CN106939031A - A kind of spirosolane type glycoalkaloid and preparation method thereof and purposes - Google Patents

Abstract

The invention provides a kind of spirosolane type glycoalkaloid (3 β, 5 α, 22 α, 25R) spirosolane 3 O β D glucopyranosyls (1 → 2) O β D glucopyranosyls (1 → 4) β D galactopyranosides and preparation method thereof and purposes, the spirosolane type glycoalkaloid is to human A549 cell lines, human large cell lung cancer cell H460 and people Lung Squamous Carcinoma Cells SK MES 1 are respectively provided with inhibitory action, and be 19.22 μ g/ml to the 503nhibiting concentration of A549 cells, 503nhibiting concentration to H460 cells is 80.03 μ g/ml, 503nhibiting concentration to the cells of SK MES 1 is 54.20 μ g/ml, it can be seen that (3 β that the present invention is prepared, 5 α, 22 α, 25R) spirosolane 3 O β D glucopyranosyls (1 → 2) O β D glucopyranosyls (1 → 4) β D galactopyranosides and its preparation can be used for treatment non-small cell lung cancer.

Description

A kind of spirosolane type glycoalkaloid and preparation method thereof and purposes

Technical field

The invention belongs to glycoalkaloid preparation method and medicinal usage technical field, and in particular to a kind of spirosolane Type glycoalkaloid and preparation method thereof and purposes.

Background technology

Glycoalkaloid is mainly distributed in Solanaceae and liliaceous plant, by hydrophobic aglycon and hydrophilic widow Sugar chain is constituted, and its aglycon includes three kinds of spirosolane, solanidane and other steroid derivatives, and oligonucleotide chain is by 3~4 monose Composition, is a kind of steroid saponin.Induction biosynthesis glycoalkaloid primarily to defence microorganism, the invasion and attack of animal and insect, Therefore, glycoalkaloid has certain toxicity, but at the same time, it also has reduction cholesterol and hypertension, anti-inflammatory, promoting blood circulation A variety of physiologically actives such as analgesia, antiallergy, enhancing immunity of organisms, resisting pathogenic microbes, with potential medical value.Solanaceae It is widespread in nature with liliaceous plant, such as bittersweet, potato and tomato plant, with very strong economic valency Value, studies of Solanum lyratum Thunb first recorded in《The grass warp of the legendary god of farming hundred》, its is sweet, cold in nature, the effects such as with heat-clearing, removing toxic substances, wind-dispelling, stagnation resolvation, research The main chemical compositions for showing bittersweet are glycoalkaloid, therefore, and new glycoalkaloid is extracted in research from studies of Solanum lyratum Thunb It is significant.

In view of said medicine effect of glycoalkaloid, the world of medicine pays much attention to it, Chinese patent literature CN 101450144A discloses a kind of Solanum dulcamara total-saponin extract and its production and use, and the extract is dulcain B, another name for Sichuan Province One or more in sheep spring glycosides C, δ-solamarine and Dioscin (structural formula is as follows), above-mentioned Solanum dulcamara total-saponin extract can use In diseases such as treatment gastritis, gastric duodenal ulcer, stomach cancer and psoriasis, and with good therapeutic effect.It is well known that Solanum dulcamara total-saponin extract is a kind of mixture, that includes the compound of various structures, but not can determine that every kind of compound It is respectively provided with identical or corresponding effect, i.e., Solanum dulcamara total-saponin extract that can not be disclosed in fully validated above-mentioned document includes Compound good synergy can be played to the disease such as treatment gastritis, gastric duodenal ulcer, stomach cancer and psoriasis, This is also the reason for Chinese medical extract has unpredictalbe side effect, therefore, medicinal plant extract is carried out further Refined and research, the compound of Various Complex is separated and extracted, and is had great importance to the progress confirmation of its effect, Therefore, it is all doctors how medicinal plant extract to be efficiently separated and obtain the compound with potential medical value The problem that medicine worker must face.

The content of the invention

The technical problems to be solved by the invention are medicinal plant extract complicated component, its all the components in the prior art Effect and influence each other can not be fully validated defect so that provide a kind of one-component compound spirosolane type sugar Glycosides alkaloid, and then offer its preparation method is preparing the purposes in treating non-small cell lung cancer drug with it.

Therefore, the present invention realizes that the technical scheme of above-mentioned purpose is：

The present invention provides a kind of spirosolane type glycoalkaloid, with structure shown in formula (I),

The present invention also provides a kind of preparation method of above-mentioned spirosolane type glycoalkaloid, comprises the following steps：

(1) take bittersweet medicinal material to add in alcoholic solution and carry out alcohol extracting, the alcohol extract of acquisition is refined, bittersweet is produced total Alkali sample, it is standby；

(2) the bittersweet total alkali sample for preparing step (1) adds heating for dissolving in alcoholic solution, with tlc silica gel Sample is mixed, is then splined in silica gel column chromatography and carries out chromatography, using volume ratio as (2~3):1 ethyl acetate and ethanol water The eluant, eluent of solution formation is eluted, and the volumetric concentration of the ethanol water is 93%~97%, is changed with aqueous hydrochloric acid solution Good bismuth potassium iodide test solution is developer, is detected with thin-layer chromatography, merges identical stream part, obtains two kinds of different groups of polarity Point；

(3) the small component of polarity is dissolved with dimethylformamide in two kinds of components for obtaining step (2), 0.45um filter membranes Filtering, is then isolated and purified, chromatographic condition is as follows using high performance liquid chromatography：C₁₈Liquid-phase chromatographic column；Using acetonitrile as mobile phase A, the 1%TFA aqueous solution are Mobile phase B, and gradient elution is carried out according to following program：0~10min, mobile phase A：The body of Mobile phase B Product is than being 24%~26%：76%~74% → 29%~31%：71%~69%；It is 25~35mL/ to control flow rate of mobile phase min；It is 20-30 DEG C to control column temperature；It is 0.5~1.5mL to control sampling volume；Composition is detected by mass detector, phase is collected To the composition that molecular mass is 901, the spirosolane type glycoalkaloid of the structure as shown in formula (I) is obtained.

In the step (1), the specific method for preparing bittersweet total alkali sample is：Bittersweet is dried into herb volumetric concentration For 70% ethanol water refluxing extraction, filtering concentrates the filtrate to the medicinal extract that 50 DEG C of relative densities are 1.05, adds distillation Moisture dissipates, filtering, takes filtrate to add to D151 large pore resin absorption columns, successively with the distilled water and 2~4 times of posts of 2~4 times of column volumes The volumetric concentration of volume elutes for 95% ethanol water, discards eluent, is then with the volumetric concentration of 3~5 times of column volumes The volumetric concentration of ethanol is 95% in 6 ‰ acidic alcohol aqueous solution elution, the acidic alcohol aqueous solution, collects acidic alcohol Eluent, neutrality is neutralized to ammoniacal liquor, filtering, concentrate the filtrate to it is dry, it is scattered with distilled water, will it is scattered after decoction add to AB-8 macroporous absorbent resins, with the distillation water elution of 6~10 times of column volumes, discard eluent, then with 3~5 times of column volumes Volumetric concentration elutes for 95% ethanol water, collects ethanol eluate, and concentrate drying produces bittersweet total alkali.

In the step (2), the parts by weight of bittersweet total alkali sample 30~40 are weighed, the body of 300-1000 parts by volume is added In temperature it is dissolving at 80~100 DEG C in the ethanol water that product concentration is 93~97%, then adds 10~20 parts by weight Tlc silica gel is well mixed, and is splined in the silica gel column chromatography of the tlc silica gel of the parts by weight of addition 780~820 and is carried out Chromatography；

The relation of the parts by weight and parts by volume is g/mL.

In the step (2), using volume ratio as 2.5:The eluant, eluent of 1 ethyl acetate and ethanol water formation is carried out Elution, the volumetric concentration of the ethanol water is 95%.

In the step (2), the specific method of the aqueous hydrochloric acid solution improvement bismuth potassium iodide test solution is：

The parts by weight of basic bismuth nitrate 0.8~0.9 are weighed, glacial acetic acid, 39~41 volumes of 9~11 parts by volume are sequentially added The water of part and the liquor kalii iodide of 19~21 parts by volume, it is well mixed to produce bismuth potassium iodide test solution；

0.6mol/L aqueous hydrochloric acid solution, the bismuth potassium iodide test solution and the salt are added into the bismuth potassium iodide test solution The volume ratio of aqueous acid is 1:2, produce the bismuth potassium iodide test solution of the aqueous hydrochloric acid solution improvement；

The relation of the parts by weight and parts by volume is g/mL.

In the step (3), mobile phase A：The volume ratio of Mobile phase B is 25%：75% → 30%：70%.

The present invention, which is also provided, includes the preparation of above-mentioned spirosolane type glycoalkaloid, or including above-mentioned preparation method The preparation of the spirosolane type glycoalkaloid prepared, using the spirosolane type glycoalkaloid as effectively into Point, customary adjuvant is added, clinically acceptable preparation is made according to common process.

The preparation includes tablet, capsule, granule, syrup, powder, pill, tincture, vina, soft extract, lozenge Or mixture.

The present invention also provides above-mentioned spirosolane type glycoalkaloid, the spiral steroid alkali that above-mentioned preparation method is prepared Alkane type glycoalkaloid, above-mentioned spirosolane type glycoalkaloid preparation prepare treatment non-small cell lung cancer drug in Purposes.

Compared with prior art, above-mentioned technical proposal of the invention has the following advantages that：

1st, spirosolane type glycoalkaloid of the present invention, its be a kind of one-component i.e. chemical name for (3 β, 5 α, 22 α, 25R)-spirosolane -3-O- β-D- glucopyranosyls-(1 → 2)-O- β-D- glucopyranosyl-(1 → 4) - β-D- galactopyranosides, it is to human A549 cell lines, human large cell lung cancer cell H460 and people's Lung Squamous Carcinoma Cells SK- MES-1 is respectively provided with inhibitory action, and half suppression of the spirosolane type glycoalkaloid of the invention prepared to A549 cells Concentration processed is 19.22 μ g/ml, and the 503nhibiting concentration to H460 cells is 80.03 μ g/ml, to the semi-inhibit of SK-MES-1 cells Concentration is 54.20 μ g/ml, it is seen that spirosolane type glycoalkaloid component of the present invention is single, and function is definite, and can For treating non-small cell lung cancer.

2nd, the preparation method of spirosolane type glycoalkaloid of the present invention, because glycoalkaloid contains polarized The less steroidal unit of larger sugar chain structure and polarity so that glycoalkaloid is water insoluble, acetoneand ethyl acetate etc. big Most organic solvents, can only be dissolved in a small number of organic solvents such as methanol, ethanol, glycoalkaloid structure and contain nitrogen-atoms, show Go out certain alkalescent, therefore can be dissolved in an acidic solution into salt, in the basic conditions Precipitation, therefore the present invention is used Acidic alcohol-ammonia water precipitating refines bittersweet total alkali, then using column chromatography, is detected with thin-layer chromatography, merges phase cocurrent flow Part, obtain two kinds of different components of polarity；By preparing after the small component dimethylformamide of polarity in two kinds of components is dissolved Chromatogram purification, mass detector detection composition, collects the composition that relative molecular mass is 901, obtains (3 β, 5 of the present invention α, 22 α, 25R)-spirosolane -3-O- β-D- glucopyranosyls-(1 → 2)-O- β-D- glucopyranosyl-(1 → 4) - β-D- galactopyranosides, the preparation method is simple to operate, scientific and reasonable.

3rd, the preparation method of spirosolane type glycoalkaloid of the present invention, with salt during column chromatography for separation The bismuth potassium iodide test solution of aqueous acid improvement is developer, and the preparation method of the developer is：Add successively into basic bismuth nitrate Enter glacial acetic acid, water and liquor kalii iodide, then add into the test solution after well mixed 0.6mol/L aqueous hydrochloric acid solution, the iodine It is 1 to change bismuth potassium test solution with the volume ratio of the aqueous hydrochloric acid solution:2, that is, the bismuth potassium iodide examination of the aqueous hydrochloric acid solution improvement is made Liquid, when the developer that the preparation method is prepared to be detected with thin-layer chromatography, for polar compound is with respect to background colour Colour developing is clear, easily determines.

Brief description of the drawings

, below will be to specific in order to illustrate more clearly of the specific embodiment of the invention or technical scheme of the prior art The accompanying drawing to be used needed for embodiment or description of the prior art is briefly described, it should be apparent that, in describing below Accompanying drawing is some embodiments of the present invention, for those of ordinary skill in the art, before creative work is not paid Put, other accompanying drawings can also be obtained according to these accompanying drawings.

Fig. 1 is that the embodiment of the present invention 1 prepares (3 β, 5 α, 22 α, 25R)-spirosolane -3-O- β-D- pyrans Portugal The structural formula of grape glycosyl-(1 → 2)-O- β-D- glucopyranosyls-(1 → 4)-β-D- galactopyranosides；

Fig. 2 is that the embodiment of the present invention 1 prepares (3 β, 5 α, 22 α, 25R)-spirosolane -3-O- β-D- pyrans Glucosyl group-(1 → 2)-O- β-D- glucopyranosyls-(1 → 4)-β-D- galactopyranosides¹HNMR spectrograms；

Fig. 3 is that the embodiment of the present invention 1 prepares (3 β, 5 α, 22 α, 25R)-spirosolane -3-O- β-D- pyrans Glucosyl group-(1 → 2)-O- β-D- glucopyranosyls-(1 → 4)-β-D- galactopyranosides¹³C NMR spectras；

Fig. 4 is that the embodiment of the present invention 1 prepares (3 β, 5 α, 22 α, 25R)-spirosolane -3-O- β-D- pyrans The H-H cosy spectrograms of glucosyl group-(1 → 2)-O- β-D- glucopyranosyls-(1 → 4)-β-D- galactopyranosides；

Fig. 5 is that the embodiment of the present invention 1 prepares (3 β, 5 α, 22 α, 25R)-spirosolane -3-O- β-D- pyrans The HMQC spectrograms of glucosyl group-(1 → 2)-O- β-D- glucopyranosyls-(1 → 4)-β-D- galactopyranosides；

Fig. 6 is that the embodiment of the present invention 1 prepares (3 β, 5 α, 22 α, 25R)-spirosolane -3-O- β-D- pyrans The HMBC spectrograms of glucosyl group-(1 → 2)-O- β-D- glucopyranosyls-(1 → 4)-β-D- galactopyranosides；

Fig. 7 is that the embodiment of the present invention 1 prepares (3 β, 5 α, 22 α, 25R)-spirosolane -3-O- β-D- pyrans The Tocsy spectrograms of glucosyl group-(1 → 2)-O- β-D- glucopyranosyls-(1 → 4)-β-D- galactopyranosides；

Fig. 8 is that the embodiment of the present invention 1 prepares (3 β, 5 α, 22 α, 25R)-spirosolane -3-O- β-D- pyrans The Noesy spectrograms of glucosyl group-(1 → 2)-O- β-D- glucopyranosyls-(1 → 4)-β-D- galactopyranosides；

Fig. 9 is that the embodiment of the present invention 1 prepares (3 β, 5 α, 22 α, 25R)-spirosolane -3-O- β-D- pyrans Glucosyl group-(1 → 2)-O- β-D- glucopyranosyls-(1 → 4)-β-D- galactopyranosides are to A549, H460 and SK- The inhibition figure of MES-1 cells.

Embodiment

Technical scheme will be clearly and completely described below, it is clear that described embodiment is this hair Bright a part of embodiment, rather than whole embodiments.Based on the embodiment in the present invention, those of ordinary skill in the art are not having There is the every other embodiment made and obtained under the premise of creative work, belong to the scope of protection of the invention.In addition, below Just can be mutual as long as the technical characteristic involved by described different embodiments of the present invention does not constitute conflict each other With reference to.

In following examples of the present invention and experimental example,

1st, reagent and consumptive material：

Tissue Culture Flask (U.S. FALCON 353014)；

Penicillin/streptomycin solution (Jiangsu Province, China Kai Ji Biotechnology Ltd. KGY002)；

0.25%Tripsin-EDTA (Jiangsu Province, China Kai Ji Biotechnology Ltd. KGY001)；

MTT (U.S. Amresco 0793)；

DMSO (U.S. SIGMA D2650)；

PBS (Jiangsu Province, China Kai Ji Biotechnology Ltd. KGB500)；

RPMI-1640 (U.S. GIBCO 31800-105)；

DMEM (U.S. GIBCO 12800-082)；

FBS (U.S. ExCell Biology FBS500)；

96wellcell culture plate (U.S. Corning Incorporated 3599)；

2nd, instrument and equipment

Superclean bench (Chinese Suzhou purifies SW-CJ-1FD)；

CO₂Incubator (Japanese SANYO XD-101)；

Biological inverted microscope (Japanese OLYMPUS IX51)；

Table-type low-speed centrifuge (Chinese Shanghai Medical Treatment Equipment Co., Ltd Medical Equipment Plant 80-2)；

2.5uL, 10uL, 200uL, 1000uL pipettor (German eppendorf)；

Vertical pressure pot (the rich news of Chinese Shanghai, YXQ-LS-50)；

Electric drying oven with forced convection (Chinese Shanghai sage is glad, 101AS-3)；

ELIASA (U.S. BioTek ELx800)；

Oscillator (Chinese Shanghai Hu Xi analytical instrument factory WH-2)；

Waters 2767/QDa prepare liquid phase mass spectrometry chromatograph：Masslynx4.1 chromatographic work stations, Waters2767 sample managers, Waters2489 ultraviolet-visible detectors, Waters2545 binary high pressure chromatogram pumps, QDa Mass detector, chromatographic column is Waters SunFire Prep C₁₈OBD (10 μm, 19 × 250mm) Column.

Embodiment 1

(3 β, 5 α, 22 α, 25R)-spirosolane -3-O- β-D- glucopyranosyls that the present embodiment is provided-(1 → 2) preparation method of-O- β-D- glucopyranosyls-(1 → 4)-β-D- galactopyranosides comprises the following steps：

(1) take 10Kg bittersweets to dry herb, add the ethanol water that 80L volumetric concentrations are 70%, refluxing extraction 3 times, 2h, filtering, merging filtrate are extracted every time；Above-mentioned filtrate is concentrated into the medicinal extract that 50 DEG C of relative densities are 1.05,10 times are added The distilled water of medicinal extract quality disperses, filtering, takes filtrate to add to D151 large pore resin absorption columns, successively with the distillation of 3 times of column volumes The volumetric concentration of water and 3 times of column volumes elutes for 95% ethanol water, discards eluent, then containing with 4 times of column volumes There is the ethanol water that volume fraction is 6 ‰ hydrochloric acid to elute, the volumetric concentration of ethanol is 95% in the acidic alcohol aqueous solution, Collect acidic alcohol eluent, neutrality be neutralized to ammoniacal liquor, filter, concentrate the filtrate to it is dry, it is scattered with distilled water, after scattered Decoction add to AB-8 macroporous absorbent resins, with the distillation water elution of 8 times of column volumes, eluent is discarded, then with 4 times of column volumes Volumetric concentration eluted for 95% ethanol water, collect ethanol eluate, concentrate drying produces 35g bittersweet total alkalis, yield For 3.5 ‰；

(2) 35g bittersweet total alkalis sample is taken to add molten in the case where temperature is 90 DEG C in the ethanol water of 650mL volumetric concentrations 95% Solution, then adds 15g tlc silica gels and is well mixed, be splined on the silica gel column chromatography for the tlc silica gel for adding 800g Middle carry out chromatography；Eluted by the eluant, eluent of 2.5: 1 ethyl acetate and ethanol water formation of volume ratio, it is described The volumetric concentration of ethanol water is 95%, and the bismuth potassium iodide test solution improved using aqueous hydrochloric acid solution is developer, with thin-layer chromatography Detected, merge identical stream part, obtain two kinds of different components of polarity；

The specific method of aqueous hydrochloric acid solution improvement bismuth potassium iodide test solution is：Basic bismuth nitrate 0.85g is weighed, successively 10mL glacial acetic acid, 40mL water and 20mL liquor kalii iodides are added, it is well mixed to produce bismuth potassium iodide test solution；Take what 1mL had been configured Bismuth potassium iodide test solution, adds 2mL0.6mol/L aqueous hydrochloric acid solution, produces the bismuth potassium iodide examination of the aqueous hydrochloric acid solution improvement Liquid.

(3) the small component of polarity is dissolved with dimethylformamide in two kinds of components for obtaining step (2), 0.45um filter membranes Filtering, is then isolated and purified, chromatographic condition is as follows using high performance liquid chromatography：C₁₈Liquid-phase chromatographic column；Using acetonitrile as mobile phase A, the 1%TFA aqueous solution are Mobile phase B, and gradient elution is carried out according to following program：0~10min, mobile phase A：The body of Mobile phase B Product is than being 25%：75% → 30%~70%；It is 30mL/min to control flow rate of mobile phase；It is 25 DEG C to control column temperature；Control sample introduction Volume is 1mL；Composition is detected by mass detector, the composition that relative molecular mass is 901 is collected, obtains as shown in formula (I) The spirosolane type glycoalkaloid of structure.

(3 β, 5 α, 22 α, 25R)-spirosolane -3-O- β-D- glucopyranosyls-(1 that the present embodiment is prepared → 2)-O- β-D- glucopyranosyls-(1 → 4)-β-D- galactopyranosides¹HNMR spectrograms,¹³C NMR spectras, H-H Cosy spectrograms, HMQC spectrograms, HMBC spectrograms, Tocsy spectrograms and Noesy spectrograms are shown in Fig. 2-8 respectively.

(3 β, 5 α, 22 α, 25R)-spirosolane -3-O- β-D- glucopyranosyls-(1 that the present embodiment is prepared → 2)-O- β-D- glucopyranosyls-(1 → 4)-β-D- galactopyranosides, its structural formula is as shown in Fig. 1, wherein carbon atom Numbering 1-27 is marked in the structural formula shown in Fig. 1.Table 1 is Fig. 2-8 analysis result.

The Fig. 2-8 of table 1 analysis result

Wherein, in table 1 hydrogen spectrum and carbon modal data be¹H-NMR (600MHz) and¹³C-NMR (150MHz), solvent is Obtained under conditions of deuterated methanol.

Embodiment 2

(3 β, 5 α, 22 α, 25R)-spirosolane -3-O- β-D- glucopyranosyls that the present embodiment is provided-(1 → 2) preparation method of-O- β-D- glucopyranosyls-(1 → 4)-β-D- galactopyranosides comprises the following steps：

(1) bittersweet total alkali is refined：10Kg bittersweets are taken to dry herb, it is 70% ethanol water to add 60L volumetric concentrations, is returned Stream is extracted 4 times, and 2h is extracted every time, is filtered, merging filtrate；Above-mentioned filtrate is concentrated into the leaching that 50 DEG C of relative densities are 1.05 Cream, the distilled water for adding 8 times of medicinal extract quality disperses, and filtering takes filtrate to add to D151 large pore resin absorption columns, successively with 2 times of posts The volumetric concentration of the distilled water of volume and 4 times of column volumes elutes for 95% ethanol water, eluent is discarded, then with 3 times of posts Volume is eluted containing volumetric concentration for the ethanol water of 6 ‰ hydrochloric acid, and the volume of ethanol is dense in the acidic alcohol aqueous solution Spend for 95%, collect acidic alcohol eluent, neutrality is neutralized to ammoniacal liquor, filter, concentrate the filtrate to it is dry, with distillation moisture Dissipate, the decoction after disperseing adds to AB-8 macroporous absorbent resins, with the distillation water elution of 6 times of column volumes, discards eluent, then Eluted with the volumetric concentration of 5 times of column volumes for 95% ethanol water, collect ethanol eluate, concentrate drying produces 35g bittersweets Total alkali, yield is 3.5 ‰；

(2) it in 93% ethanol water in temperature is 100 DEG C to take 30g bittersweet total alkalis sample to add 1000mL volumetric concentrations to be Lower dissolving, then adds 10g tlc silica gels and is well mixed, be splined on the layer of silica gel for the tlc silica gel for adding 820g Chromatography is carried out in analysis post；Eluted by the eluant, eluent of 3: 1 ethyl acetate and ethanol water formation of volume ratio, institute The volumetric concentration for stating ethanol water is 93%, and the bismuth potassium iodide test solution improved using aqueous hydrochloric acid solution is developer, with thin layer color Spectrum is detected, is merged identical stream part, is obtained two kinds of different components of polarity；

The specific method of aqueous hydrochloric acid solution improvement bismuth potassium iodide test solution is：Basic bismuth nitrate 0.8g is weighed, is added successively Enter 11mL glacial acetic acid, 39mL water and 21mL liquor kalii iodides, it is well mixed to produce bismuth potassium iodide test solution；Take the iodine that 1mL has been configured Change bismuth potassium test solution, add 2mL0.6mol/L aqueous hydrochloric acid solution, produce the bismuth potassium iodide test solution of the aqueous hydrochloric acid solution improvement.

(3) the small component of polarity is dissolved with dimethylformamide in two kinds of components for obtaining step (2), 0.45um filter membranes Filtering, is then isolated and purified, chromatographic condition is as follows using high performance liquid chromatography：C₁₈Liquid-phase chromatographic column；Using acetonitrile as mobile phase A, the 1%TFA aqueous solution are Mobile phase B, and gradient elution is carried out according to following program：0~10min, mobile phase A：The body of Mobile phase B Product is than being 24%：76% → 31%~69%；It is 25mL/min to control flow rate of mobile phase；It is 30 DEG C to control column temperature；Control sample introduction Volume is 0.5mL；Composition is detected by mass detector, the composition that relative molecular mass is 901 is collected, obtains such as formula (I) institute Show the spirosolane type glycoalkaloid of structure.

(3 β, 5 α, 22 α, 25R)-spirosolane -3-O- β-D- glucopyranosyls-(1 that the present embodiment is prepared → 2)-O- β-D- glucopyranosyls-(1 → 4)-β-D- galactopyranosides add customary adjuvant, be made according to common process Tablet.

Embodiment 3

(3 β, 5 α, 22 α, 25R)-spirosolane -3-O- β-D- glucopyranosyls that the present embodiment is provided-(1 → 2) preparation method of-O- β-D- glucopyranosyls-(1 → 4)-β-D- galactopyranosides comprises the following steps：

(1) bittersweet total alkali is refined：10Kg bittersweets are taken to dry herb, it is 70% ethanol water to add 100L volumetric concentrations, Refluxing extraction 2 times, extracts 2h every time, filtering, merging filtrate；Above-mentioned filtrate is concentrated into the leaching that 50 DEG C of relative densities are 1.05 Cream, the distilled water for adding 12 times of medicinal extract quality disperses, and filtering takes filtrate to add to D151 large pore resin absorption columns, successively with 4 times of posts The volumetric concentration of the distilled water of volume and 2 times of column volumes elutes for 95% ethanol water, eluent is discarded, then with 5 times of posts Volume is eluted containing volume fraction for the ethanol water of 6 ‰ hydrochloric acid, and the volumetric concentration of ethanol is in the acidic alcohol aqueous solution 95%, acidic alcohol eluent is collected, neutrality is neutralized to ammoniacal liquor, is filtered, dry, general scattered with distilled water is concentrated the filtrate to Decoction after scattered adds to AB-8 macroporous absorbent resins, with the distillation water elution of 10 times of column volumes, eluent is discarded, then with 3 The volumetric concentration of times column volume is the elution of 95% ethanol water, collects ethanol eluate, and concentrate drying produces 35g bittersweets total Alkali, yield is 3.5 ‰；

(2) it in 97% ethanol water in temperature is at 80 DEG C to take 40g bittersweet total alkalis sample to add 300mL volumetric concentrations to be Dissolving, then adds 20g tlc silica gels and is well mixed, be splined on the silica gel column chromatography for the tlc silica gel for adding 780g Chromatography is carried out in post；Using volume ratio as 2:The eluant, eluent of 1 ethyl acetate and ethanol water formation is eluted, described The volumetric concentration of ethanol water is 97%, and the bismuth potassium iodide test solution improved using aqueous hydrochloric acid solution is developer, with thin-layer chromatography Detected, merge identical stream part, obtain two kinds of different components of polarity；

The specific method of aqueous hydrochloric acid solution improvement bismuth potassium iodide test solution is：Basic bismuth nitrate 0.9g is weighed, is added successively Enter 9mL glacial acetic acid, 41mL water and 19mL liquor kalii iodides, it is well mixed to produce bismuth potassium iodide test solution；Take the iodate that 1mL has been configured Bismuth potassium test solution, adds 2mL0.6mol/L aqueous hydrochloric acid solution, produces the bismuth potassium iodide test solution of the aqueous hydrochloric acid solution improvement.

(3) the small component of polarity is dissolved with dimethylformamide in two kinds of components for obtaining step (2), 0.45um filter membranes Filtering, is then isolated and purified, chromatographic condition is as follows using high performance liquid chromatography：C₁₈Liquid-phase chromatographic column；Using acetonitrile as mobile phase A, the 1%TFA aqueous solution are Mobile phase B, and gradient elution is carried out according to following program：0~10min, mobile phase A：The body of Mobile phase B Product is than being 26%：74% → 29%~71%；It is 35mL/min to control flow rate of mobile phase；It is 20 DEG C to control column temperature；Control sample introduction Volume is 1.5mL；Composition is detected by mass detector, the composition that relative molecular mass is 901 is collected, obtains such as formula (I) institute Show the spirosolane type glycoalkaloid of structure.

(3 β, 5 α, 22 α, 25R)-spirosolane -3-O- β-D- glucopyranosyls-(1 that the present embodiment is prepared → 2)-O- β-D- glucopyranosyls-(1 → 4)-β-D- galactopyranosides add customary adjuvant, be made according to common process Capsule.

Embodiment 4

(3 β, 5 α, 22 α, 25R)-spirosolane -3-O- β-D- glucopyranosyls that the present embodiment is provided-(1 → 2) preparation method of-O- β-D- glucopyranosyls-(1 → 4)-β-D- galactopyranosides comprises the following steps：

(1) bittersweet total alkali is refined：10Kg bittersweets are taken to dry herb, it is 70% ethanol water to add 100L volumetric concentrations, Refluxing extraction 2 times, extracts 2h every time, filtering, merging filtrate；Above-mentioned filtrate is concentrated into the leaching that 50 DEG C of relative densities are 1.05 Cream, the distilled water for adding 12 times of medicinal extract quality disperses, and filtering takes filtrate to add to D151 large pore resin absorption columns, successively with 3 times of posts The volumetric concentration of the distilled water of volume and 2 times of column volumes elutes for 95% ethanol water, eluent is discarded, then with 4 times of posts Volume is eluted containing volume fraction for the ethanol water of 6 ‰ hydrochloric acid, and the volume of ethanol is dense in the acidic alcohol aqueous solution Spend for 95%, collect acidic alcohol eluent, neutrality is neutralized to ammoniacal liquor, filter, concentrate the filtrate to it is dry, with distillation moisture Dissipate, the decoction after disperseing adds to AB-8 macroporous absorbent resins, with the distillation water elution of 10 times of column volumes, discards eluent, so Eluted afterwards with the volumetric concentration of 4 times of column volumes for 95% ethanol water, collect ethanol eluate, concentrate drying produces 35g white English total alkali, yield is 3.5 ‰；

(2) it in 94% ethanol water in temperature is at 80 DEG C to take 38g bittersweet total alkalis sample to add 300mL volumetric concentrations to be Dissolving, then adds 16g tlc silica gels and is well mixed, be splined on the silica gel column chromatography for the tlc silica gel for adding 780g Chromatography is carried out in post；Using volume ratio as 2.6:The eluant, eluent of 1 ethyl acetate and ethanol water formation is eluted, institute The volumetric concentration for stating ethanol water is 94%, and the bismuth potassium iodide test solution improved using aqueous hydrochloric acid solution is developer, with thin layer color Spectrum is detected, is merged identical stream part, is obtained two kinds of different components of polarity；

The specific method of aqueous hydrochloric acid solution improvement bismuth potassium iodide test solution is：Basic bismuth nitrate 0.88g is weighed, successively 10.5mL glacial acetic acid, 40.5mL water and 19mL liquor kalii iodides are added, it is well mixed to produce bismuth potassium iodide test solution；1mL is taken to configure Good bismuth potassium iodide test solution, adds 2mL0.6mol/L aqueous hydrochloric acid solution, produces the bismuth iodide of the aqueous hydrochloric acid solution improvement Potassium test solution.

(3) the small component of polarity is dissolved with dimethylformamide in two kinds of components for obtaining step (2), 0.45um filter membranes Filtering, is then isolated and purified, chromatographic condition is as follows using high performance liquid chromatography：C₁₈Liquid-phase chromatographic column；Using acetonitrile as mobile phase A, the 1%TFA aqueous solution are Mobile phase B, and gradient elution is carried out according to following program：0~10min, mobile phase A：The body of Mobile phase B Product is than being 26%：74% → 29%~71%；It is 35mL/min to control flow rate of mobile phase；It is 20 DEG C to control column temperature；Control sample introduction Volume is 1.5mL；Composition is detected by mass detector, the composition that relative molecular mass is 901 is collected, obtains such as formula (I) institute Show the spirosolane type glycoalkaloid of structure.

(3 β, 5 α, 22 α, 25R)-spirosolane -3-O- β-D- glucopyranosyls-(1 that the present embodiment is prepared → 2)-O- β-D- glucopyranosyls-(1 → 4)-β-D- galactopyranosides add customary adjuvant, be made according to common process Syrup.

Embodiment 5

(3 β, 5 α, 22 α, 25R)-spirosolane -3-O- β-D- glucopyranosyls that the present embodiment is provided-(1 → 2) preparation method of-O- β-D- glucopyranosyls-(1 → 4)-β-D- galactopyranosides comprises the following steps：

(1) bittersweet total alkali is refined：10Kg bittersweets are taken to dry herb, it is 70% ethanol water to add 100L volumetric concentrations, Refluxing extraction 2 times, extracts 2h every time, filtering, merging filtrate；Above-mentioned filtrate is concentrated into the leaching that 50 DEG C of relative densities are 1.05 Cream, the distilled water for adding 12 times of medicinal extract quality disperses, and filtering takes filtrate to add to D151 large pore resin absorption columns, successively with 2 times of posts The volumetric concentration of the distilled water of volume and 3 times of column volumes elutes for 95% ethanol water, eluent is discarded, then with 4 times of posts Volume is eluted containing volume fraction for the ethanol water of 6 ‰ hydrochloric acid, and the volume of ethanol is dense in the acidic alcohol aqueous solution Spend for 95%, collect acidic alcohol eluent, neutrality is neutralized to ammoniacal liquor, filter, concentrate the filtrate to it is dry, with distillation moisture Dissipate, the decoction after disperseing adds to AB-8 macroporous absorbent resins, with the distillation water elution of 8 times of column volumes, discards eluent, then Eluted with the volumetric concentration of 5 times of column volumes for 95% ethanol water, collect ethanol eluate, concentrate drying produces 35g bittersweets Total alkali, yield is 3.5 ‰；

(2) it in 96% ethanol water in temperature is 80 DEG C to take 32g bittersweet total alkalis sample to add 1000mL volumetric concentrations to be Lower dissolving, then adds 20g tlc silica gels and is well mixed, be splined on the layer of silica gel for the tlc silica gel for adding 810g Chromatography is carried out in analysis post；Using volume ratio as 2.8:The eluant, eluent of 1 ethyl acetate and ethanol water formation is eluted, The volumetric concentration of the ethanol water is 96%, and the bismuth potassium iodide test solution improved using aqueous hydrochloric acid solution is developer, with thin layer Chromatogram is detected, is merged identical stream part, is obtained two kinds of different components of polarity；

The specific method of aqueous hydrochloric acid solution improvement bismuth potassium iodide test solution is：Basic bismuth nitrate 0.81g is weighed, successively 9.6mL glacial acetic acid, 40mL water and 19.5mL liquor kalii iodides are added, it is well mixed to produce bismuth potassium iodide test solution；1mL is taken to configure Bismuth potassium iodide test solution, add 2mL0.6mol/L aqueous hydrochloric acid solution, produce the bismuth potassium iodide examination of aqueous hydrochloric acid solution improvement Liquid.

(3) the small component of polarity is dissolved with dimethylformamide in two kinds of components for obtaining step (2), 0.45um filter membranes Filtering, is then isolated and purified, chromatographic condition is as follows using high performance liquid chromatography：C₁₈Liquid-phase chromatographic column；Using acetonitrile as mobile phase A, the 1%TFA aqueous solution are Mobile phase B, and gradient elution is carried out according to following program：0~10min, mobile phase A：The body of Mobile phase B Product is than being 26%：74% → 29%~71%；It is 35mL/min to control flow rate of mobile phase；It is 20 DEG C to control column temperature；Control sample introduction Volume is 1.5mL；Composition is detected by mass detector, the composition that relative molecular mass is 901 is collected, obtains such as formula (I) institute Show the spirosolane type glycoalkaloid of structure.

(3 β, 5 α, 22 α, 25R)-spirosolane -3-O- β-D- glucopyranosyls-(1 that the present embodiment is prepared → 2)-O- β-D- glucopyranosyls-(1 → 4)-β-D- galactopyranosides add customary adjuvant, be made according to common process Tincture.

(3 β, 5 α, 22 α, 25R)-spirosolane -3-O- β-D- glucopyranoses that the present invention of experimental example 1 is prepared Base-(1 → 2)-O- β-D- glucopyranosyls-suppression of (1 → 4)-β-D- galactopyranosides to non-small cell lung cancer cell Effect

1st, experiment purpose

(3 β, 5 α, 22 α, 25R)-spirosolane -3-O- β-D- glucopyranosyls that the research present invention is prepared - Suppression of (1 → 2)-O- β-D- glucopyranosyls-(1 → 4)-β-D- galactopyranosides to non-small cell lung cancer cell is imitated Really.

2nd, experimental method

2.1 cellular informatics

Three kinds of non-small cell lung cancer cell strains：Human A549 cell lines, human large cell lung cancer cell H460, people's lung squama Cancer cell SK-MES-1, cell is provided by Jiangsu Kai Ji Biotechnology Ltd.；A549/H460 complete mediums are 90%RPMI640+10%FBS；SK-MES-1 complete mediums are 90%DMEM+10%FBS；In 37 DEG C, 5%CO₂, saturation it is wet Cultivated in the incubator of degree.

2.2 cell culture

(1) when the cell coverage rate in blake bottle reaches 80%~90%, original culture medium is sopped up；

(2) add appropriate trypsase (0.25%), digest 1~2min；

(3) isometric culture medium containing serum is added after cell is all rounded and terminates digestion；

(4) cell is blown and beaten with liquid-transfering gun, cell is all suspended, then cell is drawn onto in 15ml centrifuge tube, 1000rpm centrifuges 5min；

(5) supernatant is outwelled, plus 1~2ml culture mediums, cell is suspended again to be transferred in blake bottle and continues to cultivate.

2.3MTT methods detection cell propagation：

(1) take the logarithm respectively growth period A549, H460, SK-MES-1 cell dissociation, count, be configured to concentration for 5 × 10⁴ 100 μ l cell suspensions are added in individual/ml cell suspension, 96 porocyte culture plates per hole；

(2) 96 porocyte culture plates are placed in 37 DEG C, 5%CO₂24h is cultivated in incubator；

(3) the spirosolane type glycoalkaloid for preparing of the embodiment of the present invention 1 is diluted to table 2 with complete medium Needed for drug concentration, the corresponding above-mentioned pastille culture mediums of 100 μ L are added per hole, while setting up blank control group (adds consubstantiality Product blank nutrient solution)；

(4) 96 porocyte culture plates are placed in 37 DEG C, 5%CO₂72h is cultivated in incubator；

(5) 96 orifice plates are subjected to MTT dyeing, λ=490nm determines OD values；

A adds 20 μ L MTT (5mg/ml) per hole, continues to cultivate 4h in incubator；

B discards culture medium, and 150 μ LDMSO dissolvings are added per hole, and shaking table 10min is gently mixed；

C λ=490nm, ELIASA reads the absorbance OD values per hole, calculates inhibiting rate；

(6) each group inhibiting rate and medicine 503nhibiting concentration (IC are counted₅₀)

Inhibiting rate (%)=(A_{Blank control group}-A_{Medicine group})/A_{Blank control group}× 100%,

In formula, A is absorbance.

Pass through probit using SPSS (Staffstical Package for the Social Science) 17.0 Weighted regression method (Bliss methods) calculates IC₅₀。

3rd, experimental result

The spirosolane type glycoalkaloid that the present invention is prepared is to human A549 cell lines, human large cell lung cancer Cell H460 and people's Lung Squamous Carcinoma Cells SK-MES-1 inhibition are as shown in table 2 and Fig. 9.

Suppression of the spirosolane type glycoalkaloid that the present invention of table 2 is prepared to A549, H460, SK-MES-1 cell Rate processed

From table 2 and Fig. 9, with the increase of spirosolane type glycoalkaloid concentration, its suppression to cell is made With gradually strengthening, (3 β, 5 α, 22 α, 25R)-spirosolane -3-O- β-D- glucopyranosyls that the present invention is prepared - (1 → 2)-O- β-D- glucopyranosyls-(1 → 4)-β-D- galactopyranosides are to the 503nhibiting concentration of A549 cells 19.22 μ g/ml, the 503nhibiting concentration to H460 cells is 80.03 μ g/ml, is to the 503nhibiting concentration of SK-MES-1 cells 54.20μg/ml。

4th, experiment conclusion

(3 β, 5 α, 22 α, 25R)-spirosolane -3-O- β-D- glucopyranosyls that the present invention is prepared-(1 → 2)-O- β-D- glucopyranosyls-(1 → 4)-β-D- galactopyranosides are to human A549 cell lines, human large cell lung cancer Cell H460 and people's Lung Squamous Carcinoma Cells SK-MES-1 have preferable inhibition, and the suppression of human A549 cell lines is imitated Fruit it is better than other two kinds of cancer cell effects, it is indicated above that the present invention prepare (3 β, 5 α, 22 α, 25R)-spirosolane- 3-O- β-D- glucopyranosyls-(1 → 2)-O- β-D- glucopyranosyls-(1 → 4)-β-D- galactopyranosides can be used for Treat non-small cell lung cancer.

Obviously, above-described embodiment is only intended to clearly illustrate example, and the not restriction to embodiment.It is right For those of ordinary skill in the art, can also make on the basis of the above description it is other it is various forms of change or Change.There is no necessity and possibility to exhaust all the enbodiments.And the obvious change thus extended out or Among changing still in the protection domain of the invention.

Claims (10)

1. a kind of spirosolane type glycoalkaloid, it is characterised in that with structure shown in formula (I),

2. the preparation method of spirosolane type glycoalkaloid described in a kind of claim 1, it is characterised in that including following step Suddenly：

(1) take bittersweet medicinal material to add in alcoholic solution and carry out alcohol extracting, the alcohol extract of acquisition is refined, bittersweet total alkali sample is produced Product, it is standby；

(2) the bittersweet total alkali sample for preparing step (1) adds heating for dissolving in alcoholic solution, is mixed with tlc silica gel Sample, is then splined in silica gel column chromatography and carries out chromatography, using volume ratio as (2~3):1 ethyl acetate and ethanol is water-soluble The eluant, eluent of liquid formation is eluted, and the volumetric concentration of the ethanol water is 93%~97%, is improved with aqueous hydrochloric acid solution Bismuth potassium iodide test solution be developer, detected that the identical stream part of merging obtains two kinds of different groups of polarity with thin-layer chromatography Point；

(3) the small component of polarity is dissolved with dimethylformamide in two kinds of components for obtaining step (2), 0.45um membrane filtrations, Then isolated and purified using high performance liquid chromatography, chromatographic condition is as follows：C₁₈Liquid-phase chromatographic column；Using acetonitrile as mobile phase A, 1% The TFA aqueous solution is Mobile phase B, and gradient elution is carried out according to following program：0~10min, mobile phase A：The volume ratio of Mobile phase B For 24%~26%：76%~74% → 29%~31%：71%~69%；It is 25~35mL/min to control flow rate of mobile phase； It is 20-30 DEG C to control column temperature；It is 0.5~1.5mL to control sampling volume；Composition is detected by mass detector, relative point is collected Protonatomic mass is 901 composition, obtains the spirosolane type glycoalkaloid of the structure as shown in formula (I).

3. preparation method according to claim 2, it is characterised in that in the step (1), prepares bittersweet total alkali sample Specific method be：Bittersweet is dried into the ethanol water refluxing extraction that herb volumetric concentration is 70%, filtering is dense by filtrate The medicinal extract that 50 DEG C of relative densities are 1.05 is reduced to, distilled water is added and disperses, filtering takes filtrate to add to D151 macroporous absorbent resins Post, is successively that 95% ethanol water is eluted with the distilled water of 2~4 times of column volumes and the volumetric concentration of 2~4 times of column volumes, abandons Eluent is removed, is then eluted with the volumetric concentration of 3~5 times of column volumes for the 6 ‰ acidic alcohol aqueous solution, the acidic alcohol water The volumetric concentration of ethanol is 95% in solution, collects acidic alcohol eluent, neutrality is neutralized to ammoniacal liquor, is filtered, and filtrate is dense Be reduced to it is dry, it is scattered with distilled water, will it is scattered after decoction add to AB-8 macroporous absorbent resins, with the distillation of 6~10 times of column volumes Water elution, discards eluent, is then eluted with the volumetric concentration of 3~5 times of column volumes for 95% ethanol water, collects ethanol and washes De- liquid, concentrate drying produces bittersweet total alkali.

4. the preparation method according to Claims 2 or 3, it is characterised in that in the step (2), weigh bittersweet total alkali It is in temperature in the parts by weight of sample 30~40, the ethanol water that the volumetric concentration for adding 300-1000 parts by volume is 93~97% Dissolved at 80~100 DEG C, the tlc silica gel for then adding 10~20 parts by weight is well mixed, and is splined on addition 780~820 Chromatography is carried out in the silica gel column chromatography of the tlc silica gel of parts by weight；

The relation of the parts by weight and parts by volume is g/mL.

5. preparation method according to claim 4, it is characterised in that in the step (2), using volume ratio as 2.5:1 Ethyl acetate and the eluant, eluent of ethanol water formation eluted, the volumetric concentration of the ethanol water is 95%.

6. preparation method according to claim 5, it is characterised in that in the step (2), the aqueous hydrochloric acid solution changes The specific method of good bismuth potassium iodide test solution is：

The parts by weight of basic bismuth nitrate 0.8~0.9 are weighed, glacial acetic acid, the water of 39~41 parts by volume of 9~11 parts by volume is sequentially added With the liquor kalii iodide of 19~21 parts by volume, it is well mixed and produces bismuth potassium iodide test solution；

0.6mol/L aqueous hydrochloric acid solution, the bismuth potassium iodide test solution and the hydrochloric acid water are added into the bismuth potassium iodide test solution The volume ratio of solution is 1:2, produce the bismuth potassium iodide test solution of the aqueous hydrochloric acid solution improvement；

The relation of the parts by weight and parts by volume is g/mL.

7. preparation method according to claim 2, it is characterised in that in the step (3), mobile phase A：Mobile phase B Volume ratio be 25%：75% → 30%：70%.

8. include the preparation of the spirosolane type glycoalkaloid described in claim 1, or it is any including claim 2-7 The preparation for the spirosolane type glycoalkaloid that preparation method described in is prepared, it is characterised in that with the spiral Steroid alkali alkane type glycoalkaloid is active ingredient, adds customary adjuvant, clinically acceptable preparation is made according to common process.

9. preparation according to claim 8, it is characterised in that the preparation includes tablet, capsule, granule, syrup Agent, powder, pill, tincture, vina, soft extract, lozenge or mixture.

10. the preparation method described in spirosolane type glycoalkaloid, claim any one of 2-7 described in claim 1 Spirosolane type glycoalkaloid described in the spirosolane type glycoalkaloid or claim 8 or 9 prepared Purposes of the preparation in treatment non-small cell lung cancer drug is prepared.