Marsdenia tenacissima polysaccharide-zinc compound as well as preparation method and application thereof

Abstract

The invention discloses a marsdenia tenacissima polysaccharide-zinc compound as well as a preparation method and application thereof, and relates to the technical field of biological medicines, and the marsdenia tenacissima polysaccharide-zinc compound is formed by compounding marsdenia tenacissima polysaccharide and zinc salt. The molecular mechanism of macrophages can be activated through a TLR4-Myd88-NF-kappa B signal channel, secretion of cell factors (TNF-alpha, IL-6, IL-1beta and IL-18) is promoted, and meanwhile generation of NO and ROS is enhanced. A modern scientific basis is provided for taking the marsdenia tenacissima polysaccharide-zinc compound as a potential immunomodulator in the field of biological medicines or functional foods.

Description

Technical Field

[0001] The present invention relates to the technical field of biomedicine, and in particular to a genus Achyranthes bidentata polysaccharide-zinc complex and a preparation method and application thereof. Background Art

[0002] Due to the diverse biological activities of natural active polysaccharides, they have received increasing attention in the fields of biomedicine and functional foods in recent years. Among these activities, the immunomodulatory activity of polysaccharides is particularly important. Polysaccharides are compounds that can interact with the immune system and enhance host-specific responses without side effects. In recent years, natural polysaccharides have great development potential as immunomodulators in the biomedical field. However, the clinical application of natural polysaccharides is still limited, mainly due to their insufficient immunoenhancing activity. Therefore, effective strategies are urgently needed to enhance the immunomodulatory effects of natural polysaccharides.

[0003] Zinc (Zn) plays an indispensable role in immune regulation processes such as cellular immunity, humoral immunity and inflammatory response, and is also an important trace element in the human body. Appropriate zinc intake can effectively improve the phagocytic ability of macrophages and promote the proliferation and differentiation of T cells, thereby activating the body's nonspecific and specific immune responses. More importantly, organic zinc (such as zinc-containing polysaccharides) has multiple advantages over inorganic zinc, such as easier absorption, reduced gastrointestinal irritation, higher bioavailability and biological activity, and lower toxicity. Therefore, introducing zinc into the polysaccharide structure is an effective method to develop new products that combine the advantages of natural active polysaccharides and zinc. This method is expected to enhance the immunomodulatory activity of natural active polysaccharides while reducing the toxicity associated with inorganic zinc.

[0004] In general, natural polysaccharides have attracted extensive attention in the fields of biomedicine and functional foods due to their diverse biological activities, especially their immunomodulatory activity. These polysaccharides from various natural products show great potential in enhancing immune function. However, their clinical application is still limited due to insufficient immunoenhancing activity. One of the promising solutions is to enhance their effects through structural modification, especially the introduction of trace elements such as zinc. Zinc-modified polysaccharides can not only enhance immunomodulatory activity, but also improve absorption rate and bioavailability. Despite these obvious advantages, the study of the structural characteristics of homogeneous polysaccharides and their immunomodulatory mechanisms, especially in traditional Chinese medicine polysaccharides including Rhizoma Coptidis, still needs further research. Summary of the invention

[0005] The first technical problem to be solved by the present invention is: in view of the deficiencies in the prior art, a polysaccharide-zinc complex of Rhizoma Cyperi is provided, which has strong immunomodulatory activity.

[0006] To solve the above-mentioned first technical problem, the technical solution of the present invention is:

[0007] A thunbergia oxyphylla polysaccharide-zinc complex, which is formed by compounding thunbergia oxyphylla polysaccharide and zinc salt.

[0008] Preferably, the Thunbergia tongguanyi polysaccharide is a homogeneous polysaccharide, the monosaccharide composition is mannose, glucose, galactose and arabinose, and the measured molecular weight range is 1kDa-100kDa.

[0009] The second technical problem to be solved by the present invention is: in view of the shortcomings of the prior art, a preparation method is provided, which has simple steps, mild reaction conditions, and the prepared Tongguanteng polysaccharide-zinc complex has strong immunomodulatory activity.

[0010] To solve the above second technical problem, the technical solution of the present invention is:

[0011] A method for preparing a radix scutellariae polysaccharide-zinc complex comprises the following steps:

[0012] S1. Preparation of crude polysaccharide of Thunbergia lappa

[0013] S1-1, washing, drying and crushing the Radix Glehniae Radix medicinal materials to obtain Radix Glehniae Radix medicinal material tablets;

[0014] S1-2, adding deionized water to the Thunb. cyperus rotundus medicinal material tablets, wherein the ratio of the added Thunb. cyperus rotundus medicinal material tablets to the added deionized water is 1-2:10 kg / L, and heating for extraction to obtain an extract;

[0015] S1-3, the extract is concentrated under reduced pressure, and then anhydrous ethanol is added to a volume concentration of 45-55% ethanol, and the supernatant I is taken after standing;

[0016] S1-4, adding anhydrous ethanol to the supernatant I until the ethanol volume concentration is 65-75%, separating the supernatant II after standing, and the remaining brown precipitate is deproteinized, dialyzed, and freeze-dried to obtain the crude polysaccharide of Thunb.

[0017] S2. Preparation of homogeneous polysaccharide of Thunbergia lappa

[0018] S2-1, the crude polysaccharide of Thunbergia tongguanensis was dissolved in deionized water and then subjected to ion exchange column chromatography, gradient elution was performed with 0-2M sodium chloride solution, the elution curve was tracked by phenol-sulfuric acid method, and the sugar part I was collected according to the elution curve, concentrated and freeze-dried, wherein the addition ratio of crude polysaccharide of Thunbergia tongguanensis to deionized water was 1-2:10 g / mL;

[0019] S2-2. The sugar part I is dissolved in water, centrifuged, and the clear liquid is subjected to molecular sieve gel column chromatography and eluted with water. The elution curve is detected by phenol-sulfuric acid method, and the sugar part II is collected according to the elution curve, concentrated, and freeze-dried to obtain the uniform polysaccharide of Thunbergia clematis.

[0020] S3. Preparation of Thunbergia oxyphylla polysaccharide-zinc complex

[0021] The prepared Thunb. Glehnia littoralis uniform polysaccharide is dissolved in deionized water, wherein the added amount ratio of Thunb. Glehnia littoralis uniform polysaccharide to deionized water is 30-50 mg / mL, and then a zinc salt solution with a concentration of 1-2 mg / mL is added, and the reaction is heated. The pH of the reaction solution is adjusted with an alkaline solution. After the reaction is completed, the pH of the solution is adjusted with an acid solution. The reaction solution is dialyzed and freeze-dried to obtain a Thunb. Glehnia littoralis polysaccharide-zinc complex.

[0022] Preferably, in step S1-2, the extraction temperature is 70-90°C, the extraction time is 2-4h, and the extraction times are 2-4 times.

[0023] Preferably, the extract in step S1-3 is concentrated under reduced pressure to 1 / 12 to 1 / 8 of the original volume;

[0024] In steps S1-3 and S1-4, the standing temperature is 3-5°C and the standing time is 12-24h.

[0025] Preferably, in step S3, the volume ratio of the added amount of the Thunbergia clematis uniform polysaccharide solution and the zinc salt solution is 2-3:1.

[0026] Preferably, the reaction temperature in step S3 is 45-55° C., and the stirring speed during the reaction is 200-500 rpm.

[0027] Preferably, after adding the zinc salt solution in step S3, the reaction is carried out for 8-15 minutes, and then an alkaline solution is added to adjust the pH to 8.0 and the reaction is continued for 20-48 hours. After the reaction is completed, the pH of the solution is adjusted to 3.0 with an acid solution.

[0028] The third technical problem to be solved by the present invention is to provide an application of a Psoralea corylifolia polysaccharide-zinc complex in regulating immunity.

[0029] The fourth technical problem to be solved by the present invention is to provide a composition for regulating immune function, comprising a polysaccharide-zinc complex of Rhizoma Coptidis as an active ingredient and a pharmaceutically acceptable carrier.

[0030] A new polysaccharide-zinc complex of Rhizoma Xanthophyllum was synthesized by modification method using homogeneous polysaccharide of Rhizoma Xanthophyllum with immunomodulatory effect and zinc salt as raw materials. Physical and chemical characterization showed that zinc was successfully introduced into the polymer chain of Rhizoma Xanthophyllum polysaccharide MTI I-1-1 without destroying the structure of Rhizoma Xanthophyllum polysaccharide MTII-1-1. The zinc in the complex mainly interacted with Rhizoma Xanthophyllum polysaccharide MTII-1-1 by forming Zn-O bond or Zn-OH bond. Further in vitro experiments confirmed that Rhizoma Xanthophyllum polysaccharide MTII-1-1 and Rhizoma Xanthophyllum polysaccharide-zinc complex MTII-1-1-Zn could effectively activate macrophages through TLR4-Myd88-NF-κB signaling pathway and induce immune response. At the same time, due to the introduction of zinc, Rhizoma Xanthophyllum polysaccharide-zinc complex MTII-1-1-Zn had a better promoting effect on macrophage activation and proliferation than Rhizoma Xanthophyllum polysaccharide MTII-1-1. The final product, the M. cymoides polysaccharide-zinc complex MTII-1-1-Zn, can be used as a potential candidate for immunomodulator in the fields of biomedicine or functional food.

[0031] Due to the adoption of the above technical solution, the beneficial effects of the present invention are:

[0032] 1. The method of the present invention has simple steps and mild reaction conditions, and the prepared Rhizoma Coptidis polysaccharide-zinc complex has a certain solubility in aqueous solution.

[0033] 2. The present invention further clarified the physical and chemical properties of Rhizoma Chuanxiong polysaccharides and Rhizoma Chuanxiong polysaccharides-zinc complexes by characterization methods, and studied the potential interaction mechanism between zinc and Rhizoma Chuanxiong homogeneous polysaccharides.

[0034] 3. In vitro experiments have shown that the polysaccharide-zinc complex of the present invention can activate the molecular mechanism of macrophages through the TLR4-My d88-NF-κB signaling pathway, promote the secretion of cytokines (TNF-α, IL-6, IL-1β and IL-18), and enhance the generation of NO and ROS. This provides a modern scientific basis for the polysaccharide-zinc complex of the present invention as a potential immunomodulator in the field of biomedicine or functional food. BRIEF DESCRIPTION OF THE DRAWINGS

[0035] Figure 1 HPGPC, relative molecular weight analysis, monosaccharide composition and D / L configuration analysis of MTII-1-1, a polysaccharide from Thunb.

[0036] Figure 2 UV and FT-IR analysis of MTII-1-1 polysaccharide from Thunb.

[0037] Figure 3 The SEM image analysis of MTII-1-1 and MTII-1-1-Zn polysaccharide-zinc complex of Thunb.

[0038] Figure 4 EDS analysis and XPS analysis of MTII-1-1 and MTII-1-1-Zn polysaccharide-zinc complex;

[0039] Figure 5 To study the effects of MTII-1-1 and MTII-1-1-Zn polysaccharide on macrophage viability and phagocytic ability.

[0040] Figure 6 To study the effects of MTII-1-1 and MTII-1-1-Zn polysaccharide on the secretion levels of TNF-α, IL-6, IL-1β and IL-18 in macrophages.

[0041] Figure 7 To study the effects of MTII-1-1 and MTII-1-1-Zn polysaccharide on the production of NO and ROS in macrophages.

[0042] Figure 8 To study the effects of MTII-1-1 and MTII-1-1-Zn polysaccharide on the mRNA expression of TNF-α, IL-6, IL-1β and IL-18 in macrophages.

[0043] Fig. 9 This is the effect of MTII-1-1, a polysaccharide from M. persica, and MTII-1-1-Zn, a polysaccharide-zinc complex from M. persica, on proteins related to the TLR4-Myd88-NF-κB pathway. DETAILED DESCRIPTION

[0044] The present invention will be further described below in conjunction with the embodiments.

[0045] Example 1

[0046] The preparation method of crude polysaccharide of Rhizoma Coptidis comprises the following steps:

[0047] 5 kg of Thunbergia cyrtonema was washed, dried and crushed to obtain Thunbergia cyrtonema tablets, 50 L of deionized water was added to the Thunbergia cyrtonema tablets, and heated and extracted at 80° C. for 3 h, and extracted 3 times, and a total of 150 L of the 3 extracts were combined;

[0048] The extract was centrifuged and the supernatant was concentrated to 1 / 10 of its original volume under reduced pressure. After concentration, anhydrous ethanol was added to the concentrate to a final volume concentration of 50%. The mixture was allowed to stand at 4°C for 18 h. Supernatant I was taken and anhydrous ethanol was added to supernatant I to a final volume concentration of 70%. The mixture was allowed to stand at 4°C for 24 h. Supernatant II was separated and the remaining brown precipitate was deproteinized with Sevag reagent, dialyzed and freeze-dried to obtain crude polysaccharide of Thunb.

[0049] Example 2

[0050] The preparation method of the homogeneous polysaccharide of Rhizoma Coptidis comprises the following steps:

[0051] (1) Ion exchange column chromatography: 300 mg of the crude polysaccharide of Thunbergia tongguanensis obtained in Example 1 was dissolved in 10 mL of deionized water and loaded onto a DEAE-FF column. Gradient elution was performed using a 0-2 M sodium chloride solution. The elution curve was tracked using the phenol-sulfuric acid method. Sugar fraction I was collected according to the elution curve, concentrated, and freeze-dried.

[0052] (2) Molecular sieve gel chromatography: The above freeze-dried sugar part I was dissolved in water, centrifuged, and the supernatant was applied to a Sephadex G-75 column and eluted with water. The elution curve was tracked using the phenol-sulfuric acid method. A single symmetrical peak appeared. The main peak was collected, concentrated, and freeze-dried to obtain the M. cyrtonema polysaccharide MTII-1-1.

[0053] Example 3

[0054] The preparation method of the polysaccharide-zinc complex of Rhizoma Coptidis comprises the following steps:

[0055] The MTII-1-1 polysaccharide obtained in Example 2 was dissolved in deionized water, wherein the ratio of MTII-1-1 polysaccharide to deionized water was 30:1 mg / mL, and then ZnCl was added. 2 solution, wherein the homogeneous polysaccharide MTII-1-1 of Tongguanteng was added: ZnCl 2 The solute mass ratio of the solution was 2.5:1, and it was stirred at 300 rpm for 10 min at 50 ° C. After stirring, the pH value of the mixed solution was adjusted to 8.0 using 0.5 M sodium hydroxide solution, and further reacted at 50 ° C for 24 h. After the reaction, the pH value of the reaction solution was adjusted to 3.0 using 0.6 M hydrochloric acid solution, and the reaction product was dialyzed in ultrapure water for 24 h to remove the unreacted product. Finally, the dialyzate was freeze-dried to obtain the Tongguanteng polysaccharide-zinc complex MTI I-1-1-Zn.

[0056] Example 4

[0057] Characterization method of radix scutellariae polysaccharides and radix scutellariae polysaccharides-zinc complex

[0058] (I) Test materials: Thunbergia oxyphylla polysaccharide MTII-1-1 (taken from Example 2), Thunbergia oxyphylla polysaccharide-zinc complex MTII-1-1-Zn (taken from Example 3).

[0059] (II) Test methods:

[0060] 1. Homogeneity analysis

[0061] 5 mg of MTII-1-1 polysaccharide from Tongguanteng and 5 mg of MTII-1-1-Zn polysaccharide-zinc complex from Tongguanteng were completely dissolved in 1 mL of distilled water, respectively, and filtered through a 0.22 μm filter membrane for analysis by a high performance liquid gel permeation chromatography (HPGPC) system.

[0062] Chromatographic conditions:

[0063] Chromatographic column: TSK gel G3000 PWXL (30cm×7.8mm, 7μm); mobile phase: 0.02M KH 2 PO 4 ; Flow rate: 0.5mL / min; Injection volume: 10μL.

[0064] 2. Monosaccharide composition analysis

[0065] Sample processing:

[0066] Accurately weigh 5.0 mg of each of the samples of Thunbergia cyrtonema polysaccharide MTII-1-1 and Thunbergia cyrtonema polysaccharide-zinc complex MTII-1-1-Zn into a stoppered test tube, add 1.8 mL of 2 M trifluoroacetic acid (TFA), and place in an oil bath at 120°C for 4 hours for hydrolysis. After the hydrolysis is completed, cool to room temperature, repeatedly add 4 mL of methanol and spin dry to remove excess TFA, then dissolve the spin-dried sample with 1 mL of deionized water, centrifuge, and take 300 μL of sample solution, add 300 μL of 0.3 M sodium hydroxide solution, and then add 300 μL of 0.5 M PMP methanol solution, mix well, and react in a 70 ° C water bath for 30 minutes. After the reaction is completed, cool, add 290 μL of 0.3 M hydrochloric acid solution for neutralization, and then add 2 times the volume of chloroform solution, shake vigorously, centrifuge, remove the chloroform phase, repeat the extraction twice, and take the aqueous phase. Filter with a 0.22 μm filter membrane for HPLC injection analysis.

[0067] Chromatographic conditions:

[0068] Chromatographic column: Agilent ZORBAX Eclipse XDB-C18, 406×25mm, 5μm; mobile phase: 0.1M phosphate (pH=6.9), buffer-acetonitrile (v / v is 83:17); detection wavelength: 250nm; flow rate: 1mL / min; injection volume: 20μL.

[0069] 3.D / L configuration analysis

[0070] Sample processing:

[0071] Accurately weigh 5.0 mg of each sample of MTII-1-1 and MTII-1-1-Zn, respectively, into a stoppered test tube, add 1.8 mL of 2M trifluoroacetic acid (TFA), and place in an oil bath to hydrolyze at 120°C for 4 hours. After the hydrolysis is completed, cool to room temperature, add 4 mL of methanol repeatedly and spin dry to remove excess TFA. Then add 2.5 mg of L-cysteine ​​methyl ester hydrochloride and 1 mL of pyridine to the spin-dried sample, and water bath at 60°C for 1 hour. After the water bath is completed, add 5 μL of o-phenylisothiocyanate and continue to react at 60°C for 1 hour. After the reaction is completed, the sample is passed through a 0.22 μm microporous filter membrane of the organic phase for HPLC injection analysis.

[0072] Chromatographic conditions:

[0073] Chromatographic column: Agilent ZORBAX Eclipse XDB-C18, 406×25 mm, 5 μm; Mobile phase: 25% CH 3 CN-H 2 O (0.1% acetic acid); detection wavelength: 250 nm; flow rate: 1 mL / min; injection volume: 20 μL.

[0074] 4. Ultraviolet spectroscopy and infrared spectroscopy

[0075] 3.0 mg of dried Thunbergia tongguanyi polysaccharide MTII-1-1 and Thunbergia tongguanyi polysaccharide-zinc complex MTII-1-1-Zn samples were placed in centrifuge tubes, 3 mL of deionized water was added for dissolution, and full wavelength scanning was performed in the range of 200-400 nm using a Spectrum SP-756P UV-visible spectrophotometer.

[0076] 3.0 mg of dried MTII-1-1 and MTII-1-1-Zn polysaccharide were respectively ground with KBr, pressed into tablets, and analyzed by Shimadzu IR Affinity-1 at 4000-400 cm -1 Scan within the range.

[0077] 5. Scanning Electron Microscopy and Energy Dispersive Spectroscopy Analysis

[0078] Scanning electron microscopy (SEM) combined with energy dispersive spectroscopy (EDS) was used to detect the morphology and elemental distribution of MTII-1-1 and MTII-1-1-Zn. The dried powders of MTII-1-1 and MTII-1-1-Zn were fixed on the sample stage and sputtered with gold. The gold-coated samples were then imaged using FE-SEM at an accelerating voltage of 15 kV. EDS was used to simultaneously analyze the Zn content and the elemental distribution of C, O, and Zn.

[0079] 6. X-ray photoelectron spectroscopy analysis

[0080] XP S spectra of MTII-1-1 and MTII-1-1-Zn were recorded using an ESCALab 250Xi (Thermo Scientific) spectrometer with a monochromatic AlKα source (1486.6 eV). The pressure, voltage, and power were set to 2.67×107 Pa, 15 kV, and 200 W, respectively. The data were collected and analyzed using Advantage software (version 5.9922) by fixing the C1s binding energy.

[0081] (III) Test results:

[0082] 1. Structural feature analysis

[0083] (1) Homogeneity analysis

[0084] like Figure 1 As shown in A, the MTII-1-1 polysaccharide from the herbaceous vine showed a uniform symmetrical peak in its HPGPC spectrum, indicating that MTII-1-1 was a uniform polysaccharide with a molecular weight range of 1 kDa-100 kDa. Figure 2 As shown in Figure C, the Tongguanteng polysaccharide-zinc complex MTII-1-1-Zn also shows a uniform symmetrical peak in its HPGPC spectrum, indicating that MTII-1-1-Zn is a uniform polysaccharide. In addition, in the HPGPC spectrum, the retention times of MTII-1-1 and MTII-1-1-Zn are almost the same, indicating that the main chain of MTII-1-1 was not destroyed during the zinc modification process.

[0085] (2) Monosaccharide composition analysis

[0086] like Figure 1 As shown in B, the HPLC spectrum shows that the MTII-1-1 polysaccharide is composed of mannose, glucose, galactose and arabinose. Figure 1 C and Figure 1As shown in D, it can be seen from the HPLC spectrum that the mannose, glucose and galactose in the MTII-1-1 polysaccharide are in the D configuration, and the arabinose is in the L configuration.

[0087] (3) Ultraviolet and infrared spectrum analysis

[0088] like Figure 2 As shown in A, the ultraviolet spectrum shows that the MTII-1-1 polysaccharide does not contain protein or nucleic acid; Figure 2 B, 3400cm -1 and 2927cm -1 The absorption bands at 1140 cm-1 are attributed to the stretching vibration of -OH and CH, respectively. -1 and 1033cm -1 The absorption bands at 937 cm-1 are attributed to the CO vibrations of COC and COH in the sugar ring; -1 、873cm -1 and 816cm -1 The absorption band at is the characteristic absorption band of α-D-Glcp, indicating that the MTII-1-1 polysaccharide from the Chinese magnolia vine contains α-D-Glcp.

[0089] (4) Scanning electron microscopy analysis

[0090] like Figure 3 As shown in AB, under 200 times magnification, the MTII-1-1-Zn complex of Thunb. tongguanyi was mainly in the form of irregular lumps with irregular gaps and grooves on the surface. In contrast, the unmodified MTII-1-1 polysaccharide was in the form of irregular particles under the same 200 times magnification.

[0091] (5) Energy dispersion spectroscopy and X-ray photoelectron spectroscopy analysis

[0092] like Figure 4 As shown in AB, zinc is evenly distributed in the MTII-1-1-Zn complex of the genus tongguanteng polysaccharide, indicating that zinc has been successfully introduced into the MTII-1-1 of the genus tongguanteng polysaccharide. Further analysis showed that the carbon, oxygen and zinc contents in the MTII-1-1-Zn complex of the genus tongguanteng polysaccharide were 42.73%, 53.17% and 4.10%, respectively.

[0093] like Figure 4As shown in Figure CF, compared with the XPS spectrum of MTII-1-1, the MTII-1-1-Zn complex showed two typical peaks at 1022.23 eV and 1045.28 eV, indicating that zinc has been successfully introduced into MTII-1-1. By further distinguishing and fitting the Zn 2P peak of MTII-1-1-Zn, it was found that the Zn 2P double peak can be decomposed into Zn 2P3 / 2 (1021.45 eV and 1022.61 eV) and Zn 2P1 / 2 (1044.59 eV and 1045.96 eV), which indicates that zinc mainly interacts with MTII-1-1 by forming Zn-O coordination bonds and Zn-OH bonds.

[0094] Example 5

[0095] Evaluation of the immunomodulatory activity of Thunb polysaccharide-zinc complex in vitro

[0096] (I) Test materials: Thunbergia oxyphylla polysaccharide MTII-1-1 (taken from Example 2), Thunbergia oxyphylla polysaccharide-zinc complex MTII-1-1-Zn (taken from Example 3).

[0097] (ii) Experimental subjects: RAW 264.7 cells.

[0098] (III) Test methods:

[0099] 1. Cell culture and treatment:

[0100] Raw 264.7 cells were cultured in RIPM 1640 medium supplemented with 10% fetal bovine serum and 1% antibiotics, including 100 U / mL penicillin and 100 μg / mL streptomycin (Invitgen, NY, USA). The incubator was an M3111 carbon dioxide incubator (Thermo Fisher Scientific, MA, USA) set at 37°C and 5% CO 2 humid environment.

[0101] 2. Cell Viability Assay

[0102] The effects of MTII-1-1 and MTII-1-1-Zn on macrophage viability were evaluated using a CCK-8 kit (Saint-bio, Shanghai, China). RAW 264.7 cells (2×104 cells / well) were seeded in 96-well plates and incubated overnight at 37°C. After incubation, different concentrations of MTII-1-1 and MTII-1-1-Zn (100μg / mL, 200μg / mL, 400μg / mL) were added for 24h, and lipopolysaccharide (2μg / mL) was used as a positive control. Then, 10μL of CCK-8 solution was added to each well and incubated at 37°C for another 3h. After incubation, the absorbance at 490nm was measured using a microplate reader (BioTek, USA) to determine the viability of macrophages.

[0103] 3. Phagocytosis Assay

[0104] RAW 264.7 cells (2×104 cells per well) were seeded into 96-well plates and incubated overnight at 37°C. Then, the cells were treated with different concentrations of MTII-1-1 and MTII-1-1-Zn (100μg / mL, 200μg / mL, 400μg / mL) for 24h, and lipopolysaccharide (2μg / mL) was used as a positive control. After incubation, the culture medium was removed and 200μL of 0.1% neutral red solution was added to each well. The cells were incubated at 37°C for 3h. After incubation, the cells were washed twice with PBS to remove the residual neutral red solution. Finally, a mixed solution of ethanol and glacial acetic acid (1:1, v / v) was added, and the absorbance value of each well at 540nm was measured using a spectrophotometer.

[0105] 4. Determination of Cytokine Levels

[0106] RAW 264.7 cells (1×105 cells per well) were seeded into 6-well plates and incubated overnight at 37°C. The cells were then treated with different concentrations of MTII-1-1 and MTII-1-1-Zn (100μg / mL, 200μg / mL, 400μg / mL) for 24h, and lipopolysaccharide (2μg / mL) was used as a positive control. After incubation, the supernatant was collected and the concentrations of TNF-α, IL-6, IL-18, and IL-1β in the supernatant were quantified using the manufacturer's instructions on the ELISA kit. The absorbance value of each well at 450nm was then measured using a spectrophotometer.

[0107] 5. Determination of NO and ROS Levels

[0108] RAW 264.7 cells (1×105 cells per well) were seeded into 6-well plates and incubated overnight at 37°C. The cells were then treated with MTII-1-1 and MTII-1-1-Zn (400 μg / mL) for 12 h, while lipopolysaccharide (2 μg / mL) was used as a positive control. After treatment, the cells were collected and washed twice with PBS solution, and then the NO and ROS levels in macrophages were determined according to the manufacturer's instructions on the NO and ROS detection kits.

[0109] (IV) Test results:

[0110] like Figure 5 As shown in A, there was no significant difference in cell viability after macrophages were treated with different concentrations of MTII-1-1 and MTII-1-1-Zn (100, 200 and 400 μg / mL), indicating that within the concentration range of 100-400 μg / mL, MTII-1-1 and MTII-1-1-Zn were not toxic to macrophages.

[0111] like Figure 5 As shown in B, after 24 hours of treatment, both MTII-1-1 and MTII-1-1-Zn can significantly enhance the phagocytic activity of macrophages (P<0.001, P<0.01, P<0.05). In addition, the macrophage phagocytic activity of the zinc-modified MTII-1-1-Zn polysaccharide-zinc complex group was significantly higher than that of the MTII-1-1 polysaccharide group (P<0.01). This result further confirmed that zinc has been successfully introduced into the polymer chain of MTII-1-1 polysaccharide, and the introduction of zinc can significantly enhance the activation of macrophages (P<0.01).

[0112] like Figure 6 As shown in A, after treatment with MTII-1-1 and MTII-1-1-Zn, both significantly enhanced the secretion of TNF-α in a dose-dependent manner compared with the control group (P<0.001). In addition, the zinc-modified MTII-1-1-Zn significantly enhanced the secretion of TNF-α, indicating that the introduction of zinc significantly improved the ability of macrophages to secrete TNF-α (P<0.001). Figure 6As shown in BD, both MTII-1-1 and MTII-1-1-Zn promoted the secretion of IL-6, IL-1β and IL-18 to varying degrees (P<0.001). For IL-6, there was no significant difference between the MTII-1-1-treated group and the control group, indicating that MTII-1-1 could not activate macrophages by regulating the expression of IL-6. However, after 24h of treatment with MTII-1-1-Zn, the secretion of IL-6 increased significantly, indicating that the introduction of zinc significantly enhanced the ability of MTII-1-1 to promote IL-6 secretion (P<0.001). Similar results also appeared in the expression of IL-1β, indicating that the introduction of zinc effectively enhanced the ability of MTII-1-1 to promote the secretion of IL-1β by macrophages (P<0.001). Regarding the expression of IL-18, there was no significant difference between the group treated with MTII-1-1 polysaccharide of the Chinese magnolia vine and the control group (P>0.05), but after zinc modification, MTII-1-1 polysaccharide of the Chinese magnolia vine significantly enhanced the ability to promote IL-18 secretion (P<0.05; P<0.001), and its activity increased by 24.49% and 48.0% (200 and 400 μg / mL), respectively.

[0113] like Figure 7 As shown in A, compared with the control group, both the MTII-1-1 polysaccharide and the MTII-1-1-Zn polysaccharide-zinc complex significantly enhanced the production of NO (P<0.001). At the same time, compared with the MTII-1-1 polysaccharide, the zinc-modified MTII-1-1-Zn polysaccharide-zinc complex further significantly enhanced the ability to generate NO (P<0.01), indicating that the introduction of zinc enhances the immunomodulatory activity of polysaccharides. Figure 7 As shown in B, compared with the control group, both MTII-1-1 and MTII-1-1-Zn polysaccharide from Rhizoma Coptidis significantly promoted the generation of ROS (P<0.001). At the same time, compared with MTII-1-1 polysaccharide from Rhizoma Coptidis, MTII-1-1-Zn polysaccharide from Rhizoma Coptidis significantly enhanced the generation of ROS (P<0.001).

[0114] Example 6

[0115] Analysis of the potential molecular mechanism of the polysaccharide-zinc complex of Rhizoma Atractylodis Macrocephalae

[0116] (I) Test materials: Thunbergia oxyphylla polysaccharide MTII-1-1 (taken from Example 2), Thunbergia oxyphylla polysaccharide-zinc complex MTII-1-1-Zn (taken from Example 3).

[0117] (ii) Experimental subjects: RAW 264.7 cells.

[0118] (III) Test methods:

[0119] RAW 264.7 cells (1×105 cells per well) were seeded into 6-well plates and treated with MTII-1-1 and MTII-1-1-Zn (400 μg / mL) of 1.4-tetracycline polysaccharide for 24 h, and lipopolysaccharide (LPS, 2 μg / mL) was used as a positive control. After treatment, all cells were collected, and total RNA was extracted using an RNA extraction kit (Vazyme, Nanjing, China), and reverse transcribed into cDNA using a HiScriP III RT SuPerMix for qPCR (+gDNA wiPer) kit (Vazyme, Nanjing, China).

[0120] RT-PCR assays were performed using Taq Pro Universal SYBR qPCR Kit (Vazyme, Nanjing, China). The mRNA expression levels of TNF-α, IL-6, IL-1β, and IL-18 were detected using the CFX ConnectTM Fast Real-Time PCR System (BIO-RAD, USA). Meanwhile, GAPDH was used as a normalization control. All primers used in this assay are listed in Table 1.

[0121] Table 1 Primer sequences used for qRT-PCR reactions

[0122]

[0123]

[0124] 2. Western Blot (WB) Assay

[0125] RAW 264.7 cells (1×105 cells per well) were seeded into 6-well plates and treated with MTII-1-1 and MTII-1-1-Zn (400 μg / mL) for 24 h, while lipopolysaccharide (LPS, 2 μg / mL) was used as a positive control. After treatment, cells were collected and lysed on ice with radioimmunoprecipitation assay buffer (RIPA). Protein concentration was calculated using a BC A kit according to the manufacturer's protocol (Zoman, China). Equal amounts of protein (15 μg) from each treatment group were then separated by 12.5% ​​sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE). Subsequently, the gel was transferred to a polyvinylidene fluoride (PVDF) membrane and blocked with 5% (w / v) skim milk powder in Tris-buffered saline containing 0.1% Tween-20 (TBST) buffer for 2 h, followed by incubation with primary antibodies overnight (12 h) at 4 °C. After incubation, the PVDF membrane was washed three times with TBST and incubated with the corresponding secondary antibody at room temperature for 1.5 h. Subsequently, TBST was used to wash three times, and then the PVDF membrane was treated with ECL luminescent liquid (Beyotime, China), followed by protein band detection. Then, the AZUREc600 gel imaging system (Azurebiosystem, CA, USA) was used for detection. The expression levels of GAPDH, TLR4, Myd88, P-NF-κB, NF-κB, IL-18 and IL-6 were analyzed and quantified using ImageJ software (NIH, Bethesda, Maryland, USA).

[0126] (IV) Experimental results:

[0127] 1.qRT-PCR Analysis

[0128] like Figure 8 As shown in AD, after treatment with MTII-1-1 and MTII-1-1-Zn, the mRNA expression levels of TNF-α, IL-6, IL-1β, and IL-18 were significantly higher than those in the control group (P<0.001). In addition, compared with MTII-1-1, MTII-1-1-Zn significantly enhanced the mRNA expression of these four proinflammatory cytokines (P<0.001). These results indicate that the introduction of zinc can further enhance the promoting effect of MTII-1-1 on the mRNA expression of proinflammatory cytokines in macrophages.

[0129] 2. Western Blot (WB) Analysis

[0130] like Fig. 9As shown in AB, after treatment with MTII-1-1 and MTII-1-1-Zn, the protein expression levels of TLR4, Myd88, P-NF-κB, IL-18 and IL-6 were significantly upregulated (P<0.001), while the expression level of NF-κB did not change significantly. In addition, compared with MTII-1-1, the expression levels of these proteins were significantly increased after treatment with MTII-1-1-Zn (P<0.001), indicating that zinc modification further enhanced the activation effect of MTII-1-1 on the TLR4-Myd88-NF-κB pathway.

[0131] In summary, the present invention has isolated a homogeneous polysaccharide of Thunbergia lappa (MTII-1-1) from Thunbergia lappa, and prepared the Thunbergia lappa polysaccharide-zinc complex MTII-1-1-Zn by zinc modification. In vitro studies have shown that Thunbergia lappa polysaccharide MTII-1-1 has certain immunomodulatory activity, and the zinc-modified Thunbergia lappa polysaccharide-zinc complex MTII-1-1-Zn exhibits stronger immunomodulatory activity. Thunbergia lappa polysaccharide-zinc complex MTII-1-1-Zn activates macrophages through the TLR4-Myd88-NF-κB signaling pathway, promotes the secretion of cytokines (TNF-α, IL-6, IL-1β and IL-18), and enhances the generation of NO and ROS. In addition, in vivo studies have shown that the Thunbergia lappa polysaccharide-zinc complex MTII-1-1-Zn can significantly promote the proliferation of macrophages and has good immunomodulatory potential. In conclusion, zinc modification not only enhances the immunomodulatory activity of MTII-1-1, but also provides valuable ideas for the development of novel polysaccharide complexes with immune-enhancing functions.

[0132] It should be understood that these embodiments are only used to illustrate the present invention and are not used to limit the scope of the present invention. In addition, it should be understood that after reading the content taught by the present invention, those skilled in the art can make various changes or modifications to the present invention, and these equivalent forms also fall within the scope limited by the appended claims of the application.

Claims

1. A polysaccharide-zinc complex of Rhizoma Coptidis, characterized in that: The Thunbergia oxyphylla polysaccharide-zinc complex is composed of Thunbergia oxyphylla polysaccharide and zinc salt.

2. The polysaccharide-zinc complex of Rhizoma Coptidis as claimed in claim 1, characterized in that: The Thunbergia tongguanensis polysaccharide is a homogeneous polysaccharide, the monosaccharide composition is mannose, glucose, galactose and arabinose, and the measured molecular weight range is 1kDa-100kDa.

3. The method for preparing the polysaccharide-zinc complex of Thunbergia lappa according to any one of claims 1 to 2, characterized in that: The following steps are involved: S1. Preparation of crude polysaccharide of Thunbergia lappa S1-1, washing, drying and crushing the Radix Glehniae Radix medicinal materials to obtain Radix Glehniae Radix medicinal material tablets; S1-2, adding deionized water to the Thunb. cyperus rotundus medicinal material tablets, wherein the ratio of the added Thunb. cyperus rotundus medicinal material tablets to the added deionized water is 1-2:10 kg / L, and heating for extraction to obtain an extract; S1-3, the extract is concentrated under reduced pressure, and then anhydrous ethanol is added to a volume concentration of 45-55% ethanol, and the supernatant I is taken after standing; S1-4, adding anhydrous ethanol to the supernatant I until the ethanol volume concentration is 65-75%, separating the supernatant II after standing, and the remaining brown precipitate is deproteinized, dialyzed, and freeze-dried to obtain the crude polysaccharide of Thunb. S2. Preparation of homogeneous polysaccharide of Thunbergia lappa S2-1, the crude polysaccharide of Thunbergia tongguanensis was dissolved in deionized water and then subjected to ion exchange column chromatography, gradient elution was performed with 0-2M sodium chloride solution, the elution curve was tracked by phenol-sulfuric acid method, and the sugar part I was collected according to the elution curve, concentrated and freeze-dried, wherein the addition ratio of crude polysaccharide of Thunbergia tongguanensis to deionized water was 1-2:10 g / mL; S2-2, the sugar part I is dissolved in water, centrifuged, the clear liquid is subjected to molecular sieve gel column chromatography, eluted with water, the elution curve is detected by phenol-sulfuric acid method, and the sugar part II is collected according to the elution curve, concentrated, and freeze-dried to obtain a uniform polysaccharide of Thunbergia clematis. S3. Preparation of Thunbergia oxyphylla polysaccharide-zinc complex The prepared Thunb. Glehnia littoralis uniform polysaccharide is dissolved in deionized water, wherein the added amount ratio of Thunb. Glehnia littoralis uniform polysaccharide to deionized water is 30-50 mg / mL, and then a zinc salt solution with a concentration of 1-2 mg / mL is added, and the reaction is heated. The pH of the reaction solution is adjusted with an alkaline solution. After the reaction is completed, the pH of the solution is adjusted with an acid solution. The reaction solution is dialyzed and freeze-dried to obtain a Thunb. Glehnia littoralis polysaccharide-zinc complex.

4. The method for preparing the polysaccharide-zinc complex of Rhizoma Coptidis as claimed in claim 3, characterized in that: In step S1-2, the extraction temperature is 70-90° C., the extraction time is 2-4 hours, and the extraction times are 2-4 times.

5. The method for preparing the polysaccharide-zinc complex of Rhizoma Coptidis as claimed in claim 3, characterized in that: The extract in step S1-3 is concentrated under reduced pressure to 1 / 12 to 1 / 8 of the original volume; In steps S1-3 and S1-4, the standing temperature is 3-5°C and the standing time is 12-24h.

6. The method for preparing the Thunbergia lappa polysaccharide-zinc complex as described in 3, characterized in that: In step S3, the volume ratio of the added amount of the homogeneous polysaccharide solution of the Radix Achyranthis Bidentatae to the zinc salt solution is 2-3:

1.

7. The method for preparing the Thunbergia lappa polysaccharide-zinc complex as described in 3, characterized in that: The reaction temperature in step S3 is 45-55° C., and the stirring speed during the reaction is 200-500 rpm.

8. The method for preparing the Thunbergia lappa polysaccharide-zinc complex as described in 3, characterized in that: In step S3, the zinc salt solution is added and reacted for 8-15 minutes, and then an alkaline solution is added to adjust the pH to 8.0 and the reaction is continued for 20-48 hours. After the reaction is completed, the pH of the solution is adjusted to 3.0 with an acid solution.

9. Use of the Thunbergia oxyphylla polysaccharide-zinc complex according to any one of claims 1 to 8 in regulating immunity.

10. A composition for regulating immune function, characterized in that: The invention comprises a polysaccharide-zinc complex of Rhizoma Coptidis as an active ingredient and a pharmaceutically acceptable carrier.

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