Preparation of neoglycans in malva verticillata flowers and new application of neoglycans in immunoregulation

Abstract

The invention discloses neoglycans in sunflower flowers in the technical field of plant extracts, and the neoglycans are mainly prepared from rhamnose, galacturonic acid, galactose and arabinose in a molar ratio of (1.7-2.2): (1.01-1.6): (20-24): (8-12). The neoglycan is obtained by adopting a method of water extraction and alcohol precipitation of malva verticillata and separation and purification of an anion exchange column and a gel column, and an activity experiment result shows that the neoglycan has an immunoregulation effect on RAW264.7 cells stimulated by LPS and can be used for preparing immunoregulation medicines and functional foods.

Description

Technical Field

[0001] The present invention relates to the technical field of plant extracts, and in particular to the preparation of a novel polysaccharide from ocotillo flower and a novel application thereof in immunomodulation. Background Art

[0002] The human immune system is a defense network covering the whole body. It is generally divided into three lines of defense. The first line of defense is the skin, mucous membranes and their secretions, cell membranes, respiratory tract, gastrointestinal tract, urethra and kidneys. The second line of defense is phagocytosis, antimicrobial proteins and inflammatory response. The third line of defense is mainly composed of immune organs (tonsils, lymph nodes, thymus, bone marrow, and spleen, etc.) and immune cells (lymphocytes, macrophages, etc.) with the help of blood circulation and lymphatic circulation. The interaction between immune molecules and immune cells in the immune system and other systems keeps the body in a healthy state.

[0003] In fact, more and more evidence shows that Chinese herbal polysaccharides have a clear role in immunomodulation and are known as natural immunomodulators. Chinese herbal polysaccharides can directly promote the growth of immune organs and immune cells, and activate multiple signaling pathways by binding to receptors on the surface of immune cells, regulating the levels of pro-inflammatory factors, anti-inflammatory factors and chemokines in the body, thereby regulating the immune system. With the deepening of polysaccharide research, studies have found that polysaccharides can bind to specific cell surface receptors including TLR, complement receptor 3 (III type complement receptors, CR3), Dectin-1 receptor, mannose receptor (mineralocorticoid receptor, MR), scavenger receptor (scavenger receptor, SR), activate receptor downstream signaling pathways, promote the release of related cytokines and the expression of related proteins, and exert immunomodulatory effects. In addition, Ca 2+ It can also activate immune-related pathways and play an immunomodulatory role.

[0004] Malva verticillate var.crispa Linnaeus is an annual herbaceous plant of the Malva genus of the Malvaceae family. Malva verticillate is a rare vegetable with high nutritional value. It is not only edible, but its roots, flowers and seeds can also be used as medicine. Polysaccharides may be one of the main bioactive substances in Malva flowers. So far, the existing technology has not yet isolated active polysaccharides from Malva flowers. Summary of the invention

[0005] Aiming at the deficiencies of the prior art, the present invention designs a method for separating polysaccharides from ocotillo flowers and using the polysaccharides for immune regulation.

[0006] One of the purposes of the present invention is to provide a new polysaccharide from okra flowers, which is mainly composed of rhamnose, galacturonic acid, galactose and arabinose, and the molar ratios thereof are 1.7-2.2:1.01-1.6:20-24:8-12 respectively; the weight average molecular weight of the new polysaccharide is 20-80 kDa, and the dispersion coefficient D is 1-1.6.

[0007] Furthermore, the number average molecular weight (Mn) is 20 to 60 kDa.

[0008] Furthermore, the residues of the new polysaccharide have the following connection patterns: terminal-linked arabinose, with a ratio of 8-18%; terminal-linked rhamnose, with a ratio of 1-5%; 1,5-linked arabinose, with a ratio of 5-15%; 1,2-linked rhamnose, with a ratio of 0.1-2%; terminal-linked galactose, with a ratio of 8-18%; 1,3,5-linked arabinose, with a ratio of 1-5%; 1,2,4-linked rhamnose, with a ratio of 1-5%; 1,4-linked galactose, with a ratio of 8-15%; 1,3-linked galactose, with a ratio of 5-20%; 1,6-linked glucose, with a ratio of 10-30%; 1,3,6-linked galactose, with a ratio of 10-30%, and the above ratios are all based on the total amount of monosaccharides constituting the polysaccharide.

[0009] The second object of the present invention is to provide a method for preparing a novel polysaccharide in okra flowers, comprising the following steps:

[0010] 1) Soaking: Immerse the winter mallow flowers in water and soak them at room temperature for 12 to 24 hours;

[0011] 2) Decoction: Heat and keep it at a slight boil for 2 to 4 hours each time, decoct twice in total, and combine the filtrates;

[0012] 3) Dialysis: The filtrate is concentrated to 1 / 10-1 / 20 of the original volume, cooled naturally to room temperature, and the concentrate is dialyzed against running water through cellophane for 2-3 days;

[0013] 4) Alcohol precipitation: The dialysate is concentrated to 1 / 5-1 / 10 of the original volume, cooled naturally to room temperature, centrifuged at 4000-8000 rpm for 10-30 min, and the supernatant is taken and 3-6 times the volume of the supernatant is added with 95% ethanol while stirring, and allowed to stand overnight;

[0014] 5) Freeze drying: centrifuge the alcohol precipitate at 4000-8000 rpm for 10-30 min, add water to the precipitate and heat to evaporate the remaining ethanol, freeze it and freeze dry it in a freeze dryer to obtain crude polysaccharide extracted from okra flower water;

[0015] 6) Separation: The crude polysaccharide extracted from the ocher flower water was separated by DEAE anion exchange, and 0.1 M NaCl was used to elute to obtain component DF1, which was then purified by gel permeation chromatography to obtain the new polysaccharide.

[0016] Furthermore, the liquid ratio of water to okra flowers is 1:10 to 1:50.

[0017] Furthermore, the material of winter melon flowers is dried or fresh winter melon flower buds.

[0018] Furthermore, the crude polysaccharide extracted from the okra flower water in step 6) is separated by an anion exchange column DEAE FAST FLOW, and the loading amount is 30-40 g and dissolved in 500-1000 mL of deionized water.

[0019] Furthermore, the DF1 in step 6) is purified by gel column and the loading amount ranges from 100 to 200 mg and is dissolved in 2.5 to 5 mL of deionized water.

[0020] The third object of the present invention is to provide the use of new polysaccharides in the preparation of immunomodulatory drugs or functional foods.

[0021] Specifically, the new polysaccharide DF111 prepared by the present invention can regulate immunity.

[0022] Furthermore, the administration concentration of the new polysaccharide is 1 ug / mL to 200 ug / mL.

[0023] A fourth object of the present invention is to provide a pharmaceutical composition comprising the novel polysaccharide mentioned above and pharmaceutically acceptable excipients.

[0024] Working principle and beneficial effects of the present invention:

[0025] The present invention adopts the method of water extraction and alcohol precipitation, anion exchange agarose gel column (DEAE SepharoseTM FastFlow) and Sephacryl gel column separation and purification, and obtains a new polysaccharide (homogeneous polysaccharide DF111) from winter mallow flowers (dried inflorescence or flower bud of winter mallow of Malvaceae). The present invention also identifies the composition, structural characteristics and related physicochemical properties of the polysaccharide components by chemical and physical methods. In vitro experiments have shown that the homogeneous polysaccharide DF111 can significantly regulate immunomodulation activity, has almost no toxicity within a certain concentration range, and is expected to become a candidate polysaccharide drug for immunomodulation. BRIEF DESCRIPTION OF THE DRAWINGS

[0026] Figure 1 It is a schematic diagram of the HPGPC test results of DF111;

[0027] Figure 2 Schematic diagram of the monosaccharide composition analysis of DF111;

[0028] Figure 3 This is the standard curve of total sugar content;

[0029] Figure 4 It is the standard curve of protein content;

[0030] Figure 5 is the standard curve of uronic acid content;

[0031] Figure 6 Schematic diagram of the effect of different concentrations of DF111 on the survival or proliferation of RAW264.7 cells;

[0032] Figure 7 Schematic diagram of the effect of different concentrations of DF111 on the production of inflammatory factors IL-6 and TNF-α by RAW264.7 cells. DETAILED DESCRIPTION

[0033] The following is further described in detail through specific implementation methods:

[0034] 1. Polysaccharide Extraction

[0035] Soaking: Soak dried / fresh winter mallow buds in water at a ratio of 1:10 to 1:30 (g / mL) at room temperature for 12 to 24 hours;

[0036] Decoction: decoct with slight boiling for 2 to 4 hours / time, decoct twice;

[0037] Dialysis: The decoction is concentrated to 1 / 10-1 / 20 of the original volume, cooled naturally to room temperature, and dialyzed against running water through cellophane for 2-3 days;

[0038] Alcohol precipitation: The dialysate is concentrated to 1 / 5-1 / 10 of the original volume, cooled naturally to room temperature, centrifuged at 4000-8000rpm for 10-30min, and the supernatant is taken and 3-6 times the volume of the supernatant is added with 95% ethanol while stirring, and allowed to stand overnight;

[0039] Freeze-drying: centrifuge the alcohol precipitate at 4000-8000rpm for 10-30min, add 0.5-3L water to dissolve the precipitate, mix well, heat to evaporate the remaining ethanol, freeze it and freeze-dry it in a freeze dryer to obtain crude polysaccharide DF extracted from okra flower water.

[0040] Separation: The crude polysaccharide DF was separated by DEAE anion exchange, and eluted with 0.1 M NaCl to obtain component DF1. DF1 was purified by gel permeation chromatography to obtain the new polysaccharide DF111.

[0041] 2. Polysaccharide purity, monosaccharide composition and methylation analysis

[0042] 1. Determination of polysaccharide purity:

[0043] Accurately weigh 2-6 mg of the new polysaccharide DF111 from wintergreen flowers, dissolve it in 300-600 μL of 0.1 M sodium nitrate, shake it to fully dissolve the sample, centrifuge it at 4000-8000 rpm for 5-10 min, and filter the supernatant through a 0.22 μM aqueous filter membrane for purity determination.

[0044] Polysaccharide-specific gel chromatography columns in series: Shodex KS 804 (8.0 mm × 300 mm, exclusion limit 4 × 10 5 Da) and Shodex KS 802 (8.0 mm × 300 mm, exclusion limit 1 × 10 4 Da). Chromatographic conditions: mobile phase is 0.1M sodium nitrate, flow rate is 0.5mL / min, injection volume is 10-20μL, column temperature is 35℃, UV absorption wavelength is 280nm, differential detector temperature is 35℃, 40-60min / sample.

[0045] 2. Analysis of polysaccharide monosaccharide composition:

[0046] Complete acid hydrolysis: Weigh 2-4 mg of sample, dissolve in 2 mL of distilled water, vortex and oscillate to dissolve as much as possible (heating or ultrasound can be used), add the sample solution to a heart-shaped bottle (specification 50 / 19), then add 2 mL of 4M trifluoroacetic acid (TFA) and mix well, add the hollow stopper of the heart-shaped bottle, and seal the stopper and the contact port of the heart-shaped bottle with medical rubber paste, and heat and hydrolyze at 110°C for 2-5 hours. After hydrolysis, cool, add methanol and evaporate under reduced pressure for several times to remove TFA. Add 200 μL of distilled water to dissolve the hydrolyzate.

[0047] PMP derivatization process: Take 50μL of the 200μL hydrolyzate in the previous step and add it to a 2mL EP tube, add 50μL of 0.6M NaOH solution and mix well. Add 200μL of freshly prepared 0.5M PMP, seal the tube and mix well, heat in a 70℃ metal bath for 100min, cool to room temperature after the reaction, then add 100μL 0.3M HCl, and then add 600μL deionized water to make the total volume of the system 1mL.

[0048] Preparation of standard solution: Prepare 10 mg / mL of each monosaccharide with deionized water, take 100 μL of each and mix them, then you will get 10 kinds of monosaccharide standard solutions, each with a concentration of 1 mg / mL. During derivatization, take out 100 μL of the standard mixture for derivatization operation.

[0049] Extraction: Oscillate on an oscillator for 5 minutes, centrifuge at 8000 rpm for 5 minutes, and stand at room temperature for 30 minutes. Keep the upper aqueous phase, repeat the extraction with chloroform three times in the same operation, and filter the upper aqueous phase through a 0.22 μm microporous membrane.

[0050] HPLC analysis: The analytical column was a C18 reverse phase column, the mobile phase was phosphate buffer (the volume ratio of pH 7.0 phosphate buffer to acetonitrile was 169:31), the column temperature was 35°C, the flow rate was 1 mL / min, the ultraviolet absorption wavelength was 245 or 254 nm, the injection volume was 10 μL, and the detection time was 75 min / sample.

[0051] like Figure 1 As shown, the chromatographic columns Shodex KS 804 and KS 802 are connected in series, A: differential detection diagram of the new polysaccharide DF111 from okra flowers; the HPGPC purity analysis determined that the weight average molecular weight (Mw) of the new polysaccharide DF111 from okra flowers was 20-80 kDa, the number average molecular weight (Mn) was 20-60 kDa, and the dispersion coefficient D was 1-1.6.

[0052] like Figure 2 As shown, 10S: the peak order of monosaccharide composition measured by PMP pre-column derivatization of 10 monosaccharide standards; DF111: monosaccharide composition analysis of DF111, a new polysaccharide from wintergreen flower (Man: mannose; Glu: gulose; Rha: rhamnose; GlcA: glucuronic acid; GalA: galacturonic acid; Glc: glucose; Gal: galactose; Xyl: xylose; Ara: arabinose).

[0053] The monosaccharide composition analysis by PMP pre-column derivatization method showed that the new polysaccharide DF111 from Olea europaea flower contained rhamnose, galacturonic acid, galactose and arabinose, and the molar ratio was 1.70-2.20:1.01-1.60:20.00-24.00:8-12.00.

[0054] 3. Determination of physical and chemical properties:

[0055] The total sugar content was determined by the phenol-sulfuric acid method using D-glucose as the standard; the protein content was determined using the BCA Protein Assay Kit; and the uronic acid content was determined by the m-phenylphenol method using glucuronic acid as the standard.

[0056] like Figure 3 As shown in the figure, it is the standard curve of total sugar content. The total sugar content of the new polysaccharide DF111 from wintergreen flowers is calculated to be 82.51% by the formula. Figure 4 As shown in the figure, it is the standard curve of protein content. The DF111 protein content is calculated by the formula to be 0, and no protein is detected. Figure 5 The figure shows the standard curve of uronic acid content. The uronic acid content of DF111 is calculated to be 5.36% by the formula.

[0057] 4. Methylation of polysaccharides

[0058] Take the new polysaccharide DF111 from winter sunflower for methylation analysis. Weigh 5-10 mg of the sample in advance and place it in a drying cabinet overnight (to ensure that the reaction is water-free). The next day, completely dissolve it in 2 mL of DMSO, add 200 mg of ground sodium hydroxide powder, stir and react for 2 hours, add 1 mL of iodomethane dropwise in an ice-water bath within 30 minutes, and then react for 3 hours in the dark. Add 1 mL of deionized water to the reaction system to quench the reaction. Concentrate the solution under reduced pressure to remove excess unreacted CH 3 I, dialyze water for 24-72h, and freeze-dry the solution. The sample after reaction is completely acid hydrolyzed, 4mL of 2M TFA is added to react at 110℃ for 4h, methanol is added several times to remove excess acid until there is no sour taste after cooling to room temperature, 2mL of water and 50mg of sodium borohydride are added for reduction after reaction, sealed and reacted at room temperature for 3h, sodium borohydride is neutralized with 25% acetic acid solution to terminate the reaction, methanol is repeatedly added to remove excess acid, placed in a 100℃ oven to dry for 15min, and then 3mL of acetic anhydride is added to a 100℃ oven for acetylation, the reaction is carried out for 1.5h, toluene is repeatedly added to remove excess acetic anhydride, and finally 15mL of chloroform and 15mL of deionized water (v / v is 1:1) are added to the reaction bottle, extraction is performed, the aqueous phase is discarded, the organic phase is washed with deionized water 3 times, anhydrous sodium sulfate is taken for drying, the chloroform is concentrated to 1mL under reduced pressure, filtered through a 0.22μm organic phase filter membrane, and loaded into a liquid phase vial. The connection mode of polysaccharides was detected and analyzed by GC MS (Thermo Fisher ISQ7000).

[0059] The results of methylation analysis are shown in Table 1 below. The results showed that the residues in the okra polysaccharide DF111 had the following connection patterns: terminal-linked arabinose, with a ratio of 8-18%; terminal-linked rhamnose, with a ratio of 1-5%; 1,5-linked arabinose, with a ratio of 5-15%; 1,2-linked rhamnose, with a ratio of 0.1-2%; terminal-linked galactose, with a ratio of 8-18%; 1,3,5-linked arabinose, with a ratio of 1-5%; 1,2,4-linked rhamnose, with a ratio of 1-5%; 1,4-linked galactose, with a ratio of 8-15%; 1,3-linked galactose, with a ratio of 5-20%; 1,6-linked glucose, with a ratio of 10-30%; 1,3,6-linked galactose, with a ratio of 10-30%; the uronic acid content was too low to make a connection pattern; the above ratios were all based on the total amount of monosaccharides constituting the okra polysaccharide DF111.

[0060] Table 1: Methylation analysis results of ocotillosum polysaccharide DF111

[0061]

[0062] 3. Determination of immunomodulatory related activities

[0063] 1. Effects of polysaccharides on the survival or proliferation of RAW264.7 cells:

[0064] Raw264.7 cells were used as the test cell line, and the MTT method was used to detect the effect of polysaccharides on cell growth and proliferation. Twelve hours before the test, cells were cultured at 3x 10 4 The number of cells was inoculated on a 96-well plate, 100 μL per well. The control group RAW264.7 cells used RMPI-1640 complete medium with 10% FBS. The concentrations of the DF111-treated group were: 1.5625, 3.125, 6.25, 12.5, 25, 50, 100, 200 μg / mL, fully mixed in RMPI-1640 complete medium containing 10% FBS, and placed at 37°C, 5% CO 2 Incubate the cells in a constant temperature cell culture incubator for 24 hours, then gently remove and discard the cell culture supernatant, add complete medium containing 0.5 mg / mL MTT, and continue culturing for 4 hours. Then, gently remove and discard the cell culture supernatant, add 100 μL DMSO, mix thoroughly, and use an enzyme reader to detect the absorbance at 570 nm within ten minutes. MTT test results ( Figure 6 ) showed that after 24 hours of incubation, the sunflower polysaccharide DF111 had no significant toxicity to RAW264.7 cells at 1.5625, 3.125, 6.25, 12.5, and 25 μg / mL (cell survival rate was greater than 90%). At a concentration of 50 μg / mL, the cell survival rate was above 85%, and at concentrations of 100 and 200 μg / mL, the cell survival rate was less than 80%. Therefore, in subsequent studies on the effect of sunflower polysaccharide DF111 on cytokine production, the culture time was set to 24 hours, and the concentrations of sunflower polysaccharide DF111 were selected to be 3.125, 6.25, and 12.5 μg / mL.

[0065] 2. Effects of polysaccharides on the production of inflammatory factors IL-6 and TNF-α by RAW264.7 cells:

[0066] RAW264.7 cells were cultured in RMPI-1640 complete medium containing 10% FBS at a density of 1 x 10 5 The cells were inoculated with the number of cells in a 24-well plate and cultured overnight. Afterwards, the cells were treated with polysaccharide solutions of different concentrations (3.125, 6.25, 12.5 μg / mL) for 2 h, and then treated with or without LPS (1 μg / mL). The cells were incubated at 37°C with 5% CO 2 After culturing for 24 hours in a constant temperature cell culture incubator, the cell supernatant was collected and the concentrations of IL-6 and TNF-α were determined by ELISA.

[0067] like Figure 7 As shown, stimulating RAW264.7 cells with 3.125, 6.25, and 12.5 μg / mL of DF111 from wintergreen aloe vera for 24 hours did not significantly induce the inflammatory factor IL-6 ( Figure 7 Left) and TNF-α( Figure 7 After treating cells with 1 μg / mL of LPS, the production of IL-6 and TNF-α can be significantly stimulated, but the new polysaccharide DF111 from wintergreen can significantly reduce the release of inflammatory factors caused by LPS stimulation, thereby weakening the stimulating effect of LPS on cells.

[0068] The above is only an embodiment of the present invention, and the common knowledge such as the known specific structure and characteristics in the scheme is not described in detail here. It should be pointed out that for those skilled in the art, several deformations and improvements can be made without departing from the structure of the present invention, which should also be regarded as the protection scope of the present invention, and these will not affect the effect of the implementation of the present invention and the practicality of the patent. The scope of protection required by this application shall be based on the content of its claims, and the specific implementation methods and other records in the specification can be used to interpret the content of the claims.

Claims

1. New polysaccharides in winter mallow flowers, It is characterized in that It is mainly composed of rhamnose, galacturonic acid, galactose and arabinose, and the molar ratios thereof are 1.7-2.2: 1.01-1.6: 20-24: 8-12. The weight average molecular weight of the new polysaccharide is 20-80 kDa, and the dispersion coefficient D is 1-1.

6.

2. The novel polysaccharide in the flower of Malva ovata according to claim 1, Features: The residues of the new polysaccharide have the following connection patterns: terminal-linked arabinose, with a ratio of 8-18%; terminal-linked rhamnose, with a ratio of 1-5%; 1,5-linked arabinose, with a ratio of 5-15%; 1,2-linked rhamnose, with a ratio of 0.1-2%; terminal-linked galactose, with a ratio of 8-18%; 1,3,5-linked arabinose, with a ratio of 1-5%; 1,2,4-linked rhamnose, with a ratio of 1-5%; 1,4-linked galactose, with a ratio of 8-15%; 1,3-linked galactose, with a ratio of 5-20%; 1,6-linked glucose, with a ratio of 10-30%; 1,3,6-linked galactose, with a ratio of 10-30%, and the above ratios are all based on the total amount of monosaccharides constituting the polysaccharide.

3. A method for preparing new polysaccharides from okra flowers according to any one of claims 1 or 2, Features: The following steps are involved: 1) Soaking: Immerse the winter mallow flowers in water and soak them at room temperature for 12 to 24 hours; 2) Decoction: Heat and keep it at a slight boil for 2 to 4 hours each time, decoct twice in total, and combine the filtrates; 3) Dialysis: The filtrate is concentrated to 1 / 10-1 / 20 of the original volume, cooled naturally to room temperature, and the concentrate is dialyzed against running water through cellophane for 2-3 days; 4) Alcohol precipitation: The dialysate is concentrated to 1 / 5-1 / 10 of the original volume, cooled naturally to room temperature, centrifuged at 4000-8000 rpm for 10-30 min, and the supernatant is taken and 3-6 times the volume of the supernatant is added with 95% ethanol while stirring, and allowed to stand overnight; 5) Freeze drying: centrifuge the alcohol precipitate at 4000-8000 rpm for 10-30 min, add water to the precipitate and heat to evaporate the remaining ethanol, freeze it and freeze dry it in a freeze dryer to obtain crude polysaccharide extracted from okra flower water; 6) Separation: The crude polysaccharide extracted from the ocher flower water was separated by DEAE anion exchange, and eluted with 0.1 M NaCl to obtain component DF1, which was purified by gel permeation chromatography to obtain the new polysaccharide.

4. The preparation method according to claim 3, Features: The liquid ratio of water to okra flowers is 1:10 to 1:

50.

5. The preparation method according to claim 4, Features: The crude polysaccharide extracted from the okra flower water in step 6) is separated by anion exchange column DEAE FAST FLOW, and the loading amount is 30-40 g and dissolved in 500-1000 mL of deionized water.

6. The preparation method according to claim 5, Features: The DF1 in step 6) was purified by gel column and the loading amount ranged from 100 to 200 mg and dissolved in 2.5 to 5 mL of deionized water.

7. Use of the novel polysaccharide according to any one of claims 1 or 2 in the preparation of immunomodulatory drugs or compositions or functional foods.

8. The use according to claim 7, Features: The administration concentration of the new polysaccharide is 1 ug / mL to 200 ug / mL.

9. A pharmaceutical composition comprising the novel polysaccharide according to any one of claims 1 or 2, and pharmaceutically acceptable excipients.

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