An anticancer composition that inhibits cancer cell growth and its application

Abstract

This application discloses an anticancer composition for inhibiting cancer cell growth and its application. The anticancer composition for inhibiting cancer cell growth comprises Ashitaba polypeptide microecological extract, anthraquinone compounds, radish seed extract, curcumin, resveratrol, fucoidan, and lycopene. This application innovatively combines Ashitaba polypeptide microecological extract, anthraquinone compounds, radish seed extract, curcumin, resveratrol, fucoidan, and lycopene in a combination that can inhibit various cancer cells, including lung cancer, gastric cancer, liver cancer, breast cancer, colon cancer, cervical cancer, bile duct cancer, esophageal cancer, floor of mouth cancer, and nasopharyngeal cancer, providing a new solution for the treatment of these cancers.

Description

Technical Field

[0001] This application relates to the field of cancer treatment technology, and in particular to an anticancer composition that inhibits the growth of cancer cells and its application. Background Technology

[0002] Cancer is one of the diseases that has the greatest impact on human health and life. The cause of cancer is usually a series of abnormal gene changes caused by the long-term influence of various single or multiple factors such as chemicals, physical agents, and viruses, which in turn lead to malignant cell proliferation.

[0003] Currently, cancer treatment methods mainly include traditional surgery, chemotherapy, and radiotherapy. With the research and development of Traditional Chinese Medicine (TCM), various Chinese herbal medicines, herbal extracts, and their combinations have been found to possess different anti-cancer functions. However, the anti-cancer effects of Chinese herbal medicines or herbal extracts are significantly affected by the quality of the herbs themselves. Therefore, further in-depth research into the active ingredients in Chinese herbal medicines, and the development of new anti-cancer drugs or drug combinations based on these effective active ingredients, is both a key focus and a challenge in the field of TCM research. This is also an effective way for TCM to gain wider recognition and use worldwide. Summary of the Invention

[0004] The purpose of this application is to provide a novel anticancer composition that inhibits the growth of cancer cells and its application.

[0005] The following technical solution is adopted in this application:

[0006] One aspect of this application discloses an anticancer composition for inhibiting the growth of cancer cells, comprising Ashitaba polypeptide microecological extract, anthraquinone compounds, radish seed extract, curcumin, resveratrol, fucoidan and lycopene.

[0007] Preferably, the total weight of Ashitaba polypeptide microecological extract is 70-88%, anthraquinone compounds are 3.9-8.3%, radish seed extract is 7-14%, curcumin is 0.5-2%, resveratrol is 0.05-4.5%, fucoidan is 0.5-1%, and lycopene is 0.05-0.2%.

[0008] Preferably, the Ashitaba polypeptide microecological extract is prepared by the following method:

[0009] Mix 20-35 parts by weight of Ashitaba peptides, 15-30 parts by weight of porcine blood peptides, 2-5 parts by weight of soybean lecithin, 6-12 parts by weight of complex vitamins, 6-12 parts by weight of complex minerals, 4-6 parts by weight of oligogentanose and 6-12 parts by weight of mannitol evenly and then pulverize and sieve to obtain mixed raw materials.

[0010] Add 0.1-1 parts by weight of Bifidobacterium lactis and 0.1-1 parts by weight of Bacillus subtilis to the mixed raw materials, and incubate at 30-35℃ for 2-4 hours to obtain a microecological fermentation broth;

[0011] The microecological fermentation broth is filtered to remove residues and the filtrate is collected. The filtrate is then sonicated at 400-500W for 10-30 minutes and centrifuged at 6000-8000rpm for 10-30 minutes. The supernatant is then sterilized and matured to obtain the Ashitaba polypeptide microecological extract of this application.

[0012] Preferably, the aging process includes placing the sterilized supernatant in a sterilized aging tank for sealed aging and preservation at a temperature of 30-35°C for 1-3 weeks.

[0013] Preferably, the anthraquinone compounds are natural hydroxyanthraquinone compounds.

[0014] Preferably, the natural hydroxyanthraquinone compounds are emodin and / or emodin methyl ether.

[0015] Preferably, emodin and / or emodin methyl ether are extracted from Polygonum cuspidatum.

[0016] Preferably, the radish seed extract is an ethanol extract of radish seeds.

[0017] Preferably, the method for preparing the ethanol extract of radish seeds includes adding 10-30 times the weight of ethanol to radish seed powder, and extracting with ultrasonic and heating aids; then, removing the residue, collecting the filtrate, and removing the solvent from the filtrate to obtain the ethanol extract of radish seeds.

[0018] Preferably, the particle size of the radish seed powder is less than 0.3 mm.

[0019] Preferably, the ethanol is 95% ethanol.

[0020] Preferably, in the preparation method of radish seed extract, the ultrasonic power is 300-400w, the heating temperature is 40-60℃, and the extraction time is 20-60min.

[0021] Another aspect of this application discloses the use of the anticancer composition of this application in the preparation of a medicament for treating cancer.

[0022] Preferably, the anticancer composition of this application is applicable to at least one of lung cancer, gastric cancer, liver cancer, breast cancer, colon cancer, cervical cancer, bile duct cancer, esophageal cancer, floor of mouth cancer, and nasopharyngeal cancer.

[0023] The beneficial effects of this application are as follows:

[0024] The anticancer composition of this application creatively combines Ashitaba polypeptide microecological extract, anthraquinone compounds, radish seed extract, curcumin, resveratrol, fucoidan, and lycopene, which can inhibit various cancer cells such as lung cancer, gastric cancer, liver cancer, breast cancer, colon cancer, cervical cancer, bile duct cancer, esophageal cancer, floor of mouth cancer, and nasopharyngeal cancer, providing a new solution for the treatment of these cancers. Attached Figure Description

[0025] Figures 1 to 3 These are the test results of the anticancer composition and its control in the embodiments of this application against the proliferation inhibitory activity of different cancer cells. Detailed Implementation

[0026] The present application will be further described in detail below through specific embodiments. The following embodiments are only for further explanation of the present application and should not be construed as limiting the present application.

[0027] Unless otherwise specified, all materials and reagents used in the following examples are commercially available, and all experimental methods used in the following examples are conventional methods.

[0028] Example

[0029] The anticancer composition used in this example to inhibit cancer cell growth consists of Ashitaba polypeptide microecological extract, anthraquinone compounds, radish seed extract, curcumin, resveratrol, fucoidan, and lycopene. Its specific preparation method is as follows:

[0030] 1. Preparation of Ashitaba polypeptide microecological extract

[0031] 1) Selection of raw materials (by weight, 1 part represents 10g)

[0032] Fresh Ashitaba leaves (32 parts Ashitaba polypeptide extracted), 20 parts porcine blood peptides, 2 parts soybean lecithin, 6 parts multivitamins, 6 parts multiminerals, 6 parts gentiosaccharides, and 6 parts mannitol (all commercially available).

[0033] 2) Preparation process

[0034] Extraction of Ashitaba polypeptides: Fresh Ashitaba leaves were pressed, and the resulting juice was mixed with pure water to prepare an Ashitaba solution. The solution was then placed in a 60°C water bath for 20 minutes. After the solution cooled to room temperature, trypsin and subtilisin were added for enzymatic hydrolysis at pH 7.5 for 1 hour at 37°C. The solution was centrifuged, and the supernatant was collected. Papain and serine protease were then added for enzymatic hydrolysis at pH 8.5 for 1 hour at 42°C. The solution was centrifuged, and the supernatant was collected. The pH was then adjusted to 5.5, and the solution was hydrolyzed for another 1 hour at 55°C. After enzyme inactivation, the solution was concentrated and dried to obtain Ashitaba polypeptide powder, which was then passed through a 50-mesh sieve for later use.

[0035] Preparation of Ashitaba polypeptide microecological extract: All raw materials except Ashitaba polypeptide were mixed evenly according to the above-mentioned weight proportions and pulverized through a 100-mesh sieve to obtain a mixed raw material; then mixed and stirred with Ashitaba polypeptide, followed by the addition of 0.1 parts by weight of Bifidobacterium lactis and 0.1 parts by weight of Bacillus subtilis, and mixed thoroughly; the mixture was incubated at 35℃ for 2 hours to obtain a microecological fermentation broth; the microecological fermentation broth was filtered to remove residues, and the filtrate was collected; the filtrate was ultrasonicated at 450W for 20 minutes, centrifuged at 7000rpm for 20 minutes, and the supernatant was sterilized. The sterilized supernatant was placed in a sterilized maturation tank and sealed for maturation at 35℃ for 2 weeks to obtain the Ashitaba polypeptide microecological extract.

[0036] Meanwhile, a mixed raw material without the addition of Ashitaba peptides was designed, meaning that all other components were exactly the same except for the absence of Ashitaba peptides. The same Bifidobacterium lactis and Bacillus were added, and the mixture was treated under the same conditions to obtain a control extract.

[0037] 2. Preparation of anthraquinone compounds

[0038] The anthraquinone compound used in this example is a natural hydroxyanthraquinone compound, specifically emodin extracted from Polygonum cuspidatum. The specific preparation method includes:

[0039] 2 kg of Polygonum cuspidatum was crushed, passed through a 20-mesh sieve, and extracted three times with 95% ethanol under reflux in a water bath. The filtrates were combined and concentrated to 300 mL. 200 mL of 3 mol / L sulfuric acid was added, and the mixture was refluxed for 1 h. 1.5 L of water was added, and the mixture was allowed to stand overnight. The mixture was then filtered and dried to obtain a pink powder. The pink powder was placed in a Soxhlet extractor and extracted with ether until the ether was colorless. The ether layer was then extracted with 5% sodium bicarbonate. The aqueous phase was discarded, and the mixture was extracted again with 5% sodium carbonate. The extracts were combined and adjusted to pH 4 with 5 mol / L hydrochloric acid. The mixture was filtered, dried, and crystallized with acetone to obtain an orange-yellow crystalline compound, namely emodin.

[0040] 3. Preparation of Radish Seed Extract

[0041] In this example, radish seed extract was obtained by extraction with 95% ethanol. The specific preparation method includes:

[0042] 2 kg of dried radish seeds were pulverized, passed through a 60-mesh sieve, and 20 times their weight of 95% ethanol were added. The mixture was extracted for 30 min at 50°C under ultrasonic conditions of 350 W. After filtration, the filtrate was collected. The residue was extracted twice more. The filtrates from the three extractions were combined, concentrated under reduced pressure to 200 mL, and then freeze-dried at -80°C to obtain a powder, which is the radish seed extract.

[0043] 4. Preparation of the composition

[0044] The composition used in this example was prepared by taking 70-88g of Ashitaba polypeptide microecological extract, 3.9-8.3g of anthraquinone compounds, 7-14g of radish seed extract, 0.5-2g of curcumin (Meryer M81102), 0.05-4.5g of resveratrol (Maclean MKL-R817262), 0.5-1g of fucoidan (Maclean MKL-F889413), and 0.05-0.2g of lycopene (Meryer M74381). The compositions for each experimental group and the control group were prepared according to the formulations in Table 1.

[0045] Table 1 Formulation of Anticancer Compositions

[0046]

[0047]

[0048] In Table 1, the control extract is a microecological extract prepared using the same method as the Ashitaba polypeptide microecological extract, but without Ashitaba polypeptide, and with the same raw materials as the control extract.

[0049] Fourteen compositions, totaling 14 compositions, were prepared according to the formulations in Table 1 for testing, including Tests 1 to 7 and Controls 1 to 7.

[0050] 5. Anticancer activity test

[0051] The anticancer activity of the above 14 compositions was characterized by their cytotoxicity against cancer cells. The cancer cells used in this experiment included: lung cancer cells A549 (Beyotime), gastric cancer cells GTL-16 (Beyotime), liver cancer cells HLE (Beyotime), breast cancer cells HCC1937 (Beyotime), colon cancer cells HCT116 (Beyotime), cervical cancer cells HeLa (Beyotime), bile duct cancer cells RBE (Beyotime), esophageal cancer cells TE-1 (Beyotime), floor of mouth cancer cells HTX2346C (Haodi Huatuo Biotechnology), and nasopharyngeal cancer cells IM-H136 (IMMOCELL). Human normal hepatocytes QSG 7701 (Beyotime) were used as the control group.

[0052] The inhibitory activity of 14 compositions against the proliferation of 10 types of cancer cells and normal human hepatocytes was determined using the standard MTT assay, specifically including:

[0053] Cells in the logarithmic growth phase were digested with 0.25% trypsin to prepare a single-cell suspension, which was then counted using a cell counter and diluted to a concentration of 5 × 10⁻⁶. 4 Single-cell suspensions of 14 different cells per mL were prepared. Then, 100 μL of each single-cell suspension was added to two 96-well plates, and 20 μL of each of the above 14 compositions was added to each well. A negative control with 20 μL of water was also added. After incubation for 72 hours, 20 μL of MTT at a concentration of 5 mg / mL was added to each well, and the plates were incubated for another 4 hours. The supernatant was discarded, and 150 μL of LDMSO was added to each well. The plates were shaken for 15 min, and the absorbance at 490 nm was measured using a Multiskan SkyHigh microplate reader. The cell proliferation inhibition rate was calculated according to the following formula.

[0054] Cell growth inhibition rate = (1 - absorbance value of cancer cells / absorbance value of normal cells) × 100%

[0055] The ratio of the absorbance value of the cancer cell negative control with added water to the absorbance value of the normal cell negative control with added water was used as a correction coefficient, which was then multiplied by the aforementioned cell growth inhibition rate to obtain the final cancer cell inhibition rate. The results of the inhibitory activity of the 14 compositions against the proliferation of 10 types of cancer cells and normal human hepatocytes are shown in Tables 2 and 3. Figures 1 to 3 As shown.

[0056] Table 2 Results of proliferation inhibition activity test in the experimental group

[0057] cell Experiment 1 Experiment 2 Experiment 3 Experiment 4 Experiment 5 Experiment 6 Experiment 7 lung cancer 66％ 78％ 69％ 71％ 70％ 77％ 76％ Stomach cancer 62％ 71％ 66％ 67％ 65％ 72％ 70％ liver cancer 56％ 67％ 58％ 62％ 61％ 66％ 66％ Breast cancer 39％ 48％ 39％ 40％ 42％ 47％ 45％ colon cancer 48％ 59％ 51％ 54％ 50％ 59％ 58％ Cervical cancer 35％ 49％ 40％ 41％ 40％ 47％ 46％ Bile duct cancer 47％ 59％ 51％ 52％ 51％ 58％ 57％ esophageal cancer 46％ 58％ 49％ 52％ 51％ 58％ 57％ floor of mouth cancer 42％ 48％ 39％ 40％ 42％ 49％ 48％ Nasopharyngeal carcinoma 44％ 57％ 49％ 50％ 49％ 56％ 53％ normal cells 0％ 0％ 0％ 0％ 0％ 0％ 0％

[0058] Table 3 Results of proliferation inhibition activity test in the control group

[0059] cell Comparison 1 Comparison 2 Comparison 3 Compare with 4 Compare with 5 Compare with 6 Compare with 7 water lung cancer 10％ 21％ 27％ 28％ 28％ 24％ 25％ 0％ Stomach cancer 8％ 17％ 21％ 20％ 19％ 15％ 14％ 0％ liver cancer 5％ 21％ 26％ 25％ 23％ 21％ 19％ 0％ Breast cancer 2％ 8％ 10％ 11％ 10％ 9％ 7％ 0％ colon cancer 5％ 11％ 15％ 18％ 16％ 13％ 11％ 0％ Cervical cancer 2％ 7％ 9％ 9％ 8％ 7％ 7％ 0％ Bile duct cancer 3％ 10％ 15％ 16％ 18％ 16％ 14％ 0％ esophageal cancer 4％ 15％ 17％ 17％ 16％ 14％ 16％ 0％ floor of mouth cancer 3％ 9％ 8％ 10％ 9％ 8％ 7％ 0％ Nasopharyngeal carcinoma 7％ 10％ 11％ 9％ 9％ 10％ 9％ 0％ normal cells 0％ 0％ 0％ 0％ 0％ 0％ 0％ 0％

[0060] Table 2, Table 3 Figures 1 to 3 The results showed that the seven compositions in experiments 1 to 7 had good growth inhibitory effects on lung cancer cells A549, gastric cancer cells GTL-16, liver cancer cells HLE, breast cancer cells HCC1937, colon cancer cells HCT 116, cervical cancer cells HeLa, bile duct cancer cells RBE, esophageal cancer cells TE-1, floor of mouth cancer cells HTX2346C, and nasopharyngeal cancer cells IM-H136, while having almost no effect on normal human liver cells QSG 7701. In contrast, the seven compositions in controls 1 to 7 showed significantly weaker growth inhibitory effects on the various cancer cells.

[0061] Morphological observation of cancer cells: A549 lung cancer cells, GTL-16 gastric cancer cells, HLE liver cancer cells, and QSG 7701 normal human liver cells were seeded into new 96-well plates. After culturing for 1 hour, 14 different compositions were added to the cultured cells, with 0.1% DMSO as a negative control. After culturing for another 72 hours, the cells were collected, washed with pre-cooled phosphate buffer, stained with DAPI, and observed under a fluorescence microscope.

[0062] The results showed that the negative control group's lung cancer cells A549, gastric cancer cells GTL-16, liver cancer cells HLE, and normal human liver cells QSG 7701 all grew well. At the same time, normal human liver cells QSG 7701, which were supplemented with 14 different compositions, also grew well. The lung cancer cells A549, gastric cancer cells GTL-16, and liver cancer cells HLE, which were supplemented with experiments 1 to 7, all showed varying degrees of nuclear rupture and chromosome aggregation, and apoptotic bodies were produced. In contrast, although a small number of nuclear ruptures also occurred in controls 1 to 7, the degree was significantly lower than that in experiments 1 to 7.

[0063] Furthermore, flow cytometry was used to detect the apoptosis rate of lung cancer cells A549, gastric cancer cells GTL-16, liver cancer cells HLE, and normal human liver cells QSG 7701. The results showed a high degree of inhibition compared to the inhibition rate results in Table 2. That is, experiments 1 to 7 could better promote apoptosis of lung cancer cells A549, gastric cancer cells GTL-16, and liver cancer cells HLE, while having almost no effect on normal human liver cells QSG7701. In contrast, the apoptosis effect of controls 1 to 7 was significantly reduced.

[0064] The above experiments demonstrate that the anticancer composition consisting of 70-88g of Ashitaba polypeptide microecological extract, 3.9-8.3g of anthraquinone compounds, 7-14g of radish seed extract, 0.5-2g of curcumin, 0.05-4.5g of resveratrol, 0.5-1g of fucoidan, and 0.05-0.2g of lycopene can effectively inhibit the growth of lung cancer cells, gastric cancer cells, liver cancer cells, breast cancer cells, colon cancer cells, cervical cancer cells, bile duct cancer cells, esophageal cancer cells, floor of mouth cancer cells, and nasopharyngeal cancer cells, and promote their apoptosis; moreover, it has almost no effect on normal human cells. Therefore, the anticancer composition of this experiment can be used to prepare drugs for the treatment of lung cancer, gastric cancer, liver cancer, breast cancer, colon cancer, cervical cancer, bile duct cancer, esophageal cancer, floor of mouth cancer, or nasopharyngeal cancer.

[0065] The above description, in conjunction with specific embodiments, provides a further detailed explanation of this application and should not be construed as limiting the specific implementation of this application to these descriptions. Those skilled in the art to which this application pertains can make several simple deductions or substitutions without departing from the concept of this application.

Claims

1. An anticancer composition for inhibiting the growth of cancer cells, characterized in that: It is composed of Ashitaba polypeptide microecological extract, anthraquinone compounds, radish seed extract, curcumin, resveratrol, fucoidan and lycopene; The Ashitaba polypeptide microecological extract accounts for 70-88% of the total weight, the anthraquinone compounds account for 3.9-8.3% of the total weight, the radish seed extract accounts for 7-14% of the total weight, the curcumin accounts for 0.5-2% of the total weight, the resveratrol accounts for 0.05-4.5% of the total weight, the fucoidan accounts for 0.5-1% of the total weight, and the lycopene accounts for 0.05-0.2% of the total weight. The anthraquinone compound is a natural hydroxyanthraquinone compound, and the natural hydroxyanthraquinone compound is emodin; The Ashitaba polypeptide microecological extract was prepared by the following method: Mix 20-35 parts by weight of Ashitaba peptides, 15-30 parts by weight of porcine blood peptides, 2-5 parts by weight of soybean lecithin, 6-12 parts by weight of complex vitamins, 6-12 parts by weight of complex minerals, 4-6 parts by weight of oligogentanose and 6-12 parts by weight of mannitol evenly and then pulverize and sieve to obtain mixed raw materials. Add 0.1-1 parts by weight of Bifidobacterium lactis and 0.1-1 parts by weight of Bacillus subtilis to the mixed raw materials, and incubate at 30-35℃ for 2-4 hours to obtain a microecological fermentation broth; The microecological fermentation broth is filtered to remove residues and the filtrate is collected. The filtrate is ultrasonicated at 400-500W for 10-30 minutes and then centrifuged at 6000-8000rpm for 10-30 minutes. The supernatant is then sterilized and matured to obtain the Ashitaba polypeptide microecological extract. The preparation method of the Ashitaba polypeptide includes: pressing fresh Ashitaba leaves as raw material, adding pure water to the obtained juice and stirring evenly to obtain Ashitaba solution, and keeping it in a 60℃ water bath for 20 minutes; after the solution temperature drops to room temperature, adding trypsin and subtilisin for enzymatic hydrolysis at pH 7.5 for 1 hour at 37℃, centrifuging, and taking the supernatant; then adding papain and serine protease for enzymatic hydrolysis at pH 8.5 for 1 hour at 42℃, centrifuging, taking the supernatant, adjusting the pH to 5.5 and hydrolyzing for 1 hour at 55℃, and after enzyme inactivation treatment, concentrating and drying to obtain Ashitaba polypeptide powder; The radish seed extract is an ethanol extract of radish seeds. The preparation method of the ethanol extract of radish seeds includes: adding 10-30 times the weight of ethanol to radish seed powder, and extracting with ultrasonic and heating aids; then, removing the residue, collecting the filtrate, and removing the solvent from the filtrate to obtain the ethanol extract of radish seeds. The cancer cells are at least one of the following: lung cancer, stomach cancer, liver cancer, breast cancer, colon cancer, cervical cancer, bile duct cancer, esophageal cancer, floor of mouth cancer, and nasopharyngeal cancer.

2. The anticancer composition according to claim 1, characterized in that: The aging process includes placing the sterilized supernatant in a sterilized aging tank for sealed aging and preservation at a temperature of 30-35°C for 1-3 weeks.

3. The anticancer composition according to claim 1, characterized in that: The emodin was extracted from Polygonum cuspidatum.

4. The anticancer composition according to any one of claims 1-3, characterized in that: The radish seed powder has a particle size of less than 0.3 mm.

5. The anticancer composition according to any one of claims 1-3, characterized in that: In the method for preparing the ethanol extract of radish seeds, the ethanol is 95% ethanol.

6. The anticancer composition according to any one of claims 1-3, characterized in that: In the preparation method of ethanol extract of radish seeds, the ultrasonic power is 300-400w, the heating temperature is 40-60℃, and the extraction time is 20-60min.

7. The use of the anticancer composition according to any one of claims 1-6 in the preparation of a medicament for treating cancer. The cancer mentioned is at least one of the following: lung cancer, stomach cancer, liver cancer, breast cancer, colon cancer, cervical cancer, bile duct cancer, esophageal cancer, floor of mouth cancer, and nasopharyngeal cancer.

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