CN109952108A - Including Artemisia annua extract as effective component for improving the composition and preparation method thereof of skin disease - Google Patents

Abstract

The disclosure provides a kind of method for separating Artemisia annua extract and the composition for preventing or treating skin disease, the method includes extracting artemisia annua with low polar solvent and extracting residue with hydrophilic solvent, the composition includes the Artemisia annua extract of the sesquiterpene lactone with low content for obtaining separation, its distillations or by its isolated compound.

Description

Including Artemisia annua extract as effective component for improving the combination of skin disease Object and preparation method thereof

Technical field

It is related to a kind of including that artemisia annua (Artemisia annua) extract is used to prevent or treat as effective component Composition of skin disease and preparation method thereof.

Background technique

Artemisia annua extract is researched and developed.Low polarity distillations are removed however, not developing from it Or extract and the extract or extract prepared therefrom that obtain are come the example for improving pruitus and dermatitis.

Skin is directly contacted with antigen to cause allergic reaction or skin irritatin is as lasting press factors makes skin The stable state of barrier is destroyed, and leads to itch and inflammation.Especially since the cause of disease of atopic dermatitis is unclear without certain Treatment method, usually using following method: only using the temporary ease symptom of steroidal anti-inflammatory drug, or will be such as moisturiser Moisturizer be applied on skin to mitigate itch.In recent years, about 10% baby and children are by atopic dermatitis bring Pain, and number of patients is in the trend continued to increase.In addition, as the xerosis of aging of population old age application on human skin is increased Meanwhile various forms of dermatitis are also increasing.Since steroid preparation can cause side effect and to its resistance, because This, there is an urgent need to develop the non-steroid natural drugs that can treat the dermatitis including atopic dermatitis.

In addition, existing to the arteannuin of artemisia annua or extract or derived essential oil including sesquiterpene lactone ingredient It is studied, but such as the extraction for removing sesquiterpene lactone ingredient and derived essential oil including arteannuin from it of the disclosure The effect that object improves dermatitis is not known.

Summary of the invention

Technical problem

The disclosure provides a kind of for preventing or treating the composition and preparation method thereof of various skin diseases, the composition Including the Artemisia annua extract by removing most of sesquiterpene lactone, derived essential oil and phytosterol not being reported in the past As effective component.

Technical solution

The one side of the disclosure provides a kind of method for separating Artemisia annua extract comprising: by sweet wormwood plant body The step that (Artemisia annua) is contacted with low polar solvent and the low polar solvent is used to extract sweet wormwood plant body ingredient Suddenly；It is separated from the mixture obtained after the contact and removes the low polar solvent come the step of obtaining residue；And The step of residue is contacted with hydrophilic solvent to extract the sweet wormwood plant ingredient from the hydrophilic solvent.

On the one hand the disclosure provides the composition for preventing or treating skin disease, the composition includes according to Separate sweet wormwood plant body extract prepared by the method for Artemisia annua extract.

On the other hand the disclosure provides a kind of for preventing or treating the composition of skin disease, the composition includes choosing From four kinds or more the compounds in ornithine, pine camphor, mucilaginous fiber alcohol, chiro-inositol, glycine betaine and chlorogenic acid.

The disclosure on the other hand provides Yi ﹑ medicine Wai Pin ﹑ food or cosmetic composition, the composition include basis Sweet wormwood plant body extract prepared by the method for the separation Artemisia annua extract is selected from ornithine, pine camphor, cement Four kinds or more compounds in inositol, chiro-inositol, glycine betaine and chlorogenic acid.

On the other hand the disclosure provides the method for the skin disease for preventing or treating individual, this method includes will be described Composition is applied to the step of individual.

Detailed description of the invention

Fig. 1 is the HPLC color that artemisia annua ethanol extract is extracted in the residue left after extracting artemisia annua with methylene chloride Spectrogram.

Fig. 2 and Fig. 3 is the HPLC chromatogram of the highly polar distillations of artemisia annua described in embodiment 2 (AAMM- fraction 1).

Fig. 4 and Fig. 5 is the chromatogram for showing the HPLC result of AAMM- fraction 4.

Fig. 6 shows in the residue after being extracted with artemisia annua dichloromethane extract (AAM) and methylene chloride and is extracted with ethyl alcohol Artemisia annua ethanol extract (AAMM) treatment rat inflammation part after use¹⁸PET-CT image of the F-FDG as tracer.

Fig. 7 be show with Artemisia annua extract AAM and AAMM treatment rat inflammation part after organize variation it is micro- Mirror photo.

Fig. 8 be with the fluoro- 2,4- dinitrobenzene (DNFB) of 1-, dexamethasone, AAMM, AAMM- fraction 1, AAMM- fraction 2, The variation organized after AAMM- fraction 3 and the inflammation part of the treatment rat of AAMM- fraction 4 passes through the histopathology of H&E dyeing display It learns microscope photo (multiplying power × 200).

Fig. 9 is display DNFB, dexamethasone, AAMM, AAMM- fraction 1, AAMM- fraction 2, AAMM- fraction 3 and AAMM- The Picro-Sirius red (Picrosirius Red) of collagen distribution after the inflammation part of the treatment rat of fraction 4 in tissue change The microscope photo (multiplying power × 100) of staining tissue slides.

Figure 10 is organized with after the inflammation part for extract (AA) and AAMM-1+4 the treatment rat for including sesquiterpene lactone Variation dyed by H&E and the sirius red stains table Pi Houdu ﹑ inflammatory cells that show is deposited in ﹑ spine skin and acanthosis nigricans (acanthosis) and collagen distribution microscope photo (multiplying power × 200).

(A) display of Figure 11 has the self inducing cell protection of the compound of any formula in chemical formula 1 to 7 by induction Activity, (B) display of Figure 11 include have the optional combined composition of the compound of any formula in chemical formula 1 to 7 self The figure of inducing cell protection activity.(rapamycin (Rapamycin) of the RPM=as positive controls and is administered to medicine at C= The negative control group treated under the identical concentration of concentration of object processing group with DMSO)

Figure 12 is shown wherein with the H&E dye of AA-M6 composition and the inflammation part of AA-M9 composition treatment DNFB induction With the result of microscope observation histotomy after color Skin slice.

Figure 13 show with methanol extract removing with methylene chloride extract in stay after the extracting solution that obtains of domestic artemisia annua Under residue extract HPLC result.

Figure 14 show with ethyl alcohol extract removing with diethyl ether extract in leave after the extracting solution that obtains of domestic artemisia annua Residue extract HPLC result.

Figure 15 show extracted with ethyl alcohol remove be extracted with ethyl acetate in stay after the extracting solution that obtains of domestic artemisia annua Under residue extract HPLC result.

Figure 16 shows extract the extracting solution that removing is obtained with domestic artemisia annua in acetone extraction with ethyl alcohol after leave The result of the HPLC of the extract of residue.

Figure 17 show with ethyl alcohol extract removing with liquid CO 2 extract in the extracting solution that obtains of domestic artemisia annua The result of the HPLC of the extract of the residue left afterwards.

Figure 18 shows to be extracted after removing extracts the extracting solution that the artemisia annua that South Korea produces obtains with methylene chloride with ethyl alcohol and stay Under residue extract HPLC result.

Figure 19 shows to be extracted after removing extracts the extracting solution that the artemisia annua that South Korea produces obtains with diethyl ether with ethyl alcohol and leave Residue extract HPLC result.

Figure 20 is stayed after showing the extracting solution for extracting the artemisia annua acquisition that removing is extracted with ethyl acetate South Korea's production with ethyl alcohol Under residue extract HPLC result.

Figure 21 leaves after showing the extracting solution for extracting the artemisia annua acquisition that removing is produced with acetone extraction South Korea with ethyl alcohol The result of the HPLC of the extract of residue.

Figure 22 shows the ethyl acetate extract (AAE1) prepared from residue and ethanol extract (AAEM1) in cell The figure of the influence of interleukin-1 beta (IL-1 β) expression.

Figure 23 shows the figure of influence of the AAE1 and AAEM1 to the LC3 expression in cell.

Specific embodiment

Hereinafter, the disclosure is more fully described in reference implementation example.However, these embodiments are merely to illustrate this public affairs It opens, and the scope of the present disclosure is not limited to these embodiments.

Embodiment 1: the preparation of artemisia annua (Artemisia annua) extract

1. the preparation of the Artemisia annua extract comprising sequiterpene and derived essential oil

The overground part dried object of purchase artemisia annua (in domestic) is simultaneously put into as extraction vessel after frittering the sample of 5kg In 50L vial, then be added 40L methylene chloride (dichloromethane) after shake up at room temperature stirring 48 hours with into Row extracts, and the use of the whatman paper filter paper and diameter with 0.34mm film thickness is the glass funnel of 30cm to the mixture Repeat the process (" extraction-filtering " 2 times) of gravity filtration twice, with the extracting solution filtered.By mentioning for the filtering Take liquid to be put into reduced pressure machine, and under reduced pressure with 30 DEG C at evaporating completely solvent be concentrated.Contain as a result, obtaining 114g Sesquiterpene lactone ingredient and derived essential oil including qinghaosu make artemisia annua dichloromethane extract as main component (in hereafter In be known as " AAM ") (yield 2.3%)

2. removing the preparation of the sequiterpene of most of artemisia annua and the artemisia annua ethanol extract of derived essential oil

To residue, (residue is remained after executing filtering to the mixture of methylene chloride and aerial part in Section 1 Residue) in 40L ethyl alcohol is added after, stirring is shaken up at 45 DEG C 48 hours and then after impregnating and being cooled to room temperature, uses tool There are whatman paper filter paper and the diameter of 0.34mm film thickness to repeat two subgravities to the mixture for the glass funnel of 30cm The process (" warm leaching-filtering " 2 times) of filtering.Then, the extracting solution of the filtering is put into reduced pressure machine, and under reduced pressure and Evaporating completely solvent is concentrated at 40 DEG C.As a result, obtain 484g remove it is most of include qinghaosu sesquiterpene lactone at Point and derived essential oil artemisia annua ethanol extract (being known as " AAMM " below).(yield 9.7%)

High performance liquid chromatography (HPLC) is carried out to the AAMM extract according to following analysis condition.As a result, it was confirmed that Residence time (residence time) is that such as sesquiterpene lactone, sequiterpene, derived essential oil are mainly observed after 14 minutes The peak of low polarity component it is very small or almost without.Specifically, the weight based on the AAMM extract, sesquiterpene lactone is about 0.001 (w/w) %, and volatile essential oil ingredient is about 0.01 (w/w) % or lower.Sesquiterpene lactone includes qinghaosu (artemisnin) ﹑ sweet wormwood Suan ﹑ arteannuin and qinghaosu (artemisitin).

Fig. 1 is to extract artemisia annua ethanol extract in the residue left after extracting artemisia annua with methylene chloride from extraction HPLC chromatogram.In Fig. 1, (A), (B) and (C) is respectively indicated by detecting 254nm, 320nm in ultraviolet light (UV) detector And 420nm wavelength and the chromatogram that obtains.In (C) in Fig. 1, pass through search and comparison reference (reference) UV light Spectrum, the low polarity component predominantly detected after 30 minutes are mainly confirmed to be carotenoid ingredient and chlorophyll content.

The HPLC analysis condition of the AAMM is as follows.

The equipment used: 1525 HPLC system of Waters

Column: RP C-18 HPLC column (4.6 × 150mm, 5 μm)

Solvent: (a) contain the acetonitrile of 0.02% trifluoroacetic acid (TFA)

(b) containing the water of 0.02%TFA

Flow phase composition: 0 minute (a) solvent: (b) solvent starts to dissolve out with the volume ratio of 10:90

0 to 30 minute (a) solvent multiplying power increases to 100% from 10%

30 minutes to 37 minutes (a) solvents are with 100% dissolution

Flow rate of mobile phase: 1.0mL/min

Detector Detection wavelength: ultraviolet light 254nm；320nm；420nm

3. the preparation of artemisia annua ethanol extract

The herb dried object of the artemisia annua of purchase China plantation, and it is put into extraction vessel after frittering the sample of 200g, then It impregnates and is cooled to room temperature after shaking up stirring at 45 DEG C 48 hours after addition 1.6L ethyl alcohol, be then repeated 2 times the process of filtering. Then, the extracting solution of filtering is put into reduced pressure machine, and under reduced pressure with 40 DEG C at evaporating completely solvent be concentrated.Knot Fruit, obtaining 23g artemisia annua ethanol extract (in hereinafter referred to as " AA "), (yield: 11.3%), artemisia annua ethanol extract includes Qinghaosu, sesquiterpene lactone and derived essential oil and the major part ingredient contained in the AAMM extract of described Section 2 preparation.

4. the preparation of the extract of different solvents

The artemisia annua that China's plantation or South Korea produce is used into methylene chloride (methylene chloride), ether respectively After (diethyl ether), ethyl acetate (ethyl acetate) acetone (acetone) or liquid CO 2 extract, Remove extracting solution and to the residue left with the method Jia Chun ﹑ ethyl alcohol according to described in Section 2 or 90% ethanol water into Row extracts to prepare analysis sample.All extractions are carried out according to method described in Section 1.But only faced by using super Boundary's extracting method carries out liquid CO 2 pretreatment to the artemisia annua of China's plantation, and (supercritical extraction method is this disclosure One of extracting method mentioned in book), removing, which is extracted, with ethyl alcohol using the method as described in Section 2 is obtained with liquid CO 2 Extracting solution after remaining residue, to prepare sample ready for use.

The analysis of the extract obtained using methanol, ethyl alcohol or 90% ethanol water is as a result, extract is shown and the 2nd The AAMM extract that section obtains has similar ingredient form.

Figure 13 show with methanol extract removing with methylene chloride extract in stay after the extracting solution that obtains of domestic artemisia annua Under residue extract HPLC result.In Figure 13, relative to the gross mass of extract, the calculated by peak area based on figure Compound 2, compound 3, compound 4, compound 5 and compound 6 content or ratio by mass be respectively 4.62%, 1.52%, 3.18%, 4.71%, 1.00%.

Figure 14 shows extract the extracting solution that removing is obtained with domestic artemisia annua in extracted by ether with ethyl alcohol after leave The result of the HPLC of the extract of residue.In Figure 14, relative to the gross mass of extract, the change of the calculated by peak area based on figure Close object 2, compound 3, compound 4, compound 5 and compound 6 content or ratio by mass be respectively 1.93%, 0.92%, 3.05%, 2.75%, 0.57%.

Figure 15 show extracted with ethyl alcohol remove be extracted with ethyl acetate in stay after the extracting solution that obtains of domestic artemisia annua Under residue extract HPLC result.In Figure 15, relative to the gross mass of extract, the calculated by peak area based on figure Compound 2, compound 3, compound 4, compound 5 and compound 6 content or ratio by mass be respectively 1.14%, 0.86%, 2.42%, 2.80%, 0.83%.

Figure 16 shows extract the extracting solution that removing is obtained with domestic artemisia annua in acetone extraction with ethyl alcohol after leave The result of the HPLC of the extract of residue.In Figure 16, relative to the gross mass of extract, the change of the calculated by peak area based on figure Close object 2, compound 3, compound 4, compound 5 and compound 6 content or ratio by mass be respectively 0.66%, 0.62%, 2.22%, 2.64%, 0.58%.

Figure 17 show with ethyl alcohol extract removing with liquid CO 2 extract in the extracting solution that obtains of domestic artemisia annua The result of the HPLC of the extract of the residue left afterwards.In Figure 17, relative to the gross mass of extract, the peak area based on figure The compound 2 of calculating, compound 3, compound 4, compound 5 and compound 6 content or ratio by mass be respectively 1.77%, 0.83%, 2.82%, 1.55%, 1.12%.

Figure 18 shows to be extracted after removing extracts the extracting solution that the artemisia annua that South Korea produces obtains with methylene chloride with methanol and stay Under residue extract HPLC result.In Figure 18, relative to the gross mass of extract, the calculated by peak area based on figure Compound 2, compound 3, compound 4, compound 5 and compound 6 content or ratio by mass be respectively 3.43%, 0.73%, 1.01%, 2.74%, 0.24%.

Figure 19 leaves after showing the extracting solution for extracting the artemisia annua acquisition that removing is produced with extracted by ether South Korea with ethyl alcohol The result of the HPLC of the extract of residue.In Figure 19, relative to the gross mass of extract, the change of the calculated by peak area based on figure Close object 2, compound 3, compound 4, compound 5 and compound 6 content or ratio by mass be respectively 2.96%, 1.14%, 2.41%, 2.84%, 1.37%.

Figure 20 is stayed after showing the extracting solution for extracting the artemisia annua acquisition that removing is extracted with ethyl acetate South Korea's production with ethyl alcohol Under residue extract HPLC result.In Figure 20, relative to the gross mass of extract, the calculated by peak area based on figure Compound 2, compound 3, compound 4, compound 5 and compound 6 content or ratio by mass be respectively 1.14%, 0.42%, 0.39%, 1.24%, 0.46%.

Figure 21 leaves after showing the extracting solution for extracting the artemisia annua acquisition that removing is produced with acetone extraction South Korea with ethyl alcohol The result of the HPLC of the extract of residue.In Figure 21, relative to the gross mass of extract, the change of the calculated by peak area based on figure Close object 2, compound 3, compound 4, compound 5 and compound 6 content or ratio by mass be respectively 1.20%, 0.25%, 0.93%, 1.58%, 1.46%.

According to Figure 13 to Figure 21, compound 2, compound 3, compound 4, compound 5 and compound 6 are relative to extract weight The content or ratio of amount be respectively 0.66% to 4.62%, 0.25% to 1.53%, 0.39% to 3.18%, 1.24% to 4.71%, 0.24% to 1.46%.

The extract contain the principle active component for the disclosure and by chemical formula 2, chemical formula 3, chemical formula 4, change There are five types of compounds for the institute that formula 5 and chemical formula 6 indicate.The four kinds of compounds of the disclosure indicated by chemical formula 2 to chemical formula 5 ((A) of the Figure 13 into Figure 21) is analyzed by following analysis method 1, the compound of the disclosure indicated by chemical formula 6 leads to It crosses following analysis method 2 and is analyzed ((B) of the Figure 13 into Figure 21).Using other solvents by extracting the extract packet obtained Include with extracted with methylene chloride after the similar ingredient of the ethanol extract that is obtained from residue.In addition, the extract tool There is the effect for the improvement inflammation skin for being similar to AAMM extract described in following tests example and its distillations.

Analysis method 1

Analysis instrument: HPLC (Young Lin, YL9100 series)

Detector: ELSD (temperature: 50 DEG C, gas: 2.5ln) (GmbH, ZAM 3000)

Analysis condition: 1mL/min, 87%A (MeCN)；13%B (D.W), isocratic 50 minutes

Column: YMC NH₂(5μm,150×4.6mm)

Column temperature: 30 DEG C

Injection rate: 10 μ L

Analysis method 2

Analysis instrument: HPLC (Waters)

Detector: 2996PDA detector (254nm) (waters)

Analysis condition: 1mL/min, A (MeCN containing 0.02%TFA)；B (D.W containing 0.02%TFA), 0 minute: 10% A- > 30 minute: 100%A

Column: Phenomenex Luna C18 (2) (5 μm, 150 × 4.6mm)

Column temperature: room temperature

Injection rate: 10 μ L

Therefore, as the methylene chloride of low polar solvent, ether, ethyl acetate, acetone, liquid CO 2 or it is similar its The solvent of structure may be used as the active solvent of the disclosure.

Embodiment 2: distillations are prepared from AAMM extract

The AAMM of the 16g in Section 2 description according to embodiment 1 is mentioned using the mixed solvent of ethyl alcohol, water or second alcohol and water Object is taken to be fractionated, to prepare the distillations of AAMM extract.It specifically, including with volume ratio is 2:8,4:6,6:4 and 10:0 The mixture or 1L ethyl alcohol of mixed second alcohol and water are used as fractionation solvent.

That is, expansion is used after the AAMM extract is contacted with the mixture of the second alcohol and water as the fractionation solvent Make 100g octadecyl-silica (ODS) resin of stationary phase and carry out the chromatography dissolved out with each solvent, obtains 4 kinds and evaporate Point object [referred to as ' AAMM- fraction 1'(is as the ethyl alcohol for being fractionated solvent: water -2:8,9.6g), ' AAMM- fraction 2'(ethyl alcohol: water -4: 6,2.4g), ' ethyl alcohol of the AAMM- fraction 3'(as fractionation solvent: water -6:4,1.3g) and ' AAMM- fraction 4'(ethyl alcohol: water - 10:0,1.5g).

After the method by test example 2 measures the efficiency of each distillations respectively, the more excellent fraction 1 of efficiency Mixed with fraction 4 according to respective yield with 9:1, come prepare ' AAMM- fraction 1+4' sample.

Fig. 2 and Fig. 3 is the chromatogram for showing the HPLC result of AAMM- fraction 1.HPLC condition in Fig. 2 and Fig. 3 respectively with Following conditions 1 and 2 are identical.In figure 2 and figure 3, (A) and (B) has used UV detector (254nm) and ELSD detector respectively.

As shown in (B) in Fig. 2, in the HPLC result according to condition 1, the ingredient that is indicated by chemical formula 1 to chemical formula 5 It is detected as main peak.In addition, as shown in figure 3, in the HPLC result according to condition 2, the chlorogenic acid that is indicated by chemical formula 6 (Chlorogenic acid) is detected as main peak.

Condition 1:

The equipment used: 9100 HPLC of Young Lin

Column: NH₂HPLC column (4.6 × 250mm, 5 μm)

Flow phase composition: acetonitrile: water volume ratio 80:20 (20 minutes)

Flow rate of mobile phase: 1.0ml/min

Detector: ultraviolet light 254nm ((A) in Fig. 2)；ELSD ((B) in Fig. 2)

Condition 2:

The equipment used: 9100 HPLC of Young Lin

Column: RP C-18 HPLC column (4.6 × 150mm, 5 μm)

Solvent: (a) containing the acetonitrile of 0.02%TFA

(b) containing the water of 0.02%TFA

Flow phase composition: 0 minute (a) solvent: (b) solvent starts to dissolve out with the volume ratio of 10:90

0 to 30 minute (a) solvent multiplying power increases to 100% from 10%

30 minutes to 37 minutes (a) solvents are with 100% dissolution

Flow rate of mobile phase: 1.0ml/min

Detector: ultraviolet light 254nm ((A) in Fig. 3)；ELSD ((B) in Fig. 3)

Fig. 4 and Fig. 5 is the chromatogram for showing the HPLC result of AAMM- fraction 4.HPLC condition in Fig. 4 and Fig. 5 respectively with Following conditions 3 and 4 are identical.In figures 4 and 5, (A) and (B) has used UV detector (420nm) and ELSD detector respectively.

As shown in (A) in Fig. 4, under UV detector (Detection wavelength 420nm), it is predicted as carotenoid or chlorophyll Ingredient be detected as main peak.As shown in (B) in Fig. 5, under ELSD detector, the α-flax indicated by chemical formula 7 is used Sour (α-linolenic acid) is used as main unsaturated fat sour component, and detects that similar fatty acid composition is main peak.

Condition 3:

Detector: in addition to using ultraviolet light 420nm ((A) in Fig. 4)；Except ELSD ((B) in Fig. 4), with 2 phase of condition Together.

Condition 4:

The equipment used: 9100 HPLC of Young Lin

Column: RP C-18 HPLC column (4.6 × 150mm, 5 μm)

Solvent: (a) containing the methanol of 0.02%TFA

(b) containing the water of 0.02%TFA

Flow phase composition: 0 minute to 10 minutes (a) solvent: (b) solvent starts to dissolve out with the volume ratio of 80:20

The ratio of solvent is increased, makes to become 100% (a) solvent

10 solvents are to 30 minutes (a) solvents with 100% dissolution

Flow rate of mobile phase: 1.0ml/min

Detector: ultraviolet light 420nm ((A) in Fig. 5)；ELSD ((B) in Fig. 5)

Embodiment 3: the separation of main component compound and Species estimation

1. including separation and the Species estimation of the ingredient in AAMM- fraction 1

After the AAMM- fraction 1 of embodiment 2 is concentrated under reduced pressure, separated by the HPLC of such as following separation method 1 Method is separated into small distillations.

As a result, isolating ornithine, pine camphor, mucilaginous fiber alcohol, chiro-inositol and glycine betaine.100mg is calculated as from dry weight AAMM- fraction 1 obtain 3mg ornithine, 29mg pine camphor, 4mg mucilaginous fiber alcohol, 7mg chiro-inositol and 3mg glycine betaine.In addition, In terms of the dry weight of AAMM- fraction 1, the fructose (fructose) that the rate of output is about 15% is separated.

Separation method 1

The equipment used: Waters 515pump

Column: YMC NH₂HPLC column (10 × 250mm, 10 μm)

Solvent: (a) acetonitrile

(b) water

Flow phase composition: 0 minute a) solvent: (b) solvent starts to dissolve out with the volume ratio of 80:20

It was dissolved out by 6 minutes to 60 minutes with same volume ratio

Flow rate of mobile phase: 4.0ml/min

Detector: RI

In addition, AAMM- fraction 1 is separated into small distillations by the HPLC separation method such as following separation method 2.

As a result, isolating the chlorogenic acid according to the disclosure.7mg chlorogenic acid is obtained from 100mg AAMM- fraction 1.

Separation method 2

The equipment used: YMC LC-Forte R

Column: Phenomenex Luna C-18 RP HPLC column (21.2 × 250mm, 10 μm)

Solvent: (a) containing the acetonitrile of 0.02%TFA

(b) containing the water of 0.02%TFA

Flow phase composition: 0 minute a) solvent: (b) solvent starts to dissolve out with the volume ratio of 10:90

0 to 40 minute (a) solvent multiplying power increases to 40% from 10%

Flow rate of mobile phase: 20.0ml/min

Detector: ultraviolet light 254nm；ELSD

NMR analysis and quality analysis are carried out to obtain data to the isolated compound, by by data and existing text It offers to make comparisons and confirms the structure.

Specifically, the molecular weight of ornithine (ornithine) is by using 1100 high performance liquid chromatography of Agilent-matter The MS measurement of amount spectrometer (HPLC-ESI-MS) is determined as 132, and by using nuclear magnetic resonance chemical analyser (Varian 500MHz NMR)¹H and¹³C-NMR spectrum (spectrum) analysis is estimated as indicating the ornithine of the structure with following formula 1.In addition, The data of NMR data and existing literature (J.C.MacDonald et al. .Can.J.Chem.1976,54,1126-1133) are carried out Compare and carrys out the Species estimation structure.

Chemical formula 1

White powder；Molecular formula C₅H₁₂N₂O₂；ESI-MS:m/z 132[M-H]^-；

¹H NMR(500MHz,D₂O): δ 3.94 (1H, dd, J=9.0,6.5Hz), 3.23 (1H, dt, J=11.5, 7.0Hz), 3.14 (1H, dt, J=11.5,7.0Hz), 2.16 (1H, m), 1.89 (1H, m), 1.82 (2H, m)¹³C NMR (125MHz,D₂O):δ174.6,61.1,45.9,28.8,23.6.

The molecular weight of pine camphor (pinitol) is by using 1100 high performance liquid chromatography of Agilent-mass spectrometer (HPLC-ESI-MS) MS measurement is determined as 194, and by using nuclear magnetic resonance chemical analyser (Varian 500MHz NMR)¹H and¹³C-NMR spectrum analysis is estimated as indicating the pine camphor of the structure with following formula 2.In addition, by NMR data and existing text Offer (M.Della greca et al. .Chem.Biodivers.2007,4,118-128；C.Sridhar et al. .Ind.J.Pharm.Sci.2006,68,111-114 data) are compared to carry out Species estimation to the structure.Change below The stereochemical structure of formula 2 shows relative stereochemistry (Relative stereochemistry) structure.

Chemical formula 2

White powder；Molecular formula C₇H₁₄O₆；ESI-MS:m/z 193[M-H]^-；

¹H NMR(500MHz,D₂O^*): δ 3.28 (1H, t, J=9.5Hz), 3.54 (3H, s), 3.59 (1H, t, J= 9.5Hz), 3.70 (1H, dd, J=9.5,3.0Hz), 3.75 (1H, dd, J=9.5,3.0Hz), 3.95 (1H, m), 3.94 (1H, m),^*D₂Oδ4.79；¹³C NMR(125MHz,D₂O):δ82.6,72.0,71.5,71.3,70.3,69.7,59.6.

The molecular weight of mucilaginous fiber alcohol (Muco-inositol) is by using 1100 high performance liquid chromatography of Agilent-matter The MS measurement of amount spectrometer (HPLC-ESI-MS) is determined as 180, and by using nuclear magnetic resonance chemical analyser (Varian 500MHz NMR)¹H and¹³C-NMR spectrum analysis is estimated as indicating the fiber 01 derivatives of symmetrical structure with following formula 3.In addition, this The NMR data of disclosed mucilaginous fiber alcohol it is similar to the NMR of existing literature (S.J.An gyal and L.Odier, Carbohydrate Res.1982,100,43-54), and the structure has been determined.The stereochemical structure of chemical formula 3 shows relatively three-dimensional Chemical structure.

Chemical formula 3

White powder；Molecular formula C₆H₁₂O₆；ESI-MS:m/z 179[M-H]^-；

¹H NMR(500MHz,D₂O^*): δ 3.95 (2H, br t, J=6.0Hz), 3.82 (4H, br d, J=6.0Hz),^*D₂O δ4.79；¹³C NMR(125MHz,D₂O):δ72.1,70.0.

The molecular weight of chiro-inositol (chiro-inositol) is by using 1100 high performance liquid chromatography of Agilent-quality The MS measurement of spectrometer (HPLC-ESI-MS) is determined as 180, and by using nuclear magnetic resonance chemical analyser (Varian 500MHz NMR)¹H and¹³C-NMR spectrum analysis is estimated as indicating the chiro-inositol of the structure with following formula 4.In addition, the disclosure The NMR data of chiro-inositol and the NMR of existing literature it is similar (R.Abraham et al. .Ma gn.Reson.Chem.2005,43, 611-624), and the structure has been determined.The stereochemical structure of chemical formula 4 shows relative stereochemistry.

Chemical formula 4

White powder；Molecular formula C₆H₁₂O₆；ESI-MS:m/z 179[M-H]^-；

¹H NMR(500MHz,D₂O^*): δ 3.96 (1H, br t, J=2.5Hz), 3.96 (1H, br t, J=2.5Hz), 3.69 (1H, br t, J=2.5Hz), 3.68 (1H, br t, J=2.5Hz), 3.53 (1H, t, J=6.5Hz), 3.51 (1H, t, J =6.5Hz)^*D₂Oδ4.79；¹³C NMR(125MHz,D₂O):δ72.6,71.5,70.3.

Glycine betaine (betaine) is by using nuclear magnetic resonance chemical analyser (Varian 500MHz NMR)¹H and¹³C-NMR Spectrum analysis is estimated as indicating the glycine betaine of the structure with following formula 5.In addition, by NMR data and existing literature (W.Gronwald et al. .Anal.Chem.2008,80,9288-9297；D.godzisz et al. .J.Mol.Struct.2002, Data 606,123-137) are compared to carry out Species estimation to the structure.

Chemical formula 5

White powder；¹H NMR(500MHz,D₂O^*):δ3.85(2H,s),3.21(6H,s)^*D₂Oδ4.79；¹³C NMR (125MHz,D₂O):δ169.1,[65.97,95.94,95.92],[53.13,53.16,53.19].

The molecular weight of chlorogenic acid is by using 1100 high performance liquid chromatography of Agilent-mass spectrometer (HPLC-ESI- MS MS measurement) is determined as 355, and by using nuclear magnetic resonance chemical analyser (Varian 500MHz NMR)¹H-NMR spectrum Analysis is estimated as indicating the chlorogenic acid of the structure with chemical formula 6.In addition, by NMR data and existing literature (T.Han et al. .Chem.Nat.Comp.2006,42,597-570 data) are compared to carry out Species estimation to the structure.

Chemical formula 6

White powder；Molecular formula C₁₆H₁₈O₉；ESI-MS:m/z 355[M+H]⁺；

¹H NMR(500MHz,CD₃OD): δ 7.56 (1H, d, J=16.0Hz), 7.05 (1H, d, J=1.5Hz), 6.95 (1H, dd, J=8.0,1.5Hz), 6.78 (1H, d, J=8.0Hz), 6.27 (1H, d, J=16.0Hz), 5.33 (1H, br td, J =9.5,3.0Hz), 4.17 (1H, br m), 3.73 (1H, br dd, J=9.5,2.5Hz), 2.18 (2H, br m), 2.04 (2H, br m).

The separation of the main component of 2.AAMM- fraction 4 and Species estimation

After the AAMM- fraction 4 of embodiment 2 is concentrated under reduced pressure, separated by the HPLC of such as following separation method Method is separated into small distillations.

As a result, isolating the alpha-linolenic acid according to the disclosure.25mg alpha-linolenic acid is obtained from 100mgAAMM- fraction 4.? During separating alpha-linolenic acid, in terms of the dry weight of fraction 4, it is further separated out the linoleic acid that yield is respectively 10% (linoleic acid) and palmitinic acid (palmitic acid).

Separation method

The equipment used: YMC LC-Forte R

Column: Phenomenex Luna C-18 RP HPLC column (21.2 × 250mm, 10 μm)

Solvent: (a) containing the methanol of 0.02%TFA

(b) containing the water of 0.02%TFA

Flow phase composition: 0 minute a) solvent: (b) solvent starts to dissolve out with the volume ratio of 90:10

Increase to 100% to 10 minutes multiplying powers by (a) solvent from 90% within 0 minute

It was dissolved out by 10 minutes to 40 minutes with same volume ratio

Flow rate of mobile phase: 20.0ml/min

Detector: ultraviolet light 210nm；ELSD

GS-MS analysis is carried out to isolated compound and NMR is analyzed to obtain data, by making data and available data Compare out and confirms the structure.

Specifically, the molecular weight of alpha-linolenic acid is measured really by using the MS of GS-MS (gas-chromatography-mass-synchrometer) It is set to 278, and by using nuclear magnetic resonance chemical analyser (Varian 500MHz NMR)¹H-NMR spectrum analysis is estimated as to change The alpha-linolenic acid of the expression of formula 7 structure.

Chemical formula 7

Alpha-linolenic acid

Colorless oil；Molecular formula C₁₈H₃₀O₂；ESI-MS:m/z 301[M+Na]⁺；

¹H NMR(500MHz,CDCl₃): δ 0.97 (3H, t, J=7.0Hz), 1.30-1.38 (8H, br m), 1.62 (2H, M), 2.06 (4H, m), 2.35 (2H, t, J=7.5Hz), 2.81 (4H, br m), 5.31-5.42 (6H, m)

Embodiment 4: by the preparation for the mixture that the compound of chemical formula 1 to chemical formula 7 forms

In order to illustrate having for the Artemisia annua extract prepared in embodiment 1 and the artemisia annua distillations prepared in embodiment 2 Ingredient is imitated, some or all of single compounds separated in embodiment 3 are formed again with similar with the yield of female distillations Mixing.

Specifically, preparation is by six kinds of changes that are separating in Section 1 in embodiment 3 and being indicated by chemical formula 1 to 6 Close object in four kinds or more compounds composition mixture, or embodiment 3 Section 2 separate alpha-linolenic acid, Asia The composition that oleic acid and palmitinic acid are mixed with same amount.In addition, by by alpha-linolenic acid or it is similar its unsaturated fatty acid or plant The compound of one of object saturated fatty acid or more is added to by selected from the six kinds of compounds indicated by chemical formula 1 to 6 In four kinds or more compounds composition mixture in prepare mixture.The composition and ratio of the mixture of preparation are illustrated in In the following table 1.

Table 1 is shown by the constituent of the mixture from the isolated compound combination of AAMM- fraction 1 and AAMM- fraction 4 And ratio of components.

[table 1]

Test example 1: by the dermatitis alleviation effects of two kinds of extract As AM and AAMM prepared by embodiment 1

The present inventor is for the artemisia annua dichloromethane extract (AAM) that obtains in embodiment 1 and extracts methylene chloride It is (every to have purchased NC/Nga atopic dermatitis mouse model for the dermatitis alleviation effects of the ethanol extract (AAMM) of residue afterwards The 6 week old male of weight 30g, central experimental animal) and it is placed in air-flowing type sterility test platform, and with Self-Feeding mark Quasi- solid feed is raised one week.All trial drugs are dissolved in a small amount of DMSO and are diluted to ultimate density with olive oil and are 0.2% uses.After the hair for removing back, on day 1, the 0.15% fluoro- 2,4-dinitrofluorobenzene of 1- (DNFB) is smeared, then The DNFB that 0.15% was smeared at the 5th day smeared 0.2% DNFB at the 9th day, DNFB is made to cause pruitus and idiocrasy inflammation Afterwards, it is treated with trial drug.Specifically, the 11st day, the 13rd day, the 15th day and the 17th day (i.e. 4 times in total), 2%DNFB and trial drug are smeared respectively.Hereafter, it uses¹⁸F- fluorodeoxyglucose (FDG) is used as tracer, is schemed with PET-CT As and tissue staining microscope photo compare DNFB inflammation induction group and AAM extract for treating group and AAMM extract for treating group State.

In order to obtain PET-CT image, other than water, all mouse fasting 12 hours.Pass through the tail intravenous administration of mouse 37MBq's (1.0mCi)¹⁸F-FDG, and after 60 minutes, with small animal position emission tomography (PET) scanner (small animal PET Scanner transmitting scanning (emission scan) 22 minutes) is carried out.

Fig. 6 shows in the residue after being extracted with artemisia annua dichloromethane extract (AAM) and methylene chloride and is extracted with ethyl alcohol Artemisia annua ethanol extract (AAMM) treatment rat inflammation part after use¹⁸PET-CT image of the F-FDG as tracer.? In Fig. 6, (A) and (B) respectively indicates transaxial plane (transaxial plane) image and coronal plane (coronal plane) Image.

Fig. 7 be show with Artemisia annua extract AAM and AAMM treatment rat inflammation part after organize variation it is micro- Mirror photo.In Fig. 7, (A), (B) and (C) is the histopathology dyed by haematoxylin and Yihong (H&E) respectively (histopathology) microscope photo (multiplying power × 200) and exempted from using the primary Keratin 10 polyclonal antibody of rabbit monoclonal Epidemic disease histochemistry microscope photo (multiplying power × 200), and the immune group using the primary substance of rabbit monoclonal (substance) P Weave chemistry microscope photo (multiplying power × 200).

PET-CT image condition:

The visual field (FOV) 38mm

Energy window 350 is to 650keV

Algorithm/reconstruction OSEM 2D/ repeatedly (2 times), subset: 8

Matrix size (mm) 128 × 128

22 minutes sweep times (minute)

60 minutes P.I time (minute)

RI/ activity (uCi)¹⁸F/1mCi

50 μm of focus size

X-ray detector pixel 2,048x 3,072

80kV, 500 μ A, 3,600 200 projection rotations, cone-beam filter

After PET-CT scanning, takes out the Skin Cell of mouse and fix cell with 4% paraformaldehyde (pH 7.4), by tissue Sample fritters into 5 μm of thickness and observes tissue with after haematoxylin and Yihong (H&E) dyeing with optical microscopy.

In addition, in order to carry out the observation of tissue change by immunohistochemical method (immunohistochemistry), By the histotomy of 10 μ m-thicks in phosphate buffered saline solution (PBS) in 3% fetal calf serum (fetal bovine serum) Culture 1 hour.In slice be added the primary Keratin 10 polyclonal antibody of rabbit monoclonal (1:100 dilution, Covance, the U.S.) or The primary Substance P antibody of rabbit monoclonal (1:100 dilution, Abcam, the U.S.) overnight incubation afterwards.Each sample is washed 3 in PBS It is secondary and cultivated together with Vectastain ABC reagent, then with 3,3'- diaminobenzidine, four hydrochloride (DAB) (Vector Laboratory, the U.S.) it is reacted.

Table 2 shows the radioactive isotope at drug therapy position¹⁸F-FDG absorbs density (UD).

[table 2]

Medication therapy groups	0.2%DNFB	AAM (0.2%)	AAMM (0.2%)
				UD	9.71	2.40	0.17

As shown in table 2 and Fig. 6 and Fig. 7, it can be confirmed and qinghaosu and arteannuin B are extracted and removed with methylene chloride Deng sesquiterpene lactone ingredient artemisia annua ethanol extract (AAMM) have it is significant mitigation DNFB induction idiocrasy inflammation and The efficiency of itch.

As shown in fig. 6, inflammation induction group (0.2%DNFB) and AAM treatment group (0.2%) are because of inflammation part¹⁸F-FDG's It accumulates into the cell and is led in PET-CT image and is marked as red (peripheral portion of bright part).On the contrary, being controlled in AAMM In the case where treatment group, there is no cellular inflammation reaction at medicament administration position, and be not observed due to¹⁸F-FDG's is intracellular It is red caused by accumulation, and only observe blue (part except the RED sector).¹⁸F-FDG PET due to it is normal Cell is hyperfunction compared to glucose metabolism, and inflammatory tissue cell or malignant cell is imaged, and in malignant tumour and inflammation Antidiastole, improved confirmation and to being widely used in the evaluation of the reaction for the treatment of.In these inflammation or malignant tumor tissue In FDG accumulate into the cell increase pass through be similar to glucose glucose transporter matter (glucose transporter Protein) it is moved to cell and by the hexokinase phosphorylation in cell, but it is different from glucose and as phosphorylation The FDG-6- phosphate of FDG generated because being no longer metabolized.In addition, FDG-6 phosphate is a kind of very strong polar substances, nothing Normal direction cell external diffusion causes to show high FDG accumulation.

As shown in (A) in Fig. 7, after being dyed to the slice for the tissue for causing inflammation with H&E, in the microscope of histotomy It observes in result, observes many navy blues on epidermis in inflammation induction group (0.2%DNFB) and AAM treatment group (0.2%) The stratum spinosum epidermidis of the elongated apperance of (darker) or round inflammatory cell.On the contrary, being observed in AAMM treatment group (0.2%) The epidermal tissue of relative clean.

(B) in Fig. 7 is the photo of the selectively keratin stain to the cutin in the tissue for causing inflammation, in inflammation In induction group (0.2%DNFB) and AAM treatment group (0.2%), in epidermis surface it can be observed that many dye dark-brown (compared with It is dark) keratin of layer.But it in AAMM treatment group (0.2%), is only observed on the epidermis of normal skin tissue's apperance Very thin keratin layer.

(C) in Fig. 7 is selectively to being known as inducing the object of inflammatory reaction and itch in the tissue for causing inflammation The photo of matter P dyeing can be seen in epidermis surface in inflammation induction group (0.2%DNFB) and AAM treatment group (0.2%) The Substance P for being much coloured to dark brown chromatograph (darker) is observed, on the contrary, in AAMM treatment group (0.2%), it has already been proven that just Normal skin histology apperance is almost without Substance P or seldom.

Test example 2: the dermatitis alleviation effects of the distillations from embodiment 2

In order to relatively and understand obtain in embodiment 1 extracted with methylene chloride after residue ethanol extract (AAMM) and in embodiment 14 kinds of distillations AAMM- fractions 1, AAMM- fraction 2, AAMM- fraction 3 and the AAMM- prepared from AAMM The efficiency of the dermatitis alleviation effects of fraction 4 and the dexamethasone as steroid antiphlogistic, has purchased NC/Nga idiocrasy Dermatitis mouse model (the 6 week old male of every weight 30g, central experimental animal) is simultaneously placed in air-flowing type sterility test Platform, and with standard Self-Feeding raising one week.Inflammation induces and drug-treated is carried out in a manner of identical with test example 1.It will Trial drug is dissolved in a small amount of DMSO and is diluted to final concentration of 0.2% with olive oil to use.By experimental animal model point Class is individually to smear the control group of olive oil alternatives to medication, induce negative control group by the dermatitis that 0.2%DNFB is handled (DNFB), the positive controls (dexamethasone) at position are induced, using 0.2%AAMM's in inflammation using 0.2% dexamethasone Comparative group (AAMM) and four kinds of distillations are applied to the medication therapy groups that inflammation induces position respectively with 0.2% dosage.? 11st day, the 13rd day, the 15th day and the 17th day (i.e. 4 times in total) smear 2%DNFB and comparative drug and trial drug respectively.So Afterwards, the Skin Cell of mouse is taken out in the same manner as example 1 and fixes cell with 4% paraformaldehyde (pH 7.4), it will After tissue sample fritters (section) into 5 μm of thickness and use haematoxylin and Yihong (H&E) dyeing, seen with optical microscopy Examine tissue.

In addition, the expression and distribution of collagen can be by picrosirius-reds (Picrosirius-Red) to show Micro mirror, which is observed, is coloured to red collagen in the histotomy at inflammation induction and drug therapy position.It is every in order to understand The degree of inflammation of a test group and improve situation, by photo compare DNFB inflammation induction group and control group, DNFB treatment group, Dexamethasone in treatment group, the state of AAMM treatment group and distillations treatment group.

Fig. 8 is with DNFB, dexamethasone, AAMM, AAMM- fraction 1, AAMM- fraction 2, AAMM- fraction 3 and AAMM- fraction 4 treatment rats inflammation part after organize variation by H&E dyeing show histopathology microscope photo (multiplying power × 200)。

As shown in figure 8, with microscope observation histotomy after the slice H&E for causing the tissue of inflammation is dyed As a result in, compared with normal tissue (control), in inflammation induction group (0.2%DNFB), many dyes are observed in epidermis surface At the prickle cell's disease (Acnathosis) and many of red thick keratin layer (darker layer) and elongated condition in epidermis Form, which collapses and is coloured to navy blue inflammatory response cell, in layer (continues many dots (dots) below the keratin layer of dyeing And the group of big Circles.On the contrary, in dexamethasone and AAMM treatment group, observe keratin in epidermis surface thinly The prickle cell's disease and inflammatory cell of distribution and minority, and inflammatory symptom improves significantly.In AAMM- fraction 1 or AAMM- fraction In 4 treatment groups, the navy blue prickle cell's disease for elongating apperance is observed in epidermis surface, although comparing dexamethasone and AAMM The efficiency for the treatment of group is weak, but keratin relatively unfertile land distribution and almost without inflammatory cell etc., compared AAMM- fraction 2 Or AAMM- fraction 3 shows relatively outstanding efficiency.On the one hand, observe that many inflammation are anti-in 2 treatment group of AAMM- fraction It answers, keratin is distributed relatively thin and seldom prickle cell's disease in 3 treatment group of AAMM- fraction, and inflammatory reaction is small, but It is to observe that collagen layer rupture is significant, and slightly weak efficiency.

Fig. 9 is with DNFB, dexamethasone, AAMM, AAMM- fraction 1, AAMM- fraction 2, AAMM- fraction 3 and AAMM- fraction Picro-Sirius red (Picrosirius Red) dyeing of collagen distribution after the inflammation part of 4 treatment rats in tissue change The microscope photo (multiplying power × 100) of histotomy.

As shown in figure 9, the state that the distribution of collagen is collapsed is observed in inflammation induction group (0.2%DNFB), and And fine and close collagen distribution can be confirmed in dexamethasone (0.2%) and AAMM (0.2%) treatment group.In addition, Also fine and close collagen distribution is observed in 4 treatment group of AAMM- fraction 1 or AAMM- fraction, on the contrary, controlling in AAMM- fraction 2 Slightly unsound collagen distribution or Anomalistic space in epidermis are observed in 3 treatment group for the treatment of group or AAMM- fraction Distribution, can be confirmed that the regular distribution of collagen is collapsed a bit.

Table 3 compares the artemisia annua ethanol extract of the embodiment 1 measured by Histological stain method and the fraction of embodiment 2 The physiological activity of each project of object, and showing will be by the tissue indexation of the micro- sem observation and each project of comparison Efficiency intensity.

[table 3]

Project	Control group	DNFB	DM	AAMM	Fraction 1	Fraction 2	Fraction 3	Fraction 4
									Hyperkeratinization (Hyperkeratosis)	0	3	1	1	1	2	1	1
Prickle cell's disease (Acanthosis)	0	3	1	0	2	3	1	1
									The quantity of inflammatory cell	0	3	0	0	0	3	1	1
Collagen	0	3	0	0	0	3	2	0

Score: without=0, slightly=1, medium=2, serious=3

Hyperkeratosis: cutin proliferation, Acanthosis: prickle cell's disease

The quantity of inflammatory cell: inflammatory cell quantity, collagen: collagen destruction

Control: DNFB does not treat noninflammatory group

The fluoro- 2,4-dinitrofluorobenzene of DNFB:0.2%1-, inflammation induce drug

DM:0.2% dexamethasone, positive controls

AAMM: in the ethanol extract (0.2%) to the residue after extraction methylene chloride prepared by embodiment 1

Fraction 1: in the highly polar distillations (0.2%) of 20% ethanol fractionation solvent prepared by embodiment 2, AAMM- fraction 1,

Fraction 2: in the middle polarity distillations (0.2%) of 40% ethanol fractionation solvent prepared by embodiment 2, AAMM- evaporates Divide 2,

Fraction 3: in the middle polarity distillations (0.2%) of 60% ethanol fractionation solvent prepared by embodiment 2, AAMM- evaporates Divide 3,

Fraction 4: in the low polarity distillations (0.2%) of 100% ethanol fractionation solvent prepared by embodiment 2, AAMM- fraction 4.

By result above, AAMM with shown under the same dosage of dexamethasone as steroid dermatitis treating medicine Out with the inflammation treatment efficiency of dexamethasone phase same level, the AAMM- fraction 1 and AAMM- fraction 4 prepared by AAMM extract exists Under the same dosage of dexamethasone, although slightly weaker than dexamethasone or AAMM, significant dermatitis therapeutic efficacy is shown.

Test example 3: the skin of the AA extract and the AAMM- fraction 1+4 fraction prepared by embodiment 2 that are prepared by embodiment 1 Skin inflammation alleviation effects

In order to confirm, the efficiency of AAMM- fraction 1 and AAMM- fraction 4 respectively is slightly weaker than as mother's extraction in test example 2 The reasons why efficiency of the AAMM of object, according to the yield obtained from AAMM extract by AAMM- fraction 1 and AAMM- fraction 4 with weight AAMM- fraction 1+4 is prepared than 9:1 mixing to be compared with the efficiency of dexamethasone and AAMM.In addition, in order to verify again The importance for preparing AAMM extract will may include AAMM extract and AAM extract in Section 3 preparation of embodiment 1 The efficiency of AA extract of most of ingredient be compared with the efficiency of AAMM extract.The concentration of all drugs is 0.2%, carrying out animal model in method identical with above-mentioned test example 1 ensures and drug therapy.It is true being treated to animal model It protects drug after two weeks, uses¹⁸F-FDG calculates use as tracerSpectrum animal imaging system (Spectrum Animal imaging system) intake (uptake) region of interest (region of interest, ROI) value, to compare The efficiency (table 4) of every kind of sample.In order to obtain PET-CT image, other than water, all mouse fasting 12 hours.Pass through mouse Tail intravenous administration 37MBq's (1.0mCi)¹⁸F-FDG, and after 60 minutes, it carries out image scanning 30 minutes.

Table 4 is shown according to usingThe chrysanthemum of the embodiment 3 of the intake zones values interested of spectrum animal imaging system The mixture (fraction 1+4, blending ratio 9:1) of wormwood artemisia ethanol extract (AA), the fraction 1 of embodiment 2 and fraction 4, and it is related The dermatitis of test specimen mitigates the comparison of efficiency (all drug concentrations are 0.2%).(unit: ROI value, × 10⁵)

[table 4]

Control group: DNFB does not treat noninflammatory group

The fluoro- 2,4- dinitrofluorobenzene of DNFB:0.2%1-, inflammation induce drug

DM:0.2% dexamethasone, positive controls

AA: in the ethanol extract (0.2%) that the dried object of artemisia annua prepared by embodiment 3 is directly extracted with ethyl alcohol

AAMM: in the ethanol extract (0.2%) to the residue after extraction methylene chloride prepared by embodiment 1

Fraction 1+4: by the highly polar distillations (AAMM- fraction 1) of the 20% ethanol fractionation solvent prepared in embodiment 2 and The sample (0.2%) that low polarity distillations (AAMM- fraction 4) are mixed with the ratio of 9:1

As shown in table 4, the ROI value of the intracellular accumulating level of the FDG in inflammation induction region is shown in dexamethasone Reduction more significant than DNFB inflammation induction group in treatment group and AAMM treatment group.On the contrary, AA extract is identified with weak efficiency, and It can reaffirm skin of the preparation method in the scytitis for being applied to improve such as artemisia annua of the AAMM extract of the disclosure It is extremely important in terms of the efficiency of skin disease.In addition, the efficiency of AAMM fraction 1+4 show the level of performance of AAMM extract and with AAMM fraction 1 and the mixing of AAMM fraction 4 come in use, confirmed the fact that show the outstanding efficiency of female extract.Therefore, according to The disclosure for preventing, improving or treating the composition of skin disease not as showing that a kind of mechanism of action is unification Object or single distillations are closed, but the substance of respective different mechanism of action acts on compoundly, to be judged as showing and ground The comparable excellent efficiency of Sai meter Song.

In addition, obtaining correspond to control group, DNFB in the same manner as in Example 1 to verify the credibility of ROI value Inflammation induction group, the mouse skin cell of AA extract for treating group and AAMM- fraction 1+4 treatment group and with 4% paraformaldehyde (pH 7.4) fixed cell, after tissue sample is frittered into 5 μm of thickness and is dyed with haematoxylin and Yihong (H&E), with optical microphotograph Sem observation tissue (top of Figure 10).In addition, the expression and distribution of collagen can pass through picrosirius-red (Picrosirius-Red) it is observed with microscope and is coloured to red in the histotomy at inflammation induction and drug therapy position Collagen (lower part of Figure 10).

As shown in the figure above Figure 10, in microscopical result after the slice dyeing to the tissue for causing inflammation, phase Phase is observed in epidermis surface in inflammation induction group (0.2%DNFB) and AA extract for treating group than normal tissue (control) Prickle cell's disease and inflammatory cell are observed to thicker keratin layer (color of epidermis outermost is deeper) and in epidermis.Phase Instead, in AAMM- fraction 1+4 treatment group, observe that the keratin distribution in epidermis surface is very thin, and it is thin to be shown with very little spine The inflammatory cell of born of the same parents' disease and very few.

As shown in the figure below Figure 10, red is being coloured to the collagen in the tissue for causing inflammation and collagen is shown In the result of albumen distribution, in inflammation induction group (0.2%DNFB) and AA extract for treating group, observe in superficial layer under The collagen state collapsed of distribution or the collagen in epidermis the consistency state that declines a lot, In AAMM- fraction 1+4 treatment group, fine and close collagen distribution is observed in epidermis.

Test example 4: the external autophagy induced activity of the mixture of the single compound of the disclosure prepared by embodiment 4

In order to reaffirm that the efficiency of fraction 1 and fraction 4 in test example 2 is slightly weaker than as female extract A AMM's The reasons why efficiency, and compared with the single compound that the disclosure is used alone, it is used in the form of complex composition to verify Validity, by single compound disclosed in embodiment 3 to the biology of the HaCaT cell as human keratinocytes strain Activity and the external autophagy of the mixture of each composition of the single compound of embodiment 3 disclosed in embodiment 4 are induced and are lived Property is compared.

HaCaT cell is in the Dole for being added to 10% fetal calf serum (FBS) and 0.1% antibiotic (penicillin, streptomysin) Culture is in shellfish section modified Eagle medium (DMEM, Hyclone, Utah, the U.S.) to be used to test.Cell is applied at 37 DEG C And it is cultivated in the 5% wet culture medium under carbon dioxide.

It is whether toxic in the cell that sample uses in test in order to be measured before measuring autophagy induced activity, it carries out It is measured using the cytotoxicity of MTT analysis.HaCaT cell is adjusted to 4 × 10 in 96 orifice plates⁴Cell/mL concentration is divided Strain is simultaneously cultivated 24 hours, with tentative adhesion and stability.After completing culture in 24 hours, by every kind of compound in culture solution Or the ultimate density of mixture is diluted to 10 μ g/mL, 50 μ g/mL and 100 μ g/mL, and supplies adherency and stable cell strain Culture 24 hours.After culture 24 hours, removal culture medium is added the MTT solution (0.5mg/mL in PBS) of 100 μ L and again 37 DEG C and 5% carbon dioxide under humidified incubator in carry out reaction 30 minutes until restore MTT.The first formed in each hole Za (formazan) crystallization is dissolved in the DMSO of 200 μ L, and uses the suction at microplate reader (Bio-Rad, the U.S.) measurement 570nm Luminosity, to measure the cell survival rate (Cell Viablity) of the non-treatment group of drug (negative control) comparison.

In result of the measurement for the MTT cytotoxicity of the test specimen of HaCaT cell strain, concentration used in test In all single compounds and mixture show 80% or more cell survival rate, and confirm that all test specimens all do not have There is significant cytotoxicity.

In order to measure autophagy induced activity, by the plant division of HaCaT cell on 60mm culture dish, and trained in the post-attachment It supports.It is 250nM by ultimate density is diluted in culture solution as the rapamycin of comparative drug after culture in 24 hours, and The ultimate density of every kind of compound or mixture is diluted to 1 μ g/mL or 10 μ g/mL in culture solution, and supply attachment is simultaneously Stable cell strain is cultivated 12 hours.Hereafter, it recycles the cell of culture and makes to dissolve after RIPA dissolution buffer is added, Protein isolate matter after 12,000rpm is centrifuged 30 minutes.Pass through bright orchid method (the Bradford) (Bio-Rad of Maas Laboratories, CA, the U.S.) isolated protein is quantified, and by quantitative protein with SDS-PA GE 12% Acrylamide gel electrophoresis is simultaneously transferred on nitrocellulose membrane.Nitrocellulose membrane and the anti-LC33 of first antibody are carried out instead at 4 DEG C After Ying Yitian, after being washed 3 times during 10 minutes with TBS-T, make the film and secondary antibody horseradish peroxidase (HRP) into Row reaction one hour.With wash cell with TBS-T solution after antibody expression after, with enhanced chemiluminescence (ECL) immunoblotting Reagent ((GE Healthcare, Cha Erfente-sage's -A Qieerxinhaodengceyan Giles-Archer lantern tests Britain) react and with Chemidoc system (Bio-Rad, it is beautiful State) confirmation, and be compared and divide as the LC3B expression degree of autophagy measurement marker and the ratio of LC3A and LC3B Analysis.

As shown in figure 11, the autophagy drug that the single compound of the disclosure is used as control group shows be equal to respectively Or (LC3B is living as autophagy lower than LC3B expression increase at the level of rapamycin (Rapamycin) (concentration 250nM) Property index), on the contrary, the unsaturated fatty acid and mixture as saturated fatty acid cetyl phosphoric acid that include alpha-linolenic acid AA-M4 composition (1 μ g/ml, 10 μ g/ml concentration), as the AA-M5 composition of chlorogenic acid and the mixture of unsaturated fatty acid (1 μ g/ml concentration), as ' ornithine '-' pine camphor '-' chiro-inositol '-' glycine betaine ' mixture AA-M6 composition (1 μ G/ml concentration), (1 μ g/ml, 10 μ g/ml is dense for the AA-M9 composition of mixing seven kinds of single compounds and AA-M10 composition Degree) show active 1.5 times of rapamycin or more of significant autophagy Skin Cell protective effect.The bioactivity AA-M9 and AA-M10 composition shows relatively excellent LC3B expression in sample, it is contemplated that the expression of LC3B and the table of LC3A Up to comparing, AA-M9 composition (1 μ g/ml concentration) and the induction of AA-M10 (1 μ g/ml and 10 μ g/ml concentration) composition can be confirmed The formation of most autophagosomes (autophagosome).

Test example 5: the dermatitis alleviation effects of the mixture of the single compound of the disclosure prepared by embodiment 4

In order to verify skin in the composition for improved body for showing relatively excellent external autophagy induced activity in test example 4 The efficiency of skin inflammation measures AA-M6 composition and AA- in the bioactivity sample in test example 4 with the DMSO of 0.3% concentration Efficiency of the M9 composition in the mouse model by DNFB induced skin inflammation.Using being shown in the mixture in the test example 4 The AA-M7 composition (0.3% concentration in DMSO) of the bioactivity of relatively weak degree is as comparative group and dexamethasone (in DMSO 0.15% concentration) it is used as positive control.In addition, control group (Control) without any solvent or drug therapy and only using The efficiency of the control group (Solvent Control) of DNFB and DMSO solvent treatment is also measured to compared with test group and true Recognize.

In order to measure the efficiency of sample, NC/Nga atopic dermatitis mouse model (6 week old of every weight 30g are had purchased Male, central experimental animal) and it is placed in air-flowing type sterility test platform, and with Self-Feeding raising one week.By all examinations It tests drug to be dissolved in a small amount of DMSO and make ultimate density 0.3% to be used in test, and as positive controls Dexamethasone is dissolved in DMSO and with the use of 0.15% concentration.Each control group and test group are made of 4 mouse.In order to DNFB is set to cause pruitus and atopic dermatitis, after the hair for removing back, on day 1, smearing is dissolved in acetone-olive oil The DNFB of 0.15% concentration of (volume ratio 3:1) in the mixed solvent, at the 5th day, after smearing 0.15% DNFB again, the 9th After it smears 0.2% DNFB, it is treated with trial drug, until the 30th day, after every two days smear 0.2% DNFB It is treated with trial drug.Then, the Skin Cell of mouse is taken out in the same manner as example 1 and with more than 4% Polyformaldehyde (pH 7.4) fixes cell, and tissue sample is frittered into 5 μm of thickness and is dyed using haematoxylin and Yihong (H&E) Afterwards, tissue (Figure 12 is observed with optical microscopy.Figure 12 is display AA-M6 composition and the treatment DNFB induction of AA-M9 composition With the result of microscope observation histotomy after inflammation part and H&E stain skin histotomy.

In Figure 12, curve (A), (B), (C), (D), (E) and (F) is shown respectively the mouse handled with 0.2%DNFB, applies Dexamethasone in treatment is used after smearing the mouse only handled with DMSO solvent after 0.2%DNFB, positive controls, smearing 0.2%DNFB Mouse, smear 0.2%DNFB after with 0.3%AA-M6 composition treat mouse, smear 0.2%DNFB after use 0.3%AA- The mouse and smear the mouse treated after 0.2%DNFB with 0.3%AA-M9 composition that M7 composition is treated.Figure 12 is shown often The pathological microscope photo of H&E stained tissue in four mouse of group respectively.

As shown in (A) and (B) in Figure 12, compare normal tissue (control), in inflammation induction group (0.2%DNFB) and In DNFB and DMSO processing group, observe in keratin layer and epidermis relatively thick in epidermis surface prickle cell's disease and and Inflammatory cell is in epidermis position with respect to navy blue lines or the aggregate of fleck.As indicated in fig. 12 c, as positive control In the dexamethasone in treatment group of group, observe that the keratin distribution in epidermis surface is very thin, almost without prickle cell's disease, and The inflammatory cell of only a few, it can be observed that the structural state that inflammation and itch turn very well.In trial drug group, such as scheme Shown in (D) (AA-M6 treatment group) and (E) (AA-M7 treatment group) in 12, dexamethasone in treatment group is compared, is observed thick Keratin layer and in prickle cell's disease form under, and weak efficiency is shown, on the contrary, as shown in (F) in Figure 12, in AA-M9 In treatment group, prickle cell's disease of relatively thin keratin layer and very little is observed, and show more excellent compared to AA-M6 and AA-M7 Elegant inflammatory symptom improvement effect.

Test example 6: the efficiency of the ethanol extract of the residue left after ethyl acetate extract and extraction ethyl acetate

According to Section 4 " preparation of the extract of 4. different solvents " of embodiment 1, the second of the artemisia annua of South Korea's production is obtained What the residue left after acetoacetic ester extract (being known as " AAE1 " below) and extraction ethyl acetate was extracted with 90% ethyl alcohol 90% ethanol extract (is known as " AAEM1 ") below.

Then, in the presence of AAE1 and AAEM1 extract, skin keratinocytes HaCaT cell strain is cultivated, and measure work For the level of the interleukin-1 ' beta ' (IL-1 β) of anti-inflammatory index.

It is whether toxic in the cell that sample uses in test in order to measure, carry out the cytotoxicity using MTT analysis Measurement.HaCaT cell is adjusted to 3 × 10 in 96 orifice plates⁴Cell/mL concentration is carried out plant division and is cultivated 24 hours, with tentative Adhesion and stability.After completing culture in 24 hours, is added and extracted with the concentration of 10 μ g/mL, 50 μ g/mL and 100 μ g/mL Object, and cultivated 24 hours in carbon dioxide incubator.After culture 24 hours, the MTT solution of 100mL is added in removal culture medium (0.5mg/mL in PBS) and reaction is carried out in the humidified incubator under 37 DEG C and 5% carbon dioxide again 1 hour until making MTT reduction.After removing MTT reagent, is dissolved and used at microplate reader (Bio-Rad, the U.S.) measurement 570nm with the DMSO of 200 μ L Absorbance, come measure the non-treatment group of drug (negative control) comparison cell survival rate.As a result, under concentration used in test All extracts show 80% or more cell survival rate, and confirm all not significant cell toxicant of all test specimens Property.

In order to measure the secretory volume of IL-1 β, by HaCaT cell with 6 × 10⁴/ mL plant division makes stabilisation 24 to 48 holes Hour.According to concentration processing extract and after a hour, activated with TNF-α (10ng/mL) and IFN-γ (10ng/mL) Afterwards, culture 24 hours is carried out in the humidified incubator under carbon dioxide.Culture solution is obtained, and is passed through according to the instruction of manufacturer ELISA kit carries out ELISA.Cytokine (Cytokine) secretory volume is under 450nm wavelength with microspectrophotometer quilt Analysis.

Figure 22 shows AAE1 and AAEM1 extract to the figure of the influence expressed of IL-1 β in cell.As shown in figure 22, AAEM1 Extract significant reduction IL-1 β compared with AAE1 extract is expressed and is had anti-inflammatory effect.In Figure 22, control (Ctrl) is Negative control simultaneously only uses culture medium, and TNF-a/INF-r is positive control and is added respectively with the concentration of 10ng/ml in the medium TNF-α/INF-r is added.

In addition, cultivating skin keratinocytes under autophagy inductive condition in the presence of AAE1 and AAEM1 extract HaCaT cell strain, and measure the level of the LC3 as autophagy marker.In test example 4, in addition to containing in the medium Have outside the extract of the 10 μ g/ml and in order to analyze, culture HaCAT cell carries out under the same conditions.The measurement of LC3 passes through Protein immunoblotting carries out.

Figure 23 shows AAE1 and AAEM1 extract to the figure of the influence expressed of LC3 in cell.As shown in figure 23, AAEM1 is mentioned Object significant increase LC3 compared with AAE1 extract is taken to express and there is cytoprotection.In Figure 23, control (Ctrl) is yin Property compares and only uses culture medium.Tub is shown for the reference value of tubulin measurement LC3 level.

The mode of invention

The one side of the disclosure provides a kind of method for separating Artemisia annua extract, which comprises plants artemisia annua Object (Artemisia annua) contacts with low polar solvent and by the constituents extraction in sweet wormwood plant body to the low polarity Step in solvent；The low polar solvent is removed from the mixture obtained after the contact come the step of obtaining residue； And the residue is contacted with hydrophilic solvent with the constituents extraction in the sweet wormwood plant is molten to the hydrophily The step of agent.

The whole or part of it of the sweet wormwood plant body can be used.Described a part can be aerial part or ground Lower part.The aerial part can be leaf, flower, fruit or combinations thereof.The under ground portion can be the stem of underground, root or its Combination.The sweet wormwood plant body can be dry.The sweet wormwood plant body can be from commercially available, or is adopting naturally It takes or cultivates to use.

The extract has the meaning for being used as crude extract (crude extract) in the art, but widely includes By the distillations for being further fractionated (fractionation).Therefore, the extract can be crude extract, distillations or its Combination.The crude extract is obtained by contacting artemisia annua with Extraction solvent, and without separating into containing special component.It is described Distillations refer to be obtained and being separated the substance containing special component to the crude extract.The extract or its fraction Object may include sweet wormwood plant body extract in the composition of the disclosure, its distillations or each distillations from childhood or it is mixed Object is closed, can only include single compound, or may include the compound as main component.The small distillations can be with It is obtained by having the ultrafiltration membrane of cut-off (cut-off) value.

In one embodiment, the separation can be impregnated, centrifugation, chromatography or combinations thereof carries out by filtering.It is described Chromatography can be ion exchange color according to various conditions (i.e. size, charge, hydrophobicity or affinity to be designed for separating) Spectrometry, affinity chromatography, exclusion chromatography, HPLC, high efficiency fluid chromatography, column chromatography, reversed-phase column chromatography method or combinations thereof. It, will be from described after separating and referring to the step of removing the low polar solvent and contact the sweet wormwood plant body with low polar solvent The sweet wormwood plant body left after extract component substance in the mixture of sweet wormwood plant body and low polar solvent, i.e. residue from It is separated in low polar solvent, and removes the low polar solvent of separation.

The extraction may include that the plant is incubated to a period of time in a solvent.The extraction can stirring or not It is carried out in the case where stirring, or may include heating.The incubation can carry out under room temperature to reflux temperature, or not It is carried out in the case where stirring.Incubation temperature can suitably be selected according to selected solvent.For example, it may be room temperature is to reflux temperature, 30 DEG C to reflux temperature, 40 DEG C to reflux temperature.The heating may include being heated to 50 DEG C, 60 DEG C, 70 DEG C, 80 DEG C or reflux temperature Degree.The heating can be heated to 50 DEG C to reflux temperature, 60 DEG C to reflux temperature, 70 DEG C to reflux temperature, 80 DEG C to times Temperature is flowed, or to reflux temperature.The extraction time can change according to selected temperature, can be 1 hour to 2 months, For example, 1 hour to 1 month, 1 hour to 15 days, 1 hour to 10 days, 1 hour to 5 days, 1 hour to 3 days, 1 hour to 2 days, 1 Hour was to 1 day, 5 hours to 1 month, 5 hours to 15 days, 5 hours to 10 days, 5 hours to 5 days, 5 hours to 3 days, 5 hours to 2 It, 5 hours to 1 day, 10 hours to 1 month, 10 hours to 15 days, 10 hours to 10 days, 10 hours to 5 days, 10 hours to 3 days Or 10 hours to 2 days.The extraction can be plant described in refluxing extraction in a solvent.The solvent can have based on institute State 1 times, 2 times, 5 times, 10 times or 15 times or more of volume of plant body weight.The solvent can have based on the plant 1 times to 15 times, 2 times to 15 times, 5 times to 15 times, 10 times to 15 times or about 15 times of volume of weight.The plant can be It dries in the cool.

Filtration method, hot water extraction, Soakage extraction, cold-maceration, Microwave Extraction, reflux cooling can be used in the extracting method The conventional method of this field of extraction, pressurised extraction, sub-critical extraction, means of supercritical extraction and ultrasonic wave extraction etc..For example, can be with It is Soakage extraction method.Soakage extraction can be temperature leaching or impregnate and can extract 1 time to 5 times at room temperature.The artemisia annua Plant can be contacted with 0.1 times of Extraction solvent to 10 times or 1 times to 6 times.Cold soaking Extracting temperature can be 20 DEG C to 40 DEG C. Temperature leaching or heating extraction temperature can be 40 DEG C to 100 DEG C.Cold soaking extraction time can be 24 hours to 120 hours, temperature leaching or Heating extraction temperature can be 0.5 hour to 48 hours.

The extraction can also include removing solvent in the extracting solution obtained by as such as the known method being concentrated under reduced pressure. The extraction can also include being prepared into dried extract for extract obtained by such as freeze-dried drying.It is described to depressurize Vacuum-concentrcted device or vacuum rotary evaporator can be used to carry out in lower concentration.In addition, dry can be is done under reduced pressure Dry, vacuum drying, fluidized drying, spray drying or freeze-dried.

The composition of the extract of sweet wormwood plant body including the disclosure includes the sesquiterpene lactone of low content It (sesquiterpene lactone) or does not include sesquiterpene lactone.Therefore, the low polar solvent can dissolve or extract sesquialter Terpene lactones, by separating low polar solvent in resulting mixture from after the step of extracting the sweet wormwood plant body ingredient, The sesquiterpene lactone can be removed from the sweet wormwood plant body extract.The sesquiterpene lactone refers to that carbon number is 15 And the structure including lactonic ring, classified according to derivative or the next of sesquiterpene lactone, the quantity of carbon can change.The sesquialter Terpene lactones includes germacranolide (Germacranolides), Heliangolides, guaianolide (Guaianolides), pseudo- guaianolide (Pesudoguaianolide), Hypocretenolides or Eudesmanolides。

The low polar solvent refers to including nonpolar solvent, and can refer to hydrophily on the contrary, when dielectric constant is less than 15, Low polarity or nonpolarity can be divided into, but not limited to this.

The sesquiterpene lactone is relatively soluble in low polar solvent or nonpolar solvent and dissolves out, and the low polar solvent can To include alkane, alkene, alkynes, cycloalkane, cycloolefin, ether, ester, ketone and the sulfoxide of linear chain or branched chain.Alkane, alkene, alkynes, Cycloalkane, cycloolefin, ether, ester, ketone or sulfoxide are respectively provided with unsubstituted or replaced by one or more substituent groups 1 to 12 A carbon atom, for example, 1 to 6,1 to 3,3 to 12,3 to 8 or 3 to 10 carbon atom.The solvent It is alkane, alkene or the alkynes of C1-C6 that is unsubstituted or being replaced by one or more substituent groups；It is unsubstituted or by one or more The cycloalkane or cycloolefin of C3 to C12, C5 to C12 or C5 to C10 that multiple substituent groups replace；It is unsubstituted or one or more A substituent group replaces and including C1 to C5 or C1 to the alkyl acetate of C3 alkyl；Or it is unsubstituted or taken by one or more For the ether or ketone of the C1-C6 that base replaces.The substituent group is halogen, can be selected from F, Cl, Br and I.The low polarity is molten Agent can be such as liquid CO 2, methane, ethane, isopropyl alkane, butane, pentane, isopentane, hexane, heptane, octane, stone Oily ether, hexamethylene, DMSO, acetonitrile, tetrahydrofuran, methyl acetate, ethyl acetate, isopropyl acetate, n- butyl acetate, acetone, Ether, t- butyl methyl ether, methyl ethyl ketone, methyl iso-butyl ketone (MIBK), acetonitrile, 1,2- dichloroethylene, methylene chloride, methylene chloride Or combinations thereof.

In addition, as the case where the sesquiterpene lactone, the group of the extract of the sweet wormwood plant body including the disclosure Close the derived essential oil or hardly include derived essential oil that object includes low content.Therefore, the extraction of the sweet wormwood plant body of the disclosure The composition of object includes or hardly including the sesquiterpene lactone of low content, derived essential oil and combinations thereof.

In the method for separating the Artemisia annua extract, the hydrophilic solvent (hydrophilic solvent) can Ornithine, pine camphor, mucilaginous fiber alcohol, chiro-inositol, glycine betaine, chlorogenic acid, alpha-linolenic acid, linoleic acid and palm fibre are selected from dissolution At least one of palmitic acid acid.In the extract or its distillations of the sweet wormwood plant body, it is dissolved in the hydrophilic solvent And dissolve out compound may include compound of following formula 1 to 6 or derivatives thereof, stereoisomer or its pharmaceutically may be used Four kinds or more in the salt form of receiving, can also include compound or derivatives thereof of chemical formula 7, stereoisomer or Its pharmaceutically acceptable salt form.That is, the hydrophilic solvent, which can be, can dissolve selected from ornithine, pine camphor, cement Four kinds or more of compound in inositol, chiro-inositol, glycine betaine and chlorogenic acid, can also dissolve selected from α-flax One of acid, linoleic acid and palmitinic acid or more.

Chemical formula 1

Chemical formula 2

Chemical formula 3

Chemical formula 4

Chemical formula 5

Chemical formula 6

Chemical formula 7

The compound of the chemical formula 1 is properly termed as ornithine, and the compound of chemical formula 2 is properly termed as pine camphor, chemical formula 3 Compound be properly termed as mucilaginous fiber alcohol, the compound of chemical formula 4 is properly termed as chiro-inositol, and the compound of chemical formula 5 can With referred to as glycine betaine, the compound of chemical formula 6 is properly termed as chlorogenic acid and the compound of chemical formula 7 is properly termed as α-flax Acid.

The hydrophilic solvent can be extracted by such as water, alcohol or combinations thereof.The alcohol can be lower alcohol, and can be Compound of the C1 to C3, C1 with one or more-OH bases to C4, C1 to C5 or C1 to C6.In one embodiment, institute It states C1 and can be methanol, ethyl alcohol, propyl alcohol, isopropanol, butanol, sec-butyl alcohol, isobutanol, tert-butyl alcohol or combinations thereof to C4 alcohol.

In one embodiment, the method can also include being carried out by contacting the extract with fractionation solvent The step of fractionation.In one embodiment, the fractionation may include contacting the extract with chromatography media and in conjunction with next With the step of being fractionated solvent dissolution.

The fractionation solvent can be hydrophilic solvent, and in one embodiment, the hydrophilic solvent can be rudimentary Alcohol or combinations thereof, the lower alcohol can be the C1 to C3, C1 to C4, C1 to C5 or C1 with one or more-OH bases extremely The compound of C6.In one embodiment, the C1 can be methanol, ethyl alcohol, propyl alcohol, isopropanol, butanol, Zhong Ding to C4 alcohol Alcohol, isobutanol, tert-butyl alcohol or combinations thereof.

Relative to the total volume of fractionation solvent, the fractionation solvent has 0 percent by volume to 100 percents by volume Alcohol, therefore, alcohol: the volume ratio of water can be 0:10,1:9,2:8,3:7,4:6,5:5,6:4,7:3,8:2,1:9 or 0:10.? In the volume ratio, when ratio is " 0 ", expression is pure water or alcohol.For example, when alcohol: when water is 2:8, can claim alcohol is 20 bodies Product percentage.In one embodiment, the total volume based on fractionation solvent with 20 percents by volume or more and can be less than 40 Percent by volume, 40 percents by volume or more and less than 60 percents by volume, 60 percents by volume or more and be less than 100 bodies Product percentage and the dissolution of the alcohol of 100 percents by volume, thus, for example, the distillations by fractionation can be with including Total volume based on fractionation solvent is 20 percents by volume, 40 percents by volume, 60 percents by volume or 100 percents by volume The distillations that the solvent fractionation of alcohol obtains.In one embodiment, the alcohol can be lower alcohol, and the lower alcohol can be tool There is compound of the C1 to C3, C1 of one or more OH bases to C4, C1 to C5 or C1 to C6.In one embodiment, described Alcohol can be ethyl alcohol.

Another aspect of the present disclosure is provided including the Huang according to prepared by the method for the extract for separating the artemisia annua The composition for being used to prevent, improve or treat skin disease of flower wormwood artemisia plant matter extracts, distillations or its isolated compound.

It include using sesquiterpene lactone can be dissolved according to the sweet wormwood plant body extract that disclosed method is extracted The step of low polarity or nonpolar solvent carry out extracting and developing and remove the sesquiterpene lactone, therefore, the artemisia annua of the disclosure plant Object Extraction object includes the sesquiterpene lactone of low content or does not include sesquiterpene lactone.According to used in disclosed method The low polarity or nonpolar solvent can also dissolve derived essential oil, therefore, can be with as the case where the sesquiterpene lactone It extracts and separates from the extract of the disclosure and remove derived essential oil.Therefore, sweet wormwood plant body extract, the fraction of the disclosure Object or its small distillations include the sesquiterpene lactone of low content, derived essential oil and combinations thereof or do not include sesquiterpene lactone, essential oil Ingredient and combinations thereof.In one embodiment, the composition of the disclosure includes the sesquiterpene lactone of following weight ratio (w/w): 1.00% to 0.001%, 0.80% to 0.001%, 0.60% to 0.001%, 0.50% to 0.001%, 0.40% to 0.001%, 0.30% to 0.001%, 0.20% to 0.001%, 0.10% to 0.001%, 0.08% to 0.001%, 0.06% to 0.001%, 0.05% to 0.001%, 0.04% to 0.001%, 0.03% to 0.001%, 0.02% to 0.001%, 0.01% to 0.001%, ﹑ 0.1% or lower, 0.05% or lower, 0.01% or lower, 0.005% or lower Or 0.001% or lower.In one embodiment, the composition of the disclosure includes the derived essential oil of following weight ratio (w/w): 1.00% to 0.01%, 0.80% to 0.01%, 0.60% to 0.01%, 0.50% to 0.01%, 0.40% to 0.01%, 0.30% to 0.01%, 0.20% to 0.01%, 0.10% to 0.01%, 0.08% to 0.01%, 0.06% to 0.01%, 0.05% to 0.01%, 0.04% to 0.01%, 0.03% to 0.01%, 0.02% to 0.01%, 0.1% or lower, 0.05% or lower, 0.01% or lower, 0.005% or lower or 0.001% or lower.The derived essential oil can be volatilization Property derived essential oil.

In one embodiment, the extract of the disclosure may include selected from ornithine, pine camphor, ﹑ mucilaginous fiber alcohol, hand Property inositol, ﹑ glycine betaine and chlorogenic acid in four kinds or more of compound, or can also include be selected from linoleic acid, palm fibre The compound of one of palmitic acid acid and alpha-linolenic acid or more.

The combination of the compound may include in distillations, and the distillations include based on fractionation solvent by using 20 percents by volume of total volume or more and less than 40 percents by volume, 40 percents by volume or more and less than 60 volumes hundred Point ratio, 60 percents by volume or more and the fractionation solvent fractionation less than the 100 volume basis when alcohol of 100 percents by volume obtain It arrives.By with including 20 percents by volume of total volume or more based on fractionation solvent and less than 40 percents by volume, 40 bodies Accumulate percentage or more and less than 60 percents by volume, 60 percents by volume or more and less than the alcohol of 100 percents by volume The distillations that fractionation solvent fractionation obtains may include ornithine, pine camphor, mucilaginous fiber alcohol, chiro-inositol, glycine betaine, chlorogenic acid Or combinations thereof be used as main component, still, the distillations obtained by the fractionation solvent fractionation of the alcohol of 100 percents by volume can be with It is used as main component including linoleic acid, palmitinic acid, alpha-linolenic acid or combinations thereof, due to including every kind of change in each distillations The content for closing object can be different, and every kind of distillations stir or mix in the proper ratio to reach optimal pharmacological effect, And it is varied as desired composition and ratio.For example, when passing through 20 percents by volume of total volume or more based on fractionation solvent And less than 40 percents by volume, 40 percents by volume or more and less than 60 percents by volume, 60 percents by volume or more and The distillations that fractionation solvent fractionation less than the 100 volume basis when alcohol of 100 percents by volume obtains are hereinafter referred to as first When distillations, the second distillations, third distillations and four distillations, the composition can be the first distillations and second evaporate Divide the mixture of object；The mixture of first distillations and third distillations；The mixture of first distillations and the 4th distillations；The The mixture of two distillations and third distillations；The mixture or third distillations and the 4th of second distillations and the 4th distillations The mixture of distillations；The mixture of first distillations, the second distillations and third distillations；First distillations, third fraction The mixture of object and the 4th distillations；Or the mixture of second distillations, third distillations and the 4th distillations；Or first fraction Object, the second distillations, third distillations and the 4th distillations mixture.When mixing two kinds of distillations, two kinds of distillations Weight ratio can be 1:9,2:8,3:7,4:6,5:5, ﹑ 6:4,7:3,8:2 or 1:9, heavy when three kinds of distillations of the mixing Amount than can be 1:1:8,1:2:7,1:3:6,1:4:5,1:5:4,1:6:3,1:7:2,1:8:1,2:1:7,2:2:6,2:3:5, 2:4:4、2:5:3、2:6:1、2:7:1、3:1:6、3:2:5、3:3:4、3:4:3、3:5:2、3:6:1、4:1:5、4:2:4、4:3: 3,4:4:2,4:5:1,5:1:4,5:2:3,5:3:2,5:4:1,6:1:3,6:2:2,6:3:1,7:1:2,7:2:1 or 8:1:1 are mixed When closing four kinds of distillations its weight ratio can be 1:1:1:7,1:2:1:6,1:3:1:5,1:4:1:4,1:5:1:3,1:6:1:2, 1:7:1:1、1:1:2:6、1:1:3:5、1:1:4:4、1:1:5:3、1:1:6:2、1:1:7:1、2:1:1:6、2:2:1:5、2:3: 1:4、2:4:1:3、2:5:1:2、2:6:1:1、2:1:2:5、2:1:3:4、2:1:4:3、2:1:5:2、2:1:6:1、3:1:1:5、 3:2:1:4、3:3:1:3、3:4:1:2、3:5:1:1、3:1:2:4、3:1:3:3、3:1:4:2、3:1:5:1、4:1:1:4、4:2: 1:3、4:3:1:2、4:4:1:1、4:1:2:3、4:1:3:2、4:1:4:1、5:1:1:3、5:2:1:2、5:3:1:1、5:1:2:2、 5:1:3:1,6:1:1:2,6:2:1:1,6:1:2:1 or 7:1:1:1.

In one embodiment, when composition include selected from ornithine, pine camphor, cement inositol (or mucilaginous fiber alcohol), Four kinds or more compounds in chiro-inositol, glycine betaine and chlorogenic acid and it is selected from linoleic acid, palmitinic acid and alpha-linolenic acid One of or more compound combination when, composition can be in the extract based on fractionation solvent total volume 20 percents by volume or more and the distillations being fractionated less than the alcoholic solvent of 40 percents by volume and 100 percents by volume The alcoholic solvent mixing or combination of the distillations that are fractionated.Therefore, the composition of the disclosure may include individual distillations It or may include the combination for two kinds of distillations that weight ratio is 1:9,2:8,3:7,4:6,5:5,6:4,7:3,8:2.At one In embodiment, the composition of the disclosure may include the combination for two kinds of distillations that weight ratio is 9:1.

The composition of the disclosure can be used for treating, ameliorating or preventing skin disease.In one embodiment, the skin Disease can be inflammatory skin diseases.In addition, the extract of the sweet wormwood plant body, its distillations or by its isolated change Inflammatory reaction can be inhibited by closing object.Therefore, the extract of the sweet wormwood plant body in the composition, its distillations or by its list From compound may include inhibit induce skin disease inflammatory reaction or restore wound effective quantity.The effective quantity can be with It is suitably selected according to the cell of those skilled in the art's selection or individual.The effective quantity can be every 1,000mg composition 0.1mg to 1000mg, for example, 0.1mg is to 500mg, 0.1mg to 100mg, 0.1mg to 50mg, 0.1mg to 25mg, 1mg to 1, 000mg, 1mg to 500mg, 1mg to 100mg, 1mg to 50mg, 1mg to 25mg, 5mg to 1000mg, 5mg to 500mg, 5mg extremely 100mg, 5mg to 50mg, 5mg to 25mg, 10mg to 1000mg, 10mg to 500mg, 10mg to 100mg, 10mg to 50mg or 10mg to 25mg.The composition can be used in the external or cell in individual inhibiting inflammatory reaction.Cell in individual Such as inflammatory reaction can be located to carry out or it is expected that the position that will do it, or allow disease from appearance by inflammatory reaction The position that property is observed.

In one embodiment, the skin disease can be dermatitis.The skin disease may include such as skin The diseases such as itch, nettle rash, idiocrasy, dry atopic dermatitis, contact atopic dermatitis.Pruitus can refer to because by insect bites Or skin may sensitivity response specific contactant and be stimulated, or because of idiocrasy, dermatitis, psoriasis, infection, stemness Skin or skin aging make cutaneous nerve faintly by lasting stimulation, make to occur that the symptom for wanting to scratch can not be endured.The nettle Rash can refer to fash caused by the reaction of the allergic inflammation as caused by strong stress, ﹑ food or medicine or anaphylactogen.The spy It is directly contacted even if answering property can refer to without anaphylactogen, body is sensitively excessively initiated immune response and caused with fash, eczema With the allergic inflammatory diseases of serious pruritus.The dry atopic dermatitis can refer to causing the dry of many skin keratins Dry disease becomes serious and deteriorates into xerotic eczema or the dermatitis symptom of senile eczema.The neurodermatitis can refer to It is scratched since neuroticism makes minimal irritation that can feel serious itch, cuticula and wrinkle of skin is made to thicken and cause itch Disease is serious and continues to scratch, and result deteriorates into dermatitis such as eczemas.The contact atopic dermatitis, which can refer to work as, is exposed to anaphylactogen Or the symptom of the local fash or stimulation and itch occurred when particular stimulation source.

The skin disease specifically can be cutaneous pruritus, prickly heat, seborrheic skin inflammation, contact atopic dermatitis, stripping De- atopic dermatitis, ichthyosis, spiradenitis, impetigo, atopic dermatitis, drug allergy, diabetic skin are scorching, have dry skin Disease, dermatodyschroia disease, bacterial cutaneous inflammation, Fungal Skin inflammation, varicella, pernio, fiery sore, eczema, edema, sudden fash, Cholinergic urticaria, epifolliculitis, acne, pore seborrheic keratosis, wart, actinic keratosis, miliaria, psoriasis, Han Senshi Disease, Behcet's disease, bedsore, keloid disease, affected part edema, perionychia, dystrophia unguium, finger tip sore, anaphylaxis skin Skin inflammation, lichen simplex chronicus, dermapostasis, neurodermatitis or combinations thereof.

On the other hand it includes selected from ornithine, ﹑ pine camphor, mucilaginous fiber alcohol, chiro-inositol, glycine betaine and green that the disclosure provides The composition of four kinds or more compounds in ortho acid.

The composition may include in the extract or distillations obtained from sweet wormwood plant body, and may include for By the isolated compound of the small fraction of the distillations or further technique, the compound can be alloisomerism The form of body, its solvate its pharmaceutically acceptable salt.The composition may include four kinds of compounds, five kinds of compounds Or six kinds of compounds.Four kinds of compounds can be ornithine, pine camphor, mucilaginous fiber alcohol and chiro-inositol；Ornithine, pine Alcohol, mucilaginous fiber alcohol and glycine betaine；Ornithine, ﹑ pine camphor, mucilaginous fiber alcohol and chlorogenic acid；Ornithine, pine camphor, chiro-inositol and Glycine betaine；Ornithine, pine camphor, chiro-inositol and chlorogenic acid；Ornithine, pine camphor, glycine betaine and chlorogenic acid；Ornithine, cement are fine Tie up alcohol, chiro-inositol and glycine betaine；Ornithine, mucilaginous fiber alcohol, chiro-inositol and chlorogenic acid；Ornithine, mucilaginous fiber alcohol, sweet tea Dish alkali and chlorogenic acid；Ornithine, chiro-inositol, glycine betaine and chlorogenic acid；Pine camphor, mucilaginous fiber alcohol, chiro-inositol and glycine betaine； Pine camphor, mucilaginous fiber alcohol, chiro-inositol and chlorogenic acid；Pine camphor, mucilaginous fiber alcohol, glycine betaine and chlorogenic acid；Pine camphor, chiral flesh Alcohol, glycine betaine and chlorogenic acid；Or mucilaginous fiber alcohol, chiro-inositol, glycine betaine and chlorogenic acid.Five kinds of compounds can be Ornithine, pine camphor, mucilaginous fiber alcohol, chiro-inositol and glycine betaine；Ornithine, pine camphor, mucilaginous fiber alcohol, chiro-inositol and green original Acid；Ornithine, mucilaginous fiber alcohol, chiro-inositol, glycine betaine and chlorogenic acid；Ornithine, pine camphor, chiro-inositol, glycine betaine and green Ortho acid；Ornithine, pine camphor, mucilaginous fiber alcohol, glycine betaine and chlorogenic acid；Or pine camphor, mucilaginous fiber alcohol, chiro-inositol, beet Alkali and chlorogenic acid.The composition include in ornithine, pine camphor, mucilaginous fiber alcohol, chiro-inositol, glycine betaine and chlorogenic acid extremely Few one kind.It may include 1 to 5:4 to 35:1 to 8:2 to 16:2 to 20:2 to 16 when six kinds of compounds include in the composition Weight ratio.The composition include in ornithine, pine camphor, mucilaginous fiber alcohol, chiro-inositol, glycine betaine and chlorogenic acid at least When a kind of, these compounds respectively include into 0.1 (w/w) % to 4 (w/w) %, 0.6 (w/w) % to 25 (w/ based on total weight W) %, 0.2 (w/w) % to 8 (w/w) %, 0.3 (w/w) % to 16 (w/w) %, 0.3 (w/w) % to 24 (w/w) % and 0.2 (w/ W) % to 12 (w/w) %.

In one embodiment, the composition of the disclosure may include selected from ornithine, ﹑ pine camphor, mucilaginous fiber alcohol, chirality Four kinds or more compounds in inositol, glycine betaine and chlorogenic acid and one in linoleic acid, palmitinic acid and alpha-linolenic acid The another plus combination of kind or more compound.Therefore, the composition of the disclosure may include fine selected from ornithine, pine camphor, cement Tie up alcohol, chiro-inositol, glycine betaine and four kinds or more of compound in chlorogenic acid and linoleic acid, palmitinic acid, alpha-linolenic acid, Linoleic acid and palmitinic acid, linoleic acid and alpha-linolenic acid, palmitinic acid and alpha-linolenic acid or linoleic acid, palmitinic acid and alpha-linolenic acid Another plus combination.The composition can include alpha-linolenic acid, linoleic acid and palm with 1 to 30:1 to 15:1 to 15 weight ratio Acid.The composition may include 1 (w/w) % to 30 (w/w) % for being based respectively on total weight, 1 (w/w) % to 15 (w/w) % And 1 (w/w) % to 15 (w/w) % alpha-linolenic acid linoleic acid and palmitinic acid.

The combination may include simply mixing in a solvent, mix, mixed melting, dissolution, suspend or colloid, when applying When with the composition, the compound simultaneously and respectively individually or can be applied in turn.

The composition of the disclosure can be the extract including the artemisia annua (Artemisia annua) plant, or Its distillations and pharmaceutical composition from its isolated compound or its pharmaceutically acceptable salt.In addition, the combination of the disclosure Object may include medicament or the preparation of the form of pharmaceutically acceptable carrier with by the extract of the sweet wormwood plant body, or Its distillations and individual is applied to from its isolated compound.Term " pharmacy " means with pharmacological activity, in addition to being used as In the narrow sense except the pharmaceutical composition for treating disease, there are also include all pharmacological activity for using disclosure composition Purpose purposes.Therefore, the composition can be the functional food for skin health, cosmetics, the outer product of medicine Or the composition of skin preparations for extenal use.

" pharmaceutically acceptable salt " with to patient's relative nontoxic and it is harmless and with useful effect concentration to this Side effect caused by salt does not damage the beneficial efficiency of the compound in the composition of the disclosure.These salt inorganic acid and can will have Machine acid is used as free acid, and hydrochloric acid can be used, bromic acid, nitric acid, sulfuric acid, cross chloric acid and phosphoric acid etc. as inorganic acid.As organic acid, Can be used citric acid, acetic acid, lactic acid, maleic acid, fumaric acid, gluconic acid, methanesulfonic acid, Glycolic acid, gluconic acid, succinic acid, Tartaric acid, galacturonic acid flutter acid, glutamic acid, aspartic acid, oxalic acid, (D) or (L) malic acid, maleic acid, methanesulfonic acid, second sulphur Acid, 4- toluenesulfonic acid, salicylic acid, citric acid, benzoic acid or malonic acid etc..In addition, these salt include alkali metal salt (such as sodium salt and Sylvite etc.) and alkali salt (such as calcium salt and magnesium salts) etc..For example, as acid-addition salts, it may include acetate, aspartic acid Salt, benzoate, benzene sulfonate, bicarbonate/carbonate, disulfate/sulfate, borate, camsilate, citric acid Salt, ethanedisulphonate, esilate, formates, fumarate, gluceptate, gluconate, glucuronate, hexafluoro Phosphate, 0- (4- oxybenzene formyl) benzoate, hydrochloride/chloride, hydrobromate/bromide, hydriodate/iodide, Isethionate, lactate, malate, maleate, malonate, mesylate, Methylsulfate, naphthoate, 2- Naphthoate, nicotinate, nitrate, Orotate, oxalates, palmitate, pamoate, phosphate/phosphor acid monohydric salt/di(2-ethylhexyl)phosphate Hydrogen salt, sugar lime, stearate, succinate, tartrate, toluene fulfonate, trifluoroacetate, aluminium, arginine, benzyl star, Calcium, choline, diethylamine, diethanol amine, glycine, lysine, magnesium, meglumine, ethanol amine, potassium, sodium, tromethamine and zinc salt Deng or in which hydrochloride or trifluoroacetate.

On the other hand the disclosure provides includes the steps that for the composition of the disclosure being applied to individual and is used to prevent or control The method for treating the skin disease of individual.

The individual can be mammal, such as people, ox, horse, pig, dog, sheep, goat or cat.For example, the individual It can be the mankind.The compound of the disclosure can be according to the age of patient, weight, gender, application to effective amount of application of human body State, health status and disease degree and change.

The application can be taken orally, or with various preparations be administered with for vein, abdominal cavity, intradermal, subcutaneous, epithelium or The non-oral administration of intramuscular delivery etc., and in the case where carrying out formulation, it (such as, is filled out using usually used diluent Fill agent, incremental agent, adhesive, wetting agent, disintegrating agent and surfactant etc.) or excipient prepare.

Solid pharmaceutical preparation for oral administration may include tablet, pill, powder, granule, capsule and tablet (troche), and this solid pharmaceutical preparation can be by by the compound of one or more of disclosure and at least one figuration Agent (such as, starch, calcium carbonate, sucrose (sucrose) or lactose (lactose) or gelatin etc.) mixes to prepare.In addition to simple Excipient outside, can also use as magnesium stearate talcum lubricant.As the liquid preparation of oral administration, can be used outstanding Floating agent, interior liquor, finish or syrup (syrup) agent etc., and in addition to common simple diluent such as water and atoleine It outside, can also include various excipient such as wetting agent, sweetener, aromatic and preservative etc..

Liquid preparation for non-oral administration may include gone out the aqueous solution of bacterium, non-aqueous solvent, suspending agent, finish, Freeze-dried preparation and suppository etc..As non-aqueous solvent and suspending agent, propylene glycol, polyethylene glycol can be used, such as olive oil The ester of the injectable of vegetable oil and such as ethyl oleate.As the matrix of suppository, can be used Acrawax (witepsol), Polyethylene glycol, emulsifier (Tween) 61, cacao butter, laurel tallow, glycerol and gelatin etc..

Particularly, for the skin preparations for extenal use of dermal administration can with patch, band (band), oil liquid, ointment, film, gel, Emulsifiable paste, lotion, the application of the form of liquid or powder, as cosmetics, can with soft toner, nutrition toner, massage cream, Nourishing cream, moisturiser, ﹑ functionality lotion, mist agent, film, gel or the application of skin adhesive type dosage form.Therefore, the external preparation for skin Agent can according to need the ingredient for properly mixing the skin preparations for extenal use for being typically used as cosmetics or drug etc., for example, water composition, Oil components, powdered ingredients, alcohols, moisturizer, thickener, ultraviolet absorbing agent, whitening agent, preservative, antioxidant, surface Activating agent, fragrance, colorant, various skin-nourishing agent.

The skin preparations for extenal use can properly mix disodium ethylene diamine tetraacetate, sodium versenate, sodium citrate, Metal-chelator, caffeine, tannin, verapamil, licorice, the light of sodium polyphosphate, sodium metaphosphate, gluconic acid etc. are sweet Grass is fixed, the hot water extract of setose thistle fruit, various crude drugs, tocopherol acetate, glycyrrhizic acid, tranexamic acid and its derivative or its The medicament of salt etc., vitamin C, magnesium ascorbyl phosphate, ascorbic acid glucoside, arbutin, kojic acid, glucose, fructose, sea The carbohydrate of algae sugar etc..

On the other hand it includes being planted according to the artemisia annua of the method preparation for the extract for separating the artemisia annua that the disclosure provides Object Extraction object, distillations or its isolated compound are used as the animal for preventing or treating skin disease of effective component Medical object.

The animal can be mammality, birds, fish or reptile class, and can be domestic animal, pet or cultivation and use fish Class, or can be the animal selected from one of chicken, pig, ox, horse, dog, rabbit, cat, duck, goat and sheep or more.

The animal may be used as individually medical object with medical object, and can be made animal feed additive come using. Animal as animal feed additive can be the Fen Ti ﹑ Ke Li ﹑ pellet (system such as pellet) ﹑ liquid phase or suspension with medical object Agent, and can individually supply or mix with feed supply.

On the other hand it includes being planted according to the artemisia annua of the method preparation for the extract for separating the artemisia annua that the disclosure provides The diet group for being used to prevent or improve skin disease of Object Extraction Wu ﹑ distillations or its isolated compound as effective component Close object.The food composition may further include acceptable excipient or carrier on food.The food composition can To be healthcare function food.

In the disclosure, defined healthcare function food crosses the law in the related healthcare function food of revision in 2008 Functionality and the safety of the related human body newly defined are sufficiently established, and can be the Room the Shi Yao bulletin of the related food drug safety Room Healthcare function food contained in the regulation that healthy functions food function raw material specified in No. 2008-72 is approved.

When including in healthcare function food by disclosed composition, can completely add the composition or and other Healthy functions food or healthy functions food ingredients are used together, and can be suitably used according to conventional methods.Effectively at Point combined amount can be according to using purpose suitably to determine.It can be with according to the effective component of the composition of the disclosure in general, constituting 0.01 weight percent including total food weight is to 15 weight percent, it preferably includes 0.2 weight percent to 10 weight Percentage, when being fabricated to beverage, can contain the 0.1g to 30g based on 100mL, the ratio of preferably 0.2g to 5g, and Entire beverage is configured to natural component.It is described but in the case where making taken long-term as purpose with health control and health Amount can be less than or equal to the range, and since there is no problem in terms of safety, effective component can be to be more than above-mentioned The amount of range uses.

It can be with routine healthcare function known to preparation any art according to the composition for healthcare function food of the disclosure The preparation of energy food.The healthcare function food can be prepared as powder, granule, tablet, pill, capsule, suspension, Emulsion, syrup, impregnating agent, liquor, extractant, chewing gum, tea, jelly or beverage etc. can preferably be prepared as beverage Form.Any carrier known in the art that can be used for preparing preparation can be used in acceptable carrier or additive on the food Or additive.In addition, acceptable carrier or additive can also include the food as animal feed on the food.

The healthcare function food according to purpose or hobby can containing nutritional agents, vitamin, electrolyte, ﹑ flavoring agent, Toner, pectic acid and its salt, alginic acid and its salt, organic acid, protecting colloid thickener, pH adjusting agent, stabilizer, preservative, Glycerol, alcohol, carbonating agent used in soda etc..It can also contain and prepare fruit juice, fruit drink and vegetable beverage Pulp.In addition, the healthcare function food composition can further include food additive, unless otherwise prescribed, " food adds Add agent " whether meet can according to the general provisions for the food additive code that food medicine object safe place recognizes and ordinary test method etc. by Specification and standard in relation to corresponding project judge.

On the other hand it includes being planted according to the artemisia annua of the method preparation for the extract for separating the artemisia annua that the disclosure provides Object Extraction Wu ﹑ distillations or its isolated compound are outer as the medicine for preventing or improving skin disease of effective component Product composition.The outer product composition of medicine may further include acceptable excipient or carrier on the outer product of medicine.

Term " the outer product of medicine " refers to the article for excluding that the purposes for medical object is used according to medicine thing method, and refers to based on health care The influence to human body for the classification standard that happiness portion is fixed is slightly in the drug of the drug for treating or preventing disease.Therefore, It may include for treating or preventing the fiber rubber product of mankind or animal disease, be mild or not to the effect of human body Directly effect and similar non-equipment or non-mechanical article, or for sterilization and the agrochemical of keeping off infection.Implement at one In example, the outer product of medicine can be used for Bao Shi ﹑ prevention or treatment cutaneous pruritus or atopic dermatitis.

On the other hand it includes being planted according to the artemisia annua of the method preparation for the extract for separating the artemisia annua that the disclosure provides Object Extraction object, distillations or its isolated compound are as effective component for preventing or improving the cosmetics of skin disease Composition.The cosmetic composition may further include acceptable excipient or carrier on cosmetics.The cosmetics Composition can be functional cosmetics.

According to food medicine object be in safely on August 11st, 2016 legislation advance notice " cosmetics law executing rule it is some Correction case adds atopic skin moisture-keeping functions according to functional cosmetics, and in one embodiment, functional cosmetics are available In prevention or treatment atopic dermatitis.

Possibility is utilized in industry

The method of the extract of the separation artemisia annua according to one aspect can be separated including with little or no including The extract of sesquiterpene lactone.

It include that sweet wormwood plant body extracts prepared by the method for the extract of the separation artemisia annua according to one aspect The composition of object may be used as alternative medicine of steroid or combinations thereof prevention or treatment Te Ying ﹑ inflammatory skin diseases or The skin disease of pruritus etc..

According to one aspect include in ornithine, pine camphor, mucilaginous fiber alcohol, chiro-inositol, glycine betaine and chlorogenic acid Combined composition of four kinds or more of the composition as single compound, may be used as the alternative medicine of steroid Or combinations thereof prevention or treatment skin disease.

According to the prevention for including the steps that for the composition being applied to individual of another aspect or treat skin disease Method may be used as the skin disease of alternative medicine of steroid or combinations thereof prevention or treatment individual.

It is outer with medical object, healthcare function food, medicine according to the medical object including the composition of another aspect, animal The spy that product or energy property cosmetics may be used as alternative medicine of steroid or combinations thereof prevention, improvement or treatment humans and animals answers The skin disease of property, inflammatory skin diseases or pruritus etc..

Claims (20)

1. a kind of method for separating Artemisia annua extract, described method includes following steps:

Sweet wormwood plant body is contacted with low polar solvent with by the constituents extraction in sweet wormwood plant body into low polar solvent The step of；

It is separated from the mixture obtained after the contact procedure and removes the low polar solvent come the step of obtaining residue； And

The residue is contacted with hydrophilic solvent with by the constituents extraction in the sweet wormwood plant body to the hydrophily Step in solvent.

2. the method for claim 1, wherein the low polar solvent is can dissolve or extract sesquiterpene lactone molten Agent.

3. the method for claim 1, wherein the low polar solvent is unsubstituted or by one or more substituent group Alkane, alkene or the alkynes of substituted C1-C6；The cycloalkane of C5-C12 that is unsubstituted or being replaced by one or more substituent groups Or cycloolefin；Alkyl acetate with C1-C5 alkyl that is unsubstituted or being replaced by one or more substituent groups；It is unsubstituted or The ether or ketone of the C1-C12 replaced by one or more substituent groups；It is unsubstituted or replaced by one or more substituent groups The sulfoxide of C1-C6；Acetonitrile；Tetrahydrofuran；Or their combination, wherein one or more substituent group is selected from F, Cl, Br And I.

4. the method for claim 1, wherein

The hydrophilic solvent can dissolve or extract selected from ornithine, pine camphor, mucilaginous fiber alcohol, chiro-inositol, glycine betaine, At least one of chlorogenic acid, alpha-linolenic acid, linoleic acid and palmitinic acid.

5. the method for claim 1, wherein the hydrophilic solvent is alcohol, water or the their combination of C1-C6.

6. the method as described in claim 1, the method also includes the Artemisia annua extract is contacted with fractionation solvent with The step of to Artemisia annua extract fractionation.

7. method as claimed in claim 6, wherein the step of fractionation includes that the Artemisia annua extract and chromatography are situated between Matter contact simultaneously dissolves out the Artemisia annua extract in conjunction with and with fractionation solvent.

8. method as claimed in claim 6, wherein the fractionation solvent is hydrophilic solvent.

9. method according to claim 8, wherein the hydrophilic solvent is alcohol, water or the their combination of C1-C6.

10. method as claimed in claim 6, wherein the step of fractionation includes with the total volume based on fractionation solvent 20 percents by volume or more and less than 40 percents by volume, 40 percents by volume or more and be less than 60 percents by volume, 60 Percent by volume or more and the alcohol dissolution for being less than 100 percents by volume or 100 percents by volume.

11. a kind of for preventing or treating the composition of skin disease, the composition includes method according to claim 1 institute The Artemisia annua extract of preparation.

12. composition as claimed in claim 11, wherein the Artemisia annua extract include less equal than 0.01 weight/ The sesquiterpene lactone of weight % does not include sesquiterpene lactone.

13. composition as claimed in claim 11, wherein the Artemisia annua extract includes selected from ornithine, pine camphor, glues Four kinds or more in matter inositol, chiro-inositol, glycine betaine and chlorogenic acid.

14. composition as claimed in claim 13, wherein the Artemisia annua extract further includes selected from linoleic acid, palm One of acid and alpha-linolenic acid or more.

15. composition as claimed in claim 11, wherein the Artemisia annua extract is with including solvent-based total volume 20 percents by volume or more and less than the distillations of the solvent fractionation of the alcohol of 40 percents by volume and with 100 volume basis The combination of the distillations of the solvent fractionation of the alcohol of ratio.

16. a kind of for preventing or treating the composition of skin disease, the composition includes selected from ornithine, pine camphor, glues Four kinds or more in matter inositol, chiro-inositol, glycine betaine and chlorogenic acid.

17. composition as claimed in claim 16, wherein the composition further includes selected from linoleic acid, palmitinic acid and α- One of linolenic acid or more.

18. the composition as described in claim 11 or 16, wherein the composition is medicinal compositions, the outer product combination of medicine Object, food composition or cosmetic composition.

19. the composition as described in claim 11 or 16, wherein the skin disease is inflammatory skin diseases.

20. composition as claimed in claim 18, wherein the skin disease is selected from cutaneous pruritus, prickly heat, seborrheica Dermatitis, contact atopic dermatitis, denuded skin inflammation, ichthyosis, spiradenitis, impetigo, atopic dermatitis, drug allergy, sugar Urinate characteristic of disease dermatitis, xerodermia, dermatodyschroia disease, bacterial cutaneous inflammation, Fungal Skin inflammation, varicella, pernio, fire sore, Eczema, edema, sudden fash, cholinergic urticaria, epifolliculitis, acne, pore seborrheic keratosis, wart, actinic keratosis, Miliaria, psoriasis, hansen's disease, Behcet's disease, bedsore, keloid disease, affected part edema, perionychia, nail nutrition are not Good disease, finger tip sore, allergic dermatitis, lichen simplex chronicus, dermapostasis and neurodermatitis.