CN114766622B - Preparation process of solid beverage of strawberry tea and cassia seed and application of solid beverage in liver protection - Google Patents
Preparation process of solid beverage of strawberry tea and cassia seed and application of solid beverage in liver protection Download PDFInfo
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- CN114766622B CN114766622B CN202210441246.7A CN202210441246A CN114766622B CN 114766622 B CN114766622 B CN 114766622B CN 202210441246 A CN202210441246 A CN 202210441246A CN 114766622 B CN114766622 B CN 114766622B
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Classifications
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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
- A23L2/00—Non-alcoholic beverages; Dry compositions or concentrates therefor; Their preparation
- A23L2/385—Concentrates of non-alcoholic beverages
- A23L2/39—Dry compositions
-
- 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
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
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- A61K36/185—Magnoliopsida (dicotyledons)
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- A61K36/18—Magnoliophyta (angiosperms)
- A61K36/185—Magnoliopsida (dicotyledons)
- A61K36/87—Vitaceae or Ampelidaceae (Vine or Grape family), e.g. wine grapes, muscadine or peppervine
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K9/00—Medicinal preparations characterised by special physical form
- A61K9/0087—Galenical forms not covered by A61K9/02 - A61K9/7023
- A61K9/0095—Drinks; Beverages; Syrups; Compositions for reconstitution thereof, e.g. powders or tablets to be dispersed in a glass of water; Veterinary drenches
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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
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- A61K2236/50—Methods involving additional extraction steps
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Abstract
The present invention belongs to the field of functional food and beverage. In particular to a production process of a compound extract or solid beverage of cassia seed with strawberry tea and application thereof in preparing a preparation for improving liver function, reducing liver inflammatory reaction and oxidation reaction or preventing or treating alcoholic liver injury. Experimental researches show that the solid beverage of the invention can relieve alcohol-induced liver injury by improving liver function, reducing liver inflammatory reaction and oxidative stress level, and has great practical value.
Description
Technical Field
The present invention belongs to the field of functional food and beverage. In particular to a production process of a compound solid beverage of strawberry tea and cassia seed and application thereof in preparing preparations for protecting liver, such as improving liver function, reducing liver inflammatory reaction and oxidation reaction, or preventing or treating alcoholic liver injury.
Background
Alcoholic liver injury (ALD) is a liver disease caused by long-term high-volume drinking, usually manifested as alcoholic fatty liver, alcoholic hepatitis, and gradually developing into liver fibrosis, cirrhosis, even liver cancer, etc. [1]. Various surveys indicate that ALD patients are growing substantially worldwide and have become an important liver disease [2] of worldwide concern. The existing methods for treating alcoholic injury include medication, nutrition support, operation treatment and the like [3], and the treatment methods can have certain treatment effects on alcoholic liver injury, but have larger side effects, and the operation treatment is mostly used for serious liver diseases, has large risk and poor prognosis, and can easily cause other viscera or intestinal injuries after long-term use of chemical medicines, so that the search of safe and efficient natural dietary supplements and plant medicines for preventing and treating alcoholic liver injury becomes a research hotspot [4] in recent years.
The berry tea is also called vine tea, has the academic name of ampelopsis grossedentata (Ampelopsis grossedentata) [5], is rich in various flavonoid compounds with functional activity, has the highest dihydromyricetin content, has shown that the berry tea has remarkable anti-inflammatory, antioxidant, antibacterial and liver-protecting effects [6-9], can effectively shorten the sobering-up time [10] of mice, has a better protection effect on acute alcoholic liver injury of mice, and can inhibit hepatic stellate cells through the effects of inducing autophagy and mediating natural killer cells to improve hepatic fibrosis [11]. Other antioxidant active ingredients such as the tea polysaccharide have remarkable inhibiting effect on the generation of liver tissue MDA in mice.
Semen cassiae (CASSIA SEMEN) is a traditional medicine and food dual-purpose resource in China, contains anthraquinone, polysaccharide, fatty acid and other substances, has the effects of moistening intestines and improving eyesight, reducing blood lipid and protecting liver, resisting inflammation, resisting oxidization and the like [12,13], and researches show that the semen cassiae ethanol extract can effectively improve the non-alcoholic fatty liver disease [14] of rats, and the components such as semen cassiae anthraquinone can play a role in protecting liver [15] by adjusting Nrf 2-mediated ERK/MAPK/JNK and other signal paths.
Alcoholism is the third major health problem in the world, the type of liver injury which is irreversible in alcoholic liver disease [26], alcohol entering the body can inhibit mitochondrial beta-oxidation, induce TG and the like to deposit in liver parenchyma to form fatty liver, and Kupffer cells are stimulated to produce tumor necrosis factors and interleukins to promote apoptosis of liver cells and participate in inflammatory infiltration [27] of neutrophils. The liver is an organ which is simultaneously supplied with blood through artery and vein, and the increase of arterial blood inflammatory factors also reflects the existence of inflammation [28] of the liver. AST, ALT, LDH is a marker reflecting the importance of liver function, and the activity of AST, ALT, LDH in blood and serum is increased when entering the blood from the liver, which indicates rupture of liver hepatocytes and mitochondrial damage [29]. MDA is taken as a final product of lipid oxidation, the expression level is increased to represent tissue lipid peroxidation, SOD and GSH can effectively remove free radicals, the expression level is reduced to represent excessive liver free radicals, and the liver injury degree [30,31] is indirectly reflected.
Transcriptomics explores the transcription and regulation rules of all genes in cells, and performs differential gene screening to define the action targets of the formula. The experiment adopts an RNA-sep high-throughput sequencing technology to carry out transcriptome sequencing on rat livers in a normal group, a model group and a solid beverage group, determines the gene content change process of the liver of a drinking rat influenced by the solid beverage of the cassia seed of the blackberry tea, and after carrying out GO function annotation and KEGG annotation analysis and enrichment analysis on genes, presumes that differential genes SPERMIDINE SYNTHASE, sirt7, RT1-A2, RT1-CE1, LOC103692716, acap2, anti-inflammatory, antioxidant, liver metabolic pathways and other pathways are related to alcoholic liver injury, and the specific action mechanism needs to be studied deeply and mined.
Semen cassiae is a traditional medicine and food homologous variety in China, and medicinal materials and extracts of semen cassiae are widely approved to be applied to the fields of health-care foods and traditional Chinese medicines. The blueberry tea has long history of folk application and high development and utilization value of flavonoid compounds such as dihydromyricetin, myricetin and the like, but the pharmacological toxicology and safety of the blueberry tea need to be further studied deeply, and the application is limited to tender stems and leaves to prepare health care tea at present.
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Disclosure of Invention
The inventor applies the solid beverage mainly comprising the strawberry tea extract and the cassia seed extract particles to the liver protection effect research, and initially explores the action mechanism of the solid beverage, so as to provide a richer theoretical basis for the deep development and utilization of corresponding resources in the fields of plant solid beverages and functional foods. The particle ratio of the solid beverage of the blackberry tea cassia seed is screened by researching and utilizing normal hepatocytes BRL3A of rats, an animal test is carried out on SD rats, liver indexes, pathological indexes, serum and liver biochemical indexes are analyzed, and transcriptome analysis shows that the solid beverage of the blackberry tea cassia seed has obvious protection effect on alcoholic liver injury of the rats. Thus, the present invention has been completed.
Therefore, the invention provides a production process for compound extraction of strawberry tea and cassia seeds, which is characterized by comprising the following steps:
1) Weighing: respectively weighing the strawberry tea and the cassia seed;
2) Crushing and sieving: respectively pulverizing and sieving the strawberry tea and the semen cassiae;
3) Extracting pure water under reflux: extracting the strawberry tea and the cassia seed with pure water at 75-80 ℃ respectively;
4) Vacuum concentration: concentrating under the condition that the vacuum pressure is-0.02 Mp to-0.095 Mp and the temperature is 60 ℃ to 80 ℃;
5) Spray drying: spray drying to obtain powder of extract of herba Myrtilli tea and semen Cassiae, preferably with inlet air temperature of 165-190 deg.C and outlet air temperature of 75-85 deg.C;
6) Sieving: respectively collecting powder of strawberry tea and semen Cassiae extract, and sieving with shaking sieve at 100%;
7) The strawberry tea and the cassia seed extract are prepared according to the following ratio of 1-2: mixing at a ratio of 1 for 20-40min.
Preferably, in step 2), a 10 mesh screen is passed.
In a specific embodiment, the reflux extraction in the step 3) is carried out for 1 hour/time and 2 times, and the extraction liquid is continuously refluxed by a centrifugal pump, so that the materials in the extraction tank are kept in a high-temperature state.
Preferably, in step 4) the concentration is carried out to a solids content of more than 40%.
In a specific embodiment, step 5) uses an LPG50 type drying tower, the air inlet temperature is set to be 165-190 ℃, the air outlet temperature is set to be 75-85 ℃, the motor frequency of the atomizer is 300-400 Hz, and the discharging frequency is 25-35 Hz for spray drying.
In a preferred embodiment, 100% of step 6) is passed through an 80 mesh shaker screen.
More preferably, in step 7), the strawberry tea and the cassia seed extract are mixed according to a ratio of 1.5:1 for 30min.
Further preferably, the specific operation of step 7) is: mixing the strawberry tea and the semen cassiae extract according to a set weight ratio, and fully mixing for 20-40 minutes in a three-dimensional motion mixer, wherein the main shaft rotating speed is 10-25r/mjn; sieving and granulating the materials in a granulator; drying the materials at 60-70deg.C until the water content is less than or equal to 5%, to obtain solid beverage.
The invention also provides a production process of the compound extract of the strawberry tea and the cassia seed, and the compound extract is preferably prepared into a solid beverage. Further provides application of the compound extract and the solid beverage in preparation of preparations for improving liver functions, reducing liver inflammatory reaction and oxidation reaction, or preventing or treating alcoholic liver injury. In particular, the formulation is a medicament.
The research on the protection effect of the solid beverage alcohol liver injury of the strawberry tea extract and the cassia seed extract particles shows that the solid beverage of the strawberry tea and the cassia seed can relieve the alcohol-induced liver injury by improving liver functions, reducing liver inflammatory reaction and oxidative stress level, and has application value in preventing or treating alcohol liver injury.
Drawings
Fig. 1 is a flow chart of a preparation process of the solid beverage of the strawberry tea and the cassia seed.
FIG. 2 cytotoxicity assay. Wherein, A, the influence of the extract of the strawberry tea on the cell survival rate; b, influence of semen cassiae extract on cell survival rate; c, effect of solid beverage on cell viability; and D, influence of alcohol on cell viability.
FIG. 3 influence of establishment of alcohol model and solid beverage on cell viability. Wherein, A, the expression level of Nrf2 after BRL3A cells are treated by alcohol with different concentrations for 48 hours; b, HO-1 expression level after BRL3A cells are treated by alcohol with different concentrations for 48 hours; and C, influence of solid beverage on survival rate of alcohol damage cells.
FIG. 4H & E staining of rat liver tissue (40X). Wherein, CON, blank group; ETOH, model set; l, solid beverage low dose group; m, dose group in solid beverage; h, high dose group of solid beverage.
FIG. 5 serum biochemical marker levels of rats. Wherein, A, glutamic pyruvic transaminase ALT; b, glutamic-oxaloacetic transaminase AST; c, lactate dehydrogenase LDH.
FIG. 6 serum inflammatory factor levels in rats. Wherein, A, interleukin 6IL-6; b, tumor necrosis factor TNF-alpha.
FIG. 7 level of liver biochemical indicators in rats. Wherein A, reducing glutathione GSH; b, malondialdehyde MDA; c, superoxide dismutase SOD; d, triglyceride TG.
FIG. 8 shows the differential expression of genes between two groups, A being the comparison of rats in the ETOH group with rats in the CON group; group B was a comparison of group H with ETOH rats.
FIG. 9 GO classification statistical diagrams of the difference genes between the ETOH group and the CON group, and between the H group and the ETOH group. Wherein A is the difference gene of the ETOH group and the CON group; b is the difference gene between the H group and the ETOH group.
FIG. 10 is a statistical diagram of the classification of the difference genes KEGG between the ETOH group and the CON group, and between the H group and the ETOH group. Wherein A is the up-regulated differential gene of the ETOH group and the CON group; b is the up-regulated differential gene of the ETOH group and the CON group; c is the up-regulated differential gene of the H group and the ETOH group; d is the down-regulated differential gene between group H and group ETOH.
Detailed Description
Example 1
The solid beverage of the strawberry tea and the cassia seed (shown in figure 1) is prepared by the following method:
100kg of qualified raw materials of the strawberry tea are crushed into 10 meshes, the crushed raw materials are sieved (10 meshes of screen), 100kg of raw materials are weighed, 1 ton of pure water is heated to 75 ℃ to 80 ℃ for 1 hour/time x2 times (continuous reflux is carried out on the materials in an extraction tank by a centrifugal pump to keep the materials in a high-temperature state), vacuum concentration (-0.02 Mp-0.095Mp, < 60 ℃ to 80 ℃ and concentration to 40% of solid matters) is adopted, and the conditions of-0.05 Mp and 70 ℃ are adopted in the embodiment. Then spray drying (LPG 50 type drying tower, air inlet temperature 165 deg.C, air outlet temperature 75-85 deg.C, atomizing motor frequency 300-400 Hz, feeding frequency 25 Hz), drying until water content is less than 5%, and sieving 100% with 80 mesh oscillating screen to obtain strawberry tea extract.
The semen Cassiae extract is extracted according to the same procedure for standby.
Mixing the strawberry tea and the semen cassiae extract according to a set weight ratio (1-2:1 in the embodiment), fully mixing for 30 minutes in a three-dimensional motion mixer, and transferring to the next procedure by using an edible PE plastic bag, wherein the main shaft rotating speed is 15 r/mjn; granulating the materials in a granulator by using a 20-mesh sieve, and transferring the materials to the next working procedure by using a stainless steel disc; drying the materials at 60-70deg.C until the water content is less than or equal to 5%, and transferring to the next process or packaging with PE plastic bag to obtain solid beverage of strawberry tea and semen Cassiae.
Example two
1 Materials and methods
1.1 Major reagents and instruments
Reagent: MEM, medium Qiao Xinzhou; FBS (fetal bovine serum), biotechnology of the racing industry; diabody (penicillin, streptomycin), EDTA, beijing cool pacing technologies limited; RNA lysate, reverse transcription kit, fluorescent quantitative PCR kit (Vazyme); glutamic pyruvic transaminase (ALT), glutamic oxaloacetic transaminase (AST), lactate Dehydrogenase (LDH), tumor necrosis factor (TNF-alpha), interleukin 6 (IL-6), triglyceride (TG), malondialdehyde (MDA), reduced Glutathione (GSH), superoxide dismutase (SOD), lipopolysaccharide (LPS) ELISA detection kit, changsha Authority Biotechnology Co., ltd; d-hanks water; aging at 43 degrees in the ox fence mountain.
Instrument: CO 2 incubator, constant temperature water bath (Shanghai Yiheng), ultra clean bench (Thermo), ultra low temperature refrigerator (Thermo company of America), refrigerated centrifuge (Hettich MIKRO-22R), inverted microscope (ZEISS), multifunctional enzyme-labeled instrument (Thermo company of America), etc.
Test material: rat normal hepatocytes BRL3A, marsupenario life technologies limited; the solid beverage of the strawberry tea extract, the cassia seed extract, the strawberry tea and the cassia seed is prepared according to the method of the first embodiment.
1.2 Cell assay
1.2.1 Cytotoxicity test
Respectively dissolving the strawberry tea extract and the semen Cassiae extract in distilled water, performing ultrasonic treatment in water bath at 37deg.C until the extract is completely dissolved, filtering with 0.45 μm bacterial filter membrane, preparing into 20mg/mL mother liquor of strawberry tea extract and mother liquor of semen Cassiae extract, and preserving at 4deg.C. The absolute ethyl alcohol is preserved by a bacterial filtration membrane with the diameter of 0.22 mu m. According to experimental study, the mother liquor is respectively diluted in half for cytotoxicity test, and the safe concentration range of the strawberry tea extract and the cassia seed extract is determined. The concentration of alcohol was diluted from 20% to 0.078% in half and tested for cytotoxicity, and the concentration range suitable for alcohol damage was determined.
Cell viability (%) = ×100%.
1.2.2 Cell RNA extraction and QPCR assay
And performing related tests according to the steps of an RNA extraction kit, a reverse transcription kit and a fluorescence quantitative PCR kit, detecting the expression levels of Nrf2 and HO-1 of alcohol damaged cells with different concentrations, and determining the alcohol concentration and time suitable for damage. And detecting the influence of the solid beverage of the strawberry tea and the cassia seed on the survival rate of BRL3A cells according to the determined concentration.
TABLE 1 primer sequences
1.3 Animal test
1.3.1 Test animals
Male SD rats, SPF grade, weighing 180-220 g, offered by Hunan Stokes Levoda laboratory animal Co.
1.3.2 Test methods
The raising environment is kept for 12 hours with alternating illumination and darkness, the room temperature is controlled at 25+/-2 ℃, and the relative humidity is 40-60%. The 40 SD rats are randomly divided into a blank group, a model group and a solid beverage group according to the health food function evaluation guiding principle (2020 edition) (solicited opinion manuscript), wherein the solid beverage group comprises a normal human body recommended amount group, a 5 times human body recommended amount group and a 7.5 times human body recommended amount group, 8 SD rats are fed with water freely, and are fed for 1 week in an adaptive manner, the weight weighing is carried out 2 times per week, the blank group is filled with 7.5mL/kg of distilled water, the model group is filled with 5mL/kg of distilled water, the solid beverage group is filled with 5mL/kg of solid beverage aqueous solution, after 1h of gastric filling, the model group and the solid beverage group are filled with 2.5mL/kg of 43 DEG ox fence mountain aging for 1-29d, and the 30 th day is filled with 4mL/kg of 43 DEG ox fence mountain aging. The grouping situation is as in table 2.
TABLE 2 grouping of rats and method of treatment
Note that: a is 50 mg/kg.bw; b, 250 mg/kg.bw; c 375 mg/kg.bw; 16.7 mg/kg.bw; 83.5 mg/kg.bw; 125.25 mg/kg.bw.
1.3.3 Sample collection and processing
After the last gastric lavage of rats, each group of rats is weighed after fasting (no water feeding) for 16 hours, the rats are dissected after anesthesia, heart arterial blood is collected, livers are taken out, the rats are weighed after being rinsed by pre-cooled normal saline, one leaf of livers is taken out and fixed by 4% paraformaldehyde fixing solution, liver tissue sections are manufactured, histopathological observation is carried out in the later stage, and the rest livers are packaged in liquid nitrogen and quickly frozen for storage in a refrigerator at-80 ℃.
1.3.4 Detection indicators and methods
(1) Liver pathology examination
Cutting liver-leaf tissue, soaking in 4% paraformaldehyde for fixation, making paraffin section, hematoxylin-eosin (HE) staining, and observing pathological changes such as morphological changes, inflammatory reaction, fatty vacuoles, etc. of liver cells under an optical microscope.
(2) Serum biochemical index
Taking heart arterial blood, standing for 2h, centrifuging for 15min at 3000r/min, subpackaging serum, detecting ALT, AST, LDH activity and IL-6 and TNF-alpha concentration according to ELISA kit specification method, and preserving at-80deg.C.
(3) Biochemical index of liver
During detection, pre-cooled physiological saline is used for washing, 10% liver tissue homogenate is prepared, supernatant is obtained after centrifugation for 10min at 4 ℃ and 3000r/min, and SOD activity and TG, MDA, GSH content are detected according to an ELISA kit instruction method.
(4) RNA extraction and transcriptome sequencing analysis
Three of each group of rat livers were randomly taken for transcriptome sequencing and differential gene expression analysis. RNA extraction and transcriptome sequencing were performed by shanghai megi biomedical technologies limited. Filtering and screening the raw data obtained by sequencing to obtain high-quality data (cleandata), comparing the CLEAN READS of each sample with a specified reference genome in sequence, calculating the gene expression quantity by adopting a TPM method, screening differential expression genes by DEseq software, wherein the screening threshold is |log2 (Fold change) |is more than or equal to 1 and the P value is less than 0.05. Screening up-regulated and down-regulated differential genes, performing GO function annotation analysis on gene enrichment conditions, and screening differential gene enrichment pathways [16] by KEGG enrichment analysis.
1.4 Data processing
All experiments were set up with at least three sets of biological replicates, data expressed as mean ± standard deviation, data analysis was performed using GRAPHPAD PRISM. The pairwise comparisons were analyzed using Dunnett-t test, and the data sets were compared using One-way ANOVA. * Compared to the normal group, #, compared to the model group, #, P < 0.05; * /#, P < 0.01; * /# #, P < 0.001; * /# # #, P < 0.0001.P <0.05 is considered to be significant in difference, and has statistical significance.
2 Results and analysis
2.1 Cell assay
2.1.1 Cytotoxicity test results
To explore the safe concentration range of the strawberry tea extract and the cassia seed extract for affecting the BRL3A activity of the cells, cytotoxicity test is carried out by determining the alcohol concentration suitable for damage, and the result shows that the safe concentration range of the strawberry tea extract for affecting the BRL3A of the cells is 0-156.25 mug/mL (A in figure 2), and the safe concentration range of the cassia seed extract for affecting the BRL3A of the cells is 0-39.063 mug/mL (B in figure 2). The maximum safe concentration of the strawberry tea extract is about 3-4 times of that of the cassia seed extract, so the proportion of the solid beverage is determined as the strawberry tea extract: semen Cassiae extract=3:1, cytotoxicity test was performed in a safe concentration range, and the safe concentration range of solid beverage-affecting cell BRL3A was determined to be 0-30:90 (μg/mL) (C in fig. 2). The alcohol concentration at which the cell viability is around 50% is suitable for injury, and thus the alcohol concentration of suitable injured cells ranges from 2.5% to 5% (D in fig. 2).
2.1.2 Establishment of alcohol model and influence of formulation on cell viability thereof
The nucleic factor-E2-related factor 2 (Nrf 2) is an important cell protection transcription factor, moderately enhances the activity of the Nrf2, can effectively inhibit the oxidative stress of cells, improves the cell survival rate [17], causes oxidative damage of the cells due to the overhigh activity of the Nrf2, promotes the proliferation of tumor cells and enhances the drug resistance [18]. heme oxygenase 1 (HO-1) is an antioxidant enzyme, and under normal physiological state, HO-1 expression quantity is low, when organism is inflamed, infected, etc., HO-1 expression quantity is increased, and injury [19] is produced to organism. To investigate the effect of the expression levels of Nrf2 and HO-1 and the formulation on BRL3A cell viability after 48h of alcohol treatment of BRL3A cells at appropriate lesion concentration (2.5% -5%), QPCR experiments were performed with alcohol concentrations of 4.5% and RNA extraction at 5% treated cells at too low concentrations, and no subsequent QPCR experiments were performed. The results showed that the expression levels of inflammatory factors Nrf2 and HO-1 were significantly increased (a in fig. 3 and B in fig. 3) when the alcohol concentration was 4%, so that an alcohol damage model could be effectively established when the alcohol concentration was 4%. In the safe concentration range, the solid beverage can effectively alleviate the problem of reduced cell viability caused by alcohol (C in FIG. 3).
2.2 Animal test
2.2.1 Rat liver coefficient case
Liver coefficient refers to the ratio of fresh weight of liver to weight of rat, and the ratio of liver to weight is constant under normal conditions, and liver coefficient is increased, which indicates liver edema, inflammation or hyperplasia; a decrease in liver coefficient indicates organ atrophy or other degenerative changes [20]. The liver coefficients of the study blank, model and solid beverage groups were not significantly different (P > 0.05), indicating that the alcohol concentration of the experimental lavage had no significant effect on the external characterization of the liver (table 3).
Table 3 liver coefficients of rats of each group
2.2.2 Liver tissue pathological forms
Liver is taken as a metabolic organ of an organism and plays an important role in alcoholic liver diseases, and ethanol and metabolic products thereof cause hepatotoxicity, cause inflammatory reaction, generate free radicals, cause cell damage, hepatocyte necrosis and the like [21]. H & E staining showed that the hepatocytes, chordae, were well aligned in the CON group rats, the liver lobule, liver sinus structures were clear, there were no oedema, steatosis, and there was little hepatocyte death (fig. 4); the liver tissue section of the rat in the ETOH group has typical pathological characteristics, the liver cells are uneven in size, balloon-like, and have more vesicular fat vacuoles, and simultaneously have inflammatory infiltration and cell necrosis. Compared with the ETOH group, the liver cell edema of the L group rat is obviously improved, the vacuolation is reduced, the inflammatory factor infiltration is reduced, and the liver cell arrangement disorder is improved. Compared with the ETOH group, the forms of the liver cells of the M group and the H group tend to be normal, the infiltration of inflammatory cells is obviously improved, the vacuolation of cells is obviously reduced, the cells are occasionally necrotized, and the arrangement of the liver cells is not greatly different from that of CON (figure 4). According to liver pathological sections, the solid beverage of the strawberry tea and the cassia seed can effectively improve the histopathological lesions of alcoholic liver injury of rats.
2.2.3 Levels of serum Biochemical indicators in rats
Clinically, AST, ALT, LDH is a sensitive indicator [20] of liver injury, and elevated activity of AST, ALT and LDH is proportional to liver injury. Compared with the CON group, the serum ALT, AST, LDH level of the ETOH group is extremely obviously increased (figure 5, P is less than 0.0001), which indicates that the alcoholic liver injury model is successfully established, and indicates that the hepatic cell necrosis and the cell membrane permeability are increased [22]. Each dosage group of the solid beverage can inhibit AST, ALT, LDH from increasing caused by drinking of rats, has obvious difference, and has dose dependency of the effect.
2.2.4 Levels of inflammatory factors IL-6 and TNF-alpha in rat serum
Inflammatory factor secretion is an important way for causing liver injury, IL-6 is a pro-inflammatory cytokine and participates in body inflammatory reaction, TNF-alpha plays a role in mediating liver injury, and can directly cause cytotoxicity, cause hepatocyte necrosis and influence microcirculation [23] in liver. Serum inflammatory factor IL-6, TNF- α levels were significantly elevated in the ETOH group compared to the CON group (fig. 6), and levels of inflammatory factor (P < 0.0001) were significantly reduced for each dose group of solid beverage relative to the ETOH group and were proportional to the dose concentration. Therefore, the solid beverage of the strawberry tea and the cassia seed can obviously reduce inflammatory response caused by drinking of rats. 2.2.5 levels of liver Biochemical indicators in rats
Long-term drinking can cause the accumulation of fatty acid and triglyceride in liver to cause fatty liver [24], active oxygen free radical is generated in alcohol metabolic process, and excessive oxidant can cause the peroxidation of free radical lipid, so that the activities of antioxidant enzyme GSH and SOD of organism are reduced, and the level of antioxidant defense system is reduced [26]. Compared with CON group, the liver of rats in ETOH group has extremely reduced GSH and SOD content (FIG. 7-A and FIG. 7-C) (P < 0.0001). MDA is a second product of lipid peroxidation and is an important index for reflecting tissue injury, and the liver TG and MDA contents of rats in the ETOH group are extremely obviously increased compared with CON (FIG. 7-B and FIG. 7-D) (P < 0.0001). Through solid beverage intervention, the content and SOD activity of each dosage group GSH, MDA, TG are obviously different from those of the ETOH group (figure 7) (P is smaller than 0.05), the MDA content of a high dosage group in the solid beverage is not obviously different from that of a CON group (P is larger than 0.05), the content of SOD and TG of a high dosage group in the solid beverage is not obviously different from that of the CON group (P is larger than 0.05), and the result shows that the activity of antioxidant enzyme of the liver of the rat can be effectively improved by the solid beverage at low, medium and high dosages, the scavenging of oxygen free radicals is accelerated, and the fatty liver formation is relieved.
2.2.6 Transcriptome sequencing data and comparative analysis
The study shows that the low, medium and high dose groups of solid beverage can obviously reduce the liver injury enzyme activity and the expression level of inflammatory factors in serum, inhibit lipid peroxidation and fatty liver generation, are dose dependent, have the most obvious effect in the high dose group, and in order to further study the influence of the solid beverage of the strawberry tea and the cassia seed on the liver function of a drinking rat from the molecular level, the experiment carries out transcriptome analysis on the liver of a CON group, an ETOH group and an H group rat, compares the sequencing result of the transcriptome with a reference genome to obtain 399553582 original reads, and obtains 395459552cleanreads after quality control. In cleanreads obtained, the unique comparison rate of all samples reached 82% or more, the Q20% values were all greater than 96%, and the Q30% values were all greater than 91%, indicating that the sequencing quantity and quality reached the requirements of subsequent analysis (Table 4).
TABLE 4 sample sequencing quality and sequence alignment
2.2.7 Differential Gene analysis
There was a significant difference in expression between 229 genes in CON group rats and ETOH group rats, 174 genes in ETOH group were significantly up-regulated, and 55 genes were significantly down-regulated (a in fig. 8). There were 106 genes significantly different in expression between ETOH group rats and H group rats, 37 genes significantly up-regulated and 69 genes significantly down-regulated in H group (B in fig. 8).
2.2.8 Influence of solid beverages on the biological Process of alcohol-damaged liver genes
(1) GO annotation
Annotation analysis is carried out on 299 differential genes of the ETOH group and the CON group and 106 differential genes of the H group and the ETOH group, and biological processes of 20 top ranks are analyzed and summarized, and molecular function grouping mainly relates to molecular function regulator, transcriptional regulation activity, catalytic activity and combination; biological process groupings relate primarily to immune system processes, cellular component tissue or biogenesis, localization, multicellular biological processes, developmental processes, responses to stimuli, metabolic processes, biological regulation, and cellular processes; the cell component groupings mainly relate to extracellular region portions, protein-containing complexes, membranes, membrane portions, organelle portions, organelles, and cell portions (fig. 9).
(2) KEGG annotation
KEGG annotation analysis was performed on ETOH group and CON group, H group and ETOH group differential genes, and the classification statistics show that the differential genes of up-and down-regulated alcohol intervention and solid beverage intervention are related to the top 20 pathway condition. The up-regulating and down-regulating genes of the alcohol trunk prognosis relate to the metabolism, genetic information processing, environmental information processing, cellular processes, organism systems and human diseases of the pathway, the up-regulating differential genes are mainly enriched in transportation and catabolism, the down-regulating differential genes are mainly enriched in transportation and catabolism, folding, classification and degradation, and the metabolism and lipid metabolism of other amino acids. After the solid beverage is dry, up-regulating genes relate to pathway metabolism, genetic information processing and human diseases, and differential genes are mainly enriched in amino acid metabolism; down-regulated genes are involved in pathways with metabolism, genetic information processing, environmental information processing, cellular processes, biological systems and human diseases, differential genes are mainly enriched in cardiovascular disease, cancer, immune system, cell growth and death (FIG. 10).
To explore key differential genes for alcohol intervention and solid beverage intervention, further KEGG enrichment analysis was performed, padjust < 0.05 was a significant enrichment, enrichment analysis indicated that amino acid biosynthesis, cysteine and methionine metabolism, metabolic pathways, arginine biosynthesis and carbon metabolism were mainly enriched (table 5).
TABLE 5KEGG enrichment results
Further screening by combining the results to obtain key differential genes SPERMIDINE SYNTHASE (spermidine synthase), sirt7 (deacetylase 7), RT1-A2 (RT 1 class Ia, locus A2), RT1-CE1 (RT 1I class, track 1), LOC103692716 (heat shock protein HSP 90-alpha), acap2 (ArfGAP) which are related to liver functions, inflammatory reactions, lipid peroxidation and the like. The alleviation of alcoholic liver injury by solid beverage of strawberry tea and cassia seed is presumed to be related to the regulation of these genes and pathways.
<110> Hunan Aijia biotechnology Co., ltd
Preparation method of <120> strawberry tea and cassia seed solid beverage and application thereof in alcoholic liver injury
<160> 6
<170> PatentIn version 3.5
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ACAGCAACAGGGTGGTGGAC 20
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TTTGAGGGTGCAGCGAACTT 20
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GAGGATGGGAAACCTTACT 19
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CTTCTTGCTCTTGGGAACA 19
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Claims (7)
1. The application of the compound extract of the blackberry tea and the cassia seed in preparing a preparation for improving liver function, reducing liver inflammatory reaction and oxidation reaction or preventing or treating alcoholic liver injury is characterized in that the production process of the compound extract of the blackberry tea and the cassia seed comprises the following steps:
1) Weighing: respectively weighing the strawberry tea and the cassia seed;
2) Crushing and sieving: respectively pulverizing and sieving the strawberry tea and the semen cassiae;
3) Extracting pure water under reflux: extracting the strawberry tea and the cassia seed with pure water at 75-80 ℃ respectively;
4) Vacuum concentration: concentrating under the conditions that the vacuum pressure is-0.02 Mp to-0.095 Mp and the temperature is 60-80 ℃;
5) Spray drying: spray drying to obtain powder of extract of strawberry tea and semen Cassiae, wherein the inlet air temperature is 165-190 ℃ and the outlet air temperature is 75-85 ℃;
6) Sieving: respectively collecting powder of strawberry tea and semen Cassiae extract, and sieving with shaking sieve at 100%;
7) The strawberry tea and the cassia seed extract are prepared according to the following ratio of 1-2: mixing at a ratio of 1 for 20-40min;
Step 5), using an LPG50 type drying tower, wherein the motor frequency of the atomizer is 300-400 Hz, and the discharging frequency is 25-35 Hz for spray drying;
The specific operation of the step 7) is as follows: mixing the strawberry tea and the semen cassiae extract according to a set weight ratio, and fully mixing for 20-40 minutes in a three-dimensional motion mixer, wherein the main shaft rotating speed is 10-25r/mjn; sieving and granulating the materials in a granulator; drying the materials at 60-70deg.C until the water content is less than or equal to 5%, to obtain solid beverage.
2. The use according to claim 1, wherein in step 2) a 10 mesh screen is passed.
3. The use according to claim 1, wherein the reflux extraction in step 3) is carried out for 1 hour/time for a total of 2 times, and the extraction liquid is continuously refluxed by a centrifugal pump, so that the material in the extraction tank is maintained at a high temperature.
4. The use according to claim 1, wherein in step 4) the concentration is carried out to a solids content of more than 40%.
5. The use according to claim 1, wherein 100% of step 6) is passed through an 80 mesh shaker screen.
6. The use according to claim 1, wherein in step 7) the extract of the raspberry tea and the extract of the cassia seed are mixed according to 1.5:1 for 30min.
7. The use according to any one of claims 1 to 6, wherein the formulation is a medicament.
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|---|---|---|---|---|
| JPH0873369A (en) * | 1994-09-01 | 1996-03-19 | Fuairudo:Kk | Tea for health |
| CN102772586A (en) * | 2011-05-13 | 2012-11-14 | 贵州省生物研究所 | Preparation with hangover alleviating and liver protection functions and preparation method thereof |
| CN105287940A (en) * | 2015-10-15 | 2016-02-03 | 李镇坚 | Alcohol-dispelling beverage and preparation method thereof |
| CN111135235A (en) * | 2019-02-01 | 2020-05-12 | 南京萨陀健康科技有限公司 | A composition for preventing and relieving acute alcoholism or alcoholic intoxication |
| CN112826876A (en) * | 2021-02-02 | 2021-05-25 | 刘洪涛 | Application of ampelopsis grossedentata aqueous extract in preparation of preparation for improving liver injury |
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| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| JPH0873369A (en) * | 1994-09-01 | 1996-03-19 | Fuairudo:Kk | Tea for health |
| CN102772586A (en) * | 2011-05-13 | 2012-11-14 | 贵州省生物研究所 | Preparation with hangover alleviating and liver protection functions and preparation method thereof |
| CN105287940A (en) * | 2015-10-15 | 2016-02-03 | 李镇坚 | Alcohol-dispelling beverage and preparation method thereof |
| CN111135235A (en) * | 2019-02-01 | 2020-05-12 | 南京萨陀健康科技有限公司 | A composition for preventing and relieving acute alcoholism or alcoholic intoxication |
| CN112826876A (en) * | 2021-02-02 | 2021-05-25 | 刘洪涛 | Application of ampelopsis grossedentata aqueous extract in preparation of preparation for improving liver injury |
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| 复方藤茶降脂颗粒提取工艺的实验研究;李跃军;曾佳;姚腊初;唐敏;李顺祥;;湖南中医杂志;20171226(第12期);全文 * |
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