CN101240005A - Method for preparing platycodin D from balloon-flower root and application thereof in anti-cancer medicament - Google Patents
Method for preparing platycodin D from balloon-flower root and application thereof in anti-cancer medicament Download PDFInfo
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- CN101240005A CN101240005A CNA200710056248XA CN200710056248A CN101240005A CN 101240005 A CN101240005 A CN 101240005A CN A200710056248X A CNA200710056248X A CN A200710056248XA CN 200710056248 A CN200710056248 A CN 200710056248A CN 101240005 A CN101240005 A CN 101240005A
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Abstract
A anti-tumor medicament of pentacyclic triterpenoid, which is anti-tumor monomeric compound extracted and separated from a traditional medicine namely root of balloonflower. The preparation of medicament is firstly extracting crushed raw root of balloonflower with 70% ethyl alcohol, consentrating it into extract; extracting with benzinum purificatum, ethyl acetate, butanol respectively, carrying out chromatography operation on butanol layer by macroporous resin column, and repeatedly carrying out chromatography operation by silica gel column after eluting of elute agent, thus pentacyclic triterpenoid is obtained. The compound has a obvious inhibition on growth of liver cancer and gastric cancer, with advantages of high efficiency, low toxin, little toxic and side effects, wide anticancer spectrum, high anticancer activity.
Description
One, technical field
The present invention designs a kind of cancer therapy drug and preparation method thereof, specifically is a kind of anti-cancer active matter pentacyclic triterpenoid of raw material extraction and preparation method thereof with traditional Chinese medicine balloonflower root.
Two, background technology
Cancer is one of three big persistent ailments that threaten human health.Annual in the world number of dying because of cancer reaches ten million people, and the treatment cancer is extremely urgent for human development.The plant amedica development in recent years is in the ascendant, and a lot of effective constituents of finding from plant have antitumour activity, and little because of its side effect, anticancer effect is good and be subject to people's attention day by day.
The inventor's systematic research saponins compound of Chinese medicine balloonflower root, show that through experimentation on animals the platycodin compounds has significant anticancer effect to cancer cells.On above-mentioned basis, the inventor separates from Radix Platycodi total saponins again and obtains active strong anticancer monomeric compound, i.e. activeconstituents.
Three, summary of the invention
1, goal of the invention: the purpose of this invention is to provide a kind of high-efficiency low-toxicity, significant monomeric compound of anticancer effect and preparation method thereof is a kind of Pentacyclic triterpene saponins compounds of extracting of raw material and in the application aspect preparation treatment liver cancer and the cancer of the stomach medicine with traditional Chinese medicine balloonflower root specifically.
2, technical scheme: the inventor has done further research on the basis of existing technology again, finds that Platycodin D is the monomeric compound that has strong antitumour activity in the platycodin class chemical ingredients.And in consulting the foreign language document, find no the report Platycodin D be used for treatment for cancer such as liver cancer and cancer of the stomach.A kind of pentacyclic triterpene compound that the present invention extracts from balloonflower root, it is 3-O-β-D-glucopyranosyl-2 β, 3 β, 16 α, 23,24-pentahydroxyolean-12-ene-28-oic acid 28-O-β-D-apiofuranosyl-(1 → 3)-β-D-xylopyranosyl-(1 → 4)-α-L-rhamnopyranosyl-(1 → 2)-α-L-arabinopyranosyl ester, molecular formula is C
57H
92O
28, its chemical structure is as follows:
A kind of preparation method who prepares Platycodin D from balloonflower root the steps include:
A, will the balloonflower root crude drug pulverize the 70% ethanol ultrasonic extraction of back with 5000mL, reclaim ethanol then extracting solution is concentrated medicinal extract.
B, medicinal extract sherwood oil, ethyl acetate, propyl carbinol extracts respectively, gets n-butanol extracting liquid.
C, n-butanol layer is crossed the NKA-12 column chromatography,, reclaim ethanol, obtain extract with 70% ethanol elution of water and 5000mL.
D, resultant extract in the C step is carried out silica gel (300-400 order) column chromatography again, with 3500mL chloroform-methanol (10: 3) drip washing, the compound that obtains spot maximum on thin layer plate is Platycodin D of the present invention, and it can be with half preparative high-performance liquid chromatographic purifying.
The present invention relates to the application of a kind of pentacyclic triterpene compound Platycodin D aspect the medicine of preparation liver cancer
Pentacyclic triterpene compound Platycodin D is to the liver cancer cell growth restraining effect in the employing mtt assay mensuration balloonflower root.Select the adherent liver cancer cell of logarithmic phase for use, after trysinization, be made into the cell suspension of desired concn again with the substratum that contains 10% calf serum RPMI1640, be seeded in 96 well culture plates, 100 μ L are inoculated in every hole, at 37 ℃ 5%CO
2Cultivate 24h in the incubator.The substratum that contains the different concns sample that experimental group renews, control group then changes the substratum that contains the equal-volume solvent, establishes 3 parallel holes for every group, and other establishes the soup control wells, at 37 ℃ 5%CO
2Cultivate 82h in the incubator.Abandoning supernatant, every hole add the substratum of 100 μ L serum-frees and the MTT of 10ul5mg/mL, cultivate 4h under these conditions, carefully abandon supernatant liquor, and add 100 μ lDMSO dissolving MTT precipitation, behind the microoscillator mixing, on microplate reader, be that 540nm measures optical density value through wavelength.Inhibiting rate %=(1-experimental group optical density value/control group optical density value) * 100%.
Experimental result shows, rising along with pentacyclic triterpene compound Platycodin D concentration in the balloonflower root, the liver cancer cell growth inhibiting rate is also raise, pentacyclic triterpene compound Platycodin D has the obvious suppression effect to the liver cancer liver cancer cell growth when 20 μ g/mL are above, wherein inhibiting rate is the highest during 20 μ g/ml, and its inhibiting rate reaches 87.39%.
The present invention relates to the application of a kind of pentacyclic triterpene compound Platycodin D aspect the medicine of preparation cancer of the stomach
Pentacyclic triterpene compound Platycodin D is to the effect of stomach cancer cell growth-inhibiting in the employing mtt assay mensuration balloonflower root.Select the adherent liver cancer cell of logarithmic phase for use, after trysinization, be made into the cell suspension of desired concn again with the substratum that contains 10% calf serum RPMI1640, be seeded in 96 well culture plates, 100 μ L are inoculated in every hole, at 37 ℃ 5%CO
2Cultivate 24h in the incubator.The substratum that contains the different concns sample that experimental group renews, control group then changes the substratum that contains the equal-volume solvent, establishes 3 parallel holes for every group, and other establishes the soup control wells, at 37 ℃ 5%CO
2Cultivate 82h in the incubator.Abandoning supernatant, every hole add the substratum and the 10Ml of 100 μ L1 serum-frees, the MTT of 5mg/mL, cultivate 4h under these conditions, carefully abandon supernatant liquor, and add 100 μ lDMSO dissolving MTT precipitation, behind the microoscillator mixing, on microplate reader, be that 540nm measures optical density value through wavelength.Inhibiting rate %=(1-experimental group optical density value/control group optical density value) * 100%.
Experimental result shows, rising along with pentacyclic triterpene compound Platycodin D concentration in the balloonflower root, the stomach cancer cell growth inhibition ratio is also raise, pentacyclic triterpene compound Platycodin D has the obvious suppression effect to the liver cancer liver cancer cell growth when 20 μ g/mL are above, wherein inhibiting rate is the highest during 20 μ g/mL, and its inhibiting rate reaches 93.64%.
Four embodiments
Embodiment 1: the extracting method of pentacyclic triterpene compound Platycodin D in the balloonflower root
(1) the balloonflower root crude drug is bought in market, recovery road, Changchun, and crushed after being dried is crossed 40 mesh sieves, the balloonflower root 2.5Kg after the pulverizing, with 70% ethanol ultrasonic extraction, reclaim concentrate behind the ethanol medicinal extract 350g.
(2) medicinal extract is used sherwood oil respectively, ethyl acetate, n-butanol extraction gets n-butanol extracting liquid.
(3) n-butanol extracting liquid is crossed NKA-12 (4Kg) column chromatography,, reclaimed ethanol, obtain extract with 7000mL water and 5000mL 70% ethanol elution.
(4) extract recycle silicon glue (300-400 order) 400g is carried out column chromatography, with 3500mL chloroform-methanol (10: 3) is eluent B drip washing, every 50mL is that a unit decibel receives, thin layer plate sprays 110 ℃ of heating of 10% vitriol oil ethanolic soln colour developing in 5 minutes, the compound that is incorporated in spot maximum on the thin layer plate is Platycodin D of the present invention, and it can be with half preparative high-performance liquid chromatographic purifying.
Embodiment 2: pentacyclic triterpene compound Platycodin D structure is identified
Platycodin D is white amorphous powder, is soluble in methyl alcohol, ethanol, and the water equal solvent, Li Baiman (Liebermann-Burchard) reaction is positive.Syncaryon Magnetic Resonance Spectrum analysis-by-synthesis, and with known references contrast, determine that Platycodin D is the pentacyclic triterpene compound.Molecular formula is C
57H
92O
28, its chemical structure is as follows:
Embodiment 3: the antihepatocarcinoma effect of Platycodin D
1, cell cultures and compound
Hepatoma cell strain (Be17402)) provided by Chinese Academy of Medical Sciences's medicine.Cell cultures in 10% calf serum RPMI1640 nutrient solution, 37 ℃ of constant temperature, 5%CO
2Cultivate 24h in the incubator.Monomeric compound is the Platycodin D of identifying through chemical structure.
2, experimental technique
Pentacyclic triterpene compound Platycodin D is to the liver cancer cell growth restraining effect in the employing mtt assay mensuration balloonflower root.Select the liver cancer cell of logarithmic phase for use, be seeded in 96 well culture plates, at 37 ℃, 5%CO
2Cultivate 24h.The substratum that contains the different concns sample that experimental group renews, control group then changes the substratum that contains the equal-volume solvent, and each concentration is established 9 multiple holes, divides 3 times replication, and other establishes the soup control wells, at 37 ℃ 5%CO
2Cultivate 82h in the incubator.Abandoning supernatant, every hole add the substratum and the 10 μ L of 100 μ L serum-frees, the MTT of 5mg/mL, cultivate 4h under these conditions, carefully abandon supernatant liquor, and add the DMSO dissolving MTT first a ceremonial jade-ladle, used in libation precipitation of 100 μ L, go up microplate reader wavelength 540nm place behind the 30min and measure optical density value.Inhibiting rate %=(1-experimental group optical density value/control group optical density value) * 100%.
3, experimental result
The result with the rising of balloonflower root pentacyclic triterpene compound concentration, also raises to the restraining effect of liver cancer cell growth as shown in Table 1, and pentacyclic triterpene compound platycodin D restraining effect when 10 μ g/mL are above is obvious.
Pentacyclic triterpene compound platycodin D is to the liver cancer cell growth restraining effect in table 1 balloonflower root
Embodiment 4: the anti-cancer of the stomach effect of Platycodin D
1, cell cultures and compound
Stomach cancer cell line (BGC823) is provided by Chinese Academy of Medical Sciences's medicine.Cell cultures in 10% calf serum RPMI1640 nutrient solution, 37 ℃ of constant temperature, 5%CO
2Cultivate 24h in the incubator.Monomeric compound is the Platycodin D of identifying through chemical structure.
2, experimental technique
Pentacyclic triterpene compound Platycodin D is to the liver cancer cell growth restraining effect in the employing mtt assay mensuration balloonflower root.Select the liver cancer cell of logarithmic phase for use, be seeded in 96 well culture plates, at 37 ℃, 5%CO
2Cultivate 24h.The substratum that contains the different concns sample that experimental group renews, control group then changes the substratum that contains the equal-volume solvent, and each concentration is established 9 multiple holes, divides 3 times replication, and other establishes the soup control wells, at 37 ℃ 5%CO
2Cultivate 60h in the incubator.Abandoning supernatant, the MTT that every hole adds the 5mg/mL of the substratum of 100 μ L serum-frees and 10 μ L cultivates 4h under these conditions, carefully abandons supernatant liquor, and the DMSO dissolving MTT that adds 100 μ L precipitates last microplate reader wavelength 540nm place mensuration optical density value behind the 30min.Inhibiting rate %=(1-experimental group optical density value/control group optical density value) * 100%.
3, experimental result
The result with the rising of balloonflower root pentacyclic triterpene compound concentration, also raises to the restraining effect of liver cancer cell growth as shown in Table 2, and pentacyclic triterpene compound platycodin D restraining effect when 10 μ g/mL are above is obvious, and inhibiting rate reaches 93.64%.
Pentacyclic triterpene compound platycodin D is to the effect of stomach cancer cell growth-inhibiting in table 2 balloonflower root
Claims (8)
1. cancer therapy drug pentacyclic triterpene compound Platycodin D, it is characterized in that having structure 3-O-β-D-glucopyranosyl-2 β, 3 β, 16 α, 23,24-pentahydroxyolean-12-ene-28-oic acid 28-O-β-D-apiofuranosyl-(1 → 3)-β-D-Xylopyranosyl-(1 → 4)-α-L-rhamnopyranosyl-(1 → 2)-α-L-arabinopyranosyl ester, molecular formula is C
57H
92O
28, its chemical structure is as follows:
2. method for preparing the described Platycodin D of claim 1, its feature comprises the following steps:
A, the balloonflower root crude drug is pulverized back ethanol ultrasonic extraction, reclaim ethanol then with extracting solution concentrated medicinal extract.
B, medicinal extract sherwood oil, ethyl acetate, propyl carbinol extracts respectively, gets n-butanol extracting liquid.
C, n-butanol extracting liquid is crossed the NKA-12 column chromatography,, reclaim eluent A, obtain extract with water and eluent A wash-out.
D, resultant extract in the C step is carried out the wet column chromatography of silica gel again, with eluent B drip washing, the compound that obtains spot maximum on thin layer plate is Platycodin D of the present invention.
3. preparation method according to claim 2 is characterized in that alcohol concn is 70% in the steps A, and consumption is 5000mL.
4. preparation method according to claim 2 is characterized in that eluent A is 70% ethanol among the step C, and consumption is 5000mL.
5. preparation method according to claim 2 is characterized in that eluent B chloroform-methanol was with 10: 3 proportionings among the step D, and consumption is 3500mL.
6. the described Platycodin D of claim 1 is in the anti-liver cancer of preparation, the application in the anti-cancer of the stomach medicine.
7. Platycodin D according to claim 6 is in the anti-liver cancer of preparation, the application in the anti-cancer of the stomach medicine, it is characterized in that Platycodin D when 20 μ g/mL to liver cancer, stomach cancer cell growth all has obvious restraining effect.
8. Platycodin D according to claim 7 the preparation anti-liver cancer, the application in the anti-cancer of the stomach medicine, it is characterized in that Platycodin D when 20 μ g/mL to liver cancer, the stomach cancer cell growth-inhibiting is all the highest, its inhibiting rate is respectively 87.39% and 93.64%.
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Cited By (9)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN101904890A (en) * | 2009-06-02 | 2010-12-08 | 中国医学科学院药用植物研究所 | Preparation method, application and QC (Quality Control) detection method of platycodon grandiflorum active site |
| CN101402666B (en) * | 2008-11-10 | 2011-07-27 | 浙江大学 | Saponin with immunoadjuvant function, preparation method, vaccine preparation containing the saponin as adjuvant and uses thereof |
| CN102264378A (en) * | 2009-01-23 | 2011-11-30 | B&C生物制药株式会社 | Pharmaceutical composition for preventing or treating hepatitis c, comprising the roots extract of platycodon grandiflorum or platycodon grandiflorum saponin components |
| CN103040901A (en) * | 2012-12-13 | 2013-04-17 | 大兴安岭林格贝有机食品有限责任公司 | Production method of total platycodin |
| CN101854909B (en) * | 2009-01-23 | 2013-05-01 | 生物光谱公司 | Skin-whitening agent containing platycodin D |
| CN104887692A (en) * | 2015-06-04 | 2015-09-09 | 中国农业科学院特产研究所 | Application of platycodin D to preparation of drug for treating melanoma metastasis |
| CN105777825A (en) * | 2016-04-26 | 2016-07-20 | 广西壮族自治区梧州食品药品检验所 | Method for extracting platycodins in platycodon grandiflorus by virtue of ionic liquid |
| CN110954644A (en) * | 2019-09-09 | 2020-04-03 | 山东琪康生物技术有限公司 | Quality control method of lung clearing powder |
| CN113116912A (en) * | 2021-06-07 | 2021-07-16 | 长春中医药大学 | Application of apiose-removed platycodin D in preparation of medicine for preventing and/or treating liver cancer |
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2007
- 2007-10-29 CN CNA200710056248XA patent/CN101240005A/en active Pending
Cited By (12)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN101402666B (en) * | 2008-11-10 | 2011-07-27 | 浙江大学 | Saponin with immunoadjuvant function, preparation method, vaccine preparation containing the saponin as adjuvant and uses thereof |
| CN102264378A (en) * | 2009-01-23 | 2011-11-30 | B&C生物制药株式会社 | Pharmaceutical composition for preventing or treating hepatitis c, comprising the roots extract of platycodon grandiflorum or platycodon grandiflorum saponin components |
| JP2012515773A (en) * | 2009-01-23 | 2012-07-12 | ビー アンド シー バイオファーマ シーオー．，エルティーディ | Pharmaceutical composition for prevention or treatment of hepatitis C, comprising a kyokyo extract or a kyo saponin compound |
| CN101854909B (en) * | 2009-01-23 | 2013-05-01 | 生物光谱公司 | Skin-whitening agent containing platycodin D |
| CN101904890A (en) * | 2009-06-02 | 2010-12-08 | 中国医学科学院药用植物研究所 | Preparation method, application and QC (Quality Control) detection method of platycodon grandiflorum active site |
| CN103040901A (en) * | 2012-12-13 | 2013-04-17 | 大兴安岭林格贝有机食品有限责任公司 | Production method of total platycodin |
| CN103040901B (en) * | 2012-12-13 | 2016-05-25 | 大兴安岭林格贝寒带生物科技股份有限公司 | A kind of production method of balloonflower root general glycoside |
| CN104887692A (en) * | 2015-06-04 | 2015-09-09 | 中国农业科学院特产研究所 | Application of platycodin D to preparation of drug for treating melanoma metastasis |
| CN105777825A (en) * | 2016-04-26 | 2016-07-20 | 广西壮族自治区梧州食品药品检验所 | Method for extracting platycodins in platycodon grandiflorus by virtue of ionic liquid |
| CN110954644A (en) * | 2019-09-09 | 2020-04-03 | 山东琪康生物技术有限公司 | Quality control method of lung clearing powder |
| CN110954644B (en) * | 2019-09-09 | 2020-10-16 | 山东琪康生物技术有限公司 | Quality control method of lung clearing powder |
| CN113116912A (en) * | 2021-06-07 | 2021-07-16 | 长春中医药大学 | Application of apiose-removed platycodin D in preparation of medicine for preventing and/or treating liver cancer |
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Open date: 20080813 |