CN102516344A - Compound with antitumor activity and preparation method and application thereof - Google Patents
Compound with antitumor activity and preparation method and application thereof Download PDFInfo
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- CN102516344A CN102516344A CN2011103602061A CN201110360206A CN102516344A CN 102516344 A CN102516344 A CN 102516344A CN 2011103602061 A CN2011103602061 A CN 2011103602061A CN 201110360206 A CN201110360206 A CN 201110360206A CN 102516344 A CN102516344 A CN 102516344A
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Abstract
The invention discloses a compound with antitumor activity, which is a compound shown as a formula I, and can be applied to preparation of an antitumor medicament and preparation of a medicament for preventing tumors. The invention further discloses a preparation method of the compound with antitumor activity. The method comprises the following steps of: performing reflux extraction on physalis pubescens L. with an extraction solvent to obtain an extracting solution; concentrating the extracting solution, diluting with water, and extracting with an organic solvent to obtain a dichloromethane extracting solution, wherein the organic solvent at least comprises dichloromethane; and concentrating the dichloromethane extracting solution, passing through a chromatographic column, eluting with an eluting system, and recrystallizing to obtain the compound with antitumor activity. The preparation method is simple and reliable, high operability and high efficiency are achieved, industrial mass production can be performed, popularization and application are facilitated, and a good application prospect is achieved.
Description
Technical field
The invention belongs to the biological chemistry field of medicaments, be specifically related to a kind of compound that from straw berry tomato, extracts with anti-tumor activity.
Background technology
WITHANOLIDE type compound is one type of steroid compound with ergostane type of 28 carbon atoms, and its agent structure is suc as formula shown in the II structure, and wherein, the T part in the formula II structure possibly be a kind of among a, b, c, d, e, f, g, the h.WITHANOLIDE type compound is divided into classic and non-classical type.Classical WITHANOLIDE type compound occupies absolute majority at nature, and these compounds mainly are distributed in several genus of Solanaceae (Solanaceae), have just comprised Physalis (Physalis) in several genus.That the biological activity of WITHANOLIDE type compound mainly embodies a concentrated reflection of is antitumor, anti-inflammatory, antibiotic and as the aspects such as antifeedant for insect of plant.Since the complex structure of this compounds, and chiral centre is a lot, and therefore, this compounds over-borrowing helps two dimensional NMR spectrum (2DNMR), circular dichroism spectrum (CD), X-single crystal diffraction modern techniquies such as (X-ray) is carried out the evaluation of structure.
At present, for treatment for cancer still with chemotherapy and radiation as first-selection, though both have obtained suitable curative effect to tumor treatment.But owing to lack specificity, thereby have bigger toxic side effect to tumour cell, simultaneously some tumour cell to chemotherapy and radiation handle insensitive, so to a great extent limit the application of chemotherapy and radiation in clinical.Therefore, people turn to Chinese medicine to sight, attempt from natural component, to seek the antitumor drug that toxic side effect is little, effect is unique.
Straw berry tomato (P.pubescens L) is the Solanaceae monkey flower, and there is product in Heilungkiang, and is best with Yian County, Qiqihar especially.Straw berry tomato, the nature and flavor bitter cold is returned lung channel, has clearing heat and detoxicating, relieve sore throat, reduces phlegm, effect such as diuresis, can be applicable to pharyngalgia hoarsen, phlegm heat cough, dysuria, controls treatment of diseases such as pemphigus, eczema outward.Modern pharmacology research shows that straw berry tomato has anti-inflammatory, antibiotic, anticancer isoreactivity, and its anti-inflammatory mechanism is to suppress generation and the release of normal people's peripheral blood polymorphonuclear leukocyte (PMN) oxyradical and play a role.Up to now, both at home and abroad the report to the anti-tumor activity of straw berry tomato is very few.
Document " the bacteriostatic activity research of straw berry tomato berry extract " (Zheng Jingtong; The bacteriostatic activity research of straw berry tomato berry extract; The Chinese experimental diagnostics; 2010 the 14th the 6th phases of volume) research of the antibacterial aspect of straw berry tomato chemical ingredients is disclosed; Comprise extraction using alcohol; Extract bacteriostatic activity test though the extract chemical ingredients has been carried out the research of antibacterial aspect, does not separate the Research of measuring with anti-tumor activity to its chemical ingredients; The research range of straw berry tomato activeconstituents is confined to the bacteriostatic activity of extract, exists technological deficiency.
Document " chemical constitution study of straw berry tomato place calyx " (Zhang Hui; The chemical constitution study of straw berry tomato place calyx; Herbal medicine; O. 11th in 2010) chemical constitution study of the dry place of straw berry tomato calyx is disclosed; The extraction separation that comprises compound; The bioactivity screening of monomeric compound obtains 4 phytosterin compounds and 11 flavonoid compounds though its chemical ingredients has been carried out separation, does not separate not obtaining WITHANOLIDE type compound; And do not find to have active compound for anti tumor efficiently yet, have technological deficiency.
For whether the plant (being straw berry tomato) of exploring this medicine-food two-purpose has antitumor effective constituent, the contriver studies the chemical ingredients that has anti-tumor activity in the calyx extract of straw berry tomato place.
Summary of the invention
The invention provides a kind of compound, belong to classical WITHANOLIDE type compound with anti-tumor activity.
A kind of compound with anti-tumor activity is the compound of formula I structure:
Formula I
The proton nmr spectra of this compound and the result of carbon-13 nmr spectra are as shown in table 1, and through the sign of proton nmr spectra and carbon-13 nmr spectra, the structure that can show the compound with anti-tumor activity that from straw berry tomato, extracts is suc as formula shown in the I.
Table 1
(in?CDCl
3，
1H?NMR?500MHz，
13C?NMR?125MHz)
| Position | C δ | H δ |
| 1 | 202.2 | |
| 2 | 132 | 6.23(1H，d，J＝10.0Hz) |
| 3 | 142.3 | 6.97(1H，dd，J＝5，10Hz) |
| 4 | 69.6 | 3.8(1H，d，J＝5Hz) |
| 5 | 63.5 | |
| 6 | 62 | 3.27(1H，s) |
| 7 | 30.2 | 2.1(1H，m)，1.5(1H，m) |
| 8 | 29 | 1.75(1H，m) |
| 9 | 43.7 | 1.13(1H，m) |
| 10 | 47.5 | |
| 11 | 21.8 | 1.9(1H，m)，1.5(1H，m) |
| 12 | 39.3 | 1.96(1H，m)，1.25(1H，m) |
| 13 | 42.9 | |
| 14 | 50 | 1.34(1H，m) |
| 15 | 75.9 | 4.84(1H，m) |
| 16 | 37 | 1.55(1H，m)，2.13(1H，m) |
| 17 | 58.7 | 1.29(1H，m) |
| 18 | 12.6 | 0.77(3H，s) |
| 19 | 17.3 | 1.45(3H，s) |
| 20 | 38.4 | 1.75(1H，m) |
| 21 | 12.5 | 0.92(3H，d，J＝5Hz) |
| 22 | 64.7 | 3.65(1H，m) |
| 23 | 29.3 | 1.7(2H，m) |
| 24 | 64.7 |
| 25 | 63.7 | |
| 26 | 91.6 | 5.03(1H，s) |
| 27 | 16.5 | 1.45(3H，s) |
| 28 | 18.8 | 1.44(3H，s) |
| 29 | 170.7 | |
| 30 | 21.3 | 2.07(3H，s) |
The present invention also provides a kind of preparation method with compound of anti-tumor activity, and it prepares simply, is easy to control, workable.
Described preparation method with compound of anti-tumor activity may further comprise the steps:
1) straw berry tomato place calyx is extracted with extracting solvent refluxing, obtain extracting solution;
Described extraction solvent perhaps contains the alcohol solution of pure percent by volume 50%～99.99% for alcohol;
2) extracting solution that obtains in the step 1) is concentrated, use organic solvent extraction behind the dilute with water again, obtain dichloromethane extraction liquid;
Described organic solvent comprises methylene dichloride at least;
3) with step 2) in the dichloromethane extraction liquid that obtains cross chromatographic column through concentrating the back, pass through the compound that recrystallization behind the eluent system wash-out obtains having anti-tumor activity;
A kind of in the eluting solvent that described eluent system is the eluting solvent be made up of sherwood oil and ETHYLE ACETATE, be made up of sherwood oil and acetone, the eluting solvent of forming by methylene dichloride and methyl alcohol, the eluting solvent formed by chloroform and methyl alcohol.
In order to obtain better invention effect, below as of the present invention preferred:
In the step 1), described alcohol is methyl alcohol or ethanol, selects these two kinds when pure, and the extraction efficiency of extracting solvent is higher.More preferably, described extraction solvent is methyl alcohol or the aqueous ethanolic solution that contains ethanol percent by volume 95%, and the character of this extraction solvent is very suitable for extracting straw berry tomato place calyx, can more fully obtain the compound with anti-tumor activity in the straw berry tomato.
Each extraction solvent that adds with respect to 6～10 times of weight of straw berry tomato place calyx weight, 90 ℃～100 ℃ refluxing extraction, each time of extracting is 2h～3h, extracts 2～3 times.Under such extraction conditions, not only saved the usage quantity of extracting solvent, and can fully the compound with anti-tumor activity in the calyx of straw berry tomato place have been extracted, improved extraction yield.
Step 2) in; Described organic solvent comprises sherwood oil, methylene dichloride and ETHYLE ACETATE; Use single organic solvent to extract successively by the polarity size during extraction; General adopt the equal-volume extraction, the volume of the solution that obtains behind promptly each volume of organic solvent of using and the extract dilute with water equates.According to the similar compatibility principle; Respectively through above-mentioned organic solvent extraction; So that the solution that obtains behind the extract dilute with water is according to the ascending separation of polarity; Compound red-purple spot through having anti-tumor activity in thin-layer chromatography (TLC) the inspection knowledge straw berry tomato almost all concentrates in the dichloromethane extraction liquid, and much more more other organic solvents mainly are to extract the compound with anti-tumor activity in the straw berry tomato or remove other polar materials to make to extract and more highly purified compound with anti-tumor activity for further.
In the step 3), the used filler of described chromatographic column is aluminum oxide or 100～200 purpose column layer chromatography silicone rubbers.The medicinal extract that described dichloromethane extraction liquid obtains after concentrating and the weight ratio of filler are 1: 1～10.
The described eluting solvent of being made up of methylene dichloride and methyl alcohol comprises that by volume ratio be the eluting solvent that 20～30: 1 methylene dichloride and methyl alcohol are formed.The described eluting solvent of being made up of methylene dichloride and methyl alcohol comprises by volume ratio and is the eluting solvent formed of 100: 1 methylene dichloride and methyl alcohol, is the eluting solvent formed of 50: 1 methylene dichloride and methyl alcohol, is the eluting solvent formed of 25: 1 methylene dichloride and methyl alcohol and is the eluting solvent that 10: 1 methylene dichloride and methyl alcohol is formed by volume ratio by volume ratio by volume ratio, and by the ascending gradient elution that carries out successively of polarity.
The described eluting solvent of being made up of chloroform and methyl alcohol comprises that by volume ratio be the eluting solvent that 40～60: 1 chloroform and methyl alcohol are formed.The described eluting solvent of being made up of chloroform and methyl alcohol comprises by volume ratio and is the eluting solvent formed of 100: 1 chloroform and methyl alcohol, is the eluting solvent formed of 50: 1 chloroform and methyl alcohol and is the eluting solvent that 25: 1 chloroform and methyl alcohol is formed by volume ratio by volume ratio, and by the ascending gradient elution that carries out successively of polarity.
The eluting solvent that the eluting solvent that employing is made up of methylene dichloride and methyl alcohol perhaps is made up of chloroform and methyl alcohol; And carry out gradient elution; This eluent system elutive power is strong; And knowing through thin-layer chromatography (TLC) inspection that the compound with anti-tumor activity in the straw berry tomato almost all is positioned at by volume ratio is that the eluting solvent formed of 25: 1 methylene dichloride and methyl alcohol is the eluting solvent that 50: 1 chloroform and methyl alcohol is formed by volume ratio perhaps; Make that the compound with anti-tumor activity in the straw berry tomato is more concentrated, and improved separation efficiency.
The described application of compound in the preparation antitumor drug with anti-tumor activity.Described tumour comprises brain tumor, lung cancer, liver cancer, mammary cancer, prostate cancer, carcinoma of the pancreas, cervical cancer, cancer of the stomach, esophagus cancer etc.The compound with anti-tumor activity of the present invention's preparation also can be applied to prepare the cancer chemoprophylactic drug and suppress in the tumor-blood-vessel growth medicine.
Carry out the screening of antitumor action with mtt assay; The result shows; The effect that the compound that the present invention has an anti-tumor activity has obvious its propagation of inhibition to tumour cell such as breast cancer cell (MCF-7), human liver cancer cell (HepG2), gastric carcinoma cells (SGC7901), human cervical carcinoma cell (Hela), and less to normal people's peripheral blood polymorphonuclear leukocyte (PMN) restraining effect.Above-mentioned cell strain can adopt the commercially available prod, as adopting the various cell strains of US mode culture collection warehousing ATCC (American type culture collection).
Phase II metabolic enzyme is significant for the generation of prophylaxis of tumours; Quinone reductase (NAD (P) H:quinone reductase as one of phase II metabolic enzyme; QR) be one type of cytoprotective enzyme with detoxifcation, the effect of removing radical, the composition that quinone reductase is had induced activity is considered to have the cancer prevention effect at the tumour initial period, and the rise of quinone reductase level is considered to the important mechanisms (reference: Hayes JD that preventing cancer takes place; McMahon M.Molecular basis for the contribution of the antioxidant responsive element to cancer chemoprevention.Cancer Letters; 2001,174 (2): 103-113.Gerhauser C, Klimo K; Heiss E; Et al.Mechanism-based in vitro screening of potential cancer chemopreventive agents.Mutat Res, 2003,523-524:163-172.).Carry out the chemoprophylactic screening of cancer with mtt assay, promptly quinone reductase is induced experiment, and the result shows, the compound that the present invention has an anti-tumor activity can be induced the expression of quinone reductase, and quinone reductase is had induced activity, has the cancer prevention effect.
The compound that the present invention has anti-tumor activity can combine with commercially available carrier or carrier commonly used, be used to prepare prevention perhaps/with the medicine of treating tumour.Described medicine can be forms such as tablet, powder injection.
Described compound with anti-tumor activity is processed tablet, is processed by the raw material of following weight part:
50 parts of compounds with anti-tumor activity;
2～10 parts of tamanoris;
6～14 parts of carboxymethylstach sodiums;
12～20 parts of starch;
0.5～1.5 part of Magnesium Stearate;
Described tamanori is that 1%～10% the hypromellose aqueous solution and amyloid mass percent are that 4%～10% starch slurry mixes according to weight ratio at 3: 10 by the mass percent that contains hypromellose.This medicinal tablet has very strong restraining effect to breast cancer cell (MCF-7), human liver cancer cell (HepG2), gastric carcinoma cells (SGC7901), human cervical carcinoma cell (Hela), and less to normal people's peripheral blood polymorphonuclear leukocyte (PMN) restraining effect.
The present invention has the compound of anti-tumor activity, when in treatment, using (administration), different effects can be provided.Usually; Can the compound that the present invention has an anti-tumor activity be formulated in nontoxic, inert and the pharmaceutically acceptable aqueous carrier medium; Wherein pH is about 5～8 usually; Preferable pH is about 6～8, although the pH value can change with being prepared Substance Properties and illness to be treated to some extent.The medicine for preparing can carry out administration through conventional route, comprising (but being not limited to): intramuscular, intraperitoneal, subcutaneous, intracutaneous or topical.
The compound that has anti-tumor activity with the present invention is an example, can be with itself and suitable pharmaceutically acceptable carrier coupling.This type carrier comprises (but being not limited to): salt solution, damping fluid, glucose, water, glycerine, ethanol and combination thereof.The form of pharmaceutical prepn should be complementary with administering mode.The present invention can be made into the injection form, for example prepares through ordinary method with the saline water or the aqueous solution that contains glucose and other assistant agents.Medicine such as tablet, capsule can prepare through ordinary method.Medicine such as injection solution, tablet and capsule should prepare under aseptic condition.The dosage of active constituents of medicine is the treatment significant quantity, for example every day 1 μ g/kg body weight～about 2000mg/kg body weight.In addition, the compound of the present invention with anti-tumor activity can also use with the other treatment agent.
When the compound that has an anti-tumor activity as the present invention is used as medicine; Can be with this compound administration of treating effective dose in Mammals; Wherein should treat effective dose usually at least about 10 micrograms/kg body weight; And in most of the cases be no more than about 8 mg/kg body weight, preferably, this effective dose is about 10 micrograms/kg body weight～about 1 mg/kg body weight.Certainly, concrete effective dose is factor such as considered route of administration, patient health situation also, and these all are within the skilled practitioners skill.
Compared with prior art, the present invention has following advantage:
The compound that the present invention has anti-tumor activity is the activeconstituents of effective antitumour medicine, and content is high in the straw berry tomato plant, and production cost is lower.The present invention has the compound of anti-tumor activity except tumour cell is had the toxic action, also has the effect of inducing quinone reductase to express.Therefore, the compound that the present invention has anti-tumor activity not only can be applicable to prepare antitumor drug, but also can be used to prepare the medicine of prophylaxis of tumours.
The present invention has the preparation method of the compound of anti-tumor activity, from the straw berry tomato plant, separates the compound monomer that obtains having anti-tumor activity with some common cheap reagent such as alcohol, methylene dichloride and silica gel, has reduced production cost.This preparation method is simple and reliable, and is workable, and efficient is high, can carry out industrialized production, helps applying, and has a good application prospect.
Description of drawings
Fig. 1 is the proton nmr spectra of the compound with anti-tumor activity of the embodiment of the invention 1 preparation;
Fig. 2 is the carbon-13 nmr spectra of the compound with anti-tumor activity of the embodiment of the invention 1 preparation.
Embodiment
Below content of the present invention is done further detailed explanation, but should embodiment be interpreted as limitation of the present invention through embodiment.Do not breaking away under the above-mentioned state of mind of the present invention, various replacement means or change according to ordinary skill knowledge and conventional means are made all are included within the present invention.
1) with the 1Kg straw berry tomato place calyx extractor of packing into; Each aqueous ethanolic solution that contains ethanol percent by volume 95% (being aqueous ethanolic solution 8Kg) that adds with respect to 8 times of medicinal material weight; 90 ℃ of refluxing extraction; Each time of extracting is 2h, extracts 2 times (aqueous ethanolic solution amounts to 16Kg) united extraction liquid altogether;
2) extracting solution that obtains in the step 1) is concentrated (recovery ethanol) and do, obtain thick medicinal extract 12g, after the dilution of 1L water, use the 1L dichloromethane extraction, coextration 3 times, combined dichloromethane extraction liquid at every turn near;
3) with step 2) in the dichloromethane extraction liquid that obtains concentrate the medicinal extract 5g that obtains through silica gel column chromatography (wet method dress post, appearance on the dry method), wherein; The used filler of chromatographic column is 100 purpose column layer chromatography silicone rubbers, and the weight ratio of medicinal extract and column layer chromatography silicone rubber is 1: 5, again through by volume ratio being the eluting solvent wash-out that 50: 1 chloroform and methyl alcohol is formed; This eluting solvent uses 6L at every turn; Co-elute 3 times merges elutriant, and recrystallization must have the compound (C of anti-tumor activity in acetone
30H
42O
8) 20mg, name PP-31J.
The proton nmr spectra of the compound with anti-tumor activity of embodiment 1 preparation is as shown in Figure 1, and carbon-13 nmr spectra is as shown in Figure 2, can characterize through Fig. 1 and Fig. 2, and the compound structure with anti-tumor activity of embodiment 1 preparation is suc as formula shown in the I.
Embodiment 2
1) with the 1Kg straw berry tomato place calyx extractor of packing into; Each aqueous ethanolic solution that contains ethanol percent by volume 95% (being aqueous ethanolic solution 8Kg) that adds with respect to 8 times of medicinal material weight; 90 ℃ of refluxing extraction; Each time of extracting is 2h, extracts 2 times (aqueous ethanolic solution amounts to 16Kg) united extraction liquid altogether;
2) extracting solution that obtains in the step 1) being concentrated (recovery ethanol) does near; Obtain thick medicinal extract 12g; After the dilution of 1L water, use single organic solvent to extract with sherwood oil, methylene dichloride and ETHYLE ACETATE successively by the polarity size respectively, every kind of organic solvent uses 1L at every turn; Every kind of organic solvent extraction 3 times; Obtain petroleum ether extraction liquid, dichloromethane extraction liquid, acetic acid ethyl acetate extract respectively, know the compound red-purple spot that has anti-tumor activity in the straw berry tomato through thin-layer chromatography (TLC) inspection and almost all concentrate in the dichloromethane extraction liquid combined dichloromethane extraction liquid;
3) with step 2) in the dichloromethane extraction liquid that obtains concentrate the medicinal extract 6g that obtains through silica gel column chromatography (wet method dress post; Appearance on the dry method); Wherein, The used filler of chromatographic column is 100 purpose column layer chromatography silicone rubbers, and the weight ratio of medicinal extract and column layer chromatography silicone rubber is 1: 5, is the eluting solvent formed of 100: 1 chloroform and methyl alcohol, is the eluting solvent formed of 50: 1 chloroform and methyl alcohol and is that the eluting solvent formed of 25: 1 chloroform and methyl alcohol is by the ascending gradient elution that carries out successively of polarity by volume ratio by volume ratio by volume ratio respectively again; Every kind of eluting solvent uses 6L at every turn, every kind of eluting solvent wash-out 3 times; Knowing through thin-layer chromatography (TLC) inspection that compound that straw berry tomato has anti-tumor activity almost all is arranged in by volume ratio is the elutriant of the eluting solvent formed of 50: 1 chloroform and methyl alcohol; Merge this elutriant, this elutriant recrystallization in acetone must be had the compound (C of anti-tumor activity
30H
42O
8) 22mg, name PP-31J.
The proton nmr spectra of the compound with anti-tumor activity of embodiment 2 preparation and carbon-13 nmr spectra are consistent with embodiment's 1.
Embodiment 3
1) with the 1Kg straw berry tomato place calyx extractor of packing into; Each aqueous ethanolic solution that contains ethanol percent by volume 95% (being aqueous ethanolic solution 10Kg) that adds with respect to 10 times of medicinal material weight; 100 ℃ of refluxing extraction; Each time of extracting is 3h, extracts 3 times (aqueous ethanolic solution amounts to 30Kg) united extraction liquid altogether;
2) extracting solution that obtains in the step 1) is concentrated (recovery ethanol) and do, obtain thick medicinal extract 16g, after the dilution of 800mL water, use the 800mL dichloromethane extraction, coextration 3 times, combined dichloromethane extraction liquid at every turn near;
3) with step 2) in the dichloromethane extraction liquid that obtains concentrate the medicinal extract 8g that obtains through silica gel column chromatography (wet method dress post, appearance on the dry method), wherein; The used filler of chromatographic column is 200 purpose column layer chromatography silicone rubbers, and the weight ratio of medicinal extract and column layer chromatography silicone rubber is 1: 10, again through by volume ratio being the eluting solvent wash-out that 25: 1 methylene dichloride and methyl alcohol is formed; This eluting solvent uses 6L at every turn; Co-elute 3 times merges elutriant, and recrystallization must have the compound (C of anti-tumor activity in acetone
30H
42O
8) 27mg, name PP-31J.
The proton nmr spectra of the compound with anti-tumor activity of embodiment 3 preparation and carbon-13 nmr spectra are consistent with embodiment's 1.
Embodiment 4
1) with the 1Kg straw berry tomato place calyx extractor of packing into; Each aqueous ethanolic solution that contains ethanol percent by volume 95% (being aqueous ethanolic solution 10Kg) that adds with respect to 10 times of medicinal material weight; 100 ℃ of refluxing extraction; Each time of extracting is 3h, extracts 3 times (aqueous ethanolic solution amounts to 30Kg) united extraction liquid altogether;
2) extracting solution that obtains in the step 1) being concentrated (recovery ethanol) does near; Obtain thick medicinal extract 16g; After the dilution of 800mL water, use single organic solvent to extract with sherwood oil, methylene dichloride and ETHYLE ACETATE successively by the polarity size respectively, every kind of organic solvent uses 800mL at every turn; Every kind of organic solvent extraction 3 times; Obtain petroleum ether extraction liquid, dichloromethane extraction liquid, acetic acid ethyl acetate extract respectively, know the compound red-purple spot that has anti-tumor activity in the straw berry tomato through thin-layer chromatography (TLC) inspection and almost all concentrate in the dichloromethane extraction liquid combined dichloromethane extraction liquid;
3) with step 2) in the dichloromethane extraction liquid that obtains concentrate the medicinal extract 8g that obtains through silica gel column chromatography (wet method dress post; Appearance on the dry method); Wherein, The used filler of chromatographic column is 200 purpose column layer chromatography silicone rubbers; The weight ratio of medicinal extract and column layer chromatography silicone rubber is 1: 10; Again respectively by volume ratio be the eluting solvent formed of 100: 1 methylene dichloride and methyl alcohol, by volume ratio be the eluting solvent formed of 50: 1 methylene dichloride and methyl alcohol, by volume ratio be the eluting solvent formed of 25: 1 methylene dichloride and methyl alcohol and by volume ratio be the eluting solvent formed of 10: 1 methylene dichloride and methyl alcohol by the ascending gradient elution that carries out successively of polarity, every kind of eluting solvent uses 6L at every turn, every kind of eluting solvent wash-out 3 times; Knowing compound that straw berry tomato has anti-tumor activity through thin-layer chromatography (TLC) inspection, almost all to be arranged in by volume ratio be the eluting solvent that 25: 1 methylene dichloride and methyl alcohol is formed; Merge this elutriant, this elutriant recrystallization in acetone must be had the compound (C of anti-tumor activity
30H
42O
8) 28mg, name PP-31J.
The proton nmr spectra of the compound with anti-tumor activity of embodiment 4 preparation and carbon-13 nmr spectra are consistent with embodiment's 1.
Embodiment 5 (Compound P P-31J extracorporeal anti-tumor cell experiment) with anti-tumor activity
The take the logarithm every hole 1 * 10 of cell of phase
4Be inoculated on 96 orifice plates, every hole adds the DMEM substratum of 200 μ L, behind the 12h, abandons supernatant, then the compound with anti-tumor activity of the embodiment of difference amount 1 preparation is dissolved in the DMSO 99.8MIN. of 50 μ L respectively; Join in the multiple hole, respectively by the compound with anti-tumor activity 0,10,20,40; 80,100 μ mol/L concentration administrations, promptly in each multiple hole, the compound concentrations with anti-tumor activity is respectively 0,10; 20,40,80,100 μ mol/L; Each concentration is established 3 multiple holes, is the compound administration group, and wherein, administration concentration is that 0 multiple hole is as blank control group; The Zorubicin of difference amount (green the skies reagent company) is dissolved in the DMSO 99.8MIN. of 50 μ L respectively, joins in the multiple hole, press Zorubicin 0,10 respectively, 20; 40,80,100 μ mol/L concentration administrations, promptly in each multiple hole, the concentration of Zorubicin is respectively 0; 10,20,40,80,100 μ mol/L; Each concentration is established 3 multiple holes, is Zorubicin administration group, and wherein, administration concentration is that 0 multiple hole is as blank control group; After cultivating 24h, abandon supernatant.The solution 50 μ L that in compound administration group, Zorubicin administration group, add band MTT respectively cultivate 4h; The solution of MTT is dissolved in phosphoric acid buffer by MTT (tetrazolium bromide, green the skies reagent company), and (PBS forms in pH=7.3); MTT concentration is 0.5mg/mL; Add 100 μ L DMSO 99.8MIN.s (DMSO) respectively to each hole again, vibration 1h, the 570nm place surveys OD (optical density(OD) optical density) value on ELIASA.Obtain the OD value under each concentration, calculate the proliferation inhibition rate under each concentration, carry out The Fitting Calculation through each concentration proliferation inhibition rate corresponding then, obtain Nonlinear regression equation, IC with it through the OD value that obtains
50Value refers to that the tumor cell line proliferation inhibition rate is 50% o'clock a administration concentration.Wherein, proliferation inhibition rate=(blank control group OD value-dosing group OD value)/blank control group OD value.
This experiment is carried out the detection of anti-tumor activity to breast cancer cell (MCF-7), human liver cancer cell (HepG2), gastric carcinoma cells (SGC7901), human cervical carcinoma cell (Hela), normal people's peripheral blood polymorphonuclear leukocyte (PMN) respectively; Above-mentioned cell strain is all purchased in ATCC, and concrete result is as shown in table 2.The result shows that after adding had the compound of anti-tumor activity, activity of tumor cells obviously descends, and was stronger to the inhibition proliferation function of tumour cell, and less to the restraining effect of normal cell PMN.
Table 2
Embodiment 6 (quinone reductase with Compound P P-31J of anti-tumor activity is induced experiment)
The take the logarithm rat liver cancer cell (Hepa 1c1c7 purchases in ATCC) of phase, every hole 1 * 10
4Be inoculated on 96 orifice plates, every hole adds the DMEM substratum of 200 μ L, behind the 12h, abandons supernatant, in 3 multiple holes, adds the compound 0.106mg with anti-tumor activity of embodiment 2 preparations then, is compound dosing group; In 3 multiple holes, add 4 '-bromine flavones 0.106mg, positive contrast dosing group; In 3 multiple holes, add DMSO 99.8MIN. 0.106mg, negative control group; Blank control group is the DMEM substratum of 200 μ L of not dosing.After cultivating 24h, abandon supernatant.In compound dosing group, positive control dosing group, negative control group and blank control group, add digitonin 10 μ g respectively and make lysis; The solution 200 μ L that add band MTT again cultivate 5min; The solution of MTT is dissolved in phosphoric acid buffer by MTT (tetrazolium bromide, green the skies reagent company), and (PBS forms in pH=7.3); MTT concentration is 0.5mg/mL, and the 550nm place surveys OD (optical density(OD) optical density) value on ELIASA.OD value through recording is calculated IR (inducing multiple) value, and the IR value is the quinone reductase inductive multiple with respect to negative control group, wherein, and IR=[(dosing group OD value-blank control group OD value)/(negative control group OD value-blank control group OD value)]/(1-proliferation inhibition rate); Wherein, proliferation inhibition rate=(blank control group OD value-dosing group OD value)/blank control group OD value.
The result shows that Compound P P-31J IR value under 1mmol/L (0.106mg) administration concentration that the present invention has anti-tumor activity is 2.54, positive control drug 4 '-the IR value of bromine flavones is 2.1; The result shows that Compound P P-31J that the present invention has an anti-tumor activity can induce the expression of quinone reductase, has cancer prophylaxis effects at the tumour initial period.
Embodiment 7 (preparation of compound medicine tablet)
1) takes by weighing the compound with anti-tumor activity, the tamanori of 0.006g, the carboxymethylstach sodium of 0.01g, the starch of 0.016g and the Magnesium Stearate of 0.001g of embodiment 3 preparation of 0.05g; Wherein, tamanori is that 8% the hypromellose aqueous solution and amyloid mass percent are that 8% starch slurry (starch and water are formed) mixes according to weight ratio at 3: 10 by the mass percent that contains hypromellose.Carboxymethylstach sodium uses as disintegrating agent.
Compound 0.05g with anti-tumor activity, starch 0.016g, the carboxymethylstach sodium 0.008g that 2) will take by weighing places and mixes 20min in the tempering tank; Add the mixed softwood of tamanori 0.006g then; After the granulation of 14 mesh sieves, the whole grain of 14 mesh sieves, Magnesium Stearate 0.001g that adding takes by weighing and the carboxymethylstach sodium of 0.002g are crossed in dry back; Mix the back compacting in flakes, promptly accomplish the preparation of compound medicine tablet; The compound medicine tablet specification of preparation is 0.083g.
The above-mentioned compound with anti-tumor activity is replaced with Zorubicin (green the skies reagent company), repeating step 1) and step 2), Zorubicin medicine tablet obtained.
Embodiment 8 (anticancer experiment in vitro of compound medicine tablet)
The take the logarithm every hole 1 * 10 of cell of phase
4Be inoculated on 96 orifice plates, every hole adds the DMEM substratum of 200 μ L, behind the 12h, abandons supernatant, then the compound medicine tablet of the embodiment of difference amount 7 preparations is dissolved in the DMSO 99.8MIN. of 50 μ L respectively; Join in the multiple hole, respectively by the compound with anti-tumor activity 0,10,20,40; 80,100 μ mol/L concentration administrations, promptly in each multiple hole, the compound concentrations with anti-tumor activity is respectively 0,10; 20,40,80,100 μ mol/L; Each concentration is established 3 multiple holes, is compound medicine tablet group, and wherein, administration concentration is that 0 multiple hole is a blank control group; The Zorubicin medicine tablet of the embodiment of difference amount 7 preparations is dissolved in the DMSO 99.8MIN. of 50 μ L respectively, joins in the multiple hole, press Zorubicin 0,10 respectively, 20; 40,80,100 μ mol/L concentration administrations, promptly in each multiple hole, the concentration of Zorubicin is respectively 0; 10,20,40,80,100 μ mol/L; Each concentration is established 3 multiple holes, is Zorubicin medicine tablet group, and wherein, administration concentration is that 0 multiple hole is a blank control group; After cultivating 24h, abandon supernatant.The solution 50 μ L that in compound medicine tablet group, Zorubicin medicine tablet group, add band MTT respectively cultivate 4h; The solution of MTT is dissolved in phosphoric acid buffer by MTT (tetrazolium bromide, green the skies reagent company), and (PBS forms in pH=7.3); MTT concentration is 0.5mg/mL; Add 100 μ L DMSO 99.8MIN.s (DMSO) respectively to each hole again, vibration 1h, the 570nm place surveys OD (optical density(OD) optical density) value on ELIASA.Record the OD value under each concentration respectively, calculate the proliferation inhibition rate under each concentration through obtaining the OD value then, carry out The Fitting Calculation through each concentration proliferation inhibition rate corresponding then, obtain Nonlinear regression equation, IC with it
50Value refers to that the tumor cell line proliferation inhibition rate is 50% o'clock a administration concentration.Wherein, proliferation inhibition rate %=(blank control group OD value-dosing group OD value)/blank control group OD value.
This experiment is carried out the detection of anti-tumor activity to breast cancer cell (MCF-7), human liver cancer cell (HepG2), gastric carcinoma cells (SGC7901), human cervical carcinoma cell (Hela), normal people's peripheral blood polymorphonuclear leukocyte (PMN) respectively; Above-mentioned cell strain is all purchased in ATCC, and concrete result is as shown in table 3.The result shows that behind the adding compound medicine tablet, activity of tumor cells obviously descends, and is stronger to the inhibition proliferation function of tumour cell, and less to the restraining effect of normal cell PMN.
Table 3
Claims (10)
1. compound with anti-tumor activity is the compound of formula I structure:
2. preparation method with compound of anti-tumor activity as claimed in claim 1 may further comprise the steps:
1) straw berry tomato place calyx is extracted with extracting solvent refluxing, obtain extracting solution;
Described extraction solvent perhaps contains the alcohol solution of pure percent by volume 50%～99.99% for alcohol;
2) extracting solution that obtains in the step 1) is concentrated, use organic solvent extraction behind the dilute with water again, obtain dichloromethane extraction liquid;
Described organic solvent comprises methylene dichloride at least;
3) with step 2) in the dichloromethane extraction liquid that obtains cross chromatographic column through concentrating the back, pass through the compound that recrystallization behind the eluent system wash-out obtains having anti-tumor activity;
A kind of in the eluting solvent that described eluent system is the eluting solvent be made up of sherwood oil and ETHYLE ACETATE, be made up of sherwood oil and acetone, the eluting solvent of forming by methylene dichloride and methyl alcohol, the eluting solvent formed by chloroform and methyl alcohol.
3. the preparation method with compound of anti-tumor activity as claimed in claim 2 is characterized in that, in the step 1), described alcohol is methyl alcohol or ethanol.
4. the preparation method with compound of anti-tumor activity as claimed in claim 2 is characterized in that, in the step 1), described extraction solvent is methyl alcohol or the aqueous ethanolic solution that contains ethanol percent by volume 95%.
5. the preparation method with compound of anti-tumor activity as claimed in claim 2; It is characterized in that; In the step 1), each extraction solvent that adds with respect to 6～10 times of weight of straw berry tomato place calyx weight is 90 ℃～100 ℃ refluxing extraction; Each time of extracting is 2h～3h, extracts 2～3 times.
6. the preparation method with compound of anti-tumor activity as claimed in claim 2; It is characterized in that; Step 2) in, described organic solvent comprises sherwood oil, methylene dichloride and ETHYLE ACETATE, uses single organic solvent to extract successively by the polarity size during extraction.
7. the preparation method with compound of anti-tumor activity as claimed in claim 2; It is characterized in that; In the step 3), the described eluting solvent of being made up of methylene dichloride and methyl alcohol comprises that by volume ratio be the eluting solvent that 20～30: 1 methylene dichloride and methyl alcohol are formed;
The described eluting solvent of being made up of chloroform and methyl alcohol comprises that by volume ratio be the eluting solvent that 40～60: 1 chloroform and methyl alcohol are formed.
8. the preparation method with compound of anti-tumor activity as claimed in claim 2; It is characterized in that; In the step 3); The described eluting solvent of being made up of methylene dichloride and methyl alcohol comprises by volume ratio and is the eluting solvent formed of 100: 1 methylene dichloride and methyl alcohol, is the eluting solvent formed of 50: 1 methylene dichloride and methyl alcohol, is the eluting solvent formed of 25: 1 methylene dichloride and methyl alcohol and is the eluting solvent that 10: 1 methylene dichloride and methyl alcohol is formed by volume ratio by volume ratio by volume ratio, and by the ascending gradient elution that carries out successively of polarity;
The described eluting solvent of being made up of chloroform and methyl alcohol comprises by volume ratio and is the eluting solvent formed of 100: 1 chloroform and methyl alcohol, is the eluting solvent formed of 50: 1 chloroform and methyl alcohol and is the eluting solvent that 25: 1 chloroform and methyl alcohol is formed by volume ratio by volume ratio, and by the ascending gradient elution that carries out successively of polarity.
9. the application of compound in the preparation antitumor drug with anti-tumor activity as claimed in claim 1.
10. application according to claim 9 is characterized in that, described compound with anti-tumor activity is processed tablet, is processed by the raw material of following weight part:
50 parts of compounds with anti-tumor activity;
2～10 parts of tamanoris;
6～14 parts of carboxymethylstach sodiums;
12～20 parts of starch;
0.5～1.5 part of Magnesium Stearate;
Described tamanori is that 1%～10% the hypromellose aqueous solution and amyloid mass percent are that 4%～10% starch slurry mixes according to weight ratio at 3: 10 by the mass percent that contains hypromellose.
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| CN105503990A (en) * | 2015-12-29 | 2016-04-20 | 吴金凤 | Novel withanolides compound as well as preparation method and medical application thereof |
| CN105524135A (en) * | 2015-12-25 | 2016-04-27 | 天津中医药大学 | Preparation method of physapubescin, and application of physapubescin in preparation of antitumor drugs |
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| CN103961362A (en) * | 2014-05-16 | 2014-08-06 | 富阳科兴生物化工有限公司 | Application of physalis pubescens lactone B in preparing anti-cancer drugs |
| CN103961362B (en) * | 2014-05-16 | 2016-08-24 | 杭州科兴生物化工有限公司 | A kind of straw berry tomato lactone B application in preparing cancer therapy drug |
| CN105524135A (en) * | 2015-12-25 | 2016-04-27 | 天津中医药大学 | Preparation method of physapubescin, and application of physapubescin in preparation of antitumor drugs |
| CN105777839A (en) * | 2015-12-25 | 2016-07-20 | 天津中医药大学 | Antitumor compound, extraction method therefor and application of antitumor compound |
| CN105777839B (en) * | 2015-12-25 | 2018-11-23 | 天津中医药大学 | A kind of antitumoral compounds, its extracting method and its application |
| CN105524135B (en) * | 2015-12-25 | 2019-11-22 | 天津中医药大学 | The preparation method and its application in preparation of anti-tumor drugs of straw berry tomato lactone |
| CN105503990A (en) * | 2015-12-29 | 2016-04-20 | 吴金凤 | Novel withanolides compound as well as preparation method and medical application thereof |
| CN105559074A (en) * | 2016-02-03 | 2016-05-11 | 华南农业大学 | Application of tea volatile essential oil to preparation of anti-cancer health care product or anti-cancer drug |
| CN111377994A (en) * | 2018-12-28 | 2020-07-07 | 南开大学 | Seven withanolides compounds from cape gooseberry and preparation method and application thereof |
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