반응표면분석법에 의한 머위(Petasites japonicus)의 bakkenolide B추출공정 최적화

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

Abstract

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, $120^{\circ}C$), 추출시간(30, 45, 60 min) 및 추출용매의 pH (5, 7, 9)를 독립변수로 하고, 추출물의 특성 즉, bakkenolide B의 함량과 항알러지의 측정지표인 비만세포의 탈과립반응 억제효과를 종속변수로 하여 분석을 실시하였다. Bakkenolide B의 추출함량은 추출온도, 시간, pH의 유의적인 영향을 받았으며, 추출온도 $127.1^{\circ}C$, 시간 46.6분, 추출용매의 pH 7.7에서 최대치 121.6 ${\mu}g/g$이 예측되었다. 비만세포의 탈과립반응 억제효과의 회귀분석 결과는 유의성이 인정되지 않았고 낮은 상관계수를 보였다.

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, $120^{\circ}C$), 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 ${\beta}$-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 ${\mu}g/g$ with extraction conditions of $127.1^{\circ}C$, 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.

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].

Fig. 1.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 (X1), time (X2) and pH (X3) on the bakkenolide B content (Y1, extracted content from the leaves, μg/g) and inhibition effects on degranulation (Y2). 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.

Table 1.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 (Y1) and inhibition effects on degranulation (Y2). 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=b0+b1X1+b2X2+b3X3+b11X12+b21X2X1+b22X22+b31X3X1+b32X3X2+b33X3:

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), C18 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 (2×105 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% CO2 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 MgCl2, 0.1% BSA, and 1 mM CaCl2 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 Na2CO3/0.05MNaHCO3). 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 CH2Cl2 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 1H, 13C, and distortionless enhancement of polarization transfer nuclear magnetic resonance spectroscopy in CDCl3 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.

Table 2.Experimental data for response parameters of Petasites japonicas leave extracts in relation to the extraction conditions

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

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 (Y1) was significant (p＜0.05) with high correlation coefficient (R2= 0.8949), and the predicted model for Y1 was

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:

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-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 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.