The Protection of Anthrodia camphorata against Acute Hepatotoxicity of Alcohol in Rats

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1 177 Journl of Food nd Drug Anlysis, Vol. 11, No. 3, 23, Pges The Protetion of Anthrodi mphort ginst Aute Heptotoxiity of Alohol in Rts YU-YUN DAI, CHENG-HUNG CHUANG, CHIN-CHUAN TSAI, HOK-MAN SIO, SHI-CHENG HUANG, JIN-CHU CHEN AND MIAO-LIN HU* Deprtment of Food Siene, Ntionl Chung-Hsing University, No.25, Guogung Rd., South Distrit, Tihung City 42, Tiwn (R.O.C.) (Reeived: Septemer 19, 22; Aepted: Jnury 22, 23) ABSTRACT Antrodi mphort is unique mushroom of Tiwn nd hs een used s folk mediine for protetion ginst liver dmge indued y lohol intoxition. However, no report hs een presented in this respet. In this rt study, we exmined whether the myelium nd spororp of Antrodi mphort protet ginst ute liver dmge indued y ethnol (EtOH). Rts were orlly dministered with myelium nd spororp of Antrodi mphort for 9 dys efore EtOH hllenge (5.5 g/kg ody wt., i.p.). Rts were divided into eight s (A-H) nd exept for s A nd H, ll rts were injeted with lohol. A: Control; B: EtOH ontrol: C: Silymrin (25 mg/kg w., p.o.); D:.5 g myelium/kg; E: 1. g myelium/kg; F:.5 g spororp/kg; G: 1. g spororp/kg; nd H: 1. g myelium/kg. The results showed tht EtOH dministrtion mrkedly inresed the tivities of glutmte-pyruvte minotrnsferse (GPT) nd glutmte-oxloette minotrnsferse (GOT). Both myelium nd spororp of Antrodi mphort signifintly deresed the tivity of GOT nd GPT, ut the effets were not dose-dependent. Myelium nd spororp of Antrodi mphort lso signifintly nd dose-dependently deresed lipid peroxidtion (mesured s TBARS) indued y EtOH. EtOH tretment signifintly inresed the tivities of hepti superoxide dismutse (SOD) nd tlse, ut did not signifintly ffet the tivity of glutthione peroxidse. Pre-tretment with either the myelium or the spororp ompletely prevented the rise in the tivity of SOD nd tlse. The histopthologil exmintion reveled tht oth myelium nd spororp mrkedly proteted ginst lipid vuole umultion nd hydropi degenertion of heptoytes indued y EtOH. Thus, the present results demonstrted tht oth myelium nd spororp of Antrodi mphort protet ginst ute liver dmge indued y EtOH. In ddition, rts fed 1. g myelium without EtOH tretment produed no oservle toxiity during the experimentl period. Key words: lohol toxiity, Anthrodi mphort, heptoprotetion, oxidtive dmge INTRODUCTION Liver is n importnt orgn for detoxifition nd metolism, nd it hs good repiring pility. Common dmge used y non-persistent or mild toxiity n e repired through ertin mehnisms. However, liver dmge indued y persistent lohol overdose or virus ttk my use hroni heptitis, irrhosis, nd even heptom to deth. In reent yers, the deth rte of mle heptom is the highest mong the ten mjor uses of deth y ner in Tiwn (1). Reports hve desried tht liver dmge used y lohol inresed with the dosge, nd the severity of dmge is different mong gender, speies, nd genes. For exmple, out 5% of Orientls lk the importnt enzyme for lohol metolism, ldehyde dehydrogense. Therefore, their tolerne of lohol is poor nd liver dmge ours esily (2-4). Antrodi mphorte, nmed A. innmome previously, is muh different from the ommon Gnoderm speies euse it hs strong mphor-like rom. Its shpe is like plte or ell. It only grows on the inner wlls of hollow Cinnmomum knehire hy woods in mountin res etween ltitudes 2 to 2 m. Cinnmomum knehire hy is n evergreen rodlef * Author for orrespondene. Tel: ; Fx: ; E-mil:mlhuhu@drgon.nhu.edu.tw ror with hevy rom nd it ould e used s good nti-inset mteril. Fungi nnot grow on it exept Antrodi mphorte whih is then treted s tresure in the ountryside. For long time, the ntives in Tiwn hve found tht liver dmge used y lohol overdose n e ured y drinking Antrodi mphorte te or holding smll piee of it in the mouth. In ddition, the pility of detoxifition, nti-ner, lohol relief, nd nti-inflmmtory is often herd (7). Therefore, it hs eome the most expensive wild fungus in the mrket, nd it will e one of the most importnt helth foods. Go hs reported studies of triterpene ompound B isolted from Antrodi mphorte (8). It ws found tht suh ompound n redue lood GPT level in mie whih hve ute liver normlity indued y tetrhloride methne. Chen reported tht the methnol extrt from Antrodi mphorte hs 3% of triterpoids whih is muh higher thn the extrt from ommon Gnoderm speies (3%) (9). Antrodi mphorte tstes more itter thn Gnoderm, possily due to the rihness of sterols nd triterpoids of multiple oxidized types. Aording to report y Hung nd ollegues, the ntioxidtive pility of Antrodi mphorte is s good s BHA (utylted hydroxynisole) (1). Therefore, it is importnt to study whether Antrodi mphorte hs the sme liver protetion pili-

2 178 Journl of Food nd Drug Anlysis, Vol. 11, No. 3, 23 ty. However, studies relted to liver-protetive effets of Antrodi mphorte ginst ethnol indued heptitis re few. The purpose of this reserh ws to study the protetive effets of myelium nd spororp of Anthrodi mphort ginst ute liver dmge indued y lohol. I. Mteril MATERIALS AND METHODS 1. The myelium ws ultured from Antrodi mphort CCRC 35398, whih ws purhsed from the Food Industry Reserh nd Development Institute, t Grpe King Biotehnology Reserh Center using potto dextrose gr (PDA) followed y deep fermenttion method. The spororp of wild Antrodi mphort olleted from mountin res in Tiwn ws provided y Grpe King Biotehnology Reserh Center. Its polyshride ontents hve een determined s 1.7% in myelium nd 6 to 8% in spororp. Both myelium nd spororp were grounded to powder nd then prepred with sline to different onentrtions of suspension for orl dministrtion to rts. 2. Silymrin, the extrt of Silyum mrinum purhsed from Aldrih Chemil Co., In. (Milwukee, WI, USA), ws used s the positive ontrol in this reserh. Its min tive ingredient is polyphenol siliinin, whih is onsidered s n effetive liver-protetive gent euse it hs protetive effet on liver dmge indued y mny drugs suh s tetrhloride methne nd etminophen. It hs een used s liver proteting gent in the US nd Europe for 2 to 3 yers. It is lso often used s ontrols for omprisons in reserh. Therefore, silymrin ws used s the drug ontrol in our reserh. The dose for rts is usully 2 to 4 mg/kg (11-13). II. Experimentl Design 1. Animls: Experimentl nimls in this reserh were S.D. white mle rts purhsed from the Ntionl Lortory Animl Center weighing 18 to 2 g. The feeding ondition ws d liitum using L 51 stndrd diet (Purin Mills, St. Louis, MO) nd deionized wter. Room temperture ws set t 22 ± 2ûC. Humidity ws set t 65 ± 5%. Light exposure nd drk were oth set t 12 hr. 2. In this reserh, high nd low doses were used ording to the instrution in the dried produt of fermented myelium (Antrodi mphorte King) produed y the Grpe King Corportion. It is 3 to 5 pills (out.42 g per pill) eh time nd 3 times per dy, tht is, 9 to 15 pills dily. Therefore, 9 pills were used s the low dose nd 15 pills s the high dose. Bsed on ody surfe res (14), it is out.34 g/kg (low dose) nd.57 g/kg (high dose) dily for rts. To lulte onveniently, out 1.5 to 1.7 times of dosge ws used in this reserh, tht is,.5 g/kg (low dose) nd 1. g/kg (high dose). 3. Grouping: Rts were dministered (p.o.) with myelium or spororp of Antrodi mphorte for 9 onseutive dys t 9.m. There were 8 s with 7 rts in eh : Group A (ontrol) ws dministered with the sme mount of sline insted of Antrodi mphorte; Group B (liver dmged) ws dministered with the sme mount of sline insted of Antrodi mphorte; Group C (positive ontrol) ws dministered with silymrin (2 mg/kg) to reple Antrodi mphorte; Group D ws dministered with low dose (.5 g/kg) of myelium of Antrodi mphorte; Group E ws dministered with high dose (1 g/kg) of myelium of Antrodi mphorte; Group F ws dministered with low dose (.5 g/kg) of spororp of Antrodi mphorte; Group G ws dministered with high dose (1 g/kg) of spororp of Antrodi mphorte; Group H ws dministered with high dose (1 g/kg) of myelium of Antrodi mphorte. 4. On the 9th dy, 6 hr fter dministrtion (3 p.m.), ethnol ws injeted to the dominl vity of rts (Sline ws injeted insted of ethnol in Group A nd H.). The design of ethnol injetion suh s dosge nd timing ws dpted from the method desried y Zhng et l. (15). Tht is, 2% ethnol solution ws used for injetion (5.5 g/kg). Eighteen hr fter ethnol injetion, rts were put unonsious using ethyl ether nd lood smples for iohemil nlysis were then tken from rotid. In ddition, liver smples were tken for determintion of lipid peroxidtion, nlysis of protein ronyl s, determintion of ntioxidnt enzymes, nd histopthologil exmintion. III. Anlytil Methods (I) The solute nd reltive weight hnge of rt liver nd kidney Asolute weights re tul weights of rt liver nd kidney. Reltive weights re weights of rt liver nd kidney reltive to 1 g weight of rt. (II) Determintion of serum iohemil indexes Blood smples tken from til nd nek of rts were entrifuged t 3 g per minute for 1 min to otin serum smples. The levels of GOT (glutmte-pyruvte minotrnsferse), GPT (glutmte-oxloette minotrnsferse), nd serum gluose were determined y utomti iohemil nlyzer (Rohe COBAS, Mir Plus). The stndrd method from IFCC (16, 17) ws used for determintion. (III) Determintion of liver protein ontents Bio-Rd Protein Assy kits were used for this nlysis. The Coomssie Brillint lue regent formed lue omplex with protein, then the sorne t 59 nm ws mesured y Hithi U-2 Spetrophotometer. Bovine

3 179 Journl of Food nd Drug Anlysis, Vol. 11, No. 3, 23 serum lumin ws used s stndrds. (IV) Determintion of liver lipid peroxidtion Aording to the method y Buege nd Aust (18), olorimetri method tht mesures the retion produt of thiorituri id (TBA) with ldehydes suh s mlondildehyde (MDA) formed y lipid peroxidtion ws used for this nlysis. The vlue ws nmed TBARS. Liver tissues were homogenized with 9 volumes of EDTA solution, nd 1.5 µl of 5 mm BHT ws dded to 1 ml of this mixture to void the interferene used y MDA generted y strong id nd het during this nlytil proess. For preipitting the protein, 2 ml of 7.5% TCA ws dded followed y ie thing for 5 min. After entrifugtion, 1 ml of.7% TBA ws dded to 2 ml of superntnt followed y heting in oiling wter for 1 min. After ooling, equl mount of n-utnol ws dded to extrt MDA followed y entrifugtion t 1, g for 1 min. Finlly, fluoresene spetrophotometer (Ex: 515 nm; Em: 555 nm) ws used for the quntittive nlysis. MDA generted y 1,1,3,3-tetrmethoxypropne (TMP) with 1 N H 2 SO 4 ws used s stndrds. (V) Anlysis of liver protein ronyl s Aording to the method y Reznik nd ollegues (19), DNPH ws used s the regent for this nlysis. One hundred µl of homogenized solution from liver tissues ws mixed with.5 ml of 1 mm DNPH (in 2 N HCl) then inuted t room temperture in drk for 1 hr with shking one every 15 min. After dding.6 ml of 2% TCA, this mixture ws entrifuged t 1, g for 1 min to preipitte protein. After disrding the superntnt, the preipitte ws wshed 3 times with ethnol-ethyl ette (1:1, v/v) to remove the residul DNPH. The preipitte ws reonstituted with 1 ml of gunidine-hcl (ph 2.3) then inuted in wter th t 37ûC for 1 hr. After entrifuged t 12, g for 15 min, the sorne t 37 nm ws mesured using Hithi U-2 spetrophotometer. E 37 (molr oeffiient) = M -1 m -1 ws used to lulte the ontent of protein ronyl s. The result ws expressed s nmoles ronyl s/g liver. (VI) Determintion of liver ntioxidnt enzymes 1. Ctlse Aording to the method y Cohen nd ollegues (2), 25 µl of homogenized solution from liver tissues ws mixed with 975 µl of 6 mm H 2 O 2 /5mM potssium phosphte, ph 7.. The derese of A 24 nm in 2 min ws reorded. The enzyme tivity ws lulted y E 24 = 43.6 M -1 m Superoxide dismutse (SOD) Aording to the method y Mrklund nd Mrklund (21), 1 µl of homogenized solution from liver tissues ws mixed with 965 µl of 1 mm Tris-HCl ontining 2 mm diethylenetriminepenteti id, ph 8.2 nd 25 µl of 8 mm pryogllol (in 3 mm HCl). The hnge of A 412 nm in 3 min ws reorded. Deionized wter ws used s lnk. The tivity of 1 unit SOD ws defined s 5% of inhiited retion. 3. GSH peroxidse (GSH-Px) Aording to the method y Lwrene nd Burk (22), 25 µl of homogenized solution from liver tissues ws mixed with 925 µl of retion solution (1 mm EDTA, 1 mm NN 3, 1 U/ml GSH-Rd, 1 mm GSH, nd 1 mm potssium phosphte, ph 7.4) nd 25 µl of 6 mm β- NADPH (in.5% NHCO 3 ). Finlly, 25 µl of 1 mm H 2 O 2 ws dded to initite the retion. The hnge of A 34 nm in 3 min ws mesured t 37ûC. The derese of β-nadph ws used to lulte the tivity of this enzyme (E 34 = M -1 m -1 ). (VII) Histopthologil exmintion A smll piee of liver ws ut from the sme lotion on liver loe in 1% formlin then put in n emedding ox. After dehydrtion overnight, this piee ws emedded with prffin t 2ûC. A 5 µm thik setion ws slied then put in n oven to dry nd fix the prffin. Finlly, the setion ws stined with Hemtoxylin-Eosin followed y mirosopi exmintion. (VIII) Sttistil nlysis The experimentl dt were indited y men ± S.D., nd were nlyzed y n nlysis of vrine using ANOVA (SAS). When the F vlue is signifint, the signifine of differene etween s ws determined y Dunn s test. Signifint differene ws ssumed for p <.5. RESULTS AND DISCUSSION I. The Asolute nd Reltive Weights of Rt Liver nd Kidney The inrese of solute nd reltive orgn weights is sensitive prmeter for toxiity, ut it is proly retion of orgn to dpt to the toxi sustne. Most sustnes tht dmge the liver will hnge the struture of smooth endoplsmi retiulum, derese the ontent of ellulr pigments, inrese the ontent of sturted yl side hins, nd use the hyperplsi nd firosis of ollgen. Finlly, the weight inreses (23). In this reserh, the solute weights of liver nd kidney tissues were not signifintly different mong ll s (Tle 1). The reltive

4 18 Journl of Food nd Drug Anlysis, Vol. 11, No. 3, 23 weights lso were not signifintly different. These results showed tht no signifint dpttion or dmge ourred under the dmge indued y ute ethnol intoxition. II. Serum Biohemil Indexes When the liver ell is dmged, the GOT nd GPT in liver will e relesed to serum. Therefore, levels of GOT nd GPT re the most ommonly used iohemil indexes for evluting the dmge of liver (24). Aording to the iohemil indexes (Figure 1), the dministrtion of ethnol used the GOT nd GPT of liver-dmged (Group B) 233% nd 37% higher thn ontrol (Group A), respetively (p <.1). Compred with Group B, ll experimentl s (Group C to G) hd mrkedly deresed GOP nd GPT (p <.5). However, there ws no signifint differene mong Group C to G. On GOT, high dose of myelium nd spororp ws etter thn low dose, ut the differene ws not signifint. On GPT, there ws no differene etween high nd low doses. GOT nd GPT of the dministered only with high dose of myelium of Antrodi mphorte (Group H) were not inresed, inditing tht the myelium is not toxi to liver. Insulin seretion indued y gluose is inhiited during the metolism of ethnol, nd susequently uses the disorder of gluose metolism suh s derese of gluose tolerne. To exmine the effet of Antrodi mphorte ginst the disorder of gluose metolism indued y ethnol, the serum gluose from ll s ws mesured. Results showed tht serum gluose inresed mrkedly in the ethnol injeted (Group B or liver dmged ). Both myelium nd spororp of Antrodi mphorte n signifintly inhiit the inrese of gluose, ut there ws no differene mong doses. These results proved tht ethnol uses the disorder of gluose metolism, nd demonstrted tht low dose of myelium nd spororp of Antrodi mphorte n signifintly prevent the disorder of gluose metolism. III. Liver Lipid Peroxidtion Mny reports hve desried tht lipid peroxidtion of liver ell memrne used y retive oxygen speies (ROS) is the min reson of liver dmge indued y ethnol (25-34). Tum et l. hve shown tht rt liver produed lrge mounts of MDA nd etldehyde fter orl dministrtion with ethnol (35). In this reserh, liver TBARS inresed mrkedly fter rts were injeted with ethnol (Figure 3-1). However, orlly dministrtion with either high dose or low dose of myelium nd spororp of Antrodi mphorte n ompletely inhiit lipid peroxidtion indued y ethnol. This effet my e due to the lerne of free rdils produed during the metolism of ethnol y the ntioxidtive mteril in Antrodi mphorte. In ddition, the liver TBARS of rts in Group H (the dministered only with high dose of myelium of Antrodi mphorte) did not inrese, inditing tht myelium itself does not use lipid peroxidtion. IV. Liver Protein Cronyl Groups GOT (U/L) In ddition to lipid peroxidtion, tive oxygen Tle 1. Effets of myelium nd spororp of Antrodi mphort on orgn weights of rts injeted with lohol 1 Orgn solute weights (g) Group Liver Kidney A: ontrol (norml sline) 1.5 ± ±.24 B: lohol ontrol 1.6 ± ±.21 C: silymrin (2mg/kg) 1.8 ± ±.17 D: myelium (.5g/kg) 11.4 ± ±.19 E: myelium (1.g/kg) 1.9 ± ±.25 F: spororp(.5g/kg) 11.7 ± ±.25 G: spororp (1.g/kg) 11.3 ± ±.2 H: myelium (1.g/kg) 1.3 ± ±.3 1: Rts were orlly dministered with the myelium or spororp of Antrodi mphort for 9 dys efore injetion (i.p.) with lohol (5.5 g/kg ody wt.). Rts exept s A nd H were injeted with lohol. Vlues re mens ± SD of 7 rts. No signifint differenes in either liver or kidney weights existed mong the vrious s (p <.5). GPT (U/L) Figure 1. Serum levels of GOT nd GPT in rts orlly dministered with the myelium or spororp of Antrodi mphort for 9 dys efore injetion (i.p.) with lohol (5.5 g/kg ody wt.). Rts exept s A nd H were injeted with lohol. A: ontrol (sline, p.o.); B: lohol ontrol; C: silymrin (2 mg/kg, p.o.); D: myelium (.5 g/kg); E: myelium (1 g/kg); F: spororp (.5 g/kg); G: spororp (1. g/kg); H: myelium (1. g/kg) ontrol. Vlues (mens ± SD of 7 rts) not shring ommon supersript re signifintly different (p <.5).

5 181 Journl of Food nd Drug Anlysis, Vol. 11, No. 3, 23 moleules indued y ethnol will use protein oxidtion (36). Aording to previous linil reports, the ontents of lipid peroxidtion produts (suh s onjugted dienes, 4-hydroxynonenl, MDA, nd F 2 -isoprostnes) nd protein ronyl s (n index of oxidtive dmge to protein) in the liver ffeted y loholi liver diseses re higher thn norml individuls (37-4). Additionl reports indited tht the lipid peroxidtion produts, 4-hydroxynonenl nd MDA, use modifitions to proteins nd the prodution of protein dduts (41, 42). Our reserh lso showed tht injetion of ethnol to rts mrkedly inresed protein ronyl s (Figure 3-2). Although ronyl ontents of Group C to G were ll lower thn Group B (the liver-dmged ), only those of the dministered with high dose of myelium of Antrodi mphorte (Group E) were signifintly different. There were no differenes etween ontrol (Group A) nd the dministered with high dose of myelium of Antrodi mphorte without injetion of ethnol (Group H). These results show tht myelium of Antrodi mphorte hs etter effet to inhiit protein dmge indued y ethnol thn spororp. Menwhile, high doses of myelium of Antrodi mphorte does not use protein dmge. 1). Silymrin s well s myelium nd spororp of Antrodi mphorte resumed the SOD tivity, tht is, they ompletely inhiited the inrese of SOD. Therefore, the ntioxidtive pility in ll experimentl s ompletely inhiited the SOD indued y ethnol, whih my e the reson why the inhiitory effet of myelium nd spororp of Antrodi mphorte ws not dose-dependent. When the SOD tivity is tively expressed, superoxide nion will trnsform to H 2 O 2 nd dditionlly tivte the expression of tlse tivity. Our reserh lso showed tht ethnol injetion inresed the tlse tivity mrkedly (Group B). Administrtion of silymrin s well s myelium nd spororp of Antrodi mphorte inhiited the inrese of tlse tivity, ut only Group E nd F were signifintly different from Group B (the liverdmged ). However, s dministered with high or low dose of myelium nd spororp were not signifintly different from ontrol (Group A) (Figure 4-2), inditing tht the ntioxidtive pility in ll experimentl s hs ompletely inhiited tlse tivity 3 1. V. Chnges of Liver Antioxidnt Enzymes Ctlse, superoxide dismutse (SOD), nd glutthione peroxidse (GSH-Px) re the mjor ntioxidnt enzymes in liver. Previous rtiles reported tht loholi liver dmge indues the expression of SOD (35, 43). It my e feedk retion of liver ells under oxidtive pressure. Our reserh lso proved tht the Cu/ZnSOD tivity inresed mrkedly in the liver dmged (Group B) (Figure 4- Gluose (mg/dl) TBARS (n moles/g liver) Cronyl n mole/g liver Figure 2. Serum levels of gluose in rts orlly dministered with the myelium or spororp of Antrodi mphort for 9 dys efore injetion (i.p.) with lohol (5.5 g/kg ody wt.). Rts exept s A nd H were injeted with lohol. A: ontrol (sline, p.o.); B: lohol ontrol; C: silymrin (2 mg/kg, p.o.); D: myelium (.5 g/kg); E: myelium (1 g/kg); F: spororp (.5 g/kg); G: spororp (1. g/kg); H: myelium (1. g/kg) ontrol. Vlues (mens ± SD of 7 rts) not shring ommon supersript re signifintly different (p <.5). Figure 3. Hepti levels of thiorituri id-retive sustnes nd protein ronyls in rts orlly dministered with the myelium or spororp of Antrodi mphort for 9 dys efore injetion (i.p.) with lohol (5.5 g/kg ody wt.). Rts exept s A nd H were injeted with lohol. A: ontrol (sline, p.o.); B: lohol ontrol; C: silymrin (2 mg/kg, p.o.); D: myelium (.5 g/kg); E: myelium (1 g/kg); F: spororp (.5 g/kg); G: spororp (1. g/kg); H: myelium (1. g/kg) ontrol. Vlues (mens ± SD of 7 rts) not shring ommon supersript re signifintly different (p <.5).

6 182 Journl of Food nd Drug Anlysis, Vol. 11, No. 3, 23 indued y ethnol. On the tivity of GSH-Px, the injetion of ethnol signifintly deresed the tivity in Group B, ut the effet ws not signifint (Figure 4-3). This result is onsistent with the result reported y Jrvelinen et l. (44). The prole reson is tht the H 2 O 2 produed during ethnol metolism is lered y tlse, so tht H 2 O 2 hs less effet on GSH-Px. This n lso explin why the GSH-Px tivity is not signifintly different mong ll s. Bsed on results from these ntioxidnt enzymes, Antrodi mphorte n inhiit SOD nd tlse ut not GSH-Px tivities indued y ethnol. U/mg prot. µ mol/min/mg prot. µ mol/min/mg prot SOD Ctlse GSH-Px Figure 4. Hepti levels of ntioxidnt enzymes (tlse, SOD, GSH-Px) in rts orlly dministered with the myelium or spororp of Antrodi mphort for 9 dys efore injetion (i.p.) with lohol (5.5 g/kg ody wt.). Rts exept s A nd H were injeted with lohol. A: ontrol (sline, p.o.); B: lohol ontrol; C: silymrin (2 mg/kg, p.o.); D: myelium (.5 g/kg); E: myelium (1 g/kg); F: spororp (.5 g/kg); G: spororp (1. g/kg); H: myelium (1. g/kg) ontrol. Vlues (mens ± SD of 7 rts) not shring ommon supersript re signifintly different (p <.5). VI. Histopthologil Exmintion As shown in Figure 5-1, the ell nd ell plte from liver tissue smple of the ontrol (Group A) hve intt struture, nd the oundry etween ells is ler. Strutures inside the ells re len without impurities nd droplets. Both ell plte nd sinusoid re entripetl from the entrl vein. Infiltrtion of inflmmtory ells does not exist in the entrl venous re. However, the ethnoldmged (Group B) hs ovious pthologil struture hnges. Figure 5-2 shows tht ells ner the entrl venous re re full of llooning degenertion nd ftty droplets, nd look like shiny droplet. These results re onsistent with findings reported y Korsrud et l. (45). In ddition, most oundries etween ells re lurred nd some even disppered to eome homogenized. Inflmmtory retion of lymphoid infiltrtion ws oserved in the entrl venous re. Hyperplsi of Kupffer ells, metplsi of liver plte strutures, nd disontinuousness of sinusoid strutures re lso oserved. Inner spe is full of ell deris nd wste. The nulei of some liver ells re swelled. Multiple nulei nd over-stin re lso oserved. Some ells re neroti (red re). Signifint liver-protetive effet ws found in the silymrin (Group C, Figure 5-3). Its liver plte nd ell struture were intt, nd the oundry etween ells ws ler. The only pthologil hnge ws tht shiny smll ftty droplets were still visile in liver ells. The dministered with high dose of myelium of Antrodi mphorte (Group E, Figure 5-4) lso showed some reovery effets. The ell fusion ondition ws improved, nd the mount of smll ftty droplets deresed. The dministered with high doses of spororp of Antrodi mphorte (Group G, Figure 5-5) gve similr results. Although the reovery effets of ll s dministered with Antrodi mphorte were not s good s the dministered with silymrin, the liver dmge indued y ethnol improved mrkedly nd the effet ws dose-dependent (dt not shown). The liver struture of the dministered only with high doses of myelium (Group H, Figure 5-6) ws norml, inditing tht myelium of Antrodi mphorte does not use liver dmge. CONCLUSION The results of serum GOT, GPT, nd gluose, liver TBARS, SOD, tlse, nd protein ronyl s, nd histopthologil exmintion illustrted tht myelium nd spororp of Antrodi mphorte hve good liver-protetive effets under the mode of single ethnol injetion. Presumly, these liver-protetive effets re relted to its ntioxidtive retions, though the tive ingredient hs not een identified. Further reserh relted to the effets of myelium nd spororp of Antrodi mphorte on hroni loholi liver dmge will e ontinued, so tht the pplition of Antrodi mphorte n e expnded.

7 183 Journl of Food nd Drug Anlysis, Vol. 11, No. 3, 23 (A) (D) (B) (E) (C) (F) Figure 5. H & E stin under light mirosope (2X) of liver tissues in rts orlly dministered with the myelium or spororp of Antrodi mphort for 9 dys efore injetion (i.p.) with lohol (5.5 g/kg ody wt.). 1: Blnk (sline, p.o., without lohol, i.p.); 2: Alohol ontrol (sline, p.o., lohol, i.p.); 3: Silymrin (2 mg/kg, p.o.) with lohol, i.p.; 4: Myelium (1 g/kg) with lohol, i.p.; 5: Spororp (1. g/kg) with lohol, i.p.; 6: Myelium (1. g/kg, p.o.) without lohol i.p. ACKNOWLEDGEMENTS We thnk Mr. Shu-Meng Lin for his trnsltion. REFERENCES 1. Deprtment of Helth, Exeutive Yun, ROC Sttistis of Puli Helth in Tiwn Are, ROC. pp Lieer, C. S Ethnol metolism, irrhosis nd loholism. Clin. Chem. At. 257: Lieer, C. S Hepti nd other medil disorders of loholism: from pthogenesis to tretment. J. Stud. Alohol. 59: Brody, T Nutritionl iohemistry. 2nd ed. pp.

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