CN109897084B

CN109897084B - Cardiac glycoside compound and preparation method and application thereof

Info

Publication number: CN109897084B
Application number: CN201711281111.4A
Authority: CN
Inventors: 姜苗苗; 曹远林; 卢云芳; 李晨阳; 张鹏; 王萌; 丁丽琴
Original assignee: Tianjin University of Traditional Chinese Medicine
Current assignee: Tianjin University of Traditional Chinese Medicine
Priority date: 2017-12-07
Filing date: 2017-12-07
Publication date: 2021-09-24
Anticipated expiration: 2037-12-07
Also published as: CN109897084A

Abstract

The cardiac glycoside compound 14 beta-hydroxy-5 beta, 14 beta-cardenolide-8, 16,20(22) -triene-3-O-beta-D-git Guttiferae glycoside is a new cardiac glycoside compound discovered by separation and extraction from Nerium oleander, and is successfully separated by methods of cold soaking extraction, macroporous resin column chromatography, silica gel column chromatography, ODS column chromatography, gel column chromatography, preparative high performance liquid phase and the like, has an anti-tumor effect, can be used as a precursor of other compounds, as a raw material for new drug development and pharmacological activity research, and can also be used for preparing anti-tumor drugs.

Description

Cardiac glycoside compound and preparation method and application thereof

Technical Field

The invention relates to a compound separated and extracted from Nerium oleander Linn, a preparation method and application thereof, in particular to a cardiac glycoside compound, and a preparation method and application thereof.

Background

The cardiac glycoside is steroid glycoside with obvious physiological activity to heart, has wide pharmacological action, cardiac, cytotoxic, anticancer, diuretic and other bioactivity, and is used in treating congestive heart failure, dysrhythmia and other cardiac diseases. In recent years, it has been found that cardiac glycosides have Na-inhibiting activity⁺/K⁺-ATPase inhibition, apoptosis and found to have better anticancer activity. Leading an increasing number of researchers to cardiac glycosides. The cardiac glycoside is mainly distributed in the genus of digitalis of Scrophulariaceae, the genus of Thevetia and Nerium of Apocynaceae, the genus of Periploca, the genus of Maries, the genus of Convallaria, the genus of Rohdea, the genus of Hyssopus, the genus of Adonis, etc., and is mainly present in the fruit, leaf or root of the plant.

Nerium oleander Linn, is a medical record of Nerium oleander in Nerium oleaceae, and leaves and skins of Nerium oleander can be used as medicines, and is bitter in taste, cold in nature and toxic. The medicine has effects of tonifying heart, relieving pain, and invigorating heart meridian, and has effects of tonifying heart, promoting urination, resisting inflammation, inhibiting bacteria, relieving pain, tranquilizing mind, and resisting tumor. The oleander contains cardiac glycosides, triterpenes, pregnanes, alkaloids and oil compounds. In addition, other compounds such as phenolic acid compounds, flavonoid glycoside compounds, sterol compounds, and the like are also included. In addition, the oleander also has the effects of greening, environmental protection, biological prevention, chemical feeling and the like. One of the major components of oleander europaea is a cardiac glycoside compound which has the effects of cardiac, bacteriostasis, analgesia, sedation and tumor resistance, and other related effects and action mechanisms are yet to be further discovered. Cardiac glycoside compounds with specific binding of Na⁺/K⁺ATPase and inhibition of its function, rendering intracellular K⁺Reduction of Na⁺Increase, in turn, cell membrane voltage-gated Ca²⁺Channel activation and Na⁺-Ca²⁺Exchanger activation, resulting in intracellular Ca²⁺The persistent rise makes the cell apoptosis.

Disclosure of Invention

The technical problem to be solved by the invention is to provide a cardiac glycoside compound.

Another object of the present invention is to provide a method for preparing the cardiac glycoside compound.

Another object of the present invention is to provide an application of the cardiac glycoside compound.

In order to solve the technical problems, the technical scheme of the invention is as follows:

a cardiac glycoside compound (cardiac glycoside compound D) is 14 beta-hydroxy-5 beta, 14 beta-cardsta-8, 16,20(22) -triene-3-O-beta-D-git Guttingoside (3 beta-O- (beta-D-diginosyl) -14 beta-hydroxy-5 beta, 14 beta-card-8, 16,20(22) -trienolide) with the structural formula of formula (I),

the cardiac glycoside compounds: white amorphous powder (chloroform/methanol),

(c0.30,MeOH)；(-)-ESI-MS:m/z513.2847[M-H]^-、(+)-ESI-MS:m/z553.3317[M+H₂O+H]⁺(ii) a The nuclear magnetic data of hydrogen spectrum and carbon spectrum are shown in the table 2 in the specific embodiment part.

The preparation method of the cardiac glycoside compound comprises the following specific steps:

(1) drying and pulverizing fresh folium Nerii (20kg per leaf), soaking in 450L (95% (v/v) ethanol for 14 days, and distilling the filtrate under reduced pressure to obtain concentrated solution 5L;

(2) adding petroleum ether with the same volume into the obtained 5L condensate for extraction, repeatedly extracting for 4 times, combining the extracts, and distilling under reduced pressure to obtain two fractions: water layer, petroleum ether layer;

(3) separating the fraction of the water layer by using a D101 macroporous resin column, performing gradient elution by using water, 30 percent ethanol, 60 percent ethanol and 95 percent ethanol respectively, and performing reduced pressure distillation respectively to obtain four parts;

(4) the 60% ethanol fraction was purified by silica gel column chromatography, dichloromethane: methanol 100:0-0: gradient elution is carried out by a 100 solvent system to obtain 11 parts Fr.1-11;

(5) petroleum ether: ethyl acetate 100:0-0: eluting with 100 solvent parts-Fr.1, gradient eluting, passing through silica gel column, detecting by thin layer chromatography, developing, and mixing the same parts to obtain 8 parts Fr.1 (1-8);

(6) dichloromethane: eluting with methanol (100: 0-0: 100) solvent (Fr.1-6), and separating by silica gel column chromatography to obtain 5 α -oleandrin A (5 α -oleaside A);

(7) petroleum ether: ethyl acetate 100:0-0: eluting with 100 solvents, collecting fraction Fr.2, detecting by silica gel column chromatography and thin layer chromatography, developing, and mixing the same fractions to obtain 5 fractions Fr.2 (1-5);

(8) and (3) performing gradient elution on the flow portion Fr.2-4 through an ODS column, eluting a solvent methanol water, and performing preparative high performance liquid chromatography on the obtained flow portion Fr.2-4: eluting with a solvent of 60:40, and separating to obtain a target compound 14 beta-hydroxy-5 beta, 14 beta-cardenolide-8, 16,20(22) -triene-3-O-beta-D-git Gugitol glycoside (3 beta-O- (beta-D-diginosyl) -14 beta-hydroxy-5 beta, 14 beta-card-8, 16,20(22) -trienolide); and (3) performing gradient elution on the flow portion Fr.2-5 through an ODS column, eluting a solvent methanol water, and performing preparative high performance liquid chromatography on the obtained flow portion Fr.2-5: eluting with a solvent of 45 parts by weight of 55 parts by weight of water, and separating to obtain 8 beta-hydroxy oleandrin 3-O-beta-D-git Gugital glucoside (8-hydroxy-oleandrigen-3-O-beta-D-diginoside); or subjecting the obtained fractions to preparative high performance liquid chromatography methanol: eluting with a solvent of 65:35 to obtain 14-carbonyl nereid (14-carbanyl-neriaside).

The cardiac glycoside compounds obtained by the above method belong to type A cardiac glycoside, C of steroid nucleus of cardiac aglycone₁₀，C₁₃Are all connected with beta CH₃,C₁₇The side chain is alpha, beta-unsaturated-gamma-pentalactone ring and is in C₃，C₁₄The positions are all substituted by hydroxyl and are all in beta-configuration; high strengthThe four rings of the steroid parent nucleus of the aglycon are fused in a way that B/C rings are all trans, C/D rings are all cis, and A/B two fusion modes are available. Trans-condensed A/B ring, D-gitter sugar and aglycone C₃-OH is combined to form glycoside (B); also cis-condensed with the A/B ring, D-gitter sugar and aglycone C₃OH to form a glycoside (A, C, D), C₁₆Substituted by acetoxy or C₈Site hydroxylation (A), C₈，C₉Form a double bond between them, C₁₆，C₁₇Form a double bond (D) therebetween, and the carbon-carbon bond at the 8, 14-position is broken to form a carbonyl group or a hydroxyl group (C). The chemical structure is characterized in that: cardiac glycoside A mother nucleus C₁₇The side chain is alpha, beta-unsaturated-gamma-five-membered lactone ring; the 8, 9-position and the 16, 17-position form a double bond; or C₈Site hydroxylation, C₁₆Substituted in position by acetoxy; the condensed mode of the A/B ring is trans or cis; the carbon-carbon bond at the 8, 14-position is broken to form a carbonyl group or a hydroxyl group; with aglycone C₃The monosaccharides that combine to form the glycoside at the-OH groups are all β -D-diginose.

The cardiac glycoside compound is applied in preparing antitumor medicine.

An antitumor pharmaceutical composition comprises a therapeutically effective amount of the cardiac glycoside compound and optionally pharmaceutically acceptable excipients.

The invention has the beneficial effects that:

the cardiac glycoside compound is obtained by successfully separating a new cardiac glycoside compound discovered by separating and extracting from Nerium oleander by adopting methods of cold immersion extraction, macroporous resin column chromatography, silica gel column chromatography, ODS column chromatography, gel column chromatography, preparative high performance liquid chromatography and the like, has an anti-tumor effect, can be used as a precursor of other compounds, and a raw material for new drug development and pharmacological activity research, and can also be used for preparing anti-tumor drugs.

Drawings

FIG. 1 is a chemical structural formula of known

compounds

1, 2, 3;

FIG. 2 is a drawing of Compound A¹H-NMR spectrum;

FIG. 3 is a drawing of Compound A¹³A C-NMR spectrum;

FIG. 4 is an HMBC spectrum of compound A;

FIG. 5 is a ROESY spectrum of Compound A;

FIG. 6 is a drawing of Compound B¹H-NMR spectrum;

FIG. 7 is a drawing of Compound B¹³A C-NMR spectrum;

FIG. 8 is an HMBC spectrum of compound B;

FIG. 9 is a NOESY spectrum of Compound B;

FIG. 10 is of Compound C¹H-NMR spectrum;

FIG. 11 is of Compound C¹³A C-NMR spectrum;

FIG. 12 is an HMBC spectrum of compound C;

FIG. 13 is a NOESY spectrum of Compound C;

FIG. 14 is of Compound D¹H-NMR spectrum;

FIG. 15 is of Compound D¹³A C-NMR spectrum;

FIG. 16 is an HMBC spectrum of compound D;

figure 17 is the NOESY spectrum of compound D.

Wherein the content of the first and second substances,

A:8-hydroxy-oleandrigenin-3-O-β-D-diginoside；

B:5α-oleaside A；

C:14-carbanyl-neriaside；

D:3β-O-(β-D-diginosyl)-14β-hydroxy-5β,14β-card-8,16,20(22)-trienolide。

Detailed Description

In order to make those skilled in the art better understand the technical solution of the present invention, the following detailed description will be given with reference to specific embodiments.

Laboratory instruments and reagents: fourier transform nuclear magnetic resonance spectrometer (Bruker, Switzerland, AVIII type 600 Hz); color developing agent: 10% sulfuric acid ethanol.

Example 1

Preparation of cardiac glycoside compounds (extraction and separation process)

Fresh leaves (20Kg) of oleander are collected from Anhui Hefei in 2014 7 months. Drying and pulverizing fresh folium Nerii (20kg per leaf), cold soaking in 450L of 95% (v/v) ethanol for 14 days, and distilling the filtrate under reduced pressure to obtain 5L of Nerium indicum concentrated solution; adding petroleum ether with the same volume into the obtained 5L condensate for extraction, repeatedly extracting for 4 times, combining the extracts, and distilling under reduced pressure to obtain two fractions: water layer (600g), petroleum ether layer (300 g); separating the fraction of the water layer (600g) by using a D101 macroporous resin column, performing gradient elution by using water, 30 percent ethanol, 60 percent ethanol and 95 percent ethanol respectively, and then performing reduced pressure distillation respectively to obtain four fractions; subjecting the 60% ethanol elution fraction (85g) to silica gel column chromatography (sample-mixed silica gel 100-; petroleum ether: ethyl acetate 100:0-0: eluting with 100 solvent parts-Fr.1, gradient eluting, passing through silica gel column, detecting by thin layer chromatography, developing, and mixing the same parts to obtain 8 parts Fr.1 (1-8); dichloromethane: methanol 100:0-0: eluting with 100 parts of solvent, namely Fr.1-6, and separating by silica gel column chromatography to obtain 5 alpha-oleaside A; petroleum ether: ethyl acetate 100:0-0: eluting with 100 solvents, collecting fraction Fr.2, detecting by silica gel column chromatography and thin layer chromatography, developing, and mixing the same fractions to obtain 5 fractions Fr.2 (1-5); and (3) performing gradient elution on the flow portion Fr.2-4 through an ODS column, eluting a solvent methanol water, and performing preparative high performance liquid chromatography on the obtained flow portion Fr.2-4: eluting with a solvent of 60:40, and separating to obtain 3 beta-O- (beta-D-diginosyl) -14 beta-hydroxy-5 beta, 14 beta-card-8, 16,20(22) -trienolide; and (3) performing gradient elution on the flow portion Fr.2-5 through an ODS column, eluting a solvent methanol water, and performing preparative high performance liquid chromatography on the obtained flow portion Fr.2-5: eluting with a solvent of 55:45, and separating to obtain 8-hydroxy-oleandrigen-3-O-beta-D-diginoside; or subjecting the obtained fractions to preparative high performance liquid chromatography methanol: eluting with 65:35 solvent, and separating to obtain 14-carbanyl-neriaside. Each structural formula is as follows:

wherein the content of the first and second substances,

a is 8 beta-hydroxyoleandrin 3-O-beta-D-git Guesticoside (8-hydroxy-oleandrigen-3-O-beta-D-diginoside);

b5 alpha-oleandrin A (5 alpha-oleaside A):

14-carbonyl nereid (14-carbanyl-neriaside);

14-hydroxy-5 beta, 14 beta-cardenolide-8, 16,20(22) -triene-3-O-beta-D-gitoxitoside (3 beta-O- (beta-D-diginosyl) -14 beta-hydroxy-5 beta, 14 beta-card-8, 16,20(22) -trienolide)

A, white crystals (chloroform/methanol),

(c 0.23,MeOH)；(-)-ESI-MS:m/z591.3161[M-H]^-、(+)-ESI-MS:m/z593.3326[M+H]⁺(ii) a The NMR data of the compound are shown in Table 1, and the molecular formula of the compound is determined to be C₃₂H₄₈O₁₀。

The NMR spectrum data of the compound A is compared with that of the compound Oleandriginin-3-O-beta-D-diginoside (known compounds are shown in figure 1 and table 3), and the main difference is that the methine carbon signal (delta 42.4) at the C-8 position disappears, and a connected oxygen quaternary carbon signal is shown at delta 77.1, which indicates that the 8-position of aglycone is hydroxylated.

The information on HSQC, HMBC, etc. further confirms the above presumption that the structure of the compound is shown below.

As shown in fig. 2-5, among the NOESY spectra, the relative signals according to the ROESY spectra: the chemical shifts of H-5/H-19, H-19/OH-8, OH-14/H-18, H-12 alpha/H-15 alpha and 19-position methyl signals are delta 24.4, so that the combination modes of aglycone are determined as cis-A/B, trans-B/C and cis-C/D. In addition, the H-3 proton shows a broad single peak, suggesting that H-3 is at the equatorial bond. The ROESY-related signals for H-18/H-21 and H-18/H-22 were observed due to the free rotation of the lactone ring, also indicating that H-17 is in the alpha position. The compound A is identified as 3 beta-O- (beta-D-diginosyl) -16 beta-acetoxy-8, 14-dihydroxy-5 beta, 14 beta-card-20 (22) -enolide, and is a novel compound which is not reported in the literature and is named as 8-hydroxy-olyandigenin-3-O-beta-D-diginoside through SciFinder search.

B, white amorphous powder (chloroform/methanol),

(c 0.29, MeOH), Kedde's reagent reaction red, suggesting possible cardiac glycoside type A compounds;

(-)-ESI-MS:m/z515.3007[M-H]^-、(+)-ESI-MS:m/z517.3162[M+H]⁺(ii) a The NMR data of the compound are shown in Table 1, and the molecular formula of the compound is determined to be C₃₀H₄₄O₇。

As shown in the figures 6-9 of the drawings,¹H-NMR(400MHz,in C₅D₅n) and¹³C-NMR(100MHz,in C₅D₅n) shows characteristic absorption signals of alpha, beta-unsaturated-gamma-pentalactone ring of type A cardiac glycoside: lactone carbonyl group (. delta.)_C174.5), one double bond (. delta.))_H 5.91,δ_C116.8,172.4) and a vicinal oxymethylene group (. delta.))_H 4.86,4.75,δ_C74.0), and a sugar end group signal (δ)_H 4.84,δ_C98.8). The compound is shown to be monosaccharide type A cardiac glycoside. And the compound comprises 2 acyl groups, 4 quaternary carbons, 10 methine groups, 11 methylene groups, 2 methyl groups and 1 methoxy carbon signal according to DEPT atlas judgment. Compared to the compound oleaside a (known compounds see fig. 1 and table 3), the main difference was found to be a shift in the chemical shift of the 19-position methyl signal (δ 26.4) to the high field (δ 14.0), suggesting that the aglycone a/B ring is trans-fused. Thus, compound B was identified as 3 β -O- (. beta. -D-diginosyl) -14-oxo-15(14 → 8) abeo-5 α -card-20(22) -enolide. Through SciFinder search, the compound is a novel compound which is not reported in the literature and is named as 5 alpha-oleaside A.

C, white amorphous powder (chloroform/methanol),

(c 0.70, MeOH), Kedde's reagent reaction red, suggesting possible cardiac glycoside type A compounds;

(-)-ESI-MS:m/z577.3015[M+COOH]^-、(+)-ESI-MS:m/z551.3187[M+H₂O+H]⁺(ii) a The NMR data of the compound are shown in Table 1, and the molecular formula of the compound is determined to be C₃₀H₄₄O₈. As shown in the figures 10-11 of the drawings,¹H-NMR(400MHz,in C₅D₅n) and¹³C-NMR(100MHz,in C₅D₅n) shows characteristic absorption signals of alpha, beta-unsaturated-gamma-pentalactone ring of type A cardiac glycoside: lactone carbonyl group (. delta.)_C174.4), one double bond (. delta.))_H 6.34,δ_C118.1,170.6) and a vicinal oxymethylene group (. delta.))_H 5.10,5.16,δ_C74.3), and sugar end group signal (. delta.))_H 4.77,δ_C99.9), suggesting that the compound is also a monosaccharide cardiac glycoside a. The compound includes 3 acyl groups, 3 quaternary carbons, 9 methine groups, 11 methylene groups, 3 methyl groups, and 1 methoxy carbon signal, as judged by binding to the DEPT profile. The main difference compared to the compound neriaside (known compounds see FIG. 1 and Table 3) is the methine signal (. delta.) of the oxygen linkage in the C-14 position_H4.20,δ_C79.9) vanish, and at δ_C219.9 shows a carbonyl signal, suggesting that the hydroxyl group at the 14-position of aglycone is oxidized to a carbonyl group. As shown in fig. 13, according to the relative signals in the NOESY profile: the chemical shifts of H-5/H-19, H-19/H-11, H-11/H-18, H-12 alpha/H-15 alpha and 19-methyl signals are delta 24.4, so that the combination modes of aglycone are trans-A/B, trans-B/C and cis-C/D. Thus, compound C was identified as 3 β -O- (. beta. -D-diginosyl) -8,14-oxo-8,14-sec-5 β -card-20(22) -enolide. Through SciFinder search, the compound is a novel compound which is not reported in the literature and is named as 14-carbanyl-neriaside.

As shown in fig. 12, the information of HSQC, HMBC and the like further confirmed the above presumption that the structures of the compounds are as follows.

D, white amorphous powder (chloroform/methanol),

(c 0.30,MeOH)；

(-)-ESI-MS:m/z513.2847[M-H]^-、(+)-ESI-MS:m/z553.3317[M+H₂O+H]⁺(ii) a The NMR data of the compound are shown in Table 1, and the molecular formula of the compound is determined to be C₃₀H₄₂O₇。

As shown in the figures 14-15 of the drawings,¹H-NMR(600MHz,C₅D₅n) and¹³C-NMR(150MHz,C₅D₅n) shows characteristic absorption signals of alpha, beta-unsaturated-gamma-pentalactone ring of type A cardiac glycoside: lactone carbonyl group (. delta.)_C175.2), one double bond (. delta.))_H6.41,δ_C112.2,160.2) and a vicinal oxymethylene group (. delta.))_H 5.06,δ_C72.4), and sugar end group signal (. delta.))_H4.80,δ_C99.6), suggesting that the compound is also a monosaccharide cardiac glycoside a.¹³C-NMR(150MHz,C₅D₅N) the spectrum shows a 30 carbon signal, where delta_C132.3(C-8), 137.7(C-9) and 135.3(C-16), 142.6(C-17) are two sets of alkene carbon signals.

As shown in FIG. 16, in the HMBC chart, the proton signals δ 2.28(H-7), δ 1.78(H-12) and δ 01.05(H-19) are respectively associated with δ 1137.1(C-9), the proton signals δ 22.92/2.81(H-15), δ 32.28(H-7) and δ 41.49(H-18) are respectively associated with δ 132.3(C-8), indicating that a double bond exists between

positions

8 and 9, the proton signals δ 6.41(H-22), δ 2.92/2.81(H-15) and δ 1.49(H-18) are respectively associated with δ 142.6(C-17), the proton signals δ 6.17(H-16), δ 2.92/2.81(H-15) are respectively associated with δ 135.3(C-16), indicating that a double bond exists between

positions

16 and 17. As shown in FIG. 17, correlation on NOESY-based mapsSignal: the chemical shift of the signals for H-5/H-19, H-19/H-11, H-11/H-18, H-18/H-15, H-7/H-12, H-12 alpha/H-15 alpha, and the methyl group at the 19-position is delta 26.7. Therefore, the combination modes of aglycone are determined to be cis-A/B, trans-B/C and cis-C/D. In addition, the H-3 proton shows a broad single peak, suggesting that H-3 is at the equatorial bond. Binding protons on sugars¹H-¹The H-COSY related signal, the ortho-coupling constant and the chemical shift value of carbon, determine that the monosaccharide part is beta-D-diginose. According to HMBC mapping, the relevant signals H-3/C-1 'and H-1'/C-3 confirm that the sugar is linked at the 3-position of the aglycone. And (4) combining the information to obtain the structure of the compound.

Thus, compound D was identified as 3 β -O- (. beta. -D-diginosyl) -14 β -hydroxy-5 β,14 β -card-8,16,20(22) -trienolide. Through SciFinder search, the compound is a novel compound which is not reported in the literature.

TABLE 1 hydrogen and carbon spectra data for A and B

TABLE 2 hydrogen and carbon spectra data for C and D

TABLE 3 data for hydrogen and carbon spectra of known

compounds

1, 2 and 3

1. oleandrin-3-O-beta-D-git Gugitale glycoside (Oleandriginin-3-O-beta-D-diginoside)

2. Nailiside (neriaside)

3. Oleandrin A (oleaside A)

Example 2

Cytotoxic activity of the compounds of the invention against tumor cells.

Materials and reagents

Human colon cancer HCT116 cell line, human colon cancer HT29 cell line, human colon cancer SW620 cell line, human colon cancer RKO cell line and human cervical cancer HeLa cell line (ATCC, rockville, MD, usa) and human gastric cancer GT cell line (obtained from gastric cancer patients in western hospital, university of sichuan), RPMI-1640 was purchased from Hyclone corporation, usa; DMEM/High Glucose is available from Hyclone, USA; foetal Bone Serum (FBS) was purchased from Gibco, USA; double antibody (penicillin streptomycin) was purchased from HyClone, usa; MTT was purchased from Solambio, Beijing; DMSO was purchased from Solarbio, Beijing.

The method comprises the following specific steps:

(1) each monomeric compound was dissolved in DMSO to prepare a 20mM stock solution, which was diluted with a basal medium (DMEM/High Glucose or RPMI-1640) for use in the experiment.

(2) Preparation of RPMI 1640: adding 1% double antibody and 10% fetal calf serum into a minimal medium RPMI 1640(Hyclone), and mixing uniformly to obtain the final product.

(3) Preparing DMEM: adding 1% double antibody and 10% fetal bovine serum into a basic culture medium DMEM/High Glucose (Hyclone), and mixing uniformly to obtain the final product.

(4) HCT116, HT29, SW620, RKO, HeLa cell lines were purchased and cultured in RPMI 1640(Hyclone) medium containing 1% of diabody and 10% of fetal bovine serum at 37 ℃ in 5% CO₂Culturing under the condition, and taking cells in logarithmic growth phase for experiment after 3 generations.

(5) GT cell line was cultured in DMEM/High Glucose (Hyclone) medium containing 1% double antibody and 10% fetal bovine serum at 37 deg.C with 5% CO₂Culturing under the condition, and taking cells in logarithmic growth phase for experiment after 3 generations.

(6) Taking cells with good logarithmic phase growth, digesting with 0.25% trypsin to obtain single cell suspension, and adjusting concentration to 1 × 10⁵cells/mL, at 100. mu.L cell suspension per well, were seeded into 96-well plates. Each plate is provided with a cell control group, a blank group and an experimental group, and each group is provided with 3 multiple wells; transferring the mixture into an incubator for culture;

(7) after about 12 hours of inoculation, the 96-well plate was removed and the old medium in the wells was discarded. Adding 100 μ L of culture solution (DMEM/High Glucose or RPMI-1640) containing different concentrations of drugs into each well of the experimental group, wherein the concentration of the compounds is 100 μ g/mL, 10 μ g/mL, 1 μ g/mL, 0.1 μ g/mL, 0.01 μ g/mL and 0.001 μ g/mL respectively, adding 100 μ L of corresponding culture solution (DMEM/High Glucose or RPMI-1640) into each well of the control group and the blank group, and placing in an incubator for continuous culture;

(8) after 48 hours of incubation, 1mg/L MTT solution (50. mu.L per well) was added and incubated at 37 ℃ for 4 hours;

(9) after the crystals are completely dissolved in 100 mu L DMSO, measuring the OD value of absorbance at the position of 570nm of wavelength on an enzyme-labeling instrument;

(10) IC of each drug on different cells was calculated using GraphPad Prism5 software₅₀。

Results of the experiment

Compound in vitro cytotoxic activity IC₅₀The data are shown in table 4 below.

TABLE 4

The in vitro cytotoxic activity experiment of tumor cells shows that the compound A, D has obvious cytotoxic effect on the tumor cells, so that the compound has a certain anticancer effect and has the potential of being further developed into antitumor drugs.

In summary, the four new cardiac glycoside compounds separated and purified from fresh leaves of Nerium oleander Linn of apocynaceae, oleander, are successfully separated by methods such as cold-leaching extraction, macroporous resin column chromatography, silica gel column chromatography, ODS column chromatography, gel column chromatography, preparative high performance liquid chromatography, and the like, and the compounds separated by the method have high purity and have the following characteristics: cardiac glycoside A mother nucleus C₁₇The side chain is alpha, beta-unsaturated-gamma-five-membered lactone ring; the 8, 9-position and the 16, 17-position form a double bond; or C₈Site hydroxylation, C₁₆Substituted in position by acetoxy; the condensed mode of the A/B ring is trans or cis; the carbon-carbon bond at the 8, 14-position is broken to form a carbonyl group or a hydroxyl group; with aglycone C₃-the monosaccharides to which the OH groups bind to form the glycoside are all β -D-diginose; the cardiac glycoside compound A, D has obvious cytotoxic activity and anti-tumor effect, can be used for apoptosis, can be used as an anti-tumor therapeutic drug, and has important drug development value.

Example 3

The preparation method comprises the following steps: mixing cardiac glycoside compound, lactose and starch at the above ratio, sieving with 200 mesh sieve, wetting with water, drying, sieving, adding magnesium stearate, and tabletting to obtain tablet with active ingredient content of 10mg and 250mg each.

Example 4

And (3) capsule preparation: cardiac glycoside compound 20mg

188mg of galactose

Magnesium stearate 2mg

The preparation method comprises the following steps: mixing cardiac glycoside compound and galactose at the above ratio, sieving with 200 mesh sieve, adding magnesium stearate into the obtained mixture, and making into No. 2 capsule.

The detailed description of the cardiac glycoside compound, the preparation method and the application thereof with reference to the specific embodiments are illustrative and not restrictive, and several examples are listed according to the limited scope, so that variations and modifications without departing from the general concept of the present invention shall fall within the protection scope of the present invention.

Claims

1.A cardiac glycoside compound, comprising: is 14 beta-hydroxy-5 beta, 14 beta-cardenolide-8, 16,20(22) -triene-3-O-beta-D-gitoxitoside, has the structural formula shown in the formula (I),

2. the process for preparing a cardiac glycoside compound of claim 1, wherein: the method comprises the following specific steps:

(1) drying and pulverizing fresh folium Nerii (20kg per leaf), soaking in 450L of 95% ethanol for 14 days, and distilling the filtrate under reduced pressure to obtain 5L concentrated solution;

(2) adding petroleum ether with the same volume into the obtained 5L oleander concentrated solution for extraction, repeatedly extracting for 4 times, combining the extracts, and distilling under reduced pressure to obtain two fractions: water layer, petroleum ether layer;

(5) petroleum ether: ethyl acetate 100:0-0: eluting with 100 solvent parts Fr.1, gradient eluting, passing through silica gel column, detecting by thin layer chromatography, developing, and mixing the same parts to obtain 8 parts Fr.1 (1-8);

(6) dichloromethane: eluting with methanol (100: 0-0: 100) solvent Fr.1-6, and separating by silica gel column chromatography to obtain 5 α -oleandrin A;

(7) petroleum ether: ethyl acetate 100:0-0: eluting with 100 solvent parts Fr.2, detecting by silica gel column chromatography and thin layer chromatography, developing, and mixing the same parts to obtain 5 parts Fr.2 (1-5);

(8) and (3) performing gradient elution on the flow portion Fr.2-4 through an ODS column, eluting a solvent methanol water, and performing preparative high performance liquid chromatography on the obtained flow portion Fr.2-4: eluting with a solvent of 60:40, and separating to obtain the target compound 14-hydroxy-5 beta, 14 beta-cardenolide-8, 16,20(22) -triene-3-O-beta-D-gitoxitoside.

3. Use of a cardiac glycoside compound of claim 1 in the preparation of a medicament with anti-tumor activity.

4. An antitumor pharmaceutical composition characterized by: comprising a therapeutically effective amount of a cardiac glycoside compound of claim 1 and optionally a pharmaceutically acceptable excipient.