JP2005247724A5 - - Google Patents

Description

NOVEL MIXTURES AND COMPOUNDS OBTAINED FROM ANTRODIA CAMPHORATA MYCES AND USE THEREOF

  The present invention relates to novel mixtures and compounds obtained from the mycelium of Antrodia Camphorata and their use. The present invention relates to a composition or mycelium comprising a compound of the present invention.

  The fruiting body of Antrodia camphorata (Sarcophyllaceae, Hirabara) is well known in Taiwan as a traditional Chinese medicine. It grows only in Taiwan on the inner core wall of the land-specific evergreen tree Cinnamomun kanehirai (hay) (Asteraceae). It is rare and not cultured. Fruiting bodies are used for the treatment of food and drug addiction, diarrhea, abdominal pain, high blood pressure, itchy skin, and liver cancer. Very few biological activity studies have been reported so far.

  Antrodia camphorata, also known as "niu-chang-chih" or "niu-chang-ku" in Taiwan, is the basidiospore cylindrical shape, weak amyloid skeleton mycelium, bitterness, and umbrella Recently reported as a novel fungal species characterized by a light cinnamon upside down on a basidiomycete, and thick spore and anthroconidia in pure culture. The growth of this new fungal species is extremely slow and is restricted to Cinnamomum kanehirai Hay, a native tree species, as the only host. Detailed features and taxonomic position of Antrodia camphorata are Wu, S.-H, et al., Antrodia cinnamomea ("niu-chang-chih"), New combination of a medicinal hungus in Taiwan, Bot. Bull. Acad. Sin. 38: 273-275 (1997).

  In Taiwanese folk remedies, the fruiting body of Antrodia camphorata is believed to have some medicinal effects. According to traditional methods, fruit bodies are crushed into dry powder or boiled with other herbs for oral consumption to treat diseases caused by addiction, diarrhea, abdominal pain, hypertension, skin itching, and liver cancer. However, little pharmacological or clinical research in these respects has appeared in the literature to date. Due to its strict host specificity, natural rarity, and artificial culture failures, "niu-chang-chih" is very effective in Taiwan. In recent years, the high purity of this fungal fruit body has been sold at a very high price of about 15,000 US dollars per kg.

The object of the present invention is to provide a novel mixture from the mycelium of Antrodia Camphorata.

Another object of the present invention is to provide a novel compound from the mycelium of Antrodia Camphorata.

  A further object of the present invention is to provide novel compositions comprising the compounds of the present invention.

A further object of the present invention is to provide a new mycelium of Antrodia Camphorata comprising the compounds of the present invention.

[式中、
XはNまたはOであり；
R₁はC_1-10アルキルオキシ、C_2-10アルケニルオキシ、またはC_2-10アルキニルオキシであり；
R₂はH、C_1-10アルキル、C_2-10アルケニル、またはC_2-10アルキニルであり；
R₃は不在であるか、H、またはヒドロキシであり;
ただしXがOである場合、R₃は不在である]。 The present invention provides a compound having the formula:

[Where
X is N or O;
R ₁ is C _1-10 alkyloxy, C _2-10 alkenyloxy, or C _2-10 alkynyloxy;
R ₂ is H, C _1-10 alkyl, C _2-10 alkenyl, or C _2-10 alkynyl;
R ₃ is absent, H, or hydroxy;
However, when X is O, R ₃ is absent].

In the compounds of the present invention, preferred R ₁ is C _2-6 alkenyloxy, or C _2-6 alkynyloxy; more preferred R ₁ is C _2-6 alkenyloxy substituted with C _1-6 alkyl. Most preferred R ₁ is butenyloxy substituted with methyl. In the compounds of the present invention, preferred R ₂ is C _1-6 alkyl, and most preferred R ₂ is isobutyl.

Accordingly, preferred compounds of the present invention are:
3-isobutyl-4- [4- (3-methyl-2-butenyloxy) phenyl] furan-2,5-dione;
3-isobutyl-4- [4- (3-methyl-2-butenyloxy) phenyl] -1H-pyrrole-2,5-dione;
3-isobutyl-4- [4- (3-methyl-2-butenyloxy) phenyl] -1H-pyrrol-1-ol-2,5-dione;
3R ^* , 4S ^* -1-hydroxy-3-isobutyl-4- [4- (3-methyl-2-butenyloxy) phenyl] pyrrolidine-2,5-dione; or
3R ^* , 4R ^* -1-hydroxy-3-isobutyl-4- [4- (3-methyl-2-butenyloxy) phenyl] pyrrolidine-2,5-dione.

  More preferred compounds of the invention include 3-isobutyl-4- [4- (3-methyl-2-butenyloxy) phenyl] -1H-pyrrole-2,5-dione, or 3-isobutyl-4- [4- ( 3-methyl-2-butenyloxy) phenyl] -1H-pyrrol-1-ol-2,5-dione. A further preferred compound of the invention is 3-isobutyl-4- [4- (3-methyl-2-butenyloxy) phenyl] -1H-pyrrol-1-ol-2,5-dione.

The present invention also provides a mixture derived from the mycelium of Antrodia Camphorata comprising a compound of the present invention. The mixtures of the present invention are prepared from water or organic solvent extracts of Antrodia Camphorata mycelium. Organic solvents include alcohols (such as CH ₃ OH, C ₂ H ₅ OH, C ₃ H ₇ OH), esters (such as acetyl acetate), alkanes (such as hexane), and halogenated alkanes (CH ₃ Cl, including such) as C ₂ H ₂ Cl ₂ are not limited thereto. Preferred organic solvents are ethanol or alcoholic solvents that do not cause any side effects in humans. The mixtures of the present invention can reduce systolic blood pressure or increase high density lipoprotein. Furthermore, the same mixture has central cholinergic agonism, liver prevention, anti-inflammatory or anti-tumor activity. In particular, the mixtures of the present invention can inhibit tumors from cells or tissues selected from the group consisting of liver, intestine, bone, blood, lymph, and breast. Subjects that receive the mixture of the present invention include, but are not limited to, humans, mammals, mice, rats, horses, pigs, chickens, ducks, dogs, and cats.

  The present invention also provides a composition comprising a compound of the present invention. The composition of the present invention can reduce systolic blood pressure or increase high density lipoprotein. Furthermore, the composition of the present invention has central cholinergic agonism, liver prevention, anti-inflammatory or anti-tumor activity. In particular, the compositions of the present invention can inhibit tumors from cells or tissues selected from the group consisting of liver, intestine, bone, blood, lymph, and breast. Subjects that receive the compositions of the present invention include, but are not limited to, humans, mammals, mice, rats, horses, pigs, chickens, ducks, dogs, and cats.

  The present invention also provides a novel mycelium of Antrodia comprising a compound of the present invention. A preferred mycelium is at least 1% by weight of the raw mycelium is Compound 1-5 of the present invention. The most preferred mycelium is Compound 1-5 of the present invention at least 3% by weight of the raw mycelium. The mycelium of Antrodia Camphorata was previously obtained by immersion fermentation such as TLM Stamford et al., Food Science "Protein enrichment of cashew wastes for animal feeds" (http://unu.edu/unupress/food/8F101e/8F101E0b.htm) Has been prepared.

  The following examples are ratio limiting and are merely representative of various aspects and features of the present invention.

General experimental method
Melting point was measured with a Yanagimoto micro hot stage melting point apparatus, and correction was not performed. The optical rotation was measured with a Jasco DIP-360 automatic polarimeter. The UV spectrum was measured with a Shimadzu UV-2200 recording spectrophotometer. IR spectra were measured with a Jasco FT / IR-230 infrared spectrometer. ¹ H- and ¹³ C-NMR spectra were measured with a Varian Unity Plus 500 spectrometer. EIMS and HR-EIMS were measured with a Jeol JMS-AX505HAD mass spectrometer at an ionization voltage of 70 eV. Column chromatography was performed on silica gel BW-820MH (normal phase) and Chromatorex-ODS DM1020T (reverse phase) (Fuji Silysia).

Extraction and isolation
Antrodia camphorata mycelium powder (ACM) (60 g) obtained from Simpson Biotech Co. Ltd., Taiwan, October 2001, was extracted three times with CHCl ₃ under reflux for 3 hours. CHCl ₃ extract (5.3 g) was chromatographed on silica gel eluting with n-hexane-acetone (19: 1-14: 6) and CHCl ₃ -MeOH (1: 1) and fractions of 9 ( Fr. 1-9) was obtained. Fraction 2 was chromatographed on silica gel to give compound 1 (8.7 mg). Fraction 4 was chromatographed on normal and reverse phase silica gel to give compound 2 (13.6 mg). Fraction 5 was chromatographed on silica gel eluting with n-hexane-acetone (8: 2) to give ergosterol peroxide (35.8 mg). For fraction 6, compound 3 (14.6 mg) was obtained by a combination of normal phase and reverse phase silica gel column chromatography. Fraction 7 gave a mixture of compounds 4 and 5 (4: 1) by column chromatography. The mixture of compounds 4 and 5 is then separated by preparative HPLC [column: Tosoh TSK-gel ODS-80YM (21.5 × 300 nm), mobile phase: CH ₃ OH-H ₂ O (70:30) with 0.1% TFA]] did.

3-isobutyl-4- [4- (3-methyl-2-butenyloxy) phenyl] furan-2,5-dione (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 _max 1763 cm ^-1 ; ¹ H-NMR Table 1 ; ¹³ C-NMR Table 2; EIMS m / z 314 [M] ⁺ (100), 246 (100), 131 (100); HR-EIMS m / z 314.1523 (calculated values for C ₁₉ H ₂₂ O ₄ , 314.1518).
3-isobutyl-4- [4- (3-methyl-2-butenyloxy) phenyl] -1H-pyrrole-2,5-dione (2):
Yellow needle crystal (n-hexane-AcOEt); mp110-111 ° C; UV (MeOH) λ _max (log ε) 230 (4.3), 272 (3.5), 355 (3.7) nm; IR (CHCl ₃ ) V _max 1724 cm ^-1 ; ¹ H-NMR Table 1; ¹³ C-NMR Table 2; EIMS m / z313 [M] ⁺ (8), 245 (100), 203 (77), 131 (28); HR-EIMS m / z313 .1681 (calculated for C ₁₉ H ₂₃ NO ₃ , 313.1678).

X-ray crystallographic analysis of Compound 2:
Yellow needles were obtained by crystallization from n-hexane-AcOEt and selected for data collection. Crystal data: C ₁₉ H ₂₃ NO ₃ ; M _r = 313.40; Dimensions 0.15 × 0.02 × 0.02mm; Triclinic system, Space group P1 (# 2), a = 6.3505 (5) Å, b = 12.205 (1) Å, c = 12.560 (2) Å, α = 64.623 (7) °, β = 75.358 (4) °, γ = 84.681 (5) °, V = 850.9 (2) Å ³ , Z = 2, D _calc = 1.223g / cm ³ , μ (MoKα) = 0.82cm ⁻¹ , F000 = 336.00 measured with Rigaku RAXIS-RAPID Imaging Plate automatic diffractometer using graphite monochromated Mo-Kα (λ = 0.71069710) radiation at 93K Carried out. Of the 8950 reflections recovered, 4745 was unique (R _int = 0.108); matched equivalent reflections. The crystal structure was dissolved by the direct method (SHEL XS86) and purified by full matrix rest square. Non-hydrogen bonds were purified anisotropically. Including but not limited to hydrogen atoms. The final indices were R = 0.074, Rw = 0.099, and GOF (Guest Observer Facility) = 1.06. The maximum and minimum peaks in the final difference Fourier map corresponded to 0.83 and -0.89e ⁻ / Å ³ respectively.

3-isobutyl-4- [4- (3-methyl-2-butenyloxy) phenyl] -1H-pyrrol-1-ol-2,5-dione (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 _max 1717 cm ^-1 ; ¹ H-NMR Table 1; ¹³ C-NMR Table 2: EIMS m / z329 [M] ⁺ (12), 261 (100), 131 (50); HR-EIMS m / z 329.1637 (calculated value for C ₁₉ H ₂₃ NO ₄ , 329.1627).
3R ^* , 4S ^* -1-hydroxy-3-isobutyl-4- [4- (3-methyl-2-butenyloxy) phenyl] pyrrolidine-2,5-dione (4):
Colorless oil; [α] _D ²³ + 2.5 ° (c 0.2, MeOH); UV (MeOH) λ _max (log ε): 225 (4.3), 275 (3.3), 183 (3.2) nm; IR (CHCl ₃ ) V _max 1715 cm ^-1 ; ¹ H-NMR Table 1; ¹³ C-NMR Table 2; EIMS m / z331 [M] ⁺ (2), 263 (67), 207 (66), 191 (30), 179 (40 ), 133 (64), 69 (100); HR-EIMS m / z 331.1747 (calculated value for C ₁₉ H ₂₅ NO ₄ , 331.1783).
3R ^* , 4R ^* -1-hydroxy-3-isobutyl-4- [4- (3-methyl-2-butenyloxy) phenyl] pyrrolidine-2,5-dione (5):
Colorless oil; [α] _D ²³ + 3.0 ° (c 0.2, MeOH); UV (MeOH) λ _max (log ε): 227 (4.3), 275 (3.4), 283 (3.3) nm; IR (CHCl ₃ ) V _max 1715 cm ^-1 ; ¹ H-NMR table 1; ¹³ C-NMR table 2; EIMS m / z331 [M] ⁺ (1), 263 (45), 207 (50), 191 (75), 179 (30 ), 133 (100), 69 (92); HR-EIMS m / z 331.1766 (calculated value for C ₁₉ H ₂₅ NO ₄ 331.1783).
Ergosterol peroxide: colorless needles (n-hexane-acetone); mp165-169 ° C (lit ² mp171-174 ° C).
Cytotoxicity assay. In vitro LLC tumor cell assay was performed by the sulforhodamine B (SRB) method. 50% growth inhibition (ED ₅₀ ) was calculated by the Probit method.

Results and discussion
Antrodia Camphorata mycelium CHCl3 extract was repeatedly chromatographed on normal and reverse phase silica gels to obtain five new maleic and succinic acid derivatives (compound 1-5) along with ergosterol peroxide. .

The structure of the new compound was determined as follows:
Compound 2 is a yellow needle crystal, has mp110-111 ° C., and the molecular formula of C ₁₉ H ₂₃ NO ₃ has been assigned by HR-EIMS. The IR spectrum showed imidocarbonyl absorption at 1724 cm- ¹ . ^{The 13} C-NMR spectrum showed 4 methyl carbons, 2 joint carbons and 1 chin carbons in the aliphatic region, and 1 benzene ring, 1 olefin group, and 2 carbonyl carbons. . ^{The 1} H-NMR spectrum shows the presence of an isobutyl moiety at δ 0.90, 2.06 and 2.51, the presence of a 3-methyl-2-butenyloxy moiety at δ 1.76, 1.81, 4.56 and 5.50, and para- at δ 6.95 and 7.50. The presence of substituted benzene moieties was shown, which was further supported by 1H-1H COZY (cooler synchrotron) and HMQC (heteronuclear multiple quantum interference) experiments. A wide range of correlations were observed by HMBC as shown in FIG. Based on molecular formula and ¹³ C-NMR spectrum, this compound was presumed to contain an additional CHNO atom containing one additional carbonyl carbon. Thus, this ambiguous part was speculated to be a maleimide group. The structure was thus established by X-ray analysis to be 3-isobutyl-4- [4- (3-methyl-2-butenyloxy) phenyl] -1H-pyrrole-2,5-dione.

The molecular formula of Compound 1 was evaluated as C ₁₉ H ₂₂ O ₄ by HR-EIMS. IR spectrum revealed carbonyl absorption of the acid anhydride at 1763 cm- ¹ . The ¹ H-NMR spectrum of Compound ¹ was the same as that of Compound 2, indicating the presence of an isobutyl moiety, a 3-methyl-2-butenyloxy moiety, and a para-substituted benzene ring. From the HMBC spectrum, compound 1 is shown to have the same partial structure as compound 2 (FIG. 1), where the maleic anhydride group is deduced based on the molecular formula of compound 1, which is later expressed as 3-isobutyl. -4- [4- (3-Methyl-2-butenyloxy) phenyl] furan-2,5-dione was determined.

The molecular formula of Compound 3 was evaluated as C ₁₉ H ₂₃ NO ₄ by HR-EIMS. IR spectrum showed carbonyl absorption at 1717 cm ⁻¹ and was assigned to hydroxyimide. ¹ H- and ¹³ C-NMR spectra were also the same as those of compounds 1 and 2, indicating the presence of an isobutyl moiety, a 3-methyl-2-butenyloxy moiety, and a para-substituted benzene ring. HMBC experiments showed that compound 3 has the same partial structure as compound 2 (FIG. 1). Compound 3 contains one more oxygen atom than Compound 2, so this compound is (3-isobutyl-4- [4- (3-methyl-2-butenyloxy) phenyl-1H-pyrrol-1-ol- It was determined to be 2,5-dione.

Compounds 4 and 5 have the same R _f value and the same molecular formula by HR-EIMS (C ₁₉ H ₂₅ NO ₄ , measured values 331.1747 and 331.1766, respectively), but they could be separated by preparative HPLC. IR spectra of both compounds showed hydroxyimidocarbonyl absorption at 1715 cm- ¹ . ^{In the 1} H- and ¹³ C-NMR spectra, both compounds showed the presence of an isobutyl moiety, a 3-methyl-2-butenyloxy moiety, and a para-substituted benzene ring, while the isobutylmethylene proton showed multiple, and compound 1 It was not as double as about -3. ¹ H- ¹ H COZY spectrum showed that the methylene group was bonded to the —CH—CH— unit. The ¹³ C-NMR spectra of compounds 4 and 5 showed two additional sp ³ carbon signals instead of the two sp ² carbon signals observed for compound 1-3. Therefore, compounds 4 and 5 were not N-hydroxymaleimide but rather N-hydroxysuccinimide and had stereocenters at the C-3 and C-4 positions in the succinimide ring. Compounds 4 and 5 were determined to be trans and cis isomers, respectively, from the coupling constant between H-3 and H-4 (4.0 and 8.0 Hz for compounds 4 and 5, respectively). In the NOESY (nuclear overhauser effect spectrum measurement) spectrum of compound 4, NOE was not observed between H-3 and H-4, while appropriate NOE was observed for compound 5. While the optical rotations of compounds 4 and 5 showed + 2.5 ° and + 3.0 °, respectively, their CD spectra showed no Cotton effect at any wavelength, and both compounds 4 and 5 are racemic mixtures. I suggest that. The resolution of these racemic mixtures by HPLC using chiral columns with some solvent systems was unsuccessful. At present, we cannot conclude clearly whether these compounds are optically active compounds or racemic mixtures. Thus their relative structures are 3R ^* , 4S ^* -and 3R ^* , 4R ^* -1-hydroxy-3-isobutyl-4- [4- (3-methyl-2-butenyloxy) phenyl] pyrrolidine-2, respectively. , 5-dione was determined.

  The isolation of these types of maleic and succinic acid derivatives from nature was followed twice by the report of Aquveque et al.

The cytotoxic activity of the chloroform extract and the isolated compound was investigated using an LLC (Lewis Lung Carcinoma) cell line (Table 3). The chloroform extract showed a mild cytotoxic effect with an ED ₅₀ value of 26.7 μg / ml. Maleic acid compounds 1 and 4 were found to have no cytotoxic activity, while compounds 2 and 3 were found to be cytotoxic to LLC cell lines with ED ₅₀ values lower than those of the chloroform extract.

X(10,30,50ppm)及びYの値を使用して、y=-0.2643Ln(x)+1.5321の相関曲線を得る。
ED50=exp[(0.97/2-1.5321)/(-0.2643)] ACM (Antrodia Camphorata Mycelium Powder) Tumor Assay
A. Cell line adherent cells:
MCF-7: Human milk carcinoma
HT-29: Human colon adenocarcinoma
KATO III: human gastric carcinoma
SW480: Human colon adenocarcinoma
SW620: Human colon adenocarcinoma
HepG2: human liver carcinoma suspension cells:
EL4: Mouse lymphoma
B. Sample Compound 1, Compound 3, ACM EtOH extract, ACM H ₂ O extract
C. Assay method
ED ₅₀ calculation (effective dose with 50% inhibition)
Adherent cells: MTT (methylthiazolyl tetrazolium) method: MCF-7, HT-29, KATO III, SW480, HepG2 cells are measured in 3 days, and SW620 is measured in 4 days.
Suspended cells: cell counting method; counts over 5 days for EL4 cells.
D. Result calculation: y = mLn (x) + b
Example

Using the X (10, 30, 50 ppm) and Y values, a correlation curve of y = −0.2643Ln (x) +1.5321 is obtained.
ED50 = exp [(0.97 / 2-1.5321) / (-0.2643)]

Sample preparation and sample description
A.ACM (Antrodia Camphorata mycelium powder) H ₂ O extract
1. Add 1 g ACM to 40 ml RO H ₂ O in a 250 ml beaker and place the beaker in an ultrasonic water bath at room temperature for 20 minutes.
2. Stir the water bath at 45 ° C for 45 minutes.
3. Place the beaker in an ultrasonic water bath for another 20 minutes.
4. Centrifuge the sample at 3000 rpm for 15 minutes.
5. Collect the supernatant and perform serial dilution with media.
B. Sample concentration measurement
1. Lighten the evaporating dish (W1).
2. Add 10 ml H ₂ O extract sample to the evaporating dish.
3. Place the evaporating dish in the oven and remove the water (W2)
Sample weight / ml = (W2-W1) / 10
C. ACM (Atrodia Camphorata mycelium powder) EtOH extract
Add 100 ml 95% alcohol to 20 g ACM in a 1.500 ml beaker and stir at room temperature for 10 minutes.
2. Filter the suspension through Advantec # 1 filter paper and collect the filtrate.
3. Concentrate the filtrate with a rotary vacuum evaporator to remove the alcohol.
D. Compound 1: Pure compound derived from ACM
E. Compound 3: ACM-derived pure compound

MTT assay
1. Discard the old medium after cell growth and wash the cells once with phosphate buffered saline.
2. Rinse cells with trypsin-EDTA.
3. Centrifuge for 5 minutes at 200rpm and discard the supernatant.
4. Suspend the pellet in 10 ml of medium.
5. Mix 100 μl cell suspension with 100 μl trypan blue and calculate viable cells.
6. Add 1 × 10 ⁴ cells / 100 μl medium to each well of the 96-well plate and incubate the plate in a CO ₂ incubator at 37 ° C. for 24 hours.
7. Discard the old medium and wash the cells once with PBS.
8. Add 100 μl sample to each well and incubate the plate in a CO ₂ incubator at 37 ° C.
On days 9.3, 4 and 5, the cells are washed once with PBS.
10. Add 57 μl MTT (0.88 mg / ml) to each well.
After 11.4 hours, discard the MTT and wash the cells once with PBS.
12. Add 50 μl DMSO / well.
13. Read OD545 with Elisa reader.
Cell counting method (EL4 cell line)
1. Discard the old medium after cell growth by centrifugation.
2. Resuspend the pellet with fresh medium.
3. Mix 100 μl cell suspension with 100 μl trypan blue and calculate viable cells.
Prepare samples of various concentrations, including 4.1 x 10 ⁵ cells / ml sample.
5. Place 100 μl of sample in each well of a 96-well plate and incubate the plate in a CO ₂ incubator at 37 ° C.
Viable cells are calculated on days 6.3, 4 and 5.
PBS
NaCl 8g
KCl 0.2g
Na ₂ HPO ₄ 1.4g
KH ₂ PO ₄ 0.2g
Prepare a volume of 1L PH7.4

Results and Discussion ACM's ED ₅₀ for cell lines

Detailed test results are as follows:
Compound 3 of the invention: HepG2 (Figure 4a), EL4 (Figure 4b), HT-29 (Figure 4c), and KatoIII (Figure 4d).
ACM H ₂ O extract: HepG2 (FIG. 5a), SW620 (FIG. 5b), and EL4 (FIG. 5c).
ACM EtOH extracts: HT-29 (Figure 6a), SW480 (Figure 6b), SW620 (Figure 6c), EL4 (Figure 6d), HepG2 (Figure 6e), and KatoIII (Figure 6f).
Compound 1 of the present invention: MCF-7 (FIG. 7a), EL4 (FIG. 7b), HT-29 (FIG. 7c), SW620 (FIG. 7d), and HepG2 (FIG. 7e).
As described above, the compounds of the present invention and the ACM extract are shown to have an inhibitory effect on various types of tumor cells.

Analyze all new compounds (1, 2 and 3) from ACM EtOH extract by high performance liquid chromatography method Objective: To determine the amount of all new compounds (1, 2 and 3) from ACM EtOH extract To measure, high performance liquid chromatography was used as our routine quantitative control method.
ACM EtOH extract sample preparation:
1) By using digital balance, accurately weigh 20.000 (g) of sample powder into a media laboratory bottle with memory containing 100 mL of 95% alcohol and do not close the lid tightly.
2) Place the sample bottle of 1) in the ultrasonic bath for 10 minutes.
3) Pour the liquid sample into a centrifuge tube, place the sample in a centrifuge, and remove coarse particles under conditions of 6500 rpm / 5 minutes.
4) Filter the liquid phase with filter paper advantec No.1.
5) Concentrate the filtered solution with a rotary vacuum evaporator to obtain a viscous yellowish alcohol-free liquid.
6) Repeat steps 1-5 three times to collect all extract products (total ACM EtOH extract = 4.60 g). Calculate the yield.

Application by water HPLC, model 2690
1) Column: Reversed phase C18
2) Mobile phase: MeOH, H ₂ O, acetonitrile
3) Injection volume: 20μL
4) Detection capacity: Photodiode Array Detector 996 at a wavelength of 254nm
5) Prepare 1.000 (g) ACM EtOH extract sample in 10 mL alcohol for HPLC analysis *.

それ故、化合物1,2及び3の全重量は、ACMサンプル中に5.92重量%である。 Results: By HPLC analysis, the extract contains pure compounds 1, 2 and 3 as shown in Table 4 below:
Table 4

Therefore, the total weight of compounds 1, 2 and 3 is 5.92% by weight in the ACM sample.

Test materials and equipment for ACM-EtOH extract
1. Test substances and dose patterns Test substances were administered orally in a 2% Tween 80 vehicle at an initial dose of 1000 mg / kg for all in vivo assays. The observation time for each assay was listed in “Methods”.
2. Animals Male or female ICR mice, male spontaneously hypotensive rats (SHR) from Wistar-Okamoto, rats from Wistar and Long Evans provided by MDS Pharma Services Taiwan Ltd. were used. The space allocation for the animals was as follows: 29 × 18 × 13 cm for 10 mice, 45 × 23 × 21 cm for 6 rats, and 45 × 23 × 21 cm for 3 guinea pigs. Mice and rats were housed in APEC ^R cages. Immunocompetent C57BL / 6J male mice, 6-8 weeks old, weighing 21 +/- 2 gm were also used in this study and were provided by the National Taiwan University Animal Center. Animals were housed in individually Ventilated Cages Racks (IVC rack, 36 Mini Isolar System). All animals were maintained in the laboratory for at least 1 week prior to use in a controlled temperature (21-23 ° C) and humidity (60-70%) environment with a 12 hour light / dark cycle. Free access to standard laboratory food (LabDiet Rodent Diet and Guinea Pig Diet, PMI Mutrition International, USA) and fresh water.
3. Cell line and culture medium The rodent melanoma cell line, B16-F0 (ATCC CRL-6322) was purchased from the American Type Culture Collection and Dulbecco's modified Eagles medium (GIBCO, USA) was used as the culture medium. Tumor cells were incubated at 37 ° C. in an atmosphere containing 5% CO ₂ .
4. General Chemical Substances:
Distilled water (in-house), dimethyl sulfoxide (DMSO, Merck, Germany), isotonic sodium chloride solution (Sintong Chemical Industry Co. Ltd., ROC), magnesium sulfate (MgSO4.7H2O, Wako, Japan), meclofenamate Sodium (Sigma, USA), methylcellulose (Sigma, USA), sodium hydroxide (NaOH, Wako, Japan), phosphate buffered saline (Sigma, USA), and Tween 80 (Wako, Japan).
Reagents Glycose-HA assay kit (Wako, Japan), alanine aminotransferase (ALT) assay kit (Wako, Japan), aspartate aminotransferase (AST) assay kit (Wako, Japan), T-cholesterol-HA and HDL assay kit (Wako, Japan), Hemolynac 3 Hemolys (Nihon Koden, Japan), Isotonic 3 Diluent (Nihon Koden, Japan).
5. Equipment general use:
Animal basket (ShinTeh, ROC), beaker 250ml and 1000ml (Kinmax, USA), disposable syringe (1ml, Top Corporation, Japan), stainless steel forceps (Klappencker, Germany), mouse scale # Z-40 (Taconic, USA), oral Dosing needle (Natsune, Japan), hypodermic needle 23G × 1 "(Top Corporation, Japan), pH meter (Suntex, USA), rat scale 500g +/- 2g (Chien-chun, ROC), glass syringe 1ml, 2ml and 5 ml (Mitsuba, Japan) and stainless scissors (Klappencker, Germany).

ビヒクル及び試験物質を経口的に投与し(PO)、一方で対照化合物を腹膜内に注射した(IP)。その後の30-60分の期間の間、累積的に測定して10秒より長い噛む挙動(口及び/または舌の動き)を示す動物の数、または唾液分泌を示す動物の数を記録した。3のラットの2以上（≧2）で観察されるポジティブの応答は、中枢のコリン作用性活性及び抹消のコリン作用性活性の可能性を示す。 Methods and results
1. Cholinergic agonism, central / deletion (Lippmann W and Pugsley TA. Arch Int Pharmacodyn. 227: 324, 1977)
The test substance was administered orally to a group of male or female rats from Wistar weighing 150 +/- 20 g. Thereafter, during the period of 30-60 minutes, the number of animals showing cumulative biting behavior (mouth and / or tongue movement) longer than 10 seconds and the number of animals showing salivation were recorded. A positive response observed in 2 or more (≧ 2) of 3 rats indicates the possibility of central cholinergic activity and peripheral cholinergic activity.
Table 5: Cholinergic agonism results, central / peripheral in rats

Vehicle and test substance were administered orally (PO) while control compound was injected intraperitoneally (IP). During the subsequent 30-60 minute period, the number of animals cumulatively measured and showing chewing behavior (mouth and / or tongue movement) longer than 10 seconds, or the number of animals showing salivation was recorded. A positive response observed in 2 or more (≧ 2) of 3 rats indicates the possibility of central cholinergic activity and peripheral cholinergic activity.

2. Cardiovascular, blood pressure and pulse (SHR 0, 1, 2, 4 hours) (Yen TT et al., Life Sci. 22: 359, 1978)
A group of 3 male spontaneously hypertensive rats (SHR) from Wistar-Okamoto weighing 250 +/- 20g was used: mean systolic blood pressure was 200 +/- 20mmHg and heart rate was 400 +/- 30 beats / minute. Blood pressure and heart rate were indirectly measured by tail cuff method in a temperature-controlled environment (32 +/- 1 ° C) before (0 hours) and 1, 2 and 4 hours after oral administration of test substance or vehicle. Recorded. Considered a significant decrease in systolic blood pressure by more than 10 percent (≧ 10%) or a decrease in heart rate by more than 20 percent (≧ 20%) at each measured time interval relative to time 0 Is done.

A SHR with a systolic blood pressure of 200 +/- 20 mmHg and a heart rate of 400 +/- 50 mmHg beats / min was used. Blood pressure was indirectly recorded by tail cuff at 0 hours (prior to administration) and 1, 2 and 4 hours after oral administration of vehicle or test substance. A decrease in blood pressure by more than 10 percent (≧ 10%) or a decrease in heart rate by more than 20 percent (≧ 20%) at each time point relative to time 0 is considered significant.
Vehicle 10 ml / kg 0 hour 229 mmHg and 403 mmHg beats / minute were taken as 100%.
ACM-EtOH extract 1000 mg / kg 0 hour 223 mmHg and 452 mmHg beats / min were taken as 100%.
Clonidine 0.1 mg / kg 0 hour 228 mmHg and 379 mmHg beats / minute were taken as 100%.

A SHR with a systolic blood pressure of 200 +/- 20 mmHg and a heart rate of 400 +/- 50 mmHg beats / min was used. Blood pressure was indirectly recorded by tail cuff at 0 hours (prior to administration) and 1, 2 and 4 hours after oral administration of vehicle or test substance. A decrease in blood pressure by more than 10 percent (≧ 10%) or a decrease in heart rate by more than 20 percent (≧ 20%) at each time point relative to time 0 is considered significant.
Vehicle 10 ml / kg 0 hour 220 mmHg and 410 mmHg beats / minute were taken as 100%.
ACM-EtOH extract 300 mg / kg 0 hour 205 mmHg and 446 mmHg beats / min were taken as 100%.
Clonidine 0.1 mg / kg 0 hour 235 mmHg and 417 mmHg beats / minute were taken as 100%.

3. Cholesterol, serum (total HDL, total / HDL, ratio), diet-induced (Schurr PE etc., Atherosclerosis Drug Discovery. Plenum, New York, pp. 215-229, 1976)
A group of 5 ICR-derived male mice weighing 22 +/- 2 g were fed a high fat diet (g / 100 g: coconut oil, 8; cholesterol, 1.0; cholic acid, 0.3; lard, 2; standard diet, 88.7). Test substances were administered orally on days 5, 6 and 7. After overnight fasting, serum was obtained from each mouse and assayed for total cholesterol (total), high density lipoprotein (HDL), and percent change in total / HDL. More than 20 percent (≧ 20%) decrease in serum total, or more than 20 percent (≧ 20%) increase in serum HDL, or more than 40% of total / HDL ratio relative to control animals treated with vehicle ( A reduction of ≧ 40%) is considered significant.

  Vehicle, test substance, or control positive compound was administered orally on days 5, 6 and 7 after feeding a high cholesterol diet (PO). Twenty-four hours after the third dose, animals fasted overnight were killed and serum total cholesterol (total) and high density lipoprotein (HDL) were evaluated. A decrease of 20% or more (≧ 20%) in total serum, or a 20% or more increase in serum HDL (≧ 20%), or a decrease of 40% or more (≧ 40%) in the total / HDL ratio is significant Is considered.

4. Liver injury, D-galactosamine (Wrobel J et al., J. Med Chem 41: 1084, 1998)
A group of 5 Wistar-derived male rats weighing 200 +/- 20 g was used. Each animal was treated with a single injection of D-galactosamine (500 mg / kg, IP). The test substance was administered orally at 0.5 hours before D-galactosamine administration, 4 hours and 8 hours in Toyokan, and the animals were killed 24 hours later. Serum alanine aminotransferase (ALT) and aspartate aminotransferase (AST) levels were measured by an optimized UV method on a HITACHI automated analyzer (model 7050). A reduction of ALT or AST activity by more than 30% (≧ 30%) relative to control animals treated with vehicle indicates significant protection.

  Test substances and vehicle were administered orally 0.5 hours before a single dose of galactosamine (500 mg / kg, IP) and 4,8 hours after administration. Rats were killed 24 hours after galactosamine injection and ALT and AST values were measured. A ≧ 30% reduction in ALT and AST relative to the vehicle group is considered significant.

5. Inflammation, carrageenan (Winter CA et al., Proc Soc Exp Biol Med. 111: 544, 1962)
A group of Long Evans-derived male or female fasted rats weighing 150 +/- 20 g was fasted overnight prior to the experiment. The test substance was administered orally 1 hour before the injection of carrageenan in the right hind leg (0.1 ml 1% suspension, in the sole of the leg). Hindlimb edema as a measure of inflammation was recorded 3 hours after carrageenan administration using a limb volumetric instrument with a water cell (25 mm diameter). A decrease in hind limb edema by more than 30 percent (≧ 30%) indicates significantly acute anti-inflammatory activity.

カラギーナンの右後脚(R.P.)への注射（0.1mlの1％懸濁物、脚の裏内に）の1時間前に、一段絶食したラットにビヒクルまたは試験物質を投与した;左の後脚(L.P.)は何も注射しなかった。カッコ内で示される３０パーセント以上(≧30%)までの後脚の浮腫の減少は、有意な急性の抗炎症活性を示す。

One hour prior to carrageenan injection into the right hind leg (RP) (0.1 ml 1% suspension, in the sole of the leg), vehicle or test substance was administered to fasted rats; left hind leg (LP) did not inject anything. A reduction in hind limb edema of greater than 30 percent (≧ 30%) shown in parentheses indicates significant acute anti-inflammatory activity.

6.Tumor, same genotype, melanoma, B16-F0 cells (Farrugia CA and Groves MJ. Anticancer Research 19: 1027-1032, 1999)
A group of 5 immunocompetent (6-8 weeks old), pathogen-free (SPF) C57BL / 6J female mice housed in an animal isolation box (IVC rack) under specific pathogen-free (SPF) conditions used. C57BL / 6J mice were injected with the syngeneic, viable B16-F0 rodent melanoma cells (ATCC CRL-6322, 1.0 × 10 ^{5 in} 0.2 ml) subcutaneously in the dorsal side of experimental mice. . Treatment was initiated 24 hours after tumor inoculation and the test compound was administered daily by oral gavage for 21 days or less if there were obvious signs of toxicity. Mice were monitored for body weight, tumor size, and onset of survival from day 1 to day 22. Further treated mice were monitored for survival to the end of the experiment on day 45.
Tumor weight (mg) was estimated by the formula for the ellipse: length (mm) × [width (mm)] ² where a specific gravity was assumed to be 1 and π was assumed to be 3. Tumor weight in animals treated with compound was calculated as T / C (treatment / control) x 100%; a T / C value of ≤42% was considered significant indicating antitumor activity . A mean survival time of T / C (treatment / control) ≧ 125% is also considered significant indicating antitumor activity.

  Animals were dosed daily with vehicle and test substance 24 hours after tumor cell transplantation with a total of 21 doses. At the same time, the control compound, mitomycin, was administered IP twice a week for a total of 6 doses. Tumor size was measured and recorded twice weekly for a period of 22 days. Tumor inhibition was calculated as T / C (treatment / control) × 100. A T / C value of ≦ 42% was considered significant indicating antitumor activity.

  Animals were dosed daily with vehicle and test substance 24 hours after tumor cell transplantation with a total of 21 doses. At the same time, the control compound, mitomycin, was administered IP twice a week for a total of 6 doses. Tumor size was measured and recorded twice weekly for a period of 22 days. A student test was used to measure significant differences in body weight changes between the test compound group and the vehicle control group.

a.動物は45日間を通じて死亡せず、生存日数は45日とした。
45日目の実験の終了日、または試験動物が死亡した日まで、生存について処理動物をモニターした。≧125％のT/C(処理/コントロール)の平均生存時間もまた、抗腫瘍活性を示す有意なものと考慮される。

a. Animals did not die during 45 days and survival was 45 days.
Treated animals were monitored for survival until the end of the experiment on day 45 or until the day the test animal died. An average survival time of T / C (treatment / control) ≧ 125% is also considered significant indicating antitumor activity.

Discussion:
According to indicators established in-house, orally administered (PO) ACM-EtOH extract caused significant activity in the following mouse and rat assays.
Central cholinergic agonism was observed in rats at 100 mg / kg; minimal and insignificant agonism was observed at 300 mg / kg; insignificant agonism or antagonism for peripheral cholinergic nerves at 100 mg / kg Observed (Table 5).
Decreased systolic blood pressure (100% at 0 hours vs. 16%, 12% and 20% respectively at 1 and 2 hours observation) and 100 mg / kg in spontaneously hypertensive (SH) rats The associated mild and insignificant decrease in heart rate (Tables 6 and 7); a dose of 300 mg / kg caused no significant changes in systolic blood pressure or heart rate (Tables 8 and 9).
Increased high density lipoprotein at 100 mg / kg in diet-induced mice (HDL, 39% relative to vehicle control) (Table 10); HDL was not significantly altered while the associated total cholesterol (total) was not significantly altered The / total ratio was significantly reduced to close to 31%; the 300 mg / kg dose did not cause significant changes in total, HDL, and HDL / total ratio (Table 10).
Liver protection from galactosamine-induced liver injury in rats at 1000 mg / kg × 3 (44% ALT reduction and 57% AST reduction compared to vehicle control); 300 mg / kg × 3 20 ALT A mild decrease of 28% and 28% of AST was observed (Table 11).
Anti-inflammatory against carrageenan-induced leg edema in rats at 1000 mg / kg (45% inhibition relative to vehicle control (Table 12); lower concentrations of 300 mg / kg showed no significant activity ( 3% inhibition against vehicle, Table 13).
Antitumor activity in the same genotype melanoma B16-F0 against C57BL / 6J mice on days 8, 11 and 15 and prolonged survival of animals at 1000 mg / kg (Table 17); There was no significant change in body weight (Table 16).

Tests for ACM, ACM-EtOH extract and compound 3 of the invention Nine groups of 5 ICR-derived female rats each (body weight 22 +/- 2 g) were used. Each animal was challenged with a single dose of carbon tetrachloride (CCl ₄ , 0.1 ml / kg in 50% olive oil, PO). ACM at doses of 300 and 1000 mg / kg and test substance of compound 3 of the invention at 30, 100 and 300 mg / kg were administered orally 30 minutes before and 4 and 8 hours after the CCl ₄ challenge. Meanwhile, orally administered ACM-EtOH extracts at 300 and 1000 mg / kg were preliminarily stored one day before (twice a day) and 30 minutes before and 4.8 hours after carbon tetrachloride. Processed. Alanine aminotransferase (ALT) and aspartate aminotransferase (AST) concentrations were measured by an optimized UV method using a HITACHI automated analyzer (model 7050). A decrease in ALT and AST concentrations by more than 30 percent (≧ 30%) relative to the vehicle group indicates significant protection from liver injury.

Discussion
ACM, ACM-EtOH extract, and Compound 3 of the present invention were evaluated for possible protective activity against liver injury induced by carbon tetrachloride in ICR mice. ACM at doses of 300 and 1000 mg / kg, Compound 3 of the present invention at doses of 30,100 and 300 mg / kg were orally administered to the test animals 0.5 hours before and 4,8 hours after the CCl4 challenge . For ACM-EtOH extracts at 300 and 1000 mg / kg, two bids (9:00 AM and 16:00 PM) were made one day before CCl4, 0.5 hours before and 4,8 hours before CCl4 challenge This was subsequently followed (total of 5 doses). The degree of liver injury was measured by increasing serum alanine aminotransferase (ALT) and aspartate aminotransferase (AST) concentrations on vehicle treated animals. ACM at 1000 mg / kg × 3 and Compound 3 of the present invention at 300 mg / kg × 3 are significant in ALT (46% and 41%) and AST (36% and 33%) on vehicle treated animals. Caused a decrease. At the same time, ACM-EtOH extracts at 300 and 1000 mg / kg × 5 also caused a significant decrease in ALT (36% and 34%) and AST (25% and 20%). Silymarin (100 mg / kg × 3, IP) tested at the same time showed a significant decrease in ALT (31%) and AST (31%) relative to the vehicle treated group.
It is concluded that ACM, ACM-EtOH, and Compound 3 of the present invention have significant hepatoprotective ability in the mouse CCl ₄ model.

  While the invention has been described and illustrated in sufficient detail to enable those skilled in the art to make and use the invention, various changes, modifications, and improvements will depart from the spirit and scope of the invention. Should be clear without.

  Those skilled in the art will readily anticipate that the present invention will be well employed to carry out its objectives and obtain the results and advantages mentioned, as well as those present. The cell lines, embryos, animals, and processes and methods for producing them represent preferred embodiments, are exemplary, and are not intended to limit the scope of the invention. Modifications and other uses to the invention will occur to those skilled in the art. These modifications are encompassed within the spirit of the invention and are defined by the scope of the claims.

  It will be readily apparent to those skilled in the art that various substitutions and modifications can be made to the invention disclosed herein without departing from the scope and spirit of the invention.

  All patents and publications mentioned in this specification are indicative of the level of those skilled in the art to which this invention pertains. To the same extent that each individual document is specifically and individually pointed to be incorporated by reference, all patents and documents are incorporated herein by reference.

  The invention described herein may be suitably practiced in the absence of any element (s), limitation (s) not specifically disclosed herein. The terms and expressions used are for descriptive purposes and are not to be used as limiting and such terms and expressions may be any of the features shown or described or any part thereof. Although not intended to be used to exclude equivalents, it will be appreciated that various modifications are possible within the scope of the claimed invention. Thus, although the invention has been specifically disclosed by way of preferred embodiments and optimal features, modifications and variations of the concepts disclosed herein will occur to those skilled in the art, and such modifications and changes may be It is to be understood that the invention is considered to be within the scope of the invention as defined by the claims.

  Other embodiments are set forth in the appended claims.

FIG. 1 is a diagram showing the HMBC correlation of Compound 2. FIG. 2 is a diagram showing a compound of the present invention. FIG. 3 is a diagram showing the NOE (nuclear overhauser effect) correlation between compounds 4 and 5 of the present invention. 4 (a)-(d) are diagrams showing test results of Compound 3 of the present invention. FIG. 5 (a)-(c) is a figure which shows the test result of the water extract of ACM (Antrodia camphorata mycelium powder). 6 (a)-(f) are diagrams showing test results of EtCM (ethyl alcohol) extract of ACM. 7 (a)-(e) are diagrams showing test results of Compound 1 of the present invention.

Claims (10)

The following formula:

[Where
X is N or O;
R ₁ is C _1-10 alkyloxy, C _2-10 alkenyloxy, or C _2-10 alkynyloxy;
R ₂ is H, C _1-10 alkyl, C _2-10 alkenyl, or C _2-10 alkynyl;
R ₃ is absent, H, or hydroxy;
However, when X is O, R ₃ is absent]
A compound having 3-isobutyl-4- [4- (3-methyl-2-butenyloxy) phenyl] furan-2,5-dione;
3-isobutyl-4- [4- (3-methyl-2-butenyloxy) phenyl] -1H-pyrrole-2,5-dione;
3-isobutyl-4- [4- (3-methyl-2-butenyloxy) phenyl] -1H-pyrrol-1-ol-2,5-dione;
3R ^* , 4S ^* -1-hydroxy-3-isobutyl-4- [4- (3-methyl-2-butenyloxy) phenyl] pyrrolidine-2,5-dione; or
3R ^* , 4R ^* -1-hydroxy-3-isobutyl-4- [4- (3-methyl-2-butenyloxy) phenyl] pyrrolidine-2,5-dione;
The compound of claim 1, wherein   Antrodia Camphorata mycelium-derived mixture prepared from an aqueous or organic solvent extract of Antrodia Camphorata.   4. The mixture of claim 3, which reduces systolic blood pressure or increases high density lipoprotein.   4. A mixture according to claim 3, having central cholinergic agonism, hepatoprotective, anti-inflammatory or anti-tumor activity. The following formula:

[Where
X is N or O;
R ₁ is C _1-10 alkyloxy, C _2-10 alkenyloxy, or C _2-10 alkynyloxy;
R ₂ is H, C _1-10 alkyl, C _2-10 alkenyl, or C _2-10 alkynyl;
R ₃ is absent, H, or hydroxy;
However, when X is O, R ₃ is absent]
A composition comprising a compound having   7. The composition of claim 6, which reduces systolic blood pressure or increases high density lipoprotein.   7. The composition of claim 6 having central cholinergic agonism, hepatoprotective, anti-inflammatory, or anti-tumor activity.   7. The composition of claim 6, wherein the tumor is derived from a cell or tissue selected from the group consisting of lung, liver, intestine, bone, blood, lymph, and breast.   An mycelium of Antrodia Camphorata comprising the compound of claim 1.