Seven lignan compounds from Zanthoxylum bungeana and their extraction method and application

Abstract

The present invention belongs to the technical field of drug extraction, and provides 7 lignan compounds in Zanthoxylum bungeana and their extraction methods and applications. The compound is a compound shown in formula 1-7. The preparation method is: extracting Zanthoxylum bungeana extract with an organic solvent; dissolving the Zanthoxylum bungeana extract in a tartaric acid solution having a mass fraction of 3%, extracting the filtrate with ethyl acetate after filtering; merging the ethyl acetate extract and the acid-water insoluble matter, extracting with petroleum ether, ethyl acetate, and n-butanol in sequence, and concentrating to obtain a petroleum ether part, an ethyl acetate part, and an n-butanol part. The purified ethyl acetate part is subjected to silica gel column chromatography, gradient elution is performed with an eluent composed of dichloromethane and methanol, the fraction is analyzed and detected, and the target fraction is collected, and the target fraction is separated with a reverse phase chromatographic column, a dextran gel column, and a preparative liquid phase. The compound obtained by the present invention has in vitro antitumor activity, has good potential medicinal value, and is expected to be used in the preparation of various antitumor drugs.

Description

Technical Field

[0001] The invention relates to the technical field of drug extraction and separation, and in particular to seven lignan compounds in Zanthoxylum bungeana and an extraction method and application thereof. Background Art

[0002] Zanthoxylum nitidum (Rutaceae) is a woody vine of the genus Zanthoxylum in the family Rutaceae. It is also known as the ground bull, double-sided thorn, mountain pepper, and mountain tiger. It is mainly distributed in Guangdong, Guangxi, Fujian, Hunan and other places in China. It is a traditional Chinese medicine in my country. Its roots, stems, leaves, and peels can all be used as medicine. It is traditionally used to treat stomachache, toothache, rheumatic joint pain, trauma, and snake bites.

[0003] Modern pharmacological studies have shown that Zanthoxylum bungeanum not only has the traditional anti-inflammatory and analgesic effects, but also has anti-inflammatory, analgesic, hemostatic, anti-tumor and antibacterial biological activities. Since the study of Zanthoxylum bungeanum in 1959, a variety of chemical components have been isolated and identified from Zanthoxylum bungeanum, including alkaloids, coumarins, lignans, flavonoids, volatile oils, steroids, alkylamides, etc. They have significant anti-tumor, anti-inflammatory, anti-traumatic, analgesic, hemostatic, anti-tumor, antibacterial and other effects. In recent years, there have been reports on the separation of lignan compounds from Zanthoxylum bungeanum, but there are no reports on the 7 lignan compounds separated by the present invention. Summary of the invention

[0004] The purpose of the present invention is to provide 7 lignan compounds in Zanthoxylum bungeanum and their extraction method and application in view of the above-mentioned problems. The present invention obtains 7 lignan compounds by chemical separation, and the 7 lignan compounds have not been publicly reported. The 7 lignan compounds have in vitro anti-tumor activity and good potential medicinal value, and are expected to be used in the preparation of various anti-tumor drugs.

[0005] In order to achieve the above object, the technical solution adopted by the present invention is as follows:

[0006] The present invention first provides 7 lignan compounds in Zanthoxylum bungeanum, and their structural formulas are as follows:

[0007]

[0008] The present invention also provides a method for extracting seven lignan compounds from Zanthoxylum bungeana, comprising the following steps:

[0009] (1) Using Zanthoxylum bungeana as a raw material and extracting with an organic solvent to obtain a Zanthoxylum bungeana extract;

[0010] (2) dissolving the Zanthoxylum bungeana extract in a 3% by mass tartaric acid solution, filtering out the insoluble matter A, and extracting the filtrate with ethyl acetate to separate the ethyl acetate extract and the acid-water insoluble matter;

[0011] (3) The ethyl acetate extract and the acid-water insoluble matter are combined, extracted with petroleum ether, ethyl acetate, and n-butanol in sequence, and concentrated to obtain a petroleum ether fraction, an ethyl acetate fraction, and an n-butanol fraction.

[0012] (4) The purified ethyl acetate fraction is subjected to silica gel column chromatography, and gradient elution is performed using a solvent system consisting of dichloromethane and methanol as an eluent. The fractions are analyzed and detected, and the target fractions containing the compounds shown in 1-7 are collected. The target fractions containing the target compounds are separated using a reverse phase chromatography column, a dextran gel column, and a preparative liquid phase column in sequence to obtain the target compounds.

[0013] In the extraction method of the present invention, the preferred step (1) is to use the root of Zanthoxylum bungeanum as raw material, and the solvent used for extraction is preferably methanol with a volume concentration of 70-100% or ethanol or acetone with a volume concentration of 75-95%; the extraction method is room temperature extraction, heating extraction, ultrasonic extraction or reflux extraction, etc., and the extracted extract is concentrated to obtain an extract, which is used for extraction and separation in subsequent steps.

[0014] In the extraction method of the present invention, preferably in step (2), after the Zanthoxylum bungeanum extract is dissolved in 3% tartaric acid solution, the pH of the solution is adjusted to 2-3; after filtering out the insoluble matter A, the filtrate is extracted with ethyl acetate 3-4 times.

[0015] In the extraction method of the present invention, in step (3), petroleum ether, ethyl acetate and n-butanol are used for extraction in sequence, and preferably each extraction liquid is used for 3-4 times.

[0016] In the extraction method of the present invention, in step (4), during silica gel column chromatography, gradient elution is performed according to the volume ratio of dichloromethane to methanol of 100:1, 80:1, 50:1, 40:1, 30:1, 20:1, 15:1, 10:1, 8:1, 5:1, 2:1, and 1:1, and the volume of the eluent of each gradient concentration is 3-4 times the column volume; every 500 ml fraction is collected and the fraction is analyzed and detected.

[0017] In the extraction method of the present invention, in step (4), when separating with a reverse phase chromatographic column, it is preferred that the eluent is composed of methanol and water in different volume ratios, and gradient elution is performed with a methanol content of 20%, 30%, 40%, 50%, 60%, 70%, 80%, and 90%. Every 300 ml fraction is collected, the fraction is tested, and the target fraction is collected.

[0018] In the extraction method of the present invention, when using a dextran gel column for separation, the preferred eluent is methanol, and each 30 ml fraction is collected, the fraction is tested, and the target fraction is collected;

[0019] In the extraction method of the present invention, when using preparative liquid phase separation, the flow rate is 7 mL / min, the detection wavelength is 210 nm, and the separation conditions of each compound are: the separation conditions of compound 4 are that the volume ratio of mobile phase MeOH:H2O:TFA is 30:70:0.01, and t R The separation conditions for compound 6 were as follows: the volume ratio of mobile phase MeOH:H2O was 60:40, and t R The separation conditions for compound 7 were as follows: the volume ratio of mobile phase MeCN:H2O was 35:65, and t R The separation conditions for compound 1 were that the mobile phase was MeOH:H2O with a volume ratio of 45:55, and t R The separation conditions for compound 5 were that the mobile phase was MeOH:H2O with a volume ratio of 45:55, and t R The separation conditions for compound 2 were that the volume ratio of mobile phase MeOH:H2O was 45:55, and t R The separation conditions for compound 3 were as follows: the volume ratio of mobile phase MeOH:H2O was 60:40, and t R It is a component of 31min.

[0020] In the extraction method of the present invention, when the fraction is analyzed and detected in step (4), the detection means can be TLC, HPLC or HPLC-MS.

[0021] The present invention also protects the use of the above compound or its pharmaceutically acceptable salt in the preparation of anti-tumor drugs.

[0022] The present invention also protects the anti-tumor drug prepared by using the above compound or its pharmaceutically acceptable salt as the active ingredient.

[0023] In summary, due to the adoption of the above technical solution, the beneficial effects of the present invention are:

[0024] The present invention provides 7 novel lignan compounds, as well as their extraction methods and applications. The extraction method of the compounds is simple and easy to operate. By examining the inhibitory effects of the compounds on various tumors, the results show that the compounds have in vitro anti-tumor activity, have good potential medicinal value, and are expected to be used in the preparation of various anti-tumor drugs. DETAILED DESCRIPTION

[0025] In order to express the present invention more clearly, the present invention is further described below through specific examples.

[0026] Example 1

[0027] The method for extracting 7 lignan compounds from Zanthoxylum bungeanum comprises the following steps:

[0028] (1) 28 kg of the dried whole plant of Zanthoxylum bungeanum was taken, crushed, and refluxed for extraction three times using 95% ethanol by volume as a solvent. The extracts were combined and the solvent was recovered under reduced pressure to obtain 3.4 kg of Zanthoxylum bungeanum extract;

[0029] (2) dissolving the extract of Zanthoxylum bungeanum in a 3% by mass tartaric acid solution, adjusting the pH of the extract solution to 2, filtering out the insoluble matter A, extracting the filtrate with ethyl acetate three times, concentrating under reduced pressure to recover the solvent, and separating the ethyl acetate extract and the acid-water insoluble matter;

[0030] (3) combining the ethyl acetate extract and the acid-water insoluble matter, extracting with petroleum ether, ethyl acetate, and n-butanol in sequence, extracting each extract 3 times, combining and concentrating them to obtain purified petroleum ether fraction, ethyl acetate fraction, and n-butanol fraction;

[0031] (4) The purified ethyl acetate fraction (700 g) was subjected to silica gel column chromatography, and dichloromethane and methanol were used as eluents. Gradient elution was performed at a volume ratio of dichloromethane to methanol of 100:1, 80:1, 50:1, 40:1, 30:1, 20:1, 15:1, 10:1, 8:1, 5:1, 2:1, and 1:1. The volume of the eluent in each gradient concentration was 3 times the column volume. Each 500 ml fraction was collected, and the samples were collected in sections. The fractions were divided and combined by TLC to obtain 11 fractions. The fractions were then analyzed and detected by HPLC-UV and HPLC-MS to find the fractions containing the target compound. The target fractions containing the compound shown in structural formula 1-7 were collected.

[0032] The obtained target fraction containing the target compound is first separated by a reverse phase chromatographic column. The eluent for separation by the reverse phase chromatographic column is composed of methanol and water in different volume ratios. The gradient elution is performed at a methanol content of 20%, 30%, 40%, 50%, 60%, 70%, 80%, and 90%. Each 300 ml fraction is collected, the fractions are tested, and the target fraction is collected.

[0033] The target fraction is then separated by a dextran gel column, the eluent of which is methanol, and 30 ml fractions are collected. The fractions are tested and the target fraction is collected.

[0034] The target fractions were separated by preparative liquid phase separation. The conditions for the preparative liquid phase were a flow rate of 7 mL / min and a detection wavelength of 210 nm. The separation conditions for each compound were: the separation conditions for compound 4 were a mobile phase of MeOH:H2O:TFA in a volume ratio of 30:70:0.01, and t R The separation conditions for compound 6 were as follows: the volume ratio of mobile phase MeOH:H2O was 60:40, and t R The separation conditions for compound 7 were as follows: the volume ratio of mobile phase MeCN:H2O was 35:65, and t R The separation conditions for compound 1 were that the mobile phase was MeOH:H2O with a volume ratio of 45:55, and t R The separation conditions for compound 5 were that the mobile phase was MeOH:H2O with a volume ratio of 45:55, and t R The separation conditions for compound 2 were that the volume ratio of mobile phase MeOH:H2O was 45:55, and t R The separation conditions for compound 3 were as follows: the volume ratio of mobile phase MeOH:H2O was 60:40, and t R It is a component of 31min.

[0035] The obtained product was structurally analyzed by NMR and HRESIMS, and was confirmed to be the target compound 1-7.

[0036] Example 2

[0037] The method for extracting 7 lignan compounds from Zanthoxylum bungeanum comprises the following steps:

[0038] (1) Take 27 kg of the dried whole plant of Zanthoxylum bungeanum, crush it, and extract it with 75% ethanol by volume for 3 times at room temperature, each time for 3 hours. Combine the extracts, and recover the solvent under reduced pressure to obtain a Zanthoxylum bungeanum extract.

[0039] (2) dissolving the extract of Zanthoxylum bungeanum in a 3% by mass tartaric acid solution, adjusting the pH of the extract solution to 3, filtering out the insoluble matter A, extracting the filtrate with ethyl acetate for 4 times, concentrating under reduced pressure to recover the solvent, and separating the ethyl acetate extract and the acid-water insoluble matter;

[0040] (3) combining the ethyl acetate extract and the acid-water insoluble matter, extracting with petroleum ether, ethyl acetate, and n-butanol in sequence, extracting each extract four times, combining and concentrating them to obtain purified petroleum ether fraction, ethyl acetate fraction, and n-butanol fraction;

[0041] (4) The purified ethyl acetate fraction was subjected to silica gel column chromatography, and dichloromethane and methanol were used as eluents. The volume ratio of dichloromethane and methanol was 100:1, 80:1, 50:1, 40:1, 30:1, 20:1, 15:1, 10:1, 8:1, 5:1, 2:1, and 1:1 for gradient elution. The volume of eluent in each gradient concentration was 4 times the volume of the column. Each 500 ml fraction was collected, and the samples were collected in sections. The fractions were divided and combined by TLC to obtain 11 fractions. After that, HPLC-UV and HPLC-MS were used for analysis and detection to find the fractions containing the target compounds. The target fractions containing the compounds shown in 1-7 were collected.

[0042] The obtained target fraction containing the target compound is first separated by a reverse phase chromatographic column. The eluent for separation by the reverse phase chromatographic column is composed of methanol and water in different volume ratios. The gradient elution is performed at a methanol content of 20%, 30%, 40%, 50%, 60%, 70%, 80%, and 90%. Each 300 ml fraction is collected, the fractions are tested, and the target fraction is collected.

[0043] The target fraction is then separated by a dextran gel column, the eluent of which is methanol, and 30 ml fractions are collected. The fractions are tested and the target fraction is collected.

[0044] The target fractions were separated by preparative liquid phase separation. The conditions for the preparative liquid phase were a flow rate of 7 mL / min and a detection wavelength of 210 nm. The separation conditions for each compound were as follows: the separation conditions for compound 4 were a mobile phase with a volume ratio of MeOH:H2O:TFA of 30:70:0.01, and t R The separation conditions for compound 6 were as follows: the volume ratio of mobile phase MeOH:H2O was 60:40, and t R The separation conditions for compound 7 were as follows: the volume ratio of mobile phase MeCN:H2O was 35:65, and t R The separation conditions for compound 1 were that the mobile phase was MeOH:H2O with a volume ratio of 45:55, and t R The separation conditions for compound 5 were that the mobile phase was MeOH:H2O with a volume ratio of 45:55, and t R The separation conditions for compound 2 were that the volume ratio of mobile phase MeOH:H2O was 45:55, and t R The separation conditions for compound 3 were as follows: the volume ratio of mobile phase MeOH:H2O was 60:40, and t R It is a component of 31min.

[0045] The obtained product was structurally analyzed by NMR and HRESIMS, and was confirmed to be the target compound 1-7.

[0046] Example 3

[0047] Example 1 was repeated, except that the extraction solvent in step (1) was changed from 95% by volume ethanol to 100% by volume methanol.

[0048] The obtained product was structurally analyzed by NMR and HRESIMS, and was confirmed to be the target compound 1-7.

[0049] Example 4

[0050] Example 2 was repeated, except that the extraction solvent in step (1) was changed from 75% by volume ethanol to 70% by volume methanol, and ultrasonic extraction was used as the extraction method.

[0051] The obtained product was structurally analyzed by NMR and HRESIMS, and was confirmed to be the target compound 1-7.

[0052] Example 5

[0053] Example 1 was repeated, except that the extraction solvent in step (1) was changed from 95% ethanol by volume to 80% methanol, and the extraction method was heating extraction at a temperature of 60° C. and an extraction time of 3 h.

[0054] The obtained product was structurally analyzed by NMR and HRESIMS, and was confirmed to be the target compound 1-7.

[0055] Example 6

[0056] Example 1 was repeated, except that the extraction solvent in step (1) was changed from ethanol with a volume concentration of 95% to acetone.

[0057] The obtained product was structurally analyzed by NMR and HRESIMS, and was confirmed to be the target compound 1-7.

[0058] 2. Product Confirmation

[0059] The products obtained in Examples 1-6 were subjected to structural analysis by NMR and HRESIMS, and the spectral data are shown below. The purity of the compounds was >95% as determined by HPLC.

[0060] Compound 1:

[0061] 1 H NMR (400 MHz, CD3OD, 25°C)δ H7.49 (1H, d, J = 1.7 Hz, H-2), 6.95 (1H, d, J = 8.2 Hz, H-5), 7.69 (1H, dd, J = 8.2, 1.7 Hz, H-6), 4.21 (1H, overlapped, H-8), 3.63 (2H, m, H-9), 7.09 (1H, d, J = 1.4 Hz, H-2'), 6.92 (2H, overlapped, H-5', H-6'), 4.66 (1H, d, J = 8.5 Hz, H-7'), 2.71 (1H, m, H-8'), 4.15 (1H, m, H-9'), 4.21 (1H, overlapped, H-9'), 4.55 (2H, d, J = 6.7 Hz, H-1”), 5.48 (1H, m, H-2”), 1.73 (3H, s, H-4”), 1.78 (3H, s, H-5”), 6.07 (2H, s, -OCH2O-), 3.86 (3H, s, -OCH3);

[0062] 13 C NMR (100 MHz, CD3OD, 25 °C) δ C 132.7 (C-1), 109.0 (C-2), 149.9 (C-3), 153.7 (C-4), 109.0 (C-5), 126.4 (C-6), 199.7 (C-7), 50.7 (C-8), 61.4 (C-9), 135.3 (C-1'), 111.8 (C-2'), 149.3 (C-3'), 151.2 (C-4'), 114.9 (C-5'), 120.6 (C-6'), 85.2 (C-7'), 54.5 (C-8'), 71.8 (C-9'), 67.0 (C-1”), 121.2 (C-2”), 138.8 (C-3”), 18.2 (C-4”), 25.9 (C-5”), 103.6 (-OCH2O-), 56.4 (-OMe);

[0063] HRESI-MS m / z 441.1894 [M+H] + , (calcd for 441.1908).

[0064] Compound 2:

[0065] 1 H NMR (400 MHz, CD3OD, 25 °C) δ H6.93 (1H, d, J = 1.8 Hz, H-2), 6.90 (1H, d, J = 8.2 Hz, H-5), 6.84 (1H, dd, J = 8.2, 1.8 Hz, H-6), 4.79 (1H, d, J = 6.6 Hz, H-7), 4.79 (1H, m, H-8), 3.83 (1H, overlapped, H-9), 3.63 (1H, dd, J = 10.9, 6.7 Hz, H-9), 6.72 (1H, overlapped, H-2'), 6.72 (1H, overlapped, H-5'), 6.66 (1H, dd, J = 8.0, 1.4 Hz, H-6'), 2.91 (1H, dd, J = 13.4, 4.9 Hz, H-7'), 2.91 (1H, dd, J = 13.4, 11.2 Hz, H-7'), 2.70 (1H, m, H-8'), 3.99 (1H, dd, J = 8.3, 6.5 Hz, H-9'), 3.71 (1H, dd, J = 8.3, 6.2 Hz, H-9'),, 4.53 (2H, d, J = 6.7 Hz, H-1”), 5.47 (1H, m, H-2”), 1.77 (3H, s, H-4”), 1.72 (3H, s, H-5”), 5.89 (2H, s, -OCH2O-), 3.82 (3H, s, -OCH3);

[0066] 13 13C NMR (100 MHz, CD3OD, 25 °C) δ C 137.5 (C-1), 110.9 (C-2), 151.1 (C-3), 148.9 (C-4), 115.1 (C-5), 119.4 (C-6), 83.9 (C-7), 54.0 (C-8), 60.4 (C-9), 135.8 (C-1'), 110.0 (C-2'), 147.3 (C-3'), 149.2 (C-4'), 109.1 (C-5'), 122.7 (C-6'), 33.8 (C-7'), 43.8 (C-8'), 73.5 (C-9'), 67.0 (C-1”), 121.3 (C-2”), 138.8 (C-3”), 25.9 (C-4”), 18.2 (C-5”), 102.1 (-OCH2O-), 56.4 (-OMe);

[0067] HRESI-MS m / z 449.1944 [M+Na] + , (calcd for 449.1935).

[0068] Compound 3:

[0069] 1 H NMR (400 MHz, CDCl3, 25 °C) δ H 7.40 (1H, d, J = 1.5 Hz, H-2), 6.90 (1H, d, J = 8.1 Hz, H-5), 7.52 (1H, dd, J = 8.1, 1.5 Hz, H-6), 7.80 (1H, s, H-9), 7.12 (1H, s, H-2'), 6.92 (1H, d, J = 1.9 Hz, H-5'), 7.14 (1H, m, H-6'), 4.70 (2H, s, H-9'), 6.09 (2H, s, -OCH2O-), 6.03 (2H, s, -OCH2O-);

[0070] 13 C NMR (100 MHz, CDCl3, 25 °C) δ C 133.1 (C-1), 109.2 (C-2), 148.4 (C-3), 152.2 (C-4), 108.2 (C-5), 125.8 (C-6), 190.1 (C-7), 127.6 (C-8), 148.0 (C-9), 123.5 (C-1'), 107.9 (C-2'), 148.2 (C-3'), 148.4 (C-4'), 108.9 (C-5'), 121.9 (C-6'), 153.2 (C-7'), 120.0 (C-8'), 55.6 (C-9'), 102.2 (-OCH2O-), 101.6 (-OCH2O-);

[0071] HRESI-MS m / z 389.0619 [M+Na] + , (calcd for C 20 H 14 O7Na + : 389.0632).

[0072] Compound 4:

[0073] 1 H NMR (400 MHz, CD3OD, 25 °C) δ H7.48 (1H, d, J = 1.6 Hz, H-2), 6.96 (1H, d, J = 8.2 Hz, H-5), 7.70 (1H, dd, J = 8.2, 1.6 Hz, H-6), 4.54 (1H, dd, J = 16.0, 7.8 Hz, H-8), 4.65 (1H, t, J = 8.7 Hz, H-9), 4.26 (1H, dd, J = 8.7, 7.8 Hz, H-9), 3.17 (1H, dt, J = 7.8, 3.9 Hz, H-11), 3.98 (1H, dd, J = 11.2, 3.9 Hz, H-12), 3.75 (1H, dd, J = 11.2, 3.9 Hz, H-12), 6.08 (2H, s, -OCH2O-);

[0074] 13 C NMR (100 MHz, CD3OD, 25 °C) δ C 131.7 (C-1), 108.9 (C-2), 154.2 (C-3), 150.1 (C-4), 109.1 (C-5), 126.7 (C-6), 196.9 (C-7), 45.7 (C-8), 69.9 (C-9), 178.9 (C-10), 47.1 (C-11), 60.4 (C-12), 103.7 (-OCH2O-);

[0075] HRESI-MS m / z 287.0526 [M+Na] + , (calcd for 287.0526).

[0076] Compound 5:

[0077] 1 H NMR (400 MHz, CD3OD, 25 °C) δ H6.97 (1H, d, J = 1.5 Hz, H-2), 6.89 (1H, overlapped, H-5), 6.84 (1H, overlapped, H-6), 4.69 (1H, d, J = 4.5 Hz, H-7), 3.12 (1H, overlapped, H-8), 4.21 (1H, overlapped, H-9), 3.84 (1H, m, H-9), 6.76 (2H, d, J = 8.5 Hz, H-2', 3'), 7.19 (2H, d, J = 8.5 Hz, H-5', 6'), 4.72 (1H, d, J = 4.5 Hz, H-7'), 3.12 (1H, overlapped, H-8'), 4.21 (1H, overlapped, H-9'), 3.81 (1H, m, H-9'), 4.53 (2H, d, J = 6.7 Hz, H-1”), 5.46 (1H, m, H-2”), 1.76 (3H, s, H-4”), 1.72 (3H, s, H-5”), 3.82 (3H, s, -OCH3);

[0078] 13 C NMR (100 MHz, CD3OD, 25 °C) δ C 135.5 (C-1), 111.3 (C-2), 151.2 (C-3), 149.1 (C-4), 115.1 (C-5), 119.7 (C-6), 87.3 (C-7), 55.5 (C-8), 72.7 (C-9), 133.0 (C-1'), 116.2 (C-2'), 128.7 (C-3'), 158.3 (C-4'), 128.7 (C-5'), 116.2 (C-6'), 87.4 (C-7'), 55.2 (C-8'), 72.5 (C-9'), 67.0 (C-1”), 121.2 (C-2”), 138.8 (C-3”), 25.9 (C-4”), 18.2 (C-5”), 56.4 (-OMe);

[0079] HRESIMS m / z 419.1846 [M+Na] + , (calcd for 419.1829).

[0080] Compound 6

[0081] 1 H NMR (400 MHz, CD3OD, 25 °C) δ H7.33(1H, d, J = 1.5 Hz, H-2), 7.20(1H, d, J = 8.1 Hz, H-5), 7.38(1H, dd, J = 8.1, 1.5 Hz, H-6), 3.67(1H, m, H-8), 4.16(1H, t, J = 8.9 Hz, H-9), 3.38(1H, t, J = 8.9 Hz, H-9), 7.23(1H, d, J = 1.5 Hz, H-2'), 7.15(1H, d, J = 8.1 Hz, H-5'), 7.20(1H, d, J = 8.1 Hz, 6'), 4.82(1H, d, J = 6.6 Hz, H-7'), 3.33(1H, m, H-8'), 4.43(2H, m, H-9'), 6.35(2H, s, -OCH2O-), 6.30(2H, s, -OCH2O-);

[0082] 13 C NMR(100 MHz, CD3OD, 25 °C) δ C 132.9(C-1), 108.5(C-2), 149.2(C-3), 148.7(C-4), 108.8(C-5), 121.7(C-6), 111.5(C-7), 58.0(C-8), 71.3(C-9), 136.3(C-1'), 107.5(C-2'), 149.4(C-3'), 149.0(C-4'), 109.0(C-5'), 120.7(C-6'), 89.2(C-7'), 54.5(C-8'), 70.5(C-9'), 102.6(-OCH2O-), 102.4(-OCH2O-);

[0083] HRESI-MS m / z m / z 393.0955[M+Na] + , (calcd for 393.0945).

[0084] Compound 7

[0085] 1 H NMR(400 MHz, CD3OD, 25 °C) δ H6.95(1H,s,H-2),7.00(1H,d,J=8.0Hz,H-5),6.83(1H,d,J=8.0Hz,H-6),3.28(1H,d,J=8.8Hz,H -8),3.76(1H,t,J=8.8Hz,H-9),2.99(1H,t,J=8.8Hz,H-9),6.79(1H,d,J=1.9Hz,H-2'),6.73(1H ,d,J=8.1Hz,H-5'),7.20(1H,dd,J=8.1,1.9Hz,6'),4.37(1H,d,J=6.7Hz,H-7'),2.96(1H,m,H- 8'),4.06(1H,dd,J=8.9,6.7Hz,H-9'),4.01(1H,dd,J=8.9,1.5Hz,H-9'),5.97(2H,s,-OCH2O-);

[0086] 13 C NMR (100 MHz, CD3OD, 25°C)δ C 133.0(C-1),108.5(C-2),149.2(C-3),149.0(C-4),108.8(C-5),121.7(C-6),111.5(C-7),58.0(C-8),71.2(C-9),133.6(C-1') ,114.4(C-2'),146.5(C-3'),146.2(C-4'),116.2(C-5'),119.0(C-6'),89.4(C-7'),54.2(C-8'),70.6(C-9'),102.6(-OCH2O-);

[0087] HRESI-MS ion at m / z 397.0673[M+K] + ,(calcd for 397.0684).

[0088] Therefore, it can be determined that the structural formula of the above compound nitidumlignan DJ is as shown in the following formula (I):

[0089]

[0090] Among them, R in compound 1 is =O, R in compound 2 is H, R1-R2 in compound 6 is -OCH2O-, and R1 and R2 in compound 7 are both -OH.

[0091] 3. Research on the anti-tumor effect of the product

[0092] The clinically used tumor treatment drug etoposide was used as the positive control drug, and blank culture medium was used as the negative control. The compounds were tested for their in vitro antitumor activity using SNU638 (human gastric cancer cells), MDA-MB-231 (human breast cancer cells), SK-Hep-1 (human liver cancer cells), A549 (human non-small cell lung cancer cells), and HCT116 (human colon cancer cells) as the test cell lines.

[0093] The tested cell lines were maintained in RPMI medium (PSF; 100 units / mL penicillin G sodium, 100 μg / mL streptomycin, and 250 ng / mL amphotericin B) supplemented with 10% fetal bovine serum and 1% antibiotics (AA). The cells were cultured at 37°C, 5% CO2, and a humidified environment.

[0094] After the cell lines were cultured for 72 h, the cell viability was determined using the sulforhodamine B (SRB) method. The cell lines in the logarithmic growth phase were inoculated into 96-well plates, and 190 μL of cell suspension was added to each well. The cell concentration of the cell suspension was 8 to 3×10 4 / mL. The dosing groups were added with 50μL, 10μL, 2μL, 0.4μL of 10μL of samples containing different concentrations of the compound of the present invention dissolved in DMSO, respectively. The positive control group was added with 20μL, 4μL, 0.8μL, 0.16μL of samples containing different concentrations of etoposide. The negative group was not added with samples and 10μL of blank DMSO consistent with the sample group was added, and further incubated for 72h. The cells were fixed with 10% trichloroacetic acid (TCA), the remaining proteins were precipitated, and stained with SRB solution in 0.1% acetic acid. The stained protein was dissolved in 10mM Tris solution (pH=10) and measured at 515nm using a Versamax enzyme-linked immunosorbent assay. The cell proliferation inhibition rate of each dosing well was calculated, and its IC 50 The results are shown in Table 1 below:

[0095] Table 1:

[0096]

[0097] As can be seen from Table 1, some of the compounds described in the present invention have good inhibitory effects on various tumor cells, indicating that the compounds described in the present invention have good anti-tumor effects and are expected to be used in the preparation of anti-tumor drugs.

[0098] The above description is a detailed description of the preferred feasible embodiments of the present invention, but the embodiments are not intended to limit the scope of the patent application of the present invention. All equivalent changes or modified changes completed under the technical spirit suggested by the present invention should fall within the patent scope covered by the present invention.

Claims

1. Five lignan compounds in Zanthoxylum bungeana, characterized in that: The structural formulas of compounds 1, 2, 5, 6 and 7 are as follows: Among them, R in compound 1 is =O, R in compound 2 is H, R1-R2 in compound 6 is -OCH2O-, and R1 and R2 in compound 7 are both -OH.

2. The method for extracting the five lignan compounds from Zanthoxylum bungeanum as claimed in claim 1, characterized in that: The following steps are involved: (1) Using Zanthoxylum bungeana as raw material, extracting with organic solvent to obtain Zanthoxylum bungeana extract; (2) dissolving the Zanthoxylum bungeanum extract in a 3% by mass tartaric acid solution, filtering out the insoluble matter A, and extracting the filtrate with ethyl acetate to separate the ethyl acetate extract and the acid-water insoluble matter; (3) combining the ethyl acetate extract and the acid-water insoluble matter, extracting with petroleum ether, ethyl acetate, and n-butanol in sequence, and concentrating to obtain a petroleum ether fraction, an ethyl acetate fraction, and an n-butanol fraction; (4) The purified ethyl acetate fraction was subjected to silica gel column chromatography, and gradient elution was performed using a solvent system consisting of dichloromethane and methanol as an eluent. The fractions were analyzed and tested, and target fractions containing compounds 1, 2, 5, 6 and 7 were collected. The target fractions containing the target compound were separated using a reverse phase chromatography column, a dextran gel column and a preparative liquid phase column, respectively, to obtain the target compound.

3. The extraction method according to claim 2, characterized in that: Step (1) uses the root of Zanthoxylum bungeanum as raw material, and the solvent used for extraction is methanol with a volume concentration of 70-100% or ethanol or acetone with a volume concentration of 75-95%; the extraction method is room temperature extraction, heating extraction, ultrasonic extraction or reflux extraction.

4. The extraction method according to claim 2, characterized in that: In step (2), after the Zanthoxylum bungeanum extract is dissolved in a 3% tartaric acid solution, the pH of the solution is adjusted to 2-3; after filtering out the insoluble matter A, the filtrate is extracted with ethyl acetate 3-4 times.

5. The extraction method according to claim 2, characterized in that: In step (3), petroleum ether, ethyl acetate and n-butanol are used for extraction in sequence, and each extraction liquid is extracted 3-4 times.

6. The extraction method according to claim 2, characterized in that: In step (4), during silica gel column chromatography, gradient elution is performed according to the volume ratio of dichloromethane and methanol of 100:1, 80:1, 50:1, 40:1, 30:1, 20:1, 15:1, 10:1, 8:1, 5:1, 2:1, and 1:1, and the volume of the eluent of each gradient concentration is 3-4 times the column volume; each 500 ml fraction is collected and the fraction is analyzed and detected.

7. The extraction method according to claim 2, characterized in that: In step (4), when using a reverse phase chromatography column for separation, the eluent is composed of methanol and water in different volume ratios, and the gradient elution is performed with a methanol content of 20%, 30%, 40%, 50%, 60%, 70%, 80%, and 90%. Each 300 ml fraction is collected, the fraction is tested, and the target fraction is collected; When using a dextran gel column for separation, the eluent is methanol, and every 30 ml fraction is collected, the fraction is tested, and the target fraction is collected; When using preparative liquid phase separation, the flow rate is 7 mL / min, the detection wavelength is 210 nm, and the collection t R The separation conditions for compound 6 were as follows: the volume ratio of mobile phase MeOH:H2O was 60:40, and the t R The separation conditions for compound 7 were as follows: the volume ratio of mobile phase MeCN:H2O was 35:65, and the t R The separation conditions for compound 1 were as follows: the mobile phase was MeOH:H2O with a volume ratio of 45:55, and the t R The separation conditions for compound 5 were as follows: the mobile phase was MeOH:H2O with a volume ratio of 45:55, and the t R The separation conditions for compound 2 were that the volume ratio of mobile phase MeOH:H2O was 45:55, and the t R It is a 26 min component.

8. Use of the compound according to claim 1 or a pharmaceutically acceptable salt thereof in the preparation of an anti-tumor drug, characterized in that: The use of compound 2 or a pharmaceutically acceptable salt thereof in the preparation of drugs for treating gastric cancer and non-small cell lung cancer, the use of compound 6 or a pharmaceutically acceptable salt thereof in the preparation of drugs for treating gastric cancer, and the use of compound 7 or a pharmaceutically acceptable salt thereof in the preparation of drugs for treating colon cancer.

9. An anti-tumor drug prepared with the compound or a pharmaceutically acceptable salt thereof as an active ingredient according to claim 1, characterized in that: The drug prepared from the compound 2 or its pharmaceutically acceptable salt is a drug for treating gastric cancer and non-small cell lung cancer, the drug prepared from the compound 6 or its pharmaceutically acceptable salt is a drug for treating gastric cancer, and the drug prepared from the compound 7 or its pharmaceutically acceptable salt is a drug for treating colon cancer.

10. Use of compound 3 or a pharmaceutically acceptable salt thereof in the preparation of drugs for treating gastric cancer, breast cancer, non-small cell lung cancer and colon cancer, wherein the structural formula of compound 3 is: 。

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