Active naphthoside components in sargentcraibia root, extraction method and application thereof

Abstract

Active naphthoside components in roots of atractylodes lancea, extraction method and application. The present application belongs to the technical field of plant separation and extraction, medicine and daily chemical products, and particularly relates to active naphthoside components in roots of atractylodes lancea, an extraction method and application thereof. The present application is aimed at solving the problem that the compounds with whitening effect in the roots of atractylodes lancea are not clear. The extraction method comprises the following steps: drying and crushing the roots of atractylodes lancea, extracting by using an ethanol reflux method, obtaining a concentrated solution after concentration, separating the concentrated solution by using macroporous resin adsorption chromatography, ODS column chromatography and preparative liquid chromatography to obtain naphthoside components, and the naphthoside components are 5-hydroxydianellose, 5-hydroxydianellin, dianellose and dianellin, which are used as active components to prepare whitening and freckle-removing cosmetics and pharmaceutical preparations. The extraction and separation method provided by the present application is simple, rapid, and the extracted compounds have high purity, and the extracted compounds can effectively scavenge DPPH free radicals and inhibit tyrosinase activity, and have good application prospect in whitening and freckle removal.

Description

Technical Field

[0001] This invention belongs to the fields of plant isolation and extraction, medicine and daily chemical products, and particularly relates to an active naphthyl glycoside component in the root of Ligusticum striatum, its extraction method and its application. Background Technology

[0002] Dianella ensifolia (L.) DC., a plant belonging to the genus Dianella in the Liliaceae family, is the dried root of a plant commonly used in traditional Chinese medicine by the Lahu ethnic group. It is also known as Shanjianlan, Shanjiaojian, and Shanmaoer. It is mainly produced in Yunnan, Guangdong, Guizhou, Sichuan, Guangxi, and Fujian provinces of my country, and is also distributed in tropical Asian regions. It has a sweet and pungent taste, and is cool in nature, possessing detoxifying, swelling-reducing, and analgesic properties. In folk medicine, it is ground into powder, mixed with a suitable amount of vinegar, and applied externally to treat boils, abscesses, tinea, lymphadenitis, and other diseases. Currently, research on the chemical composition of Dianella ensifolia is not in-depth. Its main structural types include flavanones, chromones, naphthyl glycosides, and resorcinol, exhibiting whitening, anti-tumor, and antioxidant bioactivities, but its whitening components are not clearly identified.

[0003] In recent years, frequent safety incidents involving chemically synthesized cosmetics have prompted consumers to increasingly favor all-natural plant-based cosmetics. Traditional Chinese medicine (TCM) has gained widespread consumer recognition and popularity due to its gentle efficacy and low risk of adverse reactions. However, my country boasts extremely rich TCM resources, with different types of herbs exhibiting varying medicinal properties and each containing multiple chemical components. These characteristics present significant challenges to the development of natural herbal whitening cosmetics that combine high efficacy and safety. From the precise extraction of active ingredients to ensuring product safety and efficacy, numerous difficulties exist.

[0004] Beauty is an innate pursuit for all people, especially women. However, most whitening skincare products on the market currently rely on chemical sunscreens, bleaching agents, or exfoliating agents to remove imperfections. These products often have limited effectiveness, are prone to relapse, and have certain side effects. This critical problem has long troubled those who value beauty. The depth of human skin color depends primarily on the content and distribution of melanin. Inhibiting melanin production can achieve skin whitening. Common experimental methods include experiments on inhibiting tyrosinase activity and experiments on the antioxidant effects of DPPH free radicals.

[0005] Based on existing literature, the compounds in *Ligustrum lucidum* root with whitening effects are not clearly identified. This invention has discovered naphthyl glycoside compounds in *Ligustrum lucidum* root that possess whitening effects. Summary of the Invention

[0006] The purpose of this invention is to solve the technical problem that the whitening compounds in the root of Ligustrum lucidum are not clearly identified, and to provide an active naphthyl glycoside component in the root of Ligustrum lucidum, its extraction method and its application.

[0007] The molecular formula of the active naphthyl glycosides in the root of *Ligustrum lucidum* is C2.24 H 30 O 13 Molecular weight 526, ESI-MS (negative): m / z 525.2 [MH] - The structural formula is as follows:

[0008] .

[0009] The molecular formula of the active naphthyl glycosides in the root of *Ligustrum lucidum* is C2. 24 H 30 O 12 Molecular weight 510, ESI-MS (negative): m / z 508.8 [MH] - The structural formula is as follows:

[0010] .

[0011] The extraction method of active naphthyl glycosides from the root of *Ligustrum lucidum* is as follows:

[0012] 1. After drying and pulverizing the root of *Ligustrum lucidum*, extract it using the reflux method with 70% ethanol to obtain a concentrated solution. After concentrating and drying the concentrated solution, crude extract of *Ligustrum lucidum* root is obtained.

[0013] 2. The crude extract of *Ligustrum lucidum* root was dissolved in water to obtain *Ligustrum lucidum* root solution. The *Ligustrum lucidum* root solution was loaded onto a D101 macroporous resin adsorption column and eluted sequentially with water, 15% ethanol aqueous solution, 30% ethanol aqueous solution, 60% ethanol aqueous solution, and 90% ethanol aqueous solution to obtain eluents of different concentrations of ethanol aqueous solution.

[0014] 3. The 60% ethanol-water eluent obtained in step 2 was concentrated and evaporated to dryness. Separation was performed using ODS column chromatography, with 20% methanol-water solution, 40% methanol-water solution, 60% methanol-water solution, 70% methanol-water solution and 90% methanol-water solution eluted sequentially at a flow rate of 10-20 mL / min to obtain methanol-water eluent fractions of different concentrations.

[0015] IV. The fractions eluted with 40% methanol and water and 60% ethanol and water were concentrated and evaporated to dryness, then separated by liquid chromatography. Compound 1 (5-hydroxydianellose) 1,5-dihydroxy-2-acetyl-3-methylnaphthalene-8-O-[α-L-xylosyl-(1→6)]-β-D-glucoside with a retention time of 15.5 min and compound 2 (5-hydroxydianellin) 1,5-dihydroxy-2-acetyl-3-methylnaphthalene-8-O-[α-L-rhamnosyl-(1→6)]-β-D-glucoside with a retention time of 16.5 min were separated from the 40% methanol and water eluent. Compound 3 (dianellose) with a retention time of 22.5 min was separated from the 60% methanol and water eluent. 1-Hydroxy-2-acetyl-3-methylnaphthalene-8-O-[α-L-xylosyl-(1→6)]-β-D-glucoside and compound 4 (dianellin) with a retention time of 23.5 min;

[0016] The molecular formula of 5-hydroxydianellose is C 24 H 30 O 13 Molecular weight 526, ESI-MS (negative): m / z 525.2 [MH] - The structural formula is as follows:

[0017] ;

[0018] The molecular formula of 5-Hydroxydianellin is C 25 H 32 O 13 Molecular weight 540, ESI-MS (negative): m / z 539.3 [MH] - The structural formula is as follows:

[0019] ;

[0020] The molecular formula of Dianellose is C 24 H 30 O 12 Molecular weight 510, ESI-MS (negative): m / z 508.8 [MH] - The structural formula is as follows:

[0021] ;

[0022] The molecular formula of Dianellin is C 25 H 32 O 12 Molecular weight 524, ESI-MS (negative): m / z 523.0 [MH] - The structural formula is as follows:

[0023] .

[0024] The 70% ethanol reflux method described in step one is as follows: After drying and pulverizing the root of *Ligustrum lucidum*, add 5 to 10 times its volume of 70 to 95% ethanol, reflux for 2 to 3 hours, filter while hot to remove the residue, extract 3 times, combine the filtrates and concentrate under reduced pressure at 45 to 55°C to obtain the concentrate.

[0025] The liquid chromatography described in step four was performed using a Waters 2545 high-performance liquid chromatograph with a Waters 2489 UV detector. The column packing material was C18 reversed-phase silica gel. The mobile phase consisted of a mixture of acetonitrile and water (volume ratio 22:78) and an aqueous methanol-water mixture (volume ratio 40:60). The flow rate was 10 ml / min, and the detection wavelength was 224 nm.

[0026] The active naphthyl glycosides in the root of *Ligustrum lucidum* are used as active ingredients in the preparation of whitening and freckle-removing cosmetics.

[0027] The whitening and freckle-removing cosmetics also include one or more of the following: moisturizers, antioxidants, surfactants, and thickeners.

[0028] The whitening and freckle-removing cosmetics are available in the form of water, lotion, cream, ointment, gel, or mask.

[0029] The active naphthyl glycosides in the root of *Ligustrum lucidum* are used as active ingredients in the preparation of pharmaceutical formulations.

[0030] The pharmaceutical preparation also includes one or more of stearic acid, cetyl alcohol, castor oil, triethanolamine, and propylene glycol.

[0031] The pharmaceutical preparation is a cream, granules, tablets, capsules, pills, or oral liquid.

[0032] This invention extracts four naphthyl glycosides with whitening activity from the root of *Ligustrum lucidum*. These four naphthyl glycosides exhibit DPPH free radical scavenging and tyrosinase inhibition activities, making them suitable as natural plant-based whitening agents for use in various whitening cosmetics. They show promising application prospects in whitening and freckle removal. The preparation method is rapid and simple, and the product has high purity. Attached Figure Description

[0033] Figure 1A schematic diagram of the extraction and separation process of active naphthyl glycosides from the root of Ligusticum striatum provided by the present invention;

[0034] Figure 2 The MS spectrum of compound 1 (5-hydroxydianellose) prepared in Example 1 of this invention;

[0035] Figure 3 Compound 1 (5-hydroxydianellose) prepared in Example 1 of this invention 1 H-NMR spectrum;

[0036] Figure 4 Compound 1 (5-hydroxydianellose) prepared in Example 1 of this invention 13 C-NMR spectrum;

[0037] Figure 5 The MS spectrum of compound 2 (5-hydroxydianellin) prepared in Example 1 of this invention;

[0038] Figure 6 Compound 2 (5-hydroxydianellin) prepared in Example 1 of this invention 1 H-NMR spectrum;

[0039] Figure 7 Compound 2 (5-hydroxydianellin) prepared in Example 1 of this invention 13 C-NMR spectrum;

[0040] Figure 8 The MS spectrum of compound 3 (dianellose) prepared in Example 1 of this invention;

[0041] Figure 9 Compound 3 (dianellose) prepared in Example 1 of this invention 1 H-NMR spectrum;

[0042] Figure 10 Compound 3 (dianellose) prepared in Example 1 of this invention 13 C-NMR spectrum;

[0043] Figure 11 The MS spectrum of compound 4 (dianellin) prepared in Example 1 of this invention;

[0044] Figure 12 Compound 4 (dianellin) prepared in Example 1 of this invention 1 H-NMR spectrum;

[0045] Figure 13 Compound 4 (dianellin) prepared in Example 1 of this invention 13 C-NMR spectrum. Detailed Implementation

[0046] Example 1:

[0047] The extraction method of active naphthyl glycosides from the root of *Ligustrum lucidum* is as follows:

[0048] 1. Take 1000g of dried *Ligustrum lucidum* root, crush it, put it into a round-bottom flask, add 10 times the volume of 70% ethanol aqueous solution, reflux for extraction, extract for 2 hours, extract 3 times, concentrate the filtrate under reduced pressure at 45-55℃ and evaporate to dryness to obtain crude extract.

[0049] 2. After the crude extract is dissolved in water, it is loaded onto a D101 macroporous resin column and eluted with water, 15% ethanol aqueous solution, 30% ethanol aqueous solution, 60% ethanol aqueous solution and 90% ethanol aqueous solution respectively. The 60% ethanol eluent is concentrated under reduced pressure and then evaporated to dryness to obtain the 60% ethanol eluent fraction of the Chinese privet macroporous resin.

[0050] 3. The 60% ethanol elution fraction of the macroporous resin of *Ligustrum lucidum* was separated by ODS column chromatography and eluted sequentially with methanol-water solutions of 20%, 40%, 60%, 70%, and 90% (v / v) at a flow rate of 15 mL / min to obtain methanol-water elution fractions of different concentrations.

[0051] IV. The fraction eluted with 40% methanol and water was then eluted by high-performance liquid chromatography (C18 column; methanol to water volume ratio 40:60; flow rate 10.0 mL / min; UV detection wavelength 224 nm) to obtain compound 1 (t R 15.5 min; 115.4 mg) and compound 2 (t R 16.5 min; 525.8 mg), with a purity of over 93%;

[0052] The 60% methanol eluent fraction was then eluted by high-performance liquid chromatography (HPLC) (C18 column; acetonitrile to water volume ratio 22:78; flow rate 10.0 mL / min; UV detection wavelength 224 nm) to obtain compound 3 (t R 22.5 min; 865.4 mg) and compound 4 (t R 23.5 min; 6238.5 mg), with a purity of over 95%.

[0053] Example 2: Preparation of Cream

[0054] Weigh the following raw materials (unit: g): 1g of 5-hydroxydianellose or 5-hydroxydianellin or dianellose or dianellin, 9g of stearic acid, 4.5g of hexadecyl alcohol, 12mL of castor oil, 1mL of triethanolamine, 20mL of propylene glycol, and 56mL of water. First, mix the stearic acid, hexadecyl alcohol, and castor oil thoroughly and grind continuously in a mortar to obtain the oil phase. Dissolve the 5-hydroxydianellose or 5-hydroxydianellin or dianellose or dianellin in propylene glycol. Then, add 56mL of water and 1mL of triethanolamine respectively and heat to 80℃ to dissolve, obtaining the aqueous phase. Add the aqueous phase to the oil phase and grind continuously in a mortar until emulsification is complete, then cool.

[0055] Example 3: Determination of DPPH free radical scavenging activity

[0056] Add 100 μL of sample to a 96-well plate, add 100 μL of DPPH and mix well. Incubate at room temperature in the dark for 30 min. Measure the absorbance at 517 nm using a microplate reader; this absorbance is A. 样品 100 μL of methanol solution (methanol concentration 80 μg / mL) and 100 μL of sample were placed in a 96-well plate and the absorbance was measured to be A. 空白 100 μL of methanol solution and 100 μL of DPPH were placed in a 96-well plate, and the absorbance was measured to be A. 对照 Three parallel samples were prepared for each concentration. The clearance rate was calculated, and the IC50 was determined. 50 Value. The algorithm for the clearance rate (%) is as follows:

[0057] Clearance rate (%) = [1 - (A) 样品 - A 空白 ) / A 对照 ] × 100%

[0058] Example 4: Tyrosinase inhibitory activity

[0059] The tyrosinase (T828256, MACKLIN) used in the activity assay was derived from mushrooms, and the method employed was standard. First, the tyrosinase was diluted to 125 U / mL using phosphate buffer (pH=7.4) and incubated at 37°C for 30 min. Then, 80 μL of sample (125 μM) was added to a 96-well plate, along with 80 μL of tyrosinase solution (125 U / mL) and 80 μL of levodopa (2 mM). The plate was then incubated at 37°C for 5 min, and the absorbance was measured at 475 nm using a microplate reader; this absorbance is the A value. 反应组 80 μL of tyrosinase solution, 80 μL of PBS (pH=7.4), and 80 μL of sample (125 μM) were placed in a 96-well plate, and the absorbance was measured as A. 反应对照 80 μL of tyrosinase solution, 80 μL of PBS (pH=7.4) and 80 μL of levodopa were placed in a 96-well plate, and the absorbance was measured to be A. 空白 160 μL of PBS (pH=7.4) and 80 μL of levodopa were placed in a 96-well plate, and the absorbance was measured to be A. 空白对照 Assuming three parallel samples, the activity inhibition rate (%) is calculated using the following algorithm:

[0060] Inhibition rate = [1 – (A)] 反应组 - A 反应对照组 ) / (A 空白 -A 空白对照 )]×100%

[0061] Table 1. DPPH free radical scavenging activity and tyrosinase inhibitory activity

[0062]

[0063] Note 1): The test concentration was 125 μM. Kojic acid was a positive drug in the tyrosinase inhibition test, and vitamin C was a positive drug in the DPPH clearance test.

[0064] The results in Table 1 show that the four naphthyl glycosides exhibited strong DPPH scavenging activity and tyrosinase inhibitory activity, indicating that 5-hydroxydianellose, 5-hydroxydianellin, dianellose, and dianellin have good whitening effects.

Claims

1. A method for extracting active naphthyl glycosides from the root of Ligusticum striatum, characterized in that... The extraction method of active naphthyl glycosides from the root of *Ligustrum lucidum* is as follows:

1. After drying and pulverizing the root of *Ligustrum lucidum*, extract it using the reflux method with 70% ethanol to obtain a concentrated solution. After concentrating and drying the concentrated solution, crude extract of *Ligustrum lucidum* root is obtained.

2. The crude extract of *Ligustrum lucidum* root was dissolved in water to obtain *Ligustrum lucidum* root solution. The *Ligustrum lucidum* root solution was loaded onto a D101 macroporous resin adsorption column and eluted sequentially with water, 15% ethanol aqueous solution, 30% ethanol aqueous solution, 60% ethanol aqueous solution, and 90% ethanol aqueous solution to obtain eluents of different concentrations of ethanol aqueous solution.

3. The 60% ethanol-water eluent obtained in step 2 was concentrated and evaporated to dryness. Separation was performed using ODS column chromatography, with 20% methanol-water solution, 40% methanol-water solution, 60% methanol-water solution, 70% methanol-water solution and 90% methanol-water solution eluted sequentially at a flow rate of 10-20 mL / min to obtain methanol-water eluent fractions of different concentrations. IV. The fractions washed with 40% methanol and water and the fraction washed with 60% ethanol and water were concentrated and evaporated to dryness, and then separated by liquid chromatography. Compound 1 (1,5-dihydroxy-2-acetyl-3-methylnaphthalene-8-O-[α-L-xylosyl-(1→6)]-β-D-glucoside) with a retention time of 15.5 min and compound 2 (1,5-dihydroxy-2-acetyl-3-methylnaphthalene-8-O-[α-L-rhamnosyl-(1→6)]-β-D-glucoside) with a retention time of 16.5 min were separated from the 40% methanol and water fraction. Compound 3 (1-hydroxy-2-acetyl-3-methylnaphthalene-8-O-[α-L-xylosyl-(1→6)]-β-D-glucoside) with a retention time of 22.5 min and compound 4 (23.5 min) with a retention time of 23.5 min were separated from the 60% methanol and water fraction. 1-Hydroxy-2-acetyl-3-methylnaphthalene-8-O-[α-L-rhamnosyl-(1→6)]-β-D-glucoside; Compound 1, 1,5-dihydroxy-2-acetyl-3-methylnaphthalene-8-O-[α-L-xylosyl-(1→6)]-β-D-glucoside, has the molecular formula C0. 24 H 30 O 13 It has a molecular weight of 526 and the following structural formula: ； The molecular formula of compound 2, 1,5-dihydroxy-2-acetyl-3-methylnaphthalene-8-O-[α-L-rhamnosyl-(1→6)]-β-D-glucoside, is C 25 H 32 O 13 Molecular weight 540, ESI-MS (negative): m / z 539.3 [MH] - The structural formula is as follows: ； The molecular formula of compound 3, 1-hydroxy-2-acetyl-3-methylnaphthalene-8-O-[α-L-xylosyl-(1→6)]-β-D-glucoside, is C 24 H 30 O 12 Molecular weight 510, ESI-MS (negative): m / z 508.8 [MH] - The structural formula is as follows: ； Compound 4, 1-hydroxy-2-acetyl-3-methylnaphthalene-8-O-[α-L-rhamnosyl-(1→6)]-β-D-glucoside, has the molecular formula C0. 25 H 32 O 12 Molecular weight 524, ESI-MS (negative): m / z 523.0 [MH] - The structural formula is as follows: 。 2. The method for extracting active naphthyl glycosides from the root of *Ligustrum lucidum* according to claim 1, characterized in that... The 70% ethanol reflux method described in step one is as follows: After drying and pulverizing the root of *Ligustrum lucidum*, add 5 to 10 times its volume of 70 to 95% ethanol, reflux for 2 to 3 hours, filter while hot to remove the residue, extract 3 times, combine the filtrates and concentrate under reduced pressure at 45 to 55°C to obtain the concentrate.

3. The method for extracting active naphthyl glycosides from the root of *Ligustrum lucidum* according to claim 1, characterized in that... The liquid chromatography described in step four was performed using a Waters 2545 high-performance liquid chromatograph with a Waters 2489 UV detector. The column packing material was C18 reversed-phase silica gel. The mobile phase consisted of a mixture of acetonitrile and water (volume ratio 22:78) and an aqueous methanol-water mixture (volume ratio 40:60). The flow rate was 10 ml / min, and the detection wavelength was 224 nm.

4. The application of active naphthyl glycosides in the root of Ligusticum striatum, characterized in that... The active naphthyl glycosides in the root of *Ligustrum lucidum* are compounds 1-4 extracted by the extraction method of claim 1, which are used as active ingredients in the preparation of whitening and freckle-removing cosmetics.

5. The application of the active naphthyl glycosides in the root of *Ligustrum lucidum* according to claim 4, characterized in that... The whitening and freckle-removing cosmetics also include one or more of the following: moisturizers, antioxidants, surfactants, and thickeners.

6. The application of the active naphthyl glycosides in the root of *Ligustrum lucidum* according to claim 4, characterized in that... The whitening and freckle-removing cosmetics are available in the form of water, lotion, cream, ointment, gel, or mask.

7. The application of active naphthyl glycosides in the root of *Ligustrum lucidum*, characterized in that... The active naphthyl glycosides in the root of *Ligustrum lucidum* are compounds 1-4 extracted by the extraction method of claim 1, which are used as active ingredients in the preparation of pharmaceutical formulations.

8. The application of the active naphthyl glycosides in the root of *Ligustrum lucidum* according to claim 7, characterized in that... The pharmaceutical preparation also includes one or more of stearic acid, cetyl alcohol, castor oil, triethanolamine, and propylene glycol.

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