Use of barley germ vinegar preparation in prolonging nematode life

Abstract

The application discloses application of barley germ vinegar preparation in prolonging the life of nematodes, and can be used for preparing products for prolonging the life of nematodes.

Description

Technical Field

[0001] This invention belongs to the field of bioactive extract applications, specifically relating to the application of barley germ vinegar in prolonging the lifespan of nematodes. Background Technology

[0002] Ubiquitin is a 76-amino acid polypeptide found in many different tissues and organs, excluding bacteria, and it functions to label proteins to be degraded. Ubiquitin-labeled proteins are degraded in the proteasome. Ubiquitin-controlled protein degradation has significant physiological implications, not only clearing faulty proteins but also playing a crucial regulatory role in cell cycle, DNA replication, and chromosome structure.

[0003] The active site of the proteasome, which degrades proteins, is located at the center of a barrel-shaped structure, isolated from the rest of the cell. The only pathway to the active site is a "lock" (a 19S complex), which recognizes ubiquitinated proteins, unfolds their structure, and helps the protein pass through the narrow channels of the proteasome into the active site at the center of the barrel. After the protein is degraded into short peptide fragments of 7 to 9 amino acids, it is released from another part of the proteasome. In addition, the 19S complex contains an isopeptidase that removes ubiquitin from the substrate protein. Therefore, the proteasome itself is not selective for proteins; the selective factor is the E3 enzyme, which only ubiquitinates the proteins to be degraded.

[0004] Ubiquitination regulation plays an important role in cellular life processes.

[0005] The ubiquitin-proteasome degradation pathway is the most important protein degradation pathway in eukaryotic cells, participating in various physiological processes, including at least transcriptional regulation, cell cycle, apoptosis, DNA damage repair and metabolism, and immunity.

[0006] In transcriptional regulation, several transcriptional regulators, such as nuclear factor KB (NF-κB), cell cycle regulators (E2F), and proto-oncogene products c-Fos and c-jun, can serve as substrates for regulation via the ubiquitin-proteasome pathway, leading to the activation or inactivation of downstream gene expression. In apoptosis regulation, studies have found that some apoptosis-related regulatory molecules, such as Bcl-2 and srp60, are degraded via the ubiquitin-proteasome. In cell cycle regulation, normal cell cycle function is regulated by various cyclins and cell cycle-dependent kinases (CDKs) and their inhibitors. Research has shown that the ubiquitin-proteasome can modulate the activity of CDKs and their inhibitors, and that CDK inhibitor p27 and cyclins such as cyclin D and cyclin E are important substrates for the ubiquitin-proteasome. In immunity, the ubiquitin-proteasome pathway is essential for the presentation of major histocompatibility complex (MHC) class I antigens. Endogenous proteins in antigen-presenting cells are degraded into polypeptides by the 26S proteasome and transported to the cell membrane surface by binding to MHC-I molecules in the endoplasmic reticulum, where they are recognized by cytotoxic T cells.

[0007] The ubiquitin-proteasome system (UPS) is an ATP-dependent, highly selective protein degradation system found in eukaryotic cells, capable of clearing senescent, damaged, and misfolded proteins. It participates in various important biological functions, including regulating inflammation, immune responses, cellular senescence, and signal transduction. Abnormalities in UPS can cause cellular homeostasis imbalances, leading to a variety of diseases such as tumors, neurodegenerative diseases, cardiovascular diseases, and kidney diseases.

[0008] Ubiquitination levels are closely related to cellular metabolism and aging. Literature reports that aging leads to a decrease in ubiquitination levels in nematodes, hindering protein recognition and degradation. When gene knockout therapy is used to accelerate protein clearance, the aging condition of nematodes is improved, with average lifespan extended by up to 21%.

[0009] In liver diseases, the ubiquitin-proteasome is associated with alcoholic liver disease and viral hepatitis. Studies have shown that the core protein in hepatitis C cannot be degraded due to monoubiquitination or oligoubiquitination, leading to the disease. Early-onset inflammatory bowel disease (VEOIBD) is a severe form of inflammatory bowel disease (IBD), primarily affecting infants under 6 years of age. Recent research has found that mutations in the inflammasome NLRP3 promote its binding to the highly expressed deubiquitinating enzymes BRCC3 and JOSD2 in the gut, thereby promoting NLRP3 deubiquitination, activating the NLRP3 inflammasome, and ultimately leading to VEOIBD. Cardiomyopathy is a leading cause of death in Duchenne muscular dystrophy, characterized by myocardial hypertrophy and fibrosis. A key pathogenic factor is the hypoubiquitination of the hypertrophic protein p300; when its ubiquitination level is restored, diastolic function in muscular dystrophy rats is eventually restored. Pseudoaldosteronism type 2 (PHA2) syndrome is a syndrome characterized by hypertension, hyperkalemia, and metabolic acidosis. Studies by Wakabayashi et al. have found that mutations in the Cullin3 ubiquitin ligase lead to WNK4 kinase ubiquitin dysregulation, thereby inducing hypertension. Atherosclerosis (AS) is a serious chronic inflammatory vascular disease that severely endangers human health. Ubiquitination of the transcriptional coactivator protein YAP promotes YAP nuclear translocation, increases the expression of its downstream chemokines, and accelerates the development of AS.

[0010] Protein ubiquitination plays a crucial role in the regulation of the human immune system. UPS (Ubiquitin-Proteinized Proteins) regulate the balance of the body's immune response by efficiently and selectively degrading proteins, participating in the occurrence and development of various autoimmune diseases. Systemic lupus erythematosus (SLE) is a common autoimmune disease characterized by multi-systemic damage and the formation of various autoantibodies. Enhanced antibody expression against UPS components can be detected in SLE patients. Sjögren's syndrome (SS) is a chronic inflammatory autoimmune disease primarily affecting exocrine glands. Ubiquitin ligase R052 promotes the immune-inflammatory cascade, induces apoptosis, and causes abnormal T-cell and B-cell signaling, thus participating in the pathogenesis of SLE and SS. Rheumatoid arthritis (RA) is mainly characterized by chronic symmetrical synovitis. Ubiquitin ligases c-MIR and synovin (also known as Hrd1) are highly expressed in the synovial tissue of RA patients and mice, leading to increased levels of inflammatory factors such as tumor necrosis factor (TNF)-α and IL-1β, inhibiting synovial cell apoptosis in collagen-induced arthritis mice, significantly promoting synovial tissue proliferation, and inducing arthritis. Furthermore, UPS participates in regulating antiviral immunity through the ubiquitination or deubiquitination of important proteins. For example, research from the School of Life Sciences at Wuhan University found that the deubiquitinating enzyme OTUD4 is upregulated, targeting MAVS for deubiquitination, thereby maintaining MAVS stability and promoting intracellular antiviral signaling. Another study found that the deubiquitinating enzyme OTUB1 inhibits PD-L1 degradation in the endoplasmic reticulum by regulating the ubiquitination modification of the immune checkpoint protein PD-L1, revealing the key role of the OTUB1-PD-L1 signaling pathway in regulating tumor cell immune escape.

[0011] A recent study found that the E3 ubiquitin ligase TRAF4 is highly expressed in metastatic prostate cancer. Ubiquitination modification can lead to abnormal receptor tyrosine kinase activity, promoting prostate cancer metastasis. In breast cancer cells, ERRβ (estrogen-related receptor β) ubiquitination leads to downregulated expression levels, while its high expression level in breast cancer patients is positively correlated with prognosis and prolonged recurrence-free survival. Furthermore, aberrantly expressed USP22 plays a role in promoting tumor development in malignant tumors such as colorectal cancer, gastric cancer, and small cell lung cancer.

[0012] Malt, also known as barley malt, is processed from the mature fruit of the barley plant (Hordeurn vulgare L.), a member of the Poaceae family. According to traditional Chinese medicine, malt is sweet in taste and enters the spleen and stomach meridians. It is primarily used to treat conditions such as invigorating qi and promoting digestion, strengthening the spleen and stomach, suppressing lactation, and relieving bloating. It is often used to aid digestion and treat spleen deficiency, milk accumulation, or weaning. Modern research shows that malt also has many other important effects: it is rich in dietary fiber, which aids digestion; experiments using malt decoction administered orally to mice have shown that it can influence sex hormone secretion; malt can also regulate intestinal flora, protect the liver, and inhibit platelet aggregation. Medicinally, malt is mainly used for suppressing and promoting lactation, and it also has certain health benefits.

[0013] From the perspective of Traditional Chinese Medicine (TCM) theory and drug application: The sour taste of vinegar is favored by the liver, thus it can guide medicine to the liver. For example, Corydalis, a commonly used herb in clinical practice, after being processed with vinegar, has its alkaloids combine with acetic acid to form water-soluble acetates, which are easier to dissolve during decoction, thereby improving clinical efficacy. Vinegar can also remove the fishy or foul odor of drugs and reduce their toxicity. For example, many drugs such as Euphorbia kansui, Euphorbia pekinensis, Daphne genkwa, and Phytolacca acinosa are bitter, cold, and toxic, with strong side effects. Vinegar, being warm in nature, can kill pathogens and has astringent properties; processing with vinegar can reduce their toxicity and alleviate their medicinal properties, significantly reducing the strong irritation to the digestive tract and reducing side effects such as nausea and vomiting. The saying "calcination and vinegar quenching can turn them into powder" refers to the processing of certain hard mineral drugs that do not become brittle even after high temperatures. Calcination and vinegar quenching can change the physicochemical properties of drugs, enhance their efficacy, reduce side effects, and make hard drugs such as natural copper, magnetite, and hematite brittle, making them easier to mix and pulverize. Natural copper is a natural iron sulfide ore, which is hard and difficult to break. After being repeatedly calcined and quenched in vinegar, the vinegar is absorbed completely, and the color turns blackish-brown. The surface becomes brittle and the luster disappears, and the texture becomes crisp, making it easier to prepare and extract the effective components, thus enhancing its effects of dispersing blood stasis and relieving pain. Summary of the Invention

[0014] The purpose of this invention is to address the shortcomings of existing technologies and disclose the application of barley germ vinegar in prolonging the lifespan of nematodes. Specifically, it involves the application of barley germ vinegar in the preparation of products that prolong the lifespan of nematodes. The product can be in the form of a solution, sol, emulsion, paste, tablet, capsule, or gel. In preparing the specific product, barley germ vinegar can be used as a core ingredient in combination with other active ingredients.

[0015] The preparation process of the barley germ vinegar product is as follows: barley germ is added to vinegar, refluxed at 80-100 ℃ for 2.5-3.5 h, and the extraction is repeated three times. After filtration, the supernatant is removed to obtain the final product. Preferably, the vinegar is food-grade rice vinegar with a total acidity greater than 6%; the reflux conditions are: reflux at 100 ℃ for 3 h; and the vinegar is removed by vacuum rotary evaporation.

[0016] The beneficial effects of this invention are as follows: This invention has found that barley germ vinegar can significantly prolong the lifespan of *C. elegans*, and can be used to prepare related products. The effect is definite and has been verified by experiments. Attached Figure Description

[0017] Figure 1 The results show the effects of different barley germ extracts on cell ubiquitination levels.

[0018] Figure 2 The results show the effects of different barley germ extracts on the survival rate of nematodes. Detailed Implementation

[0019] The following will provide a clear and complete description of the concept and technical effects of the present invention in conjunction with the embodiments and accompanying drawings, so as to fully understand the purpose, solution and effects of the present invention.

[0020] Example 1:

[0021] Freshly harvested barley germ was rinsed with clean water to remove surface impurities and microorganisms. 120 grams of clean, sterile barley germ was weighed and placed directly into a sterilized flask. Ten times the weight of the barley germ in distilled water was added, and the mixture was refluxed at 100°C for 3 hours, with the extraction repeated three times. A crude extract was obtained, which was filtered to remove residue, yielding a supernatant. The collected supernatants were combined and transferred to a round-bottom flask. The solvent was removed by vacuum rotary evaporation at 50°C, yielding 43.9 grams of pure barley germ aqueous extract.

[0022] Example 2:

[0023] Weigh out 30 grams of clean, sterile barley germ and place it directly into a sterilized flask. Add ten times the amount of anhydrous ethanol as solvent, and reflux at 100°C for 3 hours. Repeat the extraction three times. Filter to remove residue and obtain the supernatant. Combine the collected supernatants and transfer them to a round-bottom flask. Remove the solvent by vacuum rotary evaporation at 50°C to obtain 8.3 grams of pure barley germ ethanol extract.

[0024] Example 3:

[0025] Weigh out 39 grams of clean, sterile barley germ and place it directly into a sterilized flask. Add ten times the weight of the barley germ in 6-degree edible rice vinegar (total acidity greater than 6%). Reflux at 100°C for 3 hours, repeating the extraction process three times. Filter to remove residue, obtaining the supernatant. Combine the collected supernatants and transfer them to a round-bottom flask. Remove the solvent by vacuum rotary evaporation at 50°C to obtain 13.4 grams of pure barley germ vinegar extract.

[0026] Example 4:

[0027] Weigh out 30 grams of clean, sterile barley germ and place it directly into a sterilized flask. Add ten times the weight of the barley germ in 3-degree Shanxi aged vinegar (total acidity greater than 3%). Reflux at 100°C for 3 hours, repeating the extraction process three times. Filter to remove residue, obtaining the supernatant. Combine the collected supernatants and transfer them to a round-bottom flask. Remove the solvent by vacuum rotary evaporation at 50°C to obtain 7.2 grams of pure barley germ vinegar extract.

[0028] Example 5:

[0029] Weigh out 30 grams of clean, sterile barley germ and place it directly into a sterilized flask. Add ten times the weight of the barley germ in 6-degree Shanxi aged vinegar (total acidity greater than 6%). Reflux at 80°C for 3 hours, repeating the extraction process three times. Filter to remove residue, obtaining the supernatant. Combine the collected supernatants and transfer them to a round-bottom flask. Remove the solvent by vacuum rotary evaporation at 50°C to obtain 11.9 grams of pure barley germ vinegar extract.

[0030] Example 6:

[0031] 293 T cells (4×10) 5 Cells were seeded in 6-well plates and, after adhesion on the second day, were incubated with different concentrations of the compound (25 mg / mL for the low-dose group and 100 mg / mL for the high-dose group) for 24 h. Then, 2.5 μM MG132 was added for 3 h to inhibit proteasome degradation of ubiquitinated proteins. Total protein was then extracted by lysis in RIPA lysis buffer containing the protease inhibitor cocktail / PMSF and the dephosphorylase inhibitor NaF. Protein concentration was determined using a BCA protein assay kit. An equal volume of protein (8%) was electrophoresed by SDS-PAGE under denaturing conditions and transferred to a PDVF membrane. The membrane was blocked with 5% skim milk and then incubated overnight with ubiquitin primary antibody. After washing, the membrane was incubated with rabbit secondary antibody, and ubiquitination was detected using a chemiluminescence imaging system.

[0032] The results are as follows Figure 1 As shown, the water extract of barley germ basically did not upregulate the ubiquitination level of cells, while the high-dose group of the alcohol extract showed an upregulation of the ubiquitination level of cells. The vinegar extract of barley germ, regardless of low or high doses, significantly increased the ubiquitination level of barley germ.

[0033] Example 7:

[0034] Hermaphroditic nematodes were cultured in Nematode Growth Medium (NGM) coated with E. coli OP50 at 20°C. *C. elegans* were treated using a synchronization method. Synchronized L4 *C. elegans* were transferred to experimental plates containing different extract solutions (25 mg / mL), with each experimental sample containing at least 60 nematodes. The survival time of *C. elegans* was calculated from the time of transfer, with the day of transfer recorded as day 0 of the lifespan experiment. The nematodes were examined every other day, and the survival, death, and number of nematodes removed from the experiment were recorded. The experiment ended when all *C. elegans* died. Criteria for death of *C. elegans*: no movement or swallowing behavior, and no reaction after light touch. Removal criteria: 1) escaping to the plate wall or lid and drying out; 2) eggs hatching into cyst-like nematodes within the body; 3) burrowing into the agar; 4) accidental death, escape, and cyst-like nematodes. The experiment was divided into a control group, a water extract group, an alcohol extract group, and a vinegar extract group. A certain concentration of the filtered and sterilized test substance was added to each group's NGM plates. The final concentration of DMSO in the experiment was... <l %。

[0035] Experimental results are as follows Figure 2 As shown in the figure, compared with the control group (total survival of 17 days), the water and alcohol extracts slightly improved the survival rate of nematodes, increasing the total survival by 4 days and 3 days, respectively. The vinegar extract of barley germ significantly prolonged the survival of nematodes, increasing the total survival by 8 days.

[0036] The above description is merely a preferred embodiment of the present invention. The present invention is not limited to the above-described embodiments. Any embodiment that achieves the technical effects of the present invention using the same means should fall within the protection scope of the present invention. Within the protection scope of the present invention, various modifications and variations can be made to the technical solutions and / or implementation methods.

Claims

1. The application of barley germ vinegar in the preparation of products that extend the lifespan of *C. elegans*, characterized in that... The preparation process of the barley germ vinegar product is as follows: add barley germ to aged vinegar with a total acidity greater than 6%, reflux at 100°C for 3 hours, repeat the extraction three times, filter to obtain the supernatant, and then remove the vinegar by vacuum rotary evaporation.

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