CN101429254B - Bletilla striata polysaccharide, preparation method and new uses thereof - Google Patents
Bletilla striata polysaccharide, preparation method and new uses thereof Download PDFInfo
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Abstract
The invention discloses bletilla polysaccharide, a preparation method and novel application thereof. The bletilla polysaccharide is prepared by the following method: firstly, coarse bletilla powder is taken as a raw material, and is extracted by alcohol; secondly, herb residue is extracted by water; thirdly, resin is used for removing proteins of a concentrated solution; and fourthly, after an eluent is condensed, the bletilla polysaccharide is precipitated by the ethanol. The content of the bletilla polysaccharide prepared by the method is high, reaches 70 to 74 percent, and is improved by 14 to 20 percent compared with the prior technical method, and the bletilla polysaccharide has good protein removal effect. The preparation method can also utilize the herb residue produced in the process of production of bletilla particle medicines as the raw material, so as to save the production cost. The invention also provides the novel application of the bletilla polysaccharide to preparation of medicines for treating gastric ulcer and medicines for treating silicosis and a commonly used medical preparation formulation.
Description
Technical field
The present invention relates to field of pharmaceutical technology, the new purposes of particularly a kind of preparation of bletilla polysaccharide, and bletilla polysaccharide aspect preparation treatment stomach ulcer and silicosis medicine.
Background technology
Bletilla is the dry tuber of orchid bletilla (Bletilla Striata).The bletilla stem tuber through the water extract-alcohol precipitation gained-kind of mixed polysaccharide, this mixed polysaccharide is called Rhizoma Bletillae gel, Rhizoma Bletillae gel claims bletilla polysaccharide, bletilla mannan again.
Bletilla (being the bletilla stem tuber) has the effect of astringing to arrest bleeding, clearing heat and promoting diuresis, detumescence and promoting granulation.Be widely used in diseases such as treatment hemoptysis, haematemesis, traumatic hemorrhage, sore swollen toxin, chapped skin, pulmonary tuberculosis hemoptysis, ulcer haemorrhage clinically; Also be developed into plasma substitute, ultrasonic coupling agent, hepatic artery embolism agent etc. (Li Weiyong etc., the development of bletilla microballoon and hepatic artery embolism experimental study [J] thereof. Tongji Medical Univ's journal, 1999,1.).Bletilla polysaccharide is commonly used for film forming material of suspending agent in the liquid preparation, emulsifying agent, film etc. in preparation, also have the report that is developed to common bletilla tuber hellow capsule (Zhou Tao, non-gelatin Capsules, gelatin science and technology [J] .2005,3.).
The medicinal part of bletilla is a stem tuber, and main active ingredient has steroid class, terpene, polysaccharide etc.Bletilla polysaccharide is main water soluble component, has the artery embolization for treatment tumour, and effect such as enhancing immunity (Wang Chunhong etc., the medicinal research [J] of bletilla polysaccharide. Chinese new pharmaceutical, 2003,2 (5); And document: Zhang Yu etc., Radix Glycyrrhizae and bletilla antagonism trypterygine to the experimental study [J] of gastric stimulation and immunosuppressive action. Chinese pharmacist, 1999,2 (4) .), but not about the report of bletilla polysaccharide in the new purposes aspect preparation treatment stomach ulcer and the silicosis medicine.
The extracting method of bletilla polysaccharide mainly is to obtain through water extract-alcohol precipitation with bletilla raw medicinal herbs stem tuber in the prior art, but exist removal of impurities incomplete, the polysaccharide impurity that obtains is more, color is relatively poor, be unfavorable for the further purifying of polysaccharide, the content of polysaccharide is about about 50%, (Liu Guangbin etc., bletilla polysaccharide Study on extraction process [J]. contemporary Chinese is used pharmacy, 2007,24 (4)), and the preparation method of bletilla polysaccharide of the present invention is through alcohol extracting, water extraction, spent ion exchange resin purifying, alcohol precipitation are further refining again obtains bletilla polysaccharide, and its product impurity is few, the purity height.
The preparation method of bletilla polysaccharide of the present invention also can utilize produce in the pharmaceutical production of bletilla particle the medicine waste residue be raw material, directly use water extraction, obtain bletilla polysaccharide through purifying of the present invention, treating process.It is advantageous that and directly do not adopt the bletilla raw medicinal herbs to extract, but utilize the medicine waste residue on producing, can make full use of Bletilla striata medicinal materials, save production cost.
The pharmaceutical production of bletilla particle is the granule that is prepared into through 45%～95% extraction using alcohol with bletilla raw medicinal herbs stem tuber, the waste residue that produces during it is produced promptly is the medicine waste residue that produces in the pharmaceutical production of bletilla particle, that is to say that the medicine waste residue that produces in the pharmaceutical production of bletilla particle is the medicine residue that bletilla raw medicinal herbs stem tuber is left, and is mainly water soluble component behind 45%～95% extraction using alcohol.
Summary of the invention
One of purpose of the present invention is that will to solve in the prior art bletilla polysaccharide impure more, and color is relatively poor, and the defective of the content of polysaccharide lower (being about about 50%) provides a kind of content height of polysaccharide, and impurity is few, the bletilla polysaccharide that color is good.
Two of purpose of the present invention is that will to solve in the prior art removal of impurities that the water extraction and alcohol precipitation method of main preparation bletilla polysaccharide exists incomplete, the polysaccharide impurity that obtains is more, color is relatively poor, be unfavorable for the further purifying of polysaccharide, the defective that the content of polysaccharide is lower, and provide extracting method more simple and easy to do, the preparation method of better, the reliable bletilla polysaccharide of quality product.
The preparation method of bletilla polysaccharide of the present invention can also be a raw material with the medicine waste residue that produces in the pharmaceutical production of bletilla particle, has solved the problem that the medicine waste residue that produces in the pharmaceutical production of bletilla particle is wasted, and saves production cost.
Three of purpose of the present invention provides the new purposes of bletilla polysaccharide at preparation treatment stomach ulcer medicine.
Four of purpose of the present invention provides the new purposes of bletilla polysaccharide at preparation treatment silicosis medicine.
The pharmaceutical formulation that five of purpose of the present invention provides bletilla polysaccharide of the present invention and treats the silicosis medicine in preparation treatment stomach ulcer medicine and preparation.
The present invention's beneficial effect compared with prior art is:
1, the present invention uses earlier alcohol extracting with the meal of bletilla raw medicinal herbs stem tuber, bletilla polysaccharide is suggested hardly, and water is carried again, and the polysaccharide content that obtains is higher, its content is 70%～74%, is that water extract-alcohol precipitation improves 14～20% than the main method of bletilla polysaccharide in the prior art.
2, the present invention uses alcohol extracting earlier with the meal of bletilla raw medicinal herbs stem tuber, can remove a large amount of pigments and oil-soluble impurities, and bletilla polysaccharide is suggested hardly, helps the further purifying of polysaccharide.
3, the present invention is in preparation bletilla polysaccharide process, adopt ion exchange resin to carry out the novel method of purifying, it is more complete to remove albumen, and then further refining through alcohol precipitation, and the bletilla polysaccharide of gained is impure few, the purity height, it is more thorough that more traditional Sevag method or TCA method are removed albumen, surveys the bletilla polysaccharide of the present invention's preparation with the Coomassie brilliant blue method, can not survey protein content, and after removing albumen with Sevag method or TCA method, measuring protein content with the Coomassie brilliant blue method is 1～2%.
4, the present invention also can solve the problem that the medicine waste residue that produces in the pharmaceutical production of bletilla particle is wasted, and has saved production cost.Promptly utilize the medicine waste residue that produces in the pharmaceutical production of bletilla particle directly to prepare bletilla polysaccharide with the 2nd step in the inventive method and each later step.
New purposes of the present invention is to treat the new purposes of the stomach ulcer medicine that is caused by multiple reason and the new purposes for the treatment of the silicosis medicine in preparation with bletilla polysaccharide of the present invention in preparation, test shows: bletilla polysaccharide of the present invention can promote the ulcer healing of ulcer rat, and is certain dose-effect relationship; Can also delay or suppress the development of silicosis pathology, can obviously suppress the pulmonary fibrosis of silicosis rat, alleviate the lung weight in wet base.The bletilla polysaccharide of the present invention's preparation can be used for preparing formulations such as chewable tablet, syrup.
The present invention is through alcohol extracting, and water extraction, spent ion exchange resin purifying, alcohol precipitation are further refining again obtains bletilla polysaccharide, carries out assay then, carries out new Ergonomy test again and is processed into various formulations, and its concrete technical scheme is as follows:
One, bletilla polysaccharide of the present invention obtains with following extracting and purifying method:
1, get bletilla raw medicinal herbs stem tuber, be ground into meal, doubly measure 40-95% ethanol cold soaking or refluxing extraction 2-3 time with the 4-10 of its meal weight, the dregs of a decoction after the extraction do not dry to there not being the alcohol flavor;
2, dry to the dregs of a decoction that do not have the alcohol flavor above-mentioned, 60～80 ℃ of water temperature lixiviates of doubly measuring with the 10-30 of its weight 1-3 hour, or boil and carry 1-3 hour, extract united extraction liquid, filtration 2-3 time;
3, above-mentioned filtrate is passed through macroporous type or gel-type weak anion resin and macroporous type or gel-type acidulous cation resin, or by macroporous type or gel-type acidulous anion resin and macroporous type or gel-type weakly alkaline resin cation (R.C.), use the deionized water wash-out, collect elutriant;
4, above-mentioned elutriant is concentrated into relative density 1.1～1.5, adds ethanol and reach 30%～85%, be settled out bletilla polysaccharide to containing the alcohol amount, collecting precipitation, precipitation will precipitate further vacuum-drying and promptly get bletilla polysaccharide with 60%～80% washing with alcohol 1～3 time.
Above-mentioned steps 1 can be direct not drying to there being alcohol with the medicine waste residue that produces in the pharmaceutical production of bletilla particle and distinguishes the flavor of; Carry out the 2nd step and each later step subsequently successively, promptly do not need the medicine waste residue in the big production of bletilla particle medicine is doubly measured 40-95% ethanol cold soaking or refluxing extraction 2-3 time with the 4-10 of its weight.The medicine waste residue that produces in the pharmaceutical production of described bletilla particle is the medicine waste residue that produces behind 45%～95% extraction using alcohol with bletilla raw medicinal herbs stem tuber, below identical, repeat no more.
Above-mentioned steps 3 described filtrates are by macroporous type or gel-type weak anion resin and macroporous type or gel-type acidulous cation resin, perhaps, can or pass through macroporous type or gel-type acidulous cation resin again by macroporous type or gel-type weak anion resin earlier by macroporous type or gel-type acidulous anion resin and macroporous type or gel-type weakly alkaline resin cation (R.C.); Or pass through macroporous type or gel-type weakly alkaline resin cation (R.C.) again by macroporous type or gel-type acidulous anion resin earlier; Or pass through macroporous type or gel-type weak anion resin again by macroporous type or gel-type acidulous cation resin earlier; Or pass through macroporous type or gel-type acidulous anion resin again by macroporous type or gel-type weakly alkaline resin cation (R.C.) earlier; Or the hybrid resin post by macroporous type or gel-type weak anion resin and macroporous type or gel-type acidulous cation resin; Or the hybrid resin post by macroporous type or gel-type acidulous anion resin and macroporous type or gel-type weakly alkaline resin cation (R.C.).
Two, the preparation method of bletilla polysaccharide
1, the preparation method of bletilla polysaccharide, carry out according to the following steps:
(1) get bletilla raw medicinal herbs stem tuber, be ground into meal, doubly measure 40-95% ethanol cold soaking or refluxing extraction 2-3 time with the 4-10 of its meal weight, the dregs of a decoction after the extraction do not dry to there not being the alcohol flavor;
(2) dry to the dregs of a decoction that do not have the alcohol flavor above-mentioned, 60～80 ℃ of water temperature lixiviates of doubly measuring with the 10-30 of its weight 1-3 hour, or boil and carry 1-3 hour, extract united extraction liquid, filtration 2-3 time;
(3) above-mentioned filtrate is passed through macroporous type or gel-type weak anion resin and macroporous type or gel-type acidulous cation resin, or by macroporous type or gel-type acidulous anion resin and macroporous type or gel-type weakly alkaline resin cation (R.C.), use the deionized water wash-out, collect elutriant;
(4) above-mentioned elutriant being concentrated into relative density is 1.1～1.5, add ethanol and reach 30%～85%, be settled out bletilla polysaccharide, collecting precipitation to containing the alcohol amount, precipitation will precipitate further vacuum-drying and promptly get the bletilla polysaccharide powder with 60%～80% washing with alcohol 1～3 time.
Above-mentioned steps (1) can be direct not drying to there being alcohol with the medicine waste residue that produces in the pharmaceutical production of bletilla particle and distinguishes the flavor of, carry out the 2nd step and each later step subsequently successively, promptly do not need the medicine waste residue that to produce in the pharmaceutical production of bletilla particle doubly to measure 40-95% ethanol cold soaking or refluxing extraction 2-3 time with the 4-10 of its weight.
Above-mentioned steps 3 described filtrates are by macroporous type or gel-type weak anion resin and macroporous type or gel-type acidulous cation resin, perhaps, can or pass through macroporous type or gel-type acidulous cation resin again by macroporous type or gel-type weak anion resin earlier by macroporous type or gel-type acidulous anion resin and macroporous type or gel-type weakly alkaline resin cation (R.C.); Or pass through macroporous type or gel-type weakly alkaline resin cation (R.C.) again by macroporous type or gel-type acidulous anion resin earlier; Or pass through macroporous type or gel-type weak anion resin again by macroporous type or gel-type acidulous cation resin earlier; Or pass through macroporous type or gel-type acidulous anion resin again by macroporous type or gel-type weakly alkaline resin cation (R.C.) earlier; Or the hybrid resin post by macroporous type or gel-type weak anion resin and macroporous type or gel-type acidulous cation resin; Or the hybrid resin post by macroporous type or gel-type acidulous anion resin and macroporous type or gel-type weakly alkaline resin cation (R.C.).
2, the assay of the bletilla polysaccharide that obtains of the preparation method of above-mentioned bletilla polysaccharide: (promptly pressing the sulfuric acid-phynol ordinary method measures)
(1) preparation of reference substance solution: precision takes by weighing 100 ℃ of dextrose anhydrous 100mg that are dried to constant weight, put in the 100ml volumetric flask, be dissolved in water and be diluted to scale, shake up, precision is measured 2ml and is placed the 50ml volumetric flask again, thin up shakes up promptly to scale, and every 1ml contains dextrose anhydrous 40 μ g;
(2) making of typical curve: precision is measured reference substance solution 0.0ml, 0.2ml, 0.4ml, 0.6ml, 0.8ml, 1.0ml, puts respectively in the tool plug test tube, adds 6% phenol solution 1.2ml respectively, mixing, add sulfuric acid 6.5ml rapidly, shake up, left standstill 30 minutes, after the cooling bath cooling, with first part be blank, measure optical density (TU1800 ultraviolet spectrophotometer) at the wavelength place of 490nm, be ordinate zou with the optical density, concentration is X-coordinate, the drawing standard curve;
(3) preparation of need testing solution: precision takes by weighing 80 ℃ of testing sample 100mg that contain bletilla polysaccharide that are dried to constant weight, puts in the 100ml volumetric flask, and being dissolved in water is diluted to scale, shake up, precision is measured 2ml and is placed the 50ml volumetric flask again, and thin up shakes up promptly to scale;
(4) assay: precision is measured need testing solution 0.6ml, method according to the making of typical curve in (2), from adding 6% phenol 1.2ml, measure optical density (TU1800 ultraviolet spectrophotometer) according to aforesaid method at the wavelength place of 490nm and measure optical density, from the concentration that typical curve is read bletilla polysaccharide the need testing solution, calculate the content of bletilla polysaccharide.Polysaccharide content %=C * D * 1/W * 100%, wherein: C-sample concentration, D-extension rate, the heavy mg of W-sample.
Three, above-mentioned bletilla polysaccharide is in the new purposes of preparation treatment stomach ulcer medicine
Adopt following diverse ways to prepare the rat gastric ulcer model, observe the new purposes of bletilla polysaccharide at preparation treatment stomach ulcer medicine.
1, experiment material:
(1) laboratory animal
The SD rat, male and female half and half, body weight 180～220g is provided by unming Medical College's Experimental Animal Center, animal conformity certification SCXK (Yunnan) 2005-0008.
(2) medicine and reagent
Bletilla polysaccharide: by research and development department of Yunnan Plant Pharmaceutical Industry Co., Ltd. by the preparation method of above-mentioned bletilla polysaccharide preparation and provide.
Cimitidine Type A/AB capsule: Guangdong Taicheng Pharmaceutical Co., Ltd, lot number: 20080302;
Vetanarcol: Chemical Reagent Co., Ltd., Sinopharm Group, lot number: WB020060413.
(3) instrument
LXJ2I type whizzer: Shanghai medical analytical instrument factory;
Three use the electric heating constant water bath box: Chang'an, Beijing scientific instrument factory;
Vortex mixer: Zhejiang Le Cheng electrical apparatus factory.
2. experimental technique
(1) pyloric ligation ulcers gastric ulcer model
Get 60 of rats, male and female half and half are divided into 6 groups at random, 10 every group, are respectively: sham operated rats, model group, positive controls, high, medium and low three the dosage groups of bletilla polysaccharide.
Each organizes rat administration as follows, every day 1 time, continuous 5 days.Positive controls is irritated stomach and is given Cimitidine Type A/AB 0.1g/kg, and the high, medium and low dosage group of bletilla polysaccharide is irritated stomach respectively and given bletilla polysaccharide 400mg/kg, 200mg/kg and 100mg/kg, and model group is irritated stomach and given equivalent physiological saline (1mL/100g).1h after the last administration, and each treated animal reference literature method (Xu Shuyun, pharmacological experimental methodology [M]. the 3rd edition, People's Health Publisher, 2003; And document: Lin Ji etc., the research [J] of capsule for enterogastric disease anti-experimental character stomach ulcer effect. new Chinese medicine and clinical pharmacology, 2005,7,16 (4): 256～259.) modeling: water is can't help in the 48h fasting before the operation, with vetanarcol 30mg/kg (the administration volume is 1mL/100g) intraperitoneal injection of anesthesia, routine disinfection skin of abdomen, ventrimeson is opened abdomen in the ensiform process of sternum lower edge, exposes stomach.The descending pylorus ligation of stomach pylorus; Simultaneously through duodenal administration 1 time, model group gives isometric physiological saline, sews up stomach wall then, finishes operation.Sham operated rats is operated but not ligation pylorus equally.Prohibit the water fasting behind the pylorus ligation, take off cervical vertebra behind the 18h and put to death rat, cut open the belly and get stomach, folder closes cardiac end, injects 1% formaldehyde solution 8mL from the pylorus end, folder closes the pylorus end, and stomach immersed fixedly 10min of 1% formaldehyde solution, and cut off coat of the stomach along greater gastric curvature behind the 10min, keep the score by the Guth standard, a situation arises to observe stomach ulcer, calculates ulcer index and ulcer and suppress percentage.
(2) acetate burns the type gastric ulcer model
Get 60 of rats, male and female half and half, fasting is 24 hours before the experiment, freely drink water, with vetanarcol 30mg/kg (the administration volume is 1mL/100g) intraperitoneal injection of anesthesia, the routine disinfection skin of abdomen, ventrimeson is opened abdomen in the ensiform process of sternum lower edge, exposes stomach.The reference literature method (Qu Chunying etc., chitosan is to the influence [J] of MDA, SOD and GSH-PX in the stomach ulcer rat blood serum. Shanghai medicine, 2008,29 (5): 219～222; And document: Ye Murong etc., Zhong Jing stomach curing capsule anti-gastric-ulcer effect research [J]. Chinese patent medicine, 2002,24 (9): 692～694) modeling: 20% acetic acid solution 0.1mL is injected under the glandular stomach portion antetheca hole body intersection serous coat causes damage model, sew up stomach wall then, finish operation.Postoperative normal diet, drinking-water were divided into 5 groups at random with it on the 2nd day, 10 every group, were respectively: model group, positive controls, high, medium and low three the dosage groups of bletilla polysaccharide; Other gets 10 rats as sham operated rats, and sham operated rats is operated equally but do not injected acetate.
Each organizes rat administration as follows, every day 1 time, continuous 12 days.Positive controls is irritated stomach and is given Cimitidine Type A/AB 0.1g/kg, and the high, medium and low dosage group of bletilla polysaccharide is irritated stomach respectively and given bletilla polysaccharide 400mg/kg, 200mg/kg and 100mg/kg, and model group is irritated stomach and given equivalent physiological saline (1mL/100g).Took off cervical vertebra after the administration on the 13rd day and put to death rat, dissect and get stomach, ligation pylorus and orifice of the stomach extract full stomach, with the fixing 5min of 1% formalin solution; Stomach is cut, open and flat on sheet glass with distilled water eccysis content, observe the gastric mucosa degree of impairment, keep the score by the Guth standard, calculate ulcer index and ulcer and suppress percentage.
(3) ethanol causes the gastric mucosa injury model
Get 60 of rats, male and female half and half are divided into 6 groups at random, 10 every group, are respectively: normal control group, model group, positive controls, high, medium and low three the dosage groups of bletilla polysaccharide.
Each organizes rat administration as follows, every day 1 time, continuous 5 days.Positive controls is irritated stomach and is given Cimitidine Type A/AB 0.1g/kg, and the high, medium and low dosage group of bletilla polysaccharide is irritated stomach respectively and given bletilla polysaccharide 400mg/kg, 200mg/kg and 100mg/kg, and model group is irritated stomach and given equivalent physiological saline (1mL/100g).1h after the last administration, each treated animal reference literature method (Lin Ji etc., the research [J] of capsule for enterogastric disease anti-experimental character stomach ulcer effect. new Chinese medicine and clinical pharmacology, 2005,7,16 (4): 256～259) modeling: before the experiment, water 48h is can't help in the rat fasting, every rats gavaged 1mL dehydrated alcohol during experiment.Taking off cervical vertebra behind the 1h puts to death rat and cuts open the belly and get stomach, folder closes cardiac end, inject 1% formaldehyde solution 8mL from the pylorus end, folder closes the pylorus end, and stomach immersed fixedly 10min of 1% formaldehyde solution, and cut off coat of the stomach along greater gastric curvature behind the 10min, keep the score by the Guth standard, a situation arises to observe stomach ulcer, calculates ulcer index and ulcer and suppress percentage.
3, index detection method
Ulcer level evaluation: the Guth standard keeps the score-petechial hemorrhage note 1 minute; Streak-like hemorrhage, if width＜1mm, rotten to the corn length＜1mm note 2 minutes, 1～2mm note 3 minutes, 2～4mm note 4 minutes, 4mm note 5 minutes; If width〉1mm, above score value * 2.Ulcer inhibition rate: ulcer inhibition rate=(model control group ulcer index-treatment group ulcer index)/model control group ulcer index * 100%.
4, statistical method
Data are so that (x ± s) expression relatively adopts the SPSS11.5 statistical software between group, carry out Independent-SamplesT test.
Four, above-mentioned bletilla polysaccharide is in the new purposes of preparation treatment silicosis medicine
Adopt exposure formula tracheae injection method, between rat two tracheal rings, the aseptic silica powder suspension 1mL that injects 50mg/mL prepares rat silicosis model.Observe the drug action of bletilla polysaccharide at preparation treatment silicosis medicine.
1, experiment material:
(1) laboratory animal
The SD rat, male and female half and half, body weight 180～220g is provided by unming Medical College's Experimental Animal Center, animal conformity certification SCXK (Yunnan) 2005-0008.
(2) medicine and reagent
Bletilla polysaccharide: by research and development department of Yunnan Plant Pharmaceutical Industry Co., Ltd. by the preparation method of above-mentioned bletilla polysaccharide preparation and provide.
Vetanarcol: Chemical Reagent Co., Ltd., Sinopharm Group, lot number: WB020060413.
Silica powder: defending institute by China Preventive Medicial Science Institute's labor provides, and contains SiO
2More than 98% ,≤5um particulate accounts for 99%.
(3) instrument
LXJ2I type whizzer: Shanghai medical analytical instrument factory;
Three use the electric heating constant water bath box: Chang'an, Beijing scientific instrument factory;
Vortex mixer: Zhejiang Le Cheng electrical apparatus factory.
2, experimental technique
Get 50 of rats, male and female half and half are divided into 5 groups at random, 10 every group, are respectively: sham operated rats, model group, high, medium and low three the dosage groups of bletilla polysaccharide.
Each treated animal reference literature method (Xu Shuyun, pharmacological experimental methodology [M]. the 3rd edition, People's Health Publisher, 2003; And document: Zhang Zhongxing etc., spirulina is to the influence [J] of antioxidant levels in the animal silicosis model body. Chinese public health, 2005,1,21 (1): 8～9) modeling: with vetanarcol 30mg/kg (the administration volume is 1mL/100g) intraperitoneal injection of anesthesia, routine disinfection skin of neck, in neck medisection skin, expose tracheae, between two tracheal rings, thrust syringe needle, inject the aseptic silica powder suspension 1mL of 50mg/mL.Finish operation, skin suture then.Operation back the 3rd day, each organizes rat administration as follows, every day 1 time, continuous 10 weeks.The high, medium and low dosage group of bletilla polysaccharide is irritated stomach respectively and is given bletilla polysaccharide 400mg/kg, 200mg/kg and 100mg/kg, and model group is irritated stomach and given equivalent physiological saline (1mL/100g).After experiment finished, femoral artery sacrificed by exsanguination rat was got lung tissue and claims weight in wet base, does check pathological section then.
3, index detection method: lung weight in wet base, directly weighing.
4, statistical method
Data are so that (x ± s) expression relatively adopts the SPSS11.5 statistical software between group, carry out Independent-SamplesT test.
Five, the application of above-mentioned bletilla polysaccharide in the pharmaceutical formulation of preparation treatment stomach ulcer medicine and preparation treatment silicosis medicine
1, above-mentioned bletilla polysaccharide is used to prepare the bletilla polysaccharide syrup:
Described syrupy get above-mentioned bletilla polysaccharide 100-200 part, white sugar 50-100 part, honey 10-50 part; Bletilla polysaccharide dry powder adds 50-100
It is standby that purified water (W:V) doubly is mixed with the aqueous solution, with adding in the bletilla polysaccharide solution behind the white sugar heating and melting, adds honey again, stirs, and boils 30min, and it is promptly anticorrosion to add 3% Sodium Benzoate again.
2, above-mentioned bletilla polysaccharide is used to prepare the bletilla polysaccharide chewable tablet:
Get bletilla polysaccharide 100-500 part of the present invention, N.F,USP MANNITOL 30-60 part, sorbyl alcohol 60-150 part, lactose 30-80 part, aspartame 1-2 part, Citric Acid 2-5 part, sour Magnesium Stearate 3-6 part; Bletilla polysaccharide dry powder and auxiliary material are crossed 100 mesh sieves respectively, get above-mentioned recipe quantity bletilla polysaccharide, lactose, aspartame, Citric Acid an amount of (2-5 part) are heavy with N.F,USP MANNITOL and sorbitol mixture (1:2) adjustment sheet, supplementary material is mixed, with dehydrated alcohol system softwood, cross 16 mesh sieves and granulate 60 ℃ of dryings, the whole grain of 14 mesh sieves, add Magnesium Stearate, mix back 12mm drift compressing tablet, tablet hardness is controlled at 4kg/mm
2About.
3, above-mentioned bletilla polysaccharide is used to prepare the bletilla polysaccharide effervescent tablet:
Get above-mentioned bletilla polysaccharide 300-950 part, lactose 200-500 part, Steviosin 5-30 part, citric acid 100-200 part, sodium bicarbonate 100-250 part, polyethylene glycol 6000 50-120 part, Magnesium Stearate 5-12 part.After the polyethylene glycol 6000 fusion, add sodium bicarbonate 100-250 part, stir, cooling is pulverized, and crosses 80 mesh sieves; In addition citric acid, sweetener are crossed 80 mesh sieves,, granulate with medicinal powder, polyoxyethylene glycol wrap fine powder mixing, drying, compacting is in flakes promptly.
Embodiment
Below with embodiment the present invention is described in detail, can further be well understood to the present invention, but not be construed as limiting the invention.
Embodiment 1-6 is a bletilla polysaccharide product of the present invention, and its product is to obtain with extracting and purifying method, has four steps.Step (1) can be respectively with two kinds of raw materials in the bletilla polysaccharide product that each embodiment obtains, raw material 1 is for getting the meal that bletilla raw medicinal herbs stem tuber is pulverized, raw material 2 is the medicine waste residues that produce with in the pharmaceutical production of bletilla particle, the medicine waste residue that produces in this bletilla particle pharmaceutical production is the medicine waste residue that produces behind 45%～95% extraction using alcohol with bletilla raw medicinal herbs stem tuber, below identical, repeat no more.Do not need 4-10 with raw material 2 its weight of usefulness doubly to measure 40-95% ethanol cold soaking or refluxing extraction 2-3 time when using raw material 2 again, and directly dry to there not being pure the flavor with raw material 2, all the other steps (2), step (3), to reach step (4) all identical.
Embodiment 7-12 is the preparation method of bletilla polysaccharide of the present invention; The assay of the bletilla polysaccharide that embodiment 13 obtains for the preparation method of bletilla polysaccharide of the present invention; Embodiment 14 is preparation method's (comparing) of the bletilla polysaccharide of prior art; Embodiment 15 is the determining the protein quantity of the prepared bletilla polysaccharide of the preparation method of bletilla polysaccharide of the present invention and embodiment 14 (contrast); Embodiment 16 removes proteic effect comparison for the preparation method who removes albumen and bletilla polysaccharide of the present invention with the Sevag method; Embodiment 17 removes proteic effect comparison for the preparation method who removes albumen and bletilla polysaccharide of the present invention with the TCA method; Embodiment 18-20 is with the test of bletilla polysaccharide of the present invention in the new purposes of preparation treatment stomach ulcer medicine; Embodiment 21 is with the test of bletilla polysaccharide of the present invention in the new purposes of preparation treatment silicosis medicine; Embodiment 22-24 is the application of bletilla polysaccharide of the present invention in the pharmaceutical formulation of preparation treatment stomach ulcer medicine and preparation treatment silicosis medicine.
Embodiment 1 bletilla polysaccharide of the present invention is to obtain with following extracting and purifying method:
(1) get bletilla raw medicinal herbs stem tuber, be ground into meal, get meal 5kg (being raw material 1), with 10 times of its meal weight amount 95% ethanol cold soakings or refluxing extraction 2 times, the dregs of a decoction after the extraction do not dry to there not being the alcohol flavor;
(2) dry to the dregs of a decoction that do not have the alcohol flavor above-mentioned, use 60 ℃ of water temperature lixiviates 3 hours of 10 times of amounts of its weight again, extracts united extraction liquid, filtration 3 times;
(3) above-mentioned filtrate is passed through macroporous type weak anion resin and macroporous type acidulous cation resin hybrid resin post, use the deionized water wash-out, collect elutriant;
(4) above-mentioned elutriant is concentrated into relative density 1.1, add dehydrated alcohol and reach 60%, be settled out bletilla polysaccharide, collecting precipitation to containing the alcohol amount, precipitation promptly gets bletilla polysaccharide powder (seeing Table 2) with 80% concentration ethanol washing 2 times, the further vacuum-drying of precipitation after its washing.
Use raw material 2 in the acquisition of above-mentioned bletilla polysaccharide, during promptly with the medicine waste residue 5kg that produces in the pharmaceutical production of bletilla particle, its bletilla polysaccharide obtains with following extracting and purifying method:
(1) dries to there not being the alcohol flavor with the medicine waste residue that produces in the pharmaceutical production of bletilla particle;
(2) dry to the dregs of a decoction that do not have the alcohol flavor above-mentioned, use 60 ℃ of water temperature lixiviates 3 hours of 10 times of amounts of its weight again, extracts united extraction liquid, filtration 3 times;
(3) above-mentioned filtrate is passed through macroporous type weak anion resin and macroporous type acidulous cation resin hybrid resin post, use the deionized water wash-out, collect elutriant;
(4) above-mentioned elutriant is concentrated into relative density 1.1, add dehydrated alcohol and reach 60%, be settled out bletilla polysaccharide, collecting precipitation to containing the alcohol amount, precipitation promptly gets bletilla polysaccharide powder (seeing Table 2) with 80% concentration ethanol washing 2 times, the further vacuum-drying of precipitation after its washing.
Embodiment 2-6 also is a bletilla polysaccharide product of the present invention, and it obtains method except that the listed difference measure of table 1, and all the other measures are identical with embodiment 1.
The used macroporous type weak anion resin model of the foregoing description is D280, and macroporous type acidulous cation resin model is D113; Gel-type weak anion resin model is 330, and gel-type weak anion resin model is 312, and gel-type acidulous cation resin model is 110.
The difference measure that the bletilla polysaccharide product of the present invention of table 1 embodiment 1-6 obtains
Table 2 embodiment 1-6 bletilla polysaccharide content of the present invention and conventional physical behavior thereof
Table 1 shows: bletilla polysaccharide content is higher in the bletilla polysaccharide of the present invention, and its content is 70%～74%.
The preparation method of embodiment 7 bletilla polysaccharides of the present invention (three repetitions are all done in following test, and test-results is three multiple mean values)
The preparation method of bletilla polysaccharide of the present invention, carry out according to the following steps:
(1) get bletilla raw medicinal herbs stem tuber, be ground into meal, get meal 5kg (being raw material 1), with 10 times of its meal weight amount 95% ethanol cold soakings or refluxing extraction 2 times, the dregs of a decoction after the extraction do not dry to there not being the alcohol flavor;
(2) dry to the dregs of a decoction that do not have the alcohol flavor above-mentioned, use 60 ℃ of water temperature lixiviates 3 hours of 10 times of amounts of its weight again, extracts united extraction liquid, filtration 3 times;
(3) above-mentioned filtrate is passed through macroporous type weak anion resin and macroporous type acidulous cation resin hybrid resin post, use the deionized water wash-out, collect elutriant;
(4) above-mentioned elutriant is concentrated into relative density 1.1, add dehydrated alcohol and reach 60%, be settled out bletilla polysaccharide, collecting precipitation to containing the alcohol amount, precipitation promptly gets bletilla polysaccharide powder (seeing Table 4) with 80% concentration ethanol washing 2 times, the further vacuum-drying of precipitation after its washing.
When using raw material 2 (with measuring 5kg) among the preparation method of above-mentioned bletilla polysaccharide, be that raw material 2 is that (the medicine waste residue that produces in the pharmaceutical production of used bletilla particle is the medicine waste residue that produces behind 45%～95% extraction using alcohol with bletilla raw medicinal herbs stem tuber to the medicine waste residue that produces in the pharmaceutical production of bletilla particle, below identical, repeat no more.), the preparation method of its bletilla polysaccharide, carry out according to the following steps:
(1) dries to there not being the alcohol flavor with the medicine waste residue that produces in the big production of bletilla particle medicine;
(2) dry to the dregs of a decoction that do not have the alcohol flavor above-mentioned, use 60 ℃ of water temperature lixiviates 3 hours of 10 times of amounts of its weight again, extracts united extraction liquid, filtration 3 times;
(3) above-mentioned filtrate is passed through macroporous type weak anion resin and macroporous type acidulous cation resin hybrid resin post, use the deionized water wash-out, collect elutriant;
(4) above-mentioned elutriant is concentrated into relative density 1.1, add dehydrated alcohol and reach 60%, be settled out bletilla polysaccharide, collecting precipitation to containing the alcohol amount, precipitation promptly gets bletilla polysaccharide powder (seeing Table 4) with 80% concentration ethanol washing 2 times, the further vacuum-drying of precipitation after its washing.
Embodiment 8-12 also is the preparation method of bletilla polysaccharide of the present invention, and except that the listed measure difference of table 3, all the other steps are identical with embodiment 7, repeat no more.
The preparation method's of table 3 embodiment 7-12 bletilla polysaccharide of the present invention difference measure
The assay of the bletilla polysaccharide that the preparation method of embodiment 13 bletilla polysaccharides of the present invention obtains
The assay of this bletilla polysaccharide is pressed the sulfuric acid-phynol ordinary method and is measured, and its step is as follows:
(1) preparation of reference substance solution: precision takes by weighing 100 ℃ of dextrose anhydrous 100mg that are dried to constant weight, put in the 100ml volumetric flask, be dissolved in water and be diluted to scale, shake up, precision is measured 2ml and is placed the 50ml volumetric flask again, thin up shakes up promptly to scale, and every 1ml contains dextrose anhydrous 40 μ g;
(2) making of typical curve: precision is measured reference substance solution 0.0ml, 0.2ml, 0.4ml, 0.6ml, 0.8ml, 1.0ml, puts respectively in the tool plug test tube, adds 6% phenol solution 1.2ml respectively, mixing, add sulfuric acid 6.5ml rapidly, shake up, left standstill 30 minutes, after the cooling bath cooling, with first part be blank, measure optical density (TU1800 ultraviolet spectrophotometer) at the wavelength place of 490nm, be ordinate zou with the optical density, concentration is X-coordinate, the drawing standard curve;
(3) preparation of need testing solution: precision takes by weighing 80 ℃ of testing sample 100mg that contain bletilla polysaccharide that are dried to constant weight, puts in the 100ml volumetric flask, and being dissolved in water is diluted to scale, shake up, precision is measured 2ml and is placed the 50ml volumetric flask again, and thin up shakes up promptly to scale;
(4) assay: precision is measured need testing solution 0.6ml, method according to the making of typical curve in (2), from adding 6% phenol 1.2ml, measure optical density (TU1800 ultraviolet spectrophotometer) according to aforesaid method at the wavelength place of 490nm and measure optical density, from the concentration that typical curve is read bletilla polysaccharide the need testing solution, calculate the content of bletilla polysaccharide.Polysaccharide content %=C * D * 1/W * 100%, wherein: C-sample concentration, D-extension rate, the heavy mg of W-sample.
The content of measuring the bletilla polysaccharide of the invention described above method preparation by the said determination method sees Table 4
Preparation method's (comparing) of the bletilla polysaccharide of embodiment 14 prior aries (three repetitions are all done in following test, and test-results is three multiple mean values)
Raw materials used preparation method with bletilla polysaccharide of the present invention is identical, promptly gets bletilla raw medicinal herbs (being the bletilla stem tuber), is ground into meal, gets meal as raw material, and its preparation methods steps is as follows:
(1) getting bletilla raw medicinal herbs stem tuber, be ground into meal, is raw material with the meal, and with the water boiling and extraction of 10 times of amounts of its weight 3 times, united extraction liquid concentrates;
(2), above-mentioned concentrated solution added dehydrated alcohol reach 60% to containing the alcohol amount, be settled out bletilla polysaccharide, collecting precipitation, further vacuum-drying promptly gets the bletilla polysaccharide powder.
Above-mentioned prepared bletilla polysaccharide is still used the content assaying method of the bletilla polysaccharide of embodiment 13, measures the content of its bletilla polysaccharide, and content sees Table 4.And measure its bletilla polysaccharide protein content (seeing Table 5) with the Coomassie brilliant blue method that embodiment 15 is adopted
Represent that the 4 bletilla polysaccharide preparation methods of the present invention and the content of the prepared bletilla polysaccharide of preparation method of contrast bletilla polysaccharide contrast
Table 4 shows: the prepared prepared bletilla polysaccharide content of the preparation method of bletilla polysaccharide of the present invention is higher, its content is 70%～74%, and the prepared bletilla polysaccharide content of the preparation method of prior art bletilla polysaccharide is 50～60%, and the prepared bletilla polysaccharide content of the preparation method of bletilla polysaccharide of the present invention improves 14～20% than prior art.
The determining the protein quantity of the bletilla polysaccharide that the preparation method of the preparation method of embodiment 15 bletilla polysaccharides of the present invention and embodiment 14 (contrast) bletilla polysaccharide is prepared
Measure with the Coomassie brilliant blue method, concrete steps are as follows:
(1) gets 16 test tubes, 1 flag blank, 3 give over to unknown sample, all the other test tubes are divided into two groups by order in the table, add sample, water and reagent respectively, 0,0.01,0.02,0.04,0.06,0.08,0.1ml promptly add for each test tube respectively:, to add to 0.1ml with deionized water then with the standard protein solution of 1.0mg/m1.Add 5.0ml Coomassie brilliant blue G-250 reagent respectively in last each test tube, whenever add a pipe, on vortex mixer, mix (attention is too inviolent, is difficult in order to avoid produce a large amount of bubbles eliminate) immediately.During the application of sample amount of unknown sample sees the following form the 8th, 9,10 pipe.
(2) add reagent after 2～5 minutes, can begin to use cuvette, measure the absorbance value A595 of each sample at the 595nm place on spectrophotometer, blank is No. 1 test tube, and promptly 0.1mlH2O adds 5.0mlG-250 reagent.
(3) being X-coordinate with standard protein quality (mg), is ordinate with absorbance A595, and mapping promptly obtains a typical curve.Typical curve according to the A595 value of the unknown sample of measuring, can be found the protein content of unknown sample thus.(A595 of 0.5mg bovine serum albumin/ml solution is about 0.50)
The preparation method of the preparation method of bletilla polysaccharide of the present invention and the bletilla polysaccharide of prior art removes the albumen effect and sees table 5 for details.
Embodiment 16 usefulness Sevag methods remove albumen and bletilla polysaccharide preparation method of the present invention is as follows except that proteic concrete steps with the Sevag method except that proteic effect comparison:
In separating funnel, add the chloroform of 0.2 times of amount bletilla polysaccharide liquor capacity and the propyl carbinol mixing vibration of 0.04 times of volume and separate half an hour, till the interface of chloroform and water does not have precipitation, the protein that re-treatment could effectively be removed in the polysaccharide for 2-3 time.
The preparation method that embodiment 17 usefulness TCA methods are removed albumen and bletilla polysaccharide of the present invention removes proteic effect comparison
It is as follows to remove proteic concrete steps with the TCA method:
Take by weighing Crude polysaccharides 10g, be made into the 100mL aqueous solution, add 5% trichoroacetic acid(TCA) solution, left standstill the centrifuging and taking supernatant liquor 4 hours with the 1:1 equal-volume; Take out the part supernatant liquor, add 95% ethanol and reach 80% to containing the alcohol amount, standing over night, throw out is through absolute ethanol washing final vacuum drying, Crude polysaccharides.
The preparation method of the preparation method of table 5 bletilla polysaccharide of the present invention and prior art bletilla polysaccharide removes the albumen effect relatively
| Preparation method's (summary) | Polysaccharide protein assay (removing the albumen effect) | |
| The preparation method of prior art bletilla polysaccharide | (1) be raw material with the bletilla meal, use water boiling and extraction, united extraction liquid concentrates; (2) concentrated solution goes out bletilla polysaccharide with ethanol sedimentation; (3) Sevag method or TCA method are removed albumen. | It is 1～2% that the Coomassie brilliant blue method detects protein content |
| The preparation method of bletilla polysaccharide of the present invention | (1) is raw material with the bletilla meal, uses extraction using alcohol; (2) dregs of a decoction are used water extraction again, and united extraction liquid concentrates; (3) concentrated solution resin Deproteinization; (4) elutriant goes out bletilla polysaccharide with ethanol sedimentation after concentrating. | The Coomassie brilliant blue method can not surveyed protein content. |
Following examples 18-20 is with the new purposes experimental example of bletilla polysaccharide of the present invention at preparation treatment stomach ulcer medicine.
Adopt following three kinds of methods to prepare the rat gastric ulcer model, observe the new purposes of bletilla polysaccharide at preparation treatment stomach ulcer medicine.Used experiment material, index detection method, the statistical method of embodiment 18-20 is as follows:
1, experiment material
(1) laboratory animal
The SD rat, male and female half and half, body weight 180～220g is provided by unming Medical College's Experimental Animal Center, animal conformity certification SCXK (Yunnan) 2005-0008.
(2) medicine and reagent
Bletilla polysaccharide: by the preparation method of above-mentioned bletilla polysaccharide preparation and provide, embodiment 18 used bletilla polysaccharide content are 72.5% by research and development department of Yunnan Plant Pharmaceutical Industry Co., Ltd.; Embodiment 19 used bletilla polysaccharide content are 70.5%; Embodiment 20 used bletilla polysaccharide content are 71.5%;
Cimitidine Type A/AB capsule: Guangdong Taicheng Pharmaceutical Co., Ltd, lot number: 20080302;
Vetanarcol: Chemical Reagent Co., Ltd., Sinopharm Group, lot number: WB020060413.
(3) instrument
LXJ2I type whizzer: Shanghai medical analytical instrument factory;
Three use the electric heating constant water bath box: Chang'an, Beijing scientific instrument factory;
Vortex mixer: Zhejiang Le Cheng electrical apparatus factory.
2, index detection method
Ulcer level evaluation: the Guth standard keeps the score-petechial hemorrhage note 1 minute; Streak-like hemorrhage, if width＜1mm, rotten to the corn length＜1mm note 2 minutes, 1～2mm note 3 minutes, 2～4mm note 4 minutes, 4mm note 5 minutes; If width〉1mm, above score value * 2.Ulcer inhibition rate: ulcer inhibition rate=(model control group ulcer index-treatment group ulcer index)/model control group ulcer index * 100%.
3, statistical method
Data are so that (x ± s) expression relatively adopts the SPSS11.5 statistical software between group, carry out Independent-SamplesT test.
Embodiment 18 bletilla polysaccharides of the present invention are at the pharmacodynamics test of preparation treatment pyloric ligation ulcers stomach ulcer medicine
Get 60 of SD rats, male and female half and half are divided into 6 groups at random, 10 every group, are respectively: sham operated rats, model group, positive controls, high, medium and low three the dosage groups of bletilla polysaccharide.Each organizes rat administration as follows, every day 1 time, continuous 5 days.Positive controls is irritated stomach and is given Cimitidine Type A/AB 0.1g/kg, and the high, medium and low dosage group of bletilla polysaccharide is irritated stomach respectively and given bletilla polysaccharide 400mg/kg, 200mg/kg and 100mg/kg, and model group is irritated stomach and given equivalent physiological saline (1mL/100g).1h after the last administration, and the modeling of the equal reference literature method of each treated animal (Xu Shuyun, pharmacological experimental methodology [M]. the 3rd edition, People's Health Publisher, 2003; And document: Lin Ji etc., the research [J] of capsule for enterogastric disease anti-experimental character stomach ulcer effect. new Chinese medicine and clinical pharmacology, 2005,7,16 (4): 256～259.): water is can't help in the 48h fasting before the operation, with vetanarcol 30mg/kg (the administration volume is 1mL/100g) intraperitoneal injection of anesthesia, routine disinfection skin of abdomen, ventrimeson is opened abdomen in the ensiform process of sternum lower edge, exposes stomach.The descending pylorus ligation of stomach pylorus; Simultaneously through duodenal administration 1 time, model group gives isometric physiological saline, sews up stomach wall then, finishes operation.Sham operated rats is operated but not ligation pylorus equally.Prohibit the water fasting behind the pylorus ligation, take off cervical vertebra behind the 18h and put to death rat, cut open the belly and get stomach, folder closes cardiac end, injects 1% formaldehyde solution 8mL from the pylorus end, folder closes the pylorus end, and stomach immersed fixedly 10min of 1% formaldehyde solution, and cut off coat of the stomach along greater gastric curvature behind the 10min, keep the score by the Guth standard, a situation arises to observe stomach ulcer, calculates ulcer index and ulcer and suppress percentage (data see Table 6).
Embodiment 19 bletilla polysaccharides of the present invention burn the pharmacodynamics test of type stomach ulcer medicine at preparation treatment acetate
Get 60 of SD rats, male and female half and half, fasting is 24 hours before the experiment, freely drink water, with vetanarcol 30mg/kg (the administration volume is 1mL/100g) intraperitoneal injection of anesthesia, the routine disinfection skin of abdomen, ventrimeson is opened abdomen in the ensiform process of sternum lower edge, exposes stomach.Reference literature method (Qu Chunying etc., chitosan is to the influence [J] of MDA, SOD and GSH-PX in the stomach ulcer rat blood serum. Shanghai medicine, 2008,29 (5): 219～222. and document: Ye Murong etc., Zhong Jing stomach curing capsule anti-gastric-ulcer effect research [J]. Chinese patent medicine, 2002,24 (9): 692～694) modeling: 20% acetic acid solution 0.1mL is injected under the glandular stomach portion antetheca hole body intersection serous coat causes damage model, sew up stomach wall then, finish operation.Postoperative normal diet, drinking-water were divided into 5 groups at random with it on the 2nd day, 10 every group, were respectively: model group, positive controls, high, medium and low three the dosage groups of bletilla polysaccharide; Other gets 10 rats as sham operated rats, and sham operated rats is operated equally but do not injected acetate.Each organizes rat administration as follows, every day 1 time, continuous 12 days.Positive controls is irritated stomach and is given Cimitidine Type A/AB 0.1g/kg, and the high, medium and low dosage group of bletilla polysaccharide is irritated stomach respectively and given bletilla polysaccharide 400mg/kg, 200mg/kg and 100mg/kg, and model group is irritated stomach and given equivalent physiological saline (1mL/100g).Took off cervical vertebra after the administration on the 13rd day and put to death rat, dissect and get stomach, ligation pylorus and orifice of the stomach extract full stomach, with the fixing 5min of 1% formalin solution; Stomach is cut, open and flat on sheet glass with distilled water eccysis content, observe the gastric mucosa degree of impairment, keep the score by the Guth standard, calculate ulcer index and ulcer and suppress percentage (data see Table 7).
Embodiment 20 bletilla polysaccharides of the present invention cause the pharmacodynamics test of gastric mucosa injury medicine at preparation treatment ethanol
Get 60 of rats, male and female half and half are divided into 6 groups at random, 10 every group, are respectively: normal control group, model group, positive controls, high, medium and low three the dosage groups of bletilla polysaccharide.Each organizes rat administration as follows, every day 1 time, continuous 5 days.Positive controls is irritated stomach and is given Cimitidine Type A/AB 0.1g/kg, and the high, medium and low dosage group of bletilla polysaccharide is irritated stomach respectively and given bletilla polysaccharide 400mg/kg, 200mg/kg and 100mg/kg, and model group is irritated stomach and given equivalent physiological saline (1mL/100g).1h after the last administration, each treated animal reference literature method (Lin Ji etc., the research [J] of capsule for enterogastric disease anti-experimental character stomach ulcer effect. new Chinese medicine and clinical pharmacology, 2005,7,16 (4): 256～259.] modeling: before the experiment, water 48h is can't help in the rat fasting, every rats gavaged 1mL dehydrated alcohol during experiment.Taking off cervical vertebra behind the 1h puts to death rat and cuts open the belly and get stomach, folder closes cardiac end, inject 1% formaldehyde solution 8mL from the pylorus end, folder closes the pylorus end, and stomach immersed fixedly 10min of 1% formaldehyde solution, and cut off coat of the stomach along greater gastric curvature behind the 10min, keep the score by the Guth standard, a situation arises to observe stomach ulcer, calculates ulcer index and ulcer and suppress percentage (data see Table 8).
Table 6. bletilla polysaccharide to pyloric ligation ulcers stomach ulcer rat ulcer exponential influence (x ± s, n=10)
*P＜0.05,
*Compare with model group P＜0.01
By table 6 as seen, the sham operated rats rat does not have stomach ulcer and forms, and illustrates that operation technique can not cause stomach ulcer, and stomach ulcer is owing to pylorus ligation causes.High, medium and low three dosage groups of bletilla polysaccharide and Cimitidine Type A/AB group all can suppress the ulcer index of stomach ulcer rat, have compared utmost point significant difference (P＜0.01) with model group.Wherein, better with the effect of bletilla polysaccharide high dose group.
Table 7. bletilla polysaccharide to acetate burn type stomach ulcer rat ulcer exponential influence (x ± s, n=10)
*P＜0.05,
*Compare with model group P＜0.01
By table 7 as seen, the sham operated rats rat does not have stomach ulcer and forms, and illustrates that operation technique can not cause stomach ulcer, and stomach ulcer is owing to injection acetate causes.Bletilla polysaccharide high dose group and Cimitidine Type A/AB group all can suppress the ulcer index of stomach ulcer rat, have compared utmost point significant difference (P＜0.01) with model group.And in the bletilla polysaccharide, low dose group compares no significant difference (P〉0.05) with model group.
Table 8. bletilla polysaccharide to ethanol cause gastric mucosa injury rat ulcer exponential influence (x ± s, n=10)
*P＜0.05,
*Compare with model group P＜0.01
By table 8 as seen, to causing acute gastric mucosa damage behind the rats gavaged dehydrated alcohol, stomach ulcer appears.After the administration, high, medium and low dosage group of bletilla polysaccharide and Cimitidine Type A/AB group all can suppress the ulcer index of stomach ulcer rat, compare with model group that there were significant differences (P＜0.01 or P＜0.05); Wherein better with bletilla polysaccharide high dose group effect.
Embodiment 18-20 and table 6-8 show: adopt diverse ways to prepare the rat gastric ulcer model, observe the drug effect of the bletilla polysaccharide of the inventive method preparation at preparation treatment stomach ulcer medicine, the result confirms that bletilla polysaccharide can prevent and treat the formation of acute gastric ulcer due to the pylorus ligation, can promote the healing of chronic ulcer due to the acetate again, can also prevent and treat the gastric mucosa injury that ethanol causes, illustrate that bletilla polysaccharide has the therapeutic action that promotes ulcer healing, the stomach ulcer that different reasons are caused all has preventive and therapeutic effect.
Embodiment 21 bletilla polysaccharides of the present invention are at the pharmacodynamics test of preparation treatment silicosis medicine
1, experiment material:
(1) laboratory animal
The SD rat, male and female half and half, body weight 180～220g is provided by unming Medical College's Experimental Animal Center, animal conformity certification SCXK (Yunnan) 2005-0008.
(2) medicine and reagent
Bletilla polysaccharide: by the preparation method of above-mentioned bletilla polysaccharide preparation and provide, used bletilla polysaccharide content is 74.2% by research and development department of Yunnan Plant Pharmaceutical Industry Co., Ltd.;
Vetanarcol: Chemical Reagent Co., Ltd., Sinopharm Group, lot number: WB020060413.
Aseptic silica powder: defending institute by China Preventive Medicial Science Institute's labor provides, and contains SiO
2More than 98% ,≤5um particulate accounts for 99%.
(3) instrument
LXJ2I type whizzer: Shanghai medical analytical instrument factory;
Three use the electric heating constant water bath box: Chang'an, Beijing scientific instrument factory;
Vortex mixer: Zhejiang Le Cheng electrical apparatus factory.
2, index detection method: lung weight in wet base, directly weighing.
3, statistical method
Data are so that (x ± s) expression relatively adopts the SPSS11.5 statistical software between group, carry out Independent-SamplesT test.
4, test method: adopt exposure formula tracheae injection method, between rat two tracheal rings, the aseptic silica powder suspension 1mL that injects 50mg/mL prepares rat silicosis model, observes the drug effect of bletilla polysaccharide at preparation treatment silicosis medicine, and concrete steps are as follows:
Get 50 of rats, male and female half and half are divided into 5 groups at random, 10 every group, are respectively: sham operated rats, model group, high, medium and low three the dosage groups of bletilla polysaccharide.Each treated animal reference literature method (Xu Shuyun, pharmacological experimental methodology [M]. the 3rd edition, the People's Health Publisher, 2003. and document: Zhang Zhongxing etc., spirulina is to the influence [J] of antioxidant levels in the animal silicosis model body. Chinese public health, 2005,1,21 (1): 8～9) modeling: with vetanarcol 30mg/kg (the administration volume is 1mL/100g) intraperitoneal injection of anesthesia, the routine disinfection skin of neck in neck medisection skin, exposes tracheae, between two tracheal rings, thrust syringe needle, inject the aseptic silica powder suspension 1mL of 50mg/mL.Finish operation, skin suture then.Operation back the 3rd day, each organizes rat administration as follows, every day 1 time, continuous 10 weeks.The high, medium and low dosage group of bletilla polysaccharide is irritated stomach respectively and is given bletilla polysaccharide 400mg/kg, 200mg/kg and 100mg/kg, and model group is irritated stomach and given equivalent physiological saline (1mL/100g).After experiment finished, femoral artery sacrificed by exsanguination rat was got lung tissue and claims weight in wet base, does check pathological section (data see Table 9) then.
Table 9. bletilla polysaccharide to the influence of silicosis induced lung weight in wet base (x ± s, n=10)
#P＜0.05, compare with sham operated rats ##P＜0.01;
*P＜0.05,
*Compare with model group P＜0.01
By table 9 as seen, model group induced lung weight in wet base obviously increases, the utmost point significant difference of having compared with sham operated rats (P＜0.01).Bletilla polysaccharide height, middle dosage group can obviously suppress the pulmonary fibrosis of silicosis rat, alleviate the lung weight in wet base, compare with model group that there were significant differences (P＜0.01 or P＜0.05), and the bletilla polysaccharide low dose group there is certain restraining effect to the pulmonary fibrosis of silicosis rat, but does not have significant difference.
Embodiment 21 and table 9 show: bletilla polysaccharide can obviously suppress the pulmonary fibrosis of silicosis rat, alleviates the lung weight in wet base, illustrates that bletilla polysaccharide can delay or suppress the development of silicosis pathology to a certain extent.
Embodiment 22 usefulness bletilla polysaccharide of the present invention is used at preparation treatment stomach ulcer medicine and preparation treatment silicosis medicine preparation bletilla polysaccharide syrup
Get bletilla polysaccharide 100-200 part of the present invention, white sugar 50-100 part, honey 10-50 part; Adding purified water (W:V) that 50-100 doubly measures in bletilla polysaccharide dry powder of the present invention, to be mixed with the aqueous solution standby, with adding in the bletilla polysaccharide solution behind the white sugar heating and melting, adds honey again, stirs, and boils 30min, and it is promptly anticorrosion to add 3% Sodium Benzoate again.
Embodiment 23 usefulness bletilla polysaccharide of the present invention is used at preparation treatment stomach ulcer medicine and preparation treatment silicosis medicine preparation bletilla polysaccharide chewable tablet
Get bletilla polysaccharide 100-500 part of the present invention, N.F,USP MANNITOL 30-60 part, sorbyl alcohol 60-150 part, lactose 30-80 part, aspartame 1-2 part, Citric Acid 2-5 part, sour Magnesium Stearate 3-6 part; Bletilla polysaccharide dry powder and auxiliary material are crossed 100 mesh sieves respectively, get above-mentioned recipe quantity bletilla polysaccharide, lactose, aspartame, Citric Acid are heavy with N.F,USP MANNITOL and sorbitol mixture (1:2) adjustment sheet, supplementary material is mixed, with dehydrated alcohol system softwood, cross 16 mesh sieves and granulate 60 ℃ of dryings, the whole grain of 14 mesh sieves, add Magnesium Stearate, mix back 12mm drift compressing tablet, tablet hardness is controlled at 4kg/mm
2About.
Embodiment 24 usefulness bletilla polysaccharide of the present invention is used at preparation treatment stomach ulcer medicine and preparation treatment silicosis medicine preparation bletilla polysaccharide effervescent tablet
Get bletilla polysaccharide 300-950 part of the present invention, lactose 200-500 part, Steviosin 5-30 part, citric acid 100-200 part, sodium bicarbonate 100-250 part, polyethylene glycol 6000 50-120 part, Magnesium Stearate 5-12 part; After the polyethylene glycol 6000 fusion, add sodium bicarbonate 100-250 part, stir, cooling is pulverized, and crosses 80 mesh sieves; In addition citric acid, sweetener are crossed 80 mesh sieves,, granulate with medicinal powder, polyoxyethylene glycol wrap fine powder mixing, drying, compacting is in flakes promptly.
Claims (6)
1. the application of bletilla polysaccharide in preparation treatment silicosis medicine, described bletilla polysaccharide obtains with following extracting and purifying method:
(1) get bletilla raw medicinal herbs stem tuber, be ground into meal, doubly measure 40-95% ethanol cold soaking or refluxing extraction 2-3 time with the 4-10 of its meal weight, the dregs of a decoction after the extraction do not dry to there not being the alcohol flavor;
(2) dry to the dregs of a decoction that do not have the alcohol flavor above-mentioned, 60～80 ℃ of water temperature lixiviates of doubly measuring with the 10-30 of its weight 1-3 hour, or boil and carry 1-3 hour, extract united extraction liquid, filtration 2-3 time;
(3) above-mentioned filtrate is passed through macroporous type or gel-type weak anion resin and macroporous type or gel-type acidulous cation resin, or by macroporous type or gel-type acidulous anion resin and macroporous type or gel-type weakly alkaline resin cation (R.C.), use the deionized water wash-out, collect elutriant;
(4) above-mentioned elutriant is concentrated into relative density 1.1～1.5, adds ethanol and reach 30%～85%, be settled out bletilla polysaccharide to containing the alcohol amount, collecting precipitation, precipitation will precipitate further vacuum-drying and promptly get bletilla polysaccharide with 60%～80% washing with alcohol 1～3 time.
2. the application of bletilla polysaccharide according to claim 1 in preparation treatment silicosis medicine, described step (1) is for directly drying to there not being the alcohol flavor with the medicine waste residue that produces in the pharmaceutical production of bletilla particle.
3. the application of bletilla polysaccharide according to claim 1 and 2 in preparation treatment silicosis medicine, the described filtrate of its step (3) is by macroporous type or gel-type weak anion resin and macroporous type or gel-type acidulous cation resin, or, pass through macroporous type or gel-type acidulous cation resin again by macroporous type or gel-type weak anion resin earlier by macroporous type or gel-type acidulous anion resin and macroporous type or gel-type weakly alkaline resin cation (R.C.); Or pass through macroporous type or gel-type weakly alkaline resin cation (R.C.) again by macroporous type or gel-type acidulous anion resin earlier; Or pass through macroporous type or gel-type weak anion resin again by macroporous type or gel-type acidulous cation resin earlier; Or pass through macroporous type or gel-type acidulous anion resin again by macroporous type or gel-type weakly alkaline resin cation (R.C.) earlier; Or the hybrid resin post by macroporous type or gel-type weak anion resin and macroporous type or gel-type acidulous cation resin; Or the hybrid resin post by macroporous type or gel-type acidulous anion resin and macroporous type or gel-type weakly alkaline resin cation (R.C.).
4. a bletilla polysaccharide is used in the application for preparing the pharmaceutical formulation for the treatment of the silicosis medicine, the application of described pharmaceutical formulation is to be prepared into the bletilla polysaccharide syrup, or the bletilla polysaccharide chewable tablet, or the bletilla polysaccharide effervescent tablet, described bletilla polysaccharide obtains with following extracting and purifying method:
(1) get bletilla raw medicinal herbs stem tuber, be ground into meal, doubly measure 40-95% ethanol cold soaking or refluxing extraction 2-3 time with the 4-10 of its meal weight, the dregs of a decoction after the extraction do not dry to there not being the alcohol flavor;
(2) dry to the dregs of a decoction that do not have the alcohol flavor above-mentioned, 60～80 ℃ of water temperature lixiviates of doubly measuring with the 10-30 of its weight 1-3 hour, or boil and carry 1-3 hour, extract united extraction liquid, filtration 2-3 time;
(3) above-mentioned filtrate is passed through macroporous type or gel-type weak anion resin and macroporous type or gel-type acidulous cation resin, or by macroporous type or gel-type acidulous anion resin and macroporous type or gel-type weakly alkaline resin cation (R.C.), use the deionized water wash-out, collect elutriant;
(4) above-mentioned elutriant is concentrated into relative density 1.1～1.5, adds ethanol and reach 30%～85%, be settled out bletilla polysaccharide to containing the alcohol amount, collecting precipitation, precipitation will precipitate further vacuum-drying and promptly get bletilla polysaccharide with 60%～80% washing with alcohol 1～3 time.
5. bletilla polysaccharide according to claim 4 is used for the application in the pharmaceutical formulation of preparation treatment silicosis medicine, and its step (1) is for directly drying to there not being the alcohol flavor with the medicine waste residue that produces in the pharmaceutical production of bletilla particle.
6. be used in the application for preparing the pharmaceutical formulation for the treatment of the silicosis medicine according to claim 4 or 5 described bletilla polysaccharides, the described filtrate of its step (3) is by macroporous type or gel-type weak anion resin and macroporous type or gel-type acidulous cation resin, perhaps, pass through macroporous type or gel-type acidulous cation resin again by macroporous type or gel-type weak anion resin earlier by macroporous type or gel-type acidulous anion resin and macroporous type or gel-type weakly alkaline resin cation (R.C.); Or pass through macroporous type or gel-type weakly alkaline resin cation (R.C.) again by macroporous type or gel-type acidulous anion resin earlier; Or pass through macroporous type or gel-type weak anion resin again by macroporous type or gel-type acidulous cation resin earlier; Or pass through macroporous type or gel-type acidulous anion resin again by macroporous type or gel-type weakly alkaline resin cation (R.C.) earlier; Or the hybrid resin post by macroporous type or gel-type weak anion resin and macroporous type or gel-type acidulous cation resin; Or the hybrid resin post by macroporous type or gel-type acidulous anion resin and macroporous type or gel-type weakly alkaline resin cation (R.C.).
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