CN104220063B - Utilize the anti-cancer agent composition of thin Herba Adonidis extract or its active substance - Google Patents
Utilize the anti-cancer agent composition of thin Herba Adonidis extract or its active substance Download PDFInfo
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Abstract
The present invention is provided in cell experiment and experimentation on animals to show the anti-cancer agent composition utilizing thin Herba Adonidis extract or its active substance of antitumour activity.
Description
Technical field
The present invention relates to the anti-cancer agent composition utilizing thin Herba Adonidis (Adonisamurensis) extract or its active substance.
Background technology
Generally speaking, cancer refers to and occurs abnormal in the cycle of the cell forming tissue, cause cell can not normal differentiation, growth regulation, thus become big central malignant tumour.
According to known situation, cancer is by starting (initiation), promote (promotion) and develop three steps generations of (progression), cell mutation is caused by the carcinogenic substance being contained in environment or food, such cell proceeds improper propagation under the lasting stimulation of carcinogenic substance, thus forms cancerous tissue.
The research method being used for the treatment of the carcinostatic agent of cancer has the method exploring the direct cytotoxic substance to cancer cells, the method exploring the material of the immunological competence that can regulate human body, the method exploring the material of the transfer of known cancer cells and compares the method etc. of the material of concerned exploration angiogenesis inhibiting recently.
The carcinostatic agent used at present can be roughly divided into the biotechnological formulation such as zymin or vaccine, chemosynthesis pharmaceuticals, pharmaceuticals etc. from natural biological, wherein, the biotechnological formulation such as enzyme, vaccine is not yet in the application stage, and chemosynthesis pharmaceuticals are because of the pharmacological action difference (Gillman. of the kind according to cancer, etal., MaxwellMacmillan.18, pp1202,1986), the side effect that toxicity causes occurs, therefore, existing problems (Chung., the etal. when treating cancer, J.WonkwangMedicalSci., 3, pp13-34,1987).
For reducing the side effect of chemosynthesis carcinostatic agent to greatest extent, it is to increase result for the treatment of, lasting trial utilizes the exploitation of the carcinostatic agent of post extract.
According to report, there is the plant milk extract many (PujolM, GavilondoJ, AyalaM, RodriguezM, GonzalezEM, PerezL.2007TrendsBiotechnol25 (10): 455-459) of antitumour activity. such as Radix Angelicae Sinensis (Angelicagigas) extract is to colorectal carcinoma (KanWL, ChoCH, RuddJA, LinG.2008.120 (1): 36-43), large bowel cancer (LuJ.KimSHJiangC, LeeHGuoJ.2007.ActaPharmacolSin.28 (9): 1365-1372), the cancer of the brain (TasiNM, ChenYL, LeeCC, LinPC, ChengYL, ChangWL, LInSZ, etc. various love have anticancer effect HamHJ.2006.JNeurochem99 (4): 1251-1262), in addition, such as the root extract of Radix Glycyrrhizae (Glycyrrhizaglabra) is to prostate cancer (KanazawaM, SatomiY, MizutaniY, UkimuraO, KawauchiA, SakaiT, BabaM, OkuyamaT, NishinoH, or mastocarcinoma (JoEH MikiT.2003.EurUrol43 (5): 580-586), LimSH, RaJC, KimSR, ChoSD, JungJW, YangSR, ParkJS, HwangJW, AruomaOI, KimTY, LeeYS, there is anticancer effect KangKs.2005.CancerLett.230 (2): 239-247).
By cell experiment and/or experimentation on animals, the present invention confirms that the antitumour activity of thin Herba Adonidis extract and its active substance completes.
Summary of the invention
It is an object of the invention to provide a kind of anti-cancer agent composition.
Other objects of the present invention or concrete object become more to understand by by following content.
As confirmed by the following examples and experimental example, the present inventor is manufacturing while thin Herba Adonidis 80% ethanol extraction, above-mentioned 80% ethanol extraction is suspended in distilled water and successively with hexane (n-hexane), methylene dichloride (CH2Cl2), after ethyl acetate (EtOAc) and butanols (BuOH) carry out the thin Herba Adonidis fractionation extract of fractionation manufacture, first above-mentioned 80% ethanol extraction and the antitumour activity of each fractionation extract is observed by cell experiment, result shows the lung cancer cell types as the JEG-3 for testing, stomach cancer cell line AGS, colorectal cancer cells strain HCT-15, mammary carcinoma cells strain MDA-MB-231 and hepatoma cell strain SK-Hep1 shows cell inhibitory effect activity, the result that wherein the strongest active ethyl acetate fractionation extract carries out experimentation on animals is utilized to show, utilizing lung cancer cell types, in HF (Hollowfiber) the assay method model experiment of stomach cancer cell line AGS or hepatoma cell strain SK-Hep1, ethyl acetate divides parting to show the antitumour activity of concentration dependant in all JEG-3 for testing, especially, the naked mouse animal experimental model having transplanted SK-Hep-1 or HepG-2 as hepatoma cell strain also shows the antitumour activity of concentration dependant substantially.
Further, the present inventor is separated active substance from above-mentioned ethyl acetate fraction and the result identified is indicated as the compound shown in following<chemical formula 1>, is a kind of novel cpd.
<chemical formula 1>
The present invention provides based on above-mentioned experimental result, according to an aspect, the present invention is compound or its pharmacy acceptable salt of above-mentioned<chemical formula 1>, and according on the other hand, the present invention is included in anti-cancer agent composition wherein using compound or its pharmacy acceptable salt of thin Herba Adonidis extract, above-mentioned<chemical formula 1>as effective constituent.
In this manual, " anticancer " comprises and kills cancer cells, the propagation of anticancer, the transfer of anticancer, improves, case symptom that treatment cancer has or suppression/delay the generation of above-mentioned case symptom.
In addition, in this manual, " effective constituent " refers to and shows separately required activity or together show active composition with self not having active carrier.
In addition, in this manual, " thin Herba Adonidis extract " comprise utilized by any extracting method the carbon numbers such as methyl alcohol, ethanol, butanols be 1��4 lower alcohol, acetone, hexane, ethyl acetate, trichloromethane, methylene dichloride, water or their mixed solvent extract as extract the thin Herba Adonidis extract that grass, leaf, stem, flower, root or their mixture obtain entirely of object and utilize above-mentioned listed by the extract that obtains of a kind of fractionation said extracted thing in solvent. by any extracting method through using when extracting as the step extracting the leaf of thin Herba Adonidis of object, stem, flower, root or their mixture and being soaked in Extraction solvent, the any-modes such as extracting method can adopt cold leaching, refluxes, heats, ultrasonic wave radiation. but, goodly, above-mentioned " thin Herba Adonidis extract " is for utilizing water, ethanol or their mixed solvent extract the leaf as the thin Herba Adonidis extracting object, stem, flower, the extract that root or their mixture obtain, refer to the concentrated liquid extract or solids extraction thing of removing Extraction solvent, this solids extraction thing is utilized water and hexane, water and methylene dichloride, after this solids extraction thing is maybe suspended in water by the extract that water and ethyl acetate or water and butanols carry out certain fractionation solvent layer of fractionation gained, utilize hexane, methylene dichloride, the extract of ethyl acetate and butanols fractionation gained successively. at this, " successively fractionation " also continues to carry out fractionation with the solvent of above-mentioned listed order after referring to the evaporating point of fractionation water layer.
The compound of<chemical formula 1>of the present invention can use as a pharmaceutically acceptable salt form, and salt can use the acid salt formed by pharmaceutically acceptable free acid (freeacid).Acid salt is formed by the inorganic acids such as hydrochloric acid, nitric acid, phosphoric acid, sulfuric acid, Hydrogen bromide, hydroiodic acid HI, nitrous acid or phosphorous acid and the organic acids such as non-toxic organic, acetic acid, M-nitro benzoic acid, citric acid, lactic acid, toxilic acid, gluconic acid, methylsulfonic acid, 4-toluenesulphonic acids, tartrate, fumaric acid such as aliphatic monomer and dicarboxylate, phenyl replacement alkanoates, hydroxy alkane acid ester and chain docosandioic acid ester, aromatic acid class, aliphatics and aromatic sulphonic acid class. the salt of above-mentioned pharmaceutical innocuous comprises vitriol, pyrosulphate, hydrosulfate, sulphite, bisulfite, nitrate, phosphoric acid salt, hydrophosphate, dihydrogen phosphate, metaphosphate, tetra-sodium villaumite, bromide, iodide, fluorochemical, acetic acid, propionic salt, caprate, octylate, acrylate, formate, isobutyrate, ten carbonate, enanthate, Promolate, oxalate, malonate, succinate, suberate, sebacate, fumarate, maleate, butine-1,4-iodate, hexane-1,6-iodate, benzoate, chloro-benzoic acid, methyl benzoate, dinitro-benzoate, hydroxyl benzene acid salt, methoxybenzoic acid salt, phthalate, terephthalate, benzene sulfonate, tosylate, closilate, xylenesulfonate, phenylacetate, phenylpropionic acid, phenyl butyrate, Citrate trianion, lactic acid salt, beta-hydroxy-butanoic acid, glycollate, malate, tartrate, methylsulphonic acid, propanesulfonic acid salt, naphthalene-1-sulfonate, naphthalene-2-sulfonic acid salt or mandelate, but unrestricted. the acid salt of the present invention has been manufactured by usual method, such as, by the compound dissolution of<chemical formula 1>in machine solvent as in methyl alcohol, ethanol, acetone, methylene dichloride, cyaniding methane etc., add organic acid or organic acid generation precipitation, filter, dry obtained, or after underpressure distillation solvent and excessive acid dry or under organic solvent crystallization obtain.
In addition, the compound of<chemical formula 1>of the present invention can use base to obtain pharmaceutically acceptable metal-salt. The manufacture method of basic metal or alkaline earth salt is, such as, by compound dissolution in excessive alkali metal hydroxide or alkaline earth metal hydroxides solution, filter non-dissolved compound salt cake evaporation, dried filtrate obtain. Now, in pharmacy, metal-salt manufactures sodium, potassium or calcium salt and is advisable. Such as, now, corresponding silver salt makes basic metal or alkaline earth salt and suitable silver salt (Silver Nitrate) carry out reaction to obtain.
In addition, the form that the compound of above-mentioned<chemical formula 1>of the present invention is possible not only to its pharmacy acceptable salt uses, and, it is also possible to the forms such as the solvate that can thus manufacture, hydrate, steric isomer use.
In the anti-cancer agent composition of the present invention, when with reference to the anticancer proliferation experiment result of following experimental example, antitumour activity is that the antitumour activity to lung cancer or liver cancer is advisable, and its effective constituent is methylene dichloride fractionation extract, ethyl acetate fractionation extract or butanols fractionation extract is advisable. Especially, effective constituent is that methylene dichloride fractionation extract, ethyl acetate fractionation extract are advisable. If considering the experimentation on animals result of following experimental example, then best, antitumour activity is the antitumour activity to liver cancer, and effective constituent is ethyl acetate fractionation extract.
As long as antitumour activity can be shown according to the intention of formulation, cooperation object etc., then the effective constituent of the anti-cancer agent composition of the present invention can comprise any amount (significant quantity), and common significant quantity is 0.001��15 weight % scope relative to composition all wts.At this, " significant quantity " refers to can to the Mammals as its application, especially people is played and kill cancer cells, the propagation of anticancer, the transfer of anticancer, improves, the treatment case symptom that has of cancer or suppression/delays the amount of the effective constituent of the effect of the generation of above-mentioned case symptom. Above-mentioned significant quantity can determine by experiment within the limit of power of those skilled in the art. The object that can use the anti-cancer agent composition of the present invention is Mammals and people, especially people.
The anti-cancer agent composition of the present invention can be used as pharmaceutical composition in concrete form and uses.
The pharmaceutical composition of the present invention is except active substance, also comprise acceptable carrier, vehicle etc. in pharmaceutics, oral formulation (tablet, suspensoid, particle, emulsion, capsule, syrup etc.), parenteral formulation (sterile injectable aqueous or Oil suspensions), exterior-applied formulation (solution, breast frost, ointment, gel, emulsion, paster) etc. can be made into.
Above-mentioned " can accept in pharmaceutics " is meant to while not suppressing the activity of effective constituent, does not have the toxicity (enough low toxicity) of more than the scope using object to bear.
In pharmaceutics, acceptable carrier such as has lactose, glucose, sucrose, starch (such as W-Gum, potato starch etc.), cellulose and its derivates (such as Xylo-Mucine, ethyl cellulose etc.), Fructus Hordei Germinatus, gelatin, talcum, solid lubricant (such as stearic acid, Magnesium Stearate etc.), calcium sulfate, vegetables oil (such as peanut oil, Oleum Gossypii semen, sesame oil, sweet oil etc.), polyol (such as propylene glycol, glycerine etc.), alginic acid, emulsifying agent (such as Tweens (TWEENS)), wetting agent (such as sodium lauryl sulphate), tinting material, spices, scavenging agent, stablizer, antioxidant, sanitas, water, salt solution, phosphate buffer soln etc. above-mentioned carrier can select more than one suitable type according to the formulation of the pharmaceutical composition of the present invention.
Vehicle also can select applicable type according to the formulation of the pharmaceutical composition of the present invention, such as, when the pharmaceutical composition of the present invention manufactures aqueous suspension, applicable vehicle has suspensoid or the dispersion agents etc. such as Xylo-Mucine, methylcellulose gum, hydroxypropylcellulose, sodium alginate, polyvinylpyrrolidone. When manufacturing injection liquid, applicable vehicle has Ringer's solution, isotonic sodium-chlor etc.
The pharmaceutical composition of the present invention is by oral or parenteral mode administration, it is possible to according to circumstances by external application mode administration.
Administration amount on the one of the pharmaceutical composition of the present invention is usual 0.001��150mg/kg weight range, can divide once or administration for several times. But, the various factorss such as the severity of route of administration, age of patient, sex, body weight, patient are depended on because of the administration amount of the pharmaceutical composition of the present invention, therefore, no matter in office above-mentioned administration amount is, and where face does not should be understood to limitation of the present invention.
In the form that other are concrete, the present invention can be used as food compositions and uses.
In the food compositions of the present invention, except its effective constituent, also can comprise sweeting agent, spices, physiologically active ingredient, mineral substance etc.
Sweeting agent uses and food can be made to have the amount of suitable sweet taste, can select natural or synthetic sweetener. Goodly, when using natural sweeteners, natural sweeteners has the sugar sweeting agents such as corn jam solid, honey, sucrose, fructose, lactose, maltose.
Spices can be used for increasing taste or fragrance, can select natural or synthetic perfume. Goodly, it may also be useful to natural perfume. When using natural perfume, except increasing fragrance, also there is the effect strengthening nutrition. The spices that natural perfume has the natural perfume obtained from apple, lemon, oranges and tangerines, grape, strawberry, peach etc. or obtains from green tea, radix polygonati officinalis, the leaf of bamboo, cassia bark, Folium chrysanthemi, jasmine. In addition, the spices obtained from ginseng (red ginseng), bamboo shoots, aloe vera, ginkgo etc. can also be used. Natural perfume can be liquid concentrated solution or solid-state extract. According to circumstances can use synthetic perfume, and synthetic perfume can select ester, ethanol, aldehyde, terpene etc.
Physiologically active substance can select the vitamins etc. such as the catechins such as catechin, l-Epicatechol, l-Epigallocatechol, epigallocatechin or Vogan-Neu, xitix, tocopherol, anti-rickets element, VitB1, riboflavin.
Mineral substance can select calcium, magnesium, chromium, cobalt, copper, fluorochemical, germanium, iodine, iron, lithium, manganese, molybdenum, phosphorus, potassium, selenium, silicon, sodium, sulphur, vanadium, zinc etc.
In addition, the food compositions of the present invention, except above-mentioned sweeting agent etc., also can comprise sanitas, emulsifying agent, acid flavoring, tackifier etc. as required.
As long as foregoing preservatives, emulsifying agent etc. can reach the object added, use denier as far as possible. If with numeric representation, then denier refers to 0.0005��0.5 weight % scope relative to food compositions all wts.
The sanitas that can use has calcium sorbate, sodium sorbate, potassium sorbate, calcium benzoate, Sodium Benzoate, potassium benzoate, EDTA (ethylenediamine tetraacetic acid (EDTA)) etc.
The emulsifying agent that can select has gum Arabic, carboxymethyl cellulose, xanthan gum, pectin etc.
The acid flavoring that can select has nicotinic acid, oxysuccinic acid, fumaric acid, adipic acid, phosphoric acid, gluconic acid, tartrate, xitix, acetic acid etc. Except the effect that above-mentioned acid flavoring increases taste except having, also there is the level that the acidity adjustment of food compositions becomes suitable to suppress the effect of the propagation of microorganism.
The tackifier that can select have suspending agent, sinking agent, gel former, swelling agent etc.
In addition, functional (such as improving fragrance or palatability and increase other, preventing osteoporosis disease etc.), can various Chinese medicinal materials be added in the food compositions of the present invention, and the Chinese medicinal materials that can add has Cortex Eucommiae extract, Radix Dipsaci extract, Cornu Cervi Pantotrichum extract, Semen Flos Carthami extract, Semen Cuscutae extract, Radix Rehmanniae Preparata extract, Carapax Trionycis extract, Fructus Corni extract, wolfberry fruit extract, Radix Glycyrrhizae extract, Radix Angelicae Sinensis extract, Radix Puerariae extract, Lignum Dalbergiae Odoriferae extract, Cortex Albiziae extract, Radix Sophorae Tonkinensis extract, Flos Sophorae extract, Radix Sophorae Flavescentis extract etc.
As mentioned above, it is necessary, the present invention can provide using thin Herba Adonidis extract or its active substance as the anti-cancer agent composition of effective constituent. The symptom of lung cancer, cancer of the stomach, large bowel cancer, mastocarcinoma, liver cancer etc. is especially had curative effect by the anti-cancer agent composition of the present invention, it is possible to the mode of pharmaceutical composition or food compositions realizes commercialization.
Accompanying drawing explanation
Fig. 1 is the 1HNMR spectrum of the active substance being separated from thin Herba Adonidis;
Fig. 2 is the 13CNMR spectrum of the active substance being separated from thin Herba Adonidis;
Fig. 3 is the figure of 1HNMR and the 13CNMR spectroscopic data of the active substance that arrangement is separated from thin Herba Adonidis;
Fig. 4 is the DEPTNMR spectrum of the active substance being separated from thin Herba Adonidis;
Fig. 5 is the H-HCOSY spectrum of the active substance being separated from thin Herba Adonidis;
Fig. 6 is to the laboratory animal body weight change curve transplanted between the antitumour activity experiment periods of the SK-Hep-1 of naked mouse;
Fig. 7 is to the gross tumor volume change curve transplanted between the antitumour activity experiment periods of the SK-Hep-1 of naked mouse;
Fig. 8 is to the gross tumor volume change curve transplanted between the antitumour activity experiment periods of the SK-Hep-1 of naked mouse;
Fig. 9 is to the tumor weight change curve transplanted between the antitumour activity experiment periods of the SK-Hep-1 of naked mouse;
Figure 10 is to the laboratory animal body weight change curve transplanted between the antitumour activity experiment periods of the SHepG-2 of naked mouse;
Figure 11 is to the gross tumor volume change curve transplanted between the antitumour activity experiment periods of the HepG-2 of naked mouse;
Figure 12 is to the tumor weight change curve transplanted between the antitumour activity experiment periods of the HepG-2 of naked mouse;
Figure 13 measures the active substance being separated from thin Herba Adonidis to the result figure of the proliferation inhibition activity of SK-Hep-1 by MTT method.
Embodiment
<embodiment 1>Thin Herba Adonidis extract and the manufacture evaporated point
Dropping into immersion 48 hours in 80% ethanol and extraction three times repeatedly by cutting thin thin Herba Adonidis (full grass) 1kg, filter to obtain extracting solution, concentrating under reduced pressure extracting solution is removed solvent and is also carried out lyophilize acquisition powder shape extract.
In addition, the ethanol extraction obtained is suspended and be dissolved in 10 weight distilled water after, utilize hexane (n-hexane), methylene dichloride (CH successively2Cl2), ethyl acetate (EtOAc) butanols (BuOH) carry out fractionation, obtain evaporating point of hexane layer, dichloromethane layer, ethyl acetate layer, butanols layer and residue water layer.
Embodiment
Below, in conjunction with the embodiments and experimental example the present invention will be described. But, the scope of the present invention is not by the restriction of these embodiments and experimental example.
Being separated of<embodiment>thin Herba Adonidis extract and the manufacture evaporated point and active substance thereof
<embodiment 1>Thin Herba Adonidis extract and the manufacture evaporated point
Dropping into immersion 48 hours in 80% ethanol and extraction three times repeatedly by cutting thin thin Herba Adonidis (full grass) 1kg, filter to obtain extracting solution, concentrating under reduced pressure extracting solution is removed solvent and is also carried out lyophilize acquisition powder shape extract.
In addition, the ethanol extraction obtained is suspended and be dissolved in 10 weight distilled water after, utilize hexane (n-hexane), methylene dichloride (CH successively2Cl2), ethyl acetate (EtOAc) butanols (BuOH) carry out fractionation, obtain evaporating point of hexane layer, dichloromethane layer, ethyl acetate layer, butanols layer and residue water layer.
<embodiment 2>The Isolation and Identification of active substance
Get after above-mentioned ethyl acetate fraction 8g mixes with diatomite 545, after concentrating under reduced pressure manufactures mixture, implement open column chromatography. With n-Hex., MC (methylenechloride, methylene dichloride), DE (Diethylether, diethyl ether), EA (Ethylacetate, ethyl acetate), Methanol (MeOH, methyl alcohol) as moving phase obtain totally five little evaporate point. Wherein, DE (800mg) is evaporated a point enforcement silica gel column chromatography. With chloro-formic ester/methyl alcohol (9/2) as moving phase, flow velocity is 2.5ml/ minute, obtains and totally 18 each little evaporates point. No. 14 (DE-14) wherein are evaporated the simple purification of point enforcement and obtains single compound (compound1).
For analyzing the structure of separated material, single compound 10mg is dissolved in methyl alcohol-d and measures NMR spectra. 1HNMR spectrum (Fig. 1 and Fig. 3) is observed three methyl and it is all singlet, predict that three methyl are all combined with quaternary carbon, and, there is more than one phenyl ring according to the prediction of 4ppm signal.In addition, according to the signal of the complexity near 3.7-3.3ppm, prediction is combined with more than one sugar. Predict the carbon having more than totally 21 in 13CNMR (Fig. 2 and Fig. 3) and DEPTNMR (Fig. 4) spectrum, and observe anomeric carbon at 105.8ppm.
In addition, the proton (H-15, �� 2.2) of the proton (H-11, �� 6.40) and methyl of observing phenyl ring in H-HCOSY spectrum (Fig. 5) is coupled. Therefore, H-15 methyl is combined in phenyl ring, and observes anomeric carbon (H-1', 105) and the coupling of phenyl ring carbon (C-9, �� 146) in the analytical results of HSQC spectrum and HMBC spectrum. In addition, the methyl that prediction is connected to phenyl ring be coupled together with C-9 (�� 146), C-10 (�� 129), C-11 (�� 116) and together with being combined in C-10. In addition, observe that the hydrogen because of phenyl ring (H-11, �� 6.4) is each and C-9, C-12, C-7 are coupled together, therefore, it is positioned at Ortho position with C-9, C-7, is positioned at Meta position with C-12, be positioned at Para position with C-8. Observe because of methyl hydrogen (H-14, �� 1.7) it is coupled with phenyl ring carbon (C-7, �� 125), quaternary carbon (C-6, �� 74.4), C-5 (�� 37), therefore, together with prediction is combined in quaternary carbon, in addition, because of other methyl hydrogen (H-13, �� 1.2) and quaternary carbon (C-2, �� 74.5), C-1 (�� 29.6) be coupled, therefore, together with prediction is combined in C-2. In addition, because H-1 (�� 2.3) and C-3 (�� 75.5), C-7 (�� 125) are coupled, H-4 (�� 1.16) and C-6 (�� 74.4) are coupled, and H-5 (�� 2.02) and C-3 (�� 75.5) are coupled, therefore, prediction forms big ring from C-1 to C-8. In addition, although C-2 and C-6 is the quaternary carbon of single combining form, but moving to terrestrial magnetic field because observing chemical shift, therefore, prediction is combined with the big element of electronegativity. Therefore, the result analyzing NMR spectra shows that the compound of separation is made up of a ��-glucose and phenyl ring, anistree many rings, and the result searching for document shows, the structure of prediction is the novel substance not yet reported, thus by the novel substance called after Multioside of<chemical formula 1>.
The molecular weight of the material isolated for measuring implements HR-FABMS and analyzes. The molecular weight of novel substance is 426.46, and HR-FABMS analytical results is shown to be 449.1791 (CalcdforC21H30O9Na:449.1788) of the form being combined with sodium, thus the molecular structure confirming prediction is correct.
<experimental example>Thin Herba Adonidis extract and the antitumour activity experiment evaporated point
<experimental example 1>The anticancer proliferation activity of MTT method is utilized to test
<1>experimental technique
Utilize thin Herba Adonidis 80% ethanol extraction of MTT method Evaluation operation example and respectively evaporate point the antiproliferative effect effect to five kinds of cancer cells (lung cancer: A549, cancer of the stomach: AGS, large bowel cancer: HCT-15, mastocarcinoma: MDA-MB-231, liver cancer: SK-Hep1). For this reason, by different carcinoma cell, cell count is adjusted to 3��5 �� 104The concentration of/mL also puts into the postadhesion 24 hours in each hole of 96-orifice plate. After 24 hours, test portion is processed and after cultivating three days, adds MTT reagent and cultivate four hours again. With 1,000rpm, plate is carried out the centrifugation of 10 minutes, after careful removal substratum, after adding DMSO, measures absorbancy at 540nm.
<2>experimental result
Experimental result is as shown in Fig. 6, table 1 and table 2.
As shown in Figure 1, the anticancer cultivation effect of A549, AGS, HCT-15, MDA-MB-231 and SK-Hep1 is utilized to be, under 100 �� g/mL, in thin Herba Adonidis 80% ethanol extraction, A549 and SK-Hep1 shows the inhibition of more than 80%.As shown in table 1 and table 2, point the result that above-mentioned A549 and SK-Hep1 processes is shown with respectively evaporating of the above-mentioned thin Herba Adonidis extract of different concns, A549 and SK-Hep1 is shown the antiproliferative effect effect of concentration dependant.
Based on above-mentioned experimental result, it may also be useful to the best EtOAc of effect evaporates point (AAM-EA) and implements experimentation on animals.
Table 1
Table 2
<experimental example 2>In tubular fibre assay method (Hollowfiber (HF) assay) model of AAM-EA Antitumour activity experiment
<1>experimental technique
<1-1>medication and administration number of times
The administration of Test Materials utilizes after being attached with the transplanting of oral disposable syringe 1 tubular fibre with probe, once a day, within seven days, forces administration in stomach.
<1-2>group is formed and administration capacity
<1-2-1>group is formed
Group is constructed as follows shown in table 3.
Table 3
The setting of<1-2-2>administration capacity
The administration amount of Test Materials is set as 25,50 and 100mg/kg, and gives water for injection to negative control group. Negative control group and Test Materials administration group be set as 10mL/kg to amount of liquid medicine, and individuality be as the criterion to amount of liquid medicine to calculate according to the nearest body weight recorded for a week.
<1-3>observes and measured body weight
The observation of<1-3-1>general symptom
During whole administration, general symptom after observing once-a-day administration. General symptom observe by whether dead Different Individual record is, the kind of symptom and degree, generation day etc. information.
<1-3-2>measured body weight
To all animals time in one's hands, grouping time, start administration time, measured once in a week during administration.
<1-4>tubular fibre assay method
<1-4-1>cell cultures
After utilizing Test Materials to implement cytotoxicity experiment to five kinds of cell strains, (supplementarydata) chooses and the JEG-3 from people for tubular fibre assay method is as follows: A549 (lung cancer), AGS (cancer of the stomach), SK-Hep1 (liver cancer).
A549 and AGS is placed on RPMI1640 substratum, SK-Hep1 is placed on DMEM substratum and after adding sodium bicarbonate, L-glutaminate, penicillin/streptomycin, adds FBS so that final concentration reaches 10%, at the CO of humidifying2Incubator is cultivated. After the cell strain enough grown with Regular Insulin separation, with 1x106After the concentration of cells/ml is loaded into tubular fibre (Cellmaximplantmembrane, Spectrum), at the CO of humidifying2Incubator is cultivated 24 hours.
<1-4-2>implants
After anaesthetizing naked mouse with free from worries (zoletil) and xylazine (rompun), after open abdomen transplants the tubular fibre cultivating 24 hours a little, cross the administration that three days start Test Materials.
The measurement of the excision of<1-4-3>tubular fibre and anticancer propagation degree
After terminating experiment, naked mouse is killed by the de-bone of cervical vertebra, after extracing the fiber transplanted and removing the foreign matter on surface, put into the substratum being set in advance as 37 DEG C and stablize 30 minutes at the CO2 incubator of humidifying, carry out afterwards cultivating 4 hours after MTT (finalconc.1/Ml) processes.
After cultivation terminates, clean with 2.5% Protamine sulfates, after then soaking fiber in new Protamine sulfates, at 4 DEG C, carry out O/N. After again putting into the reaction that 2.5% new Protamine sulfates carries out four hours, erect and carry out O/N drying to cutting fiber. After confirming that fiber is completely dry, add DMSO and at room temperature shake four hours so that after the dissolving of first a ceremonial jade-ladle, used in libation salt, utilizing microplate reader (ELISAreader) to measure absorbancy.
The statistical treatment of<1-5>data
(Student-ttest) is checked to verify the comparison general student t between the vehicle control group of obtained data and Test Materials administration group. Edema rate and inhibiting rate generally represent with per-cent. Statistical method utilizes the statistics suit SPSS12.1K program as extensive commercialization.
<2>experimental result
Result is as shown in table 4.
As shown in table 4, utilize the result of the tubular fibre assay method of A549, AGS and SK-Hep1 to show, whole administration group shows meaningful antiproliferative effect effect, especially, when SK-Hep1, the effect of anticancer propagation is best.
Table 4
<experimental example 3>AAM-EA is to the antitumour activity experiment of the SK-Hep-1 transplanting naked mouse
<1>experimental technique
<1-1>medication and administration number of times
The administration of Test Materials utilizes after being attached with the oral disposable syringe 1HF with probe transplanting, once a day, forces administration totally 28 times in surrounding in stomach.
The administration of positive control substance utilizes disposable syringe (26G, 1mL, Doowonmeditec.Corp., Korea) weekly twice, and surrounding is administered to abdominal cavity totally eight times.
<1-2>group is formed and administration capacity
<1-2-1>group is formed
Group is constructed as follows shown in table 5.
Table 5
The setting of<1-2-2>administration capacity
The administration capacity of Test Materials is set as 50,200 and 500mg/kg, and cis-two chlorine two ammonia conjunction platinum (Cis-Diamineplatinum (II) dichloride) the administration amounts of positive control substance are set as 2mg/kg. Negative control group gives the water for injection as vehicle. Negative control group, Test Materials administration group and positive controls be set as 10mL/kg to amount of liquid medicine, and the amount of liquid medicine of giving of individuality is as the criterion according to the body weight measurements close to administration day and calculates.
<1-3>observes and measured body weight
The observation of<1-3-1>general symptom
During observing, check the general symptom such as outward appearance, action and movement every day, and confirm dead animal.
<1-3-2>measured body weight
Body weight self administration of medication is measured before starting date twice weekly (Tuesday, Friday) and administration.
The transplanting of<1-4>tumour
The tumor mass utilizing " Attachment.Succeedinggenerationsontransplantedtumorinnud emice ((strain) BIOTOXTECH, StudyNo.:B10999) " to be formed makes about 3 �� 3 �� mm3Section. After utilizing isofluranum (Isoflurane) that animal is carried out adsorb anesthetic, the openning of moon 4mm is cut at the position, front side of the left rear limb of animal, tumor mass is put trochar (trocar) end and makes trochar through from left rear limb incision site, thus tumor mass is filled in the back near the forelimb of left side. Then, by extracting of trochar fast rotational 360 degree simultaneously, after observing skin surface confirmation knub position, with povidone iodine (LotNo.:1005, Dongin-dangPharmCo., Ltd., Korea) the left rear limb incision site of animal is sterilized about one week.
<1-5>Tumor suppression growth result is evaluated
The skin measurement of<1-5-1>tumour
During observing, twice weekly (Tuesday, Friday) measures the major axis (maximumlength of tumour with calipers (caliper), and minor axis (perpendicularwidth L), W) and substitute into the volume (tumorvolume, TV) that following mathematical expression calculates tumour.
TV(mm3)=L (mm) �� W2(mm2)��1/2
The value measured when gross tumor volume before the administration of each individuality gets grouping.
The excision of<1-5-2>tumour and weight measurement
In the end day of the phase of observation, after utilizing isofluranum (Isoflurane, ASJ9AE, ChoongwaePharmaCorp., Korea) that animal carries out suck anesthesia, by individual tumor ablation and measure weight.
The statistical treatment of<1-6>data
The body weight of experiment acquisition, the skin of tumour, the weight of tumour SAS (Version9.1.9.2, SASInstituteInc., U.S.A.) verify.
Equal variance (significance level: 0.05) is verified by implementing Charles Bartlett inspection (Bartletttest). when equal variance, implement One-way ANOVA method (One-wayanalysisofvariance, ANOVA) (significance level: 0.05) and when observing significance, the multiple check (significance level: minor axis 0.05 and 0.01) of Dunnett'st-test is implemented for confirming the significance to each experimental group of negative control group, and when equal variance is denied, implement Cruise Ka Er-Wo Lisi and check (Kruskal-wallistest) (significance level: 0.05) and when observing significance, the multiple check of Steel'stest is implemented for confirming the significance to each experimental group of negative control group.
<2>experimental result
Result is as shown in Figure 7 to 9.
In measured body weight, the Test Materials administration group of 50mg/kg capacity does not show statistically meaningful difference than negative control group. 200 and the Test Materials administration group of 500mg/kg capacity at the 28th day, had statistically meaningful increase than negative control group. Although the positive controls of 2mg/kg capacity does not show statistically meaningful difference than negative control group, but there is the tendency of minimizing.
On the skin of tumour, 50,200 and positive controls upon administration 7th��28 days of the Test Materials administration group of 500mg/kg capacity and 2mg/kg capacity, show statistically meaningful inhibition than negative control group.
In the weight measurement of tumour, 50,200 and the Test Materials administration group of 500mg/kg capacity and the positive controls of 2mg/kg capacity, than meaningful the lacking of negative control group.
In histopathological examination result, 50,200 and necrosis (necrosis) degree of tumour of individuality of tumor survival of Test Materials administration group of 500mg/kg capacity show equal level than negative control group, but but part individual on tumor disappearance, the overall evaluation cannot be carried out. Because of tumour major part disappear and extract tumour on also because there is necrosis, therefore, apoptosis cannot compare evaluation with negative control group.
Generally speaking, to transplant in naked mouse from the anticancer experiment of the hepatoma cell strain SK-HEP-1 of human body, as the AAM-EA of Test Materials when 50,200 and 500mg/kg capacity, show the effect of obvious Tumor suppression growth.
<experimental example 4>AAM-EA is to the antitumour activity experiment of the HepG-2 transplanting naked mouse
<1>experimental technique
<1-1>medication and administration number of times
The administration of Test Materials utilizes after being attached with the oral disposable syringe 1HF with probe transplanting, once a day, forces administration totally 28 times in surrounding in stomach.
The administration of positive control substance utilizes disposable syringe (26G, 1mL, Doowonmeditec.Corp., Korea) weekly twice, and surrounding is administered to abdominal cavity totally eight times.
<1-2>group is formed and administration capacity
Group is constructed as follows shown in table 6.
Table 6
The setting of<1-2-2>administration capacity
The administration amount of Test Materials is set as 20,80 and 200mg/kg, and the administration capacity of positive controls is set as 2mg/kg. Negative control group gives the water for injection as vehicle. Negative control group, Test Materials administration group and positive controls be set as 10mL/kg to amount of liquid medicine, and the amount of liquid medicine of giving of individuality is as the criterion according to the body weight measurements close to administration day and calculates.
<1-3>observes and measured body weight
The observation of<1-3-1>general symptom
During observing, check the general symptom such as outward appearance, action and movement every day, and confirm dead animal.
The measurement of<1-3-2>body weight
Body weight self administration of medication is measured before starting date twice weekly (Tuesday, Friday) and administration.
The transplanting of<1-4>tumour
The tumor mass utilizing " Attachment.Succeedinggenerationsontransplantedtumorinnud emice ((strain) BIOTOXTECH, StudyNo.:B10999) " to be formed makes about 3 �� 3 �� mm3Section. After utilizing isofluranum (Isoflurane) that animal carries out suck anesthesia, the openning of moon 4mm is cut at the position, front side of the left rear limb of animal, tumor mass is put trochar (trocar) end and makes trochar through from left rear limb incision site, thus tumor mass is filled in the back near the forelimb of left side. Then, by extracting of trochar fast rotational 360 degree simultaneously, after observing skin surface confirmation knub position, with povidone iodine (LotNo.:1005, Dongin-dangPharmCo., Ltd., Korea) the left rear limb incision site of animal is sterilized about one week.
<1-5>Tumor suppression growth result is evaluated
The skin measurement of<1-5-1>tumour
During observing, twice weekly (Tuesday, Friday) measures the major axis (maximumlength of tumour with calipers (caliper), and minor axis (perpendicularwidth L), W) and substitute into the volume (tumorvolume, TV) that following mathematical expression calculates tumour.
TV(mm3)=L (mm) �� W2(mm2)��1/2
The value measured when gross tumor volume before the administration of each individuality gets grouping.
The excision of<1-5-2>tumour and weight measurement
In the end day of the phase of observation, after utilizing isofluranum (Isoflurane, ASJ9AE, ChoongwaePharmaCorp., Korea) that animal carries out suck anesthesia, by individual tumor ablation and measure weight.
The statistical treatment of<1-6>data
The body weight of experiment acquisition, the skin of tumour, the weight of tumour SAS (Version9.1.9.2, SASInstituteInc., U.S.A.) verify.
Equal variance (significance level: 0.05) is verified by implementing Charles Bartlett inspection (Bartletttest). when equal variance, implement One-way ANOVA method (One-wayanalysisofvariance, ANOVA) (significance level: 0.05) and when observing significance, the multiple check (significance level: minor axis 0.05 and 0.01) of Dunnett'st-test is implemented for confirming the significance to each experimental group of negative control group, and when equal variance is denied, implement Cruise Ka Er-Wo Lisi and check (Kruskal-wallistest) (significance level: 0.05) and when observing significance, the multiple check of Steel'stest is implemented for confirming the significance to each experimental group of negative control group.
<2>experimental result
Result is as shown in Figure 10 to Figure 12.
Body weight measurements shows, 20 and 80mg/kg capacity Test Materials administration group in, do not show meaningful difference than negative control group. In the Test Materials administration group of 200mg/kg capacity, 14th��21 days upon administration, there is meaningful increase than negative control group. And in the positive controls of 2mg/kg capacity, 18th��28 days upon administration, had meaningful minimizing than negative control group.
The skin measurement result of tumour shows, in the Test Materials administration group upon administration the 11st and 18��25 days of 20mg/kg capacity, in positive controls upon administration 11st��28 days of 80 and the Test Materials administration group of 200mg/kg capacity and 2mg/kg capacity, show meaningful inhibition than negative control group.
The weight measurement of tumour shows, although not showing meaningful difference in the Test Materials administration group of 20mg/kg capacity than negative control group, but 80 and the Test Materials administration group of 200mg/kg capacity and the positive controls of 2mg/kg capacity, than meaningful the lacking of negative control group.
In histopathological examination result, 20,80 and the Test Materials administration group of 200mg/kg capacity and the positive controls of 2mg/kg capacity, the calculation result of the necrosis (necrosis) of tumour, the degree of apoptosis (apoptosis) and apoptosis (apoptosis) shows similar level than negative control group.
Generally speaking, to transplanting in the anticancer experiment of the hepatoma H22 cells from human body of naked mouse, as the AAM-EA of Test Materials when 80 and 200mg/kg capacity, show the effect of obvious Tumor suppression growth.
<experimental example 5>Utilize the transplanting SK-Hep-1 of the active substance being separated from thin Herba Adonidis of MTT method thin Born of the same parents' proliferation activity is tested
With the active substance that the method evaluation identical with above-mentioned<experimental example 1>is isolated from thin Herba Adonidis, the Inhibit proliferaton of SK-Hep-1 cell is active.
As shown in figure 13, SK-Hep-1 cell is had the effect of the Inhibit proliferaton of concentration dependant to experimental result by the active substance isolated from thin Herba Adonidis.
Claims (9)
1. an anti-cancer agent composition, it is characterised in that: the compound of thin Herba Adonidis extract, following<chemical formula 1>or its pharmacy acceptable salt are included in wherein as effective constituent,
<chemical formula 1>
2. anti-cancer agent composition according to claim 1, it is characterised in that: above-mentioned thin Herba Adonidis extract is that thin Herba Adonidis is soaked in the mixed solvent of water, ethanol or water and ethanol the extract obtained.
3. anti-cancer agent composition according to claim 1, it is characterized in that: above-mentioned thin Herba Adonidis extract is thin Herba Adonidis is soaked in the mixed solvent of water, ethanol or water and ethanol the extract obtained be suspended in water, and the fractionation extract of the fractionation extract of the fractionation extract of the dichloromethane layer obtained when carrying out fractionation with hexane, methylene dichloride, ethyl acetate and butanols successively, ethyl acetate layer or butanols layer.
4. anti-cancer agent composition according to claim 1, it is characterized in that: above-mentioned thin Herba Adonidis extract is thin Herba Adonidis is soaked in the mixed solvent of water, ethanol or water and ethanol the extract obtained be suspended in water, and the fractionation extract of the ethyl acetate layer obtained when carrying out fractionation with hexane, methylene dichloride, ethyl acetate and butanols successively.
5. anti-cancer agent composition according to claim 1, it is characterised in that: above-mentioned antitumour activity is the antitumour activity to lung cancer, cancer of the stomach, large bowel cancer, mastocarcinoma or liver cancer.
6. anti-cancer agent composition according to claim 1, it is characterised in that: above-mentioned antitumour activity is the antitumour activity to liver cancer.
7. anti-cancer agent composition according to arbitrary item of claim 1 to 6, it is characterised in that: above-mentioned composition is pharmaceutical composition.
8. anti-cancer agent composition according to arbitrary item of claim 1 to 6, it is characterised in that: above-mentioned composition is food compositions.
9. compound following<chemical formula 1>or its pharmacy acceptable salt,
<chemical formula 1>
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