CN106727451B - The application of COX-2 and VEGF inhibitor and its triacontanol - Google Patents

Abstract

The present invention relates to field of medicaments, and in particular to triacontanol is as COX-2 and VEGF inhibitor, and the application in pharmacy and health care product.COX-2 of the present invention and VEGF inhibitor are made of the triacontanol of weight percent 5-95% and the pharmaceutic adjuvant of surplus.It is single active ingredient the present invention relates to triacontanol or cooperates with other medicinal ingredients as the application in the drug for preparing COX-2 and VEGF inhibitor.It is single active ingredient the present invention relates to triacontanol or cooperates with other medicinal ingredients as the application in the health care product for preparing COX-2 and VEGF inhibitor.Present invention firstly discovers that triacontanol has the activity for inhibiting COX-2 and VEGF, by inhibiting the activity of COX-2 and VEGF to can achieve the hyperplasia of prevention cancer cell and the effect of transfer.

Description

The application of COX-2 and VEGF inhibitor and its triacontanol

Technical field

The present invention relates to field of medicaments, and in particular to triacontanol as COX-2 and VEGF inhibitor, and in pharmacy and Application in health care product.

Background technique

Triacontanol, molecular formula C₃₀H₆₂O, structural formula CH₃-(CH₂)₂₈-CH₂- OH, No. CAS: 593-50-0.30 Alkanol is mainly derived from rice bran wax, beeswax, in sugarcane, is agriculturally used as broad spectrum activity plant growth promoter.But in medical applications In, in addition to Cuba, the U.S. are using the health care product that n-octacosanol is developed as main component, the report in relation to triacontanol is seldom.

Article " the Octacosanol isolated from Tinospora that G.Thippeswamy etc. is delivered cordifolia downregulates VEGF gene expression by inhibiting nuclear Translocation of NF-<kappa>B and its DNA binding activity " shows that n-octacosanol can press down Endothelial cell and ehrlich ascites cell proliferation, chick chorioallantoic membrane and cornea of rats angiogenesis and cancer ascites processed Secretion, mechanism inhibit tumor cell secretion vascular endothelial growth factor (VEGF) related with n-octacosanol, but about three There is no relevant reports for ten alkanols.

Application in relation to triacontanol in drug, application No. is the Chinese patents of CN200710066112.7 to disclose three Application of ten alkanols in preparation treatment hyperlipidaemic conditions, application No. is CN200610048846.8, CN200910146061.8 Chinese patent disclose triacontanol there is different degrees of curative effect, but its mechanism of action and target to liver cancer, intestinal cancer, lung cancer etc. Point is still not clear.

The report about triacontanol as COX-2 and VEGF inhibitor is not yet retrieved at present.

Summary of the invention

The object of the present invention is to provide a kind of COX-2 and VEGF inhibitors.

Application it is a further object of the present invention to provide triacontanol as COX-2 and VEGF inhibitor.

COX-2 of the present invention and VEGF inhibitor by weight percent 5-95% triacontanol and surplus it is medicinal Auxiliary material composition.

Since triacontanol is single active ingredient, pharmaceutic adjuvant is merely as excipients, so triacontanol is at it Middle content is not important factor, as long as energy patent medicine, generally 10-50%.The pharmaceutic adjuvant is medicinal to commonly use Auxiliary material, such as: one or both of polyethylene glycol, sodium carboxymethylcellulose.

Triacontanol can also be with other medicinal ingredients such as cyclophosphamide compatibility, inhibitor as COX-2 and VEGF.Three The addition of ten alkanols can substantially reduce the side effect of cyclophosphamide.It is specifically shown in subsequent test.

The present invention relates to triacontanol be single active ingredient or with other medicinal ingredients cooperate as preparation COX-2 and Application in the drug of VEGF inhibitor.

The present invention relates to triacontanol be single active ingredient or with other medicinal ingredients cooperate as preparation COX-2 and Application in the health care product of VEGF inhibitor.

Present invention firstly discovers that triacontanol has the activity for inhibiting COX-2 and VEGF, by inhibiting COX-2's and VEGF Activity can achieve the effect of the hyperplasia and transfer that prevent cancer cell.

COX-2 (cyclooxygenase-2) and the occurrence and development of tumour are in close relations, by promoting tumor cell proliferation, inhibiting to wither It dies, promote the mechanism such as tumor angiogenesis, participate in generation, the development of kinds of tumors, COX-2 has become a weight of tumour The treatment target molecule wanted.The overexpression of COX-2 can promote the generation of capilary in tumor tissues.Act on cox 2 inhibitor Former times dry goods can be used for preventing and treating tumour, U.S. FDA is used for human familial's adenoma in approval former times cloth anti-inflammatory drugs in 1999 Property polyp (FAP) assist in the treatment of, but its serious cardiovascular side effect limits its application, and the rofecoxib of Merck & Co., Inc. has been removed Market out.The bevacizumab for acting on VEGF inhibitor obtained the approval of FDA on 2 26th, 2004, was that first, the U.S. obtains It must ratify the medicine of the inhibition Tumor Angiongesis of listing.

Vascular endothelial growth factor (VEGF) is also known as Vascular Permeability Factor (VPF), is to have height in angiogenesis factor The special factor for promoting new vessels formation effect of degree, is the most important correlation factor that COX-2 promotees vascularization effect, swollen It plays an important role during tumor angiogenesis, is critically important factor during growth and metastasis of tumours.COX-2 may The expression of VEGF is raised by PGs approach, to participate in the generation of new vessels in tumor tissues, this may be that COX-2 promotes Tumour growth, the mechanism of infiltration, transfer.

Cancer immunotherapy is the research hotspot of field of cancer treatment, is located at first of " science " ten quantum jump in 2013.From So killing (NK) cell is the large granular lymphocyte that a group is different from T, bone-marrow-derived lymphocyte, belongs to a kind of independent lymphocyte. The cell mass is antitumor the first line of defence, without antigen presensitization can Direct Recognition and killing tumor cell, can send out The effect of specific killing target cell is waved, especially plays the role of quickly killing and dissolving to kinds of tumor cells.

The present invention illustrates triacontanol by selective depression COX-2 and VEGF, plays antitumor action.Test result Display: triacontanol can play antitumor action by selective depression COX-2, anti-new by inhibiting the expression of VEGF to play The effect that angiogenic generates.Meanwhile triacontanol can be improved body's immunity, dose-dependent activation NK cells in mice Activity, while the proliferation of mouse spleen lymphocyte and the generation of hemolytic antibody can be obviously promoted, antagonism caused by cyclophosphamide Leukocyte count reduce effect.

Present invention discloses triacontanols to cooperate as single active ingredient or with other medicinal ingredients as preparation Application in COX-2 and the drug and health care product of VEGF inhibitor.Tablet can be made, granule, hard capsule, soft capsule, do Suspension, freeze drying powder injection, oral solution, dripping pill or oral quick disintegrating tablet.Triacontanol is as its effective dose of single active ingredient Range is 5mg-2000mg.

Pharmacodynamics test:

1: the influence that triacontanol expresses nude mice COX-2

Experimental facilities: 5%CO₂Between incubator, aseptic experiment platform, centrifuge, refrigerator, ultra low temperature freezer, steril cell, surpass Pure water system, liquid nitrogen container.

Main agents: trypan blue, Tween-80 (product number: T8360), sodium carboxymethylcellulose (product number: C8621), fetal calf serum (South America blood source, product number: FBS-12A-100), trypsase (Article Number: M0043), RPMI- 1640 culture mediums (product number: C11875500bt, GIBCO), PBS, triacontanol (Kunming Longjin Pharmaceutical Co., Ltd., Lot number: 20140716) it, celecoxib (product number: 169590-42-5, Wuhan win sky Biotechnology Co., Ltd), is immunized Groupization kit: rabbit immunohistochemical kit (number: PV-9001, Beijing Zhong Shan Golden Bridge), DAB KIT 3ml (product number: ZLI-9018, Beijing Zhong Shan Golden Bridge)；Rabbit anti COX-1/Cyclooxygenase-1Polyclonal antibody (product number: 13393-1-AP, Proteintech), COX-2 epoxide hydrolase (product number: ZA-0515, China fir gold in Beijing Bridge).

Main material: human colon carcinoma HT-29 cell strain (No.1 Hospital Attached to the Chongqing Medical University's molecular weight tumor and apparent something lost Key lab, the Chongqing City Chuan Xue gives)；4~6 week old BALB/c Female nude mices 30 (weight 15-20g) are purchased from Chongqing medical courses in general University's Experimental Animal Center (credit number: SCXK (capital) 2014-0004).

The preparation of main agents:

Test-compound preparation method:

0.5% sodium carboxymethylcellulose: accurately weighing sodium carboxymethylcellulose 500mg and the distilled water 100ml boiled be added, It is stirred continuously, until liquid is clarified, room temperature stands a night.

The setting of triacontanol dosage:

High dose group: it accurately weighs 200mg triacontanol addition Tween-80 1ml and mixing is sufficiently stirred, 0.5% carboxylic is added Sodium carboxymethylcellulose pyce 12.3ml is sufficiently stirred until in uniform suspension 13.3ml, concentration 15mg/ml.

Middle dose group: it accurately weighs 200mg triacontanol addition Tween-80 1ml and mixing is sufficiently stirred, 0.5% carboxylic is added Sodium carboxymethylcellulose pyce 25.7ml is sufficiently stirred until in uniform suspension 26.7ml, concentration 7.5mg/ml.

Low dose group: it accurately weighs 200mg triacontanol addition Tween-80 1ml and mixing is sufficiently stirred, 0.5% carboxylic is added Methylcellulose 52.3ml is sufficiently stirred until in uniform suspension 53.3ml, concentration 3.75mg/ml.

The preparation of positive control medicine celecoxib:

200mg celecoxib powder is accurately weighed, Tween-80 1ml is added, mixing is sufficiently stirred, 0.5% carboxymethyl is added Sodium cellulosate 25.7ml is sufficiently stirred until in uniform suspension 26.7ml, concentration 7.5mg/ml.

Experimental procedure:

The influence that triacontanol grows transplanted tumor in nude mice

Cell culture and the foundation of Transplanted tumor model:

It is suspended with culture medium, takes 10 μ l cell suspensions, 10 μ l of trypan blue is added, cell counting board carries out living cell counting. 10mlPBS washes cell, 1000r/min, 3min, and 3 times.Single cell suspension is made in cell with the culture medium without serum, is adjusted thin Born of the same parents' concentration makes every 0.2ml cell suspension containing 3 × 10⁶A cell.

4-6 week old Female nude mice, weight 15-20g under aseptic condition, are again mixed cell suspension with 1ml syringe, Cell suspension 0.2ml is extracted, it is spare to drain air.In quasi- tumor formation point by inserting needle at caudal part 0.5-1cm, slowly by cell suspension Injection, totally 30.Close observation after kind tumor, records nude mouse weight and tumorous size after nude mice tumor formation, and observation nude mice grows shape Condition.

10th day transplanted tumor in nude mice tumor formation rate 100% after kind tumor.To transplanted tumor in nude mice size about 50-100mm³, 30 naked Mouse is divided into 5 groups, every group of 6 stomach-fillings: triacontanol low dose group (37.5mg/kg.d), middle dose group (75mg/kg.d), High dose group (150mg/kg.d), celecoxib group (75mg/kg.d), blank control group (physiological saline group), one time a day.It gives Dislocation of cervical vertebra method puts to death nude mice after medicine 4 weeks, takes tumor tissues.

With the major diameter (a) of vernier caliper measurement tumour, minor axis (b), following index: volume: V=ab is then calculated²/2；Suppression Ratio of outflow=[(V control group-V experimental group)/V control group] × 100%.

The processing of transplantable tumor sample and the expression of immunohistochemistry detection COX-1 and COX-2:

Transplantable tumor and normal tissue boundary are clear, soft, frangible in flesh of fish shape, matter, PBS cleaning, one by one number rear portion It is put into specimen tube, is saved in liquid nitrogen.A part fixes 24-48h in 4% paraformaldehyde.Dehydration after sample is sufficiently fixed, Transparent, embedding is sliced to be further processed.

Row HE dyes and presses PV-9001 immunohistochemistry (Biotin-StrePtavidin HRP Detection after slice Systems) and the operating procedure of ZLI-9018DAB Kit carry out COX-1 (antibody concentration 1:200), COX-2 (antibody concentration 1: 200) immunohistochemical staining.Mean OD value using Image Pro Plus software measurement each group high power lens picture is (total OD value/Area).

Statistical method: using 22 statistical software of IBM SPSS Statistics to 400 times of pictures of tumor size and each group Mean OD value (total OD value/Area) row one-way analysis of variance, compare between group two-by-two and analyzed using Post-hoc (LSD).With P < 0.05 is statistically significant for difference.

Experimental result: the influence (table 1) that triacontanol grows transplanted tumor in nude mice.

1 each group nude mice volume (mean ± SD mm of table³) and tumour inhibiting rate % expression

Group	Control group	Low dose group	Middle dose group	High dose group	Celecoxib group
						Nude mice n	6	6	6	6	6
Volume	799±430	458±227	656±221	429±262	729±289
						Tumour inhibiting rate (%)		42.68	17.90	46.30*	8.76

Compared with the control group, tumour growth significantly slows down triacontanol high dose group, tumour inhibiting rate 46.30%,^*(P< 0.05)。

The comparison (table 2) of each group transplantable tumor COX-1, COX-2 expression.

HE dyes visible intermediate large stretch of necrotic tissue, the visible inflammatory cell being dispersed in downright bad edge, necrosis under each group mirror Organization edge part is not downright bad tumor tissues.Transplanting oncocyte, which arranges, under light microscopic loses normal configuration, cell dense distribution, Nucleus indigo plant dye, nuclear proportion significantly increase, and cell atypia is significant.COX-1 and COX-2 dyeing, in cytoplasm visible brown color or Person's yellow pigmented section is positive staining.

The mean OD value of 2 each group immunohistochemistry of table is indicated with (mean ± SD)

OD value measurement is carried out to the immunohistochemical staining slice of COX-1, variance analysis is shown, with control group phase Than the expression difference of each group COX-1 is not statistically significant (P > 0.05).

OD value measurement is carried out to the immunohistochemical staining of COX-2 slice, variance analysis shows (table 2), and right It is compared according to group, the expression of celecoxib group, low dose group, middle dose group, high dose group COX-2 is significantly lowered,^*(P< 0.05)。

Experiment conclusion: compared with the control group, high dose group triacontanol significantly inhibits work to the growth of transplanted tumor in nude mice With.Each dosage group of triacontanol and celecoxib group have inhibiting effect to the expression of HT-29 transplanted tumor in nude mice COX-2, but right The expression unrestraint of COX-1 acts on.

The prediction of the Interactions Mode of triacontanol and COX-2, utilizes the cocrystallization structure of arachidonic acid and COX-2 1CVU, molecular docking triacontanol and COX-2 possible binding pattern (there are two types of) and with arachidonic binding pattern Compare, conclusion is that the binding pattern of the two is closely similar, and triacontanol is also likely to be the inhibitor of COX-2.

2: triacontanol inhibits the growth of LOVO colon cancer cell transplanted tumor in nude mice and shift experiment

Experimental facilities: ibid.

Major experimental reagent: ibid.Celebret (Western music fort) (batch number: M72189；Subpackage fills lot number: N07162), immunohistochemical kit: rabbit immunohistochemical kit (number: SP-9001, Beijing Zhong Shan Golden Bridge), VEGF Antibody 50ul (product number: 19003-1-AP, proteintech)；PTGS2 Rabbit polyclonal Antibody 50ul (product number: 12375-1-AP, proteintech), HE staining kit (product number: M2314), 1% yellow Jackets.

The preparation of main agents: ibid.Wherein celecoxib concentration is 6mg/ml.

Main material: human colon carcinoma LOVO cell strain (No.1 Hospital Attached to the Chongqing Medical University's molecular weight tumor and apparent something lost Key lab, the Chongqing City Chuan Xue gives)；4~6 week old BALB/c Female nude mices 50 (weight 15-20g) are purchased from Chongqing medical courses in general University's Experimental Animal Center (credit number: SCXK (capital) 2014-0004).

Experimental procedure:

Cell recovery and culture: taking cryopreservation tube, is quickly put into 37 DEG C of warm water, gently shakes and enable its fast melt, to surplus It is taken out when lower soya bean size, melts waste heat.15ml centrifuge tube one is taken out, 5ml DMEM/F12 is previously added and trains completely Base, 1000r/min centrifugation are supported, 3min removes supernatant.

It takes above-mentioned LOVO cell that 1ml DMEM/F12 complete medium is added to mix, move into culture bottle, 4ml culture is added Base polishing, is put into CO₂Stationary culture in incubator.It need to be changed night when culture medium color becomes yellow, change the liquid time 1 day, passed on Time is 3 days, is passed in the ratio of 1:3.

Nude Mouse Model is established with LOVO cell:

It is suspended with culture medium, takes 10 μ l cell suspensions, 10 μ l of trypan blue is added, cell counting board is counted.10mlPBS Wash cell, 1000r/min, 3min, 3 times.Single cell suspension is made in cell with the culture medium without serum, cell concentration is adjusted to make Every 0.2ml cell suspension contains 3 × 10⁶A cell.

4-6 week old Female nude mice, weight 15-20g under aseptic condition, are again mixed cell suspension with 1ml syringe, Cell suspension 0.2ml is extracted, in quasi- tumor formation point by inserting needle at caudal part 0.5-1cm, slowly cell suspension is injected, pulls out needle, iodine Lie prostrate cotton swab oppress entry point 2min, totally 50.

The 10-14 days transplanted tumor in nude mice tumor formation rates 80% after kind tumor.Occurs spherical, ellipse, no at the kind tumor of nude mice back The mass of rule, clear border, quality are tough compared with normal surrounding tissue.

It is 50-100mm by tumor average volume 18 days after kind tumor³35 nude mices be equally divided into 5 groups: triacontanol is high Dosage group (150mg/kg.d), middle dose group (75mg/kg.d), low dose group (37.5mg/kg.d), celecoxib group (60mg/ Kg.d), blank control group (physiological saline group).Stomach-filling is carried out according to dose requirements respectively, each stomach-filling volume is 0.2ml, often Day 1 time.Experiment terminates to calculate gross tumor volume and tumour inhibiting rate.

Transplantable tumor sample disposal: the 1% yellow Jackets intraperitoneal injection of anesthesia nude mice of 0.2ml, cervical dislocation is by nude mice Put to death, take out transplantable tumor, the fixed tissue of paraformaldehyde by dehydration, transparent, waxdip and embedding and etc. paraffin embedding is made Block.HE is dyed after slice, by SP-9001SPlink Detection Kits (Biotin-Streptavidin HRP Detection Systems) and ZLI-9018DAB Kit operating procedure carry out VEGF (antibody concentration 1:200) immune group Weave chemistry dyeing.

Statistical method: ibid.

Experimental result: the influence that triacontanol grows transplanted tumor in nude mice and shifts.Low dose group, middle dose group, high agent Amount group, celecoxib group tumour inhibiting rate be respectively 33.33%, 52.05%, 70.77%, 61.60%.Compared with the control group, low There was no significant difference (P > 0.05) for dosage group transplantable tumor volume.Middle dose group, high dose group, celecoxib group and control group ratio Compared with growth of transplanted human significantly slows down, and P < 0.05 * is shown in Table 3.

Nude mouse tumor volume size (mean ± SD mm after 3 treatment end of table³) and its tumour inhibiting rate %

Group	Control group	Low dose group	Middle dose group	High dose group	Celecoxib group
						Nude mice n	7	7	7	7	7
Volume mm3	1047±647	698±448	502±304	306±237	402±215
						Tumour inhibiting rate %	-	33.33	52.05*	70.77*	61.60*

Nude mice liver: after nude mice is put to death, liver is observed, it is seen that have the canescence tubercle to differ in size on liver, compare Group hepatic metastasis tubercle is larger, and there are many number, and 4 are almost covered with liver, and 3 more close to liver edge node number, nearly center Node number is less, can not count.Low dose group hepatic metastasis tubercle is more, has 2 nude mice livers to be covered with tubercle, 2 nearly livers Dirty marginal tubercle number is more, can not count, and remaining 3 average node numbers are 5-10.Middle dose group and celecoxib group Hepatic metastasis tubercle is smaller, and average out to 2-7/only.High dose group hepatic metastasis tubercle is smaller, and 2 have not seen tubercle, remainder 5 Average out to 2-3/only.

Transplantable tumor and the HE of liver dyeing: each group tumor tissues visible intermediate large stretch of necrotic tissue, necrosis under microscope The visible inflammatory cell being dispersed in edge, necrotic tissue marginal portion are not downright bad tumor tissues.Oncocyte row is transplanted under light microscopic Column lose normal configuration, disordered arrangements, cell dense distribution or line up glandular tube shape, and nucleus indigo plant dye, nuclear proportion significantly increases, Cell atypia is significant.There is the shape different from normal liver tissue in the visible liver organization of microscopically observation in each group hepatic tissue The irregular lumps of state or sheet cell, cell atypia is larger, and caryoplasm ratio is big, and nucleus is not of uniform size.

The ImmunohistochemistryResults Results of transplantable tumor VEGF: VEGF immunohistochemistry distribution is wider, and cytoplasm, karyon and interstitial have Expression.OD value measurement is carried out to the immunohistochemical staining slice of VEGF, variance analysis is shown, compared with the control group, The expression of small dose group VEGF reduces, but not statistically significant (P > 0.05), celecoxib group, middle dose group, high dose group The expression of VEGF is significantly lowered,^*P < 0.05 is shown in Table 4.

The mean OD value of 4 each group VEGF immunohistochemistry picture of table is indicated with mean ± SD

Group	Sample size (n)	Average IOD
			Blank control group	7	0.10750±0.06755
Low dose group	7	0.07556±0.03401
			Middle dose group	7	0.04782±0.02808*
High dose group	7	0.03952±0.02121*
			Celecoxib group	7	0.05301±0.04789*

Experiment conclusion: comparing with negative control group, and various dose group triacontanol is to metastatic Transplanted Colon Cancer in Nude Mice Growth have different degrees of inhibiting effect.High dose group and middle dose group triacontanol are to LOVO cell transplanted tumor in nude mice The expression of VEGF has inhibiting effect, can inhibit colon cancer nude mice hepatic metastases.

3: triacontanol is to transplanting in the efficacy experiment of the human tumor gastric cancer MNK-45 of nude mice

Experimental material: Tween 80,0.5%CMC-Na, physiological saline.Cyclophosphamide (CTX), the permanent auspicious limited public affairs of pharmacy in Jiangsu Department, lot number: 11030921, with physiological saline solution.Knurl source: it is passed on by Shanghai Institute of Pharmaceutical Industry's Pharmacological Evaluation research center It maintains.Knurl source: being passed on by Shanghai Institute of Pharmaceutical Industry's Pharmacological Evaluation research center and maintained, and external cell strain is converted to exempting from Epidemic disease defect nude mouse interior generation is to be grown to using after the second generation.

Experimental animal: nude mouse is provided by Shanghai Si Laike experimental animal responsibility Co., Ltd.Quality certification number: SCXK (Shanghai) 2003-0003.Nude mouse is 6 week old.Male and female all can, every time use same gender mouse.Number of animals: test group and positive control Group nude mouse every group 6, negative control group are 12.Animal feeding environment: nude mouse is placed in SPF condition in laminar-flow rack and raises, Applied feed, padding, cage tool and instrument of contact etc. should all use after autoclave sterilization.

Test key step: heteroplastic transplantation model is after the human tumor 2nd generation for taking tumor growth vigorous under aseptic condition Knurl source is prepared into tumor mass, inoculates about 2mm in nude mouse right axillary³/ mouse, next day are grouped at random, touch about to tumour 100mm³Start to be administered, tests the size and mice with tumor weight of each group mice with tumor tumour growth every 3-5 days dynamics, surveyed with slide calliper rule The minor axis (a) and major diameter (b) of mice with tumor tumour are measured, (a is pressed²× b)/2 formula calculating gross tumor volume.Experiment about surrounding or so is put to death Tumor control rate is calculated according to the following formula in groups of animals, the tumour weighing taken:

Tumor control rate %=[(control group average knurl weight-administration group average knurl weight)/control group average knurl weight] × 100%

The effect of drug combination calculates Q value according to Jin's formula:

Q=Ea+b/ (Ea+Eb-Ea × Eb)

Ea+b is the tumour inhibiting rate of drug combination, and Ea and Eb are respectively the tumour inhibiting rate of A medicine and B medicine.As Q value 0.85-1.15 is It is added (+), ﹥ 1.15 is enhancing (++).

5 triacontanol of table is to human gastric cancer MKN-45 nude mouse xenograft model antitumor curative effect

Compared with solvent control group: P < 0.01 * P < 0.05, * *.

6 triacontanol of table merges cyclophosphamide to human gastric cancer MKN-45 nude mouse xenograft model antitumor curative effect

Compared with solvent control group: P < 0.01 * P < 0.05, * *.

4: influence of the triacontanol to mice spleen lymphocytes proliferation and NK cell activity

Triacontanol preparation method: accurately weighing required sample, to be gradually added into 0.5% after a small amount of Tween 80 hydrotropy CMCNa solution is diluted to required concentration after mixing well.Administered volume is 0.5ml/20g mouse.Experimental material: tween 80,0.5%CMC-Na.Lentinan, Wuhan Dior Medicine Industry Co., Ltd, lot number 20100202.DMEM complete medium, includes 10% newborn bovine serum (GIBCO BRL), mercaptoethanol, HEPES etc..³H-TdR: radioactive concentration: 1mci/ml is purchased from Shanghai Nuclear research institute.Concanavalin A: 50 μ g/ml are purchased from Sigma company.YAC-1 cell: Shanghai Institute of Pharmaceutical Industry's pharmacology The passage of evaluation study center maintains.

Experimental animal: strain: C57BL/6 (SPF grades) is purchased from Shanghai Si Laike experimental animal responsibility Co., Ltd, animal Quality certification number: SCXK (Shanghai) 2007-0005, weight are 18-20g/6 week old.Gender: male.Number of animals: test group, feminine gender are right According to group and positive controls every group 10.

Experimental method: the measurement of NK cell activity:

C57BL/6 mouse is grouped at random, every group 10, according to dosage successive administration two weeks, after last time is administered Put to death mouse, take spleen under aseptic condition, be placed in small container, shredded spleen with surgical scissors, separate cell, through several times from Right sedimentation separation splenocyte, is made cell suspension with DMEM culture medium, splenocyte is adjusted to 1 × 10⁶/ ml is added 100 μ l and arrives Effector cell is used as in 96 orifice plates.The YAC-1 cell in growth logarithmic phase is separately taken, adjustment cell concentration is 1 × 10⁴/ ml makees For target cell, the concentration with the ratio between target cell and effector cell for 1:100 is added in 96 porocyte culture plates, while 0.5 μ is added The hole ci/³H-TdR isotope.10 multiple holes of every group of setting, cultivate 24 hours in 37 DEG C of carbon dioxide incubators, use bull Organelle collects cell, and CPM value is measured on liquid scintillation instrument, calculates specificity and percentage (Pi) is inhibited to indicate NK cell activity.Its Middle Pi=1- (experimental group mixes CPM value/control group and mixes CPM value) × 100%.

The measurement of mice spleen lymphocytes proliferation:

C57BL/6 mouse is grouped at random, every group 10, according to dosage successive administration two weeks, after last time is administered Put to death mouse, take spleen under aseptic condition, be placed in small container, shredded spleen with surgical scissors, separate cell, through several times from Right sedimentation separation splenocyte, is made cell suspension with DMEM culture medium, splenocyte is adjusted to 1 × 10⁷/ ml, sterile 96 100 μ l of splenocyte is added in well culture plate, while the concanavalin A 50ul of 1mg/ml is added, in 37 DEG C of carbon dioxide incubators 0.5 hole μ ci/ is added after 48 hours in middle culture³H-TdR isotope continues culture 24 hours.Every group of experiment setting 10 is multiple Hole, control group replace splenocyte with culture medium.Cell is collected after culture in porous cell collector, 5%TCA is fixed, Dehydrated alcohol is dehydrated, and CPM value is measured on liquid scintillation instrument.

The result shows that triacontanol sample is capable of the activity of dose-dependent activation NK cells in mice, while also can It is obviously promoted the proliferation of mouse spleen lymphocyte.See Table 7 for details~and 8.

Influence of 7 triacontanol of table to mice spleen lymphocytes proliferation

Compared with the control group: P < 0.01 * P < 0.05, * *.

8 triacontanol of table is on the active influence of NK cells in mice

Group	CPM value	Pi (%)
			Triacontanol high dose group	6856±939.3**	66
Triacontanol middle dose group	6890.2±602.8**	65
			Triacontanol low dose group	9759.8±648.0**	51
Lentinan	6276.4±559.5**	68
			Control	19919.9±622.8

Compared with the control group: P < 0.01 * P < 0.05, * *.

5: influence of the triacontanol to mouse hemolysin

Experimental material: Kunming mouse, male, 18-20g are provided by Shanghai Si Laike animal responsibility Co., Ltd.It is qualified Card number: SCXK (Shanghai) 2007-0005.Test dose: test dose design are as follows: 150mg/kg, 75mg/kg, 37.5mg/kg.Sun Property medicine: lentinan, lot number: 20100202, production unit: Wuhan Dior Medicine Industry Co., Ltd.Test dose: 50g/kg.It gives Medicine approach and capacity: gastric infusion, 0.5ml/20g.

Experimental method: mouse is grouped at random, high, medium and low each 3 dosage groups of triacontanol, positive drug group, blank model Group.Every group 10.Chicken red blood cell is made into 5% suspension with brine 3 times, and complement is mixed with 2 guinea pig serums It closes, is made into 10% concentration with physiological saline.Every mouse is injected intraperitoneally 5% physiological saline chicken red blood cell suspension 0.2ml and is immunized, Administration is started simultaneously at, 7 days after being immunized, last time administration 1h plucks eyeball and takes blood, is centrifuged, takes serum normal saline dilution 100 Times, dilute serum 1ml is taken, is mixed with 5% chicken red blood cell suspension 0.5ml, 10% complement 0.5ml, 37 DEG C of insulating box heat preservations After 30min, stopped reaction in 0 DEG C of refrigerator is set.Centrifugation takes supernatant colorimetric at 722 spectrophotometer 540nm, and it is close to survey record light It spends (OD), separately sets the blank control of not increase serum, the benchmark of tune " 0 " is using OD value as judgement blood when taking its supernatant as colorimetric The index of clear hemolysin, compares the difference of each group.

Experimental result: the high, medium and low dosage group of triacontanol, which generates mouse hemolytic antibody to have, is obviously promoted effect, Illustrate that the medicine can enhance humoral immunity, is shown in Table 9.

Influence of 9 triacontanol of table to mouse hemolysin

Group	Dosage (mg/kg)	Number of animals (only)	OD value
				Triacontanol high dose group	150	10	2.611±0.646**
Triacontanol middle dose group	75	10	2.477±0.595**
				Triacontanol low dose group	37.5	10	2.250±0.702**
Positive drug	50	10	2.398±0.789**
				Blank model group	-	10	1.692±0.257

Each group is compared with blank model group: * P < 0.05；* P < 0.01.

6: influence of the triacontanol to macrophage phagocytosis of mice

Experimental material: ibid.

Experimental method: mouse is grouped at random, and every group 10.High, medium and low each 3 dosage groups of triacontanol, positive drug, sky Each one group of white control.Start to be administered within second day after grouping, successive administration 1 week, latter hour mouse peritoneal is being administered for the last time 2% chicken red blood cell of interior injection (through removing fibrin, physiological saline is repeatedly washed) 0.2ml/ only, puts to death mouse after 4 hours, use Flushing liquor, centrifugation are collected in normal saline flushing abdominal cavity, and incline supernatant, collect cell smear, are contaminated after airing with Wright's staining liquid Color, oil is under the microscope.100 macrophage phagocytosis chicken red blood cell numbers are counted, formula calculating is pressed.(1) phagocytic percentage=gulp down Bite number of macrophages/100 number of macrophages of chicken red blood cell, chicken red blood cell number/100 of (2) phagocytic index=swallowed Number of macrophages.

Experimental result: the middle and high dosage group of triacontanol, which has, promotes Turnover of Mouse Peritoneal Macrophages phagocytosis, and has one Determine dosage correlation.Positive drug, which also has, promotes Turnover of Mouse Peritoneal Macrophages phagocytosis, is shown in Table 10.

Influence of 10 triacontanol of table to macrophage phagocytosis of mice

Each group is compared with model group: * P < 0.05；* P < 0.01.

7: the leukogenic effect that triacontanol causes Lewis murine interleukin number to reduce cyclophosphamide

Experimental animal: C57BL/6 mouse, male, 18-20g are provided by Shanghai Si Laike animal responsibility Co., Ltd.It closes Lattice card number: SCXK (Shanghai) 2007-0005.

Experimental material: Tween 80,0.5%CMC-Na.Reference substance: cyclophosphamide, Jiangsu Heng Rui pharmaceutical Co. Ltd, lot number 11030921.Knurl source: Lewis lung cancer tumor model is passed on by Shanghai Institute of Pharmaceutical Industry's Pharmacological Evaluation research center and is maintained.

Experimental method: joint cyclophosphamide causes the foundation of tumor-bearing mice bone marrow suppression model

Eugonic tumour is taken, a homogenate method is prepared into cell suspension about 5~10 × 10 under aseptic condition⁶/ ml cell Concentration.Every mouse right axillary inoculates 0.2ml.It is inoculated with the 3rd day (150mg/kg) and the 5th day (100mg/kg), just except blank Normal combined sample high dose is administered alone outside group, remaining each group gives CTX respectively and combined chemotherapy is caused to cause the suppression of tumor-bearing mice marrow Simulation.

Grouping and medication: it takes 10 mouse to begin as basic leucocyte test in experiment at random, remaining is moved Object causes transplanted tumor model with the inoculation of Lewis lung cancer and is grouped at random, every group of 10 animals, and opens in the inoculated tumour same day Begin to be administered.Triacontanol oral administration, CTX intraperitoneal injection.

(1) cyclophosphamide group (the 3rd day after inoculation, 150mg/kg；The 5th day after inoculation, 150mg/kg).

(2) triacontanol+cyclophosphamide group 150mg/kg (the inoculation same day starts to be administered, until experiment terminates).

(3) triacontanol group 150mg/kg (the inoculation same day starts to be administered, until experiment terminates).

(4) blank group (the inoculation same day starts to coordinative solvent, until experiment terminates).

Observation index: experiment takes 10 mouse to carry out retroorbital venous clump puncture blood collecting at random when starting, and haemocyte is automatic Analyzer (HEMAVET-950) tests blood routine.Hereafter start to take at random for every 3 days in the next day of cyclophosphamide after the last administration 5 mouse are taken a blood sample with above-mentioned same procedure and test blood routine, until each group leucocyte restores normal.

Experimental result: the 6th day after on-test of result, triacontanol+cyclophosphamide group animal leukocyte count are The leukocyte count of the 109.09% of intact animal, positive control cyclophosphamide modeling group animal is down to the 6.21% of intact animal. Experiment shows that the leukocyte count of triacontanol group animal is apparently higher than the leukocyte count of positive control cyclophosphamide modeling group animal, There is significant difference (P < 0.01) between two groups.Illustrate this dosage and when phase point triacontanol+cyclophosphamide group have it is obvious Antagonism cyclophosphamide cause Lewis lung cancer in mice leukocyte count reduce effect.See Table 1 for details 1, Fig. 1.

11 triacontanol of table tests the leukogenic effect that cyclophosphamide causes tumor-bearing mice leucocyte to reduce

P < 0.01 * * compared with negative control, (the range of normal value 1.8-10.7K/uL of leucocyte).

8: the anti-carrageenan proinflammatory effect research of triacontanol

Test medicine: low middle high three dosage groups are respectively 50,100,200mg/kg.Preparation method: by triacontanol sample Product are ground into powder, and weigh 2g, appropriate Tween-80 emulsification, and physiological saline solution is simultaneously settled to 50ml, and obtained concentration is The high dose test drug of 40mg/ml, doubling dilution obtain concentration be respectively 20mg/ml, 10mg/ml middle dosage and low dosage by Try object.Capacity: 1ml/200g rat is administered.

Reference substance: Indometacin Enteric-coated Tablets, source: upper Hisense's friendship the Yellow River pharmaceutical Co. Ltd, lot number: 120102, specification: 25mg, drug dose: 10mg/kg.Preparation method: taking a piece of reference substance, and grind into powder is molten with 5% sodium bicarbonate solution 12.5ml is solved and is settled to, acquisition concentration is 2mg/ml.Capacity: 1ml/200g is administered

Reagent name: carrageenan, source: Adamas-beta, lot number: preparation method: P1050992 accurately weighs angle Dish glue 0.15g is pitched, physiological saline is dissolved and is diluted to 15ml, obtains 1% Carrageenan solution.Rat LTB4ELISA kit, lot number: 20131001A.

Experimental animal: strain: SD rat, source: Shanghai Si Laike experimental animal responsibility Co., Ltd, the animal quality certification Number: SCXK (Shanghai) 2007-0005, weight: 180-200g, gender: male, animal packet: experiment before by animal according to weight with Machine is divided into 5 groups (n=8), respectively high, normal, basic three dosage groups of model group, positive drug group, triacontanol.

Experimental method: three daily stomach-fillings of dosage group of test drug are primary, successive administration five days, are administered one hour within the 6th day Afterwards, rat anesthesia, the carrageenan of intrathecal 0.3ml 1%.It causes inflammation after 5 hours, measures diffusate volume, part is taken to be placed in (aspirin containing 10M, 4U/ml heparin) 2000g centrifugation obtains supernatant after ten minutes in centrifuge tube, and ELISA method measures LTB4 Concentration.

Experimental result: as can be seen from Table 12, compared with model group, Indomethacin group can be readily apparent that reduce rat Pleural fluid exudation (^**P < 0.01), low middle high three dosage groups of triacontanol also have very the reduction of rat pleural fluid volume Obvious action (^**P < 0.01,^**P < 0.01,^**P < 0.01), wherein middle and high dosage group shows and becomes apparent than positive drug Effect.In addition, applying ELISA method twice, LTB4 concentration is not detected, thus it is speculated that reason may be that kit sensitivity is inadequate, or It is horizontal that method shown in person's kit is unsuitable for the LTB4 surveyed in animal chest fluid.The country is not bought at present uses as shown in document The kit of isotope labelling method detection.

The rat pleural effusion volume change (ml) of 12 triacontanol Carrageenan of table induction

Compared with model group:^**P〈0.01。

Experiment conclusion: three dosage groups of triacontanol can substantially reduce the exudation of rat pleural fluid, have centainly Anti-inflammatory effect.ELISA method does not measure the variation of LTB4 level.

9: influence of the triacontanol to granuloma induced by implantation of cotton pellets

Test medicine: ibid.

Reference substance: ibid.Drug dose: 3mg/kg.Compound concentration is 2mg/5ml.

Experimental article: preparing cotton balls, and each weight is 50mg, uses after sterilizing.

Experimental animal: ibid.

Experimental method: oxter implantation 50mg cotton balls (sterilized) after rat anesthesia, according to drug dose, daily stomach-filling 1 time, It is put to death after 6 days and takes out cotton balls, weighed through 60 DEG C of drying, compare weight.

Experimental result: as can be seen from Table 13, compared with model group, Indomethacin group can be readily apparent that reduce cotton balls Granuloma weight (^**P < 0.01), low middle high three dosage groups of triacontanol also have the reduction of granuloma induced by implantation of cotton pellets weight very aobvious Work effect (^**P < 0.01,^**P < 0.01,^**P < 0.01), and show that a certain amount imitates correlation.

Influence (g) of 13 triacontanol of table to granuloma induced by implantation of cotton pellets weight

Compared with model group:^**P〈0.01。

Experiment conclusion: as can be seen from Table 13, compared with model group, three dosage groups of triacontanol can reduce rat cotton balls Granuloma weight, Inhibit proliferaton reaction, triacontanol have some improvement for inflammation.

Detailed description of the invention

Fig. 1 is the leucocyte dynamic change figure that triacontanol causes tumor-bearing mice leucocyte to reduce cyclophosphamide.

Specific embodiment

Embodiment 1:

50g triacontanol and 150g polyethylene glycol, 80g sodium carboxymethylcellulose are weighed, mixing is melted at 55 DEG C, stirred It mixes, grind up after cooling.Hard capsule is made, totally 1000, every 50mg containing triacontanol.

Embodiment 2:

100g triacontanol and 250g polyethylene glycol, 120g sodium carboxymethylcellulose are weighed, mixing melts at 55 DEG C, Stirring, grind up after cooling.Hard capsule is made, totally 1000, every 100mg containing triacontanol.

Claims (1)

1. application of the triacontanol in the drug that preparation treats or prevents COX-2 and vegf expression exception related disease, described COX-2 and the extremely relevant disease of vegf expression be that gastric cancer and immune function reduce.