CN103102336A - Inula wissmannian extract and preparation thereof and application in preparation of antitumor drug - Google Patents
Inula wissmannian extract and preparation thereof and application in preparation of antitumor drug Download PDFInfo
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Abstract
The invention provides an inula wissmannian extract and preparation thereof and application in preparation of an antitumor drug. The extract is gemma alkane type sesquiterpene lactone inula wissmannian lactone methyl, ethyl, propyl, butyl and amyl or sheepear inula herb lactone. The extract has the following chemical structural formula and absolute configuration that: due to the toxicity killing function experiment of inula wissmannian lactone methyl, ethyl, propyl, butyl and amyl or sheepear inula herb lactone on human hepatoma cells HepG2, human prostatic cancer cells PC-3, human gastric carcinoma cells MGC-803, human leukemia cells K562, human nasopharynx cancer cells KB and normal human hepatocytes LO2, the result shows that the gemma alkane type sesquiterpene lactone has strong tumor cell killing function, and proliferation of tumor cells can be inhibited, so that the tumor cells are killed. The animal in-vivo experiment proves that the inula wissmannian plant extract and monomeric compounds have the anti-tumor effect and can serve as active ingredients for preparing the antitumor drug. A novel cancer treatment medicine is provided clinically, and high clinical application values and social benefits are generated.
Description
Technical field
The present invention relates to natural drug, be specifically related to extraction and separation method and the purposes of the southern regions of the Yunnan Province Sheepear Inula Herb effective constituent; Relate in particular to extraction and separation method and the application in the preparation antitumor drug of the southern regions of the Yunnan Province Sheepear Inula Herb extract Germacrane Sesquiterpenoids lactone.
Background technology
Malignant tumour has become a global public health problem.Over nearly 30 years, the Incidence number is with the speed increase of average annual 3%-5%, and 3/4 newly-increased case occurs in country of infant industry and developing country, and malignant tumour has become first cause of the death of the mankind.In China, malignant tumour has become one of major causes of death at present.Along with economical, society, environment and mode of life occur to change fast, demographic structure changes to aging fast, the variation of Chinese cancer spectrum expression characteristics.(the Zheng Rongshou such as Zheng Rongshou, Zhang Siwei, Wu Liangyou, Li Guanglin, Zhao Ping, He Jie, Chen Wanqing, China's tumour registration area Incidence in 2008 and Study on mortality [J], China's tumour, 2012,21 (1): 1-12) China tumour regional pathogenesis of cancer of registration and Study on mortality are shown: no matter city or rural area, Incidence account for former be mainly lung cancer, cancer of the stomach, colorectal cancer, liver cancer, the esophageal carcinoma, carcinoma of the pancreas, brain tumor, lymphoma, women with breast cancer and cervical cancer etc., account for 75% left and right of whole Incidences; Former of mortality of malignant tumors be mainly lung cancer, cancer of the stomach, liver cancer, the esophageal carcinoma, colorectal cancer, carcinoma of the pancreas, mammary cancer, brain tumor, leukemia and lymphoma, account for 80% left and right of whole mortality of malignant tumors.
At present, chemotherapy has become one of Main Means for the treatment of tumour.Antitumor drug commonly used usually can be divided into alkylating agent, antimetabolite, microbiotic, plant origin and act on microtubule and protein synthesis system medicine, metal complex class and other antitumour drug.These medicines have effectively extended patient's life or have improved Quality of Life, but its obvious toxic side effect has limited the performance of its curative effect.Current medical science still is in the junior stage for the control of tumour.Chinese medicine has the human toxicity experiment basis of several thousand, from natural product, seeks the lead compound of the anti-tumor activity of high-efficiency low-toxicity, is the focus of new drug research always.
The southern regions of the Yunnan Province Sheepear Inula Herb (Inula wissmanniana Hand.-Mazz.), Yunnan Province is commonly called as " torch stalk ", is the distinctive composite family Inulaplants of China, produces south of Yunnan (Yuanjiang River, screen limit), be born in low mountain and open spacious hillside fields, 1200 ~ 1650 meters of height above sea level.There is more than 10 kind of Inulaplants to have medicinal record.Chinese scholars (Zhao YM, Zhang ML, Shi QW, Kiyota H.Chemical constituents of plants from the genus Inula.Chemistry ﹠amp; Biodiversity 2006; 3:371-383) successively separate the compound that obtains the structure types such as flavones, sesquiterpene lactones, terpene from this platymiscium, sesquiterpene lactones is the characteristic chemical constituent of this platymiscium; Sesquiterpene lactones has very important biological activity, has the multiple effects such as anti-inflammatory, antitumor, antiviral, expelling parasite desinsection, immunosuppression.Sesquiterpene lactones is also simple in structure because of it, and has the various structures form, can be used as the lead compound of research and development medicine.At present, there is no the bibliographical information of relevant the southern regions of the Yunnan Province Sheepear Inula Herb chemical composition and pharmacologically active thereof, also have no the report relevant with the southern regions of the Yunnan Province Sheepear Inula Herb method for preparing extractive, purposes in the present invention.
Summary of the invention
The purpose of technical problem to be solved by this invention is to overcome above-mentioned weak point, research and design the southern regions of the Yunnan Province Sheepear Inula Herb extract and extraction and separation method and purposes.
The invention provides a kind of the southern regions of the Yunnan Province Sheepear Inula Herb extract, described extract is mainly Germacrane Sesquiterpenoids the southern regions of the Yunnan Province Sheepear Inula Herb lactone first, second, third, fourth, penta or the Sheepear Inula Herb lactone, and their structural formula and absolute configuration are as follows respectively:
The southern regions of the Yunnan Province Sheepear Inula Herb lactone first:
The southern regions of the Yunnan Province Sheepear Inula Herb lactone second:
The southern regions of the Yunnan Province Sheepear Inula Herb lactone third:
The southern regions of the Yunnan Province Sheepear Inula Herb lactone fourth:
The southern regions of the Yunnan Province Sheepear Inula Herb lactone penta:
The Sheepear Inula Herb lactone:
The southern regions of the Yunnan Province Sheepear Inula Herb lactone first, second, third, fourth, penta and the Sheepear Inula Herb lactone be colourless block crystallization, be soluble in chloroform, acetone, methyl alcohol, ethanol, molecular formula is followed successively by C
22H
30O
6, C
20H
28O
5, C
20H
28O
4, C
19H
26O
4, C
15H
22O
3, C
22H
30O
7Its chemical structure and absolute configuration by high resolution mass spectrum in conjunction with one dimension, two dimensional NMR wave spectrum and X-ray single crystal diffraction Analysis deterrmination.
Another object of the present invention has been to provide the preparation method of described the southern regions of the Yunnan Province Sheepear Inula Herb extract.
Preparation of the present invention comprises the preparation of extracting separation and monomeric compound.
Preparation method of the present invention comprises the following steps:
(1) the southern regions of the Yunnan Province Sheepear Inula Herb herb is dry, pulverize, extract 3～4 times with 1 ~ 10 times of amount 80～95% ethanol soaking at room temperature, each 12～24 hours, merge to get extracting solution;
(2) the concentrating under reduced pressure extracting solution to without the alcohol flavor, gets water suspendible fluid extract, adds 1～3 times of water dilution, uses successively 2～6 times of sherwood oils, methylene dichloride, ethyl acetate extraction, and reclaim under reduced pressure respectively extracts part, gets without aqueous extract;
(3) with above-mentioned methylene dichloride position application silica gel column chromatography, methylene chloride/methanol system gradient elution take volume ratio as 100:0～2:1, thin-layer chromatography detects, collect respectively and contain the southern regions of the Yunnan Province Sheepear Inula Herb lactone first, second, third, fourth, penta and the flow point of Sheepear Inula Herb lactone, again through dextran gel column chromatography, methylene chloride/methanol wash-out take volume ratio as 1:1 finally by liquid chromatography preparation, gets the southern regions of the Yunnan Province Sheepear Inula Herb lactone first, second, third, fourth, penta and the Sheepear Inula Herb lactone.
Another purpose of the present invention has been to provide the application of described the southern regions of the Yunnan Province Sheepear Inula Herb extract in the preparation antitumor drug.
The inventor by with the southern regions of the Yunnan Province Sheepear Inula Herb lactone first, second, third, fourth, penta and the Sheepear Inula Herb lactone toxicity lethal effect of normal liver cell LO2, human liver cancer cell HepG2, gastric carcinoma cells MGC-803, Human Prostate Cancer Cells PC-3, Leukemia K562 cell, KB cell KB is carried out research trial:
Research the southern regions of the Yunnan Province Sheepear Inula Herb lactone first, second, third, fourth, penta and the Sheepear Inula Herb lactone on the impact of normal liver cell LO2 propagation, result shows its IC
50Value is respectively 35.20,27.83,37.88,75.60,73.17,75.42 μ M, this lethal effect than respective compound to the lethal effect of liver cancer cell HepG2 a little less than 5-10 doubly, show the southern regions of the Yunnan Province Sheepear Inula Herb lactone first, second, third, fourth, penta and the Sheepear Inula Herb lactone can significantly, optionally suppress the propagation of liver cancer cell, and under the same terms on the not significant impact of the propagation of normal cell lines of human liver strain.The southern regions of the Yunnan Province Sheepear Inula Herb lactone first, second, third, fourth, penta and the Sheepear Inula Herb lactone MGC-803, PC-3, K562 and KB tumour cell are also shown powerful lethal effect, its IC
50Value is 3.49～47.76 μ M.
Show through the cell cycle analysis result, the southern regions of the Yunnan Province Sheepear Inula Herb lactone first, second, third, fourth, penta and the Sheepear Inula Herb lactone can significantly block liver cancer cell HepG2, gastric carcinoma cells MGC-803 in the fissional G2/M phase, and the propagation of inhibition tumor cell optionally causes the death of tumour cell.
At mouse H
22Find in the liver-cancer solid tumor model experiment, high density the southern regions of the Yunnan Province Sheepear Inula Herb general extractive, methylene dichloride position, the southern regions of the Yunnan Province Sheepear Inula Herb lactone first, the third all show good anti-tumor capacity, significantly suppress the growth of lump, the above-mentioned Germacrane Sesquiterpenoids lactone of presentation of results has strong killing tumor cell effect.Therefore, the southern regions of the Yunnan Province Sheepear Inula Herb extract the southern regions of the Yunnan Province Sheepear Inula Herb lactone, the southern regions of the Yunnan Province Sheepear Inula Herb lactone first, second, third, fourth or penta can be used for preparing antitumor drug.
Medicine of the present invention is the pharmaceutical composition that is comprised of as activeconstituents and pharmaceutical excipient the southern regions of the Yunnan Province Sheepear Inula Herb extract.
Medicine of the present invention is the pharmaceutical composition that is comprised of as activeconstituents and pharmaceutical excipient the southern regions of the Yunnan Province Sheepear Inula Herb lactone.
Medicine of the present invention is the pharmaceutical composition that is comprised of as activeconstituents and pharmaceutical excipient the southern regions of the Yunnan Province Sheepear Inula Herb lactone first.
Medicine of the present invention is the pharmaceutical composition that is comprised of as activeconstituents and pharmaceutical excipient the southern regions of the Yunnan Province Sheepear Inula Herb lactone second.
Medicine of the present invention is the pharmaceutical composition that is comprised of as activeconstituents and pharmaceutical excipient the southern regions of the Yunnan Province Sheepear Inula Herb lactone third.
Medicine of the present invention is the pharmaceutical composition that is comprised of as activeconstituents and pharmaceutical excipient the southern regions of the Yunnan Province Sheepear Inula Herb lactone fourth.
Medicine of the present invention is the pharmaceutical composition that is comprised of as activeconstituents and pharmaceutical excipient the southern regions of the Yunnan Province Sheepear Inula Herb lactone penta.
The present invention discloses Sheepear Inula Herb extract the southern regions of the Yunnan Province, the southern regions of the Yunnan Province Sheepear Inula Herb lactone, the southern regions of the Yunnan Province Sheepear Inula Herb lactone first, second, third, fourth or penta first; And their extraction and separation method and the application in pharmacy be provided, and provide new treatment malignant tumor medicine for clinical, bring glad tidings to the patient, larger clinical value and social benefit are arranged.
Description of drawings
Fig. 1 the southern regions of the Yunnan Province Sheepear Inula Herb lactone first X diffraction structure figure
Fig. 2 the southern regions of the Yunnan Province Sheepear Inula Herb lactone the third X diffraction structure figure
Embodiment
The present embodiment is implemented under take technical solution of the present invention as prerequisite, has provided detailed embodiment and process, but protection scope of the present invention is not limited to following embodiment.The experimental technique of unreceipted actual conditions in the following example, usually according to normal condition, or the condition of advising according to manufacturer.
Embodiment 1 the southern regions of the Yunnan Province Sheepear Inula Herb lactone first, second, third, fourth, penta and the preparation of Sheepear Inula Herb lactone
After the southern regions of the Yunnan Province Sheepear Inula Herb herb (10.0kg) drying and crushing, under 25 ℃ of room temperatures with 95% ethanol (30L) cold soaking extract 4 times (24,12,12,12h), reclaim solvent, dryly without aqueous extract (631.4g) concentrated; Total medicinal extract is suspended in 1.5L water and extracts respectively 3 times with 5L sherwood oil, methylene dichloride, ethyl acetate successively, decompression and solvent recovery obtains respectively petroleum ether part (133.2g), methylene dichloride position (179.6g) and ethyl acetate extract (28.1g).
With the upper normal phase silicagel column of the methylene dichloride position 120g of the southern regions of the Yunnan Province Sheepear Inula Herb, use successively methylene chloride-methanol (100% methylene dichloride, 100:1,50:1,20:1,10:1,5:1 and 100% methyl alcohol) gradient elution, elutriant detects through thin-layer chromatography, merges the elutriant of similar composition, and decompression and solvent recovery gets 8 wash-out parts.Get wherein the 1st wash-out part, upper silica gel column chromatography is with petroleum ether-ethyl acetate (20:1) wash-out, and through the elutriant of the similar composition of thin-layer chromatography combining data detection, decompression and solvent recovery gets 6 wash-out flow points.With the least possible methylene dichloride dissolving, standing under 25 ℃ of room temperatures the 2nd wash-out flow point (2.9g) wherein, separate out block crystallization, be accredited as the southern regions of the Yunnan Province Sheepear Inula Herb lactone third (1.52g); The 3rd flow point (3.3g) is in the least possible dichloromethane solution, and be standing under 25 ℃ of room temperatures, separates out block crystallization the southern regions of the Yunnan Province Sheepear Inula Herb lactone first (1.23g); After dissolving, filter with Chromatographic Pure Methanol (3mL), the 4th flow point (0.9g) through the preparative chromatography method, with 70% methanol-eluted fractions, obtain compound the southern regions of the Yunnan Province Sheepear Inula Herb lactone fourth (t
R40.0min, 12.5mg).In addition, then with the 2nd wash-out part (12.4g), after methyl alcohol (30mL) dissolution filter, upper MCI column chromatography with 80% methanol-eluted fractions, obtains 2 flow points.The 1st flow point (2.6g) further gone up Sephadex LH-20 gel column chromatography, with methylene chloride-methanol 1:1 wash-out, elutriant detects through thin-layer chromatography, 8 elutriants that merge similar composition, reclaim solvent and get 8 wash-out flow points, again wherein the 3rd (0.7g) and the 4th flow point (0.9g) are gone up respectively the preparative chromatography post, with 55% methyl alcohol (10L) wash-out, obtain the southern regions of the Yunnan Province Sheepear Inula Herb lactone penta (t
R32.0min, 25.6mg), Sheepear Inula Herb lactone (t
R58.2min, 31.1mg), and the southern regions of the Yunnan Province Sheepear Inula Herb lactone second (t
R75.0min, 30.0mg).
The southern regions of the Yunnan Province Sheepear Inula Herb lactone first, second, third, fourth, penta and the Sheepear Inula Herb lactone
1H-NMR and
13The C-NMR data see Table respectively 1 and table 2, and optical rotational activity spectrum, infrared spectra, mass spectrum, high resolution mass spectrum and X-ray single crystal diffraction data are as follows respectively:
The southern regions of the Yunnan Province Sheepear Inula Herb lactone first: brilliant without color lump; C
22H
30O
6;
(c0.10, CH
2Cl
2); IR (KBr) ν
max2931,1768,1726,1661,1456,1382,1314,1245,1162,1134,1071,1019,975cm
-1; ESIMS m/z413.2[M+Na]
+, m/z389.2[M-H]
-; HRESIMS m/z413.1943[M+Na]
+(calcd for C
22H
30O
6Na, 413.1940); Crystallization data: C
22H
30O
6, CH
2Cl
2, M=390.46, orthorhombic, space group P2 (1) 2 (1) 2 (1), a=6.7751 (3)
α=90 °; B=8.9058 (4)
β=90 °; C=35.0890 (14)
γ=90 °; V=2117.19 (16)
Z=4, D
Calcd=1.225mg/m
3, crystallite size 0.20 * 0.15 * 0.10mm
3.Cu K α (λ=1.54178
), F (000)=840, T=133 (2) K.R value is respectively R
1=0.0317, and wR
2=0.0873, for 22899 observed reflections[I〉2 σ (I)] the .Flack parameter is 0.05 (15).Relevant crystallization data trnascription can be in (the storage number: CCDC835957) of Cambridge crystallization Data centre Free Acquisition, address: CCDC, 12Union Road, Cambridge CB21EZ, UK (fax:+441223 336033 or e-mail:data_requestccdc.cam.ac.uk).
The southern regions of the Yunnan Province Sheepear Inula Herb lactone second: brilliant without color lump; C
20H
28O
5;
(c0.10, CH
2Cl
2); IR (KBr) ν
max3430,2928,2861,1763,1707,1650,1453,1381,1245,1150,1021,975cm
-1; ESIMS m/z371.1[M+Na]
+, m/z347.1[M-H]
-; HRESIMS m/z371.1836[M+Na]
+(calcd for C
20H
28O
5Na, 371.1834).
The southern regions of the Yunnan Province Sheepear Inula Herb lactone third: brilliant without color lump; C
20H
28O
4;
(c0.10, CH
2Cl
2); IR (KBr) ν
max2937,2923,2856,1766,1705,1643,1450,1379,1234,1153,1144,1037,973cm
-1; ESIMS m/z355.2[M+Na]
+, m/z331.2[M-H]
-; HRESIMS m/z355.1856[M+Na]
+(calcd for C
20H
28O
4Na, 355.1855); Crystallization data: C
20H
28O
4, CH
2Cl
2, M=332.42, orthorhombic, space group P2 (1) 2 (1) 2 (1), a=11.9746 (5)
α=90 °; B=12.3428 (5)
β=90 °; C=12.4853 (5)
γ=90 °; V=1845.33 (13)
Z=4, D
Calcd=1.197mg/m
3, crystallite size 0.20 * 0.15 * 0.10mm
3.Cu K α (λ=1.54178
), F (000)=720, T=273 (2) K.R value is R respectively
1=0.0276, and wR
2=0.0767, for20005 observed reflections[I〉2 σ (I)] the .Flack parameter is 0.02 (15).Relevant crystallization data trnascription can be in (the storage number: CCDC835958) of Cambridge crystallization Data centre Free Acquisition, address: CCDC, 12Union Road, Cambridge CB21EZ, UK (fax:+441223336033or e-mail:data_requestccdc.cam.ac.uk).
The southern regions of the Yunnan Province Sheepear Inula Herb lactone fourth: brilliant without color lump; C
22H
30O
6;
(c0.05, CH
2Cl
2); IR (KBr) ν
max2930,2863,1770,1713,1633,1571,1454,1383,1317,1281,1250,1141,1072,1010,976cm
-1; ESIMSm/z341.2[M+Na]
+, m/z317.2[M-H]
-; HRESIMS:m/z341.1733[M+Na]
+(calcd for C
19H
26O
4Na, 341.1729).
The southern regions of the Yunnan Province Sheepear Inula Herb lactone penta: brilliant without color lump; C
15H
22O
3;
(c0.10, CH
2Cl
2); IR (KBr) ν
max3441,2936,2865,1765,1663,1457,1382,1274,1249,1144,1079,1012,973cm
-1; ESIMSm/z273.1[M+Na]
+, m/z249.1[M-H]
-; HRESIMS:m/z273.1466[M+Na]
+(calcdfor C
15H
22O
3Na, 273.1467).
Sheepear Inula Herb lactone: brilliant without color lump; C
22H
30O
7; ESIMSm/z429.2[M+Na]
+, m/z405.2[M-H]
-
Table 1 the southern regions of the Yunnan Province Sheepear Inula Herb lactone first, second, third, fourth, penta and the Sheepear Inula Herb lactone
1H-NMR data (CDCl
3,
400MHz)
Continued 1
Compound atom numbering is seen structural formula of compound figure; Nuclear magnetisation displacement study unit is ppm; Be the coupling constant value in bracket, unit is Hz.
Table 2 the southern regions of the Yunnan Province Sheepear Inula Herb lactone first, second, third, fourth, penta and the Sheepear Inula Herb lactone
13C-NMR data (CDCl
3, 400MHz)
Compound atom numbering is seen structural formula of compound figure; Nuclear magnetisation displacement study unit is ppm.
Embodiment 2 the southern regions of the Yunnan Province Sheepear Inula Herb lactone first, second, third, fourth, penta and Sheepear Inula Herb lactone (embodiment 1 makes) to the lethal effect of 5 kinds of human tumor cells
The southern regions of the Yunnan Province Sheepear Inula Herb lactone first, second, third, fourth, penta and the Sheepear Inula Herb lactone human liver cancer cell HepG2, gastric carcinoma cells MGC-803, Human Prostate Cancer Cells PC-3, Leukemia K562 cell, KB cell KB(are purchased from Shanghai cell resource center of Sheng Ke institute) the toxicity lethal effect study.
Experimental agents the southern regions of the Yunnan Province Sheepear Inula Herb lactone first, second, third, fourth, penta and Sheepear Inula Herb lactone (embodiment 1 makes) jointly hatch 72h with tumour cell; Medicine concentration respectively is set to 0,0.5,5,20,50 μ M; Medicine dissolution is in the DMSO(methyl-sulphoxide).With the positive contrast of Zorubicin (Doxorubicin), the negative contrast of DMSO.
Experimental technique: CCK-8 method (cell counting test kit-8).It is 4 ~ 6 * 10 that 96 orifice plate every holes add concentration
4The cell suspension 100uL of individual/mL puts 37 ℃, in the 5%CO2 incubator.After 24h, add different concns medicine 1uL/ hole, establish two multiple holes, 37 ℃, 5%CO2 effect 72h.Every hole adds the CCK-8 reagent of 10uL, after cultivating 2-4h, surveys 450nm OD value with full-automatic microplate reader.
Estimate and statistical method: (the WellscanMK-2 type Labsystems) is surveyed the 450nm OD of place value, and various experimental results are carried out statistical study with SPSS software one-way ANNOVA method with full-automatic microplate reader.
Experimental result show the southern regions of the Yunnan Province Sheepear Inula Herb lactone first, second, third, fourth, penta and the Sheepear Inula Herb lactone human liver cancer cell HepG2, gastric carcinoma cells MGC-803, Leukemia K562 cell, Human Prostate Cancer Cells PC, KB cell KB are all had toxicity lethal effect (the results are shown in Table 3) preferably, illustrate that it has cytotoxicity lethal effect widely.
Table 3 the southern regions of the Yunnan Province Sheepear Inula Herb lactone first, second, third, fourth, penta and the Sheepear Inula Herb lactone to the restraining effect of 5 kinds of human tumor cells
Embodiment 3 the southern regions of the Yunnan Province Sheepear Inula Herb lactone first, second, third, fourth, penta and Sheepear Inula Herb lactone (embodiment 1 makes) on the impact of human liver cancer cell HepG2 and normal liver cell LO2 propagation
The southern regions of the Yunnan Province Sheepear Inula Herb lactone first, second, third, fourth, penta have optionally toxicity lethal effect (relative normal liver cell's strain) with the Sheepear Inula Herb lactone to human liver cancer cell, research and inquirement the impacts of these 6 compounds on human liver cancer cell HepG2 and normal liver cell LO2 propagation.
Experimental period gradient: 0,24,48,72 hours; Experimental agents the southern regions of the Yunnan Province Sheepear Inula Herb lactone first, second, third, fourth, penta and Sheepear Inula Herb lactone concentration gradient arrange: 0,1,5,10,25,50 μ M; Medicine dissolution is in DMSO.
Experimental technique: CCK-8 method.It is 4 ~ 6 * 10 that 96 orifice plate every holes add concentration
4The cell suspension 100uL of individual/mL puts 37 ℃, in the 5%CO2 incubator.After 24h, add different concns medicine 1uL/ hole, establish two multiple holes, 37 ℃, 5%CO2 cultivates.
Evaluation method: every 24 hours detection cell viabilities.Every hole adds the CCK-8 reagent of 10uL, after cultivating 2-4h, surveys 450nm OD value with full-automatic microplate reader.
Result of study shows, the southern regions of the Yunnan Province Sheepear Inula Herb lactone first, second, third, fourth, penta and the Sheepear Inula Herb lactone can significantly suppress the propagation of HepG2 cell lines, just can receive significant inhibition in 5-10 μ M, and for normal liver cell line LO2, in 50 μ M, apparent in view inhibited proliferation appears.The southern regions of the Yunnan Province Sheepear Inula Herb lactone first, second, third, fourth, penta and the Sheepear Inula Herb lactone suppress the IC of normal liver cell LO2 propagation
50Value is respectively 35.20,27.83,37.88,75.60,73.17,75.42 μ M.Therefore, this description of test the southern regions of the Yunnan Province Sheepear Inula Herb lactone first, second, third, fourth, penta and the Sheepear Inula Herb lactone can significantly, optionally suppress the propagation of human liver cancer cell, and under the same terms, the hepatocellular propagation of normal people is not had significant restraining effect.
Embodiment 4 the southern regions of the Yunnan Province Sheepear Inula Herb lactone first, second, third, fourth, penta and Sheepear Inula Herb lactone (embodiment 1 makes) to the cell cycle inhibition research of human liver cancer cell HepG2, gastric carcinoma cells MGC-803
The southern regions of the Yunnan Province Sheepear Inula Herb lactone first, second, third, fourth, penta and the concentration gradient setting respectively of Sheepear Inula Herb lactone Experimental agents: 0,1,5 μ M; Medicine dissolution is in DMSO; During drug treating, the content of DMSO lower than 0.1%, is processed the cycle of 12 hours post analysis cells.
Experimental procedure: tumour cell sucks cell conditioned medium after processing 12 hours with the different concns gradient, and centrifugal collecting cell after trysinization fixedly spends the night with 4 ℃ of 70% ethanol afterwards, adds 50 μ μ g/mL PI solution, upflowing cell instrument detection after 15min.
The cell cycle analysis result shows, the southern regions of the Yunnan Province Sheepear Inula Herb lactone first, second, third, fourth, penta and the Sheepear Inula Herb lactone can significantly block human liver cancer cell HepG2 and gastric carcinoma cells MGC-803 in the fissional G2/M phase, it is consistent that two compounds of this result and this can significantly, optionally suppress the propagation of liver cancer cell HepG2 and stomach cancer cell MGC-803.Therefore, infer the southern regions of the Yunnan Province Sheepear Inula Herb lactone first, second, third, fourth, penta and the Sheepear Inula Herb lactone may be by blocking significantly human liver cancer cell HepG2, gastric carcinoma cells MGC-803 in the fissional G2/M phase propagation of inhibition tumor cell optionally, cause the death of cancer cells.
The anti-tumor in vivo of embodiment 5 the southern regions of the Yunnan Province Sheepear Inula Herb general extractives, methylene dichloride position, the southern regions of the Yunnan Province Sheepear Inula Herb lactone first, the third (embodiment 1 makes) is active
Research the southern regions of the Yunnan Province Sheepear Inula Herb general extractive, methylene dichloride position, the southern regions of the Yunnan Province Sheepear Inula Herb lactone first, the third are at mouse H
22In vivo antitumor effect in the liver cancer cell solid tumor models, the growing state of observation tumor animal is measured inhibition rate of tumor growth.
Animal is processed and grouping: extract the H in liver cancer mouse ascites under aseptic condition
22Cell is with aseptic PBS(phosphate buffered saline buffer) be adjusted into 5 * 10
6/ ml.In the capable aseptic subcutaneous vaccination of BALB/c mouse left side abdomen, inoculum size 0.2ml cell suspension.After inoculation to hour being divided at random 14 groups, 10 every group.Administration in the 3rd day after inoculation.The 1st group of negative contrast gives physiological saline; The 2nd group of positive control group, give endoxan (Hualian Pharmaceutical Co., Ltd., Shanghai, 20mg/kg/d); The 3rd group gives high density the southern regions of the Yunnan Province Sheepear Inula Herb general extractive (500mg/kg/d); The 4th group gives middle concentration the southern regions of the Yunnan Province Sheepear Inula Herb general extractive (200mg/kg/d); The 5th group gives lower concentration the southern regions of the Yunnan Province Sheepear Inula Herb general extractive (100mg/kg/d); The 6th group gives high density Sheepear Inula Herb methylene dichloride position, the southern regions of the Yunnan Province (100mg/kg/d); The 7th group gives middle concentration Sheepear Inula Herb methylene dichloride position, the southern regions of the Yunnan Province (50mg/kg/d); The 8th group gives lower concentration Sheepear Inula Herb methylene dichloride position, the southern regions of the Yunnan Province (20mg/kg/d); The 9th group gives high density the southern regions of the Yunnan Province Sheepear Inula Herb lactone first (50mg/kg/d); The 10th group gives middle concentration the southern regions of the Yunnan Province Sheepear Inula Herb lactone first (20mg/kg/d); The 11st group gives lower concentration the southern regions of the Yunnan Province Sheepear Inula Herb lactone first (10mg/kg/d); The 12nd group gives high density the southern regions of the Yunnan Province Sheepear Inula Herb lactone third (50mg/kg/d); The 13rd group gives middle concentration the southern regions of the Yunnan Province Sheepear Inula Herb lactone third (20mg/kg/d); The 14th group gives lower concentration the southern regions of the Yunnan Province Sheepear Inula Herb lactone third (10mg/kg/d).Successive administration was observed 15 days.
Tumor-inhibiting action is estimated: the growing state of observing tumor-bearing mice every day.Weigh the next day of rising in the 5th day after administration and to measure the knurl block long-pending, with the major diameter of electronic vernier caliper measurement knurl body and minor axis.Gross tumor volume=[(knurl body major diameter+knurl body minor axis)/2]
2; Tumour inhibiting rate=(the average knurl of the average knurl weight-treatment group of control group is heavy)/average knurl of control group heavy * 100%.Draw tumor growth curve according to tumour inhibiting rate.After drug withdrawal, put to death mouse next day, and the stripping knurl is weighed.
Experimental result shows under experimental concentration, the southern regions of the Yunnan Province Sheepear Inula Herb general extractive, methylene dichloride position, the southern regions of the Yunnan Province Sheepear Inula Herb lactone first, the third all show obvious tumor suppression effect, when particularly giving high density the southern regions of the Yunnan Province Sheepear Inula Herb lactone first, the third 50mg/kg/d to tumor-bearing mice, tumour inhibiting rate and positive drug are suitable.Observation gives the growing state of the tumor-bearing mice tumour of different high concentration medicine treatments, as seen give physiological saline group tumor growth rapid, after administration the 7th day, its lump is obviously greater than other administration groups, gives positive drug endoxan, high density the southern regions of the Yunnan Province Sheepear Inula Herb general extractive, methylene dichloride position and the southern regions of the Yunnan Province Sheepear Inula Herb lactone first, the third and all can effectively suppress the growth of lump.Experimental result shows under administration concentration, and the southern regions of the Yunnan Province Sheepear Inula Herb general extractive, methylene dichloride position and the southern regions of the Yunnan Province Sheepear Inula Herb lactone first, the third all show anti-tumor activity.
The tumor inhibition effect of table 4 the southern regions of the Yunnan Province Sheepear Inula Herb general extractive, methylene dichloride position, the southern regions of the Yunnan Province Sheepear Inula Herb lactone first, the third pair tumor-bearing mice
* compare P＜0.01 with the physiological saline group.
Claims (10)
1. the southern regions of the Yunnan Province Sheepear Inula Herb extract, is characterized in that, described extract is the southern regions of the Yunnan Province Sheepear Inula Herb lactone first, second, third, fourth, penta and the Sheepear Inula Herb lactone, and their structural formula and absolute configuration are as follows respectively:
2. prepare the method for the southern regions of the Yunnan Province Sheepear Inula Herb extract as claimed in claim 1, it is characterized in that,
The method comprises the following steps:
(1) the southern regions of the Yunnan Province Sheepear Inula Herb herb is dry, pulverize, extract 3～4 times with 1 ~ 10 times of amount 80～95% ethanol soaking at room temperature, each 12～24 hours, merge to get extracting solution;
(2) the concentrating under reduced pressure extracting solution to without the alcohol flavor, gets water suspendible fluid extract, adds 1～3 times of water dilution, uses successively 2～6 times of sherwood oils, methylene dichloride, ethyl acetate extraction, and reclaim under reduced pressure respectively extracts part, gets without aqueous extract;
(3) with above-mentioned methylene dichloride position application silica gel column chromatography, methylene chloride/methanol system gradient elution take volume ratio as 100:0～2:1, thin-layer chromatography detects, collect respectively and contain the southern regions of the Yunnan Province Sheepear Inula Herb lactone first, second, third, fourth, penta and the flow point of Sheepear Inula Herb lactone, again through dextran gel column chromatography, methylene chloride/methanol wash-out take volume ratio as 1:1 finally by liquid chromatography preparation, gets the southern regions of the Yunnan Province Sheepear Inula Herb lactone first, second, third, fourth, penta and the Sheepear Inula Herb lactone.
3. the application of the southern regions of the Yunnan Province Sheepear Inula Herb extract as claimed in claim 1 in the preparation antitumor drug.
4. application as claimed in claim 3, is characterized in that, described medicine is the pharmaceutical composition that is comprised of as activeconstituents and pharmaceutical excipient the southern regions of the Yunnan Province Sheepear Inula Herb extract.
5. application as claimed in claim 3, is characterized in that, described medicine is the pharmaceutical composition that is comprised of as activeconstituents and pharmaceutical excipient the southern regions of the Yunnan Province Sheepear Inula Herb lactone.
6. application as claimed in claim 3, is characterized in that, described medicine is the pharmaceutical composition that is comprised of as activeconstituents and pharmaceutical excipient the southern regions of the Yunnan Province Sheepear Inula Herb lactone first.
7. application as claimed in claim 3, is characterized in that, described medicine is the pharmaceutical composition that is comprised of as activeconstituents and pharmaceutical excipient the southern regions of the Yunnan Province Sheepear Inula Herb lactone second.
8. application as claimed in claim 3, is characterized in that, described medicine is the pharmaceutical composition that is comprised of as activeconstituents and pharmaceutical excipient the southern regions of the Yunnan Province Sheepear Inula Herb lactone third.
9. application as claimed in claim 3, is characterized in that, described medicine is the pharmaceutical composition that is comprised of as activeconstituents and pharmaceutical excipient the southern regions of the Yunnan Province Sheepear Inula Herb lactone fourth.
10. application as claimed in claim 3, is characterized in that, described medicine is the pharmaceutical composition that is comprised of as activeconstituents and pharmaceutical excipient the southern regions of the Yunnan Province Sheepear Inula Herb lactone penta.
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| CN103664839A (en) * | 2013-11-12 | 2014-03-26 | 中国人民解放军第二军医大学 | Application of Inula wissmanniana lactone A and derivatives in preparing antineoplastic drugs |
| CN103664840A (en) * | 2013-11-12 | 2014-03-26 | 中国人民解放军第二军医大学 | Application of Inula wissmanniana lactone A and derivatives in preparing antiinflammatory drugs |
| CN104926761A (en) * | 2014-03-18 | 2015-09-23 | 中国人民解放军第二军医大学 | Isocyanate, benzoic acid and cinnamic acid substituted inula wissmanniana hand.-mazz. lactone analogues and synthesis and application thereof |
| CN105294623A (en) * | 2015-11-18 | 2016-02-03 | 浙江中医药大学 | Sesquiterpene lactone compound, preparation method and application thereof |
| CN108864015A (en) * | 2018-08-28 | 2018-11-23 | 青海大学 | It is a kind of to have effects that treat the compound and pharmaceutical composition of neurodegenerative disease |
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| CN103664839A (en) * | 2013-11-12 | 2014-03-26 | 中国人民解放军第二军医大学 | Application of Inula wissmanniana lactone A and derivatives in preparing antineoplastic drugs |
| CN103664840A (en) * | 2013-11-12 | 2014-03-26 | 中国人民解放军第二军医大学 | Application of Inula wissmanniana lactone A and derivatives in preparing antiinflammatory drugs |
| CN103664839B (en) * | 2013-11-12 | 2016-01-13 | 中国人民解放军第二军医大学 | Inula wissmanniana lactone A and derivative thereof are preparing the application in antitumor drug |
| CN103664840B (en) * | 2013-11-12 | 2016-01-13 | 中国人民解放军第二军医大学 | Inula wissmanniana lactone A and derivative thereof are preparing the application in anti-inflammatory drug |
| CN104926761A (en) * | 2014-03-18 | 2015-09-23 | 中国人民解放军第二军医大学 | Isocyanate, benzoic acid and cinnamic acid substituted inula wissmanniana hand.-mazz. lactone analogues and synthesis and application thereof |
| CN104926761B (en) * | 2014-03-18 | 2017-03-29 | 中国人民解放军第二军医大学 | The southern regions of the Yunnan Province ocean ear chrysanthemum lactone analogue and its synthesis and application that isocyanates, benzoic acid and cinnamic acid replace |
| CN105294623A (en) * | 2015-11-18 | 2016-02-03 | 浙江中医药大学 | Sesquiterpene lactone compound, preparation method and application thereof |
| CN108864015A (en) * | 2018-08-28 | 2018-11-23 | 青海大学 | It is a kind of to have effects that treat the compound and pharmaceutical composition of neurodegenerative disease |
| CN110343116A (en) * | 2018-12-21 | 2019-10-18 | 中山大学 | A kind of Flos Chrysanthemi Indici extract and preparation method thereof and the application in preparation treatment medicine for nasopharyngeal |
| CN110343116B (en) * | 2018-12-21 | 2022-02-22 | 中山大学 | Wild chrysanthemum extract, preparation method thereof and application thereof in preparation of medicine for treating nasopharyngeal carcinoma |
| CN116554136A (en) * | 2023-04-23 | 2023-08-08 | 五邑大学 | Sesquiterpene lactone with anti-prostate cancer activity and preparation method and application thereof |
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Application publication date: 20130515 |