CN111440224A - Glucoside alkaloid and application thereof - Google Patents
Glucoside alkaloid and application thereof Download PDFInfo
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Abstract
The invention discloses glycoside alkaloids and application thereof, wherein the glycoside alkaloids are compounds 1-7 respectively, and a pharmaceutical composition containing the compounds 1-7 has good inhibition and treatment effects on various tumors such as S180 sarcoma, Ehrlich ascites tumor, L ewis lung cancer transplantation tumor, mouse liver cancer cell H22 transplantation tumor, human lung cancer gland cell A549 nude mouse transplantation tumor, human lung squamous carcinoma cell NCI-H520 nude mouse transplantation tumor, human large cell lung cancer NCI-H460 nude mouse transplantation tumor and the like.
Description
Technical Field
The invention relates to the technical field of medicine preparation, in particular to glucoside alkaloid and application thereof.
Background
The glucoside alkaloid is mainly distributed in plants of Solanaceae and Liliaceae, and comprises hydrophobic aglycone and hydrophilic oligosaccharide chain, wherein the aglycone comprises spirostanyl alkane, solanesol alkane and other steroid derivatives, and the oligosaccharide chain comprises 3-4 monosaccharides and is steroid saponin. The plant synthesized glucoside alkaloid is mainly used for defending the invasion of microorganisms, animals and insects, so the glucoside alkaloid has certain toxicity, and meanwhile, the plant synthesized glucoside alkaloid also has various physiological activities of reducing cholesterol and hypertension, diminishing inflammation, promoting blood circulation, relieving pain, resisting allergy, enhancing the immunity of the organism, resisting pathogenic microorganisms and the like, and has potential medicinal value. Solanaceae and liliaceae plants widely exist in the natural world, such as solanum dulcamara, potatoes, tomatoes and other plants, have high economic value, the traditional Chinese medicine solanum dulcamara is originally carried in Shennong Baicao Jing, is sweet in taste and cold in nature, has the effects of clearing heat, detoxifying, dispelling wind, removing blood stasis and the like, and researches show that the main chemical component of solanum dulcamara is glucoside alkaloid, so that the research on extracting novel glucoside alkaloid from the traditional Chinese medicine solanum dulcamara has important significance.
In view of the medicinal efficacy of glycoside alkaloids, the medicinal world has paid great attention to it, and chinese patent document CN102319319A discloses a solanum lyratum extract and an anticancer drug comprising the same, which is prepared by the steps of extracting solanum lyratum medicinal material with ethanol, refluxing and extracting the solanum lyratum medicinal material in ethanol, recovering the solvent and evaporating to dryness to obtain an ethanol extract, refining, namely, forming the ethanol extract into a solution with water, adsorbing the solution with a weakly acidic cation exchange macroporous resin column, eluting with ethanol and hydrochloric acid ethanol in sequence, collecting the ethanol eluate of hydrochloric acid, neutralizing the eluate with ammonia water to neutrality, and desalting to obtain the solanum lyratum extract, wherein the solanum lyratum extract comprises 1-60% of steroid alkaloids, and experiments prove that the extract has good antitumor effect on S180 sarcoma, ehrlich ascites tumor, L ewis lung cancer transplantable tumor and mouse liver cancer cell H22 transplantable tumor.
Chinese patent documents CN106928309A, CN106939031A and CN106928309B disclose three glucoside alkaloids extracted from solanum dulcamara, and preparation methods and applications thereof. However, the glucoside alkaloids which can be separated and extracted are limited in types, complex in structure, single in action effect, high in energy consumption and heavy in pollution in the extraction process, and the development of the glucoside alkaloids is severely limited, so that how to provide a preparation method of glucoside alkaloids which can extract more glucoside alkaloids so as to enable the glucoside alkaloids to have better action effect and simple, convenient, energy-saving and environment-friendly process and clarify the application of the glucoside alkaloids is a problem to be solved urgently by technicians in the field.
Disclosure of Invention
In view of the above, the invention provides an energy-saving, environment-friendly and efficient preparation method of glucoside alkaloids, and the process is improved to extract more types of glucoside alkaloids and clarify the application of the glucoside alkaloids.
In order to achieve the purpose, the invention adopts the following technical scheme:
the invention firstly provides seven novel compound structures, namely glucoside alkaloid, with the molecular formula: c45H75NO17The molecular weight is 901, named (3 β,5 α,16R,20R,22S,25R) -spirostan-3-yloxy- β -D-glucopyranosyl- (1 → 2) -oxy- β -D-glucopyranosyl- (1 → 4) - β -D-galactopyranoside, hereinafter referred to as compound 1, having the following structural formula:
a glycoside alkaloid of the formula: c45H73NO17899, named (3 β,5 α,16R,20R,22S,25R) -spirostanin-5-alkenyl-3-yloxy- β -D-glucopyranosyl- (1 → 2) -oxy- β -D-glucopyranosyl- (1 → 4) - β -D-galactopyranoside, compound 2, having the following formula:
a glycoside alkaloid of the formula: c45H67NO18The compound 3 has a molecular weight of 909, namely 15 β -hydroxy- (3 β,25R) -16, 23-epoxy-23, 24-cyclic imine-cholesterol-5, 16,20,23(N) -tetraene-3 β -alcohol-3-O- β -D-glucopyranosyl- (1 → 2) - β -D-glucopyranosyl- (1 → 6) - β -D-galactopyranoside, and has the following structural formula:
a glycoside alkaloid of the formula: : c45H69NO18911 molecular weight, designated 15 β -hydroxy- (3 β,25R) -16, 23-epoxy-23, 24-cyclic imine-cholesterol-16, 20,23(N) -triene-3 β -ol-3-O- β -D-glucopyranosyl- (1 → 2) - β -D-glucopyranosyl- (1 → 6) - β -D-galactopyranoside, compound 4, having the following structural formula:
a glycoside alkaloid of the formula: c47H71NO18937, named 15 β -ethoxy- (3 β,25R) -16, 23-epoxy-23, 24-cyclic imine-cholesterol-5, 16,20,23(N) -tetraene-3 β -ol-3-O- β -D-glucopyranosyl- (1 → 2) - β -D-glucopyranosyl- (1 → 6) - β -D-galactopyranoside, having the following structural formula:
a glycoside alkaloid of the formula: c47H73NO18939, named 15 β -ethoxy- (3 β,25R) -16, 23-epoxy-23, 24-cyclic imine-cholesterol-16, 20,23(N) -triene-3 β -ol-3-O- β -D-glucopyranosyl- (1 → 2) - β -D-glucopyranosyl- (1 → 6) - β -D-galactopyranoside, having the following structural formula:
a glycoside alkaloid of the formula: c45H69NO17895, named 16, 23-epoxy-22, 26-cyclic imine-cholesterol-22 (N),23, 25-triene-3 β -ol-3-O- β -D-glucopyranosyl- (1 → 2) - β -D-glucopyranosyl- (1 → 6) - β -D-galactopyranoside, compound 7, having the following structural formula:
the preparation method of the seven compounds comprises the following steps:
(1) reflux-extracting herba Solani Lyrati dried whole plant with 70% ethanol water solution, filtering, concentrating the filtrate to obtain extract with relative density of 1.05 at 50 deg.C, adding distilled water for dispersing, filtering, adding the filtrate to D151 macroporous adsorbent resin column, eluting with 2-4 times column volume of distilled water and 2-4 times column volume of 95% ethanol water solution, discarding eluate, eluting with 3-5 times column volume of 6 ‰ hydrochloric acid ethanol water solution with ethanol volume concentration of 95%, collecting ethanol hydrochloride eluate, neutralizing with ammonia water to neutrality, filtering, concentrating the filtrate to dryness, dispersing with distilled water, adding the dispersed medicinal liquid to AB-8 macroporous adsorbent resin, eluting with 6-10 times column volume of distilled water, discarding eluate, eluting with 3-5 times column volume of 95% ethanol water solution, collecting ethanol eluate, concentrating, and drying to obtain herba Solani Lyrati total alkali;
(2) adding the solanum lyratum total alkali sample prepared in the step (1) into an alcohol solution with volume concentration of 60%, heating to 90 ℃ for dissolution, then adding thin-layer chromatography silica gel for uniform mixing, then separating by normal-phase silica gel column chromatography, performing gradient elution by using an eluent formed by ethyl acetate and an ethanol water solution with a volume ratio of (3-0): 1, wherein the volume concentration of the ethanol water solution is 93-97%, using a hydrochloric acid water solution modified bismuth potassium iodide test solution as a color developing agent, detecting by using thin-layer chromatography, and combining the same flow components;
(3) filtering different components obtained in the step (2) with 0.45um filter membranes respectively, then separating and purifying by adopting high performance liquid chromatography, and collecting eluates with different retention times to obtain glucoside alkaloid compounds 1-7.
Wherein, in the step (2), 45-55 parts by weight of solanum lyratum total alkali sample is weighed, added into 400-600 parts by volume of 60% ethanol water solution with volume concentration, heated to 80-100 ℃, then added with 10-20 parts by weight of thin layer chromatography silica gel, mixed evenly, and loaded on 700-900 parts by weight of thin layer chromatography silica gel for silica gel column chromatography separation (the relation between the parts by weight and the parts by volume is g/m L).
The preparation method of the hydrochloric acid aqueous solution modified bismuth potassium iodide test solution in the step (2) comprises the following steps:
weighing 0.8-0.9 part by weight of basic bismuth nitrate, sequentially adding 9-11 parts by volume of glacial acetic acid, 39-41 parts by volume of water and 19-21 parts by volume of potassium iodide solution, and uniformly mixing to obtain a bismuth potassium iodide test solution, adding 0.6 mol/L hydrochloric acid aqueous solution into the bismuth potassium iodide test solution, wherein the volume ratio of the bismuth potassium iodide test solution to the hydrochloric acid aqueous solution is 1:2, so as to obtain the bismuth potassium iodide test solution improved by the hydrochloric acid aqueous solution (the relationship between the parts by weight and the parts by volume is g/m L).
The liquid chromatography mobile phase in the step (3) is an acetonitrile/water/acid system, the acid is trifluoroacetic acid (TFA) with the concentration of 0.05%, and the acetonitrile/water mixing ratio is 5:95-95: 5.
Preferably, in the separation and purification process of the high performance liquid chromatography in the step (3), the chromatographic conditions are that a C18 liquid chromatographic column is 10u 250 x 10mm, acetonitrile containing 0.05% TFA is used as a mobile phase A, water containing 0.05% TFA is used as a mobile phase B, gradient elution is carried out according to the following program, the volume ratio of the mobile phase A to the mobile phase B is 5%: 95% → 95% -5%, the flow rate of the mobile phase is controlled to be 2m L/min, and the temperature of the column is controlled to be 25 ℃.
The invention also provides a pharmaceutically acceptable salt or carrier of the compounds 1-7 or a clinically acceptable preparation prepared by adding conventional auxiliary materials into the compound 1-7 serving as an active ingredient and adopting a conventional process.
Preferably, the formulation comprises a tablet, capsule, granule, syrup, powder, pill, tincture, medicated wine, soft extract, lozenge or mixture.
The invention also provides application of the compounds 1-7 in treating S180 sarcoma, Ehrlich ascites tumor, L ewis lung cancer transplantation tumor, mouse liver cancer cell H22 transplantation tumor, human lung cancer gland cell A549 nude mouse transplantation tumor, human lung squamous cancer cell NCI-H520 nude mouse transplantation tumor and human large cell lung cancer NCI-H460 nude mouse transplantation tumor.
Through the scheme, compared with the prior art, the invention has the following beneficial effects:
the invention discloses seven novel compounds 1-7 and a preparation method thereof, wherein a pharmaceutical composition containing the four novel compounds has good treatment and inhibition effects on various tumors such as S180 sarcoma, Ehrlich ascites tumor, L ewis lung cancer transplantation tumor, mouse liver cancer cell H22 transplantation tumor, human lung cancer gland cell A549 nude mouse transplantation tumor, human lung squamous carcinoma cell NCI-H520 nude mouse transplantation tumor, human large cell lung cancer NCI-H460 nude mouse transplantation tumor and the like.
Drawings
In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the drawings used in the description of the embodiments or the prior art will be briefly described below, and it is obvious that the drawings in the following description are only embodiments of the present invention, and for those skilled in the art, other drawings can be obtained according to the provided drawings without creative efforts.
FIG. 1 is a drawing showing Compound 1 obtained in example 1 of the present invention1HNMR spectrogram;
FIG. 2 is a drawing showing Compound 1 obtained in example 1 of the present invention13C NMR spectrum;
FIG. 3 is a drawing showing Compound 2 obtained in example 1 of the present invention1HNMR spectrogram;
FIG. 4 is a drawing showing Compound 2 prepared in example 1 of the present invention13C NMR spectrum;
FIG. 5 is a drawing showing Compound 3 prepared in example 1 of the present invention1HNMR spectrogram;
FIG. 6 is a drawing showing Compound 3 prepared in example 1 of the present invention13C NMR spectrum;
FIG. 7 is a drawing showing Compound 4 prepared in example 1 of the present invention1HNMR spectrogram;
FIG. 8 is a drawing showing Compound 4 prepared in example 1 of the present invention13C NMR spectrum;
FIG. 9 is a drawing showing Compound 5 prepared in example 1 of the present invention1HNMR spectrogram;
FIG. 10 is a drawing showing Compound 5 prepared in example 1 of the present invention13C NMR spectrum;
FIG. 11 is a drawing showing Compound 6 obtained in example 1 of the present invention1HNMR spectrogram;
FIG. 12 is a drawing showing Compound 6 obtained in example 1 of the present invention13C NMR spectrum;
FIG. 13 is a drawing showing Compound 7 obtained in example 1 of the present invention1HNMR spectrogram;
FIG. 14 is a drawing showing Compound 7 prepared in example 1 of the present invention13C NMR spectrum;
FIG. 15 is a graph showing the time-tumor volume curve and the time-tumor weight curve of the tumor volume and weight trend of each group of mice in Experimental example 2 of the present invention (note: P < 0.01, vs Model control group);
FIG. 16 is a graph showing the time-tumor volume curve and the time-tumor weight curve of the tumor volume and weight trend of each group of mice in Experimental example 3 of the present invention (note: P < 0.01, vs Model control group);
FIG. 17 is a graph showing the time-tumor volume curve and the time-tumor weight curve of the tumor volume and weight trend of each group of mice in Experimental example 4 of the present invention (note: P < 0.01, vs Model control group);
Detailed Description
The technical solutions in the embodiments of the present invention will be clearly and completely described below with reference to the embodiments of the present invention, and it is obvious that the described embodiments are only a part of the embodiments of the present invention, and not all of the embodiments. All other embodiments, which can be obtained by a person skilled in the art without any inventive step based on the embodiments of the present invention, are within the scope of the present invention.
In the following examples and experimental examples of the present invention,
1) medicinal materials and experimental objects:
the solanum lyratum medicinal material selected in the experiment is purchased from Jiangsu Peipi county, and identified as solanum lyratum of Solanaceae by Hoxirong Chinese institute of academy of TCM.
2) Reagents and consumables:
d151 weakly acidic propylene-based cation exchange resin (cat # 20150304);
AB-8 macroporous adsorbent resin (cat # 20150203) was purchased from Guangfu Fine chemical research institute of Tianjin;
thin layer chromatography silica gel H (chemically pure) (cat # 20151220), purchased from Qingdao ocean chemical Limited;
acetonitrile was chromatographically pure, purchased from Fisher Chemical;
95% ethanol, ammonia water, hydrochloric acid, ethyl acetate, trifluoroacetic acid and the like are analytically pure.
3) Apparatus and device
high performance liquid chromatograph (Agilent) equipped with 1329A autosampler, 1211 quaternary gradient pump, 1322 online degasser, 1316A Column oven, 1310B variable wavelength UV-detector and 2170BA chromatography station, Column diamonsil C18(10 μm, 10 × 250mm) Column.
The specific method for improving the bismuth potassium iodide test solution by the hydrochloric acid aqueous solution comprises the steps of weighing 0.85g of basic bismuth nitrate, sequentially adding 10m L glacial acetic acid, 40m L water and 20m L potassium iodide solution, uniformly mixing to obtain the bismuth potassium iodide test solution, taking 1m L prepared bismuth potassium iodide test solution, and adding 2m L of hydrochloric acid aqueous solution with the mass concentration of 0.6 mol/L to obtain the hydrochloric acid aqueous solution improved bismuth potassium iodide test solution.
Example 1
The compounds 1-4 and the preparation method thereof provided by the embodiment comprise the following steps:
(1) taking 10Kg of solanum lyratum thunb dry whole herb, adding 80L volume concentration of 70% ethanol water solution, refluxing and extracting for 3 times at 40 ℃, extracting for 2h each time, filtering, merging filtrate, concentrating the filtrate to an extract with the relative density of 1.05 at 50 ℃, adding 10 times of distilled water dispersion of the mass of the extract, filtering, taking the filtrate to a D151 macroporous adsorption resin column, eluting by using 3 times of column volume of distilled water and 3 times of column volume of 95% ethanol water solution in sequence, discarding the eluent, then eluting by using 4 times of column volume of ethanol water solution containing 6 per thousand hydrochloric acid, wherein the volume concentration of ethanol in the hydrochloric acid ethanol water solution is 95%, collecting hydrochloric acid ethanol eluent, neutralizing by using ammonia water to be neutral, filtering, concentrating the filtrate to be dry, dispersing by using distilled water, adding the dispersed medicine liquid to AB-8 macroporous adsorption resin, eluting by using 8 times of column volume of distilled water, discarding the eluent, then eluting by using 4 times of column volume of ethanol water solution with the concentration of 95% yield, collecting ethanol, drying, namely concentrating 68g of the eluent to obtain 6 per thousand of white alkali, and obtaining total alkali;
(2) dissolving 50g of solanum lyratum total alkali sample in 500m L volume percent 60% ethanol aqueous solution at the temperature of 90 ℃, adding 15g of thin layer chromatography silica gel, uniformly mixing, placing on a water bath, drying by distillation, loading on a silica gel chromatography column added with 800g of thin layer chromatography silica gel for chromatography separation, carrying out gradient elution by using an eluant formed by ethyl acetate and ethanol aqueous solution with the volume ratio of 3: 1, 2: 1, 1:2 and 1: 3 in sequence, wherein the volume concentration of the ethanol aqueous solution is 95%, using a bismuth potassium iodide test solution modified by hydrochloric acid aqueous solution as a color developing agent, carrying out detection by using thin layer chromatography, combining same fractions, replacing the next elution gradient when the detection result of the thin layer chromatography is negative in each gradient elution process, repeating the elution steps until the eluant ratio reaches the ratio of ethyl acetate and ethanol aqueous solution is 1: 3, and terminating the elution when the detection result of the final thin layer chromatography is negative.
(3) Weighing 400mg of the same flow obtained by eluting with eluent 2: 1 in the step (2), and then separating and purifying by adopting a high performance liquid chromatography, wherein the chromatographic conditions are that a C18 liquid chromatographic column is used for 10u 250 x 10mm, acetonitrile containing 0.05% TFA is used as a mobile phase A, water containing 0.05% TFA is used as a mobile phase B, gradient elution is carried out according to the following procedures of 0-60 min, the volume ratio of the mobile phase A to the mobile phase B is 5%: 95% → 95-5%, the flow rate of the mobile phase is controlled to be 2m L/min, the column temperature is controlled to be 25 ℃, components are detected by an ultraviolet detector, and the glucoside alkaloid with the structure shown in the formula compounds 1-2 is obtained by collecting.
(4) Weighing 500mg of the same flow obtained by eluting with an eluent in a ratio of 1: 3 in the step (2), and then separating and purifying by adopting a high performance liquid chromatography, wherein the chromatographic conditions are as follows, C18 liquid chromatographic column, acetonitrile containing 0.05% TFA is used as a mobile phase A, water containing 0.05% TFA is used as a mobile phase B, gradient elution is carried out according to the following procedures of 0-60 min, the volume ratio of the mobile phase A to the mobile phase B is 5%: 95% → 95%: 5%, the flow rate of the mobile phase is controlled to be 2m L/min, the column temperature is controlled to be 25 ℃, components are detected by an ultraviolet detector, and the glucoside alkaloid with the structure shown as the formula compounds 3-7 is obtained.
The compounds 1-7 prepared by the embodiment are respectively or randomly combined and added with conventional auxiliary materials to prepare tablets and capsules according to a conventional process.
FIGS. 1-2 are, in sequence, Compound 1, prepared according to example 1 of the invention1HNMR spectrogram,13C NMR spectrum chart;
FIGS. 3 to 4 are views of Compound 2 prepared in example 1 of the present invention in sequence1HNMR spectrogram,13C NMR spectrum chart; the analysis results of FIGS. 1-2 and 3-4 are shown in Table 1.
Table 1:
wherein the hydrogen spectrum and the carbon spectrum data in Table 1 are1H-NMR (600MHz) and13C-NMR (150MHz), and the solvent is deuterated methanol.
FIGS. 5 to 6 are views of Compound 3 prepared in example 1 of the present invention in sequence1HNMR spectrogram,13C NMR spectrum chart;
FIGS. 7 to 8 are views of Compound 4 prepared in example 1 of the present invention in sequence1HNMR spectrogram,13C NMR spectrum chart;
the analytical results of FIGS. 5 to 6 and 7 to 8 are shown in Table 2 below.
Table 2:
wherein the hydrogen spectrum and the carbon spectrum data in Table 2 are1H-NMR (600MHz) and13C-NMR (150MHz), and the solvent is deuterated methanol.
FIGS. 9 to 10 are views of Compound 5 prepared in example 1 of the present invention1HNMR spectrogram,13C NMR spectrum chart;
FIGS. 11 to 12 are views of Compound 6 prepared in example 1 of the present invention in sequence1HNMR spectrogram,13C NMR spectrum;
the analytical results of FIGS. 9 to 10 and 11 to 12 are shown in Table 3 below.
TABLE 3
FIGS. 13 to 14 are views of Compound 7 prepared in example 1 of the present invention in sequence1HNMR spectrogram,13C NMR spectrum;
the analytical results of FIGS. 13 to 14 are shown in Table 4 below.
TABLE 4
Wherein the hydrogen spectrum and carbon spectrum data in tables 3-4 are1H-NMR (600MHz) and13C-NMR (150MHz), and the solvent is deuterated methanol.
Example 2
The difference from the scheme of the embodiment 1 is that 100L volume percent 70 percent ethanol water solution is adopted for extraction in the step (1), 40g of solanum lyratum total alkali sample is taken in the step (2) and added into 400m L volume percent 60 percent ethanol water solution to be dissolved at the temperature of 80 ℃, then 10g of thin layer chromatography silica gel is added to be uniformly mixed, the mixture is placed on a water bath pot to be steamed to dryness, and the mixture is loaded into a silica gel chromatographic column added with 700g of thin layer chromatography silica gel to be chromatographically separated, and the rest contents are the same.
The compounds 1-7 prepared by the embodiment are respectively or randomly combined and added with conventional auxiliary materials to be prepared into granules, powder and pills according to a conventional process.
Example 3
The difference from the scheme of the embodiment 1 is that 90L vol% 70% ethanol aqueous solution is adopted for extraction in the step (1), 55g of solanum lyratum total alkali sample is taken in the step (2) and added into 600m L vol% 60% ethanol aqueous solution to be dissolved at the temperature of 100 ℃, then 20g of thin layer chromatography silica gel is added to be uniformly mixed, the mixture is placed on a water bath pot to be steamed to dryness, and the mixture is loaded into a silica gel chromatographic column added with 900g of thin layer chromatography silica gel to be subjected to chromatographic separation, and the rest contents are the same.
The compounds 1-7 prepared by the embodiment are respectively or randomly combined and added with conventional auxiliary materials to prepare syrups, tinctures and vinuses according to a conventional process.
Example 4
In this embodiment, the compounds 1 to 7 prepared in this embodiment are respectively or randomly combined and added with conventional adjuvants, and the tincture is prepared according to a conventional process.
The compounds 1 to 7 prepared in the embodiment are respectively or randomly combined and added with conventional auxiliary materials to prepare soft extract, lozenge and mixture according to the conventional process.
The effects of the compounds 1 to 7 prepared by the present invention will be described below by using specific experimental examples, in which the main drugs and reagents are prepared:
taxol (Taxol) (lot: 15100018) was purchased from Sichuan Tai Chi pharmaceutical Co., Ltd, stored at 4 ℃ in laboratory, diluted with physiological saline to 0.8mg/m L, and prepared ready for use;
compound Yew capsule (HDS) (batch number: 160402) is purchased from Chongqing Sainuo biological pharmaceutical corporation, stored at 4 deg.C in laboratory, added with physiological saline to 20mg/m L, and prepared for use.
Solanum lyratum total alkaloid (STA), prepared in example 1, is stored at 4 ℃ in a laboratory, is adjusted to the required concentration by adding physiological saline, is prepared for use, and can be prepared to the maximum concentration of 250mg/m L.
Experimental example 1
Influence of the medicinal composition solanum dulcamara total steroid alkaloid containing the compounds 1-7 on the survival time of L ewis transplanted tumor mice;
first, experiment method
1) Cell and animal:
the mouse lung cancer cells L ewis are provided by Jiangsu Kai-based biotechnology, Inc.;
60C 57B L/6 mice, female, 5W week-old, 16-18 g weight, provided by Shanghai Ling Biotechnology Limited, and the production license of the experimental animal is SCXK 2013-.
2) Preparation of transplanted tumor model
Mouse lung cancer L ewis cell suspension in logarithmic growth phase was collected, adjusted to 1 × 107/m L cell concentration, and inoculated subcutaneously in the right axilla of C57B L/6 mice at 0.1m L (i.e., 1 × 106 cells) per cell.
3) Animal grouping and administration
The method comprises the following steps of after L ewis lung cancer transplantation tumor models are successfully prepared, closely observing the growth conditions of mice, dividing the animals into 6 groups according to the size uniformity of transplantation tumors, wherein the 6 groups comprise a model control group, a paclitaxel group, a compound Chinese yew group, a solanine total alkali low-dose group, a solanine total alkali medium-dose group and a solanine total alkali high-dose group, 10 animals in each group are respectively administrated, the administration volume is 0.1m L/(10 g.BW), the administration period is 28d, the model control group is administrated with physiological saline (1 time/d) by gastric lavage, the paclitaxel group is administrated with paclitaxel 8mg/kg.BW (1 time/2 d) by intraperitoneal injection, the compound Chinese yew group is administrated with compound Chinese yew capsules 200mg/kg.BW (1 time/d), the solanine total alkali group is administrated with solanine by gastric lavage, 1 time/d, the low-dose group is 50mg/kg.BW, the medium-dose group is 100mg/kg.BW, and the high-dose group is fed with 200mg/kg.BW until the animals die naturally and the survival period of each.
4) Observation index
The survival time of each group of animals was recorded and the life extension (%) was calculated by the following formula.
Life extension rate (%) - (average survival time of administered group-survival time of model control group)/survival time of model control group × 100%
Second, experimental results
The animal survival time test results are shown in table 5, and as can be seen from table 5, compared with the model control group, the survival time of animals is obviously prolonged (P < 0.05), especially the life prolonging rate of the high-dose group of the solanum dulcamara alkaloid is equivalent to that of the paclitaxel group (P > 0.05), and the survival days of the first dead animals are longer (the survival time of the first dead mice of the paclitaxel is 37d, and the survival time of the first dead mice of the high-dose group of the solanum dulcamara alkaloid is 40 d).
TABLE 5 Effect of STA on survival of L ewis transplanted tumor mice: (
n＝10)
Note P < 0.01, vs Model control group.
Experimental example 2
Experiments on the inhibition rate of the total steroidal alkaloids of solanum dulcamara in the pharmaceutical composition containing the compounds 1-4 of the invention on human lung cancer cells A549 in nude mice xenograft tumors:
first, experiment method
1) Cell and animal:
human lung adenocarcinoma cells A549 was provided by Jiangsu Kai Biotech GmbH;
60 BA L B/c nude mice, female, 5W of week age and 18-22g of body weight are provided by Shanghai Ling Chang biological technology, the production license of the experimental animal is SCXK (Shanghai) 2013-;
2) preparation of nude mouse xenograft tumor model
Collecting human non-small cell lung cancer A549 cell sap in logarithmic growth phase, adjusting the cell sap concentration to be 1 × 107/m L, and inoculating 0.1m L (namely 1 × 106 cells) per cell to the right axilla of a BA L B/c nude mouse subcutaneously;
3) animal grouping and administration
Selecting an A549 human non-small cell lung cancer cell strain, successfully preparing a nude mouse model of the transplanted tumor, measuring the diameter of the transplanted tumor of the mouse by using a vernier caliper until the tumor grows to 100-150 mm3The animals are uniformly divided into 6 groups according to the size of transplanted tumors, and the grouping and administration mode and the administration dose are the same as those of the animals in the experimental example 1' 3); collecting corresponding data and data according to the observation index.
4) Observation index
Dynamically observing and recording the weight of the mice and the change of the activity, appearance and spirit, measuring the weight 1 time (recorded as d0) when the mice are administrated in a cage, measuring the weight 1 time every 2d after the administration is started, simultaneously observing and recording the administration process of the animals and the change of the weight, the activity, the appearance and the spirit of the mice after the administration, measuring the weight and killing the mice after the continuous administration for 28d, dissecting and taking the tumor of each group of animals by operation, weighing, and simultaneously shooting and keeping each group of transplanted tumor mice and the dissected tumor picture.
Tumor volume measurement: using the method for measuring tumor diameter, the growth of transplanted tumor in mice is dynamically observed, and the long diameter (a) and the short diameter (b) of the tumor in mice are measured by a millimeter vernier caliper, and the Tumor Volume (TV) is calculated by measuring 1 time every 2d after the administration is started, and the calculation formula is as follows:
in the formula, a and b each represent a length and a width.
Calculating the tumor inhibition rate: the mice are sacrificed immediately after the administration, the tumors of each group of animals are dissected out and weighed, and the tumor inhibition rate is calculated according to the following formula:
tumor inhibition (%) was (average tumor weight of administration group-average tumor weight of model control group)/average tumor weight of model control group was × 100%
Second, experimental results
By constructing a nude mouse xenograft tumor model of human non-small cell lung cancer, the in vivo anti-tumor effect of solanum dulcamara total alkaloid is inspected, in the administration process, all groups of mice have no obvious abnormality, and the weight overall shows a gradually increasing trend, wherein the growth rate of the solanum dulcamara total alkaloid group and the compound taxus chinensis group animals is higher than the animal weight of the paclitaxel group, and the weight change trend of all groups of mice is shown as a time-weight curve in fig. 15;
the tumor volume of the administration group is obviously inhibited and the tumor weight is obviously reduced compared with the model control group, the inhibiting effect of the solanum dulcamara total alkaloid group shows good concentration-dosage dependence, and the tumor volume and weight change trend of each group of mice are respectively shown as a time-tumor volume curve and a time-tumor weight curve in a figure 30.
In terms of tumor inhibition rate, the tumor inhibition rate of the solanum dulcamara total alkaloid also presents concentration-dose dependence, and the tumor inhibition rate of the high-dose group is equivalent to that of the paclitaxel (compared with the paclitaxel, P is more than 0.05), and the specific tumor inhibition rate value is shown in table 6;
table 6: inhibition rate of STA on human lung cancer cell A549 nude mouse xenograft tumor
n＝10)
Experimental example 3
Experiments on the inhibition rate of the solanum dulcamara total steroidal alkaloid of the pharmaceutical composition containing the compounds 1-7 of the invention on human lung cancer cells NCI-H460 nude mouse xenograft tumor;
first, experiment method
1) Cell and animal:
human large cell lung cancer cell NCI-H460 was provided by Jiangsu Kai Bio-technology GmbH;
60 BA L B/c nude mice, female, 5W of week age and 18-22g of body weight are provided by Shanghai Ling Chang biological technology, the production license of the experimental animal is SCXK (Shanghai) 2013-;
2) preparation of nude mouse xenograft tumor model
Collecting human large cell lung cancer cell NCI-H460 cell sap of logarithmic growth phase, adjusting the cell sap concentration to 1 × 107/m L, inoculating 0.1m L (namely 1 × 106 cells) per cell to the right axillary subcutaneous part of BA L B/c nude mice;
3) animal grouping and administration
NCI-H460 human large cell lung cancer cell line was selected. After the nude mouse model with the transplanted tumor is successfully prepared, the diameter of the transplanted tumor of the mouse is measured by a vernier caliper until the tumor grows to 100-150 mm3The animals are evenly divided into 6 groups according to the sizes of transplanted tumors, the grouping and administration mode and the administration dosage are the same as those of the animals in the experimental example 1, 3), and corresponding data and data collection is carried out according to observation indexes;
4) observation index
Dynamically observing and recording the weight and the changes of the activity, the appearance and the spirit of the mice, measuring the weight 1 time (marked as d0) when the mice are administrated in cages, measuring the weight 1 time every 2d after the administration is started, and simultaneously observing and recording the administration process of animals and the changes of the weight, the activity, the appearance and the spirit of the mice after the administration; after the continuous administration for 28 days, measuring the weight and killing the mice, dissecting and taking tumors of animals of each group through operation, weighing, and simultaneously shooting and reserving mice with transplanted tumors of each group and dissected tumor pictures;
tumor volume measurement: using the method for measuring tumor diameter, the growth of transplanted tumor in mice is dynamically observed, and the long diameter (a) and the short diameter (b) of the tumor in mice are measured by a millimeter vernier caliper, and the Tumor Volume (TV) is calculated by measuring 1 time every 2d after the administration is started, and the calculation formula is as follows:
in the formula, a and b each represent a length and a width.
Calculating the tumor inhibition rate: the mice are sacrificed immediately after the administration, the tumors of each group of animals are dissected out and weighed, and the tumor inhibition rate is calculated according to the following formula:
tumor inhibition (%) was (average tumor weight of administration group-average tumor weight of model control group)/average tumor weight of model control group was × 100%
Second, experimental results
By constructing a nude mouse xenograft tumor model of human non-small cell lung cancer, the in vivo anti-tumor effect of solanum dulcamara total alkaloid is inspected, in the administration process, all groups of mice have no obvious abnormality, and the weight overall shows a gradually increasing trend, wherein the growth rate of the solanum dulcamara total alkaloid group and the compound taxus chinensis group animals is higher than the animal weight of the paclitaxel group, and the weight change trend of all groups of mice is shown as a time-weight curve in fig. 16;
the tumor volume of the administration group is obviously inhibited and the tumor weight is obviously reduced compared with the model control group, and the inhibiting effect of the solanum dulcamara total alkaloid group shows good concentration-dose dependence. The tumor volume and weight trend of each group of mice are shown in the time-tumor volume curve and the time-tumor weight curve in fig. 31, respectively.
In terms of tumor inhibition rate, the tumor inhibition rate of the solanum dulcamara total alkaloid also presents concentration-dose dependence, and the tumor inhibition rate of the high-dose group is equivalent to that of the paclitaxel (compared with the paclitaxel, P is more than 0.05), and the specific tumor inhibition rate value is shown in table 7;
table 7: (STA-human Lung cancer cell NCI-H460 xenograft tumor inhibition rate in nude mice: (
n＝10)
Experimental example 4
Experiments on the inhibition rate of the solanum dulcamara total steroidal alkaloid of the pharmaceutical composition containing the compounds 1-7 of the invention on human lung cancer cells NCI-H460 nude mouse xenograft tumor;
first, experiment method
1) Cell and animal:
human squamous cell lung carcinoma cell NCI-H520 was provided by Kyowa Kayki Biotechnology, Inc.
60 BA L B/c nude mice, female, 5W of week age and 18-22g of body weight are provided by Shanghai Ling Chang biological technology, the production license of the experimental animal is SCXK (Shanghai) 2013-;
2) preparation of nude mouse xenograft tumor model
Collecting cell sap of human lung squamous carcinoma cells NCI-H520 in logarithmic growth phase, adjusting the cell sap concentration to be 1 × 107 cells/m L, and inoculating 0.1m L cells (namely 1 × 106 cells) per cell to the right axillary subcutaneous part of a BA L B/c nude mouse;
3) animal grouping and administration
Selecting a human lung squamous carcinoma cell NCI-H520 cell strain, successfully preparing a nude mouse model of the transplanted tumor, measuring the diameter of the transplanted tumor of the mouse by using a vernier caliper until the tumor grows to 100-150 mm3The animals are evenly divided into 6 groups according to the sizes of transplanted tumors, the grouping and administration mode and the administration dosage are the same as those of the animals in the experimental example 1, 3), and corresponding data and data collection is carried out according to observation indexes;
4) observation index
The weight and changes of activity, appearance and spirit of the mice were dynamically observed and recorded, the weight was measured 1 time (recorded as d0) in the case of cage administration, 1 time every 2d after the administration was started, and the animal administration process and the changes of the weight, activity, appearance and spirit of the mice after the administration were observed and recorded. After the continuous administration for 28 days, measuring the weight and killing the mice, dissecting and taking tumors of animals of each group through operation, weighing, and simultaneously shooting and reserving mice with transplanted tumors of each group and dissected tumor pictures;
tumor volume measurement: using the method for measuring tumor diameter, the growth of transplanted tumor in mice is dynamically observed, and the long diameter (a) and the short diameter (b) of the tumor in mice are measured by a millimeter vernier caliper, and the Tumor Volume (TV) is calculated by measuring 1 time every 2d after the administration is started, and the calculation formula is as follows:
in the formula, a and b each represent a length and a width.
Calculating the tumor inhibition rate: the mice are sacrificed immediately after the administration, the tumors of each group of animals are dissected out and weighed, and the tumor inhibition rate is calculated according to the following formula:
tumor inhibition (%) was (average tumor weight of administration group-average tumor weight of model control group)/average tumor weight of model control group was × 100%
Second, experimental results
By constructing a nude mouse xenograft tumor model of human non-small cell lung cancer and investigating the in vivo anti-tumor effect of solanum dulcamara total alkaloid, in the administration process, all groups of mice have no obvious abnormality and the weight overall shows a gradually increasing trend, wherein the weight of the animals of the solanum dulcamara total alkaloid group is increased faster than that of the animals of the compound taxus chinensis group, and the weight change trend of all groups of mice is shown as a time-weight curve in fig. 17.
The tumor volume of the administration group is obviously inhibited and the tumor weight is obviously reduced compared with the model control group, the inhibiting effect of the solanum dulcamara total alkaloid group shows good concentration-dosage dependence, and the tumor volume and weight change trend of each group of mice are respectively shown as a time-tumor volume curve and a time-tumor weight curve in a figure 32.
In terms of tumor inhibition rate, the tumor inhibition rate of the solanum dulcamara total alkaloid also presents concentration-dose dependence, and the tumor inhibition rate of the high-dose group is equivalent to that of the paclitaxel (compared with the paclitaxel, P is more than 0.05). Specific tumor inhibition values are shown in table 8;
table 8: (STA-human Lung cancer cell NCI-H460 xenograft tumor inhibition rate in nude mice: (
n＝10)
According to the experiments, the pharmaceutical composition of solanum dulcamara total steroidal alkaloid containing the compounds 1 to 7 has good inhibitory effect on mouse L ewis transplanted tumor, human lung adenocarcinoma cell A549 nude mouse transplanted tumor, human large cell lung cancer cell NCI-H460 nude mouse transplanted tumor and human lung squamous carcinoma cell NCI-H520 nude mouse transplanted tumor, and the early-stage research shows that solanum dulcamara total steroidal alkaloid has significant inhibitory effect on various tumor strains such as S180 sarcoma, ehrlich ascites tumor and mouse hepatoma cell H22 transplanted tumor except for the tumor strains, and the specific data are shown in Chinese patent document CN102319319A.
The embodiments in the present description are described in a progressive manner, each embodiment focuses on differences from other embodiments, and the same and similar parts among the embodiments are referred to each other. The device disclosed by the embodiment corresponds to the method disclosed by the embodiment, so that the description is simple, and the relevant points can be referred to the method part for description.
The previous description of the disclosed embodiments is provided to enable any person skilled in the art to make or use the present invention. Various modifications to these embodiments will be readily apparent to those skilled in the art, and the generic principles defined herein may be applied to other embodiments without departing from the spirit or scope of the invention. Thus, the present invention is not intended to be limited to the embodiments shown herein but is to be accorded the widest scope consistent with the principles and novel features disclosed herein.
Claims (10)
1. A glycoside alkaloid designated (3 β,5 α,16R,20R,22S,25R) -spirostanyl-3-yloxy- β -D-glucopyranosyl- (1 → 2) -oxy- β -D-glucopyranosyl- (1 → 4) - β -D-galactopyranoside, compound 1, having the following structural formula:
2. a glycoside alkaloid designated (3 β,5 α,16R,20R,22S,25R) -spirostanyl-5-alkenyl-3-yloxy- β -D-glucopyranosyl- (1 → 2) -oxy- β -D-glucopyranosyl- (1 → 4) - β -D-galactopyranoside, compound 2, having the following structural formula:
3. a glycoside alkaloid designated 15 β -hydroxy- (3 β,25R) -16, 23-epoxy-23, 24-cycloimine-cholesterol-5, 16,20,23(N) -tetraene-3 β -ol-3-O- β -D-glucopyranosyl- (1 → 2) - β -D-glucopyranosyl- (1 → 6) - β -D-galactopyranoside, compound 3 having the following structural formula:
4. a glycoside alkaloid designated 15 β -hydroxy- (3 β,25R) -16, 23-epoxy-23, 24-cyclic imine-cholesterol-16, 20,23(N) -triene-3 β -ol-3-O- β -D-glucopyranosyl- (1 → 2) - β -D-glucopyranosyl- (1 → 6) - β -D-galactopyranoside, compound 4, having the following structural formula:
5. a glycoside alkaloid designated 15 β -ethoxy- (3 β,25R) -16, 23-epoxy-23, 24-cycloimine-cholesterol-5, 16,20,23(N) -tetraene-3 β -ol-3-O- β -D-glucopyranosyl- (1 → 2) - β -D-glucopyranosyl- (1 → 6) - β -D-galactopyranoside, compound 5 having the following structural formula:
6. a glycoside alkaloid designated 15 β -ethoxy- (3 β,25R) -16, 23-epoxy-23, 24-cyclic imine-cholesterol-16, 20,23(N) -triene-3 β -ol-3-O- β -D-glucopyranosyl- (1 → 2) - β -D-glucopyranosyl- (1 → 6) - β -D-galactopyranoside, compound 6, having the following structural formula:
7. a glycoside alkaloid designated 16, 23-epoxy-22, 26-cycloimine-cholesterol-22 (N),23, 25-triene-3 β -ol-3-O- β -D-glucopyranosyl- (1 → 2) - β -D-glucopyranosyl- (1 → 6) - β -D-galactopyranoside, compound 7, having the following structural formula:
8. a glucoside alkaloid of claims 1-7 and pharmaceutically acceptable salts or carriers thereof or a clinically acceptable preparation prepared from glucoside alkaloid as effective component by adding conventional adjuvants and conventional process.
9. The formulation of claim 8, wherein the formulation comprises a tablet, capsule, granule, syrup, powder, pill, tincture, wine, electuary, lozenge or mixture.
10. Use of the glycoside alkaloid of claims 1-7 for the treatment of S180 sarcoma, Ehrlich ascites tumor, L ewis lung cancer graft tumor, mouse hepatoma cell H22 graft tumor, human lung adenocarcinoma cell A549 nude mouse graft tumor, human lung squamous carcinoma cell NCI-H520 nude mouse graft tumor, and human large cell lung carcinoma NCI-H460 nude mouse graft tumor.
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| CN106939031A (en) * | 2017-02-17 | 2017-07-11 | 中国中医科学院西苑医院 | A kind of spirosolane type glycoalkaloid and preparation method thereof and purposes |
| CN108218950A (en) * | 2018-01-17 | 2018-06-29 | 吉林省中医药科学院（吉林省中医药科学院第临床医院） | A kind of steroid saponin and steroid alkaloid class compound and preparation method and purposes |
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| CN106939031A (en) * | 2017-02-17 | 2017-07-11 | 中国中医科学院西苑医院 | A kind of spirosolane type glycoalkaloid and preparation method thereof and purposes |
| CN108218950A (en) * | 2018-01-17 | 2018-06-29 | 吉林省中医药科学院（吉林省中医药科学院第临床医院） | A kind of steroid saponin and steroid alkaloid class compound and preparation method and purposes |
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