Extraction of Luteolin and Apigenin from Leaves of Perilla Frutescens (L.) Britt

Solvent Extraction Research and Development, Japan, Vol. 21, No 1, 55 – 63 (2014)

Extraction of Luteolin and Apigenin from Leaves of Perilla frutescens (L.) Britt. with Liquid Carbon Dioxide

Kenji MISHIMA1*, Ryo KAWAKAMI1, Haruo YOKOTA1, Takunori HARADA1, Takafumi KATO1, Hirofumi KAWAMURA2, Kiyoshi MATSUYAMA3, Salim MUSTOFA4, Fauziyah HASANAH5, Yusraini Dian Inayati SIREGAR5, Lily Surayya Eka PUTRI5 and Agus SALIM5 1Department of Chemical Engineering, Faculty of Engineering, Fukuoka University, 8-19-1, Nanakuma Jonan-ku, Fukuoka 814-0180, Japan 2Department of Seasoning & Foods Division, San-Ei Gen F.F.I., Inc., 1-1-11, Sanwa-cho, Toyonaka, Osaka 561-8588, Japan 3Department of Biochemistry & Applied Chemistry, Kurume National College of Technology, Komorino 1-1-1, Kurume, Fukuoka 830-8555, Japan 4Research Center for Technology of Nuclear Industrial Material, Indonesia Nuclear Energy Agency, Gedung 42, Kawasan PUSPIPTEK Serpong, Tangerang Selatan, Banten 15419, Indonesia 5Faculty of Science and Technology, Syarif Hidayatullah State Islamic University (UIN) Jakarta, JL.Ir.H.Juanda Ciputat, Tangerang, Indonesia (Received September 2, 2013; Accepted October 12, 2013)

The extraction of luteolin and apigenin from the leaves of Perilla frutescens (L.) Britt. by liquid carbon dioxide (CO2) was carried out at 5, 20 and 25 ºC. The extraction pressure was from 8.5 to 14 MPa. The extraction yields were compared with yields obtained by other solvent extraction methods, such as supercritical CO2 extraction and conventional aqueous alcohol extraction. We conducted qualitative and quantitative analyses for luteolin and apigenin in the extract by HPLC and analyzed the extraction behavior.

The effect of two operating parameters, temperature and pressure of liquid CO2, on the extraction yield was investigated using the single-factor method. The yields of luteolin and apigenin in the extraction were significantly improved by the operating temperature, whereas a change in the selectivity of the extract was not observed.

1. Introduction Perilla frutescens (L.) Britt. var. crispa (Thunb.) W. Deane is a common mint family plant which has been cultivated in China, India, Southeast Asia and Japan. It is also called Japanese basil. It has been traditionally used not only for edible use but for treating diseases like asthma, coprostasis and sitotoxism. Previous investigations have discovered that some polyphenols extracted from P. frutescens exhibit pharmacological activities such as anti-allergy, anti-inflammatory [1] and anti-oxidant [2]. Among these components, luteolin and apigenin, the common flavones which are contained in a wide range of beneficial or dietary plants including weld (Reseda luteola L.), carrot (Daucus carota subsp. sativus), bell pepper (Capsicum annuum L.), celery (Apium graveolens var. dulce (Mill.) Pers.), garden parsley (Petroselinum

- 55 - crispum (Mill.) Fuss), chamomile (Matricaria chamomilla L.) and rosemary (Rosmarinus officinalis L.), has recently been attracting attention as anti-inflammatory, anti-allergy [3] and anti-bacterial [4] reagents. The chemical structures of luteolin and apigenin are shown in Figure 1.

(a) luteolin (b) apigenin Figure 1. Chemical structures of (a) luteolin and (b) apigenin existing in the leaves of P. frutescens.

Hitherto many researchers have reported various elution and separation methods for flavones from several types of leaves. Generally substances were prepared with organic solvents such as ethanol, methanol, acetone and toluene. Whereas some researchers developed and applied sophisticated methods [2], simple maceration at a range of temperatures is broadly accepted and common practice [1,3]. Recently, supercritical carbon dioxide (SC-CO2) extraction has provoked considerable interest as a separation technique in many fields [5–13]. In our previous paper [6,7,13], the possibility of extraction and separation of flavonoids by using SC-CO2 was demonstrated. Carbon dioxide (CO2) is nonflammable, nontoxic, inexpensive and environmentally benign and its critical condition is easily accessible. Despite these advantages, the extraction of flavones from leaves with SC-CO2 has been little reported because of the excess sensitivity of the solvent power of SC-CO2 and low solubility of flavones in SC-CO2. In general the cause of the excess sensitivity of the solvent power of the solvent is considered to be the change in density of the solvent. The density change of SC-CO2 takes place easily with a change in the operating pressure. But the change of the density of liquid CO2 is very small with a change in the operating pressure, when the gas phase and the liquid phase coexist in an extraction vessel. So the excess sensitivity of solvent power with the change of the operating pressure can be avoided, when liquid CO2 is used as the solvent instead of

SC-CO2. Here we describe the possibility of luteolin and apigenin extraction from the leaves of P. frutescens by liquid carbon dioxide (CO2). Also, we have optimized the conditions of extraction using liquid CO2, and provided a comparison between these little-used and traditional methods.

2. Experimental 2.1 Reagents Leaves of P. frutescens were purchased from local markets in Fukuoka. Luteolin and apigenin were purchased from Wako Co. Ltd. Methanol, ethanol and acetic acid were purchased from Wako Co. Ltd.

Analytical grade methanol and ethanol were used as solvents in conventional liquid extraction and as

- 56 - solvents in HPLC analysis. High-purity CO2 (more than 99 %, Fukuoka Sanso Co. Ltd.) was used as received.

2.2 Liquid CO2 Extraction Procedures Leaves of P. frutescens were ground and powdered with a freezer mill 6750 (SPEX CentriPrep Co. Ltd.). The average particle diameter was less than 1 mm. Samples were stored in a cold and dark place because of their photosensitivity. The P. frutescens leaf powders were put in a high-pressure cell (an extraction cell) of a liquid CO2 extraction apparatus (SFC; super200, JASCO Co. Ltd.). A more detailed description of the apparatus and operating procedures have been given in our previous papers [6,7]. A high-pressure cell (Akico Co., SCV50A), approximately 50 cm3 in volume, was used. The system pressure was controlled by means of a back-pressure regulator (880-81, JASCO, accurate to 0.1 MPa) and monitored by a digital pressure gauge (Shinwa Electronics Co., model DD-501, accuracy ± 0.3%). The temperature was controlled within ± 0.1 ºC with a water bath. The P. frutescens leaf powders (0.25 g) were placed in the high-pressure cell for liquid CO2 extraction and supercritical CO2 extraction. CO2 was supplied at a constant flow rate (2 cm3/min) into the extraction cell by a HPLC pump (SCF-get, JASCO), and the pump-head was cooled by a cooling unit to liquefy the CO2 from the gas cylinder. CO2 was pumped into the high-pressure cell through a stainless steel pre-heater tube (length: 3 m, diameter: 1/8 in). The pressure of the stream was controlled at 14 MPa by adjusting the backpressure regulator (880-81, JASCO). The backpressure regulator valve was warmed to avoid CO2 solidification caused by the Joule–Thomson effect. From time to time the effluent from the extraction cell was collected in a sample trap (i.e. ethanol). After the desired time had elapsed, the residue in the tubes was washed with liquid CO2 and collected in another sample trap. The extraction process was essentially affected by the solute solubility. The solute solubility was sensitive to the temperature and density of the CO2. In our study, the effects of the operating temperature and pressure were studied in order to optimize the extraction yield. The time for extraction was set at 60 min.

We took the critical temperature (Tc=31 ºC) and pressure (Pc=7.8 MPa) of CO2 into consideration and chose the extraction conditions in order to avoid the supercritical condition which is too sensitive to operate. The extraction was carried out at temperatures of 5, 20 and 25 ºC, and pressures of 8.5, 10 and 14 MPa. For comparison, supercritical CO2 extraction was carried out at 40 ºC and 10 MPa. After extraction, the extracts were collected at an expansion valve using a U-tube ethanol trap at atmospheric pressure. The extracts left behind in the high-pressure cell were also collected and separated from the P. frutescens leaf powder by filtration with an 80 % ethanol aqueous solution. The compositions of the collected samples were analyzed by HPLC (High performance liquid chromatography). 2.3 Conventional Liquid Alcohol Extraction Procedures

So as to compare the potential for liquid CO2 extraction with the conventional aqueous alcohol (methanol or ethanol) maceration more closely, we also optimized the maceration time. Extraction was performed by using a water bath (Eyela SB-24, Tokyo Rikakikai Co., Ltd.) with a shaker (Eyela SS-8) and a heating circulator (Eyela T-80) which set the temperature at 25 ºC. Sampling was carried out at 15, 30, 60 and 120 min from the starting time of the extraction. 2.4 HPLC Analysis The HPLC system consisted of a Tosoh LC-8010 system equipped with a UV detector. The detection wavelength was set at 343 nm. Separation of the extract was obtained using a TSK-GEL (Tosoh Co.)

- 57 - ODS-80Ts column (150  4.6 mm), set at 40°C. The injection volume was 100 μL. Two mobile phases were employed during the separations, (A) methanol for HPLC grade and (B) 1 % acetic acid in water. The methanol gradient profile was showed in Table 1. The flow rate of the solvent was set at 1.0 mL/min.

3. Results and Discussion We modified the HPLC profiles which Cristea et al. originally developed [14] to separate more accurately the components in the extraction medium. To estimate the validity of our elute composition, the liquid CO2 extract and standard samples of luteolin and apigenin were analyzed. For example of the results of the liquid CO2 extraction is shown with the gradient curve for the HPLC analysis in Figure 2. Through the analysis of standard luteolin and apigenin samples, we confirmed that the peaks appearing at 43 min and 47 min after extraction were luteolin and apigenin, respectively in Figure 3.

Table 1. Rate of change of the methanol gradient for HPLC analysis. Time [min] 0 25 40 50 60 65 75 Methanol [vol%] 25 40 60 90 90 5 5

100

130 80

60

80 mV 40 30

20 Gradient of methanol [% v/v] [% methanol of Gradient 0 0 20 40 60 Time [min]

Figure 2. HPLC chromatogram of the extract of leaves of P. frutescens by liquid CO2 extraction at 25 ºC and 10 MPa and the methanol gradient curve.

- 58 - 1200 1000 800

600

pigenin 46.95 pigenin

uteolin 43.43 uteolin

l a

mV 400 200 0 -200 0 20 40 60 Time [min]

Figure 3. HPLC chromatogram and retention time of pure materials.

Additionally, the most appropriate extraction time for methanol and ethanol extraction at constant temperature (25 ºC) was investigated. As described previously, the extraction was performed using a heating and shaking apparatus, and sampling was carried out at several time points. Qualitative and quantitative information was obtained by HPLC. Figure 4 shows an example. The yield of apigenin generally increased over 0-60 min, and then saturation of the extract concentration was observed. Therefore, we concluded that the appropriate time of treatment was circa 60 min.

65 60

55 50 45 40 35 30 Mass of extract [μg]ofextractMass 25 20 0 50 100 Extraction time [min]

Figure 4. Rate of change of the yield of apigenin extracted from 1 g P. frutescens leaf using 80 vol% methanol (aq.) and 80 vol% ethanol (aq.) at 25 ºC. Symbol ▲ shows extraction using methanol, whereas ■ shows extraction using ethanol.

- 59 - Next, we studied the effect of temperature on the total amount of luteolin and apigenin extracted by liquid CO2. The temperature of the water bath was set at 5, 20 and 25 ºC. The liquid CO2 density at various temperatures and pressures was checked using the physical property data available from the literature [15]. The pressure was set at 10 MPa and the extraction was carried out for 60 min. The densities of the liquid 3 CO2 at 5, 20 and 25 ºC at 10 MPa are 945, 849, and 812 kg/m , respectively. The effect of temperature on the extraction yields of each substance is showed in Figure 5 (a). In general, the extraction yield increased with increasing temperature. This tendency was observed between 5 ºC and 20 ºC for luteolin and apigenin. The yield of luteolin is greatest at 20 ºC and the yield of apigenin is greatest at 25 ºC in this work. We should take account of the high-density gas phase, which exists over the liquid phase of CO2 and is too sensitive to pressure to operate. This result shows that the eluent obtained at 20 ºC had the highest concentration, implying that lower or higher temperatures were not suitable for luteolin and apigenin extraction from P. frutescens leaves in order to have stable extraction operation in the liquid CO2 extraction.

Furthermore, we examined the effect of liquid CO2 pressure on the yields of luteolin and apigenin extracted by liquid CO2. The pressure was set at 8.5, 10 and 14 MPa. The temperature was set at 20 ºC because the temperature at which unstable phases of gas and liquid exist near the critical temperature of

CO2 should be avoided. The densities of liquid CO2 at 8.5, 10, and 14 MPa at 20 ºC are 804, 844, and 895 kg/m3, respectively. The extraction was carried out for 60 min. The yields of luteolin and apigenin in ethanol are showed in Figure 5 (b). As observed before, the yields of luteolin and apigenin were almost the same. This result shows that the eluent obtained at these pressures gave similar concentrations, implying that the yields of luteolin and apigenin were insensitive to the operating pressure of liquid CO2. The effects of operating temperature and pressure on the extraction yield are similar to the results of other liquid CO2 extractions [16,17]. Under this extraction condition (temperature = 20 ºC, pressure = 8.5, 10, and 14 MPa), the gas phase and the liquid phase coexist in the extraction vessel. The change of the density of liquid CO2 is very small with a change in the operating pressure. So the solvent power of liquid CO2 is considered to be almost constant at that condition.

(a) (b) 35 30 25 20 15 10

Mass of extract [μg] ofextract Mass 5 0 0 10 20 30 Extraction temperature [ C]

Figure 5. (a) Temperature and (b) pressure dependence of the extract yields using liquid CO2. Extraction carried out with 0.25 g of P. frutescens leaf (a) at 10 MPa, and (b) at 20 ºC. Symbol ▲ shows extracted mass of apigenin, whereas ■ shows that of luteolin.

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The yields of each extracts are also slightly changed with the extraction methods as shown in Figure 6 and Figure 7. Results of the supercritical CO2 extraction carried out at 40 ºC and 10 MPa are also shown in these Figures. SC-CO2 is a homogeneous gas phase and the density changes readily with the change in operating pressure. Both the results of SC-CO2 extraction and liquid CO2 extraction carried out with 0.25 g of P. frutescens leaf calculated to correspond to the results with 1 g of P. frutescens leaf are shown together with the results of conventional aqueous alcohol extraction carried out with 1 g of P. frutescens leaf in Figure 7. The yields of luteolin and apigenin (8.8 μg/g, 2.3 μg/g from 0.25 g of P. frutescens leaf), are calculated as 35 and 9.2μg/g from 1 g of P. frutescens leaf, respectively. The SC-CO2 extraction and liquid

CO2 extraction were carried out for 60 min and the conventional aqueous alcohol extraction was carried out for 120 min.

350

300 Liquid CO2 250

200 SC-CO2

150 mV 100 80% MeOH 50 0 80% EtOH

0 20 40 60 Time [min]

Figure 6. HPLC chromatograms of the extract using EtOH, MeOH, SC-CO2 and liquid CO2.

- 61 - luteolin

apigenin

Figure 7. Comparison of luteolin and apigenin yields extracted from 1 g of P. frutescens leaf.

The SC-CO2 extraction and liquid CO2 extraction were carried out for 60 min.

4. Conclusion

Luteolin and apigenin were successfully extracted from the leaves of P. frutescens by liquid CO2 extraction. Suitable operating conditions for the experimental system were determined by analyzing the effect of process parameters, such as temperature and pressure, as follows: T = 20 ºC, P = 10 MPa. Under optimal operating conditions, the yields of luteolin and apigenin were 32 μg, and 9.2μg from 1 g of P. frutescens leaf, respectively.

Acknowledgment This work was partially supported by a Grant-in-Aid for Scientific Research (Grant Nos. 20560707 and 23560913).

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