CN105796587A - Application of bamboo shaving polysaccharides in immunoregulation and tumor resisting - Google Patents
Application of bamboo shaving polysaccharides in immunoregulation and tumor resisting Download PDFInfo
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- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K31/00—Medicinal preparations containing organic active ingredients
- A61K31/70—Carbohydrates; Sugars; Derivatives thereof
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K36/00—Medicinal preparations of undetermined constitution containing material from algae, lichens, fungi or plants, or derivatives thereof, e.g. traditional herbal medicines
- A61K36/18—Magnoliophyta (angiosperms)
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Abstract
The invention discloses application of bamboo shaving polysaccharides in immunoregulation and tumor resisting. The bamboo shaving polysaccharides have the polysaccharide content not lower than 94.2% and are free of protein; tumors comprise gastric cancer, lung cancer, liver cancer and the like. According to the application, the bamboo shaving polysaccharides (BSPs) are applied to the preparation of anticancer drugs and immunoregulators, and the expression of Th1/Th2 cytokines is enhanced, so that the bamboo shaving polysaccharides are applied for cancer therapy.
Description
Technical field
The present invention relates to the antineoplastic immune adjustment effect of a kind of Caulis Bambusae In Taenia polysaccharide, belong to drug medical and the health product application of active components of plants.
Background technology
Cancer is one of main disease perplexing human health at present, and " the whole world cancer report 2014 " data issued according to World Health Organization (WHO) (WHO) show, the newly-increased cases of cancer of China is in first.In 4 kinds of malignant tumor such as liver, esophagus, harmonization of the stomach lung, China's new cases and death toll all occupy first place in the world.Though along with the development of science and technology, the method occurring in that a series for the treatment of cancer, operation and the radiotherapy of performing the operation adjoint, chemotherapy remain the goldstandard for the treatment of of cancer.But, these Therapeutic Method frequently can lead to patient's Nausea and vomiting, liver function injury, hypoimmunity, the serious untoward reaction such as decline and loss of consciousness that physical function even occur.
In recent years, Chinese medicine obtains many clinical practices in cancer, but itself medication taboo is more, and the property of medicine and drug effect for Chinese medicine still lacks the effective of modern medicine and proves, and there is many doubtful points, and the therapeutic effect for cancer is difficult to quantify.Currently, in order to reduce the toxic and side effects of existing treatment means, auxiliary is effective therapeutic strategy with the combined treatment of immunomodulator.Research shows, polysaccharide (Polysaccharides) is the compound that a class has extensive biological function, it is not only the material base of life entity, the biological process such as apoptosis simultaneously participating in intercellular identification, the adjustment of body's immunity, transformation, tumor cell.At present polysaccharide antitumor, immunoregulatory research are received significant attention.In China, the polysaccharide medicine put on market successively mainly has: lentinus edodes polysaccharide injecta, ZHULINGDUOTANG ZHUSHEYE, krestin capsule, astragalus polysaccharides, Grifola frondosa capsule etc..But, the source of these medicines is relatively limited, relatively costly.Therefore, development of new, efficient, inexpensive immunomodulator are significant.
China is vast in territory, is the primary growth district of bamboo, have the title of " bamboo kingdom ".Whole nation bamboo grove area is up to 7,200,000 hectares, and wherein Phyllostachys pubescens Mazei ex H.de Lebaie (Phyllostachyspubescens) distribution is the widest, economic use value is the highest.And China ancients notice the medical value of bamboo very early, the commentary for Folium Bambusae is " property is light, sweet in the mouth, hardship, has the merit of improving eyesight removing toxic substances and hemostasis ".Modern study shows, the different parts of bamboo Herb has the polysaccharide of different content and composition, and such as the Polysaccharides in Bamboo Leaves that water extraction obtains, its principal monosaccharides is glucose；The bamboo sprout polysaccharide that alkaline extraction obtains is adopted then to mainly contain glucose and xylose [Yin Jun, Ge Qing, Mao Jianwei, Fang Sheng. the component of Polysaccharides in Bamboo Leaves and antioxidant activity analysis, food industry science and technology, 2013,2:100-103.] [YusukeEdashige, TadashiIshii.Hemicellulosicpolysaccharidesfrombambooshoo tcell-walls, Phytochemistry, 1998,49 (6): 1675-1682].
Exodermis that Caulis Bambusae In Taenia (Bambooshavings) scrapes for the stem stalk of some bamboo kinds in grass family Phyllostachys, Sinobambusa and Dendrocalamus or secondly one layer, be one of large by-product of producing of bamboo processing link.Ph.D. Dissertation's " chemical constitution of Caulis Bambusae In Taenia polysaccharide and immunocompetence research " that Herba Tagetis Patulae is clear informs the preparation method of Caulis Bambusae In Taenia polysaccharide and corresponding purposes.The Caulis Bambusae In Taenia polysaccharide that the method is prepared and obtained also exists the protein of trace, and total sugar content is only 90.2%.
Summary of the invention
The technical problem to be solved in the present invention is to provide the application in immunomodulating, antitumor of a kind of Caulis Bambusae In Taenia polysaccharide；The present invention, by the Caulis Bambusae In Taenia polysaccharide (BambooShavingPolysaccharides, the BSPs) preparation for cancer therapy drug and immunomodulator, strengthens the expression of Th1/Th2 cytokine, and the treatment for cancer uses.
In order to solve above-mentioned technical problem, the present invention provides the application in immunomodulating, antitumor of a kind of Caulis Bambusae In Taenia polysaccharide, in described Caulis Bambusae In Taenia polysaccharide, polyoses content is >=94.2% (measuring according to Phenol-sulphate acid method, quality %), and does not contain protein in Caulis Bambusae In Taenia polysaccharide.
Improvement as the application in immunomodulating, antitumor of the Caulis Bambusae In Taenia polysaccharide of the present invention: described tumor includes gastric cancer, pulmonary carcinoma, hepatocarcinoma etc..
As the improvement of application in immunomodulating, antitumor of the Caulis Bambusae In Taenia polysaccharide of the present invention, described Caulis Bambusae In Taenia polysaccharide is for obtaining according to following steps preparation:
1) steam explosion pretreatment:
The Caulis Bambusae In Taenia (moisture 12～18%) of Phyllostachys pubescens Mazei ex H.de Lebaie is carried out steam explosion: moment explosion after pressurize 0.5～1.5min under the steam pressure of 2～2.5Mpa, the time of producing is 0.005075s≤T≤0.008752s；
By the Caulis Bambusae In Taenia raw material drying after steam explosion to moisture≤15%, get final product safe storage；
2) water carries:
Weigh 50g step 1) the steam explosion raw materials treated of gained, add 700～800mL distilled water, lixiviate 1.5～2.5h in the water-bath of 90～100 DEG C, filter, gained filtrate is lixiviating solution；
The lixiviating solution of 55～60 DEG C adds 4.5～5.5mg α-amylase (enzyme 50U/mg alive) and 4.5～5.5mg neutral protease (100,000 U/g) adjusts pH value to 7.0 ± 1, at 50～55 DEG C of temperature after enzymolysis 15～20min, by enzyme-deactivating；
3) precipitate with ethanol:
By step 2) feed liquid of gained is evaporated to 45～55mL, obtains concentrated solution；
Concentrated solution adds the ethanol or the edible ethanol (93%) that account for 4 times of volumes of concentrated solution, stands 10～12 hours at 3～5 DEG C, collect precipitation, and fully wash with 80% ethanol；
4) Sevag method deproteinization
By step 4) after the washing postprecipitation of gained dissolves with a certain amount of distilled water, add after mixing with the isopyknic n-butyl alcohol of distilled water, chloroform mixed solution, be centrifuged, take supernatant；In triplicate.
In described mixed solution, n-butyl alcohol: the volume ratio of chloroform is 1:4；
5) membrane separation purification
Take step 4) in supernatant, add the distilled water of a certain amount of 35～45 DEG C, the volume ratio of described supernatant and distilled water is 1:5-10, obtains diluent；
Above-mentioned diluent is carried out membrane separation purification: first filter with the microporous filter membrane of 0.2 μm, then with the poly (ether sulfone) film ultrafiltration of 30KD, gained permeate concentrates with the poly (ether sulfone) film that molecular cut off is 3KD again；
6) by step 5) concentrated solution (3KD trapped fluid) of gained carries out concentrating under reduced pressure until solid content >=30%, then lyophilization, obtains Caulis Bambusae In Taenia polysaccharide.
The BSP-New phend-sulphuric acid given period polyoses content of the present invention is 94.2%；Meanwhile, it is negative for surveying protein by Coomassie brilliant G-250 method.
The Caulis Bambusae In Taenia polysaccharide of the present invention has the ability of anticancer propagation in vitro, and internal have the immune function of regulation and control, and action effect is better than the lentinan listed.
The present invention has carried out following confirmatory experiment:
1) for multiple cancerous cell line and tumor-bearing mice thereof, Caulis Bambusae In Taenia polysaccharide (BSP-New) prepared by technique is improved respectively with the present invention, the Caulis Bambusae In Taenia polysaccharide (BSP-Old) of preparation in the Ph.D. Dissertation that Herba Tagetis Patulae is clear, lentinan (LNT) and placebo (Saline) carry out anti-tumor activity experiment:
A. in vitro cytotoxic effect detection and inhibiting tumor cell proliferation experiment；
B. the mensuration of the propagation of associated immune cells, immune factor expression and histoorgan physical signs in body: adopt euzymelinked immunosorbent assay (ELISA) (ELISA), FCM analysis, HE dyeing etc..
2) the two kinds of Caulis Bambusae In Taenia polysaccharide test sample of the oral administration gavage testing inspection adjustment effect to mouse immune system: peripheral blood lymph number, peripheral blood immunocyte subgroup, macrophages phagocytic capacity, immunoglobulin expression level, NK cell killing activity etc..
The preparation method of the Caulis Bambusae In Taenia polysaccharide test sample adopted in the present invention, compared with existing similar technique, has the advantage that
1) purity of Caulis Bambusae In Taenia polysaccharide is greatly improved；
2) utilizing large by-product that the bamboo course of processing produces is raw material, it is thus achieved that Caulis Bambusae In Taenia polysaccharide, its raw material is sufficient, with low cost；Solve the environmental problem that in bamboo production process, garbage brings, it is achieved that raw-material higher value application, achieve many things at one stroke；
3) compared with the Caulis Bambusae In Taenia polysaccharide test sample that existing immunomodulator tester (lentinan) and inventor place team early stage obtain, the Caulis Bambusae In Taenia polysaccharide of the present invention has the effect better suppressing tumor growth, extending the pattern mouse survival cycle；In vivo in immunomodulating test, also show as excellent in strengthening the expression etc. of Th1/Th2 cytokine compared with lentinan.That is, the Caulis Bambusae In Taenia polysaccharide of gained of the present invention has physiologically active more preferably；
4) Caulis Bambusae In Taenia polysaccharide can pass through oral route enhancing human body immunity activity, plays the effect of prevention and health care.
Accompanying drawing explanation
Below in conjunction with accompanying drawing, the specific embodiment of the present invention is described in further detail.
Fig. 1 be two kinds of distinct methods prepare obtain Caulis Bambusae In Taenia polysaccharide (BSP-Old and BSP-New), act on 72h as the lentinan (LNT) of positive control after, to gastric cancer (MFC, MGC-803, BGC-823), hepatocarcinoma (Huh-7, HepG2, Sk-Hep1), the contrast of the cell survival ratio of pulmonary carcinoma (NCI-H1688, NCI-H520, A549) cell line.
In figure, every column data is followed successively by Ctrl, LNT, BSP-Old, BSP-New from left to right.Ctrl represents control experiment group, only adds isopyknic drug solvent in the medium；LNT represents lentinan test group, lentinan phosphate buffered saline (PBS) is dissolved；BSP-Old represents the Caulis Bambusae In Taenia polysaccharide in comparative example 1, and BSP-New represents the Caulis Bambusae In Taenia polysaccharide that the present invention obtains, and is used that PBS dissolves.
In figure, every column data is followed successively by Ctrl (PBS), LNT (2000 μ g/mL), BSP-Old (2000 μ g/mL), BSP-New (2000 μ g/mL) from left to right.
Fig. 2 be two kinds of distinct methods prepare obtain Caulis Bambusae In Taenia polysaccharide, as the impact on tumor-bearing mice inflammatory cytokine secretion level of the lentinan (LNT) of positive control.
In figure, every column data is followed successively by Saline, LNT (50mg/kg), BSP-Old (400mg/kg), BSP-New (400mg/kg) from left to right.
Fig. 3 be two kinds of distinct methods prepare obtain Caulis Bambusae In Taenia polysaccharide, as the lentinan (LNT) of positive control to the scale effect of immunocyte subgroup in tumor tumor-bearing mice peripheral blood and spleen.
In figure, every column data is followed successively by Saline, LNT (50mg/kg), BSP-Old (400mg/kg), BSP-New (400mg/kg) from left to right.
Fig. 4 is that the Caulis Bambusae In Taenia polysaccharide prevention that two kinds of distinct methods preparations obtain is tested and Experiment on therapy tumor growth curve.
Fig. 5 is that two kinds of distinct methods prepare the Caulis Bambusae In Taenia polysaccharide the obtained impact on NK cytoactive.
Detailed description of the invention
The following is further illustrating the present invention rather than limitation of the present invention.
Embodiment 1, Caulis Bambusae In Taenia polysaccharide preparation method, be sequentially carried out following steps:
1) steam explosion pretreatment
By the Caulis Bambusae In Taenia of 1kg Phyllostachys pubescens Mazei ex H.de Lebaie (Phyllostachysheterocyclavar.pubescens (Mazel) Ohwi), (bamboo shaving is crushed to 10-20 order after natural air drying, moisture about 15%) (Hebi City right way bioenergy company limited manufactures to put into high density steam explosion machine, QBS-200B type) material cavity (5L) in, when steam pressure is 2.2MPa, dimension pressure is 1min, and moment produces (time of producing is 0.005075s≤T≤0.008752s).Wet stock after steam explosion is put in the baking oven of 80 ± 1 DEG C and be dried to moisture about 15%, it is simple to preserve.
2) water carries
Weigh the dried Caulis Bambusae In Taenia of steam explosion of Caulis Bambusae In Taenia raw material (the i.e. step 1) gained after 50g dewaxing), add 750mL distilled water, lixiviate 2h in the water-bath of 95 DEG C, obtain lixiviating solution after filtration；The filtrate of 55-60 DEG C adds 5mg α-amylase (enzyme 50U/mg alive) and 5mg neutral protease (100,000 U/g), adjust pH value to 7.0 ± 1 by the NaOH solution system of 1mol/L, at 50～55 DEG C of temperature after enzymolysis 20min, enzyme denaturing (100 DEG C, 10min).
3) precipitate with ethanol
By step 2) gains decompression (vacuum≤0.09MPa, temperature 45-50 DEG C) be concentrated into about 50mL, obtain concentrated solution；
It is slowly added to the edible ethanol (being simultaneously stirred continuously) of 4 times of concentrated solution volumes, stands at 4 DEG C overnight (about 12 hours), collect precipitation, and fully wash with 80% ethanol (about 5mL).
4) Sevag method deproteinization
By step 4) after the washing postprecipitation of gained dissolves with 10mL distilled water, mix with 10mL n-butyl alcohol, chloroform mixed solution (n-butyl alcohol: chloroform=1:4), vibration under room temperature, centrifugal (8000rpm, 15 minutes), take upper strata aqueous phase, this step is in triplicate；
5) membrane separation purification
By the sample after deproteinization (namely, aqueous phase after step 4 merging, about 10mL) with after the distilled water dilutings of 40 DEG C to 70mL, it is purified with the experiment membrane separation device of SARTORIUS Sai Duolisi company of Germany, after the microporous filter membrane coarse filtration of 0.2 μm, first with the poly (ether sulfone) film ultrafiltration of 30KD, permeate concentrates with the poly (ether sulfone) film that molecular cut off is 3KD again.
6) concentrate drying
By step 5) the trapped fluid decompression (vacuum≤0.09MPa of the 3KD of gained, temperature 45-50 DEG C) it is concentrated into admittedly containing about 30%, lyophilization (-40 DEG C, dried in vacuum overnight), obtains Caulis Bambusae In Taenia polysaccharide (called after BSP-New) about 0.48g.
It is 94.2% that BSP-New phend-sulphuric acid calculates its polyoses content；Meanwhile, it is negative for surveying protein by Coomassie brilliant G-250 method.
Comparative example 1, the step described according to the clear academic dissertation of Herba Tagetis Patulae " chemical constitution of Caulis Bambusae In Taenia polysaccharide and immunocompetence research " carry out the preparation of Caulis Bambusae In Taenia polysaccharide, i.e. for embodiment 1, make the following changes:
Cancellation step 3) in the use of 5mg neutral protease, and without Sevag method removing protein；
By step 5) " membrane separation purification " make into: diafiltration.
All the other are equal to embodiment 1.Obtain Caulis Bambusae In Taenia polysaccharide (called after BSP-Old) about 0.56g.
The method that this comparative example 1 lacks two kinds of removing protein, causes that end-product purity is not good enough；Being provided without membrane separation process, the polysaccharide molecular weight distribution obtained is relatively wide, and the mode of dialysis can not well control molecular cut off scope.
The composition contrast of the Caulis Bambusae In Taenia polysaccharide of above-described embodiment 1 and comparative example 1 gained is as shown in table 1.
The new and old extraction process of table 1. obtains the contrast of Caulis Bambusae In Taenia polysaccharide component
Experiment 1, antitumor cytolytic activity
(1) cell strain: murine fore-stomach carcinoma MFC；Human Gastric carcinoma MGC-803, BGC-823；Human liver cancer Huh-7, HepG2, Sk-Hep1；Human Lung Cancer NCI-H1688, NCI-H520, A549.
(2) testing sample: tradition extracts the Caulis Bambusae In Taenia polysaccharide (BSP-Old) of (that is, comparative example 1), complete medium is prepared, final concentration of 2000 μ g/mL；The Caulis Bambusae In Taenia polysaccharide (BSP-New) that new technology is extracted, complete medium is prepared, final concentration of 2000 μ g/mL；Positive drug: lentinan (LNT), complete medium is prepared, final concentration of 2000 μ g/mL.
(3) method: mtt assay
A. the cancer cell of exponential phase is collected with 2 × 104The concentration of/mL is inoculated in 96 orifice plates, 100 μ L/ holes, in 37 DEG C, and 5%CO2Incubator is cultivated 24h and makes cell attachment；
B. suck supernatant, add the culture fluid 200 μ L containing experiment desired concn testing sample, in 37 DEG C, 5%CO2Incubator is cultivated 72h；
C. every hole adds the MTT20 μ L containing 5mg/mL, continues to cultivate 4h；
D. terminate cultivating, abandon culture fluid in hole, add DMSO150 μ L/ hole, be placed on shaking table and shake 10min, make crystallization fully dissolve, survey each hole light absorption value in microplate reader 570nm place.Experiment arranges this bottom outlet (culture medium, MTT, DMSO), control wells (cell, the drug solvent of same concentrations, culture fluid, MTT, DMSO) simultaneously；
E. calculate: cell survival rate (%)=(administration-background)/(comparison-background) × 100；
F. analyze the known cell proliferation of experimental data and can play obvious inhibitory action (see Fig. 1), the present invention extracts the Caulis Bambusae In Taenia Polysaccharide B SP-New of acquisition, and the growth inhibited effect for cancerous cell is significantly better than lentinan LNT and traditional handicraft (comparative example 1) extracts the Caulis Bambusae In Taenia Polysaccharide B SP-Old obtained.
Experiment 2, the Caulis Bambusae In Taenia polysaccharide immunoregulation effect to tumor-bearing mice
(1) cell strain: mice model of forestomach cancerous cell MFC
(2) testing sample: new old technology extracts Caulis Bambusae In Taenia polysaccharide；Positive drug: lentinan
(3) method for establishing model:
A. 48 male 5 weeks week old cleaning grade 615 mices, every Mus left hind axil subcutaneous vaccination MFC cell 3 × 10 are taken6/ 100 μ L cell suspension.After inoculation 24h, random point 4 groups, often group 12, adopt dorsal sc injection administering mode, saline control group (Saline) every day presses 20 μ L/g (BW), lentinan for injection positive controls (LNT) is by 50mg/kg (BW), tradition extracts Caulis Bambusae In Taenia polysaccharide test group (BSP-Old) by 400mg/kg (BW), and the present invention extracts Caulis Bambusae In Taenia polysaccharide test group (BSP-New) by 400mg/kg (BW).Every day injects once, continuous two weeks, within the 14th day, gathers mouse organs, blood sample, bone marrow and tumor tissues in injectable drug；
B. mice serum inflammatory factor expression uses Multi-AnalyteELISArrayKit test kit detection (see Fig. 2)；Utilize flow cytometry, human peripheral blood and express the immunocyte subgroup of different surfaces antigen molecule in spleen and carry out statistical analysis (see Fig. 3)；
C. test result indicate that: Caulis Bambusae In Taenia polysaccharide promotes Th1 type inflammatory cytokine (IFN-γ, TNF-α etc.) secretion, big quantity research is shown in long-term lotus tumor environment, often with Th2 type inflammatory cytokine (IL-6, IL-10 etc.) to take as the leading factor, this illustrates that Caulis Bambusae In Taenia polysaccharide has the effect regulating immunologic balance；Caulis Bambusae In Taenia polysaccharide can be effectively increased effect immune cell population quantity (CD4+T cell, CD8+T cell, NK cell etc.), has promoted the mobilization of these cells.Result above shows that Caulis Bambusae In Taenia polysaccharide can effectively activate the immune system of mice.
Experiment 3, the experiment of Caulis Bambusae In Taenia many Glyco inhabiting tumor growth
(1) cell strain: mice model of forestomach cancerous cell MFC
(2) testing sample: new old technology extracts Caulis Bambusae In Taenia polysaccharide；Positive drug: lentinan
(3) experimental technique:
A. empirically purpose is divided into prevention group and treatment group.Prevention group: take 6 male 5 weeks week old cleaning grade 615 mices, press 400mg/kg (BW) new technology every day and extract Caulis Bambusae In Taenia polysaccharide (PreBSP-New) through mice antedorsal subcutaneous injection, every day injects once, in back part subcutaneous vaccination MFC cell 3 × 10 after injecting one week continuously6/ 100 μ L cell suspension；Treatment group: with prevention group in the identical time, inoculates MFC cell 3 × 10 through dorsal sc6/ 100 μ L, adopt dorsal sc injection administering mode, normal saline (Sline) matched group 20 μ L/g (BW), traditional handicraft group (BSP-Old) 400mg/kg (BW) group, new technology group (BSP-New) 800mg/kg (BW) group is in tumor volume growth to 100mm3Left and right start administration, every day inject once, continuous two weeks, every day observed and recorded Mouse Weight, life state, tumor body size；
B. tumor growth curve (see Fig. 4) is drawn according to the tumor size of record record；
C. test result indicate that: the present invention extracts the Caulis Bambusae In Taenia polysaccharide obtained can effectively play the propagation of prevention and anticancer.
Experiment 4, the immune adjustment of oral administration gavage Caulis Bambusae In Taenia Polysaccharides on Mice
(1) experiment mice: ICR mice
(2) testing sample: new old technology extracts Caulis Bambusae In Taenia polysaccharide；Positive drug: lentinan
(3) experimental technique:
A. 56 mices are divided into saline control group (NC, n=8): every three days subcutaneous injection equal-volume normal saline, every day gavage equal-volume normal saline；Cyclophosphamide immunosuppressant group (CPA, n=8): every three days subcutaneous injection 70mgCPA/kg (BW), every day gavage equal-volume normal saline；Lentinan positive drug group (CPA+LNT, n=8): every three days subcutaneous injection 70mgCPA/kg (BW), gavage 50mgLNT/kg every day (BW)；Traditional handicraft Caulis Bambusae In Taenia polysaccharide immune activation group (CPA+BSP-Old, n=8): every three days subcutaneous injection 70mgCPA/kg (BW), gavage 100mgBSP-Old/kg every day (BW)；Caulis Bambusae In Taenia polysaccharide immune activation group (CPA+BSP-New, n=8) of the present invention: every three days subcutaneous injection 70mgCPA/kg (BW), gavage 100mgBSP-New/kg every day (BW)；Traditional handicraft Caulis Bambusae In Taenia polysaccharide negative control group (BSP-Old, n=8): every three days subcutaneous injection equal-volume normal saline, gavage 200mgBSP-Old/kg every day (BW)；Caulis Bambusae In Taenia polysaccharide negative control group (BSP-New, n=8) of the present invention: every three days subcutaneous injection equal-volume normal saline, gavage 200mgBSP-New/kg every day (BW).
B., after last administration 24h (the 15th day), adopt the mode that eyeball takes blood to gather whole blood, be collected in anticoagulant tube.Adopt the quantity of Hematology analyzer detection leukocyte (PWBC) and lymphocyte (PBL):
The table 2. Caulis Bambusae In Taenia Polysaccharide B SP impact on mouse peripheral blood leukocyte and lymphocyte count
| Group | PWBC(×109/L) | PBL(×109/L) |
| NC | 7.54±1.84 | 6.03±0.98 |
| CPA | 3.61±0.48** | 2.86±0.40** |
| CPA+LNT(50) | 4.37±0.87**# | 3.59±0.80**# |
| CPA+BSP-Old(100) | 4.05±0.53** | 3.30±0.50** |
| CPA+BSP-New(100) | 5.56±0.46*# | 4.77±0.60*# |
| BSP-Old(200) | 8.10±1.49 | 6.59±1.08 |
| BSP-New(200) | 7.79±0.89 | 6.33±0.65 |
Note: NC (normalcontrol), Normal group；CPA (cyclophosphamide), cyclophosphamide；LNT (lentinan), lentinan；BSP-Old, Caulis Bambusae In Taenia polysaccharide prepared by former technique；BSP-New, the Caulis Bambusae In Taenia polysaccharide that the present invention obtains.Compare with NC group, * p < 0.05, * * p < 0.01；Compare with CPA model group, #p < 0.05, ##p < 0.01.
C., after last administration 24h (the 15th day), adopt the mode that eyeball takes blood to gather whole blood, be collected in anticoagulant tube.Utilize fluorescent antibody labelling T cell, B cell, NK cell surface antigen, adopt flow cytometry to carry out the cell colony quantity of each experimental group of statistical analysis and percentage:
The table 3. Caulis Bambusae In Taenia Polysaccharide B SP impact on Mouse Peripheral Blood Lymphocyte subset proportions and quantity
Note: NC (normalcontrol), Normal group；CPA (cyclophosphamide), cyclophosphamide；LNT (lentinan), lentinan；BSP-Old, Caulis Bambusae In Taenia polysaccharide prepared by former technique；BSP-New, the Caulis Bambusae In Taenia polysaccharide that the present invention obtains.Compare with NC group, * p < 0.05, * * p < 0.01；Compare with CPA model group, #p < 0.05, ##p < 0.01.
D. mononuclear phagocyte phagocytic activity detection: after last administration 2h (the 14th day), from the mouse tail vein injection india ink (0.1mL/10gBW) of normal saline dilution 5 times, namely injection prepared Chinese ink start timing.After injecting prepared Chinese ink, 2min and 10min takes blood 20 μ L with hemostix from angular vein clump respectively, and addition fills 2mL0.1%Na immediately2CO3In the test tube of solution, mix with vortex instrument.Sampling adds 96 orifice plates, every hole 200 μ L.Use above-mentioned 0.1%Na2CO3Solution is as blank, by microplate reader in 630nm wavelength place measurement OD value.
Table 4. mononuclear phagocyte phagocytic activity
| Group | Phagocytic index α |
| NC | 5.21±0.39 |
| CPA | 4.41±0.40** |
| CPA+LNT(50) | 5.24±0.49## |
| CPA+BSP-Old(100) | 5.18±0.44## |
| CPA+BSP-New(100) | 5.46±0.51## |
| BSP-Old(200) | 5.43±0.52 |
| BSP-New(200) | 5.67±0.37 |
Note: NC (normalcontrol), Normal group；CPA (cyclophosphamide), cyclophosphamide；LNT (lentinan), lentinan；BSP-Old, Caulis Bambusae In Taenia polysaccharide prepared by former technique；BSP-New, the Caulis Bambusae In Taenia polysaccharide that the present invention obtains.Compare with NC group, * p < 0.05, * * p < 0.01；Compare with CPA model group, #p < 0.05, ##p < 0.01.
E.NK cell killing activity detects: after last administration 24h (the 15th day), is put to death by mice cervical dislocation, prepares mouse lymphocyte suspension action effect cell (5 × 106Individual/mL), make effect target than for 50:1.Adopt lactic acid dehydrogenase (LDH) method to measure the activity of spleen NKT (NK) cell, see Fig. 5.
Finally, in addition it is also necessary to be only several specific embodiments of the present invention it is noted that listed above.It is clear that the invention is not restricted to above example, it is also possible to there are many deformation.All deformation that those of ordinary skill in the art can directly derive from present disclosure or associate, are all considered as protection scope of the present invention.
Claims (5)
1. Caulis Bambusae In Taenia polysaccharide application in immunomodulating, antitumor, is characterized in that: in described Caulis Bambusae In Taenia polysaccharide, polyoses content is >=94.2%, and does not contain protein in Caulis Bambusae In Taenia polysaccharide.
2. the Caulis Bambusae In Taenia polysaccharide according to claim 1 application in immunomodulating, antitumor, is characterized in that: described tumor includes gastric cancer, pulmonary carcinoma, hepatocarcinoma.
3. the Caulis Bambusae In Taenia polysaccharide according to claim 1 and 2 application in immunomodulating, antitumor, is characterized in that described Caulis Bambusae In Taenia polysaccharide is for obtaining according to following steps preparation:
1) steam explosion pretreatment:
The Caulis Bambusae In Taenia of Phyllostachys pubescens Mazei ex H.de Lebaie is carried out steam explosion: moment explosion after pressurize 0.5～1.5min under the steam pressure of 2～2.5Mpa, the time of producing is 0.005075s≤T≤0.008752s；
By the Caulis Bambusae In Taenia raw material drying after steam explosion to moisture≤15%, get final product safe storage；
2) water carries:
Weigh 50g step 1) the steam explosion raw materials treated of gained, add 700～800mL distilled water, lixiviate 1.5～2.5h in the water-bath of 90～100 DEG C, filter, gained filtrate is lixiviating solution；
The lixiviating solution of 55～60 DEG C adds 4.5～5.5mg α-amylase and 4.5～5.5mg neutral protease adjusts pH value to 7.0 ± 1, at 50～55 DEG C of temperature after enzymolysis 15～20min, by enzyme-deactivating；
3) precipitate with ethanol:
By step 2) feed liquid of gained is evaporated to 45～55mL, obtains concentrated solution；
Concentrated solution adds the ethanol or the edible ethanol that account for 4 times of volumes of concentrated solution, stands 10～12 hours at 3～5 DEG C, collect precipitation, and fully wash with 80% ethanol；
4) Sevag method deproteinization:
By step 4) after the washing postprecipitation of gained dissolves with distilled water, add after mixing with the isopyknic n-butyl alcohol of distilled water, chloroform mixed solution, be centrifuged, take supernatant；In triplicate；
In described mixed solution, n-butyl alcohol: the volume ratio of chloroform is 1:4；
5) membrane separation purification:
Take step 4) in supernatant, add the distilled water of 35～45 DEG C, the volume ratio of described supernatant and distilled water is 1:5-10, obtains diluent；
Above-mentioned diluent is carried out membrane separation purification: first filter with the microporous filter membrane of 0.2 μm, then with the poly (ether sulfone) film ultrafiltration of 30KD, gained permeate concentrates with the poly (ether sulfone) film that molecular cut off is 3KD again；
6) by step 5) concentrated solution of gained carries out concentrating under reduced pressure until solid content >=30%, then lyophilization, obtains Caulis Bambusae In Taenia polysaccharide.
4. the Caulis Bambusae In Taenia polysaccharide according to claim 3 application in immunomodulating, antitumor, is characterized in that:
The enzyme of described α-amylase is lived as 50U/mg；
The enzyme work of described neutral protease is 100,000 U/g.
5. the Caulis Bambusae In Taenia polysaccharide according to claim 4 application in immunomodulating, antitumor, is characterized in that:
In described edible ethanol, the volumetric concentration of ethanol is 93%.
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Cited By (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN107753929A (en) * | 2017-11-10 | 2018-03-06 | 河南工业大学 | A kind of health care oral liquid of the globulin containing wheat germ and preparation method thereof |
| CN112358553A (en) * | 2020-12-09 | 2021-02-12 | 青海大学 | Polysaccharide SM-0.2M and anti-tumor product prepared from same |
| CN112521521A (en) * | 2020-12-09 | 2021-03-19 | 青海大学 | Polysaccharide SM-W and anti-tumor product prepared from same |
-
2016
- 2016-04-26 CN CN201610269486.8A patent/CN105796587B/en active Active
Non-Patent Citations (2)
| Title |
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| 黄菊青: "竹茹多糖的化学结构和免疫活性研究", 《中国博士学位论文全文数据库医药卫生科技辑》 * |
| 齐睿婷等: "竹茹多糖提取工艺优化及其理化性质分析", 《食品工业科技》 * |
Cited By (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN107753929A (en) * | 2017-11-10 | 2018-03-06 | 河南工业大学 | A kind of health care oral liquid of the globulin containing wheat germ and preparation method thereof |
| CN112358553A (en) * | 2020-12-09 | 2021-02-12 | 青海大学 | Polysaccharide SM-0.2M and anti-tumor product prepared from same |
| CN112521521A (en) * | 2020-12-09 | 2021-03-19 | 青海大学 | Polysaccharide SM-W and anti-tumor product prepared from same |
| CN112521521B (en) * | 2020-12-09 | 2021-11-05 | 青海大学 | Polysaccharide SM-W and anti-tumor product prepared from same |
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| CN105796587B (en) | 2018-07-31 |
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