CN102114022A

CN102114022A - Application of oleanolic acid in resisting colon cancer and pharmaceutical preparations thereof

Info

Publication number: CN102114022A
Application number: CN2010102926849A
Authority: CN
Inventors: 林秀坤; 刘海洲; 廖德忠
Original assignee: Individual
Current assignee: Individual
Priority date: 2010-09-27
Filing date: 2010-09-27
Publication date: 2011-07-06

Abstract

The invention discloses a novel pharmaceutical application of oleanolic acid in resisting colon cancer, and also discloses clinically-acceptable pharmaceutical preparations made with oleanolic acid as raw material, such as tables, capsules, pills and injection. The pharmaceutical preparation of oleanolic acid contains 1-99% of oleanolic acid and 99-1% of pharmaceutical excipients (including other pharmaceuticals for compatibility). Oleanolic acid and the pharmaceutical preparations thereof provided by the invention have good effect in resisting colon cancer, which manifested by the distinct in vitro inhibitory activity to human colon cancer cells and remarkable inhibitory activity to transplantation tumor cells of nude mice. In addition, since the active components of the pharmaceutical preparations are extracts of traditional Chinese medicine, the pharmaceutical preparations have low toxic and adverse effects compared to chemotherapeutic drugs.

Description

The effect of resistive connection intestinal cancer and the pharmaceutical preparation thereof of oleanolic acid

Technical field

The present invention relates to application and the pharmaceutical preparation thereof of oleanolic acid (Oleanolic acid) aspect resistive connection intestinal cancer, belongs to the field of Chinese medicines.

Background technology

Oleanolic acid (Oleanolic acid) is a pentacyclic triterpenoid, is present in the various plants with the form of episome and glycocide.The main leaf that extracts source Oleaceae plant olive, the Fructus Ligustri Lucidi fruit; Gentianaceae plant Herba Swertiae Mileensis herb, Swertia mussotii Franch.; Leaf, the root of the big star celery of samphire; Wu adds the root bark and the peel of stem of the Zhi Wu of section Cortex araliae chinensis; The big seed Radix Hemsleyae Macrospermae of cucurbitaceous plant, lovely Radix Hemsleyae Macrospermae, the tuber of Chinese Radix Hemsleyae Macrospermae.

The pharmacologic action that oleanolic acid has been reported mainly contains hepatoprotective and falls enzyme, promotes liver cell regeneration, antiinflammatory, heart tonifying, diuresis also have effects such as blood sugar lowering, blood fat reducing, calmness, is the effective ingredient of medicines such as exploitation treatment hepatopathy and blood sugar lowering.We find that in screening antitumor drug process oleanolic acid has the effect of stronger inhibition colon cancer cell and solid tumor growth thereof.

Before the present invention, do not see with the oleanolic acid to be raw material research pharmaceutical dosage form and the report that is used to prevent and treat the colon cancer disease.Oleanolic acid is a pentacyclic triterpenoid, and is almost non-toxic, and natural resources of Chinese medicinal materials is very abundant, is a kind of very promising antitumor drug.

The chemical name of oleanolic acid (Oleanolic acid): 3 β-hydroxy-olea-12-en-28-oic acid; Molecular formula C ₃₀H ₄₈O ₃Molecular weight: 456.71.

Chemical structural formula:

Summary of the invention

One of purpose of the present invention has provided the pharmaceutical preparation of oleanolic acid;

Two of purpose of the present invention has provided the antineoplastic new usage of oleanolic acid.

The objective of the invention is to be achieved through the following technical solutions:

Pharmaceutical preparation of the present invention contains the oleanolic acid of 1%-99% and the excipient of 99%-1% (medicine that comprises other adapted), preferably contain the oleanolic acid of 20%-80% and the excipient of 80%-20% (medicine that comprises other adapted), preferably contain the oleanolic acid of 60%-70% and the excipient of 40%-30% (medicine that comprises other adapted).

Press practice of pharmacy, oleanolic acid of the present invention can be prepared into the various clinical pharmaceutical dosage form as the resistive connection bowelcancer medicine, comprise the dosage form of oral formulations or parenterai administration.Said oral formulations selects any in tablet, capsule, pill, granule, microcapsule tablet, suspensoid, drop pill, oral liquid; Said parenterai administration dosage form is selected from a kind of in the middle of injection, aerosol, suppository or the subcutaneous administration dosage form.

Adjuvant in the resistive connection bowelcancer medicine of the present invention is meant conventional excipient, as solvent, disintegrating agent, correctives, antiseptic, coloring agent, binding agent etc.The medicine that other compatibility in the antitumor drug of the present invention is used, the oleanolic acid that refers to effective dose is certain medicine material, again compatibility other allowed the Chinese medicine or the chemical drugs that share.

Oleanolic acid pharmaceutical preparation of the present invention has the effect of resistive connection intestinal cancer, is to be confirmed by following pharmacodynamics test.

Test example 1 has been investigated the inhibitory action of oleanolic acid to external colon cancer cell

Cell: human colon cancer cell (HCT-116).For ocean institute of Chinese Academy of Sciences marine drug laboratory provides.

Medicine and reagent: ocean institute of oleanolic acid (Oleanolic acid) Chinese Academy of Sciences marine drug laboratory provides white powder; 5-fluorouracil (5-FU) Nantong elite pharmaceutical Co. Ltd product, lot number is 100402; MTT(SIGMA, USA); DMSO(SIGMA, USA); Trypsin SIGMA, USA); RPMI1640(GIBCO, Invitragen Co., USA); DMEM(GIBCO, Invitragen Co., USA); The top grade hyclone (GIBCO, Invitragen Co., USA); Superfine hyclone Fetal Bovine Serum(Hyclone).

Instrument: ultra cold storage freezer (Nature, USA), cell culture incubator (SANYO, Japan), inverted microscope (NIKON, Japan), microplate reader (Bio-Tek Instruments, Inooski, VT, USA), PH counts (Thermo orion, USA), superclean bench (FLC-Harbin Dong Lian instrument plant).

Experimental technique mtt assay: tumor cell line is gone down to posterity according to the conventional method cultivation, collect the exponential phase cell, regulate concentration of cell suspension 5 * 10 ⁴About individual/ml.Cell suspension is added in 96 well culture plates, and each hole adds 180 μ l.After placing 37 ℃ of constant incubators to cultivate 24 h, experimental group adds each concentration oleanolic acid (with 5-FU as positive controls) 20 μ l/ holes, establishes 4 parallel holes for every group, and establishes blank well (only adding the medicinal solvent of dissolving) with zeroing.37 ℃ of incubators, cultivate 48 h after, with liquid-transfering gun liquid in 96 orifice plates is cleaned every hole, back and adds MTT(1 mg/ml) 30 μ l, put CO ₂Incubator is cultivated 4 h for 37 ℃, supernatant discarded, and every hole adds DMSO 150 μ l, puts shaking table and shakes up 30 min, detects under 492 nm with microplate reader, utilizes the SPSS statistical software, calculates cell mortality, asks for IC ₅₀

Result of the test shows, through the SPSS software statistics, the oleanolic acid of variable concentrations has in various degree inhibitory action to the human colon cancer cell (HCT-116) that is used to test, and is certain dose-dependence, and the oleanolic acid of variable concentrations is to suppression ratio, the IC of colon cancer cell ₅₀Value sees table 1 for details.

The oleanolic acid of table 1 variable concentrations is to the suppression ratio of colon cancer cell

Test example 2 oleanolic acid are to the tumor-inhibiting action of human colon cancer cell transplanted tumor in nude mice

Laboratory animal: the BALB/c nude mice is available from Shanghai Slac Experimental Animal Co., Ltd., and animal licence numbering: SYXK (Shanghai) 2010-0047, totally 20, in 7 ~ 9 ages in week, body weight 20 ~ 22g is male Mus.Animal feeding and experiment are raised in the Affiliated Hospital of Qingdao University Experimental Animal Center for no special pathogen level barrier system.

Tumor cell inoculation: the human colon cancer cell of the trophophase of taking the logarithm (HCT-116) is made tumor cell suspension (1 * 10 ⁷Individual/as ml), to implant BALB/c male nude mouse armpit subcutaneous (about 0.3ml/ only), situations such as inoculation back routine observation mice spirit, diet and defecation.After going down to posterity once, with the aseptic taking-up of tumor tissues and be cut into big or small identical 2mm ³Fritter, it is subcutaneous to be seeded in the nude mice armpit with the trocar.

Test grouping and method: the major diameter of vernier caliper measurement tumor nodule (a), minor axis (b), by formula V=a * b ²* 0.52 calculates gross tumor volume, when subcutaneous transplantation tumor length to 100 mm ³After, selecting tumor nude mice of the same size, nude mice is weighed is divided into 3 groups with the table of random number method, is respectively negative control group, positive controls, oleanolic acid group, every group of 5 nude mices.Drug level reaches and specifically is grouped as follows: (1) negative control group: lumbar injection 0.2ml normal saline; (2) positive drug matched group: the 5-FU of lumbar injection 30mg/kg; (3) oleanolic acid group: 40mg/kg lumbar injection.Injection in per 3 days is once injected 10 times continuously, behind last administration 24 h, takes out the tumor piece, and it is heavy to measure tumor, calculates the average tumor suppression ratio at last.Adopt the SPSS software statistics, test data is with (average ± standard deviation) expression, and P is a significant difference significance criterion of meaning less than 0.01.Result of the test sees Table 2.

Table 2 oleanolic acid is to the inhibitory action result of the test of HCT-116 transplanted tumor in nude mice (`X ± SD)

Annotate: compare * * P＜0.01 * P＜0.05 with matched group

By table 2 as seen, the transplanted tumor in nude mice of oleanolic acid treatment group and 5-FU treatment group all has been subjected to inhibition in various degree than matched group, and the oleanolic acid group to the influence of nude mice body weight all less than 1.0%, show that oleanolic acid does not have obvious toxic-side effects.

Above-mentioned isolated test and bulk testing show that oleanolic acid all has the obvious suppression effect to stripped and whole man's colon cancer cell, and does not have obvious toxic-side effects, and the prompting oleanolic acid is a kind of very promising resistive connection bowelcancer medicine.

The specific embodiment

The preparation of embodiment 1 tablet

Oleanolic acid 1000g, medical starch 100g, mix homogeneously is granulated as binding agent with an amount of ethanol, drying, through the pelletizing machine granulate, tabletting, every 0.30g, oral, each 1-2 sheet, twice of every day.

The preparation of embodiment 2 capsules

Oleanolic acid 1000g, medical starch 100g, mix homogeneously is granulated as binding agent with an amount of ethanol, and drying is crossed 120 mesh sieve granulate, dress 0# capsule, every 0.300g, each oral 1-2 grain, twice of every day.

The preparation of embodiment 3 drop pills

Polyethylene Glycol ₄₀₀₀400g, in water-bath, melt, add oleanolic acid raw material 250g, stir, in the impouring insulating tube, regulate thermostat, make medicinal liquid under 80-90 ℃, splash in the liquid paraffin that cooled off (temperature ± 4 ℃), after dripping off, to blot paraffin oil on the pill impouring filter paper, add a small amount of Pulvis Talci again, mixing gets 10000 of Olea acid dropping balls.Oral, one time four, three times on the one, one after each meal.

The preparation of embodiment 4 granules

Oleanolic acid raw material 100g, starch 1000g, Icing Sugar 500g, mix homogeneously is used an amount of alcohol granulation, and drying, granulate, packing are promptly.Oral, a 5g, twice on the one.

The preparation of embodiment 5 microencapsule tablets

Take by weighing stearic acid 12g and 18ml Herba Origani oil respectively as compound recipe capsule material, take by weighing oleanolic acid 80g as capsule core material, by the spray congealing encystation, capsule directly is 10-100 μ m with compressed air.Then with microcapsule and microcrystalline Cellulose mixing, add the mixed encystation material of 12% ethyl cellulose alcoholic solution, make granule by 18 order nylon mesh,, placed exsiccator interior 24 hours in oven dry below 50 ℃, add 1%-3% magnesium stearate tabletting, get the oleanolic acid microencapsule tablet, sheet heavily is 0.4g, and is oral, a 1-2 sheet, twice on the one.

The preparation of embodiment 6 injections

Oleanolic acid 40g, propylene glycol 50ml, PEG400 100ml, water for injection 600ml mixes heating in water bath 30 minutes, add benzyl alcohol 90ml, reuse water for injection adds to 1800ml, handles 10 minutes in ultrasound wave, heats 30 minutes in water-bath again, adjust pH is 5.5-6.5, filter, embedding, sterilization is promptly.Every 3ml, intramuscular injection, a 3ml, twice on the one.

Claims

1. pharmaceutical preparation that is used for the treatment of colon cancer is characterized in that containing the oleanolic acid for the treatment of effective dose and one or more pharmaceutically acceptable pharmaceutical excipients, or can with the other medicines of oleanolic acid prescription.

2. pharmaceutical preparation according to claim 1, but it is characterized in that containing oleanolic acid and the pharmaceutical excipient of 99%-1% or the medicine of other prescription of 1%-99%.

3. pharmaceutical preparation according to claim 2, but it is characterized in that containing oleanolic acid and the pharmaceutical excipient of 80%-20% or the medicine of other prescription of 20%-80%.

4. pharmaceutical preparation according to claim 3, but it is characterized in that containing oleanolic acid and the pharmaceutical excipient of 40%-30% or the medicine of other prescription of 60%-70%.

5. pharmaceutical preparation according to claim 1 is characterized in that said medicine is the dosage form of oral formulations or parenterai administration.

6. pharmaceutical preparation according to claim 5 is characterized in that said oral formulations is selected from any in tablet, pill, capsule, granule, microcapsule tablet, suspensoid, drop pill, the oral liquid.

7. pharmaceutical preparation according to claim 5 is characterized in that said parenterai administration dosage form is selected from any in the middle of injection, aerosol, suppository or the subcutaneous administration dosage form.

8. the application of oleanolic acid in preparation resistive connection bowelcancer medicine.

9. purposes according to Claim 8 is characterized in that said resistive connection intestinal cancer is meant the growth that suppresses human colon cancer cell (HCT-116) and nude mice transplanted tumor thereof.