CN107029089A - The application of garlic P.E and apply its product - Google Patents
The application of garlic P.E and apply its product Download PDFInfo
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- CN107029089A CN107029089A CN201710445344.7A CN201710445344A CN107029089A CN 107029089 A CN107029089 A CN 107029089A CN 201710445344 A CN201710445344 A CN 201710445344A CN 107029089 A CN107029089 A CN 107029089A
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- 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)
- A61K36/896—Liliaceae (Lily family), e.g. daylily, plantain lily, Hyacinth or narcissus
- A61K36/8962—Allium, e.g. garden onion, leek, garlic or chives
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- 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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Abstract
The invention provides a kind of application of garlic P.E and using its product, it is related to pharmaceutical technology field, the garlic P.E that the present invention is provided, certain antitumor activity can be showed by being found through experiments that, the product prepared by application by garlic P.E, can play antineoplastic action；Meanwhile, by garlic P.E combined chemotherapy medicine collective effect in tumour cell, it can reduce chemotherapeutics usage amount by shortening action time, reduce its toxic side effect；Also, garlic is food and medicament dual-purpose plant, using medicine of the garlic as adjunct antineoplastic, while can reduce toxic and side, other adverse reactions is not produced, medication is safer.
Description
Technical field
The present invention relates to pharmaceutical technology field, a kind of application more particularly, to garlic P.E and its product is applied.
Background technology
Cancer is the general designation of a major class malignant tumour.The characteristics of cancer cell is unrestricted, hyperplasia without end, makes patient's body
Interior nutriment is largely consumed.Also, cancer cell is by discharging a variety of toxin, human body can be made to produce a series of symptoms.
Meanwhile, cancer cell also can be transferred to whole body growing multiplication everywhere, cause human body to be become thin, powerless, anaemia, poor appetite, heating with
And serious organ function is impaired etc..
Wherein, hepatocellular carcinoma (HCC) is most common Primary Hepatic tumor, is the main cause of global cancer mortality.
Its development is relevant with many factors and condition, including chronic hepatitis B, hepatitis C, virus infection, chronic intake alcohol,
Carcinogen or hepatic sclerosis in food.At present, treatment liver cancer most common method is complex treatment, i.e. the combination of Chinese tradiational and Western medicine is controlled
Treat operation, radiotherapy, chemotherapy and traditional Chinese medicine chemotherapy.
Cis-platinum (DDP) is a kind of most extensive antineoplastic, and immunoblotting assay shows that cis-platinum can lower anti-apoptotic proteins
Bcl-2 and raise pro apoptotic protein Bax.However, because its drug resistance and toxic side effect, such as renal toxicity, peripheral nerve toxicity
With the side effect such as ototoxicity, considerable distress is brought to patient, and it is also larger to the immune system damage of patient's body, cause cis-platinum
Use be severely limited, cause it not enough with distribution in the concentration of non-specific systemic organs and intra-tumor, do not reach
Treat the optimal drug concentration of disease.
Polysaccharide is macromolecular carbohydrate, has not only played important function in the growing of organism, and is also weight
The BRM wanted.In recent years, many polysaccharide are separated from natural resources, and they play in cancer is resisted
Important function, with extensive biological characteristics.Garlic polysaccharide (GPS) in garlic P.E is the big of Liliaceae families
The main active of garlic.As a kind of heteroglycan, garlic polysaccharide by levulan and a small amount of glucose group into.Research shows big
Garlic polysaccharide has various active, such as strong anti-oxidation, antiviral, protects cuticula uvioresistant, and protection liver and enhancing, which are immunized, lives
Property etc..
Therefore, a kind of toxic side effect is developed small, having for infantile tumour Apoptosis can be induced simultaneously by suppressing tumour growth
The medicines resistant to liver cancer of effect is particularly important.At present, by the use of garlic P.E as the efficient adjunct antineoplastic drug candidate of low toxicity still
Have no report.
In view of this, it is special to propose the present invention.
The content of the invention
First purpose of the present invention is that providing garlic P.E is preparing the product of reduction toxic and side
In application；
Second object of the present invention is to provide a kind of for reducing the medicine of toxic and side；
Third object of the present invention is to provide a kind of application of garlic P.E in antineoplastic product is prepared；With
Alleviate toxic and side present in prior art greatly, not yet find the drug candidate of the efficient adjunct antineoplastic of low toxicity
Technical problem.
The invention provides a kind of application of garlic P.E, the application includes the garlic P.E and is preparing reduction
Application in the product of toxic and side and the application in antineoplastic product is prepared.
Further, the garlic P.E is garlic polysaccharide.
Further, the toxic side effect includes the one or more in renal toxicity, ototoxicity and neurotoxicity.
Further, the product is medicine.
Further, the tumour is liver cancer.
Further, the liver cancer includes the one or more in simple form, constrictive type and inflammatory type.
It is used to reduce the medicine of toxic and side present invention also offers a kind of, the medicine includes above-mentioned big
Garlic extract.
In addition, being used for anti-tumor drug present invention also offers one kind, the medicine includes above-mentioned garlic P.E.
Further, the medicine also includes pharmaceutically acceptable supporting agent or auxiliary material.
Further, the chemotherapeutics is cis-platinum.
The garlic P.E that the present invention is provided, certain antitumor activity can be showed by being found through experiments that, pass through application
The product prepared by garlic P.E, can play antineoplastic action；Meanwhile, garlic P.E combined chemotherapy medicine is common
Tumour cell is acted on, can be reduced chemotherapeutics usage amount by shortening action time, reduce its toxic side effect；Also, it is big
Garlic is food and medicament dual-purpose plant, using medicine of the garlic as adjunct antineoplastic, can reduce the same of toxic and side
When, other adverse reactions are not produced, and medication is safer.
Brief description of the drawings
, below will be to specific in order to illustrate more clearly of the specific embodiment of the invention or technical scheme of the prior art
The accompanying drawing used required in embodiment or description of the prior art is briefly described, it should be apparent that, in describing below
Accompanying drawing is some embodiments of the present invention, for those of ordinary skill in the art, before creative work is not paid
Put, other accompanying drawings can also be obtained according to these accompanying drawings.
Fig. 1 is the elution curve that the application Q-Sepharose anion exchange chromatography that the embodiment of the present invention 1 is provided is measured
Result figure；
Fig. 2 is the elution curve that the application Sephadex G-50 gel permeation chromatographic columns that the embodiment of the present invention 2 is provided are measured
Result figure；
Fig. 3 is the result for the inhibitory action that the garlic polysaccharide and cis-platinum that the embodiment of the present invention 5 is provided are bred to HepG2 cells
Figure；
Fig. 4 A are to be inverted fluorescence after the blank control group cell that the embodiment of the present invention 6 is provided is dyed with Hoechst 33342
Microscopy results figure；
Fig. 4 B are to be fallen after the 1mg/mL garlic polysaccharide group cells that the embodiment of the present invention 6 is provided are dyed with Hoechst 33342
Put fluorescence microscope result figure；
Fig. 4 C are that the 1mg/mL garlic polysaccharides and 5 μ g/mL cis-platinum collective effect groups cells that the embodiment of the present invention 6 is provided are used
Inverted fluorescence microscope observation result figure after Hoechst 33342 is dyed；
Fig. 5 A are the blank control group HepG2 cell line cell cycle distributed mutually result figures that the embodiment of the present invention 7 is provided；
Fig. 5 B be the embodiment of the present invention 7 provide that 0.5 48 hours HepG2 cell lines of μ g/mL cisplatin effects are used alone is thin
Born of the same parents' cycle phase distribution results figure；
Fig. 5 C are that the 0.25mg/mL garlic polysaccharides that are used alone that the embodiment of the present invention 7 is provided act on 48 hours HepG2 cells
Strain cell cycle distributed mutually result figure；
Fig. 5 D are the use in conjunction 0.25mg/mL garlic polysaccharides and 0.5 μ g/mL cisplatin effects that the embodiment of the present invention 7 is provided
48 hours HepG2 cell line cell cycle distributed mutually result figures；
Fig. 5 E are that the 0.5mg/mL garlic polysaccharides that are used alone that the embodiment of the present invention 7 is provided act on 48 hours HepG2 cells
Strain cell cycle distributed mutually result figure；
Fig. 5 F are the use in conjunction 0.5mg/mL garlic polysaccharides and 0.5 μ g/mL cisplatin effects 48 that the embodiment of the present invention 7 is provided
Hour HepG2 cell line cell cycle distributed mutually result figures；
Fig. 5 G are that the 1.0mg/mL garlic polysaccharides that are used alone that the embodiment of the present invention 7 is provided act on 48 hours HepG2 cells
Strain cell cycle distributed mutually result figure；
Fig. 5 H are the use in conjunction 1.0mg/mL garlic polysaccharides and 0.5 μ g/mL cisplatin effects 48 that the embodiment of the present invention 7 is provided
Hour HepG2 cell line cell cycle distributed mutually result figures；
Fig. 6 A are the blank control group HepG2 cell line Apoptosis situation result figures that the embodiment of the present invention 8 is provided；
For what the embodiment of the present invention 8 was provided 0.5 μ g/mL cisplatin effects are used alone 48 hours to HepG2 cell lines in Fig. 6 B
The influence result figure of Apoptosis；
Fig. 6 C be the embodiment of the present invention 8 provide that 5 μ g/mL cisplatin effects are used alone is 48 hours thin to HepG2 cell lines
The influence result figure of born of the same parents' apoptosis；
Fig. 6 D are that the 0.25mg/mL garlic polysaccharides that are used alone that the embodiment of the present invention 8 is provided act on 48 hours thin to HepG2
The influence result figure of born of the same parents' strain Apoptosis；
Fig. 6 E are that the 0.5mg/mL garlic polysaccharides that are used alone that the embodiment of the present invention 8 is provided act on 48 hours thin to HepG2
The influence result figure of born of the same parents' strain Apoptosis；
Fig. 6 F are that the 1.0mg/mL garlic polysaccharides that are used alone that the embodiment of the present invention 8 is provided act on 48 hours thin to HepG2
The influence result figure of born of the same parents' strain Apoptosis；
Fig. 6 G are the use in conjunction 0.25mg/mL garlic polysaccharides and 0.5 μ g/mL cisplatin effects that the embodiment of the present invention 8 is provided
48 hours influence result figures to HepG2 cell line Apoptosis；
Fig. 6 H are the use in conjunction 0.5mg/mL garlic polysaccharides and 0.5 μ g/mL cisplatin effects 48 that the embodiment of the present invention 8 is provided
Influence result figure of the hour to HepG2 cell line Apoptosis；
Fig. 6 I are the use in conjunction 1.0mg/mL garlic polysaccharides and 0.5 μ g/mL cisplatin effects 48 that the embodiment of the present invention 8 is provided
Influence result figure of the hour to HepG2 cell line Apoptosis；
Fig. 7 is that be used alone garlic polysaccharide and the garlic polysaccharide that the embodiment of the present invention 8 is provided are acted on cisplatin combined application
48 hours influence result figures to HepG2 cell line apoptosis rates.
Embodiment
Technical scheme is clearly and completely described below in conjunction with accompanying drawing, it is clear that described implementation
Example is a part of embodiment of the invention, rather than whole embodiments.Based on the embodiment in the present invention, ordinary skill
The every other embodiment that personnel are obtained under the premise of creative work is not made, belongs to the scope of protection of the invention.
In tumor therapeutic procedure, it will usually use broad-spectrum anti-cancer drug, such as cis-platinum.But the use of such medicine is past
Toward with serious toxicity, including the side effect such as renal toxicity, peripheral nerve toxicity and ototoxicity, bring very big pain to patient
Hardship, it is also larger to the damage of the immune system of patient's body, cause it using being severely limited, in non-specific systemic organs and
The concentration of intra-tumor is not enough with distribution, does not reach the optimal drug concentration for the treatment of disease.
Therefore, in view of the above-mentioned problems, preparing drop the invention provides the application of garlic P.E, including garlic P.E
Application in the product of low toxic and side and the application in antineoplastic product is prepared.
In the present invention, garlic P.E is garlic polysaccharide.
In the present invention, toxic side effect includes the one or more in renal toxicity, ototoxicity and neurotoxicity.
In the present invention, product is medicine.
In the present invention, tumour is liver cancer.
In the present invention, liver cancer includes the one or more in simple form, constrictive type and inflammatory type.
It is used to reduce the medicine of toxic and side present invention also offers a kind of, including above-mentioned garlic is extracted
Thing.
In addition, being used for anti-tumor drug, including above-mentioned garlic P.E present invention also offers one kind.
In the present invention, medicine also includes pharmaceutically acceptable supporting agent or auxiliary material.
Wherein, supporting agent for example can be, but one kind for being not limited in chitosan, cholesterol, liposome and nano particle or
It is a variety of；Auxiliary material for example can be, but be not limited to dextrin, starch or sucrose.
In the present invention, the formulation of medicine for example can be, but be not limited to tablet, capsule or electuary.
In the present invention, chemotherapeutics is cis-platinum.
The garlic P.E that the present invention is provided, certain antitumor activity can be showed by being found through experiments that, pass through application
The product prepared by garlic P.E, can play antineoplastic action；Meanwhile, garlic P.E combined chemotherapy medicine is common
Tumour cell is acted on, can be reduced chemotherapeutics usage amount by shortening action time, reduce its toxic side effect；Also, it is big
Garlic is food and medicament dual-purpose plant, using medicine of the garlic as adjunct antineoplastic, can reduce the same of toxic and side
When, other adverse reactions are not produced, and medication is safer.
In order to contribute to it is clearer understand present disclosure, be described in detail as follows in conjunction with specific embodiment.Such as
Not yet explicitly point out, the white garlic used in following examples is commercially available 200 gram；HepG2 cells are purchased from China Concord Medical Science University
Cell resource center；MTT is purchased from Sigma；Cis-platinum is purchased from Hao Sen pharmaceutcal corporation, Ltds；Culture medium (MEM) and hyclone are purchased
From Gibco；Nonessential amino acid solution 100 ×, trypsin-EDTA solutions 1 × (trypsase) is purchased from Gibco；Iodate third
Pyridine (PI) kit, the staining kit of Annexin V-FITC and Hoechst 33342, which are purchased from Nanjing and build up bioengineering, to be ground
Study carefully institute；Other reagents are the pure rank of analysis.
The extracting and developing of the garlic polysaccharide of embodiment 1 and purifying
Garlic peeling is ground.Garlic juice plus 4 times of 80 DEG C of volume of water after grinding are extracted 3 hours, then with 200 mesh gauzes
Filtering.Water of the filter residue again through 4 times of volumes is extracted 2 hours at 80 DEG C, filters to take supernatant again afterwards, merges supernatant.Supernatant
Concentrated with Rotary Evaporators, the pre-cooled ethanol for 95% (v/v) for adding 3 times of volumes after concentrate cooling is precipitated, and will be precipitated
Thing washs three times with 95% (v/v) ethanol and freezed.Using the protein in Sevage method Polysaccharide removings, do again afterwards
It is dry, obtain Thick many candies.
5g Thick many candies are taken, are re-dissolved in 100mL distilled water, and utilize Q-Sepharose Fast Flow chromatographic columns
(20mm ID × 350mm, Pharmacia Corp), with distilled water and the NaCl of 0.1-0.5M concentration gradients 1mL/min flow velocity
It is lower to be eluted.Detected using phend-sulphuric acid and elute the high cut (peak value 1) of sugared content and collect, dialyse, freeze, obtained greatly
Garlic polysaccharide sterling.Using Sephadex G-50 (26mm ID × 1000mm) gel permeation chromatography, existed with 0.1M NaCl
Purity checking is carried out to the garlic polysaccharide obtained in previous step under 0.5mL/min flow velocity, it is single symmetrical to obtain eluting peak
Peak, illustrates that garlic polysaccharide prepared by previous step is sterling.
In this experiment, 26.06 grams of white garlic separation garlic Thick many candies yield, yield is 13.08%.Thick many candies are through Q-
Sepharose Fast Flow chromatographic purifyings.Key component verified by gel permeation chromatography purity, elution curve such as Fig. 1 institutes
Show.
The garlic polysaccharide weight average molecular weight of embodiment 2
Garlic is determined using High Performance Gel Permeation Chromatography (HPGPC), and using liquid chromatogram instrument (Agilent, the U.S.)
The weight average molecular weight of polysaccharide.Wherein, the posts of PL aquagel-OH 30 (7.5mm ID × 300mm, 8 μm) maintain 25 DEG C, flowing
It is mutually 0.02M sodium nitrate (NaNO3) and 0.01M sodium dihydrogen phosphate (NaOH, pH7.0), flow is 0.6mL/min, and is led to
Cross the detection of RID-10A detectors.2mg samples are dissolved in mobile phase, 0.45 μm of filter membrane is crossed.The weight average molecular weight of garlic polysaccharide
Standard curve using serial dextran standard (T-10, T-40, T-70, the T-500 and T-2000) draftings of Dextran T- is ginseng
Examine and be estimated.
By observing single symmetrical peak in Sephadex G-50, it is homogeneous polysaccharide to illustrate garlic polysaccharide, such as Fig. 2 institutes
Show.The weight average molecular weight of garlic polysaccharide is detected using high productivity computing method, according to calibration curve and dextran standards, obtains big
The weight average molecular weight of garlic polysaccharide is about 2714.94Da.
The garlic polysaccharide monosaccharide composition analysis of embodiment 3
The identification of monose is with quantitatively using PMP pre-column derivatizations and high-performance liquid chromatography analysis in garlic polysaccharide.Garlic
Polysaccharide is hydrolyzed 2 hours with 2M TFA at 100 DEG C, and is converted into described alditol acetate.Resulting alditol acetate
It is high using Agilent 1280 by UV-detector (245nm) and chromatography capillary tubing string (4.6mm internal diameters × 250mm, 5 μm)
Effect liquid phase chromatogram analyzer is analyzed.Wherein, the temperature of chromatography capillary tubing string is maintained at 25 DEG C, and sample amount of refraction is 10 μ L.
Mobile phase is through potassium dihydrogen phosphate：Acetonitrile (83:17, v/v) 0.05M of dilution KH2PO4- NaOH buffer solutions, flow velocity is 1mL/
min.With mannose, rhamnose, glucuronic acid, galacturonic acid, glucose, galactolipin, xylose, arabinose and fucose
Analyzed for standard.
Gas chromatographic analysis shows, it by relative molar mass ratio is 4.6 that garlic polysaccharide, which is,：0.8：0.9：0.5 glucose
(Glc), galactolipin (Gal), xylose (Xyl) and arabinose (Ara) composition.
The cell culture of embodiment 4
Human hepatoma cell line HepG2's cell using MEM culture mediums (containing 10% hyclone, 100U/mL penicillin and
100U/mL streptomysin), in 37 DEG C, 5%CO2Cultivated in the cell culture incubator of/95% air.
The antiproliferative activity of embodiment 5
Detect garlic polysaccharide to HepG2 cell growth inhibitions using mtt assay.HepG2 cells are inoculated in 96 orifice plates,
When cell fusion degree reaches 80%, 100 μ g/mL, 250 μ g/mL and 500 μ g/mL garlic polysaccharide are separately added into, 5 μ g/mL's
Cis-platinum and garlic polysaccharide and cisplatin combined medication, therapeutic regimen are the μ g/mL of garlic polysaccharide 100,250 μ g/mL, g/mL points of 500 μ
24 hours and 48 hours are not incubated respectively under the conditions of 37 DEG C with 5 μ g/mL cis-platinums collective effects in human hepatoma cell line HepG2.
After the completion of incubation, 20 μ L MTT (5mg/mL) are added per hole, 37 DEG C are incubated 4 hours.Afterwards, discard cell culture fluid and add per hole
Enter 150 μ L DMSO.After purple crystal dissolving fully, the ELIASA (PerkinElmer, the U.S.) for setting wavelength as 490nm is examined
Survey.
In this experiment, as shown in figure 3, garlic polysaccharide can suppress human hepatoma cell line HepG2's in the range of finite concentration
Propagation, and in dose dependent.Series concentration garlic polysaccharide effect tumour cell 24 hours, when garlic polysaccharide concentration is 100 μ g/
During mL, inhibiting rate is 7.85%；When garlic polysaccharide concentration is 250 μ g/mL, inhibiting rate is 13.37%；When garlic polysaccharide concentration
During for 500 μ g/mL, inhibiting rate is 19.52%；Effect 48 hours, when garlic polysaccharide concentration is 100 μ g/mL, inhibiting rate is
14.47%；When garlic polysaccharide concentration is 250 μ g/mL, inhibiting rate is 22.38%；When garlic polysaccharide concentration is 500 μ g/mL
When, inhibiting rate is 26.18%.5 inhibiting rates of μ g/mL cisplatin effects tumour cell 24 hours are 13.68%, and inhibiting rate is within 48 hours
52.93%.For garlic polysaccharide and cisplatin combined medication, to the inhibiting rate of tumour cell apparently higher than independent medication.100μg/
+ 5 μ g/mL cisplatin effects of mL garlic polysaccharides 24 hours, its proliferation inhibition rate is 52.04%；The μ g/mL of 500 μ g/mL garlic polysaccharides+5
Cisplatin effect 24 hours, proliferation inhibition rate is up to 64.47%.Drug combination is far above cis-platinum list to the inhibiting rate of tumour cell
Medicine acts on the inhibiting rate of 48 hours 42.93%.It can be seen that garlic polysaccharide can help to increase that the antitumor activity of cis-platinum, and can be by subtracting
Few administration time reduces chemotherapeutics usage amount indirectly, reduces its toxic side effect.
HepG2 apoptosis morphologies credit analysis after the garlic polysaccharide of embodiment 6 and cisplatin induction
HepG2 cells are inoculated in 6 orifice plates, when cell fusion degree reaches 80%, 1.0mg/mL garlic are separately added into
The drug combination of polysaccharide and garlic polysaccharide and cis-platinum, therapeutic regimen is that 1mg/mL garlic polysaccharides are made jointly with 0.5 μ g/mL cis-platinums
With incubation 48 hours under the conditions of 37 DEG C.And set only plus MEM culture mediums HepG2 cells as blank control group.Afterwards, collect
Cell is simultaneously incubated 15 minutes by Hoechst 33342 under the conditions of 37 DEG C.Seen under fluorescence microscope (Nikon TS100, Japan)
Examine cell and take pictures (400 ×).
The chromosomal DNA structure of apoptotic cell changes, and dyestuff is combined with DNA highly efficient, enhances apoptosis thin
The blue-fluorescence of born of the same parents.In this experiment, as shown in Figure 4 A, the visible uniform blue elliptical erythrocyte core of blank control group, has no bright
Aobvious apoptosis.However, by combining Hoechst 33342, the visible heavier blue-fluorescence of experimental group of 1mg/mL garlic polysaccharides is such as schemed
Shown in 4B；1mg/mL garlic polysaccharides combine the experimental group of 0.5 μ g/mL cis-platinums compared with the experimental group of 1mg/mL garlic polysaccharides, show
Heavier blue-fluorescence is shown, as shown in Figure 4 C.The coloration results of Hoechst 33342 are shown, are apoptosis-induced by garlic polysaccharide
Approach realizes the apoptosis of induction HepG2 cells.Cis-platinum is a kind of antineoplastic alkylating agent thing, by making DNA is imperfect to produce carefully
Cellular toxicity, is used as a kind of nonspecific cell cycle medicine, main interference DNA duplication.By inducing cell apoptosis approach,
Its antitumor action is also confirmed, also, the garlic polysaccharide provided by embodiment 1 can cooperate with cisplatin induction HepG2
Apoptosis.
The cell cycle distribution of embodiment 7 is determined
HepG2 is inoculated in 6 orifice plates, inoculum density is 1 × 105Individual/hole, using MEM culture mediums, in 37 DEG C, 5%CO2/
Cultivated in the cell culture incubator of 95% air, 80% is reached to cell fusion degree.Afterwards, be separately added into 0.25mg/mL,
0.5mg/mL and 1.0mg/mL garlic polysaccharide, 0.5 μ g/mL cis-platinum and the drug combination of garlic polysaccharide and cis-platinum, medication
Scheme is garlic polysaccharide 0.25mg/mL, 0.5mg/mL and 1.0mg/mL respectively with 0.5 μ g/mL cis-platinum collective effects in human liver cancer
Cell line HepG2, wherein, be used alone 0.5 μ g/mL cis-platinums for positive controls, and set only plus MEM culture mediums a HepG2
Cell is blank control group, is incubated 48 hours under the conditions of 37 DEG C.Centrifugal process collects cell, PBS after Trypsin Induced
After twice, it is fixed, and is stayed overnight in 4 DEG C of storages with 70% alcohol.Cell after fixation is added after PBS to be contained
The PI solution of 0.05% ribalgilase, 37 DEG C are incubated 30 minutes.Afterwards, 5 μ L concentration are added under dark condition in room temperature is
5mg/mL PI solution.After 30 minutes, cell is analyzed by flow cytometer (Beckman Coulter Inc., the U.S.).Often
Group sets three repetitions.Flow Cytometry Assay DNA content.It is compared by the cell cycle with blank control group cell,
The cell cycle of medication experimental group is evaluated.
Acted on alone or in combination after 48 hours with PI staining evaluations garlic polysaccharide and cis-platinum, the cell cycle point of tumour cell
Cloth.As a result it is as shown in table 1.
The influence of the garlic polysaccharide of table 1 and cis-platinum to the HepG2 cell cycles
Every kind of medicine independent role reduces G1 phase cell numbers, such as shown in Fig. 5 B, 5C, 5E and 5G, garlic polysaccharide in 48 hours
Processing can increase G2 or M phase cell proportions, and cis-platinum can dramatically increase the cell proportion of S phases in the cell cycle.And combine and use
Medicine group S phases and the increase of G2 phases cell proportion, such as shown in Fig. 5 D, 5F and 5H.Blank control group is as shown in Figure 5A.Therefore, connection is illustrated
Sharing medicine and significantly inhibiting growth of tumour cell is realized by extending and preventing cell in the propagation of S and G2 phases.
The apoptosis of embodiment 8 is detected
Influence of the medicine to Apoptosis is determined, is withered using Annexin V-FITC kits and PI dyeing detection cells
Die.Annexin V-FITC and PI coloring agent can enter the incomplete cell of cell membrane, and by detecting the phosphorus on cell membrane
The cell of acyl serine, detection apoptosis or necrosis.HepG2 is inoculated in 6 orifice plates, inoculum density is 1 × 105Individual/hole, is utilized
MEM culture mediums, in 37 DEG C, 5%CO2Cultivated in the cell culture incubator of/95% air.When cell fusion degree reaches 80%
When, it is separately added into 0.25mg/mL, 0.5mg/mL and 1.0mg/mL garlic polysaccharide, 0.5 μ g/mL cis-platinum and garlic polysaccharide
With the drug combination of cis-platinum, therapeutic regimen be garlic polysaccharide 0.25mg/mL, 0.5mg/mL and 1.0mg/mL respectively with 0.5 μ g/mL
Cis-platinum collective effect in human hepatoma cell line HepG2, wherein, be used alone 0.5 μ g/mL cis-platinums for positive controls, and set
The HepG2 cells for only adding MEM culture mediums are blank control group, are incubated 48 hours under the conditions of 37 DEG C.Afterwards, cell and 5 μ L
Annexin V-FITC and 5 μ L PI are incubated altogether, and are entered by flow cytometer (California, Beckman Coulter Inc.)
Row analysis.Three repetitions of every group of setting.Apoptosis rate (%)=apoptosis cell/total cell × 100%.
According to dyeing, normal cell is Annexin V (-)/PI (-)；Viable apoptotic cell is Annexin V (+)/PI
(-)；Non-viable apoptotic cell Annexin V (+)/PI (+), non-viable non-apoptotic cell is Annexin V (-)/PI (+).Flow cytometer point
Analysis shows that individually processing can induce HepG2 Apoptosis in 48 hours to every group of medicine, as shown in Fig. 6 B, 6C, 6D, 6E and 6F.Connection
Sharing medicine also increases the early and late apoptosis rate of tumour cell, as shown in Fig. 6 G, 6H and 6I.Blank control group is as shown in Figure 6A.
The apoptosis rate (74.1%, 76.7% and 79.3%) induced by garlic polysaccharide combination with cisplatin, which is all remarkably higher than, to be used alone
0.5 μ g/mL cis-platinums (50.3%) or the apoptosis rate that garlic polysaccharide (49%, 56.8%, 63.8%) is used alone, as shown in Figure 7
(p ＜ 0.05).Wherein, garlic polysaccharide combines influence of the 0.5 μ g/mL cis-platinums medications to apoptosis rate close to 5 μ g/mL cis-platinums
Individually effect.Prompting garlic polysaccharide can reduce the consumption of chemotherapeutics 90%, and reach identical antitumous effect.Due to
Cis-platinum can interfere with DNA duplication, and with various kinds of cell toxicity, serious toxic side effect can be produced, therefore, with other
Combination therapies are a kind of increase curative effects but do not increase the good method of adverse reaction.This experiment as a result, it was confirmed that being used as low toxicity
Natural macromolecular levulan composition garlic polysaccharide, can dramatically increase anti-proliferative capacity of the cis-platinum to HepG2 cells, and in when
Between and dose dependent.
As can be seen that the garlic P.E of the invention provided is big in the experimental result of embodiment 1 to 8 provided by the present invention
Garlic polysaccharide, can show certain antitumor activity, by garlic polysaccharide combined chemotherapy drugs Cisplatin collective effect in tumour cell,
Cis-platinum usage amount can be reduced, so as to reduce its toxic side effect by shortening action time；Also, garlic is planted for food and medicament dual-purpose
Thing, using medicine of the garlic as adjunct antineoplastic, while can reduce toxic and side, other are not produced not
Good reaction, medication is safer.
Finally it should be noted that：Various embodiments above is merely illustrative of the technical solution of the present invention, rather than its limitations；To the greatest extent
The present invention is described in detail with reference to foregoing embodiments for pipe, it will be understood by those within the art that：Its according to
The technical scheme described in foregoing embodiments can so be modified, or which part or all technical characteristic are entered
Row equivalent substitution；And these modifications or replacement, the essence of appropriate technical solution is departed from various embodiments of the present invention technology
The scope of scheme.
Claims (10)
1. a kind of application of garlic P.E, it is characterised in that the application includes the garlic P.E and preparing reductionization
Treat the application in the product of poisonous side effect of medicine and the application in antineoplastic product is prepared.
2. application according to claim 1, it is characterised in that the garlic P.E is garlic polysaccharide.
3. application according to claim 1, it is characterised in that the toxic side effect includes renal toxicity, ototoxicity and nerve
One or more in toxicity.
4. application according to claim 1, it is characterised in that the product is medicine.
5. application according to claim 1, it is characterised in that the tumour is liver cancer.
6. application according to claim 5, it is characterised in that the liver cancer is included in simple form, constrictive type and inflammatory type
One or more.
7. a kind of be used to reduce the medicine of toxic and side, it is characterised in that the medicine is included described in claim 2
Garlic P.E.
8. one kind is used for anti-tumor drug, it is characterised in that the medicine includes the garlic P.E described in claim 2.
9. the medicine according to claim 7 or 8, it is characterised in that the medicine also includes pharmaceutically acceptable supporting agent
Or auxiliary material.
10. the medicine described in application according to claim 1 and claim 7, it is characterised in that the chemotherapeutics is
Cis-platinum.
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| CN201710445344.7A CN107029089A (en) | 2017-06-13 | 2017-06-13 | The application of garlic P.E and apply its product |
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Cited By (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN116509888A (en) * | 2023-04-28 | 2023-08-01 | 大连医科大学 | Application of garlic polysaccharide in preparation of medicine for treating intestinal flora disorder caused by acute lung injury |
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| Publication number | Priority date | Publication date | Assignee | Title |
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| CN102552644A (en) * | 2010-12-31 | 2012-07-11 | 中国中医科学院中医基础理论研究所 | Anti-tumor use, preparation method and composition of garlic total polysaccharide |
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Cited By (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN116509888A (en) * | 2023-04-28 | 2023-08-01 | 大连医科大学 | Application of garlic polysaccharide in preparation of medicine for treating intestinal flora disorder caused by acute lung injury |
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Application publication date: 20170811 |