Sesquiterpene dimer in artemisia sieversiana, pharmaceutical composition of sesquiterpene dimer, preparation method of sesquiterpene dimer and application of sesquiterpene dimer

Abstract

The invention provides sesquiterpene dimer compounds 1-13 in artemisia sieversiana, a pharmaceutical composition of the sesquiterpene dimer compounds 1-13 and a preparation method and application of the sesquiterpene dimer compounds 1-13, and belongs to the technical field of medicines. The 13 new sesquiterpenoid dimers shown in the structural formula of the invention, i.e., artemsiverlides A-M (1-13), have obvious inhibitory activity on human hepatic stellate cells (HSC-LX2), can form a pharmaceutical composition with a pharmaceutically acceptable carrier, and can be used for preparing anti-hepatic fibrosis drugs.

Description

Technical Field:

[0001] The present invention belongs to the field of pharmaceutical technology. Specifically, it relates to sesquiterpene dimer compounds artemsieverlides A–M (1-13), their preparation methods, pharmaceutical compositions and their applications. Background Art:

[0002] The results of epidemiological investigations show that the global average annual death toll due to liver diseases is approximately 2 million people, accounting for about 3.5% of the world's total deaths, and has become one of the main causes of human death. Liver fibrosis is a common pathological feature of advanced chronic liver diseases such as viral hepatitis, alcoholic liver disease, metabolic dysfunction-associated steatohepatitis (MASH), and cholestatic liver disease. After a large number of hepatocytes die due to liver injury, extracellular matrix proteins and fibrous connective tissues will be over-deposited in the liver, causing fibrosis. Subsequently, the excessive fibers produced by liver fibrosis further change the liver structure, forming scars and regenerative nodules, thus leading to abnormal liver function and increased liver blood flow resistance. The mechanism of liver fibrosis involves multiple complex factors, mainly including hepatocyte injury, inflammatory response, activation of hepatic stellate cells (HSC), and extracellular matrix deposition. Among them, liver injury is the basis for the occurrence of liver fibrosis. When the liver is stimulated by alcohol, drugs, viruses, or metabolic abnormalities, hepatocytes are damaged, mainly manifested as reduced membrane fluidity and increased permeability, as well as damaged mitochondria. Subsequently, liver injury causes the release of inflammatory factors such as interleukin (IL)-1β, inducing the activation of HSC. The activated HSC transforms into myofibroblasts, migrates to the injury site, accelerates proliferation and secretes extracellular matrix, resulting in the formation of fibrosis. Currently, there are few clinically effective drugs for the treatment of liver fibrosis, and they are limited to drugs for controlling the etiology and traditional Chinese medicines. The present invention aims to search for new anti-liver fibrosis active ingredients from natural products based on a screening model of human hepatic stellate cells line LX2 (HSC-LX2).

[0003] Artemisia sieversiana is a perennial herbaceous plant of the genus Artemisia in the Compositae family. It grows on slopes, grasslands, sparse forests, forest margins and other places below an altitude of 3,500 meters. It is mainly distributed in Heilongjiang, Liaoning, Inner Mongolia, Gansu, Sichuan, Yunnan, Tibet and other places. According to the "Dictionary of Chinese Materia Medica", Artemisia sieversiana is sweet and bitter in nature and is mainly used to treat wind-cold, dysentery, jaundice, etc. Mongolian medicine and Tibetan medicine record that it has the effects of detumescence, hemostasis, clearing heat and relieving summer heat. After decocting and taking it, it can treat diarrhea, colds, jaundice, etc., and can be used to treat skin burns caused by ultraviolet radiation. Modern pharmacological research shows that Artemisia sieversiana has significant effects of reducing phlegm and relieving asthma, anti-inflammatory and antibacterial. Through literature research, it is found that the main components in Artemisia sieversiana are sesquiterpenoids (guaianolide type, germacrane type, muurolane type, sesquiterpene dimer), lignans, flavonoids, volatile oils and other types of compounds. Pharmacological research shows that the compounds isolated from Artemisia sieversiana have activities such as anti-tumor, anti-inflammatory, antibacterial and antioxidant.

[0004] So far, there is no report on sesquiterpene dimer artemsieverlides A–M (1-13) in the prior art, nor is there a report on compounds 1-13 and their pharmaceutical compositions as anti-hepatic fibrosis drugs. Summary of the Invention:

[0005] The purpose of the present invention is to provide a new class of sesquiterpene dimers with medicinal value, artemsieverlides A–M (1-13), and their preparation methods and applications, pharmaceutical compositions containing artemsieverlides A–M and their applications. These compounds have obvious cytotoxic activity against hepatic stellate cells and can be used to prepare anti-hepatic fibrosis drugs.

[0006] In order to achieve the above object of the present invention, the present invention provides the following technical solutions:

[0007] The present invention provides a series of sesquiterpene dimer compounds, artemsieverlides A–M (1-13), having the structures shown in the following structural formula:

[0008]

[0009] The present invention provides a method for preparing the above sesquiterpene dimer compounds 1-13. Take the dried aerial parts of Artemisia sieversiana, crush them, and extract them twice with 95% ethanol by cold maceration for 4 days each time. Combine the ethanol extracts, recover the ethanol extract under reduced pressure. After the extract is dispersed in water, it is extracted with ethyl acetate, and then concentrated to obtain the ethyl acetate extraction part. Then, the ethyl acetate extraction part is subjected to silica gel column chromatography, and acetone-petroleum ether with a v / v ratio of 5:95, 10:90, 20:80, 30:70, and 100:0 is used as the eluent for gradient elution to obtain five fractions, Fr.A-Fr.E; Fraction Fr.D is subjected to silica gel column chromatography, and methanol-chloroform with a v / v ratio of 5:95, 10:90, and 20:80 is used as the eluent for gradient elution to obtain five fractions, Fr.D1-Fr.D5; Fraction Fr.D4 is subjected to MCI gel CHP20P column chromatography, and water-methanol with a v / v ratio of 10:90 and 0:100 is used as the eluent for gradient elution and semi-preparative HPLC to prepare compounds 1-13.

[0010] The present invention provides the use of the sesquiterpene dimer compounds 1-13 described in the above technical solution in the preparation of anti-hepatic fibrosis drugs. The present invention has no special limitation on the method of the said use, and any method well-known in the art can be selected.

[0011] The present invention provides a pharmaceutical composition, which comprises at least one of the sesquiterpene dimer compounds 1-13 described in the above technical solution and a pharmaceutically acceptable carrier.

[0012] Furthermore, the present invention also provides the use of the pharmaceutical composition described in the above technical solution in the preparation of anti-hepatic fibrosis drugs. And, a method for preparing the pharmaceutical composition, first prepare compounds 1-13 according to the preparation method of the above compounds, then take one or any combination of compounds 1-13, and add a pharmaceutically acceptable carrier.

[0013] When at least one of the compounds 1-13 is used in the preparation of anti-hepatic fibrosis drugs, the present invention preferably uses the compounds 1-13 directly or in the form of a pharmaceutical composition.

[0014] The pharmaceutical composition provided by the present invention comprises at least one of the above compounds 1-13 and a pharmaceutically acceptable carrier or excipient. In the present invention, the pharmaceutically acceptable carrier or excipient is preferably a solid, semi-solid or liquid diluent, filler, and pharmaceutical product adjuvant. The present invention has no special limitation on the pharmaceutically acceptable carrier, and any pharmaceutically acceptable carrier well-known in the art, which is non-toxic and inert to humans and animals, can be selected.

[0015] The present invention has no special limitation on the preparation method of the pharmaceutical composition, and at least one of Compounds 1-13 can be directly mixed with a pharmaceutically acceptable carrier. The present invention has no special limitation on the mixing process, and a pharmaceutical composition can be obtained by using a process well-known in the art.

[0016] The present invention provides the use of the pharmaceutical composition according to the above technical solution in the preparation of an anti-hepatic fibrosis drug. The present invention has no special limitation on the method of the use, and a method well-known in the art can be selected.

[0017] In the present invention, when the pharmaceutical composition is used for the preparation of an anti-hepatic fibrosis drug, the content of the composition in the drug is preferably 0.1-99%; in the pharmaceutical composition, the content of at least one of Compounds 1-13 in the pharmaceutical composition is preferably 0.5-90%. The pharmaceutical composition of the present invention is preferably used in the form of a dosage per unit body weight. In the present invention, the prepared drug is preferably administered by two forms: injection (intravenous injection, intramuscular injection) and oral administration.

[0018] Compared with the prior art, the present invention has the following advantages:

[0019] 1. The present invention provides a series of new sesquiterpene dimer compounds, artemsieverlides A–M (1-13).

[0020] 2. The present invention provides a new method for preparing the new compounds 1-13. The method has easily available raw materials, is easy to operate, and is suitable for industrial production.

[0021] 3. The present invention provides a pharmaceutical composition with the new compounds 1-13 as the active ingredient, providing a new drug with good medicinal effects for new anti-hepatic fibrosis drugs.

[0022] 4. The compounds 1-13 of the present invention all have strong cytotoxic activities against hepatic stellate cells (HSC-LX2). Compounds 8 and 11 have strong cytotoxic activities, and their IC 50 values are 71.6 and 58.6 μM respectively, and the activity is 2 times that of the positive drug silybin; Compounds 3, 4, 5, 6 and 9 also show certain cytotoxic activities, and their IC 50 values are 94.2, 115.2, 126.7, 99.3 and 112.4 μM respectively, and the activity is better than that of the positive drug silybin. The remaining compounds have certain HSC-LX2 cytotoxic activities, and the inhibition rate is between 30.0-62.9% when the concentration is 200 μg / mL. Description of the drawings:

[0023] Figure 1 It is a schematic structural diagram of the compounds 1-13 of the present invention;

[0024] Figure 2 Schematic diagram of the X-single crystal diffraction structure of Compounds 3, 7 and 12 of the present invention. Detailed implementation manners:

[0025] To better understand the essence of the present invention, the following combines the accompanying drawings, and uses the test examples and embodiments of the present invention to further illustrate the sesquiterpene dimer compounds of the present invention, artemsieverlides A–M (1-13), their preparation methods, structure identification, and pharmacological effects, but the present invention is not limited by these test examples and embodiments.

[0026] The following will clearly and completely describe the technical solutions in the embodiments of the present invention in conjunction with the embodiments of the present invention. Obviously, the described embodiments are only a part of the embodiments of the present invention, rather than all the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those of ordinary skill in the art without making creative efforts fall within the protection scope of the present invention.

[0027] Example 1:

[0028] Preparation of the sesquiterpene dimer compounds of the present invention, artemsieverlides A–M (1-13):

[0029] The dried aerial parts (33 kg) of Artemisia sieversiana were crushed and extracted twice by cold maceration with 95% ethanol for 4 days each time. The ethanol extracts were combined, and the ethanol was recovered under reduced pressure to obtain an ethanol extract. The extract was dispersed in water and then extracted with ethyl acetate. Subsequently, the ethyl acetate extraction part was concentrated, and then the ethyl acetate extraction part was subjected to silica gel column chromatography. Using acetone - petroleum ether with a volume ratio (v / v) of 5:95, 10:90, 20:80, 30:70, and 100:0 as eluents, five fractions, Fr.A - Fr.E (149 g, 101 g, 98 g, 153 g, and 164 g), were obtained by gradient elution; fraction Fr.D was subjected to silica gel column chromatography and eluted with methanol - chloroform with a volume ratio (v / v) of 5:95, 10:90, and 20:80 to obtain five fractions, Fr.D1 - Fr.D5 (25 g, 10 g, 60 g, 30 g, and 25 g); fraction Fr.D4 (30 g) was subjected to MCI gel CHP 20P column chromatography, gradient eluted with water - methanol at 10:90 and 0:100, and semi-preparative HPLC was used to prepare Compounds 1 (10 mg), 2 (2 mg), 3 (5 mg), 4 (10 mg), 5 (5 mg), 6 (5 mg), 7 (10 mg), 8 (5 mg), 9 (8 mg), 10 (12 mg), 11 (3 mg), 12 (15 mg), and 13 (6 mg).

[0030] Structure data of Compounds 1 - 13:

[0031] The melting point was determined by an X-4B micro melting point apparatus (Shanghai Precision Scientific Instruments Co., Ltd.); the specific rotation was determined by an Autopol VI polarimeter (Rudolph Research Analytical, Hackettstown, USA); the infrared spectrum was determined by the ATR attenuated total reflection-diamond crystal (ATR ITX-DIAMOND) method using a NICOLET iS10 infrared spectrometer (Thermo Fisher Scientific, Madison, USA); the ultraviolet spectrum was determined by a UV-2401PC ultraviolet spectrometer (Shimadzu, Kyoto, Japan); the ECD spectrum was determined by an Applied Photophysics circular dichroism spectrometer (Applied Photophysics, Surrey, UK); the nuclear magnetic resonance spectrum was determined using an Avance III 600 (Bruker, Bremerhaven, Germany) superconducting nuclear magnetic resonance spectrometer with TMS (tetramethylsilane) as the internal standard; the high-resolution mass spectrum was determined using a Shimadzu LC-MS-IT-TOF (Shimadzu, Kyoto, Japan), an Agilent UPLC / Q-TOF and a G6230 mass spectrometer (Agilent Technologies, Santa Clara, USA); the thin-layer chromatography silica gel plate HSGF254 was purchased from Yantai Jiangyou Silica Gel Development Co., Ltd.; the column chromatography silica gel (200 - 300 mesh) was produced by Linyi Haixiang Chemical Industry Co., Ltd.; the column chromatography Sephadex LH-20 was purchased from GE Healthcare Bio-Sciences AB; the high-performance liquid chromatograph was purchased from Shimadzu Corporation, the controller model was CBM-20A, the pump model was LC-20AR, the detector model was SPD-M20A, the column oven model was AT-350, and the chromatographic column model was Agilent-Eclipse XDB-C18 (5μm, 9.4×250mm); the chromatographically pure acetonitrile was purchased from Merck Millipore; the deionized water was purified by a MingCheTM-D 24UV Merk Millipore system; the medium-pressure liquid chromatography (Dr Flash-II) was a product of Shanghai Lishui Co., Ltd., the MCI column was from Mitsubishi Corporation of Japan, model CHP-20P (75 - 150μm); the analytically pure methanol and acetonitrile were purchased from Tianjin Damao Chemical Reagent Factory; the color-developing agent was 10% H 2 SO 4 -EtOH solution.

[0032]

[0033] Artemsieverlide A (1)

[0034] Molecular formula: C 30 H 38 O 7

[0035] Molecular weight: 510

[0036] Appearance: white powder

[0037] Optical rotation: (c 0.059, methanol)

[0038] HRESIMS(–) m / z: experimental value 555.2598 [M+HCOO] – , calculated value 555.2600 [M+HCOO] – .

[0039] IR ν max : 3435, 1763, 1680, 1631, 1456, 1381, 1154 cm –1 .

[0040] 1 1H NMR and 13 13C NMR (DEPT) data are shown in Tables 1 and 3.

[0041]

[0042] Artemsieverlide B(2)

[0043] Molecular formula: C 30 H 38 O 8

[0044] Molecular weight: 526

[0045] Appearance: white powder

[0046] Optical rotation: (c 0.048, methanol)

[0047] HRESIMS(+) m / z: experimental value 527.2653 [M+H] + , calculated value 527.2639 [M+H] + .

[0048] IR ν max : 3441, 1761, 1702, 1644, 1381, 1318, 1206, 1153 cm -1 .

[0049] ECD (methanol) λ max(Δε): 219 (+20.83), 316 (-2.73) nm.

[0050] 1 H NMR and 13 C NMR (DEPT) data are shown in Tables 1 and 3.

[0051]

[0052] Artemsieverlide C(3)

[0053] Molecular formula: C 30 H 40 O 6

[0054] Molecular weight: 496

[0055] Appearance: needle-like crystals

[0056] Optical rotation: (c 0.059, methanol)

[0057] HRESIMS(+) m / z: experimental value 497.2918 [M+H] + , calculated value 497.2898 [M+H] + .

[0058] IR v max : 3435, 1769, 1634, 1457, 1379, 1317, 1059, 1101 cm -1 .

[0059] ECD (methanol) λ max (Δε): 215 (-10.09) nm.

[0060] 1 H NMR and 13 C NMR (DEPT) data are shown in Tables 1 and 3.

[0061]

[0062] Artemsieverlide D(4)

[0063] Molecular formula: C 30 H 40 O 6

[0064] Molecular weight: 496

[0065] Appearance: white powder

[0066] Optical rotation: (c 0.049, methanol)

[0067] HRESIMS(+) m / z: Experimental value 497.2899 [M+H] + , Calculated value 497.2898 [M+H] + 。

[0068] IR v max : 3420, 1775, 1664, 1631, 1454, 1380, 1231, 1149 cm -1 。

[0069] ECD (methanol) λ max (Δε): 215 (-16.27) nm。

[0070] 1 H NMR and 13 C NMR (DEPT) data are shown in Tables 1 and 3.

[0071]

[0072] Artemsieverlide E (5)

[0073] Molecular formula: C 30 H 40 O 6

[0074] Molecular weight: 496

[0075] Appearance: White powder

[0076] Optical rotation: (c 0.051, methanol)

[0077] HRESIMS(+) m / z: Experimental value 497.2891 [M+H] + , Calculated value 497.2898 [M+H] + 。

[0078] IR v max : 3433, 1769, 1630, 1454, 1380, 1229 cm -1 。

[0079] 1 H NMR and 13 C NMR (DEPT) data are shown in Tables 1 and 3.

[0080]

[0081] Artemsieverlide F (6)

[0082] Molecular formula: C 30 H 40 O 6

[0083] Molecular weight: 496

[0084] Appearance: white powder

[0085] Optical rotation: (c 0.100, methanol)

[0086] HRESIMS(+) m / z: found 497.2910 [M+H] + , calculated 497.2898 [M+H] + .

[0087] IR ν max : 3437, 1763, 1633, 1455, 1379, 1238, 1147 cm –1 .

[0088] 1 1H NMR and 13 13C NMR (DEPT) data are shown in Tables 1 and 3.

[0089]

[0090] Artemsieverlide G(7)

[0091] Molecular formula: C 30 H 36 O 7

[0092] Molecular weight: 508

[0093] Appearance: needle-shaped single crystal

[0094] Optical rotation: (c 0.091, methanol)

[0095] HRESIMS(+) m / z: found 509.2543 [M+H] + , calculated 509.2534 [M+H] + .

[0096] IR ν max : 3438, 1763, 1680, 1624, 1450, 1380, 1150 cm -1 .

[0097] 1 1H NMR and 13 13C NMR (DEPT) data are shown in Tables 2 and 4.

[0098]

[0099] Artemsieverlide H(8)

[0100] Molecular formula: C 30 H 36 O 7

[0101] Molecular weight: 508

[0102] Appearance: white powder

[0103] Optical rotation: (c 0.024, methanol)

[0104] HRESIMS(+) m / z: experimental value 509.2549 [M+H] + , calculated value 509.2534 [M+H] + .

[0105] IR v max : 3439, 1761, 1678, 1635, 1625, 1376, 1178, 1153 cm -1 .

[0106] 1 H NMR and 13 C NMR (DEPT) data are shown in Tables 2 and 4.

[0107]

[0108] Artemsieverlide I(9)

[0109] Molecular formula: C 31 H 40 O 7

[0110] Molecular weight: 524

[0111] Appearance: white powder

[0112] Optical rotation: (c 0.044, methanol)

[0113] HRESIMS(+) m / z: experimental value 547.2661 [M+Na] + , calculated value 547.2666 [M+Na] + .

[0114] IR v max: 3434, 1780, 1755, 1636, 1453, 1246, 1145 cm -1 。

[0115] 1 H NMR and 13 C NMR (DEPT) data are shown in Tables 2 and 4.

[0116]

[0117] Artemsieverlide J (10)

[0118] Molecular formula: C 30 H 38 O 7

[0119] Molecular weight: 510

[0120] Appearance: White powder

[0121] Optical rotation: (c 0.071, methanol)

[0122] HRESIMS (–) m / z: Experimental value 555.2599 [M+HCOO] – , Calculated value 555.2600 [M+HCOO] – 。

[0123] IR v max : 3441, 1760, 1634, 1455, 1380, 1240, 1147, 1091 cm -1 。

[0124] 1 H NMR and 13 C NMR (DEPT) data are shown in Tables 2 and 4.

[0125]

[0126] Artemsieverlide K (11)

[0127] Molecular formula: C 30 H 36 O 7

[0128] Molecular weight: 508

[0129] Appearance: White powder

[0130] Optical rotation: (c 0.056, methanol)

[0131] HRESIMS(+) m / z: Experimental value 509.2549 [M+H] + , Calculated value 509.2534 [M+H] + .

[0132] IR ν max : 3435, 1763, 1636, 1453, 1379, 1176, 1150 cm -1 .

[0133] 1 H NMR and 13 C NMR (DEPT) data are shown in Tables 2 and 4.

[0134]

[0135] Artemsieverlide L (12)

[0136] Molecular formula: C 30 H 38 O 7

[0137] Molecular weight: 510

[0138] Appearance: Needle crystals

[0139] Optical rotation: (c 0.072, methanol)

[0140] HRESIMS(–) m / z: Experimental value 555.2583 [M+HCOO] – , Calculated value 555.2600 [M+HCOO] – .

[0141] IR ν max : 3435, 1759, 1706, 1636, 1447, 1382, 1215, 1148 cm -1 .

[0142] 1 H NMR and 13 C NMR (DEPT) data are shown in Tables 2 and 4.

[0143]

[0144] Artemsieverlide M (13)

[0145] Molecular formula: C 30 H 38 O 7

[0146] Molecular weight: 510

[0147] Appearance: white powder

[0148] Optical rotation: (c 0.071, methanol)

[0149] HRESIMS(–) m / z: found 555.2615 [M+HCOO] – , calculated 555.2600 [M+HCOO] – .

[0150] IR v max : 3436, 1758, 1631, 1448, 1380, 1210, 1151 cm -1 .

[0151] 1 1H NMR and 13 13C NMR (DEPT) data are shown in Tables 2 and 4.

[0152]

[0153]

[0154]

[0155]

[0156] Example 2:

[0157] Cytotoxic activities of sesquiterpene dimer compounds, artemsieverlides A–M (1-13) against HSC-LX2.

[0158] 1. Materials and methods

[0159] 1.1 Materials

[0160] Human hepatic stellate cell line (hepatic stellate cells line LX2, HSC-LX2) was purchased from Shanghai Jining Biotechnology Co., Ltd.; RPMI-1640 medium and fetal bovine serum were both purchased from Gibco BRL (NY, USA); MTT was purchased from Guangzhou Saiguo Biotechnology Co., Ltd.

[0161] 1.2 Instruments

[0162] Flex Station 3 benchtop multi-functional microplate reader (Bio-RAD 680, USA); analytical balance (AG135, Metler Toledo, China); incubator (DHP-9082, Shanghai).

[0163] 1.3 Experimental Procedure

[0164] The cytotoxic activity of the samples against HSC-LX2 cells was determined by the MTT method. HSC-LX2 cells were cultured in RPMI-1640 medium containing 10% fetal bovine serum. Cells in the logarithmic growth phase were seeded into 96-well plates at a density of 1×10 4 / well. After 24 h, the maintenance medium was replaced with the medium containing different concentrations of the test samples. At the same time, a cell control group with only the maintenance medium added was set up, and silybin was used as a positive drug control. After culturing for 48 h, the culture medium was discarded and 100 μL of MTT solution (concentration: 1 mg / mL) was added; it was incubated in an incubator at 37 °C for 4 h, the MTT solution was discarded and 100 μL of DMSO was added to dissolve the formazan crystals; finally, the absorbance value of each well was measured with an enzyme-linked immunosorbent assay (ELISA) reader at 490 nm. The calculation formula for the inhibition rate of HSC-LX2 cells was inhibition rate (%) = [A(blank) – A(sample)] / A(blank) × 100%. The half-maximal inhibitory concentration (50% inhibitory concentration, IC 50 ) was calculated using Graphpad Prism 5 software.

[0165] 2. Results

[0166] The in vitro cytotoxic activity of all the isolated compounds against HSC-LX2 cells was evaluated (Table 5). Compounds 1-13 all showed strong cytotoxic activity against hepatic stellate cells (HSC-LX2). Compounds 8 and 11 showed strong cytotoxic activity, with IC 50 values of 71.6 and 58.6 μM respectively, and their activity was 2 times that of the positive drug silybin; Compounds 3, 4, 5, 6 and 9 also showed certain cytotoxic activity, with IC 50 values of 94.2, 115.2, 126.7, 99.3 and 112.4 μM respectively, and their activity was better than that of the positive drug silybin. The remaining compounds had certain cytotoxic activity against HSC-LX2 cells, and their inhibition rates were between 30.0 - 62.9% at a concentration of 200 μg / mL.

[0167] Table 5. Cytotoxic Activity of Compounds against HSC-LX2 Cells

[0168]

[0169] The inhibition rate and IC 50 were obtained from three independent experiments.

[0170] 3. Conclusions

[0171] The experimental results showed that compounds 1-13 all had strong cytotoxic activities against hepatic stellate cells (HSC-LX2). Compounds 8 and 11 had strong cytotoxic activities, with their IC 50 values being 71.6 and 58.6 μM respectively, and their activities were twice that of the positive drug silybin; Compounds 3, 4, 5, 6 and 9 also showed certain cytotoxic activities, with their IC 50 values being 94.2, 115.2, 126.7, 99.3 and 112.4 μM respectively, and their activities were superior to that of the positive drug silybin. The remaining compounds had certain cytotoxic activities against HSC-LX2, and their inhibition rates were between 30.0 - 62.9% at a concentration of 200 μg / mL.

[0172] The above results indicated that the sesquiterpene dimer compounds, artemsieverlides A–M (1-13) in Artemisia sieversiana Ehrhart ex Willd. could be used as drugs for preparing drugs against liver fibrosis diseases.

[0173] Formulation Examples 1-7:

[0174] In the following formulation examples, conventional reagents were selected and formulation preparations were carried out according to existing conventional methods. These formulation examples only demonstrated that at least one of the compounds 1-13 described in the present invention could be prepared into different formulations, and specific reagents and operations were not specifically defined:

[0175] 1. At least one of compounds 1-13 was dissolved in DMSO, and then water for injection was added according to conventional methods, followed by fine filtration, filling and sterilization to prepare an injection, and the concentration of the injection was 0.5 - 5 mg / mL.

[0176] 2. At least one of compounds 1-13 was dissolved in DMSO, then it was dissolved in sterile water for injection, stirred to dissolve it, filtered through a sterile suction filter funnel, then sterile fine filtered, filled into ampoules, freeze-dried at low temperature and then hermetically sealed aseptically to obtain a powder injection.

[0177] 3. At least one of compounds 1-13 was added with an excipient in a mass ratio of 9:1 to prepare a powder.

[0178] 4. At least one of compounds 1-13 was added with an excipient in a mass ratio of 5:1 to the excipient, granulated and tabletted.

[0179] 5. At least one of compounds 1-13 was prepared into an oral liquid according to the conventional preparation method of oral liquid.

[0180] 6. At least one of compounds 1-13 was added with an excipient in a mass ratio of 5:1 to the excipient to prepare a capsule.

[0181] 7. At least one of Compounds 1-13 is added to an excipient at a mass ratio of 5:1 to the excipient to prepare a granule.

[0182] As can be seen from the above examples, the present invention provides a compound in Artemisia sieversiana, its preparation method and application, a pharmaceutical composition and its application. The 13 new sesquiterpene compounds provided by the present invention have varying degrees of cytotoxic activity against human hepatic stellate cell HSC-LX2, can form a pharmaceutical composition with a pharmaceutically acceptable carrier or excipient, and can be used to prepare an anti-hepatic fibrosis drug.

[0183] The above are only the preferred embodiments of the present invention. It should be noted that for those of ordinary skill in the art, without departing from the principle of the present invention, several improvements and modifications can be made, and these improvements and modifications should also be regarded as the protection scope of the present invention.

Claims

1. Sesquiterpene dimer compound 1-13 shown in the following structural formula, 2. The method for preparing the sesquiterpene dimer compound 1-13 represented by the structural formula according to claim 1, characterized in that: The method comprises the following steps: the dried aerial part of Artemisia grandis is crushed, and cold-extracted twice with 95% ethanol, each time for 4 days, the ethanol extract is combined, the ethanol extract is recovered under reduced pressure, the extract is dispersed in water and extracted with ethyl acetate, and then concentrated to obtain the ethyl acetate extraction part, and then the ethyl acetate extraction part is subjected to silica gel column chromatography, acetone-petroleum ether is calculated by v / v, 5:95, 10:90, 20:80, 30:70 and 100:0 as eluent, and gradient elution is performed to obtain five fractions Fr.A-Fr.E; the fraction Fr.D is subjected to silica gel column chromatography, methanol-chloroform is calculated by v / v, 5:95, 10:90 and 20:80 as eluent, and gradient elution is performed to obtain five fractions Fr.D1-Fr.D5; the fraction Fr.D4 is subjected to MCI gel CHP Compounds 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, and 13 were prepared by 20P column chromatography, water-methanol (v / v) 10:90 and 0:100 as eluent, gradient elution and semi-preparative HPLC.

3. Use of the sesquiterpene dimer compound 1-13 represented by the structural formula according to claim 1 in the preparation of anti-liver fibrosis drugs.

4. A pharmaceutical composition, characterized in that The pharmaceutical composition comprises at least one of the compounds 1-13 represented by the structural formula of claim 1 and a pharmaceutically acceptable carrier.

5. Use of the pharmaceutical composition according to claim 4 in the preparation of anti-liver fibrosis drugs.

6. The method for preparing the pharmaceutical composition according to claim 4, characterized in that: The method comprises the following steps: the dried aerial part of Artemisia grandis is crushed, and cold-extracted twice with 95% ethanol, each time for 4 days, the ethanol extract is combined, the ethanol extract is recovered under reduced pressure, the extract is dispersed in water and extracted with ethyl acetate, and then concentrated to obtain the ethyl acetate extraction part, and then the ethyl acetate extraction part is subjected to silica gel column chromatography, acetone-petroleum ether is calculated by v / v, 5:95, 10:90, 20:80, 30:70 and 100:0 as eluent, and gradient elution is performed to obtain five fractions Fr.A-Fr.E; the fraction Fr.D is subjected to silica gel column chromatography, methanol-chloroform is calculated by v / v, 5:95, 10:90 and 20:80 as eluent, and gradient elution is performed to obtain five fractions Fr.D1-Fr.D5; the fraction Fr.D4 is subjected to MCIgel CHP Compounds 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, and 13 were prepared by 20P column chromatography, water-methanol (v / v) at 10:90 and 0:100 as eluent, gradient elution, and semi-preparative HPLC; then, one of compounds 1-13 or any combination thereof was added with a pharmaceutically acceptable carrier.

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