CN104693313B - Extraction method and application of pomegranate flower polysaccharides - Google Patents

Abstract

The invention discloses a method for extracting pomegranate flower polysaccharides, using cheap pomegranate flowers as raw materials, according to pomegranate flower crushing-ethanol impurity removal-microwave cooperative extraction-protein removal-concentration-ethanol fractional precipitation-glucose Glycan gel purification process route for extraction to obtain pomegranate flower polysaccharide products of different polarities. The method is simple to operate, turns pomegranate flowers into valuables, has low cost, is beneficial to the full utilization of pomegranate resources, improves the added value of pomegranate processed products, and is suitable for industrial production. The obtained pomegranate flower polysaccharide has good antioxidant activity and immunoregulatory effect, and has good utilization and development prospects.

Description

A kind of extraction method and application of pomegranate flower polysaccharide

technical field

The invention relates to a method for rapidly extracting pomegranate flower polysaccharides with antioxidant and immune regulation activities from pomegranate flowers, and belongs to the technical field of pomegranate flower polysaccharide extraction.

Background technique

Pomegranate is a medicinal and edible resource with high nutritional value and health care function, and has the reputation of "the whole body is a treasure". At present, my country has become the world's largest pomegranate planting country, but my country's pomegranate processing industry is still in a relatively extensive stage, and the level of deep processing technology and product conversion efficiency are both low. Pomegranate blooms from May to July. It has a long flowering period and a large amount of flowers. It is generally divided into 3 batches of flowers. When budding begins, the bell-shaped flowers that cannot bear fruit must be thinned out. Most of the second and third flowers cannot bear fruit normally and fall off naturally. Pomegranate flowers are rich in resources and have great development value. In the research on the hypoglycemic effect of various organs of pomegranate, it was found that pomegranate flower has significant hypoglycemic activity, which is closely related to the ingredients it contains. According to current research reports, the active ingredients in pomegranate flowers are mainly phenols, triterpenoids and flavonoids. Betulinic acid, ursolic acid, benzolic acid, etc. Currently, there are relatively few reports on polysaccharides in pomegranate flowers, and there are no reports on the extraction and separation of pomegranate flower polysaccharides.

Polysaccharides widely exist in animals, plants, and microorganisms, and have various biological activities such as anti-tumor, anti-virus, anti-oxidation, and immune regulation. A lot of research work has confirmed that: as a research hotspot in the field of phytochemicals, plant polysaccharides have many interesting biological effects, including preventing and curing cancer, anti-oxidation (scavenging free radicals), anti-stress, hypoglycemic, anti-arteriosclerosis And so on. In addition, polysaccharides can be widely used in food processing as thickeners, stabilizers, functional factors, fat substitutes, and film-forming agents due to their excellent physical and chemical properties and biological activities. The various high and new technologies used in the production of polysaccharides have a significant impact on the molecular structure and biological activity of polysaccharides, which in turn affects the application characteristics of polysaccharides in food processing. Therefore, it is necessary to further study the influence of processing technology on the structure and biological activity of pomegranate flower polysaccharide, and develop the production technology of high activity pomegranate flower polysaccharide, so as to guide the production of pomegranate flower polysaccharide and its application in food, medicine and other fields.

By searching the literature and related patents, there are currently reports on the extraction process of polysaccharides in pomegranate peel, pomegranate seeds and guava, but there is no report on the extraction of polysaccharides in pomegranate flowers. Therefore, it is of great significance to study the extraction process of active polysaccharides in pomegranate flowers to increase the added value of pomegranate processed products, turn waste into treasure, and increase the income of farmers in resource-producing areas.

Contents of the invention

The purpose of the present invention is to provide a method for extracting pomegranate flower polysaccharide, which uses pomegranate flower as raw material, and the extracted pomegranate flower polysaccharide has high antioxidant activity and immune regulation effect, turns waste into treasure, and improves pomegranate processing. The added value of the product has a good application prospect.

Another object of the present invention is to provide an anti-oxidation active agent or a body immune regulator which uses the pomegranate flower polysaccharide obtained by the method as an active ingredient.

The present invention uses cheap pomegranate flowers as raw materials to extract the effective polysaccharide components in order to increase its utilization value, and the extraction is carried out according to the crushing of pomegranate flowers—removal of impurities by ethanol—cooperative microwave extraction—removal of protein—concentration—ethanol Fractional precipitation—the process route of dextran gel purification is carried out to obtain pomegranate flower polysaccharide products with different polarities. The specific technical scheme is as follows:

A method for extracting pomegranate flower polysaccharide is characterized in that it comprises the following steps:

(1) Wash the pomegranate flowers, dry them and crush them;

(2) Add ethanol solution to the pomegranate flower powder for extraction, filter to get the filter residue;

(3) Add water to the filter residue, microwave extraction, and combine the extracts;

(4) Add the extract to Sevag reagent to remove protein to obtain a deproteinized extract;

(5) Dialyze the deproteinized extract with a dialysis membrane to remove impurities to obtain a dialysate;

(6) Vacuum concentrate the dialysate to 1/8 to 1/4 of the original volume to obtain a concentrated solution, add absolute ethanol to the concentrated solution until the volume concentration of ethanol reaches 70%, separate after standing, and evaporate the solvent to obtain Polysaccharide A crude product, add absolute ethanol to the supernatant until the ethanol volume concentration is 80%, separate after standing, precipitate and evaporate the solvent to dry the polysaccharide B crude product, add absolute ethanol to the supernatant until the ethanol volume concentration is 95%, Separation after standing, precipitation and evaporation of solvent to dry polysaccharide C crude product;

(7) The crude products of polysaccharides A, B, and C were purified by Sephadex gel chromatography column, eluted with water as the mobile phase, the eluate was collected, and freeze-dried to obtain pomegranate flower polysaccharides A, B, and C Pure.

In the above step (1), the pomegranate flowers are dried and pulverized to 60 mesh with a high-speed pulverizer.

In the above step (2), the volume concentration of the ethanol solution is 75-95%, preferably 95%.

In the above step (2), the amount of the ethanol solution is 3-6 times, preferably 5 times, the mass of pomegranate flower powder.

In the above step (2), reflux extraction with ethanol solution for 2-4 hours.

In the above step (3), the microwave extraction is performed 3 times, and the amount of water used each time is 20-30 times, preferably 28 times, the quality of the filter residue.

In the above step (3), the microwave power is 800-1000 W, the microwave extraction temperature is 90-100°C, and the extraction time is 10 minutes.

In the above step (4), the amount of Sevag reagent used is 3-5 times the volume of the extract. Sevag reagent is chloroform:n-butanol=5:1 (volume ratio).

In the above step (5), the molecular weight of the dialysis membrane is 2000-5000Da, preferably 3500Da.

In the above step (5), the dialysis time is 2 days.

In the above step (6), when separating crude polysaccharides A, B, and C, the resting time is 8-12 hours.

In the above step (7), the dextran gel is SephadexG-100.

In the above step (7), the flow rate of water during elution is 0.05-2 mL/min, preferably 0.3 ml/min.

The pure pomegranate flower polysaccharide A, pomegranate flower polysaccharide B and pomegranate flower polysaccharide C obtained by the above extraction method all contain mannose, glucuronic acid, rhamnose, glucose, galactose, arabinose and xylose.

The obtained pomegranate flower polysaccharide A, pomegranate flower polysaccharide B and pomegranate flower polysaccharide C have been verified to have good antioxidant activity and immune regulation effect, and they can be used as active ingredients of antioxidant active agents or immune regulators for further research. application.

The invention uses pomegranate flower as raw material, and extracts three kinds of pomegranate flower polysaccharides with different polarities therefrom through a series of treatments. The method is simple to operate, turns pomegranate flowers into valuables, has low cost, is beneficial to the full utilization of pomegranate resources, improves the added value of pomegranate processed products, and is suitable for industrial production.

The pomegranate flower polysaccharides A, B, and C extracted by the present invention have been verified by experiments to have good antioxidant activity and immunoregulatory effect, and their antioxidant capacity is better than that of astragalus polysaccharides and wolfberry polysaccharides, and can significantly improve the phagocytosis of macrophages. It can significantly enhance the non-specific immunity and cellular immune system of the body, and can be used as a natural anti-oxidation active agent and an immune regulator of the body, and has good utilization and development prospects.

Description of drawings

Fig. 1 is the HPLC chromatogram of embodiment 1 gained pomegranate flower polysaccharide. Among them, (a): standard chromatogram of 9 monosaccharides; (b): chromatogram of pomegranate flower polysaccharide A monosaccharide; (c): pomegranate flower polysaccharide B monosaccharide chromatogram; (d): pomegranate flower polysaccharide C Monosaccharide chromatogram.

Fig. 2 is the determination curve of total reducing ability of pomegranate flower polysaccharide.

Figure 3 is a diagram of the scavenging ability of pomegranate flower polysaccharides on hydroxyl radicals.

Figure 4 is a diagram of the scavenging ability of pomegranate flower polysaccharides on DPPH free radicals.

detailed description

The present invention will be further described through specific examples below, and the following descriptions are only exemplary and not intended to limit the content thereof. Unless otherwise specified, the following concentrations of ethanol are volume concentrations.

Example 1

The extraction method of pomegranate flower polysaccharide is as follows:

1. Take 1000 g of pomegranate flower, wash it with tap water, dry it naturally, crush it to 60 mesh, add 5 times the mass of 95% ethanol to the pomegranate flower powder and extract it under reflux for 3 hours, and filter to obtain the filter residue;

2. Add distilled water 28 times its mass to the filter residue, extract at 100°C and microwave power of 1000 W for 10 minutes, filter, repeat the extraction twice, and combine the extracts;

3. Add 5 times the volume of Sevag reagent (chloroform: n-butanol volume ratio = 5:1) to the extract, shake vigorously for 50 minutes, then let it stand for 10 minutes, centrifuge to remove the protein in the extract; repeat This deproteinization step obtains the deproteinized extract for 3-5 times;

4. Dialyze the deproteinized extract with a 3500Da dialysis membrane for 2 days to remove small molecular impurities with a molecular weight less than 3500Da, and take the dialysate with a molecular weight greater than 3500Da;

5. Concentrate the dialysate in vacuum to 1/8 of the original volume to obtain a concentrated solution; add absolute ethanol to the concentrated solution while stirring to make the volume concentration of ethanol reach 70%, let stand for 10-12 hours, collect Precipitation, precipitation volatilization remove solvent (water and ethanol) polysaccharide A crude product; continue to add absolute ethanol to the supernatant of separated precipitation, until the volume concentration of ethanol reaches 80%, let stand for 10-12 hours, collect precipitation, precipitation volatilization Remove the solvent (water and ethanol) polysaccharide B crude product; continue to add absolute ethanol to the supernatant of the separation and precipitation until the volume concentration of ethanol reaches 95%, let it stand for 10-12 hours, collect the precipitate, and remove the solvent (water and ethanol) polysaccharide C crude product;

6. Purify the crude polysaccharides A, B, and C using SephadexG-100 chromatographic column respectively, using water as the mobile phase, and the elution flow rate is 0.3 mL/min, collect the eluents respectively, and dry to obtain the refined pomegranate flower polysaccharide A , B, C. Among them, pomegranate flower polysaccharide A weighs 20.25 g, pomegranate flower polysaccharide B weighs 25.6 g, and pomegranate flower polysaccharide C weighs 41.25 g. The total yield is 8.7% (the calculation method of the total yield is: total yield %=total polysaccharide weight/test sample weight).

Gained pomegranate flower polysaccharide A, B, C composition is analyzed, as follows:

1. Instruments and reagents:

Agilent 1100 LC/MSD Trap (Agilent, USA), equipped with a vacuum degasser (G1322A), equipped with a quaternary gradient pump (G1311A), an autosampler (G1329A), an incubator (G1316A), and a diode array detector ( DAD, G1315A), ion trap mass spectrometry (G2445D), electrospray ionization source (ESI, G1948A), derivatives were separated using HypersilODS 2 chromatographic column (200×4.6mm 5 μm, Elite Company, Dalian, China), mobile phase Filter through a 0.2 μm nylon membrane filter.

, HPLC separation conditions:

Chromatographic column Hypersil ODS ₂ (4.6 mm × 200 mm, 5 mm); A mobile phase is 30% acetonitrile (containing 15mmol/L CH ₃ COONH ₄ , pH 4.5); B mobile phase is 60% acetonitrile; gradient elution program: 0~50 min, 100%A~55%A, flow rate: 1.0 mL/min; detection wavelength: 254 nm.

,result

According to the analysis, pomegranate flower polysaccharide A, pomegranate flower polysaccharide B and pomegranate flower polysaccharide C all contain mannose, glucuronic acid, rhamnose, glucose, galactose, arabinose and xylose. The chromatogram is shown in Figure 1. The contents of these monosaccharides are shown in Table 1 below.

Example 2

The extraction method of pomegranate flower polysaccharide is as follows:

1. Take 1300g of pomegranate flowers, wash them with tap water, air-dry them naturally, and crush them to 60 mesh;

2. Add 75% ethanol 6 times its mass to the pomegranate flower powder for reflux extraction for 2 hours, and obtain a filter residue after filtering;

3. Add distilled water 30 times its mass to the filter residue, extract at 90°C and microwave power of 800 W for 10 minutes, filter, repeat the extraction twice, and combine the extracts;

4. Add 3 times the volume of Sevag reagent (chloroform:n-butanol volume ratio = 5:1) to the extract, vibrate vigorously for 20 minutes, then let it stand for 10 minutes, centrifuge to remove the protein in the extract; repeat this process The protein removal step is obtained 3-5 times to obtain the deproteinized extract;

5. Dialyze the deproteinized extract with a 2000Da dialysis membrane for 2 days to remove small molecular impurities with a molecular weight less than 2000Da, and take the dialysate with a molecular weight greater than 2000Da;

6. Concentrate the dialysate in vacuum to 1/4 of the original volume to obtain a concentrated solution; add absolute ethanol to the concentrated solution while stirring to make the volume concentration of ethanol reach 70%, let stand for 8-10 hours, collect Precipitation, precipitation volatilization to remove the solvent polysaccharide A crude product; continue to add absolute ethanol to the supernatant of the separation and precipitation, until the ethanol volume concentration is 80%, let stand for 8-10 hours, collect the precipitate, precipitation volatilization to remove the solvent polysaccharide B Crude product; continue to add dehydrated ethanol in the supernatant of separation and precipitation, reach 95% to ethanol volume concentration, leave standstill 8-10 hour, collect precipitation, precipitation volatilizes and removes the polysaccharide C crude product of solvent;

7. Purify the obtained crude polysaccharides A, B, and C using SephadexG-100 chromatographic columns, using water as the mobile phase, with an elution flow rate of 0.05mL/min, collect the eluents, and freeze-dry to obtain refined pomegranate Flower polysaccharides A, B, C. Wherein the weight of pomegranate flower polysaccharide A is 16.54 g, the weight of pomegranate flower polysaccharide B is 18.17 g, and the weight of pomegranate flower polysaccharide C is 32.02 g. The total yield was 5.1%.

According to the method of Example 1, the composition of gained pomegranate flower polysaccharide A, B, and C is analyzed, and the results are shown in Table 2 below:

Example 3

The extraction method of pomegranate flower polysaccharide is as follows:

1. Take 1300g of pomegranate flowers, wash them with tap water, air-dry them naturally, and crush them to 60 mesh;

2. Add 85% ethanol 3 times its mass to the pomegranate flower powder for reflux extraction for 4 hours, and filter to obtain the filter residue;

3. Add distilled water 20 times its mass to the filter residue, extract at 100°C and microwave power of 900 W for 10 minutes, filter, repeat the extraction twice, and combine the extracts;

4. Add 3 times the volume of Sevag reagent (chloroform:n-butanol volume ratio = 5:1) to the extract, vibrate vigorously for 20 minutes, then let it stand for 10 minutes, centrifuge to remove the protein in the extract; repeat this process The protein removal step is obtained 3-5 times to obtain the deproteinized extract;

5. Dialyze the deproteinized extract with a 5000Da dialysis membrane for 2 days to remove small molecular impurities with a molecular weight less than 5000Da, and take the dialysate with a molecular weight greater than 5000Da;

6. Concentrate the dialysate in vacuum to 1/6 of the original volume to obtain a concentrated solution; add absolute ethanol to the concentrated solution while stirring to make the volume concentration of ethanol reach 70%, let stand for 8-12 hours, collect Precipitation, precipitation volatilization to remove the solvent polysaccharide A crude product; continue to add absolute ethanol to the supernatant of the separation and precipitation until the volume concentration of ethanol is 80%, let stand for 8-12 hours, collect the precipitation, precipitation volatilization to remove the solvent polysaccharide B Crude product; continue to add dehydrated ethanol in the supernatant of separation precipitation, reach 95% to ethanol volume concentration, leave standstill 8-12 hour, collect precipitation, precipitation volatilization removes solvent polysaccharide C crude product;

7. Purify the obtained crude polysaccharides A, B, and C using SephadexG-100 chromatography column, using water as the mobile phase, and the elution flow rate is 2 mL/min, collect the eluents respectively, and freeze-dry to obtain refined pomegranate Flower polysaccharides A, B, C. Wherein the weight of pomegranate flower polysaccharide A is 17.31 g, the weight of pomegranate flower polysaccharide B is 19.31 g, and the weight of pomegranate flower polysaccharide C is 29.80 g. The total yield was 5.1%.

According to the method of Example 1, the composition of gained pomegranate flower polysaccharide A, B, and C is analyzed, and the results are shown in Table 3 below:

Example 4

The extraction method of pomegranate flower polysaccharide is as follows:

1. Take 1300g of pomegranate flowers, wash them with tap water, air-dry them naturally, and crush them to 60 mesh;

2. Add 75% ethanol 3 times its mass to the pomegranate flower powder for reflux extraction for 4 hours, and filter to obtain a filter residue;

3. Add distilled water 20 times its mass to the filter residue, extract at 90°C and microwave power 800 W for 10 minutes, filter, repeat the extraction twice, and combine the extracts;

4. Add 3 times the volume of Sevag reagent (chloroform:n-butanol volume ratio = 5:1) to the extract, vibrate vigorously for 20 minutes, then let it stand for 10 minutes, centrifuge to remove the protein in the extract; repeat this process The protein removal step is obtained 3-5 times to obtain the deproteinized extract;

5. Dialyze the deproteinized extract with a 4000Da dialysis membrane for 2 days to remove small molecular impurities with a molecular weight less than 4000Da, and take the dialysate with a molecular weight greater than 4000Da;

6. Concentrate the dialysate in vacuum to 1/4 of the original volume to obtain a concentrated solution; add absolute ethanol to the concentrated solution while stirring to make the volume concentration of ethanol reach 70%, let stand for 8-12 hours, collect Precipitation, precipitation volatilization to remove the solvent polysaccharide A crude product; continue to add absolute ethanol to the supernatant of the separation and precipitation until the volume concentration of ethanol is 80%, let stand for 8-12 hours, collect the precipitation, precipitation volatilization to remove the solvent polysaccharide B Crude product; continue to add dehydrated ethanol in the supernatant of separation precipitation, reach 95% to ethanol volume concentration, leave standstill 8-12 hour, collect precipitation, precipitation volatilization removes solvent polysaccharide C crude product;

7. Purify the obtained crude polysaccharides A, B, and C using SephadexG-100 chromatography column, using water as the mobile phase, and the elution flow rate is 1mL/min, collect the eluents respectively, and freeze-dry to obtain refined pomegranate flowers Polysaccharides A, B, C. Wherein the weight of pomegranate flower polysaccharide A is 16.23 g, the weight of pomegranate flower polysaccharide B is 19.23 g, and the weight of pomegranate flower polysaccharide C is 38.04 g. The total polysaccharide yield was 5.7%.

According to the method of Example 1, the composition of gained pomegranate flower polysaccharide A, B, and C is analyzed, and the results are shown in Table 4 below:

comparative example

The extraction method of pomegranate flower polysaccharide is as follows:

1. Take 1300g of pomegranate flowers, wash them with tap water, air-dry them naturally, and crush them to 60 mesh;

2. Add 70% ethanol 5 times its mass to the pomegranate flower powder for reflux extraction for 4 hours, and obtain the filter residue after filtration;

3. Add distilled water 28 times its mass to the filter residue, extract at 80°C and microwave power of 1000 W for 10 minutes, filter, repeat the extraction twice, and combine the extracts;

4. Add 5 times the volume of Sevag reagent (chloroform: n-butanol volume ratio = 5:1) to the extract, shake vigorously for 50 minutes, then let it stand for 10 minutes, centrifuge to remove the protein in the extract; repeat the process The protein removal step is obtained 3-5 times to obtain the deproteinized extract;

5. Dialyze the deproteinized extract with a 5500Da dialysis membrane for 2 days to remove small molecular impurities with a molecular weight less than 5500Da, and take the dialysate with a molecular weight greater than 5500Da;

6. Concentrate the dialysate in vacuum to 1/8 of its original volume to obtain a concentrated solution; add absolute ethanol to the concentrated solution while stirring to make the volume concentration of ethanol reach 65%, let stand for 8-12 hours, collect Precipitation, the precipitation is volatilized to remove the solvent to obtain crude product 1; continue to add absolute ethanol to the supernatant of the separated precipitation until the volume concentration of ethanol is 75%, let stand for 8-12 hours, collect the precipitate, and the precipitation is volatilized to remove the solvent to obtain crude product 2; Continue to add absolute ethanol to the supernatant of the separated precipitate until the ethanol volume concentration reaches 90%, let it stand for 8-12 hours, collect the precipitate, and evaporate the precipitate to remove the solvent to obtain the crude product 3;

7. The obtained crude products 1, 2, and 3 were purified by SephadexG-100 chromatography column respectively, using water as the mobile phase, and the elution flow rate was 3mL/min, and the eluents were collected respectively and freeze-dried to obtain the refined pure product 1 , 2, 3, these 3 kinds of pure products are mixed to obtain the final product, a total of 64.3g, and the total polysaccharide yield is 4.9%.

In order to verify the efficacy of the extracted pomegranate flower polysaccharide, the following experiment was carried out by taking the polysaccharide extracted in Example 1 as an example.

Antioxidant activity experiment of pomegranate flower polysaccharide

1.1 Reducing ability experiment

Experimental method: Add different concentrations of polysaccharide solutions (5-60 μg/mL) into a 10 mL centrifuge tube with stopper, and add 0.5 mL phosphate buffer (pH 6.6, 0.2 mol/L) and 0.5 mL 1 % (m/v) potassium ferricyanide [K ₃ Fe(CN) ₆ ] solution. The mixture was incubated in a water bath at 50 °C for 20 min. After the incubation, 1 mL of 10% trichloroacetic acid was added dropwise to stop the reaction, and then the contents were centrifuged at 1000 rpm for 10 min. Take 3 mL of the supernatant, add ferric chloride solution (0.5mol/L, 0.1%), shake, measure the absorbance at 700 nm, and measure the samples of each concentration three times in parallel. At the same time, the experiment was carried out under the same conditions with vitamin C as the control.

In general, the reducing power of the sample is positively correlated with the antioxidant capacity. According to the determination method of reducing ability, the greater the absorbance value measured at a wavelength of 700 nm, the stronger the reducing ability of the sample. The results are shown in Figure 2. It can be seen from the figure that the reducing ability of pomegranate flower polysaccharide increases with the increase of its concentration, suggesting that the antioxidant capacity of pomegranate flower polysaccharide increases with the increase of concentration. Among the three polysaccharides obtained by fractional precipitation, the antioxidant activity of polysaccharide A and polysaccharide B was stronger than that of polysaccharide C.

Hydroxyl free radical scavenging experiment

The experimental steps are as follows: mix 1 mL of polysaccharide solution or vitamin C solution with salicylic acid (10.0 mmol/L, 1 mL), ferrous sulfate (10 mmol/L, 1 mL) and hydrogen peroxide (0.003%, 1 mL) at 37 Incubate at ℃ for 30 min, centrifuge at 4000 r/min for 15 min, take the supernatant, adjust the zero point with purified water, and measure the absorbance (A) at a wavelength of 510 nm. The results were calculated by the following formula: hydroxyl radical scavenging activity %=[A-(A ₁ -A ₀ )]/100%. Among them, A1 is the absorbance of the sample; _A is the absorbance of the control solution (containing salicylic acid, ferrous sulfate and hydrogen peroxide); _A0 is the absorbance of the blank solution (containing salicylic acid, ferrous sulfate, polysaccharide solution and distilled water) .

Lycium barbarum polysaccharides (LBP) and astragalus polysaccharides (LBP) are well-known plant polysaccharides with strong antioxidant capacity. In this experiment, Lycium barbarum polysaccharides and Astragalus polysaccharides were set up as reference groups to verify the antioxidant capacity of pomegranate flower polysaccharides. Figure 3 shows the ability of pomegranate flower polysaccharides (A, B and C), Lycium barbarum polysaccharides (LBP), astragalus polysaccharides (AP) and vitamin C to scavenge hydroxyl radicals when the polysaccharide concentration was 200 μg/mL. Through the experimental data, it can be found that pomegranate flower polysaccharide exhibits excellent antioxidant capacity, which is stronger than that of LBP and AP. Pomegranate flower polysaccharides contain hydrogen donors, which have the ability to provide hydrogen protons, which can reduce highly oxidative free radicals, thereby terminating the free radical chain reaction and achieving the purpose of scavenging or inhibiting free radicals.

Clear DPPH·Experiment

The experimental steps are as follows: Accurately weigh 12 mg DPPH, dissolve it in ethanol to make a 62.5 μg/mL solution, take 2 mL of 200 μg/mL polysaccharide solution or vitamin C solution, and add it to a 10 mL closed test tube containing 2 mL DPPH solution . After the mixture was left to stand in the dark for 10 min, the absorbance was measured at 517 nm with a UV-vis spectrophotometer, and each sample was parallelized three times. %DPPH clearance rate = l-[(A-B)/A0] × 100%, A0: the absorption value after 2 mL ethanol replaced the sample; A: the absorption of the sample; B: the absorption of the blank sample.

Diphenylpicric acyl (DPPH·) is a very stable nitrogen-centered free radical, if the test substance can remove it, it means that the test substance has the ability to reduce hydroxyl free radicals, alkyl free radicals or hydrogen peroxide The effective concentration of free radicals and the effect of interrupting the lipid peroxidation chain reaction, as shown in Figure 4, pomegranate flower polysaccharides showed good antioxidant activity, and the pomegranate flower polysaccharides precipitated with 70% ethanol had the best scavenging rate on DPPH· Strong, the clearance rate can reach 55%; the results also show that pomegranate flower polysaccharide has a stronger ability to scavenge DPPH· than Lycium barbarum polysaccharide (LBP) and Astragalus polysaccharide (AP).

Through the above antioxidant experiments, it can be proved that the pomegranate flower polysaccharide has a strong antioxidant capacity, and the antioxidant capacity is positively correlated with the concentration. In the scavenging·OH and DPPH·experiments, the results also showed that pomegranate flower polysaccharides had stronger antioxidant capacity than Lycium barbarum polysaccharides and Astragalus polysaccharides.

Study on the Immunomodulatory Effect of Pomegranate Flower Polysaccharide

2.1 Effects of pomegranate flower polysaccharide on the immune organs of mice

Take 50 Kunming mice with a weight of about 20 g, half male and half male, and randomly divide them into 5 groups A-E, with 10 mice in each group. Polysaccharide B group (dosage 40 mg/kg×d), pomegranate flower polysaccharide group C (dosage 40 mg/kg×d), wolfberry polysaccharide group (dosage 40 mg/kg×d) and blank control group (normal saline, dosage 40mg /kg×d), and the mice in each group were gavaged according to the dosage for 14 consecutive days. The mice were sacrificed 1 h after the last administration, and the body weight, thymus and spleen mass of the mice were weighed, and the ratio of the mass of thymus and spleen to the total mass was used as the index of thymus and spleen. The experimental results showed that pomegranate flower polysaccharide A, pomegranate flower polysaccharide B, pomegranate flower polysaccharide C and Lycium barbarum polysaccharide significantly increased food intake and exercise compared with the control group (group E). The results of spleen and thymus index of experimental mice can be seen in Table 6. It can be seen from the table that pomegranate flower polysaccharides A, B, and C can all promote the increase of thymus and spleen weight in normal mice, and there is no significant difference compared with Lycium barbarum polysaccharide group. Compared with the blank control group, there was a significant difference. This shows that pomegranate flower polysaccharide A, pomegranate flower polysaccharide B, pomegranate flower polysaccharide C and Lycium barbarum polysaccharide can all promote the amount of food intake and exercise in mice.

Effects of pomegranate peel polysaccharide on phagocytosis of macrophages

Take 50 Kunming mice with a weight of about 20 g, half male and half male, and randomly divide them into 5 groups A-E, with 10 mice in each group. Polysaccharide B group (dosage 40 mg/kg×d), pomegranate flower polysaccharide group C (dosage 40 mg/kg×d), wolfberry polysaccharide group (dosage 40 mg/kg×d) and blank control group (normal saline, dosage 40mg /kg×d), mice in each group were intragastrically administered according to the dosage, and on the 7th day, 1 hour after intragastric administration, each mouse was intraperitoneally injected with 1 mL of chicken red blood cell saline suspension, and 6 hours later, the neck dislocation method was used. The mice were sacrificed, the abdominal skin of the mice was cut along the midline of the abdomen, the abdomen of the mice was rinsed with normal saline, and gently massaged for 1 min, the peritoneal fluid was drawn to make a smear, and placed in an incubator at 37°C for 30 min, and stained with Swiss staining method. Count under a microscope. The experimental results are shown in Table 7. It can be seen from the table that the pomegranate flower polysaccharides A, B, and C of the present invention can significantly improve the phagocytosis index and phagocytosis percentage, and there is no significant difference between them and the Lycium barbarum polysaccharide group, and there is a significant difference between them and the control group .

Natural plant polysaccharides can effectively enhance the body's non-specific immunity and cellular immunity. As a good immune regulator, it can promote the production of antibodies, activate cytokines, and regulate the body's immune system as a whole. Macrophages are auxiliary cells in the immune response, are an important part of the body's immune system, and play a vital role in both nonspecific and specific immunity. The above experiments prove that pomegranate flower polysaccharides A, B, and C can significantly improve the phagocytic ability of macrophages, and can significantly enhance the body's non-specific immunity and cellular immune system.

Claims (8)

1. an extracting method of pomegranate flower polysaccharide is characterized in that comprising the following steps: (1) Wash the pomegranate flowers, dry them and crush them; (2) Add ethanol solution to the pomegranate flower powder for extraction, filter to get the filter residue; (3) Add water to the filter residue, microwave extraction, and combine the extracts; (4) Add the extract to Sevag reagent to remove protein to obtain a deproteinized extract; (5) Dialyze the deproteinized extract with a dialysis membrane to remove impurities to obtain a dialysate; (6) Vacuum concentrate the dialysate to 1/8~1/4 of the original volume to obtain a concentrated solution, add absolute ethanol to the concentrated solution until the volume concentration of ethanol reaches 70%, separate after standing, and evaporate the solvent from the obtained precipitate Polysaccharide A crude product, add absolute ethanol to the supernatant until the ethanol volume concentration is 80%, separate after standing, evaporate the solvent to dry the polysaccharide B crude product, add absolute ethanol to the supernatant until the ethanol volume concentration is 95% , separated after standing, and the crude product of polysaccharide C was evaporated to dryness by precipitation; (7) The crude products of polysaccharides A, B, and C were purified by Sephadex gel chromatography column, eluted with water as the mobile phase, the eluate was collected, and freeze-dried to obtain pomegranate flower polysaccharides A, B, and C Pure; In step (2), the volume concentration of the ethanol solution is 75-95%, and the amount of the ethanol solution is 3-6 times the mass of pomegranate flower powder; In step (3), microwave extraction is performed 3 times, and the amount of water used each time is 20-30 times the quality of the filter residue; In step (3), the microwave power is 800-1000 W, the microwave extraction temperature is 90-100°C, and the extraction time is 10 minutes; In step (5), the molecular weight of the dialysis membrane is 2000-5000Da, and the dialysis time is 2 days; In step (7), the dextran gel is SephadexG-100, and the flow rate of water during elution is 0.05-2 ml/min; Pomegranate flower polysaccharide A, pomegranate flower polysaccharide B and pomegranate flower polysaccharide C all contain mannose, glucuronic acid, rhamnose, glucose, galactose, arabinose and xylose. 2. The extraction method according to claim 1, characterized in that: in step (2), the volume concentration of the ethanol solution is 95%; the amount of the ethanol solution is 5 times the mass of the pomegranate flower powder. 3. The extraction method according to claim 1, characterized in that: in step (1), pomegranate flowers are dried and crushed to 60 meshes with a high-speed pulverizer; in step (2), reflux extraction with ethanol solution for 2-4 hours. 4. The extraction method according to claim 1, characterized in that: in step (3), microwave extraction is performed three times, and the amount of water used each time is 28 times the mass of the filter residue. 5. The extraction method according to claim 1, characterized in that: in step (5), the molecular weight of the dialysis membrane is 3500Da. 6. The extraction method according to claim 1, characterized in that: in step (4), the amount of Sevag reagent is 3-5 times the volume of the extract; Sevag reagent is chloroform and n-butanol with a volume ratio of 5:1 mixed solution; in step (6), when separating crude polysaccharides A, B, and C, the resting time is 8-12 hours. 7. The extraction method according to claim 1, characterized in that: in step (7), the flow rate of water during elution is 0.3 ml/min. 8. An antioxidant active agent or a body immune regulator, characterized in that: the active ingredient comprises pomegranate flower polysaccharide A and pomegranate flower polysaccharide obtained according to the extraction method of the pomegranate flower polysaccharide described in any one of claims 1-7. At least one of B and pomegranate flower polysaccharide C.