CN102160863A - Purification preparation method of isoalantolactone and application of isoalantolactone in preparation of anti-tumor drugs - Google Patents
Purification preparation method of isoalantolactone and application of isoalantolactone in preparation of anti-tumor drugs Download PDFInfo
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Abstract
The invention discloses a purification preparation method of isoalantolactone and an application of isoalantolactone in preparation of anti-tumor drugs. The method disclosed by the invention comprises the following steps of: taking elecampane as a raw material, quenching and extracting with ethanol of 90-99%, and concentrating the filtered solution under the reduced pressure to obtain an extract; suspending the extract in saturated brine, extracting for three times with petroleum ether at 60-90 DEG C, mixing the petroleum ether extracting solutions, dehydrating with anhydrous Na2SO4, and then, concentrating under the reduced pressure for nearly drying to respectively obtain petroleum ether extracts; separating by adopting a silica gel column chromatography, carrying out gradient elution with a petroleum ether-ethyl acetate solvent system, sequentially ramming with ethyl acetate and methanol until the elution is complete, receiving eluent by volume (500mL for each fraction), concentrating each fraction under the reduced pressure, and mixing the same fractions; and obtaining the compound isoalantolactone at 8-19 fractions. The isoalantolactone prepared by the method disclosed by the invention has the advantages of high purity, good crystal form and large yield.
Description
Present patent application is to be that July 10, application number in 2008 are 200810055376.7 the applying date, and denomination of invention is divided an application for " a kind of purification preparation method of isoalantolactone and its application in the preparation antitumor drug ".
Technical field
The present invention relates to purification preparation method and its medical usage of a kind of chemical compound, specifically a kind of purification preparation method of isoalantolactone and its application in the preparation antitumor drug.
Technical background
Radix Inulae is the dry root of Compositae Radix Inulae (Inulahelenium L).The volatile oil that contains 1-3% in the radia heleii, the main component in the oil are alantolactone, isoalantolactone, dihydroalantolacton etc.In traditional medicine, the function of Radix Inulae is mainly invigorating the spleen and regulating the stomach, promoting QI circulation for relieving depression, and pain relieving is antiabortive.Be mainly used in treatment distension and fullness of the chest and abdomen pain clinically, vomiting is had loose bowels, dysentery, frequent fetal movement.It is antibiotic that modern pharmacology research has confirmed that Radix Inulae volatile oil has, anthelmintic, and analgesia influences smooth muscle, cardiovascular effect.In traditional medicine was used, the extract of Radix Inulae was generally mixture, so its uncertain therapeutic efficacy is cut, toxic and side effects is big.For example, alantolactone that Radix Inulae volatile oil is contained and derivant thereof all are soluble in alcohol, so its extract is impure and side effect is many (see institute of Materia Medica,Chinese Academy of Medical Sciences etc. for details. Chinese medicinal herbal (first) (M) Beijing: People's Health Publisher, 1979,86).Therefore, people are always at the purification preparation method of being devoted to seek better Radix Inulae effective ingredient, simultaneously also at the novel medical use of trying to explore the Radix Inulae effective ingredient for a long time.
Summary of the invention
Purpose of the present invention is exactly the purification preparation method that a kind of isoalantolactone will be provided, and a kind of new medical usage of isoalantolactone is provided simultaneously.
The object of the present invention is achieved like this:
The purification preparation method of isoalantolactone provided by the present invention may further comprise the steps:
(a) be raw material with Compositae Inula plant Radix Inulae, extract, filter with the ethanol merceration of 90--99%, filtrate decompression concentrate extractum;
(b) extractum is suspended in the saturated saline, makes its concentration reach 0.8-1.2g/ml, under 60～90 ℃ of conditions, with petroleum ether extraction 3 times, the extractant volume is respectively and is equivalent to 2/3,1/2 of aqueous solution volume, 1/3, merge petroleum ether extraction liquid, and use anhydrous Na
2SO
4Be evaporated near doing after the dehydration, get ligroin extraction respectively;
(c) adopt silica gel column chromatography to separate, the petroleum ether-ethyl acetate solvent system carries out gradient elution, and the eluent proportioning is 10: 1,8: 2,6: 4,5: 5, use ethyl acetate, methanol complete to eluting at last successively towards post, eluent by volume receives, every part of 500mL, each stream part of concentrating under reduced pressure adopts thin layer chromatography to detect, and according to the thin layer chromatography result same stream part is merged; Get the chemical compound isoalantolactone 8-19 stream part.
Used sesquiterpenoid isoalantolactone in the inventive method, its chemical constitution is as follows:
Its spectral data is:
1H-NMR δ ppm:6.13 (1H, br.s, H-13a), 5.59 (1H, br.s, H-13b), 4.77 (1H, br.s, H-15a), 4.44 (1H, br.s, H-15b), 4.50 (1H, br.S, H-8), 2.99 (1H, m, H-7), 2.34 (1H, d, J=12.0Hz, H-3b), 2.20 (1H, d, J=16.0Hz, H-9a), 2.01 (1H, m, H-3a), 1.85 (1H, J=12.5Hz, H-5), 1.75 (1H, o.m, H-6a), 1.71 (1H, o.m, H-9b), 1.60 (2H, o.m, H-2), 1.54 (1H, o.m, H-1a), 1.40 (1H, m, H-6b), 1.25 (1H, m, H-1b), 0.83 (3H, s, 14-CH
3).
13C-NMR?δppm：41.33(C-1)，22.68(C-2)，36.79(C-3)，148.97(C-4)，46.18(C-5)，27.45(C-6)，40.51(C-7)，76.82(C-8)，42.16(C-9)，34.26(C-10)，142.16(C-11)，170.67(C-12)，120.18(C-13)，17.65(C-14)，106.61(C-15)。
Its purity height of isoalantolactone of the inventive method preparation, crystal formation is good, output is big.
Test shows, adopts the inventive method to purify and prepares isoalantolactone, and its purity can reach 80--97%, and product yield is compared with general alcohol extracting method and can be improved 2-3 doubly.
The inventor has also found the new pharmacologically active of isoalantolactone through long-term a large amount of research, thereby has finished the goal of the invention of the application of isoalantolactone in the preparation antitumor drug.
Isoalantolactone of the present invention is particularly suited for using in preparation anti-alimentary tract tumor medicine.The particularly application in preparation inhibition digestive tract tumor cell proliferation medicine.
The new pharmacologically active of isoalantolactone of the present invention has obtained checking by following test.
The test cell strain:
HeLa: human cervical carcinoma cell strain; HEC-1: people's endometrial carcinoma cell strain; SHIN-3 and HOC-21: the transparent JEG-3 of people's ovary; HAC-2: people's Ovarian Cystadenocarcinoma cell strain; T-98 and U251-SP: the strain of human brain neuroglial cytoma; HLE: human hepatoma cell strain; MM1-CB and HMV-1: human melanoma cell strain; KT: cerebral transfer cell strain.
(1) isoalantolactone suppresses the tumor cell proliferation activity test:
Experimental technique: cell proliferation experiment method.
In containing the D-MEM/hams culture medium of 20% hyclone, be 2 * 10 with tested cell furnishing density
5Cell/mL.With above-mentioned tumor cell line suspension inoculation in 96 well culture plates, every hole 50ul, add blank phosphate buffer (PBS), solvent control 0.1% dimethyl sulfoxide, positive control cisplatin (DDP) and each 50ul of variable concentrations monomeric compound respectively, all establish 3 multiple holes for every group.Place saturated humidity, 37 ℃ and 5%CO2 incubator to cultivate 48 hours, finish preceding 4 hours in cultivating, each culture hole adds 5mg/ml tetrazolium (MTT) 10ul, after cultivation finishes, discard culture supernatant, suspension cell needs centrifugal back supernatant discarded, and every hole adds reaction stop solution 150 μ l, leaves standstill 1 hour, detect each hole absorbance (OD) value with enzyme-linked immunosorbent assay instrument, measure wavelength X=570nm, reference wavelength λ=630nm, and calculate the tumor cell survival rate.
The compounds of this invention is dissolved in DMSO, and the ultimate density after the interpolation is below 0.1%.With the culture medium of only adding DMSO in contrast.
Result of the test:
The inhibition tumor cell proliferation activity of The compounds of this invention is represented in following table 1, table 2 with the result with respect to contrast (adding DMSO).
Table 1 isoalantolactone gynecological tumor cell-proliferation activity
Table 2 isoalantolactone resists other tumor cell proliferation activity
(2) isoalantolactone suppresses implanted tumor cells proliferation activity test in the body:
Experimental technique: implanted tumor cells laboratory method in the body.
Get the ascitogenous sarcoma S180 and the ascitic type liver cancer H22 cell of freezing preservation, the centrifugal 5min of 2000r/min abandons supernatant, with the physiological saline solution dilution, adjusts oncocyte to 2 * 106 cells/mL; Get 5 healthy Kunming mouses, every above-mentioned cell suspension 0.4mL of usefulness, lumbar injection, the inoculation mouse web portion obviously increases after 7～10 days, protrudes.Its anesthesia is put to death, and aseptic extraction milky ascites fluid calculates viable count＞95% with normal saline 1:1 dilution back, adjusts oncocyte density and is 2 * 106 cells/mL, and is stand-by.
Hepatoma carcinoma cell (the H that is gone down to posterity in the abdominal cavity
22) and sarcoma cell (S
180) inoculation position is in mice right fore oxter, inoculum concentration is 2 * 10
6Cell/only, irritate stomach respectively and give tap water, the abdominal cavity gives cyclophosphamide (30mg/kg), irritates the isoalantolactone (100mg/kg, 10mg/kg and 1mg/kg) that stomach gives each concentration, and the administration solvent of each processed group is the 0.2mL/10g body weight, successive administration 10 days.In the 10th day of administration, take off cervical vertebra and put to death mice, strip tumor immediately, win the thymus and the spleen of tumor-bearing mice, weigh respectively, calculate tumour inhibiting rate, thymus index and spleen index.
Isoalantolactone suppresses in vivo that tumor cell proliferation is active to the results are shown in Table 3:
Implanted tumor cells proliferation activity in the table 3 isoalantolactone body
More than test shows that isoalantolactone has good tumor cell proliferation activity inhibition.It can be applied in the preparation cancer therapy drug.
Tumor is a kind of commonly encountered diseases, frequently-occurring disease, and wherein malignant tumor is the most serious class disease of present harm humans health.The medicine that is used for the treatment of at present malignant tumor such as esophageal carcinoma, gastric cancer and gynecological clinically, because of its in anticancer propagation, do not have and selectively suppress the isocellular propagation of bone marrow yet, thereby the big problem of ubiquity toxic and side effects.Clinical eager expectation research worker can be developed the anti-malignant tumor medicine that toxic and side effects is low, selectivity is high at present.
The present invention provides a kind of new selection for people prepare cancer therapy drug.
Isoalantolactone of the present invention is when the preparation antitumor drug, can active constituents of medicine according to the formulation method of routine with the isoalantolactone, add the appropriate drug carrier, make various pharmaceutical dosage forms, as tablet, drop pill, capsule, powder, injection, controlled release agent or the like.
When being used for the treatment of tumor disease, the consumption of oral formulations generally can be controlled in and takes isoalantolactone 300-600mg every day.The consumption of other dosage forms can be with reference to this consumption.Drug usage consumption of the present invention also is not limited solely to this, and the clinicist also can determine the usage and dosage of medicine according to patient's concrete condition.
The specific embodiment
The following examples can illustrate in greater detail the present invention, but do not limit the present invention in any form.
Embodiment 1
The purification preparation of isoalantolactone
A) be raw material with Hebei Compositae Inula plant Radix Inulae, extract, filter with 99% ethanol merceration, filtrate decompression concentrate extractum;
B) extractum is suspended in the saturated brine, makes its concentration reach about 1g/ml, under 75 ℃ of conditions, use petroleum ether extraction 3 times, the extractant volume is respectively and is equivalent to 2/3,1/2,1/3 of aqueous solution volume, merges petroleum ether extraction liquid, and uses anhydrous Na
2S0
4Be evaporated to after the dehydration near no moisture, get ligroin extraction respectively;
C) adopt silica gel column chromatography to separate, the petroleum ether-ethyl acetate solvent system carries out gradient elution, the eluent proportioning is 10: 1,8: 2, and 6: 4,5: 5, use ethyl acetate, methanol complete to eluting towards post at last successively, eluent by volume receives, every part of 500mL, each stream part of concentrating under reduced pressure is adopted thin layer chromatography to detect according to the thin layer chromatography result same stream part is merged; Get the chemical compound isoalantolactone 8-19 stream part.Its content is 87.6%.
Embodiment 2 medicinal granules of the present invention
The dry thing of embodiment 1 made isoalantolactone is ground into powder, adds ethanol and make adhesive, add starch and make filler, be pressed into granule.
Embodiment 3: medicine oral liquid of the present invention:
Embodiment 1 made isoalantolactone is dissolved in the 1000ml water, makes the aqueous solution of 2.5% concentration, add an amount of suspensoid, mix homogeneously in the 20ml medicine bottle of packing into, seals, sterilizes.
Embodiment 4: medicinal tablet of the present invention:
With 10 kilograms of embodiment 1 made isoalantolactone, method for preparing tablet thereof adds starch routinely, and dextrin, magnesium stearate etc. are mixed and made into wet grain, and the machine punching press is in blocks, and every contains medicine dry extract 200mg of the present invention.
Pharmaceutical dosage form of the present invention is not limited solely to this, and it can be prepared into more dosage form, as drop pill, capsule, soft capsule, sustained-release preparation, injection or the like.
Claims (3)
1. the application of isoalantolactone in the preparation cancer therapy drug.
2. the application of isoalantolactone in preparation anti-alimentary tract tumor medicine.
3. the application of isoalantolactone in preparation inhibition digestive tract tumor cell proliferation medicine.
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| CN 201110044543 CN102160863B (en) | 2008-07-10 | 2008-07-10 | Purification preparation method of isoalantolactone and application of isoalantolactone in preparation of anti-tumor drugs |
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| CN 200810055376 Division CN101624387B (en) | 2008-07-10 | 2008-07-10 | Method for purifying and preparing isoalantolactone and application thereof in preparing antineoplastic medicaments |
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Cited By (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN106496243A (en) * | 2016-10-09 | 2017-03-15 | 南开大学 | The application of isoalantolactone derivant and its salt in treatment pulmonary fibrosis medicine is prepared |
| CN108362802A (en) * | 2018-01-15 | 2018-08-03 | 广州博澳抗体生物制药有限公司 | A kind of root of Aucklandia lappa Decne reference extract and its preparation method and application |
| CN108721385A (en) * | 2018-09-04 | 2018-11-02 | 辽宁大学 | Anti-liver cancer and anti-health products containing isoalantolactone |
-
2008
- 2008-07-10 CN CN 201110044543 patent/CN102160863B/en not_active Expired - Fee Related
Cited By (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN106496243A (en) * | 2016-10-09 | 2017-03-15 | 南开大学 | The application of isoalantolactone derivant and its salt in treatment pulmonary fibrosis medicine is prepared |
| CN108362802A (en) * | 2018-01-15 | 2018-08-03 | 广州博澳抗体生物制药有限公司 | A kind of root of Aucklandia lappa Decne reference extract and its preparation method and application |
| CN108362802B (en) * | 2018-01-15 | 2022-04-19 | 广州博澳抗体生物制药有限公司 | Costus root control extract and preparation method and application thereof |
| CN108721385A (en) * | 2018-09-04 | 2018-11-02 | 辽宁大学 | Anti-liver cancer and anti-health products containing isoalantolactone |
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