CN101914168A - Medical applications and preparation methods of Pleurotus ferulae Lanzi polysaccharide and composites thereof - Google Patents
Medical applications and preparation methods of Pleurotus ferulae Lanzi polysaccharide and composites thereof Download PDFInfo
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Abstract
The invention relates to medical applications and preparation methods of Pleurotus ferulae Lanzi polysaccharide and composites thereof. The preparation methods of the invention comprise the following steps of extracting, removing protein and purifying, wherein the step of removing protein adopts the following methods of: (1) a solvent extraction method; (2) an ion exchange resin method; (3) an enzyme method; and (4) a gel chromatography; and the step of purifying adopts the following methods of: (1) the solvent extraction method; (2) an ethanol precipitation method; (3) a liquid-liquid counter-current distribution chromatography; and (4) the gel chromatography.
Description
Technical field: the present invention relates to the Pleurotus ferulae Lanzi polysaccharide with medicinal use and the pharmaceutical composition that contains the Pleurotus ferulae Lanzi polysaccharide of preparation from Pleurotus ferulae Lanzi (Pleurotus ferulae Lanzi), and their preparation, medicinal use and analytical procedure.
Background technology:
Cervical cancer is one of modal gynecologic malignant tumor, and Xinjiang is the district occurred frequently of China's cervical cancer, and M ﹠ M is all very high, and the trend of rising year by year and rejuvenation is arranged.The cervical cancer treatment means develops into whole body therapeutic by traditional topical therapeutic operation, radiotherapy---chemotherapy.Yet multiple therapy methods all can weaken anti tumor immune response.Therefore, in the therapeutic process of cervical cancer, the enhance immunity function plays an important role in the process of eliminating tumour cell.
At present existing a large amount of clinical research confirmation herbal medicine can improve the tumour patient life quality, suppress tumour, mitigation symptoms, even make the patient be with knurl to prolong life.As a kind of rare medicine-food two-purpose resource, the research of the anti-tumor aspect of Pleurotus ferulae Lanzi has some reports.The dose,optimum 500mg/kg of Pleurotus ferulae Lanzi polysaccharide in the document has been selected in this experiment for use, the result shows, the Pleurotus ferulae Lanzi polysaccharide has certain inhibition effect to mouse cervical cancer U14 transplanted tumor, inhibitory rate to 39%, clinical cervical cancer chemotherapy one line medicine cis-platinum commonly used is 46.5% to its tumour inhibiting rate, when Pleurotus ferulae Lanzi polysaccharide and cis-platinum acting in conjunction, inhibitory rate to 65.1% illustrates that the Pleurotus ferulae Lanzi polysaccharide can improve the anticancer therapeutic of cis-platinum.This polysaccharide also has certain inhibition effect, inhibitory rate to 54% to rat liver cancer H22 transplanted tumor simultaneously.According to tumor research rules regulation, the tumour inhibiting rate of herbal medicine was greater than 30% o'clock, and thinking has certain curative effect to tumour, and the Pleurotus ferulae Lanzi polysaccharide has surpassed this standard, and therefore good DEVELOPMENT PROSPECT is arranged.
The anticancer mechanism of Pleurotus ferulae Lanzi may realize by its immuno-stimulating effect.It can activate the cell of body immune system and humoral immunoresponse(HI) and bring into play antitumor action, and especially the cytokine of the activation of cellular immunization and generation thereof has important effect.
Scavenger cell is a kind of very active immune effector cell that participates in killing tumor cells, and M φ is mainly by endocytosis and some effector molecule killing tumor cells of secretion, and while activatory M φ can " respiratory burst " occur and produce a series of O rapidly after taking in tumour
2-, H
2O
2, OH
-Isoreactivity oxyradical and active nitrogen, mediated cell immunity and the cytotoxic reaction in the immunity of organism treatment of these active groups participates in killing tumor cell; The NK cell is a class has the effector cell of spontaneous cytotoxic activity to multiple target cell, but the NK cell without the tumour cell of sensitization direct killing sensitivity, as K562, YAC-1 etc. are in anti-newborn knurl, form and also play an important role aspect knurl and the metastases.
The T cell all plays an important role in the cellular immunization of body and humoral immunization are induced.It mainly contains two aspect functions as immune effector cell: promptly as TDHT mediation delayed type hypersensitivity with as Tc cell direct killing target cell.The B cell is exercised its immunologic function by excretory antibody.In addition, activatory B cell also has processing and offers the effect that antigen is given the T cell.The antibody of B emiocytosis can be carried out the panimmunity function.
The lymphocyte transformation experiment can be understood cell immune function of human body.This experiment is by measuring above-mentioned immune cell function, find: the Pleurotus ferulae Lanzi polysaccharide can significantly promote the phagocytic function of tumor-bearing mice PM φ, strengthen the effect of NK cell killing target cell K562 in the spleen, improve the activity of T, bone-marrow-derived lymphocyte, show that it can suppress the growth of transplanted tumor U14 by the cellular immune function that promotes tumor-bearing mice.The detection of T cell subsets is to observe one of important method of body cell immunologic function, CD4
+/ CD8
+Ratio can directly reflect host's t lymphocyte subset group's state, and can understand body cell immunologic function situation indirectly to a certain extent.Thereby mensuration CD4
+And CD8
+Cell per-cent, or the ratio of two kinds of cells have become a kind of ten minutes index of entry evaluation immune status easily, have only when both ratios suitable, the normal antitumor action of competence exertion.
Th1 cytokines (TNF-α, IFN-γ) has the immunoreactive ability of antitumor cell that activates body, and wherein TNF-α is a kind of effective tumor cytotoxicity factor, also is the important regulatory factor of body inflammatory reaction and immunne response.TNF-α can combine with the cell surface specific receptors and form mixture, triggers lysosomal release, causes oncolysis; In vivo, TNF-α has direct toxic action to tumor vascular endothelial cell, can increase sticking of endotheliocyte and neutrophil leucocyte, bring out the body clotting mechanism, through a series of chain reactions, produce thrombus and occluding vascular, tumor tissues necroses because of anoxic.IFN-γ is produced by the activated T cell, can strengthen the cytotoxicity of dependence of host's TXi Baoshouti and the restricted T cell of HLA, has the surperficial MHC antigen of increasing and tumour necrosis factor and expresses multiple antitumor actions such as antineoplastic vascular generation.IFN-γ can also express by the Fas/FasL of modulate tumor cell and strengthen the susceptibility of tumour cell to Fas institute mediated apoptosis approach, and the ability that makes tumour cell escape immune system attack reduces, thereby suppresses the malignant proliferation of tumour cell.So TNF-α, IFN-γ has consequence and effect in antitumor immunity of organism.
Experimental result shows in the body, the Pleurotus ferulae Lanzi polysaccharide can obviously promote the immunocompetence of NK cell in tumor-bearing mice phagocytosis of macrophages, the spleen, increase spleen index, improve splenic T, bone-marrow-derived lymphocyte transformation efficiency, impel CD3/CD8 ratio to recover normal, improve cytokine TNF-α, IFN-γ level, and can improve the influence that cis-platinum suppresses the mice with tumor immunologic function during with the cis-platinum coupling.The result shows: the Pleurotus ferulae Lanzi polysaccharide can suppress the propagation of mouse cervical cancer U14 transplanted tumor by promoting the mouse body's immunity.
Simultaneously; because the Pleurotus ferulae Lanzi polysaccharide can promote body's immunity, therefore: be expected to suppressing growth of tumour cell, strengthen body immunity, anticoagulation, protection stomach mucous membrane, antifatigue, delay senility, regulate central nervous system, improve the cardiovascular and cerebrovascular blood supply insufficiency, going greasyly to sober up, improve looks and the arteries and veins of invigorating blood circulation, anti-ageing, reducing blood-fat, decreasing cholesterol, triglyceride reducing, improve in blood viscosity and microcirculation, vasodilation, removing free radical, antitumor, cancer therapy drug, functional food and the food and be used widely.
Summary of the invention:
The objective of the invention is to, a kind of Pleurotus ferulae Lanzi polysaccharide with medicinal use of preparation from Pleurotus ferulae Lanzi (Pleurotus ferulae Lanzi) is provided, this polysaccharide does not contain protein, and its HPLC-ELSD chromatogram has characteristic spectrum as shown in Figure 1.
The present invention also provides the pharmaceutical composition that contains Pleurotus ferulae Lanzi polysaccharide of the present invention.
Pharmaceutical composition of the present invention is activeconstituents with Pleurotus ferulae Lanzi polysaccharide of the present invention, can be prepared into any pharmaceutical dosage form of taking as required as lozenge, tablet, and capsule, particle, oral liquid, injection, in generation, made tea beverage etc.According to patient's situation, determine usage and dosage in use, but obey every day three times, each 1-20 agent, as: 1-20 bag or grain or sheet, every dose of 1mg-1000mg.When being prepared into pharmaceutical preparation, as required, can add medicine or edible carrier.
The present invention also comprises the preparation method of Pleurotus ferulae Lanzi polysaccharide of the present invention, and this method comprises extraction, removes albumen, purifying.Remove albumen and adopt following method, (1) solvent extration; (2) ion-exchange-resin process; (3) enzyme process; (4) gel chromatography.Purifying adopts following method, (1) solvent extration; (2) ethanol precipitation; (3) liquid-liquid adverse current partition chromatography; (4) gel chromatography.
Preferred preparation method of the present invention, wherein extraction step is as follows:
Pleurotus ferulae Lanzi is pulverized, and extracting in water is centrifugal or remove by filter residue, the recycle-water extracting solution, the Pleurotus ferulae Lanzi concentrated solution.It is characterized by: 1) Pleurotus ferulae Lanzi is fresh or fully soaks into through water; 2) water extraction can heat extraction, and by pulverizing, fragmentation, is shortened dramatically extraction time, and the best can be to 10-60 minute; 3) in order to improve centrifugal or to remove by filter efficient in the residue process, take into account extraction efficiency simultaneously, particle size after cracking is controlled at 10-300 order scope, is the best with the 30-60 order wherein.
Preferred preparation method of the present invention, wherein remove protein process and be selected from:
(1) solvent extration is characterized by: and elder generation's adding propyl carbinol in the Pleurotus ferulae Lanzi concentrated solution (100: 1-100: 20), jolting, add again trichloromethane (100: 1-100: 50), jolting, centrifugal, protein becomes insoluble state, remains in the interface of organic solvent and water, 1-100 time.Optimum is: ((25: 1), jolting 20min add trichloromethane (25: 4) again, and jolting 20min is centrifugal, the centrifugal 15min of 4000r/min, 6-8 time repeatedly to add propyl carbinol in the Pleurotus ferulae Lanzi concentrated solution earlier.It is characterized by: because Pleurotus ferulae Lanzi concentrated solution ground singularity, the solvent-extracted together method of propyl carbinol-trichloromethane mixed solution with conventional is difficult to reach and removes proteinic purpose, must add the propyl carbinol jolting earlier, adds the trichloromethane jolting again.
(2) ion-exchange-resin process is characterized by:
A) anionite-exchange resin 200g, distilled water immersion 10h, 1mol/L hydrochloric acid dashes 10 column volumes, distilled water flushing is to neutral, and 1mol/LNaOH dashes 10 column volumes, and distilled water flushing is to neutral, it is 8-14 that Pleurotus ferulae Lanzi concentrated solution 5-500ml (being equivalent to raw material 5-500mg) regulates the pH value with NaOH, last sample, the distilled water wash-out concentrates and obtains polyoses extract; Continue and use the 0.1mol/LNaOH wash-out, remove the protein on the post.Wherein regulating pH value optimum with NaOH is 8-11, and applied sample amount is Pleurotus ferulae Lanzi concentrated solution 50-100ml (being equivalent to raw material 50-100mg).
B) Zeo-karb 1000g, distilled water immersion 10h, 1mol/LNaOH is towards 10 column volumes of post, distilled water flushing is to neutral, and 1mol/L hydrochloric acid dashes 10 column volumes, and distilled water dashes to neutral, Pleurotus ferulae Lanzi concentrated solution 5-500ml (being equivalent to raw material 5-500mg) is 1-6 with the salt acid for adjusting pH value, last sample, distilled water concentrate and obtain polyoses extract towards post; Continue with 1mol/L hydrochloric acid wash-out, remove the protein on the post.Be 3-5 with salt acid for adjusting pH value optimum wherein, applied sample amount is Pleurotus ferulae Lanzi concentrated solution 50-100ml (being equivalent to raw material 50-100mg).
(3) enzyme process is characterized by:
A) Pleurotus ferulae Lanzi concentrated solution 5-500ml (being equivalent to raw material 5-500mg) adds papoid 0.6g, transfers PH1-9, and 30-80 ℃ of water-bath put to room temperature, and be centrifugal, gets supernatant liquor, can remove deproteinize.Optimum is: Pleurotus ferulae Lanzi concentrated solution 50-100ml (being equivalent to raw material 50-100mg) adds papoid 0.6g, transfers PH5-6, and 60 ℃ of water-bath 1.5h are put to room temperature, and the centrifugal 5min of 3000r/min gets supernatant liquor, can remove deproteinize.
B) Pleurotus ferulae Lanzi concentrated solution 5-500ml (being equivalent to raw material 5-500mg) adds stomach en-0.05g, transfers PH 0-4.5, and 30-45 ℃ of water-bath put to room temperature, add NaOH and transfer to neutrality, add ethanol to containing alcohol amount 50-95%, place the vibration back, centrifugal, get precipitation, can remove deproteinize.Optimum is: Pleurotus ferulae Lanzi concentrated solution 50-100ml (being equivalent to raw material 50-100mg) adds stomach en-0.05g, transfer PH1.5-2.5,37 ℃ of water-baths, put to room temperature, add NaOH and transfer to neutrality, add ethanol to containing alcohol amount 85%, place the vibration back, the centrifugal 5min of 3000r/min gets precipitation, can remove deproteinize.
(4) gel chromatography is characterized by:
Get dextrane gel 5g powder, with distilled water immersion swelling in beaker, the dress post.With dropper Pleurotus ferulae Lanzi concentrated solution 5-500ml (being equivalent to raw material 5-500mg) is evenly joined in the gel column distilled water wash-out.Each stream part is checked protein peak through UV 200-500nm full wavelength scanner.And each stream part is detected polysaccharide with sulfuric acid-phynol method reaction back at the 490nm place.Merge and contain polysaccharide, do not contain proteinic stream part, promptly.The dextrane gel optimum is sephadex G-100 and sephadex G-50, Pleurotus ferulae Lanzi concentrated solution 1-10ml (being equivalent to raw material 1-10mg).
Preferred preparation method of the present invention, wherein purification process is selected from:
(1) solvent extration:
Adopt organic solvents such as chloroform, ethyl acetate, propyl carbinol, Virahol, sherwood oil, gasoline, or the mixed solvent of above solvent, remove proteinic Pleurotus ferulae Lanzi extracting solution with the water liquid-liquid extraction, aqueous phase is the Pleurotus ferulae Lanzi polysaccharide of purifying.
(2) ethanol precipitation:
Remove proteinic Pleurotus ferulae Lanzi extracting solution, adding ethanol to alcohol concn is 50-95%, and place the vibration back, centrifugal, gets precipitation, can be repeatedly 2-10 time, be the Pleurotus ferulae Lanzi polysaccharide of purifying.Its optimum process is: remove proteinic Pleurotus ferulae Lanzi extracting solution, adding ethanol to alcohol concn is 85%, and place the vibration back, the centrifugal 5min of 3000r/min, and it is centrifugal to add ethanol again after precipitation is dissolved in water, 3 times repeatedly.
(3) liquid-liquid adverse current partition chromatography:
Adopt organic solvents such as chloroform, ethyl acetate, propyl carbinol, Virahol, sherwood oil, gasoline, or the mixed solvent of above solvent, with water liquid-liquid adverse current partition chromatography, remove proteinic Pleurotus ferulae Lanzi extracting solution through separating, choose the Pleurotus ferulae Lanzi polysaccharide and flow out part, be the Pleurotus ferulae Lanzi polysaccharide of purifying.
(4) gel chromatography.
Get dextrane gel 5g powder, with distilled water immersion swelling in beaker, the dress post.To remove proteinic Pleurotus ferulae Lanzi extracting solution 5-500ml (being equivalent to raw material 5-500mg) with dropper and evenly join in the gel column distilled water wash-out.Each stream part through sulfuric acid-phynol method reaction back at 490nm place detection polysaccharide.Merge and contain the polysaccharide part, promptly get the Pleurotus ferulae Lanzi polysaccharide of purifying.The dextrane gel optimum is sephadex G-100 and sephadex G-50, removes proteinic Pleurotus ferulae Lanzi extracting solution 1-10ml (being equivalent to raw material 1-10mg).
Particularly preferred preparation method of the present invention is as follows: gets Pleurotus ferulae Lanzi, is cut into small pieces, add water and squeeze the juice with juice extractor, and ultrasonic, boil, cooling, the centrifuging and taking supernatant liquor, the filter paper suction filtration, it is ultrasonic that precipitation adds water, boils, cooling, centrifugal, get supernatant, the filter paper suction filtration merges supernatant liquor, concentrate, get concentrated solution, concentrated solution adds papoid, transfers PH5-6, heating in water bath is put to room temperature, and is centrifugal, gets supernatant liquor, add ethanol and place, centrifugal, get precipitation, promptly.
The present invention also comprises by discovering ramulus mori, mulberry leaf, mulberry fruit and extract, acarbose etc., can be by suppressing the gi tract metabolic enzyme, suppress the degraded of Pleurotus ferulae Lanzi polysaccharide in gi tract, thereby oral administration system preparations such as () tablets is provided and has sucked drug delivery system preparations such as () lozenges.
The present invention comprises that also Pleurotus ferulae Lanzi polysaccharide of the present invention and pharmaceutical composition thereof suppress growth of tumour cell in preparation; strengthen body immunity; anticoagulation; the protection stomach mucous membrane; antifatigue; delay senility; regulate central nervous system; improve the cardiovascular and cerebrovascular blood supply insufficiency; go greasy sobering up; improve looks and the arteries and veins of invigorating blood circulation; anti-ageing; reducing blood-fat; decreasing cholesterol; triglyceride reducing; improve blood viscosity and microcirculation; vasodilation; remove medicine or the functional food and the Application in Food of free radical.
The present invention also comprises the detection method of Pleurotus ferulae Lanzi polysaccharide, comprises spectrophotometry and chromatography.
Wherein the method for spectrophotometry is as follows:
A) preparation of typical curve:
Take by weighing the dextrose anhydrous 0.1011g of drying and be dissolved in the 100ml distilled water, be made into the standardized solution of 1.011mg/ml to constant weight.Get standardized solution 0.2,0.3,0.4,0.6 then respectively, 0.8ml puts in 5 test tubes, adding distil water 1.0ml adds sulfuric acid-phynol colour developing liquid, boiling water bath 30min is put to room temperature, at the 490nm place, surveys its absorbance A.Distilled water with same processing is done blank.With the absorbancy is ordinate zou, and sugared concentration is X-coordinate drawing standard curve.
B) sulfuric acid-phynol preparation: phenol 5g, with the less water dissolving, be transferred in the 100ml volumetric flask, be settled to scale, be made into phenol solution.
C) assay method: the 10mg sample dissolution adds the 1ml phenol solution in 10ml distilled water, 5ml sulfuric acid, and boiling water bath 30min is cooled to room temperature, and the 490nm place surveys A, utilizes calibration curve method, calculates, promptly.
Wherein chromatographic method is as follows:
(7.8 * 30cm) gel columns, the ELSD detector detects, and carries out the determination of polysaccharide method that HPLC analyzes to utilize TOSOH TSKgel G4000PWXL.Optimal conditions: TOSOH TSKgel G4000PWXL (7.8 * 30cm) gel columns, the ELSD detector detects, and water is moving phase, flow velocity 1ml/min.(color atlas is seen Fig. 1)
Below for the effect determination experiment of Pleurotus ferulae Lanzi polysaccharide of the present invention:
Test shows, Pleurotus ferulae Lanzi polyoses extract of the present invention suppresses growth of tumour cell in preparation, strengthen body immunity, anticoagulation, protection stomach mucous membrane, antifatigue, delay senility, regulate central nervous system, improve the cardiovascular and cerebrovascular blood supply insufficiency, go greasyly to sober up, improve looks and the arteries and veins of invigorating blood circulation, anti-ageing, reducing blood-fat, decreasing cholesterol, triglyceride reducing, improve blood viscosity and microcirculation, vasodilation, removing free radical, antitumor, cancer therapy drug, functional food and Application in Food.
Below experiment is used to illustrate that extract of the present invention is more superior than existing analogue.
1 toxicity test:
Medicine: the Pleurotus ferulae Lanzi polyoses extract of the embodiment of the invention 2 methods preparation
Mouse of the present invention is oral once to be given and maximum administration concentration 15g/Kg body weight, there is no toxic side effects, and blood parameters is all normal,
Result of study: the mouse of the Pleurotus ferulae Lanzi polyoses extract of the embodiment of the invention 2 methods preparation of the present invention is oral once to be given and maximum administration concentration 15g/Kg body weight, dissects corpse and sees liver redness, dysfunction of liver.
Therefore Pleurotus ferulae Lanzi polyoses extract nontoxicity of the present invention is described.
2 tests of pesticide effectiveness:
Medicine: the Pleurotus ferulae Lanzi polyoses extract of the embodiment of the invention 2 methods preparation
2.1 the Pleurotus ferulae Lanzi polysaccharide is to the influence of mice with tumor growth
2.1.1 the Pleurotus ferulae Lanzi polysaccharide is to the influence of U14 mice with tumor growth
Table 1 result shows that the Pleurotus ferulae Lanzi polysaccharide inhibitory rate to 39% of 500mg/kg shows that it has the effect of very strong inhibition mouse cervical cancer U14 growth of xenografted; The tumour inhibiting rate of clinical anti-cervical cancer medicine cis-platinum is 46.5%; Pleurotus ferulae Lanzi and during with clinical anti-cervical cancer medicine cis-platinum acting in conjunction, inhibitory rate to 65.1% illustrates that the Pleurotus ferulae Lanzi polysaccharide can improve the anticancer therapeutic of cis-platinum.
Table 1 Pleurotus ferulae Lanzi polysaccharide is to the influence of U14 mice with tumor growth
*p＜0.05VsNS
2.1.2 the Pleurotus ferulae Lanzi polysaccharide is to the influence of H22 mice with tumor growth
As can be drawn from Table 2, compare with model group, high, medium and low each the dosage group of Pleurotus ferulae Lanzi polysaccharide is all inhibited to the growth of H22 sarcoma cell, and strengthens with its restraining effect of increase of dosage.With model group knurl heavy phase ratio, the tumour inhibiting rate of each dosage group all reaches more than 30%.
Table 2 Pleurotus ferulae Lanzi polysaccharide is to the influence of H22 tumor-bearing mice knurl weight and tumour inhibiting rate
*P＜0.05,
*P＜0.01vs model control group
2.2 the Pleurotus ferulae Lanzi polysaccharide is to the influence of tumor-bearing mice immune organ
2.2.1 the Pleurotus ferulae Lanzi polysaccharide is to the influence of U14 mice with tumor immune organ
Table 3 is the result show, the Pleurotus ferulae Lanzi polysaccharide of 500mg/kg can significantly improve the U14 tumor-bearing mice spleen index (Vs NS,
* *P＜0.001), cis-platinum group mice with tumor spleen index is compared obvious reduction (Vs DDP, ###p＜0.001) with the Pleurotus ferulae Lanzi group, can improve the influence that cis-platinum suppresses the mouse immune organ when Pleurotus ferulae Lanzi and cis-platinum acting in conjunction.
Table 3 Pleurotus ferulae Lanzi polysaccharide is to the influence of U14 mice with tumor immune organ
***p＜0.01Vs?NS；
***p＜0.001Vs?NS；###p＜0.001Vs?DDP
2.2.2 the Pleurotus ferulae Lanzi polysaccharide is to the influence of H22 mice with tumor immune organ
As can be drawn from Table 4, Pleurotus ferulae Lanzi polysaccharide height, middle dosage group can significantly increase the thymus index and the spleen index of H22 tumor-bearing mice, and prompting Pleurotus ferulae Lanzi polysaccharide can improve the immunizing power of H22 tumor-bearing mice.
Table 4 Pleurotus ferulae Lanzi polysaccharide is to the influence of H22 tumor-bearing mice thymus index and spleen index
*P＜0.05,
*P＜0.01vs model control group
2.3 the Pleurotus ferulae Lanzi polysaccharide is to the influence of dinitrofluorobenzene (DNFB) inducing mouse delayed allergy (DTH)
Experimental result shows, compares with the blank group, and the swelling degree of the middle and high dosage group of Pleurotus ferulae Lanzi polysaccharide all has significant difference, and each dosage of Pleurotus ferulae Lanzi polysaccharide all can increase the thymus index of mouse; The dosage group can increase the spleen index of mouse in the Pleurotus ferulae Lanzi polysaccharide, and prompting Pleurotus ferulae Lanzi polysaccharide can improve the cellular immune function of normal mouse.The results are shown in Table 5.
Table 5 Pleurotus ferulae Lanzi polysaccharide is to DNFB induced mice delayed type hypersensitivity (DTH) Immune Effects
*P＜0.05,
*P＜0.01vs blank group
2.4 the Pleurotus ferulae Lanzi polysaccharide influences tumor-bearing mice abdominal cavity M φ phagocytic function
Fig. 2 and Fig. 3 result show that the Pleurotus ferulae Lanzi polysaccharide of 500mg/kg can obviously improve the phagocytic function of tumor-bearing mice PM φ, phagocytic percentage and phagocytic index all be higher than the NS control group (
* *P＜0.001vs NS), and the phagocytic percentage of cis-platinum group mice with tumor is compared all decline with phagocytic index with model control group, when Pleurotus ferulae Lanzi and cis-platinum acting in conjunction, can significantly improve the phagocytic function (###p＜0.001vs DDP) of the tumor-bearing mice PM φ that is subjected to the cis-platinum inhibition.
2.5 the Pleurotus ferulae Lanzi polysaccharide is to the influence of N K cell killing activity in the tumor-bearing mice spleen
Table 6 shows, compares with the NS control group, and the Pleurotus ferulae Lanzi polysaccharide of 500mg/kg can obviously strengthen the killing activity of NK cell in the spleen, its kill rate can reach 78.29% (
*P＜0.001).And the killing activity of the NK cell of cis-platinum group mice with tumor with compare with the Pleurotus ferulae Lanzi group remarkable decline (
△ △P＜0.05).When the Pleurotus ferulae Lanzi polysaccharide of this dosage and cis-platinum acting in conjunction, NK cell killing rate is when separately using cis-platinum in the spleen, and effect obviously improves (#p＜0.05), illustrates that it can recover to be subjected to NK cell activity in the spleen that cis-platinum suppresses.
Table 6 Pleurotus ferulae Lanzi polysaccharide is to the influence of NK cell killing activity in the mice with tumor spleen
**p＜0.01vs?NS；#p＜0.05，
△△p＜0.01vs?DDP
2.6 the Pleurotus ferulae Lanzi polysaccharide is to the influence of tumor-bearing mice splenic T, bone-marrow-derived lymphocyte
Table 7 result shows, the tumor-bearing mice T that handles through the Pleurotus ferulae Lanzi polysaccharide, bone-marrow-derived lymphocyte to the proliferation activity of ConA, LPS all increasing in various degree (
*P＜0.5), and DDP can not strengthen the active of T, bone-marrow-derived lymphocyte even suppress immunologic function, after DDP acts on tumor-bearing mice separately, the activity of its remarkable T, bone-marrow-derived lymphocyte significantly be lower than Pleurotus ferulae Lanzi polysaccharide group (
△ △P＜0.01).
Table 7 Pleurotus ferulae Lanzi polysaccharide is to the influence of mice with tumor splenic T, bone-marrow-derived lymphocyte
*p＜0.5vs?NS；
△△p＜0.01vs?DDP
2.7 the Pleurotus ferulae Lanzi polysaccharide is to the influence of tumor-bearing mice mouse cytokine levels
Table 8 is the result show, the Pleurotus ferulae Lanzi polysaccharide of 500mg/kg can improve tumor-bearing mice Cytokine of Serum TNF-α (
*P＜0.01vs NS) and IFN-γ (
*P＜0.01vs NS) level.And the TNF-α of cis-platinum group mice with tumor (
△ △P＜0.01vsDDP) and IFN-γ (
△P＜0.05vs DDP) level is compared remarkable decline with the Pleurotus ferulae Lanzi group.When Pleurotus ferulae Lanzi and cis-platinum acting in conjunction, can significantly improve be subjected to TNF-alpha levels in the tumor-bearing mice serum that cis-platinum suppresses (
△But IFN-γ level improves not remarkable p＜0.05vs DDP).
Table 8 Pleurotus ferulae Lanzi polysaccharide is to the influence of mice with tumor cytokine levels
**p＜0.01，
*p＜0.05vs?NS；
△△p＜0.01，
△p＜0.05vs?DDP
2.8 the Pleurotus ferulae Lanzi polysaccharide is to the influence of tumor-bearing mice periphery blood T cell hypotype
Table 9 is the result show, tumor model group peripheral blood CD4
+/ CD8
+Ratio increases, and is more obvious behind the application cis-platinum, and the Pleurotus ferulae Lanzi polysaccharide can reverse this situation, compares with tumor model group and cis-platinum group, and difference all has statistical significance.
Table 9 Pleurotus ferulae Lanzi polysaccharide is to the influence of mice with tumor peripheral blood CD4+/CD8+ ratio
**p＜0.01vs?NS；
△△p＜0.01vs?DDP
Simultaneously; because the Pleurotus ferulae Lanzi polysaccharide can promote body's immunity, therefore: be expected to suppressing growth of tumour cell, strengthen body immunity, anticoagulation, protection stomach mucous membrane, antifatigue, delay senility, regulate central nervous system, improve the cardiovascular and cerebrovascular blood supply insufficiency, going greasyly to sober up, improve looks and the arteries and veins of invigorating blood circulation, anti-ageing, reducing blood-fat, decreasing cholesterol, triglyceride reducing, improve in blood viscosity and microcirculation, vasodilation, removing free radical, antitumor, cancer therapy drug, functional food and the food and be used widely.
Description of drawings:
Fig. 1: Pleurotus ferulae Lanzi polysaccharide HPLC-ELSD collection of illustrative plates, chromatographic condition: (TOSOH TSKgel G4000PWXL (7.8 * 30cm) gel columns, the ELSD detector detects, water is moving phase, flow velocity 1ml/min)
Fig. 2 Pleurotus ferulae Lanzi polysaccharide is to the influence of tumor-bearing mice abdominal cavity M φ phagocytic function
Fig. 3 Pleurotus ferulae Lanzi polysaccharide is to the influence of tumor-bearing mice abdominal cavity M φ phagocytic index
Embodiment:
Further specify the present invention by the following examples, but not as limitation of the present invention.
Embodiment 1: the extraction preparation of Pleurotus ferulae Lanzi polysaccharide
Pleurotus ferulae Lanzi (fresh) 20kg is cut into small pieces, and every 100g adds water 400ml, and with the juice extractor 1min that squeezes the juice, ultrasonic 30min boils 3h, cooling, centrifuging and taking supernatant liquor, filter paper suction filtration.Precipitation adds 6 by the ultrasonic 30min of water gaging, boils 1h, and cooling is centrifugal, gets supernatant, and the filter paper suction filtration merges supernatant liquor, concentrates, and gets concentrated solution 9L.Paste-forming rate 7.825%.
Embodiment 2: the purifying of Pleurotus ferulae Lanzi polysaccharide
The Pleurotus ferulae Lanzi concentrated solution 50ml (being equivalent to raw material 50mg) of embodiment 1 preparation adds papoid 0.6g, transfer PH5-6,60 ℃ of water-bath 1.5h are put to room temperature, the centrifugal 5min of 3000r/min, get supernatant liquor, add ethanol to containing alcohol amount 85%, place the vibration back, the centrifugal 5min of 3000r/min, get precipitation, repeatable operation 3 times promptly gets purifying Pleurotus ferulae Lanzi polysaccharide.
Embodiment 3: papain enzymolysis technology is investigated
1, determining of the suitableeest enzyme dosage:
Get 7 tool plug Erlenmeyer flasks, every bottle adds Pleurotus ferulae Lanzi extracting solution 20ml (43.5mg/ml), add papoid (enzyme-to-substrate mass ratio) 0mg/g, 2mg/g, 4mg/g, 6mg/g, 8mg/g, 10mg/g, 12mg/g respectively, transfer PH6 with HCL, boiling water bath 10min makes enzyme-deactivating behind 50 ℃ of water-bath 2h, be cooled to room temperature, the centrifugal 5min of 3000r/min gets supernatant liquor and adds 95% ethanol to determining alcohol 80%, and 3000r/min is centrifugal, and 10min gets precipitation, with method washing precipitation three times, survey polysaccharide yield.Ratio is at the bottom of the best enzyme: 8mg/g.
| Compare mg/g at the bottom of the |
0 | 2 | 4 | 6 | 8 | 10 | 12 |
| |
0 | 1.74 | 3.54 | 5.20 | 6.99 | 8.66 | 10.43 |
| Polysaccharide yield | 26.80％ | 36.69％ | 33.47％ | 39.66％ | 41.97％ | 39.01％ | 16.13％ |
2, determining of peak enzymolysis-ability time:
Get 7 tool plug Erlenmeyer flasks, every bottle adds Pleurotus ferulae Lanzi extracting solution 20ml (43.5mg/ml), add papoid 6.96mg respectively and transfer PH6 with HCL, boiling water bath 10min makes enzyme-deactivating behind 50 ℃ of water-bath 0min, 30min, 60min, 90min, 120min, 150min, the 180min respectively, is cooled to room temperature, the centrifugal 5min of 3000r/min, get supernatant liquor and add 95% ethanol to determining alcohol 80%, 3000r/min is centrifugal, and 10min gets precipitation, with method washing precipitation three times, surveys polysaccharide yield.Best enzymolysis time is: 120min.
| Water- |
0 | 30 | 60 | 90 | 120 | 150 | 180 |
| Enzyme quality mg | 6.95 | 6.95 | 6.95 | 6.98 | 6.98 | 6.97 | 6.99 |
| Polysaccharide yield | 24.5％ | 28.9％ | 29.1％ | 36.3％ | 36.1％ | 36.7％ | 36.9％ |
3, determining of optimum pH:
Get 3 tool plug Erlenmeyer flasks, every bottle adds Pleurotus ferulae Lanzi extracting solution 20ml (43.5mg/ml), adding papoid 6.96mg respectively, to transfer PH respectively with HCL be 5,7,9, boiling water bath 10min makes enzyme-deactivating behind 50 ℃ of water-bath 120min respectively, is cooled to room temperature, the centrifugal 5min of 3000r/min, get supernatant liquor and add 95% ethanol to determining alcohol 80%, 3000r/min is centrifugal, and 10min gets precipitation, with method washing precipitation three times, surveys polysaccharide yield.Best enzymolysis pH value is: 5.
| |
5 | 7 | 9 |
| Enzyme quality mg | 6.94 | 6.98 | 6.95 |
| Polysaccharide yield % | 37.4％ | 36.5％ | 36.3％ |
4, determining of peak enzymolysis-ability temperature:
Get 7 tool plug Erlenmeyer flasks, every bottle adds Pleurotus ferulae Lanzi extracting solution 20ml (43.5mg/ml), adding papoid 6.96mg respectively, to transfer PH respectively with HCL be about 5, boiling water bath 10min makes enzyme-deactivating behind 30 ℃ respectively, 40 ℃, 50 ℃, 60 ℃, 70 ℃, 80 ℃, the 90 ℃ water-bath 120min, is cooled to room temperature, the centrifugal 5min of 3000r/min, get supernatant liquor and add 95% ethanol to determining alcohol 80%, 3000r/min is centrifugal, and 10min gets precipitation, with method washing precipitation three times, surveys polysaccharide yield.Best hydrolysis temperature is: 60 ℃.
| |
30 | 40 | 50 | 60 | 70 | 80 | 90 |
| Enzyme quality mg | 6.97 | 6.97 | 6.94 | 6.95 | 6.97 | 6.94 | 6.97 |
| Polysaccharide yield | 34.9％ | 35.5％ | 35.7％ | 36.1％ | 28.2％ | 25.8％ | 24.0％ |
Embodiment 4: Polysaccharide from Bee optimization
Orthogonal test, design L9 (33) orthogonal test is an index with the polysaccharide yield, preferred optimised process.In order to improve the reliability of statistical study, all made revision test (n=3) under each condition, measurement result is got its mean value.
Level of factor
| Level | Temperature (A) ℃ | Time (B) h | Solid-liquid ratio (C) |
| 1 | 70 | 2 | 1∶20 |
| 2 | 80 | 3 | 1∶30 |
| 3 | 90 | 4 | 1∶40 |
Orthogonal experiments and analysis
From R value, R
C＞R
A＞R
B, the primary and secondary relation that promptly influences the factors of Pleurotus ferulae Lanzi polysaccharide extract rate is solid-liquid ratio (C)＞extraction temperature (A)＞extraction time (B) successively, from k1, k2, k3, best of breed is A
1B
2C
3Be 70 ℃ of extraction temperatures, extraction time 3h, solid-liquid ratio 1: 40
Embodiment 5: the preparation of Pleurotus ferulae Lanzi polysaccharide buccal tablet
Prescription:
The proportioning of the per 5000 main supplementary materials of this medicine is as follows:
Resina Ferulae mushroom polysaccharide 1250g
Folium Mori extract 125g
Zulkovsky starch starch 625g
Microcrystalline Cellulose 125g
Dextrin 250g
10% starch slurry an amount of (2g-20ml)
Talcum powder 25g
Sodium Cyclamate 5g
Concrete preparation method may further comprise the steps and carries out:
1. after the Resina Ferulae mushroom polysaccharide freeze-drying drying, pulverized 100 mesh sieves.
2. the accurate Sodium Cyclamate that takes by weighing recipe quantity, Zulkovsky starch 2 grams add 20 milliliters of pure water and fully stir, and mix, and are placed under the 80-85 ℃ of water-bath heated and stirred and take out standby to translucent.
3. the accurate Resina Ferulae mushroom polysaccharide that takes by weighing recipe quantity, Zulkovsky starch, Microcrystalline Cellulose, dextrin fully mixes.
4. draw 10% starch slurry with dropper and add in 3, the limit edged stirs to " that holds is agglomerating, and that touches promptly looses ", crosses No. 16 sieve series and becomes wet granular, dries under the 50-60 ℃ of temperature, crosses the whole grain of sieve No. 16, compressing tablet.
Embodiment 6: the preparation of Pleurotus ferulae Lanzi polysaccharide sheet
Prescription:
The proportioning of main supplementary material in per 5000 of this medicine:,
Resina Ferulae mushroom polysaccharide 1250g
Fructus Mori extract 50g
Zulkovsky starch starch 450g
Microcrystalline Cellulose 125g
Dextrin 250g
Silicon-dioxide 25g
10% starch slurry an amount of (2g-20ml)
Magnesium Stearate 12.5g
Concrete preparation method may further comprise the steps and carries out:
After the Resina Ferulae mushroom polysaccharide freeze-drying drying, pulverized 100 mesh sieves.
Precision takes by weighing Zulkovsky starch 2 gram and adds 20 milliliters of pure water and fully stir, and mixes, and is placed under the 80-85 ℃ of water-bath heated and stirred and takes out standby to translucent.
Precision takes by weighing the Resina Ferulae mushroom polysaccharide of recipe quantity, Zulkovsky starch, and Microcrystalline Cellulose, dextrin fully mixes.
Draw 10% starch slurry with dropper and add in 3, the limit edged stirs to " that holds is agglomerating, and that touches promptly looses ", crosses No. 16 sieve series and becomes wet granular, dries under the 50-60 ℃ of temperature, crosses the whole grain of sieve No. 16, compressing tablet.
Resina Ferulae mushroom polysaccharide drying means has two kinds: direct freeze-drying, lyophilize again after 0.5%-90.0% dextrin by weight or Microcrystalline Cellulose add.Directly lyophilize is not easy to pulverize, and adds dry easier pulverizing of dextrin or Microcrystalline Cellulose.
Claims (10)
1. a Pleurotus ferulae Lanzi polysaccharide that extracts from Pleurotus ferulae Lanzi is characterized by, and does not contain protein, and its HPLC-ELSD chromatogram has characteristic spectrum as shown in Figure 1.
2. the described Pleurotus ferulae Lanzi polysaccharide of claim 1, it is characterized by, be prepared from order to the below method, Pleurotus ferulae Lanzi extracts, extract removes albumen, except that the extract purifying behind the albumen obtains, wherein said extraction step is as follows: Pleurotus ferulae Lanzi is pulverized, extracting in water, centrifugal or remove by filter residue, the recycle-water extracting solution gets the Pleurotus ferulae Lanzi concentrated solution, and the wherein said protein process that removes is selected from:
(1) solvent extration: add propyl carbinol in the Pleurotus ferulae Lanzi concentrated solution earlier, jolting adds trichloromethane again, jolting, centrifugal, protein becomes insoluble state, remains in the interface of organic solvent and water, 1-100 time, optimum is: add propyl carbinol in the Pleurotus ferulae Lanzi concentrated solution earlier, jolting 20min adds trichloromethane again, jolting 20min, centrifugal, the centrifugal 15min of 4000r/min, 6-8 time repeatedly, it is characterized by: because Pleurotus ferulae Lanzi concentrated solution ground singularity, with the solvent-extracted together method of propyl carbinol-trichloromethane mixed solution of routine, be difficult to reach and remove proteinic purpose, must add the propyl carbinol jolting earlier, add the trichloromethane jolting again
(2) ion-exchange-resin process:
A) anionite-exchange resin 200g, distilled water immersion 10h, 1mol/L hydrochloric acid dashes 10 column volumes, distilled water flushing is to neutral, and 1mol/LNaOH dashes 10 column volumes, and distilled water flushing is to neutral, it is 8-14 that Pleurotus ferulae Lanzi concentrated solution 5-500ml regulates the pH value with NaOH, last sample, the distilled water wash-out concentrates and obtains polyoses extract; Continue and use the 0.1mol/LNaOH wash-out, remove the protein on the post, wherein regulating pH value optimum with NaOH is 8-11, and applied sample amount is Pleurotus ferulae Lanzi concentrated solution 50-100ml,
B) Zeo-karb 1000g, distilled water immersion 10h, 1mol/LNaOH is towards 10 column volumes of post, distilled water flushing is to neutral, and 1mol/L hydrochloric acid dashes 10 column volumes, and distilled water dashes to neutral, Pleurotus ferulae Lanzi concentrated solution 5-500ml is 1-6 with the salt acid for adjusting pH value, last sample, distilled water concentrate and obtain polyoses extract towards post; Continuing with 1mol/L hydrochloric acid wash-out, remove the protein on the post, is 3-5 with salt acid for adjusting pH value optimum wherein, and applied sample amount is Pleurotus ferulae Lanzi concentrated solution 50-100ml,
(3) enzyme process:
A) Pleurotus ferulae Lanzi concentrated solution 5-500ml adds papoid 0.6g, transfers PH1-9,30-80 ℃ of water-bath, put to room temperature, centrifugal, get supernatant liquor, can remove deproteinize, optimum is: Pleurotus ferulae Lanzi concentrated solution 50-100ml adds papoid 0.6g, transfers PH5-6,60 ℃ of water-bath 1.5h, put to room temperature, the centrifugal 5min of 3000r/min gets supernatant liquor, can remove deproteinize
B) Pleurotus ferulae Lanzi concentrated solution 5-500ml adds stomach en-0.05g, transfers PH 0-4.5,30-45 ℃ of water-bath, put to room temperature, add NaOH and transfer to neutrality, add ethanol to containing alcohol amount 50-95%, place the vibration back, centrifugal, get precipitation, can remove deproteinize, optimum is: Pleurotus ferulae Lanzi concentrated solution 50-100ml (being equivalent to raw material 50-100mg) adds stomach en-0.05g, transfer PH1.5-2.5,37 ℃ of water-baths are put to room temperature, add NaOH and transfer to neutrality, add ethanol to containing alcohol amount 85%, place the vibration back, and the centrifugal 5min of 3000r/min gets precipitation, can remove deproteinize
(4) gel chromatography:
Get dextrane gel 5g powder, with distilled water immersion swelling in beaker, the dress post, evenly join Pleurotus ferulae Lanzi concentrated solution 5-500ml in the gel column with dropper, the distilled water wash-out, each stream part is through UV 200-500nm full wavelength scanner, check protein peak, and each stream part detects polysaccharide with sulfuric acid-phynol method reaction back at 490nm place, merges and contains polysaccharide, do not contain proteinic stream part, promptly, the dextrane gel optimum is sephadex G-100 and sephadex G-50, Pleurotus ferulae Lanzi concentrated solution 1-10ml
Wherein said purification process is selected from:
(1) solvent extration: adopt organic solvents such as chloroform, ethyl acetate, propyl carbinol, Virahol, sherwood oil, gasoline, or the mixed solvent of above solvent, remove proteinic Pleurotus ferulae Lanzi extracting solution with the water liquid-liquid extraction, aqueous phase is the Pleurotus ferulae Lanzi polysaccharide of purifying
(2) ethanol precipitation: remove proteinic Pleurotus ferulae Lanzi extracting solution, adding ethanol to alcohol concn is 50-95%, and place the vibration back, centrifugal, get precipitation, can be repeatedly 2-10 time, be the Pleurotus ferulae Lanzi polysaccharide of purifying, its optimum process is: remove proteinic Pleurotus ferulae Lanzi extracting solution, adding ethanol to alcohol concn is 85%, and place the vibration back, the centrifugal 5min of 3000r/min, it is centrifugal to add ethanol again after precipitation is dissolved in water, 3 times repeatedly
(3) liquid-liquid adverse current partition chromatography: adopt organic solvents such as chloroform, ethyl acetate, propyl carbinol, Virahol, sherwood oil, gasoline, or the mixed solvent of above solvent, with water liquid-liquid adverse current partition chromatography, remove proteinic Pleurotus ferulae Lanzi extracting solution through separating, choose the Pleurotus ferulae Lanzi polysaccharide and flow out part, be the Pleurotus ferulae Lanzi polysaccharide of purifying
(4) gel chromatography: get dextrane gel 5g powder, with distilled water immersion swelling in beaker, the dress post, to remove proteinic Pleurotus ferulae Lanzi extracting solution 5-500ml with dropper evenly joins in the gel column, the distilled water wash-out, each stream part through sulfuric acid-phynol method reaction back at 490nm place detection polysaccharide, merge and contain the polysaccharide part, promptly get the Pleurotus ferulae Lanzi polysaccharide of purifying, the dextrane gel optimum is sephadex G-100 and sephadex G-50, removes proteinic Pleurotus ferulae Lanzi extracting solution 1-10ml.
3. the described Pleurotus ferulae Lanzi polysaccharide of claim 1 is characterized by, be prepared from order to the below method,
Pleurotus ferulae Lanzi is cut into small pieces, and adds water and squeezes the juice with juice extractor, and is ultrasonic, boils, cooling, the centrifuging and taking supernatant liquor, the filter paper suction filtration, it is ultrasonic that precipitation adds water, boils, cooling, centrifugal, get supernatant, the filter paper suction filtration merges supernatant liquor, concentrate, get concentrated solution, concentrated solution adds papoid, transfers PH5-6, heating in water bath is put to room temperature, and is centrifugal, gets supernatant liquor, add ethanol and place, centrifugal, get precipitation, promptly.
4. the preparation method of the described Pleurotus ferulae Lanzi polysaccharide of claim 1 is characterized in that step is as follows: extract, except that albumen, purifying, wherein remove protein process and be selected from: (1) solvent extration; (2) ion-exchange-resin process; (3) enzyme process; (4) gel chromatography, wherein purification process is selected from: (1) solvent extration; (2) ethanol precipitation; (3) liquid-liquid adverse current partition chromatography; (4) gel chromatography.
5. by the preparation method of claim 4, it is characterized in that: wherein said extracting method, step is as follows: Pleurotus ferulae Lanzi is pulverized, extracting in water, centrifugal or remove by filter residue, recycle-water extracting solution, the Pleurotus ferulae Lanzi concentrated solution, wherein 1) Pleurotus ferulae Lanzi is fresh or fully soaks into through water; 2) water extraction can heat extraction, and by pulverizing, fragmentation, is shortened dramatically extraction time, and the best can be to 10-60 minute; 3) in order to improve centrifugal or to remove by filter efficient in the residue process, take into account extraction efficiency simultaneously, particle size after cracking is controlled at 10-300 order scope, is the best with the 30-60 order wherein.
6. by the preparation method of claim 4, it is characterized in that: the wherein said protein process that removes is selected from:
(3) solvent extration, it is characterized by: add propyl carbinol (100: 1-100: 20) in the Pleurotus ferulae Lanzi concentrated solution earlier, jolting, add again trichloromethane (100: 1-100: 50), jolting, centrifugal, protein becomes insoluble state, remain in the interface of organic solvent and water, 1-100 time, optimum is: add propyl carbinol ((25: 1) in the Pleurotus ferulae Lanzi concentrated solution earlier, jolting 20min, add trichloromethane (25: 4) again, jolting 20min is centrifugal, the centrifugal 15min of 4000r/min, 6-8 time repeatedly, it is characterized by: because Pleurotus ferulae Lanzi concentrated solution ground singularity, with the conventional solvent-extracted together method of propyl carbinol-trichloromethane mixed solution, be difficult to reach and remove proteinic purpose, must add the propyl carbinol jolting earlier, add the trichloromethane jolting again
(4) ion-exchange-resin process is characterized by:
A) anionite-exchange resin 200g, distilled water immersion 10h, 1mol/L hydrochloric acid dashes 10 column volumes, distilled water flushing is to neutral, and 1mol/LNaOH dashes 10 column volumes, and distilled water flushing is to neutral, it is 8-14 that Pleurotus ferulae Lanzi concentrated solution 5-500ml (being equivalent to raw material 5-500mg) regulates the pH value with NaOH, last sample, the distilled water wash-out concentrates and obtains polyoses extract; Continue and use the 0.1mol/LNaOH wash-out, remove the protein on the post, wherein regulating pH value optimum with NaOH is 8-11, and applied sample amount is Pleurotus ferulae Lanzi concentrated solution 50-100ml (being equivalent to raw material 50-100mg),
B) Zeo-karb 1000g, distilled water immersion 10h, 1mol/LNaOH is towards 10 column volumes of post, distilled water flushing is to neutral, and 1mol/L hydrochloric acid dashes 10 column volumes, and distilled water dashes to neutral, Pleurotus ferulae Lanzi concentrated solution 5-500ml (being equivalent to raw material 5-500mg) is 1-6 with the salt acid for adjusting pH value, last sample, distilled water concentrate and obtain polyoses extract towards post; Continuing with 1mol/L hydrochloric acid wash-out, remove the protein on the post, is 3-5 with salt acid for adjusting pH value optimum wherein, and applied sample amount is Pleurotus ferulae Lanzi concentrated solution 50-100ml (being equivalent to raw material 50-100mg),
(3) enzyme process is characterized by:
A) Pleurotus ferulae Lanzi concentrated solution 5-500ml (being equivalent to raw material 5-500mg) adds papoid 0.6g, transfers PH1-9,30-80 ℃ of water-bath, put to room temperature, centrifugal, get supernatant liquor, can remove deproteinize, optimum is: Pleurotus ferulae Lanzi concentrated solution 50-100ml (being equivalent to raw material 50-100mg) adds papoid 0.6g, transfers PH5-6,60 ℃ of water-bath 1.5h, put to room temperature, the centrifugal 5min of 3000r/min gets supernatant liquor, can remove deproteinize
B) Pleurotus ferulae Lanzi concentrated solution 5-500ml (being equivalent to raw material 5-500mg) adds stomach en-0.05g, transfers PH 0-4.5,30-45 ℃ of water-bath, put to room temperature, add NaOH and transfer to neutrality, add ethanol to containing alcohol amount 50-95%, place the vibration back, centrifugal, get precipitation, can remove deproteinize, optimum is: Pleurotus ferulae Lanzi concentrated solution 50-100ml (being equivalent to raw material 50-100mg) adds stomach en-0.05g, transfer PH1.5-2.5,37 ℃ of water-baths are put to room temperature, add NaOH and transfer to neutrality, add ethanol to containing alcohol amount 85%, place the vibration back, and the centrifugal 5min of 3000r/min gets precipitation, can remove deproteinize
(4) gel chromatography is characterized by:
Get dextrane gel 5g powder, with distilled water immersion swelling in beaker, the dress post, with dropper Pleurotus ferulae Lanzi concentrated solution 5-500ml (being equivalent to raw material 5-500mg) is evenly joined in the gel column, the distilled water wash-out, each stream part is through UV 200-500nm full wavelength scanner, check protein peak, and each stream part is detected polysaccharide with sulfuric acid-phynol method reaction back at the 490nm place, merge and contain polysaccharide, do not contain proteinic stream part, promptly, the dextrane gel optimum is sephadex G-100 and sephadex G-50, Pleurotus ferulae Lanzi concentrated solution 1-10ml (being equivalent to raw material 1-10mg).
7. by the preparation method of claim 4, it is characterized in that: wherein purification process is selected from:
(4) solvent extration:
Adopt organic solvents such as chloroform, ethyl acetate, propyl carbinol, Virahol, sherwood oil, gasoline, or the mixed solvent of above solvent, remove proteinic Pleurotus ferulae Lanzi extracting solution with the water liquid-liquid extraction, aqueous phase is the Pleurotus ferulae Lanzi polysaccharide of purifying,
(5) ethanol precipitation:
Remove proteinic Pleurotus ferulae Lanzi extracting solution, adding ethanol to alcohol concn is 50-95%, and place the vibration back, centrifugal, get precipitation, can be repeatedly 2-10 time, be the Pleurotus ferulae Lanzi polysaccharide of purifying, its optimum process is: remove proteinic Pleurotus ferulae Lanzi extracting solution, adding ethanol to alcohol concn is 85%, and place the vibration back, the centrifugal 5min of 3000r/min, it is centrifugal to add ethanol again after precipitation is dissolved in water, 3 times repeatedly
(6) liquid-liquid adverse current partition chromatography:
Adopt organic solvents such as chloroform, ethyl acetate, propyl carbinol, Virahol, sherwood oil, gasoline, or the mixed solvent of above solvent, with water liquid-liquid adverse current partition chromatography, remove proteinic Pleurotus ferulae Lanzi extracting solution through separating, choose the Pleurotus ferulae Lanzi polysaccharide and flow out part, be the Pleurotus ferulae Lanzi polysaccharide of purifying
(4) gel chromatography,
Get dextrane gel 5g powder, with distilled water immersion swelling in beaker, the dress post, to remove proteinic Pleurotus ferulae Lanzi extracting solution 5-500ml (being equivalent to raw material 5-500mg) with dropper evenly joins in the gel column, the distilled water wash-out, each stream part through sulfuric acid-phynol method reaction back at 490nm place detection polysaccharide, merge and contain the polysaccharide part, promptly get the Pleurotus ferulae Lanzi polysaccharide of purifying, the dextrane gel optimum is sephadex G-100 and sephadex G-50, removes proteinic Pleurotus ferulae Lanzi extracting solution 1-10ml (being equivalent to raw material 1-10mg).
8. the described Pleurotus ferulae Lanzi polysaccharide of claim 1 suppresses growth of tumour cell in preparation, strengthens body immunity, anticoagulation, protection stomach mucous membrane, antifatigue, delays senility, regulates central nervous system, improves the cardiovascular and cerebrovascular blood supply insufficiency, goes greasyly to sober up, improve looks and the arteries and veins of invigorating blood circulation, anti-ageing, reducing blood-fat, decreasing cholesterol, triglyceride reducing, improve medicine, functional food and the Application in Food of blood viscosity and microcirculation, vasodilation, removing free radical.
9. the composition that contains the Pleurotus ferulae Lanzi polysaccharide of claim 1; it is characterized in that; contain Pleurotus ferulae Lanzi polysaccharide and medicine or food acceptable carrier; and/or contain ramulus mori, mulberry leaf, mulberry fruit and glycosidase inhibitors such as extract, acarbose thereof, be prepared into any pharmaceutical dosage form of taking as lozenge, tablet; capsule; particle, oral liquid, injection; in generation, make tea; beverages etc. according to patient's situation, are determined usage and dosage in use; but obey every day three times; each 1-20 agent, as: 1-20 bag or grain or sheet, every dose of 1mg-1000mg.Wherein, glycosidase inhibitor comprises ramulus mori, mulberry leaf, mulberry fruit and glycosidase inhibitors such as extract, acarbose thereof, the effect that we find to add them is can be by suppressing the gi tract metabolic enzyme, suppress the degraded of Pleurotus ferulae Lanzi polysaccharide in gi tract, thereby oral administration system preparations such as () tablets is provided and has sucked drug delivery system preparations such as () lozenges.
10. the detection method of the described Pleurotus ferulae Lanzi polysaccharide of claim 1 is characterized by: comprise spectrophotometry and chromatography,
Wherein the method for spectrophotometry is as follows:
A) preparation of typical curve:
Take by weighing the dextrose anhydrous 0.1011g of drying and be dissolved in the 100ml distilled water, be made into the standardized solution of 1.011mg/ml, get standardized solution 0.2,0.3,0.4,0.6 then respectively, 0.8ml puts in 5 test tubes to constant weight, adding distil water 1.0ml, add sulfuric acid-phynol colour developing liquid, boiling water bath 30min is put to room temperature, at the 490nm place, surveying its absorbance A, do blank with the same distilled water of handling, is ordinate zou with the absorbancy, sugar concentration is X-coordinate drawing standard curve
B) sulfuric acid-phynol preparation: phenol 5g, with the less water dissolving, be transferred in the 100ml volumetric flask, be settled to scale, be made into phenol solution,
C) assay method: the 10mg sample dissolution adds the 1ml phenol solution in 10ml distilled water, 5ml sulfuric acid, and boiling water bath 30min is cooled to room temperature, and the 490nm place surveys A, utilizes calibration curve method, calculate, that is,
Wherein chromatographic method is as follows:
Utilize TOSOH TSKgel G4000PWXL (7.8 * 30cm) gel columns, the ELSD detector detects, carry out the determination of polysaccharide method that HPLC analyzes, optimal conditions: TOSOH TSKgel G4000PWXL (7.8 * 30cm) gel columns, the ELSD detector detects, water is moving phase, flow velocity 1ml/min.
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| CN107613998A (en) * | 2015-05-27 | 2018-01-19 | 庆尚北道 | Contain the prevention and treatment pharmaceutical composition or healthy food of the metabolic disease of Pleurotus ferulae water extract as active ingredient |
| CN110698567A (en) * | 2019-11-14 | 2020-01-17 | 南开大学 | Antioxidant polysaccharide extract in pleurotus ferulae as well as preparation method and application thereof |
| CN114384189A (en) * | 2022-03-23 | 2022-04-22 | 丹娜(天津)生物科技股份有限公司 | Preparation method of (1,3) -beta-D-glucan standard substance, product and application thereof |
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| CN104744601A (en) * | 2015-04-10 | 2015-07-01 | 新疆大学 | Method for extracting and purifying fleurotus ferulae polysaccharide |
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| CN107613998A (en) * | 2015-05-27 | 2018-01-19 | 庆尚北道 | Contain the prevention and treatment pharmaceutical composition or healthy food of the metabolic disease of Pleurotus ferulae water extract as active ingredient |
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| CN110698567B (en) * | 2019-11-14 | 2021-09-14 | 南开大学 | Antioxidant polysaccharide extract in pleurotus ferulae as well as preparation method and application thereof |
| CN114384189A (en) * | 2022-03-23 | 2022-04-22 | 丹娜(天津)生物科技股份有限公司 | Preparation method of (1,3) -beta-D-glucan standard substance, product and application thereof |
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