CN102093460A - Triterpenoid saponin compound as well as synthesis method and application of triterpenoid saponin - Google Patents

Abstract

The invention provides a triterpenoid saponin compound, a synthesis method of the triterpenoid saponin compound and an application of the triterpenoid saponin compound in preparing medicaments for treating cancers and dementia. The triterpenoid saponin compound is shown in the following chemical general formula, wherein any two of R1, R2 and R3 are methyl, and the other one is hydrogen atom, R4 is pent-or hexa-monosaccharide, or disaccharide formed by the pent-or hexa-monosaccharide, or disaccharide formed by the pent-or hexa-monosaccharide, or glycosyl formed by aldehyde acid derivatives of the monosaccharide or the disaccharide, and R5 is hydrogen atom, or pent-or hexa-monosaccharide, or disaccharide formed by the pent-or hexa-monosaccharide, or substituent group formed by aldehyde acid derivatives of the monosaccharide or the disaccharide. The invention also provides a medicament with the functions of treating cancers and dementia.

Description

Triterpene saponin compound, its synthetic method and application thereof

Technical field

The present invention relates to triterpene saponin compound, its synthetic method and the application in the medicine of preparation treatment cancer and dementia thereof.

Background technology

Chinese medicine is the basic substance and the carrier of traditional Chinese medical science disease preventing and treating, health care, has contained abundant scientific meaning and high practical value.The situation that goes to the world along with the continuous development and the Chinese materia medica of human medical health career becomes clear day by day, and unprecedented growth has appearred in the social demand of centering resource medicine, and imbalance between supply and demand is outstanding day by day.Reasonable development and protection natural resources of Chinese medicinal materials realize that the sustainable use of natural resources of Chinese medicinal materials has become the basic Consensus of Chinese Government and Chinese materia medica industry.

Discovery and searching new texture, new compound have become a kind of trend from wild fauna and flora.These are all at the pressure that has strengthened natural resources of Chinese medicinal materials in varying degrees.Add for a long time the understanding defective to the rational exploitation and utilization natural resources of Chinese medicinal materials, many areas cause up till now in varying degrees natural resources of Chinese medicinal materials having been carried out excessively gathering, adopting and hunt of predation formula, and a lot of natural resources of Chinese medicinal materials are endangered.Therefore, how medicinal animal and plant resource in how correctly effectively utilizing guarantees that the problem of the aspects such as sustainable development and utilization of natural resources of Chinese medicinal materials needs to be resolved hurrily.

Triterpene saponin componds is to be present in the saponin compound that a class in the multiple medicinal plant has the triterpene main structure body and be connected with sugar chain respectively by glycosidic link and ester bond on 3 hydroxyls and 28 carboxyls.For example from ground crow rhizome, separate obtaining multiple triterpenoid saponin, and (the 33rd rolls up the 14th phase, in July, 2008: 1696-1699) for Zhang Lantian etc., CHINA JOURNAL OF CHINESE MATERIA MEDICA to find to have certain antitumour activity by these compounds with Oleanolic Acid agent structure.The purposes of oleanane-type triterpene saponin compound aspect improvement memory and learning capacity of extracting from various plants disclosed among the open CN101528209A of Chinese patent application.

The source of triterpenoid saponin mainly obtains from plant at present, and the natural resources of Chinese medicinal materials shortage, the ecosystem raw medicinal material can't satisfy supply, and expectation can be with chemical synthesis process as solving the natural resources of Chinese medicinal materials shortage, the effective way of protection Chinese medicine herb resource.And by chemical synthesising technology, synthetic its active pharmaceutical ingredients should be one of important means that solves the plant resources scarcity.And with the synthetic advantage that has the resource limit of not being subjected to, is more suitable for relevant pharmaceutical industries production of chemical synthesising technology.The present inventor was that raw material successfully synthesizes the black saponin W 3 in ground (the open CN101100482A of Chinese patent application) with Oleanolic Acid and monose once.Yet naturally occurring triterpene saponin componds is connected with many polysaccharide chains usually, and every sugar chain has the sugar more than three usually.Such structure makes the resulting anomaly complexity of this compounds, and by product is many, and ultimate yield is lower, is difficult to carry out extensive commercial application.

In addition, the application of triterpene saponin componds aspect other diseases also rarely has research.

Summary of the invention

The present invention is in order to address the above problem and to propose to small part.

One object of the present invention is to provide a kind of and has equal pharmaceutical use, but the more simple triterpene saponin compound of structure.

That is, provide a kind of triterpene saponin compound, wherein, the described triterpene saponin compound triterpene saponin compound that following chemical general formula I represents of serving as reasons:

Chemical general formula I

In above chemical general formula I, R ₁, R ₂And R ₃In any two be methyl, and another is hydrogen atom, R ₄Can be for by five or hexa-atomic monose, or by described five or the disaccharides that forms of hexa-atomic monose, or the glycosyl that forms of the aldehydic acid derivative of described monose or disaccharides, R ₅Can be hydrogen atom, perhaps can be for by five or hexa-atomic monose, or by described five or the disaccharides that forms of hexa-atomic monose, or the substituting group that forms of the aldehydic acid derivative of described monose or disaccharides.

Wherein, preferred R ₁, R ₂All be methyl, and R ₃Be hydrogen atom, or R ₁And R ₂In any one be methyl, another is hydrogen atom, and R ₃Be methyl.

R ₄Be preferably a kind of glycosyl that forms that is selected from by in L-rhamnosyl, D-glucose-(1 → 2)-D-xyloside, D-glucose, L-Fucose, D-seminose, L-arabinose, D-pectinose, D-wood sugar, D-ribose and the D-glucuronic acid, most preferably be the glycosyl that L-rhamnosyl or D-glucose-(1 → 2)-D-xyloside forms; R ₅Be preferably hydrogen atom, or, most preferably be hydrogen atom, L-rhamanopyranosyl or D-xylosyl for to be selected from by a kind of substituting group that forms in L-rhamnosyl, D-glucose-(1 → 2)-D-xyloside, D-glucose, L-Fucose, D-seminose, L-arabinose, D-pectinose, D-wood sugar, D-ribose and the D-glucuronic acid; Work as R ₅During for the substituting group that forms by above-mentioned sugared or derivatives thereof, R ₄And R ₅Can be identical or different.

Preferred R ₁, R ₂All be methyl, and R ₃Be hydrogen atom, R ₄Be L-rhamnosyl, R ₅Be hydrogen atom.

Preferred R ₁, R ₂All be methyl, and R ₃Be hydrogen atom, R ₄Be L-rhamnosyl, R ₅Be the L-rhamnosyl.

Preferred R ₁, R ₂All be methyl, and R ₃Be hydrogen atom, R ₄Be the glycosyl that D-glucose-(1 → 2)-D-xyloside forms, R ₅Be hydrogen atom.

Preferred R ₁, R ₂All be methyl, and R ₃Be hydrogen atom, R ₄Be the glycosyl that D-glucose-(1 → 2)-D-xyloside forms, R ₅Be the L-rhamnosyl.

Second purpose of the present invention be to provide a kind of easy, cost is low, weak point consuming time, yield is high and be easy to the synthetic method of the described triterpene saponin compound of industrialization.

That is, provide a kind of with five or hexa-atomic sugar or its aldehydic acid derivative and Oleanolic Acid or ursolic acid be raw material, obtain the synthetic method of the triterpene saponin compound shown in the above general formula I by the convergence type method, described synthetic method specifically may further comprise the steps:

Step 1: to described five or the hydroxyl of hexa-atomic sugared or derivatives thereof protect; Make three halo nitriles or mercaptan and react, obtain at least a compd A through the hemiacetal hydroxyl of the sugared or derivatives thereof of hydroxyl protection;

Step 2: triphenylmethyl chloride and Oleanolic Acid or ursolic acid are reacted, and it is standby to obtain compd B;

Step 3: at least a compd A and compd B are reacted, and use the sodium alkoxide deprotection, obtain R ₅Compound for the chemical general formula I of hydrogen atom; Perhaps

At least a compd A and compd B are reacted, and with behind the acetate deprotection, again with step 1 at least a compd A of preparation react, use the sodium methylate deprotection then, wherein, described at least a compd A is identical or different.

Wherein, the group that carries out hydroxyl protection in the preferred steps 1 is benzyl and/or ethanoyl.

When described five in step 1 or hexa-atomic sugared or derivatives thereof are L-rhamnosyl, L-Fucose, D-seminose, L-arabinose, D-pectinose, D-ribose or D-glucuronic acid, preferably adopt benzyl to carry out hydroxyl protection.

When described five in step 1 or hexa-atomic sugared or derivatives thereof are D-glucose or D-wood sugar, preferably carry out hydroxyl protection with ethanoyl.

When being L-rhamnosyl, L-Fucose, D-seminose, L-arabinose, D-pectinose, D-ribose or D-glucuronic acid, adopt benzyl to carry out reacting with Trichloroacetonitrile and hemiacetal hydroxyl behind the hydroxyl protection for described five in the step 1 or hexa-atomic sugared or derivatives thereof.

When being D-glucose or D-wood sugar, carry out reacting with mercaptan and hemiacetal hydroxyl behind the hydroxyl protection with ethanoyl for described five in the step 1 or hexa-atomic sugared or derivatives thereof.

The 3rd purpose of the present invention is to provide the application of a kind of triterpene saponin compound in the medicine of preparation treatment cancer or dementia.

The 4th purpose of the present invention is to provide a kind of cancer or dull-witted pharmaceutical composition for the treatment of, and is liquid preparation, injection, powder, granule, capsule, pill, tablet, suppository, paste, flocculation agent, film, aerosol, sprays, powder inhalation, slowly-releasing and control-released agent, transdermal delivery system, targeting preparation, pulvis, lozenge or the cachet of the triterpene saponin compound of the above-mentioned chemical general formula I that comprises significant quantity.

The route of administration of described treatment cancer and dull-witted medicine is oral, injection, rectum or parenteral admin and local external use's administration.

Preferred vector is a starch, makes the capsule oral administration.

According to the present invention, can provide a kind of and have equal pharmaceutical use, but the more simple triterpene saponin compound of structure, provide a kind of easy, cost is low, weak point consuming time, yield is high and be easy to the synthetic method of the described triterpene saponin compound of industrialization.

Embodiment

Explain all respects of the present invention below in conjunction with embodiment.

The triterpene saponin compound of chemical general formula I

The invention provides the triterpene saponin compound of representing by following chemical general formula I.

Chemical general formula I

In above chemical general formula I, R ₁, R ₂And R ₃In any two be methyl, and another is a hydrogen atom.That is to say, with Oleanolic Acid structure (that is: R ₁, R ₂All be methyl, and R ₃Be hydrogen atom) or ursolic acid structure (that is: R ₁And R ₂In any one be methyl, another is hydrogen atom, and R ₃Be methyl) be agent structure.

R ₄(i.e. substituting group on 3 hydroxyls) can be for by five or hexa-atomic monose, or by described five or the disaccharides that forms of hexa-atomic monose, or the glycosyl that forms of the aldehydic acid derivative of described monose or disaccharides.R ₄Be preferably a kind of glycosyl that forms that is selected from by in L-rhamnosyl, D-glucose-(1 → 2)-D-xyloside, D-glucose, L-Fucose, D-seminose, L-arabinose, D-pectinose, D-wood sugar, D-ribose and the D-glucuronic acid, most preferably be the glycosyl that L-rhamnosyl or D-glucose-(1 → 2)-D-xyloside forms.

R ₅(i.e. substituting group on 28 carboxyls) can be hydrogen atom, perhaps can be for by five or hexa-atomic monose, or by described five or the disaccharides that forms of hexa-atomic monose, or the substituting group that forms of the aldehydic acid derivative of described monose or disaccharides.R ₅Be preferably hydrogen atom, or, most preferably be hydrogen atom, L-rhamanopyranosyl or D-xylosyl for to be selected from by a kind of substituting group that forms in L-rhamnosyl, D-glucose-(1 → 2)-D-xyloside, D-glucose, L-Fucose, D-seminose, L-arabinose, D-pectinose, D-wood sugar, D-ribose and the D-glucuronic acid.Work as R ₅During for the substituting group that forms by above-mentioned sugared or derivatives thereof, R ₄And R ₅Can be identical or different.

Triterpene saponin compound of the present invention can be the compound shown in following Formula I-1～I-4.

Formula I-1: Oleanolic Acid 3-O-α-L-pyrans rhamnosyl

Formula I-2: Oleanolic Acid 3-O-α-L-pyrans rhamnosyl 28-O-α-L-pyrans rhamnoside

Formula I-3: Oleanolic Acid 3-O-β-D-Glucopyranose-(1 → 2)-β-D-xylopyranoside

Compound I-4: Oleanolic Acid 3-O-β-D-Glucopyranose-(1 → 2)-β-D-xylopyranoside 28-O-α-L-pyrans rhamnoside

The inventor is surprised to find, though above-mentioned triterpene saponin compound is much simple in structure, has and natural triterpene saponin compound medicinal effect much at one, promptly can be used for the treatment of dementia, particularly senile dementia, alzheimer's disease etc.; Also can be used for treatment for cancer.Further studies show that, triterpene saponin compound shown in the chemical general formula I of the present invention is in treatment hypertension, the disease (as tuberculosis, leprosy etc.) that causes by pathogenic micro-organism, and autoimmune disorder (as transplant rejection, bronchial asthma, HBV, nephrotic syndrome, the hyperthyroidism deficiency of Yin, systemic lupus erythematous etc.) aspect also has gratifying effect.The medicinal use relevant with The compounds of this invention will describe in detail below.

In addition, the natural triterpene saponin compound that exists in the triterpene saponin compound shown in the chemical general formula I of the present invention and the plant is compared has much simple structure, thereby makes the commercial application of its synthesis pharmaceutical become possibility.Below will be described more specifically the synthetic method of triterpene saponin compound of the present invention.

The synthetic method of the triterpene saponin compound of chemical general formula I

Triterpene saponin compound shown in the above chemical general formula I adopts the convergence type semisynthesis to prepare.Alleged " semisynthesis " of this paper be meant with commercially available Oleanolic Acid or ursolic acid, and required five or hexa-atomic sugar be starting raw material carry out synthetic.Alleged " the convergence type method " of this paper is meant respectively to Oleanolic Acid or ursolic acid; and the sugar that will connect carries out the active group protection of non-reaction site; and active group is carried out in the site that will react replace; carry out glucosidesization or esterification then, go protection to obtain end product after reaction is finished.

The synthetic method of the triterpene saponin compound shown in the above chemical general formula I specifically may further comprise the steps:

Step 1: to described five or the hydroxyl of hexa-atomic sugared or derivatives thereof protect; Make Trichloroacetonitrile or mercaptan and react through the hemiacetal hydroxyl of the sugared or derivatives thereof of hydroxyl protection, it is standby to obtain at least a compd A;

Step 2: triphenylmethyl chloride and Oleanolic Acid or ursolic acid are reacted, and it is standby to obtain compd B;

Step 3: at least a compd A and compd B are reacted, and use the sodium alkoxide deprotection, obtain R ₅ Compound for the chemical general formula I of hydrogen atom; Perhaps

At least a compd A and compd B are reacted, and with behind the acetate deprotection, again with step 1 at least a compd A of preparation react, use the sodium methylate deprotection then, wherein, described at least a compd A can be identical or different.

Describe each step below respectively in detail.

Step 1: preparation compd A (activation of the hydroxyl protection of sugared or derivatives thereof and hemiacetal hydroxyl thereof replaces)

Described sugared or derivatives thereof is to prepare to be connected on 3 hydroxyls of above-mentioned chemical general formula I or 28 carboxyls so that form R ₄And/or R ₅Substituting group.The kind of sugar or derivatives thereof repeats no more as mentioned above here.Preferred hydroxy-protective group is benzyl and/or ethanoyl.Particularly, can adopt Benzoyl chloride (BzCl) or diacetyl oxide (Ac ₂O) react with sugared or derivatives thereof, but be not limited thereto.

For L-rhamnosyl, L-Fucose, D-seminose, L-arabinose, D-pectinose, D-ribose or D-glucuronic acid, preferably adopt benzyl to carry out hydroxyl protection, its advantage is the yield height of product, is easy to form crystal.

For D-glucose or D-wood sugar, preferably carry out hydroxyl protection with ethanoyl, its advantage is the yield height of product, is easy to form crystal.

Then, make Trichloroacetonitrile (Cl ₃CCN) or mercaptan (R ' SH, R ' is C ₁-C ₆Alkyl) react with hemiacetal hydroxyl through the sugared or derivatives thereof of hydroxyl protection, obtain product A, i.e. tribromo-acetyl imines ester compound or thio-ether type compounds.Mercaptan can be for example sulfur alcohol (EtSH), propylmercaptan, isopropyl mercaptan etc.

Preferably; for L-rhamnosyl, L-Fucose, D-seminose, L-arabinose, D-pectinose, D-ribose or D-glucuronic acid; adopt benzyl to carry out reacting with Trichloroacetonitrile and hemiacetal hydroxyl behind the hydroxyl protection; slough the protection of this position when being convenient to building-up reactions easily, subsequent reactions can be carried out smoothly.

Preferably,, carry out reacting with mercaptan and hemiacetal hydroxyl behind the hydroxyl protection with ethanoyl for D-glucose or D-wood sugar, be convenient to follow-up building-up reactions can slough smoothly the protection and react.

Can prepare the compd A of multiple sugar and derivative thereof in this step, so that be connected respectively to 3 and 28 of host compound; Perhaps form disaccharides at least one position in 3 and 28.

Step 2: preparation compd B (carboxy protective of Oleanolic Acid or ursolic acid)

Triphenylmethyl chloride and Oleanolic Acid or ursolic acid are reacted, on 28 carboxyls, form three benzene methyls, thereby obtain compd B.

Step 3: the compound of preparation chemical general formula I

1, preparation R ₅ Compound (replacements of 3 hydroxyls) for hydrogen atom

Compd A and compd B are reacted, use the sodium alkoxide deprotection, obtain R ₅Compound for the chemical general formula I of hydrogen atom.

At this moment, because the hydroxyl of compd A and the carboxyl of compd B protected by nonactive group, thereby have only the hemiacetal hydroxyl of compd A and three hydroxyls of compd B to react, the generation glycosidic link.This is reflected under the condition of low temperature (about zero degree), nitrogen protection and obtains coupled product, has realized sugar (or derivatives thereof) and hydroxyl link coupled feasibility.

Connect a disaccharides at 3 hydroxyl places if desired, then after the reaction of compd A and compd B is finished, do not carry out deprotection, react with additional compounds A continuing after the product separation and purification.Use the sodium alkoxide deprotection again after reaction is finished, thereby obtain the compound that 3 hydroxyl places connect the chemical general formula I of a disaccharides.

In preparation Compound I-4 o'clock, the product that above-mentioned reaction obtains beta comfiguration with the wood sugar sulphur glycosides and the Oleanolic Acid coupling selectivity of 2 hydroxyls, connect another sugar 2 again, realized that 1,2 disaccharides that connects optionally is connected with 3 hydroxyls of mode and host compound of beta comfiguration to wood sugar.Thereby, in reaction, make the unsettled α configuration of conformation be tending towards three more stable equatorial bonds, the beta comfiguration wood sugar synthetics of an axial bond.

In addition, deprotection adopts sodium alkoxide, for example sodium methylate, sodium ethylate etc.Reaction conditions gentleness during because of deprotection, thus guaranteed the stability of intermediate product ester group product.

2, preparation R ₅ Substituent compound for sugared or derivatives thereof formation

Compd A and compd B are reacted, use the acetate deprotection, obtain Compound C; The further reaction of compd A (can be identical, also can be different) that Compound C and step 1 are made with the compd A in the last step reaction, and use the sodium alkoxide deprotection, obtain R ₅Compound for the chemical general formula I of sugared or derivatives thereof.

Compd A and B react the back can slough trityl on 28 carboxyls of agent structure with acetate, and the protecting group on the sugared hydroxyl is sloughed.Can make product like this, 28 carboxyls of Compound C continue to react with other compd As.

If 3 any one sites with 28 in agent structure will be connected a disaccharides, then can carry out successive reaction with identical or different compd A, afterwards deprotection again according to mode as hereinbefore.Believe those of ordinary skill in the art under previously described instruction and, can understand and the preparation method who implements to be connected disaccharides, just repeat no more herein in conjunction with following specific embodiment.

Below with Oleanolic Acid as agent structure, according to synthetic embodiment the present invention is carried out more specific description, but the present invention is not limited by following synthetic embodiment.Those of ordinary skill in the art should be understood that in the triterpene saponin compound of chemical general formula I of the present invention, is to be reaction site with 3 hydroxyls and 28 carboxyls in the agent structure, and therefore, the Oleanolic Acid among the following synthetic embodiment all can be substituted by ursolic acid.

Synthesizing of embodiment 1 Oleanolic Acid 3-O-α-L-pyrans rhamnosyl

With natural product α-L-pyrans rhamnosyl (compound 1 is available from the Ke Bosi bio tech ltd) and Oleanolic Acid (compound 5 is available from Xi'an hat space Bioisystech Co., Ltd) as starting raw material.Synthesize according to following illustrated reaction scheme.

Specifically compound 1 (α-L-pyrans rhamnosyl) 20g is stirred under ice bath; get Benzoyl chloride (BzCl) 75ml and be dissolved among pyridine (Pyr) 150ml as solvent I; slowly splash in the above-mentioned solution; stir under the room temperature and spend the night; with the thin-layer chromatography detection reaction fully after; concentrating under reduced pressure, column chromatography (petrol ether/ethyl acetate=5/1) purifies and separates obtains compound 2 (33.8g).Compound 2 is dissolved in the mixed solvent of tetrahydrofuran (THF) (THF) and methyl alcohol (tetrahydrofuran (THF)/methyl alcohol=1/3), feed ammonia, after usefulness thin-layer chromatography detection reaction is complete, concentrating under reduced pressure, with column chromatography (petrol ether/ethyl acetate=4/1) purifies and separates, obtain compound 3 (17.6g).

Under nitrogen protection, compound 3 is dissolved among methylene dichloride (DCM) (with the dry methylene dichloride of crossing of the hydrolith) 100ml as solvent II; add Trichloroacetonitrile 26ml and 1; 8 diazabicyclos [5; 4; 0] undecylene-7 (DBU) 2.6ml, stirred overnight at room temperature, concentrating under reduced pressure then; with column chromatography (petrol ether/ethyl acetate=4/1) purifies and separates, obtain compound 4 (14.6g).

(Oleanolic Acid 10g) adds in the tetrahydrofuran (THF) (86 ℃ of tetrahydrofuran (THF) 55ml that anhydrate of oil bath), adds 1,8 diazabicyclo [5,4,0] undecylene-7 (DBU) 5ml and triphenylmethyl chloride (CCNCl with compound 5 ₃) 7.4g, 100 ℃ of stirring and refluxing, reaction is spent the night, and after usefulness thin-layer chromatography detection reaction was complete, concentrating under reduced pressure with column chromatography (petrol ether/ethyl acetate=7/1) purifies and separates, obtained compound 6 (7.5g).

Under the nitrogen protection compound 4 (2.3g) and compound 6 (3g) be dissolved among the methylene dichloride 60ml as solvent II; cryosel is bathed and is added trifluoromethyl sulfonic acid trimethylsilyl ester (TMSOTf) 100 μ l down; behind the stirring reaction 1.5～2.0 hours; with the thin-layer chromatography detection reaction fully after; add triethylamine neutralization reaction system; add methylene dichloride 500ml dilution; with saturated sodium bicarbonate washing; separating funnel separate dichloromethane part and with anhydrous sodium sulfate drying and concentrating under reduced pressure; with column chromatography (petrol ether/ethyl acetate=5/1) purifies and separates, obtain compound 7 (1.6g).

Compound 7 is dissolved in the methanol solution of sodium methylate, stirs under the room temperature, reacted 5～10 hours, after the thin-layer chromatography detection reaction is complete, extremely neutral with acidic ion exchange resin neutralization reaction system, the elimination resin, concentrating under reduced pressure is the Bio-gel P of deionized water with leacheate ₂Gel chromatographic columns (U.S. Bio-Rad company) purifying obtains compound 8, is target compound, Oleanolic Acid 3-O-α-L-pyrans rhamnosyl (0.82g, white solid, productive rate are 73%).

With Bruker ARX-400 at CDCl ₃In record ¹H and ¹³The C nuclear magnetic resonance spectrum, chemical shift is with Me ₄Si is interior mark, is unit representation with ppm.Mass detects on JEOL JMS-700 mass spectrograph.

[α] _D ²⁵+7.8°(c?1，H ₂O)； ¹H?NMR(400MHz，CDCl ₃)：δ0.72(s，6H，2CH ₃)，0.87(s，9H，3CH ₃)，1.09(s，3H，CH ₃)，1.10(s，3H，CH ₃)，2.74(dd，1H，J?3.9，10.8Hz，H-18?of?oleanolic?acid)，3.01(dd，1H，J?3.5，9.7Hz，H-3of?oleanolicacid)，3.17(m，1H)，3.40(m，1H)，3.49(m，1H)，3.62(br?s，1H)，4.52(d，1H)，4.58(s，1H)，4.70(m，2H)，5.16(br?s，1H，H-12of?oleanolic?acid)，12.05(s，1H). ¹³C?NMR(400MHz，CDCl ₃)：δ178.7，143.9，121.6，103.0，87.6，72.2，70.8，70.8，68.6，54.7，47.1，45.8，45.5，41.4，40.9，38.8，38.6，37.9，36.4，33.4，32.9，32.4，32.2，30.5，28.0，27.3，25.7，25.0，23.5，23.0，22.7，17.9，17.8，16.9，16.5，15.2.Anal.Calcd?for?C ₃₆H ₅₈O ₇：C，71.72；H，9.70；Found：C，71.90；H，9.82。

Solvent I described in the reaction is pyridine or triethylamine; Solvent II is methylene dichloride, acetonitrile, toluene or their mixture.

Synthesizing of embodiment 2 Oleanolic Acid 3-O-α-L-pyrans rhamnosyl 28-O-α-L-pyrans rhamnoside

With the compound 7 that obtains among the embodiment 1 and compound 4 as starting raw material.Synthesize according to following illustrated reaction scheme.

Compound 8 (8g) with obtaining among the embodiment 1 adds 80% acetate 20ml, after stirring 2 hours under 70 ℃, with the thin-layer chromatography detection reaction fully after, concentrating under reduced pressure with column chromatography (petrol ether/ethyl acetate=7/1) purifies and separates, obtains compound 9 (6.4g).

Under the nitrogen protection compound 9 (4g) and compound 4 (2.3g) be dissolved among the methylene dichloride 100ml as solvent II; add N-iodo succimide 1.1g under the stirring at room; be cooled to-40 ℃～-50 ℃; add trifluoromethyl sulfonic acid trimethylsilyl ester 100 μ l;-35 ℃～-45 ℃ are stirred after 1.5～2.0 hours down; with the thin-layer chromatography detection reaction fully after; add triethylamine neutralization reaction system; add methylene dichloride 500ml dilution then; with saturated sodium bicarbonate washing; separating funnel separate dichloromethane part; with anhydrous sodium sulfate drying and concentrating under reduced pressure; with column chromatography (petrol ether/ethyl acetate=7/1) purifies and separates, obtain compound 10 (5.1g).

Compound 10 (2g) is dissolved in the methanol solution of sodium methylate, stirs under the room temperature, room temperature reaction 5～10 hours, the thin-layer chromatography detection reaction is complete, and is extremely neutral with acidic ion exchange resin neutralization reaction system, the elimination resin, concentrating under reduced pressure is the Bio-gel P of deionized water with leacheate ₂Gel chromatographic columns (U.S. Bio-Rad company) purifying obtains compound 11, is target compound, Oleanolic Acid 3-O-α-L-pyrans rhamnosyl 28-O-α-L-pyrans rhamnoside (1.6g, white solid, productive rate are 72%).

With Bruker ARX-400 at CDCl ₃In record ¹H and ¹³The C nuclear magnetic resonance spectrum, chemical shift is with Me ₄Si is interior mark, is unit representation with ppm.Mass detects on JEOL JMS-700 mass spectrograph.

[α] _D ²⁵+11°(c?1.5，H ₂O)； ¹H?NMR(400MHz，CDCl ₃)：δ0.79-1.91(m，49H)，2.90(dd，1H，J?4.1，12.8Hz，H-18?of?oleanolic?acid)，3.09(dd，1H，J?4.2，11.3Hz，H-3of?oleanolic?acid)，3.35(t，1H，J?6.9，12.0Hz)，3.43(t，1H，J?9.5Hz)，3.61-3.72(m，4H)，3.75(br?s，1H)，3.82(br?s，1H)，4.72(d，1H，J?2.1Hz，H-1)，5.31(br?s，1H，H-12of?oleanolic?acid)，5.92(d，1H，J?2.3Hz，H-1). ¹³C?NMR(400MHz，CDCl ₃)：δ175.3，143.6，122.7，103.1，93.6，87.8，72.4，71.8，71.4，71.1，71.0，70.9，69.9，68.8，55.2，47.3，46.9，45.7，41.6，41.5，38.4，36.7，33.5，33.1，32.8，32.5，30.8，28.3，27.35，25.98，26.0，25.3，23.7，23.4，22.8，18.2，18.1，17.3，16.8，15.5.Anal.Calcd?for?C ₄₂H ₆₈O ₁₁：C，67.35；H，9.15；Found：C，67.60；H，9.08。

Synthesizing of embodiment 3 Oleanolic Acid 3-O-β-D-Glucopyranose-(1 → 2)-β-D-xylopyranoside

With natural product β-D-xylopyranose (compound 1, available from the strong Bioisystech Co., Ltd in sky, Zhengzhou), β-D-glucose (compound 9, available from Beijing Ge Laimengte International Trading Company Ltd) and Oleanolic Acid (compound 12 is available from Xi'an hat space Bioisystech Co., Ltd) as starting raw material.Synthesize according to following illustrated reaction scheme.

Specifically compound 1 (β-D-xylopyranose) 20g is dissolved among the pyridine 200ml as solvent I, add diacetyl oxide 81.6ml, stirring reaction is 2～4 hours under the room temperature, after the thin-layer chromatography detection reaction is complete, concentrate, column chromatography (petrol ether/ethyl acetate=5/1) purifies and separates obtains compound 2 (38 gram).Compound 2 (38g) is dissolved under nitrogen protection among the methylene dichloride 200ml as solvent II; and adding hydrogen bromide 100ml; stirring reaction is 1～2 hour under the room temperature; thin-layer chromatography detect show react completely after; concentrating under reduced pressure; with column chromatography purifies and separates (petrol ether/ethyl acetate=4/1), obtain compound 3 (20 gram).

(20g) adds 2 under nitrogen protection with compound 3; 6-lutidine (2; 6-lutidine) 10ml; sulfur alcohol (EtSH) 8.8ml and Nitromethane 99Min. 74.1ml; stirring reaction spends the night under the room temperature, and thin-layer chromatography detects and to show and react completely concentrating under reduced pressure; with column chromatography purifies and separates (petrol ether/ethyl acetate=2/1), obtain compound 4 (15.4g).

Compound 4 (15.4g) is dissolved in the methyl alcohol as solvent II I, stir under the room temperature, the methanol solution that adds sodium methylate, make the pH value between 9 to 11, room temperature reaction 2～4 hours, after the thin-layer chromatography detection reaction is complete, concentrate, with column chromatography purifies and separates (petrol ether/ethyl acetate=2/1), obtain oily compound 5 (10.2g), compound 5 (10.2g) is dissolved among the pyridine 150ml as solvent I, ice-water bath stirs down; get Benzoyl chloride 12.1ml and be dissolved among the pyridine 12ml as solvent I, slowly splash in the above-mentioned solution, stirred under the room temperature 3～6 hours; thin-layer chromatography detect show react completely after; concentrating under reduced pressure with column chromatography purifies and separates (petrol ether/ethyl acetate=10/1), obtains compound 6 (15.5g).

Under nitrogen protection, compound 6 (15.5g) is dissolved among the methylene dichloride 100ml as solvent II; add zinc chloride (1M/L) 5ml; ice-water bath stirs concentrating under reduced pressure after 3～6 hours down, with column chromatography purifies and separates (petrol ether/ethyl acetate=10/1), obtains compound 7 (15g).

With compound 7 (15g) be dissolved in as the methylene dichloride of solvent II with as in the mixed solution of the methyl alcohol of solvent IV (1: 1,150ml), ice-water bath adds Acetyl Chloride 98Min. 20ml down, 1 hour recession ice bath, stirred under the room temperature 3～6 hours, after the thin-layer chromatography detection reaction is complete, concentrating under reduced pressure, with column chromatography purifies and separates (petrol ether/ethyl acetate=6/1), obtain compound 8 (12g).

Compound 9 (β-D-glucose) 25g is added anhydrous sodium acetate (22.5g) and diacetyl oxide (100ml) and in 150 ℃ of oil bath pans, stir, reflux, after reacting half an hour, with the thin-layer chromatography detection reaction fully after, extraction concentrates, recrystallization obtains compound 10 (35g), get 10g and be dissolved in the 50ml methylene dichloride, under ice-water bath, add sulfur alcohol 2.85ml, boron trifluoride diethyl etherate 9.7ml, stirring reaction 1 hour, with the thin-layer chromatography detection reaction fully after, extraction concentrates, and with column chromatography (petrol ether/ethyl acetate=2/1) purifies and separates, obtains compound 11 (7g).

With compound 12 (Oleanolic Acids, 10g) add in the tetrahydrofuran (THF) (86 ℃ of tetrahydrofuran (THF) 55ml that anhydrate of oil bath), add 1,8 diazabicyclo [5,4,0] undecylene-7 (DBU) 5ml and triphenylmethyl chloride 7.4g, 100 ℃ of stirring and refluxing, reaction is spent the night, with the thin-layer chromatography detection reaction fully after, concentrating under reduced pressure with column chromatography (petrol ether/ethyl acetate=7/1) purifies and separates, obtains compound 13 (7.5g).

Under the nitrogen protection compound 8 (4g) and compound 13 (5.8g) be dissolved in methylene dichloride 100ml as solvent II; add N-iodo succimide (NIS) 2.2g under the stirring at room; be cooled to-40 ℃～-50 ℃; add trifluoromethyl sulfonic acid trimethylsilyl ester 200 μ l;-35 ℃～-45 ℃ are stirred after 1.5～2.0 hours down; with the thin-layer chromatography detection reaction fully after; add triethylamine neutralization reaction system; add methylene dichloride 500ml dilution then; with saturated sodium bicarbonate washing; separating funnel separate dichloromethane part; with anhydrous sodium sulfate drying and concentrating under reduced pressure; with column chromatography (petrol ether/ethyl acetate=7/1) purifies and separates, obtain compound 14 (7.1g).

Under the nitrogen protection compound 14 (4.4g) and compound 11 (2g) be dissolved in methylene dichloride 50ml as solvent II; add N-iodo succimide 1.1g under the stirring at room; be cooled to-40 ℃～-50 ℃; add trifluoromethyl sulfonic acid trimethylsilyl ester 100 μ l;-35 ℃～-45 ℃ are stirred after 1.5～2.0 hours down; with the thin-layer chromatography detection reaction fully after; add triethylamine neutralization reaction system; add methylene dichloride 500ml dilution then; with saturated sodium bicarbonate washing; separating funnel separate dichloromethane part; with anhydrous sodium sulfate drying and concentrating under reduced pressure; with column chromatography (petrol ether/ethyl acetate=7/1) purifies and separates, obtain compound 15 (3.6g).

Compound 15 (3.6g) is dissolved in the methanol solution of sodium methylate, stirs under the room temperature, room temperature reaction 5～10 hours, the thin-layer chromatography detection reaction is complete, and is extremely neutral with acidic ion exchange resin neutralization reaction system, the elimination resin, concentrating under reduced pressure is the Bio-gel P of deionized water with leacheate ₂Gel chromatographic columns (U.S. Bio-Rad company) purifying obtains compound 16, is target compound, Oleanolic Acid 3-O-β-D-Glucopyranose-(1 → 2)-β-D-xylopyranoside (1.2g, white solid, productive rate are 72%).

With Bruker ARX-400 at CDCl ₃In record ¹H and ¹³The C nuclear magnetic resonance spectrum, chemical shift is with Me ₄Si is interior mark, is unit representation with ppm.Mass detects on JEOL JMS-700 mass spectrograph.

[α] _D ²⁵+7.2°(c?1，H ₂O)； ¹H?NMR(400MHz，CDCl ₃)：δ0.35，0.82，0.88，0.90，0.91，1.11，1.29(7s，7×3H，7CH ₃)，2.83(dd，1H，J?4.1，13.0Hz，H-18?ofoleanolic?acid)，3.25(dd，1H，J?4.4，11.6Hz，H-3of?oleanolic?acid)，3.66-4.87(m，28H)，4.82(d，1H，J?7.3Hz，H-1?of?Xyl)，4.98(d，1H，J?7.8Hz，H-1ofGlc)，5.20-5.25(m，12H)，5.29(t，1H，J?3.2，H-12of?oleanolic?acid). ¹³C?NMR(400MHz，CDCl ₃)：δ176.4，144.0，122.8，104.8，101.8，95.6，88.4，79.5，78.6，78.0，77.1，76.4，75.3，74.0，72.7，72.3，70.7，69.7，69.1，66.9，56.0，48.0，46.9，46.1，42.0，41.6，39.8，36.9，33.9，33.0，32.4，30.7，27.9，28.2，26.0，23.7，23.6，23.3，17.4，17.0，15.6.Anal.Calcd?for?C ₄₁H ₆₆O ₁₂：C，65.57；H，8.86；Found：C，65.57；H，8.86。

Solvent I described in the reaction is pyridine or triethylamine; Solvent II is methylene dichloride, acetonitrile, toluene or their mixture; Solvent II I is methyl alcohol or ethanol.

Synthesizing of embodiment 4 Oleanolic Acid 3-O-β-D-Glucopyranose-(1 → 2)-β-D-xylopyranoside 28-O-α-L-pyrans rhamnoside

With the compound 15 that obtains among the embodiment 3 and compound 8 as starting raw material.Synthesize according to following illustrated reaction scheme.

Compound 15 (4g) with obtaining among the embodiment 3 adds 80% acetate 20ml, after stirring 2 hours under 70 ℃, with the thin-layer chromatography detection reaction fully after, concentrating under reduced pressure with column chromatography (petrol ether/ethyl acetate=7/1) purifies and separates, obtains compound 17 (3.1g).

Under the nitrogen protection compound 17 (2g) and compound 8 (0.9g) be dissolved in methylene dichloride 50ml as solvent II; add N-iodo succimide 0.7g under the stirring at room; be cooled to-40 ℃～-50 ℃; add trifluoromethyl sulfonic acid trimethylsilyl ester 76 μ l;-35 ℃～-45 ℃ are stirred after 1.5～2.0 hours down; with the thin-layer chromatography detection reaction fully after; add triethylamine neutralization reaction system; add methylene dichloride 500ml dilution then; with saturated sodium bicarbonate washing; separating funnel separate dichloromethane part; with anhydrous sodium sulfate drying and concentrating under reduced pressure; with column chromatography (petrol ether/ethyl acetate=7/1) purifies and separates, obtain compound 18 (1.9g).

Compound 18 (1.9g) is dissolved in the methanol solution of sodium methylate, stirs under the room temperature, room temperature reaction 5～10 hours, the thin-layer chromatography detection reaction is complete, and is extremely neutral with acidic ion exchange resin neutralization reaction system, the elimination resin, concentrating under reduced pressure is the Bio-gel P of deionized water with leacheate ₂Gel chromatographic columns (U.S. Bio-Rad company) purifying obtains compound 19, be target compound, Oleanolic Acid 3-O-β-D-Glucopyranose-(1 → 2)-β-D-xylopyranoside 28-O-α-L-pyrans rhamnoside (0.9g, white solid, productive rate are 72%).

With Bruker ARX-400 at CDCl ₃In record ¹H and ¹³The C nuclear magnetic resonance spectrum, chemical shift is with Me ₄Si is interior mark, is unit representation with ppm.Mass detects on JEOL JMS-700 mass spectrograph.

[α] _D ²⁵+6°(c?1.2，H ₂O)； ¹H?NMR(400MHz，CDCl ₃)：δ0.38，0.89，0.91，0.93，0.98，1.13，1.26，1.31(8s，8×3H，8CH ₃)，2.89(dd，1H，J?3.8.，11.2Hz，H-18of?oleanolic?acid)，3.34(dd，1H，J?4.7，11.7Hz，H-3of?oleanolic?acid)，3.57-4.89(m，32H)，4.88(d，1H，J?7.5Hz，H-1of?Xyl)，5.11(d，1H，J?7.8Hz，H-1ofGlc)，5.26-5.29(m，14H)，5.32(t，1H，J?3.2，H-12?of?oleanolic?acid)，6.53(br?s1H，H-1?of?Rha). ¹³C?NMR(400MHz，CDCl ₃)：δ177.8，172.3，170.8，162.5，158.3，151.6，149.3，145.2，143.1，139.6，133.8，128.7，125.3，122，9，114.3，111.2，108.3，102.1，98.6，92.3，86.7，79.4，73.5，69.3，62.1，58.8，52.2，48.3，43.2，40.5，38.6，35.4，33.8，31.6，29.7，26.5，25.7，23.6，22.4，21.3，20.1，19.8，18.7，16.4，15.8，15.2，14.5.Anal.Calcd?for?C ₄₇H ₇₆O ₁₆：C，62.93；H，8.54；Found：C，63.11；H，8.66。

The medicinal use of the triterpene saponin compound of chemical general formula I

Purposes in the preparation cancer therapy drug

The antitumous effect research of embodiment 5 triterpene saponin compounds

1. material and method

Material

1.1 clone

Human cervical carcinoma Siha cell strain .... available from Wuhan University cell classical collection center

People's liver cancer Hep3B cell strain .... available from Wuhan University cell classical collection center

Human hepatoma HepG2 cell's strain .... available from Wuhan University cell classical collection center

The strain of human embryo lung (HEL) hel cell .... available from Wuhan University cell classical collection center

1.2 the source of medicine

A triterpenoid saponin 1 (the Oleanolic Acid 3-O-α that obtains among the embodiment 1-L-pyrans rhamnosyl) 90%.

B triterpenoid saponin 2 (Oleanolic Acid 3-O-β-D-Glucopyranose-(1 → 2)-β-D-xylopyranoside that obtains among the embodiment 3) 90%.

The C Oleanolic Acid

The D cis-platinum

1.3 medicine and reagent

High sugared DMEM cultivates powder U.S. Sigma company product

Foetal calf serum Hangzhou folium ilicis chinensis company product

Trypsinase Wuhan Ke Rui biotech company

Tetramethyl-azo azoles salt (MTT) U.S. Sigma company product

Dimethyl sulfoxide (DMSO) (DMSO) U.S. Sigma company product

Penicillin, Streptomycin sulphate, dehydrated alcohol, NaHCO ₃, NaCI, KCI, Na ₂HPO ₄And KH ₂PO ₄Purchase in domestic each reagent company Deng chemical reagent

1.4 main equipment and reagent

Bechtop

CO ₂Incubator MODELTC 2323 CO ₂INCUBATOR

Enzyme-linked immunosorbent assay instrument

96 porocyte culture plates, COSTAR; Blood-coagulation-board

The adjustable micropipet of 10-200 μ L;

Olympus (Olympus) inverted microscope (CKX-31)

15ml import centrifuge tube, COSTAR

The 25mm inlet filter

1.5 main solution preparation

High sugared DMEM nutrient solution: get high sugared DMEM and cultivate powder one bag, be dissolved in the 800ml tri-distilled water, stirring and dissolving adds the Na of 2g ₂HCO ₃, to get 800,000 u/ and prop up penicillin and add 4ml tri-distilled water dissolving and draw 0.5ml, the substratum final concentration is 100u/ml.1,000,000 u/ prop up the dissolving of Streptomycin sulphate 5ml tri-distilled water and draw 0.5ml, substratum final concentration 100u/ml.Regulating the pH value is 7.1-7.3, is settled to 1000ml.Filtration sterilization.4 ℃ of preservations of packing.

PBS liquid: the Na that gets 2.55g ₂HPO ₄12H ₂O, the NaCl of 8g, the KCl of 0.2g, the KH of 0.2g ₂PO ₄, being dissolved in the packing of tri-distilled water 1000ml mixing, autoclaving is placed on 4 ℃ of freezer storages.

0.25% trypsin solution: get trypsinase powder 250mg and join no Ca ²⁺, Mg ²⁺PBS liquid in, earlier with a little disinfectant PBS with trypsinase powder furnishing pasty state, supply PBS liquid again to 100ml, stirring and evenly mixing, put 4 ℃ of freezer storages and spend the night, after filtering with filter paper, with 0.22 μ m filtering with microporous membrane degerming, divide the bottle of packing into, each bottle 10ml places-20 ℃ of refrigerators to preserve.

MTT liquid: take by weighing the MTT of 25mg, add the PBS liquid of 5ml, mixing is made into the MTT mother liquor of 5mg/ml, with 0.22 μ m filtering with microporous membrane degerming, lucifuge, 4 ℃ of freezer storages.

PI liquid: the PI that gets 5mg adds the Triton liquid of 1ml, adds to 100ml with PBS liquid again, and the final concentration that makes PI is 50 μ g/ml, and pH is 7.2,4 ℃ and keeps in Dark Place.

Frozen storing liquid: DMSO is mixed at 1: 9 airtight 4 ℃ of preservations with the nutrient solution that contains 20% foetal calf serum.

2. cell culture processes

2.1 the cultivation of human cervical carcinoma Siha cell

The Siha cell attachment grows in the high sugared DMEM nutrient solution that contains 10% foetal calf serum, at 37 ℃ of volume fraction 5%CO ₂With conventional cultivation the under the saturated humidity condition, 1-2d changes liquid and goes down to posterity once.

2.2 the cultivation of human embryo lung (HEL) hel cell

The hel cell adherent growth is in the high sugared DMEM nutrient solution that contains 10% foetal calf serum, at 37 ℃ of volume fraction 5%CO ₂With conventional cultivation the under the saturated humidity condition, 1-2d changes liquid and goes down to posterity once.

2.3 the cultivation of people's liver cancer Hep3B cell

The Hep3B cell attachment grows in the high sugared DMEM nutrient solution that contains 10% foetal calf serum, at 37 ℃ of volume fraction 5%CO ₂With conventional cultivation the under the saturated humidity condition, 1-2d changes liquid and goes down to posterity once.

2.4 human hepatoma HepG2 cell's cultivation

The HepG2 cell attachment grows in the high sugared DMEM nutrient solution that contains 10% foetal calf serum, at 37 ℃ of volume fraction 5%CO ₂With conventional cultivation the under the saturated humidity condition, 1-2d changes liquid and goes down to posterity once.

2.5 cell cryopreservation

The cell in vegetative period of taking the logarithm, change liquid once frozen the day before yesterday, and single cell suspension is made in conventional digestion, moves in the centrifuge tube, 1000rpm, centrifugal 10min discards nutrient solution, add frozen storing liquid, make cell suspension, move to that (freeze-stored cell density reaches 10 in the frozen pipe with suction pipe piping and druming ⁷/ ml), and airtight, mark time, title, carry out cooling stage by stage: 4 ℃ of following 3h ,-20 ℃ of following 2h ,-80 ℃ of following 1h are stored in liquid nitrogen (196 ℃) jar.

2.6 cell recovery

From liquid nitrogen container, take out required cell cryopreservation pipe rapidly, put into 37 ℃ of water baths and thaw rapidly, centrifugal then (1000rpm, 5min).Discard frozen storing liquid, add fresh medium and wash twice, cell is moved to adding proper amount of fresh nutrient solution in the culturing bottle, place in the incubator and cultivate, change liquid next day.The vegetative period cell of taking the logarithm experimentizes.

3.MTT the colorimetric method for determining medicine is to the inhibited proliferation of tumour cell

3.1 inhibited proliferation to the Siha cell

The Siha cell 1 * 10 of taking the logarithm vegetative period ⁵/ ml is inoculated in 96 well culture plates, add different concns triterpenoid saponin 1, triterpenoid saponin 2 respectively, every pore volume 100 μ g, test every group and establish 3 holes again, be total to triplicate (n=9), the medicinal DMSO dissolving of Oleanolic Acid adds 1 μ l and adds in the hole of 100 μ l substratum, provides equally different solubility are set.Establish the zeroing hole simultaneously, blank hole, positive drug cis-platinum and DMSO hole.Put 37 ℃, volume fraction 5%CO ₂After cultivating 48h in the incubator, and reference literature (Zhang Mingqing, Lian Mulan, after death cell is differentiated, Cell Biology Experiment method and technology, Beijing: press of Beijing Normal University, 1992:60; Xu Jing, Wu Feng, use the susceptibility that mtt assay detects tumor chemotherapeutic drug, China's medical science, 2005,18 (1): 140-143), every hole adds MTT liquid (5mg/ml) 20 μ l and continues to cultivate 4h, discard nutrient solution in the hole, add DMSO 50 μ l vibrators vibration 10min, in the OD value that 570nm wavelength place measures each hole, obtain inhibitory rate of cell growth by following formula with enzyme-linked immunosorbent assay instrument.

Cell inhibitory rate=(1-experimental group OD average/blank group OD average) * 100%

According to the corresponding relation of drug level and cell inhibitory rate, draw concentration-inhibiting rate curve, obtain half-inhibition concentration (IC ₅₀).

3.2 inhibited proliferation to the Hep3B cell

The Hep3B cell 1 * 10 of taking the logarithm vegetative period ⁵/ ml is inoculated in 96 well culture plates, add different concns triterpenoid saponin 1, triterpenoid saponin 2 respectively, every pore volume 100ug, test every group and establish 3 holes again, be total to triplicate (n=9), the medicinal DMSO dissolving of Oleanolic Acid adds 1 μ l and adds in the hole of 100 μ l substratum, provides equally different solubility are set.Establish the zeroing hole simultaneously, blank hole, positive drug cis-platinum and DMSO.Put 37 ℃, volume fraction 5%CO ₂After cultivating 48h in the incubator, every hole adds MTT liquid (5mg/ml) 20 μ l continues to cultivate 4h, discards nutrient solution in the hole, adds DMSO50 μ l vibrator vibration 10min, in the OD value that 570nm, wavelength place measure each hole, obtain inhibitory rate of cell growth with enzyme-linked immunosorbent assay instrument by following formula.

Cell inhibitory rate=(1-experimental group OD average/blank group OD average) * 100%

According to the corresponding relation of drug level and cell inhibitory rate, draw concentration-inhibiting rate curve, obtain half-inhibition concentration (IC ₅₀).

3.3 inhibited proliferation to the HepG2 cell

The HepG2 cell 1 * 10 of taking the logarithm vegetative period ⁵/ ml is inoculated in 96 well culture plates, add different concns triterpenoid saponin 1, triterpenoid saponin 2 respectively, every pore volume 100 μ g, test every group and establish 3 holes again, be total to triplicate (n=9), the medicinal DMSO dissolving of Oleanolic Acid adds 1 μ l and adds in the hole of 100 μ l substratum, provides equally different solubility are set.Establish the zeroing hole simultaneously, blank hole, positive drug cis-platinum and DMSO.Put 37 ℃, volume fraction 5%CO ₂After cultivating 48h in the incubator, every hole adds MTT liquid (5mg/ml) 20 μ l continues to cultivate 4h, discards nutrient solution in the hole, adds DMSO 50 μ l vibrators vibration 10min, in the OD value that 570nm, wavelength place measure each hole, obtain inhibitory rate of cell growth with enzyme-linked immunosorbent assay instrument by following formula.

Cell inhibitory rate=(1-experimental group OD average/blank group OD average) * 100%

According to the corresponding relation of drug level and cell inhibitory rate, draw concentration-inhibiting rate curve, obtain half-inhibition concentration (IC ₅₀).

3.4 inhibited proliferation to hel cell

The Hel cell 1 * 10 of taking the logarithm vegetative period ⁵/ ml is inoculated in 96 well culture plates, add different concns triterpenoid saponin 1, triterpenoid saponin 2 respectively, every pore volume 100 μ g, test every group and establish 3 holes again, be total to triplicate (n=9), the medicinal DMSO dissolving of Oleanolic Acid adds 1 μ l and adds in the hole of 100 μ l substratum, provides equally different solubility are set.Establish the zeroing hole simultaneously, blank hole, positive drug cis-platinum and DMSO.Put 37 ℃, volume fraction 5%CO ₂After cultivating 48h in the incubator, every hole adds MTT liquid (5mg/ml) 20 μ l continues to cultivate 4h, discards nutrient solution in the hole, adds DMSO50 μ l vibrator vibration 10min, in the OD value that 570nm wavelength place measures each hole, obtain inhibitory rate of cell growth with enzyme-linked immunosorbent assay instrument by following formula.

Cell inhibitory rate=(1-experimental group OD average/blank group OD average) * 100%

According to the corresponding relation of drug level and cell inhibitory rate, draw concentration-inhibiting rate curve, obtain half-inhibition concentration (IC ₅₀).

4. result

Different pharmaceutical is to the inhibited proliferation of different tumour cells

4.1 the inhibited proliferation of triterpenoid saponin 1,2 pairs of Siha cell growths of triterpenoid saponin

Each experimental group acts on the Siha cell 48h of vitro culture, and mtt assay is measured the inhibiting rate of its on cell proliferation.The result shows, Siha cell: the strongest (IC of triterpenoid saponin 2 restraining effect ₅₀=28.89 μ g/ml), the triterpenoid saponin 1 (IC that takes second place ₅₀=39.64 μ g/ml), the most weak (IC of Oleanolic Acid medicine ₅₀=14.89 μ g/ml).The results are shown in Table 1-table 3.

The inhibited proliferation of 1 pair of Siha cell of table 1 triterpenoid saponin

(IC ₅₀＝39.64μg/ml)

The inhibited proliferation of 2 pairs of Siha cells of table 2 triterpenoid saponin

(IC ₅₀＝28.89μg/ml)

Table 3 Oleanolic Acid is to the inhibited proliferation of Siha cell

(IC ₅₀＝14.89μg/ml)

4.2.Hep3B cell: the restraining effect of triterpenoid saponin 1, triterpenoid saponin 2 is suitable, (the IC of triterpenoid saponin 1 ₅₀=84.10 μ g/ml), (IC of triterpenoid saponin 2 ₅₀=21.08 μ g/ml), the most weak (IC of Oleanolic Acid medicine ₅₀=19.75 μ g/ml).The results are shown in Table 4-table 6.

The inhibited proliferation of 1 pair of Hep3B cell of table 4 triterpenoid saponin

(IC ₅₀＝84.10μg/ml)

The inhibited proliferation of 2 pairs of Hep3B cells of table 5 triterpenoid saponin

(IC ₅₀＝21.08μg/ml)

Table 6 Oleanolic Acid is to the inhibited proliferation of Hep3B cell

(IC ₅₀＝19.75μg/ml)

4.3.HepG2 cell: the results are shown in Table 7-table 9.

The inhibited proliferation of 1 pair of HepG2 cell of table 7 triterpenoid saponin

(IC ₅₀＝13.21μg/ml)

The inhibited proliferation of 2 pairs of HepG2 cells of table 8 triterpenoid saponin

(IC ₅₀＝54.21μg/ml)

Table 9 Oleanolic Acid is to the inhibited proliferation of HepG2 cell

(IC ₅₀＝31.72μg/ml)

4.4.HEL cell: the results are shown in Table 10-table 12.

The inhibited proliferation of 1 pair of hel cell of table 10 triterpenoid saponin

(IC ₅₀＝59.90μg/ml)

The inhibited proliferation of 2 pairs of hel cells of table 11 triterpenoid saponin

(IC ₅₀＝47.94μg/ml)

Table 12 Oleanolic Acid is to the inhibited proliferation of hel cell

(IC ₅₀＝38.47μg/ml)

Purposes in the preparation antidementia agent

The research of the dementia resisting effect of embodiment 6 triterpene saponin compounds (to the influence of insomnia sign rat model learning and memory)

1 material

1.1 medicine

Triterpenoid saponin 1 (the Oleanolic Acid 3-O-α that obtains among the embodiment 1-L-pyrans rhamnosyl) 90%.

Triterpenoid saponin 2 (Oleanolic Acid 3-O-β-D-Glucopyranose-(1 → 2)-β-D-xylopyranoside that obtains among the embodiment 3) 90%

SHULE ANDING PIAN, Heng Ruida pharmaceutical Co. Ltd in Shanxi produces, lot number: 070201.

1.2 reagent

The D-semi-lactosi: reagent two factories in Shanghai produce, and lot number is 070124.Being made into concentration with physiological saline is that 1.5%, 4 ℃ of refrigeration is standby.Laboratory animal is pressed 60mgkg ^-1D ^-1The subcutaneous injection of standard nape portion;

Cyclophosphamide Injection (CPA): Hengrui Medicine Co., Ltd., Jiangsu Prov., lot number 070226.Laboratory animal is pressed 100mgkg ^-1D ^-1(with reference to Miao Mingsan, Wang Zhiming, Sun Yanhong, the bosom prepared rehmannia root polysaccharide is to the influence of deficiency of blood rat serum picture and cytokine levels, Pharmacology and Clinics of Chinese Materia Medica, 2007,23 (1): 39-40.) for the standard abdominal injection.

Hydrocortisone injection: Shandong XinHua Pharmacy stock Co., Ltd, lot number 060404.Laboratory animal is pressed 50mgkg ^-1D ^-1(with reference to Hou Ying, Chen Ru, Ju rock etc., the antitoxin capsule of yin-nourishing causes the influence of deficiency of Yin rat chest gland, Mountain Western Medicine S University's journal, 2006,37 (5): 495-498.) to hydrocortisone to the standard abdominal injection.

1.3 instrument

Sleep deprivation case (self-control): be the mouse case of long 72cm, wide 48cm, high 30cm, built-in 6 disjunctor diameters are 8cm, high is the platform of 8cm (being 2 * 3 arrangements), platform interbody spacer 16cm, platform and mouse box body wall be 8cm at interval, fills with water at the platform periphery, the about 1.0cm of water surface anomaly table top, rat diet drinking-water voluntarily on platform, and can be free movable on different platform.On platform, put and shroud, shroud with parallel Stainless Steel Wire and make, discharge water above and food.Water temperature and room temperature are consistent, and are controlled at about 22-24 ℃, change water every day and sweep experimental box.

Environment contrast case (self-control): similar to the sleep deprivation case, but at placement platform not apart from its 8.0cm place, bottom, but place the fine and closely woven wire netting of one side, at the water of filling with off the net, the water surface is apart from the about 1.0cm of wire side, rat is diet drinking-water and free activity voluntarily on the net, to form the environment similar to the sleep deprivation group.Other conditions are all identical with the sleep deprivation group.

The automatic experimental record instrument of Morris water maze is the institute of Materia Medica,Chinese Academy of Medical Sciences development.

1.4 animal grouping

60 of healthy male and female half and half Wistar rats, (200g ± 20g), laboratory animal and feed provide conformity certification by Tongji Medical College, Huazhong Science and Technology Univ. experimentation on animals center to body weight: SCXK Hubei Province 2004-007.Before the experiment rat is placed on adaptation 2 weeks of raising in the same cage, is divided into 5 groups during experiment at random: environment control group, sign model group, estazolam group, 1 group of triterpenoid saponin, 2 groups of triterpenoid saponins, 12 every group.After buying 1 week (w) that conforms, with the scalping of Y type water maze method, correct number of times reaches more than 12 times the person for qualified in all 15 tests.

2 methods

2.1 model preparation and administration

The foundation of old anemia deficiency of Yin insomnia sign rat model

2.1.1 animal grouping

40 of male and female half and half Wistar rats are divided into 2 groups at random, 20 every group: the environment control group; Sign model group (old anemia YIN-deficiency type insomnia sign model group).

2.1.2 (with reference to Guo Hongyan, Luan Jing, Zhang Pengxia etc., the cornusol extract causes decline rat nonenzymatic glycosylation and the active influence of AR to the D-semi-lactosi, Heilungkiang medical science, 2004,27 (6): 1-2.) in the preparation of subacute aging model.

With the subcutaneous injection 6 all D-semi-lactosi 60mgkg of sign model group rat nape portion ^-1D ^-1, prepare subacute aged animal model.

The administration volume is 0.4ml100g ^-1, the environment control group is given the physiological saline of the capacity of grade.

2.1.3 anemia deficiency of Yin sign Preparation of model [15]

After sign model group rat is given the full 6w of D-semi-lactosi, cyclophosphamide Injection is pressed 100mgkg ^-1D ^-1Abdominal injection 5d is to make model of blood dificiency; Simultaneously hydrocortisone injection is pressed 50mgkg ^-1D ^-1Abdominal injection 5d is to make deficiency of Yin model.

The administration volume is 0.4ml100g ^-1, the environment control group is given the physiological saline of the capacity of grade.

2.1.4 sleep deprivation Preparation of model

After the sign model group is made anemia deficiency of Yin model, adopt homemade modeling device to carry out multi-platform water surrounding method and carry out sleep deprivation 2d.Phase time when rat enters REM sleep, whole-body muscle tension force reduces because this phase is followed, of flaccid muscles and rhythmicity is hung one's head, and rat can fall into the water below the platform and wake up with a start, and climbs up platform more again, and continue to keep original posture, keep under the waking state and stand.Laboratory water temperature and room temperature are consistent, and are controlled at 22-24 ℃, 40W fluorescent lamp irradiation (8:00-20:00).2w is placed on each group rat in the same cage and raises before the experiment, and 1w is placed on rat and adapts to 1h on the platform every day before the experiment, changes water every day and sweeps experimental box.Sleep deprivation is from 8:00, and light prolonged exposure during the sleep deprivation is deprived rat sleep 48h continuously.

In order to get rid of above stress the influence, set up the environment control group, the environment control group adopts the mouse case identical with the sleep deprivation group, but its bottom is placement platform not, but place the fine and closely woven wire netting of one side, rat is placed on the net, and the water of putting off the net is extremely from grid 1cm place, to form the environment similar to each sleep deprivation group.Other environment and each model group are identical.Above modeling lasts common 7w.

After 4 weeks of D-semi-lactosi modeling, the estazolam group is pressed 0.13mgkg ^-1D ^-1Dosage, 1 group of triterpenoid saponin, 2 groups of triterpenoid saponins, respectively by 200,150mgkg ^-1D ^-1Dosage begin gastric infusion 3w.The environment control group is given to wait and is held physiological saline.Above administration volume is 2ml/100g.

2.2Morris (with reference to Zhang Xiaojie, an ox outstanding person, Xing Guihua etc., vein relaxing is rescued the influence of brain oral liquid to AD learning and memory in rats ability and AChE expression, Chinese Chinese materia medica magazine, 2007,22 (6): 410-413.) to water maze laboratory

2.2.1 orientation navigation test

Water temperature is controlled at about 25 ℃, divides four quadrants to put into water towards pool wall rat morning and afternoon every day each 4 times, writes down its time that searches out platform in 2min (escape latency).If rat is not found platform in 2min, then draw it to platform with hand, allow rat stop 10 seconds (s), put back to again in the cage, calculate 120s its latent period.Last 4d.

2.2.2 space exploration test

5d removes platform, and optional 1 place of entry of rat is put into, and writes down the number of times of crossing over the original platform position in its 2min.

More than two experiments also write down rat and always account for apart from per-cent in the swimming of platform quadrant distance, pool wall 20% and 40% zone swimming are apart from per-cent.

2.3 statistical procedures

Measurement data with mean ± standard deviation (

) expression, all The data SPSS 12.0 statistical package software analysis.

3 results

3.1Morris water maze laboratory

3.1.1 orientation navigation test-results

In Morris water maze training process, originally rat is many moves about along the maze barrel wall, and along with training is carried out, rat can directly swim over to the position at platform place, and shorten latent period gradually, illustrates after whole rats are by training to have set up spatial memory in various degree.Be the analytical information acquisition capability, measured total latent period of 4d training and the back latent period of the last 2d of training.From preclinical influence, environment control group 3d before training descends rapidly latent period, tends to be steady since the 3rd day, maintains a constant level substantially, estazolam group and sign model group then all keep higher latent period always, decline are arranged slightly to the later stage.Experimental result shows, compares the equal significant prolongation in latent period (P＜0.01) of 4d and back 2d before estazolam group and the training of sign model group rat with the environment control group; Compare with the sign model group, significantly shorten the latent period of triterpenoid saponin 1,2 groups of preceding 4d of rats training of triterpenoid saponin and back 2d (P＜0.01, P＜0.05).The results are shown in Table 13.

4 days and preclinical influence in back 2 days after table 13 triterpenoid saponin 1,2 pairs of rat orientation navigation experiment of triterpenoid saponin

Annotate: ^{△ △}Compare P＜0.01 with the environment control group, ^##Compare P＜0.01 with the sign model group, ^#Compare P＜0.05 with the sign model group

In orientation navigation experiment, to have observed experimental rat and always accounted for apart from per-cent (quadrant per-cent) in the swimming distance of platform quadrant, pool wall 20% and 40% zone swimming are apart from per-cent.In experimentation; the environment control rats can rely on spatial cues to find the position of platform rapidly; its movement locus substantially all is positioned at the platform quadrant, and estazolam group and sign model group rat be then substantially around pool wall swimming, and its movement locus is and is randomly distributed among all quadrants.Experimental result shows, compares with the environment control group, and estazolam group and sign model group rat quadrant per-cent significantly reduce (P＜0.01), and pool wall 20% and 40% zone swimming are apart from the equal significant prolongation of per-cent (P＜0.01); Compare with the sign model group, triterpenoid saponin 1, triterpenoid saponin 2 rat quadrant per-cents significantly increase (P＜0.01), and pool wall 20% and 40% zone swimming all significantly reduce (P＜0.01) apart from per-cent.The results are shown in Table 14.

The influence of table 14 triterpenoid saponin 1,2 pairs of rat orientation navigation experiment of triterpenoid saponin learning and memory

Annotate: ^{△ △}Compare P＜0.01 with the environment control group, ^##Compare P＜0.01 with the sign model group

3.1.2 space exploration test-results

In the space search experiment, the swimming distance of platform quadrant always accounts for has consistent trend apart from per-cent, pool wall 20% and 40% zone swimming apart from percentage result and orientation navigation experiment, sees Table 15.After removing platform, with the environment control group relatively, estazolam group and sign model group rat stride across original platform position number of times obviously descend (P＜0.01); Compare with the sign model group, triterpenoid saponin 1, triterpenoid saponin 2 stride across original platform position number of times and obviously raise (P＜0.01, P＜0.05), see Table 16.

The influence of table 15 triterpenoid saponin 1,2 pairs of rat space search experimental learnings of triterpenoid saponin memory

Annotate: ^{△ △}Compare P＜0.01 with the environment control group, ^##Compare P＜0.01 with the sign model group, ^#Compare P＜0.05 with the sign model group

Table 16 triterpenoid saponin 1,2 pairs of rats of triterpenoid saponin stride across the influence of original platform position number of times

Annotate: ^{△ △}Compare P＜0.01 with the environment control group, ^##Compare P＜0.01 with the sign model group

The above only is preferred embodiment of the present invention, and is in order to restriction the present invention, within the spirit and principles in the present invention not all, any modification of being made, is equal to replacement, improvement etc., all should be included within the scope of protection of the invention.

Claims (10)

1. triterpene saponin compound, wherein, the described triterpene saponin compound triterpene saponin compound that following chemical general formula I represents of serving as reasons:

Chemical general formula I

In above chemical general formula I: R ₁, R ₂And R ₃In any two be methyl, and another is a hydrogen atom; R ₄For by five or hexa-atomic monose, or by described five or the disaccharides that forms of hexa-atomic monose, or the glycosyl that forms of the aldehydic acid derivative of described monose or disaccharides; R ₅Being hydrogen atom, perhaps is by five or hexa-atomic monose, or by described five or the disaccharides that forms of hexa-atomic monose, or the substituting group that forms of the aldehydic acid derivative of described monose or disaccharides.

2. triterpene saponin compound according to claim 1, wherein, R ₁, R ₂All be methyl, and R ₃Be hydrogen atom, or R ₁And R ₂In any one be methyl, another is hydrogen atom, and R ₃Be methyl.

3. triterpene saponin compound according to claim 1, wherein, R ₄For being selected from by a kind of glycosyl that forms in L-rhamnosyl, D-glucose-(1 → 2)-D-xyloside, D-glucose, L-Fucose, D-seminose, L-arabinose, D-pectinose, D-wood sugar, D-ribose and the D-glucuronic acid; R ₅Be hydrogen atom, or for to be selected from by a kind of substituting group that forms in L-rhamnosyl, D-glucose-(1 → 2)-D-xyloside, D-glucose, L-Fucose, D-seminose, L-arabinose, D-pectinose, D-wood sugar, D-ribose and the D-glucuronic acid; Work as R ₅During for the substituting group that forms by above-mentioned sugared or derivatives thereof, R ₄And R ₅Identical or different.

4. triterpene saponin compound according to claim 1, wherein, R ₁, R ₂All be methyl, and R ₃Be hydrogen atom, R ₄Be L-rhamnosyl, R ₅Be hydrogen atom; R ₁, R ₂All be methyl, and R ₃Be hydrogen atom, R ₄Be L-rhamnosyl, R ₅Be the L-rhamnosyl; R ₁, R ₂All be methyl, and R ₃Be hydrogen atom, R ₄Be the glycosyl that D-glucose-(1 → 2)-D-xyloside forms, R ₅Be hydrogen atom; Perhaps R ₁, R ₂All be methyl, and R ₃Be hydrogen atom, R ₄Be the glycosyl that D-glucose-(1 → 2)-D-xyloside forms, R ₅Be the L-rhamnosyl.

5. the synthetic method of a triterpene saponin compound, described triterpene saponin compound is each described triterpene saponin compound in the claim 1～4, the described triterpene saponin compound triterpene saponin compound that following chemical general formula I represents of serving as reasons:

Chemical general formula I

In above chemical general formula I: R ₁, R ₂And R ₃In any two be methyl, and another is a hydrogen atom; R ₄For by five or hexa-atomic monose, or by described five or the disaccharides that forms of hexa-atomic monose, or the glycosyl that forms of the aldehydic acid derivative of described monose or disaccharides; R ₅Being hydrogen atom, perhaps is by five or hexa-atomic monose, or by described five or the disaccharides that forms of hexa-atomic monose, or the substituting group that forms of the aldehydic acid derivative of described monose or disaccharides,

With five or hexa-atomic sugar or its aldehydic acid derivative and Oleanolic Acid or ursolic acid be raw material, by the synthetic described triterpene saponin compound of convergence type method, described synthetic method specifically may further comprise the steps:

Step 1: to described five or the hydroxyl of hexa-atomic sugar or its aldehydic acid derivative protect; Make three halo nitriles or mercaptan and react, obtain at least a compd A through the hemiacetal hydroxyl of the sugared or derivatives thereof of hydroxyl protection;

Step 2: triphenylmethyl chloride and Oleanolic Acid or ursolic acid are reacted, obtain compd B;

Step 3: at least a compd A and compd B are reacted, and use the sodium alkoxide deprotection, obtain R ₅Compound for the chemical general formula I of hydrogen atom; Perhaps

At least a compd A and compd B are reacted, and with behind the acetate deprotection, again with step 1 at least a compd A of preparation react, use the sodium methylate deprotection then, wherein, described at least a compd A is identical or different.

6. the synthetic method of triterpene saponin compound according to claim 5, wherein, the group that carries out hydroxyl protection in the step 1 is benzyl and/or ethanoyl.

7. the synthetic method of triterpene saponin compound according to claim 5; wherein; when being L-rhamnosyl, L-Fucose, D-seminose, L-arabinose, D-pectinose, D-ribose or D-glucuronic acid, adopt benzyl to carry out reacting with Trichloroacetonitrile and hemiacetal hydroxyl behind the hydroxyl protection for described five in the step 1 or hexa-atomic sugared or derivatives thereof.

8. the synthetic method of triterpene saponin compound according to claim 5 wherein, when being D-glucose or D-wood sugar for described five in the step 1 or hexa-atomic sugared or derivatives thereof, carries out reacting with mercaptan and hemiacetal hydroxyl behind the hydroxyl protection with ethanoyl.

9. the application of each described triterpene saponin compound in the medicine of preparation treatment cancer or dementia in the claim 1～4.

10. treat cancer or dull-witted pharmaceutical composition for one kind, be liquid preparation, injection, powder, granule, capsule, pill, tablet, suppository, paste, flocculation agent, film, aerosol, sprays, powder inhalation, slowly-releasing and control-released agent, transdermal delivery system, targeting preparation, pulvis, lozenge or the cachet of the triterpene saponin compound that each limited in the claim 1～4 that comprises significant quantity.