A polysaccharide from momordica grosvenori swingle root and a preparation method and application thereof

Abstract

The application provides a momordica root polysaccharide and a preparation method and application thereof, and belongs to the technical field of natural product development. The preparation method of the momordica root polysaccharide comprises the following steps: drying and crushing momordica roots, and removing fat by ethanol reflux; after water extraction and alcohol precipitation, and removing protein, a momordica root crude polysaccharide is obtained; and the momordica root crude polysaccharide is sequentially separated and purified by DEAE-52 cellulose column and Sephadex LH20 gel column to obtain the momordica root refined polysaccharide. The preparation method has the advantages that the polysaccharide obtained by directly extracting water in the prior art has a narrow molecular weight distribution range and better immune enhancement activity. Experimental results show that the momordica root polysaccharide can stimulate RAW264.7 cells to secrete NO, TNF-alpha and IL-6 factors, enhance the phagocytosis of RAW264.7 cells, and thus enhance the cell immune function, and can be applied to the development of products for improving immunity.

Description

TECHNICAL FIELD

[0001] The present application belongs to the technical field of natural product development, and particularly relates to a momordica grosvenori root polysaccharide and a preparation method and application thereof. BACKGROUND

[0002] Momordica grosvenori is recorded in Lingnanshaoyao Lu, is a plant of Momordica genus of Cucurbitaceae, mainly distributed in the southwest of China, is a local medicinal material in Guangxi, and was listed as a Chinese medicinal material with the same origin of food and medicine by the State Health Department in 1987. The fruit is a commonly used medicinal part, and has the effects of relieving cough and reducing sputum. The tuberous root of momordica grosvenori is large, bitter in taste and slightly cold in nature, and can be used as medicine in folk, has the effects of dampness and diarrhea, dredging collaterals and pain relief, anti-inflammatory, enzyme reduction, etc. However, in the process of planting, a large amount of roots are discarded, causing great waste of resources.

[0003] The tuberous root of momordica grosvenori is large, bitter in taste and slightly cold in nature, and has the effects of dampness and diarrhea, dredging collaterals and pain relief, anti-inflammatory, enzyme reduction, etc. However, there are few reports on the improvement of immunity of momordica grosvenori root. SUMMARY

[0004] Therefore, the present application aims to provide a momordica grosvenori root polysaccharide and a preparation method and application thereof, and the prepared momordica grosvenori root polysaccharide has the effect of enhancing immunity.

[0005] In order to achieve the above-mentioned application purposes, the present application provides the following technical solutions:

[0006] The present application provides a preparation method of a momordica grosvenori root crude polysaccharide LGP, comprising the following steps: drying and crushing the momordica grosvenori root, and removing fat by ethanol reflux, and then obtaining the residue, and then obtaining the momordica grosvenori root crude polysaccharide LGP after water extraction and alcohol precipitation and removing protein.

[0007] Preferably, the solvent for removing fat by ethanol reflux is an ethanol solution with a volume percentage of 95%, the time is 2h, and the number of times is 2; and the mass ratio of the momordica grosvenori root to the ethanol solution is 1:10-1:20.

[0008] Preferably, in the water extraction and alcohol precipitation, the solid-liquid ratio of the water extraction is 1:20-1:40, the temperature is 70-90℃, the time is 3-5h, and the number of times is 2.

[0009] Preferably, in the alcohol precipitation reaction of the water extraction and alcohol precipitation, the volume percentage of ethanol is 60%-80%, and the alcohol precipitation is carried out overnight at 4℃.

[0010] The present application also provides a preparation method of a momordica grosvenori root refined polysaccharide LGP-A, comprising: sequentially separating and purifying the above-mentioned momordica grosvenori root crude polysaccharide LGP through a DEAE-52 cellulose column and a Sephadex LH20 gel column, and then obtaining the momordica grosvenori root refined polysaccharide LGP-A after freeze-drying.

[0011] Preferably, the DEAE-52 cellulose column is eluted with water and sodium chloride with concentrations of 0.05M, 0.1M, 0.15M, 0.3M and 0.5M in sequence; and the eluent of the Sephadex LH20 gel column is 25% ethanol solution by volume.

[0012] The application further provides the crude polysaccharide LGP obtained by the preparation method.

[0013] The application further provides the refined polysaccharide LGP-A obtained by the preparation method.

[0014] Preferably, the refined polysaccharide LGP-A of the momordica root comprises arabinose and galactose, and has a molecular weight of 1.41x10 3 kDa.

[0015] The application further provides the use of the crude polysaccharide LGP or the refined polysaccharide LGP-A in the preparation of an immune-enhancing product.

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

[0017] The application provides a preparation method of polysaccharide of momordica root, comprising: drying and crushing the momordica root, and performing ethanol reflux degreasing to obtain residues; the residues are subjected to water extraction and alcohol precipitation, and then protein is removed to obtain crude polysaccharide LGP of the momordica root; and the crude polysaccharide LGP of the momordica root is sequentially subjected to DEAE-52 cellulose column separation and Sephadex LH20 gel column purification to obtain the refined polysaccharide LGP-A of the momordica root. The polysaccharide obtained by the preparation method of the application has a narrow molecular weight distribution range and better immune-enhancing activity compared with the polysaccharide obtained by direct water extraction in the prior art. The polysaccharide of the momordica root obtained by further extraction of the momordica root after degreasing has the function of enhancing human immune regulation, and can be applied to the preparation of an immune-enhancing product, so that the momordica root resources can be fully utilized and resource waste can be avoided.

[0018] The experimental results show that the polysaccharide of the momordica root of the application can stimulate RAW264.7 cells to secrete factors such as NO, TNF-α and IL-6, enhance the phagocytic ability of RAW264.7 cells, and thus enhance the cellular immune function, and can be applied to the development of an immune-enhancing product. BRIEF DESCRIPTION OF DRAWINGS

[0019] Figure 1 : Liquid chromatogram of the crude polysaccharide LGP of the momordica root and CPS;

[0020] Figure 2 : DEAE-52 cellulose column chromatography purification elution curve (A) of the crude polysaccharide of the momordica root, and Sephadex LH-20 elution curve (B) of the polysaccharide of the momordica root;

[0021] Figure 3 LGP-A UV full wavelength scan (A) and molecular weight distribution (B);

[0022] Figure 4 LGP-A monosaccharide composition analysis (A. blank control; B. monosaccharide standard; C. LGP-A; 1. PMP; 2. Mannose; 3. Ribose; 4. Glucuronic acid; 5. Rhamnose; 6. Galacturonic acid; 7. Glucose; 8. Galactose; 9. Arabinose);

[0023] Figure 5 LGP-A Fourier transform infrared spectrum;

[0024] Figure 6 LGP-A NMR analysis (A. 1 H NMR; B. 13 C NMR; C. 1 H- 1 H COSY; D. HMQC);

[0025] Figure 7 LGP-A Congo red test;

[0026] Figure 8 LGP-A scanning electron microscope image (top left. Magnification 200 times; top right. Magnification 1000 times; bottom left. Magnification 600 times; bottom right. Magnification 2000 times);

[0027] Figure 9 LGP-A on RAW264.7 cell proliferation experiment results;

[0028] Figure 10 LGP-A on RAW264.7 cell phagocytosis experiment results;

[0029] Figure 11 LGP-A on RAW264.7 cell NO, TNF-α and IL-6 secretion amount. DETAILED DESCRIPTION

[0030] The present application provides a preparation method of crude polysaccharide LGP from momordica root, comprising the following steps: drying and crushing momordica root, defatting by ethanol reflux, and then obtaining the residue, and then water extraction and alcohol precipitation, and then removing protein to obtain crude polysaccharide LGP from momordica root.

[0031] The solvent for ethanol reflux degreasing in this invention is preferably an ethanol solution with a volume percentage of 95%, the time is preferably 2 hours, and the number of times is preferably 2; the mass ratio of the monk fruit root to ethanol is preferably 1:10 to 1:20, more preferably 1:15; the temperature of ethanol reflux degreasing is about 75°C (the temperature at which the 95% ethanol solution is boiled).

[0032] In the water extraction and alcohol precipitation process of the present invention, the preferred material-to-liquid ratio is 1:20 to 1:40, more preferably 1:30; the preferred temperature is 70 to 90°C, more preferably 80°C; the preferred time is 3 to 5 hours, more preferably 4 hours; and the preferred number of times is 2. In the alcohol precipitation reaction of the water extraction and alcohol precipitation process, the preferred volume percentage of ethanol is 60% to 80%, more preferably 70%, and the alcohol precipitation is carried out overnight at 4°C.

[0033] The present invention also provides a method for preparing refined polysaccharide LGP-A from monk fruit root, comprising: separating and purifying the above-mentioned crude polysaccharide LGP from monk fruit root sequentially through a DEAE-52 cellulose column and a Sephadex LH20 gel column, and then freeze-drying it to obtain the refined polysaccharide LGP-A from monk fruit root.

[0034] The DEAE-52 cellulose column of this invention preferably elutes sequentially with water and sodium chloride at concentrations of 0.05M, 0.1M, 0.15M, 0.3M, and 0.5M. The preferred flow rate is 1 mL / min, with collection every 7 minutes. The elution curve obtained by the phenol-sulfuric acid method shows a clear and well-shaped absorption peak in the 0.3M elution fraction. This fraction is combined, concentrated under reduced pressure, dialyzed (molecular weight cutoff 1000), and freeze-dried to obtain the 0.3M eluted sample. DEAE-52 cellulose, as a basic anion exchanger, can separate acidic and neutral polysaccharides from crude polysaccharides.

[0035] The 0.3M elution sample was dissolved in water and purified using a Sephadex LH20 column (Ф1.4cm×65cm). Eluent was used for elution at a flow rate of 0.5mL / min. The eluents were combined, concentrated, and freeze-dried to obtain *Siraitia grosvenorii* root polysaccharide. The purified sample was preferably freeze-dried at -80℃ for 24–48 h. The eluent for the Sephadex LH20 gel column was preferably a 25% (v / v) ethanol solution. The Sephadex LH20 column can remove small molecule compounds from the polysaccharide, allowing for further purification of the crude *Siraitia grosvenorii* root polysaccharide (LGP) according to its molecular weight.

[0036] The preparation method of the present invention has a narrower molecular weight distribution range and better immune-enhancing activity compared with the polysaccharide obtained by direct water extraction in the prior art.

[0037] The present invention also provides crude polysaccharide LGP from the root of *Siraitia grosvenorii* obtained by the above preparation method.

[0038] The present invention also provides refined polysaccharide LGP-A from the root of Siraitia grosvenorii obtained by the above preparation method.

[0039] The refined polysaccharide LGP-A from monk fruit root of this invention comprises arabinose and galactose, with a molecular weight of 1.41 × 10⁻⁶. 3 kDa.

[0040] This invention also provides the application of the above-mentioned crude polysaccharide LGP from Luo Han Guo root or the above-mentioned refined polysaccharide LGP-A from Luo Han Guo root in the preparation of immune-enhancing products.

[0041] Experimental results show that the Siraitia grosvenorii root polysaccharide of the present invention can stimulate RAW264.7 cells to secrete factors such as NO, TNF-α, and IL-6, enhance the phagocytic capacity of RAW264.7 cells, thereby enhancing cellular immune function, and can be applied to the development of products that improve immunity.

[0042] The root of *Siraitia grosvenorii*, a plant belonging to the genus *Siraitia* of the Cucurbitaceae family, was collected from Guilin City, Guangxi Zhuang Autonomous Region. Reference standards included D-mannose (batch number MUST-23012802, mass fraction 99.52%), D-ribose (batch number MUST-23021307, mass fraction 98.00%), D-glucuronic acid (batch number MUST-23021309, mass fraction 98.00%), rhamnose (batch number MUST-22061104, mass fraction 99.99%), D-galacturonic acid (batch number MUST-23021308, mass fraction 98.00%), D(+)-anhydrous glucose (batch number MUST-22030214, mass fraction 99.61%), and D-galactose (batch number MUST-, mass fraction 9...). 8.00% ), DL-arabinose (batch number MUST-22022819, mass fraction 98.25%), and dextran standard (batch number 140637~646-201203) were all purchased from the National Institutes for Food and Drug Control; CCK-8, tumor necrosis factor-α (TNF-α), and interleukin-6 (IL-6) detection kits were purchased from Elabscience; nitric oxide (NO) detection kit (Shanghai Beyotime Biotechnology Co., Ltd.), HPD100 macroporous adsorption resin (Cypress (Beijing) Technology Co., Ltd.); DEAE-52 cellulose (Shanghai Yuanye Biotechnology Co., Ltd.); Sephadex LH20 gel (GE Healthcare, USA); MD77 dialysis bags (Scientific Research Specialty Pharmaceuticals, USA); all other reagents were of analytical grade.

[0043] Waters 515 HPLC system (equipped with a Waters 2410 differential detector, Waters Corporation); LC-2030C HPLC system (Shimadzu Corporation, Japan); Nicolet Fourier transform infrared spectrometer (Therom Fisher Scientific, USA); Brucker Avance 500MHz superconducting nuclear magnetic resonance spectrometer (Brucker GmbH, Germany); SW00101-4N401812 laboratory equipment (Tongzhou Zongheng (Xiamen) Fluid Technology Co., Ltd.); ALPHA1-2LD PLU freeze dryer (Beijing Bomeixing Instrument Co., Ltd.); T6 New Century UV-Vis spectrophotometer (Beijing Puxi General Instrument Co., Ltd.); Zeiss EVO18 scanning electron microscope (Zeiss GmbH, Germany); Spark microplate reader (Tecan GmbH, Switzerland); BSP-150 biochemical incubator (Shanghai Boxun Medical Biological Instrument Co., Ltd.); 5% CO2 cell incubator (Thermo Fisher Scientific, USA). Forma (Forma Corporation); Dmi1 inverted phase contrast microscope (Leica Corporation, USA).

[0044] The technical solutions provided by the present invention will be described in detail below with reference to the embodiments, but they should not be construed as limiting the scope of protection of the present invention.

[0045] Example 1

[0046] Preparation of crude polysaccharide LGP and refined polysaccharide LGP-A from monk fruit root

[0047] (1) Degreasing: Take 500g of dried monk fruit root, dry and crush it, add 10 times the volume of 95% ethanol solution, heat and reflux at 75℃ for 2 hours to degrease, repeat twice to obtain monk fruit root residue.

[0048] (2) Water extraction and alcohol precipitation: After drying the residue of Luo Han Guo root, add 30 times the amount of water and heat and extract in a water bath at 70℃ (4h, 2 times); after filtering the extract, combine the filtrates, concentrate under vacuum at 55℃ to 1 / 4 of the total volume, decolorize through AB-8 macroporous resin, concentrate the macroporous resin by washing with water, add 95% ethanol solution until the ethanol volume percentage reaches 70%, let stand overnight at 4℃, centrifuge the precipitated material at 4000r / min for 10min, dissolve the centrifuged precipitate in water, remove protein 5 times by sevage method to obtain Luo Han Guo root crude polysaccharide LGP;

[0049] (3) DEAE-52 cellulose column separation: Take the crude polysaccharide from the root of Siraitia grosvenorii and place it on a DEAE-52 cellulose column (Ф2.1cm×42cm). Elute with water, 0.05M, 0.1M, 0.15M, 0.3M and 0.5M sodium chloride in sequence. The flow rate is 1mL / min. Collect the 0.3M fraction for 7min / tube. Concentrate under reduced pressure, dialyze (molecular weight cutoff 1000), and freeze dry to obtain the 0.3M eluted sample.

[0050] (4) Sephadex LH20 gel column purification: The sample was dissolved in water and purified by passing it through a Sephadex LH20 column (Ф1.4cm×65cm). It was eluted with 25% ethanol solution at a flow rate of 0.5mL / min. The solutions were combined, concentrated, and freeze-dried at -80℃ for 48h to obtain refined polysaccharide LGP-A from Luo Han Guo root.

[0051] Example 2

[0052] Preparation of crude polysaccharide LGP and refined polysaccharide LGP-A from monk fruit root

[0053] (1) Degreasing: Take 500g of dried monk fruit root, dry and crush it, add 10 times the volume of 95% ethanol solution, heat and reflux at 75℃ for 2 hours to degrease, repeat twice to obtain monk fruit root residue.

[0054] (2) Water extraction and alcohol precipitation: After drying the residue of Luo Han Guo root, add 20 times the amount of water and heat and extract in a water bath at 70℃ (3h, 2 times); after filtering the extract, combine the filtrates, concentrate under vacuum at 55℃ to 1 / 4 of the total volume, decolorize through AB-8 macroporous resin, concentrate the macroporous resin by washing with water, add 95% ethanol solution by volume until the ethanol volume percentage reaches 60%, let stand overnight at 4℃, centrifuge the precipitate at 4000r / min for 10min, dissolve the centrifuged precipitate in water, remove protein 5 times by sevage method to obtain Luo Han Guo root crude polysaccharide LGP;

[0055] (3) DEAE-52 cellulose column separation: Take the crude polysaccharide from the root of Siraitia grosvenorii and place it on a DEAE-52 cellulose column (Ф2.1cm×42cm). Elute with water, 0.05M, 0.1M, 0.15M, 0.3M and 0.5M sodium chloride in sequence. The flow rate is 1mL / min. Collect the 0.3M fraction for 7min / tube. Concentrate under reduced pressure, dialyze (molecular weight cutoff 1000), and freeze dry to obtain the 0.3M eluted sample.

[0056] (4) Sephadex LH20 gel column purification: The sample was dissolved in water and purified by passing it through a Sephadex LH20 column (Ф1.4cm×65cm). It was eluted with 25% ethanol solution at a flow rate of 0.5mL / min. The solutions were combined, concentrated, and freeze-dried at -80℃ for 48h to obtain refined polysaccharide LGP-A from Luo Han Guo root.

[0057] Example 3

[0058] Preparation of crude polysaccharide LGP and refined polysaccharide LGP-A from monk fruit root

[0059] (1) Degreasing: Take 500g of dried monk fruit root, dry and crush it, add 20 times the volume of 95% ethanol solution, heat and reflux at 75℃ for 2 hours to degrease, repeat twice to obtain monk fruit root residue.

[0060] (2) Water extraction and alcohol precipitation: After drying the residue of Luo Han Guo root, add 40 times the amount of water and heat and extract in a 90℃ water bath (5h, 2 times); after filtering the extract, combine the filtrates, concentrate under vacuum at 55℃ to 1 / 4 of the total volume, decolorize through AB-8 macroporous resin, concentrate the macroporous resin by washing with water, add 95% ethanol solution until the ethanol volume percentage reaches 80%, let stand overnight at 4℃, centrifuge the precipitate at 4000r / min for 10min, dissolve the centrifuged precipitate in water, remove protein 5 times by sevage method to obtain Luo Han Guo root crude polysaccharide LGP;

[0061] (3) DEAE-52 cellulose column separation: Take the crude polysaccharide from the root of Siraitia grosvenorii and place it on a DEAE-52 cellulose column (Ф2.1cm×42cm). Elute with water, 0.05M, 0.1M, 0.15M, 0.3M and 0.5M sodium chloride in sequence. The flow rate is 1mL / min. Collect the 0.3M fraction for 7min / tube. Concentrate under reduced pressure, dialyze (molecular weight cutoff 1000), and freeze dry to obtain the 0.3M eluted sample.

[0062] (4) Sephadex LH20 gel column purification: The sample was dissolved in water and purified by passing it through a Sephadex LH20 column (Ф1.4cm×65cm). It was eluted with 25% ethanol solution at a flow rate of 0.5mL / min. The solutions were combined, concentrated, and freeze-dried at -80℃ for 48h to obtain refined polysaccharide LGP-A from Luo Han Guo root.

[0063] Comparative Example 1

[0064] Weigh 500g of dried monk fruit root powder, add water at a w / v ratio of 1:8 and decoct for 1.5h. Filter through gauze and cotton. Add another 4L of water to the residue and decoct for another 1.5h, then filter through gauze and cotton. Combine the two filtrates and centrifuge at 4000r / min for 10min. Concentrate the supernatant to 800mL under reduced pressure in a rotary evaporator at 70℃. Then add 200mL of a 4:1 mixture of chloroform and n-butanol, sonicate for 10min, centrifuge at 4000r / min for 10min, and collect the aqueous phase. Repeat this operation three times to remove impurities and proteins. Concentrate the aqueous phase to 200mL under reduced pressure in a rotary evaporator at 70℃ (concentrate). Add anhydrous ethanol to the concentrate until the ethanol content reaches 80% by volume, stirring continuously, to obtain a grayish-white flocculent precipitate. After standing overnight at 4℃, the supernatant was poured off, and the remaining portion was centrifuged at 4000 / min for 10 min to obtain a precipitate. The supernatant ethanol solution was recovered. The precipitate was washed four times with a small amount of anhydrous ethanol and dried at 60℃ to obtain crude polysaccharide (CPS) from the root of *Siraitia grosvenorii*.

[0065] Example 4

[0066] Comparison of molecular weights of crude polysaccharides LGP and CPS from monk fruit root

[0067] Comparison of molecular weight ranges of LGP and CPS: High-performance gel permeation chromatography (HPGPC) was used to compare the molecular weight ranges of polysaccharides. LGP prepared in Example 1 and CPS, D0, D2, D4, D6, D7, D8, and D200 dextran standards prepared in Comparative Example 1 were dissolved in water to prepare 1 mg / mL solutions, centrifuged, and analyzed by liquid chromatography. Liquid chromatography conditions: TSKgel G4000PWXL (7.8 mm I.D. × 30 cm, 10 μm) column; mobile phase: ultrapure water; column temperature: 30℃; flow rate: 0.5 mL / min; RID 10A time-of-flight detector; injection volume: 20 μL. Specific results are as follows: Figure 1 As shown.

[0068] from Figure 1 It can be seen that the retention time of LGP is in the range of 10-21 min, with substances in the range of 11-14 min being the main components, while the retention time of CPS is in the range of 10-22 min.

[0069] Example 5

[0070] Structural characterization

[0071] 1. Preparation of crude polysaccharide LGP and refined polysaccharide LGP-A from Siraitia grosvenorii root in Example 1

[0072] The yield of crude polysaccharide LGP (%) = m / m0 * 100;

[0073] Yield of LGP-A from Luo Han Guo root polysaccharide (%) = m1 / m*100.

[0074] In the formula, m: mass of crude polysaccharide LGP after alcohol precipitation and protein removal; m0: mass of defatted monk fruit root powder; m1: mass of monk fruit root polysaccharide LGP-A.

[0075] Depend on Figure 2 It can be seen that the crude polysaccharide from Luo Han Guo root was obtained by water extraction, alcohol precipitation, and protein removal, with a yield of 1.3% (calculated based on the mass of Luo Han Guo root raw material); further purification by Sephadex LH20 gel column chromatography yielded refined polysaccharide LGP-A from Luo Han Guo root, with a yield of 0.13% (calculated based on the mass of LGP).

[0076] 2. Purity and molecular weight of LGP-A, a refined polysaccharide from monk fruit root.

[0077] The purity of LGP-A prepared in Example 1 was verified using a full-wavelength ultraviolet (UV) scan. 2 mg of the dried sample was dissolved in pure water to prepare a 0.5 mg / mL polysaccharide solution, with pure water used as a blank control. The characteristic absorption peaks of nucleic acids and proteins in the UV spectrum are 260 nm and 280 nm, respectively; therefore, the content of impurities such as proteins, nucleic acids, and peptides in the polysaccharide can be determined by UV scanning. A full-wavelength scan was performed in the range of 190-400 nm.

[0078] The molecular weight of the polysaccharides was determined using high-performance gel permeation chromatography (HPGPC). Dried LGP-A, D0, D2, D4, D6, D7, D8, and D200 dextran standards were dissolved in water to prepare 1 mg / mL solutions, centrifuged, and analyzed by liquid chromatography. Liquid chromatography conditions: TSKgel G4000PWXL (7.8 mm I.D. × 30 cm, 10 μm) column; mobile phase: ultrapure water; column temperature: 30℃; flow rate: 0.5 mL / min; RID 10A time-of-flight detector; injection volume: 20 μL. A standard curve was plotted with retention time on the x-axis and the logarithm of molecular weight on the y-axis, and the molecular weight of LGP-A was calculated. Specific results are shown below. Figure 3 As shown.

[0079] Depend on Figure 3 It was found that LGP-A exhibits maximum absorption at 230 nm, and no characteristic absorption peaks of nucleic acids and proteins were observed in the 260–280 nm range, proving that LGP-A does not contain impurities such as proteins and nucleic acids. HPGPC analysis revealed that LGP-A presents a single chromatographic peak with good symmetry. Based on standard curves plotted using dextran standards of different molecular weights, the regression equation obtained was y = -0.2728x + 9.5237(R²). 2 =0.9977), the molecular weight of LGP-A was calculated to be 1.41 × 10⁻⁶. 3 kDa.

[0080] 3. Composition analysis of LGP-A monosaccharides

[0081] The monosaccharide composition was determined by PMP-pre-column derivatization high-performance liquid chromatography. 1.25 mg of the dried LGP-A sample prepared in Example 1 was dissolved in 1.25 mL of trifluoroacetic acid, shaken well, sealed, and hydrolyzed at 110 °C for 3 h. After cooling, 2 mL of methanol was added, and the mixture was evaporated to dryness under reduced pressure. This process was repeated 5 times to remove excess trifluoroacetic acid. 200 μL of the polysaccharide sample and monosaccharide standard were added to 200 μL of 0.5 mol / L PMP-methanol solution and 200 μL of 0.3 mol / L NaOH solution, and derivatized in a 70 °C oven in the dark for 100 min. After the reaction, the mixture was cooled to room temperature, 250 μL of 0.3 mol / L hydrochloric acid solution was added, mixed well, and extracted 5 times with chloroform. The lower layer was discarded to remove excess PMP. The mixture was centrifuged at 10000 r / min, and the supernatant was stored at 4 °C for HPLC analysis.

[0082] HPLC conditions: ZORBAX SB-C18 (4.6×250mm, 5μm) column; mobile phase: acetonitrile-phosphate buffer (pH 7.2) = 15:85; column temperature: 30℃; flow rate: 0.8mL / min; UV detection wavelength: 254nm; injection volume: 10μL.

[0083] The results of HPLC detection are as follows Figure 4 As shown.

[0084] Depend on Figure 4 It can be seen that LGP-A is mainly composed of galactose and arabinose, with a molar ratio of 17.817:16.652, indicating that LGP-A is an arabinogalactan.

[0085] 4. LGP-A Fourier Transform Infrared Chromatography Analysis

[0086] Weigh 1.5 mg of the dried LGP-A sample prepared in Example 1 and 150 mg of dried potassium bromide, grind them evenly under an infrared lamp, compress them into tablets, and perform infrared scanning. Specific results are as follows: Figure 5 As shown.

[0087] Depend on Figure 5 It can be seen that at 3302.79cm -1 and 2929.89cm -1 There are two characteristic absorption peaks of polysaccharides at 1645.12 cm⁻¹, representing the stretching vibration peak of OH and the stretching vibration peak of CH, respectively. -1 The peak at 1400-1200 cm⁻¹ is due to the asymmetric stretching vibration of the carboxyl group (C=O), indicating the presence of bound water. -1 It is the CH angular vibration peak; 1405 cm⁻¹ -1: This is the in-plane bending vibration peak of CH; 1075cm -1 1046cm -1 The peaks are formed by the overlap of stretching and ring vibrations of COC and COH, indicating the presence of pyranose; 899 cm⁻¹ -1 The absorption peaks indicate the presence of β-glycosidic bonds.

[0088] 5. LGP-A nuclear magnetic resonance analysis

[0089] Weigh 25 mg of the dried LGP-A sample prepared in Example 1, dissolve it in 0.5 mL of D2O, and then load it into an NMR tube for detection. Specific results are as follows: Figure 6 As shown.

[0090] LGP-A 1 H NMR spectrum as follows Figure 6 As shown in Figure A, the LGP-A proton signal is concentrated in the δ 3.24–5.26 range, exhibiting severe stacking. Two distinct α-anomeric proton signals are present at δ 5.26 and δ 5.10, representing α-arabinose terminal hydrogen signals; β-anomeric proton signals are present at δ 4.71, δ 4.55, and δ 4.52, representing β-galactose terminal hydrogen signals. The signal peaks located in the δ 4.2–3.2 region represent overlapping signals from hydrogen atoms at C2–C6 of the LGP-A monosaccharide ring.

[0091] LGP-A 13 C NMR spectra as follows Figure 6 As shown in B: The LGP-A carbon signal is concentrated in the range of δ48.97 to 109.29, which is consistent with... 1 HNMR offers higher resolution. δ109.29, δ108.00, and δ107.51 represent anodic carbon signals from α-arabinose; δ103.75 represents an anodic carbon signal from β-galactose. The resonance region from δ84 to 60 represents the overlap of signal shifts from C2 to C6 of the LGP-A monosaccharide residues.

[0092] 1 H- 1In the H COSY spectrum, δH 5.26 is correlated with δH 4.23, δH 4.23 is correlated with δH 3.97, δH 3.97 is correlated with δH 4.11, and δH 4.11 is correlated with δH 3.73, which are the correlation signals of arabinose H1-H2, H2-H3, H3-H4, and H4-H5a, respectively. In the HMBC spectrum, the arabinose terminal group δH 5.26 (Ara-H-1) correlates with δC 76.6 (Ara-C-3) and δC 83.9 (Ara-C-4); δH 4.23 (Ara-H-2) correlates with δC 76.6 (Ara-C-3), δC 83.9 (Ara-C-4), and δC 109.3 (Ara-C-1); δH 3.97 (Ara-H-3) correlates with δC 81.4 (Ara-C-2) and δC 61.2 (Ara-C-5); δH 3.97 (Ara-H-3), δH 3.73 (Ara-H-5a), and δH 3.83 (Ara-H-5b) correlates with δC 83.9 (Ara-C-4); δH... The δH values ​​of 3.73 (Ara-H-5a), 3.83 (Ara-H-5b), and 76.6 (Ara-C-3) correlate with each other, suggesting an arabinose ring based on the chemical shift values ​​and related signals. A strong correlation exists between δH 5.26 (Ara-H-1) and δC 83.9 (Ara-C-4), presumably indicating a 1,4 linkage of arabinose. Given the terminal hydrogen signal δH 5.26 (Gal-H-1), which is greater than 5, it is speculated to be in the α configuration. Therefore, NMR spectroscopy suggests that this polysaccharide contains galactose residues, linked in the manner →4)α-L-Araf-(1→).

[0093] 1 H- 1In the H COSY spectrum, δH 5.09 is correlated with δH 4.14, δH 4.14 is correlated with δH 3.97, δH 3.97 is correlated with δH 4.11, δH 4.11 is correlated with δH 3.80, and δH 4.11 is correlated with δH 3.89, which are the correlation signals of arabinose H1-H2, H2-H3, H3-H4, H4-H5a, and H4-H5b, respectively. In the HMBC spectrum, the arabinose terminal groups at δH 5.09 (Ara-H-1) correlate with δC 76.6 (Ara-C-3), 84.1 (Ara-C-4), and 66.9 (Ara-C-5); δH 4.13 (Ara-Ha-2) correlates with δC 76.6 (Ara-C-3); δH 4.11 (Ara-Ha-4) correlates with 76.6 (Ara-C-3); and δH 3.89 (Ara-Hb-5) correlates with δC 76.6 (Ara-C-3) and 107.6 (Ara-C-1). Based on the chemical shift values ​​and correlation signals, these groups are linked into an arabinose ring. The strong correlation between δH 5.09 (Ara-H-1) and δC 84.1 (Ara-C-4) suggests a possible 1,4 linkage of the arabinose. Since the terminal hydrogen signal δH5.09 (Ara-H-1) is greater than 5, it is speculated that it may be in the α configuration. Therefore, the NMR spectrum suggests that the polysaccharide contains arabinose residues, and its linkage is →3)α-L-Araf-(1→).

[0094] 1 H- 1In the H COSY spectrum, δH 4.52 is correlated with δH 3.38, δH 3.38 with δH 3.55, δH 3.55 with δH 3.78, δH 3.78 with δH 4.15, and δH 4.15 with δH 3.66, which are the correlation signals of galactose H1-H2, H2-H3, H3-H4, H4-H5, and H5-H6, respectively. In the HSQC spectrum, the galactose terminal group δH4.52 (Gal-H-1) is coupled with δC103.2 (Gal-C-1), and δH3.38 (Gal-H-2) is coupled with δC73.1 (Gal-C-2). In the HMBC spectrum, the galactose terminal group δH3.38 (Gal-H-2) is correlated with δC75.2 (Gal-C-3), δH3.55 (Gal-H-3) with δC73.1 (Gal-C-2), δH3.78 (Gal-H-4) with δC75.2 (Gal-C-3), δH3.72 (Gal-H-6) with δC81.2 (Gal-C-5), and δH4.15 (Gal-H-5) with δC81.2 (Gal-C-5). The correlation between δC 80.3 (Gal-C-4) and δC 69.9 (Gal-C-6) indicates a galactose ring based on chemical shift values ​​and correlation signals. A strong correlation between δH 4.52 (Gal-H-1) and δC 69.9 (Gal-C-6) suggests a possible 1,6 linkage of galactose. Since the terminal hydrogen signal δH 4.52 (Gal-H-1) is less than 5, it is speculated to be a β configuration. Therefore, NMR spectroscopy suggests that this polysaccharide contains galactose residues, linked in the manner →6)β-L-Galp-(1→). The results are summarized in Table 1.

[0095] Table 1 LGP-A sugar residue linkage modes

[0096] Sugar residues H1 / C1 H2 / C2 H3 / C3 H4 / C4 H5 / C5 H6 / C6 (A)→4) α-L-Araf-(1→ 5.26 / 109.3 4.23 / 81.4 3.97 / 76.6 4.11 / 83.9 3.83,3.73 / 61.6 (B)→3) α-L-Araf-(1→ 5.09 / 107.6 4.14 / 81.0 3.97 / 76.6 4.11 / 84.1 3.80,3.89 / 66.9 (C)→6) β-D-Galp-(1→ 4.52 / 103.2 3.38 / 73.1 3.55 / 75.16 3.78 / 80.3 4.15 / 81.2 3.66,3.77 / 69.9

[0097] 6. LGP-A Congo Red Experiment

[0098] The dried LGP-A sample prepared in Example 1 was dissolved in water to prepare a 0.5 mg / mL polysaccharide solution, which was then mixed thoroughly with a 200 μmol / L Congo red solution. An appropriate volume of 1 mol / L NaOH solution was added to achieve final NaOH concentrations of 0 mol / L, 0.1 mol / L, 0.2 mol / L, 0.3 mol / L, 0.4 mol / L, and 0.5 mol / L. The reaction was carried out at room temperature in the dark for 10 min. A full-wavelength scan was performed using a UV spectrophotometer, and a line graph was plotted with NaOH concentration on the x-axis and the maximum absorption wavelength on the y-axis. If a polysaccharide with a triple helix structure undergoes a complexation reaction with Congo red reagent, the maximum absorption wavelength (λ) of the mixed solution is [not specified]. maxThe wavelength shifts towards longer wavelengths, i.e., a redshift. Sodium hydroxide can disrupt the triple helix structure of polysaccharides; therefore, as the concentration of sodium hydroxide increases, the wavelength of the mixed solution increases. max Gradually decreasing, i.e., blue shift.

[0099] The experimental results of LGP-A Congo Red are as follows: Figure 7 As shown.

[0100] Depend on Figure 7 It can be seen that after LGP-A is mixed with Congo Red, λ max No significant red shift occurred, and the λ of the mixed solution and the single Congo red solution increased with increasing sodium hydroxide concentration. max The trends are similar, therefore it is determined that LGP-A does not contain a triple helix structure.

[0101] 7. LGP-A Scanning Electron Microscopy Analysis

[0102] 1.5 mg of the dried LGP-A sample prepared in Example 1 was weighed, and the microstructure of LGP-A was analyzed using thermal field emission scanning electron microscopy. Specific results are as follows: Figure 8 As shown.

[0103] Depend on Figure 8 It can be seen that under a scanning electron microscope, they are stacked in sheets with a rough surface and a network structure; there are also a few rod-like structures.

[0104] Example 6

[0105] Effects of LGP-A on the Immunogenic Effects of RAW264.7 Mouse Macrophages

[0106] 1. RAW264.7 cell proliferation experiment

[0107] The effect of polysaccharides on cell proliferation was detected using the CCK-8 assay. Cells in the logarithmic growth phase were selected and their cell size was adjusted to 1×10⁶ cells / year. 5 LGP-A polysaccharide solution (prepared in Example 1) at different concentrations (0.625 μg / mL, 1.25 μg / mL, 2.5 μg / mL, 5 μg / mL) was added to each well in the drug treatment group; 1 μg / mL LPS solution (prepared in Example 1) was added to the positive control group; and the same volume of complete culture medium was added to the blank group. Each group had three replicates. After drug administration, the wells were incubated for 24 hours. 10 μL of CCK-8 solution was added to each well, and the plates were incubated at 37°C for 45 min. The absorbance was measured at 450 nm using a microplate reader.

[0108] P(%)=(A-A0) / (A1-A0)×100%

[0109] In the formula: A is the average absorbance of the drug-treated group; A0 is the average absorbance of the background group; A1 is the average absorbance of the blank group.

[0110] The results of the LGP-A proliferation experiment on RAW264.7 cells are as follows: Figure 9 As shown.

[0111] Depend on Figure 9 It was found that, compared with the control group, LGP-A concentrations within the range of 0.625–5 μg / mL promoted the proliferation of RAW264.7 cells without cytotoxicity. Therefore, the following experiments used polysaccharide solutions with concentrations of 0.625–5 μg / mL for treatment.

[0112] 2. Phagocytosis assay of RAW264.7 cells

[0113] Neutral red assay for phagocytic capacity of RAW264.7 cells: Cells in logarithmic growth phase were selected and adjusted to 1×10⁻⁶ cells. 5 Cells / mL were seeded in 96-well plates and cultured at 37°C and 5% CO2 for 24 h. The same volume of different concentrations of LGP-A (prepared in Example 1) polysaccharide solution (0.625 μg / mL, 1.25 μg / mL, 2.5 μg / mL, 5 μg / mL) was added to each well in the drug treatment group; 1 μg / mL LPS solution was added to the positive control group; and the same volume of complete culture medium was added to the blank group. Each group had three replicates. After drug treatment, the plates were cultured for 24 h. The supernatant was discarded, and the plates were washed twice with PBS. 100 μL of 0.05% neutral red solution was added to each well, and the plates were cultured for 1 h. The supernatant was discarded, and the plates were washed twice with PBS. 100 μL of cell lysis buffer (glacial acetic acid: anhydrous ethanol = 1:1) was added, and the plates were incubated at room temperature for 1 h. The absorbance was measured at 540 nm using a microplate reader.

[0114] Phagocytic capacity index = (A1 / A0) × 100%;

[0115] In the formula: A1 is the average absorbance of the drug-treated group; A0 is the average absorbance of the blank group.

[0116] Phagocytosis by macrophages is the initial non-specific immune response to pathogen invasion. After engulfing pathogens, macrophages not only activate specific immunity but also play a crucial role in the resolution of inflammation. Therefore, the phagocytic capacity of macrophages can serve as one indicator of the body's immune defense level.

[0117] The phagocytic effect of LGP-A on RAW264.7 cells is as follows: Figure 10 As shown.

[0118] Depend on Figure 10The results showed that LGP-A polysaccharide significantly enhanced the phagocytic ability of RAW264.7 cells within the concentration range of 0.625 μg / mL to 5 μg / mL (P < 0.05). At a concentration of 1.25 μg / mL, the phagocytic rate increased from 100% in the control group to 138.45%. These results indicate that LGP-A can promote the phagocytic ability of RAW264.7 cells to neutral red, and the polysaccharide exhibits high immunomodulatory activity within the concentration range of 0.625–5 μg / mL.

[0119] 3. NO, TNF-α, and IL-6 secretion levels in RAW264.7 cells

[0120] The effects of LPS and different concentrations of LGP-A (prepared in Example 1) on NO secretion from mouse macrophages were detected using the Griess method. Cell culture and drug administration methods were the same as above, with cells cultured for 24 hours after drug administration. Procedures were followed according to the kit instructions. Absorbance values ​​were measured at 540 nm using a microplate reader.

[0121] The effects of LPS and different LGP-A concentrations on the secretion of TNF-α and IL-6 in mouse macrophages were detected using ELISA. Cell culture and drug administration methods were the same as above. The procedure was performed according to the kit instructions, and absorbance values ​​were measured at 450 nm using a microplate reader.

[0122] NO, as a cell-produced signal transduction mediator, can activate macrophages to initiate non-specific immunity, promote macrophage metabolism, and enhance macrophage phagocytic capacity, making it an important indicator for evaluating the level of immune response. Elevated levels of cytokines such as TNF-α and IL-6 are one of the markers of macrophage activation. Studies have shown that polysaccharides can be evaluated for their immunomodulatory activity by increasing the secretion of NO, TNF-α, and IL-6 in RAW264.7 cells to a level lower than that induced by lipopolysaccharide. Appropriate amounts of NO, TNF-α, and IL-6 can enhance the body's immune level, while excessive amounts can induce inflammatory responses.

[0123] Lipopolysaccharide (LPS) stimulates cells to produce a large number of cytokines and is generally used as a positive control in cellular immunity experiments. A drug is considered to have appropriate immunomodulatory activity if the cytokine levels in the treated group are higher than those in the blank control group but lower than those in the LPS-induced group; otherwise, it is considered excessive immunization.

[0124] The results of LGP-A on the secretion of NO, TNF-α, and IL-6 in RAW264.7 cells are as follows: Figure 11 As shown.

[0125] Depend on Figure 11The results showed that LGP-A polysaccharide could promote the secretion of cytokines such as NO, TNF-α, and IL-6 from RAW264.7 cells in a dose-dependent manner. At a concentration of 5 μg / mL, the secretion levels of NO, TNF-α, and IL-6 in the LGP-A polysaccharide solution reached their maximum values ​​of 20.64±1.29 μM, 27.16±0.17 μM, and 3.77±0.13 μM, respectively, which were lower than those in the 1 μg / mL LPS group (25.79±1.63 μM, 75.54±0.23 μM, and 18.97±0.11 μM). These results indicate that LGP-A polysaccharide can promote the secretion of NO, TNF-α, and IL-6 by macrophages, but the maximum dose group showed lower secretion levels than the LPS group, and no inflammatory response was induced. Therefore, LGP-A plays an immunomodulatory role.

[0126] Example 7

[0127] Comparison of the immunomodulatory activities of crude polysaccharide LGP from monk fruit root with CPS

[0128] The effects of LGP prepared in Example 1 and CPS prepared in Comparative Example 1 on the immune function of mouse macrophage RAW264.7 were studied using the method of Example 6. The results are shown in Table 2.

[0129] Table 2. Effects of LGP and CPS on the immune response of mouse macrophages RAW264.7

[0130] Polysaccharide LPA CPS Cell proliferation rate (%) 130±1.23 118±2.1 Cell phagocytosis rate (%) 145±1.11 132±1.21 NO secretion amount (μM) 20.33±0.98 17.64±1.29 TNF-α secretion amount (μM) 25.12±0.12 20.16±0.13 IL-6 secretion amount (μM) 4.55±0.13 3.77±0.19

[0131] Table 2 shows that both LGP and CPS polysaccharides at a concentration of 5 μg / mL can enhance the proliferation of RAW264.7 cells and promote phagocytosis. They can also promote the secretion of cytokines such as NO, TNF-α, and IL-6 in RAW264.7 cells in a dose-dependent manner, but overall, LGP has better activity than CPS.

[0132] The above description is only a preferred embodiment of the present invention. It should be noted that for those skilled in the art, several improvements and modifications can be made without departing from the principle of the present invention, and these improvements and modifications should also be considered within the scope of protection of the present invention.

Claims

1. A method for preparing refined polysaccharide LGP-A from monk fruit root, characterized in that, Includes the following steps: The roots of monk fruit were dried, pulverized, and defatted by reflux of ethanol. The residue was then extracted with water, precipitated with alcohol, and protein was removed to obtain crude polysaccharide LGP from monk fruit roots. The crude polysaccharide LGP from the root of Siraitia grosvenorii was sequentially separated and purified by DEAE-52 cellulose column and Sephadex LH20 gel column, and then freeze-dried to obtain the refined polysaccharide LGP-A from the root of Siraitia grosvenorii. The DEAE-52 cellulose column was eluted sequentially with water and sodium chloride solutions of concentrations of 0.05M, 0.1M, 0.15M, 0.3M, and 0.5M. The Sephadex LH20 gel column purification involved dissolving the 0.3M eluted sample in water, passing it through a Sephadex LH20 gel column, eluting with ethanol at a flow rate of 0.5 mL / min, combining the solutions, and concentrating. The eluent for the Sephadex LH20 gel column was a 25% (v / v) ethanol solution.

2. The preparation method according to claim 1, characterized in that, The solvent for ethanol reflux degreasing is an ethanol solution with a volume percentage of 95%, the time is 2 hours, and the number of times is 2; the mass ratio of the monk fruit root to the ethanol solution is 1:10 to 1:

20.

3. The preparation method according to claim 1, characterized in that, The ratio of material to liquid in the water extraction, alcohol precipitation, and water extraction process is 1:20 to 1:40, the temperature is 70 to 90°C, the time is 3 to 5 hours, and the number of extractions is 2.

4. The preparation method according to claim 1, characterized in that, The volume percentage of ethanol in the water extraction and alcohol precipitation reaction is 60%–80%, and the alcohol precipitation is carried out overnight at 4°C.

5. The refined polysaccharide LGP-A from the root of *Siraitia grosvenorii* obtained by the preparation method according to any one of claims 1 to 4.

6. The refined polysaccharide LGP-A from monk fruit root according to claim 5, characterized in that, The refined polysaccharide LGP-A from monk fruit root comprises arabinose and galactose, with a molecular weight of 1.41 × 10⁻⁶. 3 kDa.

7. The application of the refined polysaccharide LGP-A from the root of Monk Fruit as described in claim 5 in the preparation of immune-enhancing products.

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