A longan pro-inflammatory protein, extraction method and application

Abstract

The present invention discloses a longan pro-inflammatory protein, an extraction method and an application thereof. The pro-inflammatory protein is a longan protein extract extracted by the phenol extraction method. The longan pro-inflammatory protein of the present invention can promote the growth of oral inflammatory pathogenic bacteria. When culturing oral flora, it can specifically promote the growth of pathogenic bacteria without promoting the growth of other non-pathogenic bacteria, providing an experimental basis for simulating the environment of massive reproduction of pathogenic bacteria in the oral cavity. At the same time, the longan pro-inflammatory protein of the present invention can also promote the growth of pathogenic bacteria in the intestine and promote the expression of inflammatory factors in cells at a relatively low concentration, providing a basis for subsequent pharmaceutical research and development.

Description

Technical Field

[0001] The present invention relates to the field of biotechnology, and particularly to a longan pro-inflammatory protein, an extraction method and an application thereof. Background Art

[0002] Longan (Dimocarpus longan Lour.), also known as Guiyuan, contains various nutrients such as sugars, vitamins, and minerals required by the human body, and has high nutritional value. However, longan is warm in nature and sweet in taste, and eating too much of it is likely to generate internal heat and cause "getting angry", and it is often called a "hot fruit". Modern medicine believes that "getting angry" is an inflammatory reaction. Oranges and lychees are also common "hot fruits", and it has been found that the main components causing "getting angry" in them are water-soluble proteins. In particular, three families of 14-3-3 proteins induce an immune inflammatory response in the body through the arachidonic acid (AA) metabolic pathway of the human body.

[0003] Gingival inflammation is a typical symptom of "getting angry", mainly caused by periodontal diseases caused by oral microorganisms. Bacteria are the main type of oral microorganisms, and often grow and reproduce on the salivary film to form dental plaque. The bacteria in dental plaque metabolize to produce harmful substances that stimulate the gingival tissue to produce inflammation. Streptococcus mutans is the main type of oral flora and often forms dental plaque leading to dental caries. Its mechanism of action is mainly to synthesize exopolysaccharide (EPS) from sucrose through three glucosyltransferases (GtfsB, GtfsC, and GtfsD), so that the bacteria firmly adhere to the tooth surface and form a biofilm, providing guarantee for the growth of bacteria. Staphylococcus epidermidis is often an opportunistic pathogen in the oral cavity. Oral bacteria are prone to form biofilms in the oral cavity, and most biofilms have high tolerance to various antibiotics, resulting in the occurrence of various diseases in the oral cavity. However, there is currently no relevant research on the proteins in longan that cause gingival inflammation. At the same time, when culturing bacteria, they are basically cultured separately, and it is difficult to batch culture the inflammation-causing pathogenic bacteria specifically, so as to simulate the environment for the proliferation of inflammation-causing pathogenic bacteria. Summary of the Invention

[0004] Aiming at the above deficiencies existing in the prior art, the purpose of the present invention is to provide a longan pro-inflammatory protein to solve the problems in the prior art that there is no relevant research on the proteins in longan that cause gingival inflammation and it is difficult to specifically culture the gingival inflammation-causing pathogenic bacteria in the flora.

[0005] A further purpose of the present invention is to provide an extraction method of the above longan pro-inflammatory protein to solve the problem of difficultly obtaining a large amount of longan pro-inflammatory protein accurately.

[0006] A further object of the present invention is to provide an application of the above-mentioned longan pro-inflammatory protein, which is used to solve the problem in the prior art that it is difficult to perform targeted batch culture of inflammatory pathogenic bacteria, and provides a basis for subsequent drug research and development.

[0007] In order to solve the above technical problems, the present invention adopts the following technical solutions:

[0008] A longan pro-inflammatory protein, wherein the pro-inflammatory protein is a longan protein extract extracted by the phenol extraction method. By shotgun mass spectrometry analysis of the longan protein extract, the proteins with a peptide segment coverage rate of more than 30% detected include: cytoplasmic copper / zinc superoxide dismutase, NADP-specific isocitrate dehydrogenase, cytochrome c oxidase subunit Vb, glutathione transferase, glutathione peroxidase, ribosomal protein L22, NADP-dependent isocitrate dehydrogenase, cyclin-dependent kinase, FRIGIDA-like protein 3a-2, RNA polymerase β, auxin response factor, GTP-binding nuclear protein, NBS-LRR disease resistance protein NBS37, ascorbate peroxidase, transcription factor E2FB, phospholipase Dδ, cryptochrome 1, WRKY transcription factor 2-3, WRKY transcription factor 72-4, WRKY transcription factor 28-2, cyclin T1-4, ribosomal protein S4, maternal effect embryonic arrest protein, glutathione S-transferase, dehydroascorbate reductase; the molecular weight of the pro-inflammatory protein in the longan protein is above 30KD.

[0009] The present invention also provides an extraction method of longan pro-inflammatory protein, which specifically includes the following steps:

[0010] Step 1: After the longan pulp is juiced, it is filtered, centrifuged and the supernatant is collected. After freeze-drying, the volume is reduced, dialysis treatment is carried out at 4°C, and then freeze-dried again;

[0011] Step 2: Add the powder dried in Step 1 to a pre-cooled phenol extraction buffer, and then add a pre-cooled Tris-saturated phenol solution, shake vigorously to mix evenly, oscillate in an ice box for 2h, and centrifuge at 4°C to obtain the upper layer liquid;

[0012] Step 3: Repeat Step 2 for the upper layer liquid, take the upper layer phenol phase, add a pre-cooled methanol solution containing ammonium acetate, let the protein precipitate at -20°C, centrifuge at 4°C, and take the precipitate;

[0013] Step 4: Add acetone to the precipitate, mix evenly, place it at -20°C for 1h, and then centrifuge at 4°C;

[0014] Step 5: After repeating Step 4 twice, air-dry to obtain the longan protein.

[0015] Application of a longan pro-inflammatory protein, the longan protein obtained by the above extraction method is used to promote the growth of pathogenic bacteria in the oral flora, especially to promote the growth of Streptococcus mutans and Staphylococcus epidermidis.

[0016] Compared with the prior art, the present invention has the following beneficial effects:

[0017] 1. The longan pro-inflammatory protein of the present invention can promote the growth of pathogenic bacteria causing oral inflammation. When culturing the oral flora, it can specifically promote the growth of pathogenic bacteria without promoting the growth of other non-pathogenic bacteria, providing an experimental basis for simulating the environment of a large number of pathogenic bacteria multiplying in the oral cavity; at the same time, the longan pro-inflammatory protein of the present invention can also promote the growth of pathogenic bacteria in the intestine, and promote the expression of inflammatory factors of cells at a lower concentration, providing an experimental basis for subsequent related drug research and development.

[0018] 2. The method of the present invention can efficiently extract pro-inflammatory protein from longan, has little influence on the protein activity, has a high product purity and good pro-inflammatory activity, providing an experimental basis for subsequent research on the "getting angry" of longan. Description of the Drawings

[0019] Figure 1 It is the SDS-PAGE electrophoresis diagram of the longan protein of the present invention.

[0020] Figure 2 It is the diagram of the influence of the longan protein of the present invention on the growth of oral bacteria; among them, Figure 2 A is Streptococcus mutans, Figure 2 B is Staphylococcus epidermidis, Figure 2 C is Staphylococcus saprophyticus, and P < 0.05.

[0021] Figure 3 It is the diagram of the influence of different concentrations of longan protein on the growth of bacteria; among them, Figure 3 A is Streptococcus mutans, Figure 3 B is Staphylococcus epidermidis, Figure 3 C is Staphylococcus saprophyticus, and P < 0.05.

[0022] Figure 4 It is the diagram of the influence of longan protein on the formation of bacterial biofilm; among them, A is the biofilm morphology, B is the biofilm amount, and P < 0.05.

[0023] Figure 5 It is the diagram of the influence of longan protein on the EPS production of bacterial biofilm; among them, Figure 5 A is Streptococcus mutans, Figure 5 B is Staphylococcus epidermidis, Figure 5 C is Staphylococcus saprophyticus, and P < 0.05.

[0024] Figure 6 SDS-PAGE electrophoresis analysis of longan protein components extracted by different methods.

[0025] Figure 7 Effects of longan proteins at different concentrations on mouse intestinal pathogenic bacteria and probiotics.

[0026] Figure 8 Effects of longan proteins at different concentrations on cellular inflammatory factors.

[0027] Figure 9 Effects of longan proteins at different concentrations on inflammatory factors in blood.

[0028] Figure 10 Effects of longan proteins at different concentrations on lung inflammation. Specific implementation mode

[0029] The present invention will be further described below in conjunction with the accompanying drawings and embodiments.

[0030] I. A pro-inflammatory protein of longan

[0031] The pro-inflammatory protein described in the present invention is a longan protein extract extracted by the phenol extraction method. Based on the shotgun proteomics analysis method, the trypsin digestion products of longan proteins are analyzed using a reversed-phase liquid chromatography-tandem mass spectrometry system. Combining database retrieval, a total of 25 proteins with a peptide segment coverage rate of more than 30% are identified, including: cytoplasmic copper / zinc superoxide dismutase, NADP-specific isocitrate dehydrogenase, cytochrome c oxidase subunit Vb, glutathione transferase, glutathione peroxidase, ribosomal protein L22, NADP-dependent isocitrate dehydrogenase, cyclin-dependent kinase, FRIGIDA-like protein 3a-2, RNA polymerase β, auxin response factor, GTP-binding nuclear protein, NBS-LRR disease resistance protein NBS37, ascorbate peroxidase, transcription factor E2FB, phospholipase Dδ, cryptochrome 1, WRKY transcription factor 2-3, WRKY transcription factor 72-4, WRKY transcription factor 28-2, cyclin T1-4, ribosomal protein S4, maternal effect embryonic arrest protein, glutathione S-transferase, dehydroascorbate reductase.

[0032] II. A method for extracting a pro-inflammatory protein of longan

[0033] Example 1

[0034] The fresh longan pulp was juiced and filtered through four layers of gauze. It was centrifuged at 5000 rpm / min for 15 min at 4°C to collect the supernatant, which was then freeze-dried to reduce its volume. After dialysis at 4°C for 3 days, the sample was collected and freeze-dried again. 100 mg of the dry powder was weighed and 15 mL of pre-cooled phenol extraction buffer (pH 7.5, 500 mM Tris-base, 63.7 mM EDTA aqueous solution, 100 mM KCl, 700 mM sucrose) was added. Then 15 mL of pre-cooled Tris-saturated phenol solution was added, and it was shaken vigorously to mix evenly. It was placed in an ice box and shaken on a shaker for 2 h, and then centrifuged at 5000 rpm / min for 15 min at 4°C. The upper layer was taken, and the above steps were repeated. 35 mL of pre-cooled methanol containing 0.1 M ammonium acetate was added to the re-obtained upper phenol phase, and the protein was precipitated by standing overnight at -20°C. It was centrifuged at 5000 rpm / min for 15 min at 4°C, and the supernatant was removed. Acetone was added and shaken gently, and then it was left to stand at -20°C for 1 h and centrifuged at 5000 rpm / min for 15 min at 4°C. The above steps were repeated twice, and the longan protein was obtained by air drying and dissolved in water for quantification.

[0035] Comparative Example 1: Extraction of total longan protein by isoelectric point precipitation method

[0036] 500 g of dried longan pulp was taken, pulverized by a pulverizer, and 1600 g of distilled water was added. The pH value was adjusted to 8.0, and after soaking for 2 h, it was filtered by suction. The filtrate was adjusted to pH 3.0, 4.0, and 5.0, and after standing for 2 h, it was centrifuged (10000 r / min, 10 min). The supernatant was removed, and the precipitate was cooled and dried to obtain longan protein, which was dissolved in water for quantification.

[0037] Comparative Example 2: Extraction of total longan protein by kit

[0038] Referring to the instructions of the plant protein extraction kit (Shanghai Sangon C500053-0050): The plant tissue was frozen at -80°C, cut into pieces, and the frozen and cut tissue was placed in a pre-cooled mortar. While adding liquid nitrogen to keep it frozen, the plant tissue was ground into powder. 100 mg of tissue powder was taken, 1 mL of Solution A and 0.7 μL of Solution C were added, and after suspending the powder, it was left to stand at -20°C for 45 min and centrifuged (4°C, 15000 r / min, 15 min) to take the precipitate. Then 1 mL of Solution B, 10 μL of Solution D, and 0.7 μL of Solution C were added, re-suspended, left to stand at -20°C for 60 min, and centrifuged to take the precipitate. The same above reagents were added again, and it was centrifuged immediately to take the precipitate. The precipitate was freeze-dried to obtain longan protein, which was dissolved in water for quantification.

[0039] III. Application of longan pro-inflammatory protein

[0040] Prepare 10% separating gel, pour the gel and let it solidify; then prepare 5% stacking gel solution, pour it on top of the separating gel, and insert a comb. After standing for 30 min, pull out the comb, add 100 μg of the above-mentioned longan protein aqueous solution into the sample wells, perform electrophoresis at 120 V for 1 h, stop electrophoresis, take out the gel, put it into Coomassie Brilliant Blue R250 staining solution and shake it overnight, then put the gel into 25% methanol and 7.5% acetic acid in turn and shake it at room temperature for decolorization, and take pictures.

[0041] It can be seen from Figure 1 that after Coomassie Brilliant Blue staining, the bands of the longan protein extract are clear, with fewer impurities, and there are more bands with molecular weights in the range of 30 kDa - 250 kDa, and fewer bands below 30 kDa, indicating that the protein is complete and of high quality, and can be used in subsequent experiments.

[0042] Table 1 Absorbance of longan extracted by different methods

[0043]

[0044] After Coomassie Brilliant Blue staining, the bands of the samples extracted by the saturated phenol extraction method and the kit extraction method are basically separated clearly, as Figure 6 shown. The saturated phenol extraction method has a high quality of extracting longan protein, and the molecular weight distribution of the obtained longan protein is in the range of 250 kDa - 15 kDa, and most of the complete proteins contained in longan pulp can be extracted. The concentration of longan protein extracted by the isoelectric point precipitation method is lower than that of the longan protein extracted by the saturated phenol extraction method, and only longan proteins with specific pH values can be extracted. In summary, the saturated phenol extraction method has the best extraction effect.

[0045] (1) Effects of longan pro-inflammatory protein on oral pathogenic bacteria

[0046] 1. Bacterial strain activation

[0047] Prepare TYP medium and TSA medium, and activate the cryopreserved Streptococcus mutans, Staphylococcus saprophyticus, and Staphylococcus epidermidis.

[0048] 2. Effects of longan protein on the growth of three oral bacteria

[0049] Take 100 mL of bacterial liquid in the logarithmic growth phase and add 2 mL of 3 mg / mL longan protein sample solution, and add 2 mL of 3 mg / mL bovine serum albumin as a control. Every 2 h, take 1 mL of bacterial liquid and measure the OD value of the bacterial liquid at 600 nm with a spectrophotometer.

[0050] 3. Effects of different concentrations of longan protein on the growth of oral bacteria

[0051] Prepare longan protein solutions with concentrations of 0 mg / mL, 3 mg / mL, 6 mg / mL, and 9 mg / mL. Add 2 mL of the above-mentioned longan protein solutions with different concentrations to 100 mL of 3 kinds of bacterial culture media respectively, culture at 37 °C for 12 h, and measure the OD value at 600 nm using a spectrophotometer.

[0052] 4. Effects of longan protein with different concentrations on the biofilm formation of three oral bacteria

[0053] Add 1 mL of the bacterial suspensions of 3 kinds of bacteria in the logarithmic growth phase to a 24-well plate. Add 1 mL of longan protein with concentrations of 0 mg / mL, 3 mg / mL, 6 mg / mL, and 12 mg / mL to the bacterial suspensions of each kind of bacteria respectively, culture at 37 °C for 12 h, carefully aspirate and discard the liquid in the wells, wash the well plate with PBS buffer solution, add 1 mL of crystal violet staining solution to each well, stain for 15 min, then wash 3 times with PBS buffer solution, take pictures under a microscope, then add 1 mL of 33% acetic acid solution, shake at 50 r / min for 30 min, aspirate 200 μL of the solution, and read the value at 575 nm using an enzyme-linked immunosorbent assay (ELISA) reader.

[0054] 5. Effects of longan protein with different concentrations on the EPS production of three oral bacteria

[0055] Add 1 mL of the bacterial suspensions of 3 kinds of bacteria in the logarithmic growth phase to a 24-well plate. Add 1 mL of longan protein with concentrations of 0 mg / mL, 3 mg / mL, 6 mg / mL, and 12 mg / mL to the bacterial suspensions of each kind of bacteria respectively, culture at 37 °C for 12 h, aspirate and discard the supernatant, wash the biofilm formed at the bottom of the well with PBS buffer solution, then scrape off all the biofilm, centrifuge at 4000 r / min for 10 min, discard the supernatant after centrifugation, resuspend the precipitate with PBS solution and wash 2 - 3 times, add 1 mL of 0.4 mol / L NaOH solution to each tube to resuspend the precipitate, incubate at 37 °C for 2 h, and centrifuge at 6000 r / min for 10 min at 4 °C. Take 200 μL of the supernatant and add 600 μL of anthrone reagent, heat at 95 °C for 8 min, cool to room temperature, take 200 μL and add it to a 96-well plate, and read the absorbance value at 625 nm.

[0056] Repeat the above experiments three times for the obtained data, perform analysis of variance and multiple comparisons using SPSS 19.0 software, and P < 0.05 indicates that the difference is statistically significant.

[0057] 6. Result analysis

[0058] Analyze the effects of 3 mg / mL longan protein on the growth of the three kinds of bacteria. From Figure 2It can be seen that from 0 h to 4 h, 3 mg / mL longan protein had no significant effect on the growth of Streptococcus mutans; from 4 h to 26 h, 3 mg / mL longan protein significantly promoted its growth (P<0.05), probably because the bacteria were in the logarithmic phase and grew rapidly. From 0 to 6 h and from 22 to 26 h, 3 mg / mL longan protein had no significant effect on the growth of Staphylococcus epidermidis; from 6 to 22 h, 3 mg / mL longan protein significantly promoted its growth (P<0.05). Within 0 h to 26 h, 3 mg / mL longan protein had no significant effect on the growth of Staphylococcus saprophyticus, probably because the bacteria were in the lag phase and grew slowly. Streptococcus mutans is a common oral pathogen, Staphylococcus epidermidis is a conditional pathogen, and Staphylococcus saprophyticus is usually not pathogenic in the oral cavity. The above results indicate that longan protein can promote the growth of oral pathogens and has no significant effect on the growth of non-pathogenic bacteria.

[0059] It can be seen from Figure 3 that 3 mg / mL and 6 mg / mL longan protein can significantly promote the growth of Streptococcus mutans and Staphylococcus epidermidis (P<0.05), and their effects increase with the increase of concentration. 9 mg / mL longan protein inhibited the growth of the two bacteria, but was still higher than the control group (P<0.05), probably because the protein concentration was too high, resulting in too high cell osmotic pressure, thus causing bacterial death. Different concentrations of longan protein had no significant effect on the growth of Staphylococcus saprophyticus. The above results indicate that longan protein can significantly promote the growth of oral pathogens and conditional pathogens and has no significant effect on the growth of oral non-pathogenic bacteria.

[0060] It can be seen from Figure 4 that 3 mg / mL and 6 mg / mL longan protein significantly promoted the biofilm formation ability of Streptococcus mutans and Staphylococcus epidermidis, and the biofilm diameter increased with the increase of protein concentration (p<0.05). 9 mg / mL inhibited the biofilm formation of Streptococcus mutans, but was still significantly higher than the untreated group. Different concentrations of longan protein had no significant effect on the biofilm formation of Staphylococcus saprophyticus. It shows that longan protein with a concentration above 3 mg / mL can promote the biofilm formation of oral pathogens, but too high a concentration of longan protein will inhibit the continuous formation of biofilms, probably because the high concentration of longan protein inhibits bacterial growth, thus leading to a decrease in the ability of bacteria to form biofilms. The above results indicate that longan protein can significantly promote the biofilm formation of oral pathogens and conditional pathogens and has no significant effect on non-pathogenic bacteria.

[0061] The anthrone-sulfuric acid method was used to detect the effect of longan protein on the production of extracellular EPS by the biofilms of the three bacteria, and the results are as Figure 5As shown in the figure, longan protein at 3 mg / mL and 6 mg / mL significantly increased the amount of EPS synthesized by Streptococcus mutans and Staphylococcus epidermidis (P<0.05), and it showed an increasing trend with the increase of concentration. Longan protein at 9 mg / mL decreased the amount of EPS synthesized by the above bacteria, but there was no statistical significance, which might be due to the inhibition of bacterial growth by high-concentration longan protein, but the already synthesized EPS was still secreted into the culture medium. There was no significant difference in the amount of EPS produced by Staphylococcus saprophyticus affected by longan protein. The above results indicate that longan protein can significantly promote the production of extracellular polysaccharides by the biofilms of oral pathogenic bacteria and conditional pathogenic bacteria, and has no significant effect on non-pathogenic bacteria.

[0062] The present invention found that longan protein at 3 mg / mL and 6 mg / mL could enhance the growth ability of the oral pathogenic bacterium Streptococcus mutans and the conditional pathogenic bacterium Staphylococcus epidermidis, promote the formation of their biofilms and the secretion of extracellular polysaccharides, and showed a dose-dependent relationship. However, longan protein at 9 mg / mL had a certain inhibitory effect on the growth, biofilm formation and EPS formation of Streptococcus mutans and Staphylococcus epidermidis, which might be due to the inhibitory effect of certain components in the excessive longan protein on the growth of bacteria, thus inhibiting the production of biofilms and extracellular polysaccharides. At the same time, the present invention also found that longan protein had no significant effect on the growth, biofilm formation and EPS production of the non-pathogenic bacterium Staphylococcus saprophyticus. It was speculated that longan protein could specifically affect the growth of oral pathogenic bacteria, thus simulating the environment of the proliferation of pathogenic bacteria in the oral cavity and providing a simulation condition for the subsequent application of drugs.

[0063] (2) Effects of longan pro-inflammatory protein on intestinal pathogenic bacteria

[0064] Two C57BL / 6 mice were intragastrically administered with longan protein at 100 mg / mL·d group and 200 mg / mL·d group respectively, and a control group was set up at the same time. The culture conditions of all mice were exactly the same. After 24 hours, the feces of each mouse were collected respectively. 0.1 g of mouse feces was diluted to 10-7 under sterile conditions and coated on BBL agar, modified GAM medium, Lbs agar and Pfizer enterococcus selective agar for culture, and anaerobic culture was carried out at 36°C for 48 hours. Taking the fecal diluent of each gram of mouse (CFU / g) as the unit, the calculation formula is as follows:

[0065] The effect of longan protein on enterococci in the mouse intestine was analyzed by the spread plate method. As Figure 7 shown, compared with the control group, longan protein at 100 mg / mL·d group and 200 mg / mL·d group significantly increased the numbers of enterococci and Bacteroides in the mouse intestine (P<0.05), and significantly decreased the numbers of Lactobacillus and Bifidobacterium (P<0.01), indicating that longan protein can promote the growth of intestinal pathogenic bacteria in mice and inhibit the growth of probiotics.

[0066] (3) Effects of longan pro-inflammatory protein on cellular inflammatory factors

[0067] To analyze the effects of longan pro-inflammatory protein on the expression of cellular pro-inflammatory factors, RAW264.7 cells were treated with 0.2 mg / mL, 0.4 mg / mL, and 0.6 mg / mL longan pro-inflammatory protein for 12 h, and the expression of pro-inflammatory factors TNF-α, IL-6, and IL-8 was detected. As Figure 8 shown, after treating the cells with 0.2 mg / mL, 0.4 mg / mL, and 0.6 mg / mL longan pro-inflammatory protein for 12 h, the mRNA and protein expression levels of TNF-α, IL-6, and IL-8 in the cells increased. The above-mentioned inflammatory factors in the 0.4 mg / mL longan pro-inflammatory protein treatment group and the 0.4 mg / mL longan pro-inflammatory protein treatment group were statistically significant (P < 0.01), and their expression levels also increased with the increase in the treatment concentration, indicating that 0.4 mg / mL longan pro-inflammatory protein can promote the expression of inflammatory factors in RAW264.7 cells.

[0068] (4) Effects of longan pro-inflammatory protein on pro-inflammatory factors in blood

[0069] Male C57BL / 6 mice (20 - 22 g) were adaptively fed for 5 d under the conditions of temperature 22 ± 2°C and humidity 55 ± 5%. The mice were randomly divided into 4 groups, with 10 mice in each group, namely the blank control group, the low-dose group, and the high-dose group. The blank control group was intragastrically administered with normal saline, the low-dose group was intraperitoneally injected with 100 mg / kg·d longan protein, and the high-dose group was intraperitoneally injected with 200 mg / kg·d longan protein. The experimental period was 24 hours. Mouse serum or cells were cultured in a centrifuge tube, placed at room temperature for 10 min, and centrifuged at 3000 rpm / min for 10 min to separate the serum. The operation was carried out according to the instructions of the enzyme-linked immunosorbent assay (ELISA) kit. Using an enzyme-labeled instrument, the contents of SAA, hs-CRP, PCT, TNF-α, IL-6, and IL-8 in each sample were measured according to the standard curve.

[0070] Mouse lung tissues were fixed in 4% paraformaldehyde, routinely HE-stained, continuously sectioned with a microtome at a thickness of 4 - 6 μm, observed under a microscope, and panoramically scanned.

[0071] After intraperitoneally injecting mice with 100 mg / kg·d and 200 mg / kg·d longan protein for 24 h, the expression of inflammatory factors SAA, hs-CRP, PCT, and IL-6 in the mouse blood was detected using an ELISA detection kit. As Figure 9As shown, compared with the blank control group, intragastric administration of 100 mg / kg·d longan protein significantly increased the expression of SAA, hs-CRP, PCT, and IL-6 in the blood of mice (P<0.01). Intragastric administration of 200 mg / kg·d longan protein further increased the expression of the above inflammatory factors in mice (P<0.01), showing a dose-dependent effect.

[0072] Observation of the pathological sections of the lungs of mice stained with HE under an optical microscope revealed that, as Figure 10 can be seen, the alveolar walls in the control group were all relatively thin, with uniform size and normal structure. There was no interstitial edema, congestion, or infiltration of inflammatory cells. In the 100 mg / mL longan protein treatment group, the alveolar septum was significantly widened. Some alveolar structures were intact, with infiltration of inflammatory cells. In the 200 mg / mL longan protein treatment group, the alveolar septum was further widened, more alveolar structures were incomplete, and there was a large amount of infiltration of inflammatory cells, indicating that 100 mg / mL longan protein could cause an inflammatory response in the lungs of mice.

[0073] Finally, it should be noted that the above embodiments are only used to illustrate the technical solutions of the present invention and not to limit them. Those of ordinary skill in the art should understand that any modifications or equivalent replacements made to the technical solutions of the present invention without departing from the purpose and scope of the present technical solutions shall be covered by the scope of the claims of the present invention.

Claims

1. Application of a longan pro-inflammatory protein, characterized in that, The longan pro-inflammatory protein is used to promote the growth of pathogenic bacteria in the oral flora; the pathogenic bacteria are Streptococcus mutans and Staphylococcus epidermidis; The extraction method of the longan pro-inflammatory protein specifically includes the following steps: Step 1: After squeezing the longan pulp and filtering, perform centrifugation and collect the supernatant. After freeze-drying to reduce the volume, perform dialysis treatment at 4°C, and then freeze-dry again; Step 2: Add the powder dried in Step 1 to the pre-cooled phenol extraction buffer, and then add the pre-cooled Tris-saturated phenol solution. Shake vigorously to mix evenly, oscillate in an ice box for 2 h, and perform centrifugation at 4°C to obtain the upper liquid; Step 3: Repeat Step 2 for the upper liquid. Take the upper phenol phase, add the pre-cooled methanol solution containing ammonium acetate, let it stand at -20°C to precipitate the protein, perform centrifugation at 4°C, and take the precipitate; Step 4: Add acetone to the precipitate, mix evenly, place it at -20°C for 1 h, and then perform centrifugation at 4°C; Step 5: Repeat Step 4 twice, and then air-dry to obtain the longan pro-inflammatory protein.

2. The application according to claim 1, wherein The centrifugation treatment is centrifugation at 5000 rpm / min for 15 min.

3. The application according to claim 1, wherein In Step 2, the phenol extraction buffer is a mixed solution of 500 mM Tris-base, 63.7 mM EDTA aqueous solution, 100 mM KCl, and 700 mM sucrose, with a pH value of 7.5; 100 mg of the powder dried in Step 1 is added to 15 mL of the phenol extraction buffer solution and 15 mL of the Tris-saturated phenol solution.

4. The application according to claim 1, wherein The pre-cooling is to pre-cool to 4°C in advance.

5. The application according to claim 1, wherein The methanol solution containing ammonium acetate is 0.1 M ammonium acetate added to 35 mL of methanol.

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