Pitaya stem polysaccharide as well as preparation method and application thereof

Abstract

The invention belongs to the technical field of biological medicine, and particularly discloses a preparation method for extracting pitaya stem polysaccharide by taking pitaya stems as raw materials and application of the pitaya stem polysaccharide to regulation and control of macrophage polarization in the wound repair process to promote wound repair and inhibit the inflammatory effect. The pitaya stem polysaccharide is prepared through hot bath acid extraction of pitaya stem polysaccharide, alcohol precipitation, standing, centrifugation, redissolution, dialysis, freeze-drying and other processes. The structure of the pitaya stem polysaccharide is analyzed through various analysis methods. According to the invention, the effects of a mouse wound model and the inflammatory dragon fruit stem polysaccharide on regulating macrophage polarization, inflammatory factor level, newborn granulation level and collagen deposition level in mouse skin tissues in a wound repair process are also utilized. The results prove that the pitaya stem polysaccharide can promote the polarization of macrophages M2 / M1 in the wound skin tissue, regulate the level of inflammatory factors in the wound skin tissue and promote the deposition of new granulation and collagen in the wound skin tissue, thereby promoting the wound repair. Therefore, the dragon fruit stem polysaccharide can be applied to development and utilization of medicines related to inflammation treatment and wound repair.

Description

Technical Field

[0001] The invention belongs to the technical field of biomedicine, and specifically discloses a preparation method for extracting pitaya stem polysaccharide using pitaya stem as a main raw material, and an application of the method in regulating macrophage polarization to promote wound repair and inhibit inflammatory effects. Background Art

[0002] Pitaya (Hylocereus undatus) is a common fruit of the Cactaceae family. It is currently planted on a large scale in tropical and subtropical regions of my country. The pitaya pulp is usually eaten fresh or squeezed for drinking and contains a lot of phytonutrients. However, the development and maturation process of pitaya is usually accompanied by the rapid growth and branching of the stem. In order to ensure the growth and development of the fruit, it is generally necessary to prune the branches of the pitaya stem, leaving only the main stem. In addition, a large number of rhizomes are produced after the fruit is picked. These unused parts gradually become waste in agricultural production and are becoming a source of pollution. The recycling of these wastes is particularly important. The polysaccharides rich in pitaya stems have the advantages of sustainability, biosafety and biodegradability as plant polysaccharides, and are indispensable in production and life. Its market demand is increasing worldwide. However, due to the lack of research on active substances in pitaya stems and the lack of relevant processing technology and technology, a large number of pitaya stems are discarded, resulting in waste of resources and environmental pollution.

[0003] Wounds are acute or chronic injuries to normal skin tissue caused by various reasons, which cause serious or direct damage to the anatomical structure and function of the organ, affect the quality of life of individuals, and cause long-term life pressure and economic burden. Wound repair is affected by many factors in life, such as diet, drugs, environment, and other factors. Among them, drug intervention is the factor that has the greatest impact on wound repair. The main purpose of wound drug intervention treatment is to achieve rapid healing of wounds and leave no scars that affect the appearance. The process of skin wound healing involves complex and delicate cytological and molecular mechanisms, involving the interaction of multiple cells and cytokines. It is generally divided into four stages: hemostasis, inflammation, tissue formation and remodeling. The stages overlap and influence each other.

[0004] Macrophages are important phagocytic and antigen-presenting cells in the body. According to their functions and secreted cytokines, macrophages can be divided into M1 type (classical activation) and M2 type (selective activation). The transformation of macrophages of different phenotypes is called macrophage polarization. Macrophages participate in the entire process of wound repair, and play a vital role in regulating the polarization of macrophages M2 / M1 during wound healing. In the early stage of wound healing, pro-inflammatory M1 macrophages are dominant, which can release pro-inflammatory factors such as IL-6, IL-1β, iNOS and other mediators that help initiate the initial pro-inflammatory stage. In the late stage of wound healing, M2 macrophages are dominant, which have typical anti-inflammatory effects and can secrete a variety of growth factors and anti-inflammatory factors such as Arg-1, IL-10, etc., to reduce inflammatory responses and accelerate wound repair. According to the journal Science Translational Medicine, gradually changing the phenotype of macrophages in wounds from M1 to M2 plays an important role in dissolving inflammation, angiogenesis and re-epithelialization.

[0005] At present, the treatment methods for wounds include physical therapy, stem cell technology, cytokine therapy and gene therapy, etc. However, conventional treatments such as VSD technology have long treatment time and poor efficacy in clinical applications. Emerging treatment methods such as stem cell technology, cytokines and gene therapy are mostly in experimental exploration, and their clinical efficacy and safety need to be verified experimentally. Therefore, effective natural products with low toxicity and high economic benefits have received more and more attention. The development of natural wound repair treatment drugs and the clarification of their mechanism of action are currently urgent issues to be solved. Summary of the invention

[0006] The present invention aims to solve one of the technical problems existing in the above-mentioned existing background. To this end, the present invention proposes a preparation method and application of pitaya stem polysaccharide, aiming to establish a standardized extraction method of pitaya stem polysaccharide, analyze the physicochemical properties of pitaya stem polysaccharide, verify the role of pitaya stem polysaccharide in regulating macrophage polarization and promoting wound repair during wound repair and inhibiting inflammatory effects, in order to promote the application of pitaya stem in medicine.

[0007] The first aspect of the present invention is to provide a method for preparing pitaya stem polysaccharide.

[0008] The second aspect of the present invention is to provide pitaya stem polysaccharide prepared by the preparation method.

[0009] The third aspect of the present invention is to provide application directions of the pitaya stem polysaccharide.

[0010] The preparation method according to the first aspect of the present invention comprises the following steps: (1) Wash fresh pitaya stems, remove the outer skin fibers, and grind them into a homogenate to obtain a pitaya stem homogenate; (2) adding distilled water and dilute hydrochloric acid to the pitaya stem homogenate, and allowing to stand at room temperature to obtain a pitaya stem mixed acidic solution; (3) mixing the pitaya stem with an acid solution and performing acid extraction in a water bath; (4) centrifuging the solution obtained in step (3) and taking the supernatant; (5) Concentrating the supernatant obtained in step (4) by rotary evaporation, and adding anhydrous ethanol for precipitation; (6) Centrifuging the solution obtained in step (5), taking out the precipitate, and re-dissolving the precipitate with distilled water; (7) The reconstituted solution obtained in step (6) is dialyzed and freeze-dried to obtain pitaya stem polysaccharide.

[0011] The invention uses fresh pitaya stem as raw material and adopts processes such as acid extraction, rotary evaporation, alcohol precipitation, redissolution, dialysis and freeze drying to prepare pitaya stem polysaccharide.

[0012] In some embodiments of the first aspect of the present invention, the mass volume ratio of the pitaya stem homogenate and distilled water in step (2) is 1 g: (10-20) mL.

[0013] In some embodiments of the first aspect of the present invention, the standing condition in step (2) is: 25° C., standing for 30 minutes.

[0014] Fresh pitaya stems are rich in various enzymes. After homogenization and standing, the cell walls are initially decomposed by the enzymes in the pitaya stems, which is beneficial to the further extraction of polysaccharides.

[0015] In some embodiments of the first aspect of the present invention, in step (2), the pH value of the pitaya stem mixed acidic solution is 1-2.

[0016] In some embodiments of the first aspect of the present invention, in step (3), the process conditions of water bath acid extraction are: temperature of 80-90° C. and time of 2-3 hours.

[0017] In some embodiments of the first aspect of the present invention, in step (5), the volume ratio of the supernatant to anhydrous ethanol is 1:4.

[0018] In some embodiments of the first aspect of the present invention, in step (6), the mass volume ratio of the precipitate to the reconstituted distilled water is 1:(10-15).

[0019] In some embodiments of the first aspect of the present invention, in step (4) and / or (6), the centrifugation condition is 5000 rpm for 10 minutes.

[0020] In some embodiments of the first aspect of the present invention, in step (7), the dialysis conditions are: using a dialysis bag with a molecular weight cutoff of 3.0 kDa, placing the dialysate in the dialysis bag, and dialyzing for 48 hours at 4°C.

[0021] The pitaya stem polysaccharide described in the second aspect of the present invention is prepared by the preparation method described in the first aspect of the present invention.

[0022] Fourier transform infrared spectroscopy showed that the pitaya stem polysaccharide had an obvious polysaccharide structure signal peak: 3410 cm -1 The broad and strong absorption region near 2926 cm -1 and 2836 cm -1 The absorption peaks near 1740 cm are attributed to the stretching vibration of CH bonds, including the stretching vibration of CH, CH2, and CH3. -1 The absorption peak near 1600 cm is caused by the stretching vibration of the C=O ester group of uronic acid in the polysaccharide. This result is consistent with the results of monosaccharide composition analysis, indicating that PSP contains galacturonic acid. -1 and 1360 cm -1 The absorption peaks near 1033 cm correspond to the asymmetric and symmetric stretching vibrations of the carboxylic acid group. -1 The absorption peak near 770cm suggests the presence of a pyranose ring. -1 The nearby absorption peak is related to the stretching vibration of D-glucoside.

[0023] The application described in the third aspect of the present invention includes the use of pitaya stem polysaccharide in the preparation of drugs for regulating macrophage polarization, promoting wound repair or inhibiting inflammation.

[0024] In some application embodiments of the third aspect of the present invention, the macrophage M1 marker is CD68, the macrophage M2 marker is CD163, and the wound repair marker is inflammatory factors (IL-6, IL-1β, iNOS, Arg-1, IL-1), new granulation, and collagen deposition level.

[0025] Compared with the prior art, the above technical solution of the present invention has at least the following technical effects or advantages: 1. The present invention obtains the pitaya stem polysaccharide by crushing the fresh pitaya stem sample, acid extraction, rotary evaporation, alcohol precipitation, redissolution dialysis and freeze drying. Experiments have confirmed that the pitaya stem polysaccharide can regulate the polarization of macrophages M2 / M1 in the process of wound repair, reduce the ratio of macrophages M1 in the wound, increase the ratio of macrophages M2, reduce the expression level of pro-inflammatory factors (IL-6, IL-1β, iNOS), increase the expression of anti-inflammatory factors (Arg-1, IL-1), promote the wound from the excessive inflammation period to the proliferation and remodeling period, and promote wound repair. The new granulation and collagen deposition levels of the wound skin tissue of mice were also measured, and the results showed that the pitaya stem polysaccharide can promote cell proliferation, new blood vessel formation, granulation and collagen deposition of the wound skin tissue, and promote wound repair.

[0026] 2. The present invention provides a theoretical basis for the study of the biological activity of pitaya stem polysaccharides, which is conducive to promoting the rational development and utilization of pitaya stem resources, and is more conducive to promoting the development of pitaya stem-related drug processing and production, and provides more theoretical basis for regulating macrophage M1 / M2 polarization, regulating body inflammation, and promoting clinical treatment in wound repair. At the same time, the preparation method of the target product in the present invention is simple and easy to implement, and the target product is single and has a high yield. The results of this study can provide some theoretical basis for the development and utilization of pitaya stem waste, and have reference significance for improving the economic benefits of the pitaya stem production and processing industry. BRIEF DESCRIPTION OF THE DRAWINGS

[0027] Figure 1 is the Fourier transform infrared spectrum of pitaya stem polysaccharide prepared in Example 5; Figure 2 This is a thermogravimetric analysis diagram of pitaya stem polysaccharide prepared in Example 6; Figure 3 This is the monosaccharide composition diagram of pitaya stem polysaccharide prepared in Example 7; Figure 4 is the X-ray diffraction pattern of pitaya stem polysaccharide prepared in Example 8; Figure 5 is a scanning electron microscope image of the pitaya stem polysaccharide prepared in Example 9; Figure 6 Comparison of wound surfaces of mice on days 0, 2, 4, 6, 8, 10, 12, and 14 in Example 10; Figure 7 The figure is a bar graph of the wound healing rates of mice on days 4, 8, 10, 12 and 14 in Example 10; Figure 8 is a bar graph showing the expression level of IL-6 in mouse skin tissue in Example 11; Fig. 9is a bar graph of the expression level of IL-1β in mouse skin tissue in Example 11; Fig.10 is a bar graph showing the expression level of iNOS in mouse skin tissue in Example 11; Fig.11 is a bar graph showing the expression level of Arg-1 in mouse skin tissue in Example 11; Fig.12 is a bar graph showing the expression level of IL-10 in mouse skin tissue in Example 11; Fig.13 This is a HE-stained section of mouse skin tissue in Example 12; Fig.14 is a bar graph of the granulation tissue gaps in the mouse skin tissue in Example 12; Fig.15 This is a Masson-stained section of mouse skin tissue in Example 13; Fig.16 The immunofluorescence image of mouse skin tissue macrophage markers (M1: CD68; M2: CD163) in Example 14; Fig.17 This is a bar graph of mouse skin tissue macrophages M2 / M1 in Example 14. DETAILED DESCRIPTION

[0028] The present invention is described in detail below through examples to facilitate understanding of the present invention by persons skilled in the art. It is necessary to point out that the examples are only used to further illustrate the present invention and cannot be understood as limiting the scope of protection of the present invention. Non-essential improvements and adjustments made to the present invention by persons skilled in the art based on the above-mentioned invention content should still fall within the scope of protection of the present invention. At the same time, the raw materials mentioned below that are not described in detail are all commercially available products, and the process steps or preparation methods that are not mentioned in detail are all process steps or preparation methods known to those skilled in the art.

[0029] Example 1 The preparation method of No. 1 pitaya stem polysaccharide comprises the following steps: (1) Take fresh pitaya stems, wash them, remove the outer skin fibers, and use a grinder to grind them into a homogenate to obtain pitaya stem homogenate.

[0030] (2) Add the pitaya stem homogenate to distilled water (the mass volume ratio of the pitaya stem homogenate to distilled water is 1 g:15 mL) and dilute hydrochloric acid, and let stand at 25° C. for 30 minutes to obtain a pitaya stem mixed acidic solution (pH value is 1.5).

[0031] (3) The pitaya stem mixed with the acidic solution was placed in a water bath, maintained at 85° C. for 2.5 hours, and then centrifuged at 5000 rpm for 10 minutes to collect the supernatant.

[0032] (4) The supernatant was concentrated by rotary evaporation, 3 volumes of anhydrous ethanol were added for precipitation, and the mixture was allowed to stand at 4°C for 12 h.

[0033] (5) Centrifuge the alcohol-precipitated solution at 5000 rpm for 10 minutes, remove the lower precipitate, and add distilled water to reconstitute it. The ratio of precipitate to distilled water is 1 g:15 mL.

[0034] (6) The reconstituted solution was placed in a 3.0 KDa dialysis bag for dialyzation for 48 hours. After freeze-drying, pitaya stem polysaccharide was obtained with a wet weight yield of 1.428±0.126%.

[0035] Example 2 The preparation method of No. 2 pitaya stem polysaccharide comprises the following steps: (1) Take fresh pitaya stems, wash them, remove the outer skin fibers, and use a grinder to grind them into a homogenate to obtain pitaya stem homogenate.

[0036] (2) Add the pitaya stem homogenate to distilled water (the mass volume ratio of the pitaya stem homogenate to distilled water is 1 g:20 mL) and dilute hydrochloric acid, and let stand at 25° C. for 30 minutes to obtain a pitaya stem mixed acidic solution (pH value is 2).

[0037] (3) The pitaya stem mixed with the acidic solution was placed in a water bath, maintained at 90° C. for 3 h, and then centrifuged at 5000 rpm for 10 minutes to collect the supernatant.

[0038] (4) The supernatant was concentrated by rotary evaporation, 4 volumes of anhydrous ethanol were added for precipitation, and the mixture was allowed to stand at 4°C for 12 h.

[0039] (5) Centrifuge the alcohol-precipitated solution at 5000 rpm for 10 minutes, remove the lower precipitate, and add distilled water to reconstitute it. The ratio of precipitate to distilled water is 1 g:10 mL.

[0040] (6) The reconstituted solution was placed in a 3.0 KDa molecular weight dialysis bag and dialyzed for 48 hours. After freeze-drying, pitaya stem polysaccharide was obtained with a yield of 1.217±0.126%.

[0041] Example 3 The preparation method of No. 3 pitaya stem polysaccharide comprises the following steps: (1) Take fresh pitaya stems, wash them, remove the outer skin fibers, and use a grinder to grind them into a homogenate to obtain pitaya stem homogenate.

[0042] (2) Add the pitaya stem homogenate to distilled water (the mass volume ratio of the pitaya stem homogenate to distilled water is 1 g:10 mL) and dilute hydrochloric acid, and let stand at 25° C. for 30 minutes to obtain a pitaya stem mixed acidic solution (pH value is 3).

[0043] (3) The pitaya stem mixed with the acidic solution was placed in a water bath, maintained at 75° C. for 2 hours, and then centrifuged at 5000 rpm for 10 minutes to collect the supernatant.

[0044] (4) The supernatant was concentrated by rotary evaporation, 5 volumes of anhydrous ethanol were added for precipitation, and the mixture was allowed to stand at 4°C for 12 h.

[0045] (5) Centrifuge the alcohol-precipitated solution at 5000 rpm for 10 minutes, remove the lower precipitate, and add distilled water to reconstitute it. The ratio of precipitate to distilled water is 1 g:10 mL.

[0046] (6) The reconstituted solution was placed in a 3.0 KDa dialysis bag for dialyzation for 48 hours. After freeze-drying, pitaya stem polysaccharide was obtained with a yield of 0.959±0.126%.

[0047] Taking into account the optimal conditions for the preparation of pitaya stem polysaccharides and the differences in the yields of the prepared polysaccharides, subsequent experiments were carried out using pitaya stem polysaccharide No. 1.

[0048] Example 4 This embodiment is the determination of the Fourier Transform Infrared Spectroscopy (FTIR) of pitaya stem polysaccharides, which mainly includes the following steps: (1) Instrument preparation: Turn on, preheat, and perform self-test according to the instrument manual to ensure that the instrument is in normal condition.

[0049] (2) Parameter settings: Number of scans: 32 times; Resolution: 4.0 cm -1 ; (3) Background spectrum collection: A background spectrum should be collected before each sample spectrum collection. Place pure KBr in an infrared drying oven at 120°C for 4 hours and place it in a desiccator for later use. Wipe the mold set, mortar, and sample rack with anhydrous ethanol to ensure that their surfaces are clean. After the ethanol evaporates, collect the background spectrum.

[0050] (4) Sample spectrum collection: Pitaya stem polysaccharide and pure KBr were mixed together and ground thoroughly in a mortar. The mass ratio of pitaya stem polysaccharide to pure KBr was 1:100. The wavelength range of the FT-IR measurement was 500-4000 cm −1 Spectral data collection of pitaya stem polysaccharides.

[0051] The Fourier transform infrared spectrum of pitaya stem polysaccharide is shown in Figure 1 As shown in the figure, there is an obvious polysaccharide structure signal peak in the Fourier infrared spectrum. -1 The broad and strong absorption region near 2926 cm is caused by the stretching vibration of hydrogen bonds formed within and between hydroxyl molecules. -1 The infrared absorption near 1690 cm is attributed to the stretching vibration of the CH bond, including the stretching and bending vibration of CH, CH2, and CH3. These two groups of signals are the characteristic absorption peaks of the polysaccharide skeleton. -1 The absorption peaks near the galacturonic acid are caused by the stretching vibration of C=O on the ester group, which indicates that pitaya stem polysaccharide is an acidic polysaccharide. The asymmetric and symmetric stretching of the carboxylic acid group correspond to 1361 cm -1 The strong absorption peaks near 1180 cm -1 Weak absorption peak at 1031 cm -1 There is a peak at 891 cm, which is an asymmetric COC stretching vibration peak, indicating the presence of -O-CH3 groups in polysaccharides; -1 The characteristic absorption near indicates that the polysaccharide contains β-glycosidic bonds Example 5 This example is a thermogravimetric analysis (TGA) determination of pitaya stem polysaccharides, which mainly includes the following steps: (1) Turn on the host computer and other related instruments and introduce nitrogen.

[0052] (2) After the TG baseline is stabilized, the weight value is reset to zero. The sample of Example 1 to be tested is placed in a crucible on the sample plate, and the sample is ensured to be spread flat on the bottom of the crucible and in good contact with the bottom.

[0053] (3) The heating rate is 10 °C / min, and the detection temperature range is 30~800 °C. After setting the measurement parameters, the sample is tested.

[0054] Thermogravimetric analysis results of pitaya stem polysaccharides are shown in the figure below. Figure 2 As shown in the figure, the thermal stability of pitaya stem polysaccharides was evaluated by thermogravimetric analysis. The stability of the raw material has a profound impact on its application in industrial manufacturing. In this example, the oxidation and thermal stability of pitaya stem polysaccharides were evaluated by using the temperature fluctuation behavior of the weight obtained by TGA technology. Figure 2As shown in the figure, the TGA curve clearly shows the evolution of pitaya stem polysaccharides at different stages. It can be seen from the TGA curve that pitaya stem polysaccharides have three obvious mass changes during the high temperature process. The first weight loss occurred at 30~220℃, which may be due to the heat evaporating the water in the polysaccharide. This part of water is mainly combined with solids such as polysaccharides and proteins through hydrogen bonds. The weight loss rate of polysaccharides in this stage was 10.889%; the second weight loss occurred at 220~430℃, which may be caused by the degradation of carbohydrate long chains and the aggregation of fragments. The weight loss rate of this part was 56.616%; the third weight loss occurred after 430℃, and the mass loss was relatively slow. The reason may be that although the polysaccharide is further decomposed, the remaining polysaccharide has good thermal stability, such as some residual cellulose, or most polysaccharides have been carbonized, resulting in slow weight loss. The above research results show that jackfruit peel polysaccharides with high thermal stability have broad application prospects in functional foods, medicine and other fields.

[0055] Example 6 This example is the determination of the monosaccharide composition of pitaya stem polysaccharides, which mainly includes the following steps: (1) Hydrolysis of pitaya stem polysaccharides Take a clean chromatographic bottle, weigh an appropriate amount of polysaccharide sample, add 1 mL of 2 mol / L TFA acid solution, and heat at 121°C for 2 hours. Pass nitrogen and blow dry. Add 99.99% methanol to wash, blow dry again, and repeat the methanol washing 2 to 3 times to fully remove TFA. Add sterile water to dissolve, and transfer to the chromatographic bottle for testing.

[0056] (2) Configuration of standard products Accurately weigh the standard samples (including glucose, mannose, galactose, arabinose, rhamnose, fructose and other monosaccharides), add water to prepare 10 mg / mL standard solution stock solution, then take appropriate amount of standard stock solution to mix and prepare standard mixed standards with the highest index concentration of 60 μg / mL, 50 μg / mL or 40 μg / mL.

[0057] Experimental conditions: The chromatographic system used was the Thermo ICS 5000+ ion chromatography system (ICS 5000+, Thermo Fisher Scientific, USA), and the monosaccharide components were analyzed and detected using an electrochemical detector.

[0058] A Dionex™ CarboPac™ PA20 (150*3.0 mm, 10 μm) liquid chromatography column was used; the injection volume was 5 μL. Mobile phase A (H2O), mobile phase B (0.1 mol / L NaOH), mobile phase C (0.1 mol / L NaOH, 0.2 mol / LNaAc), flow rate 0.5 mL / min; column temperature 30°C; elution gradient: 0 min A phase / B phase / C phase (95:5:0, V / V), 26 min A phase / B phase / C phase (85:5:10, V / V), 42 min A phase / B phase / C phase (85:5:10, V / V), 42.1 min A phase / B phase / C phase (60:0:40, V / V), 52 min A phase / B phase / C phase (60:40:0, V / V), 52.1 min A phase / B phase / C phase (95:5: 0, V / V), 60 minutes of phase A / phase B / phase C (95:5:0, V / V).

[0059] The monosaccharide composition of pitaya stem polysaccharide is shown in the figure below. Figure 3 As shown, by comparison with monosaccharide standards, it is shown that pitaya stem polysaccharide is a heteropolysaccharide, mainly composed of glucose, galactose, arabinose, rhamnose, galacturonic acid and glucuronic acid, with a molar ratio of 44.16: 20.48: 16.05: 11.01: 8.01: 0.25. Among them, pitaya stem polysaccharide is mainly composed of glucose and galactose, accounting for 44.16% and 20.48% respectively.

[0060] Example 7 This example is the determination of X-ray diffraction (XRD) of pitaya stem polysaccharide, which mainly includes the following steps: (1) Sample preparation Sample grinding: The sample obtained in Example 1 was ground using a mortar until no obvious particles were present in the pitaya stem polysaccharide sample.

[0061] Tablet pressing: Use a medicine spoon to take an appropriate amount of powder sample and add it to the middle of the groove of the sample rack, so that the loose sample powder is slightly higher than the plane of the sample rack; take a glass slide and gently press the surface of the sample, and keep the entire surface even and flat and consistent with the plane of the groove when flattening.

[0062] (2) Instrument preparation Open and check the circulating water cooling system, turn on the X-ray light source, and perform the aging operation once according to the set program after starting up.

[0063] (3) Detection The operating instrument is Rigaku UltimalⅣ, and the operating conditions are (40 kV / 15 mA; 4°~70°). After setting the relevant parameters of the instrument, open the door of the sample chamber and insert the sample rack into the sample card slot, and click the test button on the computer to perform the test.

[0064] The X-ray diffraction pattern of pitaya stem polysaccharide is as follows Figure 4 As shown in the figure, dragon fruit stem polysaccharide has a broad diffusion peak between 5° and 50°, and no sharp diffraction peak. The dragon fruit stem polysaccharide sample may be mainly amorphous, and is a typical amorphous polymer in structure. That is, dragon fruit stem polysaccharide is a non-crystalline polymer.

[0065] Example 9 This example is a scanning electron microscope (SEM) determination of pitaya stem polysaccharides, which mainly includes the following steps: (1) Stick the conductive tape to the sample table, then use a sample spoon to take a small amount of the pitaya stem polysaccharide solid sample obtained in Example 1, tap or shake the sample spoon with tweezers to make the sample fall evenly on the conductive tape, then use an ear bulb to blow away the sample that is not adhered, and finally use a high-pressure air gun to blow away the sample that is not firmly adhered.

[0066] (2) Gold spraying: Place the sample to be tested in the sample chamber of the MC1000 ion sputtering instrument, set different currents and times for gold spraying as needed, and use platinum as the metal target. Finally, use SEM for observation.

[0067] SEM images of pitaya stem polysaccharides Figure 5 As shown in the figure, the scanning electron micrograph shows that the pitaya stem polysaccharide is an irregular flaky structure with an obvious pore structure, irregular edges and holes, and a relatively smooth surface. This may indicate that the polysaccharide material has a certain porosity, which may affect the adsorption performance, biocompatibility, or potential of the material as a drug delivery system. The pitaya stem polysaccharide is amorphous during the vacuum freeze-drying process. Due to its shape, the pitaya stem polysaccharide has a large specific surface area and may have better adsorption properties. The difference in the surface morphology of pitaya stem polysaccharides may be related to the molecular weight and type of the constituent monosaccharides.

[0068] Example 10 This example is a control experiment of a wound surface mouse model, which mainly includes the following contents: (1) Random grouping Six-week-old male mice (C57BL / 6 mice) weighing 20-23 g were selected. After one week of feeding (temperature 23±2℃, humidity 50±10%, light-dark cycle 12 h) to adapt to the environment, the mice were randomly divided into three groups (there was no significant difference in body weight among the groups of mice), namely, blank control group (CON group), positive control group (POS group), pitaya stem polysaccharide group (PSP group), with 20 mice in each group.

[0069] (2) Creating skin wounds The three groups of mice had their back hair cut short with electric clippers one day before skin wound preparation. Veet depilatory cream was evenly applied to the back skin preparation area. After 10 minutes, the depilatory cream was wiped off with clean wet gauze to fully expose the back skin of the mice. Each mouse was anesthetized by intraperitoneal injection of ready-to-use tribromoethanol solution at a dose of 0.1 mL / 10 g. After anesthesia took effect, a skin wound with a diameter of 10.0 mm and deep to the muscle layer was prepared using sterile surgical blades, tweezers, ophthalmic scissors and other instruments, and sterile gauze was used to compress and stop bleeding.

[0070] (3) Wound intervention and sample collection After the wounds in each group were successfully prepared, the following treatments were performed: PSP group: gauze soaked in pitaya stem polysaccharide solution (200 mg / kg, 0.1 mL per wound); POS group: 0.1 mL of recombinant bovine basic fibroblast growth factor gel (rb-bFGF) was evenly applied to each wound; CON group: gauze soaked in saline was used for wet compresses; to keep the wound moist, the back of each mouse was covered with vaseline gauze and fixed with sterile gauze and bandages. The first intervention was recorded as day 0, and the intervention was performed once a day and the dressing was changed for 14 consecutive days.

[0071] On days 0, 2, 4, 6, 8, 10, 12, and 14 of wound intervention, one mouse was randomly selected from each group as the observation object and photographed to record the wound repair status. Figure 6 shown.

[0072] On the 0th, 4th, 8th and 14th days of wound intervention, one mouse was randomly selected from each group as the observation object to take photos and record the wound repair situation. GraphPad Prism 9.0 software was used to process the wound healing rate data. The bar graph of the mouse wound healing rate is shown in Figure 2. Figure 7 shown.

[0073] On the 3rd, 7th and 14th days of wound intervention, 5 mice were randomly selected from each group as sampling objects. The details were as follows: each mouse was anesthetized by intraperitoneal injection of ready-to-use tribromoethanol solution at a dose of 0.1 mL / 10 g. After the anesthesia took effect, a skin wound tissue specimen with a diameter of about 10.0 mm deep to the muscle layer was removed from the original wound using surgical instruments. The blood scab and stains on the surface of the specimen were simply rinsed with PBS buffer, and the surface moisture was absorbed with filter paper for later use.

[0074] Embodiment 11 This example is the part of measuring the content of inflammatory factors in the wound skin tissue of each group of mice, which mainly includes the following steps: (1) Grind the wound skin tissue and pre-cooled PBS thoroughly (the mass ratio of wound skin tissue to PBS is 1:5) to obtain wound skin tissue homogenate. This process is carried out on ice. (2) using ultrasound to fully disrupt the tissue homogenate obtained in step (1); (3) Centrifuge the tissue homogenate obtained in step (2) at 5000 rpm for 10 minutes and collect the supernatant; (4) Add 300 μL of washing solution to each well of the ELISA plate and let it soak for 30 seconds. After discarding the washing solution, pat the microplate dry on absorbent paper. (5) On the ELISA plate, add the standard sample after gradient dilution to the standard sample wells; add the sample diluted with detection buffer to the sample wells; add the detection antibody (IL-6, IL-1β, iNOS, Arg-1, IL-10) to each well to be tested, seal the plate with a sealing film, shake at 200 rpm, and incubate at room temperature for 2 hours; (6) Discard the liquid, add 300 μL of washing solution to each well, and wash 6 times; (7) Add 100 μL of horseradish peroxidase-labeled streptavidin to each well, seal the plate with a new sealing film, shake at 200 rpm, and incubate at room temperature for 45 min. (8) Discard the liquid, add 300 μL of washing solution to each well, and wash 6 times; (9) Add 100 μL of chromogenic substrate to each well, protect from light, and incubate at room temperature for 20 minutes; (10) Add 100 μL of stop solution to each well to terminate the color development reaction; (11) Detection reading: Use a microplate reader for dual wavelength detection to measure the OD value at a maximum wavelength of 450 nm and a reference wavelength of 570 nm or 630 nm. The calibrated OD value is the measured value at 450 nm minus the measured value at 570 nm or 630 nm; In this example, in order to further evaluate the effect of pitaya stem polysaccharide on cytokine secretion in wound skin tissue, the expression levels of inflammatory factors (IL-6, IL-1β, iNOS, Arg-1, IL-10) in wound skin tissue on days 3 and 7 after trauma were determined using an ELISA kit. The results are as follows: Figures 8 to 12 As shown, CON is the blank control group, POS is the positive control group, and PSP is the pitaya stem polysaccharide group. The results showed that on the 7th day after trauma, the contents of pro-inflammatory factors (IL-6, IL-1β, iNOS, negatively correlated with wound repair effect) in the skin tissue of mice in the CON group were significantly increased compared with those in the POS group and the PSP group, while the contents of anti-inflammatory factors (Arg-1, IL-10, positively correlated with wound repair effect) were significantly decreased compared with those in the POS group and the PSP group. There were no significant differences in the pro-inflammatory factors (IL-6, IL-1β, iNOS) and anti-inflammatory factors (Arg-1, IL-10) between the POS group and the PSP group. The above results show that pitaya stem polysaccharides have a significant positive effect on the balance of pro-inflammatory and anti-inflammatory factors in mice during wound repair, that is, they have a promoting effect on wound repair.

[0075] Example 12 This example is the evaluation part of the granulation and epidermal formation of the wound skin tissue of each group of mice, which mainly includes the following steps: (1) Tissue fixation, dehydration, and embedding: Tissue specimens removed on days 3, 7, and 14 after intervention were fixed with 4% paraformaldehyde solution for 48 hours and placed in a dehydrator for dehydration. They were then placed in an embedding frame. The melted paraffin and xylene (1:1) were poured into the embedding frame and baked at 60°C for 1 hour. The wax blocks were then cooled in a -20°C freezer and trimmed. (2) Slicing and baking: Place the wax block on an automatic slicer, set the slice thickness to 4 μm, cut three slices intermittently, place the sliced ​​tissue on a spreader at 40°C water, flatten it, pick up the tissue with a slide, place it in a 60°C oven for baking, and when the water is dried and the wax is melted, store it at room temperature for later use; (3) Dewaxing of paraffin sections: sequentially place the sections in environmentally friendly dewaxing solution I for 20 minutes, environmentally friendly dewaxing solution II for 20 minutes, anhydrous ethanol I for 5 minutes, anhydrous ethanol II for 5 minutes, 75% alcohol for 5 minutes, and rinse with distilled water; (4) Pretreatment: Place the sections in high-definition constant stain pretreatment solution for 1 minute; (5) Hematoxylin staining: Stain the sections with hematoxylin for 3-5 minutes, wash with tap water, differentiate with differentiation solution, wash with distilled water, blue with bluing solution, and rinse with running water; (6) Eosin staining: Dehydrate the sections in 95% alcohol for 1 minute and then stain them in eosin solution for 15 seconds.

[0076] (7) Dehydration and sealing: The sections were placed in anhydrous ethanol I for 2 minutes, anhydrous ethanol II for 2 minutes, anhydrous ethanol III for 2 minutes, n-butanol I for 2 minutes, n-butanol II for 2 minutes, xylene I for 2 minutes, and xylene II for 2 minutes to make them transparent, and then sealed with neutral gum.

[0077] (7) Microscopic examination, image acquisition and analysis. The cell nucleus is blue and the cytoplasm is red.

[0078] In this example, in order to further evaluate the effect of pitaya stem polysaccharide on wound skin tissue granulation and epidermal formation, HE staining was used to evaluate the level of wound skin tissue granulation and epidermal formation on days 3, 7, and 14 after injury. The results are as follows: Figures 13 and 14 As shown, CON is the blank control group, POS is the positive control group, and PSP is the pitaya stem polysaccharide group. The results showed that the wound skin of the pitaya stem polysaccharide group (PSP) showed significantly higher epidermal and granulation structures than the control group (CON), and there was no significant difference between the pitaya stem polysaccharide group (PSP) and the positive control group (POS). Specifically, on the third day, the PSP group and the POS group began to form an epidermal layer, while no new epithelial tissue was observed in the wounds of the CON group. On the third day, the PSP group and the POS group formed a full length of epidermal layer, while less epithelial re-formation was observed in the wounds of the CON group. On the 14th day, the PSP group and the POS group were able to tightly connect the regenerated dermis and fill the appendages under the fully healed epithelial cambium, while the control group still had a large unhealed wound area with a thin granulation structure. The above results show that pitaya stem polysaccharides can effectively improve granulation tissue formation and epithelial tissue regeneration, thereby significantly accelerating skin wound healing and promoting wound repair.

[0079] Embodiment 13 This example is a part of evaluating collagen deposition in the wound skin tissue of each group of mice, which mainly includes the following steps: (1) Tissue fixation, dehydration, and embedding: Tissue specimens removed on days 3, 7, and 14 after intervention were fixed with 4% paraformaldehyde solution for 48 h and then placed in a dehydrator for dehydration. The specimens were then placed in an embedding frame. The melted paraffin and xylene (1:1) were poured into the embedding frame and baked at 60°C for 1 h. The wax blocks were then cooled in a -20°C freezer and trimmed. (2) Slicing and baking: Place the wax block on an automatic slicer, set the slice thickness to 4 μm, cut three slices intermittently, place the sliced ​​tissue on a slide spreader at 40°C, pick up the tissue with a glass slide, place it in a 60°C oven for baking, and store it at room temperature until the water is dried and the wax is melted; (3) Dewaxing of paraffin sections: sequentially place the sections in environmentally friendly dewaxing solution I for 20 minutes, environmentally friendly dewaxing solution II for 20 minutes, anhydrous ethanol I for 5 minutes, anhydrous ethanol II for 5 minutes, 75% alcohol for 5 minutes, and then wash with distilled water; (4) Soak the sections in Masson A solution overnight and rinse with distilled water; (5) Slice into a dye solution of Masson B solution and Masson C solution in equal proportions, stain for 1 minute, wash with distilled water, differentiate with differentiation solution for a few seconds, and wash with distilled water; (6) Immerse the sections in Masson D solution for 6 minutes and rinse with distilled water; (7) Immerse in Masson E solution for 1 minute; (8) Without washing, drain slightly and directly put into Masson F solution for 2 to 30 seconds; (9) The sections were rinsed and differentiated with 1% acetic acid and dehydrated with two cylinders of anhydrous ethanol; (10) Transparent sealing: Place the slices in anhydrous ethanol III for 5 minutes, then in xylene for 5 minutes to make them transparent, and then seal the slices with neutral gum.

[0080] (11) Microscopic examination, image acquisition and analysis. Collagen fibers appear blue; muscle fibers, cellulose and red blood cells appear red.

[0081] In this example, in order to further evaluate the effect of pitaya stem polysaccharide on collagen deposition in wound skin tissue, Masson staining was used to evaluate the level of collagen deposition in wound skin tissue on days 3, 7, and 14 after trauma. The results are as follows: Fig.15 As shown, CON is the blank control group, POS is the positive control group, and PSP is the pitaya stem polysaccharide group. The results showed that on the 7th day, compared with the CON group, the wounds in the POS group and the PSP group were covered with a darker blue, indicating that collagen deposition was maximally enhanced in the wounds treated with pitaya stem polysaccharides, resulting in thicker wound granulation formation and smaller wound gaps. On the 14th day, granulation tissue formation and collagen deposition were more obvious. The above results show that pitaya stem polysaccharides can effectively improve granulation tissue formation and collagen deposition, thereby significantly accelerating skin wound healing and promoting wound repair.

[0082] Embodiment 14 This example is the determination part of the M1 macrophage marker CD68 and the M2 macrophage marker CD163 in the wound skin tissue of each group of mice, which mainly includes the following steps: (1) Tissue fixation, dehydration, and embedding: Tissue specimens removed on days 3, 7, and 14 after intervention were fixed with 4% paraformaldehyde solution for 48 hours and placed in a dehydrator for dehydration. They were then placed in an embedding frame. The melted paraffin and xylene (1:1) were poured into the embedding frame and baked at 60°C for 1 hour. The wax blocks were then cooled in a -20°C freezer and trimmed.

[0083] (2) Slicing and baking: Place the wax block on an automatic slicer, set the slice thickness to 4 μm, cut three slices intermittently, place the sliced ​​tissue on a spreader with 40°C water, spread it flat, pick up the tissue with a glass slide, place it in a 60°C oven for baking, and when the water is dried and the wax is melted, store it at room temperature for later use.

[0084] (3) Dewaxing of paraffin sections: Place the sections in environmentally friendly dewaxing solution I for 20 minutes, environmentally friendly dewaxing solution II for 20 minutes, anhydrous ethanol I for 5 minutes, anhydrous ethanol II for 5 minutes, 75% alcohol for 5 minutes, and rinse with distilled water.

[0085] (4) Elimination of endogenous peroxidase activity: Incubate in 30 mL / L H2O2 at room temperature for 10 minutes, then wash with distilled water.

[0086] (5) Antigen retrieval: Perform antigen retrieval using the microwave method and wash with PBS for 5 minutes.

[0087] (6) Circle serum blocking: After the slices are slightly dried, use a histology pen to draw circles around the tissue, add BSA, and block for 30 minutes.

[0088] (7) Add primary antibody: Add the prepared primary antibody dropwise and incubate the sections flat in a humidified chamber at 4°C overnight.

[0089] (8) Adding secondary and tertiary antibodies: Place the slide in PBS (pH 7.4) and shake on a decolorizing shaker for 3 times, 5 minutes each time. Add the corresponding secondary antibody and incubate at room temperature in the dark for 50 minutes. After drying the slices with filter paper, add tertiary antibodies to the circle and incubate at room temperature for 50 minutes. Then, gently shake and wash in PBS buffer for 3 times, 8 minutes each time.

[0090] (9) DAPI counterstaining of cell nuclei: Place the slide in PBS (pH 7.4) and wash on a decolorizing shaker three times, 5 minutes each time. Add DAPI staining solution and incubate at room temperature for 10 minutes in the dark.

[0091] (10) DAB color development: Place the slices in PBS buffer and gently shake them on a decolorizing shaker for 3 times, 5 minutes each time. After drying the slices with filter paper, add an appropriate amount of DAB color development solution in the circle. Control the color development time under a microscope until the image is clear, and rinse the slices with running water to stop the color development.

[0092] (11) Dehydration and sealing: The sections were placed in anhydrous ethanol I for 2 minutes, anhydrous ethanol II for 2 minutes, anhydrous ethanol III for 2 minutes, n-butanol I for 2 minutes, n-butanol II for 2 minutes, xylene I for 2 minutes, and xylene II for 2 minutes until transparent, and then sealed with neutral gum.

[0093] (12) Microscopic examination, image acquisition and analysis. DAPI-stained cell nuclei are blue, M1 macrophage marker CD68 is red, and M2 macrophage marker CD163 is green.

[0094] In this example, in order to further evaluate the effect of pitaya stem polysaccharide on the polarization of M1 / M2 macrophages in wound skin tissue, the M1 macrophage marker CD68 and M2 macrophage marker CD163 were used to measure the M1 / M2 polarization level of wound skin tissue macrophages on the 7th day after trauma. The results of immunofluorescence assay of M1 and M2 macrophage markers CD68 and CD163 in the wound skin tissue of mice are shown in Figure 2. Fig.16 As shown, CON is the blank control group, POS is the positive control group, and PSP is the pitaya stem polysaccharide group. The results showed that the expression of CD163 in the CON group was significantly lower than that in the POS group, and the expression of CD163 in the PSP group was slightly increased compared with the POS group, but there was no significant difference between the POS group and the PSP group; the expression of CD68 in the CON group was slightly increased compared with the POS group, but there was no significant difference between the POS group and the PSP group.

[0095] The expression level ratio of M1 and M2 macrophage markers CD68 and CD163 in mouse skin tissue (M2 / M1) is shown in the following figure. Fig.17 As shown, CON is the blank control group, POS is the positive control group, and PSP is the pitaya stem polysaccharide group. The results showed that compared with the CON group, the level of M2 / M1 macrophages in the skin tissue of mice in the POS group was significantly increased, with a very significant difference, and there was no significant difference between the POS group and the PSP group (****P<0.0001, ***P<0.001, **P<0.01, *P<0.05).

[0096] The above results show that pitaya stem polysaccharides have a regulatory effect on the expression levels of M1 and M2 macrophage markers CD68 and CD163 in colon tissue, can promote the transformation of M1 macrophages in mouse skin tissue into M2, increase the M2 / M1 ratio, reduce the release of inflammatory factors, and thus promote the repair of wound tissue.

Claims

1. A method for preparing pitaya stem polysaccharide, characterized in that: The following steps are involved: (1) Wash fresh pitaya stems, remove the outer skin fibers, and grind them into a homogenate to obtain a pitaya stem homogenate; (2) adding distilled water and dilute hydrochloric acid to the pitaya stem homogenate, and allowing to stand at room temperature to obtain a pitaya stem mixed acidic solution; (3) mixing the pitaya stem with an acid solution and performing acid extraction in a water bath; (4) centrifuging the solution obtained in step (3) and taking the supernatant; (5) Concentrating the supernatant obtained in step (4) by rotary evaporation, and adding anhydrous ethanol for precipitation; (6) Centrifuging the solution obtained in step (5), taking out the precipitate, and re-dissolving the precipitate with distilled water; (7) The reconstituted solution obtained in step (6) is dialyzed and freeze-dried to obtain pitaya stem polysaccharide.

2. The preparation method according to claim 1, characterized in that: In step (2), the mass volume ratio of the pitaya stem homogenate to distilled water is 1 g: (10-20) mL; and the standing condition is: 25° C. for 30 minutes.

3. The preparation method according to claim 1, characterized in that: In step (2), the pH value of the pitaya stem mixed acidic solution is 1-2.

4. The preparation method according to claim 1, characterized in that: In step (3), the process conditions of water bath acid extraction are: temperature of 80-90°C and time of 2-3 hours.

5. The preparation method according to claim 1, characterized in that: In step (5), the volume ratio of the supernatant to anhydrous ethanol is 1:

4.

6. The preparation method according to claim 1, characterized in that: In step (6), the mass volume ratio of the precipitate to the reconstituted distilled water is 1:(10-15).

7. The preparation method according to claim 1, characterized in that: In step (4) and / or (6), the centrifugation condition is 5000 rpm for 10 minutes.

8. The preparation method according to claim 1, characterized in that: In step (7), the dialysis conditions are as follows: using a dialysis bag with a molecular weight cutoff of 3.0 kDa, placing the dialysate in the dialysis bag, and dialyzing at 4°C for 48 hours.

9. A pitaya stem polysaccharide, characterized in that: Prepared by the preparation method according to any one of claims 1 to 8.

10. Use of the pitaya stem polysaccharide according to claim 9 in the preparation of drugs for regulating macrophage polarization, promoting wound repair or inhibiting inflammation.

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