Selenium-rich dendrobium polysaccharide with antioxidant and / or anti-wrinkle effects and preparation method thereof

Abstract

The invention discloses a selenium-rich dendrobium polysaccharide and a preparation method thereof, wherein the preparation method comprises the following steps: 1) taking a dendrobium officinale extract and adding a nitric acid solution to dissolve it; 2) taking the solution obtained in step 1), adding sodium selenite and barium chloride to react for 6 to 10 hours, cooling and filtering, taking the filtrate to adjust the pH value to 4 to 7, dialyzing, taking the dialyzate and drying, and obtaining the selenium-rich dendrobium polysaccharide. The preparation method of the selenium-rich dendrobium polysaccharide of the invention, through specific synthesis process parameters and a concentration and purification method, the total polysaccharide content in the selenium-rich dendrobium polysaccharide is greater than 90%, and the selenium content is greater than 1.2 mg / g. Experiments have proved that it has significant antioxidant and anti-wrinkle activities.

Description

Technical Field

[0001] The present invention specifically relates to a selenium-rich dendrobium polysaccharide having anti-oxidation and / or anti-wrinkle effects and a preparation method thereof. Background Art

[0002] Dendrobium officinale is the fresh or dried stem of Dendrobium officinale Kimura et Migo, an orchidaceae plant. It is also known as "black knot grass" and "iron hanging plant" and has been included in the 2015 edition of the Chinese Pharmacopoeia. Dendrobium has the unique effects of nourishing the stomach and promoting fluid, nourishing yin and clearing away heat. It is praised as the first of the "Nine Great Immortal Grasses" in the Taoist medical classic "Dao Zang". It is also listed as a top-grade product in "Shennong's Herbal Classic". It is called "life-saving immortal grass" and "inch gold grass" by the people. Dendrobium officinale has extremely small seeds, no endosperm, and low reproductive capacity. In addition, over-harvesting has led to the extinction of its resources. It has been listed as a Class II protected plant in the world. It is expensive in the market and has always been a treasure among dendrobiums. With the application of tissue rapid culture technology, the artificial cultivation technology of Dendrobium officinale has become relatively mature, which has basically solved the problem of resource shortage. Modern research shows that Dendrobium candidum contains polysaccharides, alkaloids, phenanthrene, bibenzyl, amino acids and other active ingredients. Polysaccharides are the main active ingredient and the substance with the highest content. It has the effects of lowering blood sugar, blood lipids, blood pressure, enhancing immunity, anti-fatigue, antibacterial, anti-tumor and antioxidant. Due to its wide range of biological activities, it has attracted much attention from scientific researchers and has become a hot topic of research today.

[0003] Selenium is an essential trace element for the human body. Modern research shows that selenium can improve human immunity, promote the proliferation of lymphocytes and the synthesis of antibodies and immunoglobulins; selenium has a significant inhibitory and protective effect on various cancers such as colon cancer, skin cancer, liver cancer, and breast cancer. Its intermediate metabolite methylselenol in the body has a strong anti-cancer activity; selenium has a synergistic antioxidant effect with nutrients such as vitamin E, allicin, linoleic acid, germanium, and zinc, increasing antioxidant activity; at the same time, selenium has the effect of reducing and alleviating the toxicity of heavy metals. At the same time, the scientific community also recognizes that selenium has a cancer prevention effect and is the "king of cancer prevention" among human trace elements. Selenium is essential to the human body and cannot be self-produced. Therefore, the World Health Organization recommends supplementing 200μg of selenium every day to effectively prevent the high incidence of various diseases. Selenium is divided into two types: plant active selenium and inorganic selenium. Inorganic selenium generally refers to sodium selenite and sodium selenate, including yeast selenium and malt selenium with a large amount of inorganic selenium residues, which are obtained from by-products of metal mines. Inorganic selenium is highly toxic and not easily absorbed, and is not suitable for use by humans and animals. Plant active selenium is formed by biotransformation and combined with amino acids, generally in the form of selenomethionine. Plant active selenium is a source of selenium permitted for use by humans and animals. The main selenium-rich foods on the market can be divided into: 1. Selenium-rich health foods; 2. Selenium yeast nutritional enhancers; 3. Processed selenium-rich foods - many local governments in non-selenium-rich production areas will cultivate selenium-rich foods by applying selenium-rich fertilizers; 4. Natural plant active selenium - mainly agricultural crops grown from selenium-rich soil.

[0004] At present, the selenium content in Dendrobium officinale is mainly increased by plant transformation technology, such as patent CN107827672A, which is to obtain selenium-enriched Dendrobium officinale by adding selenium-containing compound fertilizers during the cultivation of Dendrobium officinale. Existing research reports have not yet achieved selenium enrichment in the Dendrobium officinale extraction process and the conversion of inorganic selenium to organic selenium, and thereby prepared selenium-enriched Dendrobium officinale polysaccharides to expand its application. Summary of the invention

[0005] In order to solve the above problems, the present invention provides a selenium-rich dendrobium polysaccharide, which is a selenium polysaccharide formed by combining dendrobium officinale polysaccharide and sodium selenite; the mass ratio of polysaccharide to selenium in the selenium polysaccharide is 93-96:

[0006] 1.3~1.5×10 -1 .

[0007] The present invention also provides a use of the selenium-rich Dendrobium polysaccharide in preparing antioxidant medicines or health foods.

[0008] The present invention also provides a use of the selenium-rich Dendrobium polysaccharide in preparing anti-wrinkle medicines.

[0009] Finally, the present invention provides a method for preparing selenium-enriched dendrobium polysaccharide, which comprises the following steps:

[0010] 1) Take the Dendrobium officinale extract and add nitric acid solution to dissolve it;

[0011] 2) taking the solution obtained in step 1), adding sodium selenite and barium chloride to react for 6 to 10 hours, cooling and filtering, adjusting the pH value of the filtrate to 4 to 7, dialyzing, taking the dialyzate and drying it to obtain selenium-enriched Dendrobium polysaccharide.

[0012] Furthermore, the content of Dendrobium officinale polysaccharide in the Dendrobium officinale extract in step 1) is 50-80%, preferably 80%.

[0013] Furthermore, the mass volume ratio of the Dendrobium officinale extract to the nitric acid solution is 7-13g:1000mL, preferably 10g:1000mL.

[0014] Furthermore, the concentration of the nitric acid solution is 0.1-1% v / v, ml / ml, preferably 0.5%.

[0015] Furthermore, in step 2), the amount of sodium selenite added is 0.5 to 1 times, preferably 0.8 times, of the weight of the Dendrobium officinale extract; and the amount of barium chloride added is 0.8 to 1.4 times, preferably 1.2 times, of the weight of the Dendrobium officinale extract.

[0016] Furthermore, in step 2), the reaction time is 6 hours, the temperature is 50-80° C., preferably 60° C.; and the cooling is performed to room temperature.

[0017] Furthermore, in step 2), the pH value of the filtrate is adjusted to 5-6 using anhydrous sodium carbonate.

[0018] Furthermore, in step 2), the dialysis uses a 3500MW dialysis bag, and the dialysis is performed 5 times, each time for 12 hours.

[0019] Furthermore, the drying in step 2) is freeze drying, and the freeze drying procedure is pre-freezing at 40°C for 6 hours, evacuating until the vacuum degree of the freeze drying chamber is less than 20Pa, and then starting the drying procedure: drying at -30°C for 6 hours, and then drying at 30°C for 8 hours.

[0020] The preparation method of the selenium-enriched dendrobium polysaccharide of the present invention, through specific synthesis process parameters and concentration and purification methods, makes the total polysaccharide content in the selenium-enriched dendrobium polysaccharide greater than 90%, and the selenium content greater than 1.2 mg / g. Experiments have proved that the polysaccharide has significant antioxidant and anti-wrinkle activities.

[0021] Through the research on process, quality standard and efficacy, it is confirmed that the present invention synthesizes the selenium-rich polysaccharide in a combined state by combining Dendrobium officinale polysaccharide and sodium selenite, which can not only synergistically increase the efficacy of Dendrobium officinale polysaccharide and selenium, but also solve the toxicity of inorganic selenium. It can provide the market with a safe and efficient selenium-rich Dendrobium officinale polysaccharide, which can greatly promote the comprehensive development and utilization of Dendrobium officinale and meet the needs of the people for healthy life.

[0022] Obviously, according to the above contents of the present invention, in accordance with common technical knowledge and customary means in the art, without departing from the above basic technical ideas of the present invention, other various forms of modification, replacement or change may be made.

[0023] The above contents of the present invention are further described in detail below through specific implementation methods in the form of embodiments. However, this should not be understood as the scope of the above subject matter of the present invention being limited to the following examples. All technologies realized based on the above contents of the present invention belong to the scope of the present invention. BRIEF DESCRIPTION OF THE DRAWINGS

[0024] Figure 1 Spectral scanning curve of selenium standard

[0025] Figure 2 Spectrum scanning curve of the test sample

[0026] Figure 3 Selenium standard curve

[0027] Figure 4 Effect of selenium-enriched Dendrobium polysaccharide on the relative fluorescence intensity of ROS in zebrafish (A: Reactive oxygen species in zebrafish after staining with fluorescent probe DCF-DA; B: Relative fluorescence intensity analyzed by Image J) DETAILED DESCRIPTION

[0028] Example 1: Preparation of selenium-enriched dendrobium polysaccharide of the present invention

[0029] Take 10g of Dendrobium candidum extract (total polysaccharide>80%), add 0.5% HNO 3 1000mL solution was heated and stirred to dissolve all the polysaccharides to obtain a 10mg / L polysaccharide solution. 0.8 times the weight of the polysaccharide was added to each 2 SeO 3 and 1.2 times the weight of polysaccharide BaCl 2, placed on a magnetic stirrer at 60°C for 6 hours, cooled to room temperature, filtered, adjusted the pH value of the filtrate to 5-6 with anhydrous sodium carbonate, loaded into a 3500MW dialysis bag, dialyzed 5 times at room temperature, each time for 12 hours, and then took the liquid in the dialysis bag into a drying plate with a thickness of no more than 6mm, placed in a freeze dryer, pre-frozen at -40°C for 6 hours, and evacuated until the vacuum degree of the freeze drying chamber was less than 20Pa, and then started the drying program: drying at -30°C for 6 hours, and then drying at 30°C for 8 hours to obtain selenized Dendrobium officinale polysaccharide.

[0030] The beneficial effects of the present invention are further demonstrated by the following experimental examples:

[0031] Experimental Example 1 Study on the Selenium Content in Selenium-enriched Dendrobium Officinale Polysaccharides

[0032] (1) Measurement method

[0033] Preparation of test solution: Take 0.05 g of selenium-enriched Dendrobium selenium polysaccharide, accurately weigh, add 4 mL of concentrated sulfuric acid-perchloric acid-nitric acid mixture (concentrated sulfuric acid: perchloric acid: nitric acid = 1:1:4), leave overnight, heat to evaporate the strong acid, transfer quantitatively to a 10 mL volumetric flask with distilled water, make up to the scale, shake well, and obtain.

[0034] Preparation of standard curve: accurately measure 0.5, 1.0, 2.0, 3.0 and 4.0 mL of selenium standard solution (10 μg / mL), place in a separatory funnel, add water to 30 mL, adjust pH to 2 with dilute hydrochloric acid solution, add 3.0 mL of 1% o-phenylenediamine hydrochloride test solution, shake well, place in a dark place for 2 hours, add 10.0 mL of toluene, extract, let stand, separate the toluene layer, and add toluene to make up to 10 mL. Use the corresponding reagent as blank, measure the absorbance at 334 nm, and draw a standard curve with absorbance as the ordinate and concentration as the abscissa.

[0035] Determination method: Accurately pipette 1.0 mL of the test solution, add water to 30 mL, adjust the pH to 2 with dilute hydrochloric acid or sodium hydroxide solution, and follow the method under the standard curve preparation, starting from "add 3.0 mL of 1% o-phenylenediamine hydrochloride test solution", measure the absorbance value according to the law, read the selenium content in the test solution from the standard curve, and calculate it.

[0036] (2) Methodological research

[0037] 2.1 Specificity test and determination of maximum absorption wavelength

[0038] Take the test sample and selenium standard solution, add 3.0 mL of 1% o-phenylenediamine hydrochloride test solution according to the method under (1), shake well, place in the dark for 2 h, add 10.0 mL of toluene, extract, let stand, separate the toluene layer, and add toluene to make up to 10 mL. After color development, scan under UV in the wavelength range of 190 nm to 400 nm. Figure 1-2 .

[0039] from Figures 1-2 The results show that after the test sample and selenium standard solution are colored, the maximum absorption is at 334nm, so 334nm is determined as the detection wavelength of selenium content.

[0040] 2.2 Linear range investigation

[0041] According to the method under standard curve under item (1), draw a standard curve with absorbance (A) as the ordinate and concentration (C) as the abscissa. Figure 3 The regression equation was: A = 0.2241C + 0.0033, r = 0.9998. The results showed that when the selenium content was in the range of 0.5 to 4.0 μg / mL, the concentration and absorbance showed a good linear relationship.

[0042] 2.3 Instrument precision experiment

[0043] The selenium standard solution (2 μg / mL) after color development was taken and measured 6 times at a wavelength of 334 nm, the absorbance value was recorded, and the RSD value was calculated. The measurement results are shown in Table 1. The results show that the instrument has good precision.

[0044] Table 1 Instrument precision test results

[0045]

[0046]

[0047] 2.4 Solution stability test

[0048] Stability of selenium standard solution: Accurately measure 2.0 ml of standard solution (10 μg / mL) and place it in a separatory funnel. Prepare the standard curve according to the method in item (1), starting from "add water to 30 mL". Take toluene solution and measure the absorbance at a wavelength of 334 nm every 20 minutes to calculate the RSD value.

[0049] Stability of the test solution: Take selenium-rich Dendrobium officinale polysaccharide, prepare the test solution according to the method under (1) and develop the color, then take the toluene solution and measure the absorbance at a wavelength of 334 nm every 20 minutes, and calculate the RSD value.

[0050] The determination results are shown in Table 2. The results show that the selenium standard solution and the test solution are stable within 2 hours after color development.

[0051] Table 2 Solution stability test results

[0052]

[0053] 2.5 Repeatability Experiment

[0054] Six portions of selenium polysaccharide from Dendrobium officinale of the same batch, each portion weighing about 0.15 g, were accurately weighed, and the test solution was prepared and determined according to the method under (1), and the selenium content was calculated. The results are shown in Table 3. The results show that the method has good repeatability.

[0055] Table 3 Repeatability test results

[0056]

[0057] 2.6 Sample recovery experiment

[0058] Weigh 6 portions of selenium-rich Dendrobium officinale polysaccharide (1.26 mg / g) with a known content, each portion is about 0.075 g, and 9.0 mL of selenium standard solution (10 μg / mL) is accurately added to each portion. Prepare the test solution and measure it according to the method under (1), calculate the selenium content, and calculate the sample recovery rate according to the following formula. The measurement results are shown in Table 4.

[0059] Sample recovery rate = (measured amount - selenium weight in sample) / added reference substance × 100%

[0060] The results showed that the average recovery rate of 6 times was 99.1%, ranging from 95% to 105%, and the RSD was less than 2%, indicating that the method had high accuracy.

[0061] Table 4 Sample recovery test results

[0062]

[0063] Experimental Example 2 Single Factor Investigation on the Preparation Process of Selenium-enriched Dendrobium Officinale Polysaccharide

[0064] Selenium polysaccharides are mainly prepared by artificial chemical synthesis, HNO 3 and BaCl 2 Can catalyze selenization reaction, HNO 3 It promotes the reaction between selenite and the active groups on polysaccharides; BaCl 2 Middle Ba 2+ Ba has a strong coordination effect with -OH on polysaccharides. 2+ The presence of is conducive to the formation of the complex, enhances the nucleophilicity of -OH and forms a stable single complex. The present invention conducts single factor investigation from reaction time, reaction temperature, nitric acid volume fraction, sodium selenite and barium chloride, and screens out the main influencing factors by calculating the yield and measuring the selenium content.

[0065] (1) Effect of different reaction times on the synthesis process of selenium polysaccharides

[0066] Accurately weigh 6 portions of Dendrobium officinale polysaccharide, 0.5 g each, add 50 mL of 0.5% nitric acid solution, heat to dissolve, add 0.5 g of sodium selenite pentahydrate and BaCl 2 0.7 g, placed on a magnetic stirrer, and reacted at 70 °C for 2 h, 4 h, 6 h, 8 h, 12 h, and 14 h. The reaction solution was cooled to room temperature, filtered, and the supernatant was added with anhydrous Na 2 CO 3 The pH value was adjusted to 5-6, and dialyzed with a 3500MW dialysis bag. A small amount of dialyzate was added with ascorbic acid for detection. When no red color was found, the dialysis was stopped, and freeze-dried to obtain Dendrobium candidum selenium polysaccharide. The selenium content and selenium polysaccharide yield in the obtained selenium polysaccharide were determined according to the following method. The results are shown in Table 5.

[0067] Determination of selenium content: Same as the method in Test Example 1 above.

[0068] Selenium polysaccharide yield: Take the freeze-dried Dendrobium officinale selenium polysaccharide, weigh it, and calculate it according to the following formula.

[0069]

[0070] The experimental results show that under the above reaction conditions, the selenium content increases rapidly from 2 to 4 hours, increases slowly from 4 to 12 hours, and begins to decrease from 12 to 14 hours; while the yield of selenium polysaccharides decreases from 2 to 14 hours. Considering the selenium content and the yield of selenium polysaccharides comprehensively, a reaction time of 6 hours can be selected.

[0071] Table 5 Determination results of selenium polysaccharides obtained at different reaction times

[0072]

[0073] (2) Effect of different reaction temperatures on the synthesis process of selenium polysaccharides

[0074] Weigh 5 portions of Dendrobium candidum polysaccharide, 0.5 g each, add 50 mL of 0.5% nitric acid solution, heat to dissolve, add 0.5 g of sodium selenite pentahydrate and BaCl 2 0.7g, placed on a magnetic stirrer, reacted at 30℃, 40℃, 60℃, 70℃, 80℃ for 6h. Follow the procedure in item (1) from "the reaction solution is cooled to room temperature". The results are shown in Table 6.

[0075] The experimental results show that under the above reaction conditions, the selenium content at 30-60°C and the yield of selenium polysaccharides at 30-40°C show an upward trend, the selenium content in the range of 60-80°C and the yield of selenium polysaccharides at 40-80°C show a downward trend, the selenium content reaches the highest value when the reaction temperature is 60°C, and the selenium polysaccharide yield reaches the highest value at 40°C. Because the yields are not much different, the selenium content factor is mainly considered, and 60°C is selected for preparation.

[0076] Table 6 Determination results of selenium polysaccharides obtained at different reaction temperatures

[0077]

[0078] (3) Effect of nitric acid concentration on the synthesis process of selenium polysaccharides

[0079] Weigh 5 portions of Dendrobium candidum polysaccharide, 0.5 g each, add 50 mL of 0.1%, 0.3%, 0.5%, 0.7%, and 0.9% nitric acid solution in sequence, heat to dissolve, add 0.5 g of sodium selenite pentahydrate and BaCl 2 0.7 g, placed on a magnetic stirrer, reacted at 70°C for 6 h. Follow the procedure in (1) from "the reaction solution is cooled to room temperature". The results are shown in Table 7.

[0080] The experimental results show that under the above reaction conditions, the selenium content shows an upward trend when the nitric acid concentration is 0.1%-0.5%, and the selenium content is not much different when it is 0.5%-0.9%; while the yield of selenium polysaccharides shows a downward trend when the nitric acid concentration is 0.1%-0.9%. Considering the selenium content and selenium polysaccharide yield, 0.5% nitric acid can be selected for the reaction.

[0081] Table 7 Determination results of selenium polysaccharides obtained at different nitric acid concentrations

[0082]

[0083] (4) Effect of the dosage of sodium selenite pentahydrate on the synthesis process of selenium polysaccharides

[0084] Weigh 5 portions of Dendrobium officinale polysaccharide, 0.5 g each, add 50 mL of 0.5% nitric acid solution to each, heat to dissolve, add sodium selenite pentahydrate (0.4, 0.6, 0.8, 1.0, 1.2 times the weight of the polysaccharide) and Bacl in sequence. 2 0.7g, placed on a magnetic stirrer, reacted at 70℃ for 6h. Follow the procedure in item (1) from "the reaction solution is cooled to room temperature". The results are shown in Table 8.

[0085] The experimental results show that under the above reaction conditions, the final selenium content obtained when 0.8 to 1.0 times the amount of sodium selenite added is the highest and the difference between the two is not large; while the addition of 0.4 to 1.2 times the amount of sodium selenite has little effect on the final selenium polysaccharide yield, and is relatively higher at 0.6 to 0.8 times, so 0.8 times sodium selenite can be selected for the preparation of polysaccharides.

[0086] Table 8 Determination results of selenium polysaccharides obtained by adding different sodium selenite multiples

[0087]

[0088] (5) Effect of barium chloride dosage on the synthesis process of selenium polysaccharides

[0089] Weigh 5 portions of Dendrobium candidum polysaccharide, 0.5 g each, add 50 mL of 0.5% nitric acid solution to each portion, heat to dissolve, and add 0.5 g of sodium selenite pentahydrate and BaCl 2 (0.6, 0.8, 1.0, 1.2, 1.4 times the weight of the polysaccharide), placed on a magnetic stirrer, and reacted at 70°C for 6 hours. Follow the procedure in item (1) from "the reaction solution is cooled to room temperature". The results are shown in Table 9.

[0090] The experimental results show that under the above reaction conditions, the selenium content reaches the highest value when 1.2 times the weight of the polysaccharide is added to prepare the barium chloride, and the addition of 0.4 to 1.4 times the weight of the polysaccharide has little effect on the yield of selenium polysaccharide. Therefore, the amount of barium chloride added is mainly considered to consider the selenium content factor. When preparing selenium polysaccharides, it is chosen to add 1.2 times the weight of the polysaccharide.

[0091] Table 9 Determination results of different barium chloride selenium polysaccharides

[0092]

[0093] Experimental Example 3 Screening experiment of preparation process of selenium-rich Dendrobium officinale polysaccharide

[0094] According to the results of single factor investigation, reaction temperature, reaction time and amount of sodium selenite added are the main influencing factors in the preparation of selenium polysaccharides from Dendrobium officinale. Therefore, in this process study, the yield of selenium polysaccharides and selenium content were used as evaluation indicators, reaction time (A), reaction temperature (B) and amount of sodium selenite added (C) were selected as investigation factors, and 3 levels were selected for each factor, and the experiment was designed, as shown in Table 10.

[0095] Table 10 Synthesis process design table

[0096]

[0097] (1) Sample preparation

[0098] Weigh 9 portions of selenium-rich Dendrobium officinale polysaccharide, 0.5 g each, and prepare according to the test parameters in Table 10. Add 50 mL of 0.5% nitric acid solution to each portion, heat to dissolve, and add the specified amount of sodium selenite pentahydrate and BaCl in sequence. 2 (1.2 times the weight of polysaccharide), placed on a magnetic stirrer, reacted at the specified reaction temperature for the specified time. The reaction solution was cooled to room temperature, centrifuged, and the supernatant was added with anhydrous Na 2 CO 3 Adjust the pH value to 5-6, dialyze with a 3500MW dialysis bag, take a small amount of the dialysate and add ascorbic acid for detection, stop dialysis when there is no red color, freeze-dry, and obtain Dendrobium officinale selenium polysaccharide.

[0099] (2) Determination of evaluation indicators and data processing methods

[0100] In this experiment, the yield of selenium polysaccharides and selenium content were used as evaluation indicators, and the weighted scoring method was used to comprehensively evaluate the synthesis process of selenium polysaccharides from Dendrobium officinale. Scoring criteria: Each indicator was divided by its maximum value and then multiplied by 100 to obtain its comprehensive score in a certain process. According to the role of each component, the weight coefficients of selenium content and selenium polysaccharide yield were determined to be 0.7 and 0.3, respectively. The weighted sum of the two components was used to obtain a comprehensive score, that is, comprehensive score = (selenium content / maximum selenium content) × 100 × 0.7 + (selenium polysaccharide yield / maximum selenium polysaccharide yield) × 100 × 0.3.

[0101] (3) Result analysis and determination of the optimal process

[0102] The test results are shown in Table 11.

[0103] Table 11 Experimental design and results

[0104]

[0105] Based on the comprehensive consideration of the experimental results, the optimal process parameters for the synthesis of selenium polysaccharides from Dendrobium officinale were determined to be A 2 B 2 C 1 That is: weigh Dendrobium officinale polysaccharide, add 50mL of 0.5% nitric acid solution, heat to dissolve, add 0.8 times amount of sodium selenite pentahydrate and barium chloride, place on a magnetic stirrer, react for 6 hours at 60℃. The reaction solution is cooled to room temperature, filtered, and the supernatant is adjusted to pH 5-6 by adding anhydrous sodium carbonate, dialyzed with a 3500MW dialysis bag, and a small amount of dialyzate is added with ascorbic acid for detection. When there is no red color, stop dialysis, freeze-dry, and obtain Dendrobium officinale selenium polysaccharide.

[0106] Experimental Example 4 Study on the purification process of selenium-rich Dendrobium officinale polysaccharide

[0107] The main purpose of dialysis purification is to remove the remaining selenium ions in the synthesis reaction to avoid the presence of free selenium ions affecting the selenium content in the selenized polysaccharide measured later. At the same time, it can remove other small molecular impurities to ensure the quality and efficacy of selenium polysaccharides.

[0108] (1) Different dialysis molecular weight

[0109] Weigh 3 portions of Dendrobium candidum polysaccharide, 0.5g each, add 50mL of 0.5% nitric acid solution, heat to dissolve, add 0.8 times of sodium selenite pentahydrate and barium chloride, place on a magnetic stirrer, and react for 6 hours at 60°C. The reaction solution is cooled to room temperature, filtered, and the supernatant is adjusted to pH 5-6 by adding anhydrous sodium carbonate, and then loaded into 1000MW, 3500MW, and 7000MW dialysis bags, respectively, and dialyzed 5 times at room temperature, each for 12 hours. The last time, a small amount of dialysate is added to ascorbic acid for detection. When there is no red color, stop dialysis and freeze-dry. The yield of selenium polysaccharide is determined according to the following formula, and the dialysis is compared to see if it is complete. The results are shown in Table 12.

[0110]

[0111] Where: W1 is the weight of Dendrobium polysaccharide g; W2 is the weight of selenium polysaccharide after dialysis g

[0112] The results showed that the 1000MW dialysis bag had the highest yield, and the 1000MW dialysis bag could only remove molecules below 1000MW, resulting in a lower purity of selenium polysaccharides; the 7000MW dialysis bag had a lower yield, and some selenium polysaccharides would be precipitated during the filtration process, resulting in waste. Therefore, a 3500MW dialysis bag was selected for purification.

[0113] Table 12 Dialysis yield of selenium-enriched Dendrobium with different molecular weights

[0114]

[0115] (2) Water changes

[0116] Weigh 5 portions of Dendrobium candidum polysaccharide, 0.5g each, add 50mL of 0.5% nitric acid solution, heat to dissolve, add 0.8 times of sodium selenite pentahydrate and barium chloride, place on a magnetic stirrer, and react for 6 hours at 60°C. The reaction solution is cooled to room temperature, filtered, and the supernatant is adjusted to pH 5-6 by adding anhydrous sodium carbonate, and then loaded into 3500MW dialysis bags, and dialyzed 3, 4, 5, 6, and 7 times at room temperature, each for 12 hours. The last time, a small amount of dialysate is added to ascorbic acid for detection, and freeze-dried. The yield of selenium polysaccharide is determined, and the dialysis is compared to see if it is complete. The results are shown in Table 13.

[0117] The results showed that after changing the water 5 times, the ascorbic acid test was negative, and the free selenium ions were completely dialyzed. However, with the increase in the number of dialysis, the yield of Dendrobium selenium polysaccharides tended to decrease. At the same time, the dialysis time was too long, which increased the production cost. Taking all factors into consideration, the number of water changes was confirmed to be 5 times.

[0118] Table 13 Dialysis yield of selenium-enriched Dendrobium at different water changes

[0119]

[0120] (3) Dialysis time

[0121] Weigh 4 portions of Dendrobium candidum polysaccharide, 0.5g each, add 50mL of 0.5% nitric acid solution, heat to dissolve, add 0.8 times of sodium selenite pentahydrate and barium chloride, place on a magnetic stirrer, and react for 6 hours at 60°C. The reaction solution is cooled to room temperature, filtered, and the supernatant is adjusted to pH 5-6 by adding anhydrous sodium carbonate, and then loaded into 3500MW dialysis bags, and dialyzed 5 times at room temperature, each dialysis time is 8, 10, 12, and 14 hours, and a small amount of ascorbic acid is added to the dialysate for the last time for detection, and freeze-dried. Determine the yield of selenium polysaccharide, and compare whether the dialysis is complete. The results are shown in Table 14.

[0122] The results showed that the yield of selenium-enriched dendrobium polysaccharides decreased with the increase of dialysis time, but the ascorbic acid test was positive at 8 hours, indicating incomplete dialysis. In order to ensure complete dialysis and take into account the polysaccharide yield, the dialysis time was determined to be 12 hours.

[0123] Table 14 Dialysis yield of selenium-enriched Dendrobium at different water change days

[0124]

[0125] In summary, the purification process of selenium-rich Dendrobium officinale polysaccharide was determined as follows: the reaction solution was cooled to room temperature, filtered, anhydrous sodium carbonate was added to the supernatant to adjust the pH value to 5-6, and the supernatant was loaded into a 3500MW dialysis bag, and dialyzed 5 times at room temperature, each time for 12 hours.

[0126] Experimental Example 5 Study on the Drying Process of Selenium-enriched Dendrobium Officinale Polysaccharide

[0127] Freeze drying is one of the drying technologies. Compared with traditional heat drying technology, freeze drying is carried out under vacuum and low temperature conditions, so it can reduce oxidation, effectively protect the active ingredients, greatly protect the drugs, and reduce the loss of nutrients after rehydration. The present invention studies three aspects: sublimation drying time, analytical drying temperature, and analytical drying time.

[0128] (1) Sublimation drying time

[0129] Selenium-enriched Dendrobium officinale polysaccharide dialysate was placed in a drying tray with a thickness not exceeding 6 mm, placed in a freeze dryer, pre-frozen at -40°C for 6 hours, and vacuumed until the vacuum degree of the freeze drying chamber was less than 20 Pa, and then the drying program was started: sublimation drying temperature: -30°C; sublimation drying time: 4h, 5h, 6h; analytical drying temperature: 40°C; analytical drying time: 8h. The water content of freeze-dried selenium-enriched Dendrobium officinale polysaccharide was determined, and the results are shown in Table 15.

[0130] The results showed that after 6 hours of sublimation drying, the water content of selenium-rich Dendrobium officinale polysaccharide was less than 5%, so the sublimation drying time was determined to be 6 hours.

[0131] Table 15 Effect of moisture content at different sublimation times

[0132]

[0133] (2) Analysis of drying temperature

[0134] Selenium-enriched Dendrobium officinale polysaccharide dialysate was placed in a drying tray with a thickness of no more than 6 mm, placed in a freeze dryer, pre-frozen at -40°C for 6 hours, and vacuumed until the vacuum degree of the freeze drying chamber was less than 20 Pa, and then the drying program was started: sublimation drying temperature: -30°C; sublimation drying time: 6 hours; analytical drying temperature: 20°C, 30°C, 40°C; analytical drying time: 8 hours. The water content of freeze-dried selenium-enriched Dendrobium officinale polysaccharide was determined, and the results are shown in Table 16.

[0135] The results showed that the moisture content of selenium-rich Dendrobium officinale polysaccharides could be lower than 5% when the drying temperature was 30℃ and 40℃. Considering that low temperature is conducive to the preservation of effective ingredients, the drying temperature was determined to be 30℃.

[0136] Table 16 Effect of different analytical drying temperatures on moisture content

[0137]

[0138] (3) Analysis and drying time

[0139] Selenium-enriched Dendrobium officinale polysaccharide dialysate was placed in a drying tray with a thickness of no more than 6 mm, placed in a freeze dryer, pre-frozen at -40°C for 6 hours, and vacuumed until the vacuum degree of the freeze drying chamber was less than 20 Pa, and then the drying program was started: sublimation drying temperature: -30°C; sublimation drying time: 6 hours; analytical drying temperature: 30°C; analytical drying time: 4, 6, 8, 10 hours. The water content of freeze-dried selenium-enriched Dendrobium officinale polysaccharide was determined, and the results are shown in Table 17.

[0140] The results showed that the water content of selenium-rich Dendrobium officinale polysaccharide could be lower than 5% after drying for more than 6 hours. To ensure the drying efficiency, the drying time was determined to be 8 hours.

[0141] Table 17 Effect of different analytical drying times on moisture content

[0142]

[0143] In summary: the drying process of the selenium-rich Dendrobium officinale polysaccharide of the present invention is: take the selenium-rich Dendrobium officinale polysaccharide dialysate in a drying plate with a thickness not exceeding 6 mm, place it in a freeze dryer, pre-freeze at -40°C for 6 hours, evacuate until the vacuum degree of the freeze drying chamber is less than 20Pa, and start the drying program: dry at -30°C for 6 hours, and then dry at 30°C for 8 hours.

[0144] Experimental Example 6: Research and Verification of Selenium-enriched Dendrobium Officinale Polysaccharide Preparation Process

[0145] In order to verify the repeatability of the preparation process of selenium-rich Dendrobium officinale polysaccharide determined by the present invention, three verification tests were carried out. Process conditions: 10 g of Dendrobium officinale polysaccharide was taken, 0.5% HNO 3 1000mL solution was heated and stirred to dissolve all the polysaccharides to obtain a 10mg / L polysaccharide solution. 0.8 times the weight of the polysaccharide was added to each 2 SeO 3 and 1.2 times the weight of polysaccharide BaCl 2 , placed on a magnetic stirrer at 60°C for 6 hours, cooled to room temperature, filtered, adjusted the pH of the filtrate to 5-6 with anhydrous sodium carbonate, put into a 3500MW dialysis bag, dialyzed 5 times at room temperature, each time for 12 hours, and then took the dialysate in a drying plate with a thickness of no more than 6mm, placed in a freeze dryer, pre-frozen at -40°C for 6 hours, vacuumed, and after the vacuum degree of the freeze drying chamber was less than 20Pa, the drying program was started: drying at -30°C for 6 hours, and then drying at 30°C for 8 hours, and selenized Dendrobium polysaccharide was obtained. The yield and selenium content were calculated, and the determination results are shown in Table 18.

[0146] The results of the verification test showed that the determined preparation process of selenium-rich Dendrobium officinale polysaccharide was stable, feasible and reproducible.

[0147] Table 18 Selenium polysaccharide synthesis process verification test

[0148]

[0149] Test Example 7 Study on the quality of selenium-rich Dendrobium officinale polysaccharides of the present invention

[0150] Three batches of selenium-enriched Dendrobium officinale polysaccharides prepared under the process validation were taken and their moisture content, total polysaccharide content and selenium content were determined respectively.

[0151] (1) Measurement method

[0152] 1.1 Moisture determination: According to the second method of drying method of Part IV 0832 Moisture Determination of the Chinese Pharmacopoeia 2020 edition.

[0153] 1.2 Determination of total polysaccharide content (phenol-concentrated sulfuric acid method)

[0154] Preparation of reference solution Take an appropriate amount of anhydrous glucose reference, weigh accurately, and add water to make a 100 μg / mL standard solution.

[0155] Preparation of the test solution: Weigh 10 mg of selenium-rich Dendrobium officinale polysaccharide, accurately weigh it, place it in a beaker, add 50 ml of water, heat, stir to dissolve, cool to room temperature, make up to 100 ml, shake well, filter, and take the filtrate to obtain the solution.

[0156] Drawing of standard curve: Accurately measure 0.2ml, 0.4ml, 0.6ml, 0.8ml and 1.0ml of reference solution, place in stoppered test tubes, add water to make up to 1.0ml, accurately add 1ml of 5% phenol solution (prepared before use), shake well, then accurately add 5ml of sulfuric acid, shake well, heat in a boiling water bath for 20 minutes, take out, cool in an ice bath for 5 minutes, use the corresponding reagent as blank, measure the absorbance at a wavelength of 488nm according to the UV-Vis spectrophotometry (General Rule 0401), and draw a standard curve.

[0157] Determination method: Accurately pipette 1.0 mL of the test solution and place it in a 10 mL stoppered test tube. Develop the color according to the above method. Starting from "accurately add 1 mL of 5% phenol solution (prepared before use)", determine the absorbance value according to the law, read the polysaccharide content in the test solution from the standard curve, and calculate it.

[0158] 1.3 Determination of selenium content

[0159] Preparation of test solution: Take 0.05 g of Dendrobium selenium polysaccharide, accurately weigh, add 4 mL of concentrated sulfuric acid-perchloric acid-nitric acid mixture (concentrated sulfuric acid: perchloric acid: nitric acid = 1:1:4), leave overnight, heat to evaporate the strong acid, transfer quantitatively to a 10 mL volumetric flask with distilled water, make up to the scale, shake well, and obtain.

[0160] Preparation of standard curve: 0.5, 1.0, 2.0, 3.0 and 4.0 mL of selenium standard solution (10 μg / mL) were accurately measured and placed in a separatory funnel, and water was added to 30 mL. The pH was adjusted to 2 with dilute hydrochloric acid solution, and 3.0 mL of 1% o-phenylenediamine hydrochloride test solution was added, shaken, and placed in a dark place for 2 h. 10.0 mL of toluene was added, extracted, allowed to stand, the toluene layer was separated, and toluene was added to make up to 10 mL. The absorbance was measured at 334 nm with the corresponding reagent as blank, and the standard curve was drawn with absorbance as ordinate and concentration as abscissa.

[0161] Determination method: Accurately pipette 1.0 mL of the test solution, add water to 30 mL, adjust the pH to 2 with dilute hydrochloric acid or sodium hydroxide solution, and follow the method under the standard curve preparation, starting from "add 3.0 mL of 1% o-phenylenediamine hydrochloride test solution", measure the absorbance value according to the law, read the selenium content in the test solution from the standard curve, and calculate it.

[0162] (2) Measurement results

[0163] The measurement results are shown in Table 19.

[0164] Table 19 Quality determination results of selenium-enriched Dendrobium officinale polysaccharides

[0165]

[0166] Experimental Example 8 Study on the Antioxidant Activity of Selenium-enriched Dendrobium Polysaccharides

[0167] 1. Experimental Materials

[0168] 1.1 Instruments: ZA-D5 five-layer single-row independent culture unit (Shanghai Haisheng Biological Experiment Equipment Co., Ltd.); SZ680 continuous zoom stereo microscope (Chongqing Aote Optical Instrument Co., Ltd.); ZXSD-A1090 biochemical incubator (Shanghai Zhicheng Analytical Instrument Manufacturing Co., Ltd.); SQP 1 / 10,000 electronic scale (Sartorius Scientific Instrument (Beijing) Co., Ltd.); fluorescence microscope (Leica Microsystems Co., Ltd.)

[0169] 1.2 Reagents: Lipopolysaccharide (LPS, analytical grade) (EC: 297-473-0SIGMA); 2',7'-Dichlorofluorescent yellow diacetate (DCFH-DA) (CAS: 4091-99-0SIGMA); Tricaine (analytical grade); Sodium chloride (NaCl); Potassium chloride (KCl); Calcium chloride (CaCl 2 ·2H 2 O); magnesium sulfate (MgSO 4 7H 2 O).

[0170] 1.3 Sample: selenium-rich Dendrobium officinale polysaccharide (prepared according to Example 1, batch number 210401, total polysaccharide 93.7%, selenium content 1.38 mg / g); Dendrobium officinale polysaccharide (prepared as follows: take Dendrobium officinale medicinal material, crush it into coarse powder, add 12 times the amount of water to extract 3 times, each time for 1 h, filter, combine the filtrate, concentrate under reduced pressure, add ethanol to the alcohol content of 80%, let it stand overnight, filter with suction, wash the precipitate with a small amount of ethanol 3 times, add appropriate amount of water to the precipitate, heat and stir to dissolve, add ethanol again to the alcohol content of 80%, let it stand overnight, filter with suction, wash the precipitate with a small amount of ethanol 3 times, and dry at 60°C to obtain. Total polysaccharide 92.4%); Sodium selenite pentahydrate (AR,>98.0%, Chengdu Aikoda Chemical Reagent Co., Ltd.).

[0171] 1.4 Solution preparation

[0172] 1.4.1X H-buffer water: weigh sodium chloride (NaCl) (3.5 g), NaHCO 3 (0.2 g), KCl (0.05 g) and CaCl 2 (0.1175 g), add distilled water to make up to 1000.0 ml, filter with a vacuum filter (pore size 0.2 μM), sterilize in an autoclave, and keep at room temperature for use.

[0173] 1.4.2 LPS stock solution (100.0 μg / ml): Weigh 2.0 mg of LPS, add 20.0 ml of buffer water to dissolve, and store at -20°C for later use.

[0174] 1.4.3 DCHF-DA (100.0 μg / ml): Weigh 2.0 mg DCFH-DA, add 20.0 ml buffer water to dissolve, and store at -20°C away from light.

[0175] 1.4.4 Tricaine (0.2 mg / ml): Weigh 2.0 mg of tricaine, add 10.0 ml of buffer water to dissolve, and store at 4°C.

[0176] 1.4.5 Sample solution: dilute with buffer water to the experimental concentration.

[0177] (1) Selenium-enriched Dendrobium officinale polysaccharide solution: Weigh 10 mg of selenium-enriched Dendrobium officinale polysaccharide and add 20.0 ml of buffer water to dissolve. (0.50 mg / ml, selenium concentration: 0.69 μg / ml)

[0178] (2) Dendrobium officinale polysaccharide solution: Weigh 10 mg of Dendrobium officinale polysaccharide and add 20.0 ml of buffer water to dissolve. (0.50 mg / ml)

[0179] (3) Sodium selenite solution: Take 23 mg of sodium selenite pentahydrate, add 100 ml of buffer water to dissolve, then take 1 ml and place it in a 10 ml volumetric flask, add buffer water to dilute to the mark, shake well, and you have it. (Selenium concentration: 0.69 μg / ml)

[0180] 2. Experimental Methods

[0181] Under a stereomicroscope, 9hpf wild-type zebrafish embryos with normal development were randomly selected and placed in a 6-well cell culture plate, 20 embryos / well, and blank control group (or solvent control group), model control group and test sample group (selenium-enriched Dendrobium officinale polysaccharide group, Dendrobium officinale polysaccharide group, sodium selenite group) were set up respectively. The culture water in the 6-well plate was drained, 5.0ml buffer water was added to each well of the blank control group, 4500.0μl buffer water was added to each well of the model control group, and 4500.0μl sample solution was added to each well of the test sample group. After pretreatment for 40min, 500.0μl LPS stock solution was added to each well of the model control group and the test sample group (to make the final concentration of LPS 10μg / ml), and the oxidative stress model was constructed, and the cells were placed in a 28℃ constant temperature incubator for incubation for 24h. After 24 hours of incubation, the solution in each well was aspirated, and after washing with culture water 2-3 times, 5.0 ml of culture water was added to each well, and cultured in a constant temperature incubator at 28.5°C until 3 dpf. After 3 dpf, the egg membranes in each well were aspirated, and 5.0 ml (20.0 ug / ml) of fluorescent probe (DCHF-DA) was added to each well to detect the ROS content, and cultured for 1 hour in the dark. The embryos were then washed three times with culture water, anesthetized with tricaine, observed and recorded under a fluorescence microscope, and the fluorescence intensity was analyzed using Image J.

[0182] 3. Experimental Data Collection and Analysis

[0183] 3.1 Experimental data collection

[0184] The relative fluorescence intensity of ROS and the ROS inhibition rate of zebrafish embryos in each group were analyzed.

[0185] (1) The calculation formula of ROS relative fluorescence intensity is as follows:

[0186] ROS relative fluorescence intensity = FI 空白组、模型组、待测样品组 / MFI 模型组

[0187] Where: FI—fluorescence intensity; MFI (Mean Fluorescence Intensity)—mean fluorescence intensity. The result is rounded to one decimal place.

[0188] (2) ROS inhibition rate

[0189] ROS inhibition rate (%) = [1-ROS relative fluorescence intensity (model group, test sample group)] * 100%

[0190] 3.2 Experimental Results Analysis

[0191] The experimental data were analyzed using GraphPad Prism 8 statistical analysis software (or other analysis software) for the ROS inhibition rate in each group, and the differences between the sample group and the model group were compared. The values ​​were expressed as mean ± standard error (Mean ± SEM).

[0192] 4. Experimental Data

[0193] The experimental results are shown in Table 20. Figure 4 .

[0194] Table 20 Effect of selenium-enriched Dendrobium polysaccharide on the relative fluorescence intensity of ROS in zebrafish (n=20)

[0195]

[0196] # :Indicates that there is a statistical difference between the blank group and the model group. # P<0.05, ## P<0.01, ### P < 0.001;

[0197] *: Indicates that there is a statistically significant difference between the sample group and the model group, *P<0.05, **P<0.01, ***P<0.001.

[0198] 5. Experimental Conclusion

[0199] LPS was used to induce oxidative stress in zebrafish, and the fluorescent probe DCF-DA was used to detect the ROS content in zebrafish. Figure 4 As shown, the blank group without LPS was used as the control, and its relative fluorescence intensity was 0.33±0.06%. The model group induced by LPS was used as the control, and its relative fluorescence intensity was 1.00±0.13%, which was increased compared with the blank control group (0.33±0.06), and ##p<0.01, the difference (very significant), indicating that the zebrafish oxidative stress model was successfully constructed. At the same time, the results of the selenium-enriched Dendrobium polysaccharide group were statistically different from those of the model control group *p<0.05, indicating that 0.50mg / mL of selenium-enriched Dendrobium polysaccharide can reduce the content of reactive oxygen, that is, it can inhibit oxidative stress in zebrafish. However, the antioxidant activity of the same dose of Dendrobium officinale polysaccharide group and sodium selenite group was not obvious.

[0200] Experimental Example 9 Study on the Anti-wrinkle Activity of Selenium-enriched Dendrobium Polysaccharide (Zebrafish Type I Collagen Content Detection Method)

[0201] 1. Experimental Materials

[0202] 1.1 Instruments: ZA-D5 five-layer single-row independent culture unit (Shanghai Haisheng Biological Experiment Equipment Co., Ltd.); SZ680 continuous zoom stereo microscope (Chongqing Aote Optical Instrument Co., Ltd.); ZXSD-A1090 biochemical incubator (Shanghai Zhicheng Analytical Instrument Manufacturing Co., Ltd.); SQP 1 / 10,000 electronic scale (Sartorius Scientific Instrument (Beijing) Co., Ltd.); ultrasonic disruptor (Xinzhi / JY92-IIN); refrigerated centrifuge.

[0203] 1.2 Reagents: sodium chloride; potassium chloride; calcium chloride; magnesium sulfate; ELIAS (type I collagen) Jianglai Technology kit.

[0204] 1.3 Samples: selenium-rich Dendrobium officinale polysaccharide (prepared according to Example 1, batch number 210401, total polysaccharides 93.7%, selenium content 1.38 mg / g); Dendrobium officinale polysaccharide (prepared, same as Test Example 8, total polysaccharides 92.4%); sodium selenite pentahydrate (AR, >98.0%, Chengdu Aikoda Chemical Reagent Co., Ltd.).

[0205] 1.4 Solution preparation

[0206] 1.4.1X H-buffer water: weigh sodium chloride (NaCl) (3.5 g), NaHCO 3 (0.2 g), KCl (0.05 g) and CaCl 2 (0.1175 g), add distilled water to make up to 1000.0 ml, filter with a vacuum filter (pore size 0.2 μM), sterilize in an autoclave, and keep at room temperature for use.

[0207] 1.4.2 Sample solution

[0208] (1) Selenium-enriched Dendrobium officinale polysaccharide solution: Weigh 10 mg of selenium-enriched Dendrobium officinale polysaccharide and dissolve it in 20.0 ml of buffer water to obtain (0.50 mg / ml, selenium concentration: 0.69 μg / ml). Dilute it in equal proportions to 2 times (0.25 mg / ml) and 4 times (0.125 mg / ml) for later use.

[0209] (2) Dendrobium officinale polysaccharide solution: Weigh 10 mg of Dendrobium officinale polysaccharide and add 20.0 ml of buffer water to dissolve. (0.50 mg / ml)

[0210] (3) Sodium selenite solution: Take 23 mg of sodium selenite pentahydrate, add 100 ml of buffer water to dissolve, then take 1 ml and put it in a 10 ml volumetric flask, add buffer water to dilute to the scale, shake well, and you have it. (Selenium concentration: 0.69 μg / ml)

[0211] 2. Experimental Methods

[0212] Under a stereomicroscope, select 6-9 hpf wild-type zebrafish embryos with normal development in a 96-well plate, 1 per well. Drain the excess liquid in the well, add 200 μL of standard dilution water to each well of the blank control group, and add 200 μL of sample solution to each well of the sample group. Place the zebrafish embryos in a 28°C biochemical incubator and incubate for 72 hours. After incubation, count the number of zebrafish larvae with normal development in each group, and collect 95 zebrafish larvae. Aspirate the zebrafish larvae in the wells one by one and transfer them to a 6-well plate, 1 group per well. Aspirate the excess liquid in the wells, fill them with double distilled water, let stand for 1 minute, discard the liquid, and repeat the washing 3 times. Transfer the zebrafish larvae to a 1.5 mL EP tube, 1 group per tube, and place them on ice. After the zebrafish fry cooled and sank to the bottom, use a flattened 10μL pipette tip to absorb the excess liquid in the tube (absorb it thoroughly), wipe the residual liquid on the tube wall with a clean paper towel, and add 500μL pre-cooled PBS to each tube. Place the EP tube in ice water, crush it with an ultrasonic cell crusher for 10 minutes (power 20%, ultrasound on time 30s, ultrasound off time 30s), centrifuge at 4℃, 5000g for 10 minutes, and take the supernatant for zebrafish type I collagen content detection.

[0213] The operation was performed according to the instructions of the zebrafish type I collagen kit. The specific steps are as follows:

[0214] ① Take out the required strips from the aluminum foil bag after equilibration at room temperature for 20 minutes, and seal the remaining strips in a ziplock bag and return them to 4°C.

[0215] ② Set up standard wells and sample wells, and add 50 μL of standard of different concentrations to each standard well.

[0216] ③ First add 10μL of the sample to be tested into the sample well, and then add 40μL of sample diluent.

[0217] ④ Then add 100 μL of horseradish peroxidase (HRP)-labeled detection antibody to each of the standard wells and sample wells, seal the reaction wells with a sealing film, and incubate in a 37°C water bath or incubator for 60 min.

[0218] ⑤Discard the liquid, pat dry with absorbent paper, fill each well with washing solution, let it stand for 1 minute, shake off the washing solution, pat dry with absorbent paper, and repeat this washing process 5 times (the residual bubbles in the last washing well plate can be sucked dry with a vacuum pump).

[0219] ⑥ Add 50 μL of substrate A and B to each well and incubate at 37°C in the dark for 15 min.

[0220] ⑦ Add 50 μL of stop solution to each well and measure the OD value of each well at a wavelength of 450 nm within 15 minutes.

[0221] Draw a standard curve: In an Excel worksheet, use the standard concentration as the horizontal axis and the corresponding OD value as the vertical axis to draw a linear regression curve for the standard, and calculate the concentration value of each sample according to the curve equation.

[0222] 3. Experimental results analysis

[0223] The experimental data were analyzed using GraphPad Prism 8 statistical analysis software (or other analysis software) on the content of zebrafish type I collagen in each group, and the differences between the sample group and the blank control group were compared. The values ​​were expressed as mean ± standard error (Mean ± SEM).

[0224] 4. Experimental Data

[0225] The experimental results are shown in Table 21.

[0226] Table 21 Effect of selenium-enriched Dendrobium polysaccharide on type Ⅰ collagen content in zebrafish (n=95)

[0227]

[0228] Note: * indicates that there is a statistically significant difference between the sample group and the blank group, *P<0.05, **P<0.01, ***P<0.001.

[0229] # It showed that there was a statistically significant difference compared with the Dendrobium officinale polysaccharide group. # P<0.05, ## P<0.01, ### P<0.001

[0230] 5. Experimental Conclusion

[0231] The type I collagen content in zebrafish of different concentration groups of selenium-rich Dendrobium polysaccharide was significantly increased compared with the blank control group, and there were statistical differences, indicating that selenium-rich Dendrobium polysaccharide can promote the increase of type I collagen content and has significant anti-wrinkle effect.

[0232] Compared with the blank control group, the sodium selenite group had no significant effect on increasing the content of type I collagen in zebrafish. Compared with the blank control group, the Dendrobium officinale polysaccharide group had a certain effect on increasing the content of type I collagen in zebrafish, but the effect was significantly worse than that of selenium-enriched Dendrobium polysaccharide.

Claims

1. A method for preparing selenium-rich dendrobium polysaccharide with anti-wrinkle effect, characterized in that: It includes the following steps: 1) Take a Dendrobium officinale extract with a polysaccharide content of 80%, add 0.5% v / v, ml / ml nitric acid solution to dissolve it at a mass volume ratio of 10 g: 1000 mL; 2) taking the solution obtained in step 1), adding 0.8 times the weight of sodium selenite and 1.2 times the weight of barium chloride of the Dendrobium officinale extract at 60°C for 6h, cooling to room temperature and filtering, adjusting the pH value of the filtrate to 5-6, dialyzing 5 times with a 3500MW dialysis bag, each time for 12 hours, taking the dialysate and freeze-drying to obtain selenium-enriched Dendrobium polysaccharide; The drying is drying, and the freeze drying procedure is pre-freezing at 40°C for 6 hours, evacuating until the vacuum degree of the freeze drying chamber is less than 20Pa, and then starting the drying procedure: drying at -30°C for 6 hours, and then drying at 30°C for 8 hours.

Images