A naphthol glycoside compound in qinglongyi and a preparation method and application thereof

Abstract

This invention belongs to the field of pharmaceutical technology, specifically relating to a naphthol glycoside compound, juglone-naphthol glycoside I, extracted and isolated from *Clerodendrum trichotomum*, its preparation method, and its application. The preparation method specifically includes the following steps: First, *Clerodendrum trichotomum* powder is extracted by heating and reflux with 70% ethanol. After filtration, 10% (w / v) CaCl2 powder is added to the extract, stirred thoroughly, and allowed to stand. The ethanol is recovered from the supernatant to obtain a concentrated extract. After water dispersion, the extract is extracted five times each with dichloromethane and n-butanol. The n-butanol layer is separated, and then subjected to reversed-phase silica gel column chromatography and recrystallization to obtain the target compound. The preparation method provided by this invention only requires salting out impurities, solvent extraction, reversed-phase column chromatography separation, and recrystallization, achieving efficient enrichment and separation of the target compound. The steps are simplified and easy to operate. Furthermore, the naphthol glycoside prepared by this invention has been shown in in vitro activity experiments to have good anti-ethanol-induced gastric ulcer damage, providing a valuable pharmacodynamic substance for the future development of novel anti-gastric ulcer drugs, with broad application prospects.

Description

Technical Field

[0001] This invention belongs to the field of pharmaceutical technology, specifically relating to a naphthol glycoside compound obtained from scabra, its preparation method, and its application. Background Technology

[0002] Qinglongyi is a type of walnut (Manchurian walnut) Juglans mandshurica The green pericarp (peel) that covers the pit of the unripe fruit (Maxim). This medicine was first recorded in the "Kaibao Materia Medica". Traditional medicine believes that it has the effects of clearing heat and detoxifying, reducing swelling and relieving pain, and dispelling wind and treating tinea. Modern pharmacological research has confirmed that the green pericarp has biological activities such as anti-tumor, antibacterial, antioxidant and analgesic effects. In particular, clinical research on tumor prevention and treatment and skin disease intervention is deepening year by year, and it has become a research hotspot.

[0003] The structural characteristic of naphthol glycosides is that the naphthol core is linked to a glycosyl group via a glycosidic bond, and the core often has a series of derivatives formed by the substitution of phenolic hydroxyl groups in varying numbers and positions. The distribution of these compounds in the plant kingdom exhibits some family and genus specificity, with current reports concentrated in the Juglandaceae and Ebenaceae families, particularly the Juglandaceae genus, which is a typical source of these components. Current research on these compounds is in the exploratory stage, with significant limitations in both breadth and depth. For example, pharmacological activity mainly focuses on antitumor activity. Although structure-activity relationships have shown that monosaccharide derivatives generally exhibit superior activity compared to disaccharide derivatives, further studies on antitumor mechanisms and other pharmacological effects are still relatively few, limiting a comprehensive understanding of the bioactivity spectrum of naphthol glycosides. These compounds possess significant development potential and value. Summary of the Invention

[0004] The purpose of this invention is to provide a method for preparing a novel naphthol glycoside compound from *Cyclocarya paliurus*.

[0005] Another objective of this invention is to explore its application in treating gastric ulcers, providing new and valuable pharmacological substances for the development of anti-gastric ulcer drugs.

[0006] To achieve the above objectives, the present invention employs the following technical solution:

[0007] This invention discloses a naphthol glycoside, the molecular formula of which is: C 17 H 18 O9 has the following structural formula:

[0008]

[0009] This invention also provides a method for preparing the compound, the specific preparation steps of which are as follows:

[0010] (1) Ethanol extraction: Using the green dragon skin as raw material, it was appropriately crushed and extracted three times by reflux with 70% ethanol for 2 hours each time. After the extraction was completed, the filtrates were filtered and the three filtrates were combined.

[0011] (2) Salting out impurities: Add 10% (W / V) CaCl2 powder (i.e., add 10 g CaCl2 powder per 100 mL of 70% ethanol solution) to the filtrate obtained in step (1), stir thoroughly and let stand, and recover the ethanol from the supernatant to obtain concentrated extract.

[0012] (3) Solvent extraction: The concentrated extract obtained in step (2) is dispersed in water to a certain concentration, and then extracted 5 times each with dichloromethane and water-saturated n-butanol. The n-butanol layers are combined, the solvent is recovered, and n-butanol extract is obtained.

[0013] (4) Reversed-phase column chromatography separation: The n-butanol paste obtained in step (3) was separated by reversed-phase silica gel ODS column chromatography. The mixture was eluted sequentially with a methanol-water mixed solvent with a volume ratio of 10:90 and 30:70, eluting for 3 column volumes for each ratio and discarding the eluent. Then, the mixture was eluted with a methanol-water mixed solvent with a volume ratio of 40:60, eluting for the first 2 column volumes and discarding the eluent. After eluting for another 1.5 column volumes, the eluent was collected, the solvent was recovered, and the crude product was obtained.

[0014] (5) Recrystallization treatment: The crude product obtained in (4) is recrystallized twice using a dichloromethane-methanol mixed solvent to obtain the compound juglone I of the present invention.

[0015] This invention relates to the application of the above-mentioned naphthol glycoside compound, juglone-naphthol glycoside I, in the preparation of anti-gastric ulcer drugs.

[0016] The beneficial effects of this invention are as follows:

[0017] (1) This invention prepares a new naphthol glycoside compound from scabra, which has not been reported in the prior art; the preparation method provided by this invention can achieve efficient enrichment and separation of the target compound, and the steps are simple and easy to operate;

[0018] (2) The compounds prepared by this invention have good anti-gastric ulcer activity, which not only provides potential candidate molecules for anti-gastric ulcer drugs, but also expands the scope of active applications of naphthol compounds. Attached Figure Description

[0019] Figure 1 Here is the chemical structural formula of the compound of this invention;

[0020] Figure 2 The above are the positive HR-ESI-MS spectra of the compounds of this invention.

[0021] Figure 3 The compounds of this invention1 H-NMR spectrum;

[0022] Figure 4 The compounds of this invention 13 C-NMR spectrum;

[0023] Figure 5 The DEPT spectrum of the compound of this invention;

[0024] Figure 6 The HSQC spectrum of the compound of this invention;

[0025] Figure 7 The HMBC spectrum of the compound of this invention;

[0026] Figure 8 The compounds of this invention 1 H- 1 H COSY spectrum. Detailed Implementation

[0027] Based on the technical content disclosed in this invention, those skilled in the art will clearly understand other embodiments of this invention. The following embodiments are merely examples. Various adjustments and improvements can be made to this invention without violating its spirit and scope. These changes should be within the protection scope of this invention. The substantive content of this invention is described in detail below with reference to the embodiments. Example 1

[0028] Preparation method of the compound of the present invention:

[0029] (1) Ethanol extraction: Take 5 kg of raw material of Qinglongyi, crush it into 60-80 mesh powder, and extract it three times by reflux with 70% ethanol. The ratio of the weight of the medicinal material to the volume of ethanol is 1:8 for the first extraction and 1:6 for the other two extractions. The reflux time is 2 h each time. After the extraction is completed, filter and combine the filtrates from the three extractions.

[0030] (2) Salting out impurities: Add 10% (W / V) CaCl2 powder (i.e., add 10 g CaCl2 powder per 100 mL of 70% ethanol solution) to the filtrate obtained in step (1), stir thoroughly, let stand at room temperature for 16 h, siphon the supernatant with a rubber tube, and recover the ethanol in the supernatant until there is no alcohol smell, to obtain 412 g of concentrated extract;

[0031] (3) Solvent extraction: The concentrated extract obtained in step (2) was dispersed in pure water to form an aqueous solution with a concentration of 1.55 ± 0.05 g / mL. Then, it was extracted 5 times each with dichloromethane and water-saturated n-butanol. The n-butanol layers were combined, and the solvent was recovered to obtain 56 g of n-butanol extract.

[0032] (4) Reversed-phase column chromatography separation: The n-butanol paste obtained in step (3) was separated by reversed-phase silica gel ODS column chromatography (a medium-pressure column Buchi Pump Module C-601 was selected, with an inner diameter of 50 mm, an effective height of 550 mm of reversed-phase silica gel packed inside, a flow rate of 5 mL / min, and an ODS particle size of 50 μm). The column was eluted sequentially with a methanol-water mixed solvent gradient with a volume ratio of 10:90 and 30:70, eluting for 3 column volumes for each ratio and discarding the eluent; then eluted with a methanol-water mixed solvent with a volume ratio of 40:60, eluting for the first 2 column volumes and discarding the eluent, and then eluting for 1.5 column volumes and collecting the eluent, recovering the solvent, and obtaining 0.5 g of crude product;

[0033] (5) Recrystallization treatment: The crude product obtained in (4) is recrystallized twice using a mixed solvent of dichloromethane and methanol with a volume ratio of 1:2 to obtain 26 mg of the compound juglone glycoside I of the present invention (purity ≥98%). Example 2

[0034] Identification of the compound: The compound obtained in this invention is a red needle-like crystal, showing a positive Molish reaction. After acid hydrolysis and acetylation, GC detection revealed D-glucose and [α-glucose]. 25 D = +12.3°(c = 0.10, MeOH). HR-ESI-MS m / z 389.0834 [M+Na] + (calcd for 389.0849), indicating a molecular weight of 366. (Combined) 1 H-NMR, 13 Based on C-NMR and DEPT spectroscopy, the molecular formula of the compound of this invention is deduced to be C. 17 H 18 O9 has an unsaturation degree of 9.

[0035] exist 1 An aldehyde matrix signal was observed in H NMR (600 MHz, CD3OD). d H 9.92 (1H, s (H-11); four aromatic proton signals d H 7.53 (1H, s , H-3), 7.06 (1H, d , J = 8.0 Hz, H-6), 7.43 (1H, t , J = 8.0 Hz, H-7), 7.90 (1H,d , J = 8.0 Hz, H-8), where d H 7.06 (1H, d , J =8.0 Hz, H-6), 7.43 (1H, t , J = 8.0 Hz, H-7), 7.90 (1H, d , J = 8.0 Hz, H-8) represents a set of aromatic proton signals on an ABC-coupled aromatic ring; a set of glucose proton signals d H 5.06 (1H, d , J = 7.8 Hz, H-1'), 3.55 (1H, m , H-2'), 3.49 (1H, t , J = 9.1 Hz, H-3'), 3.40 (1H, t , J = 9.1 Hz, H-4'), 3.55 (1H, m , H-5'), 3.74 (1H, dd , J = 6.4, 12.0 Hz H-6' α ), 3.99 (1H, d , J = 12.0 Hz, H-6' β ),in d H 5.06 (1H, d , J = 7.8 Hz, H-1') is a terminal proton signal of a sugar. Based on its terminal proton coupling constant, the sugar is inferred to be... β - Glucose. Comprehensive analysis of the above data indicates that the structural characteristics of this compound belong to the naphthol glycoside class of compounds.

[0036] exist 13 Seventeen carbon signals were observed in the C NMR (150 MHz, CD3OD) and DEPT spectra. d C158.4 (C-1), 114.6 (C-2), 111.5 (C-3), 148.6 (C-4), 155.3 (C-5), 117.3 (C-6), 128.9 (C-7), 116.4 (C-8), 128.5 (C-9), and 120.4 (C-10) represent the carbon signals of the 10 benzene rings. d C 198.0 (C-11) represents a single aldehyde carbon signal; d C 105.1 (C-1'), 75.0 (C-2'), 78.2 (C-3'), 71.5 (C-4'), 78.9 (C-5'), and 62.7 (C-6') represent six glucose carbon signals. d C 105.1 (C-1') is the terminal carbon signal of glucose, and the above are the characteristic carbon signals of naphthol glycosides.

[0037] exist 1 H- 1 In the H COSY spectrum, H-1′ was found in the sugar region. d 5.06) and H-2′ ( d 3.55) related, H-2′ ( d 3.55) and H-3′ ( d 3.49) related, H-3′ ( d 3.49) and H-4′ ( d 3.40) related, H-4′ ( d 3.40) and H-5′ d 3.55) related, H-5′ ( d 3.55) and H2-6′ ( d (3.74, 3.99) are related, and further verification shows that... β- Glucose. The absolute configuration of the sugar was determined through acid hydrolysis and GC. Comprehensive analysis revealed that this sugar is... β- D-glucose. H-1'( was observed in the HMBC spectrum.) d 5.06) and C-4 ( d The correlation with 148.6 indicates that the glucose is linked to C-4. Furthermore, H-11 ( d The correlation between C-1 (158.4) and C-3 (111.5) suggests that the C-2 position is substituted with an aldehyde group. The structure of this compound was identified as a new compound through a SciFinder online literature search and named juglone glycoside I. The specific NMR signal assignments of the compounds of this invention are shown in Table 1.

[0038] Table 1. NMR signal assignments of the compounds of this invention.

[0039]

[0040] Effect Example

[0041] The CCK8 assay was used to investigate the anti-ethanol-induced GES-1 cell damage activity of this compound. The specific experimental details are as follows:

[0042] (1) Experimental cells: GES-1 cells were purchased from Orex Biotech (Shanghai) Co., Ltd., catalog number: ORC0320; teprenone was purchased from Shanghai Aladdin Biochemical Technology Co., Ltd., catalog number: T137670;

[0043] (2) Investigation of cell experimental conditions:

[0044] 1) To examine the cytotoxicity of the drug: GES-1 cells were inoculated at a rate of 1×10⁻⁶. 5 GES-1 cells were seeded at a concentration of 100 μL / mL in 96-well plates. Different concentrations of the compound (6.25, 12.5, 25, 50, 100, and 200 μmol / L) were used to treat GES-1 cells for 24 h. Cell viability was determined using the CCK8 assay to assess drug toxicity. Results showed that the cell viability at 6.25, 12.5, 25, 50, and 100 μmol / L was nearly 100%, indicating no significant cytotoxicity at these concentrations. However, at 200 μmol / L, cell viability decreased to 85%, suggesting potential toxicity.

[0045] 2) Establishment of a gastric ulcer cell model: GES-1 cells were treated with 0.6%, 3%, 5%, 7%, and 9% ethanol for 3 h and 5 h, respectively. Cell viability was detected by CCK8 assay to screen for optimal modeling conditions. The experimental results are shown in Table 2.

[0046] Table 2 Screening results of ethanol concentration during 3 h and 5 h modeling ( ±s, n= 6)

[0047]

[0048] Based on the above results, the cell viability rate was selected as 50%–60%, and the conditions for establishing the ethanol-induced GES-1 cell damage model were 5% ethanol concentration and 3 h treatment time.

[0049] 3) Determine the optimal positive control concentration: Teprenone (GGA) was selected as the positive control, and GES-1 cells in the logarithmic growth phase were inoculated at a concentration of 1 × 10⁻⁶. 5GES-1 cells were seeded at a concentration of 100 μL / mL in 96-well plates. The cells were divided into a control group, a model group, and different concentrations of positive control drug groups (10, 20, 40, 80, and 160 μmol / L GGA groups). Except for the control group, the model group was treated with 5% ethanol for 3 h, and the different concentrations of positive control drug groups were co-treated with 5% ethanol for 3 h. Cell viability was determined by CCK8 assay to identify the optimal therapeutic concentration of the positive control drug. The experimental results are shown in Table 3.

[0050] Table 3 Screening for positive drug concentrations ( ±s, n= 6)

[0051]

[0052] Experimental results showed that, compared with the control group, the cell viability in the model group was significantly reduced. P <0.01. Compared with the model group, the cell viability was significantly increased after treatment with different concentrations of positive drug groups (10, 20, 40, 80, and 160 μmol / L GGA groups). P <0.01), and there was no statistically significant difference in cell survival between the 80 μmol / L and 160 μmol / L GGA treatment groups. Therefore, the optimal therapeutic concentration of GGA was determined to be 80 μmol / L.

[0053] (3) Drug efficacy experiment:

[0054] 1) Drug preparation: Take the compound of the present invention, weigh it accurately on an analytical balance, and then dissolve the monomer compound to be tested in a small amount of DMSO solution (final concentration ≤ 0.1%). Then add an appropriate amount of complete culture medium to prepare the concentration of the compound to 6.25, 12.5, 25, 50, 100 and 200 μmol / L. After sterilization, store at 4ºC for later use.

[0055] 2) Protective effect of the drug on ethanol-damaged GES-1 cells: Except for the control group, 100 μL of logarithmic growth phase cells (density 1×10⁻⁶) were seeded in each well of the 96-well plate. 5 / mL). Cells were cultured at 37 ˚C, 5% CO2 until adherence was achieved to establish a cell model. After 3 h of culture, different concentrations (3.125, 6.25, 12.5, 25, 50, 100, 200 μmol / L) of the compound were added to the model medium for further culture. After 24 h, 10 μL of CCK-8 was added to each well for an additional 2 h of culture. The absorbance (OD) of each well was measured at 450 nm using a microplate reader, and cell viability was calculated.

[0056] Cell viability (%) = [(Experimental group - Blank group) / (Control group - Blank group)] × 100%

[0057] Experimental data are presented in the form of ( Mean ± standard deviation (S) indicates the mean. Intergroup comparisons were performed using GraphPad Prism Version 7.0 software and the One-way analysis of variance (ANOVA) method.

[0058] 3) Experimental Results: The experimental results showed that, compared with the control group, the cell survival rate in the model group was significantly reduced. P <0.01). Compared with the model group, treatment with the positive control drug and the 6.25, 12.5, 25, 50, and 100 μmol / L drug groups for 24 h significantly improved cell viability (the 6.25 μmol / L drug group showed significantly higher viability). P <0.05, other groups P <0.01), and the cell survival rate was highest in the 50 μmol / L drug group, which was close to that of the 80 μmol / L positive control drug, showing a significant anti-ethanol-induced GES-1 cell damage effect. However, cell survival rate decreased with increasing concentration, as shown in Table 4:

[0059] Table 4 Results of the drug efficacy experiment ( ±s, n= 6)

[0060]

[0061] In summary, this invention provides a novel naphthol glycoside compound, juglone-naphthol glycoside I, obtained from *Cynanchum paniculatum*, including its preparation method and its effect on ethanol-induced GES-1 cell damage. This not only breaks through the research limitations of naphthol glycosides in the field of anti-tumor therapy, but also provides a valuable natural compound for the development of anti-gastric ulcer drugs, showing good development prospects.

Claims

1. A compound, juglone-naphthol glycoside I, isolated from *Cynanchum paniculatum*, characterized in that... Includes the following steps: (1) Ethanol extraction: Using the green dragon skin as raw material, it was appropriately crushed and extracted three times by reflux with 70% ethanol for 2 hours each time. After the extraction was completed, the filtrates were filtered and the three filtrates were combined. (2) Salting out impurities: Add 10% W / V CaCl2 powder to the filtrate obtained in step (1), that is, add 10 g CaCl2 powder to every 100 mL of 70% ethanol solution, stir thoroughly and let stand, and recover the ethanol from the supernatant to obtain concentrated extract. (3) Solvent extraction: The concentrated extract obtained in step (2) is dispersed in water to a certain concentration, and then extracted 5 times each with dichloromethane and water-saturated n-butanol. The n-butanol layers are combined, the solvent is recovered, and n-butanol extract is obtained. (4) Reversed-phase column chromatography separation: The n-butanol paste obtained in step (3) was separated by reversed-phase silica gel ODS column chromatography. The mixture was eluted sequentially with a methanol-water mixed solvent with a volume ratio of 10:90 and 30:70, eluting for 3 column volumes for each ratio and discarding the eluent. Then, the mixture was eluted with a methanol-water mixed solvent with a volume ratio of 40:60, eluting for the first 2 column volumes and discarding the eluent. After eluting for another 1.5 column volumes, the eluent was collected, the solvent was recovered, and the crude product was obtained. (5) Recrystallization treatment: The crude product obtained in (4) is recrystallized twice using a dichloromethane-methanol mixed solvent to obtain the compound juglone glycoside I; The molecular formula of the compound juglone glycoside I is C 17 H 18 O9 has the following structural formula:

2. The use of the compound juglone-naphthol I according to claim 1 in the preparation of a drug for the prevention and treatment of ethanol-induced gastric ulcers.

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