Selenized lycium barbarum polysaccharide and preparation method thereof

Abstract

The invention discloses selenized lycium barbarum polysaccharide and a preparation method thereof, and belongs to the technical field of structural modification of traditional Chinese medicine polysaccharide. The selenium content of the selenized lycium barbarum polysaccharide is 1.64 mg / g to 7.6 mg / g. The preparation method comprises the step of carrying out selenylation modification on the lycium barbarum polysaccharide by using a nitric acid-sodium selenite system. The anti-stress ability of the lycium barbarum polysaccharide can be remarkably improved, and materials and demonstration are provided for developing novel anti-stress polysaccharide drugs.

Description

Technical Field

[0001] The present invention belongs to the structural modification of Chinese herbal polysaccharides, and particularly relates to a selenium-enriched wolfberry polysaccharide and a preparation method thereof. Background Art

[0002] Wolfberry belongs to the genus Lycium of the Solanaceae family in plant taxonomy. It was first recorded for its medicinal function in "Shennong's Herbal Classic" and listed as a top-grade herb, and has been included in all editions of the "Chinese Pharmacopoeia". It is flat in nature and sweet in taste, and belongs to the liver, kidney, and lung meridians. It has the effects of nourishing the liver and kidney, improving eyesight, and moistening the lungs, and is mainly used to treat various diseases such as liver-kidney yin deficiency, dizziness, blurred vision, weakness of the waist and knees, enuresis, and consumptive thirst. Modern pharmacological studies have confirmed that wolfberry polysaccharide is the main active ingredient of wolfberry, with biological activities such as antioxidant, anti-tumor, enhancing the body's immunity, prolonging the lifespan of the body, protecting the liver, and improving reproductive function, but its bioavailability is low.

[0003] Selenium is an essential trace element for the body, with functions such as antioxidant, anti-aging, and immune regulation. It participates in the formation of the active center of the body's peroxidase. Appropriate use can enhance the body's resistance and antioxidant ability. As a kind of organic selenium, selenium polysaccharide maintains the basic structure of polysaccharide, while optimizing the effective bioavailability of trace element selenium and improving the pharmacological activity of polysaccharide, which is more conducive to the body's digestion and absorption, and plays the dual roles of plant polysaccharide and trace element selenium. Summary of the Invention

[0004] Object of the Invention: The object of the present invention is to provide a selenium-enriched wolfberry polysaccharide that can significantly enhance the anti-stress ability of wolfberry polysaccharide; another object of the present invention is to provide a preparation method of the selenium-enriched wolfberry polysaccharide.

[0005] Technical Solution: The selenium-enriched wolfberry polysaccharide described in the present invention has a selenium content of 1.64 - 7.6 mg / g.

[0006] The preparation method of the selenium-enriched wolfberry polysaccharide described in the present invention includes selenium-modifying wolfberry polysaccharide with a nitric acid-sodium selenite system.

[0007] Preferably, the specific steps include: mixing wolfberry polysaccharide and sodium selenite, and the mass ratio of wolfberry polysaccharide to sodium selenite is 0.8 - 1.2:1; adding a catalyst to react in a reaction system containing nitric acid; controlling the reaction temperature at 70 - 90 °C and the reaction time at 6 - 8 hours; removing unreacted substances by dialysis to obtain the selenium-enriched wolfberry polysaccharide.

[0008] Preferably, the mass ratio of wolfberry polysaccharide to the catalyst is 3:1 - 2.

[0009] Preferably, the mass ratio of wolfberry polysaccharide to the catalyst is 2:1.

[0010] Preferably, the mass ratio of wolfberry polysaccharide to sodium selenite is 1:1.

[0011] Preferably, the reaction temperature is 80 °C.

[0012] Preferably, the reaction time is 7 hours.

[0013] Preferably, the catalyst is barium chloride.

[0014] Preferably, after the reaction is completed, the pH is adjusted to 6 - 7, and then dialysis is carried out.

[0015] Preferably, the reaction system contains 0.5% nitric acid.

[0016] Beneficial effects: Compared with the prior art, the present invention has the following remarkable advantages: The present invention provides a selenization modification method for improving the anti-stress ability of wolfberry polysaccharide and its optimized modification conditions, and proves that selenization modification can significantly improve the anti-stress activity of wolfberry polysaccharide, providing materials and demonstrations for the development of new anti-stress polysaccharide drugs. Description of the Drawings

[0017] Figure 1 Survival rate (%) of nematodes in each group after treatment with different concentrations of LBP for 48 h;

[0018] Figure 2 Survival rate (%) of nematodes in each group after treatment with different concentrations of sLBP for 48 h;

[0019] Figure 3 Effects of wolfberry polysaccharide and selenized wolfberry polysaccharide on the locomotor ability of Caenorhabditis elegans;

[0020] Figure 4 Effects of wolfberry polysaccharide and selenized wolfberry polysaccharide on the body length of Caenorhabditis elegans. Detailed Embodiments

[0021] The technical solution of the present invention will be further described below with reference to the drawings.

[0022] 1. Wolfberry polysaccharide is obtained by purchase or self-extraction in the laboratory

[0023] The laboratory extraction method of wolfberry polysaccharide is as follows:

[0024] Using the water decoction - ethanol precipitation method. Take 1000 g of wolfberries, wash them, place them in an oven at 60 °C for drying, add 30 times the amount of water, soak for 3 hours, decoct twice, each time for 1 h, combine the filtrates and concentrate to 1000 mL, centrifuge at 3000 rpm / min for 20 min, discard the precipitate, carry out ethanol precipitation with 95% ethanol to a final concentration of 80%, let stand at 4 °C for 12 h, recover ethanol, centrifuge, precipitate, and freeze-dry to obtain crude wolfberry polysaccharide. Remove proteins under the conditions of 3.5% cellulase, pH 6, temperature 45 °C, and time 0.5 h to obtain wolfberry polysaccharide (LBP).

[0025] 2. Selenization modification of Lycium barbarum polysaccharide

[0026] Using the nitric acid-sodium selenite method

[0027] (1) Modification condition design: Since the mass ratio, reaction time, reaction temperature, and catalyst mass are the main factors affecting polysaccharide modification, based on preliminary experiments, four factors, namely reaction temperature (A), reaction time (B), mass ratio (sodium selenite: Lycium barbarum polysaccharide, C), and catalyst mass (D), are used as independent variables. Each factor is set at three levels. With the selenium yield as the response value, the Box-Behnken design method in the response surface software Design-Expert 8.0.6.1 is adopted to conduct a four-factor and three-level design, with a total of 29 experiments.

[0028] Table 1 Factor and level table for the corresponding surface experiment design

[0029]

[0030] (2) Modification operation: Take 29 portions of 300 mg of LBP and slowly add them to reaction flasks containing 50 mL of 0.5% nitric acid respectively, stirring while adding until completely dissolved. Add the sodium selenite dosage dropwise according to the reaction temperature, reaction time, and catalyst dosage designed in Table 1, and at the same time conduct magnetic stirring for 30 min. After stirring evenly, place it in a constant temperature water bath to react for the corresponding time, cool to room temperature, add a certain amount of sodium sulfate, adjust the pH to 6 - 7 with anhydrous sodium carbonate, centrifuge at 3000 rpm / min for 10 min, collect the supernatant, put it into a dialysis bag with a molecular weight cut-off of 1000, sample and detect whether it contains sodium selenite every 6 hours, and end dialysis until there is no sodium selenite residue. Collect the dialysis solution, concentrate it under reduced pressure, and freeze-dry it under vacuum to obtain 29 selenized Lycium barbarum polysaccharides. Determine the selenium content by atomic fluorescence spectrometry, calculate the selenium yield of the selenized Lycium barbarum polysaccharide, and determine the infrared spectrum by the potassium bromide tablet method.

[0031] Taking the selenium yield of sLBP as the response value, the experimental design conditions and results are shown in Table 2.

[0032] The range of selenium yield is between 1.64 and 7.76. Analyzing the experimental results of the response surface curve, through quadratic regression fitting, the fitting equation of the relationship between the selenium yield (Y) and the influencing factors of reaction temperature (A), reaction time (B), mass ratio (sodium selenite: Lycium barbarum polysaccharide, C), and catalyst mass (D) is obtained as follows:

[0033] Y = 7.62 - 0.39A - 0.23B + 0.35C + 0.14D + 0.70AB - 0.43AC - 0.25AD - 1.28BC - 1.16BD + 0.17CD - 3.66A² - 1.46B² - 1.70C² - 1.55D²

[0034] Table 2. Response surface design and results

[0035]

[0036]

[0037] Analysis of variance of the response surface model is shown in Table 3. The F value of the equation is 310.28, and the P value is <0.0001, indicating that only less than 0.0001% of the total variation cannot be explained by this model. The model is extremely significant, reliable and effective. In the model, the P values of A, B, C, D, AB, AD, BC, A 2 , B 2 , C 2 , D 2 are all less than 0.05, and the P value of the lack-of-fit term is 0.1426, P>0.05, indicating that there is no significance between the lack-of-fit term and the pure error, and the model has a good fitting. It can be used to predict and analyze the selenium yield.

[0038] Table 3. Analysis of variance table of the response surface model

[0039]

[0040]

[0041] It can be seen that under the conditions of the mass ratio of wolfberry polysaccharide to sodium selenite of 0.8-1.2:1, the reaction temperature controlled at 70-90 °C, the reaction time of 6-8 hours, and the mass ratio of wolfberry polysaccharide to catalyst of 3:1-2, selenium-enriched wolfberry polysaccharide can be constructed. The best modification conditions are: the mass ratio of wolfberry polysaccharide to sodium selenite of 1.0:1, the reaction time of 7 h, the reaction temperature of 80 °C, and the mass ratio of wolfberry polysaccharide to catalyst of 2:1. Under the optimal conditions, the average selenium yield of the polysaccharide is 7.6012 mg / g.

[0042] Table 4. Optimal preparation condition combination after response surface optimization

[0043] Factors and response values Optimal preparation conditions Adjusted preparation conditions Reaction temperature (°C) 79.02 80 Reaction time (h) 6.73 7 Mass ratio (w:w) 1.04：1 1.0：1 Catalyst (g) 0.16 0.15 Selenium yield (mg / g) 7.72 7.60

[0044] The results of infrared spectroscopy measurement show that in the infrared spectrogram of sLBP, in addition to the characteristic absorption peaks of polysaccharides, there are vibration absorption peaks at 1002.70 cm -1 , 1002.41 cm -1 that coincide with the vibration mode of selenate ester; there are characteristic absorption peaks at 802.51 cm -1 , 811.93 cm -1 that coincide with the vibration mode of selenite ester, suggesting the presence of selenate ester and selenite ester bonds in sLBP, indicating that wolfberry polysaccharide has been successfully modified.

[0045] 3. Comparison of the stress activities of selenium-enriched wolfberry polysaccharides

[0046] (1) Detection of the tolerance of test nematodes to wolfberry polysaccharide (LBP) and selenium-enriched wolfberry polysaccharide (sLBP)

[0047] On NGM medium, synchronous cultivation of wild-type nematode N2 was carried out. When the nematodes developed to the first day of adulthood, the nematodes were picked into a 48-well plate with a pick needle, and each well was added with S.complete liquid medium. The total volume of each well was 200 μL, which contained 5 mg / mL (wet weight) E.coli OP50. The different concentration drug groups were serially diluted from 4000 μg / mL to 0.061 μg / mL. A total of 17 concentrations of wolfberry polysaccharide extract (LBP) or selenium-enriched wolfberry polysaccharide extract (sLBP) diluted in a ratio of 4000 - 0.061 μg / mL were added to each well, and the control group was added with the same volume of distilled water. There were 3 wells in each concentration group, and there were about 30 nematodes in each well. After culturing at 20 °C for 48 h, the survival of nematodes in each well was counted. The criterion for judging nematode death was: when touching the nematode with a pick needle, if the nematode did not show avoidance or wriggling behavior, it was judged to be dead.

[0048] The effects of different concentrations of LBP and sLBP on the survival rate of Caenorhabditis elegans are as Figure 1 and Figure 2 shown. After treating wild-type nematode N2 for 48 h, the survival rate of nematodes in the control group and all different concentration drug groups was 100%.

[0049] (2) Lifespan experiment

[0050] Synchronous operation of nematodes (wild-type N2) was carried out on NGM plates. On the first day of the adult stage, the nematodes were picked into a 96-well plate containing S.complete liquid medium (containing 4 mM FUDR), and 1 - 2 nematodes were placed in each well. The total volume of the culture solution in each well was 80 μL, which contained 1 mg / mL (wet weight) E.coli OP50. The administration groups were added with 500 μg / mL, 250 μg / mL, 125 μg / mL, 62.50 μg / mL, and 31.25 μg / mL of LBP and sLBP at a total of 5 concentrations respectively, and the control group was added with the same volume of distilled water. The number of nematodes in each group was about 90. The nematodes were observed every other day, and the death of nematodes was counted until all of them died.

[0051] The average lifespan of nematodes in the control group was 24 d. The maximum lifespan of nematodes in the 500 μg / mL LBP group was 28 d, with a 20.3% lifespan extension; the maximum lifespan of nematodes in the 125 μg / mL sLBP group was 30 d, with a 26.4% extension, showing the best lifespan extension effect. In the sLBP group, as the drug concentration increased, the maximum lifespan days of nematodes showed a trend of increasing first and then decreasing.

[0052] Table 5 Effects of Lycium barbarum polysaccharide and selenium-enriched Lycium barbarum polysaccharide on the lifespan of Caenorhabditis elegans (days)

[0053]

[0054]

[0055] Note: Data with inconsistent superscripts in the same column are significantly different, P < 0.05. The same below.

[0056] (3) Determination of locomotor ability

[0057] Locomotor ability is an indicator of the vitality of Caenorhabditis elegans and thus a useful phenotype of this model organism. Its locomotor ability can be mainly manifested in the following aspects: locomotor speed, coordination, amplitude, number of body bends, etc. Synchronization operation was performed on nematodes (wild-type N2) on NGM plates. 500 μg / mL LBP and 125 μg / mL sLBP were added to the culture medium respectively, and the locomotor ability was measured on the 3rd day, 7th day, and 11th day after administration. The locomotor ability of nematodes was divided into three categories: A, B, or C. Nematodes that could perform spontaneous sinusoidal movement were classified as category A. Nematodes that could not perform sinusoidal movement but could still move when stimulated were classified as category B. Nematodes that could only move their heads or wag their tails after being stimulated were classified as category C. The classification of the locomotor ability of Caenorhabditis elegans was observed and recorded.

[0058] The results showed that synchronized wild-type nematodes N2 were treated with 500 μg / mL LBP and 125 μg / mL sLBP respectively, and the locomotor ability was measured on the 3rd day, 7th day, and 11th day. The results were as Figure 3 shown. On the 3rd day, the locomotor ability of the control group, LBP group, and sLBP group was all category A; on the 7th day, both category A and B locomotor abilities appeared in each group. Compared with the control group, the proportion of category B locomotor ability in the LBP group and sLBP group was lower, and the proportion of category A locomotor ability was higher, and that in the sLBP group was less than that in the LBP group; on the 11th day, category A, B, and C locomotor abilities all appeared in each group. Compared with the control group, the proportion of category C locomotor ability in the LBP group and sLBP group was lower, and the proportion of category A locomotor ability was higher, and the category C locomotor ability in the sLBP group was less than that in the LBP group. The results indicated that LBP and sLBP could significantly enhance the locomotor ability of nematodes, and the enhancing effect of sLBP was better.

[0059] (4) Determination of body length

[0060] Synchronize nematodes (wild-type N2) on NGM plates. On the first day of the adult stage, pick the nematodes into a 96-well plate containing S. complete liquid medium (containing 4 mM FUDR), with 1-2 nematodes in each well. The total volume of the culture medium in each well is 80 μL, which contains 1 mg / mL (wet weight) E. coli OP50. In the treatment groups, 500 μg / mL LBP and 125 μg / mL sLBP are added respectively, and the control group is added with the same volume of distilled water, with about 90 nematodes in each well. Place it in a biochemical incubator at 20 °C for 48 h. Pick the nematodes onto a blank NGM solid medium, and place the medium in an oven at 50 °C. After heating for 5 min, the nematodes die. Take pictures with a stereomicroscope and measure the body length of the nematodes, with 30 nematodes measured in each group.

[0061] In terms of growth status, body length is the most intuitive manifestation. Treat synchronized wild-type nematodes N2 with 500 μg / mL LBP and 125 μg / mL sLBP respectively, and measure the body length on the 3rd, 7th, and 11th days. The results are as Figure 4 shown. On the 3rd, 7th, and 11th days, compared with the control group, the body lengths of the LBP group and the sLBP group have a tendency to increase but there are no significant differences (P>0.05). The results indicate that there are no obvious changes in the body lengths of the control group, the LBP group, and the sLBP group, suggesting that both LBP and sLBP have no significant effects on the normal growth and development activities of Caenorhabditis elegans.

[0062] Heat stress test

[0063] Synchronize nematodes (wild-type N2) on NGM plates. On the first day of the adult stage, pick the nematodes into a 3 mL culture dish containing S. complete liquid medium, and the total volume of each culture dish is 2 mL. Pick about 200 nematodes into each culture dish. In the treatment groups, 500 μg / mL LBP and 125 μg / mL sLBP are added respectively, and the control group is added with an equal volume of distilled water. Culture in an incubator for 72 h. Rinse the nematodes into a 1.5 mL EP tube with M9 buffer, and rinse repeatedly for 3 times to remove the drugs and E. coli OP50. Transfer the nematodes to an NGM medium without OP50 coated. Pick the nematodes into a 48-well plate containing M9 buffer, with 3 wells in each group and about 30 nematodes in each well. Transfer the nematodes to a 35 °C incubator and count the number of dead nematodes every 1 h until all are dead, and count the number of dead nematodes in each group. Use a picking needle to touch the nematodes, and if its body does not move, regard this phenomenon as the death criterion. Each group is parallel three times.

[0064] The results of the heat stress test are shown in Table 6. Under the heat stress condition of 35 °C, the maximum lifespan of the control group was 22 h. The maximum lifespan of wild-type nematode N2 treated with 500 μg / mL LBP was 22 h, which was increased by 16.9% compared with the control group. The maximum lifespan of wild-type nematode N2 treated with 125 μg / mL sLBP was 24 h, which was significantly higher than that of the control group (P<0.05), and was increased by 27.8% compared with the control group and by 10.9% compared with the LBP group. The results indicate that LBP and sLBP can significantly extend the maximum lifespan of Caenorhabditis elegans under heat stress conditions, and both have a significant promoting effect on improving the heat stress resistance of nematodes, and the promoting effect of sLBP is stronger than that of LBP.

[0065] Table 6 Effects of Lycium barbarum polysaccharide and selenium-enriched Lycium barbarum polysaccharide on the lifespan of Caenorhabditis elegans under heat stress conditions (hours)

[0066] Group Mean lifespan (hours) % of control Maximum lifespans Control <![CDATA[11.92±0.97 b > 100 22 LBP (500 μg / mL) <![CDATA[13.91±1.14 ab > 116.9 22 sLBP (125 μg / mL) <![CDATA[15.23±1.03 a > 127.8 24

[0067] The above results show that selenium modification can significantly improve the stress resistance of Lycium barbarum polysaccharide. Selenium-enriched Lycium barbarum polysaccharide can be used as a component drug of anti-stress agents.

Claims

1. A selenized wolfberry polysaccharide, characterized in that: The selenium content is 1.64~7.6mg / g.

2. A method for preparing the selenized wolfberry polysaccharide according to claim 1, characterized in that: The method comprises selenization modification of wolfberry polysaccharide using a nitric acid-sodium selenite system.

3. The method for preparing selenized wolfberry polysaccharide according to claim 2, characterized in that: The specific steps include: mixing wolfberry polysaccharides with sodium selenite, wherein the mass ratio of wolfberry polysaccharides to sodium selenite is 0.8-1.2:1; adding a catalyst to a reaction system containing nitric acid; controlling the reaction temperature to 70-90°C and the reaction time to 6-8 hours; and removing unreacted substances by dialysis to obtain selenized wolfberry polysaccharides.

4. The method for preparing selenized wolfberry polysaccharide according to claim 3, characterized in that: The mass ratio of wolfberry polysaccharide to catalyst is 3:1-2.

5. The method for preparing selenized wolfberry polysaccharide according to claim 3, characterized in that: The mass ratio of Lycium barbarum polysaccharide to catalyst is 2:

1.

6. The method for preparing selenized wolfberry polysaccharide according to claim 3, characterized in that: The mass ratio of wolfberry polysaccharides to sodium selenite is 1:

1.

7. The method for preparing selenized wolfberry polysaccharide according to claim 3, characterized in that: The reaction temperature was 80°C.

8. The method for preparing selenized wolfberry polysaccharide according to claim 3, characterized in that: The reaction time was 7 hours.

9. The method for preparing selenized wolfberry polysaccharide according to claim 3, characterized in that: The catalyst is barium chloride.

10. The method for preparing selenized wolfberry polysaccharide according to claim 3, characterized in that: After the reaction was completed, the pH was adjusted to 6-7 and then dialyzed.

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