CN106749107A - Terpenoid in Roots of Euphorbia soongarica and its production and use - Google Patents
Terpenoid in Roots of Euphorbia soongarica and its production and use Download PDFInfo
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Abstract
The present invention relates to terpenoid in a kind of Roots of Euphorbia soongarica and its production and use, the method is with Roots of Euphorbia soongarica（E. soongarica）Herb be raw material, with Solvent Extract methods, separated by three kinds to four kinds methods in solvent extraction, normal-phase silica gel column chromatography method, reversed-phase silica gel column chromatography method, the gel filtration chromatography methods of Sephadex LH 20, obtain 10 new terpenoids（Including 5 Diterpeneses, 3 carbon drop triterpenes and 2 triterpene compounds）, and multi-medicine tolerant reversal activity measure has been carried out to obtaining 10 new terpenoids, as a result show：The new terpenoid has different degrees of multi-medicine tolerant reversal activity, can be used to prepare the application in multi-drug resistance reversing medicaments, or it is prepared into antineoplastic combined medicament with antitumor combination.
Description
Technical field
The present invention relates to pharmaceutical technology field, and in particular to terpenoid in Roots of Euphorbia soongarica and preparation method thereof and
Purposes.
Background technology
Euphorbia (Euphorbia) is a big category in Euphorbiaceae (Euphorbiaceae), and the whole world there are about more than 2000 kinds
Plant, is distributed widely in the torrid zone and Temperate Region in China.Euphorbia has important medical value, is used for defaecation, diuresis, controls
Treat the illness such as tuberculosis, oedema, psoriasis and scabies.Roots of Euphorbia soongarica (Euphorbia soongarica Boiss.) is the root of Beijing euphorbia
Category herbaceos perennial, originates in In The North of Xinjiang and West Part of Gansu, is distributed in China, West Siberia to the Central Asia and Mongolia.Its
Root is used as medicine, and is had functions that to relieve oedema or abdominal distension through diuresis or purgation, is subsided a swelling, dissipates the stasis of blood, and the substitute for also serving as the root of Beijing euphorbia in Xinjiang region is used.Report at present
The compound obtained from Roots of Euphorbia soongarica have more than 50, predominantly flavonoids, Polyphenols and triterpene compound, but relevant
The research of its bioactivity is very few, only finds that 3 triterpene compounds have certain cytotoxicity, and the feature of Euphorbia into
Point --- diterpene-kind compound does not appear in the newspapers.Therefore, to the further Research on Mining of chemical composition of Roots of Euphorbia soongarica, to clear and definite
Roots of Euphorbia soongarica secondary metabolite provides reference, also provides foundation to disclose the material base of its drug action.
Tumor multi-medicine drug-resistant (multidrug resistance, MDR), i.e., a kind of medicine act on tumour be allowed to produce it is resistance to
After the property of medicine, the tumour to from also have not in contact with, structure is unrelated, target spot is different, mechanism is different various antineoplastics intersect it is resistance to
The phenomenon of the property of medicine.MDR has various Forming Mechanisms, wherein one of topmost mechanism is that (research at present is most for ABC families transport protein
Extensively, most deep is the P- glycoprotein of ABCB1 gene codes, P-gp) overexpression, cause drug efflux to increase, formed resistance to
The property of medicine.In recent years, many isolated diterpene-kind compounds from plant are found to have significant MDR Reversal activities, because
And turn into one of study hotspot.
The content of the invention
It is an object of the present invention to provide terpenoid in a kind of Roots of Euphorbia soongarica and its production and use,
The method with the herb of Roots of Euphorbia soongarica (E.soongarica) as raw material, with Solvent Extract methods, by solvent extraction, just
Three kinds to four kinds methods in phase silica gel column chromatography, reversed-phase silica gel column chromatography method, Sephadex LH-20 gel filtration chromatography methods
Separated, obtained 10 new terpenoids (including 5 Diterpeneses, 3 carbon drop triterpenes and 2 triterpenes chemical combination
Thing), and multi-medicine tolerant reversal activity measure has been carried out to obtaining 10 new terpenoids, as a result show：The new terpene
Class compound has different degrees of multi-medicine tolerant reversal activity, can be used to prepare the application in multi-drug resistance reversing medicaments, or
It is prepared into antineoplastic combined medicament with antitumor combination.
Terpenoid in a kind of Roots of Euphorbia soongarica of the present invention, the compound is diterpene-kind compound and triterpene
The structural formula of class compound, wherein diterpene-kind compound is：
Formula (I) compound is 3 β-benzoyloxy -5,6- epoxies-with the continuous sub- β of alkane -12E- alkene -7,15 beta-diol -14-
Ketone；
Formula (II) compound is 3 β-benzoyloxy-with continuous sub- alkane -5E, the β of 12E- diene -7,15 beta-diol -14- ketone；
Formula (III) compound is Jatropha curcas alkane -4E, the β of 6 (17)-diene -13,14 beta-diol -3- ketone；
Formula (IV) compound be the sweet blue or green α of euphorbia B alkane -5 of 12 α-benzene acryloyl amido -3 β-acetoxyl groups -, 15 beta-diols -
14- ketone；
Formula (V) compound is 12 α-benzoyloxy-sweet blue or green β of euphorbia B alkane -4Z- alkene -3,5 salmefamol -14- ketone；
The structural formula of triterpene compound is：
Formula (VI) compound drops the-β -ol -7- ketone -24- aldehyde of euphane -8Z, 22E- diene -3 for 25,26,27- tri-；
Formula (VII) compound is the β -ol -7,25- diketone of 27- drop-euphanes -8Z, 23E- diene -3；
Formula (VIII) compound is 24R- euphanes -8Z, 25E- diene -3 β, 24- glycol -7- ketone；
Formula (Ⅸ) compound drops the-β -ol -7,20- diketone of euphane -8Z- alkene -3 for 22,23,24,25,26,27- six；
Formula (Ⅹ) compound is 7,15- dioxies-D:The β -ol of C-friedo- oleanane -8Z- alkene -3.
The preparation method of the terpenoid in the Roots of Euphorbia soongarica, follow these steps to carry out：
A, with 5-10 times to measure the ethanol that volumetric concentration is 50-99% with Roots of Euphorbia soongarica herb as raw material, after crushing water-soluble
Liquid, absolute ethyl alcohol, volumetric concentration are methanol aqueous solution, absolute methanol, the acetone water that volumetric concentration is 50-99% of 50-99%
Solution or acetone carry out diacolation or cold soaking extraction at room temperature, or heating and refluxing extraction, are concentrated to give Roots of Euphorbia soongarica crude extract；
B, the crude extract for obtaining step a are disperseed with acetonitrile, add hexamethylene, n-hexane or petroleum ether extraction, or will be thick
The dispersion of extract hexamethylene, n-hexane or petroleum ether, adds acetonitrile extraction 1-5 times, by the concentration of acetonitrile extraction liquid, obtains acetonitrile
Extract medicinal extract；
C, the acetonitrile extraction thing medicinal extract for obtaining step b are through normal-phase silica gel column chromatography, reversed-phase silica gel column chromatography, Sephadex
Separated for two or three in LH-20 gel filtration chromatography methods, that is, obtained formula (I)-formula (Ⅹ) compound.
Normal-phase silica gel column chromatography method used is normal pressure or pressurized column chromatography in step c, and filler used is silica gel, wash-out used
Agent is the mixture of at least two solvents in petroleum ether, hexamethylene, n-hexane, acetone, chloroform, ethyl acetate, methyl alcohol, is adopted
With isocratic elution or gradient elution.
Reversed-phase silica gel column chromatography method used is normal pressure or pressurized column chromatography in step c, and eluant, eluent is that volumetric concentration is 30-
Acetonitrile-methanol-the water mixed solution of 99% methanol aqueous solution, the acetonitrile solution of 10-99% or 10-99%, using isocratic
Wash-out or gradient elution.
In step c Sephadex LH-20 gel filtration chromatographies method used be normal pressure column chromatography, eluant, eluent be methyl alcohol, acetone,
Dichloromethane, chloroform or in them at least two solvents mixture, using isocratic elution or gradient elution.
Formula (IV) and formula (V) compound in terpenoid in the Roots of Euphorbia soongarica, formula (VI), formula (VII) and
Purposes of formula (VIII) compound in multi-drug resistance reversing medicaments are prepared.
Formula (IV) and formula (V) compound in terpenoid in the Roots of Euphorbia soongarica, formula (VI), formula (VII) and
Purposes of formula (VIII) compound in the medicine of preparation and antitumor combination.
Terpenoid in Roots of Euphorbia soongarica of the present invention and its production and use, is obtained by methods described
The 10 new terpenoids for obtaining are determined by multi-medicine tolerant reversal activity, test result indicate that：The formula (IV), formula
(V), formula (VI), formula (VII) and formula (VIII) compound can to some extent suppress the medicine of P- glycoprotein mediation in mdr cell
The exclusive effect of thing, so as to show multi-medicine tolerant reversal activity, can be used to prepare multi-drug resistance reversing medicaments or and antineoplastic
Thing combination prepares antineoplastic combined medicament.
Terpenoid in Roots of Euphorbia soongarica of the present invention, can be obtained by being isolated and purified from plant, also may be used
Obtained with synthesizing through chemical modification method well known to those skilled in the art.
Terpenoid in Roots of Euphorbia soongarica of the present invention, is total to using high resolution mass spectrum, a peacekeeping two-dimensional nucleus magnetic
The Modern spectroscopy means such as spectrum of shaking determine its structure, and Structural Identification process is as follows：
Formula (I) compound is white amorphous powder, [α]D 20+44.0(c 0.1,MeOH)；UV(MeOH)λmax(logε)
229(4.21),267(4.19)nm；ECD(MeOH)202(Δε-2.71),231(Δε-8.34),272(Δε+8.15),348
(Δε+0.53)nm；HRESI (+) MS provides quasi-molecular ion peak m/z 455.2422 [M+H]+(calculated value is C27H35O6
455.2434), determine that its molecular formula is C27H34O6；According to1H,13C NMR and two dimensional NMR data determine that its structure is 3
β-benzoyloxy -5,6- epoxy-and with the continuous sub- β of alkane -12E- alkene -7,15 beta-diol -14- ketone, its1H and13C NMR datas belong to
Be shown in Table 1 [600MHz (1H), 150MHz (13C), solvent：CDCl3]。
Formula (II) compound is white amorphous powder, [α]D 20+90.0(c 0.1,MeOH)；UV(MeOH)λmax(logε)
229(4.20),274(4.13)nm；ECD(MeOH)216(Δε-16.13),237(Δε-14.58),278(Δε+16.85),
345(Δε+2.53)nm.According to HRESI (+) MS (m/z 439.2498 [M+H]+, calculated value is C27H35O5439.2484) really
Fixed its molecular formula is C27H34O5；According to1H,13C NMR and two dimensional NMR data determine that its structure is 3 β-benzoxy
Base-with continuous sub- alkane -5E, the β of 12E- diene -7,15 beta-diol -14- ketone.Its1H and13C NMR datas ownership is shown in Table 1 [400MHz
(1H), 100MHz (13C), solvent：CDCl3]。
Formula (III) compound is white amorphous powder, [α]D 20-75.6(c 0.09,MeOH)；UV(MeOH)λmax(log
ε)231(3.88)nm；ECD(MeOH)231(Δε-12.86)nm；According to it13C NMR and HRESI (+) MS (m/z 317.2101
[M+H]+, theoretical value C20H29O3317.2117) data determine that its molecular formula is C20H28O3；According to1H,13C NMR and two-dimensional nucleus
MR data determines that its structure is Jatropha curcas alkane -4E, the β of 6 (17)-diene -13,14 beta-diol -3- ketone.Its1H and13C NMR numbers
Be shown in Table 1 according to ownership [600MHz (1H), 150MHz (13C), solvent：CDCl3]。
The formula of table 1. (I), formula (II), formula (III) compound1H and13C NMR datas [δ (ppm), J (Hz)]
Formula (IV) compound is white amorphous powder, [α]D 20+1.0(c 0.1,MeOH)；UV(MeOH)λmax(logε)
216(4.23),277(4.29)nm；ECD(MeOH)203(Δε-10.54),260(Δε-1.43),311(Δε+4.49)nm；
By its HRESI (+) MS data (m/z 524.2996 [M+H]+, calculated value is C31H42NO6524.3012) it is and true with reference to nitrogen rate
Contain a nitrogen-atoms in fixed its molecule, molecular formula is C31H41NO6；According to1H,13C NMR and two dimensional NMR data are true
Fixed its structure is the sweet blue or green α of euphorbia B alkane -5 of 12 α-benzene acryloyl amido -3 β-acetoxyl groups -, 15 beta-diol -14- ketone.Its1H and13C
NMR ownership be shown in Table 2 [400MHz (1H), 100MHz (13C), CDCl3]。
Formula (V) compound is colorless oil, [α]D 20-31.0(c 0.1,MeOH)；UV(MeOH)λmax(logε)230
(4.01)nm；ECD(MeOH)250(Δε-7.37),333(Δε+0.67)nm；According to it13C NMR and HRESI (+) MS data
(m/z 439.2469[M+H]+, theoretical value C27H35O5439.2484) determine that its molecular formula is C27H34O5.According to1H,13C NMR
And two dimensional NMR data determine its structure for 12 α-benzoyloxy-sweet blue or green β of euphorbia B alkane -4Z- alkene -3,5 salmefamols -
14- ketone；Its1H and13C NMR ownership be shown in Table 2 [400MHz (1H), 100MHz (13C), CDCl3]。
The formula of table 2. (IV), formula (V) compound1H and13C NMR datas [δ (ppm), J (Hz)]
Formula (VI) compound is white amorphous powder, [α]D 20+7.8(c 0.09,MeOH)；UV(MeOH)λmax(logε)
216(3.94)nm；ECD(MeOH)219(Δε+1.37),247(Δε-0.27),280(Δε+1.34),337(Δε+0.65)
nm；According to its HRESI (+) MS data (m/z 413.3050 [M+H]+, theoretical value C27H41O3413.3056) its molecular formula is judged
It is C27H40O3；According to1H,13C NMR and two dimensional NMR data determine that its structure drops-euphane-for 25,26,27- tri-
β -ol -7- ketone -24- the aldehyde of 8Z, 22E- diene -3.Its1H and13C NMR datas ownership be shown in Table 3 [600MHz (1H), 150MHz (13C),
Solvent：CDCl3]。
Formula (VII) compound is white amorphous powder, [α]D 20+7.0(c 0.1,MeOH)；UV(MeOH)λmax(logε)
224(3.96)nm；ECD(MeOH)233(Δε+0.75),257(Δε-1.13),284(Δε+0.49),335(Δε+1.09)
nm；HRESI (+) MS provides m/z 441.3353 [M+H]+(theoretical value C29H45O3441.3369), determine that its molecular formula is
C29H44O3；According to1H,13C NMR and two dimensional NMR data determine its structure for 27- drop-euphanes -8Z, 23E- diene -
3 β -ol -7,25- diketone.Its1H and13C NMR datas ownership be shown in Table 3 [400MHz (1H), 100MHz (13C), solvent：CDCl3]。
The formula of table 3. (VI), formula (VII) compound1H and13C NMR datas [δ (ppm), J (Hz)]
Formula (VIII) compound is white amorphous powder, [α]D 20+15.0(c 0.1,MeOH)；UV(MeOH)λmax(logε)
254(3.97)nm；ECD(MeOH)220(Δε-2.73),260(Δε-2.44),338(Δε+1.86)nm；HRESI (+) MS gives
Go out quasi-molecular ion peak m/z 457.3662 [M+H]+(theoretical value C30H49O3457.3682), determine that its molecular formula is C30H48O3；
By parsing1H,13C NMR and two dimensional NMR data determine its planar structure, and determine its with known compound [(+)-
(24S)-eupha-8,25-diene-3 β, 24-diol-7-one] there is different C-24 absolute configurations.Further using deuterated
Acetone is carried out to above-mentioned two compound13C NMR are determined and are carried out comparing, as a result show the C-20 of formula (VIII) compound,
C-23, C-24 differs -0.22 with known compound respectively, -0.20, -0.34ppm, and other data are consistent, it is thus determined that formula
(VIII) structure of compound is 24R- euphanes -8Z, 25E- diene -3 β, 24- glycol -7- ketone.Formula (VIII) compound1H and13C
NMR data be shown in Table 3 [400MHz (1H), 100MHz (13C), solvent：CDCl3]；Formula (VIII) compound and known compound [(+)-
(24S)-eupha-8,25-diene-3 β, 24-diol-7-one]13C (document report and that we get) NMR data is shown in
Table 4 [150MHz (13C)]。
The formula of table 4. (VIII) compound and [(+)-(24S)-eupha-8,25-diene-3 β, 24-diol-7-one] (document
It is report and that we get)13C NMR datas contrast [acetone-d6,δ(ppm)]
* the ND in document is to be not detected by the signal.
Formula (Ⅸ) compound is white amorphous powder, [α]D 20-24.2(c 0.03,MeOH)；UV(MeOH)λmax(log
ε)252(3.89)nm；ECD(MeOH)257(Δε-2.75),287(Δε-1.41),339(Δε+1.35)nm.By it13C
NMR and HRESI (+) MS data (m/z 373.2750 [M+H]+, theoretical value C24H37O3373.2743) determine that its molecular formula is
C24H36O3.According to1H,13C NMR and two dimensional NMR data determine its structure drop for 22,23,24,25,26,27- six-it is big
β -ol -7,20- the diketone of halberd alkane -8Z- alkene -3；Its1H and13C NMR datas ownership be shown in Table 5 [600MHz (1H), 150MHz (13C), it is molten
Agent：CDCl3]。
Formula (Ⅹ) compound is white amorphous powder, [α]D 20+1.0(c 0.1,MeOH)；UV(MeOH)λmax(logε)
245(3.96)nm；ECD(MeOH)209(Δε-4.22),254(Δε-4.76),284(Δε+0.53),334(Δε+0.74)
nm.According to its HRESI (+) MS data (m/z 455.3544 [M+H]+, theoretical value C30H47O3455.3525) its molecular formula is judged
It is C30H46O3；According to1H,13C NMR and two dimensional NMR data determine that its structure is 7,15- dioxies-D:C-friedo- is neat
The β -ol of pier fruit alkane -8Z- alkene -3.Its1H and13C NMR datas ownership be shown in Table 5 [400MHz (1H), 100MHz (13C), solvent：
CDCl3]。
The formula of table 5. (Ⅸ), formula (Ⅹ) compound1H and13C NMR datas [δ (ppm), J (Hz)]
Brief description of the drawings
Fig. 1-Fig. 2 is formula of the present invention (I) compound1H and13C nmr spectrums；
Fig. 3-Fig. 4 is formula of the present invention (II) compound1H and13C nmr spectrums；
Fig. 5-Fig. 6 is formula of the present invention (III) compound1H and13C nmr spectrums；
Fig. 7-Fig. 8 is formula of the present invention (IV) compound1H and13C nmr spectrums；
Fig. 9-Figure 10 is formula of the present invention (V) compound1H and13C nmr spectrums；
Figure 11-Figure 12 is formula of the present invention (VI) compound1H and13C nmr spectrums；
Figure 13-Figure 14 is formula of the present invention (VII) compound1H and13C nmr spectrums；
Figure 15-Figure 16 is formula of the present invention (VIII) compound1H and13C nmr spectrums；
Figure 17-Figure 18 is formula of the present invention (Ⅸ) compound1H and13C nmr spectrums；
Figure 19-Figure 20 is formula of the present invention (Ⅹ) compound1H and13C nmr spectrums.
Specific embodiment
Embodiment 1.
A, Roots of Euphorbia soongarica herb 10kg is taken, with 50L acetone seepage pressure effects at room temperature after crushing, evaporated under reduced pressure solvent is obtained
Roots of Euphorbia soongarica crude extract；
B, the crude extract for obtaining step a are disperseed with acetonitrile, add hexamethylene to be extracted, and merge acetonitrile layer and steaming of depressurizing
It is dry to obtain acetonitrile extraction thing medicinal extract；
C, the acetonitrile extraction thing medicinal extract purification on normal-phase silica gel post separation for obtaining step b, with volume ratio 100:1-0:1 just oneself
Alkane-ethyl acetate carries out gradient elution, and flow point is analyzed through silica gel thin-layer chromatography (TLC), merges identical flow point, obtains 12 components
(F1-F12)；Component F6 is carried out into purification on normal-phase silica gel post separation, is 40 with volume ratio:1-0:1 n-hexane-ethyl acetate carries out ladder
Degree wash-out, obtains component F6A-F6F；By component F6C through the anti-phase post separations of RP-18, the methanol-water with concentration as 50%-100%
Solution gradient is eluted, and collects the eluent of 70% methanol-water solution, is used after evaporated under reduced pressure and is prepared reversed-phase column (C18 5μm10×
250mm) separate, with the acetonitrile-aqueous solution isocratic elution that concentration is 45%, obtain formula (I) and formula (III) compound；F7 sections is entered
Row purification on normal-phase silica gel post separation, using volume ratio 100:9:1-0:0:1 n-hexane-chloroform-acetone gradient elution, obtains component
F7A-F7H；F7F sections, first through Sephadex LH-20 gel post separations, is eluted with absolute methanol, and the flow point for obtaining is again through RP-18
Anti-phase post separation, the methanol-water solution gradient elution with concentration as 30%-100% collects the wash-out of 70% methanol-water solution
Liquid, uses after evaporated under reduced pressure and prepares reversed-phase column (C185 μm of 10 × 250mm) separate, with acetonitrile-aqueous solution that concentration is 50% etc.
Degree wash-out, obtains formula (II) and formula (VI) compound；F7G sections, first through Sephadex LH-20 gel post separations, is used absolute methanol
Wash-out, the flow point for obtaining again through purification on normal-phase silica gel post separation, using volume ratio 10:1-0:1 n-hexane-ethyl acetate gradient is washed
It is de-, obtain component F7G3A-F7G3H；F7G3E sections through the anti-phase post separations of RP-18, the methanol-water with concentration as 50%-100% is molten
Liquid gradient elution, collects the eluent of 70% methanol-water solution (F7G3E3) and 80% methanol-water solution (F7G3E4), decompression
It is evaporated, F7G3E3 sections of use prepares reversed-phase column (C185 μm of 10 × 250mm) separate, with acetonitrile-aqueous solution that concentration is 45% etc.
Degree wash-out, obtains formula (IV) and formula (Ⅸ) compound；F7G3E4 is used and is prepared reversed-phase column (C185 μm of 10 × 250mm) separate, with
Concentration is 47% acetonitrile-aqueous solution isocratic elution, obtains formula (VII), formula (VIII) and formula (Ⅹ) compound；To F7H sections using just
Phase silica gel post separation, it is 10 to use volume ratio:1-0:1 n-hexane-acetone carries out gradient elution, obtains component F7H1-F7H8；
F7H2 sections again through the anti-phase post separations of RP-18, the methanol-water solution gradient elution with concentration as 50%-100%, 70% first of collection
Alcohol-water solution, uses after evaporated under reduced pressure and prepares reversed-phase column (C185 μm of 10 × 250mm) separate, with acetonitrile that concentration is 47%-
Aqueous solution isocratic elution, obtains formula (V) compound.
Embodiment 2
A, Roots of Euphorbia soongarica herb 10kg is taken, with the acetone-water solution that the concentration of 60L is 80% cold soaking at room temperature after crushing
Extract, evaporated under reduced pressure solvent obtains Roots of Euphorbia soongarica crude extract；
B, the crude extract for obtaining step a are disperseed with acetonitrile, add n-hexane to be extracted, and merge acetonitrile layer and steaming of depressurizing
It is dry to obtain acetonitrile extraction thing medicinal extract；
C, the acetonitrile extraction thing medicinal extract purification on normal-phase silica gel post separation for obtaining step b, are 100 with volume ratio:1-0:1 stone
Oily ether-ethyl acetate carries out gradient elution, and flow point is analyzed through TLC, merges identical flow point, obtains 12 components (F1-F12)；Will
Component F6 carries out purification on normal-phase silica gel post separation, is 40 with volume ratio:1-0:1 cyclohexane-ethyl acetate gradient elution, obtains component
F6A-F6F；By F6C components through the anti-phase post separations of RP-18, the acetonitrile-aqueous solution gradient elution with concentration as 30%-100% is received
Collect the eluent of 50% acetonitrile-aqueous solution, used after evaporated under reduced pressure and prepare reversed-phase column (C185 μm of 10 × 250mm) separate, with dense
The acetonitrile-aqueous solution isocratic elution for 45% is spent, formula (I) and formula (III) compound is obtained；Normal phase silicagel column point is carried out by F7 sections
From using volume ratio 100:9:1-0:0:1 n-hexane-dichloromethane-acetone gradient elution, obtains component F7A-F7H；F7F
Duan Xianjing Sephadex LH-20 gel post separations, are 1 with volume ratio:1 chloroform-methanol wash-out, the flow point for obtaining is again through RP-
18 anti-phase post separations, the methanol-water solution gradient elution with concentration as 30-100% collects the wash-out of 70% methanol-water solution
Liquid, uses after evaporated under reduced pressure and prepares reversed-phase column (C185 μm of 10 × 250mm) separate, with acetonitrile-aqueous solution that concentration is 50% etc.
Degree wash-out, obtains formula (II) and formula (VI) compound；F7G sections first through Sephadex LH-20 gel post separations, be with volume ratio
1:1 chloroform-methanol wash-out, the flow point for obtaining again through purification on normal-phase silica gel post separation, using volume ratio 10:1-0:1 hexamethylene-second
Acetoacetic ester gradient elution, obtains component F7G3A-F7G3H；F7G3E sections through the anti-phase post separations of RP-18, with concentration as 50-100%
Methanol-water solution gradient elution, collect 70% methanol-water solution (F7G3E3) and 80% methanol-water solution (F7G3E4)
Eluent, evaporated under reduced pressure, F7G3E3 is used and is prepared reversed-phase column (C185 μm of 10 × 250mm) separate, with the first that concentration is 65%
Alcohol-water solution isocratic elution, obtains formula (IV) and formula (Ⅸ) compound；F7G3E4 is used and is prepared reversed-phase column (C18 5μm 10×
250mm) separate, with the methanol-water solution isocratic elution that concentration is 67%, obtain formula (VII), formula (VIII) and formula (Ⅹ) compound；
Purification on normal-phase silica gel post separation is used to F7H sections, using volume ratio 10:1-0:1 cyclohexane-acetone gradient elution, obtains component
F7H1-F7H8；F7H2 sections again through the anti-phase post separations of RP-18, the acetonitrile-aqueous solution gradient elution with concentration as 30%-100%,
50% acetonitrile-aqueous solution is collected, is used after evaporated under reduced pressure and is prepared reversed-phase column (C185 μm of 10 × 250mm) separate, be with concentration
47% acetonitrile-aqueous solution isocratic elution obtains formula (V) compound.
Embodiment 3
A, Roots of Euphorbia soongarica herb 10kg is taken, with 70L ethanol seepage pressure effects at room temperature after crushing, evaporated under reduced pressure solvent is obtained
Roots of Euphorbia soongarica crude extract；
B, the crude extract for obtaining step a are disperseed with acetonitrile, add petroleum ether to be extracted, and merge acetonitrile layer and steaming of depressurizing
It is dry to obtain acetonitrile extraction thing medicinal extract；
C, the acetonitrile extraction thing medicinal extract purification on normal-phase silica gel post separation for obtaining step b, are 100 with volume ratio:1-0:1 ring
Hexane-ethylacetate carries out gradient elution, and flow point merges identical flow point, obtains 12 components (F1-F12) through TLC analyses；By group
Dividing F6 carries out purification on normal-phase silica gel post separation, is 40 with volume ratio:1-0:1 petroleum ether-ethyl acetate gradient elution, obtains component
F6A-F6F；By F6C components through the anti-phase post separations of RP-18, the methanol-water solution gradient elution with concentration as 50-100% is collected
The eluent of 70% methanol-water solution, uses after evaporated under reduced pressure and prepares reversed-phase column (C185 μm of 10 × 250mm) separate, with concentration
It is 65% methanol-water solution isocratic elution, obtains formula (I) and formula (III) compound；Purification on normal-phase silica gel post separation is carried out by F7 sections,
Using volume ratio 100:9:1-0:0:1 hexamethylene-chloroform-acetone gradient elution, obtains component F7A-F7H；F7F sections through RP-
18 anti-phase post separations, the acetonitrile-aqueous solution gradient elution with concentration as 10-100% collects the wash-out of 50% acetonitrile-aqueous solution
Liquid, with preparing reversed-phase column (C after evaporated under reduced pressure185 μm of 10 × 250mm) separate, it is isocratic with acetonitrile-aqueous solution that concentration is 50%
Wash-out, obtains formula (II) and formula (VI) compound；F7G sections first through Sephadex LH-20 gel post separations, be 1 with volume ratio:1
Methylene chloride-methanol wash-out, the flow point for obtaining again through purification on normal-phase silica gel post separation, using volume ratio 10:1-0:1 petroleum ether-
Ethyl acetate gradient, obtains component F7G3A-F7G3H；F7G3E sections through the anti-phase post separations of RP-18, with concentration as 50-
100% methanol-water solution gradient elution, collects the eluent of 70-80% methanol-water solutions, is with concentration after evaporated under reduced pressure
45% acetonitrile-aqueous solution isocratic elution, obtains formula (IV), formula (VII), formula (VIII), formula (Ⅸ) and formula (Ⅹ) compound；To F7H
Duan Caiyong purification on normal-phase silica gel post separations, using volume ratio 10:1-0:1 petroleum ether-acetone gradient elution, obtains component F7H1-
F7H8；F7H2 sections is separated through RP-18 reversed-phase column chromatographies again, and the methanol-water solution gradient elution with concentration as 50-100% is collected
70% methanol-water solution, uses after evaporated under reduced pressure and prepares reversed-phase column (C185 μm of 10 × 250mm) separate, it is 45% with concentration
Acetonitrile-aqueous solution isocratic elution, obtains formula (V) compound.
Embodiment 4.
A, Roots of Euphorbia soongarica herb 10kg is taken, with 80 DEG C of the ethanol-water solution temperature that the concentration of 80L is 90% after crushing
Refluxing extraction, evaporated under reduced pressure solvent obtains Roots of Euphorbia soongarica crude extract；
B, the crude extract for obtaining step a are disperseed with hexamethylene, add acetonitrile to be extracted, and merge acetonitrile layer and steaming of depressurizing
It is dry to obtain acetonitrile extraction thing medicinal extract；
C, the acetonitrile extraction thing medicinal extract purification on normal-phase silica gel post separation for obtaining step b, with volume ratio 100:1-0:1 just oneself
Alkane-ethyl acetate carries out gradient elution, and flow point merges identical flow point, obtains 12 components (F1-F12) through TLC analyses；By component
F6 carries out purification on normal-phase silica gel post separation, is 40 with volume ratio:1-0:1 petroleum ether-ethyl acetate gradient elution, obtains component F6A-
F6F；By F6C sections of component through the anti-phase post separations of RP-18, the acetonitrile-aqueous solution gradient elution with concentration as 30-100% is collected
The eluent of 50% acetonitrile-aqueous solution, uses after evaporated under reduced pressure and prepares reversed-phase column (C185 μm of 10 × 250mm) separate, with concentration
It is 65% methanol-water solution isocratic elution, obtains formula (I) and formula (III) compound；Purification on normal-phase silica gel post separation is carried out by F7 sections,
Using volume ratio 100:9:1-0:0:1 petroleum ether-chloroform-acetone carries out gradient elution, obtains component F7A-F7H；F7F sections first passes through
Sephadex LH-20 gel post separations, the flow point afforded with absolute methanol is with concentration again through the anti-phase post separations of RP-18
The methanol-water solution gradient elution of 30-100%, collects the eluent of 70% methanol-water solution, using preparation after evaporated under reduced pressure
Reversed-phase column (C185 μm of 10 × 250mm) separate, with the methanol-water solution isocratic elution that concentration is 70%, obtain formula (II) and formula
(VI) compound；F7G sections uses volume ratio 10:1-0:1 cyclohexane-ethyl acetate gradient elution, obtains component F7G1-F7G8；
F7G5 sections is collected 40-60% second through the anti-phase post separations of RP-18, the acetonitrile-aqueous solution gradient elution with concentration as 30-100%
The eluent of nitrile-aqueous solution, with the acetonitrile-aqueous solution isocratic elution that concentration is 45% after evaporated under reduced pressure, obtains formula (IV), formula
(VII), formula (VIII), formula (Ⅸ) and formula (Ⅹ) compound；Purification on normal-phase silica gel post separation is used to F7H sections, using volume ratio 10:1-0:1
N-hexane-acetone gradient elution, obtain component F7H1-F7H8；F7H2 sections again through the anti-phase post separations of RP-18, with concentration as 30-
100% acetonitrile-aqueous solution gradient elution, collects 40-60% acetonitrile-aqueous solutions, is used after evaporated under reduced pressure and prepares reversed-phase column (C18
5 μm of 10 × 250mm) separate, with the acetonitrile-aqueous solution isocratic elution that concentration is 47%, obtain formula (V) compound.
Embodiment 5
A, Roots of Euphorbia soongarica herb 10kg is taken, with 90L absolute methanols seepage pressure effects, evaporated under reduced pressure solvent at room temperature after crushing
Obtain Roots of Euphorbia soongarica crude extract；
B, the crude extract for obtaining step a are disperseed with n-hexane, add acetonitrile to be extracted, and merge acetonitrile layer and steaming of depressurizing
It is dry to obtain acetonitrile extraction thing medicinal extract；
C, the acetonitrile extraction thing medicinal extract purification on normal-phase silica gel post separation for obtaining step b, with volume ratio 100:1-0:1 oil
Ether-ethyl acetate carries out gradient elution, and flow point is analyzed through TLC, merges identical flow point, obtains 12 components (F1-F12)；By group
Dividing F6 carries out purification on normal-phase silica gel post separation, is 40 with volume ratio:1-0:1 n-hexane-ethyl acetate gradient elution, obtains component
F6A-F6F；By F6C sections of component through preparing reversed-phase column (C185 μm of 10 × 250mm) separate, the acetonitrile with concentration as 45-100%-
Aqueous solution gradient elution, obtains formula (I) and formula (III) compound；Purification on normal-phase silica gel post separation is carried out by F7 sections, using volume ratio
100:9:1-0:0:1 hexamethylene-dichloromethane-acetone gradient elution, obtains component F7A-F7H；F7F sections first through Sephadex
LH-20 gel post separations, with volume ratio 1:The flow point that 1 chloroform-methanol is afforded again through the anti-phase post separations of RP-18, with dense
The methanol-water solution gradient elution for 30-100% is spent, the eluent of 70% methanol-water solution is collected, used after evaporated under reduced pressure
Silica gel post separation, is 10 with volume ratio:1-0:1 n-hexane-ethyl acetate gradient elution, obtains component F7F2A-F7F2H；It is right
F7F2E components are prepared reversed-phase column (C185 μm of 10 × 250mm) separate, washed so that acetonitrile-aqueous solution that concentration is 50% is isocratic
It is de-, obtain formula (II) and formula (VI) compound；F7G sections, first through Sephadex LH-20 gel post separations, is eluted with absolute methanol
The flow point for obtaining again through purification on normal-phase silica gel post separation, using volume ratio 10:1-0:1 cyclohexane-ethyl acetate gradient elution, obtains
Component F7G3A-F7G3H；F7G3E sections is washed through the anti-phase post separations of RP-18, the methanol-water solution gradient with concentration as 50-100%
It is de-, the eluent of 70-80% methanol-water solutions is collected, washed so that acetonitrile-aqueous solution that concentration is 45% is isocratic after evaporated under reduced pressure
It is de-, obtain formula (IV), formula (VII), formula (VIII I), formula (Ⅸ) and formula (Ⅹ) compound；Purification on normal-phase silica gel post separation is used to F7H sections,
It is 10 to use volume ratio:1-0:1 cyclohexane-acetone gradient elution, obtains component F7H1-F7H8；F7H2 sections anti-through RP-18 again
Phase post separation, the methanol-water solution gradient elution with concentration as 50-100% collects the methanol-water solution of 60-80%, decompression
Used after being evaporated and prepare reversed-phase column (C185 μm of 10 × 250mm) separate, with the acetonitrile-aqueous solution isocratic elution that concentration is 46%,
Obtain formula (V) compound.
Embodiment 6
A, Roots of Euphorbia soongarica herb 10kg is taken, with the methanol-water solution that 100L concentration is 90% cold soaking at room temperature after crushing
Extract, evaporated under reduced pressure solvent obtains Roots of Euphorbia soongarica crude extract；
B, the crude extract for obtaining step a are disperseed with petroleum ether, add acetonitrile to be extracted, and merge acetonitrile layer and steaming of depressurizing
It is dry to obtain acetonitrile extraction thing medicinal extract；
C, the acetonitrile extraction thing medicinal extract purification on normal-phase silica gel post separation for obtaining step b, are 100 with volume ratio:1-0:1 ring
Hexane-ethylacetate carries out gradient elution, and flow point merges identical flow point, obtains 12 components (F1-F12) through TLC analyses；By group
Dividing F6 carries out purification on normal-phase silica gel post separation, is 40 with volume ratio:1-0:1 n-hexane-ethyl acetate gradient elution, obtains component
F6A-F6F；By F6C sections of component through preparing reversed-phase column (C185 μm of 10 × 250mm) separate, the methyl alcohol with concentration as 60-100%-
Aqueous solution gradient elution, obtains formula (I) and formula (III) compound；Purification on normal-phase silica gel post separation is carried out by F7 sections, using volume ratio
100:9:1-0:0:1 petroleum ether-dichloromethane-acetone gradient elution, obtains component F7A-F7H；F7F sections anti-phase through RP-18
Post separation, the methanol-water solution gradient elution with concentration as 30-100% collects the eluent of 70% methanol-water solution, decompression
Used after being evaporated and prepare reversed-phase column (C185 μm of 10 × 250mm) separate, with the acetonitrile-aqueous solution isocratic elution that concentration is 50%,
Obtain formula (II) and formula (VI) compound；F7G sections first through Sephadex LH-20 gel post separations, with volume ratio 1:1 chloroform-
The flow point that methyl alcohol is afforded again through purification on normal-phase silica gel post separation, using volume ratio 10:1-0:1 n-hexane-ethyl acetate gradient is washed
It is de-, obtain component F7G3A-F7G3H；F7G3E sections through the anti-phase post separations of RP-18, the methanol-water solution with concentration as 30-100%
Gradient elution, collects the eluent of 70-80% methanol-water solutions, with acetonitrile-aqueous solution that concentration is 44% etc. after evaporated under reduced pressure
Degree wash-out, obtains formula (IV), formula (VII), formula (VIII), formula (Ⅸ) and formula (Ⅹ) compound；To F7H sections using normal phase silicagel column point
From it is 10 to use volume ratio:1-0:1 petroleum ether-acetone gradient elution, obtains component F7H1-F7H8；F7H2 sections again through RP-
18 anti-phase post separations, the acetonitrile-aqueous solution gradient elution with concentration as 30-100% collects the acetonitrile-aqueous solution of 40-60%,
Used after evaporated under reduced pressure and prepare reversed-phase column (C185 μm of 10 × 250mm) separate, it is isocratic with methanol-water solution that concentration is 65%
Wash-out, obtains formula (V) compound.
Embodiment 7
Cell toxicant and the anti-drug-resistance activity test of formula (I)-formula (Ⅹ) compound：
Material and reagent：The culture mediums of RPMI 1640 are purchased from Invitrogen companies；Dual anti-and hyclone is purchased from
Hyclone companies；Trypsase is purchased from Gibco companies；Tetrazolium bromide (MTT) is purchased from Shanghai Yi Fei bio tech ltd；Two
Methyl sulfoxide (DMSO) is purchased from Chinese medicines group Chemical Co., Ltd.；Rhodamine 123 is purchased from SIGMA companies；Verapamil hydrochloride is purchased
From Aladdin Chemical Co., Ltd.；Taxol is purchased from Biocompounds companies；Vinorelbine tartrate wins auspiciousization purchased from Shanghai
Learn Science and Technology Ltd..
Cell line：Human mouth squamous cell carcinoma KB cells and its vincristine persister KBv200 cells are (public purchased from ATCC
Department).
Cell culture：Human mouth squamous cell carcinoma KB cells and its vincristine persister KBv200 cells are in RPMI
Culture in 1640 complete mediums (culture medium+10%FBS+1% of RPMI 1640 are dual anti-).All cells are placed in CO2Incubator
(37 DEG C of temperature, 5%CO2) maintain Secondary Culture.Persister (KBv200 cells) is trained in the complete medium without antineoplastic
For testing after supporting two weeks.
Experimental technique：, be inoculated with for KB the or KBv200 cells in exponential phase with the density in 2500/hole by mtt assay
In in 96 hole microtest plates, added for examination monomeric compound (20 μ L/ holes) after 37 DEG C of incubators of temperature are incubated 12h；Separately set
Blank control group, solvent (DMSO) control group and positive drug control group；Tumour cell is in 37 DEG C of temperature, 5%CO2Under the conditions of train
Abandoning supernatant after 72h is supported, MTT liquid (5mg/mL, with normal saline, 20 μ L/ holes) is added, in 37 DEG C of temperature, 5%CO2
Under the conditions of continue cultivate 1h；Abandoning supernatant, 100 μ L DMSO are added per hole, after after formazan dissolving, each hole are detected with ELIASA
The absorbance (A) of 550nm；Inhibiting rate (the half suppression for examination monomeric compound to growth of tumour cell is calculated by following equation
Amount IC processed50Value is calculated using Logit methods) and resistance coefficient：Inhibiting rate (%)=(A control group-A administration groups)/A control groups ×
100%；Resistance coefficient (RF)=IC50(KBv200)/IC50(KB)。
Experimental result：The half growth inhibition ratio of the formula (I) got in Roots of Euphorbia soongarica-formula (Ⅹ) compound, is shown in Table 6：
Half growth inhibition ratio of the Roots of Euphorbia soongarica Chinese style (I) of table 6.-formula (Ⅹ) compound to KB and KBv200 cells
As seen from the above table, formula (II), formula (V), formula (VII), formula (VIII), formula (Ⅹ) compound are equal to KB and KBv200 cells
There is weaker cytotoxicity (IC5010.34-22.94 μM of scope), and it is little to the inhibitory activity difference of KB and KBv200 cells
(resistance coefficient range is in 0.90-1.19), shows that mdr cell KBv200 is equally sensitive to these compounds.Other compounds are not
Show cytotoxicity (IC5030 μM of ＞).
Embodiment 8.
The multi-medicine tolerant reversal activity test of formula (I)-formula (Ⅹ) compound：
Experimental technique：Rhodamine 123 intracellular accumulation tests (Flow cytometry), and the model can show with P- sugar indirectly
Protein drug efflux pump is the Reversal activity of the tumor multi-drug resistance reversal agents of target spot.If Rhodamine 123 intracellular accumulation is relative
Value is bigger, shows that it accumulates more in the cell, then its multi-medicine tolerant reversal activity is stronger.KBv200 cells are pressed 15 × 104/
The density of mL is inoculated in 24 orifice plates, per hole 1mL, overnight so that adherent；When cell growth to 70-80% is merged, added per hole
After monomeric compound (final concentration of 10 μM) to be measured 4h is incubated in 37 DEG C of incubators of temperature；If blank control group, solvent (DMSO)
Control group and positive drug control group (Verapamil, 10 μM)；Then Rhodamine 123 (10 μM of final concentration) is added per hole after temperature
37 DEG C of incubator lucifuges of degree are incubated 1h；Culture medium is discarded, after washing 2 times with 1 × PBS of 1mL ice (PBS) per hole,
Cell is collected in digestion；Per hole with 300 μ L PBS it is resuspended be placed on ice, lucifuge, with flow cytomery (excitation wavelength 488nm,
Launch wavelength 530nm).Rhodamine 123 intracellular accumulation relative value is calculated by following equation.
Rhodamine 123 intracellular accumulation relative value=(administration group average fluorescent strength-blank control group mean fluorecence is strong
Degree)/(solvent control group average fluorescent strength-blank control group average fluorescent strength).
Experimental result：The Rhodamine 123 intracellular accumulation phase of the formula (I) got in Roots of Euphorbia soongarica-formula (Ⅹ) compound
7 are shown in Table to value：
The Rhodamine 123 intracellular accumulation relative value of the formula (I) got in the Roots of Euphorbia soongarica of table 7-formula (Ⅹ) compound
As shown above, formula (IV), formula (V), formula (VI), formula (VII), formula (VIII) compound Rhodamine 123 it is intracellular
Accumulation relative value is all higher than 1, i.e. these compounds can to some extent suppress the drug efflux effect of P-gp mediations, Ye Jixian
Different degrees of multi-medicine tolerant reversal activity is shown.
Embodiment 9
Formula (VIII) compound reverse multiple drug resistance of tumor active testing：
The best formula (VIII) of P- glycoprotein inhibitory activity in the terpenoid got in this experimental selection Roots of Euphorbia soongarica
Compound is combined with antineoplastic vinorelbine (NVB), to the growth inhibition of mdr cell before and after detection combination, carries out multiple medicine resistance to
Medicine Reversal activity is tested.
Experimental technique：KBv200 cells in exponential phase are inoculated in 96 holes with the density in 2500/hole micro
Culture plate (per the μ L of hole 160), adds vinorelbine (20 μ L/ holes) and for examination monomer chemical combination after being incubated 24h in 37 DEG C of incubators
Thing or positive control drug Verapamil (20 μ L/ holes), each concentration is 3 multiple holes, and sets blank control group and solvent
(DMSO) control group；Tumour cell is in 37 DEG C of temperature, 5%CO2Under the conditions of cultivate 72h after abandoning supernatant, MTT is complete with cell
Full nutrient solution presses 1:After 10 ratio is mixed, 100 μ L are added per hole, in 37 DEG C of temperature, 5%CO2Under the conditions of continue cultivate 1h after
Abandoning supernatant, 100 μ L DMSO are added per hole, after after formazan dissolving, the absorbance of each hole 570nm are detected with ELIASA
(A) inhibiting rate, is calculated by the formula in 5.2.2, and reversal index is calculated by following equation.
Reversal index=IC50(vinorelbine)/IC50(vinorelbine+diterpene-kind compound)
Experimental result：Formula (VIII) compound and vinorelbine are combined to the half growth inhibition ratio of KBv200 cells and its inverse
Turn multiple and be shown in Table 8：
The formula of table 8 (VIII) compound is combined the half growth inhibition ratio to KBv200 cells with vinorelbine
As seen from the above table, vinorelbine with formula (VIII) compound combination after compared with vinorelbine independent role, IC50Value is aobvious
Writing reduces, and reduction degree represents that the reversal index (225.35 times) of 3 μM formula (VIII) compound is and 10 μM of sun with reversal index
Property comparison medicine Verapamil quite (259.46 times), i.e. formula (VIII) compound there is the work of very strong reverse multiple drug resistance of tumor
Property.
Claims (7)
1. the terpenoid in a kind of Roots of Euphorbia soongarica, it is characterised in that the compound is diterpene-kind compound and triterpenes
The structural formula of compound, wherein diterpene-kind compound is：
Formula (I) compound is 3 β-benzoyloxy -5,6- epoxies-with the continuous sub- β of alkane -12E- alkene -7,15 beta-diol -14- ketone；
Formula (II) compound is 3 β-benzoyloxy-with continuous sub- alkane -5E, the β of 12E- diene -7,15 beta-diol -14- ketone；
Formula (III) compound is Jatropha curcas alkane -4E, the β of 6 (17)-diene -13,14 beta-diol -3- ketone；
Formula (IV) compound is the sweet blue or green α of euphorbia B alkane -5 of 12 α-benzene acryloyl amido -3 β-acetoxyl groups -, 15 beta-diol -14- ketone；
Formula (V) compound is 12 α-benzoyloxy-sweet blue or green β of euphorbia B alkane -4Z- alkene -3,5 salmefamol -14- ketone；Triterpenes
The structural formula of compound is：
Formula (VI) compound drops the-β -ol -7- ketone -24- aldehyde of euphane -8Z, 22E- diene -3 for 25,26,27- tri-；
Formula (VII) compound is the β -ol -7,25- diketone of 27- drop-euphanes -8Z, 23E- diene -3；
Formula (VIII) compound is 24R- euphanes -8Z, 25E- diene -3 β, 24- glycol -7- ketone；
Formula (Ⅸ) compound drops the-β -ol -7,20- diketone of euphane -8Z- alkene -3 for 22,23,24,25,26,27- six；
Formula (Ⅹ) compound is 7,15- dioxies-D:The β -ol of C-friedo- oleanane -8Z- alkene -3.
2. the preparation method of terpenoid according to claim 1 in Roots of Euphorbia soongarica, it is characterised in that by following step
Suddenly carry out：
A, with Roots of Euphorbia soongarica herb as raw material, it is the ethanol water of 50-99%, nothing to measure volumetric concentration with 5-10 times after crushing
Water-ethanol, volumetric concentration are the methanol aqueous solution of 50-99%, absolute methanol, volumetric concentration for 50-99% aqueous acetone solution or
Acetone carries out diacolation or cold soaking extraction at room temperature, or heating and refluxing extraction, is concentrated to give Roots of Euphorbia soongarica crude extract；
B, the crude extract for obtaining step a are disperseed with acetonitrile, add hexamethylene, n-hexane or petroleum ether extraction, or by crude extract
Disperseed with hexamethylene, n-hexane or petroleum ether, add acetonitrile extraction 1-5 times, by the concentration of acetonitrile extraction liquid, obtain acetonitrile extraction
Thing medicinal extract；
C, the acetonitrile extraction thing medicinal extract for obtaining step b are through normal-phase silica gel column chromatography, reversed-phase silica gel column chromatography, Sephadex LH-
Separated for two or three in 20 gel filtration chromatography methods, that is, obtained formula (I)-formula (Ⅹ) compound.
3. the preparation method of terpenoid according to claim 2 in Roots of Euphorbia soongarica, it is characterised in that institute in step c
It is normal pressure or pressurized column chromatography with normal-phase silica gel column chromatography method, filler used is silica gel, and eluant, eluent used is petroleum ether, hexamethylene
The mixture of at least two solvents in alkane, n-hexane, acetone, chloroform, ethyl acetate, methyl alcohol, using isocratic elution or ladder
Degree wash-out.
4. the preparation method of terpenoid according to claim 2 in Roots of Euphorbia soongarica, it is characterised in that institute in step c
Be normal pressure or pressurized column chromatography with reversed-phase silica gel column chromatography method, eluant, eluent be volumetric concentration for 30-99% methanol aqueous solution,
The acetonitrile solution of 10-99% or the acetonitrile-methanol-water mixed solution of 10-99%, using isocratic elution or gradient elution.
5. the preparation method of terpenoid according to claim 2 in Roots of Euphorbia soongarica, it is characterised in that institute in step c
It is normal pressure column chromatography with Sephadex LH-20 gel filtration chromatography methods, eluant, eluent is methyl alcohol, acetone, dichloromethane, chloroform
Or in them at least two solvents mixture, using isocratic elution or gradient elution.
6. formula (IV) in terpenoid according to claim 1 in Roots of Euphorbia soongarica and formula (V) compound, formula
(VI), the purposes of formula (VII) and formula (VIII) compound in multi-drug resistance reversing medicaments are prepared.
7. formula (IV) in terpenoid according to claim 1 in Roots of Euphorbia soongarica and formula (V) compound, formula
(VI), the purposes of formula (VII) and formula (VIII) compound in the medicine for preparing antitumor combination.
Priority Applications (1)
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Cited By (6)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN107827752A (en) * | 2017-11-02 | 2018-03-23 | 中国科学院新疆理化技术研究所 | To the macrocyclic diterpene compounds in the fruit of the leaf root of Beijing euphorbia and preparation method and multi-medicine tolerant reversal purposes |
| CN108358993A (en) * | 2018-03-26 | 2018-08-03 | 海南师范大学 | A kind of new diterpene-kind compound and its preparation method and application |
| CN108992443A (en) * | 2018-07-23 | 2018-12-14 | 昆明理工大学 | Application of Jolkinol type (II -2a) a thousand pieces of gold alkane type diterpene in reverse multiple drug resistance of tumor |
| CN112266401A (en) * | 2020-09-24 | 2021-01-26 | 中国科学院昆明植物研究所 | Compound euphosphorin F and application thereof in preparation of tomato yellow leaf curl virus resistant drugs |
| CN113248384A (en) * | 2021-05-20 | 2021-08-13 | 沈阳药科大学 | Euphorbia lathyris diterpene alkane type compound and preparation method and application thereof |
| CN113968837A (en) * | 2021-11-12 | 2022-01-25 | 中国科学院兰州化学物理研究所 | Compound with antiepileptic activity and application thereof in preparing antiepileptic medicine |
Citations (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| WO2006116897A1 (en) * | 2005-04-30 | 2006-11-09 | Nihon University | Diterpenes from euphorbia kansui and their use |
| CN104292186A (en) * | 2014-09-27 | 2015-01-21 | 中国科学院新疆理化技术研究所 | Macrocyclic diterpene compound separated from euphorbia macrorrhiza C.A.Mey and multi-drug resistance reversing application |
| CN104387344A (en) * | 2014-09-27 | 2015-03-04 | 中国科学院新疆理化技术研究所 | Macrocyclic diterpenoid compounds separated from euphorbia macrorrhiza C.A.Mey and application |
| CN104622865A (en) * | 2013-11-14 | 2015-05-20 | 中国科学院上海药物研究所 | Application of ingenane diterpene compound in preparation of antitumor drug |
-
2016
- 2016-11-24 CN CN201611042551.XA patent/CN106749107B/en active Active
Patent Citations (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| WO2006116897A1 (en) * | 2005-04-30 | 2006-11-09 | Nihon University | Diterpenes from euphorbia kansui and their use |
| CN104622865A (en) * | 2013-11-14 | 2015-05-20 | 中国科学院上海药物研究所 | Application of ingenane diterpene compound in preparation of antitumor drug |
| CN104292186A (en) * | 2014-09-27 | 2015-01-21 | 中国科学院新疆理化技术研究所 | Macrocyclic diterpene compound separated from euphorbia macrorrhiza C.A.Mey and multi-drug resistance reversing application |
| CN104387344A (en) * | 2014-09-27 | 2015-03-04 | 中国科学院新疆理化技术研究所 | Macrocyclic diterpenoid compounds separated from euphorbia macrorrhiza C.A.Mey and application |
Non-Patent Citations (3)
| Title |
|---|
| JIE GAO 等: "Terpenoids from Euphorbia soongarica and Their Multidrug Resistance Reversal Activity", 《J. NAT. PROD.》 * |
| WEI LI 等: "New lanostane-type triterpenoids from the fruiting body of Ganoderma hainanense", 《FITOTERAPIA》 * |
| YE TIAN 等: "Lathyrane Diterpenoids from the Roots of Euphorbia micractina and Their Biological Activities", 《J. NAT. PROD.》 * |
Cited By (7)
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| CN107827752A (en) * | 2017-11-02 | 2018-03-23 | 中国科学院新疆理化技术研究所 | To the macrocyclic diterpene compounds in the fruit of the leaf root of Beijing euphorbia and preparation method and multi-medicine tolerant reversal purposes |
| CN107827752B (en) * | 2017-11-02 | 2020-07-28 | 中国科学院新疆理化技术研究所 | Macrocyclic diterpenoid compound in euphorbia foliosa fruits, preparation method and multi-drug resistance reversing application |
| CN108358993A (en) * | 2018-03-26 | 2018-08-03 | 海南师范大学 | A kind of new diterpene-kind compound and its preparation method and application |
| CN108992443A (en) * | 2018-07-23 | 2018-12-14 | 昆明理工大学 | Application of Jolkinol type (II -2a) a thousand pieces of gold alkane type diterpene in reverse multiple drug resistance of tumor |
| CN112266401A (en) * | 2020-09-24 | 2021-01-26 | 中国科学院昆明植物研究所 | Compound euphosphorin F and application thereof in preparation of tomato yellow leaf curl virus resistant drugs |
| CN113248384A (en) * | 2021-05-20 | 2021-08-13 | 沈阳药科大学 | Euphorbia lathyris diterpene alkane type compound and preparation method and application thereof |
| CN113968837A (en) * | 2021-11-12 | 2022-01-25 | 中国科学院兰州化学物理研究所 | Compound with antiepileptic activity and application thereof in preparing antiepileptic medicine |
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