Optimization of Extraction Conditions for Bakkenolide B from the Leaves of Petasites japonicus by Using Response Surface Methodology

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Optimization of Extraction Conditions for Bakkenolide B from the Leaves of Petasites japonicus by Using Response Surface Methodology

Jung-Hun Kim

¹

, Yong-Min Kim

¹

, Hun-Sik Chung

¹

, Young-Whan Choi

²

, Kyoung-Pil Lee

³

, Dong-Soon Im

³

and Young-Guen Lee

¹

*

1

Department of Food Science and Technology, College of Natural Resources & Life Science, Pusan National University, Miryang 627-706, Korea

2

Department of Horticultural Bioscience, College of Natural Resources & Life Science, Pusan National University, Miryang 627-706, Korea

3

Molecular Inflammation Research Center for Aging Intervention (MIRCA) and College of Pharmacy, Pusan National University, Busan 609- 735, Korea

Received January 9, 2014 /Revised February 10, 2014 /Accepted February 14, 2014

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 ex- traction 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-hexosami- dase assay by comparing both the bakkenolide B content and its anti-allergic activity expressed as ex- tract 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 bakke- nolide B content. Using regression analysis, correlation between the inhibition effect of mast cell de- granulation and bakkenolide B content was found to be low.

Key words : Anti-allergy, bakkenolide-B, optimum extraction conditions, Petasites japonicus, response surface methodology

*Corresponding author

*Tel : +82-55-350-5354, Fax : +82-55-350-5359

*E-mail : [email protected]

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.

Journal of Life Science 2014 Vol. 24. No. 2. 167~172 DOI : http://dx.doi.org/10.5352/JLS.2014.24.2.167

Fig. 1. Structure of bakkenolide B.

Introduction

Petasites genus has been reported to have many sesqui- terpene 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 pharmaco- logical studies have suggested that petasin and s-petasin were active ingredients in this plant [1, 5, 16, 17], fur- thermore, we found that bakkenolide B (Fig. 1), a major com- ponent in the leaves of Petasites japonicus, showed significant effects in an ovalbumin-induced asthma model in our pre- vious studies [11].

Recently, the use of herbs containing Petasites genus as dietary supplements and medicines in a form of the ex- tractable 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 func- tional 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 con-

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Table 1. Box-Behnken experimental design used for the extraction procedure of

Petasites japonicas

Experiment

number

Extraction temperature (℃, X1) Extraction time (min, X2) Extraction pH (X3)

Coded Actual Coded Actual Coded Actual

12 34 5 67 89 1011 1213 14 15

-1-1 11 0 00 -10 -11 10 0 0

8080 120120 100 100100 10080 12080 120100 100 100

-11 -11 -1 -11 10 00 00 0 0

3060 3060 30 3060 6045 4545 4545 45 45

00 00 -1 -11 -11 -11 10 0 0

7.07.0 7.07.0 5.0 9.05.0 9.05.0 5.09.0 9.07.0 7.0 7.0

ditions of water extraction for bakkenolide B and its anti-al-

lergic effects (described as inhibition effects on de- granulation) 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.

Analysis of regression

Triplicate tests were performed at all experimental points in randomized order. Each extracts were analyzed for de- pendent parameters (response variables), bakkenolide B con- tent (Y

1

) and inhibition effects on degranulation (Y

2

). Mean values were analyzed to fit the following second order poly-

nomial models to response Y variables.

The model proposed for each response of Y is

where X

1

and X

2

correspond to independent parameters, and b

n

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 re- sponse 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 ana-

lyzed 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

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Table 2. Experimental data for response parameters of

Petasites japonicas

leave extracts in relation to the extraction conditions

Extraction condition Response variables Temperature

(℃, X1) Time (min, X2) pH

(X3)

Bakkenolid B content (μg/g, Y1)

Inhition effect on degranulation

(Y2) 8080

120120 100 100100 10080 12080 120100 100 100

3060 3060 30 3060 6045 4545 4545 45 45

7.07.0 7.07.0 5.0 9.05.0 9.05.0 5.09.0 9.07.0 7.0 7.0

117.146.1 84.689.6 95.4 60.686.3 120.6 66.0 109.2 75.7 103.5 85.9 94.6 81.6

5.167.38 4.664.85 5.62 7.304.01 4.164.85 2.483.50 1.968.58 7.56 2.83

B content was calculated by application the data from HPLC

analysis to standard curve which established using bakkeno- lide B isolated and identified from Petasites japonicas leaves.

Measurement of degranulation

Degranulation was estimated by measuring β-hexosami- dase release, as previously described by Dearman et al. [3].

RBL-2H3 cells (2×10

⁵

cells/well in 24-well plates) were sensi- tized with 0.5 mg/ml monoclonal anti-dinitrophenyl specific mouse IgE (DNP-IgE, D8406, Sigma, St. Louis, MO) over- night at 37

^o

C 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 stim- ulation, and then incubated in PIPES buffer or extract-con- taining PIPES buffer in 500 µl at 37

^o

C 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

^o

C to induce degranulation. Aliquots (50 μl) of medium were then trans- ferred 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

^o

C 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

). Absorban- ces (OD) at 410 nm were measured using a microplate reader. Degranulation (%) was calculated by the ratio of re- leased β-hexosamidase in stimulated cells to total β-hexosa- midase 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 temper- ature 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 silica- gel (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 en- hancement of polarization transfer nuclear magnetic reso- nance spectroscopy in CDCl

3

by comparison with previously reported spectral data [4].

Results and Discussion

Bakkenolide B content (Y

1

) and inhibition effects on de-

granulation (Y

2

) of extracts for each extraction of variable

combinations were shown as Table 2. The regression co-

efficients were calculated by employing a least squares tech-

nique to predict second order polynomial models for Y

1

and

Y

2

.

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A B C

Fig. 2. 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.

A B C

Fig. 3. 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 (Y

1

) was significant (p<0.05) with high correlation coefficient (R

²

= 0.8949), and the predicted model for Y

1

was

Y

1

=-134.626250+3.954125X

1

+2.163000X

2

-23.451250X

3

-0.005 131X

12

-0.043933X

2

X

1

-0.001567X

22

-0.077312X

3

X

1

+0.4595 83X

3

X

2

+0.762500X

32

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 μ g/g at the extraction conditions of 127.1℃, 46.6 min, and pH 7.76. The extraction temperature and time were im- portant factors, whereas extraction pH had the lowest sig- nificant effects on bakkenolide B content. These results part- ly 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 com- pared 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 (Y

2

) had no significant (p>0.05) and showed low correlation coefficient (R

²

=0.5095). The model for Y

2

was predicted as follows:

Y

2

=-47.441563+0.7178960X

1

-0.013917X

2

+5.9427080X

3

-0.003607X

12

-0.001692X

2

X

1

+0.002809X

22

+0.005188X

3

X

1

-0.012750X

3

X

2

-0.420729X

32

The maximum responses predicted peak for the variables

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about inhibition effects on degranulation indicated that the value of the saddle point was 6.44% at the extraction temper- ature (93.61℃), the extraction time (46.40 min), and the ex- traction 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 bakkeno- lide B inhibited antigen-induced degranulation in RBL-2H3 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 concen- trations 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 ho- mogeneous in extract at room temperature, so it had showed irregular effects. In future manufacturing of beverage by us- ing Petasites japonicas, it will be remains as a next work that the sub-ingredients having a role of surfactant must be add- ed in the objective products.

Acknowledgement

This work was supported by a 2-Year Research Grant of Pusan National University.

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초록：반응표면분석법에 의한 머위( Petasites japonicus )의 bakkenolide B추출공정 최적화 김정헌

¹

․김용민

¹

․정헌식

¹

․최영환

²

․이경필

³

․임동순

³

․이영근

¹

*

(

¹

부산대학교 식품공학과 ,

²

부산대학교 원예생명과학과 ,

³

부산대학교 약학대 MIRCA)

머위 (Petasites japonicus) 잎에서 알레르기성 천식 억제 효능이 있는 bakkenolide B의 최적 추출공정을 반응표면 분석법을 이용하여 결정하였다 . Box-Behnken design에 따라 설정한 추출온도(80, 100, 120℃), 추출시간(30, 45, 60 min) 및 추출용매의 pH (5, 7, 9)를 독립변수로 하고, 추출물의 특성 즉, bakkenolide B의 함량과 항알러지의 측정