Radix asparagi extract and anti-tumor application thereof

Abstract

The invention discloses a radix asparagi extract and an application thereof in tumor resistance, and belongs to the field of biological medicine, radix asparagi is heated and extracted with ethanol with the concentration of 60% to obtain an alcohol extract, then the alcohol extract is added into macroporous resin, then elution is performed with ethanol with the concentration of 30% and ethanol with the concentration of 60% in sequence, ethanol eluate with the concentration of 60% is collected, and concentration and drying are performed to obtain the radix asparagi extract. The radix asparagi extract disclosed by the invention has a relatively good inhibition effect on various cancer cells.

Description

Technical Field

[0001] The invention belongs to the field of biomedicine, and specifically relates to an asparagus extract and an application thereof in anti-tumor. Background Art

[0002] Asparagus cochinchinensis is a plant of the Liliaceae family. Asparagus cochinchinensis (Lour.) Merr.] The dried root tuber tastes sweet and slightly bitter, and is cold in nature. It has the effects of nourishing yin, clearing heat, moistening the lungs and nourishing the kidneys. It is used to treat yin deficiency fever, lung abscess, thirst and other diseases. Domestic and foreign studies have found that asparagus mainly contains steroidal saponins, oligosaccharides and polysaccharides, and has anti-tumor, antioxidant, anti-aging, immunity enhancement, anti-inflammatory and other pharmacological effects. At present, the research on the anti-tumor effect of asparagus mainly focuses on the inhibition of tumor cell proliferation and apoptosis by monomers and crude extracts, and its anti-tumor efficacy site has not been clarified, and there are no relevant patents or literature reports. Summary of the invention

[0003] The technical problem to be solved by the present invention is to provide an asparagus extract and its application in anti-tumor.

[0004] In order to solve the above technical problems, the technical solutions adopted by the present invention are as follows: A method for preparing an asparagus extract comprises the following steps: heating and extracting the asparagus with 60% ethanol to obtain an alcohol extract, adding the alcohol extract to a macroporous resin, eluting the extract with 30% and 60% ethanol in sequence, collecting the 60% ethanol eluate, concentrating and drying the eluate to obtain the asparagus extract.

[0005] Furthermore, before the heating extraction, enzyme treatment is required, and the method of enzyme treatment is: weigh the dried medicinal material of Asparagus cochinchinensis, add 1.5% cellulase and 1.2% pectinase according to a solid-liquid ratio of 1:5, soak at 30°C for 2h, filter, and collect the filter residue.

[0006] Furthermore, the heating extraction method is: adding 60% ethanol at a concentration of 1:20 to the collected filter residue and heating reflux extraction twice, each time for 1 hour, collecting the filtrate, and concentrating by rotary evaporation to obtain the alcohol extract.

[0007] Furthermore, the elution method is: adding the alcohol extract to the HPD-300 macroporous resin, and then eluting with ethanol at concentrations of 30% and 60% in sequence.

[0008] Furthermore, the concentration and drying method is rotary evaporation concentration followed by freeze drying.

[0009] The application of the asparagus extract obtained by the above-mentioned preparation method in the preparation of anti-tumor drugs.

[0010] Further, the tumor is any one of human breast cancer, human cervical cancer, human liver cancer, human lung cancer, human colon cancer, human brain astrocytic glioblastoma or human glioma. Preferably, the tumor is human breast cancer.

[0011] Compared with the prior art, the present invention has the following beneficial effects: The invention provides a method for preparing an asparagus cochinchinensis extract. The method is simple and has high extraction efficiency. The obtained extract has good anti-tumor effect. BRIEF DESCRIPTION OF THE DRAWINGS

[0012] Figure 1 This is the total ion current (ESI positive ion) of the asparagus extract eluted with 60% ethanol. DETAILED DESCRIPTION

[0013] The experimental methods in the following examples are all conventional methods unless otherwise specified. The experimental materials used in the following examples are all purchased from commercial channels unless otherwise specified.

[0014] Example 1 Preparation of Asparagus cochinchinensis Extract Weigh the coarse particles of dried Asparagus cochinchinensis, add 1.5% cellulase and 1.2% pectinase according to a 1:5 solid-liquid ratio, soak at 30℃ for 2h and filter. Add 60% ethanol at a ratio of 1:20 and heat under reflux for extraction twice, each time for 1h. Collect the filtrate and concentrate the eluent by rotary evaporation. Use HPD-300 macroporous resin for adsorption and loading, the sample concentration is 0.1g / ml, and the loading volume is 10BV. Then use 30%, 60%, and 90% ethanol as eluents, and elute in turn to obtain extracts of different fractions. The 30% ethanol eluent, 60% ethanol eluent, and 90% ethanol eluent were concentrated by rotary evaporation and freeze-dried to obtain drug dry powder (AcTS) of different ethanol elution groups.

[0015] Example 2 Study on anti-tumor activity of different elution groups The different dry powders obtained in Example 1 were subjected to cell experiments to screen their anti-tumor activities. Human breast cancer cell line MDA-MB-231, human colon cancer cell line HCT116, and human cervical cancer cell line Hela were used for preliminary screening of their activities, and the drug action time on cells was 24 hours.

[0016] Cell culture and screening methods: 1. Cell culture Cell recovery: Take the cell line out of the liquid nitrogen biological container in the laboratory, thaw it in a 37℃ constant temperature water bath, and then put it on ice. Process the cells on an ultra-clean workbench, transfer them to a centrifuge tube with a Pasteur pipette, centrifuge at 1200rpm for 2min, discard the supernatant, add 3 mL of complete culture medium to the tube to form a cell suspension, transfer the cell suspension to a cell culture bottle, and culture it in a 37℃, 5% CO2 incubator.

[0017] Cell passaging: When the cells grow to 80% in the culture flask, discard the culture medium and wash the cells with 2 mL PBS. Add 2 mL trypsin to the flask and digest in the incubator. When the cells become round and float under an inverted microscope, add complete culture medium to stop digestion. Pipette the cell suspension into a sterile centrifuge tube, centrifuge at 1200 rpm for 2 min, discard the supernatant, add 6 mL complete culture medium, transfer to 2 culture flasks, and culture in a CO2 incubator.

[0018] Cell freezing: When the cells grow to 80% in the culture flask, discard the culture medium and wash the cells with 2 mL PBS. Add 2 mL of trypsin to the flask and digest in the incubator. When the cells become round and float under an inverted microscope, add complete culture medium to stop digestion. Pipette the cell suspension into a sterile centrifuge tube, centrifuge at 1200 rpm for 2 min, discard the supernatant, add 1 mL of serum-free freezing solution to form a cell suspension, use a program cooling box to freeze at -80°C overnight, and transfer to a liquid nitrogen biological container for long-term storage the next day.

[0019] 2. CCK8 assay to detect the effect of AcTS on tumor cell proliferation Cell plate: When the cell growth is stable, take the tumor cells in the logarithmic growth phase, digest the cells with trypsin, and add complete culture medium to adjust the cell density to 0.8×10 5 ~1.2×10 5 The cell suspension was inoculated into 96-well plates at a concentration of 100 μL per well and cultured in a CO2 incubator overnight.

[0020] Cell administration: When the cells grow to a density of about 80%, discard the cell culture medium, and add 100 μL of AcTS drug-containing culture medium with different concentration gradients into each well. The drug concentration gradient is: 10, 20, 40, 80, 160 μg / mL, and culture for 24 h and 48 h, respectively.

[0021] Activity detection: Add 100 μL CCK-8 reagent (containing 10% CCK8, 90% culture medium) to each well and incubate in a CO2 incubator for 1 h. Use a microplate reader to detect the absorbance of the cell well plate at different drug treatment concentrations at 450 nm. Calculate cell viability.

[0022] The calculation formula is as follows: Cell survival rate = (A1-A0) / (A2-A0) x 100%; Among them, A0 is the absorbance measured by the blank group, A1 is the absorbance measured by the experimental group, and A2 is the absorbance measured by the control group.

[0023] The experimental results showed that the 30% ethanol elution site had no anti-tumor effect, the 60% ethanol elution site had a good inhibitory effect on the three cell lines, and the 90% ethanol elution site had a certain anti-tumor effect. 50 See Table 1 for values.

[0024] Table 1 IC values ​​of three ethanol elution sites on three tumor cells 50 value Note: All values ​​are expressed as mean ± standard deviation (n=3). * Compared with the control group, each drug group: ** P<0.01, *** P<0.01, **** The difference was statistically significant when P<0.001.

[0025] Example 3 Component detection and anti-tumor activity study of the extract from the 60% ethanol elution fraction The mother sapogenin was used as a reference substance, and the content of the extract from the 60% ethanol elution part was determined. The results showed that the total saponin content of this part reached 66.48%. The total ion current of the 60% ethanol elution part was analyzed by UHPLC-Q-TOF MS. Figure 1 .

[0026] The anti-tumor activity of the extract from the 60% ethanol elution fraction was further investigated. Cell lines (human breast cancer cell lines MDA-MB-231, MCF-7, human cervical cancer cell line Hela, human liver cancer cell line HepG2, human lung cancer cell line A549, human colon cancer cell line HCT116, human brain astrocytic glioblastoma cell line U87MG, human glioma cell line U251MG) were used for screening of their anti-tumor activity. The cell culture and screening methods were the same as those in Example 2.

[0027] The results are shown in Table 2. The extracts from the 60% ethanol elution site have different inhibitory effects on the proliferation of 8 tumor cells. With the increase of drug concentration and the extension of action time, the lower the activity of tumor cells, the stronger the inhibitory effect is and the dose-dependent. Table 2 shows that the IC values ​​of the extracts from the 60% ethanol elution site for the inhibition of the growth of five tumor cells are 50 The values ​​were all less than 100 μg / mL, and the inhibitory effect on breast cancer cells (MDA-MB-231 and MCF-7) was the best. The IC 50 The values ​​were 16.110 μg / mL and 17.903 μg / mL, respectively. It had good inhibitory effects on HepG2, U251, Hela and A549 cells, and its IC 50The values ​​were 32.120 μg / mL, 41.745 μg / mL, 46.134 μg / mL, and 48.293 μg / mL, respectively. The inhibitory effect on U87 and HCT116 cells was slightly weaker, and its IC 50 The values ​​were 64.400 and 66.565 μg / mL, respectively.

[0028] The above results all indicate that the total saponins extracted from the 60% ethanol elution part of Asparagus cochinchinensis have a certain inhibitory effect on different tumor cells and have the best inhibitory effect on MDA-MB-231 cells.

[0029] Table 2 IC2 of total saponins extracted from 60% ethanol elution of Asparagus cochinchinensis on different tumor cells after 24 and 48 hours 50 Values ​​(n=3) Note: All values ​​are expressed as mean ± standard deviation (n=3). * Compared with the control group, each drug group: ** P<0.01, *** P<0.01, **** The difference was statistically significant when P<0.001.

Claims

1. A method for preparing an asparagus extract, characterized in that: The asparagus was extracted by heating with 60% ethanol to obtain an alcohol extract, which was then added to a macroporous resin and then eluted with 30% and 60% ethanol in sequence, the 60% ethanol eluate was collected, concentrated and dried to obtain the asparagus extract.

2. The preparation method according to claim 1, characterized in that: Before the heating extraction, enzyme treatment is required. The method of enzyme treatment is as follows: weigh the dried medicinal material of Asparagus cochinchinensis, mix it with water at a ratio of 1g medicinal material to 5ml water, then add 1.5g cellulase and 1.2g pectinase per 100ml water, soak at 30°C for 2h, filter, and collect the residue.

3. The preparation method according to claim 2, characterized in that The heating extraction method is: mixing the filter residue collected in claim 2 with 60% ethanol in a ratio of 1g of filter residue to 20ml of ethanol solution, heating and refluxing extraction twice, each time for 1 hour, collecting the filtrate, and concentrating by rotary evaporation to obtain an alcohol extract.

4. The preparation method according to claim 3, characterized in that: The elution method is: adding the alcohol extract to the HPD-300 macroporous resin, and then eluting with ethanol at concentrations of 30% and 60% in sequence.

5. The preparation method according to claim 1, characterized in that: The concentration and drying method is rotary evaporation concentration followed by freeze drying.

6. Use of the asparagus extract obtained by the preparation method according to any one of claims 1 to 5 in the preparation of anti-tumor drugs.

7. The use according to claim 6, characterized in that: The tumor is any one of human breast cancer, human cervical cancer, human liver cancer, human lung cancer, human colon cancer, human brain astrocytic glioblastoma or human glioma.

8. The use according to claim 7, characterized in that: The tumor is human breast cancer.

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