CN113713007B - Licorice root food-regeneration functional part for improving chronic liver diseases and preparation method and application thereof - Google Patents
Licorice root food-regeneration functional part for improving chronic liver diseases and preparation method and application thereof Download PDFInfo
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- 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)
- A61K36/185—Magnoliopsida (dicotyledons)
- A61K36/48—Fabaceae or Leguminosae (Pea or Legume family); Caesalpiniaceae; Mimosaceae; Papilionaceae
- A61K36/484—Glycyrrhiza (licorice)
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- A—HUMAN NECESSITIES
- A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
- A23L—FOODS, FOODSTUFFS, OR NON-ALCOHOLIC BEVERAGES, NOT COVERED BY SUBCLASSES A21D OR A23B-A23J; THEIR PREPARATION OR TREATMENT, e.g. COOKING, MODIFICATION OF NUTRITIVE QUALITIES, PHYSICAL TREATMENT; PRESERVATION OF FOODS OR FOODSTUFFS, IN GENERAL
- A23L33/00—Modifying nutritive qualities of foods; Dietetic products; Preparation or treatment thereof
- A23L33/10—Modifying nutritive qualities of foods; Dietetic products; Preparation or treatment thereof using additives
- A23L33/105—Plant extracts, their artificial duplicates or their derivatives
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P1/00—Drugs for disorders of the alimentary tract or the digestive system
- A61P1/16—Drugs for disorders of the alimentary tract or the digestive system for liver or gallbladder disorders, e.g. hepatoprotective agents, cholagogues, litholytics
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- A—HUMAN NECESSITIES
- A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
- A23V—INDEXING SCHEME RELATING TO FOODS, FOODSTUFFS OR NON-ALCOHOLIC BEVERAGES AND LACTIC OR PROPIONIC ACID BACTERIA USED IN FOODSTUFFS OR FOOD PREPARATION
- A23V2002/00—Food compositions, function of food ingredients or processes for food or foodstuffs
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- A—HUMAN NECESSITIES
- A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
- A23V—INDEXING SCHEME RELATING TO FOODS, FOODSTUFFS OR NON-ALCOHOLIC BEVERAGES AND LACTIC OR PROPIONIC ACID BACTERIA USED IN FOODSTUFFS OR FOOD PREPARATION
- A23V2400/00—Lactic or propionic acid bacteria
- A23V2400/11—Lactobacillus
- A23V2400/169—Plantarum
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- 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
- A61K2236/10—Preparation or pretreatment of starting material
- A61K2236/19—Preparation or pretreatment of starting material involving fermentation using yeast, bacteria or both; enzymatic treatment
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- 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
- A61K2236/51—Concentration or drying of the extract, e.g. Lyophilisation, freeze-drying or spray-drying
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- Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
- Y02—TECHNOLOGIES OR APPLICATIONS FOR MITIGATION OR ADAPTATION AGAINST CLIMATE CHANGE
- Y02A—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE
- Y02A50/00—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE in human health protection, e.g. against extreme weather
- Y02A50/30—Against vector-borne diseases, e.g. mosquito-borne, fly-borne, tick-borne or waterborne diseases whose impact is exacerbated by climate change
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Abstract
The invention discloses a liquorice food-regeneration functional part for improving chronic liver diseases, and a preparation method and application thereof, wherein the preparation method of the liquorice food-regeneration functional part comprises the following steps: (1) pulverizing dried Glycyrrhrizae radix decoction pieces to obtain Glycyrrhrizae radix powder; (2) mixing Glycyrrhrizae radix powder with water to obtain fermentation culture medium; (3) sequentially adding activated cellulose decomposing bacteria, lactobacillus plantarum and bacillus subtilis into a fermentation medium, and fermenting for a period of time respectively; (4) and finally, freeze drying to obtain the product. Compared with the prior art, the liquorice food source functional site for improving chronic liver diseases can inhibit exogenous HBx expression and endogenous HBx expression, and has a remarkable improvement effect on chronic liver diseases, especially chronic liver diseases related to HBx. In addition, the product has simple preparation process and low cost, can effectively extract specific components in the liquorice, and is worthy of further popularization.
Description
Technical Field
The invention belongs to the technical field of medicines or foods for improving chronic liver diseases, and particularly relates to a liquorice food-regeneration functional part for improving chronic liver diseases and a preparation method and application thereof.
Background
Chronic liver diseases (chronic hepatitis and liver cirrhosis) belong to the categories of traditional Chinese medicine accumulation, hypochondriac pain, jaundice, tympanites and the like. Is prepared from the liver meridian and liver collateral damaged by epidemic toxin, emotional depression, overstrain, improper diet, and the like, and has the effects of lasting time and gradual accumulation. Is caused by the complex pathogenesis of the combination of poison, phlegm, heat and stasis, and the interweaving of a plurality of pathogenesis runs through the whole process of the disease, but is only emphasized in different stages and specific symptoms. Clinical syndromes of deficiency and excess are complicated, and liver function damage and liver fibrosis are the main pathological changes; the pathogenesis of the liver meridian is that the liver meridian is blocked by the phlegm-heat-phlegm toxin due to deficiency and excess. The majority of patients with chronic liver disease do not have any symptoms, and a small number of patients develop the following symptoms of chronic liver disease: 1. easy fatigue and unsmooth toxin expelling. 2. Yellowish face, white and turbid eyes, and other chronic liver diseases. 3. Debilitation and no semen collection. 4. Fever, weakness, nausea, vomiting, myalgia, dizziness, headache and abdominal pain, as well as jaundice, the symptoms of chronic liver diseases are similar to those of cold, and patients with chronic liver diseases have mild hepatomegaly, aversion to oil, continuous and obvious abdominal distension, and gingival bleeding and epistaxis. The skin has dilated capillaries, and the palms of the hands and feet can be seen with dense erythema like cinnabar, which is marked by the size and color space and is called as the liver palms. Some people often have low fever, menstrual disorder, sexual dysfunction or decline, and some patients with chronic liver disease symptoms also have arthritis, nephritis and diabetes. Chronic liver disease patients especially have obvious causes such as fatigue, drug influence, alcohol action and the like, and the reason should be quickly found out when the chronic liver disease patients have symptoms such as anorexia, nausea and vomiting, diarrhea and loose stool, abdominal distension, hepatosplenomegaly and the like, so as to prevent liver cirrhosis transformation.
HBV is a lysogenic virus of a hepadnavirus family, the genome of HBV is in a relaxed circular double-stranded structure and contains 4 Open Reading Frames (ORFs), wherein a product (HBx) protein coded by an X region is a multifunctional regulatory protein, has a wide transactivation function and has definite effects on intracellular signal transduction, virus replication and transcription, cell cycle process, cell proliferation and apoptosis, protein degradation, genetic stability of hepatocytes and the like. In view of the important role played by the HBx protein in the development of chronic liver disease, HBx has gradually become the focus of research in recent years. The medicine or food aiming at the HBx-related chronic liver disease is worthy of further development.
Disclosure of Invention
The purpose of the invention is as follows: in order to solve the technical problems, the invention provides a liquorice food source functional part for improving chronic liver diseases and a preparation method and application thereof.
The technical scheme is as follows: in order to achieve the purpose, the invention adopts the following technical scheme:
a preparation method of a liquorice food source functional part for improving chronic liver diseases comprises the following steps:
(1) pulverizing dried Glycyrrhrizae radix decoction pieces to obtain Glycyrrhrizae radix powder;
(2) mixing Glycyrrhrizae radix powder with water to obtain fermentation culture medium;
(3) sequentially adding activated cellulose decomposing bacteria, lactobacillus plantarum and bacillus subtilis into a fermentation medium, and fermenting for a period of time respectively;
(4) and finally, freeze drying to obtain the product.
Preferably, the particle size of the powder is 200 mesh or larger.
Preferably, the mass ratio of the liquorice powder to the water is 1: (2-3).
Preferably, in the step (3), the amount of the activated cellulolytic bacteria inoculated is 10 4 -10 5 CFU/g, fermentation temperature of 30-40 ℃ and fermentation time of 24-36 h.
Preferably, in step (3), the activated Lactobacillus plantarum is inoculatedThe amount of the seed is 10 6 -10 7 CFU/g, fermentation temperature of 30-40 deg.C, and fermentation time of 12-24 h.
Preferably, in step (3), the amount of the activated Bacillus subtilis to be inoculated is 10 5 -10 6 CFU/g, fermentation temperature of 25-35 deg.C, and fermentation time of 20-30 h.
Preferably, in the step (4), the freeze-drying time is 24-36 h.
A liquorice food source functional part for improving chronic liver diseases is prepared by the preparation method.
The invention finally provides application of the liquorice food source functional site in preparation of medicines or foods for improving chronic liver diseases.
Preferably, the glycyrrhiza uralensis humanization functional site is applied to preparation of medicines or foods for improving HBx-related chronic liver diseases.
Has the advantages that: compared with the prior art, the liquorice cannibalism functional site for improving chronic liver diseases can inhibit exogenous HBx expression and endogenous HBx expression, and has a remarkable improvement effect on chronic liver diseases, especially on HBx-related chronic liver diseases. Meanwhile, the specific fungi are fermented and extracted in a fixed sequence, so that the effect of the obtained product can be obviously improved. In addition, the product has simple preparation process and low cost, can effectively extract specific components in the liquorice, and is worthy of further popularization.
Detailed Description
The following is a comprehensive description of the solution of the present invention, which is the most preferred embodiment of the present invention, but the present invention is not limited to the following examples.
Example 1
A liquorice food source functional part for improving chronic liver diseases is prepared by the following method:
(1) pulverizing dried Glycyrrhrizae radix decoction pieces to 200 mesh or larger to obtain Glycyrrhrizae radix powder;
(2) mixing licorice powder and water according to a mass ratio of 1: 2, mixing to obtain a fermentation medium;
(3) in the above-mentioned fermentation medium, firstly inoculatingInoculating with activated cellulolytic bacteria in an inoculum size of 10 4 CFU/g, the fermentation temperature is 30 ℃, and the fermentation time is 24 h; then inoculating activated lactobacillus plantarum with the inoculation amount of 10 6 CFU/g, the fermentation temperature is 30 ℃, and the fermentation time is 12 h; finally inoculating activated bacillus subtilis with the inoculation amount of 10 5 CFU/g, the fermentation temperature is 25 ℃, and the fermentation time is 20 h.
(4) And finally, freeze-drying for 24 hours to obtain the product.
Example 2
A liquorice food source functional part for improving chronic liver diseases is prepared by the following method:
(1) pulverizing dried Glycyrrhrizae radix decoction pieces to 200 mesh or larger to obtain Glycyrrhrizae radix powder;
(2) mixing licorice powder and water according to a mass ratio of 1: 3, mixing to obtain a fermentation medium;
(3) inoculating activated cellulolytic bacteria in the fermentation medium with an inoculum size of 10 5 CFU/g, the fermentation temperature is 40 ℃, and the fermentation time is 36 h; then inoculating activated lactobacillus plantarum with the inoculation amount of 10 7 CFU/g, the fermentation temperature is 40 ℃, and the fermentation time is 24 hours; finally inoculating activated bacillus subtilis with the inoculation amount of 10 6 CFU/g, the fermentation temperature is 35 ℃, and the fermentation time is 30 h.
(4) And finally, freeze-drying for 36h to obtain the product.
Example 3
A liquorice food source functional part for improving chronic liver diseases is prepared by the following method:
(1) pulverizing dried Glycyrrhrizae radix decoction pieces to 200 mesh or larger to obtain Glycyrrhrizae radix powder;
(2) mixing licorice powder and water according to a mass ratio of 1: 2.5, mixing to obtain a fermentation medium;
(3) inoculating activated cellulolytic bacteria in the fermentation medium with an inoculum size of 10 5 CFU/g, the fermentation temperature is 35 ℃, and the fermentation time is 30 h; then inoculating activated lactobacillus plantarum with the inoculation amount of 10 6 CFU/g, the fermentation temperature is 35 ℃, and the fermentation time is 18 h; finally inoculating activated Bacillus subtilis, inoculatingAmount of 10 6 CFU/g, fermentation temperature of 32 ℃ and fermentation time of 25 h.
(4) And finally, freeze-drying for 30 hours to obtain the product.
Example 4
A liquorice food source functional part for improving chronic liver diseases is prepared by the following method:
(1) pulverizing dried Glycyrrhrizae radix decoction pieces to 200 mesh or larger to obtain Glycyrrhrizae radix powder;
(2) mixing licorice powder and water according to a mass ratio of 1: 3, mixing to obtain a fermentation medium;
(3) inoculating activated cellulolytic bacteria in the fermentation medium with an inoculum size of 10 5 CFU/g, the fermentation temperature is 35 ℃, and the fermentation time is 28 h; then inoculating activated lactobacillus plantarum with the inoculation amount of 10 6 CFU/g, the fermentation temperature is 35 ℃, and the fermentation time is 15 h; finally inoculating activated bacillus subtilis with the inoculation amount of 10 5 CFU/g, fermentation temperature of 32 ℃ and fermentation time of 22 h.
(4) And finally, freeze-drying for 28 hours to obtain the product.
Example 5
A liquorice food source functional part for improving chronic liver diseases is prepared by the following method:
(1) pulverizing dried Glycyrrhrizae radix decoction pieces to 200 mesh or larger to obtain Glycyrrhrizae radix powder;
(2) mixing licorice powder and water according to a mass ratio of 1: 2, mixing to obtain a fermentation medium;
(3) inoculating activated cellulolytic bacteria in the fermentation medium with an inoculum size of 10 5 CFU/g, the fermentation temperature is 35 ℃, and the fermentation time is 32 h; then inoculating activated lactobacillus plantarum with the inoculation amount of 10 7 CFU/g, the fermentation temperature is 35 ℃, and the fermentation time is 20 h; finally inoculating activated bacillus subtilis with the inoculation amount of 10 6 CFU/g, fermentation temperature of 32 ℃ and fermentation time of 28 h.
(4) And finally, freeze-drying for 32h to obtain the product.
Comparative example 1
The same as example 3, except that the order of fermentation of the strains was different, and the activated lactobacillus plantarum strain was inoculated for fermentation, then the activated bacillus subtilis strain was inoculated for fermentation, and finally the activated cellulolytic bacteria were inoculated for fermentation, and the inoculation amount and the fermentation time were the same as those of example 3.
Comparative example 2
The same as example 3, except that the order of fermentation of the strains was different, the activated Bacillus subtilis was inoculated for fermentation, then the activated cellulolytic bacteria was inoculated for fermentation, and finally the activated Lactobacillus plantarum was inoculated for fermentation, and the inoculum size and fermentation time were the same as those of example 3.
Examples of the experiments
1. Cell culture and transfection
HepG2-NTCP cell line was cultured in DMEM medium containing 10% fetal calf serum, 2.5ug/mL puromycin; culturing PH cells in HM culture medium; the Huh-7 cell line was cultured in DMEM medium containing 10% fetal bovine serum. All cells were in 5% CO 2 Culturing at 37 deg.C in incubator. Plasmids were transfected according to the instructions of Li pofectamin 3000TM (Invi trogen).
2. Mouse model establishment
In order to investigate whether the glycyrrhiza uralensis feeding functional part can inhibit the expression of HBx in a mouse body, preccDNA is firstly injected into a Cre transgenic mouse body to enable the preccDNA to express HBV, the mouse is randomly divided into 3 groups for drug treatment after modeling for one week, and the virus protein HBx expression level in the liver tissue of the mouse is detected by adopting an immunohistochemical technology.
The specific method comprises the following steps:
propagating and culturing Cre transgenic mice (C57BL/6-Tg [ Al b-Cre ]21Mgn/J) and preparing 96 mice with 6-8 weeks of age (the weight is about 20 g/mouse); dissolving the extracted prcccDNA plasmid in PBS to make the final concentration of the prcccDNA plasmid be 2.5 ug/mL; the prcccDNA plasmid was injected into mice at a dose of 4 ug/mouse by tail vein high pressure injection to construct HBV infection model. After modeling for one week, collecting blood through a retroorbital venous plexus, and detecting the HBVDNA level of serum; mice with similar HBV replication levels were selected for subsequent experiments.
3. Cell experiments-inhibition of exogenous HBx expression and endogenous expression
In order to further explore the effect of the glycyrrhiza uralensis food-derived functional site (prepared in example 3) on HBx expression, 3 Xflag-HBx and 2 XHA-HBx were transfected into HepG2 cells, respectively, and then different concentrations of glycyrrhiza uralensis food-derived functional site were used for treatment, and the specific operation method was as follows:
taking out the cell to be detected, adding a proper amount of RIPA (including PI) cracked cell to extract total protein; taking 30ug of total protein for denaturation at 95 ℃, and then separating the protein by adopting SDS-PAGE gel; transferring the protein to a PVDF membrane by a BioRad wet transfer method, and sealing the PVDF membrane for 2 hours at room temperature by using 5% skimmed milk; adding primary antibody according to experimental purposes and incubating overnight at 4 ℃; the next day, after washing the membrane, adding the corresponding secondary antibody and incubating for 2h at room temperature; finally, ECL was developed and the data analyzed. GAPDH was used as an internal reference.
The western blot detection of HBx protein level shows that the glycyrrhiza uralensis gourmet functional site can obviously inhibit the expression of exogenous HBx protein.
To further confirm the inhibitory effect of the glycyrrhiza uralensis feeding function site on HBx, HBV expression plasmid pCH9/3091 containing 1.1-fold HBV genome was transfected in HepG2 cells, and then different concentrations of glycyrrhiza uralensis feeding function site were added to the medium for treatment. Westernblot results show that the glycyrrhiza uralensis feeding functional site can also obviously inhibit the expression of endogenous HBx protein.
4. Animal experiments-inhibition of HBx expression in mice
The modeled mice were randomly divided into four groups: control group (200 uL of physiological saline per injection), group of the licorice cannibalisation functional part obtained in example 3 (dose calculated by 20mg/kg/2days, administered intraperitoneally), group of comparative example 1 (dose calculated by 20mg/kg/2days, administered intraperitoneally), and group of comparative example 2 (dose calculated by 20mg/kg/2days, administered intraperitoneally). The mouse liver was isolated bluntly, and large intact tissue blocks were excised and sectioned for paraffin embedding.
The paraffin embedded sections were placed in a 55C oven overnight; placing in an oven at 95 deg.C for 10min the next day; immediately placing the mixture in dimethylbenzene and ethanol with concentration gradient to carry out dewaxing treatment. And (3) after the high-temperature antigen is repaired, the membrane is placed in 0.1% Triton-100/PBS for 20min, endogenous peroxidase is removed by an endogenous peroxidase blocker, goat working serum is dripped to seal non-specific sites, and then HBx antibody is added for incubation overnight. And on the next day, sequentially dripping a biotin-labeled secondary antibody and a streptavidin-peroxidase reagent after washing the film, finally dripping a freshly prepared DAB color development reagent for color development for 3-5min, carrying out counter-staining on the film for 10min at room temperature by hematoxylin, washing the film with tap water to turn blue, dehydrating and sealing the film, and observing the film under a mirror.
The expression levels of HBx were significantly reduced in the example 3, comparative example 1 and comparative example 2 treated groups compared to the control group, and the expression levels of HBx were also significantly reduced in the example 1 group compared to the comparative example 1 and comparative example 2 groups. The results show that the licorice digestion functional part obtained by the invention can effectively inhibit the expression of HBx in a mouse infected by HBV, and the specific fermentation strain and fermentation sequence are adopted, so that the activity of the licorice digestion functional part can be obviously improved.
Claims (7)
1. A preparation method of a liquorice food source functional part for improving chronic liver diseases is characterized by comprising the following steps:
(1) pulverizing dried Glycyrrhrizae radix decoction pieces to obtain Glycyrrhrizae radix powder;
(2) mixing Glycyrrhrizae radix powder with water to obtain fermentation culture medium;
(3) sequentially adding activated cellulose decomposing bacteria, lactobacillus plantarum and bacillus subtilis into a fermentation medium, and fermenting for a period of time respectively; the inoculum size of the activated cellulolytic bacteria was 10 4 -10 5 CFU/g, fermentation temperature of 30-40 ℃, and fermentation time of 24-36 h; the inoculum size of the activated lactobacillus plantarum was 10 6 -10 7 CFU/g, fermentation temperature of 30-40 ℃, and fermentation time of 12-24 h; the inoculum size of the activated Bacillus subtilis was 10 5 -10 6 CFU/g, fermentation temperature of 25-35 deg.C, fermentation time of 20-30 h;
(4) and finally, freeze drying to obtain the product.
2. The method of preparing a glycyrrhiza uralensis-derived functional site for improving chronic liver disease according to claim 1, wherein in the step (1), the particle size of the powder is 200 mesh or more.
3. The method of preparing a glycyrrhiza uralensis ized functional site for improving chronic liver diseases according to claim 1, wherein in the step (2), the mass ratio of glycyrrhiza uralensis powder to water is 1: (2-3).
4. The method of preparing a glycyrrhiza uralensis-derived functional site for improving chronic liver diseases according to claim 1, wherein the freeze-drying time in step (4) is 24 to 36 hours.
5. A glycyrrhiza uralensis humanized functional site for improving chronic liver diseases, which is prepared by the preparation method according to any one of claims 1 to 4.
6. The use of the glycyrrhiza uralensis humanized functional site according to claim 5 in the preparation of a medicament for improving chronic liver disease.
7. The use of the glycyrrhiza uralensis humanization functional site according to claim 5 in the preparation of a medicament for improving HBx-related chronic liver disease.
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