Extraction method and application of a rhodiola-based polygalacturonic acid-methyl galacturonate polysaccharide and an anti-fatigue functional beverage containing the polysaccharide

Abstract

The present invention relates to the technical fields of polysaccharide extraction and functional beverage preparation, and relates to a method for extracting and applying rhodiola-based polygalacturonic acid-methyl galacturonate polysaccharide and an anti-fatigue functional beverage containing the polysaccharide. The present invention uses rhodiola as a raw material, and prepares polygalacturonic acid-methyl galacturonate polysaccharide with anti-fatigue effect through low-temperature alkaline extraction, alcohol precipitation, purification, concentration and drying. Then, based on this polysaccharide, an anti-fatigue functional beverage is prepared by mixing fruit and vegetable juice, sweetener and ethyl paraben. It is suitable for long-term consumption, realizing the reuse of rhodiola residues and developing an efficient anti-fatigue and delicious functional beverage, solving the problem of physical fatigue that troubles people.

Description

Technical Field

[0001] The present invention relates to the technical field of polysaccharide extraction and functional beverage preparation, and particularly relates to a method for extracting rhodiola-based polygalacturonic acid-methyl galacturonate polysaccharide, its application, and an anti-fatigue functional beverage containing the polysaccharide. Background Art

[0002] Rhodiola crenulata (Hook.f.et Thoms.) H.Ohba is a perennial herb of the Crassulaceae family, widely distributed in regions such as Northeast China, Tibet, Xinjiang, Yunnan, Guizhou, and Sichuan. It grows in the understory of mountain slopes or the crevices of rock shrubs in alpine pollution-free areas. Due to its harsh growth environment, such as hypoxia, low temperature and dryness, strong winds, ultraviolet radiation, and large temperature differences between day and night, it has strong vitality and special adaptability. The whole plant of Rhodiola can be used as medicine, with effects such as anti-fatigue, intelligence nourishing and heart nourishing, moisture absorption and hemostasis, etc. Currently, there are relatively few anti-fatigue products prepared from Rhodiola polysaccharides, and many are still in the primary stage. Polysaccharides are considered superior to other polymers in many fields due to their easy tailoring, biological activity, biocompatibility, and uniformity. It is widely present in plants, microbial cells, and animals, and plays an important role in expressing antioxidant, anti-tumor, antibacterial, anti-inflammatory, and immunomodulatory activities, etc.

[0003] Currently, there are already beverages on the market with Rhodiola as the main raw material, including functional and health-care types, etc., each with its own characteristics, having effects such as anti-hypoxia, anti-fatigue, nourishing qi and tonifying deficiency, strengthening the spleen and kidney, and enhancing immunity. However, there are few anti-fatigue beverages made from polysaccharides extracted from Rhodiola medicinal residues, and this beverage can be taken for a long time without any toxic and side effects. The present invention improves the high-efficiency utilization rate of the effective components of Rhodiola and is suitable for industrial production. Summary of the Invention

[0004] The purpose of the present invention is to provide a method for extracting rhodiola-based polygalacturonic acid-methyl galacturonate polysaccharide, its application, and an anti-fatigue functional beverage containing the polysaccharide. By steps such as low-temperature alkaline extraction, etc., polygalacturonic acid-methyl galacturonate polysaccharide with anti-fatigue efficacy is prepared, and an anti-fatigue functional beverage is further prepared from this polysaccharide to solve the problems existing in the above-mentioned prior art.

[0005] To achieve the above purpose, the present invention provides the following solutions:

[0006] One of the technical solutions of the present invention: provides a method for extracting rhodiola-based polygalacturonic acid-methyl galacturonate polysaccharide, and the steps include:

[0007] [[ID=

[0008] Further, the rhodiola is the rhodiola residue dried and then crushed and sieved through a 40-80 mesh sieve.

[0009] Further, the steps of the low-temperature alkaline extraction include:

[0010] Mix the rhodiola with water, add NaOH to a final concentration of 0.1 mol / L, magnetically stir at 45 °C for 4 h, and filter to obtain a filter residue and a filtrate;

[0011] The filter residue is repeatedly extracted at least twice, and after the filtrates are combined and concentrated, the pH value of the concentrated solution is adjusted to 9-10 to complete the low-temperature alkaline extraction.

[0012] Preferably, the mass / volume ratio of the rhodiola to water is 300 g:2000 mL.

[0013] Preferably, the concentration is concentrated to 1 / 4 of the volume before concentration.

[0014] Preferably, the pH value is adjusted using nitric acid.

[0015] In the low-temperature alkaline extraction step, the acidity and alkalinity need to be strictly controlled to ensure that no colloid precipitates, because the precipitation of colloid is irreversible and affects the extraction effect.

[0016] Further, the steps of the alcohol precipitation include:

[0017] Add ethanol with twice the volume of the concentrated solution for alcohol precipitation, let it stand overnight (12 h), and collect the precipitate product to complete the alcohol precipitation step.

[0018] Preferably, the volume fraction of the ethanol is 95%-100%, and more preferably 95%.

[0019] Since there are more salts in the concentrated solution, when there are more salts, it affects the purity of dialysis. Therefore, directly performing alcohol precipitation can remove most of the salts, small molecular sugars, and pigment impurities.

[0020] Further, the steps of the purification, concentration, and drying include:

[0021] Dialyze the precipitate product to remove salts, small molecular sugars, and impurities to obtain crude polysaccharides;

[0022] Use an ultrafiltration membrane to remove proteins from the crude polysaccharides, and after concentration and freeze-drying, the rhodiola-based polygalacturonic acid-methyl galacturonate polysaccharide is obtained.

[0023] Preferably, a 3000 Da membrane is used for dialysis.

[0024] Preferably, the pore size of the ultrafiltration membrane used for removing proteins is 100 kDa.

[0025] Preferably, the concentration is carried out using a 1000 Da ultrafiltration membrane.

[0026] The second technical solution of the present invention: Provide a rhodiola-based polygalacturonic acid-methyl galacturonate polysaccharide extracted by the above method.

[0027] The third technical solution of the present invention: Provide an application of the above rhodiola-based polygalacturonic acid-methyl galacturonate polysaccharide in the preparation of anti-fatigue drugs, foods or beverages.

[0028] The fourth technical solution of the present invention: An anti-fatigue functional beverage, the raw materials of which include the above rhodiola-based polygalacturonic acid-methyl galacturonate polysaccharide.

[0029] The fifth technical solution of the present invention: A preparation method of the above anti-fatigue functional beverage, the steps of which include: mixing rhodiola-based polygalacturonic acid-methyl galacturonate polysaccharide, fruit and vegetable juice, sweetener and ethyl paraben, and then adding water for homogenization to obtain the anti-fatigue functional beverage.

[0030] Further, the mass / volume ratio of the rhodiola-based polygalacturonic acid-methyl galacturonate polysaccharide, fruit and vegetable juice, sweetener and ethyl paraben is 1-2 g: 200-400 mL: 100-150 g: 0.15 g.

[0031] Further, the fruit and vegetable juice is prepared by juicing fruits and / or vegetables and then passing through a 200-mesh filter screen.

[0032] Preferably, the fruit is at least one of mango, apple, honeydew melon, and citrus.

[0033] Preferably, the vegetable is at least one of cabbage, cucumber, radish, and potato.

[0034] Further, the sweetener includes at least one of honey, sucrose, stevia sugar, and high fructose syrup.

[0035] The present invention discloses the following technical effects:

[0036] In the present invention, low-temperature alkaline extraction is used for rhodiola medicinal residues to obtain polygalacturonic acid-methyl galacturonate polysaccharide. Different from the polysaccharides extracted by methods such as decoction, it is a pectin-like polysaccharide. When it is applied to the preparation of anti-fatigue drinks, the prepared functional drinks have significant anti-fatigue performance, and the anti-fatigue performance it brings is not the pseudo-anti-fatigue characteristic shown by the improvement of exercise ability caused by short-term stimulation of the body.

[0037] The present invention can remove the peculiar smell of rhodiola itself by adding fruit and vegetable juices, and can also enhance the overall taste of the beverage in combination with sweeteners, providing a highly effective anti-fatigue and delicious functional beverage for the general population. By drinking it for a period of time, physical fatigue can be relieved, thus realizing the reuse of the active ingredients in the medicinal residues and developing a new type of functional beverage that is easy to process and produce, solving the problem of physical fatigue that plagues people. BRIEF DESCRIPTION OF THE DRAWINGS

[0038] In order to more clearly illustrate the technical solutions in the embodiments of the present invention or the prior art, the following will briefly introduce the drawings required for use in the embodiments. Obviously, the drawings described below are only some embodiments of the present invention. For those of ordinary skill in the art, without creative efforts, other drawings can be obtained based on these drawings.

[0039] Figure 1 High performance liquid chromatography (HPLC) chromatogram of the PRC extracted in Example 1;

[0040] Figure 2 Infrared spectrum of the PRC extracted in Example 1;

[0041] Figure 3 NMR spectrum of the PRC extracted in Example 1, where A is 1 1H-NMR, B is 13 13C-NMR, and C is the HSQC spectrum;

[0042] Figure 4 Bar chart of the exhaustive swimming time of mice after the last gavage;

[0043] Figure 5 Biochemical indices of mice after exhaustive swimming at a dose of 200 mg / kg and the control group. Among them, A is the content of whole blood lactic acid, B is the glucose content, C is the content of serum urea nitrogen, and D is the iCa content;

[0044] Figure 6 Finished product diagrams of the anti-fatigue functional beverages prepared in Examples 2 to 9. Among them, S1 is Example 2, S2 is Example 3, S3 is Example 4, S4 is Example 5, S5 is Example 6, S6 is Example 7, S7 is Example 8, and S8 is Example 9. DETAILED DESCRIPTION OF THE EMBODIMENTS

[0045] The various exemplary embodiments of the present invention will now be described in detail. This detailed description should not be considered as a limitation of the present invention, but rather as a more detailed description of certain aspects, characteristics, and implementation schemes of the present invention.

[0046] It should be understood that the terms described in the present invention are only for describing specific embodiments and are not used to limit the present invention. In addition, for the numerical ranges in the present invention, it should be understood that each intermediate value between the upper and lower limits of the range is also specifically disclosed. Each intermediate value within any stated value or stated range, as well as each smaller range between any other stated value or intermediate value within the stated range, is also included in the present invention. The upper and lower limits of these smaller ranges may be independently included or excluded from the range.

[0047] Unless otherwise specified, all technical and scientific terms used herein have the same meaning as commonly understood by those of ordinary skill in the art to which the present invention pertains. Although the present invention only describes preferred methods and materials, any methods and materials similar or equivalent to those described herein can also be used in the implementation or testing of the present invention. All documents mentioned in this specification are incorporated by reference to disclose and describe the methods and / or materials related to the documents. In case of conflict with any incorporated document, the content of this specification shall prevail.

[0048] Without departing from the scope or spirit of the present invention, various improvements and changes can be made to the specific embodiments of the present invention specification, which are obvious to those skilled in the art. Other embodiments obtained from the present invention specification are obvious to those skilled in the art. The present invention specification and examples are only exemplary.

[0049] Regarding the use of "comprising", "including", "having", "containing", etc. herein, they are all open-ended terms, meaning including but not limited to.

[0050] The Rhodiola residue used in the present invention is provided by the formula granule workshop of New Green Company and is the residue after alcohol-water extraction.

[0051] Unless otherwise specified, the overnight standing described in the specific embodiments of the present invention refers to standing for 12 h.

[0052] In the specific embodiments of the present invention, the sweetener is selected as honey and the fruit and vegetable juice is selected as mango juice. Among them, the fruit and vegetable juice is used to remove the peculiar smell of Rhodiola itself, and the sweetener is used to increase the overall taste of the beverage. Therefore, they can be equivalently replaced during specific implementation without affecting the realization of the specific effect.

[0053] Example 1

[0054] The extraction steps of Rhodiola-based polygalacturonic acid-methyl galacturonate polysaccharide are as follows:

[0055] S1. Take the dried Rhodiola residue, crush it and pass through a 40-80 mesh sieve. Weigh 300 g of the powder and add it to 2 L of distilled water. Use NaOH to adjust the pH value to 9-10, stir magnetically at 45 °C for 4 h and filter to obtain the filter residue and the filtrate;

[0056] S2. Repeat the steps of S1 twice for the filter residue, combine the filtrates, and concentrate the filtrate to 500 mL using a rotary evaporator.

[0057] S3. Add ethanol (volume fraction 95%) with a volume twice that of the concentrated filtrate for alcohol precipitation, let it stand overnight, centrifuge to collect the solid product, wash it twice with an equal volume of ethanol solution with a mass fraction of 60%, and obtain the precipitate product after centrifugation.

[0058] S4. Add 30 mL of water to the precipitate product for dialysis (3000 Da membrane) to remove salts, small molecular sugars, and impurities to obtain crude polysaccharide.

[0059] S5. Dissolve the crude polysaccharide in 1000 mL of water, use a 100 kDa ultrafiltration membrane to remove the protein component, then use a 1000 Da ultrafiltration membrane to concentrate it to 200 mL, and finally perform freeze-drying to obtain rhodiola-based polygalacturonic acid-methyl galacturonate polysaccharide, denoted as PRC.

[0060] Examples 2 - 9

[0061] Prepare the PRC prepared in Example 1 into an anti-fatigue functional beverage, and the steps are as follows:

[0062] Mix PRC, mango juice, honey, and ethyl paraben evenly, and add purified water to 1 L for homogenization to obtain the anti-fatigue functional beverage.

[0063] The mango juice is obtained by peeling and pitting fresh mangoes to get the pulp, juicing it, and then filtering it through a 200-mesh sieve.

[0064] The dosage of each component is shown in Table 1.

[0065] Table 1

[0066]

[0067] Comparative Example 1

[0068] The extraction steps of rhodiola polysaccharide are as follows: (Reference: Wu Yaru, Research on the Extraction, Structure Characterization and Antitumor Activity of Rhodiola Polysaccharide, Tianjin University of Science and Technology, 2021)

[0069] Accurately weigh 500 g of the defatted powder, add 5 times the amount of deionized water, stir evenly, and extract it in a 4°C refrigerator for 12 h. After centrifuging to take the supernatant, freeze-thaw it repeatedly 3 - 5 times. Add 95% anhydrous ethanol and let it precipitate at 4°C overnight. After deproteinizing by the Sevag method, dialyze it in distilled water for 48 h (4°C) using a 10 kDa dialysis bag, and obtain rhodiola crude polysaccharide by freeze-drying.

[0070] Comparative Example 2

[0071] The extraction steps of rhodiola polysaccharide are as follows: (Refer to the method described in the paper "Study on the Structural Characteristics and TLR4 Agonistic Activity of Two Rhodiola Polysaccharides" by Teng Fei, Gu Yangqin, Shou Panting, etc. in the Chinese Pharmaceutical Journal in 2021)

[0072] 500 g of the dried residue after defatting was soaked in 5 times the volume of distilled water for 2 h, reflux-extracted 3 times, the extraction solutions were combined, concentrated, then precipitated with 3 times the volume of 95% ethanol, deproteinized by Sevag method, and freeze-dried for later use.

[0073] Comparative Example 3

[0074] The extraction steps of rhodiola polysaccharide are as follows: (Refer to Wissam Farhat, Richard Venditti, Ashley Quick, et al. Hemicellulose extraction and characterization for applications in paper coatings and adhesives[J]. Industrial Crops and Products, 2017)

[0075] The dried residue after defatting was soaked in 5 times the volume of distilled water, added with NaOH with a final concentration of 1 M, magnetically stirred at 80 °C for 2 h, then filtered. Extraction was carried out 3 times, the filtrates were combined, concentrated, then precipitated with 3 times the volume of 95% ethanol overnight, the precipitate was deproteinized by Sevag method, and freeze-dried for later use.

[0076] Test Example

[0077] The polysaccharides extracted in Example 1 and Comparative Examples 1 - 3 were analyzed, and the results are shown in Table 2.

[0078] Table 2

[0079]

[0080] It can be seen from the component comparison data in Table 2 that the components of the polysaccharides extracted by the extraction method of the present invention are significantly different from those of the polysaccharides extracted in Comparative Examples 1 - 3.

[0081] The PRC extracted in Example 1 was analyzed.

[0082] Monosaccharide composition: Hydrolysis and derivatization methods, as well as a two-step hydrolysis method for polysaccharides rich in uronic acid, were adopted. 5 mg of the dried polysaccharide was hydrolyzed with 0.5 mL of 2 M trifluoroacetic acid (TFA) solution at 120 °C for 2 h under N2 sealing conditions. After drying with N2, the residue was obtained, which was the first step of hydrolysis. 0.5 mL of 10% H2SO4 was added to the residue, transferred to a 2 mL ampoule, sealed with N2, reacted at 100 °C for 2 h, and then neutralized with NaOH solution for the excess. 0.5 M PMP solution was added for derivatization at 70 °C for 1 h. After neutralization with hydrochloric acid, chloroform was used for extraction 4 times. The aqueous solution was filtered through a 0.22 μm filter membrane, and 0.2 mL was taken and added to a liquid phase injection vial, diluted to 1 mL.

[0083] High-performance liquid chromatography (HPLC) analysis was performed using an Agilent 1260 chromatograph equipped with ZORBAX Eclipse Plus C18 (4.6 mm × 250 mm × 5 μm; Agilent Technologies, USA).

[0084] Figure 1 It is the high-performance liquid chromatogram of the PRC extracted in Example 1. From Figure 1 It can be seen that the PRC extracted in Example 1 is mainly composed of galacturonic acid (GalA) and its methyl ester (92.8% ± 4.5%), among which arabinose (Ara) accounts for 4.79% ± 0.62%.

[0085] Degree of methylation (DM) and acetylation (DAc): The degree of methoxylation (DM) was detected by ultraviolet spectrophotometry using methanol as the standard, and the degree of acetylation (DAc) was detected by the acetate kinase method. The degree of esterification (DM) and the degree of acetylation (DAc) were 65.6% ± 5.3% and 6.79% ± 0.74% respectively.

[0086] Figure 2 It is the infrared spectrogram of the PRC extracted in Example 1. From Figure 2 It can be seen that a broad and strong absorption peak appears at 3500 - 3000 cm -1 , which is caused by the stretching vibration of O-H in the polysaccharide. A weak peak appears between 3000 - 2750 cm -1 , which is caused by the stretching and bending vibrations of the C-H bond of CH2. The characteristic peak at 1740 cm -1 indicates the presence of an ester (COOR) group, and the characteristic peak at 1610 cm -1 belongs to the presence of a carboxylic acid group, indicating the presence of uronic acid in the polysaccharide from the rhodiola residue. The degree of methyl esterification of the rhodiola polysaccharide can be calculated from these two peaks. The specific calculation method is the ratio of the absorption peak area at 1740 cm -1 to the sum of the absorption peak areas at 1740 cm -1 and 1610 cm-1. The calculated degree of methyl esterification is 20.5%. The characteristic peak at 1150 cm -1The characteristic peak indicates the presence of C-C single bonds, 1100 cm -1 The characteristic peak indicates the stretching vibration of the pyranose ring. 1020 cm -1 The characteristic peak is due to the stretching vibration of the C-O-C glycosidic bond, indicating that the polysaccharide from the Rhodiola residue has a pyran configuration. 836 cm -1 It is caused by the bending vibration of H-C, 761 cm -1 An absorption peak appears, which is caused by the α-ring breathing vibration.

[0087] Figure 3 It is the NMR spectrum of the PRC extracted in Example 1, where A is 1 1H-NMR, B is 13 13C-NMR, and C is the HSQC spectrum. From Figure 3 It can be found that in A, the differential signals of the α-Araf residue are most obvious at δ5.03 and δ5.09 ppm, while the differential signals of the esterified group increase sharply at δ3.27 and the acetylated group at δ2.03 ppm; in B, the signal peak at δ174.55 ppm combined with the signal peak at δ16.75 ppm infers the →4)-α-GalpA-(1→(A) resonance absorption peak. The signal peak of the acetylated α-GalpA methyl carbon appears significantly at δ23.11 ppm; combined with C, the results show that there is a pectin structure in PRC, and this structural domain may be attached to the HG backbone in the form of a T-Ara side chain.

[0088] The main 1H NMR and 13 13C-NMR chemical shifts of PRC are listed in Table 3.

[0089] Table 3

[0090]

[0091] Therefore, it can be determined that the polysaccharide extracted in the present invention is different from the polysaccharides extracted in Comparative Examples 1 to 3, and it is a polygalacturonic acid (GalA)-methyl galacturonate (GalA-6-OCH3) polysaccharide.

[0092] Through the observation experiments of large-dose infusion and anti-fatigue on mice, as well as the biochemical index tests of forced swimming and after exercise of mice, safety and efficacy evaluations were carried out (Animal Ethics Approval: Academic Committee of Southwest Forestry University - SWFU-2022117).

[0093] The gavage method was used for drug administration. Kunming mice, with an equal number of males and females, were divided into two groups of 10 each. The administration volume was 0.2 mL / 10 g. The beverage of Example 8 was prepared into a lyophilized powder, redissolved, and gavaged at the maximum concentration of 2.0 g / mL. The control group was given normal saline. After a single administration, the mice were observed for seven days, and the number of mice showing positive reactions was recorded. At the same time, poisoning symptoms were observed. Animal death was used as the index of positive reaction. The results are shown in Table 4.

[0094] Table 4

[0095]

[0096] The animal models were gavaged at high and low (200 mg / kg, 50 mg / kg) doses according to the polysaccharide amount, and the control group was given ultrapure water. There were 10 mice in each group. After continuous gavage twice a day for 14 days, the indexes were measured.

[0097] The control group and the experimental group of mice needed to undergo swimming adaptation training, which started 2 weeks after gavage and was carried out every day. It started from 10 min / d and gradually increased by 5 - 10 min every day until the end.

[0098] Method for measuring the index of exhaustive swimming time: 30 min after the last gavage, a metal wire with a weight of 5% of the body weight was attached to the tail of the mouse, and the mouse was placed in a swimming tank with a water depth of 30 cm. The water temperature was about 32 °C. The mouse was forced to exercise until exhaustion, and the entire swimming time was recorded based on the criterion that it did not float up for 8 s after sinking to the bottom. The results are as Figure 4 shown. It can be seen that after the mice continuously took different doses of polysaccharides for 14 days, the forced swimming time of the mice in the 200 mg / kg and 50 mg / kg dose groups was significantly improved compared with the control group, and they had excellent anti - fatigue ability. Among them, the forced swimming time of the 200 mg / kg dose group was significantly increased to more than 2 h (P < 0.01), and there was a very significant difference in anti - fatigue ability compared with the control group.

[0099] Determination of serum biochemistry and ion content after exercise:

[0100] Serum preparation: 30 min after the last gavage, the mice in each group were placed in a constant - temperature water tank to swim for 90 min, rested for 30 min, and then immediately had their eyes enucleated to collect 0.5 mL of blood. The blood was refrigerated at 4 °C for about 3 h. After coagulation, it was centrifuged at 2000 r / min at low temperature for 10 min, and the serum was transferred to a centrifuge tube. 60 μL of the removed whole blood was respectively injected into the CG4+ and Chem8+ cards, and the lactic acid value was read on a portable blood analyzer.

[0101] In the analysis of the whole blood lactic acid content, the most direct and objective manifestation of fatigue is the decline in exercise endurance. Swimming is a whole-body consumptive exercise. Swimming can consume a large amount of energy and oxygen to a certain extent, and at the same time, a large amount of lactic acid can accumulate in the body. The exhaustive swimming time can be used as an important indicator reflecting exercise endurance and is applied to various tests; the glucose content can reflect that after the body exercises, the excitability of the sympathetic nerve increases, leading to an increase in the secretion of catecholamines, adrenaline, and norepinephrine by the adrenal medulla, and ultimately leading to the decomposition of liver glycogen, increasing the body's glucose content; urea nitrogen is one of the final products of amino acid catabolism in the body. The blood urea nitrogen mainly comes from the liver and is excreted through the kidneys with urine.

[0102] The content of urea nitrogen in the body's serum will increase with the prolongation of exercise time, that is, the more urea nitrogen is produced, the worse the body's anti-fatigue ability is. The level of serum uric acid nitrogen can reflect the body's anti-fatigue ability; as the exercise time increases, more and more energy of the mouse body is consumed, and the calcium element in the blood will be affected to a certain extent. The specific tests of the content of various biochemical indexes after exercise are as Figure 5 shown. Figure 5 The biochemical indexes of the mice after exhaustive swimming at a dose of 200 mg / kg and the control group are shown. Among them, A is the whole blood lactic acid content, B is the glucose content, C is the serum urea nitrogen content, and D is the iCa content. It can be Figure 5 seen that after administering rhodiola polysaccharide to the mice, the contents of glucose, urea nitrogen, and iCa in the body have no obvious changes compared with the control group, while the lactic acid secretion of the mice is significantly reduced compared with the control group. First, it shows that the polysaccharide from rhodiola residue has high safety, and second, it shows that the polysaccharide from rhodiola residue has an obvious inhibitory effect on lactic acid secretion, increasing the anti-fatigue time of the mice, and also proving that the polysaccharide in rhodiola residue plays a great role in the anti-fatigue of the human body.

[0103] Figure 6 The finished product diagrams of the anti-fatigue functional beverages prepared in Examples 2 to 9 are shown. Among them, S1 is Example 2, S2 is Example 3, S3 is Example 4, S4 is Example 5, S5 is Example 6, S6 is Example 7, S7 is Example 8, and S8 is Example 9.

[0104] For the anti-fatigue functional beverages prepared in Examples 2 to 9, the pH value and viscosity were measured, and a sensory evaluation score was carried out. The results are shown in Table 5.

[0105] For the sensory evaluation, according to GB-T21733, the well-mixed tested sample was taken in a colorless and transparent glass bottle. In a good light outdoors, observe its state, clarity, etc. facing the light, and smell its odor and taste it at room temperature, and record the evaluation results of 10 people.

[0106] Table 5

[0107]

[0108] From Figure 6 and Table 5, it can be seen that after adding mango juice and honey, the light yellow color not only enhances the visual appearance of the beverage itself but also masks the odor of the rhodiola polysaccharide itself. Adding polysaccharides can enhance flocculation stability and protect the color. The S6 and S7 formulations are the best, and finally, the S7 formulation is determined considering economic efficiency.

[0109] In the present invention, the discarded residue after rhodiola extraction is used to obtain polysaccharides by low-temperature alkaline extraction, and it is determined to be a pectin-like acidic polysaccharide through characterization. After being formulated with fruit and vegetable juices with natural aromas and colors, and supplemented with natural honey as a sweetener and pH regulator, a healthy beverage with excellent color, aroma, and taste and no additives and having an anti-fatigue effect is jointly produced. Through animal safety and in vivo efficacy experiments, it is finally determined that the finished product of the formulation has a significant anti-fatigue effect and will not produce side effects. Through continuous testing for up to two weeks, the improvement in exercise ability caused by short-term stimulation of the beverage to the body is excluded, reflecting the long-term safety of the beverage from the side. The present invention has high potential economic value in terms of raw material sources, extraction technology, formulated finished products, and in vivo efficacy.

[0110] The embodiments described above are only descriptions of the preferred embodiments of the present invention and do not limit the scope of the present invention. Without departing from the design spirit of the present invention, various deformations and improvements made by those of ordinary skill in the art to the technical solutions of the present invention shall fall within the protection scope determined by the claims of the present invention.

Claims

1. A method for extracting rhodiola-based polygalacturonic acid-methyl galacturonate polysaccharide, characterized in that the steps Comprising: Using Rhodiola as raw material, through low-temperature alkaline extraction, alcohol precipitation, purification, concentration and drying to obtain the polygalacturonic acid-methyl galacturonate polysaccharide; The steps of the low-temperature alkaline extraction include: Mix Rhodiola with water, use NaOH to adjust the pH value to 9 - 10, magnetically stir at 45°C for 4 h, and filter to obtain filter residue and filtrate; The filter residue is extracted at least two more times, and after combining the filtrates and concentrating to obtain a concentrated solution, adjust the pH value to 9 - 10 to complete the low-temperature alkaline extraction.

2. The extraction method according to claim 1, wherein The mass / volume ratio of Rhodiola to water is 300 g:2000 mL.

3. The extraction method according to claim 1, wherein The steps of the alcohol precipitation include: Add twice the volume of ethanol to the concentrated solution for alcohol precipitation, let it stand for 12 h, and collect the precipitate product to complete the alcohol precipitation step.

4. The extraction method according to claim 3, characterized in that, The steps of the purification, concentration and drying include: Dialyze the precipitate product to remove salts, small molecule sugars and impurities to obtain crude polysaccharide; Use an ultrafiltration membrane to remove proteins from the crude polysaccharide, and after concentration and freeze-drying, obtain the Rhodiola-based polygalacturonic acid-methyl galacturonate polysaccharide.

5. A Rhodiola-based polygalacturonic acid-methyl galacturonate polysaccharide extracted by the extraction method according to any one of claims 1 - 4.

6. An application of the Rhodiola-based polygalacturonic acid-methyl galacturonate polysaccharide according to claim 5 in the preparation of anti-fatigue drugs, food or beverages.

7. A fatigue-resistant functional beverage, characterized in that, The raw material includes the Rhodiola-based polygalacturonic acid-methyl galacturonate polysaccharide according to claim 5.

8. A method for preparing an anti-fatigue functional beverage as claimed in claim 7, characterized in that the steps Comprising: After mixing the Rhodiola-based polygalacturonic acid-methyl galacturonate polysaccharide, fruit and vegetable juice, sweetener and ethyl paraben, add water for homogenization to obtain the anti-fatigue functional beverage.

9. The preparation method according to claim 8, characterized in that, The mass / volume ratio of the Rhodiola-based polygalacturonic acid-methyl galacturonate polysaccharide, fruit and vegetable juice, sweetener and ethyl paraben is 1 - 2 g:200 - 400 mL:100 - 150 g:0.15 g; the fruit and vegetable juice is prepared by juicing fruits and / or vegetables and passing through a 200-mesh filter; the sweetener includes at least one of honey, sucrose, stevioside and high fructose syrup.

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