CN102232941B - Cyclohexenone Compounds of Antrodia Antrodia for Inhibiting the Growth of Ovarian Cancer Tumor Cells - Google Patents

Abstract

The invention relates to a new application of a compound, which is 4-hydroxy-2, 3-dimethoxy-6-methyl-5 (3, 7, 11-trimethyl-2, 6, 10-dodecatriene) -2-cyclohexenone (4-hydroxy-2, 3-dimethoxy-6-methyl-5 (3, 7, 11-trimethyl-dodeca-2, 6, 10-trienyl) -cyclohexenone) obtained by separating and purifying antrodia camphorata extract.

Description

Be used for suppressing the antrodia camphorata cyclohexenone compound of ovarian cancer tumor cell growth

Technical field

The invention relates to a kind of new application of chemical compound, especially about a kind of purposes that is suppressed ovarian cancer tumor cell growth by the chemical compound of institute's separation and purification in Antrodia Camphorata (Antrodia cinnamomea) extract of utilizing.

Background technology

Human ovarian carcinoma (ovarian cancer) is one of common gynecological cancer, and research points out that possible the pathogenesis of ovarian cancer comprises: hormone and ovulation factor, environmental factors such as ovary long term exposure are at carcinogen, family's medical history, obesity, age and suffer from that danger that breast carcinoma causes the generation ovarian cancer increases etc.Because ovarian cancer early symptom: lower abdomen is uncomfortable, feel sick and the symptom such as anorexia to generally gastrointestinal disease is similar, there is no discernible manifest symptom, and there is not general and simple inspection method, easier mode is through blood letting cancer antigen CA-125 or utilizes ultrasound at present, X-ray or laparoscopy pelvic cavity are diagnosed, yet these methods of inspection can not be effectively and are diagnosed out exactly early stage ovarian cancer, therefore most case just was diagnosed in the cancer later stage, about 70% ovarian cancer has belonged to the III-IV phase when medical, usually this moment, cancerous cell shifted diffusion, poor prognosis, therefore cause suitable low of the survival rate of suffering from the cancer women, so ovarian cancer has become one of main gynecological cancer that causes death in the developed countries.

The treatment of ovarian cancer is that to perform the operation be main, and chemotherapy and radiation cure are auxiliary Comprehensive Treatment, get final product with operative treatment for early stage ovarian cancer, sometimes add preventative chemotherapy; The ovarian cancer in other stage spreads everywhere for preventing cell, must append complete chemotherapy or radiation cure, and as for the ovarian cancer of recurrence, except actively removing resectable focus, the replacing of chemotherapy medication also may promote curative effect.Yet owing to lack the method for early stage Accurate Diagnosis, so that the treatment effect of ovarian cancer is not so good as expection, and its 5 annual survival rate only has 30%; In addition, no matter be radiation cure or chemotherapy, often can cause many side effect or malaise symptoms, therefore research and development can suppress ovarian cancer cell growth and very urgent without the material of harmful side effect.

Antrodia Camphorata (Antrodia cinnamomea), among the peoplely in Taiwan be called again wild rice, Antrodia camphorata or red Camphor tree in Camphor tree mushroom, Camphor tree wild rice, the Camphor tree, be the exclusive medicinal mushrooms of this province, it belongs to the perennial mushroom fungus class of Aphyllophorales (Aphyllophorales), Polyporaceae (Polyporaceae).Because Antrodia camphorata only parasitizes on the dried hollow heartwood interior wall tissue of Taiwan distinctive child care class Cinnamomum kanahirai hay ebon at occurring in nature, add artificial felling trees unlawfully, more shape is rare so that parasitize the wild Antrodia Camphorata quantity that wherein can grow, and because the growth phase of Antrodia camphorata sporophore is worked as slowly under naturalness, so wild Antrodia camphorata quantity is rare and expensive.

The sporophore of Antrodia Camphorata is perennial, and stockless is suberin to wooden, and the Lignum cinnamomi camphorae fragrance that its tool is strong, and changeableization of form have tabular, mitriform, horse-hof shape or tower-like.Nascent is platypelloid type and is cerise that its periphery can present radiation warp shape afterwards, and to expansion growth all around, color also changes light red brown or khaki into, and many pores are arranged, and it is the abundantest position of medical value of Antrodia Camphorata.

In the Taiwan folk custom medically, Antrodia Camphorata has that the circulation of qi promoting of dispeling the wind, blood stasis dispelling are invigorated blood circulation, the effect of warming middle-JIAO removing food stagnancy, removing toxic substances and promoting subsidence of swelling and sedation-analgesia, and be considered as first-class antidote, all alimentary toxicosis, diarrhoea, vomiting, pesticide intoxication all has Detoxication, all has the auxiliary Treatment effect of controlling to improving liver, stomach malfunction and blood circulation disease in addition.Antrodia Camphorata as the gill fungus mushrooms of edible medicinal, composition with many complexity, in the known physiologically active ingredient, comprise: triterpenoid compound (triterpenoids), polysaccharide body (polysaccharides, such as callose), adenosine (adenosine), vitamin is (such as vitamin B, niacin), protein (containing immunoglobulin), super oxygen disproportionation ferment (superoxide dismutase, SOD), trace element (as: calcium, phosphorus, germanium), nucleic acid, steroid and blood pressure stabilization material (such as antodia acid) etc., these a little physiologically active ingredients are considered to have antitumor, increase immunocompetence, antiallergic, Anti-bacterium, resisting hypertension, the multiple efficacies such as blood sugar lowering and cholesterol reducing, and help the treatment of hepatoprotective and liver related disease.

The composition Study of relevant Antrodia camphorata, mostly focus on macromolecular polysaccharide body (polysaccharides) and micromolecular triterpenes (triterpenoids) and steroid (steroids), wherein, Antrodia camphorata contains macromolecular polysaccharide body, form with different monosaccharide and to be present in its sporophore and the mycelium, but after spectrum analysis, all contain the callose (β-D-glucans) of tool physiologically active; Triterpenoid compound is the general name that is combined into hexagon or pentagon native compound by 30 carbons, and the bitterness of Antrodia Camphorata institute tool is namely mainly from this composition of triterpenes, and it also is studied maximum composition.The triterpenoid compound that obtains from sporophore has antrocin, 4,7-dimethoxy-5-methyl isophthalic acid, 3-benzo dioxy ring (4,7-dimethoxy-5-methy-1,3-benzodioxole) and 2,2 ', 5,5 '-tetramethoxy-3,4,3 ', 4 '-two-methylene-dioxy-6,6 '-dimethyl diphenyl (2,2 ', 5,5 '-teramethoxy-3,4,3 ', 4 '-bi-methyl enedioxy-6,6 '-dimethyl biphenyl) (Chiang et al., 1995), the new triterpenoid compound antcinA take lumistane (ergostane) as skeleton, antcin B, antcin C antcin E, antcin F, methyl antcinate G and methyl antcinate H (Cherng et al., 1995,1996).The chemical compound that sporophore contains in addition take lumistane as skeleton comprises Zhankuic acid A, B and C zhankuic acid D and zhankuic acid E (Chen and Yang, 1995; Yang 1996), noval chemical compound 15 α take lanostane (lanostane) as skeleton-acetyl-dehydrosulphurenic acid (15 α-acetyl-dehydrosulphurenic acid), dehydroeburicoic acid (dehydroeburicoic acid) and the sulfurenic acid (dehydrasulphurenic acid) that anhydrates.

Although can learn that by at present many experiments the Antrodia Camphorata extract has aforementioned effect, and its ingredient is also analyzed successively to be gone out, but which kind of effective ingredient in the extract can be facilitated the inhibition cancer effect of Antrodia Camphorata actually, do not deliver concrete relevant effective ingredient, remaining further experiment research differentiates, if so can find out the composition of contained real establishment tumor growth in this extract, to be conducive to Antrodia Camphorata and press down the research that cancer is shut down mutually and turned, and Antrodia Camphorata is applied to cancer, and for example treatment and the prevention of ovarian cancer there are greatest help.

Summary of the invention

For be that what composition has the effect that presses down cancer in the clear Antrodia Camphorata extract actually, the present invention provides the chemical compound of following structural (1) by separation and purification in the Antrodia Camphorata extract;

Wherein, X is oxygen (O) or sulfur (S), and Y is oxygen or sulfur; R ₁Hydrogen base (H), methyl (CH ₃) or (CH ₂) m-CH ₃, R ₂Hydrogen base, methyl or (CH ₂) m-CH ₃, R ₃Hydrogen base, methyl or (CH ₂) m-CH ₃, m=1～12; N=1～12.

In the chemical compound suc as formula (1) structural formula, the preferably is the chemical compound of formula as follows (2):

The chemical compound of formula (2), its chemistry 4-hydroxyl-2 by name, 3-dimethoxy-6-methyl-5 (3,7,11-trimethyl-2,6,10-12 carbon triolefins)-2-cyclonene (4-hydroxy-2,3-dimethoxy-6-methy-5 (3,7,11-trimethyl-dodeca-2,6,10-trienyl)-cyclohex-2-enone), molecular formula is C ₂₄H ₃₈O ₄, outward appearance is the pale yellow powder shape, molecular weight is 390.

(1), the compound separation purification of formula (2) is from Antrodia Camphorata water extract or organic solvent extraction thing, organic solvent can comprise alcohols (for example methanol, ethanol or propanol), esters (for example ethyl acetate), alkanes (for example hexane) or alkyl halide (for example chloromethanes, ethyl chloride), but not as limit, wherein the preferably is alcohols, and better person is ethanol.

(2) 4-hydroxyl-2,3-dimethoxy-6-methyl-5 (3,7,11-trimethyl-2,6,10-12 carbon triolefins)-2-cyclonene.

(1) or/and the chemical compound of formula (2) be used in the composition of the medical composition that suppresses ovarian cancer tumor cell growth.Aforementioned medical composition still can comprise pharmaceutically acceptable carrier except the chemical compound of the formula that comprises effective dose (1) or/and formula (2).Carrier can be excipient (such as water), filler (such as sucrose or starch), adhesive (such as cellulose derivative), diluent, disintegrating agent, absorption enhancer or sweeting agent, but does not only limit to this.Medical composition of the present invention can be manufactured according to the preparation method of general known pharmacy, with formula (1) or/mix mutually with more than one carrier with formula (2) effective ingredient dosage, prepare required dosage form, this dosage form can comprise lozenge, powder, granule, capsule or other liquid preparation, but not as limit.

Below will further specify embodiments of the present invention; following cited embodiment illustrates the present invention; be not to limit scope of the present invention; anyly have the knack of this skill person; without departing from the spirit and scope of the present invention; when can doing a little change and retouching, so protection scope of the present invention is as the criterion when looking accompanying the claim person of defining.

The specific embodiment

Through extraction Antrodia Camphorata water extract or organic solvent extraction thing later, can be further by in addition separation and purification of high performance liquid chroma-tography, again each separatory (fraction) is carried out the test of cancer resistant effect afterwards.At last, then the separatory for the tool cancer resistant effect carries out component analysis, and the composition that may produce cancer resistant effect is further done respectively the inhibition test of ovarian cancer tumor cell.Find namely that finally the chemical compound suc as formula (1)/formula (2) is to have the effect that suppresses ovarian cancer tumor cell growth among the present invention.

For convenience of description the present invention below will be with the 4-hydroxyl-2 of formula (2), and 3-dimethoxy-6-methyl-5 (3,7,11-trimethyl-2,6,10-12 carbon triolefins)-2-pimelie kelone compounds describes.In addition, for confirming 4-hydroxyl-2,3-dimethoxy-6-methyl-5 (3,7,11-trimethyl-2,6,10-12 carbon triolefins)-the 2-pimelie kelone compounds is to the inhibition of growth of tumour cell, is with the MTT analytic process among the present invention, according to National Cancer Institute (National Cancer Institute, NCI) antitumor drug screening pattern is carried out the test of cell survival rate to ovarian cancer tumor cell.Confirmed by those tests, 4-hydroxyl-2,3-dimethoxy-6-methyl-5 (3,7,11-trimethyl-2,6,10-, 12 carbon triolefins)-the 2-cyclonene is for ovarian cancer tumor cell: ES-2 cell line can reduce its survival rate, relatively under and can to reduce simultaneously growth half suppression ratio desired concn (be IC ₅₀Value), therefore must be by 4-hydroxyl-2,3-dimethoxy-6-methyl-5 (3,7,11-trimethyl-2,6,10-12 carbon triolefins)-2-cyclonene is applied on the growth inhibited of ovarian cancer tumor cell, and further can be used in the treatment of ovarian cancer.Hereby aforementioned embodiments is elaborated as follows:

Embodiment 1:

4-hydroxyl-2, the separation of 3-dimethoxy-6-methyl-5 (3,7,11-trimethyl-2,6,10-12 carbon triolefins)-2-cyclonene

With Antrodia Camphorata mycelium, sporophore or the mixture of the two about 100 grams, insert in the triangular pyramidal bottle, the water and the alcohols (70%～100% alcohol solution) that add proper proportion, wherein this alcohols is preferably ethanol, extract more than at least 1 hour in 20～25 ℃ of lower stirrings, with filter paper and 0.45 μ m membrane filtration, collect filtrate and namely get the Antrodia Camphorata extract afterwards.

Antrodia Camphorata extract with aforementioned collection, utilize high-effect liquid chromatography (LC) instrument (High PerformanceLiquid chromatography), chromatography pipe (column) with RP18 is analyzed, and with methanol (A) and 0.1%～0.5% aqueous acetic acid (B) (its solution proportion is: 0～10 minute, the B ratio was 95%～20% as mobile phase (mobile phase); 10～20 minutes, the B ratio was 20%～10%; 20～35 minutes, the B ratio was 10%～90%; 35～40 minutes, the B ratio was 10%～95%), carry in the speed undershoot of per minute 1ml, simultaneously with the long detector analysis of ultraviolet-visible light all-wave.

25 minutes to 30 minutes the extract that rushes is collected the concentrated solid product of pale yellow powder shape of getting final product to get, and this is 4-hydroxyl-2,3-dimethoxy-6-methyl-5 (3,7,11-trimethyl-2,6,10-12 carbon triolefins)-2-cyclonene.By analysis, its molecular formula is C ₂₄H ₃₈O ₄, molecular weight 390, fusing point (m.p.) are 48 ℃～52 ℃.Nuclear magnetic resonance, NMR (NMR) assay value is then as follows: ¹H-NMR (CDCl ₃) δ (ppm): 1.51,1.67,1.71,1.75,1.94,2.03,2.07,2.22,2.25,3.68,4.05,5.07 and 5.14. ¹³C-NMR (CDCl ₃) δ (ppm): 12.31,16.1,16.12,17.67,25.67,26.44,26.74,27.00,39.71,39.81,4.027,43.34,59.22,60.59,120.97,123.84,124.30,131.32,135.35,135.92,138.05,160.45 and 197.12.

Embodiment 2:

The active testing of In Vitro Anti ovarian cancer tumor cell

By in the further test implementation example 1 the discovery chemical compound to the inhibition of tumor cell, present embodiment will be according to National Cancer Institute (National Cancer Institute, NCI) antitumor drug screening pattern, at first get the 4-hydroxyl-2 that separates among the embodiment 1,3-dimethoxy-6-methyl-5 (3,7,11-trimethyl-2,6,10-12 carbon triolefins)-the 2-pimelie kelone compounds, add and contain in the culture fluid of human ovarian carcinoma tumor cell ES-2, carry out the test of tumor cell viability.Wherein, the test of cell survival can be adopted known MTT analytic process and be analyzed, and ovarian cancer tumor cell ES-2 is human Ovarian Cancer Cells (humanovarian cancer cell line).

The MTT analytic process is a kind of common analytical method for analysis of cells hypertrophy (cell proliferation), survival rate (percent of viable cells) and cytotoxicity (cytotoxicity).Wherein, MTT (3-[4,5-dimethylthiazol-2-yl] 2,5-diphenyltetrazolium bromide) is a yellow stain, it can be absorbed by living cells and is reduced into water insoluble by the succinic acid tetrazolium reductase (succinate tetrazoliumreductase) in the Mitochondria and be hepatic formazan, therefore whether form by formazan, can judge and calculate the survival rate of cell.

At first human ovarian cancer cell ES-2 is cultivated in McCoy ' the 5A culture medium that contains 10% hyclone (fetal bovine serum), this culture medium still comprises the dish Ni Xilin (Penicillin) of 10U/ml, and the streptomycin (Streptomycin) of 100 μ g/ml, and in 5%CO ₂, cultivated 24 hours in 37 ℃ of environment.Cell after the hypertrophy is cleaned once with PBS, and process cell with 1 times trypsin-EDTA, under 1,200rpm centrifugal 5 minutes subsequently, with cell precipitation and abandon supernatant.Add afterwards the new culture fluid of 10ml, slight wobble suspends cell again, in the 96 holes trace dish that again cell is placed in.During test, organize in contrast (without total extract of purifies and separates) respectively at the Antrodia Camphorata extract that adds 30,10,3,1,0.3,0.1 and 0.03 μ g/ml in each hole; And the 4-hydroxyl-2 that in each hole, adds 30,10,3,1,0.3,0.1 and 0.03 μ g/ml, 3-dimethoxy-6-methyl-5 (3,7,11-trimethyl-2,6,10-12 carbon triolefins)-2-cyclonene is as test group, in 37 ℃, 5%CO ₂Lower cultivation 48 hours.Thereafter, the MTT in add 2.5mg/ml under the environment of lucifuge in each hole reacts the lysis buffer cessation reaction that adds again 100 μ l after 4 hours in each hole.Under 570nm extinction wavelength, measure its light absorption value with the ferment immunity analysis instrument at last, use the survival rate of calculating cell, and to extrapolate its half suppression ratio desired concn of growing (be IC ₅₀Value), its result as shown in Table 1.

Table one: external test result to the ovarian cancer tumor cell survival rate

By in the table one as can be known, by 4-hydroxyl-2, the effect of 3-dimethoxy-6-methyl-5 (3,7,11-trimethyl-2,6,10-12 carbon triolefins)-2-cyclonene, it is for the IC of ES-2 human ovarian carcinoma tumor cell ₅₀Value is 0.80 μ g/ml, compared to the measured IC of matched group Antrodia Camphorata extraction mixture ₅₀Value (not showing in the table) is low many, so the 4-hydroxyl-2 in the susceptible of proof Antrodia Camphorata extract, 3-dimethoxy-6-methyl-5 (3,7,11-trimethyl-2,6,10-12 carbon triolefins)-the 2-cyclonene can be used in the inhibition of ovarian cancer tumor cell growth really.

In sum, the 4-hydroxyl-2 of separated from Antrodia camphorate of the present invention, 3-dimethoxy-6-methyl-5 (3,7,11-trimethyl-2,6,10-12 carbon triolefins)-2-pimelie kelone compounds, but the growth of establishment ovarian cancer tumor cell.On the other hand, be the material of natural extraction because of antrodia camphorata cyclohexenone compound, when so it is applied to suppress ovarian cancer, can't cause that the patient is uncomfortable or produce other side effect such as toxicity, complication, and its also can with chemotherapeutic agents and usefulness, to reduce the chemotherapeutics using dosage and to reduce the side effect that those chemotherapeutic agents are caused; In addition, also it can be prepared into the medical composition for the treatment of ovarian cancer, wherein, this medical composition still can comprise pharmaceutically acceptable carrier except the antrodia camphorata cyclohexenone compound that comprises effective dose.Carrier can be excipient (such as water), filler (such as sucrose or starch), adhesive (such as cellulose derivative), diluent, disintegrating agent, absorption enhancer or sweeting agent, but does not only limit to this.Medical composition of the present invention can be manufactured according to the preparation method of general known pharmacy, the antrodia camphorata cyclohexenone compound of effective ingredient dosage is mixed mutually with more than one carrier, prepare required dosage form, this dosage form can comprise lozenge, powder, granule, capsule or other liquid preparation, but not as limit.Use the purpose that reaches treatment oophoroma tumor disease.

Claims (2)

1. one kind with compound 4-hydroxy base-2, and 3-dimethoxy-6-methyl-5 (3,7,11-trimethyl-2,6,10-12 carbon triolefins)-2-cyclonene is used in the application that preparation suppresses the medicine of ovarian cancer tumor cell growth.

2. application according to claim 1, wherein, described ovarian cancer tumor cell is ES-2 cell line.