CN103655559B - Horned artemisia ester alkali compounds is preparing the application in anti-breast cancer medicines - Google Patents

Abstract

The invention relates to the technical field of medicine, in particular to the application of cartilage artemisinin compounds in the preparation of anti-breast cancer drugs. Currently, there are two methods for treating breast cancer, surgical treatment and drug treatment, but surgical treatment is usually mastectomy, which seriously damages women's body and mind, and the method of drug treatment has great side effects. Carrageenate base compounds have an inhibitory effect on the activity of breast cancer cell MCF-7 and RhoGTPs family target cells, and the application of carberene base compounds in the preparation of anti-breast cancer drugs can effectively slow down the growth of tumors and prolong the life expectancy of patients. Life expectancy, side effects are mild.

Description

Application of carotene base compounds in the preparation of anti-breast cancer drugs

technical field

The invention relates to the technical field of medicine, in particular to the application of cartilage artemisinin compounds in the preparation of anti-breast cancer drugs.

Background technique

Breast cancer is one of the common malignant tumors in women. According to statistics, the incidence rate accounts for 7%-10% of all kinds of malignant tumors in the whole body. About 500,000 people die of breast cancer in the world every year, which seriously threatens the health of women.

The methods for the treatment of breast cancer include surgical treatment and drug treatment, and surgical treatment is often mastectomy, which seriously damages women's body and mind. Drug therapy is the same as other cancer treatments. Most of them are chemotherapy. Commonly used drugs include cisplatin, bleomycin, vincristine, mitomycin, fluorouracil, paclitaxel, etc. These drugs effectively prolong the life of patients Or improve the patient's quality of life. However, the side effects of antitumor drugs are obvious, which limits their curative effect. The prevention and treatment of tumors in modern medicine is still in its infancy. Traditional Chinese medicine has thousands of years of human toxicity experiment basis. From natural products, searching for high-efficiency and low-toxicity anti-tumor active lead compounds has always been a hot spot in new drug research.

Artemisia annua (English name: Incarvilleasinensis), a plant of the genus Artemisia in the family Lagerstroemia, also known as "Crancas horns" has the effect of reducing swelling and relieving pain. It is mainly used to treat bruises and rheumatic joint pains, and has good anti-inflammatory properties. Analgesic effect (see literature [1] ChiYM, YanWM, LiJS. Original botanical statistics and commercial investigation of Chinese crudedrug "Tougucao". China Journal of Chinese Meteria Medica, 1990, 15(5): 262-265.). Scientists have started research on the main active substances (alkaloids) of Artemisia argyi since 1990, and up to now, more than 20 alkaloids have been isolated from it. Through pharmacological activity studies, it was found that Incarvillateine, one of the monoterpene alkaloids, has strong analgesic activity (see literature [2] NakamuraM, ChiYM, YanWM, et al. 1293-1294).

RhoGTPase is involved in a variety of important cell life activities, such as the remodeling of actin cytoskeleton, cell adhesion, cell movement, vesicle transport, etc. ). Studies have shown that Rho family proteins are abnormally expressed in a variety of cancer cells, especially in breast cancer cells (see literature [4]HernandezE, DeLaMota-PeynadoA, DharmawardhaneSVlaarCP.NovelinhibitorsofRac1inmetastaticbreastcancer.PRHealthSciJ.2010;29:348-356.), and They have been proven to be a good anti-breast cancer target (see [5] RosenblattAE, GarciaMI, LyonsL, XieY, MaiorinoC, DesireL, et al. Inhibition of the RhoGTPase, Rac1, decrease sestrogen receptor levels and disanovel therapeutic strategy in breast cancer. EndocrRelatCancer.2011;18:207-219).

There is no relevant literature report on the study of carartemisinin against breast cancer.

Contents of the invention

The object of the present invention is to provide a new application of carartesinate base.

The inventors of the present invention systematically studied the main chemical components of Artemisia annua, and screened for anti-tumor activity, and screened out several artemisinin base compounds with good anti-tumor activity.

The invention provides the application of the carbasartee base compound in the preparation of anti-breast cancer drugs.

Tests of the present invention show that the administration of cartilage artemisinin can effectively inhibit the activity of breast cancer cell MCF-7 and breast cancer treatment target RhoGTPs. Through experiments on nude mouse models of solid tumors, the development of tumors can be slowed down and the days of survival can be prolonged.

The artemisinine compounds described in the present invention are Incarvine A (IncarvineA), Artemisinin B (IncarvineB), Artemisinin C (IncarvineB), Artemisinin D (IncarvineD), Artemisinin Ester base E (IncarvineE), carotene base F (IncarvineF). Their structural formulas are as follows:

The artemisinin base provided by the invention is prepared by the following method:

Artemisia annua is dried and crushed, refluxed with 80% and 95% ethanol, concentrated under reduced pressure to form an extract, diluted with water, adjusted to pH 2-3 with 2% HCl, then extracted with petroleum ether and ethyl acetate successively to obtain Petroleum ether and ethyl acetate parts; then add 2% NaOH to the aqueous layer to adjust the pH to 11, extract with chloroform to obtain the chloroform parts; the chloroform parts are separated by repeated silica gel (200-300 mesh) column chromatography and SephadexLH-20 column chromatography Carrageenate bases A-F.

The present invention also provides the application of the above six carartemisinic compounds in the preparation of RhoGTPs protein inhibitors.

The application of the cartilage base compound of the present invention in the preparation of an anti-breast cancer drug is a pharmaceutical composition composed of the active ingredient cartilage base compound and pharmaceutically acceptable auxiliary materials.

The pharmaceutically acceptable auxiliary materials refer to conventional pharmaceutical auxiliary materials in the field of pharmacy, wherein, diluents, excipients such as water, etc.; binders such as cellulose derivatives, gelatin or polyvinylpyrrolidone, etc.; fillers such as starch, etc. ; disintegrants such as calcium carbonate or sodium bicarbonate; other adjuvants such as flavoring and/or sweetening agents can also be added to the composition.

The pharmaceutical composition can be made into various dosage forms by adopting conventional methods in the medical field, using the carrageenate base compound as an active ingredient and pharmaceutically acceptable auxiliary materials. When used for oral administration, it can be prepared into conventional solid preparations such as tablets, powders or capsules, etc.; when used for injection, it can be prepared as injection solution. In various preparations, the weight content of the active ingredient is 0.1% to 99.9%, preferably 0.5 to 90%.

The pharmaceutical composition can be used to prepare anti-breast cancer drugs. According to dosage forms, it can be applied to individuals in need of treatment through oral administration, intraperitoneal injection, subcutaneous injection, intravenous injection, intramuscular injection, intralymphatic injection, and mucosal administration. An individual may be a human or an animal. The dose is generally 1-1000 mg/kg body weight/day, which can be changed according to the age and condition of the individual.

By carrying out the inhibitory activity of cartilage artemisinic compounds to breast cancer cell MCF-7, the experiment of the inhibitory activity of cartilage artemisinic compounds to RhoGTPs protein shows that 6 horn artemisinic compounds in the present invention have the effect on breast cancer cells It has good inhibitory activity, and tested the in vivo activity of 6 carotene base compounds through the breast cancer solid tumor animal model established by MCF-7 cells. From the experimental results, it was found that the 6 carotene base compounds not only It can inhibit tumor growth and prolong the life cycle of tumor models.

Compared with the prior art, the beneficial effects of the present invention are as follows: the present invention provides the application of cartilage artemisinin in the preparation of anti-breast cancer drugs. Carteroartemisinin can effectively slow down the growth of tumors, prolong the life of patients, and have mild side effects .

detailed description

Now, the present invention will be described in detail in conjunction with the embodiments, but the implementation of the present invention is not limited thereto.

For the experimental methods without specific conditions indicated in the following examples, the conventional conditions or the conditions suggested by the manufacturer are usually followed.

Example 1: Preparation of Carotene Alkaline Compounds

Grind the dried Artemisia annua, extract twice with 80% and 95% ethanol under reflux, each time for two hours, concentrate the extract under reduced pressure to form an extract, dilute with water, adjust the pH to 2-3 with 2% HCl, and then Sequentially extract with petroleum ether and ethyl acetate to obtain petroleum ether and ethyl acetate parts; then add 2% NaOH to the water layer to adjust the pH to 11, extract with chloroform to obtain chloroform parts; the chloroform part is a silica gel column (200-300 mesh) , eluted with 6 gradients of chloroform:methanol (20:0, 20:1, 10:1, 5:1, 2:1, 0:1) to obtain a fraction of Fr.1-Fr.6, Fr. .1 fraction was subjected to repeated silica gel (200-300 mesh) column chromatography, gradient elution with cyclohexane:methanol:diethylamine (30:1:1～5:1:1), and SephadexLH-20 column chromatography After separation, two compounds were obtained, which were identified as carartemisinin A and caroartemisinin C by comparison with references. Fraction Fr.2 was subjected to repeated silica gel (200-300 mesh) column chromatography, gradient elution with cyclohexane:methanol:diethylamine (6:4:1-5:3:1), and SephadexLH-20 column chromatography Four compounds were obtained after separation, and compared with the references, they were identified as carotene B, carotene D, carotene E, and carotene F.

Amonoterpenealkaloid from Incarvilleasinensis.Phytochemistry.1995;40:353-354; ChiY-M, HashimotoF, YanW-MNoharaT.

Artemisinin B, Artemisinin C: ChiY-M, HashimotoF, YanW-MNoharaT.TwoalkaloidsfromIncarvilleasinensisPhytochemistry.1995;39:1485-1487;

Artemisinin D: ChiY-M, HashimotoF, YanW-MNoharaT.FourmonoterpenealkaloidderivativesfromIncarvilleasinensis.Phytochemistry.1997;46:763-769;

Artemisinin E, Artemisinin F: HuangDS, ZhangWD, PeiYH, PengXY, HuangZS, LiHL, etal.TwonewaloidsfromIncarvilleasinensis.HelveticaChimicaActa.2009;92:1558-1561.

Example 2: Inhibitory activity of carotene base compounds on breast cancer cell MCF-7 (purchased from Shanghai Institute of Biological Sciences, Chinese Academy of Sciences)

Dissolve 6 carrageenate base compounds in DMSO (1000×), and set the concentration gradient of the 6 carrageenate base compounds: 0, 1, 5, 10, 25, 50, 100 μM, respectively in the experimental Cell viability was detected at 0, 24, 48, and 72 hours after the start, and the DMSO content was lower than 0.1% during drug treatment.

Cell viability detection method: MTT assay, adding 100 μl of cell suspension with a concentration of 3-5×10 ⁴ cells/ml to each well of a 96-well plate, and placing it in a 37° C., 5% CO2 incubator. After 12 hours, drugs with a gradient concentration were added, 100 μl/well, and triplicate wells were set up. 37°C, 5% CO2 for 24 hours. Add 20 μl of 5 mg/ml MTT solution to each well, stop the culture after 4 hours of action, and carefully absorb the culture solution in the well. Add 150 Ul DMSO to each well and place on a shaker for 10 minutes at low speed to fully dissolve the crystals, then measure the OD value at 570 nm with a microplate reader.

The experimental results are shown in Table 1.

Inhibitory activity of 16 compounds on breast cancer cell line MCF-7 (IC ₅₀ , μM)

It can be seen from the above experimental results that the six carrageenate base compounds have very good inhibitory effects on MCF-7 cells, and their activity is comparable to that of the positive drug Tamoxifen (Tamoxifen).

Example 3: Inhibitory activity of hornartemisinic compounds on RhoGTPs protein

Carrageenate base compounds have obvious inhibitory effects on RhoGTPs family targets. Therefore, it is concluded that these compounds play their role by acting on RhoGTPs. Table 2 shows the inhibitory activity of these compounds on the Rac1 protein in the RhoGTPs family (purchased from Cytoskeleton).

MCF-7 cells were starved for 48 hours without FBS in MEM culture medium, and then the compound of 0, 1, 5, 10, 25, 50, and 100 μM was used to act on the cells for 24 hours, and then EGF was used Stimulate the cells for 2 minutes, finally extract the total protein, detect the inhibitory activity with G-LISA kit, and calculate the IC ₅₀ , the results are shown in Table 2

The inhibitory activity of the 26 compounds on the Rac1 protein in the RhoGTPs family (IC ₅₀ , μM)

It can be seen from the above results that the six artemisinin compounds have significant inhibitory activity on the Rac1 protein in the RhoGTPs family, and their inhibitory activity is comparable to that of the positive drug EHT1864.

Example 4: Inhibitory activity of cartilage artemisinin compounds on nude mouse model of breast cancer solid tumor

In this experiment, the leukemia solid tumor nude mouse model established by injecting human MCF-7 cells on the right side was used to test the anti-breast cancer activity of the above six compounds in vivo. Each nude mouse (purchased from Shanghai Experimental Animal Center) was subcutaneously inoculated with 0.1mL cell suspension (5x106cell/mouse) in the right underarm under sterile conditions. When the tumor grows to about 150 mm ³ , select 48 nude mice with similar tumor volume and good shape (the shape should be a single spherical shape as far as possible, without irregular shape or multiple tumors clustered together), and divide them into 7 groups. Group of 8. They are control group, IncarvineA group, IncarvineB group, IncarvineC group, IncarvineD group, IncarvineE group, IncarvineF group. The day of grouping was defined as Day0, and the day after grouping was administered with a dosage of 20 mg/kg for 20 days. To investigate the growth inhibitory effect on human leukemia K562 nude mouse xenograft tumor under the experimental conditions. Body weight and tumor volume were measured twice a week. The formula for calculating tumor volume (TV) is: TV=1/2×a×b ² , where a and b represent length and width, respectively. The relative tumor volume (RTV) was calculated according to the measured results, and the calculation formula was: RTV=Vt/V ₀ . Wherein, V ₀ is the tumor volume measured at the time of cage administration (ie, d ₀ ), and Vt is the tumor volume at each measurement.

The experimental results are shown in Table 3 and Table 4. The results showed that the six compounds had obvious activity of inhibiting tumor growth, and could prolong the lifespan of tumor nude mice. It shows that these 6 compounds have the potential to be developed into anti-breast cancer drugs.

Table 3 The average tumor size (volume, mm ³ ) of solid tumor animal models treated with the above compounds

Table 4. The average survival time (days) of solid tumor animal models treated with the above compounds

As can be seen from Table 3, 6 keratinyl compounds can significantly inhibit the growth of breast cancer cells in animals. Compared with the control group, after treatment with 6 keratinyl compounds, breast cancer cells in nude mice The growth rate of solid tumors was significantly lower than that of the control group. It can be seen that the six carotene base compounds can inhibit the growth of breast cancer cells in vivo. At the same time, Table 4 shows that the life cycle of nude mice with solid tumors is greatly prolonged after the treatment of 6 carrageenate base compounds, which also illustrates the therapeutic effect of the compounds on tumor models.

The above experiments show that the 6 keratinyl compounds of the present invention have better inhibitory activity on breast cancer cells, and the 6 keratinyl compounds have been tested by the breast cancer solid tumor animal model established by MCF-7 cells. According to the in vivo activity of the compounds, it was found from the experimental results that the 6 carotene base compounds could not only inhibit the growth of tumors, but also prolong the life cycle of tumor models. Therefore, these six carotene base compounds have the prospect of being developed into drugs for the treatment of leukemia.

The above examples are preferred implementations of the present invention, but the implementation of the present invention is not limited by the above examples. Any other modifications made without departing from the spirit and principle of the present invention shall be considered within the protection scope of the present invention.

Claims (6)

1. horned artemisia ester alkali compounds is preparing the application in anti-breast cancer medicines,

Wherein, the structure of described horned artemisia ester alkali compounds is as follows:

2. apply as claimed in claim 1, it is characterized in that, wherein said horned artemisia ester alkali compounds obtains by the following method: the dry herb of Herba Incarvilleae sinensis is pulverized, with 80% with 95% alcohol reflux and concentrating under reduced pressure becomes extractum, pH to 2-3 is adjusted with 2%HCl after thin up, then use petroleum ether successively, extraction into ethyl acetate, obtain petroleum ether and ethyl acetate extract; Then water layer adds 2%NaOH tune pH to 11, with chloroform extraction, obtains chloroform extract; Chloroform extract, through 200-300 object silica gel column chromatography and SephadexLH-20 pillar layer separation repeatedly, obtains object.

3. apply as claimed in claim 1, it is characterized in that, the pharmaceutical composition that wherein said anti-breast cancer medicines is made up of horned artemisia ester alkali compounds and excipient substance described in claim 1.

4. apply as claimed in claim 3, it is characterized in that, described excipient substance is for being selected from: one or more in binding agent, filler, agent of bursting apart, flavouring agent or sweeting agent.

5. apply as claimed in claim 4, it is characterized in that, wherein, described binding agent is selected from: one or more in cellulose derivative, gelatin or polyvinylpyrrolidone; Described filler is starch; Described agent of bursting apart is that calcium carbonate is or/and sodium bicarbonate.

6. the application of horned artemisia ester alkali compounds in preparation RhoGTPs protein inhibitor, it is characterized in that, the structure of described horned artemisia ester alkali compounds is as follows: