CN101475458A - Mitissimol with antineoplastic activity, preparation and use thereof - Google Patents
Mitissimol with antineoplastic activity, preparation and use thereof Download PDFInfo
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- CN101475458A CN101475458A CNA2008100801339A CN200810080133A CN101475458A CN 101475458 A CN101475458 A CN 101475458A CN A2008100801339 A CNA2008100801339 A CN A2008100801339A CN 200810080133 A CN200810080133 A CN 200810080133A CN 101475458 A CN101475458 A CN 101475458A
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Abstract
The invention provides Luctarius rhzomorph with anti-tumor activity. The method for preparing the Luctarius rhzomorph is to extract from higher fungi Lactarius mitissimus, Lctarius hirtipes J.Z.Ying and other fruiting bodies by one or more solvents of ethanol, methanol, chloroform, ethyl acetate, acetone and sherwood oil, and chromatographically separate the prepared extractive by a silica gel column repeatedly. The invention also provides application direction of the Luctarius rhzomorph in preparation of an anti-tumor medicine, including preparation of an anti-tumor medicine taking the Luctarius rhzomorph as an effective ingredient clinically. The Luctarius rhzomorph has stronger anti-tumor activity through in-vitro experiments, wherein the activity of Luctarius rhzomorph Mitissimol D is the strongest with half inhibition concentration IC50 to A549 (lung adenocarcinoma cell line), HCT-8 (human colon cancer cell line), Bel-7402 (human hepatoma cell line) and Hela (human cervical carcinoma cell line) of 2.5, 11.0, 3.6 and 4.6 mu g/ml respectively, and is almost equivalent to positive control drug cisplatin. Therefore, the compounds are expected to be novel anti-tumor medicines.
Description
Technical field
The present invention relates to a kind of Mitissimol and its production and application, belong to extraction separation and the preparation antitumor drug applied technical field of fungi with anti-tumor activity.
Background technology
Cancer has become one of main threat of human health, and is obvious ascendant trend, and cancer has become topmost dead factor in the U.S., accounts for the first place of the various causes of the death in the city in China's cancer mortality.Thereby China classifies the research of cancer therapy drug one of as most important strategic problem of new drug development, and studying novel cancer therapy drug with efficient, low toxicity, highly selective becomes the secular objectives and tasks of drug research worker.
Nature provides extensive and single-minded effective bioactive ingredients, and many organisms have developed a whole set of defense mechanism person that comes the contest competition in the long-term evolution process.Organism employed " weapon " is more and more exquisiter, and therefore seeking new activeconstituents or basic structure from natural resources is a very wise way as medicine or agricultural chemicals.
In various Biological resources, fungi belongs to " creation coefficient " extra high biology, contains the various secondary metabolite of a large amount of structures.People contrast the people by experiment for making it injured and int sporophore, find sporophore a large amount of secondary metabolite of very fast generation after injured, various active such as that these products have is antimycotic, desinsection.One of them typical example is: be converted at once after the impaired reaction of the stearoylvelutinal of non-activity (1) among the Lactarius Lactarious and have very strong active microbiotic isovelleral (2).Higher fungi more and more causes people's attention in recent years.Be because from higher fungi, constantly find novel structure and compound on the one hand, and structural changes is big with notable biological activity.This structure diversity is significant for the discovery of medicine (or agricultural chemicals).On the other hand, higher fungi not only can directly be adopted and be used as research material usefulness, and can pass through mycelium or spore fermentation culture more conveniently, and a large amount of raw material resources are provided, and for possible from now on suitability for industrialized production provides assurance, this also is a potential advantage.Sesquiterpenoid in the higher fungi is a big compounds type in the higher fungi, and its structural changes is very abundant, and biological activity is remarkable.Mainly comprise tremulane, caryophyllane, collybial, protoilludanes, marasmanes, lactaranes, cucumanes, fomannosanes, illudanes, isoilludanes, hirsutanes, pleurotellanes, cerapicanes, bulleranes, isolactaranes and merulanes type sesquiterpene.This existence in fungi of Humulane sesquiterpene is very rare, has only applicant's research group to find a series of new humulane Humulane sesquiterpenes at present, and has stronger anti-tumor activity, yet there are no other reports at present both at home and abroad.
Summary of the invention
The purpose of this invention is to provide the new humulane Humulane sesquiterpenoid Mitissimol (mitissiols) of a class and, extract the method for Mitissimol in the dry foot breast mushroom sporophores such as (Lactarius hirtipes J.Z.Ying) from thin matter breast mushroom (Lactariusmitissimus) with anti-tumor activity; Further providing with the Mitissimol simultaneously is the application direction of activeconstituents preparation treatment and prevention antitumor drug aspect medicine.
Technical scheme of the present invention is achieved in that this Mitissimol with anti-tumor activity, and its feature comprises the Mitissimol of following general structural formula (I):
Wherein: R
1Be hydrogen, carboxyl, hydroxyl or aldehyde radical; R
2Be hydrogen, hydroxyl, ester group or acyl ammonia; R
3Be hydrogen, carboxyl, hydroxyl or aldehyde radical; R
4Be hydrogen, carboxyl, hydroxyl or aldehyde radical; R
5For hydrogen or hydroxyl or 5 are methylene radical or carbonyl; 3,4 or 6,7 or 8,9 or 10,11 form epoxy or form singly-bound or the two keys of formation.
Described Mitissimol with anti-tumor activity comprises that the humulane sesquiterpene Mitissimol mitissiols A-H structure of (I) feature that has general formula is as follows:
Described Mitissimol with anti-tumor activity comprises the humulane sesquiterpene Mitissimol 11 β-19 α-epoxy-4Z of (I) feature that has general formula, and 7Z-humuladiene-5 α-ol structure is as follows:
Described preparation method with Mitissimol of anti-tumor activity: described Mitissimol with anti-tumor activity is from the sporophore of the thin matter breast mushroom of higher fungi (Lactarius mitissimus), with wherein one or more solvent extractions of ethanol, methyl alcohol, chloroform, ethyl acetate, acetone, sherwood oil, obtain extract obtained the separation with silica gel column chromatography repeatedly.
Described preparation method with Mitissimol of anti-tumor activity comprises the steps:
A, extraction: the thin matter of 1.6kg higher fungi breast mushroom (Lactariusmitissimus) is pulverized, and with 95% extraction using alcohol of 15L 3 times, again with ethyl acetate 4.5L extraction, solvent recuperation is to doing, weigh 33.5g;
B, enter 200-300 order silica gel column chromatography and separate:
A, use CHCl
3-MeOH solvent is with 100:0,98:2, and 95:5,90:10 (Vol/Vol) gradient elution is at CHCl
3-MeOH100:0 wash-out Duan Deyi component is further carried out silica gel column chromatography, uses sherwood oil-acetone solvent with 25:1,15:1, and 10:1, the 5:1 gradient elution gets pure product Mitissimol mitissimol A 66mg at the 15:1 position;
B, at a step CHCl
3-MeOH 95:5 wash-out Duan Deyi component is further carried out silica gel column chromatography, uses sherwood oil-acetone solvent with 10:1,8:1, and 6:1,4:1,5:1,2:1, the 1:1 gradient elution gets pure product Mitissimol mitissimol B 8mg at the 10:1 position;
C, at b step sherwood oil-further silica gel column chromatography in acetone 2:1 position, petroleum ether-ethyl acetate is with 20:1,15:1,10:1,5:1,1:1, the 1:5 gradient elution gets pure product Mitissimol mitissimol C 26mg in petroleum ether-ethyl acetate 20:1 component;
D, further utilize SephadexLH-20 gel filtration chromatography, CHCl in step c petroleum ether-ethyl acetate 15:1 component
3-MeOH 1:1 is an eluent, and purifying obtains Mitissimol mitissimol D 5mg;
E, further utilize the thin layer preparative chromatography in step c petroleum ether-ethyl acetate 10:1 component, sherwood oil-acetone 4:1 is a developping agent, and purifying obtains Mitissimol mitissimolE 8mg;
F, obtain Mitissimol mitissimol F 17mg in step c petroleum ether-ethyl acetate 5:1 component;
G, obtain Mitissimol mitissimol G 4mg in step c petroleum ether-ethyl acetate 1:1 component;
H, get pure product Mitissimol mitissimol 7mg at sherwood oil-acetone 4:1 position in the b step.
Described preparation method with Mitissimol of anti-tumor activity, described Mitissimol with anti-tumor activity is from the sporophore of higher fungi dry foot breast mushroom (Lactarius hirtipes J.Z.Ying), with wherein one or more solvent extractions of ethanol, methyl alcohol, chloroform, ethyl acetate, acetone, sherwood oil, obtain extract obtained the separation with silica gel column chromatography repeatedly.
Described preparation method with Mitissimol of anti-tumor activity comprises the steps:
A, extraction: with 6kg higher fungi dry foot breast mushroom (Lactarius hirtipes J.Z.Ying), pulverize, with 95% extraction using alcohol of 20L 3 times, again with ethyl acetate 5L extraction, solvent recuperation is to doing, weigh 8.2g;
B, 200-300 order silica gel column chromatography separate: with sherwood oil-acetone gradient elution 50:1,20:1,9:1,5:1,1:1 gets 5 components, gets sherwood oil-acetone 9:1 component and uses the RP-C18 column chromatography repeatedly, with sherwood oil-chloroform recrystallization, obtain Mitissimol 2 β-3 α-epoxy-6Z, 9Z-humuladiene-8 α-ol 22mg.
Described application with Mitissimol of anti-tumor activity as the preparation antitumor drug comprises that being used to clinically prepare with the Mitissimol is the antitumor drug of effective constituent.
Described application with Mitissimol of anti-tumor activity as the preparation antitumor drug, comprise being used to clinically prepare and contain composition or the compound that Mitissimol is an effective constituent, the material that preparation contains Mitissimol pharmacy acceptable salt, solvate or corresponding crystal habit is an effective constituent, and adds the antitumor drug of medicine acceptable carrier or ancillary component.
Mitissimol of the present invention shows to have stronger anti-tumor activity through in vitro tests, wherein Mitissimol Mitissimol D activity is the strongest, to A549 (human lung adenocarcinoma cell line), HCT-8 (human colon cancer cell strain), Bel-7402 (human hepatoma cell strain), the half-inhibition concentration IC of Hela (human cervical carcinoma cell strain)
50Be respectively 2.5,11.0,3.6,4.6 μ g/ml, almost suitable with positive control medicament cis-platinum, this compounds is expected to become new antitumor drug.
Description of drawings
Fig. 1 is the crystallogram of Mitissimol Mitissimol A
Fig. 2 is Mitissimol (S)-MTPA ester 1b and Mitissimol (R)-MTPA ester 1a chemical shift difference
Embodiment
Embodiment 1: the preparation of Mitissimol mitissiols A-H
A, extraction:
To pick up from the thin matter breast mushroom of 1.6kg higher fungi (Lactarius mitissimus) on the firm mountain of sorrow, Yunnan Province (its bacterial classification is kept at Kunming Inst. of Botany, Chinese Academy of Sciences's Specimen Room) pulverizes, with 95% extraction using alcohol of 15L 3 times, again with ethyl acetate 4.5L extraction, solvent recuperation is to doing, weigh 33.5g;
B, enter 200-300 order silica gel column chromatography and separate:
A, use CHCl
3-MeOH solvent is with 100:0,98:2, and 95:5,90:10 (Vol/Vol) gradient elution is at CHCl
3-MeOH 100:0 wash-out Duan Deyi component is further carried out silica gel column chromatography, uses sherwood oil-acetone solvent with 25:1,15:1, and 10:1, the 5:1 gradient elution gets pure product Mitissimol mitissimol A 66mg at the 15:1 position;
B, at a step CHCl
3-MeOH 95:5 wash-out Duan Deyi component is further carried out silica gel column chromatography, uses sherwood oil-acetone solvent with 10:1,8:1, and 6:1,4:1,5:1,2:1, the 1:1 gradient elution gets pure product Mitissimol mitissimol B 8mg at the 10:1 position;
C, at b step sherwood oil-further silica gel column chromatography in acetone 2:1 position, petroleum ether-ethyl acetate is with 20:1,15:1,10:1,5:1,1:1, the 1:5 gradient elution gets pure product Mitissimol mitissimol C 26mg in petroleum ether-ethyl acetate 20:1 component;
D, further utilize SephadexLH-20 gel filtration chromatography, CHCl in step c petroleum ether-ethyl acetate 15:1 component
3-MeOH 1:1 is an eluent, and purifying obtains Mitissimol mitissimol D 5mg;
E, further utilize the thin layer preparative chromatography in step c petroleum ether-ethyl acetate 10:1 component, sherwood oil-acetone 4:1 is a developping agent, and purifying obtains Mitissimol mitissimolE8mg;
F, obtain Mitissimol mitissimol F 17mg in step c petroleum ether-ethyl acetate 5:1 component;
G, obtain Mitissimol mitissimol G 4mg in step c petroleum ether-ethyl acetate 1:1 component;
H, get pure product Mitissimol mitissimol 7mg at 4: 1 positions of sherwood oil-acetone in the b step.
C, Mitissimol mitissiols A-H physico-chemical property
Material and instrument:
Fusing point is measured with the micro-fusing point instrument of XRC-1 type that Sichuan scientific instrument factory produces, and temperature is not proofreaied and correct; Optically-active is measured with the SEPA-300 instrument; Infrared at Bio-Rad FTS-135 determination of infrared spectroscopy; Ultraviolet is measured on the UV-210A ultraviolet spectrometer; NMR is by Bruker AM-400 and Bruker AM-500 nmr determination, and tetramethylsilane (TMS) is interior mark; Mass spectrum is measured by VGA Auto Spec-3000 type mass spectrograph.Thin-layer chromatography and column chromatography silica gel are Haiyang Chemical Plant, Qingdao's product, and Sephadex LH-20 is a Pharmacia company product.With 10% sulfuric acid-ethanol heating colour developing and iodo steam displaing color.
Mitissimol mitissimol A:
Colourless crystallization (CHCl
3); Mp169-171 ℃; [α]
D 25.2-42.0 (c0.40, CHCl
3); UV (CHCl
3) UV (CHCl
3) λ
Max(log ε) 252 (4.30) nm; IR (KBr): 3481,2988,2959,2925,2881,1638,1445,1388,1297,1273,1045,968,900cm
-1 1H-NMR (500MHz, CDCl
3) δ: 4.22 (d, 1H, J=10.0,1-H), 5.69 (d, 1H, J=16.4,3-H), 6.06 (d, 1H, J=16.4,4-H), 5.90 (brd, 1H, J=10.6,7-H), 2.47 (m, 1H, 8-H
α), 2.28 (m, 1H, 8-H
β α), 2.37 (m, 1H, 9-H
α), 2.23 (m, 1H, 9-H
β), 5.25 (brd, 1H, J=10.6,11-H), 1.14 (s, 3H, 12-CH
3), 1.14 (s, 3H, 13-CH
3), 1.79 (brs, 3H, 14-CH
3), 1.62 (s, 3H, 15-CH
3);
13C NMR (CDCl
3, 500MHz)) and δ: 75.6 (d), 42.0 (s), 157.0 (d), 127.2 (d), 203.7 (s), 137.8 (s), 148.1 (d), 24.6 (t), 39.5 (t), 138.6 (s), 128.0 (d), 26.6 (q), 17.1 (q), 11.7 (q), 16.0 (q); FABMS (pos) m/z235[M+1]
+, 469[2M+1]
+HRESIM (pos) m/z257.1516 (measured value) (C
15H
22O
2Na, theoretical value 257.1517).
The X crystalline diffraction structural analysis of Mitissimol Mitissimol A shown in Figure 1: C
15H
22O
2, M is 234.33, spacer is P2 (1) 2 (1) 2 (1) unit cell parameters a=9.1505 (18)
B=9.5977 (19)
C=6.152 (3)
Unit cell volume: V=418.5 (5)
Molecule number Z=4 in the structure cell; Structure cell density d=1.097Mg/m
3R
fAnd R
wValue is respectively 0.0423 and 0.1049.Visit instrument with the MAC-DIP-2030K face and collect diffracted intensity data, MOK α radiation, graphite monochromator.Use the ω scan mode, obtain independent point diffraction 7350 and 2517.On microcomputer, resolve crystalline structure with direct method (SHELXS86).CCDC number is 601319.
The atomic coordinate of table 1, Mitissimol Mitissimol A
The bond distance of table 2, Mitissimol Mitissimol A, bond angle value
Mitissimol mitissimol B:
Colourless acicular crystal (CHCl
3); Mp200-201 ℃; [α]
D 25.5-1.0 (c0.59, CHCl
3); UV (CHCl
3) λ
Max(log ε) 244 (3.43) nm; IR (KBr): 3507,3011,2995,2962,2924,1651,1641,1466,1386,1294,1271,1061,975,911cm
-1 1HNMR (500MHz, CDCl
3) δ: 3.30 (d, 1H, J=9.7,1-H), 5.93 (d, 1H, J=16.4,3-H), 6.25 (d, 1H, J=16.4,4-H), 6.05 (brd, 1H, J=8.7,7-H), 2.41 (m, 2H, 8-H), 2.28 (ddd, 1H, J=13.4,3.9,2.8,9-H
α), 1.38 (td, 1H, J=13.4,7.1,9-H
β), 2.75 (d, 1H, J=9.7,11-H), 1.16 (s, 3H, 12-CH
3), 1.24 (s, 3H, 13-CH
3), 1.85 (brs, 3H, 14-CH
3), 1.29 (s, 3H, 15-CH
3);
13CNMR (CDCl
3, 500MHz)) and δ: 76.1 (d), 40.4 (s), 155.9 (d), 129.2 (d), 202.5 (s), 139.2 (s), 147.0 (d), 24.5 (t), 38.0 (t), 63.5 (s), 65.8 (d), 26.8 (q), 17.1 (q), 12.0 (q), 16.7 (q); ESIMS (pos) m/z 273[M+Na]
+, 523[2M+Na]
+HRESIMS (pos) m/z273.1474 (measured value) (C
15H
22O
3Na, theoretical value 273.1466).
Mitissimol mitissimol C:
Colourless crystallization (MeOH); Mp193-196 ℃; [α]
D 25.1+ 99.0 (c0.64, MeOH); UV (MeOH) λ
Max(log ε) 252 (4.11) nm; IR (KBr): 3424 (OH), 3037,2962,2928,1647 (C=CC=OC=C), 1477,1386,1099,1026,994,901cm
-1 1H NMR (400MHz, CD
3OD) δ: 4.19 (d, 1H, J=10.7,1-H), 5.84 (d, 1H, J=16.5,3-H), 6.07 (d, 1H, J=16.5,4-H), 5.73 (brd, 1H, J=10.0,7-H), 4.61 (ddd, 1H, J=10.4,10.0,5.5,8-H), 2.70 (dd, 1H, J=11.4,5.5,9-H
α), 2.12 (dd, 1H, J=11.4,10.4,9-H
β), 5.30 (brd, 1H, J=10.7,11-H), 1.11 (s, 3H, 12-CH
3), 1.13 (s, 3H, 13-CH
3), 1.858 (brs, 3H, 14-CH
3), 1.65 (s, 3H, 15-CH
3);
13CNMR (CD
3OD, 400MHz)) δ: 76.0 (d), 43.8 (s), 161.4 (d), 128.0 (d), 206.5 (s), 140.7 (s), 147.8 (d), 65.6 (d), 52.0 (t), 135.8 (s), 130.9 (d), 26.9 (q), 17.6 (q), 12.1 (q), 17.3 (q); ESIMS (pos) [M+Na]
+273, [2M+Na]
+523; HRESIMS (pos) m/z273.1467[M+Na]
+(measured value) (C
15H
22O
3Na, theoretical value 273.1466).
Mitissimol mitissimol D:
White powder; M.p.193-196 ℃ (MeOH); [α]
D 25+ 138.0 (c0.51, MeOH); UV (MeOH) λ max (log ε) 230 (3.93); IR (KBr): 3338 (OH), 3039,2961,2928,1665 (C=CCOC=C), 1388,1359,1126,1031,982,909cm
1 1H NMR (500MHz, CD
3OD) δ: 3.25 (d, 1H, J=9.7,1-H), 6.12 (d, 1H, J=16.4,3-H), 6.28 (d, 1H, J=16.4,4-H), 5.92 (brd, 1H, J=9.9,7-H), 4.53 (ddd, 1H, J=12.1,9.9,5.3,8-H), 2.51 (dd, 1H, J=12.7,5.3,8-H
α), 1.27 (dd, 1H, J=12.7,12.1,8-H
α), 2.79 (d, 1H, J=9.7,11-H), 1.22 (s, 3H, 12-CH
3), 1.11 (s, 3H, 13-CH
3), 1.95 (brs, 3H, 14-CH
3), 1.29 (s, 3H, 15-CH
3);
13CNMR (CD
3OD) δ: 76.9 (d), 41.9 (s), 160.1 (d), 130.5 (d), 205.3 (s), 142.8 (s), 146.8 (d), 65.2 (d), 48.4 (t), 61.8 (s), 66.7 (d), 27.2 (q), 17.7 (q), 12.5 (q), 18.0 (q); ESI-MS (pos) m/z[M+Na]
+289, [2M+Na]
+555; HRESI-MS (pos) m/z 289.1408 (measured values) (calcd for C
15H
22O
4Na, theoretical value 289.1415).
Mitissimol mitissimol E:
White powder; M.p.80-82 ℃ (MeOH); [α]
D 18-41.5 (c0.5, MeOH); UV (MeOH) λ max (log ε) 233 (3.84); IR (KBr): 3442 (OH), 2967,2929,1687,1637 (C=OC=C), 1467,1389,1365,1055,966,916cm
-1 1H NMR (400MHz, CD
3OD) δ: 3.39 (d, 1H, J=9.8,1-H), 6.08 (d, 1H, J=17.2,3-H), 6.80 (d, 1H, J=17.2,4-H), 2.81 (d, 1H, J=9.1,7-H), 3.77 (ddd, 1H, J=11.6,9.1,3.6,8-H), 2.44 (dd, 1H, J=13.4,3.6,8-H
α), 1.40 (dd, 1H, J=13.4,11.6,8-HH
α), 2.77 (d, 1H, J=9.8,11-H), 1.20 (s, 3H, 12-CH
3), 1.18 (s, 3H, 13-CH
3), 1.69 (s, 3H, 14-CH
3), 1.46 (s, 3H, 15-CH
3);
13CNMR (CD
3OD) δ: 76.1 (d), 41.8 (s), 158.9 (d), 128.1 (d), 200.4 (s), 67.0 (s), 66.9 (d), 66.0.4 (d), 46.7 (t), 60.6 (s), 66.0 (d), 27.5 (q), 18.1 (q), 16.7 (q), 18.9 (q); ESI-MS (pos) m/z[M+Na]
+305, [2M+Na]
+587; HRESI-MS (pos) m/z305.1365 (measured value) (C
15H
22O
5Na, theoretical value, 305.1364).
Mitissimol mitissimol F:
Colourless gelatinoid, m.p.148-150 ℃ (MeOH); [α]
D 25.1-37.7 (c0.55, MeOH); UV (MeOH) λ
Max(log ε) 235 (3.48); IR (KBr): 3441,2968,2927,1676,1617,1453,1395,1150,1116,1064,994,901cm
-1Cm
-1 1H NMR (CD
3OD) δ: 3.34 (d, 1H, J=10.0,1-H), 6.20 (d, 1H, J=16.1,3-H), 7.11 (d, 1H, J=16.1,4-H), 2.52 (dd, 1H, J=13.4,9.2,7-H
α), 1.23 (dd, 1H, J=13.4,5.2,7-H
β), 2.43 (ddd, 1H, J=9.2,5.2,2.4,8-H), 2.68 (d, 1H, J=2.4,9-H), 2.81 (d, 1H, J=10.0,11-H), 1.22 (s, 3H, 12-CH
3), 1.13 (s, 3H, 13-CH
3), 1.40 (s, 3H, 14-CH
3), 0.95 (s, 3H, 15-CH
3);
13C NMR (CD
3OD) δ: 72.9 (d), 43.2 (s), 150.7 (d), 129.8 (d), 209.5 (s), 79.4 (s), 44.8 (t), 52.4 (d), 60.3 (d), 61.8 (s), 62.3 (d), 27.1 (q), 18.3 (q), 27.9 (q), 11.9 (q); ESI-MS (pos) m/z[M+Na]
+305, [2M+Na]
+587; HR-ESI-MS (pos) m/z305.1373 (measured value) (C
15H
22O
5Na, theoretical value 305.1364).
Utilize the Mosher compositions and methods to measure the absolute configuration of Mitissimol mitissimol F: the Mosher method is the usefulness that development in recent years is got up
1H NMR measures the method for secondary alcohol compounds absolute configuration.It is R configuration and S configuration of compound and the secondary alcohol formation ester that utilizes 2-methoxyl group-2-(trifluoromethyl)-2-phenyl-acetic acid (MTPA), according to
1The last Δ of HNMR (Δ=S-R) on the occasion of or the molecular model of negative value and possible two isomer determine the absolute configuration of secondary alcohol.According to experimental result shown in Figure 2, the H-C (3) of Mitissimol (R)-MTPA ((2-methoxyl group-2-(trifluoromethyl)-2-phenyl-acetic acid) (2-methoxyl group-2-(trifluoromethyl)-2-phenyl-acetic acid)) ester 1a, H-C (4), CH
2(7), H-C (8), Me (12), the chemical shift of Me (13) and Me (14) than Mitissimol (S)-MTPA ((2-methoxyl group-2-(trifluoromethyl)-2-phenyl-acetic acid) (2-methoxyl group-2-(trifluoromethyl)-2-phenyl-acetic acid)) ester 1b at High-Field more, and H-C (9), the chemical shift of H-C (11) and Me (15) is at lower.Therefore, Mitissimol mitissimol F is the S configuration in the absolute configuration of C-1.Concrete grammar is as follows:
MTPA (2-methoxyl group-2-(trifluoromethyl)-2-phenyl-acetic acid) and mitissimol F esterification: get 2.82mg Mitissimol mitissimol F, 11.2mg (S)-MTPA is at CHCl
2Dissolve in the 10ml solution, add (S)-MTPA 11.2mg again, N, N-dicyclohexylcarbodiimide N, N '-dicyclohexylcarbodiimide (DCC) 9.9mg and 4-Dimethylamino pyridine 4-dimethylaminopyridine (DMAP) 2mg are in the 50ml round-bottomed flask.Round-bottomed flask is positioned on the magnetic stirring apparatus stirs, at room temperature react 12h.Reaction solution gets Mitissimol (S)-MTPA ester 1a 3.1mg with preparation TLC (thin-layer chromatography); Get 2.60mg Mitissimol mitissimol F, 11.2mg (R)-MTPA is at CHCl
2Dissolve in the 10ml solution, add (R)-MTPA 10.8mg again, N, N-dicyclohexylcarbodiimide N, N '-dicyclohexylcarbodiimide (DCC) 8.7mg and 4-Dimethylamino pyridine 4-dimethylaminopyridine (DMAP) 2mg are in the 50ml round-bottomed flask.Round-bottomed flask is positioned on the magnetic stirring apparatus stirs, at room temperature react 12h.Reaction solution gets Mitissimol (R)-MTPA ester 1a 3.1mg with preparation TLC (thin-layer chromatography), gets Mitissimol (R)-MPTA ester 1b 2.6mg.
Mitissimol (R)-MTPA (2-methoxyl group-2-(trifluoromethyl)-2-phenyl-acetic acid) ester 1a:
Colourless amorphous powder;
1HNMR δ 7.180 (1H, d, J=16.3Hz, H-4), 6.231 (1H, d, J=16.1Hz, H-3), 5.087 (1H, d, J=10.0Hz, H-1), 2.947 (1H, d, J=10.0Hz, H-11), 2.696 (1H, d, J=2.4Hz, H-9), (2.540 1H, dd, J=13.2,4.9Hz, H-7 α), (1.224 1H, br d, J=9.7Hz, H-7 β), 2.474 (1H, br d, J=8.2Hz, H-8), 1.403 (3H, s, H-14), 1.244 (3H, s, H-12), 1.117 (3H, s, H-13), 0.896 (3H, s, H-15); FABMS m/z499
Mitissimol (S)-MTPA (2-methoxyl group-2-(trifluoromethyl)-2-phenyl-acetic acid)) ester 1b:
Colourless amorphous powder;
1HNMR δ 7.149 (1H, d, J=16.2Hz, H-4), 6.197 (1H, d, J=16.2Hz, H-3), 5.075 (1H, d, J=10.1Hz, H-1), 3.015 (1H, d, J=10.1Hz, H-11), 2.732 (1H, brd, J=2.4Hz, H-9), (2.538 1H, dd, J=13.3,5.0Hz, H-7 α), (1.221 1H, br d, J=9.2Hz, H-7 β), 2.472 (1H, br d, J=8.9Hz, H-8), 1.397 (3H, s, H-14), 1.239 (3H, s, H-12), 1.112 (3H, s, H-13), 0.905 (3H, s, H-15); FABMSm/z499
Mitissimol mitissimol G:
Colourless gelatinoid, m.p.205-207 ℃ of (MeOH) .[α]
D 24.2-2.b (c0.11, CHCl
3) .UV (CHCl
3) λ
Max(log ε) 241 (3.63); IR (KBr): 3552,3391,2990,2967,2927,1696,1630,1464,1387,1338,1127,1081,1049,999,911cm
-1.
1H NMR (pyridine-d
5) δ: 3.62 (d, 1H, J=9.5,1-H), 6.77 (d, 1H, J=16.2,3-H), 7.58 (d, 1H, J=16.2,4-H), 2.67 (brd, 1H, J=14.6,7-H
α), 2.57 (dd, 1H, J=14.6,6.8,7-H
β), 3.80 (dd, 1H, J=9.7,6.8,8-H), 2.54 (brd, 1H, J=13.1,9-H
α), 1.80 (dd, 1H, J=13.1,9.7,9-H
β), 3.25 (d, 1H, J=9.5,11-H), 1.43 (s, 3H, 12-CH
3), 1.32 (s, 3H, 13-CH
3), 2.05 (s, 3H, 14-CH
3), 1.34 (s, 3H, 15-CH
3);
13C NMR (pyridine-d
5) δ: 74.6 (d), 42.1 (s), 150.8 (d), 127.2 (d), 204.1 (s), 77.6 (s), 51.6 (t), 64.6 (d), 52.9 (t), 60.7 (s), 65.1 (d), 26.7 (q), 17.6 (q), 22.6 (q), 18.8 (q); ESI-MS (pos) m/z[M+Na]
+307, [2M+Na]
+591.HR-ESI-MS (pos) m/z307.1522 (measured value) C
15H
24O
5Na, theoretical value 307.1521).
Mitissimol mitissimol H:
White powder; M.p.63-65 ℃ (MeOH); [α]
D 19.5+ 3.08 (c0.06, MeOH); UV (MeOH) λ max (log ε) 202 (3.54); IR (KBr): 3440,2932,1709,1456,1015,975,911cm
-1 1H-NMR (CD
3OD) δ: 3.35 (d, 1H, J=10.0,1-H), 5.43 (t, 1H, J=5.6,3-H), 1.97 (d, 1H, J=2.67,4-H
α), 1.70 (overlapped, 1H, 4-H
β), 3.01 (m, 1H, 6-H), 4.37 (d, 1H, J=8.2,7-H), 3.75 (m, 1H, 8-H), 2.02 (d, 1H, J=1.3,9-H
α), 1.70 (overlapped, 1H, 9-H
β), 5.38 (d, 1H, J=9.8,11-H), 1.71 (overlapped, 3H, 12-CH
3), 1.61 (brs, 3H, 13-CH
3), 0.99 (brs, 3H, 14-CH
3), 0.82 (s, 3H, 15-CH
3);
13CNMR (CD
3OD) δ: 60.5 (d), 47.6 (s), 97.9 (d), 41.4 (t), 215.4 (s), 41.9 (d), 89.8 (d), 70.5 (d), 50.2 (t), 135.3 (s), 120.1 (d), 26.2 (q), 18.5 (q), 17.8 (q), 17.0 (q); FAB-MS (pos) m/z[M+Gly]+361, [M+2Gly]+453; HR-ESI-MS (pos) m/z269.1643 (measured value) (C
15H
25O
3, theoretical value 269.1647).
Embodiment 2: Mitissimol 11 β-19 α-epoxy-4Z, the preparation of 7Z-h umuladiene-5 α-ol
A, extraction:
The higher fungi dry foot breast mushroom 6kg that will pick up from the firm mountain of Yunnan Province sorrow (its bacterial classification is kept at Kunming Inst. of Botany, Chinese Academy of Sciences's Specimen Room) pulverizes, and with 95% extraction using alcohol of 20L 3 times, again with ethyl acetate 5L extraction, solvent recuperation is to doing, weigh 8.2g;
B, 200-300 order silica gel column chromatography separate: with sherwood oil-acetone gradient elution 50:1,20:1,9:1,5:1,1:1 gets 5 components, gets sherwood oil-acetone 9:1 component and uses the RP-C18 column chromatography repeatedly, with sherwood oil-chloroform recrystallization, obtain Mitissimol 2 β-3 α-epoxy-6Z, 9Z-humuladiene-8 α-ol22mg.
C, Mitissimol 11 β-19 α-epoxy-4Z, physico-chemical property Mitissimol 11 β-19 α-epoxy-4Z of 7Z-humuladiene-5 α-ol, 7Z-humuladiene-5 α-ol:
Colourless acicular crystal; Mp189-191 ℃; [α]
D 25-21.3 (c0.32, CHCl
3); IR (KBr): 3296,2594,1640,1610,1452,1387,1360,1297,972cm
-1 1HNMR (500MHz, CDCl
3) δ: 1.59 (d, 1H, J=14.3,1-H α), 1.34 (dd, 1H, J=14.3,9.8,1-H β), 5.37 (d, 1H, J=16.1,3-H), 5.72 (dd, 1H, J=16.1,6.9,4-H), 4.69 (m, 1H, 5-H), 5.44 (m, 1H, 7-H), 2.46 (m, 1H, 8-H α), 2.08 (m, 1H, 8-H β), 2.02,1.16 (m, 2H, 9-2H), 2.56 (d, 1H, J=9.8,11-H), 1.70 (s, 3H, 14-CH
3), 1.16 (s, 3H, 13-CH
3), 1.02 (s, 3H, 12-CH
3), 1.16 (s, 3H, 15-CH
3);
13CNMR (CDCl
3, 500MHz)) and δ: 40.4 (t), 35.1 (s), 139.3 (d), 132.1 (d), 78.1 (d), 143.1 (s), 124.9 (d), 22.7 (t), 37.9 (t), 61.4 (s), 63.3 (d), 30.5 (q), 16.1 (q), 12.9 (q), 23.0 (q); HRESIMS (pos) m/z236.1757 (measured value) (C
15H
24O
2, theoretical value 236.1776).
Attached: tetrazolium salts (MTT) reduction method detects the anti-tumor activity test of above-mentioned Mitissimol
1, experiment material and reagent
1-1, tetrazolium salts (MTT): (3-(4 for the phosphate buffered saline buffer dissolving Thiazolyl blue MTT of usefulness 0.01mol/L, 5-dimrthythiazol-z-yl) 2,5-diphenytetrazolium bromide, SIGMA company) concentration is 5mg/ml, filtration sterilization, 4 ℃ keep in Dark Place after the packing.
The sodium laurylsulfonate of the configuration of 1-2, tetrazolium salts lysate: 80g (SDS, Huamei Bio-Engrg Co.)
Be dissolved in the N-N-dimethyl diamide (Beijing Chemical Plant) of 200ml, the heating in water bath hydrotropy adds 200ml distilled water, mixes with 1N hydrochloric acid (1:1) with 80% acetate and transfers pH to 4.7.
1-3, cell strain are selected for use: A549 human lung adenocarcinoma cell line, the strain of HCT-8 human colon cancer cell, Bel-7402 human hepatoma cell strain, the strain of Hela human cervical carcinoma cell
2, experimental technique
2-1, get and cultivated 4-5 days, be in one bottle of the cell cultures of exponential phase of growth, add 0.05%Trypsin-EDTA liquid, attached cell is come off, RPMI1640 (available from the U.S. Gibco company) nutrient solution that contains 5% foetal calf serum with 10ml is made into suspension;
2-2, on blood cell counting plate, do the cell numeration with trypan blue dyeing back;
2-3, usefulness cell culture medium (available from U.S. Gibco company) diluting cells suspension are made into every 100ml and contain 10000 or 20000 cells.
2-4, get 96 hole flat boards, every hole adds cell suspension 100 μ l, places 37 ℃ of CO2gas incubator to cultivate 24 hours flat board,
2-5, test compound are made as 5 concentration gradients, are followed successively by 6.25ug/ml, 12.5ug/ml, 25ug/ml, 50ug/ml, 100ug/ml;
2-6, with flat board at 37 ℃, contain 5%CO
2Cultivated 2-3 days in the baking oven of air and 100% humidity;
2-7, tetrazolium salts are configured to 1mg/ml with serum-free RPMI1640 (available from U.S. Gibco company) nutrient solution, and every hole adds 50 μ l, and 37 ℃ of incubations 4 hours make MTT be reduced to Jia Za (Formazan);
2-8, absorption supernatant liquor add the dissolving of 150 μ l dimethyl sulfoxide (DMSO) Shi Jia Za, shake up with dull and stereotyped shaking table;
2-9, microplate reader detect each hole optical density OD value (λ=570nm);
2-10, drafting cell viability graphic representation are obtained half-inhibition concentration IC
50Value.μg/ml
3, experimental result
Above-mentioned data show that Mitissimol of the present invention shows that through in vitro tests cancer cells is had had strong inhibitory effects, 9 humulane type Mitissimols being tested all have stronger anti-tumor activity, wherein Mitissimol Mitissimol D activity is the strongest, to A549 (human lung adenocarcinoma cell line), HCT-8 (human colon cancer cell strain), Bel-7402 (human hepatoma cell strain), the half-inhibition concentration IC of Hela (human cervical carcinoma cell strain)
50Be respectively 2.5,11.0,3.6,4.6 μ g/ml, almost suitable with positive control medicament cis-platinum, this compounds is expected to become new antitumor drug, demonstrates excellent application value.
The invention provides this application at above-mentioned conclusion (of pressure testing), comprise that being used to clinically prepare with the Mitissimol is the antitumor drug of effective constituent with Mitissimol of anti-tumor activity as the preparation antitumor drug.The material that preparation contains Mitissimol pharmacy acceptable salt, solvate or corresponding crystal habit is an effective constituent, and adds the antitumor drug of medicine acceptable carrier or ancillary component.
The Mitissimol that has an anti-tumor activity with the present invention is that the pharmaceutical preparation of main effective constituent can comprise that also adding the medicine acceptable carrier makes the drug combination preparation form, comprising: tablet, sugar coated tablet, film coated tablet, enteric coated tablet, capsule, hard capsule, soft capsule, oral liquid, suck agent, granule, electuary, pill, powder, paste, sublimed preparation, suspensoid, solution, injection, suppository, ointment, plaster, creme, sprays, drops, patch, sustained release preparation, controlled release preparation.Preparation of the present invention, injection type preferably, as: lyophilized injectable powder, injection liquid, infusion solutions, little liquid drugs injection, injectable emulsion etc.
The Mitissimol that has anti-tumor activity with the present invention is the pharmaceutical preparation of main effective constituent, can prepare according to the technology of pharmaceutics routine techniques.
The Mitissimol that has anti-tumor activity with the present invention is the pharmaceutical preparation of main effective constituent, the preparation of its oral administration can contain vehicle commonly used, such as tackiness agent, weighting agent, thinner, tablet agent, lubricant, disintegrating agent, tinting material, seasonings and wetting agent, can carry out dressing to tablet in case of necessity.
The weighting agent that is suitable for comprises Mierocrystalline cellulose, mannitol, lactose and other similar weighting agent.Suitable disintegrating agent comprises starch, polyvinylpyrrolidone and starch derivative, for example sodium starch glycollate.Suitable lubricant, for example Magnesium Stearate.The acceptable wetting agent of appropriate drug comprises sodium lauryl sulphate.
Can fill by mixing, the method that compressing tablet etc. are commonly used prepares solid oral composition.Mix repeatedly active substance is distributed in those compositions of a large amount of weighting agents of whole use.
The Mitissimol that has anti-tumor activity with the present invention is the pharmaceutical preparation of main effective constituent, can be water-based or oily suspensions, solution, emulsion, syrup or elixir, perhaps can be a kind of used water before use or other suitable composite drying products of carrier.This liquid preparation can contain conventional additive, such as suspension agent, for example sorbyl alcohol, syrup, methylcellulose gum, gelatin, Natvosol, carboxymethyl cellulose, aluminium stearate gel or hydrogenation edible-fat, emulsifying agent, for example Yelkin TTS, anhydro sorbitol-oleic acid ester or gum arabic; Non-aqueous carrier (they can comprise edible oil), for example Prunus amygdalus oil, fractionated coconut oil, such as oily ester, propylene glycol or the ethanol of the ester of glycerine; Sanitas, for example para hydroxybenzene methyl esters or propylparaben or Sorbic Acid, and if desired, can contain conventional flavouring agent or tinting material.
The Mitissimol that has anti-tumor activity with the present invention is the injection of main effective constituent, this title complex can be suspended or dissolving, the preparation of solution is normally by being dissolved in active substance in a kind of carrier, filter-sterilized before it is packed into a kind of suitable bottle or ampoule, sealing then.For example a kind of local anesthetic of auxiliary material, sanitas and buffer reagent also can be dissolved in this carrier.In order to improve its stability, can be after the bottle of packing into that this composition is freezing, and under vacuum, water is removed.
The Mitissimol that has anti-tumor activity with the present invention is the pharmaceutical preparation of main effective constituent, optionally adding the medicine acceptable carrier when being prepared into medicament is selected from: N.F,USP MANNITOL, sorbyl alcohol, Sodium Pyrosulfite, sodium bisulfite, Sulfothiorine, cysteine hydrochloride, Thiovanic acid, methionine(Met), vitamins C, the EDTA disodium, EDTA calcium sodium, the alkali-metal carbonate of-valency, acetate, phosphoric acid salt or its aqueous solution, hydrochloric acid, acetic acid, sulfuric acid, phosphoric acid, amino acid, sodium-chlor, Repone K, Sodium.alpha.-hydroxypropionate, Xylitol, maltose, glucose, fructose, dextran, glycine, starch, sucrose, lactose, mannitol, silicon derivative, Mierocrystalline cellulose and derivative thereof, alginate, gelatin, polyvinylpyrrolidone, glycerine, soil temperature 80, agar, lime carbonate, Calcium hydrogen carbonate, tensio-active agent, polyoxyethylene glycol, cyclodextrin, β-cyclodextrin, the phospholipid material, kaolin, talcum powder, calcium stearate, Magnesium Stearate etc.
The Mitissimol that has anti-tumor activity with the present invention is the pharmaceutical preparation of main effective constituent, determines usage and dosage according to patient's situation in use.
The Mitissimol that has anti-tumor activity with the present invention is the pharmaceutical preparation of main effective constituent, can be
Unit dosage form, when making medicament, the medicament of unitary dose can contain the have Mitissimol of anti-tumor activity or the material 0.1-1000mg of its pharmacy acceptable salt, solvate and corresponding crystal habit of the present invention, account for the 0.1-99.9% of unit formulation gross weight, all the other are pharmaceutically acceptable carrier, and pharmaceutically acceptable carrier can be the 0.1-99.9% of total formulation weight amount by weight.
Listed examples of the present invention is intended to further illustrate structure, preparation method, formulation method and the application direction that this class has the Mitissimol of anti-tumor activity, and protection scope of the present invention is not constituted any restriction.
Claims (9)
1, the Mitissimol that has anti-tumor activity, its feature comprises the Mitissimol of following general structural formula (I):
Wherein: R
1Be hydrogen, carboxyl, hydroxyl or aldehyde radical; R
2Be hydrogen, hydroxyl, ester group or acyl ammonia; R
3Be hydrogen, carboxyl, hydroxyl or aldehyde radical; R
4Be hydrogen, carboxyl, hydroxyl or aldehyde radical; R
5For hydrogen or hydroxyl or 5 are methylene radical or carbonyl; 3,4 or 6,7 or 8,9 or 10,11 form epoxy or form singly-bound or the two keys of formation.
4, according to claim 1 or 2 described preparation methods with Mitissimol of anti-tumor activity, its feature comprises: described Mitissimol with anti-tumor activity is from the sporophore of the thin matter breast mushroom of higher fungi (Lactariusmitissimus), with wherein one or more solvent extractions of ethanol, methyl alcohol, chloroform, ethyl acetate, acetone, sherwood oil, obtain extract obtained the separation with silica gel column chromatography repeatedly.
5, according to the described preparation method with Mitissimol of anti-tumor activity of claim 4, its feature comprises the steps:
A, extraction: the thin matter of 1.6Kg higher fungi breast mushroom (Lactarius mitissimus) is pulverized, and with 95% extraction using alcohol of 15L 3 times, again with ethyl acetate 4.5L extraction, solvent recuperation is to doing, weigh 33.5g;
B, enter 200-300 order silica gel column chromatography and separate:
A, use CHCl
3-MeOH solvent is with 100:0,98:2, and 95:5,90:10 (vol/vol) gradient elution is at CHCl
3-MeOH 100:0 wash-out Duan Deyi component is further carried out silica gel column chromatography, uses sherwood oil-acetone solvent with 25:1,15:1, and 10:1, the 5:1 gradient elution gets pure product Mitissimol mitiSsimol A 66mg at the 15:1 position;
B, at a step CHCl
3-MeOH 95:5 wash-out Duan Deyi component is further carried out silica gel column chromatography, uses sherwood oil-acetone solvent with 10:1,8:1, and 6:1,4:1,5:1,2:1, the 1:1 gradient elution gets pure product Mitissimol mitissimol B 8mg at the 10:1 position;
C, at b step sherwood oil-further silica gel column chromatography in acetone 2:1 position, petroleum ether-ethyl acetate is with 20:1,15:1,10:1,5:1,1:1, the 1:5 gradient elution gets pure product Mitissimol mitissimol C 26mg in petroleum ether-ethyl acetate 20:1 component;
D, further utilize Sephadex LH-20 gel filtration chromatography, CHCl in step C petroleum ether-ethyl acetate 15:1 component
3-MeOH 1:1 is an eluent, and purifying obtains Mitissimol mitissimol D 5mg;
E, further utilize the thin layer preparative chromatography in step C petroleum ether-ethyl acetate 10:1 component, sherwood oil-acetone 4:1 is a developping agent, and purifying obtains Mitissimol mitissimol E 8mg;
F, obtain Mitissimol mitissimol F 17mg in step C petroleum ether-ethyl acetate 5:1 component;
9, obtain Mitissimol mitissimol G 4mg in step C petroleum ether-ethyl acetate 1:1 component;
H, get pure product Mitissimol mitissimo 17mg at sherwood oil-acetone 4:1 position in the b step.
6, according to the described preparation method of claim 3 with Mitissimol of anti-tumor activity, its feature comprises: described Mitissimol with anti-tumor activity is from the sporophore of higher fungi dry foot breast mushroom (Lactariushirtipes J.Z.Ying), with wherein one or more solvent extractions of ethanol, methyl alcohol, chloroform, ethyl acetate, acetone, sherwood oil, obtain extract obtained the separation with silica gel column chromatography repeatedly.
7, according to the described preparation method with Mitissimol of anti-tumor activity of claim 6, its feature comprises the steps:
A, extraction: with 6Kg higher fungi dry foot breast mushroom (Lactarius hirtipes J.Z.Ying), pulverize, with 95% extraction using alcohol of 20L 3 times, again with ethyl acetate 5L extraction, solvent recuperation is to doing, weigh 8.2g;
B, 200-300 order silica gel column chromatography separate: with sherwood oil-acetone gradient elution 50:1,20:1,9:1,5:1,1:1 gets 5 components, gets sherwood oil-acetone 9:1 component and uses the RP-C18 column chromatography repeatedly, with sherwood oil-chloroform recrystallization, obtain Mitissimol 2 β-3 α-epoxy-6Z, 9Z-humuladiene-8 α-ol22mg.
8, according to the described application with Mitissimol of anti-tumor activity as the preparation antitumor drug of claim 1, its feature comprises: being used to clinically prepare with the Mitissimol is the antitumor drug of effective constituent.
9, described according to Claim 8 application with Mitissimol of anti-tumor activity as the preparation antitumor drug, its feature comprises: be used to clinically prepare and contain composition or the compound that Mitissimol is an effective constituent, the material that preparation contains Mitissimol pharmacy acceptable salt, solvate or corresponding crystal habit is an effective constituent, and adds the antitumor drug of medicine acceptable carrier or ancillary component.
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CN113185393A (en) * | 2021-04-30 | 2021-07-30 | 中南林业科技大学 | Guaiane type sesquiterpene compound, preparation method and application thereof |
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CN112121060A (en) * | 2020-09-24 | 2020-12-25 | 安徽大学 | A Pleurotus Imperatae polysaccharide-selenium nanoparticle composition with tumor cell growth inhibiting effect, and its preparation method and application |
CN112121060B (en) * | 2020-09-24 | 2021-11-09 | 安徽大学 | A Pleurotus Imperatae polysaccharide-selenium nanoparticle composition with tumor cell growth inhibiting effect, and its preparation method and application |
CN113185393A (en) * | 2021-04-30 | 2021-07-30 | 中南林业科技大学 | Guaiane type sesquiterpene compound, preparation method and application thereof |
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