A sesquiterpene acetyl glycoside in rhizoma et radix ligustici wallichii and preparation method and application thereof

Abstract

A sesquiterpene acyl glycoside in rhizoma artemisiae japonicae and preparation method and application thereof. The present application belongs to the technical field of medicine, and relates to extraction and separation of a sesquiterpene acyl glycoside compound rhizoma artemisiae japonicae cembranoid I from rhizome of rhizoma artemisiae japonicae by using macroporous resin, silica gel and preparation chromatography and the like, and the preparation method is simple and easy to implement. 23 H 38 O 10 In vitro human cancer cell inhibition activity research shows that the compound has anticancer activity, including obvious inhibition on human colon cancer cells HT-29 and human breast cancer MCF-7 cells, and is expected to be developed into a new anticancer drug.

Description

Technical Field

[0001] The present invention specifically relates to a sesquiterpene acetyl glycoside compound having an inhibitory effect on cancer cells. Background Art

[0002] Atractylodes japonica Koidz.ex Kitam. Atractylodes japonica is a perennial herb of the genus Atractylodes in the Compositae family. Its medicinal part is the rhizome, which is pungent, bitter, and warm in nature and non-toxic. It is mainly produced in the three northeastern provinces of China. Since 2005, the medicinal material of Atractylodes japonica has been a key protected object of the medicinal material varieties in Jilin Province (the authentic producing area).历代医学著作对其药用价值的评价都很高,其药用部位根茎具有健脾燥湿、发汗解表明目、祛风等功效,主要用于治疗食欲不振,消化不良;内外翳障,夜盲症等病症。关苍术与苍术的主要化学成分及功效相似,且关苍术在东北拥有丰富的野生资源,为了资源利用更加合理,将其开发成苍术替代品,充分开发其药用价值。The medicinal value of Atractylodes japonica has been highly evaluated in previous medical works. Its rhizome, the medicinal part, has the effects of strengthening the spleen and drying dampness, inducing sweating and relieving exterior syndrome, improving eyesight, dispelling wind, etc. It is mainly used to treat loss of appetite, indigestion, internal and external nebula, night blindness and other diseases. The main chemical components and effects of Atractylodes japonica are similar to those of Atractylodes lancea. Moreover, Atractylodes japonica has rich wild resources in Northeast China. For more reasonable utilization of resources, it is developed as a substitute for Atractylodes lancea to fully develop its medicinal value.

[0003] Sesquiterpene compounds are one of the main chemical component types of Atractylodes japonica. Classified from the structural parent nucleus, they mainly include 4 types: eudesmane type, guaiane type, costunolide type and eremophilane type. Most of them have a wide range of biological activities, including anti-cancer, anti-inflammatory, antibacterial, neuroprotective, liver-protecting and other activities, and have good development prospects and values. Summary of the Invention

[0004] The purpose of the present invention is to provide a new sesquiterpene acetyl glycoside compound, its preparation method and medical use, in order to expand the resources and sources of cancer treatment drugs; at the same time, to improve the resource development and utilization value of Atractylodes japonica.

[0005] To achieve the above purpose, the present invention extracts and isolates a sesquiterpene compound, Atractylodes japonica guaianoside I, having anti-colorectal cancer and breast cancer activities from Atractylodes japonica. Its structural formula is as follows:

[0006] .

[0007] The present invention also provides a preparation method of Atractylodes japonica guaianoside I: using Atractylodes japonica as the raw material, and obtaining it by alcohol extraction and column chromatography in sequence.

[0008] The above column chromatography sequentially includes a macroporous resin column, a normal-phase silica gel column, a reversed-phase silica gel column and preparative HPLC.

[0009] The specific preparation steps of the compound Atractylodes japonica guaianoside I of the present invention are as follows:

[0010] (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.

[0011] (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.

[0012] (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.

[0013] (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.

[0014] (5) Preparative HPLC purification: The Fr.2 fraction obtained in step (4) was dissolved in methanol and introduced into a preparative HPLC system. The mobile phase was a mixture of methanol and water with a volume ratio of 36:64, and the flow rate was 3 mL / min. The fraction was collected to obtain the compound of the present invention.

[0015] This invention also provides the use of Atractylodes guaiacide 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 colon cancer and breast cancer.

[0016] Compared with the prior art, the present invention has the following advantages: First, the compounds of the present invention have the prospect of preparing clinical cancer prevention and treatment drugs, thus expanding the drug sources; Second, using Atractylodes lancea as raw material, alternatives to the endangered medicinal herb Atractylodes lancea can be developed through multiple medicinal pathways. 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.

[0027] Example 1: Method for preparing 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) Preparative HPLC purification: The Fr.2 fraction obtained in step (4) was dissolved in methanol and purified by preparative HPLC (Waters, 515-2414, SunFire). TM Prep C 18 (250 mm × 10 mm id, 5 μm), injection concentration not exceeding 35 mg / mL, mobile phase being a methanol-water mixture with a volume ratio of 36:64, flow rate 3 mL / min, t R After collecting the fraction for 26.5~26.9 min, the compound of the present invention (8.8 mg) was obtained.

[0033] Example 2: Characterization of the compounds of the present invention:

[0034] The compound obtained in Example 1 was a yellow oily substance, soluble in methanol, showed a positive Molish reaction, and was detected by acid hydrolysis thin-layer chromatography as D-glucose. Its specific rotation at 25°C was +12° (c = 0.15 MeOH). In HR-ESI-MS, m / z [M+NH4] is visible at position 492.28027. + The ion peak (calcd for 492.2809) 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. 23 H38 O 10 Its degree of unsaturation is calculated to be 5.

[0035] exist 1 H-NMR (600 MHz, Pyridine- d 5 See in the score Figure 3 Four methyl proton signals were observed. δ H 1.41 (3H, s , H-12), 1.44 (3H, s , H-13), 1.15 (3H, d , J = 7.2 Hz, H-15), 2.02(3H, s , H-2''), 5 methylene proton signals δ H 2.63 (1H, dd , J = 18.6, 9.0 Hz, H-2 α ), 3.09 (1H, dd , J = 18.6, 10.2 Hz, H-2 β ), 1.24 (1H, m H-6 α ), 2.49 (1H, m H-6 β ), 1.63 (1H, m H-8 α ), 2.34 (1H, m H-8 β ), 1.97 (1H, m H-9 α ), 2.68 (1H, m H-9 β ), 3.77(2H, br.s (H-14), 2 methylene proton signals δ H 1.89 (1H, m , H-4), 2.04 (1H, m (, H-7), and a set of glycoproton signals δ H 4.99 (1H, d , J = 7.2 Hz, H-1'), 3.99 (1H, m, H-2'), 4.20 (1H, m , H-3'), 4.02 (1H, m , H-4'), 3.97 (1H, m , H-5'), 4.70 (1H, dd , J = 11.4, 6.6Hz, H-6' α ), 4.91 (1H, d , J = 11.0 Hz, H-6' β ),in δ H 4.99 (1H, d , J = 7.2 Hz, H-1') is the terminal matrix signal, indicating that the sugar is β Configuration.

[0036] exist 13 C-NMR (150 MHz, Pyridine- d 5 (See score) Figure 4 23 carbon signals were observed, including one carbonyl carbon signal. δ C 219.4, 1 ester group carbon signal δ C 170.7. See also the DEPT spectrum. Figure 5 It was determined that there were 4 methyl carbon signals. δ C 24.2, 24.2, 12.8, 20.8, six methylene carbon signals δ C 39.3, 36.6, 23.4, 37.1, 69.6, 64.9, 9 methylene carbon signals δ C 46.8, 44.0, 52.2, 50.6, 98.6, 75.2, 78.6, 71.7, 74.7, two seasonal carbon signals δ C 74.4, 80.5, among which δ C 98.6, 75.2, 78.6, 71.7, 74.7, and 64.9 are six carbon signals on sugars.

[0037] exist 1 H- 1 In the H COSY spectrum, see Figure 8H2-2 is associated with H-1, H-1 with H-5, H-5 with H-4 and H2-6, H-4 with H3-15, H2-6 with H-7, H-7 with H2-8, and H2-8 with H2-9. Therefore, it is inferred that the parent nucleus structure contains the segments "-CH2-CH-CH-CH-CH3" and "-CH-CH2-CH-CH2-CH2-", which are linked at C-5. Furthermore, 1 H- 1 The H COSY spectrum also shows correlations between H-1' and H-2', H-2' and H-3', H-3' and H-4', and H-4' and H-5', suggesting the presence of the segment "-CH-CH-CH-CH-" in the structure. 1 H-NMR, 13 C-NMR and HSQC spectra are shown in [reference 1] and [reference 2] respectively. Figure 3 , Figure 4 , Figure 6 This suggests that the fragment represents the upper portion of the sugar. The core structure of this compound 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 compound (1S,4S,5S,7R,10R)-10,11,14-trihydroxyguai-3-one 11-O- β The structure of -D-glucopyranoside is similar, and it can be seen from DEPT, HSQC, and HMBC spectra, respectively. Figure 5 , Figure 6 , Figure 7 This led to the conclusion that the main difference between the two is that the hydroxyl hydrogen at the 6' position of the sugar is replaced by an acetyl group. See the NOESY spectrum. Figure 9 H-1 β With H-4, H-6 ( δ H 1.24), H2-14 related signals, H-5 and Me-15 related signals, H-12 and H-6 ( δ H 2.49), the relevant signals of H-7 indicate that H-4, C-10 hydroxymethyl moiety, and C-7 dimethylmethanol moiety are... β Configuration, H-5, H-7, Me-15, OH-10 are α Configuration.

[0038] Based on the above structural analysis, the main NMR data are shown in Table 1. A search of the SciFinder database confirmed it as a new compound, named Guan Cangzhu Guaichuanmu glycoside I, with the following structural formula: Figure 1 As shown.

[0039]

[0040] Effect Example

[0041] (1) Materials and methods

[0042] The compounds prepared in Example 1 were subjected to in vitro anticancer activity experiments. The cells used in the experiments were human colon cancer HT-29 cells and human breast cancer MCF-7 cells. Both tumor lines were purchased from Cell Reagent Company, and the activity was tested using the MTT assay.

[0043] The specific method is as follows:

[0044] Take HT-29 and MCF-7 cells in good logarithmic growth phase and adjust the cell density to 102. 4 Cells were seeded per well in a 96-well plate, with 100 μL of cell suspension per well. After inoculation, the human cancer cells were placed in RPMI 1640 medium containing 10% L-glutamine and fetal bovine serum (also containing 100 μg / mL penicillin and 100 μg / mL streptomycin), and then cultured at 37°C in a 5% CO2 incubator for 24 h before drug administration. Different drug concentrations were tested: 5, 10, 20, 40, 80, and 160 μmol / L. Blank wells and control wells were also included (blank wells were uninoculated, and control wells contained drug-free medium). Cisplatin served as a positive control. Each group had three replicates. After incubation for another 48 h, 20 μL of 5 mg / mL MTT was added to each well. After another 4 h of culture, the cells were centrifuged, and the supernatant in the 96-well plate was discarded. Then, 150 μL of DMSO was added to each well, and the plate was shaken at low temperature for 10 min. The cells were then analyzed using an ELISA reader at a wavelength of 570 nm. The absorbance of each well was measured at a wavelength of nm, and the inhibition rate of the test drug on cancer cell growth was calculated. Cell survival inhibition rate % = [1 - (Experimental group A - Blank group A) / (Control group A - Blank group A)] × 100%. The IC50 of the drug was calculated using the Logit method. 50 Values. The results are shown in Table 2 below:

[0045] (2) Results

[0046] IC calculated by linear regression 50 The values ​​show that the sesquiterpene compound Guaifenesin I involved in this invention has an IC50 effect on human colon cancer HT-29 cells and human breast cancer MCF-7 cells. 50The values ​​were 69.17±1.27 μmol / L and 45.62±2.19 μmol / L, respectively. Cisplatin, as a positive control, showed IC50 values ​​against human colon cancer HT-29 cells and human breast cancer MCF-7 cells. 50 The values ​​were 7.52±0.83 μmol / L and 5.16±0.64 μmol / L, respectively.

[0047] In summary, the novel compound Guaicinone I isolated from the rhizome of Atractylodes lancea in this invention shows promise for the preparation of clinical cancer prevention and treatment drugs.

Claims

1. A sesquiterpene acetylglycoside compound isolated from the rhizome of Atractylodes lancea, the molecular formula of which is C2. 23 H 38 O 10 It is named Guan Cangzhu Guaichuanmu glycoside I, and its structural formula is as follows: 。 2. The method for preparing the compound according to claim 1, characterized in that: The guaiac glycoside I of Atractylodes lancea was prepared from the rhizome of Atractylodes lancea using macroporous resin column chromatography, normal-phase silica gel column chromatography, reverse-phase silica gel column chromatography, and HPLC. 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) 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) Preparative HPLC purification: The Fr.2 fraction obtained in step (4) was dissolved in methanol and introduced into a preparative HPLC system. The mobile phase was a mixture of methanol and water with a volume ratio of 36:64, and the flow rate was 3 mL / min. The fraction was collected to obtain the compound of the present invention.

3. The use of the compound Guan Cangzhu Guaichuanan Glycoside I according to claim 1 in the preparation of drugs for treating colon cancer and breast cancer.

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