CN102579425B - Caulis Spatholobi extract, application thereof and new application of isoliquiritigenin - Google Patents
Caulis Spatholobi extract, application thereof and new application of isoliquiritigenin Download PDFInfo
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Abstract
The invention provides Caulis Spatholobi extract, an application thereof and a new application of isoliquiritigenin, and particularly provides Caulis Spatholobi extract containing isoliquiritigenin, an active part and a monomer compound isoliquiritigenin as well as an application of a composition in preparing medicine for treating and preventing triple-negative breast cancer. The composition consists of isoliquiritigenin and one or more active ingredients in gallocatechin, epicatechin and catechin.
Description
Technical field
The present invention relates to the new purposes of Caulis Spatholobi extract and isoliquiritigenin, be specifically related to Caulis Spatholobi extract and the application of isoliquiritigenin in preparation prevention and treatment three negative breast cancer medicines containing isoliquiritigenin.
Background technology
Caulis Spatholobi is the dry rattan that pulse family (Leguminosae) spatholobus suberectus belongs to (Spatholobus) plant spatholobus suberectus (Spatholobus suberectus Dunn).Be the nourshing blood and promoting blood circulation effect that Song Yihou finds, have in recent years a small amount of bibliographical information, this medicine has antitumor action, and we carry out systematic research to this medicine.
In recent years studies have found that Caulis Spatholobi and extract thereof have antineoplastic activity, but to breast carcinoma, particularly the breast carcinoma of three feminine genders (the type breast carcinoma that this is special) has no report.
As everyone knows, breast carcinoma is the relatively special tumor of a quasi-biology feature, is one of modal malignant tumor of women, and its sickness rate is high, serious threat women's psychosomatic health.Dependence situation according to tumor to hormone, estrogen receptor (estrogen receptor after SABC detects, ER), progesterone receptor (progesterone receptor, PR) and human epidermal growth factor receptor 2 (HER2) be all expressed as negative patient with breast cancer owing to lacking special targeted therapy, prognosis is poor, this is three negative breast cancer (triple negative breast cancer, TNBC).
The statistical datas such as Anders show, annual approximately 1,000,000 women in the whole world are diagnosed as breast carcinoma, and wherein approximately 17% is three negative breast cancer.2 times of left and right that sickness rate that there are some researches show African American women three negative breast cancer is pink toes, three negative breast cancer ratios of Korea S's report are 14.7%, Japan is 15%, China there is no the correlational study data report of multicenter, large sample at present, but reports that its ratio should be lower than black race and higher than white people.Interethnic morbidity difference is that gene and sudden change thereof are the basic reasons that causes these differences.
Three negative breast cancer, taking invasive clinical manifestation as feature, compared with other types breast carcinoma, have poor overall survival and disease free survival phase; Axillary lymphatic metastasis rate is low, and the generation that internal organs (as lung) shift early; Local relapse is higher, the risk of relapse 1-3 after treatment that mostly occurs.Aspect clinical pathology, three negative breast cancer have that diameter of tumor is large, tumor cell exist more central authorities downright bad, have a higher proliferation activity.
The research in early stage finds that this medicine can suppress lactic acid dehydrogenase-A (LDH-A, it is the key enzyme of final step in glycolytic pathway, it does not substantially express in the normal cell of oxygen supply abundance, but in various tumor cells, due to the impact of its mitochondrial function defect and anoxia microenvironment, LDH-A expresses obviously to be increased).LDH-A down-regulated expression, LDH-A activity decreased, can effectively stop aerobic glycolysis, allows tumor cannot obtain energy, thus dodar suppresses the effect of tumor.
In view of breast carcinoma to carbohydrate metabolism by way of special dependency, suppressing aerobic glycolysis has become one of the important channel that suppresses breast cancer cell, and becomes one of targeting important means that anti-breast carcinoma is new.Suppress the New Policy that glycolysis can be used as breast cancer tumor treatment.
Summary of the invention
One object of the present invention is to provide a kind of Caulis Spatholobi extract containing isoliquiritigenin and the application of active site in preparation prevention and treatment three negative breast cancer medicines;
Another object of the present invention is to provide the application of isoliquiritigenin in preparation prevention and treatment three negative breast cancer medicines.
Another object of the present invention is to provide the pharmaceutical composition of a kind for the treatment of three negative breast cancer.
Another object of the present invention is to provide the application in a kind of pharmaceutical composition preparation treatment three negative breast cancer medicines.
Caulis Spatholobi extract of the present invention is Caulis Spatholobi water extract or ethanol extraction;
The preferred 60-120 of described Caulis Spatholobi water extract DEG C water extraction 1-8 time; More preferably extract 2-5 time;
Described Caulis Spatholobi ethanol extraction, preferably 10-95% ethanol extraction, more preferably 20-80% ethanol extraction, more preferably 30-70% ethanol extraction, more preferably 40-70% ethanol extraction; Extracting mode can select to adopt percolation, backflow, dipping or supersound extraction; Preferably extract 1-8 time; More preferably extract 2-5 time;
The preferred above-mentioned Aqueous Extracts From Caulis Spatholobi of Caulis Spatholobi extract of the present invention, for ethanol extraction, petroleum ether, ethyl acetate, n-butyl alcohol successively extract or select the extract that an extraction obtains successively;
The extract that obtains of extraction is preferably upper macroporous resin further, first wash with water, after the ethanol eluting that increases gradually by concentration successively, collect high concentration ethanol eluate and be Caulis Spatholobi active site;
The invention provides a kind of new Caulis Spatholobi extract containing isoliquiritigenin, this extract is prepared by the following method: after Caulis Spatholobi water extraction or ethanol extraction, use n-butanol extraction, extract is macroporous resin in conventional method, first wash with water, after the ethanol eluting that increases gradually by concentration successively within the scope of 10-95% concentration of alcohol, comprising using 60-90% ethanol elution, collect 60-90% ethanol elution thing, to obtain final product;
Preferably, described macroporous resin is low pole, more preferably D101 macroporous resin;
The preferred 60-120 of described Caulis Spatholobi water extraction DEG C water extraction 1-8 time; More preferably extract 2-5 time;
Described Caulis Spatholobi ethanol extraction, preferably 10-95% ethanol extraction, more preferably 20-80% ethanol extraction, more preferably 30-70% ethanol extraction, more preferably 40-70% ethanol extraction; Extracting mode can select to adopt percolation, backflow, dipping or supersound extraction; Preferably extract 1-8 time; More preferably extract 2-5 time;
The preparation method of Caulis Spatholobi active site of the present invention is preferably: by Aqueous Extracts From Caulis Spatholobi or ethanol extract dispersing and dissolving in water, with n-butanol extraction; Butanol extraction liquid obtains n-butyl alcohol extract after being evaporated to and doing, and n-butyl alcohol extract adsorbs through D101 macroporous adsorptive resins, be washed to eluent colourless after, use successively 15-25%, 3545%, 60-90% alcoholic solution eluting, collect 80% ethanol elution thing, be evaporated to dryly, obtain final product.More preferably comprise 80% ethanol elution, collect 80% ethanol elution thing.
The present invention is the openly application of isoliquiritigenin in preparation prevention and treatment three negative breast cancer medicines further;
Described isoliquiritigenin can be bought from market, also can from Caulis Spatholobi, extract and obtain, can also from other plant, separation and purification obtain, also can obtain by chemosynthesis or biological synthesis method, its structural formula is as follows: the invention provides the pharmaceutical composition of a kind of prevention or treatment three negative breast cancer, the active component of said composition is mainly made up of one or more active component in isoliquiritigenin and optional nutgall catechin, epicatechin and catechin.
Described pharmaceutical composition, is preferably made up of one or more active component in isoliquiritigenin 1-9 weight portion and optional nutgall catechin 5-15 weight portion, epicatechin 150-250 weight portion, catechin 150-250 weight portion.
In described compositions, preferably formed by one or more active component of isoliquiritigenin 3-7 weight portion and optional nutgall catechin 8-12 weight portion, epicatechin 180-230 weight portion, catechin 180-230 weight portion.
Pharmaceutical composition of the present invention, is preferably made up of following active component:
Isoliquiritigenin 1-9 weight portion nutgall catechin 5-15 weight portion
Epicatechin 150-250 weight portion.
Pharmaceutical composition of the present invention, is preferably made up of following active component:
Isoliquiritigenin 3-7 weight portion nutgall catechin 8-12 weight portion
Epicatechin 180-230 weight portion.
Pharmaceutical composition of the present invention, is more preferably made up of following active component:
Isoliquiritigenin 5 weight portion nutgall catechin 10 weight portions
Epicatechin 200 weight portions.
Pharmaceutical composition of the present invention, is preferably made up of following active component:
Isoliquiritigenin 1-9 weight portion nutgall catechin 5-15 weight portion
Epicatechin 150-250 weight portion catechin 150-250 weight portion;
Pharmaceutical composition of the present invention, is preferably made up of following active component:
Isoliquiritigenin 3-7 weight portion nutgall catechin 8-12 weight portion
Epicatechin 180-230 weight portion catechin 180-230 weight portion.
Pharmaceutical composition of the present invention, is more preferably made up of following active component:
Isoliquiritigenin 5 weight portion nutgall catechin 10 weight portions
Epicatechin 200 weight portion catechin 200 weight portions.
Isoliquiritigenin of the present invention, nutgall catechin, epicatechin and catechin can be extracted and obtain from Caulis Spatholobi, can also from other plant, separation and purification obtain, and also can obtain by chemosynthesis or biological synthesis method.
Caulis Spatholobi extract of the present invention, active site, monomeric compound and compositions can add conventional adjuvant to make the acceptable any preparation of pharmaceutics according to common process, as medicinal tea, capsule, tablet, granule, gel, slow releasing agent, oral liquid, drop pill or nanometer formulation; Also can use with other related drugs compatibilities, make corresponding preparation.
Experimental example
Experimental example 1 Caulis Spatholobi active site of the present invention and the research of monomeric compound inside and outside antitumor
1, the experiment in vitro of Caulis Spatholobi active site and monomeric compound
1.1 experimental drugs: 80% eluate (making by embodiment 4), isoliquiritigenin (Isoliquiritigenin) (being purchased from Nat'l Pharmaceutical & Biological Products Control Institute),
1.2 experimental techniques: the cell MCF-7 (cell strain that estrogen receptor relies on) of the trophophase of taking the logarithm respectively, MDA-MB-231 (three negative breast cancer cell strains, be the cell strain that non-estrogen receptor relies on) and MCF-10 (matched group, the normal mammary glandular cell strain of people) every hole 100 μ L are inoculated in 96 orifice plates, and cell concentration is 5 × 10
4/ mL, cultivate 24h after cell attachment, change serum-free medium, after 24h, absorb old culture medium, add respectively again 90 μ L serum-free mediums to add the medicinal liquid (containing 2 ‰ DMSO) of Caulis Spatholobi 80% eluate (SS) 10-100 μ g/mL and isoliquiritigenin (ISO) 20-80uM, make medicinal liquid final concentration be respectively 10-100 μ g/mL; 0-80uM; Blank group adds the phosphate buffer of 10 μ L containing 2 ‰ DMSO, establishes 6 parallel holes for every group, is placed in 37 DEG C, 5%CO
2in cell culture incubator, cultivate 48h.Collecting cell, measures the inhibition of medicine cell growth, the impact of apoptosis rate.
1.3 experimental results: in table 1,2.
The 80% alcohol eluate of table 1. Caulis Spatholobi, the impact of isoliquiritigenin on two kinds of growth of tumour cell
Experimental result: carry out cell culture with 80% eluate and isoliquiritigenin (ISO), observation of cell growth rate, result shows that the two has the effect that suppresses tumor growth, and has the relation (with the increase to drug dose, curative effect increases) that dose-effect relies on.Isoliquiritigenin (ISO) and 80% eluate comparison, the better effects if of ISO.The inhibition of three negative cells strains is better than to the cell strain that estrogen is relied on.
The 80% alcohol eluate of table 2. Caulis Spatholobi, the impact of isoliquiritigenin on two kinds of apoptosis of tumor cells rates
Experimental result shows that 80% eluate and isoliquiritigenin (ISO) have the effect that promotes apoptosis of tumor cells, and has the relation (with the increase to drug dose, curative effect increases) that dose-effect relies on.ISO and 80% eluate comparison, the better effects if of ISO.And the two is better than to the inhibition of three negative cells strains the cell strain that estrogen relies on.
2, Caulis Spatholobi active site and the isoliquiritigenin impact on the different tumor cell line protein expressions of two classes
2.1 experimental drugs: 80% eluate (making by embodiment 4), isoliquiritigenin (being purchased from Nat'l Pharmaceutical & Biological Products Control Institute)
2.2 experimental techniques: each cell MCF-7 of the trophophase of taking the logarithm respectively, the every hole 100 μ L of MDA-MB-231 are inoculated in 96 orifice plates, and cell concentration is 5 × 10
4/ mL, cultivate 24h after cell attachment, change serum-free medium, after 24h, absorb old culture medium, add respectively again 90 μ L serum-free mediums to add the medicinal liquid (containing 2 ‰ DMSO) of Caulis Spatholobi 80% eluate (SS) 20-80 μ g/mL and isoliquiritigenin (ISO) 20-80uM, make medicinal liquid final concentration be respectively 10-10 μ g/mL; 0-80uM; Be placed in 37 DEG C, 5%CO
2in cell culture incubator, cultivate 48h.Collecting cell, extracts albumen, the impact with Western blot method mensuration medicine on tumor cell protein expression.
2.3 experimental results: see Fig. 1,2.Experimental result shows that 80% eluate and isoliquiritigenin can pass through inducing cell mitochondria pathway apoptosis, has obvious dose-effect dependence, thus the effect of the anti-two class breast tumor of performance.
3, in body, suppress the effect of tumor growth
3.1 experimental drugs: 80% eluate (making by embodiment 4), isoliquiritigenin (being purchased from Nat'l Pharmaceutical & Biological Products Control Institute)
3.2 experimental techniques: adopt the method for MDA-MB-231 and MCF-7 tumor cell transplantation to set up Breast Carcinoma in nude mice animal model.At the position of mammary gland injection 3*10
6, dosage: 80% eluate 5mg/kg, isoliquiritigenin low dose group 20ug/kg, isoliquiritigenin high dose group 40ug/kg.The each medicine group of observation and comparison and the negative blank group tumor size of nude mice and the variation of weight.After experiment finishes, collect the tumor of each animal groups, the impact in observation and comparison Caulis Spatholobi extract body, tumor growth being suppressed.
3.3 experimental result
The impact in the 80% alcohol eluate (SS) of table 3 Caulis Spatholobi, isoliquiritigenin body, tumor growth being suppressed
Result shows, 80% eluate, isoliquiritigenin are grown in vivo and are all presented inhibitory action the breast carcinoma of two types, and the inhibition of MDA-MS-231 is slightly better than to MCF-7 and illustrates that (rate of increase MDA-MS-231 of its tumor is relatively lower, tumor proliferation rate=RTVT/RTVC × 100% compared with the better effects if of estrogen receptor dependence to the inhibitory action of three negative breast carcinomas.RTVT=administration group relative tumour volume; RTVC=matched group relative tumour volume).Isoliquiritigenin is strong to the ability of neoplasm growth compared with 80% eluate, and apoptosis rate is also compared with eluting object height.
Experimental example 2 Caulis Spatholobi extract of the present invention, active site and the impact of monomeric compound body on LDH
1, Caulis Spatholobi extract and monomeric compound thereof suppress the effect of the expression of breast tumor cell LDH-A
1.1 experimental drugs: Aqueous Extracts From Caulis Spatholobi (H
2o) (make) ethanol extract (60%EtOH) (making by embodiment 2), isoliquiritigenin (being purchased from Nat'l Pharmaceutical & Biological Products Control Institute) by embodiment 1;
Different dosage form solvent extract: Caulis Spatholobi medicinal material coarse powder 1000g, with 60% ethanol ultrasonic extraction of 10 times of amounts (W/V) 2 times, each 1 hour, merge extracted twice liquid, be evaporated to without after alcohol taste, extract successively with petroleum ether, ethyl acetate, n-butyl alcohol respectively, obtain 4 extracts that polarity is different: petroleum ether extract (PE) 1.1g, acetic acid ethyl ester extract (EtOAC) 11.5g, n-butyl alcohol extract (BUOH) 24.4g and water layer stay excess (DW) 79.3g.
1.2 experimental techniques: the MCF-7 of the trophophase of taking the logarithm respectively, the every hole 100 μ L of MDA-MB-231 and MCF-10 are inoculated in 96 orifice plates, and cell concentration is 5 × 10
4/ mL, under normal pressure and anoxia condition, cultivate 24h after cell attachment, change serum-free medium, after 24h, absorb old culture medium, add respectively again 90 μ L serum-free mediums to add respectively the medicinal liquid (containing 2 ‰ DMSO) of Aqueous Extracts From Caulis Spatholobi, ethanol extract, different solvents extract 10-100 μ g/mL and isoliquiritigenin 10-50uM, make medicinal liquid final concentration be respectively 10-100 μ g/mL, 10-50uM; Blank group adds the phosphate buffer of 10 μ L containing 2 ‰ DMSO, establishes 6 parallel holes for every group, is placed in 37 DEG C, 5%CO
2in cell culture incubator, cultivate 48h.After collecting cell, extract total protein, for measuring the impact of medicine on cell LDH-A activity, protein expression.With the apoptosis rate of its cell of cells were tested by flow cytometry.
1.3 experimental results: in table 4.
The different extracts of table 4 are on cell LDH activity (U/mg) (dosage is 50ug/ml) and apoptotic impact
Experimental result shows n-butyl alcohol, and acetic acid ethyl ester extract and water layer stay the inhibitory action of LDH-A of excess better, but water extract, ethanol extract also have certain effect, but the having better effect of n-butanol extraction position.Also explanation simultaneously, observing different pharmaceutical is also the best results at n-butanol extraction position to the apoptosis rate of inducing tumor cell.
2,80% eluate and isoliquiritigenin are on the active impact on breast tumor cell LDH-A under normal oxygen and anoxia bar
1.1 experimental drugs: 80% eluate (making by embodiment 4), isoliquiritigenin (being purchased from Nat'l Pharmaceutical & Biological Products Control Institute).
1.2 experimental techniques: because the generation of LDH-A produces conventionally under anaerobic environment, tumor tissues is exactly anaerobic environment, therefore with 80% eluate and isoliquiritigenin impact on LDH-A expression under normal oxygen and anaerobic environment.Take the logarithm the respectively MCF-7 of trophophase, the every hole 100 μ L of MDA-MB-231 are inoculated in 96 orifice plates, and cell concentration is 5 × 10
4/ mL, under normal pressure and anoxia condition, cultivate 24h after cell attachment, change serum-free medium, after 24h, absorb old culture medium, add respectively again 90 μ L serum-free mediums to add the medicinal liquid (containing 2 ‰ DMSO) of Caulis Spatholobi 80% eluate 10-10 μ g/mL and isoliquiritigenin 10-50uM, make medicinal liquid final concentration be respectively 10-100 μ g/mL, 10-50uM; Blank group adds the phosphate buffer of 10 μ L containing 2 ‰ DMSO, establishes 6 parallel holes for every group, is placed in 37 DEG C, 5%CO
2in cell culture incubator, cultivate 48h.After collecting cell, extract total protein, for measuring medicine to cell DH-A activity.
1.3 experimental results: in table 5,6.
Table 5 80% eluate is in normal oxygen and the impact (uM concentration) of anaerobic environment on LDH-A
Table 6 isoliquiritigenin is in normal oxygen and the impact (uM concentration) of anaerobic environment on LDH-A
80% eluate and isoliquiritigenin have obvious inhibitory action to the activity of LDH-A under normal oxygen and anoxia condition, and have the relation that obvious dose-effect relies on.The effect of isoliquiritigenin is better compared with the effect of 80% eluate.
3,80% alcohol eluate and isoliquiritigenin are in body tumor tissues LDH-A protein expression and the impact on its apoptosis
Adopt the method for tumor cell transplantation to set up Breast Carcinoma in nude mice animal model, at the position of mammary gland injection 3*10
6, give 80% alcohol eluting agent amount 60mg/kg, isoliquiritigenin dosage 20,40mg/kg, inferior on every Wendesdays, successive administration 26 days, gets tumor tissues, measure the expression of LDH-A in each tissue by the method for SABC, and the impact of medicine on tumor tissues apoptosis, the results are shown in Figure 3,4.
Result shows that 80% ethanol extract and isoliquiritigenin can suppress the expression of the LDH-A albumen in tumor tissues, the inspection of LDH-A tissue staining finds that the LDH-A protein expression of matched group is apparently higher than administration group, illustrate that administration (SS or ISO) can obviously suppress the expression of LDH-A in tumor tissues, painted obvious reduction (P < 0.05) in two groups of different cell transplantation tumor tissues.
80% ethanol extract and isoliquiritigenin energy inducing apoptosis of tumour cell, by the level of apoptosis of cell in TUNEL (the original position enzyme labelling assay for determining of apoptosis) method detection transplanted tumor in nude mice tissue.In the nucleus of apoptotic cell, there is brown yellow granule in this dyeing, administration group comprises SS, and the positive cell number of ISO group apoptosis obviously increases, in transplanted tumor tissue, the positive cell number of apoptosis obviously increases (P < 0.05) compared with Normal group, is combined into tumor size, the whole weight of tumor body and final volume and obviously reduces (P < 0.05).
Conclusion: SS and ISO can obviously suppress the growth of transplanted tumor in MCF-7 and MDA-MB-231 cell nude mouse, its mechanism of action may be the expression by lowering LDH-A, and then reduce tumor cell energy for growth in vivo, there is the effect that suppresses breast tumor cell growth in body.
Experimental example 3 the present invention carry out composition of prescription to the monomeric compound having separated and observe its inhibitory action to LDH-A
1.1 experimental drugs: isoliquiritigenin (ISO, be purchased from Nat'l Pharmaceutical & Biological Products Control Institute), nutgall catechin (GC), epicatechin (EC) and catechin (C) (being all purchased from Hiroad standing grain medical sci-tech Development Co., Ltd)
1.2 experimental techniques: drug component is: blank group; ISO group; GC group; EC group; C group; ISO+GC group (ISO: GC=5: 10); ISO+GC+EC group (ISO: GC: EC=5: 10: 200); And ISO+GC+EC+C group (5: 10: 200: 200).Take the logarithm the respectively MCF-7 of trophophase, the every hole 100 μ L of MDA-MB-231 are inoculated in 96 orifice plates, and cell concentration is 5 × 10
4/ mL, under condition of normal pressure, cultivate 24h after cell attachment, change serum-free medium, after 24h, absorb old culture medium, add respectively again 90 μ L serum-free mediums to add respectively above-mentioned medicine, blank group adds the phosphate buffer of 10 μ L containing 2 ‰ DMSO, establishes 6 parallel holes for every group, is placed in 37 DEG C, 5%CO
2in cell culture incubator, cultivate 48h.After collecting cell, extract total protein, the impact with Western blot (β-actin is internal reference) mensuration medicine on cell LDH-A protein expression.
1.3 experimental results: result is as table 7, Fig. 5.
Table 7 C, EC, GC, ISO be the impact on LDH-A separately
| Matched group | C | EC | GC | ISO | |
| MDA-MB-231 | 0.9±0.06 | 0.8±0.06 | 0.8±0.05 | 0.8±0.05 | 0.35±0.04 |
| MCF-7 | 0.8±0.04 | 0.8±0.04 | 0.78±0.03 | 0.76±0.03 | 0.56±0.04 |
As can be seen from Table 7, ISO shows inhibitory action to the expression of LDH-A separately; Separately GC, EC, C, all to the expression of LDH-A without obvious inhibitory action; As seen from Figure 5, GC, EC, C three combine use, to the expression unrestraint effect of LDH-A; And in the time that ISO and GC, EC, C compatibility use, obviously strengthen the inhibitory action (protein band is obviously thin out, and protein expression is suppressed) to LDH-A.As can be seen here, the relevant monomer compatibility of ISO and Caulis Spatholobi can obviously strengthen the inhibitory action to LDH-A, thereby increases the effect that it suppresses tumor.
Brief description of the drawings:
Fig. 1: the impact that 80% eluate is expressed the different tumor cell globin of two classes matter.
Fig. 2: the impact that isoliquiritigenin is expressed the different tumor cell globin of two classes matter.
Fig. 3: the impact of 80% eluate on tumor tissues LDH-A and apoptosis.
Fig. 4: the impact of isoliquiritigenin on tumor tissues LDH-A and apoptosis.
Fig. 5: monomeric compound compatibility uses the impact on tumor LDH-A activity.
Detailed description of the invention
Embodiment 1
Caulis Spatholobi medicinal material coarse powder 100g, decocts and extracts 2 times with 10 times of water (W/V), and each 1 hour, merge extracted twice liquid, be evaporated to dryly, obtain Aqueous Extracts From Caulis Spatholobi 9.7g.Water extract adds conventional adjuvant according to the agent of common process granulation.
Caulis Spatholobi medicinal material coarse powder 100g, with 60% alcohol reflux of 10 times of amounts (W/V) times 2 times, each 1 hour, merge extracted twice liquid, be evaporated to dryly, obtain Caulis Spatholobi 60% ethanol extract 15.5g.Ethanol extract adds conventional adjuvant to make drop pill according to common process.
Embodiment 3
Caulis Spatholobi medicinal material coarse powder 1000g, with 60% ethanol ultrasonic extraction of 10 times of amounts (W/V) 2 times, each 1 hour, merge extracted twice liquid, be evaporated to without after alcohol taste, extract successively with petroleum ether, ethyl acetate, n-butyl alcohol respectively, obtain 4 extracts that polarity is different: petroleum ether extract (PE) 1.1g, acetic acid ethyl ester extract (ETOAC) 11.5g, n-butyl alcohol extract-I (BUOH-I) 24.4g and water layer stay excess (DW) 79.3g.
Embodiment 4
Caulis Spatholobi medicinal material coarse powder, with 60% ethanol ultrasonic extraction of 10 times of amounts (W/V) 2 times, each 1 hour, merge extracted twice liquid, be evaporated to without after alcohol taste, with appropriate n-butanol extraction 3 times, combining extraction liquid, be concentrated into dryly, obtain n-butyl alcohol extract-II (BuOH-II).Get BuOH-II, after water dispersing and dissolving, upper D101 macroporous adsorptive resins adsorbs.First wash with water colourless to effluent after, more successively with respectively 3 times of column volumes of eluting of 20%, 40% ethanol water, finally use 3 times of column volumes of 80% ethanol elution, eluent is evaporated to dry, obtains 80% ethanol elution thing.
Embodiment 5
Caulis Spatholobi medicinal material coarse powder, with 60% ethanol ultrasonic extraction of 10 times of amounts (W/V) 2 times, each 1 hour, merge extracted twice liquid, be evaporated to without after alcohol taste, with appropriate n-butanol extraction 3 times, combining extraction liquid, be concentrated into dryly, obtain n-butyl alcohol extract-II (BuOH-II).Get BuOH-II, after water dispersing and dissolving, upper D101 macroporous adsorptive resins adsorbs.First wash with water colourless to effluent after, more successively with respectively 3 times of column volumes of eluting of 20%, 40% ethanol water, finally use 3 times of column volumes of 60% ethanol elution, eluent is evaporated to dry, 60% ethanol elution thing, obtains extract.
According to the methods experiment of experimental example 2, result shows that this extract has inhibitory action to the expression of LDH-A, can inducing apoptosis of tumour cell.
Embodiment 6
Caulis Spatholobi medicinal material coarse powder, with 60% ethanol ultrasonic extraction of 10 times of amounts (W/V) 2 times, each 1 hour, merge extracted twice liquid, be evaporated to without after alcohol taste, with appropriate n-butanol extraction 3 times, combining extraction liquid, be concentrated into dryly, obtain n-butyl alcohol extract-II (BuOH-II).Get BuOH-II, after water dispersing and dissolving, upper D101 macroporous adsorptive resins adsorbs.First wash with water colourless to effluent after, more successively with respectively 3 times of column volumes of eluting of 20%, 40% ethanol water, finally use 3 times of column volumes of 90% ethanol elution, eluent is evaporated to dry, 90% ethanol elution thing, obtains extract.
According to the methods experiment of experimental example 2, result shows that this extract has inhibitory action to the expression of LDH-A, can inducing apoptosis of tumour cell.
Caulis Spatholobi medicinal material coarse powder, with 60% ethanol ultrasonic extraction of 10 times of amounts (W/V) 2 times, each 1 hour, merge extracted twice liquid, be evaporated to without after alcohol taste, with appropriate n-butanol extraction 3 times, combining extraction liquid, be concentrated into dryly, obtain n-butyl alcohol extract-II (BUOH-II).Get BUOH-II, after water dispersing and dissolving, upper D101 macroporous adsorptive resins adsorbs.First wash with water colourless to effluent after, more successively with respectively 3 times of column volumes of eluting of 20%, 40% ethanol water, finally use 3 times of column volumes of 80% ethanol elution, eluent is evaporated to dry, obtains 80% ethanol elution thing.
Embodiment 8
Get isoliquiritigenin (ISO) and nutgall catechin (GC), epicatechin (EC) and catechin (C) in quality ISO: GC: EC: C=5: 10: 200: 200 ratios totally 100 grams be mixed to get monomer composition.
Get isoliquiritigenin (ISO) and nutgall catechin (GC), epicatechin (EC) and catechin (C) in quality ISO: GC: EC: C=2: 14: 160: 240 ratios totally 100 grams be mixed to get monomer composition.
Embodiment 10
Get isoliquiritigenin (ISO) and nutgall catechin (GC), epicatechin (EC) and catechin (C) in quality ISO: GC: EC: C=9: 6: 240: 140 ratios totally 100 grams be mixed to get monomer composition.
Embodiment 11
Caulis Spatholobi medicinal material coarse powder, with 60% ethanol ultrasonic extraction of 10 times of amounts (W/V) 2 times, each 1 hour, merge extracted twice liquid, be evaporated to without after alcohol taste, with appropriate n-butanol extraction 3 times, combining extraction liquid, be concentrated into dryly, obtain n-butyl alcohol extract-II (BUOH-II).Get 3UOH-II, after water dispersing and dissolving, upper D101 macroporous adsorptive resins adsorbs.First wash with water colourless to effluent after, more successively with respectively 3 times of column volumes of eluting of 20%, 40% ethanol water, finally use 3 times of column volumes of 80% ethanol elution, eluent is evaporated to dry, 80% ethanol elution thing.
Getting 80% eluate adds conventional adjuvant to make nanometer formulation according to common process.
Embodiment 12
Caulis Spatholobi medicinal material coarse powder 200g, with 60% ethanol ultrasonic extraction of 10 times of amounts (W/V) 2 times, each 1 hour, merge extracted twice liquid, be evaporated to without after alcohol taste, with appropriate n-butanol extraction 3 times, combining extraction liquid, be concentrated into dryly, obtain n-butyl alcohol extract-II (BUOH-II) 38.2g.Take BUOH-II5g, after water dispersing and dissolving, upper D101 macroporous adsorptive resins adsorbs.First wash with water colourless to effluent after, again successively with 20%, 40%, 80% ethanol water, 3 times of column volumes of eluting respectively, collect each eluting stream part, be evaporated to dryly, obtain 20% ethanol elution thing (Fr.20%) 1.12,40% ethanol elution thing (Fr.40%) 0.56g and 80% ethanol elution thing (Fr.80%) 2.33g.
Gained Fr.80%102mg separates through silica gel column chromatography, ODS preparative high-performance liquid chromatographic, obtains compound isoliquiritigenin (2.8mg).
Isoliquiritigenin (isoliquiritigenin, ISO)
ESI-MS：m/z 255[M-H]
-，
UV λ max:205nm, 239nm (acromion), 368.5nm.
1H-NMR(400MHz，DMSO-d
6)：δ13.51(1H，2’-OH)，10.56(1H，4’-OH)，10.08(1H，4-OH)，8.15(1H，d，J＝8.7Hz，H-6’)，7.77(1H，d，J＝15.8Hz，H-)，7.75(2H，d，J＝8.0Hz，H-2，6)，7.72(1H，d，J＝15.8Hz，H-)，6.82(2H，d，J＝8.0Hz，H-3，5)，6.38(1H，dd，J＝8.7，1.5Hz，H-5’)，6.25(1H，d，J＝1.5Hz，H-3’)。
13C-NMR(100MHz，DMSO-d6)：δ126.0(C-1)，130.6(C-2)，116.1(C-3)，160.1(C-4)，116.0(C-5)，130.6(C-6)，144.2(C-)，117.8(C-)，191.7(C＝O)，113.8(C-1’)，164.6(C-2’)，102.5(C-3’)，165.6(C-4’)，107.5(C-5’)，132.1(C-6’)。
Embodiment 13
Caulis Spatholobi medicinal material coarse powder, with 50% ethanol percolate extraction of 10 times of amounts (W/V) 2 times, each 1 hour, merge extracted twice liquid, be evaporated to without after alcohol taste, with appropriate n-butanol extraction 3 times, combining extraction liquid, be concentrated into dryly, obtain n-butyl alcohol extract-II (BuOH-II).Get BuOH-II, after water dispersing and dissolving, upper D101 macroporous adsorptive resins adsorbs.First wash with water colourless to effluent after, more successively with respectively 3 times of column volumes of eluting of 20%, 40% ethanol water, finally use 3 times of column volumes of 80% ethanol elution, eluent is evaporated to dry, 80% ethanol elution thing;
According to the methods experiment of experimental example 2, result shows the growth of this extract energy inhibition tumor cell, and the expression of LDH-A is had to inhibitory action, can inducing apoptosis of tumour cell.
Embodiment 14
Caulis Spatholobi medicinal material coarse powder, extracts 2 times with 10 times of water gagings, and each 1 hour, merge extracted twice liquid, with appropriate n-butanol extraction 3 times, combining extraction liquid, be concentrated into dryly, obtain n-butyl alcohol extract-II (BUOH-II).Get BUOH-II, after water dispersing and dissolving, upper D101 macroporous adsorptive resins adsorbs.First wash with water colourless to effluent after, more successively with respectively 3 times of column volumes of eluting of 20%, 40% ethanol water, finally use 3 times of column volumes of 80% ethanol elution, eluent is evaporated to dry, 80% ethanol elution thing;
According to the methods experiment of experimental example 2, result shows the growth of this extract energy inhibition tumor cell, and the expression of LDH-A is had to inhibitory action, can inducing apoptosis of tumour cell.
Embodiment 15
Caulis Spatholobi medicinal material coarse powder, with 70% ethanol percolate extraction of 10 times of amounts (W/V) 2 times, each 1 hour, merge extracted twice liquid, be evaporated to without after alcohol taste, with appropriate n-butanol extraction 3 times, combining extraction liquid, be concentrated into dryly, obtain n-butyl alcohol extract-II (BUOH-II).Get BUOH-II, after water dispersing and dissolving, upper D101 macroporous adsorptive resins adsorbs.First wash with water colourless to effluent after, more successively with respectively 3 times of column volumes of eluting of 20%, 40% ethanol water, finally use 3 times of column volumes of 80% ethanol elution, eluent is evaporated to dry, 80% ethanol elution thing;
According to the methods experiment of experimental example 2, result shows the growth of this extract energy inhibition tumor cell, and the expression of LDH-A is had to inhibitory action, can inducing apoptosis of tumour cell.
Embodiment 16
Get isoliquiritigenin (ISO) and nutgall catechin (GC), epicatechin (EC) in ISO: GC: EC=5: within 10: 200: 200, ratio is mixed to get monomer composition.
Embodiment 17
Get isoliquiritigenin (ISO) and nutgall catechin (GC) in ISO: GC=2: 14 ratios totally 100 grams be mixed to get monomer composition.
Embodiment 18
Get isoliquiritigenin (ISO), epicatechin (EC) by ISO: EC=9: 240 mass ratios totally 100 grams be mixed to get monomer composition.
Claims (6)
1. the Caulis Spatholobi extract that contains isoliquiritigenin prevents or treats the application in three negative breast cancer medicines in preparation, it is characterized in that, described Caulis Spatholobi extract is prepared by the following method: after Caulis Spatholobi water extraction or ethanol extraction, use n-butanol extraction, extract is macroporous resin in conventional method, first wash with water, after the ethanol eluting that increases gradually by concentration successively within the scope of 10-95% concentration of alcohol, comprising using 60-90% ethanol elution, collect 60-90% ethanol elution thing, to obtain final product.
2. application as claimed in claim 1, is characterized in that, described macroporous resin is low pole.
3. the application as described in claim 1 or 2 any one, is characterized in that ethanol extraction 10-95% ethanol extraction.
4. application as claimed in claim 3, is characterized in that, ethanol extraction mode is percolation, backflow, dipping or supersound extraction.
5. a pharmaceutical composition that prevents or treat three negative breast cancer, it is characterized in that, said composition is made up of one or more active component in isoliquiritigenin 1-9 weight portion and optional nutgall catechin 5-15 weight portion, epicatechin 150-250 weight portion, catechin 150-250 weight portion.
6. the application of pharmaceutical composition as claimed in claim 5 in preparation prevention or treatment three negative breast cancer medicines.
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| CN106138186A (en) * | 2015-04-16 | 2016-11-23 | 陈晓萍 | A kind of prevention or the external preparation for the treatment of breast cancer |
| CN108685998A (en) * | 2018-08-02 | 2018-10-23 | 广西壮族自治区药用植物园 | Treat the preparation method of breast cancer Caulis Spatholobi tablet |
| CN108969731A (en) * | 2018-10-12 | 2018-12-11 | 广西壮族自治区药用植物园 | The preparation method for treating the Caulis Spatholobi ointment of breast cancer |
| CN109200086A (en) * | 2018-10-12 | 2019-01-15 | 广西壮族自治区药用植物园 | The preparation method for treating the Caulis Spatholobi tablet of oophoroma |
| CN114099528B (en) * | 2021-11-25 | 2023-05-02 | 广西壮族自治区中医药研究院 | Anti-depression quality marker for spatholobus stem, and preparation method and application thereof |
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| CN1939396A (en) * | 2006-10-26 | 2007-04-04 | 首都医科大学附属北京中医医院 | Suberect spatholobus stem extract with antineoplastic function and its making method |
| CN101658513A (en) * | 2008-08-26 | 2010-03-03 | 石河子大学 | Use of isoliquiritigenin as medicament for preventing and treating or curing postoperative metastasis and relapse of malignant tumors |
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| CN1939396A (en) * | 2006-10-26 | 2007-04-04 | 首都医科大学附属北京中医医院 | Suberect spatholobus stem extract with antineoplastic function and its making method |
| CN101658513A (en) * | 2008-08-26 | 2010-03-03 | 石河子大学 | Use of isoliquiritigenin as medicament for preventing and treating or curing postoperative metastasis and relapse of malignant tumors |
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