CN104817575B - Compound, its extraction method, pharmaceutical composition containing the same and application thereof - Google Patents

Abstract

The embodiment of the present invention discloses a compound with the following chemical structural formula, and the compound is called vinblastine. At the same time, the present invention also provides an extraction method of the compound, a pharmaceutical composition containing the compound, and uses of the compound and the pharmaceutical composition in preparing antitumor drugs.

Description

Compound, its extraction method, pharmaceutical composition containing it and use thereof

technical field

The invention relates to a compound, in particular to a natural compound extracted from periwinkle plant, an extraction method of the compound, a pharmaceutical composition containing the compound, and an application of the compound and the pharmaceutical composition in preparing antitumor drugs.

Background technique

Tumor is one of the important causes of human death. With the development of industrial society, diseases such as liver cancer, breast cancer, and lung cancer have seriously affected people's healthy living standards. It can be seen that the prevention and treatment of tumors is very important. Chemotherapy is an effective way to treat tumors. At present, there are dozens of tumor chemotherapy drugs commonly used in clinical practice, which can effectively prolong human life. However, synthetic anti-tumor drugs have severe side effects, which have severely limited the efficacy of these drugs. play. Traditional Chinese medicine and natural medicine are the great wealth of our country, which have the characteristics of less toxic and side effects, so finding anti-tumor drugs from traditional Chinese medicine and natural medicine is an effective way to solve these problems.

Periwinkle Catharanthus roseus (L.) G.Don is the whole plant of periwinkle and yellow periwinkle in the genus Catharanthus of the family Apocynaceae. Periwinkle is native to the east coast of Africa and the tropical regions of America, and is currently widely cultivated in tropical and subtropical regions. It is cultivated in the south of the Five Ridges of China and in Jiangsu and Zhejiang. Periwinkle is bitter, cold and poisonous in nature. It has the effects of detoxifying and anti-cancer, clearing away heat and calming the liver. Modern pharmacological studies have shown that periwinkle has the effects of anti-tumor, lowering blood pressure, lowering blood fat, lowering blood sugar and expanding blood vessels.

Contents of the invention

The present inventor obtained the compound vinblastine (cathachunine) extracted from the dried whole plant of periwinkle, and the present inventor also found that the above-mentioned vinblastine (cathachunine) can be used for anti-tumor. The present invention also provides a pharmaceutical composition containing the above-mentioned cathachunine, and the use of the pharmaceutical composition in the preparation of antitumor drugs. The specific technical scheme is as follows:

The first aspect of the present invention provides compound, the chemical structural formula of this compound is as follows:

The above compound is called vinblastine (cathachunine).

The second aspect of the present invention provides the extraction method of above-mentioned compound vinblastine, comprises the following steps:

(1) Extraction: Dried the whole plant of periwinkle, crushed, extracted with ethanol aqueous solution under reflux, obtained extract, diluted with water, adjusted pH to 2-3 with HCl solution, filtered to obtain filtrate and filter residue, added NH ₃ ·H to the filtrate ₂ O to adjust the pH to 9-10, and extract with chloroform to obtain the chloroform site;

(2) Separation: Apply silica gel column chromatography to the above-mentioned chloroform part, elute with a gradient of chloroform-methanol system with a volume of 100:1 to 2:1, detect by thin-layer chromatography, and collect and obtain a stream containing vinblastine (cathachunine). After separation, ODS column chromatography, eluting with a methanol-water system with a volume ratio of 20:80, and detection by thin-layer chromatography, the pure product of vinblastine cathachunine was obtained.

In a preferred embodiment of the second aspect of the present invention, the above-mentioned reflux extraction with ethanol aqueous solution is used to obtain the extract, specifically: 2-4 times of reflux extraction with 95% ethanol, each time for 2-3 hours, and the extract is decompressed Concentrate to extract.

The third aspect of the present invention provides a pharmaceutical composition, which comprises the above-mentioned compound vinblastine (cathachunine) and optional pharmaceutically acceptable carriers or excipients.

In a preferred embodiment of the third aspect of the present invention, based on the total weight of the pharmaceutical composition, the content of the compound vinblastine (cathachunine) is 1-99%, preferably 20-80%, more preferably 40-60%.

In another preferred embodiment of the third aspect of the present invention, the pharmaceutically acceptable carrier or excipient is selected from solvents, diluents, dispersants, suspending agents, surfactants, isotonic agents, thickeners , emulsifiers, preservatives, binders, lubricants, stabilizers, hydrating agents, emulsification accelerators, buffers, absorbents, colorants, fragrances, sweeteners, ion exchangers, release agents, coatings agents, flavoring agents, and antioxidants.

In yet another preferred embodiment of the third aspect of the present invention, the dosage form of the pharmaceutical composition is tablet, capsule, powder, granule, lozenge, pill, solution, suspension, emulsion, syrup, Powders, granules, pellets, elixirs, injections, medicinal drops, ointments, lotions, gels, creams, sprays, suppositories, or patches.

The fourth aspect of the present invention provides the use of the above-mentioned compound vinblastine (cathachunine) or the above-mentioned pharmaceutical composition in the preparation of antitumor drugs.

In a preferred embodiment of the fourth aspect of the present invention, the daily dose of the drug administered to a subject in need is 0.01-1000 mg/kg body weight based on the compound vinblastine, preferably 0.1-100 mg/kg body weight, more preferably 1-100mg/kg body weight.

The inventor obtained the compound vinblastine (cathachunine) from the dried whole plant of periwinkle, which is a white powder, [α] 20D+22.5 (c 0.2, CHCl ₃ ); :709.33605[M+H] ⁺ , its molecular formula is deduced as C ₄₁ H ₄₈ N ₄ O ₇ ; and its structure is determined by ¹ H and ¹³ C NMR and UV spectroscopy.

As used herein, the term "ODS column" refers to an octadecylsilane column.

The active ingredient of the raw material of the pharmaceutical composition of the present invention can be obtained by the extraction method according to the present invention by using conventional techniques in the field of preparation, mixed with one or more pharmaceutically acceptable carriers or excipients, and then formed into the desired dosage form to prepare the pharmaceutical composition of the present invention.

As used herein, "pharmaceutically acceptable" means that it has no substantial toxic effect when used in the usual pharmaceutical doses, and thus is approved by a government or its equivalent international organization or has been approved for use in animals, more particularly in humans. , or be registered in the Pharmacopoeia.

The "pharmaceutically acceptable carrier or excipient" available in the pharmaceutical composition of the present invention can be any conventional carrier in the field of pharmaceutical preparations, and the selection of a specific carrier will depend on the mode of administration or the type of disease used to treat a specific patient and state. The preparation of suitable pharmaceutical compositions for a particular mode of administration is well within the knowledge of those skilled in the pharmaceutical art. For example, pharmaceutically acceptable carriers include conventional solvents, diluents, dispersants, suspending agents, surfactants, isotonic agents, thickeners, emulsifiers, binders, lubricants, and stabilizers in the pharmaceutical field. , hydrating agent, emulsification accelerator, buffering agent, absorbent, coloring agent, ion exchanger, release agent, coating agent, flavoring agent, and antioxidant, etc. Flavoring agents, preservatives and sweeteners, etc. can also be added to the pharmaceutical compositions when necessary.

As used herein, the term "pharmaceutical composition" has its ordinary meaning. In addition, the "pharmaceutical composition" of the present invention can also exist or be provided in the form of health products, functional foods, foods, food additives, etc. The active ingredient of the raw material of the pharmaceutical composition of the present invention can be obtained by using conventional techniques in the field of pharmacy, especially in the field of preparation, by means of extraction, separation and purification commonly used in drug production, optionally combined with one or more pharmaceutically acceptable The pharmaceutical composition of the present invention is prepared by mixing the carrier and then forming the desired dosage form. According to the pharmaceutical composition of the present invention, it is a pharmaceutical preparation suitable for oral administration, parenteral administration, local administration, and external administration. The pharmaceutical composition of the present invention can be made into various forms such as tablet, powder, granule, capsule, oral liquid and the like. The above-mentioned medicines in various dosage forms can be prepared according to conventional methods in the field of pharmacy. Specifically, according to the pharmaceutical composition of the present invention, the pharmaceutical dosage form includes but not limited to: tablet, capsule, granule, powder, injection, powder for injection, transdermal patch, ointment, gel formulations, suppositories, oral solutions, oral suspensions, injection emulsions, oral emulsions, etc., sustained-release tablets, controlled-release tablets. The above-mentioned medicines in various dosage forms can be prepared according to conventional methods in the field of pharmacy.

Dosage forms for oral administration may include, for example, tablets, pills, hard or soft capsules, solutions, suspensions, emulsions, syrups, powders, powders, fine granules, granules, pellets, elixirs, etc., It is not limited to this. These formulations may contain, in addition to the active ingredient, diluents (such as lactose, dextrose, sucrose, mannitol, sorbitol, cellulose, and glycine), lubricants (such as silicon dioxide, talc, stearic acid, or its magnesium salt, calcium salt and polyethylene glycol). Tablets may also contain binders such as magnesium aluminum silicate, starch paste, gelatin, tragacanth, methylcellulose, sodium carboxymethylcellulose and polyvinylpyrrolidine. If necessary, it may also contain pharmaceutical additives such as disintegrating agents (such as starch, agar, alginic acid or its sodium salt), absorbing agents, coloring agents, flavoring agents, sweetening agents and the like. Tablets can be prepared according to the usual methods of mixing, granulating or coating.

Dosage forms for parenteral administration may include, for example, injections, medicinal drops, ointments, lotions, gels, creams, sprays, suspensions, emulsions, suppositories, patches, etc., but are not limited thereto.

Pharmaceutical compositions according to the present disclosure may be administered orally or parenterally, eg rectally, topically, dermally, intravenously, intramuscularly, intraperitoneally or subcutaneously.

The pharmaceutically acceptable dose of the active ingredient, i.e., the administered dose, can be determined according to the age, sex, and body weight of the subject to be treated, the specific disease or pathological state to be treated, the severity of the disease or pathological state, the route of administration, and the judgment of the diagnoser. Change. It is within the level of those skilled in the art to determine the dosage to be administered taking these factors into consideration. The general dosage may be 0.01-1000 mg/kg/day, preferably 0.1-100 mg/kg/day, more preferably 1-100 mg/kg/day. However, the scope of the present disclosure is not limited in any way by the dosages administered.

Description of drawings

In order to more clearly illustrate the technical solutions in the embodiments of the present invention or the prior art, the following will briefly introduce the drawings that need to be used in the description of the embodiments or the prior art. Obviously, the accompanying drawings in the following description are only These are some embodiments of the present invention. Those skilled in the art can also obtain other drawings based on these drawings without creative work.

Fig. 1 is the fluorescence intensity after 48h of vinblastine acting on HL-60 cells under different concentrations;

Fig. 2 Comparison of ROS at 48 hours after vinblastine acting on HL-60 cells at different concentrations.

detailed description

The following will clearly and completely describe the technical solutions in the embodiments of the present invention with reference to the accompanying drawings in the embodiments of the present invention. Obviously, the described embodiments are only some, not all, embodiments of the present invention. Based on the embodiments of the present invention, all other embodiments obtained by persons of ordinary skill in the art without creative efforts fall within the protection scope of the present invention.

Extraction and characterization of embodiment 1 compound vinblastine (cathachunine)

1. Instruments and materials

X-5 microscopic melting point tester (Beijing Tektronix Instrument Co.); Jasco FT/IR—480Plus FourierTransform infrared spectrometer (JASCO Corporation); Jasco V-550 ultraviolet/visible spectrometer (JASCO Corporation); Bruker AV- 400MHz nuclear magnetic resonance instrument, TMS is internal standard (Germany Bruker company); Thermo FinniganLCQ Advantage MAX mass spectrometer (U.S. Thermo company); Dionex analytical high performance liquid chromatography (U.S. Dionex company); Cosmosil C-18 chromatographic column (250mm× 4.6mm, 5μm); Varian preparative high-performance liquid chromatography (Varian Company, USA); silica gel for column chromatography (Qingdao Ocean Chemical Factory); silica gel GF254 thin-layer prefabricated plate (Yantai Institute of Chemical Industry); Sephadex LH-20 (Pharmacia Company); ODS column chromatographic material (Merck Company, Germany); reagents used are analytically pure and chromatographically pure.

The periwinkle medicinal material is collected from Haikou City, Hainan Province, and is identified as the whole plant of Catharanthus roseus (L.) G.Don by Dr. Wang Chunhua from Tianjin University of Traditional Chinese Medicine. Plant specimens (No.2012CH0014) are deposited in Binhai Modern Chinese Medicine Laboratory, Tianjin University of Traditional Chinese Medicine.

2. Extraction of vinblastine (cathachunine)

Dried 3 kg of periwinkle herb, crushed, reflux extracted with 20 L of 95% ethanol for 3 times, each time for 2 hours, the extract was concentrated under reduced pressure to 500 g of extract, diluted with water, adjusted to pH 2-3 with HCl solution, filtered to obtain Filtrate and filter residue, the filtrate was added with NH ₃ ·H ₂ O to adjust the pH to 9-10, extracted with chloroform to obtain the chloroform fraction, which was concentrated under reduced pressure to obtain 115 g of the crude product; the above chloroform fraction was subjected to silica gel column chromatography, and the volume was 100:1 To 2:1 chloroform-methanol system gradient elution, thin-layer chromatography detection, collect and obtain the fraction 20g containing vinblastine (cathachunine), and then through ODS column chromatography, the volume ratio is 20:80 methanol- The water system was eluted and detected by thin layer chromatography to obtain 40 mg of pure vinblastine (cathachunine).

3. Characterization

Compound vinblastine (cathachunine): white powder, [α]20D+22.5(c 0.2, CHCl ₃ ); according to the electrospray ion mass spectrometry signal m/z: 709.33605[M+H] ⁺ , its molecular formula is estimated to be C ₄₁ H ₄₈ N ₄ O ₇ ; the ultraviolet spectrum has strong absorption at 220nm and 260nm, indicating that the compound has the characteristics of an indole ring structure; the infrared spectrum has absorption peaks at 3452cm ^-1 , 1737cm ^-1 , and 1688cm ^-1 , suggesting that the compound contains hydroxyl , carbonyl and amido groups. The ¹ H NMR spectrum shows four aromatic hydrogen proton signals on the indole ring [δ7.53(1H,d,J=7.6Hz,7.16(1H,m),7.13(1H,m) and 7.11(1H,d ,J=7.6Hz)] and the characteristic hydrogen proton signals on the four indoline rings [δ6.52(1H,s),6.08(1H,s),5.67(1H,dd,J=9.6,4.0Hz ) and 5.46 (1H, d, J=9.6Hz)], the above NMR data suggest that the compound is a vinblastine (VLB) type alkaloid. From the HMBC spectrum, it can be seen that H-3'a (δ3.99)/H -5'b(δ2.99)/H-14'(δ1.54) is correlated with C-18(δ179.0), indicating that C-20 and N-4 are connected by amide bond, combined with ROESY spectrum and CD The spectrum can determine the absolute stereo configuration of vinblastine. The ¹ H and ¹³ C NMR data are shown in Table 1. The chemical structural formula of the compound vinblastine (cathachunine) can be determined from the above characterization.

Table 1 ¹ H and ¹³ C NMR data of vinblastine (cathachunine)

The ¹ H and ¹³ C NMR data were measured at 400MHz and 100MHz, respectively, and J is the coupling constant.

Example 2 Cytotoxic activity test of vinblastine (Cathachunine)

1. Experimental materials

1.1 Test sample

After the vinblastine (cathachunine) extracted in Example 1 was dissolved in DMSO (Merck), PBS (-) was added to form a 100 μM/ml solution or a uniform mixture, and then diluted with DMSO-containing PBS (-).

1.2 Cell lines

HL-60 (human leukemia cells, American Type Culture Collection, (ATCC))

K562 (human leukemia cells, American Type Culture Collection, (ATCC))

EA.hy926 (Human endothelial cells, American Type Culture Collection, (ATCC))

1.3 Culture medium

PRMI1640 (Gibco, USA) + 15% NBS (Gibco, USA) + double antibody (HyClone, USA)

1.4 Other materials

WST-1 cell proliferation and toxicity test package (Beyotime institute of biotechnology, China), automatic microplate reader: (BioTek, U.S.A.), imported 96-well culture plate, etc.

2. Experimental method

Cell viability experiments were performed using the WST-1 cell proliferation and toxicity assay package, and the standard operating procedures of the assay package were used. Add cell suspension with a concentration of 4-6×104 cells/ml to each well of the 96-well plate, and place in a 37° C., 5% CO2 incubator. 10 μl of WST solution (containing 100 μl of medium) was added to each well, and after incubation for 2 hours, the OD value at 450 nm was measured with a microplate reader.

3. Test results

The results are shown in Table 2. The results show that vinblastine has significant cytotoxic activity on human leukemia cells HL-60 and K562, and has weak cytotoxicity on human endothelial cells EAhy926.

Table 2. Cytotoxic effects of vinblastine

Example 3 Effect of vinblastine (Cathachunine) on the level of reactive oxygen species (Reactive oxygen species, ROS) in tumor cells

1. Experimental materials

1.1 Test sample

After vinblastine (cathachunine) was dissolved in DMSO (Merck), PBS (-) was added to make a 100 μM/ml solution or a uniform mixture, and then diluted with DMSO-containing PBS (-).

1.2 Cell lines

HL-60 (human leukemia cells, American Type Culture Collection, (ATCC))

1.3 Culture medium

PRMI1640 (Gibco, USA) + 15% NBS (Gibco, USA) + double antibody (HyClone, USA)

1.4 Other materials

Fluorescence Microscope XSP-BM21AY (Shanghai No. 6 Optical Instrument Factory)

2. Test method

The common fluorescent probe DCFH-DA can be deacetylated in the cell to become non-fluorescent DCFH. When there are ROS in the cell, DCFH can continue to be oxidized into fluorescent DCF. His fluorescence represents the intracellular ROS levels. In this experiment, DCFH-DA was used to evaluate the ROS production of vinblastine (cathachunine) in HL-60 cells. The specific test method is as follows: HL-60 cells are washed once with PBS, added with DCFH-DA, and stored at 37° C. for 30 minutes in the dark. Cells were then washed twice and kept in 1 ml medium. ROS production was measured using fluorescence microscopy at excitation and emission wavelengths of 488 and 530 nm, respectively. The fluorescence intensity was further analyzed with a fluorescent microplate reader at excitation and emission wavelengths of 488 and 525 nm.

3. Test results, as shown in Figure 1 and Figure 2.

The four panels a-d in Figure 1 respectively represent the effects of vinblastine on the ROS levels in HL-60 cells at concentrations of 1, 3, 10, and 30 μM. The more fluorescent spots, the higher the ROS level; Figure 2 also illustrates the effect of vinblastine on the ROS level of HL-60 at concentrations of 1, 3, 10, and 30 μM in the form of a histogram. The above two figures both illustrate that the compound vinblastine (cathachunine) can significantly increase the production of ROS in HL-60 cells after cultured for 48 hours at a concentration of 30 μM.

Embodiment 4: prepare the tablet of pharmaceutical composition of the present invention

The above ingredients are mixed and formulated into tablets according to a usual method.

Embodiment 5: prepare the capsule of pharmaceutical composition of the present invention

The above ingredients are mixed according to the usual method and packed into gelatin capsules.

The above descriptions are only preferred embodiments of the present invention, and are not intended to limit the protection scope of the present invention. Any modification, equivalent replacement, improvement, etc. made within the spirit and principles of the present invention are included in the protection scope of the present invention.

Claims (12)

1. A compound, characterized in that the chemical structural formula is as follows: The compound is called vinblastine. 2. an extraction method of compound vinblastine according to claim 1, is characterized in that, comprises the following steps: (1) Extraction: Dried the whole plant of periwinkle, crushed, extracted with ethanol aqueous solution under reflux, obtained extract, diluted with water, adjusted pH to 2-3 with HCl solution, filtered to obtain filtrate and filter residue, added NH ₃ ·H to the filtrate ₂ O to adjust the pH to 9-10, and extract with chloroform to obtain the chloroform site; (2) Separation: apply silica gel column chromatography to the above chloroform part, and use gradient elution in the chloroform-methanol system with a volume of 100:1 to 2:1, detect by thin layer chromatography, collect and obtain the fraction containing vinblastine, and then After ODS column chromatography, eluting with a methanol-water system with a volume ratio of 20:80, and detecting by thin layer chromatography, the pure product of vinblastine was obtained. 3. The method according to claim 2, characterized in that, the extract is obtained by reflux extraction with ethanol aqueous solution, specifically: 2-4 times with 95% ethanol reflux extraction, each 2-3 hours, the extract Concentrate under reduced pressure to extract. 4. A pharmaceutical composition, characterized in that the pharmaceutical composition comprises the compound vinblastine according to claim 1 and optional pharmaceutically acceptable carriers or excipients. 5. The pharmaceutical composition according to claim 4, characterized in that, based on the total weight of the pharmaceutical composition, the content of the compound vinblastine is 1-99%. 6. The pharmaceutical composition according to claim 5, characterized in that, based on the total weight of the pharmaceutical composition, the content of the compound vinblastine is 20-80%. 7. The pharmaceutical composition according to claim 6, characterized in that, based on the total weight of the pharmaceutical composition, the content of the compound vinblastine is 40-60%. 8. The pharmaceutical composition according to claim 4, wherein the pharmaceutically acceptable carrier or excipient is selected from diluents, dispersants, suspending agents, surfactants, isotonic agents, thickeners Agents, emulsifiers, preservatives, binders, lubricants, stabilizers, hydrating agents, emulsification accelerators, buffers, absorbents, colorants, fragrances, sweeteners, ion exchangers, release agents, coatings Cloth agents, flavoring agents, and antioxidants. 9. The compound vinblastine as claimed in claim 1 or the pharmaceutical composition as claimed in claim 4 are used in the preparation of antitumor drugs. 10. The use according to claim 9, characterized in that, the daily dose of the drug administered to a subject in need is 0.01-1000 mg/kg body weight based on the compound vinblastine. 11. The use according to claim 10, characterized in that, the daily dose of the drug administered to a subject in need is 0.1-100 mg/kg body weight based on the compound vinblastine. 12. The use according to claim 11, characterized in that, the daily dose of the drug administered to a subject in need is 1-100 mg/kg body weight based on the compound vinblastine.