CN107474027A - The propionic acid of straw mushroom fructification active component 2 (5H) furanone 4 and application - Google Patents

The propionic acid of straw mushroom fructification active component 2 (5H) furanone 4 and application Download PDF

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CN107474027A
CN107474027A CN201710635324.6A CN201710635324A CN107474027A CN 107474027 A CN107474027 A CN 107474027A CN 201710635324 A CN201710635324 A CN 201710635324A CN 107474027 A CN107474027 A CN 107474027A
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compound
furanone
propionic acid
straw mushroom
silica gel
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王�琦
姜建新
陈屏
秦惠娟
李元伟
杨建华
张晶
李玉
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Jilin Agricultural University
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Jilin Agricultural University
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    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07D—HETEROCYCLIC COMPOUNDS
    • C07D307/00—Heterocyclic compounds containing five-membered rings having one oxygen atom as the only ring hetero atom
    • C07D307/02—Heterocyclic compounds containing five-membered rings having one oxygen atom as the only ring hetero atom not condensed with other rings
    • C07D307/34—Heterocyclic compounds containing five-membered rings having one oxygen atom as the only ring hetero atom not condensed with other rings having two or three double bonds between ring members or between ring members and non-ring members
    • C07D307/56—Heterocyclic compounds containing five-membered rings having one oxygen atom as the only ring hetero atom not condensed with other rings having two or three double bonds between ring members or between ring members and non-ring members with hetero atoms or with carbon atoms having three bonds to hetero atoms with at the most one bond to halogen, e.g. ester or nitrile radicals, directly attached to ring carbon atoms
    • C07D307/58—One oxygen atom, e.g. butenolide

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Abstract

The invention discloses straw mushroom fructification active component 2 (5H) furanone 4 propionic acid and application.It is that straw mushroom is dried into fructification to crush, and adds ethanol solution to obtain ethanol extract after ultrasonic extraction, concentration, then with 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 10:1 methylene chloride acetone elution, noval chemical compound 2 (5 is obtained after the purifying of Sephadex LH 20 and HPLC processingH) 4 propionic acid of furanone, test result indicates that 2 (5H) 4 propionic acid significant effect in terms of the activity of tumor cells of stomach cancer SGC 7901 is suppressed of furanone.

Description

(5H)-furanone -4- propionic acid of straw mushroom fructification active component 2 and application
Technical field
The invention belongs to Chemistry for Chinese Traditional Medicine field, and in particular to (5H)-furanone -4- propionic acid of straw mushroom fructification active component 2 and Using.
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 invention to provide the safe and reliable monomeric compound separated from straw mushroom fructification, and extraction separation The application of method and compound in anti-tumor aspect.
Straw mushroom fructification active component 2 (5H)-furanone -4- propionic acid, its chemical constitution are：
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Straw mushroom fructification active component 2 (5HThe preparation method of)-furanone -4- propionic acid, 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, 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, acquisition ethanol extract is recovered under reduced pressure.
2）Ethanol extract dichloromethane, methanol are dissolved, with silica gel（100-200 mesh）By volume 1:1 dry method mixes sample, Dry column-packing, cross the silica gel chromatographic column of 100-200 mesh（150 mm × 305 mm）, with petroleum ether-chloroform（100:1、20:1、 10:1、9:1、8:2、7:3、6:4、1:1）, chloroform-methanol（100:1、20:1、9:1、8:2、7:3、0:1）Gradient elution, through solidifying Glue column chromatography and high performance liquid chromatography separation obtain 15 components.Idiographic flow is as shown in Figure 1.
2）By step 1）After described ethanol extract dichloromethane, methanol dissolving, with silica gel by volume 1:1 amount is entered Row dry method mixes 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（9：1）Elution fraction；3）By step 2）Described The silica gel of elution fraction and 200-300 mesh by volume 1:2 ratio mixes sample, dry column-packing, with volume ratio 9:1 dichloromethane Alkane-acetone elution, is purified through hydroxypropyl sephadex Sephadex LH-20, and condition is methanol, through preparing HPLC separation, Condition is 20% methanol, 203 nm, 10 ml/min, obtains compound 2 (5H)-furanone -4- propionic acid.
The invention provides straw mushroom fructification active component 2 (5H)-furanone -4- propionic acid and application.It is to do straw mushroom Dry fructification crushes, and adds ethanol solution to obtain ethanol extract after ultrasonic extraction, concentration, then with 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, the group after elution Point and silica gel（200-300 mesh）By volume 1:2 ratio mixes sample, dry column-packing, with volume ratio 9:1 dichloromethane-acetone Elution, 2 (5 are obtained after Sephadex LH-20 purifying and HPLC processingH)-furanone -4- propionic acid, test result indicates that 2 (5H)-furanone -4- propionic acid significant effect in terms of stomach cancer SGC-7901 activity of tumor cells is suppressed.
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 portion（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 27.3 min, 254 nm）, obtain compound 4, HPLC such as Fig. 2 It is shown.
By chloroform-methanol（9:1）Part（12.0 mg）By 1:2 ratio（200-300 mesh）Silica gel mixed sample, dry column-packing Son, use dichloromethane-acetone（50:1、20:1、10:1、8:2、7:3、6:4）Elution, obtain 5 components.Wherein dichloromethane- Acetone（8:2）Part, purified through Sephadex LH-20, through preparing HPLC（80% ~ 100% methanol, 254 nm, 10 ml/ min-1）Obtain compound 6（12.5 mg, 24.344min）, 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, purified through Sephadex LH-20, warp Prepare 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 7 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 compound 52 (5 of embodiment 3HThe extraction of)-furanone -4- propionic acid
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）After described ethanol extract dichloromethane, methanol dissolving, with silica gel by volume 1:1 amount is entered Row dry method mixes 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（9：1）Elution fraction；3）By step 2）Described The silica gel of elution fraction and 200-300 mesh by volume 1:2 ratio mixes sample, dry column-packing, with volume ratio 9:1 dichloromethane Alkane-acetone elution, is purified, condition is methanol, through HPLC through hydroxypropyl sephadex Sephadex LH-20（20% methanol, 203 nm, 10 ml/min）Prepare compound 52 (5H)-furanone -4- propionic acid, HPLC are as shown in Figure 3.
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（600 MHz, 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.
The compound effects of table 1 are in the measure of the h of HEK-293 cells 24 inhibiting rate
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 24 h 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 (3)

1. straw mushroom fructification active component 2 (5H)-furanone -4- propionic acid, other chemical constitutions are：
。
2. straw mushroom fructification active component 2 (5HThe preparation method of)-furanone -4- propionic acid, it includes：
1）Take straw mushroom to dry fructification, crush, add ethanol solution, ultrasonic extraction；
2）Step 1 is extracted into solution and carries out concentration acquisition ethanol extract, after dichloromethane, methanol dissolving medicinal extract, is pressed with silica gel Volume ratio 1:1 amount dry method mixes sample, dry column-packing, crosses the mm silica gel chromatographic columns of 150 mm of 200-300 mesh × 305, uses chlorine Imitation-carbinol elutes, then with silica gel by volume 1:2 ratio mixes sample, dry column-packing, with volume ratio 10:1 dichloromethane-the third After ketone elution, purified through hydroxypropyl sephadex Sephadex LH-20, then through high-efficient liquid phase chromatogram HPLC separationization Compound is 2 (5H)-furanone -4- propionic acid.
3.2(5H)-furanone -4- propionic acid is preparing the application in treating gastric cancer medicament.
CN201710635324.6A 2017-07-30 2017-07-30 The propionic acid of straw mushroom fructification active component 2 (5H) furanone 4 and application Pending CN107474027A (en)

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CN113952342A (en) * 2021-12-06 2022-01-21 上海市农业科学院 Application of small molecule compound

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JONGHYUN HAN ET AL: ""Upregulation of death receptor 5 and activation of caspase 8/3 play a critical role in ergosterol peroxide induced apoptosis in DU 145 prostate cancer cells"", 《CANCER CELL INTERNATIONAL》 *
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CN108383811A (en) * 2018-04-09 2018-08-10 陕西科技大学 A kind of furanone derivatives and its extracting method and application
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CN113952342A (en) * 2021-12-06 2022-01-21 上海市农业科学院 Application of small molecule compound

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Application publication date: 20171215