Flavonoid glycoside compound as well as preparation method and application thereof

Abstract

The invention discloses a flavonoid glycoside compound as well as a preparation method and application thereof. The invention provides a flavonoid glycoside compound 2 ''-O-benzoyl isoorientin. The compound is a new flavonoid glycoside compound which is separated from gentiana rigescens for the first time. The invention further provides a preparation method of the 2 ''-O-benzoyl isoorientin, and further provides application of the flavonoid glycoside compound 2 ''-O-benzoyl isoorientin in preparation of anti-aging cosmetics or drugs and application of the flavonoid glycoside compound 2 ''-O-benzoyl isoorientin in preparation of anti-tumor drugs.

Description

TECHNICAL FIELD

[0001] The present application relates to the technical field of medicinal compounds, in particular to a flavonoid glycoside compound, a preparation method and application thereof, more particularly to a new flavonoid glycoside compound extracted and separated from Gentiana rigescens Franch., a preparation method thereof and application of the flavonoid glycoside compound in preparation of anti-aging cosmetics or drugs and anti-tumor drugs. BACKGROUND

[0002] Gentiana rigescens Franch. Gentiana rigescens As a traditional medicinal plant in China, Gentiana rigescens Franch. has been widely recognized for its medicinal value, but there is still a lot of exploration space for the systematic study of its active ingredients. Modern research shows that Gentiana rigescens Franch. mainly contains iridoid, flavonoids, triterpenoids and phenolic acids and other active ingredients. Among them, flavonoids have attracted much attention due to their unique structural characteristics and wide pharmacological activities.

[0003] In the field of anti-aging research, with the intensification of population aging, the development of safe and effective anti-aging active ingredients has become a research hotspot. The existing synthetic anti-aging ingredients generally have problems such as strong irritation and poor stability, while the compounds from natural sources show unique advantages such as multi-target action mechanism and low toxicity, becoming an important source for the development of anti-aging products.

[0004] In the research and development of anti-tumor drugs, non-small cell lung cancer (NSCLC) is the most common type of lung cancer in clinical, and its treatment still faces challenges such as chemotherapy resistance and limited indications for targeted therapy. Natural products have become an important source for the research and development of anti-tumor drugs due to their structural diversity and low toxicity.

[0005] At present, the research on flavonoid glycosides isolated from Gentiana rigescens Franch. is relatively insufficient, and its anti-aging and anti-tumor activities have not been fully explored. In particular, the research on anti-NSCLC is still in its infancy, and the related active ingredients and their mechanisms of action need to be further explored. Therefore, discovering flavonoid glycosides with novel structures from Gentiana rigescens Franch. and elucidating their molecular mechanisms of anti-aging and anti-NSCLC will not only help to expand the medicinal value of Gentiana rigescens Franch., but also provide important candidate molecules for the development of new anti-aging and anti-tumor drugs. SUMMARY

[0006] In view of the problems in the prior art, the present application provides a flavonoid glycoside compound isolated from Gentiana rigescens Franch., a preparation method and application thereof.

[0007] To achieve the purpose of the present application, the following technical solutions are adopted: In a first aspect, the present application provides a flavonoid glycoside compound, which is 2''-O-benzoyl isohorslinin, and its chemical structural formula is as shown in Figure 1 ​

[0008] In a second aspect, the present application provides a preparation method of the flavonoid glycosides, comprising the following steps: (1) adding Gentiana rigescens to a solvent to extract to obtain an extract, and concentrating the extract under reduced pressure to obtain a first extract; (2) dispersing the first extract obtained in step (1) in water, and sequentially extracting with ethyl acetate and n-butanol, collecting the n-butanol phase, and concentrating under reduced pressure to obtain a second extract; (3) purifying the second extract obtained in step (2) by macroporous adsorption resin column chromatography to obtain a column chromatography sample I; (4) purifying the column chromatography sample I obtained in step (3) by MCI microporous resin column chromatography to obtain a column chromatography sample II; (5) purifying the column chromatography sample II obtained in step (4) by preparative high performance liquid chromatography to obtain the flavonoid glycosides.

[0009] The Gentiana rigescens is a perennial herb of Gentianaceae Gentiana, mainly produced in Yunnan, Sichuan and Guizhou. Gentiana rigescens Franch. The Gentiana rigescens is a perennial herb of Gentianaceae Gentiana, mainly produced in Yunnan, Sichuan and Guizhou.

[0010] Preferably, in step (1), the mass-volume ratio of the Gentiana rigescens to the solvent is 1:3-10, the extraction is hot reflux extraction, and the extraction is performed 2-4 times, each time for 1.5-2.5 h.

[0011] Preferably, in step (2), the volume ratio of the extract to water is 1:1-1.5, the volume ratio of water to ethyl acetate is 1:1-3, the volume ratio of water to n-butanol is 1:1-3, and the extraction is performed 1-3 times.

[0012] Preferably, in steps (1) and (2), the concentration under reduced pressure is to the point that there is no odor of organic solvent.

[0013] Preferably, in step (3), the macroporous adsorption resin column chromatography purification uses LX-T28 type macroporous adsorption resin, the mass ratio of the second extract sample to the macroporous adsorption resin is 1:3-4, the height-diameter ratio of the chromatography column is 6-8:1, the elution is performed by sequentially using elution solvents with methanol-water volume ratios of 0:100, 30:70, 60:40, 70:30 and 100:0, each gradient elution is performed for 3-5 column volumes, the part with a methanol-water volume ratio of 70:30 is collected, and the column chromatography sample I is obtained by concentrating under reduced pressure and drying. Preferably, in the step (4), the MCI microporous resin column chromatography separation and purification, the MCI microporous resin is CHP20 / P20; the mass ratio of the sample I and the MCI microporous resin is 1:4-6; the height-diameter ratio of the column is 14-16:1, the elution mode is: the elution flow rate is 20-30 mL / min, and the elution solvents with the methanol-water volume ratio of 40:60 and 50:50 are sequentially used for elution, each gradient elution is 3-5 column volumes, the 40:60 part eluent is collected, and the column chromatography sample II is obtained by drying under reduced pressure. Preferably, in the step (5), the preparative high performance liquid chromatography separation and purification, the chromatographic column is a C18 reversed-phase chromatographic column, the elution mode is isocratic elution using the elution solvent with the methanol-water volume ratio of 36:64, the flow rate is 10 mL / min, the eluent collected is 6-7 min, and the flavonoid glycosides are obtained by drying under reduced pressure.

[0014] Preferably, the chromatographic column specifications are 250 mm*20 mm, 10 μm.

[0015] In a third aspect, the application provides the use of the flavonoid glycosides in the preparation of anti-aging cosmetics or drugs.

[0016] Preferably, the anti-aging cosmetics or drugs inhibit the expression of p21 and p16 genes.

[0017] In a fourth aspect, the application provides the use of the flavonoid glycosides in the preparation of anti-tumor drugs.

[0018] Preferably, the tumor is lung cancer. Preferably, the lung cancer is non-small cell lung cancer.

[0019] Preferably, the drug further comprises a pharmaceutically acceptable excipient.

[0020] Preferably, the dosage form of the drug comprises any one of a tablet, a capsule, a solution, an aerosol, a spray, an ointment or a film.

[0021] Preferably, the drug further comprises a pharmaceutically acceptable excipient.

[0022] Preferably, the excipient comprises any one of a diluent, a flavoring agent, a binder or a filler, or a combination of at least two, such as a combination of a binder and a diluent, a combination of a binder and a flavoring agent, a combination of a binder and a filler, and the like, and other optional combinations can also be selected, which will not be described here one by one.

[0023] The application has the following beneficial effects: The application discloses a new flavonoid glycoside compound 2''-O-benzoylisohamnetin from Gentiana rigescens, and provides a method for separating and purifying the compound from Gentiana rigescens, which is simple in operation and easy for industrialized preparation. The application further discloses that the flavonoid glycoside compound 2''-O-benzoylisohamnetin can inhibit the expression of p21 and p16 genes, and can be used for preparing anti-aging cosmetics or medicines. Furthermore, the flavonoid glycoside compound 2''-O-benzoylisohamnetin can inhibit the proliferation of non-small cell lung cancer cells A549, and can be used for preparing anti-tumor medicines, especially non-small cell lung cancer medicines, thereby providing a new selection for anti-aging and anti-cancer medicine development. BRIEF DESCRIPTION OF DRAWINGS

[0024] Figure 1 is a chemical structural formula of 2''-O-benzoylisohamnetin; Figure 2 is a chemical structural formula of 2''-O-benzoylisohamnetin 1 H-NMR spectrum; Figure 3 is a chemical structural formula of 2''-O-benzoylisohamnetin 13 C-NMR spectrum and DEPT spectrum; Figure 4 is a UV spectrum of 2''-O-benzoylisohamnetin; Figure 5 is an IR spectrum of 2''-O-benzoylisohamnetin; Figure 6 is a chemical structural formula of 2''-O-benzoylisohamnetin 1 H- 1 H COSY spectrum; Figure 7 is a HSQC spectrum of 2''-O-benzoylisohamnetin; Figure 8 is a HMBC spectrum of 2''-O-benzoylisohamnetin; Figure 9 is a toxicity evaluation diagram of 2''-O-benzoylisohamnetin on HaCaT cells; Figure 10 is a p21 expression level diagram of HaCaT cells treated by 2''-O-benzoylisohamnetin with different concentrations; Figure 11 is a p16 expression level diagram of HaCaT cells treated by 2''-O-benzoylisohamnetin with different concentrations; Figure 12 is a proliferation inhibition rate diagram of 2''-O-benzoylisohamnetin on A549 cells. DETAILED DESCRIPTION

[0025] The application will be further described in detail below with reference to the accompanying drawings and examples, but in no way limits the application, any transformation or improvement based on the teaching of the application falls into the protection scope of the application.

[0026] The process, conditions, reagents, experimental methods, etc. for implementing the application, except for the following specifically mentioned contents, are the general knowledge and common sense in the art, and the application has no special limitations. The experimental methods not specified in the specific conditions in each example are usually carried out according to the conventional conditions or according to the conditions recommended by the manufacturer.

[0027] The following are the sources of some main raw materials and reagents in the examples: Gentiana rigescens Diels: collected in Yun County, Lincang City, Yunnan Province; Human immortalized keratinocytes (HaCaT): Wuhan Ponsay Life Science and Technology Co., Ltd.; Non-small cell lung cancer cells (A549): Wuhan Ponsay Life Science and Technology Co., Ltd.; Macroporous adsorption resin: LXT-28 type (Xi'an Lanxiao Science and Technology New Material Co., Ltd.); MCI microporous resin: CHP20 / P20 type (Mitsubishi Chemical, Japan); High performance liquid chromatograph: Agilent 1260 type high performance liquid chromatograph (Agilent, USA).

[0028] Example 1 Preparation of flavonoid glycoside compound 2''-O-benzoyl isohorskinin (1) The raw material of Gentiana rigescens Diels was added into 5 times of 90% methanol aqueous solvent for hot reflux extraction, each time for 2 h, and the extraction was carried out twice. The extract was combined and concentrated under reduced pressure to obtain a first extract; (2) The first extract obtained in step 1 was dispersed in water (the volume ratio of the first extract to water was 1:1), and then extracted with ethyl acetate (the volume ratio of water to ethyl acetate was 1:2.5) and n-butanol (the volume ratio of water to n-butanol was 1:2.5) in sequence, each solvent extraction was carried out twice. The n-butanol phase was collected and concentrated under reduced pressure to obtain a second extract; (3) The second extract obtained in step (2) was separated and purified by macroporous adsorption resin column chromatography: the macroporous adsorption resin was LX-T28, the mass ratio of the second extract sample to the macroporous adsorption resin was 1:3, the height-diameter ratio of the chromatography column was 6:1, and the elution mode was to elute with methanol-water elution solvents with volume ratios of 0:100, 30:70, 60:40, 70:30 and 100:0 in sequence, each gradient elution was carried out for 3 column volumes. The part with a methanol-water volume ratio of 70:30 was collected, concentrated under reduced pressure and dried to obtain a column chromatography sample I; (4) The column chromatography sample I obtained in step (3) was separated and purified by MCI microporous resin column chromatography: the MCI microporous resin was CPH20 / P20, the mass ratio of the column chromatography sample I to the MCI microporous resin was 1:5; the height-diameter ratio of the column was 14:1, and the elution mode was as follows: the elution flow rate was 20 mL / min, and the elution solvents with methanol-water volume ratios of 40:60 and 50:50 were used for elution in sequence, 5 column volumes for each gradient elution, the eluate in the 40:60 part was collected, and column chromatography sample II was obtained by drying under reduced pressure; The column chromatography sample II obtained in step (4) was separated and purified by preparative high performance liquid chromatography: the chromatographic column was a reversed-phase chromatographic column (C18, 250 mm×20 mm, 10 μm, Daisogel, Japan), isocratic elution was performed using an elution solvent with a methanol-water volume ratio of 36:64, the flow rate was 10 mL / min, the eluate with a peak eluted at 6-7 min was collected, and the flavonoid glycoside compound was obtained by drying under reduced pressure, with a purity of 95 %.

[0029] The flavonoid glycoside compound was a yellow powder: it was identified by 1 H-NMR (500 MHz, CD3OD), 13 C-NMR (125 MHz, CD3OD), mass spectrometry, infrared spectroscopy, etc. Figure 1 , and the molecular formula was C 28 H 24 O 12 , and it was named as 2''-O-benzoylisoorientin, with structure identification data of ESI-MS m / z 551 [M-H] - , 1 H-NMR (500 MHz, CD3OD), 13 C-NMR (125 MHz, CD3OD), data assignment is shown in Table 1 and Figures 2-3 , the UV spectrum is shown in Figure 4 , the IR spectrum is shown in Figure 5 , the COSY spectrum is shown in Figure 6 , the HSQC spectrum Figure 7 , and the HMBC spectrum is shown in Figure 8 .

[0030] Table 1 shows the 1 H and 13 C NMR data assignment of 2''-O-benzoylisoorientin (CD3OD) (δH 3.30 ppm, δC 49.0 ppm)

[0031] Preparation of the flavonoid glycoside compound 2''-O-benzoyl isohispidulin (1) The Gentiana rigescens H. Smith raw material was added to 10 times 85% methanol aqueous solvent for hot reflux extraction, each time for 2.5 h, and the extraction was performed twice. The extract was combined and concentrated under reduced pressure to obtain a first extract; (2) The first extract obtained in step 1 was dispersed in water (the volume ratio of the first extract to water was 1:1), and was sequentially extracted with ethyl acetate (the volume ratio of water to ethyl acetate was 1:1) and n-butanol (the volume ratio of water to n-butanol was 1:1), each solvent for 1 time. The n-butanol phase was collected and concentrated under reduced pressure to obtain a second extract; (3) The second extract obtained in step (2) was separated and purified by macroporous adsorption resin column chromatography. The macroporous adsorption resin was LX-T28, and the mass ratio of the second extract sample to the macroporous adsorption resin was 1:4. The height-diameter ratio of the column was 8:1. The elution mode was sequential elution with methanol-water elution solvents with volume ratios of 0:100, 30:70, 60:40, 70:30 and 100:0, each gradient elution for 5 column volumes. The methanol-water fraction with a volume ratio of 70:30 was collected, and the column chromatography sample I was obtained by drying under reduced pressure; (4) The column chromatography sample I obtained in step (3) was separated and purified by MCI microporous resin column chromatography. The MCI microporous resin was CHP20 / P20, and the mass ratio of the column chromatography sample I to the MCI microporous resin was 1:6. The height-diameter ratio of the column was 16:1. The elution mode was sequential elution with methanol-water elution solvents with volume ratios of 40:60 and 50:50 at a flow rate of 30 mL / min, each gradient elution for 3 column volumes. The 40:60 fraction eluent was collected, and the column chromatography sample II was obtained by drying under reduced pressure; (5) The column chromatography sample II obtained in step (4) was separated and purified by preparative high performance liquid chromatography. The chromatographic column was a reversed-phase chromatographic column (C18, 250 mm x 20 mm, 10 μm, Daisogel, Japan). Isocratic elution was performed with a methanol-water elution solvent with a volume ratio of 36:64 at a flow rate of 10 mL / min. The eluent was collected at a peak elution time of 6-7 min, and the flavonoid glycoside compound was obtained by drying under reduced pressure.

[0032] The purity was 98%, and the structure was the same as that of Example 1.

[0033] Preparation of the flavonoid glycoside compound 2''-O-benzoyl isohispidulin (1) The Gentiana rigescens H. Smith raw material was added to 10 times 85% methanol aqueous solvent for hot reflux extraction, each time for 2.5 h, and the extraction was performed twice. The extract was combined and concentrated under reduced pressure to obtain a first extract; (2) The first extract obtained in step 1 was dispersed in water (the volume ratio of the first extract to water was 1:1.5), and was sequentially extracted with ethyl acetate (the volume ratio of water to ethyl acetate was 1:3) and n-butanol (the volume ratio of water to n-butanol was 1:3), each solvent extraction was performed for 3 times, and the n-butanol phase was collected, and after concentration under reduced pressure, a second extract was obtained; (3) The second extract obtained in step (2) was separated and purified by macroporous adsorption resin column chromatography: the macroporous adsorption resin was LX-T28, the mass ratio of the second extract sample to the macroporous adsorption resin was 1:3.5, the height-diameter ratio of the chromatography column was 7:1, the elution mode was sequential elution with methanol-water elution solvents with a volume ratio of 0:100, 30:70, 60:40, 70:30 and 100:0, each gradient elution was performed for 4 column volumes, the fraction with a methanol-water volume ratio of 70:30 was collected, and after concentration under reduced pressure and drying, a column chromatography sample I was obtained; (4) The column chromatography sample I obtained in step (3) was separated and purified by MCI microporous resin column chromatography: the MCI microporous resin was CHP20 / P20, the mass ratio of the column chromatography sample I to the MCI microporous resin was 1:4; the height-diameter ratio of the chromatography column was 15:1, the elution mode was: the elution flow rate was 25 mL / min, and sequential elution was performed with methanol-water elution solvents with a volume ratio of 40:60 and 50:50, each gradient elution was performed for 4 column volumes, the 40:60 fraction eluent was collected, and after concentration under reduced pressure and drying, a column chromatography sample II was obtained; (5) The column chromatography sample II obtained in step (4) was separated and purified by preparative high performance liquid chromatography: the chromatography column was a reversed-phase chromatography column (C18, 250 mm x 20 mm, 10 μm, Daisogel, Japan), isocratic elution was performed with a methanol-water elution solvent with a volume ratio of 36:64, the flow rate was 10 mL / min, the eluent with a peak eluted at 6-7 min was collected, and after concentration under reduced pressure and drying, the flavonoid glycoside compound was obtained.

[0034] It was detected that the purity was 97 %, and the structure was the same as that in Example 1.

[0035] Cell toxicity experiment of 2''-O-benzoyl-isohamnetin on human immortalized keratinocytes (HaCaT) Human immortalized keratinocytes (HaCaT) were inoculated in a 96-well plate, the inoculation density was 5 x 10 5 After 24 hours of culture, cells were treated with different concentrations (3.9063, 7.8125, 15.625, 31.25, 62.5, 125, 250 μg / mL) of 2''-O-benzoyl-isohamnetin for 24 h, and a blank control group was set up, the cells were treated with CCK8 for 1 h, the absorbance was measured at 450 nm, and the cell survival rate was calculated, and each group was performed in triplicate.

[0036] Results are shown in Figure 9 As shown in the results, the results showed that 2''-O-benzoyl isohorskinin exhibited different degrees of cytotoxicity at different concentrations. When the concentration was 250 μg / mL, the cytotoxicity reached 87%; 125 μg / mL was 77%; 62.5 μg / mL was 37%; 31.25 μg / mL was 47%; 15.625 μg / mL was 5%. At lower concentrations, 7.8125 μg / mL and 3.9063 μg / mL had no cytotoxicity. The experimental results showed that when the concentration was less than 15.625 μg / mL, the cytotoxicity was less than 5%, completely meeting the safety standards of cell experiments. Based on this, we selected 15.625 μg / mL and its half concentration 7.8125 μg / mL as the two safe and effective concentration gradients for subsequent experimental research. Such concentration setting can not only ensure the reliability of the experimental results, but also effectively avoid the interference of cytotoxicity on the experimental data.

[0037] Example 5 Effect of 2''-O-benzoyl isohorskinin on cell senescence This example first establishes a cell senescence model and sets up a blank control group and a model group for comparative analysis. The expression level changes of senescence-related marker genes p21 and p16 mRNA after 2''-O-benzoyl isohorskinin treatment at different concentrations (7.8125 and 15.625 μg / mL) for 24 h were detected by RT-PCR technology. The determination method is as follows: 1) Construction of senescence model After HaCaT cells inoculated in 6 plates for 24 h, they were randomly divided into blank control group and model group: discard the culture medium in each well, wash with PBS for three times, add 100 μl or 1 mL PBS to each well, the distance between cells and UV is 15 cm, irradiate for 15 s, the blank control group is covered with aluminum foil, then discard the PBS, add fresh culture medium to each well and continue to culture for 24 h.

[0038] 2) Extraction of total RNA Cells were collected after 24 hours treatment with 7.8125 μg / mL and 15.625 μg / mL of 2''-O-benzoyl-isobavirein, respectively, and digested with 0.25% trypsin, and the digestion reaction was terminated by adding complete medium. The cells were completely detached by gently blowing the six-well plate, and the cell suspension was transferred to a 1.5 mL centrifuge tube and centrifuged at 12000 rpm at 4°C for 15 minutes. After the supernatant was discarded, 200 μL of pre-cooled chloroform was added, mixed thoroughly by inverting, and the solution was allowed to separate completely at ice for 10 minutes, and then centrifuged at 12000 rpm at 4°C for 15 minutes. 200 μL of the upper aqueous phase was carefully transferred to a new centrifuge tube, taking care not to touch the protein precipitate in the middle layer. Then an equal volume of pre-cooled isopropanol was added, mixed, and the RNA was precipitated at ice for 10 minutes, and then centrifuged at 12000 rpm at 4°C for 15 minutes to collect the precipitate. After the supernatant was discarded, the precipitate was washed twice with 1 mL of 75% ethanol (centrifuged at 12000 rpm at 4°C for 15 minutes each time). Finally, the ethanol was discarded, and the precipitate was dried at room temperature for 10 minutes, and then an appropriate amount of DEPC water was added to dissolve the RNA precipitate. After the concentration of the RNA was determined, it was aliquoted and stored in a -80°C ultra-low temperature freezer for later use. The entire operation was performed on ice and in an RNase-free environment to ensure the integrity and purity of the RNA.

[0039] 3) Reverse transcription to obtain cDNA The reverse transcription to obtain cDNA was performed according to the requirements of the kit (Hifair® II1 st strand cDNA, Yixing Biotech Co., Ltd.).

[0040] 4) qPCR The system was prepared according to the requirements of the kit (Synthesis SuperMix for qPCR, Yixing Biotech Co., Ltd.), added to an eight-tube tube, 20 μL of the system per well, and three replicates were set. After centrifugation, the program was set according to the instructions, and the machine was detected.

[0041] The results are shown in Figure 10 and 11, the results showed that the expression levels of p21 and p16 mRNA in the model group were significantly up-regulated compared with the blank control group (p<0.01), which confirmed the success of the construction of the aging model. After the intervention of 2''-O-benzoyl isohorskinin, the expression of p21 and p16 showed a concentration-dependent downward trend, and the differences between each drug group and the model group were statistically significant (p<0.05). Notably, under the same concentration conditions, 2''-O-benzoyl isohorskinin had a more significant inhibitory effect on the expression of p21 gene. These results showed that 2''-O-benzoyl isohorskinin could effectively down-regulate the expression of key marker genes in senescent cells, and especially showed a stronger regulatory effect on p21 gene, suggesting that the compound might play an anti-aging role by specifically regulating the p21-related signaling pathway.

[0042] Example 6 Proliferation inhibition effect of 2''-O-benzoyl isohorskinin on non-small cell lung cancer (A549) Non-small cell lung cancer cells (A549) were inoculated in 96-well plates at a density of 5×10 5 cells / well, and cultured for 24 hours. Different concentrations (3.9063, 7.8125, 15.625, 31.25, 62.5, 125, 250, and 500 μg / mL) of 2''-O-benzoyl isohorskinin were added to treat the cells for 24 h, and a blank control group was set up. The cells were treated with CCK8 for 1 h, and the absorbance was measured at 450 nm. The cell survival rate (%) was calculated, and 3 replicates were performed for each group.

[0043] The results are shown in Figure 12 , and the results showed that the inhibition of 2''-O-benzoyl isohorskinin on the proliferation of A549 cells showed a significant concentration-dependent effect. When the drug concentration reached 500 μg / mL, the cell survival rate was significantly reduced to 35%; with decreasing concentration, the inhibitory effect gradually weakened, and at concentrations of 250 μg / mL, 125 μg / mL and 62.5 μg / mL, the cell survival rates were 38%, 67% and 78%, respectively. These results showed that the compound might inhibit the growth of non-small cell lung cancer (A549) in a dose-dependent manner.

[0044] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this application belongs. However, if there is a discrepancy between the definitions of terms in the present specification and those of the patent application filing office, the former shall prevail. Finally, it should be noted that the above examples are merely used to describe the technical solutions of the present application, but not to limit the present application; even though the present application has been described in detail with reference to the foregoing examples, those of ordinary skill in the art should understand that modifications can be made to the technical solutions recorded in the foregoing examples, or some of the technical features thereof can be equivalently replaced; and these modifications or replacements 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 application.

Claims

1. A flavonoid glycoside compound, characterized in that, The flavonoid glycoside is 2''-O-benzoyl isohorscinetin, and its chemical structural formula is shown as Formula I: 。 2. A preparation method of the flavonoid glycoside in claim 1, comprising the following steps: (1) adding the Gentiana rigescens Diels raw material into a solvent for extraction to obtain an extract, and concentrating the extract under reduced pressure to obtain a first extract; the solvent is 85 vol% to 95 vol% methanol or ethanol aqueous solution; (2) dispersing the first extract obtained in step 1 in water, sequentially extracting with ethyl acetate and n-butanol, collecting the n-butanol phase, and concentrating under reduced pressure to obtain a second extract; (3) purifying the second extract obtained in step (2) by macroporous adsorption resin column chromatography to obtain a column chromatography sample I; (4) purifying the column chromatography sample I obtained in step (3) by MCI microporous resin column chromatography to obtain a column chromatography sample II; (5) purifying the column chromatography sample II obtained in step (4) by preparative high performance liquid chromatography to obtain the flavonoid glycoside.

3. The preparation method according to claim 2, characterized in that, In step (1), the mass-volume ratio of the Gentiana rigescens Diels raw material to the solvent is 1:3 to 10, in units of g / ml, and the extraction method is hot reflux extraction, and the extraction is performed 2 to 4 times, and each extraction is performed for 1.5 to 2.5 hours.

4. The preparation method according to claim 2, characterized in that, In step (2), the volume ratio of the extract to water is 1:1 to 1.5, the volume ratio of water to ethyl acetate is 1:1 to 3, and the volume ratio of water to n-butanol is 1:1 to 3, and the extraction is performed 1 to 3 times.

5. The preparation method according to claim 2, characterized in that, In step (3), in the macroporous adsorption resin column chromatography purification, the macroporous adsorption resin is LX-T28, the mass ratio of the second extract sample to the macroporous adsorption resin is 1:3 to 4, the height-diameter ratio of the chromatography column is 6 to 8:1, the elution method is to sequentially elute with methanol-water elution solvents with a volume ratio of 0:100, 30:70, 60:40, 70:30 and 100:0, 3 to 5 column volumes for each gradient elution, the methanol-water elution liquid with a volume ratio of 70:30 is collected, and the column chromatography sample I is obtained by concentrating and drying under reduced pressure; Preferably, in the MCI microporous resin column chromatography purification in step (4), the MCI microporous resin is CHP20 / P20; the mass ratio of the column chromatography sample I to the MCI microporous resin is 1:4 to 6; the height-diameter ratio of the chromatography column is 14 to 16:1, the elution method is to sequentially elute with methanol-water elution solvents with a volume ratio of 40:60 and 50:50 at a flow rate of 20 to 30 mL / min, 3 to 5 column volumes for each gradient elution, the 40:60 part of the elution liquid is collected, and the column chromatography sample II is obtained by concentrating and drying under reduced pressure; Preferably, in the preparative high performance liquid chromatography separation and purification in step (5), the chromatography column is a C18 reverse-phase chromatography column, the elution method is isocratic elution with a methanol-water elution solvent with a volume ratio of 36:64 at a flow rate of 10 mL / min, the elution liquid collected is 6 to 7 min, and the flavonoid glycoside is obtained by concentrating and drying under reduced pressure.

6. The flavonoid glycoside in claim 1 is used for preparing anti-aging cosmetics or drugs.

7. Use according to claim 6, characterized in that, The anti-aging cosmetics or drugs inhibit the expression of p21 and p16 genes.

8. Use of the flavonoid glycoside compound of claim 1 in the preparation of an antitumor drug.

9. Use according to claim 8, characterized in that, The tumor is lung cancer; Preferably, the lung cancer is non-small cell lung cancer.

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