Preparation method and application of medicine guaijaverin for regulating macrophage polarization and inhibiting inflammatory response
Through the preparation and application of guavacin, the polarization direction of macrophages was successfully regulated, thereby inhibiting the secretion of inflammatory factors, solving the inflammation problem caused by excessive polarization of macrophages, and achieving effective regulation of the inflammatory response.
Patent Information
- Application Number
- CN202411420725.6
- Authority / Receiving Office
- CN · China
- Patent Type
- Applications(China)
- Current Assignee / Owner
- Filing Date
- 2024-10-11
- Publication Date
- 2025-01-14
- Estimated Expiration
- Not applicable · inactive patent
AI Technical Summary
When macrophages are overpolarized to M1 type under lipopolysaccharide induced, it leads to excessive secretion of inflammatory factors and causes tissue and organ damage. The prior art is difficult to effectively regulate their polarization and inhibit inflammatory response.
Guava glycoside was prepared by crude extraction, concentration and purification, chromatography separation and purification identification, and guava glycoside was used to regulate macrophages from M1 to M2 in LPS-induced macrophage models. Polarization inhibits the secretion of inflammatory factors.
Guava glycoside effectively inhibits the expression of macrophage nitric oxide synthase (iNOS) and other proinflammatory factors, promotes anti-inflammatory factors secretion, reduces inflammatory responses, and shows temporal and dose effects.
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Figure CN119306778A_ABST
Abstract
Description
Technical field:
[0001] The present invention relates to the field of medical technology, and in particular to a preparation method and application of guajava glycoside, a drug for regulating macrophage polarization and inhibiting inflammatory response. Background technology:
[0002] Macrophages are one of the body's innate immune cells and important immune cells that make up the body's immune system. They are the body's first line of defense against pathogen invasion. Macrophages play an important role in identifying pathogens, presenting antigens, clearing pathogens, regulating inflammatory responses and tissue repair. Macrophages are usually divided into two different subgroups: M1 and M2. Under the action of lipopolysaccharide (LPS), macrophages polarize into M1 macrophages and express inducible nitric oxide synthase (iNOS), secreting pro-inflammatory factors such as NO, IL-1β, IL-6, IL-12, IL-27, and TNF-α. Appropriate amounts of pro-inflammatory factors play an important role in clearing pathogens, but excessive secretion of pro-inflammatory factors can cause "inflammatory factor storms" that can cause tissue and organ damage and lead to patient death. Macrophages polarize to M2 type under the action of cytokines (such as IL-4 and IL-13) secreted by helper T (Th2) cells, and exert anti-inflammatory effects by secreting anti-inflammatory factors such as IL-10 and TGF-β; at the same time, M2 macrophages have tissue repair, remodeling and angiogenesis effects. In order to solve the above technical problems, a new technical solution is proposed. Summary of the invention:
[0003] The purpose of the present invention is to overcome the shortcomings of the above-mentioned prior art, and proposes a preparation method and application of guava glycosides, a drug for regulating macrophage polarization and inhibiting inflammatory response. Macrophages are divided into two different subgroups, M1 and M2. Macrophages are polarized into M1 macrophages under the induction of lipopolysaccharide (LPS), secrete pro-inflammatory factors, and excessive inflammation can cause tissue and organ damage, leading to patient death; macrophages are polarized into M2 macrophages under the action of IL-4 and IL-13, secrete anti-inflammatory factors such as IL-10 and TGF-β, and exert anti-inflammatory effects. The present discovery provides that in the LPS-induced macrophage inflammation model, guava glycosides have the effect of regulating macrophage polarization from M1 to M2 and inhibiting the secretion of inflammatory factors.
[0004] The technical solution adopted by the present invention is: a method for preparing guava glycosides, a drug for regulating macrophage polarization and inhibiting inflammatory response, the method comprising the following steps:
[0005] Step 1: Rough extraction;
[0006] Step 2: Concentration and purification;
[0007] Step 3, chromatographic separation;
[0008] Step 4: purification and identification;
[0009] As a preferred technical solution, the crude extraction first cleans the guava peel, removes impurities, and cuts it into small pieces to increase the contact area. Secondly, the cut guava peel is placed in a hot reflux extraction device, and a sufficient amount of 70% ethanol is added to cover the material. Hot reflux extraction is performed again at boiling temperature. The extraction takes 4-6 hours. Finally, after the extraction is completed, the solution is cooled to room temperature and filtered to remove residual plant solids.
[0010] As a preferred technical solution, the concentration and essence first uses a rotary evaporator to remove the solvent to obtain a crude extract; secondly, an appropriate amount of distilled water is added to the crude extract, and then transferred to a separatory funnel; the crude extract is extracted again with ethyl acetate and the organic phase is collected; finally, the organic phase is dried with anhydrous sodium sulfate to remove moisture.
[0011] As a preferred technical solution, the chromatographic separation first prepares a glass column filled with silica gel for chromatographic separation; secondly, the dried extract is loaded onto the column and eluted with a suitable eluent, which is usually carried out in order of increasing polarity; finally, the eluted components are collected and the components containing guajava glycosides are monitored using an ultraviolet or visible spectrometer.
[0012] As a preferred technical solution, the purification and identification first combines the components containing guajava glycosides and removes the eluent by a rotary evaporator; then the structure of the purified guajava glycosides is identified by nuclear magnetic resonance (NMR), mass spectrometry (MS) or infrared spectroscopy (IR), and finally guajava glycosides are obtained.
[0013] A drug for regulating macrophage polarization and inhibiting inflammatory response, guava glycoside, the chemical formula of guava glycoside is C 20 H 18 O 11 , molecular weight is 434.35, guava glycoside is light yellow powder, the specific molecular formula is
[0014]
[0015] An application of guajava glycosides, a drug for regulating macrophage polarization and inhibiting inflammatory response, wherein the specific application steps are as follows:
[0016] Step 1: Extracting and culturing macrophages;
[0017] Step 2: Drug preparation;
[0018] Step 3: Drug dosage effect;
[0019] Step 4: Drug time effect;
[0020] As a preferred technical solution, the extraction and culture of macrophages described above, firstly, 1 ml of 4% thioglycolate broth was injected into the peritoneal cavity of mice 3 days in advance; secondly, the mice were killed by cervical dislocation, immersed in 75% alcohol for disinfection, and 4 ml of ice saline was injected into the peritoneal cavity to lavage the peritoneal macrophages; the peritoneal lavage fluid was transferred into a 10 ml EP tube with a pipette; after centrifugation (4°C, 300g, 5min), the supernatant was discarded, 1 ml of 1× red blood cell lysis was added to lyse the red blood cells for 3min, and 1 ml of RPMI-1640 medium containing 10% FBS was added to terminate the lysis of red blood cells; after centrifugation again (4°C, 300g, 5min), the supernatant was discarded. Add an appropriate amount of RPMI-1640 medium containing 10% FBS to mix well, and count the cells; after culturing in a cell culture incubator at 37°C and 5% CO2 for 2 hours, the supernatant was removed, and the cells were washed twice with PBS. The obtained adherent cells were macrophages (2×10 per well 6 The RPMI-1640 containing 10% FBS is a cell culture medium, wherein FBS stands for fetal bovine serum (Fetal Bovine Serum), and RPMI-1640 stands for RPMI-1640 medium (RPMI-1640 Medium).
[0021] As a preferred technical solution, the prepared drug first prepares guava glycosides, and the specific preparation method is to dissolve 20 mg of guava glycosides in 1.535 ml of DMSO to prepare a 30 mM drug solution. The prepared drug solution with a molar concentration of 30 mM is diluted to 15 mM and 7.5 mM drug solutions. Take 100 ul of drug solvent DMSO as a control drug; wherein DMSO is the full name of dimethyl sulfoxide (Dimethyl sulfoxide). Then prepare LPS solution, and the specific preparation method is to dissolve 1 mg of LPS in 1 ml of RPMI-1640 culture medium, vortex mix, and make it into a concentration of 1 mg / ml LPS solution. Take 1 ml of RPMI-1640 culture medium as a control drug. wherein LPS is the full name of lipopolysaccharide (Lipopolysaccharide). RPMI-1640 is a widely used cell culture medium suitable for the culture of a variety of mammalian cells.
[0022] As a preferred technical solution, the drug dose effect includes grouping, guava glycoside pretreatment, LPS induction of macrophages and sample collection. The grouping is to divide the obtained macrophages into Con, LPS, LPS+Gua 7.5mM, LPS+Gua 15mM LPS+Gua 30mM groups. The guava glycoside pretreatment LPS+Gua 7.5mM, LPS+Gua 15mM LPS+Gua 30mM groups are respectively added with 2ul of guava glycoside drug solution with a molar concentration of 7.5mM, 15mM, and 30mM for pretreatment for 1h, and the Con and LPS groups are pretreated with 2ul of control drug DMSO for 1h. The LPS-induced macrophages, LPS, LPS+Gua7.5mM, LPS+Gua 15mM, LPS+Gua 30mM groups added 20ul of 1mg / ml LPS to induce macrophages, and the Con group added 20ul of RPMI-1640 culture medium as a control drug to induce macrophages. The collected samples were collected 8 hours after LPS induction; Con was the control group; LPS was lipopolysaccharide solution; Gua was guava glycosides.
[0023] As a preferred technical solution, the time effect includes grouping, guava glycoside pretreatment, LPS induction of macrophages and sample collection. The grouping is to divide the obtained macrophages into Con 4h, LPS 4h, Gua 4h, LPS+Gua4h, Con 8h, LPS 8h, Gua 8h, LPS+Gua 8h groups. The guava glycoside pretreatment Gua 4h, LPS+Gua 4h, Gua 8h, LPS+Gua 8h groups are pretreated with 2ul of guava glycoside drug solution with a molar concentration of 30mM for 1h; Con4h, LPS 4h, Con8h, LPS 8h groups are pretreated with 2ul of control drug DMSO for 1h. The LPS-induced macrophages, LPS4h, LPS+Gua 4h, LPS 8h, LPS+Gua 8h groups added 20ul of 1mg / ml LPS to induce macrophages, and Con 4h, Gua 4h, Con 8h, Gua 8h groups added 20ul of RPMI-1640 culture medium as a control drug to induce macrophages. The collected samples, collected Con 4h, LPS 4h, Gua 4h, LPS+Gua 4h samples at 4h of LPS induction; collected Con 8h, LPS8h, Gua 8h, LPS+Gua 8h group samples at 8h of LPS induction.
[0024] As a preferred technical solution, the mRNA transcription level of AhR was detected by the fluorescence quantitative PCR method established in the laboratory. The main steps are: RNA extraction, cDNA synthesis, real-time quantitative PCR, and calculation and analysis of the target gene amount of each sample with internal reference correction.
[0025] The beneficial effects of the present invention are:
[0026] 1. Regulate macrophage polarization. Guava glycosides (Gua) inhibit the mRNA expression of nitric oxide synthase (iNOS) in macrophages induced by lipopolysaccharide (LPS) and promote the mRNA expression of arginase 1 (Arg-1), that is, regulate the transformation of LPS-induced M1 macrophages to M2 macrophages.
[0027] 2. Anti-inflammatory effect: guava glycosides can inhibit the release of inflammatory factors by macrophages, such as tumor necrosis factor-α (TNF-α), interleukin-6 (IL-6), interleukin-27 (IL-27) and interleukin-1β (IL-1β), thereby reducing the inflammatory response. Description of the drawings:
[0028] Figure 1 This is a control diagram of guajardin in the present invention regulating macrophage polarization;
[0029] Figure 2 This is a schematic diagram of the guajava glycosides of the present invention inhibiting the expression of pro-inflammatory factors in macrophages; Specific implementation method:
[0030] The present invention is further described with reference to specific embodiments:
[0031] A preparation method of guava glycosides for regulating macrophage polarization, the method comprising the following steps: step 1, crude extraction; step 2, concentration and purification; step 3, chromatographic separation; step 4, purification and identification; the crude extraction first cleans the guava peel, removes impurities, and cuts it into small pieces to increase the contact area, then puts the cut guava peel into a hot reflux extraction device, adds a sufficient amount of 70% ethanol covering material, and performs hot reflux extraction again at boiling temperature, which takes 4-6 hours, and finally after the extraction is completed, the solution is cooled to room temperature and filtered to remove the residual solids of the plant. The concentration and essence first use a rotary evaporator to remove the solvent to obtain a crude extract; secondly, add an appropriate amount of distilled water to the crude extract, and then transfer it to a separatory funnel; extract the crude extract again with ethyl acetate and collect the organic phase; finally, dry the organic phase with anhydrous sodium sulfate to remove moisture. The chromatographic separation first prepares a glass column filled with silica gel for chromatographic separation; secondly, the dried extract is loaded onto the column and eluted with an appropriate eluent, which is usually carried out in the order of increasing polarity; finally, the eluted components are collected and the components containing guava glycosides are monitored by an ultraviolet or visible spectrometer. The purification and identification first combine the components containing guava glycosides and remove the eluent by a rotary evaporator; then the purified guava glycosides are structurally identified by using nuclear magnetic resonance (NMR), mass spectrometry (MS) or infrared spectroscopy (IR) to obtain guava glycosides.
[0032] A guava glycoside for regulating macrophage polarization, the chemical formula of guava glycoside is C 20 H 18 O 11 , molecular weight is 434.35, guava glycoside is light yellow powder, the specific molecular formula is
[0033]
[0034] An application of guava glycosides in a drug for inhibiting macrophage inflammatory response, the specific application steps are as follows: step 1, extracting and culturing macrophages; step 2, drug preparation; step 3, drug dosage effect; step 4, drug time effect. For the extraction and culturing of macrophages, firstly, 1 ml of 4% thioglycolate broth is injected into the peritoneum of mice 3 days in advance; secondly, the mice are killed by cervical dislocation, immersed in 75% alcohol for disinfection, and 4 ml of ice saline is injected into the peritoneum to lavage peritoneal macrophages; the peritoneal lavage fluid is transferred into a 10 ml EP tube with a pipette; after centrifugation (4°C, 300g, 5min), the supernatant is discarded, 1 ml of 1× red blood cell lysis is added to lyse the red blood cells for 3min, and 1 ml of RPMI-1640 culture medium containing 10% FBS is added to stop the lysis of red blood cells; after centrifugation again (4°C, 300g, 5min), the supernatant is discarded. Add appropriate amount of RPMI-1640 medium containing 10% FBS, blow and mix, and count the cells; after culturing in a cell culture incubator at 37°C and 5% CO2 for 2 hours, remove the supernatant, wash twice with PBS, and obtain adherent cells as macrophages (2×10 per well). 6 The RPMI-1640 containing 10% FBS is a cell culture medium, wherein FBS stands for fetal bovine serum (Fetal Bovine Serum), and RPMI-1640 stands for RPMI-1640 medium (RPMI-1640 Medium).
[0035] The prepared drug is first prepared by dissolving 20 mg of guava glycoside in 1.535 ml of DMSO to prepare a 30 mM drug solution. The prepared drug solution with a molar concentration of 30 mM is diluted to 15 mM and 7.5 mM drug solutions. 100 ul of drug solvent DMSO is taken as a control drug; wherein DMSO is called dimethyl sulfoxide. Then, LPS solution is prepared by dissolving 1 mg of LPS in 1 ml of RPMI-1640 culture medium, vortexing and mixing to make a concentration of 1 mg / ml LPS solution. 1 ml of RPMI-1640 culture medium is taken as a control drug. wherein LPS is called lipopolysaccharide. RPMI-1640 is a widely used cell culture medium suitable for the culture of a variety of mammalian cells.
[0036] The drug dose effect includes grouping, guava glycoside pretreatment, LPS induction of macrophages and sample collection. The grouping is to divide the obtained macrophages into Con, LPS, LPS+Gua 7.5mM, LPS+Gua 15mM LPS+Gua30mM groups. The guava glycoside pretreatment LPS+Gua 7.5mM, LPS+Gua 15mM LPS+Gua 30mM groups are respectively pretreated with 2ul of guava glycoside drug solution with a molar concentration of 7.5mM, 15mM, and 30mM for 1h, and the Con and LPS groups are pretreated with 2ul of control drug DMSO for 1h. The LPS-induced macrophages, LPS, LPS+Gua 7.5mM, LPS+Gua15mM, LPS+Gua 30mM groups added 20ul of 1mg / ml LPS to induce macrophages, and the Con group added 20ul of RPMI-1640 culture medium as a control drug to induce macrophages. The collected samples were collected 8 hours after LPS induction; Con was the control group; LPS was lipopolysaccharide solution; Gua was guava glycosides.
[0037] The time effect includes grouping, guava glycoside pretreatment, LPS induction of macrophages and sample collection. The grouping is to divide the obtained macrophages into Con 4h, LPS 4h, Gua 4h, LPS+Gua4h, Con 8h, LPS 8h, Gua8h, LPS+Gua 8h groups. The guava glycoside pretreatment Gua4h, LPS+Gua 4h, Gua 8h, LPS+Gua 8h groups are pretreated with 2ul of 30mM guava glycoside drug solution for 1h; Con 4h, LPS 4h, Con 8h, LPS 8h groups are pretreated with 2ul of control drug DMSO for 1h. The LPS-induced macrophages, LPS 4h, LPS+Gua 4h, LPS 8h, LPS+Gua 8h groups added 20ul of 1mg / ml LPS to induce macrophages, and Con 4h, Gua 4h, Con 8h, Gua 8h groups added 20ul of RPMI-1640 culture medium as a control drug to induce macrophages. The collected samples, collected Con4h, LPS 4h, Gua 4h, LPS+Gua4h samples at 4h of LPS induction; collected Con 8h, LPS 8h, Gua 8h, LPS+Gua 8h group samples at 8h of LPS induction.
[0038] The mRNA transcription level of AhR was detected by the fluorescence quantitative PCR method established in the laboratory. The main steps are: RNA extraction, cDNA synthesis, real-time quantitative PCR, and calculation and analysis of the target gene amount of each sample with internal reference correction. For details, see the primer sequence table:
[0039]
[0040] The experimental data and results were analyzed using Prism 8.0 statistical software, and the data were expressed as mean ± standard error of the mean. One-way analysis of variance was used. P < 0.05 indicated that the difference was statistically significant.
[0041] 0.05; P<0.01; P<0.001; NS P>0.05, there is no statistical difference.
[0042] In the LPS-induced macrophage inflammation model, guava glycosides have the effect of regulating the polarization of LPS-induced macrophages from M1 to M2, and have time effect and dose effect. In the LPS-induced macrophage inflammation model, guava glycosides have the effect of inhibiting the secretion of proinflammatory factors, and have time effect and dose effect. Figure 1 It can be concluded that in the LPS (10ug / ml)-induced macrophage inflammation model, guava glycosides (7.5uM, 15uM, 30uM) inhibited the expression level of iNOS mRNA, a marker of macrophage M1 type. Compared with LPS induced for 4h, iNOS mRNA expression was higher after 8h of LPS induction, and guava glycosides (30uM) had a more obvious effect in inhibiting iNOS mRNA expression ( Figure 2 A). In macrophages induced by LPS for 8 hours, 30uM guava glycosides had a more significant inhibitory effect on iNOS mRNA expression than 15uM and 7.5uM guava glycosides ( Figure 2 B). LPS induction for 4h and 8h failed to promote the expression of Arg-1 mRNA; the expression level of Arg-1 mRNA was higher after 8h of guava glycosides treatment than after 4h ( Figure 2 C). In macrophages induced by LPS for 8 hours, 30uM guava glycosides promoted the expression of Arg-1 mRNA more significantly than 15uM and 7.5uM guava glycosides ( Figure 2 D) The above results show that guava glycosides can regulate the polarization of macrophages from M1 to M2, and there is a time effect and a dose effect.
[0043] Reference Figure 2It can be concluded that in the LPS (10ug / ml)-induced macrophage inflammation model, guava glycosides (7.5uM, 15uM, 30uM) inhibited the expression levels of proinflammatory factors IL-6, TNF-α, IL-1β, and IL-27mRNA. Guava glycosides with a molar concentration of 30uM inhibited the expression of proinflammatory factors IL-6, TNF-α, IL-1β, and IL-27mRNA more significantly than guava glycosides with molar concentrations of 15uM and 7.5uM. Compared with LPS induction for 4h, the expression of proinflammatory factors IL-6, TNF-α, IL-1β, and IL-27mRNA was higher after 8h of LPS induction; the effect of guava glycosides on inhibiting the expression of proinflammatory factors IL-6, TNF-α, IL-1β, and IL-27mRNA was more obvious after 8h of guava glycosides than after 4h. The above results show that guava glycosides have the effect of inhibiting the expression of LPS-induced macrophage inflammatory factors, and there is a time effect and a dose effect.
[0044] For those skilled in the art, various other corresponding changes and modifications can be made according to the above technical solutions and concepts, and all of these changes and modifications should fall within the protection scope of the claims of the present invention.
Claims
1. A method for preparing guava glycosides, a drug for regulating macrophage polarization and inhibiting inflammatory response, characterized in that: The method comprises the following steps: Step 1: Rough extraction; Step 2: Concentration and purification; Step 3, chromatographic separation; Step 4: Purification and identification.
2. The method for preparing guava glycosides for regulating macrophage polarization and inhibiting inflammatory response according to claim 1, characterized in that: The crude extraction first cleans the guava peel, removes impurities, and cuts it into small pieces to increase the contact area. Secondly, the cut guava peel is placed in a hot reflux extraction device, and a sufficient amount of 70% ethanol is added to cover the material. Hot reflux extraction is performed again at boiling temperature. The extraction takes 4-6 hours. Finally, after the extraction is completed, the solution is cooled to room temperature and filtered to remove residual plant solids.
3. The method for preparing guava glycosides for regulating macrophage polarization and inhibiting inflammatory response according to claim 1, characterized in that: The concentration and essence are firstly carried out by removing the solvent using a rotary evaporator to obtain a crude extract; secondly, an appropriate amount of distilled water is added to the crude extract, and then the crude extract is transferred to a separatory funnel; the crude extract is extracted again with ethyl acetate, and the organic phase is collected; finally, the organic phase is dried with anhydrous sodium sulfate to remove moisture.
4. The method for preparing guajaroside, a drug for regulating macrophage polarization and inhibiting inflammatory response, according to claim 1, characterized in that: The chromatographic separation first prepares a glass column filled with silica gel for chromatographic separation; secondly, the dried extract is loaded onto the column and eluted with a suitable eluent, which is usually carried out in the order of increasing polarity; finally, the eluted components are collected and the components containing guajava glycosides are monitored with an ultraviolet or visible spectrometer.
5. The method for preparing guava glycosides for regulating macrophage polarization and inhibiting inflammatory response according to claim 1, characterized in that: The purification and identification first combines the components containing guajava glycosides and removes the eluent by a rotary evaporator; then the structure of the purified guajava glycosides is identified by nuclear magnetic resonance (NMR), mass spectrometry (MS) or infrared spectroscopy (IR), and finally guajava glycosides are obtained.
6. A guava glycoside for regulating macrophage polarization, characterized in that: The chemical formula of guava glycoside is C 20 H 18 O 11 , molecular weight is 434.35, guava glycoside is light yellow powder, the specific molecular formula is 7. An application of guava glycosides in a drug for regulating macrophage polarization and inhibiting inflammatory response, characterized in that: The specific application steps are as follows: Step 1: Extraction and culture of macrophages; Step 2: preparing the medicine; Step 3: Drug dosage effect; Step 4, drug time effect; the time effect includes grouping, guava glycoside pretreatment, LPS induction of macrophages and sample collection. The grouping is to divide the obtained macrophages into Con 4h, LPS 4h, Gua 4h, LPS+Gua 4h, Con 8h, LPS 8h, Gua 8h, LPS+Gua 8h groups. The guava glycoside pretreatment Gua 4h, LPS+Gua 4h, Gua8h, LPS+Gua 8h groups are pretreated with 2ul of 30mM guava glycoside drug solution for 1h; Con 4h, LPS4h, Con8h, LPS 8h groups are pretreated with 2ul of control drug DMSO for 1h. The LPS-induced macrophages, LPS 4h, LPS+Gua 4h, LPS 8h, LPS+Gua 8h groups added 20ul of 1mg / ml LPS to induce macrophages, and Con 4h, Gua 4h, Con 8h, Gua 8h groups added 20ul of RPMI-1640 culture medium as a control drug to induce macrophages. The collected samples, collected Con 4h, LPS4h, Gua 4h, LPS+Gua 4h samples at 4h of LPS induction; collected Con 8h, LPS 8h, Gua8h, LPS+Gua 8h group samples at 8h of LPS induction.
8. The use of guava glycosides in a drug for regulating macrophage polarization and inhibiting inflammatory response according to claim 7, characterized in that: The extraction and culture of macrophages described above firstly inject 1 ml of 4% thioglycolate broth into the peritoneum of mice 3 days in advance; secondly, kill the mice by cervical dislocation, immerse in 75% alcohol for disinfection, and inject 4 ml of ice saline into the peritoneum to lavage the peritoneal macrophages; transfer the peritoneal lavage fluid into a 10 ml EP tube with a pipette; centrifuge (4°C, 300g, 5min) and discard the supernatant, add 1 ml of 1× red blood cell lysis to lyse the red blood cells for 3min, and add 1 ml of RPMI-1640 medium containing 10% FBS to stop the lysis of red blood cells; centrifuge again (4°C, 300g, 5min) and discard the supernatant. Add an appropriate amount of RPMI-1640 medium containing 10% FBS to mix well, and count the cells; after culturing in a cell culture incubator at 37°C and 5% CO2 for 2 hours, remove the supernatant, wash twice with PBS, and the obtained adherent cells are macrophages (2×10 per well 6 The RPMI-1640 containing 10% FBS is a cell culture medium, wherein FBS stands for fetal bovine serum (Fetal Bovine Serum), and RPMI-1640 stands for RPMI-1640 medium (RPMI-1640 Medium).
9. The use of guava glycosides in a drug for regulating macrophage polarization and inhibiting inflammatory response according to claim 7, characterized in that: The prepared drug is first prepared by dissolving 20 mg of guava glycoside in 1.535 ml of DMSO to prepare a 30 mM drug solution. The prepared drug solution with a molar concentration of 30 mM is diluted to 15 mM and 7.5 mM drug solutions. 100 ul of drug solvent DMSO is taken as a control drug; wherein DMSO is called dimethyl sulfoxide. Then, LPS solution is prepared by dissolving 1 mg of LPS in 1 ml of RPMI-1640 culture medium, vortexing and mixing to make a concentration of 1 mg / ml LPS solution. 1 ml of RPMI-1640 culture medium is taken as a control drug. wherein LPS is called lipopolysaccharide. RPMI-1640 is a widely used cell culture medium suitable for the culture of a variety of mammalian cells.
10. The use of guava glycosides in a drug for inhibiting macrophage inflammatory response according to claim 7, characterized in that: The drug dose effect includes grouping, guava glycoside pretreatment, LPS induction of macrophages and sample collection. The grouping is to divide the obtained macrophages into Con, LPS, LPS+Gua 7.5mM, LPS+Gua 15mM LPS+Gua 30mM groups. The guava glycoside pretreatment LPS+Gua 7.5mM, LPS+Gua 15mM LPS+Gua 30mM groups are respectively pretreated with 2ul of guava glycoside drug solution with a molar concentration of 7.5mM, 15mM, and 30mM for 1h, and the Con and LPS groups are pretreated with 2ul of control drug DMSO for 1h. The LPS-induced macrophages, LPS, LPS+Gua7.5mM, LPS+Gua 15mM, LPS+Gua 30mM groups added 20ul of 1mg / ml LPS to induce macrophages, and the Con group added 20ul of RPMI-1640 culture medium as a control drug to induce macrophages. The collected samples were collected 8 hours after LPS induction; Con was the control group; LPS was lipopolysaccharide solution; Gua was guava glycoside.