CN113968917B - A kind of Poria cocos polysaccharide complex and preparation method thereof - Google Patents

Abstract

The invention discloses a pachyman compound and a preparation method thereof, wherein the preparation method comprises the following steps: (1) Mixing pachymaran, hydroxyethylidene diphosphonic acid, zinc chloride and hydrazine hydrate with water to obtain a mixed solution, and adjusting the pH value to 5.0-8.0; (2) Transferring the mixed solution into a closed polytetrafluoroethylene high-pressure reaction kettle, and carrying out microwave hydrothermal reaction for 10-30 min at 110-140 ℃; (3) Centrifuging, and sequentially precipitating the supernatant with ethanol, centrifuging, washing and drying. The method adopts a one-step in-situ microwave hydrothermal reaction method to prepare the pachymaran compound, has the advantages of wide sources of reaction raw materials, simple and easy operation process, short synthesis time and low temperature, and more importantly, can effectively improve the phosphorus content and the zinc content in the pachymaran compound.

Description

A kind of Poria cocos polysaccharide complex and preparation method thereof

technical field

The invention relates to a polysaccharide complex and a preparation method thereof, in particular to a pachyphyll polysaccharide complex and a preparation method thereof, and belongs to the technical field of polysaccharide derivatives.

Background technique

The traditional Chinese medicine Poria cocos is a kind of medicinal fungus, which has the functions of dispelling dampness and diuresis, invigorating the spleen and stomach, calming the nerves and calming the mind. It can not only be used as medicine, but also a traditional health food. A large number of experiments and clinical studies have shown that the main biologically active substance Poria cocos contained in Poria cocos has pharmacological effects such as enhancing the body's immunity, anti-tumor, anti-virus, anti-inflammation, and hypnosis. However, due to the presence of strong intermolecular and intramolecular hydrogen bonds, the activity and solubility of pachyranan are reduced, so that its clinical administration route is limited. Therefore, it is necessary to modify the structure of Poria cocos polysaccharide to improve its solubility, enhance its biological activity, and reduce possible adverse reactions and side effects in clinical trials.

The activity of polysaccharides is directly or indirectly affected by its molecular structure. Appropriate chemical modification of the molecular structure of polysaccharides by certain methods can effectively improve the biological activity of polysaccharides, and even prepare polysaccharide derivatives with multiple biological activities. After molecular modification of polysaccharides, a variety of polysaccharide derivatives with new structures and functions have been designed, which is of great significance for the study of polysaccharide structure and performance, and provides a basis for the research and development of polysaccharide food additives, functional foods, health products and natural medicines. Provide experimental basis and theoretical basis.

At present, there are sulfated pachyranan, carboxymethyl pachyranan, acetylated pachyranan and sulfonylated pachyranan in literature reports, but there is no report on phosphoramidated pachyranan derivatives. There are many methods for polysaccharide structural modification, among which phosphorylation graft modification is a covalent modification in polysaccharide molecular modification, the hydroxyl on the branch chain is replaced by phosphate, which can enhance or increase the anti-tumor and anti-viral properties of polysaccharides. and immunomodulatory biological activities. The phosphate group is mainly attached to the carbon-6 and carbon-3 positions of the glucose unit, and the phosphorylation at the carbon-3 position can significantly improve the biological activity of the polysaccharide. At present, there are relatively few reports on phosphorylated polysaccharide derivatives in the literature, and there is no report on the phosphorylated polysaccharide derivatives of Poria cocos polysaccharides in the search on the Internet. According to reports, the process operation mentioned in these reports is relatively complicated, and the phosphate content of the polysaccharide derivatives obtained is not high.

Zinc is one of the essential trace elements for the human body. At least 85 kinds of enzymes are metalloenzymes containing zinc. Zinc is related to almost all protein and amino acid metabolism. Zinc plays a very important role in cell growth and proliferation, normal biological growth and development, improving immunity, preventing diseases, promoting physical and intellectual development, etc. It is also a component or enzyme activator of many metalloenzymes, and it also has obvious antioxidant effects . However, inorganic zinc supplementation is very irritating to the gastric mucosa, and most zinc ions are excreted from the body without being absorbed, so it is not conducive to human body absorption, and the effect of zinc supplementation is poor. Since organic zinc is closer to its function in the body, it is easier to be absorbed by the body. Over the years, researchers from various countries have been searching for an organic zinc nutritional enhancer with few side effects and high bioavailability. Trace elements and polysaccharides form complexes that can be directly digested and absorbed through the small intestine absorption route, greatly improving the absorption rate of trace elements, and can supplement polysaccharides needed by the human body while supplementing zinc. There are reports in the literature on the preparation of seaweed polysaccharide zinc complexes, Pleurotus eryngii polysaccharide complexes, Flammulina velutipes polysaccharide zinc complexes, Polyporus polysaccharide zinc complexes, etc., but there is no report on the zinc complex coordination derivatives of phosphoric acid amide-poria cocos polysaccharides .

Contents of the invention

The object of the present invention is to provide a kind of complex polysaccharide compound of phosphate group and zinc content both higher in phosphate root and zinc content——phosphoric acid amido-pachyan polysaccharide zinc complex coordination derivative and its preparation method.

In order to achieve the above object, the present invention adopts the following technical solutions:

A preparation method of a pachyrhin compound, characterized in that the pachyrhin compound is prepared by reacting hydroxyethylidene diphosphonic acid and zinc chloride with pachyrhin under microwave hydrothermal conditions, specifically comprising the following steps:

(1) Mixing polysaccharides, hydroxyethylidene diphosphonic acid, zinc chloride and hydrazine hydrate with water to obtain a mixed solution, and adjusting the pH value of the mixed solution to 5.0-8.0;

(2) Transfer the mixed solution into a closed polytetrafluoroethylene high-pressure reactor, and react with microwave hydrothermal reaction at 110-140°C for 10-30 minutes;

(3) Centrifuge to take the supernatant, and then carry out alcohol precipitation, centrifugation, washing and drying on the supernatant in sequence to obtain the complex of polysaccharides.

The preparation method of the aforementioned polysaccharide complex is characterized in that, in step (1), the mass ratio of polysaccharide, hydroxyethylidene diphosphonic acid, zinc chloride and hydrazine hydrate is 1.0: 1.0～3.0: 2.0～4.0 : 0.002～0.006.

The benefit of the present invention is that: the preparation method of the polysaccharide complex provided by the present invention adopts a one-step in-situ microwave hydrothermal reaction method, which not only has a wide source of reaction raw materials, but also has a simple and easy operation process and a short synthesis time , low temperature, low energy consumption, can effectively save energy, is green and environmentally friendly, has the potential of development and utilization, is convenient for industrialization and technology promotion, and has significant advantages in the actual application process. More importantly, this method can effectively improve The content of phosphorus and zinc in the polysaccharide complex can be popularized and used in the fields of food additives, functional health foods, medicines and the like.

Detailed ways

The present invention will be specifically introduced below in conjunction with specific embodiments.

The first part, the method for determining the phosphate content and zinc content in the sample

1. The method for determining the content of phosphate in the sample

Ammonium persulfate was used as an oxidant to convert organic phosphorus into inorganic phosphorus, molybdovanadate was used as a color reagent, and the molybdenum blue colorimetric method was used to determine the content of phosphate.

The determination steps are as follows:

(1) Slowly add 100mL concentrated sulfuric acid to 500mL water, cool to room temperature to obtain a sulfuric acid solution, dissolve 10g sodium molybdate in 400mL water to obtain a sodium molybdate solution, add the above sulfuric acid solution to the sodium molybdate solution , mix well to obtain sodium molybdate-sulfuric acid solution;

(2) Weigh 0.8g of ammonium persulfate and 4.2g of anhydrous sodium sulfate and mix them evenly to obtain ammonium persulfate-sodium sulfate decomposer;

(3) Weigh an appropriate amount of sample into a 100mL Erlenmeyer flask, first add 10mL of distilled water to the Erlenmeyer flask, then add 1mL of 1M sulfuric acid solution and 50mg of ammonium persulfate-sodium sulfate decomposition agent into the Erlenmeyer flask, and place the Erlenmeyer flask Heat evenly on a small electric furnace with asbestos net until the solution is just dry and thick and thick white smoke is just coming out;

(4) When it is slightly cold, add 10mL of water to the Erlenmeyer flask first, then add 4-40mg of sodium sulfite powder or 10 drops of methanol, then boil it on the electric furnace for 30-60s, then carefully transfer the solution to a 50mL colorimetric tube , and rinse the original Erlenmeyer flask several times with a small amount of water, merge the washing solution into the colorimetric tube, and finally control the solution in the colorimetric tube to about 25mL;

(5) Add 4mL sodium molybdate-sulfuric acid solution and 1mL hydrazine sulfate solution with a mass concentration of 0.15% to the colorimetric tube, put it in a boiled water bath for 10 minutes, take it out, cool it in running water, dilute it to the mark with water, and immediately dilute it with 1cm Measure the absorbance of the cuvette at a wavelength of 580nm with the reagent blank as a control, and check the corresponding phosphate content from the standard curve.

The formula for calculating the phosphate content is as follows:

PPO ₄ ^3- = CPO ₄ ^3- V/m×100%

In the formula, PPO ₄ ^3- is the phosphate content in the sample (%), CPO ₄ ^3- is the absorbance measured at 580nm and the corresponding phosphate concentration is found on the standard curve, V _sample is the volume of sample dilution, m _sample is the quality of the sample.

2. Method for Determination of Zinc Content in Samples

The content of zinc in the samples was determined by EDTA titration.

The determination steps are as follows:

(1) Weigh 1.0g of the sample (accurate to 0.0002g) into a 250mL Erlenmeyer flask, slowly add 10mL of acetic acid solution with a volume concentration of 25% with a dropper, add 50mL of distilled water after dissolution, boil the solution for 5 min, remove CO ₂ ;

(2) After cooling, adjust the pH value to 5~6 with a hexamethylenetetramine solution with a mass concentration of 20%, add acetic acid-sodium acetate buffer solution, add 3 drops of xylenol orange indicator, and use standard EDTA The solution was titrated, and the solution changed from purple red to bright yellow as the end point, and a blank control was made at the same time.

The formula for calculating the zinc content is as follows:

PZn ²⁺ = (C _EDTA ´ V _EDTA ´ M _Zn /m _sample ) ´ 100%

In the formula, PZn2 ⁺ is the zinc content (%) in the sample, C _EDTA is the concentration of the standard EDTA solution, V _EDTA is the volume of the standard EDTA solution, M _Zn is the molar amount of zinc, and _msample is the mass of the sample.

The second part, preparation of Poria cocos polysaccharide complex

Example 1

Mix polysaccharide, hydroxyethylidene diphosphonic acid, zinc chloride, hydrazine hydrate and water according to the mass ratio of 1.0: 2.5: 3.0:0.005: 20.0, adjust the pH value of the mixed solution to 6.5, and transfer the mixed solution into a closed polyquaternary In a vinyl fluoride high-pressure reaction kettle, microwave hydrothermal reaction at 130 ⁰ C for 20 minutes, centrifuge to take the supernatant, and then carry out alcohol precipitation, centrifugation, washing and drying on the supernatant in sequence to obtain the complex I of pachyrhin.

After the detection of the method for measuring the phosphate content and zinc content in the sample recorded in the first part, the phosphate content in the prepared Poria cocos polysaccharide complex I was 13.48%, and the zinc content was 10.07%.

Example 2

Poria cocos, hydroxyethylidene diphosphonic acid, zinc chloride, hydrazine hydrate and water are mixed according to the mass ratio of 1.0: 1.5: 2.0:0.002: 20.0, the pH value of the mixed solution is adjusted to 5.0, and the mixed solution is transferred to a closed poly In a tetrafluoroethylene high-pressure reactor, microwave hydrothermal reaction at 110 ⁰ C for 10 minutes, centrifuge to take the supernatant, and then carry out alcohol precipitation, centrifugation, washing and drying on the supernatant in sequence to obtain the complex II of polysaccharide.

After the detection of the method for measuring the phosphate content and zinc content in the sample recorded in the first part, the phosphate content in the prepared Poria cocos polysaccharide complex II is 7.03%, and the zinc content is 6.14%.

Example 3

Poria cocos, hydroxyethylidene diphosphonic acid, zinc chloride, hydrazine hydrate and water are mixed according to the mass ratio of 1.0: 2.0: 3.0:0.004: 20.0, the pH value of the mixed solution is adjusted to 8.0, and the mixed solution is transferred to a closed poly In a tetrafluoroethylene high-pressure reactor, microwave hydrothermal reaction at 120 ⁰ C for 30 minutes, centrifuge to take the supernatant, and then carry out alcohol precipitation, centrifugation, washing and drying on the supernatant in sequence to obtain the complex III of polysaccharide.

Through the detection of the method for measuring the phosphate content and zinc content in the sample recorded in the first part, the phosphate content in the prepared Poria cocos polysaccharide complex III is 8.24%, and the zinc content is 6.92%.

Example 4

Poria cocos, hydroxyethylidene diphosphonic acid, zinc chloride, hydrazine hydrate and water are mixed according to the mass ratio of 1.0: 2.5: 3.0:0.004: 20.0, the pH value of the mixed solution is adjusted to 7.0, and the mixed solution is transferred to a closed poly In a tetrafluoroethylene high-pressure reactor, microwave hydrothermal reaction at 140 ⁰ C for 30 minutes, centrifuge to take the supernatant, and then carry out alcohol precipitation, centrifugation, washing and drying on the supernatant in sequence to obtain the complex IV of pachyphyllum polysaccharide.

Through the detection of the method for measuring the phosphate content and zinc content in the sample recorded in the first part, the phosphate content in the prepared Poria cocos polysaccharide complex IV is 12.17%, and the zinc content is 8.94%.

Example 5

In comparative example 1, only microwave hydrothermal heating was changed to reflux heating, and other experimental conditions were the same.

Mix polysaccharides, hydroxyethylidene diphosphonic acid, zinc chloride, hydrazine hydrate and water according to the mass ratio of 1.0: 2.5: 3.0:0.005: 20.0, adjust the pH value of the mixture to 6.5, reflux heating reaction for 20min, and centrifuge to take the supernatant liquid, and then the supernatant was subjected to alcohol precipitation, centrifugation, washing, and drying in sequence to obtain the complex V of pachyphyllin.

Through the detection of the method for measuring the phosphate content and zinc content in the sample recorded in the first part, the phosphate content in the prepared Poria cocos polysaccharide complex V is 6.37%, and the zinc content is 4.26%, both of which are significantly lower than those using microwave water. The content of the phosphate radical and the content of zinc in the polysaccharide complex obtained by thermal method.

From the above five examples, it can be known that the preparation method of the pachyrhiza complex provided by the present invention (one-step in-situ microwave hydrothermal reaction method) can effectively increase the content of phosphorus and zinc in the polysaccharide complex.

To sum up, the present invention increases the coordination ability of the polysaccharide by performing one-step phosphoric acid hydrazide modification on the polysaccharide, realizes the microwave hydrothermal in-situ reaction, effectively improves the reaction efficiency, and makes the polysaccharide compound prepared The phosphorus content and the zinc content in it are all higher.

The third part, comparing the fluidity and permeability of pachymansin and pachymansin complex

The introduction of the hydrazide phosphate functional group builds a negatively charged hydrophilic surface structure for the polysaccharide, which can effectively improve the water solubility and activity of the polysaccharide, and the modified polysaccharide will have biological activities such as immune regulation. It shows potential application prospects in aspects and development of new materials. The polysaccharides and phosphohydrazide functional groups in the polysaccharide complexes are chelated and coordinated with zinc ions through oxygen, nitrogen or oxygen-nitrogen combinations, and the structure and functional modification technology can be used to transform pachyphyllan into more fluid and permeable polysaccharides. A good active polysaccharide is the key to the efficient development and utilization of Poria cocos polysaccharide. The polysaccharides and phosphate hydrazide functional groups in the polysaccharide complex tend to increase the biological activity of the polysaccharide when combined with zinc ions, and can exert a stronger synergistic effect, increase the permeability of the intestinal mucosa, and then improve various The digestion and absorption of nutrients is expected to improve the bioavailability of polysaccharides, and has a good development and application prospect.

1. Liquidity experiment

The fluidity test is mainly to investigate the difference in the fluidity of the polysaccharides and polysaccharide complexes.

Take by weighing 10.0 g pachyphyllan and the polysaccharide compound prepared by 10.0 g of Example 1 with a balance, place them in two beakers respectively, add 90 mL of distilled water to each of the two beakers, and make a pachyphyllan solution and a polysaccharide complex Add the substance solution and stir gently until it is completely dissolved (note: try not to generate bubbles when dissolving). A constant capillary viscometer with d = 1.0 mm was used to measure the flow time of pachyphyllan and polysaccharide complexes at different temperatures.

Table 1 The flow time of pachymansin at different temperatures

temperature 1st 2nd the 3rd time 4th Average time 25°C 112s 110s 111s 112s 111.25s 30℃ 97s 97s 98s 97s 97.25s 35°C 84s 85s 85s 83s 84.25s 40℃ 77s 76s 77s 77s 76.75s 45°C 67s 66s 66s 67s 66.50s

Table 2 Flow time of Poria cocos polysaccharide complex at different temperatures

temperature 1st 2nd the 3rd time 4th Average time 25°C 22s 22s 23s 22s 22.25s 30℃ 18s 19s 18s 19s 18.50s 35°C 15s 16s 15s 15s 15.25s 40℃ 13s 13s 13s 12s 12.75s 45°C 10s 10s 11s 11s 10.50s

The above experimental results show that the flow time of the polysaccharide complex is significantly shorter than that of the polysaccharide, indicating that the complex polysaccharide has better fluidity in comparison.

2. Permeability experiment

Accurately weigh 100 mg of pachyphyllin sample and 100 mg of pachyphyllin complex sample, respectively dissolve them in distilled water and set the volume to a 100 mL volumetric flask, shake evenly to obtain the sample solution. Use a pipette to draw 5 mL of the sample solution into a 50 mL volumetric flask, make up the volume with distilled water, shake evenly to obtain a diluted sample solution, and transfer the diluted sample solution to a stoppered Erlenmeyer flask. Cut a certain length of MD44 dialysis bag, fasten one end with a rubber band, add 10 mL of distilled water, then fasten the other end, put the tied dialysis bag into the above-mentioned conical flask with a stopper, and place it on a shaker at 100 Shake at r/min for 30min. Take 1 mL of the liquid in the dialysis bag and put it into a 10 mL graduated test tube with a stopper, add 1 mL of 5% phenol solution, then slowly drop in 5 mL of concentrated sulfuric acid, shake well, use distilled water as a blank control, and pass UV- Measure the absorbance value at 490 nm with a visible light spectrophotometer, and calculate the permeability according to the polysaccharide content of the standard curve by the following formula:

Permeability/%=( _{inside C bag} / _total C)*100%

In the formula, C is the content of polysaccharides _in the dialysis bag (mg/mL), and C _is the total content of polysaccharides (mg/mL).

The results showed that the permeability rate of the polysaccharide complex sample was 68.12%, and that of the polysaccharide sample was 49.26%.

From the results of the permeability experiment, it can be seen that the permeability of the polysaccharide compound is better than that of the polysaccharide.

It should be noted that the above embodiments do not limit the present invention in any form, and all technical solutions obtained by means of equivalent replacement or equivalent transformation fall within the protection scope of the present invention.

Claims (2)

1. a preparation method of polysaccharide compound of poria cocos, is characterized in that, under microwave hydrothermal condition, uses hydroxyethylidene diphosphonic acid and zinc chloride and polysaccharide reaction preparation of polysaccharide of cocos cocos, specifically comprises the following steps: (1) Mix Poria cocos polysaccharide, hydroxyethylidene diphosphonic acid, zinc chloride and hydrazine hydrate with water according to the mass ratio of 1.0:1.0～3.0:2.0～4.0:0.002～0.006:20 to obtain a mixed solution, adjust the ratio of the mixed solution pH value to 5.0-8.0; (2) Transfer the mixed solution into a closed polytetrafluoroethylene autoclave, and react with microwave hydrothermal reaction at 110-140°C for 10-30 minutes; (3) Centrifuge to take the supernatant, and then carry out alcohol precipitation, centrifugation, washing and drying on the supernatant in sequence to obtain the polysaccharide complex. 2. A polysaccharide complex, characterized in that, the polysaccharide complex is a phosphoamide polysaccharide zinc complex coordination derivative, which is specifically prepared by the method described in claim 1.