TW200528093A - Novel mixture and compounds from mycelia of antrodia camphorata and use thereof - Google Patents

Abstract

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

Description

200528093 发明 Description of the invention: [Technical field to which the invention belongs] This description relates to a novel mixture and compound derived from Antrodia cinnamomea (such as ^ G ^^) mycelium and its medical use. The invention further relates to a composition or mycelium comprising a compound of the invention. [Prior art] In the mouth 4 cattle; f Zaozhi (polyporaceae, 1 l0phoraies) fruit bodies are often used as traditional Chinese medicine. It grows only in Taiwan's endemic evergreen camphor tree 67/7, such as 卯 阴 / 7々Grasscatcher /, the inner layer of the heartwood of Hay (Uuraceae). It is very dry and uncultivated. This fruiting body has been used to treat food and medicine, diarrhea, abdominal pain, high blood pressure, skin ringworm, and liver cancer. Few related biological activity studies have been reported so far. In Mouth Bay, Antrodia cinnamomea ”is also called“ Antrodia cinnamomea ”, according to the cylindrical spores of its fruiting body, the soft amyloid skeleton hyphae, the bitterness, and the dipsum of the day camphor tree. The characteristics of membrane spores and anthroconidia in pure culture have recently been reported as new strains. The growth of this new strain is quite slow and is limited to the host of the local tree species Cinnamomum kanehirai Η "(Lauraceae). A detailed description and classification of Antrodia camphorata can be found in Wu, s.-H. et al. 1 997 Published in New combination 〇fa medicinal fungus in Taiwan 'Bot. Bull. Acad. Sin. 38: 273-275. Cinnamomea Λ. 200528093 In Taiwan folk medicine, the fruiting body of Antrodia cinnamomea is considered to have some degree of medical treatment. Effect. The fruit body is made into dry powder or boiled with other herbs according to traditional methods and taken orally to treat symptoms caused by poisoning, thirst, abdominal pain, high blood pressure, skin squeeze and liver cancer. However, and No pharmacological or clinical research has been documented to support these points. Because of its clear host and thinness in nature, and the failure of artificial cultivation, " Antrodia cinnamomea " is very expensive in Taiwan. High-quality fruiting bodies have been sold to extremely high prices, which is approximately 15,000 US dollars per kilogram. [Summary of the Invention] Summary of the Invention Novel mixture from Antrodia cinnamomea mycelium. Another subject matter of the present invention provides novel compounds from Antrodia cinnamomea mycelium. The subject matter of the present invention provides a novel composition comprising the compound of the present invention. The subject matter of the present invention is improved The present invention provides a novel antrodia mycelium containing a compound of the present invention. DETAILED DESCRIPTION OF THE INVENTION The present invention provides a compound having the formula

6 200528093 where X is N or 0;

Ri is Cho alkoxy and Ch. Alkenyl or ". Alkynyloxy; R2 is fluorene, Ch. Alkenyl, c2-1G alkenyl or & ". Alkynyl; and Chi 3 is absent, fluorene or meridian; but when X is 0, R3 is absent. Among the compounds of the present invention, R1 is preferably c Oxy or C2 ** 6 fast oxy; more preferably, C2-6 熝 ϋ f τ π is substituted by Cl-6 alkyl. "The best Rl is butyloxy substituted by f. Among the compounds of the present invention, P 2 is preferred, R 2 is Ch alkyl, and L is isobutyl. Therefore, the preferred compound of the present invention is 3-isobutyl 3-isobutyl-4- [4- (3-methyl-octabutenyloxy) phenyl] ... pyrrol-2 dione; 3- Isobutyl-4- [4- (3-methyl its 9 LUT group ~ 2 ~ butenyloxy) phenyl] -If-2,5-dione; from hydrazone; l-hydroxy-3-isobutyl ~ 4- "4 L4 (3-fluorenyl-2-butenyloxy) phenyl] 0-pyrrolidine-2,5-dione; or -Ethyl-3-isobutyl ~ 4-" 4 L4 -(3-fluorenyl_2-butenoxy) phenyl] 0 is better than pyrrolidine-2,5-dione. A more preferred compound of the present invention is 3-isobutyl-4- [4- (3- Methylbutenyloxy) phenylpropyrrol-2 5-di200528093 ketone; or 3-isobutyl-4- [4- (3-methyl_2-butaneoxy) phenyl] _ι # 十Each-butanol-2,5-diaster. The best compound of the present invention is 3_isobutyl-4_ [4_ (3_methyl_2 butenoxy) phenyl] -1 million 1-alcohol-2,5-dione. The present invention also provides a mixture of Antrodia cinnamomea mycelium, including a compound of the present invention. The mixture of the present invention is used to extract water or an organic solvent from the antrodia cinnamomea mycelium mixture. C2H5〇HCsH.OH) ^ s type (such as ethyl acetate), silk (such as hexane) and Burn (e.g., m, C2H2ci2). Koda qe good organic solvent-based ethanol or humans without causing side effects of alcohol-based solvent. The mixture of the present invention can reduce systolic blood pressure "heightening density lipoprotein. In addition, the same mixture has a central choline agonistic effect, a hepatoprotective effect, an anti-inflammatory or anti-tumor activity. The mixture of the present invention can inhibit tumors of cells or tissues selected from liver, intestine, bone, blood, lymph, and breast. Individuals receiving the mixture of the invention include, but are not limited to, dogs and cats. Humans, mammals, rats, mice, horses, pigs, chickens, ducks. Also mentioned in this article are compositions containing the compounds of the present invention. The composition of the present invention can reduce systolic blood pressure or increase high density lipoprotein. In addition, the composition of the present invention has a central nervous system biliary test agonistic effect, a hepatoprotective effect, an anti-inflammatory or anti-tumor activity. In particular, the composition of the present invention can inhibit tumors of cells or tissues selected from liver, intestine, bone, blood, lymph, and breast. Individuals receiving the compositions of the present invention include, but are not limited to, humans, mammals, mess, mice, horses, pigs, chickens, ducks, dogs, and cats. 200528093 The present invention also provides antrodia cinnamomea mycelium comprising a compound of the present invention. Preferably, the weight of the mycelium of at least 1% of the promycelium is the total weight of the compound i-5 of the present invention. More preferably, the mycelium has a weight of at least 3% of the promycelium based on the total weight of the compounds 1 to 5 of the present invention. The burdock mycelium system was prepared according to the previous submerged 1 iquid fermentation, such as τ · L · M · Stamford et al. Food Science " Protein enrichment of cashew wastes for animal feeds "from the website http: / /www·unu.edu/unupress/food/8F101e/8F101E0b.htm. [Embodiments] The following examples only represent various aspects and features of the present invention, and are not intended to limit the scope of the present invention. The melting point of the general experimental steps is based on Yanagimoto micro Melting point heating device to measure without modification. Optical rotation is measured with Jasco DIP-360 automatic polarimeter. Ultraviolet spectrum is measured with Shimadzu UV-2200 spectrophotometer. Infrared spectrum is measured with jasco FT7IR-230 Measured with an infrared spectrometer. 13C-NMR spectra were measured with a Varian Unity Plus 500 spectrometer. EIMS and HR-EIMS were measured with a Jeol JMS-AX 50 5 HAD mass spectrometer at an ionization voltage of 70 eV. Tube layer The analysis system uses BW-820MH (normal phase) and Chromatorex-〇DS DM1 020T (reverse phase) (Fuji Silysia) silica gel. Extraction and separation 200528093 Taken from 2 years October 6. Antrodia cinnamomea powder mycelium from Taiwan Shanshang Biotech Co., Ltd., extracted three times with chloroform under 3 hours countercurrent. Aeroform extract (5.3 g) was chromatographed with Shixi gel, and Nine propane (19: 1-14: 6) and aerosol_methanol (1: 1) eluted nine fractions (Fr · Η). The second part was calibrated and calibrated by Shixi gel chromatography. For 1 (8.7 mg), the fourth part was calibrated to 2 (13.6 mg) after normal-phase and reverse-phase silica gel chromatography, and the fifth part was subjected to silica gel chromatography and n-hexaneacetone (8: 2) Calibrated to 6 (35 · 8 mg) after elution, the sixth part was calibrated to 3 (14 · 6 mg) after combined normal phase and reverse phase silica gel column chromatography, and the seventh part was After column chromatography, mixtures 4 and 5 (4: 1) were produced. The mixtures 4 and 5 were then separated by the prepared HPLC [column: Tosoh TSK_gei 〇ds_8〇Tm (21.5 x 300 mm), moved Phase: methanol monowater (70:30) containing 0.1% TFA. 3-isobutyl-4- [4- (3-methyl-2-butenoxy) phenyl] pyrrole— 2, 5-dione (compound 1) · yellow oil; UV (Me〇H) max (log 227 (4 · υ, 258 (3 · 9), 275 (3.8), 355 (3.4) nm; IR (CHCh) max Π 63 cm 1, Table 1 is 1H-NMR; Table 2 is earned; EIMS heart 314 ⑷ + _ (100) ^ 246 (100) ^ 131 (100); HR-EIMS m / z 314. 1523 (calculated value of Ci9H22〇4: 314.11518). 3_isobutyl-4- [4- (3-methyl-2-butenoxy) phenyl] _1 # —pyrrole-2, 5-dione · (2): ^ yellow crystal (/?- Hexane-ethyl acetate); mp 11 (M11 uv (methanol) max O〇g ε) 230 (4.3), 272 (3.5), 355 (3.7) nm; IR (CHCl3) 10 200528093 max 1724 cm1; Table 1 is 1H-NMR; Table 2 is 13C-NMR; EIMS π / ζ313 [M] + (8), 245 (100), 203 (77), 131 (28); HR-EIMS you / z 313 · 1681 (calculated for C19H23N03: 31 3.1 678). X-Ray Crystal Diffraction of Compound 2: A yellow needle was obtained by crystallization from n-hexane-ethyl acetate and was selected for data collection. Crystallization data: Ci9H23N03; .313.40; volume 0.15 x χ 0.02 x 0.02mm triclinic, space group PI (# 2), a = 6. 3505 (5) A, b = 12.52 (1) A, c = 12 · 560 (2) A, a = 64. 623 (7). , /3=75.358(4). , R = 84 · 681 (5). , V = 850 · 9 (2) A3, Z = 2,

Dcaic = 1.223 g / cm3 »^ (MoKa) = 0.82 cm- * F_ = 336. 00. Emitted at 93 K and measured with a Rigaku RAXIS-RAPID image plate diffractometer with monochromatic graphite (MOF) (10.Π 069 Α). Collect 895. 'Reflection' is a unique (— ο ”. Merging equivalent reflections. Solve the crystal structure using the direct method (SHELXS86) and actuate the least squares of the full matrix. Non-hydrogen atoms are calculated heterogeneously. Hydrogen atoms include but do not estimate final parameters G74, HG99, and ⑽ (object observation facilities) = 10.6, the maximum and minimum peaks of the difference Fourier diagram correspond to 0.83 and -〇89 e- / A3 〇 * isobutyl — 4- [4 -(3-methyl_2-butenoxy) phenyl], pyrrole + alcohol-2,5-difluorene (compound 3): yellow oil; uv (Me〇H) 232.5 (4.3), 296 ( 3.7), 374 (3.7) nm; we ·)> 200528093 1717cm1; Table 1 is 1H-NMR; Table 2 is 13C-NMR; EIMS see / ζ 329 [ΜΓ (12), 261 (1 00), 131 (50 ); HR-EIMS: 329.1 637 (calculated for C19H23N04: 329.1627). 45 ^ -Hydroxy-3-isobutyl-4- [4- (3-methyl-2-butenoxy) phenyl] pyrrolidine-2, 5-dione (4): colorless oil body; [a] D23 + 2.5 ° (c 0.2, methanol); UV (methanol) max (10 g ε): 225 (4.3), 275 (3.3), 283 (3.2) nm; IR (CHC13) y_1715 cm-1; Table 1 is 1H-NMR; Table 2 is 13C-NMR; EI MS π / ζ 331 [M] + (2), 263 (67), 207 (66), 191 ( 30), 1 79 (40) > 1 33 (64) > 69 (1 00); HR-EIMS m / z: 331.1 747 (calculated value of C19H25N04: 331.1 783). ^ -Buhydroxy-3-isobutyl-4- [4- (3-methyl-2-butenoxy) phenyl] pyrrolidine-2, 5-dione (5): colorless oil Body; [a] d23 +3.0.2, MeOH); UV (MeOH) λ max (log ε): 227 (4.3), 275 (3.4), 283 (3.3) nm ; IR (CHCl〇> max 1715 cm-1; Table 1 is 1H-NMR; Table 2 is 13C-NMR; EIMS yz7 / z 331 [M] + (1), 263 (45), 207 (50), 191 (75), 1 79 (30), 1 33 (1 00), 69 (92); HR-EIMS π //: 331. 1 766 (calculated value of Ci9H25N04: 33ΐ · ΐ783). Kerosterol: colorless crystal (n-hexane-acetone); mp 1 65-1 69 ° C (lit2 mp 171-174. Cytotoxicity test: in vitro LLC tumor cell test using sul f orhodamin B (SRB) method 50% growth inhibition (ED5G) was calculated using the PrObit method. 200528093 Results and discussion The aerosol extract of Antrodia cinnamomea mycelium was repeatedly chromatographed in normal phase and reverse phase to obtain sufficient amounts of cis Adipic acid, succinic acid derivatives (compounds 1 to 5) and ergosterol peroxide. Table 1: 1H-NMR spectral data of compound BU 5 (ppm, J = Hz) (500 MHz, CDC13) ___H 1 2 3 4 5 3 — — — 2.87 (1¾ m) 3.08 (1H, m) 4 — 3.52 (lH, d, 4.07 (lH, d, /=4.0) J = 8.0) Γ 2.59 (2H, d, 2.51 (2H, d, 2.50 (2H, d, 1.51 (lH, m) 1.02 (1H, m) • 7 = 7.0) J = 1.0) /=7.0) 1.72-1,84 (1H) 1.42 ~ 1.48 (1H) 2, 2.12 (1¾ sep, • 7 = 7.0) 2.06 (1H, sep, 7 = 7.0) 2.05 (1H, sep, J = 7.0) 1.72-1.84 (1H) 1.42 ~ 1.48 (1H) 3, 0.70 (3H, d, 0.66 (3H, d, 0.94 (6H, d, 0.90 (6H, d, 0.88 (6H, d, /=6.5) • 7 = 6.5) 4, /=7.0) 7 = 7.0) J = 1.0) 0.89 (3H, d, 0.80 (3H, d, J = 6.5) J = 6.5) 2 ”, 6” 7.50 (2H, d, 7.50 (2H, d, 7.50 (2H, d5 7.07 (2H, d, 6.96 (2H , D, 7 = 9.0) J = 9.0) • 7 = 9.0) • 7 = 8.5) «7 = 9.0) 3”, 5 ”7.02 (2H, d, 6.95 (2H, d, 6.98 (2H, d, 6.87 (2H, d, 6.84 (2H, d, J = 9.0) J = 9.0) /=9.0) /=8.5) • 7 = 9.0) 1 ”, 4.57 (2H, d, 4.56 (2H, d, 4.55 (2H, d, 4.47 (2H, d, 4.47 (2H, d, J = 6.6) J = 6.5) 7 = 6.9) J = 6.5) J = 6.5) 2 ”, 5.50 (1H, brt, 5.50 ( 1H, brt, 5.49 (1H, brt, 5.47 (1HM 5.47 (1H, br t, J = 6.6) J = 6.5) J = 6.9) J = 6.5) J = 6.5) 4, 1.81 (3 H, s) 1.81 (3H, s) 1.81 (3H, s) 1.79 (3 H, s) 1.79 (3 H, s) __5 ,,, 1.76 (3H, s) 1.76 (3 H, s) 1.76 (3H, s) 1.73 (3 H, s) 1.73 (3H, s)

Table 2: 13C-NMR spectral data of compound 1-5 (δ ppm) (125 MHz, CDC13) C 1 2 3 4 5 2 166.4 (s) 171.7 (s) 168.8 (s) 174.8 (s) 175.1 (s) 3 139.8 (s) 138.8 ⑻a) 135.9 (s) a) 44.6 (d) 40.3 (d) 4 140.2 (s) 139.2 (s) a) 136.0 ⑻a) 49.8 (d) 47.5 (d) 5 165.4 (s) 171.1 (s) 168.1 (s) 173.2 (s) 173.6 (s) 1, 33.6 (t) 32.8 (t) 33.2 (t) 40.4 (t) 35.3 (t) 2, 27.9 (d) 28.1 (d) 28.4 (d ) 25.3 (d) 25.2 (d) 13 200528093 3, 4, 22.7 (q) 22.7 (q) 1, 11, 119.9 (s) 121.2 (s) 2 ”, 6, 131.1 (d) 130.9 (d) 3” , 5 ,, 115.1 (d) 114.9 (d) 4, 160.9 (s) 160.1 (s) 1 ”, 65.0 (t) 64.9 (t) 2”, 118.7 (d) 119.3 (d) 3 139.1 (s) 138.6 (s) a) 4 ,,, 25.2 (q) 25.8 (q) 5 ”, 18.2 (q) 18.2 (q) a) Transfers can be changed interactively. 23.0 (q) 21.3 (q) 21.8 (q) 23.0 ( q) 22.4 (q) 120.8 (s) 127.9 (s) 125.1 (s) 131.0 (d) 128.8 (d) 130.2 (d) 115.0 (d) 115.4 (d) 115.0 (d) 160.2 (s) 158.7 (s) 158.7 (s) 65.1 (t) 64.1 (t) 64.8 (t) 119.2 (d) 119.4 (d) 119.3 (d) 138.9 (s) 138.3 (s) 138.4 (s) 26.1 (q) 25.8 (q) 25.8 (q) 18.5 (q) 18.1 (q) 18.2 (q) The structure of the new compound was determined by the following method: Compound 2 was crystallized in yellow under the conditions of mp 11〇_11KC 'and analyzed by hr eims. The molecular formula is c19h23n03. Infrared spectrum at 1 724 cm- ^ shows the absorption of the sulfhydryl group. The% -NMR spectrum shows that there are signals of four methyl carbons, two methylene carbons, and one methyleneimine carbon in the aliphatic region, and there are also a benzene ring, a dilute hydrocarbon, and two several groups. carbon. 1H_NMR spectrum shows isobutyl moiety composition at 5090, 206, and 251 'with 3-methyl 2-butenoxy moiety composition at 4.50 and 5.5, and at 6.95 and 7.50. These results with para-substitution are subsequently supported by iH, cos ". Qing experiment is further supported. The correlation between the length of the obscure circle and the circle iii is shown in Figure i. Based on the molecular formula and 13.-plane spectrum results, this compound is speculated to contain more Multi-atoms, including more than a few carbons. Therefore, the part of the 3 pastes / months is speculated to have a cis-buteneimine group. This chemical structure was then determined by X-ray analysis to select the 3-isobutyl group of the gi system. 4- [4- (3-methyl-2-butenoxy) phenyl] -1 and -pyrrole ~ 2,5-dione (Figure 2). 14 200528093 Compound 1 molecular formula, analyzed by HR-EIMS Its molecular formula is Cl9H22〇4. Infrared spectrum shows the absorption of carbonyl group of anhydrous acid at 1 76 3 cm 1. The 1H-NMR spectrum of compound 1 is similar to that of 2 and shows isobutyl, 3-methyl 2-butane. Partial composition of alkenyloxy and para-substituted benzene ring. From the results of HMBC spectroscopy, based on the molecular formula, compound 1 has the same structure as that of part 2 (Figure 丨), that is, there are all butenyl The result of Compound 1 was determined to be 3-isobutyl-4- [4- (3-methyl-2-butenoxy) phenyl] σfuran-2,5-dione. The molecular formula of Compound 3 is given by The molecular formula was analyzed by HR-EIMS. Infrared spectroscopy showed that the carbonyl group of hydroxyimine was absorbed at 1717 cnT1. The results of 1Η and C-NMR spectra of compound 3 were similar to those of compound! And 2 showed isobutyl, 3-methyl The composition of the 2-butenyloxy group and the para-substituted benzene ring. From the hmbc experiment, compound 3 has part of the same structure as 2 (Figure n, compound 3 contains more than one oxygen atom. Therefore, this The compound was identified as 3-isobutyl-4- [4- (3-methyl-2-2-butoxy) phenyl] -1 and -furan-butanol-2,5-dihydrazone. Analysis by HR-EIMS It is found that compounds 4 and 5 have the same h value and molecular formula (c19h25N04, found at 331.1 747 and 331 1 766 respectively), but the two can be separated by HPLC. Infrared spectrum shows that the two compounds have hydroxyimine groups in the absorption In the 1H- and "C_spectrum results, the two compounds showed the presence of isobutyl, 3-methyl 2-butanyloxy, para-substituted benzene ring but isobutyl methylene The protons are shown to be multiples rather than doubled of compound B3. The fluorescein spectrum shows that the subf group is attached to the -CH-CH_ unit. Compounds 4 and 5 of 15 200528093

Compound constants (compounds 4 and 5 are not known to be cis-butanimidine, compounds 4 and 5 have a couple 5 between H-3 and η-4 of 4.0 and 8 · 0Ηζ, respectively), The decisions were the isomers of trans and cis, respectively. Compound 4 was found by NOESY (Nuclear Overhauser Effect Photoelectron) to have no NOE between H 3 and H-4, while compound 5 was found to have NOE. The optical rotations of compounds 4 and 5 were +2.5. And + 3 · 〇. However, its CD spectrum is obviously not at any wavelength and there is no Carton effect, indicating that compounds 4 and 5 may be a racemic mixture. HPLC analysis of these racemic mixtures using optical isomer chromatography tubes with different solvent systems was unsuccessful. At this time we cannot fully infer whether these compounds are optically active or racemic mixtures. Therefore, their relative structures are 45 ^-and%, respectively, with 1-hydroxy-3-isobutyl-4- [4- (3-methyl-2-butenoxy) benzyl] each of -2, 5-Erji. These natural maleic acid and succinic acid derivatives were again isolated according to the report of Aquveque et al. The cytotoxic activity of aerobic extracts and isolated compounds was performed using LLC (Lewis lung cancer) cell beads (Table 3). The aerobic extract showed moderate cytotoxicity, with a value of 26.7 g / ml. Maleic acid compounds 1 and 4 were not cytotoxic. However, 2 and 3 were found to be cytotoxic to LLC cell beads, and their EDw values were low. Table 3 50% growth inhibition value of chloroform extract and compound 1 from Antrodia cinnamomea mycelium on LLC cell beads (ED50) ED50 (// g / ml) aeroform extract 26.7 1 > 20 2 3. 6

3 7. 5 4 > 10

Adriamycina) 〇 · 14 -------· a) Positive control group Antrodia cinnamomea mycelium tumor test (powder of Antrodia cinnamomea mycelium) A · Cell line Attached cells:

MCF-7: Human breast tumor HT-29: Human colon gland tumor KATO III: Human gastric gland tumor SW480: Human colon gland tumor SW620: Human colon gland tumor HepG2: Human liver gland tumor suspension cell: 17 200528093 EL4: Mouse lymphoma B. Samples of mouthpieces 1, mouthpieces 3, alcohol extract of Antrodia cinnamomea mycelium, water extract of Antrodia camphorata mycelium C. Test method calculation of EDsq (50% inhibition of effective dose) Attached cells: MTT Method; MCF-7, HT 9Q τττ w, ΚΑΤΟ 111, SW480,

HepG2 cells were performed for 3 days. SW620 was performed for 4 days. Suspension cells: Cell counting method; EL4 cells were counted for 5 days. D. Results Calculation: y = mLn (x) + b Example X Y 0 0. 97 10 ppm 〇 941 30 ppm 0 · 6 100 ppm 0. 331

The current X value (10, 30, 50 ppm) and Y value can be calculated using the formula y = _0 · 2643Ln (x) + 1.5321 ED5〇 = exp [(〇 · 97/2 —L 532i) ―—〇 · 2643 )] Sample preparation and sample description 18 200528093 A · ACM (Hand Takaba Milling Ceremony Powder) Water Extraction Method 1. Take 1 g of ACM and add it to a 250 ml coiled cup containing 40 ml of reverse osmosis water at room temperature. Place the beaker in the ultrasonic water bath for 20 minutes. 2. Then stir the water bath for 45 minutes at 45 ° C. 3. Place the beaker in the ultrasonic water bath for 20 minutes. 4. Centrifuge the sample at 3000 rpm for 15 minutes. 5. Serial dilution of the suspension with cell culture. B. Measure the sample concentration 1 · Evaporation pan weighing (W1). 2 · Take 10 liters of water extract into the evaporation pan 3 · Place the evaporation pan into the oven to remove the moisture (W2) Sample weight / ml = (W2-W1) / 10 C · ACM Silk powder) Alcohol extraction 1. Take 20 g of ACM and put it into a 5 ml beaker containing 100 ml of 95% alcohol and stir at room temperature for 10 minutes. 2. Filter the suspension with Advantecl filter paper and collect the filtrate. 3. Concentrate the liquid with a rotary vacuum evaporator and remove all alcohol. D · Compound 1 · Pure compound obtained from ACM E · Compound 3: Pure compound obtained from ACM MTT test method

After cell proliferation, the old culture medium was removed and the cells were washed once with PBS. 19 200528093 2. Cells were treated with 1X trypsin-EDTA. Centrifuge at 3.120 rpm for 5 minutes and discard the supernatant. 4. Resuspend the cells in 10 liters of culture medium. 5 · Mix 100 μ 1 cell suspension with 〇 μ 1 cone blue stain to calculate

V viable cells. 6. In a 96-well plate, put ιχ104 cells / 100μ1 culture medium in each well 'and place the plate in 37. (: Incubate for 24 hours in a carbon dioxide incubator. 7. Remove the old culture medium and wash the cells once with PBS. ® 8. Add a sample of ΙΟμ1 to each well, and place the dish in a 3rc carbon dioxide incubator. 9 · In On days 3, 4, and 5, the old culture medium was removed and the cells were washed once with PBS. 〇 · Add 57 μ1 of MTT (0. 88 mg / liter) to each well. 11. 4 hours later, remove the MTT and wash with PBS Cells once. 12. Add 50 μΐ DMS0 to each well. 13. Read the results with ELISA at 0D545. Spring cell counting method (EL4 cell line) 1. Remove the old culture solution by centrifugation after cell proliferation. 2. Fresh culture The cells were mixed with liquid solution. · 3 · 100 μ 1 cell suspension was mixed with 100 μ 1 cone blue stain to calculate viable cells. 4. Prepare samples of different concentrations containing 1 × 105 cells / ml. 20 200528093 5. In a 96-well plate, place 100 μΐ of sample in each well, and place the plate in a carbon dioxide incubator at 37 ° C. 6. Count the number of viable cells on days 3, 4, and 5.

PBS

NaCl 8 g KC1 0 2 g

Na2HP〇4 1. 4 g KH2PO4 0.2 g volume adjusted to 1 liter pH 7. 4 Results and discussion Antrodia cinnamomea mycelium in cell line ED50 cell line HepG2 HT-29 KATO III EL4 SW480 SW620 MCF-7 Compound 1 21 ppm 5 2 ppm 38 ppm 3.5 15 ppm 6 ppm ppm Compound 3 35 ppm 42 ppm 6 9 ppm 2.6 20 ppm 27 ppm 0. 02 ppm ppm Antrodia cinnamomea 32 ppm 5 2 ppm 156 2.6 71 ppm 4 ppm corpuscle ppm ppm ( (ACM) Alcoholic extract Antrodia 295 ppm 707 20 207 ppm 132 ppm 318 ppm 200528093

Compound 3 of the invention: HepG2 (Figure 4a), EL4 (Figure 4b), HT-29C Figure 4c) and Kato III (Figure 4d) ACM water extract · HepG2 (Figure 5a), SW620 (Figure 5b) and EL4 (Figure

5c) 〇 ACM alcohol extract: HT-29 (Figure 6a), SW480 (Figure 6b), SW620 (Figure 6c), EL4 (Figure 6d), HepG2 (Figure 6e) and Kato III (Figure 6f). Compound 1 of the present invention: MCF-7 (Figure 7a), EL4 (Figure 7b), HT-29 (Figure 7c), SW620 (Figure 7d) and HepG2 (Figure 7e). From the above figure, it can be proved that the compound of the present invention and the ACM extract have an inhibitory effect on different tumor cells. Analysis of all new compounds (1, 2 and 3) from ACM alcohol extract by high performance liquid chromatography Purpose: To measure all new compounds (1, 2 and 3) from alcohol extract of Antrodia cinnamomea mycelium (ACM) ), We prepared the routine quality control procedures using ineffective liquid chromatography on the mother's day.丨 Preparation of Antrodia cinnamomea mycelium alcohol extract samples ··

D. Accurately weigh the sample powder with an electronic balance 2Q. Place it in a graduated inspection bottle containing 100 22 200528093 liters of 95% alcohol. Do not screw the cap 2). Place the above sample bottle on the ultrasonic wave. Zhensheng sink for ten minutes. 3). Pour the liquid sample into a centrifuge tube, and then centrifuge for 5 minutes at 650 1 ρΐί 丨 to remove the natural particles of the sample. 4) Filter the liquid layer with No. 1 filter paper. 5) Concentrate the filtrate with a rotary vacuum evapotranspiration machine until a viscous, alcohol-free yellow liquid appears. 6) Repeat steps 1 to 5 three times, and coke the percentage of ancient poverty complaints π 1 (and all the products taken from the total (antrodia antrodia mycelium alcohol extract = 4.60 g), calculate the yield. Model 2690 Wo Conditions of use of the Tes system 1), column: reverse phase C18 2), mobile phase: methanol, water, acetonitrile

3) 、 Injection volume: 20L 4) Detection · Photodiode array detector 996 at a wavelength of 254 nm Ready to extract alcohol from Antrodia cinnamomea mycelium containing 1 · 0 0 0 (g) in 10 liters of alcohol Samples for HPLC analysis. According to HPLC analysis, the extracted products containing pure compounds 1, 2, and 3 are shown in Table 4 and Table 4:

___ ^ Composition of two standard compounds " '--- one -__ 23 200528093: 0.0100 (g) in 1 ml of alcohol Standard name Compound 1 Compound 2 Compound 3 Concentration (g / ml) 0.01 0.01 0.01 Peak area 49,315, 783 129,327,136 136,255,406 Retention time (minutes) 134.8 124.3 119.8 Concentration (g / ml) 8.59 X 10-3 5.59 X 10'4 1.659 X 10-2 Peak area 42, 374, 766 7, 226, 937 226,102, 223 Production Rate% (w / w) 8. 59 0.559 16. 59 Therefore, the total weight of compounds 1, 2 and 3 is 5.92% of the weight of the ACM sample. Cow Mycelium Mycelium-Alcohol Test Materials and Equipment 1. Test Substances and Administration Forms All in vitro tests use 2% Tween 80 as a carrier, which contains the test substance at an initial dose of 1000 mg / kg. The observation time for each experiment is detailed in the method. 2. Experimental animals Male or female ICR mice, Wi star-Okamoto naturally derived male hypotensive rats (SHR), using Wi provided by the Taiwan Panglobal Pharmacological Institute star and Long 200528093

Evans-derived rat. The animal breeding space is as follows: 10 mice are housed in a 29 × 18 × 3 cm space, 6 rats are housed in a 45 × 23 × 21 cm space, and 3 mice are housed in a 45 × 23 × 21 cm space. Mice and rats are housed in aPECr cages. C57BL / 6J immunized male mice weighing 21 ± 2 grams and 6-8 weeks old provided by the National Taiwan University Animal Center were also used in this study. Experimental animals were housed in individually ventilated cages (IVC racks, 36 mini isolation systems). Each cage was sterilized and 5 mice were housed (in a space of 26 · 7 × 20 · 7 × 14 cm). All animals are temperature-controlled (21.-23.〇) and humidity-controlled (60% -70%), and exposed to light for at least one week before the experimental use. Provide standard laboratory feed (LabD 丨 et

Rodent Diet and Guinea Pig Diet, PMI Nutrition

International, USA) with free access to water. 3. Cell lines and culture fluid Murine melanoma cell line B1 6-FO (ATCC CRL-6322) was prepared by American

Provided by Type Culture Collection and dulbecco ’s Modified

Eagle ’s Medium (GIBC0, USA) was used as the cell culture medium. Tumor cells were cultured in air at 37 ° C and 5% CO2. 4. Chemicals in general ... Sterilized water (made in-house), dimethyl sulfoxide (DMS0, Merck, Germany), isotonic sodium chloride solution (Taiwan Xindong Chemical Industry Co., Ltd.), magnesium sulfide 25 200528093 ( MgSOi 7H2O, Wako, Japan), Meclamine Sodium (Sigma, USA), Methyl Cellulose (Signa, USA), Sodium Hydroxide (NaOH, Wako, Japan), Phosphate Buffer (Sigma, USA) ) And Tween 80 (Wako, Japan). Reagents

Glicose-HA Test Reagent Set (wako, Japan), Alanine Transaminase (ALT) · Test Reagent Set (Wako, Japan), Aspartic Acid Transaminase (AST) Test Reagent Set (Wako, Japan), T-Cholestero HA and HDL test reagent kits (Wako, Japan), Hemolynac 3 Hemolys (Nihon Koden, Japan), ·

Isotonic 3 Diluent (Nihon Koden, Japan). 5. General equipment use: animal box (Sindh 'Republic of China), 250ml and 1000ml beakers (Kinmax, USA)' disposable syringe (1L, Top Corporaion, Japan), stainless steel tweezers ( klappencker, Germany), rat scale #Z — 4〇 (Taconic, USA), oral feeding syringe (Natsune, Japan), hypodermic needle 23 GX 1 ,, (Top Φ

Corporation, Japan), pH meter (Suntex, USA), mouse scale 500 g ± 2 g (Chien-chun, Republic of China), glass syringe j ml, 2 ml and 5 ml (Mitsuba, Japan), and stainless steel Scissors (nappencker, Germany). -Methods and results: 1. Experiments on central / peripheral choline synergists with reference to Lippmann and

Pugsley TA was published in Arch 丨 Discuss pharmac〇dyn. 227: 324 26 200528093 in 1977. The test substance was orally administered to three Wistar-derived male or female experimental rats weighing 150 ± 20 g. After another 30-60 minutes, the experimental animals showed a cumulative chewing behavior (mouth and / or tongue movement) of more than 10 seconds, as well as salivation. The observation of a normal phase response in 2 or more (-2) 3 rats indicates that it may have a role in the central and peripheral cholera test. Table 5: Results of acetylcholine stimulating action in the central / peripheral nerves of rats in the treatment group. The central nerve peripheral nerve diameter σ and chewing fraction saliva secretion fraction carrier P0 10 ml / kg 1 2 3-0/3- 0/3 ACM alcohol extract P0 1000 1 + one extract mg / kg 2 + one 3 + one 0/3 1 — (3/3)-300 2 one one mg / kg 3 + 1/3-0/3 27 200528093

Arecoline- IP 30 mg / kg 1 + + HBR 2 + + 3 + + (3/3) (3/3)

The vehicle and the test substance were administered orally (P0), while the positive reference compound was administered intraperitoneally (IP). After 30-60 minutes, the cumulative measurement of the experimental animals showed more than 10 seconds of chewing behavior (mouth and / or tongue movement), and the occurrence of salivation. The observation of a normal phase response in 2 or more (-2) 3 rats indicates that acetylcholine may have a role in the center and periphery. 2. Cardiovascular, blood pressure and heart rate (SHR 〇,], 2, 4 hours) experiments refer to the literature published by Yen TT et al. 1 978 in Life Sci 22: 359 using 3 Wistar- weighing 250 ± 20 grams- Okamoto-derived natural hypertensive male (SHR) experimental group has an average systolic blood pressure of 200 ± 20, and a heart rate of 400.

-30-Man " Knife Face. Blood pressure and heart rate were indirectly recorded by the cuf f method in a temperature-controlled environment (32 ± 1 ° C) at 0 and 1, 2, and 4 hours before oral administration of the test substance or vehicle. It is considered meaningful when the systolic blood pressure drops by 10% or more (^ 10%) or the heart rate decreases by 20% or more ($ 20%) when compared with Q at each interval measurement. Cardiovascular and blood pressure results of watch rats (SHR 0, 1, 2, 4 hours)% of the control group treated (compared with 0 o'clock) 28 200528093 1 hour 2 hours 4 hours Vehicle P0 10 1 100 96 90 m 1 / kg 2 97 100 91 3 90 92 92 flat 96 96 91 all ACM- P0 1000 1 78 85 71 alcohol extraction mg / kg 2 86 89 80 substance 3 89 89 89 flat (84) (88) (80) all Clonidine P0 0 · 1 1 71 67 71 mg / kg 2 95 86 88 3 72 85 69 flat (79) (79) (76) Table 7: Results of cardiovascular and heart rate (SHR 0, 1, 2, 4 hours) Control group in treatment group% (compared with 0) 1 hour 2 hours 4 hours Vehicle P0 10 1 87 100 99

29 200528093 ml / kg 2 116 103 107 3 108 104 121 flat 104 102 '^ ---- 109 all ACM- P0 1000 1 98 93 ^-95 alcohol extraction mg / kg 2 81 100 88 product 3 83 78 92 flat 87 90 —_ 92 All Clonidine P0 0 · 1 1 62 97 112 mg / kg 2 84 87 104 3 68 86 78 Ping (71) 90 ^ -— 98 All use systolic blood pressure 200 ± 20 mmHg and heart rate 400 ± 50 mmHg times / minute of SHR rats. Blood pressure is administered via the viai tai 1 cuff method

Indirect records were made at 0 and 1, 2, and 4 hours after the carrier. When the nucleus is compared with 0 at each measurement time point (see the description below), its blood pressure drops by 10% or more (^ 10%), and the heart rate decreases by 20% or more (^ 20%). Brother Xi is meaningful. Carrier lOml / kg η η * Find 229mmHg and 403 mmHg times / minute as 100% ^ 30 200528093 ACM-alcoholic extract 1 000mg / kg 0 223mmHg and 452 mmHg times / minute as 100%.

When Clonidine 0.1 mg / kg 0, 228mmHg and 379 mmHg times / minute are regarded as 100%.

Table 8: Cardiovascular and blood pressure results in rats (SHR0, 1, 2, 4 hours)% of the control group in the treatment group (compared with 0 hours) 1 hour 2 hours 4 hours Vehicle P0 10 1 94 97 97 ml / kg 2 88 97 94 3 94 97 103 flat 92 97 98 all ACM- P0 300 1 111 102 103 alcohol extraction mg / kg 2 94 84 100 substance 3 112 110 112 flat 106 99 105 all Clonidine P0 0 1 1 86 73 81 mg / kg 2 63 73 90 3 62 68 80 31 200528093

Mean (70) (71) (85) Table 9: Cardiovascular and heart rate results (SHR 0, 1, 2, 4 hours)% of the control group in the treatment group (compared to 0) 1 hour 2 hours 4-hour carrier P0 10 1 82 85 84 ml / kg 2 88 115 102 3 109 111 119 flat 93 104 102 all ACM- P0 300 1 97 96 92 alcohol extraction mg / kg 2 105 108 98 product 3 85 96 82 flat 96 100 91 All Clonidine P0 0 · 1 1 77 85 102 mg / kg 2 78 78 100 3 62 94 104 Ping (72) 86 102 All 32 200528093 The use of systolic blood pressure 200 ± 20 mmHg and heart rate 400 ± 50 mmHg times / minute SHR rats. Blood pressure was recorded indirectly at 0 (before) and 1, 2, 4 hours after oral administration of the test substance or vehicle via the Viai taii cuff method. When compared with 0 each time after the measurement time point (see the supplement below), the blood pressure drops by 10% or more (-10%), or the heart rate decreases by 20% or more (-20%), Is considered meaningful. When the carrier is 10ml / kg, 220mmHg and 410mmHg times / minute are regarded as 100%. ACM-alcoholic extract 300mg / kg at 205mmHg and 446mmHg times / min was regarded as 100%.

Clonidine 0.1 mg / kg at 235 mmHg and 417 mmHg times / minute was regarded as 1000/0. 3. Cholesterol and serum (total HDL, total / HDL ratio) experiments caused by diet Refer to the literature published by SchurrPE et al. 1 976 in Atherosclerosis Drug Discovery. Plenum, New York, ρρ 21 5-229. Five ICR-derived male mice weighing 22 ± 2 grams were fed high-fat food (g / 10 0g: coconut oil, 8; cholesterol, 1.0; choline acid, 0.3; lard, 2, standard food 8 8 7) 7 days to trigger hypercholesterolemia. The test articles were administered orally on days 5, 6, and 7. After fasting overnight, the serum of each mouse was used to measure the total cholesterol (Total), high-density lipoprotein (HDL), and total / HDL change rates. 200528093 When compared with the vehicle-treated control animals, the total serum volume decreased by 20% or more (2 20%), or the serum HDL increased by 20% or more 20%), or its total volume / HDL A decrease of 40% or more (-40%) is considered significant. Table 10: Diet-induced cholesterol (total / HDL, total / HDL ratio) results in mice. Total HDL in total dose group / HDL diameter Indiv.% Dce Indiv.% Dce Indiv% Dce Carrier PO 10 1 361 70 5. 16 ml / kg 2 316 82 3. 85 X 3 3 379 79 4. 80 4 392 78 5. 03 5 367 86 4. 27 flat 363 — 79 one one 4. 59-both ACM-alcoholic extraction PO 1000 1 420 117 3. 95 mg / kg 2 327 115 2. 84 x 3 3 332 104 3.19 4 363 98 3. 70 5 294 117 2. 51 flat 347 4 110 3. 15 31 both (39) 34 200528093 P0 300 mg / kg x 3 1 2 3 4 5 370 301 217 379 328 66 65 74 76 98 5. 61 4. 63 2. 93 4. 99 3. 35 flat 319 12 76 -4 4. 20 8 both Benzaf ibrate P0 100 1 230 91 2. 53 mg / kg 2 214 120 1. 78 x 3 3 225 133 1. 69 4 231 123 1. 88 5 242 97 2. 49 flat 228 (37) 113 2. 02 both (43 ) (56) The carrier, test substance or reference positive compound is administered orally (p0) on the 5th, 6th, and 7th days after feeding with high cholesterol food. Twenty-four hours after the third feeding, all-night experimental animals were sacrificed to evaluate their total serum cholesterol (Ding & 1) and high density lipoprotein (HDL). When its blood residues and oils are reduced by 20% or 20% or more, or serum HDL is increased by 20% or more (whether ?, °), or its total / HDL ratio is reduced by 40% % Or more 40%). 4. D-galactosamine's liver injury test in rats refers to the literature published by Wrobel J et al. 1998 35 200528093 in J. Med Chem 41: 1084. Five Wistar-derived male mice weighing 200 ± 20 grams were used. Each animal was treated with a single injection of D-galactosamine (500 mg / kg, Ip). The test article was administered orally 0.5 hours and 4 hours and 8 hours before D galactosamine injection, and the animals were sacrificed after 24 hours. The amount of alanine transaminase (ALT) and aspartate transaminase (AST) was determined using a HITACHI analyzer (model 7050) according to the best UV method. A 30% or more (230%) decrease in ALT or AST activity compared to vehicle-treated control animals is considered protective. Table 11: Results of galactosamine-induced liver injury in rats

36 200528093 Serum serum size of treatment group ALT (X soil SEM) AST (X soil SEM) U / L Dec.% U / L Dec.% carrier 1 816 1628 P0 10 2 1044 1716 3 652 888 ml / kg 4 656 828 X 3 5 644 956 X 762.4-12032 SEM 77.4 193.0 __ ACM-Wine 1 364 516 P0 1000 2 376 532 Fine extraction 3 596 672 mg / kg 4 452 524 x 3 5 336 356 (57) X 424.8 ( 44) 520.0 SEM 46.9 50.1 1 460 852 300 2 656 880 3 640 876 mg / kg 4 752 1004 x 3 5 536 692 28 X 608.8 20 860.8 SEM 50.6 49.8 1 508 656 Guanine P0 300 2 532 912 3 412 776 mg / kg 4 436 652 x 3 5 636 1028 (33) X 504 8 (34) 804 8 SEM 39.6 73.4

The test substance and vehicle were administered orally 0.5 and 4 hours and 8 hours before injection of a single dose of galactosamine (500 mg / kg, IP), and then the animals were sacrificed after 24 hours. 37 200528093 And AST values. Compared with the carrier group, a -30% decrease in alt and phantom is considered significant. 5. Carrageenan's inflammation experiments refer to Winter CA et al. 1 962 Yu Hua called soc

Literature published in Exp Biol Med. 1 1 1: 544. Three Long Evans-derived male or female mice of the reset line of 150 ± 20 g were fasted overnight before the experiment. The test substance was administered orally before the injection of carrageenan (1% intraplantar suspension in 0.5 milliliters) to the right hind paw} hours, and the hindpaw edema was regarded as a judgement of inflammation. Water cells were used after the administration of carrageenan The sire official's fullness measurer (diameter 25 meters) measures 3 hours. A 30% or more (-30%) decrease in hindpaw edema indicates that its acute anti-inflammatory effect is significant. Table 12 · Inflammation test results of carrageenan in rats Treatment route Dose volume (X 0.01 ml) RP. LP Diff 槪 P0 10 ml / kg 1 194 103 91 2 202 108 94 3 199 104 95- ------------ Average 198 105 93 AQH Extraction P0 1000 mg / kg 1 146 101 45 Substances 2 147 95 52 3 160 104 56 — Average 151 100 51 (45) Aspirin P0 150 ing / kg 1 152 102 50 2 146 102 106 44 3 163 57 —_ average 154 103 50 (46) Table 13: Results of rat inflammation test with carrageenan 38 200528093 Treatment route dose Fine paw volume (x 0. 01 ml) RPLP Diff carrier P0 10 ml / kg 1 193 105 88 ~-2 198 107 91 3 199 102 97 average 197 105 92 ACM-alcoholic extraction P0 300 mg / kg 1 195 104 91 product 2 187 103 84 3 196 103 93 average 193 103 89 3 Aspirin P0 150 ng / kg 1 146 103 43 2 149 101 104 48 3 169 65 average 155 103 52 (—43) Inject carrageenan (1% intraplan tar suspension 0.1%) in Right hind paw (R. P.) of rats fasted all night 1 hour ago I carrier and test article; left hind paw (L · P ·) is not injected. Hind paw edema is reduced by 30% or more (30% or more), see the supplement below, indicating that its acute anti-inflammatory effect is significant.

For tumor experiments of the homologous melanoma B16-F0 cell line, reference is made to the literature published by Farrugia CA and Groves MJ. 1999 in Anticancer Research 1 9: 1 027-1 032. Five C57BL / 6J male mice without pathogens (SPF) and immunity (6-8 weeks old) were housed in an animal isolation room (iVC rack) in a pathogen-free (SPF) environment. A murine B16-F0 melanoma living cell line (ATCC CRL-6322, 1.0 × 105 cells in 0.2 ml) homologous to C57BL / 6J mice was injected subcutaneously into the backs of experimental mice side. Treatment was started 24 hours after tumor inoculation, and the test compound was administered daily as an oral feed for 21 days. The number of days was reduced when symptoms of significant toxicity appeared. The mice were monitored for weight, tumor size, and survival from day 1 to day 22 of 2005200528093. In addition, experimental mice were monitored for survival until the end of the study day 45. The tumor weight (mg) was evaluated according to the formula: length (mm) x [width (mm)] 2 x 0 · 5 ′ where the specific gravity is assumed to be 1 and 3 to 3. The tumor proliferation of the compound-treated animals was calculated by T / C (experimental group / control group) χ 100%; if the T / c value is $ 42%, it is regarded as evidence of significant antitumor activity. Mean survival time of T / c (experimental group / control group) ^

125% is also considered as evidence of significant antitumor activity. Table 14: Tumor experimental results of the homologous melanoma B16_F0 cell line. Treatment route dose group tumor weight i K mg) T / C, mean ± SEM. T / C (° / 〇) T: / C (° / 〇) Eleventh T / C (° / 〇) P0 10 ml / kg 1 0 0 60 x21 2 0 39 298 3 0 0 49 4 0 54 541 5 0 21 117 0 100 23 ± 11 100 213 + 93 100 ACM-alcohol extraction ilL · P0 100〇mg / kg 1 0 0 0 substance χ 21 2 0 0 0 3 0 0 4 0 0 14 5 0 0 32 0 100 0 ± 0 0 * 9 ± 6 4 * Mitair ^ cin Ip ~ 2 ng / kg 1 0 0 0 χ 6 2 0 0 64 3 0 0 4 0 0 68 5 0 0 41 0 100 0 + 0 0 * 34 + 15 16 * ^ 15: homologous melanin Tumor experiment results of tumor B16-F0 cell line Is pathway dose group tumor weight (mg) and% T / C, mean ± SEM

40 200528093

15th T / C (%) 18th T / C (%) 22nd T / C (%) Carrier P0 10 ml / kg 1 211 746 2054 x 21 2 657 1597 2870 3 216 669 1419 4 835 2455 3688 5 240 726 1682 432 soil 100 1239 soil 100 2343 ± 100 131 349 416 ACM-alcohol extract P0 1000 1 49 280 913 _ Do mg / kg 2 62 630 1545 x 21 3 388 1079 2560 4 148 435 1514 5 229 535 1637 175 soil 62 41 氺 592 + 135 48 1634 ± 70 265 Mitomycin IP 2 mg / kg 1 36 256 437 x 6 2 136 849 1248 3 0 0 0 4 213 525 663 5 207 327 Died 41 200528093 119 ± 44 27 * 391 +142 32 * 587 ± 25 * 260 For a total of 21 days, the same reference compound and mitomycin were given twice a week in a manner of p, for a total of 6 times. Tumor size was measured and recorded twice a week for 22 days. Inhibition of tumor growth was calculated as τ / c (experimental group / control group) χ 100. A T / c value of $ 42% is considered evidence of significant antitumor activity. Table 16. Tumor experimental results of homologous tumor B16-F0 cell line. Tumor weight in the treatment group dose group (Taixiang \ half; ^ Hi ± SEM) No. 1st 8th 11th 15th 18th 22 said wit P0 10 ml / kg 1 21 20 20 21 22 25 X 21 2 22 22 21 22 26 30 3 21 21 20 20 22 21 4 21 20 20 20 21 24 5 22 21 20 19 20 23 21.4 + 0.2 20.8+ 0.4 20.2 + 0.2 20.4 + 0.5 22.2 ± 1.0 24 6 + 1.5 AGH quince extract P0 1000 ng / kg 1 2 20 20 21 19 21 19 22 21 22 22 23 24 x 21 3 20 18 18 19 21 26 4 21 20 19 21 20 21 5 20 20 21 22 23 25 20.2 + 0.2 19.6 soil 0.5 19.6 + 0.6 21.0 + 0.5 21.6 + 0.5 23 · 8 ± 0 · 9 Mitomycin IP 2 mg / kg 1 25 25 26 25 24 27 x 6 2 22 22 22 25 27 30 3 19 21 21 21 22 21 4 22 22 22 24 26 27 5 19 20 19 20 21 Died 21.4 + 1.1 22.2+ 22.0 + 1.1 23.0 ± 1.0 24.0 ± 1 · 1 26.3 + 1.9 0.8 Twenty-four hours after tumor cell implantation, experimental animals were given vehicle and test substance daily for 21 days. At the same time, the reference compound and mitomycin (Mitomycin) were administered by IP in total 42 200528093 twice a week, A total of 6 times. Tumor size was measured and recorded twice a week for 22 days. The Student, st test was used to determine whether there was a significant difference in body weight change between the test compound and the vehicle control group. Table 17 · Homologous melanoma B16-F0 Tumor test results of cell lines i Live time ~ ri ~ T, treatment dose

a: If the animal does not die after 45 days, its survival period is regarded as 45 days. Survival of experimental animals during the 45-day study period or until the death of the experimental animals is

The average survival time of T / C (experimental group / control group)] was also considered as evidence of significant antitumor activity. Discussion: In accordance with established standards within the company, daily preparations were given orally-alcoholic extracts, according to the following mouse and rat tests to produce significant effects: 43 200528093 Rats were given a central choline synergist at 1 000 mg / kg ; Non-significant synergists are given a minimum of 300 mg / kg; non-significant synergists or antagonists acting on peripheral cholinergic nerves are given 1,000 mg / kg (Table 5). Spontaneous SBP rats (SH) at a dose of 1000 g / kg reduced their systolic blood pressure (16%, 12%, and 20% 'observed vs. 100% at time points of 1, 2, and 4 hours, 0 ), And the heart rate was moderate but not significantly reduced (Tables 6 and 7); the 300 mg / kg dose did not cause significant changes in systolic blood pressure and heart rate (Tables 8 and 9). A dose of 1000 mg / kg increased dietary induced high-density lipoprotein (HDL, more than 39% of the vehicle control group) in mice (Table 10); when the η DL / 1 / 10ta 1 ratio decreased significantly to close to 31% 'but there was no significant change in total cholesterol; the 300 mg / kg dose did not cause changes in Total, HDL and HDL / Total ratios. After galactosamine-induced liver injury in rats, treatment with a dose of 1000 mg / kg x 3 resulted in a hepatoprotective effect (compared with the vehicle control group with a 44% decrease in ALT and a 57% decrease in AST), and at 300 mg At a dose of 3 kg / kg, ALT decreased by 200/0 and AST decreased by 28% (Table 11). An anti-inflammatory response to rat paw edema induced by carrageenan at a dose of 1000t g / kg (45% inhibition compared to the vehicle control group) (Table 12); a lower level of 300 mg / kg and No significant effect (3% inhibition compared to vehicle, Table 13). C57BL / 6J mouse homologous melanoma β16-F0 cell line experiment, at a dose of 1000 milli44 200528093 g / kg, was used at 8, π, and i to extend the survival time of animals (Table 16). Days (Tables 14 and 15) with anti-tumor effect U); no significant difference in animal weight (Table [0050] ACM, then-alcoholic extract and compound 3 of the present invention using nine groups of KR-derived male rats (weight For example, each animal was given a single-dose of four gasified carbons 4,5_ stick oil containing 0 ml / kg (P0). ACM test substance at qn, 1 λλJ A substance at 30, 100 and 300 mg / kg dose,

Orally administered after treatment of Sihua ^ 30 minutes and 4 and 8 hours; ㈣—Alcoholic extracts 30 minutes before and 4 and 8 hours after treatment of gasified carbon, in a dose of 亳 g / kg daily ( Twice daily) repeated oral administration. Animals were sacrificed at 24 small β temples after being treated with tetragas fossils. The values of alanine transaminase (alt) and aspartate transaminase (AST) were measured by the HITACHI automatic analyzer (the model is measured by the best method. Compared with the carrier group, the ALT or AST value decreased by 30% Or more (^ 30%), which means it has a protective effect on liver injury.

Table 18: Liver injury of mice with tetracarbonated carbon

45 200528093

ACM P0 1000 1 1440 888 mg / kg 2 2720 1520 x 3 3 2272 1328 4 1272 792 5 1320 880 X 180. 5 (46) 1082 (36) SEM 292 144 1 2336 1256 300 2 1552 1072 mg / kg 3 3720 1512 x 3 4 3816 2336 5 3952 2792 X 3075 8 1794 -6 SEM 480 330 ACM-alcohol extract P0 1000 1 1936 1232 Extract mg / kg 2 1528 768 x 5 3 1896 1136 4 2752 1656 5 2472 1592 X 2117 (36) 1277 25 SEM 219 162 1 1656 976 300 2 3536 1712 mg / kg 3 2328 1808 x 5 4 1736 1416 5 1792 888 X 2210 (34) 1360 20 SEM 352 187 Compound 3 P0 300 1 1368 776 mg / kg 2 1576 896 x 3 3 1440 896 4 2728 1352 5 2720 1728 X 1966 (41) 1130 (33) SEM 311 179 1 3200 2256 100 2 4576 2976 mg / kg 3 2512 1536 x 3 4 2728 1552 5 3696 1600 46 200528093 30 mg / kg x 3 X SEM 1 2 3 4 5 3342 — 370 4296 3696 2152 2400 4256 0 1984 282 2136 2288 1096 1792 2496 -17 ~ -16 X SEM 3360 457 1962 245 Silymarin PO 100 1 2856 1 2QR (Silymarin) mg / kg 2 1832 Λ. L · ZJ \ J 1152 x 3 3 1296 952 4 2792 1072 5 2728 1336 X 2301 (31) 1162 (31) SEM 313 71 Discussion · The present invention ACM, ACM-alcoholic extract, and compound 3 have been evaluated as potentially protective against liver damage in ICR mice induced by tetracarbonated carbon. The ACM test article was administered orally at 0.5, 400, and 300 mg / kg doses after the animals were treated with carbon tetrachloride for 0.5 and 4 hours and 8 hours. ACM-alcoholic extracts were given twice (b.i.d.) at a dose of 300 and 1000 mg / kg on the day before (9:00 AM and 16:00 pM) before carbon tetraoxide treatment, And 0.5 hours before and 4 hours and 8 hours after the start of the process of the four gasification carbon (total 5 doses). The degree of liver damage is determined by comparison with the alanine aminotransferase (ALT) and aspartate aminotransferase (AST) in the serum of the carrier animal. In the present invention, ACM at a dose of 1,000 mg / kg X 3 and Compound 3 at a dose of 300 mg / kg X 3 resulted in ALTUe% and 41%) and AST (36% and 33%) compared to the treatment of a carrier animal. Significant decline. ACM-alcoholic extracts at the same dose of 300 and 1qqq mg / kg X 5 also caused significant reductions in ALT (36% and 34%) 47 200528093 and AST (25% and 20%). Simultaneous trials of silymarin (100 mg / kg χ3, Ιρ) showed a significant decrease in ALT (31%) and AST (31%) compared to the treated vehicle animals. The ACM, ACM-alcoholic extract, and compound 3 of the present invention have significant liver protective effects in the four-gasification carbon model of mice. The present invention has been described and illustrated in detail so that those skilled in the art can implement and use it, but any substitution, change and modification should not depart from the spirit and scope of the present invention.

Within. Those skilled in the art will soon find that the present invention can easily achieve the objective, and the results and advantages described herein. Cell lines, embryos, animals, and their manufacturing processes and methods are only representative of exemplary preferred embodiments and are not intended to limit the scope of the invention. Those skilled in the art will think of modifications and other uses of the present invention, but those modifications should be included in the spirit of the present invention and limited to the scope of patent application.

Obviously, those skilled in the art can easily make changes and modifications to the present invention without departing from the spirit and scope of the present invention. All patents and published works described in this specification are general technology related to inventions in this field. When each individual publication is specifically and individually displayed and merged with the reference, all patents and publications merged with the reference have the same scope. The invention map described herein < can be implemented without any requirements or restrictions, not specifically what is disclosed herein. The terms and expressions used are for illustration only, not for limitation, and they are not intended to exclude any shown or described or part of it. 48 200528093 Features are considered to be various changes and modifications It may still be within the scope of the invention patent application. Therefore, it should be understood that although the preferred embodiments and optional features have been used to specifically disclose the present invention, those skilled in the art may modify and change according to the concepts disclosed herein, but these modifications and changes should be Within the scope defined by the claims attached to the present application. [Brief Description of the Drawings] Figure 1 shows the HMBC injection of Compound 2 of the present invention. φ Figure 2 shows the compounds of the invention. Figure 3 shows the noOE (Nuclear Overhauser Effect) injection of compounds 4 and 5 of the present invention. Figures 4 (a)-(d) show the test results of Compound 3 of the present invention. Figures 5 (a)-(c) show the test results of water extract of Antrodia cinnamomea mycelium powder [ACM (An t rod i a αmycelia powder)]. Figures 6 (a)-(f) show the test results of Antrodia cinnamomea mycelium powder alcohol extract. Figures 7 (a)-(e) show the test results of Compound 1 of the present invention. Lu 49

Claims (1)

200528093 Patent application scope: 1. A compound with the formula f Wherein X is N or 0; Rl is Cl-1. Burnt oxygen, C2-1. Feminine or C2-1. Fast oxygen; R2 is H, Ci-i. Alkenyl, C2-1G dilute or C2-1. Fast radical; and R3 is absent, fluorene or hydroxyl; but when X is 0, R3 is absent. 2. The compound according to item 1 of the scope of patent application, wherein 1 is C2-6 alkenyloxy or C2-6 alkynyloxy. 3. The compound according to item 2 of the scope of patent application, wherein I is a C alkyl substituted by C 2-6 alkenyloxy. 4. The compound according to item 1 of the scope of patent application, wherein R2 is isobutyl. · 5. The compound according to item 1 of the scope of patent application, which is 3-isobutyl-4- [4- (3-methyl-2-butenoxy) phenyl] furan-2, 5-dione ; 50 200528093 3-isobutyl-4- [4- (3-fluorenyl-2-butenoxy) phenyl] -1 and -0-pyrrole-2, 5-dione; 3-isobutyl -4- [4- (3-fluorenyl-2-butenyloxy) phenyl] -1 # -pyrrole-butanol-2,5-division; 5 and gas liS1 times -1-via radical_3 -Isobutyl-4- [4- (3-methyl-2 -butadienyl) benzyl] pyrrolidine-2,5-dione; or from hydroxy-3-isobutyl-4- [ 4- (3-methyl-2-butenoxy) phenyl] pyrrolidine-2,5-dione. 6. — A mixture of Antrodia cinnamomea mycelium prepared from water or organic solvent extract of Antrodia cinnamomea mycelium. 7. The mixture according to item 6 of the scope of patent application, wherein the organic solvent is an alcohol, an ester, an alkane or a haloalkane. 8. The mixture according to item 7 of the scope of patent application, wherein the alcohol is alcohol. 9. The mixture according to item 6 of the scope of patent application, wherein the compound is 3-isobutyl-4- [4- (3-methyl-2-butenoxy) phenyl] furan ~ 2, 5-di Ketone; · 3-isobutyl-4- [4- (3-methyl-2-butenoxy) phenyl] -pyrrole-2 5_dione; 3-isobutyl-4- [4- (3-methyl-2-butenyloxy) phenyl]-winter. Pyrrol-2-ol-2, 5-dione; mono-2 butenyloxy) phenyl] hydroxy-3 —isobutyl-4 4- [4-methylpyrrolidine-2, 5-dione; or 51 200528093 汔 汔 次--1 -Cyclo-3-isobutyl-4- [4- (3 -methyl-2-butanyloxy) phenyl] mouth ratio σ each -2,5 -di _. I 0 · The mixture according to item 6 of the scope of patent application, which reduces systolic blood pressure or increases high-density lipoprotein. · II · A mixture according to item 6 of the scope of patent application, which has central cholinergic action, hepatoprotective effect, anti-inflammatory or antitumor activity. · 12 · The mixture according to item 11 of the scope of the patent application, wherein the tumor is a tumor selected from cells or tissues of liver, intestine, bone, blood, lymph and breast. 13. · A composition comprising a compound of the formula Wherein X is N or 0; is C ^ o alkoxy, GB alkenyloxy or 1Q alkynyloxy; R2 is fluorene, Cl, alkyl, C2-H alkenyl or C2-1Q alkynyl; and R3 is not , Η, or meridian; but when X is 0, R3 is absent. 14. The composition according to scope 13 of the application for patent, wherein the compound is 52 200528093 3-isobutyl-4- [4- (3-methylbutenoxy) phenyl] furan_2, 5-dione ; 3-isobutyl-4- [4- (3-methyl-2-butenoxy) phenyl] in the case of 2,5-dione; 3-isobutyl-4- [4 -(3-methyl-2-butenoxy) phenyl] — aramidol-butanol-2, 5-dione; state M ^ -1-hydroxy-3-isobutyl-4- [4 ~ (3-methyl-2-butenoxy) phenyl] mouth ratio of 17 to 2-5,5-di_; or the state of wish-b-hydroxy-3-isobutyl-4- [4- (3-Methyl-2-butenyloxy) phenyl] Biao Mingming * shao "-2,5-digang. 15 · The composition according to item 4 of the scope of the patent application, wherein the compound is 3-isobutyl-4- [4- (3-methyl-2-butenoxy) phenyl] -ΐ77_σ 比比 _2 , 5-dione; or 3-isobutyl-4- [4- (3-methyl-2-butenoxy) phenyl] -1V7-pyrrole-1; [_alcohol-2,5-diamine. 16. The composition according to item 3 of the scope of patent application, which reduces systolic blood pressure or increases high-density lipoprotein. 17. A composition according to item 13 of the scope of patent application, which has a central choline agonizing effect, a liver-protecting effect, an anti-inflammatory or anti-tumor activity. 18. The composition according to item 3 of the patent claim, wherein the tumor is a tumor selected from the cells or tissues of the liver, intestine, moon, ash, lymph, and breast. 19. An Antrodia cinnamomea mycelium, which contains the compound in the scope of patent application No. 1. 200528093 20. The mycelium according to item 19 of the scope of application for patent, wherein the compound is 3-isobutyl-4- [4- (3-fluorenyl-2-butenoxy) phenyl] furan-2, 5-dione; 3-isobutyl-4- [4- (3-methyl-2-butenyloxy) phenyl] -1) 7-pyrrole_2, 5-dione; Gasoline) phenyl ] -1 and -σ ratios 各 7 each _1 monool 3-isobutyl-4- [4- (3-methylbutan-2, 5-difluorene; hydroxy-3-isobutyl 3- Methyl-2-butenoxy) phenyl] Pyrrolidine-2, 5-dione; or (3- · fluorenyl_2-butoxymethyl) phenyl] from pyrene-hydroxy-3-isobutyl ~ 4 ~ [4 pyrrolidine-2, 5 -Dione. 54