New Lanostanes and Naphthoquinones Isolated from Antrodia salmonea and their Antioxidative Burst Activity in Human Leukocytes

• Abstract
• Introduction
• Materials and Methods
• General experiment procedures
• Plant materials
• Extraction and isolation
• Characterization of compounds 1, 2, 3, and 4
• Measurement of PMA- or fMLP-induced oxidative burst by human leukocytes
• Estimation of cell viability
• Statistical analysis
• Results and Discussion
• Acknowledgements
• References

Abstract

Four new compounds were isolated from the basidiomata of the fungus Antrodia salmonea, a newly identified species of Antrodia (Aphyllophorales) in Taiwan. These new compounds are named as lanosta-8,24-diene-3β,15α,21-triol (1), 24-methylenelanost-8-ene-3β,15α,21-triol (2), 2,3-dimethoxy-5-(2′,5′-dimethoxy-3′,4′-methylenedioxyphenyl)-7-methyl-[1] [4]-naphthoquinone (3), and 2,3-dimethoxy-6-(2′,5′-dimethoxy-3′,4′-methylenedioxyphenyl)-7-methyl-[1] [4]-naphthoquinone (4), respectively. Their structures were elucidated by spectroscopic methods. An in vitro cellular functional assay was performed to evaluate their anti-oxidative burst activity in human leukocytes. They showed inhibitory effects against phorbol 12-myristate-13-acetate (PMA), a direct protein kinase C activator, induced oxidative burst in neutrophils (PMN) and mononuclear cells (MNC) with 50 % inhibitory concentration (IC₅₀) ranging from 3.5 to 25.8 μM. The potency order of these compounds in PMA-activated leukocytes was as 1 > 3 > 4 > 2. They were relatively less effective in formyl-Met-Leu-Phe (fMLP), a G-protein coupled receptor agonist, induced oxidative burst, except for compounds 3 and 4 in fMLP-activated PMN. These results indicated that three (1, 3, and 4) of these four newly identified compounds displayed anti-oxidative effect in human leukocytes with different potency and might confer anti-inflammatory activity to these drugs.

Introduction

Antrodia salmonea T. T. Chang et W. N. Chou (Polyporaceae, Aphyllophorales), the cause of brown heart rot of Cunninghamia konishii Hayata (Cunninghamieae) in Taiwan, is a basidiomycete first identified in 2004 as a new species of the genus Antrodia [1]. The resupinate salmon-pink basidiomata of A. salmonea grows on the empty rotten trunk of the endemic coniferous tree C. konishii and has vernacularly been called shiang-shan-chih. This fungus is similar to Antrodia cinnamomea T. T. Chang et W. N. Chou, which bears the vernacular name niu-chang-chih, and has only been collected from the endemic aromatic tree Cinnamomum kanehirai Hay. (Lauraceae) in Taiwan, but it has a different color on the pore surface of its basidiomata. The basidiomata of A. cinnamomea has been used for the treatment of food and drug intoxication, diarrhea, abdominal pain, hypertension, skin itching, and cancer [2]. In the previous studies, chemical investigation revealed that niu-chang-chih contained triterpenes, steroids, biphenyl compounds and a sesquiterpene [3], [4], [5], [6], [7], [8], [9], [10], [11] and pharmacological studies of this fungus showed cytotoxicity against P-388 murine leukaemia cells [4], anti-inflammatory [12], [13], and antiviral [14] properties.

Both A. salmonea and A. cinnamomea have a strong bitter taste, which is believed to indicate the presence of potent ingredients. Therefore, it is said that the former fungus can substitute for the later as the medicinal fungus. This paper deals with the structure elucidation of new lanostane and naphthoquinone derivatives isolated from A. salmonea by spectroscopic analysis. Moreover, their anti-oxidative burst activities in human leukocytes are also presented.[]

Materials and Methods

General experiment procedures

Melting points were determined on a Yanaco MP-13 micro-melting point apparatus and are uncorrected. Optical rotations were taken on a JASCO DIP-370 polarimeter. UV spectra were recorded on a Hitachi U-3310 spectrophotometer. IR spectra were obtained on a Nicolet Avatar 320 FT-IR spectrometer. HR-EI-MS were measured on a Finnigan MAT-95XL spectrometer. ¹H-, ¹³C-, and 2D-NMR spectra were obtained on a Varian UNITY INOVA NMR spectrometer. Chemical shifts are shown in δ values (ppm) with deuterated solvents as internal standards.

Plant materials

Antrodia salmonea T. T. Chang et W. N. Chou, was purchased from a Chinese medicinal store in Taipei, and identified by the corresponding author. A voucher specimen (TFRI-1126) was deposited in the herbarium of Taiwan Forestry Research Institute, Taipei, Taiwan.

Extraction and isolation

The chipped fruiting bodies of A. salmonea (1012 g) were extracted four times with distilled water (4 × 10 L) at 85 °C. The residue of fruiting bodies was then heated under reflux five times with ethanol (5 × 20 L) for 6 h and the ethanolic extracts were evaporated. The concentrate (354.2 g) was suspended in 2 L of distilled water and partitioned between dichloromethane and water (1 : 1, v/v) to afford organic and aqueous parts. Methanol (1.5 L) was added to the evaporated organic part (311.5 g) to provide methanol-soluble (296.4 g) and methanol-insoluble (15.1 g) portions. The methanol-soluble portion was chromatographed on a silica gel column using two solvent systems (ethyl acetate gradient in hexane, and then methanol gradient in dichloromethane) as eluents. The fractions were collected in 500 mL portions and pooled according to their TLC profile in toluene-ethyl acetate-acetic acid (10 : 1:0.5) solvent system to give ten fractions (I - X). Fr-IV (53.1 g) was retreated with methanol and divided into methanol-soluble and methanol-insoluble portions. The methanol-soluble portion (Fr-IV-MS, 51.6 g) was chromatographed on silica gel MPLC (acetone gradient in hexane) to give thirty-eight sub-fractions (Fr-IV-MS-1 to Fr-IV-MS-38). After re-chromatographic separation of Fr-IV-MS-26 on silica gel MPLC (acetone gradient in hexane) and HPLC (Cosmosil 5C₁₈-AR-II column; 20 × 250 mm; 70 - 100 % MeOH in H₂O (with 0.5 % HOAc) as gradient solvent system; flow rate 16 mL/min; UV detector set at 210 nm), lanosta-8,24-diene-3β,15α,21-triol (1, 12 mg) and 24-methylenelanost-8-ene-3β,15α,21-triol (2, 20 mg) were obtained. Fr-IV-MS-25 and Fr-IV-MS-24 were further purified on a Sephadex LH-20 column eluting with methanol to afford 2,3-dimethoxy-5-(2′,5′-dimethoxy-3′,4′-methylenedioxyphenyl)-7-methyl-[1] [4]-naphthoquinone (3, 25 mg) and 2,3-dimethoxy-6-(2′,5′-dimethoxy-3′,4′-methylenedioxyphenyl)-7-methyl-[1] [4]-naphthoquinone (4, 17 mg), respectively.

Characterization of compounds 1, 2, 3, and 4

Lanosta-8,24-diene-3β,15α,21-triol (1): White amorphous powder; m. p. 168 - 170 °C; [α]_D ²⁵: + 41.9 ° (MeOH, c 0.62); ¹H- and ¹³C-NMR: see Table [1]; HR-EI-MS: m/z = 458.3758 [M]⁺ (calcd. for C₃₀H₅₀O₃ : 458.3756).

24-Methylenelanost-8-ene-3β,15α,21-triol (2): White amorphous powder; m. p. 184 - 185 °C; [α]_D ²⁵: + 63.8 ° (CHCl₃, c 0.94); ¹H- and ¹³-NMR: see Table [1]; HR-EI-MS: m/z = 472.3912 [M]⁺ (calcd. for C₃₁H₅₂O₃ : 472.3908).

2,3-Dimethoxy-5-(2′,5′-dimethoxy-3′,4′-methylenedioxyphenyl)-7-methyl- [1] [4] -naphthoquinone (3): Brown amorphous powder; m. p. 191 - 193 °C; UV (MeOH): λ_max (log ε) = 285 (4.4), 259 (4.5), 207 (5.0) nm; IR (KBr): ν_max = 2946, 2833, 1666, 1617, 1596, 1507, 1453, 1430, 1363, 1319, 1288, 1244, 1216, 1149, 1056, 954 cm^-1; ¹H-NMR (CDCl₃, 500 MHz): δ = 7.24 (1H, d, J = 1.5 Hz, H-6), 7.89 (1H, d, J = 1.5 Hz, H-8), 6.27 (1H, s, H-6′), 5.99 (1H, d, J = 1.5 Hz, H-7′), 5.98 (1H, d, J = 1.5 Hz, H-7′), 4.04 (3H, s, 2-OCH₃), 3.99 (3H, s, 3-OCH₃), 2.44 (3H, s, 7-CH₃), 3.66 (3H, s, 2′-OCH₃), 3.83 (3H, s, 5′-OCH₃); ¹³C-NMR (CDCl₃, 125 MHz): δ = 182.4 (C-1), 145.9 (C-2), 148.1 (C-3), 181.6 (C-4), 138.6 (C-5), 138.0 (C-6), 143.5 (C-7), 126.6 (C-8), 131.6 (C-9), 127.1 (C-10), 127.2 (C-1′), 135.1 (C-2′), 138.2 (C-3′), 136.5 (C-4′), 139.0 (C-5′), 107.8 (C-6′), 101.7 (C-7′), 61.2 (2-OCH₃ or 3-OCH₃), 61.1 (2-OCH₃ or 3-OCH₃), 21.5 (7-CH₃), 59.8 (2′-OCH₃), 56.9 (5′-OCH₃); HR-EI-MS: m/z = 412.1157 [M]⁺ (calcd. for C₂₂H₂₀O₈ : 412.1156).

2,3-Dimethoxy-6-(2′,5′-dimethoxy-3′,4′-methylenedioxyphenyl)-7-methyl- [1] [4] -naphthoquinone (4): Yellow amorphous powder; m. p. 188 - 190 °C; UV (MeOH): λ_max (log ε) = 285 (sh), 259 (4.0), 206 (4.3) nm; IR (KBr): ν_max = 2949, 2839, 1665, 1610, 1594, 1509, 1453, 1430, 1352, 1290, 1234, 1217, 1189, 1138, 1100, 1063, 1040, 956 cm^-1; ¹H-NMR (CDCl₃, 500 MHz): δ = 7.84 (1H, s, H-5), 7.90 (1H, s, H-8), 6.26 (1H, s, H-6′), 6.02 (2H, s, H-7′), 4.09 (6H, s, 2-OCH₃, 3-OCH₃), 2.28 (3H, s, 7-CH₃), 3.71 (3H, s, 2′-OCH₃), 3.85 (3H, s, 5′-OCH₃); ¹³C-NMR (CDCl₃, 125 MHz): δ = 182.1 (C-1), 147.6 (C-2 or C-3), 147.5 (C-2 or C-3), 182.0 (C-4), 128.1 (C-5), 144.3 (C-6), 144.5 (C-7), 127.6 (C-8), 128.4 (C-9), 125.7 (C-10), 129.6 (C-1′), 135.5 (C-2′), 138.6 (C-3′), 136.9 (C-4′), 139.1 (C-5′), 108.7 (C-6′), 101.9 (C-7′), 61.4 (2-OCH₃, 3-OCH₃), 20.5 (7-CH₃), 60.1 (2′-OCH₃), 57.0 (5′-OCH₃); HR-EI-MS: m/z = 412.1155 [M]⁺ (calcd. for C₂₂H₂₀O₈ : 412.1156).

Measurement of PMA- or fMLP-induced oxidative burst by human leukocytes

Oxidative burst (free radical production) is a major immuno-defense mechanism by human leukocytes. It was measured as mentioned in our previous study [13]. Briefly, in a white 96-well, flat-bottom microplate (Costar, NY, USA), quercetin (a flavonoid, included as a positive control) or compounds 1 - 4 were serially diluted to final concentrations ranging from 1 to 50 μM with a volume of 10 μL. To each well, 50 μL of neutrophil (PMN) or mononuclear cells (MNC) suspension (1 × 10⁷cells/mL) and 50 μL of lucigenin (180 μM) solution were added. After incubation for 10 min with test drugs, the cell suspension was triggered by adding 50 μL of PMA (0.66 μM, final concentration) or fMLP (1 μM, final concentration), and chemiluminescence was monitored every 1 min for 1 s during a 30-min observation period using a microplate luminometer reader (Orion,® Germany) and represented as relative light units (RLU). Peak levels were recoded to calculate the activity of test compounds in relation to their corresponding solvent controls (0.1 % DMSO). The 50 % inhibitory doses (IC₅₀) in response to PMA- or fMLP-triggered chemiluminescence by test compounds were calculated.

Estimation of cell viability

For the examination of cell viability the previous method was followed [13]. Briefly, after incubation of test compound(s) with cells for 2 hours, cell viability was determined by adding propidium iodide (PI, 10 μg/mL) and fluorescein diacetate (FDA, 100 ng/mL). After incubation with PI (for dead cell staining) and FDA (for viable cell staining) at room temperature for 10 min, the cell suspension was analyzed on a flow cytometer (FACSCalibur^TM; Becton Dickinson) by recording forward and light scatter, red (for PI) and green (for FDA) fluorescence. After gating on light scatter to include single cells and to exclude clumps and debris, cell populations were displayed by green (viable) versus red (dead) fluorescence. Cell viability (%) was calculated by the CellQuest® software (Becton Dickinson) on a Power Macintosh 7300/200 computer. In some experiments, estimation of cell viability was further confirmed by trypan blue exclusion assay.

Statistical analysis

Data are analyzed by analysis of variance (ANOVA) followed by post-hoc Dunnett’s t-test for multiple comparisons. Concentration dependence is analyzed by simple linear regression analysis of response levels against concentration of the test drug and testing the slope of the regression line against 0 by Student’s t test at an α level equal to 0.05.

Results and Discussion

Compound 1 was obtained as a white amorphous solid and showed a molecular ion peak at m/z = 458.3758 by HR-EI-MS, which corresponded to the molecular formula C₃₀H₅₀O₃. Its ¹³C- and DEPT-NMR spectra (Table [1]) displayed seven methyl, ten methylene, six methine, and seven quaternary carbons. One methine (δ_C = 126.1) and three quaternary carbon (δ_C = 135.2, 135.1, and 130.8) signals were in the olefinic region, which suggested one trisubstituted and one tetrasubstituted double bond in compound 1. Besides, two methine (δ_C = 78.1 and 72.6) and one methylene (δ_C = 62.0) groups were oxygenated, which also showed four protons at δ_H = 3.46 (1H, t, J = 8.0 Hz), 4.60 (1H, t, J = 7.0 Hz), 4.08 (1H, d, J = 9.0 Hz), and 3.92 (1H, dd, J = 9.0, 5.0 Hz) by analysis of the HMQC spectrum. In the ¹H-NMR spectrum of 1, one olefinic proton (δ = 5.27, 1H, t, J = 7.0 Hz) and seven methyl groups (δ = 0.98, 1.07, 1.08, 1.22, 1.34, 1.58, and 1.65) at quaternary carbons were observed. Based on the above data and COSY, HMQC, and HMBC experiments, it was suggested that compound 1 was a lanostane-type triterpene with a structure similar to 15α-hydroxytrametenolic acid [15]. In contrast to 15α-hydroxytrametenolic acid, which displayed a carboxyl carbon signal at δ = 178.9, compound 1 showed an oxygenated methylene group at δ_C = 62.0 corresponding to the signals at δ_H = 4.08 and 3.92. The relative configuration of 1 was confirmed by a NOESY experiment. Cross-peaks of CH₃ - 18/H-20, H-21 and CH₃ - 28/H-17 revealed that the C-17 side chain was in a β-position. In addition, the NOE correlations between H-15 and CH₃ - 18, as well as H-3 and CH₃ - 29, indicated that the hydroxy groups at C-15 and C-3 were located at α and β positions, respectively. Hence, the structure of triterpene 1 was determined as lanosta-8,24-diene-3β,15α,21-triol.

Compound 2 gave a molecular ion peak at m/z = 472.3912 in its HR-EI-MS. It corresponded to the molecular formula C₃₁H₅₂O₃, 14 mass units (CH₂) higher than that of 1. The ¹³C-NMR spectrum of 2 (Table [1]) was similar to that of 1 except for the signals of C-23 - C-27 and C-31. In its ¹H-NMR spectrum, the triplet of H-24 at δ = 5.27 in compound 1 disappeared and two signals at δ = 4.83 (1H, br s) and 4.86 (1H, d, J = 1.5 Hz) were observed. These two signals correlated with the carbon signal at δ = 106.5 (C-31) in the HMQC spectrum and correlated with C-24 (δ = 157.0) and a methine carbon C-25 (δ = 34.1) in the HMBC spectrum, which indicated that an olefinic methylene group was located at C-24 and constituted a terminal double bond with C-24 instead of an internal double bond between C-24 and C-25 in 1. Furthermore, compound 2 showed NOE correlations very similar to those of 1 except for the cross-peaks between H-31 at δ = 4.83 and CH₃ - 26 and CH₃ - 27, indicating a terminal double bond at C-24. Except for C-17, C-20, C-21, and C-22, the ¹³C-NMR chemical shifts of compound 2 also closely resembled those of sulphurenic acid, which contained a carboxylic acid functionality at C-21 [11], [15]. From the above evidence, the structure of 2 was accordingly assigned as 24-methylenelanost-8-ene-3β,15α,21-triol.

The HR-EI-MS of compound 3 showed a molecular ion peak at m/z = 412.1157, which corresponded to the molecular formula C₂₂H₂₀O₈. Its ¹H-NMR spectrum exhibited two doublets at δ = 7.27 (1H, J = 1.5 Hz) and 7.92 (1H, J = 1.5 Hz) and a singlet at δ = 6.31 (1H) for aromatic protons in addition to four methoxy groups (δ = 4.08, 4.02, 3.69, and 3.86) and one tertiary methyl group (δ = 2.47). It also displayed two close doublets at δ = 5.99 (1H, J = 1.5 Hz) and 5.98 (1H, J = 1.5 Hz), which correlated with a carbon signal at δ = 101.7 in the HMQC spectrum. Thus, the presence of a dioxymethylene group was deduced. The ¹³C- and DEPT-NMR spectra of 3 showed three methine and eleven quaternary carbon signals in the aromatic region and two signals at δ = 182.4 and 181.6 due to the conjugated carbonyl groups, suggesting a naphthoquinone-type compound with an additional aromatic ring attached. The INADEQUATE (incredible natural abundance double quantum transfer experiment) of 3 revealed the connectivities of C-3-C-4-C-10-C-5-C-6-C-7-C-8-C-9-C-1-C-2, C-2′-C-1′-C-6′-C-5′, and C-5-C-1′, which demonstrated that the additional aromatic ring was attached to C-5 of the naphthoquinone compound. The locations of the dioxymethylene and methoxy groups were determined by the HMBC spectrum. The dioxymethylene protons showed correlations with C-3′ and C-4′ and the protons of four methoxy groups correlated with C-2, C-3, C-2′ and C-5′, respectively, which indicated that the dioxymethylene group was attached to C-3′ and C-4′ and four methoxy groups were located at C-2, C-3, C-2′, and C-5′. Furthermore, cross-peaks of 7-CH ₃/C-6, C-7, C-8, H-6/7-CH₃, and H-8/7-CH₃ revealed that the methyl group was located at C-7. The structure of 3 was further confirmed by NOE difference experiments. The signals of H-6′ and the methyl group at C-7 were enhanced by irradiation of H-6, indicating that the methyl group was located at C-7 and the aromatic substituent at C-5. The NOE correlations of 2′-OCH₃/H-6, H-7′ and H-6′/5′-OCH₃ showed that two methoxy groups in the aromatic substituent were at 2′ and 5′ positions and the dioxymethylene group was located at C-3′ and C-4′. Therefore, the structure of 3 was established as 2,3-dimethoxy-5-(2′,5′-dimethoxy-3′,4′-methylenedioxyphenyl)-7-methyl-[1] [4]-naphthoquinone.

Compound 4 gave the same molecular formula C₂₂H₂₀O₈ as compound 3 from a molecular ion peak at m/z = 412.1155 in its HR-EI-MS. The ¹³C-NMR spectrum of 4 showed two unsaturated carbonyl signals at δ = 182.1 and 182.0, suggesting a naphthoquinone-type compound. Similar to 3, the ¹H-NMR spectrum of 4 exhibited four methoxy groups at δ = 4.09, 4.08, 3.85, and 3.71 and a tertiary methyl group at δ = 2.28. It also displayed one dioxymethylene group at δ = 6.02 and three singlets at δ = 7.90, 7.84, and 6.26 due to the aromatic protons. Its HMBC spectrum revealed that H-5 at δ_H = 7.84 correlated with C-4, C-7, C-9, and C-1′, and H-8 at δ_H = 7.90 correlated with C-1, C-6, C-10, and 7-CH₃, which indicated that the aromatic ring and methyl group were linked to C-6 and C-7, respectively. The structure of 4 was further confirmed by NOE difference experiments. The irradiation of methyl group at C-7 enhanced the signals of H-8, H-6′, and 2′-OCH₃, which revealed that the aromatic substituent was located at C-6 and a proton and a methoxy group were at C-6′ and C-2′, respectively. Besides, another methoxy group located at C-5′ was supported by the NOE correlation observed from H-6′ to 5′-OCH₃. Therefore, the structure of 4 was assigned as 2,3-dimethoxy-6-(2′,5′-dimethoxy-3′,4′-methylenedioxyphenyl)-7-methyl-[1] [4]-naphthoquinone.

Evaluations of biological activity using human leukocytes indicated that three of these four new compounds displayed antioxidative activity depending on stimulant or cell type examined. The rapid production of reactive oxygen species (ROS), i. e., oxidative burst, by activated leukocytes plays as a potent microbicidal mechanism, while over-production of these free radicals also mediates tissue damage during inflammatory responses [16], [17]. Many antioxidants of botanic sources have been reported to exhibit anti-inflammatory effects by inhibiting ROS production in leukocytes. We have previously reported that some ergostanes (zhankuic acids and antcin K) isolated from the basidiomata of A. cinnamomea could decrease ROS production in activated human neutrophils by a lucigenin-amplified chemiluminescence method [13]. Using the same method, we detected extracellular ROS production both in formyl-Met-Leu-Phe (fMLP, a receptor-mediated activator) and phorbol 12-myristate-13-acetate [PMA, a protein kinase c (PKC)-mediated activator] stimulated neutrophils (PMN) and mononuclear cells (MNC) in the presence of these four compounds. fMLP and PMA induced exuberant ROS production up to 5 - 10 fold more than that in resting cells and which was concentration-dependently inhibited by compounds 1, 3, and 4 with IC₅₀ ranging from 3.5 to 25.8 (μM) (Table [2]). Quercetin, an inhibitor for ROS production of plant origin [13], was included as a positive control, and also significantly inhibited fMLP- or PMA-induced ROS production by PMN or MNC (Table [2]). Some of the potency of these compounds was comparable to that of zhankuic acids isolated from A. cinnamomea [13]. For example, compound 1 was the most effective one in the inhibition of PMA-induced oxidative burst both in PMN and MNC with similar efficacy as quercetin. On the contrary, they were all relatively less effective in fMLP-induced oxidative burst (except for compounds 3 and 4 in fMLP-induced PMN activation), indicating that these compounds were more selective in the inhibition of PKC-dependent signaling pathway in PMN activation. Besides, the antioxidative property of these compounds was not due to cytotoxic effects, since no significant cell death could be detected at the end of individual experiments as compared to solvent control. These results indicated that these four newly identified compounds displayed immunomodulating effects in human leukocytes. The anti-inflammatory potency of these compounds depended upon different stimuli (receptor- or non-receptor mediated activator) or cellular model (PMN or MNC) examined.

Acknowledgements

The authors thank Mr. C. H. Hsu for his assistance in the preparation of neutrophil functions. This study was supported, in part, by grants of NSC-93-2320-B-077-004 (Y.C.S), NRICM93-DBCMR-09 (Y.C.S.), and 94-DBCMR9 (W.Y.W.) from National Science Council, National Research Institute of Chinese Medicine, and Tao-Yuan General Hospital, Department of Health, Taiwan, respectively.

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Tun-Tschu Chang

Division of Forest Protection

Taiwan Forestry Research Institute

No. 53, Nai-Hai Road

Taipei 100

Taiwan

Republic of China

Phone: +886-2-23078901

Fax: +886-2-23078755

Email: ttchang@serv.tfri.gov.tw

References

• 1 Chang T T, Chou W N. Antrodia cinnamomea reconsidered and A. salmonea sp. nov. on Cunninghamia konishii in Taiwan.  Bot Bull Acad Sin. 2004;  45 347-52
  PubMedSearch in Google Scholar
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Tun-Tschu Chang

Division of Forest Protection

Taiwan Forestry Research Institute

No. 53, Nai-Hai Road

Taipei 100

Taiwan

Republic of China

Phone: +886-2-23078901

Fax: +886-2-23078755

Email: ttchang@serv.tfri.gov.tw

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