WO2024251002A1 - Salidroside derivative, and preparation method therefor and use thereof - Google Patents

Abstract

The present invention relates to a salidroside derivative, and particularly relates to a hydroxy-α-salidroside compound. The hydroxy-α-salidroside compound is superior to α-salidroside in the aspects of scavenging DPPH free radicals, whitening and ameliorating learning and memory disorders, and can be added as a new functional component to cosmetics, functional foods or pharmaceutical compositions. According to the preparation method for hydroxy-α-salidroside of the present invention, α-salidroside is used as a substrate, and hydroxy-α-salidroside is synthesized under the action of hydroxylase.

Description

Salidroside derivatives, preparation methods and applications

CROSS-REFERENCE TO RELATED APPLICATIONS

The present invention claims priority to Chinese patent application CN202310670529.3, entitled “A derivative of salidroside, preparation method and application”, filed on June 8, 2023, and claims priority to Chinese patent application CN202410159803.5, entitled “Derivatives of salidroside, preparation method and application”, filed on February 24, 2024. The entire contents of the above applications are incorporated herein by reference for all purposes.

Technical Field

The invention relates to a salidroside derivative, in particular to a hydroxy-alpha-salidroside compound.

Background Art

The information disclosed in this background technology section is only intended to enhance the understanding of the overall background of the invention, and should not necessarily be regarded as an admission or any form of suggestion that the information constitutes the prior art already known to a person skilled in the art.

Rhodiola rosea is a precious 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. Drugs 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.

Chinese patent 202211359878.5 discloses an α-salidroside and its preparation method and application. The α-salidroside has a chemical name: 2-(4-hydroxyphenyl)ethyl-α-D-glucoside, a molecular weight of 300.23, and a chemical formula as follows. It has excellent free radical scavenging activity and can be used as the main raw material for cosmetics, anti-fatigue health products and other products.

Summary of the invention

Purpose of the invention: In order to meet the demand for product iteration, the applicant studied salidroside and its derivatives and obtained hydroxy-α-salidroside. In addition, the present invention also provides a preparation method and application of hydroxy-α-salidroside.

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

Technical solution:

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

The molecular weight of the hydroxy-α-salidroside of the present invention is 316.108 after LC-MS analysis.

The H-NMR spectrum of the hydroxy-α-salidroside described in the present invention: 1HNMR (600MHz, 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=3.6Hz, 1H:β-H), 4.73 (d, 1H), 4.83 (d, 1H), 8.64 (s, 1H:OH), 8.69 (s, 1H:OH).

The C-NMR spectrum of the hydroxy-α-salidroside: 13C NMR (151 MHz, 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.

In a second aspect, the present invention provides a method for preparing the hydroxy-α-salidroside compound.

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

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

Further preferably, the commercially available hydroxylase enzyme preparations are derived from Merck & Co., Ltd., Shanghai McLean Biochemical Technology Co., Ltd. and Shanghai Yuanye Biotechnology Co., Ltd., including but not limited to monophenol monooxygenase (Monophenol monooxygenase), polyphenol oxidase (Polyphenol Oxidase), tyrosinase (Tyrosinase) and the like.

Preferably, a surfactant that does not affect the enzymatic reaction, such as polysorbate (Tween-20) or sorbitan fatty acid ester (Span), may be added to the reaction system.

In the present invention, the preparation of 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.

Preferably, the reaction system contains ammonium salt.

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

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

In a third aspect, the present invention provides the use of the hydroxy-α-salidroside compound in the preparation of a product having a free radical scavenging function; or the use of the hydroxy-α-salidroside compound in cosmetics having a whitening function.

Preferably, the free radical is a 1,1-diphenyl-2-trinitrophenylhydrazine (DPPH) free radical.

In the application, preferably, the product is a cosmetic having the function of scavenging free radicals.

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

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

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

Preferably, the free radical is a DPPH free radical.

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.

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

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

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

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

Learning and memory disorders are a group of symptoms that may occur in many diseases. There are many causes of learning and memory disorders, such as neurodegenerative diseases including Alzheimer's disease and Parkinson's disease, stress, aging, cerebral ischemic damage, brain trauma, hypertension, diabetes, menopausal syndrome and depression. Among them, Alzheimer's disease is the most common learning and memory disorder disease. Clinical drugs can only alleviate symptoms, but cannot delay or terminate its progress. The efficacy produced in different patients is inconsistent and there are many adverse reactions. In addition, people's work and life pace is accelerated, competitive pressure is increased, and they are in a state of chronic stress for a long time, which leads to imbalance of body coordination and functional disorders, especially affecting the cognitive function of the brain, leading to a decline in learning and memory ability. Therefore, there is an urgent need for drugs to improve learning and memory disorders. Studies have found that the hydroxy-α-salidroside described in the present invention has the effect of improving the learning and memory level of model mice, which is manifested in that it can significantly improve the relative discrimination index of rats for objects in new locations, shorten the platform-finding latency in a water maze experiment, increase the number of platform crossings, and reduce the number of errors and dark room time in a dark avoidance experiment, indicating that hydroxy-α-salidroside can better improve learning and memory disorders.

In a fifth aspect, a method for scavenging free radicals is provided, comprising applying an effective amount of hydroxy-α-salidroside or a composition comprising an effective amount of hydroxy-α-salidroside to a target area or a subject. The free radical is a DPPH free radical.

In a sixth aspect, a method for whitening skin is provided, comprising applying an effective amount of hydroxy-α-salidroside or a composition comprising an effective amount of hydroxy-α-salidroside to the skin.

In a seventh aspect, a method for improving learning and memory disorders is provided, comprising administering to a subject an effective amount of hydroxy-α-salidroside or a composition comprising an effective amount of hydroxy-α-salidroside.

The "subject" of the present invention refers to any organism that needs treatment, prevention or diagnosis, preferably a mammal, and more preferably a human.

The "effective amount" of the present invention refers to the amount of the compound sufficient to achieve the desired therapeutic, preventive or diagnostic effect. The amount can be adjusted according to the age, weight, health status, gender and specific therapeutic, preventive or diagnostic purpose of the subject. The effective amount can be measured by conventional pharmacological test methods, in vitro tests, animal model tests or clinical trials. Usually, the effective amount is in the range of about 0.01 mg/kg to 1000 mg/kg body weight, more preferably in the range of about 0.1 mg/kg to 100 mg/kg body weight.

Beneficial effects:

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 into cosmetics or health products as a new functional ingredient; the hydroxy-α-salidroside also has the excellent performance of improving the learning and memory level of model mice, and can be used for the development and application of preparing Alzheimer's disease drugs or Parkinson's disease drugs.

BRIEF DESCRIPTION OF THE DRAWINGS

The accompanying drawings constituting a part of the present invention are used to provide a further understanding of the present invention. The exemplary embodiments of the present invention and their descriptions are used to explain the present invention and do not constitute an improper limitation of the present invention. The embodiments of the present invention are described in detail below in conjunction with the accompanying drawings, wherein:

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.

Figure 2: HPLC spectrum of the enzyme-catalyzed reaction solution of Example 1.

Figure 3: LC-MS spectrum of the substance at rt = 6.97 min in the HPLC spectrum of the enzyme-catalyzed reaction liquid of Example 1.

Figure 4: H-NMR spectrum of the substance at rt = 6.97 min in the HPLC spectrum of the enzyme-catalyzed reaction solution of Example 1.

Figure 5: C-NMR spectrum of the substance at rt = 6.97 min in the HPLC spectrum of the enzyme-catalyzed reaction solution of Example 1.

DETAILED DESCRIPTION

The present invention will be further described below in conjunction with specific examples. It should be understood that these examples are only used to illustrate the present invention and are not intended to limit the scope of the present invention. The experimental methods in the following examples without specifying specific conditions are usually carried out under conventional conditions or under conditions recommended by the manufacturer.

Unless otherwise defined, all professional and scientific terms used herein have the same meanings as those familiar to those skilled in the art. The reagents or raw materials used in the present invention can be purchased through conventional channels. Unless otherwise specified, the reagents or raw materials used in the present invention are used in a conventional manner in the art or in accordance with the product instructions. In addition, any method and material similar to or equivalent to the described content can be applied to the method of the present invention. The preferred implementation methods and materials described in the text are for demonstration purposes only.

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 I.

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.

The hydroxylase may be a commercially available hydroxylase preparation 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 and the like.

The method of using the hydroxylase enzyme preparation includes adding it to the reaction system in the form of powder or liquid, or 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.

The preparation method of hydroxy-α-salidroside of the present invention is to add hydroxylase to α-salidroside phosphate buffer to carry out hydroxylase reaction. Surfactants that do not affect the enzymatic reaction, such as Tween-20 and Span, can be added to the enzyme reaction solution.

When using the above-mentioned hydroxylase to prepare hydroxy-α-salidroside compounds, the amount of hydroxylase and the 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.

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

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

The mixture containing the hydroxy-α-salidroside compound, the hydrolyzate of the mixture, the enzyme-inactivated product of the mixture, the purified product of the mixture, and the dry powder product thereof can be used in foods, beverages and cosmetics containing the hydroxy-α-salidroside compound, and can also be used as an ingredient of food, special medical food, health products or medicines, and used in the production of food and beverages, cosmetics, special medical food, health products or medicines.

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

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

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.

The document "Enzymatic routes for the production of mono-and di-glucosylated derivatives of hydroxytyrosol", Antonio Trincone et al. "Bioresource Technology", Vol. 115, is an article on "Enzymatic production of mono- and di-glucosylated derivatives of hydroxytyrosol". By adding the visceral material homogenate from Aplysia.fasciata to 1.5 mL of 10 mM receptor solution in 50 mM potassium acetate buffer (pH 5.5, containing 150 mM maltose), (3,4-dihydroxyphenyl)ethyl-α-D-pyranoglucoside compound was obtained, and the target compound was obtained by inferring 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. There is no chemical shift data for the key hydrogen atom of α-glucoside, the chemical structure of the specific compound is not disclosed, and the stereo configuration of the compound cannot be confirmed.

The inventors of the present invention carried out acetylation treatment on the hydroxy-α-salidroside compound of the present invention according to the method described in the document, and then carried out carbon spectrum and hydrogen spectrum analysis, but could not obtain the carbon spectrum and hydrogen spectrum data of the acetylated product and its supplementary document. Therefore, the hydroxy-α-salidroside compound obtained by the present invention is different from the compound obtained in the document.

Example 1. Tyrosinase hydroxylation experiment

Take 500 mg of α-salidroside (molecular weight 300.23), add 50 mL of 0.2 M phosphate buffer (and adjust the pH to 10), add 500 mg of ascorbic acid, and then add 3 mg of tyrosinase (T828256-100KU, McLean), add water to make the volume to 100 mL, stir the reaction at 25°C, react for 24 hours, and obtain an enzyme-catalyzed reaction solution.

Example 2. Polyphenol oxidase hydroxylation experiment

Take 100 mg of α-salidroside (molecular weight 300.23), add 50 mL of 0.2 M phosphate buffer (and adjust the pH to 10), add 100 mg of ascorbic acid, and then add 3 mg of tyrosinase (S10171-100KU, Shanghai Yuanye Biotechnology Co., Ltd.), add water to make the volume to 100 mL, stir the reaction at 15°C, and react for 50 hours to obtain an enzyme-catalyzed reaction solution.

Example 3. Monophenol monooxygenase hydroxylation experiment

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 the reaction at 35°C, react for 24 hours, and obtain an enzyme-catalyzed reaction solution.

Example 4. Monophenol monooxygenase hydroxylation experiment

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

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

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

(1) TLC analysis

The TLC analysis spectrum of the reaction solution of Example 1 is shown in Figure 1, which shows that new substances are produced in the enzyme-catalyzed reaction solution. In the figure, from left to right are lane 1 and lane 2; lane 1 is α-salidroside, and lane 2 is the new substance produced by the enzyme-catalyzed reaction.

(2) HPLC analysis

According to the above chromatographic conditions, the purified reaction solution of Example 1 was subjected to HPLC analysis. The HPLC spectrum is shown in FIG2 .

The HPLC spectrum shown in FIG2 shows that the reaction solution after purification in Example 1 mainly has two peaks, and the elution times are rt=6.97 min and rt=9.98 min, respectively. The peak at rt=9.98 min is α-salidroside, and the elution time of the peak at rt=6.97 min is significantly different from that of α-salidroside.

(3) LC-MS analysis

The substance at rt=6.97 min in the HPLC spectrum of the reaction solution in Example 1 was analyzed by LC-MS, and the measured value of the molecular ion peak [M-1] was 315.108. The LC-MS spectrum is shown in FIG3 .

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

(4) Separation and purification

The enzyme catalytic reaction solution obtained in Example 1-4 was separated and purified by HPLC, and the material with rt=6.97min was collected, wherein the equipment and conditions of the HPLC method included:

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

The collected substances with rt=6.97 min from Examples 1-4 were concentrated and freeze-dried to obtain freeze-dried solids, which were used as the target substances of Examples 1-4 for subsequent experiments.

(5) H-NMR and C-NMR spectrum analysis

The above freeze-dried samples were taken for H-NMR and C-NMR analysis.

The H-NMR spectrum of the substance at rt=6.97 min in the HPLC spectrum of the reaction solution of Example 1 is shown in FIG4 , and the C-NMR spectrum is shown in FIG5 .

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).

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.

According to the H-NMR spectrum and C-NMR spectrum of the product in 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 in Example 1 has two single peaks (s, 1H: OH) at 8.64ppm and 8.69ppm, which is the "characteristic of bisphenol hydroxyl". α-Salidroside is a single peak at 9.10ppm, which is the "characteristic of monophenol hydroxyl", and the C-NMR spectrum of the product in Example 1 has 14 characteristic peaks, while α-Salidroside has only 12 characteristic peaks, which are obviously different.

In summary, it can be seen from the TLC spectrum and HPLC spectrum 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.

The HPLC spectrum, LC-MS spectrum, H-NMR spectrum, and C-NMR spectrum of the product of the enzyme-catalyzed reaction of Example 2-4 are the same as those of the reaction solution of Example 1. Therefore, the substance obtained by the enzyme-catalyzed reaction of Example 2-4 is also hydroxy-α-salidroside.

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

Table 1

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:

500 mg of α-salidroside (molecular weight 300.23) was taken, added with 50 mL of 0.2 M phosphate buffer (pH 9), 500 mg of ascorbic acid, 10 mL of 0.2 M ammonium salt (specific ammonium salt varieties are shown in Table 2), and then 1.5 g of monophenol monooxygenase (T3824-50KU, Merck) was added, and water was added to make the volume to 100 mL. The mixture 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.

Table 2

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.

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

Table 3. Materials and reagents

DPPH free radical scavenging experiment: Free radicals directly or indirectly play a strong oxidative role and are widely involved in the physiological and pathological processes of the body. When there are excessive free radicals in the body, they can damage the body through oxidation. Salvianolic acid compounds are donors of phenolic hydroxyl groups and have a structural basis for antioxidant activity. This experiment uses the DPPH free radical scavenging reaction to study the scavenging efficiency of hydroxy-α-salidroside, α-salidroside, and β-salidroside on DPPH free radicals.

Solution preparation:

(1) Preparation of Hydroxy-α-Salidroside Solution: The target substances of Examples 1-3 were prepared respectively according to the method of Example 5, and were prepared into 500 mg/L aqueous solutions respectively according to conventional methods.

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

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

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

2.DPPH free radical scavenging experiment:

(1) Take 0.9 mL of hydroxy-α-salidroside, α-salidroside, and β-salidroside aqueous solutions respectively, add 0.9 mL of DPPH anhydrous ethanol solution respectively, mix well, place in a dark place and stand, take samples at 0.5 h, 1 h, 2 h, 3 h, 4 h, and 5 h, measure the absorbance at a wavelength of 517 nm, and calculate the average value of three parallel solutions, which is recorded as Ai.

(2) Take 0.9 mL of hydroxy-α-salidroside, α-salidroside, and β-salidroside aqueous solutions respectively, add 0.9 mL of anhydrous ethanol respectively, mix well, and place in a dark place to stand. Samples are taken at 0.5 h, 1 h, 2 h, 3 h, 4 h, and 5 h, and the absorbance is measured at a wavelength of 517 nm. The average value of three parallel solutions is calculated and recorded as Aj.

(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 value of three parallel measurements, which is recorded as A4.

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

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

Table 4. DPPH free radical scavenging rate

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

Example 10. Tyrosinase activity inhibition experiment

Materials and reagents are shown in Table 5.

Table 5. Materials and reagents

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

Solution preparation:

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

(2) Preparation of Hydroxy-α-Salidroside Solution: The target substances of Examples 1-3 were prepared respectively according to the method of Example 5, and were prepared into 1 mg/mL aqueous solutions respectively according to conventional methods.

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

1) Prepare 0.2 mol/L disodium hydrogen phosphate and 0.1 mol/L citric acid aqueous solution.

2) Take 154.5 mL of sodium dihydrogen phosphate aqueous solution and 45.5 mL of citric acid aqueous solution, mix well, and adjust the pH to 6.8 (measured by a pH meter, the pH value can be adjusted with 0.2 mol/L of sodium dihydrogen phosphate or 0.1 mol/L of citric acid).

(4) Preparation of tyrosinase solution: Use the above-mentioned sodium hydrogen phosphate-citrate buffer to prepare a 100 u/mL tyrosinase solution.

(5) Preparation of levodopa solution: Use the above sodium hydrogen phosphate-citrate buffer to prepare a 1 mg/mL levodopa solution.

Experimental protocol:

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

(2) Sample background T ₀ : Take 1 mL of hydroxy-α-salidroside solution and 1 mL of α-salidroside, add 0.5 mL of sodium dihydrogen phosphate-citrate buffer, mix well, incubate in a 37°C water bath for 10 min, then add 2 mL of L-dopa, control the reaction time to 5 min, and immediately measure the absorbance at 475 nm.

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

(4) Solvent background C ₀ : Take 1.5 mL of sodium hydrogen phosphate-citrate buffer, incubate in a 37°C water bath for 10 min, add 2 mL of L-DOPA, control the reaction time to 5 min, and immediately measure the absorbance at 475 nm.

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

Table 6

Data in Table 6 illustrate: 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.

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

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.

Hydroxy-α-salidroside sample solution: The target substance of Example 4 was prepared according to the method described in Example 5, and prepared into a 10 mg/mL solution.

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

Experimental methods

Modeling: Establishment of AD model in Wistar male rats

AD model group 1-3 modeling method: Wistar male rats were intraperitoneally injected with 120 mg/kg D-galactose and 80 mg/kg 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.

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

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

AD model group 3: Each rat was intragastrically administered with α-salidroside sample solution once a day, with a dose of 5 mg per kg body weight.

AD model group 2 and blank control group: each rat was intragastrically administered with the sample solution once a day, with the same volume of distilled water as that of the AD model group 1.

1. Water maze experiment

Starting from the 31st day, Morris water maze test was performed, and medication was continued according to the above scheme during the test.

The constant temperature swimming pool used in the experiment was 120 cm in diameter and 50 cm in height, with a water depth of 30 cm and a water temperature maintained at (22±1)℃. Two objects were randomly hung on the wall of the pool as close visual cues. The built-in platform was 7 cm in diameter and was submerged 2 cm under water. There were several small holes on it to provide a surface for rats to stand easily.

The environment information was kept fixed throughout the experiment. Milk was added to the pool to make the platform invisible. The maze was divided into 4 quadrants, and the platform was placed in the center of one of the quadrants as the only way for the animals to escape the water environment in the experiment.

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.

Starting from the second day of the Morris water maze test, the platform was placed in a quadrant, and the positioning navigation test was performed, and the escape latency was recorded. The test was repeated for 3 consecutive days, 3 times a day, and the entry point was changed each time. The order of the entry points for all rats was kept consistent every day (clockwise). During the test, care should be taken to keep the platform position fixed, and the other 3 quadrants without the platform were selected as the entry points for the rats. 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 for 10s, and the escape latency was recorded as 120s.

The water maze escape latency of rats in each group was recorded, and the average escape latency of each group per day is recorded in Table 7.

Table 7 Results of rat water maze escape latency measurement

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).

Escape latency: Escape latency refers to the time it takes for an animal to swim from the starting position in the water maze to find the hidden platform. As the number of experiments increases, the time it takes for the animal to find the platform should gradually decrease, indicating that they have made progress in learning and remembering the location of the platform. A longer escape latency may indicate that the animal's spatial learning and memory abilities are impaired.

Table 7 Data Description:

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

(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 AD model rats in the AD3 group 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.

It should be noted that the above examples are only used to illustrate the technical solution of the present invention rather than to limit it. Although the present invention is described in detail with reference to the given examples, those skilled in the art may modify or replace the technical solution of the present invention as needed without departing from the spirit and scope of the technical solution of the present invention.

Claims (10)

A hydroxy-α-salidroside, chemically named: 2-(3,4-dihydroxyphenyl)ethyl-α-D-glucoside, with a structural formula as shown in Formula I,
The use of hydroxy-α-salidroside as described in claim 1 in the preparation of a product having the function of scavenging free radicals; Preferably, the free radical is a DPPH free radical, and the product is a cosmetic or a functional food. The use of hydroxy-α-salidroside as described in claim 1 in the preparation of products with whitening function. The use of hydroxy-α-salidroside as claimed in claim 1 in the preparation of a medicine for improving learning and memory disorders; Preferably, the medicine for improving learning and memory disorders includes but is not limited to medicine for treating Alzheimer's disease; Preferably, the medicines for improving learning and memory disorders include but are not limited to medicines for treating Parkinson's disease. A composition, characterized in that it contains the hydroxy-α-salidroside according to claim 1. Use of the composition as claimed in claim 5 in scavenging free radicals. Use of the composition as claimed in claim 5 in the preparation of whitening cosmetics. Use of the composition of claim 5 in the preparation of a medicine for improving learning and memory disorders; Preferably, the medicine for improving learning and memory disorders includes but is not limited to medicine for treating Alzheimer's disease; Preferably, the medicines for improving learning and memory disorders include but are not limited to medicines for treating Parkinson's disease. The method for preparing hydroxy-α-salidroside according to claim 1, characterized in that hydroxylase is added to a solution containing α-salidroside to carry out a reaction; Preferably, the hydroxylase is selected from monophenol monooxygenase, polyphenol oxidase or tyrosinase. The preparation method according to claim 9, characterized in that an ammonium salt is also added in the reaction; Preferably, the ammonium salt is ammonium chloride, ammonium acetate or ammonium sulfate.