Preparation method and application of nutmeg 0.2 M salt washing polysaccharide with multiple biological activities

Abstract

The invention relates to a preparation method and application of nutmeg 0.2 M salt washing polysaccharide with various biological activities. The decolored and degreased myristica fragrans polysaccharide is obtained by purifying myristica fragrans, crushing, sieving, carrying out ethanol reflux decoloration, carrying out petroleum ether reflux degreasing, carrying out microwave-assisted heat reflux extraction, carrying out suction filtration to obtain filtrate, concentrating, carrying out alcohol precipitation and freeze-drying. According to the microwave extraction process, a single factor is combined with a response surface experiment, extraction temperature, extraction time, liquid-to-material ratio and microwave power are taken as investigation factors, comprehensive scores of polysaccharide extraction rate, decolorization rate and purity are taken as investigation indexes, and the optimal process is as follows: the extraction time is 15 minutes, the extraction temperature is 80 DEG C, the liquid-to-material ratio is 25: 1 mL / g, the microwave power is 325 W, and the extraction rate, the decolorization rate and the purity are 6.28%, 57.40% and 70.12% respectively. The myristica fragrans polysaccharide is used as a raw material, cotton-like DEAE purification and DEAE-52 separation are adopted to obtain three components, UV, IR, HPLC and other instruments are adopted for structural analysis, the myristica fragrans polysaccharide has immunoregulation, antioxidation and hypoglycemic activities, the three activities are better than those of 0.2 M salt washing polysaccharide, and development and utilization of the myristica fragrans polysaccharide in the field of medicine are facilitated.

Description

Technical Field

[0001] The invention relates to the field of extraction technology of active ingredients of traditional Chinese medicine and drug development, and specifically to a preparation method and application of nutmeg 0.2M salt-washed polysaccharide with multiple biological activities. Background Art

[0002] Nutmeg (Myristica fragrans Houtt.), also known as meat fruit or jade fruit. It has a strong aroma and sweetness, and is often used in baking, pastries, coffee, tea, seasonings, perfumes, bath products, etc. In addition, as a traditional medicinal and edible plant, the seeds and skin of nutmeg are taken orally by the people for diarrhea, stomach cramps, flatulence, and gastric mucosal inflammation, and it is used externally as an analgesic. Modern pharmacological research mainly focuses on anti-inflammatory, analgesic, antibacterial, anti-cancer, and liver protection. Nutmeg is often boiled in water for medicinal use, but its polysaccharide as one of the main active ingredients has rarely been studied, especially microwave water extraction.

[0003] Regarding microwave extraction of polysaccharides: A method for extracting Xanthoceras sorbifolia polysaccharides from Xanthoceras sorbifolia (CN115286722A), crushing the Xanthoceras sorbifolia shell, adding petroleum ether in a mass ratio of 1:10-40 and stirring evenly, standing for stratification, filtering, and obtaining defatted Xanthoceras sorbifolia shell; adding a pretreatment agent and anhydrous ethanol (the mass ratio of defatted Xanthoceras sorbifolia shell to the pretreatment agent and anhydrous ethanol is 1:2-7:10-30) to the defatted Xanthoceras sorbifolia shell, stirring, filtering, and obtaining a decolorized Xanthoceras sorbifolia shell, wherein the pretreatment agent is a mixture of 1:10-40 and a mixture of 1:10-30. The invention discloses a method of preparing a polysaccharide comprising mixing L-ascorbyl palmitate and stevioside in a ratio of 5:10; adding distilled water (mass ratio of 1:10-25) to the decolorized Xanthoceras sorbifolia shell, using a microwave frequency of 2400-2500 MHz, a microwave power of 1100-1500 W, an extraction time of 20-50 min, and an extraction temperature of 30-90°C to obtain an extract, which is then filtered and concentrated to one fifth of the extract to obtain a concentrated solution; adding an anhydrous ethanol solution in a ratio of 1:2-5 by volume to the concentrated solution, filtering and drying to obtain Xanthoceras sorbifolia polysaccharide.

[0004] A method for microwave-assisted extraction of polysaccharides from Osmunda oleracea (CN108084290A): fresh Osmunda oleracea is washed, dried at 80°C to constant weight, crushed and sieved through a 60-mesh sieve, degreased by methanol reflux for 3 times, filtered and dried to constant weight, an appropriate amount of water is added, microwave-assisted treatment is performed at 240W for 1.5 minutes, the extraction is repeated 3 times, the supernatant is filtered and concentrated by stirring, three times of 95% ethanol is added to filter the precipitate, the precipitate is vacuum-freezed and centrifuged to obtain crude Osmunda oleracea polysaccharide powder; and then the Osmunda oleracea pure polysaccharide product is obtained through deproteinization, decolorization, macroporous resin, dialysis purification and freeze drying; the crude polysaccharide extraction rate is 21.96%, and the pure polysaccharide extraction rate is 3.08%.

[0005] The existing microwave-assisted polysaccharide extraction methods are mostly optimized for the extraction rate. The present invention targets decolorized and defatted nutmeg, investigates the comprehensive scores of the decolorization rate, extraction rate and purity of the polysaccharide through the hierarchical analysis method, optimizes the microwave extraction process, purifies with cotton-like DEAE, separates with DEAE-52, and determines the immunomodulatory, antioxidant and hypoglycemic activities of nutmeg monomer polysaccharides, thereby providing a theoretical basis for the development and utilization of nutmeg polysaccharides, improving the development and utilization of nutmeg in medicine and health food, and increasing the added value of nutmeg. Summary of the invention

[0006] In view of the characteristics of the existing polysaccharide extraction time, low efficiency, and high dissolution of pigments and impurities, the purpose of the present invention is to provide a preparation method and application of nutmeg 0.2M salt-washed polysaccharide with multiple biological activities, and the decolorization rate, extraction rate and purity of the polysaccharide are weighted by using a hierarchical analysis method combined with a response surface experiment, and the comprehensive score is calculated. The extraction temperature, extraction time, microwave power, and liquid-to-solid ratio are investigated, and the components are purified by DEAE and separated by DEAE-52, and the structure and activity of each component are determined. The steps are as follows:

[0007] (1) Take the original medicinal material nutmeg, wash it, crush it with a grinder, sieve it, and dry it to constant weight;

[0008] (2) using ethanol to reflux and decolorize to obtain decolorized nutmeg powder;

[0009] (3) using petroleum ether for reflux degreasing to obtain decolorized and defatted nutmeg powder;

[0010] (4) Decolorized and defatted nutmeg powder was extracted with microwave-assisted hot water reflux, concentrated, precipitated with alcohol, filtered and freeze-dried to obtain crude nutmeg polysaccharide.

[0011] Preferably, in step (4), the extraction rate, decolorization rate and purity of microwave-assisted hot water reflux extraction are weightedly scored by analytic hierarchy process, and the calculated weights are 59.07%, 33.38% and 7.55% respectively.

[0012] Preferably, the extraction temperature of the microwave-assisted hot water reflux extraction in step (4) is 50-90°C, more preferably 80°C.

[0013] Preferably, the extraction time of microwave-assisted hot water reflux extraction in step (4) is 10 to 30 minutes, more preferably 15 minutes.

[0014] Preferably, the liquid-to-solid ratio of microwave-assisted hot water reflux extraction in step (4) is 10:1 to 30:1 mL / g, and more preferably 25:1 mL / g.

[0015] Preferably, the power of the microwave-assisted hot water reflux extraction in step (4) is 275 to 475 W, more preferably 325 W.

[0016] Preferably, the average decolorization rate of the nutmeg crude polysaccharide obtained under the optimal process of microwave-assisted hot water reflux extraction in step (4) is 57.40%, the average extraction rate is 6.28%, the average purity is 70.12%, the average comprehensive score is 90.95, and the RSD with the model prediction is 1.16%.

[0017] (5) Purification by cotton-like DEAE cellulose chromatography column: the crude polysaccharide loading concentration is 20 mg / mL, the loading volume is 10% of the column volume, the eluent is 0.5 M NaCl solution, the flow rate is 10 mL / min, the eluate is collected, dialyzed for 24 h in a dialysis bag with a cut-off flow rate of 3500 Da, and then transferred to distilled water for dialyzation for 24 h. The water is changed every 1, 2, 4, 6, and 8 h, the dialysate is concentrated to 100-200 mL, and lyophilized to obtain purified polysaccharide.

[0018] (6) Separation by DEAE-52 cellulose chromatography column: the purified polysaccharide loading concentration was 10 mg / mL, the loading volume was 10% of the column volume, the eluent was distilled water, 0.1, 0.2, 0.3, 0.4, and 0.5 M NaCl solution, the flow rate was 1 mL / min, and the elution peaks of three components, namely, water wash, 0.1 M salt wash, and 0.2 M salt wash, were collected, dialyzed, concentrated, and freeze-dried, respectively, to obtain monomer polysaccharides of the three components with yields of 8.09%, 9.40%, and 11.91%, respectively.

[0019] (7) UV analysis results: The three component polysaccharides contain no or low amounts of nucleic acids and proteins; infrared spectra show that the spectra of the three polysaccharides are basically the same.

[0020] (8) The main components of 0.2 M salt-washed polysaccharide determined by high performance liquid chromatography were 30.41% Gal, 25.87% Arab, and 13.36% Rha, and two acidic sugars GluA and GalA accounted for 5.91% and 13.02% respectively, and minor components were 5.46% Man, 4.79% Glu, and 1.20% Xyl; the molecular weight was 7.9×10 5 Da, number average molecular weight is 6.4×10 5 Da, weight average molecular weight is 9.8×10 5 Da, the dispersion coefficient is 1.54.

[0021] (9) The results of the immunological activity of the three component polysaccharides: the results of the activity of stimulating NO secretion and the activity of phagocytosis of neutral red were 0.2 M salt washing>0.1 M salt washing>water washing.

[0022] (10) The antioxidant activities for scavenging ABTS, DPPH, and OH free radicals were all in the order of 0.2 M salt washing > 0.1 M salt washing > water washing.

[0023] (11) The results of the inhibition of α-glucosidase and α-amylase showed that the glucose-lowering activities of the two systems were 0.2 M salt washing > 0.1 M salt washing > water washing.

[0024] Compared with the prior art, the present invention has the following significant advantages:

[0025] (1) Microwave extraction is used on the basis of decolorizing and defatting nutmeg, and the optimized process time is short, the efficiency is high, and the dissolution of pigments and impurities can be avoided; the general decolorization method is to decolorize the extract by physical adsorption and other methods, which inevitably causes the loss of polysaccharides. The present invention directly decolorizes the original medicinal material and then extracts it, without the need for subsequent decolorization steps, thereby reducing the loss of polysaccharides.

[0026] (2) In the extraction process, the polysaccharide extraction rate, decolorization rate and purity were comprehensively evaluated to improve the extraction rate while ensuring the quality of the polysaccharide.

[0027] (3) The microwave extraction rate was lower than that of hot water extraction, but the extraction time was reduced from 150 min to 15 min, which was shortened tenfold. The purity was increased by 2.84%, and the decolorization rate was increased by 42.32%, which greatly improved the quality of the obtained polysaccharides.

[0028] (4) The 0.2M salt-washed polysaccharide involved in the invention: the content of protein and nucleic acid is low or non-existent; the main monosaccharides in the monomeric polysaccharide composition are 30.41% Gal, 25.87% Arab and 13.36% Rha, and the two acidic sugars GluA and GalA account for 5.91% and 13.02% respectively, and are composed of 5.46% Man, 4.79% Glu and 1.20% Xyl; the molecular weight is 7.9×10 5 Da, number average molecular weight is 6.4×10 5 Da, weight average molecular weight is 9.8×10 5 Da, dispersion coefficient 1.54.

[0029] (5) The experimental results of immunomodulatory, antioxidant and hypoglycemic activities of the components separated from nutmeg polysaccharide showed that 0.2M salt-washed polysaccharide has multiple good biological activities. BRIEF DESCRIPTION OF THE DRAWINGS

[0030] Figure 1 These are the experimental results of weighted scoring of (a) extraction temperature, (b) extraction time, (c) liquid-to-solid ratio, and (d) microwave power;

[0031] Figure 2are the interaction diagrams of (a) extraction temperature and extraction time, (b) extraction temperature and microwave power, (c) extraction temperature and liquid-to-solid ratio, (d) extraction time and microwave power, (e) extraction time and liquid-to-solid ratio, and (f) liquid-to-solid ratio and microwave power;

[0032] Figure 3 This is the elution curve of DEAE-52 cellulose separation of purified nutmeg polysaccharide;

[0033] Figure 4 (a) UV spectra and (b) IR spectra of three isolated polysaccharides;

[0034] Figure 5 It is the liquid chromatogram of the monosaccharide composition of three separated polysaccharides;

[0035] Figure 6 The graphs are (a) the results of the NO secretion experiment of RAW264.7 cells (ns: p>0.05, **: p<0.01, ***: p<0.001, ****: p<0.0001), and (b) the results of the neutral red phagocytosis experiment (*: p<0.0001).

[0036] Figure 7 Results of (a) ABTS free radical scavenging experiment, (b) DPPH free radical scavenging experiment, (c) OH free radical scavenging experiment, (d) α-glucosidase inhibition experiment, and (e) α-amylase inhibition experiment DETAILED DESCRIPTION

[0037] Example 1 This example provides a method for decolorizing and defatting nutmeg powder

[0038] Wash the nutmeg raw material, grind it, pass it through a 60-mesh sieve, and dry it at 60℃ with air blowing to constant weight.

[0039] Weigh 300g of nutmeg powder, add 80% ethanol at a liquid-to-solid ratio of 9:1mL / g, heat to a slight boiling point, reflux for decolorization for 2h, filter, take the filter residue, and air-dry at 40°C to obtain decolorized nutmeg powder; take 100g of decolorized nutmeg powder, add petroleum ether at a liquid-to-solid ratio of 5:1, heat to a slight boiling point, reflux for degreasing for 2h, filter, take the filter residue, repeat the degreasing twice, and let it stand overnight to obtain decolorized and defatted nutmeg powder.

[0040] Example 2 Polysaccharide content determination method using phenol-sulfuric acid method

[0041] The regression equation is y = 9.185x + 0.0643, R 2 =0.9997. When the glucose concentration ranges from 20 to 100 μg / mL, there is a good linear relationship between the concentration and the absorbance result. The absorbance of the sample is brought into the standard curve to calculate the polysaccharide content.

[0042]

[0043] Where C is the polysaccharide concentration in the sample (mg / mL); V is the extraction solvent volume (mL); N is the dilution factor; m is the mass of nutmeg powder (g)

[0044] Example 3 This example provides a method for calculating the decolorization rate

[0045] Nutmeg water extract is orange-yellow. According to the principle of complementary colors, the extract mainly absorbs visible blue light in the wavelength range of 446-464nm. The UV-visible spectrophotometer scans within this range and obtains a maximum absorption wavelength of 450nm, which is the detection wavelength. The decolorization rate is calculated as follows.

[0046] Decolorization rate (%) = (A0-A1) / A0×100

[0047] Where A0 and A1 are the absorbance of the polysaccharide extract at 450 nm before and after decolorization, respectively.

[0048] Example 4 This example provides a method for optimizing microwave extraction of nutmeg polysaccharides using a response surface method

[0049] 1. Analytical Hierarchy Process

[0050] Decolorization rate, extraction rate and purity were selected as indicators, and the priority order was determined through subjective analysis: extraction rate > decolorization rate > purity. The evaluation matrix of the three inspection indicators of decolorization rate, extraction rate and purity was constructed as shown in Table 1.

[0051] Table 1 Ratio scale table

[0052]

[0053] The results showed that the maximum characteristic root was 3.014 and the RI value was 0.525, so CR = CI / RI = 0.013 < 0.1, passing the consistency test. The characteristic vectors of extraction rate, decolorization rate and purity were 1.772, 1.001, and 0.226, respectively, and the weight values ​​were 59.07%, 33.38%, and 7.55%, respectively.

[0054] Overall score = 59.07A / A max +33.38B / B max +7.55C / C max

[0055] Wherein A is the polysaccharide extraction rate (%); B is the decolorization rate (%); C is the purity (%).

[0056] 2. Single Factor Experiment

[0057] Five portions of decolorized and defatted nutmeg powder, 2 g each, were taken and accurately weighed. The fixed factors were extraction temperature of 80℃, extraction time of 15 min, liquid-to-solid ratio of 25:1 mL / g, and microwave power of 325 W. The effects of extraction temperature (50, 60, 70, 80, 90℃), time (10, 15, 20, 25, 30 min), liquid-to-solid ratio (10:1, 15:1, 20:1, 25:1, 30:1 mL / g), and microwave power (275, 325, 375, 425, 475 W) on the decolorization rate, extraction rate, and comprehensive purity score of polysaccharides were studied.

[0058] like Figure 1 As shown in (a), within the extraction temperature range of 50-90℃, the comprehensive score changes in a trend of first increasing and then decreasing, reaching the highest value at 80℃. As the temperature increases, the viscosity of the extraction solvent decreases and the rate of intermolecular movement increases, which leads to an increase in the transfer rate of nutmeg polysaccharides from the cell interior to the extraction solvent. Excessively high temperatures may lead to the degradation of polysaccharides and the dissolution of impurities, resulting in a decrease in the extraction rate. Finally, an extraction temperature of 80℃ was selected as the center point of the response surface experiment.

[0059] like Figure 1 As shown in (b), within the extraction time range of 10 to 30 minutes, as the extraction time increases, the comprehensive score first increases and then decreases, reaching a maximum value at 15 minutes. This is because the extraction time is too long, which causes impurities and pigments to dissolve, affecting the comprehensive score. Therefore, the extraction time of 15 minutes is selected as a central point of the response surface experiment.

[0060] like Figure 1 As shown in (c), in the range of liquid-to-solid ratio of 10:1 to 30:1 mL / g, as the liquid-to-solid ratio increases, the comprehensive score first increases and then decreases. More solvent can make the contact surface between nutmeg powder and extraction solvent larger, and make the material in the raw material cell and the extraction solvent produce a higher density difference, which is more conducive to the transfer of polysaccharides from the cell to the solvent. However, too large a liquid-to-liquid ratio will cause many impurity molecules to occupy the space of polysaccharides, thereby reducing the extraction rate, and too much extraction solvent will lead to waste of resources and increase post-processing costs. Finally, a liquid-to-solid ratio of 25:1 mL / g was selected as a central point of the response surface experiment.

[0061] like Figure 1As shown in (d), within the microwave power range of 275-475 W, with the increase of microwave power, the comprehensive score shows a trend of first increasing and then decreasing. The reason may be that higher microwave power can generate stronger electromagnetic energy, accelerate the diffusion rate of polysaccharides into aqueous solution, and thus enhance the dissolution efficiency of polysaccharides. In addition, water molecules and other polar components in raw materials have the characteristics of absorbing electromagnetic radiation. The higher the microwave power, the faster the temperature of the material rises, resulting in a sharp increase in the internal pressure of the material, which will promote cell lysis and accelerate the outflow of polysaccharides from the cell wall. However, when the power is too large, some nutmeg polysaccharide molecules are degraded during extraction, resulting in a decrease in polysaccharide yield and a decrease in extraction rate. Finally, a microwave power of 325 W was selected as a central point of the response surface experiment.

[0062] 3. Response surface experiment

[0063] On the basis of the single factor experiment, the "Box-Behnken" design method in Design-Expert8.0.6 software was used to design a four-factor three-level experiment with decolorization rate, extraction rate, and purity comprehensive score as the response variables to optimize the microwave extraction process of nutmeg polysaccharide. The level settings of the response surface factors are shown in Table 2.

[0064] Table 2. Response surface experiment factor level table

[0065]

[0066] A response surface experiment was conducted based on the factor level table designed in Table 2, and the results are shown in Table 3.

[0067] Table 3 Response surface experimental design and results

[0068]

[0069]

[0070] Based on the results of the response surface experiment, the Design-Expert.8.0 software was used for response surface analysis. The nutmeg decolorization rate, polysaccharide extraction rate, and purity comprehensive score were used as response variables, and multiple linear regression analysis was performed on each independent factor in the experiment. The following regression equation model was obtained:

[0071] Y=91.70+1.49A-1.76B-1.43C+3.04D-0.2AB-1.12AC-4.99AD-0.51BC+5.25BD+9.10CD-13.08A 2 -15.77B 2 -15.56C 2 -9.25D 2

[0072] The variance analysis of the regression model is shown in Table 4. Model F = 23.78, P < 0.0001, indicating that the constructed model is extremely significant; the lack of fit term F = 4.27, P = 0.0874, which indicates that the unknown factors have no significant effect on the experimental results and the model is relatively stable. Model R 2 =0.9596, correction factor R 2 adj =0.9193, indicating that the model has a good fit with the actual experimental results, and the experimental error is relatively low; the signal-to-noise ratio is 15.501>4, indicating that the model not only has high precision, but also has significant response signals, and the experimental results are reliable. Based on the above data, it is shown that the model can be used to analyze and predict the microwave extraction process of nutmeg polysaccharide. And the comparison results of the effects of various influencing factors are: liquid-to-solid ratio (D)> extraction time (B)> extraction temperature (A)> microwave power (C).

[0073] Table 4 Analysis of variance of regression model

[0074]

[0075] Note: *** indicates extremely significant difference (P<0.001); ** indicates highly significant difference (P<0.01); * indicates significant difference (P<0.05).

[0076] The interactive effects of operating parameters on the comprehensive score were explored using a three-dimensional response surface. Figure 2 As shown in (a~f), the interaction between extraction temperature and microwave power is significant, and the interactions between extraction time and microwave power, and between liquid-to-solid ratio and microwave power are highly significant. This may be because when the microwave power is appropriate, the comprehensive score is more sensitive to changes in extraction time and temperature.

[0077] The optimal process conditions simulated by response surface analysis were as follows: extraction time 14.84min, extraction temperature 80.29℃, liquid-to-solid ratio 25.73:1mL / g, microwave power 324.81W; combined with the actual experimental operation, the actual process parameters were simplified to set extraction time 15min, extraction temperature 80℃, liquid-to-solid ratio 25:1mL / g, microwave power 325W, and three parallel extractions. The average extraction rate of nutmeg polysaccharide was 6.28%, the average decolorization rate was 57.40%, the average purity was 70.12%, and the average comprehensive score was 90.95, with an RSD of 1.16% compared with the model prediction, indicating that this model has good predictive power and can be used as a prediction model for optimizing the extraction process of nutmeg polysaccharide.

[0078] The present invention is compared with a hot water reflux extraction method: decolorized and defatted nutmeg powder is extracted with hot water reflux at 80°C for 2.5 hours at a liquid-to-solid ratio of 15:1 mL / g, and the extraction rate is 8.86%, the decolorization rate is 15.08%, and the purity is 67.28%. The microwave extraction rate is lower than that of the hot water extraction, but the extraction time is reduced from 150 min to 15 min, which is shortened by ten times, the purity is increased by 2.84%, the decolorization rate is increased by 42.32%, and the quality of the obtained polysaccharides is greatly improved.

[0079] Example 5 This example provides a method for purifying and separating nutmeg polysaccharide

[0080] Weigh 1000 mg of nutmeg crude polysaccharide, dissolve it in 50 mL of distilled water, and slowly add it along the wall of the chromatography column. Elute with 0.5 M NaCl solution at a flow rate of 10 mL / min, collect the eluate, dialyze it with flowing water through a dialysis bag with a cut-off flow rate of 3500 Da for 24 hours, and then transfer it to distilled water for 24 hours. Change the water every 1, 2, 4, 6, and 8 hours, concentrate the dialysate to 100-200 mL, and freeze-dry to obtain purified polysaccharide.

[0081] The purified polysaccharide was further separated by DEAE-52 cellulose chromatography column. Specifically, 100 mg of purified polysaccharide was weighed, dissolved in 10 mL of distilled water, and wet sample was added. It was eluted with pure water, 0.1, 0.2, 0.3, 0.4, and 0.5 M NaCl aqueous solution in sequence, with a flow rate of 1 mL / min, and one bottle was collected every 5 minutes. The absorbance was determined by the phenol-sulfuric acid method, and the elution curve was drawn with the number of bottles as the horizontal axis and the absorbance A as the vertical axis. The results are shown in Figure 3 As shown, it can be seen from the elution curve that the purified nutmeg polysaccharide mainly appears in 6 elution peaks on the DEAE-52 cellulose column, and the elution peaks of the first three components are collected; the 0.3, 0.4, and 0.5M NaCl components with less samples are not subjected to subsequent experiments in the present invention, and are dialyzed, concentrated, and freeze-dried according to the above method, and the yields of the three components are 8.09%, 9.40%, and 11.91%, respectively.

[0082] Example 6 This example analyzes the structure of nutmeg polysaccharide

[0083] 1. UV spectrum measurement and analysis

[0084] Take 5.00 mg of each of the three separated polysaccharides, dissolve them in 5 ml of distilled water, and then scan them with an ultraviolet spectrophotometer in the range of 200-400 nm to record the ultraviolet absorption spectrum. Figure 4 As shown in (a), the spectral curve is relatively smooth, indicating that the protein and nucleic acid content in the three component polysaccharides is low or absent.

[0085] 2. Infrared spectrum measurement and analysis

[0086] 1 mg of each purified polysaccharide component was mixed with 150 mg of KBr crystals in a mortar and ground into powder. After mixing evenly, the mixture was pressed into transparent sheets by a tablet press. The scanning range was set to 4000-400 cm in the infrared spectrometer. -1 The number of scans was set to 32. The purpose was to determine the functional groups and glycosidic bond linkage of nutmeg monomer polysaccharides. Figure 4 (b) as shown.

[0087] 3400cm -1 The peak intensity of 0.2M salt-washed polysaccharide is the highest, indicating that it contains more hydroxyl groups, may have higher hydrophilicity, and is more soluble in water. The peak intensity of 2931cm-1 is caused by CH stretching vibration. The peak intensity of water-washed polysaccharide is the highest, indicating that it has more methyl groups. The peak intensity of 0.1M salt-washed and 0.2M salt-washed polysaccharides here gradually decreases, which may be related to the increase in acidic sugar content and the corresponding decrease in the proportion of methyl groups in the molecule. -1 The COO stretching vibration is 1420 cm -1 The absorption peaks at 1000-1200cm are caused by the symmetrical vibration of C=O. The peak intensities of these two locations are the smallest for water-washed polysaccharides. As the ionic strength of the eluent increases, the peak intensities of the separated polysaccharide components also gradually increase. This is related to the increase in the acidic sugar content, which is consistent with the changes in the above-mentioned methyl peak intensity. -1 The relatively large absorption peak in this area is caused by CO stretching vibration. The water-washed polysaccharide shows three adjacent peaks, which is a typical pyran structure. The absorption peak at 850cm-1 proves that it contains α-glycosidic bonds, and it is speculated that it contains α-D-(+)-pyranose glucose. However, it is weaker in 0.1M and 0.2M salt-washed polysaccharides.

[0088] Weigh 5 mg of each polysaccharide component, add 2 mL of 2M trifluoroacetic acid, react at 110°C for 5 h, transfer the solution to an evaporating dish, add methanol several times to evaporate to remove trifluoroacetic acid. Add 2 mL of distilled water to re-dissolve, transfer 0.5 mL, add 0.5 mL of 0.5 M PMP and 0.5 mL of 0.3 M NaOH solution in sequence, place in a 70°C water bath for 1 h, cool to room temperature, and add 0.5 mL of 0.3 M HCl solution. Extract with chloroform, repeat 3 times, collect the aqueous phase, filter through a 0.22 μm microporous membrane, and analyze using HPLC.

[0089] Chromatographic conditions: Separation column: ODS2 C 18 Column (250×4.6mm 2, 5mm), mobile phase: acetonitrile-0.05mol / mL phosphate buffer pH=6.8 (18:82), flow rate: 1mL / min, column temperature: 30°C, DAD detector, detection wavelength is 245nm, injection volume is 20μL.

[0090] The experimental results are as follows Figure 5 As shown, the monosaccharide molar ratio was further calculated, and the results are shown in Table 5. The molar mass proportion of the monosaccharide composition of each polysaccharide is shown in Table 6. The results showed that nutmeg polysaccharide was composed of a variety of monosaccharides. The water-washed polysaccharide did not contain uronic acid, and was mainly composed of neutral sugar Glu (94.84%), and also contained a small amount of Rib (1.12%), Gal (2.51%), and Arab (1.53%); 0.1M salt-washed polysaccharide was mainly composed of Glu (25.27%), Gal (31.87%), and Arab (16.79%), and two acidic sugars GluA (4.45%) and GalA (5.18%) appeared; 0.2M salt-washed polysaccharide was mainly composed of Gal (30.41%) and Arab (25.87%), and the proportion of acidic sugars further increased to GluA (5.91%) and GalA (13.02%). The changes in the content of acidic sugars in nutmeg polysaccharide and the changes in the intensity of the carboxyl peak in infrared can corroborate each other.

[0091] Table 5 Molar ratio of monosaccharide composition of three polysaccharides

[0092]

[0093] Table 6 Molar mass ratio of monosaccharide composition of three polysaccharides (%)

[0094]

[0095] 4. Molecular Weight Analysis

[0096] The polysaccharide sample was prepared into a solution with a concentration of 2 mg / mL, filtered through a 0.22 μm microporous membrane, and in the HPLC analysis, a differential refractive index detector (RID) was used to determine the molecular weight of the polysaccharide. Dextran with different known molecular weights was used as a standard to draw a calibration curve. The calibration curve is:

[0097] Log(M)=17.67504-10.07584x+2.82763x 2 -0.29707x 3

[0098] The molecular weights of the three polysaccharide components are shown in Table 7. The molecular weights are arranged in the order of 0.2M salt washing>0.1M salt washing>water washing. The dispersion index refers to the ratio of weight average / number average. The closer the index is to 1, the narrower the molecular weight distribution is and the more uniform and symmetrical it is. The results show that the polysaccharides of the three components are uniform monomeric polysaccharides, and the molecular weight distribution of nutmeg polysaccharide shows a certain regularity. With the increase of the ionic strength of the eluent, the molecular weight of the separated polysaccharide gradually increases.

[0099] Table 7 Molecular weight of polysaccharide components

[0100]

[0101] Example 7 Immunoactivity Experiment

[0102] 1. Cell Culture

[0103] The RAW264.7 cell line is a mouse macrophage cell line obtained from the type culture specimen of the Chinese Academy of Sciences (Shanghai). The cells were cultured in complete medium and maintained in a humidified 5% CO2 incubator at 37°C. The cells were cultured for 36-48h to reach the logarithmic growth phase and then used in the experiment.

[0104] 2. Effect of nutmeg polysaccharide on NO secretion in RAW264.7 cells

[0105] In the study of the immunomodulatory activity of polysaccharides, the immunomodulatory properties of polysaccharides are generally evaluated by stimulating macrophages to release NO and promoting cell phagocytic ability.

[0106] NO production was determined by measuring the nitrite concentration in the supernatant of cultured RAW264.7 cells using the Griess reaction. 6 cells / mL) were pre-incubated in a 24-well plate for 24 h, and three components of nutmeg polysaccharide (800, 400, 200, 100, 50 μg / mL) were added respectively. The incubation continued for 24 h, and the supernatant was taken to measure the NO concentration. Lipopolysaccharide (2.5 μg / mL) was used as a positive control. The experimental results are shown in Figure 6As shown in (a), within the concentration range of 50-800 μg / mL, the activity of stimulating NO secretion is 0.2M salt-washed polysaccharide>0.1M salt-washed polysaccharide>water-washed. Compared with the blank control, 0.2M salt-washed polysaccharide has the best activity in stimulating NO secretion, and each concentration has significant stimulating activity (p<0.0001), which are 49.23±1.20, 50.39±0.89, 51.16±0.75, 48.39±0.62, 41.09±0.41μM, respectively, which are higher than 36.34±2.04μM of LPS. Water-washed polysaccharide has no significant stimulating activity on NO release at concentrations below 400μg / mL. The results show that 200μg / mL of 0.2M salt-washed polysaccharide has the best activity in stimulating NO secretion and has good immunomodulatory activity.

[0107] 3. Effect of nutmeg polysaccharide on the phagocytic ability of RAW264.7 cells

[0108] Wash the 24-well plate where NO release was measured in step 5.2 twice with PBS to remove non-adherent cells, and add 200 μL of neutral red solution (0.1%, g / g) to each well. Incubate for 1 hour, then discard the supernatant, wash twice with PBS, add 200 μL of cell lysis solution (ethanol: 1.0 mol / L acetic acid = 1:1, v / v), and place the plate at room temperature overnight. Measure its absorbance at 490 nm with an enzyme reader. The calculation formula of the phagocytic index is as follows:

[0109]

[0110] Neutral red test results Figure 6 As shown in (b), within the concentration range of 50-800 μg / mL, the three components all significantly promoted the phagocytic ability of RAW264.7 cells. Compared with the blank control, the phagocytic effect of the three components of polysaccharides was 0.2 M salt-washed polysaccharide > 0.1 M salt-washed polysaccharide > water-washed. 400 μg / mL of 0.2 M salt-washed polysaccharide had the strongest phagocytic effect, with a phagocytic index of 2.73±0.077.

[0111] In summary, the experiments of the two systems proved that 0.2M salt-washed nutmeg polysaccharide had better activity.

[0112] Example 8 Antioxidant Activity Experiment

[0113] (1) ABTS free radical scavenging experiment

[0114] Accurately weigh 0.189 g of potassium persulfate and dilute to volume in a 5 mL volumetric flask; accurately weigh 0.0192 g of ABTS and dilute to volume in a 5 mL volumetric flask; pipette 88 μL of potassium persulfate solution into the ABTS solution and keep it in the dark overnight for 12 h, dilute the ABTS solution kept in the dark overnight with water until the absorbance at 734 nm using a UV spectrophotometer is about 0.7.

[0115] Experimental group: 2 mL of ABTS solution was mixed with 2 mL of sample solution (2, 1, 0.5, 0.25, 0.125 mg / mL). Blank control group: 2 mL of ABTS solution was mixed with 2 mL of distilled water. The mixture was kept in dark place for 6 minutes and the absorbance of the mixture was measured at 734 nm using a UV spectrophotometer. Vc was used as a positive control.

[0116] Clearance rate (%) = [(A1–A2) / A1] × 100

[0117] A1 is the absorbance of the blank control group, and A2 is the absorbance of the experimental group.

[0118] (2) DPPH free radical scavenging experiment

[0119] Weigh 0.0039g of DPPH accurately into a 100mL volumetric flask and adjust the volume with anhydrous ethanol. Experimental group: 2mL of sample (4, 2, 1, 0.5, 0.25mg / mL) mixed with 2mL of DPPH solution; blank control group: 2mL of samples of different concentrations mixed with 2mL of anhydrous ethanol solution; model control group: 2mL of DPPH mixed with 2mL of anhydrous ethanol; react in the dark for 30min, and measure the absorbance at 517nm. Vc is used as the positive control.

[0120] Clearance (%) = [1-(A1-A2) / A0] × 100

[0121] A1 is the absorbance of the experimental group, A2 is the absorbance of the blank control group, and A0 is the absorbance of the model control group.

[0122] (3) Hydroxyl radical scavenging experiment

[0123] Accurately weigh 0.125 g of Fe2SO4 into a 50 mL volumetric flask and make up to volume with distilled water. Accurately weigh 0.1243 g of salicylic acid into a 100 mL volumetric flask and make up to volume with 70% ethanol. Accurately transfer 0.1 mL of H2O2 into a 100 mL volumetric flask and make up to volume with pure water.

[0124] Experimental group: 2 mL of polysaccharide sample ((2, 1, 0.5, 0.25, 0.125 mg / mL), added with 2 mL of Fe2SO4 solution and 2 mL of salicylic acid-ethanol, and added with 2 mL of H2O2; background control group: 2 mL of sample, added with 2 mL of Fe2SO4 solution and 2 mL of salicylic acid-ethanol, and added with 2 mL of H2O2; blank control group: 2 mL of distilled water, added with 2 mL of Fe2SO4 solution and 2 mL of salicylic acid-ethanol, and added with 2 mL of H2O2, mixed thoroughly, kept in a constant temperature water bath at 37°C for 30 min, cooled to room temperature, and measured the absorbance at 510 nm. Vc was used as the positive control.

[0125] Clearance (%) = [1-(A1-A2) / A0] × 100

[0126] A1 is the absorbance of the experimental group, A2 is the absorbance of the background control group, and A0 is the absorbance of the blank control group.

[0127] (4) Antioxidant test results

[0128] The results of ABTS free radical scavenging ability test are as follows Figure 7 As shown in (a), when the sample concentration is 2 mg / mL, the removal rates of water washing, 0.1 M salt washing, and 0.2 M salt washing are 16.41%, 52.71%, and 89.92%, respectively. The results of the DPPH free radical scavenging ability test are shown in Figure 7 As shown in (b), when the sample concentration is 4 mg / mL, the removal rates of water washing, 0.1 M salt washing, and 0.2 M salt washing are 17.31%, 50.15%, and 72.67%, respectively. The results of the OH radical scavenging ability test are shown in Figure 7 As shown in (c), when the sample concentration was 2 mg / mL, the removal rates of water washing, 0.1 M salt washing, and 0.2 M salt washing were 19.24%, 39.34%, and 54.60%, respectively.

[0129] In summary, the antioxidant activity results of the three systems were 0.2 M salt washing > 0.1 M salt washing > water washing.

[0130] Example 9 Hypoglycemic Activity Experiment

[0131] (1) α-glucosidase inhibition test

[0132] Experimental group: 400 μL of PBS was pipetted, and 400 μL of samples of different concentrations (10, 5, 2.5, 1.25, 0.625 mg / mL) and 200 μL of α-glucosidase were added and mixed, and heated in a water bath at 37°C for 15 min. After completion, 200 μL of pNPG solution was added, and heated in a water bath at 37°C for 20 min; Negative control group: The same amount of PBS was used instead of the sample for the experiment; Background control group: The same amount of PBS was used instead of α-glucosidase for the experiment. The absorbance was measured at 405 nm. The positive control was acarbose.

[0133] Inhibition rate (%) = [1-(A1-A2) / A0] × 100

[0134] A1 is the absorbance of the experimental group, A0 is the absorbance of the negative control group, and A2 is the absorbance of the background control group.

[0135] (2) α-amylase inhibition test

[0136] Experimental group: Take 0.5mL of samples of different concentrations (8, 4, 2, 1, 0.5mg / mL), add 0.5mL of 50U / L α-amylase, water bath at 70℃ for 10min, add starch solution, water bath at 70℃ for 10min, add 0.5mL of 0.1M hydrochloric acid solution and 2mL of dilute iodine solution; Control group: Use an equal amount of pure water instead of α-amylase for the experiment; Blank group: Use an equal amount of pure water instead of the sample for the experiment. The positive control is acarbose.

[0137] Inhibition rate (%) = A1 / (A2-A3) × 100

[0138] A1 is the absorbance of the experimental group, A2 is the absorbance of the control group, and A3 is the absorbance of the blank group.

[0139] (3) Results of hypoglycemic experiments

[0140] The results of α-glucosidase inhibition activity assay were as follows Figure 7 As shown in (d), when the sample concentration was 10 mg / mL, the inhibition rates of water washing, 0.1 M salt washing, and 0.2 M salt washing were 37.64%, 42.95%, and 71.81%, respectively. Figure 7 As shown in (e), when the sample concentration was 10 mg / mL, the inhibition rates of water washing, 0.1 M salt washing, and 0.2 M salt washing were 23.94%, 40.60%, and 48.31%, respectively.

[0141] In summary, the hypoglycemic activity results of the two systems were 0.2M salt washing > 0.1M salt washing > water washing, and the 0.2M salt-washed polysaccharide had the best hypoglycemic activity.

[0142] The above embodiments are preferred implementation modes of the present invention, but the protection scope of the present invention is not limited thereto, and any other changes, modifications, substitutions, combinations, and simplifications made without departing from the spirit and principles of the present invention shall be considered as equivalent replacement methods and shall be included in the protection scope of the present invention.

Claims

1. A preparation method and application of 0.2M salt-washed nutmeg polysaccharide with multiple biological activities, characterized by the following: (1) Microwave-assisted hot water reflux extraction was used to optimize the extraction process by using the analytic hierarchy process based on the comprehensive scores of polysaccharide extraction rate, decolorization rate, and purity to obtain crude polysaccharides; (2) The crude polysaccharide is purified by a cotton-like DEAE chromatography column, and then the purified polysaccharide is further separated by a DEAE-52 cellulose chromatography column to obtain three components of monomeric polysaccharides; (3) Among them, 0.2M salt-washed polysaccharide has good immunomodulatory, antioxidant and hypoglycemic activities.

2. The method according to claim 1, characterized in that: In step (1), for microwave-assisted hot water reflux extraction, the extraction process was optimized by analytic hierarchy process based on the comprehensive scores of polysaccharide extraction rate, decolorization rate and purity, and the calculated weight values ​​were 59.07%, 33.38% and 7.55% respectively.

3. The method according to claim 1, characterized in that: In step (1), the extraction temperature was optimized to 80°C, the extraction time was 15min, the liquid-to-solid ratio was optimized to 25:1mL / g, and the power was 325W. The results were a decolorization rate of 57.40%, an extraction rate of 6.28%, a purity of 70.12% and a comprehensive score of 90.95, and nutmeg crude polysaccharide was obtained by freeze drying.

4. The method according to claim 1, characterized in that: The purification described in step (2) is carried out using a cotton-like DEAE cellulose chromatography column: the crude polysaccharide loading concentration is 20 mg / mL, the loading volume is 10% of the column volume, the eluent is a 0.5 M NaCl solution, the flow rate is 10 mL / min, the eluate is collected, dialyzed for 24 hours through a dialysis bag with a cut-off flow of 3500 Da, and then transferred to distilled water for dialysis. The water is changed every 1, 2, 4, 6, and 8 hours, and the dialyzate is concentrated to 100-200 mL, and lyophilized to obtain purified polysaccharide.

5. The method according to claim 1, characterized in that: The separation described in step (2) is carried out using a DEAE-52 cellulose chromatography column: the purified polysaccharide loading concentration is 10 mg / mL, the loading volume is 10% of the column volume, the eluent is distilled water, 0.1, 0.2, 0.3, 0.4, and 0.5 M NaCl solution, the flow rate is 1 mL / min, and the eluate is dialyzed, concentrated, and freeze-dried in the same manner as above to obtain monomer polysaccharides of three components: water wash, 0.1 M salt wash, and 0.2 M salt wash.

6. The method according to claim 5, characterized in that: The water-washed polysaccharide is mainly composed of Glu with a high proportion of 94.84%; the 0.1M salt-washed polysaccharide is composed of 31.87% Gal, 25.27% Glu, 16.79% Arab, 11.35% Man, 5.09% Xyl, and two acidic sugars 4.45% GluA and 5.18% GalA; the main components of the 0.2M salt-washed polysaccharide are 30.41% Gal, 25.87% Arab and 13.36% Rha, and the proportions of the two acidic sugars GluA and GalA are further increased to 5.91% and 13.02%; the molecular weights are 3.8×10 5 , 6.5×10 5 ,7.9×10 5 Da.

7. The method according to claim 5, characterized in that: The immunomodulatory, antioxidant and hypoglycemic activities of the three component polysaccharides are 0.2M salt-washed>0.1M salt-washed>water-washed, and the 0.2M salt-washed polysaccharide has all three good biological activities.

8. The method according to claim 1, characterized in that: The extracted sample was decolorized and defatted nutmeg powder.

9. The method according to claim 1, characterized in that: The prepared 0.2M salt-washed polysaccharide is used in drugs with immunomodulatory, antioxidant and hypoglycemic activities.

Images