A new alcohol glycoside compound in ligusticum chuanxiong and preparation method and application thereof

Abstract

The application belongs to the technical field of medicine, and relates to an aglycone compound with in-vitro anti-tumor activity extracted and separated from dry rhizomes of Atractylodes japonica (Koidz.ex Kitam.). Atractylodes japonica The application has the advantages of clear process flow, simple operation method, few impurities of the obtained target substance, high purity, in-vitro anti-tumor activity research showing that the compound has anti-tumor activity, including certain inhibition on human colon cancer cells HT-29, human breast cancer cells MCF-7 and human cervical cancer cells Hela, and can provide raw materials for development of anti-tumor drugs, and has important practical application value.

Description

Technical Field

[0001] This invention specifically relates to an atractylodes glycoside compound with tumor cell inhibitory effects. Background Technology

[0002] Guan Cangshu ( Atractylodes japonica Koidz. ex Kitam. is a herbaceous plant belonging to the genus Atractylodes in the family Asteraceae. It commonly grows on grassy slopes, under forests, in thickets, and in sandy loam rich in humus. Its rhizome is large and nodular, and its medicinal use has a long history. Medical works throughout history, such as *Ben Cao Zheng*, *Ben Cao Tong Xuan*, and *Yi Xue Qi Yuan*, all highly praise its medicinal value. Koidz. ex Kitam. has a pungent, bitter, and warm nature. It possesses the effects of strengthening the spleen and drying dampness, relieving depression and dispelling foulness, improving eyesight and dispelling wind. It is mainly used to treat symptoms such as dampness affecting the spleen, loss of appetite, vomiting and diarrhea, phlegm retention and edema, blurred vision, and night blindness. Koidz. ex Kitam. contains various components, including sesquiterpenes, enynes, triterpenes, flavonoids, lignans, sterols, and polysaccharides. Among these, enynes, polysaccharides, and terpenes constitute a large proportion. Current pharmacological studies have found that Koidz. ex Kitam. and its active ingredients have anti-tumor, antibacterial, anti-inflammatory, anti-osteoporosis, and immunomodulatory effects.

[0003] Atractylodes lancea is mainly distributed in Heilongjiang, Jilin, and Liaoning provinces of China, and is a traditional Chinese medicine native to Northeast China with abundant wild resources. Because it is similar to Atractylodes lancea in chemical composition and efficacy, it can serve as a substitute, addressing the practical problems of Atractylodes lancea's increasing depletion and sharp decline in production, leading to high clinical prices and supply shortages. Therefore, it has promising development prospects. Summary of the Invention

[0004] The purpose of this invention is to provide a novel glycoside compound and develop its pharmaceutical uses for the prevention and treatment of tumor diseases.

[0005] To achieve the above objectives, the present invention employs the following technical solution: This invention discloses an alcohol glycoside compound obtained from Atractylodes lancea, wherein the molecular formula of the alcohol glycoside compound is: C 22 H 34 O 11 Its common name is Atractylodes alcoholic glycoside I, and its structural formula is as follows: .

[0006] This invention also provides a method for preparing atractylodes glycoside I, using Atractylodes lancea rhizome as raw material, and obtaining it through systematic solvent extraction, normal-phase silica gel column chromatography, reverse-phase silica gel column chromatography, and preparative HPLC separation and purification. The specific preparation steps are as follows: (1) Alcohol extraction: The dried rhizome of Atractylodes lancea was used as raw material, and was appropriately crushed. It was extracted three times by reflux with 70% ethanol, filtered, and the three filtrates were combined. The solvent was recovered under reduced pressure and dried to obtain an extract in the form of paste. (2) System solvent extraction: The paste-like extract obtained in step (1) is dispersed in water to form a solution of appropriate concentration, and then extracted with dichloromethane and n-butanol in sequence. The n-butanol extract is collected, and the solvent is recovered under reduced pressure to obtain the n-butanol extract. (3) Normal phase silica gel column chromatography: The n-butanol extract obtained in step (2) was subjected to a normal phase silica gel column and a system gradient elution was performed sequentially with a mixed solution of dichloromethane and methanol with a volume ratio of 10:1 and a mixed solution of dichloromethane and methanol with a volume ratio of 3.5:1. The fraction with a volume ratio of 3.5:1 was collected, and the solvent was recovered under reduced pressure to obtain the normal phase silica gel fraction. (4) Reversed-phase silica gel column chromatography: Take the fraction prepared in step (3) and elute it sequentially with a methanol-water mixture with a volume ratio of 1:3 and a methanol-water mixture with a volume ratio of 1:1. Collect the eluted fraction of the methanol-water mixture with a volume ratio of 1:1 and recover the solvent under reduced pressure to obtain the reversed-phase silica gel fraction. (5) Preparative HPLC separation and purification: The Fr.2 fraction obtained in step (4) above was dissolved in methanol and introduced into a preparative HPLC system. The mobile phase was a methanol-water mixture with a volume ratio of 63:37. The elution flow rate was 1.5 mL / min, and the retention time was t. R After collecting the fraction within the range of 21.3 min to 22.2 min, the fraction was recovered and dried to obtain the pure compound.

[0007] This invention also provides the application of atractylodes glycoside I in the preparation of drugs for the prevention and treatment of cancer. Preferably, it is used in the preparation of drugs for the prevention and treatment of human colon cancer cells, human breast cancer cells, and human cervical cancer cells.

[0008] The beneficial effects and significance of this invention are as follows: the process flow of this invention is clear, the operation method is simple, the target substance obtained has few impurities and high purity. At the same time, this novel glycoside compound with anti-tumor activity was found in the rhizome of Atractylodes lancea, which can be widely used in the pharmaceutical, health product and other industries and has high economic value. Attached Figure Description

[0009] Figure 1 Here is a chemical structure diagram of the compound of this invention; Figure 2 The above are the positive HR-ESI-MS spectra of the compounds of this invention. Figure 3 The compounds of this invention 1 H-NMR spectrum; Figure 4 The compounds of this invention 13 C-NMR spectrum; Figure 5 The DEPT spectrum of the compound of this invention; Figure 6 The HSQC spectrum of the compound of this invention; Figure 7 The HMBC spectrum of the compound of this invention; Figure 8 The compounds of this invention 1 H- 1 H COSY spectrum. Detailed Implementation

[0010] To facilitate a further understanding of the present invention, the following embodiments are provided for more detailed and specific description. These embodiments are only for a better understanding of the invention and are not intended to limit the scope or implementation principles of the invention. Those skilled in the art can make adjustments and improvements to the present invention without departing from its spirit and scope, and these adjustments and improvements should also be considered within the protection scope of the present invention. The present invention will be described in detail below with reference to specific embodiments.

[0011] Example

[0012] Preparation method of the compound of the present invention: (1) Alcohol extraction: 10 kg of dried rhizome of Atractylodes lancea was used as raw material. It was appropriately crushed into coarse powder and passed through a No. 1 sieve. It was extracted three times by reflux with 70% ethanol. The first and second extractions were each 2 hours, and the third extraction was 1 hour. The ratio of the weight of the medicinal material to the volume of ethanol was 1:8. After the extraction was completed, the extract was filtered, the three filtrates were combined, the solvent was recovered under reduced pressure, and after drying, 2.25 kg of extract in the form of paste was obtained. (2) System solvent extraction: The extract obtained in step (1) was dispersed in water to form a solution of 1.45±0.05 g / mL, and then extracted 10 times with an equal volume of dichloromethane to the dispersion. Then it was extracted 8 times with an equal volume of n-butanol to the dispersion. The n-butanol extract was collected and the solvent was recovered under reduced pressure to obtain the n-butanol extract. (3) Normal phase silica gel column chromatography separation: The n-butanol extract obtained in step (2) was subjected to normal phase silica gel column chromatography separation. The solvent was first recovered from the extract and then dissolved in methanol. The extract was then mixed with 80-100 mesh silica gel at a mass ratio of 2:1 and the solvent was evaporated. 200-300 mesh silica gel was selected as the column chromatography silica gel. The mass ratio of the column chromatography silica gel to the n-butanol extract was 55:1-35:1 and the column diameter-to-height ratio was 8:1. Gradient elution was performed with dichloromethane-methanol mixed solvent at volume ratios of 10:1 and 3.5:1. The elution was performed at a volume ratio of 10:1 for 6 column volumes and at a volume ratio of 3.5:1 for 8 column volumes. The eluted portion of the dichloromethane-methanol mixed solvent at a volume ratio of 3.5:1 was recovered under reduced pressure to obtain 18.6 g of the separated product. (4) Reversed-phase silica gel column chromatography: The fraction prepared in step (3) was separated by chromatography using a medium-pressure column packed with reversed-phase silica gel ODS. The column was selected with an inner diameter of 70 mm, a length of 470 mm, and an effective height of 400 mm. It was eluted with a methanol-water mixture of 1:3 for 2.5 column volumes and a methanol-water mixture of 1:1 for 3.5 column volumes. The eluted fraction of the methanol-water mixture of 1:1 was collected. After identification by reversed-phase silica gel thin-layer chromatography, similar fractions were combined to obtain three fractions: Fr.1, F.2, and Fr.3. (5) Preparative HPLC separation and purification: The reversed-phase silica gel fraction Fr.2 obtained after purification in step (4) above was dissolved in methanol and introduced into a preparative HPLC system (Waters, 515-2414, SunFire). TM Prep C 18 The sample was separated and purified using a 250 mm × 10 mm (id, 5 μm) membrane. The mobile phase was a methanol-water mixture with a volume ratio of 63:37, the elution flow rate was 1.5 mL / min, and the retention time was t. R After collecting the fraction within the period of 21.3 min to 22.2 min, the fraction was recovered and dried to obtain 16.8 mg of the pure compound.

[0013] Example

[0014] The compound obtained in this invention is a white powder, shows a positive Molish reaction, and D-glucose and D-celery are detected by acid hydrolysis thin-layer chromatography. In HR-ESI-MS, m / z [M+Na] is visible at position 497.1988. + The ion peak indicates that the molecular weight of the compound of this invention is 474. (Combined with...) 1 H-NMR, 13 C-NMR and DEPT spectra suggest that the molecular formula of this compound is C. 22 H34 O 11 Its unsaturation degree is calculated to be 6.

[0015] 1 In the H-NMR (600MHz, CD3OD) spectrum, d 7.39 (2H, d , J =8.4 Hz) and 7.41 (2H, d , J The signal at 8.4 Hz is the proton signal of an AA'BB' coupling system on a benzene ring. d 1.51 (3H, s It shows two methyl proton signals. d 3.04 (3H, s It shows one methoxy signal. d 4.66 (2H, d , J =12.0 (Hz) shows a single hydroxymethylene proton signal. Furthermore, in d 4.33 (1H, d , J = 7.8 Hz, H-1'), 5.05 (1H, d , J Two sugar-terminal proton signals appeared at 3.0 Hz (H-1''). d 3.24~4.01 d The regions 3.60–4.00 are attributed to other proton signals on the sugar, and based on their data characteristics and terminal proton coupling constants, they are preliminarily determined to be... β- D - Glucoside and β- D - Apigenin.

[0016] exist 13 The C-NMR (150 MHz, CD3OD) and DEPT spectra showed 22 carbon signals, including 3 methyl groups, 4 methylene groups, 11 methine groups, and 4 quaternary carbons. d 146.4 (C-1), 126.8 (C-2), 129.3 (C-3), 137.7 (C-4), 129.3 (C-5), and 126.8 (C-6) are carbon signals on the benzene ring; d 103.2 (C-1'), 75.1 (C-2'), 78.0 (C-3'), 71.8 (C-4'), 77.0 (C-5'), and 68.7 (C-6') are carbon signals of the glucose group; d 111.0 (C-1''), 78.2 (C-2''), 80.6 (C-3''), 75.0 (C-4''), and 65.5 (C-5'') are the carbon signals of the apigenin group; in d Carbon signals of two methyl groups at positions 28.2 and 28.3; d A methoxy carbon signal appeared at 50.7.4.

[0017] Displayed in HMBC spectrum d 4.33 (H-1') and C-10 oxygenated carbon ( d 71.5) Related d 5.05 (H-1'') and glucose C-6' ( d 68.7) related d 4.66 (H-10) and the 3, 5 (H-10) on the benzene ring d 129.3), 4 bits ( d 137.7) and glucose terminal carbon ( d The correlation between 103.2) indicates that celery is linked to glucose at position 1→6, the terminal group of glucose is linked to the 10 position of the aglycone, and position 10 is the benzyl position of the benzene ring. 1 H- 1 The H COSY spectrum simultaneously shows the correlation signals on two sugar molecules.

[0018] Based on the analysis of 1D and 2D-NMR spectra, the structure of this compound was determined as follows: Figure 1 As shown in Table 1, the main NMR data were determined by SciFinder database search to be a new compound and named Atractylodesalcoholic glycoside I.

[0019]

[0020] Example

[0021] The in vitro tumor cell inhibition screening test of Atractylodes lancea glycoside I prepared in Example 1 was carried out using the MTT assay. The results showed that the compound of the present invention had a good inhibitory effect on human colon cancer HT-29, human breast cancer MCF-7 and human cervical cancer HeLa cells. The above-mentioned human colon cancer cells HT-29, human breast cancer cells MCF-7 and human cervical cancer cells HeLa were all purchased from Cell Reagent Company.

[0022] The specific method is as follows: Tumor cells were cultured in RPMI 1640 medium (containing 100 μg / mL penicillin and 100 μg / mL streptomycin). Cells in the logarithmic growth phase and in good condition were digested with 0.25% trypsin and seeded in 96-well plates at 10 cells per well. 4 Cell density was 100 μL / well. After seeding, the tumor cells were cultured in RPMI 1640 medium containing 10% fetal bovine serum at 37°C in a 5% CO2 incubator for 24 h. Drug was then administered for 48 h at concentrations of 5, 10, 20, 40, 80, and 160 μM, with three replicates per group. Blank and control wells were also included. Blank wells contained no cells, and control wells contained no drug. Cisplatin was the positive control group. After 48 h of drug treatment, 20 μL / well of 5 mg / mL MTT was added, and the cells were cultured for another 4 h. After centrifugation, the supernatant in 96 wells was discarded, and 150 μL / well of DMSO was added. The wells were then shaken at low temperature for 10 min to completely dissolve any crystals. The absorbance (OD value) of each well was measured at a wavelength of 570 nm using an enzyme-linked immunosorbent assay (ELISA) reader. The results were recorded, and the inhibition rate of tumor cell growth by the test drug was calculated using the following formula: Cell inhibition rate % = [1 - (Experimental group A - Blank group A) / (Control group A - Blank group A)] × 100%. Data were processed using SPSS software. The results are shown in Table 2 below:

[0023] (2) Results: IC was calculated by linear regression. 50 The values ​​show that the compound Atractylodes lancea glycoside I prepared from the rhizome of Atractylodes lancea in this invention has an IC50 effect on human colon cancer HT-29 cells, human breast cancer MCF-7 cells, and human cervical cancer HeLa cells. 50 The values ​​were 43.15±2.21 μmol / L, 52.11±1.94 μmol / L, and 66.21±1.51 μmol / L, respectively. Cisplatin, as a positive control, showed IC50 values ​​against human colon cancer HT-29 cells, human breast cancer MCF-7 cells, and human cervical cancer HeLa cells. 50 The values ​​were 9.12±0.97 μmol / L, 10.99±1.11, and 10.32±1.69 μmol / L, respectively. The results indicate that Atractylodes lancea glycoside I has a good inhibitory effect on the growth of human colon cancer HT-29 cells, human breast cancer MCF-7 cells, and human cervical cancer HeLa cells, and shows promise for development into a clinical drug for tumor prevention and treatment.

Claims

1. A glycoside compound, characterized in that, The molecular formula of the compound is C 22 H 34 O 11 Its structural formula is as follows:

2. A method for preparing the glycoside compound according to claim 1, characterized in that: The compound was obtained from the rhizome of Atractylodes lancea as raw material, and was purified by systematic solvent extraction, normal-phase silica gel column chromatography, reversed-phase silica gel column chromatography, and preparative HPLC separation and purification. The specific preparation steps are as follows: (1) Alcohol extraction: The dried rhizome of Atractylodes lancea was used as raw material, and was appropriately crushed. It was extracted three times by reflux with 70% ethanol, filtered, and the three filtrates were combined. The solvent was recovered under reduced pressure and dried to obtain an extract in the form of paste. (2) System solvent extraction: The paste-like extract obtained in step (1) is dispersed in water to form a solution of appropriate concentration, and then extracted with dichloromethane and n-butanol in sequence. The n-butanol extract is collected, and the solvent is recovered under reduced pressure to obtain the n-butanol extract. (3) Normal phase silica gel column chromatography: The n-butanol extract obtained in step (2) was subjected to a normal phase silica gel column and a system gradient elution was performed sequentially with a mixed solution of dichloromethane and methanol with a volume ratio of 10:1 and a mixed solution of dichloromethane and methanol with a volume ratio of 3.5:

1. The fraction with a volume ratio of 3.5:1 was collected, and the solvent was recovered under reduced pressure to obtain the normal phase silica gel fraction. (4) Reversed-phase silica gel column chromatography: Take the fraction prepared in step (3) and elute it sequentially with a methanol-water mixture with a volume ratio of 1:3 and a methanol-water mixture with a volume ratio of 1:

1. Collect the eluted fraction of the methanol-water mixture with a volume ratio of 1:1 and recover the solvent under reduced pressure to obtain the reversed-phase silica gel fraction. (5) Preparative HPLC separation and purification: The reversed-phase silica gel fraction obtained in step (4) above was dissolved in methanol and introduced into a preparative HPLC system. The mobile phase was a methanol-water mixture with a volume ratio of 63:

37. The elution flow rate was 1.5 mL / min, and the retention time was t. R After collecting the fraction within the range of 21.3 min to 22.2 min, the fraction was recovered and dried to obtain the pure compound.

3. The use of the glycoside compound of claim 1 in the preparation of drugs for the prevention and treatment of tumors, characterized in that, The tumors described are human colon cancer cells, human breast cancer cells, and human cervical cancer cells.

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