Extraction of Quercetin from Nothopanax scutellarium Leaves via Ionic Liquid-based Microwave-assisted Extraction

Quercetin is a major flavonoid in most plant extracts.5 Quercetin is known for anti-oxidant, anti-inflammatory, anti-obesity, anti-hypertensive, vasodilatory, anti-hypercholesterolemic, antiatherosclerotic, anti-bacterial, and anti-biofilm effects.6-9 However, the conventional method for extracting this flavonoid uses large volumes of organic solvent. Furthermore, this process is limited by long extraction times and low active compound levels. In ethanol extracts of mangkokan Leaf an average at 1.53%, in ethyl fractions acetate an average at 4.79%, and ethyl acetate extract at 1.64%, ethyl acetate fraction at 5.83%.2-3


INTRODUCTION
Mangkokan (Nothopanax scutellarium Merr.) leaves have been used traditionally to treat various conditions such as alopecia, wounds, anuria, and body odor. 1 Based on prior studies, mangkokan leaves can promote hair growth. [2][3][4] Although the mechanism of this effect is unclear, the activity of mangkokan leaves was believed to be attributable to its flavonoid content.
Quercetin is a major flavonoid in most plant extracts. 5 Quercetin is known for anti-oxidant, anti-inflammatory, anti-obesity, anti-hypertensive, vasodilatory, anti-hypercholesterolemic, antiatherosclerotic, anti-bacterial, and anti-biofilm effects. [6][7][8][9] However, the conventional method for extracting this flavonoid uses large volumes of organic solvent. Furthermore, this process is limited by long extraction times and low active compound levels. In ethanol extracts of mangkokan Leaf an average at 1.53%, in ethyl fractions acetate an average at 4.79%, and ethyl acetate extract at 1.64%, ethyl acetate fraction at 5.83%. [2][3] Several studies used ionic liquid (IL)-based microwave-assisted extraction (MAE), which has great advantages for extracting plant with high extraction levels and short extraction times. [10][11] The main advantages of MAE compared with conventional extraction techniques include the use of smaller amounts of solvents, lower costs and improved extraction quality because of the reduced degradation of thermolabile constituents. [12][13][14][15][16]

Extraction methods
One gram of the dried mangkokan leaf sample was placed in a boiling flask and mixed with ILs, followed by MAE. The operating conditions included a running time of 10 min, IL concentration of 1.5 mol, 10 mL/g ratio, and power of 10 W. The extracts were filtered using filter paper.

Fractionation and separation with salt
The filtrates were fractioned using ethyl acetate. The extract was separated using 0.01 mol/L NaHCO 3 , K 2 HPO 4 , or NaCl (1:1 ratio). Each solution was vortexed for 20 s and then centrifuged for 15 min at 3000 rpm. The organic phase containing quercetin was the top layer, which was dried in a water bath and hydrolyzed using 4 N HCl. The dried extract was weighed, diluted with deionized water to 5 mL, and used for further analysis.

Flavonoid identification
Flavonoids were identified using TLC. Extracts were spotted on TLC plates using 5-µL microcapillary tubes. Quercetin standard was used for comparison. Elution was conducted in a closed chamber that was already saturated with n-hexane:ethyl acetate:acetic acid (7:2.5:0.5) as the mobile phase. The plate was dried, sprayed using AlCl 3 , and visualized under UV light for fluorescence. The Rf value was calculated.

Total flavonoid determination
The total flavonoid contents of extracts were determined using quercetin as a standard. Various amounts of quercetin (up to 10 mg) were dissolved in 10 mL of methanol to prepare concentrations of 10-100 ppm to determine the linearity of quercetin standard. After preparing the extract, as much as 1 mL was removed and mixed with 3 mL of 96% methanol, 0.1 mL of AlCl 3 , 0.1 mL of 1 M sodium acetate, and 2.8 mL of double-distilled water. The solution was incubated at room temperature for 30 min, and the absorbance was measured using a UV-Vis spectrophotometer at 410 nm. The total flavonoid content was calculated from the calibration curve, and the results are presented as mg/g.

Quercetin determination using HPLC
The quercetin contents of the extracts were analyzed using HPLC. HPLC was performed in the isocratic mode using a C18 bonded-silica gel column (5 µm, 150 × 4.6 mm, GL Sciences, Japan) and an LC-20AT HPLC system equipped with a UV-Vis detector (SPD-20A). The mobile phase was methanol:Aqua Pro Injection:glacial acetic acid (65:34:1). The UV detector wavelength was 370 nm. The flow rate was 1 mL/ min, the run time was 10 min, and the injection volume was 20 µL. The sample was filtered using a 0.45-micron syringe filter before analysis.

Flavonoid identification
The results of TLC analysis are presented in Figure 2. Rf for each extract was compared with that of quercetin standard (0.38).

Total flavonoid determination
The standard solution was analyzed for linearity. A linear response was identified (r 2 = 0.9955), and the equation was y = 0.0078x − 0.0052. Extracts obtained using different ILs and three different salts were analyzed via UV-Vis spectrophotometry. The [BMIM] Cl extract using NaCl as the separation salt had the highest flavonoid content (360.57 mg/g), as shown in Table 2.

Quercetin determination using HPLC
A linear response was observed (y = 65,196x − 270952, r 2 = 0.9998). The [BMIM] BF 4 extract separated using NaCl exhibited the highest quercetin content among the solvents (26.13 mg/g), as shown in Table  3. The chromatogram is presented in Figure 3.

DISCUSSION
Mangkokan leaves were extracted via ILMAE, and the extracts were fractionated using ethyl acetate. ILs have non-volatile characteristics. Organic solvents such as ethyl acetate can be used to separate nonvolatile compounds. 17 Then, the extracts were separated using different salts. This process precipitates residues in ILs, resulting in significant increases in extraction yields. 18 The determination of the total flavonoid content was conducted by a colorimetric method using a UV-Vis spectrophotometer. In this study, the highest flavonoid content in mangkokan leaf extract was achieved using [BMIM]Cl, whereas [BMIM]BF 4 provided the highest quercetin content. This could be attributable to the presence of other flavonoid contents or differences in the principle of analysis, but further research is needed to clarify this issue. UV-Vis spectrophotometry is one of the most widely used methods for the determination of total flavonoid content because of its low implementation costs and availability in the laboratory. [19][20] However, HPLC is a more sensitive and selective analytical procedure for measuring isolated compounds, and it is widely used for all classes of flavonoids. 20 The use of [BMIM]BF 4 for the success extraction of natural products was previously reported. [21][22] This finding is attributable to the fact that ILs can be used as both polar and non-polar solvents. 23 ILs can also be used in inorganic, organic, and polymer-solvent systems. 24 In addition, ILs are more environmentally benign solvents than the volatile organic solvents commonly used for extraction. 25 From this study, anion and cation is strongly influence an extraction result. Different anion and cation were studied and differences in their extraction yield were readily apparent, as shown in Table 3. The results showed that the ionic liquids based on Clwere the more efficient for total flavonoid, with BF 4 − being the most efficient for quercetin. The different of cations including Bmim+ and Hmim+ were evaluated and the results are shown in Table 3. The extraction yield decreased when the alkyl chain length of the cation was increased from butyl to hexyl. The results suggested that the increasing alkyl chain length influences the extraction yield. Proton acidity and hydrophobicity for cations increased from buthyl to hexyl at the 1-position of the 1-alkyl-3-methylimidalizium ring. Both the proper hydrogen bonding and hydrophobic interactions of Bmim+ resulted in stronger solvation interactions with flavonoids followed by higher extraction yields than [Hmim] + .
The different of salt using in partition affects extraction yields that different for each ionic liquids. Sodium cloride obtains the highest extraction quercetin compared with other salt in mangkokan leaves extraction using [BMIM]BF 4 solvent. Each ionic liquids has different optimum partition salt. It is because the mechanism of the saltingout effect is very complex due to the interplay of different types of interaction between the solutes and the solvent. Hydration theories and the related water-structuring effects (kosmotropy) and semiquantitative way the intensity of the salting-out effects.
There were several factors that affect ionic liquid in this extraction process that has not been conducted in this study; extraction time and microwave power, temperature, and plant characteristics that must be consider for further study. The quantity of the extracted compound can be increased by increasing the extraction time, but it increase the risks of thermolable component. 26 The extraction time and microwave power was very sensitive parameters and both was needed to be handled simultaneously. 27 A combination of low or medium power with longer exposure is generally chosen to optimize MAE procedures, but study showed that various strengths from 500 W to 1000 W have no significant effect on flavonoid yields. 26 Temperatures in the range 60-90 °C have been used most often in MAEs carried out at atmospheric pressure. 27 The choice of extraction temperature depends on the stability and extraction results of the active compound target. Sample characteristics affect MAE performance. The small particle size will cause difficulties in separating the extract from the residue and additional cleaning steps may have to be used. In addition, fine samples treated with solvents for 90 minutes before extraction can improve MAE heating efficiency, increase diffusion and increase the mass transfer of active compounds to the solvent. However, in a number of reported cases, a long pretreatment time did not improve the extraction results because the active ingredients might have left the sample matrix before extraction. 28

CONCLUSIONS
The highest quercetin content was observed for mangkokan leaves extracted using [BMIM]BF 4 combined with NaCl as the separation salt (26.13 mg/g), confirming its utility for extracting quercetin from this medicinal plant.