CN110078841B - Dragon-pearl fruit polysaccharide with immunoregulation function and preparation method and application thereof - Google Patents
Dragon-pearl fruit polysaccharide with immunoregulation function and preparation method and application thereof Download PDFInfo
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- A—HUMAN NECESSITIES
- A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
- A23L—FOODS, FOODSTUFFS, OR NON-ALCOHOLIC BEVERAGES, NOT COVERED BY SUBCLASSES A21D OR A23B-A23J; THEIR PREPARATION OR TREATMENT, e.g. COOKING, MODIFICATION OF NUTRITIVE QUALITIES, PHYSICAL TREATMENT; PRESERVATION OF FOODS OR FOODSTUFFS, IN GENERAL
- A23L33/00—Modifying nutritive qualities of foods; Dietetic products; Preparation or treatment thereof
- A23L33/10—Modifying nutritive qualities of foods; Dietetic products; Preparation or treatment thereof using additives
- A23L33/125—Modifying nutritive qualities of foods; Dietetic products; Preparation or treatment thereof using additives containing carbohydrate syrups; containing sugars; containing sugar alcohols; containing starch hydrolysates
-
- C—CHEMISTRY; METALLURGY
- C08—ORGANIC MACROMOLECULAR COMPOUNDS; THEIR PREPARATION OR CHEMICAL WORKING-UP; COMPOSITIONS BASED THEREON
- C08B—POLYSACCHARIDES; DERIVATIVES THEREOF
- C08B37/00—Preparation of polysaccharides not provided for in groups C08B1/00 - C08B35/00; Derivatives thereof
-
- C—CHEMISTRY; METALLURGY
- C08—ORGANIC MACROMOLECULAR COMPOUNDS; THEIR PREPARATION OR CHEMICAL WORKING-UP; COMPOSITIONS BASED THEREON
- C08B—POLYSACCHARIDES; DERIVATIVES THEREOF
- C08B37/00—Preparation of polysaccharides not provided for in groups C08B1/00 - C08B35/00; Derivatives thereof
- C08B37/0003—General processes for their isolation or fractionation, e.g. purification or extraction from biomass
-
- A—HUMAN NECESSITIES
- A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
- A23V—INDEXING SCHEME RELATING TO FOODS, FOODSTUFFS OR NON-ALCOHOLIC BEVERAGES AND LACTIC OR PROPIONIC ACID BACTERIA USED IN FOODSTUFFS OR FOOD PREPARATION
- A23V2002/00—Food compositions, function of food ingredients or processes for food or foodstuffs
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- Chemical Kinetics & Catalysis (AREA)
- General Health & Medical Sciences (AREA)
- Biochemistry (AREA)
- Materials Engineering (AREA)
- Medicinal Chemistry (AREA)
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- Food Science & Technology (AREA)
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- Medicines Containing Plant Substances (AREA)
Abstract
The invention discloses a longzhuguo polysaccharide with immunoregulation function, a preparation method and application thereof, wherein the preparation method comprises the following steps: extracting the powder of the longzhuguo with water, and taking the supernatant; adding absolute ethyl alcohol, standing, and collecting precipitate; removing protein by adopting a Sevag method after dissolving and precipitating; removing pigment by macroporous resin column chromatography; removing small molecular impurities, and drying. The invention establishes the longzhuguo polysaccharide extraction process, and the process method is simple to operate, convenient, reasonable, stable and quantifiable to use, and has important application value. The polysaccharide of the dragon fruit extracted by the method is evaluated by in vitro immunoregulation activity, so that the multiplication capacity of macrophages is obviously improved, NO, IL-6 and TNF-alpha can be obviously promoted to be secreted, and the polysaccharide is a natural polysaccharide with good immunoregulation effect and has important development and application values.
Description
Technical Field
The invention relates to the technical field of functional foods, in particular to a dragon-pearl fruit polysaccharide with immunoregulation function and a preparation method and application thereof.
Background
Longzhu fruit (passflora foetida L.), another name: pseudo-bitter fruits, dragon beard fruits, longan fruits and the like, passion fruit plants and herbaceous vines. The berry egg is spherical, sour and sweet, and the seed is edible. Meanwhile, the whole plant of the longzhu fruit can be used as a medicine and is decocted in water for treating lung diseases, cough and asthma. In addition, the longzhu fruit also has the effects of clearing away heat and toxic materials, diminishing inflammation, relieving pain, resisting depression, resisting bacteria, improving osteoporosis and the like, but reports on the biological activity of the longzhu fruit related to the immune system are not found so far.
Polysaccharides are polar macromolecular compounds. Generally extracted from animal, plant, microorganism, and seaweed. Up to now, almost ten thousand kinds of polysaccharide compounds have been extracted. Many experiments prove that the polysaccharide has close relation with infection, tumor, inflammation and some autoimmune diseases. Therefore, research and development of polysaccharides have attracted more and more attention. The research on the biological activity of polysaccharide drugs is the most active and most advanced field in the research on polysaccharides. Polysaccharides from a variety of different sources have been found in recent years to play a critical role in the immune system. The extraction of polysaccharide generally comprises the steps of degreasing, enzyme hydrolysis, hot water extraction, alcohol precipitation, drying, crushing and the like, and parameters of each process are adjusted according to the characteristics of an extraction object, but no report related to the extraction method of the polysaccharide of the longzhuguo exists so far.
Disclosure of Invention
The invention aims to overcome the defect that no method for extracting polysaccharide with immunoregulation effect from longzhuguo exists in the prior art, and provides polysaccharide with immunoregulation effect and a preparation method and application thereof.
The first purpose of the invention is to provide a preparation method of polysaccharide of longzhuguo.
The second purpose of the invention is to provide the longzhuguo polysaccharide prepared by any one of the preparation methods.
The third purpose of the invention is to provide the application of the polysaccharide of the dragon fruit in preparing functional food.
In order to achieve the purpose, the invention is realized by the following technical scheme:
according to the invention, the extraction process of the polysaccharide of the dragon fruit is optimized by combining a response surface method through a single-factor experiment, so that the optimal preparation process parameters of the polysaccharide of the dragon fruit are obtained, and the extraction process method of the polysaccharide of the dragon fruit is obtained. Meanwhile, the influence of the polysaccharide on the proliferation capacity of macrophage RAW264.7, the NO release amount and the secretion amounts of several immune related cytokines (IL-6 and TNF-alpha) is observed by adopting a method of intervening by the polysaccharide of the dragon fruits with different concentrations, so that the active immunoregulation activity function of the polysaccharide extracted from the dragon fruits is evaluated.
The invention therefore claims a preparation method of polysaccharide of pitaya, which comprises the following steps:
s1, adding water into the dragon pearl fruit powder for extraction, centrifuging to obtain a supernatant, repeatedly extracting, and combining the supernatants to obtain a crude dragon pearl fruit polysaccharide extract;
s2, concentrating the crude polysaccharide extract of the dragon ball fruits, adding absolute ethyl alcohol, standing, and collecting precipitates;
s3, removing protein by adopting a Sevag method after dissolving the precipitate;
s4, removing pigments by macroporous resin column chromatography;
and S5, removing small molecular impurities and drying.
Preferably, in step S1, the dragon fruit powder is fresh and mature dragon fruit, and is obtained by washing, airing, drying, crushing and sieving.
More preferably, in step S1, the drying conditions are 45 ℃ for 24 h.
Further preferably, in step S1, the size of the dragon fruit powder is 40 mesh.
Preferably, in step S1, the weight ratio of the dragon fruit powder to the water is 1: 5-1: 30.
more preferably, in step S1, the weight ratio of the dragon fruit powder to the water is 1: 5-1: 25.
further preferably, in step S1, the weight ratio of the dragon fruit powder to the water is 1: 15.5.
Preferably, in step S1, the extraction time is 10-60 min.
More preferably, in step S1, the extraction time is 10-50 min.
Further preferably, in step S1, the extraction time is 31 min.
Preferably, in the step S1, the extraction temperature is 30-90 ℃.
More preferably, in step S1, the extraction temperature is 35-65 ℃.
More preferably, in step S1, the extraction temperature is 55 deg.C
More preferably, in step S1, the extraction is repeated 3 times.
More preferably, in step S1, centrifugation is carried out at 12000r/min for 20 min.
Preferably, in step S2, the crude extract of polysaccharide from garcinia mangostana is concentrated, added with absolute ethanol, left overnight at 4 ℃, centrifuged, and the precipitate is collected.
Preferably, in step S2, concentration is performed by rotary evaporation.
Preferably, in step S2, the mixture is concentrated to 1/10 of the original volume, and 3 times of anhydrous ethanol is added.
Preferably, in step S2, it is left to stand at 4 ℃ overnight.
Preferably, in step S2, centrifugation is carried out at 12000r/min for 15 min.
Preferably, in step S3, the aqueous solution of the precipitate of step S2 is mixed with Sevag solution, solid-liquid separation is performed, the upper aqueous solution is collected, the precipitate is removed, and the above operation is repeated to remove the organic solvent.
More preferably, in step S3, the volume ratio of the precipitated aqueous solution of step S2 to the Sevag solution is 4: 1.
more preferably, in step S3, it is repeated 3 times.
More preferably, in step S3, centrifugation is carried out at 12000r/min for 15min for solid-liquid separation.
Preferably, in step S3, the organic solvent is removed by vacuum rotary evaporation.
More preferably, in step S3, the Sevag solution is mixed in a volume ratio of 4:1 chloroform and n-butanol.
Preferably, in step S4, pigment is removed by AB-8 macroporous resin column chromatography.
More preferably, in step S4, the aqueous solution of the product of step S3 is loaded onto a macroporous resin chromatographic column, eluted through macroporous resin, then eluted with distilled water, and the eluate is collected.
Further preferably, in step S4, a flow rate of 2mL/min is eluted through the macroporous resin.
Further preferably, in step S4, 3 column volumes of distilled water are eluted.
Further preferably, in step S4, the eluent is concentrated to 1/10 of the total volume.
Preferably, in step S5, the product of step S4 is added into a dialysis bag and dialyzed in distilled water.
More preferably, in step S5, the dialysis bag is 5kDa in size.
Further preferably, in step S5, the dialysis is performed for 48h, and the dialysate is changed every 6 h.
More preferably, in step S5, freeze-drying is performed.
Most preferably, the preparation method of the dragon pearl fruit polysaccharide comprises the following steps:
s1, adding water into the dragon pearl fruit powder for extraction, extracting at 30-90 ℃ for 10-60 min, centrifuging, taking supernatant, repeatedly extracting, and combining supernatant to obtain a dragon pearl fruit polysaccharide crude extract, wherein the dragon pearl fruit powder is fresh and mature dragon pearl fruit and is obtained by cleaning, airing, drying, crushing and sieving, and the weight ratio of the dragon pearl fruit powder to water is 1: 5-1: 30, of a nitrogen-containing gas;
s2, concentrating the crude polysaccharide extract of the dragon fruit, adding absolute ethyl alcohol, standing overnight at 4 ℃, centrifuging, and collecting precipitate;
s3, removing protein by adopting a Sevag method after dissolving and precipitating: in step S3, uniformly mixing the aqueous solution of the precipitate in step S2 with Sevag solution, carrying out solid-liquid separation, collecting the upper-layer aqueous solution, removing the precipitate, and repeating the operation to remove the organic solvent;
s4, removing pigments by AB-8 macroporous resin column chromatography: adding AB-8 into the aqueous solution of the product obtained in the step S3, putting the aqueous solution into a macroporous resin chromatographic column, eluting the aqueous solution through macroporous resin, eluting the aqueous solution with distilled water, and collecting the eluent;
s5, removing small molecular impurities, and drying: adding the water solution of the product of step S4 into dialysis bag, dialyzing in distilled water, and freeze drying.
Most preferably, the preparation method of the dragon pearl fruit polysaccharide comprises the following steps:
s1.40-mesh dragon fruit powder is extracted by adding water, the extraction is carried out at 55 ℃ for 31min, the centrifugation is carried out at 12000r/min for 20min, supernatant is taken, the extraction is repeated for 3 times, and the supernatant is combined to obtain a crude dragon fruit polysaccharide extract, wherein the dragon fruit powder is fresh and mature dragon fruit and is obtained by cleaning, airing, drying at 45 ℃ for 24h, crushing and sieving, and the weight ratio of the dragon fruit powder to the water is 1: 15.5;
s2, concentrating the crude polysaccharide extract of the dragon ball fruits to 1/10 of the original volume through rotary evaporation, adding 3 times of volume of absolute ethyl alcohol, standing overnight at 4 ℃, centrifuging for 15min at 12000r/min, and collecting precipitates;
s3, removing protein by adopting a Sevage method after dissolving the precipitate: in step S3, the aqueous solution of precipitate obtained in step S2 is mixed with Sevage solution at a volume ratio of 4:1,12000 r/min, centrifuging for 15min, collecting supernatant and precipitate, collecting precipitate, repeating the above operation for 3 times, combining supernatants, and vacuum rotary evaporating to remove organic solvent, wherein the volume ratio of Sevage solution is 4:1 chloroform and n-butanol;
s4, removing pigments by AB-8 macroporous resin column chromatography: diluting the product obtained in the step S3 by 5-10 times with water, adding the diluted product into a macroporous resin chromatographic column, eluting the product at a flow rate of 2mL/min through macroporous resin, eluting the product with distilled water of 2 times of the volume of the column, collecting eluent, and concentrating the eluent to 1/10 of the total volume;
s5, removing small molecular impurities, and drying: adding the product of step S4 into dialysis bag with specification of 5kDa, dialyzing in distilled water for 72h, changing dialysate every 6h, and freeze drying.
Meanwhile, the invention also claims the longzhuguo polysaccharide prepared by the preparation method.
Further, the invention claims the application of the polysaccharide of the dragon fruit in the preparation of functional food.
The invention also claims the functional food containing the polysaccharide of the dragon fruit.
Preferably, the functional food is a functional food for improving an immunoregulatory ability.
More preferably, the immunoregulation capability is one or more of increasing the proliferation capability of macrophages, promoting the secretion of NO by the macrophages, promoting the secretion of IL-6 by the macrophages or promoting the secretion of TNF-alpha by the macrophages.
Compared with the prior art, the invention has the following beneficial effects:
the invention establishes the longzhuguo polysaccharide extraction process, and the process method is simple to operate, convenient, reasonable, stable and quantifiable to use, and has important application value. The polysaccharide of the dragon fruit extracted by the method is evaluated by in vitro immunoregulation activity, so that the multiplication capacity of macrophages is obviously improved, NO, IL-6 and TNF-alpha can be obviously promoted to be secreted, and the polysaccharide is a natural polysaccharide with good immunoregulation effect and has important development and application values.
Drawings
FIG. 1 is a technical scheme of the present invention.
FIG. 2 is a glucose standard curve.
FIG. 3 is a graph showing the effect of extraction time on polysaccharide extraction amount from strobilus Juniperi Rigidae.
FIG. 4 is a graph showing the effect of extraction temperature on the amount of polysaccharide extracted from Ficus tikoua.
FIG. 5 is a graph showing the effect of feed liquid ratio on the amount of polysaccharide extracted from Ficus tikoua.
FIG. 6 is a response surface plot of a quadratic regression equation for extraction time (A) and extraction temperature (B).
FIG. 7 is a response surface plot of a quadratic regression equation for extraction time (A) and feed-liquid ratio (C).
FIG. 8 is a response surface plot of a quadratic regression equation for extraction temperature (B) and feed-to-liquid ratio (C).
FIG. 9 is a graph of the effect of PFP on the proliferation of RAW264.7 macrophages.
FIG. 10 is a graph of the effect of PFP on NO secretion from RAW264.7 macrophages.
FIG. 11 is a graph showing the effect of PFP on IL-6 secretion from RAW264.7 macrophages.
FIG. 12 is a graph of the effect of PFP on TNF- α secretion by RAW264.7 macrophages.
Detailed Description
The invention is described in further detail below with reference to the drawings and specific examples, which are provided for illustration only and are not intended to limit the scope of the invention. The test methods used in the following examples are all conventional methods unless otherwise specified; the materials, reagents and the like used are, unless otherwise specified, commercially available reagents and materials.
Example 1A method for preparing polysaccharide of longzhuguo
Extraction of polysaccharide from Yilongzhuguo
1. Cleaning fresh and mature fructus Pyracanthae, naturally drying surface water, oven drying in oven (45 deg.C, 24 hr), pulverizing, and sieving (40 mesh);
2. adding distilled water into dried longzhuguo powder (400g) according to the ratio of material to liquid of 1:15.5, and leaching in a water bath at 55 ℃ for 31 min. After centrifugation at 12000r/min for 20min, the supernatant was collected. Adding distilled water into the centrifuged precipitate according to the method, repeatedly extracting for three times, and mixing the supernate to obtain the extract of the total sugar of the dragon pearl fruit;
3. concentrating the extract of the total sugar of the dragon pearl fruits to 1/10 times of the original volume by rotary evaporation, adding 3 times of absolute ethyl alcohol, putting the mixture into a refrigerator at 4 ℃, standing overnight, taking out the mixture, centrifuging the mixture for 15min at 12000r/min, collecting precipitates, and freeze-drying the precipitates to obtain the crude polysaccharide of the dragon pearl fruits.
Secondly, purifying the crude polysaccharide of the dragon pearl fruit
1. The Sevag method, a classical and highly efficient protein removal method, was used.
And (3) collecting the precipitate in the last step according to the weight ratio of 1: 10 adding distilled water for re-dissolving and mixing, uniformly mixing with Sevag solution (prepared by mixing chloroform and n-butanol according to the ratio of 4:1 (v/v)) according to the ratio of 4:1(v/v), centrifuging for 15min under the condition of 12000r/min, locating the denatured protein at the junction of the water layer and the organic solvent layer, and taking the upper polysaccharide aqueous solution. Repeating the operation for three times, rotationally evaporating the organic solvent in vacuum, and freeze-drying to obtain the protein-removed polysaccharide of the longzhu fruit.
2. The protein-removed longzhuguo polysaccharide water solution is dark brown liquid, and for further purification, the invention adopts AB-8 macroporous resin column chromatography to remove pigments.
Removing protein from the longzhuguo polysaccharide according to the weight ratio of 1: adding 100 times of distilled water for redissolution, mixing, slowly adding into macroporous resin chromatographic column, eluting with macroporous resin at flow rate of about 2mL/min, and slowly eluting with 2 times of column volume of distilled water. Collecting all eluates, concentrating to 1/10 of total volume, and freeze drying to obtain protein-removed and pigment-removed polysaccharide.
3. Removing protein and pigment of the longzhuguo polysaccharide according to the weight ratio of 1: 5 adding distilled water, redissolving, mixing, adding into dialysis bag (5kDa), dialyzing in distilled water for 72 hr (changing dialysate every 6 hr), and freeze drying to obtain white powder, i.e. polysaccharide (PFP).
Determination of polysaccharide content of longzhuguo
Determining the absorbance of glucose with different concentrations by a phenol-sulfuric acid method to prepare a concentration-absorbance standard curve, preparing each sample of the longzhuguo polysaccharide into a solution with a certain concentration, measuring the absorbance of the solution according to the standard curve term, substituting the solution into a concentration-absorbance standard curve equation, and calculating to obtain the polysaccharide content.
Preparation of a standard curve: accurately weighing 4mg of standard glucose into a 100mL volumetric flask, adding distilled water, uniformly mixing and fixing the volume to obtain a glucose standard solution. Accurately sucking the glucose standard solution into a clean test tube, gradually increasing the volume in a certain gradient manner to 0.4 mL, 0.6 mL, 0.8 mL, 1.0mL, 1.2 mL, 1.4 mL, 1.6 mL and 1.8mL respectively, adding distilled water to a uniform volume of 2mL, and simultaneously using 2mL of distilled water as a blank control. Then, 1.0mL of 6% phenol and 5.0mL of concentrated sulfuric acid were added to each tube. Shaking the mixture, standing for 20min, and measuring absorbance of the solution at 490nm to obtain regression equation20.732x+0.0006(r20.9921). The results showed that the concentration of 8.0 to 36.0. mu.g/mL had a good linear relationship (FIG. 2).
The method is adopted to respectively detect the content and the proportion of polysaccharide in crude polysaccharide, deproteinized and pigment polysaccharide of longzhuguo and a final product (PFP).
Third, experimental results
Freeze-drying the extract of the total sugar of the dragon pearl fruits to obtain 11.56g of crude polysaccharide of the dragon pearl fruits, wherein the yield of the crude polysaccharide of the dragon pearl fruits is 2.89 percent;
after freeze drying, 8.65g of deproteinized longzhuguo polysaccharide, 5.84g of deproteinized and pigmented longzhuguo polysaccharide and 2.38g of final product (PFP) are obtained respectively. The content of polysaccharide in each product is determined by adopting a phenol-sulfuric acid method, and the content of polysaccharide in the product accounts for 42.3 percent, 65.1 percent and 83.6 percent of the total mass of each product respectively.
Example 4 Effect of extraction time on polysaccharide extraction from Ficus tikoua
First, experiment method
The feeding ratio is 1: 20(g/mL) and 50 ℃ of extraction temperature are fixed, and the influence of different extraction time (10, 20, 30, 40, 50 and 60min) on the extraction amount of the polysaccharide of the dragon fruit is researched under the condition of unified other experimental operations.
Second, experimental results
As shown in FIG. 3, the extraction amount of polysaccharide from Fructrs Callicarpa at 30-60 min is significantly higher than that at 10 and 20min (P < 0.05). Therefore, in the extraction condition, the extraction time is the best time for extracting the polysaccharide from the dragon fruits within 30-60 min.
Example 5 Effect of extraction temperature on the amount of polysaccharide extracted from Ficus tikoua
First, experiment method
Extracting for 30min, wherein the material-liquid ratio is 1: 20(g/mL) is fixed, and under the condition of uniform other experimental operations, the change of the extraction amount of the polysaccharide of the dragon fruit at the extraction temperature of 30, 40, 50, 60, 70, 80, 90 and 100 ℃ is respectively researched.
Second, experimental results
As shown in FIG. 4, the extraction amount of polysaccharide from Fructrs Callicarpa at 50 deg.C is significantly higher than that at 30, 60, 70, 80, 90, 100 deg.C (P < 0.05). Therefore, in the extraction conditions, 50 ℃ is the optimal temperature for extracting the polysaccharide from the dragon fruits.
Example 6 Effect of feed liquid ratio on polysaccharide extraction amount of Ficus simplicissima lour
First, experiment method
Under the conditions that the extraction time is 30min and the extraction temperature is 50 ℃, the other extraction conditions are kept consistent, and the extraction temperature is respectively detected in the range of 1: 5. 1: 10. 1:15. 1: 20. 1: 25. 1: the extraction amount of polysaccharide from the dragon fruit changes under the condition of a feed-liquid ratio of 30 (g/mL).
Second, experimental results
The results are shown in fig. 5, when the ratio of the feed to the liquid is 1: the extraction amount of the polysaccharide of the dragon fruit is obviously higher than that of the polysaccharide of the dragon fruit with the material-liquid ratio of 1: 5. 1: 10. 1: 20. 1: 25. 1: polysaccharide extraction at 30(g/mL) (P < 0.05). Thus, in this extraction condition, 1:15 g/mL is the optimal feed-liquid ratio for extracting the polysaccharide from the dragon fruits.
Example 7 optimization of polysaccharide extraction from Polygala Longzhuguensis by response surface analysis
First, experiment method
On the basis of a single-factor experiment, the optimal feed ratio, the optimal extraction time and the optimal extraction temperature of the polysaccharide extraction of the dragon fruits are further optimized by using a response surface analysis method. Experiments were designed according to the Box-Behnken principle: selecting three factors of extraction time (A), extraction temperature (B) and material-liquid ratio (C) as independent variables, and taking the extracted polysaccharide content (Y) of the dragon fruit as a response value. The test factors and the horizontal design are shown in table 1, and the corresponding data analysis software is used for carrying out multi-factor and multi-level response surface test analysis and calculating to obtain the optimal extraction process conditions of the dragon fruit polysaccharide. And finally, extracting the polysaccharide of the dragon fruit under the optimal extraction process condition of the polysaccharide of the dragon fruit obtained by a response surface analysis method. The polysaccharide extraction amount is determined by 3 parallel verification experiments, and the reliability of the adopted analysis method is verified.
The polysaccharide content (mg/g) is equal to crude polysaccharide of longzhuguo/longzhuguo dry powder.
Table 1 test factors and level design:
in order to realize the further optimization of the longzhuguo polysaccharide extraction process, a corresponding optimization experiment is designed according to the Box-Behnken principle. The experimental protocol design and results are shown in table 2.
Table 2 response surface optimization experimental design:
second, experimental results
The response surface optimization experiment is shown in table 3.
Table 3:
establishing extraction time by using corresponding data analysis software, wherein a three-factor mathematical regression model of extraction temperature and feed-liquid ratio is as follows:
y＝17.50.219A+0.169B+9.41C-0.0037A2-0.00157B2-1.19C2+0.0032AB-0.0076A C-0.0960BC。
analysis of variance was performed on the regression equation, and the results are shown in table 4.
Table 4 analysis of variance of quadratic regression model:
from the analysis in Table 4, the significance of the regression equation coefficients, the extraction time (A) and the square term of the extraction time (A) can be obtained2) Has a highly significant effect on the extraction of polysaccharide from the dragon fruit (P)<0.001); extraction temperature (B), feed-liquid ratio (C), square term of extraction temperature (B)2) And the interaction term (BC) of the extraction temperature and the material-liquid ratio has a significant influence on the extraction amount of the polysaccharide of the dragon fruit (P)<0.05)。
The P value of the regression model is less than 0.001, which shows that the selected model is highly significant, and the P value of the mismatching term is 0.412 and more than 0.05, i.e. the mismatching term is not significantly different. Analyzing the resulting coefficient of determination (R) of the regression model2 adj) The value of 0.980 indicates that the model can cover 98.0% of change, so the model has certain reference value for analyzing the experimental result and can predict the polysaccharide extraction amount.
And performing quadratic multiple regression fitting on the data in the table 4 to obtain a response surface curved surface diagram of the quadratic regression equation, wherein the response surface curved surface diagram is shown in fig. 6-8.
A response surface 3D curve graph and a contour analysis graph of interaction of various factors can be obtained from the response surface curve graph. The highest point of the response curve formed by the interaction of the two factors in the figure is also the central point of the smallest ellipse in the contour line, so that it can be seen that the response value (Y) has an extreme value in the selected range. Whether the influence of a certain factor on the response value (Y) is obvious or not can be observed through the change of the color of the curved surface from blue to red, and the influence is more obvious when the change is faster and the gradient is larger. In addition, the shape of the contour line can reflect the strength of interaction of the two factors, the closer the contour line shape is to the ellipse, the more the contour line shape is to the circle, the opposite is true.
As can be seen from FIGS. 6 to 8, the curve of the extraction time (A) on the extraction amount of polysaccharide from strobilus Pini is steep, the contour lines are dense, and the influence is most obvious; the curve of the extraction temperature (B) on the extraction amount of the polysaccharide of the dragon fruit is smooth, the contour lines are sparse, and the influence is relatively small. Among the three factors studied, the effect on the extraction amount of polysaccharide from big to small is as follows: the extraction time (A) > the material-liquid ratio (C) > the extraction temperature (B). Observing the shape of the contour line, the interaction of the extraction time (A) and the extraction temperature (B) and the interaction of the extraction temperature (B) and the feed-liquid ratio (C) are stronger, and the interaction of the extraction time (A) and the feed-liquid ratio (C) is weaker. This is consistent with the results of the ANOVA analysis.
Performing calculation analysis on the obtained extraction amount of the polysaccharide of the dragon fruit and a multivariate quadratic regression equation of the influence factors of the polysaccharide of the dragon fruit to obtain the optimal extraction process conditions of the polysaccharide of the dragon fruit as follows: the extraction time is 31min, the extraction temperature is 55 ℃, and the feed-liquid ratio is 1:15.5 (g/mL). The maximum value of Y in the regression equation, i.e., the theoretical value of polysaccharide extraction from Pogostemon cablin under the optimum extraction conditions, was 29.372 mg/g.
Third, response surface verification experiment
A verification experiment is carried out, 6 groups of parallel data are averaged, the obtained experimental value of the polysaccharide extraction amount of the dragon fruit is 28.886mg/g, and the relative error of the two is 1.65%. The experimental value is basically consistent with the theoretical value, and the obtained regression model has certain feasibility for optimizing the extraction process conditions of the polysaccharide of the dragon fruits. Therefore, the extraction time is 31min, the extraction temperature is 55 ℃, and the feeding ratio is 1:15.5, which is the optimal extraction process parameter of the polysaccharide of the dragon fruit. Under the condition, the obtained extract of the total sugar of the dragon pearl fruits is frozen and dried to obtain 11.56g of crude polysaccharide of the dragon pearl fruits, and the yield of the polysaccharide of the dragon pearl fruits is 2.89 percent.
Example 8 evaluation of polysaccharide of Polygala Longzhuguensis in vitro immunomodulatory Activity
First, culture and passage of RAW264.7 macrophage
Culturing RAW264.7 macrophage cell in complete culture medium containing 10% fetal bovine serum and 1% double-antibody DEME at 37 deg.C and 5% CO2And (5) carrying out adherent culture in an incubator. When the cells were attached to the bottom of the flask, passaging was required, and the cells were digested with pancreatin digest containing 0.25% EDTA.
Second, the Effect of PFP on RAW264.7 macrophage proliferation
1. Experimental methods
The MTT method is adopted to determine the influence of PFP on the proliferation capacity of RAW264.7 macrophage.
The method comprises the following specific steps: RAW264.7 macrophages in logarithmic growth phase at 1X 105one/mL of the cells were plated in 96-well cell culture plates, one well for each well100 μ L of cell culture medium was added. After 24h of culture, the cells adhered to the wall, and 100. mu.L of medium containing 0, 0.064, 0.32, 1.6, 8, 40. mu.g/mL of polysaccharide from the dragon fruit was added to each concentration gradient treatment group (final concentrations were 0, 0.032, 0.16, 0.8, 4, 20. mu.g/mL). 3 replicate wells per group, 5% CO at 37 ℃2After the incubator continues to culture for 24h, the medium is discarded, 10 mu of LMTT solution is added into each hole, and the temperature is 37 ℃ and the CO content is 5 percent2The incubator continues to culture for 4h, and the absorbance is detected at 490nm of the microplate reader.
2. Results of the experiment
As shown in FIG. 9, PFP has no cytotoxicity in the concentration range of 0.032-20. mu.g/mL, and can promote the proliferation of RAW264.7 macrophages, and the promotion effect is more obvious with the increase of PFP concentration.
Third, the effect of PFP on NO secretion from RAW264.7 macrophage
1. Experimental methods
When acting on RAW264.7 macrophages, polysaccharides can induce the occurrence of various cellular immune response reactions, and achieve immune regulation by promoting cells to proliferate and produce cytokines. Among them, NO is an active substance in the living body, and the amount of NO released is an important index for judging whether the immunoregulatory activity of macrophages is enhanced. Lipopolysaccharide (LPS) is a major component in the cell wall of gram-negative bacteria, is toxic to the host, has the effect of inducing a cellular immune response, and is commonly used as a positive control for the evaluation of immunomodulatory activity. The NO content secreted by RAW264.7 macrophages of each treatment group is measured by a nitrite kit (Griess method).
The method comprises the following specific steps: RAW264.7 macrophages in logarithmic growth phase at 1X 105one/mL was plated in 96-well cell culture plates, and 200. mu.L of cell culture medium was added per well. After 24h of culture, the cells were attached to the wall and the cell culture medium was changed. Adding 200 μ L of culture medium into blank control group; adding 200 μ L of culture medium containing 0.032, 0.16, 0.8, 4, 20 μ g/mL polysaccharide into each concentration gradient treatment group; LPS group was added to 200. mu.L of LPS (1. mu.g/mL) containing medium, and each group was replicated in 3 wells at 37 ℃ with 5% CO2After the incubator continues to culture for 24h, 10. mu.L of the upper layer is sucked out of each holeThe culture medium was assayed for NO content in the supernatant of each group of cells according to the kit instructions, and absorbance at 540nm was measured on a microplate reader.
2. Results of the experiment
As shown in FIG. 10, PFP can significantly promote NO secretion from macrophages in the concentration range of 0.032-20 μ g/mL, and is dose-dependent. When the PFP concentration reached 20. mu.g/mL, the amount of NO released was about 6 times that of the blank.
Fourth, the Effect of PFP on IL-6 and TNF-alpha secretion by RAW264.7 macrophages
1. Experimental methods
Macrophages stimulated by polysaccharides can secrete cytokines such as Tumor Necrosis Factor (TNF) and Interleukin (IL), which can regulate the immune function of the body to a certain extent and play an important role in immune response
The method comprises the following specific steps: RAW264.7 macrophages in logarithmic growth phase at 1X 105one/mL was plated in 96-well cell culture plates, and 200. mu.L of cell culture medium was added per well. After 24h of culture, the cells were attached to the wall and the cell culture medium was changed. The experimental treatment method and grouping are as above. Each experimental group was replicated 3 times in 3 duplicate wells. And 24, taking cell culture supernatant, and determining the content of IL-6 and TNF-alpha by an enzyme-linked immunosorbent assay (ELISA). The specific operation method is carried out according to the specification of the IL-6 and TNF-alpha kit, the absorbance value is measured at 450nm, a standard curve is established, and the content of the IL-6 and the content of the TNF-alpha are calculated.
2. Results of the experiment
As shown in FIG. 11, the effect of PFP on IL-6 production by RAW264.7 macrophages was not significant at concentrations below 0.16. mu.g/mL, and the promotion was gradually significant with increasing PFP concentration. When the PFP concentration reaches 20. mu.g/mL, the IL-6 secretion is increased by 94.7% compared with the blank control group.
As shown in FIG. 12, compared with the blank control group, PFP can significantly promote TNF-alpha secretion from RAW264.7 macrophage in the concentration range of 0.032-20 μ g/mL, and is in dose-dependent relationship. When the PFP concentration reached 20. mu.g/mL, the secreted TNF-. alpha.was 30.76 times higher than that of the blank control.
Claims (5)
1. The application of the dragon pearl fruit polysaccharide in preparing functional food for immunoregulation is characterized in that the preparation method of the dragon pearl fruit polysaccharide comprises the following steps:
s1, adding water into the dragon pearl fruit powder for extraction, centrifuging to obtain a supernatant, repeatedly extracting, and combining the supernatants to obtain a crude dragon pearl fruit polysaccharide extract;
s2, concentrating the crude polysaccharide extract of the dragon ball fruits, adding absolute ethyl alcohol, standing, and collecting precipitates;
s3, dissolving the precipitate, and removing protein by a Sevag method;
s4, removing pigments by macroporous resin column chromatography;
s5, removing small molecular impurities, and drying;
in step S1, the weight ratio of the dragon fruit powder to the water is 1: 5-1: 30, of a nitrogen-containing gas;
the extraction time is 5-30 min;
the extraction temperature is 30-90 ℃.
2. The use according to claim 1, wherein in step S2, the crude extract of polysaccharide from garcinia mangostana is concentrated, added with absolute ethanol, left overnight at 4 ℃, centrifuged, and the precipitate collected.
3. The use according to claim 2, wherein in step S3, the aqueous solution of the precipitate obtained in step S2 is mixed with Sevag solution, subjected to solid-liquid separation, the upper aqueous solution is collected, the precipitate is removed, and the above operation is repeated to remove the organic solvent.
4. The use of claim 3, wherein the aqueous solution of the product of step S3 is applied to an AB-8 macroporous resin chromatography column, eluted through macroporous resin, eluted with distilled water, and the eluate is collected.
5. The use of claim 4, wherein in step S5, the product of step S4 is added to a dialysis bag and dialyzed against distilled water.
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