CN113321696B - Preparation method of cardiac glycoside and application of cardiac glycoside in preparation of antitumor drugs - Google Patents

Abstract

The invention discloses a preparation method of cardiac glycoside and application thereof in preparing antitumor drugs, wherein the cardiac glycoside is prepared by the following operation steps: 1) taking the whole plant of the dried streblus petiolata, adding 75% ethanol water solution, sequentially heating, refluxing and extracting, filtering, combining the filtrates, and recovering the solvent ethanol under reduced pressure to obtain an extract; 2) suspending the extract in water, adsorbing with macroporous adsorbent resin chromatographic column, washing with water, and eluting with ethanol water solution to obtain eluate rich in streblus indica cardiac glycoside. Most of the cardiac glycoside compounds provided by the invention can obviously inhibit the growth of tumor cells, have concentration dependence and can be used for developing related medicaments for inhibiting the growth of tumor cells.

Description

Preparation method of cardiac glycoside and application of cardiac glycoside in preparation of antitumor drugs

Technical Field

The invention belongs to the technical field of biological medicines, and particularly relates to a preparation method of cardiac glycoside and application of the cardiac glycoside in preparation of antitumor drugs.

Background

Cancer is a risk factor which seriously affects the health of people, the burden of cancer is continuously increased in the world in recent years, and the tumor death accounts for 1/4 of all causes of death in China and is the first death. The cancer morbidity and mortality of China will continuously rise, and the prevention and treatment tasks are extremely difficult. Cancer, as the first killer threatening human life and health, not only brings heavy burden to the family and society of patients, but also affects public safety and social stability, and is receiving increasing attention from all the world of society. Research and development and application of antitumor drugs have become an important field of rapid development of biomedical science.

The Streblus asper is a plant of the Moraceae (Moraceae) Streblus genus (Streblus Lour), and is a folk medicinal plant with multiple medicinal values. Research shows that the streblus gigas has the effects of resisting cancer, inhibiting bacteria, resisting inflammation, killing pests and the like. The chemical components of cardiac glycosides are the main active substances of streblus streblumea, and the compounds show good antitumor activity.

The cardiac glycoside obtained from the whole streblus streblumea (roots, stems, leaves) has the activity of obviously inhibiting MCC-803, T24, SKOV-3, HepG2, Wi-38 and A549 tumor cells, and can be used for developing related medicaments for inhibiting the growth of the tumor cells.

Disclosure of Invention

The invention aims to provide a preparation method of cardiac glycoside and application thereof in preparing anti-tumor drugs aiming at the defects of the prior art. The cardiac glycoside compound prepared by the method is an active monomer compound with the function of inhibiting the growth of tumor cells, has concentration dependence, and can be used for developing related medicaments for inhibiting the growth of tumor cells.

The technical scheme for realizing the purpose of the invention is as follows:

a method for preparing cardiac glycoside comprises the following steps:

1) taking a whole plant of dry streblus indica leaves, wherein the whole plant comprises roots, stems and leaves, adding an ethanol water solution, sequentially carrying out heating reflux extraction, combining filtrates, and recovering ethanol under reduced pressure to obtain an extract;

2) suspending the extract prepared in the step 1) in water, adsorbing by a macroporous adsorption resin chromatographic column, washing by water, and eluting by using an ethanol water solution to obtain an eluate rich in streblus indica cardiac glycoside.

The application of the cardiac glycoside prepared by the preparation method in preparing the antitumor drug comprises the cardiac glycoside shown in the general formula (I) or the medicinal salt thereof:

in the general formula (I), R₁、R₂、R₃Each independently represents a hydrogen atom, C₁～C₆Alkyl radical, C₁～C₆Haloalkyl, C₃～C₆Cycloalkyl, C₁～C₆Alkylcarbonyl group, R₄、R₅、R₆、R₇、R₈、R₉、R₁₀Each independently represents a hydrogen atom, C₁～C₆Alkyl radical, C₁～C₆Haloalkyl, C₃～C₆Cycloalkyl or glycosyl.

The glycosyl is monosaccharide or disaccharide.

The monosaccharide is at least one selected from the group consisting of arabinosyl, ribosyl, xylosyl, lyxosyl, glucosyl, allosyl, mannosyl, rhamnosyl, cinchona-glycosyl, fructosyl, galactosyl, apisyl, glucuronyl, and galacturonyl.

The disaccharide base is at least one selected from the group consisting of lactose base, sucrose base and maltose base.

The cardiac glycoside is any one of the compounds shown in the following structural formula 1:

wherein, the compound 1 is (3 beta, 5 alpha, 17R) -streblus streblumea heart-3-O- (2',3' -O-dimethyl) -beta-D-pyranosyl glucoside, i.e. streblus streblumea cardiac glycoside A;

the compound 2 is (3 beta, 5 beta, 17R) -streblus streblumea cardiotonic-3-O-alpha-L-rhamnopyranosyl-4' -O-beta-D-glucopyranoside, i.e. streblus cardiotonic glycoside B;

compound 3 is (3 β,5 β,17R) -streblus streblumea cardiotonic-3-O- (3' -O-methyl) - α -L-rhamnopyranosyl-4 "-O- β -D-glucopyranoside, streblus streblumea cardiac glycoside C;

compound 4 is (3 β,5 β,17S,18 α) -streblus streblumea cardiotonic-3-O- (3 '-O-methyl) - β -D-glucopyranosyl-4' -O- (3 "-O-methyl) - β -D-glucopyranosyl-6" -O- β -D-glucopyranoside, streblus blumea cardiotonic glycoside D;

compound 5 is (3 β,5 β,17R) -streblus indica cardiotonic-3-O- (3 '-O-methyl) - β -D-glucopyranosyl-2' -O- (3 "-O-methyl) - β -D-glucopyranosyl-6" -O- β -D-glucopyranoside, streblus indica cardiotonic glycoside E.

The application of the cardiac glycoside in preparing the antitumor drugs comprises effective treatment dose of the cardiac glycoside or pharmaceutically acceptable salt thereof, or pharmaceutically acceptable excipient and at least one physiologically acceptable excipient.

The technical scheme has the advantages that:

the invention has the technical effects and advantages that: in the 5 compounds of cardiac glycoside provided by the technical scheme, especially the compound selected from the group consisting of streblus indica cardiac glycoside A, streblus indica cardiac glycoside B, streblus indica cardiac glycoside C, streblus indica cardiac glycoside D and streblus indica cardiac glycoside E, all the compounds can inhibit the growth of MCC-803, T24, SKOV-3, HepG2, Wi-38 and A549 tumor cells, have concentration dependence and can be used for developing related medicaments for inhibiting the growth of the tumor cells.

The cardiac glycoside compound prepared by the method is an active monomer compound with the function of inhibiting the growth of tumor cells, has concentration dependence, and can be used for developing related medicaments for inhibiting the growth of tumor cells.

Detailed Description

The present invention will be further illustrated with reference to the following examples, but is not limited thereto.

Example (b):

a method for preparing cardiac glycoside comprises the following steps:

1) taking a whole plant of dry streblus indica leaves, wherein the whole plant comprises roots, stems and leaves, adding an ethanol water solution, sequentially carrying out heating reflux extraction, combining filtrates, and recovering ethanol under reduced pressure to obtain an extract;

2) suspending the extract prepared in the step 1) in water, adsorbing by a macroporous adsorption resin chromatographic column, washing by water, and eluting by using an ethanol water solution to obtain an eluate rich in streblus indica cardiac glycoside.

The application of the cardiac glycoside prepared by the preparation method in preparing the antitumor drug comprises the cardiac glycoside shown in the general formula (I) or the medicinal salt thereof:

in the general formula (I), R₁、R₂、R₃Each independently represents a hydrogen atom, C₁～C₆Alkyl radical, C₁～C₆Haloalkyl, C₃～C₆Cycloalkyl radical, C₁～C₆Alkylcarbonyl group, R₄、R₅、R₆、R₇、R₈、R₉、R₁₀Each independently represents a hydrogen atom, C₁～C₆Alkyl radical, C₁～C₆Haloalkyl, C₃～C₆Cycloalkyl or glycosyl.

The glycosyl is monosaccharide or disaccharide.

The monosaccharide is at least one selected from the group consisting of arabinosyl, ribosyl, xylosyl, lyxosyl, glucosyl, allosyl, mannosyl, rhamnosyl, cinchona-glycosyl, fructosyl, galactosyl, apisyl, glucuronyl, and galacturonyl.

The disaccharide base is at least one selected from the group consisting of lactose base, sucrose base and maltose base.

The cardiac glycoside is any one of the compounds shown in the following structural formula 1:

wherein, the compound 1 is (3 beta, 5 alpha, 17R) -streblus indica heart-3-O- (2',3' -O-dimethyl) -beta-D-pyranyl quinine glucoside, namely streblus indica heart glucoside A;

the compound 2 is (3 beta, 5 beta, 17R) -streblus streblumea cardiotonic-3-O-alpha-L-rhamnopyranosyl-4' -O-beta-D-glucopyranoside, i.e. streblus cardiotonic glycoside B;

compound 3 is (3 β,5 β,17R) -streblus streblumea cardiotonic-3-O- (3' -O-methyl) - α -L-rhamnopyranosyl-4 "-O- β -D-glucopyranoside, streblus streblumea cardiac glycoside C;

compound 4 is (3 β,5 β,17S,18 α) -streblus indica cardiac-3-O- (3 '-O-methyl) - β -D-glucopyranosyl-4' -O- (3 "-O-methyl) - β -D-glucopyranosyl-6" -O- β -D-glucopyranoside, i.e. streblus indica cardiac glycoside D;

compound 5 is (3 β,5 β,17R) -streblus indica cardiotonic-3-O- (3 '-O-methyl) - β -D-glucopyranosyl-2' -O- (3 "-O-methyl) - β -D-glucopyranosyl-6" -O- β -D-glucopyranoside, streblus indica cardiotonic glycoside E.

In this example:

"halogen atom" means fluorine atom, chlorine atom, bromine atom, iodine atom;

"C1-C6" represents that the number of carbon atoms of the subsequent substituent is 1-6;

"C1-C6 alkyl group" means a C1-6 straight-chain or branched-chain alkyl group, methyl group, ethyl group, n-propyl group, isopropyl group, n-butyl group, sec-butyl group, isobutyl group, tert-butyl group, n-pentyl group, neopentyl group, 1-methylbutyl group, 2-methylbutyl group, 3-methylbutyl group, 1-ethylpropyl group, 1-dimethylpropyl group, 1, 2-dimethylpropyl group, n-hexyl group, 1-methylpentyl group, 2-methylpentyl group, 3-methylpentyl group, 4-methylpentyl group, 1-ethylbutyl group, 2-ethylbutyl group, 1-dimethylbutyl group, 1, 2-dimethylbutyl group, 1, 3-dimethylbutyl group, 2-dimethylbutyl group, 2, 3-dimethylbutyl group, 3-dimethylbutyl group, 1, 2-trimethylpropyl, 1,2, 2-trimethylpropyl, 1-ethyl-1-methylpropyl or 1-ethyl-2-methylpropyl and the like;

"C2-C6 alkenyl" means a straight-chain or branched alkenyl group having 2 to 6 carbon atoms, vinyl group, 1-propenyl group, 2-propenyl group, 1-butenyl group, 2-butenyl group, 3-butenyl group, 2-methyl-1-propenyl group, 1, 3-butadienyl group, 1-pentenyl group, 2-pentenyl group, 1-methyl-1-butenyl group, 3-pentenyl group, 1-methyl-2-butenyl group, 4-pentenyl group, 1-methyl-3-butenyl group, 1, 2-dimethyl-2-propenyl group, 2-methyl-2-butenyl group, 1, 2-dimethyl-1-propenyl group, 1, 3-pentadienyl group, 1-hexenyl group, 2-hexenyl, 1-methyl-1-pentenyl, 3-hexenyl, 4-hexenyl, 5-hexenyl, 1-methyl-4-pentenyl, 1-ethyl-3-butenyl, 1- (isobutyl) ethenyl, 1-ethyl-1-methyl-2-propenyl, 1- (isopropyl) -2-propenyl, 1, 3-dimethyl-2-butenyl, 3-methyl-4-pentenyl, 1, 2-trimethyl-2-propenyl, or 1, 5-hexadienyl;

"C2-C6 alkynyl" represents a straight-chain or branched alkynyl group having 2 to 6 carbon atoms, such as an ethynyl group, 1-propynyl group, 2-propynyl group, 1-butynyl group, 2-butynyl group, 3-butynyl group, 1-pentynyl group, 2-pentynyl group, 3-pentynyl group, 1-methyl-2-butynyl group, 4-pentynyl group, 1-methyl-3-butynyl group, 1-hexynyl group, 2-hexynyl group, 3-hexynyl group, 1-methyl-2-pentynyl group, 5-hexynyl group, 1-ethyl-3-butynyl group, 1-ethyl-1-methyl-2-propynyl group or 1, 1-dimethyl-2-butynyl group;

"C1-C6 haloalkyl" represents a C1-6 straight-chain or branched alkyl group, fluoromethyl group, difluoromethyl group, bromomethyl group, dibromomethyl group, iodomethyl group, chlorodifluoromethyl group, 1-fluoroethyl group, 1-difluoroethyl group, 2,2, 2-trifluoroethyl group, 1,2, 2-tetrafluoroethyl group, pentafluoroethyl group, 1-fluoropropyl group, 1-difluoropropyl group, 3,3, 3-trifluoropropyl group, 1-bromopropyl group, 1-fluorobutyl group, 4, 4-difluorobutyl group, 4,4, 4-trifluoro-3-methylbutyl group, 2,2,3,4, 4-hexafluorobutyl group, 1-chlorobutyl group, 1-bromobutyl group, 1-iodobutyl group, 1-fluoropentyl group, 2, 1-trifluoromethyl group, 2,3,4, 4-tetrafluoro-ethyl group, 1-fluoropropyl group, 1,2, 3,4, 3-difluoropropyl group, 3, 3-trifluoropropyl group, 1-bromobutyl group, 1-fluoropentyl group, 2,2, and the like, A group such as 5,5, 5-trifluoropentyl group, 1-chloropentyl group, 5,5, 5-trichloropentyl group, 1-bromopentyl group, 1-fluorohexyl group, 6,6, 6-trifluorohexyl group, 1-chlorohexyl group or 6-iodohexyl group;

"C1-C6 alkoxy" means that the alkyl moiety is a group as defined above for (C1-C6 alkyl) -O-, methoxy, ethoxy, n-propoxy, isopropoxy, n-butoxy, isobutoxy, sec-butoxy, tert-butoxy, n-pentoxy, 1-methylbutoxy, 2-methylbutoxy, 3-methylbutoxy, 1-ethylpropoxy, 1-dimethylpropoxy, 1, 2-dimethylpropoxy, or n-hexoxy;

"C1-C6 haloalkoxy" means a haloalkane moiety of (C1-C6 haloalkane) -O-group, difluoromethoxy, dichloromethoxy, trifluoromethoxy, trichloromethoxy, tribromomethoxy, 2-fluoroethoxy, 1-chloroethoxy, 1-bromoethoxy, 2, 2-difluoroethoxy, 2, 2-dichloroethoxy, 2,2, 2-trifluoroethoxy, 2,2, 2-trichloroethoxy, 1,2, 2-tetrafluoroethoxy, 1-chloropropoxy, 2-bromopropoxy, 2-bromo-1-methylethoxy, 3-iodopropoxy, 2, 3-dichloropropoxy, 2, 3-dibromopropoxy, 3,3, 3-trifluoropropoxy, 1-bromo-3, 3, 3-trifluoropropoxy, 2,2,3,3, 3-pentafluoropropoxy, 2,2, 2-trifluoro-1-trifluoromethylethoxy, 2-chlorobutoxy, 4-bromobutoxy, 3-bromo-2-methylpropoxy, 4,4, 4-trifluorobutoxy, 5,5, 5-trifluoropentyloxy, or 4,4,5,5, 5-pentafluoropentyloxy;

"C3-C6 cycloalkyl" represents a C3-6 cycloalkyl, cyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl or other group;

"C1 to C6 alkylcarbonyl" represents a group having an alkyl moiety of (C1 to C6 alkyl) -C (═ O) -yl, methylcarbonyl, ethylcarbonyl, n-propylcarbonyl, isopropylcarbonyl, n-butylcarbonyl, isobutylcarbonyl, sec-butylcarbonyl, tert-butylcarbonyl, n-pentylcarbonyl, 2-methylbutylcarbonyl, 2-dimethylpropylcarbonyl, n-hexylcarbonyl, or the like, as defined above;

"aldehyde C1 to C6 alkyl" means that the alkyl moiety is a group defined as above, such as H — C (═ O) - (C1 to C6 alkyl) -group, carboxaldehyde group, aldehyde methyl group, aldehyde ethyl group, aldehyde n-propyl group, aldehyde isopropyl group, aldehyde n-butyl group, aldehyde isobutyl group, aldehyde sec-butyl group, aldehyde tert-butyl group, aldehyde n-pentyl group, aldehyde 2-methylbutyl group, aldehyde 2, 2-dimethylpropyl group, or aldehyde n-hexyl group;

"carboxy C1-C6 alkyl" means that the alkyl moiety is a group as defined above for HO — C (═ O) - (C1-C6 alkyl) -based, methylcarboxyl, carboxymethyl, carboxyethyl, carboxy-n-propyl, carboxyisopropyl, carboxy-n-butyl, carboxyisobutyl, carboxy-sec-butyl, carboxy-tert-butyl, carboxy-n-pentyl, or carboxy-n-hexyl.

In this example, the glycosyl group is a monosaccharide or disaccharide group, the monosaccharide is a polyhydroxy aldehyde or ketone compound, most commonly a pentose and a hexose, and the pentose includes L-arabinose, D-xylose, D-lyxose and D-ribose. The hexose comprises L-rhamnose, D-cinchona sugar, D-glucose, D-mannose, D-allose, and D-galactose. The hexose includes D-fructose and L-sorbose, preferably L-rhamnose, D-glucose, D-xylose, and L-arabinose, and 6-deoxy sugar (methyl pentose) is a monosaccharide having one hydroxyl group of monosaccharide molecule substituted by a hydrogen atom, and commonly includes D-fructose and L-rhamnose.

Primary alcohol hydroxyl groups in the monosaccharide molecules are oxidized to carboxyl groups, thereby forming uronic acids, including glucuronic acid and galacturonic acid, for example.

Monosaccharides having a branch in the sugar chain are called branched chain sugars, and include, for example, D-apiose.

Disaccharides, also known as disaccharides, are formed from two molecules of a monosaccharide via a glycosidic bond, and include, for example, lactose, sucrose, and maltose.

In this example, the pharmaceutically acceptable salts refer to salts formed with a metal or an organic base, such as a hydroxyl group, a carboxyl group, or an amino group, or salts formed with an inorganic acid or an organic acid, among the compounds of the present invention represented by the general formula (I), as metals, alkali metals such as sodium or potassium, or alkaline earth metals such as magnesium or calcium; as the organic base, triethylamine, diisopropylamine, or the like; as the inorganic acid, phosphoric acid, hydrochloric acid, hydrobromic acid, hydroiodic acid, boric acid, sulfuric acid or the like; as the organic acid, formic acid, acetic acid, lactic acid, ascorbic acid, succinic acid, fumaric acid, maleic acid, oxalic acid, citric acid, benzoic acid, salicylic acid, tartaric acid, methanesulfonic acid, 4-toluenesulfonic acid, trifluoromethanesulfonic acid or the like is exemplified.

In the method, the whole plant (roots, stems and leaves) of streblus indica lam leaves is extracted by aqueous ethanol solution in a reflux way, the solvent is recovered under reduced pressure to obtain extract, the extract is suspended in water and is separated by a macroporous adsorption resin chromatographic column to obtain eluate rich in streblus indica lam cardiac glycoside, then the eluate rich in streblus indica lam cardiac glycoside is chromatographed by a silica gel column to obtain a plurality of fractions, and the fractions are refined by positive and negative silica gel column chromatography, preparative HPLC and the like respectively to obtain the pure compounds 1-5.

Can adopt¹H-NMR、¹³One-dimensional nuclear magnetic resonance spectrum such as C-NMR and the like, and¹H-¹HCOSY、DEPT、HSQC、HMBC、ROEand (3) identifying the structure of the obtained compound 1-5 by using two-dimensional nuclear magnetic resonance spectrum and other spectrum technologies such as SY and the like, HRESIMS mass spectrum and chemical reaction.

The excipients acceptable in this case may be any conventional excipients in the pharmaceutical formulation, health care product arts, the selection of a particular excipient will depend on the mode of administration or disease type and state to be used in the treatment of a particular patient, and the method of preparation of a suitable pharmaceutical composition for a particular mode of administration is well within the knowledge of those skilled in the pharmaceutical arts, and may include, for example, diluents, carriers, fillers, binders, wetting agents, disintegrants, etc. conventional in the pharmaceutical arts as pharmaceutically acceptable excipients.

The pharmaceutical composition can be prepared into various forms such as tablets, powder, granules, capsules, oral liquid, injection emulsion, sterile powder for injection and the like, and the medicines of various formulations can be prepared according to a conventional method in the pharmaceutical field.

In the present case, a cosmetic composition is provided comprising the above mentioned cardiac glycoside and optionally at least one physiologically acceptable excipient, by "physiologically acceptable excipient" is meant a substance suitable for topical or oral use and which does not present any risk of toxicity, incompatibility, etc., when in contact with the mucous membranes, nails, scalp, hair, body hair and skin, etc., of mammals, in particular humans, and such excipients are well known to the person skilled in the art, where applicable, and include, for example, organic solvents, chelating agents, penetrants, thickeners, fillers, emulsifiers or surfactants, preservatives, colorants, fragrances and mixtures thereof.

Specifically, the method comprises the following steps:

example 1:

a method for preparing cardiac glycoside comprises the following steps:

1) taking 10kg of dry leaves (roots, stems, leaves) of streblus streblumea, adding 75% ethanol water solution, sequentially heating and refluxing for 3 times (3 hr, 2 hr), mixing filtrates, and recovering ethanol under reduced pressure to obtain 2.10kg of extract;

2) suspending 1.5kg of the extract in 20L of water, adsorbing with D101 macroporous adsorbent resin chromatographic column, washing with water, and eluting with 20% and 95% ethanol to obtain 20% and 95% ethanol eluate 250g and 625.0g, wherein the 95% ethanol eluate is rich in streblus indica glucoside.

Example 2:

separation preparation and structure identification of cardiac glycoside compounds 1-5:

the isolation of compounds 1-5 is performed simultaneously, and thus is described in one example,

isolation preparation of compounds 1-5:

300.0g of the 95% ethanol eluate obtained in example 1 was subjected to silica gel column chromatography, and gradient elution using methylene chloride-methanol (100:0, 60:1, 20:1, 10:1, 8:1, 4:1, 2:1, 0:100) as an eluent, to give Fr.1(32g), Fr.2(46g), Fr.3(43g), Fr.4(35g), Fr.5(47g), Fr.6(30g), Fr.7(28g), and Fr.8(26 g); fraction Fr.3(43g) was subjected to reverse phase C₁₈Performing silica gel column chromatography, performing gradient elution with water-methanol (100:0-50:50) as eluent to obtain 6 fractions Fr.3.1(5.1g), Fr.3.2(13.5g), Fr.3.3(14.2g), Fr.3.4(6.5g), Fr.3.5(1.5g) and Fr.3.6(1.1g), and subjecting Fr.3.3(14.2g) to reversed phase C₁₈Silica gel column chromatography (water-methanol, 100:0-50:50) to give Fr.3.3.1(1330mg), Fr.3.3.2(2315mg), Fr.3.3.3(3345mg), Fr.3.3.4(4870mg), Fr.3.3.5(2460mg) and Fr.3.3.6(1234 mg); fr.3.2(13.5g) by reverse phase C₁₈Silica gel column chromatography preparative chromatography, semi-preparative liquid chromatography (30% methanol) to give compound 1(50.7mg), fraction Fr.5(47g) was subjected to reverse phase C₁₈Performing silica gel column chromatography, and performing gradient elution with water-methanol (100:0-50:50) as eluent to obtain 6 components of Fr.5.1(6.6g), Fr.5.2(8.4g), Fr.5.3(12.4g), Fr.5.4(10.1g), Fr.5.5(6.5g) and Fr.5.6(4.1 g); component Fr.5.3(12.4g) is passed through phase reversal C₁₈Silica gel column chromatography (water-methanol, 100:0-50:50) to give Fr.5.3.1(2860mg), Fr.5.3.2(3125mg), Fr.5.3.3(3309mg), Fr.5.3.4(807mg), Fr.5.3.5(523mg) and Fr.5.3.6(304 mg); fr.5.3.2(3125mg) by reverse phase C₁₈Silica gel column chromatography preparative chromatography, semipreparative liquid chromatography (25% methanol) to give compounds 2(43.7mg) and 3(47.8 mg); subjecting fraction Fr.5.3.5(523mg) to Sephadex LH-20 and reversed phase C₁₈Column chromatography on silica gel and semi-preparative liquid purification (25% acetonitrile) gave compounds 4(33.7mg) and 5(24.6 mg).

And (3) structure identification: by using infrared, mass, nuclear magnetic resonance spectroscopy (¹HNMR、¹³CNMR、DEPT、¹H-¹The structures of the obtained compounds 1-5 are identified by spectrum techniques such as HCOSY, HSQC, HMBC, ROESY), Mass Spectrum (MS) and optical rotation, the positions of substituents and the connection positions of sugars are determined by using a two-dimensional nuclear magnetic resonance spectrum technology, and all carbon signals and hydrogen signals are accurately assigned by combining with a high-resolution mass spectrum.

The physicochemical data for compounds 1-5 are shown below:

compound 1: streblunolide A:

white powder, easily soluble in methanol, pyridine.

HRESIMS m/z[M+Na]⁺603.3134(calculated for C₃₁H₄₈O₁₀Na,found:603.3145)；¹H NMR(600MHz,CD₃OD)δ_H2.14(1H,m,H-1a),δ_H 1.70(1H,m,H-1b),δ_H 1.65(1H,m,H-2a),δ_H 1.88(1H,m,H-2b),δ_H4.21(1H,m,H-3),δ_H 1.68(1H,m,H-4a),δ_H 2.18(1H,m,H-4b),δ_H 0.97(1H,m,H-6a),δ_H1.54(1H,m,H-6b),δ_H 1.68(1H,m,H-7a),δ_H 2.14(1H,m,H-7b),δ_H 1.98(1H,m,H-8),δ_H1.52(1H,m,H-9),δ_H 1.66(1H,m,H-11a),δ_H 1.90(1H,m,H-11b),δ_H 1.44(1H,m,H-12a),δ_H1.54(1H,m,H-12b),δ_H 1.71(1H,m,H-15a),δ_H 2.19(1H,m,H-15b),δ_H 2.18(1H,m,H-16a),δ_H 1.88(1H,m,H-16b),δ_H 2.85(1H,m,H-17),δ_H 0.87(3H,s,H-18),δ_H 3.46(2H,s,H-19),δ_H5.03(1H,J＝18.5,1.6Hz,H-21a),δ_H 4.92(1H,J＝18.5,1.6Hz,H-21b),δ_H 5.91(1H,brs,H-22),δ_H 4.42(1H,d,H-1′),δ_H 3.08(1H,m,H-2′),δ_H 2.95(1H,m,H-3′),δ_H 3.09(1H,m,H-4′),δ_H 3.29(1H,m,H-5′),δ_H 1.27(3H,d,H-6′),δ_H 3.54(3H,s,H-2′-OCH₃),δ_H 3.62(3H,s,H-3′-OCH₃)；¹³C NMR(150MHz,CD₃OD)δ_C 18.9(C-1),δ_C 25.3(C-2),δ_C 75.0(C-3),δ_C 35.8(C-4),δ_C 75.0(C-5),δ_C 23.2(C-6),δ_C 37.5(C-7),δ_C 42.6(C-8),δ_C 40.4(C-9),δ_C 40.4(C-10),δ_C 26.0(C-11),δ_C 40.5(C-12),δ_C 50.7(C-13),δ_C 85.0(C-14),δ_C 32.4(C-15),δ_C27.9(C-16),δ_C 51.8(C-17),δ_C 16.2(C-18),δ_C 56.2(C-19),δ_C 178.2(C-20),δ_C 75.3(C-21),δ_C 117.9(C-22),δ_C 177.2(C-23),δ_C 101.4(C-1′),δ_C 87.8(C-2′),δ_C 85.9(C-3′),δ_C76.9(C-4′),δ_C 73.2(C-5′),δ_C 18.0(C-6′),δ_C 61.0(C-2′-OCH₃),δ_C 61.1(C-3′-OCH₃)。

Compound 2: streblunolide B:

white powder, easily soluble in methanol, pyridine.

HRESIMS m/z[M+Na]⁺721.3396(calculated for C₃₅H₅₄O₁₄Na,found:721.3411)；¹H NMR(600MHz,CD₃OD)δ_H 1.26(1H,m,H-1a),δ_H 1.46(1H,m,H-1b),δ_H 2.01(1H,m,H-2a),δ_H 2.21(1H,m,H-2b),δ_H 4.12(1H,m,H-3),δ_H 1.68(1H,m,H-4a),δ_H 2.10(1H,m,H-4b),δ_H 1.12(1H,m,H-6a),δ_H 1.91(1H,m,H-6b),δ_H 1.35(1H,m,H-7a),δ_H 1.71(1H,m,H-7b),δ_H 1.60(1H,m,H-8),δ_H 1.70(1H,m,H-9),δ_H 1.56(1H,m,H-11a),δ_H 1.68(1H,m,H-11b),δ_H 1.49(1H,m,H-12a),δ_H 1.60(1H,m,H-12b),δ_H 1.64(1H,m,H-15a),δ_H 2.13(1H,m,H-15b),δ_H 1.82(1H,m,H-16a),δ_H1.86(1H,m,H-16b),δ_H 2.83(1H,m,H-17),δ_H 0.85(3H,s,H-18),δ_H 0.90(2H,s,H-19),δ_H5.02(1H,J＝18.5,1.6Hz,H-21a),δ_H 4.90(1H,J＝18.5,1.6Hz,H-21b),δ_H 5.87(1H,brs,H-22),δ_H 4.69(1H,d,H-1′),δ_H 3.81(1H,m,H-2′),δ_H 4.29(1H,m,H-3′),δ_H 3.30(1H,m,H-4′),δ_H 3.29(1H,m,H-5′),δ_H 1.27(3H,d,H-6′),δ_H 4.37(1H,d,H-1″),δ_H 3.20(1H,m,H-2″),δ_H 3.29(1H,m,H-3″),δ_H 3.31(1H,m,H-4″),δ_H 3.28(1H,m,H-5″),δ_H 3.64(3H,d,H-6″a),δ_H 3.85(3H,d,H-6″b)；¹³C NMR(150MHz,CD₃OD)δ_C 22.6(C-1),δ_C 28.0(C-2),δ_C 75.4(C-3),δ_C 33.4(C-4),δ_C 77.4(C-5),δ_C 25.0(C-6),δ_C 36.2(C-7),δ_C 40.1(C-8),δ_C 41.8(C-9),δ_C 40.9(C-10),δ_C 26.6(C-11),δ_C 41.6(C-12),δ_C 51.9(C-13),δ_C 86.3(C-14),δ_C34.8(C-15),δ_C 26.9(C-16),δ_C 50.9(C-17),δ_C 16.3(C-18),δ_C 17.2(C-19),δ_C 178.4(C-20),δ_C 75.1(C-21),δ_C 117.8(C-22),δ_C 177.3(C-23),δ_C 99.3(C-1′),δ_C 72.3(C-2′),δ_C72.1(C-3′),δ_C 83.8(C-4′),δ_C 69.6(C-5′),δ_C 18.1(C-6′),δ_C 106.0(C-1″),δ_C 76.6(C-2″),δ_C 78.7(C-3″),δ_C 71.1(C-4″),δ_C 78.3(C-5″),δ_C 62.3(C-6″)。

Compound 3: streblunolide C (streblonolide C):

white powder, easily soluble in methanol, pyridine.

HRESIMS m/z[M+Na]⁺735.3563(calculated for C₃₆H₅₆O₁₄Na,found:735.3568)；¹H NMR(600MHz,CD₃OD)δ_H 1.27(1H,m,H-1a),δ_H 1.47(1H,m,H-1b),δ_H 2.01(1H,m,H-2a),δ_H 2.19(1H,m,H-2b),δ_H 4.13(1H,m,H-3),δ_H 1.69(1H,m,H-4a),δ_H 2.14(1H,m,H-4b),δ_H 1.13(1H,m,H-6a),δ_H 1.92(1H,m,H-6b),δ_H 1.38(1H,m,H-7a),δ_H 1.74(1H,m,H-7b),δ_H 1.61(1H,m,H-8),δ_H 1.72(1H,m,H-9),δ_H 1.58(1H,m,H-11a),δ_H 1.69(1H,m,H-11b),δ_H 1.49(1H,m,H-12a),δ_H 1.59(1H,m,H-12b),δ_H 1.64(1H,m,H-15a),δ_H 2.13(1H,m,H-15b),δ_H 1.83(1H,m,H-16a),δ_H1.86(1H,m,H-16b),δ_H 2.81(1H,m,H-17),δ_H 0.86(3H,s,H-18),δ_H 0.91(2H,s,H-19),δ_H4.99(1H,J＝18.5,1.6Hz,H-21a),δ_H 5.02(1H,J＝18.5,1.6Hz,H-21b),δ_H 5.87(1H,brs,H-22),δ_H 4.75(1H,d,H-1′),δ_H 3.81(1H,m,H-2′),δ_H 4.13(1H,m,H-3′),δ_H 3.30(1H,m,H-4′),δ_H 3.21(1H,m,H-5′),δ_H 1.27(3H,d,H-6′),δ_H 4.37(1H,d,H-1″),δ_H 3.22(1H,m,H-2″),δ_H 3.29(1H,m,H-3″),δ_H 3.05(1H,m,H-4″),δ_H 3.39(1H,m,H-5″),δ_H 3.65(3H,d,H-6″a),δ_H 3.84(3H,d,H-6″b),δ_H 3.39(3H,s,H-3′-OCH₃)；¹³C NMR(150MHz,CD₃OD)δ_C 22.6(C-1),δ_C 28.0(C-2),δ_C 75.4(C-3),δ_C 33.4(C-4),δ_C 77.8(C-5),δ_C 24.9(C-6),δ_C 36.1(C-7),δ_C 40.1(C-8),δ_C 41.8(C-9),δ_C 40.9(C-10),δ_C 26.6(C-11),δ_C 41.6(C-12),δ_C 51.9(C-13),δ_C 86.3(C-14),δ_C 33.1(C-15),δ_C 26.9(C-16),δ_C 50.9(C-17),δ_C 16.3(C-18),δ_C17.2(C-19),δ_C 178.4(C-20),δ_C 75.2(C-21),δ_C 117.8(C-22),δ_C 177.3(C-23),δ_C 98.0(C-1′),δ_C 75.0(C-2′),δ_C 71.3(C-3′),δ_C 83.4(C-4′),δ_C 69.6(C-5′),δ_C 18.0(C-6′),δ_C106.0(C-1″),δ_C 77.8(C-2″),δ_C 81.0(C-3″),δ_C 68.3(C-4″),δ_C 77.8(C-5″),δ_C 62.5(C-6″),δ_C 56.8(C-3′-OCH₃)。

Compound 4: streblunolide D:

white powder, easily soluble in methanol, pyridine.

HRESIMS m/z[M+Na]⁺911.4252(calculated for C₄₃H₆₈O₁₀Na,found:911.4253)；¹H NMR(600MHz,CD₃OD)δ_H 1.27(1H,m,H-1a),δ_H 1.48(1H,m,H-1b),δ_H 1.38(1H,m,H-2a),δ_H 1.58(1H,m,H-2b),δ_H 4.10(1H,m,H-3),δ_H 1.70(1H,m,H-4a),δ_H 2.17(1H,m,H-4b),δ_H 2.00(1H,m,H-5),δ_H 1.26(1H,m,H-6a),δ_H 1.99(1H,m,H-6b),δ_H 1.28(1H,m,H-7a),δ_H 1.87(1H,m,H-7b),δ_H 1.61(1H,m,H-8),δ_H 1.71(1H,m,H-9),δ_H 1.59(1H,m,H-11a),δ_H 1.70(1H,m,H-11b),δ_H 1.72(1H,m,H-12a),δ_H 2.17(1H,m,H-12b),δ_H 1.70(1H,m,H-15a),δ_H 2.14(1H,m,H-15b),δ_H 1.87(1H,m,H-16a),δ_H 1.98(1H,m,H-16b),δ_H 3.32(1H,m,H-17),δ_H 0.85(3H,s,H-18),δ_H 0.93(3H,s,H-19),δ_H 5.02(1H,J＝18.5,1.6Hz,H-21a),δ_H 4.99(1H,J＝18.5,1.6Hz,H-21b),δ_H 5.87(1H,brs,H-22),δ_H 4.36(1H,d,H-1′),δ_H 3.19(1H,m,H-2′),δ_H 3.13(1H,m,H-3′),δ_H 3.36(1H,m,H-4′),δ_H 3.75(1H,m,H-5′),δ_H 3.61(3H,dd,H-6′a),δ_H 3.83(3H,dd,H-6′b),δ_H4.43(1H,d,H-1″),δ_H 3.26(1H,m,H-2″),δ_H 3.74(1H,m,H-3″),δ_H 4.60(1H,m,H-4″),δ_H3.81(1H,m,H-5″),δ_H 3.75(3H,d,H-6″a),δ_H 4.16(3H,d,H-6″b),δ_H 4.58(1H,d,H-1″′),δ_H3.21(1H,m,H-2″′),δ_H 3.42(1H,m,H-3″′),δ_H 3.32(1H,m,H-4″′),δ_H 3.58(1H,m,H-5″′),δ_H 3.63(3H,d,H-6″′a),δ_H 3.83(3H,d,H-6″′b),δ_H 3.58(3H,s,H-3′-OCH₃),δ_H 3.56(3H,s,H-3″-OCH₃)；¹³C NMR(150MHz,CD₃OD)δ_C 22.4(C-1),δ_C 30.6(C-2),δ_C 74.9(C-3),δ_C 36.3(C-4),δ_C 30.3(C-5),δ_C 24.4(C-6),δ_C 36.3(C-7),δ_C 41.0(C-8),δ_C 36.9(C-9),δ_C 42.7(C-10),δ_C 26.6(C-11),δ_C 41.8(C-12),δ_C 52.1(C-13),δ_C 86.1(C-14),δ_C 37.9(C-15),δ_C30.6(C-16),δ_C 51.1(C-17),δ_C 16.4(C-18),δ_C 24.4(C-19),δ_C 178.5(C-20),δ_C 75.6(C-21),δ_C 117.8(C-22),δ_C 177.3(C-23),δ_C 102.1(C-1′),δ_C 75.3(C-2′),δ_C 84.8(C-3′),δ_C77.9(C-4′),δ_C 71.7(C-5′),δ_C 61.8(C-6′),δ_C 104.2(C-1″),δ_C 74.9(C-2″),δ_C 86.4(C-3″),δ_C 71.8(C-4″),δ_C 78.0(C-5″),δ_C 69.6(C-6″),δ_C 104.4(C-1″′),δ_C 76.5(C-2″′),δ_C78.0(C-3″′),δ_C 71.1(C-4″′),δ_C 78.3(C-5″′),δ_C 62.9(C-6″′),δ_C 61.3(C-3′-OCH₃),δ_C61.2(C-3″-OCH₃)。

Compound 5: streblunolide e (strebllsolide e):

white powder, easily soluble in methanol, pyridine.

HRESIMS m/z[M+Na]⁺927.4208(calculated for C₄₃H₆₈O₁₀Na,found:927.4202)；¹H NMR(600MHz,CD₃OD)δ_H 1.22(1H,m,H-1a),δ_H 1.50(1H,m,H-1b),δ_H 1.37(1H,m,H-2a),δ_H 1.57(1H,m,H-2b),δ_H 4.08(1H,m,H-3),δ_H 1.66(1H,m,H-4a),δ_H 2.01(1H,m,H-4b),δ_H 1.23(1H,m,H-6a),δ_H 1.83(1H,m,H-6b),δ_H 1.39(1H,m,H-7a),δ_H 1.90(1H,m,H-7b),δ_H 1.52(1H,m,H-8),δ_H 1.70(1H,m,H-9),δ_H 1.51(1H,m,H-11a),δ_H 1.67(1H,m,H-11b),δ_H 1.79(1H,m,H-12a),δ_H 1.94(1H,m,H-12b),δ_H 1.62(1H,m,H-15a),δ_H 2.54(1H,m,H-15b),δ_H 1.81(1H,m,H-16a),δ_H1.83(1H,m,H-16b),δ_H 3.16(1H,m,H-17),δ_H 0.90(3H,s,H-18),δ_H 0.93(3H,s,H-19),δ_H5.10(1H,J＝18.5,1.6Hz,H-21a),δ_H 5.13(1H,J＝18.5,1.6Hz,H-21b),δ_H 5.91(1H,brs,H-22),δ_H 4.36(1H,d,H-1′),δ_H 3.20(1H,m,H-2′),δ_H 3.10(1H,m,H-3′),δ_H 4.13(1H,m,H-4′),δ_H 3.32(1H,m,H-5′),δ_H 3.86(3H,dd,H-6′a),δ_H 4.13(3H,dd,H-6′b),δ_H 4.44(1H,d,H-1″),δ_H 3.25(1H,m,H-2″),δ_H 3.76(1H,m,H-3″),δ_H 4.59(1H,m,H-4″),δ_H 3.73(1H,m,H-5″),δ_H 3.73(3H,d,H-6″a),δ_H 4.09(3H,d,H-6″b),δ_H 4.58(1H,d,H-1″′),δ_H 3.23(1H,m,H-2″′),δ_H 3.79(1H,m,H-3″′),δ_H 3.35(1H,m,H-4″′),δ_H 3.75(1H,m,H-5″′),δ_H 3.62(3H,d,H-6″′a),δ_H 3.86(3H,d,H-6″′b),δ_H 3.59(3H,s,H-3′-OCH₃),δ_H 3.57(3H,s,H-3″-OCH₃)；¹³C NMR(150MHz,CD₃OD)δ_C 22.1(C-1),δ_C 30.8(C-2),δ_C 74.9(C-3),δ_C 36.3(C-4),δ_C77.5(C-5),δ_C 24.0(C-6),δ_C 36.8(C-7),δ_C 40.9(C-8),δ_C 36.3(C-9),δ_C 43.8(C-10),δ_C31.5(C-11),δ_C 42.9(C-12),δ_C 51.3(C-13),δ_C 86.1(C-14),δ_C 37.9(C-15),δ_C 30.6(C-16),δ_C 59.7(C-17),δ_C 16.1(C-18),δ_C 24.4(C-19),δ_C 178.5(C-20),δ_C 77.9(C-21),δ_C117.8(C-22),δ_C 177.3(C-23),δ_C 102.1(C-1′),δ_C 74.9(C-2′),δ_C 84.8(C-3′),δ_C 74.9(C-4′),δ_C 71.7(C-5′),δ_C 61.8(C-6′),δ_C 104.2(C-1″),δ_C 74.9(C-2″),δ_C 85.6(C-3″),δ_C 71.8(C-4″),δ_C 78.0(C-5″),δ_C 69.6(C-6″),δ_C 104.4(C-1″′),δ_C 76.6(C-2″′),δ_C78.0(C-3″′),δ_C 71.7(C-4″′),δ_C 77.9(C-5″′),δ_C 62.9(C-6″′),δ_C 61.3(C-3′-OCH₃),δ_C61.3(C-3″-OCH₃)。

Example 3:

determination of cytotoxic Activity of streblus streblume cardiac glycoside 1-5:

3.1 determination of the cytotoxic Activity of streblus streblumea cardiac glycoside 1-5:

the experimental procedure for determining the cytotoxic activity of streblus streblumea cardiac glycoside 1-5 was as follows: (1) taking cells in exponential growth phase, adding pancreatin digestive juice to make adherent cells fall off, preparing cell suspension with culture solution containing 10% newborn calf serum, counting 2 × 10⁴Per milliliter; (2) inoculating the cell suspension on 96-well plate, placing at constant temperature CO at 180 microliter/well₂Culturing for 24h in an incubator; (3) changing the solution, adding the tested compound, culturing for 48h at 20 microliter/hole; (4) adding MTT into a 96-well plate, wherein the volume per well is 20 microliter, and reacting in an incubator for 4 hours; (5) removing clear liquid, adding 150 microliters/hole DMSO, and shaking for 5 min; (6) measuring absorbance of each well at 570nm wavelength with microplate reader, and calculating MCC-803. The inhibition rates of T24, SKOV-3, HepG2, Wi-38 and A549 tumor cells and the compound with the inhibition rate of more than 50 percent are calculated by the graph pad, prism, v5.0 software to obtain the IC of the compound on the tumor cells₅₀The value is obtained.

3.2 Experimental results:

the results of cytotoxic activity of streblus streblumea glycosides 1-5 are shown in Table 1, and it can be seen from Table 1 that streblus streblumea glycosides 1-5 show significant inhibitory activity against MCC-803, T24, SKOV-3, HepG2, Wi-38 and A549 tumor cell growth, their IC' s₅₀Between 0.001 μ M and 0.116 μ M, the activity of streblus tonic glycosides 1-5 against these tumor cells was comparable to the activity of the positive control ouabain (G-ouabain).

TABLE 1 cytotoxic Activity of streblus streblumea cardiac glycoside 1-5 (IC)₅₀,μM)^a

a mean of three experiments, b positive control.

Claims (7)

1. A method for preparing cardiac glycoside, which is characterized by comprising the following steps:

1) taking a whole plant of dry streblus indica leaves, wherein the whole plant comprises roots, stems and leaves, adding an ethanol water solution, sequentially carrying out heating reflux extraction, combining filtrates, and recovering ethanol under reduced pressure to obtain an extract;

2) suspending the extract prepared in the step 1) in water, adsorbing by a macroporous adsorption resin chromatographic column, washing by water, and eluting by using an ethanol water solution to obtain an eluate rich in streblus indica cardiac glycoside.

2. The use of cardiac glycosides obtained by the process of claim 1 in the preparation of an anti-neoplastic agent,

comprising a cardiac glycoside of the general formula (I):

in the general formula (I), R₁、R₂、R₃Each independently represents a hydrogen atom, C₁～C₆Alkyl radical, C₁～C₆Haloalkyl, C₃～C₆Cycloalkyl radical, C₁～C₆Alkylcarbonyl group, R₄、R₅、R₆、R₇、R₈、R₉、R₁₀Each independently represents a hydrogen atom, C₁～C₆Alkyl radical, C₁～C₆Haloalkyl, C₃～C₆Cycloalkyl or glycosyl.

3. The use of a cardiac glycoside as claimed in claim 2 in the preparation of an antineoplastic medicament, wherein the glycosyl is mono-glycosyl or di-glycosyl.

4. The use of a cardiac glycoside according to claim 3, wherein the monosaccharide is at least one member selected from the group consisting of arabinosyl, ribosyl, xylosyl, lyxosyl, glucosyl, allosyl, mannosyl, rhamnosyl, cinchona-syl, fructosyl, galactosyl, apisyl, glucuronyl, and galacturonyl.

5. The use of a cardiac glycoside according to claim 3 in the preparation of an antineoplastic medicament, wherein the disaccharide base is selected from at least one of the group consisting of lactose base, sucrose base, and maltosyl.

6. The use of a cardiac glycoside as claimed in claim 2 in the preparation of an antineoplastic medicament, wherein the cardiac glycoside is any one of the compounds of formula 1:

wherein, the compound 1 is (3 beta, 5 alpha, 17R) -streblus streblumea heart-3-O- (2',3' -O-dimethyl) -beta-D-pyranosyl glucoside, i.e. streblus streblumea cardiac glycoside A;

the compound 2 is (3 beta, 5 beta, 17R) -streblus streblumea cardiotonic-3-O-alpha-L-rhamnopyranosyl-4' -O-beta-D-glucopyranoside, i.e. streblus cardiotonic glycoside B;

compound 3 is (3 β,5 β,17R) -streblus streblumea cardiotonic-3-O- (3' -O-methyl) - α -L-rhamnopyranosyl-4 "-O- β -D-glucopyranoside, streblus streblumea cardiac glycoside C;

compound 4 is (3 β,5 β,17S,18 α) -streblus streblumea cardiotonic-3-O- (3 '-O-methyl) - β -D-glucopyranosyl-4' -O- (3 "-O-methyl) - β -D-glucopyranosyl-6" -O- β -D-glucopyranoside, streblus blumea cardiotonic glycoside D;

compound 5 is (3 β,5 β,17R) -streblus indica cardiotonic-3-O- (3 '-O-methyl) - β -D-glucopyranosyl-2' -O- (3 "-O-methyl) - β -D-glucopyranosyl-6" -O- β -D-glucopyranoside, streblus indica cardiotonic glycoside E.

7. Use of a cardiac glycoside for the preparation of an anti-tumor medicament, comprising a therapeutically effective amount of a cardiac glycoside or a pharmaceutically acceptable salt thereof, or a pharmaceutically acceptable excipient, as defined in any one of claims 2 to 6, and at least one physiologically acceptable excipient.