CN102504043A - Active dendrobe polysaccharide capable of protecting liver and resisting liver fibrosis and preparation method of antibody affinity chromatography - Google Patents
Active dendrobe polysaccharide capable of protecting liver and resisting liver fibrosis and preparation method of antibody affinity chromatography Download PDFInfo
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- CN102504043A CN102504043A CN2011103690240A CN201110369024A CN102504043A CN 102504043 A CN102504043 A CN 102504043A CN 2011103690240 A CN2011103690240 A CN 2011103690240A CN 201110369024 A CN201110369024 A CN 201110369024A CN 102504043 A CN102504043 A CN 102504043A
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- dendrobium polysaccharide
- active
- polysaccharide
- antibody
- dendrobium
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- 238000002360 preparation method Methods 0.000 title claims abstract description 26
- 238000001042 affinity chromatography Methods 0.000 title claims abstract description 14
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Abstract
The invention discloses active dendrobe polysaccharide capable of protecting liver and resisting liver fibrosis, whose chemical structural formula is seen in the figure. The active dendrobe polysaccharide is gala glucomannan taking glucose and mannose as a main chain and galactose as a branch, the molecular weight of the gala glucomannan is about 2.2*104dalton, and the mole ratio of glucose to mannose to galactose in the composition is 31:10:8. The invention further discloses extraction, separation and purification methods of the active dendrobe polysaccharide as well as the preparation method of antibody affinity chromatography of the active dendrobe polysaccharide. The active dendrobe polysaccharide prepared by the method has remarkable physiological activity of protecting liver and resisting liver fibrosis and can be used for preparing liver-protecting health care products and medicines for curing liver fibrosis.
Description
Technical field
The invention belongs to the preparation method field of plant milk extract and plant milk extract, be specifically related to active dendrobium polysaccharide extract of a kind of anti-hepatic fibrosis and antibody affinity chromatography preparation method thereof.
Background technology
Liver is a most important detoxifcation organ in the human body, and harmful toxic matters such as virokine, alcohol, medicine can cause liver injury with frequent contact of liver.The liver injury meeting causes pathological changes such as necrocytosis, apoptosis, inflammation, fibrosis, finally causes various hepatic diseases such as hepatic fibrosis.Hepatic fibrosis is that various hepatopathys worsen the common pathologic basis that develops into liver cirrhosis and liver cancer, is to cause liver problem sufferer's main causes of death, and human health and quality of life are had great hazardness.Treatment to liver injury at present mainly depends on medicine, but their curative effect is limited, and taking for a long time of these medicines can produce new liver injury; And the current anti-hepatic fibrosis medicines that still lacks determined curative effect, no obvious toxic-side effects clinically.Therefore, early prevention is carried out in liver injury, hepatic fibrosis and seem particularly necessary.Research shows; Contain the prevention liver injury in many edible animal-plant materials, suppress the fibrosis activity chemical ingredients; If with these active chemical componentses is that healthy ingredient is developed dissolving liver-protecting healthy food, to effective prevention of liver injury, hepatic fibrosis with significant.
The stem of noble dendrobium be Ministry of Health regulation can be used for one of article of protective foods, as traditional drink in the edible history in existing more than 2000 year of China.It is reported that Dendrobium officinale polysaccharide causes rats'liver damage to ciclosporin A has provide protection, the Herba Dendrobii polysaccharide to Sodium Selenite cause the rats'liver damage, hepatic fibrosis has provide protection.Current, dendrobium polysaccharide mainly obtains through water extraction and alcohol precipitation method, as, the patent of invention that publication number is respectively CN 101015649A, CN 101407558A, CN 101407557A discloses Herba Dendrobii and the golden water extract-alcohol precipitation preparation method who pitches dendrobium polysaccharide.The Herba Dendrobii polysaccharide is the basic substance of its performance physiologically active.At present, the acquisition of Herba Dendrobii polysaccharide is through further refining through ion exchange column again behind water extract-alcohol precipitation or the water extract-alcohol precipitation.The Herba Dendrobii polysaccharide product purity that water extraction and alcohol precipitation method obtains is low, is purity less than 30%? Though improved purity after further refining through ion exchange column, need repeatable operation, the product yield is low, generally has only about 40%.
Summary of the invention
In order to solve deficiencies such as existing Herba Dendrobii polyoses producing method yield is low, purity is low, the object of the present invention is to provide the antibody affinity chromatography preparation method who protects the liver anti-hepatic fibrosis Herba Dendrobii polysaccharide that has that a kind of product purity is high, quality controllable, purification efficiency is high.
It is following that the present invention protects the liver the chemical structural formula of the active dendrobium polysaccharide of anti-hepatic fibrosis:
The molecular weight of this activity dendrobium polysaccharide is 2.2 * 10
4Dalton, it is to be that main chain, semi-lactosi are ramose gala konjac glucomanna with glucose and seminose, wherein, the mol ratio of glucose, seminose and semi-lactosi is 31 ︰, 10 ︰ 8.
The concrete operations step of extraction, separation purification method that protects the liver the active dendrobium polysaccharide of anti-hepatic fibrosis is following:
(1) raw materials pretreatment: get the stem of noble dendrobium of pulverizing, in 50 ℃ of backflows of temperature, 5 h, degreasing, decolouring are positioned over 25 ℃ of environment of temperature, remove organic solvent with volatilization, obtain the stem of noble dendrobium dregs of a decoction with sherwood oil;
(2) hot water lixiviate: get the stem of noble dendrobium dregs of a decoction, add zero(ppm) water,, obtain vat liquor in 80 ℃ of lixiviate 60 min of temperature by feed liquid mass volume ratio 1 g ︰ 30ml; With vat liquor spinning 15 min under rotating speed 15000 r/min conditions, the centrifugal sediment that obtains is with twice of the lixiviate again of 80 ℃ of hot water; Merge the centrifuged supernatant of three collections, obtain stem of noble dendrobium vat liquor; Under 60 ℃ of conditions of temperature, concentrating under reduced pressure is 1 ml ︰ 3g to the ratio of liquid concentrator volume and stem of noble dendrobium dregs of a decoction quality, obtains stem of noble dendrobium liquid concentrator with stem of noble dendrobium vat liquor; It is that 3000 daltonian dialysis tubings are dialysed 3 days to remove small-molecule substance that stem of noble dendrobium liquid concentrator uses molecular weight cut-off, gets stem of noble dendrobium dialysis trapped fluid;
(3) alcohol precipitation: in stem of noble dendrobium dialysis trapped fluid, add volume(tric)fraction and be 95% ethanol, make that ethanol final volume mark reaches 80% in the stem of noble dendrobium dialysis trapped fluid, left standstill 72 hours in 4 ℃ of temperature; Under 4 ℃ of temperature, rotating speed 12000 r/min conditions, deposition is got in spinning 15 minutes, obtains stem of noble dendrobium throw out;
(4) deproteinated: get stem of noble dendrobium throw out, add zero(ppm) water, be mixed with the dendrobium polysaccharide water extract A of 100 mg/ml; Get dendrobium polysaccharide water extract A, chloroform is pressed dendrobium polysaccharide water extract A 1/5 volume add, add the butanols of chloroform volume 1/5 thereupon again; Mix, shaken 20 min are then under 10 ℃ of temperature, rotating speed 12000 r/min conditions; Spinning 15 minutes, the denatured protein of branch vibration layer and solution layer intersection gets dendrobium polysaccharide water extract B;
Get dendrobium polysaccharide water extract B, chloroform is pressed dendrobium polysaccharide water extract B 1/5 volume add, add the butanols of chloroform volume 1/5 thereupon again; Mix; Shaken 20 min, then under 10 ℃ of temperature, rotating speed 12000 r/min conditions, spinning 15 minutes; The denatured protein of branch vibration layer and solution layer intersection gets dendrobium polysaccharide water extract C;
Get dendrobium polysaccharide water extract C, chloroform is pressed dendrobium polysaccharide water extract C 1/5 volume add, add the butanols of chloroform volume 1/5 thereupon again; Mix; Shaken 20 min, then under 10 ℃ of temperature, rotating speed 12000 r/min conditions, spinning 15 minutes; The denatured protein of branch vibration layer and solution layer intersection gets dendrobium polysaccharide water extract;
(5) purifying: 1. DEAE-52 ion exchange resin column classification: get dendrobium polysaccharide water extract,, follow the tracks of the detection effluent, collect the main peak effluent A of zero(ppm) water wash-out part with the phenol sulfuric acid process through DEAE-52 high-efficiency anion exchange resin column; It is 2000 daltonian dialysis tubing dialysis 3 days that main peak effluent A uses molecular weight cut-off, collects dialysis liquid concentrator A; 2. Sephacryl S-200 gel resin post classification: get dialysis liquid concentrator A,, follow the tracks of the detection effluent, collect the main peak effluent B of zero(ppm) water wash-out part with the phenol sulfuric acid process through Sephacryl S-200 gel resin post; It is 2000 daltonian dialysis tubing dialysis 3 days that main peak effluent B uses molecular weight cut-off, collects dialysis liquid concentrator B;
Get dialysis liquid concentrator B; Successively through classification of DEAE-52 ion exchange resin column and the classification of Sephacryl S-200 gel resin post; Repeat DEAE-52 ion exchange resin column classification and Sephacryl S-200 gel resin post progressive operation step 8 time; Collect last dialysis liquid concentrator through lyophilize, obtain active dendrobium polysaccharide;
(6) chemical structure of active dendrobium polysaccharide characterizes: through all-hydrolytic analysis, Smith DeR, methylation analysis, IR spectroscopy, and the spectral analysis of the nuclear magnetic resonance method has confirmed that the chemical structural formula of active dendrobium polysaccharide is following:
。
The said stem of noble dendrobium is fresh Herba Dendrobii or exsiccant Herba Dendrobii.
The antibody affinity chromatography preparation protects the liver the method for the active dendrobium polysaccharide of anti-hepatic fibrosis, and its concrete operations step is following:
(1) preparation immunogen: get in the carbonate buffer solution that active dendrobium polysaccharide is dissolved in pH value 9.2; Being mixed with massfraction is the active dendrobium polysaccharide solution of 10 mg/ml; Add bovine serum albumin then, the massfraction that makes the bovine serum albumin in the active dendrobium polysaccharide solution is 10 mg/ml, stirring and evenly mixing; Place the thermostat container of 37 ℃ of temperature to react 3d, obtain reaction system; Add thanomin, make that the thanomin massfraction reaches 1% o'clock termination reaction in the reaction system, reaction solution; Reaction solution separates through Sephadex G-150 gel column, fraction collection; Adopt phenolsulfuric acid method and Bradford method to measure active dendrobium polysaccharide and bovine serum albumin content in each pipe respectively, calculate the bovine serum albumin mole molecule number that every mole of active dendrobium polysaccharide molecule connects; Getting the conjugate that every mole of active dendrobium polysaccharide molecule is connected with 5 moles of bovine serum albumin molecules is active dendrobium polysaccharide-bovine serum albumin immunogen, obtains the polysaccharide compound immunogen;
(2) immune mouse: 5 mg polysaccharide compound immunogens are dissolved in the 5 mL saline water,, obtain immunogen solution with the two pushing manipulation mixings of isopyknic Freund's complete adjuvant; Adopt the abdominal injection immunity female mice in 8 ages in week, every injected in mice immunogen solution 0.2 ml; After 1 week the 7th day got 5 mg polysaccharide compound immunogens and is dissolved in the 5 mL saline water, with equal-volume Freund's incomplete adjuvant mixing, the immunogen mixed solution; Every female mice abdominal injection 0.2 ml immunogen mixed solution carries out booster immunization, and booster immunization repeats 3 times, 1 time weekly; After last 1 immunity the 7th day cut tail and got blood, measures tiring of active dendrobium polysaccharide specific antibody in the mice serum with enzyme-linked immunosorbent assay (ELISA); Select the mouse that active dendrobium polysaccharide specific antibody titres is higher than 1 ︰ 10000 in the serum to be used for cytogamy; And before cytogamy 4-5 days; It is that the polysaccharide compound immunogen aqueous solution of 1.0 mg/mL impacts immunity that massfraction that 0.2 ml do not contain adjuvant is inoculated in the abdominal cavity, obtains immune mouse;
(3) cytogamy: the SPL of under aseptic condition, getting immune mouse is processed every milliliter and is contained 1 * 10
3～1 * 10
4The cell suspension of individual cell with cell suspension under the condition of 4 ℃ of temperature, rotating speed 1000 r/min centrifugal 10 minutes, is got centrifugation, obtains the SPL of immune mouse; Get 1 * 10
8The SPL of individual immune mouse and 2 * 10
7Individual Balb/c mouse SP2/0 tumour cell mixes, and is preheated to 37 ℃, and slowly adding 1ml, to be preheated to 37 ℃ massfraction be 50% polyglycol solution; Slowly add 10 ml again and be preheated to 37 ℃ the DMEM perfect medium that massfraction is 20% foetal calf serum that contains; Cell suspension under 4 ℃ of temperature, rotating speed 1000 r/min conditions centrifugal 10 minutes, is abandoned supernatant; In deposition, add DMEM perfect medium 5ml; Cell suspends again, is sub-packed in the 96 porocyte culture plates, is 5% carbonic acid gas (CO in 37 ℃ of temperature, volume(tric)fraction
2) cultivated 1 day under the condition of gas, 95% humidity; Add 4 * HAT nutrient solution, 50 μ l then; Displace the l/2DMEM perfect medium in the culture hole of 96 porocyte culture plates on the 6th day with the HAT nutrient solution, displaced the HAT nutrient solution with the HT nutrient solution on the 11st day, obtain fused cell;
(4) the active dendrobium polysaccharide monoclonal antibody hybridoma cell strain of preparation: fused cell is inoculated in HAT selects on the substratum; With the polysaccharide compound immunogen is antigen; Adopt the indirect enzyme-linked immunosorbent analytical method to detect tiring of the active dendrobium polysaccharide specific antibody of fused cell excretory; The selection antibody titer is higher than the fused cell of 1 ︰ 1000, obtains the hybrid cell of ability secretion activity dendrobium polysaccharide specific antibody; Adopt limiting dilution assay that said hybrid cell is carried out cloning in 96 porocyte culture plates; Adopt the indirect enzyme-linked immunosorbent analytical method to detect tiring of the clone's active dendrobium polysaccharide specific antibody of secretion in culture supernatant liquid on the 96 porocyte culture plates; Select antibody titer to be higher than the single cell clone of 1 ︰ 100, obtain positive colony; Get positive colony and adopt limiting dilution assay in 96 porocyte culture plates, to carry out three time cloningizations, select the positive colony of ability secretion activity dendrobium polysaccharide specific antibody, obtain hybridoma cell clone; Selecting strong, the well-grown hybridoma cell clone of antibody response to be inoculated on the HAT substratum, is 5% carbonic acid gas (CO in 37 ℃ of temperature, volume(tric)fraction
2) cultivated three days under the condition of gas, 95% humidity, obtain active dendrobium polysaccharide monoclonal antibody hybridoma cell strain;
(5) the active dendrobium polysaccharide monoclonal antibody of preparation: get the female mice in 10 ages in week, abdominal injection 0.5 ml pristane; Every female mice inoculation in the 7th day is in the active dendrobium polysaccharide monoclonal antibody hybridoma cell strain 5 * 10 of logarithmic phase
6Individual; Fortnight is gathered the ascites of female mice, and ascites under the condition of 4 ℃ of temperature, rotating speed 5000 r/min, centrifugal 15 minutes, is collected supernatant; Adopt the antibody A in sad-ammonium sulfate precipitation method deposition supernatant, antibody A obtains antibody B through Sephadex G-150 gel filtration chromatography; Adopt PAGE and SDS-PAGE method to identify the purity of antibody B respectively, use the specificity of indirect enzyme-linked immunosorbent assay antibody B simultaneously active dendrobium polysaccharide; Collect band homogeneous, apparent antibody B, lyophilize obtains active dendrobium polysaccharide monoclonal antibody;
(6) the active dendrobium polysaccharide monoclonal antibody immune affinity chromatographic column of preparation: (A) coupling of agarose (Sepharose) 4B carrier and antibody: get the active dendrobium polysaccharide monoclonal antibody of 10g and be dissolved in the coupling buffer; Placing molecular weight cut-off then is 2000 daltonian dialysis tubing dialysis 24 hours; Get the dialysis trapped fluid and be mixed with the active dendrobium polysaccharide monoclonal antibody aqueous solution that massfraction is 0.5 mg/ml; The pH value of said coupling buffer is 8.2, and coupling buffer is to contain 0.5 Mol/L sodium-chlor (NaCl) and 0.1 Mol/L sodium hydrogencarbonate (NaHCO
3) the aqueous solution; The sepharose 4B 15g of the cyanogen bromide-activated of learning from else's experience takes out fast with said coupling buffer and to wash 5 times, pours into rapidly in the active dendrobium polysaccharide monoclonal anti liquid solution and carries out coupling, and UV scanning is with the monitoring coupling process; With the coupling buffer eccysis active dendrobium polysaccharide monoclonal antibody of link coupled not, obtain agarose-antibody coupling mixture; (B) sealing activating group: agarose-antibody coupling mixture is joined in Tutofusin tris-hydrochloric acid (Tris-HCl) damping fluid of 6 times of volumes, keep 3 hours with unreacted radical in the sealing sepharose 4B; The pH value of said tris-HCI buffer is 8.0, and tris-HCI buffer is for containing 0.5 Mol/L sodium-chlor and 0.1 Mol/L Tutofusin tris-aqueous solution of hydrochloric acid; (C) dress post: void column is washed with the pH value 7.2 of 10 times of column volumes, the phosphoric acid buffer of concentration 0.01 Mol/L; Place a hydrophobic sieve plate then in the bottom; And put into the pH value 7.2 of void column 1/8 volume, the phosphoric acid buffer of concentration 0.1 Mol/L, obtain the pre-treatment void column; Agarose-antibody coupling mixture that step (A) is obtained washs three times with the 0.1 Mol/L phosphoric acid buffer that contains 0.5Mol/L sodium-chlor 10 times of agaroses-antibody coupling mixture volume, pH value 4.5 earlier; Then with the 0.1 Mol/L tris-HCI buffer washing that contains 0.5 Mol/L sodium-chlor 10 times of agaroses-antibody coupling mixture volume, pH value 8.0 three times; With phosphoric acid buffer washing 5 times of agaroses-antibody coupling mixture volume, pH value 7.2, concentration 0.1 Mol/L three times, obtain agarose-antibody coupling composite colloids again; Agarose-antibody coupling composite colloids is poured in the pre-treatment void column; Wait; Add a filter paper at agarose-antibody coupling composite colloids top; And with the pH value 7.2 of 10 times of column volumes, the phosphoric acid buffer flushing of concentration 0.1 Mol/L; To get rid of the bubble in the post bed, use the 0.1 Mol/L phosphoric acid buffer preservation that massfraction is 0.01% sodiumazide that contains of pH value 7.2 at last, obtain active dendrobium polysaccharide monoclonal antibody immunoaffinity chromatography prepacked column;
(7) antibody affinity chromatography prepares active dendrobium polysaccharide: (a) balance: with the flow velocity equilibrium activity dendrobium polysaccharide monoclonal antibody immunoaffinity chromatography prepacked column of level pad with 2 ml/min; Obtain the balance chromatography column through ultraviolet detection 280 nm places light absorption value monitoring prepacked column balance to baseline; Said level pad is the 0.1 Mol/L Tris-HCl aqueous solution that contains 0.5 Mol/L sodium-chlor of pH value 8.0; (b) go up appearance: get stem of noble dendrobium Crude polysaccharides and be mixed with the sample solution that mass concentration is 0.2 mg/ml, with 50 ml sample solutions with the flow velocity of 2 ml/min through the balance chromatography column, obtain chromatography and carry a kind post; (c) wash-out: clean chromatography with level pad with the flow velocity of 2 ml/min and carry a kind post, carry a kind post through ultraviolet detection 280 nm places light absorption values monitoring and cleaned to baseline; With the flow velocity of 1 ml/min dendrobium polysaccharide is carried a kind post from chromatography with the 0.1 Mol/L phosphoric acid elutriant that contains 0.5 Mol/L sodium-chlor of pH value 4.5 and elute, effluent volume is 5 times that chromatography carries kind column volume; Follow the tracks of detection with the phenol sulfuric acid process and collect dendrobium polysaccharide content in each pipe, collect dendrobium polysaccharide main peak effluent; Effluent lyophilize of dendrobium polysaccharide main peak or spraying drying obtain active dendrobium polysaccharide; The chemical structural formula of active dendrobium polysaccharide is following:
Said active dendrobium polysaccharide have protect the liver, anti-hepatic fibrosis is active, can be used for preparing the medicine of liver health product and treatment hepatic fibrosis.
Compared with prior art, useful technique effect of the present invention embodies in the following areas:
(1) the present invention preparation method of protecting the liver the active dendrobium polysaccharide of anti-hepatic fibrosis has remedied the deficiency of Herba Dendrobii polyoses producing method, provides that a kind of Herba Dendrobii purity of polysaccharide can reach more than 98%, the recovery is the antibody affinity chromatography preparation method of the active dendrobium polysaccharide that specificity is strong, product purity is high, purification efficiency is high more than 95%;
(2) the antibody affinity chromatography method is based on specificity, the reversibility combination principle between antigen and antibody, from the system of complicacy, separates obtaining required title product, has that selectivity is strong, product purity is high, purification efficiency is high, the sweeping characteristics of treatment samples.On the basis of clear and definite chemical structure, the present invention prepares polysaccharide antibody, have through antibody affinity chromatography method preparation protect the liver, the active Herba Dendrobii polysaccharide of anti-hepatic fibrosis, the sample size of every batch processed is more than 50 times of conventional resin column classification preparation method;
(3) the active dendrobium polysaccharide structure of batch preparations of the present invention is clear and definite, have protect the liver, anti-hepatic fibrosis is active, can be used for the 4th generation healthcare products and medicine field.
Description of drawings
Fig. 1 is active dendrobium polysaccharide anionite-exchange resin elution profile.
Fig. 2 is active dendrobium polysaccharide efficient gel permeation chromatography figure.
Fig. 3 is active dendrobium polysaccharide IR-FT spectrogram.
Fig. 4 is the methylated sugar alcohol acetic ester TIC of active dendrobium polysaccharide.
Fig. 5 is the hydrogen nuclear magnetic resonance spectrogram of active dendrobium polysaccharide.
Embodiment
Below in conjunction with accompanying drawing and concrete embodiment the present invention is done explanation further.Characteristics of the present invention and advantage will be clearer along with description, but these exemplary embodiments only are used for explaining the present invention, scope of the present invention are not constituted any restriction.
The raw materials used manufacturing enterprise of following examples is following: Herba Dendrobii (what stone old and well-known family Chinese medicinal materials development corporation, Ltd. of Anhui), sherwood oil (Yancheng Rong Sheng chemical industry), dialysis tubing (source, Shanghai leaf bio tech ltd); Tetracol phenixin (Huasheng Chemical Co Ltd, Danyang), DEAE-52 ion exchange resin (Shanghai grace is faced Environmental Protection Technology Co., Ltd), Sephacryl S-200 gel resin (Beijing winter song bio tech ltd); Tutofusin tris (Shanghai is along sulfone commerce and trade ltd), hydrochloric acid (Yancheng City three is along Fine Chemical Co., Ltd), Biphenylylmethylcarbinol (emerging laboratory equipment ltd of Shanghai section); SP2/0 tumour cell (Shanghai grind living biochemical reagents ltd), polyoxyethylene glycol (with the occasion chemical industry), foetal calf serum (Shanghai strength horse bio tech ltd); DMEM perfect medium (going up the graceful bio tech ltd of Hypon); Tissue Culture Plate (Hangzhou Anpu Bioengineering Co., Ltd), HAT nutrient solution (Beijing bispin microbiological culture media products factory), HAT selects substratum (Beijing bispin microbiological culture media products factory); Pristane (Guizhou is Bioisystech Co., Ltd all one's life); HT nutrient solution (Beijing bispin microbiological culture media products factory), vitamin E (Jia Kang source, Beijing development in science and technology ltd), SD rat (SCXK of Medical University Of Anhui (Anhui) 2011-002); Phenol (Shantou Xilong Chemical Factory); The vitriol oil (Shanghai pilot scale chemical industry), Sephadex G-150 gel (Beijing is auspicious reach permanent brightness development in science and technology ltd), gpt test kit (biological study institute is built up in Nanjing); Glutamic-oxal(o)acetic transaminase test kit (biological study institute is built up in Nanjing); Kunming mice (Medical University Of Anhui experimentation on animals center (credit number: SCXK (Anhui) 2005-001)), thanomin (roc chemical industry ltd in the Guangdong), serum albumin (Shanghai Ze Mai Bioisystech Co., Ltd); Balb/c mouse (Institute of Experimental Animals, Chinese Academy of Medical Sciences/Beijing China Fukang biotech inc); Sepharose 4B (Shanghai Ya Ji bio tech ltd), Freund's complete adjuvant (source, Shanghai consor thing Science and Technology Ltd.), Freund's incomplete adjuvant (source, Shanghai consor thing Science and Technology Ltd.); Trichloromethane (Shantou Xilong Chemical Factory), propyl carbinol (Chemical Reagent Co., Ltd., Sinopharm Group).
The manufacturing enterprise of the used instrument of following examples is following: CO2gas incubator (Shanghai new talent medicine equipment Manufacturing Co., Ltd); High performance liquid chromatograph (Qingdao Shenghan Chromatograph Technology Co., Ltd.); IR (Shanghai Dumet company); Nuclear magnetic resonance apparatus (nucleus magnetic resonance company of peace section), gas chromatograph (Qingdao Shenghan Chromatograph Technology Co., Ltd.), R-201 rotatory evaporator (Shanghai Shen Sheng Bioisystech Co., Ltd); CT15RT whizzer (the U.S. biochemical instrument plant engineering in sky, Shanghai ltd), Model 680 type ELIASAs (BIO-RAD).
Embodiment 1: the extraction of active dendrobium polysaccharide, separation and purification and structural characterization
The concrete operations step of extraction, separation purification method that protects the liver the active dendrobium polysaccharide of anti-hepatic fibrosis is following:
(1) raw materials pretreatment: get the Herba Dendrobii of pulverizing, in 50 ℃ of backflows of temperature, 5 h degreasings, decolouring, be positioned over 25 ℃ of environment of temperature, remove organic solvent, obtain the stem of noble dendrobium dregs of a decoction with volatilization with sherwood oil;
(2) hot water lixiviate: get the stem of noble dendrobium dregs of a decoction, add zero(ppm) water,, obtain vat liquor in 80 ℃ of lixiviate 60 min of temperature by feed liquid mass volume ratio 1 g ︰ 30ml; With vat liquor spinning 15 min under rotating speed 15000 r/min conditions, the centrifugal sediment that obtains is with twice of the lixiviate again of 80 ℃ of hot water; The centrifuged supernatant that merges twice collection obtains stem of noble dendrobium vat liquor; Under 60 ℃ of conditions of temperature, concentrating under reduced pressure is 1 ml ︰ 3g to the ratio of liquid concentrator volume and stem of noble dendrobium dregs of a decoction quality, obtains stem of noble dendrobium liquid concentrator with stem of noble dendrobium vat liquor; It is that 3000 daltonian dialysis tubings are dialysed 3 days to remove little amalyzing substances that stem of noble dendrobium liquid concentrator uses molecular weight cut-off, gets stem of noble dendrobium dialysis trapped fluid;
(3) alcohol precipitation: in stem of noble dendrobium dialysis trapped fluid, add volume(tric)fraction and be 95% ethanol, make that ethanol final volume mark reaches 80% in the stem of noble dendrobium dialysis trapped fluid, left standstill 72 hours in 4 ℃ of temperature; Under 4 ℃ of temperature, rotating speed 12000 r/min conditions, deposition is got in spinning 15 minutes, obtains stem of noble dendrobium throw out;
(4) deproteinated: get stem of noble dendrobium throw out, add zero(ppm) water, be mixed with the dendrobium polysaccharide water extract A of 100 mg/ml; Get dendrobium polysaccharide water extract A, chloroform is pressed dendrobium polysaccharide water extract A 1/5 volume add, add the butanols of chloroform volume 1/5 thereupon again; Mix; Shaken 20 min, then under 10 ℃ of temperature, rotating speed 12000 r/min conditions, spinning 15 minutes; The denatured protein of branch vibration layer and solution layer intersection gets dendrobium polysaccharide water extract B;
Get dendrobium polysaccharide water extract B, chloroform is pressed dendrobium polysaccharide water extract B 1/5 volume add, add the butanols of chloroform volume 1/5 thereupon again; Mix; Shaken 20 min, then under 10 ℃ of temperature, rotating speed 12000 r/min conditions, spinning 15 minutes; The denatured protein of branch vibration layer and solution layer intersection gets dendrobium polysaccharide water extract C;
Get dendrobium polysaccharide water extract C, chloroform is pressed dendrobium polysaccharide water extract C 1/5 volume add, add the butanols of chloroform volume 1/5 thereupon again; Mix; Shaken 20 min, then under 10 ℃ of temperature, rotating speed 12000 r/min conditions, spinning 15 minutes; The denatured protein of branch vibration layer and solution layer intersection gets dendrobium polysaccharide water extract;
(5) purifying: 1. DEAE-52 ion exchange resin column classification: get dendrobium polysaccharide water extract,, follow the tracks of the detection effluent, collect the main peak effluent A of zero(ppm) water wash-out part with the phenol sulfuric acid process through DEAE-52 high-efficiency anion exchange resin column; It is 2000 daltonian dialysis tubing dialysis 3 days that main peak effluent A uses molecular weight cut-off, collects dialysis liquid concentrator A; 2. Sephacryl S-200 gel resin post classification: get dialysis liquid concentrator A,, follow the tracks of the detection effluent, collect the main peak effluent B of zero(ppm) water wash-out part with the phenol sulfuric acid process through Sephacryl S-200 gel resin post; It is 2000 daltonian dialysis tubing dialysis 3 days that main peak effluent B uses molecular weight cut-off, collects dialysis liquid concentrator B;
Get dialysis liquid concentrator B; Successively through classification of DEAE-52 ion exchange resin column and the classification of Sephacryl S-200 gel resin post; Repeat DEAE-52 ion exchange resin column classification and Sephacryl S-200 gel resin post progressive operation step 8 time; Collect last dialysis liquid concentrator through lyophilize, obtain active dendrobium polysaccharide; Last DEAE-52 ion exchange resin column fractionated result sees accompanying drawing 1, and the result of accompanying drawing 2 shows that this activity dendrobium polysaccharide is a homogeneous polysaccharide.
(6) chemical structure of active dendrobium polysaccharide characterizes: the all-hydrolytic analytical results shows that this activity dendrobium polysaccharide is made up of glucose, seminose and three kinds of monose of semi-lactosi; Accompanying drawing 3 presentation of results should have typical glucide structure by the activity dendrobium polysaccharide; 4 methylation analysis results can know from Smith DeR amount of money accompanying drawing, and this activity dendrobium polysaccharide contains the described fragment that methylates of table 1; Accompanying drawing 5 NMR spectrums have further confirmed to have the described mode of connection of table 1 by the activity dendrobium polysaccharide.The result of consolidated statement 1 and accompanying drawing 1～accompanying drawing 5, confirm that the chemical structural formula of active dendrobium polysaccharide is following:
Embodiment 2: the antibody affinity chromatography preparation method of active dendrobium polysaccharide
Step 3 cytogamy: the SPL of under aseptic condition, getting immune mouse is processed every milliliter and is contained 1 * 10
3～1 * 10
4The cell suspension of individual cell with cell suspension under the condition of 4 ℃ of temperature, rotating speed 1000 r/min centrifugal 10 minutes, is got centrifugation, obtains the SPL of immune mouse; Get 1 * 10
8The SPL of individual immune mouse and 2 * 10
7Individual Balb/c mouse SP2/0 tumour cell mixes, and is preheated to 37 ℃, and slowly adding 1ml, to be preheated to 37 ℃ massfraction be 50% polyglycol solution; Slowly add 10 ml again and be preheated to 37 ℃ the DMEM perfect medium that massfraction is 20% foetal calf serum that contains; Cell suspension under 4 ℃ of temperature, rotating speed 1000 r/min conditions centrifugal 10 minutes, is abandoned supernatant; In deposition, add DMEM perfect medium 5ml; Cell suspends again, is sub-packed in the 96 porocyte culture plates, is 5% carbonic acid gas (CO in 37 ℃ of temperature, volume(tric)fraction
2) cultivated 1 day under the condition of gas, 95% humidity; Add 4 * HAT nutrient solution, 50 μ l then; Displace the l/2 substratum in the culture hole of 96 porocyte culture plates on the 6th day with the HAT nutrient solution, displaced the HAT nutrient solution with the HT nutrient solution on the 11st day, obtain fused cell;
The active dendrobium polysaccharide monoclonal antibody hybridoma cell strain of step 4 preparation: fused cell is inoculated in HAT selects on the substratum; With the polysaccharide compound immunogen is antigen; Adopt the indirect enzyme-linked immunosorbent analytical method to detect tiring of the active dendrobium polysaccharide specific antibody of fused cell excretory; The selection antibody titer is higher than the fused cell of 1 ︰ 1000, obtains the hybrid cell of ability secretion activity dendrobium polysaccharide specific antibody; Adopt limiting dilution assay that said hybrid cell is carried out cloning in 96 porocyte culture plates; Adopt the indirect enzyme-linked immunosorbent analytical method to detect tiring of active dendrobium polysaccharide specific antibody in the clone's supernatant on the 96 porocyte culture plates; Select antibody titer to be higher than the single cell clone of 1 ︰ 100, obtain positive colony; Get positive colony and adopt limiting dilution assay in 96 porocyte culture plates, to carry out three time cloningizations, select the positive colony of ability secretion activity dendrobium polysaccharide specific antibody, obtain hybridoma cell clone; Selecting strong, the well-grown hybridoma cell clone of antibody response to be inoculated on the HAT substratum, is 5% carbonic acid gas (CO in 37 ℃ of temperature, volume(tric)fraction
2) cultivated three days under the condition of gas, 95% humidity, obtain active dendrobium polysaccharide monoclonal antibody hybridoma cell strain;
The active dendrobium polysaccharide monoclonal antibody of step 5 preparation: get the female mice in 10 ages in week, abdominal injection 0.5 ml pristane; Every female mice inoculation in the 7th day is in the active dendrobium polysaccharide monoclonal antibody hybridoma cell strain 5 * 10 of logarithmic phase
6Individual; Fortnight is gathered the ascites of female mice, and ascites under the condition of 4 ℃ of temperature, rotating speed 5000 r/min, centrifugal 15 minutes, is collected supernatant; Adopt the antibody A in sad-ammonium sulfate precipitation method deposition supernatant, antibody A obtains antibody B through Sephadex G-150 gel filtration chromatography; Adopt PAGE and SDS-PAGE method to identify the purity of antibody B respectively, use the specificity of indirect enzyme-linked immunosorbent assay antibody B simultaneously active dendrobium polysaccharide; Collect band homogeneous, apparent antibody B, lyophilize obtains active dendrobium polysaccharide monoclonal antibody;
The coupling of step 6 (A) agarose (Sepharose) 4B carrier and antibody: get the active dendrobium polysaccharide monoclonal antibody of 10g and be dissolved in the coupling buffer; Placing molecular weight cut-off then is 2000 daltonian dialysis tubing dialysis 24 hours; Get the dialysis trapped fluid and be mixed with the active dendrobium polysaccharide monoclonal antibody aqueous solution that massfraction is 0.5 mg/ml; The pH value of said coupling buffer is 8.2, and coupling buffer is to contain 0.5 Mol/L sodium-chlor (NaCl) and 0.1 Mol/L sodium hydrogencarbonate (NaHCO
3) the aqueous solution; The sepharose 4B 15g of the cyanogen bromide-activated of learning from else's experience takes out fast with said coupling buffer and to wash 5 times, pours into rapidly in the active dendrobium polysaccharide monoclonal anti liquid solution and carries out coupling, and UV scanning is with the monitoring coupling process; With the coupling buffer eccysis active dendrobium polysaccharide monoclonal antibody of link coupled not, obtain agarose-antibody coupling mixture; (B) sealing activating group: agarose-antibody coupling mixture is joined in Tutofusin tris-hydrochloric acid (Tris-HCl) damping fluid of 6 times of volumes, keep 3 hours with unreacted radical in the sealing sepharose 4B; The pH value of said tris-HCI buffer is 8.0, and tris-HCI buffer is for containing 0.5 Mol/L sodium-chlor and 0.1 Mol/L Tutofusin tris-aqueous solution of hydrochloric acid; (C) dress post: void column is washed with the pH value 7.2 of 10 times of column volumes, the phosphoric acid buffer of concentration 0.01 Mol/L; Place a hydrophobic sieve plate then in the bottom; And put into the pH value 7.2 of void column 1/8 volume, the phosphoric acid buffer of concentration 0.1 Mol/L, obtain the pre-treatment void column; Agarose-antibody coupling mixture that step (A) is obtained washs three times with the 0.1 Mol/L phosphoric acid buffer that contains 0.5Mol/L sodium-chlor 10 times of agaroses-antibody coupling mixture volume, pH value 4.5 earlier; Then with the 0.1 Mol/L tris-HCI buffer washing that contains 0.5 Mol/L sodium-chlor 10 times of agaroses-antibody coupling mixture volume, pH value 8.0 three times; With phosphoric acid buffer washing 5 times of agaroses-antibody coupling mixture volume, pH value 7.2, concentration 0.1 Mol/L three times, obtain agarose-antibody coupling composite colloids again; Agarose-antibody coupling composite colloids is poured in the pre-treatment void column; Wait; Add a filter paper at agarose-antibody coupling composite colloids top; And with the pH value 7.2 of 10 times of column volumes, the phosphoric acid buffer flushing of concentration 0.1 Mol/L; To get rid of the bubble in the post bed, use the 0.1 Mol/L phosphoric acid buffer preservation that massfraction is 0.01% sodiumazide that contains of pH value 7.2 at last, obtain active dendrobium polysaccharide monoclonal antibody immunoaffinity chromatography prepacked column;
Step 7 antibody affinity chromatography prepares active dendrobium polysaccharide: (a) balance: with the flow velocity equilibrium activity dendrobium polysaccharide monoclonal antibody immunoaffinity chromatography prepacked column of level pad with 2 ml/min; Obtain the balance chromatography column through ultraviolet detection 280 nm places light absorption value monitoring prepacked column balance to baseline; Said level pad is the 0.1 Mol/L Tris-HCl aqueous solution that contains 0.5 Mol/L sodium-chlor of pH value 8.0; (b) go up appearance: get stem of noble dendrobium Crude polysaccharides and be mixed with the sample solution that mass concentration is 0.2 mg/ml; With 50 ml sample solutions with the flow velocity of 2 ml/min through the balance chromatography column, obtain chromatography and carry a kind post; (c) wash-out: clean chromatography with level pad with the flow velocity of 2 ml/min and carry a kind post, carry a kind post through ultraviolet detection 280 nm places light absorption values monitoring and cleaned to baseline; With the flow velocity of 1 ml/min dendrobium polysaccharide is carried a kind post from chromatography with the 0.1 Mol/L phosphoric acid elutriant that contains 0.5 Mol/L sodium-chlor of pH value 4.5 and elute, effluent volume is 5 times that chromatography carries kind column volume; Follow the tracks of detection with the phenol sulfuric acid process and collect dendrobium polysaccharide content in each pipe, collect dendrobium polysaccharide main peak effluent; Effluent lyophilize of dendrobium polysaccharide main peak or spraying drying obtain active dendrobium polysaccharide;
The dendrobium polysaccharide that makes through above-mentioned steps shows that through the fluorescence assisted carbohydrate electrophoresis interpretation of result dendrobium polysaccharide purity can reach more than 98%, and the recovery is 95%.
Embodiment 3: dendrobium polysaccharide causes the provide protection of acute liver damage and hepatic fibrosis rats to tetracol phenixin
After SD rat flexibility fed a week; Weigh and be divided into 6 groups at random: dose groups (200 mg/kg.bw), dendrobium polysaccharide high dose group (400 mg/kg.bw), Biphenylylmethylcarbinol positive controls (200 mg/kg.bw) in normal control group, model control group, dendrobium polysaccharide low dose group (100 mg/kg.bw), the dendrobium polysaccharide, 10 every group.Except that the normal control group, all the other each groups all adopt subcutaneous injection CCl
4, peanut oil mixed solution (1 ︰ 9) causes the rat liver fibrosis model, 2 times weekly, each 4.0 ml, continuous 12 weeks.Since the 5th week, irritate clothes relative medicine (10.0 ml/kg), every day 1 time, continuous 9 weeks respectively by group.The last administration was put to death rat after 24 hours, and separation of serum is measured serum glutamic pyruvic transminase (ALT), glutamic-oxal(o)acetic transaminase (AST) activity, and the result sees table 2.
Table 2 dendrobium polysaccharide is to CCl
4The influence of inductive acute liver damage and hepatic fibrosis rats Serum ALT and AST (x scholar s, n=10)
Annotate: * P<0.05 common group of vs; * P<0.01 common group of vs;
#P<0.05 vs CCl
4Group;
##P<0.01 vs CCl
4Group.
Embodiment 4: dendrobium polysaccharide is to the provide protection of chronic alcoholic liver injury and hepatic fibrosis mouse
After will cleaning level kunming mice flexibility and feeding a week; Weigh and be divided into 6 groups at random: 1. negative control group: give 20ml/kg.bw zero(ppm) water every day and irritate stomach; Give zero(ppm) water 15ml/kg.bw after 1 hour again and irritate stomach; 2. alcoholic liver model group: give 20ml/kg.bw zero(ppm) water every day and irritate stomach, give 50% ethanol 15ml/kg.bw after 1 hour again and irritate stomach; 3. low dosage dendrobium polysaccharide group: give the 100mg/kg dendrobium polysaccharide every day and irritate stomach, give 50% ethanol 15ml/kg.bw after 1 hour again and irritate stomach; 4. middle dosage dendrobium polysaccharide group: give the 200mg/kg dendrobium polysaccharide every day and irritate stomach, give 50% ethanol 15ml/kg.bw after 1 hour again and irritate stomach; 5. high dosage dendrobium polysaccharide group: give the 400mg/kg dendrobium polysaccharide every day and irritate stomach, give 50% ethanol 15ml/kg.bw after 1 hour again and irritate stomach; 6. vitamin E intervention group: give the 500mg/kg vitamin E every day and irritate stomach, give 50% ethanol 15ml/kg.bw after 1 hour again and irritate stomach.12 week of continuous irrigation stomach, mouse was put to death in the back, and separation of serum is measured serum glutamic pyruvic transminase (ALT), glutamic-oxal(o)acetic transaminase (AST) activity, and the result sees table 3.
Table 3 dendrobium polysaccharide to the influence of chronic alcoholic liver injury and hepatic fibrosis mice serum ALT and AST (x scholar s, n=10)
Annotate: * P<0.05 vs negative control group;
#P<0.05 vs alcoholic liver model group;
Embodiment 5: dendrobium polysaccharide is to the provide protection of chronic non-alcoholic liver injury rat
After SD rat flexibility fed a week, weigh and random packet, be divided into common group, hyperlipidemia model group earlier; Feed with normal diet for common group, the hyperlipidemia model group is fed with high lipid food (high lipid food prescription: 10% lard+2% SUV+0.4% propylthiouracil+5% yolk powder+82.6% normal diet); The 6th weekend in week; Randomly draw 1, model group from common group and randomly draw 3 rats; After water 12h is can't help in fasting, prepare serum through aorta abdominalis negative pressure blood sampling tube, serum is with automatical analysis biochemical instruments analysis of biochemical index; And get the same position of right lobe of liver; Carry out techtology through HE dyeing and observe, after the affirmation modeling success, the hyperlipidemia model group is divided into model group, diet improvement group, polysaccharide improvement group, polysaccharide+diet improvement group, nicotinic acid+diet improvement group (table 4) (8 every group) at random again.Give corresponding diet and filling stomach polysaccharide according to table 3 every day.
Table 4 animal divides into groups and administration
Annotate: "-" expression does not process in the table 4.
Every separated 3d weighs and writes down rat hair, animation, body weight and food ration.Can't help water 12h in the 8th week SD rat fasting at weekend.With no antithrombotics negative pressure pipe abdominal aortic blood, separation of serum, measure serum glutamic pyruvic transminase (ALT), glutamic-oxal(o)acetic transaminase (AST) activity, the result sees table 5.
Table 5 dendrobium polysaccharide to the influence of chronic non-alcoholic liver injury and hepatic fibrosis rats Serum ALT and AST (x scholar s, n=8)
Annotate: < common group of 0.05 vs of * P in the table 5; * P < common group of 0.01 vs.
Embodiment 6: dendrobium polysaccharide is to the provide protection of immunological liver injury and hepatic fibrosis mouse
After will cleaning level kunming mice flexibility and feeding a week, weigh and be divided into 5 groups at random: normal group, model group [BCG-CWS (BCG)+LPS (LPS)] and the basic, normal, high dose groups of dendrobium polysaccharide.Tested first day, every tail vein injection saline 0.2ml of normal group, other respectively organize every mouse tail vein injection mass concentration is 1% BCG solution 0.2ml.Rise next day, normal group and model group intravenous injection every day saline water 0.2ml, continuous 6 weeks; The basic, normal, high dose groups difference of dendrobium polysaccharide tail vein injection dendrobium polysaccharide 100mg/kg every day, 200mg/kg, 400mg/kg, continuous 6 weeks.After the last administration, except that the normal group tail vein injection saline, all the other respectively organize every mouse tail vein injection LPS 4 μ g; Behind the 12h, mouse is put to death in the back, and separation of serum is measured serum glutamic pyruvic transminase (ALT), glutamic-oxal(o)acetic transaminase (AST) activity, and the result sees table 6.
Table 6 dendrobium polysaccharide to the influence of immunological liver injury and hepatic fibrosis mice serum ALT and AST (x scholar s, n=10)
Annotate: * P in the table 6<0.05 vs normal group; * P<0.01 vs normal group;
#P<0.05 vs model group;
##P<0.01 vs model group.
Claims (5)
1. one kind protects the liver the active dendrobium polysaccharide of anti-hepatic fibrosis, and it is characterized in that: the chemical structural formula of this activity dendrobium polysaccharide is following:
The molecular weight of this activity dendrobium polysaccharide is 2.2 * 10
4Dalton, it is to be that main chain, semi-lactosi are ramose gala konjac glucomanna with glucose and seminose, wherein, the mol ratio of glucose, seminose and semi-lactosi is 31 ︰, 10 ︰ 8.
2. protect the liver extraction, the separation purification method of the active dendrobium polysaccharide of anti-hepatic fibrosis, it is characterized in that the concrete operations step is following:
(1) raw materials pretreatment: get the stem of noble dendrobium of pulverizing, in 50 ℃ of backflow 5h of temperature, degreasing, decolouring are positioned over 25 ℃ of environment of temperature, remove organic solvent with volatilization, obtain the stem of noble dendrobium dregs of a decoction with sherwood oil;
(2) hot water lixiviate: get the stem of noble dendrobium dregs of a decoction, add zero(ppm) water,, obtain vat liquor in 80 ℃ of lixiviate 60 min of temperature by feed liquid mass volume ratio 1 g ︰ 30ml; With vat liquor spinning 15 min under rotating speed 15000 r/min conditions, the centrifugal sediment that obtains is with twice of the lixiviate again of 80 ℃ of hot water; Merge the centrifuged supernatant of three collections, obtain stem of noble dendrobium vat liquor; Under 60 ℃ of conditions of temperature, concentrating under reduced pressure is 1 ml ︰ 3g to the ratio of liquid concentrator volume and stem of noble dendrobium dregs of a decoction quality, obtains stem of noble dendrobium liquid concentrator with stem of noble dendrobium vat liquor; It is that 3000 daltonian dialysis tubings are dialysed 3 days to remove small-molecule substance that stem of noble dendrobium liquid concentrator uses molecular weight cut-off, gets stem of noble dendrobium dialysis trapped fluid;
(3) alcohol precipitation: in stem of noble dendrobium dialysis trapped fluid, add volume(tric)fraction and be 95% ethanol, make that ethanol final volume mark reaches 80% in the stem of noble dendrobium dialysis trapped fluid, left standstill 72 hours in 4 ℃ of temperature; Under 4 ℃ of temperature, rotating speed 12000 r/min conditions, deposition is got in spinning 15 minutes, obtains stem of noble dendrobium throw out;
(4) deproteinated: get stem of noble dendrobium throw out, add zero(ppm) water, be mixed with the dendrobium polysaccharide water extract A of 100 mg/ml; Get dendrobium polysaccharide water extract A, chloroform is pressed dendrobium polysaccharide water extract A 1/5 volume add, add the butanols of chloroform volume 1/5 thereupon again; Mix, shaken 20 min are then under 10 ℃ of temperature, rotating speed 12000 r/min conditions; Spinning 15 minutes, the denatured protein of branch vibration layer and solution layer intersection gets dendrobium polysaccharide water extract B;
Get dendrobium polysaccharide water extract B, chloroform is pressed dendrobium polysaccharide water extract B 1/5 volume add, add the butanols of chloroform volume 1/5 thereupon again; Mix; Shaken 20 min, then under 10 ℃ of temperature, rotating speed 12000 r/min conditions, spinning 15 minutes; The denatured protein of branch vibration layer and solution layer intersection gets dendrobium polysaccharide water extract C;
Get dendrobium polysaccharide water extract C, chloroform is pressed dendrobium polysaccharide water extract C 1/5 volume add, add the butanols of chloroform volume 1/5 thereupon again; Mix; Shaken 20 min, then under 10 ℃ of temperature, rotating speed 12000 r/min conditions, spinning 15 minutes; The denatured protein of branch vibration layer and solution layer intersection gets dendrobium polysaccharide water extract;
(5) purifying: 1. DEAE-52 ion exchange resin column classification: get dendrobium polysaccharide water extract,, follow the tracks of the detection effluent, collect the main peak effluent A of zero(ppm) water wash-out part with the phenol sulfuric acid process through DEAE-52 high-efficiency anion exchange resin column; It is 2000 daltonian dialysis tubing dialysis 3 days that main peak effluent A uses molecular weight cut-off, collects dialysis liquid concentrator A; 2. Sephacryl S-200 gel resin post classification: get dialysis liquid concentrator A,, follow the tracks of the detection effluent, collect the main peak effluent B of zero(ppm) water wash-out part with the phenol sulfuric acid process through Sephacryl S-200 gel resin post; It is 2000 daltonian dialysis tubing dialysis 3 days that main peak effluent B uses molecular weight cut-off, collects dialysis liquid concentrator B;
Get dialysis liquid concentrator B; Successively through classification of DEAE-52 ion exchange resin column and the classification of Sephacryl S-200 gel resin post; Repeat DEAE-52 ion exchange resin column classification and Sephacryl S-200 gel resin post progressive operation step 8 time; Collect last dialysis liquid concentrator through lyophilize, obtain active dendrobium polysaccharide;
(6) chemical structure of active dendrobium polysaccharide characterizes: through all-hydrolytic analysis, Smith DeR, methylation analysis, IR spectroscopy, and the spectral analysis of the nuclear magnetic resonance method has confirmed that the chemical structural formula of active dendrobium polysaccharide is following:
3. extraction, the separation purification method that protects the liver the active dendrobium polysaccharide of anti-hepatic fibrosis according to claim 2, it is characterized in that: the said stem of noble dendrobium is fresh Herba Dendrobii or exsiccant Herba Dendrobii.
4. the antibody affinity chromatography preparation protects the liver the method for the active dendrobium polysaccharide of anti-hepatic fibrosis, it is characterized in that the concrete operations step is following:
(1) preparation immunogen: get in the carbonate buffer solution that active dendrobium polysaccharide is dissolved in pH value 9.2; Being mixed with massfraction is the active dendrobium polysaccharide solution of 10 mg/ml; Add bovine serum albumin then, the massfraction that makes the bovine serum albumin in the active dendrobium polysaccharide solution is 10 mg/ml, stirring and evenly mixing; Place the thermostat container of 37 ℃ of temperature to react 3d, obtain reaction system; Add thanomin, make that the thanomin massfraction reaches 1% o'clock termination reaction in the reaction system, reaction solution; Reaction solution separates through Sephadex G-150 gel column, fraction collection; Adopt phenolsulfuric acid method and Bradford method to measure active dendrobium polysaccharide and bovine serum albumin content in each pipe respectively, calculate the bovine serum albumin mole molecule number that every mole of active dendrobium polysaccharide molecule connects; Getting the conjugate that every mole of active dendrobium polysaccharide molecule is connected with 5 moles of bovine serum albumin molecules is active dendrobium polysaccharide-bovine serum albumin immunogen, obtains the polysaccharide compound immunogen;
(2) immune mouse: 5 mg polysaccharide compound immunogens are dissolved in the 5 mL saline water,, obtain immunogen solution with the two pushing manipulation mixings of isopyknic Freund's complete adjuvant; Adopt the abdominal injection immunity female mice in 8 ages in week, every injected in mice immunogen solution 0.2 ml; After 1 week the 7th day got 5 mg polysaccharide compound immunogens and is dissolved in the 5 mL saline water, with equal-volume Freund's incomplete adjuvant mixing, the immunogen mixed solution; Every female mice abdominal injection 0.2 ml immunogen mixed solution carries out booster immunization, and booster immunization repeats 3 times, 1 time weekly; After last 1 immunity the 7th day cut tail and got blood, measures tiring of active dendrobium polysaccharide specific antibody in the mice serum with enzyme-linked immunosorbent assay (ELISA); Select the mouse that active dendrobium polysaccharide specific antibody titres is higher than 1 ︰ 10000 in the serum to be used for cytogamy; And before cytogamy 4-5 days; It is that the polysaccharide compound immunogen aqueous solution of 1.0 mg/mL impacts immunity that massfraction that 0.2 ml do not contain adjuvant is inoculated in the abdominal cavity, obtains immune mouse;
(3) cytogamy: the SPL of under aseptic condition, getting immune mouse is processed every milliliter and is contained 1 * 10
3～1 * 10
4The cell suspension of individual cell with cell suspension under the condition of 4 ℃ of temperature, rotating speed 1000 r/min centrifugal 10 minutes, is got centrifugation, obtains the SPL of immune mouse; Get 1 * 10
8The SPL of individual immune mouse and 2 * 10
7Individual Balb/c mouse SP2/0 tumour cell mixes, and is preheated to 37 ℃, and slowly adding 1ml, to be preheated to 37 ℃ massfraction be 50% polyglycol solution; Slowly add 10 ml again and be preheated to 37 ℃ the DMEM perfect medium that massfraction is 20% foetal calf serum that contains; Cell suspension under 4 ℃ of temperature, rotating speed 1000 r/min conditions centrifugal 10 minutes, is abandoned supernatant; In deposition, add DMEM perfect medium 5ml; Cell suspends again, is sub-packed in the 96 porocyte culture plates, is 5% carbonic acid gas (CO in 37 ℃ of temperature, volume(tric)fraction
2) cultivated 1 day under the condition of gas, 95% humidity; Add 4 * HAT nutrient solution, 50 μ l then; Displace the l/2DMEM perfect medium in the culture hole of 96 porocyte culture plates on the 6th day with the HAT nutrient solution, displaced the HAT nutrient solution with the HT nutrient solution on the 11st day, obtain fused cell;
(4) the active dendrobium polysaccharide monoclonal antibody hybridoma cell strain of preparation: fused cell is inoculated in HAT selects on the substratum; With the polysaccharide compound immunogen is antigen; Adopt the indirect enzyme-linked immunosorbent analytical method to detect tiring of the active dendrobium polysaccharide specific antibody of fused cell excretory; The selection antibody titer is higher than the fused cell of 1 ︰ 1000, obtains the hybrid cell of ability secretion activity dendrobium polysaccharide specific antibody; Adopt limiting dilution assay that said hybrid cell is carried out cloning in 96 porocyte culture plates; Adopt the indirect enzyme-linked immunosorbent analytical method to detect tiring of the clone's active dendrobium polysaccharide specific antibody of secretion in culture supernatant liquid on the 96 porocyte culture plates; Select antibody titer to be higher than the single cell clone of 1 ︰ 100, obtain positive colony; Get positive colony and adopt limiting dilution assay in 96 porocyte culture plates, to carry out three time cloningizations, select the positive colony of ability secretion activity dendrobium polysaccharide specific antibody, obtain hybridoma cell clone; Selecting strong, the well-grown hybridoma cell clone of antibody response to be inoculated on the HAT substratum, is 5% carbonic acid gas (CO in 37 ℃ of temperature, volume(tric)fraction
2) cultivated three days under the condition of gas, 95% humidity, obtain active dendrobium polysaccharide monoclonal antibody hybridoma cell strain;
(5) the active dendrobium polysaccharide monoclonal antibody of preparation: get the female mice in 10 ages in week, abdominal injection 0.5 ml pristane; Every female mice inoculation in the 7th day is in the active dendrobium polysaccharide monoclonal antibody hybridoma cell strain 5 * 10 of logarithmic phase
6Individual; Fortnight is gathered the ascites of female mice, and ascites under the condition of 4 ℃ of temperature, rotating speed 5000 r/min, centrifugal 15 minutes, is collected supernatant; Adopt the antibody A in sad-ammonium sulfate precipitation method deposition supernatant, antibody A obtains antibody B through Sephadex G-150 gel filtration chromatography; Adopt PAGE and SDS-PAGE method to identify the purity of antibody B respectively, use the specificity of indirect enzyme-linked immunosorbent assay antibody B simultaneously active dendrobium polysaccharide; Collect band homogeneous, apparent antibody B, lyophilize obtains active dendrobium polysaccharide monoclonal antibody;
(6) the active dendrobium polysaccharide monoclonal antibody immune affinity chromatographic column of preparation: (A) coupling of agarose (Sepharose) 4B carrier and antibody: get the active dendrobium polysaccharide monoclonal antibody of 10g and be dissolved in the coupling buffer; Placing molecular weight cut-off then is 2000 daltonian dialysis tubing dialysis 24 hours; Get the dialysis trapped fluid and be mixed with the active dendrobium polysaccharide monoclonal antibody aqueous solution that massfraction is 0.5 mg/ml; The pH value of said coupling buffer is 8.2, and coupling buffer is to contain 0.5 Mol/L sodium-chlor (NaCl) and 0.1 Mol/L sodium hydrogencarbonate (NaHCO
3) the aqueous solution; The sepharose 4B 15g of the cyanogen bromide-activated of learning from else's experience takes out fast with said coupling buffer and to wash 5 times, pours into rapidly in the active dendrobium polysaccharide monoclonal anti liquid solution and carries out coupling, and UV scanning is with the monitoring coupling process; With the coupling buffer eccysis active dendrobium polysaccharide monoclonal antibody of link coupled not, obtain agarose-antibody coupling mixture; (B) sealing activating group: agarose-antibody coupling mixture is joined in Tutofusin tris-hydrochloric acid (Tris-HCl) damping fluid of 6 times of volumes, keep 3 hours with unreacted radical in the sealing sepharose 4B; The pH value of said tris-HCI buffer is 8.0, and tris-HCI buffer is for containing 0.5 Mol/L sodium-chlor and 0.1 Mol/L Tutofusin tris-aqueous solution of hydrochloric acid; (C) dress post: void column is washed with the pH value 7.2 of 10 times of column volumes, the phosphoric acid buffer of concentration 0.01 Mol/L; Place a hydrophobic sieve plate then in the bottom; And put into the pH value 7.2 of void column 1/8 volume, the phosphoric acid buffer of concentration 0.1 Mol/L, obtain the pre-treatment void column; Agarose-antibody coupling mixture that step (A) is obtained washs three times with the 0.1 Mol/L phosphoric acid buffer that contains 0.5Mol/L sodium-chlor 10 times of agaroses-antibody coupling mixture volume, pH value 4.5 earlier; Then with the 0.1 Mol/L tris-HCI buffer washing that contains 0.5 Mol/L sodium-chlor 10 times of agaroses-antibody coupling mixture volume, pH value 8.0 three times; With phosphoric acid buffer washing 5 times of agaroses-antibody coupling mixture volume, pH value 7.2, concentration 0.1 Mol/L three times, obtain agarose-antibody coupling composite colloids again; Agarose-antibody coupling composite colloids is poured in the pre-treatment void column; Wait; Add a filter paper at agarose-antibody coupling composite colloids top; And with the pH value 7.2 of 10 times of column volumes, the phosphoric acid buffer flushing of concentration 0.1 Mol/L; To get rid of the bubble in the post bed, use the 0.1 Mol/L phosphoric acid buffer preservation that massfraction is 0.01% sodiumazide that contains of pH value 7.2 at last, obtain active dendrobium polysaccharide monoclonal antibody immunoaffinity chromatography prepacked column;
(7) antibody affinity chromatography prepares active dendrobium polysaccharide: (a) balance: with the flow velocity equilibrium activity dendrobium polysaccharide monoclonal antibody immunoaffinity chromatography prepacked column of level pad with 2 ml/min; Obtain the balance chromatography column through ultraviolet detection 280 nm places light absorption value monitoring prepacked column balance to baseline; Said level pad is the 0.1 Mol/L Tris-HCl aqueous solution that contains 0.5 Mol/L sodium-chlor of pH value 8.0; (b) go up appearance: get stem of noble dendrobium Crude polysaccharides and be mixed with the sample solution that mass concentration is 0.2 mg/ml, with 50 ml sample solutions with the flow velocity of 2 ml/min through the balance chromatography column, obtain chromatography and carry a kind post; (c) wash-out: clean chromatography with level pad with the flow velocity of 2 ml/min and carry a kind post, carry a kind post through ultraviolet detection 280 nm places light absorption values monitoring and cleaned to baseline; With the flow velocity of 1 ml/min dendrobium polysaccharide is carried a kind post from chromatography with the 0.1 Mol/L phosphoric acid elutriant that contains 0.5 Mol/L sodium-chlor of pH value 4.5 and elute, effluent volume is 5 times that chromatography carries kind column volume; Follow the tracks of detection with the phenol sulfuric acid process and collect dendrobium polysaccharide content in each pipe, collect dendrobium polysaccharide main peak effluent; Effluent lyophilize of dendrobium polysaccharide main peak or spraying drying obtain active dendrobium polysaccharide; The chemical structural formula of active dendrobium polysaccharide is following:
5. a purposes that protects the liver the active dendrobium polysaccharide of anti-hepatic fibrosis is characterized in that, this activity dendrobium polysaccharide have protect the liver, anti-hepatic fibrosis is active, can be used for preparing the medicine of liver health product and treatment hepatic fibrosis.
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| CN105906739A (en) * | 2016-07-05 | 2016-08-31 | 镇江劲草堂生物科技有限公司 | Purification method of dendrobium candidum protocorm polysaccharide (DCPP) |
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| CN107188980A (en) * | 2017-05-24 | 2017-09-22 | 中国科学院上海药物研究所 | The different xylans of β 1,4, its sulfated derivative and its production and use |
| CN108341887A (en) * | 2018-03-14 | 2018-07-31 | 漯河中德双成功能食品研究院有限公司 | A kind of preparation method of Dendrobidium huoshanness polysaccharide |
| CN108341887B (en) * | 2018-03-14 | 2020-08-07 | 河南夕阳蓝食品科技有限公司 | Preparation method of dendrobium huoshanense polysaccharide |
| CN108912236A (en) * | 2018-09-29 | 2018-11-30 | 霍山县天下泽雨生物科技发展有限公司 | A kind of Dendrobidium huoshanness refined polysaccharide and preparation method thereof with antitumor action |
| CN109400742A (en) * | 2018-11-09 | 2019-03-01 | 浙江省医学科学院 | A kind of dendrobium devonianum refined polysaccharide and its preparation method and application |
| CN109400742B (en) * | 2018-11-09 | 2021-10-29 | 浙江省医学科学院 | Dendrobium devonianum refined polysaccharide and preparation method and application thereof |
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