Extraction and determination of triterpenoids from Phaeoporus
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- Σουσάννα Μαλαξός
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1 ~ Mycosystema ( ) 95% 24h 6mL100mg mg/g 94.92mg/g Q939.5 A 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 China; 2 Graduate School of Chinese Academy of Sciences, Beijing , 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.O * Corresponding author. guold@sun.im.ac.cn
2 3 397 Phaeoporus obliquus (Pers.:Fr.) J. Schroet.chaga BasidiomycotaHymenomycetes Aphyllophorales Hymenochaetaceae Phaeoporus Rzymowska 1998 Wasser & Weis 1999 Kim et al. 2005Rasina 2002 Raghuvar Gopal et al Ichimura et al Kashiwada et al Aiken & Chen 2005Pisha et al Liu et al g/L g/L Kim et al HPLC HPLC BioSpec-1601
3 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 mL 5% g 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 PDA 28 7d cm 0.4cm 50mL 250mL r/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 g VB 1 ph mL 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 r/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 g VB 1 ph mg 6mL 95% 24h 0.1mL 3
4 mg 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 nm 900nm 1 551nm 1n 3 y x % S.D % S.D LOD 8µg/mL 8µg/mL 88µg/mL OD r ε L/mol cm S.D R.S.D. 1.4% n min OD p<0.05 S.D R.S.D. 3.9% 70min 70min µg/mL 50µg/mL 75µg/mL R.S.D. n 6 3.3% Table 1 Linearity and repeatability of UV-Vis spectrophotometry Regression equation y = S.D. of slope (n = 3) S.D. of intercept (n = 3) % 95% Confidence intervals of slope to % 95% Confidence intervals of intercept to r (Correlation coefficient) ε (L/mol cm) LOD (µg/ml) 8 Linearity range (µg/ml) 8 to 88 S.D. & R.S.D. (%) of repeatability (n = 6) & 1.4 %
5 % 94.9% % 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. (%) (n = 3) Table 3 Recoveries (%) of betulin from samples (n = 3) (µg) Sample (µg) Amount added (µg) x ± S.D. Recovery (%) R.S.D. (%) ± ± ± ± % h 36h 12h 24h mg/g 94.92mg/g
6 (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) a* b c a b c c S.D R.S.D. (%) * 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) Amount of extraction (mg/g) a* ab b S.D R.S.D. (%) * 5. Note: * Values in the same line with different letters indicate significant difference at p < 0.05 level g h 1g 100mg
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