Sesquiterpene dimers in artemisia annua, pharmaceutical compositions thereof, and methods of making and using the same

Abstract

The application provides sesquiterpene dimmer compounds 1-13 in artemisia, a pharmaceutical composition of the sesquiterpene dimmer compounds 1-13, a preparation method of the sesquiterpene dimmer compounds 1-13 and application thereof, and belongs to the technical field of medicines. The 13 new sesquiterpene dimers shown in the structural formula of the application, artemisieverlides A-M (1-13), have obvious inhibitory activity on human liver stellate cells (HSC-LX2), can be combined with a pharmaceutically acceptable carrier to form a pharmaceutical composition, and can be used for preparing an anti-liver fibrosis drug.

Description

Technical fields:

[0001] This invention belongs to the field of pharmaceutical technology. Specifically, it relates to the sesquiterpene dimer compound artemsieverlides A–M(1-13), its preparation method, pharmaceutical composition, and applications. Background technology:

[0002] Epidemiological surveys indicate that approximately 2 million people worldwide die from liver disease annually, accounting for about 3.5% of all deaths globally, making it one of the leading causes of death worldwide. 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 liver injury causes massive hepatocyte death, extracellular matrix proteins and fibrous connective tissue excessively deposit in the liver, leading to fibrosis. Subsequently, the excessive fibrosis further alters the liver structure, forming scars and regenerative nodules, resulting in abnormal liver function and increased hepatic blood flow resistance. The mechanisms of liver fibrosis involve multiple complex factors, primarily including hepatocyte injury, inflammatory response, hepatic stellate cell (HSC) activation, and extracellular matrix deposition. Liver injury is the foundation of liver fibrosis. When the liver is stimulated by alcohol, drugs, viruses, or metabolic abnormalities, hepatocytes are damaged, mainly manifested as decreased cell membrane fluidity and increased permeability, as well as mitochondrial damage. Subsequently, liver injury causes the release of inflammatory factors such as interleukin (IL)-1β, inducing HSC activation. Activated HSCs transform into myofibroblasts, migrate to the site of injury, accelerate proliferation, and secrete extracellular matrix, leading to fibrosis. Currently, there are few clinically effective drugs for treating liver fibrosis, and they are limited to drugs that control the cause and traditional Chinese medicine. This invention aims to identify new anti-liver fibrosis active ingredients from natural products based on a human hepatic stellate cell line LX2 (HSC-LX2) screening model.

[0003] *Artemisia sieversiana* is a perennial herb belonging to the genus *Artemisia* in the family Asteraceae. It grows on mountain slopes, grasslands, sparse forests, and forest edges below altitudes of 3500 meters. It is mainly distributed in Heilongjiang, Liaoning, Inner Mongolia, Gansu, Sichuan, Yunnan, and Tibet. According to the *Dictionary of Traditional Chinese Medicine*, *Artemisia sieversiana* is sweet and bitter in nature and is used to treat colds, dysentery, and jaundice. Mongolian and Tibetan medicine records its effects of reducing swelling, stopping bleeding, and clearing heat and relieving summer heat. A decoction of it can treat diarrhea, colds, and jaundice, and can also be used to treat skin burns caused by ultraviolet radiation. Modern pharmacological studies have shown that *Artemisia sieversiana* has significant expectorant, antiasthmatic, anti-inflammatory, and antibacterial effects. Literature review has found that the main components of *Artemisia sieversiana* are sesquiterpenes (guaiacane, gemmaconane, murinene, and sesquiterpene dimers), lignans, flavonoids, and volatile oils. Pharmacological studies have shown that compounds isolated from Artemisia annua have antitumor, anti-inflammatory, antibacterial and antioxidant activities.

[0004] To date, there are no reports of the sesquiterpene dimer artemsieverlides A–M(1-13) in the prior art, nor are there any reports of compound 1-13 and its pharmaceutical compositions as anti-hepatic fibrosis drugs. Summary of the Invention:

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

[0006] To achieve the above-mentioned objectives of the present invention, the present invention provides the following technical solution:

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

[0008]

[0009] This invention provides a method for preparing the above-mentioned sesquiterpene dimer compounds 1-13. The dried aerial parts of Artemisia annua are pulverized and extracted twice with 95% ethanol for 4 days each time. The ethanol extracts are combined, and the ethanol extract is recovered under reduced pressure. The extract is dispersed in water and extracted with ethyl acetate, then concentrated to obtain the ethyl acetate extract. The ethyl acetate extract is then subjected to silica gel column chromatography using acetone-petroleum ether (v / v) at ratios of 5:95, 10:90, 20:80, 30:70, and 100:0 as eluents to obtain five fractions Fr.A-Fr.E. Fraction Fr.D is subjected to silica gel column chromatography using methanol-chloroform (v / v) at ratios of 5:95, 10:90, and 20:80 as eluents to obtain five fractions Fr.D1-Fr.D5. Fraction Fr.D4 is subjected to MCI chromatography. Compounds 1-13 were obtained by gradient elution and semi-preparative HPLC using gelCHP20P column chromatography with water-methanol (v / v) at ratios of 10:90 and 0:100.

[0010] This invention provides the application of the sesquiterpene dimer compounds 1-13 described in the above-mentioned technical solution in the preparation of anti-liver fibrosis drugs. This invention does not specifically limit the method for this application; any method well-known in the art can be used.

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

[0012] Furthermore, the application of the pharmaceutical composition described in the above-mentioned technical solution in the preparation of an anti-hepatic fibrosis drug is also provided. Additionally, the preparation method of the pharmaceutical composition involves first preparing compounds 1-13 according to the above-mentioned compound preparation method, then taking one of compounds 1-13 or any combination thereof, and adding a pharmaceutically acceptable carrier.

[0013] When at least one of the compounds 1-13 is used to prepare an anti-liver fibrosis drug, the present invention preferably uses the compounds 1-13 directly or in the form of a pharmaceutical composition.

[0014] The pharmaceutical composition provided by this invention comprises at least one of compounds 1-13 described above and a pharmaceutically acceptable carrier or excipient. In this invention, the pharmaceutically acceptable carrier or excipient is preferably a solid, semi-solid, or liquid diluent, filler, or pharmaceutical excipient. This invention does not impose any particular limitation on the pharmaceutically acceptable carrier; any pharmaceutically acceptable carrier well-known in the art that is non-toxic to humans and animals and inert may be selected.

[0015] The present invention does not impose any particular limitation on the preparation method of the pharmaceutical composition. At least one of compounds 1-13 can be directly mixed with a pharmaceutically acceptable carrier. The present invention does not impose any particular limitation on the mixing process. Any process well known in the art that can obtain the pharmaceutical composition can be selected.

[0016] This invention provides the application of the pharmaceutical composition described above in the preparation of anti-liver fibrosis drugs. This invention does not specifically limit the method of application; any method well-known in the art can be used.

[0017] In this invention, when the pharmaceutical composition is used to prepare an anti-liver fibrosis drug, the content of the composition in the drug is preferably 0.1% to 99%; in the pharmaceutical composition, the content of at least one of compounds 1-13 is preferably 0.5% to 90%. The pharmaceutical composition of this invention is preferably used in the form of a dose per unit body weight. In this invention, the prepared drug is preferably administered by both injection (intravenous injection, intramuscular injection) and oral administration.

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

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

[0020] 2. This invention provides a new method for preparing new compounds 1-13, which uses readily available raw materials, is easy to operate, and is suitable for industrial production.

[0021] 3. This invention provides a pharmaceutical composition with novel compounds 1-13 as active ingredients, providing a new drug with good pharmaceutical effects for new anti-hepatic fibrosis drugs.

[0022] 4. Compounds 1-13 of the present invention all exhibit strong cytotoxic activity against hepatic stellate cells (HSC-LX2), while compounds 8 and 11 exhibit strong cytotoxic activity with IC50 values ​​of [missing information]. 50 The values ​​were 71.6 and 58.6 μM, respectively, indicating that the activity was twice that of the positive control drug silymarin; compounds 3, 4, 5, 6, and 9 also showed certain cytotoxic activity, with IC50 values ​​of 71.6 and 58.6 μM, respectively. 50 The concentrations were 94.2, 115.2, 126.7, 99.3 and 112.4 μM, respectively, showing superior activity compared to the positive control drug silymarin. The remaining compounds exhibited certain HSC-LX2 cytotoxic activity, with inhibition rates ranging from 30.0% to 62.9% at a concentration of 200 μg / mL. Attached image description:

[0023] Figure 1 Here are schematic diagrams of the structural formulas of compounds 1-13 of this invention;

[0024] Figure 2 This is a schematic diagram of the X-ray single-crystal diffraction structure of compounds 3, 7 and 12 of the present invention. Detailed implementation method:

[0025] To better understand the essence of the present invention, the following description, in conjunction with the accompanying drawings, uses experimental examples and embodiments of the present invention to further illustrate the sesquiterpene dimer compound, artemsieverlides A–M(1-13), its preparation method, structural identification, and pharmacological effects, but these experimental examples and embodiments are not intended to limit the present invention.

[0026] The technical solutions of this invention will be clearly and completely described below with reference to the embodiments thereof. Obviously, the described embodiments are only a part of the embodiments of this invention, and not all of them. All other embodiments obtained by those skilled in the art based on the embodiments of this invention without creative effort are within the scope of protection of this invention.

[0027] Example 1:

[0028] Preparation of the sesquiterpene dimer compound artemsieverlides A–M(1-13) of this invention:

[0029] The dried aerial parts of Artemisia annua (33 kg) were pulverized and extracted twice with 95% ethanol for 4 days each time. The ethanol extracts were combined, and the ethanol extract was recovered under reduced pressure. The extract was dispersed in water and extracted with ethyl acetate, followed by concentration to obtain the ethyl acetate extract. The ethyl acetate extract was then subjected to silica gel column chromatography, with acetone-petroleum ether ratios of 5:95, 10:90, 20:80, 30:70, and 100:0 (v / v). The eluent was used for gradient elution to obtain five fractions: Fr. A-Fr. E (149 g, 101 g, 98 g, 153 g, and 164 g); fraction Fr. D was eluted by silica gel column chromatography with methanol-chloroform at v / v ratios 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 chromatography. 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) were prepared by gel CHP 20P column chromatography, elution with water-methanol gradients of 10:90 and 0:100, and semi-preparative HPLC.

[0030] Structural data of compounds 1-13:

[0031] Melting point adopted The melting point was determined using an X-4B microscopic melting point apparatus (Shanghai Precision Scientific Instruments Co., Ltd.); specific rotation was determined using an Autopol VI polarimeter (Rudolph Research Analytical, Hackettstown, USA); infrared spectroscopy was performed using the ATR attenuated total reflectance-diamond crystal (ATR ITX-DIAMOND) method on a NICOLET iS10 infrared spectrometer (Thermo Fisher Scientific, Madison, USA); ultraviolet spectroscopy was performed using a UV-2401PC ultraviolet spectrometer (Shimadzu, Kyoto, Japan); ECD spectroscopy was performed using an Applied Photophysics circular dichroism spectroscopy system (Applied Photophysics, Surrey, UK); and nuclear magnetic resonance spectroscopy was performed using an Avance III. 600 (Bruker, Bremerhaven, Germany) superconducting nuclear magnetic resonance spectrometer was used for analysis, with TMS (tetramethylsilane) as an internal standard; high-resolution mass spectrometry was performed using a Shimadzu LC-MS-IT-TOF (Shimadzu, Kyoto, Japan) and an Agilent UPLC / Q-TOF and G6230 mass spectrometer (Agilent Technologies, Santa Clara, USA); thin-layer chromatography silica gel plates HSGF254 were purchased from Yantai Jiangyou Silica Gel Development Co., Ltd.; column chromatography silica gel (200-300 mesh) was produced by Linyi Haixiang Chemical Co., Ltd.; column chromatography dextran gel LH-20 was purchased from GE Healthcare Bio-Sciences AB; high-performance liquid chromatography was purchased from Shimadzu, with a controller model CBM-20A, a pump model LC-20AR, a detector model SPD-M20A, a column oven model AT-350, and a column model Agilent-Eclipse. XDB-C18 (5μm, 9.4×250mm); chromatographic grade acetonitrile was purchased from Merida Corporation; deionized water was purified using a MingChe™-D 24UV Merk Millipore system; medium-pressure liquid chromatography (Dr Flash-Ⅱ) was a product of Shanghai Lishui Corporation; the MCI column was from Mitsubishi Corporation of Japan, model CHP-20P (75~150μm); analytical grade methanol and acetonitrile were purchased from Tianjin Damao Chemical Reagent Factory; the colorimetric reagent was 10% H2SO4-EtOH solution.

[0032]

[0033] Artemsieverlide A(1)

[0034] Molecular formula: C 30 H 38 O7

[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 v max :3435,1763,1680,1631,1456,1381,1154cm –1 .

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

[0041]

[0042] Artemsieverlide B(2)

[0043] Molecular formula: C 30 H 38 O8

[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 v max :3441,1761,1702,1644,1381,1318,1206,1153cm -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 O6

[0054] Molecular weight: 496

[0055] Properties: 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 :3435,1769,1634,1457,1379,1317,1059,1101cm -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 O6

[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 :3420,1775,1664,1631,1454,1380,1231,1149cm -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 O6

[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 :3433,1769,1630,1454,1380,1229cm -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 O6

[0083] Molecular weight: 496

[0084] Appearance: White powder

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

[0086] HRESIMS(+) m / z: Experimental value 497.2910 [M+H] + Calculated value: 497.2898 [M+H] + .

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

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

[0089]

[0090] Artemsieverlide G(7)

[0091] Molecular formula: C 30 H 36 O7

[0092] Molecular weight: 508

[0093] Properties: Needle-like single crystals

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

[0095] HRESIMS(+) m / z: Experimental value 509.2543 [M+H] + Calculated value: 509.2534 [M+H] + .

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

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

[0098]

[0099] Artemsieverlide H(8)

[0100] Molecular formula: C30 H 36 O7

[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 :3439,1761,1678,1635,1625,1376,1178,1153cm -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 O7

[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] + The calculated value is 547.2666 [M+Na]. + .

[0114] IR v max :3434,1780,1755,1636,1453,1246,1145cm -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 O7

[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 :3441,1760,1634,1455,1380,1240,1147,1091cm -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 O7

[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 v max :3435,1763,1636,1453,1379,1176,1150cm -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 O7

[0137] Molecular weight: 510

[0138] Properties: Needle-like 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 v max :3435,1759,1706,1636,1447,1382,1215,1148cm -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 O7

[0146] Molecular weight: 510

[0147] Appearance: White powder

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

[0149] HRESIMS(–)m / z: Experimental value 555.2615 [M+HCOO] – Calculated value: 555.2600[M+HCOO] – .

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

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

[0152]

[0153]

[0154]

[0155]

[0156] Example 2:

[0157] The sesquiterpene dimer, artemsieverlides A–M(1-13), exhibits cytotoxic activity against HSC-LX2.

[0158] 1. Materials and Methods

[0159] 1.1 Materials

[0160] Human hepatic stellate cell line (HSC-LX2) was purchased from Shanghai Jining Biotechnology Co., Ltd.; RPMI-1640 medium and fetal bovine serum were 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 using the MTT assay. HSC-LX2 cells were cultured in RPMI-1640 medium containing 10% fetal bovine serum. Cells in the logarithmic growth phase were harvested and cultured at 1×10⁻⁶ cells / cells. 4The cells were seeded at a density of 1 / 2 well in 96-well plates. After 24 hours, the maintenance medium was replaced with culture medium containing different concentrations of the test samples. A control group with only maintenance medium was also included, and silymarin was used as a positive control. After 48 hours of culture, the culture medium was discarded and 100 μL of MTT solution (1 mg / mL) was added. The plates were then incubated at 37°C for 4 hours. The MTT solution was discarded and 100 μL of DMSO was added to dissolve the formazan crystals. Finally, the absorbance of each well was measured at 490 nm using a microplate reader. The HSC-LX2 cell inhibition rate was calculated as: inhibition rate (%) = [A(blank) – A(sample)] / A(blank) × 100%. The half-maximal inhibitory concentration (IC50) is the concentration of cells in the microplate. 50 The calculations were performed using Graphpad Prism 5 software.

[0165] 2. Results

[0166] The in vitro cytotoxic activity of all isolated compounds against hepatic stellate cells (HSC-LX2) was evaluated (Table 5). Compounds 1-13 all exhibited strong cytotoxic activity against hepatic stellate cells (HSC-LX2), while compounds 8 and 11 showed particularly strong cytotoxic activity with IC50 values ​​of [missing information]. 50 The values ​​were 71.6 and 58.6 μM, respectively, indicating that the activity was twice that of the positive control drug silymarin; compounds 3, 4, 5, 6, and 9 also showed certain cytotoxic activity, with IC50 values ​​of 71.6 and 58.6 μM, respectively. 50 The concentrations were 94.2, 115.2, 126.7, 99.3 and 112.4 μM, respectively, showing superior activity compared to the positive control drug silymarin. The remaining compounds exhibited certain HSC-LX2 cytotoxic activity, with inhibition rates ranging from 30.0% to 62.9% at a concentration of 200 μg / mL.

[0167] Table 5. HSC-LX2 cytotoxic activity of the compounds

[0168]

[0169] Inhibition rate and IC 50 All results were obtained from three independent experiments.

[0170] 3. Conclusion

[0171] Experimental results showed that compounds 1-13 all exhibited strong cytotoxic activity against hepatic stellate cells (HSC-LX2), while compounds 8 and 11 showed strong cytotoxic activity with IC50 values ​​of [missing information]. 50 The values ​​were 71.6 and 58.6 μM, respectively, indicating that the activity was twice that of the positive control drug silymarin; compounds 3, 4, 5, 6, and 9 also showed certain cytotoxic activity, with IC50 values ​​of 71.6 and 58.6 μM, respectively. 50The concentrations were 94.2, 115.2, 126.7, 99.3 and 112.4 μM, respectively, showing superior activity compared to the positive control drug silymarin. The remaining compounds exhibited certain HSC-LX2 cytotoxic activity, with inhibition rates ranging from 30.0% to 62.9% at a concentration of 200 μg / mL.

[0172] The above results indicate that the sesquiterpene dimer compound artemsieverlides A–M(1-13) in Artemisia annua can be used as a drug in the preparation of drugs for treating liver fibrosis.

[0173] Formulation Examples 1-7:

[0174] In the following formulation examples, conventional reagents were selected and the formulations were prepared according to existing conventional methods. These formulation examples only demonstrate that at least one of the compounds 1-13 described in this invention can be prepared into different formulations, and no specific limitations are made on the specific reagents and operations:

[0175] 1. Dissolve at least one of compounds 1-13 in DMSO, add water for injection according to conventional methods, filter, fill and sterilize to prepare an injection solution with a concentration of 0.5-5 mg / mL.

[0176] 2. Dissolve at least one of compounds 1-13 in DMSO, then dissolve it in sterile water for injection, stir until dissolved, filter using a sterile suction funnel, then filter aseptically, dispense into ampoules, freeze-dry at low temperature, and then seal aseptically to obtain a powder for injection.

[0177] 3. Add at least one of compounds 1-13 to the excipient at a mass ratio of 9:1 to prepare a powder.

[0178] 4. Add at least one of compounds 1-13 to the excipient at a mass ratio of 5:1, and granulate and compress the mixture into tablets.

[0179] 5. Prepare an oral liquid from at least one of compounds 1-13 using conventional oral liquid preparation methods.

[0180] 6. Add at least one of compounds 1-13 to the excipient at a mass ratio of 5:1 to make a capsule.

[0181] 7. Add at least one of compounds 1-13 to the excipient at a mass ratio of 5:1 to prepare granules.

[0182] As can be seen from the above embodiments, the present invention provides a compound from Artemisia annua, its preparation method and application, and a pharmaceutical composition and its application. The 13 novel sesquiterpenoid compounds provided by the present invention exhibit varying degrees of cytotoxic activity against human hepatic stellate cells (HSC-LX2), and can be combined with pharmaceutically acceptable carriers or excipients to form pharmaceutical compositions, which can be used to prepare anti-hepatic fibrosis drugs.

[0183] The above description is only a preferred embodiment of the present invention. It should be noted that for those skilled in the art, several improvements and modifications can be made without departing from the principle of the present invention, and these improvements and modifications should also be considered within the scope of protection of the present invention.

Claims

1. Sesquiterpene dimer compounds 1-13, as shown in the following structural formulas, ， 。 2. The method for preparing the sesquiterpene dimer compound 1-13 as shown in claim 1, characterized in that, The method includes the following steps: The dried aerial parts of Artemisia annua are pulverized and extracted twice with 95% ethanol for 4 days each time. The ethanol extracts are combined, and the ethanol extract is recovered under reduced pressure. The extract is dispersed in water and extracted with ethyl acetate, followed by concentration to obtain the ethyl acetate extract. The ethyl acetate extract is then subjected to silica gel column chromatography with acetone-petroleum ether (v / v) at ratios of 5:95, 10:90, 20:80, 30:70, and 100:0 as eluents, yielding five fractions Fr. A–Fr. E. Fraction Fr. D is subjected to silica gel column chromatography with methanol-chloroform (v / v) at ratios of 5:95, 10:90, and 20:80 as eluents, yielding five fractions Fr. D1–Fr. D5. Fraction Fr. D4 is subjected to MCI gel CHP chromatography. Compounds 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, and 13 were obtained by gradient elution and semi-preparative HPLC with water-methanol (v / v) at ratios of 10:90 and 0:

100.

3. The use of the sesquiterpene dimer compound 1−13 of the structural formula shown in claim 1 in the preparation of an anti-hepatic fibrosis drug.

4. A pharmaceutical composition, characterized in that, The pharmaceutical composition comprises at least one of compounds 1-13 as shown in claim 1 and a pharmaceutically acceptable carrier.

5. The use of the pharmaceutical composition of claim 4 in the preparation of an anti-hepatic fibrosis drug.

6. The method for preparing the pharmaceutical composition according to claim 4, characterized in that, The method includes the following steps: The dried aerial parts of Artemisia annua are pulverized and extracted twice with 95% ethanol for 4 days each time. The ethanol extracts are combined, and the ethanol extract is recovered under reduced pressure. The extract is dispersed in water and extracted with ethyl acetate, followed by concentration to obtain the ethyl acetate extract. The ethyl acetate extract is then subjected to silica gel column chromatography with acetone-petroleum ether (v / v) at ratios of 5:95, 10:90, 20:80, 30:70, and 100:0 as eluents, yielding five fractions Fr. A–Fr. E. Fraction Fr. D is subjected to silica gel column chromatography with methanol-chloroform (v / v) at ratios of 5:95, 10:90, and 20:80 as eluents, yielding five fractions Fr. D1–Fr. D5. Fraction Fr. D4 is subjected to MCI gel CHP chromatography. Compounds 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, and 13 were prepared by gradient elution and semi-preparative HPLC with water-methanol (v / v) at ratios of 10:90 and 0:

100. Then, one of compounds 1–13 or any combination thereof was taken and a pharmaceutically acceptable carrier was added.

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