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Optimization of Extraction Conditions for Bakkenolide B from the Leaves of Petasites japonicus by Using Response Surface Methodology
Optimization of Extraction Conditions for Bakkenolide B from the Leaves of Petasites japonicus by Using Response Surface Methodology
Journal of Life Science. 2014. Feb, 24(2): 167-172
Copyright © 2014, Korean Society of Life Science
This is an Open-Access article distributed under the terms of the Creative Commons Attribution Non-Commercial License (http://creativecommons.org/licenses/by-nc/3.0) which permits unrestricted non-commercial use, distribution, and reproduction in any medium, provided the original work is properly cited.
  • Received : January 09, 2014
  • Accepted : February 14, 2014
  • Published : February 28, 2014
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About the Authors
정헌 김
Department of Food Science and Technology, College of Natural Resources & Life Science, Pusan National University, Miryang 627-706, Korea
용민 김
Department of Food Science and Technology, College of Natural Resources & Life Science, Pusan National University, Miryang 627-706, Korea
헌식 정
Department of Food Science and Technology, College of Natural Resources & Life Science, Pusan National University, Miryang 627-706, Korea
영환 최
Department of Horticultural Bioscience, College of Natural Resources & Life Science, Pusan National University, Miryang 627-706, Korea
경필 이
Molecular Inflammation Research Center for Aging Intervention (MIRCA) and College of Pharmacy, Pusan National University, Busan 609- 735, Korea
동순 임
Molecular Inflammation Research Center for Aging Intervention (MIRCA) and College of Pharmacy, Pusan National University, Busan 609- 735, Korea
영근 이
Department of Food Science and Technology, College of Natural Resources & Life Science, Pusan National University, Miryang 627-706, Korea
lyg5354@pusan.ac.kr

Abstract
Optimal conditions for extraction of bakkenolide B from Petasites japonicus leaves were determined by using response surface methodology. A second-order Box-Behnken design representing three extraction temperatures (80, 100, 120℃), three extraction times (30, 45, 60 min), and three solvent pH’s (5, 7, 9) was executed. The efficiency of the extraction conditions was defined using the β-hexosamidase assay by comparing both the bakkenolide B content and its anti-allergic activity expressed as extract inhibition on degranulation. The response surface plot described for the bakkenolide B content showed that the maximum content was predicted as 121.6 μg/g with extraction conditions of 127.1℃, 46.6 min, and pH 7.7. Extraction temperature and time were important factors in determining bakkenolide B content. Using regression analysis, correlation between the inhibition effect of mast cell degranulation and bakkenolide B content was found to be low.
Keywords
Introduction
Petasites genus has been reported to have many sesquiterpene lactones, especially petasin-type and bakkenolide type [4] , and the extracts from this plant have been shown to have anti-allergic and anti-inflammatory effects [5 , 10 , 18] and to inhibit mast cell degranulation [15] . Some pharmacological studies have suggested that petasin and s-petasin were active ingredients in this plant [1 , 5 , 16 , 17] , furthermore, we found that bakkenolide B ( Fig. 1 ), a major component in the leaves of Petasites japonicus , showed significant effects in an ovalbumin-induced asthma model in our previous studies [11] .
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Structure of bakkenolide B.
Recently, the use of herbs containing Petasites genus as dietary supplements and medicines in a form of the extractable material has increased in many countries. In many previous information, only a few solvents such as methanol, ethanol and acetone were generally used [6 , 7 , 14] , but they were must been eliminated from the final food or medicine products. Water extraction is profitable in developing a functional beverage using the extracts derived from Petasites japo-nicus , even though the most target components have low water solubility. Extraction efficiency is affected by multiple parameters, including temperature, time, solvent polarity, and etc. Response surface methodology (RSM) described originally by Box and Wilson [2] is effective for optimizing multiple, interrelated parameters.
This study was designed to determine the optimum conditions of water extraction for bakkenolide B and its anti-allergic effects (described as inhibition effects on degranulation) of extracts from the leaves of Petasites japonicus by using RSM.
Materials and Methods
- Materials
Petasites japonicus leaves were collected in June 2012 in Chungdo province (South Korea) and a voucher specimen (accession number MW-PRDR-11) was deposited at the Herbarium of Pusan National University.
- Experimental design
Box-behnken design was used to investigate the effects of three independent parameters, extraction temperature (X 1 ), time (X 2 ) and pH (X 3 ) on the bakkenolide B content (Y 1 , extracted content from the leaves, μg/g) and inhibition effects on degranulation (Y 2 ). The independent parameters were coded at three levels (−1, 0, and 1), and the complete design consisted of 15 experimental points including three replicates of center points as shown in Table 1 .
Box-Behnken experimental design used for the extraction procedure ofPetasites japonicas
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Box-Behnken experimental design used for the extraction procedure of Petasites japonicas
- Analysis of regression
Triplicate tests were performed at all experimental points in randomized order. Each extracts were analyzed for dependent parameters (response variables), bakkenolide B content (Y 1 ) and inhibition effects on degranulation (Y 2 ). Mean values were analyzed to fit the following second order polynomial models to response Y variables.
The model proposed for each response of Y is
Y=b 0 +b 1 X 1 +b 2 X 2 +b 3 X 3 +b 11 X 1 2 +b 21 X 2 X 1 +b 22 X 2 2 +b 31 X 3 X 1 +b 32 X 3 X 2 +b 33 X 3 :
where X1 and X2 correspond to independent parameters, and bn values represent corresponding regression coefficients [12] . SAS statistical analysis system was used to predict models through regression analysis and variance analysis (ANOVA). When the results showed a saddle point in response surfaces, optimal conditions were determined using analysis of ridge.
- Extraction
The leaves (400 g) of Petasites japonicas were blended and then extracted with 800 ml of distilled water on the basis of pre-established conditions, followed by filteration using Whatman No. 2.
- Determination of bakkenolide B content
The extracts (100 ml) from 15 experimental conditions were extracted with 100 ml of n-hexane in separation funnels and evaporated using a rotary vacuum evaporator at 50℃ to 20 ml of volume. The solution filtered by membrane filter (Dismic-13JP, 0.50 μm PTFE, ADVANTEC, Japan) was analyzed by HPLC system. The system consisted of HPLC (9600, Younglin, Korea), C 18 column (250×4.6 mm, 5 μm, Agilent, USA), mobile solvent consisted of 28% THF, 12% acetonitrile and 60% water, and detector at 215 nm. The bakkenolide B content was calculated by application the data from HPLC analysis to standard curve which established using bakkenolide B isolated and identified from Petasites japonicas leaves.
- Measurement of degranulation
Degranulation was estimated by measuring β-hexosamidase release, as previously described by Dearman et al. [3] . RBL-2H3 cells (2×10 5 cells/well in 24-well plates) were sensitized with 0.5 mg/ml monoclonal anti-dinitrophenyl specific mouse IgE (DNP-IgE, D8406, Sigma, St. Louis, MO) overnight at 37℃ in a 5% CO 2 incubator. Cells were washed twice with PIPES buffer (pH 7.2), containing 25 mM PIPES, 110 mM NaCl, 5 m M KCl, 5.6 mM glucose, 0.4 mM MgCl 2, 0.1% BSA, and 1 mM CaCl 2 to remove DNP-IgE before stimulation, and then incubated in PIPES buffer or extract-containing PIPES buffer in 500 μl at 37℃ for 30 min. Extracts were diluted 10 times by the PIPES buffer. After washing with PIPES buffer, cells were incubated with 10 mg/ml of human dinitrophenyl albumin (DNP-hAb, A6661 Sigma, St. Louis, MO) for a further 30 min at 37℃ to induce degranulation. Aliquots (50 μl) of medium were then transferred to a 96-well microplate and incubated for 60 min with 50 ml of 1 mM 4-nitrophenyl N-acetyl-β-D-glucosaminide (N9376, Sigma, St. Louis) in 0.1 M citrate buffer (pH 4.5). Cells were lysed with an equal volume of 0.5% Triton X-100 at 37℃ for 1 h. Total β-hexosamidase activity in RBL-2H3 mast cells was measured at the same rate. The reaction was terminated by adding 250 ml of 0.05 M sodium carbonate buffer (pH 10.0; 0.05 M Na 2 CO 3 /0.05MNaHCO 3 ). Absorbances (OD) at 410 nm were measured using a microplate reader. Degranulation (%) was calculated by the ratio of released β-hexosamidase in stimulated cells to total β-hexosamidase activity [11 , 13] .
- Isolation and identification of bakkenolide B
The fresh leaves of P. japonicus (425.36 g) were chopped to a fine particle with an electric mixer (HMF-3100S, Hanil Electric, Seoul, Korea) and then extracted at room temperature with 75% EtOH. The EtOH was then removed using a rotary evaporator and the remaining aqueous extract was fractionated successively with BuOH, EtOAc, and n-hexane. The hexane extract (2.6728 g) so obtained was evaporated in vacuo, and the residue was chromatographed on a silicagel (40 μm, Baker, NJ) column (100×4.0 cm) using a step gradient 2.5%, 15%, 25% acetone in dichloromethylene and 15% and 25% MeOH in chloroform to obtain 62 fractions Fraction 9 (MWLSH9, 304.9 mg) was separated on a Sephadex column (100×3.0 cm) using MeOH as eluant to obtain 7 fractions. The fraction 3 (MWLSH9IC, 209.7 mg) was further separated on a Sephadex column (100×3.0 cm) using MeOH to obtain five fractions. Fractions 2 and 3 (MWLSH9ICIB, 202.3 mg) were passed through a silicagel column (100×4.0 cm) using 1.5% acetone in CH 2 Cl 2 as eluant to yield bakkenolide B (173.8 mg). Pure bakkenolide B was identified by HPLC on a Phenomenex Luna C18 column (Phenomenex, 150×4.6 mm ID; 5 μm particle size) using an acetonitrile-water reagent alcohol gradient at a flow rate of 1.0 ml per minute. Bakkenolide B isolated from P. japonicus leaves was identified using 1 H, 13 C, and distortionless enhancement of polarization transfer nuclear magnetic resonance spectroscopy in CDCl 3 by comparison with previously reported spectral data [4] .
Results and Discussion
Bakkenolide B content ( Y1 ) and inhibition effects on degranulation ( Y2 ) of extracts for each extraction of variable combinations were shown as Table 2 . The regression coefficients were calculated by employing a least squares technique to predict second order polynomial models for Y1 and Y2 .
Experimental data for response parameters ofPetasites japonicasleave extracts in relation to the extraction conditions
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Experimental data for response parameters of Petasites japonicas leave extracts in relation to the extraction conditions
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Response surface plots for the effects of extraction conditions on bakkenolide B content of extracts from Petasites japonicus: (A) Time and temperature; (B) pH and time; (C) pH and temperature
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Response surface plots for the effects of extraction conditions on the inhibition effect on degranulation of extracts from Petasites japonicus : (A) Time and temperature; (B) pH and time; (C) pH and temperature. *Inhibition effects are expressed as the remained number from that 10% minus β-hexosamidase release rate (%).
- Bakkenolide B content
The analysis of variance of bakkenolide B content ( Y1 ) was significant ( p <0.05) with high correlation coefficient ( R2 = 0.8949), and the predicted model for Y1 was
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The model indicated that extraction temperature had the most linear effect on bakkenolide B content as it showed the largest positive linear coefficient. The response surface plot described for bakkenolide B content ( Fig. 1 ) showed that the maximum bakkenolide B content was predicted as 121.6 μg/g at the extraction conditions of 127.1℃, 46.6 min, and pH 7.76. The extraction temperature and time were important factors, whereas extraction pH had the lowest significant effects on bakkenolide B content. These results partly disagreed the previous studies that the extraction time had no significant effect on the extraction of soluble solids from various natural products [2 , 6 , 14] . On the other hand, the bakkenolide B content of extracts were very low as compared with s-petasin (up to 680 μg/g) which extracted by ethanol in our previous studies [8] . It may be originated from the different polarities of water and ethanol, while both bakkenolide B and s-petasin were nonpolar compounds.
- Inhibition effects on degranulation
Inhibition effects are expressed as the remained number from that 10% minus β-hexosamidase release rate (%). The regression equation of inhibition effects on degranulation ( Y2 ) had no significant ( p >0.05) and showed low correlation coefficient ( R2 =0.5095). The model for Y2 was predicted as follows:
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The maximum responses predicted peak for the variables about inhibition effects on degranulation indicated that the value of the saddle point was 6.44% at the extraction temperature (93.61℃), the extraction time (46.40 min), and the extraction pH 6.93. In the present study, 400 g of leaves were used as materials and adjusted to volume of 800 ml, so the bakkenolide B content of the leaves ( Table 2 ) was diluted to half (briefly summed up 30 μg/ml) in experimental extracts. In our previous study [11] , we found that bakkenolide B inhibited antigen-induced degranulation in RBL-2H 3 mast cells and the induction of inducible NOS and COX-2 in mouse peritoneal macrophages, and observed significant effects of bakkenolide B in 10 μg/ml and lower concentrations in RBL-2H3 mast cells. Therefore, bakkenolide B concentration of most experimental extracts were upper than minimum concentration level which could be effective on degranulation, though the extracts had no significant effects in the analysis of variance. These results may be inferred that the bakkenolide B was extracted at hot state from the leaves of Petasites japonicas, after then insoluble and not homogeneous in extract at room temperature, so it had showed irregular effects. In future manufacturing of beverage by using Petasites japonicas , it will be remains as a next work that the sub-ingredients having a role of surfactant must be added in the objective products.
Acknowledgements
This work was supported by a 2-Year Research Grant of Pusan National University
References
Bickel D. , Roder T. , Bestmann H. J. , Brune K. 1994 Identification and characterization of inhibitors of peptido-leukotriene-synthesis from Petasites hybridus Planta Medica 60 318 - 322    DOI : 10.1055/s-2006-959492
Box G. E. P. , Wilson K. G. 1951 On the experimental attainment of optimum conditions J R Stat Soc 13 1 - 45
Dearman R. J. , Skinner R. A. , Deakin N. , Shaw D. , Kimber I. 2005 Evaluation of an in vitro method for the measurement of specific IgE antibody responses: the rat basophilic leukemia (RBL) cell assay Toxicology 206 195 - 205    DOI : 10.1016/j.tox.2004.08.007
Dong X. W. , Li R. J. , Gao X. , Row K. H. 2010 Bakkenolides from Petasites tatewakianus Fitoterapia 81 153 - 156    DOI : 10.1016/j.fitote.2009.08.013
Fiebich B. L. , Grozdeva M. , Hess S. , Hull M. , Danesch U. , Bodensieck A. 2005 Petasites hybridus extracts in vitro inhibit COX-2 and PGE2 release by direct interaction with the enzyme and by preventing p42/44 MAP kinase activation in rat primary microglial cells Planta Medica 71 12 - 19    DOI : 10.1055/s-2005-837744
Ko S. R. , Kim S. C. , Choi K. J. 1992 Extract yields and saponin comtent of red ginseng extracts prepared with various concentrations of ethanol Korean J Pharmacogn 23 24 - 28
Kwon J. H. , Bélanger J. M. R. , Jocelyn Pare J. R. 2003 Optimization of microwave-assisted extraction (MAP) for ginseng components by response surface methodology J Agric Food Chem 51 1807 - 1810    DOI : 10.1021/jf026068a
Lee D. W. , Lee S. Y. , Chung H. S. , Choi Y. W. , Im D. S. , Lee Y. G. 2013 Optimization of a process for extraction of petasin from Petasites japonicus leaves by response surface methodology J Life Sci 23 (11) 1360 - 1364    DOI : 10.5352/JLS.2013.23.11.1360
Lee H. N. , Kundu J. K. , Cha Y. N. , Surh Y. J. 2013 Resolvin D1 stimulates efferocytosis through p50/p50-mediated suppression of tumor necrosis factor-alpha expression J Cell Sci 126 4037 - 4047    DOI : 10.1242/jcs.131003
Lee J. S. , Yang E. J. , Yun C. Y. , Kim D. H. , Kim I. S. 2011 Suppressive effect of Petasites japonicus extract on ovalbumin-induced airway inflammation in an asthmatic mouse model J Ethnopharmacol 133 551 - 557    DOI : 10.1016/j.jep.2010.10.038
Lee K. P. , Kang S. , Park S. J. , Choi Y. W. , Lee Y. G. , Im D. S. 2013 Anti-allergic and anti-inflammatory effects of bakkenolide B isolated from Petasites japonicus leaves J Ethnopharmacol 148 890 - 894    DOI : 10.1016/j.jep.2013.05.037
Liu F. F. , Ang C. Y. W. , Springer D. 2000 Optimization of extraction conditions for active components in Hypericum perforatum using response surface methodology J Agric Food Chem 48 3364 - 3371    DOI : 10.1021/jf991086m
Lu Y. , Son J. K. , Chang H. W. 2012 Saucerneol F, a new lignan isolated from Saururus chinensis, attenuates degranulation via phospholipase Cg1 inhibition and eicosanoid generation by suppressing MAP kinases in mast cells Biomol Ther 20 526 - 531    DOI : 10.4062/biomolther.2012.20.6.526
Park N. Y. , Lee G. D. , Jeong Y. J. , Kwon J. H. 1998 Optimization of extraction conditions for Physicochemical properties of ethanol extracts from Chrysanthemum boreale J Korean Soc Food Sci Nutr 27 (4) 585 - 590
Shimoda H. , Tanaka J. , Yamada E. , Morikawa T. , Kasajima N. , Yoshikawa M. 2006 Anti type I allergic property of Japanese butterbur extract and its mast cell degranulation inhibitory ingredients J Agric Food Chem 54 2915 - 2920    DOI : 10.1021/jf052994o
Thomet O. A. , Wiesmann U. N. , Schapowal A. , Bizer C. , Simon H. U. 2001 Role of petasin in the potential anti-inflammatory activity of a plant extract of Petasites hybridus Biochem Pharmacol 61 1041 - 1047    DOI : 10.1016/S0006-2952(01)00552-4
Wang G. J. , Wu X. C. , Lin Y. L. , Ren J. , Shum A. Y. , Wu Y. Y. 2002 Ca2+ channel blocking effect of iso-S-petasin in rat aortic smooth muscle cells Eur J Pharmacol 445 239 - 245    DOI : 10.1016/S0014-2999(02)01764-8
Zhang F. J. , Wang Q. , Guo M. L. 2011 Anti-allergic effects of total bakkenolides from Petasites tricholobus in ovalbumin-sensitized rats Phytother Res 25 116 - 121    DOI : 10.1002/ptr.3237