CN102516344B - 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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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 from straw berry tomato, extract have compound of anti-tumor activity and its preparation method and application.
Background technology
WITHANOLIDE type compound is the steroid compound that a class has the 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 may 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 the specificity that lacks tumour cell, thereby have bigger toxic side effect, some tumour cell of while is insensitive to the chemotherapy and radiation processing, has limited the application of chemotherapy and radiation in clinical so to a great extent.Therefore, people turn to Chinese medicine to sight, attempt to seek from natural component 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 studies show that straw berry tomato has anti-inflammatory, antibiotic, anticancer isoreactivity, and its anti-inflammatory mechanism is to suppress the generation of normal people's peripheral blood polymorphonuclear leukocyte (PMN) oxyradical and release 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, the test of extract bacteriostatic activity, though the extract chemical ingredients has been carried out the research of antibacterial aspect, but its chemical ingredients is not separated Research of measuring with anti-tumor activity, 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, though having been carried out separation, its chemical ingredients obtains 4 phytosterin compounds and 11 flavonoid compounds, but do not separate and obtain 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 with anti-tumor activity, belong to classical WITHANOLIDE type compound.
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, by the sign of proton nmr spectra and carbon-13 nmr spectra, can show that the structure of the compound with anti-tumor activity that extracts is suc as formula shown in the I from straw berry tomato.
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 is alcohol or the alcohol solution that contains pure percent by volume 50%～99.99%;
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 ethyl acetate, be made up of sherwood oil and acetone, the eluting solvent of being formed 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 ethyl acetate, use single organic solvent to extract successively by the polarity size during extraction, general adopt the equal-volume extraction, namely the volume of the solution that obtains behind each volume of organic solvent of using and the extract dilute with water equates.According to the similar compatibility principle, respectively by 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, know the compound red-purple spot that has anti-tumor activity in the straw berry tomato by thin-layer chromatography (TLC) inspection and almost all concentrate in the dichloromethane extraction liquid, other organic solvents mainly are to make and extract more and more highly purified compound with anti-tumor activity in order further to extract the compound with anti-tumor activity in the straw berry tomato or to remove other polar materials.
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 are 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 are formed by volume ratio by volume ratio, and by the ascending gradient elution that carries out successively of polarity.
The eluting solvent that employing is made up of methylene dichloride and methyl alcohol or the eluting solvent of being formed by chloroform and methyl alcohol, and carry out gradient elution, this eluent system elutive power is strong, and knowing the compound with anti-tumor activity in the straw berry tomato by thin-layer chromatography (TLC) inspection almost all is positioned at by volume ratio and is the eluting solvent formed of 25: 1 methylene dichloride and methyl alcohol or is the eluting solvent that 50: 1 chloroform and methyl alcohol are formed by volume ratio, 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 that a class has detoxifcation; remove the cytoprotective enzyme of free radical effect; the composition that quinone reductase is had induced activity is considered to have the cancer prevention effect at the tumour initial period; 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, namely 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 an anti-tumor activity can be combined with commercially available carrier or carrier commonly used, for the preparation of prevention or treat the medicine of tumour.Described medicine can be forms such as tablet, powder injection.
Described compound with anti-tumor activity is made tablet, is made 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 when (administration) used in treatment, can provide different effects.Usually, that the compound that the present invention can be had an anti-tumor activity is formulated in is nontoxic, inertia with pharmaceutically acceptable aqueous carrier medium in, wherein pH is about 5～8 usually, preferable pH is about 6～8, although the pH value can change to some extent with being formulated Substance Properties and illness to be treated.The medicine for preparing can carry out administration by 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 example, can be with itself and suitable pharmaceutically acceptable carrier coupling.This class carrier comprises (but being not limited to): salt solution, damping fluid, glucose, water, glycerine, ethanol and combination thereof.The form of pharmaceutical preparation should be complementary with administering mode.The present invention can be made into the injection form, for example is prepared by ordinary method with physiological saline or the aqueous solution that contains glucose and other assistant agents.Medicine such as tablet, capsule can be prepared by 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 will treat this compound administration of 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 also should be considered factors such as route of administration, patient health situation, 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, content height 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 for the preparation of the medicine of prophylaxis of tumours.
The present invention has the preparation method of the compound of anti-tumor activity, separates the compound monomer that obtains having anti-tumor activity with some common cheap reagent such as alcohol, methylene dichloride and silica gel from the straw berry tomato plant, has reduced production cost.This preparation method is simple and reliable, and is workable, and the efficient height can carry out industrialized production, is conducive to apply, 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
By the following examples content of the present invention is described in further detail, but embodiment should be interpreted as limitation of the present invention.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.
Embodiment 1
1) 1Kg straw berry tomato place calyx is packed into extractor, 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, extract 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 near, obtain thick medicinal extract 12g, after the dilution of 1L water, use the 1L dichloromethane extraction, coextraction 3 times, combined dichloromethane extraction liquid at every turn;
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, sample 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 being the eluting solvent wash-out that 50: 1 chloroform and methyl alcohol are formed by volume ratio, 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 as shown in Figure 1, carbon-13 nmr spectra can characterize by Fig. 1 and Fig. 2 as shown in Figure 2, the compound structure with anti-tumor activity of embodiment 1 preparation is suc as formula shown in the I.
Embodiment 2
1) 1Kg straw berry tomato place calyx is packed into extractor, 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, extract 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 sherwood oil respectively, methylene dichloride and ethyl acetate use single organic solvent to extract by the polarity size successively, every sample organic solvent uses 1L at every turn, every sample organic solvent extraction 3 times, obtain petroleum ether extraction liquid respectively, dichloromethane extraction liquid, acetic acid ethyl acetate extract, know the compound red-purple spot that has anti-tumor activity in the straw berry tomato by 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, sample on the dry method), wherein, the used filler of chromatographic column is 100 purpose column layer chromatography silicone rubbers, the weight ratio of medicinal extract and column layer chromatography silicone rubber is 1: 5, be the eluting solvent that 100: 1 chloroform and methyl alcohol are formed by volume ratio respectively again, be the eluting solvent formed of 50: 1 chloroform and methyl alcohol and be 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, every sample eluting solvent uses 6L at every turn, every sample eluting solvent wash-out 3 times; Knowing by 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) 1Kg straw berry tomato place calyx is packed into extractor, 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, extract 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 near, obtain thick medicinal extract 16g, after the dilution of 800mL water, use the 800mL dichloromethane extraction, coextraction 3 times, combined dichloromethane extraction liquid at every turn;
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, sample 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 through being the eluting solvent wash-out that 25: 1 methylene dichloride and methyl alcohol are formed by volume ratio, this eluting solvent uses 6L at every turn, co-elute 3 times, merge elutriant, 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) 1Kg straw berry tomato place calyx is packed into extractor, 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, extract 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 sherwood oil respectively, methylene dichloride and ethyl acetate use single organic solvent to extract by the polarity size successively, every sample organic solvent uses 800mL at every turn, every sample organic solvent extraction 3 times, obtain petroleum ether extraction liquid respectively, dichloromethane extraction liquid, acetic acid ethyl acetate extract, know the compound red-purple spot that has anti-tumor activity in the straw berry tomato by 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, sample 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, be the eluting solvent that 100: 1 methylene dichloride and methyl alcohol are formed by volume ratio respectively again, be the eluting solvent that 50: 1 methylene dichloride and methyl alcohol are formed by volume ratio, be the eluting solvent formed of 25: 1 methylene dichloride and methyl alcohol and be that the eluting solvent formed of 10: 1 methylene dichloride and methyl alcohol is by the ascending gradient elution that carries out successively of polarity by volume ratio by volume ratio, every sample eluting solvent uses 6L at every turn, every sample eluting solvent wash-out 3 times; Knowing compound that straw berry tomato has anti-tumor activity by 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 are 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 (the 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, abandon supernatant liquor, then the compound with anti-tumor activity of embodiment 1 preparation of difference amount is dissolved in the dimethyl sulfoxide (DMSO) of 50 μ L respectively, join in the multiple hole, respectively by the compound 0,10,20 with anti-tumor activity, 40,80,100 μ mol/L concentration administrations are namely in each multiple hole, 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, wherein, administration concentration is that 0 multiple hole is organized as blank; The Zorubicin of difference amount (green the skies reagent company) is dissolved in the dimethyl sulfoxide (DMSO) of 50 μ L respectively, joins in the multiple hole, press Zorubicin 0,10 respectively, 20,40,80,100 μ mol/L concentration administrations, namely 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, be Zorubicin administration group, wherein, administration concentration is that 0 multiple hole is organized as blank; After cultivating 24h, abandon supernatant liquor.In compound administration group, Zorubicin administration group, add the solution 50 μ L cultivation 4h that is with MTT respectively, the solution of MTT is by MTT (tetrazolium bromide, green skies reagent company) is dissolved in phosphoric acid buffer (PBS, pH=7.3) form in, MTT concentration is 0.5mg/mL, add 100 μ L dimethyl sulfoxide (DMSO) (DMSO) more respectively to each hole, vibration 1h, the 570nm place surveys OD (optical density(OD) optical density) value on microplate reader.Obtain the OD value under each concentration, the proliferation inhibition rate by under each concentration of OD value calculating that obtains carries out The Fitting Calculation by each concentration proliferation inhibition rate corresponding with it then, obtains Nonlinear regression equation, IC
50Value refers to that the tumor cell line proliferation inhibition rate is 50% o'clock administration concentration.Wherein, proliferation inhibition rate=(blank group OD value-dosing group OD value)/blank 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, adds the compound 0.106mg with anti-tumor activity of embodiment 2 preparations then in 3 multiple holes, is compound dosing group; In 3 multiple holes, add 4 '-bromine flavones 0.106mg, positive contrast dosing group; In 3 multiple holes, add dimethyl sulfoxide (DMSO) 0.106mg, negative control group; Blank group is the DMEM substratum of 200 μ L of not dosing.After cultivating 24h, abandon supernatant liquor.Adding digitonin 10 μ g make lysis in organizing to compound dosing group, positive control dosing group, negative control group and blank respectively, the solution 200 μ L that add band MTT again cultivate 5min, the solution of MTT is by MTT (tetrazolium bromide, green skies reagent company) is dissolved in phosphoric acid buffer (PBS, pH=7.3) form in, MTT concentration is 0.5mg/mL, and the 550nm place surveys OD (optical density(OD) optical density) value on microplate reader.Calculate IR (inducing multiple) value by the OD value that records, the multiple of IR value for inducing with respect to the quinone reductase of negative control group, wherein, IR=[(dosing group OD value-blank group OD value)/(negative control group OD value-blank group OD value)]/(1-proliferation inhibition rate); Wherein, proliferation inhibition rate=(blank group OD value-dosing group OD value)/blank 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 the preventing cancer effect 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, granulate after 14 mesh sieves, the whole grain of 14 mesh sieves is crossed in dry back, the Magnesium Stearate 0.001g that adding takes by weighing and the carboxymethylstach sodium of 0.002g, mix the back compacting in flakes, namely finish 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, abandon supernatant liquor, then the compound medicine tablet of embodiment 7 preparation of difference amount is dissolved in the dimethyl sulfoxide (DMSO) of 50 μ L respectively, join in the multiple hole, respectively by the compound 0,10,20 with anti-tumor activity, 40,80,100 μ mol/L concentration administrations are namely in each multiple hole, 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, wherein, administration concentration is that 0 multiple hole is blank group; The Zorubicin medicine tablet of embodiment 7 preparation of difference amount is dissolved in the dimethyl sulfoxide (DMSO) of 50 μ L respectively, joins in the multiple hole, press Zorubicin 0,10 respectively, 20,40,80,100 μ mol/L concentration administrations, namely 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, be Zorubicin medicine tablet group, wherein, administration concentration is that 0 multiple hole is blank group; After cultivating 24h, abandon supernatant liquor.In compound medicine tablet group, Zorubicin medicine tablet group, add the solution 50 μ L cultivation 4h that is with MTT respectively, the solution of MTT is by MTT (tetrazolium bromide, green skies reagent company) is dissolved in phosphoric acid buffer (PBS, pH=7.3) form in, MTT concentration is 0.5mg/mL, add 100 μ L dimethyl sulfoxide (DMSO) (DMSO) more respectively to each hole, vibration 1h, the 570nm place surveys OD (optical density(OD) optical density) value on microplate reader.Record the OD value under each concentration respectively, by obtaining the proliferation inhibition rate under each concentration of OD value calculating, carry out The Fitting Calculation by each concentration proliferation inhibition rate corresponding with it then then, obtain Nonlinear regression equation, IC
50Value refers to that the tumor cell line proliferation inhibition rate is 50% o'clock administration concentration.Wherein, proliferation inhibition rate %=(blank group OD value-dosing group OD value)/blank 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 is alcohol or the alcohol solution that contains pure percent by volume 50%～99.99%;
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 ethyl acetate, be made up of sherwood oil and acetone, the eluting solvent of being formed 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, 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 ethyl 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 methylene dichloride of 20～30:1 and the eluting solvent that methyl alcohol is formed;
The described eluting solvent of being made up of chloroform and methyl alcohol comprises that by volume ratio be the chloroform of 40～60:1 and the eluting solvent that methyl alcohol is 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 methylene dichloride of 100:1 and eluting solvent that methyl alcohol is formed, is the methylene dichloride of 50:1 and eluting solvent that methyl alcohol is formed, is the methylene dichloride of 25:1 and eluting solvent that methyl alcohol is formed and is the methylene dichloride of 10:1 and the eluting solvent that 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 chloroform of 100:1 and eluting solvent that methyl alcohol is formed, is the chloroform of 50:1 and eluting solvent that methyl alcohol is formed and is the chloroform of 25:1 and the eluting solvent that 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 is characterized in that 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.
10. application according to claim 9 is characterized in that, described compound with anti-tumor activity is made tablet, is made by the raw material of following weight part:
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 3:10 by the mass percent that contains hypromellose.
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| 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 |
| CN105559074B (en) * | 2016-02-03 | 2019-02-12 | 华南农业大学 | A kind of tealeaves volatile essential oil is preparing the application in cancer-preventing health product or anticancer drug |
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