CN101298482B - Dextran - Google Patents

Abstract

The invention relates to a glucose, in particular to an anti-lung cancer glucose. The main chain of the glucose of the invention is constituted of the following repeating structural units with the molecular weight of 1.5 million to 2.5 million Dalton. The glucose of the invention is mainly used for preparing anti-lung cancer drugs.

Description

A kind of dextran

Technical field

The present invention relates to a kind of glucose polymer, relate in particular to a kind of anti-lung cancer dextran.

Background technology

This malignant tumour of lung cancer has become the No.1 killer who threatens human health, and the sickness rate of lung cancer is along with the aggravation of environmental pollution, and the speed with 11.90% since nearly 10 years rises.Global malignant tumour neopathy in 2000 has surpassed more than 1,000 ten thousand, dead 6,200,000 examples, and lung cancer new cases 123.9 ten thousand examples wherein account for the cancer example 12.32% of always falling ill, and lung cancer death 110.3 ten thousand examples account for 17.77% of total death toll, all rank first.The whole world has 1,500,000-1,800,000 people to die from lung cancer every year at present, and China's lung cancer harm situation is even more serious, by per 100,000 people's 83.43 sickness rate calculating.The annual New Development lung cancer patient of China does not descend 1,000,000, dies from lung cancer person every year and has reached 600,000 more than! This malignant tumour of lung cancer has become the No.1 killer who threatens human health.Therefore, the special medicine of development effectively preventing lung cancer has become the task of top priority of domestic and international the world of medicine, emphasis and focus.

All there is certain limitation in the method for current treatment lung cancer with relevant medicine, as the traditional method of treatment lung cancer excision, radiotherapy and chemotherapy (pharmacological agent) is arranged.But be lung cancer through clinical definite in a single day, 80% patient has just lost the chance of excision; And there be partial restricted and ray damaging to healthy tissues in radiotherapy; There is the restricted of ubiquity cytotoxic effect and dosage thereof in chemistry (medicine) treatment; Serious to liver, kidney, marrow and Digestive tract toxic side effect especially, restricted the result of treatment of lung cancer greatly.Emerging interventional therapy has certain effect to primary tumor, but is difficult to tackle the metastasis of constantly distributing; Modern molecular targeted property (gene) treatment brings dawn to patients with lung cancer, but the interlinkage of its vector construction, carrier and bullet and the problems such as variation in body distribution, metabolic process thereof are also among discussion.

The inventor had before had research explanation, and slug PE-40 glycoprotein all has direct repression and chemotherapeutics such as DDP and 5-FU and cytokine TNF-a are had synergism small cell lung cancer and non-small cell lung cancer cell, and does not have obvious toxic-side effects; Population effect is better than other Chinese medicine, but owing to contain the protein composition, the problem that exists sensitization and stability to be difficult to Quality Control has limited subject range to a certain extent.The present invention is intended to further, on the effective slug glycoprotein basis to lung cancer, removes deproteinize, extracts polysaccharide, and keeps drug effect not subtract.The slug polysaccharide is by the eubolism of DNA in the interfere with cancer cells and RNA and promotes cancer cell-apoptosis, prevents the mechanism of cancer kitchen range vasa vasorum growth to realize result of treatment.

Now further, by high-flux cell screening active ingredients technology, determined optimum activity slug Crude polysaccharides component (KY-1) in the above-mentioned slug polysaccharide.And the molecular structure of the slug dextran (KY-1-1) of one of clear and definite composition among the KY-1.

Summary of the invention

The main chain of dextran of the present invention is made of following repeated structural unit, and molecular weight is 1,500,000-2,500,000 daltonian dextran.

Dextran of the present invention, it extracts from slug.

Research had before been arranged, extract slug glycoprotein PE-40 from slug, described glycoprotein is not only to small cell lung cancer with non-small cell lung cancer cell all has direct repression and to multiple chemotherapeutics such as DDP and 5-FU, and cytokine TNF-a, the efficacy enhancing and toxicity reducing effect is arranged, and population effect is better than other Chinese medicine, but owing to contain the protein composition, the problem that exists sensitization to be difficult to Quality Control has limited applicable scope to a certain extent.In order to address this problem, after again on the basis of slug glycoprotein, remove deproteinize, extract polysaccharide, and keep drug effect not subtract.The slug polysaccharide of this moment is a kind of mixing polysaccharide (Crude polysaccharides) that contains multiple polysaccharide, the eubolism that these many raw sugar can be by DNA in the interfere with cancer cells and RNA and promote cancer cell-apoptosis, prevent the mechanism of cancer kitchen range vasa vasorum growth to reach the effect of treatment.The present invention is in order further to study the medical mechanism and the action effect of above-mentioned mixing polysaccharide; from mixing polysaccharide (Crude polysaccharides), further separate various components such as KY-1, KY-2 and KY-3 etc.; further from KY-1, separate KY-1-1 and KY-1-2 again; polysaccharide fraction (KY-1-1) through the protection of experimental verification the present patent application is one of them homogeneous component of studying at present among the KY-1 with best activity and medicinal effect that obtains; and the experimental technique by science, determined that the KY-1-1 molecular structure is a kind of dextran.

Dextran of the present invention is mainly used in the preparation anti-lung-cancer medicament.Dextran of the present invention is added pharmaceutically acceptable excipient substance again, can make various forms of anti-lung-cancer medicaments.As the common formulation of lyophilized injectable powder, injection, tablet, capsule or the like.

The preparation method of dextran of the present invention (KY-1-1) comprises, two main processes of the extraction of Crude polysaccharides and the separation and purification of Crude polysaccharides.Crude polysaccharides prepares by technologies such as alcohol precipitation and trichoroacetic acid(TCA) deproteinated and dialysis thereof.Its concrete affirmation process will describe in detail in the following embodiments.

Dextran of the present invention is wherein a kind of homogeneous polysaccharide at active the strongest position in the slug Crude polysaccharides of discovering at present, is one of best component of present anti-lung cancer effect.The present invention has determined that the molecular structure of this dextran is that synthetic the further investigation with pharmacological effect of later medicine laid a good foundation.

Introduce preparation method, the molecular structure of dextran of the present invention in detail determines and pharmacodynamic study below with reference to accompanying drawing.

Description of drawings

Fig. 1 is the typical curve of dextran molecule amount and retention time relational expression;

Fig. 2 is that the water component is separated the HPLC figure that obtains two kinds of components through chromatographic system (S-1000);

Fig. 3 is that component KY-1 separates the HPLC figure that obtains two kinds of components through the S-400 gel column again;

Fig. 4 is the HPLC detected result figure of component KY-1-1;

Fig. 5 is the HPLC detected result figure of component KY-1-2;

Fig. 6 is the HPLC detected result figure of component KY-2;

Fig. 7 is the ultraviolet full scan figure of component KY-1-1;

Fig. 8 is the gas chromatogram of monose in the standard sugar sample;

Fig. 9 is the gas chromatogram of component KY-1-1;

Figure 10 is the total ion current figure after component KY-1-1 methylates;

Figure 11 is that retention time is the mass spectrum of 3.44min;

The 1H nuclear magnetic resonance map of KY-1-1 when Figure 12 is 27 ℃;

KY-1-1 when Figure 13 is 27 ℃ ¹³The C nuclear magnetic resonance map;

KY-1-1 when Figure 14 is 27 ℃ ¹³The nuclear magnetic resonance map of C DEPT-135;

Figure 15 is KY-1-1 ¹H- ¹H COSY nuclear magnetic resonance map;

Figure 16 is the TOCSY nuclear magnetic resonance map of KY-1-1;

Figure 17 is the HMQC part nuclear magnetic resonance map of KY-1-1;

Figure 18 is the HMBC nuclear magnetic resonance map of KY-1-1;

Figure 19 is the NOESY part nuclear magnetic resonance map of KY-1-1.

Embodiment

Dextran provided by the invention extracts in the slug body, experimental results show that it has good effect of anti-lung cancer.Below introduce extracting method, structural confirmation and the curative effect checking of dextran of the present invention in detail.

One, the preparation method of dextran of the present invention

1. the extraction of Crude polysaccharides

Material: the high prominent full worm of sufficient folds in a garment slug (Vaginulus alte Ferussac, 1821) is subordinate to Mollusca, Gastropoda, Pulmonata, Stylommatophora, the pleased Yu of sufficient folds in a garment Kuo section, sufficient folds in a garment Limax.

Take by weighing bright slug (1604g) and add 1000mL water, homogenate, centrifugal 6000r/min, 15min gets supernatant liquor, residue is as above operation repeatedly, and last supernatant liquor merges, and concentrates, get supernatant liquor 1000mL, add ethanol then in the supernatant liquor that merges, transferring ultimate density is 60%, standing over night, centrifugal, must precipitate, lyophilize, get bright slug crude extract (40g, yield 2.5%, polysaccharide content 16.9%).

Above bright slug crude extract is dissolved in water, behind 30% the trichoroacetic acid(TCA) mixing 15min according to volume ratio 1: 10, leaves standstill 30min, centrifugal, waste gets supernatant liquor, use tap water, distilled water, the dialysis (molecular weight that dams is the film of 10,000 Dalton) of flowing concentrates, lyophilize, get slug Crude polysaccharides (24g, yield 1.5%, polysaccharide content 19.8%).Through pharmacologically active screening, this slug Crude polysaccharides component is active principle.

2, the separation and purification of Crude polysaccharides

With the slowly dissolving at room temperature of refrigerated Crude polysaccharides, centrifugal (12000r/min 10min) removes precipitation.Trichoroacetic acid(TCA) (1000D dialysis tubing) is dialysed, taken off to supernatant liquor, with [DEAESepharose fastflow (the room temperature of ion exchange column on the trapped fluid, nature pH value)], the distilled water wash-out gets the water component, concentrate after gel column (S-1000) separates and to receive two kinds of components, respectively called after KY-1 and KY-2.Subsequently, KY-1 is continued separation through gel column (S-400) can obtain other two kinds of components, respectively called after KY-1-1 and KY-1-2.Wherein KY-1-1 then is a dextran of the present invention.

Two, the structural confirmation of dextran in the extract of the present invention

1. the mensuration of purity detecting and polysaccharide molecular weight

(1) dextran typical curve:

Taking by weighing Dextran T-2000, Dextran T-500, Dextran T-70, Dextran T-40 and each 0.01g of DextranT-10 is dissolved in respectively in the 5mL deionized water, measure corresponding retention time with HPLC (SHODEX SB-804 gel column, 200nm detects).With the retention time is X-coordinate, and the logarithmic value of molecular weight is an ordinate zou, makes dextran typical curve (as shown in Figure 1), simulates the regression equation of the regression equation curve of curve again from figure:

y＝-0.6647x+7.6644

R ²＝0.9961

The working parameter of HPLC is as follows:

Single injected sampling amount: 20 μ l

Moving phase: secondary redistilled water

Flow velocity: 0.8mL/min

Column temperature: 40 ℃

Detector: UV-detector (206nm, 280nm)

(2) high performance liquid chromatography (HPLC) detects

Component KY-1, KY-1-1, KY-1-2, KY-2 analyze through HPLC (condition is the same), obtain Fig. 2-6 (wherein among Fig. 2, the 4-6 pipe is KY-1, and the 13-16 pipe is KY-2 (red 190nm, blue 280nm)).As seen from the figure, a little less than the higher and 280nm of KY-1-1 component purity absorbed, molecular weight was greater than 2,000,000.KY-2 contains three components at least, and first component has certain absorption at the 280nm place.By analysis as can be known: KY-1-1 is the homogeneous component, and all the other are blending ingredients.

2. determination of polysaccharide

(1) drafting of typical curve

The analytically pure dextrose anhydrous 0.5000g of sample thief (105 ℃ of dry constant weights) is diluted to 100ml, and taking out solution 1ml again, to be dissolved in 50ml volumetric flask adding distil water fixed molten to 50ml.Get 0.2ml, 0.4ml, 0.6ml, 0.8ml, 1.0ml, 1.2ml respectively, every sample is used three test tubes respectively, add distilled water respectively to 2ml, add phenol (5%) 1ml then, add the 5ml vitriol oil again, vibrate, be placed on and heat 15min in the boiling water bath, taking-up is placed on cooling rapidly in the frozen water, measures its light absorption value at the 490nm place with ultraviolet spectrophotometer then.

(2) mensuration of sample

Get two kinds of samples of KY-1, KY-2 respectively and be mixed with 1mg/ml solution respectively, be diluted to 20,40,60,80 μ g/ml then successively, getting 1 milliliter of phenol solution of 1ml sample liquid adds in the 15ml test tube magnetic force vibration and shakes up, add the 5ml vitriol oil, be placed on and heat 15min in the boiling water bath, taking-up is placed on cooling rapidly in the frozen water, detects its light absorption value at the 490nm place with ultraviolet spectrophotometer then.

Measurement result is as shown in table 1, and as can be seen from the table, the polysaccharide content of KY-1 is more than 90%.

Each fraction polysaccharide Determination on content of table 1

3, the ultra-violet analysis of KY-1-1

Take by weighing sample 3mg, be mixed with the solution of 1mg/mL, under 190～600nm, carry out the ultraviolet full scan with distilled water.The result shows that (as Fig. 7) all do not have absorption peak at 280nm and 260nm place, illustrates that this homogeneous polysaccharide does not contain protein and nucleic acid.

4.KY-1-1 sugared compositional analysis

(1) acetylize of standard monose sample

Precision such as takes by weighing at semi-lactosi, Fucose, wood sugar, rhamnosyl, glucose, seminose and the pectinose of mole (2mmol/L), be dissolved in respectively in the 3mL distilled water, add 20～30mg sodium borohydride (NaBH4), under room temperature, interrupted oscillation, reduction 3h uses in the Glacial acetic acid and excessive N aBH then ₄, no longer produce bubble to solution till, pH adds 3mL methyl alcohol between 4～5, the concentrating under reduced pressure evaporate to dryness repeats 4～5 times, to remove byproduct of reaction boric acid and moisture, places vacuum drier to spend the night then.Next day, heat 15min in 110 ℃ of baking ovens, fully remove residual moisture after, add the 4mL aceticanhydride, 100 ℃ of reaction 1h, cooling adds 3mL toluene then, the concentrating under reduced pressure evaporate to dryness repeats 4～5 times, to remove unnecessary aceticanhydride.Product after the acetylize is transferred to separating funnel with after the 3mL chloroform dissolving, adds after a small amount of distilled water fully shakes, remove upper water solution, so repeat 4 times.Chloroform layer is settled to 10mL with an amount of anhydrous sodium sulfate drying, carries out GC and detects.The results are shown in Figure 8, six kinds of standard monose and all obtain good ultimate separation.

(2) acetylize of sample is handled

Get 2mg KY-1-1 sample, put into the long test tube of thin-walled, add trifluoroacetic acid (TFA) 4mL of 2mol/L, at 110 ℃ of hydrolysis 2h.Hydrolyzed solution is lower than 40 ℃ of evaporated under reduced pressure, adds 3mL methyl alcohol evaporate to dryness then, repeat aforesaid operations 4～5 times, to remove TFA fully.Then according to reduce with quadrat method, acetylize, be settled to 5ml with chloroform, carry out GC and detect.The results are shown in Figure 9, this polysaccharide fraction mainly contains the glucose monosaccharide component, also contains some non-carbohydrate impurity in addition, may be the cause that some by products of reacting in operating process of experiment are not removed fully.

(3) instrument test condition

Gas chromatograph (GC) is equipped with DB-23 quartz capillary column, 30m * 0.25mm * 0.25 μ m.Flame ionization ditector (FID), high purity nitrogen is done carrier gas.Temperature programming: 120 ℃ of post initial temperature rise to 240 ℃ with 15 ℃/min, permanent 6.5min.250 ℃ of injector temperatures, splitting ratio 1: 50.250 ℃ of detector temperatures, hydrogen 35ml/min, air 350mL/min, make-up gas 30mL/min.Column flow rate is 1mL/min.

5.KY-1-1 methylation analysis

(1) preparation of NaOH-DMSO (0.025g/mL) reagent

Getting 0.5g NaOH is dissolved in the 1mL redistilled water, get 0.2mL NaOH (50%) and 0.2mL methyl alcohol mixing, then with 6mL DMSO dilution, thermal agitation NaOH-DMSO suspension on vortex mixer, ultrasonic bath 3～5min then, centrifugal collection NaOH precipitation, triplicate, last NaOH precipitation is suspended among the 4mL standby.

(2) step that methylates

Sample thief KY-1-1 2mg places test tube with cover dry, add 0.5mL DMSO in test tube, ultrasonic bath 2min, room temperature is placed 30min then, in above-mentioned test tube, add 0.6mL NaOH-DMSO suspension and 0.6ml methyl iodide, cover tight lid, carry out repeatedly mixing on ultrasonic bath and the vortex mixer, add 4mL water behind the 7min and in said mixture, stop methylation reaction, methylated polysaccharide extracts with the chloroform of equivalent, if layering is bad, can carry out of short duration centrifugal to help layering, the water on upper strata discards, and the organic phase of lower floor extracts 5 times with the water of equivalent.Organic phase is at 40 ℃ of following concentrating under reduced pressure.Repeat aforesaid operations once.DMSO 0.2mL, NaOH-DMSO 0.2mL, methyl iodide 0.3mL, water 3mL.IR detects.

The sample of exhaustive methylation is dissolved in the formic acid solution of 3mL 88% close plug, 100 ℃ of following depolymerization 3h.Add 3mL methyl alcohol in reaction flask, 40 ℃ of following concentrating under reduced pressure evaporates to dryness repeat 3 times.Polysaccharide sample to depolymerization adds 2mol/L trifluoracetic acid (TFA) 4mL, 100 ℃ of following tube sealing hydrolysis 6h, and 40 ℃ of following concentrating under reduced pressure evaporates to dryness repeat 5 times.

Complete acid-hydrolyzed sample is dissolved in the distilled water about 3mL, adds 20mg NaBH4, close plug reduces 3h under room temperature.The back adds 3mL methyl alcohol and handles concentrating under reduced pressure evaporate to dryness, 4～5 times repeatedly between 4～5 with Glacial acetic acid neutralization, pH value.Place then to vacuumize under the P2O5 vacuum drier room temperature and spend the night, in 100 ℃ of baking ovens, heat 15min again.

The sample of above-mentioned processing adds the 3mL aceticanhydride, close plug, and 100 ℃ of reaction 1h add 3mL toluene then, and the concentrating under reduced pressure evaporate to dryness repeats 4～5 times.Product after the acetylize replaces solution transfer in separating funnel with isopyknic chloroform and distilled water, and is fully after the concussion, static.After the layering, remove upper water solution, use isopyknic distilled water wash 4 times again, remove water layer, the chloroform layer anhydrous sodium sulfate drying, 10min is placed in concussion, refilters and removes the sodium sulfate solid, and the concentrating under reduced pressure evaporate to dryness is dissolved in the 0.5mL chloroform again, last GC-MS.Analytical results is shown in Figure 10 and 11, and retention time is 1,4 dextran that connects at the fragment ion peak 43,58,71,101,117,127,159,186,201 etc. of 3.44min as seen from the figure.

(3) chromatographic condition

Chromatographic condition: gas phase-mass spectrometry (GC-MS) is equipped with DB-5MS quartz capillary column, 30m * 0.25mm * 0.25 μ m.Temperature programming: 80 ℃ of post initial temperature, keep 1min, with 5 ℃/min to 200 ℃, again with 2 ℃/min to 215 ℃, with 20 ℃/min to 270 ℃, helium is done carrier gas at last, 250 ℃ of injector temperatures, splitting ratio 1: 50, column flow rate is 1mL/min.EI (70eV), Multiplier voltage 350v, heater current 250 μ A, 200 ℃ of interface temperature, 250 ℃ of ion source temperatures, total mass number sweep limit 42-462amu, scanning speed 2.5scan/s.

6.KY-1-1 nuclear magnetic resonance spectroscopy

Sample thief KY-1-1 20mg is dissolved in 0.5mL D ₂Among the O, after the freeze-drying 3 times, be dissolved in 0.5mL D repeatedly ₂Among the O.At the 500MHz nmr determination, ¹Be interior mark with HDO δ 4.78 during 25 ℃ of H NMR (25 ℃ and 60 ℃), ¹³The chemical shift of C NMR is with the heavy aqueous solution (D of saturated trimethyl silicane propane sulfonic acid sodium ₂O+DSS) δ=0.00ppm at peak is an external standard.Right down at 60 ℃ ¹H- ¹The relevant spectrum of H ( ¹H- ¹H COSY, ¹H- ¹H correlated spectroscopy), total correlation spectrum (TOCSY, total correlation spectroscopy), the relevant spectrum of heteronuclear volume (HMQC, heteronuclear multiple quantum coherence), relevant spectrum of heteronuclear multikey (HMBC, heteronuclear multiple-bond correlation spectroscopy) and NOESY spectrum (Overhauser effect spectroscopy) is measured.

¹27 ℃ of collection of illustrative plates of H NMR as shown in figure 12 in, mainly contain the resonance peak of an anomer hydrogen at the resonance zone δ 5.443 in anomer hydrogen district, at δ 5.02 some little resonance peaks are arranged, but the resonance peak that this little resonance peak is different Head Section this residue content trace in this polysaccharide are in the possible sample due to the contained trace impurity.Seriously overlapping at δ 3.50～4.41 place's resonance peaks, can not provide accurate information.

¹³C NMR composes as shown in figure 13, and as seen from the figure: anomeric carbon district δ 102.47 is the anomeric carbon signal; At δ 63.21 places are 6 resonance peaks that do not take place to replace of glucose; Negative peak (as shown in figure 14) do not occur at δ about 69.0, illustrate that this component does not contain the residue of 6 replacements.Company's oxygen carbon signal of sugar is at δ 63.21～79.56.

¹H- ¹H COSY spectrum (as shown in figure 15), TOCSY spectrum (as shown in figure 16) can be released the chemical shift of hydrogen, and the chemical shift of carbon is released (table 2) by HMQC spectrum (as shown in figure 17) by the chemical shift of known hydrogen.

The chemical shift of table 2 KY-1-1

Residue → 4)-chemical shift of α-D-Glcp is all greater than 5.00ppm, and H-1 exists ¹In the H NMR spectrum is unimodal a, J _{H-1, H-2}Less than 3Hz, H-1/H-2 has the intersection peak in NOESY, and these can show that in different head region be α-configuration.Residue → 4)-and the chemical shift of the C-4 of α-D-Glcp moves an about 7ppm with the chemical shift on the site to low than the monosaccharide residue that takes place to replace, and illustrates that these sites are sites that replacement takes place, and the analytical results of these results and GC-MS is consistent.

The H1 of residue in HMBC spectrum (as shown in figure 18) has relevant resonance peak with C4 in the KY-1-1 repeating unit, compose (as shown in figure 19) in the NOESY spectrum with NOESY, residue H1 also has relevant resonance peak with H4, illustrates that this repeating unit is the dextran that is connected with α-1,4.

In sum, KY-1-1 is one and is made up of glucose, is configured as 1, the 4 pyranose form dextran that connects of α-configuration, and its concrete repeat unit structure is:

→4)-α-D-Glcp-(1→4)-α-D-Glcp-(1→

Its stereochemical formula is:

Three, the pharmacodynamic experiment of KY-1-1

1. materials and methods

(1) material

Human lung adenocarcinoma (A549) cell is purchased in the Xiangya Medical College, Zhongnan Univ cell centre, human small cell lung carcinoma (NCI-H446) cell and Chinese hamster lung epithelial (CHL) cell are purchased the Shanghai cell biological institute in the Chinese Academy of Sciences, with containing 10% calf serum RPMI RPMI-1640, put 37 ℃, 5%CO ₂Incubator is cultivated, and routine goes down to posterity, and the vegetative period cell of taking the logarithm experimentizes.

Electronic analytical balance takes by weighing 3.8g KY-1-1 and packs in the 10mL cleaning cillin bottle, after an amount of DMSO dissolving, add physiological saline and be diluted to 300.0 μ g/mL respectively, 100.0 μ g/mL, 30.0 μ g/mL, 10.0 μ g/mL and 3.0 μ g/mL, 1.0 μ g/mL, be sub-packed in the 2mL EP pipe, place 4 ℃ of refrigerators to preserve.

Cisplatin vial (DDP), Qilu Pharmaceutical Co., Ltd., specification: the 10mg/ bottle, lot number: 6030052DB with the NS dissolving, is diluted to 100.0 μ g/mL, 10.0 μ g/mL and 1.0 μ g/mL.

Taxol (TAX), the Beijing Sihuan Medicine Science and Technology Co., Ltd produces, and specification: 30mg/5ml/ props up, lot number: 060830, with the NS dissolving, be diluted to 100.0 μ g/mL, 10.0 μ g/mL and 1.0 μ g/mL.

(2) method

1) the KY-1-1 selectivity suppresses the human lung carcinoma cell proliferation activity

Preparation human lung adenocarcinoma (A549) cell, human small cell lung carcinoma (NCI-H446) cell and Chinese hamster lung epithelial (CHL) cell suspension, regulating concentration is 0.5 * 104/mL, be inoculated in 96 hole plastic culture plates, every hole liquid measure is 180 μ l, treats (about 12 hours) behind the cell attachment, adds respectively to be tried thing and contrast medicine experiment liquid 20 μ L/ holes, make the final concentration of KY-1-1 be respectively 0.1 μ g/mL, 0.3 μ g/mL, 1.0 μ g/mL, 3.0 μ g/mL, 10.0 μ g/mL and 30.0 μ g/mL, Platinol and taxol final concentration are 0.1 μ g/mL, cultivate 48h, add MTT (5mg/mL, MTT:PBS) 20 μ l/ holes, continue to cultivate 6 hours, absorb each hole nutrient solution, add 100 μ L/ hole DMSO, jolting makes the fully dissolving of hyacinthine precipitation, measures absorbance (A) with EXL-800 type microplate reader under the 570nm wavelength.By formula: IR (%)=(1-drug treating group A average/control group A average) * 100% calculates inhibiting rate; Half-inhibition concentration IC50 adopts the CalcusZn program to calculate.The cytotoxicity selectivity index of being tried thing is by formula: SI=IC50 (CHL cell)/IC50 (A549 cell or NCI-H446) calculates.More than experiment repeats 2 times.

2) KY-1-1 is to the influence of human lung carcinoma cell grappling dependency growth

Preparation human lung adenocarcinoma (A549) cell and human small cell lung carcinoma (NCI-H446) cell single cell suspension, regulating concentration is 0.3 * 103/mL.The culture system in every hole: single cell suspension 1.8mL, tried thing or contrast medicine experiment liquid 0.2ml.The KY-1-1 final concentration is respectively 0.1 μ g/mL, 0.3 μ g/mL, 1.0 μ g/mL, 3.0 μ g/mL, 10.0 μ g/mL and 30.0 μ g/mL; DDP (0.1 μ g/mL) and Tax (0.1 μ g/mL), the blank group adds the equivalent substratum; Every group 3 hole is inoculated in 24 orifice plates, puts in the CO2 incubator and cultivates 7 days.Every hole adds 95% methyl alcohol 0.5mL, fixing 15min, Giemsa staining 10-30min.Is a colony greater than 50 or diameter greater than 75 μ m with cell count, counts every hole colony number separately by 3 people under inverted microscope, gets mean and record.By formula: colony forms inhibiting rate (%)=[1-(treatment group colony mean/blank group colony mean)] * 100% and calculates colony inhibiting rate and plate efficient=colony mean/inoculating cell number * 100% calculating plate efficient.Half-inhibition concentration IC50 adopts the CalcusZn program to calculate.More than experiment repeats 2 times.

3) KY-1-1 is to the influence of human lung carcinoma cell grappling dependent/non-dependent growth

Get 24 well culture plates, shop fixtures layer agar, every hole adds 0.6% nutrient agar 0.5mL.The single cell suspension of preparation human lung adenocarcinoma (A549) cell and human small cell lung carcinoma (NCI-H446), regulating concentration is 1.6 * 103/mL; Every group top-layer agar culture system: single cell suspension 1.6ml, tried thing or contrast medicine 0.2ml.The KY-1-1 final concentration is respectively 0.1 μ g/mL, 0.3 μ g/mL, 1.0 μ g/mL, 3.0 μ g/mL, 10.0 μ g/mL, 30.0 μ g/mL; DDP (0.1 μ g/mL) and Tax (0.1 μ g/mL) 0.2mL, the blank group adds the equivalent substratum; With spherical calibrated pipet fast and 3% agar solution 0.2mL mixing of heating in water bath.Be inoculated in 24 orifice plates of shop fixtures layer agar with the 0.5mL/ hole immediately, every group is provided with 3 multiple holes.Place the CO2 incubator to cultivate 7 days.Is a colony greater than 50 or diameter greater than 75 μ m with cell count, and each comfortable inverted microscope of 3 people is the colony number in the every hole of counting down, gets mean and record.By formula: colony forms inhibiting rate (%)=[1-(treatment group colony mean/blank group colony mean)] * 100% and calculates colony inhibiting rate and plate efficient=colony mean/inoculating cell number * 100% calculating plate efficient.Half-inhibition concentration IC50 adopts the CalcusZn program to calculate.More than experiment repeats 2 times.

4) statistical method

Experimental data is represented with X ± SD, the capable One Way of relatively employing SPSS 15.0 Evaluation forwindows softwares ANOVA between many group means analyzes, the variance post analysis adopts LSD and SNK method, Student ' the s t that relatively adopts in twos of mean checks, and is statistical significance significance standard with P＜0.05.

2. experimental procedure and result

(1) KY-1-1 is to the influence of Chinese hamster lung epithelial (CHL) cell proliferation

As shown in table 3, KY-1-1 to Chinese hamster lung epithelial (CHL) cell proliferation do not have the obvious suppression effect, its IC ₅₀Value is 411.98 μ g/mL.

Table 3 KY-1-1 is to the influence of Chinese hamster lung epithelial (CHL) cell proliferation (n=9, X ± SD)

^*P＜0.05 VS contrast; ^*P＜0.01 VS contrast;

(2) KY-1-1 is to the influence of human small cell lung carcinoma NCI-H446 cell proliferation

As shown in table 4, cell proliferation has the obvious suppression effect to KY-1-1 to human small cell lung carcinoma NCI-H446, is dose-dependently, its IC ₅₀Value is 7.22 μ g/mL.KY-1-1 has selective inhibitory to human small cell lung carcinoma (NCI-H446), and is low to normal cytotoxicity, and its cytotoxicity selectivity index to human small cell lung carcinoma NCI-H446 clone is 57.06 (411.98/7.22).

Table 4 KY-1-1 is to the influence of human small cell lung carcinoma (NCI-H446) cell proliferation (n=9, X ± SD)

^*P＜0.01 VS contrast;

(3) KY-1-1 is to the influence of human small cell lung carcinoma (A549) cell proliferation

As shown in table 5, cell proliferation has the obvious suppression effect to KY-1-1 to human lung adenocarcinoma (A549), is dose-dependently., its IC ₅₀Value is 9.44 μ g/mL.KY-1-1 has selective inhibitory to human small cell lung carcinoma (A549) cell, and is low to normal cytotoxicity, and its cytotoxicity selectivity index to human lung adenocarcinoma A549 clone is 43.64 (411.98/9.44).

Table 5 KY-1-1 is to the influence of human lung adenocarcinoma (A549) cell proliferation (n=9, X ± SD)

^*P＜0.01 VS contrast;

(4) KY-1-1 is to the restraining effect of human small cell lung carcinoma (NCI-H446) cell grappling dependency growth

As shown in table 6, growth has obvious restraining effect to KY-1-1 to human small cell lung carcinoma (NCI-H446) cell grappling dependency, is dose-dependently., its IC ₅₀Value is 7.01 μ g/mL.

Table 6 plate clone forming method is measured KY-1-1 to the influence of NCI-H446 cell grappling dependency energy for growth (n=9, X ± SD)

^*P＜0.01 vs contrast

(5) KY-1-1 is to the restraining effect of human lung adenocarcinoma (A549) cell grappling dependency energy for growth

As shown in table 7, KY-1-1 has the obvious suppression effect to human lung adenocarcinoma (A549) cell, is dose-dependently, its IC ₅₀Value is 8.94 μ g/mL.

Table 7 plate clone forming method is measured KY-1-1 to the influence of A549 cell grappling dependency energy for growth (n=9, X ± SD)

^*P＜0.01 VS contrast

(6) KY-1-1 is to the restraining effect of the non-grappling dependency of human small cell lung carcinoma (NCI-H446) cell energy for growth

As shown in table 8, KY-1-1 has obvious restraining effect to human small cell lung carcinoma (NCI-H446) cell colony formation ability, is dose-dependently, its IC ₅₀Value is 7.60 μ g/mL.

The influence that table 8 KY-1-1 forms human small cell lung carcinoma (NCI-H446) cell colony (n=9, X ± SD)

^*P＜0.01 VS contrast

(7) KY-1-1 is to the restraining effect of the non-grappling dependency of human small cell lung carcinoma (A549) cell energy for growth

As shown in table 9, KY-1-1 has obvious restraining effect to human lung adenocarcinoma (A549) cell colony formation ability, is dose-dependently, its IC ₅₀Value is 9.11 μ g/mL.

The influence that table 9 KY-1-1 forms the human lung adenocarcinoma cell colony (n=9, X ± SD)

^*P＜0.05 VS contrast; ^*P＜0.01 VS contrast;

Four, anti-lung-cancer medicament composition

(1) by the preparation of following component and method: KY-1-1 10g, N.F,USP MANNITOL 1000g (excipient) adds the injection water and adds to mixing behind the 1000ml, adjusting PH is 5.0-5.5, add 0.05% gac in 40-45 ℃ of decolouring 15 minutes, with 0.45um millipore filtration coarse filtration, filter to clarification with the 0.22um millipore filtration more earlier, repetition measurement content and pH value, divide the injection vial of packing into, 5ml/ bottle, frozen vacuum dryer freeze-drying, gland, conventional packing, labeling, sterilization.Specification: 100mg/ bottle.

Usage and consumption: dextran 100mg adds in 5% glucose solution, and intravenous drip dripped off in 30 minutes.Every day 2 times, 21 days courses of treatment.Can take the circumstances into consideration to use continuously several courses of treatment.

(2) preparation method of slug polysaccharide injection

Prepare by following component and method: KY-1-1 10g, N.F,USP MANNITOL 1000g (excipient), add the injection water and add to mixing behind the 1000m, adjusting PH is 5.0-5.5, adds 0.05% gac in 40-45 ℃ of decolouring 15 minutes, earlier with 0.45um millipore filtration coarse filtration, again with extremely clarification of 0.22um millipore filtration filter, repetition measurement content and pH value aseptic subpackagedly go in the injection 5ml ampoule to seal conventional packing, labeling, sterilization, warehouse-in.Obtain the injection liquid of 100mg/ ampoule.Specification: 100mg/ ampoule (5ml/ ampoule)

Usage and dosage: slug polysaccharide 100mg (100mg/ ampoule) adds in 5% glucose solution, and intravenous drip dripped off in 30 minutes.

Every day 2 times, 21 days courses of treatment.Can take the circumstances into consideration to use continuously several courses of treatment.

In other embodiments of the invention, add pharmaceutically proper supplementary material, be prepared into formulations such as tablet, capsule.

Claims (3)

1. A main chain by following repeated structural unit constitute, molecular weight is the application of 1,500,000-2,500,000 daltonian dextran in the preparation anti-lung-cancer medicament.
2. 2. application as claimed in claim 1 is characterized in that described dextran extracts from slug.
3. 3. treat the lung cancer drugs composition for one kind, it contains the described dextran of claim 1 of effective dose.

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