A method for preparing polysaccharide iron of Choerospondias axillaris

Abstract

The present invention discloses a preparation method of polysaccharide iron of Choerospondias axillaris, and the preparation method is as follows: prepare polysaccharide of Choerospondias axillaris from fresh pulp of Choerospondias axillaris, then conduct orthogonal experiments with the polysaccharide of Choerospondias axillaris, and finally obtain that the factor order affecting the content of Fe<supgt;3+< / supgt; in the polysaccharide iron complex is: mass ratio of polysaccharide of Choerospondias axillaris to trisodium citrate > reaction temperature > reaction time > addition amount of ferric chloride. The optimal preparation process conditions of the polysaccharide iron complex are determined as pH 8-9, reaction time 1 h, mass ratio of polysaccharide of Choerospondias axillaris CAP30 to trisodium citrate 0.5, reaction temperature 60 °C, and addition amount of 2 mol / L ferric chloride 0.6 mL. The beneficial effects of the present invention are: under the optimal synthesis process conditions, the iron content of the polysaccharide iron complex of Choerospondias axillaris is about 27.09%, which helps to improve the added value of Choerospondias axillaris, extend the deep processing industrial chain of Choerospondias axillaris, and has good application value and market prospects.

Description

Technical Field

[0001] The present invention relates to the field of iron supplements, and specifically to a preparation method of polysaccharide iron from Choerospondias axillaris. Background Art

[0002] Iron is the essential trace element with the highest content in the human body. It is an essential component of hemoglobin and myoglobin, and participates in the metabolism and immune function of the human body. Iron deficiency in the body will cause various dysfunctions and iron deficiency anemia (IDA). IDA is one of the most common nutritional deficiencies in the world. With the economic development and the improvement of living standards, its incidence rate gradually decreases, but it still exists generally, mainly occurring in children and women of childbearing age.

[0003] Among the main anemic populations such as pregnant women and children, the anemia caused by iron deficiency accounts for more than 50%. For pregnant women with IDA, the cesarean section delivery rate and the incidence of postpartum hemorrhage are significantly increased, and the incidence of low birth weight and premature birth of the fetus also increases with the aggravation of the anemia degree of the patient. For child patients, IDA will affect the development of the important organ functions of infants and young children, cause movement disorders and decreased immunity in children, and affect the cognitive development of children. IDA is also a common complication of chronic kidney disease (CKD). Approximately 42.7% of CKD patients suffer from IDA. For CKD patients with IDA, anemia can accelerate the progression of CKD, increase the dialysis risk and the risk of cardiovascular events of the patient. In addition, due to insufficient oxygen in the blood, patients are prone to symptoms such as fatigue, irritability, and memory loss, which seriously affect physical health, reduce the quality of life and labor ability.

[0004] The threat of iron deficiency to health lies in its concealment. Only when iron deficiency reaches a certain level will the body show obvious symptoms, and at this time, nutritional deficiency diseases have already occurred. Long-term and severe iron deficiency may even cause lifelong disabilities and brain dullness that are difficult to completely eliminate. Therefore, the timely diagnosis and treatment of IDA have important clinical significance.

[0005] Iron supplements are specific drugs for treating iron deficiency, and are a safe, effective and economical common method for preventing and treating IDA. Therefore, it is urgently necessary to develop a new type of nutritional iron supplement with high efficiency and low toxicity. At present, oral iron agents mainly include the first-generation iron agents represented by ferrous sulfate and the second-generation iron agents represented by ferrous succinate. Both the first-generation and second-generation iron agents are absorbed in the form of ions, with relatively large adverse reactions, many gastrointestinal adverse events, and low bioavailability.

[0006] In recent years, the third-generation iron agent - polysaccharide iron complex has emerged. It not only has ideal stability, water solubility, absorption rate, etc., but also has no gastrointestinal irritation caused by free iron ions, and can play a good role in preventing and treating IDA. On the other hand, after the iron in the polysaccharide iron complex is released, the ligand polysaccharide can still exert various biological activities in the body, such as immunomodulation, antivirus, hypoglycemic, etc. Therefore, polysaccharide iron (III) complexes with trivalent iron (Fe 3+ )as the core are a class of new iron-supplementing substances with great potential, and are expected to be developed into a biological iron supplement with dual effects, and have become a hot topic in current research.

[0007] Choerospondias axillaris is a plant of the genus Choerospondias in the family Anacardiaceae. The fruit of Choerospondias axillaris is also known as five-eye fruit, acetic acid fruit, snot fruit, etc. It is mainly distributed in Jiangxi, Hubei, Hunan, Sichuan and other places in China, and also in countries such as India and Japan. Recognized by the State Forestry Administration, Chongyi County, Ganzhou City, Jiangxi Province officially obtained the title of "Hometown of Choerospondias axillaris in China" in December 2004. The pulp of Choerospondias axillaris is rich in a large amount of nutritional components such as plant flavonoids, natural pectin, vitamins, etc., and has the functions of antioxidant, anti-aging, protecting cardiovascular, anti-tumor, antivirus and nourishing the heart and calming the mind. In the past for a long time, Choerospondias axillaris was often regarded as a wild fruit and abandoned in the mountains because of its sour taste when eaten raw; with the continuous development of modern processing industry and scientific research, this "wild fruit" shows high application value whether as a natural medicine or processed into a health food. Polysaccharide is an important macromolecular active ingredient in jujube fruits.

[0008] The technical team studied the extraction process conditions of Choerospondias axillaris crude polysaccharide CAP. Under the optimal extraction process, the extraction rate of Choerospondias axillaris crude polysaccharide CAP is about 27%, indicating that the pulp of Choerospondias axillaris is rich in natural polysaccharides, which will help to increase the added value of Choerospondias axillaris. Summary of the Invention

[0009] The purpose of the present invention is to provide a preparation method of Choerospondias axillaris polysaccharide iron, which helps to increase the added value of Choerospondias axillaris, extend the deep processing industrial chain of Choerospondias axillaris, and has good application value and market prospect.

[0010] The technical scheme adopted by the present invention is as follows: A preparation method of Choerospondias axillaris polysaccharide, and the method steps are as follows:

[0011] Step S1, Wash the fresh Choerospondias axillaris, remove the skin and seeds to obtain the pulp of Choerospondias axillaris, and dry and crush the pulp of Choerospondias axillaris to obtain Choerospondias axillaris powder;

[0012] Step S2, Extract the Choerospondias axillaris powder under extraction conditions to obtain a crude extract of Choerospondias axillaris. The extraction conditions are: distilled water: the material-liquid ratio of the Choerospondias axillaris powder in step S1 is 1:40 g / mL, the extraction temperature is 100 °C, and the extraction time is 5 h;

[0013] Step S3: After filtering and concentrating the Choerospondias axillaris extract, add anhydrous ethanol to make the ethanol concentration of the solution 20% v / v. Let it stand in a refrigerator at 4°C for more than 24 hours and then centrifuge at a rotational speed of 4,800 r / min for 10 min. Discard the precipitate at the lower layer, and the supernatant obtained is the Choerospondias axillaris polysaccharide solution.

[0014] Step S4: Continue to add anhydrous ethanol to the Choerospondias axillaris polysaccharide solution to make the ethanol concentration of the Choerospondias axillaris solution reach 30% v / v. Again, let it stand in a refrigerator at 4°C for more than 24 hours and then centrifuge at a rotational speed of 4,800 r / min for 10 min. After discarding the supernatant at the upper layer, the precipitate part at the lower layer is the Choerospondias axillaris polysaccharide CAP30, which is dried in vacuum for later use.

[0015] Furthermore, calculate the polysaccharide yield. The Choerospondias axillaris polysaccharide CAP30 contains neutral sugars, uronic acids, and proteins. The phenol-sulfuric acid method is used to determine the content of neutral sugars in the Choerospondias axillaris polysaccharide CAP30, the m-hydroxybiphenyl colorimetric method is used to determine the content of uronic acids in the Choerospondias axillaris polysaccharide CAP30, and the Coomassie brilliant blue method is used to determine the proteins in the Choerospondias axillaris polysaccharide CAP30.

[0016] Furthermore, the phenol-sulfuric acid method is used to determine the content of neutral sugars in the Choerospondias axillaris polysaccharide CAP30. The specific steps are as follows:

[0017] Step (1): Prepare the standard curve: Accurately weigh 0.0100 g of glucose standard product dried to constant weight at 105°C, place it in a 10 mL volumetric flask, dissolve it with distilled water and dilute to the scale. The resulting mother liquor, i.e., the concentration of the glucose standard solution, is 1 mg / mL.

[0018] Dilute the mother liquor to 20, 40, 60, 80, and 100 μg / mL respectively; respectively pipette 1 mL of the diluted glucose standard solutions of the above series of concentrations, add 1 mL of 5% phenol and 5 mL of concentrated sulfuric acid, mix well, and measure the absorbance at 490 nm after 35 min.

[0019] Step (2): Sample determination: Weigh 0.0100 g of Choerospondias axillaris polysaccharide CAP30 respectively, dissolve it by ultrasonic treatment, and make the volume constant in a 100 mL volumetric flask; pipette 1 mL of the Choerospondias axillaris polysaccharide CAP30 solution into a test tube, add 1 mL of 5% phenol and 5 mL of concentrated sulfuric acid respectively, mix well, and measure the absorbance at 490 nm after 35 min.

[0020] Furthermore, the m-hydroxybiphenyl colorimetric method is used to determine the content of uronic acids in the Choerospondias axillaris polysaccharide CAP30. The specific steps are as follows:

[0021] Step (1), prepare a standard curve using galacturonic acid: Weigh 10 mg of galacturonic acid standard and dissolve it in 10 mL of distilled water to prepare a stock solution of 1 mg / mL, which is the galacturonic acid standard solution. Then dilute it to solutions of 20, 40, 60, 80, and 100 μg / mL respectively.

[0022] Take 0.5 mL of the above series of diluted stock solutions respectively. Add 0.5 mL of distilled water to the blank test tubes. Add 3 mL of reagent A to each blank test tube, mix well, heat in a water bath at 100 °C for 5 min, then cool in an ice bath. Then add 50 μL of reagent B and let it stand at room temperature for 20 min, and measure the absorbance at 520 nm.

[0023] Step (2), sample determination: Weigh 0.0100 g of Choerospondias axillaris polysaccharide CAP30, dissolve it by ultrasonic treatment, and make the volume up to 100 mL in a volumetric flask. Take 0.5 mL of the Choerospondias axillaris polysaccharide CAP30 solution in a test tube, add 3 mL of reagent A respectively, mix well, heat in a water bath at 100 °C for 5 min, then cool in an ice bath. Then add 50 μL of reagent B respectively and let it stand at room temperature for 20 min, and measure the absorbance at 520 nm.

[0024] Among them, reagent A: Dissolve 0.25 g of sodium tetraborate in 100 mL of concentrated sulfuric acid solution.

[0025] Reagent B: Dissolve 0.15 g of 3-phenylphenol and 0.5 g of NaOH in 100 mL of water, wrap it with tin foil and store it in a refrigerator at 4 °C in the dark. It is valid within one month.

[0026] Furthermore, the Coomassie brilliant blue method is used to determine the protein in Choerospondias axillaris polysaccharide CAP30. The specific steps are as follows:

[0027] Step (1), prepare a standard curve: Weigh 0.0100 g of bovine serum albumin (BSA) as a standard substance, and prepare a stock solution of 1.00 mg / mL, which is the bovine serum albumin standard solution. Dilute the 1.00 mg / mL bovine serum albumin standard solution to standard solutions of 10, 20, 40, 60, 80, and 100 μg / mL respectively.

[0028] Take 1.0 mL of the above series of diluted bovine serum albumin standard solutions in a test tube, add 5.0 mL of Coomassie brilliant blue solution, mix well immediately and react for 5 min, and measure the absorbance at 595 nm.

[0029] Step (2), sample determination: Weigh 10 mg of Choerospondias axillaris polysaccharide CAP30 and dissolve it in 1 mL of water, add 5 mL of Coomassie brilliant blue G-250 staining agent, mix well immediately and react for 5 min, and measure the absorbance at 595 nm.

[0030] Preparation of Coomassie Brilliant Blue G-250 Dye Reagent: Weigh 100 mg of Coomassie Brilliant Blue, dissolve it in 50 mL of 95% ethanol, then add 120 mL of 85% phosphoric acid, and make up the volume to 1 L with distilled water.

[0031] Furthermore, a preparation method of polysaccharide iron of Choerospondias axillaris is as follows:

[0032] Step S5: Weigh the reserved Choerospondias axillaris polysaccharide CAP30 and trisodium citrate. Dissolve Choerospondias axillaris polysaccharide CAP30 and trisodium citrate in 10 mL of deionized water. Fix the mass of Choerospondias axillaris polysaccharide CAP30 at 0.1 g, and make the mass ratio of Choerospondias axillaris polysaccharide CAP30 to trisodium citrate be 0 (the mass of trisodium citrate is 0), 0.5, 1, 1.5, 2 respectively. Stir magnetically for 10 min to mix evenly to obtain a mixed solution.

[0033] Step S6: Continuously stir the mixed solution magnetically in a constant temperature water bath at different temperature gradients of 50 °C, 60 °C, 70 °C, 80 °C, 90 °C. Slowly and evenly add FeCl3 solutions with volumes of 0 mL, 0.3 mL, 0.6 mL, 0.9 mL, 1.2 mL at 2 mol / L respectively. Control the pH of the mixed solution to be 5 - 6, 6 - 7, 7 - 8, 8 - 9, 9 - 10 respectively by dropping 20% NaOH solution.

[0034] Step S7: The reaction time of the mixed solution at different temperature gradients is 0.5 h, 1 h, 1.5 h, 2 h, 2.5 h respectively. Centrifuge the mixed solution at a centrifugal speed of 4000 rpm for 10 min to remove the reddish-brown precipitate and obtain the supernatant.

[0035] Step S8: Add 4 times the volume of absolute ethanol to the supernatant, let it stand for more than 24 hours, centrifuge again at a centrifugal speed of 4000 rpm for 10 min to obtain the lower precipitate. Take the lower precipitate and wash it 2 times with absolute ethanol, centrifuge at a centrifugal speed of 4000 rpm for 10 min, and then vacuum dry it to obtain the crude polysaccharide iron of Choerospondias axillaris.

[0036] Step S9: Dissolve the crude polysaccharide iron of Choerospondias axillaris in water, dialyze it against deionized water in a dialysis bag for 3 days to obtain a dialysis solution. Vacuum concentrate the dialysis solution to about one-fourth of its volume to obtain a concentrated solution, add 4 times the volume of ethanol for alcohol precipitation, centrifuge at a centrifugal speed of 4000 rpm for 10 min, remove the supernatant, and finally vacuum dry it to obtain the polysaccharide iron complex of Choerospondias axillaris.

[0037] Furthermore, for the polysaccharide iron complex Fe 3+Using the iron content as the evaluation index, with the pH fixed at 8 - 9, an orthogonal experiment was designed using an orthogonal table with four factors and three levels to conduct a range analysis on four factors: the mass ratio of Choerospondias axillaris polysaccharide CAP30 to sodium citrate, the addition amount of ferric chloride, the reaction temperature, and the reaction time. The optimal preparation process conditions were obtained based on the magnitude of the range;

[0038] The factors affecting the iron 3+ content were in the order of: the mass ratio of Choerospondias axillaris polysaccharide CAP30 to sodium citrate > reaction temperature > reaction time > addition amount of ferric chloride. The optimal preparation process conditions for the polysaccharide iron complex were determined as follows: the reaction time was 1 h, the mass ratio of Choerospondias axillaris polysaccharide CAP30 to sodium citrate was 0.5, the reaction temperature was 60 °C, and the addition amount of ferric chloride was 0.6 mL.

[0039] Furthermore, to determine the iron content in Choerospondias axillaris polysaccharide iron, a method for determining the iron content in Choerospondias axillaris polysaccharide iron by phenanthroline colorimetry was adopted. Specifically:

[0040] Step (1), making the standard curve:

[0041] Prepare ammonium ferrous sulfate hexahydrate (Fe(NH4)2·(SO4)2·6H2O) at 100 mg / L, and dilute it 10 times to make a working solution of ammonium ferrous sulfate hexahydrate at 10 mg / L. Take 7 50 - mL volumetric flasks, accurately add different volumes (0.00, 1.00, 2.00, 4.00, 6.00, 8.00, 10.00 mL) of the above 10 mg / L ammonium ferrous sulfate hexahydrate working solution into 50 - mL volumetric flasks. Then, add 1 mL of hydroxylamine hydrochloride (10%) and 2.5 mL of phenanthroline color reagent with a concentration of 0.1% [2.5 mL of phenanthroline color reagent (0.1%, w / v)] in sequence. After shaking well, add a small piece of congo red test paper, slowly add ammonia water until the congo red test paper just changes from blue to red - violet. Then, add 5 mL of acetic acid - sodium acetate buffer solution (6.8 g of sodium acetate is added to 2.88 mL of acetic acid and made up to 100 mL in a volumetric flask), shake well and make up the volume with distilled water, and let it stand for 15 min. Measure the absorbance at 510 nm. Using distilled water instead of the Choerospondias axillaris polysaccharide iron sample solution as the blank control;

[0042] Step (2): Take 20 mg of the polysaccharide iron complex of Choerospondias axillaris sample and dissolve it in 50 mL of distilled water. Take 10 mL of the above sample solution and make up the volume to 50 mL with distilled water. Add 1 mL of sulfuric acid solution (concentrated sulfuric acid: water = 1:35, v / v), 5.0 mL of potassium persulfate solution (40 g / L), place it on an electric furnace, slowly boil for 15 min, keep the volume not less than 20 mL, cool to room temperature, and adjust the pH to be close to 2 with ammonia water solution (ammonia: water = 1:3, v / v) or sulfuric acid solution (concentrated sulfuric acid: water = 1:35, v / v) for standby.

[0043] Step (3): Determination of the iron content in the polysaccharide iron complex of Choerospondias axillaris sample: Take 5 mL of the sample in

Step S42

[0044] Step (4): Through the standard curve y = 0.04506x + 0.0012 (R 2 = 0.9995) prepared by ammonium ferrous sulfate hexahydrate standard solution, calculate the iron content in the polysaccharide iron complex of Choerospondias axillaris, where y is the absorbance, x is the concentration of ammonium ferrous sulfate hexahydrate working solution, and R is the linear correlation coefficient.

[0045] The beneficial effects of the present invention are as follows: Using orthogonal experiments to optimize the preparation process of the polysaccharide iron complex of Choerospondias axillaris and developing a new type of high-efficiency and low-toxic nutritional iron supplement will help to increase the added value of Choerospondias axillaris, extend the deep processing industrial chain of Choerospondias axillaris, and have good application value and market prospects. The beneficial effects of the present invention are as follows: Under the optimal synthesis process conditions, the iron content of the polysaccharide iron complex of Choerospondias axillaris is about 27.09 ± 0.66%, which helps to increase the added value of Choerospondias axillaris, extend the deep processing industrial chain of Choerospondias axillaris, and have good application value and market prospects. Description of the Drawings

[0046] Figure 1 It is a diagram showing the influence of the addition amount of ferric chloride in the present invention on the iron content of the polysaccharide iron complex of Choerospondias axillaris.

[0047] Figure 2 It is a diagram showing the influence of the mass ratio of Choerospondias axillaris polysaccharide CAP30 to sodium citrate on the iron content of the polysaccharide iron complex of Choerospondias axillaris in the present invention.

[0048] Figure 3This is a graph showing the effect of the pH of the reaction system of the present invention on the iron content of polysaccharide iron complex from Choerospondias axillaris.

[0049] Figure 4 This is a graph showing the effect of the reaction temperature of the present invention on the iron content of polysaccharide iron complex from Choerospondias axillaris.

[0050] Figure 5 This is a graph showing the effect of the reaction time of the present invention on the iron content of polysaccharide iron complex from Choerospondias axillaris. Detailed implementation manners

[0051] The fruits of Choerospondias axillaris were purchased from Chongyi County, Ganzhou City, Jiangxi Province.

[0052] The present invention works and is implemented as follows. The optimization method for the preparation process of polysaccharide from Choerospondias axillaris is as follows: Fresh Choerospondias axillaris fruits are washed, peeled, and pitted, and the pulp is dried and pulverized. Polysaccharide from Choerospondias axillaris is extracted under the optimal extraction process with a solid-liquid ratio of 1:40 (g / mL), an extraction temperature of 100 °C, and an extraction time of 5 h. After the extract is filtered and concentrated, absolute ethanol is added. When the ethanol concentration is 20% (v / v), it is left to stand at 4 °C for more than 24 hours, and then centrifuged (4800 r / min, 10 min) to discard the precipitate impurities; absolute ethanol is continuously added to the supernatant to make the ethanol concentration of the solution reach 30% (v / v), and it is again left to stand at 4 °C for more than 24 hours and centrifuged (4800 r / min, 10 min). At this time, if absolute ethanol is continuously added to the supernatant, no new precipitate will be produced. Therefore, the obtained precipitate is polysaccharide from Choerospondias axillaris (polysaccharide CAP30 from Choerospondias axillaris), which is dried in vacuo for later use.

[0053] The polysaccharide yield is calculated, and the neutral sugar content is determined by the phenol-sulfuric acid method, the uronic acid content is measured by the m-hydroxybiphenyl colorimetric method, and the protein content is measured by the Coomassie brilliant blue method.

[0054] The orthogonal experiment of the present invention is carried out, and the orthogonal experiment method is as follows: Weigh 0.1 g of the Choerospondias axillaris polysaccharide CAP30 prepared in steps S1 - S4, add sodium citrate, dissolve it in 10 mL of distilled water, and stir magnetically for 10 min to make it evenly mixed. Continuously stir magnetically in a water bath at a certain temperature, and slowly and evenly drop a certain volume of 2 mol / L ferric chloride solution into the mixed solution. Control the pH value of the mixed solution by dropping 20% sodium hydroxide solution. After the dropping is completed, react the mixed solution in a heated water bath for a certain time. Centrifuge the reaction mixture (4000 rpm, 10 min) to remove the reddish - brown precipitate. Add 4 times the volume of absolute ethanol to the supernatant, let it stand overnight, centrifuge (4000 rpm, 10 min), take the precipitate, wash it 2 times with absolute ethanol, centrifuge (4000 rpm, 10 min), and vacuum - dry the lower - layer precipitate to obtain the crude Choerospondias axillaris polysaccharide iron. Dissolve the dried crude Choerospondias axillaris polysaccharide iron in water, dialyze it against deionized water in a dialysis bag for 3 days, take the inner dialysis solution, vacuum - concentrate it to about one - quarter of its volume, add 4 times the volume of ethanol to the concentrated solution for alcohol precipitation, centrifuge (4000 rpm, 10 min), remove the supernatant, and vacuum - dry it. Finally, the Choerospondias axillaris polysaccharide iron complex is prepared.

[0055] According to the results of single - factor experiments, taking the Fe 3+ content as the evaluation index, fixing the pH value at 8 - 9, using a four - factor and three - level orthogonal table (Table 1) to design the experiment, optimize the preparation process of the complex. In a 10 - mL reaction system, conduct a range analysis on four factors: the mass ratio of Choerospondias axillaris polysaccharide CAP30 to sodium citrate (A), the addition amount of ferric chloride (B), the reaction temperature (C), and the reaction time (D). Determine the optimal preparation process according to the magnitude of the range (K value). If the optimal factor combination is not in the orthogonal experiment design, further verification experiments need to be carried out.

[0056] Table 1 Orthogonal experiment design factor and level table

[0057]

[0058] For the data processing and analysis of the present invention, the data are expressed as mean ± standard deviation, with 3 replicates set. Use SPSS software for single - factor variance analysis, and P < 0.05 is determined to be statistically significant.

[0059] Results and analysis: Determination results of the physical and chemical indexes of Choerospondias axillaris polysaccharide; The yield of Choerospondias axillaris polysaccharide CAP30 is 16.51 ± 0.55%, the neutral sugar content is (76.15 ± 0.86)%, the uronic acid content is (51.93 ± 3.68), and the protein content is (1.17 ± 0.18)%.

[0060] Single - factor experiment results of the preparation of Choerospondias axillaris polysaccharide iron in the present invention

[0061] (1)Effect of the amount of ferric chloride added on iron content: Fix the mass ratio of Choerospondias axillaris polysaccharide CAP30 to trisodium citrate at 2:1, reaction temperature at 70 °C, reaction time at 1 h, control the pH of the reaction solution at 8 - 9, and investigate the mass ratio of Choerospondias axillaris polysaccharide (Choerospondias axillaris polysaccharide CAP30) to trisodium citrate. From Figure 1 It can be seen that when the addition amount of ferric chloride solution (2 mol / L) reaches 0.9 mL, the iron content reaches the maximum value. As the addition amount of ferric chloride further increases, the iron content decreases instead. This may be because when the addition amount of ferric chloride solution is too large, it will cause the formation of by-product ferrous oxide, reduce the pH of the reaction system, and is not conducive to the complexation reaction of Choerospondias axillaris polysaccharide and iron element.

[0062] (2)Effect of the mass ratio of Choerospondias axillaris polysaccharide CAP30 to trisodium citrate on iron content; Fix the addition amount of ferric chloride (2 mol / L) at 0.9 mL, reaction temperature at 70 °C, reaction time at 1 h, control the pH of the reaction solution at 8 - 9, and investigate the effect of the mass ratio of Choerospondias axillaris polysaccharide CAP30 to trisodium citrate on iron content. Trisodium citrate acts as a catalyst in the reaction. From Figure 2 It can be seen that the iron content with the addition of trisodium citrate is significantly higher than that without the addition of trisodium citrate. When the mass ratio of the two (Choerospondias axillaris polysaccharide: trisodium citrate) is 1.5, the iron content reaches the maximum value. As the mass ratio increases, the iron content decreases. This may be because when the addition amount is too large, the ion movement intensity in the reaction system is too large, which is not conducive to the complexation reaction of Choerospondias axillaris polysaccharide and iron element and affects the reaction efficiency.

[0063] (3)Effect of the pH of the reaction system on iron content; Fix the addition amount of ferric chloride at 0.9 mL, the mass ratio of Choerospondias axillaris polysaccharide CAP30 to trisodium citrate at 1.5, reaction temperature at 70 °C, reaction time at 1 h, and investigate the effect of the pH value of the solution on iron content. From Figure 3 It can be seen that as the pH value of the reaction system increases, the iron content also shows an upward trend. When the pH of the reaction system is in the range of 8.0 - 9.0, the iron content reaches the maximum value. However, when the pH of the reaction system reaches 10, the iron content of the Choerospondias axillaris polysaccharide - iron complex decreases significantly. This may be because the increase in the pH of the reaction system promotes the polymerization of iron elements through O - bridges or OH - bridges to form polymeric β - FOOH iron nuclei. When the pH of the reaction system is too high, the hydroxyl concentration in the reaction system is too high, and iron hydroxide precipitation will occur, thus reducing the iron content. Therefore, the pH of the reaction system is fixed at 8 - 9 for the orthogonal experiment.

[0064] (4)Effect of reaction temperature on iron content: Fix the addition amount of ferric chloride at 0.9 mL, the mass ratio of Choerospondias axillaris polysaccharide CAP30 to trisodium citrate at 1.5, the reaction time at 1 h, control the pH of the reaction solution at 8 - 9, and investigate the effect of reaction temperature on iron content. From Figure 4 It can be seen that when the reaction temperature is in the range of 50 - 70 °C, the iron content of Choerospondias axillaris polysaccharide iron increases slowly with the increase of temperature and reaches the maximum value at 70 °C. However, when the temperature exceeds 70 °C, the iron complex of Choerospondias axillaris polysaccharide decreases significantly. This may be due to the too high reaction temperature, which causes violent ion vibration in the reaction system and affects the progress of the complexation reaction.

[0065] (5)Effect of reaction time on iron content: Fix the addition amount of ferric chloride at 0.9 mL, the mass ratio of Choerospondias axillaris polysaccharide CAP30 to trisodium citrate at 1.5, the reaction temperature at 70 °C, control the pH of the reaction solution at 8 - 9, and investigate the effect of reaction time on iron content. From Figure 5 It can be seen that when the reaction time is in the range of 0.5 - 2.5 h, the iron content of Choerospondias axillaris polysaccharide iron first increases and then decreases, reaching the maximum value at 1 h. However, when the time exceeds 1 h, the iron complex of Choerospondias axillaris polysaccharide decreases significantly. This may be because during the reaction process, the complex formed by the complexation reaction of Choerospondias axillaris polysaccharide and iron element is unstable. Therefore, when the reaction time increases, it affects the iron content of Choerospondias axillaris polysaccharide iron.

[0066] The results of the orthogonal experiment are shown in Table 2. The order of factors affecting the iron content of the polysaccharide iron complex Fe 3+ is the mass ratio of Choerospondias axillaris polysaccharide CAP30 to trisodium citrate > reaction temperature > reaction time > addition amount of ferric chloride. The complex prepared by the orthogonal experiment No. 1 A1B1C1D1 (pH 8 - 9, reaction time 0.5 h, mass ratio of CAP to trisodium citrate 0.5, reaction temperature 50 °C, addition amount of ferric chloride 0.6 mL) has the highest Fe 3 + content, which is (26.02 ± 0.933)%. However, the optimal preparation process optimized according to the range of extreme differences is A1B1C2D2 (pH 8 - 9, reaction time 1 h, mass ratio of Choerospondias axillaris polysaccharide CAP30 to trisodium citrate 0.5, reaction temperature 60 °C, addition amount of ferric chloride 0.6 mL). Verifying this process, the results show that the Fe 3 + content of the obtained complex is (27.09 ± 0.66)%, which is higher than that of Process No. 1. Therefore, the optimal preparation process of the complex is determined as pH 8 - 9, reaction time 1 h, mass ratio of Choerospondias axillaris polysaccharide CAP30 to trisodium citrate 0.5, reaction temperature 60 °C, addition amount of ferric chloride 0.6 mL.

[0067] Table 3. Perform variance analysis on the results of the orthogonal experiment, and the analysis results are shown in Table 3 (in Table 3, * is P< 0.05 indicates a significant effect; ** is P < 0.01 indicates a highly significant effect)

[0068] Table 2 Orthogonal experiment table

[0069]

[0070] Table 3 Variance analysis of orthogonal experiment

[0071] 。

Claims

1. A preparation method of polysaccharide iron of Choerospondias axillaris, characterized in that: The method steps are as follows: Step S1: Wash fresh Choerospondias axillaris fruits, remove the skin and pits to obtain Choerospondias axillaris fruit pulp, and dry and crush the Choerospondias axillaris fruit pulp to obtain Choerospondias axillaris powder. Step S2: Under extraction conditions, extract the Choerospondias axillaris powder to obtain a crude extract of Choerospondias axillaris polysaccharide. The extraction conditions are as follows: distilled water: the material-liquid ratio of the Choerospondias axillaris powder in Step S1 is 1:40 g / mL, the extraction temperature is 100 °C, and the extraction time is 5 h. Step S3: After filtering and concentrating the above crude extract of Choerospondias axillaris polysaccharide, add absolute ethanol to make the ethanol concentration of the solution 20% v / v. Let it stand in a refrigerator at 4 °C for more than 24 hours and then centrifuge. The centrifugation speed is 4800 r / min and the time is 10 min. Discard the lower precipitate, and the obtained supernatant is the Choerospondias axillaris polysaccharide solution. Step S4: Continue to add absolute ethanol to the Choerospondias axillaris solution to make the ethanol concentration of the Choerospondias axillaris supernatant reach 30% v / v. Again, let it stand in a refrigerator at 4 °C for more than 24 hours and then centrifuge. The centrifugation speed is 4800 r / min and the time is 10 min. After discarding the upper supernatant, the lower precipitate part is Choerospondias axillaris polysaccharide CAP30, which is vacuum-dried and reserved for use. Step S5: Weigh the reserved Choerospondias axillaris polysaccharide CAP30 and trisodium citrate. Dissolve the Choerospondias axillaris polysaccharide CAP30 and trisodium citrate in 10 mL of deionized water. Fix the mass of Choerospondias axillaris polysaccharide CAP30 at 0.1 g, and make the mass ratios of Choerospondias axillaris polysaccharide CAP30 and trisodium citrate 0.5, 1, 1.5, and 2 respectively. Stir magnetically for 10 min to mix evenly to obtain a mixed solution. Step S6: Continuously stir the mixed solution magnetically in a constant temperature water bath at different temperature gradients of 50 °C, 60 °C, 70 °C, 80 °C, and 90 °C, and slowly and evenly add FeCl3 solutions with volumes of 0.3 mL, 0.6 mL, 0.9 mL, and 1.2 mL at 2 mol / L. Control the pH of the mixed solution to be 5-6, 6-7, 7-8, 8-9, and 9-10 respectively by dropping 20% NaOH solution. Step S7: The reaction time of the mixed solution at different temperature gradients is 0.5 h, 1 h, 1.5 h, 2 h, and 2.5 h respectively. Centrifuge the mixed solution at a centrifugation speed of 4000 rpm for 10 min to remove the reddish-brown precipitate and obtain the supernatant. Step S8: Add 4 times the amount of absolute ethanol to the supernatant, let it stand for more than 24 hours, and centrifuge again at a centrifugation speed of 4000 rpm for 10 min to obtain the lower precipitate. Take the lower precipitate and wash it 2 times with absolute ethanol, and then centrifuge at a centrifugation speed of 4000 rpm for 10 min and vacuum-dry it to obtain the crude product of Choerospondias axillaris polysaccharide iron. Step S9: Dissolve the crude product of Choerospondias axillaris polysaccharide iron in water, tie it in a bag and dialyze it against deionized water to remove salts for 3 days to obtain a dialysate. Vacuum-concentrate the dialysate to one-fourth of its volume to obtain a concentrated solution, add 4 times the amount of ethanol for alcohol precipitation, centrifuge at a centrifugation speed of 4000 rpm for 10 min, remove the supernatant, and finally vacuum-dry it to obtain the Choerospondias axillaris polysaccharide iron complex.

2. The preparation method of polysaccharide iron of Choerospondias axillaris according to claim 1, characterized in that: Using the polysaccharide iron complex Fe 3+ content as the evaluation index, an orthogonal experiment was designed using an orthogonal table with four factors and three levels. Range analysis was carried out on four factors: the mass ratio of Choerospondias axillaris polysaccharide CAP30 to trisodium citrate, the addition amount of ferric chloride, the reaction temperature, and the reaction time. The preparation process conditions were obtained according to the magnitude of the range. Factors affecting the Fe content of polysaccharide iron complex are in the following order: mass ratio of Choerospondias axillaris polysaccharide CAP30 to trisodium citrate > reaction temperature > reaction time > addition amount of ferric chloride. The preparation process conditions of polysaccharide iron complex are determined as follows: reaction time is 1 h, mass ratio of Choerospondias axillaris polysaccharide CAP30 to trisodium citrate is 0.5, reaction temperature is 60 °C, and addition amount of ferric chloride is 0.6 mL. 3+ ​

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