Phenylethanoid glycoside compound as well as preparation method and application thereof

Abstract

The invention discloses a phenylethanoid glycoside compound as well as a preparation method and application thereof. The phenylethanoid glycoside compound is hydroxytyrosyl-1-O-beta-D-glucoside, and is a new compound separated from hance brandisia herb for the first time. The invention further discloses a preparation method of the compound. The preparation method comprises the steps of alcohol solvent extraction, liquid-liquid extraction, multiple times of silica gel column chromatography, Sephadex LH-20 gel chromatography purification and the like. Finally, the invention discloses an anti-tumor application of the compound and a pharmaceutical composition containing the compound. An in-vitro antitumor activity test shows that the compound has a remarkable growth inhibition effect on tumor cells (especially liver cancer cells). Therefore, the compound provided by the invention can be used for preparing antitumor drugs and has important development and application values.

Description

Technical Field

[0001] This invention relates to the fields of natural product chemistry and pharmacology, specifically to a phenylethanol glycoside compound, its preparation method and application, and more specifically to a novel phenylethanol glycoside compound isolated from honeysuckle, its preparation method, its application in antitumor activity, and a pharmaceutical composition containing the compound. Background Technology

[0002] Honey Bucket Flower is a plant of the Scrophulariaceae family (Honey Bucket Flower). Brandisia hancei Hook. F. The entire plant of *Forsythia suspensa* has many folk names, such as Cat Flower, Honeycomb Flower, Honey Jar, Wild Forsythia, Leaf Flower, Honey Fruit, and Ironwood. Clinically, this plant is mainly used to treat acute and chronic hepatitis and has a long history of medicinal use. Its traditional folk applications are even wider, often used to treat rheumatic pain, edema, diarrhea, jaundice, tuberculosis with hemoptysis, and other diseases. It also has some efficacy in treating osteomyelitis, periostitis, and boils. However, current research on the active ingredients in *Forsythia suspensa* is still insufficient, especially systematic research on its phenylethanoid glycosides.

[0003] Therefore, searching for new phenylethanol glycosides with antitumor activity from honeysuckle is of great significance for expanding the medicinal value of honeysuckle and developing new antitumor drugs. Summary of the Invention

[0004] One of the objectives of this invention is to provide a novel phenylethanol glycoside compound—hydroxytyrosol-1-O-β-D-glucoside—isolated from honeydew flowers.

[0005] The second objective of this invention is to provide a method for preparing the above-mentioned phenylethanol glycosides.

[0006] The third objective of this invention is to provide the application of the above-mentioned phenylethanol glycosides in the preparation of antitumor drugs.

[0007] The fourth objective of this invention is to provide an anticancer drug composition comprising the above-mentioned phenylethanol glycosides.

[0008] To achieve the above objectives, the present invention adopts the following technical solution: In a first aspect, the present invention provides a phenylethanol glycoside compound, which is a hydroxytyrosol-1-O-β-D-glucoside, and its chemical structural formula is shown in Formula I: .

[0009] The compound's English name is: 3',4'dihydroxyphenethyl alcohol 1-O-β-D-glucopyranoside, with the molecular formula C1. 14 H20 O8 is a new natural product isolated from honeydew flowers for the first time.

[0010] Secondly, the present invention provides a method for preparing the phenylethanol glycosides, comprising the following steps: (1) Extraction: The honeydew flower medicinal material was added to an alcohol solvent for extraction to obtain an extract, and the extract was concentrated under reduced pressure to obtain a paste; (2) Extraction: The extract obtained in step (1) is dispersed in water and extracted with petroleum ether and ethyl acetate in sequence. The ethyl acetate phase is collected and concentrated under reduced pressure to obtain the ethyl acetate extract. (3) Silica gel column chromatography purification: The ethyl acetate extract obtained in step (2) was separated and purified by secondary silica gel column chromatography to obtain column chromatography sample I rich in the target compound; (4) Gel chromatography purification: The column chromatography sample I obtained in step (3) is separated and purified by Sephadex LH-20 gel chromatography, and the target fraction is collected to obtain the hydroxytyrosol-1-O-β-D-glucoside.

[0011] Preferably, the alcohol solvent in step (1) is an aqueous solution of methanol or ethanol at a concentration of 85 vol% to 95 vol%. This concentration range can balance the contradiction between the dissolution efficiency of the target substance and the amount of impurities dissolved.

[0012] Preferably, the extraction method in step (1) can be immersion extraction, hot reflux extraction, ultrasonic extraction or any combination of two of them, such as immersion extraction and ultrasonic combination, immersion extraction and hot reflux extraction combination, etc. Other arbitrary combinations will not be described in detail here.

[0013] Preferably, in step (1), the mass-to-volume ratio of the honeysuckle herb to the solvent is 1:3-10, expressed in g / ml. The extraction method is immersion extraction, performed 2-4 times, with each extraction lasting 3-4 days. Immersion extraction slowly releases the target compound through static osmosis, reducing the risk of thermal degradation compared to dynamic extraction. The 2-4 repeated extractions combined with the long immersion time of 3-4 days each time create a multi-stage concentration gradient diffusion, allowing the target compound within the cell wall to gradually migrate into the solvent. The synergistic effect of these parameters achieves efficient enrichment of the target component in honeysuckle while maintaining the structural integrity of the compound.

[0014] The extraction process employs a solvent system with increasing polarity. After petroleum ether is used to preferentially remove lipid-soluble impurities, ethyl acetate can selectively enrich the target analytes with isopolarity.

[0015] Preferably, in step (2), the mass-to-volume ratio of extract to water is 1:1 to 1.5 (g / ml), the volume ratio of water to petroleum ether is 1:1 to 3, and the extraction is performed 2 to 3 times. The volume ratio of water to ethyl acetate is 1:1 to 3.

[0016] In step (3), the secondary column chromatography adjusts the polarity ratio of the elution solvent. The first chromatography uses a dichloromethane-methanol gradient with a wide polarity range to achieve coarse separation, and the second chromatography uses a petroleum ether-ethyl acetate gradient to achieve fine separation, forming a process route for step-by-step purification.

[0017] Preferably, in the silica gel column chromatography described in step (3), the packing material is 200-300 mesh silica gel, and the first silica gel column chromatography elution method is to elute with a solvent gradient of dichloromethane to methanol at volume ratios of 100:0, 95:5, 90:10, 80:20, and 100:0, and the second silica gel column chromatography elution method is to elute with a solvent gradient of petroleum ether to ethyl acetate at volume ratios of 99:1, 95:5, 50:50, and 40:60. In step (4), the gel chromatography stage can effectively separate trace impurities with molecular weights similar to the target analyte based on differences in molecular size.

[0018] Preferably, the elution solvent used in step (4) for Sephadex LH-20 gel chromatography purification is methanol.

[0019] Thirdly, the present invention provides the use of the hydroxytyrosol-1-O-β-D-glucoside or a pharmaceutically acceptable salt thereof in the preparation of antitumor drugs.

[0020] Preferably, the drug exerts its anti-tumor effect by inhibiting the growth of tumor cells.

[0021] Preferably, the tumor includes, but is not limited to, liver cancer, lung cancer, gastrointestinal cancer, or brain cancer.

[0022] Preferably, the dosage form of the drug includes any one of tablets, capsules, solutions, aerosols, sprays, ointments, or films.

[0023] Fourthly, the present invention provides an anticancer pharmaceutical composition comprising a therapeutically effective amount of the hydroxytyrosol-1-O-β-D-glucoside or a pharmaceutically acceptable salt thereof, and pharmaceutically acceptable excipients.

[0024] Preferably, the pharmaceutically acceptable excipients include any one or a combination of at least two of the following: sustained-release agents, excipients, fillers, binders, wetting agents, disintegrants, absorption promoters, surfactants, and lubricants. The combination of at least two is, for example, a combination of binders and excipients, a combination of binders and flavoring agents, a combination of binders and fillers, etc. Other combinations are also possible and will not be elaborated here.

[0025] Beneficial effects This invention is the first to isolate a novel phenylethanol glycoside compound, hydroxytyrosol-1-O-β-D-glucoside, from honeydew flowers. This compound has not been reported in the prior art and is a novel compound.

[0026] The preparation method provided by this invention is stable and reproducible. By combining solvent extraction, liquid-liquid extraction, multiple silica gel column chromatography and gel chromatography, the compound can be separated from honeysuckle flowers with high efficiency and high purity.

[0027] The compounds of this invention have been shown in in vitro antitumor activity tests to have significant inhibitory effects on the growth of various human tumor cell lines (such as liver cancer HepG2, Huh-7, etc.), demonstrating good prospects for antitumor applications.

[0028] This invention provides new lead compounds and material basis for the development of novel antitumor drugs and expands the medicinal value of honeydew flowers. Attached Figure Description

[0029] Figure 1 Example 1: Preparation of phenylethanol glycosides 1 H-NMR spectrum; Figure 2 Example 1: Preparation of phenylethanol glycosides 13 C-NMR spectrum; Figure 3 The results of the experiment on the inhibitory activity of hydroxylamine against HepG2 liver cancer cells. * in the figure indicates that there is a statistically significant difference between the control group and the treatment group (*: p<0.05, **: p<0.01, ***: p<0.001). Figure 4 Experimental results of the inhibitory activity of hydroxylamine against Huh-7 liver cancer cells. * in the figure indicates that there is a statistically significant difference between the control group and the treatment group (*: p<0.05, **: p<0.01, ***: p<0.001). Figure 5 Experimental results of the inhibitory activity of hydroxytyrosol-1-O-β-D-glucoside on HepG2 liver cancer cells. * in the figure indicates that there is a statistically significant difference between the control group and the treatment group (*: p<0.05, **: p<0.01, ***: p<0.001). Figure 6 Experimental results of the inhibitory activity of hydroxytyrosol-1-O-β-D-glucoside on Huh-7 liver cancer cells. * in the figure indicates that the control group and the treatment group are statistically significant (*: p<0.05, **: p<0.01, ***: p<0.001). Detailed Implementation

[0030] The present invention will now be described in further detail with reference to the accompanying drawings and embodiments, but this does not limit the present invention in any way. Any modifications or improvements made based on the teachings of the present invention shall fall within the protection scope of the present invention.

[0031] The processes, conditions, reagents, and experimental methods used in implementing this invention, except as specifically mentioned below, are all common knowledge and general knowledge in the field, and this invention does not have any particular limitations. Experimental methods in the embodiments that do not specify specific conditions are generally performed under conventional conditions or as recommended by the manufacturer.

[0032] Unless otherwise stated, all technical terms and scientific terms used in this specification have the same meaning as commonly understood by one of ordinary skill in the art to which this invention pertains. However, in the event of any conflict, the specification containing the definitions shall prevail.

[0033] The following are some of the main raw materials and reagents used in the examples: Honeycomb Flower ( Brandisia hancei Hook. F. Purchased from the pharmacy of Yunnan Huangjia Medical Circle Traditional Chinese Medicine Oncology Hospital; Hydroxytyrosol was purchased from Shanghai Hanxiang Biotechnology Co., Ltd., with a purity of 98%. HepG2 and Huh-7 cells were purchased from the Cell Bank of the Chinese Academy of Sciences.

[0034] Example 1: Preparation of Hydroxytyrosol-1-O-β-D-Glucoside (1) Extraction: Take 29 kg of dried honeydew flower medicinal material, crush it, and extract it three times at room temperature with 95% ethanol aqueous solution (material-liquid ratio 1:8, g / ml), each time for 4 days. Combine the extracts and concentrate them under reduced pressure at 50℃ to recover the solvent and obtain the extract.

[0035] (2) Extraction: The above extract was suspended in 1.5 times its volume of distilled water and extracted three times each with equal volumes of petroleum ether and ethyl acetate. The ethyl acetate phase was collected and concentrated under reduced pressure to obtain the ethyl acetate extract.

[0036] (3) Silica gel column chromatography purification: First column chromatography: The ethyl acetate extract was packed into a 200-300 mesh silica gel column (ethyl acetate extract to silica gel mass ratio of 1:6) using a dry method. Gradient elution was performed using a dichloromethane-methanol system (100:0~95:5~90:10~80:20~100:0, v / v), with 5-8 column volumes eluted per gradient. The results were detected by thin-layer chromatography (TLC). Eluents with similar components were combined and concentrated under reduced pressure to obtain five fractions, B1 to B5.

[0037] Second column chromatography: Take fraction B4 and elute with a petroleum ether-ethyl acetate system (99:1~95:5~50:50~40:60, v / v). Elute 5~8 column volumes for each gradient and detect by thin-layer chromatography (TLC). Combine the eluents containing the target compound to obtain column chromatography sample I. (4) Gel chromatography purification: The above column chromatography sample I was purified by passing it through a Sephadex LH-20 gel column with methanol as the eluent. The fraction containing the target compound was collected by thin-layer chromatography (TLC), concentrated under reduced pressure and dried under vacuum to obtain approximately 3.7 mg of pure hydroxytyrosol-1-O-β-D-glucoside with a purity of 90%.

[0038] Compound structure characterization The compound is a white powder; its NMR spectrum is shown below. Figure 1-2 The data is attributed to the following: 1 H-NMR (400 MHz, CD3OD) δ: 2.82 (2H, t, J = 7.5 Hz, H-2), 4.40 (1H,d, J = 7.9 Hz, H-1''), 6.60 (1H, dd, J = 8.0, 2.1 Hz, H-6'), 6.70 (1H, d, J =8.0 Hz, H-5'), 6.73 (1H, d, J = 2.2 Hz, H-2'); 13 C-NMR (100 MHz, CD3OD) δ: 146.1 (C-3'), 144.9 (C-4'), 131.5 (C-1'), 121.3 (C-6'), 117.1 (C-2'), 116.3 (C-5'), 103.0 (C-1''), 77.6 (C-3''), 76.1(C-5''), 74.9 (C2''), 72.4 (C-1), 70.5 (C-4''), 62.0 (C-6''), 36.6 (C-2). The above spectral data are consistent with the structure of hydroxytyrosol-1-O-β-D-glucoside, confirming its structure as shown in Formula I:

[0039] Example 2: Preparation of Hydroxytyrosol-1-O-β-D-Glucoside (1) Extraction: Take 29 kg of dried honeydew flower medicinal material, crush it, and extract it 4 times with 85% ethanol aqueous solution (material-liquid ratio 1:3, g / ml) at room temperature, each time for 4 days. Combine the extracts and concentrate them under reduced pressure at 50℃ to recover the solvent and obtain the extract.

[0040] (2) Extraction: The above extract was suspended in one volume of distilled water and extracted twice each with three volumes of petroleum ether and ethyl acetate. The ethyl acetate phase was collected and concentrated under reduced pressure to obtain the ethyl acetate extract.

[0041] (3) Silica gel column chromatography purification: First column chromatography: The ethyl acetate extract was packed into a 200-300 mesh silica gel column (ethyl acetate extract to silica gel mass ratio of 1:6) using a dry method. Gradient elution was performed using a dichloromethane-methanol system (100:0~95:5~90:10~80:20~100:0, v / v), with 5-8 column volumes eluted per gradient. The results were detected by thin-layer chromatography (TLC). Eluents with similar components were combined and concentrated under reduced pressure to obtain five fractions, B1 to B5.

[0042] Second column chromatography: Take fraction B4 and elute with a petroleum ether-ethyl acetate system (99:1~95:5~50:50~40:60, v / v). Elute 5~8 column volumes for each gradient and detect by thin-layer chromatography (TLC). Combine the eluents containing the target compound to obtain column chromatography sample I. (4) Gel chromatography purification: The above column chromatography sample I was purified by passing it through a Sephadex LH-20 gel column with methanol as the eluent. The fraction containing the target compound was collected by thin-layer chromatography (TLC), concentrated under reduced pressure and dried under vacuum to obtain about 3.5 mg of pure hydroxytyrosol-1-O-β-D-glucoside with a purity of 91%. The structure was the same as in Example 1.

[0043] Example 3: Preparation of Hydroxytyrosol-1-O-β-D-Glucoside (1) Extraction: Take 29 kg of dried honeydew flower medicinal material, crush it, and extract it twice with 90% methanol aqueous solution (material-liquid ratio 1:10, g / ml) at room temperature for 3 days each time. Combine the extracts and concentrate them under reduced pressure at 50℃ to recover the solvent and obtain the extract.

[0044] (2) Extraction: The above extract was suspended in 1.5 times its volume of distilled water and extracted three times each with 2 times its volume of petroleum ether and ethyl acetate. The ethyl acetate phase was collected and concentrated under reduced pressure to obtain the ethyl acetate extract.

[0045] (3) Silica gel column chromatography purification: First column chromatography: The ethyl acetate extract was packed into a 200-300 mesh silica gel column (ethyl acetate extract to silica gel mass ratio of 1:6) using a dry method. Gradient elution was performed using a dichloromethane-methanol system (100:0~95:5~90:10~80:20~100:0, v / v), with 5-8 column volumes eluted per gradient. The results were analyzed by thin-layer chromatography (TLC). Eluents with similar components were combined and concentrated under reduced pressure to obtain five fractions, B1 to B5.

[0046] Second column chromatography: Take fraction B4 and elute with a petroleum ether-ethyl acetate system (99:1~95:5~50:50~40:60, v / v). Elute 5~8 column volumes for each gradient and detect by thin-layer chromatography (TLC). Combine the eluents containing the target compound to obtain column chromatography sample I. (4) Gel chromatography purification: The above column chromatography sample I was purified by passing it through a Sephadex LH-20 gel column with methanol as the eluent. The fraction containing the target compound was collected by thin-layer chromatography (TLC), concentrated under reduced pressure and dried under vacuum to obtain approximately 3.9 mg of pure hydroxytyrosol-1-O-β-D-glucoside with a purity of 89%. The structure was the same as in Example 1.

[0047] Example 4: In vitro antitumor activity test The liver cancer cell lines HepG and Huh-72, stored at -80℃, were thawed, resuspended, and cultured in a cell culture incubator with the medium changed every two days. After passage, cells in the logarithmic growth phase were used for experiments. Cells were cultured at 4 × 10⁻⁶ cells / day. 4 Cells were seeded at the appropriate density in 96-well plates, with 5 accessory wells per cell group. The remaining wells were filled with an equal volume of PBS. Three plates were seeded in parallel for detection at 24h, 48h, and 72h. 100 μL of medium containing different concentrations of hydroxytyrosol-1-O-β-D-glucoside (final concentrations of 1, 3, 13, 30, and 100 μM) and hydroxytyrosol (final concentrations of 150, 300, 600, 1200, and 2400 μM) were added to each well, and the plates were incubated for 24h. After 24h, the 96-well plates were removed, and 10 μL of 5 mg / mL MTT solution was added to each well. The plates were incubated for another 4–6h, the liquid was discarded, and DMSO was added in the dark. The plates were then shaken for 10 min to dissolve any crystals. The absorbance was measured at 570 nm. The same procedure was repeated for the 24h plates at 48h and 72h. The obtained absorbance values ​​were processed and analyzed.

[0048] Human hepatocellular carcinoma cell lines HepG2 and Huh-7, frozen at -80℃, were thawed and then cultured in DMEM complete medium containing 10% fetal bovine serum and 1% penicillin-streptomycin at 37℃ with 5% CO2. The medium was replaced with fresh medium every two days. Once the cells reached the logarithmic growth phase, they were digested with trypsin and resuspended for subsequent experiments.

[0049] Cells in the logarithmic growth phase were spaced at 4 × 10⁴ cells per well. 4 Cells were seeded at a density of 100 μL in each well of a 96-well plate. Five replicates were set up for each experiment, and an equal volume of PBS was added to the remaining blank wells to maintain humidity balance. Simultaneously, three 96-well plates were independently seeded for each time point (24 h, 48 h, 72 h).

[0050] After 24 hours of cell attachment, the original culture medium was discarded. Fresh culture medium containing different concentrations of the drug was added to each experimental group. Test compound group: Hydroxytyrosol-1-O-β-D-glucoside (sample prepared in Example 1), with final concentrations of 1, 3, 13, 30, and 100 μM.

[0051] Positive control group: Hydroxytyrosol, with final concentrations of 150, 300, 600, 1200, and 2400 μM.

[0052] Blank control group: Culture medium containing only an equal volume of drug solvent.

[0053] Each well contains 100 μL. The 96-well plate is then returned to the incubator for further incubation.

[0054] Detection was performed at 24 h, 48 h, and 72 h after drug addition. At each detection time point, 10 μL of MTT solution (5 mg / mL) was added to each well, and the cells were incubated for another 4 hours. Subsequently, the supernatant in the wells was carefully discarded, and 150 μL of LDMSO was added to each well. The cells were then placed on a shaker and shaken at low speed for 10 minutes to ensure that the formazan crystals were fully dissolved.

[0055] The absorbance of each well was measured at 570 nm using a microplate reader. Cell viability and proliferation inhibition rate were calculated by comparing the absorbance values ​​of each group with the blank control group. All experiments were independently repeated three times. Data are expressed as mean ± standard deviation and analyzed using appropriate statistical methods.

[0056] The results showed that, compared with the control group, the cell viability of HepG2 and Huh-7 liver cancer cells decreased in a time- and drug concentration-dependent manner after hydroxytyrosol intervention (see [link to study]). Figure 3-4The 48-hour IC50 values ​​for HepG2 and Huh-7 cells were 4390 μM and 3610 μM, respectively. After intervention with the compound hydroxytyrosol-1-O-β-D-glucoside, compared with the control group, the cell viability of HepG2 and Huh-7 liver cancer cells was significantly reduced in a time- and drug concentration-dependent manner (see...). Figure 5-6 Furthermore, the 48-hour IC50 values ​​for HepG2 and Huh-7 cells were 43.623 μM and 25.19 μM, respectively. These results indicate that the inhibitory activity of the compound hydroxytyrosol-1-O-β-D-glucoside of this invention on HepG2 and Huh-7 cells is significantly higher than that of hydroxytyrosol, suggesting that it has good anticancer activity and is promising for development into an antitumor drug.

[0057] Finally, it should be noted that the above embodiments are only used to illustrate the technical solutions of the present invention, and not to limit them; although the present invention has been described in detail with reference to the foregoing embodiments, those skilled in the art should understand that modifications can still be made to the technical solutions described in the foregoing embodiments, or equivalent substitutions can be made to some of the technical features; and these modifications or substitutions do not cause the essence of the corresponding technical solutions to deviate from the spirit and scope of the technical solutions of the embodiments of the present invention.

Claims

1. A phenylethanol glycoside compound, characterized in that, The phenylethanol glycoside is hydroxytyrosol-1-O-β-D-glucoside, and its chemical structural formula is shown in Formula I: 。 2. The method for preparing the phenylethanol glycoside compound according to claim 1, characterized in that, It includes the following steps: (1) The honeydew flower medicinal material was extracted with an alcohol solvent to obtain an extract, and the extract was concentrated under reduced pressure to obtain a paste; (2) Disperse the extract obtained in step 1 in water, extract with petroleum ether and ethyl acetate in sequence, collect the ethyl acetate phase, and concentrate under reduced pressure to obtain the ethyl acetate extract; (3) The ethyl acetate extract obtained in step (2) was purified by secondary silica gel column chromatography to obtain column chromatography sample I; (4) The column chromatography sample I obtained in step (3) was separated and purified by Sephadex LH-20 gel chromatography to obtain phenylethanol glycosides.

3. The preparation method according to claim 2, characterized in that, The alcohol solvent in step (1) is an aqueous solution of methanol or ethanol at a concentration of 85 vol% to 95 vol%.

4. The preparation method according to claim 2, characterized in that, In step (3), the silica gel column chromatography uses 200-300 mesh silica gel as the packing material. The first silica gel column chromatography elution method is to use a solvent gradient elution with dichloromethane and methanol in volume ratios of 100:0, 95:5, 90:10, 80:20, and 100:

0. The second silica gel column chromatography elution method is to use a solvent gradient elution with petroleum ether and ethyl acetate in volume ratios of 99:1, 95:5, 50:50, and 40:

60.

5. The preparation method according to claim 2, characterized in that, In step (4), the elution solvent in the Sephadex LH-20 gel chromatography purification is methanol.

6. The use of the phenylethanol glycoside compound of claim 1 or a pharmaceutically acceptable salt thereof in the preparation of an antitumor drug.

7. The application according to claim 6, characterized in that, The drug inhibits the growth of tumor cells.

8. The application according to claim 6, characterized in that, The tumor is liver cancer, lung cancer, gastrointestinal cancer, or brain cancer.

9. The application according to claims 6-8, characterized in that, The dosage form of the drug includes any one of tablets, capsules, solutions, aerosols, sprays, ointments, or films.

10. An anticancer drug composition, characterized in that, Includes the phenylethanol glycosides of claim 1 or their pharmaceutically acceptable salts and pharmaceutically acceptable excipients.

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