Salidroside derivatives, preparation methods and applications

Abstract

The present invention relates to a salidroside derivative, specifically a hydroxy-α-salidroside compound. The hydroxy-α-salidroside compound is superior to α-salidroside in scavenging DPPH free radicals, whitening skin, and improving learning and memory disorders. The compound can be added as a new functional ingredient to cosmetics, functional foods, or pharmaceutical compositions. The preparation method of the hydroxy-α-salidroside of the present invention uses α-salidroside as a substrate and synthesizes hydroxy-α-salidroside under the action of hydroxylase.

Description

[0001] CROSS-REFERENCE TO RELATED APPLICATIONS

[0002] This application claims priority to Chinese patent application No. 2023106705293, filed on June 8, 2023, entitled “A derivative of salidroside, its preparation method and application”, the entire contents of which are incorporated by reference into this application and constitute a part of this application for all purposes. Technical Field

[0003] The present invention relates to a salidroside derivative, in particular to a hydroxy-α-salidroside compound. Background Art

[0004] Rhodiola rosea is a precious traditional Chinese medicine. The 2020 edition of the Chinese Pharmacopoeia includes the dried roots and rhizomes of Rhodiola rosea of the Crassulaceae family. It is produced in Tibet and other places in China and grows in areas with an altitude of about 4050 to 5400 meters. Medicines such as Rhodiola rosea tablets and Xinnaoxin capsules prepared from Rhodiola rosea have the effects of promoting blood circulation and removing blood stasis, dredge meridians and relieve pain. The functions of health foods such as Rhodiola rosea capsules are to enhance immunity and relieve physical fatigue. Salidroside is the effective active ingredient of the traditional Chinese medicine Rhodiola rosea. It is reported that salidroside has physiological activities such as protecting cardiovascular and cerebrovascular vessels, anti-fatigue, anti-depression, anti-aging, anti-hypoxia, anti-radiation, anti-tumor, immune regulation, whitening and freckle removal. Salidroside in its natural state (plant-derived salidroside) is β-salidroside, and its glycosidic bond is β-type.

[0005] Chinese Patent No. 202211359878.5 discloses α-salidroside, its preparation method, and application. The α-salidroside, chemically named 2-(4-hydroxyphenyl)ethyl-α-D-glucoside, has a molecular weight of 300.23 and a chemical formula shown in Formula I. It has excellent free radical scavenging activity and can be used as a primary raw material for cosmetics, anti-fatigue health products, and other products.

[0006] Summary of the Invention

[0007] Purpose of the Invention: Driven by the need for product iteration, the applicant conducted research on salidroside and its derivatives, obtaining hydroxy-α-salidroside. Furthermore, the present invention provides a preparation method and application of hydroxy-α-salidroside.

[0008] There is no report on hydroxy-α-salidroside in the prior art.

[0009] Technical solution:

[0010] In a first aspect, the present application provides a hydroxy-α-salidroside compound, chemical name: 2-(3,4-dihydroxyphenyl)ethyl-α-D-glucoside, chemical formula shown in Formula II, which has an α-glucoside bond on the alcoholic hydroxyl group of hydroxytyrosol.

[0011]

[0012] Preferably, according to the present invention, the molecular weight of the hydroxy-α-salidroside is 316.108 as analyzed by LC-MS.

[0013] Preferably, according to the present invention, the H-NMR spectrum of the hydroxy-α-salidroside is: 1H NMR (600 MHz, DMSO): δ2.64-2.72 (m, 2H), 3.02-3.08 (m, 1H), 3.17-3.21 (m, 1H), 3.31-3.37 (m, 4H), 3.37-3.46 (m, 2H), 3.46-3.51 (m, 1H), 3.57-3.63 (m, 1H), 3.67-3.73 (m, 1H), 4.44 (t, 1H), 4.57 (d, 1H), 4.68 (d, J = 3.6 Hz, 1H: β-H), 4.73 (d, 1H), 4.83 (d, 1H), 8.64 (s, 1H: OH), 8.69 (s, 1H: OH).

[0014] The C-NMR spectrum of the hydroxy-α-salidroside: 13C NMR (151 MHz, DMSO): δ 35.5, 61.4, 68.9, 70.8, 72.5, 73.3, 73.8, 99.1, 115.9, 116.7, 120.0, 129.9, 144.0, 145.5.

[0015] In a second aspect, the present application provides a method for preparing the hydroxy-α-salidroside compound.

[0016] The method comprises dissolving α-salidroside in a buffer solution, adding hydroxylase, and performing a hydroxylation reaction in a reaction system to prepare the salidroside.

[0017] Preferably, the hydroxylase can be selected from commercially available hydroxylase preparations.

[0018] Further preferably, the commercially available hydroxylase enzyme preparations are derived from Merck & Co., Ltd., Shanghai MacLean Biochemical Technology Co., Ltd. and Shanghai Yuanye Biotechnology Co., Ltd., including but not limited to monophenol monooxygenase, polyphenol oxidase, tyrosinase, etc.

[0019] Preferably, a surfactant that does not affect the enzymatic reaction, such as Tween-20 or Span, may be added to the reaction system.

[0020] In the present invention, the preparation of the hydroxy-α-salidroside can also be carried out in an organic solvent. The organic solvent used in the reaction can be any type and concentration range that does not affect the reaction, specifically including one solvent selected from DMSO, 2-propanol, and ethanol, or a mixed solvent of two or more.

[0021] Preferably, the reaction system contains ammonium salt.

[0022] The ammonium salt preferably includes but is not limited to ammonium chloride, ammonium acetate, or ammonium sulfate.

[0023] Ammonium salt can improve the conversion rate of substrate α-salidroside.

[0024] In a third aspect, the present application provides the use of the hydroxy-α-salidroside compound in the preparation of products with free radical scavenging function; or the use of the hydroxy-α-salidroside compound in cosmetics with whitening function.

[0025] Preferably, the free radical is a DPPH free radical.

[0026] In the application, preferably, the product is a cosmetic with the function of scavenging free radicals.

[0027] Preferably, the product with free radical scavenging function is a functional food.

[0028] The present invention further provides a composition containing hydroxy-α-salidroside.

[0029] Preferably, the composition has the function of scavenging free radicals or whitening skin.

[0030] Preferably, the free radical is a DPPH free radical.

[0031] The cosmetic composition containing hydroxy-α-salidroside is selected from aqueous solutions, oils, emulsions, gel products, paste products, or others, and can be produced according to existing cosmetic technical specifications.

[0032] The functional food composition containing hydroxy-α-salidroside has a dosage form selected from capsules, tablets, ointments, aqueous solutions, or others, and can be produced according to existing functional food technical specifications.

[0033] In a fourth aspect, the present application also provides the use of the hydroxy-α-salidroside compound in the preparation of a drug for improving learning and memory disorders.

[0034] Preferably, the drugs for improving learning and memory disorders include but are not limited to drugs for treating Alzheimer's disease.

[0035] Preferably, the drugs for improving learning and memory disorders include but are not limited to drugs for treating Parkinson's disease.

[0036] Learning and memory impairments are a symptom cluster that can occur in many diseases. They can be caused by a variety of factors, including neurodegenerative diseases such as Alzheimer's disease and Parkinson's disease, stress, aging, ischemic brain injury, brain trauma, hypertension, diabetes, menopausal syndrome, and depression. Alzheimer's disease is the most common learning and memory impairment. Clinically available medications can only alleviate symptoms but cannot slow or halt its progression. Their efficacy varies from patient to patient and they are associated with numerous adverse reactions. Furthermore, the accelerated pace of work and life, increased competitive pressure, and chronic stress can lead to imbalances in coordination and function, particularly affecting cognitive function in the brain and resulting in decreased learning and memory abilities. Therefore, there is an urgent need for medications to improve learning and memory impairments. Studies have found that the hydroxy-α-salidroside described in this application has the ability to improve the learning and memory levels of model mice, as manifested in significantly improving the relative discrimination index of rats for objects in new locations, shortening the platform-finding latency in the water maze experiment, increasing the number of platform crossings, and reducing the number of errors and dark room time in the dark avoidance experiment, indicating that hydroxy-α-salidroside can effectively improve learning and memory disorders.

[0037] Beneficial effects:

[0038] The present invention provides a salidroside derivative, namely hydroxy-α-salidroside, which has higher activity in scavenging DPPH free radicals than α-salidroside and can be added to cosmetics or health products as a new functional ingredient; the hydroxy-α-salidroside also has the excellent performance of improving the learning and memory levels of model mice and can be developed and applied as a drug for the preparation of Alzheimer's disease or Parkinson's disease drugs. BRIEF DESCRIPTION OF THE DRAWINGS

[0039] Figure 1 .TLC spectrum of the enzyme-catalyzed reaction solution of Example 1; in the figure, from left to right are lane 1 and lane 2; lane 1 is α-salidroside, and lane 2 is hydroxy-α-salidroside.

[0040] Figure 2 .HPLC spectrum of the enzyme-catalyzed reaction solution of Example 1.

[0041] Figure 3 . In the HPLC spectrum of the enzyme-catalyzed reaction liquid of Example 1, the LC-MS spectrum of the substance at rt = 6.97 min.

[0042] Figure 4. In the HPLC spectrum of the enzyme-catalyzed reaction liquid of Example 1, the H-NMR spectrum of the substance at rt = 6.97 min.

[0043] Figure 5 . In the HPLC spectrum of the enzyme-catalyzed reaction liquid of Example 1, the C-NMR spectrum of the substance at rt = 6.97 min. DETAILED DESCRIPTION

[0044] The present invention will be further described below with reference to specific embodiments. The embodiments are not intended to limit the present invention, and the protection scope of the present invention is not limited thereto.

[0045] The hydroxy-α-salidroside of the present invention has a chemical name: 2-(3,4-dihydroxyphenyl)ethyl-α-D-glucoside, and its structural formula is shown in Formula II.

[0046] The preparation method of hydroxy-α-salidroside of the present invention uses α-salidroside as a substrate and realizes hydroxylation under the action of hydroxylase.

[0047] The hydroxylase can be selected from commercially available hydroxylase enzyme preparations from Merck & Co., Ltd., Shanghai MacLean Biochemical Technology Co., Ltd. and Shanghai Yuanye Biotechnology Co., Ltd., including catechol oxidase but not limited to monophenol monooxygenase, polyphenol oxidase, tyrosinase, etc.

[0048] The method of using the hydroxylase enzyme preparation includes adding it to the reaction system in the form of powder or liquid, and also fixing the enzyme preparation on a resin to make an immobilized enzyme preparation and adding it to the reaction system. The immobilized enzyme preparation can be reused.

[0049] The method for preparing hydroxy-α-salidroside of the present invention comprises adding hydroxylase to α-salidroside phosphate buffer to perform a hydroxylase reaction. A surfactant that does not affect the enzymatic reaction, such as Tween-20 and Span, may be added to the enzyme reaction solution.

[0050] When using the above-mentioned hydroxylase to prepare hydroxy-α-salidroside compounds, the amount of hydroxylase and reaction conditions have a great influence on the production efficiency, so it is crucial to select appropriate reaction conditions such as the amount of enzyme and reaction time.

[0051] The reaction can be carried out in the presence of a solvent. The solvent used in the reaction can be any solvent as long as it does not affect the reaction and its concentration range. Specifically, it can include common reagents such as DMSO, 2-propanol, and ethanol. These solvents can be used alone or in combination of two or more.

[0052] The enzyme used in the present invention can be inactivated by heating or changing the pH value, thereby stopping the enzyme catalytic reaction.

[0053] The mixture containing the hydroxy-α-salidroside compound, the hydrolyzate of the mixture, the enzyme-inactivated product of the mixture, and the purified product of the mixture, as well as their dried powder products, can be used in foods, beverages, and cosmetics containing the hydroxy-α-salidroside compound, and can also be used as ingredients in foods, special medical foods, health products, or medicines, and used in the production of foods and beverages, cosmetics, special medical foods, health products, or medicines.

[0054] TLC analysis conditions: The enzyme reaction solution was rapidly analyzed by thin layer chromatography. The specific analysis conditions were as follows: a TLC plate (Merck Kieselgel 60F254) produced by Merck was used, and a solution of chloroform:methanol:acetic acid = 5:1:1 was used as the developing solvent.

[0055] HPLC analysis conditions: high performance liquid chromatograph: Hitachi HPLC 5440 chromatograph, chromatographic column: Kromasil 100-5C18 (250*4.6 mm), detector: photodiode array (DAD 280 nm), detection wavelength: UV 280 nm, injection volume: 10 μL, flow rate: 1 mL / min, column temperature: 30°C, mobile phase: acetonitrile: 0.1% formic acid aqueous solution (v / v) isocratic elution.

[0056] LC-MS analysis conditions: chromatographic column: Kromasil 100-5C18 (250*4.6mm), detector: photodiode array (DAD 280nm), detection wavelength: 280nm, injection volume: 10μL, flow rate: 1mL / min, column temperature: 30℃, mobile phase with detection time of 5-15min; H-ESI mode, molecular weight scanning range: 50-800.

[0057] The document "Enzymatic routes for the production of mono- and di-glucosylated derivatives of hydroxytyrosol" by Antonio Trincone et al., Bioresource Technology, Volume 115, is an article on "Enzymatic production of mono- and di-glucosylated derivatives of hydroxytyrosol." The article describes the preparation of (3,4-dihydroxyphenyl)ethyl-α-D-glucopyranoside by adding a homogenate of visceral material from Aplysia fasciata to 1.5 ml of a 10 mM receptor solution in 50 mM potassium acetate buffer (pH 5.5, containing 150 mM maltose). The target compound was then inferred from the NMR data obtained by acetylation of the reaction product. However, the document lacks mass spectrometry identification data and does not disclose the molecular weight of the specific compound. It also lacks chemical shift data for the key hydrogen atoms of the α-glucoside, and the chemical structure of the specific compound is not disclosed, making it impossible to confirm the stereoconfiguration of the compound.

[0058] The inventors of the present application acetylated the hydroxy-α-salidroside compound described in the present application according to the method described in the document, and then performed carbon spectrum and hydrogen spectrum analysis. However, they were unable to obtain the carbon spectrum and hydrogen spectrum data of the acetylated product and its supplementary document. Therefore, the hydroxy-α-salidroside compound obtained in the present application is different from the compound obtained in the document.

[0059] Example 1. Tyrosinase hydroxylation experiment

[0060] 500 mg of α-salidroside (molecular weight 300.23) was added to 50 mL of 0.2 M phosphate buffer (and adjusted to pH 10), 500 mg of ascorbic acid, and 3 mg of tyrosinase (T828256-100KU, McLean), and water was added to make the volume to 100 mL. The mixture was stirred at 25°C for 24 hours to obtain an enzyme-catalyzed reaction solution.

[0061] Example 2. Polyphenol oxidase hydroxylation experiment

[0062] 100 mg of α-salidroside (molecular weight 300.23) was added to 50 mL of 0.2 M phosphate buffer (and adjusted to pH 10), 100 mg of ascorbic acid, and 3 mg of tyrosinase (S10171-100KU, Shanghai Yuanye Biotechnology Co., Ltd.). Water was added to make the volume to 100 mL, and the reaction was stirred at 15°C for 50 hours to obtain an enzyme-catalyzed reaction solution.

[0063] Example 3. Monophenol monooxygenase hydroxylation experiment

[0064] Take 500 mg of α-salidroside (molecular weight 300.23), add 50 mL of 0.2 M phosphate buffer (pH 9), add 500 mg of ascorbic acid, and then add 1.5 g of monophenol monooxygenase (T3824-50KU, Merck). Add water to make the volume to 100 mL, stir and react at 35°C for 24 hours to obtain an enzyme-catalyzed reaction solution.

[0065] Example 4. Monophenol monooxygenase hydroxylation experiment

[0066] According to the method described in Example 3, the feed amount was simultaneously increased by 10 times, and the reaction was stirred at 35° C. for 24 hours to obtain an enzyme-catalyzed reaction solution.

[0067] Example 5. Purification and structural analysis of hydroxy-α-salidroside

[0068] The reaction solutions of Examples 1-4 were analyzed by TLC, HPLC, LC-MS, and NMR.

[0069] (1) TLC analysis

[0070] Example 1 reaction solution TLC analysis spectrum see Figure 1 , indicating that new substances are produced in the enzyme-catalyzed reaction solution. In the figure, from left to right are lanes 1 and 2; lane 1 is α-salidroside, and lane 2 is the new substance produced by the enzyme-catalyzed reaction.

[0071] (2) HPLC analysis

[0072] According to the above chromatographic conditions, the purified reaction solution of Example 1 was subjected to HPLC analysis. The HPLC spectrum is shown in Figure 2 .

[0073] Figure 2 The HPLC spectrum shown shows that the reaction solution after purification in Example 1 mainly has two peaks, with peak elution times of rt = 6.97 min and rt = 9.98 min, respectively. The peak at rt = 9.98 min is α-salidroside, and the peak at rt = 6.97 min has a significant difference in elution time from that of α-salidroside.

[0074] (3) LC-MS analysis

[0075] The substance with rt=6.97min in the HPLC spectrum of the reaction solution of Example 1 was subjected to LC-MS analysis, and the measured value of the molecular ion peak [M-1] was 315.108. The LC-MS spectrum is shown in FIG. Figure 3 .

[0076] According to the UV spectrum and LC-MS spectrum displayed by the photodiode array detector, preliminary analysis of the new peak substance with a retention time of rt = 6.97 min in the HPLC spectrum showed a molecular weight of 316.108, which may be a glycoside formed by the hydroxylation of α-salidroside, possibly hydroxy-α-salidroside.

[0077] (4) Separation and purification

[0078] A semi-preparative high performance liquid chromatograph (BRIX 2850, Beijing Chengda Instrument Co., Ltd.) was used with a COSMOSIL 5C18-MS-II column (250*20 mm). The mobile phase was acetonitrile: 0.1% formic acid aqueous solution (v / v) at a ratio of 1:9 for isocratic elution. A photodiode array detector was used with a UV detection wavelength of 280 nm. The injection volume was 100 μL and the flow rate was 18 mL / min.

[0079] The substances with rt=6.97 min in the HPLC spectra of the reaction solutions of Examples 1-4 were collected respectively, concentrated and freeze-dried, and used for subsequent experiments.

[0080] (5) H-NMR and C-NMR spectrum analysis

[0081] The freeze-dried samples were subjected to H-NMR and C-NMR analyses.

[0082] The H-NMR spectrum of the substance with rt=6.97min in the HPLC spectrum of the reaction solution of Example 1 is shown in FIG. Figure 4 , C-NMR spectrum see Figure 5 .

[0083] 1H-NMR (600MHz, DMSO): δ2.64-2.72(m,2H),3.02-3.08(m,1H),3.17-3.2 1(m,1H),3.31-3.37(m,4H),3.37-3.46(m,2H),3.46-3.51(m,1H),3.57- 3.63(m,1H),3.67-3.73(m,1H),4.44(t,1H),4.57(d,1H),4.68(d,J=3.6 Hz,1H:β-H),4.73(d,1H),4.83(d,1H),8.64(s,1H:OH),8.69(s,1H:OH).

[0084] 13C-NMR (151MHz, DMSO): δ35.5,61.4,68.9,70.8,72.5,73.3,73.8,99.1,115.9,116.7,120.0,129.9,144.0,145.5.

[0085] According to the H-NMR spectrum and C-NMR spectrum of the product of Example 1, combined with the H-NMR spectrum and C-NMR spectrum of α-salidroside disclosed in Chinese patent application 202211359878.5, the H-NMR spectrum of the product of Example 1 has two single peaks (s, 1H: OH) at 8.64ppm and 8.69ppm, which is the "characteristic of bisphenol hydroxyl". α-salidroside has a single peak at 9.10ppm, which is the "characteristic of monophenol hydroxyl", and the C-NMR spectrum of the product of Example 1 shows There are 14 characteristic peaks. There are only 12 characteristic peaks of α-salidroside, which are obviously different from the two.

[0086] In summary, from the TLC spectrum and HPLC spectrum, it can be seen that the enzyme-catalyzed reaction produces a new substance; from the LC-MS analysis, the molecular weight of the new substance is 316.108; combined with the H-NMR and C-NMR spectra, it is confirmed that the substance obtained by the enzyme-catalyzed reaction in Example 1 is hydroxy-α-salidroside.

[0087] The HPLC spectrum, LC-MS spectrum, H-NMR spectrum, and C-NMR spectrum of the product of the enzymatic reaction of Example 2-4 were the same as those of the reaction solution of Example 1. Therefore, the substance obtained by the enzymatic reaction of Example 2-4 was also hydroxy-α-salidroside.

[0088] The concentration of hydroxy-α-salidroside in the reaction solution of Example 1-4 was determined by HPLC. The results are recorded in Table 1.

[0089] Table 1

[0090] Hydroxy-α-salidroside, g / L α-Salidroside conversion rate, % Example 1 1.375 26.1 Example 2 0.146 13.9 Example 3 1.960 37.2 Example 4 4.150 39.4

[0091] Example 6-8. Based on Example 3, an ammonium salt, such as ammonium chloride, ammonium acetate, or ammonium sulfate, is added to the reaction system to carry out an enzymatic reaction. The details are as follows:

[0092] 500 mg of α-salidroside (molecular weight 300.23) was taken, added to 50 mL of 0.2 M phosphate buffer (pH 9), added to 500 mg of ascorbic acid, and added to 10 mL of 0.2 M ammonium salt (specific ammonium salt types are shown in Table 2). Then, 1.5 g of monophenol monooxygenase (T3824-50KU, Merck) was added, and the volume was made up to 100 mL with water. The reaction was stirred at 35° C. for 24 hours to obtain an enzyme-catalyzed reaction solution. The hydroxy-α-salidroside in the reaction solution was detected by HPLC. The results are recorded in Table 2.

[0093] Table 2

[0094] Example 6 Example 7 Example 8 ammonium salts Ammonium chloride Ammonium acetate ammonium sulfate Hydroxy-α-salidroside, g / L 2.280 2.012 1.907 α-Salidroside conversion rate, % 43.3 38.2 36.2

[0095] The data in Table 2 show that both ammonium chloride and ammonium acetate can improve the conversion rate of the substrate α-salidroside in the enzymatic reaction.

[0096] Example 9. Free radical scavenging experiment. The materials and reagents used are shown in Table 3.

[0097] Table 3. Materials and reagents

[0098]

[0099] DPPH free radical scavenging experiment: Free radicals exert strong oxidative effects, directly or indirectly, and are widely involved in physiological and pathological processes. Excessive free radicals can damage the body through oxidation. Salvianolic acid compounds are donors of phenolic hydroxyl groups and have the structural basis for antioxidant activity. This experiment used the DPPH [1,1-diphenyl-2-trinitrophenylhydrazine, also known as 1,1-diphenyl-2-picrylhydrazyl, (free radical)] free radical scavenging reaction to study the DPPH free radical scavenging efficiency of hydroxy-α-salidroside, α-salidroside, and β-salidroside.

[0100] Solution preparation:

[0101] (1) Preparation of Hydroxy-α-Salidroside Solution: The freeze-dried solids obtained after separation and purification of the reaction solutions of Examples 1-3 were respectively taken and prepared into 500 mg / L aqueous solutions according to conventional methods.

[0102] (2) Preparation of α-salidroside solution: Prepare a 500 mg / L aqueous solution according to conventional methods.

[0103] (3) Preparation of β-salidroside solution: Prepare a 500 mg / L aqueous solution according to conventional methods.

[0104] (4) Preparation of DPPH ethanol solution: Prepare a 0.04 mg / ml anhydrous ethanol solution according to conventional methods.

[0105] 2.DPPH free radical scavenging experiment:

[0106] (1) Take 0.9 mL of hydroxy-α-salidroside, α-salidroside, and β-salidroside aqueous solutions, add 0.9 mL of DPPH anhydrous ethanol solution, mix well, and place in a dark place for static. Samples are taken at 0.5 h, 1 h, 2 h, 3 h, 4 h, and 5 h, respectively, and the absorbance is measured at a wavelength of 517 nm. The average value of three replicates is calculated and recorded as Ai;

[0107] (2) Take 0.9 mL of hydroxy-α-salidroside, α-salidroside, and β-salidroside aqueous solutions, add 0.9 mL of anhydrous ethanol, mix well, and place in a dark place for standing. Samples were taken at 0.5 h, 1 h, 2 h, 3 h, 4 h, and 5 h, and the absorbance was measured at a wavelength of 517 nm. The average value of three replicates was calculated and recorded as Aj;

[0108] (3) Take 0.9 mL of distilled water, add 0.9 mL of DPPH ethanol solution, mix well, measure the absorbance at a wavelength of 517 nm, and calculate the average of three parallel measurements, which is recorded as A4;

[0109] (4) Add 0.9 mL of anhydrous ethanol to 0.9 mL of distilled water and mix well. This is used as a blank.

[0110] The DPPH free radical scavenging rate was calculated according to the following formula. The results are shown in Table 4.

[0111]

[0112] Table 4. DPPH free radical scavenging rate

[0113]

[0114] The data in Table 4 illustrate that the free radical scavenging ability of hydroxy α-salidroside reaches its highest value in 0.5 hours, which is more than 4 times that of α-salidroside and β-salidroside.

[0115] Example 10. Tyrosinase activity inhibition experiment

[0116] Materials and reagents are shown in Table 5.

[0117] Table 5. Materials and reagents

[0118] Reagent name Reagent type purity factory Tyrosinase / >1350 u / mg Beijing Coolaibo Technology Co., Ltd. Levodopa / 99％ Shanghai MacLean Biochemical Technology Co., Ltd. Disodium hydrogen phosphate analytically pure / Sinopharm Chemical Reagent Co., Ltd. citric acid analytically pure / Sinopharm Chemical Reagent Co., Ltd. α-Salidroside / / Shandong Henglu Biotechnology Co., Ltd.

[0119] Instrument: 752N UV-visible spectrophotometer, Shanghai Youke Instrument Co., Ltd.

[0120] Solution preparation:

[0121] (1) Preparation of α-salidroside solution: Prepare a 1 mg / mL aqueous solution according to conventional methods.

[0122] (2) Preparation of Hydroxy-α-Salidroside Solution: The freeze-dried solids obtained after separation and purification of the reaction solutions of Examples 1-3 were respectively prepared into 1 mg / mL aqueous solutions according to conventional methods.

[0123] (3) Preparation of disodium hydrogen phosphate-citrate buffer:

[0124] 1) Prepare a 0.2 mol / L sodium hydrogen phosphate and 0.1 mol / L citric acid aqueous solution;

[0125] 2) Take 154.5 mL of sodium hydrogen phosphate aqueous solution and 45.5 mL of citric acid aqueous solution, mix well, and adjust the pH to 6.8 (measure with a pH meter; 0.2 mol / L sodium hydrogen phosphate or 0.1 mol / L citric acid can be used to adjust the pH).

[0126] (4) Preparation of tyrosinase solution: Prepare 100 u / mL tyrosinase solution using the above-mentioned sodium hydrogen phosphate-citrate buffer.

[0127] (5) Preparation of levodopa solution: Prepare 1 mg / mL levodopa solution using the above-mentioned sodium hydrogen phosphate-citrate buffer.

[0128] Experimental plan:

[0129] (1) Take 1 mL of hydroxy-α-salidroside solution and 1 mL of α-salidroside solution, add 0.5 mL of tyrosinase to each, mix well, and incubate in a 37°C water bath for 10 min. Then add 2 mL of levodopa to each solution, control the reaction time to 5 min, and immediately measure the absorbance T at 475 nm.

[0130] (2) Sample background T0: Take 1 mL of hydroxy-α-salidroside solution and 1 mL of α-salidroside solution, add 0.5 mL of sodium hydrogen phosphate-citrate buffer solution, mix well, and incubate in a 37°C water bath for 10 min. Then add 2 mL of levodopa, control the reaction time to 5 min, and immediately measure the absorbance at 475 nm.

[0131] (3) Enzyme reaction tube C: Take 1 mL of sodium hydrogen phosphate-citrate buffer, add 0.5 mL of tyrosinase, mix well, and incubate in a 37°C water bath for 10 min. Then add 2 mL of levodopa, control the reaction time to 5 min, and immediately measure the absorbance at 475 nm.

[0132] (4) Solvent background C0: Take 1.5 mL of sodium hydrogen phosphate-citrate buffer, incubate in a 37°C water bath for 10 min, add 2 mL of levodopa, control the reaction time to 5 min, and immediately measure the absorbance at 475 nm.

[0133] The inhibition rate of tyrosinase activity was calculated according to the following formula, and the results are recorded in Table 6.

[0134]

[0135] Table 6

[0136] sample Inhibition rate of tyrosinase activity, % α-Salidroside 1.01 Example 1 Hydroxy-α-Salidroside 46.3 Example 3 Hydroxy-α-Salidroside 47.0

[0137] The data in Table 6 illustrate that the tyrosinase activity inhibition rate of the hydroxy-α-salidroside solution obtained in Example 1 and Example 3 is 46 times or more than that of α-salidroside.

[0138] Example 11. Experimental study on the effect of hydroxy-α-salidroside on learning and memory in Wistar rats

[0139] Experimental animals: 56 young male Wistar rats, weighing 290-315 g, were randomly divided into 4 groups, 14 rats in each group, and randomly divided into blank control group, AD model group 1, AD model group 2, and AD model group 3.

[0140] Hydroxy-α-salidroside sample solution: According to the method described in Example 5, the substance with rt=6.97 min in the HPLC spectrum of the fermentation broth of Example 4 was collected and prepared into a 10 mg / ml solution.

[0141] α-Salidroside sample solution: α-Salidroside is the raw material for preparing hydroxy-α-Salidroside in the examples, and is prepared into a 10 mg / ml solution.

[0142] Experimental methods

[0143] Modeling: Establishment of AD model in Wistar male rats

[0144] AD model group 1-3 modeling method: Wistar male rats were intraperitoneally injected with 120 mg / kg of D-galactose and 80 mg / kg of sodium nitrite daily for 7 consecutive weeks to complete the modeling; the blank control group was intraperitoneally injected with an equal volume of normal saline.

[0145] The drugs were administered according to the following schedule starting on the second day after the end of the 7th week of modeling and continued for 30 days.

[0146] AD model group 1: Each rat was orally administered with hydroxy-α-salidroside sample solution once a day, with a dose of 5 mg per kg body weight.

[0147] AD model group 3: Each rat was orally gavaged with α-salidroside sample solution once a day, with a dose of 5 mg per kg body weight.

[0148] AD model group 2 and blank control group: rats were gavaged with the sample solution once a day, with the same volume of distilled water as that of AD model group 1.

[0149] 1. Water Maze Experiment

[0150] Starting from the 31st day, the Morris water maze test was performed, and the drug administration was continued according to the above scheme during the test.

[0151] The experimental heated swimming pool was 120 cm in diameter, 50 cm high, 30 cm deep, and maintained at a temperature of (22 ± 1)°C. Two objects were randomly hung on the pool wall as visual cues of close proximity. A built-in platform was 7 cm in diameter and submerged 2 cm below the surface of the water. Several small holes were placed on the platform to provide a stable surface for the rats.

[0152] The environmental information remained constant throughout the experiment. Milk was added to the pool to make the platform invisible. The maze was divided into four equal quadrants, with the platform placed in the center of one quadrant, providing the animals with only one way to escape the water.

[0153] On the first day of the Morris water maze test, rats were placed in a water pool (without a platform) and allowed to swim freely for 2 minutes to familiarize themselves with the environment in the maze.

[0154] Starting from the second day of the Morris water maze test, the platform was placed in a quadrant, and a positioning navigation test was performed, and the escape latency was recorded. The test was repeated for three consecutive days, three times a day, and the entry point was changed each time. The order of the entry points for all rats remained the same every day (clockwise). During the test, care should be taken to keep the platform position fixed. The other three quadrants without the platform were selected as the entry points for the rats, and the rats were gently placed in the water toward the pool wall. 120s was set as the longest escape latency, and the recording stopped automatically after 120s. If the rat found the platform within 120s, its actual escape latency was recorded; if the platform was not found within 120s, the experimenter led it to the platform and stayed there for 10s, and the escape latency was recorded as 120s.

[0155] The water maze escape latency of rats in each group was recorded, and the average daily escape latency of each group is recorded in Table 7.

[0156] Table 7 Results of rat water maze escape latency measurement

[0157]

[0158]

[0159] The Morris water maze test is an experiment that forces experimental animals (rats and mice) to swim and learn to find a platform hidden in the water. It is mainly used to test the learning and memory ability of experimental animals for spatial position and direction (spatial positioning).

[0160] Escape latency: Escape latency is the time it takes an animal to swim from its starting position in the water maze to locate the hidden platform. As the number of trials increases, the time it takes for an animal to find the platform should decrease, indicating progress in learning and remembering the platform's location. Longer escape latency may indicate impaired spatial learning and memory.

[0161] Table 7 Data Description:

[0162] (1) On the 2nd to 4th day of the navigation experiment, the escape latency of the AD1-3 group model rats was longer than that of the blank control group rats (P<0.05), indicating that the AD1-3 group model rats needed a longer time to find the underwater hidden platform.

[0163] (2) The AD model group 1 was the hydroxy-α-salidroside treatment group, and its escape latency was shorter than that of the AD model group 3 (α-salidroside treatment group) and the AD model group 2 (blank model group), indicating that the AD3 group model rats needed a shorter time to find the underwater hidden platform, and that hydroxy-α-salidroside had a certain effect on improving the spatial learning and memory ability of the AD model rats.

[0164] It should be noted that the above examples are only intended to illustrate the technical solutions of the present invention and are not intended to limit the same. Although the present invention has been described in detail with reference to the examples given, those skilled in the art may modify or replace the technical solutions of the present invention as needed without departing from the spirit and scope of the technical solutions of the present invention.

Claims

1. Use of the hydroxy-α-salidroside of formula II in the preparation of a drug for improving learning and memory disorders, wherein the drug for improving learning and memory disorders is a drug for treating Alzheimer's disease.

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