WO2008049265A1

WO2008049265A1 - Compositions for providing hepatoprotective effect

Info

Publication number: WO2008049265A1
Application number: PCT/CN2006/002828
Authority: WO
Inventors: Hattori Masao; Chia Chin Sheu
Original assignee: Simpson Biotech Co., Ltd.
Priority date: 2006-10-23
Filing date: 2006-10-23
Publication date: 2008-05-02
Also published as: CN101553238A

Abstract

This invention relates to a composition for injection comprising a neutral polysaccharide of Antrodia camphorata having characteristics as follows: (a) appearance: colorless and shapeless powder, (b) pH: neutral, (c) molecular weight: 899.5~1670.5 kDa determined by HPLC as shown in (4), (d) rotatory power: (α) D +115.0 º (c = 0.4433, H2O), (e) intrinsic viscosity: (ŋ) = 0.03971~0.06255 dl ·g-1, (f) specific heat Cp: 0.2536~0.3995 Cal/g · ºC, (g) IR spectrum: as shown in (5), (h) 1H-NMR spectrum: as shown in (6), (i) 13C-NMR spectrum: as shown in (7), and (j) GC-MS analysis: as shown in (8). This invention also relates to a composition for oral administering comprising a neutral polysaccharide of Antrodia camphorata having characteristics (a)-(j) as above.

Description

COMPOSITIONS FOR PROVIDING HEPATOPROTECTIVE

EFFECT

FIELD OFTHE INVENTION

This invention relates to compositions for injection and oral administering.

DESCRIPTION OF PRIOR ART

Antrodia camphorata (Chinese name, niu-chang-chih or niu-chang-ku) is a new species of the genus Antrodia (family poly-poraceae, Aphyllophorales) that is parasitic on the inner cavity of the endemic species cinnamomum Kanehirai Hey. It is endangered species in Taiwan. The fruit body of Antrodia camphorata is perennial and has a strong smell. It differs a lot from general reishi mushroom in its plate-shaped or bell-shaped appearance. The plate-shaped one is orange red (yellow) with ostioles all over its surface and has light yellow white phellem in bottom layer. It grows by adhering phellem to the inner wall inside a hollow Antrodia camphorata. The bell-shaped one also shows orange (yellow) color in fruit body layer (bell surface) that is completely filled with ostioles inside, which are, spores of bitter taste in orange red for fresh state and in orange brown or brown afterward. Bell body is a shell that appears in dark green brown color. The spores look smooth and transparent in slightly curved column shape under the investigation by microscope.

Antrodia camphorata is traditionally used for treatment of toxication caused by food, alcohol or drugs, as well as diarrhea, abdominal pain, hypertension, skin itching and cancer (Shen et al., 2004, FEMS Microbiol. Lett., 231: 137-143). In the past, phytochemical investigations have resulted in the isolation of a series of new steroid acids, triterpene acids and polysaccharides (Lieu et al., 2004, Toxicol. Appl. Pharmacol. 201 :186-193).

Polysaccharides are common structural and storage polymers in living organisms, representing more than 75 % of the dry weight of plants. Compositional analysis of glycoconjugates is important in structural studies of these compounds. Polysaccharides are potentially useful, biologically active ingredients for pharmaceutical uses due to a variety of biological activities, such as mitogenic activity, activation of alternative-pathway complement (APCs) and plasma-clotting activity (Lee et al., 2002, FEMS Microbiol. Lett., 209:63-67; Chen et al., 2005, Life Sciences, 76: 3029-3042).

The effects of the polysaccharides extracted from Antrodia camphorata are anti-hepatitis B virus effects, anti-inflammatory activity, anti-angiogenic activities and antitumor effects (Lee et al., 2002, FEMS Microbiol. Lett., 209:63-67; Shen et al., 2004, FEMS Microbiol. Lett, 231: 137-143; Chen et al., 2005, Life Sciences, 76: 3029-3042; Lieu et al., 2004, Toxicol. Appl. Pharmacol. 201:186-193). However, the component, structure or other characteristics of the polysaccharides extracted from Antrodia camphorata are not yet clear identified.

Hepatitis is a common disease in the world especially in developing countries. However, there are no effective drugs for the treatment of this disease. In recent years, scientists have carried out a considerable amount of research on traditional medicine in an attempt to develop new drugs for hepatitis. Compounds that can either decrease the necrotic damage to hepatocytes via enhanced defense mechanisms against toxic insult or improve the repair of damaged hepatocyte are considered potentially useful in the treatment of human hepatitis.

BRIEF DESCRIPTION OF THE DRAWINGS

Figure 1 illustrates elution profile of Antrodia camphorata (ACN) by ion exchange column chromatography on DE-52 (Detection was performed by phenol-H₂Sθ₄ method).

Figure 2 illustrates elution profile of ACN by gel filtration column chromatography on HW-65 (Detection was performed by phenol-H₂SO₄ method).

Figure 3 illustrates elution profile of ACN2 by gel filtration column chromatography on HW-65 (Detection was performed by phenol-H₂Sθ₄ method).

Figure 4 illustrates elution profile of ACN2-1 by gel filtration column chromatography on HW-65 (Detection was performed by phenol-H₂SO₄ method).

Figure 5 illustrates IR spectrum ofACN2a (measured with KBr method).

Figure 6 illustrates ¹H-NMR spectrum of ACN2a (measured in D₂O).

Figure 7 illustrates ¹³C-NMR spectrum oϊACN2a (measured in D₂O). Figure 8 illustrates GC-MS analysis of sugar composition of ACN2a.

Figure 9 illustrates determination of the absolute configurations (D/L) of the component sugars of the ACN2a.

Figure 10 illustrates serum AST levels dependence of hour. Nor: normal control; Con6: the blood samples are collected 6 hrs after intravenous injection of LPS; Conl2: the blood samples are collected 12 hrs after intravenous injection of LPS; Con 18: the blood samples are collected 18 hrs after intravenous injection of LPS. The results represent the mean ± S.D. of the values obtained 10 ICR mice per group. *: P<0.05 and **: p<0.01 compare to corresponding P acnes-LPS control group as determined with student's t-test.

Figure 11 illustrates serum ALT levels dependence of hour. Nor: normal control; Con6: the blood samples are collected 6 hrs after intravenous injection of LPS; Conl2: the blood samples are collected 12 hrs after intravenous injection of LPS; Conlδ: the blood samples are collected 18 hrs after intravenous injection of LPS. The results represent the mean ± S.D. of the values obtained 10 ICR mice per group. *: PO.05 and **: pO.Ol compare to corresponding /! acnes-LPS control group as determined with student's t-test.

Figure 12 illustrates effect of ACN2a given by oral administration on P acnes-LPS induced liver injury of mice. A: normal control, B: P. acnes-LPS, C: FK506 lmg/kg + P acnes-LPS, D: ACN2a 0.2 g/kg + P. acnes-UPS, E: ACN2a 0.4 g/kg + P acnes-LPS, and F: ACN2a 0.8 g/kg + P acnes-LPS. Figure 13 illustrates effect of ACN2a on serum ALT levels in ICR mice with P acnes-LPS induced liver injury. Nor: normal control, Con: P acnes-LPS, A0.2: ACN2a 0.2 mg/kg (b.w., i.v.) + P. acnes-LPS, A0.4: ACN2a 0.4 mg/kg (b.w., i.v.) + P. acnes-LPS, A0.8: ACN2a 0.8 mg/kg (b.w., i.v.) + P acnes-LPS, and FK506: FK506 1 mg/kg (b.w., RO.) + P acnes-LPS. The results represent the mean ± S.D. of the values obtained 10 ICR mice per group. *: P<0.05 and **: pO.Ol compare to corresponding P acnes-LPS control group as determined with student's t-test.

Figure 14 illustrates effect of ACN2a given by intravenous injection on P acnes-LPS induced liver injury of mice. A: normal control, B: P acnes-LPS, C: FK506 lmg/kg + P acnes-LPS, and (G: ACN2a 0.2 mg/kg, H: ACN2a 0.4 mg/kg, I: ACN2a 0.8 mg/kg)(b.w., i.v.) + P acnes-LPS.

Figure 15 illustrates effect of ACN2a on liver proliferating cell. Nor: normal control, Con: P acnes-LPS, A0.4g: ACN2a 0.4 g/kg (p.o.) + P acnes-LPS, A0.4mg: ACN2a 0.4 mg/kg (b.w., i.v.) + P acnes-LPS, and proliferating cell (arrow).

Figure 16 illustrates effect of ACN2a on serum ALT levels in ICR mice with P acnes-LPS induced liver injury. Nor: normal control, Con: P acnes-LPS, A0.2: ACN2a 0.2 g/kg (b.w., p.o.) or ACN2a 0.2 mg/kg (b.w., i.v.) + £ acnes-LPS, A0.4: ACN2a 0.4 g/kg (b.w., p.o.) or ACN2a 0.4 mg/kg (b.w, i.v.) + P acnes-LPS, A0.8: ACN2a 0.8 g/kg (b.w, p.o.) or ACN2a 0.8 mg/kg (b.w, i.v.) + P. acnes-LPS, and FK506: FK506 lmg/kg (b.w., p.o.) + P acnes-LPS. The results represent the mean ± S.D. of the values obtained 10 ICR mice per group. *: P<0.05 and **: p<0.01 compare to corresponding P acnes-LPS control group as determined with student's t-test. Figure 17 shows that the hot water extract of the Antrodia camphorata was fractionated.

Figure 18 shows animal experiment (by oral administration) schedule. Figure 19 shows animal experiment (by intravenous injection) schedule. Figure 20 shows animal experiment (therapeutic effect) schedule. Figure 21 shows mechanism of P. acnes -LPS induced hepatic toxicity.

SUMMARY OF THE INVENTION

This invention provides a composition for injection comprising a neutral polysaccharide of Antrodia camphorata having characteristics as follows: (a) appearance: colorless and shapeless powder, (b) pH: neutral, (c) molecular weight: 899.5~1670.5 kDa determined by FDPLC as shown in Figure 4, (d) rotatory power: C α ) _D +115.0° (c =0.4433, H₂O), (e) intrinsic viscosity: C η ] =0.03971-0.06255 dl ^• g^"1, (f) specific heat Cp: 0.2536-0.3995 CaI/ g • ⁰C ₅ (g) IR spectrum: as shown in Figure 5, (h) ¹H-NMR spectrum: as shown in Figure 6, (i) ¹³C-NMR spectrum: as shown in Figure 7, and (j) GC-MS analysis: as shown in Figure 8.

This invention also provides a composition for oral administering comprising a neutral polysaccharide of Antrodia camphorata having characteristics as follows: (a) appearance: colorless and shapeless powder, (b) pH: neutral, (c) molecular weight: 899.5-1670.5 kDa determined by HPLC as shown in Figure 4, (d) rotatory power: C α J _D +115.0° (c =0.4433, H₂O), (e) intrinsic viscosity: ( η ) =0.03971-0.06255 dl ^• g^'1, (f) specific heat Cp: 0.2536-0.3995 CaI/ g • °C, (g) IR spectrum: as shown in Figure 5, (h) ¹H-NMR spectrum: as shown in Figure 6, (i) --.,„ _-v_Uu w u ^■ i _/, • ^/ y y (_jj

¹³C-NMR spectrum: as shown in Figure 7, and (j) GC-MS analysis: as shown in Figure 8.

DETAILED DESCRIPTION OF THE INVENTION

This invention provides a composition for injection comprising a neutral polysaccharide of Antrodia camphorata having characteristics as follows: (a) appearance: colorless and shapeless powder, (b) pH: neutral, (c) molecular weight: 899.5-1670.5 kDa determined by HPLC as shown in Figure 4, (d) rotatory power: C α 3 _D +115.0° (c =0.4433, H₂O), (e) intrinsic viscosity: C η ) =0.03971-0.06255 dl • g^'1, (f) specific heat Cp: 0.2536-0.3995 CaI/ g • °C, (g) IR spectrum: as shown in Figure 5, (h) ¹H-NMR spectrum: as shown in Figure 6, (i) ¹³C-NMR spectrum: as shown in Figure 7, and (j) GC-MS analysis: as shown in Figure 8.

In a preferred embodiment, the the molecular weight of the polysaccharide is 1092.25-1477.75 kDa determined by HPLC. In a more preferred embodiment, the molecular weight of the polysaccharide is 1285 kDa determined by HPLC, the intrinsic viscosity of the polysaccharide is 0.0417 dl • g^"1 ,and the specific heat Cp of the polysaccharide is 0.2663 CaI/ g • °C .

The IR spectrum of the polysaccharide shows the component sugars comprising galactose, glucose, fucose, mannose and galatosamine. The ¹H-NMR spectrum of the polysaccharide shows the component sugars comprising D-galactose, D-glucose, L-fucose and D-mannose. The ratio of the component sugars comprising galactose, glucose, fucose, mannose and galatosamine is 1 : 0.46: 0.035: 0.016: 0.092. The component sugars have main chain consisting of: (a) terminal residue: fucose or glucose, and (b) middle residue: 1,3- linked glucose, 1,4-linked glucose, 1,6-linked and 1,2,6-linked galactose, wherein the 1,2,6-linked galactose residue is attached by the branch chain at 2-0 site. The polysaccharide of this invention has galactose in main backbone and can be linear or branch form. The polysaccharide of this invention is extracted from Antrodia camphorata by water.

The polysaccharide has therapeutic effective amount 0.01-5.0 mg per kg body weight of a patient. In a preferred embodiment, the polysaccharide has therapeutic effective amount 0.05-3.0 mg per kg body weight of a patient. In a more preferred embodiment, the polysaccharide has therapeutic effective amount 0.1-2.0 mg per kg body weight of a patient. In a most preferred embodiment, the polysaccharide has therapeutic effective amount 0.1-1.0 mg per kg body weight of a patient. The injection composition can against hepatoxicity or hepatic injury.

The composition further comprises a pharmaceutical carrier. In preferred embodiment, the carrier is liquid or semi-solid form for injection use.

The suitable carriers are polar solvents, such as water, alcohol, ketones, esters and mixtures of the above solvents, preferably water, alcohol and water/alcohol mixture. For the preferable embodiment, the suitable carriers are water, normal saline, buffering aqueous solution and buffering saline etc. The carrier used with the composition of this invention also can be lactose, dextrin, and starch and sodium stearate.

This invention also provides a composition for oral administering comprising a neutral polysaccharide of Antrodia camphorata having characteristics as follows: (a) appearance: colorless and shapeless powder, (b) pH: neutral, (c) molecular weight: 899.5-1670.5 IcDa determined by HPLC as shown in Figure 4, (d) rotatory power: C a ) ₀ +115.0° (c =0.4433, H₂O)₅ (e) intrinsic viscosity: C η ] =0.03971-0.06255 dl • g^"1, (f) specific heat Cp: 0.2536-0.3995 CaI/ g ^• °C, (g) IR spectrum: as shown in Figure 5, (h) ¹H-NMR spectrum: as shown in Figure 6, (i) ¹³C-NMR spectrum: as shown in Figure 7, and (j) GC-MS analysis: as shown in Figure 8.

In a preferred embodiment, the the molecular weight of the polysaccharide is 1092.25-1477.75 kDa determined by HPLC. In a more preferred embodiment, the molecular weight of the polysaccharide is 1285 kDa determined by HPLC, the intrinsic viscosity of the polysaccharide is 0.0417 dl • g^"1 ,and the specific heat Cp of the polysaccharide is 0.2663 CaI/ g * °C .

The polysaccharide has therapeutic effective amount 0.01-5.0 g per kg body weight of a patient. In a preferred embodiment, the polysaccharide has therapeutic effective amount 0.05-3.0 g per kg body weight of a patient. In a more preferred embodiment, the polysaccharide has therapeutic effective amount 0.1-2.0 g per kg body weight of a patient. In a most preferred embodiment, the polysaccharide has therapeutic effective amount 0.1-1.0 g per kg body weight of a patient The oral taken composition can against hepatoxicity or hepatic injury.

The composition further comprises a pharmaceutical carrier. In preferred embodiment, the carrier is solid, liquid, or semi-solid form for oral administering.

The suitable carriers are polar solvents, such as water, alcohol, ketones, esters and mixtures of the above solvents, preferably water, alcohol and water/alcohol mixture. For the preferable embodiment, the suitable carriers are water, normal saline, buffering aqueous solution and buffering saline etc. The carrier used with the composition of this invention also can be lactose, dextrin, and starch and sodium stearate. The liquid carriers used for oral administering include water, soybean oil, wine and juices etc.

EXAMPLE

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

Example 1

(A)MateriaIs

Antrodia camphorata mycelium was provided by Simpson Biotech Co. Ltd. (Taiwan). A standard molecular weight market of pullulans (Shodex Standard P-82) was purchased from Showa Denko Co. Ltd., (Japan).

(B) General Experimental Procedures

Optical rotation was determined in H₂O with a JASCO DIP-360 automatic polarmater. UV absorptions were measured with a SHIMADZU UV-2200 UV-VIS recording spectrometer. IR spectra were recorded in a KBr disk or liquid film using a JASCO FT/IR-230 infrared spectrometer. NMR spectra were recorded on Varian Unity Plus 500 (H was at 500MHz, C was at 125MHz) and Varian GEMINI 300 (H was at 300MHz, C was at 75MHz). A solution of polysaccharide in D₂O was measured with 1,4-diozane as an external reference. GC-MS analysis was carried out on a SHIMADZU GC- 17A gas chromatography equipped with JEOL mass Spectrometer. TLC was carried out on pre-coated silica-gel 60 F254 plates (Merck, 0.25mm), cellulose F plates (Merck, 0.1mm), and spots were detected by spraying with 10%H2SO4 or AHP by heating at 100 ^°C . Carbohydrates were determined by the phenol- H2SO4 method.

Example 2 Preparation of neutral polysaccharide from Antrodia Camphorata

(A) Extraction and Fractionation of polysaccharides

The freeze-dried power of Antrodia Camphorata (1.5kg) was extracted with CHCl₃ (41x3 times) at room temperature for 1 day, then filtered and dried. The residue was dipped into H₂O at room temperature for 1 h and extracted (3 times) at 100 ^°C for 2h. After the hot water extract were combined and concentrated to 800 ml, and 3200 ml of EtOH was added to the extract. The mixture was stirred and left in the refrigerator for one night. The precipitate was filtered and washed with cold EtOH, then dried. After treatment of the precipitate with 10% trichloroacetic acid (TCA), the TCA-soluble fraction obtained by centrifugation (3000rpm x lOmin) was extensively dialyzed for 3 d against distilled water. The nondialyzed portion was lyophilized to give a brownish residue (AC). Yield • 14.25g.

(B) Ion-Exchange Column Chromatography of AC

AC (lOOmg) dissolved in H₂O was applied to a column of DE-52 (Whatman international Ltd. England.2.0 x 20cm) The column was eluted with 60ml OfH₂O, 60ml of 0.5M NaCl, 60ml of IM NaCl, 60ml of 2M NaCl, anf fractions of every 2 ml were collected. H₂O fraction (ACN) was concentrated and lyophilized to yield 68.3 mg. (C)GeI Filtration of ACN

ACN (68.3mg) was dissolved in 0.2M NaCl solution and applied to a column of Toyopearl HW-65(Tosoh, Tokyo, Japan.2.0 x 90cm). The column was eluted with the same solution, and fractions of every 5ml were collected. The eluted fractions were separated into two fractions (ACNl and ACN2) according to the elution profile prepared on the basis of the phenol- H₂SO₄ method at 480nm. Yield : ACNl, 19mg ; ACN2, 49mg. ACN2 was farther purified by the column of HW-65 at the same condition as described above. A colorless polysaccharide was got (named ACN2a, yield : 41 mg).

The hot water extract of the Antrodia camphorata was fractionated as shown in Figure 17. The non-dialyzable portion (AC) of the 10% TCA soluble fraction had hepatoprotective active and contained polysaccharide because the phenol- H₂SO₄ reaction was positive. As shown in Figure 1, AC was separated by ion-exchange column chromatography on DE-52 cellulose. The most potent water fraction (CAN) was then separated by gel filtration (Figure 2).The second fraction ACN2a was further purified by gel filtration on HW-65 to yield a colorless polysaccharide (ACN2a) as hepato-protective component (Figure 3).

Example 3

The structure analysis of neutral polysaccharide from Antrodia Camphorata

(A) Estimation of Molecular Weight

The average molecular weight of the polysaccharide (ACN2a) was estimated by HPLC analysis. The sample was applied on a TSK-GMPWXL gel filtration

12 rr rurrr / DI lI C OE \ column (7.8 x 300mm i.d., Tosoh Corp., Tokyo, Japan) and eluted with 0.2M NaCl at lml/min. Commercial available pullulans (Shodex Standard P-82) were used as standard molecular markers.

This polysaccharide (ACN2a) was proved a single fraction by HPLC (Figure 4), and its apparent molecular weight was estimated to be 1285320 by HPLC. The polysaccharide is colorless and shapeless powder, and has C a ] D + 115.0° (c = 0.4433, H₂O) ; Intrinsic viscosity [ η ) = 0.0417dl g^{" l} (measured with Ostwald viscometer), and Specific heat Cp : 0.2663 Cal/g °C (measured by DSC method (differential scanning calorimeter). There are 0.20% protein (measured by Bradford method) and 0.12% nitrogen (by elementary analysis method) in the ACN2a ; Sulfate is not present in the ACN2a (measured by Barium rhodizonate method).

(B) Identification of Component Sugars

The polysaccharide (2mg) was dissolved in 2ml of 2N trifluoroacetic acid (TFA) and sealed. After being hydrolyzed for Ih at 125 ^°C in a steam autoclave, TFA was removed by evaporation of the reaction mixture to dryness. The hydrolysates were reduced with NaBH₄. Trimethyl-silylation was prepared with silblender-HTP for GC-MS analysis. (Column, DB-I, J&W Scientific, 0.25mm i.d. x 30m ; column temperature, 50^°C~190^°C, 5°C/min I then 190^°C, 12min ; Helium carrier flow 4.25kgf/cm).

According to identification of component sugars (Figure 5), the polysaccharide consisted of galactose> glucose_^ fucose_N mannose and galatosamine (1 : 0.46 • 0.035 : 0.016 : 0.092). About 62.38 % sugar of component sugar is galactose.

The rotatory power of ACN2a is + 115.0° (c = 0.4433 » H₂O). This result suggests that component sugars had α- D - or β- L -configuration possibly (by the isorotation law of Hudson). According to determination of the absolute configuration of component sugars (Figure 6), the absolute configuration of component sugars was L-fucose, D-galactose, D-glucose and D-mannose respectively.

(C) Determination of the Absolute Configuration of Component Sugars

Determination of the absolute configuration of component sugars was performed as reported by Hara et al. polysaccharide (Img) was hydrolyzed in 2N trifluoroacetic acid (TFA) at 125^°C for Ih. TFA was removed by evaporation to give a sugar fraction. Pyridine solutions (0.5ml) of the sugar fraction (2mg) and L-cysteine methyl ester hydrochloride (3mg) were mixed, and warmed at 6O⁰C for 1.5h, then dried with N2. The dried sample was trimethylsilylated with silblender-HTP (0.4ml) at 60 ^°C for Ih. After partitioning with CHCl₃ (3ml) and H₂O (3ml) , the CHCl₃ extract was analyzed by GC-MS (Column, DB-wax, J&W Scientific, 30m x 0.25mm ; column temperature, 50^°C~230^°C, 10^°C/min ; then 23O⁰C, 12min I Helium carrier flow 4.25kgf/cm)

(D) Methylation Analysis

The polysaccharide (5mg) was methylated with methyl iodide by Anumula and Taylor's method. Methylated polysaccharides were hydrolyzed with 4N trifluoroacetic acid (TFA) for 90 min at 125^°C in a steam autoclave. After TFA was removed by evaporation, the hydrolysates were converted to alditols with IM NH₄OH containing 3mg/ml NaBH₄ then acetylated. The partially methylated alditol acetates were analyzed by GC and GC-MS (Column, Sp-2330, Supelco, Bellefnte, Pa., 60m x 0.25mm, 0.20um film thickness. Helium was used as a carrier gas, and column temperature was 160^°C to 210°C at 2°C/min, then 210°C to 240 "Cat 5°C/min and 240 ^°C, 14min). Peak areas were corrected using published molar response factors. The derivatized compounds were identified by comparison of their relative retention time to l,5-di-O-acetyl-2,3₅4,6-tetra-O-methylglucitol and their GC-EI-MS fragmentation patterns.

In the FT-IR spectrum, as shown in the Figure 6, pyranoid form was suggested to be present because of the obserbation of three absorption bands at 1153.22 cm^{" l}, 1079.94 cm^" ' and 1033.66 cm^{" ]} (Furanose form has only two absorption bands in the region) . D-Glucopyranose was suggested to be present because of the absorption band at 917.95 cm^" \ In addition, the band at 873.6 cm^{" l} is a special absorption band of manno-pyranoid and galactopyranoid. Aminosugar was suggested to be present because of the observation of a-NH2 absorption band at 1637.27 cm^{" l}. It is the same as analysis conclusion of elementary analysis.

In the HNMR spectrum (Figure 7) , H^{" l} signals were observed at more than 4.8ppm (4.885, 4.909, 4.963ppm) ,which suggest that component sugars have α-configuration. It is the same as analysis conclusion of rotatory power. In addition, at less than 4.8ppm (4.738, 4.663ppm) , H^{" l} signals were also observed. This results suggest that component sugars have also little β-configuration. Methyl proton signal was observed at 1.134 ppm, which was assigned to the methyl of fucose residues. Anomeric signal was detected at less than 5.0 ppm as singlet. These results suggest that fucose residue have a β-L -configuration. (Anomeric signal of α- L- fucose was observed at more than 5.0 ppm).

In the CNMR spectrum (Figure 8), C-4 and C-5 signals were observed at less

15 ivi irr M ICCT (Ol II C 0R\ than 80ppm. This result suggests that component sugars are pyranoid form (The chemical shifts of C-4 and C-5 for furanose form are present in the region 80~85ppm) It is the same as analysis conclusion of IR. In addition, methyl signal was observed at 13.7ppm, which was assigned to the methyl of fucose residues. This result suggest that fucose residues are L- fucoses (C-6 signal of _D - fucose is observed in the region 60~65 ppm). It is the same as analysis conclusion of HNMR spectrum.

The results of methylation analysis, as summarized in Table 1, showed that ACN2a was composed of terminal-Fucose, 1,4-linked glucose, 1-6 linked and 1,2,6- linked galactose residues, and little terminal and 1,3 -linked glucose residues, and little terminal and 1,3- linked glucose residues. By the methylation analysis, ACN2a contained a backbone composed of α- D -1, 6-Gal (α- D -1,6- and α-D -1,2,6-) Gal, it is about 72.82 %. And the number of branch points were about 15.75 % of total residues' numbers, the branch was attached to 2-O of a galactosy residues of the main chain.

Table 1: The results of methylated analysis of ACN2a

Methylated Molar TR MS main fragments (M/Z) Linkages sugar ratio type

2,3,4-Me₃-FuC 0.209 0.789 71,89,101,117,131,161,175 FuC-(I^

2,3,4,6-Me₄-GIc 0.084 1 71,87,101,117,129,145,161,205 GIc-(I^

2,4,6-Me₃-GIc 0.026 1.31 71,87,101,117,129,161,233 ->3> GIc-(I^

2,3,6-Me₃-GIc 0.157 1.489 87,99,101,113,117,233 GIc-(I ^■»

2 ,3,4-Me₃-GIc 1 1 .6 71 ,87 ,99,101,117,129,161,189 ^■»6)-

GIc-(I-*

3 ,4-Me₃-GaI 0.276 1 .881 87 ,99 ,129,189 ^■*2,6> GIc-(I-*

T_R is the relation time of each component, relative to that of 1, 5-0-2, 3, 4, 6-Me₄-GIc

Example 4

Protective effect of the neutral polysaccharide (ACN2a) against P. acnes-hPS induced hepatoxicity

(A) Preparation of P. acnes and reagent

P. acnes (ATCC 6919) was cultured with brain heart infusion medium (Wako Pure Chemical Industries, Ltd. Osaka, Japan), supplemented with L-cysteine (0.03%) and Tween 80 (0.03%) under anaerobic conditions for 48 h at 37 °C . The culture was centrifuged at 5500 x g for 15 min at 4 ⁰C and washed with phosphate — buffered saline (PBS). The bacterial pellet was resuspended with 300 ml of PBS and the cells were killed by heat treatment at 80 °C for 30 min, then lyophilized to prepare a heat-killed P. acnes powder. LPS from Escherichia coli 055: B5 was purchased from Sigma- Aldrich, Inc. (Steinheim, Germany). FK506 (tacrolimus hydrate) was provided by Fujisawa Pharmaceutical Co., Ltd. (Osaka, Japan).

(B) Animals Four- week-old male ICR mice (18-20 g), male BALB/C (18-20 g) and male wistar rat (106-180 g) (SLC, Hamamatu, Japan) were used for the experiment of the protective effect against hepatoxicity induced by P. acnes-LPS.⁵⁵' ⁵⁶⁾ The animals were acclimatized for one week before the study.

(C) Hepatoprotective experiment (by oral administration)

The hepatoprotective activity of ACN2 a was investigated using: A: (1) normal control (untreated); (2) P. acnes— LPS (0.15 mg- 0.05 μg/mouse) treated control; [(3) 0.2 g/kg/day, (4) 0.4 g/kg/day, (5) 0.8 g/kg/day of body weight (b.w.), P.O.] plus a P. acnes -LPS treatment; and (6) FK506 (1 mg/kg, b.w., P.O.) plus P. acnes— LPS treatment in the ICR mice.

B: (1) normal control (untreated); (2) P. acnes- -LPS (0.20 mg- 0.075 μg/mouse) treated control; (3) ACN2a (0.4 g/kg/day, b.w., P.O.); (4) ACN2a (0.4 g/kg/day, b.w., P.O.) plus a P. acnes— LPS treatment in the BALB/C mice.

C: (1) normal control (untreated); (2) P acnes — LPS (5 mg- 5 μg/rat) treated control; (3) ACNIa (0.2 g/kg/day, b.w., P.O.) plus a P. acnes— LPS treatment in the wistar rat.

Heat-killed P. acnes cells suspended in saline were injected via a tail vein (Figure 18). Seven day later, acute liver damage was induced by intravenous injection of LPS. ACN2a was given once daily through a gastric tube to the animals for 7 consecutive days. One hour after of ACN2 'a was given on the 8^th day, LPS was injected. FK506 was used as a positive control drug and administered through a gastric tube 48, 36, 24, 12 and 1 h before intravenous injection of LPS. Blood samples were taken into tubes for analysis of liver injury 6 h after LPS injection, and these animals were sacrificed. The tubes were centrifuged at 3500 x g for 15 min and the serum was used as a sample. All samples were stored at -20 °C until the assay. The serum aspartate aminotransferase (AST) and alanine aminotransferase (ALT) activities, which are markers of hepatocyte injury, were determined using kits (transaminase C II - test Wako) for the measurement of enzyme activity (Wako Pure Chemical Industries, Ltd. Osaka, Japan).

(D) Liver histology

Mice were sacrificed after blood was collected, and the livers were freshly and quickly obtained. Liver slices were made, and immediately fixed in a 10% buffered formalin phosphate solution, dehydrated with 50-100% ethanol solution, and embedded in paraffin. Four to five micrometer sections were cut and stained with hematoxylin-eosin.

(E) Hepatoprotective experiment (by intravenous injection)

The hepatoprotective activity of ACN2a was investigated using:

A (1) normal control (untreated); (2) P. acnes— LPS (0.15 mg- 0.05 μg/mouse) treated control; [(3) 0.2 g/kg/day, (4) 0.4 g/kg/day, (5) 0.8 g/kg/day, b. w., i. v.] plus a P. acnes -LPS treatment; and (6) FK506 (1 mg/kg, b.w., P.O.) plus P. acnes -LPS treatment in the ICR mice.

B (1) normal control (untreated); (2) P. acnes— LPS (0.15 mg- 0.05 μg/mouse) treated control; [(3) 0.4 g/kg/day, (4) 0.8 g/kg/day b. w., i. v.), P.O.] plus a P. acnes— LPS treatment.

The experimental model of acute liver injury was induced as above described. A i. v. method is same as an oral administration. B i. v. method is that on the 8 day, ACN2a was injected with LPS in saline via a tail vein only once (Figure 19). Blood samples were taken into tubes for analysis of liver injury 6 h after LPS injection, then these animals were sacrificed. The tubes were centrifuged at 3500 x g for 15 min and the serum was used as a sample. All samples were stored at -20 °C until the assay. The serum aspartate aminotransferase (AST) and alanine aminotransferase (ALT) activities were determined using kits for the measurement of enzyme acitivity.

(F) Therapeutic Effect of ACN2a on P. acnes -LPS Induced Hepatic Injury in Mice

The therapeutic effect of ACN2a was investigated using:

(1) normal control (untreated); (2) R acnes — LPS (0.15 mg- 0.05 μg/mouse) treated control; [(3) 0.4 g/kg/day, (4) 0.8 g/kg/day, (5) 1.6 g/kg/day, (6) 3.2 mg/kg/day (7) 6.4 mg/kg/day of body weight (b. w.), i. v.] plus a R acnes— XPS treatment; and (6) FK506 (1 mg/kg, b.w., P.O.) plus P. acnes— -LPS treatment.

The experiment model of acute liver injury was induced as above described. On the 8^th day, after 3h of LPS was given, ACNIa was injected in saline via a tail vein only once (Figure 20). Blood samples were taken into tubes after 3h of LPS injection for one P. acnes— LPS treated group and the other blood samples were taken into tubes for analysis of liver injury after 6h of LPS injection, then these animals were sacrificed. The tubes were centrifuged at 3500 x g for 15 min and the serum was used as a sample. All samples were stored at -20 °C until the assay. The serum aspartate aminotransferase (AST) and alanine aminotransferase (ALT) activities, which are markers of hepatocyte injury, were determined using kits for the measurement of enzyme activity.

20 ie \ (G) Result a. Estimation of model animals

Injection of P. acnes followed by LPS is useful for the creation of experimental models of acute hepatic damage. Most of the animals died from severe liver injury within 24 hr of LPS injection. In this study, we found the best dose of P acnes -LPS (0.15 mg- 0.05 ug/mouse). All of animals survived from severe liver injury, and liver injury was the severest 12 hr after intravenous injection of LPS (Figures 10 and 11). So in this invention, the blood samples were collected for analysis of liver injury 6 hr after LPS injection.

b. Hepato-protective effect of ACN2a given by oral administering

Table 2 showed the effect of ACN2a on ALT and AST levels in serum of mice treated with P. acnes -LPS. The acute hepatoxicity reaction was significantly (P <0.05) suppressed in all of the animals pretreated with 0.4 and 0.8 g/kg of body weight of ACN2a. So ACN2a had protective effect against P. acnes -LPS induced hepatic toxicity in mice; moreover, these protective effect was found to be dose dependent. Furthermore, the acute hepatotoxicity reactions were also significantly (ρ<0.05) suppressed both in all of the BALB/c mice pretreated with 0.4 g/kg of body weight of ACN2a and in all of Wistar rats pretreated with 0.25 g/kg of body weight of ACN2 a, indicating that ACN2a had protective effect on P. acnes - LPS induced hepatic toxicity in murine animals.

Table 2. Effect of ACN2a on serum ALT and AST levels in murine animals with P. acnes - IPS induced liver injury.

Animal Groups Dose (g/kg) AST (IU/L) ^• ALT (IUZL)

ICR mouse Control / 397.3 ± 201.35** 49.2 ± 18.64**

(10 mice per group) 0 15 me 0 05

P. acnes _~ L?S u.ⁱo mg u.uo _{± g ±} ^_{5 3} μg/mouse

LVslTcma °^{2 2M6Λ} ± ⁷⁷⁶"^{37 im6 ± 521}'⁷²

P. acnes - 0.4 1748.3 ± 439.79* 1156.55 ± 517.09* L?S+ACN2a

P. acnes — 0.8 1392.1 ± 460.36* 948.6 ± 555.30* LΫS+ACN2a

P, acnes -

1 mg/kg 1236.0 ± 625.00** 685.0 ± 344.28* LPS+FK506

Control / 195.4 ± 56.56* 38.9 ± 6.63**

BALB/c mouse 0.2 mg-0.075

P. acnes - LPS 208.0 ± 41.30 (8 mice per group) μg/mouse 519.0 ± 128.25

P. acnes —

0.4 431.9 ± 119.71* 129.0 ± 45.28* LPS+ACN2a

Control / 104.2 ± 70.54** 65.4 + 9.02*

Wistar rat P. acnes - LPS 05 mg-5 μg/rat 1095.7 ± 240.34 975.3 ± 644.97 (8 rats per group)

P. acnes - 0.2 439.5 ± 253.26* 306.0 ± 130.76* WS+ACN2a

Value are expressed as means ± S.D. of 10 or 8 animals per group. *, p<0.05 and **, /κθ.001 compared with corresponding P. acnes - LPS determined by Student's t-test.

The hepatoprotective effect of ACN2 a was confirmed by histological observation (Figure 12). Injection of P. acnes - LPS caused granulomatosis, bleeding necrosis and cellular infiltration of ICR mouse livers. However, ACN2a pretreatment reduced the injury score of cellular infiltration and inhibited necrosis of liver cells (Table 3). The ballooned hepatocytes were of different sizes and much larger than normal hepatocytes.

Table 3. Histological injury score of liver under different doses of ACN2a.

Injury score

Animal Groups Cellular

Granulomatosis Bleeding necrosis infiltration

Control 0 0 0

P. acnes - LPS 3 4 3

ICR mouse P. acnes - L¥S+ACN2a (0.2 g/kg) 4 2 3

(10 mice per

P. acnes - LPS+ACN2a (0.4 g/kg) 4 1 3 group)

P. acnes - WS+ACN2a (0.8 g/kg) 3 0 2

P. acnes - LPS+FK506 (1.0 2 0 2 mg/kg)

Livers were scored for hepatic injury via light microscopy: 0 = no visible cell damage; 1= focal hepatocyte damage on < 25 of the tissue; 2 = focal hepatocyte damage on < 25 - 50% of tissue; 3 = extensive, but focal, hepatocyte lesions; 4 = global hepatocyte necrosis. c. Hepato-protective effect of ACN2a given by intravenous injection

Figure 13 shows the effect of ACN2a on ALT levels in serum of ICR mice treated with P. acnes — LPS. The acute hepatotoxicity reaction was significantly (p<0.05) suppressed dose-dependently in all of the ICR mice treated with 0.2 and 0.4 mg/kg of body weight of ACN2 a injected by a tail vein, indicated that ACN2a had also protective effect on P. acnes - LPS induced hepatic toxicity in ICR mice by intravenous injection.

The hepatoprotective effect of ACN 2 a given by intravenous injection was confirmed by histological observation (Figure 14). Injection of P. acnes - LPS caused ballooning degeneration, bleeding necrosis and cellular infiltration of ICR mouse liver. However, ACN2a pretreatment reduced the injury score of cellular infiltration and inhibited necrosis of liver cells (Table 4).

Table 4. Histolo^ ;ical injury score of liver under different doses of ACN2a.

Injury score

Animal Groups Bleeding Cellular

Granulomatosis necrosis infiltration

Control

P acnes - LPS 4 3

hepatocyte lesions; 4 = global hepatocyte necrosis.

Furthermore, the proliferating phase of liver cell were found by histological observation (Figure 15) in the mice pretreated with ACN2a could promote the proliferation of liver cell and improve repair of damaged hepatocytes. d. Comparison of hepatoprotective effect between oral administration and intravenous injection

Effect of ACN2 'a on ALT levels in serum of ICR mice between two different administration routesby in ICR mice treated with P. acnes - LPS was compared (Fig. 16). The acute hepatotoxicity reaction was significantly (/?<0.05) suppressed dose-dependently in all of the ICR mice pretreated with both 0.2, 0.4 and 0.8 ACN2a g/kg (b. w., p. o.), and 0.2 and 0.4 ACN2a mg/kg (b. w., p. o.), indicating that ACN2a had protective effect on P. acnes - LPS induced hepatic toxicity in ICR mice by both oral administration and by intravenous injection. Furthermore, although the doses of ACN2a given by intravenous injection was only 1/1000 of those given by oral administration, the hepatoprotective effect of the latter was 100 times stronger than the former administration, indicating that intravenous injection was the most preferable method.

In summary, ACN2a showed a hepatoprotective effect on P. acnes - LPS induced hepatic injury in murine animals (ICR mice, BALB/c mice and Wistar rats) by both oral administration and intravenous injection.

Example 5 Mechanism of the hepaprotective model

Mechanism of the experimental model induced by P. acnes -LPS was shown in Figure 21. Injection of P. acnes into mice via a tail vein results in monocytic infiltration of the liver, so hepatic macrophages were increase, and subsequent intravenous injection of a small amount of LPS activated hepatic macrophage. Cytokines of tumor necrosis factor (TNF) IL-K soluble IL-2 receptor etc., were gone out of hepatic macrophage and increased. Then liver was injured via three ways by these cytokines: 1). TNF and IL-I broadly necrosised hepatocyte via platelet activating factor (PAF) and leukotriene etc. 2) TNF and IL-I broadly necrosised hepatocyte via neutrophi and microcirculation lesion. In this way, oxygen free radicals played a major role. 3) IL-2 was decreased because of combining with soluble IL-2 receptor, results in suppressor T cell decreasing and cytotoxic T cell (CTL) increasing. Broad hepatocyte was necrosised by CTL.

The crude polysaccharide of Antrodia camphorata was effective in scavenging oxygen free radical formation and increasing IL-2. In this invention, it was found that both crude polysaccharide and neutral polysaccharide (ACN2a) had protective effect against P. acnes -LPS induced hepatic toxicity in mice. It was conceivable that the polysaccharide of Antrodia camphorata exerted its hepatoprotectie activity by, at least partly, scavenging oxygen free radical formation, resulting in obstructing the 2) way of P. acnes -LPS induced hepatic toxicity or by increasing IL-2, resulting in decreasing CTL and protecting liver(Figure 21).

While the invention has been described and exemplified in sufficient detail for those skilled in this art to make and use it, various alternatives, modifications, and improvements should be apparent without departing from the spirit and scope of the invention.

One skilled in the art readily appreciates that the present invention is well adapted to carry out the objects and obtain the ends and advantages mentioned, as well as those inherent therein. The embryos, animals, and processes and methods for producing them are representative of preferred embodiments, are exemplary, and are not intended as limitations on the scope of the invention. Modifications therein and other uses will occur to those skilled in the art. These modifications are encompassed within the spirit of the invention and are defined by the scope of the claims.

rui/uN ZUUO / u u z o z ft gone out of hepatic macrophage and increased. Then liver was injured via three ways by these cytokines: 1). TNF and IL-I broadly necrosised hepatocyte via platelet activating factor (PAF) and leukotriene etc. 2) TNF and IL-I broadly necrosised hepatocyte via neutrophi and microcirculation lesion. In this way, oxygen free radicals played a major role. 3) IL-2 was decreased because of combining with soluble IL-2 receptor, results in suppressor T cell decreasing and cytotoxic T cell (CTL) increasing. Broad hepatocyte was necrosised by CTL.

The crude polysaccharide of Antrodia camphorata was effective in scavenging oxygen free radical formation and increasing IL-2. In this invention, it was found that both crude polysaccharide and neutral polysaccharide (ACN2a) had protective effect against R acnes -LPS induced hepatic toxicity in mice. It was conceivable that the polysaccharide of Antrodia camphorata exerted its hepatoprotectie activity by, at least partly, scavenging oxygen free radical formation, resulting in obstructing the 2) way of P. acnes -LPS induced hepatic toxicity or by increasing IL-2, resulting in decreasing CTL and protecting liver.

Endotoxemia (P. acnes)

Chart 5 Mechanism of P. acnes -LPS induced hepatic toxicity.

Claims

WHAT IS CLAIMED IS:

1. A composition for injection comprising a neutral polysaccharide of Antrodia camphorata having characteristics as follows:

(a) appearance: colorless and shapeless powder,

(b) pH: neutral,

(c) molecular weight: 899.5-1670.5 kDa determined by HPLC as shown in

Figure 4,

(d) rotatory power: C α ) _D +115.0° (c =0.4433, H₂O),

(e) intrinsic viscosity: C η 3 =0.03971-0.06255 dl ^• g^"1,

(f) specific heat Cp: 0.2536-0.3995 CaI/ g • °C ,

(g) IR spectrum: as shown in Figure 5,

(h) ¹H-NMR spectrum: as shown in Figure 6,

(i) ¹³C-NMR spectrum: as shown in Figure 7, and

(j) GC-MS analysis: as shown in Figure 8.

2. The composition of Claim 1, wherein the polysaccharide has therapeutic effective amount 0.01-5.0 mg per kg body weight of a patient.

3. The composition of Claim 2, wherein the polysaccharide has therapeutic effective amount 0.05-3.0 mg per kg body weight of a patient.

4. The composition of Claim 3, wherein the polysaccharide has therapeutic effective amount 0.1-2.0 mg per kg body weight of a patient.

5. The composition of Claim 4, wherein the polysaccharide has therapeutic effective amount 0.1-1.0 mg per kg body weight of a patient.

6. The composition of Claim 1, wherein the molecular weight of the polysaccharide is 1092.25-1477.75 kDa determined by HPLC.

7. The composition of Claim 6, wherein the molecular weight of the polysaccharide is 1285 kDa determined by HPLC.

8. The composition of Claim 1, wherein the intrinsic viscosity of the polysaccharide is 0.0417 dl • g^"1.

9. The composition of Claim 1, wherein the specific heat Cp of the polysaccharide is 0.2663 CaI/ g • °C .

10. The composition of Claim 1, which can against hepatoxicity or hepatic injury.

11. The composition of Claim 1, which further comprises a pharmaceutically acceptable carrier.

12. The composition of Claim 11, wherein the carrier is liquid or semi-solid form.

13. A composition for oral administering comprising a neutral polysaccharide of Antrodia camphorata having characteristics as follows:

(a) appearance: colorless and shapeless powder,

(b) pH: neutral,

(c) molecular weight: 899.5-1670.5 kDa determined by HPLC as shown in

Figure 4,

(d) rotatory power: C a ] ₀ +115.0° (c =0.4433, H₂O),

(e) intrinsic viscosity: C η ] =0.03971-0.06255 dl • g^"1,

(f) specific heat Cp: 0.2536-0.3995 CaI/ g • °C ,

(g) IR spectrum: as shown in Figure 5,

(h) ¹H-NMR spectrum: as shown in Figure 6,

(i) ¹³C-NMR spectrum: as shown in Figure 7, and

(j) GC-MS analysis: as shown in Figure 8.

14. The composition of Claim 13, the polysaccharide has therapeutic effective

3 i amount 0.01-5.O g per kg body weight of a patient.

15. The composition of Claim 14, wherein the polysaccharide has therapeutic effective amount 0.05-3.0 g per kg body weight of a patient.

16. The composition of Claim 15, wherein the polysaccharide has therapeutic effective amount 0.1-2.O g per kg body weight of a patient.

17. The composition of Claim 16, wherein the polysaccharide has therapeutic effective amount 0.1-1.0 g per kg body weight of a patient

18. The composition of Claim 13, wherein the molecular weight of the polysaccharide is 1092.25-1477.75 kDa determined by HPLC.

19.The composition of Claim 18, wherein the molecular weight of the polysaccharide is 1285 kDa determined by HPLC.

20. The composition of Claim 13, wherein the intrinsic viscosity of the polysaccharide is 0.0417 dl • g^"1.

21. The composition of Claim 13, wherein the specific heat Cp of the polysaccharide is 0.2663 CaI/ g • °C .

22. The composition of Claim 13, which can against hepatoxicity or hepatic injury.

23. The composition of Claim 13, which further comprises a pharmaceutically acceptable carrier.

24. The composition of Claim 23, wherein the carrier is solid, liquid, or semi-solid form.