CN101914168B - 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 Polysaccharides of Pleurotus Ferulae with medicinal use and the pharmaceutical composition that contains Polysaccharides of Pleurotus Ferulae 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 treatment of human cervical cancer means develop 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, improve body's immunity and play an important role in the process of tumors destroyed cell.
At present existing a large amount of clinical research confirmation herbal medicine can improve Quality of Life of Patients with Tumor, suppress tumour, and mitigation symptoms even makes 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 Polysaccharides of Pleurotus Ferulae in the document has been selected in this experiment, result shows, Polysaccharides of Pleurotus Ferulae has certain inhibition to mouse cervical cancer U14 transplanted tumor, inhibitory rate to 39%, clinical Chemotherapy of Cervical Cancer one line medicine cis-platinum commonly used is 46.5% to its tumour inhibiting rate, when Polysaccharides of Pleurotus Ferulae and cis-platinum acting in conjunction, inhibitory rate to 65.1% illustrates that Polysaccharides of Pleurotus Ferulae can improve the anticancer therapeutic of cis-platinum.This Polysaccharides on Mice liver cancer H22 transplanted tumor also has certain inhibition, inhibitory rate to 54% simultaneously.According to tumor research rules regulations, the tumour inhibiting rate of herbal medicine is greater than 30% the time, and thinking has certain curative effect to tumour, and Polysaccharides of Pleurotus Ferulae has surpassed this standard, and therefore good DEVELOPMENT PROSPECT is arranged.
The anticancer mechanism of Pleurotus ferulae Lanzi may realize by its Immunestimulatory effect.It can activate the immune cell of body 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 the M φ that activates simultaneously can " respiratory burst " occur and produce rapidly a series of O 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 that 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 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: namely as TDHT mediation delayed type hypersensitivity with as Tc cell direct killing target cell.The B cell is exercised its immunologic function by the antibody of secretion.In addition, the B cell of activation also has processing and offers antigen to the effect of T cell.The antibody of B emiocytosis can be carried out the panimmunity function.
The lymphocyte transformation experiment can be understood the cellular immune function of body.This experiment is by measuring above-mentioned immune cell function, find: Polysaccharides of Pleurotus Ferulae can significantly promote the phagocytic function of tumor-bearing mice PM φ, strengthen the effect of NK cell killing target cell K562 in spleen, improve the activity of T, bone-marrow-derived lymphocyte, show that it can suppress by the cellular immune function that promotes tumor-bearing mice the growth of transplanted tumor U14.The detection of T cell subsets is to observe one of important method of Immune Function, CD4
+/ CD8
+Ratio can directly reflect host's t lymphocyte subset group's state, and can indirectly understand the Immune Function situation 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 index of the immune status of entry evaluation very easily, only have 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, is also the important regulatory factor of body inflammatory reaction and immunne response.TNF-α can be combined with the cell surface specific receptors and be formed 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 T cell of activation, can strengthen the cytotoxicity of the dependence of host's φt cell receptor and the restricted T cell of HLA, has the surperficial MHC antigen of increasing and expression of tumor necrosis factor, the multiple antitumor actions such as antineoplastic vascular generation.IFN-γ can also the Fas/FasL by the modulate tumor cell expresses and strengthens tumour cell to the susceptibility of Fas institute mediated apoptosis approach, and the ability of tumour cell escape from immune system attack is reduced, thus the malignant proliferation of inhibition tumor cell.So TNF-α, IFN-γ has consequence and effect in antitumor immunity of organism.
In body, experimental result shows, Polysaccharides of Pleurotus Ferulae can obviously promote the immunocompetence of NK cell in the phagocytic function, spleen of tumor-bearing mice peritoneal macrophage, 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 impact that cis-platinum suppresses the mice with tumor immunologic function during with Cisplatin.Result shows: Polysaccharides of Pleurotus Ferulae can suppress the propagation of mouse cervical cancer U14 transplanted tumor by promoting the mouse body's immunity.
Simultaneously; because Polysaccharides of Pleurotus Ferulae can promote body's immunity, therefore: be expected in inhibition tumor cell growth, 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 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 food and be used widely.
Summary of the invention:
The object of the invention is to, a kind of Polysaccharides of Pleurotus Ferulae 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 Polysaccharides of Pleurotus Ferulae of the present invention.
Pharmaceutical composition of the present invention take Polysaccharides of Pleurotus Ferulae of the present invention as activeconstituents, can be prepared into any pharmaceutical dosage form of taking as required as lozenge, tablet, and capsule, particle, oral liquid, injection, in generation, make tea, beverage etc.According to patient's situation, determine usage and dosage in use, but take 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 Polysaccharides of Pleurotus Ferulae of the present invention, and the method comprises extraction, except albumen, and purifying.Except albumen adopts following methods, (1) solvent extration; (2) ion-exchange-resin process; (3) enzyme process; (4) gel chromatography.Purifying adopts following methods, (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, and the recycle-water extracting solution gets the Pleurotus ferulae Lanzi concentrated solution.It is characterized by: 1) Pleurotus ferulae Lanzi is fresh or fully infiltrates 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 simultaneously extraction efficiency, particle size after cracking is controlled at 10-300 order scope, wherein take the 30-60 order as best.
Preferred preparation method of the present invention, wherein method of removing protein is selected from:
(1) solvent extration, it is characterized by: first add propyl carbinol (100: 1-100: 20) in the Pleurotus ferulae Lanzi concentrated solution, 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: first add in the Pleurotus ferulae Lanzi concentrated solution propyl carbinol ((25: 1), jolting 20min, then add trichloromethane (25: 4), 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 propyl carbinol of routine-trichloromethane mixed solution solvent-extracted method together, be difficult to reach the purpose except deproteinize, must first add the propyl carbinol jolting, then add the trichloromethane jolting.
(2) ion-exchange-resin process is characterized by:
A) anionite-exchange resin 200g, distilled water immersion 10h, 10 column volumes of 1mol/L hydrochloric acid punching, distilled water flushing is to neutral, and 1mol/LNaOH rushes 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, loading, distilled water wash-out, the concentrated polyoses extract that obtains; Continue and use the 0.1mol/LNaOH wash-out, remove the protein on 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 rushes 10 column volumes of post, distilled water flushing is to neutral, and 10 column volumes of 1mol/L hydrochloric acid punching, distilled water rush 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, loading, distilled water rushes post, the concentrated polyoses extract that obtains; Continue with 1mol/L hydrochloric acid wash-out, remove the protein on post.Be wherein 3-5 with salt acid for adjusting pH value optimum, 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 after vibration, 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 after vibration, 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 gel column the distilled water wash-out.Each stream part checks protein peak through UV 200-500nm full wavelength scanner.And each stream part is rear at 490nm place's detection polysaccharide with the sulfuric acid-phynol method reaction.Merge and contain polysaccharide, do not contain stream part of protein, and get final product.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 the organic solvents such as chloroform, ethyl acetate, propyl carbinol, Virahol, sherwood oil, gasoline, or the mixed solvent of above solvent, with the Pleurotus ferulae Lanzi extracting solution of water liquid-liquid extraction except deproteinize, aqueous phase is the Polysaccharides of Pleurotus Ferulae of purifying.
(2) ethanol precipitation:
Except the Pleurotus ferulae Lanzi extracting solution of deproteinize, adding ethanol to alcohol concn is 50-95%, places after vibration, centrifugal, gets precipitation, can be repeatedly 2-10 time, be the Polysaccharides of Pleurotus Ferulae of purifying.Its optimum process is: except the Pleurotus ferulae Lanzi extracting solution of deproteinize, adding ethanol to alcohol concn is 85%, places after vibration, and the centrifugal 5min of 3000r/min adds ethanol after precipitation is dissolved in water centrifugal, 3 times repeatedly again.
(3) liquid-liquid adverse current partition chromatography:
Adopt the 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 the Pleurotus ferulae Lanzi extracting solution of deproteinize through separating, choose Polysaccharides of Pleurotus Ferulae and flow out part, be the Polysaccharides of Pleurotus Ferulae of purifying.
(4) gel chromatography.
Get dextrane gel 5g powder, with distilled water immersion swelling in beaker, the dress post.To evenly join in gel column except the Pleurotus ferulae Lanzi extracting solution 5-500ml (being equivalent to raw material 5-500mg) of deproteinize the distilled water wash-out with dropper.Each stream part after sulfuric acid-phynol method reaction at 490nm place detection polysaccharide.Merge and contain the polysaccharide part, namely get the Polysaccharides of Pleurotus Ferulae of purifying.The dextrane gel optimum is sephadex G-100 and sephadex G-50, except the Pleurotus ferulae Lanzi extracting solution 1-10ml (being equivalent to raw material 1-10mg) of deproteinize.
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, concentrated, 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, and get final product.
The present invention also comprises by research and finds ramulus mori, mulberry leaf, mulberry fruit and extract, acarbose etc., can be by suppressing the gi tract metabolic enzyme, suppress the degraded of Polysaccharides of Pleurotus Ferulae 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 Polysaccharides of Pleurotus Ferulae of the present invention and pharmaceutical composition thereof are in the growth of preparation inhibition tumor cell, 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 the medicine of free radical or the application in functional food and food.
The present invention also comprises the detection method of Polysaccharides of Pleurotus Ferulae, comprises spectrophotometry and chromatography.
Wherein the method for spectrophotometry is as follows:
A) preparation of typical curve:
Take the dextrose anhydrous 0.1011g of drying to constant weight and be dissolved in 100ml distilled water, be made into the standardized solution of 1.011mg/ml.Then the accurate solution 0.2 of label taking, 0.3,0.4,0.6,0.8ml put in 5 test tubes respectively, and adding distil water 1.0ml adds the sulfuric acid-phynol nitrite ion, and boiling water bath 30min is put to room temperature, at the 490nm place, survey its absorbance A.Distilled water with same processing is done blank.Take absorbancy as ordinate zou, sugared concentration is X-coordinate drawing standard curve.
B) sulfuric acid-phynol preparation: phenol 5g, use a small amount of water dissolution, 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 490nm surveys A in the place, utilizes calibration curve method, calculates, and get final product.
Wherein chromatographic method is as follows:
(7.8 * 30cm) gel columns, the ELSD detector detects, and carries out the Methods in Determination of Polysaccaride Content 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 Polysaccharides of Pleurotus Ferulae of the present invention:
Test shows, Polysaccharides of Pleurotus Ferulae extract of the present invention in the growth of preparation inhibition tumor cell, 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 sober up, improve looks and the arteries and veins of invigorating blood circulation, anti-ageing, reducing blood-fat, decreasing cholesterol, triglyceride reducing, improve the application in blood viscosity and microcirculation, vasodilation, removing free radical, antitumor, cancer therapy drug, functional food and food.
It is more superior than existing analogue that below experiment is used for explanation extract of the present invention.
1 toxicity test:
Medicine: the Polysaccharides of Pleurotus Ferulae extract of the embodiment of the present invention 2 method preparations
Mouse of the present invention is oral once to be given and maximum administration concentration 15g/Kg body weight, is showed no toxic side effects, and blood parameters is all normal,
Result of study: the mouse of the Polysaccharides of Pleurotus Ferulae extract of the embodiment of the present invention 2 method preparations of the present invention is oral once to be given and maximum administration concentration 15g/Kg body weight, dissects corpse and sees that liver is red and swollen, dysfunction of liver.
Therefore Polysaccharides of Pleurotus Ferulae extract nontoxicity of the present invention is described.
2 tests of pesticide effectiveness:
Medicine: the Polysaccharides of Pleurotus Ferulae extract of the embodiment of the present invention 2 method preparations
2.1 Polysaccharides of Pleurotus Ferulae is to the mice with tumor affects on the growth
2.1.1 Polysaccharides of Pleurotus Ferulae is to U14 mice with tumor affects on the growth
The demonstration of table 1 result, the Polysaccharides of Pleurotus Ferulae 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 Polysaccharides of Pleurotus Ferulae can improve the anticancer therapeutic of cis-platinum.
Table 1 Polysaccharides of Pleurotus Ferulae is to U14 mice with tumor affects on the growth
*p＜0.05VsNS
2.1.2 Polysaccharides of Pleurotus Ferulae is to H22 mice with tumor affects on the growth
As can be drawn from Table 2, compare with model group, high, medium and low each dosage group of Polysaccharides of Pleurotus Ferulae 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%.
The impact of table 2 Polysaccharides of Pleurotus Ferulae on H22 tumor weight and tumour inhibiting rate
*P＜0.05,
*P＜0.01vs model control group
2.2 the impact of Polysaccharides of Pleurotus Ferulae on the tumor-bearing mice immune organ
2.2.1 the impact of Polysaccharides of Pleurotus Ferulae on U14 mice with tumor immune organ
Table 3 result shows, the Polysaccharides of Pleurotus Ferulae 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 impact that cis-platinum suppresses the mouse immune organ when Pleurotus ferulae Lanzi and cis-platinum acting in conjunction.
The impact of table 3 Polysaccharides of Pleurotus Ferulae on U14 mice with tumor immune organ
***p＜0.01Vs NS；
***p＜0.001Vs NS；###p＜0.001Vs DDP
2.2.2 the impact of Polysaccharides of Pleurotus Ferulae on H22 mice with tumor immune organ
As can be drawn from Table 4, thymus index and spleen index that Polysaccharides of Pleurotus Ferulae is high, middle dosage group can significantly increase the H22 tumor-bearing mice, the prompting Polysaccharides of Pleurotus Ferulae can improve the immunizing power of H22 tumor-bearing mice.
The impact of table 4 Polysaccharides of Pleurotus Ferulae on H22 tumor-bearing mice thymus index and spleen index
*P＜0.05,
*P＜0.01vs model control group
2.3 the impact of Polysaccharides of Pleurotus Ferulae on dinitrofluorobenzene (DNFB) inducing mouse delayed allergy (DTH)
Experimental result shows, compares with the blank group, and the swelling of the middle and high dosage group of Polysaccharides of Pleurotus Ferulae all has significant difference, and each dosage of Polysaccharides of Pleurotus Ferulae all can increase the thymus index of mouse; In Polysaccharides of Pleurotus Ferulae, the dosage group can increase the spleen index of mouse, and the prompting Polysaccharides of Pleurotus Ferulae can improve the cellular immune function of normal mouse.The results are shown in Table 5.
The impact of table 5 Polysaccharides of Pleurotus Ferulae on DNFB induced mice delayed type hypersensitivity (DTH) immunologic function
*P＜0.05,
*P＜0.01vs blank group
2.4 Polysaccharides of Pleurotus Ferulae is on tumor-bearing mice abdominal cavity M φ phagocytic function impact
Fig. 2 and Fig. 3 result show, the Polysaccharides of Pleurotus Ferulae of 500mg/kg can obviously improve the phagocytic function of tumor-bearing mice PM φ, phagocytic percentage and phagocytic index all 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 model control group with phagocytic index, 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 impact of Polysaccharides of Pleurotus Ferulae on N K cell killing activity in the tumor-bearing mice spleen
Table 6 shows, compares with the NS control group, and the Polysaccharides of Pleurotus Ferulae of 500mg/kg can obviously strengthen the killing activity of NK cell in 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 Polysaccharides of Pleurotus Ferulae of this dosage and cis-platinum acting in conjunction, in spleen, NK cell killing rate is when separately using cis-platinum, and effect obviously improves (#p＜0.05), and the activity that it can recover to be subjected to NK cell in spleen that cis-platinum suppresses is described.
The impact of table 6 Polysaccharides of Pleurotus Ferulae on NK cell killing activity in the mice with tumor spleen
**p＜0.01vs NS；#p＜0.05，
△△p＜0.01vs DDP
2.6 the impact of Polysaccharides of Pleurotus Ferulae on tumor-bearing mice splenic T, bone-marrow-derived lymphocyte
Table 7 result shows, the tumor-bearing mice T that processes through Polysaccharides of Pleurotus Ferulae, bone-marrow-derived lymphocyte to the proliferation activity of ConA, LPS all increasing in various degree (
*P＜0.5), and the activity that DDP can not strengthen T, bone-marrow-derived lymphocyte Immunosuppression function even, after DDP independent role tumor-bearing mice, the activity of its remarkable T, bone-marrow-derived lymphocyte significantly lower than the Polysaccharides of Pleurotus Ferulae group (
△ △P＜0.01).
The impact of table 7 Polysaccharides of Pleurotus Ferulae on mice with tumor splenic T, bone-marrow-derived lymphocyte
*p＜0.5vs NS；
△△p＜0.01vs DDP
2.7 the impact of Polysaccharides of Pleurotus Ferulae on tumor-bearing mice mouse cytokine levels
Table 8 result shows, the Polysaccharides of Pleurotus Ferulae 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 tumor-bearing mice serum that cis-platinum suppresses (
△But IFN-γ level improves not remarkable p＜0.05vs DDP).
The impact of table 8 Polysaccharides of Pleurotus Ferulae on the mice with tumor cytokine levels
**p＜0.01，
*p＜0.05vs NS；
△△p＜0.01，
△p＜0.05vs DDP
2.8 the impact of Polysaccharides of Pleurotus Ferulae on tumor-bearing mice periphery blood T cell hypotype
Table 9 result shows, tumor model group peripheral blood CD4
+/ CD8
+Ratio increases, and is more obvious after the application cis-platinum, and Polysaccharides of Pleurotus Ferulae can reverse this situation, compares with tumor model group and cis-platinum group, and difference all has statistical significance.
The impact of table 9 Polysaccharides of Pleurotus Ferulae on mice with tumor peripheral blood CD4+/CD8+ ratio
**p＜0.01vs NS；
△△p＜0.01vs DDP
Simultaneously; because Polysaccharides of Pleurotus Ferulae can promote body's immunity, therefore: be expected in inhibition tumor cell growth, 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 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 food and be used widely.
Description of drawings:
Fig. 1: Polysaccharides of Pleurotus Ferulae 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)
The impact of Fig. 2 Polysaccharides of Pleurotus Ferulae on tumor-bearing mice abdominal cavity M φ phagocytic function
The impact of Fig. 3 Polysaccharides of Pleurotus Ferulae on tumor-bearing mice abdominal cavity M φ phagocytic index
Embodiment:
Further illustrate by the following examples the present invention, but not as limitation of the present invention.
Embodiment 1: the extraction preparation of Polysaccharides of Pleurotus Ferulae
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, and is cooling, centrifuging and taking supernatant liquor, filter paper suction filtration.Precipitation adds 6 by the ultrasonic 30min of water gaging, boils 1h, and is cooling, centrifugal, gets supernatant, and the filter paper suction filtration merges supernatant liquor, and is concentrated, gets concentrated solution 9L.Paste-forming rate 7.825%.
Embodiment 2: the purifying of Polysaccharides of Pleurotus Ferulae
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 after vibration, the centrifugal 5min of 3000r/min, get precipitation, repeatable operation 3 times namely gets the purifying Polysaccharides of Pleurotus Ferulae.
Embodiment 3: papain enzymolysis technique 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 respectively papoid (enzyme-to-substrate mass ratio) 0mg/g, 2mg/g, 4mg/g, 6mg/g, 8mg/g, 10mg/g, 12mg/g, transfer PH6 with HCL, after 50 ℃ of water-bath 2h, boiling water bath 10min makes enzyme-deactivating, be 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, survey polysaccharide yield.At the bottom of best enzyme, ratio is: 8mg/g.
| Compare mg/g at the bottom of |
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 respectively papoid 6.96mg to transfer PH6 with HCL, after 50 ℃ of water-bath 0min, 30min, 60min, 90min, 120min, 150min, 180min, boiling water bath 10min makes enzyme-deactivating 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 respectively papoid 6.96mg to transfer respectively PH with HCL is 5,7,9, after 50 ℃ of water-bath 120min, boiling water bath 10min makes enzyme-deactivating 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 respectively papoid 6.96mg to transfer respectively PH with HCL is 5 left and right, after 30 ℃ respectively, 40 ℃, 50 ℃, 60 ℃, 70 ℃, 80 ℃, 90 ℃ water-bath 120min, boiling water bath 10min makes enzyme-deactivating, 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, take polysaccharide yield as index, 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 namely affects the factors of Polysaccharides of Pleurotus Ferulae extraction yield 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 Polysaccharides of Pleurotus Ferulae lozenge
Prescription:
The proportioning of the every 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 appropriate (2g-20ml)
Talcum powder 25g
Sodium Cyclamate 5g
Concrete preparation method comprises the following steps and carries out:
1. after Resina Ferulae mushroom polysaccharide freeze-drying drying, pulverized 100 mesh sieves.
2. the accurate Sodium Cyclamate that takes recipe quantity, Zulkovsky starch 2 grams add 20 milliliters of pure water fully to stir, and mix, and are placed under 80-85 ℃ of water-bath heated and stirred and to translucent, take out standby.
3. precision takes the Resina Ferulae mushroom polysaccharide of recipe quantity, Zulkovsky starch, and 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 is namely loose ", crosses No. 16 sieve series and becomes wet granular, dries at 50-60 ℃ of temperature, crosses the whole grain of sieve No. 16, compressing tablet.
Embodiment 6: the preparation of Polysaccharides of Pleurotus Ferulae sheet
Prescription:
The proportioning of main supplementary material in every 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 appropriate (2g-20ml)
Magnesium Stearate 12.5g
Concrete preparation method comprises the following steps and carries out:
After Resina Ferulae mushroom polysaccharide freeze-drying drying, pulverized 100 mesh sieves.
Precision takes Zulkovsky starch 2 grams and adds 20 milliliters of pure water fully to stir, and mixes, and is placed under 80-85 ℃ of water-bath heated and stirred and to translucent, takes out standby.
Precision takes 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 is namely loose ", crosses No. 16 sieve series and becomes wet granular, dries at 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 the dry easily pulverizing again of dextrin or Microcrystalline Cellulose.
Claims (1)
1. the preparation method of a Polysaccharides of Pleurotus Ferulae that extracts from Pleurotus ferulae Lanzi, is characterized by, and step is as follows, Pleurotus ferulae Lanzi 20kg, be cut into small pieces, every 100g adds water 400ml, with the juice extractor 1min that squeezes the juice, ultrasonic 30min, boil 3h, cooling, centrifuging and taking supernatant liquor, filter paper suction filtration; Precipitation adds 6 times of ultrasonic 30min of water gaging, boils 1h, and is cooling, centrifugal, gets supernatant, and the filter paper suction filtration merges supernatant liquor, and is concentrated, gets concentrated solution 9L, paste-forming rate 7.825%,
Get Pleurotus ferulae Lanzi concentrated solution 50ml and add 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 after vibration, the centrifugal 5min of 3000r/min, get precipitation, repeatable operation 3 times namely gets the purifying Polysaccharides of Pleurotus Ferulae.
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