Roxburgh rose extract capable of improving inflammation level of sepsis and preparation method of roxburgh rose extract

Abstract

The invention belongs to the technical field of plant extraction, and discloses a roxburgh rose extract capable of improving the sepsis inflammation level and a preparation method thereof.The process comprises the steps of raw material treatment, freezing, drying, auxiliary material mixing, extraction, concentration and drying.The roxburgh rose extract capable of improving the sepsis inflammation level and the preparation method of the roxburgh rose extract capable of improving the sepsis inflammation level are provided; the obtained food-grade extract can be used for recovery and improvement of sepsis, and the roxburgh rose extract reduces the inflammation level of sepsis and alleviates organ injury by a multi-path combined regulation mechanism. The invention provides a safe and effective new means for the existing method for treating sepsis by drugs, supplements and perfects the preparation technology, functional activity and active action mechanism of the roxburgh rose extract, expands the application limitation of roxburgh rose in the clinical medicine field, and has a good application prospect by applying the roxburgh rose extract to sepsis intervention. The method is suitable for preparing the rosa roxburghii tratt extract, and the prepared rosa roxburghii tratt extract is suitable for adjuvant therapy of sepsis.

Description

Technical Field

[0001] The invention relates to the technical field of plant extraction, and in particular to a roxburghii roxburghii extract capable of improving the inflammation level of sepsis and a preparation method thereof. Background Art

[0002] Sepsis is a systemic inflammatory response syndrome caused by infection with pathogenic microorganisms such as bacteria and viruses. The mortality rate of septic shock caused by improper diagnosis and treatment is as high as 40%, which seriously endangers human health. During the occurrence of sepsis, neutrophils in the immune system can produce a special extracellular structure, namely neutrophil extracellular trap (NETs), which has an important impact on severe infections including sepsis. NETs are DNA network structures formed by the stimulation of multiple factors, intracellular histone citrullination, chromatin depolymerization and release in neutrophils, in which a variety of proteins and enzymes including histones, MPO, NE, MMP-9, and serine proteases are anchored. NETs can cause coagulation disorders and vascular occlusion, and have obvious damaging effects on tissues, especially epithelial and endothelial cells, and can also trigger the production of inflammatory factors in other immune cells. On the other hand, in the pathophysiological process of sepsis, overactivation of macrophages is also one of the important mechanisms. They are activated under various stimuli, express and secrete a large number of proinflammatory factors, trigger cytokine storms, and cause adverse consequences such as extensive thrombosis, increased vascular permeability, decreased effective blood volume, and organ failure. At present, there are limited reports on treatment methods and related drugs for inhibiting the level of sepsis inflammation by regulating NETs and related immune cell pathways, especially in the control of abnormal immune response of sepsis based on natural food extracts. There are no specific reports. The current treatment of sepsis mainly relies on comprehensive treatment methods such as drug therapy to control infection, correct metabolic disorders and symptomatic supportive therapy, which can stabilize the patient's vital signs and alleviate the progression of this acute course to a certain extent. However, the limitations of treatment methods and the side effects of treatment cannot be ignored. Therefore, starting from the perspective of regulating the immune response of sepsis, finding specific targets or drugs that can effectively control inflammation and reduce inflammatory damage has important clinical application value for improving disease progression.

[0003] Rosa roxburghii is a local precious fruit resource unique to Guizhou, China. It has the characteristics of being both edible and medicinal. Its fruit is rich in a variety of nutritional and health active ingredients. In the field of traditional Chinese medicine, the use of rosa roxburghii has a certain history. It is usually used in the combination of Chinese medicinal materials and medicinal and edible prescriptions. It is boiled and taken after direct combination of multiple medicinal materials. At present, there are related reports on the research and application of rosa roxburghii extracts, but the preparation methods, products, effects and mechanisms of action are relatively single and limited. In the intervention of sepsis, there is a lack of relevant technical applications and basic research results. Summary of the invention

[0004] The present invention aims to provide a roxburghii fruit extract that can improve the level of sepsis inflammation and a preparation method thereof. The present invention adopts a processing pretreatment method of roxburghii fruit, supplemented by green tea for extraction, to obtain a food-grade extract. The extract achieves the effect of reducing the level of sepsis inflammation and alleviating organ damage through a multi-path combined regulation mechanism.

[0005] The present invention provides the following technical solutions:

[0006] A roxburghii extract capable of improving the inflammation level of sepsis and a preparation method thereof, comprising the following steps:

[0007] S1. Raw material processing: fresh roxburghii fruit is used, washed, cut into pieces, sieved to remove seeds and set aside;

[0008] S2, freezing: Spread the roxburghii fruit pieces flat on a container, and slowly freeze them at low temperatures until the fruit is completely crystallized inside and outside;

[0009] S3, drying: the frozen roxburghii fruit pieces are spread on a baking tray, and placed in a far-infrared drying device to dry to constant weight, and the residual seeds are further sieved;

[0010] S4, mixing ingredients: mixing the dried roxburghii fruit pieces with the dried green tea at a mass ratio of 5:1;

[0011] S5, extraction: the mixed raw material and 60% ethanol were ultrasonically extracted at a ratio of 1:15 (g / ml) for 2h, filtered, the residue was re-extracted once in the same way, filtered again, and the filtrates produced twice were combined to obtain a roxburghii extract;

[0012] S6, concentration: the obtained roxburghii extract was subjected to rotary evaporation at 45°C to evaporate ethanol; further rotary evaporation was continued at 55°C to evaporate water and residual ethanol until the extract reached a thick state, thereby obtaining a roxburghii concentrate;

[0013] S7, drying: the obtained roxburghii concentrate is divided into drying containers with a material layer thickness of about 0.5 cm, and freeze-dried at -20°C to obtain the roxburghii extract.

[0014] Furthermore, in step S1, the roxburghii fruit pieces are cut into pieces of about 0.5-1.0 cm in size.

[0015] Furthermore, in step S2, the mixture is placed at a freezing temperature of about -10°C for slow freezing.

[0016] Further, in step S3, the product is dried to a constant weight in a far-infrared drying device at 55-60°C.

[0017] The roxburghii extract is used for auxiliary treatment of sepsis.

[0018] The beneficial effects of the technical solution are: the roxburghii extract of the present invention exerts its efficacy by improving the blood biochemical indicators of mice, reducing the formation of NETs and macrophage activation in mouse tissues, inhibiting the expression of neutrophil inflammatory factors and NETs formation, and inhibiting the expression of macrophage inflammatory factors and cell pyroptosis and other multi-path regulation, reducing the expression level of inflammatory factors in cells, thereby being able to achieve the effect of reducing the level of sepsis inflammation and reducing organ damage. Compared with the current surgery and drug treatment of patients with sepsis, a systemic inflammation, this technology and product are prepared by natural and safe edible raw materials and technical means, and the preparation process is not too complicated. The natural extract can significantly improve the overall symptoms of sepsis and promote comprehensive and continuous physical recovery, effectively supplementing and enriching the existing conventional drug treatment methods, and has good application prospects. At the same time, the process of slow freezing and far-infrared drying of the roxburghii raw material of the technical solution of the present invention is significantly improved in terms of drying efficiency, product functional activity indicators and other preservation rates compared to the existing conventional technology of direct far-infrared drying of fresh fruits. BRIEF DESCRIPTION OF THE DRAWINGS

[0019] Figure 1 This is a graph showing the relative fold changes in the expression of inflammatory expression factors in each group of cells in the third experimental process of the present invention;

[0020] Figure 2 This is a graph showing the detection of various indicators in the orbital blood of mice in the fourth experimental process of the present invention;

[0021] Figure 3 The formation of NETs in each group under confocal microscope in the fifth experimental process of the present invention;

[0022] Figure 4 This is a graph of the relative multiple changes in the expression of inflammatory factors in each group of macrophages and neutrophils in the sixth experimental process of the present invention, as well as pictures taken under a microscope. DETAILED DESCRIPTION

[0023] The present invention is further described in detail below in conjunction with the accompanying drawings and embodiments:

[0024] 1. Preparation of Rosa roxburghii Extract

[0025] A roxburghii extract capable of improving the inflammation level of sepsis and a preparation method thereof, comprising the following steps:

[0026] 1. Raw material processing: Use fresh roxburghii fruit, remove rotten and spoiled fruit and impurities, wash with clean water and drain the water, cut the washed roxburghii fruit into pieces of about 0.5-1.0 cm in size, sieve to remove seeds and set aside.

[0027] 2. Freezing: Spread the cut pieces of sea buckthorn fruit flat in a container and slowly freeze them at a low temperature of about -10°C until the fruit is completely frozen and crystallized inside and out.

[0028] 3. Drying: Spread the frozen fruit pieces flat on a baking tray and place them in a far-infrared drying device at 55℃-60℃ to dry to constant weight, and further sieve to remove residual seeds.

[0029] 4. Mixing ingredients: Mix the dried roxburghii fruit pieces and the dried green tea in a mass percentage of (5:1).

[0030] 5. Extraction: Mix the raw materials and 60% ethanol at a ratio of 1:15 (g / ml) and perform ultrasonic extraction for 2 h. Filter and repeat the extraction once with the residue in the same way. Filter again and combine the filtrate to obtain the roxburghii extract.

[0031] 6. Concentration: The roxburghii extract was subjected to rotary evaporation at 45°C to evaporate a large amount of ethanol; further rotary evaporation was continued at 55°C to evaporate a large amount of water and residual ethanol until the extract reached a thick state hanging on the wall, thereby obtaining the roxburghii concentrate.

[0032] 7. Drying: The roxburghii concentrate is divided into drying containers with a material layer thickness of about 0.5 cm, and freeze-dried at -20°C to obtain the roxburghii extract.

[0033] In the whole preparation process of the invention, the roxburghii raw material is subjected to slow freezing and then far-infrared drying, the drying time is 6 hours, the roxburghii dry moisture content is 12%, the browning degree is 0.12, the VC content is 1200 mg / 100 g, and the total flavonoids content is 390 mg / 100 g.

[0034] Compared with the current conventional direct far-infrared drying technology for fresh fruit raw materials, the raw material drying time is 9h, the product moisture is 16%, the browning degree is 0.15, the VC content is 670mg / 100g, and the total flavonoids content is 360mg / 100g.

[0035] The drying efficiency (drying time and product moisture content), product sensory quality, physical and chemical nutritional indicators, especially the preservation rate of natural active substances of this technology are significantly improved compared with the existing products of direct drying of fresh fruits. At the same time, the slow freezing pretreatment of the raw materials can realize the migration of water in the tissue, the formation and growth of ice crystals. The larger ice crystals formed in the pulp tissue destroy the physical structure of the tissue cells, which is beneficial to the release and outward migration of the water contents of the cells during the drying and extraction process, and improves the evaporation and drying rate of water in the fruit cells and the extraction efficiency of the effective ingredients.

[0036] 2. Establishment of mouse endotoxemia model by intraperitoneal injection of LPS

[0037] Male or female wild-type C57BL / 6 mice of 6-8 weeks and weighing 20-25g were used. The mice were treated with the roxburghii extract of the present invention: 500 mg / kg, gavage once a day, 200 μL / mouse, and the control group was gavaged with an equal volume of normal saline. One week later, the mice were intraperitoneally injected with LPS (15 mg / kg, Sigma, L2630) for endotoxemia modeling, and the control group mice were injected with an equal volume of PBS. The state of the mice was observed every 12 hours after the operation, and the observation time was 7 days. The mice were blood sampled from the orbits 24 hours after the injection, and the liver, lung, kidney and other tissues were taken to evaluate the level of inflammation and organ damage in the body.

[0038] 3. RNA extraction and real-time fluorescence quantitative PCR (qPCR)

[0039] 1. Collect liver, lung, and kidney tissues from each group of mice, as well as the treated macrophages and neutrophils;

[0040] 2. Use RNA rapid extraction kit to extract total cell RNA by column centrifugation and measure RNA concentration;

[0041] 3. Prepare 10 μL RNA reverse transcription system (RR036A, TaKaRa), use 500 ng RNA and 2 μL reaction mixture for each sample, perform reverse transcription at 37°C for 15 min, perform reverse transcriptase inactivation at 85°C for 5 s, and store at 4°C;

[0042] 4. The total system in each well is 10 μL. Add 5 μL of TB Green Premix Ex Taq II, 1 μL of cDNA obtained by reverse transcription, 3.2 μL of water, 0.8 μL of the internal reference gene (GAPDH) and the upstream and downstream primers of the target gene to a 384-well qPCR plate. Make 3 replicate wells for each gene in each sample.

[0043] 5. Test on the machine. After pre-denaturation, deformation, annealing and extension, measure the fluorescence intensity of the amplified DNA to obtain the Ct value;

[0044] 6. Normalize each group, subtract the GAPDH value from the target gene value, and compare it with the control group. Use the 2-ΔΔCt method to calculate the relative multiple of each group. (The gene names and primer sequences of each group are shown in Table 1, and the relative multiple of each group is shown in Figure 1 shown).

[0045] Table 1 Gene names and primer sequences of each group

[0046]

[0047] like Figure 1As shown, in the liver and lung tissues, after adding the roxburghii extract of the present invention, the mRNA fold change of inflammatory factors (IL6, IL-1β, TNF-α) was significantly reduced compared with the endotoxemia model group injected with LPS only, indicating that the expression of inflammatory factors was inhibited and thus reduced.

[0048] 4. Serum alanine aminotransferase (ALT), aspartate aminotransferase (AST), creatinine (Cr), and creatine kinase (CK) detection

[0049] Blood was collected from the mouse orbits and placed in EP tubes. The blood was placed in a 4°C refrigerator overnight to allow the blood to coagulate and the serum to precipitate. The blood was then centrifuged at 4°C, 2000 g for 20 min, and the light yellow supernatant was taken and tested on an automatic biochemical analyzer for various indicators. Figure 2 shown.

[0050] like Figure 2 As shown, after mice were treated with the roxburghii extract of the present invention, the levels of alanine aminotransferase (ALT), aspartate aminotransferase (AST) and creatinine (Cr) in their serum were significantly reduced.

[0051] 5. Immunofluorescence staining and confocal microscopy observation

[0052] 1. Take the liver, lungs, kidneys and other organs of the mouse and fix them in 4% paraformaldehyde;

[0053] 2. Wax-embed the slices, dewax them after baking, put them into xylene I and II for dewaxing, and then pass through anhydrous ethanol, different gradient alcohols and distilled water;

[0054] 3. Repair the sections in pH 8.0 EDTA antigen repair solution in a rice cooker at high pressure for 6 minutes. After cooling to room temperature, block with 5% BSA at room temperature for 20 minutes, and then dilute rabbit-H3Cit, goat-MPO, rabbit-F4 / 80 and rat-CD80 primary antibodies with 1% BSA and incubate at 4°C overnight.

[0055] 4. For cell immunofluorescence, after the cell stimulation reaches the predetermined time, the culture supernatant is aspirated, the cells are washed with PBS, 500 μL of 4% paraformaldehyde is added to each well for fixation at room temperature for 20 min; PBS is washed, 300 μL of 5% BSA is added to each well for blocking at room temperature for 20 min; rabbit-H3Cit and goat-MPO primary antibodies are diluted with 1% BSA and dropped into the slides for incubation at 4°C overnight;

[0056] 5. Wash the samples with 1×PBST for 3 times, 5 min each time, incubate with 1% BSA, diluted fluorescent secondary antibody at a ratio of 1:200, at room temperature for 1-2 h, and wash again with 1×PBST for 3 times, 5 min each time;

[0057] 6. Add DAPI working solution to stain the cell nucleus and incubate at room temperature for 15 minutes;

[0058] 7. Wash with 1×PBST 3 times, 5 min each time, seal with anti-fluorescence quenching sealant, and observe under a laser confocal microscope at 63 times magnification. Figure 3 shown.

[0059] VI. Isolation and Culture of Mouse Bone Marrow Macrophages and Neutrophils

[0060] Macrophage isolation and culture

[0061] 1. Use 8-10 week old male or female WT mice weighing 25-30g for sampling. Kill the mice and soak them in 75% ethanol for 5-10 minutes. Use ophthalmic scissors to separate the femur and tibia of the mice, remove the skin and muscles, and cut off a small part of the two ends of the long bones to expose the bone marrow cavity.

[0062] 2. Use a syringe to absorb DMEM culture medium and insert the needle into the bone marrow to flush until the bone turns white;

[0063] 3. Collect the washed cell suspension into a centrifuge tube, mix it by blowing, filter it, and centrifuge it at 350g for 5 minutes;

[0064] 4. Remove the supernatant, add 1 mL of red blood cell lysis buffer and lyse the red blood cells at 4°C for 5 minutes, then add saline to terminate the lysis.

[0065] 5. Centrifuge at 350 g for 5 min to obtain bone marrow cell pellet;

[0066] 6. Resuspend the cells in DMEM complete medium containing 10% FBS, 2% penicillin / streptomycin and 10 ng / mL macrophage colony stimulating factor at a concentration of 1×10 6 / mL density was inoculated into a 12-well culture plate and placed in a 37°C, 5% CO2 incubator for adherent culture; the medium was changed on the third and fifth days, and bone marrow-derived macrophages were induced after one week.

[0067] Neutrophil isolation and culture

[0068] 1. Use the mouse bone marrow neutrophil isolation kit and follow the operating instructions to centrifuge bone marrow cells at 800g for 20 minutes using density gradient centrifugation, with both the ascending and descending speeds set to 3 to isolate bone marrow neutrophils;

[0069] 2. After centrifugation, carefully aspirate the neutrophil layer, wash it, and then use complete medium at 5-10×10 7 The cells were seeded at a density of 1.544 × 10 / mL in a 24-well culture plate and cultured at 37°C in 5% CO2.

[0070] VII. Cell Treatment and Observation of Pyroptotic Cell Morphology

[0071] Cell treatment: To verify the extract effect and induce the secretion of inflammatory factors by neutrophils and macrophages, cells were cultured at a rate of approximately 1×10 6 / mL were inoculated in 12-well or 24-well plates; the extract (400μg / mL) was first added to mouse neutrophils and macrophages for pretreatment for 12h, and then LPS (1μg / mL) was added to stimulate for 6h to induce inflammation; if cell pyroptosis was to be continued, nigericin (10μM) was added to the cells and stimulated for another 30min, and the cells were subsequently collected for downstream experiments.

[0072] Morphological observation of pyroptotic cells: Mouse macrophages were cultured at a rate of 1×10 5 / mL density was inoculated into a 24-well glass-bottom culture plate; corresponding stimulation was added according to the cell treatment plan; after the predetermined time, the cells were observed under a light microscope to see if cell death occurred, and the typical pyroptosis morphology of cell swelling and swelling, and the appearance of cell membrane blebbing and protrusion was found; and pictures of each group of cells were taken under a phase contrast microscope.

[0073] The relative multiples of each group of macrophages and neutrophils, as well as the pictures taken under the microscope Figure 4 shown.

[0074] like Figure 4 As shown, in neutrophils and macrophages, after adding the roxburghii extract of the present invention, the fold change of mRNA of inflammatory factors (IL6, IL-1β, TNF-α) was significantly reduced relative to the experimental group treated with LPS only, indicating that the expression of inflammatory factors was inhibited and thus reduced.

[0075] The above experiments show that the roxburghii extract obtained by this technology, and the results of analyzing the effect of inflammation produced by sepsis show that the roxburghii extract improves the prognosis of mouse endotoxemia (IL-6, IL-1b, TNF-aqPCR in tissues and cells, and serum AST, ALT, creatinine levels) and shows good effects. At the same time, through the above experiments, it can be concluded that the present invention is to reduce NETs formation and macrophage activation in mouse tissues, inhibit the expression of neutrophil inflammatory factors and NETs formation, and inhibit the expression of macrophage inflammatory factors and cell pyroptosis and other multi-path regulation to exert its efficacy. The extract raw material processing preparation method, raw material formula combination and extract effect and mechanism of action of the present invention have obvious difference characteristics and advantages compared with the prior art methods, and have good application prospects.

[0076] The above is only an embodiment of the present invention, and the common knowledge such as the known specific technical solutions or characteristics in the solution is not described in detail here. It should be pointed out that for those skilled in the art, several modifications and improvements can be made without departing from the technical solution of the present invention, which should also be regarded as the protection scope of the present invention, and these will not affect the effect of the implementation of the present invention and the practicality of the patent. The scope of protection required by this application shall be based on the content of its claims, and the specific implementation methods and other records in the specification can be used to interpret the content of the claims.

Claims

1. A roxburghii extract capable of improving the level of sepsis inflammation and a preparation method thereof, It is characterized in that The following steps are involved: S1. Raw material processing: fresh roxburghii fruit is used, washed, cut into pieces, sieved to remove seeds and set aside; S2, freezing: Spread the roxburghii fruit pieces flat on a container, and slowly freeze them at low temperatures until the fruit is completely crystallized inside and outside; S3, drying: the frozen roxburghii fruit pieces are spread on a baking tray, and placed in a far-infrared drying device to dry to constant weight, and the residual seeds are further sieved; S4, mixing ingredients: mixing the dried roxburghii fruit pieces with the dried green tea at a mass ratio of 5:1; S5, extraction: the mixed raw material and 60% ethanol were ultrasonically extracted at a ratio of 1:15 (g / ml) for 2h, filtered, the residue was re-extracted once in the same way, filtered again, and the filtrates produced twice were combined to obtain a roxburghii extract; S6, concentration: the obtained roxburghii extract was subjected to rotary evaporation at 45°C to evaporate ethanol; further rotary evaporation was continued at 55°C to evaporate water and residual ethanol until the extract reached a thick state, thereby obtaining a roxburghii concentrate; S7, drying: the obtained roxburghii concentrate is divided into drying containers with a material layer thickness of about 0.5 cm, and freeze-dried at -20°C to obtain the roxburghii extract.

2. A roxburghii extract capable of improving the level of sepsis inflammation and a preparation method thereof according to claim 1, Features: In the step S1, the roxburghii fruit pieces are cut into pieces with a size of about 0.5-1.0 cm.

3. The roxburghii extract capable of improving the inflammation level of sepsis according to claim 1 and the preparation method thereof, Features: In the step S2, the mixture is placed at a freezing temperature of about -10°C and slowly frozen.

4. The roxburghii extract capable of improving the inflammation level of sepsis according to claim 1 and the preparation method thereof, Features: In the step S3, the far-infrared drying equipment is used to dry the product to a constant weight at 55-60°C.

5. A roxburghii extract capable of improving the inflammation level of sepsis according to claim 1, wherein the extract is used as an adjuvant treatment for sepsis.

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