CN104257715B

CN104257715B - Herba Artemisiae extract and its preparation method and application

Info

Publication number: CN104257715B
Application number: CN201410549512.3A
Authority: CN
Inventors: 朴光春; 元海丹
Original assignee: Individual
Current assignee: Individual
Priority date: 2014-10-17
Filing date: 2014-10-17
Publication date: 2018-12-25
Anticipated expiration: 2034-10-17
Also published as: CN104257715A

Abstract

The invention discloses a kind of Herba Artemisiae extracts, wherein containing general flavone compound.General flavone compound includes following one or more: 6- methoxyl group-quercetagetin -7-O- β-D-Glucose glycosides, Quercetin -3-O- β-D glucoside, Isorhamnetin, patuletin -3-O- β-D-Glucose glycosides, acacetin7OβDglucoside, rutin, 5, 7, 4 '-trihydroxyflavones, 3, 3 ', 4 ', 5, 7- pentahydroxyflavone, 5, 7, 4 '--3 '-methoxy flavones of trihydroxy, Kaempferol, 3, 5, 7, 4 '-tetrahydroxys -6-methoxy-2-phenyl-4H-chromen-4-one, 3 ', 4 ', 5, 7- kaempferol, 5, 7, 4 '-trihydroxies -6, 3 '-dimethoxy flavones, 5, 7, 4 '-trihydroxies -6-methoxy-2-phenyl-4H-chromen-4-one, quercitin, 7, 3 ', 4 '-trimethyl Quercetins, 5 7,4 '-dimethoxy flavone of hydroxyl -, 5,4 '-dihydroxy -7- methoxy flavone grassland florigen, 5,7- dihydroxy -4 '-methoxy flavone, 5,7,3 '-dihydroxy -4- methoxy flavones and derivative.Herba Artemisiae extract of the invention has significant prevention and treatment effect to hepatic injury.

Description

Artemisia annua extract and preparation method and application thereof

Technical Field

The invention relates to the technical field of medicines, in particular to an artemisia annua extract and a preparation method and application thereof.

Background

The artemisia plants of the compositae are more than 350, are widely distributed in temperate regions of northern hemisphere, Europe, Asia and North America, and are distributed in tropical regions of Africa, south Asia and Central America, more than 170 kinds of Chinese (without separated sethoxydim (Bess) Poljak), and are produced all over the regions, wherein, for example, artemisia annua A.annua L, artemisia apiacea A.apiacea Hance, artemisia capillaris A.capitalis Thunb, A.argyi Levl.Vant and the like are used as medicines, particularly, the artemisia annua has the widest application, and the artemisia annua can treat malaria and can also be used for preparing wine cakes.

Artemisia annua, herb, subshrubes or shrubs often have fragrance when kneaded; leaf intergrowth, normal division; the head-shaped inflorescence is small, is anisotropic, is disc-shaped, is bent and drooped frequently, and is arranged into a cone inflorescence, a spike-shaped inflorescence or a total inflorescence; the flower is all tubular; female limbus, row 1, slender, 2-3 fissures; panicle amphiprotic, fructious or infertile; the inflorescence support is naked or has support hair; small fruit, furrows and no crown hair. The head-like inflorescence contains the same-like flower species which have been classified as the genus Calsium.

Liver injury is a pathological state with prominent common characteristics of various liver diseases, and seriously threatens human health. Various harmful factors such as drugs, viruses, alcohol, organisms, etc. may cause liver functions to be damaged in various degrees, thereby reducing detoxification, excretion, storage and regeneration of the liver, reducing liver blood flow, aggravating metabolic load, causing internal environment disorders, hepatocyte necrosis and apoptosis, and further causing liver damage. The liver damage caused by various harmful factors mainly comprises drug liver damage, virus liver damage, alcoholic liver damage, immune liver damage and the like, and billions of people all over the world suffer from liver damage once or just suffer from the liver damage. Taking hepatitis b virus as an example of the cause of viral liver injury, 20 million people worldwide have been infected with Hepatitis B Virus (HBV), of which 3.78 million people transform into chronic infection and 0.5-1.2 million people die of cirrhosis, liver failure, liver cancer, etc. There are also countless patients with drug-induced liver injury and alcoholic liver injury. The long-term existence of liver injury often leads to liver fibrosis, which is an important initiating factor for further inducing liver cirrhosis, liver failure and even liver cancer. Therefore, the prevention and treatment of hepatocyte injury is one of the important links in clinical treatment of liver diseases, and is the basis for inhibiting the occurrence and development of diseases such as hepatic fibrosis, hepatic necrosis, fatty liver, cholestasis, liver cirrhosis, liver cancer and the like.

There is no particularly good method for treating and preventing liver injury in clinic. Therefore, the current clinical demand for the anti-liver injury medicine with definite curative effect and low side effect is very strong. The evergreen wormwood is a medicine with bright characteristics and has wide application foundation in extended areas of Jilin province. Artemisia sacrorum is dry aerial part of Artemisia sacrorum Ledeb of Artemisia of Compositae, and Artemisia sacrorum paste is applied in Changbai mountain area for preventing and treating various liver injury diseases, and has a long folk application history. When treating symptoms of hepatitis such as hypochondriac pain, common people prefer to use artemisia annua as the first choice.

Disclosure of Invention

The invention provides an artemisia annua extract and a preparation method and application thereof.

The invention adopts the following technical scheme:

the artemisia annua extract contains total flavone compounds. The total flavone content in the Artemisia Rohdea extract is more than 50% (weight percentage content).

The total flavone compounds include one or more of 6-methoxy-quercetagetin-7-O- β -D-glucoside, quercetin-3-O- β -D-glucoside, isorhamnetin, quercetagetin-3-O- β -D-glucoside, acacetin-7-O- β -D-glucoside, rutin, 5, 7, 4 '-trihydroxyflavone, 3', 4 ', 5, 7-pentahydroxyflavone, 5, 7, 4' -trihydroxy-3 '-methoxyflavone, kaempferol, 3, 5, 7, 4' -tetrahydroxy-6-methoxyflavone, 3 ', 4', 5, 7-tetrahydroxyflavone, 5, 7, 4 '-trihydroxy-6, 3' -dimethoxyflavone, 5, 7, 4 '-trihydroxy-6-methoxyflavone, glycoside, 7, 3', 4 '-trimethylquercetin, 5-hydroxy-7, 4' -dimethoxyflavone, 5, 4 '-dihydroxyflavone, 5' -dihydroxyflavone, 7 '-methoxyflavone, 5, 7' -dihydroxyflavone, 5-4 '-methoxyflavone, 5-dihydroxyflavone, 5-4' -dihydroxyflavone, 5-4-hydroxyflavone, 5-dihydroxyflavone, 5-4-dihydroxyflavone, 5-4-hydroxyflavone, 5-dihydroxyflavone, 5-.

The preparation method of the artemisia annua extract comprises the following steps: taking dried artemisia annua or freshly collected overground parts of artemisia annua as raw materials, adopting one or more of a solvent extraction method, a macroporous adsorption resin method, a polyamide column chromatography method, a normal-phase column chromatography method, a reverse-phase column chromatography method and a gel process, and combining concentration drying, freeze drying and spray drying to prepare the artemisia annua extract.

In the solvent extraction method and solvent extraction method, one or more of water, methanol, ethanol, n-butanol, or other lower aliphatic alcohol is selected as solvent, and the extraction is carried out at room temperature or under ultrasonic extraction, or under heating or under reflux, wherein the extraction frequency is one or more times.

In the macroporous adsorption resin method, normal pressure or pressure column chromatography is selected, the resin adopts HPD-100, HPD-300, D-101, X-5, H103, AB-8, DA-201, HPD-400, NKA-9, S-8 and HPD-500 type resin, the elution solvent adopts one or more of water, methanol, ethanol, acetone or hydrous methanol, hydrous ethanol or hydrous acetone, and elution with fixed concentration or gradient elution is carried out during elution.

In the polyamide column chromatography, normal pressure or pressurized column chromatography is selected, the used column filler is polyamide, the eluting solvent is one or more of water, methanol, ethanol, acetone or hydrous methanol, hydrous ethanol or hydrous acetone, and elution with fixed concentration or gradient elution is carried out during elution.

In the reversed phase column chromatography, normal pressure or pressure column chromatography is selected, eluting solvent is one or more of water, methanol, ethanol, acetone or hydrous methanol, hydrous ethanol or hydrous acetone, and elution with fixed concentration or gradient elution is carried out during elution.

In normal phase column chromatography, normal pressure or pressure column chromatography is selected, eluting solvent is one or more of methanol, ethanol, acetone, ethyl acetate, petroleum ether, n-butanol, dichloromethane and chloroform, and elution with fixed concentration or gradient elution is carried out during elution.

The artemisia annua extract has the effects of preventing and treating fatty liver, drug-induced liver injury, viral liver injury, alcoholic liver injury and/or immunological liver injury diseases.

The artemisia annua extract can be prepared into health-care products or medicines for treating liver injury, and the medicines are prepared into any pharmaceutically acceptable dosage forms such as liquid, solid, semisolid, oral or non-oral administration, capsules, powder, pills, tablets, oral liquid, injection and the like by using the compound and the pharmaceutically acceptable medicines.

The medicine of the present invention can inhibit the occurrence and the development of diseases such as hepatic fibrosis, hepatic necrosis, fatty liver, cholestasis, liver cirrhosis, liver cancer, etc.

The active ingredients of the medicine obtained by the invention are extracted from artemisia annua, the sources of the active ingredients are wide, and the medicine has obvious curative effect on liver injury.

Drawings

Figure 1 is a schematic representation of the effect of 95% EE (a part of artemisia annua extract) cell proliferation and lipid accumulation.

HepG2 cells were treated with different concentrations (0, 25, 50, 100. mu.g/ml) of 95% EE for 24 h. Cell proliferation was tested by the MTS method (A). HepG2 cells were treated with different concentrations (0, 25, 50, 100. mu.g/ml) of 95% EE for 24h and then stained with OilRed O. HepG2 cells were treated with different concentrations (0, 25, 50, 100. mu.g/ml) of 95% EE for 24h, and the triglyceride content was measured spectrophotometrically and expressed as. mu.g triglyceride/mg protein (C). Data are expressed as mean ± s.e. P < 0.01 and p < 0.001 for the untreated control groups, respectively.

FIG. 2 is a graphical representation of the effect of 50% EE, 95% EE and WE (each part of Artemisia iwayomogi extract) on AMPK and ACC phosphorylation in HepG2 cells.

Cells were treated with 50% EE, 95% EE and WE at the indicated concentrations for 24h, proteins were extracted and subjected to Western blot analysis using antibodies to pAMPK, AMPK, pACC, ACC, and β -actin β -actin protein levels were used as internnalcotrol.

FIG. 3 is a graphical representation of the effect of 95% EE on AMPK and ACC phosphorylation in HepG2 cells.

Cells were treated with 100 μ g/ml 95% EE at different indicated times (a) cells were treated with different indicated concentrations of 95% EE for 24h (b) cells were pretreated with compound C for 2h, then treated with 95% EE for 24h (C, D), protein was extracted and subjected to western blot analysis with pAMPK, AMPK, pACC, ACC and β -actin. β -actin protein levels were expressed as internal control data to mean ± s.e (n ═ 3), representing p < 0.01 compared to untreated controls.

Detailed Description

The following examples are further detailed descriptions of the present invention.

Example 1 preparation of Artemisia Rohdea extract

Soaking 500g dry herba Artemisiae Annuae in water for 0.5-3 hr, heating for 0.5-3 hr, filtering to obtain filtrate I, adding water into the residue, heating for 0.5-3 hr to obtain filtrate II, and mixing filtrates I and II to obtain herba Artemisiae Annuae water extractive solution. Loading the water extract of Artemisia iwayomogi to a pretreated macroporous adsorbent resin column, and sequentially eluting with water and 10% -95% ethanol. Concentrating 10% -95% ethanol eluate, and freeze drying to obtain herba Artemisiae Annuae extract.

Example 2 determination of Total Flavonoids in Artemisia Rohdea extract

Standard curve of flavone:

reagent: rutin, methanol

Preparation of rutin control solution

Precisely weighing 5.0mg rutin, adding methanol, dissolving, and metering to 100ml to obtain 50 μ g/ml.

Sucking rutin control solution 0, 1.0ml, 2.0ml, 3.0ml, 4.0ml, 5.0ml, 6.0ml and 7.0ml, adding methanol to the scale mark in a 10ml volumetric flask, shaking up, measuring the wavelength: 360 nm. The results are as follows:

y＝0.1371x+0.0093 R²＝0.9999

preparation of test sample (artemisia annua 50% ethanol eluate) solution:

putting 1ml of sample solution into an evaporation dish, adding 1g of polyamide powder for adsorption, putting the sample solution into a constant-temperature drying box, volatilizing the solvent at 50 ℃, transferring the solvent into a chromatographic column, eluting the chromatographic column by using 30ml of petroleum ether, discarding the petroleum ether, eluting the total flavonoids by using 100ml of methanol, collecting the eluent, drying the eluent by evaporation under pressure, dissolving the residue by using the methanol, and fixing the volume to a volumetric flask of 25ml to obtain the finished product.

And (3) measuring the absorbance of the test sample:

taking 1ml of polyamide powder eluate of a test article, putting the polyamide powder eluate into a 25ml volumetric flask, adding methanol to reach a constant volume of 25ml, taking out 1ml from 25ml, adding methanol to dilute by 10 times, measuring the absorbance to be 0.316, and calculating to obtain the total flavone content of 28.115 mg/ml.

And (3) conversion result:

the content of total flavone is 68.2%.

Example 3 isolation and structural characterization of chemical Components in Artemisia Rohdea extract

The Artemisia annua extract is extracted by using solutions such as dichloromethane, ethyl acetate and the like, the extract is subjected to various separation methods such as a reverse phase silica gel column, a SephadexLH-20 gel column, a preparative HPLC, an ODS low pressure column and the like, and is subjected to gradient elution by a methanol-water system and the like to obtain 20 flavonoid compounds, and the chemical structures of the flavonoid compounds are determined to be 1) 6-methoxy-quercetol-7-O- β -D-glucoside, 2) quercetin-3-O- β -D-glucoside, 3) isorhamnetin, 4) tagetin-3-O- β -D-glucoside, 5) farnesol-7-O- β -D-glucoside, 6) rutin, 7)5, 7, 4 '-trihydroxyflavone, 8)3, 3', 4 ', 5, 7-pentahydroxyflavone, 9)5, 7, 4' -trihydroxy-3 '-methoxyflavone, 10) kaempferol, 11)3, 5, 7, 4' -hydroxyflavone, 7 '-hydroxyflavone, 7', 4 '-hydroxyflavone, 7', 5, 7 '-hydroxyflavone, 7-5, 7' -hydroxyflavone, 7 '-trihydroxy-4' -hydroxyflavone, 7-5-4 '-hydroxyflavone, 7' -hydroxy.

The spectrum data of the flavonoid compounds in the artemisia annua extract are respectively as follows:

compound 1: 6-methoxy-quercetagetin-7-O- β -D-glucoside

¹H-NMR(300MHz，(CD₃)₂CO)δppm：12.42(1H，s，5-OH)，7.89(1H，d，J＝2.01Hz，H-2’)，7.46(1H，dd，J＝2.04，8.34Hz，H-6’)，6.82(1H，d，J＝8.46Hz，H-5’)，6.47(1H，s，H-8)，5.12(1H，d，J＝7.26Hz，H-1”)，3.75(3H，s，6-OCH₃).

¹³C-NMR(75MHz，DMSO-d₆)δppm：145.26(C-2)，133.43(C-3)，178.11(C-4)，156.72(C-5)，131.70(C-6)，152.84(C-7)，94.22(C-8)，151.98(C-9)，104.76(C-10)，122.06(C-1′)，116.64(C-2′)，157.95(C-3′)，148.91(C-4′)，115.65(C-5′)，121.67(C-6′)，101.31(C-1″)，74.55(C-2″)，76.96(C-3″)，70.41(C-4″)，78.02(C-5″)，61.45(C-6″)，60.43(6-OCH₃).

Compound 2 Quercetin-3-O- β -D glucoside

¹H-NMR(300MHz，CD₃OD)δppm：7.68(1H，dd，J＝2.10Hz，H-2’)，7.56(1H，dd，J＝2.1，9.00Hz，H-6’)，6.83(1H，d，J＝8.40Hz，H-5’)，6.36(1H，d，J＝2.10Hz，H-8)，6.17(1H，d，J＝2.10Hz，H-6)，5.23(1H，d，J＝7.20Hz，3-O-glu)，3.30-3.71(m-sugar-H).

¹³C-NMR(75MHz，CD₃OD)δppm：58.46(C-2)，135.60(C-3)，179.50(C-4)，163.06(C-5)，99.87(C-6)，166.02(C-7)，94.69(C-8)，159.00(C-9)，105.68(C-10)，123.06(C-1′)，115.99(C-2′)，145.91(C-3′)，149.85(C-4′)，117.54(C-5′)，123.19(C-6′)，104.20(C-1″)，75.72(C-2″)，78.10(C-3″)，71.20(C-4″)，78.39(C-5″)，62.53(C-6″).

Compound 3: isorhamnetin

¹H-NMR(300MHz，CD₃OD)δppm：7.73(1H，d，J＝2.10Hz，H-2′)，7.63(1H，dd，J＝2.10，8.70Hz，H-6′)，6.88(1H，d，J＝8.70Hz，H-5′)，6.38(1H，d，J＝2.10Hz，H-8)，6.18(1H，d，J＝2.10Hz，H-6)，3.59(3H，s，-OCH₃).

¹³C-NMR(75MHz，CD₃OD)：148.78(C-2)，137.23(C-3)，177.34(C-4)，162.52(C-5)，99.28(C-6)，165.72(C-7)，94.43(C-8)，158.25(C-9)，104.49(C-10)，124.15(C-1’)，116.22(C-2’)，147.99(C-3’)，146.24(C-4’)，115.98(C-5’)，121.66(C-6’)，65.10(3’-OCH₃)。

Compound 4, marigold-3-O- β -D-glucoside

¹H-NMR(500MHz，CD₃OD)δppm：6.49(1H，s)，3.87(3H，s)，7.70(1H，d，J＝2.00Hz)，7.58(1H，dd，J＝2.00，2.00Hz)，6.86(1H，d，J＝8.50Hz)，5.27(1H，d，J＝7.50Hz)，5.27(1H，d，J＝7.50Hz)，3.26～3.57(m，suger-H)。

¹³C-NMR(125MHz，CD₃OD)：159.19(C-2)，135.34(C-3)，179.76(C-4)，153.87(C-5)，132.80(C-6)，159.07(C-7)，95.03(C-8)，153.87(C-9)，106.12(C-10)，123.22(C-1’)，117.60(C-2’)，145.92(C-3’)，149.66(C-4’)，116.03(C-5’)，123.15(C-6’)，104.37(C-1″)，75.75(C-2″)，78.39(C-3″)，71.29(C-4″)，78.14(C-5″)，62.62(C-6″)，60.96(6-OCH₃)。

Compound 5 farnesoid-7-O- β -D-glucoside

¹H-NMR(500MHz，CD₃OD)δppm：8.04(2H，d，J＝8.00Hz)，7.11(2H，d，J＝8.00Hz)，5.08(1H，d，J＝7.50Hz)，3.87(3H，s，OCH₃)，3.22～3.81(m，sugar-H)。

¹³C-NMR(125MHz，CD₃OD)：158.94(C-2)，104.83(C-3)，179.14(C-4)，149.12(C-5)，103.58(C-6)，153.82(C-7)，77.57(C-8)，144.87(C-9)，1005.30(C-10)，123.00(C-1’)，134.77(C-2’)，117.49(C-3’)，145.19(C-4’)，116.41(C-5’)，123.40(C-6’)，104.73(C-1″)，70.51(C-2″)，75.05(C-3″)，61.82(C-4″)，77.14(C-5″)，61.40(C-6″)，49.83(4’-OCH₃)。

Compound 6: rutin

¹H-NMR(500MHz，CD₃OD)δppm：8.03(1H，d，J＝9.00Hz)，7.58(1H，dd，J＝1.00，1.00Hz)，6.95(1H，d，J＝8.50Hz)，4.98(1H，d，J＝8.00Hz)，4.54(1H，br s)，1.08(3H，d，J＝6.0Hz，OCH₃)，3.35～4.52(m，sugar-H)。

¹³C-NMR(125MHz，CD₃OD)：149.40(C-2)，133.30(C-3)，178.87(C-4)，154.01(C-5)，101.88(C-6)，158.90(C-7)，77.18(C-8)，152.56(C-9)，106.89(C-10)，123.59(C-1’)，116.40(C-2’)，134.54(C-3’)，145.07(C-4’)，117.60(C-5’)，123.10(C-6’)，104.78(C-1″)，73.19(C-2″)，76.31(C-3″)，71.42(C-4″)，75.00(C-5″)，61.37(C-6″)，103.65(C-1′″)，69.60(C-2′″)，70.84(C-3′″)，71.57(C-4′″)，68.29(C-5′″)，17.54(C-6′″)。

Compound 7: 5, 7, 4' -Trihydroxyflavone

¹H-NMR(500MHz，DMSO-d₆)δppm：6.19(1H，d，J＝2.1Hz，H-6)，6.48(1H，d，J＝2.1Hz，H-8)，6.74(1H，s，H-3)，6.92(2H，d，J＝8.8Hz，H-3′，H-5′)，，7.90(2H，d，J＝8.8Hz，H-2′，H-6′).

¹³C-NMR(125MHz，DMSO-d₆)δppm：164.17(C-2)，103.04(C-3)，181.77(C-4)，157.48(C-5)，98.90(C-6)，163.88(C-7)，94.16(C-8)，161.22(C-9)，103.77(C-10)，121.38(C-1′)，128.56(C-2′)，116.05(C-3′)，161.06(C-4′)，116.05(C-5′)，128.56(C-6′).

Compound 8: 3, 3 ', 4', 5, 7-pentahydroxyflavone

¹H-NMR(300MHz，CD₃OD)δppm：6.18(1H，d，J＝2.0Hz，H-6)，6.38(1H，d，J＝2.0Hz，H-8)，7.73(1H，d，J＝2.1Hz，H-2′)，6.88(1H，d，J＝8.52Hz，H-5′)，7.63(1H，dd，J＝2.19，8.52Hz，H-6′).

¹³C-NMR(75MHz，CD₃OD)δppm：147.99(C-2)，137.24(C-3)，177.34(C-4)，158.23(C-5)，99.22(C-6)，162.52(C-7)，94.39(C-8)，165.58(C-9)，104.52(C-10)，148.77(C-1′)，115.98(C-2′)，124.14(C-3′)，146.22(C-4′)，116.22(C-5′)，121.67(C-6′).

Compound 9: 5, 7, 4 '-trihydroxy-3' -methoxyflavone

¹H-NMR(500MHz，CD₃OD)δppm：3.97(3H，s，-OCH₃)，6.19(1H，d，J＝2.0Hz，H-6)，6.44(1H，d，J＝2.0Hz，H-8)，6.61(1H，s，H-3)，6.90(1H，d，J＝8.5Hz，H-5′)，7.48(1H，d，J＝2.0Hz，H-2′)，7.52(1H，dd，J＝8.5，2.0Hz，H-6′).

¹³C-NMR(125MHz，CD₃OD)δppm：160.89(C-2)，104.15(C-3)，183.79(C-4)，163.13(C-5)，123.75(C-6)，166.09(C-7)，95.31(C-8)，156.19(C-9)，105.39(C-10)，124.69(C-1′)，110.70(C-2′)，149.58(C-3′)，152.52(C-4′)，116.86(C-5′)，121.77(C-6′)，56.72(-OCH₃).

Compound 10: kaempferol

¹H-NMR(500MHz，CD₃OD)δppm：6.18(1H，d，J＝2.0Hz，H-6)，6.40(1H，d，J＝2.0Hz，H-8)，6.90(2H，d，J＝9.0Hz，H-3′，H-5′)，8.09(2H，d，J＝9.0Hz，H-2′，H-6′).

¹³C-NMR(125MHz，CD₃OD)δppm：148.15(C-2)，137.17(C-3)，177.44(C-4)，158.35(C-5)，99.38(C-6)，165.77(C-7)，94.55(C-8)，162.53(C-9)，104.56(C-10)，123.81(C-1′)，130.69(C-2′)，116.35(C-3′)，160.57(C-4′)，116.35(C-5′)，130.69(C-6′).

Compound 11: 3, 5, 7, 4' -tetrahydroxy-6-methoxyflavone

¹H-NMR(500MHz，CD₃OD)δppm：3.87(3H，s，-OCH₃)，6.48(1H，s，H-8)，6.89(2H，d，J＝8.8Hz，H-3′，H-5′)，8.07(2H，d，J＝8.8Hz，H-2′，H-6′).

¹³C-NMR(125MHz，CD₃OD)δppm：148.39(C-2)，136.85(C-3)，177.64(C-4)，153.67(C-5)，132.27(C-6)，158.50(C-7)，94.80(C-8)，152.98(C-9)，105.01(C-10)，123.77(C-1′)，130.72(C-2′)，116.33(C-3′)，160.60(C-4′)，116.33(C-5′)，130.72(C-6′)，60.99(-OCH₃).

Compound 12: 3 ', 4', 5, 7-tetrahydroxyflavone

¹H-NMR(500MHz，CD₃OD)δppm：7.42(1H，dd，J＝2.0，8.0Hz，H-6′)，7.39(1H，d，J＝2.0Hz，H-2′)，6.94(1H，d，J＝8.5Hz，H-5′)，6.75(1H，s，H-3)，6.44(1H，d，J＝2.0Hz，H-8)，6.20(1H，d，J＝2.0Hz，H-6).

¹³C-NMR(125MHz，CD₃OD)δppm：164.73(C-2)，104.62(C-3)，183.66(C-4)，162.33(C-5)，99.78(C-6)，165.61(C-7)，94.75(C-8)，158.25(C-9)，103.89(C-10)，122.31(C-1′)，114.22(C-2′)，146.78(C-3′)，150.71(C-4′)，116.01(C-5′)，120.46(C-6′).

Compound 13: 5, 7, 4 '-trihydroxy-6, 3' -dimethoxyflavone

¹H-NMR(500MHz，CD₃OD)δppm：3.96(3H，s，-OCH₃)，3.88(3H，s，-OCH₃)，6.56(1H，s，H-3)，6.61(1H，s，H-8)，7.45(1H，d，J＝2.0Hz，H-2′)，6.92(1H，d，J＝8.35Hz，H-5′)，7.49(1H，dd，J＝2.0，8.35Hz，H-6′).

¹³C-NMR(125MHz，CD₃OD)δppm：166.24(C-2)，103.80(C-3)，184.23(C-4)，154.70(C-5)，132.97(C-6)，158.90(C-7)，95.38(C-8)，154.70(C-9)，105.78(C-10)，123.78(C-1′)，110.75(C-2′)，149.52(C-3′)，152.13(C-4′)，116.81(C-5′)，121.76(C-6′)，56.71(-OCH₃)，60.94(-OCH₃).

Compound 14: 5, 7, 4' -trihydroxy-6-methoxyflavone

¹H-NMR(500MHz，CD₃OD)δppm：3.89(3H，s，-OCH₃)，6.58(1H，s，H-3)，6.67(1H，s，H-8)，6.92(2H，d，J＝8.75Hz，H-3′，H-5′)，7.84(2H，d，J＝8.75Hz，H-2′，H-6′).

¹³C-NMR(125MHz，CD₃OD)δppm：164.46(C-2)，102.46(C-3)，184.31(C-4)，152.69(C-5)，131.70(C-6)，，157.66(C-7)，94.17(C-8)，152.69(C-9)，104.87(C-10)，122.14(C-1′)，128.45(C-2′)，1116.15(C-3′)，161.32(C-4′)，116.15(C-5′)，128.45(C-6′).

Compound 15: quercetin

¹H-NMR(300MHz，Acetone-d₆)δppm：6.25(1H，d，J＝2.07Hz，H-6)，6.46(1H，d，J＝2.07Hz，H-8)，7.49(1H，d，J＝2.07Hz，H-2′)，7.39(1H，dd，J＝2.1，8.34Hz，H-6′)，6.98(1H，d，J＝8.34Hz，H-5′)，5.28(1H，d，J＝1.5Hz，H-1″)，3.72(1H，dd，J＝3.4，9.4Hz，H-3″)，3.34-3.43(2H，m，H-4″，H-5″)，0.91((3H，d，J＝5.7Hz，-CH3).

¹³C-NMR(75MHz，Acetone-d₆)δppm：158.15(C-2)，135.63(C-3)，179.18(C-4)，163.06(C-5)，99.35(C-6)，164.77(C-7)，94.35(C-8)，157.63(C-9)，105.75(C-10)，122.43(C-1′)，115.99(C-2′)，145.68(C-3′)，148.85(C-4′)，116.56(C-5′)，122.73(C-6′)，102.57(C-1″)，71.26(C-2″)，71.97(C-3″)，72.86(C-4″)，71.19(C-5″)，17.63(C-6″).

Compound 16: 7, 3 ', 4' -trimethylquercetin

¹H-NMR(300MHz，CDCl₃)δppm：13.08(1H，s，5-OH)，7.53(1H，dd，J＝8.50，2.00Hz，H-6′)，7.34(1H，d，J＝2.00Hz，H-2′)，6.98(1H，d，J＝8.50Hz，H-5′)，6.61(1H，s，H-8)，6.58(1H，s，H-6)，4.05(3H，s，OCH₃)，3.98(3H，s，OCH₃)，3.97(3H，s，OCH₃).

Compound 17: 5-hydroxy-7, 4' -dimethoxyflavone

¹H-NMR(300MHz，CDCl₃)δppm：7.87(2H，d，J＝8.85Hz，H-2′，6′)，7.04(2H，d，J＝8.85Hz，H-3′，5′)，6.60(1H，s，H-3)，6.51(1H，d，J＝2.10Hz，H-8)，6.39(1H，d，J＝2.10Hz，H-6)，3.91(3H，s，OCH₃)，3.90(3H，s，OCH₃).

¹³C-NMR(75MHz，DMSO-d₆)δppm：164.51(C-2)，104.41(C-3)，182.47(C-4)，162.24(C-5)，98.04(C-6)，165.29(C-7)，92.64(C-8)，157.06(C-9)，104.92(C-10)，123.63(C-1′)，128.06(C-2′)，114.65(C-3′)，162.61(C-4′)，114.52(C-5′)，128.06(C-6′)，55.79(OCH₃)，55.54(OCH₃).

Compound 18: 5, 4' -dihydroxy-7-methoxyflavone

¹H-NMR(300MHz，DMSO-d₆)δppm：3.86(3H，s，OCH₃)，6.37(1H，d，J＝2.08Hz，H-6)，6.76(1H，d，J＝2.08Hz，H-8)，6.86(1H，s，H-3)，7.11(2H，d，J＝8.85Hz，H-3′，5′)，8.02(2H，d，J＝8.85Hz，H-2′，6′)，10.63(1H，br.s，4′-OH)，12.93(1H，s，5-OH).

Compound 19: 5, 7-dihydroxy-4' -methoxyflavone

¹H-NMR(300MHz，DMSO-d₆)δppm：3.87(3H，s，OCH₃)，6.20(1H，d，J＝1.80Hz，H-6)，6.50(1H，d，J＝1.80Hz，H-8)，6.84(1H，s，H-3)，6.93(2H，d，J＝8.70Hz，H-3′，5′)，7.95(2H，d，J＝8.70Hz，H-2′，6′)，10.63(1H，br.s，4′-OH)，12.97(1H，s，5-OH).

Compound 20: 5, 7, 3' -dihydroxy-4-methoxyflavone

¹H-NMR(300M Hz，ACETONE)δppm：6.14(1H，s，6-H)，6.44(1H，s，8-H)，6.50(1H，s，3-H)，7.00(1H，d，J＝8.40Hz，5′-H)，7.37(1H，d，J＝1.80Hz，2′-H)，7.41(1H，m，6′-H)，12.86(1H，s，5-OH)，3.82(3H，s，4′-OCH₃).

The chemical structural formula of the flavonoid compounds in the artemisia annua extract is as follows:

example 4 Activity test and therapeutic Effect on liver injury of Artemisia Rohderiana extract

1) Effect of 95% EE fraction of Artemisia annua extract on lipopexia

To observe the effect of 95% EE on lipid accumulation, HepG2 cells were treated with different concentrations of 95% EE (0, 25, 50, 100. mu.g/ml) for 24hr and then stained with Oil Red O. As shown in fig. 1C, 95% EE inhibited lipid accumulation and exhibited dose-dependence. In addition, triglyceride levels in the cells were tested after treating HepG2 cells with different concentrations of 95% EE (0, 25, 50, 100. mu.g/ml) for 24 hr. As shown in fig. 1B, 95% EE significantly reduced triglyceride content and exhibited dose dependence. Different concentrations of 95% EE (25. mu.g/ml, 50. mu.g/ml, 100. mu.g/ml) reduced fat reserves by 13.2% (p < 0.01), 14.6% (p < 0.01), 19.0% (p < 0.001), respectively, compared to the non-dosed group. These results indicate that 95% EE is able to inhibit lipid accumulation.

2) Effect of 95% EE fraction of Artemisia annua extract on AMPK phosphorylation

In order to screen the effective site for AMPK phosphorylation, WE, 50% EE, and 95% EE were tested on AMPK activity by western blotting, 95% EE significantly promoted phosphorylation of AMPK and ACC and showed dose-dependence (fig. 2), therefore, in this experiment, 95% EE was selected for further study, to investigate the effect of 95% EE on AMPK phosphorylation, HepG2 cells were treated with 95% EE at a concentration of 100 μ g/ml, respectively, at different labeled times, after each treatment, cells were collected and cell lysates were extracted, AMPK α and ACC activation states were tested by western blotting using phosphorylated antibodies, and the results showed that 95% EE activated AMPK α -Thr172 in HepG2 cells and showed time-dependence (fig. 3A), consistent with increased AMPK activity, ACC-Ser79 (the most phosphorylated site) was also characterized and showed time-dependence.

Then, HepG2 cells were treated with different concentrations of 95% EE for 24 hr. As shown in fig. 3B, 95% EE promoted phosphorylation of AMPK and ACC and was dose-dependent. Therefore, to demonstrate the effect of 95% EE on AMPK activity, we attempted to inhibit AMPK and ACC activity by pharmacological means. When HepG2 cells were pretreated with an AMPK inhibitor, compound C (10 μ M), the extent of AMPK and ACC phosphorylation by 95% EE was significantly reduced (fig. 3C, 3D). These results indicate that 95% EE functions by activating AMPK activity of HepG2 cells.

Although embodiments of the present invention have been shown and described, it will be appreciated by those skilled in the art that changes, modifications, substitutions and alterations can be made in these embodiments without departing from the principles and spirit of the invention, the scope of which is defined in the appended claims and their equivalents.

Claims

1. The artemisia annua extract is characterized in that: the artemisia annua extract contains total flavone compounds; wherein,

the content of total flavone compounds in the artemisia annua extract is more than 50%;

the total flavone compounds include 6-methoxy-quercetagetin-7-O- β -D-glucoside, quercetin-3-O- β -D-glucoside, isorhamnetin, marigold-3-O- β -D-glucoside, farnesin-7-O- β -D-glucoside, rutin, 5, 7, 4 ' -trihydroxyflavone, 3 ', 4 ', 5, 7-pentahydroxyflavone, 5, 7, 4 ' -trihydroxy-3 ' -methoxyflavone, kaempferol, 3, 5, 7, 4 ' -tetrahydroxy-6-methoxyflavone, 3 ', 4 ', 5, 7-tetrahydroxyflavone, 5, 7, 4 ' -trihydroxy-6, 3 ' -dimethoxyflavone, 5, 7, 4 ' -trihydroxy-6-methoxyflavone, quercitrin, 7, 3 ', 4 ' -trimethylquercetin, 5-hydroxy-7, 4 ' -dimethoxyflavone, 5, 4 ' -dihydroxy-7-methoxy-7, 5 ' -dihydroxyl-7, 4 ' -methoxyflavone, 5, 7 ' -dihydroxyl-3 ' -methoxyflavone, 5, 7 ' -dihydroxyl-4 ' -methoxyflavone.

2. The method for preparing artemisia annua extract as claimed in claim 1, wherein the method comprises the following steps:

soaking dried Artemisia Rotundra Hance in water for 0.5-3 hr, heating for 0.5-3 hr, filtering to obtain filtrate I, adding water into the residue, heating for 0.5-3 hr to obtain filtrate II, and mixing the filtrates I and II to obtain water extract of Artemisia Rotundra Hance;

loading the artemisia rupestris water extract into a pretreated macroporous adsorption resin column, sequentially eluting with water and 10% -95% ethanol, concentrating the eluent, and freeze-drying to obtain an artemisia rupestris extract;

extracting herba Artemisiae Annuae extract with dichloromethane, ethyl acetate, etc., separating the extractive solution with reverse phase silica gel column, SephadexLH-20 gel column, preparative HPLC, ODS low pressure column, gradient eluting with methanol-water system, and separating to obtain 20 flavonoids.

3. Use of the artemisia annua extract as claimed in claim 1 in the preparation of a medicament for preventing and treating fatty liver, drug-induced liver injury, viral liver injury, alcoholic liver injury and/or immune liver injury diseases.