CN115505014A - Callicarpa nudiflora extract, preparation method and application thereof - Google Patents

Callicarpa nudiflora extract, preparation method and application thereof Download PDF

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CN115505014A
CN115505014A CN202211280994.8A CN202211280994A CN115505014A CN 115505014 A CN115505014 A CN 115505014A CN 202211280994 A CN202211280994 A CN 202211280994A CN 115505014 A CN115505014 A CN 115505014A
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methanol
extract
dichloromethane
silica gel
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杨国栋
刘昱甫
刘永林
马瑜
李诒光
陈杰
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Jiangzhong Pharmaceutical Co Ltd
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Abstract

The invention discloses a callicarpa nudiflora extract, a preparation method and application thereof, and relates to the technical field of traditional Chinese medicine extraction. The extract comprises cistanoside D, digitonin and cosmosiin. Compared with the prior art, the obtained callicarpa nudiflora extract has clear components, greatly improves the use safety, simultaneously plays a synergistic anti-inflammatory role, has good anti-inflammatory effect and good application prospect.

Description

Callicarpa nudiflora extract, preparation method and application thereof
Technical Field
The invention relates to the technical field of traditional Chinese medicine extraction, and particularly relates to a callicarpa nudiflora extract, and a preparation method and application thereof.
Background
Callicarpa nudiflora hook et arn. Is Callicarpa L. Plant of Callicarpa of Verbenaceae (Verbenaceae). The callicarpa nudiflora is used as a medicine by stems, branches and leaves, and has the effects of removing blood stasis and swelling, resisting bacteria and stopping bleeding, diminishing inflammation and detoxifying, and dispelling wind and eliminating dampness. It can be used for treating suppurative inflammation, acute infectious hepatitis, hemorrhage of respiratory tract and digestive tract, and hemorrhage due to wound, and can be used for treating burn, scald, and traumatic hemorrhage. Research shows that the callicarpa nudiflora mainly contains components such as flavonoids, terpenes, phenylpropanoids, volatile oil and the like, and has pharmacological activities such as hemostasis, anti-thrombosis, anti-inflammation, bacteriostasis, cytotoxic activity, immunity enhancement and the like.
Chinese patent application CN201610139149.7 discloses an application of callicarpa nudiflora extract in preparing a medicament for treating glioma, wherein the callicarpa nudiflora extract is obtained by separating and extracting aerial parts of callicarpa nudiflora, and the extraction steps comprise: (a) Drying and pulverizing aerial parts of Callicarpa nudiflora, extracting with ethanol under reflux, mixing extractive solutions, concentrating until no alcohol smell exists, sequentially extracting with petroleum ether, ethyl acetate and water saturated n-butanol to obtain petroleum ether extract, ethyl acetate extract and n-butanol extract; (b) Removing impurities from n-butanol extract, eluting with 10% ethanol for 6 column volumes, eluting with 70% ethanol for 8 column volumes, collecting 70% ethanol eluate, and concentrating under reduced pressure to obtain 70% ethanol eluate concentrate; (c) Separating the 70% ethanol elution concentrate in the step (b) by using normal phase silica gel, performing gradient elution by using dichloromethane-methanol, and collecting eluent; separating with reverse phase silica gel bonded with octadecylsilane, isocratically eluting with 70% methanol water solution, collecting eluate, and concentrating the Callicarpa nudiflora extract under reduced pressure.
Chinese patent application CN201510306705.0 discloses an anti-inflammatory callicarpa nudiflora extract, which contains 24-36% of flavonoids, 0.8-3% of phenylethanoid glycosides, and 15-27% of diterpene compounds. It is prepared by the following steps: (1) Pulverizing folium Callicarpae Formosanae into coarse powder, adding 90-95% ethanol, heating and reflux extracting for 1-3 times to obtain extractive solution, filtering, and concentrating the filtrate to obtain extract A; (2) Uniformly dispersing the extract A obtained in the step (1) in water, sequentially adding petroleum ether and ethyl acetate for extraction, and removing the solvent to respectively obtain a petroleum ether extraction effective part B and an ethyl acetate extraction part C; (3) Mixing the effective part B and the effective part C according to the weight ratio of 1-3.
At present, the callicarpa nudiflora extract can only determine a few compounds in the callicarpa nudiflora extract, and all ingredients in the extract cannot be determined, so that the drug effect is uncertain; in addition, a specific extraction method for a specific compound having pharmacological activity in callicarpa nudiflora is still lacking and needs to be perfected.
Disclosure of Invention
The invention aims to provide a callicarpa nudiflora extract, a preparation method and application thereof, and provides a callicarpa nudiflora extract with definite ingredients and good anti-inflammatory activity and a method for extracting specific ingredients from callicarpa nudiflora aiming at the problems in the prior art.
Interpretation of terms:
in the invention, the proportions of the mobile phases are volume ratios, and the purity percentages are mass fractions.
In the present invention, the numerical ranges include both end values and any real number therein, such as "30 to 90%" including but not limited to 30%, 31%, 32%, 32.5%, 33.1%, 33.333%, 60%, 70%, 80%, 90%, etc.
In the invention, the molecular structural formulas of the monomer compounds are respectively as follows:
(6S,7R)-3-oxo-megastigma-4,8-dien-7-O-β-D-glucoside：
Figure BDA0003898227050000021
phoebenoside A：
Figure BDA0003898227050000022
(6R,9R)-3-oxo-α-ionol-9-O-β-D-glucopyranoside：
Figure BDA0003898227050000023
blumenol C glucoside：
Figure BDA0003898227050000031
isoverbascoside:
Figure BDA0003898227050000032
myricoside：
Figure BDA0003898227050000033
cistanoside D:
Figure BDA0003898227050000034
and (3) digitonin:
Figure BDA0003898227050000035
calceolariosideB：
Figure BDA0003898227050000036
4,4'-dimethoxy-3'-hydroxy-7',9-diepoxylignan-3-O-β-D-glucopyranoside：
Figure BDA0003898227050000041
luteolin-7-O-glucuronide：
Figure BDA0003898227050000042
luteolin-7-O-beta-D-glucoside:
Figure BDA0003898227050000043
luteolin-4' -O- β -D-glucoside:
Figure BDA0003898227050000044
cosmosiin:
Figure BDA0003898227050000045
luteolin-7-O-β-L-rhamnopyranosyl(1,2)-β-D-glucopyranoside：
Figure BDA0003898227050000046
celery-7-O-beta-D-neohesperidin:
Figure BDA0003898227050000047
2-O-butyl-1-O-(2'-ethylhexyl)benzene-1,8-dicarboxylate：
Figure BDA0003898227050000051
in order to realize the purpose of the invention, the technical scheme of the invention is as follows:
in one aspect, the invention provides a callicarpa nudiflora extract, which is characterized by comprising cistanoside D, digitonin and cosmosiin.
Preferably, in the callicarpa nudiflora extract, the mass ratio of cistanoside D, digitonin and cosmosiin is 1-10.
On the other hand, the invention provides a preparation method of the callicarpa nudiflora extract, which is characterized by comprising the following steps of:
s1, adding water to folium callicarpae nudiflorae for cold soaking, then heating and decocting, combining filtrates, concentrating under reduced pressure to obtain an extract 1, suspending and dispersing the extract with water, adding ethyl acetate for extraction, and recovering a solvent under reduced pressure to obtain an extract 2;
s2, mixing the extract 2 with silica gel, performing silica gel column chromatography coarse separation, and performing gradient elution with dichloromethane-methanol 600 in a ratio of 1-1: 1 to obtain 9 fractions Fr 1-9;
s3 and Fr 6 are separated on an MCI column, and are subjected to gradient elution by a methanol-water solvent system of 30-90% methanol, and then are combined to obtain Fr 6-1-6-5;
wherein, the Fr 6-1 is subjected to silica gel column chromatography and gradient elution by dichloromethane-methanol 30: 1-1: 1 to obtain 6 components of Fr 6-1-6-1-6. Performing ODS column chromatography on Fr 6-1-3, performing isocratic elution with methanol-water of 45-50, and separating the prepared liquid phase with methanol of 42-48% and methanol of 40-45% respectively to obtain the digitonin; fr 6-4 is separated by normal phase silica gel column chromatography, dichloromethane-methanol is subjected to gradient elution with the ratio of 80: 1-1: 1 to obtain 6 components Fr 6-4-1-6-4-6; separating the prepared liquid phase of Fr 6-4-2 with 52-58% methanol to obtain cistanoside D; selecting 3 components with the most extraction amount, wherein the polarity is from high to low, and the components are Fr 6, fr4 and Fr 3 in sequence;
s4, fr4 is subjected to reversed phase packing YMC column chromatography pressure separation, gradient elution is carried out by using a methanol-water solvent system of 60-95% methanol, and 3 components with the largest extraction amount are selected, wherein the polarity is from high to low, and Fr 4-1, fr4-2 and Fr4-3 in sequence;
wherein, the component Fr4-3 is separated by Sephadex LH-20 column and methanol, 2 components with the largest extraction amount are selected, and Fr 4-3-1 and Fr4-3-3 are sequentially arranged according to polarity from high to low; fr4-3-3 is separated by normal phase silica gel column chromatography, and the content of dichloromethane-methanol is 30:1 to 1: gradient elution is carried out on 1 to obtain 4 components Fr 4-3-3-1 to 4-3-3-5, and isocratic elution is carried out on Fr 4-3-3-1 through a preparation liquid phase by 42-48 percent methanol to obtain the cosmosiin.
In still another aspect, the present invention provides a callicarpa nudiflora extract comprising (6S, 7R) -3-oxo-megastigma-4, 8-dien-7-O-beta-D-glucoside, phoebenide A, (6R, 9R) -3-oxo-alpha-ionol-9-O-beta-D-glucopyranoside, blunol C glucoside, isorhodanoside, myrosine, cistanoside D, digitoside, calceolaroside B, 4' -dimethoxy-3' -hydroxy-7', 9-diopoxyignan-3-O-beta-D-glucopyranoside, luteolin-7-O-glucuronide, luteolin-7-O-beta-D-glucoside, luteolin-4 ' -O-beta-D-glucoside, cosmosiin, luteolin-7-O-beta-L-rhamnopyranosyl (1, 2) -beta-D-glucopyranoside, celery-7-O-beta-D-neohesperidin and 2-O-butyl-1-O- (2 ' -ethylhexyl) benzene-1,8-dicarboxylate.
Preferably, in the callicarpa nudiflora extract, (6S, 7R) -3-oxo-megastigma-4, 8-dien-7-O-beta-D-glucoside, phoebenoside A, (6R, 9R) -3-oxo-alpha-ionol-9-O-beta-D-glucopyranoside, blumenol C glucoside, acteoside, myrosine, cistanoside D, digitoside, caleroside B, 4' -dimethoxy-3' -hydroxy-7', the mass ratio of 9-diazoxyignan-3-O- β -D-glucopyranoside, luteolin-7-O-glucuronide, luteolin-7-O- β -D-glucoside, luteolin-4 ' -O- β -D-glucoside, cosmosin, luteolin-7-O- β -L-rhamnopyranosyl (1, 2) - β -D-glucopyranoside, apium-7-O- β -D-neohesperidin and 2-O-butyl-1-O- (2 ' -ethylhexyl) benzene-1,8-dicarboxylate is 1-5; further preferred are 3.2.
In another aspect, the present invention provides a method for preparing the beautyberry extract, comprising the following steps:
s1, adding water to folium callicarpae nudiflorae for cold soaking, then heating and decocting, combining filtrates, concentrating under reduced pressure to obtain an extract 1, suspending and dispersing the extract with water, adding ethyl acetate for extraction, and recovering a solvent under reduced pressure to obtain an extract 2;
s2, mixing the extract 2 with silica gel, performing coarse separation by silica gel column chromatography, performing gradient elution by using dichloromethane-methanol 600:1, and selecting 3 components with the largest extraction amount, wherein the components are Fr 6, fr4 and Fr 3 in sequence from high to low according to polarity;
separating S3 and Fr 6 on MCI column, gradient eluting with methanol-water solvent system of 30-90% methanol, selecting 2 components with maximum extraction amount, sequentially Fr 6-1-4 and Fr 6-1-3 according to polarity from high to low;
wherein Fr 6-1 is subjected to silica gel column chromatography, gradient elution is carried out by dichloromethane-methanol 30: 1-1: 1,2 components with the largest extraction amount are selected, and Fr 6-1-4 and Fr 6-1-3 are sequentially carried out according to polarity from high to low;
performing ODS column chromatography on Fr 6-1-3, performing isocratic elution with methanol-water of 45-50, and separating the prepared liquid phase by isocratic elution with 42-48% methanol and 40-45% methanol respectively to obtain compounds 8 and 11; fr 6-1-4, preparing liquid phase, and separating with 43-46% methanol and 45-50% methanol to obtain compounds 15 and 16;
fr 6-4 is separated by normal phase silica gel column chromatography, 2 components with the largest extraction amount are selected by dichloromethane-methanol gradient elution from 80:1 to 1:1, and Fr 6-4-2 and Fr 6-4-1 are sequentially selected according to polarity from high to low;
fr 6-4-1 is separated from the liquid phase by 50-55% methanol to obtain compound 1; fr 6-4-2 is separated by 52-58% methanol through a preparation liquid phase to obtain a compound 4 and a compound 7;
s4 and Fr4 are subjected to reversed phase packing YMC column chromatography pressure separation, gradient elution is carried out by using a methanol-water solvent system of 60-95% methanol, and 3 components with the largest extraction amount are selected, wherein the polarity is from high to low, and Fr 4-1, fr4-2 and Fr4-3 in sequence;
wherein Fr 4-1 is separated by normal phase silica gel column chromatography, and dichloromethane-methanol is eluted in a gradient manner with the ratio of 20: 1-1: 1 to obtain compounds 2 and 6; separating the component Fr4-2 in a silica gel column, and performing gradient elution on dichloromethane-methanol at a ratio of 50: 1-5: 1; then preparing liquid phase and passing through 52-58% methanol to obtain compounds 3 and 10;
separating the component Fr4-3 with Sephadex LH-20 column and methanol, selecting 2 components with the most extraction amount, sequentially Fr 4-3-1 and Fr4-3-3 according to polarity from high to low;
fr 4-3-1 is separated by normal phase silica gel column chromatography, and dichloromethane-methanol is eluted in a gradient way with the ratio of 20: 1-5: 1 to obtain compounds 9 and 12; elution ratio dichloromethane: methanol 10: 1.7: 1; fr4-3-3 is separated by normal phase silica gel column chromatography, and the content of dichloromethane-methanol is 30:1 to 1: gradient elution is carried out by 1,2 components with the largest extraction amount are selected, and according to the polarity from high to low, fr4-3-3-3 and Fr 4-3-3-1 are sequentially selected;
fr 4-3-3-1 is subjected to liquid phase preparation and isocratic elution by 42-48% methanol to obtain compounds 13 and 14; fr4-3-3-3 is purified by a preparative liquid phase through 42-50% methanol to obtain a compound 5;
separating S5 and Fr 3 on MCI column, gradient eluting with 40-95% methanol-water solvent system, separating with 45-55% methanol to obtain compound 17,
wherein, the compounds 1 to 17 are respectively (6S, 7R) -3-oxo-megastigma-4, 8-dien-7-O-beta-D-glucoside, phoebenoside A, (6R, 9R) -3-oxo-alpha-ionol-9-O-beta-D-glucopyranoside, blumenol C glucoside, isoverbascoside, myrricoside, cistanoside D, digitoside, calcoelariosidic B, 4' -dimethoxy-3' -hydroxy-7', 9-diethoxyignan-3-O-beta-D-glucopyranoside, luteolin-7-O-glucuronide, luteolin-7-O-beta-D-glucoside, luteolin-4 ' -O-beta-D-glucoside, cosmosin, luteolin-7-O-beta-L-rhamnopyranosyl (1, 2) -beta-D-glucopyranoside, apium graveolens-7-O-beta-D-neohesperidin, 2-O-butyl-1-O- (2 ' -ethylhexyl) bezene-1, 8-dicarboxylate.
Preferably, in step S1, the water is added for cold soaking for 6-18h, the times of heating and decocting are 2-5 times, each time is 1-5h, and the times of ethyl acetate extraction are 1-5 times.
Preferably, in step S2, the volume ratio of dichloromethane to methanol is 400-600: 1. 200-400: 1. 100-300: 1. 50-150: 1. 50-100 parts of: 1. 15-40: 1. 10-20: 1. 5-15: 1. 1-8:1. as a specific example of the present invention, gradient elution was performed according to the following volume ratio: 500: 1. 300, and (2) 300: 1. 200: 1. 100, and (2) a step of: 1. 70: 1. 20: 1. 15: 1. 10: 1.5: 1.
preferably, in step S3, the methanol-water solvent system is 30-60% methanol, 50-70% methanol, 60-80% methanol, 70-90% methanol, 85-90% methanol. As a specific example of the present invention, gradient elution is performed according to the following volume ratio: 50% methanol, 60% methanol, 70% methanol, 80% methanol, 90% methanol.
The volume ratio of dichloromethane to methanol for eluting Fr 6-1 is 10-30: 1. 10-20: 1. 10-15: 1. 5-12: 1. 3-10: 1. 1-8:1. as a specific example of the present invention, gradient elution was performed according to the following volume ratio: 20: 1. 15: 1. 12: 1. 10: 1.8: 1.6:1.
the volume ratio of dichloromethane to methanol for eluting Fr 6-4 is 60-80: 1. 20-40: 1. 15-30: 1. 10-20: 1. 5-15: 1. 1-8:1. as a specific example of the present invention, gradient elution was performed according to the following volume ratio: 70: 1. 30: 1. 20: 1. 15: 1. 10: 1.5: 1.
preferably, in step S4, the methanol-water solvent system is 60-70% methanol, 70-80% methanol, 80-90% methanol, 90-95% methanol. As a specific example of the present invention, gradient elution was performed according to the following volume ratio: 65% methanol, 75% methanol, 85% methanol, 95% methanol.
The volume ratio of dichloromethane to methanol for eluting Fr 4-1 is 10-20: 1. 1-10:1. as a specific example of the present invention, gradient elution was performed according to the following volume ratio: 10: 1.8: 1.
eluting Fr4-2 with dichloromethane-methanol at a volume ratio of 25-50: 1. 15-25: 1. 10-20: 1. 8-15: 1. 5-10:1. as a specific example of the present invention, gradient elution was performed according to the following volume ratio: 30: 1. 20: 1. 15: 1. 10: 1.5: 1.
the volume ratio of dichloromethane to methanol for eluting Fr 4-3-1 is 10-20:1 and 5-10: 1; as a specific example of the present invention, gradient elution is performed according to the following volume ratio: 10: 1.7: 1.
the volume ratio of dichloromethane to methanol for eluting Fr4-3-3 is 15-30: 1. 10-20: 1. 5-15: 1. 1-10:1; as a specific example of the present invention, gradient elution is performed according to the following volume ratio: 20: 1. 15: 1. 10: 1.5: 1.
preferably, in step S5, the methanol-water solvent system is 40-60% methanol, 50-70% methanol, 60-80% methanol, 70-85% methanol, 80-95% methanol. As a specific example of the present invention, gradient elution is performed according to the following volume ratio: 50% methanol, 60% methanol, 70% methanol, 80% methanol, 90% methanol.
In another aspect, the present invention provides a pharmaceutical composition comprising the above-mentioned callicarpa nudiflora extract.
In still another aspect, the present invention provides a method for extracting (6S, 7R) -3-oxo-megastigma-4, 8-dien-7-O-beta-D-glucoside from Callicarpa nudiflora, comprising the steps of:
s1, adding water to folium callicarpae nudiflorae for cold soaking, then heating and decocting, combining filtrates, concentrating under reduced pressure to obtain an extract 1, suspending and dispersing the extract with water, adding ethyl acetate for extraction, and recovering a solvent under reduced pressure to obtain an extract 2;
s2, mixing the extract 2 with silica gel, performing silica gel column chromatography coarse separation, and performing gradient elution with dichloromethane-methanol 600 in a ratio of 1-1: 1 to obtain 9 fractions Fr 1-9;
s3 and Fr 6 are separated on an MCI column, gradient elution is carried out by using a methanol-water solvent system of 30-90 percent methanol, and Fr 6-1-6-5 is obtained by combination;
wherein, the Fr 6-1 is subjected to silica gel column chromatography and gradient elution by dichloromethane-methanol 30: 1-1: 1 to obtain 6 components Fr 6-1-6-1-6; fr 6-1-4 is subjected to preparative liquid phase purification to obtain compounds 15 and 16;
separating Fr 6-4 with normal phase silica gel column chromatography, gradient eluting with dichloromethane-methanol 80: 1-1: 1 to obtain 6 components Fr 6-4-1-6-4-4, and purifying Fr 6-4-1 with liquid phase to obtain (6S, 7R) -3-oxo-megastigma-4, 8-dien-7-O-beta-D-glucoside.
In another aspect, the present invention provides a method for extracting phoebenoside a from callicarpa nudiflora, comprising the following steps:
s1, adding water to folium callicarpae nudiflorae for cold soaking, then heating and decocting, combining filtrates, concentrating under reduced pressure to obtain an extract 1, suspending and dispersing the extract with water, adding ethyl acetate for extraction, and recovering a solvent under reduced pressure to obtain an extract 2;
s2, mixing the extract 2 with silica gel, performing coarse separation by silica gel column chromatography, and performing gradient elution by using dichloromethane-methanol 600 in a ratio of 1-1: 1 to obtain 9 fractions Fr 1-9;
s3, fr4 is subjected to reversed-phase packing YMC column chromatography pressure separation and gradient elution by a methanol-water solvent system of 60-95% methanol to obtain 4 components Fr 4-1-4; wherein, fr 4-1 is separated by normal phase silica gel column chromatography, and dichloromethane-methanol is eluted in a gradient way of 20: 1-1: 1; collecting dichloromethane: methanol 7:1, removing segment to obtain phoebenoside A.
In another aspect, the present invention provides a method for extracting (6R, 9R) -3-oxo- α -ionol-9-O- β -D-glucopyranoside from callicarpa nudiflora, comprising the steps of:
s1, adding water to folium callicarpae nudiflorae for cold soaking, then heating and decocting, combining filtrates, concentrating under reduced pressure to obtain an extract 1, suspending and dispersing the extract with water, adding ethyl acetate for extraction, and recovering a solvent under reduced pressure to obtain an extract 2;
s2, mixing the extract 2 with silica gel, performing coarse separation by silica gel column chromatography, and performing gradient elution by using dichloromethane-methanol 600 in a ratio of 1-1: 1 to obtain 9 fractions Fr 1-9;
s3, fr4 is subjected to reversed phase packing YMC column chromatography pressure separation and gradient elution by a methanol-water solvent system of 60-95% methanol to obtain 4 components Fr 4-1-4;
wherein, the component Fr4-2 is separated by a silica gel column, after dichloromethane-methanol gradient elution is carried out at 50: 1-5: 1, 10:1, and then (6R, 9R) -3-oxo-alpha-ionol-9-O-beta-D-glucopyranoside is obtained by high performance liquid purification.
In another aspect, the present invention provides a method for extracting blumenol C glucoside from callicarpa nudiflora, comprising the steps of:
s1, adding water to folium callicarpae nudiflorae for cold soaking, then heating and decocting, combining filtrates, concentrating under reduced pressure to obtain an extract 1, suspending and dispersing the extract with water, adding ethyl acetate for extraction, and recovering a solvent under reduced pressure to obtain an extract 2;
s2, mixing the extract 2 with silica gel, performing coarse separation by silica gel column chromatography, and performing gradient elution by using dichloromethane-methanol 600 in a ratio of 1-1: 1 to obtain 9 fractions Fr 1-9;
s3 and Fr 6 are separated on an MCI column, gradient elution is carried out by using a methanol-water solvent system of 30-90 percent methanol, and Fr 6-1-6-5 is obtained by combination;
wherein Fr 6-4 is separated by normal phase silica gel column chromatography, 6 components of Fr 6-4-1-6-4-4 are obtained by dichloromethane-methanol gradient elution at the ratio of 80: 1-1: 1, and blumenol Cglcoside is obtained by high performance liquid chromatography purification of Fr 6-4-2.
In another aspect, the present invention provides a method for extracting calcoelarioside B from Callicarpa nudiflora, comprising the steps of:
s1, adding water to folium callicarpae nudiflorae for cold soaking, then heating and decocting, combining filtrates, concentrating under reduced pressure to obtain an extract 1, suspending and dispersing the extract with water, adding ethyl acetate for extraction, and recovering a solvent under reduced pressure to obtain an extract 2;
s2, mixing the extract 2 with silica gel, performing silica gel column chromatography coarse separation, and performing gradient elution with dichloromethane-methanol 600 in a ratio of 1-1: 1 to obtain 9 fractions Fr 1-9;
s3, fr4 is subjected to reversed phase packing YMC column chromatography pressure separation and gradient elution by a methanol-water solvent system of 60-95% methanol to obtain 4 components Fr 4-1-4;
wherein, the component Fr4-3 is separated by Sephadex LH-20 column and methanol, 5 components Fr 4-3-1-4-3-5 are obtained by combination, the Fr 4-3-1 adopts normal phase silica gel column chromatography for separation, and the calcoelarioside B is obtained by dichloromethane-methanol 20: 1-5: 1 gradient elution.
In another aspect, the present invention provides a method for extracting 4,4' -methoxy-3 ' -hydroxy-7', 9-diazoxyignan-3-O- β -D-glucopyranoside from callicarpa nudiflora, comprising the steps of:
s1, adding water to folium callicarpae nudiflorae for cold soaking, then heating and decocting, combining filtrates, concentrating under reduced pressure to obtain an extract 1, suspending and dispersing the extract with water, adding ethyl acetate for extraction, and recovering a solvent under reduced pressure to obtain an extract 2;
s2, mixing the extract 2 with silica gel, performing silica gel column chromatography coarse separation, and performing gradient elution with dichloromethane-methanol 600 in a ratio of 1-1: 1 to obtain 9 fractions Fr 1-9;
s3, fr4 is subjected to reversed-phase packing YMC column chromatography pressure separation and gradient elution by a methanol-water solvent system of 60-95% methanol to obtain 4 components Fr 4-1-4;
wherein, the component Fr4-2 is separated by a silica gel column, and after dichloromethane-methanol gradient elution is carried out at a ratio of 50: 1-5: 1, the compound 4,4' -methoxy-3 ' -hydroxy-7', 9-diazoxyignan-3-O-beta-D-glucopyranoside is obtained by preparative liquid phase purification.
In still another aspect, the present invention provides a method for extracting luteolin-7-O-glucuronide from Callicarpa nudiflora, comprising the steps of:
s1, adding water to folium callicarpae nudiflorae for cold soaking, then heating and decocting, combining filtrates, concentrating under reduced pressure to obtain an extract 1, suspending and dispersing the extract with water, adding ethyl acetate for extraction, and recovering a solvent under reduced pressure to obtain an extract 2;
s2, mixing the extract 2 with silica gel, performing coarse separation by silica gel column chromatography, and performing gradient elution by using dichloromethane-methanol 600 in a ratio of 1-1: 1 to obtain 9 fractions Fr 1-9;
s3 and Fr 6 are separated on an MCI column, gradient elution is carried out by using a methanol-water solvent system of 30-90 percent methanol, and Fr 6-1-6-5 is obtained by combination;
wherein Fr 6-1 is subjected to silica gel column chromatography, and dichloromethane-methanol gradient elution is carried out to obtain 6 components Fr 6-1-6-1-6; performing ODS column chromatography on Fr 6-1-3, performing 45-50 methanol-water isocratic elution, and preparing a liquid phase for separation to obtain luteolin-7-O-glucuronide.
In still another aspect, the present invention provides a method for extracting 2-O-butyl-1-O- (2' -ethylhexyl) benzene-1,8-dicarboxylate from Callicarpa nudiflora, comprising the steps of:
s1, adding water to folium callicarpae nudiflorae for cold soaking, then heating and decocting, combining filtrates, concentrating under reduced pressure to obtain an extract 1, suspending and dispersing the extract with water, adding ethyl acetate for extraction, and recovering a solvent under reduced pressure to obtain an extract 2;
s2, mixing the extract 2 with silica gel, performing coarse separation by silica gel column chromatography, and performing gradient elution by using dichloromethane-methanol 600 in a ratio of 1-1: 1 to obtain 9 fractions Fr 1-9;
separating S3 and Fr 3 on MCI column, gradient eluting with 40-95% methanol-water solvent system, separating with 45-55% methanol to obtain 2-O-butyl-1-O- (2' -ethylhexyl) bezene-1, 8-dicarboxlate.
In another aspect, the invention provides the application of the beautyberry extract, or the beautyberry extract prepared by the preparation method, or the extract prepared by the extraction method in preparing anti-inflammatory drugs.
Finally, the invention provides the application of cistanoside D in the preparation of anti-inflammatory drugs.
In the invention, the applications of the cistanoside D and the callicarpa nudiflora extract respectively comprise the application of the cistanoside D and the callicarpa nudiflora extract as unique active ingredients, or the combination of the cistanoside D and the callicarpa nudiflora extract and at least one other anti-inflammatory active ingredient.
The active ingredient may be administered orally in solid dosage forms such as capsules, tablets, lozenges, troches, granules and powders, or in liquid dosage forms such as elixirs, syrups, emulsions, dispersions and suspensions. The active ingredient may also be administered parenterally in sterile liquid dosage forms, such as dispersions, suspensions or solutions. Other dosage forms which can be used for administering the active ingredient are ointments, creams, drops, transdermal patches or powders for topical administration; ophthalmic solutions or suspensions for ocular administration, i.e., eye drops; a spray or powder composition for inhalation or intranasal administration, or a cream, ointment, spray or suppository for rectal or vaginal administration. Gelatin capsules contain the active ingredient and powdered carriers such as lactose, starch, cellulose derivatives, magnesium stearate, stearic acid, and the like. Similar diluents can be used to prepare compressed tablets. Both tablets and capsules can be prepared as sustained release products to provide sustained release of the drug over a period of hours. Compressed tablets may be sugar or film coated to mask any unpleasant taste and protect the tablet from the air, or may be enteric coated for selective disintegration in the gastrointestinal tract. Liquid dosage forms for oral administration may contain coloring and flavoring agents to increase patient acceptance. In general, water, suitable oils, saline, aqueous dextrose (glucose), and related sugar solutions and glycols such as propylene glycol or polyethylene glycol are suitable carriers for parenteral solutions. Solutions for parenteral administration preferably contain water-soluble salts of the active ingredient, suitable stabilizers and buffer substances as required. Antioxidants such as sodium bisulfite, sodium sulfite or ascorbic acid, alone or in combination, are suitable stabilizers. Citric acid and its salts and sodium EDTA may also be used. In addition, parenteral solutions may also contain preservatives, such as benzalkonium chloride, methyl or propyl paraben, and chlorobutanol. For administration by inhalation, the compounds of the invention may conveniently be delivered in the form of a spray from a pressurized pack or a nebulizer. The compounds may also be delivered in the form of a powder for formulation, which powder composition may be inhaled with the aid of an insufflation powder inhaler device. A preferred delivery system for inhalation is a Metered Dose Inhalation (MDI) aerosol which may be formulated as a suspension or solution of the active ingredient in a suitable propellant, such as a fluorocarbon or hydrocarbon. For ocular administration, ophthalmic formulations can be formulated with a solution or suspension of the active ingredient in a suitable ophthalmic vehicle in an appropriate weight percent to maintain contact of the compound with the ocular surface for a time sufficient to allow penetration of the compound into the cornea and interior regions of the eye.
The invention has the beneficial effects that:
aiming at the problems in the prior art, the callicarpa nudiflora extract with definite ingredients and good anti-inflammatory activity and the preparation method thereof are provided, and the method for extracting the specific ingredients from the callicarpa nudiflora is provided. Compared with the prior art, the obtained callicarpa nudiflora extract has the advantages of clear components, greatly improved use safety, good anti-inflammatory effect and good application prospect.
Drawings
FIG. 1 is a flow chart of the extraction process of the present invention.
Detailed Description
The present invention will be further explained with reference to specific examples in order to make the technical means, the technical features, the technical objectives and the effects of the present invention easier to understand, but the following examples are only preferred embodiments of the present invention, and not all embodiments of the present invention. Other embodiments obtained by persons skilled in the art without making creative efforts based on the embodiments in the implementation belong to the protection scope of the invention. In the following examples, unless otherwise specified, all the operations were performed by conventional methods, all the equipments were performed by conventional methods, and the materials of the equipments used in the respective examples were the same.
In the following examples, beautyberry medicinal material was purchased from Henan Baisha Hensheng Source planting professional cooperative, identified as beautyberry C.nudiflora by Prof teaching of Vanzuche, university of traditional Chinese medicine, jiangxi, and certificate specimens (No. Y202101G) were stored in Jiangxi traditional Chinese medicine grain scientific research center.
Bruker Avance-III HD 600MHz type nuclear magnetic resonance spectrometer (Bruker, germany); triple TOF 5600 high resolution time-of-flight mass spectrometer (AB Sciex, USA); thermoUITIMate type 3000 high performance liquid phase (Sammer Feishell technologies, USA); elette P3500 semi-preparative liquid chromatograph (dalianelit analytical instruments ltd); YMC-Triart C18 semi-preparative chromatography columns (10 mm. Times.250mm, 5 μm, japan YMC Co.); MCI-gel (75-150 μm, mitsubishi chemical corporation, japan), sephadex LH-20 Sephadex (GE Healthcare, USA); column chromatography silica gel (100-200 mesh, 200-300 mesh, qingdao ocean plant); analytical balance model MS204S/01 (Mettler Toledo, switzerland); milli-Q ultra pure water machine (Millipore, USA); lipopolysaccharide, dexamethasone (Sigma company, usa); phosphate buffer, DMEM high-glucose medium (Solarbio, china); fetal bovine serum (Gibco, USA); NO and CCK8 kits (shanghai bi yuntian biotechnology limited); the reagents used are either chromatographically pure or analytically pure.
Example 1
3.0kg of callicarpa nudiflora dry leaves are soaked in water for 12h, then are heated and decocted for 3 times, each time lasts for 3h, the hot filtrate is filtered and combined, the filtrate is concentrated under reduced pressure to form an extract, the extract is suspended and dispersed by using a proper amount of water, a proper amount of ethyl acetate is added, the extraction is carried out for 3 times, and the solvent is recovered under reduced pressure to obtain 314g of the extract. Mixing the sample with silica gel, performing silica gel column chromatography for coarse separation, and performing silica gel column chromatography with dichloromethane: methanol 500: 1. 300: 1. 200: 1. 100: 1. 70: 1. 20: 1. 15: 1. 10: 1.5:1, gradient elution is carried out, the height of the fractions expanded on a silica gel plate is taken as a selection standard through preliminary judgment of thin-layer chromatography, the fractions with similar expansion height are merged, and finally 9 fractions Fr 1-9 are obtained; the developing agent selected from the section Fr 1 is dichloromethane-methanol 30, the developing agent selected from the section Fr 2 is dichloromethane-methanol 25, the developing agent selected from the section Fr 3 is dichloromethane-methanol 22;
selecting 3 components with the maximum extraction amount, and sequentially selecting Fr 6, fr4 and Fr 3 according to polarity from high to low.
Fr 6 (30.14 g) is separated on an MCI column, after gradient elution is carried out on a methanol-water solvent system (50% methanol, 60% methanol, 70% methanol, 80% methanol and 90% methanol), the height of the fractions spread on a silica gel plate is taken as a selection standard through preliminary judgment of thin layer chromatography, the fractions with similar spread height are combined, and Fr 6-1-6-5 is obtained through combination; the developing solvent selected from the section Fr 6-1 is dichloromethane-methanol 12, the developing solvent selected from the section Fr 6-2 is dichloromethane-methanol 15;
selecting 2 components with the maximum extraction amount, sequentially from high polarity to low polarity, fr 6-1 and Fr 6-4.
Wherein Fr 6-1 (8.11 g) is subjected to silica gel column chromatography and is subjected to gradient elution by dichloromethane-methanol to obtain 6 components Fr 6-1-6-1-6; wherein the ratio of dichloromethane: 20 parts of methanol: 1. 15: 1. 12: 1. 10: 1.8: 1.6:1, preliminarily judging by thin-layer chromatography that the height of fractions spreading on a silica gel plate is a selection standard, combining fractions spreading to close heights to obtain 6 components Fr 6-1-6-1-6, wherein a spreading agent selected from the Fr 6-1-1 part is dichloromethane-methanol 12; the developing solvent selected from the Fr 6-1-6 part is dichloromethane-methanol 10;
selecting 2 components with the maximum extraction amount, sequentially from high polarity to low polarity, fr 6-1-4 and Fr 6-1-3.
Fr 6-1-3 (1.04 g) was subjected to ODS column chromatography, methanol-water (48. Fr 6-1-4 (0.95 g) was further purified by semi-preparative high performance liquid chromatography using 54% methanol to give compounds 15 (1.8 mg) and 16 (2.3 mg).
Fr 6-4 (5.81 g) is separated by normal phase silica gel column chromatography, and 6 components Fr 6-1-1 to 6-1-6 are obtained by dichloromethane-methanol gradient elution; respectively dichloromethane: 20 parts of methanol: 1. 15: 1. 12: 1. 10: 1.8: 1.6:1. according to the preliminary judgment of thin-layer chromatography, taking the height of the fractions developed on a silica gel plate as a selection standard, combining the fractions with similar development heights, selecting a developing agent from a Fr 6-4-1 part as dichloromethane-methanol 10; the developing solvent selected from the Fr 6-4-6 part is dichloromethane-methanol 8;
selecting 2 components with the most extraction amount, sequentially from high polarity to low polarity, fr 6-4-2 and Fr 6-4-1.
Fr 6-4-1 was purified by semi-preparative high performance liquid chromatography with 49% methanol to give compound 1 (3.2 mg). Fr 6-4-2 was purified by semi-preparative high performance liquid chromatography on 44% methanol to give compounds 4 (3.8 mg) and 7 (4.2 mg).
Fr4 (86.82 g) is subjected to pressure separation by reversed phase packing YMC column chromatography, then methanol-water (95% methanol, 85% methanol, 75% methanol and 65% methanol) is used for gradient elution, the initial judgment by thin layer chromatography is carried out by taking the height of the fraction spread on a silica gel plate as a selection standard, and the fractions with close spread height are combined to obtain 4 components Fr 4-1-4; section Fr 4-1 selects a developing solvent that is dichloromethane-methanol 12, section Fr4-2 selects a developing solvent that is dichloromethane-methanol 12;
selecting 3 components with the maximum extraction amount, sequentially from high polarity to low polarity, fr 4-1, fr4-2, and Fr 4-3.
Fr 4-1 (12.37 g) was separated by normal phase silica gel column chromatography eluting with dichloromethane in the ratio: methanol 10: 1.8: 1, compound 2 (8 mg) and 6 (12 mg) were obtained. Fraction Fr4-2 (9.12 g) was isolated on a silica gel column eluting with a dichloromethane-methanol gradient with the elution ratio dichloromethane: 30 parts of methanol: 1. 20: 1. 15: 1. 10: 1.5: 1; further purification by semi-preparative HPLC with 51% methanol afforded compounds 3 (2.2 mg) and 10 (5.4 mg).
Separating a component Fr4-3 (6.34 mg) by a Sephadex LH-20 column (1: 1), combining the fractions which are closely developed according to the preliminary judgment of thin layer chromatography by taking the height of the fraction developed on a silica gel plate as a selection standard, and combining to obtain 5 components in total, wherein a developing agent selected from a Fr 4-3-1 part is dichloromethane-methanol 10, a developing agent selected from a Fr 4-3-2 part is dichloromethane-methanol 10, a developing agent selected from a Fr4-3-3 part is dichloromethane-methanol 10;
selecting 2 components with the most extraction amount, sequentially from high polarity to low polarity, fr 4-3-1 and Fr 4-3-3.
Fr 4-3-1 (1.34 g) was separated by normal phase silica gel column chromatography, gradient eluted with dichloromethane-methanol (20: 1-5: 1) in the ratio dichloromethane: 30 parts of methanol: 1. 20: 1. 15: 1. 10: 1.5: 1; compound 9 (2.2 mg) (10 eluted fraction) and 12 (5.4 mg) (5 eluted fraction).
Fr4-3-3 (2.54 g) was separated by normal phase silica gel column chromatography, and eluted with a dichloromethane-methanol (20: 1 to 5: 1) gradient at a ratio of dichloromethane: 30 parts of methanol: 1. 20: 1. 15: 1. 10: 1.5: 1; according to the preliminary judgment of thin layer chromatography, the height of the fractions developed on a silica gel plate is taken as a selection standard, the fractions with similar development heights are combined to obtain 4 components Fr 4-3-3-1 to 4-3-4, a developing agent selected from Fr 4-3-3-1 part is dichloromethane-methanol 10, a developing agent selected from Fr 4-3-3-2 part is dichloromethane-methanol 10, a developing agent selected from Fr4-3-3-3 part is dichloromethane-methanol 10, a developing agent selected from Fr 4-3-3-4 part is dichloromethane-methanol 10;
selecting 2 components with the maximum extraction amount, sequentially from high polarity to low polarity, fr4-3-3-3 and Fr 4-3-3-1.
Fr 4-3-3-1 was further purified by semi-preparative high performance liquid chromatography with 40% methanol to give compounds 13 (1.6 mg) and 14 (1.1 mg). Fr4-3-3-3 was further purified by semi-preparative HPLC using 45% methanol to give compound 5 (1.7 mg).
Fr 3 (61.15 g) was separated on an MCI column and eluted with a gradient of methanol-water solvent system (50% methanol, 60% methanol, 70% methanol, 80% methanol, 90% methanol); the 70% methanol eluted fraction was further purified by preparative high performance liquid chromatography with 39% methanol to give compound 17 (1.3 mg).
Mixing the compound 1-17 monomers obtained in the above steps to obtain Callicarpa nudiflora extract A.
Example 2
The preparation method is the same as example 1, 3.4mg of compound 7, 1.8mg of compound 8 and 2.1mg of compound 14 are obtained, and the obtained compounds 7, 8 and 14 are mixed to obtain nakedflower beautyberry extract B.
Example 3 structural identification
Compound 1: the mixture is a colorless oily substance,molecular formula C 19 H 30 O 7 Na，ESI-MS m/z:393.1[M+Na]+.1H-NMR (600MHz, CD3OD) delta: 5.89 (1H, br s, H-4), 5.79 (1H, dd, J =15.4,6.5Hz, H-8), 5.65 (1H, dd, J =15.4,9.2Hz, H-9), 4.41 (1H, t, J =6.5Hz, H-1 '), 4.36 (1H, d, J =7.8Hz, H-7), 3.83 (1H, m, H-6 '), 3.67 (1H, dd, J =11.9,5.4Hz, H-6 '), 3.36 (1H, m, H-5 '), 3.29 (m, 6H, H-4 '), 3.22 (1H, m, H-3 '), 3.18 (1H, m, H-2 '), 2.69 (1H, d, J =9.3Hz, H-6), 2.44 (1H, d, J =16.8Hz, H-2), 2.06 (1H, d, J =16.8Hz, H-2), 1.95 (3H, s, H-11), 1.30 (3H, d, J =4.8Hz, H-10), 1.04 (3H, s, H-11), 1.01 (3H, s, H-12); 13C-NMR (150MHz, CD3OD) delta: 37.1 (C-1), 48.3 (C-2), 202.1 (C-3), 126.1 (C-4), 165.9 (C-5), 56.8 (C-6), 77.0 (C-7), 138.2 (C-8), 128.8 (C-9), 21.0 (C-10), 23.8 (C-11), 28.0 (C-12), 27.6 (C-13), 102.5 (C-1 '), 75.3 (C-2'), 78.0 (C-3 '), 71.5 (C-4'), 78.1 (C-5 '), 62.7 (C-6'). The above data are consistent with literature reports (Zhu LH, bao TH, deng Y, et al. Constitutents from Apium graveolens and the same anti-inflammatory effects [ J]J Asian Nat Prod Res,2017,19, 1079-1086), so this compound was identified as (6s, 7r) -3-oxo-megastigma-4,8-dien-7-O- β -D-glucoside.
Compound 2: amorphous powder of formula C 19 H 32 O 7 Na，ESI-MS m/z:395.2[M+Na]+.1H-NMR (600MHz, CD3OD) delta: 4.37 (1H, d, J =7.8Hz, H-1 '), 3.92 (1H, m, H-9), 3.87 (1H, dd, J =5.1,2.4Hz, H-6 '), 3.67 (1H, dd, J =11.8,5.3Hz, H-6 '), 3.36 (1H, d, J =8.7Hz, H-3 '), 3.34 (1H, d, J =1.3Hz, H-4 '), 3.28 (1H, m, H-5 '), 3.19 (1H, dd, J =4.3,1.5Hz, H-2 '), 2.55 (1H, m, H-7), 2.45 (1H, m, H-3), 2.32 (1H, td, J =12.1,5.9Hz, H-7), 1.83 (1H, m, H-2), 1.77 (3H, s, H-13), 1.68 (1H, m, H-8), 1.31 (1H, d, J =6.3Hz, H-10), 1.21 (3H, s, H-11), 1.21 (3H, s, H-12); 13C-NMR (150MHz, CD3OD) delta: 37.7 (C-1), 38.4 (C-2), 35.1 (C-3), 201.6 (C-4), 131.7 (C-5), 168.5 (C-6), 27.1 (C-7), 36.4 (C-8), 77.7 (C-9), 21.8 (C-10), 27.2 (C-11), 27.6 (C-12), 11.8 (C-13), 104.1 (C-1 '), 75.4 (C-2'), 77.9 (C-3 '), 71.7 (C-4'), 78.3 (C-5 '), 62.8 (C-6'). The above data are consistent with literature reports (Nguyen TVT, tran T M, nguyen TL, et al]Natural Product Communications,2019,14 (6), 1-4), and the compound was identified as phoebenoside A.
Compound 3: white powder of formula C 19 H 30 O7Na，FAB-MS m/z:393.2[M+Na]+.1H-NMR (600MHz, CD3OD) delta: 5.89 (1H, br s, H-4), 5.79 (1H, dd, J =15.4,6.5Hz, H-8), 5.66 (1H, dd, J =15.4,9.3Hz, H-7), 4.41 (1H, m, H-9), 4.36 (1H, d, J =7.8Hz, H-1 '), 3.83 (1H, dd, J =11.8,2.4Hz, H-6 '), 3.67 (1H, dd, J =11.7,5.4Hz, H-6 '), 3.37 (1H, m, H-3 '), 3.34 (1H, d, J =8.9Hz, H-4 '), 3.30 (1h, d, j =9.2hz, H-2 '), 3.18 (1h, m, H-5 '), 2.69 (1h, d, j =9.3hz, H-6), 2.44 (1h, d, j =16.8hz, H-2), 2.06 (1h, d, j =16.8hz, H-2), 1.95 (3h, d, j =1.3hz, ch3-13), 1.30 (3h, d, j =6.4hz, ch3-10), 1.04 (3h, s, ch3-12), 1.02 (3h, s, ch3-11); 13C-NMR (150MHz, CD3OD) delta: 37.1 (C-1), 48.3 (C-2), 202.1 (C-3), 126.1 (C-4), 165.9 (C-5), 56.8 (C-6), 128.8 (C-7), 138.2 (C-8), 77.0 (C-9), 21.0 (C-10), 27.6 (C-11), 28.0 (C-12), 23.8 (C-13), 102.5 (C-1 '), 75.3 (C-2'), 78.1 (C-3 '), 71.5 (C-4'), 78.0 (C-5 '), 62.7 (C-6'). The above data are consistent with literature reports (Kuang H X, yang B Y, xia Y G, et al chemical constraints from the flow of Datura meter L [ J ]]Arch Pharm Res,2008,31, 1094-7.), so the compound was identified as (6R, 9R) -3-oxo- α -ionol-9-O- β -D-glucopyranoside.
Compound 4: reddish brown oily solid of formula C 19 H 31 O 7 ，FAB-MS m/z:371.2[M－H]-.1H-NMR (600MHz, CD3OD) delta: 5.81 (1H, s, H-4), 4.33 (1H, d, J =7.8Hz, H-1 '), 3.85 (1H, d, J =1.6Hz, H-6 '), 3.83 (1H, m, H-9), 3.66 (1H, m, H-6 '), 2.50 (1H, d, J =17.4Hz, H-2), 2.06 (3H, d, J =1.4Hz, H-13), 2.00 (1H, m, H-6), 1.98 (1H, d, J =5.8Hz, H-2), 1.81 (1H, m, H-7), 1.70 (1H, m, H-8), 1.68 (1H, m, H-7), 1.62 (1H, 1,8, 1.11H-8, 3H-12, 11H-12H-8), 1.11H-13); 13C-NMR (150MHz, CD3OD) delta: 37.4 (C-1), 48.1 (C-2), 202.4 (C-3), 125.4 (C-4), 169.9 (C-5), 52.6 (C-6), 26.7 (C-7), 37.4 (C-8), 77.9 (C-9), 21.9 (C-10), 29.0 (C-11), 27.5 (C-12), 25.0 (C-13), 104.0 (C-1 '), 75.3 (C-2'), 78.2 (C-3 '), 71.7 (C-4'), 77.6 (C-5 '), 62.8 (C-6'). The above spectral data are consistent with literature reports (Matsunami K, otsuka H, takeda Y. Structural derivatives of blumenol C glucoside and byzantioside B [ J]Chem Pharm Bull (Tokyo), 2010,58, 438-41), so the compound was identified as blumenol C glucoside.
Compound 5: white powder with molecular formula C 29 H 37 O 15 ，ESI-MS m/z:625.2[M+H]+.1H-NMR (600MHz, CD3OD) delta: 1.11 (3H, d, J =6.2Hz, rha-H-6 '), 2.81 (2H, m, H-7), 3.31-4.07 (12H, m, sugar-H, H-8), 4.40 (1H, d, J =7.9Hz, glc-H-1'), 5.21 (1H, s, rha-H-1 '), 6.29 (1H, d, J =15.9Hz, H-alpha), 6.58 (1H, dd, J =2.1,8.0Hz, H-6), 6.69 (1H, d, J =8.0Hz, H-5), 6.72 (1H, d, J =2.0Hz, H-2), 6.80 (1H, d, J =8.2Hz, H-5'), 6.97 (1H, dd, J =2.1,8.2Hz, H-6 '), 7.07 (1H, d, J =2.1Hz, H-2'), 7.61 (1H, d, J =15.6Hz, H-beta); <xnotran> 13C-NMR (150MHz,CD3OD) δ:131.5 (C-1), 117.1 (C-2), 146.8 (C-3), 146.1 (C-4), 116.5 (C-5), 121.3 (C-6), 36.6 (C-7), 72.3 (C-8), 127.7 (C-1 '), 114.7 (C-2'), 146.8 (C-3 '), 149.8 (C-4'), 116.5 (C-5 '), 123.2 (C-6'), 115.2 (C- α), 144.7 (C- β), 168.3 (C = O), 104.2 (C-1 ″), 76.0 (C-2 ″), 81.6 (C-3 ″), 70.4 (C-4 ″), 76.2 (C-5 ″), 62.3 (C-6 ″), 103.0 (C-1 ″ '), 72.3 (C-2 ″'), 72.0 (C-3 ″ '), 74.0 (C-4 ″'), 70.6 (C-5 ″ '), 18.5 (C-6 ″'). </xnotran> The above data are consistent with literature reports (Su B N, ma L P, jia Z J. Iridoid and phenyl propanoid glycosides from Pediculararisalaeri [ J ]]Planta Med,1998,64 (8): 720-723.) the compound was identified as an isoverbascoside.
Compound 6: amorphous powder of formula C 34 H 43 O 19 ；FAB-MS m/z:755.2[M–H]-。1H-NMR(600MHz,CD3OD)δ:7.60(1H,d,J＝15.9Hz,H-β'),7.08(1H,d,J＝2.1Hz,H-2””),6.99(1H,dd,J＝8.2,2.1Hz,H-6””),6.82(1H,d,J＝8.2Hz,H-5””),6.71(1H,d,J＝2.1Hz,H-2),6.69(1H,d,J＝8.0Hz,H-5),6.57(1H,dd,J＝8.1,2.1Hz,H-6),6.26(1H,d,J＝15.9Hz,H-α'),5.29(1H,d,J＝1.8Hz,H-1”),5.22(1H,d,J＝2.7Hz,H-1”'),4.94(1H,t,J＝9.6Hz,H-4'),4.39(1H,d,J＝7.9Hz,H-1'),4.07(1H,m,H-α),3.88(1H,m,H-3'),3.85(1H,d,J＝9.2Hz,H-4”'),3.74(1H,m,H-α),3.68(1H,dd,J＝9.4,3.3Hz,H-3”),3.63(1H,s,H-5”'),3.56(1H,m,H-5'),3.42(1H,m,H-4”),2.80(2H,m,H-β),1.13(3H,d,J＝6.2Hz,H-6”)；13C-NMR(150MHz,CD3OD)δ:131.5(C-1),116.2(C-2),146.1(C-3),144.6(C-4),117.0(C-5),121.2(C-6),72.2(C-α),36.5(C-β),104.1(C-1′),75.9(C-2′),80.4(C-3′),70.3(C-4′),76.4(C-5′),62.2(C-6′),102.1(C-1″),72.4(C-2″),80.1(C-3″),72.5(C-4″),68.8(C-5″),18.7(C-6″),111.4 <xnotran> (C-1 ″ '), 78.5 (C-2 ″ '), 80.0 (C-3 ″ '), 74.8 (C-4 ″ '), 65.7 (C-5 ″ '), 127.6 (C-1 ″ ″), 115.2 (C-2 ″ ″), 146.9 (C-3 ″ ″), 149.9 (C-4 ″ ″), 116.6 (C-5 ″ ″), 123.3 (C-6 ″ ″), 114.6 (C- α '), 148.0 (C- β '), 168.2 (C = O). </xnotran> The spectral data are consistent with literature reports (
Figure BDA0003898227050000201
,Bedir E,Kirmizibekmez H,et al.Secondary metabolites from Phlomisoppositiflora[J]Nat Prod Res,2005, 19.
Compound 7: white powder; molecular formula C 31 H 40 O 15 Na；ESI-MS m/z:675.2[M+Na]+.1H-NMR (500MHz, CD3OD) delta: 7.67 (1H, d, J =15.9Hz, H-alpha '), 7.20 (1H, d, J =1.9Hz, H-2 '), 7.09 (1H, dd, J =8.2,2.0Hz, H-6 '), 6.83 (1H, d, J =7.8Hz, H-5 '), 6.82 (1H, d, J =2.4Hz, H-2), 6.74 (1H, d, J =8.1Hz, H-5), 6.69 (1H, dd, J =8.2,2.1Hz, H-6), 6.38 (1H, d, J =15.9Hz, H-beta '), 5.21 (1H, s, H-1), 4.93 (1H, t, J =9.3Hz, H-4 "), 4.39 (1H, d, J =7.9Hz, H-1"), 4.07 (1H, t, J =9.7Hz, H-3 "), 3.89 (3H, s, OCH3), 3.85 (1H, m, H-3), 3.82 (3H, s, OCH3), 2.83 (2H, m, H- α), 1.11 (3H, d, J =6.2Hz, H-6); <xnotran> 13C-NMR (150MHz,CD3OD) δ:132.9 (C-1), 112.8 (C-2), 147.9 (C-3), 147.5 (C-4), 117.1 (C-5), 121.1 (C-6), 36.5 (C-7), 72.3 (C-8), 127.6 (C-1 '), 111.8 (C-2'), 147.4 (C-3 '), 149.4 (C-4'), 116.5 (C-5 '), 124.4 (C-6'), 149.4 (C-7 '), 115.1 (C-8'), 168.3 (C-9 '), 104.2 (C-1 ″), 76.0 (C-2 ″), 81.5 (C-3 ″), 70.4 (C-4 ″), 76.0 (C-5 ″), 62.4 (C-6 ″), 103.0 (C-1 ″'), 72.1 (C-2 ″ '), 72.3 (C-3 ″'), 73.8 (C-4 ″ '), 70.6 (C-5 ″'), 18.4 (C-6 ″ '), 56.4 (3' -OCH 3), 56.5 (3-OCH 3). </xnotran> The data are consistent with the literature reports (Chenyi, leaf color cloud, zhaoyong, guangfeng, chemical component research of phenethyl alcohol in Fangfeng) [ J]Chinese herbal medicine, 2017,48 (19): 3941-3944), so that the compound is identified as cistanoside D.
Compound 8: a brown-yellow oil; molecular formula C 31 H 40 O 15 Na；ESI-MS:m/z:675.2[M+Na]+。1H-NMR(CD3OD,600MHz)δ:7.67(1H,d,J＝15.9Hz,H-7”'),7.21(1H,d,J＝2.0Hz,H-2”'),7.09(1H,dd,J＝8.2,2.0Hz,H-6”'),6.82(2H,dd,J＝8.2,10.0Hz,H-5,5”'),6.75(1H,d,J＝2.1Hz,H-2), 6.70 (1H, dd, J =8.2,2.2Hz, H-6), 6.38 (1H, d, J =15.9Hz, H-8' "), 5.21 (1H, brs, H-1"), 4.93 (1H, t, J =9.4Hz, H-4 '), 4.39 (1H, d, J =7.9Hz, H-1 '), 3.90 (3H, s, OCH3), 3.82 (3H, s, OCH3), 2.83 (2H, m, H-8), 1.11 (3H, d, J =6.2Hz, H-6 "); <xnotran> 13C-NMR (CD 3OD,150 MHz) δ:168.3 (C-9 "'), 151.1 (C-4"'), 149.5 (C-3 "'), 147.9 (C-7"'), 147.6 (C-3), 147.4 (C-4), 132.9 (C-1), 127.5 (C-1 "'), 124.4 (C-6"'), 121.1 (C-6), 117.1 (C-5), 116.5 (C-5 "'), 115.0 (C-8"'), 112.8 (C-2), 111.7 (C-2 "'), 104.2 (C-1'), 103.0 (C-1"), 81.5 (C-3 '), 76.2 (C-2'), 76.1 (C-5 '), 73.8 (C-4 "), 72.4 (C-2"), 72.1 (C-7), 72.1 (C-3 "), 70.6 (C-4'), 70.4 (C-5"), 62.4 (C-6 '), 56.5 (4-OCH 3), 56.4 (4 "' -OCH 3), 36.6 (C-8), 18.4 (C-6"). </xnotran> The above data are consistent with literature reports (TeborgD, junior P. Martynoside and the novel dimeric openchain glucoside digipen-strand from Penstemondigitas [ J)]Planta Med,1989,55, 474-476), so the compound was identified as digitonin.
Compound 9: a brown oil; molecular formula C 23 H 25 O 11 ；ESI-MS m/z 477[M-H]-.1H-NMR (CD 3OD,600 MHz) delta: 7.58 (1H, d, J =2.3Hz, H-7 "), 7.04 (1H, d, J =2.3Hz, H-2"), 6.90 (1H, dd, J =8.2,2.3Hz, H-6 "), 6.79 (1H, d, J =8.2Hz, H-5"), 6.68 (1H, d, J =2.3Hz, H-2), 6.65 (1H, d, J =8.1Hz, H-5), 6.64 (1H, d, J =15.4Hz, H-8 '), 6.55 (1H, dd, J =8.0,2.3Hz, H-6), 4.51 (1H, dd, J =2.4,12.0Hz, H-6' a), 4.35 (m, 1H), 4.33 (1H, t, H-6' b), 3.96 (1H, m, H-alpha), 3.72 (1H, m, H-alpha), 2.80 (2H, m, H-beta); <xnotran> 13C-NMR (CD 3OD,150 MHz) δ:131.4 (C-1), 116.5 (C-2), 146.8 (C-3), 146.1 (C-4), 116.3 (C-5), 121.2 (C-6), 72.4 (C- α), 36.7 (C- β), 104.6 (C-1 '), 75.1 (C-2'), 77.9 (C-3 '), 71.7 (C-4'), 75.5 (C-5 '), 64.6 (C-6'), 127.7 (C-1 ″), 114.8 (C-2 ″), 147.2 (C-3 ″), 149.6 (C-4 ″), 117.1 (C-5 ″), 123.2 (C-6 ″), 144.7 (C-7 ″), 115.0 (C-8 ″), 169.2 (C-9 ″). </xnotran> The above data are substantially consistent with the data reported in the literature (
Figure BDA0003898227050000213
T,
Figure BDA0003898227050000211
D,
Figure BDA0003898227050000212
Phenylethanoid glycosides fromScutellariagalericulata[J]Turk J Chem,2002,26 (4): 465-471.), and the compound was identified as calcoelarioside B.
Compound 10: white powder of formula C 26 H 31 O 11 ，FAB-MS m/z:519.2[M－H]-.1H-NMR (600MHz, CD3OD) delta: 7.15 (1H, dd, J =8.3,1.5Hz, H-5), 7.04 (1H, d, J =2.0Hz, H-2), 6.95 (1H, d, J =1.9Hz, H-2 '), 6.93 (1H, dd, J =8.4,2.0Hz, H-6), 6.82 (1H, dd, J =8.1,2.0Hz, H-6 '), 6.77 (1H, d, J =8.1Hz, H-5 '), 4.72 (1H, d, J =4.4Hz, H-1 '), 4.24-4.275 (2H, m, H-9 '), 3.88 (3H, s, OCH3), 3.86 (1H, s, OCH3), 3.85 (s, 0H), 3.69 (2H, m, H-9 '), 3.49 (1H, m, H-6' a), 3.47 (1H, m, H-6' b), 3.39-3.40 (4H, m, H-2' -5 '), 3.39 (d, J =1.3Hz, 0H), 3.14 (2H, m, H-8 '); <xnotran> 13C-NMR (150MHz,CD3OD) δ:137.5 (C-1), 111.6 (C-2), 147.5 (C-3), 151.0 (C-4), 118.0 (C-5), 119.8 (C-6), 87.1 (C-7), 55.5 (C-8), 72.7 (C-9), 133.7 (C-1 '), 111.0 (C-2'), 147.4 (C-3 '), 149.1 (C-4'), 116.1 (C-5 '), 120.1 (C-6'), 87.5 (C-7 '), 55.4 (C-8'), 72.7 (C-9 '), 56.4 (4-OCH 3), 56.7 (4' -OCH 3), 102.9 (C-1 ″), 74.9 (C-2 ″), 77.8 (C-3 ″), 71.3 (C-4 ″), 78.2 (C-5 ″), 62.5 (C-6 ″). </xnotran> The above data are consistent with the literature reports (Lining, tan Ninghua, zhoujun. Curculigo orchioides A new lignan glycoside (English) [ J ]]Yunnan plant research, 2003 (06): 711-716.), so that the compound was identified as 4,4' -methoxy-3 ' -hydroxy-7', 9-diazoxyignan-3-O-beta-D-glucopyranoside.
Compound 11 is a pale yellow powder of formula C 21 H 17 O 12 ，ESI-MS m/z:462.1[M-H] - 。 1 H-NMR(600MHz,CD 3 OD)δ:7.45(1H,dd,J＝8.4,2.1Hz,H-6′),7.43(1H,d,J＝2.2Hz,H-2′),6.94(1H,d,J＝8.3Hz,H-5′),6.79(1H,d,J＝2.2Hz,H-8),6.64(1H,s,H-3),6.51(1H,d,J＝2.2Hz,H-6),5.21(1H,d,J＝7.9Hz,H-1″),3.56–4.19(4H,m,H-2″-H-5″)； 13 C-NMR(150MHz,CD 3 OD)δ:166.9(C-2),104.2(C-3),184.1(C-4),163.0(C-5),101.4(C-6),164.4(C-7),96.0(C-8),158.9(C-9),107.3(C-10),123.5(C-1′),114.3(C-2′),147.1(C-3′),151.2(C-4′),116.8(C-5′),120.5(C-6′),101.0(C-1″),74.4(C-2 '), 77.0 (C-3 '), 72.8 (C-4 '), 76.7 (C-5 '), 170.8 (C-6 '). The above data are consistent with literature reports (Rabelo A S, oliveira I)
Figure BDA0003898227050000221
A G et al.Antinociceptive,anti-inflammatory and antioxidant activities of aqueous extract from Remirea maritima(Cyperaceae).[J]J Ethnopharmacol,2013, 145), so that the compound was identified as luteolin-7-O-glucuronide.
Compound 12: pale yellow powder, molecular formula C 21 H 19 O 11 ，ESI-MS m/z:447[M－H]-.1H-NMR (600MHz, CD3OD) delta: 12.99 (1H, s, 5-OH), 7.47 (1H, dd, J =8.5,2.3Hz, H-6 '), 7.45 (1H, d, J =2.2Hz, H-2'), 7.33 (1H, d, J =8.5Hz, H-5 '), 6.62 (1H, s, H-3), 6.46 (1H, d, J =2.1Hz, H-8), 6.22 (1H, d, J =2.1Hz, H-6), 4.95 (1H, d, J =7.6Hz, H-1'), 3.93-3.44 (1H, m, sugar-H); 13C-NMR (150MHz, CD3OD) delta: 165.5 (C-2), 103.2 (C-3), 183.8 (C-4), 159.5 (C-5), 100.3 (C-6), 166.3 (C-7), 95.1 (C-8), 163.3 (C-9), 105.4 (C-10), 119.8 (C-1 '), 114.9 (C-2 '), 148.7 (C-3 '), 150.0 (C-4 '), 118.0 (C-5 '), 127.3 (C-6 '), 105.1 (C-1 "), 71.3 (C-2 '), 74.8 (C-3 '), 78.5 (C-4 '), 77.5 (C-5 '), 62.4 (C-6.4 '). The data are consistent with the literature reports (Lepidium minor, haoyang, and so on. Research on chemical compositions of duckweed [ J ]]Chinese herbal medicine, 1999 (2): 88-90.), so the compound was identified as luteolin-7-O-beta-D-glucoside.
Compound 13: light yellow powder, formula C 21 H 19 O 11 ，ESI-MS m/z:447[M－H]-.1H-NMR (600MHz, CD3OD) delta: 12.92 (1H, s, 5-OH), 7.47 (1H, d, J =8.5Hz, H-6 '), 7.45 (1H, brs, H-2'), 7.33 (1H, d, J =8.5Hz, H-5 '), 6.62 (1H, s, H-3), 6.46 (1H, d, J =2.1Hz, H-8), 6.22 (1H, d, J =2.1Hz, H-6), 4.95 (1H, d, J =7.6Hz, H-1'), 3.93-3.44 (6H, m, H-2 '-6'); <xnotran> 13C-NMR (150MHz,CD3OD) δ:183.8 (C-4), 166.3 (C-2), 165.5 (C-7), 163.3 (C-5), 159.5 (C-9), 150.0 (C-4 '), 148.7 (C-3'), 127.3 (C-1 '), 119.8 (C-6'), 118.0 (C-5 '), 114.9 (C-2'), 105.4 (C-10), 105.1 (C-3), 103.2 (C-1 ″), 100.3 (C-6), 95.1 (C-8), 78.5 (C-3 ″), 77.5 (C-5 ″), 74.8 (C-2 ″), 71.3 (C-4 ″), 62.4 (C-6 ″). </xnotran> The above data are consistent with the reports in the literature (Jimin, li Shujuan, ma super beautiful, narrow leaf blue basin flower)Research on sequential chemical components and their antioxidant and alpha-glucosidase inhibitory activities [ J]The university of inner Mongolia journal (Nature science edition), 2014,45 (4): 398-403), so the compound was identified as luteolin-4' -O-beta-D-glucoside.
Compound 14: light yellow powder, formula C 21 H 19 O 10 ，ESI-MS m/z:447[M－H]-.1H-NMR (600MHz, CD3OD) delta 7.90 (2H, d, J =8.4Hz, H-6', H-2'), 6.94 (2H, d, J =8.4Hz, H-5', H-3'), 6.84 (1H, s, H-3), 6.67 (1H, s, H-8), 6.51 (1H, d, J =2.0Hz, H-6), 5.08 (1H, d, J =6.3Hz, H-1 "); 13C-NMR (150MHz, CD3OD) delta: 166.8 (C-2), 104.1 (C-3), 184.1 (C-4), 159.0 (C-5), 101.2 (C-6), 164.8 (C-7), 96.1 (C-8), 162.9 (C-9), 107.1 (C-10), 123.0 (C-1 '), 129.7 (C-2 ',6 '), 117.1 (C-3 ',5 '), 163.2 (C-4 '), 101.6 (C-1 "), 74.7 (C-2"), 77.9 (C-3 '), 71.3 (C-4 '), 78.4 (C-5 '), 62.5 (C-6). The above data are consistent with the literature reports (Dongchun, yangyun, qianshui, etc.. The study of chemical composition of aerial part of Ligusticum wallichii [ J)]China journal of Chinese materia medica, 2007,2 (4): 1418.), so the compound is identified as cosmosiin.
Compound 15: pale yellow powder, molecular formula C 23 H 21 O 12 ，ESI-MS m/z:489[M－H]-.1H-NMR (600mhz, cd3od) δ:7.43 (1H, dd, j =8.3,2.3hz, H-6 '), 7.41 (1H, d, j =2.2hz, H-2 '), 6.91 (1H, d, j =8.3hz, H-5 '), 6.77 (1H, d, j =2.2hz, H-8), 6.61 (1H, s, H-3), 6.47 (1H, d, j =2.2hz, H-6), 5.30 (1H d, j =1.8hz, H-1 "'), 5.21 (1H, d, j 7.7hz, H-1"), 1.34 (3h, d, j =6.2hz, H-6 "'); <xnotran> 13C-NMR (150MHz,CD3OD) δ:184.0 (C-4), 166.9 (C-2), 164,4 (C-7), 163,0 (C-5), 159,1 (C-9), 151,3 (C-4 '), 147,1 (C-3'), 123,5 (C-1 '), 120,5 (C-6'), 116,8 (C-5 '), 114,3 (C-2'), 107,1 (C-10), 104,2 (C-3), 102,6 (C-1 ″ '), 100,9 (C-6), 99,8 (C-1 ″), 95,9 (C-8), 79,1 (C-5 ″), 79,0 (C-3 ″), 78,3 (C-2 ″), 74,0 (C-4 ″'), 72,2 (C-3 ″ '), 72,2 (C-2 ″'), 71,4 (C-4 ″), 70,0 (C-5 ″ '), 62,4 (C-6 ″), 18,3 (C-6 ″'). </xnotran> The above data are consistent with literature reports (Raimundo R G, nascimento, jackelyne A, et al. New flovones from Margaritopsis scarrasoana with antioxidant activity [ J)]Quinimica Nova,2015,38 (1), 60-65.), so the compound was identified as luteolin-7-O- β -L-rhamnopyranosyl (1, 2) - β -D-glucopyranoside.
Compound 16: yellow powder, formula C 27 H 29 O 14 ，ESI-MS m/z:577.2[M－H]-.1H-NMR (CD 3OD,600 MHz) delta: 7.78 (2H, d, H-2',6 '), 6.94 (2H, m, H-3',5 '), 6.79 (1H, d, J =2.2Hz, H-8), 6.66 (1H, s, H-3), 6.46 (1H, d, J =2.2Hz, H-6), 5.30 (1H, d, J =1.8Hz, H-1 '), 5.21 (1H, d, J =7.6Hz, H-1 '), 1.34 (3H, d, J =6.2Hz, H-6 '); <xnotran> 13C-NMR (CD 3OD,150 MHz) δ:184.0 (C-4), 166.8 (C-7), 164.4 (C-2), 163.0 (C-5), 162.9 (C-4 '), 159.0 (C-9), 129.6 (C-2', 6 '), 123.0 (C-1'), 117.1 (C-3 ', 5'), 107.0 (C-10), 104.1 (C-3), 102.5 (C-6), 101.0 (C-1 ″ '), 99.8 (C-1 ″), 95.9 (C-8), 79.1 (C-2 ″), 79.0 (C-3 ″), 78.3 (C-5 ″), 74.0 (C-4 ″'), 72.2 (C-2 ″ '), 72.2 (C-3 ″'), 71.4 (C-4 ″), 70.0 (C-5 ″ '), 62.4 (C-6 ″), 18.3 (C-6 ″'). </xnotran> The above data are basically consistent with the literature reports (Chenlin, tocaipeng, hanjiaxin, etc.. Chemical compositions of Piper methysticum and its cholinesterase activity study [ J)]The research and development of natural products, 2018,30 (9): 1569), so the compound is identified to be apigenin-7-O-beta-D-neohesperidin.
Compound 17: yellow powder, formula C 20 H 29 O 4 ，ESI-MS m/z:333.2[M－H]-.1H-NMR (CD 3OD,600 MHz) delta: 7.73 (2H, dd, J =5.7,3.3Hz, H-3,6), 7.63 (2H, dd, J =5.8,3.3Hz, H-4, 5), 4.30 (4H, m, H-1 '), 4.23 (2H, m, H-1 "), 1.72 (3H, m, H-2', 2"), 1.45 (4H, m, H-3', a "), 1.36 (6H, m, H-3",4", 5"), 0.95 (9H, m,3 XCH 3, H-4',6", b"); 13C-NMR (CD 3OD,150 MHz) delta 169.3 (CO), 133.6 (C-4), 132.4 (C-5), 130.6 (C-1, 2), 129.9 (C-3, 6), 69.1 (C-1 "), 66.7 (C-1 '), 40.2 (C-2"), 31.7 (C-2'), 31.6 (C-3 "), 30.1 (C-4"), 25.0 (C-a "), 24.0 (C-5"), 20.3 (C-3 '), 14.4 (C-4', 6 "), 11.4 (C-b"). The above-mentioned hydrogen spectrum and carbon spectrum data are consistent with the literature (high sea, yixiangqian, linlin, etc.. Studies on chemical components and structure-activity relationship of marine-derived phthalates [ J]Natural product research and development, 2013,25 (10): 1320-1324.), so the compound was identified as 2-O-butyl-1-O- (2' -ethylhexyl) bezene-1, 8-dicarboxylate.
And (3) purity identification:
Figure BDA0003898227050000241
Figure BDA0003898227050000251
example 3 evaluation of anti-inflammatory Activity
3.1 culture of cells and evaluation of toxicity
BV2 microglia in DMEM medium containing 10% fetal calf serum at 37 ℃ and 5% CO 2 Culturing under the condition, and carrying out passage when the growth reaches 90%. The CCK8 method was used to examine the effect of compounds on BV2 cell survival. Meanwhile, BV2 cells in logarithmic growth phase are inoculated into a 96-well plate with the density of 9 multiplied by 10 per well 3 100 μ L per well, after cells were adherent, the experiment was divided into blank groups; a dexamethasone group; administration groups (25. Mu. Mol/L), 3 multiple wells per group, at 37 ℃ 5% 2 Culturing under the condition for 24h. Adding 10% CCK-8 medium, and culturing at 37 deg.C for 2 hr. Thereafter, the absorbance was measured at 450 nm.
3.2 determination of the NO content
BV2 cells were seeded in 96-well plates at a density of 9X 10 3 A hole. After the cells are attached, the experiment is divided into a blank group, an LPS group, a dexamethasone group and administration groups with different concentrations, and each group is provided with 3 multiple wells. Adding fresh culture solution into blank group, pretreating dexamethasone group and administration group for 3.5h before adding LPS for stimulation, adding final concentration of 1 μ g/mLLPS together with LPS group, culturing for 24 hr, collecting supernatant, and determining strictly according to NO detection kit instruction.
3.3 statistical analysis
Data processing and statistical analysis are carried out by using software GraphPad Prism 8.0; the intensity of the anti-inflammatory activity of the compounds in vitro is expressed as NO inhibition, and each experiment is repeated 3 times, all values being expressed as mean. + -. SD.
3.4 statistics of results
The macrophage plays an important role as an important role in the generation and development process of inflammation, secretes certain proinflammatory factors and anti-inflammatory factors after being invaded by exogenous bodies and the like, and plays an important role in the inflammatory reaction process of an organism, so that inflammation mediators are inhibitedAnd the activation of the associated signaling pathways are important means of alleviating inflammation. NO is a notable inflammatory mediator, and excessive amounts of NO in the central nervous system activate microglia and cause an inflammatory response, with neurotoxicity. It is closely related to the occurrence and development of various inflammatory diseases, and is generally considered as a marker of inflammation generation and used as an in vitro anti-inflammatory drug screening model. We used LPS to stimulate BV2 cells to produce the inflammatory mediator NO to primary screen the isolated compounds for anti-inflammatory activity. First, the CCK8 method was used to determine the effect of compounds on BV2 cell survival to ensure that subsequent experiments were performed under non-toxic conditions. The experimental results show that when the concentration of the compound is 25 mu mol.L -1 All compounds showed a BV2 cell survival rate of more than 80%. Afterwards, we further tested the inhibition effect of different extracts on the generation of NO by BV2 cells induced by LPS using Griess method, and the results are as follows:
Figure BDA0003898227050000261
as can be seen from the data in the table above, compounds 1-17 all exhibited certain anti-inflammatory effects, with compound 7 having significantly better anti-inflammatory effects than the other compounds and dexamethasone. Meanwhile, the anti-inflammatory effect of the callicarpa nudiflora extract A (mixture of compounds 1-17) reaches 50.97 +/-0.04 (25 mu mol. L) -1 ) (%), the anti-inflammatory effect of the nakedflower beautyberry extract A (the mixture of the compounds 7, 8 and 14) reaches 48.64 +/-0.02 (25 mu mol. L) -1 ) (%), which is superior to any monomer compound, proves that the synergistic anti-inflammatory effect is realized after the combination, and the extract has clear components and has obvious advantages compared with the nakedflower beautyberry extract in the prior art.
The above description is only for the purpose of illustrating the preferred embodiments of the present invention and is not to be construed as limiting the invention, and any modifications, equivalents, improvements and the like that fall within the spirit and principle of the present invention are intended to be included therein.

Claims (10)

1. A Callicarpa nudiflora extract is characterized by comprising cistanoside D, diosgenin and cosmosiin.
2. The callicarpa nudiflora extract as claimed in claim 1, wherein the mass ratio of cistanoside D, digitonin and cosmosiin in the callicarpa nudiflora extract is 1-10.
3. The method for preparing the callicarpa nudiflora extract as claimed in any one of claims 1-2, comprising the steps of:
s1, adding water to folium callicarpae nudiflorae for cold soaking, then heating and decocting, combining filtrates, concentrating under reduced pressure to obtain an extract 1, suspending and dispersing the extract with water, adding ethyl acetate for extraction, and recovering a solvent under reduced pressure to obtain an extract 2;
s2, mixing the extract 2 with silica gel, performing silica gel column chromatography coarse separation, and performing gradient elution with dichloromethane-methanol 600 in a ratio of 1-1: 1 to obtain 9 fractions Fr 1-9;
s3 and Fr 6 are separated on an MCI column, gradient elution is carried out by using a methanol-water solvent system of 30-90% methanol, and Fr 6-1-6-5 is obtained by combination;
wherein, fr 6-1 is subjected to silica gel column chromatography, dichloromethane-methanol 30: 1-1: 1 gradient elution is used for obtaining 6 components of Fr 6-1-6-1-6, fr 6-1-3 is subjected to ODS column chromatography, methanol-water 45-50 isocratic elution, and the prepared liquid phase is respectively subjected to isocratic separation by 42-48% methanol and 40-45% methanol to obtain the digitonin; fr 6-4 is separated by normal phase silica gel column chromatography, dichloromethane-methanol is eluted in a gradient way with the ratio of 80: 1-1: 1 to obtain 6 components Fr 6-4-1-6-4-6; separating the prepared liquid phase of Fr 6-4-2 with 52-58% methanol to obtain cistanoside D; selecting 3 components with the most extraction amount, wherein the polarity is from high to low, and the components are Fr 6, fr4 and Fr 3 in sequence;
s4, fr4 is subjected to reversed phase packing YMC column chromatography pressure separation, gradient elution is carried out by using a methanol-water solvent system of 60-95% methanol, and 3 components with the largest extraction amount are selected, wherein the polarity is from high to low, and Fr 4-1, fr4-2 and Fr4-3 in sequence;
wherein, the component Fr4-3 is separated by Sephadex LH-20 column and methanol, 2 components with the largest extraction amount are selected, and Fr 4-3-1 and Fr4-3-3 are sequentially arranged according to polarity from high to low; fr4-3-3 is separated by normal phase silica gel column chromatography, and dichloromethane-methanol is 30:1 to 1: gradient elution is carried out on 1 to obtain 4 components Fr 4-3-3-1 to 4-3-3-5, and isocratic elution is carried out on Fr 4-3-3-1 through a preparation liquid phase by 42-48 percent methanol to obtain the cosmosiin.
4. A callicarpa nudiflora extract is characterized by comprising (6S, 7R) -3-oxo-megastigma-4, 8-dien-7-O-beta-D-glucoside, phoebenoside A, (6R, 9R) -3-oxo-alpha-ionol-9-O-beta-D-glucopyranoside, blunol C-glucoside, isoverbascoside, myricoside, cistanoside D, diosgenin, calceoside B, 4'-dimethoxy-3' -hydroxy-7', 9-dioxolignan-3-O-beta-D-glucopyranoside, luteolin-7-O-glucuronide, xylodin-7-O-beta-D-glucoside, xylodin-4' -O-beta-D-glucoside, neohesperidin-1-O-beta-2-oxypyridyl-1, 1-O-beta-1-isoquercitrin-D-glucoside and neorhinocerin (1, 8-O-beta-xyloside).
5. The callicarpa nudiflora extract as claimed in claim 4, wherein in the callicarpa nudiflora extract, (6S, 7R) -3-oxo-megastigma-4,8-dien-7-O- β -D-glucoside, phoebenoside A, (6R, 9R) -3-oxo- α -ionol-9-O- β -D-glucopyranoside, blunol C-glucoside, acteoside, myricoside, cisoside D, diglucoside, calcolioside B, 4' -dimethoxy-3' -hydroxy-7', the mass ratio of 9-diopoxyignan-3-O- β -D-glucopyranoside, luteolin-7-O- β -D-glucoside, luteolin-4 ' -O- β -D-glucoside, cosmosiin, luteolin-7-O- β -L-rhamnopyranosyl (1, 2) - β -D-glucopyranoside, celery-7-O- β -D-neohesperidin and 2-O-butyl-1-O- (2 ' -ethylhexyl) bezene-1, 8-dicarboxylate is 1-5 to 10-1-10, 0.1-1-5 to 3.5-3-3.5.
6. The method for preparing the callicarpa nudiflora extract as claimed in any one of claims 4 to 5, comprising the steps of:
s1, adding water to folium callicarpae nudiflorae for cold soaking, then heating and decocting, combining filtrates, concentrating under reduced pressure to obtain an extract 1, suspending and dispersing the extract with water, adding ethyl acetate for extraction, and recovering a solvent under reduced pressure to obtain an extract 2;
s2, mixing the extract 2 with silica gel, performing coarse separation by silica gel column chromatography, performing gradient elution by using dichloromethane-methanol 600:1, and selecting 3 components with the largest extraction amount, wherein the components are Fr 6, fr4 and Fr 3 in sequence from high to low according to polarity;
separating S3 and Fr 6 on MCI column, gradient eluting with methanol-water solvent system of 30-90% methanol, selecting 2 components with maximum extraction amount, sequentially Fr 6-1-4 and Fr 6-1-3 according to polarity from high to low;
wherein Fr 6-1 is subjected to silica gel column chromatography, gradient elution is carried out by dichloromethane-methanol 30: 1-1: 1,2 components with the largest extraction amount are selected, and Fr 6-1-4 and Fr 6-1-3 are sequentially carried out according to polarity from high to low;
subjecting Fr 6-1-3 to ODS column chromatography, eluting with methanol-water at 45-50 isocratic, separating the preparative liquid phase with 42-48% methanol and 40-45% methanol at isocratic to obtain compounds 8 and 11; fr 6-1-4, separating the prepared liquid phase with 43-46% methanol and 45-50% methanol respectively to obtain compounds 15 and 16;
fr 6-4 is separated by normal phase silica gel column chromatography, 2 components with the largest extraction amount are selected by dichloromethane-methanol gradient elution from 80:1 to 1:1, and Fr 6-4-2 and Fr 6-4-1 are sequentially selected according to polarity from high to low;
fr 6-4-1 is separated from the liquid phase by 50-55% methanol to obtain compound 1; fr 6-4-2 is separated by preparing liquid phase and separating with 52-58% methanol to obtain compounds 4 and 7;
s4, fr4 is subjected to reversed phase packing YMC column chromatography pressure separation, gradient elution is carried out by using a methanol-water solvent system of 60-95% methanol, and 3 components with the largest extraction amount are selected, wherein the polarity is from high to low, and Fr 4-1, fr4-2 and Fr4-3 in sequence;
wherein Fr 4-1 is separated by normal phase silica gel column chromatography, and dichloromethane-methanol is eluted in a gradient manner with the ratio of 20: 1-1: 1 to obtain compounds 2 and 6; separating the component Fr4-2 in a silica gel column, and performing gradient elution on dichloromethane-methanol at a ratio of 50: 1-5: 1; then passing through a preparation liquid phase with 52-58% methanol to obtain compounds 3 and 10;
separating the component Fr4-3 with Sephadex LH-20 column and methanol, selecting 2 components with the most extraction amount, sequentially Fr 4-3-1 and Fr4-3-3 according to polarity from high to low;
fr 4-3-1 is separated by normal phase silica gel column chromatography, and dichloromethane-methanol is eluted in a gradient way with the ratio of 20: 1-5: 1 to obtain compounds 9 and 12; elution ratio dichloromethane: methanol 10: 1.7: 1; fr4-3-3 is separated by normal phase silica gel column chromatography, and dichloromethane-methanol is 30:1 to 1: gradient elution is carried out by 1,2 components with the largest extraction amount are selected, and according to the polarity from high to low, fr4-3-3-3 and Fr 4-3-3-1 are sequentially selected;
fr 4-3-3-1 is subjected to liquid phase preparation and isocratic elution by 42-48% methanol to obtain compounds 13 and 14; fr4-3-3-3 is purified by a preparative liquid phase through 42-50% methanol to obtain a compound 5;
separating S5 and Fr 3 on MCI column, gradient eluting with methanol-water solvent system containing 40-95% methanol, separating with 45-55% methanol to obtain compound 17;
wherein, the compounds 1 to 17 are respectively (6S, 7R) -3-oxo-megastigma-4, 8-dien-7-O-beta-D-glucoside, phoebenoside A, (6R, 9R) -3-oxo-alpha-ionol-9-O-beta-D-glucopyranoside, blumenol C glucoside, isoverbascoside, myrricoside, cistanoside D, digitoside, calcoelariosidic B, 4' -dimethoxy-3' -hydroxy-7', 9-diethoxyignan-3-O-beta-D-glucopyranoside, luteolin-7-O-glucuronide, luteolin-7-O-beta-D-glucoside, luteolin-4 ' -O-beta-D-glucoside, cosmosin, luteolin-7-O-beta-L-rhamnopyranosyl (1, 2) -beta-D-glucopyranoside, apium graveolens-7-O-beta-D-neohesperidin, 2-O-butyl-1-O- (2 ' -ethylhexyl) bezene-1, 8-dicarboxylate.
7. A pharmaceutical comprising the nakedflower beautyberry extract of any one of claims 1 to 2 and 4 to 5 or the nakedflower beautyberry extract prepared by the preparation method of claim 3 or 6.
8. A method for extracting (6S, 7R) -3-oxo-megastigma-4, 8-dien-7-O-beta-D-glucoside from callicarpa nudiflora is characterized by comprising the following steps:
s1, adding water to folium callicarpae nudiflorae for cold soaking, then heating and decocting, combining filtrates, concentrating under reduced pressure to obtain an extract 1, suspending and dispersing the extract with water, adding ethyl acetate for extraction, and recovering a solvent under reduced pressure to obtain an extract 2;
s2, mixing the extract 2 with silica gel, performing silica gel column chromatography coarse separation, and performing gradient elution with dichloromethane-methanol 600 in a ratio of 1-1: 1 to obtain 9 fractions Fr 1-9;
s3 and Fr 6 are separated on an MCI column, gradient elution is carried out by using a methanol-water solvent system of 30-90% methanol, and Fr 6-1-6-5 is obtained by combination;
wherein, fr 6-1 is subjected to silica gel column chromatography, and dichloromethane-methanol is subjected to gradient elution at a ratio of 30: 1-1: 1 to obtain 6 components Fr 6-1-6-1-6; fr 6-1-4 is purified by a preparation liquid phase to obtain compounds 15 and 16;
separating Fr 6-4 by normal phase silica gel column chromatography, gradient eluting with dichloromethane-methanol 80: 1-1: 1 to obtain 6 components Fr 6-4-1-6-4-4, and purifying Fr 6-4-1 by preparative liquid phase to obtain (6S, 7R) -3-oxo-megastigma-4, 8-dien-7-O-beta-D-glucoside.
9. Use of the callicarpa nudiflora extract as defined in any one of claims 1-2, 4-5 or the callicarpa nudiflora extract prepared by the preparation method as defined in claim 3 or 6, or the extract prepared by the extraction method as defined in claim 8, for the preparation of a medicament for the treatment of inflammation.
10. Application of cistanoside D in preparing antiinflammatory medicine is provided.
CN202211280994.8A 2022-10-19 2022-10-19 Callicarpa nudiflora extract, preparation method and application thereof Pending CN115505014A (en)

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