CN102344454A - Wikstroemia indica (L.) C.A.Mey extract as well as preparation method and application thereof - Google Patents
Wikstroemia indica (L.) C.A.Mey extract as well as preparation method and application thereof Download PDFInfo
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Abstract
The invention discloses a wikstroemia indica (L.) C.A.Mey extract as well as a preparation method and application thereof. The wikstroemia indica (L.) C.A.Mey extract has the effective compositions of: naringenin, 5, 6, 7- trihydroxy-4'-methoxyflavanone, p-hydroxybenzoic acid, benzoic acid, quercetin glycoside,genkwano1 B, daphnoretin, Lirioresinol, Sikokianin and the like. The invention simultaneously provides application of the wikstroemia indica (L.) C.A.Mey extract, in particular the favorable application in preparing anti-cancer or anti-inflammation analgesia drug. A cytotoxicity experiment shows that the wikstroemia indica (L.) C.A.Mey extract disclosed by the invention has cytotoxicity functions on breast cancer of human, breast cancer drugresistant cell strain, mouse breast adenocarcinoma, which are in linear relation.
Description
Technical field
The invention belongs to medical technical field, be specifically related to a kind of Radix Wikstroemae extract.
Background technology
Root of Indian Stringbush (Wikstroemia indlca (L.) C.A.Mey); Be herbal medicine commonly used among the people, nature and flavor bitter cold, little suffering, poisonous; Cure mainly wound; Blood stasis removing analgesic, the decocting liquid of Root of Indian Stringbush leaf is extremely sensitive to pneumococcus, streptococcus aureus, to Pseudomonas aeruginosa, Corynebacterium diphtheriae medium sensitivity; The decocting liquid of rhizome skin then has obvious restraining effect to streptococcus aureus in vitro; The ETHYLE ACETATE soluble part of root skin ethanol soluble extraction also has external bacteriostatic action, and its contained flavonoid glycoside composition preliminary experiment all has restraining effect to streptococcus aureus, Hemolytic streptococcus, streptococcus pneumoniae, intestinal bacteria; The Root of Indian Stringbush tablet is 25mg/ml to the minimum inhibitory concentration (MIC) of beta hemolytic streptococcus, pneumococcus; To streptococcus aureus, Pseudomonas aeruginosa and colibacillary MIC is 50mg/ml.The Radix Wikstroemae extract extracorporeal bacteria inhibitor test shows that Root of Indian Stringbush has stronger bacteriostatic action.
The Root of Indian Stringbush clinical application is extensive, but effective substance is indeterminate, because Chinese medicine onset composition is various, prior art research does not have the effective constituent of clear and definite Radix Wikstroemae extract, fails to give full play to the applications well of Root of Indian Stringbush.
Summary of the invention
An object of the present invention is to overcome the deficiency of existing Root of Indian Stringbush plant resources utilization technology, a kind of new Radix Wikstroemae extract is provided.
Another object of the present invention provides the preparation method of said Radix Wikstroemae extract.
A further object of the invention provides the application of said Radix Wikstroemae extract.
The object of the invention is achieved through following technical scheme:
A kind of Radix Wikstroemae extract is provided; Its effective constituent is naringenin, 5; 6,7-trihydroxy--4 '-melonia flavones, PHB, phenylformic acid, Quercetin, genkwanol B, Thymelol, Lirioresinol, Sikokianin, said naringenin, 5; 6,7-trihydroxy--4 '-melonia flavones, PHB, phenylformic acid, Quercetin, genkwanol B have the structure shown in the formula (I)～(VI) respectively:
Compound 1 (naringenin): white crystal (petroleum ether-ethyl acetate), mp253-255 ℃, the reaction of HCl-Mg powder shows positive, TOF-MS m/z:272 [M]
+ 1H-NMR(500MHz，CD
3OD)δ：2.71(1H，dd，J＝3.0，17.0Hz，H-3)，3.12(1H，dd，J＝13.0，17.0Hz，H-3)，5.35(1H，dd，J＝3.0，13.0Hz，H-2)，5.88(1H，s，H-8)，5.89(1H，s，H-6)，6.82(2H，d，J＝8.5Hz，H-3′，5′)，7.30(2H，d，J＝8.5Hz，H-2′，6′)。
Compound 2 (5,6,7-trihydroxy--4 '-the melonia flavones): light yellow crystal (petroleum ether-ethyl acetate), HCl-Mg powder reacting positive.TOF-MS?m/z：299[M]
+。
1H-NMR(500MHz，CD
3OD)δ：2.55(1H，dd，J＝8，16.5Hz，H-3)，2.82(1H，dd，J＝5.5，16.5Hz，H-3)，3.8(3H，s，-OCH
3)，3.98(1H，dd，J＝8.0，13Hz，H-2)，5.98(1H，s，H-8)，6.79(2H，d，J＝8.5Hz，H-3′，5′)，7.25(2H，d，J＝8.5Hz，H-2′，6′)。
Compound 3 (PHB): white powder crystallization (petroleum ether-ethyl acetate), mp214-217 ℃, TOF-MS m/z:137 [M]
+ 1H-NMR(500MHz，CD
3OD)δ：7.97(2H，d，J＝7Hz，H-3，H-5)，6.91(2H，d，J＝7.0Hz，H-2，H-6)。
Compound 4 (phenylformic acid): colourless tabular crystal (petroleum ether-ethyl acetate), 122 ℃ of mp, TOF-MS m/z:121 [M-H]
+ 1H-NMR(500MHz，CD
3OD)δ：7.77(2H，d，J＝8.0Hz，H-2，H-6)，7.91(2H，d，J＝8.8Hz，H-3，H-5)，7.39(1H，d，J＝8.0Hz，H-4)。
Compound 4 (Quercetin): yellow needle-like crystal (petroleum ether-ethyl acetate), after the TLC Development of Thin-Layer Chromatography, 254nm has yellow fluorescence under the uv lamp, displaing yellow spot under 15% sulfuric acid-ethanolic soln, HCl-Mg powder reacting positive.mp?255～256℃EI-MS?m/z：283[M-H]
+。
1H-NMR(500MHz，DMSO-d
6)δ：3.86(3H，s，-OCH
3)，6.37(1H，d，J＝2.5Hz，H-6)，6.77(1H，d，J＝2.0Hz，H-8)，6.85(1H，s，H-3)，6.93(2H，d，J＝8.5Hz，H-2′，H-6′)，7.97(2H，d，J＝8.5Hz，H-3′，H-5′)，10.39(1H，s，7-OH)，12.96(1H，s，5-OH)。
Compound 6 (genkwanol B): light yellow crystal (methyl alcohol), HR-ESI-MS m/z:557 [M-H]-.1H-NMR(500MHz，CD3OD)δ：4.61(1H，d，J＝8.5Hz，H-2)，3.58(1H，m，H-3)，2.06(1H，dd，J＝5.0，16.5Hz，H-4)，2.61(1H，dd，J＝8.0，16.5Hz，H-4)，6.05(1H，s，H-6)，6.33(1H，d，J＝2.0Hz，H-3′，5′)，6.75(2H，d，J＝8.5Hz，H-2′，6′)，6.20(1H，d，J＝2.0Hz，H-6″)，6.30(1H，d，J＝2.0Hz，H-8″)，6.86(2H，d，J＝8.5Hz，H-3′″，5′″)，7.28(2H，d，J＝8.5Hz，H-2′″，6′″)。13C-NMR(500MHz，CD3OD)δ：83.1(C-2)，69.0(C-3)，29.4(C-4)，186.7(C-5)，100.3(C-6)，167.7(C-7)，85.0(C-8)，156.8(C-9)，103.5(C-10)，128.6(C-1′)，129.3(C-2′，6′)，156.6(C-4′)，115.7(C-3′，5′)，89.6(C-2″)，69.5(C-3″)，200.7(C-4″)，163.1(C-5″)，97.0(C-6″)，165.6(C-7″)，96.1(C-8″)，158.4(C-9″)，99.8(C-10″)，126.1(C-1′″)，114.5(C-3′″，5′″)，158.9(C-4′″)，131.5(C-2′″，6′″)。
Thymelol: pale yellow powder shape crystal (chloroform-methanol), after the Development of Thin-Layer Chromatography under the 365nm uv lamp apparent blue-fluorescence, FeCl
3Colour developing is deep green.mp?250～252℃，TOF-MS?m/z：352[M]
+。
1H-NMR(500MHz，DMSO-d
6)δ：3.83(3H，s，-OCH
3)，6.39(1H，d，J＝9.5Hz，H-3′)，6.87(1H，s，H-8)，7.13(1H，dd，J＝8.5，2.5Hz，H-6′)，7.19(1H，d，J＝2.5Hz，H-8′)，7.22(1H，s，H-5)，7.71(1H，d，J＝8.5Hz，H-5′)，7.88(1H，s，H-4)，8.05(1H，d，J＝9.5Hz，H-4′)，10.30(1H，s，-OH)。
13C-NMR(125MHz，DMSO-d
6)δ：160.4(C-2)，136.4(C-3)，131.6(C-4)，110.1(C-5)，146.4(C-6)，151.1(C-7)，103.5(C-8)，148.1(C-9)，110.9(C-10)，160.7(C-2′)，114.2(C-3′)，144.8(C-4′)，130.6(C-5′)，114.6(C-6′)，157.7(C-7′)，104.7(C-8′)，155.7(C-9′)，115.1(C-10′)，56.7(OCH
3-6′)。
Lirioresinol: light yellow crystal (methyl alcohol), HR-ESI-MS m/z:557 [M-H]
- 1H-NMR(500MHz，CD
3OD)δ：4.61(1H，d，J＝8.5Hz，H-2)，3.58(1H，m，H-3)，2.06(1H，dd，J＝5.0，16.5Hz，H-4)，2.61(1H，dd，J＝8.0，16.5Hz，H-4)，6.05(1H，s，H-6)，6.33(1H，d，J＝2.0Hz，H-3′，5′)，6.75(2H，d，J＝8.5Hz，H-2′，6′)，6.20(1H，d，J＝2.0Hz，H-6″)，6.30(1H，d，J＝2.0Hz，H-8″)，6.86(2H，d，J＝8.5Hz，H-3′″，5′″)，7.28(2H，d，J＝8.5Hz，H-2′″，6′″)。
13C-NMR(500MHz，CD
3OD)δ：83.1(C-2)，69.0(C-3)，29.4(C-4)，186.7(C-5)，100.3(C-6)，167.7(C-7)，85.0(C-8)，156.8(C-9)，103.5(C-10)，128.6(C-1′)，129.3(C-2′，6′)，156.6(C-4′)，115.7(C-3′，5′)，89.6(C-2″)，69.5(C-3″)，200.7(C-4″)，163.1(C-5″)，97.0(C-6″)，165.6(C-7″)，96.1(C-8″)，158.4(C-9″)，99.8(C-10″)，126.1(C-1′″)，114.5(C-3′″，5′″)，158.9(C-4′″)，131.5(C-2′″，6′″)。
Sikokianin: colourless amorphous (methyl alcohol), FAB-MS m/z:557 [M+H]
+ 1H-NMR(500MHz，CD
3OD)δ：3.62(3H，s，-OCH
3)，6.04(2H，d，J＝2.5Hz，H-6，6″)，6.09(2H，d，J＝2.5Hz，H-8，8″)，6.77(2H，d，J＝8.5Hz，H-3′″，5′″)，6.91(2H，d，J＝8.5Hz，H-3′，5′)，7.02(2H，d，J＝8.5Hz，H-2′，6′)，7.12(2H，d，J＝8.5Hz，H-2′″，6′″)。
13C-NMR(125MHz，CD
3OD)δ：81.5(C-2)，46.7(C-3)，192.2(C-4，4″)，162.1(C-5，5″)，97.4(C-6)，165.5(C-7，7″)，92.4(C-8)，159.7(C-9)，101.4(C-10)，125.9(C-1′)，128.9(C-2′，6′，2′″，6′″)，115.7(C-3′，C-5′)，158.1(C-4′)，81.6(C-2″)，46.1(C-3″)，97.5(C-6″)，92.6(C-8″)，160.4(C-9″)，101.6(C-10″)，124.0(C-1′″)，116.0(C-3′″，C-5′″)，158.7(C-4′″)，57.4(-OCH
3)。
The present invention provides the preparation method of said Radix Wikstroemae extract simultaneously, may further comprise the steps:
(1) Root of Indian Stringbush medicinal material (over-ground part) 1000g that gets Ex-all impurity pulverizes back (being crushed to the about 1cm of length); The volume by volume concentration that adds 8～10 times of amounts (volume multiple) is 1～2 hour after-filtration of extraction using alcohol of 95%; Filtrate decompression is concentrated into nothing alcohol flavor, gets thick medicinal extract;
(2) the thick medicinal extract that step (1) is made is scattered in (the quality of the thick medicinal extract of Root of Indian Stringbush: the volume=0.25Kg of water: 0.5～1L) in the water; Through sherwood oil, chloroform, ETHYLE ACETATE, n-butanol extraction; Before and after extraction is regardless of in proper order; Perhaps singly extract respectively, extract respectively 3 times, extract and (disperse thick medicinal extract volume of water: extraction solvent volume=1ml: 0.3～1ml) with the each consumption of solvent with a kind of or two or more mixed solvents wherein.
(3) get acetic acid ethyl acetate extract or butanol extraction liquid and carry out silica gel column chromatography, through chloroform: methyl alcohol (100: 1-1: 1, volume ratio) wash-out obtains efficient part; Efficient part continues through chloroform: methyl alcohol (100: 1-1: 1, volume ratio) wash-out, use normal hexane again: acetone (5: 1-1: 1, volume ratio), the gained cut obtains the purpose compound through sephadex LH-20 with methanol-eluted fractions.
The present invention provides the application of Radix Wikstroemae extract simultaneously, especially has good application aspect the anticancer or anti-inflammatory and analgesic effect medicine of preparation.Cellulotoxic experiment shows that Radix Wikstroemae extract of the present invention has CDCC to human breast carcinoma (MCF-7), mammary cancer drug-resistant cell strain (MCF-7/ADR), mouse breast cancer (MA782 tumour) cell, and linear.
The present invention has following beneficial effect:
Though the existing clinical application of Root of Indian Stringbush is extensive, effective substance is indeterminate, because Chinese medicine onset composition is various, efficient part is indeterminate, has restricted the further clinical application of Root of Indian Stringbush in addition.The present invention adopt suitable method clear and definite the efficient part of Radix Wikstroemae extract, and system's medical chemistry achievement in research of its efficient part further is provided, clear and definite its Application Areas.
Though separate a sesquiterpenoid that obtains in the existing bibliographical information Root of Indian Stringbush good activity is arranged; But because the curative effect of Chinese medicine is the characteristic of multipath and many target spots; Therefore more can embody the characteristic of Chinese medicine to the research of efficient part, have even more important and urgent meaning.The present invention is through the optimization of extraction and purification process technology; Success obtains the ETHYLE ACETATE and the propyl carbinol part of Root of Indian Stringbush alcohol extract, has good anti-inflammatory and analgesic effect, and the present invention simultaneously is behind the efficient part of confirming the Root of Indian Stringbush anti-inflammatory and analgesic effect; Its chemical ingredients systematic research and creationary analysis summary have been carried out; Analysis obtains a plurality of compounds, and wherein four compounds obtain for from this plant, separating first, have filled up the technological gap in present technique field.
Description of drawings
Fig. 1 naringenin
1The H-NMR spectrum;
Figure 25,6,7-trihydroxy--4 '-the melonia flavones
1The H-NMR spectrum;
Fig. 3 PHB
1The H-NMR spectrum;
Fig. 4 is benzoic
1The H-NMR spectrum;
Fig. 5 Quercetin
1The H-NMR spectrum;
Fig. 6 genkwanol B's
1The H-NMR spectrum;
Fig. 7 Root of Indian Stringbush opposed polarity position causes the result that influences of pain to the mouse hot plate method; Among each group post figure, square column is from left to right represented the threshold of pain of blank group, petroleum ether extraction position (A group), chloroform extraction position (B group), ethyl acetate extraction position (C group), n-butanol extraction position (D group), water position (E group), positive controls respectively;
Fig. 8 Root of Indian Stringbush opposed polarity position Dichlorodiphenyl Acetate causes the result that influences of mouse pain;
Fig. 9 Root of Indian Stringbush opposed polarity position p-Xylol induced mice auricle edema influence the result.
Embodiment
Further specify the present invention below in conjunction with accompanying drawing and specific embodiment.Unless stated otherwise, the reagent that the embodiment of the invention adopts, raw material etc. are conventional reagent, raw material etc., and the method that embodiment adopts specifies, is all present technique field ordinary method.
The preparation of embodiment 1 Radix Wikstroemae extract
Prepare Radix Wikstroemae extract according to the following steps:
(1) get Root of Indian Stringbush over-ground part 1000g and pulverize back (approximately long 1cm), the adding volume by volume concentration is 95% an ethanol 8L extract at room temperature after 2 hours, and extracting solution is obtained extract 80g in 40 ℃ of concentrating under reduced pressure, dryings;
(2) with the 80g extract respectively with sherwood oil 150mL, chloroform 150mL, ETHYLE ACETATE 150mL, propyl carbinol 150mL extraction 3 times, before and after extraction is regardless of in proper order, perhaps singly extract respectively with a kind of or two or more extraction solvents wherein;
(3) get acetic acid ethyl acetate extract or butanol extraction liquid and carry out silica gel column chromatography, through chloroform: methyl alcohol (100: 1-1: 1, volume ratio) gradient elution; Collect chloroform: methyl alcohol: water volume ratio is 50: 1 to 1: 1 a elution fraction, obtains efficient part; Efficient part continues through chloroform: methyl alcohol (100: 1-1: 1; Volume ratio) gradient elution; Collect chloroform: the methyl alcohol volume ratio is 30: 1 to 1: 1 a elution fraction, uses normal hexane again: acetone (5: 1-1: 1, volume ratio); Collect 4: 1 to 2: 1 cut, the gained cut obtains product through sephadex LH-20 with methanol-eluted fractions.The structural formula or the spectral data of products therefrom are following:
Naringenin: have the structure shown in the formula (I):
Compound 1 (naringenin): white crystal (petroleum ether-ethyl acetate), mp253-255 ℃, the reaction of HCl-Mg powder shows positive, TOF-MS m/z:272 [M]
+ 1H-NMR(500MHz，CD
3OD)δ：2.71(1H，dd，J＝3.0，17.0Hz，H-3)，3.12(1H，dd，J＝13.0，17.0Hz，H-3)，5.35(1H，dd，J＝3.0，13.0Hz，H-2)，5.88(1H，s，H-8)，5.89(1H，s，H-6)，6.82(2H，d，J＝8.5Hz，H-3′，5′)，7.30(2H，d，J＝8.5Hz，H-2′，6′)。
5,6,7-trihydroxy--4 '-the melonia flavones: have the structure shown in the formula (II):
Compound 2 (5,6,7-trihydroxy--4 '-the melonia flavones): light yellow crystal (petroleum ether-ethyl acetate), HCl-Mg powder reacting positive.TOF-MS?m/z：299[M]
+。
1H-NMR(500MHz，CD
3OD)δ：2.55(1H，dd，J＝8，16.5Hz，H-3)，2.82(1H，dd，J＝5.5，16.5Hz，H-3)，3.8(3H，s，-OCH
3)，3.98(1H，dd，J＝8.0，13Hz，H-2)，5.98(1H，s，H-8)，6.79(2H，d，J＝8.5Hz，H-3′，5′)，7.25(2H，d，J＝8.5Hz，H-2′，6′)。
PHB: have the structure shown in the formula (III):
(III)
Compound 3 (PHB): white powder crystallization (petroleum ether-ethyl acetate), mp214-217 ℃, TOF-MS m/z:137 [M]
+ 1H-NMR(500MHz，CD
3OD)δ：7.97(2H，d，J＝7Hz，H-3，H-5)，6.91(2H，d，J＝7.0Hz，H-2，H-6)。
Phenylformic acid: have the structure shown in the formula (IV):
Compound 4 (phenylformic acid): colourless tabular crystal (petroleum ether-ethyl acetate), 122 ℃ of mp, TOF-MS m/z:121 [M-H]
+ 1H-NMR(500MHz，CD
3OD)δ：7.77(2H，d，J＝8.0Hz，H-2，H-6)，7.91(2H，d，J＝8.8Hz，H-3，H-5)，7.39(1H，d，J＝8.0Hz，H-4)。
The preparation of embodiment 2 Radix Wikstroemae extracts
Prepare Radix Wikstroemae extract according to the following steps:
(1) get Root of Indian Stringbush over-ground part 1000g and pulverize back (approximately long 1cm), the adding volume by volume concentration is 95% an ethanol 8L extract at room temperature after 2 hours, and extracting solution is obtained extract 85g in 40 ℃ of concentrating under reduced pressure, dryings;
(2) the 80g extract is used sherwood oil 300mL, chloroform 300mL, ETHYLE ACETATE 300mL, propyl carbinol 300mL respectively, extract respectively 4 times, before and after extraction is regardless of in proper order, perhaps singly extract respectively with a kind of or two or more extraction solvents wherein;
(3) get acetic acid ethyl acetate extract or butanol extraction liquid and carry out silica gel column chromatography, through chloroform: methyl alcohol: water (100: 1: 0-1: 1: 1, volume ratio) gradient elution; Collect chloroform: methyl alcohol: water volume ratio is 100: 1 to 50: 1 a elution fraction, obtains efficient part; Efficient part continues through chloroform: methyl alcohol (100: 1-1: 1; Volume ratio) gradient elution; Collect chloroform: methyl alcohol: water volume ratio is 60: 1 to 50: 1 a elution fraction, uses normal hexane again: acetone (5: 1-1: 1, volume ratio); Collect 5: 1 cut, the gained cut obtains product through sephadex LH-20 with methanol-eluted fractions.The structural formula or the spectral data of products therefrom are following:
Quercetin: have the structure shown in the formula V:
Compound 4 (Quercetin): yellow needle-like crystal (petroleum ether-ethyl acetate), after the TLC Development of Thin-Layer Chromatography, 254nm has yellow fluorescence under the uv lamp, displaing yellow spot under 15% sulfuric acid-ethanolic soln, HCl-Mg powder reacting positive.mp?255～256℃?EI-MS?m/z：283[M-H]
+。
1H-NMR(500MHz，DMSO-d
6)δ：3.86(3H，s，-OCH
3)，6.37(1H，d，J＝2.5Hz，H-6)，6.77(1H，d，J＝2.0Hz，H-8)，6.85(1H，s，H-3)，6.93(2H，d，J＝8.5Hz，H-2′，H-6′)，7.97(2H，d，J＝8.5Hz，H-3′，H-5′)，10.39(1H，s，7-OH)，12.96(1H，s，5-OH)。
The preparation of embodiment 3 Radix Wikstroemae extracts
Prepare Radix Wikstroemae extract according to the following steps:
(1) get Root of Indian Stringbush over-ground part 1000g and pulverize back (approximately long 1cm), the adding volume by volume concentration is 95% an ethanol 8L extract at room temperature after 2 hours, and extracting solution is obtained extract 80g in 40 ℃ of concentrating under reduced pressure, dryings;
(2) the 80g extract is used sherwood oil 500mL, chloroform 500mL, ETHYLE ACETATE 500mL, propyl carbinol 500mL respectively, extract respectively 4 times, before and after extraction is regardless of in proper order, perhaps singly extract respectively with a kind of or two or more extraction solvents wherein;
(3) get acetic acid ethyl acetate extract or butanol extraction liquid and carry out silica gel column chromatography, through chloroform: methyl alcohol: water (100: 1: 0-1: 1: 1, volume ratio) gradient elution; Collect chloroform: methyl alcohol: water volume ratio is 50: 1 to 1: 1 a elution fraction, obtains efficient part; Efficient part continues through chloroform: methyl alcohol (30: 1-1: 1; Volume ratio) gradient elution; Collect chloroform: methyl alcohol: water volume ratio is 20: 1 to 1: 1 a elution fraction, uses (normal hexane: acetone (5: 1-1: 1, volume ratio) again; Collect 2: 1 to 1: 1 cut, the gained cut obtains product through sephadex LH-20 with methanol-eluted fractions.The structural formula or the spectral data of products therefrom are following:
Genkwanol B: have the structure shown in the formula (VI):
Compound 6 (genkwanol B): light yellow crystal (methyl alcohol), HR-ESI-MS m/z:557 [M-H]-.1H-NMR(500MHz，CD3OD)δ：4.61(1H，d，J＝8.5Hz，H-2)，3.58(1H，m，H-3)，2.06(1H，dd，J＝5.0，16.5Hz，H-4)，2.61(1H，dd，J＝8.0，16.5Hz，H-4)，6.05(1H，s，H-6)，6.33(1H，d，J＝2.0Hz，H-3′，5′)，6.75(2H，d，J＝8.5Hz，H-2′，6′)，6.20(1H，d，J＝2.0Hz，H-6″)，6.30(1H，d，J＝2.0Hz，H-8″)，6.86(2H，d，J＝8.5Hz，H-3′″，5′″)，7.28(2H，d，J＝8.5Hz，H-2′″，6′″)。13C-NMR(500MHz，CD3OD)δ：83.1(C-2)，69.0(C-3)，29.4(C-4)，186.7(C-5)，100.3(C-6)，167.7(C-7)，85.0(C-8)，156.8(C-9)，103.5(C-10)，128.6(C-1′)，129.3(C-2′，6′)，156.6(C-4′)，115.7(C-3′，5′)，89.6(C-2″)，69.5(C-3″)，200.7(C-4″)，163.1(C-5″)，97.0(C-6″)，165.6(C-7″)，96.1(C-8″)，158.4(C-9″)，99.8(C-10″)，126.1(C-1′″)，114.5(C-3′″，5′″)，158.9(C-4′″)，131.5(C-2′″，6′″)。
The different extraction of the Root of Indian Stringbush alcohol extract position that present embodiment adopts embodiment 1 to prepare is carried out analgesic experiment to the research (hot plate method) of thermal stimulus induced mice pain to the extraction position of Root of Indian Stringbush.Mouse is placed the pain sensation reaction that can stimulate mouse metapedes to occur licking on the hot plate.Experimental result is seen shown in the accompanying drawing 7.In the accompanying drawing 7; Among each group post figure, square column is from left to right represented the threshold of pain of blank group, petroleum ether extraction position (A group), chloroform extraction position (B group), ethyl acetate extraction position (C group), n-butanol extraction position (D group), water position (E group), positive controls respectively.Visible by figure, in the different extraction of the Root of Indian Stringbush alcohol extract position, A group mortality ratio in experimentation is higher, does not constitute statistical significance; And B group and E organize the not statistically significant (P＞0.05) of comparing with the blank group.C group, D group and positive controls are compared with the blank group, and each group all can improve the mouse threshold of pain, has statistical significance (P＜0.05).It is inhibited to show that ethyl acetate extraction position and n-butanol extraction position cause mouse pain to thermal stimulus, and the effect of n-butanol extraction position group is more obvious.
Employing to the method (writhing method) of Glacial acetic acid min. 99.5 induced mice pain to carrying out the Root of Indian Stringbush analgesic experiment.Experimental result is seen shown in the accompanying drawing 8.In the accompanying drawing 8, what square column was from left to right represented blank group, petroleum ether extraction position (A group), chloroform extraction position (B group), ethyl acetate extraction position (C group), n-butanol extraction position (D group), water position (E group), positive controls (Glacial acetic acid min. 99.5) respectively turns round the body number of times.Visible by figure, the Glacial acetic acid min. 99.5 of abdominal injection 0.7% can make mouse tangible writhing response occur.Wherein A group mortality ratio in experimentation is higher, does not constitute statistical significance; B group and E organize the not statistically significant (P＞0.05) of comparing with the blank group.C group, D group and positive controls are compared with the blank group; All can resist the pain reaction of acetic acid due to stimulating, the mouse writhing number of times is obviously reduced, statistical significance (P＜0.05) is arranged); Inhibiting rate to mouse is respectively 50.28%; 46.83%, and compare between the two and do not have too big-difference (P＞0.05), but more obvious with the effect of ethyl acetate extraction position.Prompting ethyl acetate extraction position group, n-butanol extraction position group is turned round on the phantom type at the mouse Glacial acetic acid min. 99.5 has analgesic activity.
The application experiment of embodiment 5 Radix Wikstroemae extracts of the present invention aspect anti-inflammatory
Adopt the influence (mice ear method) of p-Xylol induced mice auricle edema to carry out the anti-inflammatory experiment.Experimental result is seen shown in the accompanying drawing 9.In the accompanying drawing 9, square column is from left to right represented the swelling degree (mg) of blank group, petroleum ether extraction position (A group), chloroform extraction position (B group), ethyl acetate extraction position (C group), n-butanol extraction position (D group), water position (E group), positive controls respectively.Redden, become big rapidly in various degree through the generalized case of modeling animal is observed visible each experimental group animal YLENE coated side mouse ear, mouse ear portions blood vessel is high-visible.B group, C group, D group and the caused mice auricle swelling of positive controls p-Xylol all have certain restraining effect, with the blank group significant difference are arranged more all.Inhibiting rate is respectively 68.67%, 72.80%, and 69.87%.Wherein C group and its antiphlogistic effects of D group effect are the most near positive controls (indomethacin), and prompting ethyl acetate extraction position group, n-butanol extraction position group have obvious restraining effect on the model of YLENE induced mice ear swelling.
The application test of the anti-tumor aspect of embodiment 6 Radix Wikstroemae extracts of the present invention
Present embodiment is an example with human breast carcinoma (MCF-7), mammary cancer drug-resistant cell strain (MCF-7/ADR), mouse breast cancer (MA782 tumour) cell, the CDCC of research Radix Wikstroemae extract of the present invention.
Cytotoxic activity adopts mtt assay research, and reagent adopts 95% ethanol extraction of Root of Indian Stringbush over-ground part of the present invention respectively, the polar fraction of sherwood oil, chloroform, ETHYLE ACETATE, n-butanol extraction, and the MTT determination of cytotoxic activity is carried out at each polarity extraction position.Measure the result and see table 1.
The determination of cytotoxic activity result of each extract of table 1 Root of Indian Stringbush over-ground part ethanol extraction
Experimental result shows: ETHYLE ACETATE and n-butyl alcohol extract have cytotoxic activity to MCF-7, and MCF-7/ADR, MA782 are had the optionally cytotoxic activity of varying strength.
Claims (5)
1. Radix Wikstroemae extract; Its effective constituent is naringenin, 5 for its effective constituent; 6,7-trihydroxy--4 '-melonia flavones, PHB, phenylformic acid, Quercetin, genkwanol B, Thymelol, Lirioresinol, Sikokianin; Said naringenin, 5,6,7-trihydroxy--4 '-melonia flavones, PHB, phenylformic acid, Quercetin, genkwanol B have the structure shown in the formula (I)～(VI) respectively:
The structure spectra data of said Thymelol, Lirioresinol, Sikokianin are following:
250～252 ℃ of Thymelol: mp, TOF-MS m/z:352 [M]
+ 1H-NMR(500MHz，DMSO-d
6)δ：3.83(3H，s，-OCH
3)，6.39(1H，d，J＝9.5Hz，H-3′)，6.87(1H，s，H-8)，7.13(1H，dd，J＝8.5，2.5Hz，H-6′)，7.19(1H，d，J＝2.5Hz，H-8′)，7.22(1H，s，H-5)，7.71(1H，d，J＝8.5Hz，H-5′)，7.88(1H，s，H-4)，8.05(1H，d，J＝9.5Hz，H-4′)，10.30(1H，s，-OH)。
13C-NMR(125MHz，DMSO-d
6)δ：160.4(C-2)，136.4(C-3)，131.6(C-4)，110.1(C-5)，146.4(C-6)，151.1(C-7)，103.5(C-8)，148.1(C-9)，110.9(C-10)，160.7(C-2′)，114.2(C-3′)，144.8(C-4′)，130.6(C-5′)，114.6(C-6′)，157.7(C-7′)，104.7(C-8′)，155.7(C-9′)，115.1(C-10′)，56.7(OCH
3-6′)；
Lirioresinol：HR-ESI-MS?m/z：557[M-H]
-。
1H-NMR(500MHz，CD
3OD)δ：4.61(1H，d，J＝8.5Hz，H-2)，3.58(1H，m，H-3)，2.06(1H，dd，J＝5.0，16.5Hz，H-4)，2.61(1H，dd，J＝8.0，16.5Hz，H-4)，6.05(1H，s，H-6)，6.33(1H，d，J＝2.0Hz，H-3′，5′)，6.75(2H，d，J＝8.5Hz，H-2′，6′)，6.20(1H，d，J＝2.0Hz，H-6″)，6.30(1H，d，J＝2.0Hz，H-8″)，6.86(2H，d，J＝8.5Hz，H-3′″，5′″)，7.28(2H，d，J＝8.5Hz，H-2′″，6′″)。
13C-NMR(500MHz，CD
3OD)δ：83.1(C-2)，69.0(C-3)，29.4(C-4)，186.7(C-5)，100.3(C-6)，167.7(C-7)，85.0(C-8)，156.8(C-9)，103.5(C-10)，128.6(C-1′)，129.3(C-2′，6′)，156.6(C-4′)，115.7(C-3′，5′)，89.6(C-2″)，69.5(C-3″)，200.7(C-4″)，163.1(C-5″)，97.0(C-6″)，165.6(C-7″)，96.1(C-8″)，158.4(C-9″)，99.8(C-10″)，126.1(C-1′″)，114.5(C-3′″，5′″)，158.9(C-4′″)，131.5(C-2′″，6′″)；
Sikokianin：FAB-MS?m/z：557[M+H]
+。
1H-NMR(500MHz，CD
3OD)δ：3.62(3H，s，-OCH
3)，6.04(2H，d，J＝2.5Hz，H-6，6″)，6.09(2H，d，J＝2.5Hz，H-8，8″)，6.77(2H，d，J＝8.5Hz，H-3′″，5′″)，6.91(2H，d，J＝8.5Hz，H-3′，5′)，7.02(2H，d，J＝8.5Hz，H-2′，6′)，7.12(2H，d，J＝8.5Hz，H-2′″，6′″)。
13C-NMR(125MHz，CD
3OD)δ：81.5(C-2)，46.7(C-3)，192.2(C-4，4″)，162.1(C-5，5″)，97.4(C-6)，165.5(C-7，7″)，92.4(C-8)，159.7(C-9)，101.4(C-10)，125.9(C-1′)，128.9(C-2′，6′，2′″，6′″)，115.7(C-3′，C-5′)，158.1(C-4′)，81.6(C-2″)，46.1(C-3″)，97.5(C-6″)，92.6(C-8″)，160.4(C-9″)，101.6(C-10″)，124.0(C-1′″)，116.0(C-3′″，C-5′″)，158.7(C-4′″)，57.4(-OCH
3)。
2. the preparation method of the said Radix Wikstroemae extract of claim 1 is characterized in that may further comprise the steps:
(1) get the Root of Indian Stringbush pulverizing medicinal materials of Ex-all impurity after, 95% the ethanol that adds that 8～10 volumes doubly measure extracted 1～2 hour, filtered, filtrate decompression is concentrated into does not have the alcohol flavor, thick medicinal extract;
(2) the thick medicinal extract that makes of step (1) is scattered in the water, through sherwood oil, chloroform, ETHYLE ACETATE, n-butanol extraction;
(3) get acetic acid ethyl acetate extract or butanol extraction liquid and carry out silica gel column chromatography, through chloroform: methanol-eluted fractions obtains efficient part; Efficient part continues through chloroform: methanol-eluted fractions, use normal hexane again: acetone, the gained cut obtains the purpose compound through sephadex LH-20 with methanol-eluted fractions.
3. the application of the said Radix Wikstroemae extract of claim 1 is characterized in that being applied to prepare medicine aspect, relieving inflammation and relaxing pain aspect.
4. the application of the said Radix Wikstroemae extract of claim 1 is characterized in that being applied to prepare the cancer therapy drug aspect.
5. according to the application of the said Radix Wikstroemae extract of claim 4, it is characterized in that being applied to prepare anti-human breast carcinoma, anti-breast cancer drug-resistant cell strain or anti-mouse breast cancer medicine.
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