Black sesame extract freeze-dried powder as well as preparation method and application thereof

Abstract

The invention relates to the technical field of medicines, in particular to black sesame extract freeze-dried powder as well as a preparation method and application thereof. The preparation method comprises the following steps: frying and crushing black sesame to obtain a black sesame pigment extraction raw material, extracting the black sesame pigment in the black sesame pigment extraction raw material by adopting an alkali-solution and acid-isolation method, uniformly mixing the black sesame pigment with distilled water, further adding an alpha-amylase / calcium chloride solution for incubation, centrifuging, taking supernatant, adjusting the pH value to 1.0-3.0, and filtering to obtain the black sesame pigment. Adding an artificial gastric juice containing 0.01-0.02 g / mL of pepsin, incubating, centrifuging, taking the supernatant, adjusting the pH value, adding an artificial small intestine solution containing 0.01-0.02 g / mL of pancreatin, adjusting the pH value, incubating, centrifuging, taking the supernatant, adjusting the pH value, and freeze-drying to obtain black sesame extract freeze-dried powder; the black sesame extract freeze-dried powder prepared by the invention has an inhibition effect on tumors in a zebra fish PDX model, and can be applied to antitumor drugs or foods.

Description

Technical Field

[0001] The present invention relates to the field of medical technology, and in particular to a freeze-dried black sesame extract powder and a preparation method and application thereof. Background Art

[0003] Promoting the traditional Chinese medicine culture of both medicine and food, discovering valuable bioactive substances from traditional plants that are both medicine and food and using them to prevent and treat liver diseases such as HCC has become a hot topic in the fields of food, medicine and health.

[0004] Black sesame (BS) tastes sweet and is neutral in nature. It enters the liver, kidney, and large intestine meridians. It nourishes the liver and kidney, benefits the essence and blood, and moisturizes the intestines. As a traditional plant with both medicinal and edible properties, BS contains a variety of bioactive substances such as polysaccharides, pigments, and lignans, and has biological activities such as antioxidant, anti-proliferative, and anti-tumor. Black sesame pigment (BSP) is an important active ingredient in black sesame. It is a high-molecular polymer heterogeneous structure and belongs to the catechol-type biomacromolecule. It has monomer structures such as phenol, catechol, guaiacol, vanillin, and coniferyl alcohol. Modern research has confirmed that black sesame pigment has a variety of biological activities such as antioxidant, anti-tumor, and hypoglycemic, which may be related to signal pathways such as oxidative stress. BSP can significantly improve non-alcoholic fatty liver disease in animal models and significantly inhibit oxidative stress, lipid accumulation, and the secretion of inflammatory factors during the development of non-alcoholic fatty liver organoids.

[0005] Traditional Chinese medicine theory believes that black foods have functions such as nourishing the liver and kidneys. Currently, research on black sesame pigment is mostly focused on its chemical structure and in vitro biological activity, and there are no reports on the anti-tumor activity of black sesame pigment in vivo. Summary of the invention

[0006] In view of the above-mentioned deficiencies and gaps in the prior art, the present invention aims to provide a black sesame extract freeze-dried powder and a preparation method and application thereof, so as to provide an application of the black sesame extract in anti-tumor.

[0007] In order to solve the above problems, the present invention adopts the following technical solutions:

[0008] In a first aspect, the present invention provides a method for preparing a freeze-dried powder of black sesame extract, comprising:

[0009] The black sesame seeds are fried and crushed to obtain a raw material for extracting black sesame pigment;

[0010] Extracting the black sesame pigment from the black sesame pigment extraction raw material by alkaline dissolution and acid precipitation method;

[0011] The black sesame pigment is dissolved in a mixed solution of distilled water and α-amylase / calcium chloride solution and incubated, the supernatant is centrifuged to adjust the pH to 1.0-3.0, artificial gastric juice containing 0.01-0.02 g / mL of pepsin is added and then incubated, the supernatant is centrifuged to adjust the pH to 6.0-7.0, artificial small intestine solution containing 0.01-0.02 g / mL of pancreatic enzyme is added, and then the pH is adjusted to 6.0-7.0, incubated, the supernatant is centrifuged to adjust the pH to 7.0-8.0, and freeze-dried to obtain freeze-dried black sesame extract powder.

[0012] Furthermore, the method of frying and crushing black sesame to obtain raw materials for extracting black sesame pigment includes: frying black sesame at a temperature of 200-220° C. for 20-30 min, heating in an oven at 100-120° C. for 2-3 h, and crushing to obtain raw materials for extracting black sesame pigment.

[0013] Further, the black sesame pigment extracted from the black sesame pigment extraction raw material by alkali dissolution and acid precipitation method comprises:

[0014] Weigh the black sesame pigment extraction raw material into a conical flask, add 5-10 g / L NaOH solution as the extraction solution, shake and stir at 100-150 rpm in a shaker at 10-40° C. for 20-40 min, filter and collect the filtrate, centrifuge and collect the supernatant, remove the upper layer of oil after standing, collect the lower layer into a new conical flask, add 2-3 mol / L hydrochloric acid solution to adjust the pH to 1.0-3.0, stand for 1-3 h, centrifuge and collect the black sesame pigment precipitate, place the black sesame pigment precipitate in an oven and dry it to constant weight to obtain black sesame pigment.

[0015] Furthermore, the volume ratio of the mass of the black sesame pigment extraction raw material to the NaOH solution is 1:(10-30).

[0016] Furthermore, the α-amylase / calcium chloride solution is obtained by dissolving 32.5 mg of α-amylase in 25 mL of 1 mmol / L calcium chloride.

[0017] Furthermore, the volume ratio of the distilled water to the α-amylase / calcium chloride solution is (15-30):0.5.

[0018] Furthermore, the pH is adjusted to 1-3 using HCl with a concentration of 5-8 mol / L; the pH is adjusted to 6-7 using NaHCO3 solution with a concentration of 0.5-1.0 mol / L.

[0019] In a second aspect, the present invention provides freeze-dried black sesame extract powder prepared by the method for preparing freeze-dried black sesame extract powder.

[0020] In a third aspect, the present invention provides the use of the black sesame extract freeze-dried powder in the preparation of anti-tumor drugs.

[0021] Furthermore, the tumor is a liver cancer cell.

[0022] Furthermore, the concentration of the black sesame extract freeze-dried powder is not less than 19.5 μg / mL.

[0023] The beneficial effects of the present invention are as follows: the present invention extracts black sesame pigment by an alkali dissolution and acid precipitation method, and finally prepares a black sesame extract freeze-dried powder by digestion and freeze-drying. The black sesame extract freeze-dried powder of the present invention has an anti-tumor effect and can be used to prepare health products, special medical foods or drugs for preventing and treating HCC or for patients with liver damage. BRIEF DESCRIPTION OF THE DRAWINGS

[0024] Figure 1 Graph showing the hydroxyl radical and DPPH radical contents of the black sesame pigment extract digested in the small intestine in an embodiment of the present invention.

[0025] Figure 2 This is a comparison of the fluorescence intensity of zebrafish tumor cells (HC2023052901) after different sample treatments in the examples of the present invention.

[0026] Figure 3 This is a histogram of the fluorescence intensity of zebrafish tumor cells (HC2023052901) after different sample treatments in the examples of the present invention.

[0027] Figure 4 This is a comparison of the fluorescence intensity of zebrafish tumor cells (HC2023060801) after different sample treatments in the examples of the present invention.

[0028] Figure 5 This is a histogram of the fluorescence intensity of zebrafish tumor cells (HC2023060801) after different sample treatments in the examples of the present invention.

[0029] Figure 6 This is a comparison chart of the fluorescence intensity of zebrafish tumor cells (HC2023061901) after different sample treatments in the examples of the present invention.

[0030] Figure 7 This is a histogram of the fluorescence intensity of zebrafish tumor cells (HC2023061901) after different sample treatments in the examples of the present invention. DETAILED DESCRIPTION

[0031] The present invention is further described in detail below in conjunction with specific embodiments.

[0032] It should be noted that these embodiments are only used to illustrate the present invention rather than to limit the present invention. Simple improvements to the method based on the concept of the present invention all fall within the scope of protection claimed by the present invention.

[0033] Example 1

[0034] Preparation of black sesame pigment

[0035] Weigh appropriate black sesame seeds and put them into a heating frying machine for frying at 220°C for 30 minutes until there is a popping sound, the black sesame seeds swell slightly, or there are cracks on the surface, and the aroma escapes. The fried black sesame seeds are placed in a 120°C oven for 2 hours, crushed for 30 seconds, interrupted for 1 minute, and repeated 3 times until the black sesame seeds are powdered as the raw material for extracting black sesame pigment.

[0036] The black sesame pigment was extracted by alkali dissolution and acid precipitation. Weigh 200g of crushed black sesame into a conical flask, add 3000mL of freshly prepared 10g / L NaOH solution as the extraction solution, the solid-liquid ratio is 1:15 (W / V), place the above black sesame solution in a shaker at 30℃, shake and stir at 140rpm for 40min to fully dissolve the black sesame pigment. After the black sesame solution is shaken, filter once with four layers of gauze, collect the filtrate, centrifuge at 6000rpm for 25min to remove the precipitate, and collect the black supernatant; place the supernatant in a separatory funnel, let it stand for 30min, and then the oil layer is separated, the upper oil layer is removed, and the lower layer is collected into a new conical flask. The solution is stirred at 150rpm and 1.5mol / L hydrochloric acid solution is added to adjust the pH to 2.0, and the stirring is continued for 20min to fully precipitate the pigment. After standing for 2h, centrifuge at 6000rpm for 25min to collect the black sesame pigment precipitate. The black sesame pigment precipitate is placed in an oven and dried at 60°C to a constant weight to obtain the black sesame pigment.

[0037] Example 2

[0038] In vitro digestion of black sesame pigment

[0039] Take 2 g of the black sesame pigment prepared in Example 1 and dissolve it in 20 mL of distilled water and 500 μL of α-amylase / calcium chloride solution (32.5 mg of α-amylase is dissolved in 25 mL of 1 mmol / L calcium chloride, pH 7), incubate in a constant temperature oscillator at 37 ° C for 10 min, about 120 rpm, and take the supernatant; use 6 mol / L HCl to adjust the pH of the supernatant to 2, then add 10 mL of artificial gastric juice containing 0.1 g of pepsin, mix well, and incubate in a constant temperature oscillating incubator at 37 ° C. 1h, about 120rpm, take the supernatant; use 0.9mol / L NaHCO3 solution to adjust the pH of the supernatant to 6, add 10mL of artificial small intestine solution containing 0.1g pancreatic enzyme, mix well, and then use 0.9mol / L NaHCO3 solution to adjust the pH to 7.4, incubate in a constant temperature oscillator at 37℃ for 2h, about 120rpm, take the supernatant, adjust the pH to 7.0, use it as a small intestine digestion sample and lyophilize to obtain black sesame extract lyophilized powder, which is stored at -80℃ for use.

[0040] The preparation method of the freeze-dried powder of black sesame extract of the present invention effectively prevents the change of the physical, chemical and biological characteristics of the product, and can effectively protect the stability of the effective ingredients of the product. Secondly, the freeze-dried product is loose in shape and the color does not change substantially after drying, and can be quickly dissolved after adding water. In addition, since the drying is carried out under vacuum conditions, it has a good protective effect on some easily oxidizable phenolic substances. Finally, the moisture content of the product after freeze-drying is very low, which improves the stability of the product and reduces the chance of contamination, which not only facilitates transportation but also extends the shelf life of the product.

[0041] After oral and gastric digestion in vitro, the scavenging rates of hydroxyl radicals and DPPH radicals of black sesame pigment extract digested in the small intestine were significantly increased. Figure 1 .

[0042] Example 3

[0043] Anti-hepatocarcinogenic activity of freeze-dried black sesame extract powder

[0044] Test materials: freeze-dried powder of black sesame extract prepared in Example 2, positive control: sorafenib, white powder, batch number H2124463, Shanghai Aladdin Biochemical Technology Co., Ltd., solvent: DMSO.

[0045] Experimental subjects: Zebrafish with human liver cancer xenografts

[0046] Test method:

[0047] Maximum detectable concentration (MTC) determination

[0048] 3dpf wild-type AB strain zebrafish were randomly selected in a 6-well plate, and 30 zebrafish were treated in each well (experimental group). The samples were dissolved in water (concentrations shown in Table 1) and a normal control group was set up at the same time, with a volume of 3mL per well. After 2 days of treatment at 35℃, the MTC of the samples on normal zebrafish was measured.

[0049] Anti-tumor efficacy evaluation

[0050] After washing the hepatocellular carcinoma tumor tissue with PBS, cut it into small pieces and put them into 15mL centrifuge tubes for digestion. Add 5mL of mixed enzyme solution, shake it manually in a 37℃ water bath for 5min after adding 1mL each time, and then take out the supernatant and put it into 10mL of DMEM medium containing 10% FBS to terminate digestion. After passing through a 70μm sieve, centrifuge it at 2000rpm for 5min, remove the supernatant, and precipitate the tumor cells.

[0051] Preparation of mixed enzyme solution:

[0052] Collagenase IV: 1 g

[0053] Hyaluronidase: 100mg

[0054] DNase I: 20,000U

[0055] The solution was added to HBSS (Hanks balanced salt solution) buffer, fully dissolved and mixed, filtered through a 0.22 μm filter membrane, and aliquoted and stored at -20°C.

[0056] The cells were resuspended in PBS, and the tumor cells were labeled with 4.00 μL / mL CM-DiI. Then, the cells were transplanted into the yolk sac of wild-type AB zebrafish at 2 dpf by microinjection. About 200 cells were transplanted per tail to establish a zebrafish tumor transplantation model. The model zebrafish were cultured at 35°C until 3 dpf. At 3 dpf, zebrafish with good consistency of transplanted tumor cells were selected under a microscope and randomly assigned to 6-well plates, with 30 tails per well. The sample and positive control sorafenib (concentrations are shown in Tables 2, 3, and 4) were given in water, and a model control group was set up at the same time, with a capacity of 3 mL per well. After 2 days of treatment at 35°C, 10 zebrafish were randomly selected from each experimental group and photographed under a fluorescence microscope. The data were collected using NIS-Elements D 3.20 advanced image processing software, and the fluorescence intensity of tumor cells was analyzed. The statistical analysis results of this indicator were used to evaluate the anti-tumor efficacy of the sample. The statistical processing results are expressed as mean ± SE. SPSS26.0 software was used for statistical analysis, and p<0.05 indicated that the difference was statistically significant.

[0057] Test results

[0058] Maximum Detectable Concentration (MTC)

[0059] Under the experimental conditions, the anti-tumor efficacy MTC of black sesame pigment extract freeze-dried powder was 78.1 μg / mL. See Table 1.

[0060] Table 1 Results of MTC exploratory experiments on the anti-tumor efficacy of samples (n=30)

[0061]

[0062] Anti-tumor efficacy evaluation

[0063] Under the experimental conditions, the freeze-dried powder of black sesame pigment extract has anti-tumor effect. See Tables 2, 3, 4 and Figure 2-7 .

[0064] Table 2 Sample anti-tumor (HC2023052901) efficacy evaluation test results (n=10)

[0065]

[0066] Compared with the model control group, *p<0.05, **p<0.01

[0067] Table 3 Sample anti-tumor (HC2023060801) efficacy evaluation test results (n=10)

[0068]

[0069] Compared with the model control group, *p<0.05, **p<0.01, ***p<0.001

[0070] Table 4 Sample anti-tumor (HC2023061901) efficacy evaluation test results (n=10)

[0071]

[0072] Compared with the model control group, *p<0.05, **p<0.01, ***p<0.001

[0073] Therefore, the freeze-dried powder of black sesame extract after in vitro gastrointestinal simulated digestion and freeze drying showed significant anti-liver cancer activity. The black sesame pigment prepared by this method can be used to prepare health products, special medical foods or drugs for preventing and treating HCC or for patients with liver damage.

[0074] Finally, it should be noted that the above embodiments are only used to illustrate the technical solutions of the present invention rather than to limit it. Although the present invention has been described with reference to the preferred embodiments of the present invention, it should be understood by those skilled in the art that various changes may be made in form and details without departing from the spirit and scope of the present invention as defined in the appended claims.

Claims

1. A method for preparing freeze-dried black sesame extract powder, characterized in that: include: The black sesame seeds are fried and crushed to obtain a raw material for extracting black sesame pigment; Extracting the black sesame pigment from the black sesame pigment extraction raw material by alkaline dissolution and acid precipitation method; The black sesame pigment is dissolved in a mixed solution of distilled water and α-amylase / calcium chloride solution and incubated, the supernatant is centrifuged to adjust the pH to 1.0-3.0, artificial gastric juice containing 0.01-0.02 g / mL of pepsin is added and then incubated, the supernatant is centrifuged to adjust the pH to 6.0-7.0, artificial small intestine solution containing 0.01-0.02 g / mL of pancreatic enzyme is added, and then the pH is adjusted to 6.0-7.0, incubated, the supernatant is centrifuged to adjust the pH to 7.0-8.0, and freeze-dried to obtain freeze-dried black sesame extract powder.

2. The method for preparing freeze-dried black sesame extract powder according to claim 1, characterized in that: The method of frying and crushing black sesame to obtain raw materials for extracting black sesame pigment comprises: frying black sesame at a temperature of 200-220° C. for 20-30 minutes, heating in an oven at 100-120° C. for 2-3 hours, and crushing to obtain raw materials for extracting black sesame pigment.

3. The method for preparing freeze-dried black sesame extract powder according to claim 1, characterized in that: The black sesame pigment extracted from the black sesame pigment extraction raw material by alkali dissolution and acid precipitation method comprises: Weigh the black sesame pigment extraction raw material into a conical flask, add 5-10 g / L NaOH solution as the extraction solution, shake and stir at 100-150 rpm in a shaker at 10-40° C. for 20-40 min, filter and collect the filtrate, centrifuge and collect the supernatant, remove the upper layer of oil after standing, collect the lower layer into a new conical flask, add 2-3 mol / L hydrochloric acid solution to adjust the pH to 1.0-3.0, stand for 1-3 h, centrifuge and collect the black sesame pigment precipitate, place the black sesame pigment precipitate in an oven and dry it to constant weight to obtain black sesame pigment.

4. The method for preparing freeze-dried black sesame extract powder according to claim 1, characterized in that: The volume ratio of the black sesame pigment extraction raw material to the NaOH solution is 1:10-30.

5. The method for preparing freeze-dried black sesame extract powder according to claim 1, characterized in that: The α-amylase / calcium chloride solution is obtained by dissolving 32.5 mg of α-amylase in 25 mL of 1 mmol / L calcium chloride.

6. The method for preparing freeze-dried black sesame extract powder according to claim 1, characterized in that: The volume ratio of the distilled water to the α-amylase / calcium chloride solution is 15-30:0.

5.

7. The method for preparing freeze-dried black sesame extract powder according to claim 1, characterized in that: The pH value is adjusted to 1-3 using HCl with a concentration of 5-8 mol / L; the pH value is adjusted to 6-7 using NaHCO3 solution with a concentration of 0.5-1.0 mol / L.

8. The freeze-dried black sesame extract powder prepared by the method for preparing freeze-dried black sesame extract powder according to any one of claims 1 to 7.

9. Use of the freeze-dried black sesame extract powder according to claim 8 in the preparation of anti-tumor drugs, health products or foods.

10. The use according to claim 9, characterized in that: The concentration of the black sesame extract freeze-dried powder is not less than 19.5 μg / mL.

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