CN100386316C - Mixtures and compounds from Antrodia camphorate myceliums and use thereof - Google Patents
Mixtures and compounds from Antrodia camphorate myceliums and use thereof Download PDFInfo
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- CN100386316C CN100386316C CNB2004100080464A CN200410008046A CN100386316C CN 100386316 C CN100386316 C CN 100386316C CN B2004100080464 A CNB2004100080464 A CN B2004100080464A CN 200410008046 A CN200410008046 A CN 200410008046A CN 100386316 C CN100386316 C CN 100386316C
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- isobutyl
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Abstract
The present invention relates to a new compound from Antrodia Camphorata mycelium, a derivative of maleic acid and succinic acid and the medicinal application thereof. The present invention comprises composition or mycelium of the compound of the present invention.
Description
Technical field
The present invention relates to from Antrodia camphorata (Antrodia Camphorata) mycelial new blend and compound and uses thereof.The present invention relates to comprise compound compositions of the present invention or mycelium.
Background technology
In Taiwan, (Polyporaceae, sporophore Aphyllophorales) is well-known as Chinese medicine to Antrodia camphorata.It only is grown in (Lauraceae) heartwood wall internal layer of the evergreen Cinnamomun kanehirai of Taiwan region (Hay).It is very rare and do not planted.This sporophore has been used to treat food and drug intoxication, diarrhoea, stomachache, hypertension, skin mange and liver cancer.So far almost do not report bioactivity research.
In Taiwan, Antrodia camphorata is called the Cinnamomum kanahirai hay mushroom again, reporting it recently is the following new fungi kind of feature: the cylinder shape that comes across its sporidium in the sporophore, empty soft amyloplast skeletal hyphae, bitter taste and bright orange brown living, and chlamydospore in the pure growth and anthroconidia to shape for hat (pileate) basidioma.The growth of this new fungi kind is very slow, and to be confined to region trees kind Cinnamomum kanehirai Hay (Lauraceae) be unique host.The detailed description of Antrodia camphorata and taxonomic position are seen people such as being set forth in Wu, S.-H., Antrodiacinnamomea (Antrodia camphorata), New combination of a medicinal fungus inTaiwan, Bot.Bull.Acad.Sin.38:273-275.
In the folk medicine of Taiwan, think that the sporophore of Antrodia camphorata has some medical effect.According to traditional method sporophore is ground into dried powder or oral with the stewed back of boiling of other herbal medicine, be used for treatment because of poisoning, diarrhoea, stomachache, hypertension, skin mange and the caused symptom of liver cancer.Yet almost there are not pharmacology or clinical study to occur in the literature so far.Because have strict host specificity and rare in nature, and the artificial culture failure so, " Antrodia camphorata " is very expensive in the price in Taiwan.In recent years, this high-quality fungus sporophore of tool is sold to high price, is 15000 dollars every kilogram approximately.
Summary of the invention
Therefore, the purpose of this invention is to provide from the mycelial new blend of Antrodia camphorata.
Another object of the present invention provides from the mycelial new compound of Antrodia camphorata.
A further object of the present invention provides the novel composition that comprises compound of the present invention.
A further object of the present invention provides the new Antrodia camphorata mycelium that comprises compound of the present invention.
Description of drawings
Fig. 1 shows that the HMBC of compound 2 is relevant.
Fig. 2 shows compound of the present invention.
Fig. 3 shows that the NOE (nuclear Overhauser effect) of compound 4 of the present invention and 5 is relevant.
Fig. 4 a-d shows the test-results of compound 3 of the present invention.
Fig. 5 a-c shows the test-results of ACM (Antrodia camphorata mycelium powder) water extract.
Fig. 6 a-f shows the test-results of ACM ethanolic extract.
Fig. 7 a-e shows the test-results of compound 1 of the present invention.
Embodiment
The invention provides the compound of following formula
Wherein
X is N or O;
R
1Be C
1-10Alkoxyl group, C
2-10Alkenyloxy or C
2-10Alkynyloxy group;
R
2Be H, C
1-10Alkyl, C
2-10Alkenyl or C
2-10Alkynyl; And
R
3Do not exist, or be H or hydroxyl;
Condition is if X is O, then R
3Do not exist.
In compound of the present invention, preferred R
1Be C
2-6Alkenyloxy or C
2-6Alkynyloxy group; More preferably R
1For through C
1-6The C that alkyl replaces
2-6Alkenyloxy, and R most preferably
1For through methyl substituted butenyloxy.In compound of the present invention, preferred R
2Be C
1-6Alkyl, most preferably R
2Be isobutyl-.
Therefore, preferred compound of the present invention is 3-isobutyl--4-[4-(3-methyl-2-butene oxygen base) phenyl] furans-2,5-diketone, 3-isobutyl--4-[4-(3-methyl-2-butene oxygen base) phenyl]-1H-pyrroles-2,5-diketone, 3-isobutyl--4-[4-(3-methyl-2-butene oxygen base) phenyl]-1H-pyrroles-1-alcohol-2,5-diketone, 3R
*, 4S
*-1-hydroxyl-3-isobutyl--4-[4-(3-methyl-2-butene oxygen base) phenyl] tetramethyleneimine-2,5-diketone, or 3R
*, 4R
*-1-hydroxyl-3-isobutyl--4-[4-(3-methyl-2-butene oxygen base) phenyl] tetramethyleneimine-2, the 5-diketone.
Preferred compound of the present invention is 3-isobutyl--4-[4-(3-methyl-2-butene oxygen base) phenyl]-1H-pyrroles-2,5-diketone or 3-isobutyl--4-[4-(3-methyl-2-butene oxygen base) phenyl]-1H-pyrroles-1-alcohol-2, the 5-diketone.
The compound of present invention further optimization is 3-isobutyl--4-[4-(3-methyl-2-butene oxygen base) phenyl]-1H-pyrroles-1-alcohol-2, the 5-diketone.
The present invention also provides from the mycelial mixture of Antrodia camphorata, and it comprises compound of the present invention.Mixture preparation of the present invention is from mycelial water of Antrodia camphorata or organic solvent extraction thing.This organic solvent includes but not limited to that alcohol is (as CH
3OH, C
2H
5OH or C
3H
7OH), ester (as ethyl acetate), alkane (as hexane) and haloalkane are (as CH
3Cl, C
2H
2Cl
2).Preferred organic be ethanol or any side effect that does not cause the people contain the alcoholic acid solvent.Mixture of the present invention can lower systolic pressure or increase high-density lipoprotein (HDL).In addition, identical mixture has the central cholinergic system excitement, protects the liver, anti-inflammatory or anti-tumor activity.In detail, mixture of the present invention can suppress to be selected from the tumour of the cell or tissue of liver, intestines, bone, blood, lymph and breast.The experimenter who accepts mixture of the present invention includes but not limited to people, Mammals, mouse, rat, horse, pig, chicken, duck, dog and cat.
The present invention also provides and comprises compound compositions of the present invention.Composition of the present invention can lower systolic pressure or increase high-density lipoprotein (HDL).In addition, composition of the present invention have the central cholinergic system excitement, protect the liver, anti-inflammatory or anti-tumor activity.In detail, composition of the present invention can suppress to be selected from the tumour of the cell or tissue of liver, intestines, bone, blood, lymph and breast.The experimenter who accepts composition of the present invention includes but not limited to people, Mammals, mouse, rat, horse, pig, chicken, duck, dog and cat.
The present invention also provides the Antrodia camphorata (Antrodia) that comprises compound of the present invention mycelium.The weight of preferred its at least 1% promycelium of mycelium is the gross weight of compound 1-5 of the present invention.The weight of its at least 3% promycelium of most preferred mycelium is the gross weight of compound 1-5 of the present invention.According to previous deep fermentation (submerged liquid fermentation) preparation Antrodia camphorata mycelium, people such as deep fermentation such as T.L.M.Stamford, Food Science " Protein enrichment of cashew wastes for animal feeds " is from http://www.unu.edu/unupress/food/8F10le/8F10lE0b.htm.
Embodiment
The following example is nonrestrictive, and only represents various aspect of the present invention and feature.
General experimental procedure
Measure fusing point with Yanagimoto low profile thermal platform melting point apparatus, and do not proofread and correct.Measure opticity with the automatic polarimeter of JascoDIP-360.Measure UV spectrum with Shimadzu UV-2200 recording spectrophotometer.Measure infrared spectra with Jasco FT/IR-230 infrared spectrometer.With Varian Unity Plus 500 spectrometer measurements
1H-and
13The C-NMR spectrum.Under the ionization voltage of 70eV, measure EIMS and HR-EIMS with JeolJMS-AX 505 HAD mass spectrographs.Adopt the silica gel of BW-820MH (positive) and Chromatorex-ODS DM1020T (anti-phase) (Fuji Silysia) to carry out column chromatography.
Extraction with separate
Under refluxing, will be from Taiwan Simpson Biotech Co., Ltd., the Antrodia camphorata mycelium powder in October calendar year 2001 (60 gram) is used chloroform extraction 3 hours, extracts three times.6) and chloroform-methanol (1: 1) wash-out chloroform extract (5.3 gram) is carried out silica gel chromatography, with normal hexane-acetone (19: 1-14:, obtain nine parts (Fr.1-9).Second section is carried out silica gel chromatography, obtain compound 1 (8.7 milligrams).The 4th part is carried out positive and reverse phase silica gel chromatogram, obtain compound 2 (13.6 milligrams).The 5th part is carried out silica gel chromatography,, obtain ergosterol peroxide (35.8 milligrams) with normal hexane-acetone (8: 2) elution.To positive and the reversed-phase silica gel column chromatography that the 6th part makes up, obtain compound 3 (14.6 milligrams).The 7th part is carried out column chromatography, obtain the mixture of compound 4 and 5 (4: 1), be prepared HPLC to 4 with 5 mixture subsequently and separate [post: Tosoh TSK-gel ODS-80T
M(21.5 * 300 millimeters), moving phase: the methanol-water (70: 30) that contains 0.1%TFA].
3-isobutyl--4-[4-(3-methyl-2-butene oxygen base) phenyl]-furans-2,5-diketone (compound 1): yellow oil; UV (MeOH) λ
Max(log ε) 227 (4.1), 258 (3.9), 275 (3.8), 355 (3.4) nm; IR (CHCl
3) v
Max1763cm
-1,
1H-NMR table 1;
13C-NMR table 2; EIMS m/z 314[M]
+(100), 246 (100), 131 (100); HR-EIMSm/z314.1523 (C
19H
22O
4Calculated value: 314.1518).
3-isobutyl--4-[4-(3-methyl-2-butene oxygen base) phenyl]-1H-pyrroles-2,5-diketone (2): yellow needle crystal (normal hexane-ethyl acetate); Mp 110-111 ℃; UV (methyl alcohol) λ
Max(log ε) 230 (4.3), 272 (3.5), 355 (3.7) nm; IR (CHCl
3) v
Max1724cm
-1 1H-NMR table 1;
13C-NMR table 2; EIMS m/z 313[M]
+(8), 245 (100), 203 (77), 131 (28); HR-EIMS m/z 313.1681 (C
19H
23NO
3Calculated value: 313.1678).
The X-radiocrystallography of compound 2:
Obtain yellow needle crystal by normal hexane-ethyl acetate crystallization, after selecting, supply data gathering.Crystal data: C
19H
23NO
3Mr=313.40; 0.15 * 0.02 * 0.02 millimeter of size; Three oblique brilliant spacer P1 (#2), a=6.3505 (5)
B=12.205 (1)
C=12.560 (2)
α=64.623 (7) °, β=75.358 (4) °, γ=84.681 (5) °, V=850.9 (2)
Z=2, D
Calc=1.223g/cm
3, μ (MoK α)=0.82cm
-1, F000=336.00.In 93K radiation down, to have the unicolor Mo-K α of graphite (λ=0.71069
) Rigaku RAXIS-RAPID video plate diffractometer measure.In 8950 reflections of gathering, 4745 is unique (R
Int=0.108); Merge and equate reflection.Resolve crystalline structure and with the young waiter in a wineshop or an inn side of the holding actuarial of complete matrix with direct method (SHELXS86).(anisotropically) actuarial non-hydrogen atom anisotropically.Hydrogen atom is included in interior but not actuarial.Final index R=0.074, R
w=0.099, and GOF (Guest Observer Facility, object is observed facility)=1.06.The maximum of final difference fourier figure and smallest peaks correspond respectively to 0.83 and reach-0.89e
-/
3-isobutyl--4-[4-(3-methyl-2-butene oxygen base) phenyl]-1H-pyrroles-1-alcohol-2,5-diketone (compound 3): yellow oil; UV (MeOH) λ
Max(log ε): 232.5 (4.3), 296 (3.7), 374 (3.7) nm; IR (CHCl
3) v
Max1717cm
-1 1H-NMR table 1;
13C-NMR table 2; EIMS m/z 329[M]
+(12), 261 (100), 131 (50); HR-EIMS m/z:329.1637 (C
19H
23NO
4Calculated value: 329.1627).
3R
*, 4S
*-1-hydroxyl-3-isobutyl--4-[4-(3-methyl-2-butene oxygen base) phenyl] tetramethyleneimine-2,5-diketone (4): water white oil; [α]
D 23+ 2.5 ° (c0.2, methyl alcohol); UV (methyl alcohol) λ
Max(log ε): 225 (4.3), 275 (3.3), 283 (3.2) nm; IR (CHCl
3) v
Max1715cm
-1 1H-NMR table 1;
13C-NMR table 2; EIMS m/z 331[M]
+(2), 263 (67), 207 (66), 191 (30), 179 (40), 133 (64), 69 (100); HR-EIMS m/z:331.1747 (C
19H
25NO
4Calculated value: 331.1783).
3R
*, 4R
*-1-hydroxyl-3-isobutyl--4-[4-(3-methyl-2-butene oxygen base) phenyl] tetramethyleneimine-2,5-diketone (5): water white oil; [α]
D 23+ 3.0 ° (c0.2, MeOH); UV (MeOH) λ
Max(log ε): 227 (4.3), 275 (3.4), 283 (3.3) nm; IR (CHCl
3) v
Max1715cm
-1 1H-NMR table 1;
13C-NMR table 2; EIMS m/z 331[M]
+(1), 263 (45), 207 (50), 191 (75), 179 (30), 133 (100), 69 (92); HR-EIMS m/z:331.1766 (C
19H
25NO
4Calculated value: 331.1783).
Ergosterol superoxide: colourless needle crystal (normal hexane-acetone); Mp 165-169 ℃ (lit
2Mp 171-174 ℃).
Cytotoxic assay: adopt sulforhodamin B (SRB) to carry out the outer LLC tumour cell of body of laws and measure.Calculate 50% growth-inhibiting (ED with the Probit method
50).
Result and discussion
The mycelial chloroform extract of Antrodia camphorata is repeated positive and reverse phase silica gel chromatogram, to obtain five kinds of new toxilic acids and succinic acid derivative (compound 1-5) and ergosterol superoxide.
Table 1
Compound 1-5's
1H-NMR spectrum data (δ ppm, J=Hz) (500MHz, CDCl
3)
Table 2
Compound 1-5's
13C-NMR spectrum data (δ ppm) (125MHz, CDCl
3)
A) ownership is interchangeable.
All new compounds is following to be determined:
It is C that the molecular formula of compound 1 is analyzed by HR-EIMS
19H
22O
4Infrared spectra is presented at 1763cm
-1There is the carbonyl absorption of acid anhydrides at the place.Compound 1
1The H-NMR spectrum is similar with compound 2, shows the existence of the phenyl ring of isobutyl-part, 3-methyl 2-butylene oxygen base section, para-orientation.The bright compound 1 of HMBC stave has the structure (Fig. 1) identical with compound 2 parts, wherein infers the existence of maleic anhydride according to molecular formula.Therefore, determine that compound 1 is 3-isobutyl--4-[4-(3-methyl-2-butene oxygen base) phenyl] furans-2, the 5-diketone.
It is C that the molecular formula of compound 3 is analyzed by HR-EIMS
19H
23NO
4Infrared spectra is presented at 1717cm
-1There is carbonyl absorption at the place, can belong to be hydroxy diimide.
1H-and
13C-NMR spectrum is similar with compound 1 and 2 also, shows the existence of the phenyl ring of isobutyl-part, 3-methyl 2-butylene oxygen base section, para-orientation.In the HMBC experiment, prove that compound 3 has part-structure and compound 2 identical (Fig. 1).Compound 3 contains a Sauerstoffatom than compound more than 2, therefore, determines that this compound is 3-isobutyl--4-[4-(3-methyl-2-butene oxygen base) phenyl]-1H-pyrroles-1-alcohol-2, the 5-diketone.
According to people's such as Aquveque report separate for the second time these types from natural toxilic acid and succinic acid derivative.
Use the cytotoxic activity (table 3) of LLC (Lewis lung cancer) clone research chloroform extract and isolated compound.Chloroform extract shows medium cytotoxicity, its ED
50Value is 26.7 mcg/ ml.Maleinization compound 1 and 4 is not had a cytotoxic activity, and finds compound 2 and 3 for LLC cell cording cytotoxicity, its ED
50Value is lower than chloroform extract.Table 3 is from the mycelial chloroform extract of Antrodia camphorata and compound 1-4 50% growth-inhibiting (ED to LLC clone
50) value
A) positive control
The tumour of ACM (Antrodia camphorata mycelium powder) is measured
A. clone
Adherent cell:
MCF-7: human breast carcinoma
HT-29: human colon adenocarcinoma
KATO III: people's adenocarcinoma of stomach
SW480: human colon adenocarcinoma
SW620: human colon adenocarcinoma
HepG2: people liver adenoncus knurl
Suspension cell:
EL4: mouse lymph lymphoma
B. sample
C. measuring method
Calculate ED
50(50% of effective dose suppresses)
Adherent cell: MTT (methyl thiazolyl tetrazolium) method: for MCF-7, HT-29, KATO III, SW480, HepG2,3 days definite cells.SW6204 days.
Suspension cell: cell counting; EL4 cell counting 5 days.
D. result
Calculate: y=m Ln (x)+b
Example
| | Y | |
| 0 | 0.97 | |
| 10ppm | 0.941 | |
| 30ppm | 0.6 | |
| 100 ppm | 0.331 |
Use X (10,30,50ppm) to obtain correlation curve with the Y value
y＝-0.2643Ln(x)+1.5321
ED
50＝exp【(0.97/2-1.5321)/(-0.2643)】
Specimen preparation and sample description
The water extract of A.ACM (Antrodia camphorata mycelium powder)
1. 1 gram ACM is added 40 milliliters of RO H are housed
2In 250 ml beakers of O, under the room temperature beaker is placed ultrasonic water bath, continue 20 minutes.
2. stir water-bath 45 minutes down at 45 ℃.
3. beaker is placed ultrasonic water bath to continue 20 minutes again.
Under 3000rpm with centrifugal 15 minutes of sample.
5. collection supernatant liquor, and carry out serial dilution with substratum.
B. working sample concentration
1. evaporating pan weighing (W1).
2. 10 ml water extracts are added in the evaporating pan
3. evaporating pan is inserted in the baking oven to remove moisture (W2)
Sample weight/milliliter=(W2-W1)/10
C.ACM (Antrodia camphorata mycelium powder) ethanolic extract
1. 100 milliliter of 95% ethanol is added in 500 ml beakers that 20 gram ACM are housed, and at room temperature stirred 10 minutes.
2. with No. 1 filter paper filtering suspension of Advantec, and collect filtrate.
3. with rotary type vacuum evaporimeter concentrated filtrate, remove ethanol.
D. compound 1: from the pure compound of ACM
E. compound 3: from the pure compound of ACM
The MTT assay method
1. discard old substratum after the cell proliferation, clean cell once with phosphate buffered saline (PBS) (PBS) then.
2. wash cell with trypsinase-EDTA.
3. under 1200rpm centrifugal 5 minutes, abandoning supernatant then.
4. with 10 milliliters of substratum suspension granules.
5. 100 microlitre cell suspending liquids are mixed with 100 microlitre trypan blues, with calculating survivaling cell.
6. in every hole of 96 porose discs, add 1 * 10
4Individual cell/100 microlitre substratum will coil under 37 ℃ and cultivate 24 hours in CO2gas incubator.
7. discard old substratum, clean cell once with PBS.
8. in every hole, add 100 microlitre samples, will coil under 37 ℃ and in CO2gas incubator, cultivate.
9. cleaned cell once at the 3rd, 4 and 5 day with PBS.
10. in every hole, add 57 microlitre MTT (0.88 mg/ml).
11.4 discard MTT after hour, and clean cell once with PBS.
12. every hole adds 50 microlitre DMSO.
13. under OD545, read the result with the ELISA instrument.
Cell counting (EL4 clone)
1. discard old substratum by centrifugal after the cell proliferation.
With fresh culture with the granule resuspending.
3. 100 microlitre cell suspending liquids are mixed with 100 microlitre trypan blues, with calculating survivaling cell.
4. preparation contains 1 * 10
5The sample of the different concns of individual cells/ml sample.
5. in every hole of 96 porose discs, put into 100 microlitre samples, will coil under 37 ℃ and in CO2gas incubator, cultivate.
6. at the 3rd, 4 and 5 day calculating survivaling cell.
PBS
NaCl 8 grams
KCl 0.2 gram
Na
2HPO
41.4 gram
KH
2PO
40.2 gram
Volume is adjusted to 1 liter of PH 7.4
Result and discussion
ACM is in the ED of clone
50
| Clone | HepG2 | HT-29 | KATO III | EL4 | SW480 | SW620 | MCF-7 |
| |
21ppm | 52ppm | 38ppm | 3.5ppm | | 6ppm | |
| Compound | |||||||
| 3 | 35ppm | 42ppm | 69ppm | 2.6ppm | 20ppm | 27ppm | 0.02ppm |
| The ACM ethanolic extract | 32ppm | 52ppm | 156ppm | 2.6ppm | 71ppm | 4ppm | |
| The ACM water extract | 295ppm | 707ppm | 20ppm | 207ppm | 132ppm | 318ppm |
Experimental result is as follows in detail:
Compound 3:HepG2 of the present invention (Fig. 4 a), EL4 (Fig. 4 b), HT-29 (Fig. 4 c) and Kato III (Fig. 4 d).
ACM water extract: HepG2 (Fig. 5 a), SW620 (Fig. 5 b) and EL4 (Fig. 5 c).
ACM ethanolic extract: HT-29 (Fig. 6 a), SW480 (Fig. 6 b), SW620 (Fig. 6 c), EL4 (Fig. 6 d), HepG2 (Fig. 6 e) and Kato III (Fig. 6 f).
Compound 1:MCF-7 of the present invention (Fig. 7 a), EL4 (Fig. 7 b), HT-29 (Fig. 7 c), SW620 (Fig. 7 d) and HepG2 (Fig. 7 e).
Above result proves that compound of the present invention and ACM extract are inhibited to various types of tumour cells.
By high-efficient liquid phase chromatogram technique analysis all new compounds (1,2 and 3) from the ACM ethanolic extract
Purpose:, adopt the conventional quality control procedure of high performance liquid chromatography as us in order to measure the amount of all new compounds (1,2 and 3) from the ACM ethanolic extract.
Preparation ACM alcohol extraction matter sample:
1), with the accurate weighing sample powder 20.000 of electronic balance (gram), insert and contain in 100 milliliter of 95% alcoholic acid scale experiment bottle, its bottle cap does not screw.
2), the sample bottle with above-mentioned steps placed ultrasonic water bath 10 minutes.
3), liquid sample is poured in the centrifuge tube, subsequently with those samples under the 6500rpm condition centrifugal 5 minutes, to remove coarse particles.
4), with No. 1 filter paper filtering liquid level of advantec.
5), with rotary type vacuum evaporimeter concentrated filtrate, until thickness occurring, not containing the alcoholic acid weak yellow liquid.
6), repeating step 1 to 5 three time, and collect all extraction products (total ACM ethanolic extract=4.60 grams), calculated yield.
The application of 2690 type Water HPLC:
1), post: anti-phase C18
2), moving phase: methyl alcohol, water, acetonitrile
3), volume injected: 20 microlitres
4), detect: photodiode array detector 996 is measured down in wavelength 254nm
5), be ready for 1.000 (gram) ACM alcohol extraction matter sample in 10 milliliters of ethanol, to carry out the HPLC analysis
*:
The result: analyze according to HPLC, the extraction product that contains pure compound 1,2 and 3 is shown in the following table 4.
Table 4:
Therefore, compound 1,2 and 3 gross weight are 5.92% of ACM example weight.
The test of ACM ethanolic extract
Material and equipment
1. substances and give pattern
All in vivo test all with oral in 2%Tween 80 carriers the predose substances of 1000 mg/kg give.Be described in the method observing time of each test.
2. animal
It is that male spontaneous ypotension rat (SHR), Wistar and Long Evans derive is rat that male or female ICR mouse, the Wistar-Okamoto that use is provided by the general ball in Taiwan institute of pharmacology derives.The animal rearing space is as follows: 10 mouse are raised in 29 * 18 * 13 centimetres of spaces, and 6 rats are raised in 45 * 23 * 21 centimetres of spaces, and 3 cavys are raised in 45 * 23 * 21 centimetres of spaces.Mouse and rat feeding are at APEC
RIn the cage.By heavy 21 ± 2 grams that Univ Nat Taiwan's animal center provides, 6-8 week, big C57BL/6J immunocompetence male mice also used in this research.With the cage (IVC frame, 36 small-sized isolated systems) of experimental animal feeding at independent ventilation.Each cage gives autoclaving, and contains 5 mouse (in 26.7 * 20.7 * 14 centimetres space).All animals all in temperature control (21 °-23 ℃) and wet (60%-70%) environment of control, in the laboratory, keep at least one week down in 12 hours periodicity of illuminations before use.Standard test feed (LabDiet Rodent Diet and Guinea Pig Diet, PMI Nutrition International, the U.S.) and tap water ad lib are provided.
3. clone and substratum
Mouse melanoma cells, B16-F0 (ATCC CRL-6322) be available from American TypeCulture Collection, and with Dulbecco ' s Modified Eagle ' s Medium (GIBCO, the U.S.) as cell culture medium.With tumor cell culture in 37 ℃ and contain 5%CO
2Air in.
4. pharmaceutical chemicals
Generally:
Distilled water (self-control), dimethyl sulfoxide (DMSO) (DMSO, Merck, Germany) waits a sodium chloride solution (Xindong Chemical Industry Co., Ltd., Taiwan), sal epsom (MgSO
47H
2O, Wako, Japan), Sodium meclophenamate (Sigma, the U.S.), methylcellulose gum (Signa, the U.S.), sodium hydroxide (NaOH, Wako, Japan), phosphate buffered saline (PBS) (Sigma, the U.S.) and Tween 80 (Wako, Japan).
Reagent
Glicose-HA measures test kit (Wako, Japan), alanine aminotransferase (ALT) is measured test kit (Wako, Japan), aspartate amino transferase (AST) is measured test kit (Wako, Japan), T-cholesterol-HA and HDL measure test kit (Wako, Japan), Hemolynac 3Hemolys (Nihon Koden, Japan), Isotonic 3 Diluent (NihonKoden, Japan).
5. equipment
The general use:
Cattle container (letter moral, Taiwan), 250 milliliters and 1000 ml beaker (Kinmax, the U.S.), and disposable syringe (1 milliliter, Top Corporation, Japan), antimagnetic type tweezer (klappencker, Germany), mouse scale (mouse scale) #Z-40 (Taconic, the U.S.), oral feeding injection tube (Natsune, Japan), hypodermic needle 23G x 1 " (Top Corporation; Japan), pH meter (Suntex, the U.S.); mouse scale 500 gram ± 2 grams (Chien-chun; Taiwan), 1 milliliter of glass syringe; 2 milliliters and 5 milliliters (Mitsuba, Japan); and stainless steel scissors (Klappencker, Germany).
Method and result:
1. cholinergic excitement, maincenter/periphery (Lippmann W and Pugsley TA, Arch IntPharmacodyn.227:324,1977).
It is male or the female rats group that 3 heavy 150 ± 20 Wistar that restrain of test substances orally give are derived.In subsequently 30-60 minute, the appearance of record cumulative measurement surpasses the animal number of chewing behavior (mouth and/or tongue motion) in 10 seconds, and the animal number that salivation occurs.The positive reaction of observing in 3 rats 2 or more (〉=2) the active and periphery cholinergic activity of central cholinergic system that expressed possibility.
Table 5: the exciting result of cholinergic, maincenter/periphery is in rat
Carrier and test substances give with oral (PO), and positive control compound peritoneal injection (IP) gives.In 30-60 minute time subsequently, the appearance of record cumulative measurement surpasses the animal number of the behavior of chewing for 10 seconds (mouth and/or tongue motion), and the animal number that salivation occurs.The positive reaction of observing in 3 rats 2 or more (〉=2) expressed possibility cholinergic activity or periphery cholinergic activity.
2. cardiovascular, blood pressure and heart rate ( SHR 0,1,2,4 hour) (people such as Yen TT, Lite Sci.22:359,1978)
Using 3 heavy 250 ± 20 Wistar-Okamoto that restrain to derive is spontaneously hypertensive male mouse (SHR) group; Average systolic is 200 ± 20mmHg, and heart rate is 400 ± 30 times/minute.Blood pressure and heart rate pass through tail cuff method under temperature control environment (32 ± 1 ℃), (0 o'clock) and record indirectly in 1,2,4 hour afterwards before orally give test substances or carrier.Compare with 0 the time when measure the back pitch time at every turn, systolic pressure descends 10% or more (〉=10%), or heart rate descends 20% or more (〉=20%), then thinks significant.
Table 6: rat central vessel, blood pressure result ( SHR 0,1,2,4 hour)
Table 7: cardiovascular, heart rate result ( SHR 0,1,2,4 hour)
Using systolic pressure is that 200 ± 20mmHg and heart rate are 400 ± 50mmHg time/minute SHR.Blood pressure is via vial tail cuff method 0 o'clock and do record indirectly in 1,2,4 hour behind orally give test substances or carrier.When each Measuring Time point with in bracket, provide 0 the time compare, blood pressure drops 10% or more (〉=10%), or heart rate descends 20% or more (〉=20%), it is significant then being considered as.
229mmHg and 403mmHg time during carrier 10ml/kg 0/
Minute be used as 100%.
223mmHg and 452mmHg time during ACM-ethanolic extract 1000mg/kg 0/
Minute be used as 100%.
228mmHg and 379mmHg time during clonidine 0.1mg/kg 0/
Minute be used as 100%.
Table 8: cardiovascular, blood pressure result ( SHR 0,1,2,4 hour) in rat
Table 9: cardiovascular, heart rate result ( SHR 0,1,2,4 hour)
Using systolic pressure is that 200 ± 20mmHg and heart rate are 400 ± 50mmHg time/minute SHR.Blood pressure is in orally give test substances or carrier (before) 0 o'clock and do record indirectly in 1,2,4 hour afterwards via vial tail cuff method.Each Measuring Time point was compared with 0 o'clock that provides in bracket, blood pressure drops 10% or more (〉=10%), or heart rate decline 20% or more (〉=20%), and it is significant then being considered as.
220mmHg and 410mmHg time during carrier 10ml/kg 0/
Minute be used as 100%.
205mmHg and 446mmHg time during ACM-ethanolic extract 300mg/kg 0/
Minute be used as 100%.
235mmHg and 417mmHg time during clonidine 0.1mg/kg 0/
Minute be used as 100%.
3. people such as (, Atherosclerosis Drug Discovery.Plenum, New York, pp.215-229,1976) the Schurr PE that cholesterol, serum (total HDL, total amount/HDL than), diet are brought out.
To weight is that to derive be male mice group feeding high lipid food (g/100g: Oleum Cocois, 8 for 5 ICR of 22 ± 2 grams; Cholesterol, 1.0; Cholic acid, 0.3; Lard, 2; Standard food 88.7) 7 days to cause hypercholesterolemia.The the 5th, 6,7 day orally give test substances.After overnight fasted, obtain the change per-cent that serum is used for measuring total cholesterol (total amount), high-density lipoprotein (HDL) (HDL) and total amount/HDL from every mouse.Compare with control animal with vehicle treatment, the decline 20% of serum total amount or more (〉=20%), or serum hdl rising 20% or more (〉=20%), or total amount/HDL decline 40% or more (〉=40%), it is significant being considered as.
Table 10: the cholesterol that in mouse, causes (total amount/HDL, total amount/HDL ratio) result by diet
The the 5th, 6,7 day oral (PO) after the feeding high-cholesterol diet gives carrier, test substances or contrast positive compound.Twenty four hours after administration is for the third time put to death the laboratory animal of overnight fasted, with assessment serum total cholesterol (total amount) and high-density lipoprotein (HDL) (HDL).The decline 20% of serum total amount or more (〉=20%), or serum hdl rising 20% or more (〉=20%), or total amount/HDL then thinks significant than decline 40% or more (〉=40%).
4. liver injury, D-galactosamine (people such as Wrobel J, J.Med Chem 41:1084,1998).
Operating weight is that to derive be the male rat group for 5 Wistar of 200 ± 20 grams.Every animal is with single injection of d-galactose amine (500mg/kg, IP) treatment.0.5 hour and 4 hours afterwards and 8 hours orally give test substances were put to death animal after 24 hours before the D-galactosamine administration.Serum alanine aminotransferase (ALT) and aspartate amino transferase (AST) level are measured with HITACHI automatic analyser (7050 type), measure by optimized UV method.Compare with the control animal with vehicle treatment, the active decline 30% of ALT or AST or more (〉=30%) then show significant protection.
Table 11: the result of liver injury, GalN is in rat
The GalN single administration (500mg/kg, IP) 0.5 hour before with 4 hours afterwards and 8 hours orally give testers and carrier, put to death rat in the GalN injection after 24 hours, and mensuration ALT and AST value.Compare with vehicle group, it is significant that ALT and AST decline 〉=30% are considered as.
5. inflammation, carrageenan (people such as Winter CA, Proc Soc Exp Biol Med.111:544,1962)
Weight be 3 Long Evans of 150 ± 20 grams to derive be male or female group, first fasting is the whole night before research.Accept injection carrageenan (0.1 milliliter of 1% built-in (intraplantar) suspension) orally give test substances before 1 hour at right back pawl.The rear solid end oedema is measured as inflammation, uses the plethysmometer record that has tank (25 millimeters of diameters) after 3 hours giving carrageenan.The decline 30% of rear solid end oedema or more (〉=30%) show significant anti-inflammatory activity.
Table 12: inflammation result, carrageenan is in rat
Table 13: inflammation result, carrageenan is in rat
Gave overnight fasted rat test substances and carrier in preceding 1 hour at right back pawl (R.P.) injection carrageenan (0.1 milliliter of 1% built-in (intraplantar) suspension); Left back pawl (L.P.) is not then injected.The decline 30% of rear solid end oedema or more (〉=30%) shown in the bracket show significant acute anti-inflammatory activity.
6. tumour, homology melanoma B 16-F0 cell (Farrugia CA and Groves MJ., Anticancer Research 19:1027-1032,1999)
Use the C57BL/6J male mice group of (SPF), the immunocompetence (6-8 week is big) of 5 specific-pathogen frees, it is raised between animal isolation in (SPF) of specific-pathogen free environment under in (IVC frame).Will (ATCC CRL-6322 has 1.0 * 10 in 0.2 milliliter with C57BL/6J mouse homologous B16-F0 mouse melanoma viable cell
5Individual cell) dorsal part of experiment mice is gone in subcutaneous injection.24 hours begin treatments behind the tumor inoculation, give test compounds 21 day in oral tube feed mode every day, perhaps reduces fate when the overt toxicity symptom occurring.Body weight, tumour size and survival from the 1st day to the 22nd day monitoring mouse.The survival of monitoring experiment mouse in addition finished until the research back on the 45th day.
Assess tumor weight (milligram) according to the prolate ellipsoid formula: long (millimeter) * [wide (millimeter)]
2* 0.5, suppose that proportion is 1, π is 3.Tumor growth in the animal of compounds for treating calculates with T/C (treatment/contrast) * 100%; T/C value≤42% then shows notable antitumor activity.Mean survival time 〉=125% of T/C (treatment/contrast) also is considered as having notable antitumor activity.
Table 14: tumour, homology melanoma b16-F0 cell result
Table 15: tumour, homology melanoma b16-F0 cell result
Tumour cell was implanted after 24 hours, gave laboratory animal carrier and test substances every day, altogether 21 administrations.Simultaneously, control compound, mitomycin gives twice weekly in the IP mode, altogether 6 administrations.The tumour size is measured weekly and is noted down twice, 22 days by a definite date.Calculating tumor growth with T/C (weight/contrast) * 100 suppresses.T/C value≤42% is considered as having notable antitumor activity.
Table 16: tumour, the result of homology melanoma b16-F0 cell
Tumour cell was implanted after 24 hours, gave laboratory animal carrier and test substances every day, altogether 21 administrations.Simultaneously, control compound, mitomycin gives twice weekly in the IP mode, altogether 6 administrations.The tumour size is measured weekly and is noted down twice, 22 days by a definite date.Use Si Shi t check to determine the significant difference of the body weight change between test compounds and the vehicle Control group.
Table 17: tumour, homology melanoma b16-F0 cell result
A: if animal is not dead after 45 days, then its survival time is used as 45 days.
The survival to 45 of monitor therapy mouse day research phase, or till the laboratory animal death.Mean survival time 〉=125% of T/C (treatment/contrast) also is considered as having notable antitumor activity.
Discuss:
According to making standard by oneself, oral (PO) gives ACM-ethanolic extract, causes remarkable activity in following mouse and rat mensuration:
The central cholinergic system excitement of 1000 mg/kg in rat; Minimum and inapparent excitement under 300 mg/kg as seen; Under 1000 mg/kg, periphery choline nerve there are not significant excitement or antagonism (table 5).
In spontaneously hypertensive (SH) rat, under 1000 mg/kg, systolic pressure reduces (to be observed in 1,2 and 4 hour time point, with 0 o'clock 100% compare, be respectively 16%, 12% and 20%), and with the heart rate moderate but inapparent decline (table 6 and 7); The dosage of 300 mg/kg does not cause the noticeable change (table 8 and 9) of systolic pressure and heart rate.
Under 1000 mg/kg, in the mouse that diet brings out, increase high-density lipoprotein (HDL) (HDL Duos 39% than vehicle Control) (table 10); Relevant total cholesterol (total amount) does not significantly change, and the HDL/ total amount drops near 31% than significantly; The dosage of 300 mg/kg does not cause total amount, HDL and HDL/ total amount than significantly changing.
The rats'liver damage that GalN is brought out, time visible hepatoprotective effect in 1000 mg/kg * 3 (compare with vehicle Control, ALT descend 44% and AST descends 57%); The moderate of time visible ALT 20% descends and AST decline 28% (table 11) in 300 mg/kg * 3.
The anti-inflammatory action of the rat pawl oedema of under 1000 mg/kg, carrageenan being brought out (comparing 45% with vehicle Control suppresses) (table 12); 300 lower mg/kg levels do not have remarkable activity (compare 3% with carrier and suppress table 13).
Under 1000 mg/kg, in the 8th, the 11 and 15 day anti-tumor activity (table 14 and 15) in C57BL/6J mouse homology melanoma b16-F0 cell, and the prolongation (table 17) of animals survived time; The weight of animals does not significantly change (table 16).
The test of ACM of the present invention, ACM-ethanolic extract and compound 3
Use nine groups, it is male mice (weighing 22 ± 2 grams) that every group of 5 ICR derive.Every animal is with the tetracol phenixin (CCl of single dose
4, in 50% sweet oil, 0.1 ml/kg, PO) challenge.30 minutes and 4 and 8 hours afterwards orally give test substances before tetracol phenixin excites, dosage is the ACM of 300 and 1000 mg/kg, or dosage is the compound of the present invention 3 of 30,100 and 300 mg/kg; And before tetracol phenixin 1 day (twice of every day) and 30 minutes and 4 and 8 hours afterwards 300 and 1000 mg/kg ACM ethanolic extracts of orally give are carried out pre-treatment.24 hours execution animals behind tetracol phenixin.Use HITACHI automatic analyser (7050 type) to measure alanine aminotransferase (ALT) and aspartate amino transferase (AST) level with optimized UV method.Compare with carrier, ALT or the decline 30% of AST level or more (〉=30%), showing has significant provide protection to liver injury.
The result
Table 18: measure liver injury, tetracol phenixin is in mouse
Discuss:
Estimated the ICR mouse that 3 pairs of ACM of the present invention, ACM-ethanolic extract and compounds bring out by tetracol phenixin liver injury may protect activity.Before tetracol phenixin excites 0.5 hour and 4 and 8 hours afterwards, with test substances, dosage was the ACM-ethanol of 300 and 1000 mg/kg and the compound of the present invention 3 orally give experimental animals that dosage is 30,100 and 300 mg/kg.ACM ethanolic extract for 300 and 1000 mg/kg, before tetracol phenixin, carried out two treatments (9:00AM and 16:00PM) (b.i.d.) in 1 day, and then before tetracol phenixin excites 0.5 hour with treated (amounting to 5 administrations) in 4 hours afterwards and 8 hours.Determine the degree of liver injury by the increase of serum alanine aminotransferase for the animal of vehicle treatment (ALT) and aspartate amino transferase (AST).Dosage is that the ACM of 1000 mg/kg * 3 and compound of the present invention 3 that dosage is 300 mg/kg * 3 cause, for the animal of vehicle treatment, and the remarkable minimizing of ALT (46% and 41%) and AST (36% and 33%).While dosage is the remarkable minimizing that the ACM-ethanolic extract of 300 and 1000 mg/kg * 5 also causes ALT (36% and 34%) and AST (25% and 20%).
(100 mg/kg * 3 IP) show with respect to the vehicle treatment group the remarkable minimizing of ALT (31%) and AST (31%) to test silymarin simultaneously.
Reach a conclusion, ACM of the present invention, ACM-ethanolic extract and compound 3 have significant liver-protecting activity in the mouse carbon tetrachloride model.
Although description that the present invention is carried out and illustration are made those skilled in the art and are used it to being enough in detail, under the prerequisite that does not deviate from essence of the present invention and scope, variously substitute, change with improvement should be tangible.
One of ordinary skill in the art will readily recognize that the present invention is suitable for achieving the goal, and obtain described herein and inherent result and advantage.Clone, embryo, animal and manufacturing processed thereof and method are represented embodiment preferred, are exemplary, are not desire restriction scope of invention.Those skilled in the art will envision that modification and other purposes to it.These are revised and all to be encompassed in the essence of the present invention and by the scope definition of claim.
Those skilled in the art will easily know, under the prerequisite that does not deviate from scope of the present invention and essence the present invention be changed and will revise.
All patents mentioned in the specification sheets and works are all represented general technical staff of the technical field of the invention's level.Be incorporated herein by reference just as every single works specifically and individually indicates, all patents and works all are incorporated herein by reference.
The present invention that this paper exemplarily describes can obtain under one or more elements herein, the one or more restriction implementing specifically not being disclosed in.The term that is adopted and express and only to be the non-limiting term of illustrative, and non-desire is with these terms and express to get rid of and reach the Equivalent of described or its Partial Feature shown in any, but think that various changes are possible in the claimed invention scope.Therefore, should understand, though specifically described the present invention by preferred embodiment and optional feature, those skilled in the art may adopt the change and the variant of design disclosed herein, think that these modifications and variant are in the invention scope by the claims definition.
Other embodiment illustrates in claims.
Claims (10)
1. following compound:
3-isobutyl--4-[4-(3-methyl-2-butene oxygen base) phenyl] furans-2, the 5-diketone,
3-isobutyl--4-[4-(3-methyl-2-butene oxygen base) phenyl]-1H-pyrroles-2, the 5-diketone,
3-isobutyl--4-[4-(3-methyl-2-butene oxygen base) phenyl]-1H-pyrroles-1-alcohol-2, the 5-diketone,
3R
*, 4S
*-1-hydroxyl-3-isobutyl--4-[4-(3-methyl-2-butene oxygen base) phenyl] tetramethyleneimine-2, the 5-diketone, or
3R
*, 4R
*-1-hydroxyl-3-isobutyl--4-[4-(3-methyl-2-butene oxygen base) phenyl] tetramethyleneimine-2, the 5-diketone
2. from the mycelial mixture of Antrodia camphorata, it prepares from mycelial water of Antrodia camphorata or organic solvent extraction thing; Described mixture contains 3-isobutyl--4-[4-(3-methyl-2-butene oxygen base) phenyl] furans-2,5-diketone, 3-isobutyl--4-[4-(3-methyl-2-butene oxygen base) phenyl]-1H-pyrroles-2,5-diketone, 3-isobutyl--4-[4-(3-methyl-2-butene oxygen base) phenyl]-1H-pyrroles-1-alcohol-2,5-diketone, 3R
*, 4S
*-1-hydroxyl-3-isobutyl--4-[4-(3-methyl-2-butene oxygen base) phenyl] tetramethyleneimine-2,5-diketone or 3R
*, 4R
*-1-hydroxyl-3-isobutyl--4-[4-(3-methyl-2-butene oxygen base) phenyl] tetramethyleneimine-2, the 5-diketone.
3. the mixture of claim 2, wherein this organic solvent is alcohol, ester, alkane or haloalkane.
4. the mixture of claim 3 should alcohol be an ethanol wherein.
The mixture of claim 2 be used for lowering systolic pressure, increase high-density lipoprotein (HDL) or be used for the central cholinergic system excitement, protect the liver in preparation, the purposes of anti-inflammatory or anti-tumor drug.
6. the purposes of claim 5, wherein tumour is from the cell or tissue that is selected from liver, intestines, bone, blood, lymph and breast.
7. composition, its inclusion compound 3-isobutyl--4-[4-(3-methyl-2-butene oxygen base) phenyl] furans-2,5-diketone, 3-isobutyl--4-[4-(3-methyl-2-butene oxygen base) phenyl]-1H-pyrroles-2,5-diketone, 3-isobutyl--4-[4-(3-methyl-2-butene oxygen base) phenyl]-1H-pyrroles-1-alcohol-2,5-diketone, 3R
*, 4S
*-1-hydroxyl-3-isobutyl--4-[4-(3-methyl-2-butene oxygen base) phenyl] tetramethyleneimine-2,5-diketone or 3R
*, 4R
*-1-hydroxyl-3-isobutyl--4-[4-(3-methyl-2-butene oxygen base) phenyl] tetramethyleneimine-2, the 5-diketone.
The composition of claim 7 be used for lowering systolic pressure, increase high-density lipoprotein (HDL) or be used for the central cholinergic system excitement, protect the liver in preparation, the purposes of anti-inflammatory or anti-tumor drug.
9. the purposes of claim 8, wherein tumour is from the cell or tissue that is selected from liver, intestines, bone, blood, lymph and breast.
10. Antrodia camphorata mycelium, its inclusion compound 3-isobutyl--4-[4-(3-methyl-2-butene oxygen base) phenyl] furans-2,5-diketone, 3-isobutyl--4-[4-(3-methyl-2-butene oxygen base) phenyl]-1H-pyrroles-2,5-diketone, 3-isobutyl--4-[4-(3-methyl-2-butene oxygen base) phenyl]-1H-pyrroles-1-alcohol-2,5-diketone, 3R
*, 4S
*-1-hydroxyl-3-isobutyl--4-[4-(3-methyl-2-butene oxygen base) phenyl] tetramethyleneimine-2,5-diketone or 3R
*, 4R
*-1-hydroxyl-3-isobutyl-4-[4-(3-methyl-2-butene oxygen base) phenyl] tetramethyleneimine-2, the 5-diketone.
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| CN101357883B (en) * | 2007-07-30 | 2012-09-26 | 国鼎生物科技股份有限公司 | Antrodia camphoratea pimelie kelone compound for treating autoimmune disease and medicine composition |
| CN101362679B (en) * | 2007-08-08 | 2012-05-30 | 国鼎生物科技股份有限公司 | Antrodia camphoratea pimelie kelone compound for inhibiting B type hepatitis virus |
| CN101376624B (en) * | 2007-08-29 | 2011-06-01 | 国鼎生物科技股份有限公司 | Cinnamomum kanehirae Hayata pimelie kelone compound for liver protection |
| US8101792B2 (en) * | 2007-09-17 | 2012-01-24 | Simpson Biotech Co., Ltd. | Compounds isolated from Antrodia cinnamomea and use thereof |
| CN101417934B (en) * | 2007-10-24 | 2012-04-18 | 国鼎生物科技股份有限公司 | New compounds separated from Antrodia camphorate extract |
| TWI442931B (en) * | 2011-06-07 | 2014-07-01 | New Bellus Entpr Co Ltd | Auxiliary pharmaceutical composition for use in radiation treatment |
| CN103202863A (en) * | 2011-11-18 | 2013-07-17 | 善笙生物科技股份有限公司 | Ameliorative Or Preventive Effect Of Antrodia Cinnamomea In Arthritis, Cartilage Destruction, Or Chondrocyte Death |
| CN104546830B (en) * | 2013-10-23 | 2018-03-09 | 善笙生物科技股份有限公司 | Purposes from the mycelial compound of Antrodia camphorata and mixture |
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Non-Patent Citations (4)
| Title |
|---|
| . Chen,C.H.J.Nat.Product,Vol.58 . 1995 |
| . 陈劲初.Fung,Sci,Vol.16 . 2001 |
| . Chen,C.H.J.Nat.Product,Vol.58 . 1995 * |
| . 陈劲初.Fung,Sci,Vol.16 . 2001 * |
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