CN101732383A - Total sesquiterpene lactone extract of centipeda minima, preparation method and application thereof - Google Patents
Total sesquiterpene lactone extract of centipeda minima, preparation method and application thereof Download PDFInfo
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Abstract
The invention provides a total sesquiterpene lactone extract of centipeda minima, a preparation method and application thereof. The preparation method comprises the following steps: crushing dried total centipeda minima, and extracting centipeda minima volatile oil by a supercritical carbon dioxide extracting method; and enriching the centipeda minima volatile oil through macroporous adsorption resin column chromatography or silica gel column chromatography to obtain the total sesquiterpene lactone extract of the centipeda minima. According to the method, the total sesquiterpene lactone with effect of inhibiting tumor proliferation is extracted from Chinese medicament centipeda minima and is enriched for the first time; the extract is rich in short-leaf geraniin; the extract contains at least nine sesquiterpene lactone compounds, wherein the angelic acid centipeda minima and the isobutyric acid centipeda minima are new active sesquiterpene lactone compounds; by the preparation method, the yield is high; the method is suitable for industrialized production; and the extract has the effect of inhibiting tumor cell proliferation, and can be used for preparing various medicaments or health-care products with anti-tumor effect.
Description
Technical field
Pharmaceutically active ingredient preparation field, particularly a kind of total sesquiterpene lactone extract of centipeda minima and its production and application in the invention belongs to.
Background technology
Herba Centipedae is the wounded in the battle herb of feverfew Myriogyne minuta Centipeda minima (L.) A.Br.et.Aschers, and Herba Centipedae is used as medicine and begins to be stated from Southern Tang Dynasty's " feeding habits book on Chinese herbal medicine ", and is also on the books in the Compendium of Material Medica.In China, the Herba Centipedae main product in Zhejiang, ground such as Hubei, Jiangsu, Guangdong, also there is distribution on ground such as Guangxi, Henan, Jiangxi, Fujian, Anhui.By 2000 edition and 2005 edition " record, function with cure mainly be " clearing the nasal passage, cough-relieving be used for headche due to wind-cold, cough with copious phlegm, have a stuffy nose obstructed, nasal sinusitis watery nasal discharge " respectively by Chinese pharmacopoeia for this medical herbs.
The chemical constituent of Herba Centipedae mainly contains volatile oil, terpenoid, flavonoid and phytosterin compound, wherein flavonoid and the sesquiterpenoids effective ingredient pharmacological research that is considered to Herba Centipedae shows, Herba Centipedae tool antiinflammatory, antibiotic, antiallergic and antitumor isoreactivity
[1]Wherein the research of Herba Centipedae anti-tumor active ingredient mainly comprises two aspects: the antitumor action of (1) centipeda volatile oil: Wu and Zhen etc. adopts the method for supercritical extraction and vapor distillation respectively; extract centipeda volatile oil; prove that two volatile oil all have anti-tumor activity, this achievement in research has been carried out patent protection
[2](2) anti-tumor activity of sesquiterpene lactones compounds in the Herba Centipedae, wherein the anti-tumor activity of geraniin has the inside and outside anti-tumor activity of stronger body in the Herba Centipedae, and this achievement in research is also by patent
[3]
Although much studies show that, the sesquiterpene lactones compounds is the anti-tumor active ingredient of Herba Centipedae, but, in the retrieval of State Intellectual Property Office's coordinate indexing and CNKI, Scifinder Scholar, total sesquiterpene lactone compounds in discovery extraction, the separation Herba Centipedae does not more contain the patent and the document of the Herba Centipedae anti-tumor effective component of new active sesquiterpene lactones as the technical study of its anti-tumor effective component.
Summary of the invention
In order to overcome the shortcoming and defect of prior art, primary and foremost purpose of the present invention is to provide a kind of Herba Centipedae (Centipeda minima) total sesquiterpene lactone extract that contains noval chemical compound.
Another object of the present invention is to provide a kind of preparation method of above-mentioned total sesquiterpene lactone extract of centipeda minima.
A further object of the present invention is to provide above-mentioned total sesquiterpene lactone extract of centipeda minima to have the purposes of the medicine that suppresses tumor proliferation as preparation.
Purpose of the present invention is achieved through the following technical solutions: a kind of total sesquiterpene lactone extract of centipeda minima is characterized in that: this extract contains angelic acid Herba Centipedae element (formula I) and isopropylformic acid. Herba Centipedae element (formula II):
(formula I) (formula II)
The preparation method of above-mentioned total sesquiterpene lactone extract of centipeda minima comprises following operating procedure: the dry herb of Herba Centipedae is pulverized, adopted supercritical carbon dioxide (CO
2) extracting process extracts and to obtain centipeda volatile oil; Again centipeda volatile oil is obtained total sesquiterpene lactone extract of centipeda minima through enrichment of macroporous adsorbent resin column chromatography method or silica gel column chromatography enrichment;
The enrichment of described macroporous adsorbent resin column chromatography method, be that described centipeda volatile oil is crossed macroporous resin column, with the ethanol water gradient elution, adopting gas chromatography-mass spectrum (GC-MS) methods analyst to follow the tracks of detects, collection contains stream part of sesquiterpene lactones chemical compound, merges stream part, concentrating under reduced pressure, drying obtains total sesquiterpene lactone extract of centipeda minima;
Described silica gel column chromatography method enrichment, be that described centipeda volatile oil is crossed silicagel column, with the mixed organic solvents gradient elution, adopting gas chromatography-mass spectrum (GC-MS) methods analyst to follow the tracks of detects, collection contains stream part of sesquiterpene lactones chemical compound, merges stream part, concentrating under reduced pressure, drying gets total sesquiterpene lactone extract of centipeda minima.
Described Herba Centipedae is the wounded in the battle herb of feverfew Myriogyne minuta.
The extracting pressure of described supercritical carbon dioxide method for extracting is 20～40 MPas, and extraction temperature is 30～50 ℃, and flow velocity is 20 liters/hour, and extraction time is 1～4 hour.
Added in the described supercritical carbon dioxide method for extracting and guided and supported agent.
It is described that to guide and support agent be ethanol.
Described is that to adopt mass percent concentration successively be that 20～30% and 50～80% ethanol water carries out gradient elution with the ethanol water gradient elution, be that 20～30% ethanol aqueous wash removes decontamination with mass percent concentration earlier promptly, the reuse mass percent concentration is 50～80% ethanol water eluting.
Described mixed organic solvents is counted petroleum ether with volume ratio: acetone=9: 1～1: 9, petroleum ether: ethyl acetate=9: 1～1: 9, hexane: ethyl acetate=9: 1～1: 9 or hexane: acetone=9: 1～1: 9.
The quality percentage composition of geraniin is no less than 50% in the described total sesquiterpene lactone extract of centipeda minima.
Above-mentioned total sesquiterpene lactone extract of centipeda minima suppresses the purposes of the medicine of tumor proliferation as preparation.
The relative prior art of the present invention has following advantage and beneficial effect: (1) extracts from the Chinese medicine Herba Centipedae first and enrichment has effective site---the total sesquiterpene lactone that suppresses the tumor proliferation effect; (2) this extract contains and is rich in geraniin; (3) this extract contains at least 9 sesquiterpene lactones compounds (comprising geraniin), and wherein angelic acid Herba Centipedae element and isopropylformic acid. Herba Centipedae element are new active sesquiterpene lactones compounds; (4) this preparation technology's yield height, suitable suitability for industrialized production have the effect of the tumor cell proliferation of inhibition, can be used for preparing various medicines or health product with antitumor action.
Description of drawings
Fig. 1 is the GC-MS analysis result figure of geraniin standard sample.
Fig. 2 is for adopting the GC-MS analysis result figure that does not add the centipeda volatile oil of the method gained of guiding and supporting agent.
Fig. 3 is for adopting the GC-MS analysis result figure of the centipeda volatile oil that adds the method gained of guiding and supporting agent.
Fig. 4 is the GC-MS analysis result figure of the volatile oil of steam distillation.
Fig. 5 is for adopting the GC-MS analysis result figure of macroporous absorption adsorbent resin column chromatography enrichment gained total sesquiterpene lactone extract of centipeda minima.
Fig. 6 is for adopting the GC-MS analysis result figure of silica gel column chromatography method enrichment gained total sesquiterpene lactone extract of centipeda minima.
The specific embodiment
The present invention is described in further detail below in conjunction with embodiment and accompanying drawing, but embodiments of the present invention are not limited thereto.
The condition that the gas chromatography-mass spectrum (GC-MS) that following embodiment adopts is analyzed is as follows:
Experimental apparatus is an Agilent 6890N series gas chromatograph-mass spectrometer, and pillar is HP-5 capillary column (30m * 0.25mm * 25 μ m), and carrier gas is a helium, and flow is 1.5mL/min, and detector temperature is 300 ℃.Analysis condition is as follows: 60 ℃ of chromatographic column initial temperatures, be warmed up to 150 ℃ with the speed program of 3 ℃/min and keep 5min, be warmed up to 240 ℃ with the speed program of 1.5 ℃/min again and keep 10min, be warmed up to 300 ℃ with the speed program of 2 ℃/min at last and keep 10min.Ionization mode: EI, energy: 70eV.
Embodiment 1
Centipeda volatile oil preparation: the dry herb of 300g Herba Centipedae (pick up from Guangdong, identified by the Guangzhou medical material senior engineer of the company wheat ball that shakes) is pulverized, crossed 60 mesh sieves, place supercritical CO
2In the abstraction instrument, extracting pressure is that 20 MPas, extraction temperature are that 40 ℃, flow velocity are that 20 liters/hour, extraction time are 2 hours, obtains centipeda volatile oil 15.2g.
Embodiment 2:
Centipeda volatile oil preparation: the dry herb of 300g Herba Centipedae is pulverized, crossed 60 mesh sieves, place supercritical CO
2In the abstraction instrument, extracting pressure is that 25 MPas, extraction temperature are that 38 ℃, flow velocity are that 20 liters/hour, 100mL mass percent concentration be 95% ethanol for guiding and supporting agent, extraction time are 2 hours, obtains centipeda volatile oil 16.7g.
Embodiment 3:
Total sesquiterpene lactone extract of centipeda minima preparation: take by weighing D101 macroporous adsorbent resin 150g, adding distil water 800mL stirs, and the water floatation is removed resin monomer or fragment, at room temperature places 24h then, treat the abundant swelling of resin after, resin is drained; Reuse ethanol wet method is with the resin pillar (internal diameter * length=6 * 100cm of packing into, after having adorned post, the retention volume of pillar is 550mL) in, mix the muddiness that is not white in color with mobile pillar to the effluent that cleans of ethanol with water (effluent is 1: 5 with the volume ratio of water), there is not alcoholic acid taste with the abundant drip washing pillar of distilled water to effluent then, the placement balance pillar that spends the night obtains macroporous resin column.
Take by weighing the centipeda volatile oil 3.8g of gained among the embodiment 1, the mass percent concentration that is dissolved in 30mL is in 95% ethanol, behind the ultrasonic degas, add in the macroporous resin column that above-mentioned processing and balance cross, be that 20% ethanol elution pillar is colourless to effluent with mass percent concentration earlier, the reuse mass percent concentration is that 50% ethanol water eluting pillar is colourless to effluent, collecting mass percent concentration is stream part of 50% ethanol water eluting gained, analyzing tracking with GC-MS detects, collect retention time stream part between 70min～120min, concentrating under reduced pressure, drying obtains total sesquiterpene lactone extract of centipeda minima 0.8g.
Embodiment 4:
The total sesquiterpene lactone extract of centipeda minima preparation: the method for pressing embodiment 3 is handled the D101 macroporous adsorbent resin; Take by weighing prepared centipeda volatile oil 4.0g among the embodiment 1, the mass percent concentration that is dissolved in 30mL is in 95% ethanol, behind the ultrasonic degas, add macroporous resin column (internal diameter * length=6 * 100cm that above-mentioned processing and balance are crossed, retention volume 550mL) in, be that 20% ethanol elution pillar is colourless to effluent with mass percent concentration earlier, the reuse mass percent concentration is that 80% alcoholic solution eluting pillar is colourless to effluent, collecting mass percent concentration is the eluent of 80% alcoholic solution eluting gained, analyzing tracking with GC-MS detects, collect retention time stream part between 70min～120min, concentrating under reduced pressure, drying obtains total sesquiterpene lactone extract of centipeda minima 1.2g.
Embodiment 5:
Take by weighing prepared centipeda volatile oil 9.5g among the embodiment 1, behind acetic acid ethyl dissolution, mix sample with 12g silica gel (100～200 order), evaporated under reduced pressure obtains sample; Other gets 90g silica gel (200～300 order) dry column-packing (internal diameter * length=3.5 * 50cm), sample is packed on the post, be covered with one deck silica gel again, use petroleum ether: the acetone gradient elution, collect petroleum ether: the acetone volume ratio is eluting stream part of 9: 1 to 1: 9, analyzes to follow the tracks of with GC-MS and detects, collect retention time stream part between 70min～120min, concentrating under reduced pressure, drying obtains total sesquiterpene lactone extract of centipeda minima 1.8g.
Embodiment 6:
Take by weighing prepared centipeda volatile oil 9.5g among the embodiment 1, behind acetic acid ethyl dissolution, mix sample with 12g silica gel (100～200 order), evaporated under reduced pressure obtains sample; Other gets 90g silica gel (200～300 order) dry column-packing (internal diameter * length=3.5 * 50cm), sample is packed on the post, be covered with one deck silica gel again, use petroleum ether: ethyl acetate system gradient elution, collect petroleum ether: the ethyl acetate volume ratio is 9: 1 to 1: 9 an eluting flow point, analyzes to follow the tracks of with GC-MS and detects, collect retention time stream part between 70min～120min, concentrating under reduced pressure, drying obtains total sesquiterpene lactone extract of centipeda minima 1.6g.
Embodiment 7:
Take by weighing prepared centipeda volatile oil 9.5g among the embodiment 1, behind acetic acid ethyl dissolution, mix sample with 12g silica gel (100～200 order), evaporated under reduced pressure obtains sample; Other gets 90g silica gel (200～300 order) dry column-packing (internal diameter * length=3.5 * 50cm), sample is packed on the post, be covered with one deck silica gel again, use hexane: ethyl acetate system gradient elution, collect hexane: the ethyl acetate volume ratio is 9: 1 to 1: 9 eluting flow points, analyzes to follow the tracks of with GC-MS and detects, and collects retention time stream part between 70min～120min, concentrating under reduced pressure, drying obtain total sesquiterpene lactone extract of centipeda minima 1.3g.
Embodiment 8:
Take by weighing prepared centipeda volatile oil 9.5g among the embodiment 1, behind acetic acid ethyl dissolution, mix sample with 12g silica gel (100～200 order), evaporated under reduced pressure obtains sample; Other gets 90g silica gel (200～300 order) dry column-packing (internal diameter * length=3.5 * 50cm), sample is packed on the post, be covered with one deck silica gel again, use hexane: acetone system gradient elution, collect hexane: the acetone volume ratio is 9: 1 to 1: 9 an eluting flow point, analyzes to follow the tracks of with GC-MS and detects, and collects retention time stream part between 70min～120min, concentrating under reduced pressure, drying obtain total sesquiterpene lactone extract of centipeda minima 1.5g.
Embodiment 9: the content assaying method of geraniin in centipeda volatile oil and the total sesquiterpene lactone extract:
The dry herb of 308g Herba Centipedae is pulverized, crossed 60 mesh sieves, add 8 times of quality water, use steam distillation, added thermal distillation 5 hours, gained volatile oil anhydrous sodium sulfate drying obtains the volatile oil of 0.4g steam distillation.
Adopt the centipeda volatile oil, the volatile oil of steam distillation, the embodiment 3 that add the method gained of guiding and supporting agent to adopt macroporous absorption adsorbent resin column chromatography enrichment gained total sesquiterpene lactone extract of centipeda minima and embodiment 5 to adopt silica gel column chromatography method enrichment gained total sesquiterpene lactone extract of centipeda minima to carry out gas chromatography-mass spectrum (GC-MS) analysis respectively among the centipeda volatile oil of the method gained of agent, the embodiment 2 with adopting not interpolation to guide and support among geraniin standard sample, the embodiment 1, the result is shown in Fig. 1～5.
The purity of the geraniin in the geraniin standard sample is 96.0% as seen from Figure 1.
As seen from Figure 2, adopting the relative mass content of geraniin in the centipeda volatile oil that does not add the method gained of guiding and supporting agent is 22.8%; In addition, according to the MS analysis result, the chemical compound that goes out the peak between retention time 70min～120min has 231 mostly, 247 (246), 264 (263) fragment ion, the chemical compound that explanation goes out the peak in this time period mostly is pseudo-guaianolide type sesquiterpene greatly, and the relative amount that this time period goes out the chemical compound at peak is 42.3%.
As seen from Figure 3, adopting the relative mass content of geraniin in the centipeda volatile oil that adds the method gained of guiding and supporting agent is 28.7%; Equally, the chemical compound that goes out the peak between retention time 70min～120min has 231 mostly, 247 (246), 264 (263) fragment ion, the chemical compound that explanation goes out the peak in this time period mostly is pseudo-guaianolide type sesquiterpene greatly, and retention time is that the relative amount that goes out the chemical compound at peak between 70min～120min is 45.9% in GC figure.
As seen from Figure 4, steam distillation gained volatile oil does not contain geraniin, and retention time is that the relative amount that goes out the chemical compound at peak between 70min～120min is 0% in GC figure.
As seen from Figure 5, adopting the relative mass content of geraniin in the macroporous absorption adsorbent resin column chromatography enrichment gained total sesquiterpene lactone extract of centipeda minima is 51.9%; The relative amount that goes out the chemical compound at peak between GC figure retention time 70min～120min is 99.0%, has 231,247 (246) mostly, and 264 (263) fragment ion illustrates mostly to be pseudo-guaianolide type sesquiterpene greatly.
As seen from Figure 6, adopting the relative mass content of geraniin in the silica gel column chromatography method enrichment gained total sesquiterpene lactone extract of centipeda minima is 63.7%; The relative amount that goes out the chemical compound at peak between GC figure retention time 70min～120min is 97.9%, has 231,247 (246) mostly, and 264 (263) fragment ion illustrates mostly to be pseudo-guaianolide type sesquiterpene greatly.
Whether conclusion: adopt the method for supercritical extraction to extract centipeda volatile oil, no matter add and guide and support agent, the relative amount of geraniin all＞20% in the centipeda volatile oil of gained; The content of geraniin is 0 in the centipeda volatile oil of employing steam distillation method gained.Adopt supercritical extraction method to extract the centipeda volatile oil of gained, no matter be through the macroporous adsorbent resin column chromatography enrichment or through the silica gel column chromatography enrichment, the relative amount of geraniin all＞50% in the total sesquiterpene lactone extract of centipeda minima of gained.
Embodiment 10: the separation of sesquiterpene lactones compounds and structure are identified in the total sesquiterpene lactone extract of centipeda minima:
Total sesquiterpene lactone extract of centipeda minima is crossed silica gel column chromatography, use petroleum ether: ethyl acetate, petroleum ether: acetone, chloroform: methanol, chloroform: acetone, dichloromethane: acetone equal solvent system eluting, and then through sephadex LH-20 gel filtration chromatography purification, obtain 9 sesquiterpene lactones compounds, their chemical constitution adopts spectrum analysis to identify in conjunction with the method for chemical reaction.
Result: from this volatile oil, separate obtaining 9 sesquiterpene lactones compounds, be respectively geraniin, obstruct baileyin more, helenalin, isopropylformic acid. autumnolide, isovaleric acid autumnolide, angelic acid autumnolide, isovaleric acid pulchellin, angelic acid Herba Centipedae element and isopropylformic acid. Herba Centipedae element, wherein plain the and isopropylformic acid. Herba Centipedae element of goose angelic acid Herba Centipedae is a noval chemical compound.GC-MS analyzes (term harmonization of analysis condition and volatile oil) result and shows that these 9 sesquiterpene lactones compounds all go out the peak between retention time 70min～120min.Their structure is as shown below:
The many stalks of geraniin (Brevilin-A) baileyin (Plenolin) helenalin (Helenalin)
Isopropylformic acid. autumnolide isovaleric acid autumnolide angelic acid autumnolide
(Florilenalin-2-O-isobutyrate)??(Florilenalin-2-O-isovalerate)??(Florilenalin-2-O-angelate)
The plain isopropylformic acid. Herba Centipedae of isovaleric acid pulchellin angelic acid Herba Centipedae element
(Pulchellin-2-O-isovalerate)????(Centipedin-2-O-angelate)???????(Centipedin-2-O-isobutyrate)
Angelic acid Herba Centipedae spectroscopic data and physicochemical property plain and isopropylformic acid. Herba Centipedae element is as follows:
Angelic acid Herba Centipedae element: colorless oil, vanillin strong sulfuric acid response show red.UV (MeOH): 241nm; HR-ESI-MS:369.1671 ([M+Na]
+, C
20H
26O
5Na, calc.369.1678);
1HNMR,
13CNMR and 2DNMR data see Table 1.
Table 1 angelic acid Herba Centipedae element
1HNMR,
13CNMR and 2DNMR data
| The position | ?C | ??H | ??HMBC | ??COSY | ??ROESY |
| ??1 | ??54.8 | ??1.94(1H，m) | ??H-14 | ??H-10 | ??H-7，H-8，H-10，??H-14 |
| ??2 | ??76.3 | ??4.91(1H，d，J＝7.1Hz) | ??H-1，H-3 | ??H-3 | ??H-3α，H-10，H-14 |
| ??3α??β | ??39.2 | ??2.20(1H，dd，??J＝15.7，7Hz)??2.02(1H，m) | ??H-15 | ??H-2 | ??H-2，H-6α，H-15??- |
| ??4 | ??68.3 | ??- | ??H-2，H-15 | ??- | ??- |
| ??5 | ??69.6 | ??- | ??H-1，H-3 | ??- | ??- |
| ??6α??β | ??32.3 | ??1.82(1H，m)??2.26(1H，m) | ??- | ??H-7 | ??-??H-3α |
| ??7 | ??38.0 | ??3.51(1H，m) | ??- | ??H-6，H-8，??H-13 | ??H-1，H-8 |
| ??8 | ??75.9 | ??4.86(1H，m) | ??H-6，H-9 | ??H-7，H-9 | ??H-1，H-7 |
| ??9α??β | ??34.9 | ??2.03(1H，m)??1.90(1H，m) | ??H-14 | ??H-10 | |
| ??10 | ??28.7 | ??1.61(1H，m) | ??H-1，H-2，H-14 | ??H-1，H-14 | ??H-1，H-2，H-9α |
| ??11 | ??138.9 | ??- | ??H-13，H-7 | ??- | |
| ??12 | ??169.4 | ??- | ??H-13 | ??- | |
| ??13 | ??123.5 | ??6.31(1H，d，J＝2.7Hz)??5.64(1H，d，J＝2.3Hz) | ??- | ??H-7 | ??- |
| ??14 | ??21.6 | ??1.21(3H，d，J＝6，8Hz) | ??H-1 | ??H-10 | ??H-1，H-2 |
| ??15 | ??15.5 | ??1.39(3H，s) | ??- | ??- | ??H-3α |
| ??16 | ??167.7 | ??- | ??H-2，H-20，??H-18 | ??- | |
| ??17 | ??127.8 | ??- | ??H-19，H-20 | ??- | |
| ??18 | ??137.9 | ??6.04(1H，qd，??J＝7.3，1.4Hz) | ??H-19，H-20 | ??H-19 | ??- |
| ??19 | ??15.8 | ??1.98(3H，dd，??J＝7.4，1.6Hz) | ??- | ??H-18 | ??- |
| ??20 | ??20.6 | ??1.86(3H，m) | ??- | ??- |
Isopropylformic acid. Herba Centipedae element: colorless oil, vanillin strong sulfuric acid response show red.UV (MeOH): 241nm; HR-ESI-MS:357.1662 ([M+Na]
+, C
19H
26O
5Na, calc.357.1678);
1HNMR,
13CNMR and 2DNMR data see Table 2.
Table 2 isopropylformic acid. Herba Centipedae element
1HNMR,
13CNMR and 2DNMR data
| The position | ??C | ??H | ??HMBC | ??COSY | ??ROESY |
| ??1 | ??54.7 | ??1.86(1H，m) | ??H-3，H-14 | ??H-10 | ??H-7，H-8，??H-14，H-15 |
| ??2 | ??76.2 | ??4.77(1H，d，J＝7.3Hz) | ??H-1 | ??H-3 | ??H-3α，H-10，??H-14 |
| ??3α??β | ??39.0 | ??2.11(1H，dd，J＝15.5，7.2Hz)??1.89(1H，m) | ??H-15 | ??H-2 | ??H-2，H-15??- |
| ??4 | ??68.3 | ??- | ??H-1，H-2，H-15 | ??- | ??- |
| ??5 | ??69.6 | ??- | ??H-6 | ??- | ??- |
| ??6α??β | ??32.0 | ??1.78(1H，m，)??2.21(1H，dd，J＝15.5，12.1Hz) | ??- | ??H-7 | ??-??- |
| ??7 | ??37.8 | ??3.46(1H，m) | ??H-6，H-9，H-13 | ??H-6，H-8 | ??H-1，H-8 |
| ??8 | ??75.9 | ??4.82(1H，m) | ??H-6 | ??H-7，H-9 | ??H-1，H-7 |
| ??9α??β | ??34.7 | ??1.98(1H，m)??1.85(1H，m) | ??H-14 | ??H-8 | ??H-10??- |
| ??10 | ??28.5 | ??1.54(1H，m) | ??H-1，H-2，H-14 | ??H-1，H-14 | ??H-2，H-9α |
| The position | ??C | ??H | ??HMBC | ??COSY | ??ROESY |
| ??11 | ??138.7 | ??- | ??H-13 | ??- | ??- |
| ??12 | ??169.3 | ??- | ??H-13 | ??- | ??- |
| ??13 | ??123.4 | ??6.26(1H，d，J＝2.7Hz)??5.60(1H，d，J＝2.4Hz) | ??H-7 | ??- | ??- |
| ??14 | ??21.4 | ??1.13(3H，d，J＝6，8Hz) | ??H-1 | ??H-10 | ??H-1，H-2 |
| ??15 | ??15.4 | ??1.34(3H，s) | ??- | ??- | ??H-1，H-3α |
| ??16 | ??176.7 | ??- | ??H-18，H-19 | ??- | ??- |
| ??17 | ??33.7 | ??2.45(1H，m，J＝7.0Hz) | ??H-18，H-19 | ??H-18，??H-19 | ??- |
| ??18 | ??18.7 | ??1.08(3H，d，J＝7.0Hz) | ??H-19 | ??H-17 | ??- |
| ??19 | ??18.7 | ??1.08(3H，d，J＝7.0Hz) | ??H-18 | ??H-17 | ??- |
Conclusion: total sesquiterpene lactone extract of centipeda minima is made up of at least 9 sesquiterpene lactones compounds, wherein the relative amount of geraniin in extract is no less than 50%, also contains the plain and isopropylformic acid. Herba Centipedae element of 2 noval chemical compound angelic acid Herba Centipedae.
Embodiment 11: each sample of Herba Centipedae suppresses the tumor cell proliferation determination of activity:
Cell strain: hepatoma carcinoma cell HepG-2, prostate gland cancer cell PC-3, breast cancer cell MCF7, leukaemia HL-60, nasopharyngeal carcinoma cell CNE, except that the CNE cell is purchased in the Shanghai Inst. of Life Science, CAS cell bank, ATCC (being provided by biology department of Hong Kong Chinese University) all is provided other cell.
Adopt not interpolation to guide and support among geraniin, the embodiment 1 to adopt the centipeda volatile oil, the volatile oil of steam distillation, the embodiment 3 that add the method gained of guiding and supporting agent to adopt macroporous absorption adsorbent resin column chromatography enrichment gained total sesquiterpene lactone extract of centipeda minima, embodiment 5 to adopt silica gel column chromatography method enrichment gained total sesquiterpene lactone extract of centipeda minima, angelic acid Herba Centipedae element and isopropylformic acid. Herba Centipedae element among the centipeda volatile oil of the method gained of agent, the embodiment 2 is sample, with the positive contrast medicine of cisplatin.
With the inhibited proliferation of tetrazolium salts (MTT) colorimetric method for determining sample to tumor cell: with cell culture in 96 well culture plates, Deng cell monolayer grow good after, add with the good sample (concentration is 10～0.08 μ g/mL) of growth-promoting media (containing mass fraction 10% calf serum) dilution, at 37 ℃, 5%CO
2Cultivated 3 days in the incubator.The MTT solution (5mg/mL disposes with phosphate buffer solution) that adds 20 μ L continues to cultivate 4 hours.The sucking-off sample solution adds the dimethyl sulfoxine dissolving, under the room temperature, 96 orifice plates is placed microvoid orifice plate agitator vibration 10 minutes.Measure the OD value in each hole with microplate reader, the measurement wavelength is 570nm, and reference wavelength is 630nm, the half-inhibition concentration (IC of calculation sample cell growth
50).Establish 4 balance pipes for every group, every group of experiment repeats 3 times.The results are shown in Table 3.
Each sample extracorporeal suppression tumor cell proliferation activity measurement result (IC of table 3 Herba Centipedae
50, μ g/mL)
"-" is undetermined.
Whether conclusion: (1) adopts the volatile oil of supercritical extraction gained, no matter add in the leaching process and guide and support agent, and tumor cell is all had fine proliferation inhibition activity; (2) the total sesquiterpene lactone extract increased activity through obtaining after the effective site enrichment; (3) geraniin in the total sesquiterpene lactone extract and two new sesquiterpene lactones monomeric compounds all have good active.
The foregoing description is a preferred implementation of the present invention; but embodiments of the present invention are not restricted to the described embodiments; other any do not deviate from change, the modification done under spirit of the present invention and the principle, substitutes, combination, simplify; all should be the substitute mode of equivalence, be included within protection scope of the present invention.
Claims (10)
1. total sesquiterpene lactone extract of centipeda minima, it is characterized in that: this extract contains angelic acid Herba Centipedae element (formula I) and isopropylformic acid. Herba Centipedae element (formula II):
(formula I) (formula II)
2. the preparation method of a kind of total sesquiterpene lactone extract of centipeda minima according to claim 1, it is characterized in that comprising following operating procedure: the dry herb of Herba Centipedae is pulverized, adopted supercritical carbon dioxide method for extracting to extract and obtain centipeda volatile oil; Again centipeda volatile oil is obtained total sesquiterpene lactone extract of centipeda minima through enrichment of macroporous adsorbent resin column chromatography method or silica gel column chromatography enrichment;
The enrichment of described macroporous adsorbent resin column chromatography method, be that described centipeda volatile oil is crossed macroporous resin column, with the ethanol water gradient elution, adopting the gas chromatography-mass spectrum methods analyst to follow the tracks of detects, collection contains stream part of sesquiterpene lactones chemical compound, merges stream part, concentrating under reduced pressure, drying obtains total sesquiterpene lactone extract of centipeda minima;
Described silica gel column chromatography method enrichment, be that described centipeda volatile oil is crossed silicagel column, with the mixed organic solvents gradient elution, adopting the gas chromatography-mass spectrum methods analyst to follow the tracks of detects, collection contains stream part of sesquiterpene lactones chemical compound, merges stream part, concentrating under reduced pressure, drying gets total sesquiterpene lactone extract of centipeda minima.
3. the preparation method of a kind of total sesquiterpene lactone extract of centipeda minima according to claim 2, it is characterized in that: described Herba Centipedae is the wounded in the battle herb of feverfew Myriogyne minuta.
4. the preparation method of a kind of total sesquiterpene lactone extract of centipeda minima according to claim 2, it is characterized in that: the extracting pressure of described supercritical carbon dioxide method for extracting is 20～40 MPas, extraction temperature is 30～50 ℃, and flow velocity is 20 liters/hour, and extraction time is 1～4 hour.
5. the preparation method of a kind of total sesquiterpene lactone extract of centipeda minima according to claim 2 is characterized in that: added in the described supercritical carbon dioxide method for extracting and guided and supported agent.
6. the preparation method of a kind of total sesquiterpene lactone extract of centipeda minima according to claim 5 is characterized in that: described to guide and support agent be ethanol.
7. the preparation method of a kind of total sesquiterpene lactone extract of centipeda minima according to claim 2 is characterized in that: described is that to adopt mass percent concentration successively be that 20～30% and 50～80% ethanol water carries out gradient elution with the ethanol water gradient elution.
8. the preparation method of a kind of total sesquiterpene lactone extract of centipeda minima according to claim 2, it is characterized in that: described mixed organic solvents is counted petroleum ether with volume ratio: acetone=9: 1～1: 9, petroleum ether: ethyl acetate=9: 1～1: 9, hexane: ethyl acetate=9: 1～1: 9 or hexane: acetone=9: 1～1: 9.
9. the preparation method of a kind of total sesquiterpene lactone extract of centipeda minima according to claim 2, it is characterized in that: the quality percentage composition of geraniin is no less than 50% in the described total sesquiterpene lactone extract of centipeda minima.
10. total sesquiterpene lactone extract of centipeda minima according to claim 1 suppresses the purposes of the medicine of tumor proliferation as preparation.
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