Naphthol glycoside compound with antifungal activity in cortex juglandis mandshuricae and preparation method of naphthol glycoside compound
By extracting and purifying the naphthol glycoside 1-butoxynaphthalene-5-O-β-D-glucoside from *Trichophyton rubrum*, the problems of high toxicity and drug resistance of existing antifungal drugs were solved, achieving a highly effective inhibition of *Trichophyton rubrum* and expanding the application of *Trichophyton rubrum* in antifungal drugs.
Patent Information
- Application Number
- CN202511445906.9
- Authority / Receiving Office
- CN · China
- Patent Type
- Applications(China)
- Current Assignee / Owner
- Filing Date
- 2025-10-11
- Publication Date
- 2026-01-13
AI Technical Summary
Existing antifungal drugs have significant toxic side effects and are prone to drug resistance, making it difficult to meet clinical treatment needs. Research on the antifungal activity of *Clerodendrum trichotomum* is not in-depth, and there is a lack of novel, highly effective, and low-toxicity antifungal lead compounds.
1-Butoxynaphthalene-5-O-β-D-glucoside, a naphthol glycoside, was extracted from *Cyclocarya paliurus*. The compound was then purified using a combination of 60% ethanol reflux extraction, polar solvent extraction, macroporous resin purification, and reversed-phase column chromatography, resulting in a high-purity HPLC-based preparation method.
The obtained compound has high purity and high yield, and significantly inhibits Trichophyton rubrum, which provides a good foundation for it to become an antifungal drug candidate and expands the application prospects of gentian root coat in antifungal drugs.
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Abstract
Description
TECHNICAL FIELD
[0001] The present application relates to the technical field of medicine, in particular to a naphtholigoside compound with antifungal activity in Juglans mandshurica and a preparation method thereof. BACKGROUND
[0002] In recent years, the problem of clinical fungal infection is increasingly serious. Mild infection caused by fungi not only invades the skin and mucous membrane, but also can cause severe infection, invade deep tissues and organs, and even endanger life in severe cases. However, the existing antifungal drugs generally have problems such as large toxic and side effects, easy drug resistance, and are difficult to meet the clinical treatment needs. Under this background, screening of antifungal lead compounds with novel structure, high efficiency and low toxicity from natural drugs has become an important direction to solve the current antifungal treatment dilemma. Natural drugs show broad prospects in the field of antifungal drug research and development due to their rich chemical components, diverse mechanisms of action and lower toxic and side effects. Juglans mandshurica is the pericarp of unripe fruit of Juglans regia L. Juglans mandshurica Modern research has confirmed that Juglans mandshurica has various pharmacological activities such as anti-inflammatory, antibacterial and antitumor, and it has shown good potential in antifungal effect. It is commonly used externally in folk to treat tinea pedis, tinea capitis, tinea cruris and other fungal infectious diseases, and has achieved certain effect. However, there are few and insufficient in-depth reports on the antifungal activity related to the pharmacodynamic substances of Juglans mandshurica. Therefore, it is expected to have good prospects for drug development to further explore this medicinal resource. SUMMARY
[0003] The present application aims to provide a new naphtholigoside compound in Juglans mandshurica and a preparation method and application thereof.
[0004] The above-mentioned purpose of the present application is achieved by the following technical solutions. The present application discloses a naphtholigoside 1-butoxy naphthalene-5- O - β -D-glucoside, the molecular formula of the naphtholigoside is: C 20 H 26 O7, and the chemical structural formula is as follows:
[0005] The present application also provides a preparation method of the compound: taking Juglans mandshurica as raw material, refluxing extraction with 60% ethanol, taking the n-butanol layer after extraction with different polarity solvents, then purifying and enriching by macroporous resin, and then purifying by reverse phase column chromatography and preparation HPLC chromatography, and the specific steps are as follows: (1) Ethanol extraction: taking Qinglongyi as raw material, crushing appropriately, using 60% ethanol reflux extraction for 3 times, 2 hours each time, filtering after extraction, combining 3 times of filtrate, recovering ethanol to obtain ethanol extract; (2) Solvent extraction: dispersing the ethanol extract obtained in step (1) with distilled water to a certain concentration, then sequentially extracting with dichloromethane and water-saturated n-butanol for 8 times, combining n-butanol layers, recovering solvent to obtain n-butanol extract; (3) Macroporous resin enrichment and purification: taking the n-butanol extract obtained in step (2) and dispersing with water to a certain concentration, enriching and purifying through D101 macroporous resin column chromatography, gradient eluting with water, 30% and 50% ethanol respectively, collecting each gradient eluent, recovering 50% ethanol elution part under reduced pressure to obtain 50% ethanol elution part extract; (4) Reverse phase column chromatography separation: taking the 50% ethanol elution part extract obtained in step (3), separating by reverse phase silica gel ODS column chromatography. Gradient eluting with methanol-water mixed solvent with volume ratio of 10:90 and 20:80 respectively, each ratio eluting for 3.5 column volumes, discarding; then eluting with methanol-water mixed solvent with volume ratio of 45:55, discarding the first 1.5 column volumes, collecting the eluent after eluting for 2.5 column volumes, recovering solvent to obtain a crude product; (5) HPLC preparation and purification: separating and purifying the crude product obtained in step (4) by preparative HPLC. Using methanol-water with volume ratio of 1:1 as mobile phase, elution flow rate is 1 mL / min, collecting the fraction in the stage of t R =23.8min~24.2min, recovering and drying to obtain a finished product.
[0006] The present application relates to the above naphthoside compound 1-butoxy naphthalene-5- O - β -D-glucoside in the preparation of antifungal drugs, the fungus is trichophyton rubrum.
[0007] The present application has the advantages that: (1) the operation steps of the whole preparation process are clear, the purity and yield of the obtained compound are higher, especially the combination of solvent extraction and macroporous resin chromatography effectively enriches the target component and improves the separation efficiency; (2) in vitro antifungal activity experiment shows that the compound of the present application can effectively inhibit pathogenic fungi, has a good foundation to become an antifungal candidate drug, and expands its application prospect. BRIEF DESCRIPTION OF DRAWINGS
[0008] Figure 1 is the chemical structure and number diagram of the compound of the present application; Figure 2 is the positive HR-ESI-MS spectrum of the compound of the present application; Figure 3H-NMR spectrum of the compound of the present application 1 H-NMR spectrum of the compound of the present application Figure 4 H-NMR spectrum of the compound of the present application 13 H-NMR spectrum of the compound of the present application Figure 5 H-NMR spectrum of the compound of the present application Figure 6 H-NMR spectrum of the compound of the present application Figure 7 H-NMR spectrum of the compound of the present application Figure 8 H-NMR spectrum of the compound of the present application 1 H- 1 H COSY spectrum of the compound of the present application DETAILED DESCRIPTION
[0009] The present application is described below in detail and completely, which is helpful for the person skilled in the art to better understand the present application, but does not limit the present application in any way. Based on the examples in the present application, the person skilled in the art can modify and polish the specific details of the present application, but the other examples obtained without the innovation breakthrough are within the protection scope of the present application.
[0010] Example 1 Preparation method of the compound of the present application (1) Ethanol extraction: 6 kg of Qinglongyi was taken as raw material, crushed into 60-80 mesh powder, and extracted with 60% ethanol for 3 times, the weight of the medicinal material and the volume of ethanol were 1:8, 2 h each time, and the extraction was filtered after the extraction was completed. After recovering ethanol, 324 g of ethanol extract was obtained; (2) Solvent extraction: the ethanol extract obtained in step (1) was dispersed with distilled water to a concentration of 1.45 ± 0.05 g / mL, and then extracted with dichloromethane and water-saturated n-butanol for 8 times respectively, and the n-butanol layer was combined and the solvent was recovered to obtain 164 g of n-butanol extract; (3) Macroporous resin enrichment and purification: the n-butanol extract obtained in step (2) was dispersed with water to a concentration of 0.40 ± 0.05 g / mL, and then enriched and purified by D101 macroporous resin column chromatography (the inner diameter of the chromatographic column was 5 cm, the height was 100 cm, and the filler height was 70 cm), and then eluted with water, 30% and 50% ethanol respectively, 3 column volumes, 2.5 column volumes and 3 column volumes respectively, and the gradient eluate was collected, and the 50% ethanol elution part was recovered under reduced pressure to obtain 41 g of 50% ethanol elution extract; (4) Reversed-phase column chromatography separation: Take the 50% ethanol elution part of the extract obtained in step (3), and separate by reversed-phase silica gel ODS column chromatography (in which the chromatographic column is selected from a Buchi Pump Module C-601 medium-pressure chromatographic column, the column inner diameter is 30 mm, the effective height of the reversed-phase silica gel is 450 mm, the flow rate is 4 mL / min, and the ODS particle size is 50 μm), and gradient elution is carried out with methanol-water mixed solvents with volume ratios of 10:90 and 20:80, respectively, and each ratio is eluted for 3.5 column volumes, and discarded; then eluted with methanol-water mixed solvents with a volume ratio of 45:55, and the first 1.5 column volumes are discarded, and then 2.5 column volumes are eluted, and the eluent is collected, the solvent is recovered, and 0.12 g of a crude product is obtained; (5) HPLC preparation and purification: the crude product obtained in step (4) is separated and purified by semi-preparative HPLC. Methanol-water with a volume ratio of 1:1 is used as the mobile phase, the elution flow rate is 1.5 mL / min, and the retention time t R =23.8 min~24.2 min stage, and the fraction is collected, and the product is obtained by drying and recovering.
[0011] Example 2: Compound identification: the prepared compound of the application is a brownish yellow powder, the Molish reaction is positive, after acid hydrolysis and acetylation, D-glucose is detected by GC, [α]25D= +9.8° (c = 0.05, MeOH). HR-ESI-MS m / z: 401.1568 [M+Na] + (calcd for 401.1576), indicating that the molecular weight is 378. Combined with the results of H-NMR, 1 H-NMR, 13 C-NMR and DEPT spectrum, it is speculated that the molecular formula of the compound of the application is C 20 H 26 O7, and the unsaturation degree is calculated to be 8.
[0012] In 1 H NMR (600 MHz, CD3OD), one methyleneoxy proton signal δ H 4.16 (1H, t , J = 6.3 Hz, H-11); six aromatic proton signals, in which δ H 6.91 (1H, d , J = 8.1 Hz, H-2), 7.36 (1H, t , J = 8.1 Hz, H-3), 7.94 (1H,d J = 8.1 Hz, H-4) is a set of ABC coupled aromatic protons on the aromatic ring, δ H 7.24 (1H, d J = 8.1 Hz, H-6), 7.36 (1H, t J = 8.1 Hz, H-7), 7.89 (1H, d J = 8.1 Hz, H-8) is another set of ABC coupled aromatic protons on the aromatic ring; a set of glucose proton signals δ H 5.10 (1H, d J = 7.8 Hz, H-1'), 3.66 (1H, t J = 7.8 Hz, H-2'), 3.51 (1H, m , H-3'), 3.46 (1H, m , H-4'), 3.52 (1H, m , H-5'), 3.93 (1H, dd J = 12.0, 2.1 Hz, H-6'α), 3.74 (1H, dd J = 12.0, 5.3 Hz, H-6'β), wherein δ H 5.10 (1H, d J = 7.8 Hz, H-1') is the anomeric proton signal of the sugar, which is deduced to be β -glucose according to the coupling constant. The above data analysis indicates that the structural characteristics of the compound belong to naphtholigoside compounds.
[0013] In 13 C NMR (150 MHz, CD3OD) and DEPT spectrum, 20 carbon signals can be observed. Among them, δ C 155.9 (C-1), 106.3 (C-2), 125.9 (C-3), 115.3 (C-4), 154.5 (C-5), 110.0 (C-6), 126.5 (C-7), 117.0 (C-8), 128.2 (C-9), 128.4 (C-10) are 10 benzene ring carbon signals; δ C 102.6 (C-1′), 75.1 (C-2′), 78.2 (C-3′), 71.5 (C-4′), 78.2 (C-5′), and 62.5 (C-6′) represent six glucose carbon signals. δ C 102.6 (C-1′) is the terminal carbon signal of glucose. The above are the characteristic carbon signals of naphthol glycosides. In addition, there are four hydrocarbon carbon signals: 69.0 (C-11), 32.7 (C-12), 20.7 (C-13), and 14.2 (C-14). Among them, position 11 is the oxygen-linked hydrocarbon carbon signal, and position 14 is the methyl carbon signal.
[0014] exist 1 H- 1 In H COSY, H2-11 ( δ 4.16) and H2-12 ( δ 1.92) related, H2-12 ( δ 1.92) and H2-13 ( δ 1.65) related, H2-13 ( δ 1.65) and H3-14 ( δ 1.06) is related, suggesting the existence of a butyl oxygen structural segment, i.e., -O-CH2-CH2-CH2-CH3; in HMBC, H1-1′ ( δ 5.10) and C-5 ( δ (154.5) is related, suggesting that the sugar is linked at the 5-position of the naphthalene ring, H2-11 ( δ 4.16) and C-1 ( δ Based on the correlation with 155.9), it was determined that the butyl oxygen structure is attached to the 1-position of the naphthalene ring. The structure of this compound was identified as a new compound through a SciFinder online literature search and named 1-butoxynaphthalene-5- O - β -D-glucoside.
[0015] Table 1. NMR signal assignment of the compounds of this invention
[0016] Example of efficacy: In vitro inhibition of Trichophyton rubrum.
[0017] (1) Experimental supplies Fungal strain: Trichophyton rubrum (Beijing Bio-Biotechnology Co., Ltd., product number: bio-68036); Main experimental materials and devices; sabouraud agar culture medium (Beijing Land Bridge Technology Co., Ltd., item number: CM102), RPMI-1640 liquid culture medium (Wuhan Punuo Life Science and Technology Co., Ltd., item number: PM150110), PDA culture medium (Beijing Aobos Technology Co., Ltd., item number: 02-002); dimethyl sulfoxide / DMSO (National Pharmaceutical Group Chemical Reagent Co., Ltd., item number: 201-030-3); fluconazole (Araladin, item number: F113133); 96-well cell culture plate (Corning Corporation, item number: 3599); constant temperature incubator (Thermo Fisher Corporation, item number: 3110); blood cell counting plate (Suzhou Well Experimental Products Co., Ltd., item number: WELL-XB); the compound of the application prepared in Example 1.
[0018] (2) Experimental method i. Drug preparation Precisely take 10 mg of the compound of the application, dissolve it in 1 mL of DMSO to prepare a drug stock solution with a concentration of 12.5 mg / mL. Before the experiment, 0.08 mL is taken and diluted with RPMI-1640 liquid culture medium to prepare a 1 mg / mL solution for standby; ii. Bacteria solution preparation Inoculate the Trichophyton rubrum into the test tube slant culture medium, and place it in the constant temperature incubator for 28℃ culture for 6 days. Wash out the mycelium and spores with normal saline, and count the bacteria concentration to 1.0 × 10 6 ~4.0 × 10 6 cfu / mL; the working bacteria solution for the experiment is diluted 100 times with the culture medium, and the final concentration is 1.0 × 10 4 ~4.0 × 10 4 cfu / mL; iii. Activity determination Take a sterile 96-well plate, add 200 μL of diluted sample solution (drug concentration 1 mg / mL) to the first well; add 100 μL of RPMI1640 medium to wells 2-10, pipette 100 μL from the first well into the second well, mix well and pipette 100 μL into the third well, and so on to achieve 10-fold dilution, mix the drug in the tenth well and pipette 100 μL into the eleventh well, add 100 μL of bacterial suspension and 100 μL of medium to the eleventh well as a negative control; add 200 μL of medium to the twelfth well as a blank control. Repeat three groups (the concentration of solvent DMSO in each drug well is controlled to be ≤1% to avoid interfering with fungal growth). Read the OD value of each well at a wavelength of 630 nm using a microplate reader. The calculation method of the percentage of fungal growth inhibition is: percentage of fungal growth inhibition = 1 - [(OD value in each well - OD value in blank control well) / (OD value in growth control well - OD value in blank control well)] x 100%. The lowest concentration with a bacteriostatic rate ≥80% is defined as the MIC to determine the concentration; MFC is set according to the test method of MIC, respectively MIC, 2MIC, 4MIC, 8MIC, and red hair trichophyton is cultured at 28°C for 6 days, after the culture is completed, 20 μL of the culture solution is inoculated in fresh PDA medium and cultured for 5-7 days, and the viable bacteria count method is used for evaluation, and the lowest drug concentration that can reduce the viable bacteria count by 99.9% is defined as the minimum bactericidal concentration MFC.
[0019] (3) Results The MIC of the compound of the present application for trichophyton rubrum is 0.0078 mg / mL, and the MFC is 0.0313 mg / mL, the MIC of the positive drug group is 0.00049 mg / mL, and the MFC is 0.0020 mg / mL, the negative control bacteria grow normally, and the blank control bacterial solution is turbid, and the experimental results show that the compound has significant inhibition and killing effect on trichophyton rubrum, and has the development prospect of preparing antibacterial drugs.
Claims
1. A method for preparing naphthol glycosides from *Cyclocarya paliurus*, characterized in that: Using *Clerodendrum bungei* as raw material, the extract was obtained by reflux extraction with 60% ethanol, followed by extraction with solvents of different polarities to separate the n-butanol layer, purification and enrichment with macroporous resin, and then separation by reversed-phase column chromatography combined with preparative HPLC. The specific steps are as follows: (1) Ethanol extraction: Using the green dragon skin as raw material, it was appropriately crushed and extracted three times by reflux with 60% ethanol for 2 hours each time. After the extraction was completed, the extract was filtered, the filtrates from the three extractions were combined, and the ethanol was recovered to obtain the ethanol extract. (2) Solvent extraction: The ethanol extract obtained in step (1) is dispersed in distilled water to a certain concentration, and then extracted 8 times in sequence with dichloromethane and water-saturated n-butanol. The n-butanol layers are combined, the solvent is recovered, and n-butanol extract is obtained. (3) Macroporous resin enrichment and purification: Take the n-butanol extract obtained in step (2) and disperse it with water to a certain concentration. Then, enrich and purify it by D101 macroporous resin column chromatography. Use water, 30% ethanol and 50% ethanol for gradient elution respectively. Collect the eluents of each gradient and recover the 50% ethanol elution fraction under reduced pressure to obtain the 50% ethanol elution fraction extract. (4) Reversed-phase column chromatography separation: Take the 50% ethanol eluted extract obtained in step (3) and separate it by reversed-phase silica gel ODS column chromatography. Elute sequentially with a methanol-water mixed solvent with a volume ratio of 10:90 and 20:80, eluting for 3.5 column volumes for each ratio and discarding the eluent; then elute with a methanol-water mixed solvent with a volume ratio of 45:55, eluting for the first 1.5 column volumes and discarding the eluent, then eluting for another 2.5 column volumes and collecting the eluent, recovering the solvent, and obtaining the crude product; (5) Preparative purification by HPLC: The crude product obtained in step (4) was purified by preparative HPLC. A methanol-water mixture with a volume ratio of 1:1 was used as the mobile phase, the elution flow rate was 1 mL / min, and the retention time was t. R The fraction was collected during the period of 23.8 min to 24.2 min, and the recovered and dried fractions were used to obtain the naphthol glycoside. The above-mentioned naphthol glycoside compound is 1-butoxynaphthalene-5- O - β -D-glucoside, with the molecular formula C 20 H 26 O7, the structural formula is as follows:
2. The application of the naphthol glycoside compound according to claim 1 in the preparation of anti-Trichophyton rubrum.
3. The application as described in claim 2, characterized in that: It can be used to prepare drugs against pathogenic fungi.