CN103467293B - 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 divisional application of the application's denomination of invention in be the applying date be on February 17th, 2011 to be the application number of " novel cpd, antineoplastic agent and pharmaceuticals, food or makeup " be 201110040985.7 patent applications.
Technical field
The present invention relates to novel cpd and novel cpd and there is in preparation application in the pharmaceuticals of antitumor action, food or makeup.
Background technology
Antineoplastic agent carcinostatic agent carcinostatic agent is select optionally to act on tumour cell and the preparation less to normal cytotoxicity substantially, but also have the problem on a lot of use to be resolved not yet, the appearance of the fatal side effects such as the leukopenia such as caused by bone marrow depression, thrombopenia, Neutrophilic granulocytopenia, 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 being difficult to oral administration, route of administration is restricted.Therefore, expect to develop and optionally act 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, there will be a known a large amount of compounds, such as, have the report that 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) etc. are a large amount of.In addition, as the antineoplastic agent taking natural goods as lead compound, there will be a known D51-7059 (such as with reference to patent documentation 3), Taxane derivative (such as with reference to patent documentation 4) etc.The content of the antineoplastic agent of naphthoquinones system that is relevant to this compound or that be correlated with the biosynthesizing system of this compound, decomposing solution as phone predicts, has the report (such as with reference to non-patent literature 2) about rhinacanthin A, B, C, D, G, H, I, K, M, N, the Q from rhinacanthus nasuta Kurz (Rhinacanthus nasutus (L.) Kurz).In addition, as the synthetic compound relevant to naphthoquinones system antineoplastic agent, there will be a known the antineoplastic agent (such as with reference to patent documentation 5 ~ 8) relevant to furans naphthoquinone derivatives.
On the other hand, prove containing " following novel cpd A according to the present invention, 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 (below sometimes also referred to as rhinacanthus nasuta Kurz, Rhinacanthus nasutus (L.) Kurz), belong to the Twig and leaf of Bignose Rhinacanthus being considered to originate in South India Deccan Traps (Deccan Plateau) to belong to acanthaceous evergreen brushwood, its herb known has expelling parasite, anti-inflammatory, to the anti-microbial effect (such as with reference to non-patent literature 3) of dermatophytes, main in China's Mainland, the ground such as TaiWan, China use as Chinese medicine, and also use as Chinese medicine in Japan recently.In addition, disclose in applicant application in the past Twig and leaf of Bignose Rhinacanthus have Scavenger of ROS ability (such as with reference to patent documentation 9), there is promotion Excretion (such as with reference to patent documentation 10), there is anti-allergic effects (such as with reference to patent documentation 11) and antitumor action (such as with reference to patent documentation 12).
Prior art document
Patent documentation 1 Japanese Patent No. 3483257 publication
Patent documentation 2 Japanese Patent No. 4296312 publication
Patent documentation 3 Japanese Patent Publication 6-51689 publication
Patent documentation 4 Japanese Patent No. 3942197 publication
Patent documentation 5 Japanese Unexamined Patent Publication 9-235280 publication
Patent documentation 6 Japanese Unexamined Patent Publication 9-252753 publication
Patent documentation 7 Japanese Unexamined Patent Publication 11-21284 publication
Patent documentation 8 Japanese Unexamined Patent Publication 2005-220037 publication
Patent documentation 9 Japanese Unexamined Patent Publication 9-143091 publication
Patent documentation 10 Japanese Unexamined Patent Publication 9-169662 publication
Patent documentation 11 Japanese Unexamined Patent Publication 2001-10964 publication
Patent documentation 12 Japanese Unexamined Patent Publication 2002-53481 publication
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 herd the large illustrated handbook of wild oriental drug grass (Japanese original name: primary colors herds 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), containing 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 containing 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 object of this invention is to provide: do not worry its side effect, safety and there are novel cpd, the antineoplastic agent containing this novel cpd and the pharmaceuticals with antitumor action containing this novel cpd, food or the makeup of excellent antitumor action.
Present inventor carries out the result of searching for for the novel cpd with antitumor action, find, in 7 novel cpds (novel cpd A, novel cpd B, novel cpd C, novel cpd D, novel cpd E, novel cpd F or novel cpd G) obtained from Twig and leaf of Bignose Rhinacanthus, there is the excellent inhibited proliferation for the melanoma cell strain (HMV-II) from human body, complete the present invention.In order to solve above-mentioned problem, the present invention is made up of following item.
The present invention relates to " novel cpd (below this novel cpd being called novel cpd A) represented by following formula (1) ", " antineoplastic agent containing novel cpd A " and " pharmaceuticals with antitumor action containing novel cpd A, food or makeup ".
In addition, the present invention relates to " novel cpd (below this novel cpd being called novel cpd B) represented by following formula (2) ", " antineoplastic agent containing novel cpd B " and " pharmaceuticals with antitumor action containing novel cpd B, food or makeup ".
In addition, the present invention relates to " novel cpd (below this novel cpd being called novel cpd C) represented by following formula (3) ", " antineoplastic agent containing novel cpd C " and " pharmaceuticals with antitumor action containing novel cpd C, food or makeup ".
In addition, the present invention relates to " novel cpd (below this novel cpd being called novel cpd D) represented by following formula (4) " and " novel cpd D has the application in the pharmaceuticals of antitumor action, food or makeup in preparation ".
In addition, the present invention relates to " novel cpd (below this novel cpd being called novel cpd E) represented by following formula (5) " and " novel cpd E has the application in the pharmaceuticals of antitumor action, food or makeup in preparation ".
In addition, the present invention relates to " novel cpd (below this novel cpd being called novel cpd F) represented by following formula (6) " and " novel cpd F has the application in the pharmaceuticals of antitumor action, food or makeup in preparation ".
And then, the invention still further relates to " novel cpd (below this novel cpd being called novel cpd G) represented by following formula (7) " and " novel cpd G has the application in the pharmaceuticals of antitumor action, food or makeup 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), containing 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 containing 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 composition of the natural goods (Twig and leaf of Bignose Rhinacanthus) that the raw material of makeup just used from the past always, so do not worry its side effect, safety, and can distinguish that from following test example there is excellent antitumor action.
Accompanying drawing explanation
Fig. 1 is the schema distinguished from Twig and leaf of Bignose Rhinacanthus root when being separated 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).
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), Twig and leaf of Bignose Rhinacanthus can be obtained by extraction purification operation as raw material, also can obtain in other plant materials.In addition, also can use by synthesizing 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.But also rhinacanthus nasuta Kurz can be used, from other plant materials containing novel cpd (novel cpd A, novel cpd B, novel cpd C, novel cpd D, novel cpd E, novel cpd F or novel cpd G), extract the extract obtained, thick purified or the dry thing of plant materials, the slurry of plant materials.
From the plant materialss such as rhinacanthus nasuta Kurz time extraction purification 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), any extraction purification operation of usual industrial use can be used.After as the leaf, stem, root, flower etc. of raw material, the phase gathers in due course, directly or implement the drying process of usual air seasoning etc., extraction raw material is made.When extracting 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, extract with solvent, as Extraction solvent, can by water, the alcohols such as ethanol, methyl alcohol, Virahol, the ketone such as acetone, methyl ethyl ketone, the ester such as methyl acetate, ethyl acetate class, the oil loving solvent such as hexane, chloroform is used alone or is formed mixed solvent and uses.Extracting temperature is generally 0 ~ 100 DEG C, is preferably 5 ~ 50 DEG C.Extraction time is 1 hour ~ about 10 days, and quantity of solvent is that on average every part of dried feed is generally 1 ~ 30 times of weight, is preferably 5 ~ 10 times of weight.Extraction operation, by stirring, is also undertaken by dipping placement.Extraction operation repeatedly can carry out 2 ~ 3 times.By filtering or centrifugation and removing 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 from the crude extract obtained with aforesaid operations, as long as known crude drug separation purification method, any method can be adopted, but be preferably used alone or in combination two-phase solvent apportion design, counter-current distribution method, column chromatography, preparative high performance liquid chromatography etc.Such as two-phase solvent apportion design, can exemplify by distributing in normal hexane, chloroform, methyl ethyl ketone, ethyl acetate, methyl acetate equal solvent and water, thus target compound is recovered to the method etc. in solvent phase from said extracted liquid.As column chromatography, ion exchange column chromatography method, use positive or the reverse phase silica gel modification dextrane gel as the method for carrier, the Adsorption column chro-matography using DIAION HP-20 etc., use SephadexLH-20 etc. can be exemplified as the gel filtration method etc. of carrier, these methods can be used alone or in combination or use repeatedly.As preparative high performance liquid chromatography, the method for the reversed-phase column using octadecyl silicon etc. can be exemplified, use the method etc. of the normal phase column of silica gel etc.
As the route of administration of antineoplastic agent of the present invention, be not particularly limited, such as, can exemplify administration in the intestines such as oral administration drop rectum with drug, the mucosa delivery of nose administration etc., the drug administration by injection etc. of intravenous administration subcutaneous administration etc.As the formulation of antineoplastic agent of the present invention, form that is any and the matched preparation of medication can be taked, 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 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 excipient pharmacologically allowed together administration.As excipient, as long as monose, disaccharides, polyose, inorganic salts, grease, distilled water etc. are as the usual spendable excipient of preparation, any excipient can be used.When carrying out formulation, 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), different according to the difference at the symptom of route of administration, formulation, disease, the age of object etc., be generally and be 0.1 ~ 1000mg, be preferably 0.5 ~ 300mg, more preferably 1 ~ 100mg every day for each person.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 oral antineoplastic agent of the present invention, can set and the active constituent content in the optimal preparation of each administration form according to the data of the effective dosage of the form of preparation as the dosage of preparation.
As the form of food, can exemplify by rhinacanthus nasuta Kurz and containing 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, from the crude extract containing this novel cpd extracted the plant of this novel cpd, plant materials slurry containing this novel cpd, the food etc. of the dry thing of the plant materials containing this novel cpd.
As tea, can separately or used in combination with other tea raw materials.As other tea raw material, as 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. usually used as the edible raw material of tea, any tea raw material can be used.
When obtaining plant milk extract, as long as by hot water extraction, by spendable method in ethanol, aqueous ethanol extraction etc. usually food extraction, any method can be used.From plant materials, 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) are obtained by well-established law.
As the form with the food of antitumor action of the present invention, except tea, as long as nourishing drink, jelly, biscuit, tablet, pill, soft capsule, hard capsule, powder, granula subtilis, granule etc., usually used as the available form of food, can use any form.As auxiliary material, also the additives such as excipient, tackiness agent, lubricant, dispersion agent, suspension agent, emulsifying agent, thinner, buffer reagent, antioxidant, bacterial inhibitor can be used.
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, different according to the difference at picked-up form, the state of health of object, the age of object etc., but being generally every day is for each person 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, different from the difference of food form, but be generally 0.0001 ~ 1wt%, be preferably 0.001 ~ 0.5wt%, be more preferably 0.01 ~ 0.1wt%.
As the example of 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, can exemplify 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), other medical components can be contained as required, in addition, also the additives such as tackiness agent, dispersion agent, suspension agent, emulsifying agent, thinner, buffer reagent, antioxidant, bacterial inhibitor can be used.
As the form of makeup, astringent, beauty liquid, emulsion, breast frost, gel, facial mask, beauty treatment muffin, cleansing milk, baths etc. can be used as the spendable any form of external preparation cosmetic formulations.In above-mentioned makeup, on the basis of the required compositions such as 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, the crude extract of this novel cpd extracted from plant or the plant materials containing this novel cpd, also can as required containing the composition that can coordinate in cosmetic formulations.As gradation composition, such as, can exemplify 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.
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) used time outside, according to the symptom of object, the difference at the age of object and different, but being generally every day is for each person 0.001 ~ 100mg, be preferably 0.01 ~ 10mg, more preferably 0.1 ~ 1mg.
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 antineoplastic agent of the present invention, pharmaceuticals, makeup, separately or be generally 0.0001 ~ 1wt% as during mixture, be preferably 0.001 ~ 0.5wt%, be more preferably 0.01 ~ 0.1wt%.
Below exemplify each novel cpd of the present invention and as the differentiation separation example of the Rhinacanthin C of comparative example, the test example of antitumor action and embodiment, the present invention is illustrated in further detail, but the present invention be not limited to these.
1. each novel cpd of the present invention and the differentiation as the Rhinacanthin C of comparative example are separated example
1-1. novel cpd A
(1) differentiation of novel cpd A is separated
The differentiation of novel cpd A is separated 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, the extraction that each 24 hours amount to 3 times is carried out to Twig and leaf of Bignose Rhinacanthus (Rhinacanthus nasutus (L.) Kurz) dry root (5Kg), concentrate after these are combined, obtain dry substance (407g).Then, after making its dissolving that suspends in 90% (v/v) methyl alcohol of 7L, carry out 3 sub-distribution with the hexane of equivalent, then take out 90% (v/v) methanol phase and carry out 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.Then the chloroform obtained by this operation is combined mutually, obtain dry substance 69.3g.
By wherein 69.0g supply using hexane/ethyl acetate as eluting solvent silica gel column chromatography (
kanto Kagaku K. K.'s system).That is: after successively silicagel column being washed by the eluting solvent hexane/ethyl acetate (7:3) of the eluting solvent hexane/ethyl acetate (9:1) of 3 bed volumes (BV), the eluting solvent hexane/ethyl acetate (8:2) of 2BV and 2BV, use eluting solvent hexane/ethyl acetate (6:4) wash-out of 2BV again, obtain component C (dry substance 3.73g).
Then, for component C, implement using methyl alcohol as eluting solvent Sephadex LH-20 column chromatography (
pharmacia Inc.).That is:, after being washed by post with the methyl alcohol of 2BV, by the methanol-eluted fractions of 1BV, component F (dry substance 369mg) is obtained.
Then, by component F (dry substance 369mg) supply using water/methyl alcohol as eluting solvent Flash ODS column chromatography (
chemical company of wild village system).That is: after Flash ODS post being washed with 50% (v/v) methyl alcohol of 180ml, after using 60% (v/v) methyl alcohol, 70% (v/v) methanol wash step by step successively, by 80% (v/v) methanol-eluted fractions, obtain the component G (dry substance 75.6mg) containing target novel cpd A.
And then, with preparative high performance liquid chromatography (ODS post,
chemical company of wild village system, moving phase: 45% (v/v) acetonitrile, detection: 254nm UV detector) purifying is carried out to component G (dry substance 75.6mg), obtain novel cpd A (dry substance 9.8mg).
In addition, in above-mentioned, high performance liquid chromatography employs Waters 515 system and Waters 600 system (being Japanese Waters Co., Ltd. system).In addition, silica gel column chromatography, Sephadex LH-20 column chromatography and Flash ODS chromatography use usual experimental apparatus and experimental installation.
(2) structural analysis of novel cpd A
The structural analysis of novel cpd A use high resolution mass spectrum analytical method (HRFABMS) and nuclear magnetic resonance spectrometry (
1h NMR,
13c NMR) carry out.Below represent result.
(2-1) result of high resolution mass spectrum analytical method (HRFABMS)
In high resolution mass spectrum analytical method (HRFABMS), there is " m/z 272.1039 [M]
+(calcd.272.1049 Δ 0.9mmu). ", known molecular formula is " C
16h
16o
4".In addition, in Low Resolution Mass Spectra analytical method (LRFABMS), occurred " m/z 272. ".
(2-2) nuclear magnetic resonance spectrometry (
1h NMR) result
Nuclear magnetic resonance spectrometry (
1h NMR) in, there is following spectrum peak.
“
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, there is following spectrum peak.
“
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 high resolution mass spectrum analytical equipment, JEOL JMS SX-102 type mass spectrometer (Jeol Ltd.'s system) is used.In addition, as NMR (Nuclear Magnetic Resonance) spectrum device (
1h NMR and
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) represents.
1-2. novel cpd B, C
(1) differentiation of novel cpd B, C is separated
The differentiation of novel cpd B, C is separated, and carries out according to the flow process shown in Fig. 1.That is: at room temperature, with 90% (v/v) ethanol of 25L, the extraction that each 24 hours amount to 3 times is carried out to Twig and leaf of Bignose Rhinacanthus (Rhinacanthus nasutus (L.) Kurz) dry root (5Kg), concentrate after these are combined, obtain dry substance (407g).Then, after making its dissolving that suspends in 90% (v/v) methyl alcohol of 7L, carry out 3 sub-distribution with the hexane of equivalent, then take out 90% (v/v) methanol phase and carry out 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.Then the chloroform obtained by this operation is combined mutually, obtain dry substance 69.3g.
By wherein 69.0g supply using hexane/ethyl acetate as eluting solvent silica gel column chromatography (
kanto Kagaku K. K.'s system).That is:, after being washed by silicagel column by the eluting solvent hexane/ethyl acetate (9:1) of 3BV, with eluting solvent hexane/ethyl acetate (8:2) wash-out of 1BV, component A (dry substance 4.71g) is obtained.
Then, by component A supply using methyl alcohol as eluting solvent Sephadex LH-20 column chromatography (
pharmacia Inc.).That is:, after being washed by Sephadex LH-20 post with the methyl alcohol of 1.5BV, by the methanol-eluted fractions of 0.5BV, component A-2 (1.34g) is obtained.
And then, with using water/methyl alcohol as eluting solvent Flash ODS column chromatography (
with Guang Chun medicine Inc.) distinguish component A-2.That is: after Flash ODS post being washed with 50% (v/v) methyl alcohol of 180ml, after using 60% (v/v) methyl alcohol, 70% (v/v) methanol wash step by step successively, by 80% (v/v) methanol-eluted fractions, obtain the component A-2-1 (dry substance 261mg) containing target novel cpd B, C.
Component A-2-1 further with using hexane/ethyl acetate as eluting solvent silica gel column chromatography (
kanto Kagaku K. K.'s system) distinguish.That is:, after being washed by silicagel column with hexane/ethyl acetate (9:1) eluting solvent of 3BV, with hexane/ethyl acetate (8:2) the eluting solvent wash-out of 1BV, component D (dry substance 92.4mg) is obtained.
And then, with preparative high performance liquid chromatography (ODS post,
chemical company of wild village system, moving phase: 58% (v/v) acetonitrile/water/0.1% (v/v) formic acid, detect: 254nm UV detector) purifying is carried out to component D (dry substance 92.4mg), 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 of novel cpd B, C use high resolution mass spectrum analytical method (HRFABMS) and nuclear magnetic resonance spectrometry (
1h-NMR,
13c-NNMR) carry out.Below represent its result.
(2-1) result of high resolution mass spectrum analytical method (HRFABMS)
(2-1-1) novel cpd B
For novel cpd B, there is " m/z 256.1099 [M]
+(calcd.256.1010 Δ 0mmu). ", known molecular formula is " C
16h
16o
3".In addition, in LRFABMS, occurred " m/z 256. ", in LREIMS, occurred " m/z 256. ".
(2-1-2) novel cpd C
For novel cpd C, there is " m/z 270.1266 [M]
+(calcd.270.1256 Δ 1.0mmu). ", its molecular formula known is " C
17h
18o
3".In addition, in LRFABMS, occurred " m/z 270. ".(2-2) nuclear magnetic resonance spectrometry (
1h NMR) result
(2-2-1) novel cpd B
For novel cpd B, there is following spectrum peak.
“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, there is following spectrum peak.
“
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, there is following spectrum peak.
“
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, there is following spectrum peak.
“
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) represents, novel cpd C is the novel cpd that above-mentioned formula (3) represents.
1-3. novel cpd D, E
(1) differentiation of novel cpd D, E is separated
The differentiation of novel cpd D, E is separated 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, the extraction that each 24 hours amount to 3 times is carried out to Twig and leaf of Bignose Rhinacanthus (Rhinacanthus nasutus (L.) Kurz) dry root (5Kg), concentrate after these are combined, obtain dry substance (407g).Then, after making its dissolving that suspends in 90% (v/v) methyl alcohol of 7L, after carrying out 3 sub-distribution with the hexane of equivalent, take out 90% (v/v) methanol phase and carry out 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.Then the chloroform obtained by this operation is combined mutually, obtain dry substance 69.3g.
By wherein 69.0g supply using hexane/ethyl acetate as eluting solvent silica gel column chromatography (
kanto Kagaku K. K.'s system).That is: after successively post being washed by the eluting solvent hexane/ethyl acetate (9:1) of 3BV and the eluting solvent hexane/ethyl acetate (8:2) of 2BV, use eluting solvent hexane/ethyl acetate (7:3) wash-out of 2BV again, obtain B component (dry substance 5.66g).
Then, for B component, methyl alcohol is implemented as eluting solvent Sephadex LH-20 column chromatography (
pharmacia Inc.).That is:, after being washed by post with the methyl alcohol of 2.5BV, by the methanol-eluted fractions of 1BV, B component-2 (dry substance 480mg) is obtained.
Then, by B component-2 (dry substance 480mg) supply using water/methyl alcohol as eluting solvent Flash ODS column chromatography (
chemical company of wild village system, that is: after Flash ODS post being washed with 50% (v/v) methyl alcohol of 180ml, after using 60% (v/v) methyl alcohol, 70% (v/v) methyl alcohol, 80% (v/v) methanol wash step by step successively, use 90% (v/v) methanol-eluted fractions again, obtain the component X (dry substance 143mg) containing target novel cpd D, E.
And then, with silica gel column chromatography (
kanto Kagaku K. K.'s system) component X (dry substance 143mg) is distinguished.That is: after silicagel column being washed by the eluting solvent hexane/ethyl acetate (8:2) of 2BV, with eluting solvent hexane/ethyl acetate (7:3) wash-out of 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 (ODS post,
chemical company of wild village system, moving phase: 58% (v/v) acetonitrile, detects: 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 (ODS post,
chemical company of wild village system, moving phase: 70% (v/v) methyl alcohol, detects: 280nm UV detector), use further preparative high performance liquid chromatography (ODS post,
chemical company of wild village system, moving phase: 55% (v/v) acetonitrile, detects: 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 of novel cpd D, E use high resolution mass spectrum analytical method (HRFABMS) and nuclear magnetic resonance spectrometry (
1h NMR,
13c NMR) carry out.Below represent its result.
(2-1) result of high resolution mass spectrum analytical method (HRFABMS)
(2-1-1) novel cpd D
For novel cpd D, there is " m/z 397.1651 [M+H]
+(calcd.397.1651 Δ 0.0mmu). ", known molecular formula is " C
23h
24o
6".
(2-1-2) novel cpd E
For novel cpd E, there is " m/z 441.1919 [M+H]
+(calcd.441.1913 Δ 0.5mmu). ", 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, there is following spectrum peak.
“
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, there is following spectrum peak.
“
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) represents, novel cpd E is the novel cpd that above-mentioned formula (5) represents.
1-4. novel cpd F
(1) differentiation of novel cpd F is separated
The differentiation of novel cpd F is separated 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, the extraction that each 24 hours amount to 3 times is carried out to Twig and leaf of Bignose Rhinacanthus (Rhinacanthus nasutus (L.) Kurz) dry root (5Kg), concentrate after these are combined, obtain dry substance (407g).Then, after making its dissolving that suspends in 90% (v/v) methyl alcohol of 7L, after carrying out 3 sub-distribution with the hexane of equivalent, take out 90% (v/v) methanol phase and carry out 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.Then the chloroform obtained by this operation is combined mutually, obtain dry substance 69.3g.
By wherein 69.0g supply using hexane/ethyl acetate as eluting solvent silica gel column chromatography (
kanto Kagaku K. K.'s system).That is: after successively post being washed by the eluting solvent hexane/ethyl acetate (9:1) of 3BV and the eluting solvent hexane/ethyl acetate (8:2) of 2BV, with eluting solvent hexane/ethyl acetate (7:3) wash-out of 2BV, obtain B component (dry substance 5.66g).
Then, for B component (dry substance 2.8g), methyl alcohol is implemented as eluting solvent SephadexLH-20 column chromatography (
pharmacia Inc.).That is:, after being washed by post with the methyl alcohol of 1.5BV, by the methanol-eluted fractions of 0.5BV, B component-1 (dry substance 1.16g) is obtained.
Then, by B component-1 (dry substance 1.1g) supply using water/methyl alcohol as eluting solvent Flash ODS column chromatography (
chemical company of wild village system).That is: after Flash ODS post being washed with 50% (v/v) methyl alcohol of 180ml, use 60% (v/v) methyl alcohol, 70% (v/v) methanol wash step by step successively, then use 80% (v/v) methanol-eluted fractions, obtain the B component-1-2 (dry substance 429mg) containing target novel cpd F.
Then, with using hexane/ethyl acetate as eluting solvent silica gel column chromatography (
kanto Kagaku K. K.'s system) B component-1-2 (dry substance 429mg) is distinguished.That is:, after being washed by silicagel column by the eluting solvent hexane/ethyl acetate (8:2) of 2BV, with eluting solvent hexane/ethyl acetate (7:3) wash-out of 1BV, B component-1-2-2 (dry substance 32.9mg) is obtained.
With preparative high performance liquid chromatography (ODS post,
chemical company of wild village system, moving phase: 55% (v/v) acetonitrile, detect: 280nm UV detector) purifying is carried out to B component-1-2-2 (dry substance 32.9mg), by use further preparative high performance liquid chromatography (C-30 post,
chemical company of wild village system, moving phase: 55% (v/v) acetonitrile, detects: 254nm UV detector) carry out purifying, obtain novel cpd F (dry substance 10.3mg).
(2) structural analysis of novel cpd F
The structural analysis of novel cpd F use high resolution mass spectrum analytical method (HRFABMS) and nuclear magnetic resonance spectrometry (
1h NMR,
13c NMR) carry out.Below represent its result.
(2-1) result of high resolution mass spectrum analytical method (HRFABMS)
In high resolution mass spectrum analytical method (HRFABMS), there is " m/z 459.2379 [M+H]
+(calcd.459.2383 Δ 0.4mmu). ", known molecular formula is " C
26h
34o
7".
(2-2) nuclear magnetic resonance spectrometry (
1h NMR) result
Nuclear magnetic resonance spectrometry (
1h NMR) in, there is following spectrum peak.
“
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, there is following spectrum peak.
“
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) represents.
1-5. novel cpd G
(1) differentiation of novel cpd G is separated
The differentiation of novel cpd G is separated 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, the extraction that each 24 hours amount to 3 times is carried out to Twig and leaf of Bignose Rhinacanthus (Rhinacanthus nasutus (L.) Kurz) dry root (5Kg), concentrate after these are combined, obtain dry substance (407g).Then, after making its dissolving that suspends in 90% (v/v) methyl alcohol of 7L, after carrying out 3 sub-distribution with the hexane of equivalent, take out 90% (v/v) methanol phase and carry out 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.Then the chloroform obtained by this operation is combined mutually, obtain dry substance 69.3g.
By wherein 69.0g supply using hexane/ethyl acetate as eluting solvent silica gel column chromatography (
kanto Kagaku K. K.'s system).That is: after successively post being washed by the eluting solvent hexane/ethyl acetate (9:1) of 3BV and the eluting solvent hexane/ethyl acetate (8:2) of 2BV, with eluting solvent hexane/ethyl acetate (7:3) wash-out of 2BV, obtain B component (dry substance 5.66g).
Then, for B component (dry substance 2.8g), implement using methyl alcohol as eluting solvent SephadexLH-20 column chromatography (
pharmacia Inc.).That is:, after being washed by post with the methyl alcohol of 1.5BV, by the methanol-eluted fractions of 0.5BV, B component-1 (dry substance 1.16g) is obtained.
And then, by B component-1 (dry substance 1.16g) supply using water/methyl alcohol as eluting solvent FlashODS column chromatography (
chemical company of wild village system).That is: after Flash ODS post being washed with 50% (v/v) methyl alcohol of 180ml, after using 60% (v/v) methyl alcohol, 70% (v/v) methanol wash step by step successively, by 80% (v/v) methanol-eluted fractions, obtain the B component-1-1 (dry substance 319mg) containing target novel cpd G.
And then, with using hexane/ethyl acetate as eluting solvent silica gel column chromatography (
kanto Kagaku K. K.'s system) B component-1-1 (dry substance 319mg) is distinguished.That is:, after being washed by silicagel column by the eluting solvent hexane/ethyl acetate (9:1) of 2BV, with eluting solvent hexane/ethyl acetate (8:2) wash-out of 1BV, component E (dry substance 114mg) is obtained.
And then, with preparative high performance liquid chromatography (ODS post,
chemical company of wild village system, moving phase: 68% (v/v) methyl alcohol, detects: 254nm UV detector) purifying is carried out to component E (dry substance 114mg), obtain novel cpd G (dry substance 3.6mg).
(2) structural analysis of novel cpd G
The structural analysis of novel cpd G use high resolution mass spectrum analytical method (HRFABMS) and nuclear magnetic resonance spectrometry (
1h NMR,
13c-NNMR) carry out.Below represent its result.
(2-1) result of high resolution mass spectrum analytical method (HRFABMS)
In high resolution mass spectrum analytical method (HRFABMS), there is " m/z 399.1813 [M+H]
+(calcd.399.1808 Δ 0.5mmu). ", known molecular formula is " C
23h
26o
6".
(2-2) nuclear magnetic resonance spectrometry (
1h NMR) result
Nuclear magnetic resonance spectrometry (
1h NMR) in, there is following spectrum peak.
“
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, there is following spectrum peak.
“
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) represents.
1-6. Rhinacanthin C (comparative example)
(1) differentiation of Rhinacanthin C is separated
The differentiation of Rhinacanthin C is separated 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, the extraction that each 24 hours amount to 3 times is carried out to Twig and leaf of Bignose Rhinacanthus (Rhinacanthus nasutus (L.) Kurz) dry root (5Kg), concentrate after these are combined, obtain dry substance (407g).Then, after making its dissolving that suspends in 90% (v/v) methyl alcohol of 7L, after carrying out 3 sub-distribution with the hexane of equivalent, take out 90% (v/v) methanol phase and carry out 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.Then the chloroform obtained by this operation is combined mutually, obtain dry substance 69.3g.
By wherein 69.0g supply using hexane/ethyl acetate as eluting solvent silica gel column chromatography (
kanto Kagaku K. K.'s system).That is: from the component eluted by the eluting solvent hexane/ethyl acetate (9:1) of 3BV, obtain the Rhinacanthin C (dry substance 5.66g) represented with following formula (8).
In addition, for being distinguished the Rhinacanthin C be separated by aforesaid method, nuclear magnetic resonance spectrometry is utilized to obtain
1h NMR spectroscopic data (CDCl
3), there is following spectrum peak 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 measured for the melanoma cell strain HMV-II from human body.
Use the Ham F12 substratum containing 10% (v/v) foetal calf serum, with 1 × 10 on 96 hole culture plates (NUNC)
4the cell concn inoculation of cells/90 μ l from the melanoma cell strain HMV-II of human body, 37 DEG C, under 5% carbon dioxide exists, carry out cultivating for 24 hours.Cultivate after 24 hours, adding novel cpd A of the present invention, novel cpd B, novel cpd D, novel cpd F or novel cpd G (is dissolved in DMSO, final DMSO concentration=0.1% (v/v) in substratum, in addition, by cultured cells strain on the substratum of DMSO only adding 1/1000 volume as a control group), so 37 DEG C, 5% carbon dioxide exist under cultivate 24 hours.The mensuration of relative growth rate uses tetrazolium bromide (MTT) [3-(4,5-dimethylthiazole-2-base)-2,5-phenylbenzene bromination tetrazole Nacalai Tesque] [(3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide)] method carry out [with reference to Co., Ltd. Tokyo chemistry with people's distribution, new biochemical experiment lecture 12 molecular immunology I immune cells factor 358-359 page].
That is: add after above-mentioned each novel cpd cultivates 24 hours, at 96 hole culture plate (0.33cm
2/ well) each hole nutrient solution 90 μ l in add the MTT solution { 5mg/ml of 10 μ l; After dissolving in without calcium magnesium PBS [Du Shi phosphoric acid buffer (Dulbecco ' s Phosphate-Buffered Saline)] solution, solution after film filter (0.22 μm) filters }, carrying out vibration makes it even, 37 DEG C, 5% carbon dioxide cultivates 4 hours under existing.Cultivate after 4 hours in each hole, add 10% (w/v) SDS-50% (v/v) N, dinethylformamide-0.005N hydrochloric acid soln 100 μ l, 37 DEG C, 5% carbon dioxide leaves standstill after 18 hours under existing, use immune detector (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.
Each novel cpd (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.
[table 1]
For the proliferation inhibition activity from human melanoma's 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 " the excellent proliferation inhibition activity to the melanoma cell strain (HMV-II) from human body " equal with the Rhinacanthin C with excellent antitumor action.
Embodiment
the making of embodiment 1. tablet
Use and carry out distinguishing the novel cpd A be separated according to described method in above-mentioned " differentiation of 1-1. novel cpd A (1) novel cpd A is separated ", make tablet (every 1 500mg) with following prescription.
(preparation method)
In lactose (95.8g), interpolation novel cpd A (0.2g), dried corn starch (2g), talcum (1.8g), calcium stearate (0.2g) mix.Then, use Singlepunchtabletpress, make tablet according to well-established law.
the making of embodiment 2. hard capsule
Using the method according to recording in above-mentioned " differentiation of 1-2. novel cpd B, C (1) novel cpd B, C is separated " to carry out distinguishing the novel cpd B be separated, making hard capsule (on average every 1 360mg) with following prescription.
(preparation method)
Add lactose (220g) and W-Gum (110g) in the novel cpd B (5g) and mix, mediating after adding the aqueous solution of hydroxypropylcellulose (25g) wherein.Then, Squeezinggranulator is used to make particle by well-established law.By this is particles filledly made hard capsule in snap fit capsule.
the making of embodiment 3. soft capsule
Using the method according to recording in above-mentioned " differentiation of 1-2. novel cpd B, C (1) novel cpd B, C is separated " to carry out distinguishing the novel cpd C be separated, making soft capsule (on average every 1 170mg) with following prescription.
Novel cpd C 0.5mg
Soybean oil 169.5mg
(preparation method)
In soybean oil (169.5g), add novel cpd C (0.5g) mix.Then soft capsule is made by using rotary-die type automatic molder to be filled in soft capsule according to well-established law.
the making of embodiment 4. pill
Using the method according to recording in above-mentioned " differentiation of 1-3. novel cpd D, E (1) novel cpd D, E is separated " to carry out distinguishing the novel cpd D be separated, making pill (on average every 1 100mg) with following prescription.
(preparation method)
With said ratio, raw material is mixed, after adding water in right amount, with the uniform kneaded material of kneader manufacture, use rotary pellet press will to carry out drying after the kneaded material pill obtained thus make pill.
the making of embodiment 5. pill
Using the method according to recording in above-mentioned " differentiation of 1-3. novel cpd D, E (1) novel cpd D, E is separated " to carry out distinguishing the novel cpd E be separated, making pill (on average every 1 100mg) with following prescription.
(preparation method)
With said ratio, raw material is mixed, after adding water in right amount, with the uniform kneaded material of kneader manufacture, use rotary pellet press will to carry out drying after the kneaded material pill obtained thus make pill.
the making of embodiment 6. ointment
Using the method according to recording in above-mentioned " differentiation of 1-4. novel cpd F (1) novel cpd F is separated " to carry out distinguishing the novel cpd F be separated, making ointment with following prescription.
(oil-phase component)
(preparation method)
Oil-phase component and water-phase component are heated to respectively at 80 DEG C evenly, in oil phase, add aqueous phase while stirring, cool after emulsification, make ointment thus.
the making of embodiment 7. astringent
Using the method according to recording in above-mentioned " differentiation of 1-5. novel cpd G (1) novel cpd G is separated " to carry out distinguishing the novel cpd G be separated, making astringent with following prescription.
(oil-phase component)
(preparation method)
By oil-phase component and water-phase component uniform dissolution respectively, by adding oil phase while stirring in aqueous phase, prepare astringent.
Above according to above-mentioned embodiment to novel cpd of the present invention, antineoplastic agent and there are the pharmaceuticals of antitumor action, food or makeup be illustrated, but the present invention is not limited to above-mentioned embodiment, not departing from the scope of its main idea, can implement in various mode.
Claims (12)
1. compound, is characterized in that, represents by following formula (4),
2. compound, is characterized in that, represents by following formula (5),
3. compound, is characterized in that, represents by following formula (6),
4. compound, is characterized in that, represents by following formula (7),
5. compound according to claim 1 is preparing the application in pharmaceuticals, food or the makeup with antitumor action.
6. application according to claim 5, is characterized in that, described in there is antitumor action pharmaceuticals are antineoplastic agents.
7. compound according to claim 2 is preparing the application in pharmaceuticals, food or the makeup with antitumor action.
8. application according to claim 7, is characterized in that, described in there is antitumor action pharmaceuticals are antineoplastic agents.
9. compound according to claim 3 is preparing the application in pharmaceuticals, food or the makeup with antitumor action.
10. application according to claim 9, is characterized in that, described in there is antitumor action pharmaceuticals are antineoplastic agents.
11. compounds according to claim 4 have the application in the pharmaceuticals of antitumor action, food or makeup in preparation.
12. application according to claim 11, is characterized in that, described in there is antitumor action pharmaceuticals are antineoplastic agents.
Applications Claiming Priority (2)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| JP2010-034621 | 2010-02-19 | ||
| JP2010034621 | 2010-02-19 |
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| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| 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 CN103467293A (en) | 2013-12-25 |
| CN103467293B true 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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| Country | Link |
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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 |
| CN107708717B (en) * | 2015-07-04 | 2021-05-14 | 株式会社奥碧慧央集团 | Application of rhinacanthin quinone C as nerve cell apoptosis 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 |
| 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 |
| US7618662B2 (en) * | 2004-12-22 | 2009-11-17 | Avon Products, Inc | Use of natural plant extracts in cosmetic compositions |
-
2011
- 2011-02-17 CN CN201110040985.7A patent/CN102167687B/en not_active Expired - Fee Related
- 2011-02-17 JP JP2011032672A patent/JP5620847B2/en active Active
- 2011-02-17 CN CN201310273558.2A patent/CN103467293B/en not_active Expired - Fee Related
- 2011-02-18 TW TW100105402A patent/TWI436764B/en active
Non-Patent Citations (1)
| Title |
|---|
| 白鹤灵芝的研究进展;王乃平等;《上海中医药杂志》;20070430;第41卷(第4期);第77-79页 * |
Also Published As
| Publication number | Publication date |
|---|---|
| CN103467293A (en) | 2013-12-25 |
| JP5620847B2 (en) | 2014-11-05 |
| TW201129357A (en) | 2011-09-01 |
| CN102167687A (en) | 2011-08-31 |
| TWI436764B (en) | 2014-05-11 |
| JP2011190250A (en) | 2011-09-29 |
| CN102167687B (en) | 2014-03-12 |
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