note
Penetrate with water) matched group, positive drug group, medicine low dose group, medicine high dose group；Often group 6, by design dosage from operation day
Being coated with step 2 medicine outside wound surface, surface sterile gauze is wrapped up, and changes medicine every day；Wound surface size is measured, after logistic when changing dressings
Wound surface area and healing rate, with wound healing 95% for healing standard, record the healing days of each wound surface；
4, method is analyzed: representing with mean ± S.D, statistical method is one factor analysis of variance, with P < 0.05 as significance
Difference；
5, experimental result part table 1；
Table 1: each group rabbit wound model wound healing situation (mean ± S.D)
Note: compare with Matrix controls group,*P<0.05, **P<0.01。
6, conclusion
Compare with Matrix controls group, Herba Dendrobii extract each dosage group and positive drug group rabbit postoperative 10d wound surface size, more
Closing express delivery and the average healing all has significant difference, Herba Dendrobii extract can accelerate the healing of wound model rabbit wound.
Embodiment 2: the extraction separation method step of this Herba Dendrobii extract is as follows:
(1) adding pure water in the fresh bar of Herba Dendrobii after cleaning and smash squeezing to pieces, amount of water is 11 times of fresh bar quality,
The fresh juice of Herba Dendrobii is filtered to obtain after squeezing the juice；
(2) in step (1) crosses filtering residue, add the water of filtration slag amount 4 times and extract 2 times at 90 DEG C, extraction time 50
Filter merging filtrate after minute, abandon filtering residue；
(3) the fresh juice of combining step (1) Herba Dendrobii and step (2) filtrate, adds amalgamation liquid quality 0.04% in amalgamation liquid
Neutral protease 50 DEG C is incubated 1.5 hours, then room temperature standing for fermentation 14 hours；
(4) will the most naturally thaw after step (3) fermented liquid quick freeze, make age of starch precipitation in extract；Collect
Supernatant, remainder is centrifuged, and collects centrifugal liquid and is incorporated in supernatant；
(5) step (4) liquid is crossed strong acid cation exchange resin column (model 001 × 7), collect trickle；
(6) step (5) effluent is crossed weak-base anion-exchange resin post (model D900), collect colourless effluent；
(7) step (6) effluent uses titanium rod filter to concentrate, till being concentrated to concentrate front liquid volume 1/3；
(8) ethanol that mass percent concentration is 95% adding concentrated solution volume 5 times in step (7) concentrated solution enters
Row precipitate with ethanol overnight, centrifugal, collect supernatant, supernatant lyophilization prepares Herba Dendrobii extract.
Use following experiment that the present embodiment extract carries out therapeutic wound healing:
1, laboratory animal: Japan large ear rabbit, 1.8 ± 0.2kg, male and female half and half, experimental animal is supported by Beijing KeYu animal
Grow center to provide, the quality certification number: SCXK(capital) 2012-0004；
2, the preparation of medicine: take Herba Dendrobii extract freeze-drying powder end 5g, with water for injection by Herba Dendrobii extract lyophilized powder
Stirring and evenly mixing becomes paste standby；
3, experimental technique: family's rabbit back unhairing, at back rostral 1/3, at the other 1.5cm of spinal column, symmetrical design 3cm
The rectangle wound surface of × 4cm size, major axis and spine parallel；After 1% procaine local anaesthesia, by design complete resection skin, superficial fascia
Tissue, until deep fascia superficial surface.By the above-mentioned rabbit 30 making wound model, it is randomly divided into 5 groups, i.e. blank group, substrate (note
Penetrate with water) matched group, positive drug group, medicine low dose group, medicine high dose group；Often group 6, by design dosage from operation day
Being coated with step 2 medicine outside wound surface, surface sterile gauze is wrapped up, and changes medicine every day；Wound surface size is measured, after logistic when changing dressings
Wound surface area and healing rate, with wound healing 95% for healing standard, record the healing days of each wound surface；
4, method is analyzed: representing with mean ± S.D, statistical method is one factor analysis of variance, with P < 0.05 as significance
Difference；
5, experimental result part table 2；
Table 2: each group rabbit wound model wound healing situation (mean ± S.D)
Note: compare with Matrix controls group,*P<0.05, **P<0.01。
6, conclusion
Compare with Matrix controls group, Herba Dendrobii extract each dosage group and positive drug group rabbit postoperative 10d wound surface size, more
Closing express delivery and the average healing all has significant difference, Herba Dendrobii extract can accelerate the healing of wound model rabbit wound.
Embodiment 3: the extraction separation method step of this Herba Dendrobii extract is as follows:
(1) adding pure water in the fresh bar of Herba Dendrobii after cleaning and smash squeezing to pieces, amount of water is 12 times of fresh bar quality,
The fresh juice of Herba Dendrobii is filtered to obtain after squeezing the juice；
(2) in step (1) crosses filtering residue, add the water of filtration slag amount 5 times and extract 1 time at 95 DEG C, extraction time 60
Filter merging filtrate after minute, abandon filtering residue；
(3) the fresh juice of combining step (1) Herba Dendrobii and step (2) filtrate, adds amalgamation liquid quality 0.05% in amalgamation liquid
Neutral protease 60 DEG C is incubated 1 hour, then room temperature standing for fermentation 16 hours；
(4) will the most naturally thaw after step (3) fermented liquid quick freeze, make age of starch precipitation in extract；Collect
Supernatant, remainder is centrifuged, and collects centrifugal liquid and is incorporated in supernatant；
(5) step (4) liquid is crossed strong acid cation exchange resin column (model C D350), collect trickle；
(6) step (5) effluent is crossed weak-base anion-exchange resin post (model 700B), collect colourless effluent；
(7) step (6) effluent uses titanium rod filter to concentrate, till being concentrated to concentrate front liquid volume 1/4；
(8) ethanol that mass percent concentration is 95% adding concentrated solution volume 4 times in step (7) concentrated solution enters
Row precipitate with ethanol overnight, centrifugal, collect supernatant, supernatant lyophilization prepares Herba Dendrobii extract.
Use following experiment that the present embodiment extract carries out therapeutic wound healing:
1, laboratory animal: Japan large ear rabbit, 1.8 ± 0.2kg, male and female half and half, experimental animal is supported by Beijing KeYu animal
Grow center to provide, the quality certification number: SCXK(capital) 2012-0004；
2, the preparation of medicine: take Herba Dendrobii extract freeze-drying powder end 5g, with water for injection by Herba Dendrobii extract lyophilized powder
Stirring and evenly mixing becomes paste standby；
3, experimental technique: family's rabbit back unhairing, at back rostral 1/3, at the other 1.5cm of spinal column, symmetrical design 3cm
The rectangle wound surface of × 4cm size, major axis and spine parallel；After 1% procaine local anaesthesia, by design complete resection skin, superficial fascia
Tissue, until deep fascia superficial surface.By the above-mentioned rabbit 30 making wound model, it is randomly divided into 5 groups, i.e. blank group, substrate (note
Penetrate with water) matched group, positive drug group, medicine low dose group, medicine high dose group；Often group 6, by design dosage from operation day
Being coated with step 2 medicine outside wound surface, surface sterile gauze is wrapped up, and changes medicine every day；Wound surface size is measured, after logistic when changing dressings
Wound surface area and healing rate, with wound healing 95% for healing standard, record the healing days of each wound surface；
4, method is analyzed: representing with mean ± S.D, statistical method is one factor analysis of variance, with P < 0.05 as significance
Difference；
5, experimental result part table 3；
Table 3: each group rabbit wound model wound healing situation (mean ± S.D)
Note: compare with Matrix controls group,*P<0.05, **P<0.01。
6, conclusion
Compare with Matrix controls group, Herba Dendrobii extract each dosage group and positive drug group rabbit postoperative 10d wound surface size, more
Closing express delivery and the average healing all has significant difference, Herba Dendrobii extract can accelerate the healing of wound model rabbit wound.
Claims (3)
1. the preparation method of a Herba Dendrobii extract, it is characterised in that step is as follows:
(1) adding pure water in the fresh bar of Herba Dendrobii after cleaning and smash squeezing to pieces, amount of water is 10～12 times of fresh bar quality,
The fresh juice of Herba Dendrobii is filtered to obtain after squeezing the juice；
(2) in step (1) crosses filtering residue, add filtration slag amount 3～the water of 5 times and extract 1～2 time at 90～95 DEG C, extracting
Filter merging filtrate after time 40～60 minutes, abandon filtering residue；
(3) the fresh juice of combining step (1) Herba Dendrobii and step (2) filtrate, in amalgamation liquid add amalgamation liquid quality 0.03%～
The neutral protease of 0.05% is incubated 1～2 hour in 45～60 DEG C, then room temperature standing for fermentation 12～16 hours；
(4) will the most naturally thaw after step (3) fermentation liquid quick freeze, make age of starch precipitation in extract；Collect supernatant,
Remainder is centrifuged, and collects centrifugal liquid and is incorporated in supernatant；
(5) step (4) liquid is crossed strong acid cation exchange resin column, collect trickle；
(6) step (5) effluent is crossed weak-base anion-exchange resin post, collect colourless effluent；
(7) step (6) effluent uses titanium rod filter to concentrate, till being concentrated to concentrate front liquid volume 1/3～1/4；
(8) ethanol that mass percent concentration is 95% adding concentrated solution volume 4～5 times in step (7) concentrated solution is carried out
Precipitate with ethanol overnight, centrifugal, collect supernatant, supernatant lyophilization prepares Herba Dendrobii extract.
The preparation method of Herba Dendrobii extract the most according to claim 1, it is characterised in that: titanium rod filter element hole
Footpath 0.2～3 microns.
3. the Herba Dendrobii extract that the preparation method of the Herba Dendrobii extract described in claim 1 prepares is in preparation treatment wound
Application in mouth healing drug.
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| CN100497396C (en) * | 2006-11-23 | 2009-06-10 | 天津市轩宏医药技术有限公司 | Dendrobium candidum polysaccharide extractive, medicine composition thereof and its preparation and use |
| CN102229677B (en) * | 2011-05-12 | 2012-11-14 | 芜湖华信生物药业股份有限公司 | Method for preparing dendrobium huoshanense water-soluble extractive |
| CN103113483B (en) * | 2013-01-24 | 2015-05-20 | 北京航空航天大学 | Method for extracting various active ingredients from dendrobium officinale |
| CN103772521B (en) * | 2014-01-09 | 2016-06-01 | 云南云尚生物技术有限公司 | The extracting method of a kind of Dendrobium officinale polysaccharide |
| CN104448013A (en) * | 2014-10-27 | 2015-03-25 | 霍山县天下泽雨生物科技发展有限公司 | Process for extracting dendrobium polysaccharide and dendrobine from dendrobium officinale |
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