CN103772334B - Method for extracting stilbene compound from penoy seed husk - Google Patents

Method for extracting stilbene compound from penoy seed husk Download PDF

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CN103772334B
CN103772334B CN201410012555.8A CN201410012555A CN103772334B CN 103772334 B CN103772334 B CN 103772334B CN 201410012555 A CN201410012555 A CN 201410012555A CN 103772334 B CN103772334 B CN 103772334B
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ethyl acetate
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acetone
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CN103772334A (en
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刘普
邓瑞雪
梁菊
杨丹蕾
刘欣
尹卫平
段文录
柴元武
时清亮
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Henan University of Science and Technology
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    • C07D307/00—Heterocyclic compounds containing five-membered rings having one oxygen atom as the only ring hetero atom
    • C07D307/77—Heterocyclic compounds containing five-membered rings having one oxygen atom as the only ring hetero atom ortho- or peri-condensed with carbocyclic rings or ring systems
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Abstract

The invention mainly relates to a method for extracting a stilbene compound from penoy seed husk. The method comprises the following steps: (1) ultrasonically extracting penoy seed husk serving as a raw material, filtering after extraction, and evaporating and concentrating filtrate to obtain an extract; (2) adding the extract into the water for dispersing, and adding water-saturated ethyl acetate for extracting; (3) dissolving the ethyl acetate extract by using acetone, filtering, stirring by using silica gel to obtain a separated sample, adding into a silica gel column, performing gradient elution by using a petroleum ether-acetic acid mixed solvent, combining similar fractions to obtain fraction segments 1-30; (4) separating a fraction segment 14 by using preparation HPLC (High Performance Liquid Chromatography) to obtain compounds I and II; separating a fraction segment 18 by using preparation HPLC to obtain a compound III. The extracted stilbene compound high anti-bacterial and anti-tumor activities, and can be taken as an anti-bacterial or ant-tumor medicinal active component. The method has the advantage of high extraction rate, rapid, simple and convenient process and high economical benefit.

Description

A kind of method extracting stilbene compound from peony seeds skin
Technical field
The invention belongs to natural product chemistry and tree peony deep processing studying technological domain, relate generally to a kind of method extracting stilbene compound from peony seeds skin.
Background technology
Tree peony originates in the western part of China Qinling Mountains and Daba Mountain one is with mountain area, is perennial machaka.Tree peony is the distinctive woody rare flower of China, has the self-sow of thousands of years and the artificial culture history of more than 2,000 year.Peony seed oil contains multiple unsaturated fatty acids, wherein three kinds of important unsaturated fatty acidss: linolenic acid, the content of linoleic plus oleic acid is respectively: 35.7%, 22.19%, 27.24%, in addition in peony seed oil still containing the odd-numbered fatty acid with better antitumour activity, be a kind of food oils source with important Development volue.On March 22nd, 2011, Ministry of Health of the People's Republic of China approval from phoenix tree peony ( paeonia ostiit.Hong et J.X.Zhang) and Paeonia papaveracea ( paeonia rockii) seed benevolence through the peony seed oil made through techniques such as squeezing, decolouring, deodorizations can to produce as new resource food and edible [Ministry of Health of the People's Republic of China announces, 2011 No. 9: the Ministry of Health is about ratifying the bulletin of the oily and peony seed oil of acer truncatum seed as new resource food].
Stilbene compound (stilbeniods) refers to have the monomer of stilbene skeleton and the general name of polymkeric substance thereof.Oligomeric stilbene class belongs to phenolic acid compound.This compounds has biological activity widely, as antibacterium and anti-mycotic activity, and anti-inflammatory activity, anti-oxidant activity, anti-tumor activity, anti-HIV activity and liver-protecting activity etc.[Li little Mei, Li Na, Huang Kaisheng, Lin Mao. the spectral signature of Oligostilbenes, source of students and biological activity, Acta Pharmaceutica Sinica, 2002,37 (1): 69-74; Chen Li, Chen Jianbo. the pharmacological research summary of stilbene compound, Guangdong pharmacy, 2005,15 (3): 84-86]
Due to the high nutritive value of peony seed oil, the plantation amount of China's oil peony seeds is increasing gradually, and the output of oil peony seeds also increases year by year.At present, the preparation technology of peony seed oil is first by peony seeds drying shelling.According to current output, oil peony seeds output is 30,000 tons, the peony seeds skin about shelling can produce about 3000-5000 ton, and the exploitation of these tree peony seedcakes will be extremely important and research topics for arduous task.
Summary of the invention
The object of this invention is to provide a kind of method extracting stilbene compound from peony seeds skin, from peony seeds skin, extract stilbene class chemical combination there is antibacterial and antineoplastic activity preferably, can as antibacterial or anti-tumor drug activeconstituents.Extracting method extraction efficiency of the present invention is high, and extract under lower temperature condition, both can ensure that effective constituent proposes, can avoid again stilbene compound under the high temperature conditions long-time refluxing extraction time poor stability shortcoming, whole leaching process is quick, easy, high financial profit.
The technical scheme that the present invention realizes above-mentioned purpose employing is: a kind of method extracting stilbene compound from peony seeds skin, comprises the following steps:
1) peony seeds skin raw material is got, drying, pulverizing are placed in supersonic extractors, add its weight 10-20 acetone doubly, supersound extraction 30-40min under 30-40 DEG C of condition, extraction terminates rear filtration, collect filtrate, in filter residue, add its weight 10-20 acetone doubly again, supersound extraction 30-40min under 30-40 DEG C of condition, extract and terminate rear filtration, collect filtrate, repeat to add acetone, supersound extraction and filter operation at least twice in filter residue, merge all filtrate, and by filtrate evaporation concentration under-0.01Mpa condition, obtain soaking paste, for subsequent use; 2) it is even that leaching paste step 1) obtained adds water-dispersion, and obtain dispersion liquid, add and extract with the water saturation ethyl acetate of dispersion liquid with weight, then reclaim under reduced pressure ethyl acetate, obtains ethyl acetate extract, for subsequent use;
3) by step 2) the ethyl acetate extract acetone solution that obtains, the consumption of acetone be the 5-9 of ethyl acetate extract quality doubly, filter, in the filtrate obtained, add filtrate weight 1-2 silica gel mixed sample doubly, volatilize solvent, obtain sample separation, for subsequent use; Then sample separation is joined in silicagel column, silica gel column chromatography is adopted to be separated, the amount of the separation silica gel of filling in silicagel column is 20-40 times of sample separation weight, gradient elution is carried out with the sherwood oil-acetic acid mixed solvent of Different Weight ratio, the elution amount of each gradient is 2 times of column volumes, wherein in each wash-out sherwood oil used-acetic acid mixed solvent, both weight ratios are followed successively by 9:1,7:1,5:1,3:1,1:1,1:2 and 1:3, TLC is adopted to analyze, merge similar cut, obtain fraction section 1-30, for subsequent use;
4) fraction section 14 step 3) obtained is separated with preparative HPLC, obtains chemical compounds I and II; Equally fraction section 18 is separated with preparative HPLC, obtains compound III, namely complete the extraction of three kinds of stilbene compounds in peony seeds skin;
Three kinds of described stilbene compounds are respectively chemical compounds I, compound ii and compound III, and its structural formula is respectively:
。
Described peony seeds skin raw material is the seed skin of Paeonia papaveracea.
In described step 4), the separation condition of preparative HPLC is: chromatographic column is YMC-C18 reverse-phase chromatographic column; Flow velocity is 3.0mL/min; The methanol-water solution of moving phase to be volume ratio be 4:6.
Beneficial effect of the present invention
The stilbene compound that the present invention extracts from peony seeds skin, proves through pharmacology test, has antibacterial and antineoplastic activity preferably, can as antibacterial or anti-tumor drug activeconstituents.Extracting method of the present invention adopts acetone extraction in addition, the stilbene constituents that acetone can make polarity relatively large is as much as possible to be extracted, when adopting supersound extraction, carry out under lower temperature (30-40 DEG C) condition, both can ensure that effective constituent proposed, can avoid again stilbene compound under the high temperature conditions long-time refluxing extraction time poor stability shortcoming, whole leaching process is quick, easy, high financial profit.
Accompanying drawing explanation
Fig. 1 is chemical compounds I 1h NMR spectrogram;
Fig. 2 is chemical compounds I 13c NMR spectrogram;
Fig. 3 is the DEPT 2D-NMR spectrogram of chemical compounds I;
Fig. 4 is chemical compounds I 1h- 1h COSY spectrogram;
Fig. 5 is chemical compounds I 1h- 1h hsqc spectrum figure;
Fig. 6 is chemical compounds I 1h- 1h HMBC spectrogram;
Fig. 7 is chemical compounds I 1h- 1h NOESY spectrogram;
Fig. 8 is compound ii 1h NMR spectrogram;
Fig. 9 is compound ii 13c NMR spectrogram;
Figure 10 is the DEPT 2D-NMR spectrogram of compound ii;
Figure 11 is compound ii 1h- 1h COSY spectrogram;
Figure 12 is compound ii 1h- 1h hsqc spectrum figure;
Figure 13 is compound ii 1h- 1h HMBC spectrogram;
Figure 14 is compound ii 1h- 1h NOESY spectrogram;
Figure 15 is chemical compounds I 1h NMR spectrogram;
Figure 16 is chemical compounds I 13c NMR spectrogram;
Figure 17 is the DEPT 2D-NMR spectrogram of chemical compounds I;
Figure 18 is chemical compounds I 1h- 1h COSY spectrogram;
Figure 19 is chemical compounds I 1h- 1h hsqc spectrum figure;
Figure 20 is chemical compounds I 1h- 1h HMBC spectrogram;
Figure 21 is chemical compounds I 1h- 1h NOESY spectrogram;
Embodiment
From peony seeds skin, extract a method for stilbene compound, comprise the following steps:
1) paeonia rockii seed skin raw material is got, drying, pulverizing is placed in supersonic extractors, add its weight 10-20 acetone doubly, supersound extraction 30-40min under 30-40 DEG C of condition, extraction terminates rear filtration, collect filtrate, its weight 10-20 acetone is doubly added again in filter residue, supersound extraction 30-40min under 30-40 DEG C of condition, extraction terminates rear filtration, collect filtrate, repeat to add acetone in filter residue, supersound extraction and filter operation at least twice, merge all filtrate, and by filtrate evaporation concentration under-0.01Mpa condition, obtain soaking paste, for subsequent use, 2) it is even that leaching paste step 1) obtained adds water-dispersion, and obtain dispersion liquid, add and extract with the water saturation ethyl acetate of dispersion liquid with weight, then reclaim under reduced pressure ethyl acetate, obtains ethyl acetate extract, for subsequent use,
3) by step 2) the ethyl acetate extract acetone solution that obtains, the consumption of acetone be the 5-9 of ethyl acetate extract quality doubly, filter, in the filtrate obtained, add filtrate weight 1-2 silica gel mixed sample doubly, volatilize solvent, obtain sample separation, for subsequent use; Then sample separation is joined in silicagel column, silica gel column chromatography is adopted to be separated, the amount of the separation silica gel of filling in silicagel column is 20-40 times of sample separation weight, gradient elution is carried out with the sherwood oil-acetic acid mixed solvent of Different Weight ratio, the elution amount of each gradient is 2 times of column volumes, wherein in each wash-out sherwood oil used-acetic acid mixed solvent, both weight ratios are followed successively by 9:1,7:1,5:1,3:1,1:1,1:2 and 1:3, TLC is adopted to analyze, merge similar cut, obtain fraction section 1-30, for subsequent use;
4) fraction section 14 step 3) obtained is separated with preparative HPLC, obtains chemical compounds I and II; Equally fraction section 18 is separated with preparative HPLC, obtains compound III, namely complete the extraction of three kinds of stilbene compounds in peony seeds skin;
Three kinds of described stilbene compounds are respectively chemical compounds I, compound ii and compound III, and its structural formula is respectively:
。
Described peony seeds skin raw material is the seed skin of Paeonia papaveracea.
In described step 4), the separation condition of preparative HPLC is: chromatographic column is YMC-C10 reverse-phase chromatographic column; Flow velocity is 3.0mL/min; The methanol-water solution of moving phase to be volume ratio be 4:6.
The title of three new compounds is respectively: chemical compounds I: English name Rockiol A, Chinese purple plague purpura alcohol A; Compound ii: English name Rockiol B, Chinese purple plague purpura alcohol B; Compound III: English name Rockiol C, Chinese purple plague purpura alcohol C.
The Structural Identification of above-mentioned three new compounds be by organic spectroscopy analysis ( 1h NMR, 13c NMR, DEPT 2D-NMR, 1h- 1h COSY, HSQC, HMBC, NOESY) and compare with the spectral data of similar compound and obtain the structure of its unknown compound, see Fig. 1-2 1.
The spectral data of compound as:
Chemical compounds I, faint yellow amorphous powder. UV λ max(MeOH) (log ε): 286 (3.41), 226 (4.04). IR ν max(KBr) cm -1: 3340,1630,1517 and 1452. compounds 1h-NMR (CD 3oD, 400MHz) and 13c-NMR (CD 3oD, 100MHz) be shown in Table 1.HRESIMS:m/z 681.2124 [M+H] +(calculated value 681.2127), molecular composition is C 42h 32o 9.
Compound ii, faint yellow amorphous powder. UV λ max(MeOH) (log ε): 286 (3.33), 224 (4.16). IR ν max(KBr) cm -1: 3340,1630,1516 and 1452. compounds 1h-NMR (CD 3oD, 400MHz) and 13c-NMR (CD 3oD, 100MHz) be shown in Table 2.HRESIMS:m/z 681.2123 [M+H] +(calculated value 681.2127), molecular composition is C 42h 32o 9.
Compound III, faint yellow amorphous powder. UV λ max(MeOH) (log ε): 288 (3.90), 223 (4.46). IR ν max(KBr) cm -1: 3330,1624,1516 and 1451. compounds 1h-NMR (CD 3oD, 400MHz) and 13c-NMR (CD 3oD, 100MHz) be shown in Table 2. HRESIMS:m/z 679.1973 [M+H] +(calculated value 679.1977), molecular composition is C 42h 30o 9.
table 1:the nuclear magnetic resonance data of chemical compounds I
table 2:the nuclear magnetic resonance data of compound ii
table 3:the nuclear magnetic resonance data of compound III
Below in conjunction with specific embodiment, the present invention will be further described:
embodiment 1:
From peony seeds skin, extract a method for stilbene compound, comprise the following steps:
1) paeonia rockii seed skin raw material 7.5 ㎏ is got, drying, pulverizing are placed in supersonic extractors, add the acetone of its weight 10 times, supersound extraction 30min under 30 DEG C of conditions, extraction terminates rear filtration, collect filtrate, in filter residue, add the acetone of its weight 10 times again, supersound extraction 30min under 30 DEG C of conditions, extract and terminate rear filtration, collect filtrate, repeat to add acetone, supersound extraction and filter operation at least twice in filter residue, merge all filtrate, and by filtrate evaporation concentration under-0.01Mpa condition, obtain leaching paste 926g, for subsequent use; 2) it is even that leaching paste step 1) obtained adds water-dispersion, and obtain dispersion liquid, add and extract with the water saturation ethyl acetate of dispersion liquid with weight, then reclaim under reduced pressure ethyl acetate, obtains ethyl acetate extract 267g, for subsequent use;
3) by step 2) the ethyl acetate extract acetone solution that obtains, the consumption of acetone is 5 times of ethyl acetate extract quality, filters, adds the silica gel mixed sample of filtrate weight 1 times, volatilize solvent, obtain sample separation in the filtrate obtained, for subsequent use; Then sample separation is joined in silicagel column, silica gel column chromatography is adopted to be separated, the amount of the separation silica gel of filling in silicagel column is 20 times of sample separation weight, gradient elution is carried out with the sherwood oil-acetic acid mixed solvent of Different Weight ratio, the elution amount of each gradient is 2 times of column volumes, wherein in each wash-out sherwood oil used-acetic acid mixed solvent, both weight ratios are followed successively by 9:1,7:1,5:1,3:1,1:1,1:2 and 1:3, TLC is adopted to analyze, merge similar cut, obtain fraction section 1-30, for subsequent use;
4) fraction section 14 step 3) obtained is separated with preparative HPLC, obtains chemical compounds I 11.8mg and II 9.9mg; Fraction section 18 be separated with preparative HPLC equally, obtain compound III 14.8mg, the separation condition of preparative HPLC is: chromatographic column is YMC-C10 reverse-phase chromatographic column; Flow velocity is 3.0mL/min; The methanol-water solution of moving phase to be volume ratio be 4:6, namely completes the separation of three kinds of stilbene compounds in peony seeds skin.
embodiment 2:
From paeonia rockii seed skin, extract a method for stilbene compound, comprise the following steps:
1) peony seeds skin raw material 7.5kg is got, drying, pulverizing are placed in supersonic extractors, add the acetone of its weight 20 times, supersound extraction 40min under 40 DEG C of conditions, extraction terminates rear filtration, collect filtrate, in filter residue, add the acetone of its weight 20 times again, supersound extraction 40min under 40 DEG C of conditions, extract and terminate rear filtration, collect filtrate, repeat to add acetone, supersound extraction and filter operation at least twice in filter residue, merge all filtrate, and by filtrate evaporation concentration under-0.01Mpa condition, obtain leaching paste 930g, for subsequent use; 2) it is even that leaching paste step 1) obtained adds water-dispersion, and obtain dispersion liquid, add and extract with the water saturation ethyl acetate of dispersion liquid with weight, then reclaim under reduced pressure ethyl acetate, obtains ethyl acetate extract 279g, for subsequent use;
3) by step 2) the ethyl acetate extract acetone solution that obtains, the consumption of acetone is 9 times of ethyl acetate extract quality, filters, adds the silica gel mixed sample of filtrate weight 2 times, volatilize solvent, obtain sample separation in the filtrate obtained, for subsequent use; Then sample separation is joined in silicagel column, silica gel column chromatography is adopted to be separated, the amount of the separation silica gel of filling in silicagel column is 40 times of sample separation weight, gradient elution is carried out with the sherwood oil-acetic acid mixed solvent of Different Weight ratio, the elution amount of each gradient is 2 times of column volumes, wherein in each wash-out sherwood oil used-acetic acid mixed solvent, both weight ratios are followed successively by 9:1,7:1,5:1,3:1,1:1,1:2 and 1:3, TLC is adopted to analyze, merge similar cut, obtain fraction section 1-30, for subsequent use;
4) fraction section 14 step 3) obtained is separated with preparative HPLC, obtains chemical compounds I 12.5mg and II 9.3mg; Fraction section 18 be separated with preparative HPLC equally, obtain compound III 15.0mg, the separation condition of preparative HPLC is: chromatographic column is YMC-C10 reverse-phase chromatographic column; Flow velocity is 3.0mL/min; The methanol-water solution of moving phase to be volume ratio be 4:6, namely completes the separation of three kinds of stilbene compounds in peony seeds skin.
embodiment 3:
From paeonia rockii seed skin, extract a method for stilbene compound, comprise the following steps:
1) peony seeds skin raw material 7.5 ㎏ is got, drying, pulverizing are placed in supersonic extractors, add the acetone of its weight 15 times, supersound extraction 35min under 35 DEG C of conditions, extraction terminates rear filtration, collect filtrate, in filter residue, add the acetone of its weight 15 times again, supersound extraction 35min under 35 DEG C of conditions, extract and terminate rear filtration, collect filtrate, repeat to add acetone, supersound extraction and filter operation at least twice in filter residue, merge all filtrate, and by filtrate evaporation concentration under-0.01Mpa condition, obtain leaching paste 935g, for subsequent use; 2) it is even that leaching paste step 1) obtained adds water-dispersion, and obtain dispersion liquid, add and extract with the water saturation ethyl acetate of dispersion liquid with weight, then reclaim under reduced pressure ethyl acetate, obtains ethyl acetate extract 285g, for subsequent use;
3) by step 2) the ethyl acetate extract acetone solution that obtains, the consumption of acetone is 7 times of ethyl acetate extract quality, filters, adds the silica gel mixed sample of filtrate weight 1.5 times, volatilize solvent, obtain sample separation in the filtrate obtained, for subsequent use; Then sample separation is joined in silicagel column, silica gel column chromatography is adopted to be separated, the amount of the separation silica gel of filling in silicagel column is 30 times of sample separation weight, gradient elution is carried out with the sherwood oil-acetic acid mixed solvent of Different Weight ratio, the elution amount of each gradient is 2 times of column volumes, wherein in each wash-out sherwood oil used-acetic acid mixed solvent, both weight ratios are followed successively by 9:1,7:1,5:1,3:1,1:1,1:2 and 1:3, TLC is adopted to analyze, merge similar cut, obtain fraction section 1-30, for subsequent use;
4) fraction section 14 step 3) obtained, weight is 500mg, is separated with preparative HPLC, obtains chemical compounds I 12.3mg and II 9.8mg; Same by fraction section 18, weight is 1200mg, is separated with preparative HPLC, obtains compound III 14.6mg, and the separation condition of preparative HPLC is: chromatographic column is YMC-C10 reverse-phase chromatographic column; Flow velocity is 3.0 mL/min; The methanol-water solution of moving phase to be volume ratio be 4:6, namely completes the separation of three kinds of stilbene compounds in peony seeds skin.
antibacterial pharmacological testing
1. test materials
Paeonia rockii seed skin stilbene compound: chemical compounds I, compound ii and compound III.
Bacterial strain: streptococcus aureus s. aureas, streptococcus uberis s. uberis, subtilis bacillus subtilis, intestinal bacteria e. agalactiae, Salmonellas samonellalthered is provided by The First Affiliated Hospital of Henan University of Science and Technology.
method
The mensuration of minimum inhibitory concentration (MIC).Adopt document [Li Jianqiang, Li Liujin. veterinary microbiology test practice instructs [M]. Xi'an: Shaanxi science tech publishing house, 1999:29-30] middle test tube turbidimetry, by the sterilizing test tubes numbering of 10 band tampons, No. 1 pipe adds cultured solution of broth 1.5 mL, and all the other each pipes add 1 mL.Get 10 mg/mL sample solutions (solvent is the mixed solution of V (methyl alcohol): V (water)=1: 1) 0.5 mL to add in No. 1 pipe and mix, from No. 1 pipe, get 1 mL again add No. 2 pipes, serial dilution to like this No. 8 pipe, No. 8 pipes discard 1 mL containing liquid meat soup.Draw 50mL bacteria containing amount and be equivalent to the bacterial suspension (bacterial concentration 1.5 hundred million/mL) of Maxwell opacity tube the 1st pipe 1/2 to 1 ~ No. 9 pipe, 50 mL meat soups added by No. 10 pipes.Take out after putting 37 DEG C of cultivation 12 ~ 15 h and observe turbidity.No. 9 pipes are muddy positive control pipe, and No. 10 pipes are muddy negative control pipe.Manage than No. 9 limpid and respectively manage thereafter the test tube consistent with No. 9 pipe turbidity, its medicament contg is minimum inhibitory concentration.
test-results
Research finds that peony seeds skin stilbene compound is to gram-positive microorganism (streptococcus aureus, streptococcus uberis, subtilis) and Gram-negative bacteria (intestinal bacteria, Salmonellas) all there is good restraining effect, and drug effect is relatively better.Experimental result is as follows:
Table 4: peony seeds stilbene compound minimum inhibitory concentration (MIC) (mg/mL)
inhibition tumor cell active testing
1, test materials: the stilbene compound obtained is extracted by embodiment 3.
, method:
Cytotoxic activity adopts mtt assay test, and test concrete operation step is as follows:
1. to take the logarithm cell in vegetative period, with the RPM1640 nutrient solution containing 10% foetal calf serum, make single cell suspension 5 × 10 4individual/mL, be added to by this suspension in 96 well culture plates, every hole adds 1 × 10 4individual cell.After cultivating 24 h, add test medicine, chemical monomer concentration is respectively 30 g/mL, 10 g/mL.
2. by flat board at 37 DEG C, containing 5%CO 2hatch 3 days in the incubator of air and 100% humidity, remove nutrient solution.
3. the RPM1640 nutrient solution of MTT serum-free is made into 1 mg/mL solution, and every hole adds 100 L, and 37 DEG C of incubation 4 h, make MTT be reduced to Jia Za.
4. sucking-off supernatant liquid, adds 150L DMSO Shi Jia Za and dissolves, survey OD value in ELISA instrument 570 nm place.
Table 5: stilbene compound cytotoxic activity experimental data (n=6)
。

Claims (2)

1. from peony seeds skin, extract a method for stilbene compound, it is characterized in that: comprise the following steps:
1) peony seeds skin raw material is got, drying, pulverizing are placed in supersonic extractors, add its weight 10-20 acetone doubly, supersound extraction 30-40min under 30-40 DEG C of condition, extraction terminates rear filtration, collect filtrate, in filter residue, add its weight 10-20 acetone doubly again, supersound extraction 30-40min under 30-40 DEG C of condition, extract and terminate rear filtration, collect filtrate, repeat to add acetone, supersound extraction and filter operation at least twice in filter residue, merge all filtrate, and by filtrate evaporation concentration under-0.01Mpa condition, obtain soaking paste, for subsequent use;
2) it is even that leaching paste step 1) obtained adds water-dispersion, and obtain dispersion liquid, add and extract with the water saturation ethyl acetate of dispersion liquid with weight, then reclaim under reduced pressure ethyl acetate, obtains ethyl acetate extract, for subsequent use;
3) by step 2) the ethyl acetate extract acetone solution that obtains, the consumption of acetone be the 5-9 of ethyl acetate extract quality doubly, filter, in the filtrate obtained, add filtrate weight 1-2 silica gel mixed sample doubly, volatilize solvent, obtain sample separation, for subsequent use; Then sample separation is joined in silicagel column, silica gel column chromatography is adopted to be separated, the amount of the separation silica gel of filling in silicagel column is 20-40 times of sample separation weight, gradient elution is carried out with the sherwood oil-acetic acid mixed solvent of Different Weight ratio, the elution amount of each gradient is 2 times of column volumes, wherein in each wash-out sherwood oil used-acetic acid mixed solvent, both weight ratios are followed successively by 9:1,7:1,5:1,3:1,1:1,1:2 and 1:3, TLC is adopted to analyze, merge similar cut, obtain fraction section 1-30, for subsequent use;
4) fraction section 14 step 3) obtained is separated with preparative HPLC, obtains chemical compounds I and II; Equally fraction section 18 is separated with preparative HPLC, obtains compound III, namely complete the extraction of three kinds of stilbene compounds in peony seeds skin; The separation condition of described preparative HPLC is: chromatographic column is YMC-C10 reverse-phase chromatographic column; Flow velocity is 3.0mL/min; The methanol-water solution of moving phase to be volume ratio be 4:6;
Three kinds of described stilbene compounds are respectively chemical compounds I, compound ii and compound III, and its structural formula is respectively:
。
2. a kind of method extracting stilbene compound from peony seeds skin as claimed in claim 1, is characterized in that: described peony seeds skin raw material is the seed skin of Paeonia papaveracea.
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Title
Cytotoxic and AnUmutagenic Stilbenes from Seeds of Paeonia lactiflora;Hyo Jin Kim et al.;《Arch. Pharm. Res.》;20020630;第25卷(第3期);293-299 *
Identification and Ecdysteroid Antagonist Activity of Three Resveratroi Trimers (Suffruticosois A, B and C) from Paeonia suffruticosa;Satyajit D. Sarker et al.;《Tetrahydron》;19990303;第55卷(第2期);513-524 *
Three New Oligostilbenes from the Seeds of Paeonia suffruticosa;Chun-Nian He et al.;《Chem. Pharm. Bull.》;20100330;第58卷(第6期);843-847 *

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