26 3 396~403 2007 Mycosystema ( 1 100080 2 100049) 95% 24h 6mL100mg -- 59.86mg/g 94.92mg/g Q939.5 A 1672-6472 2007 03-0396-0403 Extraction and determination of triterpenoids from Phaeoporus obliquus LIN Geng-Shen 1, 2 ZHANG Xiao-Qing 1 WEI Tie-Zheng 1 GUO Liang-Dong 1* ( 1 Systematic Mycology and Lichenology Laboratory, Institute of Microbiology, Chinese Academy of Sciences, Beijing 100080 China; 2 Graduate School of Chinese Academy of Sciences, Beijing 100049, China) Abstract: A procedure of triterpenoid (TTP) extraction from Phaeoporus obliquus was developed. The result of screening of seven solvents proved that the optimal solvent for extraction of total TTP from P. obliquus was isopropanol with the extraction time of 24h. In this extraction method less isopropanol (6mL) and dry mycelia (100mg) were used and lots of samples were able to be extracted simultaneously. Using vanillin-acetic acid-perchloric acid spectrophotometry to detect total TTP in P. obliquus has proved to be simple, speedy, accurate, and repeatable. The detection result showed that TTP contents in natural sclerotia and fermentative mycelia were 59.86mg/g and 94.92mg/g respectively showing the TTP content in fermentative mycelia was higher than that in natural sclerotia. The results suggest that the product development of TTP from fermentative mycelia of P. obliquus can be an alternative to the collection of natural sclerotia of the fungus. Key words: Betulin, mycelia, sclerotia, spectrophotometry No.O654041011 * Corresponding author. E-mail: guold@sun.im.ac.cn 2007-01-12 2007-04-19
3 397 Phaeoporus obliquus (Pers.:Fr.) J. Schroet.chaga BasidiomycotaHymenomycetes Aphyllophorales Hymenochaetaceae Phaeoporus 2005 16 2002 200 Rzymowska 1998 Wasser & Weis 1999 Kim et al. 2005Rasina 2002 Raghuvar Gopal et al. 2005 Ichimura et al. 1998 Kashiwada et al. 2004 Aiken & Chen 2005Pisha et al. 1995 Liu et al. 2004 0.83 19.80g/L20062006 0.87g/L Kim et al. 2005 HPLC HPLC 2004 2005 1 1.1 1.1.1 BioSpec-1601
398 26 1.1.2 2004 8 4243 4451 1.2 Betulin Sigma Chem. Co. St. Louis USA 160µg/mL 0.10mL 0.15mL 0.20mL 0.25mL, 0.30mL 0.35mL 0.40mL 0.45mL 0.50mL 0.55mL 5mL 100 0.20mL 5%- 0.500g 10mL 0.80mL 70 15min 5mL 400nm 900nm 551nm 1cm OD OD n 3 r ε limit of detection LOD µg/ml 3 δ/s, 3 δ s 6 150min 10min 551nm OD 25µg/mL 50µg/mL 75µg/mL 6 0.1mL 16µg OD % Recovery %Found - M 0 100/Added M 0 Added Found 3 1.3 PDA 28 7d 20 0.4cm 0.4cm 50mL 250mL 28 150r/min 10d 1000mL 20g 6g 5g 3g 1.5g MgSO 4 7H 2 O 0.2g K 2 HPO 4 0.4g KH 2 PO 4 0.01g VB 1 ph 6.0 0.5mL 1mol/L 13.20g CaCl 2 2H 2 O 2.40g MnSO 4 4H 2 O 2.40g ZnSO 4 7H 2 O 0.48g CuSO 4 5H 2 O 8mL 30mg 72mL 250mL 28 150r/min 12d 1000mL 80g 2.5g 2.5g 1.5g MgSO 4 7H 2 O 0.2g K 2 HPO 4 0.4g KH 2 PO 4 0.01g VB 1 ph 6.0 60 40 100mg 6mL 95% 24h 0.1mL 3
3 399 100mg 12h 24h 36h 1.4 SPSS 11.5 SPSS Inc. Chicago IL USA ANOVA xstandard deviation S.D. Relative standard deviation R.S.D. % 100 S.D./x 2 2.1 400nm 900nm 1 551nm 1n 3 y 0.0104x - 0.0765 95% 0.0091 0.0116 S.D. 0.0005 95%-0.1457-0.0073 S.D. 0.0279 LOD 8µg/mL 8µg/mL 88µg/mL OD r 0.9805 ε 1.8815 10 4 L/mol cm S.D. 0.414 R.S.D. 1.4% n 6 0 70min OD p<0.05 S.D. 0.017 R.S.D. 3.9% 70min 70min 2 3 25µg/mL 50µg/mL 75µg/mL R.S.D. n 6 3.3% Table 1 Linearity and repeatability of UV-Vis spectrophotometry Regression equation y = 0.0104-0.0765 S.D. of slope (n = 3) 0.0005 S.D. of intercept (n = 3) 0.0279 95% 95% Confidence intervals of slope 0.0091 to 0.0116 95% 95% Confidence intervals of intercept -0.1457 to -0.0073 r (Correlation coefficient) 0.9805 ε (L/mol cm) 1.8815 10 4 LOD (µg/ml) 8 Linearity range (µg/ml) 8 to 88 S.D. & R.S.D. (%) of repeatability (n = 6) 0.414 & 1.4 %
400 26 3 99.9% 94.9% 12 97.3% R.S.D. 2.2% 2 -(n = 6) Table 2 Intra- and inter-day precision in different concentrations of standard solution by UV-Vis spectrophotometry (n = 6) Sample concentration (µg/ml) n OD x Intra-day S.D. (%)R.S.D. (%) OD x Inter-day S.D. (%)R.S.D. (%) 25 6 0.131 0.004 2.7 0.136 0.004 2.6 50 6 0.451 0.009 2.0 0.441 0.009 2.0 75 6 0.681 0.021 3.1 0.656 0.021 3.3 3 (n = 3) Table 3 Recoveries (%) of betulin from samples (n = 3) (µg) Sample (µg) Amount added (µg) x ± S.D. Recovery (%) R.S.D. (%) 15.57 16 31.52 ± 1.15 99.9 3.7 31.14 16 46.11 ± 0.82 97.8 1.8 46.71 16 60.62 ± 0.75 96.7 1.2 62.28 16 74.31 ± 1.45 94.9 1.9 2.2 95% 4 5 24h 36h 12h 24h 2.3 59.86mg/g 94.92mg/g
3 401 4 (n = 3) Table 4 Contents of triterpenoids extracted from mycelia of Phaeoporus obliquus with different extraction solvents (n = 3) (95%) Extraction solvents Ethanol (95%) Isopropanol Ethyl acetate Acetone Methanol Hexane Chloroform Amount of extract (mg/g) 2.803 a* 5.974 b 0.699 c 2.430 a 5.618 b 0.497 c 0.518 c S.D. 0.012 0.133 0.007 0.012 0.058 0.002 0.009 R.S.D. (%) 0.4 2.2 1.1 0.5 1.0 0.4 1.7 * 5 Note: * Values in the same line with different letters indicate significant difference at p < 0.05 level. 5 (n = 3) Table 5 Contents of triterpenoids extracted from mycelia of Phaeoporus obliquus with isopropanol in different extraction time (n = 3) Extraction time (h) 12 24 36 Amount of extraction (mg/g) 4.774 a* 5.982 ab 6.874 b S.D. 0.065 0.127 0.129 R.S.D. (%) 1.4 2.1 1.9 * 5. Note: * Values in the same line with different letters indicate significant difference at p < 0.05 level. 3 2004 20052003 2005 1 10g 0.5 1.5h 1g 100mg
402 26 210nm -- 551nm 210nm - - 100mg --2005 14.8mg/g 0.1 2mg/g 20032004 2006 59.86mg/g 94.92mg/g 30mg/g Shih et al. 2006 9.67mg/g 2005 14.8 mg/g 2004 0.1 2mg/g 2003 [REFERENCES] Aiken C, Chen CH, 2005. Betulinic acid derivatives as HIV-1 antivirals. TRENDS in Molecular Medicine, 11: 31~36 Chen YQ Li Y Zhou LJ, 2006. Study on the conditions of liquid culture of Inonotus obliquus. Journal of Jilin Agricultural University, 28: 358~360,368 (in Chinese) Huang NL, 2002. Mysterious folk medicinal fungi in Russia - Inonotus obliquus. Edible Fungi of China, 21: 7~8 (in Chinese) Huang SM, Yang XL, Huang J, Xu JL, Zhu HS, 2004. Method for the rapid quantification of triterpenoids in Ganoderma lucidum by ultraviolet spectrometry. Transactions of Beijing Institute of Technology, 24: 555~558 (in Chinese) Ichimura T, Watanabe O, Maruyama S, 1998. Inhibition of HIV-1 protease by water-soluble lignin-like substance from an edible mushroom, Fuscoporia obliqua. Bioscience Biotechnology and Biochemistry, 62: 575~577 Kashiwada Y, Sekiya M, Ikeshiro Y, Fujioka T, Kilqore NR, Wild CT, Allaway GP, Lee KH, 2004. 3-O-Glutaryl-dihydrobetulin and related monoacyl derivatives as potent anti-hiv agents. Bioorganic & Medicinal Chemistry Letters, 14: 5851~5853 Kim YO, Han SB, Lee HW, Ahn HJ, Yoon YD, Jung JK, Kim HM, Shin CS, 2005. Immuno-stimulating effect of the endo-polysaccharide produced by submerged culture of Inonotus obliquus. Life Sciences, 77: 2438~2456 Liu WK, Ho JC, Cheung FW, Liu BP, Ye WC, Che CT, 2004. Apoptotic activity of betulinic acid derivatives on murine melanoma B16 cell line. European Journal of Pharmacology, 498: 71~78
3 403 Ma L, Wu F, Chen RY, 2003. Analysis of triterpene constituents from Ganoderma lucidum. Acta Pharmaceutica Sinica, 38: 50~52 (in Chinese) Pisha E, Chai H, Lee IS, Chagwedera TE, Farnsworth NR, Cordell GA, Beecher CW, Fong HH, Kinghorn AD, Brown DM, Wani MC, Wall ME, Hieken TJ, Gupta TK, Pezzuto JM, 1995. Discovery of betulinic acid as a selective inhibitor of human melanoma that functions by induction of apoptosis. Nature Medicine, 1: 1046~1051 Raghuvar Gopal DV, Narkar AA, Badrinath Y, Mishra KP, Joshi DS, 2005. Betulinic acid induces apoptosis in human chronic myelogenous leukemia (CML) cell line K-562 without altering the levels of Bcr-Abl. Toxicology Letters, 155: 343~351 Rasina LN, 2002. Effect of cryosubstance Chagi on deposition or isolation of 90Sr and on the effect of prolonged external exposure to gamma-radiation. Radiatsionnaia Biologiia Radioecologiia, 42: 399~403 (in Russian) Rzymowska J, 1998. The effect of aqueous extracts from Inonotus obliquus on the mitotic index and enzyme activities. Bollettino Chimico Farmaceutico, 137: 13~15 Shih IL, Pan K, Hsieh C, 2006. Influence of nutritional components and oxygen supply on the mycelial growth and bioactive metabolites production in submerged culture of Antrodia cinnamomea. Process Biochemistry, 41: 1129~1135 Sun H, Zhang Z, 2005. Spectrophotometric methods to determine the grand total of the triterpenoids in Betula. Journal of Nanjing Forestry University (Natural Sciences Edition), 29: 110~112 (in Chinese) Wang ZH, Huo YF, Wang B, Shen JW, 2006. Submerged fermentation of Phaeoporus obliquus. Mycosystema, 25: 461~467 (in Chinese) Wasser SP, Weis AL, 1999. Therapeutic effect of substances occurring in higher Basidiomycetes mushrooms: a modern perspective. Critical Review in Immunology, 19: 65~96 Xiang XY, Gu Q, Zheng WF, Zhang XP, Wei JC, 2006. Non-polar constituents of Inonotus obliquus in wild and submerge culture. Chinese Traditional and Herbal Drug, 37: 670~672 (in Chinese) Xu R, Xu J, 2005. UV Spectrophotometric determination of triterpene in Poria cocos (Schw.) Wolf. Chinese Journal of Pharmaceutical Analysis, 25: 449~451 (in Chinese) Zhang XQ, Dai YC, 2005. Flora Fungorum Sinicorum Vol. 29 Hymenochaetaceae. Beijing: Science Press. 92~93 (in Chinese) Zhang Z, Sun H, 2004. Determination of betulin content from bark of Betula platyphylla Suk. by HPLC. Chemistry and Industry of Forest Products, 24: 61~63 (in Chinese) 2006.. 28: 358~360 368 2002.. 21: 7~8 2004.. 24: 555~558 2003.. 38: 50~52 2005.. ( ) 29: 110~112 2006.. 25: 461~467 2006.. 37: 670~672 2005.. 25: 449~451 2005.... 92~93 2004.. 24: 61~63