CN104398532B - Application of cardiac glycoside compound 12beta-hydroxycalotropin - Google Patents

Abstract

The invention discloses the use of a cardiac glycoside compound 12β-hydroxycalotropin in the preparation of a drug capable of treating and preventing tumors. The cardiac glycoside compound 12β-hydroxycalotropin has significant anti-tumor activity and can be used as a lead compound for treating tumors; meanwhile, The extraction and separation method of the cardiac glycoside compound 12β-hydroxycalotropin provided by the invention is simple and easy, has low cost, high efficiency and yield, and good quality of the extract.

Description

Use of a cardiac glycoside compound

This application is a divisional application of "a new use of a cardiac glycoside compound". The filing date of the original application is April 11, 2013, and the application number is: 201310124216.4.

technical field

The invention relates to a cardiac glycoside compound, in particular to a cardiac glycoside 12β-hydroxycalotropin and its application.

Background technique

Tumor is a great threat to human health and life. It and cardiovascular disease have become two major difficulties in medicine, and constitute the first two causes of death in the world. Among the world's 5.2 billion population, about 7 million people are newly diagnosed with cancer every year, and more than 5 million people die of cancer every year, almost every 6 seconds, one cancer patient dies. In my country, an average of 1.5 million people are newly diagnosed with cancer every year, and about 800,000 people die of cancer every year. None of the existing antineoplastic drugs can completely and effectively cure the disease. At the same time, due to the continuous discovery of new tumors, people are forced to find new anti-tumor drugs. For a long time, finding anti-tumor drugs from medicinal plants, especially tropical medicinal plants, has become a common wish of medicine, pharmacy, chemistry and biologists from all over the world. Studies have shown that 70% of anti-tumor drugs are derived from tropical or subtropical plants. After vincristine, the discovery of paclitaxel and its derivatives Epothilones made a breakthrough in this work. However, due to the extremely low content of some anti-tumor components in plants, some plants are endangered and rare species, and the plants grow slowly, and over-harvesting may lead to the extinction of the species. These factors limit the development of anti-tumor herbal medicines.

Calotropis gigantea (L.) Dryand ex Ait.f.] is a plant of the genus Calotropis in the family Asclepiadaceae. In my country, it is mainly distributed in Hainan, Guangdong, Sichuan and Yunnan. The whole plant of croissant melon is poisonous, and its roots, stems, leaves, flowers, fruits, and white juices from various parts can be used medicinally. It has the functions of antibacterial, anti-inflammatory, anthelmintic, phlegm-reducing, and detoxification. It is a commonly used folk medicine. Studies at home and abroad have shown that crocodile melon mainly contains cardiac glycosides, which are toxic components. However, it is still unknown whether the cardiac glycoside compound 12β-hydroxycalotropin in croissanthus melon has anti-tumor effect.

Contents of the invention

The purpose of the present invention is to provide a use of cardiac glycoside compound 12β-hydroxycalotropin in the preparation of medicines capable of treating and preventing tumors.

The inventors of the present application extracted, separated, purified and tested the anti-tumor activity of the cardiac glycoside compound 12β-hydroxycalotropin in the horned melon, and found that the cardiac glycoside compound 12β-hydroxycalotropin in the horned melon has a new anti-tumor application.

The above object of the present invention is achieved through the following technical scheme: the use of the cardiac glycoside compound 12β-hydroxycalotropin in the preparation of drugs with therapeutic and preventive effects on tumors, the molecular formula of the cardiac glycoside compound 12β-hydroxycalotropin is C ₂₉ H ₄₀ O ₁₀ , the structural formula is as follows:

The tumors described in the present invention are mainly cholangiocarcinoma, renal cancer, pancreatic cancer, colorectal cancer, bladder cancer, breast cancer, uterine cancer, ovarian cancer, colorectal cancer, liver cancer, biliary cancer, biliary tract cancer, acute leukemia, malignant lymphoma , brain tumor, bone tumor, melanoma, glioma, oral cancer, nasopharyngeal cancer, lung cancer or gastric cancer, etc.

Cardiac glycoside compound 12β-hydroxycalotropin of the present invention can utilize various separation means including adsorption resin column chromatography, silica gel column chromatography, reversed phase silica gel column chromatography, gel column chromatography, decompression column chromatography etc. Obtained, or obtained through synthetic and semi-synthetic means. Combined with suitable excipients, it can be made into various dosage forms according to conventional methods.

The preferred preparation method of the cardiac glycoside compound 12β-hydroxycalotropin of the present invention is as follows:

A, select the stem of the horned melon, after crushing, add ethanol, extract 3 to 4 times, each time for 6 to 8 days, combine the extracts, concentrate under reduced pressure until there is no alcohol smell to obtain the ethanol extract of the horned melon;

B. Disperse the ethanol extract of croissant melon in water to make crocus melon suspension, respectively press the volume ratio of crocus melon suspension and sherwood oil to 1:3, 1:2 and 1:1, use petroleum ether to After the croissant melon suspension is repeatedly extracted, the aqueous phase after petroleum ether extraction is retained;

C. Filter the aqueous phase after petroleum ether extraction, separate the filtrate by D-101 macroporous adsorption resin column chromatography, and elute with water and methanol in turn, collect the methanol eluate, concentrate under reduced pressure and recover methanol, and obtain methanol leaching paste;

D. The methanol extract is subjected to vacuum silica gel column chromatography, and the volume ratios of chloroform and methanol are 100:1, 50:1, 25:1, 10:1, 5:1, 2:1 and 0:100 respectively As an eluent, carry out gradient elution on a decompression silica gel column to obtain crude extracts Fr.1-Fr.7;

E. The crude extract Fr.4 obtained in step D is subjected to silica gel column chromatography, and the volume ratio of chloroform and methanol is 10:1, 8:1, 6:1, 4:1, 2:1, 0:1, respectively As an eluent, gradient elution was performed on the silica gel column to obtain 6 fractions Fr.4-1～Fr.4-6;

F. The Fr.4-2 in the E step is subjected to silica gel column chromatography, and chloroform and acetone are used as eluents for eluting. During elution, the same components are collected by TLC thin layer chromatography to obtain 6 parts of Fr.4- 2-1～Fr.4-2-6; Fr.4-2-3 was subjected to Sephadex LH-20 gel column chromatography, reverse phase column chromatography, and silica gel column chromatography again in sequence to obtain cardiac glycosides Compound 12β-hydroxycalotropin.

In the step A of the present invention, the mass volume ratio of the horn melon stem to the ethanol is 1kg:1-3L; the volume percentage of the ethanol is 92-98%.

In the step B of the present invention, after repeated extraction of the croissant melon suspension with petroleum ether for 3 times, the aqueous phase after petroleum ether extraction is retained.

In step F of the present invention, the volume ratio of chloroform and acetone is 4:5; the gel column adopted during gel column chromatography is Sephadex LH-20 gel column, and the eluting solvent adopts volume percentage content during gel column chromatography 95% ethanol; the elution solvent used in the silica gel column chromatography again is a mixed solvent of chloroform, methanol and acetone with a volume ratio of 11:1:1.

The solvents adopted in the above-mentioned steps A, B, C, D, E, F, such as sherwood oil, chloroform, methanol, ethanol, etc. are industrial grade solvents, which are used after redistillation.

The silica gel column, gel column and reversed-phase column used in the above steps D, E and F are all conventional equipment in the prior art.

The present invention has the following advantages: the cardiac glycoside compound 12β-hydroxycalotropin provided by the present invention has significant anti-tumor activity, and can be used as a lead compound for treating tumors; meanwhile, the extraction and separation method provided by the present invention is simple and easy, with low cost and high efficiency , high yield, good quality extract.

detailed description

The present invention will be further elaborated below in conjunction with specific examples, but the present invention is not limited.

Example 1

A. After 25.4 kg of calotropis gigantea stems are naturally air-dried and pulverized, by the calotropis gigantea sample: the mass/volume ratio (kg/L) of ethanol aqueous solution is about 1:2, with 50L volume fraction of ethanol aqueous solution of 95% Fully mixed with 25.4kg of horn melon samples, sealed, leached at room temperature for 3 times, each time for 7 days, filtered and combined the extracts, concentrated under reduced pressure until there was no alcohol smell to obtain the ethanol extract;

B. At room temperature, disperse the ethanol extract in water to make a suspension, according to the volume ratio of ethanol extract: petroleum ether = 1:3, 1:2, 1:1, repeat the ethanol extract with petroleum ether After extracting 3 times, and concentrating under reduced pressure to recover petroleum ether, petroleum ether extract (254.1 g) was obtained;

C. Filter the water liquid after petroleum ether extraction, separate the filtrate by D-101 macroporous adsorption resin column chromatography, elute with water and methanol in turn, collect the methanol eluate, concentrate and recover methanol under reduced pressure, and obtain methanol immersion Paste (255.7g);

D, the methanol extract is subjected to vacuum silica gel column chromatography, washed with chloroform:methanol=100:1, 50:1, 25:1, 10:1, 5:1, 2:1, 0:100 volume ratio Removal of reagents, gradient elution, respectively to obtain 7 parts Fr.1 ~ Fr.7;

E. The Fr.4 (38.4g) obtained by eluting step D with chloroform:methanol=10:1 was subjected to silica gel column chromatography, and chloroform:methanol=10:1, 8:1, 6:1, 4:1 , 2:1 volume ratio eluent for gradient elution, and finally eluted with methanol to obtain 6 parts Fr.4-1～Fr.4-6;

F, the Fr.4-2 (16.7g) of step E was subjected to silica gel column chromatography, eluted with an eluent with a volume ratio of chloroform:acetone=4:5, and then the same components were collected by TLC thin layer chromatography, Obtain 6 parts, the 3rd part in these 6 parts, namely Fr.4-2-3, Fr.4-2-3 (2.0g) carries out Sephadex LH-20 gel column chromatography ( Volume fraction is 95% ethanol elution), reverse phase column chromatography, and silica gel column chromatography (chloroform:methanol:acetone=11:1:1 elution), finally obtain cardiac glycoside compound 12β-hydroxycalotropin (98.2mg ).

Example 2

After column chromatography, Fr.4-1 (3.2 g) in step E in Example 1 was eluted with an eluent with a volume ratio of chloroform:methanol=25:1, and the same components were collected by TLC thin layer chromatography , to obtain 7 parts Fr.4-1-1～Fr.4-1-7; Fr.4-1-4 (869.5mg) was subjected to silica gel column chromatography, followed by chloroform:methanol=40:1, chloroform : Acetone=6:2 volume ratio of the eluent for elution to finally obtain the cardiac glycoside compound calotropin (109.9mg). The study found that the structural difference between this compound and the target compound of the present invention is that there is no hydroxyl group on the 12-position carbon, while the 12-position carbon of the target compound of the present invention is substituted by a hydroxyl group. In the further in vitro anti-tumor activity test, it was found that the anti-IC ₅₀ values of the two compounds were significantly different (see Table 2).

Example 3

Structural identification of cardiac glycoside compound 12β-hydroxycalotropin:

The structure of the cardiac glycoside compound 12β-hydroxycalotropin in Example 1 was identified by spectroscopic techniques, including ultraviolet, infrared, nuclear magnetic resonance and high-resolution mass spectrometry. Using 2D-NMR technology, the ¹³ C-NMR and ¹ H-NMR data of the cardiac glycoside compound 12β-hydroxycalotropin firstly found in the stem of the horned melon were assigned (see Table 1 below).

The following are the physical and chemical constants of the cardiac glycoside compound 12β-hydroxycalotropin:

12β-hydroxycalotropin: C ₂₉ H ₄₀ O ₁₀ , white amorphous powder (methanol), brown in 10% sulfuric acid ethanol solution. mp214-216℃; [α] _D +6.4° (c=0.045, MeOH); HR-ESI-MS: m/z [M+Na] ⁺ 583.2318 (calcd.For C ₂₉ H ₄₀ O ₁₀ Cl, 583.2310) ；IRλ _max (cm ^-1 ):3433cm ^-1 ,2957cm ^-1 ,2923cm ^-1 ,2852cm ^-1 ,1712cm ^-1 ,1655cm ^-1 ,1648cm ^-1 ,1638cm ^-1 ,1630cm ^-1 ,1459cm ^-1 ,1262cm ^-1 , 1105cm ^-1 , 1025cm ^-1 ; UVλ _max nm (MeOH): 218nm. See Table 1 below for ¹³ C-NMR and ¹ H-NMR.

Table 1 Carbon spectrum and hydrogen spectrum data of cardiac glycoside compound 12β-hydroxycalotropin

Example 4

Comparative experiment of cardiac glycoside compound 12β-hydroxycalotropin and calotropin in vitro anti-tumor activity test:

Cardiac glycoside compounds 12β-hydroxycalotropin and calotropin were tested by MTT method on human gastric cancer cells (SGC-7901), human gastric adenocarcinoma cells (KKLS, MKN-28, MKN-45), human chronic myelogenous leukemia cells (K562) , human oral epidermal carcinoma cells (KB), human breast cancer cells (MCF7), human lung cancer cells (NCI-H187), human osteofibrosarcoma cells (HT-1080), human osteosarcoma cells (G-292), human osteosarcoma cells ( KHOS/NP), human prostate cancer cell line (LNCap) and other cell lines in vitro anti-tumor activity (results are shown in Table 2).

The activity test method is as follows:

The experiment set up negative control group (water), DMSO solvent control group, positive control group (mitomycin C) and 8 different concentrations (0.1, 0.3, 0.9, 2.7, 8.1, 24.3 μg·mL ^-1 ) Samples with IC ₅₀ values <0.1μg·mL ^-1 continued to be diluted downwards, and then 6 different concentrations (0.0003, 0.001, 0.004, 0.011, 0.033 and 0.100μg·mL ^-1 ) were set. 3 in parallel. Cells in logarithmic growth phase were collected, counted by hemocytometer, inoculated into 96-well flat-bottomed cell culture plates according to the amount of 4500 cancer cells per well, and cultured in a 37°C incubator with 5% CO ₂ and humidity above 90%. After 24 hours, take out and add a certain amount of sample to be tested, continue to cultivate for 72 hours, take out and place under a microscope to observe the cell morphology of each well, record the changes in cell morphology, and then add 5 mg/mL MTT solution (dissolved in balanced salt solution PBS) to each well 15 μL, after reacting at 37°C for 4 hours, suck out the cell culture medium, add 100 μL DMSO to each well to fully dissolve Formazane, place the cell culture plate on a MK3 microplate reader, measure the absorbance (A) of each well with a wavelength of 570nm, and follow the steps below Formula for growth inhibition rate.

Take the sample concentration as the abscissa and the inhibition rate as the ordinate, and use the origin software to fit the inhibition rate curve according to the concentration gradient. The sample concentration when the inhibition rate is 50% is the IC ₅₀ value of the cytotoxic activity, and the sample activity The results were expressed as half inhibitory concentration (IC ₅₀ ).

It can be seen from Table 2 that the cardiac glycoside compound 12β-hydroxycalotropin and calotropin have structural differences and thus significant differences in antitumor activity. The antitumor activity of the cardiac glycoside compound of the invention is obviously stronger than that of calotropin.

Table 2 In vitro tumor inhibition results of cardiac glycoside compound 12β-hydroxycalotropin

Example 5

8 g of cardiac glycoside compound 12β-hydroxycalotropin prepared in Example 1 was mixed with 75 g of microcrystalline cellulose and 7 g of magnesium stearate, and the mixture was punched into tablets with a diameter of 5 mm and a weight of 100 mg with a single-punch tablet machine. Each tablet contains 8mg of 12β-hydroxycalotropin. Combined with symptoms, take 1-2 tablets each time, 2-3 times a day.

Example 6

Mix 20 g of the cardiac glycoside compound 12β-hydroxycalotropin prepared in Example 1 with 95 g of lactose and 6 g of magnesium stearate, and fill capsules at a rate of 300 mg. In this capsule, each capsule contains 25mg of 12β-hydroxycalotropin. Combined with symptoms, take 1-2 capsules each time, 3-4 times a day.

The specific embodiments listed above are to illustrate the present invention. It should be pointed out that the above examples are only used to further illustrate the present invention, and do not represent the protection scope of the present invention. Non-essential modifications and adjustments made by others according to the hints of the present invention still belong to the protection scope of the present invention.

Claims (5)

1. The use of the cardiac glycoside compound 12β-hydroxycalotropin in the preparation of a drug for treating tumors, the molecular formula of the cardiac glycoside compound 12β-hydroxycalotropin is C ₂₉ H ₄₀ O ₁₀ , and the structural formula is as follows: The tumor is oral cavity cancer, lung cancer or human prostate cancer cell LNCap. 2. the purposes of the cardiac glycoside compound 12β-hydroxycalotropin according to claim 1 in the preparation of the medicine with therapeutic effect, it is characterized in that: the preparation method of the cardiac glycoside compound 12β-hydroxycalotropin is as follows: A, select the stem of the horned melon, after crushing, add ethanol, extract 3 to 4 times, each time for 6 to 8 days, combine the extracts, concentrate under reduced pressure until there is no alcohol smell to obtain the ethanol extract of the horned melon; B. Disperse the ethanol extract of croissant melon in water to make crocus melon suspension, respectively press the volume ratio of crocus melon suspension and sherwood oil to 1:3, 1:2 and 1:1, use petroleum ether to After the croissant melon suspension is repeatedly extracted, the aqueous phase after petroleum ether extraction is retained; C. Filter the aqueous phase after petroleum ether extraction, separate the filtrate by D-101 macroporous adsorption resin column chromatography, and elute with water and methanol in turn, collect the methanol eluate, concentrate under reduced pressure and recover methanol, and obtain methanol leaching paste; D. The methanol extract is subjected to vacuum silica gel column chromatography, and the volume ratios of chloroform and methanol are 100:1, 50:1, 25:1, 10:1, 5:1, 2:1 and 0:100 respectively As an eluent, carry out gradient elution on a decompression silica gel column to obtain crude extracts Fr.1-Fr.7; E. The crude extract Fr.4 obtained in step D is subjected to silica gel column chromatography, and the volume ratio of chloroform and methanol is 10:1, 8:1, 6:1, 4:1, 2:1, 0:1, respectively As an eluent, gradient elution was performed on the silica gel column to obtain 6 fractions Fr.4-1～Fr.4-6; F. The Fr.4-2 in the E step is subjected to silica gel column chromatography, and chloroform and acetone are used as eluents for eluting. During elution, the same components are collected by TLC thin layer chromatography to obtain 6 parts of Fr.4- 2-1～Fr.4-2-6; Fr.4-2-3 was subjected to Sephadex LH-20 gel column chromatography, reverse phase column chromatography, and silica gel column chromatography again in sequence to obtain cardiac glycosides Compound 12β-hydroxycalotropin. 3. The use of the cardiac glycoside compound 12β-hydroxycalotropin according to claim 2 in the preparation of a drug capable of treating tumors, characterized in that: in step A, the mass-to-volume ratio of the horned melon stem to ethanol is 1kg: 1-3L ; The volume percentage of the ethanol is 92-98%. 4. The use of the cardiac glycoside compound 12β-hydroxycalotropin according to claim 2 in the preparation of a drug for treating tumors, characterized in that: in the step B, after repeated extraction of the horn melon suspension with petroleum ether for 3 times, Keep the aqueous phase extracted with petroleum ether. 5. The purposes of the cardiac glycoside compound 12β-hydroxycalotropin according to claim 2 in the preparation of medicines with therapeutic tumor effect is characterized in that: the volume ratio of chloroform and acetone is 4:5 in the step F; gel column layer The gel column that adopts during analyzing is Sephadex LH-20 gel column, and the elution solvent adopts the ethanol that volume percent content is 95% during gel column chromatography; It is a 11:1:1 mixed solvent of chloroform, methanol and acetone.