CN103450314B - There is plant milk extract and the activeconstituents thereof of anti-tumor activity - Google Patents
There is plant milk extract and the activeconstituents thereof of anti-tumor activity Download PDFInfo
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Abstract
The invention belongs to medical art, relate to from Korea Chinese littleleaf box (<i>Buxus</iGreat T.GreaT.GT<i>? microphylla? </i>var.<iGreatT .GreaT.GT? koreana</i>? Nakai) in leaf, extraction and isolation obtains total alkaloids with anti tumor activity in vitro and preparation method thereof, and separation obtains five kinds of buxus alkaloids compounds with anti tumor activity in vitro and preparation method thereof from Korea's Chinese littleleaf box leaf total alkaloids.The raw material that extract involved in the present invention adopts easily obtains in a large number, the monomeric compound novel structure obtained, and there is anti tumor activity in vitro, the extraction and separation method of total alkaloids and wherein active monomer compound is simple and easy, be convenient to carry out further pharmacology and clinical study to it, for exploitation good effect and the little new type antineoplastic medicine of toxic side effect create conditions.
Description
Technical field
The invention belongs to medical art, relate to and obtain total alkaloids with anti tumor activity in vitro and preparation method thereof from the leaf extraction and isolation of Korea Chinese littleleaf box (Buxusmicrophyllavar.koreanaNakai), and separation obtains five kinds of buxus alkaloids compounds with anti tumor activity in vitro and preparation method thereof from Korea's Chinese littleleaf box leaf total alkaloids.
Background technology
Buxus alkaloids has for be only distributed in Buxaceae (Buxaceae) Buxus (Buxus) plant a series of the alkaloid compound falling cycloartane triterpenoids basic framework.This compounds reports that existing more than 220 plant [Yu-xinYan, CurrentBioactiveCompound, 2011,47-64] at present, mostly has treatment myocardial ischemia, anti-senile dementia and antibacterial isoreactivity.Wherein cyclovirobuxinum D (cyclovirobuxinD) is included in 2010 " Chinese Pharmacopoeia ", and be used for the treatment of coronary heart disease, irregular pulse etc., clinical preparation has Buxine Tablet.
The Buxus plant whole world has more than 70 to plant.Be distributed in Asia, Europe, Tropical Africa and the place such as Cuba, Jamaica.China known about 17 kinds and several subspecies and mutation, west is from Tibet, and to the east of Taiwan, south is from Hainan Island, and northwest is all produced to SOUTH OF GANSU, but it is western and the west and south [Chinese Plants will] to be mainly distributed in China.The mutation that Korea Chinese littleleaf box (Buxusmicrophyllavar.koreanaNakai) is little leaf boxwood, originates in the Korea peninsula, introduces a fine variety in various places, Liaoning Province of China in generation twentieth century 30 or 40 years.Be different from the Buxus other plant being distributed in China, it has the characteristic of highly cold-resistant (-35 DEG C), thus is widely used in the afforestation in Liaoning and Jilin Province, has a large amount of artificial culture in surrounding area, Shenyang City, Liaoning Province at present.And only have one section of document of 1966 [NakanoT., JournaloftheChemicalSocietyC:Organic, 1966,1805-1810] to the alkaloidal report of triterpenes in Korea Chinese littleleaf box, obtain 8 alkaloids from being wherein separated.
In recent years, there is report from little leaf boxwood, be separated the buxus alkaloids [Yu-xinYan with extracorporeal anti-tumor function obtained, JournalofNaturalProducts, 2009, 308-311], but according to document [Wang Lingling, Harbin Teachers' Univ.'s natural science journal, 2008, 74-76] in the Korea Chinese littleleaf box of report on peroxidase and Isoenzyme Patterns of Esterase and little leaf boxwood and Buxus sempervirens/common box exist notable difference and Korea Chinese littleleaf box on growing environment with the otherness of this genus other plant distinctness, Korea Chinese littleleaf box can be predicted distinct with this genus other plant in chemical composition and biological activity.
The total alkaloids that in Korea's Chinese littleleaf box leaf involved in the present invention, extraction and isolation obtains and anti-tumor activity thereof, there is not yet Patents identical therewith or bibliographical information up to now both at home and abroad.Involved in the present invention is separated 5 the new buxus alkaloids compounds obtained from Korea's Chinese littleleaf box leaf total alkaloids, there is not yet Patents or the bibliographical information of compound identical therewith and anti-tumor activity so far at home and abroad.
Summary of the invention
The invention provides a kind of total alkaloids of preparing for raw material with Korea's Chinese littleleaf box leaf and preparation method, and it is in the strain of preparation HeLa Cells, MCF-7 Human Breast Cancer Cells strain, typeⅡ pneumocyte strain, the application in the strain of people's HepG-2 cell line and K562 cell strain inhibitor.
New buxus alkaloids compounds and preparation method that the present invention provides again five kinds to be only separated from Korea's Chinese littleleaf box leaf total alkaloids to obtain, and its application in preparation HeLa Cells strain inhibitor.
Five kinds of structures being only separated the new buxus alkaloids compounds obtained from Korea's Chinese littleleaf box leaf total alkaloids of the present invention are such as formula shown in I ~ V:
V
Technology of preparing scheme of the present invention comprises the steps:
With the dry leaf 1kg of Korea Chinese littleleaf box for raw material, with 15 ~ 20 liter of 0.1% ~ 0.3% aqueous hydrochloric acid seepage pressure effects, extracting solution 1% ~ 5% aqueous sodium hydroxide solution alkalizes to pH=10 ~ 11, adopt macroporous adsorbent resin 1 ~ 2kg dynamic adsorption, after aqueous sodium hydroxide solution wash-out 3 ~ 5 retention volume of pH=10 ~ 11, by 95% ethanol elution, 4 ~ 6 retention volume, the partially recycled solvent of 95% ethanol elution obtains the total alkaloids extract of Korea's Chinese littleleaf box leaf.
With the total alkaloids extract of Korea's Chinese littleleaf box leaf for raw material, be separated through 200 ~ 300 order silica gel column chromatographies, be 3:1 respectively with volume proportion, the mixed solvent gradient elution of the sherwood oil-acetone of 1:1 and 0:1, be that the elution fraction of 3:1 is separated through 200 ~ 300 order aluminum oxide column chromatographies again by wherein sherwood oil-acetone volume proportion, be that the mixed solvent wash-out of the sherwood oil-acetone of 100:2 obtains such as formula IV compound with volume proportion, be that the mixed solvent wash-out of the sherwood oil-acetone of 100:10 obtains as formula III compound with volume proportion, be that the elution fraction of 1:1 is again through open ODS pillar layer separation by wherein sherwood oil-acetone volume proportion, with the aqueous methanol gradient wash-out of 5% ~ 80%, wherein obtain formula II compound in 35% ~ 50% methanol aqueous solution eluate, be that the elution fraction of 0:1 is separated through HPD-100 type macroporous adsorbent resin column chromatography by wherein sherwood oil-acetone volume proportion, with the aqueous ethanolic solution gradient elution of 35% and 80%, wherein obtain formula V compound in 35% aqueous ethanolic solution wash-out thing, formula I is obtained in 85% aqueous ethanolic solution wash-out thing.
The Structural Identification of formula I described above ~ V compound adopts various spectroscopic technique: main utilize comprise high resolution mass spectrum, nuclear magnetic resonance spectrum (
1hNMR,
13cNMR, 2D-NMR).
Mtt assay is adopted to test the total alkaloids of Korea described above Chinese littleleaf box leaf to HeLa Cells strain, MCF-7 Human Breast Cancer Cells strain, typeⅡ pneumocyte strain, the extracorporeal anti-tumor function of the strain of people's HepG-2 cell line and K562 cell strain, test-results shows that the total alkaloids of Korea's Chinese littleleaf box leaf has remarkable cytotoxicity, its IC to above-mentioned five kinds of cell strains in vitro
50value is respectively 16.54 ± 1.86 μ g/mL, 30.45 ± 2.68 μ g/mL, 9.40 ± 1.06 μ g/mL, 40.56 ± 4.23 μ g/mL and 9.55 ± 1.56 μ g/mL, therefore, the total alkaloids of Korea of the present invention Chinese littleleaf box leaf has the prospect preparing clinical tumor prevention and therapy medicine.
Adopt the mtt assay test compound such as formula I ~ V that separation obtains from the total alkaloids of Korea's Chinese littleleaf box leaf to the extracorporeal anti-tumor function of HeLa Cells strain, test-results shows to have cytotoxicity to HeLa Cells strain in vitro, its IC such as formula the compound of I ~ V
50value is respectively 13.27 ± 1.68 μMs, 14.85 ± 1.39 μMs, 29.49 ± 9.32 μMs, 31.20 ± 4.07 μMs and 36.29 ± 4.12 μMs, therefore, the compound such as formula I ~ V obtained that is separated from the total alkaloids of Korea's Chinese littleleaf box leaf of the present invention has the prospect preparing clinical tumor prevention and therapy medicine.
The invention has the advantages that, the raw material adopted easily obtains in a large number, gained total alkaloids has more significant anti-tumor activity, the monomeric compound novel structure obtained, and there is anti tumor activity in vitro, the extraction and separation method of total alkaloids and wherein active monomer compound is simple and easy, is convenient to carry out further pharmacology and clinical study to it, for exploitation good effect and the little new type antineoplastic medicine of toxic side effect create conditions.
Embodiment:
Listed embodiment contributes to those skilled in the art and understands the present invention better below, but does not limit the present invention in any way.
Embodiment 1: the preparation of Korea's Chinese littleleaf box leaf total alkaloids:
Korea Chinese littleleaf box leaf (1600g) shreds, after soaking 12h with the 0.2%HCl aqueous solution (5L), with 0.2%HCl aqueous solution seepage pressure effects, obtain acid water extracting liquid 29L altogether, the acid liquid 5%NaOH aqueous solution alkalizes to pH=11, obtain alkaline solution and be about 30L, by alkaline solution HPD-100 type macroporous adsorbent resin 1.5kg dynamic adsorption, after by NaOH aqueous solution wash-out 4 retention volume of pH=11, by 95% ethanol elution, 5 retention volume, the partially recycled solvent of 95% ethanol elution obtains total alkaloids (18g).
Embodiment 2: the anti tumor activity in vitro of Korea's Chinese littleleaf box leaf total alkaloids (embodiment 1)
The subject cell of taking the logarithm vegetative period, with (2-3) × 10
4the density of cells/mL is inoculated in 96 well culture plates, 100 μ L in every hole, after making its adherent 24h, adds the testing compound 100 μ L being diluted to different concns with nutrient solution, continues to cultivate 96h in 37 DEG C.Then every hole adds after 50 μ LMTT solution hatch 4h in 37 DEG C, and abandon or adopt supernatant liquor, every hole adds 200 μ LDMSO, after shaken at room temperature 10min, the absorbance in each hole is measured, if A1(is containing 200 μ LDMSO at microplate reader 570nm place) be blank control wells, with cis-platinum (CP) for positive control.Following formula is utilized to try to achieve growth of tumour cell inhibiting rate: 1-(medicine feeding hole absorbance/control wells absorbance) × 100%.Required result substitutes into IC
50software for calculation, obtains IC
50value.Its IC
50result is as shown in table 1.
The anti tumor activity in vitro of table 1 Korea Chinese littleleaf box leaf total alkaloids (CXHY-YS)
Vitro Experimental Results shows that Korea's Chinese littleleaf box leaf total alkaloids demonstrates cytotoxicity to various human JEG-3 in vitro, especially to MCF-7 Human Breast Cancer Cells strain, the strain of people's HepG-2 cell line and K562 cell strain, there is the activity with the same order of magnitude of positive control drug cis-platinum, be expected to develop it in the strain of preparation HeLa Cells, MCF-7 Human Breast Cancer Cells strain, typeⅡ pneumocyte strain, the application in the strain of people's HepG-2 cell line and K562 cell strain inhibitor.
Embodiment 3: such as formula the preparation method of five kinds of monomeric compounds of I ~ V in Korea's Chinese littleleaf box leaf total alkaloids (embodiment 1)
With the total alkaloids extract 18g of Korea's Chinese littleleaf box leaf for raw material, be separated through 200 ~ 300 order silica gel column chromatographies, be 3:1 respectively with volume proportion, the mixed solvent gradient elution of the sherwood oil-acetone of 1:1 and 0:1, be that the elution fraction of 3:1 is separated through 200 ~ 300 order aluminum oxide column chromatographies again by wherein sherwood oil-acetone volume proportion, be that the mixed solvent wash-out of the sherwood oil-acetone of 100:2 obtains such as formula IV compound (6.0mg) with volume proportion, be that the mixed solvent wash-out of the sherwood oil-acetone of 100:10 obtains as formula III compound (5.2mg) with volume proportion, be that the elution fraction of 1:1 is again through open ODS pillar layer separation by wherein sherwood oil-acetone volume proportion, with the aqueous methanol gradient wash-out of 5% ~ 80%, wherein obtain formula II compound (3.0mg) in 35% ~ 50% methanol aqueous solution eluate, be that the elution fraction of 0:1 is separated through HPD-100 type macroporous adsorbent resin column chromatography by wherein sherwood oil-acetone volume proportion, with the aqueous ethanolic solution gradient elution of 35% and 80%, wherein obtain formula V compound (13mg) in 35% aqueous ethanolic solution wash-out thing, formula I (20mg) is obtained in 85% aqueous ethanolic solution wash-out thing.
Embodiment 4: such as formula the Structural Identification of five kinds of monomeric compounds of I ~ V in Korea's Chinese littleleaf box leaf total alkaloids
Formula I: colorless needle crystals (sherwood oil-acetone), bismuth potassium iodide reacting positive, HR-ESI-MS:[M+H]
+m/z356.2945(calcd356.2953), determine that molecular formula is C
24h
37nO.
1hNMR(600MHz, CDCl
3),
13cNMR(150MHz, CDCl
3) and HMBC attribution data in table 2.
The NMR signals assignment of table 2 formula I
Formula II compound: colorless needle crystals (sherwood oil-acetone), bismuth potassium iodide reacting positive, HR-ESI-MS:[M+H]
+m/z368.2942(calcd368.2953), determine that molecular formula is C
25h
37nO.
1hNMR(600MHz, CDCl
3),
13cNMR(150MHz, CDCl
3) and HMBC attribution data in table 3.
The NMR signals assignment of table 3 formula II compound
Formula III compound: colorless needle crystals (sherwood oil-acetone), bismuth potassium iodide reacting positive, HR-ESI-MS:[M+H]
+m/z368.2940(calcd368.2953), determine that molecular formula is C
25h
37nO.
1hNMR(600MHz, CDCl
3),
13cNMR(150MHz, CDCl
3) and HMBC attribution data in table 4.
The NMR signals assignment of table 4 formula III compound
Formula IV compound: colorless needle crystals (sherwood oil-acetone), bismuth potassium iodide reacting positive, HR-ESI-MS:[M+H]
+m/z370.3107(calcd370.3110), determine that molecular formula is C
25h
39nO.
1hNMR(600MHz, CDCl
3),
13cNMR(150MHz, CDCl
3) and HMBC attribution data in table 5.
The NMR signals assignment of table 5 formula IV compound
Formula V compound: colorless needle crystals (sherwood oil-acetone), bismuth potassium iodide reacting positive, HR-ESI-MS:[M+H]
+m/z385.3578(calcd385.3583), determine that molecular formula is C
26h
44n
2.
1hNMR(600MHz, pyridine-d
5),
13cNMR(150MHz, pyridine-d
5) and HMBC attribution data in table 6.
The NMR signals assignment of table 6 formula V compound
Embodiment 5: such as formula the anti tumor activity in vitro of five kinds of monomeric compounds of I ~ V in Korea's Chinese littleleaf box leaf total alkaloids
Concrete test method is with described in embodiment 2, the cyclokoreanineB(CB obtained from Korea Chinese littleleaf box with bibliographical information) [NakanoT., JournaloftheChemicalSocietyC:Organic, 1966,1805-1810] be positive control, test formula I ~ V five monomeric compounds to the cytotoxicity of HeLa Cells strain, its IC
50result is as shown in table 7.
Such as formula the cytotoxicity to HeLa cell strain of five kinds of monomeric compounds of I ~ V in table 7 Korea Chinese littleleaf box leaf total alkaloids
Vitro Experimental Results shows that five kinds of monomeric compounds such as formula I ~ V in Korea's Chinese littleleaf box leaf total alkaloids have cytotoxicity to HeLa Cells strain, IC in vitro
50value scope is 13 ~ 37 μMs, is expected to develop it and is preparing the application in HeLa Cells strain inhibitor.
Claims (5)
1. buxus alkaloids compounds, is characterized in that, shown in its structural formula as I ~ V:
2. prepare the method for compound according to claim 1 for one kind, it is characterized in that, with the dry leaf 1kg of Korea Chinese littleleaf box (Buxusmicrophyllavar.koreana) for raw material, with 15 ~ 20 liter of 0.1% ~ 0.3% aqueous hydrochloric acid seepage pressure effects, the extracting solution aqueous sodium hydroxide solution of 1% ~ 5% alkalizes to pH=10 ~ 11, adopt macroporous adsorbent resin 1 ~ 2kg dynamic adsorption, after aqueous sodium hydroxide solution wash-out 3 ~ 5 retention volume of pH=10 ~ 11, by 95% ethanol elution, 4 ~ 6 retention volume, the partially recycled solvent of 95% ethanol elution obtains the total alkaloids extract of Korea's Chinese littleleaf box leaf, with the total alkaloids extract of Korea's Chinese littleleaf box leaf of preparation for raw material, be separated through 200 ~ 300 order silica gel column chromatographies, be 3:1 respectively with volume proportion, the mixed solvent gradient elution of the sherwood oil-acetone of 1:1 and 0:1, be that the elution fraction of 3:1 is separated through 200 ~ 300 order aluminum oxide column chromatographies again by wherein sherwood oil-acetone volume proportion, be that the mixed solvent wash-out of the sherwood oil-acetone of 100:2 obtains such as formula IV compound with volume proportion, be that the mixed solvent wash-out of the sherwood oil-acetone of 100:10 obtains as formula III compound with volume proportion, be that the elution fraction of 1:1 is again through open ODS pillar layer separation by wherein sherwood oil-acetone volume proportion, with the aqueous methanol gradient wash-out of 5% ~ 80%, wherein obtain formula II compound in 35% ~ 50% methanol aqueous solution eluate, be that the elution fraction of 0:1 is separated through HPD-100 type macroporous adsorbent resin column chromatography by wherein sherwood oil-acetone volume proportion, with the aqueous ethanolic solution gradient elution of 35% and 80%, wherein obtain formula V compound in 35% aqueous ethanolic solution wash-out thing, formula I is obtained in 85% aqueous ethanolic solution wash-out thing.
3. a pharmaceutical composition, comprises compound according to claim 1.
4. compound according to claim 1 or composition according to claim 3 are preparing the application in cancer therapy drug.
5. application according to claim 4, is characterized in that, described cancer is HeLa Cells strain, MCF-7 Human Breast Cancer Cells strain, typeⅡ pneumocyte strain, the strain of people's HepG-2 cell line and K562 cell strain.
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