CN104844723A - Preparation method and application of dendrobium officinale extract - Google Patents
Preparation method and application of dendrobium officinale extract Download PDFInfo
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Abstract
The invention discloses a preparation method of a dendrobium officinale extract. The method comprises the following steps: by taking fresh dendrobium officinale strips as a raw material, performing fermentation with neutral protease after juice squeezing; quickly freezing fermentation liquor, naturally unfreezing the fermentation liquor, and removing starch and other impurities from the fermentation liquor; removing a starch solution through treatment with strongly acidic cation exchange resin and weakly basic anion exchange resin; and finally performing concentration with a titanium stick filter, performing alcohol precipitation and centrifugation, collecting supernatant, and freeze-drying the supernatant to prepare the dendrobium officinale extract. The preparation method is simple to operate, low in cost and high in industrialization degree. The dendrobium officinale extract provided by the invention is used for preparing medicines for treating wounds. Compared with a control group, the dendrobium officinale extract is remarkably different in wound size, healing speed and mean healing time within 10 days after operation of rabbits; and the dendrobium officinale extract can speed up the healing of a wound of a wound model rabbit.
Description
Technical field
The present invention relates to a kind of preparation method and application of Herba Dendrobii extract, belong to active substance of plant extraction and isolation field.
Background technology
Herba Dendrobii (formal name used at school: Dendrobium officinale Kimura et Migo) is the orchid family Dendrobium Sw, and 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 " Chinese nine immortal grass "; Iron sheet stone solution firmly originates on the south China Qinling Mountains and the Yangtze valley, be mainly distributed in 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 etc.Artificial organ is cultivated Herba Dendrobii and is mainly rich in the effective constituents such as polysaccharide, and dendrobine, bibenzyl, luxuriant and rich with fragrance constituents content are extremely low; Polysaccharide is topmost effective constituent in the stem of noble dendrobium; 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 heat, the beneficial stomach that promotes the production of body fluid, moistens the lung and relieve the cough, kidney tonifying benefit power, the effect such as to promote longevity, in vivo test finds, to laryngopharyngeal diseases, gastroenteropathy, cataract, diabetes, antitumor, anti-ageing, to strengthen body immunity and have significant curative effect.
More the mainly containing of the method report that dendrobium polysaccharide extracts adopts dry pulverizing medicinal materials, degreasing, water extraction, ethanol precipitation, deproteinated, drying to obtain total polysaccharides.Gained Herba Dendrobii total polysaccharides composition is mainly high physiologically active polysaccharide, starch-polysaccharides, pectin polysaccharide, protein etc., and the high reactivity polysaccharide content wherein with drug action is not very high.These report methods are more but still be weak, and still need to improve in extraction yield and purity.The present invention extracts and adopts the fresh stem of noble dendrobium to be that raw material extracts, and improves yield.Dry dendrobium polysaccharide in the stem of noble dendrobium to be heated generation intermolecular condensation, par-tial polysaccharide component damage water-soluble reduces, and extraction yield reduces.
Summary of the invention
The object of the present invention is to provide a kind of preparation method of Herba Dendrobii extract, the method with the fresh bar of Herba Dendrobii for raw material, juice adopts neutral protein enzymic fermentation, after fermented liquid first removes the impurity such as starch after quick-frozen is thawed naturally, use storng-acid cation exchange resin and weak base anion-exchange resin process to remove the solution of starch, finally adopt that titanium rod strainer is concentrated, alcohol precipitation, centrifugal, collect supernatant liquor lyophilize and obtain Herba Dendrobii extract.
The concrete operations of aforesaid method are as follows:
(1) add pure water in the fresh bar of the Herba Dendrobii after cleaning and smash squeezing to pieces, amount of water is 10 ~ 12 times of fresh bar quality, filters to obtain the fresh juice of the stem of noble dendrobium after squeezing the juice;
(2) in step (1) excessively filter residue, add the water filtering slag amount 3 ~ 5 times and extract 1 ~ 2 time at 90 ~ 95 DEG C, filtering merging filtrate after 40 ~ 60 minutes extraction times, abandoned filter residue;
(3) the fresh juice of combining step (1) stem of noble dendrobium and step (2) filtrate, adds the neutral protease of amalgamation liquid quality 0.03 ~ 0.05% in 45 ~ 60 DEG C of insulations 1 ~ 2 hour, then normal temperature standing for fermentation 12 ~ 16 hours in amalgamation liquid;
(4) naturally thaw after step (3) fermented liquid quick freeze again, make age of starch precipitation in extract; Collect supernatant liquor, remainder is centrifugal, collects centrifugal liquid and is incorporated in supernatant liquor;
(5) step (4) liquid is crossed strong acid cation exchange resin column, collect flowing liquid;
(6) step (5) effluent liquid is crossed weak base anion-exchange resin post, collect colourless effluent liquid;
(7) step (6) effluent liquid adopts titanium rod strainer to concentrate, till being concentrated to concentrated front liquid volume 1/3 ~ 1/4;
(8) mass percent concentration adding concentrated solution volume 4 ~ 5 times in step (7) concentrated solution be 95% ethanol carry out alcohol precipitation spend the night, centrifugal, collect supernatant liquor, supernatant liquor lyophilize obtains Herba Dendrobii extract.
Described titanium rod 0.2 ~ 3 micron, filter element aperture.
Another object of the present invention is applied to by the Herba Dendrobii extract that aforesaid method is obtained in preparation treatment wound healing medicine.
Application of the present invention is for activeconstituents is in preparation treatment wound healing medicine with above-mentioned extract.
One or more pharmaceutically acceptable auxiliary materials can also be added in application of the present invention, described auxiliary material comprises the weighting agent of pharmaceutical field routine, thinner, tackiness agent, vehicle, absorption enhancer, weighting agent, tensio-active agent and stablizer etc., also can add flavouring agent, pigment and sweeting agent etc. if desired.
Application of the present invention, except making capsule, can also make the various ways such as pill, pulvis, tablet, granula, oral liquid and injection liquid.
The application's crude extract employing enzymic fermentation spends the night, freeze thawing is degraded into small molecules class impurity centrifugal segregation impurity constituents such as macro-molecular protein, pectin, phlegmatic temperament, starch-polysaccharides; Cation seperation column is used to slough amino acid in centrifugate, part fat-soluble pigment, ash content; Use anionite-exchange resin to remove water colo(u)r and residual small protein impurity in the stem of noble dendrobium, substitute traditional organic solvent SAVAGE method deproteinated technique and there is better industrial operation; Adopt titanium candle filter to substitute traditional heat to concentrate and expensive membrane separation plant, reduce production cost simultaneously retentive activity polysaccharide there will not be the concentrated destroyed phenomenon of heat; The extract obtained protein after testing of alcohol precipitation lyophilize, starch are residual extremely low, and this technological operation is simple, and cost is low, and industrialization level is high.
The present invention is extract obtained, and to be used for the treatment of wound healing effect better, and protein in extract, starch remove comparatively thoroughly external application spreads on the surface of a wound and breed bacteria can not become inflammatory mediator; Experimental result shows extract of the present invention and compares with Matrix controls group, and rabbit postoperative 10d surface of a wound 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.
Embodiment
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 and separation method step of this Herba Dendrobii extract is as follows:
(1) add pure water in the fresh bar of the Herba Dendrobii after cleaning and smash squeezing to pieces, amount of water is 10 times of fresh bar quality, filters to obtain the fresh juice of the stem of noble dendrobium after squeezing the juice;
(2) in step (1) excessively filter residue, add the water filtering slag amount 3 times and extract 1 time at 93 DEG C, filtering merging filtrate after 40 minutes extraction times, abandoned filter residue;
(3) the fresh juice of combining step (1) stem of noble dendrobium and step (2) filtrate, the neutral protease 45 DEG C adding amalgamation liquid quality 0.03% in amalgamation liquid is incubated 2 hours, then normal temperature standing for fermentation 12 hours;
(4) naturally thaw after step (3) fermented liq quick freeze again, make age of starch precipitation in extract; Collect supernatant liquor, remainder is centrifugal, collects centrifugal liquid and is incorporated in supernatant liquor;
(5) step (4) liquid is crossed strong acid cation exchange resin column (model D001), collect flowing liquid;
(6) step (5) effluent liquid is crossed weak base anion-exchange resin post (model D941), collect colourless effluent liquid;
(7) step (6) effluent liquid adopts titanium rod strainer to concentrate, till being concentrated to concentrated front liquid volume 1/3 (titanium rod 2 microns, filter element aperture);
(8) mass percent concentration adding concentrated solution volume 4 times in step (7) concentrated solution be 95% ethanol carry out alcohol precipitation spend the night, centrifugal, collect supernatant liquor, supernatant liquor lyophilize obtains Herba Dendrobii extract.
Following experiment is adopted to carry out therapeutic wound healing to the present embodiment extract:
1, laboratory animal: Japan large ear rabbit, 1.8 ± 0.2kg, male and female half and half, experimental animal is provided by KeYu animal cultivation center, Beijing, conformity certification number: SCXK(capital) 2012-0004;
2, the preparation of medicine: get Herba Dendrobii extract freeze-drying powder end 5g, become paste for subsequent use Herba Dendrobii extract lyophilized powder stirring and evenly mixing with water for injection;
3, experimental technique: family's rabbit back unhairing, at head side 1/3, back place, the other 1.5cm place of backbone, symmetrical design 3cm × 4cm size, the rectangle surface of a wound of major axis and spine parallel; After 1% PROCAINE HCL, PHARMA GRADE local anaesthesia, by design complete resection skin, fascia superficialis tissue, until deep fascia superficial surface.By above-mentioned rabbit 30 of making wound model, be divided into 5 groups at random, namely blank group, matrix (water for injection) control group, positive drug group, medicine low dose group, medicine high dose group; Often organize 6, from operation day, be coated with step 2 medicine outward by the design dosage surface of a wound, surface sterile gauze wrapping, every day changes medicine; Measure the surface of a wound size when changing dressings, calculate postoperative wound surface area and healing rate, with wound healing 95% for healing standard, record the healing days of each surface of a wound;
4, analytical procedure: represent with mean ± S.D, statistical method is one-way analysis of variance, take P<0.05 as significant 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 surface of a wound 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.
Embodiment 2: the extraction and separation method step of this Herba Dendrobii extract is as follows:
(1) add pure water in the fresh bar of the Herba Dendrobii after cleaning and smash squeezing to pieces, amount of water is 11 times of fresh bar quality, filters to obtain the fresh juice of the stem of noble dendrobium after squeezing the juice;
(2) in step (1) excessively filter residue, add the water filtering slag amount 4 times and extract 2 times at 90 DEG C, filtering merging filtrate after 50 minutes extraction times, abandoned filter residue;
(3) the fresh juice of combining step (1) stem of noble dendrobium and step (2) filtrate, the neutral protease 50 DEG C adding amalgamation liquid quality 0.04% in amalgamation liquid is incubated 1.5 hours, then normal temperature standing for fermentation 14 hours;
(4) naturally thaw after step (3) fermented liq quick freeze again, make age of starch precipitation in extract; Collect supernatant liquor, remainder is centrifugal, collects centrifugal liquid and is incorporated in supernatant liquor;
(5) step (4) liquid is crossed strong acid cation exchange resin column (model 001 × 7), collect flowing liquid;
(6) step (5) effluent liquid is crossed weak base anion-exchange resin post (model D900), collect colourless effluent liquid;
(7) step (6) effluent liquid adopts titanium rod strainer to concentrate, till being concentrated to concentrated front liquid volume 1/3;
(8) mass percent concentration adding concentrated solution volume 5 times in step (7) concentrated solution be 95% ethanol carry out alcohol precipitation spend the night, centrifugal, collect supernatant liquor, supernatant liquor lyophilize obtains Herba Dendrobii extract.
Following experiment is adopted to carry out therapeutic wound healing to the present embodiment extract:
1, laboratory animal: Japan large ear rabbit, 1.8 ± 0.2kg, male and female half and half, experimental animal is provided by KeYu animal cultivation center, Beijing, conformity certification number: SCXK(capital) 2012-0004;
2, the preparation of medicine: get Herba Dendrobii extract freeze-drying powder end 5g, become paste for subsequent use Herba Dendrobii extract lyophilized powder stirring and evenly mixing with water for injection;
3, experimental technique: family's rabbit back unhairing, at head side 1/3, back place, the other 1.5cm place of backbone, symmetrical design 3cm × 4cm size, the rectangle surface of a wound of major axis and spine parallel; After 1% PROCAINE HCL, PHARMA GRADE local anaesthesia, by design complete resection skin, fascia superficialis tissue, until deep fascia superficial surface.By above-mentioned rabbit 30 of making wound model, be divided into 5 groups at random, namely blank group, matrix (water for injection) control group, positive drug group, medicine low dose group, medicine high dose group; Often organize 6, from operation day, be coated with step 2 medicine outward by the design dosage surface of a wound, surface sterile gauze wrapping, every day changes medicine; Measure the surface of a wound size when changing dressings, calculate postoperative wound surface area and healing rate, with wound healing 95% for healing standard, record the healing days of each surface of a wound;
4, analytical procedure: represent with mean ± S.D, statistical method is one-way analysis of variance, take P<0.05 as significant 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 surface of a wound 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.
Embodiment 3: the extraction and separation method step of this Herba Dendrobii extract is as follows:
(1) add pure water in the fresh bar of the Herba Dendrobii after cleaning and smash squeezing to pieces, amount of water is 12 times of fresh bar quality, filters to obtain the fresh juice of the stem of noble dendrobium after squeezing the juice;
(2) in step (1) excessively filter residue, add the water filtering slag amount 5 times and extract 1 time at 95 DEG C, filtering merging filtrate after 60 minutes extraction times, abandoned filter residue;
(3) the fresh juice of combining step (1) stem of noble dendrobium and step (2) filtrate, the neutral protease 60 DEG C adding amalgamation liquid quality 0.05% in amalgamation liquid is incubated 1 hour, then normal temperature standing for fermentation 16 hours;
(4) naturally thaw after step (3) fermented liq quick freeze again, make age of starch precipitation in extract; Collect supernatant liquor, remainder is centrifugal, collects centrifugal liquid and is incorporated in supernatant liquor;
(5) step (4) liquid is crossed strong acid cation exchange resin column (model C D350), collect flowing liquid;
(6) step (5) effluent liquid is crossed weak base anion-exchange resin post (model 700B), collect colourless effluent liquid;
(7) step (6) effluent liquid adopts titanium rod strainer to concentrate, till being concentrated to concentrated front liquid volume 1/4;
(8) mass percent concentration adding concentrated solution volume 4 times in step (7) concentrated solution be 95% ethanol carry out alcohol precipitation spend the night, centrifugal, collect supernatant liquor, supernatant liquor lyophilize obtains Herba Dendrobii extract.
Following experiment is adopted to carry out therapeutic wound healing to the present embodiment extract:
1, laboratory animal: Japan large ear rabbit, 1.8 ± 0.2kg, male and female half and half, experimental animal is provided by KeYu animal cultivation center, Beijing, conformity certification number: SCXK(capital) 2012-0004;
2, the preparation of medicine: get Herba Dendrobii extract freeze-drying powder end 5g, become paste for subsequent use Herba Dendrobii extract lyophilized powder stirring and evenly mixing with water for injection;
3, experimental technique: family's rabbit back unhairing, at head side 1/3, back place, the other 1.5cm place of backbone, symmetrical design 3cm × 4cm size, the rectangle surface of a wound of major axis and spine parallel; After 1% PROCAINE HCL, PHARMA GRADE local anaesthesia, by design complete resection skin, fascia superficialis tissue, until deep fascia superficial surface.By above-mentioned rabbit 30 of making wound model, be divided into 5 groups at random, namely blank group, matrix (water for injection) control group, positive drug group, medicine low dose group, medicine high dose group; Often organize 6, from operation day, be coated with step 2 medicine outward by the design dosage surface of a wound, surface sterile gauze wrapping, every day changes medicine; Measure the surface of a wound size when changing dressings, calculate postoperative wound surface area and healing rate, with wound healing 95% for healing standard, record the healing days of each surface of a wound;
4, analytical procedure: represent with mean ± S.D, statistical method is one-way analysis of variance, take P<0.05 as significant 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 surface of a wound 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.
Claims (3)
1. a preparation method for Herba Dendrobii extract, is characterized in that step is as follows:
(1) add pure water in the fresh bar of the Herba Dendrobii after cleaning and smash squeezing to pieces, amount of water is 10 ~ 12 times of fresh bar quality, filters to obtain the fresh juice of the stem of noble dendrobium after squeezing the juice;
(2) in step (1) excessively filter residue, add the water filtering slag amount 3 ~ 5 times and extract 1 ~ 2 time at 90 ~ 95 DEG C, filtering merging filtrate after 40 ~ 60 minutes extraction times, abandoned filter residue;
(3) the fresh juice of combining step (1) stem of noble dendrobium and step (2) filtrate, adds the neutral protease of amalgamation liquid quality 0.03 ~ 0.05% in 45 ~ 60 DEG C of insulations 1 ~ 2 hour, then normal temperature standing for fermentation 12 ~ 16 hours in amalgamation liquid;
(4) naturally thaw after step (3) fermented liquid quick freeze again, make age of starch precipitation in extract; Collect supernatant liquor, remainder is centrifugal, collects centrifugal liquid and is incorporated in supernatant liquor;
(5) step (4) liquid is crossed strong acid cation exchange resin column, collect flowing liquid;
(6) step (5) effluent liquid is crossed weak base anion-exchange resin post, collect colourless effluent liquid;
(7) step (6) effluent liquid adopts titanium rod strainer to concentrate, till being concentrated to concentrated front liquid volume 1/3 ~ 1/4;
(8) mass percent concentration adding concentrated solution volume 4 ~ 5 times in step (7) concentrated solution be 95% ethanol carry out alcohol precipitation spend the night, centrifugal, collect supernatant liquor, supernatant liquor lyophilize obtains Herba Dendrobii extract.
2. the preparation method of Herba Dendrobii extract according to claim 1, is characterized in that: titanium rod 0.2 ~ 3 micron, filter element aperture.
3. the Herba Dendrobii extract that the preparation method of Herba Dendrobii extract according to claim 1 obtains treats the application in wound healing medicine in preparation.
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