CN104479037A

CN104479037A - Compound with anti-tumour activity, preparation method and application

Info

Publication number: CN104479037A
Application number: CN201410716919.0A
Authority: CN
Inventors: 张满锦
Original assignee: Individual
Current assignee: Individual
Priority date: 2014-12-02
Filing date: 2014-12-02
Publication date: 2015-04-01

Abstract

The invention provides a compound extracted from porphyridium as well as a preparation method and an application thereof. The structural formula of the compound provided by the invention is defined in the specification, and the compound has an anti-tumour activity, can be applied to preparation of anti-tumour medicines, and has a wide clinical application prospect.

Description

A kind of compound, preparation method and purposes with anti-tumor activity

Technical field

The present invention relates to field of medicaments, be specifically related to a kind of compound, preparation method and the purposes with anti-tumor activity.

Background technology

Cancer and diabetes, cardiovascular disorder are called as " worldwide three large diseases ".The World Health Organization announces, and the annual cancer stricken number in the whole world increases substantially, and the newly-increased cancer patients's number checked out every year so far, more than 1,400 ten thousand, has become the first killer of health of people.At present, the treatment of tumour, based on chemicals and chemotherapy of tumors, not only has severe side effect, and fails to reach satisfied effect to harm humans life and health, the treatment that accounts for the solid tumor of malignant tumour more than 90%.Therefore, the current urgency to new type antineoplastic medicine pharmaceutical requirements is self-evident.

Summary of the invention

The invention provides a kind of compound extracted from Porphyridium cruentum, its structural formula is as follows:

The present invention also provides the preparation method of described compound, comprises following steps:

(1) Porphyridium cruentum Crude polysaccharides is prepared;

(2) the Porphyridium cruentum Crude polysaccharides obtained by step 1 is by column chromatography (ion exchange column and gel column), and separation and purification obtains compound according to claim 1.

The ion exchange column that above-mentioned steps 2 adopts is DE52 cellulose ion exchange column (3.5cm × 25cm), and the gel column of employing is SePhacry1S 1 gel column (l.6cm × 80cm).

The present invention also provides described compound preparing the application in antitumor drug.Research sets up mouse model by transplanted tumor organon, after gastric infusion, detect Transplanted murine tumors euphorbia egg decoctum growing state, spleen index, thymus index and spleen lymphocyte proliferation with the compound of various dose, the anti-tumor activity of compound is evaluated.

Embodiment

The invention discloses a kind of novel cpd and preparation method thereof and application, those skilled in the art can use for reference present disclosure, and suitable improving technique parameter realizes.Special needs to be pointed out is, all similar replacements and change apparent to those skilled in the art, they are all deemed to be included in the present invention.Compound of the present invention, preparation method and application are described by preferred embodiment, related personnel obviously can not depart from content of the present invention, spirit and scope methods and applications as herein described are changed or suitably change with combination, realize and apply the technology of the present invention.

Below in conjunction with embodiment, the present invention is specifically described.

Embodiment one: the preparation of compound

(1) preparation of Porphyridium cruentum Crude polysaccharides

By fresh algae liquid with the centrifugal skilful min of 4000r/min, get its supernatant liquor 40 ~ 50 DEG C of rotary evaporations to concentrate, algae liquid after concentrated adds long-pending 95% ethanol, the 4 DEG C of alcohol precipitations of triploid and spends the night, picking next day top floss is Porphyridium cruentum exocellular polysaccharide, this polysaccharide is redissolved in the distilled water that equal volume algae liquid is long-pending, add trichoroacetic acid(TCA) deproteinated, after loading dialysis tubing (through molecular weight 8000) tap water flowing water dialysis 48h, distill water dialysis 24h.The liquid glucose of having dialysed amasss alcohol settling with triploid again, and method is the same.Throw out vacuum lyophilization, gained is white flock Porphyridium cruentum Crude polysaccharides.

(2) gel filtration chromatography of Porphyridium cruentum polysaccharide

1. the ion-exchange chromatography of Porphyridium cruentum polysaccharide

Porphyridium cruentum polysaccharide is made into the aqueous solution of 10mg/mL, the DE52 cellulose ion exchange column (3.5cm × 25cm) that upper tris damping fluid pre-equilibration is good.First use distilled water wash-out, then use the NaCI solution gradient wash-out of O ~ 2.Omol/L, flow velocity 1.0mL/min, often pipe 5mL fraction collection, phenol---sulfuric acid process measures sugared content by pipe, draws elution curve, merges same composition.

2. the gel filtration chromatography of Porphyridium cruentum polysaccharide

Sugar component ion-exchange collected is further purified through Sephacry1S-400 column chromatography (l.6cm × 80cm), the NaCI eluant solution of 0.1mol/L, flow velocity 0.2mL/min, automatically collect with the amount of 5mL/ pipe, detection method is the same, with pipe number for X-coordinate, absorbancy is ordinate zou mapping.Collect, merge the elutriant at main sugared peak, vacuum concentration, flowing water is dialysed 48 hours, distill water dialysis 48 hours, obtains White Flocculus, obtain required compound after lyophilize.

Embodiment two: the anticancer experiment in vitro of compound---adopt srb assay to measure

(1) cell cultures: by good for propagation and be in the tumour cell of logarithmic phase, counting after the Digestive system dispersion of 0.25% pancreatin and EDTA half and half, make cell suspension, adjustment cell concn is (0.5-1) × 10 ⁵individual/mL.Be inoculated in 96 orifice plates, 180 μ L/ holes, at 37 DEG C, 5%CO ₂after cultivating preculture 24h in cell culture incubator, every hole adds different concns compound 20 μ L, the total liquid measure in every hole is 200 μ L, each drug level establishes 6 multiple holes, and establish blank (RPMll640 nutrient solution) and normal control hole (not to add medicine, add normal saline), be placed in 37 DEG C, 5%CO ₂incubator cultivates 48h under complete wet condition, and inhibiting tumor assay nutrient solution used is RPMll640, includes 5% bovine serum, 100Iu/mL penicillin and 100 μ g/mL Streptomycin sulphates.

(2) SRB colour developing: after cell cultures terminates, take out culture plate, every hole adds trichoroacetic acid(TCA) (TCA) the 50 μ L of 50% (quality, volume), fixed cell.The final concentration of TCA is 10%, is gently added on the liquid level of every hole, then in 4 DEG C of refrigerators, places 1h.Culture plate each hole deionized water wash 5 times, to remove TCA, in atmosphere after drying, every hole adds the SRB100 μ L of 0.4%, and ambient temperatare puts 10 ~ 30min, discard in each hole and wash 5 times with 1% Glacial acetic acid after liquid, remove unconjugated dyestuff, after air drying, add Tris-100 150 μ L/ hole and dissolve, on micro oscillator after vibration fully, measure 530nm absorbance at enzyme-linked immunosorbent assay instrument.

Growth of tumour cell inhibiting rate (IR)=(1-A ₁/ A ₂) × l00%

Wherein, A _lfor test holes is according to group light absorption value, A ₂for control wells light absorption value.

(3) result

What table 1 showed is the effect of compound to hepatoma cell strain.Result is visible, compound shows good restraining effect to hepatoma cell strain (SMMC7721), but there is larger difference in the sample of different molecular weight, be wherein observed when sample concentration is 51.5 μ g/mL Hep-2 maximum suppression effect, inhibiting rate is 37.5%.

Table 1 compound is to the inhibition of each tumour cell

Embodiment three: the anti-tumor in vivo experiment of compound

(1) foundation of mouse tumor model and administration

Mouse model is set up by transplanted tumor organon.Select the mouse after inoculation S180 7d, sterilization skin of abdomen, by aseptic empty needle suction ascites, normal saline dilution, counts under inverted microscope with blood counting chamber after Trypan Blue, and viable count is more than 98%, and adjustment cell concn is l × 10 ⁷/ mL.Get mouse 60, be divided into 6 groups at random, namely Normal group, model group, ring phosphorus phthalein amine positive controls and the high, medium and low dosage group of compound, weigh and record.Except Normal group, respectively organize mouse right fore armpit subcutaneous vaccination 0.2mL cell suspension in other.Start administration after inoculation 24h, Normal group and model group are all to the physiological saline of equivalent, and ring phosphorus phthalein amine group dosage is 20mg/kg, high, medium and low three the dosage groups of compound, be respectively 200,100,50mg/kg/d, administering mode is 0.02mL/g gastric infusion, continuous 10d.

(2) compound is to the restraining effect of mice-transplanted tumor S180

Weigh after last administration 24h, cervical dislocation puts to death mouse, wins knurl block, spleen and thymus gland and weighs, and calculates tumour inhibiting rate, spleen index and thymus index.Formula is as follows:

Tumour inhibiting rate=[the average knurl heavy (g) of heavy (the g)/tumour control group of average knurl of 1-administration group mouse] × 100%

Heavy (the mg)/Mouse Weight (g) of spleen of spleen index=mouse

The body weight (g) of chest gland weight (the mg)/mouse of thymus index=mouse

(3) compound is on the impact of tumor-bearing mice spleen lymphocytic hyperplasia

Cleaning grade mouse oxter inoculation S180 sarcoma, by foundation and the administration 7d of (1) mouse tumor model, after last administration 24h, cervical dislocation puts to death mouse, spleen is got under aseptic condition, preparation splenic lymphocyte suspension is 8 ~ 10 × 1O with the RPMI mono-RPMI-1640 adjustment cell concn containing 10% calf serum ⁶/ mL.In 96 well culture plates, every hole adds 100 μ g/mL cell suspensions, every multiple hole of mouse 6, then in every hole, add each 100 μ L of nutrient solution containing 10 μ g/mLConA, puts 5%CO ₂, cultivate 48h for 37 DEG C.Cultivation terminates front 4h, adds the MTT20 μ L of 5mg/mL in every hole, and cultivate after terminating, the SDs solution 120 μ L lysing cell that every hole adds 20% of acidifying spends the night, and microplate reader is surveyed the light absorption value (measuring wavelength 57Onm reference wavelength 630nm) in each hole.

(4) result

1. the growth-inhibiting effect of Porphyridium cruentum Polysaccharides on Mice transplantability 5180 solid tumor

From table 2, compared with model group, the compound of various dose all can suppress the growth of S180 solid tumor significantly, and the tumour inhibiting rate of high, medium and low dosage is respectively 53.3%, 47.5% and 40.5%.Compared with Normal group, the body weight of model group mouse, spleen index, thymus index all change (P>0.05) without obvious.Chemotherapeutics ring phosphorus phthalein amine significantly can suppress the growth of animal transplanting tumor, but makes the body weight of mouse significantly decline (p<0.01); Also inhibits the immunologic function of animal simultaneously, the spleen index of mouse and thymus index are all declined comparatively significantly (p<0.05).And the body weight of compound to tumor-bearing mice has no significant effect, and its spleen index is increased to some extent.This illustrates that the enhancing of immunologic function is a kind of vital role mode of compound antitumor.

The impact that table 2 polysaccharide weighs transplantability 5180 solid tumor Mouse Weight, index of immunity, knurl

Group

Dosage (mg/kg/d)

Body weight (g)

Spleen index (mg/g)

Thymus index (mg/g)

Tumor weight (g)

Tumour inhibiting rate (%)

Normal group

——

6.08±0.94

6.26±1.48

3.34±0.22

Model group

——

5.42±1.74

6.11±1.33

3.33±0.25

1.157±0.421

Compound high dose group

200

5.85±2.72

7.54±2.12 ^**

3.53±0.58

0.540±0.238 ^△△

53.33%

Dosage group in compound

100

6.74±1.23 ^*

7.91±2.12 ^**△△

3.45±0.21

0.608±0.326 ^△△

47.45%

Compound low dose group

50

5.30±1.19

7.79±1.68 ^**△△

3.50±0.54

0.689±0.112 ^△

40.45%

Ring phosphorus phthalein amine positive controls

20

3.80±1.12 ^**△△

4.83±1.23 ^**△△

2.62±0.82

0.470±0.178 ^△△

59.38%

2. Porphyridium cruentum polysaccharide is on the impact of tumor-bearing mice spleen lymphocyte proliferation

Compared with Normal group, the multiplication capacity of the splenic lymphocyte of tumor-bearing mice obviously declines (p<0.01), and the multiplication capacity of the splenic lymphocyte of ring phosphorus phthalein amine group mouse also declines (p<0.01) significantly; And compare with ring phosphorus phthalein amine treatment group with model group, the multiplication capacity of the splenic lymphocyte of compound three dosage groups strengthens (p<0.01) all significantly, and has good dosage---effect relation.When concentration is 200 μ g/mL, administration group is 2.41 relative to the proliferation index of model group, is 2.15 relative to the proliferation index of CTX positive controls.Illustrate that immunomodulatory is one of important mechanisms of compound antitumor.

Claims

1. an antineoplastic compound, is characterized in that: structure is as follows:

A preparation method for antineoplastic compound, is characterized in that: comprise the following steps:

(1) Porphyridium cruentum Crude polysaccharides is prepared;

2. preparation method according to claim 2, it is characterized in that, the ion exchange column that step 2 adopts is DE52 cellulose ion exchange column (3.5cm × 25cm), and the gel column of employing is SePhacry1S 1 gel column (l.6cm × 80cm).

3. a compound is preparing the application in antitumor drug.