CN100386316C - Compounds and mixtures or compositions from Antrodia camphorata mycelium and uses thereof - Google Patents

Abstract

The present invention relates to a novel mixture and maleic and succinic acid derivatives from Antrodia Camphorata mycelium, and their medical use. The present invention relates to a composition or mycelium comprising a compound of the invention.

Description

Compounds and mixtures or compositions from mycelia of Antrodia camphorata and uses thereof

technical field

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

Background technique

In Taiwan, the fruiting body of Antrodia camphorata (Polyporaceae, Aphyllophorales) is well known as a traditional Chinese medicine. It only grows in the inner layer of the heartwood wall of the endemic Taiwanese evergreen Cinnamomun kanehirai (Hay) (Lauraceae). It is rare and not cultivated. The fruiting body has been used to treat food and drug poisoning, diarrhea, abdominal pain, hypertension, skin mange and liver cancer. Few biological activity studies have been reported to date.

In Taiwan, Antrodia Cinnamomea, also known as Antrodia cinnamomea, has recently been reported as a new fungal species characterized by the cylindrical shape of its basidiospores present in the fruiting body, mycelium with a soft starchy skeleton, bitter taste and bright yellow-brown inversion Born to cap (pileate) basidiocarps, and chlamydospores and anthroconidia in pure culture. The new fungal species grows extremely slowly and is restricted to the endemic tree species Cinnamomum kanehirai Hay (Lauraceae) as the sole host. The detailed description and taxonomic position of Antrodia cinnamomea are described in Wu, S.-H. et al., Antrodiacinnamomea (Antrodia cinnamomea), New combination of a medicinal fungus in Taiwan, Bot.Bull.Acad.Sin.38:273-275.

In Taiwanese folk medicine, the fruiting bodies of Antrodia Cinnamomea are believed to have certain medical effects. According to the traditional method, the fruit body is ground into dry powder or stewed with other herbs and taken orally to treat symptoms caused by poisoning, diarrhea, abdominal pain, high blood pressure, skin mange and liver cancer. However, few pharmacological or clinical studies have appeared in the literature to date. Because of its strict host specificity, its rarity in nature, and the failure of artificial cultivation, "Antrodia cinnamomea" is very expensive in Taiwan. In recent years, high-quality fruiting bodies of the fungus have been sold for a very high price, about 15,000 US dollars per kilogram.

Contents of the invention

Therefore, the object of the present invention is to provide a new mixture from the mycelium of Antrodia camphorata.

Another object of the present invention is to provide new compounds from the mycelium of Antrodia camphorata.

Yet another object of the present invention is to provide novel compositions comprising the compounds of the present invention.

Another object of the present invention is to provide new mycelium of Antrodia camphorata comprising the compound of the present invention.

Description of drawings

Figure 1 shows the HMBC correlation of Compound 2.

Figure 2 shows compounds of the invention.

Figure 3 shows the NOE (Nuclear Overhouse Effect) correlation of compounds 4 and 5 of the present invention.

Figures 4a-d show the test results for compound 3 of the present invention.

Figures 5a-c show the test results of ACM (Antrodia camphorata mycelium powder) water extract.

Figures 6a-f show the experimental results of ACM ethanol extracts.

Figures 7a-e show the test results for Compound 1 of the present invention.

Detailed ways

The present invention provides compounds of the formula

in

X is N or O;

R ₁ is C _1-10 alkoxy, 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; and

R ₃ does not exist, or is H or hydroxyl;

The condition is that if X is O, then _R3 is absent.

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

A more preferred compound of the present invention is 3-isobutyl-4-[4-(3-methyl-2-butenyloxy)phenyl]-1H-pyrrole-2,5-dione or 3-isobutyl Butyl-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 mycelium of Antrodia camphorata comprising the compound of the present invention. The mixture of the present invention is prepared from the water or organic solvent extract of the mycelia of Antrodia camphorata. The organic solvents include but are not limited to alcohols (such as CH ₃ OH, C ₂ H ₅ OH or C ₃ H ₇ OH), esters (such as ethyl acetate), alkanes (such as hexane) and haloalkanes (such as CH ₃ Cl, C ₂ H ₂ Cl ₂ ). A preferred organic solvent is ethanol or a solvent containing ethanol that does not cause any side effects in humans. The mixture of the present invention can reduce systolic blood pressure or increase high density lipoprotein. Furthermore, the same mixture has central cholinergic agonistic, hepatoprotective, anti-inflammatory or antitumor activity. Specifically, the mixture of the present invention can inhibit tumors of cells or tissues selected from liver, intestine, bone, blood, lymph and breast. Subjects receiving the mixture 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 a compound of the invention. The composition of the present invention can reduce systolic blood pressure or increase high-density lipoprotein. In addition, the compositions of the invention have central cholinergic agonistic, hepatoprotective, anti-inflammatory or anti-tumor activity. Specifically, the composition of the present invention can inhibit tumors in cells or tissues selected from liver, intestine, bone, blood, lymph and breast. Subjects receiving 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 Antrodia mycelium comprising a compound of the present invention. Preferred mycelia have at least 1% of the weight of the original mycelium of the total weight of compounds 1-5 of the present invention. The most preferred mycelium has at least 3% of the weight of the original mycelium of the total weight of compounds 1-5 of the present invention. According to the previous submerged liquid fermentation (submerged liquid fermentation) to prepare Antrodia camphorata mycelia, such as T.L.M.Stamford et al., Food Science "Protein enrichment of cashew wastes for animal feeds", from http://www.unu.edu/unupress /food/8F10le/8F10lE0b.htm.

Example

The following examples are non-limiting and merely represent various aspects and characteristics of the invention.

General Experimental Procedure

Melting points were measured with a Yanagimoto mini hot stage melting point apparatus and were uncorrected. Optical rotation was measured with a JascoDIP-360 automatic polarimeter. UV spectra were measured with a Shimadzu UV-2200 self-recording spectrophotometer. Infrared 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 JeolJMS-AX 505 HAD mass spectrometer at an ionization voltage of 70eV. Column chromatography was performed using silica gel of BW-820MH (normal phase) and Chromatorex-ODS DM1020T (reverse phase) (Fuji Silysia).

Extraction and Separation

Under reflux, Antrodia camphorata mycelium powder (60 g) from Taiwan Shansheng Biotechnology Co., Ltd., October 2001 was extracted three times with chloroform for 3 hours. Chloroform extract (5.3 g) was subjected to silica gel chromatography eluting with n-hexane-acetone (19:1-14:6) and chloroform-methanol (1:1) to obtain nine fractions (Fr. 1-9). Silica gel chromatography on the second fraction afforded compound 1 (8.7 mg). The fourth fraction was subjected to normal and reverse phase silica gel chromatography to afford compound 2 (13.6 mg). The fifth fraction was chromatographed on silica gel eluting with n-hexane-acetone (8:2) to give ergosterol peroxide (35.8 mg). Combined normal and reverse phase silica gel column chromatography on the sixth fraction afforded compound 3 (14.6 mg). The seventh part was subjected to column chromatography to obtain a mixture of compounds 4 and 5 (4:1), followed by preparative HPLC separation of the mixture of 4 and 5 [column: Tosoh TSK-gel ODS- _80TM (21.5 × 300 mm), Mobile phase: methanol-water (70:30) with 0.1% TFA].

3-Isobutyl-4-[4-(3-methyl-2-butenyloxy)phenyl]-furan-2,5-dione (compound 1): yellow oil; UV(MeOH) λ _max (logε)227(4.1), 258(3.9), 275(3.8), 355(3.4)nm; IR(CHCl ₃ )v _max 1763cm ^-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} (calcd for _C19H22O4 : 314.1518).

3-isobutyl-4-[4-(3-methyl-2-butenyloxy)phenyl]-1H-pyrrole-2,5-dione (2): yellow needle crystals (n-hexane- ethyl acetate); mp 110-111°C; UV(methanol) λ _max (logε) 230(4.3), 272(3.5), 355(3.7)nm; IR(CHCl ₃ )v _max 1724cm ^-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 (C ₁₉ H ₂₃ Calculated for NO ₃ : 313.1678).

X-ray crystallography of compound 2:

b＝12.205(1)c＝12.560(2)

α＝64.623(7)°，β＝75.358(4)°，γ＝84.681(5)°，V＝850.9(2)Z＝2，D_calc＝1.223g/cm³，μ(MoKα)＝0.82cm^-1，F000＝336.00。于93K下放射，以具有石墨单色的Mo-Kα(λ＝0.71069

)的Rigaku RAXIS-RAPID影象板衍射计进行测量。在采集的8950个反射中，4745个为独特的(R_int＝0.108)；合并相等反射。以直接法(SHELXS86)解析晶体结构并以全矩阵最小二承方精算。非均质地(anisotropically)精算非氢原子。氢原子被包括在内但不精算。最终指数R＝0.074，R_w＝0.099，而GOF(Guest Observer Facility，客体观察设施)＝1.06。最终差值傅立叶图的最大与最小峰分别对应于0.83及-0.89e^-/

Crystallization from n-hexane-ethyl acetate gave yellow needles which were selected for data collection. Crystal data: C ₁₉ H ₂₃ NO ₃ ; Mr=313.40; size 0.15×0.02×0.02 mm; triclinic space group P1 (#2), a=6.3505(5)

b=12.205(1) c=12.560(2)

α=64.623(7)°, β=75.358(4)°, γ=84.681(5)°, V=850.9(2) Z=2, D _calc =1.223 g/cm ³ , μ(MoKα)=0.82 cm ⁻¹ , F000=336.00. Emit at 93K to have graphite monochromatic Mo-Kα (λ=0.71069

) Rigaku RAXIS-RAPID image plate diffractometer for measurement. Of the 8950 reflections collected, 4745 were unique ( _Rint = 0.108); equal reflections were merged. The crystal structure was analyzed by direct method (SHELXS86) and actuarial calculation was performed by full matrix least squares. Non-hydrogen atoms are anisotropically actuarial. Hydrogen atoms are included but not actuarial. The final index R=0.074, R _w =0.099, and GOF (Guest Observer Facility, object observation facility)=1.06. The maximum and minimum peaks of the final difference Fourier map correspond to 0.83 and -0.89e ^- /

3-isobutyl-4-[4-(3-methyl-2-butenyloxy)phenyl]-1H-pyrrol-1-ol-2,5-dione (compound 3): yellow oil; UV(MeOH)λ _max (logε): 232.5(4.3), 296(3.7), 374(3.7)nm; IR(CHCl ₃ )v _max 1717cm ^-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 (calcd 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° (c0.2, methanol); UV (methanol) λ _max (logε): 225(4.3), 275(3.3), 283(3.2)nm; IR(CHCl ₃ )v _max 1715cm ^-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 (calcd for _C19H25NO4 : 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°(c0.2, MeOH); UV(MeOH)λ _max (logε): 227(4.3), 275(3.4), 283(3.3)nm; IR(CHCl ₃ )v _max 1715cm ^-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 (calcd for _C19H25NO4 : 331.1783).

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

Cytotoxicity Assay: In vitro LLC tumor cell assay using sulforhodamin B (SRB). 50% growth inhibition (ED ₅₀ ) was calculated by Probit method.

Results and discussion

Normal-phase and reverse-phase silica gel chromatography were repeated on chloroform extracts of Antrodia mycelia to obtain five new maleic and succinic acid derivatives (compounds 1-5) and ergosterol peroxides.

Table 1

¹ H-NMR Spectrum Data of Compound 1-5 (δppm, J=Hz) (500MHz, CDCl ₃ )

Table 2

¹³ C-NMR spectral data (δppm) of compound 1-5 (125MHz, CDCl ₃ )

a) Attribution is interchangeable.

The structure of the new compound was determined as follows:

Compound 2 is a yellow needle crystal, mp 110-111°C, and its molecular formula is C ₁₉ H ₂₃ NO ₃ analyzed by HR-EIMS. Infrared spectrum shows imide carbonyl absorption at 1724cm ^-1 . ^{The 13} C-NMR spectrum shows that there are four methyl carbons, two methylene carbons and one methine carbon signal in the aliphatic region, as well as one benzene ring, one alkenyl and two carbonyl carbons. ^{The 1} H-NMR spectrum shows isobutyl moieties at δ0.90, 2.06 and 2.51, 3-methyl 2-butenyloxy moieties at δ1.76, 1.81, 4.56 and 5.50, and para The para-substituted benzene moiety is further supported by ¹ H- ¹ H COZY (cooled synchrotron) and HMQC (heteronuclear multiple quantum correlation) experiments. Long-range correlations were observed by HMBC, as shown in Figure 1. Based on the molecular formula ^and13C -NMR spectrum, it was concluded that this compound contained additional CHNO atoms, including more than one carbonyl carbon. Therefore, it is presumed that this unclear portion is a maleimide group. This structure was then determined to be 3-isobutyl-4-[4-(3-methyl-2-butenyloxy)phenyl]-1H-pyrrole-2,5-dione by X-ray analysis.

The molecular formula of compound 1 was analyzed by HR-EIMS as C ₁₉ H ₂₂ O ₄ . Infrared spectrum shows that there is carbonyl absorption of acid anhydride at 1763cm ^-1 . ^{The 1} H-NMR spectrum of compound 1 is similar to that of compound 2, showing the existence of isobutyl moiety, 3-methyl 2-butenyloxy moiety, and para-substituted benzene ring. The HMBC spectrum indicated that compound 1 had a partially identical structure to compound 2 (Figure 1), where the presence of maleic anhydride was deduced from the molecular formula. Therefore, Compound 1 was identified as 3-isobutyl-4-[4-(3-methyl-2-butenyloxy)phenyl]furan-2,5-dione.

The molecular formula of compound 3 was analyzed by HR-EIMS as C ₁₉ H ₂₃ NO ₄ . Infrared spectrum shows that there is carbonyl absorption at 1717cm ^-1 , which can be attributed to hydroxyimide. ^{The 1} H- and ¹³ C-NMR spectra were also similar to those of compounds 1 and 2, showing the presence of isobutyl moieties, 3-methyl 2-butenyloxy moieties, and para-substituted benzene rings. In the HMBC experiment, it was proved that compound 3 had the same partial structure as compound 2 (Fig. 1). Compound 3 contains one more oxygen atom than compound 2, therefore, this compound is determined to be 3-isobutyl-4-[4-(3-methyl-2-butenyloxy)phenyl]-1H-pyrrole-1 - Alcohol-2,5-dione.

Compounds 4 and 5 have the same R _f value and the same molecular formula (C ₁₉ H ₂₅ NO ₄ , measured values are 331.1747 and 331.1766, respectively) by HR-EIMS analysis, but they can be separated by preparative HPLC. The infrared spectrum of the two compounds shows that there is absorption of hydroxyl diimide carbonyl at 1715cm ^-1 . In the ¹ H- and ¹³ C-NMR spectra, both compounds showed the existence of isobutyl moiety, 3-methyl 2-butenyloxy moiety, and para-substituted benzene ring, but isobutylmethylene The protons showed multiplets rather than doublets like compounds 1-3. ^{The 1} H- ¹ H COZY spectrum shows that the methylene group is attached to the -CH-CH- unit. The ¹³ C-NMR spectra of compounds 4 and 5 showed two additional sp ³ carbon signals in place of the sp ² carbon signals found in compounds 1-3. Therefore, compounds 4 and 5 are not N-hydroxymaleimides, but N-hydroxysuccinimides with stereocenters at the C-3 and C-4 positions of the succinimide ring. Compounds 4 and 5 were identified as trans and cis isomers by the coupling constants between H-3 and H-4 (4.0 and 8.0 Hz for compounds 4 and 5, respectively). No NOE between H-3 and H-4 was observed in the NOESY (nuclear Overhaus effect) spectrum of compound 4, while a significant NOE was observed in the NOESY spectrum of compound 5. The optical rotations of compounds 4 and 5 were +2.5° and +3.0°, respectively, and their CD spectra showed no Carten effect at any wavelength, indicating that compounds 4 and 5 were racemic mixtures. Resolution of these racemic mixtures by HPLC with several solvent systems using chiral columns was unsuccessful. Currently, we cannot deduce definitively whether these compounds are optically active compounds or racemic mixtures. Therefore, their relative structures were determined as 3R ^* , 4S ^* - and 3R ^* , 4R ^* -1-hydroxy-3-isobutyl-4-[4-(3-methyl-2-butenyloxy) Phenyl]pyrrolidine-2,5-dione.

These types of naturally derived maleic and succinic acid derivatives were isolated for the second time according to the report of Aquveque et al.

Cytotoxic activity of chloroform extracts and isolated compounds was studied using LLC (Lewis lung cancer) cell line (Table 3). The chloroform extract showed moderate cytotoxicity with an _ED50 value of 26.7 μg/ml. Maleic compounds 1 and 4 were not cytotoxic, whereas compounds 2 and 3 were found to be cytotoxic to LLC cell lines with _ED50 values lower than those of chloroform extracts. Table 3 The 50% growth inhibition (ED ₅₀ ) value of the chloroform extract from the mycelia of Antrodia camphorata and compounds 1-4 on LLC cell lines

a) positive control

Tumor Assay of ACM (Antrodia Cinnamomum Mycelium Powder)

A. Cell lines

Adherent cells:

MCF-7: human breast cancer

HT-29: Human colon adenocarcinoma

KATO III: Human gastric adenocarcinoma

SW480: Human colon adenocarcinoma

SW620: Human colon adenocarcinoma

HepG2: human hepatic adenocarcinoma

Suspension cells:

EL4: mouse lymphoma

B. Sample

Compound 1, Compound 3, ACM ethanol extract, ACM water extract

C. Determination method

Calculation of _ED50 (50% inhibition of the effective dose)

Adherent cells: MTT (methylthiazolyl tetrazolium) method: For MCF-7, HT-29, KATO III, SW480, HepG2, 3 days to determine the cells. SW6204 days.

Suspension cells: cell counting method; EL4 cell counting for 5 days.

D. Results

Calculation: y=m Ln(x)+b

example

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

Use X (10, 30, 50ppm) and Y value to get the correlation curve

y=-0.2643Ln(x)+1.5321

ED ₅₀ ＝exp【(0.97/2-1.5321)/(-0.2643)】

Sample preparation and sample description

A. Water extract of ACM (Antrodia mycelium powder)

1. Add 1 g of ACM into a 250 ml beaker filled with 40 ml of RO H ₂ O, 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 the ultrasonic water bath for another 20 minutes.

4. Centrifuge the sample at 3000 rpm for 15 minutes.

5. Collect the supernatant and make serial dilutions with culture medium.

B. Determination of sample concentration

1. Weigh the evaporator (W1).

2. Add 10ml of water extract to the evaporator

3. Place the evaporator in the oven to remove moisture (W2)

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

C. ACM (Antrodia mycelium powder) ethanol extract

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

2. Filter the suspension with Advantec No. 1 filter paper, and collect the filtrate.

3. Concentrate the filtrate with a rotary vacuum evaporator to remove ethanol.

D. Compound 1: Pure compound from ACM

E. Compound 3: Pure compound from ACM

MTT assay

1. Discard the old medium after cell proliferation, and then wash the cells once with phosphate-buffered saline (PBS).

2. Rinse the cells with trypsin-EDTA.

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

4. Suspend the pellet with 10 ml medium.

5. Mix 100 microliters of cell suspension with 100 microliters of trypan blue to count viable cells.

6. Add 1×10 ⁴ cells/100 microliters of medium to each well of a 96-well plate, and incubate the plate in a carbon dioxide incubator at 37°C for 24 hours.

7. Discard the old medium and wash the cells once with PBS.

8. Add 100 microliters of sample to each well and incubate the plate in a carbon dioxide incubator at 37°C.

9. Wash the cells once with PBS on days 3, 4 and 5.

10. Add 57 microliters of MTT (0.88 mg/ml) to each well.

After 11.4 hours the MTT was discarded and the cells were washed once with PBS.

12. Add 50 microliters of DMSO to each well.

13. Read the result at OD545 with an ELISA instrument.

Cytometry (EL4 cell line)

1. Discard the old medium by centrifugation after cell proliferation.

2. Resuspend the pellet with fresh medium.

3. Mix 100 microliters of cell suspension with 100 microliters of trypan blue to count viable cells.

4. Prepare samples at different concentrations containing 1×10 ⁵ cells/ml of sample.

5. Put 100 microliters of sample into each well of a 96-well plate, and incubate the plate in a carbon dioxide incubator at 37°C.

6. Count viable cells at days 3, 4 and 5.

PBS

NaCl 8 grams

KCl 0.2 g

_{Na2HPO4 1.4} g

KH ₂ PO ₄ 0.2 g

Adjust the volume to 1 liter pH 7.4

Results and discussion

ED ₅₀ of ACM in cell lines

cell line HepG2 HT-29 KATO III EL4 SW480 SW620 MCF-7 Compound 1 21ppm 52ppm 38ppm 3.5ppm 15ppm 6ppm Compound 3 35ppm 42ppm 69ppm 2.6ppm 20ppm 27ppm 0.02ppm ACM ethanol extract 32ppm 52ppm 156ppm 2.6ppm 71ppm 4ppm ACM water extract 295ppm 707ppm 20ppm 207ppm 132ppm 318ppm

The detailed experimental results are as follows:

Compound 3 of the present invention: HepG2 (Fig. 4a), EL4 (Fig. 4b), HT-29 (Fig. 4c) and Kato III (Fig. 4d).

ACM water extracts: HepG2 (Fig. 5a), SW620 (Fig. 5b) and EL4 (Fig. 5c).

ACM ethanol extracts: 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).

The above results prove that the compounds of the present invention and the ACM extract have inhibitory effects on various types of tumor cells.

Analysis of all novel compounds (1, 2 and 3) from ethanol extracts of ACM by HPLC

Purpose: To measure the amount of all novel compounds (1, 2 and 3) from the ethanol extracts of ACM, high performance liquid chromatography was employed as our routine quality control procedure.

Prepare ACM ethanol extract samples:

1) Accurately weigh 20.000 (grams) of the sample powder with an electronic balance, put it into a graduated test bottle containing 100 milliliters of 95% ethanol, and keep the bottle cap tight.

2), the sample bottle of the above step is placed in an ultrasonic water bath for 10 minutes.

3) Pour liquid samples into centrifuge tubes, and then centrifuge those samples at 6500 rpm for 5 minutes to remove coarse particles.

4), filter the liquid layer with advantec No. 1 filter paper.

5) Concentrate the filtrate with a rotary vacuum evaporator until a viscous, ethanol-free light yellow liquid appears.

6), repeat steps 1 to 5 three times, and collect all the extracted products (total ACM ethanol extract = 4.60 g), and calculate the yield.

Application of 2690 Water HPLC:

1), column: reversed phase C18

2), mobile phase: methanol, water, acetonitrile

3), injection volume: 20 microliters

4) Detection: Photodiode array detector 996 measures at a wavelength of 254nm

5) Prepare a sample of 1.000 (grams) of ACM ethanol extract in 10 ml of ethanol for HPLC analysis ^* :

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

Table 4:

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

ACM ethanol extract test

Materials and Equipment

1. Test Substances and Modes of Administration

All in vivo tests were administered orally with an initial dose of 1000 mg/kg of test substance in a 2% Tween 80 vehicle. The observation time for each trial is described in Methods.

2. Animals

Male or female ICR mice, male spontaneously hypotensive rats (SHR) derived from Wistar-Okamoto, and rats derived from Wistar and Long Evans were used provided by Taiwan Pan Globe Institute of Pharmacology. The animal breeding space was as follows: 10 mice were kept in a space of 29 × 18 × 13 cm, 6 rats were raised in a space of 45 × 23 × 21 cm, and 3 guinea pigs were raised in a space of 45 × 23 × 21 cm. Mice and rats were housed in APEC ^R cages. C57BL/6J immunocompetent male mice weighing 21±2 g and 6–8 weeks old provided by the Animal Center of National Taiwan University were also used in this study. The experimental animals were housed in individually ventilated cages (IVC racks, 36 small isolation systems). Each cage was autoclaved and contained 5 mice (in a space of 26.7 x 20.7 x 14 cm). All animals were kept in a controlled temperature (21°-23° C.) and humidity (60%-70%) environment in the laboratory under a 12-hour light cycle for at least one week prior to use. Standard experimental feed (LabDiet Rodent Diet and Guinea Pig Diet, PMI Nutrition International, USA) and tap water were provided ad libitum.

3. Cell lines and media

Murine melanoma cells, B16-F0 (ATCC CRL-6322) were purchased from American TypeCulture Collection, and Dulbecco's Modified Eagle's Medium (GIBCO, USA) was used as the cell culture medium. Tumor cells were cultured at 37 °C in an atmosphere containing 5% _CO2 .

4. Chemicals

generally:

Distilled water (self-made), dimethyl sulfoxide (DMSO, Merck, Germany), isotonic sodium chloride solution (Sindong Chemical Industry Co., Ltd., Taiwan), magnesium sulfate (MgSO ₄ 7H ₂ O, Wako, Japan) , Sodium Meclofenac (Sigma, USA), Methylcellulose (Signa, USA), Sodium Hydroxide (NaOH, Wako, Japan), Phosphate Buffered Saline (Sigma, USA) and Tween 80 (Wako, Japan) .

Reagent

Glicose-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 3Hemolys (Nihon Koden, Japan), Isotonic 3 Diluent (NihonKoden, Japan).

5. Equipment

General use:

Animal box (Xinde, Taiwan), 250ml and 1000ml beakers (Kinmax, USA), disposable syringe (1ml, Top Corporation, Japan), stainless steel tweezers (klappencker, Germany), mouse scale (mouse scale) #Z- 40 (Taconic, U.S.), oral feeding syringe (Natsune, Japan), hypodermic needle 23G x 1” (Top Corporation, Japan), pH meter (Suntex, U.S.), rat scale 500 g ± 2 g (Chien-chun , Taiwan), glass syringes 1 ml, 2 ml, and 5 ml (Mitsuba, Japan), and stainless steel scissors (Klappencker, Germany).

Method and Results:

1. Cholinergic agonism, central/peripheral (Lippmann W and Pugsley TA, Arch Int Pharmacodyn. 227:324, 1977).

The test substances were administered orally to groups of 3 Wistar-derived male or female rats weighing 150±20 g. Over the next 30-60 minutes, cumulative measurements are recorded of the number of animals exhibiting chewing behavior (mouth and/or tongue movements) for more than 10 seconds, as well as the number of animals exhibiting salivation. The observation of a positive response in 2 or more (≥2) of 3 rats indicates possible central and peripheral cholinergic activity.

Table 5: Cholinergic Agonism Results, Central/Peripheral, in Rats

Vehicle and test substances are administered orally (PO), while the positive control compound is administered intraperitoneally (IP). Over a subsequent 30-60 minute period, cumulative measurements are recorded of the number of animals exhibiting chewing behavior (mouth and/or tongue movements) over 10 seconds, as well as the number of animals exhibiting salivation. The observation of a positive response in 2 or more (≥2) of 3 rats indicates possible cholinergic activity or peripheral cholinergic activity.

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

A group of 3 Wistar-Okamoto-derived spontaneously hypertensive male mice (SHR) weighing 250±20 grams were used; the average systolic blood pressure was 200±20 mmHg, and the heart rate was 400±30 beats/min. Blood pressure and heart rate were recorded indirectly by the tail cuff method in a temperature-controlled environment (32±1°C) before (0:00) and 1, 2, and 4 hours after oral administration of the test substance or vehicle. A decrease in systolic blood pressure of 10% or more (≥10%), or a decrease in heart rate of 20% or more (≥20%) compared to time 0 after each measurement at the interval was considered significant.

Table 6: Cardiovascular and blood pressure results in rats (SHR 0, 1, 2, 4 hours)

Table 7: Cardiovascular, heart rate results (SHR 0, 1, 2, 4 hours)

Use an SHR with a systolic blood pressure of 200±20 mmHg and a heart rate of 400±50 mmHg beats/min. Blood pressure was recorded indirectly via vial tail cuff at 0 and 1, 2, and 4 hours after oral administration of the test substance or vehicle. A decrease in blood pressure of 10% or more (≥10%), or a decrease in heart rate of 20% or more (≥20%) at each measurement time point compared to 0 given in parentheses is considered yes obviously.

Carrier 10ml/kg 0:229mmHg and 403mmHg times/

Minutes count as 100%.

ACM-ethanol extract 1000mg/kg 0:223mmHg and 452mmHg times/

Minutes count as 100%.

Clonidine 0.1mg/kg 0:228mmHg and 379mmHg times/

Minutes count as 100%.

Table 8: Cardiovascular, blood pressure results in rats (SHR 0, 1, 2, 4 hours)

Table 9: Cardiovascular, heart rate results (SHR 0, 1, 2, 4 hours)

Use an SHR with a systolic blood pressure of 200±20 mmHg and a heart rate of 400±50 mmHg beats/min. Blood pressure was recorded indirectly via vial tail cuff at 0 hours (before) and 1, 2, 4 hours after oral administration of test substance or vehicle. A decrease in blood pressure of 10% or more (≥10%), or a decrease in heart rate of 20% or more (≥20%) compared to 0 given in parentheses at each measurement time point was considered significant .

Carrier 10ml/kg 0:220mmHg and 410mmHg times/

Minutes count as 100%.

ACM-ethanol extract 300mg/kg 0:205mmHg and 446mmHg times/

Minutes count as 100%.

Clonidine 0.1mg/kg 0 hours 235mmHg and 417mmHg times/

Minutes count as 100%.

3. Cholesterol, serum (total HDL, total/HDL ratio), diet-induced (Schurr PE et al., Atherosclerosis Drug Discovery. Plenum, New York, pp.215-229, 1976).

Groups of 5 ICR-derived male mice weighing 22±2 g were fed high-fat diet (g/100 g: coconut oil, 8; cholesterol, 1.0; cholic acid, 0.3; lard, 2; standard diet, 88.7)7 day to cause hypercholesterolemia. The test substances were administered orally on days 5, 6, and 7. After an overnight fast, serum was obtained from each mouse to determine percent changes in total cholesterol (total), high-density lipoprotein (HDL) and total/HDL. Decrease in total serum by 20% or more (≥20%), or increase in serum HDL by 20% or more (≥20%), or decrease in total/HDL by 40% or more, compared to vehicle-treated control animals More (≥40%), considered significant.

Table 10: Diet-induced cholesterol (total/HDL, total/HDL ratio) results in mice

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

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

Groups of 5 Wistar-derived male rats weighing 200±20 g were used. Each animal was treated with a single injection of D-galactosamine (500 mg/kg, IP). The test substances were orally administered 0.5 hours before and 4 hours and 8 hours after the administration of D-galactosamine, and the animals were sacrificed 24 hours later. Serum alanine aminotransferase (ALT) and aspartate aminotransferase (AST) levels were measured with a HITACHI automatic analyzer (model 7050) by an optimized UV method. A reduction in ALT or AST activity of 30% or more (>30%) compared to vehicle-treated control animals is indicative of significant protection.

Table 11: Results of Liver Injury, Galactosamine, in Rats

The test substance and vehicle were orally administered 0.5 hours before and 4 hours and 8 hours after a single administration of galactosamine (500 mg/kg, IP), and the rats were sacrificed 24 hours after galactosamine injection, and the ALT and AST values were determined. A > 30% reduction in ALT and AST compared to the vehicle group was considered significant.

5. Inflammation, carrageenan (Winter CA et al., Proc Soc Exp Biol Med. 111:544, 1962)

Groups of 3 Long Evans-derived males or females weighing 150±20 g were fasted overnight prior to the study. The test substances were administered orally 1 hour before the right hind paw received an injection of carrageenan (0.1 ml of a 1% intraplantar suspension). Hind paw edema, as a measure of inflammation, was recorded 3 hours after carrageenan administration using a plethysmometer with a sink (25 mm diameter). A reduction in hind paw edema of 30% or more (≧30%) indicates significant anti-inflammatory activity.

Table 12: Inflammation Results, Carrageenan, in Rats

Table 13: Inflammation Results, Carrageenan, in Rats

Overnight fasted rats were given test substances and vehicle 1 hour before injection of carrageenan (0.1 ml of 1% intraplantar suspension) in the right hind paw (R.P.); the left hind paw (L.P.) was not injected. A 30% or greater (>30%) decrease in hindpaw edema indicated in parentheses indicates significant acute anti-inflammatory activity.

6. Tumors, syngeneic melanoma B 16-F0 cells (Farrugia CA and Groves MJ., Anticancer Research 19:1027-1032, 1999)

Use a group of 5 specific pathogen free (SPF), immunocompetent (6-8 week old) C57BL/6J male mice housed in animal isolation rooms (IVC racks) in a specific pathogen free (SPF) environment . Live B16-F0 murine melanoma cells (ATCC CRL-6322, 1.0×10 ⁵ cells in 0.2 ml) syngeneic to C57BL/6J mice were injected subcutaneously into the dorsal side of the experimental mice. Treatment was initiated 24 hours after tumor inoculation, and test compounds were administered daily by oral gavage for 21 days, or a reduced number of days when overt symptoms of toxicity occurred. Body weight, tumor size and survival of mice were monitored from day 1 to day 22. In addition, the survival of the experimental mice was monitored until the end of day 45 post-study.

Tumor weight (mg) was estimated according to the prolate ellipsoid formula: length (mm) × [width (mm)] ² × 0.5, assuming a specific gravity of 1 and π of 3. Tumor growth in compound-treated animals is calculated as T/C (Treatment/Control) x 100%; T/C values ≤ 42% indicate significant antitumor activity. A T/C (Treatment/Control) mean survival time ≥ 125% was also considered to have significant antitumor activity.

Table 14: Tumor, Syngeneic Melanoma B16-F0 Cell Results

Table 15: Tumor, Syngeneic Melanoma B16-F0 Cell Results

24 hours after tumor cell implantation, the experimental animals were administered with vehicle and test substances every day for a total of 21 administrations. Meanwhile, the control compound, mitomycin, was administered IP twice a week for a total of 6 administrations. Tumor size was measured and recorded twice a week for 22 days. Tumor growth inhibition was calculated as T/C (weight/control) x 100. T/C value ≤ 42% is considered to have significant anti-tumor activity.

Table 16: Tumor, Syngeneic Melanoma B16-F0 Cell Results

24 hours after tumor cell implantation, the experimental animals were administered with vehicle and test substances every day for a total of 21 administrations. Meanwhile, the control compound, mitomycin, was administered IP twice a week for a total of 6 administrations. Tumor size was measured and recorded twice a week for 22 days. Significant differences in body weight changes between test compounds and vehicle controls were determined using Student's t-test.

Table 17: Tumor, Syngeneic Melanoma B16-F0 Cell Results

a: If the animal did not die after 45 days, its survival period was regarded as 45 days.

Survival of treated mice was monitored through the 45-day study period, or until the experimental animal died. A T/C (Treatment/Control) mean survival time ≥ 125% was also considered to have significant antitumor activity.

discuss:

Oral (PO) administration of the ACM-ethanol extract resulted in significant activity in the following mouse and rat assays according to self-defined criteria:

Central cholinergic agonism at 1000 mg/kg in rats; minimal and insignificant agonism was seen at 300 mg/kg; no significant agonism or antagonism of peripheral cholinergic nerves at 1000 mg/kg (Table 5 ).

In spontaneously hypertensive (SH) rats, at 1000 mg/kg, systolic blood pressure was reduced (16%, 12% compared to 100% at time 0, observed at 1, 2, and 4 hours). % and 20%) with moderate but not significant decreases in heart rate (Tables 6 and 7); the 300 mg/kg dose did not result in significant changes in systolic blood pressure and heart rate (Tables 8 and 9).

At 1000 mg/kg, high-density lipoprotein (HDL, 39% more than vehicle control) was increased in diet-induced mice (Table 10); associated total cholesterol (total) was not significantly changed, while HDL/total The amount ratio decreased significantly to nearly 31%; the 300 mg/kg dose did not cause significant changes in total, HDL, and HDL/total ratios.

For rat liver injury induced by galactosamine, hepatoprotective effect was seen at 1000 mg/kg×3 (compared with vehicle control, ALT decreased by 44% and AST decreased by 57%); it was observed at 300 mg/kg×3 There was a moderate decrease of 20% in ALT and 28% in AST (Table 11).

Anti-inflammatory effect on carrageenan-induced rat paw edema at 1000 mg/kg (45% inhibition compared to vehicle control) (Table 12); lower 300 mg/kg level had no significant activity (compared to 3% inhibition compared to vehicle, Table 13).

At 1000 mg/kg, antitumor activity in C57BL/6J mouse syngeneic melanoma B16-F0 cells on day 8, 11 and 15 (Tables 14 and 15), and prolongation of animal survival time (Table 17); animal body weight did not change significantly (Table 16).

ACM of the present invention, ACM-ethanol extract and the test of compound 3

Nine groups of 5 ICR-derived male mice (weight 22±2 grams) were used. Each animal was challenged with a single dose of carbon tetrachloride (CCl ₄ , in 50% olive oil, 0.1 ml/kg, PO). The test substance was orally administered at doses of 300 and 1000 mg/kg of ACM, or at doses of 30, 100 and 300 mg/kg of compound 3 of the invention, 30 minutes before and 4 and 8 hours after the carbon tetrachloride challenge; and Orally administered ethanol extracts of 300 and 1000 mg/kg ACM were pretreated 1 day (twice a day) and 30 minutes before carbon tetrachloride and 4 and 8 hours after. Animals were sacrificed 24 hours after carbon tetrachloride. Alanine aminotransferase (ALT) and aspartate aminotransferase (AST) levels were measured with an optimized UV method using a HITACHI automatic analyzer (Model 7050). ALT or AST levels were reduced by 30% or more (≥30%) compared to vehicle, indicating significant protection against liver damage.

result

Table 18: Determination of liver injury, carbon tetrachloride, in mice

discuss:

The possible protective activity of ACM, ACM-ethanol extract and compound 3 of the present invention on liver damage induced by carbon tetrachloride in ICR mice was evaluated. The test substances, ACM-ethanol at doses of 300 and 1000 mg/kg, and compound 3 of the invention at doses of 30, 100 and 300 mg/kg were administered 0.5 hours before and 4 and 8 hours after carbon tetrachloride challenge. Orally administered to test animals. For 300 and 1000 mg/kg ethanol extracts of ACM, two treatments (9:00AM and 16:00PM) were given 1 day before CTC (b.i.d.), followed by 0.5 hours before CTC challenge with Treatments were given 4 hours and 8 hours later (a total of 5 doses). The extent of liver injury was determined by the increase in serum alanine aminotransferase (ALT) and aspartate aminotransferase (AST) relative to vehicle-treated animals. ACM at a dose of 1000 mg/kg×3 and compound 3 of the invention at a dose of 300 mg/kg×3 resulted in ALT (46% and 41%) and AST (36% and 33%) was significantly reduced. Simultaneous doses of 300 and 1000 mg/kg x 5 of ACM-ethanol extract also resulted in significant reductions in ALT (36% and 34%) and AST (25% and 20%).

Simultaneous testing of silymarin (100 mg/kg x 3, IP) showed significant reductions in ALT (31%) and AST (31%) relative to the vehicle treated group.

It is concluded that ACM, ACM-ethanol extract and compound 3 of the present invention have significant hepatoprotective activity in the mouse carbon tetrachloride model.

While the invention has been described and illustrated in sufficient detail to enable those skilled in the art to make and use it, various alternatives, changes and modifications will be apparent without departing from the spirit and scope of the invention.

Those skilled in the art will readily appreciate that the present invention is adapted to carry out the ends and obtain the results and advantages described herein and inherent therein. Cell lines, embryos, animals, and processes and methods for their manufacture represent preferred embodiments, are exemplary, and are not intended to limit the scope of the invention. Modifications and other uses thereof will occur to those skilled in the art. These modifications are encompassed within the spirit of the invention and defined by the scope of the claims.

Those skilled in the art will readily recognize that changes and modifications of this invention can be made without departing from the scope and spirit of this invention.

All patents and works mentioned in the specification represent the level of ordinary skilled persons in the technical field to which the present invention belongs. All patents and works are incorporated by reference as if each individual work was specifically and individually indicated to be incorporated by reference.

The invention exemplarily described herein may be practiced without one or more elements, one or more limitations, specifically disclosed herein. The terms and expressions employed are merely illustrative rather than restrictive and are not intended to exclude any equivalents of any features shown and described or some of them, but rather to consider that all within the scope of the claimed invention A change is possible. It is therefore to be understood that while the invention has been described in detail by way of preferred embodiments and optional features, those skilled in the art may employ alterations and variations of the concepts disclosed herein which are considered within the scope of the invention as defined by the claims.

Other embodiments are set forth in the appended claims.

Claims (10)

1. The following compounds: 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

2. A mixture from the mycelium of Antrodia camphorata, which is prepared from a water or organic solvent extract of the mycelia of Antrodia camphorata; said mixture contains 3-isobutyl-4-[4-(3-methyl-2-butene Oxy)phenyl]furan-2,5-dione, 3-isobutyl-4-[4-(3-methyl-2-butenyloxy)phenyl]-1H-pyrrole-2,5 -diketone, 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. 3. The mixture of claim 2, wherein the organic solvent is an alcohol, an ester, an alkane or an alkyl halide. 4. The mixture of claim 3, wherein the alcohol is ethanol. 5. The use of the mixture of claim 2 in the preparation of drugs for reducing systolic blood pressure, increasing high-density lipoprotein, or for central cholinergic stimulation, liver protection, anti-inflammatory or anti-tumor. 6. The use of claim 5, wherein the tumor is derived from cells or tissues selected from the group consisting of liver, intestine, bone, blood, lymph and breast. 7. A composition comprising the compounds 3-isobutyl-4-[4-(3-methyl-2-butenyloxy)phenyl]furan-2,5-dione, 3-isobutyl Base-4-[4-(3-methyl-2-butenyloxy)phenyl]-1H-pyrrole-2,5-dione, 3-isobutyl-4-[4-(3-methyl Base-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 yl-2-butenyloxy)phenyl]pyrrolidine-2,5-dione. 8. The use of the composition according to claim 7 in the preparation of drugs for lowering systolic blood pressure, increasing high-density lipoprotein, or for stimulating central cholinergic, hepatoprotective, anti-inflammatory or anti-tumor. 9. The use of claim 8, wherein the tumor is derived from cells or tissues selected from the group consisting of liver, intestine, bone, blood, lymph and breast. 10. A mycelium of Antrodia camphorata, which comprises compound 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.

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