CN101612180B - Preparation method of polysaccharide extract of oleander and medical application of the polysaccharide extract - Google Patents
Preparation method of polysaccharide extract of oleander and medical application of the polysaccharide extract Download PDFInfo
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Abstract
The invention relates to a preparation method of polysaccharide extract of oleander and an application of the polysaccharide extract in preparing antitumor drugs. The preparation method comprises the following steps of: washing and drying fresh leaves of the oleander, drying and grinding; using alcohol to reflow and remove color, taking filter residue hot water, extracting and taking a supernatant; concentrating the supernatant, centrifuging, removing precipitates, putting the supernatant into a dialysis bag so as to further remove small molecular components in the solution and obtaining the polysaccharide extract of oleander; adding alcohol, carrying out centrifugation and precipitation, after carrying out precipitation for three times, freezing and drying and obtaining crude polysaccharide (NP) extract powder of the oleander. The extract can be applied to preparing drug composites for treating animals and tumor diseases of the body, including the composition of the extract and other drugs, drug carriers or excipients for preparing the drug for treating animals and tumor diseases of the body. The two types of single-component polysaccharide obtained by separation and purification in the extract can directly restrict human tumor cell proliferation and enhance apoptosis so as to show the direct kill function to the tumor cells.
Description
Technical field
The present invention relates to a kind of Folium seu Cortex Nerii polysaccharide (NP) preparation method of extract and medical usage thereof, specifically, relate to a kind of Folium seu Cortex Nerii polysaccharide (NP) preparation method of extract and this polyoses extract purposes aspect the preparation antitumor drug.
Background technology
Folium seu Cortex Nerii (Nerium indicum Mill.) belongs to Apocynaceae Alstonia plant, is the common a kind of ornamental plant of China.Leaf and peel of stem contain a large amount of cardenolides, are used as medicine to fry in shallow oil soup or grind into powder, can the heart tonifying diuresis, and the Dingchuan town tries out in heart failure, the cough of panting, epilepsy, traumatic injury swell and ache etc.Also be used to prepare the pesticide of oncomelania extremely.
Antitumor action about the Alstonia plant has United States Patent (USP) 5135745 " extract sof Nerium species; methods of preparation; and use therefore " at present, antitumous effect of a kind of apocynaceae plant Folium seu Cortex Nerii oleanderis (Neriumoleander) extract and preparation method thereof has been described, by vegetable material being boiled the extract on cell proliferation disease that obtained in several hours mitigation is arranged, also have the cardenolide in the report Folium seu Cortex Nerii oleanderis can suppress tumor cell proliferation promotion apoptosis of tumor cells.Also there is report Folium seu Cortex Nerii oleanderis (Nerium oleander) extract can induce the generation of γ-IFN, thereby plays antiviral effect (Chinese patent application number 200410058927.7).(research of (Nerium indicumMill.) extract antineoplastic is reported and there is no Folium seu Cortex Nerii both at home and abroad.
Vegetable polysaccharides has the adjusting human body immune function, has antiviral, antibiotic, parasiticide, antitumor, radioprotective, anti-ageing many-sided biological activity of waiting for a long time, thereby more and more is subjected to the attention of researcher.In recent years, a large amount of studies show that herbal polysaccharide has antitumor action, and toxic and side effects is little, thereby has become the focus of antitumor research.The little polyose medicaments of a collection of steady quality, determined curative effect, toxicity and untoward reaction such as lentinan, Radix Angelicae Sinensis polysaccharide, polyporusum bellatus, krestin have been widely used in clinical as antitumor drug.
Summary of the invention
The object of the present invention is to provide a kind of Folium seu Cortex Nerii ((Nerium indicum Mill.) polysaccharide (NP) preparation method of extract.
Another object of the present invention provides the application of this Folium seu Cortex Nerii polysaccharide (NP) extract in the medicine of preparation treatment humans and animals neoplastic disease.
The invention still further relates to this Folium seu Cortex Nerii polysaccharide (NP) extract as the application in the pharmaceutical composition of preparation treatment animal and human's tumor, comprise that the combination of this extract and other medicines, pharmaceutical carrier or excipient is used to prepare the medicine of treatment animal and human neoplastic disease.
For the extracting method of realizing the said Folium seu Cortex Nerii polysaccharide of the present invention (NP) extract by the following technical solutions:
(1) gather the bright leaf of Folium seu Cortex Nerii, washing is clean, dries moisture, 50～65 ℃ of oven dry, and the pulverizing back is crossed 60 mesh sieves and is made the sweetscented oleander leaf powder.
(2) reflux 3 times down at 60 ℃ with 70% ethanol, each 3h removes sweetscented oleander leaf powder pigment and oils and fats, crosses the leaching filtering residue, volatilizes solvent.
(3) get the filtering residue that volatilizes behind the solvent, by filtering residue: water is volume ratio extracting in 60～99 ℃ of hot water of 1: 6～10, each 4h, repeat extracting 3 times after, centrifugal or sucking filtration removes slag, and gets supernatant; The preferred temperature of extracting is 95 ℃ in the hot water.
(4) adopt the concentrated supernatant that obtains by step (3) of rotary evaporator to 1/2～1/4 of original volume, obtain concentrated solution; Concentrated solution adds 10% trichloroacetic acid to pH=4.0,4 ℃ of refrigerators are placed behind the 24h with the centrifugal 15min of 8000r/min, abandon precipitation, getting supernatant again transfers the pH value of solution to neutral with the NaOH liquid of 0.5mol/L, adopt the albumen in a kind of or this several method coupling removal concentrated solution in sevage method, TCA method, the enzyme process, get supernatant again.
(5) will remove the proteic supernatant bag filter dialysis 3d that packs into, wherein first flowing water dialysis 2d, back distill water dialysis 1d, the micromolecule composition of further removing in the solution obtains the clean extract of Folium seu Cortex Nerii polysaccharide.
(6) 95% ethanol of 3 times of volumes of adding in the clean extract of Folium seu Cortex Nerii polysaccharide that obtains, under 4 ℃, after leaving standstill 1d, with the centrifugal 15min of 8000r/min, the collecting precipitation thing, after this precipitate washed three times with dehydrated alcohol, acetone and ether successively, lyophilization promptly got the said Folium seu Cortex Nerii crude polysaccharides of the present invention (NP) extract powder.
(7), add the 1ml distilled water with 100mg and make injection with Folium seu Cortex Nerii crude polysaccharides (NP) extract powder; Divide three grades of dosed administrations, the first order: 50mg/kg, the second level: 100mg/kg according to the go down to posterity body weight (kg) of the well-grown S180 tumor-bearing mice of 7d of abdominal cavity, the third level: 150mg/kg group, every group of 10 S180 tumor-bearing mices, every day intraperitoneal injection, behind the successive administration 10d, weigh next day; Mice is put to death in cervical vertebra dislocation, peels off every Mus lump, weighs after blotting the tumor surface blood stains with filter paper, calculates tumour inhibiting rate.The tumour inhibiting rate of three grades of dosed administrations is respectively first group 40.00%, second group 50.83% 3rd group 44.17%, show that NP has significant inhibitory effect to the S180 sarcoma, wherein the tumor killing effect of NP 100mg/kg group is best, tumour inhibiting rate can reach 50.83%, illustrates that Folium seu Cortex Nerii polysaccharide (NP) extract can use in the medicine of preparation treatment humans and animals neoplastic disease.Folium seu Cortex Nerii polysaccharide (NP) extract can be used as the application in the pharmaceutical composition for preparing the tumor for the treatment of the animal and human simultaneously, comprises that the combination of this extract and other medicines, pharmaceutical carrier or excipient is used to prepare the medicine of treatment animal and human neoplastic disease.
The present invention adopts and is applicable to the Folium seu Cortex Nerii (extraction separation method of polyoses extract in (Nerium indicum Mill.) leaf, ((Nerium indicum Mill.) leaf carries out obtaining Folium seu Cortex Nerii ((Nerium indicum Mill.) polyoses extract behind the extraction separation to Folium seu Cortex Nerii, studies show that the extract that obtains with this method has murine sarcoma S180 suppresses effect preferably, and with clinical important antineoplastic chemotherapy medicine significant synergy is arranged; But this extract is the life-span of significant prolongation S180 ascites tumor mice also.The antitumor action of this extract may be relevant with its enhancing body's immunological function.The polysaccharide of two kinds of single components that separation and purification obtains from this extract can directly suppress the propagation of people source tumor cell and promote apoptosis, illustrates that this extract also has direct lethal effect to tumor cell.
Description of drawings
Fig. 1 is Folium seu Cortex Nerii polysaccharide NP3Sephadex G-100 gel filtration chromatography figure of the present invention.
Fig. 2 Folium seu Cortex Nerii polysaccharide NP4Sephadex G-100 gel filtration chromatography figure;
Fig. 3 is the NP3 of Folium seu Cortex Nerii polysaccharide of the present invention (NP) extract and the polyacrylamide gel electrophoresis figure of NP4;
Fig. 4 is that Folium seu Cortex Nerii polysaccharide of the present invention (NP) extract variable concentrations NP4 handles K562 cell 96h dna fragmentation testing result (M:Maker; A: negative control group; B:80 μ g/mL; C:160 μ g/mL; D:320 μ g/mL).
The specific embodiment
Following examples are used for that the present invention is further elaborated, but scope not thereby limiting the invention.
Embodiment 1:
After gathering sweetscented oleander leaf and cleaning 60 ℃ of oven dry, pulverized 60 mesh sieves, took by weighing 200g, with 70% ethanol 60 ℃ of diffluence pigment, defats next time 3 times, 3h at every turn, 50 ℃ of oven dry of residue; Take by weighing the medical material after the oven dry, according to matter liquor ratio medical material: water=1: 10 hot-water extraction in 95 ℃ of water-baths, each 4h repeats extracting 3 times, sucking filtration go behind the residue supernatant; Adopt the rotary evaporator concentrated supernatant to 1/2 of original volume, obtain concentrated solution; (the polysaccharide concentrated solution adds 10% trichloroacetic acid to pH=4.0 to concentrated solution with the trichloroacetic acid method Deproteinization earlier, 4 ℃ of refrigerators are placed behind the 24h with the centrifugal 15min of 8000r/min, abandon precipitation), get supernatant transfers solution with the NaOH liquid of 0.5mol/L pH value again to neutral, add again 1/3 volume sevage (chloroform: n-butyl alcohol=4: 1), behind the vibration 20min, the centrifugal 15min of 8000rpm, go precipitation, repeat 1 time, remove residual protein.The supernatant bag filter (molecular weight 7000-14000D) of packing into, circulating water dialysis 2d, distill water dialysis 1d, dialysis solution is concentrated into behind 1/2 volume 95% ethanol by volume: polysaccharide liquid=3: 1 carries out the precipitate with ethanol polysaccharide, leave standstill 24h (4 ℃), with the centrifugal 15min of 8000r/min, get precipitation with absolute ethanol washing number time, lyophilization gets Folium seu Cortex Nerii crude polysaccharides (NP) 7.9g.
Embodiment 2:
Press the inhibitory action of Folium seu Cortex Nerii polyoses extract (NP) lumbar injection of embodiment 1 described method extraction to murine sarcoma S180.
Get the abdominal cavity well-grown S180 tumor-bearing mice of 7d that goes down to posterity, the cervical vertebra dislocation is put to death, and aseptic condition extracts ascites down; With the normal saline dilution of sterilization, mixing is made tumor cell suspension (1.0 * 10
7/ mL); Press 0.2mL/ only, it is subcutaneous to be inoculated in the right side of mice axillary fossa.Mice is divided into 5 groups at random after will inoculating 24h: 50mg/kg, the 100mg/kg of normal saline negative control group, cyclophosphamide (CTX) positive controls (20mg/kg), NP, 150mg/kg group, every group 10, every day intraperitoneal injection, behind the successive administration 10d, weigh next day, and mice is put to death in the cervical vertebra dislocation, peels off every Mus lump, weigh after blotting the tumor surface blood stains with filter paper, calculate tumour inhibiting rate.The results are shown in Table 1.
Tumour inhibiting rate (%)=(the average tumor of the average tumor weight/matched group of 1-experimental group is heavy) * 100%
The inhibitory action that table 1 NP grows to mice S180 sarcoma (x ± s, n=10)
Compare with negative control group:
*P＜0.01; Compare with CTX group matched group:
#P＜0.05
The tumor-bearing mice hair that NP respectively organizes after the administration is glossy, and is movable normal with feed; Negative control group tumor-bearing mice hair is withered, fluffy, and jaundice is slow in action slightly; CTX group tumor-bearing mice hair color and active situation are organized but better than negative control group not as NP, and body weight obviously alleviates, and food-intake reduces to some extent.Negative control group tumor piece is big, boundary is unclear, quality is softer, be difficult to peel off, have in addition soak into to breastbone, clavicle and axillary fossa rear side etc. and locate; Each group of NP and CTX organize that the tumor body is less, boundary is clear, quality more firmly, peel off easily.Compare with the normal saline negative control group by the tumor of visible NP 50,100, the 150mg/kg group of table 1 is heavy, significant differences (P＜0.01) is all arranged, wherein 100mg/kg NP group relatively has significant difference (P＜0.05) with the CTX group.The tumour inhibiting rate of NP 50,100,150mg/kg group is respectively 40.00%, 50.83%, 44.17%, show that NP has significant inhibitory effect to the S180 sarcoma, wherein the tumor killing effect of NP 100mg/kg group is best, as if tumour inhibiting rate can reach 50.83%, and NP is not dose-dependence to the inhibition of S180 sarcoma.
Embodiment 3:
Press of the influence of Folium seu Cortex Nerii polyoses extract (NP) tail vein injection of embodiment 1 described method extraction to S180 ascites tumor mice increase in life span.
Abdominal cavity inoculation S180 tumor cell, every injected in mice S180 tumor cell 1.0 * 10
6Individual, the mice behind the inoculation 24h is divided into 5 groups, 10 every group at random, 24h post processing group injection NP is respectively 50mg/kg, 100mg/kg, 150mg/kg group, negative control group injection equivalent normal saline, the CTX of positive controls injection 20mg/kg, successive administration 10d, tail intravenously administrable.With administration was the 1st day record mice life span the same day, calculated the tumor-bearing mice increase in life span.The results are shown in Table 2.
Increase in life span (%)=(the average life span-1 of life span/matched group that the administration group is average) * 100%
Table 2NP to the influence of ascites tumor mice increase in life span (x ± s, n=10)
Compare with negative control group:
*P＜0.01
Embodiment 4:
After stripping the tumor tissue of respectively organizing mice with tumor among the embodiment 2, rapidly after 10% neutral formalin solution is fixing, carry out routine dehydration, saturating wax, embedding in the 24h, gained wax stone serial section adopts the SABC method to detect tumor tissue PCNA, Bcl-2 and the proteic expression of Bax.
(1) dyeing course is mainly as follows: paraffin section is after routine dewaxing and aquation, PBS (pH=7.4) flushing 3min * 3 time, requirement according to each antibody, tissue antigen is repaired (Bcl-2 and Bax employing microwave antigen retrieval accordingly, PCNA adopts the high pressure antigen retrieval), take out under room temperature and cool off, distilled water flushing 3min * 2 time, PBS washes 2min * 2 time, every section Dropwise 50 μ L peroxidase blocking solution (reagent A), hatch 10min under the room temperature, PBS washes 3min * 3 time, remove PBS liquid, add the normal non-immune serum of 50 μ L (reagent B), hatch 10min under the room temperature, remove serum deprivation and drip first antibody, hatch 60min under the room temperature, PBS washes 5min * 3 time, remove PBS liquid, the Dropwise 5 biotin labeled second antibody of 0 μ L (reagent C) is hatched 10min under the room temperature, PBS washes 3min * 3 time, remove PBS liquid, Dropwise 50 μ L biotin-peroxide enzymatic solution (reagent D) is hatched 10min under the room temperature, PBS washes 3min * 3 time, remove PBS liquid, add fresh configuration DAB solution, microscopically is observed 3-10min, the tap water flushing, haematoxylin is redyed, the 0.1%HCl differentiation, and indigo plant is returned in the PBS flushing, through the gradient alcohol dehydration drying, dimethylbenzene is transparent, neutral gum mounting, microscopy.
(2) result judges: the PCNA positive staining is positioned at nucleus, and the Bcl-2 positive staining is positioned at cytoplasm, and the Bax positive staining is positioned at cytoplasm.Positive cell dyeing is pale brown color or sepia.Each specimen low power lens (* 100) is observed down, selects 5 visuals field at random; High power lens (* 400) is chosen 200 tumor cells in each visual field down, and the positive cell number of 1000 cells of counting is represented with percent, calculates the cell positive rate of expressing PCNA, Bcl-2 and Bax in the sarcoma tissue.
NP 50,100,150mg/kg group is compared with negative control group as seen from Table 3, after the administration in the sarcoma tissue positive rate of PCNA cell obviously reduce, learn processing difference highly significant (P＜0.01) by statistics; The positive rate of Bcl-2 cell obviously reduces, and learns by statistics and handles difference highly significant (P＜0.01); The positive rate of Bax cell obviously raises, and learns by statistics and handles difference highly significant (P＜0.01), wherein 100mg/kg NP group and CTX group comparing difference highly significant (P＜0.01).Illustrated that NP can significantly suppress S180 sarcoma cell PCNA, the proteic expression of Bcl-2 and promote the proteic expression of Bax simultaneously.
Table 3 each experimental group PCNA, Bcl-2, the proteic expression of results of Bax (x ± s, n=10)
Compare with negative control group:
*P＜0.01; Compare with CTX group matched group:
#P＜0.01
Embodiment 5: Folium seu Cortex Nerii polysaccharide (NP) and the inhibitory action of cyclophosphamide (CTX) coupling to S180 subcutaneous transplantation sarcoma
Press the Folium seu Cortex Nerii polyoses extract (NP) that embodiment 1 described method is extracted, it is identical with embodiment 2 to connect the tumor method, mice behind the inoculation 24h is divided into 6 groups at random: the normal saline negative control group, CTX is single to give CTX 10mg/kg with treatment group 1, CTX is single to give CTX 20mg/kg with treatment group 2, NP is single to give NP 100mg/kg with group, NP combined with CT X treatment group 1 gives NP 100mg/kg+CTX 10mg/kg, NP combined with CT X treatment group 2 gives NP 100mg/kg+CTX 20mg/kg, every group 10, the NP intraperitoneal injection, CTX tail intravenously administrable, administration every day of every mice, successive administration 10d.Tumor-bearing mice is put to death in 24h cervical vertebra dislocation after the last administration, strips the tumor piece and with weighing behind the blood stains of absorbent paper exhaustion tumor piece surface.With CTX treatment group and the difference that adds with tumour inhibiting rate between NP therapeutic alliance group, observation NP and CTX coupling are to the inhibitory action effect of S180 subcutaneous transplantation sarcoma relatively merely.The medication combined evaluation of effect of NP and CTX coupling carries out medication combined evaluation of effect by being evaluation criterion with drug interaction index (CDI) to the effect of NP and cyclophosphamide (CTX) use in conjunction treatment S180 murine sarcoma.
Tumour inhibiting rate (the %)=heavy g of (the average tumor of the average tumor weight-experimental group of matched group is heavy) the average tumor of g/ matched group * 100%
The computing formula of drug interaction index (CDI) is: (A * B) in this experiment, AB is two coupling groups and the heavy ratio of normal saline matched group tumor to CDI=AB/, and A or B are private medicine group of each prescription and the heavy ratio of normal saline matched group tumor.When CDI＜1, represent that two medicine couplings have synergism; When CDI＜0.7, represent the synergism highly significant of two medicine couplings.
By the tumor of each medication group shown in the table 4 heavy with the equal highly significant of negative control group comparing difference (P＜0.01=, tumor killing effect is obvious, tumour inhibiting rate is all＞30%.Wherein CTX 10mg/kg group tumour inhibiting rate is 31.85%, and tumor killing effect is obvious, and with NP 100mg/kg group coupling inhibitory rate to 62.22%, CDI=0.95 represents that these two kinds of dose drug couplings have synergism.CTX 20mg/kg group tumour inhibiting rate is 40.00%, with NP 100mg/kg group coupling inhibitory rate 63.70%, but the tumor-inhibiting action of enhanced CT X 20mg/kg group, CDI=1.10＞1.0 these two kinds of dose drug couplings of expression do not have synergism.Show that the coupling of NP 100mg/kg and CTX10mg/kg group has synergism.
Table 4NP and CTX coupling to the collaborative inhibition effect of S180 nude mice subcutaneous transplantation tumor (x ± s, n=10)
Compare with negative control group:
*P＜0.01
Embodiment 6: Folium seu Cortex Nerii polysaccharide (NP) is cleaned up ability and the exponential influence of immune organ to mouse macrophage carbon
Normal experiment mice is divided into 4 groups at random, every group 10, be respectively the normal saline negative control group, NP 50,100,150mg/kg group, the 8d lumbar injection is pressed the Folium seu Cortex Nerii polyoses extract (NP) that embodiment 1 described method is extracted continuously, behind the last administration 24h, claim its body weight, dosage tail vein injection by every 10g body weight 0.1mL gives mice prepared Chinese ink (india inks that the physiological saline solution dilution is 5 times), timing immediately, 2min, 10min get blood 30 μ L from the eye socket venous plexus respectively after injecting prepared Chinese ink, exist side by side to be about to it to join 3mL concentration be 0.1% Na
2CO
3In the solution, the Na with 0.1%
2CO
3Solution is made blank, measures absorbance (A) with spectrophotometer at the 600nm wavelength.Mice is put to death in the cervical vertebra dislocation then, gets liver, thymus and spleen, blots the organ surface blood stains with filter paper, weighs respectively.Be calculated as follows phagocytic index a, carbon is cleaned up index K, index and spleen index, thymus index.
Phagocytic index a=body weight/(liver weight+spleen is heavy) * K
1/3Carbon is cleaned up index K=(LgA
1-LgA
2)/(t
2-t
1)
Index and spleen index (%)=spleen weight mg/ the weight of animals g * 100%
Thymus index (%)=thymic weight mg/ the weight of animals g * 100%
A in the formula: phagocytic index, the activate the phagocytic capacity of reflection per unit tissue weight; K: clean up index, speed is engulfed in expression; A
1: t
1The time absorbance; A
2: t
2The time absorbance; t
1: to the time that the 1st time is got blood behind the prepared Chinese ink; t
2: to the time that the 2nd time is got blood behind the prepared Chinese ink.
The carbon of NP 100,150mg/kg group is cleaned up exponential sum phagocytic index and negative control group comparing difference highly significant (P＜0.01) as seen from table, the phagocytic index of 50mg/kg group and negative control group comparing difference be (P＜0.05) significantly, show that NP can significantly strengthen the phagocytic function of macrophage, significantly improves the carbon Cl.Wherein to clean up the exponential sum phagocytic index the most outstanding for the carbon of NP 100mg/kg dosage group, and visible NP is not dose-dependence to the phagocytic activity of mouse macrophage.
Remarkable or the highly significant (P＜0.05 or P＜0.01) of the thymus index of NP 50,100,150mg/kg group and index and spleen index and negative control group comparing difference shows that NP can significantly increase the organ index of mice as seen from Table 5.Wherein the thymus index of NP 100mg/kg dosage group and index and spleen index are the most outstanding.
Table 5 NP to mouse macrophage carbon clean up ability influence (x ± s, n=10)
Compare with negative control group
*P＜0.05,
*P＜0.01
Embodiment 7: Folium seu Cortex Nerii polysaccharide (NP) is to the influence of Turnover of Mouse Peritoneal Macrophages activate the phagocytic capacity
1. chicken erythrocyte suspension preparation
Heparin moistening syringe needle tube with preparing extracts live chickens blood then, puts the people immediately and fills 5 times in the conical flask of 1% anticoagulant heparin agent of amount for taking blood, and mixing is preserved in 4 ℃ of refrigerators.Before the use, with physiological saline solution centrifuge washing 3 times, preceding 1 centrifugal speed is 150O rpm, centrifugal 5min, abandon supernatant and interface GCL, last continuous centrifugal 2 times (2000rpm5min), it is standby to be made into 5% chicken erythrocyte suspension according to hematocrit with normal saline.
2. mice group and administration
Experiment mice is divided into 4 groups at random, every group 10, be respectively the normal saline negative control group, NP 50,100, the 150mg/kg group, the 8d lumbar injection is pressed the Folium seu Cortex Nerii polyoses extract (NP) that embodiment 1 described method is extracted continuously, starch meat soup (the 0.3g Carnis Bovis seu Bubali cream of every mouse peritoneal injection 6% in the 2nd day after drug withdrawal, 1.0g peptone, 0.5g sodium chloride, 6.0g soluble starch is dissolved in the 100mL distilled water, boiling sterilization, put in 4 ℃ of refrigerators preserve standby), every Mus 1mL, every Mus 1mL of 48h pneumoretroperitoneum injection chicken erythrocyte suspension, behind the 1h again the normal saline 1mL to lumbar injection 0.9% rub its abdominal part slightly, mice is put to death in the cervical vertebra dislocation, every Mus is got the peritoneal fluid smear, and cold wind dries up, and methanol is 5min fixedly, Ji's nurse Sa dye liquor dyeing 10min, flushing is dried, and high power lens (* 400) is observed, count 100 phagocyte, calculate phagocytic rate and phagocytic index by following formula:
Phagocytic rate (%)=engulf macrophage number/100 macrophage * 100% of chicken red blood cell
Summation/100 that phagocytic index=100 macrophage is engulfed the chicken red blood cell number
Phagocytic percentage and the phagocytic index and the negative control group comparing difference highly significant (P＜0.01) of NP 50,100,150mg/kg group show that NP can significantly strengthen the peritoneal macrophage activate the phagocytic capacity as seen from Table 6.Wherein the phagocytic percentage and the phagocytic index of NP 100mg/kg group are the most outstanding.
Table 6 NP to the influence of Turnover of Mouse Peritoneal Macrophages phagocytic activity (x ± s, n=10)
Compare with negative control group
*P＜0.01
Embodiment 8: Folium seu Cortex Nerii polysaccharide (NP) is to the inductive normal mouse splenic T of ConA proliferation of lymphocytes
Experiment mice is divided into 4 groups at random, every group 5, be respectively the normal saline negative control group, NP 50,100,150mg/kg group, the 8d lumbar injection is pressed the Folium seu Cortex Nerii polyoses extract (NP) that embodiment 1 described method is extracted continuously, the 4h mice is put to death with the cervical vertebra dislocation after the last administration, 75% soak with ethanol 5min, the aseptic spleen of getting shreds, add serum-free RPMI-1640 culture medium 5mL, grind with grinding rod, cross 200 order rustless steel cells sieve, the supernatant after absorption is ground is in vitro, the centrifugal 5min of 1500rpm adds 0.83%Tris-NH after abandoning supernatant
4Cl 5mL leaves standstill 5min, so that erythrocyte destroys fully, the centrifugal 5min of 1500rpm abandons behind the supernatant with serum-free RPMI-1640 culture fluid washed cell 2 times, makes 1 * 10 with the RPMI-1640 culture fluid that contains 10% hyclone again
6/ mL splenocyte suspension adds 96 well culture plates, every hole 180 μ L, 4 every group multiple holes, every hole add ConA 20 μ L to final concentration be 5 μ g/mL, put 5%CO
2Incubator is cultivated 48h for 37 ℃, adds MTT 20 μ L, places 5%CO
237 ℃ of incubators reaction 4h, sucking-off supernatant gently, every hole adds dimethyl sulfoxine 150 μ L, the 30s that on agitator, vibrates, microplate reader 595nm reads at the place A value, calculates cell proliferation rate (establishing 4 zeroing holes that only add culture fluid in addition) as follows.
Cell proliferation rate (%)=test sample group mean light absorbency value/matched group mean light absorbency value * 100%
As seen from Table 7 NP to ConA in the inductive mouse spleen T lymphproliferation response NP 50,100,150mg/kg group at absorbance and the negative control group comparing difference highly significant (P＜0.01) of 595nm, the lymphocytic proliferation rate of each group is respectively 130%, 174%, 139%, illustrate that NP can obviously improve the lymphocytic multiplication capacity of the inductive mouse spleen T of ConA, wherein the absorbance of NP 100mg/kg dosage group and lymphocytic proliferation rate are the most outstanding.
Table 7 NP to the influence of mouse spleen T lymphproliferation response (x ± s, n=5)
Compare with negative control group
*P＜0.01
Embodiment 9: Folium seu Cortex Nerii polysaccharide (NP) is to the breeder reaction of the inductive normal mouse spleen of LPS bone-marrow-derived lymphocyte
Experiment mice is divided into 4 groups at random, every group 5, be respectively the normal saline negative control group, NP 50,100,150mg/kg group, the 8d lumbar injection is pressed the Folium seu Cortex Nerii polyoses extract (NP) that embodiment 1 described method is extracted continuously, the 4h mice is put to death with the cervical vertebra dislocation after the last administration, 75% soak with ethanol 5min, the aseptic spleen of getting shreds, add serum-free RPMI-1640 culture medium 5mL, grind with grinding rod, cross 200 order rustless steel cells sieve, the supernatant after absorption is ground is in vitro, the centrifugal 5min of 1500rpm adds 0.83%Tris-NH after abandoning supernatant
4Cl 5mL leaves standstill 5min, so that erythrocyte destroys fully, the centrifugal 5min of 1500rpm abandons behind the supernatant with serum-free RPMI-1640 culture fluid washed cell 2 times, makes 1 * 10 with the RPMI-1640 culture fluid that contains 10% hyclone again
6/ mL splenocyte suspension adds 96 well culture plates, every hole 180 μ L, and 4 every group multiple holes, every hole adds LPS 20 μ L to final concentration 10 μ g/mL, puts 5%CO
2Incubator is cultivated 48h for 37 ℃, adds MTT 20 μ L, places 5%CO
237 ℃ of incubators reaction 4h, sucking-off supernatant gently, every hole adds dimethyl sulfoxine 150 μ L, the 30s that on agitator, vibrates, microplate reader 595nm reads at the place A value, calculates cell proliferation rate (establishing 4 zeroing holes that only add culture fluid in addition) as follows.
Cell proliferation rate (%)=test sample group mean light absorbency value/matched group mean light absorbency value * 100%
As seen from Table 8 NP to LPS in the breeder reaction of inductive mouse spleen bone-marrow-derived lymphocyte NP 50,100,150mg/kg group at absorbance and the negative control group comparing difference highly significant (P＜0.01) of 595nm, the lymphocytic proliferation rate of each group is respectively 150%, 199%, 142%, illustrates that NP can obviously improve the multiplication capacity of the inductive mouse spleen bone-marrow-derived lymphocyte of LPS.Wherein the absorbance of NP 100mg/kg dosage group and lymphocytic proliferation rate are the most outstanding.
Table 8NP to the influence of mouse spleen bone-marrow-derived lymphocyte breeder reaction (x ± s, n=5)
Compare with negative control group
*P＜0.01
Embodiment 10: the purification of Folium seu Cortex Nerii polysaccharide
Adopt DEAE-SephadexA-25 ion exchange column and glucosan Sephadex G-100 gel column to be further purified the Folium seu Cortex Nerii polysaccharide that embodiment 1 obtains.Adopt the following step that separation gel is carried out pretreatment.
(1) pretreatment of DEAE-SephadexA-25 ion exchange column: 25g DEAE-SephadexA-25 is with the abundant swelling of distilled water, soak 30min with 0.5mol/L HCl, be washed to neutrality, soak 30min with 0.5mol/L NaOH, be washed to neutrality, reuse 0.5mol/L HCl soaks 30min, is washed to neutrality, get C1-type DEAE-SephadexA-25, adding distil water soaks standby then.
(2) pretreatment of glucosan Sephadex G-100: add an amount of (being slightly larger than water absorption rate) distilled water, heated and boiled 1h removes the bubble in the gel particle.
The polysaccharide purification step
(1) DEAE-SephadexA-25 dress post: (1.6cm * 50cm), pillar vertically installs wet method dress post, gets distilled water and pours 1/3 place in the post into when buffer, and the ion adsorbent after will outgasing is then slowly poured in the post along wall carefully with Glass rod.
(2) balance: treat to open screw clamp after the ion adsorbent sedimentation, come the balance chromatographic column as buffer, make the gel distribution, swelling degree, ion distribution etc. of gel column reach an equilibrium state with 3-5 distilled water doubly.
(3) go up sample: before the application of sample, put the clean filter paper of a slice on the good ion exchange column surface of balance.Get the 50mg crude polysaccharides, be made into 10mg/mL solution, after the filtration, slowly add, inject eluent and begin eluting along post jamb.
(4) eluting: use distilled water, 0.1,0.2,0.4,0.8,1.0,2.0mol/L NaCl segmentation gradient elution respectively, flow velocity 2min/mL, automatic collector collect stream part, every pipe 5mL.
(5) collect: follow the tracks of with the phenolsulfuric acid method and detect liquid glucose, merge the positive mountain portions of eluent polysaccharide.
(6) concentrate, dialysis, drying: concentrate eluant, the 48h that dialyses in distilled water uses a small amount of AgNO
3After the no chloride ion of solution check existed, lyophilization got N1, two components of N2.
(7) get N1, each 50mg of N2 is made into 10mg/mL solution respectively, behind the filter and remove residue, cross Sephadex G-100 pillar, the distilled water eluting, flow velocity 2min/mL, automatic collector collect stream part, every pipe 5mL, the phenolsulfuric acid method is followed the tracks of the content that detects sugared peak, merges the positive mountain portions of eluent polysaccharide, and concentrated, lyophilization gets Folium seu Cortex Nerii holosaccharide NP3 and NP4.Through SephadexG-100 column chromatography (Fig. 1,2) and (Fig. 3) purity evaluation of polyacrylamide gel electrophoresis (SDS-PAGE), confirm that NP3 and NP4 are the polysaccharide of molecular weight homogeneous.
Embodiment 11:MTT method is measured the proliferation function of NP3 and NP4 inhibition K562 cell
Human leukemia cell line K562 with the RPMI-1640 culture fluid that contains 10% hyclone, puts 37 ℃, contains 5%CO
2, cultivate in the incubator of saturated humidity, changed liquid and go down to posterity 1 time in 2-3 days.The experiment cell that is in exponential phase.The K562 cell density is adjusted into 1 * 10
5/ mL, be inoculated in 96 well culture plates, every hole 180 μ L, every then hole adds the NP3 and NP4 (with the preparation of serum-free RPMI-1640 culture fluid) the 20 μ L of variable concentrations respectively, make the polysaccharide final concentration be respectively 640 μ g/mL, 320 μ g/mL, 160 μ g/mL, 80 μ g/mL, 40 μ g/mL, 20 μ g/mL, negative control group adds isopyknic serum-free medium, if blank group (do not add cell and only add culture medium), establish 4 parallel holes for every group, placing 37 ℃, saturated humidity, volume fraction is 5% CO
2After cultivating in the incubator, hatching 48h, every hole adds the MTT solution 20 μ L of 5mg/mL, after continuing to hatch 4h, the careful suction removed supernatant, and every hole adds dimethyl sulfoxide (DMSO) 150 μ L, vibration 10min, put and measure 490nm and 630nm double wave strong point absorbance (A) on the microplate reader, experiment repeats 3 times, averages, logarithm value with drug level pair cell increment suppression ratio is made rectilinear regression, calculates IC
50Value.
Cell proliferation inhibition rate (%)=(the average OD value of the average OD value/matched group of 1-medication group) * 100%
IC
50=lg-1[Xm-i (∑ p-0.5) is Xm wherein: the logarithm value of the Cmax of design; I: the logarithm value of each concentration multiple proportions number; ∑ p: each organizes the growth inhibition ratio sum; 0.5: empirical.
Behind the NP3 of variable concentrations, the NP4 effect K562 cell 48h, the growth of K562 cell all is subjected to inhibition in various degree, and along with the rising inhibitory action of drug level is strengthened gradually, all presents tangible dose-effect relationship, the IC of NP3 effect K562 cell 48h
50Be 131.21 μ g/mL, the IC of NP4 effect K562 cell 48h
50Be 113.88 μ g/mL, the results are shown in Table 9,10.
MTT testing result behind the NP3 effect K562 cell 48h of table 9 variable concentrations (x ± s)
Compare with negative control group
*P＜0.05,
*P＜0.01
MTT testing result behind the NP4 effect K562 cell 48h of table 10 variable concentrations (x ± s)
Compare with negative control group
*P＜0.05,
*P＜0.01
Embodiment 12: the influence that cell colony formation test detection NP3 and NP4 form the K562 cell colony
The cumulative volume of cultivating system is 1mL, the RPMI-1640 culture fluid contains 10% hyclone and 0.8% methylcellulose, the final concentration of experimental group polysaccharide is respectively 120 μ g/mL, 80 μ g/mL, 40 μ g/mL, negative control group adds the serum-free RPMI-1640 of serum-free equivalent, and the K562 final concentration of cells is 3 * 10
2/ mL plants in 24 orifice plates, establishes 3 parallel holes for every group.Cultivate 11d, counting colony number (cell mass of forming more than or equal to 20 cells is a colony) is calculated colony and is formed suppression ratio.Experiment repeats 3 times.
Colony formation suppression ratio (%)=(1-experimental group colony number/to array colony number) * 100%
NP3, NP4 processed group are compared colony and are counted significant difference or highly significant (P＜0.05 or P＜0.01) with negative control group.Increase along with NP3, NP4 concentration, the K562 cell colony forms suppression ratio and all obviously rises, the suppression ratio of NP3 is followed successively by 20.9%, 48.9%, 88.1%, the suppression ratio of NP4 is followed successively by 24.5%, 54.9%, 90.8%, and the drug treating group is compared with negative control group, obviously little many of colony, and born of the same parents' endoparticle increases and becomes big, see Table 11,12.
The influence that the NP3 of table 11 variable concentrations forms the K562 cell colony (x ± s)
Compare with negative control group
*P＜0.05,
*P＜0.01
The influence that the NP4 of table 12 variable concentrations forms the K562 cell colony (x ± s)
Compare with negative control group
*P＜0.05,
*P＜0.01
Embodiment 13:N4 is to the detection of K562 cell induction apoptosis
Adopt the dna fragmentation method to detect, concise and to the point step is as follows:
(1) gets the K562 cell (10 that negative control group and NP4 final concentration are respectively 80 μ g/mL, 160 μ g/mL, 320 μ g/mL processing 96h
7About individual cell);
(2) the PBS washing is 2 times, adds the abundant cracking of 50 μ L cell pyrolysis liquids;
(3) the centrifugal 5min of 12000rpm sucts clearly in another EP pipe;
(4) add SDS (final concentration is 1%), RnaseA (final concentration is 1 μ g/ μ L), put 56 ℃ and hatch 120min;
(5) add E.C. 3.4.21.64 (final concentration is 2.5 μ g/ μ L) again, 37 ℃ of effect 120min;
(6) the 10M ammonium acetate of adding 1/10 volume, 2.5 volume dehydrated alcohol deposit D NA ,-20 ℃ of placements are spent the night;
(7) the centrifugal 5min of 12000rpm abandons supernatant;
(8) DNA of Ti Quing air-dry after, be dissolved in 20 μ LTE buffer;
(9) through the 10g/L agarose gel electrophoresis, 35V, behind 3～4h with 0.5 μ g/mL ethidium bromide staining 30min;
(10) uviol lamp is observed down, with picked-up of gel images analyser and analysis result.
After NP4 handles K562 cell 96h, the DNA that obvious apoptosis feature all appears in 320 μ g/mL, 160 μ g/mL processed group changes, promptly be typical dna degradation " scalariform " band fragment, and along with the rising apoptosis scalariform band of drugs with function concentration is more obvious, 80 μ g/mL group phenomenon is not obvious, and dna fragmentation phenomenon (Fig. 4) does not appear in negative control group NP4 processed group.
Claims (2)
1. the preparation method of a Folium seu Cortex Nerii (Nerium indicum Mill.) polyoses extract is characterized in that it obtains in order to method down:
(1) gather the sweetscented oleander leaf washing totally, dry moisture, 50～65 ℃ of oven dry are pulverized the back and are crossed 60 mesh sieves;
(2) medicinal powder refluxes 3 times down at 60 ℃ with 70% ethanol, and each 3h crosses the leaching filtering residue, volatilizes solvent;
(3) get the filtering residue that volatilizes behind the solvent, by filtering residue: water is that 1: 6～10 volume ratio extracts in 60～99 ℃ of hot water, each 4h, repeat extracting 3 times after, centrifugal or sucking filtration removes slag, and gets supernatant;
(4) adopt the concentrated extracting solution that obtains by step (3) of rotary evaporator to 1/2～1/4 of original volume, obtain concentrated solution; Concentrated solution adds 10% trichloroacetic acid to pH=4.0,4 ℃ of refrigerators are placed behind the 24h with the centrifugal 15min of 8000r/min, abandon precipitation, get supernatant again and transfer the pH value of solution extremely neutral with the NaOH liquid of 0.5mol/L, the albumen in the solution is removed in employing sevage method, TCA method, enzyme process or this several method coupling;
(5) will remove pack into the bag filter dialysis of proteic supernatant, flowing water dialysis 2d, back distill water dialysis 1d;
(6) by 95% ethanol that adds 3 times of volumes in the supernatant of step (5) gained, under 4 ℃, leave standstill 1d after, with the centrifugal 15min of 8000r/min, the collecting precipitation thing, this precipitate is through successively with dehydrated alcohol, acetone and ether washing postlyophilization.
2. the purposes of Folium seu Cortex Nerii polyoses extract as claimed in claim 1 in the medicine of preparation treatment animal and human neoplastic disease, wherein said medicine comprises that this oleander extract uses separately and being used in combination as effective ingredient and cyclophosphamide (CTX), pharmaceutical carrier or excipient.
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