CN105796587B - Caulis bambusae in taenian polysaccharide immunological regulation, it is antitumor in application - Google Patents
Caulis bambusae in taenian polysaccharide immunological regulation, it is antitumor in application Download PDFInfo
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- CN105796587B CN105796587B CN201610269486.8A CN201610269486A CN105796587B CN 105796587 B CN105796587 B CN 105796587B CN 201610269486 A CN201610269486 A CN 201610269486A CN 105796587 B CN105796587 B CN 105796587B
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- A—HUMAN NECESSITIES
- 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
- A61K31/715—Polysaccharides, i.e. having more than five saccharide radicals attached to each other by glycosidic linkages; Derivatives thereof, e.g. ethers, esters
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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)
- A61K36/88—Liliopsida (monocotyledons)
- A61K36/899—Poaceae or Gramineae (Grass family), e.g. bamboo, corn or sugar cane
Abstract
The invention discloses a kind of caulis bambusae in taenian polysaccharide immunological regulation, it is antitumor in application, polyoses content is >=94.2% in the caulis bambusae in taenian polysaccharide, and protein is not contained in caulis bambusae in taenian polysaccharide；Tumour includes gastric cancer, lung cancer, liver cancer etc..Preparation of the present invention by caulis bambusae in taenian polysaccharide (Bamboo Shaving Polysaccharides, BSPs) for anticancer drug and immunomodulator, enhances the expression of Th1/Th2 cell factors, is used for the treatment of cancer.
Description
Technical field
The present invention relates to a kind of antineoplastic immune adjustment effects of caulis bambusae in taenian polysaccharide, belong to the drug medical of active components of plants
With health products application.
Background technology
Cancer is one of the main disease of current puzzlement human health, according to the World Health Organization (WHO) publication《The whole world
Cancer report 2014》Data show that the newly-increased cases of cancer in China is in first.It is pernicious in 4 kinds of liver, esophagus, stomach and lung etc.
In tumour, Chinese new cases and death toll occupy first place in the world.Although with the development of science and technology, occurring a series of
The method for the treatment of cancer, but operation and adjoint radiotherapy of performing the operation, chemotherapy are still the gold for the treatment of of cancer
Standard.However, these therapies frequently can lead to patient's Nausea and vomiting, hepatic disorder, hypoimmunity, or even occur
The serious adverse reaction such as the decline of physical function and the loss of consciousness.
In recent years, Chinese medicine obtains many clinical applications in cancer, but itself medication taboo is more, and for
The pharmacological property and drug effect of traditional Chinese medicine still lack effective demonstration of modern medicine, there are many doubtful points, the treatment for cancer
Effect is difficult to quantify.Currently, it in order to reduce the toxic side effect of existing treatment means, assists with the combination therapy side of immunomodulator
Case is effective therapeutic strategy.Studies have shown that polysaccharide (Polysaccharides), which is one kind, has extensive biological function
Compound, it is not only the material base of life entity, simultaneously participate in it is intercellular identification, body's immunity adjusting,
Transformation, the biological processes such as apoptosis of tumour cell.Antitumor for polysaccharide at present, immunoregulatory research is by wide
General concern.In China, the polysaccharide medicine launched successively mainly has：Lentinus edodes polysaccharide injecta, polyporus polysaccharide injection, rainbow conk
Polyoses capsule, astragalus polyose, grifola frondosus capsule etc..However, the source of these drugs is relatively limited, cost is higher.Therefore, it develops
Novel, efficient, inexpensive immunomodulator is significant.
China has a vast territory, is the main vitellarium of bamboo, is known as the title of " bamboo kingdom ".National bamboo grove area is up to
7200000 hectares, wherein moso bamboo (Phyllostachys pubescens) distribution is most wide, economic use value highest.In and
State ancients notice the medical value of bamboo very early, and the commentary for the leaf of bamboo is that " property is light, sweet in flavor, bitter, has improving eyesight to detoxify and stop
The work(of blood ".Modern research shows that the different parts of bamboo complete stool have the polysaccharide of different content and composition, as water extraction obtains
Polysaccharides in Bamboo Leaves, principal monosaccharides be glucose；The bamboo shoots polysaccharide obtained using alkaline extraction then mainly contains glucose and xylose
[Yin Jun, Ge Qing, Mao Jianwei, the component of the bright Polysaccharides in Bamboo Leaves in side and antioxidant activity analysis, food industry science and technology, 2013,2:
100-103.][Yusuke Edashige,Tadashi Ishii.Hemicellulosic polysaccharides from
bamboo shoot cell-walls,Phytochemistry,1998,49(6):1675-1682]。
Caulis bambusae in taenian (Bamboo shavings) is grass family Phyllostachys, in Bambusa blumeana category and Dendrocalamus some bamboo kinds stalk institute
The exodermis that scrapes or secondly one layer, are one of large by-products that bamboo processing link generates.The clear doctorate opinion of yellow chrysanthemum
Text《The chemical constitution of caulis bambusae in taenian polysaccharide and immunocompetence research》Inform the preparation method of caulis bambusae in taenian polysaccharide and corresponding purposes.The party
There is micro protein in caulis bambusae in taenian polysaccharide obtained by prepared by method, and total sugar content is only 90.2%.
Invention content
The technical problem to be solved in the present invention is to provide a kind of caulis bambusae in taenian polysaccharide immunological regulation, it is antitumor in application；This
Caulis bambusae in taenian polysaccharide (Bamboo Shaving Polysaccharides, BSPs) is used for anticancer drug and immunomodulator by invention
Preparation, enhance Th1/Th2 cell factors expression, for cancer treatment use.
In order to solve the above technical problem, the present invention provides a kind of caulis bambusae in taenian polysaccharide immunological regulation, it is antitumor in application,
Polyoses content is >=94.2% (being measured according to Phenol-sulphate acid method, quality %) in the caulis bambusae in taenian polysaccharide, and is free of in caulis bambusae in taenian polysaccharide
There is protein.
As the present invention caulis bambusae in taenian polysaccharide immunological regulation, it is antitumor in application improvement：The tumour include gastric cancer,
Lung cancer, liver cancer etc..
As the present invention caulis bambusae in taenian polysaccharide immunological regulation, it is antitumor in application improvement, the caulis bambusae in taenian polysaccharide be by
It is prepared according to following steps：
1) steam blasting pre-processes：
The caulis bambusae in taenian (moisture 12~18%) of moso bamboo is subjected to steam blasting：It is protected under the steam pressure of 2~2.5Mpa
Moment explosion after 0.5~1.5min of pressure, it is 0.005075s≤T≤0.008752s to produce the time；
By the caulis bambusae in taenian raw material drying after steam explosion to moisture≤15%, you can safe storage；
2) water carries：
The steam explosion raw materials treated obtained by 50g steps 1) is weighed, 700~800mL distilled water is added, in 90~100 DEG C
1.5~2.5h, filtering are extracted in water-bath, gained filtrate is leaching liquor；
It is added in 4.5~5.5mg alpha-amylases (enzyme activity 50U/mg) and 4.5~5.5mg in 55~60 DEG C of leaching liquor
Property protease (100,000 U/g) adjust pH value to 7.0 ± 1, at a temperature of 50~55 DEG C digest 15~20min after, by enzyme-deactivating；
3) alcohol precipitation：
Feed liquid obtained by step 2) is concentrated under reduced pressure into 45~55mL, obtains concentrate；
It is added in concentrate and accounts for the ethyl alcohol or edible alcohol (93%) of 4 times of volumes of concentrate, stand 10 at 3~5 DEG C~
12 hours, precipitation is collected, 80% ethyl alcohol is used in combination fully to be washed；
4) Sevag methods take off albumen
After precipitating a certain amount of distillation water dissolution after the washing obtained by step 4), it is added isometric just with distilled water
After butanol, the mixing of chloroform mixed solution, centrifugation takes supernatant；In triplicate.
In the mixed solution, n-butanol：The volume ratio of chloroform is 1：4；
5) membrane separating and purifying
It takes supernatant in step 4), is added a certain amount of 35~45 DEG C of distilled water, the volume ratio of the supernatant and distilled water is
1：5-10 obtains dilution；
Above-mentioned dilution is subjected to membrane separating and purifying：It is first filtered with 0.2 μm of miillpore filter, then super with the poly (ether sulfone) film of 30KD
Filter, the poly (ether sulfone) film that gained permeate is again 3KD with molecular cut off are concentrated；
6) concentrate (3KD trapped fluids) obtained by step 5) be concentrated under reduced pressure up to solid content >=30%, then
Freeze-drying, obtains caulis bambusae in taenian polysaccharide.
It is 94.2% that the BSP-New phend-sulphuric acids of the present invention, which calculate phase polyoses content,；Meanwhile using Coomassie brilliant blue
It is feminine gender that G-250 methods, which survey protein,.
The caulis bambusae in taenian polysaccharide of the present invention has the ability for inhibiting cancer cell multiplication in vitro, has regulation and control immune system in vivo
Function, function and effect are better than the lentinan listed.
The present invention has carried out following confirmatory experiment：
1) it is directed to a variety of cancerous cell lines and its tumor-bearing mice, the caulis bambusae in taenian polysaccharide prepared respectively with modified technique of the present invention
(BSP-New), the caulis bambusae in taenian polysaccharide (BSP-Old) prepared in the Ph.D. Dissertation of Huang Juqing, lentinan (LNT) and placebo
(Saline) antitumor activity experiment is carried out：
A. in vitro cytotoxic effect detection and inhibiting tumor cell proliferation experiment；
B. the proliferation of associated immune cells, the measurement of immune factor expression and histoorgan physical signs in body：It adopts
With enzyme-linked immunization (ELISA), FCM analysis, HE dyeing etc..
2) adjustment effect of the two kinds of caulis bambusae in taenian polysaccharide test samples of oral administration gavage testing inspection to mouse immune system：Periphery hemolymph
Number, peripheral blood immunocyte subgroup, macrophages phagocytic capacity, immunoglobulin expression level, NK cell killing activities etc..
The preparation method of the caulis bambusae in taenian polysaccharide test sample used in the present invention has the following advantages that compared with existing similar technique：
1) purity of caulis bambusae in taenian polysaccharide is greatly improved；
2) it is raw material to utilize large by-product that bamboo process generates, the caulis bambusae in taenian polysaccharide of acquisition, raw material is sufficient, at
This is cheap；It solves the environmental problem that waste is brought in bamboo production process, realizes the higher value application of raw material, at one stroke
More；
3) the caulis bambusae in taenian polysaccharide obtained early period with team where existing immunomodulator reference material (lentinan) and inventor
Sample is compared, and caulis bambusae in taenian polysaccharide of the invention has the function of preferably inhibiting tumour growth, extends the pattern mouse survival period；
Vivo immunization is adjusted in experiment, is also shown as compared with lentinan in the expression etc. of enhancing Th1/Th2 cell factors excellent.
That is, the caulis bambusae in taenian polysaccharide of present invention gained has more preferably physiological activity；
4) caulis bambusae in taenian polysaccharide can enhance immunity of organism activity by oral route, play the role of prevention and health care.
Description of the drawings
The specific implementation mode of the present invention is described in further detail below in conjunction with the accompanying drawings.
Fig. 1 be two kinds of distinct methods be prepared caulis bambusae in taenian polysaccharide (BSP-Old and BSP-New), as positive control
Lentinan (LNT) act on 72h after, to gastric cancer (MFC, MGC-803, BGC-823), liver cancer (Huh-7, HepG2, Sk-Hep1),
The comparison of the cell survival ratio of lung cancer (NCI-H1688, NCI-H520, A549) cell line.
In figure, it is followed successively by Ctrl, LNT, BSP-Old, BSP-New from left to right in every column data.Ctrl is represented to according to the facts
Group is tested, isometric drug solvent is only added in the medium；LNT represents lentinan test group, by lentinan phosphoric acid
Salt buffer salting liquid (PBS) dissolves；BSP-Old represents the caulis bambusae in taenian polysaccharide in comparative example 1, and BSP-New represents what the present invention obtained
Caulis bambusae in taenian polysaccharide, is dissolved with PBS.
In figure, it is followed successively by Ctrl (PBS), LNT (2000 μ g/mL), BSP-Old (2000 μ g/ from left to right in every column data
mL)、BSP-New(2000μg/mL)。
Fig. 2 be two kinds of distinct methods be prepared caulis bambusae in taenian polysaccharide, as positive control lentinan (LNT) to lotus knurl
The influence of mouse inflammatory cytokine secretion level.
In figure, per be followed successively by from left to right in column data Saline, LNT (50mg/kg), BSP-Old (400mg/kg),
BSP-New(400mg/kg)。
Fig. 3 be two kinds of distinct methods be prepared caulis bambusae in taenian polysaccharide, as positive control lentinan (LNT) to tumour
The scale effect of immunocyte subgroup in tumor-bearing mice peripheral blood and spleen.
In figure, per be followed successively by from left to right in column data Saline, LNT (50mg/kg), BSP-Old (400mg/kg),
BSP-New(400mg/kg)。
Fig. 4 is that the caulis bambusae in taenian polysaccharide that two kinds of distinct methods are prepared prevents experiment and Experiment on therapy tumor growth curve.
Fig. 5 is influence of the caulis bambusae in taenian polysaccharide that is prepared of two kinds of distinct methods to NK cell activity.
Specific implementation mode
It is the further explanation to the present invention below, rather than limiting the invention.
The preparation method of embodiment 1, caulis bambusae in taenian polysaccharide, follows the steps below successively：
1) steam blasting pre-processes
By the caulis bambusae in taenian of 1k g moso bamboos (Phyllostachys heterocycla var.pubescens (Mazel) Ohwi)
(bamboo shaving is crushed to 10-20 mesh, moisture 15% or so after natural air drying) is put into high density steam blasting machine (Hebi City
Right way bioenergy Co., Ltd manufacture, QBS-200B types) material cavity (5L) in, when steam pressure be 2.2MPa when, dimension pressure
For 1min, moment produces and (produces the time as 0.005075s≤T≤0.008752s).Wet stock after steam explosion is put into 80 ± 1
DEG C baking oven in dry to moisture 15% or so, convenient for preserving.
2) water carries
The caulis bambusae in taenian raw material after 50g dewaxings (caulis bambusae in taenian after the steam explosion drying i.e. obtained by step 1)) is weighed, 750mL distilled water is added,
2h is extracted in 95 DEG C of water-bath, and leaching liquor is obtained after filtering；5mg alpha-amylase (enzyme activity is added in 55-60 DEG C of filtrate
50U/mg) and 5mg neutral proteinases (100,000 U/g), with the NaOH solution system tune pH value of 1mol/L to 7.0 ± 1, in 50~55
After 20min being digested at a temperature of DEG C, enzyme deactivation (100 DEG C, 10min).
3) alcohol precipitation
Step 2) gains decompression (vacuum degree≤0.09MPa, 45-50 DEG C of temperature) is concentrated into 50mL or so, must be concentrated
Liquid；
It is slowly added to the edible alcohol (while being stirred continuously) of 4 times of volume of the concentrated liquid, stands overnight that (about 12 is small at 4 DEG C
When), precipitation is collected, 80% ethyl alcohol (about 5mL) is used in combination fully to wash.
4) Sevag methods take off albumen
After precipitation distills water dissolution with 10mL after the washing obtained by step 4), with 10mL n-butanols, chloroform mixed solution
(n-butanol：Chloroform=1：4) it mixes, is vibrated under room temperature, centrifuge (8000rpm, 15 minutes), take upper strata aqueous phase, this step repeats
Three times；
5) membrane separating and purifying
Sample (that is, the water phase after step 4 merging, about 10mL) after de- albumen is diluted to 70mL with 40 DEG C of distilled water
Afterwards, purified with membrane separation device with the experiment of German SARTORIUS Sai Duolisi companies, the miillpore filter with 0.2 μm is thick
After filter, the poly (ether sulfone) film ultrafiltration of 30KD, the poly (ether sulfone) film that permeate is again 3KD with molecular cut off is first used to be concentrated.
6) it is concentrated and dried
The trapped fluid decompression (vacuum degree≤0.09MPa, 45-50 DEG C of temperature) of 3KD obtained by step 5) is concentrated into and is contained admittedly
30% or so, it is freeze-dried (- 40 DEG C, be dried in vacuum overnight), obtains caulis bambusae in taenian polysaccharide (being named as BSP-New) about 0.48g.
It is 94.2% that BSP-New calculates its polyoses content with phend-sulphuric acid；Meanwhile it being surveyed with Coomassie brilliant G-250 method
Protein is feminine gender.
Comparative example 1, according to the clear academic dissertation of yellow chrysanthemum《The chemical constitution of caulis bambusae in taenian polysaccharide and immunocompetence research》The step of description
The rapid preparation for carrying out caulis bambusae in taenian polysaccharide, that is, for embodiment 1, make the following changes：
Cancellation step 3) in 5mg neutral proteinases use, and without Sevag method removing proteins；
" membrane separating and purifying " of step 5) is made into：Diafiltration.
Remaining is equal to embodiment 1.Obtain caulis bambusae in taenian polysaccharide (being named as BSP-Old) about 0.56g.
The comparative example 1 lacks the method for two kinds of removing proteins, causes final product purity not good enough；Membrane separation process is not used, is obtained
Polysaccharide molecular weight distribution it is relatively wide, the mode of dialysis can not control molecular cut off range well.
The ingredient comparison of the caulis bambusae in taenian polysaccharide of 1 gained of above-described embodiment 1 and comparative example is as shown in table 1.
1. new and old extraction process of table obtains the comparison of caulis bambusae in taenian 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, Hep G2,
Sk-Hep 1；Human Lung Cancer NCI-H1688, NCI-H520, A549.
(2) sample to be tested：The caulis bambusae in taenian polysaccharide (BSP-Old) of tradition extraction (that is, comparative example 1), complete medium are prepared, eventually
A concentration of 2000 μ g/mL；The caulis bambusae in taenian polysaccharide (BSP-New) of new process extraction, complete medium are prepared, final concentration of 2000 μ g/
mL；Positive drug：Lentinan (LNT), complete medium are 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 holes μ L/, in
37 DEG C, 5%CO2Being cultivated in incubator for 24 hours keeps cell adherent；
B. it sucks supernatant, the 200 μ L of culture solution containing concentration sample to be tested needed for experiment is added, in 37 DEG C, 5%CO2Training
It supports in case and cultivates 72h；
C. the 20 μ L of MTT containing 5mg/mL are added per hole, continue to cultivate 4h；
D. culture is terminated, culture solution in hole is abandoned, adds 150 holes μ L/ DMSO, be placed on shaking table and shake 10min, crystallization is made to fill
Divide dissolving, each hole light absorption value is surveyed at microplate reader 570nm.It tests while this bottom outlet (culture medium, MTT, DMSO), control wells is set
(cell, the drug solvent of same concentrations, culture solution, MTT, DMSO)；
E. it calculates：Cell survival rate (%)=(administration-background)/(control-background) × 100；
F. cell Proliferation known to analysis experimental data can play apparent inhibiting effect (see Fig. 1), and the present invention, which extracts, to be obtained
Caulis bambusae in taenian Polysaccharide B SP-New, (the comparison of lentinan LNT and traditional handicraft is significantly better than for the growth inhibition effect of cancer cell
Example 1) extract obtained caulis bambusae in taenian Polysaccharide B SP-Old.
Test immunoregulation effect of 2, the caulis bambusae in taenian polysaccharide to tumor-bearing mice
(1) cell strain：Mice model of forestomach cancer cell MFC
(2) sample to be tested：New old technology extracts caulis bambusae in taenian polysaccharide；Positive drug：Lentinan
(3) method for establishing model：
A. 48 males, 5 weeks 615 mouse of week old cleaning grade are taken, MFC cells 3 × 10 are inoculated per mouse left hind armpit6/
100 μ L cell suspensions.After inoculation for 24 hours, random point 4 groups, every group 12, using dorsal sc injection administering mode, physiological saline
Control group (Saline) presses 20 μ L/g (BW) daily, and lentinan for injection positive controls (LNT) press 50mg/kg (BW), pass
System extraction caulis bambusae in taenian polysaccharide test group (BSP-Old) presses 400mg/kg (BW), and the present invention extracts caulis bambusae in taenian polysaccharide test group (BSP-New)
By 400mg/kg (BW).Daily injection is primary, continuous two weeks, in injection drug the 14th day acquisition mouse organs, blood sample, bone
Marrow and tumor tissues；
B. mice serum inflammatory factor expression is detected using Multi-Analyte ELISArray Kit kits
(see Fig. 2)；Using flow cytometry, the immunocyte subgroup that different surfaces antigen molecule is expressed in human peripheral blood and spleen carries out
Statistical analysis (see Fig. 3)；
C. the experimental results showed that：Caulis bambusae in taenian polysaccharide promotes the secretion of Th1 types inflammatory cytokine (IFN-γ, TNF-α etc.), greatly
Quantity research is shown in long-term lotus knurl environment, is often leading with Th2 types inflammatory cytokine (IL-6, IL-10 etc.), this says
Bright caulis bambusae in taenian polysaccharide has the effect of adjusting immunologic balance；Caulis bambusae in taenian polysaccharide can be effectively increased effect immune cell population quantity (CD4
+ T cell, CD8+T cells, NK cells etc.), promote the mobilization of these cells.The above result shows that caulis bambusae in taenian polysaccharide can be effective
Activation mouse immune system.
Experiment 3, caulis bambusae in taenian polysaccharide inhibit tumour growth experiment
(1) cell strain：Mice model of forestomach cancer cell MFC
(2) sample to be tested：New old technology extracts caulis bambusae in taenian polysaccharide；Positive drug：Lentinan
(3) experimental method：
A. empirically purpose is divided into prevention group and treatment group.Prevention group：6 males, 5 weeks 615 mouse of week old cleaning grade are taken,
It is subcutaneously injected daily through mouse antedorsal by 400mg/kg (BW) new process extraction caulis bambusae in taenian polysaccharide (PreBSP-New), it is daily to inject
Once, continuous injection inoculates MFC cells 3 × 10 in back part after a week6/ 100 μ L cell suspensions；Treatment group：With prevention
Group is in same time, through dorsal sc inoculation MFC cells 3 × 106/ 100 μ L, using dorsal sc injection administering mode, physiology
20 μ L/g (BW) of brine (Sline) control group, traditional handicraft group (BSP-Old) 400mg/kg (BW) group, new process group (BSP-
New) 800mg/kg (BW) groups are in tumor volume growth to 100mm3Left and right starts to be administered, and daily injection is primary, continuous two weeks, often
Day observes and records mouse weight, life state, knurl size；
B. draw tumor growth curve according to the tumor size of record record (see Fig. 4)；
C. the experimental results showed that：The caulis bambusae in taenian polysaccharide that the present invention extracts can effectively play prevention and inhibit cancer cell
Proliferation.
Experiment 4, the adjusting of oral administration gavage caulis bambusae in taenian Polysaccharides on Mice immune system
(1) experiment mice：ICR mouse
(2) sample to be tested：New old technology extracts caulis bambusae in taenian polysaccharide；Positive drug：Lentinan
(3) experimental method：
A. 56 mouse are divided into saline control group (NC, n=8)：Isometric physiology salt was subcutaneously injected every three days
Water, the daily isometric physiological saline of gavage；Cyclophosphamide immunosupress group (CPA, n=8)：70mg was subcutaneously injected every three days
CPA/kg (BW), the daily isometric physiological saline of gavage；Lentinan positive drug group (CPA+LNT, n=8)：Every three days skins
Lower injection 70mg CPA/kg (BW), daily gavage 50mg LNT/kg (BW)；Traditional handicraft caulis bambusae in taenian polysaccharide immune activation group (CPA+
BSP-Old, n=8)：70mg CPA/kg (BW), daily gavage 100mg BSP-Old/kg (BW) was subcutaneously injected every three days；This
Invention caulis bambusae in taenian polysaccharide immune activation group (CPA+BSP-New, n=8)：70mg CPA/kg (BW) were subcutaneously injected every three days, daily
Gavage 100mg BSP-New/kg (BW)；Traditional handicraft caulis bambusae in taenian polysaccharide negative control group (BSP-Old, n=8)：Every three days skins
The lower isometric physiological saline of injection, daily gavage 200mg BSP-Old/kg (BW)；Caulis bambusae in taenian polysaccharide negative control group of the present invention
(BSP-New, n=8)：Isometric physiological saline, daily gavage 200mg BSP-New/kg (BW) were subcutaneously injected every three days.
B. last dose for 24 hours rear (the 15th day), acquires whole blood in such a way that eyeball takes blood, is collected in anticoagulant tube.It adopts
With the quantity of Hematology analyzer detection leucocyte (PWBC) and lymphocyte (PBL)：
Influences of the 2. caulis bambusae in taenian Polysaccharide B SP of table to mouse peripheral blood leucocyte 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 (normal control), Normal group；CPA (cyclophosphamide), cyclophosphamide；LNT
(lentinan), lentinan；BSP-Old, caulis bambusae in taenian polysaccharide prepared by former technique；BSP-New, the caulis bambusae in taenian that the present invention obtains are more
Sugar.Compared with NC groups, * p ＜ 0.05, * * p ＜ 0.01；Compared with CPA model groups, #p ＜ 0.05, ##p ＜ 0.01.
C. last dose for 24 hours rear (the 15th day), acquires whole blood in such a way that eyeball takes blood, is collected in anticoagulant tube.Profit
T cell, B cell, NK cell surface antigens are marked with fluorescence antibody, using flow cytometry each experimental group for statistical analysis
Cell colony quantity and percentage：
Influences of the 3. caulis bambusae in taenian Polysaccharide B SP of table to Mouse Peripheral Blood Lymphocyte subset proportions and quantity
Note：NC (normal control), Normal group；CPA (cyclophosphamide), cyclophosphamide；LNT
(lentinan), lentinan；BSP-Old, caulis bambusae in taenian polysaccharide prepared by former technique；BSP-New, the caulis bambusae in taenian that the present invention obtains are more
Sugar.Compared with NC groups, * p ＜ 0.05, * * p ＜ 0.01；Compared with CPA model groups, #p ＜ 0.05, ##p ＜ 0.01.
D. mononuclear macrophage phagocytic activity detects：After last dose 2h (the 14th day), given birth to from mouse tail vein injection
The india ink (0.1mL/10g BW) that brine dilutes 5 times is managed, injection prepared Chinese ink starts timing.After injecting prepared Chinese ink 2min and
10min takes 20 μ L of blood with hemostix from angular vein clump respectively, and is added fills 2mL 0.1%Na immediately2CO3The test tube of solution
In, with vortex instrument mixing.96 orifice plates are added in sampling, per 200 μ L of hole.With above-mentioned 0.1%Na2CO3Solution is used as blank control
Microplate reader measures OD values at 630nm wavelength.
4. mononuclear macrophage phagocytic activity of table
| 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 (normal control), Normal group；CPA (cyclophosphamide), cyclophosphamide；LNT
(lentinan), lentinan；BSP-Old, caulis bambusae in taenian polysaccharide prepared by former technique；BSP-New, the caulis bambusae in taenian that the present invention obtains are more
Sugar.Compared with NC groups, * p ＜ 0.05, * * p ＜ 0.01；Compared with CPA model groups, #p ＜ 0.05, ##p ＜ 0.01.
E.NK cell killing activities detect：Last dose for 24 hours rear (the 15th day), mouse cervical dislocation is put to death, is prepared small
Mouse lymphotactin suspension is as effector cell (5 × 106A/mL), it is 50 to make effect target ratio:1.Using lactic dehydrogenase (LDH) method
The activity for measuring spleen natural kill (NK) cell, is shown in Fig. 5.
Finally, it should also be noted that it is listed above be only the present invention several specific embodiments.Obviously, this hair
Bright to be not limited to above example, acceptable there are many deformations.Those skilled in the art can be from present disclosure
All deformations for directly exporting or associating, are considered as protection scope of the present invention.
Claims (4)
1. application of the caulis bambusae in taenian polysaccharide in preparing immunomodulator, anti-tumor agent, it is characterized in that：It is more in the caulis bambusae in taenian polysaccharide
Sugared content is >=94.2%, and protein is not contained in caulis bambusae in taenian polysaccharide；
The caulis bambusae in taenian polysaccharide is to be prepared according to the following steps：
1) steam blasting pre-processes：
The caulis bambusae in taenian of moso bamboo is subjected to steam blasting：Moment is quick-fried after 0.5~1.5min of pressurize under the steam pressure of 2~2.5Mpa
Broken, it is 0.005075s≤T≤0.008752s to produce the time；
By the caulis bambusae in taenian raw material drying after steam explosion to moisture≤15%, you can safe storage；
2) water carries：
The steam explosion raw materials treated obtained by 50g steps 1) is weighed, 700~800mL distilled water is added, in 90~100 DEG C of water-bath
1.5~2.5h of middle extraction, filtering, gained filtrate are leaching liquor；
4.5~5.5mg alpha-amylases and 4.5~5.5mg neutral proteinase tune pH value is added extremely in 55~60 DEG C of leaching liquor
7.0 ± 1, after digesting 15~20min at a temperature of 50~55 DEG C, by enzyme-deactivating；
3) alcohol precipitation：
Feed liquid obtained by step 2) is concentrated under reduced pressure into 45~55mL, obtains concentrate；
The ethyl alcohol or edible alcohol for accounting for 4 times of volumes of concentrate are added in concentrate, 10~12 hours are stood at 3~5 DEG C, collects
Precipitation, is used in combination 80% ethyl alcohol fully to be washed；
4) Sevag methods take off albumen：
After the distillation water dissolution of precipitation after the washing obtained by step 4), it is added mixed with the isometric n-butanol of distilled water, chloroform
After closing solution mixing, centrifugation takes supernatant；In triplicate；
In the mixed solution, n-butanol：The volume ratio of chloroform is 1：4；
5) membrane separating and purifying：
It takes supernatant in step 4), is added 35~45 DEG C of distilled water, the volume ratio of the supernatant and distilled water is 1：5-10 is obtained dilute
Release liquid；
Above-mentioned dilution is subjected to membrane separating and purifying：It is first filtered with 0.2 μm of miillpore filter, then with the poly (ether sulfone) film ultrafiltration of 30KD,
The poly (ether sulfone) film that gained permeate is again 3KD with molecular cut off is concentrated；
6) concentrate obtained by step 5) be concentrated under reduced pressure up to solid content >=30%, then be freeze-dried, obtain caulis bambusae in taenian
Polysaccharide.
2. application of the caulis bambusae in taenian polysaccharide according to claim 1 in preparing immunomodulator, anti-tumor agent, feature
It is：The tumour includes gastric cancer, lung cancer, liver cancer.
3. application of the caulis bambusae in taenian polysaccharide according to claim 1 or 2 in preparing immunomodulator, anti-tumor agent,
It is characterized in：
The enzyme activity of the alpha-amylase is 50U/mg；
The enzyme activity of the neutral proteinase is 100,000 U/g.
4. application of the caulis bambusae in taenian polysaccharide according to claim 3 in preparing immunomodulator, anti-tumor agent, feature
It is：
The volumetric concentration of ethyl alcohol is 93% in the edible alcohol.
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| 竹茹多糖的化学结构和免疫活性研究;黄菊青;《中国博士学位论文全文数据库医药卫生科技辑》;20151015(第10期);E057-11 * |
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