HK1081195B - Compounds and mixture or compositions from mycelia of antrodia camphorata and use thereof - Google Patents

Description

Compounds and mixtures or compositions from Antrodia camphorata mycelium and uses thereof

Technical Field

The present invention relates to novel mixtures and compounds from Antrodia Camphorata (Antrodia Camphora) mycelium and uses thereof. The present invention relates to a composition or mycelium comprising a compound of the invention.

Background

In taiwan, the fruiting body of antrodia camphorata (Polyporaceae, aphylophorrales) is well known as a traditional Chinese medicine. It grows only in the intramural layer of heartwood of the Taiwan endemic evergreen Cinnamun kanehirai (Hay) (Lauraceae). It is rare and has not been cultivated. The fruiting body has been used for treating food and drug poisoning, diarrhea, abdominal pain, hypertension, scabies of skin and liver cancer. Few studies of biological activity have been reported to date.

In taiwan, antrodia camphorata, also called antrodia camphorata, has recently been reported to be a new fungal species characterized by: its spore-bearing cylindrical, weak and soft starchy skeleton hyphae, bitter and bright yellow-brown inverted to cap-shaped (pileate) basidiocarps, and chlamydospores and anthraconidia in pure culture appear in the fruiting body. The growth of this new fungal species is extremely slow and limited to the endemic tree species, Cinnamomum kanehirai Hay (Lauraceae), as the only host. The detailed description and taxonomic location of antrodia camphorata is described in Wu, s. -h. et al, antrodiacannamomea (antrodia camphorata), New combination of a medial funcus inTaiwan, bot. 273-275.

In taiwan folk medicine, the fruiting body of antrodia camphorata is considered to have some medical effects. The fruiting body is ground into dry powder or decocted with other herbs according to conventional method for oral administration, and can be used for treating symptoms caused by poisoning, diarrhea, abdominal pain, hypertension, scabies and hepatocarcinoma. However, to date, little pharmacological or clinical research has emerged in the literature. The price of "antrodia camphorata" in taiwan is very expensive because of its strict host specificity and scarcity in nature, and failure of artificial cultivation. In recent years, high quality fungal fruit bodies have been sold to extremely high prices, about $ 15000 per kg.

Disclosure of Invention

Accordingly, it is an object of the present invention to provide a novel mixture from mycelia of antrodia camphorata.

It is another object of the present invention to provide novel compounds derived from Antrodia camphorata mycelium.

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

It is still another object of the present invention to provide novel antrodia camphorata mycelia comprising the compound of the present invention.

Drawings

Figure 1 shows HMBC correlation of compound 2.

Figure 2 shows a compound of the invention.

FIG. 3 shows NOE (Nuclear Ouhauser effect) correlation of compounds 4 and 5 of the present invention.

FIGS. 4a-d show the results of the assay for Compound 3 of the present invention.

FIGS. 5a-c show the results of an ACM (Antrodia camphorata mycelium powder) aqueous extract test.

FIGS. 6a-f show the results of the ACM ethanolic extract.

FIGS. 7a-e show the results of the assay for Compound 1 of the present invention.

Detailed Description

The present invention provides compounds of the formula

Wherein

X is N or O;

R₁is C_1-10Alkoxy radical, C_2-10Alkenoxy or C_2-10An alkynyloxy group;

R₂is H, C_1-10Alkyl radical, C_2-10Alkenyl or C_2-10An alkynyl group; and is

R₃Absent, or is H or hydroxy;

with the proviso that if X is O, then R₃Is absent.

Among the compounds of the present invention, R is preferred₁Is C_2-6Alkenoxy or C_2-6An alkynyloxy group; more preferably R₁Is warp C_1-6Alkyl substituted C_2-6Alkenyloxy, and most preferably R₁Is a butenyloxy group substituted with a methyl group. Among the compounds of the present invention, R is preferred₂Is C_1-6Alkyl, most preferably R₂Is an isobutyl group.

Thus, a preferred compound of the invention is 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-diones, or 3R^*，4R^*-1-hydroxy-3-isobutyl-4- [4- (3-methyl-2-butenyloxy) phenyl]Pyrrolidine-2, 5-dione.

More preferred compounds of the invention are 3-isobutyl-4- [4- (3-methyl-2-butenyloxy) phenyl ] -1H-pyrrole-2, 5-dione or 3-isobutyl-4- [4- (3-methyl-2-butenyloxy) phenyl ] -1H-pyrrole-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 invention also provides mixtures from antrodia camphorata mycelia comprising the compounds of the invention. The mixture of the present invention is prepared from water or organic solvent extracts of antrodia camphorata mycelia. The organic solvent includes, but is not limited to, alcohol (e.g., CH)₃OH、C₂H₅OH or C₃H₇OH), esters (e.g. ethyl acetate), alkanes (e.g. hexane) and haloalkanes (e.g. CH)₃Cl、C₂H₂Cl₂). Preferred organic solvents are ethanol or ethanol-containing solvents that do not cause any side effects in humans. The mixture of the present invention can reduce systolic blood pressure or increase high density lipoprotein. In addition, the same mixture has central cholinergic agonistic, hepatoprotective, anti-inflammatory or anti-tumor activity. In particular, the mixture of the invention inhibits tumors selected from cells or tissues of the liver, intestine, bone, blood, lymph and breast. Subjects receiving the mixture of the 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 can reduce systolic blood pressure or increase high density lipoprotein. In addition, the compositions of the present invention have central cholinergic agonistic, hepatoprotective, anti-inflammatory or anti-tumor activity. In particular, the compositions of the present invention inhibit tumors selected from cells or tissues of the 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 camphorata (Antrodia) mycelium comprising the compounds of the present invention. Preferred mycelia have a weight of at least 1% of the raw mycelia of the total weight of the compounds 1-5 of the present invention. Most preferred mycelium is mycelium wherein at least 3% of the weight of the mycelium of the raw material is the total weight of compounds 1-5 of the present invention. Antrodia camphorata mycelium was prepared by previous submerged liquid fermentation (submerged fermentation), such as T.L. M.Stamford et al, Food Science "Protein engineering of case waters for animal feeds" from http:// www.unu.edu/unepress/Food/8F 10le/8 F10E0b.htm.

Examples

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

General Experimental procedures

Melting points were measured with a Yanagimoto mini-hotplate melting point tester and were uncorrected. Optical rotation was measured with a JascoDIP-360 autosclerometer. The UV spectrum was measured with a Shimadzu UV-2200 self-recording spectrophotometer. The infrared spectra were measured on a Jasco FT/IR-230 infrared spectrometer. Measured with a Varian Unity Plus 500 spectrometer¹H-and¹³C-NMR spectrum. EIMS and HR-EIMS were measured with a JeolJMS-AX 505 HAD mass spectrometer at an ionization voltage of 70 eV. Column chromatography was carried out on silica gel using BW-820MH (normal phase) and Chromatorex-ODS DM1020T (reversed phase) (Fuji Silysia).

Extraction and separation

The powder of mycelia of Antrodia camphorata (60 g) from Taiwan Shansheng Biotechnology, Inc., 10 months 2001 was extracted with chloroform for 3 hours and three times under reflux. The chloroform extract (5.3 g) was chromatographed on silica gel, eluting with n-hexane-acetone (19: 1-14: 6) and chloroform-methanol (1: 1) to give nine fractions (Fr.1-9). The second portion was subjected to silica gel chromatography to give compound 1(8.7 mg). Performing normal phase and reverse phase silica gel on the fourth partChromatography gave compound 2(13.6 mg). The fifth fraction was subjected to silica gel chromatography and eluted with n-hexane-acetone (8: 2) to give ergosterol peroxide (35.8 mg). Combined normal and reverse phase silica gel column chromatography of the sixth fraction afforded compound 3(14.6 mg). Column chromatography of the seventh fraction gave 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-80T_M(21.5 × 300 mm), mobile phase: methanol-Water with 0.1% TFA (70: 30)]。

3-isobutyl-4- [4- (3-methyl-2-butenyloxy) phenyl]-furan-2, 5-dione (compound 1): a yellow oil; UV (MeOH) lambda_max(logε)227(4.1)，258(3.9)，275(3.8)，355(3.4)nm；IR(CHCl₃)v_max 1763cm^-1，¹H-NMR Table 1;¹³C-NMR Table 2; EIMS M/z 314[ M ]]⁺(100)，246(100)，131(100)；HR-EIMSm/z314.1523(C₁₉H₂₂O₄The calculated value of (a): 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-; UV (methanol) lambda_max(logε)230(4.3)，272(3.5)，355(3.7)nm；IR(CHCl₃)v_max 1724cm^-1；¹H-NMR Table 1;¹³C-NMR Table 2; EIMS M/z 313[ M ]]⁺(8)，245(100)，203(77)，131(28)；HR-EIMS m/z 313.1681(C₁₉H₂₃NO₃The calculated value of (a): 313.1678).

X-ray crystallography of compound 2:

the yellow needle crystal is obtained by n-hexane-ethyl acetate crystallization, and the yellow needle crystal is selected for data collection. Crystal data: c₁₉H₂₃NO₃(ii) a Mr 313.40; the size is 0.15 multiplied by 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＝1.223g/cm³，μ(MoKα)＝0.82cm^-1And F000 is 336.00. Irradiated at 93K with Mo-Ka (λ. 0.71069) having a graphite monochromic color) The Rigaku RAXIS-RAPID shadowgraph diffractometer performs the measurement. Of the 8950 reflections collected, 4745 were unique (R)_int0.108); the equal reflections are combined. The crystal structure was resolved by direct method (SHELXS86) and refined with the full matrix minimum squares. Non-hydrogen atoms are unequivocally (anistropic). Hydrogen atoms are included but not accounted for. Final index R ═ 0.074, R_w0.099 and GOF (Guest Observer Facility) 1.06. The maximum and minimum peaks of the final difference Fourier map correspond to 0.83 and-0.89 e, respectively^-/

3-isobutyl-4- [4- (3-methyl-2-butenyloxy) phenyl]-1H-pyrrol-1-ol-2, 5-dione (compound 3): a yellow oil; UV (MeOH) lambda_max(logε)：232.5(4.3)，296(3.7)，374(3.7)nm；IR(CHCl₃)v_max 1717cm^-1；¹H-NMR Table 1;¹³C-NMR Table 2; EIMS M/z 329[ M ]]⁺(12)，261(100)，131(50)；HR-EIMS m/z：329.1637(C₁₉H₂₃NO₄The calculated value of (a): 329.1627).

3R^*，4S^*-1-hydroxy-3-isobutyl-4-, [ 2 ]4- (3-methyl-2-butenyloxy) phenyl]Pyrrolidine-2, 5-dione (4): a colorless oil; [ alpha ] to]_D ²³+2.5 ° (c0.2, methanol); UV (methanol) lambda_max(logε)：225(4.3)，275(3.3)，283(3.2)nm；IR(CHCl₃)v_max 1715cm^-1；¹H-NMR Table 1;¹³C-NMR Table 2; EIMS M/z 331[ M ]]⁺(2)，263(67)，207(66)，191(30)，179(40)，133(64)，69(100)；HR-EIMS m/z：331.1747(C₁₉H₂₅NO₄The calculated value of (a): 331.1783).

3R^*，4R^*-1-hydroxy-3-isobutyl-4- [4- (3-methyl-2-butenyloxy) phenyl]Pyrrolidine-2, 5-dione (5): a colorless oil; [ alpha ] to]_D ²³+3.0°(c0.2，MeOH)；UV(MeOH)λ_max(logε)：227(4.3)，275(3.4)、283(3.3)nm；IR(CHCl₃)v_max 1715cm^-1；¹H-NMR Table 1;¹³C-NMR Table 2; EIMS M/z 331[ M ]]⁺(1)，263(45)，207(50)，191(75)，179(30)，133(100)，69(92)；HR-EIMS m/z：331.1766(C₁₉H₂₅NO₄The calculated value of (a): 331.1783).

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

Cytotoxicity assay: in vitro LLC tumor cell assays were performed using sulfofordamin B (SRB). 50% growth inhibition (ED) was calculated by Probit method₅₀)。

Results and discussion

The chloroform extracts of Antrodia camphorata mycelium were subjected to repeated normal phase and reverse phase silica gel chromatography to obtain five new maleic and succinic acid derivatives (compounds 1-5) and ergosterol peroxide.

TABLE 1

Of Compounds 1 to 5¹H-NMR spectral data (. delta.ppm, J. ═ Hz) (500MHz, CDCl)₃)

TABLE 2

Of Compounds 1 to 5¹³C-NMR spectral data (. delta.ppm) (125MHz, CDCl)₃)

a) Attribution is interchangeable.

The structure of the novel compounds is determined as follows:

the compound 2 is yellow needle-shaped crystal, mp 110-₁₉H₂₃NO₃. Infrared spectrum at 1724cm^-1Showing imide carbonyl absorption.¹³C-NMR spectra show four methyl carbons, two methylene carbons and one methine carbon signal in the aliphatic region, as well as one phenyl ring, one alkenyl group and two carbonyl carbons.¹H-NMR spectra showed isobutyl moieties at delta 0.90, 2.06 and 2.51, 3-methyl-2-butenyloxy moieties at delta 1.76, 1.81, 4.56 and 5.50, and para-substituted benzene moieties at 6.95 and 7.50, consisting of¹H-¹H COSY (cooling synchrotron) and HMQC (heteronuclear multiple quantum correlation) experiments are further supported. Long range correlation was observed by HMBC as shown in figure 1. Based on the molecular formula and¹³C-NMR spectra, conclude that this compound contains additional CHNO atoms, including more than one carbonyl carbon. Therefore, the unclear portion is presumed to be a maleimide group. The structure was then determined to be 3-isobutyl-4- [4- (3-methyl-2-butenyloxy) phenyl by X-ray analysis]-1H-pyrrole-2, 5-dione.

The molecular formula of the compound 1 is C by HR-EIMS analysis₁₉H₂₂O₄. The infrared spectrum shows 1763cm^-1With carboxylic acid anhydridesAnd (4) absorbing the base. Process for preparation of Compound 1¹The H-NMR spectrum was similar to that of Compound 2, showing the presence of an isobutyl moiety, a 3-methyl-2-butenyloxy moiety, a para-substituted benzene ring. The HMBC spectrum indicated that compound 1 had a structure partially identical to compound 2 (fig. 1), where the presence of maleic anhydride was inferred from the molecular formula. Thus, compound 1 was identified as 3-isobutyl-4- [4- (3-methyl-2-butenyloxy) phenyl]Furan-2, 5-dione.

The molecular formula of the compound 3 is C by HR-EIMS analysis₁₉H₂₃NO₄. The infrared spectrum showed 1717cm^-1The carbonyl group is absorbed and can be classified as hydroxyl diimide.¹H-and¹³the C-NMR spectrum was also similar to that of compounds 1 and 2, showing the presence of an isobutyl moiety, a 3-methyl-2-butenyloxy moiety, a para-substituted benzene ring. In the HMBC experiment, compound 3 was demonstrated to have a partial structure identical to compound 2 (fig. 1). Compound 3 contains one more oxygen atom than compound 2, and therefore, this compound was identified as 3-isobutyl-4- [4- (3-methyl-2-butenyloxy) phenyl]-1H-pyrrol-1-ol-2, 5-dione.

Analysis by HR-EIMS revealed that Compounds 4 and 5 have the same R_fThe same value of formula (C)₁₉H₂₅NO₄Observed 331.1747 and 331.1766, respectively), however they can be separated by preparative HPLC. The infrared spectrum of the two compounds is 1715cm^-1The hydroxyl diimide carbonyl is absorbed. In that¹H-and¹³in the C-NMR spectrum, both compounds showed the presence of an isobutyl moiety, a 3-methyl-2-butenyloxy moiety, a para-substituted benzene ring, but the isobutylmethylene proton showed multiple peaks rather than two peaks as in compounds 1-3.¹H-¹The H COSY spectrum shows that the methylene group is linked to the-CH-unit. Of compounds 4 and 5¹³The C-NMR spectrum shows two additional sp³Carbon signal, instead of sp found in Compounds 1-3²Carbon signal. Thus, compounds 4 and 5 are not N-hydroxymaleimides, but are N-hydroxysuccinimides having stereogenic centers at the C-3 and C-4 positions of the succinimide ring. Compounds 4 and 5 are based on the coupling constant between H-3 and H-4 (Compounds 4 and 5 min)4.0 and 8.0Hz, respectively) were determined as trans and cis isomers, respectively. No NOE between H-3 and H-4 was observed in the NOESY (nuclear Vouhause effect) spectrum of compound 4, whereas 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, while their CD spectra showed no catamenial effect at any wavelength, indicating that compounds 4 and 5 are both racemic mixtures. Resolution of these racemic mixtures by HPLC using chiral columns with several solvent systems has not been successful. At present, we cannot clearly infer whether these compounds are optically active compounds or racemic mixtures. Therefore, their relative structures are respectively determined to be 3R^*，4S^*-and 3R^*，4R^*-1-hydroxy-3-isobutyl-4- [4- (3-methyl-2-butenyloxy) phenyl]Pyrrolidine-2, 5-dione.

These types of maleic and succinic acid derivatives from natural sources were isolated a second time according to the report of aquvque et al.

The cytotoxic activity of the chloroform extracts and the isolated compounds was investigated using LLC (Lewis lung carcinoma) cell line (table 3). Chloroform extract shows moderate cytotoxic effect, its ED₅₀The value was 26.7. mu.g/ml. Maleic acid compounds 1 and 4 had no cytotoxic activity, whereas compounds 2 and 3 were found to be cytotoxic to the LLC cell line, with ED₅₀Lower than that of the chloroform extract. TABLE 3 50% growth inhibition (ED) of LLC cell lines by chloroform extracts from Antrodia camphorata mycelium and compounds 1-4₅₀) Value of

a) Positive control

Tumor determination of ACM (Antrodia camphorata mycelium powder)

A. Cell lines

Adhesion of cells:

MCF-7: human breast cancer

HT-29: human colon adenocarcinoma

KATO III: human gastric adenocarcinoma

SW 480: human colon adenocarcinoma

SW 620: human colon adenocarcinoma

HepG 2: tumor of human liver gland

Suspension of cells:

EL 4: lymphoma of mouse

B. Sample (I)

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

C. Measurement method

Calculating ED₅₀(50% inhibition of effective dose)

Adhesion of cells: MTT (methylthiazolyltetrazolium) method: cells were identified for MCF-7, HT-29, KATO III, SW480, HepG2, day 3. SW6204 days.

Suspension of cells: a cell counting method; EL4 cells were counted for 5 days.

D. Results

And (3) calculating: y is m Ln (x) + b

Examples of the invention

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

Using X (10, 30, 50ppm) and Y values, correlation curves were obtained

y＝-0.2643Ln(x)+1.5321

ED₅₀＝exp【(0.97/2-1.5321)/(-0.2643)】

Sample preparation and sample description

Aqueous extract of acm (antrodia camphorata mycelium powder)

1.1 g of ACM was added to a solution containing 40 ml of RO H₂O in a 250 ml beaker, place the beaker in an ultrasonic water bath at room temperature for 20 minutes.

2. The water bath was stirred at 45 ℃ for 45 minutes.

3. The beaker was placed in an ultrasonic water bath for an additional 20 minutes.

4. The samples were centrifuged at 3000rpm for 15 minutes.

5. The supernatant was collected and serially diluted with medium.

B. Determination of sample concentration

1. The evaporation pan was weighed (W1).

2. 10ml of the aqueous extract was added to the evaporation pan

3. The evaporation pan was placed in an oven to remove water (W2)

Sample weight/ml ═ (W2-W1)/10

ACM (Antrodia Camphorata mycelium powder) ethanol extract

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

2. The suspension was filtered through Advantec No. 1 filter paper and the filtrate was collected.

3. The filtrate was concentrated by rotary vacuum evaporator to remove ethanol.

D. Compound 1: pure compounds from ACM

E. Compound 3: pure compounds from ACM

MTT assay

1. After proliferation of the cells, the old medium was discarded, and the cells were washed once with Phosphate Buffered Saline (PBS).

2. Cells were washed with trypsin-EDTA.

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

4. The pellet was suspended in 10ml of medium.

5. 100 microliters of the cell suspension was mixed with 100 microliters trypan blue to count viable cells.

6. Add 1X 10 to each well of a 96 well plate⁴Cells/100. mu.l of medium, and the plate was incubated at 37 ℃ for 24 hours in a carbon dioxide incubator.

7. Old medium was discarded and cells were washed once with PBS.

8. A100. mu.l sample was added to each well and the plates were incubated at 37 ℃ in a carbon dioxide incubator.

9. Cells were washed once with PBS on days 3, 4 and 5.

10. To each well was added 57. mu.l MTT (0.88 mg/ml).

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

12. Add 50. mu.l DMSO per well.

13. Results were read in an ELISA instrument at OD 545.

Cell counting method (EL4 cell line)

1. After proliferation of the cells, the old medium was discarded by centrifugation.

2. The pellet was resuspended in fresh medium.

3. 100 microliters of the cell suspension was mixed with 100 microliters trypan blue to count viable cells.

4. Preparation of a catalyst containing 1X 10⁵Samples of different concentrations of individual cells per milliliter of sample.

5. A100. mu.l sample was placed in each well of a 96-well plate, and the plate was incubated at 37 ℃ in a carbon dioxide incubator.

6. Surviving cells were counted on days 3, 4 and 5.

PBS

NaCl 8 g

KCl 0.2 g

Na₂HPO₄1.4 g

KH₂PO₄0.2 g

The volume was adjusted to 1 liter pH 7.4

Results and discussion

ED of ACM in cell lines₅₀

Cell lines	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 aqueous 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. 4 d).

ACM aqueous extract: HepG2 (fig. 5a), SW620 (fig. 5b) and EL4 (fig. 5 c).

Ethanol extract of ACM: HT-29 (FIG. 6a), SW480 (FIG. 6b), SW620 (FIG. 6c), EL4 (FIG. 6d), HepG2 (FIG. 6e) and Kato III (FIG. 6 f).

Compound 1 of the present invention: MCF-7 (FIG. 7a), EL4 (FIG. 7b), HT-29 (FIG. 7c), SW620 (FIG. 7d) and HepG2 (FIG. 7 e).

The results prove that the compound and the ACM extract have the inhibiting effect on various tumor cells.

Analysis of all novel compounds (1, 2 and 3) from ACM ethanol extract by high performance liquid chromatography

The purpose is as follows: to measure the amount of all new compounds (1, 2 and 3) from the ACM ethanolic extract, high performance liquid chromatography was employed as our conventional quality control procedure.

Preparation of ACM ethanol extract sample:

1) the sample powder was precisely weighed at 20.000 g with an electronic balance and placed in a graduated test bottle containing 100 ml of 95% ethanol, and the cap was not screwed.

2) And placing the sample bottle in the ultrasonic water bath for 10 minutes.

3) The liquid samples were poured into centrifuge tubes and those samples were subsequently centrifuged at 6500rpm for 5 minutes to remove coarse particles.

4) The liquid layer was filtered through an advantec No. 1 filter paper.

5) The filtrate was concentrated by rotary vacuum evaporator until a viscous, ethanol-free, pale yellow liquid appeared.

6) Steps 1 to 5 were repeated three times and all the extracted products were collected (total ACM ethanolic extract ═ 4.60 g) and the yields calculated.

Application of model 2690 Water HPLC:

1) a column: inverse C18

2) And a mobile phase: methanol, water and acetonitrile

3) Injection volume: 20 microliter

4) And detecting: measurement at a wavelength of 254nm by a photodiode array detector 996

5) A sample of 1.000 g ACM ethanolic extract in 10ml ethanol was prepared for HPLC analysis^*：

As a result: the extract products containing pure compounds 1, 2 and 3 according to HPLC analysis are shown in table 4 below.

Table 4:

thus, the total weight of compounds 1, 2, and 3 was 5.92% of the weight of the ACM sample.

Testing of ACM ethanol extracts

Materials and apparatus

1. Test substances and modes of administration

All in vivo tests were given with an initial dose of test substance of 1000mg/kg orally in 2% Tween 80 vehicle. The observation time for each experiment is described in the methods.

2. Animal(s) production

Male or female ICR mice, Wistar-Okamoto-derived male Spontaneous Hypotension Rats (SHR), Wistar and Long Evans-derived rats, provided by taiwan pantoball pharmacology studies, were used. The animal raising space was as follows: 10 mice were housed in a space of 29X 18X 13 cm, 6 rats were housed in a space of 45X 23X 21 cm, and 3 guinea pigs were housed in a space of 45X 23X 21 cm. Mice and rats were housed in APEC^RIn the cage. C57BL/6J immunocompetent male mice weighing 21 + -2 grams, 6-8 weeks old, provided by the national Taiwan university animal center, were also used in this study. Experimental animals were housed in independently ventilated cages (IVC racks, 36 mini-isolation system). Each cage was autoclaved and contained 5 mice (in a space of 26.7X 20.7X 14 cm). All animals were kept in a temperature-controlled (21 ℃ -23 ℃) and humidity-controlled (60% -70%) environment for at least one week in a 12 hour light cycle in the laboratory prior to use. Standard experimental diets (LabDiet Rodent Diet and Guinea Pig Diet, PMI Nutrition International, USA) were provided free access to tap water.

3. Cell lines and culture media

Murine melanoma cells, B16-F0(ATCC CRL-6322) were purchased from American type culture Collection and Dulbecco's Modified Eagle's Medium (GIBCO, USA) was used as the cell culture Medium. Tumor cells were cultured at 37 ℃ and 5% CO₂In the air of (2).

4. Chemical medicine

In general:

distilled water (homemade), dimethyl sulfoxide (DMSO, Merck, germany), isotonic sodium chloride solution (taiwan, product of the chemical industry of trusted east), magnesium sulfate (MgSO)₄·7H₂O, Wako, japan), meclofenamate sodium (Sigma, usa), methylcellulose (signala, 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 (Nihon Koden, Japan).

5. Device

Generally, the following are used:

animal box (Xinde, Taiwan), 250 ml and 1000 ml beaker (Kinmax, USA), disposable syringe (1 ml, Top Corporation, Japan), stainless steel forceps (klappencer, Germany), mouse scale (mouse scale) # Z-40 (Taonic, USA), oral feeding syringe (Natsune, Japan), hypodermic needle 23G x 1 "(Top Corporation, Japan), pH meter (Suntex, USA), mouse scale 500 g + -2 g (Chien-chun, Taiwan), glass syringe 1 ml, 2 ml and 5 ml (Mitsuba, Japan), and stainless steel scissors (appncker, Klebsiel).

The method and the result are as follows:

1. cholinergic agonism, central/peripheral (Lippmann W and Pugsley TA, Arch IntPharmacodyn.227: 324, 1977).

The test substance was orally administered to a group of 3 male or female rats of the Wistar-derived line weighing 150 ± 20 grams. Within the following 30-60 minutes, the number of animals exhibiting a cumulative measured chewing behaviour (mouth and/or tongue movements) of more than 10 seconds, as well as the number of animals exhibiting salivation, was recorded. The observation of positive reactions of 2 or more (. gtoreq.2) in 3 rats indicates the possibility of central cholinergic activity and peripheral cholinergic activity.

Table 5: cholinergic agonism results, central/peripheral, in rats

The vehicle and test substance were administered orally (PO), while the positive control compound was administered by intraperitoneal Injection (IP). The number of animals exhibiting a cumulative measured chewing behaviour (mouth and/or tongue movement) of more than 10 seconds, as well as the number of animals exhibiting salivation, was recorded over the following period of 30-60 minutes. The observation of positive reactions of 2 or more (. gtoreq.2) in 3 rats indicates the possibility of cholinergic 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)

3 Wistar-Okamoto-derived spontaneous hypertensive male mouse (SHR) groups weighing 250. + -.20 g were used; the mean systolic pressure is 200 + -20 mmHg and the heart rate is 400 + -30 times/min. Blood pressure and heart rate were recorded indirectly by tail cuff method under a temperature controlled environment (32 ± 1 ℃) before (0 hours) and 1, 2, 4 hours after oral administration of the test substance or vehicle. A10% or more (10%) reduction in systolic blood pressure or a 20% or more (20%) reduction in heart rate is considered significant when compared to 0 after each inter-time measurement.

Table 6: results of blood pressure and central blood vessel in rat (SHR 0, 1, 2, 4 hours)

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

SHR with a systolic blood pressure of 200 + -20 mmHg and a heart rate of 400 + -50 mmHg per minute was used. Blood pressure was recorded indirectly via the visual tail cuff method at 0 hours and 1, 2, 4 hours after oral administration of the test substance or vehicle. Blood pressure drops by 10% or more (. gtoreq.10%) or heart rate drops by 20% or more (. gtoreq.20%) compared to the time point of each measurement at 0 given in parentheses are considered significant.

229mmHg and 403mmHg times at 10ml/kg 0 of the carrier

The minutes are taken as 100%.

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

The minutes are taken as 100%.

0.1mg/kg 0 of clonidine is combined with 228mmHg and 379mmHg times

The minutes are taken 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)

SHR with a systolic blood pressure of 200 + -20 mmHg and a heart rate of 400 + -50 mmHg per minute was used. Blood pressure was recorded indirectly via the visual tail cuff method at 0 and 1, 2, 4 hours after (prior) oral administration of the test substance or vehicle. A10% or more (. gtoreq.10%) decrease in blood pressure or a 20% or more (. gtoreq.20%) decrease in heart rate per measurement time point compared to 0 given in brackets is considered significant.

220mmHg and 410mmHg times when the carrier is 10ml/kg 0

The minutes are taken as 100%.

300mg/kg 0 of ACM-ethanol extract is treated with 205mmHg and 446mmHg times

The minutes are taken as 100%.

0.1mg/kg 0 of clonidine 235mmHg and 417mmHg times-

The minutes are taken 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).

A group of 5 ICR-derived male mice weighing 22 + -2 grams was fed a high-fat diet (g/100 g: coconut oil, 8; cholesterol, 1.0; cholic acid, 0.3; lard, 2; standard diet 88.7) for 7 days to induce hypercholesterolemia. Test substances were administered orally on days 5, 6, and 7. After an overnight fast, serum was obtained from each mouse to determine total cholesterol (total), High Density Lipoprotein (HDL), and percent change in total/HDL. A decrease of 20% or more (. gtoreq.20%) in total serum, or a 20% or more (. gtoreq.20%) increase in serum HDL, or a 40% or more (. gtoreq.40%) decrease in total/HDL as compared to vehicle-treated control animals was considered significant.

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

Vehicle, test substance or control positive compound was administered orally (PO) on days 5, 6, 7 after feeding high cholesterol diet. Twenty-four hours after the third dose, overnight fasted experimental animals were sacrificed to assess serum total cholesterol (total) and High Density Lipoprotein (HDL). A decrease of 20% or more (. gtoreq.20%) in serum total volume, or a 20% or more (. gtoreq.20%) increase in serum HDL, or a 40% or more decrease (. gtoreq.40%) in total volume/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 derived male rats weighing 200. + -.20 grams was used. Each animal was treated with a single injection of D-galactosamine (500mg/kg, IP). The test substances were given orally 0.5 hours before and 4 and 8 hours after the administration of D-galactosamine, and the animals were sacrificed after 24 hours. Serum alanine Aminotransferase (ALT) and aspartate Aminotransferase (AST) levels were measured with a HITACHI automatic analyzer (model 7050) by an optimized UV method. A30% or more (30% or more) reduction in ALT or AST activity compared to vehicle-treated control animals indicates significant protection.

Table 11: results of liver injury, galactosamine, in rats

The test substances and vehicle were orally administered 0.5 hours before and 4 hours and 8 hours after one administration of galactosamine (500mg/kg, IP), the rats were sacrificed 24 hours after galactosamine injection, and ALT and AST values were determined. ALT and AST reductions of > 30% compared to the vector group were considered significant.

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

3 Long Evans derived lines, male or female groups weighing 150 + -20 grams, were fasted overnight prior to the study. Test substance was administered orally 1 hour before the right hind paw received an injection of carrageenan (0.1 ml of a 1% internal (intraplantar) suspension). Hind paw edema was used as a measure of inflammation and was recorded 3 hours after carrageenan administration using an plethysmometer with a water bath (25 mm diameter). A drop of 30% or more (. gtoreq.30%) in hindpaw edema indicates significant anti-inflammatory activity.

Table 12: inflammation results, carrageenan, in rats

Table 13: inflammation results, carrageenan, in rats

Test substance and vehicle were administered to fasted rats overnight 1 hour before the injection of carrageenan (0.1 ml of 1% intrapallar suspension) in the right hind paw (R.P.); left hind paw (l.p.) was not injected. A30% or more (. gtoreq.30%) decrease in hindpaw edema shown in brackets indicates significant acute anti-inflammatory activity.

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

A group of 5 Special Pathogen Free (SPF), immunocompetent (6-8 week old) C57BL/6J male mice were used, housed in an animal isolation room (IVC rack) in a Special Pathogen Free (SPF) environment. Viable B16-F0 murine melanoma cells (ATCC CRL-6322, 1.0X 10 in 0.2 ml) homologous to C57BL/6J mice⁵Individual cells) were injected subcutaneously into the dorsal side of experimental mice. Treatment was started 24 hours after tumor inoculation and test compounds were given daily by oral gavage for 21 days or decreased days when overt toxicity symptoms occurred. Mice were monitored for weight, tumor size and survival from day 1 to day 22. The survival of the experimental mice was also monitored until the end of the 45 th day after the study.

Tumor weight (mg) was estimated according to the prolate ellipsoid formula: length (mm) × [ width (mm)]²X 0.5, assuming a specific gravity of 1, pi is 3. Tumor growth in compound-treated animals was calculated as T/C (treatment/control) x 100%; the T/C value is less than or equal to 42 percent, indicating remarkable anti-tumor activity. T/C (treatment/control) survival mean of 125% or more 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 given vehicle and test substance daily 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 a period of 22 days. Tumor growth inhibition was calculated as T/C (weight/control) × 100. T/C value 42% or less is regarded as having significant antitumor activity.

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

24 hours after tumor cell implantation, the experimental animals were given vehicle and test substance daily 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 a period of 22 days. The student's t-test was used to determine the significant difference in body weight change between the test compound and the vehicle control group.

Table 17: tumor, syngeneic melanoma B16-F0 cell results

a: if the animal did not die after 45 days, its survival time was considered 45 days.

The treated mice were monitored for survival for the 45 day study period, or until the experimental animals died. T/C (treatment/control) survival mean of 125% or more was also considered to have significant antitumor activity.

Discussion:

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

central cholinergic agonism at 1000mg/kg in rats; minimal and insignificant agonism was visible at 300 mg/kg; there was no significant agonism or antagonism of peripheral biliary alkaline nerves at 1000mg/kg (table 5).

In Spontaneous Hypertensive (SH) rats, a reduction in systolic blood pressure at 1000mg/kg (16%, 12% and 20% respectively, observed at time points of 1, 2 and 4 hours, compared to 100% at 0) with a moderate but insignificant drop in heart rate (tables 6 and 7); the 300mg/kg dose did not result in significant changes in systolic blood pressure and heart rate (tables 8 and 9).

High density lipoprotein (HDL, 39% more than vehicle control) was increased in diet-induced mice at 1000mg/kg (table 10); the associated total cholesterol (total) was not significantly altered, while the HDL/total ratio decreased significantly to nearly 31%; the 300mg/kg dose did not result in significant changes in total, HDL and HDL/total ratio.

In semi-lactosamine induced liver injury in rats, hepatoprotective effect was seen at 1000mg/kg x 3 (44% ALT reduction and 57% AST reduction compared to vehicle control); a moderate decrease of ALT 20% and an AST 28% was seen at 300 mg/kg.times.3 (Table 11).

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

Antitumor activity in C57BL/6J mouse syngeneic melanoma B16-F0 cells at 1000mg/kg, days 8, 11 and 15 (tables 14 and 15), and prolongation of animal survival time (table 17); animal body weights were not significantly changed (table 16).

Testing of ACM, ACM-ethanol extract and Compound 3 of the present invention

Nine groups of 5 ICR-derived male mice (weighing 22 ± 2 grams) were used per group. Each animal was dosed with a single dose of carbon tetrachloride (CCl)₄PO) in 50% olive oil, 0.1 ml/kg, elicits an immune response. Orally administering a test substance 30 minutes before and 4 and 8 hours after carbon tetrachloride challenge at doses of 300 and 1000mg/kg ACM, or at doses of 30, 100 and 300mg/kg compound 3 of the invention; whereas 300 and 1000mg/kg of ACM ethanolic extract administered orally was pretreated 1 day (twice daily) 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 by optimized UV using a HITACHI automatic analyzer (model 7050). Compared with the carrier, the ALT or AST level is reduced by 30 percent or more (more than or equal to 30 percent), which indicates that the liver injury is remarkably protected.

Results

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

Discussion:

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

The simultaneous test of silymarin (100 mg/kg x 3, IP) showed a significant reduction of ALT (31%) and AST (31%) relative to the vehicle treated group.

It was concluded that the 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, modifications, and improvements should 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 well adapted to carry out the objects and obtain the ends and advantages mentioned, as well as those inherent therein. Cell lines, embryos, animals, and processes and methods for making them represent preferred embodiments, are exemplary, and are not intended to limit the scope of the invention. Modifications thereof and other uses will occur to those skilled in the art. Such modifications are intended to be included within the spirit of the present invention and defined by the scope of the appended claims.

Those skilled in the art will readily appreciate that variations and modifications may be made to the present invention without departing from the scope and spirit thereof.

All patents and publications mentioned in the specification are indicative of the levels of those of ordinary skill in the art to which the invention pertains. All patents and publications are herein incorporated by reference to the same extent as if each individual publication was specifically and individually indicated to be incorporated by reference.

The invention illustratively described herein suitably may be practiced in the absence of any element or elements, limitation or limitations, which is not specifically disclosed herein. The terms and expressions which have been employed are used as terms of description and not of limitation, and there is no intention in the use of such terms and expressions of excluding any equivalents of the features shown and described or portions thereof, but it is recognized that various modifications are possible within the scope of the invention claimed. Thus, it should be understood that although the present invention has been specifically described by preferred embodiments and optional features, modification and variation of the concepts herein disclosed may be resorted to by those skilled in the art, and that such modifications and variations are considered to be within the scope of this invention as defined by the appended claims.

Other embodiments are set forth in the following 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-diones

2. A mixture from antrodia camphorata mycelia prepared from an aqueous or organic solvent extract of antrodia camphorata mycelia; said mixture containing 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-diones 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. Use of a mixture according to claim 2 for the preparation of a medicament for reducing systolic blood pressure, increasing high density lipoprotein, or for central cholinergic agonism, hepatoprotection, anti-inflammatory or anti-tumor.

6. The use of claim 5, wherein the tumor is from a cell or tissue selected from the group consisting of liver, intestine, bone, blood, lymph and breast.

7. A composition comprising the 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-diones or 3R^*，4R^*-1-hydroxy-3-isobutyl-4- [4- (3-methyl-2-butenyloxy) phenyl]Pyrrolidine-2, 5-dione.

8. Use of a composition according to claim 7 for the preparation of a medicament for reducing systolic blood pressure, increasing high density lipoprotein, or for central cholinergic agonism, hepatoprotection, anti-inflammatory, or anti-tumor.

9. The use of claim 8, wherein the tumor is from a cell or tissue selected from the group consisting of liver, intestine, bone, blood, lymph and breast.

10. Antrodia camphorata mycelium comprising the 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-diones or 3R^*，4R^*-1-hydroxy-3-isobutyl 4- [4- (3-methyl-2-butenyloxy) phenyl]Pyrrolidine-2, 5-dione.