TW201018462A - Novel mixture and compounds from mycelia of antrodia camphorata having hepatoprotection, anti-inflammatory and anti-tumor activities - 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

201018462 VI. Description of the invention: [Technical field to which the invention pertains] The present invention relates to an article from Niobium

Antrodia Cinna/nonwa Wu, S ugly a a ) New combination of compounds and compounds and their medical uses. The invention further relates to a composition or mycelium comprising a compound of the invention. [Prior Art] The Taiwanese scorpion (Polyporaceae, Aphyl lophor ales) is often used as a traditional Chinese medicine. It grows only in the inner layer of the heartwood wall of the Taiwanese evergreen tree Cinnamomun kanehirai (Hay) (Lauraceae), which is rare and uncultivated. This fruiting body has been used to treat food and drug poisoning, diarrhea, abdominal pain, high blood pressure, skin blemishes, and liver cancer. Few related biological activity studies have been reported to date. In Taiwan, "Niuzhizhi" is also called "Niutian mushroom". According to the cylindrical spores of its fruiting body, the soft and soft amylopectin hyphae, the bitterness and the drawing of the eucalyptus pillat, the fruit of the plume, and the thick The characteristics of membrane scorpion and anthroconidia in pure culture have recently been reported as new strains. The growth of this new species is rather slow and limited to the local tree species Cinnamomum kanehirai Hay (Lauraceae). The detailed description and classification of burdock is described in Wu, S.-Η. et al., 1997, in New combination of a medicinal fungus in Taiwan, Bot. Bull. Acad. Sin. 38: 273-275 201018462 cinnamomea j Among Taiwanese folk medicines, the fruiting body of Antrodia camphorata is considered to have a certain degree of medical effect. According to the traditional method, Zizi is made into a dry powder or used with other herbal auxins and used orally to treat symptoms caused by poisoning, thirst, abdominal pain, high blood pressure, phlegm and liver cancer. However, no pharmacological or clinical studies have been documented to support these views. Because of the clear host and rare in nature' and the failure of artificial cultivation, "Niuzhizhi" is very expensive in Taiwan. In recent years, high-quality fruit entities have been sold to extremely high prices of about every kilogram. SUMMARY OF THE INVENTION The subject matter of the present invention provides a novel mixture from Antrodia camphorata g. The present invention provides a novel compound from the mycelium of Antrodia camphorata. The subject matter provides a novel composition comprising a compound of the invention. The subject of the invention provides a novel mycelium mycelium comprising a compound of the invention. DETAILED DESCRIPTION OF THE INVENTION The present invention provides a compound of the formula 201018462

Wherein X is N or 0; _ Ri is C 〇 〇 alkoxy, Cno olefinoxy or C 2 ι () alkynyloxy; R 2 is hydrazine, Cl-l. An alkyl group, a (V!. alkenyl group or a c2 ιβ alkynyl group; and a no group, a hydrazine or a hydroxy group; but when the X system is 0, the R3 system is absent. Among the compounds of the present invention, a 1 system Cm.oxy group or CM is preferred. More preferably, I is substituted by a C alkyl group for a Cz-e alkenyloxy group, and most preferably R1 is a mercapto group substituted with a butenyloxy group. Among the compounds of the present invention, a preferred one is a Cl-6 alkyl group. Rz is isobutyl. Spring is a preferred compound of the invention 3-isobutyl-4-[4-(3-methyl-2-butoxy)phenyl]furan_2 5_2 3-isobutyl-4-[4-(3-indolyl-2-butoxy)phenyl]pyrrole-2,5-dione; 3-isobutyl_4_[4_(3_ Indenyl-2-butenyloxy)phenyl]pyrrole q-alcohol-2,5-diindole; methyl-2-butenyloxy)phenyl]« ratio <? no secondary, 45^-1 - benzyl-3-isobutyl-4-[4-(3- rosin-2, 5-dione; or 201018462 弋 - - - hydroxy-3-isobutyl-4-[4-( 3_Methyl-2-butenyloxy) stupid] 〇 is slightly dazzling·-2, 5-dimen. The more preferred compound of the present invention is 3-isobutyl_4-[4-(3-A 2-butenyloxy)phenyl]-1 and _pyrrole-25-dione; or 3-isobutyl-4-[4-(3-methyl-2-butoxy)phenyl Divination and -D ratio The preferred compound of the present invention is 3-isobutyl-4-[4-(3-methyl-2-butenoxy)benzene. The invention also provides Antrodia camphorata a mixture of filaments, including a compound of the invention. The mixture of the invention comprises an aqueous or organic solvent extract of a mixture of mycelium of Antrodia camphorata, including but not limited to alcohols (eg CH3〇H, C2H5〇h or C3h7〇h). ), esters (such as ethyl acetate), alkanes (such as hexane) and halogens (such as CH3C1, C2H2Cl2). Preferred organic solvents are ethanol or alcohol-based solvents that do not cause human side effects. Reduce systolic blood pressure or increase high-density lipoprotein. In addition, the same mixture has central nervous choline agonism 'hepatoprotective effect, anti-inflammatory or anti-tumor activity. In detail, the mixture of the present invention can be inhibited from liver, intestine, bone , tumors of cells or tissues of blood, lymph, and breast. Individuals receiving the mixture of the invention include, but are not limited to, humans, mammals, rats, mice, horses, pigs, dinosaurs, ducks, dogs, and depictions. A composition comprising a compound of the invention. The composition of the present invention can reduce contraction I or increase high-density lipoprotein. Further, the composition of the present invention has central 201018462 neurocholinergic agonism, hepatoprotective action, anti-inflammatory or anti-tumor activity. In detail, the combination of the present invention The tumor can inhibit tumors of cells or tissues selected from the group consisting of liver, intestine, and breast. Individuals receiving the composition of the present invention include, but are not limited to, human mammals, rats, mice 'horses, pigs, chickens, ducks, dogs and still. The invention also provides a mycelium of Antrodia camphorata comprising a compound of the invention. Preferably, the mycelium is at least 1% by weight of the original mycelium and is the total weight of the compound 1 _ 5 of the present invention. More preferably, the weight of at least 3% of the original mycelium is the total weight of the compound j _5 of the present invention. The sorghum mycelium is intended to be prepared by submerged 1 iquid fermentation, immersed in liquid fermentation, such as Τ·········································· Www.unu.edu/unupress/food/8F101e/8F101E0b.htm [Embodiment] Φ The following examples are merely representative of various aspects and features of the present invention and are not intended to limit the scope of the present invention. The general experimental procedure melting point is Yanagi mo To the micro-melting point heating device to measure and not modify it. The optical rotation is measured by Jasco DIP-360 automatic polarometer. The ultraviolet spectrum is measured by Shimadzu UV-2200 spectrophotometer. The infrared spectrum is Jasco The FT/IR-230 infrared spectrometer measures the spectrum of 咜- and 13C-NMR measured by a Varian Unity Plus 500 spectrometer. EIMS and HR-EIMS are based on 201018462 > JMS-AX 505 HAD mass spectrometer at 7〇# The column chromatography was performed using BW-820MH (normal phase) and chr〇matorex-0DSDM1 020T (reverse phase) (Fuji Silysia) ruthenium gel. Extraction and separation were taken from 2001. In the month, 6 grams of Antrodia camphorata powder hyphae from Taiwan Shanshao Biotech Co., Ltd. was extracted three times with chloroform under a countercurrent of 3 hours. The air-extracted extract (5.3 g) was chromatographed with ruthenium gel and n-hexane acetone (19: 1—14:6) and the gas-like _methanol (1:1) washes out nine parts (Fr. ig). The second part is calibrated with ruthenium gel to 1 (8.7 mg), the fourth part. The fractions were normalized to reverse phase and the gel was chromatographed to 2 (13.6 mg). The fifth fraction was eluted with ruthenium gel chromatography and n-hexane-propyl mesh (8:2) and then calibrated to 6 (35). · 8 mg), the sixth part is calibrated to 3 (14.6 mg) by combined normal phase and reverse phase gelation column chromatography, and the seventh part is subjected to column chromatography to produce mixtures 4 and 5 (4: 1), mixtures 4 and 5 were subsequently separated by preparative HPLC [column: Tosoh TSK-gel ODS-80TH (21. 5 X 30 〇 mm), mobile phase: methanol-water containing 〇.l% TFA ( 70··30)] 3-isobutyl-4-[4-(3-methyl-2-butoxy)phenyl]-furan-2, 5-dione (compound 1): yellow oil Body; UV (MeOH) .ax (log ε ) 227 (4. 1) ' 258 (3·9), 275 (3. 8), 355 ( 3. 4) nm; IR (CHCh) .ax 1763 cml' Table 1 is 1H-NMR; Table 2 is 13C-NMR; EIMS/z^314[M] + 〇〇〇) ' 246 (100) > 131 (100) ; HR-EIMS m/z 314. 1523 (calculated for C19H22O4: 314. 1518). 201018462 3-Isobutyl-4-[4-(3-methyl-2-butoxy)phenyl]-1#-pyrrole-2, 5-dione (2): yellow crystal Alkane-ethyl acetate); mp 110-111 °C; UV (methanol)... (log ε) 230 (4.3), 272 (3.5), 355 (3.77) nm; IR (CHC13) ax 1724 cm'1; Table 1 is 1H-NMR; Table 2 is 13C-NMR; EIMS 茁/z313 [M]+ (8) > 245 (1 00) ^ 203 (77) ' 131 (28) ; HR- EIMS m/z 313. 1681 (calculated for C19H23N〇3: 313.7678). ® X-ray crystallographic diffraction of compound 2: Crystallization from n-hexane-ethyl acetate gave a yellow needle and was selected for data collection. Crystallization data: C19H23NO3; ^=313.40; volume 0.15 X 〇.〇2 X 〇.〇2mm; slant, space group P1 (#2), a=6.3505(5) A, b=12.205(1) A, c=12. 560(2) A, α = 64. 623(7)°, =75. 358(4). , r =84· 681 (5) °, V=850. 9(2) A3, Z=2, DcaU=l. 223 g/cm3, ^ (MoK a ) = 0. 82 cm > Fdd〇= 336.00 . It was irradiated at 93 K and measured by a Rigaku RAXIS-RAPID image plate diffractometer with monochromatic graphite Μ〇 _κ α (λ = 0.71079 Α). Collect 8950 reflections ' 4745 series unique (Rint = 〇 1〇 8); merge the equivalent reflections. The crystal structure was solved by the direct method (SHELXS86) and the full matrix least squares actuarial. Non-hydrogen atoms are calculated heterogeneously. Hydrogen atoms include but are not inferred. The final parameters are 0. 074, core = 〇. 099 ' and G0F (object observation facility) = 1 〇 6. The maximum and minimum peaks of the final difference Fourier map correspond to 0.83 and -0.89 201018462, e-/A3, respectively. 3_Isobutyl_4_[4_(3 -methyl_2-butoxyoxy)phenyl]-H-«tb-l-enzyme-2,5-dione (compound 3): yellow oil body ; UV (MeOH) A. ax (loge): 232. 5 (4.33), 296 (3. 7), 374 (3·7) nm; IR (CHCh) v.ax 1717CHT1; Table 1 is 1H- NMR; Table 2 is 13C-NMR; EIMS/i7/z 329 [M] + (12), 261 (100), 131 (50); HR-EIMS/ff/z: 329.1637 (calculated value of C19H23N〇4: 329. 1627). Said, only - hydroxy-3-isobutyl-4-[4-(3-methyl-2-butoxy)phenyl]pyrrolidine-2, 5-dione (4): colorless Oil body; [a ]»23 +2. 5° (c 0. 2, methanol); UV (methanol) in... (log ε): 225 (4.3), 275 (3.3), 283 (3.2) nm; IR (CHCh) v ... 1715 cm1; Table 1 is 1H-NMR; Table 2 is 13C-NMR; EIMS2»/z331[M; T(2), 263 (67), 207 (66), 191(30), 179 (40), 133 (64), 69 (100); HR-EIJIS Double 々: 331.1747 (calculated value of C19H25N〇4: 331.1783). Hydroxy-3-isobutyl-4-[4-(3-methyl-2-butoxy)phenyl]pyrrolidine-2,5-dione (5): colorless oil; a ]D23 +3. 0° (c 0. 2, MeOH); UV (MeOH) λ (log ε ) : 227 (4.3), 275 (3.4), 283 (3.33) nm; IR (CHCl〇Bax Π15 cm.1; Table 1 is 1H-NMR; Table 2 is 13C-NMR; EIMS i»/z331 [M] + (1), 263 (45), 207 (50), 191 (75) , 179(30), 133(100), 69(92); HR-EIMSiff/: 331. 1766 (calculated value of Ci9H25N〇4: 331. 1783). 201018462

Peroxidized ergot lyophilized: colorless crystals (n-hexane-acetone); mp 165-169 °C (1 it2 mp 171-174. 〇. Fine toxicity test: suiforhodamin B (SRB) Method. 50% growth inhibition (EDsd) is calculated by the Pr〇bit method. Results and discussion The gas-like extracts of A. angustifolia mycelium are repeated with normal phase and reverse phase gel chromatography to obtain a sufficient amount of Butenedioic acid, succinic acid derivative (Compound 5) and ergosterol peroxide. Table 1: 1H-NMR spectral data of compound 1-5 (ppm, J = Hz) (500 MHz, CDCh) Η 1 2 3 4 5 3 _ a 2.87 (13⁄4 m) 3.08 (lH,m) 4 ..... 3.52 (lH,d, 4.07 (13⁄4 d, ^=4.0) ^8.0) Γ 2.59 (2Η, d, 2.51 (2H,d, 2.50 (2H, d, 1.51 (13⁄4 m) 1.02 (lH,m) J=1.Q) J=7.0) J=TO) 1.72-1,84 (1H) 1.42-1.48 (1H) 2, 2.12 (1H, sep, 7=7.0) 2.06 (1H, sep, J=7.0) 2.05 (1H, sep, 7=7.0) 1.72-1.84 (1H) 1.42-1.48 (1H) 3, 0.70 (33⁄4 4 0.66 (3H, d, 0.94 (6H, d, 0.80 (6H, d, 0.88 (63⁄4 d, J=6.5) 7=6.5) 4, •7=7.0) J=7.0) •7=7.0) 0.89 (3H, d, 0.80 (33⁄4 d, Household 6.5) /=6.5) 2",6,, 7.50 (2H, d, 7.50 (2H, d, 7.50 (2H, d, 6.96 (2H, d, •7=9.0) /= 9.0) J=9_0) •7=8.5) J=9.0) 3”,5” 7.02 (2H, d, 6.95 (2H, d, 6.87 (2H, d, 6.84 (2H, d, • 7=9.0) /=9.0) •7=9.0) J=8.5) /=9.0) Γ” 4.57 (2H, d, 4.56 (2H, d, 4.55 (2H, d, 4.47 (2H, d, 4.47 ( 2H, d, /=6.6) /=6.5) J=6.9) J=6.S) /=6.5) 2", 5.50 (1H, brt, 5.50 (1H, brt, 5.49 (1H, brt, 5.47 (lH ,brt, 5.47 (lH,brt, /=6.6) J=6.5) •7=6.9) •7=6.5) J=6.5) 4,” 1.81(33⁄4 s) 1.81 (3H,s) 1.81 (3H,s ) 1.79 (3H, s) 1.79 (3H, s) 5", 1.76 (3H, s) 1.76 (3H, s) 1.76 (3H, s) 1.73 (3H, s) 1.73 (3H, s)

Table 2: 13C-NMR spectral data of compound 1-5 (δ ppm) (125 MHz, CDC13) 11 201018462 C 1 2 2 166.4 (8) 171.7 (s) 3 139.8 (8) 138.8 (s) a) 4 140.2 (s) 139.2 (s) a) 5 165.4 (s) 171.1 (s) Γ 33.6 (t) 32.8 (t) 2, 27.9 (d) 28.1 (d) 3, 4, 22.7 (q) 22.7 (q) 1, 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"5 139.1 (s) 138.6 (s)a) 4,,, 25.2 (q) 25.8 (q) 5", 18.2(q) 18.2(q a) The transfer can be changed interactively. 3__4 5 168.8 (8) 174.8 (8) 175.1 (8) 135.9 (s) a) 44.6 (d) 40.3 (d) 136.0 (s) a) 49.8 (d) 47.5 (d) 168.1 (8) 173.2 (8) 173.6 (s) 33.2 (t) 40.4 ( t) 35.3 (1) 28.4 (d) 25.3 (d) 25.2 (4) 23.0 (q) 21,3 ^ 21.8 (q) 23.0 (6) 22.4 (q) 120.8(8) 127.9(8) (8) 131.0(d) 128.8(d) 130.2 (d) 115.0 (d) 115.4(d) 115.0(d) 160.2 (8) 158J (8) 158 7 (8) 65.1 (t) 64.1 (t) 64.8 (t) 119.2(d) 119.4(d) 119.3(d) 138.9 (8) 138.3 (s) 138.4 (8) 26.1 (q) 25.8 (q) 25.8 (q) 18.5 (q) 18.1 (q) — 18 2 (4)

The structure of the new compound was determined by the following method: Compound 2 was crystallized in yellow crystals under the conditions of mp U0_lirc, and its molecular formula CleH23N〇3 was separated from the hr side. The absorption spectrum of the infrared spectrum at 1 m(3), the thiosemicarbazide group shows four signals of methyl carbon, two methylene sulphide, and - methine carbon in the aliphatic region. There is a benzene ring, an olefin and two carbonyl carbons. The 1H-NMR spectrum showed an isobutyl moiety at δ 〇9〇2 〇6 and 2 51, with a 3-methyl-2-butenyl moiety at 5 um, 5 5 ' and a 6.9... .5. There are para-substituted benzenes, and these results are followed by mc〇SY and T-C experiments. According to the molecular formula and the results of the bile spectrum, this compound is presumed to contain more C_ atoms, including more than, basic. Therefore 12 201018462 This ambiguous part is speculated to have sleepy butadiene. This chemical structure was determined by X-ray analysis as 3-isobutyl-4-[4_(3-methyl-2-butenyloxy)phenyl]-1 pico-pyrrole-2,5-dione (Fig. 2 ). The molecular formula d of Compound 1 was analyzed by HR_EIMS. Infrared spectroscopy showed carbonyl absorption of the anhydrous acid at 1763 cm-1. The 1H-NMR spectrum of the compound i was similar to that of 2, and showed a partial composition of an isobutyl group, a 3-methyl-2-butenyloxy group, and a para-substituted benzene ring. From the HMBC spectroscopy results based on the molecular formula ❹ Compound 1 has the same structure as the two parts (Fig!), # all have a cis-butyl group. The result of Compound 1 was determined to be 3-isobutyl-4-[4_(3-methyl-2-butenyloxy)phenyl] anthracene-2, 5-dim. The molecular formula of Compound 3 was analyzed by HR_EIMS to determine its molecular formula θα′′4. Infrared spectroscopy showed carbonyl absorption of hydroxyimine at 1717 cm1. The results of 1H- and 13C-NMR spectroscopy of Compound 3 were similar to those of Compounds i and 2, showing the isobutyl, 3-methyl-2-butenyloxy, and the partial composition of the para-substituted benzene ring from the HMBc experiment. ® Compound 3 has a partial structure identical to 2 (圚1), and Compound 3 contains more than one oxygen atom. Therefore, this compound was designated as 3-isobutyl-4-[4(3methyl-2-butyloxy)phenyl]-1 and _furan-1-alcohol-2,5-dione. Compounds 4 and 5 were analyzed by HR-E IMS to have the same enthalpy and molecular formula (Cl9H2sN〇4, found at 331.1747 and 331. 1766, respectively), however both could be separated by HPLC. Infrared spectroscopy showed the carbonyl absorption of the hydroxyimine at the 1715 cnfl of the di-compound. As a result of 1H_ and uc_ NMR spectroscopy, the di-compound showed 13 I · 201018462 isobutyl, 3-mercapto 2-butenyloxy, the presence of a para-substituted alkene ring but the proton of the isobutyl methylene group It is a multiple of compound ^ instead of double. The 1h_1hc〇sy* spectrum shows that the methylene group is attached to the -CH-CH- unit. The 13C-NMR spectrum of compounds 4 and 5 indicates that there are two additional sp3 carbon signals to replace the sp2 carbon signal found by compound 3. Thus, compounds 4 and 5 are known to be at the C-3 and C-4 stereocenter positions of the succinimide ring, and are not hydroxy maleimide or hydrazine hydroxybutadiimide. Compounds 4 and 5 were determined by the coupling constants between H-3 and H-4 (compounds 4 and 5 of 4.0 and 8.0 Hz, respectively), which were determined to be the inverse of the trans- and cis isomers, respectively. Compound 4 was found by N〇ESY (nuclear Overhauser effect spectrum) to have no n〇E between H-3 and H-4, while compound 5 was found to have Ν0Ε. 5。 The optical rotation of the compounds 4 and 5 were respectively +2.5. And +3.0. However, its CD spectrum shows no C〇tton effect at any wavelength, indicating that compounds 4 and 5 may be racemic mixtures. The resolution of these racemic mixtures using HPLC with optical isomer chromatography with different solvent systems was not successful. At the moment we cannot fully speculate whether these compounds are optically active compounds or racemic mixtures. Because of this, its relative structure is 弋 and Ρ 状-;; ι-hydroxy-3-isobutyl-4-[4-(3-methyl-2-butoxy)phenyl] Strontium-2,5-dione. These natural maleic acid and succinic acid derivatives were again isolated according to the report of Aquveque et al. The cytotoxic activity of the gas-like extract and the isolated compound was performed using the LLCC Lewis Lewis 14 201018462 cancer cell line (Table 3). Argon you & extract showed moderate cytotoxicity with an ed50 value of 26.7 g/ml. Cisuccinic acid compounds i and 4 were not cytotoxic, whereas 2 and 3 were found to be cytotoxic to LLC cell lines with ED5. The value is lower than the chloroform extract. Table 3: A gas-like extract from the mycelium of Antrodia camphorata and compound ι_4 on LLC cells

50% growth inhibition value of the strain (ED5〇) ED5〇(β g/ml) Air imitation extract 26. 7 1 >20 2 3. 6 3 7. 5 4 >10 Adr iamycina) 0. 14 a) Tumor test of A. faecalis mycelium in positive control group (powder of Astragalus membranaceus mycelium) A. Cell line attached to cells: MCF-7 : Human breast tumor HT-29 : Human colon gland tumor KATO III : Human gastric gland tumor SW480 : Human colon Adenocarcinoma 15 201018462 SW620 : Human colon gland tumor HepG2 : Human hepatic gland tumor Suspension cells: EL4 : Mouse lymphoma B. Sample compound 1, compound 3, Antrodia camphorata mycelium alcohol extract, Antrodia camphorata mycelium water extract

C. Test Method Calculate ED5. (50% inhibition of effective dose) Attached cells: MTT assay; cells of MCF-7, HT-29, KATO 111, SW480, HepG2 were performed for 3 days. SW620 is carried out for 4 days. Suspended cells: cytometry; EL4 cells counted for 5 days. D. Result Calculation: y = mLn(x) + b

Example XY 0 0. 97 10 ppm 0. 941 30 ppm 0 .6 100 ppm 0. 331 Contemporary X values (10, 30, 50 ppm) and Y values can be calculated as Equation 16 201018462 y = -0.2643Ln(x) + 1.5321 ED5〇=exp [( 0.97/2-1.5321 ) /(-0.2643)] Sample Preparation and Sample Description A. ACM (Handbag Zhiguang. Silk Powder) Water Extraction Method 1. Take 1 gram of ACM to add In a 250 ml beaker with 40 ml of reverse osmosis water, place the beaker in an ultrasonic water bath for 20 minutes at room temperature. 2. The water bath was then stirred at 45 ° C for 45 minutes. 3. Place the beaker in the ultrasonic bath for 2 minutes. 4. Centrifuge the sample for 15 minutes at 3000 rpm. 5. Serially dilute the suspension in cell culture. B. Measuring sample concentration 1. Evaporate pan weight (W1). 2. Take 10 ml of water extract in the evaporation tray 3. Place the evaporation tray in the oven to remove moisture (W2) Sample weight / ml = (W2-W1) /10 C. ACM (子#芝丝丝船Powder) Alcohol extraction method 1. Take 20 g of ACM into a 500 ml beaker containing 100 ml of 95% alcohol and stir at room temperature for 10 minutes. 2. Filter the suspension with Advantec No. 1 filter paper and collect the filtrate. 3. Concentrate the filtrate with a rotary vacuum evaporator and remove all alcohol. D. Compound 1: pure compound obtained from ACM 17 201018462 E. Compound 3: pure compound obtained from ACM MTT assay 1. After the cell proliferation, the old culture solution was removed, and the cells were washed once with PBS. 2. Treat cells with IX trypsin-EDTa. 3. Centrifuge at 1200 rpm for 5 minutes and pour off the supernatant. 4. Resuspend the cells in 1 ml of medium. 5. Mix 100 μl of the cell suspension with 1 μ μ of cone blue stain to calculate viable cells. 6. In a 96-well plate, put ιχ1〇4 cells/1〇〇μ1 medium in each well, and place the plate in a carbon dioxide incubator at 37 °C for 24 hours. 7. Remove old culture solution. Wash cells once with PBS. 8. Add ΐ〇〇μΐ sample to each well and place the plate in 37β (: carbon dioxide incubator culture. 9. Remove the old culture solution on days 3, 4 and 5 and wash the cells once with pBS. 10. Each The wells were added with 57 μM of MTT (0.88 mg/ml). After 4 hours, the sputum was removed and the cells were washed once with PBS 12. Add 5 μM DMS0 per well 13. Read the results by ELISA at 0D545. Method (EL4 cell line) 1. After cell proliferation, remove the old culture solution by centrifugation. 2. Mix the cells evenly with fresh culture medium. 201018462 3. Mix 1〇〇"1 cells. Mix cell suspension l〇Q ul Cone blue dye to calculate survival 4. 5. Prepare containing lx I. 5 cells / ml in a 96-well plate, put 10 in each well. Incubate in a carbon dioxide incubator. Samples of different concentrations. Place the plate in 37 ° C of 6. Awkward live

PBS

NaCl 8 g KC1 0.2 g Na2HP〇4 1·4 g KH2PO4 〇. 2 g Volume adjusted to 1 liter

RESULTS AND DISCUSSION EDs 〇 cell line HepG2 HT-29 KATO III EL4 SW480 SW620 MCF-7 Compound 1 21 ppm 52 ppm 38 ppm 3. 5 ppm 15 ppm 6 ppm Compound 3 35 ppm 42 ppm 69 Ppm 2. 6 ppm 20 ppm 27 ppm 0. 02 ppm Astragalus membranaceus mycelium 32 ppm 52 ppm 156 ppm 2. 6 ppm 71 ppm 4 ppm 19 201018462 (ACM) Alcohol extract Astragalus mycelium (ACM) water extract. 295 ppm 707 ppm 20 ppm 207 ppm 132 ppm 318 ppm Detailed experimental results are as follows: Compound 3 of the invention: HepG2 (Fig. 4a), EL4 (Fig. 4b), HT-29 (Fig. 4c) and Kato III (Fig. 4d) ACM water extracts: HepG2 (Fig. 5a), SW620 (Fig. 5b) and EL4 (Fig. 5c). ACM alcohol extracts: HT-29 (Fig. 6a), SW480 (Fig. 6b), SW620 (Fig. 6c), EL4 (Fig. 6d), HepG2 (Fig. 6e) and Kato III (Fig. 6). Compound 1 of the invention: MCF-7 (Fig. 7a), EL4 (Fig. 7b), HT-29 (Fig. 7c), SW620 (圊7d) and HepG2 (Fig. 7e). ® The compound of the present invention and ACM extract can be proved by the above figures. Inhibition of different tumor cells. Analysis of all new compounds (1, 2 and 3) from ACM alcohol extracts by high performance liquid chromatography. Purpose. To measure alcohol extracts from A. sinensis mycelium (ACM) For the total amount of all new substances (Bu 2 and 3), we use high performance liquid chromatography as an example. 20 201018462 Qualitative quality control procedure. Preparation of Astragalus membranaceus mycelium alcohol extract samples: 1) The electronic balance accurately weighs the sample powder 2 〇. 〇〇〇 (g), placed in a graduated test bottle containing ιOOmL of 95% alcohol, the cap is not tightened. 2) Place the above sample vial in an ultrasonic shock tank for ten minutes. 3) Pour the liquid sample into the centrifuge tube and centrifuge at 5500 r pm for five minutes to remove the natural particles from the sample. ® 4), filter the liquid layer with a - filter paper. 5) Concentrate the filtrate with a rotary vacuum evapotranspiration until a thick, alcohol-free yellow liquid appears. 6), repeat steps 1 to 5 three times, and collect all the extracted products (total burdock mycelium alcohol extract = 4.60 g), and calculate the yield. Model 2690 Waters system conditions: 1), column · reverse phase C18 2), mobile phase: methanol, water, acetonitrile

3), injection volume: 20L 4) 'Detection: Photodiode array detector 996 measured at wavelength 254 ηιη 5), prepared in 10 ml of alcohol containing 丨.000 (g) of Antrodia camphorata mycelium alcohol Extract sample 'for HPLC analysis results: analysis by HPLC' extraction product containing pure compounds 1, 2, and 3 201018462 The substance is shown in Table 4 Table 4: Composition of three standard compounds: 1 ml of alcohol 0.0100 (gram weight) Standard name Compound 1 Compound 2 Compound 3 Concentration (g/ml) 0.01 0.01 0.01 Crest area 49,315,783 129,327,136 136, 255, 406 Indwelling time (minutes) 134.8 124.3 119.8 *Astragalus membranaceus II alcohol extraction sample: 10 ml Alcohol contains 1·000 (g) Concentration (g/ml) 8.59 X 10'3 5.59 X 10'4 1.659 X 10_2 Crest area 42, 374, 766 7, 226, 937 226,102, 223 Yield% (w/ 。 。 。 。 。 。 。 。 。 。 。 。 。 。 。 。 。 。 。 。 。 。 。 。 。 。 。 。. Antrodia camphorata mycelium-alcohol test Materials and equipment 1. Test substance and form of administration All in vitro tests were carried out under the oral administration of 2% Tween 80, which contained the test substance at the initial dose of 1 000 mg/kg. The observation time of each test is detailed in the method 〇22 201018462 2. Experimental animal male or female ICR mice, Wistar_〇kam〇t〇 naturally derived male hypotensive rats (SHR), using Taiwan pan-global pharmacology Wistar and Evans-derived rats were provided by the Institute. The animal feeding space was as follows: 29 X Μ X 10 mice were housed in 13 cm space, 6 rats were housed in 45 X 23 X 21 cm space, and 3 cultivars were raised in 45 X 23 X 21 cm space. Mice and rats were housed in aPecr dragons. The C57BL/6J immunized male mice, which were supplied by the National Taiwan University Animal Center and weighed 21 ± 2 g and 6-8 weeks old β, were also used in this study. The experimental animals were housed in a independently ventilated cage (IVC rack, 36 mini isolation system). Each cage was sterilized and 5 mice were housed (in a space of 26.7 x 20. 7 x 14 cm). All animals were temperature-controlled (21 ° -23 °C) and controlled (60%-70%), and were exposed to light during at least one week prior to use. Provide standard experimental feed (LabDiet

Rodent Diet with Guinea Pig Diet, PMI Nutrition

International, USA) Free to eat with water. 3. Cell line and culture medium Murine melanoma cell line B16-F0 (ATCC CRL-6322) by American

Provided by Type Culture Collection, and with Duibecco, s Modified

Eagle's Medium (GIBCO, USA) is used as a cell culture fluid. Tumor cells were cultured at 37. (: and contains 5% CCh in the air. 4. Chemicals 23 201018462 General: Sterilized water (in-house), dimethyl sub-milling (DMS0, Merck, Germany), isotonic sodium solution (Taiwan Xindong Chemical Industry Co., Ltd., Magnesium Sulfide (MgSCh. 7H2〇, Wako, Japan), Samarium Amphetamine (Sigma, USA), Methyl Vividin (Signa, USA), Sodium Hydroxide (Na0H, Wak) 〇, Japan), phosphate buffer (Sigma, USA) and Tween 80 (Wako, Japan).

Glicose-HA test kit (Wako, Japan), alanine transaminase (ALT) ❹ test reagent kit (Wako, Japan) 'Tianmenwort acid transaminase (AST) test kit (Wako, Japan), T-Cholesterol-HA and HDL test reagent kits (Wako, Japan), Hemolynac 3 Hemolys (Nihon Koden, Japan),

Isotonic 3 Diluent (Nihon Koden, Japan). 5. Equipment is generally used: animal box (Shunde 'Republic of China'), 250 ml and 1000 ml beaker (Kinmax, G USA), discarded syringe (1 ml, Top Corporation, Japan), stainless steel tweezers (klappencker, Germany) , rat scale #Z-40 (Taconic, USA), oral feeding syringe (Natsune, 曰本), hypodermic needle 23Gxl,, (Top

Corporation, Japan), pH meter (Suntex, USA), rat scale 500 g ± 2 g (Chien-chun, Republic of China), glass syringe 1 ml, 2 ml and 5 ml (Mitsuba, Japan) 'and stainless steel scissors ( Klappencker, Germany). 24 201018462 Methods and Results: 1. Experimental study of the central/peripheral bile basic synergy with reference to Lippmann W and

Pugsley TA was published in 1977 on Arch Int Pharmacodyn. 227:324. The test subjects were orally administered to three Wistar-derived male or female experimental groups weighing 150 ± 20 g. After another 30-60 minutes, the experimental animals showed cumulative chewing behavior (mouth and/or tongue action) for more than 10 seconds, as well as salivation. ® A positive phase reaction of 2 or more (22) 3 rats was observed to indicate that it may have an effect on the central and peripheral sputum. Table 5: Results of the acetylcholine stimulating action of the central/peripheral nerve of the rat. The dose of the central nervous system peripheral nerves, the dumbing fraction, the salivary secretion fraction ❷ Carrier P0 10 ml/kg 1 2 3 — 0/3 - 0/3 25 201018462

Mode (IP) given. After 30-60 minutes, the experimental animals were cumulatively measured for more than 10 seconds of chewing behavior (mouth and/or tongue action) and salivation. It was observed that a positive phase reaction of 2 or more than (22) 3 rats indicates that acetaminophen may have an effect at the central and peripheral tissues. 2. Cardiovascular, blood pressure and heart rate (SHR〇, 1, 2, 4 hours) experimental reference Yen TT et al. 1978 published in Life Sci. 22: 359 using three Wistar_0kam〇t weighing 250±20 grams The sputum-derived natural hypertension male (SHR) experimental group had an average systolic blood pressure of 200 ± 20 mmHg and a heart rate of 4 〇〇 26 201018462 ± 30 beats / min. Blood pressure, rate 疋 by the tai 1 cuff method in a temperature-controlled environment) before oral administration of the test substance or carrier 0 hours and 2, 4 hours for indirect recording each time after the interval measurement compared with G, the contraction A drop of 10% or more 10%) or a decrease in heart rate of 2% or more is considered meaningful. ❹

Table 6: Cardiovascular and blood pressure results of rats (SHR 0, 1, 2, 4 hours) Treatment dose group control group % (compared with sputum) Path ____ - ~ ^ 1 hour 2 hours 4 hours Carrier P0 10 1 100 96 90 ml/kg 2 97 100 91 3 90 92 92 flat 96 96 91 ACM- P0 1000 1 78 85 71 Alcohol extraction mg/kg 2 86 89 80 3 89 89 89 flat (84) (88) (80) One-to-one Clonidine P0 0. 1 1 71 67 71 mg/kg 2 95 86 88 3 72 85 69 27 201018462 Flat (79) (79) (76) Table 7: Results of cardiovascular and heart rate (SHR 0, 1, 2, 4 hours) Treatment dose group control group % (compared with 0) Diameter 1 hour 2 hours 4 hours Carrier P0 10 1 87 100 99 ml /kg 2 116 103 107 3 108 104 121 Flat 104 102 109 ACM- P0 1000 1 98 93 95 Alcohol extraction mg/kg 2 81 100 88 3 83 78 92 flat 87 90 92 Clonidine P0 0.1 1 62 97 112 mg/kg 2 84 87 104 3 68 86 78 Ping (71) 90 98

28 201018462 SHR rats with systolic blood pressure of 200±20 mmHg and heart rate of 400±50 mmHg/min were used. Blood pressure is an indirect record at 0:00 and 1, 2, and 4 hours after oral administration of the test substance or vehicle via the vial tail cuff method. When each time after the measurement time point is compared with 0 (see below), its blood pressure drops by 10% or more (210%), or the heart rate decreases by 20% or more (20%), it is considered meaningful. When the carrier was 10 ml/kg 0, 229 mmHg and 403 mmHg times/min were regarded as 100%. ® ACM-Alcohol Extract 1000mg/kg 0 223mmHg and 452 mmHg times/min as 100%.

Clonidine 0.lmg/kg 0 228mmHg and 379 mmHg times/min as 1〇〇%. Table 8: Cardiovascular, such as pressure results of rats (SHR 0, 1, 2, 4 hours) Treatment route Dose group control group % (compared with 0) 1 hour 2 hours 4 hours Carrier P0 10 1 94 97 97 Ml/kg 2 88 97 94 3 94 97 103 Flat 92 97 98 Average 29 201018462 ACM- P0 300 1 111 102 103 Alcohol extraction mg/kg 2 94 84 100 3 112 110 112 Flat 106 99 105 Clonidine P0 0.1 1 86 73 81 mg/kg 2 63 73 90 3 62 68 80 Level (70) (71) (85) Table 9 · Results of cardiovascular and heart rate (SHR 0 > 1 ' 2, 4 hours) Treatment dose Group control group % (compared with 0) diameter 1 hour 2 hours 4 hours carrier P0 10 1 82 85 84 ml / kg 2 88 115 102 3 109 111 119 flat 93 104 102

30 201018462 ACM- P0 300 1 97 96 92 Alcohol extraction mg/kg 2 105 108 98 Substance 3 85 96 82 Flat 96 100 91 Clonidine P0 0. 1 1 77 85 102 mg/kg 2 78 78 100 3 62 94 104 Flat (72) 86 102 SHR rats with systolic blood pressure of 200 ± 20 mmHg and heart rate of 400 ± 50 mmHg/min were used. Blood pressure was administered orally to the test substance via the vial tail cuff method or indirectly recorded at 0 (pre) and 1, 2, and 4 hours after exposure to Zhao. When each time after the measurement time point is compared with 0 (see supplement below), the blood pressure drops by 10% or more (2 φ 10%), or the heart rate decreases by 20% or more 20%) It makes sense. Carrier ACM-Alcohol Extract

Clonidine 10ml/kg 220 220mmHg and 410 mmHg times/min as 100%. At 300 mg/kg 〇 205 mmHg and 446 mmHg times/min were taken as 100%. 0.lmg/kg 235235mmHg and 417 31 201018462 mmHg times/min as 1003⁄4 3. Diet-induced gallbladder, serum (HDL total, total/hdl ratio) experimental reference Schurr PE et al. 1976 in Atherosclerosis Drug Discovery. Plenum, New York, pp. 215-229. Five ICR-derived male mice weighing 22±2 grams were fed high-fat food (g/100g. coconut oil '8; steroids, 1. 〇; cholesteric acid, 0.3; lard, 2; standard food 88.7 ) 7 days to trigger hypercholesterolemia. The test article was orally administered on days 5, 6, and 7 days. After overnight fasting, the serum of each mouse was used to detect the change in total cholesterol (Hall), high density lipoprotein (HDL), and total/HDL. The total serum decreased by 2% or more (220%), or the serum HDL increased by 20% or more (220%), or the total amount/HDL decreased, compared to the vehicle treated with the vehicle. When 40% or more 40%), it is considered meaningful. Table 10: Diet-induced cholesterol in mice (total/HDL, total/HDL ratio) Results Reference route Dose group Total amount HDL Total/HDL Indiv. %Dce Indiv. %Dce Indi v %Dce Vector P0 10 1 361 70 5. 16 32 201018462

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 -- average ACM - alcohol extraction P0 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) P0 300 1 370 66 5. 61 Mg/kg 2 301 65 4. 63 x 3 3 217 74 2. 93 4 379 76 4. 99 5 328 98 3. 35 flat 319 12 76 -4 4. 20 8 both 33 201018462

Hemoglobin (HDL). When the total serum level drops by 2% or more (& 2%), or the bloody HDL increases by 203⁄4 or more (2 20%), or its total/HDL ratio decreases by 4% or more ( 2 40%), it is considered meaningful.

4*D-galactosamine for liver injury in rats. Reference is made to the literature published by Wr〇beij et al., 1998, 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 the injection of galactosamine, and the animals were sacrificed after 24 hours. The amount of alanine alanine transaminase (alt) and aspartate transaminase (AST) was measured by an HITACHI analyzer (Model 7050) according to the best UV method. A decrease in ALT or AST activity compared to vehicle-treated control animals 34 201018462 30% or more (2 30%) is considered protective. Table 11: Experimental results of liver injury induced by galactosamine in rats

35 201018462 Treatment dose group Jinqing serum diameter ALT (X soil SEM) AST (X soil SEM) U/L )ec.% U/L Dec.% Carrier 1 816 1628 PO 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 PO 1000 2 376 532 Fine extraction mg/kg 3 4 596 452 672 524 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 PO 300 2 532 912 3 412 776 mg/ Kg 4 436 652 x 3 5 636 1028 (33) X 5048 (34) 8048 SEM 39.6 73.4

The test article and vehicle were administered orally 0.5 hours and 4 hours and 8 hours before the injection of a single dose of galactosamine (500 mg/kg, IP), and the animals were sacrificed after 24 hours. 36 201018462

Animals and test ALT and AST values. A decrease of 2 30% in ALT and AST was considered significant compared to the vehicle group. 5. The inflammatory test of carrageenan refers to the literature published by Winter CA et al. 1962 on Proc Soc Exp Biol Med. 1 1 1:544. Three Long Evans-derived male or female mice weighing 150 ± 20 grams were fasted overnight before the experiment. The test substance was administered orally by injection of carrageenan (1% intraplantar suspension 0.1 ml) 1 hour before the right hind paw, and hind paw edema was regarded as an inflammation judgment, and the aqueous cells were used after the administration of carrageenan. The organ fullness measurer (25 mm in diameter) was recorded for 3 hours. A decrease in hind paw edema of 30% or more (30%) indicates a significant anti-inflammatory effect. Table 12: Inflammation of the rat carrageenan results of the treatment route Dose J (X 0.01 ml) RP. LP. Diff P0 10 ml/kg 1 194 103 91 2 202 108 94 3 199 104 95 Average 198 105 93 — Winning P0 1000 ng/kg 1 146 101 45 Object 2 147 95 52 3 160 104 56 Average 151 100 51 (45) Aspirin P0 150 1 152 102 50 2 146 102 106 44 3 163 57 Average 154 103 50 (46) Table 13: Inflammation test results of carrageenan rats 37 201018462 Treatment route doses of claw claws (x 0.01 ml) RP LP. Wff m Ρ0 10 ml/kg 1 193 105 88 - 2 198 107 91 3 199 102 97 Average 197 105 92 toe thorns 0 300 qg/kg 1 195 104 91 objects 2 187 103 84 3 196 103 93 average 193 103 89 1 ~~ ΑφίΓΪπ Η) 150 / / kg 1 146 103 43 2 149 101 104 48 3 169 65 155 103 52 The test substance and the carrier were given 1 hour before the injection of carrageenan (1% intraplanar tar suspension oi ml) to the right hind paw (R·P.) of the fasting rats overnight; The left hind paw (L·P.) is not injected. The hind paw edema decreased by 30% or more by 30%), as indicated below, indicating that its acute anti-inflammatory effect is meaningful.

Tumor experiments with homologous melanoma B16-F0 cell lines are based on the literature published by Farrugia CA and Groves MJ. 1959, Anticancer Research 19: 1027-1032 。. Five C57BL/6J male mice without pathogen (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 viable cell strain homologous to C57BL/6J mice (ATCC CRL-6322 '0.2 ml in 0.2.〇 X 1〇5 cells) was subcutaneously injected into the dorsal side of the experimental mice. Treatment was started 24 hours after tumor inoculation, and the test compound was administered as an oral feed daily for 21 days' days when the apparent toxicity symptoms were observed. Mice were monitored for their body weight, tumor size and survival from day 1 to day 22, 2010. The mice were monitored for survival until the end of the 45th day of the study. According to the formula to assess tumor weight (mg) · long (mm [width (mm)

^ J X 0.5, where the specific gravity is assumed to be 丨 and π is 3. Tumor proliferation in compound-treated animals was calculated as tvc (experimental group/control group) χ 1 () 0%; if T/c value was '42% as evidence of significant anti-tumor activity ^ T/c (experimental group/control) The mean survival time of the group of $125% was also considered to be evidence of significant anti-tumor activity.

Table 14. Treatment of abdomen tumor results of homologous melanoma B16-F0 cell line - Route dose group Τ/ς Mean ± SEM No. 1 T/C (%) No. 8 T/C (%) No. 11曰T/C(%) K) 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 2 Delete 100 P0 1000ng/kg 1 0 0 "〇物x21 2 0 0 0 3 0 0 0 4 0 0 14 5 0 0 32 0 100 0±0 0* 9±6 4* Mitanycin IP 2 n®/kg 1 0 0 0 x 6 2 0 0 64 3 0 0 0 4 0 0 68 5 0 0 41 0 100 0+0 0* 34+15 16* Table 15 · Tumor test results of homologous melanoma B16-F0 cell line

^ It diameter dose group tumor weight (mg) and % ΤΛ; mean ± SEM 39 201018462 别第15曰T/C(%) 18曰T/C(%) 22nd 曰T/C(%) m. P0 10 ml/kg 1 211 746 2054 x21 2 657 1597 2870 3 216 669 1419 4 835 2455 3688 5 240 726 1682 432+ 100 1239+ 100 2343 soil 100 131 349 416 ACM-alcohol extract PO 1000 1 49 280 913 mg/ Kg 2 62 630 1545 x 21 3 388 1079 2560 4 148 435 1514 5 229 535 1637 175±62 41 wood 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

40 201018462 119+44 27 wood 391±142 32* 587 soil 25* 260 After 24 hours of tumor cell implantation, the experimental animal vehicle and test substance were administered daily for 21 days. At the same time, the reference compound and mitomycin were administered twice a week in an ip manner for a total of 6 times. Tumor size was measured and recorded twice a week for 22 days. Inhibition of tumor growth was calculated by T/C (experimental group/control group) xl〇〇. The T/C value S 42% was considered to be evidence of significant anti-tumor activity. • Table 16: Tumor test results of homologous melanoma B16-F0 cell line Treatment route Dose group _ weight (mg), mean soil SEM No. 1 8th, 11th, 15th, 18th, 22nd P0 10 rnl/Ί^ 1 21 20 20 21 22 25 x21 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 21.4t0.2 20.8+ 20.2 +0.2 20.4+0.5 22.2tl.O 24.fel.5 0.4 ACM-Linnon P0 1000 1 20 21 21 22 22 23 Object 2 20 19 19 21 22 24 x21 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+ 19.6+0.6 21.0±0.5 21.6+0.5 23.8±0.9 0.5 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±U 22.2t 22. Oil. 1 23.Q+1.0 24 0+1.1 26.3±1.9 0.8 After 24 hours of tumor cell implantation, The experimental animal vehicle and test article were administered daily for a total of 21 days. At the same time, the reference compound and mitomyc i η were administered twice a week in IP mode 41 201018462 for a total of 6 times. The tumor size was recorded twice per four times for 22 days. Student's, st test was used to determine whether the body weight changes of the test compound and the vehicle control group were significantly different. Table 17: Tumor test results of homologous melanoma B16-F0 cell line Treatment time group survival time (曰), + SEM path Do not follow-up process Τ / γ η carrier F0 10 ml / kg 1 2 23 28 _________V \ 70 JX 21 3 30 4 28 5 27 27.2 ± 1.2 100 ACM - alcohol extract P0 1000 mg / kg 1 2 44 31 x 21 3 25 4 42 5 32 34. 8+3. 6 128 Mutomycin IP 2 mg/kg 1 2 45* 28 x 6 3 45a 4 30 5 22 34. 0±4. 7 125 wood a: If the animal is in 45 days After not dying, the survival period is taken as 45 days. The survival of the experimental animals was monitored at the 45-day study period or until the death of the experimental animals. The average survival time of T/C (experimental group/control group) was (4) purely evidence of significant antitumor activity. Discussion: According to the established standardization within the company, Fuping daily oral (Ρ0) to ACM-alcohol extract, according to the following mouse sputum test to produce significant effects: 42 201018462 Rats give central biliary and alkaline synergy 1000 mg/kg; a non-significant synergy was administered at least 300 mg/kg; a non-significant synergist or antagonist acting on peripheral choline nerves was administered at 1 mg/kg (Table 5). In spontaneously hypertensive rats (SH), the systolic blood pressure was reduced at 16 mg/kg (16%, 12%, and 20%, observed at 1, 2, and 4 hours, respectively, vs. 100%). '〇 time), and the heart rate was moderate but not significantly reduced (Tables 6 and 7); φ 300 mg/kg did not cause significant changes in systolic blood pressure and heart rate (Tables 8 and 9). A dose of 1000 mg/kg increased the high-density lipoprotein (HDL, vector control group, more than 39%) in diet-induced mice (Table 1〇); when the HDL/t〇tai ratio decreased significantly to nearly 31%, However, total cholesterol (T〇tai) did not change significantly; the dose of 3〇〇 mg/a kg did not cause a change in the ratio of Total, HDL and HDL/Total. _ After liver injury induced by galactosamine, liver protection was achieved at a dose of 1 mg/kg (3 (44% reduction in ALT and 57% ast reduction compared with the vehicle control group), and 300 mg At a dose of /kg χ3, the sputum decreased by 2% and the AST decreased by 28% (Table u). The anti-inflammatory response of 1000 mg/a catty to the rat paw edema induced by carrageenan (45% inhibition compared with the vehicle control group) (Table 12); the lower level of mg/kg had no significant effect (Table 12) There is a 3% inhibition compared to the carrier, Table 13). The homologous melanoma B16F 〇 cell line of C57BL/6J mice was tested at 丨〇(10)mmol 43 201018462 g/kg dose at 8th, U and u, and prolonged animal survival (Table 16). Days (Tables 14 and 15) with anti-tumor effect 17); animal body weight was not significantly different (Table [0050] ACM, ACM alcohol extract and compound 3 of the present invention used nine groups (10) derived male rats (weight 22 ± 2 g 5 of them. Each animal was given a single-dose of four gasified carbon (cci4, 5 earning oil containing Q i ml / kg 'P0). ACM test substances at 3〇, 1〇〇 and 3〇 At a dose of 〇mg/kg, administered orally after 4 and 8 hours before treatment of the four gasified carbons; then _ alcoholic sputum extract '3 与 and 4 and 8 hours before the treatment of the four gasified carbons Oral administration was repeated daily (twice daily) at a dose of 3 〇〇 and 1 〇〇〇 mg/kg. The animals were sacrificed 24 hours after treatment with four gasified carbon. Alanine transaminase (ALT) and aspartame The value of the aminotransferase (AST) is measured by the HITACHI automatic analyzer (model 7050) in the best UV method. Compared with the carrier group, the 'ALT or AST value drops by 30% or more 30%, which represents the liver. Damage has a protective effect. Results ❹ Table 18: Four-gasified carbon on liver injury in mice Treatment route Dose group ALT AST U/L Dec. % U/L Dec. % Carrier (2% PO 10 ml/kg 1 3936 2056 Tween 80) X 3 2 3456 1856 3 3712 1528 4 2560 1328 5 2968 1696 X 3326 0 1693 0 SEM 250 126 44 201018462 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 Take 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 45 201018462

The ACM, ACM-alcohol tomb enemy, such as a refined extract and compound 3 of the present invention, have been evaluated to have a protective effect on liver damage caused by four-carbonized carbon-induced ICR mice. The ACM test substance was 3 〇 1 after 0.5 hours and 4 hours and 8 hours before the treatment of the four gasified carbon. . Orally administered at a dose of 3 mg/kg. (10) One alcohol extract with 3 〇〇 and round

The mg/kg dose was administered twice (bl:·〇〇Mand 16:〇〇pM) twice before the treatment of the four gasified carbons (bl d·), and the treatment of the four gasified carbon front 开始. 5 hours and 4 hours After 8 hours (total of 5 doses), the degree of liver damage was determined by alanine transaminase (ALT) and aspartate transaminase (AST) in the serum of the treated animals. In the present invention, ACM at a dose of 1 mg/kg X 3 and Compound 3 at a dose of 300 mg/kg x 3 resulted in ALT (46% and 41%) and AST (36% and 33%) compared to vehicle-treated animals. ) significantly decreased. At the same time, ACM-alcohol extracts at doses of 300 and 1000 mg/kg x5 also caused a significant decrease in ALT (36% and 34%) 46 201018462 and AST (25% and 20%). Simultaneous testing of silymarin (100 mg/kg X 3 'IP) showed a significant decrease in ALT (31%) and AST (31%) compared to vehicle-treated animals. The ACM, ACM-alcohol extract and Compound 3 of the present invention have significant hepatoprotective effects in the treatment of a four-carbonized carbon model in mice. The present invention has been described and illustrated in detail so that this invention can be implemented and utilized in the light of the scope of the invention. Those skilled in the art will soon recognize that the present invention can easily achieve the goal and achieve the results and advantages described herein. The cell line 'embryo, animal and its manufacturing process and method are only exemplary. The examples are not intended to limit the scope of the invention. Modifications and other uses of the present invention will occur to those skilled in the art, and such modifications are intended to be included within the scope of the invention and are limited to the scope of the invention. It will be apparent to those skilled in the art that the present invention may be modified and modified without departing from the spirit and scope of the invention. All of the patents & published work described in this specification are the general techniques related to the invention. When each individual published work is carried out and the data is merged one by one, all references to the reference materials are incorporated in the same scope as the patents and publications. The hair styles described herein may be performed without any element or singularity and are not specifically disclosed herein. The nouns and expressions used are for illustrative purposes only and are not intended to be limiting, and are not intended to be construed as a limitation. It is possible that within the scope of the invention of the invention, it is to be understood that the invention may be modified by the preferred embodiments and the features of the invention. Modifications of the present invention are intended to be within the scope of the appended claims. BRIEF DESCRIPTION OF THE DRAWINGS Figure 1 shows the HMBC pairing of Compound 2 of the present invention. Figure 2 shows the compound of the invention. Figure 3 shows the N〇E (nuclear Overhauser effect) of the compounds 4 and 5 of the present invention. Figures 4 (a) - (d) show the test results of the compound 3 of the present invention. Figure 5 (a)-(c) shows the test results of the aqueous extract of ACM (Antrodia mycelia powder). Figure 6 (a)-(f) shows the test results of the Nitrogen extract of Mycorrhiza bovis. Figure 7 (a) - (e) shows the test results of the compound 1 of the present invention. ® [Main component symbol description] None 0 48

Claims (1)

201018462 VII. Patent application scope: 1. A compound with the following formula Wherein X is N or hydrazine; R1 is Cuu alkoxy, (ν1()alkenyloxy or Ca alkynyloxy; R2 is hydrazine, alkyl, C2_lfl alkenyl or C2_le alkynyl; and R3 is free, hydrazine or a hydroxyl group; but when the X system is 0, the r3 system is not. According to the compound of the scope of the application, the "thirty oxy or C2-e- alkynyloxy group. 3. According to the scope of claim 2 a compound in which Ri is substituted with an alkenyloxy group. 4. A compound according to the scope of claim 4, which is "2 isisobutyl. 5. According to the compound of claim 1 It is isobutyl-4-[4-(3-methyl-2-butenyl)phenyl]furan-2,5-dione; 49 201018462 3-isobutyl-4-[4-( 3-methyl-2-butenyloxy)phenyl]-i-t-buto-2, 5-dione; 3-isobutyl-4-[4-(3-methyl-2-buteneoxy) Phenyl]-1 and -indole-polyol-2, 5-dione; 汔45^-1-hydroxy-3-isobutyl-4-[4-(3-indolyl-2 -butenyloxy)phenyl]pyrrolidine-2,5-dione; or -1-hydroxy-3-isobutyl-4-[4-(3-methyl-2-butene) Oxy)phenyl] 0 is more specific than oxalate-2,5-dione. A mixture of the mycelium of A. angustifolia, which comprises the compound 3-isobutyl-4-[4-(3-mercapto-2-butoxy)phenyl]furan-2, 5-dione, 3-iso Butyl-4-[4-(3-methyl-2-butoxy)phenyl]-1 in pyrrole-2,5-dione, 3-isobutyl-4-[4-(3- Methyl-2-butenyloxy)phenyl]-1 and pyrrol-1-ol-2,5-dione, (^^-buhydroxy-3-isobutyl-4-[4-( 3-methyl-2-butenyloxy)phenyl]pyrrolidine-2,5-dione, or hydroxy-3-isobutyl-4-[4-(3-methyl-2-buteneoxy) Phenyl]-pyrrolidine-2, 5-dione, which is prepared from water or an organic solvent extract of Antrodia camphorata mycelium. 7. A mixture according to claim 6 of the patent application, wherein the tumor is selected from the group consisting of Tumors of cells or tissues of the liver, intestines, bones, blood, lymph, and breasts. 8. Compositions, including compounds of the formula 201018462 Wherein X is N or 0; R1 is Cl-1° alkoxy, Cho alkenyloxy or C2-i decynyloxy; R2 is hydrazine, Ci-io phenotype, C2_1D dilute or c2-ie fast radical; And Ra is no, sputum or warp group; but when X is sputum, then R3 is not, wherein the composition does not contain for reducing systolic blood pressure, increasing high density lipoprotein, central nervous choline stimulating effect, liver protection Or anti-inflammatory use. 9. The composition according to item 8 of the patent application wherein the compound is 3-isobutyl-4-[4-(3-methyl-2-butenyloxy)phenyl]furan-2,5-diindole 3-isobutyl-4-[4-(3-methyl-2-butenyloxy)phenylpyrrole-2,5-dione; 3-isobutyl-4-[4-(3- Base 2 -butenyloxy)phenyl b 1)5Γ_pyrrole-丨-ol-2, 5-diindole; said <hydroxy-3-isobutyl_4_[4_(3methyl_2 Alkenyloxy)phenyl]pyrrolidine-2,5-dione; or 51 201018462 tl-hydroxy-3_isobutyl-4_[4_(3-methyl-2-butenyloxy)phenyl] &quot Biluo Hyun - 2,5 - II. 10. The composition according to item 8 of the patent application, wherein the compound is 3-isobutyl-4-[4-(3-methyl-2-butoxy)phenyl]_1#_pyrrole_2, 5_dione; or 3-isobutyl-4-[4-(3-methyl-2-butoxy)phenyl]pyrrole-4-ol-2, 5-dione. 11. The composition according to item 8 of the patent application, which has antitumor activity. 12. The composition according to claim 8 wherein the tumor is selected from the group consisting of tumors of cells or tissues of the liver, intestine, bone, blood, lymph and breast. 13. A mycelium of Antrodia camphorata for use in anti-tumor, comprising the compound 3-isobutyl-4-[4-(3-methyl-2-butoxy)phenyl]furan-2, 5 - Di-, 3-isobutyl-4-[4-(3-methyl-2-butenyloxy)phenyl]-1#-pyrrole-2,5-di-, isobutyl-4- [4-(3-Methyl-2-butenyloxy)phenyl]-1#-pyrrole-bupropan-2,5-dione, 弋^9-di-hydroxy-_3-isobutyl 4-[4-(3-methyl_2-butoxyoxy)phenyl]'* is slightly more entertaining (-2,5-dioxime, or <^^>>*^-1- Peryl-3-isobutyl-4-[4-(3-methyl-2-butenyloxy)phenyl]pyrrolidine-2, 5-dioxime. 14. According to the scope of claim 13 Mycelium, wherein the tumor is selected from the group consisting of liver, intestine, bone, blood, lymph, and tumors of cells or tissues of the breast.