A kind of lignan glycoside compound in ligusticum chuanxiong and its preparation method and application

Abstract

The application discloses a kind of to utilize 70% ethanol reflux extraction, macroporous resin, normal phase silica gel, reverse phase silica gel multiple column chromatography combination separation and preparation type HPLC purification method from the rhizome of Atractylodes lancea in the field of medicine, and a kind of lignan glycosides compound is obtained, and its molecular formula is C 43 H 56 O 21 The raw material involved in the application is easy to obtain in large quantities, and the compound obtained therein has in vitro anti-tumor activity, the extraction and separation method is simple to operate, and further pharmacological activity and clinical development research can be carried out, which creates conditions for developing new anti-tumor drugs with good curative effect.

Description

Technical Field

[0001] This invention relates to the field of pharmacodynamic material basis of traditional Chinese medicine, specifically to the extraction and isolation of a cyclolignan glycoside compound from Atractylodes lancea, its preparation method, and the application of the compound in anti-tumor activity. Background Technology

[0002] Guan Cangshu ( Atractylodes japonica Koidz. ex Kitam. is a perennial herb belonging to the genus Atractylodes in the family Asteraceae. Its rhizome is primarily used medicinally and is a common traditional Chinese medicine. It is mainly produced in the three northeastern provinces of my country, and is a specialty of Jilin Province. It has a pungent and bitter taste, and is warm in nature. It possesses anti-inflammatory, antibacterial, diaphoretic, and diuretic properties, and is often used clinically to treat acute and chronic hepatitis, indigestion, diabetes, hyperlipidemia, and pruritus. It is also worth noting that due to severe damage to its habitat and over-harvesting of wild resources, its wild population is dwindling. Since *Atractylodes lancea* has similar effects, we have been considering using it as a substitute to fully utilize its medicinal value.

[0003] According to literature reports, many plants in the Atractylodes genus contain lignans, mainly including simple lignans, monoepoxy lignans, and benzofuran lignans, which possess antiviral, hepatoprotective, and antioxidant properties. However, research on lignans in Atractylodes lancea is currently limited. Therefore, exploring these components from this medicinal resource holds potential application value and promising development prospects. Summary of the Invention

[0004] The purpose of this invention is to provide a new cyclic lignan glycoside compound, its preparation method, and its medical applications, in order to expand the sources of anti-tumor drugs; and at the same time, to further strengthen the research on the resource development and utilization value of Atractylodes lancea.

[0005] To achieve the above objectives, this invention extracts and isolates a cyclic lignan glycoside compound, Guancang cyclic lignan glycoside I, which has anti-tumor properties, from Atractylodes lancea, and its structural formula is as follows: .

[0006] This invention also provides a method for preparing Atractylodes cyclolignan I: using Atractylodes cyclolignan rhizome as raw material, it is prepared by sequentially extracting with alcohol and performing various chromatographic column chromatography.

[0007] The column chromatography described above includes, in sequence, macroporous resin column, normal-phase silica column, reverse-phase silica column, and preparative HPLC.

[0008] The specific preparation steps of the compound Atractylodes cyclolignin I of this invention are as follows: (1) Alcohol extraction: Take 10 kg of dried rhizome of Atractylodes lancea as raw material, crush it appropriately, extract it by reflux with 70% ethanol for 3 hours, repeat 3 times, filter, combine the 3 filtrates, recover the extract under reduced pressure, and dry to obtain the extract. (2) Enrichment and purification: The extract obtained by ethanol extraction in step (1) above is dispersed in water to a solution with a relative density of 1.35±0.05 g / mL, enriched and purified by AB-8 macroporous resin column chromatography, and eluted with water and 35% ethanol respectively. The 35% ethanol eluent is collected, and the solvent is recovered under reduced pressure to obtain the 35% ethanol eluent. (3) Normal phase silica gel column chromatography: Take the 35% ethanol elution fraction obtained in step (2) and perform normal phase silica gel column chromatography. The system is sequentially eluted with a mixed solution of dichloromethane and methanol with a volume ratio of 15:1, 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. Collect the eluent 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 perform reversed-phase silica gel ODS column chromatography. Elute sequentially with a methanol-water mixture with a volume ratio of 1:2 and a methanol-water mixture with a volume ratio of 1:1. Collect the specified eluted fraction of the methanol-water mixture with a volume ratio of 1:1, recover the solvent, and obtain the crude product. (5) Preparative HPLC purification: The crude product obtained in step (4) is dissolved in methanol and introduced into a preparative HPLC. The mobile phase is a mixture of methanol and water with a volume ratio of 30:70. The flow rate is 3 mL / min. After collecting the fraction, the solvent is recovered and dried to obtain the compound of the present invention.

[0009] This invention also provides the application of atractylodes cyclolignan I in the preparation of antitumor drugs.

[0010] Compared with the prior art, the present invention has the following advantages: First, the compounds of the present invention have the prospect of preparing anti-tumor drugs, thus expanding the sources of anti-tumor drugs; Second, Atractylodes lancea can be used as a substitute to replace the endangered Chinese herb Atractylodes lancea.

[0011] Figure 1 Here is the chemical structural formula 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 Figure 9 The main correlation diagram of the HMBC spectrum of the compounds of this invention. Implementation

[0012] 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 1

[0013] Preparation method of the compound of the present invention: (1) Alcohol extraction: Take 10 kg of dried rhizome of Atractylodes lancea as raw material, grind it into coarse powder and pass it through No. 1 sieve. Extract it three times with 70% ethanol for 3 hours each time. The ratio of the weight of the medicinal material to the volume of ethanol is 1:8. After the extraction is completed, filter it, combine the three filtrates, recover the solvent under reduced pressure, and dry it to obtain 2.2 kg of extract. (2) Enrichment and purification: The extract obtained in step (1) was dispersed in water to a solution with a relative density of 1.35±0.05 g / mL, and enriched and purified by chromatographic chromatography using an AB-8 type macroporous resin column (inner diameter of the column is 10 cm, length is 2.30 m, and effective height of the resin is 1.55 m). The column was eluted with water for 5 column volumes and then discarded. The column was then eluted with 35% ethanol for 3 column volumes, and 352 g of the 35% ethanol eluent was collected. (3) Normal phase silica gel column chromatography: The 35% ethanol elution fraction obtained in step (2) was subjected to normal phase silica gel column chromatography (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). The column was eluted sequentially with a 15:1 mixture of dichloromethane and methanol for 3.5 column volumes, a 5:1 mixture of dichloromethane and methanol for 2 column volumes, and a 3:1 mixture of dichloromethane and methanol for 3 column volumes. The eluent with a volume ratio of 3:1 was collected, and the solvent was recovered under reduced pressure to obtain 32 g of fraction. (4) Reversed-phase silica gel column chromatography: Take the fraction prepared in step (3) and pass it through a reversed-phase silica gel ODS column (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 with a volume ratio of 1:2 for 3 column volumes, then with a methanol and water mixture with a volume ratio of 1:1 for 1 column volume and discard the elution. Then elute with a methanol and water mixture with a volume ratio of 1:1 for 2 column volumes and collect the elution. Recover the solvent to obtain 0.68 g of crude product. (5) Preparative HPLC purification: The crude product obtained in step (4) was dissolved in methanol and injected into a preparative HPLC system (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 30:70, flow rate 3 mL / min, t R After collecting the fraction within a time period of 26.5 to 26.9 minutes, the solvent was recovered and dried to obtain 38 mg of the compound of the present invention. Example 2

[0014] Characterization of the compounds of this invention: HR-ESI-MS m / z: 909.3425 [M+H] + This indicates that the molecular weight of this compound is 908. 1 H-NMR (CD3OD, 600 MHz) δ H : 7.30 (1H, s, H-2‴), 7.30 (1H, s, H-6‴), 6.53 (1H, s, H-5), 6.41 (2H, overlapped, J = 0.6 Hz, H-2′, H-6′), 5.35 (1H, s, H-1""), 4.65 (1H, d, J = 11.2Hz, H-6″α), 4.38 (1H, dd, J = 11.2, 6.6 Hz, H-6″β), 4.37 (1H, d, J =6.0 Hz, H-1), 4.34 (1H, d, J = 7.8 Hz, H-1″), 4.24 (1H, m, H-5""), 4.14 (1H, m, H-2""), 3.90 (1H, dd, J= 3.0, 9.6 Hz, H-3""),3.85 (1H, m, H-2a1),3.83 (3H, s, 6-OCH3),3.79(3H, s, 3‴, 5‴-OCH3),3.72 (6H, s, 3′, 5′-OCH3),3.61 (1H, m, H-3a1),3.59 (1H,m,H-5″),3.51 (1H, m, H-3a2),3.45 (1H, m, H-2a2),3.45 (1H, m, H-4""),3.42 (1H, m,H-3″),3.40 (1H, m, H-4″),3.34 (3H, s, 8-OCH3),3.29 (1H, m, H-2″),2.66 (1H,dd, J = 4.2, 15.0 Hz, H-4α),2.56 (1H, t, J = 15.0 Hz, H-4β),2.16 (1H, m, H-2),1.62 (1H, m, H-3),1.20 (3H, d, J = 6.6 Hz, H-6""); 13 C-NMR(CD3OD,150 MHz) δ C:167.4 (C-7‴), 154.5 (C-5‴), 154.5 (C-3‴), 148.9 (C-3′), 148.9 (C-5′), 148.5 (C-6), 147.5 (C-8), 140.0 (C-4‴), 139.2 (C-4′), 138.8 (C-7), 134.4 (C-1′), 130.2 (C-4a), 127.0 (C-1‴), 126.2 (C-8a), 107.7 (C-2‴), 107.7 (C-6‴), 107.7 (C-5), 106.8 (C-2′), 106.8 (C-6′), 104.7 (C-1″), 103.4 (C-1″”), 78.0 (C-3″), 75.3 (C-5″), 75.1 (C-2″), 73.6 (C-4″”), 72.2 (C-3″”), 72.0 (C-2″”), 71.9 (C-4″), 71.7 (C-2a), 71.3 (C-5""), 66.1 (C-3a), 65.5 (C-6″), 60.2 (C-8-OCH3), 56.8 (C-5′-OCH3), 56.8 (C-3′-OCH3), 56.6 (C-5‴-OCH3), 56.6 (C-3‴-OCH3), 56.6 (C-6-OCH3), 46.3 (C-2), 42.7 (C-1), 40.8 (C-3), 33.7 (C-4), 17.9 (C-6"").

[0015] The main NMR data and correlations are shown in Table 1. A search of the SciFinder database confirmed it as a new compound with the chemical name (-)-Nancanthol 2a- O -{6- O -[(3′,5′-dimethoxy)benzoyl4′- O [α-L-rhamnopyranoside]-β-D-glucopyranoside, commonly known as Guan Cangzhu cyclolignan glycoside I.

[0016]

[0017] Example of efficacy – In vitro inhibition of tumor cell proliferation (1) Materials and methods The compound prepared in Example 1 was subjected to in vitro antitumor experiments. The activity of the compound against six tumor cell lines (HeLa, HepG-2, A549, MCF-7, SGC-7901, and HT-29) was screened using the conventional MTT assay. The results showed that the compound had a good inhibitory effect on MCF-7, SGC-7901, and HT-29.

[0018] The specific method is as follows: The aforementioned tumor cells were cultured in RPMI 1640 substrate (fetal bovine serum containing 10% L-glutamine, 100 μg / mL penicillin, and 100 μg / mL streptomycin). Tumor cells in good logarithmic growth phase were taken, digested with 0.25% trypsin, and cultured at a cell density of 102. 3 ~10 4 Cells were seeded per well in 96-well plates, with 100 μL of cell suspension per well. After 24 h of incubation at 37°C and 5% CO2, the test sample (dissolved in DMSO) was added to the culture medium to achieve final cell concentrations of 5, 10, 20, 40, 80, and 160 μM. A blank control group was prepared by adding 100 μL of RPMI 1640 with 10 μL of drug-free solvent. Each group had three replicates. After incubation for another 48 h, 20 μL of 5 mg / mL MTT was added to each well, and the plates were incubated at 37°C for another 4 h. After centrifugation, the culture medium in the wells was discarded, and 150 μL of DMSO solution was added to each well. The plates were then shaken at low temperature for 10 min. The absorbance (OD) value of each well was read using an ELISA reader at a wavelength of 570 nm. The results were recorded, and the inhibition rate of tumor cell growth by the test drug was calculated using the following formula. Cell growth inhibition rate % = [1 - (mean OD value of drug-treated wells - OD value of zeroing wells) / (mean OD value of cell control wells - OD value of zeroing wells)] × 100%. The IC50 of the drug is calculated using the Logit method. 50 Values. The results are shown in Table 2 below:

[0019] (2) Results IC calculated by linear regression 50 The values ​​show that the cyclic lignan glycosides involved in this invention have an IC50 effect on human breast cancer cells MCF-7, human gastric cancer cells SGC-7901, and human colon cancer cells HT-29. 50The values ​​were 62.11±1.18 μM, 38.09±1.52 μM, and 45.44±2.16 μM, respectively. Cisplatin, as a positive control, showed IC50 values ​​against human breast cancer cells MCF-7, human gastric cancer cells SGC-7901, and human colon cancer cells HT-29. 50 The values ​​were 6.11±0.77 μM, 5.87±1.01 μM, and 18.78±1.29 μM, respectively.

[0020] The results showed that the compounds of the present invention had a certain inhibitory effect on the growth of human breast cancer cells MCF-7, human gastric cancer cells SGC-7901 and human colon cancer cells HT-29, and have good development prospects for the preparation of clinical tumor prevention and treatment drugs.

Claims

1. A cyclolignan glycoside compound isolated from the rhizome of Atractylodes lancea, characterized in that: The molecular formula of the compound is C 43 H 56 O 21 Its chemical name is (-)-candelilla resin phenol 2a- O -{6- O -[(3′,5′-dimethoxy)benzoyl4′- O [α-L-rhamnopyranoside]-β-D-glucopyranoside, which has the following structure: 。 2. The method for preparing the compound according to claim 1, characterized in that: The compound was prepared from the rhizome of Atractylodes lancea using macroporous resin column, normal-phase silica gel column, reverse-phase silica gel column, and preparative HPLC. The specific preparation steps are as follows: (1) Alcohol extraction: Take 10 kg of dried rhizome of Atractylodes lancea as raw material, crush it appropriately, extract it by reflux with 70% ethanol for 3 h, repeat 3 times, filter, combine the 3 filtrates, recover the extract under reduced pressure, and dry to obtain the extract. (2) Enrichment and purification: The extract obtained by ethanol extraction in step (1) above is dispersed in water to a solution with a relative density of 1.35±0.05 g / mL, enriched and purified by AB-8 macroporous resin column chromatography, and eluted with water and 35% ethanol respectively. The 35% ethanol eluent is collected, and the solvent is recovered under reduced pressure to obtain the 35% ethanol eluent. (3) Normal phase silica gel column chromatography: Take the 35% ethanol elution fraction obtained in step (2) and perform normal phase silica gel column chromatography. The system is sequentially eluted with a mixed solution of dichloromethane and methanol with a volume ratio of 15:1, 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. Collect the eluent 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 perform reversed-phase silica gel ODS column chromatography. Elute sequentially with a methanol-water mixture with a volume ratio of 1:2 and a methanol-water mixture with a volume ratio of 1:

1. Collect the specified eluted fraction of the methanol-water mixture with a volume ratio of 1:1, recover the solvent, and obtain the crude product. (5) Preparative HPLC purification: The crude product obtained in step (4) is dissolved in methanol and introduced into a preparative HPLC. The mobile phase is a mixture of methanol and water with a volume ratio of 30:

70. The flow rate is 3 mL / min. After collecting the fraction, the solvent is recovered and dried to obtain the compound of the present invention.

3. The use of the compound of claim 1 in the preparation of antitumor drugs, characterized in that, The tumors are breast cancer, stomach cancer, and colon cancer.

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