CN1224628C - Preparation of solanine sulfurate and its use in medicine - Google Patents

Abstract

The present invention provides a new compound solasodine sulfate, a preparation method and the application thereof. The new compound has obvious anticancer, asthma relieving and antiinflammatory action, and the molecule structure of the compound is the right formula. Black nightshade, Australian eggplants or yellow-fruited nightshade which have rich resources and low price are used as raw materials; acetic acid is used as an extraction solvent in the preparation; the method has the advantages of low cost, convenient preparation process and equipment, short period, high purity of product and strong drug effect and activity. The compound can be used as a new medicine source for new traditional Chinese medicines for resisting cancer and inflammation and relieving asthma.

Description

Solasodine vitriol and preparation method thereof and in pharmaceutically application

Technical field

The present invention relates to solasodine vitriol and preparation method thereof and in pharmaceutically application.

Background technology

The domestic and international chemical ingredients of existing black nightshade, Australian eggplant and Fruit of yellowfruit nightshade, the detection method of solasodine and have anti-S ₁₈₀, effects such as anti-inflammatory, rising blood sugar report, but do not find the preparation method of solasodine vitriol, solasodine vitriol and solasodine vitriol relevant information as yet in pharmaceutically application.

Summary of the invention

The invention provides a kind of anticancer, relieving asthma, the anti-inflammatory drug activity is strong, long-acting toxic side effect is little, solvability is strong, stable in properties, production method is easy, and purity height, solasodine vitriol that drug activity is strong and preparation method thereof and compound that this is new be anticancer as a class, relieving asthma, the source new drugs of anti-inflammatory drug is in pharmaceutically application, to replace weak curative effect, toxic side effect is big, production method is complicated, poor product quality can not enter the world market existing medicine, bring benefit to the mankind.

The preparation principle of solasodine vitriol of the present invention be with no anticancer, relieving asthma, the mixture hydrolysis of antiinflammatory active compound α-solasonine, β-solasonine, γ-solasonine, solamargine, will with sugared bonded alkaloid---have 3-hydroxyl anticancer, anti-inflammatory action in the solasonine molecule and become unbound state by bonding state, and be present among the molecule with the form of drug activity group, make molecule produce anticancer, anti-inflammatory activity, reach the purpose that no drug activity mixture of ingredients is all changed into drug activity monomer solasodine.But this monomeric antitumour activity relatively a little less than, 20 μ g/ml are to HeLa cell inhibitory rate (isolated experiment) only 40%～42%; To human hepatocellular SMMC7701 cell, lung cancer MCI446 cell, the almost unrestraint effect of leukemia HL60 cell, in addition, there is not antiasthmatic effect yet; Outstanding feature of the present invention is with strong polar molecule drug activity group, strengthen its molecule drug activity, drug activity group with the 6th secondary amino group that encircles and sulfuric acid salify in the molecule are strengthened the decision antitumour activity power on its molecule precursor structure makes its molecule keep and strengthen anticancer, the antiphlogistic drug activity of 3-hydroxyl; Its drug activity strengthened after solasodine was transformed into solasodine vitriol, and 20 μ g/ml reach 100% to HeLa cell inhibitory rate (isolated experiment); Behind the solasodine salify, two active groups of parahelium alkali that make it to form on 3-hydroxyl and the 6th ring coexist as with in a part, because two active groups interact, make its molecule produce new drug activity again, having strengthened has also increased antiasthmatic effect simultaneously to liver cancer, lung cancer, leukemic anticancer drug activity: 5～20 μ g/ml are 100% to human hepatocellular SMMC7701 stem cell, lung cancer NCI446 stem cell, cancer of the stomach SGC7901 stem cell inhibiting rate; 10～20 μ g/ml are to leukemia HL60 stem cell inhibiting rate＞75%; Drug administration by injection 12～24mg/kg is to mice transplanted tumor inhibiting rate 54～72%; Gastric infusion 100～200mg/kg is to mice transplanted tumor inhibiting rate 40～68%; Cavy asthma P＜0.05 that 50～75mg/kg gastric infusion can obviously bring out antihistaminic.

Its reaction is as follows:

Solasodine solasodine vitriol

Solanidine-S R=H

α-solasonine β-solasonine

Solamargine γ-solasonine

1, the molecular structure that the purpose of this invention is to provide a kind of solasodine vitriol solasodine vitriol:

Molecular formula is: C ₂₇H ₄₅NSO ₆, molecular weight: 511.6,

Physico-chemical property: solasodine vitriol is white needle-like crystals, and is water-soluble, is insoluble in ether, ethanol.

2, another object of the present invention provides the preparation method of solasodine vitriol

Solasodine vitriol of the present invention is with plant of Solanaceae black nightshade (Solanum Nigrum L), Australian eggplant (Solanum aviculare parst) and Fruit of yellowfruit nightshade (Solanum xanthocar pum Schrad, etwendl) living fruit or its herb are raw material, extract solasodine, be feedstock production solasodine vitriol with the solasodine again, its preparation method is as follows:

(1) extraction of raw material processing and thick total alkaloids

Black nightshade, Australian eggplant or Fruit of yellowfruit nightshade given birth to fruit or its herb after screening, pulverize and press the juice solid-liquid separation to get fruit juice, pomace, the aqueous acetic acid leaching pomace with 2～6%, solid-liquid weight ratio 1: 1, leach 2～3 times, solid-liquid separation gets immersion liquid, merges fruit juice and immersion liquid, places sedimentation, getting supernatant liquor is heated to boil and removes supernatant, the filtration cooling is placed sedimentation with filtrate, gets to add strong aqua or NaOH, Na when supernatant liquor is heated to 80～85 ℃ ₂CO ₃, KOH the aqueous solution to pH8～9, cooling-sedimentation removes supernatant liquor, down turbid liquid centrifugal solid-liquid is separated, the thick total alkaloids of paste;

(2) thick total alkaloids is refining

With the thick total alkaloids of 2～6% aqueous acetic acids dissolving paste, solid-liquid weight ratio 1: 10～30, being heated to boils removes supernatant, filter cooling-sedimentation, when getting supernatant liquor and being heated to 80～85 ℃, the aqueous solution that adds strong aqua or alkali is to pH8～9, cooling-sedimentation removes supernatant liquor, to following turbid liquid centrifugal solid-liquid separate the paste total alkaloids, 80～100 ℃ of following freeze-day with constant temperature, pulverize the total alkaloids in black nightshade, Australian eggplant or the Fruit of yellowfruit nightshade, its main component is α, β, γ-solasonine, four kinds of alkaloidal mixtures of solamargine;

(3) extraction of thick solasodine

With 80～95% dissolve with ethanol solution total alkaloidss that contain 3～10% hydrochloric acid, solid-liquid weight ratio 1: 30～40, hydrolysis reflux 2～3 hours, cold filtration, with 95% ethanol, water, be washed till neutrality, the dry solasodine hydrochloride that gets is with the 95% dissolve with ethanol solution solasodine hydrochloride that contains 3～10%NaOH, reflux 1～2 hour, heat filtering, the cooling suction strainer is washed till neutrality, dry thick solasodine;

(4) solasodine is refining

Thick solasodine is dissolved in methyl alcohol, ethanol or the chloroform to saturated solution, mix with thick solasodine solution with the silica gel G after the activation, the immobilising paste of furnishing, at 80～90 ℃ of following freeze-day with constant temperature, pack into after the cooling in the chromatography column, jolt ramming, with chloroform or chloroform: methyl alcohol volume ratio 10: 1～2 or chloroform: ethanol: one of acetone volume ratio 3: 1: 2 is carried out wash-out and is got elutriant, after the check wash-out finishes, elutriant normal pressure or underpressure distillation are concentrated, behind concentrated solution crystallisation by cooling filtration drying, be crushed to 0.1～1.0 micron powder and get solasodine;

(5) prepare solasodine vitriol with solasodine

Solasodine is dissolved in being heated in 95% ethanol boils, solid-liquid weight ratio 1: 20-40 stirs and slowly adds 10～50% aqueous sulfuric acid in the thermotropism solasodine ethanolic soln down, to pH=1, crystallisation by cooling, suction strainer is washed till neutrality with ethanol, the dry solasodine vitriol that gets.

Check among the preparation method (4) whether wash-out finishes with 1～5%SbCl ₃Dehydrated alcohol or chloroformic solution be developer, its judgement is to check on the filter paper or on the silica gel G thin layer chromatography (tlc) plate.Concrete operations are: get firm effusive elutriant of certain moment with kapillary, put on filter paper or silica gel G plate, oven dry is with 1～5%SbCl ₃Solution evenly is sprayed on the filter paper or silica gel G plate of point sample, oven dry, as the end as yet of punctation explanation wash-out appears, and complete as redfree spot explanation wash-out, should stop wash-out.

The solasodine vitriol that obtains with preparation method of the present invention is white needle-like crystals, solasodine vitriol purity＞97%.Raw material sources of the present invention are abundant cheap, and it is low that the preparation method adopts acetic acid to do to extract solvent cost, and preparation technology and simple equipments are with short production cycle, and product purity height, drug activity are strong.

3, another purpose of the present invention provide solasodine vitriol anticancer, relieving asthma, application on the anti-inflammatory pharmaceutical

Prove that through pharmacodynamic experiment and clinical trial solasodine vitriol has following effect:

(1) antitumous effect

Pharmacodynamic experiment is the result prove, solasodine vitriol 8～30mg/kg intraperitoneal injection is 60～72% to mouse transplantability solid-type tumor control rate; Gastric infusion is 40～68% to mouse transplantability solid-type tumor control rate; Solasodine vitriol 5～20 μ g/ml are 100% to human hepatocellular SMMC7701 stem cell, lung cancer NCI446 stem cell, cancer of the stomach SGC7901 stem cell, HeLa stem cell inhibiting rate; During 10～20 μ g/ml, be 60～80% to the sick HL60 stem cell of human white blood inhibiting rate.Anticancer mechanism experiment showed, that the antitumous effect mechanism of solasodine vitriol is, can significantly reduce cancer cells C-myc gene and H-ras genetic expression, suppresses the differentiation of cell proliferation inducing cell; Can significantly reduce cancer cells hTRT expression of gene, suppress telomerase activation, the anticancer growth; Can block its DNA from G ₁Phase transformed to the S phase, thereby suppressed the synthetic antitumous effect that plays of DNA.Clinical test results proves that drug administration by injection solasodine sulfate concentration is 1.0mg/ml, 20～30ml/ time; Oral medication capsule, tablet, dosage 100mg/ grain (sheet), 2 (sheet)/day, 10th be a course of treatment to liver cancer, lung cancer, cancer of the stomach, mammary cancer, cervical cancer, leukemia medication, there is 60% patient in 1～2 course of treatment, to obtain obvious curative effects, cooperates operation medication effect better.

(2) antiasthmatic effect

Pharmacodynamic experiment is the result prove, solasodine vitriol abdominal injection 10～30mg/kg, cavy asthma P＜0.05 that gastric infusion 50～100mg/kg can obviously bring out antihistaminic, guinea pig bronchial spasm P＜0.01 that can significantly cause antiovalbumin; 1 * 10 ^-6Mol/ml can significantly expand guinea pig bronchial unstriated muscle P＜0.01.Clinical test results proves, solasodine vitriol drug administration by injection 1.0mg/ml, 30～50ml/ time, 2 times/day; Oral medication capsule or tablet, dosage 100mg/ grain (sheet), 2 (sheet)/time, 2 times/day, there is 85～98% heavily trouble symptoms of asthma in 20～40 minutes to disappear or obviously alleviate, vital capacity increases, and wheezing sound disappears, and breathes to be tending towards normal, 7 days/course of treatment, 1～2 life course of treatment of medication can be taken care of oneself, and in addition, blood pressure, heart is also made moderate progress.

(3) anti-inflammatory action

Pharmacodynamic experiment is the result prove, solasodine vitriol drug administration by injection 10～30mg/kg, gastric infusion 60～200mg/kg can significantly resist mouse foot swelling P＜0.01 that carrageenin causes; Can significantly resist the swollen P of rat granuloma＜0.001.Clinical test results proves, solasodine vitriol drug administration by injection 1.0mg/ml, 30～50ml/ time, 2 times/day; Oral medication capsule or tablet, dosage 100mg/ grain (sheet), 2 times/day, 2 (sheet)/time, white corpuscle in the pneumonia, pulmonary tuberculosis, mazoitis, tonsillitis, pharyngitis, trachitis medication 3 days is obviously reduced, inflammation obviously disappears, and has 1～3 of patient's medication of 85～98% every detection index trend course of treatment normal.

Solasodine vitriol be a kind of have significantly anticancer, relieving asthma, the Chinese medicine one kind new medicine source of anti-inflammatory action, its purposes is mainly as the medicine manufacturing of the following disease of treatment:

1, the manufacturing of cancer therapy drug

(1) manufacturing of injections such as treatment liver cancer, lung cancer, cancer of the stomach, mammary cancer, cervical cancer, leukemia.

(2) manufacturing of oral preparations such as treatment liver cancer, lung cancer, cancer of the stomach, mammary cancer, cervical cancer, leukemia.

2, the manufacturing of suppressing panting calming medicine

(1) manufacturing of treatment immunity asthma injection.

(2) manufacturing of treatment immunity asthma oral preparations.

3, the manufacturing of anti-inflammatory drug

(1) manufacturing of injections such as treatment pneumonia, pulmonary tuberculosis, trachitis, tonsillitis, mazoitis, pharyngitis.

(2) manufacturing of oral preparations such as treatment pneumonia, pulmonary tuberculosis, trachitis, tonsillitis, mazoitis, pharyngitis.

Description of drawings

Fig. 1 is preparation technology's schema of solasodine vitriol of the present invention.

Fig. 2 is the uv absorption spectra of embodiment of the invention gained solasodine vitriol.

Fig. 3 is the DSC figure of embodiment of the invention gained solasodine vitriol.

Fig. 4 is the infrared spectrogram of embodiment of the invention gained solasodine vitriol.

Fig. 5 is the proton NMR spectrum figure of embodiment of the invention gained solasodine vitriol.

Fig. 6 is the heavy water exchange proton NMR spectrum figure of embodiment of the invention gained solasodine vitriol.

Fig. 7 is the two dimension of embodiment of the invention gained solasodine vitriol ¹H- ¹The H proton nmr spectrum of being correlated with.

Fig. 8 is the counter-rotating gated decoupling of embodiment of the invention gained solasodine vitriol ¹³The C nmr spectrum.

Fig. 9 is an embodiment of the invention gained solasodine vitriol ¹³C DEPT nmr spectrum.

Figure 10 is the two dimension of embodiment of the invention gained solasodine vitriol ¹³C- ¹The H heteronuclear nmr spectrum of being correlated with.

Figure 11 is the LCQ positive and negative ion electrospray ionization mass spectrum figure of embodiment of the invention gained solasodine vitriol.

Figure 12 is the x-ray diffraction pattern of embodiment of the invention gained solasodine vitriol.

Embodiment

Below in conjunction with drawings and Examples, test example the present invention is further described, but the present invention is not limited to embodiment, this professional those of ordinary skill is done some modification, all in protection domain of the present invention.

Embodiment 1 is a feedstock production solasodine vitriol with the black nightshade

With reference to Fig. 1, it is as follows to give birth to the preparation method who really prepares solasodine vitriol with black nightshade:

(1) taking by weighing black nightshade that the 500kg10 middle of the month to late October gathers gives birth to fruit and removes foreign material, pulverize solid-liquid separation with paste roller mill and get fruit juice, pomace, the black nightshade pomace is leached 2 times with 3% aqueous acetic acid, solid-liquid weight ratio 1: 1, solid-liquid separation gets immersion liquid, merge fruit juice and immersion liquid, place sedimentation, get supernatant liquor and be heated to and boil, remove the green colloid thing of come-up, filter cooling, filtrate is placed sedimentation, get when supernatant liquor is heated to 80 ℃ and add strong aqua to pH8, cooling-sedimentation is removed supernatant liquor, to turbid liquid down through centrifugal solid-liquid separate the thick total alkaloids of paste black nightshade.

(2) with the thick total alkaloids of 3% aqueous acetic acid dissolving paste black nightshade, solid-liquid weight ratio 1: 30, being heated to boils removes the green colloid thing of come-up, filter cooling-sedimentation, when getting supernatant liquor and being heated to 80 ℃, add strong aqua to pH8, cooling-sedimentation, make it precipitation and remove supernatant liquor fully, to turbid liquid precipitate thing centrifugal solid-liquid down separate paste black nightshade total alkaloids, 80～100 ℃ of following freeze-day with constant temperature, pulverize the black nightshade total alkaloids.

(3) the black nightshade total alkaloids is dissolved in 95% ethanolic soln that contains 5% hydrochloric acid, solid-liquid weight ratio 1: 40 is heated to complete molten suction strainer, the filtrate water-bath is heated to boils, hydrolysis 2 hours, cooling suction strainer, be washed to neutrality, get the solasodine hydrochloride, the heating of solasodine hydrochloride is dissolved in 95% ethanolic soln that contains 3%NaOH with 3 after drying of 80% washing with alcohol, being heated to boils refluxed 1 hour, heat filtering, crystallisation by cooling, suction strainer, be washed to neutrality, dry thick solasodine plate crystal.

(4) with thick solasodine with 95% dissolve with ethanol to saturated solution, in solution, add activated silica gel G, being stirred to the paste that do not flow constantly ends, after 80～90 ℃ of freeze-day with constant temperature coolings, be loaded in the stainless steel chromatography post under the vibration, jolt ramming mixes the liquid wash-out with chloroform, methyl alcohol at 10: 2, flow velocity 1ml/ second, use SbCl ₃Ethanol solution is a developer, judge with spot color reaction on the filter paper whether wash-out finishes, when the redfree spot reaction, stop wash-out, air distillation has been concentrated into a little crystallization and has separated out to elutriant, emits concentrated solution crystallisation by cooling suction strainer, the filtrate redistillation concentrates the crystallisation by cooling suction strainer, repeatable operation merges the powder that crystallizing and drying is crushed to 0.1～1.0 micron, gets solasodine.

(5) above-mentioned solasodine is dissolved in 95% ethanol, solid-liquid weight ratio 1: 30, being heated to the stirring of boiling descends slowly to add 50% sulfuric acid complete to crystalline deposit, make solution to pH=1, fully cool off suction strainer, be washed till neutrality with ethanol, the dry solasodine vitriol 216g that gets is 98.9% through high effective liquid chromatography for measuring purity.

Embodiment 2 is a feedstock production solasodine vitriol with the Fruit of yellowfruit nightshade

Get the 500kg Fruit of yellowfruit nightshade and give birth to fruit, get solasodine by embodiment 1 (1)-(4) preparation method, take by weighing solasodine 182g, with 95% ethanol it was all dissolved the solid-liquid weight ratio 1: 30, being heated to the stirring of boiling and slowly adding 36% aqueous sulfuric acid down to acid, placement is fully cooled off, make crystallization separate out suction strainer, be washed till neutrality with 95% ethanol, the dry solasodine vitriol 254g that gets is 97.4% through high effective liquid chromatography for measuring purity.

Embodiment 3 is a feedstock production solasodine vitriol with the Australian eggplant

Get the 500kg Australian eggplant and give birth to fruit, get solasodine by embodiment 1 (1)-(4) preparation method, take by weighing solasodine 100g, with 3000ml95% ethanol heating for dissolving to boiling, slowly add 40% aqueous sulfuric acid under stirring while hot, place the cool overnight suction strainer, be washed till neutrality with 95% ethanol, the dry solasodine vitriol 103g that gets is 98.1% through high effective liquid chromatography for measuring purity.

Test example 1: with thin layer chromatography check embodiment gained solasodine vitriol is with a kind of monomeric compound

Concrete operations are as follows: 5% solasodine vitriol sample solution is put in silica gel G F respectively with kapillary ₂₅₄On the plate initial point, the available three kinds of developping agents in dry back launch the salt of solasodine sulfate radical: benzene: methyl alcohol (4: 3); Benzene: methyl alcohol: acetone (2: 1: 2); Chloroform: ethanol: acetone (3: 1: 2), a spot all appears in λ=254nm under UV-light, proves that sample all is with a kind of compound.

Test example 2: with the purity of high effective liquid chromatography for measuring embodiment gained solasodine vitriol

Accurately take by weighing a certain amount of solasodine vitriol standard (sample that content has been demarcated) and gained detected sample of the present invention, 3%NaOH95% ethanolic soln with 40 times of weightmeasurement ratios mixes, reflux 2 hours, cooling adds the water of 2 times of volumes, with the chloroform extraction of 2 times of cumulative volumes 3 times, combining extraction liquid, quantitatively be diluted to scale with benzene, get the standardized solution and the corresponding detected sample solution of solasodine vitriol, (30CM * 3.9mmi.d.) is with methyl alcohol-0.01Mtris buffered soln (75: 25) post moving phase at wBondapaK18, flow velocity 2ml/min, 25 ℃ of temperature, detect at UV205, maximum absorption is arranged, record solasodine vitriol purity＞97% at the 15min place.

Test 3: the chemical structure test analysis of solasodine vitriol

Should change institute's test department test through Institute of Analysis of Changchun Branch of the Chinese Academy of Sciences, the uv absorption spectra of embodiment gained solasodine vitriol, DSC figure, infrared spectrogram, proton NMR spectrum figure, heavy water exchange proton NMR spectrum figure, two dimension ¹H- ¹The H proton be correlated with nmr spectrum, the counter-rotating gated decoupling ¹³The C nmr spectrum, ¹³C DEPT nmr spectrum, two dimension ¹³C- ¹H heteronuclear be correlated with nmr spectrum, LCQ negative ions electrospray ionization mass spectrum figure, x-ray diffraction pattern are seen Fig. 2,3,4,5,6,7,8,9,10,11,12 respectively.Test-results is a solasodine vitriol through spectrum elucidation, full structure ownership proof embodiment gained.Spectrum elucidation, full structure ownership " analytical test report " are seen the specification sheets rear attachment.

The manufacturing of embodiment 4 capsules

Accurately take by weighing the solasodine vitriol 43.0g of purity＞97% respectively, starch small grain 110.0g is mixed respectively in molten device, get the solasodine vitriol capsule contents of 1000 recipe quantities, the blue white glues capsule of 2# is put on the capsule board that at every turn can carry out 400, take by weighing the 60.0g capsule contents, be loaded in the capsule that is fixed on the capsule board envelope capsule.Press above-mentioned prescription batching and produce 3 recipe quantities, its qualification rate gets 3000 of solasodine vitriol capsules 99.25～99.62%, the plastic-aluminum shrouding, and the dress box gets 300 box capsules.

The manufacturing of embodiment 5 lyophilized injectable powders

Solasodine vitriol is down handled particulate to 0.1～1.0 micron through particulate aseptic, accurately take by weighing 24.5g, add the aseptic injection dissolved in distilled water and be 1000 bottles recipe quantity (containing industrial waste) to 24500ml, it is sub-packed in 1000 injection peace bottles, every bottle of 24ml handles through freeze-drying, the envelope bottle, the dress box gets lyophilized powder injection 100 boxes (10 in every box).

The manufacturing of embodiment 6 powder injection

Solasodine vitriol is handled to 0.1～1.0 micron through particulate down aseptic, accurately taking by weighing 24.5g is 1000 bottles of 1 recipe quantities (containing industrial waste), it is loaded in 1000 injection peace bottles with every bottle of 24mg, aseptic envelope bottle, the dress box, get solasodine sulfate powder injection 100 boxes, 10 bottle/boxes, qualification rate 〉=99.84%.

Annex:

Changchun Branch of the Chinese Academy of Sciences should change at the analyzing and testing center institute's test department

The analytical test report

One, molecular formula, molecular weight, chemical structural formula

Data 1 (one), reference substance solasodine

Molecular formula: C ₂₇H ₄₃NO ₂

Molecular weight: 413.64

Structural formula:

(2) the base chemical structural formula of detected sample solasodine vitriol

Two, ultra-violet absorption spectrum

Data 2 instruments: U.S. Cary 1E ultraviolet-visible spectrophotometer

Solvent: methyl alcohol

Condition: test solution concentration is respectively 11 μ g/ml, 12 μ g/ml, sweep length 190-600nm

1. ultra-violet absorption spectrum

A. reference substance ultra-violet absorption spectrum

B. test sample ultra-violet absorption spectrum

2. reference substance determination data:

λ _Max=203nm, uptake factor is: ${\mathrm{<figure-callout\; id="1"\; label="E"\; filenames="C20031011585800311.png,C20031011585800351.png"\; state="\{\{state\}\}">E</figure-callout>}}_{1\mathrm{<figure-callout\; id="1"\; label="cm"\; filenames="C20031011585800311.png,C20031011585800351.png"\; state="\{\{state\}\}">cm</figure-callout>}}^{1\%}=412$

3. test sample determination data:

λ _Max=204nm, uptake factor is: ${\mathrm{<figure-callout\; id="1"\; label="E"\; filenames="C20031011585800311.png,C20031011585800351.png"\; state="\{\{state\}\}">E</figure-callout>}}_{1\mathrm{<figure-callout\; id="1"\; label="cm"\; filenames="C20031011585800311.png,C20031011585800351.png"\; state="\{\{state\}\}">cm</figure-callout>}}^{1\%}=418$

4. resolve: exist the K band to absorb among the UV, show and contain double bond structure in the compound.

Three, DSC experiment

Data 3 instrument models: the DSC of PE company 7 types

Test condition: 50～300 ℃, heat up with 5 ℃/min

1. reference substance

A.DSC figure

B. fusing point: 199.5 ℃

2. test sample

A.DSC figure

B. do not observe tangible fusing point

Four, infrared absorption spectrum (IR)

Data 4 instrument models: the U.S. FTS-135 of BIO-RAD company type fourier transform infrared spectrometer

Test condition: adopt the KBr pressed disc method

1. infrared spectrogram

A. reference substance infrared spectrogram

B. test sample infrared spectrogram

2. test sample examination of infrared spectrum data sheet:

Frequency (cm -1)	Intensity	Oscillatory type	The group ownership
				3380，3302 2943 2931 2872 2846 2759 1656 1606，1594 1464 1453 1432 1378 1351 1297，1181 1154，1252 1195 1132，1095 1064，1054 1022，983 961，921，900 836 800 795 739 619	vs，br vs s，sh s s s m m-ms sh，s，ms ms m m m ms s m m vw w vw m	ν OHν as CH3ν as CH2ν s CH3ν s CH2ν NH2 +ν C-Cδ NH2 +δ as CH3，δ CH2 δ s CH3δ OH ω CH2ν C-Nν asC-O-C ν OP C-O(H)Ring vibration absorbs ρ CH3，ν s C-O-Cν ip C-O(H)Γ NH2 +γ -CHΓ CH2γ OH	-OH -CH 3 -CH 2 -CH 3 -CH 2 ＞NH 2 + C＝C ＞NH 2 + -CH 3，-CH 2- -CH 3 -OH -CH 2- C-N C-O-C，C 3-OH six-ring, five-ring-CH 3，-C-O-C C 3-OH ＞NH 2 + ＞C＝CH- -CH 2CH 2- -OH

Annotate: intensity: vs: extremely strong, s: strong, m: medium, w: a little less than

3. analysis result:

A.3380 and 3302cm ^-1Extremely strong wide absorption derive from the hydroxyl stretching vibration of hydrogen bonded, 1351cm ^-1For its flexural vibration absorption band, with 619cm ^-1The wide uptake zone of beginning is the OH out-of-plane deformation vibration, 1132～1054cm ^-1And 836cm ^-1Be respectively C ₃C-O out-phase on the-OH and homophase stretching vibration, these all prove C on the cyclopentanoperhydro-phenanthrene ₃The existence of-OH.

B.2943 and 2872cm ^-1Be respectively CH ₃Unsymmetrically and symmetrical stretching vibration, 2931 and 2846cm ^-1Be respectively CH ₂Unsymmetrically and symmetrical stretching vibration, and 1464,1453 and 1432cm ^-1Then be CH ₃Unsymmetrically angle vibration and CH ₂Scissoring vibration, 1378cm ^-1Be CH ₃Symmetric deformation vibration, 1297～1252cm simultaneously ^-1Interval absorption peak can belong to and is CH ₂Wagging vibration, 961～900cm ^-1Scope overlapping CH ₃Rocking vibration, these have all confirmed-CH ₃,-CH ₂-existence, and because 739 and 783cm ^-1Extremely weak CH ₂The appearance of rocking vibration further specifies-CH ₂-CH ₂-and isolated CH ₂Existence.

C.2759cm ^-1Strong peak be NH ₂ ⁺The stretching vibration characteristic peak, 1606 and 1594cm ^-1For its angle vibration, add 1195cm ^-1C-N stretching vibration and 800cm ^-1Extremely weak NH ₂ ⁺The existence of on-plane surface angle vibration shows existence＞NH in the sample molecule ₂ ⁺

D.1651cm ^-1The medium tenacity absorption band be the C=C stretching vibration, 795cm ^-1Weak peak be CH on-plane surface angle vibration on two keys, can represent has in the molecule＞existence of C=CH-.

E. at 1132～1054cm ^-1With 961～900cm ^-1The unsymmetrically and the symmetrical stretching vibration that also have C-O-C in two intervals respectively, 1022 and 983cm ^-1Strong absorption band may absorb relevant with the ring vibration of five-ring and six-ring.

F. infrared spectra confirms that the test sample base is consistent with the reference substance structure.

Five, ¹The H nucleus magnetic resonance ( ¹H NMR)

Data 5 instruments: Unity-400 nuclear magnetic resonance spectrometer

Solvent: reference substance: deuterochloroform (CDCl ₃), reference standard δ _TMS=0.00

Test sample: deuterated methanol (CD ₃OD), reference standard δ _TMS=0.00

(1), one dimension ¹The H nuclear magnetic resonance experiment

1. ¹The H nuclear magnetic resonance experiment

A. reference substance ¹H nuclear magnetic resonance spectrum and spectrogram

B. test sample ¹H nuclear magnetic resonance spectrum and spectrogram

C. test sample heavy water exchange ¹The H nmr spectrum

2. test sample ¹The data list of H nuclear magnetic resonance measuring

The peak sequence number	Chemical shift (δ)	Multiplicity	Coupling constant	Proton number	Corresponding proton
							1 2 3 4 5 6 7 8 9 10 11 12 13 14	5.35 4.88 4.62 3.39 3.30 3.05 2.87 2.36 2.30～1.19 1.17 1.15～1.05 1.04 0.98 0.86	d br dm m m dd t m m d m s d s	5.2 6.0 12.4，2.9 12.4 7.2 6.8	1 3 1 1 1 1 1 21 3 2 3 3 3	＝CH- NH，-OH，HCl ＞CH- ＞CH- CH 3OD ＞CH- ＞CH- ＞CH- ＞CH-，-CH 2- -CH 3 -CH 2- -CH 3 -CH 3 -CH 3

(2), two dimension ¹H- ¹H be correlated with nuclear magnetic resonance experiment ( ¹H- ¹H COSY)

1. test sample two dimension ¹H- ¹The H NMR spectrogram of being correlated with

2. test sample two dimension ¹H- ¹The relevant NMR experimental data tabulation of H

Sequence number	Chemical shift (δ)	Relevant peaks displacement (δ)	The intersection peak intensity
				1 2 3 4 5 6 7 8 9 10	5.35 4.62 3.39 3.05 2.87 2.36 2.20 2.08 1.94 1.80	2.00 2.10，1.92 2.20，1.46 2.87 3.05 1.17 3.39，1.14 1.40 1.52 1.45，1.22，1.08	m m，ms ms，w s s s ms，s s s s

(3), proton N MR spectrum is resolved

1. calculated by one dimension proton N MR spectrum peak relative integral area, test sample has 44 protons to be formed, and conforms to proton number in the solasodine salinization credit minor.

2. a low δ 5.35 is a proton contribution, has intersection information with High-Field δ 2.00 places, and δ 5.35 chemical shift peaks belong to the contribution of olefinic carbon proton, belong to H with its relevant peaks δ 2.00 ₁₈

3. δ 4.62 splits and is divided into multiplet, and with δ 2.10 and δ 1.92 coupling mutually, drop on oxygen carbonaceous subarea again, corresponding to the contribution of 1 proton, therefore, this proton carbon is subjected to ortho position proton coupling again in abutting connection with the stronger Sauerstoffatom of electronegativity, belongs to H ₈The proton contribution.

4. High-Field δ 0.86 and δ 1.04 be for unimodal by force, and correspond respectively to 3 protons contributions, belongs to H respectively ₂₉And H ₂₇The methyl proton contribution.

5. High-Field δ 1.17 and δ 0.98 split and are divided into two spectral lines, and the J value corresponds respectively to 3 protons about 7Hz, belong to H ₂₆, H ₂₈The methyl proton contribution.

δ the last 4.88 peak in the heavy water exchange spectrum, weaken, widen, displacement, belonging to is active proton contribution in the compound.

7. ¹H- ¹There are strong correlation information in δ 2.87 and δ 3.05 in the H COSY spectrum, and δ 2.87 splits and is divided into triplet, J=12.4Hz, 3.05 of δ split branch for dd, 1 proton of the corresponding respectively son in two peaks, and two peaks all drop on middle place, analyze proton carbon and are connected to the N atom, and two peaks belong to the chiral carbon two of diving with the contribution of carbonaceous.

8. test sample is become the influence of salt effect, except that the part peak position be equipped with move, other peak types and reference substance basically identical.

Six, ¹³The C nuclear magnetic resonance spectrum ( ¹³C NMR)

Data 6 instruments: Unity-400 nuclear magnetic resonance spectrometer

Solvent: reference substance: deuterochloroform (CDCl ₃), reference standard δ _CDCl3=77.00

Test sample: deuterated methanol (CD ₃OD), reference standard δ _CD3OD=49.30

(1), one dimension ¹³The C nuclear magnetic resonance experiment

1. ¹³The C nuclear magnetic resonance experiment

A. reference substance ¹³C NNE (counter-rotating gated decoupling, as follows) nuclear magnetic resonance spectrum

B. reference substance ¹³The CDEPT nuclear magnetic resonance spectrum

C. test sample ¹³C NNE nuclear magnetic resonance spectrum

D. test sample ¹³C DEPT nuclear magnetic resonance spectrum

2. test sample ¹³The tabulation of C NMR determination data

The peak preface	Chemical shift (δ)	Multiplicity	The C number	Corresponding C type
					1 2 3 4 5 6 7 8 9 10 11 12 13 14 15	142.66 122.33 100.54 85.11 72.63 63.29 57.94 51.79 46.96 43.28 43.14 42.43 40.64 38.78 38.09	s d s d d d d d t t d s t t s	1 1 1 1 1 1 1 1 1 1 1 1 1 1 1	＞C＝ -CH＝ ＞C＜ ＞CH- ＞CH- ＞CH- ＞CH- ＞CH- -CH 2- -CH 2- ＞CH- ＞C＜ -CH 2- -CH 2- ＞C＜

16 17 18 19 20 21 22 23 24 25 26 27

33.52 33.40 33.32 33.09 32.56 29.68 29.19 22.19 20.15 18.95 16.84 15.18

t t t d t d t t q q q q

1 1 1 1 1 1 1 1 1 1 1 1

-CH 2- -CH 2- -CH 2- ＞CH- -CH 2- ＞CH- -CH 2- -CH 2- -CH 3 -CH 3 -CH 3 -CH 3

(2), two dimension ¹³C- ¹H be correlated with nuclear magnetic resonance experiment ( ¹³C- ¹H HETCOR)

1. test sample two dimension ¹³C- ¹The H NMR spectrogram of being correlated with

2. test sample two dimension ¹³C- ¹The H NMR experimental data of being correlated with

Sequence number	13C chemical shift (δ)	Relevant 1H chemical shift (δ)	Relevant peaks intensity
				1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23	122.33 85.11 72.63 63.29 57.94 51.79 46.96 43.28 43.14 40.64 38.78 33.52 33.40 33.32 33.09 32.56 29.68 29.19 22.19 20.15 18.95 16.84 15.18	5.35 4.62 3.39 1.91 1.20 0.99 2.87，3.05 2.21 2.36 1.80，1.21 1.84 1.87 2.00 1.70 1.80 1.78 1.87 1.75 1.58 1.04 0.98 0.86 1.17	s s s s s s w s s w w ms s s w w s w w s s s s

(3), ¹³The C nuclear magnetic resonance spectrum is resolved

1. one dimension ¹³C NMR composes distinct 27 spectral lines, the zero lap phenomenon, and the DEPT spectrum presents 23 proton carbon peaks, methyl carbon (CH ₃) 4, secondary carbon(atom) (CH ₂-) 10,9 of primary carbons (CH), consistent with C atom number and type in the solasodine hydrochloride chemical structural formula.

2. a low olefinic carbon district has only two spectral lines, and δ 142.66 is a quaternary carbon, and δ 122.33 is the contribution of proton olefinic carbon, so contains in the compound-CH=C＜structure.

3. four spectral lines of High-Field are all the contribution of methyl carbon, and are consistent with proton N MR spectrum analysis, contain 4 methyl in this compound.

4. low δ 100.54 is a quaternary carbon, and by substituent effect as can be known, therefore this quaternary carbon belongs to C in abutting connection with two nitrogen, Sauerstoffatoms that above electronegativity is stronger ₂The carbon contribution.

5. δ 42.43 and δ 38.09 are all quaternary carbon, and by the compound characteristic as can be known, these two quaternary carbons all are link point carbon, and its node carbonaceous is respectively by-CH ₃Replace, because two keys are to β position carbon deshielding effect, δ 38.09 should belong to C ₂₁, δ 42.43 belongs to C ₁₀The carbon contribution.

6. δ 72.63 displacement peaks drop on oxygen carbon district, ¹³C- ¹There are strong coupling information in this peak and proton δ 3.39 in the relevant spectrum of H heteronuclear two-dimensional, and hence one can see that, and this carbon belongs to C in abutting connection with Sauerstoffatom ₂₄The carbon contribution.

7. δ 63.29, δ 57.94, δ 51.79 drop on oxygen carbon district, but ¹³C- ¹Above-mentioned three peaks are relevant with high field proton δ 1.91, δ 1.20 and δ 0.99 respectively in the H two-dimensional correlation spectrum, and three peaks are all d and split branch, and therefore above-mentioned three peaks should be the contribution of link point carbon.

8. δ 85.11 drops on low of oxygen carbon district, and multiplicity is d, and relevant proton peak is δ 4.62, and this peak should belong to the link point carbon in abutting connection with Sauerstoffatom, i.e. C ₈The carbon contribution.

Seven, full structure ownership

Data 7 1. solasodine salt base portion skeletal atom label chemical structural formulas

2. chemical structure-NMR composes full ownership

Sequence number	13C chemical shift (δ)	1The H chemical shift (δ) of being correlated with	The structure label
				1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27	142.66 122.33 100.54 85.11 72.63 63.29 57.94 51.79 46.96 43.28 43.14 42.43 40.64 38.78 38.09 33.52 33.40 33.32 33.09 32.56 29.68 29.19 22.19 20.15 18.95 16.84 15.18	5.35 4.62 3.39 1.91 1.20 0.99 2.87，3.05 2.21 2.36 1.80，1.21 1.84 1.87 2.00 1.70 1.80 1.78 1.87 1.75 1.58 1.04 0.98 0.86 1.17	20 19 2 8 24 9 11 13 4 25 16 10 22 15 21 17 18 7 12 23 5 6 14 27 28 29 26

Eight, mass spectrum

Data 8 instruments: LCQ electrospray mass spectrometer

Condition: the LCQ electric spray ion source, spray voltage 2kV, atomization gas is a nitrogen N ₂, 200 ℃ of metal capillary temperature, dissolve with methanol, the injection pump sample introduction that flows, sample introduction speed 5 μ l.

1.LCQ mass spectrum

A. the LCQ electrospray ionization mass spectrum figure of reference substance

B. the LCQ electrospray ionization mass spectrum figure of test sample

2. test sample mass spectroscopy data (EI-MS data):

m/z	Relative abundance (%)
		414 396 271 253	100.0 16.5 12.3 11.4

3. lytic pathway:

4. resolve:

A. sample is identical with the quasi-molecular ion peak of reference substance among the figure, and its cracked rule is consistent.Wherein the m/z414 peak in the positive ion electrospray ionization mass spectrum of sample is the quasi-molecular ion peak that sample loses HCl, and is identical with the quasi-molecular ion peak of reference substance.

The b.m/z396 peak is the dehydration peak of quasi-molecular ion peak, and the m/z271 peak is that m/z396 peak ion loses The fragment peak that obtains, m/z253 ion are the fragment peak that the m/z271 ion is further sloughed a part water.

Nine, X-ray diffraction experiment

Data 9 instrument models: D/max-IIB-type x-ray diffractometer

Test condition: CuK _αSource of radiation, operating voltage 40kV, working current 20mA.

1.X-x ray diffration pattern x

A. reference substance x-ray diffraction pattern

B. test sample x-ray diffraction pattern

2.2 θ data list

A. reference substance data sheet

B. test sample data sheet

Ten, integration analysis:

It is 7 that data 10 1. are calculated degree of unsaturation by solasodine salinization credit minor.

2. Australian eggplant amine salt sample is identical with the quasi-molecular ion peak of reference substance, and its cracked rule is consistent.Wherein the m/z414 peak in the positive ion electrospray ionization mass spectrum of sample is the quasi-molecular ion peak that sample loses HCl, and is identical with the quasi-molecular ion peak of reference substance.The molecular weight of base portion is 414 in the proof compound.

3. one dimension ¹H NMR spectrum peak relative area can get, and test sample has 44 protons to be formed, and conforms to proton number in the solasodine salinization credit minor. ¹³C NMR quantitative experiment and ¹³C DEPT NMR experimental result represents that this compound has 27 carbon atoms, the mutual zero lap image in absorption resonance peak, wherein-CH ₃4 at carbon peak, CH ₂10 at carbon peak, 9 at tertiary carbon peak, 4 at quaternary carbon peak proves that this compound is made up of 27 carbon, and is consistent with carbon atom number in the Australian eggplant amine salt.

4.IR in the spectrum 2943 and 2872cm ^-1Be respectively unsymmetrically and the symmetrical stretching vibration of CH3,2931 and 2846cm ^-1Be respectively CH ₂Unsymmetrically and symmetrical stretching vibration, and 1464,1453 and 1432cm ^-1Then be CH ₃Unsymmetrically angle vibration and CH ₂Scissoring vibration, 1378cm ^-1Be CH ₃Symmetric deformation vibration, 1297～1252cm simultaneously ^-1Interval absorption peak can belong to and is CH ₂Wagging vibration, 961～900cm ^-1Scope overlapping CH ₃Rocking vibration, these have all confirmed-CH ₃,-CH ₂-existence, and because 739 and 783cm ^-1Extremely weak CH ₂The appearance of rocking vibration further specifies-CH ₂-CH ₂-and isolated CH ₂Existence.

5.IR in the spectrum 3380 and 3302cm ^-1Extremely strong wide absorption derive from the hydroxyl stretching vibration of hydrogen bonded, 1351cm ^-1For its flexural vibration absorption band, with 619cm ^-1The wide uptake zone of beginning is the vibration of OH and outside sweep, 1132～1054cm ^-1And 836cm ^-1Be respectively C ₃C-O out-phase on the-OH and homophase stretching vibration, these all prove C on the cyclopentanoperhydro-phenanthrene ₃The existence of-OH.

In the proton N MR spectrum δ the last 4.88 peak in the heavy water exchange spectrum, weaken, widen, displacement.Prove this peak for＞NH ,-active proton contributions such as OH and HCl.

7. δ 4.61 splits and is divided into multiplet in the proton N MR spectrum, and with δ 2.10 and δ 1.92 coupling mutually, drop on oxygen carbonaceous subarea again, corresponding to 1 proton contribution, therefore, this proton carbon is in abutting connection with the stronger Sauerstoffatom of electronegativity, be subjected to ortho position proton coupling again, belong to the contribution of OCH proton.

8. ¹³C NMR experiment shows and only contains two double key carbon peaks in this product that UV composes λ _MaxBe 204nm, constitute the monoene structure.

9. ¹³C NMR spectrum δ 42.43 and δ 38.09 are all quaternary carbon, and by the compound characteristic as can be known, these two quaternary carbons all are link point carbon, and its node carbonaceous is respectively by-CH ₃Replace, because two keys are to β position carbon deshielding effect, δ 38.09 should belong to C ₂₁, δ 42.43 belongs to C ₁₀The carbon contribution.

10. ¹³C NMR composes four spectral lines of High-Field, is all the contribution of methyl carbon, and is consistent with proton N MR spectrum analysis, contains 4 methyl in this compound.Low δ 100.54 is a quaternary carbon, and by substituent effect as can be known, therefore this quaternary carbon belongs to＞contribution of C-O carbon in abutting connection with two nitrogen, Sauerstoffatoms that above electronegativity is stronger.

11.IR 2759cm in the spectrum ^-1Strong peak be NH ₂ ⁺The stretching vibration characteristic peak, 1606 and 1594cm ^-1For its angle vibration, add 1195cm ^-1C-N stretching vibration and 800cm ^-1Extremely weak NH ₂ ^-The existence of on-plane surface angle vibration shows to have NH in the sample molecule ₂ ⁺

12. comprehensive each spectrum analysis, test sample is the salt compound of reference substance.The test sample chemical structural formula is:

Claims (5)

1, the solasodine vitriol of a kind of following general formula (I):

2, the preparation method of the compound of claim 1 is characterized in that this method may further comprise the steps:

(1) extraction of raw material processing and thick total alkaloids

Black nightshade, Australian eggplant or Fruit of yellowfruit nightshade given birth to fruit or its herb after screening, pulverize and press the juice solid-liquid separation to get fruit juice, pomace, the aqueous acetic acid leaching pomace with 2～6%, solid-liquid weight ratio 1: 1, leach 2～3 times, solid-liquid separation gets immersion liquid, merges fruit juice and immersion liquid, places sedimentation, getting supernatant liquor is heated to boil and removes supernatant, the filtration cooling is placed sedimentation with filtrate, gets to add strong aqua or NaOH, Na when supernatant liquor is heated to 80～85 ℃ ₂CO ₃, KOH the aqueous solution to pH8～9, cooling-sedimentation removes supernatant liquor, down turbid liquid centrifugal solid-liquid is separated, the thick total alkaloids of paste;

(2) thick total alkaloids is refining

With the thick total alkaloids of 2～6% aqueous acetic acids dissolving paste, solid-liquid weight ratio 1: 10～30, being heated to boils removes supernatant, filter cooling-sedimentation, when getting supernatant liquor and being heated to 80～85 ℃, the aqueous solution that adds strong aqua or alkali is to pH8～9, and cooling-sedimentation removes supernatant liquor, to following turbid liquid centrifugal solid-liquid separate the paste total alkaloids, 80～100 ℃ of following freeze-day with constant temperature, pulverize the total alkaloids in black nightshade, Australian eggplant or the Fruit of yellowfruit nightshade;

(3) extraction of thick solasodine

With 80～95% dissolve with ethanol solution total alkaloidss that contain 3～10% hydrochloric acid, solid-liquid weight ratio 1: 30～40, hydrolysis reflux 2～3 hours, cold filtration, with 95% ethanol, water, be washed till neutrality, the dry solasodine hydrochloride that gets is with the 95% dissolve with ethanol solution solasodine hydrochloride that contains 3～10%NaOH, reflux 1～2 hour, heat filtering, the cooling suction strainer is washed till neutrality, dry thick solasodine;

(4) solasodine is refining

Thick solasodine is dissolved in methyl alcohol, ethanol or the chloroform to saturated solution, mix with thick solasodine solution with the silica gel G after the activation, the immobilising paste of furnishing, at 80～90 ℃ of following freeze-day with constant temperature, pack into after the cooling in the chromatography column, jolt ramming, with chloroform or chloroform: methyl alcohol volume ratio 10: 1～2 or chloroform: ethanol: one of acetone volume ratio 3: 1: 2 is carried out wash-out and is got elutriant, after the check wash-out finishes, elutriant normal pressure or underpressure distillation are concentrated, behind concentrated solution crystallisation by cooling filtration drying, be crushed to 0.1～1.0 micron powder and get solasodine;

(5) prepare solasodine vitriol with solasodine

Solasodine is dissolved in being heated in 95% ethanol boils, solid-liquid weight ratio 1: 20-40 stirs and slowly adds 10～50% aqueous sulfuric acid in the thermotropism solasodine ethanolic soln down, to pH=1, crystallisation by cooling, suction strainer is washed till neutrality with ethanol, the dry solasodine vitriol that gets.

3, the application of the compound of claim 1 in the preparation cancer therapy drug.

4, the application of the compound of claim 1 in the preparation suppressing panting calming medicine.

5, the application of the compound of claim 1 in the preparation anti-inflammatory drug.

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