CN107474092A - A kind of straw mushroom fructification active component and its application - Google Patents
A kind of straw mushroom fructification active component and its application Download PDFInfo
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Abstract
The invention discloses a kind of straw mushroom fructification active component preparation method and application.Straw mushroom is dried fructification and crushed by the present invention, adds ethanol solution to obtain ethanol extract after ultrasonic extraction, concentration, after dichloromethane, methanol dissolving, with silica gel by volume 1:1 amount dry method mixes sample, dry column-packing, crosses silica gel chromatographic column and is eluted with chloroform methanol, component and silica gel after elution(200 300 mesh)By volume 1:2 ratio mixes sample, dry column-packing, with volume ratio 8:2 methylene chloride acetone elution, 3 β are obtained after the purifying of Sephadex LH 20 and HPLC processing, 5 α, 9 α trihydroxy ergots steroids 7, the ketone of 22 diene 6, test result indicates that 3 β, 5 α, the ketone of 97,22 diene of α trihydroxy ergot steroids 6 significant effect in terms of the activity of tumor cells such as stomach cancer SGC 7901, human breast carcinoma MCF 7 and human liver cancer HepG 2 are suppressed.
Description
Technical field
The invention belongs to Chemistry for Chinese Traditional Medicine field, and in particular to a kind of straw mushroom fructification active component and preparation method thereof and should
With.
Background technology
Straw mushroom(Volvariella volvacea(Bull. ex Fr.) Sing.)Also known as Nanhua mushroom, straw mushroom, in the world really
Recognize the origin that Nanhua straw mushroom is world artificial cultivation straw mushroom, spread out of by overseas Chinese it is external, therefore also known as " Chinese mushroom ", " Guangdong mushroom ", into
For the third-largest culturing edible fungus in the world.China is one of straw mushroom main producing region in the world, is distributed in Guangdong, Guangxi, Sichuan, good fortune
Build, Hunan, the area such as Jiangxi and Taiwan.Straw mushroom is divided into two kinds of strains:One kind is black grass mushroom, and one kind is white straw mushroom.Straw mushroom is happiness
The saprophytic fungus of high temperature, high humidity, China is as one of straw mushroom main producing region, and China's straw mushroom yield accounts for the whole world up to 150,000 tons within 95 years
The 60% of yield, is occupied first of the world.Straw mushroom mouthfeel is fresh and tender, and meat is fine and smooth, delicious flavour, bacterium soup such as milk, has " meat or fish in element " good
Reputation.Straw mushroom health value is high, has clearing away summerheat, invigorates the spleen and benefits qi, strengthen immunity, accelerating wound healing and other effects, it also has
Having reduces the effect such as cholesterol and anticancer.Straw mushroom contains abundant protein, essential amino acid, fat, polysaccharide, vitamin
And several mineral materials, wherein nucleic acid material account for 8.8%, content highest, have the effect of antiviral, hypoglycemic, reducing blood lipid;Often
The 100 g fresh mushrooms mg containing vitamin C 207.7, the g of sugar 2.6, the g of crude protein 2.68, fat 2.24 g, the g of ash content 0.91;Often
100 g dried agaric mushrooms are containing the g of crude protein 28.03, the g of crude fat 1.24, the g of soluble polysaccharide 0.50, the g of crude fibre 18.9, moisture
10.65 g, the g of ash content 9.01, the mg of vitamin C 206.28;Straw mushroom protein contains essential amino acid, accounts for total amino acid content
38.2 %.In addition, from the category fungi it is separated obtain straw mushroom polysaccharide, agglutinin, albumen, amino acid, triterpene compound,
The Multiple components such as sterol.There are some researches show straw mushroom plays the role of antitumor, anti-oxidant, anti-sepsis acid and regulation is immune, but to it
The research of cycle chemistry composition is less, as edible and medicinal bacterium famous in the world, there is higher nutritive value and medicinal valency
Value, has wide DEVELOPMENT PROSPECT.
Recent domestic to multiple eating bacterium, as White mushroom, grifola frondosus, pixie stool, phoenix-tail mushroom, black fungus, white fungus,
Armillaria luteo-virens etc. have carried out the research in terms of the extraction purification of polysaccharide, separation identification and feature, and substantial amounts of data show more
Sugar has antitumor action, but the research of the chemical composition and bioactivity of fungi is still short of;Medicinal fungi active component removes
Outside polysaccharide and glycoprotein macromolecular substances, another important activity composition is exactly fungi micromolecular compound, fungi small molecule chemical combination
Thing mainly has phenols, polyketone class, terpene, alkaloids and steroidal to have Synergistic action more, and current research focuses mostly in terpene
With the in vitro study of steroid compound, mainly based on terpenoid and steroidal;Ergot steroid compound is present in medicinal true
In bacterium, also known as steroids can be divided into ergosterol, ergot sterone and its derivative;Ergot steroid has a variety of pharmacology
Activity, wherein peroxide ergot steroid have 15 kinds, and because containing peroxide bridge, its bioactivity is strong, mainly have antitumor, immunological regulation,
The effect such as anti-oxidant, antibacterial.At present, the research of straw mushroom focuses mostly in the drug effect of crude extract, secondary metabolite and straw mushroom polysaccharide
Experiment and the breeding and cultivation of straw mushroom, it is less to the relative molecular weight and chemical constitution research of its chemical composition and active material.
In living now, tumour getting worse threatens the life and health of the mankind, be directed to the research of cancer therapy drug with
Exploitation is global active demand.The treatment of cancer is more at present torments patient based on chemical drug from body & mind, because
It is imperative that this finds the small medicine of good effect, toxic side effect.Safe and reliable PTS is developed from fungus metabolite, is
A kind of one of rapid effective new way, by the use in conjunction with traditional anti-cancer chemical drug, the raising to chemotherapy patients' immunity
There is profound influence.
The content of the invention
It is an object of the present invention to provide a kind of straw mushroom fructification active component and its preparation method and application.
A kind of straw mushroom fructification active component, its chemical name are 3 β, 5 α, 9 α-trihydroxy ergot steroid -7,22- bis-
Alkene -6- ketone;
Its chemical constitution is:
A kind of preparation method of straw mushroom fructification active component, it includes:
1)Straw mushroom is taken to dry fructification coarse powder, using 95% ethanol as solvent, by solid-liquid ratio 1:4 amount adds round-bottomed flask, 30
30 min of ultrasound, stand overnight at DEG C, are recovered under reduced pressure extract solution, residue 95% ethanol heating and refluxing extraction 3 times, each 3h, close
And extract solution, acquisition ethanol extract is recovered under reduced pressure;;
2)By step 1)Described ethanol extract dichloromethane, methanol dissolving, with silica gel by volume 1:1 amount carries out dry method
Mix sample, dry column-packing, the mm of the mm of 100-200 mesh 150 × 305 silica gel chromatographic column is crossed, with chloroform-methanol volume ratio(9:1、
8:2、7:3、6:4、1:1)Gradient elution is carried out, obtains chloroform-methanol(8:2)Elution fraction; 3)By step 2)Described elution
The silica gel of component and 200-300 mesh by volume 1:2 ratio mixes sample, dry column-packing, with volume ratio 8:2 dichloromethane-the third
Ketone elutes, and is purified through hydroxypropyl sephadex Sephadex LH-20, through HPLC preparative separations, condition is 80% ~ 100% first
Alcohol, 254 nm, 10 ml/min, obtain compound 3 β, 5 α, 9 α-trihydroxy ergot steroid -7,22- diene -6- ketone.3β, 5α,
9 α-trihydroxy ergot steroid -7,22- diene -6- ketone, the application in treatment stomach cancer, human breast carcinoma or human liver cancer medicine is prepared.
The invention provides a kind of straw mushroom fructification activity preparation method and application, and it is that straw mushroom is dried into fructification powder
It is broken, add ethanol solution to obtain ethanol extract after ultrasonic extraction, concentration, after dichloromethane, methanol dissolving, volume is pressed with silica gel
Than 1:1 amount dry method mixes sample, dry column-packing, crosses silica gel chromatographic column chloroform-methanol gradient elution, component and silica gel after elution
(200-300 mesh)By volume 1:2 ratio mixes sample, dry column-packing, with volume ratio 8:2 dichloromethane-acetone elution, warp
3 β are obtained after Sephadex LH-20 purifying and HPLC processing, 5 α, 9 α-trihydroxy ergot steroid -7,22- diene -6- ketone are real
Test result and show 3 β, 5 α, 9 α-trihydroxy ergot steroid -7,22- diene -6- ketone are suppressing stomach cancer SGC-7901, human breast carcinoma
MCF-7 and human liver cancer HepG-2 activity of tumor cells aspect significant effect.
Brief description of the drawings
Fig. 1 straw mushrooms extract flow chart;
The HPLC figures of Fig. 2 compounds 4;
The HPLC figures of Fig. 3 compounds 5;
Fig. 4 compounds 6-8 HPLC figures;
The HPLC figures of Fig. 5 compounds 9;
The HPLC figures of Fig. 6 compounds 10;
The HPLC figures of Fig. 7 compounds 11;
Fig. 8 ergosterol chemical constitutions;
Fig. 9 ergosterol peroxide chemical constitutions;
Figure 10 2 (5H)-furanone -4- propionic acid chemical constitutions;
Figure 11 3 β, 5 α, 9 α-trihydroxy ergot steroid -7,22- diene -6- ketone chemical constitutions;
Figure 12 3 β, 5 α, 9 α, 14 α-tetrahydroxy ergot steroid -7,22- diene -6- ketone chemical constitutions;
Figure 13 5 α, 6 α-epoxy -24- methyl cholesteric -8 (14), the β of 22- diene -3,7 α -ol chemical constitutions;
Inhibiting rate of Figure 14 compounds to SGC-7901 cells;
Inhibiting rate of Figure 15 compounds to Human Prostate Cancer Cells PC-3M;
Inhibiting rate of Figure 16 compounds to human lung carcinoma cell NCI-H460;
Inhibiting rate of Figure 17 compounds to human breast cancer cell line Bcap-37;
Inhibiting rate of Figure 18 compounds to human liver cancer cell HepG-2.
Embodiment
The extraction of the straw mushroom chemical composition of embodiment 1
Dried straw mush-room is purchased from Jiangsu Jiangnan Biology Technology Co., Ltd. in May, 2015.Through straw mushroom used in Molecular Identification this experiment
(Volvariella volvacea)It is of the same race with the straw mushroom in Genebank databases
Straw mushroom is taken to dry fructification coarse powder 5kg, using 95% ethanol as solvent, by solid-liquid ratio 1:4 amount adds round-bottomed flask, 30
30 min of ultrasound, are stood overnight at DEG C, and extract solution is recovered under reduced pressure, and residue is extracted 3 times with 95% alcohol reflux, and each 3h, merging carries
Liquid is taken, is recovered under reduced pressure and obtains the g of ethanol extract 725.Ethanol extract is dissolved with organic reagent, with silica gel(100-200 mesh)Press body
Product ratio 1:1 dry method mixes sample, the silica gel chromatographic column of dry column-packing, excessively 100-200 mesh(150 mm × 305 mm), with petroleum ether-
Chloroform(1:0、20:1、10:1、9:1、8:2、7:3、6:4、1:1), chloroform-methanol(1:0、20:1、9:1、8:2、7:3、0:1)Ladder
Degree elution, obtains 15 components.Idiographic flow is as shown in Figure 1.
The separation of the straw mushroom chemical composition of embodiment 2
By chloroform extract(13.1 g)Dissolving, with silica gel(100-200 mesh)By 1:2 ratio mixes sample, silicagel column in dry method(73
mm × 305 mm), it is that eluent gradient elutes with petroleum ether-ethyl acetate, is divided into 5 parts, wherein petroleum ether-acetic acid second
Ester(8:2)Elute position silica gel mixed sample dress post(200-300 mesh), use petroleum ether-ethyl acetate(15:1)Elution obtains compound 1
(500.4 mg).
By chloroform-methanol(20:1)Part(9.5 g)Dissolving mixes sample and carries out silica gel column chromatography(100-200 mesh), with oil
Ether-ethyl acetate(100:1、50:1、20:1、10:1、8:2、7:3)Gradient elution is carried out, to petroleum ether-ethyl acetate(50:1、
7:3)Part carries out silica gel column chromatography(200-300 mesh), obtain compound 2 and compound 3;Gel layer is carried out to methanol fractions
Analysis, then carry out preparing HPLC preparations(Condition 15%-95% methanol, 254 nm), compound 4 is obtained, HPLC is as shown in Figure 2.
By chloroform-methanol(9:1)Part(12.0 mg)By 1:2 ratio(200-300 mesh)Silica gel mixed sample, dry method load,
Use dichloromethane-acetone(50:1、20:1、10:1、8:2、7:3、6:4)Elution, obtain 5 components.Wherein dichloromethane-acetone
(10:1)Component, purified through Sephadex LH-20, through preparing HPLC(20% methanol, 203 nm, 10 ml min-1)Obtain compound
5(16.0 mg), HPLC is as shown in Figure 3;Wherein dichloromethane-acetone(8:2)Part, purified through Sephadex LH-20, warp
Prepare HPLC(80% ~ 100% methanol, 254 nm, 10 ml/min-1)Obtain compound 7(5.9 mg, 20.293 min), compound 8
(61.3 mg, 28.119 min), HPLC is as shown in Figure 4;Wherein, dichloromethane-acetone(6:4)Part, through Sephadex LH-
20 purifying, through preparing HPLC(15% ~ 40%, 30 min methanol, 230 nm, 10 ml of flow velocity/min-1)Obtain compound 9(124.8
Mg, 13.02 min), HPLC is as shown in Figure 5;Wherein, dichloromethane-acetone(1:1)Component, through preparing HPLC(15% methanol,
254 nm, flow velocity 10ml/min) obtain compound 10(20.1 mg, 12.617 min), HPLC is as shown in Figure 6.
By chloroform-methanol(8:2)Part(17.1 g)Dissolved with methanol, by silica gel 1:3 ratio mixes sample, crosses silica gel
(100-200 mesh)Column chromatography, use methylene chloride-methanol(100:1、50:1、20:1、10:1、9:1、8:2、0:1), obtain 3 groups
Point;Wherein methylene chloride-methanol(20:1)12-16 components be dissolved in methanol, through gel column, eluted through methanol, through prepare HPLC
(20% methanol, 254 nm, 10 ml/min-1), obtain compound 11(15.0 mg、11.78min), HPLC is as shown in Figure 7;Its
Middle methylene chloride-methanol(10:1、8:2)22-27 components and 10-17 components, through Sephadex LH-20 gel columns, through methanol
Elution, obtain compound 12(18.5 mg)With compound 13(13.4 mg).
The extraction of 3 compound of embodiment 63 β, 5 α, 9 α-trihydroxy ergot steroid -7,22- diene -6- ketone
1)Straw mushroom is taken to dry fructification coarse powder, using 95% ethanol as solvent, by solid-liquid ratio 1:4 amount adds round-bottomed flask, 30
30 min of ultrasound, stand overnight at DEG C, are recovered under reduced pressure extract solution, residue 95% ethanol heating and refluxing extraction 3 times, each 3h, close
And extract solution, acquisition ethanol extract is recovered under reduced pressure;2)By step 1)Described ethanol extract dichloromethane, methanol dissolving, with
Silica gel by volume 1:1 amount carries out dry method and mixes sample, dry column-packing, crosses the mm of the mm of 100-200 mesh 150 × 305 silica gel color
Post is composed, with chloroform-methanol volume ratio(9:1、8:2、7:3、6:4、1:1)Gradient elution is carried out, obtains chloroform-methanol(8:2)Wash
De- component; 3)By step 2)Described elution fraction and the silica gel of 200-300 mesh by volume 1:2 ratio mixes sample, dry method dress
Post, with volume ratio 8:2 dichloromethane-acetone elution, is purified, condition through hydroxypropyl sephadex Sephadex LH-20
100% methanol, through HPLC preparative separations, condition is 80% ~ 100% methanol, 254 nm, 10 ml/min, obtains compound 3 β, 5 α,
9 α-trihydroxy ergot steroid -7,22- diene -6- ketone.
The Structural Identification of the straw mushroom chemical composition of embodiment 4
The compound separated to embodiment 2 carries out Structural Identification.
Compound 1:White needle-like crystals(Chloroform).The nm of thin-layer chromatography 254 has UV absorption, and 365 nm unstressed configurations absorb,
10% H2SO4Solution displaing amaranth.1H-NMR (600 MHz, CDCl3) δ:3.62 (1H, m, H-3), 5.18 (1H,
m, H-6), 5.57 (1H, m, H-7), 0.63 (3H, s, H-18), 0.95 (3H, s, H-19), 1.05 (3H,
d, J=6.8 Hz, H-21), 5.18 (1H, m, H-22), 5.20 (1H, m, H-23), 1.80 (1H, m, H-
24), 1.40 (1H, m, H-25), 0.82 (3H, d, J=4.5 Hz, H-26), 0.81 (3H, d, J=4.5 Hz,
H-27), 0.91 (3H, d, J=3.9 Hz, H-28);13C-NMR (150 MHz, CDCl3)δ: 39.3 (C-1),
32.1 (C-2), 70.6 (C-3), 40.6 (C-4), 139.9 (C-5), 119.9 (C-6), 116.5 (C-7),
141.5 (C-8), 46.2 (C-9), 35.5 (C-10), 21.3 (C-11), 38.5 (C-12), 43.0 (C-13),
54.7 (C-14), 21.9 (C-15), 28.4 (C-16), 55.9 (C-17), 12.2 (C-18), 16.4 (C-19),
40.5 (C-20), 21.2(C-21), 132.9 (C-22), 135.7 (C-23), 42.8 (C-24), 33.3 (C-
25), 19.8 (C-26), 20.1 (C-27), 17.7 (C-28) .Authenticating compound 1 is ergosterol
(ergosterol), chemical constitution is as shown in Figure 8.
Compound 2:White powder.ESI-MSm/s:284[M+], 1H-NMR(600MHz, DMSO-d)δ:2.17(2H, t,J=7.0 Hz, CH2-2), 1.47(2H, m, CH2-3), 1.17(28H, m, CH2-4、7), 0.84(3H, t, J=7.0
Hz, CH3-18);13C-NMR(150Hz, DMSO-d)δ: 173.89(C=O-1), 33.55(CH-2), 31.26(CH2-
16), 29.01(CH2-15), 24.46(CH3-3), 22.00(CH2-17), 14.00(CH3-18), 28-28.58(CH2-
14、4).Authenticating compound 2 is octadecanoid acid(Octadecanoic acid).
Compound 3:White powder.The nm of thin-layer chromatography 254 absorbs without UV absorption, 365 nm unstressed configurations, 10% H2SO4It is molten
Spot is kermesinus after liquid colour developing.1H-NMR (600 MHz, CDCl3) δ: 6.26 (1H, d, J=9.6 Hz, H-6),
6.49 (1H, d, J=9.6 Hz, H-7), 5.14(1H, dd, J=15.2, 8.1 Hz, H-22), 5.42 (1H,
dd, J=15.2, 7.4 Hz, H-23), 0.82 (3H, d, J=4.5 Hz, H-26), 0.81 (3H, d, J=4.5
Hz, H-27), 0.91 (3H, d, J=3.9 Hz, H-28);13C-NMR (150 MHz, CDCl3) δ: 36.9(C-1),
30.1 (C-2), 66.4 (C-3), 34.6 (C-4), 82.1 (C-5),135.1 (C-6), 130.7 (C-7), 79.4
(C-8), 51.0 (C-9), 36.9(C-10), 20.6 (C-11), 39.3 (C-12), 44.5 (C-13), 51.6
(C-14), 23.3 (C-15), 28.6 (C-16), 56.1 (C-17), 12.9 (C-18), 18.1 (C-19), 39.7
(C-20), 20.9 (C-21), 135.3 (C-22), 132.3 (C-23), 42.7 (C-24), 33.0 (C-25),
19.6 (C-26), 19.9 (C-27), 17.5 (C-28).Authenticating compound 3 is ergot steroid -5 α, 8 α-epidioxy -6,
The β -ol of 22- diene -3, i.e. ergosterol peroxide(ergosterol peroxide), chemical constitution is as shown in Figure 9.
Compound 4:Faint yellow amorphous powder.ESI-MS m/z: 243.2[M+H]+With 507.3 [2 M+Na]+ 。1H-
NMR (600 MHz, C5D5N) δ: 8.06 (1H, s, H-9), 7.89 (1H, s, H-6), 2.50 (3H, s, 7-
CH3), 2.49 (3H, s, 8-CH3). 13C-NMR (150 MHz, C5D5N) δ: 19.87 (7-CH3), 20.44 (8-
CH3), 147.7 (C-10a), 143.0 (C-9a), 127.2 (C-9), 139.8 (C-8), 144.8 (C-7),
129.7 (C-6), 139.1 (C-5a), 131.0 (C-4a), 162.1 (C-4), 151.8 (C-2).Authenticating compound 4
For 7,8- lumichromes(lumichrome).
Compound 5:White powder, ESI-MSm/z: 157.2[M+H]+。1H-NMR (600 MHz, D2O) δ: 5.81
(1H, s, H-2), 4.83 (2H, s, H-4), 2.59 (2H, t, J = 7.2 Hz, H-5), 2.39 (2H, t,J = 7.2 Hz, H-6). 13C-NMR (150 MHz, D2O) δ: 181.1 (s, C-8), 178.2 (s, C-2),
174.5 (s, C-4), 113.6 (d, C-3), 74.7 (t, C-5), 34.5 (t, C-7), 24.8 (t, C-6).,
The final determination compound 5 of HMBC, HSQC and COSY spectrum in conjunction with the compound is 2 (5H)-furanone -4- propionic acid(2(5H)-
ficifuranone-4-propionic acid), chemical constitution is as shown in Figure 10.
Compound 6:White powder.ESI-MS m/z: 467.3 [M+Na]+;1H-NMR (600 MHz, CD3OD) δ:
5.49 (1H, s, H-7), 5.14 (1H, dd, J=15.2, 8.1 Hz, H-22), 3.83 (1H, m, H-3),
1.2 (3H, d, J=7.2 Hz, H-21), 0.76 (3H, dd, J=11.7, 6.8 Hz, H-26), 0.85 (3H,
d, J=6.8 Hz, H-27), 0.90 (3H, s, H-28), 0.57 (3H, s, H-18), 096 (3H, d, J=6.8
Hz, H-19)。13C-NMR (150 MHz, CD3OD) δ: 36.1 (C-1), 29.1 (C-2), 67.8 (C-3), 26.6
(C-3), 75.1 (C-4), 200.1 (C-5), 120.9 (C-7), 165.0 (C-8), 80.1 (C-9), 41.6
(C-10), 26.6 (C-11), 34.3 (C-12), 44.3 (C-13), 52.8 (C-14), 21.6 (C-15), 23.4
(C-16), 57.4 (C-17), 12.6 (C-18), 20.1 (C-19), 37.1 (C-20), 18.2 (C-21),
136.6 (C-22), 133.6 (C-23), 42.8 (C-24), 31.0 (C-25), 18.7 (C-26), 19.0 (C-
27), 16.7 (C-28).Authenticating compound 5 is 3 β, 5 α, 9 α-trihydroxy ergot steroid -7,22- diene -6- ketone(3β, 5α,
9α-trihydroxyergosta-7, 22-dien-6-one), chemical constitution is as shown in figure 11.
Compound 7:White powder.1H-NMR (600 MHz, CD3OD) δ: 4.78 (1H, m, H-3), 5.96
(2H, s, H-7), 0.76 (3H, dd, J=11.3, 6.8 Hz, H-18), 1.19 (3H, s, H-19), 1.93
(1H, m, H-20), 1.03 (3H, s, H-21), 5.17 (1H, dd, J=15.4, 7.7 Hz, H-22), 0.85
(3H, d, J=6.4Hz, H-26), 0.88 (3H, s, H-27), 0.95 (3H, d, J=6.4Hz, H-29), 6.36
(1H, s, 3-OH), 8.44 (1H, s, 5-OH), 6.63 (1H, s, 9-OH)。13C-NMR (150 MHz, CD3OD)δ: 25.7 (C-1), 31.1 (C-2), 67.1 (C-3), 36.7 (C-4), 79.9 (C-5), 199.1 (C-6),
122.6 (C-7), 159.1 (C-8), 77.8 (C-9), 42.7 (C-10), 31.1 (C-11), 28.8 (C-12),
47.9 (C-13), 87.1 (C-14), 27.5 (C-15), 28.8 (C-16), 51.2 (C-17), 16.8 (C-18),
20.2 (C-19), 40.65 (C-20), 21.6 (C-21), 136.1 (C-22), 133.4 (C-23), 43.2 (C-
24), 34.2 (C-25), 19.9 (C-24), 20.2 (C-27), 18.0 (C-24).Authenticating compound 6 is 3 β, 5 α,
9 α, 14 α-ketone of tetrahydroxy ergot steroid -7,22- diene -6(3β, 5α, 9α, 14α-tetrahydroxyergosta-7,
22-dien-6-one), chemical constitution is as shown in figure 12.
Compound 8:White powder.ESI-MS m/z: 428[M]+,1H-NMR (600 MHz, CD3OD) δ: 1.41
(2H, s, H-1), 1.52 (2H, m, H-2), 2.11 (2H, m, H-4), 3.07 (1H, d, J=3.0 Hz, H-
6), 4.43 (1H, d, J=2.3 Hz, H-7), 2.44 (1H, d, J=7.9 Hz, H-9), 1.43 (2H, d, J=
2.6 Hz, H-11), 1.22 (2H, s, H-12), 2.13 (2H, m, H-15), 1.43 (2H, d, J=2.6 Hz,
H-16), 1.24 (1H, m, H-17), 0.89 (3H, d, J=3.7 Hz, H-18), 2.13 (1H, m, H-20),
1.06 (3H, d, J=6.4 Hz, H-21), 5.24 (1H, m, H-23), 1.89 (1H, m, H-24), 1.43
(1H, d, J=2.6 Hz, H-25), 0.86 (3H, s, H-26), 0.89 (3H, d, J=3.7 Hz, H-27)。13C-
NMR (150 MHz, CD3OD) δ: 33.4 (C-1), 31.9 (C-2), 69.2 (C-3), 40.4 (C-4), 68.0
(C-5), 62.6 (C-6), 65.8 (C-7), 126.6 (C-8), 40.5 (C-9), 37.1 (C-10), 18.5 (C-
11), 37.9 (C-12), 44.3 (C-13), 152.8 (C-14), 25.7 (C-15), 28.4 (C-16), 58.2
(C-17), 18.2 (C-18), 16.9 (C-19), 40.6 (C-20), 21.7 (C-21), 136.8 (C-22),
133.29 (C-23),44.3 (C-24), 20.4 (C-25), 34.4 (C-26), 20.2 (C-27), 20.1 (C-
28).Authenticating compound 7 is 5 α, 6 α-epoxy -24- methyl cholesteric -8 (14), the β of 22- diene -3,7 α -ol(5α, 6α-
epoxy-24-methylcholesta-8(14), 22-diene-3β, 7α-diol), chemical constitution is as shown in figure 13.
Compound 9:White, amorphous solid(Methanol).1H-NMR (600 MHz, CD3OD) δ: 9.02 (1H, d, J
=2.0 Hz, H-2), 8.68 (1H, dd, J=15.5, 5.0 Hz, H-6), 8.27 (1H, m, H-4), 7.53
(1H, dd, J=5.0, 8.0 Hz, H-5)。13C-NMR (150 MHz, CD3OD) δ: 169.8 (C=O), 152.8
(C-6), 149.5 (C-2), 137.3 (C-4), 131.4 (C-3), 125.4 (C-5).Authenticating compound 8 is niacinamide
(nicotinamide).
Compound 10:White amorphous powder.ESI-MS m/z:[268.7 M+H] +。1H-NMR (600 MHz,
D2O) δ: 8.31 (1H, s, H-2), 8.14 (1H, s, H-8), 5.88 (1H, d, J=6.0 Hz, H-1′),
5.18 (3H, m, 2′-OH, 3′-OH, 5′-OH), 4.62 (1H, J=6.0 Hz, H-2′), 4.14 (H, s, H-
3′), 3.68 (1H, d, J=12 Hz, H-5′), 3.35 (1H, d, J=12 Hz, H-5′)。13C-NMR (150
MHz, D2O) δ: 152.4 (C-2), 148.3 (C-4), 119.0 (C-5), 155.5 (C-6), 140.5 (C-8),
88.2 (C-1′), 73.6 (C-2′), 70.6 (C-3′), 85.7 (C-4′), 61.4 (C-5′).Authenticating compound 10
For adenosine(Adenosine).
Compound 11:Colorless needle crystals(Methanol).1H-NMR (600 MHz, CD3OD) δ: 9.12 (1H, d, J
= 1.8 Hz, H-2), 8.73 (1H, dd, J = 1.2, 4.8 Hz, H-6), 8.41 (1H, dt, J = 1.8,
3.6, 7.8 Hz, H-4), 7.56(1H, dd, J= 4.8, 7.8 Hz, H-5);13C-NMR (150 MHz, CD3OD)δ: 167.7 (C=O), 153.6 (C-2), 151.2 (C-6), 139.2 (C-4), 128.7 (C-3), 125.2 (C-
5).Authenticating compound 11 is nicotinic acid(Nicotinic acid).
Compound 12:White, needle-shaped crystals (methanol).1H-NMR (600 MHz, CD3OD)δ: 7.36 (1H, d, J =
2.0 Hz, H-3), 7.29 (1H, dd, J = 8.0, 2.0 Hz, H-7), 6.79 (1H, d, J = 8.0 Hz,H-
6)。13C-NMR (150 MHz, CD3OD)δ:123.9(C-6),115.8(C-2),151.5(C-4),146.4(C-3), 23.0
(C-1), 117.7(C-5), 170.3(COOH).Authenticating compound 12 is protocatechuic acid(protocatechuic acid).
Compound 13:Clear crystal (methanol).EI-MSm/z:118[M+], 1H-NMR(600 MHz, CD3OD) δ:
2.53 (4H, -2CH2 ), 12.2(2H, -2COOH)。13C-NMR(150 MHz, CD3OD)δ: 28.8(C-2, 3),
173.8(C-1, 4).It is butanedioic acid (succinic acid) to determine the compound.
The anti tumor activity in vitro experiment of the monomeric compound of embodiment 5
Six isolated compounds, ergosterol from straw mushroom ethanol extract(Compound 1), peroxide ergosterol
(Compound 3), 2 (5H)-furanone -4- propionic acid(Compound 5), 3 β, 5 α, 9 α-trihydroxy ergot steroid -7,22- diene -6-
Ketone(Compound 6), 3 β, 5 α, 9 α, 14 α-ketone of tetrahydroxy ergot steroid -7,22- diene -6(Compound 7), 5 α, 6 α-epoxy-
24- methyl cholesteric -8 (14), the β of 22- diene -3,7 α -ol(Compound 8), wherein compound 1,3,8 is ergosterol class chemical combination
Thing, compound 6,7 are ergot sterone class compounds, and compound 5 is Furanones compound;Detected using CCK-8 cell viabilities
Method carries out cancer cell multiplication inhibitory action experiment to above-mentioned six compounds.
1. the configuration of sample
Take compound 1,3,6,7,8 each 4mg to add 100 μ L DMSO dissolvings respectively, with PBS constant volume to 10 mL, be made into
The μ gmL of mother liquor 400-1, then doubling dilution is into 200,100,50,25,12.5,6.25,3.125,1.562 μ gmL-1.Take
4 mg compounds 5 are dissolved in 10 mL PBS, be made into concentration 400,200,100,50,25,12.5,6.25,3.125,
1.562 μg·mL-1, choose the μ gmL of 5 FU 5 fluorouracil 10-1For positive control, all samples are through 0.25 μm of sterilised membrane filter
Filtering.
2. the determination of cell concentration
Take the logarithm four kinds of cancer cells in growth period, count the cancer cell number in four kinds of cell suspending liquids with cell counting count board, match somebody with somebody
Into 1mL mother liquor, concentration is 1.6 × 105 cell·mL-1, then with culture medium proportional diluted into concentration gradient, 1.6 × 105、0.8
×105、0.4×105、0.2×105、0.1×105、0.05×105 cell·mL-1, then inoculating cell is into 96 orifice plates, often
Group repeats 5 multiple holes, cultivates micro- Microscopic observation cell attachment after 4 h, then plus after the h of CCK-8 reagents culture 4 determines OD values,
One is produced using cell concentration as abscissa(X-axis), OD values are ordinate(Y-axis)Standard curve, according to this standard curve
Cell quantity can be determined.
3.CCK-8 method cell inhibitory effect screening active ingredients
Take the logarithm SGC-7901gastriccarcinomacellline, human liver cancer HpepG2 cells, human lung cancer NCI-H460 cells, the human milk in growth period
Gland cancer MCF-7 cells, human prostata cancer PC-3M cells and people kidney embryo HEK-293 cells respectively with 0.25% Trypsin Induced into
Individual cells, SGC-7901 cells, NCI-H460 cells, MCF-7 cells, PC-3M cells are cultivated completely with 10%RPMI-1640
Basigamy is made into individual cells with 10%DMEM complete mediums and hanged into individual cells suspension, HpepG2 cells, HEK-293 cells
Supernatant liquid;Cell count is carried out, measure cell density is 4 × 104Individual/hole, it is inoculated in 96 orifice plates, per the μ L of hole 100, by 96 holes
Plate moves into 37 DEG C, 5% CO2Cultivated in incubator.After 24 h, micro- sem observation finds that cell is completely adherent, and supernatant is discarded,
The incomplete culture medium of equivalent is changed, adds 10 μ L various concentrations(400、200、100、50、25、12.5、6.25、3.125、
1.562 μg·mL-1)Sample and 10 μ L 5 FU 5 fluorouracil(5-FU), not contain the hole of cell and sample as blank pair
According to, using the hole containing cell n.s as control cell, 4 multiple holes of repetition.After culture terminates, culture supernatant is carefully drawn,
Discard, by 10:1 amount mixing incomplete culture medium and CCK-8 reagents, 110 μ L are added per hole, continue to be incubated 4 h, automatic
The light absorption value in 450 nm measure hole on ELIASA(OD values), record experimental result.(Experiment is repeated once)It is calculated as follows each
Proliferation inhibition activity of the kind acute drug to cell:
Inhibiting rate(%)=(control wells mean OD value-medicine feeding hole mean OD value)/control wells mean OD value × 100%.
Cell inhibitory effect activity IC50Measure:The inhibiting rate of above-mentioned cancer cell is acted on according to compound various concentrations
Relation, the IC of compound is obtained by regression equation50Value.Handled with SPSS statistical softwares,P>0.05 indicates that no conspicuousness is poor
It is different,P<0. 05 indicate significant difference.
Determine compound first influences on normal cell HEK-293 Proliferation Ability(It is shown in Table 1).From the statistical analysis of result
Draw, compound 1,3,5,6,7,8 compared with blank group, there was no significant difference (P>0.05), illustrate in set concentration model
Compound is enclosed to act on normal cell acellular poison;It is wherein maximum with the difference of compound 6, illustrate compound 6 to normal cell
Toxicity is minimum.
With 5-FU(10 μg·mL-1)For positive control, compound is studied respectively to SGC-7901gastriccarcinomacellline, people forefront
Adenocarcinoma cell PC-3M, human lung carcinoma cell NCI-H460, human breast cell MCF-7 and human liver cancer cell HepG-2 are through various concentrations
Act on the influence of cell inhibitory effect after 24 h(Such as Figure 14-18).Figure 14 results show that compound 1,3,5,6 is in 200-400 μ
g·mL-1To the inhibiting rate of cancer cell compared with positive drug, both have pole significant difference (P<0.01), in 12.5-50 μ g
mL-1In the range of, for compound 6,8 compared with positive drug, difference is not notable(P>0.05), in the range of finite concentration, suppress effect
Fruit strengthens as medicine activity increases.Wherein, it is best with the inhibitory action of compound 5.
Influence of the compound to Human Prostate Cancer Cells PC-3M cell inhibitory effects after various concentrations act on 24 h is as schemed
Shown in 15, compound plays the role of to suppress PC-3M cancer cell multiplications in concentration range, and as compound concentration raises,
The effect for suppressing PC-3M cells propagation is stronger.Compound 1 is compared with positive drug 5-FU, in 200-400 μ gmL-1,(P<
0.05)Significant difference, in 12.5 μ gmL-1, inhibiting rate 50.22%, in 100 μ gmL-1It is inhibiting rate 78.06%,
In 200 μ gmL-1When, inhibiting rate reaches 80.48%, and the inhibition of compound 1 is best, next to that compound 3,6, and chemical combination
The difference compared with positive drug of thing 5,7 is not notable(P>0.05).
Influence of the compound to human lung carcinoma cell NCI-H460 cell inhibitory effects after various concentrations act on 24 h is as schemed
Shown in 16,5-FU is in 10 μ gmL-1When, inhibiting rate reaches 47.82%, and the inhibiting rate pole of compound is substantially less than positive drug(P<
0.01), the inhibiting rate of compound is not linear in set concentration range, illustrates compound to human lung carcinoma cell NCI-
H460 unrestraints act on.
Compound to human breast cell MCF-7 through various concentrations act on 24 h after cell inhibitory effect influence such as Figure 17,
5-FU is in 10 μ gmL-1When, inhibiting rate 78.9%, compound 3 is in 100-400 μ gmL-1In the range of with positive medicine phases
Than inhibiting rate is significantly higher than positive drug(P<0.05), compound 1,6 is in 50-400 μ gmL-1In the range of compared with positive drug,
Inhibiting rate is significantly higher than positive drug(P<0.05), there was no significant difference compared with positive drug for compound 5,7,8.
Compound to human liver cancer cell HepG-2 through various concentrations act on 24h after cell inhibitory effect influence such as Figure 18,
5-FU is in 10 μ gmL-1When, inhibiting rate 70.21%, compound 6,7 is in 200-400 μ gmL-1Compared with positive drug, suppression
Rate processed is significantly higher than positive drug(P<0.05), there was no significant difference compared with positive drug for compound 1,3,8(P>0.05), compound
Strengthen in set concentration range inhibiting rate with the increase of concentration.
The IC of compound on intracellular proliferation inhibition activity50It is as shown in table 2 to be worth result, from table 2 it can be seen that compound is to four
Planting tumor cell proliferation inhibition activity size is:SGC-7901gastriccarcinomacellline:Compound 6>Compound 5>Compound
3>Compound 1>Compound 8>Compound 7;Human prostata cancer PC-3M cells:Compound 3>Compound 1>Compound 6>Compound 7
>Compound 5>Compound 8;Human breast carcinoma MCF-7:Compound 6>Compound 1>Compound 3>Compound 7>Compound 5>Compound
8;Human liver cancer HepG-2 cells:Compound 6>Compound 7>Compound 3>Compound 8>Compound 1>Compound 5.
Claims (4)
1. a kind of straw mushroom fructification active component, its chemical name is 3 β, 5 α, 9 α-trihydroxy ergot steroid -7,22- diene -
6- ketone.
A kind of 2. straw mushroom fructification active component described in claim 1, it is characterised in that:Its chemical constitution is:
。
3. a kind of preparation method of straw mushroom fructification active component, it includes:
1)Straw mushroom is taken to dry fructification coarse powder, using 95% ethanol as solvent, by solid-liquid ratio 1:4 amount adds round-bottomed flask, 30
30 min of ultrasound, stand overnight at DEG C, are recovered under reduced pressure extract solution, residue 95% ethanol heating and refluxing extraction 3 times, each 3h, close
And extract solution, acquisition ethanol extract is recovered under reduced pressure;
2)By step 1)Described ethanol extract dichloromethane, methanol dissolving, with silica gel by volume 1:1 amount carries out dry method
Mix sample, dry column-packing, the mm of the mm of 100-200 mesh 150 × 305 silica gel chromatographic column is crossed, with chloroform-methanol volume ratio(9:1、
8:2、7:3、6:4、1:1)Gradient elution is carried out, obtains chloroform-methanol(8:2)Elution fraction;3)By step 2)Described elution
The silica gel of component and 200-300 mesh by volume 1:2 ratio mixes sample, dry column-packing, with volume ratio 8:2 dichloromethane-the third
Ketone elutes, and is purified through hydroxypropyl sephadex Sephadex LH-20, through HPLC preparative separations, condition is 80% ~ 100% first
Alcohol, 254 nm, 10 ml/min, obtain compound 3 β, 5 α, 9 α-trihydroxy ergot steroid -7,22- diene -6- ketone.
4. 3 β, 5 α, 9 α-trihydroxy ergot steroid -7,22- diene -6- ketone are in treatment stomach cancer, human breast carcinoma or human liver cancer medicine
In application.
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