A conjugated mulberry polyphenol, its extraction method and application

Abstract

The present invention relates to the technical field of polyphenol extraction, and specifically discloses a mulberry bound polyphenol, its extraction method and application. The mulberry fruits are freeze-dried and crushed, and an 80% acetone solution is added at a solid-liquid ratio of 1:10 (w:v). After mixing, the supernatant is discarded by centrifugation. The precipitate is hydrolyzed with NaOH solution, neutralized with hydrochloric acid to neutrality, and then ethyl acetate is added continuously for extraction 5 times to obtain a crude extract of mulberry bound polyphenol. The mulberry bound polyphenol is obtained after purification by macroporous resin D101. The mulberry bound polyphenol extracted by the present invention can significantly inhibit the proliferation and viability of various cancer cells, and at the same time has no obvious effect on the growth of normal mammary epithelial cells and liver cells, and has antibacterial activity against Escherichia coli and Staphylococcus aureus. Therefore, it has application potential in the preparation of anti-tumor and antibacterial drugs.

Description

Technical Field

[0001] The present invention relates to the technical field of polyphenol extraction, and particularly relates to a mulberry conjugated polyphenol, an extraction method and an application thereof. Background Art

[0002] Mulberry, also known as mulberry fruit, is the fruit spike of the mulberry plant (Latin name: Morus alba L.) of the Moraceae family. Mulberry has a history of thousands of years of edible and medicinal use in China and enjoys the reputation of "folk holy fruit". "Compendium of Materia Medica" records that it has the effects of "benefiting the five internal organs and joints, promoting blood circulation; making people intelligent and preventing graying and aging". Mulberry is a classic plant that is both edible and medicinal. Modern nutritional research shows that mulberry is rich in various bioactive components, such as vitamins, fatty acids, minerals, polyphenols, and polysaccharides. Epidemiological research shows that mulberry can reduce the incidence risk of chronic diseases such as cardiovascular diseases, diabetes, and cancer.

[0003] Mulberry contains a large amount and a rich variety of polyphenolic compounds, up to (1515.9 ± 5.7 mg GAE / 100 g) fresh weight, which is the highest polyphenol content among all dark fruits. Mulberry polyphenols have the functions of antioxidant, anti-inflammatory, and immune regulation, and have preventive and health-care effects on atherosclerosis, neurodegenerative diseases, diabetes, tumors, etc. Currently, more research has been conducted on free mulberry polyphenols, and there are few reports on conjugated polyphenols and their applications. Summary of the Invention

[0004] In order to extract conjugated polyphenols from mulberry, the present invention provides a mulberry conjugated polyphenol, an extraction method and an application thereof. The mulberry conjugated polyphenol obtained by extraction in the present invention can significantly inhibit the proliferation of various cancer cells, has no obvious effect on the growth of normal mammary epithelial cells and liver cells, and has the potential to prepare anti-tumor drugs. The mulberry conjugated polyphenol provided by the present invention has antibacterial activity against both Escherichia coli and Staphylococcus aureus and can be used for the preparation of antibacterial products.

[0005] The present invention provides an extraction method for mulberry conjugated polyphenol, comprising the following steps:

[0006] S1, mixing mulberry powder and acetone solution according to a solid-liquid ratio of 1 g: 8 - 12 mL, centrifuging to discard the supernatant, and retaining the precipitate;

[0007] S2, hydrolyzing the precipitate obtained by centrifugation in S1 with NaOH solution, and neutralizing with hydrochloric acid to neutrality to obtain a neutral solution;

[0008] S3, adding ethyl acetate to the neutral solution obtained in S2 for extraction, and then removing the organic solvent by rotary evaporation under reduced pressure to obtain a crude extract of mulberry conjugated polyphenol, and obtaining mulberry conjugated polyphenol after purification.

[0009] Further, in S1, the preparation process of the mulberry powder is as follows: the mulberry fruits are crushed, freeze-dried, ground and sieved to obtain the mulberry powder.

[0010] Further, in S1, the material-liquid ratio of the mulberry powder to the acetone solution is 1 g: 10 mL.

[0011] Further, in S2, the molar concentration of the NaOH solution is 1.6 - 2 mol / L.

[0012] Further, in S2, the hydrochloric acid neutralization step is carried out under liquid nitrogen protection to avoid the denaturation of bound polyphenols.

[0013] Further, in S3, macroporous resin D101 is used for purification.

[0014] The present invention also provides bound mulberry polyphenols obtained by extraction with the above method.

[0015] The present invention also provides the application of the described bound mulberry polyphenols in the preparation of anti-tumor drugs, and the bound mulberry polyphenols can inhibit the proliferation and viability of tumor cells.

[0016] Further, the tumor cells include human lung cancer cells, liver cancer cells and breast cancer cells.

[0017] The present invention also provides the application of the described bound mulberry polyphenols in the preparation of bacteriostatic agents, and the bound mulberry polyphenols can inhibit Escherichia coli and Staphylococcus aureus.

[0018] Further, the MIC of the bound mulberry polyphenols against Escherichia coli is 2.5 mg / mL, and the MIC against Staphylococcus aureus is 10 mg / mL.

[0019] Compared with the prior art, the beneficial effects of the present invention are as follows:

[0020] 1. The present invention uses mulberry fruits as experimental materials, and the obtained bound polyphenols can significantly inhibit the proliferation of various cancer cells, and have no obvious effect on normal breast epithelial cells and liver cells. Therefore, the bound polyphenols prepared by the present invention have the potential to prepare anti-tumor drugs, laying a material foundation for the development of anti-cancer drugs and health foods.

[0021] 2. The bound mulberry polyphenols extracted by the present invention have significant antibacterial activity against both Escherichia coli and Staphylococcus aureus, and can be applied to the preparation of bacteriostatic agents and antibacterial drugs.

[0022] 3. The bound mulberry polyphenols extracted by the present invention have both anti-tumor and antibacterial activities, which can promote the in-depth development and utilization of mulberry resources. Description of the Drawings

[0023] 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 required for use in the description of the embodiments or the prior art. Obviously, the drawings in the following description are only some embodiments of the present invention. For those of ordinary skill in the art, without creative efforts, other drawings can also be obtained based on these drawings.

[0024] Figure 1 It is the appearance diagram of the mulberry combined polyphenol sample prepared in Example 1 of the present invention.

[0025] Figure 2 It is the influence of mulberry combined polyphenol on the viability of different tumor cells.

[0026] Figure 3 It is the cell imaging of the influence of mulberry combined polyphenol on the proliferation of different tumor cells.

[0027] Figure 4 It is the statistical chart of the influence of mulberry combined polyphenol on the proliferation of different tumor cells.

[0028] Figure 5 It is the influence of mulberry combined polyphenol on normal cells;

[0029] In the figure, A is the influence of mulberry combined polyphenol on the viability of normal mammary epithelial cells MCF-10A;

[0030] B is the influence of mulberry combined polyphenol on the viability of normal liver cells HL7702.

[0031] Figure 6 It is the influence of different resin purifications on the anti-tumor activity of mulberry combined polyphenol. Specific Embodiments

[0032] The following will describe the specific embodiments of the present invention in detail, but it should be understood that the protection scope of the present invention is not limited by the specific embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those of ordinary skill in the art without creative efforts belong to the protection scope of the present invention. The experimental methods described in the embodiments of the present invention are all conventional methods unless otherwise specified. The materials, reagents, etc. used in the following embodiments can be obtained from commercial channels unless otherwise specified.

[0033] Example 1: A kind of mulberry combined polyphenol and its extraction method.

[0034] I. Experimental Materials

[0035] Mulberry fruits were purchased from Alibaba Health Pharmacy.

[0036] II. Experimental Methods

[0037] 1. Extraction of conjugated polyphenols from mulberries

[0038] (1) Add liquid nitrogen to mulberry fruits and freeze-dry them. Grind them to a powder at low temperature with a grinder, and pass through a 200-mesh sieve to obtain mulberry powder.

[0039] (2) Mix the mulberry powder with 80% (v / v) acetone solution according to a solid-liquid ratio of 1 g:10 mL, shake well for 1 h, centrifuge at 4500 rpm for 15 min, and take the supernatant. Repeat the extraction three times, combine the supernatants, concentrate under reduced pressure to recover the organic solvent, discard the supernatant containing free polyphenols, and retain the precipitate containing conjugated polyphenols.

[0040] (3) Hydrolyze the precipitate by adding 2 mol / L NaOH solution according to a dosage ratio of 1 g:10 mL, stir for 1 h under liquid nitrogen protection, and then slowly neutralize it to neutral with 37% (w / w) hydrochloric acid in small portions, obtaining a neutral solution.

[0041] (4) Extraction: Add ethyl acetate to the neutral solution according to a volume ratio of 1:1, extract repeatedly 5 times, and remove the organic solvent by rotary evaporation under reduced pressure to obtain the crude extract of conjugated polyphenols from mulberries.

[0042] (5) Purify the crude extract of conjugated polyphenols from mulberries with D101 macroporous resin, elute with 95% ethanol, and vacuum-dry the eluate to obtain conjugated polyphenols from mulberries.

[0043] 2. Effects of conjugated polyphenols from mulberries on the viability of different tumor cells

[0044] Select MCF-7, A549, H1299, HepG2, and MDA-MB-231 cells in the logarithmic growth phase, adjust the cell density to 8×10 4 cells / mL, add 100 μL to each well of a 96-well culture plate, and incubate overnight in a 37 °C, 5% CO2 incubator. After the cells adhere to the wall, take out the culture plate, replace the medium with different concentrations (0, 10, 15, 20, 30, 50 μg / mL) of conjugated polyphenols from mulberries, and set 5 parallel wells. After continuing to culture for 48 h, aspirate the liquid in the wells, wash with PBS, add a new medium, add 20 μL of MTT solution (5 mg / mL) to each well, continue to incubate for 4 h, and then terminate the culture. Carefully aspirate the culture supernatant in the wells, add 150 μL of DMSO to each well, and shake for 10 min to fully dissolve the crystals. Measure the light absorption value of each well at a wavelength of 490 nm on an enzyme-linked immunosorbent assay monitor.

[0045] 3. Inhibition of tumor cell proliferation by conjugated polyphenols from mulberries

[0046] Seed well-grown HepG2 and MCF-7 cells at 8×10 3Inoculate at a density of cells per well in a 96-well plate. Wait for the cells to adhere, then add mulberry-bound polyphenols to treat the cells for 24 h. Dilute the EdU solution with cell culture medium at a ratio of (1000:1), add 100 μL of the diluted solution to each well, and incubate in a 37 °C CO₂ incubator for 2 h. Discard the culture medium, wash with PBS, add an appropriate amount of cell staining fixative, incubate at room temperature for 30 min, and then discard the fixative.

[0047] Add 50 μL of 2 mg / mL glycine to each well, incubate on a shaker for 5 min, discard the liquid, and wash with PBS. Add 100 μL of 1× Apollo staining reaction solution to each well, incubate on a shaker at room temperature in the dark for 30 min, discard the liquid, and add 100 μL of PBS containing 0.5% Triton X-100 to wash 3 times. Add 50 μL of DAPI working solution to each well, incubate on a shaker for 10 min, discard the liquid, and wash with PBS. Observe and take pictures under a fluorescence microscope.

[0048] 4. Effects of mulberry-bound polyphenols on the growth of normal mammary epithelial cells and hepatocytes

[0049] Select normal human mammary epithelial cells MCF-10A and human liver cells HL7702 in the logarithmic growth phase, adjust the cell density to 8×10 4 cells / mL, add 100 μL to each well of a 96-well culture plate, and incubate overnight in a 37 °C, 5% CO₂ incubator. After the cells adhere, take out the culture plate, replace the medium with medium containing different concentrations (0, 10, 15, 20, 30, 50 μg / mL) of mulberry-bound polyphenols, and set 5 parallel wells. After continuing to culture for 48 h, aspirate the liquid in the wells, wash with PBS, add fresh medium, add 20 μL of MTT solution (5 mg / mL) to each well, continue to incubate for 4 h, and then terminate the culture. Carefully aspirate the culture supernatant in the wells, add 150 μL of DMSO to each well, and shake for 10 min to fully dissolve the crystals. Measure the optical absorption value of each well at a wavelength of 490 nm on an enzyme-linked immunosorbent assay monitor.

[0050] 5. Study on the antibacterial activity of mulberry-bound polyphenols

[0051] (1) Inhibition zone experiment

[0052] Prepare a 100 mg·mL -1 mulberry-bound polyphenol solution with the obtained mulberry-bound polyphenols using sterile water. Immerse filter paper in the 100 mg·mL -1 mulberry-bound polyphenol solution for 30 min. Take 200 μL of Escherichia coli and Staphylococcus aureus bacterial solutions respectively, spread them evenly on the surface of an LB solid medium plate. After air-drying for 5 min, place the soaked filter paper on the surface of the LB solid medium coated with the bacterial solution. Use filter paper soaked with sterile water as a blank control, and incubate in a 37 °C constant temperature incubator for 24 h.

[0053] (2) Determination of minimum inhibitory concentration (MIC)

[0054] Preparation of bacterial suspension: Escherichia coli or Staphylococcus aureus was activated on an LB solid medium plate, and a single colony was picked and transferred into 100 mL of LB liquid medium. It was cultured with shaking at 37 °C and 150 r / min for 24 h to obtain a bacterial suspension.

[0055] The final mass concentration of mulberry-bound polyphenols was adjusted to 10 mg / mL, 5 mg / mL, 2.5 mg / mL, 1.25 mg / mL, 0.625 mg / mL, and 0.312 mg / mL with LB medium. 500 μL of the Escherichia coli or Staphylococcus aureus bacterial suspension was added to each tube. Sterile water was added to the LB medium as a blank control, and Escherichia coli or Staphylococcus aureus without adding mulberry-bound polyphenols was used as a negative control. After culturing with shaking at 37 °C and 150 r / min for 24 h, the lowest mass concentration of mulberry-bound polyphenols required to significantly inhibit the growth of Escherichia coli or Staphylococcus aureus was recorded as the MIC.

[0056] III. Experimental results

[0057] 1. The appearance of the prepared mulberry-bound polyphenol sample is as Figure 1 shown.

[0058] 2. Effects of mulberry-bound polyphenols on the viability of different tumor cells

[0059] The results are as Figure 2 shown. The CCK-8 experiment showed that the mulberry-bound polyphenols prepared by the present invention had significant inhibitory effects on the cell viability of breast cancer cells MCF-7, liver cancer cells HepG2, lung cancer cells A549, lung cancer cells H1299, and breast cancer cells MDA-MB-231.

[0060] 3. Effects of mulberry-bound polyphenols on the proliferation of tumor cells

[0061] The results are as Figure 3 and Figure 4 shown. The EdU experiment further showed that the mulberry fruit-bound polyphenols significantly inhibited the proliferation of liver cancer and breast cancer cells.

[0062] 4. Effects of mulberry-bound polyphenols on the growth of normal mammary epithelial cells and hepatocytes

[0063] As Figure 5 shown, the mulberry-bound polyphenols had no significant effect on the growth of normal mammary epithelial cells MCF-10A and hepatocytes HL7702.

[0064] 5. Study on the antibacterial activity of mulberry-bound polyphenols

[0065] Table 1 Antibacterial effect of bound polyphenols from mulberry fruits extracted in Example 1

[0066]

[0067] Table 2 Minimum inhibitory concentration (MIC) of bound polyphenols from mulberry fruits against Escherichia coli and Staphylococcus aureus

[0068]

[0069]

[0070] Note: — indicates no colony growth; + indicates colony growth

[0071] As can be seen from Table 1, the bound polyphenols from mulberry fruits extracted in Example 1 of the present invention have significant antibacterial activities against both Escherichia coli and Staphylococcus aureus.

[0072] As can be seen from Table 2, the MIC of the bound polyphenols from mulberry fruits prepared in Example 1 of the present invention against Escherichia coli is 2.5 mg / mL, and the MIC against Staphylococcus aureus is 10 mg / mL.

[0073] Example 2: A bound polyphenol from mulberry fruits and its extraction method

[0074] The remaining steps are basically the same as those in Example 1, except that:

[0075] The extraction steps of the bound polyphenols from mulberry fruits are as follows:

[0076] (1) Add liquid nitrogen to the mulberry fruits for freeze-drying, grind them to powder at low temperature with a grinder, and pass through a 250-mesh sieve to obtain mulberry powder.

[0077] (2) Mix the mulberry powder with 80% (v / v) acetone solution according to the solid-liquid ratio of 1 g:8 mL, shake well for 1 h, centrifuge at 5000 rpm for 12 min, and take the supernatant; repeat the extraction three times, combine the supernatants, concentrate under reduced pressure to recover the organic solvent, discard the supernatant containing free polyphenols, and retain the precipitate containing bound polyphenols.

[0078] (3) Add 2 mol / L NaOH solution to the precipitate according to the dosage ratio of 1 g:11 mL for hydrolysis, stir for 1 h under liquid nitrogen protection, and then slowly add 37% (w / w) hydrochloric acid dropwise in small amounts to neutralize to neutrality to obtain a neutral solution.

[0079] (4) Extraction: Add ethyl acetate to the neutral solution according to the volume ratio of 1:1, extract repeatedly for 5 times, and remove the organic solvent by rotary evaporation under reduced pressure to obtain the crude extract of the bound polyphenols from mulberry fruits.

[0080] (5) The crude extract of mulberry bound polyphenols was purified by D101 macroporous resin, eluted with 95% ethanol, and the eluate was dried in vacuo to obtain mulberry bound polyphenols.

[0081] According to the method steps in Example 1, the inhibitory effects of the mulberry bound polyphenols prepared in Example 2 on Escherichia coli and Staphylococcus aureus were studied, and the results are shown in Table 3.

[0082] Table 3 Antibacterial effects of the mulberry bound polyphenols prepared in Example 2

[0083]

[0084] As can be seen from Table 3, the mulberry bound polyphenols prepared in Example 2 can also inhibit Escherichia coli and Staphylococcus aureus.

[0085] Example 3: A kind of mulberry bound polyphenols and its extraction method.

[0086] The remaining steps are basically the same as those in Example 1, except that:

[0087] The extraction steps of mulberry bound polyphenols are as follows:

[0088] (1) Mulberry fruits were added with liquid nitrogen and freeze-dried, ground to powder with a grinder at low temperature, and passed through a 200-mesh sieve to obtain mulberry powder.

[0089] (2) According to the solid-liquid ratio of 1 g: 12 mL, the mulberry powder was mixed with 80% (v / v) acetone solution and shaken well for 1 h, centrifuged at 4800 rpm for 15 min, and the supernatant was taken; after repeating the extraction three times, the supernatants were combined, concentrated under reduced pressure to recover the organic solvent, the supernatant containing free polyphenols was discarded, and the precipitate containing bound polyphenols was retained.

[0090] (3) According to the dosage ratio of 1 g: 12 mL, 2 mol / L NaOH solution was added to the precipitate for hydrolysis, stirred for 1 h under liquid nitrogen protection, and then neutralized to neutrality by slowly adding 37% (w / w) hydrochloric acid in small amounts and many times to obtain a neutral solution.

[0091] (4) Extraction: Ethyl acetate was added to the neutral solution according to the volume ratio of 1:1, and extracted repeatedly for 5 times, and the organic solvent was removed by rotary evaporation under reduced pressure to obtain the crude extract of mulberry bound polyphenols.

[0092] (5) The crude extract of mulberry bound polyphenols was purified by D101 macroporous resin, eluted with 95% ethanol, and the eluate was dried in vacuo to obtain mulberry bound polyphenols.

[0093] According to the method steps in Example 1, the inhibitory effects of the mulberry bound polyphenols prepared in Example 3 on Escherichia coli and Staphylococcus aureus were studied, and the results are shown in Table 4.

[0094] Antibacterial effect of bound polyphenols from mulberry fruits prepared in Example 3 (Table 4)

[0095]

[0096] As can be seen from Table 4, the bound polyphenols from mulberry fruits prepared in Example 3 can also inhibit Escherichia coli and Staphylococcus aureus.

[0097] Example 4: Effect of purification of bound polyphenols from mulberry fruits using different resins on their antitumor activity

[0098] I. Experimental materials

[0099] Macroporous resins selected were AB-8, HPD826, D101 and HPD600.

[0100] II. Effect of bound polyphenols from mulberry fruits purified using different resins on human lung cancer cell line A549

[0101] 1. Pretreatment of macroporous resins: Macroporous resins were soaked in 95% ethanol overnight for sufficient swelling, then washed with double-distilled water until no white turbidity and no alcohol smell remained, and reserved for use.

[0102] 2. The pretreated macroporous resins (AB-8, HPD826, D101 and HPD600) were packed into columns by wet method, and the crude extract of bound polyphenols from mulberry fruits (obtained after extraction in Example 1) was added respectively, and then separation and purification of MFBPs were carried out to obtain purified bound polyphenols from mulberry fruits respectively.

[0103] The bound polyphenols from mulberry fruits purified using AB-8, HPD826, D101 and HPD600 resins were used to intervene in human lung cancer cell line A549 for 24 h respectively.

[0104] The results of CCK-8 assay showed that the bound polyphenols from mulberry fruits purified using D101 had significant antitumor activity, and the effect was significantly better than that of the bound polyphenols from mulberry fruits purified using the other three resins ( Figure 6 ).

[0105] In summary, the bound polyphenols from mulberry fruits extracted in this invention can significantly inhibit the proliferation of various cancer cells, have no obvious effect on normal mammary epithelial cells and liver cells, and have the potential to be developed into antitumor drugs. The bound polyphenols from mulberry fruits extracted in this invention also show significant antibacterial activity against Escherichia coli and Staphylococcus aureus, and can be applied to the preparation of bacteriostatic agents and antibacterial drugs.

[0106] Although the preferred embodiments of the present invention have been described, additional changes and modifications can be made to these embodiments by those skilled in the art once they learn of the basic inventive concept. Therefore, the appended claims are intended to be construed to include the preferred embodiments as well as all changes and modifications that fall within the scope of the present invention.

[0107] Obviously, those skilled in the art can make various changes and modifications to the present invention without departing from the spirit and scope of the present invention. Thus, if these modifications and variations of the present invention fall within the scope of the claims of the present invention and their equivalent technologies, the present invention is also intended to include these modifications and variations.

Claims

1. Use of mulberry combined polyphenols in the preparation of anti-tumor drugs, characterized in that, The mulberry combined polyphenols can inhibit the proliferation and viability of tumor cells; the tumor cells are human lung cancer cells; The preparation of the mulberry combined polyphenols comprises the following steps: S1, mixing mulberry powder and acetone solution according to the material-liquid ratio of 1 g: 8-12 mL, centrifuging, discarding the supernatant, and retaining the precipitate; S2, hydrolyzing the precipitate in S1 with NaOH solution and neutralizing with hydrochloric acid to obtain a neutral solution; S3, adding ethyl acetate to the neutral solution obtained in S2 for extraction, removing the organic solvent by rotary evaporation under reduced pressure to obtain a crude extract of mulberry combined polyphenols, and purifying with macroporous resin D101 to obtain mulberry combined polyphenols.

2. Use of the mulberry combined polyphenols according to claim 1 in the preparation of anti-tumor drugs, characterized in that, In S1, the dosage ratio of the mulberry powder to the acetone solution is 1 g: 10 mL.

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