CN118178574A - Preparation method and application of anti-tumor active component of distillers' grains alcohol extract - Google Patents
Preparation method and application of anti-tumor active component of distillers' grains alcohol extract Download PDFInfo
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K36/00—Medicinal preparations of undetermined constitution containing material from algae, lichens, fungi or plants, or derivatives thereof, e.g. traditional herbal medicines
- A61K36/18—Magnoliophyta (angiosperms)
- A61K36/88—Liliopsida (monocotyledons)
- A61K36/899—Poaceae or Gramineae (Grass family), e.g. bamboo, corn or sugar cane
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P35/00—Antineoplastic agents
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- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K2236/00—Isolation or extraction methods of medicinal preparations of undetermined constitution containing material from algae, lichens, fungi or plants, or derivatives thereof, e.g. traditional herbal medicine
- A61K2236/30—Extraction of the material
- A61K2236/33—Extraction of the material involving extraction with hydrophilic solvents, e.g. lower alcohols, esters or ketones
- A61K2236/333—Extraction of the material involving extraction with hydrophilic solvents, e.g. lower alcohols, esters or ketones using mixed solvents, e.g. 70% EtOH
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K2236/00—Isolation or extraction methods of medicinal preparations of undetermined constitution containing material from algae, lichens, fungi or plants, or derivatives thereof, e.g. traditional herbal medicine
- A61K2236/50—Methods involving additional extraction steps
- A61K2236/53—Liquid-solid separation, e.g. centrifugation, sedimentation or crystallization
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K2236/00—Isolation or extraction methods of medicinal preparations of undetermined constitution containing material from algae, lichens, fungi or plants, or derivatives thereof, e.g. traditional herbal medicine
- A61K2236/50—Methods involving additional extraction steps
- A61K2236/55—Liquid-liquid separation; Phase separation
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Abstract
The invention discloses a preparation method and application of an anti-tumor active component of a vinasse alcohol extract, and belongs to the technical field of biological medicines. Aiming at the problems of low utilization rate and low application added value of the distillers ' grains in the medicine field at present, 60% ethanol is added into the distillers ' grains and assisted by ultrasonic leaching, and the supernatant is collected and subjected to rotary evaporation to obtain the distillers ' grains ethanol extract. Adsorbing DGAE% of resin material on AB-8 column, eluting with ultrapure water, 30%, 60% and 90% ethanol, and rotary evaporating to obtain AB-8 penetrating component; the components are further separated by SP207 macroporous resin material, ultrapure water and 90% ethanol are sequentially used for eluting, the ultrapure water eluting components are subjected to rotary evaporation and vacuum freeze drying, and the active component of the distilled grain alcohol extract is obtained and named DGAE-1. The research results prove that: DGAE-1 can significantly inhibit proliferation and clonogenic capacity of colorectal cancer cells and promote apoptosis. The active component of the distiller's grains ethanol extract can be used for preparing antitumor drug preparation.
Description
Technical Field
The invention belongs to the technical field of biological medicines, and particularly relates to a preparation method and application of an anti-tumor active component of a vinasse alcohol extract.
Background
Distillers' grains (stillage) is the solid waste remaining after a series of steps such as crushing, steaming, fermenting and distilling the starchy material. As the largest byproduct in the wine industry, the utilization value of the wine is improved, and the exploration of a new application way of the wine is a problem faced by the wine industry. The distillers 'grains are byproducts in the brewing process, but the distillers' grains contain common proteins, starch, fibers, fat, aldehydes, acids, esters, alcohols, aromatic compounds, various metabolites and the like, and have high development value and application prospect.
At present, research on reasonable utilization of distillers 'grains in China has been advanced, and the distillers' grains resources are widely utilized, so that the research on multiple aspects of biology, chemical industry, agriculture and the like are covered. Most of them are used as feeds, which play an important role in animal husbandry, but the added value of the application is low. In recent years, the distillers 'grains extract contains various functional components, and the utilization value of the distillers' grains extract can be greatly improved by deep excavation. The research finds that: the active substances in the distillers 'grains extract have the effects of resisting oxidation, resisting inflammation, reducing blood fat and the like, which indicates that the distillers' grains extract has certain application potential in the aspect of medical efficacy. At present, the research on the distillers grains is mainly focused on the aspects of nutritional ingredients and functional values, and no report on antitumor activity components of the distillers grains is yet seen.
Disclosure of Invention
Aiming at the problems of low utilization rate and low application added value of the distillers 'grains in the field of medicines at present, the invention provides the application of the distillers' grain alcohol extract active component in preparing an anti-tumor pharmaceutical preparation and a preparation method of the active component.
The invention takes the distilled grain as a material, and obtains the active component of the distilled grain alcohol extract by optimizing the preparation process, and the active component DGAE-1 can obviously inhibit the proliferation and the cell clone formation capacity of colorectal cancer cells, obviously promote the apoptosis of the cells and has no toxic or side effect on normal cells. Therefore, the invention provides new development and utilization value for the vinasse, and can be applied to development of antitumor pharmaceutical preparations.
In order to achieve the above purpose, the present invention adopts the following technical scheme:
A preparation method of an anti-tumor active component of a vinasse alcohol extract comprises the following steps:
Step 1, crushing vinasse, adding an ethanol solution for ultrasonic auxiliary leaching, and centrifuging to obtain a supernatant; filtering, adding ethanol solution into the filter residue, performing ultrasonic-assisted extraction, centrifuging, taking the supernatant again, and preserving the supernatant obtained by two centrifugations at 4 ℃ for later use;
Step 2, combining the two supernatants obtained in the step 1, performing rotary evaporation until the ethanol solution volatilizes, and filtering the obtained solution by a water-based filter membrane to remove impurities to obtain a distilled grain ethanol extract solution (alcohol extracts from distillers' grains, DGAE);
step 3, separating the distilled grain alcohol extract by using weak-polarity AB-8 macroporous resin, and eluting the extract by sequentially using ultrapure water and ethanol solution with gradient concentration; taking an elution component of ultrapure water, performing rotary evaporation to obtain an AB-8 penetrating component, and eluting with ethanol solution with gradient concentration to obtain an elution component;
and 4, separating the AB-8 penetrating component by using a medium-polarity SP207 macroporous resin, eluting by sequentially using ultrapure water and 90% ethanol, taking the eluting component of the ultrapure water for rotary evaporation, and performing vacuum freeze drying to obtain the active component (DGAE-1) of the distillers' grains ethanol extract, and preserving at the temperature of minus 20 ℃.
Further, the ratio of the distilled grain to the ethanol solution in the step1 is 1 g:20 mL-30 mL.
Further, the conditions of the ultrasonic-assisted leaching in the step 1 are as follows: leaching for 20-40 min under the condition of 300W.
Further, the speed of centrifugation in the step 1 is 11000 rpm, the temperature is 4 ℃, and the time is 10-15 min.
Further, the concentration of the ethanol solution in the step 1 is 60%.
Further, the pore diameter of the water-based filter membrane in the step 2 is 0.22 mu m.
Further, the mass of the distilled grain alcohol extract liquid in the step 3 is less than 5% of the mass of the resin.
Further, the gradient concentration of ethanol in step3 is 30%, 60% and 90% ethanol.
Further, the temperature of the rotary evaporation in step 3 and step 4 was 65 ℃.
The distillers' grains ethanol extract active component with antitumor activity prepared by the preparation method is provided.
The application of the distillers' grains ethanol extract antitumor active component prepared by the preparation method in preparing antitumor drugs.
Compared with the prior art, the invention has the following advantages:
The active component of the distillers' grains alcohol extract has obvious anti-tumor activity, and the anti-tumor activity detection shows that the active component can obviously inhibit tumor cells such as: proliferation and clonogenic capacity of colorectal cancer cells HT-29, SW620 promote apoptosis. Proved that the active component of the distillers' grains alcohol extract has potential to be developed into a novel anti-tumor pharmaceutical preparation.
Drawings
FIG. 1 is a schematic diagram of test results of the effect of distillers' grains alcohol extract active components on tumor cell proliferation;
FIG. 2 is a schematic diagram of the test results of the effect of distillers' grains alcohol extract active components on tumor cell cloning;
FIG. 3 is a schematic diagram of the results of the test of the effect of distillers' grains alcohol extract active components on tumor cell apoptosis;
FIG. 4 is a schematic diagram of the results of a test of the effect of distillers' alcoholic extract active on tumor growth in mice.
Detailed Description
For an appreciation of the invention, we will describe it in full detail. However, the present invention has a variety of implementations and is not limited to the specific examples listed herein. These examples are presented in order to enhance a thorough understanding of the present disclosure.
Example 1: preparation method of active component (DGAE-1) of distilled grain alcohol extract
Step 1, taking 100g of crushed vinasse powder, adding 2500 mL of 60% ethanol, 300W, carrying out ultrasonic-assisted extraction for 30min, centrifuging at 11000 rpm for 10min, taking supernatant, adding 2500 mL of 60% ethanol into filter residues again, carrying out the same operation as above, and combining the supernatants.
And 2, rotationally evaporating the supernatant obtained in the step 1 at 65 ℃ until ethanol volatilizes, thus obtaining the distilled grain ethanol extract DGAE.
And 3, carrying out suction filtration on DGAE obtained in the step 2 through a water system filter membrane with the aperture of 0.22 mu m to remove impurities.
Step 4, adding DGAE% of the mass of the resin material into the AB-8 resin material with weak polarity, eluting with ultrapure water (1 BV/h,3 h), and collecting penetrating components; elution was then performed with a gradient of ethanol (1 BV/h,3 h) and the eluted fractions were collected. The eluted fractions and the penetrated fractions were collected by rotary evaporation at 65℃and the antitumor activities of the fractions were compared at the cellular level by the MTT method.
And 5, separating the AB-8 penetrating component obtained in the step 4 by using a medium-polarity SP207 macroporous resin, eluting by sequentially using ultrapure water and 90% ethanol (1 BV/h,3 h), and taking the ultrapure water eluting component for rotary evaporation at 65 ℃. After vacuum freeze drying, obtaining distilled grain alcohol extract active component (DGAE-1) 3.273 mg with concentration of 0.425 mg/mL, and preserving at-20deg.C.
Example 2: identification of antitumor Activity of active component (DGAE-1) of distiller's grains alcohol extract
HT-29 and SW620 cells, which had a cell density of 90% and were in logarithmic growth phase, were selected, and after digestion with 1X trypsin, the number of cells was adjusted to 5X 103 cells per well and plated in 96-well cell culture plates, each group was plated with 5 duplicate wells, while a control group was plated. After cell attachment, the experimental group was treated 48 h with DGAE-1 at different concentrations. Old culture solution is discarded, PBS is used for washing twice, the culture medium is replaced, 10 mu L of MTT (thiazole blue) is added into each hole, and the culture medium is placed in a cell culture box for incubation of 4 h. The medium in the well plate was discarded and 150 μl dimethyl sulfoxide (DMSO) was added. Placed on a microplate shaker to oscillate 10min and absorbance was measured for each well at 570: 570 nm using a microplate reader.
The active component DGAE-1 of the distillers' grains ethanol extract obtained by the invention is used for detecting the in-vitro anti-tumor activity of the human colorectal cancer cell strain. The results show that: CRC cells 48 h were treated with DGAE-1 at the different concentrations described above, with HT-29 cells having an IC50 value of 2.673 μg/mL and SW620 cells having an IC50 value of 2.968 μg/mL, with little effect on the growth of human normal colorectal mucosal cells (FHC) (as shown in FIG. 1).
Example 3: effect of distillers' grains alcohol extract active ingredient (DGAE-1) on colorectal cancer cell cloning
Experimental group: the log phase cells were selected, the number of cells was adjusted to 5×10 3 cells per well and plated in 24 well cell culture plates, 4 duplicate wells were placed in each group, and a blank group was placed. After the cells are attached, DGAE-1 with different concentrations is added into the experimental group for continuous treatment for 5-7 days. After the treatment is finished, the sample is washed 3 times by PBS, 600 mu L of fixing liquid is added into each hole, and the sample is fixed by 20 min. And then the mixture is washed for 3 times by PBS, and 500 mu L of 0.1% crystal violet dye solution is added into each hole for dyeing 15min. And then washed 3 times with PBS, dried and placed under a microscope for photographing. 600 μl of 1% sds solution was added to each well, placed on a microplate shaker to oscillate 10 min, and the absorbance of each well was measured by an microplate reader at 570: 570 nm.
The results of the cell cloning experiments show that: as the concentration of DGAE-1 increases, the number of cell clones formed by HT-29 and SW620 cells decreases significantly and the activity of the cells shows a dose gradient decrease. The results show that: the active component DGAE-1 of the distillers' grains ethanol extract can obviously inhibit the proliferation activity of colorectal cancer cells (shown in figure 2).
Example 4: effect of distillers' grains alcohol extract active ingredient (DGAE-1) on colorectal cancer apoptosis
The log phase cells were selected, the number of cells was adjusted to 3X 10 5, and the cells were inoculated into a cell culture dish, and after the cells were attached, the experimental group was treated with DGAE-1 at different concentrations 48 and h. After the cell treatment is finished, the culture solution is discarded and washed for 2-3 times by PBS. Pre-chilled PBS was added, the cells were scraped with cells, transferred to a centrifuge tube, repeated once, and collected by centrifugation. The supernatant was discarded, PBS was added to resuspend the cells, and the supernatant was centrifuged again. Adding a proper amount of cell lysate (WBIP:PMSF=100:1) according to the cell number, and performing ice lysis for 30: 30 min; the supernatant was centrifuged at 13000 rpm at 4℃for 15: 15 min, and the total protein concentration of the cells was measured by BCA method (biquinolinecarboxylic acid method). Proteins were separated by SDS-PAGE gel electrophoresis, and the separated protein bands were transferred to PVDF membrane. Immunoblots were performed using the corresponding primary antibodies as follows: caspase-3, cleaved-Caspase-3, caspase-8, cleaved-Caspase-8, caspase-9, cleaved-Caspase-9, were co-incubated with horseradish peroxidase conjugated species-specific secondary antibodies 2 h. Finally, the protein bands were visualized by chemiluminescent imager.
Western blot experiment results show that: DGAE-1 treatment of the cells can significantly increase the expression levels of Cleaved-caspase-3, cleaved-caspase-8 and Cleaved-caspase-9 in the cells. The following is indicated: the active component DGAE-1 of the distillers' grains ethanol extract can activate Caspase-dependent apoptosis pathway in cells and finally induce apoptosis (shown in figure 3).
Example 5: active component (DGAE-1) of distiller's grains ethanol extract can inhibit tumor growth in vivo
1. Mouse name: BALB/C nude mice
2. Grade: SPF stage
3. Gender: male male
4. Age: 4 weeks of age
5. Feeding conditions: the temperature is controlled at 23-25 ℃, the humidity is controlled at 55-65%, and the indoor lamplight simulates day and night and 12 h light and shade circulation.
HT-29 cells in logarithmic growth phase were taken and digested to give a cell suspension. Using the xenograft tumor model, 3X10 6 HT-29 cells suspended in PBS buffer were subcutaneously injected into each nude mouse, and the subcutaneous tumor formation of the nude mice was periodically observed. When nude mice tumors grew to about 100mm 3, the nude mice were randomly divided into two groups: DGAE-1 treatment group and physiological saline control group. The nude mice were given by gavage at a dose of 25 mg/kg DGAE-1 based on their body weight, and the control group was given an equivalent dose of physiological saline by gavage every day for two weeks. During the experiment, the length and width of the subcutaneous tumor of the nude mice were measured every three days with a vernier caliper, and the weight of the nude mice was weighed by an electronic scale. Tumor volume was calculated according to the formula (tumor volume = 0.5 x tumor length x tumor width 2). After the end of the administration, the nude mice were euthanized, the subcutaneous tumor tissue and other tissue organs were dissected, weighed and stored in a-80 ℃ freezer for later use.
The results show that: compared with the normal saline control group, the tumor volume of the nude mice in the DGAE-1 treatment group is obviously reduced, and the weight is obviously reduced. The following is indicated: the active component DGAE-1 of the distilled grain alcohol extract has obvious inhibition effect on the tumor growth of nude mice (shown in figure 4).
What is not described in detail in the present specification belongs to the prior art known to those skilled in the art. While the foregoing describes illustrative embodiments of the present invention to facilitate an understanding of the present invention by those skilled in the art, it should be understood that the present invention is not limited to the scope of the embodiments, but is to be construed as protected by the accompanying claims insofar as various changes are within the spirit and scope of the present invention as defined and defined by the appended claims.
Claims (10)
1. The preparation method of the anti-tumor active component of the distillers' grains ethanol extract is characterized by comprising the following steps:
Step 1, crushing vinasse, adding an ethanol solution for ultrasonic auxiliary leaching, and centrifuging to obtain a supernatant; filtering, adding ethanol solution into the filter residue, performing ultrasonic-assisted extraction, centrifuging, taking the supernatant again, and preserving the supernatant obtained by two centrifugations at 4 ℃ for later use;
Step 2, combining the two supernatants obtained in the step 1, performing rotary evaporation until the ethanol solution volatilizes, and filtering the obtained solution by a water-based filter membrane to remove impurities and obtain a vinasse ethanol extract;
step 3, separating the distilled grain alcohol extract by using weak-polarity AB-8 macroporous resin, and eluting the extract by sequentially using ultrapure water and ethanol solution with gradient concentration; taking an elution component of ultrapure water, performing rotary evaporation to obtain an AB-8 penetrating component, and eluting with ethanol solution with gradient concentration to obtain an elution component;
And 4, separating the AB-8 penetrating component by using a medium-polarity SP207 macroporous resin, eluting by sequentially using ultrapure water and 90% ethanol, taking the eluting component of the ultrapure water for rotary evaporation, and performing vacuum freeze drying to obtain the active component of the distillers' grains ethanol extract, and preserving at the temperature of minus 20 ℃.
2. The method for preparing the anti-tumor active component of the distillers' grains ethanol extract according to claim 1, which is characterized in that: the ratio of the distilled grain to the ethanol solution in the step 1 is 1 g:20 mL-30 mL.
3. The method for preparing the anti-tumor active component of the distillers' grains ethanol extract according to claim 1, which is characterized in that: the conditions of the ultrasonic assisted leaching in the step 1 are as follows: leaching for 20-40 min under the condition of 300W.
4. The method for preparing the anti-tumor active component of the distillers' grains ethanol extract according to claim 1, which is characterized in that: the centrifugation speed in the step1 is 11000 rpm, the temperature is 4 ℃, and the time is 10-15 min.
5. The method for preparing the anti-tumor active component of the distillers' grains ethanol extract according to claim 1, which is characterized in that: the concentration of the ethanol solution in the step 1 is 60%; the temperature of the rotary evaporation in the step 3 and the step 4 was 65 ℃.
6. The method for preparing the anti-tumor active component of the distillers' grains ethanol extract according to claim 1, which is characterized in that: and (2) the aperture of the water-based filter membrane in the step (2) is 0.22 mu m.
7. The method for preparing the anti-tumor active component of the distillers' grains ethanol extract according to claim 1, which is characterized in that: the mass of the distilled grain alcohol extract liquid in the step 3 is less than 5% of the mass of the resin.
8. The method for preparing the anti-tumor active component of the distillers' grains ethanol extract according to claim 1, which is characterized in that: the ethanol with gradient concentration in the step 3 is ethanol with concentration of 30%, 60% and 90%.
9. A distillers' grains ethanol extract active ingredient with anti-tumor activity prepared by the preparation method of any one of claims 1-8.
10. Use of the distillers' grains ethanol extract anti-tumor active component prepared by the preparation method of any of claims 1-8 in preparation of anti-tumor drugs.
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