CN101940738A - Coix seed anticancer Chinese medicament and preparation method thereof - Google Patents

Abstract

A coix seed anticancer traditional Chinese medicine, the chemical substance components with anticancer effect extracted from coix seed, the chemical substance components with anticancer effect mainly include: trilinoleic acid glyceride, dilinoleic acid oleic acid Glycerides, Glyceryl Palmitate Dilinoleate, Glyceryl Linoleate Dioleate, Glyceryl Palmitate Linoleate, Glyceryl Dipalmitate Linoleate, Glyceryl Trioleate, Dioleate Palmitate Glyceryl Dioleate, Glyceryl Dioleate, Glyceryl Dioleate. The effective chemical substance group extracted from coix seed is made into the following preparations or dosage forms: oral liquid, tablet, capsule, buccal tablet, pill, injection or spray preparation. The advantages of the present invention are that the extraction process is simple and can be made into A variety of dosage forms, with good anticancer effect.

Description

Coix seed anticancer traditional Chinese medicine and preparation method thereof

field of invention

The invention relates to a traditional Chinese medicine with an anti-tumor (anti-cancer) effect, in particular to a method for preparing an effective chemical substance group with an anti-tumor (anti-cancer) effect from a compound extracted from coix seed. the

Background technique

Coix seed is the dry mature seed kernel of Coix lacryma-jobi L.var.ma-yuen (Roman.) Stapf, a grass plant, which is broadly oval or oblong, 4-8mm long and 3-6mm wide. The surface is milky white, smooth, with occasional remnants of yellowish-brown testa; one end is blunt and round, the other end is wider and slightly concave, with a light brown dotted hilum; the back is round and convex, and the ventral side has a wide and deep longitudinal groove . The quality is solid, the section is white, powdery. It is produced in most parts of my country, mainly in Fujian, Hebei, Liaoning and other places. the

Coix seed was first recorded in "Shen Nong's Materia Medica". It is sweet in nature and light in taste. It contains coixenolide, crude protein 13%-14%, and lipid 2%-8%. In lipids, triacylglycerol is 61%-64%, diacylglycerol is 6%-7%, monoacylglycerol is 4%, sterol ester is 9%, and free fatty acid is 17%-18%. The content of linoleic acid in triacylglycerol can reach 25%-28%, and the content of linoleic acid in free fatty acids is 27%-28%. Free fatty acids also include palmitic acid and stearic acid. (stearic acid), cis-8-octadecenoic acid (cis-8-oc-tadecenoic acid) is oleic acid, etc. Monoacylglycerol has α-monoolein which has anti-tumor effect, and sterol ester has cis-, trans-feruloylstigmasterol and cis- , trans-feruloyl campesterol (cis-, trans-feruloyl campesterol), etc. It also contains anti-complement dextran, acidic polysaccharides CA-1, CA-2 and coixan A, B, C for hypoglycemic effect. Seed volatile oil contains 69 components, the main ones are hexanal, hexanoic acid, 2-ethyl-3-hydroxy-hexylbutrate, γ-nonyl Lactone (γ-nonalactone), nonanoic acid (nonanoic acid), octanoic acid (octanoic aid), ethylpalmitate (ethylpalmitate), methyllinoleate (methyllinoleate), vanillin (vanillin) and ethyl linoleate ( ethyllinoleate) and so on. the

In the study of traditional Chinese medicine, coix seed is used to treat edema, dysuria, beriberi, spleen deficiency and diarrhea, damp arthralgia, lung abscess, intestinal abscess and other diseases. Modern studies believe that coix seed has the effects of anti-tumor, immune regulation, lowering blood sugar and blood calcium, lowering blood pressure, lowering fat and losing weight, anti-malarial, and anti-virus. the

In recent years, published literature has reported some research contents on the chemical constituents and pharmacological effects of coix seed. The chemical composition and pharmacological research progress of Jennifer were reviewed, including the main components of the traditional Chinese medicine such as fatty acids and esters, sterols, triterpenoids, alkaloids, polysaccharides, and related antiviral, immune regulation, and tumor vascular inhibition. Generative, anti-inflammatory, analgesic and sedative effects. the

Liu Jin, Tang Anzhou, Kang Min, etc. published the article "Research on the Effect of Coix Seed Extract on Nasopharyngeal Cancer Cells" in "Guangxi Medicine", studying the effect of Coix Seed Extract on nasopharyngeal cancer cell lines CNE1, CNE2, TWO3 and C666- 1 Inhibitory effect of cells cultured in vitro. Observe the cell morphology changes after it acts on nasopharyngeal carcinoma cells under an inverted microscope. MTT method was used to detect the inhibitory effect of drug extracts and drug-containing serum on cell proliferation. The results proved that coix seed could inhibit the proliferation of CNE1, CNE2, TWO3 and C666-1 cells, and its effect was time- and concentration-dependent. Conclusion Coix seed extract has anti-proliferation effect on nasopharyngeal carcinoma cells in vitro. the

Contents of the invention

The purpose of the present invention is to determine the pharmacological action of the traditional Chinese medicine coix seed - anti-tumor (anti-cancer) effect, to extract anti-tumor effective chemical components and extraction methods from coix seed, and to use these effective chemical components as a preparation Raw materials of anti-tumor (anti-cancer) drugs, so as to make better use of Chinese herbal medicine resources in China and enrich the field of Chinese herbal medicine for treating diseases. the

The present inventor has conducted research on the extract of the Coix seed medicinal material, and the in vivo and in vitro anticancer test studies have shown that the extract has a good antitumor effect. the

The technical solution adopted is:

An anticancer traditional Chinese medicine of coix seed is characterized in that it is a chemical substance component with antitumor (anticancer) effect extracted from coix seed. The chemical substance components with anti-tumor effect mainly include:

1. Glyceryl trilinoleate, molecular formula: C ₅₇ H ₉₈ O ₆ , molecular weight: 878.7 {retention time (min) in the liquid chromatogram: 9.721}

2. Dilinoleic acid glyceryl oleate, molecular formula: C ₅₇ H ₁₀₀ O ₆ , molecular weight: 880.8 {retention time (min) in the liquid chromatogram: 11.679}

3. Glyceryl palmitate, molecular formula: C ₅₅ H ₉₈ O ₆ , molecular weight: 854.7 {retention time (min) in the liquid chromatogram: 12.228}

4. Glyceryl linoleate, molecular formula: C ₅₇ H ₁₀₂ O ₆ , molecular weight: 882.8 {retention time (min) in the liquid chromatogram: 14.188}

5. Palmitic acid linoleic acid glyceryl oleate, molecular formula: C ₅₅ H ₁₀₀ O ₆ , molecular weight: 856.8 {retention time (min) in the liquid chromatogram: 14.858}

6. Dipalmitic linoleic acid glyceride, molecular formula: C ₅₃ H ₉₈ O ₆ , molecular weight: 830.7 {retention time (min) in the liquid chromatogram: 15.584}

7. Glyceryl trioleate, molecular formula: C ₅₇ H ₁₀₄ O ₆ , molecular weight: 884.8 {retention time (min) in the liquid chromatogram: 17.370}

8. Glyceryl palmitate, molecular formula: C ₅₅ H ₁₀₂ O ₆ , molecular weight: 858.8 {retention time (min) in the liquid chromatogram: 18.327}

9. Dipalmitic acid glyceryl oleate, molecular formula: C ₅₃ H ₁₀₀ O ₆ , molecular weight: 832.8 {retention time (min) in the liquid chromatogram: 19.317}

10. Glyceryl stearate dioleate, molecular formula: C ₅₇ H ₁₀₆ O ₆ , molecular weight: 886.8 {retention time (min) in the liquid chromatogram: 22.140}

(See Attachment 1)

The chemical components with anti-tumor effect, under the liquid phase chromatographic conditions: the chromatographic column is a Dima C18 column (4.6mm × 250mm, 5 μm), and the mobile phase is acetonitrile-dichloromethane (60:40); Flow rate: 1mL/min; column temperature: 25°C; ELSD parameters: drift tube temperature 35°C. the

The effective chemical substance group extracted from coix seed is made into the following preparations or dosage forms: oral administration preparations: oral liquid, tablets, capsules, buccal tablets, pills; injection administration preparations: injections; spray administration preparations. the

The effective anti-cancer components extracted from coix seed have been proved by experiments to inhibit the growth of cancer cells both in vivo and in vitro, and can be applied to the treatment of cancer. the

The method for extracting effective chemical components with anti-cancer effect is to add an appropriate amount of ethyl acetate to the coix seed coarse powder and reflux extraction for 3 to 4 times, combine the extracts, filter, recover the solvent, and dry under reduced pressure until no solvent is obtained. Coix Seed Effective Substance Group. the

The invention has the advantages of simple extraction process, can be made into various dosage forms, and has good anticancer effect. the

Description of drawings

Figure 1 is a liquid chromatogram of effective chemical components extracted from Coix Seed that have anti-tumor (anti-cancer) effects. the

Fig. 2 is a graph showing the inhibitory effect of 10% drug-containing serum on human liver cancer cell HepG2. the

Fig. 3 is a graph showing the inhibitory effect of 10% drug-containing serum on human liver cancer cell SMMC-7721. the

为：阳性对照 Numbers in Figure 2 and Figure 3

For: positive control

For: high dose

为：中剂量 

For: medium dose

为：低剂量 

For: low dose

Detailed ways

Extraction of effective components of coix seed:

Pre-extraction treatment: crush the coix seed medicinal material first, and pass through a 20-mesh sieve. the

Extraction method: add 3 times the amount of ethyl acetate, reflux extraction at 70°C for 3 times, combine the extracts, filter, recover the solvent, dry under reduced pressure until there is no solvent, and obtain the effective components of coix seed. the

Pharmacological study on the effective substance group of coix seed

Mice inoculated with H22 liver cancer solid tumor were gavaged with coix seed extract, and its inhibitory effect was determined. the

In vitro experiments, MTT method combined with serum pharmacology methods were used to determine the growth inhibition of mouse serum containing Coix seed extract on human liver cancer cell lines HepG2 and SMMC-7721, which is the proof of the anti-tumor (anti-cancer) effect of Coix seed extract. Foundation for further research. the

in conclusion

In the in vivo and in vitro experiments, the effective substance group of coix seed has obvious inhibitory effect on the growth of tumor cells. the

Preparation of anti-liver cancer drugs

The effective chemical substance group with anti-cancer effect in coix seed can be prepared into anti-liver cancer drugs, for example, can be prepared into different dosage forms according to needs: oral administration preparations, such as oral liquid, tablet, capsule, buccal tablet, pill ; Injection preparations, such as injections; in the anti-cancer tests of spray preparations, etc., it has a good anti-liver cancer effect. the

1. Materials and methods

1.1 Materials Human liver cancer cell lines HepG2 and SMMC-7721, and mouse liver cancer H22 cell lines were purchased from Shanghai Fumeng Biological Co., Ltd. and preserved in our laboratory. Clean-grade Kunming mice, body weight (18-20g), female (purchased from the Experimental Animal Center of Dalian Medical University, certificate number: SCXX (Liao) 2008-0002). Coix seed was purchased from a supermarket. the

1.2 Reagents and instruments DMEM medium, trypsin, and tetramethylazolium salt (MTT) were all produced by GIBCO in the United States; dimethyl sulfoxide (DMSO): Beijing Solaibao Technology Co., Ltd.; calf serum (NBCS ): Hangzhou Sijiqing Bioengineering Materials Co., Ltd.; Penicillin: Harbin Pharmaceutical Group Pharmaceutical Factory Batch No.: A081100207; Streptomycin: Dalian Merro Pharmaceutical Factory Batch No.: 65081216; Cyclophosphamide: Jiangsu Hengrui Pharmaceutical Co., Ltd. Batch No. : 09040721

ACCULAB ALC-11C.4 electronic balance: Sartorius Group, Germany; NUAIRETM US AUTOFLOW CO ₂ incubator: NUAIRE, Germany; BCN-1360B biological clean bench: Beijing Donglian Haer Instrument Manufacturing Co., Ltd.; AE31 Inverted phase contrast microscope: Motic; SUNRISE microplate reader: TECAN, Switzerland; Milli-Q ultrapure water treatment device: Millipore, USA; stainless steel positive pressure filter (500mL): Haining Yatai Pharmaceutical Machinery Co., Ltd.; 96 wells sterile Culture plate: American Costar Company; portable pressure steam sterilizer: Dandong Daily Hardware Factory; blood counting plate: Shanghai Qiujing Biochemical Instrument Factory

1.3 Experimental method

1.3.1 Preparation of drug Coix seed coarse powder, reflux extraction with ethyl acetate, recovery of solvent. the

1.3.2 Grouping of animals and dosage The mice were randomly divided into 6 groups, 10 in each group. They were blank control group; negative control group, intragastric administration of normal saline, 0.2ml per day; cyclophosphamide control group (CTX), dose 20mg·kg ^-1 ·d ^-1 ; large, medium and small doses of coix seed extract The groups are 3, 1, and 0.3 g·kg ^-1 ·d ^-1 , respectively.

1.3.3 Administration method and sample preparation Each group was inoculated with H22 tumor strain according to conventional methods, tumor cells (ice bath) 2×10 ⁷ /mL, each mouse was inoculated with 0.2mL in the right anterior armpit, and the administration was started on the next day after inoculation. 1 time a day, 10 days of medication. The mice in each group were subjected to the following test 1 hour after the last administration: after weighing, the eyeballs were removed to take blood, which was dropped into a centrifuge tube, and the whole blood was incubated at 37°C for 30 minutes, centrifuged at 3500r·min ^-1 for 10 minutes, and the upper serum was drawn , and the same group of sera were combined, 0.45um, 0.22um microporous membranes were filtered and sterilized in turn, and stored in a -80°C refrigerator for later use. They were sacrificed by cervical dislocation, the tumor mass was completely stripped off, weighed and recorded with a precision electronic balance, and the tumor inhibition rate was calculated.

Tumor inhibition rate (%)=[(average tumor weight in the control group-average tumor weight in the administration group)/average tumor weight in the control group]×100%

1.3.4 MTT (thiazolium) test Human liver cancer cells HepG2 and SMMC-7721 were inoculated in DMEM culture medium containing 10% inactivated newborn calf serum, and cultured in an incubator at 37°C and 5% CO ₂ . Digest and passage with 0.25% trypsin on 2-3 days. After 2 to 3 passages, take a bottle of cells in logarithmic growth phase and in good growth state, wash with PBS, digest with 0.25% trypsin, and when the cells tend to become round, wash and blow the cells with culture medium to make cells Suspension. Count with a hemocytometer, add culture medium to dilute to a concentration of 5×10 ⁴ cells/mL, inoculate in a 96-well culture plate, 100 uL per well. After culturing for 24 hours, culture medium containing different concentrations of serum from different experimental groups was added, and 5 replicate wells were set up in each group. Cultivate continuously in the incubator for 24h, 48h, 72h, and 96h, add 20uL of MTT (5mg/mL) solution respectively, continue to cultivate for 4h, suck off the supernatant, add 150uL of dimethyl sulfoxide to each well, shake until bromo The thiazolyl blue was completely dissolved, and its absorbance (OD) was measured with a microplate reader at a wavelength of 492nm, and the absorbance (OD) of the blank control was subtracted during the measurement.

Cell growth inhibition rate (IC%)=[(control group OD-administration group OD)/control group OD]×100%

表示，数据用SPSS15.0软件分析。 1.3.5 Statistical processing

Indicates that the data were analyzed with SPSS15.0 software.

2 result

2.1 Inhibitory effect of coix seed extract on mouse H22 liver cancer solid tumor The inhibition rate of each dose is higher than 30%, which has obvious inhibitory effect. The tumor inhibition rate has a linear relationship with the administered dose. Compared with the negative control group, the average There was a significant difference in weight (P<0.05), but no significant difference compared with the cyclophosphamide control group (P>0.05) (see Table 1). the

2.2 The in vitro inhibitory effect of drug-containing serum on human tumor cell lines The OD values of drug-containing serum in CTX serum, Coix seed large, medium and small dose groups were significantly different from those of blank serum at each time of action (P<0.05) (See Table 2). the

Table 1 Inhibitory effect of coix seed extract on H22 liver cancer solid tumor in mice

Note: Compared with the control group *P<0.05

Table 2 In vitro inhibitory effect of 10% drug-containing serum on human tumor cell lines

Claims (3)

1. A kind of coix seed anticancer Chinese medicine, it is characterized in that the chemical substance component with anticancer effect extracted from coix seed, the chemical substance component with anticancer effect mainly contains: ①. Glyceryl trilinoleate, molecular formula: C ₅₇ H ₉₈ O ₆ , molecular weight: 878.7, retention time in the liquid chromatogram is 9.721 minutes, ②. Dilinoleic acid glyceryl oleate, molecular formula: C ₅₇ H ₁₀₀ O ₆ , molecular weight: 880.8, retention time in the liquid chromatogram is 11.679 minutes, ③. Glyceryl palmitate dilinoleate, molecular formula: C ₅₅ H ₉₈ O ₆ , molecular weight: 854.7, retention time in the liquid chromatogram is 12.228 minutes, ④. Glyceryl linoleate, molecular formula: C ₅₇ H ₁₀₂ O ₆ , molecular weight: 882.8, retention time in the liquid chromatogram is 14.188 minutes, ⑤. Palmitic acid linoleic acid glyceryl oleate, molecular formula: C ₅₅ H ₁₀₀ O ₆ , molecular weight: 856.8, retention time in the liquid chromatogram is 14.858 minutes, ⑥. Dipalmitic acid linoleic acid glyceride, molecular formula: C ₅₃ H ₉₈ O ₆ , molecular weight: 830.7, retention time in the liquid chromatogram is 15.584 minutes, ⑦. Glyceryl trioleate, molecular formula: C ₅₇ H ₁₀₄ O ₆ , molecular weight: 884.8, retention time in the liquid chromatogram is 17.370 minutes, ⑧. Glyceryl palmitate, molecular formula: C ₅₅ H ₁₀₂ O ₆ , molecular weight: 858.8, retention time in the liquid chromatogram is 18.327 minutes, ⑨. Dipalmitic acid glyceryl oleate, molecular formula: C ₅₃ H ₁₀₀ O ₆ , molecular weight: 832.8, retention time in the liquid chromatogram is 19.317 minutes, ⑩. Glyceryl stearate dioleate, molecular formula: C ₅₇ H ₁₀₆ O ₆ , molecular weight: 886.8, retention time in liquid chromatogram: 22.140 minutes. 2. a kind of coix seed anticancer Chinese medicine according to claim 1, is characterized in that described chemical component with anticancer effect, under liquid phase chromatographic conditions: chromatographic column is Dima C18 column (4.6 mm×250mm, 5μm), the mobile phase is acetonitrile-dichloromethane (60:40); the flow rate is 1mL/min; the column temperature is 25°C; the ELSD parameter is the drift tube temperature 35°C. When the chromatographic conditions for the analysis of chemical substances with anti-cancer effects are changed, the order and time of elution of chromatographic peaks (compounds) will change accordingly. 3. A kind of coix seed anticancer traditional Chinese medicine according to claim 1, characterized in that the effective chemical substance group extracted from coix seed is made into the following preparations or dosage forms: oral liquid, tablet, capsule, buccal tablet, pill, Preparations for injection or spray administration.

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