CN103467293A - Novel compound and application thereof in pharmaceuticals, food or cosmetics with anti-tumor function - Google Patents
Novel compound and application thereof in pharmaceuticals, food or cosmetics with anti-tumor function Download PDFInfo
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Abstract
The invention relates to a novel compound and application thereof in pharmaceuticals, food or cosmetics with anti-tumor function. The invention provides a novel compound which is safe without side effect and has a good anti-tumor function, and application thereof in pharmaceuticals, food or cosmetics, which is safe without side effect and has a good anti-tumor function. The invention provides a novel compound (D) expressed in the following formula (4), application of the compound in pharmaceuticals, food or cosmetics with anti-tumor function.
Description
The application is to be that the application number that on February 17th, 2011, denomination of invention was " novel cpd, antineoplastic agent and pharmaceuticals, food or makeup " is dividing an application of 201110040985.7 patent applications the applying date.
Technical field
The present invention relates to novel cpd and novel cpd and there is the application in pharmaceuticals, food or the makeup of antitumor action in preparation.
Background technology
Antineoplastic agent carcinostatic agent carcinostatic agent be substantially select optionally to act on tumour cell and to normal cytotoxicity less preparation, but also have the problem on a lot of the use not to be resolved yet, the appearance of fatal side effects such as the leukopenia caused by bone marrow depression, thrombopenia, neutrophil leucocyte minimizing, the appearance of the side effect of the Digestive tract obstacles such as nausea and vomiting, and the appearance of the side effect such as alopecia, drug-fast appearance, and because the medicament that is difficult to oral administration, route of administration is restricted etc.Therefore, expectation is developed and is optionally acted on tumour cell and, route of administration few to normal cytotoxicity limits few antineoplastic agent carcinostatic agent carcinostatic agent.
As the antineoplastic agent obtained from natural goods, known have a large amount of compounds, such as a large amount of reports such as ametycin (such as with reference to non-patent literature 1), camptothecine (such as with reference to patent documentation 1), triterpenoid (such as with reference to patent documentation 2) are arranged.In addition, as take the antineoplastic agent that natural goods is lead compound, known have D51-7059 (such as with reference to patent documentation 3), a Taxane derivative (such as with reference to patent documentation 4) etc.As the content of the antineoplastic agent about predicting relevant to this compound or relevant with biosynthesizing system, the decomposing solution of this compound naphthoquinones system, relevant for the report (for example, with reference to non-patent literature 2) of rhinacanthin A, B from rhinacanthus nasuta Kurz (Rhinacanthus nasutus (L.) Kurz), C, D, G, H, I, K, M, N, Q.In addition, as being the synthetic compound that antineoplastic agent is relevant to naphthoquinones, known have an antineoplastic agent relevant to the furans naphthoquinone derivatives (for example, with reference to patent documentation 5～8).
On the other hand, prove according to the present invention and contain " following novel cpd A, novel cpd B, novel cpd C, novel cpd D, novel cpd E, novel cpd F and novel cpd G " Twig and leaf of Bignose Rhinacanthus (following sometimes also referred to as rhinacanthus nasuta Kurz, Rhinacanthus nasutus (L.) Kurz), to belong to the Twig and leaf of Bignose Rhinacanthus that is considered to originate in South India Deccan Traps (Deccan Plateau) to belong to acanthaceous evergreen brushwood, known its herb has expelling parasite, anti-inflammatory, anti-microbial effect (for example, with reference to non-patent literature 3) to dermatophytes, mainly in China, the ground such as Taiwan are used as Chinese medicine, and in Japan, also as Chinese medicine, use recently.In addition, disclose ability (for example, with reference to patent documentation 9) that Twig and leaf of Bignose Rhinacanthus has Scavenger of ROS in application in the past in applicant, there is promotion Excretion (for example, with reference to patent documentation 10), there is anti-allergic effects (for example, with reference to patent documentation 11) and antitumor action (for example, with reference to patent documentation 12).
The prior art document
No. 3483257 communique of patent documentation 1 Japanese Patent
No. 4296312 communique of patent documentation 2 Japanese Patents
Patent documentation 3 Japanese Patent Publication 6-51689 communiques
No. 3942197 communique of patent documentation 4 Japanese Patents
Patent documentation 5 Japanese kokai publication hei 9-235280 communiques
Patent documentation 6 Japanese kokai publication hei 9-252753 communiques
Patent documentation 7 Japanese kokai publication hei 11-21284 communiques
Patent documentation 8 TOHKEMY 2005-220037 communiques
Patent documentation 9 Japanese kokai publication hei 9-143091 communiques
Patent documentation 10 Japanese kokai publication hei 9-169662 communiques
Patent documentation 11 TOHKEMY 2001-10964 communiques
Patent documentation 12 TOHKEMY 2002-53481 communiques
Non-patent literature
Non-patent literature 1The Journal of Antibiotics the 9th volume, 141-146 page, 1956
Non-patent literature 2Fight Medicine the 49th volume, 2001～2003 pages, 1988 years
Non-patent literature 3 primary colors are herded the large illustrated handbook of wild oriental drug grass (the Japanese original name: primary colors is herded the wild and large figure Monitoring of Han medicinal herbs), 492 pages, Bei Longguan, 1988
Summary of the invention
But, still do not know above-mentioned novel cpd (novel cpd A, novel cpd B, novel cpd C, novel cpd D, novel cpd E, novel cpd F or novel cpd G), contain this novel cpd (novel cpd A, novel cpd B, novel cpd C, novel cpd D, novel cpd E, novel cpd F or novel cpd G) antineoplastic agent and contain this novel cpd (novel cpd A, novel cpd B, novel cpd C, novel cpd D, novel cpd E, novel cpd F or novel cpd G) the pharmaceuticals with antitumor action, food or makeup.
Therefore, the purpose of this invention is to provide: do not worry its side effect, safety and there are novel cpd, the antineoplastic agent that contains this novel cpd and the pharmaceuticals with antitumor action, food or the makeup that contain this novel cpd of excellent antitumor action.
The result that the present inventor is searched for for the novel cpd with antitumor action, there is the excellent inhibited proliferation for the melanoma cell strain from human body (HMV-II) in 7 novel cpds that discovery obtains from Twig and leaf of Bignose Rhinacanthus (novel cpd A, novel cpd B, novel cpd C, novel cpd D, novel cpd E, novel cpd F or novel cpd G), completed the present invention.In order to solve above-mentioned problem, the present invention consists of following item.
The present invention relates to " novel cpd meaned by following formula (1) (following this novel cpd is called to novel cpd A) ", " antineoplastic agent that contains novel cpd A " and " pharmaceuticals with antitumor action, food or the makeup that contain novel cpd A ".
In addition, the present invention relates to " novel cpd meaned by following formula (2) (following this novel cpd is called to novel cpd B) ", " antineoplastic agent that contains novel cpd B " and " pharmaceuticals with antitumor action, food or the makeup that contain novel cpd B ".
In addition, the present invention relates to " novel cpd meaned by following formula (3) (following this novel cpd is called to novel cpd C) ", " antineoplastic agent that contains novel cpd C " and " pharmaceuticals with antitumor action, food or the makeup that contain novel cpd C ".
In addition, the present invention relates to " novel cpd meaned by following formula (4) (following this novel cpd is called to novel cpd D) " and " novel cpd D has the application in pharmaceuticals, food or the makeup of antitumor action in preparation ".
In addition, the present invention relates to " novel cpd meaned by following formula (5) (following this novel cpd is called to novel cpd E) " and " novel cpd E has the application in pharmaceuticals, food or the makeup of antitumor action in preparation ".
In addition, the present invention relates to " novel cpd meaned by following formula (6) (following this novel cpd is called to novel cpd F) " and " novel cpd F has the application in pharmaceuticals, food or the makeup of antitumor action in preparation ".
And then, the invention still further relates to " novel cpd meaned by following formula (7) (following this novel cpd is called to novel cpd G) " and " novel cpd G has the application in pharmaceuticals, food or the makeup of antitumor action in preparation ".
According to the present invention, novel cpd (novel cpd A can be provided, novel cpd B, novel cpd C, novel cpd D, novel cpd E, novel cpd F or novel cpd G), contain this novel cpd (novel cpd A, novel cpd B, novel cpd C, novel cpd D, novel cpd E, novel cpd F or novel cpd G) antineoplastic agent and contain this novel cpd (novel cpd A, novel cpd B, novel cpd C, novel cpd D, novel cpd E, novel cpd F or novel cpd G) the pharmaceuticals with antitumor action, food or makeup, above-mentioned novel cpd is because be from as food, the raw material of makeup was from just started the composition of the natural goods (Twig and leaf of Bignose Rhinacanthus) of use always in the past, so do not worry its side effect, safety, and can distinguish from following test example and there is excellent antitumor action.
The accompanying drawing explanation
Fig. 1 distinguishes the schema while separating each novel cpd (novel cpd A, novel cpd B, novel cpd C, novel cpd D, novel cpd E, novel cpd F or novel cpd G) from the Twig and leaf of Bignose Rhinacanthus root.
Embodiment
Each novel cpd of the present invention (novel cpd A, novel cpd B, novel cpd C, novel cpd D, novel cpd E, novel cpd F or novel cpd G), can obtain by extracting purification procedures Twig and leaf of Bignose Rhinacanthus as raw material, also can obtain in other plant materials.In addition, also can use the novel cpd (novel cpd A, novel cpd B, novel cpd C, novel cpd D, novel cpd E, novel cpd F or novel cpd G) obtained by synthetic.But also can use rhinacanthus nasuta Kurz, extract the extract obtain, thick purified or the dry thing of plant materials, the slurry of plant materials from other plant materials that contains novel cpd (novel cpd A, novel cpd B, novel cpd C, novel cpd D, novel cpd E, novel cpd F or novel cpd G).
While from the plant materialss such as rhinacanthus nasuta Kurz, extracting purifying novel cpd of the present invention (novel cpd A, novel cpd B, novel cpd C, novel cpd D, novel cpd E, novel cpd F or novel cpd G), can use any extraction purification procedures of common industrial use.After will the phase gathers in due course as the leaf of raw material, stem, root, flower etc., directly or implement the drying process of common air seasoning etc., make the extraction raw material.While being extracted from above-mentioned dried plant materials, can carry out with reference to known method (Fight Medicine the 16th volume, 929～934 pages 2009).
That is: by after raw material pulverizing or chopping, with solvent, extracted, as extracting solvent, can be by water, the alcohols such as ethanol, methyl alcohol, Virahol, the ketone such as acetone, methyl ethyl ketone, the ester classes such as methyl acetate, ethyl acetate, the oil loving solvents such as hexane, chloroform are used separately or form mixed solvent and use.Extract temperature and be generally 0～100 ℃, be preferably 5～50 ℃.Extraction time is about 1 hour～10 days, and quantity of solvent is that average every part of dried feed is generally 1～30 times of weight, is preferably 5～10 times of weight.Extracting operation can, by stirring, also can place and carry out by dipping.Extracting operation can repeat 2～3 times as required.From the crude extract obtained with aforesaid operations, by filtration or centrifugation, remove in the extracting solution after insoluble sludge or the method for each novel cpd of purifying (novel cpd A, novel cpd B, novel cpd C, novel cpd D, novel cpd E, novel cpd F or novel cpd G) from the squeezeding juice of plant, so long as known crude drug separation purification method, any method be can adopt, but two-phase solvent apportion design, counter-current distribution method, column chromatography, preparative high performance liquid chromatography etc. preferably are used alone or in combination.For example, as the two-phase solvent apportion design, can exemplify by being distributed in normal hexane, chloroform, methyl ethyl ketone, ethyl acetate, methyl acetate equal solvent and water, thereby target compound is recovered to from said extracted liquid to method in solvent phase etc.As column chromatography, can exemplify the ion exchange column chromatography method, use positive or reverse phase silica gel as the method for carrier, use DIAION HP-20 etc. the adsorpting column chromatogram method, use SephadexLH-20 etc. the modification dextrane gel as the gel filtration method of carrier etc., these methods can be used alone or in combination or use repeatedly.As preparative high performance liquid chromatography, can exemplify the method for the reversed-phase column that uses octadecyl silicon etc., use the method etc. of the normal phase column of silica gel etc.
Route of administration as antineoplastic agent of the present invention, be not particularly limited, such as exemplifying administration in the intestines such as oral administration drop rectum with drug, the mucosa delivery of nose administration etc., the drug administration by injection of intravenous administration subcutaneous administration etc. etc.Formulation as antineoplastic agent of the present invention, can take any and form matched preparation of medication, such as can exemplify tablet, powder, granula subtilis, granule, capsule, powder, pill, containing solid preparations such as tablets, the liquid preparations such as solution, suspension, emulsion, syrup, injection, gel preparation etc.Can be by directly administrations such as the sterling of each novel cpd (novel cpd A, novel cpd B, novel cpd C, novel cpd D, novel cpd E, novel cpd F or novel cpd G), purified, thick purifieds, also can with the together administration of excipient that allows on pharmacology.As excipient, so long as monose, disaccharides, polyose, inorganic salts, grease, distilled water etc. can be used any excipient as the common spendable excipient of preparation.While carrying out preparation, also can use the additives such as tackiness agent, lubricant, dispersion agent, suspension agent, emulsifying agent, thinner, buffer reagent, antioxidant, bacterial inhibitor.
Effective dosage of each novel cpd (novel cpd A, novel cpd B, novel cpd C, novel cpd D, novel cpd E, novel cpd F or novel cpd G), according to the difference at the symptom of route of administration, formulation, disease, the age of object etc. and difference, to be generally every day for each person be 0.1～1000mg, be preferably 0.5～300mg, 1～100mg more preferably.The content of each novel cpd in oral antineoplastic agent of the present invention (novel cpd A, novel cpd B, novel cpd C, novel cpd D, novel cpd E, novel cpd F or novel cpd G), can be according to the form of preparation effective dosage as the data of the dosage of preparation set with the optimal preparation of each administration form in active constituent content.
Form as food, can exemplify rhinacanthus nasuta Kurz and contain each novel cpd (novel cpd A, novel cpd B, novel cpd C, novel cpd D, novel cpd E, novel cpd F or novel cpd G) the dry thing of plant materials make the form of tea, or be combined with each novel cpd (novel cpd A, novel cpd B, novel cpd C, novel cpd D, novel cpd E, novel cpd F or novel cpd G) sterling, the partial purification product of this novel cpd, the crude extract of this novel cpd extracted from the plant that contains this novel cpd, the plant materials slurry that contains this novel cpd, the food of the dry thing of the plant materials that contains this novel cpd etc.
As tea, can mix use separately or with other tea raw materials.As other tea raw material, so long as green tea, oolong tea, Leaf of Assam Tea, black tea, simmer tea, brown rice tea, Eucommia Tea, persimmon-leaf tea, mulberry tea etc. be usually used as the edible raw material of tea, can use any tea raw material.
While obtaining plant milk extract, so long as use hot water extraction, spendable method in extracting with the common food such as ethanol, aqueous ethanol extraction, can be used any method.Can obtain by well-established law crude extract, the partial purification product of each novel cpd (novel cpd A, novel cpd B, novel cpd C, novel cpd D, novel cpd E, novel cpd F or novel cpd G) from plant materials.
As the form with food of antitumor action of the present invention, except tea, so long as nourishing drink, jelly, biscuit, tablet, pill, soft capsule, hard capsule, powder, granula subtilis, granule etc. can be used any form usually used as the available form of food.As auxiliary material, also can use the additives such as excipient, tackiness agent, lubricant, dispersion agent, suspension agent, emulsifying agent, thinner, buffer reagent, antioxidant, bacterial inhibitor.
The present invention has effective intake of each novel cpd (novel cpd A, novel cpd B, novel cpd C, novel cpd D, novel cpd E, novel cpd F or novel cpd G) of the food of antitumor action, according to the difference at picked-up form, the state of health of object, the age of object etc. and difference, but being generally every day for each person is 0.001～100mg, be preferably 0.01～10mg, more preferably 0.1～1mg.
The present invention has the content of each novel cpd (novel cpd A, novel cpd B, novel cpd C, novel cpd D, novel cpd E, novel cpd F or novel cpd G) in the food of antitumor action, according to the difference of food form and difference, but be generally 0.0001～1wt%, be preferably 0.001～0.5wt%, more preferably 0.01～0.1wt%.
Example as the form of external application pharmaceuticals of the present invention and makeup, be not particularly limited.
As the form of external application pharmaceuticals, such as exemplifying ointment, creme, paste, adhesive tape agent, external preparation etc.Pharmaceuticals of the present invention, on the basis of each novel cpd (novel cpd A, novel cpd B, novel cpd C, novel cpd D, novel cpd E, novel cpd F or novel cpd G), can contain as required other medical components, in addition, also can use the additives such as tackiness agent, dispersion agent, suspension agent, emulsifying agent, thinner, buffer reagent, antioxidant, bacterial inhibitor.
As the form of makeup, can use astringent, beauty liquid, emulsion, breast frost, gel, facial mask, beauty treatment muffin, cleansing milk, baths etc. as the spendable any form of external preparation cosmetic formulations.In above-mentioned makeup, on the basis of the essential compositions such as the crude extract of the partial purification product of the sterling of each novel cpd (novel cpd A, novel cpd B, novel cpd C, novel cpd D, novel cpd E, novel cpd F or novel cpd G), this novel cpd, this novel cpd of extracting or the plant materials that contains this novel cpd, also can contain as required the composition that can coordinate in cosmetic formulations from plant.As gradation composition, such as exemplifying solid oil, lard, liquid oils, low molecule wetting Agent for Printing Inks, polymer wetting Agent for Printing Inks, fat-soluble wetting Agent for Printing Inks, softener, tensio-active agent, sanitas, antioxidant, pH adjusting agent, ethanol, water etc.
Each novel cpd (novel cpd A, novel cpd B, novel cpd C, novel cpd D, novel cpd E, novel cpd F or novel cpd G) is effective dosage of used time outside, according to the difference at age of the symptom of object, object and difference, but being generally every day for each person is 0.001～100mg, be preferably 0.01～10mg, more preferably 0.1～1mg.
The content of each novel cpd in antineoplastic agent of the present invention, pharmaceuticals, makeup (novel cpd A, novel cpd B, novel cpd C, novel cpd D, novel cpd E, novel cpd F or novel cpd G), be generally 0.0001～1wt% during separately or as mixture, be preferably 0.001～0.5wt%, more preferably 0.01～0.1wt%.
The differentiation that below exemplifies each novel cpd of the present invention and Rhinacanthin C as a comparative example separates test example and the embodiment of example, antitumor action, the present invention is illustrated in further detail, but the present invention is not limited to these.
1. the differentiation of each novel cpd of the present invention and Rhinacanthin C as a comparative example separates example
1-1. novel cpd A
(1) differentiation of novel cpd A separates
The differentiation of novel cpd A separates to be carried out according to the flow process shown in Fig. 1.That is: at room temperature, with 90% (v/v) ethanol of 25L, Twig and leaf of Bignose Rhinacanthus (Rhinacanthus nasutus (L.) Kurz) dry root (5Kg) is carried out amounting in each 24 hours the extraction of 3 times, concentrated after these are combined, obtained dry substance (407g).Then, making in its 90% (v/v) at 7L methyl alcohol to suspend after dissolving, with the hexane of equivalent, carrying out 3 sub-distribution, then taking out 90% (v/v) methyl alcohol and carry out mutually concentrating under reduced pressure.In this concentrating under reduced pressure thing, add pure water to 5L, move in separating funnel and carry out 3 two-phase solvents distribution with chloroform.The chloroform that then will obtain by this operation combines mutually, obtains dry substance 69.3g.
By 69.0g wherein supply with silica gel column chromatography using hexane/ethyl acetate as eluting solvent (
kanto Kagaku K. K.'s system).That is: by the eluting solvent hexane/ethyl acetate (7:3) of the eluting solvent hexane/ethyl acetate (8:2) of the eluting solvent hexane/ethyl acetate (9:1) of 3 bed volumes (BV), 2BV and 2BV successively by after the silicagel column washing, use again eluting solvent hexane/ethyl acetate (6:4) wash-out of 2BV, obtain component C(dry substance 3.73g).
Then, for component C, implement Sephadex LH-20 column chromatography using methyl alcohol as eluting solvent (
, Pharmacia company system).That is: the methyl alcohol of using 2BV is by after the post washing, and the methanol-eluted fractions with 1BV, obtain component F (dry substance 369mg).
Then, component F (dry substance 369mg) is supplied with Flash ODS column chromatography using water/methyl alcohol as eluting solvent (
,Ye village chemical company system).That is: use 50% (v/v) methyl alcohol of 180ml by after the washing of Flash ODS post, after using successively step by step 60% (v/v) methyl alcohol, 70% (v/v) methanol wash, by 80% (v/v) methanol-eluted fractions, obtain the component G (dry substance 75.6mg) that contains target novel cpd A.
And then, the use preparative high performance liquid chromatography (the ODS post,
wild village chemical company system, moving phase: 45% (v/v) acetonitrile, detection: 254nm UV detector) component G (dry substance 75.6mg) is carried out to purifying, obtain novel cpd A(dry substance 9.8mg).
In addition, in above-mentioned, high performance liquid chromatography has been used Waters515 system and Waters600 system (be Japanese Waters Co., Ltd. system).In addition, silica gel column chromatography, Sephadex LH-20 column chromatography and Flash ODS chromatography are used experimental apparatus and the experimental installation of common use.
(2) structural analysis of novel cpd A
The structural analysis use high resolution mass spectrum analytical method (HRFABMS) of novel cpd A and nuclear magnetic resonance spectrometry (
1h NMR,
13c NMR) carry out.Below mean result.
(2-1) result of high resolution mass spectrum analytical method (HRFABMS)
In high resolution mass spectrum analytical method (HRFABMS), occurred " m/z272.1039[M]
+(calcd.272.1049 Δ 0.9mmu). ", the known molecular formula is " C
16h
16o
4".In addition, in Low Resolution Mass Spectra analytical method (LRFABMS), " m/z272. " appearred.
(2-2) nuclear magnetic resonance spectrometry (
1h NMR) result
Nuclear magnetic resonance spectrometry (
1h NMR), in, following spectrum peak has appearred.
“
1H?NMR(CDCl
3,500MHz)：δ3.69(H-14,J=11.2Hz,1H,d)、3.75(H-14,J=11.2Hz,1H,d)、3.95(8-OMe,3H,s)、4.22(H-2,J=11.7Hz,1H,d)、4.31(H-2,J=11.7Hz,1H,d)、5.93(H-4,J=11.7Hz,1H,d)、6.43(H-5,J=11.7Hz,1H,d)、6.51(H-7,1H,s)、7.47(H-10,J=1.5,6.7,7.5Hz,1H,m)、7.50(H-11,J=1.5,6.7,7.5Hz,1H,m)、8.14(H-9,J=1.5,7.5Hz,1H,d)、8.24(H-12,J=1.5,7.5Hz,1H,d).”
(2-3) nuclear magnetic resonance spectrometry (
13c NMR) result
Nuclear magnetic resonance spectrometry (
13c NMR), in, following spectrum peak has appearred.
“
13C?NMR(CDCl
3,125MHz)：δ55.7(8-OMe)、65.9(C-14)、74.4(C-2)、74.9(C-3)、106.8(C-7)、119.1(C-6)、121.6(C-9)、122.7(C-12)、126.0(C-12a)、126.3(C-10)、126.7(C-11)、127.6(C-8a)、130.1(C-5)、132.1(C-4)、148.9(C-13)、150.6(C-8).”
In addition, in above-mentioned, as the high resolution mass spectrum analytical equipment, use JEOL JMS SX-102 type mass spectrometer (Jeol Ltd.'s system).In addition, as the NMR (Nuclear Magnetic Resonance) spectrum device (
1h NMR reaches
13c NMR), use JEOL JNM-GSX500 type core NMR (Nuclear Magnetic Resonance) spectrum device (Jeol Ltd.'s system).
From the above results and HMQC spectrum, HMBC spectrum, novel cpd A is the novel cpd that above-mentioned formula (1) means.
1-2. novel cpd B, C
(1) differentiation of novel cpd B, C separates
The differentiation of novel cpd B, C separates, and according to the flow process shown in Fig. 1, carries out.That is: at room temperature, with 90% (v/v) ethanol of 25L, Twig and leaf of Bignose Rhinacanthus (Rhinacanthus nasutus (L.) Kurz) dry root (5Kg) is carried out amounting in each 24 hours the extraction of 3 times, concentrated after these are combined, obtained dry substance (407g).Then, making in its 90% (v/v) at 7L methyl alcohol to suspend after dissolving, with the hexane of equivalent, carrying out 3 sub-distribution, then taking out 90% (v/v) methyl alcohol and carry out mutually concentrating under reduced pressure.In this concentrating under reduced pressure thing, add pure water to 5L, move in separating funnel and carry out 3 two-phase solvents distribution with chloroform.The chloroform that then will obtain by this operation combines mutually, obtains dry substance 69.3g.
By 69.0g wherein supply with silica gel column chromatography using hexane/ethyl acetate as eluting solvent (
kanto Kagaku K. K.'s system).That is: the eluting solvent hexane/ethyl acetate (9:1) of using 3BV is by after the silicagel column washing, and eluting solvent hexane/ethyl acetate (8:2) wash-out with 1BV, obtain component A(dry substance 4.71g).
Then, component A is supplied with Sephadex LH-20 column chromatography using methyl alcohol as eluting solvent (
pharmacia company system).That is: the methyl alcohol of using 1.5BV is by after the washing of Sephadex LH-20 post, and the methanol-eluted fractions with 0.5BV, obtain component A-2 (1.34g).
And then, use Flash ODS column chromatography using water/methyl alcohol as eluting solvent (
with the pure medicine of light company system) differentiation component A-2.That is: use 50% (v/v) methyl alcohol of 180ml by after the washing of Flash ODS post, after using successively step by step 60% (v/v) methyl alcohol, 70% (v/v) methanol wash, by 80% (v/v) methanol-eluted fractions, obtain the component A-2-1 (dry substance 261mg) that contains target novel cpd B, C.
Component A-2-1 further use silica gel column chromatography using hexane/ethyl acetate as eluting solvent (
kanto Kagaku K. K.'s system) distinguish.That is: hexane/ethyl acetate (9:1) eluting solvent of using 3BV is by after the silicagel column washing, and hexane/ethyl acetate (8:2) the eluting solvent wash-out with 1BV, obtain component D(dry substance 92.4mg).
And then, the use preparative high performance liquid chromatography (the ODS post,
wild village chemical company system, moving phase: 58% (v/v) acetonitrile/water/0.1%(v/v) formic acid, detect: 254nm UV detector) component D (dry substance 92.4mg) is carried out to purifying, obtain novel cpd B(dry substance 4.6mg) and novel cpd C(dry substance 8.0mg).
(2) structural analysis of novel cpd B, C
The structural analysis use high resolution mass spectrum analytical method (HRFABMS) of novel cpd B, C and nuclear magnetic resonance spectrometry (
1h-NMR,
13c-NNMR) carry out.Below mean its result.
(2-1) result of high resolution mass spectrum analytical method (HRFABMS)
(2-1-1) novel cpd B
For novel cpd B, occurred " m/z256.1099[M]
+(calcd.256.1010 Δ 0mmu). ", the known molecular formula is " C
16h
16o
3".In addition, " m/z256. " in LRFABMS, occurred, " m/z256. " in LREIMS, occurred.
(2-1-2) novel cpd C
For novel cpd C, occurred " m/z270.1266[M]
+(calcd.270.1256 Δ 1.0mmu). ", known its molecular formula is " C
17h
18o
3".In addition, " m/z270. ", in LRFABMS, appearred.(2-2) nuclear magnetic resonance spectrometry (
1h NMR) result
(2-2-1) novel cpd B
For novel cpd B, following spectrum peak has appearred.
“
1H?NMR(CDCl
3,500MHz)：δ1.31(H-14,3H,s)、3.89(H-2,J=11.7Hz,1H,d)、3.90(8-OMe,3H,s)、4.29(H-2,J=2.0,11.7Hz,1H,d)、5.97(H-4,J=2.0,11.7Hz,1H,dd)、6.26(H-5,J=11.7Hz,1H,d)、6.48(H-7,1H,s)、7.42(H-10,J=6.3,7.8Hz,1H,m)、7.46(H-11,J=6.3,7.8Hz,1H,m)、8.10(H-9,J=7.8Hz,1H,d)、8.20(H-12,J=7.8Hz,1H,d).”
(2-2-2) novel cpd C
For novel cpd C, following spectrum peak has appearred.
“
1H?NMR(CDCl
3,500MHz)：δ1.32(H-14,3H,s)、3.38(3-OMe,3H,s)、3.91(8-OMe,3H,s)、4.08(H-2,J=11.7Hz,1H,d)、4.33(H-2,J=11.7Hz,1H,d)、5.90(H-4,J=11.7Hz,1H,d)、6.42(H-5,J=11.7Hz,1H,d)、6.50(H-7,1H,s)、7.40(H-10,1H,m)、7.44(H-11,1H,m)、8.08(H-9,J=7.8Hz,1H,d)、8.20(H-12,J=7.8Hz,1H,d).”
(2-3) nuclear magnetic resonance spectrometry (
13c NMR) result
(2-3-1) novel cpd B
For novel cpd B, following spectrum peak has appearred.
“
13C?NMR(CDCl
3,125MHz)：δ23.9(C-14)、55.7(8-OMe)、72.9(C-3)、79.5(C-2)、106.55(C-7)、119.9(C-6)、121.7(C-9)、122.6(C-12)、125.8(C-12a)、126.2(C-10)、126.6(C-11)、127.2(C-5)、127.6(C-8a)、136.8(C-4)、149.2(C-13)、150.8(C-8).”
(2-3-2) novel cpd C
For novel cpd C, following spectrum peak has appearred.
“
13C?NMR(CDCl
3,125MHz)：δ24.3(C-14)、52.1(3-OMe)、55.7(8-OMe)、77.3(C-3)、77.5(C-2)、106.9(C-7)、119.5(C-6)、121.6(C-9)、122.8(C-12)、126.1(C-12a)、126.5(C-10)、127.2(C-11)、127.7(C-8a)、129.2(C-5)、134.8(C-4)、148.9(C-13)、150.5(C-8).”
From above result and HMQC spectrum, HMBC spectrum, novel cpd B is the novel cpd that above-mentioned formula (2) means, novel cpd C is the novel cpd that above-mentioned formula (3) means.
1-3. novel cpd D, E
(1) differentiation of novel cpd D, E separates
The differentiation of novel cpd D, E separates to be carried out according to the flow process shown in Fig. 1.That is: at room temperature, with 90% (v/v) ethanol of 25L, Twig and leaf of Bignose Rhinacanthus (Rhinacanthus nasutus (L.) Kurz) dry root (5Kg) is carried out amounting in each 24 hours the extraction of 3 times, concentrated after these are combined, obtained dry substance (407g).Then, after making in its 90% (v/v) at 7L methyl alcohol to suspend and dissolving, after carrying out 3 sub-distribution with the hexane of equivalent, take out 90% (v/v) methyl alcohol and carry out mutually concentrating under reduced pressure.In this concentrating under reduced pressure thing, add pure water to 5L, move in separating funnel and carry out 3 two-phase solvents distribution with chloroform.The chloroform that then will obtain by this operation combines mutually, obtains dry substance 69.3g.
By 69.0g wherein supply with silica gel column chromatography using hexane/ethyl acetate as eluting solvent (
kanto Kagaku K. K.'s system).That is: with the eluting solvent hexane/ethyl acetate (9:1) of 3BV, and the eluting solvent hexane/ethyl acetate (8:2) of 2BV successively by after the post washing, use again eluting solvent hexane/ethyl acetate (7:3) wash-out of 2BV, obtain B component (dry substance 5.66g).
Then, for B component, using methyl alcohol as eluting solvent, implement Sephadex LH-20 column chromatography (
pharmacia company system).That is: the methyl alcohol of using 2.5BV is by after the post washing, and the methanol-eluted fractions with 1BV, obtain B component-2 (dry substance 480mg).
Then, B component-2 (dry substance 480mg) supplied with Flash ODS column chromatography using water/methyl alcohol as eluting solvent (
wild village chemical company system, that is: use 50% (v/v) methyl alcohol of 180ml by after the washing of Flash ODS post, after using successively step by step 60% (v/v) methyl alcohol, 70% (v/v) methyl alcohol, 80% (v/v) methanol wash, use again 90% (v/v) methanol-eluted fractions, obtain the component X (dry substance 143mg) that contains target novel cpd D, E.
And then, the use silica gel column chromatography (
kanto Kagaku K. K.'s system) by component X(dry substance 143mg) distinguish.That is: use the eluting solvent hexane/ethyl acetate (8:2) of 2BV by after the silicagel column washing, eluting solvent hexane/ethyl acetate (7:3) wash-out with 1BV, obtain B component-2-1(dry substance 32.3mg) and B component-2-2(dry substance 18.8mg).
With preparative high performance liquid chromatography (the ODS post,
wild village chemical company system, moving phase: 58% (v/v) acetonitrile, detect: 280nm UV detector) carry out purifying, from B component-2-1(dry substance 32.3mg) obtain novel cpd D(dry substance 5.0mg).
With preparative high performance liquid chromatography (the ODS post,
wild village chemical company system, moving phase: 70% (v/v) methyl alcohol, detect: 280nm UV detector), further use preparative high performance liquid chromatography (the ODS post,
wild village chemical company system, moving phase: 55% (v/v) acetonitrile, detect: 254nm UV detector) carry out purifying, from B component-2-2(dry substance 18.8mg) obtain novel cpd E(dry substance 1.6mg).
(2) structural analysis of novel cpd D, E
The structural analysis use high resolution mass spectrum analytical method (HRFABMS) of novel cpd D, E and nuclear magnetic resonance spectrometry (
1h NMR,
13c NMR) carry out.Below mean its result.
(2-1) result of high resolution mass spectrum analytical method (HRFABMS)
(2-1-1) novel cpd D
For novel cpd D, occurred " m/z397.1651[M+H]
+(calcd.397.1651 Δ 0.0mmu). ", the known molecular formula is " C
23h
24o
6".
(2-1-2) novel cpd E
For novel cpd E, occurred " m/z441.1919[M+H]
+(calcd.441.1913 Δ 0.5mmu). ", the known molecular formula is " C
25h
28o
7".
(2-2) nuclear magnetic resonance spectrometry (
1h NMR) result
(2-2-1) novel cpd D
In novel cpd D, following spectrum peak has appearred.
“
1H?NMR(CDCl
3,500MHz)：δ0.98(H-12and?H-13,6H,s)、1.98(H-8’,3H,s)、2.24(H-7’,3H,s)、2.65(H-9,2H,s)、3.90(H-11,2H,s)、6.21(H-5’,1H,d)、7.08(H-3’,1H,d)、7.25(H-4’,1H,dd)、7.60(H-7,1H,t)、7.67(H-6,1H,t)、7.99(H-8,1H,d)、8.03(H-5,1H,d).”
(2-2-2) novel cpd E
In novel cpd E, following spectrum peak has appearred.
“
1H?NMR(CDCl
3,500MHz)：δ1.01(H-12and?H-13,6H,s)、1.48(H-10’,3H,s)、1.90(H-9’,3H,s)、2.26(H-8’,3H,s)、2.70(H-9,2H,s)、3.91(H-11,2H,s)、6.02(H-5’,1H,d)、6.67(H-4’,1H,dd)、7.09(H-3’,1H,d)、7.66(H-7,1H,t)、7.73(H-6,1H,t)、8.04(H-8,1H,d)、8.08(H-5,1H,d).”
(2-3) result of nuclear magnetic resonance spectrometry (13C-NMR)
(2-3-1) novel cpd D
In novel cpd D, observed following spectrum peak.
“
13C?NMR(CDCl
3,125MHz)：δ13.4(C-8’)、25.3(C-12and?C-13)、28.1(C-7’)、32.2(C-9)、37.0(C-10)、73.4(C-11)、121.6(C-3)、126.1(C-8)、127.0(C-5)、129.3(C-8a)、132.9(C-4a)、132.9(C-7)、134.8(C-5’)、134.9(C-3’)、135.0(C-6)、136.2(C-2’)、136.4(C-4’)、154.4(C-2)、167.3(C-1’)、181.3(C-1)、184.8(C-4)、198.0(C-6’).”
(2-3-2) novel cpd E
In novel cpd E, observed following spectrum peak.
“
13C?NMR(CDCl
3,125MHz)：δ12.8(C-9’)、22.6(C-8’)、25.2(C-12and?C-13)、25.3(C-10’)、32.1(C-9)、37.2(C-10)、73.0(C-11)、79.4(C-6’)、121.4(C-3)、126.1(C-8)、126.4(C-4’)、127.0(C-5)、129.1(C-8a)、132.9(C-7)、133.3(C-4a)、134.9(C-6)、136.3(C-3’)、136.5(C-2’)、139.8(C-5’)、153.4(C-2)、167.7(C-1’)、181.6(C-1)、184.2(C-4)、207.3(C-7’).”
From above result and HMQC spectrum, HMBC spectrum, novel cpd D is the novel cpd that above-mentioned formula (4) means, novel cpd E is the novel cpd that above-mentioned formula (5) means.
1-4. novel cpd F
(1) differentiation of novel cpd F separates
The differentiation of novel cpd F separates to be carried out according to the flow process shown in Fig. 1.That is: at room temperature, with 90% (v/v) ethanol of 25L, Twig and leaf of Bignose Rhinacanthus (Rhinacanthus nasutus (L.) Kurz) dry root (5Kg) is carried out amounting in each 24 hours the extraction of 3 times, concentrated after these are combined, obtained dry substance (407g).Then, after making in its 90% (v/v) at 7L methyl alcohol to suspend and dissolving, after carrying out 3 sub-distribution with the hexane of equivalent, take out 90% (v/v) methyl alcohol and carry out mutually concentrating under reduced pressure.In this concentrating under reduced pressure thing, add pure water to 5L, move in separating funnel and carry out 3 two-phase solvents distribution with chloroform.The chloroform that then will obtain by this operation combines mutually, obtains dry substance 69.3g.
By 69.0g wherein supply with silica gel column chromatography using hexane/ethyl acetate as eluting solvent (
kanto Kagaku K. K.'s system).That is: with the eluting solvent hexane/ethyl acetate (9:1) of 3BV, and the eluting solvent hexane/ethyl acetate (8:2) of 2BV successively by after the post washing, eluting solvent hexane/ethyl acetate (7:3) wash-out with 2BV, obtain B component (dry substance 5.66g).
Then, for B component (dry substance 2.8g), using methyl alcohol as eluting solvent, implement the SephadexLH-20 column chromatography (
pharmacia company system).That is: the methyl alcohol of using 1.5BV is by after the post washing, and the methanol-eluted fractions with 0.5BV, obtain B component-1 (dry substance 1.16g).
Then, B component-1 (dry substance 1.1g) supplied with Flash ODS column chromatography using water/methyl alcohol as eluting solvent (
wild village chemical company system).That is: use 50% (v/v) methyl alcohol of 180ml by after the washing of Flash ODS post, use successively step by step 60% (v/v) methyl alcohol, 70% (v/v) methanol wash, then use 80% (v/v) methanol-eluted fractions, obtain the B component-1-2 (dry substance 429mg) that contains target novel cpd F.
Then, use silica gel column chromatography using hexane/ethyl acetate as eluting solvent (
kanto Kagaku K. K.'s system) B component-1-2 (dry substance 429mg) is distinguished.That is: the eluting solvent hexane/ethyl acetate (8:2) of using 2BV is by after the silicagel column washing, and eluting solvent hexane/ethyl acetate (7:3) wash-out with 1BV, obtain B component-1-2-2(dry substance 32.9mg).
With preparative high performance liquid chromatography (the ODS post,
wild village chemical company system, moving phase: 55% (v/v) acetonitrile, detect: 280nm UV detector) to B component-1-2-2(dry substance 32.9mg) carry out purifying, by further use preparative high performance liquid chromatography (the C-30 post,
wild village chemical company system, moving phase: 55% (v/v) acetonitrile, detect: 254nm UV detector) carry out purifying, obtain novel cpd F(dry substance 10.3mg).
(2) structural analysis of novel cpd F
The structural analysis use high resolution mass spectrum analytical method (HRFABMS) of novel cpd F and nuclear magnetic resonance spectrometry (
1h NMR,
13c NMR) carry out.Below mean its result.
(2-1) result of high resolution mass spectrum analytical method (HRFABMS)
In high resolution mass spectrum analytical method (HRFABMS), occurred " m/z459.2379[M+H]
+(calcd.459.2383 Δ 0.4mmu). ", the known molecular formula is " C
26h
34o
7".
(2-2) nuclear magnetic resonance spectrometry (
1h NMR) result
Nuclear magnetic resonance spectrometry (
1h NMR), in, following spectrum peak has appearred.
“
1H?NMR(CDCl
3,500MHz)：δ1.00(H-12and?H-13,6H,s)、1.08(H-10’,3H,s)、1.13(H-8’,3H,d)、1.46(H-5’,1H,m)、1.66(H-5’,1H,m)、1.79(H-9’,3H,s)、2.08(H-4’,1H,m)、2.23(H-4’,1H,m)、2.69(H-9,2H,s)、3.20(6’-OMe,3H,s)、3.80(H-7’,1H,q)、3.89(H-11,2H,s)、6.70(H-3’,1H,t)、7.66(H-7,1H,t)、7.73(H-6,1H,t)、8.06(H-8,1H,d)、8.09(H-5,1H,d).”
(2-3) nuclear magnetic resonance spectrometry (
13c NMR) result
Nuclear magnetic resonance spectrometry (
13c NMR), in, following spectrum peak has appearred.
“
13C?NMR(CDCl
3,125MHz)：δ12.3(C-9’)、17.0(C-8’)、18.6(C-10’)、22.7(C-4’)、25.2(C-12)、25.2(C-13)、31.9(C-5’)、32.2(C-9)、37.0(C-10)、49.1(6’-OMe)、70.6(C-7’)、72.8(C-11)、78.4(C-6’)、121.8(C-3)、126.1(C-8)、127.0(C-5)、127.7(C-2’)、129.4(C-8a)、132.9(C-7)、133.0(C-4a)、134.9(C-6)、142.4(C-3’)、154.2(C-2)、168.1(C-1’)、181.2(C-1)、184.8(C-4).”
From above result and HMQC spectrum, HMBC spectrum, novel cpd F is the novel cpd that above-mentioned formula (6) means.
1-5. novel cpd G
(1) differentiation of novel cpd G separates
The differentiation of novel cpd G separates to be carried out according to the flow process shown in Fig. 1.That is: at room temperature, with 90% (v/v) ethanol of 25L, Twig and leaf of Bignose Rhinacanthus (Rhinacanthus nasutus (L.) Kurz) dry root (5Kg) is carried out amounting in each 24 hours the extraction of 3 times, concentrated after these are combined, obtained dry substance (407g).Then, after making in its 90% (v/v) at 7L methyl alcohol to suspend and dissolving, after carrying out 3 sub-distribution with the hexane of equivalent, take out 90% (v/v) methyl alcohol and carry out mutually concentrating under reduced pressure.In this concentrating under reduced pressure thing, add pure water to 5L, move in separating funnel and carry out 3 two-phase solvents distribution with chloroform.The chloroform that then will obtain by this operation combines mutually, obtains dry substance 69.3g.
By 69.0g wherein supply with silica gel column chromatography using hexane/ethyl acetate as eluting solvent (
kanto Kagaku K. K.'s system).That is: with the eluting solvent hexane/ethyl acetate (9:1) of 3BV, and the eluting solvent hexane/ethyl acetate (8:2) of 2BV successively by after the post washing, eluting solvent hexane/ethyl acetate (7:3) wash-out with 2BV, obtain B component (dry substance 5.66g).
Then, for B component (dry substance 2.8g), implement SephadexLH-20 column chromatography using methyl alcohol as eluting solvent (
pharmacia company system).That is: the methyl alcohol of using 1.5BV is by after the post washing, and the methanol-eluted fractions with 0.5BV, obtain B component-1 (dry substance 1.16g).
And then, the FlashODS column chromatography by B component-1 (dry substance 1.16g) supply using water/methyl alcohol as eluting solvent (
wild village chemical company system).That is: use 50% (v/v) methyl alcohol of 180ml by after the washing of Flash ODS post, after using successively step by step 60% (v/v) methyl alcohol, 70% (v/v) methanol wash, by 80% (v/v) methanol-eluted fractions, obtain the B component-1-1 (dry substance 319mg) that contains target novel cpd G.
And then, use silica gel column chromatography using hexane/ethyl acetate as eluting solvent (
kanto Kagaku K. K.'s system) B component-1-1 (dry substance 319mg) is distinguished.That is: the eluting solvent hexane/ethyl acetate (9:1) of using 2BV is by after the silicagel column washing, and eluting solvent hexane/ethyl acetate (8:2) wash-out with 1BV, obtain component E(dry substance 114mg).
And then, the use preparative high performance liquid chromatography (the ODS post,
wild village chemical company system, moving phase: 68% (v/v) methyl alcohol, detect: 254nm UV detector) component E (dry substance 114mg) is carried out to purifying, obtain novel cpd G(dry substance 3.6mg).
(2) structural analysis of novel cpd G
The structural analysis use high resolution mass spectrum analytical method (HRFABMS) of novel cpd G and nuclear magnetic resonance spectrometry (
1h NMR,
13c-NNMR) carry out.Below mean its result.
(2-1) result of high resolution mass spectrum analytical method (HRFABMS)
In high resolution mass spectrum analytical method (HRFABMS), occurred " m/z399.1813[M+H]
+(calcd.399.1808 Δ 0.5mmu). ", the known molecular formula is " C
23h
26o
6".
(2-2) nuclear magnetic resonance spectrometry (
1h NMR) result
Nuclear magnetic resonance spectrometry (
1h NMR), in, following spectrum peak has appearred.
“
1H?NMR(CDCl
3,500MHz)：δ0.99(H-12and?H-13,6H,s)、1.80(H-8’,3H,s)、2.13(H-7’,3H,s)、2.34(H-4’,2H,dd)、2.50(H-5’,2H,t)、2.68(H-9,2H,s)、3.89(H-11,2H,s)、6.62(H-3’,1H,t)、7.67(H-7,1H,t)、7.74(H-6,1H,t)、8.07(H-8,1H,d)、8.09(H-5,1H,d).”
(2-3) nuclear magnetic resonance spectrometry (
13c NMR) result
Nuclear magnetic resonance spectrometry (
13c NMR), in, following spectrum peak has appearred.
“
13C?NMR(CDCl
3,125MHz)：δ12.3(C-8’)、22.7(C-4’)、25.2(C-12and?C-13)、29.9(C-7’)、32.1(C-9)、37.0(C-10)、42.1(C-5’)、72.9(C-11)、121.8(C-3)、126.1(C-8)、127.0(C-5)、128.9(C-2’)、129.4(C-8a)、132.9(C-4a)、133.0(C-7)、135.0(C-6)、140.0(C-3’)、154.3(C-2)、167.9(C-1’)、181.3(C-1)、184.8(C-4)、207.3(C-6’).”
From above result and HMQC spectrum, HMBC spectrum, novel cpd G is the novel cpd that above-mentioned formula (7) means.
1-6. Rhinacanthin C (comparative example)
(1) differentiation of Rhinacanthin C separates
The differentiation of Rhinacanthin C separates to be carried out according to the flow process shown in Fig. 1.That is: at room temperature, with 90% (v/v) ethanol of 25L, Twig and leaf of Bignose Rhinacanthus (Rhinacanthus nasutus (L.) Kurz) dry root (5Kg) is carried out amounting in each 24 hours the extraction of 3 times, concentrated after these are combined, obtained dry substance (407g).Then, after making in its 90% (v/v) at 7L methyl alcohol to suspend and dissolving, after carrying out 3 sub-distribution with the hexane of equivalent, take out 90% (v/v) methyl alcohol and carry out mutually concentrating under reduced pressure.In this concentrating under reduced pressure thing, add pure water to 5L, move in separating funnel and carry out 3 two-phase solvents distribution with chloroform.The chloroform that then will obtain by this operation combines mutually, obtains dry substance 69.3g.
By 69.0g wherein supply with silica gel column chromatography using hexane/ethyl acetate as eluting solvent (
kanto Kagaku K. K.'s system).That is: obtain the Rhinacanthin C (dry substance 5.66g) meaned with following formula (8) the component eluted from the eluting solvent hexane/ethyl acetate (9:1) with 3BV.
In addition, for distinguish the Rhinacanthin C separated by aforesaid method, utilize nuclear magnetic resonance spectrometry to obtain
1h NMR spectroscopic data (CDCl
3), following spectrum peak has appearred in result, consistent with the numerical value of document (Biol.Pharm.Bull.Vol.27,1070-1074 (2004)).
“
1H?NMR(CDCl
3,500MHz)：δ0.99(H-12and?H-13,6H,s)、1.52(H-8’,2H,d)、1.55(H-10’,2H,s)、1.76(H-9’,2H,s)、1.98(H-5’,2H,t)、2.13(H-4’,2H,dd)、2.67(H-9,2H,s)、3.88(H-11,2H,s)、5.17(H-7’,1H,dd)、6.66(H-3’,1H,t)、7.64(H-7,1H,t)、7.71(H-6,1H,t)、8.04(H-8,1H,d)、8.07(H-5,1H,d).”
2. the test example of antitumor action
Study on antitumor effect is undertaken by the proliferation inhibition activity of measuring for the melanoma cell strain HMV-II from human body.
The Ham F12 substratum that use contains 10% (v/v) foetal calf serum, upper with 1 * 10 at 96 hole culture plates (NUNC)
4the inoculation of the cell concn of cells/90 μ l is from the melanoma cell strain HMV-II of human body, under 37 ℃, 5% carbon dioxide exist, carries out cultivating in 24 hours.Cultivate after 24 hours, add novel cpd A of the present invention, novel cpd B, novel cpd D, novel cpd F or novel cpd G(to be dissolved in DMSO, final DMSO concentration in substratum=0.1% (v/v), in addition, under existing, cultivate 24 hours by cultured cells strain on the substratum of the DMSO only adding 1/1000 volume as a control group), and then at 37 ℃, 5% carbon dioxide.The mensuration of relative growth rate is used tetrazolium bromide (MTT), and [3-(4,5-dimethylthiazole-2-yl)-2,5-phenylbenzene bromination tetrazole Nacalai Tesque] ［ (3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide) ］ method is carried out ［ issuing true tumor chemical experiment lecture 12 molecular immunology I immunocyte cytokine 358-359 pages with reference to Co., Ltd.'s Tokyo chemistry with the people ］.
That is: add above-mentioned each novel cpd to cultivate after 24 hours, at 96 hole culture plate (0.33cm
2mTT solution { the 5mg/ml that adds 10 μ l in the nutrient solution 90 μ l in each hole/well); Without calcium magnesium PBS[Du Shi phosphoric acid buffer (Dulbecco ' s Phosphate-Buffered Saline)] dissolve in solution after, solution after film filter (0.22 μ m) filters }, vibrate and make it even, cultivate 4 hours under existing at 37 ℃, 5% carbon dioxide.Cultivate after 4 hours and add 10% (w/v) SDS-50% (v/v) N in each hole, dinethylformamide-0.005N hydrochloric acid soln 100 μ l, under 37 ℃, 5% carbon dioxide exist after standing 18 hours, use immunodetection instrument (immuno reader) (Dainippon Pharmaceutical Co., Ltd's system), with 750nm in contrast, measure the absorbancy at 590nm place, as the index of relative growth rate.
(novel cpd A, novel cpd B, novel cpd D, novel cpd F or novel cpd G) is as shown in table 1 for the cell inhibitory effect activity of melanoma cell strain HMV-II for each novel cpd.[table 1]
For the proliferation inhibition activity from human body melanoma cell strain (HMV-II)
As shown in table 1, can confirm that each novel cpd (novel cpd A, novel cpd B, novel cpd D, novel cpd F or novel cpd G) has " to the excellent proliferation inhibition activity of the melanoma cell strain from human body (HMV-II) " equal with the Rhinacanthin C with excellent antitumor action.
Embodiment
the making of embodiment 1. tablets
Use distinguishes according to the method for putting down in writing in above-mentioned " 1-1. novel cpd A(1) differentiation of novel cpd A separate " the novel cpd A separated, with following prescription making tablet (every 1 500mg).
(preparation method)
Interpolation novel cpd A(0.2g in lactose (95.8g)), dried corn starch (2g), talcum (1.8g), calcium stearate (0.2g) are mixed.Then, use Singlepunchtabletpress, according to well-established law, make tablet.
the making of embodiment 2. hard capsules
Use distinguishes according to the method for record in above-mentioned " differentiation of 1-2. novel cpd B, C (1) novel cpd B, C separates " the novel cpd B separated, and with following prescription, makes hard capsule (average every 1 360mg).
(preparation method)
At novel cpd B(5g) in add lactose (220g) and W-Gum (110g) and mixed, mediated after adding therein the aqueous solution of hydroxypropylcellulose (25g).Then, use Squeezinggranulator to make particle by well-established law.By this is particles filledly made to hard capsule in snap fit capsule.
the making of embodiment 3. soft capsules
Use distinguishes according to the method for record in above-mentioned " differentiation of 1-2. novel cpd B, C (1) novel cpd B, C separates " the novel cpd C separated, and with following prescription, makes soft capsule (average every 1 170mg).
Novel cpd C 0.5mg
Soybean oil 169.5mg
(preparation method)
Add novel cpd C(0.5g in soybean oil (169.5g)) mixed.Then by with the rotary-die type automatic molder, according to well-established law, being filled in soft capsule and making soft capsule.
the making of embodiment 4. pills
Use distinguishes according to the method for record in above-mentioned " differentiation of 1-3. novel cpd D, E (1) novel cpd D, E separates " the novel cpd D separated, and with following prescription, makes pill (average every 1 100mg).
(preparation method)
With said ratio, raw material is mixed, after adding water in right amount, with the uniform kneaded material of kneader manufacture, thereby use rotary pellet press to make pill by carrying out drying after the kneaded material pill obtained.
the making of embodiment 5. pills
Use distinguishes according to the method for record in above-mentioned " differentiation of 1-3. novel cpd D, E (1) novel cpd D, E separates " the novel cpd E separated, and with following prescription, makes pill (average every 1 100mg).
(preparation method)
With said ratio, raw material is mixed, after adding water in right amount, with the uniform kneaded material of kneader manufacture, thereby use rotary pellet press to make pill by carrying out drying after the kneaded material pill obtained.
the making of embodiment 6. ointment
Use distinguishes according to the method for record in above-mentioned " differentiation of 1-4. novel cpd F (1) novel cpd F separates " the novel cpd F separated, and with following prescription, makes ointment.
(oil-phase component)
(water-phase component)
(preparation method)
By oil-phase component and water-phase component be heated to respectively 80 ℃ evenly, add while stirring water in oil phase, after emulsification, carry out coolingly, make thus ointment.
the making of embodiment 7. astringents
Use distinguishes according to the method for record in above-mentioned " 1-5. novel cpd G(1) differentiation of novel cpd G separate " the novel cpd G separated, with following prescription making astringent.
(oil-phase component)
Novel cpd G 0.1g
Polyoxyethylene (60 moles) hydrogenated castor oil 2.0g
1,3 butylene glycol 5.0g
(water-phase component)
(preparation method)
By oil-phase component and water-phase component difference uniform dissolution, by add while stirring oil phase in water, prepare astringent.
Abovely according to above-mentioned embodiment, novel cpd of the present invention, antineoplastic agent and pharmaceuticals, food or makeup with antitumor action are illustrated, but the present invention is not limited to above-mentioned embodiment, in the scope that does not break away from its main idea, can in variety of way, implement.
Claims (12)
1. novel cpd, is characterized in that, by following formula (4), mean,
2. novel cpd, is characterized in that, by following formula (5), mean,
3. novel cpd, is characterized in that, by following formula (6), mean,
4. novel cpd, is characterized in that, by following formula (7), mean,
5. novel cpd claimed in claim 1 has the application in pharmaceuticals, food or the makeup of antitumor action in preparation.
6. application according to claim 5, is characterized in that, the described pharmaceuticals with antitumor action are antineoplastic agents.
7. novel cpd claimed in claim 2 has the application in pharmaceuticals, food or the makeup of antitumor action in preparation.
8. application according to claim 7, is characterized in that, the described pharmaceuticals with antitumor action are antineoplastic agents.
9. novel cpd claimed in claim 3 has the application in pharmaceuticals, food or the makeup of antitumor action in preparation.
10. application according to claim 9, is characterized in that, the described pharmaceuticals with antitumor action are antineoplastic agents.
11. novel cpd claimed in claim 4 has the application in pharmaceuticals, food or the makeup of antitumor action in preparation.
12. application according to claim 11, is characterized in that, the described pharmaceuticals with antitumor action are antineoplastic agents.
Applications Claiming Priority (2)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| JP2010034621 | 2010-02-19 | ||
| JP2010-034621 | 2010-02-19 |
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| Application Number | Title | Priority Date | Filing Date |
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| CN201110040985.7A Division CN102167687B (en) | 2010-02-19 | 2011-02-17 | Compound and application thereof in preparation of anti-tumor pharmaceutical, food or cosmetic |
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| Publication Number | Publication Date |
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| CN103467293A true CN103467293A (en) | 2013-12-25 |
| CN103467293B CN103467293B (en) | 2015-06-10 |
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| CN201110040985.7A Expired - Fee Related CN102167687B (en) | 2010-02-19 | 2011-02-17 | Compound and application thereof in preparation of anti-tumor pharmaceutical, food or cosmetic |
| CN201310273558.2A Expired - Fee Related CN103467293B (en) | 2010-02-19 | 2011-02-17 | Novel compound and application thereof in pharmaceuticals, food or cosmetics with anti-tumor function |
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| CN201110040985.7A Expired - Fee Related CN102167687B (en) | 2010-02-19 | 2011-02-17 | Compound and application thereof in preparation of anti-tumor pharmaceutical, food or cosmetic |
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| JP (1) | JP5620847B2 (en) |
| CN (2) | CN102167687B (en) |
| TW (1) | TWI436764B (en) |
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| CN103030620B (en) * | 2010-02-19 | 2014-10-22 | 株式会社安露莎 | Drug, food, or cosmetic which has anti-obesity agent and fat accumulation inhibiting action |
| WO2017006407A1 (en) * | 2015-07-04 | 2017-01-12 | 株式会社Ａｏｂ慧央グループ | Nerve cell death inhibitor, anti-alzheimer's disease agent, brain hypofunction inhibitor, drug or food having anti-alzheimer's disease effect or brain hypofunction-inhibiting effect, and method for producing nerve cell death inhibitor |
| CN115385795B (en) * | 2022-10-11 | 2023-11-17 | 广西中医药大学赛恩斯新医药学院 | Preparation method and application of Rhinacanthus nasuta (L.) kurz naphthoquinone monomer Rhinacanthin-C |
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| JP2002053481A (en) * | 2000-08-10 | 2002-02-19 | Arsoa Honsya Corp | Anti-malignant tumor agent |
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| US7618662B2 (en) * | 2004-12-22 | 2009-11-17 | Avon Products, Inc | Use of natural plant extracts in cosmetic compositions |
| US8221766B2 (en) * | 2004-12-22 | 2012-07-17 | Avon Products, Inc. | Use of plant extracts to prevent and/or reduce the signs of subjective discomfort and/or irritation in the topical application of cosmetic products |
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| JP2002053481A (en) * | 2000-08-10 | 2002-02-19 | Arsoa Honsya Corp | Anti-malignant tumor agent |
Non-Patent Citations (1)
| Title |
|---|
| 王乃平等: "白鹤灵芝的研究进展", 《上海中医药杂志》, vol. 41, no. 4, 30 April 2007 (2007-04-30), pages 77 - 79 * |
Also Published As
| Publication number | Publication date |
|---|---|
| CN102167687B (en) | 2014-03-12 |
| CN102167687A (en) | 2011-08-31 |
| TWI436764B (en) | 2014-05-11 |
| CN103467293B (en) | 2015-06-10 |
| JP5620847B2 (en) | 2014-11-05 |
| TW201129357A (en) | 2011-09-01 |
| JP2011190250A (en) | 2011-09-29 |
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