CN116621800A - Artemisinin A-C and its pharmaceutical composition, preparation method and application - Google Patents

Artemisinin A-C and its pharmaceutical composition, preparation method and application Download PDF

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CN116621800A
CN116621800A CN202310575536.5A CN202310575536A CN116621800A CN 116621800 A CN116621800 A CN 116621800A CN 202310575536 A CN202310575536 A CN 202310575536A CN 116621800 A CN116621800 A CN 116621800A
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column chromatography
meoh
frs
fractions
water
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CN116621800B (en
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陈纪军
何小凤
李天泽
耿长安
马云保
张雪梅
黄晓燕
胡敬
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Kunming Institute of Botany of CAS
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    • C07—ORGANIC CHEMISTRY
    • C07D—HETEROCYCLIC COMPOUNDS
    • C07D311/00—Heterocyclic compounds containing six-membered rings having one oxygen atom as the only hetero atom, condensed with other rings
    • C07D311/02—Heterocyclic compounds containing six-membered rings having one oxygen atom as the only hetero atom, condensed with other rings ortho- or peri-condensed with carbocyclic rings or ring systems
    • C07D311/04—Benzo[b]pyrans, not hydrogenated in the carbocyclic ring
    • C07D311/22—Benzo[b]pyrans, not hydrogenated in the carbocyclic ring with oxygen or sulfur atoms directly attached in position 4
    • C07D311/26—Benzo[b]pyrans, not hydrogenated in the carbocyclic ring with oxygen or sulfur atoms directly attached in position 4 with aromatic rings attached in position 2 or 3
    • C07D311/28—Benzo[b]pyrans, not hydrogenated in the carbocyclic ring with oxygen or sulfur atoms directly attached in position 4 with aromatic rings attached in position 2 or 3 with aromatic rings attached in position 2 only
    • C07D311/30—Benzo[b]pyrans, not hydrogenated in the carbocyclic ring with oxygen or sulfur atoms directly attached in position 4 with aromatic rings attached in position 2 or 3 with aromatic rings attached in position 2 only not hydrogenated in the hetero ring, e.g. flavones
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    • A61P1/16—Drugs for disorders of the alimentary tract or the digestive system for liver or gallbladder disorders, e.g. hepatoprotective agents, cholagogues, litholytics
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P35/00—Antineoplastic agents
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07D—HETEROCYCLIC COMPOUNDS
    • C07D311/00—Heterocyclic compounds containing six-membered rings having one oxygen atom as the only hetero atom, condensed with other rings
    • C07D311/02—Heterocyclic compounds containing six-membered rings having one oxygen atom as the only hetero atom, condensed with other rings ortho- or peri-condensed with carbocyclic rings or ring systems
    • C07D311/04—Benzo[b]pyrans, not hydrogenated in the carbocyclic ring
    • C07D311/22—Benzo[b]pyrans, not hydrogenated in the carbocyclic ring with oxygen or sulfur atoms directly attached in position 4
    • C07D311/26—Benzo[b]pyrans, not hydrogenated in the carbocyclic ring with oxygen or sulfur atoms directly attached in position 4 with aromatic rings attached in position 2 or 3
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Abstract

The invention provides artemisia annua alcohol A-C, a pharmaceutical composition thereof, a preparation method and application thereof, and belongs to the technical field of medicines. The 3 sesquiterpene-flavonol hybrids shown in the structural formula have an inhibitory effect on human liver cancer cell lines HepG2, huh7 and SK-Hep-1 by using the Artemisininols A-C (arteannuols A ‒ C, 1-3), can form a pharmaceutical composition with a pharmaceutically acceptable carrier, and can be used for preparing anti-liver cancer drugs.

Description

Artemisinin A-C and its pharmaceutical composition, preparation method and application
Technical field:
the invention belongs to the technical field of medicines. In particular to 3 sesquiterpene-flavonol hybrids with novel structures, artemisininols A-C (1-3), a preparation method and application thereof, and application of a pharmaceutical composition taking the compounds 1-3 as active ingredients in preparing anti-liver cancer drugs.
The background technology is as follows:
primary liver cancer has become the third leading cause of cancer death worldwide. Hepatitis B Virus (HBV) and Hepatitis C Virus (HCV) are the main causative agents, and at least 75% of all HCC cases are caused by them. To date, a total of five synthetic drugs (sorafenib, regorafenib, lenvatinib, cabozantinib, and donafinib) and three monoclonal antibody drugs (nivorunib, peng Boli bead mab, and la Mo Jilu mab) are clinically used to treat HCC. However, the relatively single structural type and target site of action, adverse reactions greatly limit their therapeutic efficacy. Natural products have attracted considerable attention because of their diverse structures and their diverse biological activities. Icaritin is an isopentenyl-substituted flavonoid compound obtained by separating icariin from Epimedium plant through enzymolysis, is a multi-target immunoregulatory small molecule, and is approved as a first innovative traditional Chinese medicine in China for treating HCC.
Artemisia annua (Artemisia annua) is also called sweet wormwood, artemisia rupestris and Artemisia annua, is annual herb of Artemisia of Compositae, and is widely used as medicine in dry overground parts, and is widely distributed in North America, europe, africa and Asia, wherein 70% of the Artemisia annua is concentrated in China. The artemisia annua is a traditional antimalarial traditional Chinese medicine, has the effects of clearing deficiency heat, cooling blood, removing steam, relieving summer heat and checking malaria, has the characteristics of cold nature, bitter and pungent taste, and can be used for treating symptoms such as warm evil hurting yin, night heat, early cooling, yin deficiency fever, bone steaming and fatigue fever, summer heat evil fever, malaria chill and fever, damp-heat jaundice, and the like, and is externally used for treating mosquito bite, sore swelling, scald and the like. Sweet wormwood is initially carried in the war state, "fifty two diseases prescription: "Artemisia apiacea person, jing's name is Securinega suffruticosa 5432, mainly treats hemorrhoids", jin Daige He "elbow backup Ji Fang" is carried out on the formula for treating malaria: herba Artemisiae Annuae is held firstly, soaked in water and then is minced to obtain juice for taking, and later, all the traditional Chinese herbs are recorded, for example, herba Artemisiae Annuae is called herba Artemisiae Annuae for treating malaria and chill in Ming dynasty, and herba Artemisiae Annuae and carapax Trionycis are decocted for treating malaria in Qing dynasty. Along with the deep research of sweet wormwood, the sweet wormwood herb can be used for treating diseases such as lupus erythematosus, bronchial asthma, chronic bronchial asthma, autumn diarrhea, skin itch, urticaria, caterpillar allergy and the like, and has the effects of resisting viruses, fungi, inflammation, cancer and the like. The literature researches show that the structural types of non-volatile components in the artemisia annua are mainly sesquiterpenes, diterpenes, flavones, coumarin, flavones, phenylpropionic acid and the like except volatile components, and some compounds have pharmacological activities of malaria resistance, tumor resistance, bacteriostasis, insect disinfestation, fever resistance, inflammation resistance, immunoregulation, fibrosis resistance, steatosis inhibition and the like.
Until now, no report exists in the prior art on arteannuols A-C (arteannuols A-C, 1-3) and pharmacological activity of the arteannuols A-C, nor on pharmaceutical compositions of the arteannuols A-C as active ingredients, nor on application of the pharmaceutical compositions of the arteannuols A-C in preparation of drugs for treating liver cancer.
The invention comprises the following steps:
the invention aims to provide a novel Artemisininol A-C (arteannuols A-C, 1-3) with a medicinal value shown in a structural formula, a preparation method thereof, a pharmaceutical composition taking Artemisininol A-C as an active ingredient and application thereof. According to the invention, 3 sesquiterpene-flavonol hybrids with novel structures are separated from artemisia annua, and artemisia annua alcohol A-C (arteannuols A-C, 1-3) are obtained. The compound has an inhibiting effect on human liver cancer cell strains HepG2, huh7 and SK-Hep-1, and can be used for preparing anti-liver cancer drugs.
In order to achieve the above object of the present invention, the present invention provides the following technical solutions:
the invention provides a series of sesquiterpene-flavonol hybrids, namely, arteannuols A-C (1-3), and the structures of the arteannuols A-C are shown as the following formulas:
the invention also provides a preparation method of the Artemisinin A-C (compound 1-3), which comprises the following steps:
pulverizing dried aerial parts of herba Artemisiae Annuae, extracting with 3 times of 90% ethanol twice, mixing extractive solutions, concentrating under reduced pressure to obtain crude extract, dispersing in water, extracting with ethyl acetate to obtain ethyl acetate extract, subjecting the ethyl acetate extract to silica gel column chromatography, and gradient eluting with acetone-petroleum ether at volume ratio of 10:90,20:80, 30:70 and ethyl acetate to obtain 4 fractions Frs.A-D; LC-MS analysis shows that fractions A and B contain sesquiterpene dimer compounds, and TLC detection shows that the fractions A and B have more cross components, and therefore, after being combined, the fractions A and B are subjected to silica gel column chromatography, and MeOH-CHCl is used for preparing the fraction A and B 3 Gradient elution with MeOH in a volume ratio of 1:99,2:98,5:95,10:90,20:80,40:60, yielded 5 fractions Frs.B-1-B-5; fr.B-2 was chromatographed on an MCI gel CHP20P column with 50:50, 70:30, 90:10 MeOH-H 2 O and MeOH eluted to give four subfractions Frs.B-2a-B-2d; fr.B-2C via Rp-C 18 Column chromatography with MeOH-H at 50:50, 60:40, 70:30 and 80:20 2 O gradient elution to obtain subfraction Frs.B-2c-1-B-2c-5; fr.B-2c-3 was purified by column chromatography on silica gel with MeOH-CHCl at 2:98,5:95 and 10:90 3 Eluting the eluent to obtain 5 subfractions Frs.B-2c-3a-B-2c-3e; fr.B-2c-3c was subjected to Sephadex LH-20 (column chromatography with 50:50 MeOH-CHCl) 3 Eluting, and then separating with semi-preparative HPLC in Agilent XDB-C 18 Purifying the mixture on a column by using 78:22 methanol-water to obtain compounds 1 and 2; fr.B-4 was subjected to MCI gel CHP20P column chromatography eluting with 50:50, 70:30, 90:10 and 100:0 methanol-water to give four subfractions Frs.B-4a-B-4d; fr.B-4b first uses Rp-C 18 Column chromatography, treating with methanol-water 50:50, 60:40 and 70:30, eluting with methanol-chloroform 50:50 by Sephadex LH-20 column chromatography, and separating with semi-preparative high performance liquid HPLC in Agilent XDB-C 18 Purification on a column with 50:50 acetonitrile-water afforded compound 3.
The invention provides application of compounds 1-3 shown in structural formula in preparation of anti-liver cancer drugs, and the application method is not particularly limited, and can be selected by methods well known in the art.
The invention also provides a pharmaceutical composition which comprises at least one of the compounds 1-3 shown in the structural formula and a pharmaceutically acceptable carrier.
And also provides application of the pharmaceutical composition in preparing anti-liver cancer drugs. And simultaneously provides a preparation method of the pharmaceutical composition: the compounds 1-3 of the present invention are prepared by the above-described method of preparing compounds, and then a pharmaceutically acceptable carrier is added.
When at least one of the compounds 1 to 3 is used for preparing an anti-liver cancer drug, the present invention preferably uses the compounds 1 to 3 directly or in the form of a pharmaceutical composition
The invention provides a pharmaceutical composition comprising at least one of the above compounds 1-3 and a pharmaceutically acceptable carrier. In the present invention, the pharmaceutically acceptable carrier is preferably a solid, semi-solid or liquid diluent, filler and pharmaceutical preparation adjuvant. The pharmaceutically acceptable carrier is not particularly limited, and pharmaceutically acceptable carriers which are well known in the art, nontoxic and inert to human and animals can be selected.
The preparation method of the pharmaceutical composition is not particularly limited, at least one of the compounds 1-3 is directly mixed with a pharmaceutically acceptable carrier, the mixing process is not particularly limited, and the pharmaceutical composition can be obtained by adopting the processes well known in the art.
The invention provides application of the pharmaceutical composition in preparing anti-liver cancer drugs, the application method is not particularly limited, and methods well known in the art can be selected.
In the present invention, when the pharmaceutical composition is used for preparing an anti-liver cancer 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 the compounds 1 to 3 in the pharmaceutical composition is preferably 0.5 to 90%. The pharmaceutical composition of the present invention is preferably used in the form of a unit weight dose. In the present invention, the prepared medicine may be preferably administered in both injection (intravenous injection, intramuscular injection) and oral administration.
Compared with the prior art, the invention has the following advantages:
1. the invention provides 3 sesquiterpene-flavonol hybrids with novel structures, namely, arteannuols A-C (1-3).
2. The invention provides a novel method for preparing novel compounds 1-3, which has the advantages of easily available raw materials, easy operation and suitability for industrial production.
3. The invention provides a pharmaceutical composition with novel compounds 1-3 as active ingredients, and provides a novel drug with better medicinal effect for a novel anti-liver cancer drug.
4. The compound 1-3 of the invention has inhibitory activity on three liver cancer cells HepG2, huh7 and SK-Hep-1, and IC thereof 50 32.7-70.4. Mu.M. Wherein, the compounds 1 and 3 have inhibition effect on HepG2 and Huh7 cells, and IC thereof 50 49.1 and 48.2, 41.1 and 32.7 μm, respectively; compound 2 has moderate inhibitory activity on HepG2 and Huh7 cells, IC thereof 50 59.1 and 59.3. Mu.M. Compounds 1-3 have inhibitory activity in the hand against SK-Hep-1 cells, IC thereof 50 Between 57.7 and 70.4 μm. The results show that the compounds 1-3 separated from the artemisia annua can be used as medicaments for treating liver cancer related diseases.
Description of the drawings:
FIG. 1 is a schematic representation of the structural formula of compounds 1-3 of the present invention.
The specific embodiment is as follows:
in order to better understand the essence of the present invention, the method for preparing artemia A-C (artemia A-C, 1-3) of the present invention, the structure identification, the pharmacological effect, the method for preparing the present invention and the pharmaceutical composition will be further described with reference to the accompanying drawings, but the present invention is not limited to the experimental examples and examples.
The technical solutions of the present invention will be clearly and completely described in the following in connection with the embodiments of the present invention. It will be apparent that the described embodiments are only some, but not all, embodiments of the invention. All other embodiments, which can be made by those skilled in the art based on the embodiments of the invention without making any inventive effort, are intended to be within the scope of the invention.
Example 1:
preparation of sesquiterpene dimer of the present invention, artemisininol A-C (arteannuols A-C, 1-3):
48kg of dried aerial parts of artemisia annua are crushed, extracted twice with 3 times of 90% ethanol, the obtained crude extract is dispersed in water after the decompression concentration of the combined extract, and 3.1kg of ethyl acetate extract is obtained by extraction with ethyl acetate. Subsequently, the ethyl acetate extract fraction was subjected to silica gel column chromatography and eluted with a gradient of acetone-petroleum ether volume ratio of 10:90,20:80, 30:70 and ethyl acetate to give 4 fractions Frs.A-D. LC-MS analysis shows that fractions A and B contain sesquiterpene dimer compounds, and TLC detection shows that the fractions A and B have more cross components, and therefore, after being combined, the fractions A and B are subjected to silica gel column chromatography, and MeOH-CHCl is used for preparing the fraction A and B 3 Gradient elution with MeOH in a volume ratio of 1:99,2:98,5:95,10:90,20:80,40:60 yielded 5 fractions Frs.B-1-B-5. 51.6g Fr.B-2 were purified by column chromatography on MCI gel CHP20P with 50:50, 70:30, 90:10 MeOH-H 2 O and MeOH elution gave four subfractions Frs.B-2a-B-2d.10.3g Fr.B-2C pass Rp-C 18 Column chromatography with MeOH-H at 50:50, 60:40, 70:30 and 80:20 2 O gradient elution to obtain subfraction Frs.B-2c-1-B-2c-5.4.2g Fr.B-2c-3 were purified by column chromatography on silica gel with MeOH-CHCl at 2:98,5:95 and 10:90 3 The eluent was eluted to give 5 subfractions Frs.B-2c-3a-B-2c-3e.910mg Fr.B-2c-3c was subjected to Sephadex LH-20 (column chromatography with 50:50 MeOH-CHCl) 3 Eluting, and then separating with semi-preparative HPLC in Agilent XDB-C 18 Purification on a column with 78:22 methanol-water afforded compounds 1 (18 mg) and 2 (18 mg). 8g of Fr.B-4 were subjected to MCI gel CHP20P column chromatography eluting with 50:50, 70:30, 90:10 and 100:0 methanol-water to give four subfractions Frs.B-4a-B-4d.2.3g Fr.B-4b first with Rp-C 18 Column chromatography, treating with methanol-water 50:50, 60:40 and 70:30, and subjecting to Sephadex LEluting with methanol-chloroform 50:50, and separating with semi-preparative High Performance Liquid Chromatography (HPLC) in Agilent XDB-C 18 Purification on a column with 50:50 acetonitrile-water afforded compound 3 (22 mg).
Structural data for compounds 1-3:
the optical rotation was determined by an Autopol VI polarimeter (Rudolph Research Analytical, hackettstown, USA); infrared spectroscopy (IR) was performed using KBr tabletting and was performed by a Bio-Rad FTS-135 infrared spectrometer (Hercules, california, USA); the UV spectrum was determined by UV-2401PC type UV spectrometer (Shimadzu, kyoto, japan); ECD spectra were measured by a Applied Photophysics round dichroscope (Agilent, santa Clara, united States); nuclear magnetic resonance spectroscopy (1D and 2D NMR) was performed using an Avance III-600 superconducting nuclear magnetic resonance apparatus (Bruker, bremerhaven, germany) with deuterated chloroform as solvent; high resolution mass spectrometry (hresis) was determined using a Shimadzu LCMS-IT-TOF mass spectrometer (Shimadzu, kyoto, japan); the thin layer chromatography silica gel plate HSGF254 is a product of Nicotiana tabacum Jiang you silica gel development Co., ltd; column chromatography silica gel (200-300 meshes) is produced by Yi Ling Shang Hai Xiang chemical industry Co., ltd; sephadex LH-20 (Sephadex LH-20) is available from GE Healthcare Bio-Sciences AB company; the high performance liquid chromatograph is manufactured by Shimadzu corporation, the controller model is CBM-20A, the pump model is LC-20AR, the detector model is SPD-M20A, the column temperature box model is AT-350, the used chromatographic column model is Agilent-Eclipse XDB-C18 (5 μm, 9.4X1250 mm); chromatographic pure acetonitrile was purchased from merida; MCI gel CHP20P (75-150 μm) was purchased from Mitsubishi Chemical Corporation (Tokyo, japan); the color-developing agent is 10% H 2 SO 4 -EtOH solution.
Artemisininol A (artemannol A, 1)
The molecular formula: c (C) 33 H 42 O 11
Molecular weight: 614
Traits: yellow powder;
HRESIMS m/z:615.2796[M+H] + (calcd.for C 33 H 43 O 11 ,615.2800)；
IR(KBr)ν max :3433,1722,1652,1597,1513,1461,1353,1290,1274,1218,1165,1132,1007cm –1 ；
ECD(c 0.27,MeOH)λ max (Δε):203(+13.11),252(+1.72),351(+0.78)nm；
UV(MeOH)λ max (logε):257(3.21),270(3.16),350(3.26)nm；
1 h NMR 13 The C NMR (DEPT) data are shown in Table 1.
Artemisininol B (arteannuol B, 2)
The molecular formula: c (C) 33 H 42 O 11
Molecular weight: 614
Traits: yellow powder;
HRESIMS m/z:615.2791[M+H] + (calcd.for C 33 H 43 O 11 ,615.2800)；
IR(KBr)ν max :3434,1723,1650,1598,1560,1511,1460,1362,1270,1219,1165,1143,1006cm –1 ；
ECD(c 0.22,MeOH)λ max (Δε):203(+8.16),248(+1.52),352(+0.89)n；
UV(MeOH)λ max (logε):257(3.16),272(3.11),348(3.20)nm；
1 h NMR 13 The C NMR (DEPT) data are shown in Table 1.
Artemisininol C (artemannol C, 3)
The molecular formula: c (C) 33 H 40 O 1
Molecular weight: 596
Traits: yellow powder;
HRESIMS m/z:597.2687[M+H] + (calcd.for C 33 H 41 O 10 ,597.2694)；
IR(KBr)ν max :3431,1714,1651,1601,1562,1511,1460,1361,1270,1219,1165,1142,1065,1008cm –1 ；
UV(MeOH)λ max (logε):257(3.13),269(3.07),348(3.14)nm；
1 h NMR 13 The C NMR (DEPT) data are shown in Table 1.
TABLE 1 Compounds 1-3 13 C NMR data (150 MHz, CDCl) 3 δin ppm, J in Hz) and 1 h NMR data (600 MHz, CDCl) 3 ,δin ppm,J in Hz)
a Recorded in acetone-d 6 ； b recorded in CD 3 OD.
Example 2:
inhibitory activity of Compounds 1-3 against three liver cancer cell lines.
1. Materials and methods
1.1 materials
HepG2 cell lines were given by the Kunming plant institute of China academy of sciences activity screening center, SK-Hep-1 and Huh7 cell lines were purchased from Shanghai Ji Ning Biotechnology Co., ltd; medium (Dulbecco's Modified Eagle Medium, DMEM) was purchased from Thermo Fisher Scientific (Suzhou, china); serum (fetal bovine serum, FBS) was purchased from Life Technologies (NY, USA); RPMI-1640 was purchased from ThermoFisher Biochemical Products (Beijing, china).
1.2 instruments
Flex Station 3 bench-top multifunctional microplate reader (Bio-RAD 680, USA); analytical balances (AG 135, metler Toledo, china); incubator (DHP-9082, shanghai).
1.3 Experimental procedure
1) Taking liver cancer cells growing in log phase, discarding old culture medium, washing twice with PBS, discarding PBS;
2) Digesting the cells with 0.25% trypsin, and rapidly absorbing trypsin when the outline of the cells is deepened and the rounding trend is observed under a microscope;
3) The cells were stopped and resuspended in DMEM complete medium containing 10% FBS, 10 μl of cell suspension was taken, counted with a cytometer, and the cell concentration was adjusted to 1×10 with medium 4 Per mL, plated on 96-well plates with 100. Mu.L of cell suspension added to each well at 37℃with 5% CO 2 Incubating for 24 hours in an incubator of (2) to adhere cells;
4) Sucking the culture medium, adding diluted samples into the plate, adding 100 mu L of the diluted samples into each hole, setting 3 compound holes for each concentration, and continuously incubating in an incubator for 48 hours;
5) Sucking out the culture medium, adding the prepared MTT solution (1 mg/mL), adding 100 mu L of the solution into each hole, and incubating in an incubator for 4 hours;
6) Sucking MTT solution, adding DMSO, adding 100 μl per well, and incubating in incubator for 10min;
7) Measuring absorbance at 490nm wavelength using enzyme-labeled instrument, calculating cell inhibition ratio by formula inhibition ratio = (negative-experimental group)/(negative-blank group) ×100%, and calculating IC by statistical software GraphPad prism 5 50 Experiments were repeated 3 times.
Table 2 results of cytotoxic Activity of Compounds 1-3 against three liver cancer cells
2. Results
The anti-hepatoma cell toxicity activity of all the isolated compounds was evaluated, and the experimental results are shown in Table 2, wherein the compounds 1-3 have inhibitory activity on three hepatoma cells HepG2, huh7 and SK-Hep-1, and the IC thereof 50 32.7-70.4. Mu.M. Wherein, the compound 1 and 3 have inhibiting effect on HepG2 and Huh7 cells, and IC thereof 50 49.1 and 48.2, 41.1 and 32.7 μm, respectively; compound 2 has moderate inhibitory activity on HepG2 and Huh7 cells, IC thereof 50 59.1 and 59.3. Mu.M. Compounds 1-3 have inhibitory activity in the hand against SK-Hep-1 cells, IC thereof 50 Between 57.7 and 70.4 μm.
3. Conclusion(s)
Experimental results show that the compound 1-3 has inhibitory activity on three liver cancer cells HepG2, huh7 and SK-Hep-1, and IC thereof 50 32.7-70.4. Mu.M. Wherein, the compound 1 and 3 have inhibiting effect on HepG2 and Huh7 cells, and IC thereof 50 49.1 and 48.2, 41.1 and 32.7 μm, respectively; compound 2 has moderate inhibitory activity on HepG2 and Huh7 cells, IC thereof 50 59.1 and 59.3. Mu.M. Compounds 1-3 have inhibitory activity in the hand against SK-Hep-1 cells, IC thereof 50 Between 57.7 and 70.4 μm. The results show that the compounds 1-3 separated from the artemisia annua can be used as medicaments for treating liver cancer related diseases.
Formulation examples
In the following formulation examples, conventional reagents are selected and formulation preparation is performed according to the conventional methods, and this application example only embodies that at least one of the compounds 1 to 3 of the present invention can be prepared into different formulations, and specific reagents and operations are not particularly limited:
1. taking any one or any combination of the compounds 1-3, adding water for injection according to the conventional method, finely filtering, filling and sterilizing to prepare injection.
2. Dissolving compound 1-3 or their combination in sterile injectable water, stirring to dissolve, filtering with sterile suction filter funnel, sterile fine filtering, packaging in 2 ampoule, lyophilizing at low temperature, and sealing to obtain powder for injection.
3. Taking any one or any combination of the compounds 1-3, adding the excipient according to the weight ratio of 9:1 with the excipient, and preparing the powder.
4. Taking any one or any combination of the compounds 1-3, adding the excipient according to the weight ratio of the compound to the excipient of 1:5-1:10, granulating and tabletting.
5. Taking any one or any combination of the compounds 1-3, and preparing the oral liquid according to the conventional oral liquid preparation method.
6. Taking any one or any combination of the compounds 1-3, adding excipient according to the weight ratio of the compound to the excipient of 5:1, and preparing into capsules, granules or medicinal granules.
From the above examples, the present invention provides a compound of Artemisia annua, its preparation method and application, pharmaceutical composition and its application. The artemisia annua alcohol provided by the invention mainly comprises 3 sesquiterpene-flavonol hybrids with novel structures, has different degrees of inhibition effects on liver cancer cells, can be combined with a pharmaceutically acceptable carrier or excipient to form a pharmaceutical composition, and can be used for preparing anti-liver cancer drugs.
The foregoing is merely a preferred embodiment of the present invention and it should be noted that modifications and adaptations to those skilled in the art may be made without departing from the principles of the present invention, which are intended to be comprehended within the scope of the present invention.

Claims (6)

1. Artemisinin A-C shown in the following structural formula, namely compounds 1-3,
2. the method for preparing the Artemisinin A-C, namely the compound 1-3, shown in the structural formula of claim 1, is characterized by comprising the following steps:
pulverizing dried aerial parts of herba Artemisiae Annuae, extracting with 3 times of 90% ethanol twice, mixing extractive solutions, concentrating under reduced pressure to obtain crude extract, dispersing in water, extracting with ethyl acetate to obtain ethyl acetateThe ester extraction part, then, the ethyl acetate extraction part is subjected to silica gel column chromatography and is eluted with acetone-petroleum ether in a volume ratio of 10:90,20:80, 30:70 and ethyl acetate gradient to obtain 4 fractions Frs.A-D; LC-MS analysis shows that fractions A and B contain sesquiterpene dimer compounds, and TLC detection shows that the fractions A and B have more cross components, and therefore, after being combined, the fractions A and B are subjected to silica gel column chromatography, and MeOH-CHCl is used for preparing the fraction A and B 3 Gradient elution with MeOH in a volume ratio of 1:99,2:98,5:95,10:90,20:80,40:60, yielded 5 fractions Frs.B-1-B-5; fr.B-2 was chromatographed on an MCI gel CHP20P column with 50:50, 70:30, 90:10 MeOH-H 2 O and MeOH eluted to give four subfractions Frs.B-2a-B-2d; fr.B-2C via Rp-C 18 Column chromatography with MeOH-H at 50:50, 60:40, 70:30 and 80:20 2 O gradient elution to obtain subfraction Frs.B-2c-1-B-2c-5; fr.B-2c-3 was purified by column chromatography on silica gel with MeOH-CHCl at 2:98,5:95 and 10:90 3 Eluting the eluent to obtain 5 subfractions Frs.B-2c-3a-B-2c-3e; fr.B-2c-3c was subjected to Sephadex LH-20 (column chromatography with 50:50 MeOH-CHCl) 3 Eluting, and then separating with semi-preparative HPLC in Agilent XDB-C 18 Purifying the mixture on a column by using 78:22 methanol-water to obtain compounds 1 and 2; fr.B-4 was subjected to MCI gel CHP20P column chromatography eluting with 50:50, 70:30, 90:10 and 100:0 methanol-water to give four subfractions Frs.B-4a-B-4d; fr.B-4b first uses Rp-C 18 Column chromatography, treating with methanol-water 50:50, 60:40 and 70:30, eluting with methanol-chloroform 50:50 by Sephadex LH-20 column chromatography, and separating with semi-preparative high performance liquid HPLC in Agilent XDB-C 18 Purification on a column with 50:50 acetonitrile-water afforded compound 3.
3. The application of the Artemisinin A-C shown in the structural formula of claim 1 in preparing anti-liver cancer drugs.
4. A pharmaceutical composition comprising at least one of the artemisia annua alcohols a-C of the structural formula of claim 1 and a pharmaceutically acceptable carrier.
5. The use of the pharmaceutical composition of claim 4 in the preparation of an anti-liver cancer drug.
6. The method of preparing a pharmaceutical composition of claim 4: characterized in that the method comprises the following steps: pulverizing dried aerial parts of herba Artemisiae Annuae, extracting with 3 times of 90% ethanol twice, mixing extractive solutions, concentrating under reduced pressure to obtain crude extract, dispersing in water, extracting with ethyl acetate to obtain ethyl acetate extract, subjecting the ethyl acetate extract to silica gel column chromatography, and gradient eluting with acetone-petroleum ether at volume ratio of 10:90,20:80, 30:70 and ethyl acetate to obtain 4 fractions Frs.A-D; LC-MS analysis shows that fractions A and B contain sesquiterpene dimer compounds, and TLC detection shows that the fractions A and B have more cross components, and therefore, after being combined, the fractions A and B are subjected to silica gel column chromatography, and MeOH-CHCl is used for preparing the fraction A and B 3 Gradient elution with MeOH in a volume ratio of 1:99,2:98,5:95,10:90,20:80,40:60, yielded 5 fractions Frs.B-1-B-5; fr.B-2 was chromatographed on an MCI gel CHP20P column with 50:50, 70:30, 90:10 MeOH-H 2 O and MeOH eluted to give four subfractions Frs.B-2a-B-2d; fr.B-2C via Rp-C 18 Column chromatography with MeOH-H at 50:50, 60:40, 70:30 and 80:20 2 O gradient elution to obtain subfraction Frs.B-2c-1-B-2c-5; fr.B-2c-3 was purified by column chromatography on silica gel with MeOH-CHCl at 2:98,5:95 and 10:90 3 Eluting the eluent to obtain 5 subfractions Frs.B-2c-3a-B-2c-3e; fr.B-2c-3c was subjected to Sephadex LH-20 (column chromatography with 50:50 MeOH-CHCl) 3 Eluting, and then separating with semi-preparative HPLC in Agilent XDB-C 18 Purifying the mixture on a column by using 78:22 methanol-water to obtain compounds 1 and 2; fr.B-4 was subjected to MCI gel CHP20P column chromatography eluting with 50:50, 70:30, 90:10 and 100:0 methanol-water to give four subfractions Frs.B-4a-B-4d; fr.B-4b first uses Rp-C 18 Column chromatography, treating with methanol-water 50:50, 60:40 and 70:30, eluting with methanol-chloroform 50:50 by Sephadex LH-20 column chromatography, and separating with semi-preparative high performance liquid HPLC in Agilent XDB-C 18 Purifying the mixture on a column by using acetonitrile-water in a ratio of 50:50 to obtain a compound 3; and then taking one or any combination of the compounds 1-3, and adding a pharmaceutically acceptable carrier.
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CN103420967A (en) * 2013-08-19 2013-12-04 南京标科生物科技有限公司 Preparation method for marina thistle flavin
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CN104257715A (en) * 2014-10-17 2015-01-07 朴光春 Artemisia sacrorum extract as well as preparation method and application thereof
CN108888644A (en) * 2018-04-08 2018-11-27 青岛康庆和医药科技有限责任公司 A kind of Chinese medical extract and application thereof can be used for preparing treatment asthmatic medicament
CN115521955A (en) * 2022-07-17 2022-12-27 李玉山 Preparation method of flavonoid compound

Patent Citations (6)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN102633761A (en) * 2012-03-27 2012-08-15 宁夏医科大学 Method for extracting three poly-methoxy-group flavones from industrial production waste
CN103420966A (en) * 2013-08-13 2013-12-04 南京标科生物科技有限公司 Method for purifying chrysosplenium phenol D
CN103420967A (en) * 2013-08-19 2013-12-04 南京标科生物科技有限公司 Preparation method for marina thistle flavin
CN104257715A (en) * 2014-10-17 2015-01-07 朴光春 Artemisia sacrorum extract as well as preparation method and application thereof
CN108888644A (en) * 2018-04-08 2018-11-27 青岛康庆和医药科技有限责任公司 A kind of Chinese medical extract and application thereof can be used for preparing treatment asthmatic medicament
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