A guaian-type sesquiterpene glycoside in sileris millefolium and preparation method and application thereof

Abstract

The application belongs to the technical field of medicine, and relates to extraction and separation of a guaiane sesquiterpene glycoside compound guaiane sesquiterpene glycoside II from rhizome of rabdosia japonica by using macroporous resin, normal-phase silica gel, reverse-phase silica gel and dextran gel column chromatography and recrystallization and the like. 21 H 36 O8, in-vitro anti-tumor activity research shows that the compound has good anti-tumor activity, including obvious inhibition on human gastric cancer cells BGC-823, human hepatoma cells HepG-2 and human lung cancer cells A549. The application can provide a source of pharmacodynamic material basis for development of anti-tumor drugs, and has development and application prospects.

Description

Technical Field

[0001] This invention specifically relates to a guaiacol-type sesquiterpene glycoside compound with tumor cell inhibitory effects. Background Technology

[0002] Guan Cangshu ( Atractylodes japonica Koidz. ex Kitam. is a herbaceous plant belonging to the Asteraceae family. Its dried rhizome is used medicinally. It is abundant in wild resources in Northeast my country, growing mainly in humus-rich sandy loam soils along forest edges, under forest canopies, on hillsides, and terraces at altitudes of 200-800 m. Koidz. ex Kitam. has the effects of strengthening the spleen and drying dampness, drying the soil and promoting diuresis, improving eyesight and dispelling wind, and eliminating foul odors. It is mainly used to treat various clinical symptoms such as loss of appetite, indigestion, rheumatoid arthritis, and colds. Modern pharmacological studies have shown that it has great potential for development in anti-tumor, anti-inflammatory, antibacterial, and protective and repairing effects on gastric mucosa tissue. In Japan and South Korea, the rhizome of Koidz. ex Kitam. is not only commonly used medicinally as Atractylodes macrocephala, but is also made into health foods.

[0003] The volatile oil components of Atractylodes lancea are mainly sesquiterpenes, which can be classified into several types, including eucalyptane and guaiacolane. Some representative compounds have been reported to have relevant pharmacological activities. For example, atractylone has the effects of protecting the liver, inhibiting viruses, and eliminating inflammation; apigenide diene can inhibit capillary permeability and eliminate inflammation; atractylodes lactone I can increase amylase activity, enhance intestinal digestion and absorption, and regulate the spleen and stomach; atractylodes lactone II has an inhibitory effect on human colorectal cancer cells Lovo; and atractylodes lactone III also has antiviral effects. Currently, there are not many reported compounds of this type in Atractylodes lancea. Summary of the Invention

[0004] The purpose of this invention is to provide a novel guaiacol-type sesquiterpene glycoside compound, its preparation method, and its application.

[0005] To achieve the above objectives, the present invention employs the following technical solution:

[0006] This invention discloses a guaiacane-type sesquiterpene glycoside, the molecular formula of which is: C 21 H 36 O8, commonly known as Guan Cangshu Guaichuanmu glycoside II, has the following structural formula:

[0007]

[0008] This invention also provides a method for preparing guaiac glycoside II: it is obtained by macroporous resin, normal-phase silica gel, reverse-phase silica gel and dextran gel column chromatography and recrystallization. The specific preparation steps are as follows:

[0009] (1) Alcohol extraction: The dried rhizome of Atractylodes lancea was used as raw material, and after appropriate crushing, it was extracted three times by reflux with 70% ethanol. After filtration, the three filtrates were combined, the solvent was recovered under reduced pressure, and the extract was dried to obtain a paste-like extract.

[0010] (2) Enrichment and purification: The extract obtained in step (1) was dispersed in water to a solution with a relative density of 1.25±0.05 g / mL, and enriched and purified by AB-8 macroporous resin column chromatography. It was eluted with water, 30% ethanol, 50% ethanol and 95% ethanol respectively. The 50% ethanol eluent was collected and the solvent was recovered under reduced pressure to obtain the 50% ethanol eluent.

[0011] (3) Normal phase silica gel column chromatography: Take the 50% ethanol elution fraction obtained in step (2) and use a normal phase silica gel column. Use a mixed solution of dichloromethane and methanol with a volume ratio of 8:1, a mixed solution of dichloromethane and methanol with a volume ratio of 5:1, and a mixed solution of dichloromethane and methanol with a volume ratio of 3:1 for systematic gradient elution. Collect the fraction with a volume ratio of 3:1 and recover the solvent under reduced pressure.

[0012] (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 1:2, a methanol-water mixture with a volume ratio of 1:1, and a methanol-water mixture with a volume ratio of 2:1. Collect the elution fraction with a volume ratio of 1:1 methanol-water mixture. After identification by reversed-phase silica gel thin-layer chromatography, similar fractions are combined to obtain three fractions: Fr.1, F.2, and Fr.3.

[0013] (5) Dextran gel column chromatography: Take the Fr.2 portion obtained in step (4), pass it through a dextran gel column, and elute isocratically with methanol-water at a volume ratio of 1:1.5 as the mobile phase. Collect the eluent, recover the solvent, and obtain the crude product.

[0014] (6) Recrystallization treatment: The crude product obtained in step (5) is recrystallized twice using a two-phase solvent system of methanol:dichloromethane=5:1 to obtain the pure product of the compound.

[0015] This invention also provides the application of Atractylodes guaiacide glycoside II 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 gastric cancer, liver cancer, and lung cancer.

[0016] The beneficial effects and significance of this invention are as follows: This study conducted in-depth component research and development on the rhizome of Atractylodes lancea, and found compounds with unique chemical structures and strong activity, providing novel anti-tumor drugs for clinical research. Attached Figure Description

[0017] Figure 1 Here is the chemical structural formula of the compound of this invention;

[0018] Figure 2 The above are the positive HR-ESI-MS spectra of the compounds of this invention.

[0019] Figure 3 The compounds of this invention 1 H-NMR spectrum;

[0020] Figure 4 The compounds of this invention 13 C-NMR spectrum;

[0021] Figure 5 The DEPT spectrum of the compound of this invention;

[0022] Figure 6 The HSQC spectrum of the compound of this invention;

[0023] Figure 7 The HMBC spectrum of the compound of this invention;

[0024] Figure 8 The compounds of this invention 1 H- 1 H COSY spectrum

[0025] Figure 9 This is the NOESY spectrum of the compound of this invention. Implementation

[0026] Based on the technical content disclosed in this invention, those skilled in the art will clearly understand other embodiments of this invention. The following embodiments are merely examples. Various adjustments and improvements can be made to this invention without violating its spirit and scope. These changes should be within the protection scope of this invention. The invention will be described in detail below with reference to specific embodiments. Example

[0027] Preparation method of the compound of the present invention:

[0028] (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 No. 1 sieve. It was extracted three times by reflux with 70% ethanol for 2 hours each time. The ratio of the weight of the medicinal material to the volume of ethanol was 1:6. After the extraction was completed, the extract was filtered, the filtrates from the three extractions were combined, the solvent was recovered under reduced pressure, and after drying, 2.4 kg of extract in paste form was obtained.

[0029] (2) Enrichment and purification: The extract obtained in step (1) was dispersed in water to a solution with a relative density of 1.25±0.05 g / mL, and enriched and purified by AB-8 macroporous resin column chromatography (the inner diameter of the column is 10 cm, the length is 1.80 m, and the effective height of the resin is 1.35 m). The column was eluted with water, 30% ethanol, 50% ethanol, and 95% ethanol for 3 column volumes, 2 column volumes, 3 column volumes, and 2 column volumes respectively. The 50% ethanol eluent was collected, and the solvent was recovered under reduced pressure to obtain 386 g of the 50% ethanol eluent.

[0030] (3) Normal phase silica gel column chromatography: Take the 50% ethanol elution fraction obtained in step (2) and use a normal phase silica gel column (the inner diameter of the column is 6.5 cm and the length is 1.8 m, of which the effective height of silica gel is 1.2 m). Elute 3 column volumes with a mixed solution of dichloromethane and methanol with a volume ratio of 8:1, 2.5 column volumes with a mixed solution of dichloromethane and methanol with a volume ratio of 5:1, and 3 column volumes with a mixed solution of dichloromethane and methanol with a volume ratio of 3:1. Collect the fraction with a volume ratio of 3:1, and recover the solvent under reduced pressure to obtain 36 g of collected fraction.

[0031] (4) Reversed-phase silica gel column chromatography: Take the fraction prepared in step (3) and perform reversed-phase silica gel ODS column chromatography (the inner diameter of the column is 5 cm, the length is 1.5 m, and the effective height of the reversed-phase silica gel is 0.8 m). Elute with a methanol and water mixture of volume ratio of 1:2 for 3 column volumes, a methanol and water mixture of volume ratio of 1:1 for 2 column volumes, and a methanol and water mixture of volume ratio of 2:1 for 2.5 column volumes. Collect the elution fraction of the methanol and water mixture of volume ratio of 1:1. After identification by reversed-phase silica gel thin-layer chromatography, similar fractions are combined to obtain three fractions: Fr.1, F.2, and Fr.3.

[0032] (5) Dextran gel column chromatography: Take the Fr.2 fraction obtained in step (4) and pass it through a dextran gel column. Use methanol-water with a volume ratio of 1:1.5 as the mobile phase for isocratic elution. Elute for 4 column volumes, discard the first 2 column volumes of eluent, collect the last 2 column volumes of eluent, recover the solvent, and obtain 42.5 mg of crude product.

[0033] (6) Recrystallization treatment: The crude product obtained in step (5) is recrystallized twice using a two-phase solvent system of methanol:dichloromethane=5:1 to obtain 12.2 mg of the pure product of the compound. Example

[0034] Identification of the compound: The compound obtained in this invention is a yellow, flaky crystal (methanol:dichloromethane = 5:1), showing a positive Molish reaction. Acid hydrolysis and thin-layer chromatography revealed D-glucose, [α]25D+26° (c = 0.15 MeOH). In HR-ESI-MS, m / z [M+NH4] is visible at position 434.27472. + The ion peak (calcd for 434.2754) indicates that the molecular weight of the compound of this invention is 416. (Combined with...) 1 H-NMR, 13 C-NMR and DEPT spectra suggest that the molecular formula of this compound is C. 21 H 36 O8 has an unsaturation degree of 4.

[0035] exist 1 H-NMR (600 MHz, Pyridine- d 5 Four methyl proton signals were observed in the spectrum. δ H 1.46(6H, s , H-12, H-13), 1.28 (3H, s , H-14), 1.11 (3H, d , J = 6.6 Hz, H-15), 4 methylene proton signals δ H 2.51 (1H, dd , J = 18.6, 9.6 Hz, H-2 α ), 2.98 (1H, dd , J = 18.6, 9.6 Hz, H-2 β ), 1.19 (1H, m H-6 α ), 1.33 (1H, m H-6 β ), 2.41 (1H, m H-8 α ), 1.52 (1H, m H-8 β ), 2.12 (1H, m H-9 α ), 1.95 (1H, o H-9 β ), 4 methine proton signals δ H1.93(1H, t , J = 9.6 Hz, H-1), 1.87 (1H, m , H-4), 1.97 (1H, o , H-5), 2.02 (1H, m (, H-7), and a set of glycoproton signals δ H 5.06 (1H, d , J = 7.8 Hz, H-1'), 4.01 (1H, t , J = 8.0Hz, H-2'), 4.26 (1H, m , H-3'), 4.23 (1H, m , H-4'), 3.94 (1H, m , H-5'), 4.35 (1H, m , H-6' α ), 4.52 (1H, dd , J = 2.4, 17.1 Hz, H-6' β ),in δ H 5.06 (1H, d , J = 8.0Hz, H-1') is the terminal matrix signal, indicating that the sugar is β Configuration.

[0036] exist 13 C-NMR (150 MHz, Pyridine- d 5 In the spectrum, 21 carbon signals were observed, including one carbonyl carbon signal. δ C 219.2, 4 methyl carbon signals δ C 23.8, 24.5, 30.7, 12.8, five methylene carbon signals δ C 39.2, 36.3, 23.0, 42.5, 63.0, 9 methylene carbon signals δ C 50.9, 44.0, 52.5, 50.3, 98.6, 75.4, 78.9, 71.9, 78.1, two seasonal carbon signals δ C 71.4, 80.4, among which δC 98.6, 75.4, 78.9, 71.9, 78.1, and 63.0 are six carbon signals on sugars.

[0037] exist 1 H- 1 In the H COSY spectrum, H2-2 is correlated with H-1, H-4 with H3-15, H-5 with H2-6, H2-6 with H-7, H-7 with H2-8, and H2-8 with H2-9, suggesting the presence of segments "-CH-CH3-", "-CH2-CH-", and "-CH-CH2-CH-CH2-CH2-" in the parent nucleus structure; H-1' is correlated with H-2', H-2' with H-3', H-4' with H-5', and H-5' with H-6', suggesting the presence of "-CH-CH-CH-" and "-CH-CH-CH2-" in the structure. 1 H-NMR, 13 C-NMR and HSQC spectra indicate that this fragment is the upper sugar portion. The core structure of the compound of this invention is consistent with that in the reference [Kitajima J, Kamoshit A, Ishikawa T, et al. Glycosides of Atractylodes Japonica [Chem Pharm Bull, 2003, 51(2): 152-157] The known compound (1S,4S,5S,7R,10R)-10,11,14-trihydroxyguai-3-one 11-O- β -D-glucopyranoside is highly similar to that of other methyl groups, with the main difference being the absence of a hydroxyl group at position 14. DEPT and HSQC spectra confirmed that position 14 is a methyl group. In the NOESY spectrum, H-1... β With H-6 ( δ H 1.19), Me-14 related signals, H-6 ( δ H 2.23) The correlation signals with H-4, H-12 / H-13, H-7 and H-12 / H-13, and H-5 and Me-15 indicate that H-5, Me-14, Me-15, and C-7 are partially dimethylmethanol. β Configuration, H-4, H-7, OH-10 are α Configuration.

[0038] Based on the comprehensive analysis of the 2D-NMR spectrum, 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, named Guan Cangzhu Guaichuanmu glycoside II.

[0039] Example

[0040] The MTT (tetrazolate salt) method was used to determine the killing effect of Atractylodes lancea guaiac glycoside II on three types of human tumor cells.

[0041] (1) Tumor cells: human gastric cancer cells BGC-823, human liver cancer cells HepG-2 and human lung cancer cells A549.

[0042] (2) Specific experimental procedures: Human gastric cancer cells BGC-823, human liver cancer cells HepG-2, and human lung cancer cells A549 were cultured in RPMI 1640 substrate (fetal bovine serum containing 10% L-glutamine, 100 μg / mL penicillin, and 100 μg / mL streptomycin). The tumor cells in the logarithmic growth phase were digested with 0.25% trypsin at a concentration of 10 × 10⁻⁶. 4 Cells were cultured at a concentration of 180 μL / mL in 96-well plates, with three replicates per group. Blank and control wells were also included. Blank wells contained no cells, and control wells contained culture medium without the drug. Cisplatin was used as the positive control. After culturing at 37°C and 5% CO2 for 24 h, the test sample was added to the culture medium to achieve final drug concentrations of 5, 10, 20, 40, 80, and 160 μmol / L. The solutions were then incubated at 37°C and 5% CO2 for another 48 h. 20 μL of 5 mg / mL MTT solution was added to each well, and the plates were incubated for another 4 h before termination. The supernatant was carefully discarded, and 150 μL of DMSO was added to each well. The plates were shaken for 10 min to completely dissolve any crystals. The absorbance (A) of each well was measured at 570 nm using an ELISA reader, and the cell survival inhibition rate was calculated as follows: Cell survival inhibition rate % = [1 - (Experimental group A - Blank group A) / (Control group A - Blank group A)] × 100%. Data were processed using SPSS software.

[0043] (3) Experimental results: The experimental data on the survival inhibition rate of tumor cells by different concentrations of the new compound and the positive control drug cisplatin are shown in Table 2.

[0044]

[0045] The results in the table above show that the IC calculated by linear regression... 50 The values ​​show that the sesquiterpene compound Guaifenesin II involved in this invention has an IC50 effect on human gastric cancer BGC-823 cells, human hepatocellular carcinoma HepG-2 cells, and human lung cancer A549 cells. 50The values ​​were 66.15±0.97 μmol / L, 68.06±1.11 μmol / L, and 43.44±1.82 μmol / L, respectively. Cisplatin, as a positive control, showed IC50 values ​​against human gastric cancer BGC-823 cells, human hepatocellular carcinoma HepG-2 cells, and human lung cancer A549 cells. 50 The values ​​were 5.11±1.53 μmol / L, 4.86±0.96 μmol / L, and 10.05±1.25 μmol / L, respectively.

[0046] In summary, the guaiac sesquiterpene glycoside guaiac glycoside II isolated from the rhizome of Atractylodes lancea described in this invention has the potential to be used in the preparation of clinical tumor treatment drugs.

Claims

1. A guaiacane-type compound isolated from the rhizome of Atractylodes lancea, the molecular formula of which is C2. 21 H 36 O8, named Guan Cangzhu Guaichuanmu glycoside II, has the following structural formula: 。 2. The method for preparing the compound according to claim 1, characterized in that: The guaiac glycoside II of Atractylodes lancea was obtained from the rhizome of Atractylodes lancea through macroporous resin, normal-phase silica gel, reverse-phase silica gel, and dextran gel column chromatography and recrystallization. The specific preparation steps are as follows: (1) Alcohol extraction: The dried rhizome of Atractylodes lancea was used as raw material, and after appropriate crushing, it was extracted three times by reflux with 70% ethanol. After filtration, the three filtrates were combined, the solvent was recovered under reduced pressure, and the extract was dried to obtain a paste-like extract. (2) Enrichment and purification: The extract obtained in step (1) was dispersed in water to a solution with a relative density of 1.25±0.05 g / mL, and enriched and purified by AB-8 macroporous resin column chromatography. It was eluted with water, 30% ethanol, 50% ethanol and 95% ethanol respectively. The 50% ethanol eluent was collected and the solvent was recovered under reduced pressure to obtain the 50% ethanol eluent. (3) Normal phase silica gel column chromatography: Take the 50% ethanol elution fraction obtained in step (2) and use a normal phase silica gel column. Use a mixed solution of dichloromethane and methanol with a volume ratio of 8:1, a mixed solution of dichloromethane and methanol with a volume ratio of 5:1, and a mixed solution of dichloromethane and methanol with a volume ratio of 3:1 for systematic gradient elution. Collect the fraction with a volume ratio of 3:1 and recover the solvent under reduced pressure. (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 1:2, a methanol-water mixture with a volume ratio of 1:1, and a methanol-water mixture with a volume ratio of 2:

1. Collect the elution fraction with a volume ratio of 1:1 methanol-water mixture. After identification by reversed-phase silica gel thin-layer chromatography, similar fractions are combined to obtain three fractions: Fr.1, F.2, and Fr.

3. (5) Dextran gel column chromatography: Take the Fr.2 portion obtained in step (4), pass it through a dextran gel column, and elute isocratically with methanol-water at a volume ratio of 1:1.5 as the mobile phase. Collect the eluent, recover the solvent, and obtain the crude product. (6) Recrystallization treatment: The crude product obtained in step (5) is recrystallized twice using a two-phase solvent system of methanol:dichloromethane=5:1 to obtain the pure product of the compound.

3. The use of the compound Guaifenesin II of claim 1 in the preparation of a drug for treating tumors, wherein the tumor is gastric cancer, liver cancer, or lung cancer.

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