KR20050091464A - Novel mixture and compounds from mycelia of antrodia camphorata and use thereof - Google Patents

Abstract

The present invention relates to novel mixtures and maleic and succinic acid derivatives derived from Antrodia Camporata mycelium, and to medical uses thereof. The present invention relates to a composition or mycelium comprising the compounds of the present invention.

Description

NOVEL MIXTURE AND COMPOUNDS FROM MYCELIA OF ANTRODIA CAMPHORATA AND USE THEREOF}

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

The fruiting body of the Antrodia camphorata (Polyporaceae, Aphyllophorales) is well known as a traditional herbal medicine in Taiwan. This fruiting body only grows on the inner wall of redwood in Cinnamomun kanehirai (Lauraceae), a veritable evergreen tree in Taiwan. This is a rare species and has not been cultivated. This fruiting body has been used for the treatment of food and drug intoxication, diarrhea, abdominal pain, high blood pressure, itching of the skin, and liver cancer. To date, there have been few reports of biological activity studies.

Also known as "niu-chang-chih" or "niu-chang-ku" in Taiwan, Antrodia camphorata is a cylindrical basidiospores, slender amyloid skeleton found recently in fruiting bodies. Mycelia, bitter and light cinnamon resupinate leaves are inverted, characterized by pilates (pileate basidiocarps), and chlamydospore and anthroconidia in pure culture Has been reported as a new fungal species. This new fungus grows very slowly and grows only in Cinnamomum kanehirai Hay (Lauraceae), the endemic tree species as its sole host. Detailed features and taxonomic locations for Antrodia camphorata can be found in Wu, SH, et al ., Antrodia cinnamomea ("niu-chang-chih"), New combination of a medicinal fungus in Taiwan, Bot. Bull. Acad. Sin. 38: 273-275 (1997).

In Taiwan folk medicine, Antrodia camphorata fruiting bodies are believed to have specific medical effects. According to the traditional method, this fruiting body is ground or dehydrated with other herbs for oral ingestion for the treatment of symptoms caused by poisoning, diarrhea, abdominal pain, high blood pressure, skin itching, and liver cancer. However, to date, very little literature has been reported on cases of pharmacological or clinical studies in this regard. Due to the stringent host specificity and scarcity in nature, and the failure of artificial cultivation, "recipients" are very expensive in Taiwan. In recent years, the high quality fruiting bodies of fungi have been sold at extremely high prices of US $ 15,000 per kg.

It is an object of the present invention to provide a novel mixture derived from Antrodia camphorata mycelium.

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

Another object of the present invention is to provide a novel composition comprising the compound of the present invention.

Another object of the present invention is to provide a novel mycelium of antrodia camphorata comprising the compound of the present invention.

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 or H or hydroxy;

Provided that 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.

Thus, preferred compounds of the invention are 3-isobutyl-4- [4- (3-methyl-2-butenyloxy) phenyl] furan-2,5-dione, 3-isobutyl-4- [4- ( 3-methyl-2-butenyloxy) phenyl] -1 H -pyrrole-2,5-dione, 3-isobutyl-4- [4- (3-methyl-2-butenyloxy) phenyl] -1 H -pyrrole-1-ol-2,5-dione, 3R ^*, 4S ^* - 1- hydroxy-3-isobutyl-4- [4- (3-methyl-2-butenyl) phenyl] pyrrolidine 2,5-dione, or a 3R ^*, 4R ^* - 1- hydroxy-3-isobutyl-4- [4- (3-methyl-2-butenyl) phenyl] pyrrolidine-2,5- Dion.

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

The present invention also provides novel mixtures derived from Antrodia camphorata mycelium comprising the compounds of the present invention. The mixture of the present invention is prepared from H ₂ O or organic solvent extracts of the Antrodia camphorata mycelium. Organic solvents include, but are not limited to, alcohols (eg, CH ₃ OH, C ₂ H ₅ OH, C ₂ H ₇ OH), esters (eg, acetyl acetate), alkanes (eg, hexane), and Halogenated alkanes (eg, CH ₃ Cl, C ₂ H ₂ Cl ₂ ). Preferred organic solvents are ethanol or alcoholic solvents which do not cause any side effects in humans. The mixtures of the present invention may reduce cardiac contraction blood pressure or increase high density lipoproteins. Furthermore, the mixtures of the present invention have central cholinergic agonism, hepatoprotection, anti-inflammatory or anti-tumor activity. More specifically, the mixtures of the present invention can inhibit tumors from cells or tissues selected from the group consisting of liver, intestine, bone, lymph, and breast. Acceptable subjects of the mixtures of the present invention include, but are not limited to, humans, mammals, mice, rats, horses, pigs, chickens, ducks, dogs, and cats.

The invention also provides compositions comprising the compounds of the invention. The composition of the present invention may reduce cardiac contraction blood pressure or increase high density lipoprotein. Furthermore, the composition of the present invention has central cholinergic promoting action, hepatoprotective, anti-inflammatory or anti-tumor activity. More specifically, the compositions of the present invention can inhibit tumors from cells or tissues selected from the group consisting of liver, intestine, bone, lymph, and breast. Acceptable subjects of the compositions of the present invention include, but are not limited to, humans, mammals, mice, rats, horses, pigs, chickens, ducks, dogs, and cats.

The invention also provides a novel mycelium of not Trojan dia containing the compound of the present invention. Preferred mycelium contains at least 1% by weight natural mycelium, which is the total weight of compounds 1-5 of the present invention. Most preferred mycelium contains at least 3% by weight natural mycelium, which is the total weight of compounds 1-5 of the present invention. Mycelia of Antrodia camphorata are found in the water presented in TLM Stamford et al., Food Science "Protein enrichment of cashew wastes for animal feeds" by http://www.unu.edu/unupress/food/8F101e/8F101E0b.htm It is already prepared by submerged liquid fermentation.

Example

The following examples are non-limiting and merely present various aspects and features of the present invention.

Overall Experimental Procedure

Melting points were measured on a Yanagimoto micro hot-stage melting point apparatus and no correction was made. Optical rotation was measured using a Jasco DIP-360 automated polarimeter. UV spectra were measured using a Shimadzu UV-2200 read spectrophotometer. IR spectra were measured using a Jasco FT / IR-230 infrared spectrometer. ¹ H-NMR and ¹³ C-NMR spectra were measured using a Varian Unity Plus 500 spectrometer. EIMS and HR-EIMS were measured using a Jeol JMS-AX 505 HAD mass spectrometer under 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 separation

Simpson Biotech Co., Taiwan Antrodia Camphorata mycelium powder (ACM) (60 g) provided in October 2001 by Ltd. was extracted three times with CHCl ₃ for 3 hours under reflux. CHCl ₃ extract (5.3 g) was chromatographed on silica gel with n -hexane-acetone (19: 114: 6) and CHCl ₃ -MeOH (1: 1) as eluent to obtain 9 fractions (fraction 1-9). Got. Fraction 2 was chromatographed on silica gel to give compound 1 (8.7 mg). Fraction 4 was chromatographed on normal and reversed phase silica gel to give compound 2 (13.6 mg). Fraction 5 was chromatographed on silica gel with n -hexane-acetone (8: 2) as eluent to afford ergosterol peroxide (35.8 mg). Fraction 6 was chromatographed on normal and reversed phase silica gel columns to give compound 3 (14.6 mg). Fraction 7 was column chromatographed to give a mixture of compounds 4 and 5 (4: 1). Then, a mixture of compounds 4 and 5 was prepared by prep HPLC [column: Tosoh TSK-gel ODS-80TM (21.5 × 300 mm), mobile phase: CH ₃ OHH ₂ O (70:30) containing 0.1% TFA] Separated through.

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) ν _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 for C ₁₉ H ₂₂ O ₄ , 314.1518).

3-isobutyl-4- [4- (3-methyl- 2- butenyloxy ) phenyl] -1 H -pyrrole-2,5-dione (2): yellow needle ( n -hexane-AcOEt) ; mp 110-111 ° C .; UV (MeOH) λ _max (log ε) 230 (4.3), 272 (3.5), 355 (3.7) nm; IR (CHCl ₃ ) ν _max 1724 cm ⁻¹ ; ¹ H-NMR Table 1; ¹³ C-NMR Table 2; EIMS m / z 313 [M] ⁺ (8), 245 (100), 203 (77), 131 (28); HR-EIMS m / z 313.1681 (calculated for C ₁₉ H ₂₃ NO ₃ , 313.1678).

X-ray crystallographic analysis of compound 2: Yellow precipitate was obtained by crystallization from n -hexane-AcOEt and selected for data collection. Determination data: C ₁₉ H ₂₃ NO ₃ ; M _r = 313.40; Size 0.15x0.02x0.02 mm; Triclinic system, spacer 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 _calculation = 1.223 g / cm ³ , μ (MoKα) = 0.82 cm ⁻¹ , F ₀₀₀ = 336.00. Measurements were performed on a Rigaku RAXIS-RAPID Imaging Plate diffractometer using graphite monochromated Mo-Kα (λ = 0.71069 μs) radioactivity at 93K. In the collected 8950 reflections, 4745 was unique (R _int = 0.108) and equivalent reflectances were merged. The crystal structure was interpreted by the direct method (SHELXS86) and refined to the full-matrix least-square. Atoms other than hydrogen atoms were anisotropically corrected. Hydrogen atoms were included without correction. The final indices were R = 0.074, R _w = 0.099, with GOF (Guest Observer Facility) = 1.06. The maximum and minimum peaks on the final difference Fourier map corresponded to 0.83 and 0.89 e ⁻ / Å ³ , respectively.

3-isobutyl-4- [4- (3-methyl-2-butenyloxy) phenyl] -1 H -pyrrole-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 ) ν _max 1717 cm ⁻¹ ; ¹ H-NMR Table 1; ¹³ C-NMR Table 2; EIMS m / z 329 [M] ⁺ (12), 261 (100), 131 (50); HR-EIMS m / z : 329.1637 (calculated for C ₁₉ H ₂₃ NO ₄ , 329.1627).

3R ^*, 4S ^* - 1- hydroxy-3-isobutyl-4- [4- (3-methyl-2-butenyl) phenyl] pyrrolidine-2,5-dione (4): colorless oil; [a] _D ²³ + 2.5 ° ( c 0.2, MeOH); UV (MeOH) λ _max (log ε): 225 (4.3), 275 (3.3), 283 (3.2) nm; IR (CHCl ₃ ) ν _max 1715 cm ⁻¹ ; ¹ H-NMR Table 1; ¹³ C-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 (calculated for C ₁₉ H ₂₅ NO ₄ , 331.1783).

3R ^*, 4R ^* - 1- hydroxy-3-isobutyl-4- [4- (3-methyl-2-butenyl) phenyl] pyrrolidine-2,5-dione (5): colorless oil; [a] _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 ) ν _max 1715 cm ⁻¹ ; ¹ H-NMR Table 1; ¹³ C-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 (calculated for C ₁₉ H ₂₅ NO ₄ , 331.1783).

Ergosterol peroxide: colorless needles ( n -hexane-acetone); mp 165-169 ° C. (lit ² mp 171-174 ° C.).

Cytotoxicity Analysis: In vitro LLC tumor cell analysis was performed using the sulforhodamine B (SRB) method. 50% growth inhibition rate (ED ₅₀ ) was measured by the Probit method (Probit method).

Results and results

Not Trojan dia Campo rata mycelium of CHCl ₃ extracts a normal phase and a new maleic and succinic acid derivatives of the five kinds with a result of repeated chromatography on reverse phase silica gel, ergosterol peroxide - is (compound 15) was obtained.

Table 1: Compounds 1-5 ¹ H-NMR spectral data (δ ppm, J = Hz) (500 MHz, CDCl ₃ )

Table 2: of compounds 1-5 ¹³ C-NMR spectral data (δ ppm) (125 MHz, CDCl ₃ )

a) Designations may be exchangeable.

The structure of the new compounds was determined as follows:

Compound 2 gave a yellow needle (mp 110-111 ° C.) and the molecular formula C ₁₉ H ₂₃ NO ₃ was designated by HR-EIMS. The IR spectrum showed imide carbonyl adsorption at 1724 cm ⁻¹ . ^{The 13} C-NMR spectrum showed signals for four methyl carbons, two methylene carbons, and one methine carbon in the aliphatic region, and one signal for one benzene ring, one olefin group, and two carbonyl carbons. Appeared. ^{In the 1} H-NMR spectrum, isobutyl moieties were present at δ 0.90, 2.06, and 2.51, and 3-methyl-2-butenyloxy moieties were present at δ 1.76, 1.81, 4.56, and 5.50 It was found that para -substituted benzene moieties were present at δ 6.95 and 7.50. This was further demonstrated by ¹ H- ¹ H COZY (cooler synchrotron) and HMQC (heteronuclear multiple quantum coherence) experiments. Long range correlations were observed by HMBC as shown in FIG. 1. Based on the molecular formula and ¹³ C-NMR spectra, it was estimated that this compound contains additional CHNO atoms containing one or more carbonyl carbons. Therefore, it is assumed that this unclear part is a maleimide group. Subsequently, this structure was confirmed to be 3-isobutyl-4- [4- (3-methyl-2-butenyloxy) phenyl] -1 H -pyrrole-2,5-dione by X-ray analysis.

The molecular formula of compound 1 was designated C ₁₉ H ₂₂ NO ₄ by HR-EIMS. In the IR spectrum, carbonyl adsorption of acid anhydride was found at 1763 cm ⁻¹ . ¹ H-NMR spectrum of the compound 1 was similar to that of compound 2, an isobutyl moiety, 3-methyl-2-butenyl oxy moiety, and para-substituted benzene ring is shown to exist. Judging from the HMBC spectrum, Compound 1 proved to have the same partial structure as Compound 2 (FIG. 1), where the presence of maleic anhydride groups indicated that Compound 1 was 3-isobutyl-4- [4- (3- It was estimated on the basis that it was determined as methyl-2-butenyloxy) phenyl] furan-2,5-dione.

The molecular formula of compound 3 was designated C ₁₉ H ₂₃ NO ₄ by HR-EIMS. The number in the IR spectrum indicated carbonyl adsorption that can be designated as hydroxy imide at 1717 cm ⁻¹ . ¹ H-NMR and ¹³ C-NMR spectra were also similar to those of compounds 1 and 2 , with the presence of an isobutyl moiety, a 3-methyl-2-butenyloxy moiety, and a para -substituted benzene ring. In the HMBC experiment, compound 3 was found to have the same partial structure as compound 2 (FIG. 1). Compound 3 contains one more oxygen atom than Compound 2 , thus compound (3-isobutyl-4- [4- (3-methyl-2-butenyloxy) phenyl-1 H -pyrrole-1- It was determined to be all-2,5-dione.

Compounds 4 and 5 had the same R _f value and the same molecular formula (as observed at C ₁₉ H ₂₅ NO ₄ , 331.1747 and 331.1766, respectively) by HR-EIMS, but they could be separated by preparative HPLC. IR spectra of these two compounds showed hydroxy imide carbonyl adsorption at 1747 cm ⁻¹ . ^{In the 1} H-NMR 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, but with isobutyl methylene both showed the multi-split, compound 1 did not show a doublet, as in the case of three. ^{The 1} H- ¹ H COSY spectrum suggests that this methylene group is attached to the CH-CH- unit. ¹³ C-NMR spectrum of the compound 4 and 5 was characterized by the two additional sp ³ carbon signal in the second instead of sp ³ carbon signal observed in the compound 13. Thus, compounds 4 and 5 were not N -hydroxy maleimide, but N -hydroxy succinimide having a stereocenter at positions C-3 and C-4 in the succinimide ring. Compounds 4 and 5 were determined to be trans and cis isomers from the binding constants between H-3 and H-4, respectively (4.0 and 8.0 for compounds 4 and 5, respectively). No NOE was observed between H-3 and H- 4 in the NOESY (Nuclear Overhauser Effect Spectroscopy) spectrum of Compound 4 , while an acceptable NOE was observed in Compound 5 . The optical rotations of compounds 4 and 5 were + 2.5 ° and + 3.0 °, respectively, but their CD spectra showed no cotton effect at any wavelength length, suggesting that both compounds 4 and 5 are racemic mixtures. HPLC using chiral columns with several solvent systems, but the resolution of these racemic mixtures was not successful. At present, we cannot clearly conclude whether these compounds are optically active compounds or racemic mixtures. Thus, 3R ^* their respective structures, respectively, 4S ^* - and 3R ^*, 4R ^* - 1- hydroxy-3-isobutyl-4- [4- (3-methyl-2-butenyl) phenyl] P Determined by Ralidin-2,5-dione.

As reported by Aquveque et al., This type of natural maleic acid and succinic acid derivative was isolated twice.

Using the Lewis lung carcinoma (LLC) cell line, the cytotoxic activity of the chloroform extract and the isolated compound was examined (Table 3). Chloroform extract showed moderate cytotoxic activity with ED ₅₀ value of 26.7 μg / ml. Maleic acid based compounds 1 and 4 showed no cytotoxicity, while compounds 2 and 3 showed lower ED ₅₀ values than chloroform extracts and were found to be cytotoxic to LLC cell lines.

Table 3: For LLC cell lines Antrodia Camphorata  CHCl from Mycelium ₃  50% growth inhibition of extracts and compounds 1-4 (ED ₅₀ ) Value

a) positive control.

ACM ( Antrodia Camphorata Tumor Analysis of Mycelia Powder)

A. Cell Line

Adherent cells:

MCF-7: Human Breast 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. Analysis Method

ED ₅₀ (50% inhibition of effective amount) calculation

Adherent cells: MTT (methyl thiazolyl tetrazolium) method; MCF-7, HT-29, KATO III, SW480 and HepG2 cells were measured on day 3 and SW620 on day 4.

Suspension cells: cell counting method; EL4 cells counted on day 5.

D. Results

Calculation: y = mLn (x) + b

<Example>

X Y 0 0.97 10 ppm 0.941 30 ppm 0.6 100 ppm 0.331

A correlation curve is obtained using the values of X (10, 30, 50 ppm) and Y.

y = -0.2643Ln (x) + 1.5321

ED ₅₀ = 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 of ACM to 40 ml RO H ₂ O in a 250 ml beaker and leave the beaker in an ultrasonic bath at room temperature for 20 minutes.

2. Stir for 45 minutes in 45 ℃ water bath

3. Leave the beaker in the ultrasonic bath for an additional 20 minutes

4. Centrifuge the sample at 3000 rpm for 15 minutes

5. Collect supernatant and perform serial dilution with medium.

B. Sample Concentration Measurement

1.Weighing of the evaporating dish (W1)

2. Add 10 ml of H ₂ O extract sample to the evaporation dish

3. Put the evaporating dish in the oven to remove moisture (W2)

Sample weight / ml = (W2-W1) / 10

C. ACM ( Antrodia Camphorata  Mycelium Powder) EtOH Extract

1. Add 100 ml of 95% alcohol to 20 g ACM in 500 ml beaker and stir at room temperature for 10 minutes

2. Filter the suspension with Advantec ＃ 1 filter paper and collect the filtrate.

3. Concentrate the filtrate using a rotary vacuum evaporator to remove alcohol.

D. Compound 1: Pure Compound from ACM

E. Compound 3: Pure Compound from ACM

MTT analysis method

1. After cell proliferation, remove the existing medium and wash cells once with phosphate-buffered saline (PBS).

2. Cell Washing with Trypsin-EDTA

3. Centrifuge at 1200 rpm for 5 minutes, then remove supernatant

4. Pellet Suspension Using 10 ml Medium

5. Calculate viable cells by mixing 100 μl Trypan Blue into 100 μl cell suspension

6. Add 1 × 10 ⁴ cells / 100 μl of medium to each well of a 96 well plate and incubate the plate for 24 hours in a CO ₂ incubator at 37 ° C.

7. After removing the existing medium, wash cells once with PBS

8. Add 100 μl of sample to each well and incubate the plate in CO ₂ incubator at 37 ° C.

9. Cell washing with PBS after 3, 4 and 5 days

10. Add 57 μl MTT (0.88 mg / mL) to each well

11. After 4 hours, remove MTT and wash cells once with PBS.

12. Add 50 μl DMSO / well

13.Read OD545 on ELISA Reader

Cell counting method (EL4 cell line)

1. Removal of existing medium after cell proliferation by centrifugation

2. Resuspend pellet using fresh medium

3. Calculate viable cells by mixing 100 μl Trypan Blue into 100 μl cell suspension

4. Sample preparation of different concentrations containing 1 × 10 ⁴ cells per μl of sample

5. Load 100 μl of sample into each well of 96 well plates and incubate the plate in CO ₂ incubator at 37 ° C.

7. Viable Cell Counting After 3, 4, and 5 Days

PBS

NaCl 8g

KCl 0.2g

Na ₂ HPO ₄ 1.4 g

KH ₂ PO ₄ 0.2g

Volume to 1 l (pH 7.4)

Results and analysis

ED _{50 of} ACM for Cell Line

Detailed test results are as follows:

Compounds 3 of the invention: HepG2 (FIG. 4A), EL4 (FIG. 4B), HT-29 (FIG. 4C) and Kato III (FIG. 4D).

ACM H2O extracts: HepG2 (FIG. 5A), SW620 (FIG. 5B) and EL4 (FIG. 5C).

ACM EtOH extract: HT-29 (FIG. 6A), SW480 (FIG. 6B), SW620 (FIG. 6C), EL4 (FIG. 6D), HepG2 (FIG. 6E) and Kato III (FIG. 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).

From the above, it is demonstrated that the compound of the present invention and the ACM extract have an inhibitory effect on various types of tumor cells.

Analysis of all new compounds (1, 2 and 3) derived from ACM EtoH extract by liquid chromatography method

Purpose: To quantify all new compounds ( 1 , 2 and 3 ) derived from ACM EtOH extract, high performance liquid chromatography was used as a conventional quality control procedure.

Preparation of ACM EtOH Extract Samples:

1) Using a digital balance, exactly 20.000 g of sample powder was weighed into a graduated incubation bottle containing 100 ml of 95% alcohol and the lid was not tightly locked.

2) The sample bottle of the step is left for 10 minutes in an ultrasonic bath.

3) The liquid sample is placed in a centrifuge tube and centrifuged at 6500 rpm for 5 minutes to remove coarse particles.

4) The liquid layer was advantec no. 1 Filter by filter paper.

5) Concentrate the filtration solution using a rotary vacuum evaporator to obtain a dark yellow non-alcoholic liquid.

6) Repeat steps 1-5 five times, then collect all extraction products (total ACM EtOH extract = 4.60 g) and calculate the yield.

H ₂ Utilizes HPLC, Model 2690:

1) column: reverse phase C18

2) Mobile phase: MeOH ?? H ₂ O ?? acetonitrile

3) Injection volume: 20 μl

4) Detection: Photodiode Array Detector 996, Wavelength 254 nm

5) Prepare 1.000 g of ACM EtOH extract sample in 10 ml alcohol for HPLC analysis ^*

Results : According to HPLC analysis, the extraction products containing pure compounds 1 , 2 , and 3 are shown in Table 4 below.

Table 4:

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

Test for ACM-EtOH Extract

Materials and utensils

1. Test substance and dosage pattern

Test substances were administered orally at an initial dose of 1000 mg / kg in 2% Tween 80 excipients for all in vitro assays. Observation times for each assay are listed in the “Methods” section.

2. Animal

Male or female ICR mouse, MDS Pharma Services Taiwan Ltd. Spontaneous hypertensive male rats (SHR), Wistar and Long Evans rats of Wistar-Okamoto family provided were used. The space allocation for the animals is as follows: 29 × 18 × 13 cm per 10 mice, 45 × 23 × 21 cm per 6 rats, and 45 × 23 × 21 cm per 3 guinea pigs. Mice and rats were housed in APECR cages. 6-8 week old immunocompetent C57BL / 6J male mice weighing 21 ± 2 mg were also used in this study, which was provided by the National Taiwan University Animal Center. Animals were housed in individually ventilated cage racks (IVC rack, 36 Mini Isolator System). The cage was sterilized by autoclave and housed 5 mice (26.7 × 20.7 × 14 cm) per cage. Prior to use in the experiments, all animals were maintained for at least 1 week under controlled temperature (21 ° C.-23 ° C.) and humidity (60% -70%) environments with a 12 hour contrast cycle. Standard laboratory feed (LabDiet Rodent Diet and Guinea Pig Diet, PMI Nutrition International, USA) and tap water were freely available.

3. Cell Lines and Medium

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 medium. Tumor cells were incubated at 37 ° C. in an atmosphere containing 5% CO ₂ .

4. Chemical

Overall:

Distilled water (domestic), dimethyl sulfoxide (DMSO, Merck, Germany), sodium chloride isotonic solution (Sintong Chemical Industry Co. Ltd., ROC), magnesium sulphate _{(MgSO 4 .7H 2 0, Wako} , Japan), methoxy phenacyl chloride formate Sodium (Sigma, USA), methylcellulose (Signa, USA), sodium hydroxide (NaOH, Wako, Japan), phosphate buffered saline (Sigma, USA) and Tween 80 (Wako, Japan).

sample:

Glucose-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. Appliance

Overall use:

Breeding case (ShinTeh, ROC), beaker 250 ml and 1000 ml (Kinmax, USA), disposable syringe (1 ml, Top Corporation, Japan), stainless steel tweezers (klappencker, Germany), mouse grade # Z-40 (Taconic, USA), needle for oral administration (Natsune, Japan), needle hypodermic syringe 23 Gx1 (Top Corpoation, Japan), pH meter (Suntex, USA), rat grade 500 g ± 2 g (Chien-chun, ROC), glass syringe 1 Ml, 2 ml and 5 ml (Mitsuba, Japan), and stainless steel scissors (Klappencker, Germany).

Method and result:

Central / peripheral cholinergic action (Lippmann W and Pugsley TA. Arch Int Pharmacodyn. 227: 324, 1977)

Test substances were administered orally to three Wistar male or female rats weighing 150 ± 20 g. Over the next 30-60 minutes, the number of animals exhibiting chewing behavior (mouth and / or tongue movement) of 10 seconds or more and the number of animals exhibiting saliva secretion were measured and recorded. A positive response observed in two or more of 3 rats (≧ 2) indicates that there may be central choline activity and peripheral choline activity.

Table 5: Results of Central / Peripheral Cholinergic Promotion in Rats

Excipients and test agents were administered orally (PO) and injected with a positive reference compound intraperitoneally (IP). Over the next 30-60 minutes, the number of animals showing at least 10 seconds of chewing behavior (mouth and / or tongue movement) and the number of animals showing saliva secretion were measured and recorded. Positive reactions observed in two or more of three rats indicate that there may be central choline activity and peripheral choline activity (≧ 2).

2. Cardiovascular blood pressure and heart rate (SHR 0, 1, 2, 4 hours) (Yen TT et al. Life Sci. 22: 359, 1978)

A group of three spontaneous hypertensive male rats (SHR) of the Wistar-Okamoto system weighing 250 ± 20 g were used. Mean heart contraction blood pressure was 200 ± 20 mmHg and heart rate was 400 ± 30 beats / minute. Blood pressure and heart rate were indirectly measured using the tail cuff method 1 hour, 2 hours and 4 hours prior to oral administration of a test substance or excipient under a temperature controlled environment (32 ± 1 ° C.). A significant reduction in cardiac contraction pressure of 10% or more (≧ 10%), or 20% or more (≧ 20%) reduction in cardiac contraction at each measurement point, immediately before dosing (0 hour), is considered significant.

Table 6: Results of Cardiovascular Blood Pressure in Rats (SHR 0, 1, 2, 4 hours)

Table 7: Results of cardiovascular heart rate (SHR 0, 1, 2, 4 hours)

SHR with a heart rate of 400 ± 50 mmHg beats / minute and a heart contraction blood pressure of 200 ± 20 mmHg was used. Blood pressure was indirectly recorded at the tail tip immediately before (0 hour) and 1 hour, 2 hours, and 4 hours after administration of the excipient or test substance. As indicated in parentheses, significant decreases in blood pressure at the time of each measurement at least 10% (≥10%) or at least 20% (≥20%) reduction in heart rate at the time prior to dosing (at 0 o'clock) are significant. To be considered.

Excipients: 229 mmHg and 403 mmHg immediately before (10 o'clock) 10 ml / kg administration

Beats / min-set to 100%.

ACM-EtOH extract: 223 mmHg and 452 mmHg immediately before (1000 hours) 1000 mg / kg dose

Beats / min-set to 100%.

Clonidine: 228 mmHg and 379 mmHg immediately before 0.1 mg / kg administration

Beats / min-set to 100%.

Table 8: Results of cardiovascular blood pressure in rats (SHR 0, 1, 2, 4 hours)

Table 9: Results of Cardiovascular Heart Rate (SHR 0, 1, 2, 4 Hours)

SHR with a heart rate of 400 ± 50 mmHg beats / minute and a heart contraction blood pressure of 200 ± 20 mmHg was used. Blood pressure was indirectly recorded at the tail tip immediately before (0 hour) and 1 hour, 2 hours, and 4 hours after administration of the excipient or test substance. As indicated in parentheses, blood pressure reduction of 10% or more (≧ 10%) or heart rate reduction of 20% or more (≧ 20%) is considered significant when measured at regular time intervals from administration (0 hour).

Excipients: 220 mmHg and 410 mmHg immediately before (10 o'clock) 10 ml / kg administration

Beats / min-set to 100%.

ACM-EtOH extract: 205 mmHg and 446 mmHg immediately before (at 0 o'clock) 300 mg / kg

Beats / min-set to 100%.

Clonidine: 235 mmHg and 417 mmHg immediately before 0.1 mg / kg dose

Beats / min-set to 100%.

3. Dietary Cholesterol Serum (Total HDL, Total / HDL Ratio) (Schurr PE et al., Atherosclerosis Drug Discovery.Plenum, New York, pp. 215-229, 1976)

A group of five ICR male mice weighing 22 ± 2 g were fed a high fat diet (g / 100g: coconut oil, 8; cholesterol, 1.0; cholic acid, 0.3; lard 2; standard feed 88.7) for 7 days, Hypercholesterolemia was induced. Test substances were administered orally on days 5, 6 and 7. After fasting overnight, serum was collected from each mouse and evaluated for total cholesterol (total), high density lipoprotein (HDL) and total / HDL percent change (%). A reduction of at least 20% (≧ 20%) of serum total or at least 40% (≧ 40%) of total / HDL ratio is considered significant based on excipient treated control animals.

Table 10: Cholesterol induced by diet in mice, (total / HDL, total / HDL ratio)

After ingesting a high cholesterol diet, 5 days, 6 days and 7 days later, it was administered via excipients, test substances, and positive reference compounds (PO). Twenty four hours after the third dose, the test animals fasted overnight were sacrificed and evaluated for total cholesterol (total) and high density lipoprotein (HDL). A reduction of at least 20% (≧ 20%) of serum total or an increase of at least 20% (≧ 20%) of serum HDL or a reduction of at least 40% (≧ 40%) of total / HDL ratio is considered significant. .

4. Liver damage, D-galactosamine (Wrobel J et al., J. Med Chem 41: 1084, 1998)

A group of 5 Wistar male mice weighing 220 ± 20 g were used. Each animal was treated once with injection of D-galactosamine (500 mg / kg, IP). The test substances were administered 0.5 hours before and 4 and 8 hours after D-galactosamine, and animals were sacrificed 24 hours later. Serum allamine aminotransferase (ALT) and aspartate aminotransferase (AST) levels were measured by an optimized UV method using a HITACHI automated analyzer (model 7050). A 30% or more (≧ 30%) reduction in ALT or AST activity relative to excipient treated animals suggests significant protective effects.

Table 11: Results of Liver Injury Induced by Galactosamine in Rats

Test substances and excipients were administered orally 0.5 hours prior to galactosamine (500 mg / kg, IP) and 4 and 8 hours after administration. Animals were sacrificed 24 hours after galactosamine injection and ALT and AST values were measured. A 30% or more (≧ 30%) reduction in ALT or AST activity relative to the excipient treatment group is considered significant.

5. Inflammation Caused by Carrageenan (Winter CA et al., Proc SocExp Biol Med. 111: 544, 1962)

Groups of three Long Evans male or female mice weighing 150 ± 20 g that were fasted overnight were fasted overnight before the start of the study. One hour after the test substance was administered orally, carrageenan was injected into the right hind paw (0.1 ml 1% suspension, intraplantar injection). Inflammation was measured by recording hind edema 3 hours after carrageenan administration using a volumetric plethysmometer equipped with a water cell (25 mm diameter). A 30% or more reduction in hind edema (≥30%) suggests significant anti-inflammatory activity.

Table 12: Results of Inflammation Induced by Carrageenan in Rats

Table 13: Results of Inflammation Induced by Carrageenan in Rats

Rats fasted overnight were dosed with excipients or test substances one hour after injection of carrageenan (R.P.) in the right hind (R.P.) and no injection in the left hind paw. As shown in parentheses, a reduction of 30% or more (≧ 30%) of hind edema suggests significant anti-inflammatory activity.

6. Tumors, syngeneic (syngeneic), melanoma, B16-F0 cells (Farrugia CA and Groves MJ.Anticancer Research 19: 1027-1032, 1999)

Immunogenic (SPF) C57BL / 6J male mice bred in animal separators (IVC racks) under specific pathogen free (SPF) conditions were used. Viable B16-F0 rodent melanoma cells (ATCC CRL-6322, 1.0 × 10 5 in 0.2 mL) syngeneic with C57BL / 6J mice were injected through the dorsal subcutaneous of the experimental animals. Treatment commenced 24 hours after tumor inoculation and test compounds were administered daily via oral gavage for 21 days or until clear signs of toxicity appeared. Mice were monitored for body weight, tumor size, and survival from day 1 to day 22. In addition, survival of surviving mice was monitored by day 45 at the end of the study.

Tumor weight (mg) was calculated by estimating specific gravity as 1 and π (3) according to the formula length (mm) × [width (mm)] ² × 0.5 for the spheroids. Tumor growth in the compound-treated animals was calculated as T / C (treatment / control) x 100%, and when the T / C value was ≤42%, it was significant in demonstrating antitumor activity. Consider.

An average survival time of ≧ 125% of T / C (treatment / control) is also considered significant in demonstrating antitumor activity.

Table 14: Results of Tumor, Syngeneic, Melanoma B16-F0 Cells

Table 15: Results of tumor, syngeneic, melanoma B16-F0 cells

Twenty four hours after tumor cell transplantation, the test animals received a total of 21 excipients and test substances daily. At the same time, the reference compound mitomycin was administered IP totally six times, twice a week. Tumor size was measured and recorded twice a week for 22 days. Inhibition rate of tumor growth was calculated as T / C (treatment / control) × 100. A value of 42% of T / C is considered significant in demonstrating antitumor activity.

Table 16: Results of tumor, syngeneic, melanoma B16-F0 cells

Twenty four hours after tumor cell transplantation, the test animals received a total of 21 excipients and test substances daily. At the same time, a total of six IP administrations of the reference compound, mitomycin, was performed twice a week. Tumor size was measured and recorded twice a week for 22 days. Student t tests were used to determine significant differences in body weight changes between test compound groups and excipient controls.

Table 17: Results of tumor, syngeneic, melanoma B16-F0 cells

a: The animals did not survive until 45 days, so the survival date was 45 days.

Survival of the treated mice was monitored until 45 days at the end of the study or until the test animals died. A mean survival time of T / C (treatment / control) ≧ 125% is also considered significant in demonstrating antitumor activity.

Analyze :

ACM-EtOH extract administered orally (PO) according to established criteria in the art induced significant activity in the following mouse and rat assays:

Central cholinergic promotion at the 1000 mg / kg dose in rats; Minimal, insignificant facilitation was observed at the 300 mg / kg dose and action on peripheral cholinergic nerves at the dose of 1000 mg / kg (Table 5).

In spontaneous hypertension (SH) rats, reductions in cardiac contraction blood pressure at the 1000 mg / kg dose (16%, 12% and 20, respectively, at 1 hour, 2 hours, and 4 hours later at 100% at 0 hours) %) And associated moderately insignificant decreases in heart rate (Tables 6 and 7); At the 300 mg / kg dose, cardiac contraction blood pressure and heart rate did not change significantly (Tables 8 and 9).

In mice induced by diet, high density lipoprotein increase (HDL, 39% over excipient control) at 1000 mg / kg dose (Table 10); The associated total cholesterol (total) did not change significantly, but the HDL / total ratio decreased to almost significant 31%. There was no significant change in total, HDL and HDL / total ratios at the 300 mg / kg dose (Table 10).

In rats, hepatoprotective action from liver damage caused by galactosamine at 1000 mg / kg dose (44% decrease in ALT and 57% decrease in AST, based on excipient controls); At the 300 mg / kg × 3 dose, there was a modest decrease of 20% for ALT and 28% for AST (Table 11).

Anti-inflammatory action (45% inhibition vs. excipient control) on forefoot edema caused by carrageenan (1000 mg / kg) (Table 12); At low levels of 300 mg / kg no significant activity was seen (3% inhibition vs. excipients, Table 13).

Extending animal survival time at 1000 mg / kg dose (Table 17), and antitumor activity in syngeneic melanoma B16-F0 cells for C57BL / 6J mice after 8, 11 and 15 days (Tables 14 and 15) ); The body weight of the animals did not change significantly (Table 16).

Compound 3 , ACM, and ACM-ETOH extracts of the invention:

Nine groups of ICR male mice (22 ± 2 g body weight) consisting of 5 mice each were used. Each animal received one dose of carbon tetrachloride (CCl ₄ , 0.1 mL / kg in 50% olive oil, PO). 300 and 1000 mg / kg of ACM test substance, or 30, 100 and 300 mg / kg of the compound of the present invention were administered orally 30 minutes prior to CCl ₄ and 4 and 8 hours after administration. ACM-EtOH extract was administered orally 300 and 1000 mg / kg doses a day (twice a day) and 30 minutes before, and 4 and 8 hours after the administration of CCl ₄ . Animals were sacrificed 24 hours after CCl ₄ administration. Alanine aminotransferase (ALT) and aspartate aminotransferase (AST) levels were measured using an optimized UV method using a HITACHI automated analyzer (model 7050). A 30% or more (≧ 30%) reduction in ALT or AST levels relative to the excipient group is considered to have a significant protective effect from liver damage.

result

Table 18: Liver Damage Induced by Carbon Tetrachloride in Mice

Analyze :

Compound 3 , ACM, and ACM-EtOH extracts of the invention were evaluated for possible protective activity against liver damage caused by carbon tetrachloride in ICR mice. 300 and 1000 mg / kg of ACM test substance, and 30, 100 and 300 mg / kg of the compound of the present invention were administered orally 0.5 hours prior to CCl ₄ administration and 4 hours and 8 hours after administration. The ACM-EtOH extract was treated with two doses (bids 9 and 16 pm) at 300 and 1000 mg / kg doses one day before and 30 minutes before and 4 and 8 hours after administration of CCl ₄ . The degree of liver injury was measured by increasing serum alanine aminotransferase (ALT) and aspartate aminotransferase (AST) levels in excipient treated animals. ACM at 1000 mg / kg × 3 doses, and Compound 3 of the present invention at 300 mg / kg × 3 doses of ALT (46% and 41%) and AST (36% and 33%) compared to excipient treated animals. Significant reduction was induced. At the same time, ACM-EtOH extract induced a significant reduction of ALT (36% and 34%) and AST (25% and 20%) at 300 and 1000 mg / kg × 5 doses.

Silymarin (100 mg / kg × 3, IP) tested at the same time significantly reduced ALT (31%) and AST (31%) compared to the excipient treatment group.

Compounds 3 , ACM, and ACM-EtOH extracts of the present invention have significant hepatoprotective activity 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, it is to be understood that various changes, modifications, and improvements may be made without departing from the spirit and scope of the invention.

Those skilled in the art will understand that the present invention can be well adapted to carry out the above-described objects as well as the features inherent to the present invention and to obtain the results and advantages of the present invention. Cell lines, fetuses, animals, and procedures and methods for making them are merely exemplary of the preferred embodiments and are not intended to limit the scope of the invention. Those skilled in the art will be able to implement the modifications and other uses illustrated herein. These modifications are included within the scope of the present invention and defined by the claims of the present invention.

Those skilled in the art will appreciate that the invention disclosed herein may be variously substituted and modified without departing from the scope and spirit of the invention.

All patents and publications mentioned in the specification are indicative of the level of ordinary skill in the art, the contents of which are incorporated herein by reference.

The invention may be practiced in the absence of elements or elements, limitations or limitations not specifically described herein. The terms and expressions used are for the purpose of description and are not used in a limiting sense, and are not intended to use such terms and expressions, excluding any equivalents of the features presented and described and any part thereof, but various modifications may be made. It will be understood that the invention is possible within the scope of the claims. Thus, although the invention has been described in detail by its preferred embodiments and optional features, those skilled in the art can employ variations and modifications of the concepts disclosed herein, which variations and modifications are defined in the appended claims. It should be understood that it is included within the scope of the present invention.

Other embodiments are presented in the following claims.

The present invention provides novel mixtures and novel compounds derived from Antrodia camphorata mycelium, and novel compositions comprising the compounds of the present invention and novel mycelium of Antrodia camphorata .

1 shows the HMBC correlation of compound 2. FIG.

2 shows compounds of the invention.

FIG. 3 is a diagram showing the correlation of the clear overhauser effect (NOE) of compounds 4 and 5 of the present invention.

4 shows the test results of the compound 3 of the present invention.

Figure 5 is a view showing the test results of the ACM ( Antrodia camphorata mycelium powder) H ₂ O extract.

6 shows the test results of ACM EtOH (ethyl alcohol) extract.

7 shows the test results of Compound 1 of the present invention.

Claims (20)

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 or H or hydroxy; Provided that when X is O, R ₃ is absent. The method of claim 1, R ₁ is C _2-6 alkenyloxy or C _2-6 alkynyloxy. The method of claim 2, C _2-6 alkenyloxy substituted with C _1-6 alkyl. The method of claim 1, R ₂ is isobutyl. The method of claim 1, 3-isobutyl-4- [4- (3-methyl-2-butenyloxy) phenyl] furan-2,5-dione, 3-isobutyl-4- [4- (3-methyl-2-butenyl Oxy) phenyl] -1 H -pyrrole-2,5-dione, 3-isobutyl-4- [4- (3-methyl-2-butenyloxy) phenyl] -1 H -pyrrole-1-ol-2 , 5-dione, 3R ^*, 4S ^* - 1- hydroxy-3-isobutyl-4- [4- (3-methyl-2-butenyl) phenyl] pyrrolidine-2,5-dione, or 3R ^*, 4R ^* - 1- hydroxy-3-isobutyl-4- [4- (3-methyl-2-butenyl) phenyl] pyrrolidine-2,5-dione compounds. Not Trojan dia Campo rata (Antrodia Camphorata) mixture of dia not Trojan Campo rata mycelium derived prepared from mycelium water extract or organic solvent extract of. The method of claim 6, A mixture derived from antrodia camphorata mycelium , characterized in that the organic solvent is alcohol, ester, alkanes or halogenated alkanes. The method of claim 7, wherein A mixture derived from the Antrodia camphorata mycelium, wherein the alcohol is ethanol. The method of claim 6, The compound is 3-isobutyl-4- [4- (3-methyl-2-butenyloxy) phenyl] furan-2,5-dione, 3-isobutyl-4- [4- (3-methyl-2 -Butenyloxy) phenyl] -1 H -pyrrole-2,5-dione, 3-isobutyl-4- [4- (3-methyl-2-butenyloxy) phenyl] -1 H -pyrrole-1- ol-2,5-dione, 3R ^*, 4S ^* - 1- hydroxy-3-isobutyl-4- [4- (3-methyl-2-butenyl) phenyl] pyrrolidine-2,5- characterized in that 1-hydroxy-3-isobutyl-4- [4- (3-methyl-2-butenyl) phenyl] pyrrolidine-2,5-dione in-dione, or a 3R ^*, 4R ^* A mixture derived from the Antrodia camphorata mycelium. The method of claim 6, A mixture derived from Antrodia camphorata mycelium that reduces heart contraction blood pressure or increases high density lipoprotein. The method of claim 6, A mixture derived from Antrodia camphorata mycelium with central cholinergic agonism, hepatoprotection, anti-inflammatory, or anti-tumor activity. The method of claim 11, A mixture derived from Antrodia camphorata mycelium, wherein said tumor is derived from a cell or tissue selected from the group consisting of liver, intestine, bone, lymph, and breast. A composition comprising 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 or H or hydroxy; Provided that when X is O, R ₃ is absent. The method of claim 13, The compound is 3-isobutyl-4- [4- (3-methyl-2-butenyloxy) phenyl] furan-2,5-dione, 3-isobutyl-4- [4- (3-methyl-2 -Butenyloxy) phenyl] -1 H -pyrrole-2,5-dione, 3-isobutyl-4- [4- (3-methyl-2-butenyloxy) phenyl] -1 H -pyrrole-1- ol-2,5-dione, 3R ^*, 4S ^* - 1- hydroxy-3-isobutyl-4- [4- (3-methyl-2-butenyl) phenyl] pyrrolidine-2,5- characterized in that 1-hydroxy-3-isobutyl-4- [4- (3-methyl-2-butenyl) phenyl] pyrrolidine-2,5-dione in-dione, or a 3R ^*, 4R ^* Composition. The method of claim 14, The compound is 3-isobutyl-4- [4- (3-methyl-2-butenyloxy) phenyl] -1 H -pyrrole-2,5-dione or 3-isobutyl-4- [4- (3 -Methyl-2-butenyloxy) phenyl] -1 H -pyrrole-1-ol-2,5-dione. The method of claim 13, A composition that reduces heart contraction blood pressure or increases high density lipoprotein. The method of claim 13, A composition having central cholinergic promoting, hepatoprotective, anti-inflammatory, or anti-tumor activity. The method of claim 13, Wherein said tumor is derived from a cell or tissue selected from the group consisting of liver, intestine, bone, lymph, and breast. Mycelium of Antrodia camphorata containing the compound of claim 1. The method of claim 19, The compound is 3-isobutyl-4- [4- (3-methyl-2-butenyloxy) phenyl] furan-2,5-dione, 3-isobutyl-4- [4- (3-methyl-2 -Butenyloxy) phenyl] -1 H -pyrrole-2,5-dione, 3-isobutyl-4- [4- (3-methyl-2-butenyloxy) phenyl] -1 H -pyrrole-1- ol-2,5-dione, 3R ^*, 4S ^* - 1- hydroxy-3-isobutyl-4- [4- (3-methyl-2-butenyl) phenyl] pyrrolidine-2,5- characterized in that 1-hydroxy-3-isobutyl-4- [4- (3-methyl-2-butenyl) phenyl] pyrrolidine-2,5-dione in-dione, or a 3R ^*, 4R ^* Mycelia of Antrodia camphorata .