CN102408465B - A kind of largeflower-like honeysuckle flower chlorogenic acid Ester Saponin and its production and use - Google Patents
A kind of largeflower-like honeysuckle flower chlorogenic acid Ester Saponin and its production and use Download PDFInfo
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- CN102408465B CN102408465B CN201110284745.1A CN201110284745A CN102408465B CN 102408465 B CN102408465 B CN 102408465B CN 201110284745 A CN201110284745 A CN 201110284745A CN 102408465 B CN102408465 B CN 102408465B
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- 229930182490 saponin Natural products 0.000 title claims abstract description 38
- 239000001397 quillaja saponaria molina bark Substances 0.000 title claims abstract description 19
- PZIRUHCJZBGLDY-UHFFFAOYSA-N Caffeoylquinic acid Natural products CC(CCC(=O)C(C)C1C(=O)CC2C3CC(O)C4CC(O)CCC4(C)C3CCC12C)C(=O)O PZIRUHCJZBGLDY-UHFFFAOYSA-N 0.000 title claims abstract description 17
- -1 chlorogenic acid Ester Saponin Chemical class 0.000 title claims abstract description 16
- CWVRJTMFETXNAD-FWCWNIRPSA-N 3-O-Caffeoylquinic acid Natural products O[C@H]1[C@@H](O)C[C@@](O)(C(O)=O)C[C@H]1OC(=O)\C=C\C1=CC=C(O)C(O)=C1 CWVRJTMFETXNAD-FWCWNIRPSA-N 0.000 title claims abstract description 15
- CWVRJTMFETXNAD-KLZCAUPSSA-N Neochlorogenin-saeure Natural products O[C@H]1C[C@@](O)(C[C@@H](OC(=O)C=Cc2ccc(O)c(O)c2)[C@@H]1O)C(=O)O CWVRJTMFETXNAD-KLZCAUPSSA-N 0.000 title claims abstract description 15
- 229940074393 chlorogenic acid Drugs 0.000 title claims abstract description 15
- FFQSDFBBSXGVKF-KHSQJDLVSA-N chlorogenic acid Natural products O[C@@H]1C[C@](O)(C[C@@H](CC(=O)C=Cc2ccc(O)c(O)c2)[C@@H]1O)C(=O)O FFQSDFBBSXGVKF-KHSQJDLVSA-N 0.000 title claims abstract description 15
- 235000001368 chlorogenic acid Nutrition 0.000 title claims abstract description 15
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Abstract
The present invention relates to a kind of saponin compound and its production and use, belong to field of natural medicinal chemistry, it is characterized in that the chemical process by water or organic solvent, extracting and developing, purifying from Buds of Lonicera macranthoides, obtain one chlorogenic acid Ester Saponin of new honeysuckle, called after Lonimacranthoide VII.This compound lower concentration can suppress MMP-2,9 activity in inside and outside, and Tumor suppression shifts; High density has stronger cytotoxic activity, and Tumor suppression is bred, and proves that Lonimacranthoide VII has the novelty teabag preparing antitumor drug in field of medicaments.
Description
One, technical field:
The present invention relates to field of natural medicinal chemistry, disclose the one of extraction and isolation chlorogenic acid Ester Saponin of new honeysuckle compounds from Buds of Lonicera macranthoides, and the preparation method of this compound and the purposes in field of medicaments, especially in the purposes preparing MMP-2/9 inhibitor antitumor drug.
Two, technical background:
Chlorogenic acid and oleanolic acid type saponin constituents is rich in woodbine.Chlorogenic acid have antibacterial, antiviral, increase white cell, hepatic cholagogic, antitumor, hypotensive, reducing blood-fat, the effect such as scavenging free radicals and stimulating central nervous system system; In recent years report is had to show that chlorogenic acid has the activity (Un-Ho, J., et al.Life Sciences, 2005,77:2760-2769) of stronger suppression MMP-2/9 (MMP-9).Oleanolic acid type saponin constituents is also a kind of important biologically active substance, and these saponin(es report has tumoristatic activity (Bang SC, et al.Chem Pharm Bull, 2005,53:1451-1454) active.Largeflower-like honeysuckle flower (Lonicera macranthoides Hand.-Mazz.) is Caprifoliaceae woodbine, there is effect that is clearing heat and detoxicating, antisepsis and anti-inflammation, to swell furunculosis at tcm clinical practice and the carbuncle that is widely used among the people, larynx numbness, erysipelas, toxic-heat and blood stasis, common cold due to wind-heat, the treatment of the diseases such as warm morbidity.2005 version " Chinese Pharmacopoeia " record, under together listing itself and lonicera hypoglauca miq, Lonicera confusa in Lonicera confusa DC. item.In the research process of Institute of Botany's saponin component in Buds of Lonicera macranthoides, obtain a kind of new chlorogenic acid Ester Saponin compounds, called after Lonimacranthoide VII, there is Tumor suppression propagation (IC simultaneously
50=41.37 μMs), MMP2 (MMP-2) (IC
50=6.23 μMs) and GELB (MMP-9) (IC
50=7.05 μMs) active; In vivo in test, Lonimacranthoide VII senior middle school low dose group (2.5,5.0 and 7.5mg/kg) all obviously can suppress mouse melanoma B16/BL6 Pulmonary metastasis focuses number.Be expected to be developed to a class new while the cancer therapy drug of control growth and metastasis of tumours.
Three, summary of the invention:
The invention discloses a kind of chlorogenic acid Ester Saponin compounds, be called for short Lonimacranthoide VII, molecular formula is: C
57h
82o
20, molecular weight: 1086, chemistry is by name:
3-O-α-L-rhamnopyranosyl-(1 → 2)-α-L-arabinopyranosyl hederagenin 23-O-chlorogenic acylester, chemical structural formula is:
The preparation method of above-claimed cpd, is characterized in that, with largeflower-like honeysuckle flower (Lonicera macranthoidesHand.-Mazz.) bud for raw material, extracting through water, methyl alcohol, ethanol, methanol-water mixing solutions or alcohol-water mixing solutions; Aqueous extract directly crosses macroporous resin adsorption, and be dissolved in water after the extracting solution of methyl alcohol, ethanol, methanol-water mixing solutions or alcohol-water mixing solutions is concentrated macroporous resin adsorption; Macroporous resin adsorption thing obtains through column chromatography for separation.Wherein, macroporous resin comprises D101, AB-8 or HP-20; One or more being selected from silica gel, gel and reverse phase silica gel of column chromatography carrier obtain.
Neochlorogenic acid Ester Saponin Lonimacranthoide VII of the present invention and medically acceptable pharmaceutical excipient form pharmaceutical composition and preparation thereof.As, tablet, capsule and injection.
The invention provides the purposes that neochlorogenic acid Ester Saponin Lonimacranthoide VII prepares MMP-2/9 inhibitor and antitumor drug.In vitro, the IC of LonimacranthoideVII Tumor suppression propagation
50be 41.37 μMs, suppress the IC of MMP2 (MMP-2)
50be 6.23 μMs, suppress the IC of GELB (MMP-9)
50it is 7.05 μMs.In body, when Lonimacranthoide VII dosage be 2.5,5 and 7.5mg/kg time to mouse B16/BL6 cells i injection Lung metastases inhibiting rate be respectively 43.5%, 52.9% and 75.2%.
Four, accompanying drawing illustrates:
Can be used as APPENDIX MATERIALSThe with figure below to report.
The structural formula of Fig. 1, new honeysuckle chlorogenic acid Ester Saponin Lonimacranthoide VII
The main HMBC correlationship of Fig. 2, compound (I)
The ESI-MS spectrum of Fig. 3, compound (I)
Fig. 4, compound (I)
1h-NMR composes
Fig. 5, compound (I)
13c-NMR composes
The HMQC spectrum of Fig. 6, compound (I)
The HMBC spectrum of Fig. 7, compound (I)
Five, embodiment:
In conjunction with embodiment, the invention will be further described, but content of the present invention is not restricted to cited embodiment.
Embodiment 1. is separation andpreconcentration compound L onimacranthoide VII from largeflower-like honeysuckle flower
By largeflower-like honeysuckle flower dry flower 10kg, by 100kg 95% (volume ratio) ethanol-water solution refluxing extraction 3 times, each 2 hours, be concentrated into and obtain medicinal extract (dry weight 1.7kg) without alcohol taste; Gained medicinal extract adds the water dissolution of 10 times of volumes, and filter paper filtering removing water-insoluble, filtrate is adsorbed with macroporous resin HP-20, then with water, 20%, 70% ethanolic soln wash-out, merges 70% ethanolic soln elutriant, concentrates to obtain largeflower-like honeysuckle flower total saponins 860g.Gained largeflower-like honeysuckle flower total saponins carries out silica gel column chromatography, and eluent is followed successively by chloroform-methanol-water (17: 3: 0.2 → 4: 1: 0.1 → 7: 3: 0.5 → 3: 3: 0.5), methyl alcohol; Wherein chloroform-methanol-water (3: 3: 0.5) part carry out repeatedly reverse phase silica gel C-18 column chromatography (eluent is 50% ethanol-water solution) be separated and gel Sephdex LH-20 column chromatography purification (eluent is 55% ethanol-water solution) obtain compound L onimacranthoide VII 0.8g, yield is 0.008%, and purity is 96.99% (HPLC area normalization method).
The qualification of compound L onimacranthoide VII, white powder, TLC Vanillin-vitriol oil test solution heating displaing amaranth, becomes blue after placement.Molish reaction and Liebermann-Burchard reacting positive.Be insoluble in chloroform, soluble in water-methanol mixed solution.This compound of above information indicating is saponins compound.By ESI-MS:[M-1]
-at m/z 1085.8; [M+Na]
+at m/z 1109.8 (see accompanying drawing 3) and
13c-NMR spectrum determines that its molecular formula is C
57h
82o
20, molecular weight 1086.By
1h,
13c-NMR composes (C
5d
5n, 500MHz) (see accompanying drawing 4,5) and completely after acid hydrolysis co-TLC experimental result show that its sapogenin is hederagenin.
13c-NMR:C-3 (δ 81.3) shows that this compound is connected to sugar chain in C-3 position.
1h-NMR spectrum and HMQC compose 2 sugared end group signal: δ 4.92 (1H, d, J=6.6Hz) in (see accompanying drawing 6), 6.17 (1H, brs); And
13c-NMR: δ 101.6,104.9 display compounds have 2 sugar and replace on C-3 position.Kind and the ratio of sugar are defined as pectinose, rhamnosyl (1: 1) by GC and 2D-NMR.The order of connection of sugar chain is composed (see accompanying drawing 2,7) by HMBC and is determined: relevant with the C-2 (δ 76.0) of Ara by the H-1 (δ 6.17) of Rha, the H-1 (δ 4.92) of Ara is relevant to the C-3 (δ 81.3) of aglycon, determines sugar chain 3-O-α-L-rhamnopyranosyl-(1 → 2)-α-L-arabinopyranosyl of C-3.
1except the hydrogen signal of aglycon and sugar also has in H-NMR: trans double bond hydrogen signal [δ 6.55 (1H, d, J=15.9Hz, H-8 "), 7.97 (1H, d, J=15.9Hz, H-7 ")], 1, 3, 4-trisubstituted benzene hydrogen signal [δ 7.18 (1H, dd, J=2.3, 8.3Hz, H-6 "), 7.21 (1H, d, J=8.3Hz, H-5 ") and 7.60 (1H, d, J=2.3Hz, H-2 ")], three meet oxygen methyl proton [δ 4.65 (1H, m, H-3 '), δ 4.36 (1H, m, H-4 ') and δ 6.24 (1H, m, H-5 ')], four methene proton [δ 2.70, 3.01 (2H, m, H-2 ') and 2.74, 3.02 (2H, m, H-6 ')], these signal combination carbon spectrum signals show that this saponin(e structure exists chlorogenic acid ester.Composed by HMBC and determine that chlorogenic acid ester is connected on the C-23 position of aglycon.Comprehensive each data and contrast authenticating compound Lonimacranthoide VII with document and be: 3-O-α-L-rhamnopyranosyl-(1 → 2)-α-L-arabinopyranosyl hederagenin 23-O-chlorogenic acyl ester.New compound is retrieved as through the CA network edition.
The aglycon of table 1 compound L onimacranthoide VII and NMR data (δ, ppm, 0=TMS, the C of sugar chain portion
5d
5n)
aValues in parantheses are
1H-
1H splittings in cases where these are clearly resolved.
bAra＝α-L-arabinopyranose；
cRha＝α-L-rhamnopyranose.
The chlorogenic acid ester moiety of table 2 compound L onimacranthoide VII and NMR data (δ, ppm, 0=TMS, the C of chlorogenic acid
5d
5n)
HPLC method measures the purity of Lonimacranthoide VII, and chromatographic condition is: chromatographic column C
18(Agilent EclipseXDB-C
18, 4.6 × 250mm, 5 μm); Column temperature: 35 DEG C; Moving phase: methanol-water (68: 32) eluant solution; Flow velocity: 0.8mL/min; Sample size 5 μ L; UV determined wavelength: 332nm; Theoretical plate number calculates should be not less than 1000 by Lonimacranthoide VII peak.The retention time of Lonimacranthoide VII is 8.85min, and the purity calculating product by area normalization method is 96.99%.
Embodiment 2, from largeflower-like honeysuckle flower, prepare Lonimacranthoide VII with water extraction
Largeflower-like honeysuckle flower dry flower 1kg, with water heating extraction three times, water consumption is 10 liters, extraction time is 2 hours, and Extracting temperature is 100 DEG C, and extracting solution adsorbs through macroporous resin D101, use water again, 15%, 75% ethanol elution, 75% ethanol eluate decompression and solvent recovery obtains largeflower-like honeysuckle flower total saponins 87g.Gained largeflower-like honeysuckle flower total saponins carries out silica gel column chromatography, and moving phase is followed successively by chloroform-methanol-water (17: 3: 0.2 → 4: 1: 0.1 → 7: 3: 0.5 → 3: 3: 0.5), methyl alcohol; Wherein chloroform-methanol-water (3: 3: 0.5) carry out repeatedly reverse phase silica gel C-18 column chromatography (eluent is 50% ethanol-water solution) be separated and gel column Sephdex LH-20 chromatography purification (eluent is 55% ethanol-water solution) obtain compound L onimacranthoideVII 0.08 gram, yield is 0.008%, and purity is 95% (external standard method).The qualification of products therefrom and purity test in the present embodiment are for reference substance adopts HPLC method detection computations to obtain with embodiment 1 gained Lonimacranthoide VII.The chromatographic condition of HPLC measuring method is: chromatographic column C
18(Agilent Eclipse XDB-C
18, 4.6 × 250mm, 5 μm); Column temperature: 35 DEG C; Moving phase: methanol-water (68: 32) eluant solution; Flow velocity: 0.8mL/min; Sample size 5 μ L; UV determined wavelength: 332nm; Theoretical plate number calculates should be not less than 1000 by Lonimacranthoide VII peak.The retention time of Lonimacranthoide VII is 8.85min.
Embodiment 3, from largeflower-like honeysuckle flower, prepare Lonimacranthoide VII by methyl alcohol cold soaking extraction method
Largeflower-like honeysuckle flower dry flower 1Kg, extracts three times with methyl alcohol cold soaking, and methanol usage is 20 liters, and extraction time is 20 days, and Extracting temperature is room temperature, and extracting solution is concentrated into and obtains medicinal extract (dry weight is 120g) without alcohol taste; Gained medicinal extract adds the water dissolution of 10 times of volumes, and filter paper filtering removing water-insoluble, filtrate is adsorbed with macroporous resin AB-8, then with water, 10%, 80% ethanolic soln wash-out, merges 80% ethanolic soln elutriant, concentrates to obtain largeflower-like honeysuckle flower total saponins 81g.Gained largeflower-like honeysuckle flower total saponins is again through column chromatography (silica gel column chromatography: chloroform-methanol-water gradient elution system, RP-C18 column chromatography: water-methanol system, gel filtration chromatography: water-methanol system) be separated after, obtain compound L onimacranthoide VII 0.07 gram, yield is 0.007%, and purity is 98% (external standard method).The qualification of products therefrom and purity test in the present embodiment are for reference substance adopts HPLC method detection computations to obtain with embodiment 1 gained Lonimacranthoide VII.The chromatographic condition of HPLC measuring method is: chromatographic column C
18(Agilent Eclipse XDB-C
18, 4.6 × 250mm, 5 μm); Column temperature: 35 DEG C; Moving phase: methanol-water (68: 32) eluant solution; Flow velocity: 0.8mL/min; Sample size 5 μ L; UV determined wavelength: 332nm; Theoretical plate number calculates should be not less than 1000 by Lonimacranthoide VII peak.The retention time of Lonimacranthoide VII is 8.85min.
Embodiment 4, from largeflower-like honeysuckle flower, prepare Lonimacranthoide VII by methyl alcohol heating and refluxing extraction method
Largeflower-like honeysuckle flower dry flower 1kg, extracts 3 times with 8kg methanol eddy, each 2 hours, and extracting solution is concentrated into and obtains medicinal extract (dry weight is 150g) without alcohol taste; Gained medicinal extract adds the water dissolution of 10 times of volumes, and filter paper filtering removing water-insoluble, filtrate is adsorbed with macroporous resin D101, then with water, 10%, 75% ethanolic soln wash-out, merges 75% ethanolic soln elutriant, concentrates to obtain largeflower-like honeysuckle flower total saponins 89g.Gained largeflower-like honeysuckle flower total saponins is again through column chromatography (silica gel column chromatography: chloroform-methanol-water gradient elution system, RP-C18 column chromatography: water-methanol system, gel filtration chromatography: water-methanol system) be separated after, obtain compound L onimacranthoide VII 0.09 gram, yield is 0.009%, and purity is 96% (external standard method).The qualification of products therefrom and purity test in the present embodiment are for reference substance adopts HPLC method detection computations to obtain with embodiment 1 gained Lonimacranthoide VII.The chromatographic condition of HPLC measuring method is: chromatographic column C
18(AgilentEclipse XDB-C
18, 4.6 × 250mm, 5 μm); Column temperature: 35 DEG C; Moving phase: methanol-water (68: 32) eluant solution; Flow velocity: 0.8mL/min; Sample size 5 μ L; UV determined wavelength: 332nm; Theoretical plate number calculates should be not less than 1000 by Lonimacranthoide VII peak.The retention time of Lonimacranthoide VII is 8.85min.
Embodiment 5, from largeflower-like honeysuckle flower, prepare Lonimacranthoide VII by the methanol-water mixing solutions extraction method of high density
Largeflower-like honeysuckle flower dry flower 1kg, by the mixing solutions refluxing extraction 3 times of 10kg 95% (volume ratio) methanol-water, each 2 hours, extracting solution was concentrated into and obtains medicinal extract (dry weight is 170g) without alcohol taste; Gained medicinal extract adds the water dissolution of 9 times of volumes, and filter paper filtering removing water-insoluble, filtrate is adsorbed with macroporous resin HP-20, then with water, 10%, 75% ethanolic soln wash-out, merges 75% ethanolic soln elutriant, concentrates to obtain largeflower-like honeysuckle flower total saponins 78g.Gained largeflower-like honeysuckle flower total saponins is again through column chromatography (silica gel column chromatography: chloroform-methanol-water gradient elution system, RP-C18 column chromatography: water-methanol system, gel filtration chromatography: water-methanol system) be separated after, obtain compound L onimacranthoide VII 0.10 gram, yield is 0.010%, and purity is 97% (external standard method).The qualification of products therefrom and purity test in the present embodiment are for reference substance adopts HPLC method detection computations to obtain with embodiment 1 gained Lonimacranthoide VII.The chromatographic condition of HPLC measuring method is: chromatographic column C
18(Agilent Eclipse XDB-C
18, 4.6 × 250mm, 5 μm); Column temperature: 35 DEG C; Moving phase: methanol-water (68: 32) eluant solution; Flow velocity: 0.8mL/min; Sample size 5 μ L; UV determined wavelength: 332nm; Theoretical plate number calculates should be not less than 1000 by Lonimacranthoide VII peak.The retention time of Lonimacranthoide VII is 8.85min.
Embodiment 6, from largeflower-like honeysuckle flower, prepare LonimacranthoideVII by the methanol-water mixing solutions extraction method of lower concentration
Largeflower-like honeysuckle flower dry flower 1kg, by the mixing solutions refluxing extraction 3 times of 8kg 40% (volume ratio) methanol-water, each 2 hours, extracting solution was concentrated into and obtains medicinal extract (dry weight is 180g) without alcohol taste; Gained medicinal extract adds the water dissolution of 4 times of volumes, and filter paper filtering removing water-insoluble, filtrate is adsorbed with macroporous resin HP-20, then with water, 10%, 75% ethanolic soln wash-out, merges 75% ethanolic soln elutriant, concentrates to obtain largeflower-like honeysuckle flower total saponins 79g.Gained largeflower-like honeysuckle flower total saponins is again through column chromatography (silica gel column chromatography: chloroform-methanol-water gradient elution system, RP-C18 column chromatography: water-methanol system, gel filtration chromatography: water-methanol system) be separated after, obtain compound L onimacranthoide VII 0.09 gram, yield is 0.009%, and purity is 98% (external standard method).The qualification of products therefrom and purity test in the present embodiment are for reference substance adopts HPLC method detection computations to obtain with embodiment 1 gained Lonimacranthoide VII.The chromatographic condition of HPLC measuring method is: chromatographic column C
18(Agilent Eclipse XDB-C
18, 4.6 × 250mm, 5 μm); Column temperature: 35 DEG C; Moving phase: methanol-water (68: 32) eluant solution; Flow velocity: 0.8mL/min; Sample size 5 μ L; UV determined wavelength: 332nm; Theoretical plate number calculates should be not less than 1000 by Lonimacranthoide VII peak.The retention time of Lonimacranthoide VII is 8.85min.
Embodiment 7, from largeflower-like honeysuckle flower, prepare Lonimacranthoide VII by ethanol cold soaking extraction method
Largeflower-like honeysuckle flower dry flower 1kg, extracts three times with ethanol cold soaking, and ethanol consumption is 20 liters, and extraction time is 20 days, and Extracting temperature is room temperature, and extracting solution is concentrated into and obtains medicinal extract (dry weight is 110g) without alcohol taste; Gained medicinal extract adds the water dissolution of 10 times of volumes, and filter paper filtering removing water-insoluble, filtrate is adsorbed with macroporous resin D-101, then with water, 10%, 75% ethanolic soln wash-out, merges 75% ethanolic soln elutriant, concentrates to obtain largeflower-like honeysuckle flower total saponins 75g.Gained largeflower-like honeysuckle flower total saponins is again through column chromatography (silica gel column chromatography: chloroform-methanol-water gradient elution system, RP-C18 column chromatography: water-methanol system, gel filtration chromatography: water-methanol system) be separated after, obtain compound L onimacranthoide VII 0.11 gram, yield is 0.011%, and purity is 98% (external standard method).The qualification of products therefrom and purity test in the present embodiment are for reference substance adopts HPLC method detection computations to obtain with embodiment 1 gained Lonimacranthoide VII.The chromatographic condition of HPLC measuring method is: chromatographic column C
18(Agilent Eclipse XDB-C
18, 4.6 × 250mm, 5 μm); Column temperature: 35 DEG C; Moving phase: methanol-water (68: 32) eluant solution; Flow velocity: 0.8mL/min; Sample size 5 μ L; UV determined wavelength: 332nm; Theoretical plate number calculates should be not less than 1000 by Lonimacranthoide VII peak.The retention time of Lonimacranthoide VII is 8.85min.
Embodiment 8, from largeflower-like honeysuckle flower, prepare Lonimacranthoide VII by the alcohol-water mixing solutions extraction method of lower concentration
Largeflower-like honeysuckle flower dry flower 1kg, by the mixing solutions refluxing extraction 3 times of 10kg 30% (volume ratio) alcohol-water, each 2 hours, extracting solution was concentrated into and obtains medicinal extract (dry weight is about 170g) without alcohol taste; Gained medicinal extract adds the water dissolution of 4 times of volumes, and filter paper filtering removing water-insoluble, filtrate is adsorbed with macroporous resin HP-20, then with water, 10%, 75% ethanolic soln wash-out, merges 75% ethanolic soln elutriant, concentrates to obtain largeflower-like honeysuckle flower total saponins 8.2g.Gained largeflower-like honeysuckle flower total saponins is again through column chromatography (silica gel column chromatography: chloroform-methanol-water gradient elution system, RP-C18 column chromatography: water-methanol system, gel filtration chromatography: water-methanol system) be separated after, obtain compound L onimacranthoide VII 0.07 gram, yield is 0.007%, and purity is 97% (external standard method).The qualification of products therefrom and purity test in the present embodiment are for reference substance adopts HPLC method detection computations to obtain with embodiment 1 gained Lonimacranthoide VII.The chromatographic condition of HPLC measuring method is: chromatographic column C
18(Agilent Eclipse XDB-C
18, 4.6 × 250mm, 5 μm); Column temperature: 35 DEG C; Moving phase: methanol-water (68: 32) eluant solution; Flow velocity: 0.8mL/min; Sample size 5 μ L; UV determined wavelength: 332nm; Theoretical plate number calculates should be not less than 1000 by Lonimacranthoide VII peak.The retention time of Lonimacranthoide VII is 8.85min.
Embodiment 9, compound L onimacranthoide VII extracorporeal suppression tumor cell proliferation function
Select people acute myeloblastic leukemia clone HL-60 and human hepatoma cell line HepG2, take Zorubicin as positive drug, mtt assay tests the anti-tumor activity of the new compound Lonimacranthoide VII that the present invention prepares, result shows: LonimacranthoideVII has anti-tumor activity, to the IC of HL-60 cell
50it is 41.37 μMs; To the IC of HepG2 cell
50it is 45.38 μMs.
The IC of table 2 compound on tumor cell strain
50value
| Compound | HL-60 | HepG2 |
| Salicornia Bigelovii Torr. saponin(e first (μM) | 41.37 | 45.38 |
| Zorubicin (μM) | 1.68 | 1.56 |
The active testing of embodiment 10, compound L onimacranthoide VII vitro inhibition MMP2/9 (MMP-2/9)
Collecting cell, with 10
524 orifice plates are inoculated in individual/hole, overnight incubation.PBS washes twice, and change serum free medium and cultivate 1h, after removing substratum, PBS washes 2 times, and the serum free medium of the different concns Lonimacranthoide VII and solvent control that add 200 μ l cultivates 24h.Centrifugal collecting cell supernatant liquor, SDS-PAGE electrophoresis.100ml 2.5%Triton-X-100 wash-out half an hour, then add 100ml gelatinase damping fluid, incubation 18-24h in 37 degree of constant-temperature tables.Dyeing, decolouring is until band is clear.
Table 4 gelatin zymography detect Lonimacranthoide VII to MMP-2,9 inhibit activities
Result shows, relatively non-administration control group, MMP-2 after administration, 9 suppressed, IC
50be respectively 6.23,7.05 μMs.
Embodiment 11, compound L onimacranthoide VII are to the restraining effect of mouse melanoma B16/BL6 transfer
C57BL/6 mouse 80, body weight 18 ~ 22g, each mouse is in tail vein injection B16/BL6 tumour cell (purchased from Beijing institute of materia medica of the Chinese Academy of Medical Sciences) suspension 1 × 10
6/ only.Mouse was divided into 7 groups at random by body weight in second day, be respectively negative control group, Lonimacranthoide VII high (7.5mg/kg), in (5.0mg/kg), low (2.5mg/kg) each dosage group.Each group of tail vein gives relative medicine, and negative control group gives injection physiological saline, dosage 0.2mL/10g body weight.Successive administration 21 days.After last administration 24 hours, disconnected neck put to death mouse, strips the full lung of mouse, dissected Microscopic observation branch on count Lung neoplasm number, calculated each group of mouse lung metastasis mean, and between utilizing student t to test to organize, statistics compares.
Metastasis inhibition rate (%)=(negative control group mean transferred stove number-administration group mean transferred stove number)/negative control group mean transferred stove number × 100%
Experimental result shows, compound L onimacranthoide VII senior middle school low dose group all can the transfer of obvious Tumor suppression.Dosage be 2.5,5.0 and 7.5mg/kg time to mouse B16/BL6 cells i injection Lung metastases restraining effect be respectively 43.5%, 52.9% and 75.2%.High dose group is compared with negative control group, and Pulmonary metastasis focuses digital display work reduces (P < 0.01).
Embodiment 12, tablet containing the new saponin monomer LonimacranthoideVII of the present invention
The Lonimacranthoide VII 10g that Example 1 obtains mixes with Microcrystalline Cellulose 50g and dextrin 5g, makees wetting agent, make softwood with appropriate 30% ethanol, and ordinary method is granulated, and adds the mixing of appropriate Magnesium Stearate, makes tablet.In this tablet, every sheet is containing Lonimacranthoide VII 50mg.
Embodiment 13, capsule containing the present invention new saponin monomer Lonimacranthoide VII
Get Lonimacranthoide VII 5g and starch 7g, dextrin 10g, Icing Sugar 10g mixes, and makees wetting agent, make softwood with appropriate 30% ethanol, and ordinary method is granulated, and loads in hard capsule.In this capsule, every contains LonimacranthoideVII 70mg.
Embodiment 14, injection containing the present invention new saponin monomer Lonimacranthoide VII
Get Lonimacranthoide VII 5g with normal injection agent preparation method, dissolve with 250ml 60 DEG C of distilled water for injection, regulate to wait with NaCl and open, enclose ampulla.This injection 10ml is containing Lonimacranthoide VII 200mg.
Claims (3)
1. a chlorogenic acid Ester Saponin compounds Lonimacranthoide VII, it is characterized in that chemistry 3-O-α-L-rhamnopyranosyl-(1 → 2)-α-L-arabinopyranosyl hederagenin 23-O-chlorogenic acyl ester by name, chemical structural formula is:
2. prepare the method for compound described in claim 1, it is characterized in that the technique of extraction using alcohol, concrete steps are as follows: largeflower-like honeysuckle flower dry flower 1kg, 3 times are extracted with ethanol cold soaking, ethanol consumption is 20 liters, and extraction time is 20 days, and Extracting temperature is room temperature, extracting solution is concentrated into and obtains medicinal extract without alcohol taste, and dry weight is 110g; Gained medicinal extract adds the water dissolution of 10 times of volumes, filter paper filtering removing water-insoluble, and filtrate is adsorbed with macroporous resin D-101, then with water, 10%, 75% ethanolic soln wash-out, merges 75% ethanolic soln elutriant, concentrates to obtain largeflower-like honeysuckle flower total saponins 75g; Gained largeflower-like honeysuckle flower total saponins is again after silica gel column chromatography, RP-C18 column chromatography, gel filtration chromatography are separated, obtain compound L onimacranthoide VII 0.11g, wherein said silica gel column chromatography elution system is chloroform-methanol-water, RP-C18 column chromatography elution system is water-methanol, and gel filtration chromatography elution system is water-methanol.
3. compound according to claim 1 is preparing the purposes in antitumor drug.
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| CN102050862A (en) * | 2009-10-30 | 2011-05-11 | 江苏省中国科学院植物研究所 | New honeysuckle chlorogenic acid ester saponin as well as preparation method and use thereof |
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| CN102050862A (en) * | 2009-10-30 | 2011-05-11 | 江苏省中国科学院植物研究所 | New honeysuckle chlorogenic acid ester saponin as well as preparation method and use thereof |
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| Title |
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| 灰毡毛忍冬花蕾的化学成分;陈君 等;《中国天然药物》;20060930;第4卷(第5期);第347-351页 * |
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