Optimization of nutmeg polysaccharide extraction method using response surface methodology

Abstract

The invention discloses a nutmeg polysaccharide extraction process optimized by a response surface method. The process comprises the following steps: crushing clean nutmeg, sieving, defatting, hot water reflux extraction, concentrating, ethanol precipitation, suction filtration and freeze drying. The invention adopts a petroleum ether defatting-hot water extraction method for extraction, optimizes the polysaccharide extraction process by combining a single factor with a response surface test, compares the polysaccharide extraction rate, oil content, polysaccharide purity, color, antioxidant and hypoglycemic activity of nutmeg and non-defatted nutmeg under an optimal extraction process, and the nutmeg polysaccharide obtained by the optimal extraction process has white color, high extraction rate and improved activity, thereby providing a theoretical basis for the study of nutmeg polysaccharide.

Description

Technical Field

[0001] The invention relates to the technical field of biotechnology, and in particular to a method for extracting nutmeg polysaccharide optimized using a response surface methodology. Background Art

[0002] Nutmeg (Myristica fragrans Houtt.) is an evergreen tree plant of the genus Myristica in the family Myristicaceae. It is a plant that can be used as both medicine and food. Its seeds are used as medicine to treat cold diarrhea, abdominal pain, vomiting, etc.; it can be used externally as a parasite repellent and to treat rheumatism. In addition, it can also be used as a condiment, raw material for industrial oil, etc. Myristica fragrans is an important spice and medicinal plant.

[0003] Common extraction methods for natural product polysaccharides include water extraction, enzymatic hydrolysis, ultrasonic extraction, and microwave extraction. Nutmeg is a plant that contains a lot of lipids, so in the process of polysaccharide extraction, defatting is required first to improve the extraction rate and purity of polysaccharides.

[0004] Through searching, the prior art reported:

[0005] A method for optimizing the extraction conditions of yam polysaccharides using response surface methodology (CN112266423A), comprising the following steps: (1) washing, peeling and slicing the yam, freeze-drying it overnight in a freeze dryer, and crushing it with a grinder to pass through a 30-50 mesh sieve; quantitatively weighing the sieved yam powder, adding 3-4 times the volume of anhydrous petroleum ether to defatting, and then adding 4-5 times the volume of ethanol solution to remove 80%-100% of the pigment and oligosaccharide; dissolving the defatted and depigmented powder in distilled water at a material-liquid ratio of 1:10-1:11, and leaching it three times at 70°C, each time for 2-2.5 hours; combining the extracts, and concentrating them in vacuum to 1 / 5-1 / 6 of the original volume; adding 3-4 times the volume of distilled water; The ethanol was added to the solution and precipitated overnight at 4°C. The precipitate was centrifuged, and the precipitate was dissolved in water. The protein in the solution was removed by enzyme + sevage method. The salt and small molecules were removed by dialysis. The crude yam polysaccharide was obtained by freeze drying. (2) The variable parameters of yam polysaccharide extraction were determined by response surface methodology. A three-factor three-level experimental method was set up to conduct repeated experiments to obtain experimental results. The extraction rate of yam polysaccharide was calculated. Then, the software was used for multiple regression analysis. The regression equation of the influence of experimental factors on the response value was obtained as follows: R (%) = 12.00 + 0.24A + 0.34B + 0.34C - 0.65AB - 0.064AC - 0.26BC - 1.55A 2 -1.18B 2 -1.60C 2Wherein R is the extraction rate of yam polysaccharide, variable parameter A is the extraction temperature, variable parameter B is the extraction time, and variable parameter C is the solid-liquid ratio; the final extraction conditions are: temperature is 70.51℃, time is 2.06h, solid-liquid ratio is 1:10.48, and the polysaccharide extraction rate is 12.04%.

[0006] There are many studies on nutmeg volatile oil, but few studies on the extraction and activity of nutmeg polysaccharides. This study used single factor preliminary screening and response surface experiment optimization to investigate the effects of defatting times, extraction time, extraction temperature, and solid-liquid ratio on the polysaccharide extraction rate, and compared the polysaccharide extraction rate, oil content, polysaccharide purity, color, antioxidant and hypoglycemic activity of nutmeg and non-defatted nutmeg under the optimal extraction process. Summary of the invention

[0007] The present invention aims to extract nutmeg polysaccharide to the maximum extent, and provides a response surface methodology to optimize the extraction process of nutmeg polysaccharide, the steps of which are as follows:

[0008] (1) Degreasing process: Take the raw medicinal material of nutmeg, wash it, dry it to constant weight, grind it with a grinder, and sieve it; weigh a certain amount of nutmeg powder, add petroleum ether to defat it, and filter it to obtain defatted nutmeg powder;

[0009] (2) Decolorization process: adding anhydrous ethanol to remove residual petroleum ether and pigment, filtering and evaporating the ethanol to obtain defatted and decolorized nutmeg powder;

[0010] (3) Water extraction process: accurately measure the defatted and decolorized nutmeg powder, add water and heat extraction to obtain a nutmeg polysaccharide water extract; concentrate the water extract, add an appropriate amount of anhydrous ethanol, precipitate overnight, filter, evaporate the ethanol, and freeze-dry the residue to obtain nutmeg polysaccharide.

[0011] The present invention adopts fixed three factors and changes one factor to carry out a single factor experiment, and explores the influence of changes in the number of defatting times (0, 1, 2, 3, 4 times), the extraction temperature (60, 70, 80, 90, 100 DEG C) in the water extraction step, the extraction time (1.5, 2, 2.5, 3, 3.5h), and the solid-liquid ratio (1:10, 1:15, 1:20, 1:25, 1:30g:mL) on the extraction rate of nutmeg polysaccharide.

[0012] Preferably, the defatting of nutmeg powder in step (1) is repeated 1-4 times, more preferably 3 times.

[0013] Preferably, the extraction temperature of the hydrothermal extraction in step (3) is 60-100°C, more preferably 75-85°C.

[0014] Preferably, the hydrothermal extraction time in step (3) is 1.5-3.5 h, more preferably 2.5 h.

[0015] Preferably, the solid-liquid ratio of defatted nutmeg powder to water in step (3) is 1:10-30 g:ml, more preferably 1:15 g:ml.

[0016] Preferably, the specific steps of the extraction process are:

[0017] (1) Degreasing process: Take the raw material of nutmeg, wash it, place it in an oven at 60°C to dry it to constant weight, grind it with a grinder, and pass it through a 40-mesh sieve; weigh a certain amount of nutmeg powder, add petroleum ether at a solid-liquid ratio of 1:5 (g:mL), heat it to slightly boil to defat it, and filter it to obtain defatted nutmeg powder;

[0018] (2) adding anhydrous ethanol at a solid-liquid ratio of 1:3 (g:mL) to remove residual petroleum ether and pigment, filtering and placing in a water bath to evaporate the ethanol, thereby obtaining defatted and decolorized nutmeg powder;

[0019] (3) Water extraction process: accurately weigh the defatted and decolorized nutmeg powder, add water at a solid-liquid ratio of 1:15 (g:mL), and perform hot reflux extraction at an extraction temperature of 80°C for 2.5 h to obtain a nutmeg polysaccharide water extract; concentrate the water extract to 1 / 3 of its volume, add an appropriate amount of anhydrous ethanol to an alcohol concentration of 80% by volume, precipitate overnight with alcohol, filter with suction, place the filter residue in a water bath to evaporate the ethanol, and freeze-dry to obtain nutmeg polysaccharide.

[0020] The present invention adopts the response surface method, and on the basis of the single factor experiment, takes the number of degreasing, extraction temperature, extraction time, and solid-liquid ratio as the investigation factors, designs a four-factor three-level response surface analysis experiment, takes the polysaccharide extraction rate as the investigation index, and obtains the optimal extraction process as follows: degreasing 3.11 times, extraction temperature 82.37°C, extraction time 2.86h (171.6min), solid-liquid ratio 1:14.99 (g:mL). In order to facilitate the actual operation, the obtained extraction process is corrected to: degreasing 3 times, extraction temperature 82°C, extraction time 170min, solid-liquid ratio 1:15 (g:mL).

[0021] The invention compares the oil content of the optimal process of defatting 3 times with that of undefatted nutmeg, and extracts the polysaccharide in terms of extraction rate, purity, color and antioxidant activity; the oil content is 0.7% and 30.0%, the polysaccharide extraction rate is 9.13% and 3.35%, the polysaccharide purity is 50.4% and 43.8%, and the color is milky white and brown, respectively. The IC values ​​of antioxidant activity ABTS scavenging ability of polysaccharides extracted from the optimal process, defatting twice, defatting once and undefatted nutmeg are compared. 50 The IC values ​​of DPPH scavenging ability were 1.26, 1.42, 1.51 and 1.63 mg / mL respectively. 50 The IC values ​​of α-glucosidase inhibition were 1.49, 1.71, 1.90, and 2.40 mg / mL, respectively.50 They were 4.74, 5.57, 7.23 and 8.91 mg / mL respectively. It can be seen that the optimal extraction process significantly improved the extraction rate, antioxidant and hypoglycemic activities of polysaccharides.

[0022] Compared with the prior art, the present invention has the following significant advantages:

[0023] (1) This application is designed to meet existing needs, using water as the extraction agent, taking the number of degreasing times, extraction temperature, extraction time, and solid-liquid ratio as the investigation factors, investigating the effects of different parameters on the extraction rate of nutmeg polysaccharide, and using the response surface methodology to optimize the nutmeg polysaccharide extraction process;

[0024] (2) The present invention adopts ethanol decolorization, which is simple and easy to operate and has obvious decolorization effect;

[0025] (3) The present invention investigated the effect of defatting times on the extraction rate of nutmeg polysaccharide by single factor combined with response surface experiment, and found that the defatting effect was best when defatting three times, and the extraction rate of nutmeg polysaccharide was the highest;

[0026] (4) The antioxidant activity of the extracted nutmeg polysaccharide was determined and it was found that the antioxidant activity of the nutmeg polysaccharide extracted by the optimal process was better than that of the nutmeg polysaccharide extracted without defatting;

[0027] (5) The present invention measured the hypoglycemic activity of the extracted nutmeg polysaccharide and found that the antioxidant activity of the nutmeg polysaccharide extracted by the optimal process was better than that of the nutmeg polysaccharide extracted without defatting. BRIEF DESCRIPTION OF THE DRAWINGS

[0028] Figure 1 It is a schematic diagram of the effect of defatting times on the extraction rate of nutmeg polysaccharide;

[0029] Figure 2 is a schematic diagram of the effect of extraction temperature on the extraction rate of nutmeg polysaccharide;

[0030] Figure 3 is a schematic diagram showing the effect of extraction time on the extraction rate of nutmeg polysaccharide;

[0031] Figure 4 It is a schematic diagram of the effect of the solid-liquid ratio on the extraction rate of nutmeg polysaccharide;

[0032] Figure 5 is the response surface and contour map of the effects of degreasing times and extraction temperature on polysaccharide extraction rate;

[0033] Figure 6 It is the response surface and contour map of the effects of degreasing times and material-liquid ratio on polysaccharide extraction rate;

[0034] Figure 7 It is the response surface and contour map of the effects of extraction temperature and material-liquid ratio on polysaccharide extraction rate;

[0035] Figure 8 is a scatter plot of actual values ​​and predicted values;

[0036] Fig. 9 is a scatter plot of actual values ​​and residuals;

[0037] Fig.10 is a scatter plot of predicted values ​​and residuals;

[0038] Fig.11 It is a schematic diagram of the optimal extraction process of nutmeg polysaccharide and undefatted nutmeg polysaccharide;

[0039] Fig.12 This is the ABTS scavenging ability graph of nutmeg polysaccharide extracted by the optimal extraction process, defatted twice, defatted once, and not defatted;

[0040] Fig.13 This is the DPPH scavenging ability graph of nutmeg polysaccharide extracted by the optimal extraction process, defatted twice, defatted once, and not defatted;

[0041] Fig.14 This is the α-glucosidase inhibition chart of nutmeg polysaccharide extracted by the optimal extraction process, defatted twice, defatted once, and not defatted. DETAILED DESCRIPTION

[0042] Embodiment 1:

[0043] This embodiment provides a method for defatting and decolorizing nutmeg, the steps are as follows:

[0044] Take the original nutmeg material, wash it, place it in a 60℃ oven to dry to constant weight, grind it with a grinder, and pass it through a 40-mesh sieve; weigh 100g of nutmeg powder, place it in a round-bottom flask, add 500mL of anhydrous petroleum ether, heat it in a water bath until it is slightly boiling, start timing, and reflux and degrease for 1.5h; filter it with suction, take the filter residue and filter cake, and continue to add 500mL of anhydrous petroleum ether to reflux and degrease. The obtained defatted petroleum ether powder (petroleum ether is defatted 1 time, 2 times, 3 times and 4 times respectively) is decolorized by adding 300mL of anhydrous ethanol, filter it with suction, take the filter residue and place it in an evaporating dish, heat it in a 60℃ water bath to evaporate the ethanol, and dry it at 60℃ to constant weight to obtain defatted and decolorized nutmeg powder.

[0045] Embodiment 2:

[0046] This embodiment provides a method for determining the content of nutmeg polysaccharide, and the steps are as follows:

[0047] (1) Preparation of reference solution

[0048] Accurately weigh 33 mg of anhydrous glucose reference substance dried to constant weight at 105°C, place it in a 100 mL volumetric flask, add water to dissolve and dilute to the scale, shake well, and obtain (0.33 mg of anhydrous glucose per 1 ml).

[0049] (2) Preparation of anthrone-sulfuric acid

[0050] Accurately weigh 0.2 g of anthrone, add 80% sulfuric acid solution, dissolve it, make up to volume in a 100 mL brown volumetric flask, shake well, and you have it.

[0051] (3) Preparation of standard curve

[0052] Accurately measure 0.1, 0.2, 0.3, 0.4, 0.5, and 0.6 mL of the reference solution, respectively, and place them in 10 mL stoppered graduated test tubes, add water to 2.0 mL each, shake well, slowly drop 0.2% anthrone-sulfuric acid solution to the scale in an ice water bath, mix well, cool and place in a water bath to keep warm for 10 minutes, take out, immediately place in an ice water bath to cool for 10 minutes, take out, and replace the anthrone-sulfuric acid solution with distilled water as a blank. Measure the absorbance at a wavelength of 635 nm. Draw a standard curve with absorbance as the ordinate and concentration as the abscissa.

[0053] (4) Determination of polysaccharide content

[0054] Weigh 4g of defatted nutmeg powder accurately, add 60mL of water to a 150mL round-bottom flask, place in a water bath at 80℃, reflux extract for 2.5h, filter, and make up the filtrate to 60mL to obtain the test solution. Concentrate the water extract to 1 / 4 volume, add an appropriate amount of anhydrous ethanol to an alcohol concentration of 80%, precipitate overnight, filter, and freeze-dry the residue to obtain nutmeg polysaccharide.

[0055] The polysaccharide content in nutmeg was determined by anthrone-sulfuric acid colorimetric method. The absorbance of nutmeg polysaccharide was substituted into the standard curve to calculate the polysaccharide content and extraction rate.

[0056]

[0057] Where: C——polysaccharide concentration (mg / mL); N——dilution factor; V——water extract volume (mL); M——mass of nutmeg powder (g).

[0058] Embodiment 3:

[0059] This embodiment provides a method for optimizing the extraction process of nutmeg polysaccharide using response surface methodology, the steps are as follows:

[0060] 1. Single Factor Experiment

[0061] Four factors, namely, the number of defatting times, the extraction temperature in the water extraction step, the extraction time and the solid-liquid ratio, were selected to determine the polysaccharide extraction rate of nutmeg water extract.

[0062] (1) The extraction temperature was fixed at 80 °C, the extraction time was 2 h, and the solid-liquid ratio was 1:15 (g:mL). The number of degreasing times was changed to 0, 1, 2, 3, and 4 times, and the extraction rate of nutmeg polysaccharide was determined. The experimental results are shown in the attached figure. Figure 1 As shown, when the number of defatting times is 3 times, the polysaccharide extraction rate is higher, so 3 times of defatting is selected.

[0063] (2) The number of defatting times was fixed at 3 times, the extraction time was 2 h, the solid-liquid ratio was 1:15 (g:mL), and the extraction temperature was changed to 60, 70, 80, 90, and 100 °C. The extraction rate of nutmeg polysaccharide was determined. The experimental results are shown in the attached figure. Figure 2 As shown in the figure, within the extraction temperature range of 60-100°C, the polysaccharide extraction rate is higher when the extraction temperature is 80°C. Therefore, the extraction temperature is selected as 80°C.

[0064] (3) The number of defatting times was fixed at 3 times, the extraction temperature was 80 °C, the solid-liquid ratio was 1:15 (g:mL), and the extraction time was changed to 1.5, 2, 2.5, 3, and 3.5 h. The extraction rate of nutmeg polysaccharide was determined. The experimental results are shown in the attached figure. Figure 3 As shown in the figure, within the extraction time range of 1.5-3.5h, the polysaccharide extraction rate was higher when the extraction time was 2.5h. Therefore, the extraction time was selected as 2.5h.

[0065] (4) The number of defatting times was fixed at 3 times, the extraction time was 2 h, and the extraction temperature was 80 °C. The solid-liquid ratio was changed to 1:10, 1:15, 1:20, 1:25, and 1:30 (g:mL). The extraction rate of nutmeg polysaccharide was determined. The experimental results are shown in the attached figure. Figure 4 As shown in the figure, the polysaccharide extraction rate was higher when the solid-liquid ratio was 1:15. Therefore, the solid-liquid ratio was selected as 1:15 (g:mL).

[0066] 2. Response surface experiment

[0067] (1) Response surface experimental design and results

[0068] Based on the single factor experiment, a response surface experiment with four factors and three levels was established. The factor level table is shown in Table 1.

[0069] Table 1. Response surface experiment factor level table

[0070]

[0071] A total of 29 groups of experiments were responded to, with the number of degreasing times (times), extraction temperature (℃), extraction time (h) and solid-liquid ratio (g:mL) as influencing factors, and the polysaccharide extraction rate as the investigation index. The design combinations and their corresponding experimental results are shown in Table 2.

[0072] Table 2 Response surface design and experimental results

[0073]

[0074]

[0075] (2) Regression equation fitting and variance analysis

[0076] The regression equation of nutmeg polysaccharide extraction rate Y was established by fitting data with degreasing times (A), extraction temperature (B), extraction time (C) and solid-liquid ratio (D) as independent variables:

[0077] Y=+9.00+1.03×A+1.40×B+0.13×C-0.31×D-0.24×A×B+0.037×A×C+0.25×A×D-0.053×B×C+1.05×B×D+0.034×C×D-4.08×A 2 -2.39×B 2 +0.35×C 2 -2.16×D 2

[0078] The variance analysis is shown in Table 3. According to Table 3, the P value of the regression equation model is extremely significant, indicating that the model has a good fit and the experimental method is feasible; the P value of the lack of fit term is not significant, indicating that the model error is small; the larger the F value of the factor and the smaller the P value, the greater the influence of the factor on the result, so the order of the influence of each factor on the extraction rate of nutmeg polysaccharide is: extraction time> number of degreasing times> solid-liquid ratio> extraction time.

[0079] Table 3 Variance results of response surface experiment

[0080]

[0081]

[0082] Note: **: P < 0.01 is extremely significant; *: P < 0.05 is significant

[0083] (3) Response surface plot analysis

[0084] Response surface plots are used to analyze certain response target values ​​and independent variables. Together, they form a three-dimensional space diagram. By analyzing the three-dimensional response surface diagram, the influence of different factors on the response target value can be significantly reflected. In order to obtain the effect of the corresponding interaction factor of each factor in the reaction process. The effectiveness of the influence can be determined and the extreme value can be found through the response value parameters under different conditions. Response surface experimental surface plots and contour lines are as follows Figure 5-7As shown, it can be seen that the curves of the interaction terms AB, AD, and BD are steeper and have a greater impact on the results. Figure 8 This is a scatter plot of the predicted and actual values ​​in this experiment. Fig. 9 is the normal probability distribution plot of the residual, Fig.10 The scatter plot of residuals and predicted values ​​shows that the linear relationship of data distribution is good.

[0085] (4) Prediction model verification experiment

[0086] The optimal extraction process calculated by response surface software was: defatting 3.11 times, extraction temperature 82.37℃, extraction time 2.86h, solid-liquid ratio 1:14.99 (g:mL). In order to facilitate actual operation, the obtained extraction process was revised to: defatting 3 times, extraction temperature 82℃, extraction time 170min, solid-liquid ratio 1:15 (g:mL). Three verification experiments were carried out, and the average polysaccharide extraction rate was 9.13%, with a relative deviation of 5.38% from the theoretical value, proving that the extraction process obtained by the response surface experiment has a high credibility.

[0087] Example 4

[0088] This experiment compared the polysaccharide extraction rate, oil content, polysaccharide purity, color, antioxidant and hypoglycemic activity of nutmeg and non-defatted nutmeg under the optimal extraction process (defatted 3 times). The steps are as follows:

[0089] (1) Extraction rate

[0090] Accurately weigh 4 g of nutmeg powder and undefatted nutmeg powder under the optimal process, place them in a 150 mL round-bottom flask, add 60 mL of water, place it in an 82 ° C water bath, reflux extraction for 170 min, filter, make up the filtrate to 60 mL, determine the polysaccharide content by anthrone-sulfuric acid method and calculate the extraction rate, the extraction rates are 9.13% and 3.35%, respectively.

[0091] (2) Oil content

[0092] Accurately weigh 10 g of nutmeg powder and undefatted nutmeg powder under the optimal process, add 50 mL of anhydrous petroleum ether respectively, and defatting is performed according to the method provided in Example 1; accurately weigh two round-bottom flasks dried to constant weight, and the masses are 59.4279 g and 58.7095 g respectively; pour the filtrate obtained by suction filtration into the round-bottom flask while hot, use a rotary evaporator to recover the petroleum ether, place the round-bottom flask containing oil in an oven and dry to constant weight, accurately weigh, and the masses are: 59.4983 g and 61.7120 g respectively, substitute into the formula:

[0093]

[0094] In the formula: m1 is the mass of the round-bottom flask; m2 is the total mass of the round-bottom flask and the oil; m is the mass of the nutmeg powder.

[0095] The calculated oil content of defatted nutmeg is 0.7%, and the oil content of undefatted nutmeg is 30%.

[0096] (3) Polysaccharide purity

[0097] According to the method of determining the polysaccharide content in Example 2 (4), polysaccharides were extracted from nutmeg powder and non-defatted nutmeg powder under the optimal process. 5 mg of polysaccharides extracted from defatted nutmeg and polysaccharides extracted from non-defatted nutmeg were accurately weighed, dissolved in distilled water, and dissolved in 10 mL volumetric flasks to prepare a 0.5 mg / mL test solution. The polysaccharide content was determined by the anthrone-sulfuric acid method. The concentrations of defatted and non-defatted nutmeg polysaccharides were 0.252 mg / mL and 0.219 mg / mL, respectively, and the calculated purities were 50.4% and 43.8%, respectively.

[0098] (4) Polysaccharide color

[0099] According to the method for determining the polysaccharide content in Example 2 (4), the nutmeg powder and the undefatted nutmeg powder under the optimal process were extracted respectively. The results are as follows Fig.11 As shown, the nutmeg polysaccharide with the optimal extraction process is pure white (left side), and the nutmeg polysaccharide without defatting is brown (right side).

[0100] (5) Antioxidant activity test

[0101] 1. ABTS scavenging ability assay

[0102] Preparation of ABTS assay solution: ① Accurately weigh 0.189 g K2S2O4 and dilute to a 5 mL volumetric flask; ② Accurately weigh 0.0192 g ABTS and dilute to a 5 mL volumetric flask; Accurately pipette 88 μL of solution ① into solution ②, protect from light overnight for 12 hours, and obtain ABTS assay solution.

[0103] Precisely weigh the nutmeg polysaccharides extracted by the optimal process, defatted twice, defatted once, and not defatted, and prepare them into five concentrations of 0.1, 0.5, 1.0, 1.5, and 2.0 mg / mL. Precisely pipette 2.0 mL of each nutmeg polysaccharide sample solution of different concentrations into a test tube, add 2.0 mL of ABTS assay solution, shake for 10 seconds, keep it in the dark for 6 minutes, and measure the absorbance A at 734 nm. 实验 ; For the control group, 2 mL of distilled water was precisely pipetted into a test tube, and 2.0 mL of ABTS solution was added, respectively, and the tube was shaken for 10 seconds, kept in the dark for 6 minutes, and the absorbance A was measured at 734 nm. 对照 , calculate the clearance rate.

[0104]

[0105] The clearance results are as follows Fig.12 As shown, the IC values ​​of ABTS removal ability of nutmeg polysaccharide extracted by optimal process, double defatting, single defatting and non-defatting were calculated. 50 They are 1.26, 1.42,

[0106] 1.51 and 1.63 mg / mL, indicating that the antioxidant activity of nutmeg polysaccharide optimized by the optimal extraction process was significantly improved.

[0107] 2. DPPH scavenging ability determination

[0108] Preparation of DPPH assay solution: Accurately weigh 0.0039 g DPPH, add anhydrous ethanol to a 100 mL volumetric flask to obtain DPPH assay solution.

[0109] Precisely weigh the nutmeg polysaccharides extracted by the optimal process, defatted twice, defatted once, and not defatted, and prepare them into five concentrations of 0.2, 0.4, 0.6, 0.8, and 1.0 mg / mL. Precisely pipette 2.0 mL of each nutmeg polysaccharide sample solution of different concentrations into a test tube, add 2.0 mL of DPPH determination solution, shake for 10 seconds, keep it away from light for 30 minutes, and measure the absorbance A at 517 nm. 实验 ; For the blank control group, 2 mL of each concentration sample was precisely pipetted into a test tube, and 2.0 mL of distilled water was added to each tube. The absorbance A was measured at 517 nm. 空白 ; In the model control group, 2 mL of DPPH was precisely pipetted into a test tube, 2 mL of anhydrous ethanol was added, and the absorbance A was measured at 517 nm. 模型 Calculate the clearance rate.

[0110]

[0111] The clearance results are as follows Fig.13 As shown, the IC values ​​of DPPH scavenging ability of nutmeg polysaccharides extracted by optimal process, double defatting, single defatting and non-defatting were calculated. 50 They were 1.49, 1.71, 1.90 and 2.40 mg / mL, respectively, indicating that the antioxidant activity of nutmeg polysaccharide optimized by the optimal extraction process was significantly improved.

[0112] (6) Hypoglycemic activity test

[0113] α-Glucosidase inhibition assay

[0114] Preparation of phosphate buffer solution (PBS): Accurately weigh 15.292 g of K2HPO4 and 9.118 g of KH2PO4, dilute to 1000 mL volumetric flask with ultrapure water, mix well, and the solution is ready.

[0115] Preparation of α-glucosidase solution: Add an appropriate amount of α-glucosidase to a 10 mL volumetric flask with PBS until the enzyme activity reaches 1 U / mL, mix well, and the solution is ready.

[0116] Preparation of (4-nitrophenyl)-aD-pyranoglucoside (pNPG) solution: Accurately weigh 0.0301 g of pNPG and dilute to 10 mL volumetric flask with PBS, mix well, and obtain the solution.

[0117] The nutmeg polysaccharides extracted by the optimal process, defatted twice, defatted once, and not defatted were accurately weighed, and prepared into five concentrations of 0.5, 1.0, 2.0, 4.0, and 8.0 mg / mL with PBS solution. 800 μL of nutmeg polysaccharide sample solutions of different concentrations were accurately pipetted into test tubes, and 800 μL of PBS solution and 400 μL of α-glucosidase solution were added respectively. The samples were heated in a water bath at 37°C for 15 min, and 400 μL of pNPG solution was added. The samples were heated in a water bath at 37°C for 20 min, and the absorbance A was measured at a wavelength of 405 nm. 样品 ; Add an equal amount of PBS instead of α-glucosidase solution to carry out the above experiment and measure the absorbance A 背景 ; Add an equal amount of PBS instead of the nutmeg polysaccharide sample solution to carry out the above experiment and measure the absorbance A 空白 , calculate the clearance rate.

[0118]

[0119] The clearance results are as follows Fig.14 As shown in the figure, the IC values ​​of α-glucosidase inhibition of nutmeg polysaccharide extracted by optimal process, twice defatted, once defatted and without defatted were calculated. 50 They were 4.74, 5.57, 7.23 and 8.91 mg / mL, respectively, indicating that the hypoglycemic activity of nutmeg polysaccharide optimized by the optimal extraction process was significantly improved.

[0120] Through comparative experiments, it was found that this process can better optimize the extraction and activity of nutmeg polysaccharide.

[0121] The above embodiments are preferred implementation modes of the present invention, but the implementation modes of the present invention are not limited to the above embodiments. Any other changes, modifications, substitutions, combinations, and simplifications made without departing from the spirit and principles of the present invention shall be equivalent replacement modes and shall be included in the protection scope of the present invention.

Claims

1. A response surface methodology was used to optimize the extraction process of nutmeg polysaccharide, the steps are as follows: (1) Degreasing process: Wash the raw nutmeg, dry it in an oven at 60°C to constant weight, grind it with a grinder, and pass it through a 40-mesh sieve; weigh a certain amount of nutmeg powder, add petroleum ether at a solid-liquid ratio of 1:5 g / mL, heat to slightly boil to defat, and filter to obtain defatted nutmeg powder; (2) Decolorization process: Add anhydrous ethanol to the defatted nutmeg powder at a solid-liquid ratio of 1:3 g / mL to remove residual petroleum ether and pigment, filter and place in a water bath to evaporate the ethanol, and obtain defatted and decolorized nutmeg powder; (3) Water extraction process: accurately weigh the defatted and decolorized nutmeg powder, add water at a solid-liquid ratio of 1:15 g / mL, perform hot reflux extraction, and extract at 82°C for 170 min to obtain a nutmeg polysaccharide water extract; concentrate the water extract to 1 / 3 of its volume, add an appropriate amount of anhydrous ethanol to an alcohol concentration of 80 V%, precipitate overnight, filter with suction, and freeze-dry the residue to obtain nutmeg polysaccharide; In step (1), the nutmeg powder is defatted three times using petroleum ether.

2. A method for improving the antioxidant and hypoglycemic activities of nutmeg extract, comprising the following steps: (1) Degreasing process: Wash the raw nutmeg, dry it in an oven at 60°C to constant weight, grind it with a grinder, and pass it through a 40-mesh sieve; weigh a certain amount of nutmeg powder, add petroleum ether at a material-liquid ratio of 1:5 g / mL, heat to slightly boil to defat, and filter to obtain defatted nutmeg powder; (2) Decolorization process: Add anhydrous ethanol to the defatted nutmeg powder at a solid-liquid ratio of 1:3 g / mL to remove residual petroleum ether and pigment, filter and place in a water bath to evaporate the ethanol, and obtain defatted and decolorized nutmeg powder; (3) Water extraction process: accurately weigh the defatted and decolorized nutmeg powder, add water at a solid-liquid ratio of 1:15 g / mL, perform hot reflux extraction, and extract at 82°C for 170 min to obtain a nutmeg polysaccharide water extract; concentrate the water extract to 1 / 3 of its volume, add an appropriate amount of anhydrous ethanol to an alcohol concentration of 80 V%, precipitate overnight, filter with suction, and freeze-dry the residue to obtain nutmeg polysaccharide; In step (1), the nutmeg powder is defatted three times using petroleum ether; The IC value of the antioxidant activity ABTS scavenging ability of the obtained nutmeg polysaccharide 50 The IC value of DPPH scavenging ability is 1.26 mg / mL. 50 The IC value of α-glucosidase inhibition rate is 1.49 mg / mL. 50 It is 4.74 mg / mL.

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