Development of Supercritical Fluid Extraction of Glabridin from Glycyrrhiza glabra

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1 Development of Supercritical Fluid Extraction of Glabridin from Glycyrrhiza glabra On-line Number 939 Yun-Kyoung Cho, 1 Sang-Yun Lee, 1,2 Hyun-Seok Kim, 3 Jong-Hoon Ryu, 1 and Gio-Bin Lim 1 1 Department of Chemical and Biochemical Engineering, The University of Suwon, Kyunggi , Korea gblim@suwon.ac.kr 2 Department of Chemical Engineering, Yonsei University, Seoul 1-749, Korea sang-yun@suwon.ac.kr 3 R&D Center, BINo Tech Co., Kyunggi , Korea binotech@binotech.co.kr ABSTRACT The aim of this study is to investigate the feasibility of supercritical CO 2 extraction of glabridin, one of many bioactive components in licorice (Glycyrrhiza glabra). Such operating parameters as the type and amount of co-solvents, temperatures ranging from 4 to 8 o C, and pressures ranging from 12 to 5 bar were examined at a constant flow rate of 1 ml/min. The organic solvent extraction with methanol, ethanol and acetone as an extraction solvent was conducted for the comparison to the supercritical CO 2 extraction, producing.198 wt% of glabridin from licorice. The amount of glabridin within crude extracts was analyzed by high-performance liquid chromatography. The extraction yield of glabridin was of extremely small quantity, when using pure CO 2 as an extraction solvent at 34 bar and 8 o C. On the other hand, the yield of glabridin increased dramatically as the concentration of acetone in SCCO 2 increased up to 25% (v/v). Under a constant temperature, the yield of glabridin increased with increasing pressure from 12 to 5 bar; also, the decrease of temperature from 4 to 8 o C made the recovery of glabridin from licorice increased at a constant pressure. In addition, the purity of glabridin in extracts prepared by the supercritical CO 2 extraction increased about 2 ~ 4 times compared with the organic solvent extraction. KEYWORDS supercritical fluid extraction, glabridin, Glycyrrhiza glabra INTRODUCTION The root of the plant Glycyrrhiza glabra (licorice) is a herb of Leguminosae family. Licorice roots contain many bioactive components such as glycyrrhizin, liquiritin, liquiritenin, glabridin, and so on. Among these functional components, glabridin, a trace hydrophobic flavonoid found in the cork layer and decayed part of thickening roots (Hiroaki Hayashi, et al., 1996). has various bioactive properties: antiinflammatory, anti-oxidative effect of low density liprotein (LDL), skin whitening effect (melanogenesis inhibiting), etc. and, therefore, has been widely used in pharmaceutical, food, and cosmetic industries. Especially in the cosmetic industry, the demand of glabridin as a melanogenesis-inhibiting agent has been increased significantly. The conventional methods for the extraction of glabridin from licorice have several problems such as the occurrence of a huge amount of organic solvent waste, high energy consumption due to the long extraction time and the high extraction temperature employed. Therefore, developing alternative extraction techniques with better selectivity and efficiency is required. Since supercritical fluid extraction (SFE) as an environmentally responsible and efficient extraction technique was introduced in 1

2 the 197s, many researchers have extensively studied the SFE for the extraction and separation of active compounds from herbs and other plants (Mokey, et al., 1996). SFE is a technique that makes use of the high solvent power of supercritical fluids (SFs). SFs have both the solvent capacity of liquids and the mobility of gases due to the fact that the density and viscosity of SFs lie midway between those of a gas and a liquid. Moreover, the diffusivity of SFs, which is much higher than that of liquids, results in a considerable improvement of the extraction rate. The most important characteristic of SFs is to make it easy to adjust their solvating power by changing pressure and temperature. Carbon dioxide is one of the most widely used SFs because of its non-toxicity, non-flammability, non-explosiveness, low cost, and ease of removal from the extracted materials (Chiu, et al., 2). In this work, the effects of pressure, temperature, flow rate, and types and amount of co-solvents on the supercritical CO 2 extraction of glabridin from licorice were investigated. EXPERIMENTAL Materials Licorice roots native to Uzbekistan were ground to be below 15 mesh and stored at 4 o C. Highly pure carbon dioxide (99.995%) and glabridin (97%) as a standard material were obtained from Dongmin Co. (Korea) and from Wako (Japan), respectively. All solvents (HPLC garde) for HPLC analysis and extraction were purchased from Fisher (USA). Acetic acid (HPLC grade) was obtained from J.T. Baker (USA). Structure of glabridin Organic solvent extraction (OSE) Organic solvent extraction (OSE) of licorice roots powers was performed for 2 hr at 45 o C, while continuously stirring at 1rpm. During extraction, the extraction solvent was altered in an interval of 1 hr. Licorice extracts were centrifuged for 15min at 3rpm, and then the supernatant was collected. 8 ~ 1% aqueous methanol, 8 ~ 1% aqueous ethanol, and 1% acetone were employed as extraction solvents. The concentration (wt%) of glabridin within extracts was calculated as the ratio of weight of glabridin to that of licorice powder. All extractions were conducted in triplication, and the results were presented as mean±standard deviation. Supercritical fluid extraction (SFE) The SFE was carried out with an SFX 356 extractor (ISCO Co., Lincoln, USA) with two ISCO syringe pumps (26D & 1DX) as shown in Figure 1. Licorice powders (2.5g) were accurately weighed, and then placed into a 1 ml sample cartridge. The void volume in the sample cartridge was filled with glass wool. Each syringe pump to an extractor supplied both CO 2 and an aqueous co-solvent, and each extract was collected in a vial with around 1 ml of co-solvent. Various operating parameters such as temperatures (4 ~ 8 o C), pressures (12 ~ 5 bar), flow rates of supercritical fluids (.5 ~ 1.5 ml/min), and types and amount of co-solvents were examined for the supercritical CO 2 extraction of 2

3 glabridin from licorice. The efficiency of the SFE of on glabridin was expressed as a recovery (%) defined as the ratio of concentration of glabridin obtained by SFE to that by OSE. HPLC analysis 5 8 All licorice extracts were filtered with a.45 μm syringe filter (Whatman Co., USA), and then analyzed by HPLC. The HPLC system (Waters, USA) consists of a 6E multisolvent delivery pump, a 486 tunable absorbance detector, and an autosampler. The column used was a Shiseido RP-C18 (25(H) 4.6(D.I.) mm i.d.;5 μm, Toyko, Japan). Glabridin within licorice extracts was eluted with acetonitile /2% acetic acid (5/5 (v/v)) at a flow rate of 1.8 ml/min, and monitored at 282nm. Purity determination of glabridin Figure 1. ISCO model SFX TM 356 supercritical CO 2 extractor. (1) CO 2 storage system with dip tube, (2) syringe pump for CO 2, (3) syringe pump for modifier, (4) Control unit, (5) Mixer, (6) Modifier storage flask, (7) Waste container, (8) SFE extractor To determine the purity of glabridin, licorice extracts was evaporated using a rotary evaporator (RE 111, BÜCHI, Switzerland). After complete evaporation, concentrated licorice extracts were dried at 15 o C until dried licorice extracts maintained a constant weight, and then weighed accurately. The purity (wt%) of glabridin within licorice was defined as the ratio of weight of glabridin to that of completely dried licorice extracts. RESULTS Organic solvent extraction (OSE) Organic solvent extraction (OSE) was carried out for a comparison to the supercritical CO 2 extraction. The concentration and purity of glabridin obtained by various extraction solvents were listed in Table 1, and ranged from.175±.1 to.198±.4wt% and from.55±.5 to 4.27±.27wt%, respectively. The effect of extraction solvents on the concentration of glabridin had no great difference, even though the highest concentration Table 1. Concentration and purity of glabridin extracted from licorice by OSE using various solvents. Extraction solvent Concentration a Purity a 1% methanol.197±.4.72±. 8% aqueous methanol.195±..55±.5 1% ethanol.182± ±.8 95% aqueous ethanol.198±.4.96±.9 9% aqueous ethanol.175±.1.56±.4 8% aqueous ethanol.185±.1.53±.1 1% acetone.185± ±.27 a [mean±s.d.:wt%] Concentration; mg-glabridin / g-licorice 1 Purity; g-glabridin / g-licorice extract 1 3

4 of glabridin was found to be.198±.4wt% when using 95% aqueous ethanol as an extraction solvent. However, the purity of glabridin significantly decreased as the concentration of alcohol decreased, whereas 1% acetone, different from other aqueous alcohols, resulted in the highest purity (4.27±.27wt%) of glabridin. Supercritical fluid extraction (SFE) When used pure CO 2, the extraction recovery of glabridin was extremely small (about 4%). In order to enhance the extraction efficiency, ethanol or acetone were introduced as a co-solvent. The extraction recovery of glabridin from licorice was increased with the content of co-solvent in CO 2 as illustrated in Figure 2. Figure 2 shows the extraction recovery of glabridin with various concentrations (2.5% ~ 25% (v/v)) of acetone in CO 2 at 3 bar, 4 o C. From these results, we could know that the use of co-solvent is a major factor to enhance the extraction efficiency of glabridin from licorice and therefore used ethanol and acetone as a cosolvent for all experiments. The effect of temperature on the recovery of glabridin from licorice, when used 1% acetone and ethanol as a co-solvent to SCCO 2, was investigated at 3 bar and at a flow rate of 1 ml/min, and shown in Figures 3 and 4, respectively. For 1% ethanol (Figure 3), the extraction rate and recovery of glabridin decreased with increasing temperature, whereas the addition of 1% acetone to SCCO 2 had no significant influence on the extraction rate and recovery of glabridin Amount of co-solvent (%) Figure 2. Effect of modifier contents on the glabridin extraction recovery from licorice (operating conditions; 3 bar, 4 o C, 1% acetone and 1 ml /min) o C 6 o C 8 o C 4 o C 6 o C 8 o C Figure 3. Effect of temperature on the glabridin from licorice (operating conditions; 3 bar, 1% (v/v) of 1% ethanol and 1 ml /min). Figure 4. Effect of temperature on the glabridin from licorice (operating conditions; bar, 1% (v/v) of 1% acetone and 1 ml /min). 4

5 Our experimental results are different from those of Floch et al. (Floch, et al., 1998). They reported that the increase of temperature at a constant pressure enhances the extraction efficiency and rate, making desorption of target materials from sample matrices easier by increasing vapor pressure of target materials. In addition, 1% ethanol as a co-solvent had a greater effect on enhancing the recovery of glabridin than 1% acetone did. Figures 5 and 6 show the effects of pressure on the recovery of glabridin at a constant temperature. The recovery and extraction rate of glabridin from licorice increased with increasing pressure from 12 to 5bar, because the increase in pressure improved the solvating power of supercritical fluid. These experimental results show that the supercritical CO 2 extraction of glabridin was carried out near a retrograde region (Turner, et al., 1) bar bar 3bar 4bar 5bar bar bar 3bar 4bar 5bar Figure 5. Effect of pressure on the glabridin extraction rates from licorice (operating conditions; 6 o C, 1% (v/v) of 1% ethanol and 1 ml /min). Figure 7 shows the extraction profiles of glabridin for various flow rates of SCCO 2, at a operating condition of 3 bar and 4 o C with 1% (v/v) acetone. It is apparent that the increase of the flow rate leads to the increase of the recovery of glabridin extracted. The extraction recovery of glabridin was about 6% for 1min with a low flow rate (.5ml/min), but the extraction time was reduced 3 times for a higher flow rate (1.5ml/min) and final extraction recovery was increased about 25% for the extraction time of 1min. Table 2 compares the concentrations and purities of glabridin in the extracts obtained by OSE and SFE, respectively Figure 6. Effect of pressure on the glabridin extraction rates from licorice (operating conditions; 4 o C, 1% (v/v) of 1% acetone and 1 ml /min)..5 ml/min 1mL/min 1.5mL/min Figure 7. Effect of SCCO 2 flow rate on the glabridin extraction rates from licorice (operating conditions; 3 bar, 4 o C, 1% (v/v) of 1% acetone and 1 ml /min). 5

6 Table 2. Comparison of the concentration and purity of glabridin from licorice by organic solvent extraction and supercritical fluid extraction, respectively. Extraction method Extraction solvent Concentration a Purity a Organic Solvent Extraction Supercritical Fluid Extraction 1% acetone % ethanol CO CO 2 +1% acetone b CO 2 +1% ethanol b a Unit; wt% b Experimental condition; bar, 6 o C, 1 ml /min and 1vol% of modifier For the SFE the recovery of glabridin was about 82wt% and 89wt%, respectively, when used and ethanol as co-solvent compared to that of the OSE. However, the purity of glabridin in the extracts were improved 2~4 times compared with the OSE. CONCLUSIONS The content of glabridin in licorice was found to be.175 ~.198wt% from the results of various organic solvent extractions, and the highest recovery of glabridin was.198wt% in the case of 95% ethanol. The recovery of glabridin was extremely small, when used pure SCCO 2, but the addition of ethanol or acetone as modifier to SCCO 2 enhanced significantly the recovery of glabridin from licorice. When ethanol was used as a modifier (1% (v/v)), 7 ~ 87% of glabridin in the licorice was recovered and the purity of glabridin in the extracts was enhanced about 5 times, compared to that of organic solvent extraction at the range of 4 ~ 8 o C and 12 ~ 5bar. When acetone was used as a co-solvent the extraction rate was increased with the co-solvent content in SCCO 2 at 4 o C, 3bar. The addition of acetone to SCCO 2 increased the purity of glabridin in the extracts 2 ~ 4 times, compared to that of organic solvent extraction. In conclusion, the proposed SFE method has been found to be quicker and as efficient as the OSE, whilst being less toxic to the environment. In addition, it has been confirmed that the type of co-solvent is a major factor to the selective extraction of glabridin. REFERENCES Charolotta Turner, Jerry W. king, Lennart Mathiasson ; Supercritical fluid extraction and chromatography for fat-soluble vitamin analysis, J. Chromatography A, 936, 215~237 (1). F.L. Floch, M.T. Tena, A. Rios, M. Valcarcel ; Supercritical fluid extraction of phenol compounds from olive leaves. Talanta 46, 1123 (1998). Hiroaki Hayashi, Noboru Hiraoka, Yasumasa Ikeshiro, Hirobumi Yamamoto ; Organ specific localization of flavonoids in Glycyrrhiza glabra L., Plant Sci., 116, 233~238 (1996). K. L. Chiu, Y. C. Cheng, J. H. Chen, C. J. Chang, P. W. Yang ; Supercritical fluid extraction of Ginkgo ginkgolides and flavonoids, J. Supercrit. Fluids, 24, 77~87 (2). W.K. Mokey, D.A. Mulholland, M.W. Raynor, Phytochem. Anal. 7, 1 (1996). 6