CN101880306A - Stauntonia brachyanthera Hand-Mazz saponins components as well as preparation method and application thereof - Google Patents
Stauntonia brachyanthera Hand-Mazz saponins components as well as preparation method and application thereof Download PDFInfo
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- CN101880306A CN101880306A CN2010101965155A CN201010196515A CN101880306A CN 101880306 A CN101880306 A CN 101880306A CN 2010101965155 A CN2010101965155 A CN 2010101965155A CN 201010196515 A CN201010196515 A CN 201010196515A CN 101880306 A CN101880306 A CN 101880306A
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- 230000029087 digestion Effects 0.000 description 1
- LOKCTEFSRHRXRJ-UHFFFAOYSA-I dipotassium trisodium dihydrogen phosphate hydrogen phosphate dichloride Chemical compound P(=O)(O)(O)[O-].[K+].P(=O)(O)([O-])[O-].[Na+].[Na+].[Cl-].[K+].[Cl-].[Na+] LOKCTEFSRHRXRJ-UHFFFAOYSA-I 0.000 description 1
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Landscapes
- Pharmaceuticals Containing Other Organic And Inorganic Compounds (AREA)
- Medicines Containing Plant Substances (AREA)
Abstract
The invention relates to a Stauntonia brachyanthera Hand-Mazz component as well as a preparation method and application thereof, belonging to the technical field of medicines. The method comprises the following steps of: extracting Stauntonia brachyanthera Hand-Mazz by using water or organic solvents with different concentrations, filtering and recovering the solvents to obtain a concentrated solution; eluting the concentrated solution by using water and an organic solvent through macroporous absorption resin, recovering the solvent to obtain a saponin, or obtaining the saponin from an n-butyl alcohol layer by using an extracting method; then further separating and purifying the saponin by using a normal-phase silicagel column, a reversed phase column and a high efficient liquid phase preparation system; and finally obtaining a monomer or compound with remarkable activity. The Stauntonia brachyanthera Hand-Mazz component of the invention can be singly or combined with other drugs to be used as a raw drug applied to a drug for resisting tumour, resisting inflammation, promoting urination, and treating other diseases. The Stauntonia brachyanthera Hand-Mazz component prepared with the method has high purity, remarkable pharmacological action, simple and convenient method, practicability, low price, safety, suitability for industrialized production.
Description
Technical field
The invention belongs to medical technical field, relate to a kind of extraction preparation method and application thereof of medicine material, be specifically related to the preparation method and the application thereof of Stauntonia brachyanthera Hand-Mazz saponin component.
Background technology
Stauntonia brachyanthera Hand-Mazz (Stauntonia brachyanthera Hand-Mazz) is a Lardizabalaceae Stem and leaf of False Lychee platymiscium, in provinces (district) such as China Guizhou, Guangxi distribution is arranged, be evergreen woody climber, mainly to do akebi medicinal among the people for its stem, have effects such as clearing away heat and promoting diuresis, clearing and activating the channels and collaterals, analgesia apocenosis, logical breast, identical with the akebi effect; Fruit morphology and Fruit of Threeleaf Akebia are basic identical, and difference is to ftracture after the Fruit of Threeleaf Akebia maturation, so have another name called Fructus Akebiae, YANGKAIKOU, do not ftracture after the Stauntonia brachyanthera Hand-Mazz maturation, and the look Huang, it is among the people that also it to be used as Fruit of Threeleaf Akebia medicinal; Pericarp contains numerous stone cells, the calculus that is used among the people.Leaf also is used as medicine among the people, is used to catch cold, and it is uncomfortable in chest to have a headache, vomiting and diarrhoea abdominal distension, and the Stauntonia brachyanthera Hand-Mazz whole plants all has certain pharmaceutical use.(Xie Zongwan, Yu Youqin. national herbal medicine name mirror (first volume) [M]. Beijing: the People's Health Publisher, 1996:176.)
The development and utilization of relevant Stauntonia brachyanthera Hand-Mazz more and more is subject to people's attention in recent years.Because it not only has good pharmaceutical use, and its fruit also is a kind of delicate fruit that high nutritive value is arranged.At present there being report that its fruit is developed to Stauntonia brachyanthera Hand-Mazz vinegar, its stem is as akebi be used as medicine (Wang Ye, Chen Chao, plum mode.The pharmacognosy identification of Dong's medicine Ren sus domestica (Stauntonia brachyanthera Hand-Mazz) rattan.) but up to now, it is still blank out in the chemical research field, and this patent will be first this plant to be carried out chemical ingredients to separate, and analyze and research.
Summary of the invention
The purpose of this invention is to provide a series of with oleanane type, the methyl oleanane type falls and the lupinane type is the triterpene aglycon and the glucoside compound thereof of parent nucleus, its mother nucleus structure is as follows:
Another object of the present invention is to use easy, safe, inexpensive and practical method and prepares the Stauntonia brachyanthera Hand-Mazz saponin component, and illustrating that it uses, this method can prepare higher a series of of purity with oleanane type with fall the triterpene compound that the methyl oleanane type is a parent nucleus.
The implementation of the object of the invention is as follows:
(1) Stauntonia brachyanthera Hand-Mazz raw material meal is 1 kilogram, and with 12 times, 80 ℃ of 20%～95% ethanolic solns of 10 times and 8 times warm lixiviates were down got three times (3 hours for the first time, 2 hours for the second time, 2 hours for the third time) or supersound extraction, filter, merging filtrate is concentrated into 1 liter of total extracting solution.
(2) total extracting solution is adopted following a) or b) one of method carries out enrichment to efficient part:
A with above-mentioned total extracting solution in macroporous adsorbent resin: medicinal material is that the nonpolar or low-pole macroporous adsorbent resin column chromatography of having handled well on 1: 1 the ratio separates, adopt linear gradient elution method, water, 40%, 70%, 90% aqueous ethanol or methanol-eluted fractions successively, 4～10 column volumes of each gradient elution, merge elutriant, filter, be concentrated into 1 liter, get the Stauntonia brachyanthera Hand-Mazz ethanol eluate.
B. with above-mentioned total extracting solution in medicinal material: propyl carbinol is 1: 1 a ratio, with the water-saturated n-butanol extraction of 0.5-3 times of volume 3-8 time, merges propyl carbinol, must the Stauntonia brachyanthera Hand-Mazz butanol extraction liquid.
(3) above-mentioned ethanol eluate or butanol extraction liquid are decoloured through magnesium oxide, added the magnesium oxide reflux decolour 1 hour by every liter 80 gram, filter, filtrate gets the Stauntonia brachyanthera Hand-Mazz total saponins through concentrating under reduced pressure, vacuum-drying.
(4) a kind of method or the several different methods of above-mentioned Stauntonia brachyanthera Hand-Mazz total saponins in normal phase silica gel column chromatography method, reversed-phase column chromatography method, preparative high-performance liquid chromatographic method united use, can remove impurity, purifying obtains the higher total saponins mixture of required triterpenes monomeric compound and purity thereof.Generally with not on year-on-year basis the chloroform-methanol of row or acetone-methyl alcohol as the eluent wash-out, can obtain part triterpene monomer and triterpene mixture, as with Stauntonia brachyanthera Hand-Mazz total saponins extracting solution after the decolouring of n-butanol extraction, magnesium oxide, is that eluent carries out gradient elution with silica gel column chromatography with methylene dichloride and methyl alcohol, at methylene dichloride: methyl alcohol=100: 5 obtains a new natural product 3-O-α-L-arabopyranose-3-O and removes first olea-12 20 (29)-two rare-28-carboxylic acids, and at methylene dichloride: methyl alcohol=100: 8 obtains a monomeric compound daucosterol.
(5) will separate the saponins monomer that obtains or mixture from Stauntonia brachyanthera Hand-Mazz is applied in the medicament of antitumor, anti-inflammatory, diuresis and treating correlative diseases as bulk drug separately or with the other medicines combination.
Above-mentioned concentration operation can adopt conventional concentrating and concentrating under reduced pressure, and drying can adopt 50 ℃ of following conventional dryings, vacuum-drying, spraying drying or lyophilizes.
Macroporous adsorbent resin in the step (2) is selected non-polar resin (XAD-4 for use, Diaion HP-20, D101, D102, D401, D1, D2, D3, D4, HPD-100, X-5), the low-pole resin (D-201, HPD-300, AB-8) and the polymeric adsorbent of middle polarity (XAD-6, XAD-7, XAD-8 etc.).
The employed moving phase of normal phase silica gel chromatography post can be chloroform or methylene dichloride and methyl alcohol in the step (4), adopts chloroform or methylene dichloride: the ratio gradient elution of methyl alcohol=50: 1～1: 1; The filler of reverse-phase chromatographic column can be RP-18, RP-8 or RP-2.; The employed moving phase of described high performance liquid phase preparation method is 5%～95% aqueous methanol; The filler of performance liquid chromatographic column is eight alkyl silane bonded silica gel or octadecylsilane chemically bonded silicas.
Present method for first to the chemical ingredients in the Stauntonia brachyanthera Hand-Mazz plant separate, the research of aspects such as purifying and medicinal use, the yield of the Stauntonia brachyanthera Hand-Mazz total saponin extracts that application present method obtains is more than 23%, the content of total saponins is greater than 35%, and content assaying method can adopt the vanillin-sulfuric acid method.
The present invention adopts water or aqueous ethanol extracts, through macroporous resin or propyl carbinol enrichment, the complete processing method of such cover of decolouring purifying and magnesium oxide realizes the preparation of Stauntonia brachyanthera Hand-Mazz total saponin extracts, simultaneously, also can use the normal phase silica gel column chromatography method, ODS, with in the preparative high-performance liquid chromatographic method one or more, its total saponins is further separated and purifying, to improve saponin(e purity, further obtain a series of with oleanane type, falling methyl oleanane type and lupinane type is the triterpene aglycon monomer of parent nucleus and the saponins mixture and the monomeric compound of glucosides class thereof.Extract can be separately or is applied in the medicament of antitumor, anti-inflammatory, diuresis and treating correlative diseases as bulk drug with the other medicines combination.
In addition, have two kinds of diverse ways to extract the Stauntonia brachyanthera Hand-Mazz total saponins in the step of the present invention (2) from Stauntonia brachyanthera Hand-Mazz, every kind of method all has characteristics and usable condition separately, can select inexpensive according to what of existing device condition and extracted amount during extraction as far as possible, safety, suitable extracting method.
Stauntonia brachyanthera Hand-Mazz saponin component preparation method's of the present invention extraction separation efficient height, Stauntonia brachyanthera Hand-Mazz total saponin content height also can further obtain monomeric compound and keep high physiologically active, and method is simple, workable, is easy to realize industrialized production.
Embodiment
The following examples will give further instruction to the present invention, but not thereby limiting the invention.
Embodiment one
1 kilogram of Stauntonia brachyanthera Hand-Mazz root meal, with 12 times, 80 ℃ of 70% alcohol of 10 times and 8 times warm lixiviates are down got three times, (3 hours for the first time, 2 hours for the second time, 2 hours for the third time) filter, merging filtrate, reclaim solvent or evaporate to dryness to being concentrated into 1 liter, according to macroporous adsorbent resin and medicinal material amount in macroporous adsorbent resin (HPD100) column chromatography for separation on 1: 1 the ratio, adopt linear gradient elution method, water successively, 40% aqueous alcohol, 70% aqueous alcohol, 90% aqueous ethanol wash-out, each gradient are washed 8 retention volume, merge ethanol eluate, with the magnesium oxide decolouring, added the magnesium oxide reflux decolour 1 hour by every liter 80 gram, filter, filtrate is through concentrating under reduced pressure, the dry Stauntonia brachyanthera Hand-Mazz total saponins 170g (yield 17%) that gets, adopt the normal phase silica gel column chromatography method further to separate and purifying the Stauntonia brachyanthera Hand-Mazz total saponins of above-mentioned enrichment, so that the chloroform-methanol of row or acetone-methyl alcohol can not obtain part triterpene monomer and triterpene mixture as the eluent wash-out on year-on-year basis.As carrying out gradient elution with chloroform-methyl alcohol, at chloroform: methyl alcohol=place obtained a monomeric compound in 5: 1, was a known compound, and white powder is soluble in methyl alcohol, and ultraviolet lamp no fluorescence does not down have blackening yet, and the sulfuric acid colour developing is a red-purple afterwards,
1Five unimodal methyl signals δ 0.85,0.93,1.07,1.14,1.19 (5 * CH appear in the high field region of H-NMR
3),
13Among the C-NMR, 29 carbon signals occur, wherein contain two groups of double key carbon signals and a carbonyl signal of falling the oleanane type feature, warp
1H-NMR and
13C-NMR is accredited as 3-O-α-L-arabopyranose-30-and removes first olea-12 20 (29)-two rare-28-carboxylic acid-28-O-β-D-Glucopyranose (1-6)-β-D-glucopyranosyl esters.Further adopt the reverse chromatograms post (with RP-18 (ODS) in above-mentioned triterpene mixture, RP-8 or RP-2.RP are filler) or high performance liquid phase preparative column (is filler with RP-18 or RP-8) be that moving phase is further separated and purifying with the aqueous methanol, active a series of with oleanane type with fall the triterpene compound that the methyl oleanane type is a parent nucleus to obtain having.Stauntonia brachyanthera Hand-Mazz saponins monomer component or its compound are applied in the medicament of antitumor, anti-inflammatory, diuresis and treating correlative diseases as bulk drug separately or with the other medicines combination.
Embodiment two
1 kilogram of Stauntonia brachyanthera Hand-Mazz stem meal, with 10 times, 60 ℃ of 60% alcohol of 8 times and 6 times warm lixiviates are down got (3 hours for the first time three times, 2 hours for the second time, 2 hours for the third time) filter, merging filtrate reclaims solvent or evaporate to dryness to being concentrated into 1 liter, with above-mentioned total extracting solution in medicinal material: propyl carbinol is 1: 1 a ratio, water-saturated n-butanol with 0.5-3 times of volume extracts 3-8 time, merges propyl carbinol, gets the Stauntonia brachyanthera Hand-Mazz butanol extraction liquid.Decolour with magnesium oxide, added the magnesium oxide reflux decolour 1 hour by every liter 80 gram, filter, filtrate is through concentrating under reduced pressure, the dry Stauntonia brachyanthera Hand-Mazz total saponins 182g (yield 18.2%) that gets, adopt the normal phase silica gel column chromatography method further to separate and purifying the Stauntonia brachyanthera Hand-Mazz total saponins of above-mentioned enrichment, so that the chloroform-methanol of row or acetone-methyl alcohol can not obtain part triterpene monomer and triterpene mixture as the eluent wash-out on year-on-year basis.As with Stauntonia brachyanthera Hand-Mazz total saponins extracting solution after the decolouring of n-butanol extraction, magnesium oxide, with silica gel column chromatography is that eluent carries out gradient elution with the methylene chloride-methanol, at methylene dichloride: methyl alcohol=100: 5 obtains a new compound, white powder, be dissolved in the mixed solution of chloroform and methyl alcohol, ultraviolet lamp no fluorescence does not down have blackening yet, and sulfuric acid colour developing back is a red-purple,
1Five unimodal methyl signals δ 0.90,0.97,1.10,1.22,1.28 (5 * CH appear among the H-NMR
3),
13Among the C-NMR, 29 carbon signals occur, wherein contain two groups of double key carbon signals and a carbonyl signal of falling the oleanane type feature, warp
1H-NMR and
13C-NMR is accredited as 3-O-α-L-arabopyranose-3-O and removes first olea-12,20 (29)-two rare-28-carboxylic acids, at methylene dichloride: methyl alcohol=100: 8 obtains a monomeric compound, white powder, the Molish reacting positive, the Liebermann-Burchard reaction is positive, be daucosterol through comparing with known compound, with methylene dichloride: a flow point of methyl alcohol=100: 5 is that moving phase is carried out gradient elution with silica gel column chromatography with sherwood oil-acetone once more, at sherwood oil: acetone=place obtained a monomeric compound in 100: 5, it is a known compound, white, needle-shaped crystals (CHCl3), mp.252～253 ℃, the Liebermann-Burchard reaction is positive
1H-NMR (400MHz, CDCl
3) d:4.68 (1H, s, H-29), 4.58 (1H, s, H-29), 3.18, (1H, dd, J=11.5Hz, H-3), 3.81 (1H, ABdd, J=10.8Hz, H-28), 3.33 (1H, ABdd, J=10.8Hz, H-28), 2.38 (1H, m, H-19), 1.68 (3H, s, 30-Me), 0.75,0.82,0.98,0.99,1.02 (3H * 5, s, 23,24,25,26,27-Me);
13C-NMR (75MHz, DMSO-d
6) in the spectrum, provide 30 carbon signals altogether, 79.0 (C-3) 60.5 (C-28) wherein, δ 150.5,109.8 is a pair of double key carbon signal, comprehensive above physico-chemical property and spectral data analysis are accredited as trochol.At sherwood oil: acetone=place obtained a monomeric compound in 100: 6, was a known compound, the brown amorphous powder.Sulfuric acid colour developing back is a red-purple.ESI-MS provides quasi-molecular ion peak m/z456 ([M]
+, 5)
1H-NMR (500MHz, C
5D
5N) d: provide 5.45 in the spectrum (1H, m, H-12), 3.47 (1, t, J=7.8Hz, H-3), 3.30 (1H, dd, J=4.5,13.9Hz, H-18), 1.30 (3H, s, Me-27), 0.86,0.97,1.02,1.04,1.20 (3H * 6, s, 23,24,25,26,29,30Me)
13C-NMR (125MHz, C
5D
5N) d:30 carbon signal comprises 8 quaternary carbon signals, 9 CH carbon signals, 12 CH carbon signals, 6 CH
3Carbon signal.δ 180.1 is for connecting oxygen carbonyl carbon signal.δ 144.8,122.6 constitutes ethylene linkage.In conjunction with tuberculosis carbon spectrum, the chemical shift of hydrogen spectrum, its nuclear magnetic data of analysis-by-synthesis is accredited as Oleanolic Acid.Adopt the reverse chromatograms post (with RP-18 (ODS) in above-mentioned triterpene mixture, RP-8 or RP-2.RP are filler) or high performance liquid phase preparative column (is filler with RP-18 or RP-8) be that moving phase is further separated and purifying with the aqueous methanol, active a series of with oleanane type with fall the triterpene compound that the methyl oleanane type is a parent nucleus to obtain having.Stauntonia brachyanthera Hand-Mazz saponins monomer component or its compound are applied in the medicament of antitumor, anti-inflammatory, diuresis and treating correlative diseases as bulk drug separately or with the other medicines combination.
Embodiment three
1 kilogram of Stauntonia brachyanthera Hand-Mazz leaf meal, with 8 times, 80 ℃ of 65% alcohol of 6 times and 6 times warm lixiviates are down got three times, (3 hours for the first time, 2 hours for the second time, 2 hours for the third time) filter, merging filtrate, reclaim solvent or evaporate to dryness to being concentrated into 1 liter, according to macroporous adsorbent resin and medicinal material amount in macroporous adsorbent resin (HPD100) column chromatography for separation on 1: 1 the ratio, adopt linear gradient elution method, water successively, 40% aqueous alcohol, 70% aqueous alcohol, 90% aqueous ethanol wash-out, each gradient are washed 9 retention volume, merge ethanol eluate, with the magnesium oxide decolouring, added the magnesium oxide reflux decolour 1 hour by every liter 80 gram, filter, filtrate is through concentrating under reduced pressure, the dry Stauntonia brachyanthera Hand-Mazz total saponins 120g (yield 12%) that gets, adopt the normal phase silica gel column chromatography method further to separate and purifying the Stauntonia brachyanthera Hand-Mazz total saponins of above-mentioned enrichment, so that the chloroform-methanol of row or acetone-methyl alcohol can not obtain part triterpene monomer and triterpene mixture as the eluent wash-out on year-on-year basis.Adopt the reverse chromatograms post (with RP-18 (ODS) in above-mentioned triterpene mixture, RP-8 or RP-2.RP are filler) or high performance liquid phase preparative column (is filler with RP-18 or RP-8) be that moving phase is further separated and purifying with the aqueous methanol, active a series of with oleanane type with fall the triterpene compound that the methyl oleanane type is a parent nucleus to obtain having.Stauntonia brachyanthera Hand-Mazz saponins monomer component or its compound are applied in the medicament of antitumor, anti-inflammatory, diuresis and treating correlative diseases as bulk drug separately or with the other medicines combination.
Embodiment four
1 kilogram of Stauntonia brachyanthera Hand-Mazz pericarp meal, with 12 times, 60 ℃ of 70% alcohol of 10 times and 6 times warm lixiviates are down got three times, (3 hours for the first time, 2 hours for the second time, 2 hours for the third time) filter, merging filtrate, reclaim solvent or evaporate to dryness to being concentrated into 1 liter, according to macroporous adsorbent resin and medicinal material amount in macroporous adsorbent resin (HPD100) column chromatography for separation on 1: 1 the ratio, adopt linear gradient elution method, water successively, 40% aqueous alcohol, 70% aqueous alcohol, 90% aqueous ethanol wash-out, each gradient are washed 9 retention volume, merge ethanol eluate, with the magnesium oxide decolouring, added the magnesium oxide reflux decolour 1 hour by every liter 80 gram, filter, filtrate is through concentrating under reduced pressure, the dry Stauntonia brachyanthera Hand-Mazz total saponins 143g (yield 14.3%) that gets, adopt the normal phase silica gel column chromatography method further to separate and purifying the Stauntonia brachyanthera Hand-Mazz total saponins of above-mentioned enrichment, so that the chloroform-methanol of row or acetone-methyl alcohol can not obtain part triterpene monomer and triterpene mixture as the eluent wash-out on year-on-year basis.Adopt the reverse chromatograms post (with RP-18 (ODS) in above-mentioned triterpene mixture, RP-8 or RP-2.RP are filler) or high performance liquid phase preparative column (is filler with RP-18 or RP-8) be that moving phase is further separated and purifying with the aqueous methanol, active a series of with oleanane type with fall the triterpene compound that the methyl oleanane type is a parent nucleus to obtain having.Stauntonia brachyanthera Hand-Mazz saponins monomer component or its compound are applied in the medicament of antitumor, anti-inflammatory, diuresis and treating correlative diseases as bulk drug separately or with the other medicines combination.
Embodiment five
1 kilogram of Stauntonia brachyanthera Hand-Mazz pulp meal, with 12 times, 60 ℃ of 70% alcohol of 10 times and 6 times warm lixiviates are down got three times, (3 hours for the first time, 2 hours for the second time, 2 hours for the third time) filter, merging filtrate, reclaim solvent or evaporate to dryness to being concentrated into 1 liter, according to macroporous adsorbent resin and medicinal material amount in macroporous adsorbent resin (HPD100) column chromatography for separation on 1: 1 the ratio, adopt linear gradient elution method, water successively, 40% aqueous alcohol, 70% aqueous alcohol, 90% aqueous ethanol wash-out, each gradient are washed 9 retention volume, merge ethanol eluate, with the magnesium oxide decolouring, added the magnesium oxide reflux decolour 1 hour by every liter 80 gram, filter, filtrate is through concentrating under reduced pressure, the dry Stauntonia brachyanthera Hand-Mazz total saponins 150g (yield 15%) that gets, adopt the normal phase silica gel column chromatography method further to separate and purifying the Stauntonia brachyanthera Hand-Mazz total saponins of above-mentioned enrichment, so that the chloroform-methanol of row or acetone-methyl alcohol can not obtain part triterpene monomer and triterpene mixture as the eluent wash-out on year-on-year basis.Adopt the reverse chromatograms post (generally with RP-18 (ODS) in above-mentioned triterpene mixture, RP-8 or RP-2.RP are filler) or high performance liquid phase preparative column (being filler generally) with RP-18 or RP-8 be that moving phase is further separated and purifying with the aqueous methanol, active a series of with oleanane type with fall the triterpene compound that the methyl oleanane type is a parent nucleus to obtain having.Stauntonia brachyanthera Hand-Mazz saponins monomer component or its compound are applied in the medicament of antitumor, anti-inflammatory, diuresis and treating correlative diseases as bulk drug separately or with the other medicines combination.
Saponin component, preparation method and application thereof in the embodiment six Stauntonia brachyanthera Hand-Mazz seeds
1 kilogram in Stauntonia brachyanthera Hand-Mazz seed, with 12 times, 80 ℃ of 70% alcohol of 10 times and 8 times warm lixiviates are down got three times, (3 hours for the first time, 2 hours for the second time, 2 hours for the third time) filter, merging filtrate, reclaiming solvent or evaporate to dryness to being concentrated into 1 liter, use n-butanol extraction, is that 1: 1 ratio extracts 5 times in medicinal material amount and propyl carbinol usage ratio, the Stauntonia brachyanthera Hand-Mazz total saponins mainly is enriched in n-butanol layer, merge butanol extraction liquid,, added the magnesium oxide reflux decolour 1 hour by every liter 80 gram with the magnesium oxide decolouring, filter, filtrate is through concentrating under reduced pressure, dry that Stauntonia brachyanthera Hand-Mazz total saponins 80g (yield 8%) further separates the Stauntonia brachyanthera Hand-Mazz total saponins employing normal phase silica gel column chromatography method of above-mentioned enrichment and purifying, so that the chloroform-methanol of row or acetone-methyl alcohol can not obtain part triterpene monomer and triterpene mixture as the eluent wash-out on year-on-year basis.Adopt the reverse chromatograms post (with RP-18 (ODS) in above-mentioned triterpene mixture, RP-8 or RP-2.RP are filler) or high performance liquid phase preparative column (is filler with RP-18 or RP-8) be that moving phase is further separated and purifying with the aqueous methanol, active a series of with oleanane type with fall the triterpene compound that the methyl oleanane type is a parent nucleus to obtain having.Stauntonia brachyanthera Hand-Mazz saponins monomer component or its compound are applied in the medicament of antitumor, anti-inflammatory, diuresis and treating correlative diseases as bulk drug separately or with the other medicines combination.
Embodiment seven
1 kilogram of Stauntonia brachyanthera Hand-Mazz meal, with 10 times, 40 ℃ of following temperature of the water of 8 times and 8 times amounts are soaked, 75% ethanol-extracted three times, each 2 hours, filter merging filtrate, static 12 hours, the leaching supernatant liquor is concentrated into 1 liter, last macroporous adsorbent resin AB8 post, water successively, 30% aqueous alcohol, 60% aqueous alcohol, 90% aqueous ethanol wash-out, each gradient is washed 8 retention volume, merges ethanol eluate, decolours with magnesium oxide, added the magnesium oxide reflux decolour 1 hour by every liter 80 gram, filter, filtrate is through concentrating under reduced pressure, the dry Stauntonia brachyanthera Hand-Mazz total saponins 180g (yield 18%) that gets further separates the Stauntonia brachyanthera Hand-Mazz total saponins employing normal phase silica gel column chromatography method of above-mentioned enrichment and purifying, generally with not on year-on-year basis the chloroform-methanol of row or acetone-methyl alcohol as the eluent wash-out, can to part triterpene monomer and triterpene mixture.Adopt the reverse chromatograms post (with RP-18 (ODS) in above-mentioned triterpene mixture, RP-8 or RP-2.RP are filler) or high performance liquid phase preparative column (is filler with RP-18 or RP-8) be that moving phase is further separated and purifying with the aqueous methanol, active a series of with oleanane type with fall the triterpene compound that the methyl oleanane type is a parent nucleus to obtain having.Stauntonia brachyanthera Hand-Mazz saponins monomer component or its compound are applied in the medicament of antitumor, anti-inflammatory, diuresis and treating correlative diseases as bulk drug separately or with the other medicines combination.
Embodiment eight
1 kilogram of Stauntonia brachyanthera Hand-Mazz meal, with 12 times, 80 ℃ of 70% ethanol of 10 times and 8 times warm lixiviates are down got three times, (3 hours for the first time, 2 hours for the second time, 2 hours for the third time) filter, merging filtrate, reclaiming solvent or evaporate to dryness to being concentrated into 1 liter, use n-butanol extraction, is that 1: 1 ratio extracts 4 times in medicinal material amount and propyl carbinol usage ratio, the Stauntonia brachyanthera Hand-Mazz total saponins mainly is enriched in n-butanol layer, merge butanol extraction liquid,, added the magnesium oxide reflux decolour 1 hour by every liter 80 gram with the magnesium oxide decolouring, filter, filtrate is through concentrating under reduced pressure, dry Stauntonia brachyanthera Hand-Mazz total saponins 165g (yield 16.5%) adopts the normal phase silica gel column chromatography method further to separate and purifying the Stauntonia brachyanthera Hand-Mazz total saponins of above-mentioned enrichment, generally with not on year-on-year basis the chloroform-methanol of row or acetone-methyl alcohol as the eluent wash-out, can to part triterpene monomer and triterpene mixture.Adopt the reverse chromatograms post (with RP-18 (ODS) in above-mentioned triterpene mixture, RP-8 or RP-2.RP are filler) or high performance liquid phase preparative column (is filler with RP-18 or RP-8) be that moving phase is further separated and purifying with the aqueous methanol, with a series of with oleanane type with fall the triterpene compound that the methyl oleanane type is a parent nucleus.Stauntonia brachyanthera Hand-Mazz saponins monomer component or its compound are applied in the medicament of antitumor, anti-inflammatory, diuresis and treating correlative diseases as bulk drug separately or with the other medicines combination.
The mensuration of embodiment nine anti tumor activity in vitro of isolating total saponins and part triterpene compound from Stauntonia brachyanthera Hand-Mazz
Six kinds of human body tumour cells of taking the logarithm vegetative period (people's acute myeloid leukaemia cell strain (HL-60), Human Prostate Cancer Cells strain (PC-3MIE8), human stomach cancer cell line (BGC-823), human breast cancer cell strain (MDA-MB-435), human hepatoma cell strain (Bel-7402), human cervical carcinoma cell strain (HeLa)) are 5 * 10 with the RPMI-1640 nutrient solution dilution that includes 10% (v/v) foetal calf serum (fetal bovine serum)
4The cell suspending liquid of individual/L is inoculated in 96 orifice plates, and 100 μ L/ holes place 37 ℃, saturated humidity, 5%CO
2Cultivate 24h in the incubator.After sample dissolves with DMSO, with the RPMI-1640 nutrient solution dilution that contains 10% (v/v) foetal calf serum.Above-mentioned pastille nutrient solution is added in 96 orifice plates, be positioned in the incubator with cell cultures the same terms and cultivate 48h.
The mtt assay operating process is as follows: inoculating cell is cultivated (24h) in advance, and drug treating (48h) adds MTT dyeing (4h), and the centrifugal supernatant liquor of removing adds the DMSO dissolving, and microplate reader 570nm measures the OD value down.
Data analysis is calculated in accordance with the following methods: inhibitory rate of cell growth=[1-administration group OD value/control group OD value] * 100%.
Adopt the LOGIT method to calculate medicine half-inhibition concentration (IC
50).Test-results is as shown in table 1.
Table 1
Remarks: sample is the total saponins of different purity.The saponin(e (oleanane type, fall methyl oleanane type and lupinane type) that comprises the position of different wash-outs with column chromatography, the different wash-out position of macroporous adsorbent resin.
The active mensuration of extracorporeal anti-inflammatory of embodiment ten isolating total saponins and part triterpene compound from Stauntonia brachyanthera Hand-Mazz
Mouse peritoneal injection 15mg/ml W-Gum salt brine solution 1.5ml/ only gets after 3 days that the abdominal cavity is huge has a liking for cell, is 1 * 10 with the RPMI-1640 nutrient solution dilution that includes 10% (v/v) foetal calf serum (FCS)
6The cell suspension of individual/ml is inoculated in the flat culture plate in 96 holes, and 100 μ l/well place 37 ℃, saturated humidity, 0.05CO
2Cultivate in the incubator.Behind the 2h cell attachment, remove not adherent cell, add 20 μ l/ml lipopolysaccharides (LPS), 10 μ l/well, and then add the medicine 0.1ml/well to be measured of different concns, 3 multiple holes of every concentration, and blank group (LPS).Place 37 ℃, saturated humidity, 0.05CO afterwards
2After cultivating 48h in the incubator, get supernatant liquor, to be measured.
(1) LPS inductive Turnover of Mouse Peritoneal Macrophages NO is synthesized and the restraining effect that discharges
Get supernatant to be measured and be inoculated in the flat culture plate in 96 holes 100 μ l/well; The NaNO of 100 μ M
2Being diluted in the flat culture plate in 96 holes Deng doubly is standard control (positive control); To contain the negative contrast of RPMI-1640 nutrient solution of 10% (v/v) foetal calf serum (FCS).All establish two multiple holes.The two chloride solns (NED) of 0.2% N-1-naphthyl ethylenediamine mix with 0.2% sulfanilamide (SN) solution (SA) equal-volume and are made into Gress reagent, add 0.1mlGress reagent in every hole, mix, and descend survey OD value in the 550nm wavelength behind the 10min.
Test-results: calculate inhibiting rate (IR) by following formula.Experimental result sees Table 2.
Inhibition?rate(IR)％＝(OD
LPS-OD
sample)/(OD
LPS-OD
negative?control)×100％；
Table 2
Remarks: sample is the total saponins of different purity.The saponin(e (oleanane type, fall methyl oleanane type and lupinane type) that comprises the position of different wash-outs with column chromatography, the different wash-out position of macroporous adsorbent resin.
(2) LPS inductive Turnover of Mouse Peritoneal Macrophages TNF-α is synthesized and the restraining effect that discharges
The L929 cell of taking the logarithm vegetative period is with 0.1% trypsinase and the digestion of 0.02%EDTA mixed solution.Adjust cell concn to 2 * 10 with the RPMI-1640 nutrient solution that contains 10% calf serum
5The cell suspension of individual/ml is inoculated in the flat culture plate in 96 holes, and 100 μ l/well place 37 ℃, saturated humidity, 0.05CO
2After cultivating 24h in the incubator, supernatant liquor is removed in suction, every hole adds each concentration TNF standard substance and the supernatant to be measured behind the 0.1ml doubling dilution respectively, respectively establish 3 multiple holes, negative control group is a RPMI-1640, the blank group is phosphate buffered saline buffer (PBS), adds simultaneously the dactinomycin (0.5-1 μ g/ml) of 0.05ml to each hole, 37 ℃, saturated humidity, 0.05CO
2Cultivate in the incubator.The centrifugal supernatant liquor of abandoning behind the cultivation 24h, rinse, MTT dyeing, 570nm surveys the OD value down.
Test-results: calculate inhibiting rate (IR) by following formula.Experimental result sees Table 3.
Inhibition?rate(IR)％＝(OD
LPS-OD
sample)/(OD
LPS-
OD negative?control)×100％
Table 3
Remarks: sample is the total saponins of different purity.The saponin(e (oleanane type, fall methyl oleanane type and lupinane type) that comprises the position of different wash-outs with column chromatography, the different wash-out position of macroporous adsorbent resin.
Embodiment 11 is isolating total saponins interior anti-inflammatory activity from Stauntonia brachyanthera Hand-Mazz
(1) Stauntonia brachyanthera Hand-Mazz total saponins p-Xylol causes the restraining effect of mice ear
60 of Kunming mouses, 18-22g, male and female half and half are divided into 5 groups at random, 12 every group, are respectively blank group, total saponins, indomethacin group; Experiment is respectively organized dosage and is seen Table 4.9, ig administration respectively, continuous 7 days, 1h after the administration in the 7th day was coated with dimethylbenzene 25 μ l in the mouse right ear two sides, left side ear is not painted with normal ear, behind the 2h, take off cervical vertebra and put to death, the disk of laying left ear and the same position of auris dextra with diameter 8mm punch tool, on torsion(type)balance, weigh, calculate ear swelling rate and inhibiting rate.The results are shown in Table 4Edema Rate (ER) (%)=[Right Ear (mg)-Left ear (mg)]/Left Ear (mg) * 100%Inhibitory Rate (IR) (%)=(ER
Control-ER
Samples)/ER
Control* 100%
Table 4
*P＜0.05，
**P＜0.01?vs?control，Duncan?test
(2) the Stauntonia brachyanthera Hand-Mazz total saponins is to the outgrowth restraining effect of mouse granuloma induced by implantation of cotton pellets
60 of Kunming mouses, 18-22g, male and female half and half are divided into 5 groups at random, and 12 every group, grouping and dosage are seen Table 4.10; The ig administration respectively of each group of experiment continuous 7 days, is implanted 10mg sterilization cotton balls (each cotton balls contains 1000U penicillin and 1000U Streptomycin sulphate) in administration operation on the firstth, took off cervical vertebra on the 8th day and put to death mouse, peel off granulation tissue, and in 60 ℃ of baking ovens, dry by the fire 12h, weigh, it is heavy to calculate granuloma.Test-results sees Table 5
Table 5
**P＜0.01，
***P＜0.001?vs?control，Duncan?test
(3) the Stauntonia brachyanthera Hand-Mazz total saponins is to increasing the effect of mouse peritoneal capillary permeability
60 of Kunming mouses, 18-22g, male and female half and half are divided into 5 groups at random, 12 every group, press Table4.11 grouping and administration; The ig administration respectively of each group of experiment, continuous 7 days, 1h after the administration in the 7th day, the blue NS solution 0.1ml/10g (2%) of iv ivens, ip0.6% acetic acid 0.2ml/ only behind the 20min, takes off cervical vertebra and puts to death mouse immediately, cut off the abdominal cavity, with 5mlNS solution flushing abdominal cavity for several times, collect washings, constant volume is 10ml, centrifugal 1000rpm * 5min measures absorbance at 721 spectrophotometer 590nm places.Test-results sees Table 6
Table 6
**P＜0.01，
***P＜0.001?vs?control，Duncan?test
Embodiment 12 is isolating total saponins diuretic activity from the yellow wax fruit
Get 180～200g male rat, prerun chooses that voided volume reaches 40 of the rat of filling stomach amount more than 40% in water is irritated stomach (2.5mL/100g) back 2h, be divided into physiological saline group, the high, medium and low dosage group of medicine at random, each group rat is put into metabolic cage respectively, 2 in every cage, body weight is close.Fasting 24h before the experiment, the filling stomach water lifting capacity of physiological saline group and each administration group is the 2.5mL/100g body weight.Place graduated cylinder after the administration under metabolic cage, collect 6h urine amount continuously, record urine amount volume (mL) the results are shown in Table 7.
The diuretic properties of table 7 Stauntonia brachyanthera Hand-Mazz total saponins rat (n=10, x ± s)
**P＜0.01
Claims (17)
1. a class Stauntonia brachyanthera Hand-Mazz saponin component is characterized in that: this composition be a series of with oleanane type, the methyl oleanane type falls and the lupinane type is the triterpene aglycon and the glucoside compound thereof of parent nucleus, its mother nucleus structure is as follows:
2. a class Stauntonia brachyanthera Hand-Mazz saponin component according to claim 1 is characterized in that: when being oleanane type as if parent nucleus, and R
1Be α-OH, β-OH, α-OAC or β-OAC, R
2Be-CH
3,-CHO ,-CH
2OH, or-COOH, R
3Be-CH
3,-COOH ,-CH
2OH, R
4Be-H or α/β-OH; 11,12 form epoxy construction; 13 and 28 form lactone structures.
3. a class Stauntonia brachyanthera Hand-Mazz saponin component according to claim 1 is characterized in that: if parent nucleus is when falling the methyl oleanane type, and R
1Be α-OH, β-OH, α-OAC or β-OAC, R
2Be-CH
3,-CHO ,-CH
2OH, or-COOH, R
3Be-CH
3,-COOH, or-CH
2OH.
4. a class Stauntonia brachyanthera Hand-Mazz saponin component according to claim 1 is characterized in that: when being the lupinane type as if parent nucleus, and R
1Be α-OH, β-OH, α-OAC, β-OAC, or at 3 formation ketone carbonyls, R
2Be-CH
3,-CHO ,-CH
2OH, or-COOH, R
3Be-CH
3,-COOH, or-CH
2OH.
5. according to claim 2 or 3 or 4 described class Stauntonia brachyanthera Hand-Mazz saponin components, it is characterized in that: at C-3, C-23, C-24, the C-28 position connects bglii fragment and becomes glycosides, and the number of sugar is 1-7, the kind of sugar is a furan sugar, glucuronic acid, glucose, pectinose, semi-lactosi, wood sugar, rhamnosyl, seminose, allose, lyxose, ribose, fructose, the type of sugar is furanose or pyranose, mode of connection between the sugar has the 1-2 position to connect, and the 1-4 position connects, and the 1-3 position connects one or more that are connected with the 1-6 position; One or two acyl substituted can be arranged on the furan sugar, and the acyl substituted type is mainly ethanoyl and/or angeloyl groups and/or cautious scrupulously and respectfully acyl group.
6. the preparation method of a Stauntonia brachyanthera Hand-Mazz saponin component as claimed in claim 1, it is characterized in that: the preparation method of described Stauntonia brachyanthera Hand-Mazz total saponins is as follows:
(1) with fresh or exsiccant Stauntonia brachyanthera Hand-Mazz raw material, is ground into meal, uses the aqueous ethanol solution heat of 10%-95% to extract or the ethanolic soln supersound extraction, filter, concentrate, get total extracting solution.
(2) total extracting solution is adopted following a) or b) one of method carries out enrichment to efficient part:
A. above-mentioned total extracting solution is crossed nonpolar or low-pole macroporous adsorbent resin to handle well, the aqueous ethanol solution of water and different concns or aqueous methanol solution gradient elution successively, each gradient elution 4-10 column volume merges elutriant, concentrate, get ethanol eluate;
B. the water-saturated n-butanol of above-mentioned total extracting solution with 0.5-3 times of volume extracted 3-8 time, merge propyl carbinol, get butanol extraction liquid;
(3) above-mentioned ethanol eluate or butanol extraction liquid are decoloured through magnesium oxide, filter, filtrate gets the Stauntonia brachyanthera Hand-Mazz total saponins through concentrating under reduced pressure, vacuum-drying;
(4) a kind of method or the several different methods of above-mentioned Stauntonia brachyanthera Hand-Mazz total saponins in normal phase silica gel column chromatography method, reversed-phase column chromatography method, preparative high-performance liquid chromatographic method united use, can remove impurity, purifying obtains required triterpenes monomeric compound and composition thereof.
7. the preparation method of Stauntonia brachyanthera Hand-Mazz saponin component according to claim 6 is characterized in that the Stauntonia brachyanthera Hand-Mazz raw material comprises root, stem, leaf, fruit and the seed of yellow wax fruit.
8. the preparation method of Stauntonia brachyanthera Hand-Mazz saponin component according to claim 6 is characterized in that: the used organic solvent of step (1) comprises one or more in methyl alcohol, ethanol, aqueous methanol, aqueous ethanol or the aqueous acetone.
9. according to the preparation method of the described Stauntonia brachyanthera Hand-Mazz saponin component of claim 6, it is characterized in that: the used extracting method of step (1) has heating to extract and supersound extraction.
10. the preparation method of Stauntonia brachyanthera Hand-Mazz saponin component according to claim 6 and application thereof, it is characterized in that: step (2) macroporous adsorbent resin is selected from XAD-4, Diaion HP-20, D101, D102, D401, D1, D2, D3, D4, HPD-100, X-5, D-201, HPD-300, a kind of among AB-8XAD-6, XAD-7, XAD-8, HPD400, the HPD 600.
11. the preparation method of Stauntonia brachyanthera Hand-Mazz saponin component according to claim 6 is characterized in that: selected magnesium oxide is light magnesium oxide in the step (3).
12. the preparation method of Stauntonia brachyanthera Hand-Mazz saponin component according to claim 6, it is characterized in that: the employed moving phase of normal phase silica gel chromatography post can be chloroform or methylene dichloride and methyl alcohol in the step (4), adopts chloroform or methylene dichloride: the ratio gradient elution of methyl alcohol=50: 1～1: 1.
13. the preparation method of Stauntonia brachyanthera Hand-Mazz saponin component according to claim 2 is characterized in that: the filler of reverse-phase chromatographic column can be RP-18, RP-8 or RP-2. in the step (4)
14. the preparation method of Stauntonia brachyanthera Hand-Mazz saponin component according to claim 2 is characterized in that: the employed moving phase of high performance liquid phase preparation method described in the step (4) is 5%～95% aqueous methanol.
15. the preparation method of Stauntonia brachyanthera Hand-Mazz saponin component according to claim 2 is characterized in that: the filler of performance liquid chromatographic column is eight alkyl silane bonded silica gel or octadecylsilane chemically bonded silicas in the step (4).
16. the preparation method of Stauntonia brachyanthera Hand-Mazz saponin component according to claim 6 is characterized in that concentrating and can adopt conventional concentrating and concentrating under reduced pressure, drying can adopt 50 ℃ of following conventional dryings, vacuum-drying, spraying drying or lyophilizes.
17. the application of the described Stauntonia brachyanthera Hand-Mazz saponin component of claim 1 in the medicine of antitumor, the anti-inflammatory of preparation, diuresis and treating correlative diseases.
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| CN104910237A (en) * | 2014-12-31 | 2015-09-16 | 沈阳药科大学 | Methyl reduced oleanane triterpenoid saponin, preparation method of its active composition and application thereof |
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| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN102485741A (en) * | 2010-12-03 | 2012-06-06 | 沈阳药科大学 | Methyl reduced oleanane triterpenoid, its preparation method and application |
| CN102911242A (en) * | 2011-08-02 | 2013-02-06 | 苏州宝泽堂医药科技有限公司 | Preparation method of akebia quinata acid |
| CN102408464A (en) * | 2011-09-06 | 2012-04-11 | 江苏省中国科学院植物研究所 | Novel notriterpenoid saponin compound and preparation method and application thereof |
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| CN103232518A (en) * | 2013-04-17 | 2013-08-07 | 江苏省中国科学院植物研究所 | New salicornia bigelovii torr nortriterpenoid saponin compound, preparation method and uses thereof |
| CN103232518B (en) * | 2013-04-17 | 2015-09-16 | 江苏省中国科学院植物研究所 | Triterpene saponins compound and its production and use falls in a kind of new Salicornia Bigelovii Torr. |
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Inventor after: Meng Dali Inventor after: Chen Chao Inventor after: Geng Jianliang Inventor after: Li Xi Inventor after: Li Ning Inventor before: Meng Dali Inventor before: Chen Chao Inventor before: Geng Jianliang Inventor before: Li Xi Inventor before: Li Ning |
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