4'-hydroxy-3,3'-dimethoxy-9,9'-epoxylignan-4-o-β-glucoside, and a preparation method and application thereof

Abstract

The application discloses 4'-hydroxy-3,3'-dimethoxy-9,9'-epoxy lignan-4-O-beta-glucoside and a preparation method and application thereof, belongs to the technical field of medicine extraction, and a subformula thereof is C 26 H 34 O 10 There are two 1,3,4-trisubstituted benzene rings, and there is a 9-O-9' monocyclic epoxy structure. The compound is extracted from an ethyl acetate part extract of box fruit vine; can improve cell viability or improve cell survival rate or promote cell proliferation, can also reduce cell NO content or inhibit cell NO release, thereby improving anti-inflammatory capacity, and can be applied to preparation of related medicines. The application promotes development and application of the box fruit vine in Zhanjiang, and when the prepared compound is applied as a medicine, cell survival rate is as high as 177.71±5.64 **** The release inhibition rate of cell NO reaches 65.11% at a concentration of 32 μg / mL.

Description

Technical Field

[0001] This invention relates to the field of chemical drug extraction technology, and more specifically, to a 4'-hydroxy-3,3'-dimethoxy-9,9'-epoxylignan-4-O-β-glucoside, its preparation method, and its application. Background Technology

[0002] The Zhuang medicine Operculinaturpethum (Linn.) S. Manso is the dried aerial part of the plant Operculinaturpethum (Linn.) S. Manso, which is distributed in Taiwan, Yunnan and Guangxi, China. Traditional Chinese medicine believes that it has the effects of promoting diuresis and reducing swelling, relaxing muscles and tendons, and purging and eliminating stagnation. It is used for edema, difficulty in urination, difficulty in bending and stretching muscles and bones, and contracture of hands and feet.

[0003] Currently, research on *Heterocarpus santalinus* is limited, and its effective chemical components and mechanisms of action remain unclear, hindering large-scale extraction and application. Chinese patent CN112110967B, filed earlier by the applicant, describes the separation of a yellow powder compound, luteolin-4′-O-(6”-O-acetyl)-β-D-glucoside, from the n-butanol fraction of *Heterocarpus santalinus*. This compound has potential applications in the preparation of anti-inflammatory drugs. However, further research is needed to explore the effective chemical components of *Heterocarpus santalinus* and promote its development and application. Summary of the Invention

[0004] One object of the present invention is to address at least the aforementioned deficiencies and to provide at least the advantages that will be described later.

[0005] Another object of the present invention is to provide a novel compound, 4'-hydroxy-3,3'-dimethoxy-9,9'-epoxylignan-4-O-β-glucoside, extracted from the fruit of the vine.

[0006] Another object of the present invention is to provide a method for preparing the above-mentioned novel compound 4'-hydroxy-3,3'-dimethoxy-9,9'-epoxylignan-4-O-β-glucoside.

[0007] Another object of the present invention is to provide an application of the above-mentioned novel compound 4'-hydroxy-3,3'-dimethoxy-9,9'-epoxylignan-4-O-β-glucoside.

[0008] The 4'-hydroxy-3,3'-dimethoxy-9,9'-epoxylignan-4-O-β-glucoside provided by this invention has the following structure:

[0009]

[0010] The molecular formula of this compound is C2. 26 H34 O 10 It has an unsaturation degree of 10, is a transparent solid, is readily soluble in methanol, contains two 1,3,4-trisubstituted benzene rings, and has a 9-O-9' monoepoxy structure.

[0011] This invention provides a method for preparing 4'-hydroxy-3,3'-dimethoxy-9,9'-epoxylignan-4-O-β-glucoside, comprising:

[0012] Take the coarse powder of the fruit vine, and extract it several times by cold soaking in several times the amount of 95% and 70% ethanol solutions. Combine the extracts and concentrate them under reduced pressure to obtain the total extract.

[0013] The total extract was suspended in water and extracted sequentially with petroleum ether, ethyl acetate and n-butanol. The solvents were recovered to obtain petroleum ether fraction extract, ethyl acetate fraction extract and n-butanol fraction extract, respectively.

[0014] The ethyl acetate fraction was separated by macroporous resin column chromatography, eluting with a methanol-water gradient (0%→30%→50%→70%→100%). The methanol-water (50%) eluted fraction was then separated by silica gel column chromatography, eluting with a dichloromethane-methanol gradient (30:0→30:1→25:1→20:1→15:1→10:1→5:1→2:1→1:1→0:1). After TLC identification and HPLC analysis, similar components were combined to obtain fractions B1–B9.

[0015] The B7 fraction was separated by C18 column chromatography with a methanol-water gradient elution (20%-65%). The methanol-water (50%) fraction was collected and purified by semi-preparative high-performance liquid chromatography to obtain the compound.

[0016] This invention provides the application of the above-mentioned 4'-hydroxy-3,3'-dimethoxy-9,9'-epoxylignan-4-O-β-glucoside in the preparation of drugs that improve cell viability, increase cell survival rate, or promote cell proliferation.

[0017] Preferably, the concentration of 4'-hydroxy-3,3'-dimethoxy-9,9'-epoxylignan-4-O-β-glucoside is greater than or equal to 16 μg / mL.

[0018] Preferably, the concentration of 4'-hydroxy-3,3'-dimethoxy-9,9'-epoxylignan-4-O-β-glucoside is greater than or equal to 64 μg / mL.

[0019] Preferably, the concentration of 4'-hydroxy-3,3'-dimethoxy-9,9'-epoxylignan-4-O-β-glucoside is 16, 32, 64, 128, 256 or 512 μg / mL.

[0020] This invention provides the application of the above-mentioned 4'-hydroxy-3,3'-dimethoxy-9,9'-epoxylignan-4-O-β-glucoside in the preparation of drugs that reduce cellular NO content or inhibit cellular NO release.

[0021] Preferably, the concentration of 4'-hydroxy-3,3'-dimethoxy-9,9'-epoxylignan-4-O-β-glucoside is greater than or equal to 4 μg / mL.

[0022] Preferably, the concentration of 4'-hydroxy-3,3'-dimethoxy-9,9'-epoxylignan-4-O-β-glucoside is 4, 8, 16 or 32 μg / mL.

[0023] This invention provides the application of the above-mentioned 4'-hydroxy-3,3'-dimethoxy-9,9'-epoxylignan-4-O-β-glucoside in the preparation of an anti-inflammatory drug, which exerts its effect by increasing cell viability and / or reducing cell NO content.

[0024] The present invention has at least the following beneficial effects:

[0025] 1. The novel compound 4'-hydroxy-3,3'-dimethoxy-9,9'-epoxylignan-4-O-β-glucoside of the present invention is a new compound that can improve cell viability or cell survival rate or promote cell proliferation, and can also reduce cell NO content or inhibit cell NO release, thereby improving anti-inflammatory ability.

[0026] 2. The novel compound 4'-hydroxy-3,3'-dimethoxy-9,9'-epoxylignan-4-O-β-glucoside of the present invention was obtained from the ethyl acetate extract of the Chinese herb *Cistanche deserticola*, which promotes the development and application of the drug.

[0027] 3. The novel compound 4'-hydroxy-3,3'-dimethoxy-9,9'-epoxylignan-4-O-β-glucoside of the present invention can be used in the preparation of drugs that improve cell viability, increase cell survival rate, or promote cell proliferation. It can significantly improve cell survival rate, promote cell proliferation, and enhance cell viability. In one embodiment, when the drug concentration reaches 16 μg / mL, the cell survival rate (%) reaches 121.99 ± 4.99%. ** The effect was significant; when the drug concentration reached 64 μg / mL or higher, the cell viability rate (%) reached 127.50 ± 3.86%. **The above results show significant effects; at a concentration of 512 μg / mL, the cell viability rate (%) reached 177.71 ± 5.64%, which is highly significant. However, the yellow powder compound luteolin-4′-O-(6”-O-acetyl)-β-D-glucoside obtained from the n-butanol extract of *Gnaphalium affine*, as described in Chinese Patent Publication No. CN112110967B, exhibits cytotoxic effects.

[0028] 4. The novel compound 4'-hydroxy-3,3'-dimethoxy-9,9'-epoxylignan-4-O-β-glucoside of the present invention can be used as a drug for preparing drugs that reduce cellular NO content or inhibit cellular NO release. It can reduce NO content and inhibit LPS-induced NO release from RAW264.7 cells. In one embodiment, the inhibition rates at drug concentrations of 4, 8, 16, and 32 μg / mL were 44.89%, 21.99%, 34.01%, 45.12%, and 65.11%, respectively, showing significant effects. In contrast, the yellow powder compound luteolin-4′-O-(6”-O-acetyl)-β-D-glucoside obtained from the n-butanol fraction extract of *Hedysarum heterotropoides* in Chinese Patent Publication No. CN112110967B showed an inhibition rate of only 23.29% at a drug concentration of 31.25 μg / mL, and only reached 70.13% at a drug concentration of 125 μg / mL.

[0029] The novel compound 4'-hydroxy-3,3'-dimethoxy-9,9'-epoxylignan-4-O-β-glucoside of the present invention can be used in the preparation of anti-inflammatory drugs that exert their anti-inflammatory effects by increasing cell viability or cell survival rate or promoting cell proliferation, and by reducing cell NO content or inhibiting cell NO release, or by a combination of both mechanisms.

[0030] Other advantages, objectives and features of the present invention will become apparent in part from the following description, and in part from those skilled in the art through study and practice of the invention. Attached Figure Description

[0031] Figure 1 The structure of the compound of the present invention- 1 H- 1 H COSY Related to HMBC (→);

[0032] Figure 2 The HR-ESI-MS spectrum of the compound of this invention;

[0033] Figure 3 The HMBC spectrum of the compound of this invention;

[0034] Figure 4This is a comparison of the survival rates of RAW264.7 cells in each group in Example 2 of the present invention;

[0035] Figure 5 This is a comparison of NO content in each group of RAW264.7 cells in Example 2 of the present invention; Detailed Implementation

[0036] The present invention will be further described in detail below with reference to embodiments, so that those skilled in the art can implement it based on the description.

[0037] It should be noted that, unless otherwise specified, the experimental methods described in the following embodiments are conventional methods, and the reagents and materials described are commercially available.

[0038] The structure of the 4'-hydroxy-3,3'-dimethoxy-9,9'-epoxylignan-4-O-β-glucoside of the present invention is as follows:

[0039]

[0040] compound 1 H- 1 H COSY Related to HMBC (→)

[0041] Example 1

[0042] Preparation method of 4'-hydroxy-3,3'-dimethoxy-9,9'-epoxylignan-4-O-β-glucoside

[0043] Take 10 kg of coarse powder of *He Guoteng* vine stored in a dry and cool place, and extract it by cold soaking in 10 times the amount of 95% and 70% ethanol solutions, repeating the process 3 times. After combining the extracts, concentrate them under reduced pressure to obtain a total extract of 2225 g.

[0044] The total extract was suspended in water and extracted sequentially with petroleum ether, ethyl acetate and n-butanol. The solvents were recovered to obtain 258g of petroleum ether extract, 823g of ethyl acetate extract and 809g of n-butanol extract, respectively.

[0045] The ethyl acetate fraction was separated by macroporous resin column chromatography with methanol-water gradient elution (0%→30%→50%→70%→100%).

[0046] 103 g of the methanol-water (50%) eluent fraction was separated by silica gel column chromatography using a dichloromethane-methanol gradient (30:0→30:1→25:1→20:1→15:1→10:1→5:1→2:1→1:1→0:1). After TLC identification and HPLC analysis, similar components were combined to obtain fractions B1–B9.

[0047] The B7 fraction was separated by C18 column chromatography with a methanol-water gradient elution (20%-65%). The methanol-water (40%) fraction was collected and purified by Sephadex LH-20 gel column chromatography (methanol elution) followed by semi-preparative high-performance liquid chromatography (60% methanol-40% water isocratic elution) to give the compound (clear solid, 43.3 mg, tR = 16.72 min).

[0048] Compound structure identification:

[0049] The compound is a transparent solid, readily soluble in methanol. It showed a positive Molish reaction, suggesting it is a glycoside.

[0050] ESI-MS (m / z): 529 [M+Na] + High-resolution mass spectrometry (see instruction manual) Figure 2 )HR-ESI-MS(m / z):529.2051[M+Na] + (clacd for C 26 H 34 O 10 , 529.2051), combined 1 H-NMR, 13 C10-NMR determined the molecular formula of the compound to be C10. 26 H 34 O 10 The degree of unsaturation is 10.

[0051] 1 1H-NMR (500MHz, CD3OD): δ 6.63 (1H,d,J=1.9Hz), 7.05 (1H,d,J=8.2Hz), 6.55 (1H,dd,J=1.8,8.0Hz), δ 6.70 (1H,d,J=2.7Hz), 6.71 (1H,d,J=8.1Hz), 6.62 (1H,dd,J=2.5,7.5Hz) are typical ABX spin system signals, indicating the presence of two 1,3,4-trisubstituted benzene rings in the compound. δ 3.82 (3H,s) and 3.80 (3H,s) are signals from two oxygen-bound methyl groups. δ 3.53 (2H,m) and 3.91 (2H,m) are signals from two oxygen-bound methylene groups. δ3.51(1H,m), 3.51(1H,m), 3.43(1H,m), and 3.43(1H,m) represent four oxygen-bound methine methyl signals. δ2.55(2H,m) and 2.55(2H,m) represent two methylene signals with the same chemical environment. δ2.22(1H,m) and 2.22(1H,m) represent two methine methyl signals with the same chemical environment. δ4.88(1H,d,J=7.5Hz) represents the signal of the proton at the upper end of the sugar, and the glycosidic bond configuration is β-type.

[0052] 13 C-NMR and DEPT spectra (125 MHz, CD3OD) showed 26 carbon signals. δ150.6, 149.0, 145.9, 146.4, 136.9, and 133.3 represent aromatic quaternary carbon signals. δ122.4, 118.0, 116.2, 114.3, 113.4, and 122.3 represent aromatic tertiary carbon signals. δ74.5 and 74.4 represent two hydroxymethyl carbon signals. δ40.0 and 40.0 represent two methylene carbon signals. δ47.7 and 47.5 represent two methine carbon signals. δ56.8 and 56.5 represent two methoxy carbon signals. δ103.1, 75.0, 77.9, 71.4, 78.2, and 62.6 represent glycosyl carbon signals.

[0053] With the compound secoisolariciresin-4-ylβ-D-glucopyranoside 13 A comparison of C-NMR data (Table 1) shows that the C-7 and 7' atoms of the compound of this invention are shifted to the lower field by 7.8 and 7.8 ppm, respectively; the C-8 and 8' atoms are shifted to the lower field by 2.7 and 2.5 ppm, respectively; and the C-9 and 9' atoms are shifted to the lower field by 12.5 and 12.4 ppm, respectively. The chemical shifts at other positions are basically consistent. Furthermore, the molecular weight of the compound of this invention is 18 greater than that of the compounds in the literature; therefore, it is speculated that the compound of this invention may be a 9-O-9' monoepoxy lignan glycoside. Compound secoisolariciresin-4-ylβ-D-glucopyranoside Source: Document 29:

[29] Buske A, Jürgen Schmidt, Porzel A, et al. Alkaloidal, Megastigmane and LignanGlucosides from Antidesma membranaceum (Euphorbiaceae) [J]. European Journal ofOrganic Chemistry.2010,2001(18):3537-3543.

[0054] Further analysis was performed using two-dimensional spectra (e.g.) Figure 1 ), HMBC map (such as Figure 3 The results show that the methine signal 3.53 (2H, m) of H-9 is remotely correlated with the carbon signal δ74.4 of C-9', and the methine signal 3.91 (2H, m) of H-9' is remotely correlated with the carbon signal δ74.5 of C-9. This indicates the presence of an oxygen atom between C-9 and C-9' in the compound, confirming the existence of a 9-O-9' monocyclic oxide structure.

[0055] Therefore, the compound was identified as 4'-hydroxy-3,3'-dimethoxy-9,9'-epoxylignan-4-O-β-glucopyranoside. A search of the SciFinder database revealed no reports of this compound, thus it is considered a novel compound.

[0056] The structure of compound 4'-hydroxy-3,3'-dimethoxy-9,9'-epoxylignan-4-O-β-glucoside is as follows:

[0057]

[0058] compound 1 H- 1 H COSY Related to HMBC (→)

[0059] Table 1 Compounds 1 H-NMR, 13 C-NMR data

[0060]

[0061]

[0062] Example 2

[0063] Research on the application of 4'-hydroxy-3,3'-dimethoxy-9,9'-epoxylignan-4-O-β-glucoside as a drug

[0064] 2.1 Preparation of drug solution

[0065] The test drug and lipopolysaccharide solution were diluted with cell culture medium to the required concentrations (2, 4, 8, 16, 32, 64, 128, 256, 512 μg / mL) before use.

[0066] 2.2 CCK8 assay for cell viability

[0067] RAW264.7 cells in the logarithmic growth phase were harvested and treated with 1×10⁻⁶ cells. 4Cells were seeded at a density of 100 μL / mL in 96-well plates, with 100 μL added to each well. Two control groups were established: a blank group (equal volume of basal medium), a dexamethasone group (1 μg / mL), and different concentrations of the drug (4, 8, 16, 32, 64, 128, 256, 512 μg / mL), with three replicates per group. Cells were incubated at 37°C in a 5% CO2 incubator. After 24 h, the supernatant was discarded, and 100 μL of each drug concentration was added to three replicates per group. Cells were incubated for another 24 h, followed by the addition of 10 μL of CCK8 to each well. Cells were incubated for another 4 h, then shaken for 10 min on a shaker to ensure complete dissolution. The OD value of each well was measured at 450 nm using a microplate reader. Cell viability was calculated.

[0068] 2.3 Griess method for detecting NO levels in cell supernatant

[0069] RAW264.7 cells in the logarithmic growth phase were harvested and their concentration adjusted to 5 × 10⁻⁶ cells. 5 Cells were seeded at a density of 1 mL / well in 6-well plates and cultured overnight. Experimental cells were divided into a control group (equal volume of basal medium), a model group (1 μg / mL LPS), a dexamethasone group (1 μg / mL), and drug-treated groups (1, 2, 4, 8, 16, and 32 μg / mL). One hour after drug treatment, except for the control group, all other groups were stimulated with 1 μg / mL LPS to establish an inflammatory response model. Twenty-four hours after drug treatment, 50 μL of cell supernatant from each group was transferred to 96-well plates, with three replicates per group. The OD value was measured at 540 nm using a microplate reader according to the Griess kit instructions, and the NO content and inhibition rate (%) of each group were calculated.

[0070] Results and Analysis

[0071] I. Effects of the compound on the viability of RAW264.7 cells

[0072] The effects of the compounds of this invention at eight concentrations on the viability of RAW264.7 cells were detected, as shown in Table 2. Figure 4 It can be seen that, compared with the control group, low concentrations have little effect on cell proliferation, while concentrations above 64 μg / mL significantly promote cell proliferation. Therefore, the compounds of this invention can be used as drugs to improve cell viability, promote cell proliferation, and increase cell survival rate.

[0073] Table 2 Effects of compounds on RAW264.7 cell viability ( n=3)

[0074]

[0075] Note: Compared with the control group, * p<0.05, ** p<0.01,*** p<0.001, **** p<0.0001

[0076] II. Effects of the compound on NO release from RAW264.7 cells

[0077] From Table 3 and Figure 5 As can be seen, compared with the blank group, the NO level of RAW 264.7 cells in the model group was significantly increased (p < 0.01), indicating that the cell inflammation model was successfully constructed. Compared with the model group, the NO content in the cell supernatant of the dexamethasone group and the drug groups (4, 8, 16, 32 μg / mL) was significantly reduced (p < 0.05, p < 0.01), showing a certain concentration dependence, with inhibition rates of 44.89%, 21.99%, 34.01%, 45.12%, and 65.11%, respectively, indicating that the compound of the present invention can inhibit LPS-induced NO release from RAW264.7 cells. Therefore, the compound of the present invention can be used as a drug for preparing drugs that reduce cellular NO content or inhibit cellular NO release.

[0078] Table 3 Effects of compounds on NO content in RAW264.7 cells ( n=3)

[0079]

[0080] Note: Compared with the blank group, ## p<0.01; compared with the model group, * p<0.05, ** p<0.01

[0081] As can be seen from the above results and analysis, the novel compound 4'-hydroxy-3,3'-dimethoxy-9,9'-epoxylignan-4-O-β-glucoside of the present invention can improve cell viability or cell survival rate or promote cell proliferation, and can also reduce cell NO content or inhibit cell NO release. It can be used to prepare anti-inflammatory drugs. These anti-inflammatory drugs exert their anti-inflammatory effects through mechanisms such as improving cell viability or cell survival rate or promoting cell proliferation, and reducing cell NO content or inhibiting cell NO release, or through a combination of both mechanisms.

[0082] Although embodiments of the present invention have been disclosed above, they are not limited to the applications listed in the specification and embodiments. It can be applied to various fields suitable for the present invention. Further modifications can be readily implemented by those skilled in the art.

Claims

1. A 4'-hydroxy-3,3'-dimethoxy-9,9'-epoxylignan-4-O-β-glucoside, characterized in that, The structure is as follows: 。 2. A method for preparing the 4'-hydroxy-3,3'-dimethoxy-9,9'-epoxylignan-4-O-β-glucoside according to claim 1, characterized in that, include: Take the coarse powder of the fruit vine, and extract it several times by cold soaking in several times the amount of 95% and 70% ethanol solutions. Combine the extracts and concentrate them under reduced pressure to obtain the total extract. The total extract was suspended in water and extracted sequentially with petroleum ether, ethyl acetate and n-butanol. The solvents were recovered to obtain petroleum ether fraction extract, ethyl acetate fraction extract and n-butanol fraction extract, respectively. The ethyl acetate fraction was separated by macroporous resin column chromatography with a methanol-water gradient of 0% → 30% → 50% → 70% → 100%. The fraction eluted with methanol-water at 50% was then separated by silica gel column chromatography with a dichloromethane-methanol gradient of 30:0 → 30:1 → 25:1 → 20:1 → 15:1 → 10:1 → 5:1 → 2:1 → 1:1 → 0:

1. After TLC identification and HPLC analysis, similar components were combined to obtain fractions B1–B9. The B7 fraction was separated by C18 column chromatography with methanol-water gradient elution of 20%-65%; The methanol-water fraction (50%) was collected, eluted by Sephadex LH-20 gel column chromatography, and purified by semi-preparative high-performance liquid chromatography to obtain the 4'-hydroxy-3,3'-dimethoxy-9,9'-epoxylignan-4-O-β-glucoside compound.

3. The use of the 4'-hydroxy-3,3'-dimethoxy-9,9'-epoxylignan-4-O-β-glucoside according to claim 1 in the preparation of a drug for improving cell viability or cell survival rate or promoting cell proliferation.

4. The use of the 4'-hydroxy-3,3'-dimethoxy-9,9'-epoxylignan-4-O-β-glucoside according to claim 1 in the preparation of a drug for reducing cellular NO content or inhibiting cellular NO release.

5. The use of the 4'-hydroxy-3,3'-dimethoxy-9,9'-epoxylignan-4-O-β-glucoside of claim 1 in the preparation of an anti-inflammatory drug, wherein the anti-inflammatory drug exerts its effect by increasing cell viability and / or reducing cell NO content.

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